key: cord-002705-ntokyoai authors: nasir, nazrila hairin; mohamad, mohazmi; lum, lucy chai see; ng, chirk jenn title: effectiveness of a fluid chart in outpatient management of suspected dengue fever: a pilot study date: 2017-10-04 journal: plos one doi: 10.1371/journal.pone.0183544 sha: doc_id: 2705 cord_uid: ntokyoai introduction: dengue infection is the fastest spreading mosquito-borne viral disease in the world. one of the complications of dengue is dehydration which, if not carefully monitored and treated, may lead to shock, particularly in those with dengue haemorrhagic fever. who has recommended oral fluid intake of five glasses or more for adults who are suspected to have dengue fever. however, there have been no published studies looking at self-care intervention measures to improve oral fluid intake among patients suspected of dengue fever. objective: to assess the feasibility and effectiveness of using a fluid chart to improve oral fluid intake in patients with suspected dengue fever in a primary care setting. methods: this feasibility study used a randomized controlled study design. the data was collected over two months at a primary care clinic in a teaching hospital. the inclusion criteria were: age > 12 years, patients who were suspected to have dengue fever based on the assessment by the primary healthcare clinician, fever for > three days, and thrombocytopenia (platelets < 150 x 10(9)/l). both groups received a dengue home care card. the intervention group received the fluid chart and a cup (200ml). baseline clinical and laboratory data, 24-hour fluid recall (control group), and fluid chart were collected. the main outcomes were: hospitalization rates, intravenous fluid requirement and total oral fluid intake. findings: among the 138 participants who were included in the final analysis, there were fewer hospital admissions in the intervention group (n = 7, 10.0%) than the control group (n = 12, 17.6%) (p = 0.192). similarly, fewer patients (n = 9, 12.9%) in the intervention group required intravenous fluid compared to the control group (n = 15, 22.1%), (p = 0.154). there was an increase in the amount of daily oral fluid intake in the intervention group (about 3,000 ml) compared to the control group (about 2,500 ml, p = 0.521). however, these differences did not reach statistical significance. conclusion: this is a feasible and acceptable study to perform in a primary care setting. the fluid chart is a simple, inexpensive tool that may reduce hospitalization and intravenous fluid requirement in suspected dengue patients. a randomized controlled trial with larger sample size is needed to determine this conclusively. trial registration: international standard randomized controlled trial number (isrctn) registry isrctn25394628 http://www.isrctn.com/isrctn25394628 to assess the feasibility and effectiveness of using a fluid chart to improve oral fluid intake in patients with suspected dengue fever in a primary care setting. this feasibility study used a randomized controlled study design. the data was collected over two months at a primary care clinic in a teaching hospital. the inclusion criteria were: age > 12 years, patients who were suspected to have dengue fever based on the assessment by the primary healthcare clinician, fever for > three days, and thrombocytopenia (platelets < 150 x 10 9 /l). both groups received a dengue home care card. the intervention group received the fluid chart and a cup (200ml). baseline clinical and laboratory data, 24hour fluid recall (control group), and fluid chart were collected. the main outcomes were: hospitalization rates, intravenous fluid requirement and total oral fluid intake. among the 138 participants who were included in the final analysis, there were fewer hospital admissions in the intervention group (n = 7, 10.0%) than the control group (n = 12, 17.6%) (p = 0.192). similarly, fewer patients (n = 9, 12.9%) in the intervention group required intravenous fluid compared to the control group (n = 15, 22.1%), (p = 0.154). there was an plos approximately 50 million dengue infections occur yearly, making it the fastest spreading mosquito-borne viral disease in the world [1, 2] . in 2002, dengue deaths amounted to 12,000 in south-east asia, 4,000 in the western pacific and 2,000 in america [3] . dengue has a wide spectrum of clinical presentations, ranging from a non-severe illness whereby outpatient treatment would suffice, to severe disease, hallmarked by plasma leakage requiring hospitalization. with increasing severity of plasma leakage, hypovolaemic shock ensues and the morbidity and mortality rises sharply. according to one study, the estimated cost of a non-fatal ambulatory case was us$ 514, and the cost for a non-fatal hospitalized case was us$ 1,491 [4] . thus, a hospitalized dengue case cost on average three times more than an ambulatory case. moreover, dengue has considerable impact on the quality of life (qol). one study found that all dengue patients experienced a drastic fall in their quality of life starting from the onset of symptoms [5] . furthermore, dengue patients are more likely to be susceptible to dehydration caused by the high fever, vomiting, diarrhea and associated anorexia, which, if not carefully monitored and treated, may lead to shock, particularly in those with dengue haemorrhagic fever. [6] . intravenous fluid is part of the therapy given for suspected dengue patients with dehydration. however, fluid overload frequently occurs during the management of dengue patients and is a risk which is not present when fluid is consumed orally. despite the fact that oral rehydration therapy is incorporated in the standard outpatient management of dengue, the discussion on the use of such therapy for dengue is scarce. the study conducted in nicaragua was the first study to examine the effect of fluid intake at home for dengue cases [6] . the results from this study showed that an oral fluid intake of 5 glasses per day decreases the risk for hospitalization of dengue patients [6] . in fact, based on this study, who had revised their dengue guidelines in 2009 and it includes the recommendation of oral fluid intake of five glasses or more for adults who are suspected to have dengue fever (e.g. milk, fruit juice, oral rehydration solution, barley, rice water or coconut water) [1] . the authors also postulated that oral fluid intake may have an effect on the severity of dengue [6] . however, causality cannot be confirmed due to the observational nature of this study. thus, the authors of this study suggested prospective interventional studies be carried out to definitively demonstrate this effect [6] . it is important to confirm this because there are many advantages in encouraging oral fluid intake for suspected dengue patients, in terms of averting the risk of fluid overload with all its complications, cost savings in preventing intravenous fluid and the need for admission and possibly better quality of life when admission is avoided. since the study conducted in nicaragua, other studies have been conducted with regards to oral fluid therapy in dengue. a non-randomized controlled study was conducted in taiwan comparing the effects of oral rehydration with intravenous fluid replacement in adult patients with non-shock dengue haemorrhagic fever [7] . the study found that both interventions were equally effective. however, the sample size of this study was small and the participants were recruited from the hospital in-patient. there was one interventional, randomized trial on syndromic approaches for undifferentiated fever and dengue done at the primary health care setting in vietnam [8] . however, although this study was conducted in primary care, it did not focus on oral fluid intake in dengue patients. most importantly, there have been no published studies looking at self-care intervention measures to improve oral fluid intake among patients suspected of dengue fever. should there be an association between increased oral fluid intake and a decreased risk of hospitalization, it would be beneficial to explore practical methods of improving the oral fluid intake in patients with dengue fever. the study should be conducted in the primary care setting where there is immense potential for early intervention. thus, this pilot randomized controlled study aimed to assess the feasibility of using a fluid chart to improve oral fluid intake in patients with suspected dengue fever in a primary care setting. this was an unblinded, parallel-group study with balanced randomization (1:1 ratio) for the two groups of patients. the study was conducted from 1 st june 2010 -30 th july 2010 (including recruitment and follow up) in a single centre i.e. primary health care clinic in the university malaya medical centre (ummc). this study was part of a master dissertation. although the study was completed in 2010, there was a delay by the author in writing it up as a full manuscript. ethical approval was obtained from the ummc medical ethics committee. written informed consent was obtained from the participants. for child participants, written consent was obtained from the participants' parents or guardian. this method was approved by the ethics committee. the inclusion criteria were: age > 12 years, patients who were suspected to have dengue fever based on the assessment by the primary healthcare clinician, history of fever for three days or more, and thrombocytopenia (platelets < 150 x 10 9 /l) [9] [10] [11] . the exclusion criteria were: patients with any type of current or active malignancy, human immunodeficiency virus (hiv) and other immunodeficiency states due to complicated and multi-factorial aetiologies. patients who were not given a follow-up appointment to review the clinical progression and to repeat the full blood count test were also excluded. this was to ensure that the patients enrolled in this study were similar to the target population, which were suspected dengue patients who were being followed up for outpatient monitoring. similarly, those who were assessed on the first outpatient clinic visit and found to require hospital admission were excluded from this study. the process of recruitment and follow-up of the participants is shown in fig 1 while the data collection process of the trial is shown in fig 2. all patients presenting to the clinic with a history of fever for 3 days duration or more were identified by the triage counter nurses. the phases of dengue fever were based on the day of illness: febrile phase-day 1 till day 3 of illness; critical phase-day 4 to day 6 of illness; recovery phase-day 7 of illness onwards. it was standard practice for the clinic that such patients would have a full blood count test done mostly before the patient's consultation with their doctors. online results of clinical investigations of patients were traced and those fulfilling the inclusion criteria were invited to participate in the study. the initial assessment of the patients and the subsequent follow-up consultations were conducted by the respective doctors working at the clinic. after the consultation, the researcher and trained research assistants obtained consent from the patient before they were randomized into either the control or intervention group. simple randomization was done, with a 1:1 allocation ratio, using the table of random numbers. the table of random numbers was a standard table obtained from a statistics textbook. the researchers referred directly to the table during the randomization process (no opaque envelope was used). all patients recruited into the study, regardless of randomization group, were given the standard care in the form of the dengue home care card. the information on this card was read out to all participants. the control group would be asked to recall their twenty four hour fluid intake, and details on what type of fluids was consumed. this information was obtained during recruitment and each follow-up. the intervention group participants were given a plastic cup of uniform size (200ml), along with a twenty-four hour fluid chart. these patients were instructed to use the cup for drinking any form of fluid they wished to consume for the duration of their participation in the study. a fluid chart was used to record the detailed fluid intake (s1 file) and verbal instructions were given on how to fill in the fluid chart. should the patient receive intravenous fluid during the following twenty-four hours either from a clinic or from the emergency department, they would record in the fluid chart given the time and amount of bottles received. in addition, demographic, clinical and laboratory data were collected. all the patients recruited into the study were followed up until they had improved clinically and/or biochemically as determined by their doctors. this means that the duration of follow up varies between different participants. data collection involved 24-hour fluid recall for the control participants, and the completed fluid charts from the intervention group. the research team would give out a new fluid chart if another appointment had been given to the participant. for both groups, the following data were recorded for each subsequent clinic follow-up: blood investigations (haemoglobin, haematocrit levels, total white cell count, platelets, and dengue serology) and temperature readings. the participants were managed by the respective doctors. as long as some data were collected from the participant, they would be included in the analysis. only those participants who defaulted directly after recruitment into the study (meaning that they only came to clinic during recruitment) were excluded from the analysis as no clinical and oral fluid consumption data could be collected from them. for those who defaulted their follow-up with the research team, an attempt to contact the participant via telephone was made, with a maximum of three attempts. the reasons for defaulting follow-up were elicited and if the patient was admitted, details regarding the admission were obtained. the only published study on oral hydration in dengue patients [7] did not state their estimated sample size. for this pilot study the sample size was time driven, and the pre-specified time limit for data collection was two months. the minimum recruited number of participants required for each study group was 30 patients, giving a minimum number of 60 patients in total. however, the research team strived to recruit as many patients as possible during those two months. this is to determine the actual treatment effect size, and the participant recruitment and retention rates to be used in the sample size calculation for the main evaluation trial. the data for this pilot study were expressed either as the mean ± standard deviation or as frequencies and proportions. differences in data between the control and intervention group were analyzed using the student's t test for continuous variables. the chi-squared test (χ 2 test) or the fisher's exact test was used for categorical variables, whichever was appropriate. a p value < 0.05 was considered as statistically significant. the statistical software utilized for the analysis was spss version 16.0 (spss inc. cary, nc). the main outcomes were hospitalization rates, need for intravenous fluid treatment and amount of oral fluid intake. the baseline socio demographic and clinical profiles were similar in both the intervention and control groups (table 1) . most participants were in the critical phase of dengue infection, with slightly more participants in the recovery phase in the intervention group. however, there were more participants who had a documented body temperature > 38˚c in the control group than the intervention group. more than half of the participants were confirmed to have dengue fever. the pilot study found a positive trend towards a reduction in the need for hospitalization and intravenous fluid treatment for the participants in the intervention group compared with those in the control group (table 2) . among the 138 participants who were included in the final analysis, there were fewer hospital admissions in the intervention group (n = 7, 10.0%) than the control group (n = 12, 17.6%) (p = 0.192). similarly, fewer patients in the intervention group (n = 9, 12.9%) required intravenous fluid compared to the control group (n = 15, 22.1%) (p = 0.154). the mean peak haematocrit, the difference in haematocrit values between the first two clinic visits, cut off points reached and the haematocrit fluctuation ! 20% for both groups were similar. the mean nadir platelet count and the mean difference in platelet levels between the first two clinic visits were also similar between both groups. the average daily fluid intake was higher in the intervention group (about 3,000 ml) compared to the control group (about 2,500 ml), but the difference did not reach statistical significance. this is the first study that investigated the use of a fluid chart in the management of suspected dengue patients in a primary care setting. in this pilot study, the intervention group had 7.6% fewer hospital admission and 9.2% fewer participants requiring intravenous fluid therapy compared with the control group. the mean daily fluid intake in the intervention group was 500 ml more than that in study group but this increase was not statistically significant. although the differences observed did not reach statistical significance, there is a positive trend favouring the intervention group. moreover, even a small reduction in hospital admission and the need for intravenous fluid for suspected dengue patients can potentially improve the quality of life of patients and cost savings to the economy of dengue endemic countries. the hospitalization rates of patients with suspected dengue fever in this study were 17.6% and 10% in the control and intervention groups respectively. these hospitalization rates were much lower than those during the preceding three months to the recruitment phase: 55.4% in march, 40% in april, and 63.4% in may 2010. [12] a prospective observational study in the same clinic in 2008 enrolled 214 patients aged 16 years and above with 72 hours of undifferentiated fever, 65% of whom had a laboratory confirmed diagnosis of dengue. of the 140 patients with dengue, 11.4% developed plasma leakage and 37.1% required hospitalization. [13] the marked reduction in hospitalization rates in both the control and intervention groups compared to previous observational studies might be due to the fact that the participants from both groups received instructions from the research team who had read out the advice written on the dengue home care card to each participant. the instruction included specific advice encouraging patients to increase their fluid intake to at least 5 cups of water a day. this practice of reading out the advice written on the dengue home care card was not the standard practice of the doctors practicing in the study centre, neither is it a practice in most primary care clinics in malaysia. the usual standard practice in primary care would be to give the dengue home care card to the patient with no verbal advice accompanying it, and it is up to the patient to read the card themselves. it is possible that reading out the dengue home care card became an active form of intervention in the control group, thus resulting in the control group participants to drink more fluids than they normally would had they received standard care. this could be a form of double intervention, and the difference in fluid intake between the two groups might be more pronounced if the dengue home care card was not read out to the participants, simulating the standard practice in primary care more closely. thus, the true impact of the fluid chart on the fluid intake for suspected dengue patients might be underestimated in the current results. however, the intervention group seemed to have consistently maintained a higher mean fluid intake from day 1 to day 6 of the clinic consultations. another possibility is that there might be other factors influencing the amount of fluid intake consumed by each participant, not just the fluid chart. however, this was not explored in this pilot study. the fluid chart is an intervention tool which was designed to measure and document accurately the patient's fluid intake which they should fill prospectively. by doing this, the patient's awareness of his or her own oral fluid intake should be increased. this increased awareness could be the catalyst for a behavioural change, motivating the patients to increase their oral fluid intake. the improved oral fluid intake could translate into a reduction in morbidity. the reduction in morbidity is evidenced by the observable reduction seen in hospital admission and the reduced requirement for intravenous fluid therapy in the group that was given the fluid chart. this study also found that the fluid chart was acceptable to the participants in the intervention group. there were only a few participants who did not fill the fluid chart prospectively, while the majority filled it as instructed. moreover, almost all of the participants in the intervention group were able to understand the instructions given on how to fill it correctly. it could be concluded from this pilot study that the fluid charts were feasible to be used in the primary care setting. the drop-out rates in the pilot study was 11.8% in the control group and 14.7% in the intervention group. this is much lower than the default rate of the general out-patient clinic at ummc, which was found to be 33.8% in a previous study [14] . the same study found that the default rate was 16.4% amongst patients who were suspected to have dengue infection [14] . thus, there were good participant retention rates, attesting to the good acceptability of the intervention. thus, this pilot study showed that the experimental intervention tool in the form of the fluid chart was able to produce a positive effect on the measured outcome. a larger randomized controlled trial might be able to produce results which are statistically significant. thus, based on the preliminary results obtained during this pilot study, the fluid chart as a complex intervention should be evaluated further using a larger randomized controlled trial. future studies should be conducted to examine the relationship between the amount of oral fluid intake and the clinical outcome of dengue patients in primary care. by doing so, this would provide better evidence in order to recommend how much fluids patients with suspected dengue fever should consume orally. future studies measuring the amount of fluid intake required to deflect the necessity of intravenous fluid and admission would provide a more accurate basis to change the current standard primary care practice of giving general advice. further research in this area would then provide evidence whether five glasses of fluids consumed orally per day for those suspected with dengue fever would prove to be sufficient, or otherwise. in this study, the participants were chosen on the basis that they were clinically suspected to have dengue fever, which is how the majority of dengue patients are managed and treated in primary care. this makes the participants in this study comparable to suspected dengue patients treated at the primary care level in malaysia specifically and primary care in an asian setting, generally. however, this pilot study had several limitations. due to the time constraint during the data collection, the sample size was small. nevertheless, based on the findings from this study, using hospital admissions as the outcome measure (intervention 10.0% vs control 17.6%), with a power of 80% and p = 0.05, the estimated sample size needed for each arm would be 348. the dengue home care card was also read out to all participants, despite this not being the standard practice. this possibly became an active form of intervention in the control group, and a 'double intervention' in the intervention group, thus underestimating the true impact of the fluid chart on the fluid intake of the study participants. the randomization process in this pilot study using the table of random numbers with no allocation concealment might lead to bias. thus, a computer generated sequence with block randomization and varied block size should be applied. allocation concealment methods should also be used to avoid bias. we did not include other clinically relevant parameters such as dengue severity, shock and bleeding, as outcomes measures. these variables should be measured in the definitive trial. this study was carried out in 2010, a few months after the introduction of the revised dengue classification of 2009 [15] which had yet to be disseminated to all primary health care facilities in malaysia. the 1997 who dengue case classification [16] was not applied in primary health care settings because of inherent difficulties as reviewed by bandyopadhyay et al, 2006 [17] . in the absence of clear guidelines for admission, the need for admission was left to the clinical judgment of the clinician managing the patient in the primary care setting. due to a limited number of available beds in the infectious disease wards, it was an established practice for primary care clinicians to discuss with their colleagues in the medical department before patients were allowed to be admitted. this gate-keeping practice could reduce the number of unnecessary hospitalizations. it could be possible that a number of patients who were admitted would be classified as dengue fever (df) according to the 1997 who dengue case classification [16] . however, this would be in keeping with the observations of a multicentre study that recorded 52% of df patients (447 out of 861) needing intermediate interventions, including admission for intravenous fluid therapy. [18] this pilot study has ascertained that it is a feasible and acceptable study to perform in a primary care setting. the fluid chart is a simple, inexpensive, acceptable intervention that may reduce hospitalization and the requirement for intravenous fluid treatment in suspected dengue patients. a randomized controlled trial with larger sample size is needed to determine this conclusively. dengue: guidelines for diagnosis, treatment, prevention and control dengue and dengue haemorrhagic fever fact sheet no. 117 cost of dengue cases in eight countries in the americas and asia: a prospective study quality of life of dengue patients fluid intake and decreased risk for hospitalization for dengue fever comparison of the effects of oral hydration and intravenous fluid replacement in adult patients with non-shock dengue hemorrhagic fever in taiwan randomised primary health center based interventions to improve the diagnosis and treatment of undifferentiated fever and dengue in vietnam clinical and laboratory features that distinguish dengue from other febrile illnesses in endemic populations use of simple laboratory features to distinguish the early stage of severe acute respiratory syndrome from dengue fever early clinical and laboratory indicators of acute dengue illness evaluating the effectiveness of a fluid chart to improve oral intake in suspected dengue fever patients: a feasibility study early predictors of dengue infection in adults (epod)-a malaysian outpatient experience protocol for out-patient management of dengue illness in young adults dengue guidelines for diagnosis, treatment, prevention and control new edition. geneva; who dengue haemorrhagic fever: diagnosis, treatment, prevention and control 2nd edition. geneva; who classifying dengue: a review of the difficulties in using the who case classification for dengue haemorrhagic fever multicentre prospective study on dengue classification in four south-east asian and three latin american countries we would like to thank miss azimah zamani and miss wen ting tong for their assistance in preparation of this manuscript. key: cord-003551-jzfl4xuk authors: ciejka, justyna; botwina, paweł; nowakowska, maria; szczubiałka, krzysztof; pyrc, krzysztof title: synthetic sulfonated derivatives of poly(allylamine hydrochloride) as inhibitors of human metapneumovirus date: 2019-03-28 journal: plos one doi: 10.1371/journal.pone.0214646 sha: doc_id: 3551 cord_uid: jzfl4xuk human metapneumovirus (hmpv) is a widely distributed pathogen responsible for acute upper and lower respiratory infections of varying severity. previously, we reported that n-sulfonated derivatives of poly(allylamine hydrochloride) (nspahs) efficiently inhibit replication of the influenza virus in vitro and ex vivo. here, we show a dose dependent inhibition of hmpv infection by nspahs in llc-mk2 cells. the results showed strong antiviral properties of nspahs. while the activity of nspahs is comparable to those of carrageenans, they show better physicochemical properties and may be delivered at high concentrations. the functional assays showed that tested polymers block hmpv release from infected cells and, consequently, constrain virus spread. moreover, further studies on viruses utilizing different egress mechanisms suggest that observed antiviral effect depend on selective inhibition of viruses budding from the cell surface. human metapneumovirus (hmpv) is associated with acute infections of the respiratory tract, with increased severity in children, infants, elderly, and immunocompromised patients [1] [2] [3] [4] . hmpv was identified in 2001 in the netherlands [5] and sorted to the pneumoviridae family. its prevalence in children and infants requiring hospitalization worldwide reaches 4-25% [6, 7] . moreover, serological studies showed that~70% of children are seropositive at the age of five [8] . symptoms associated with the infection are usually mild (e.g., cough, subfebrile temperature), but more severe disease also occurs (e.g., bronchitis, pneumonia) [9, 10] . hmpv f protein utilizes heparan sulfate (hs), a negatively charged glycosaminoglycan (gag) present on the cellular membrane, as an attachment factor [11] . previous studies have shown that natural polysaccharides containing sulfonate groups efficiently inhibit infections caused by viruses employing hs during cell entry [12] [13] [14] [15] [16] [17] [18] [19] and it was proposed that carrageenans inhibit the attachment of the hmpv to hs by interacting with the f protein [19] . it is, plos however, worth to mention that replication of some hs independent viruses is also hampered in the presence of these polymers [20] [21] [22] [23] . currently, in some countries, ι-carrageenan is the main component of nasal spray used to prevent respiratory viral infections [24, 25] . in our previous report we showed that n-sulfonated derivatives of poly(allylamine) hydrochloride (nspahs) effectively inhibit influenza virus in vitro and ex vivo by inhibition of virion release from infected cells [26] . surprisingly, we showed that new compounds share the mechanism of action with ι-carrageenan, and it is very different from previously proposed. although the antiviral activity of nspahs is comparable to the one observed for carrageenans, physicochemical properties of nspahs seem to be superior [27] . the aim of this study was to examine the anti-hmpv activity of nspahs. for this, a series of in vitro experiments were conducted. two nspah derivatives were selected for the study: nspah-15-94 (molecular mass (mw) of 15 kda, degree of substitution with sulfonate groups (ds) of 94%) and nspah-65-96 (mw = 65 kda, ds = 96%), as these were the most potent inhibitors of hmpv replication in the preliminary studies. as reference, two sulfonated polysaccharides with well-established antiviral properties (ι-carrageenan, and λ-carrageenan) were used. nspah-15-94 and nspah-65-96 were synthesized, analyzed and purified by dialysis as described before [26] . the purification process was carried out against deionized water for 1 week (molecular mass cutoff of 14 kda). afterward, the water was removed by freeze drying to give the product as pale-yellow crystals. iota carrageenan (ι-car) and lambda carrageenan (λcar) were obtained from sigma-aldrich, poland. rhesus monkey kidney epithelial cells (llc-mk2, atcc ccl-7) were maintained in minimal essential medium (mem) containing 1 part of earle's mem and two parts of hanks mem (thermo scientific, poland) supplemented with 3% heat-inactivated fetal bovine serum (fbs; paa laboratories, austria), penicillin (100 u/ml) and streptomycin (100 μg/ml) (sigma-aldrich, poland). cells were propagated in t75 flasks (tpp, switzerland) at 37˚c in atmosphere containing 5% co 2 . hmpv virus stock (clinical isolate, clade b2) was kindly provided by dr. oliver schildgen (institute of pathology, witten/herdecke university). hmpv a1 virus stock (strain: nl/1/00) was acquired from the european virus archive (011v-00930). viruses were propagated as described before [28] . human coronavirus nl63 (hcov-nl63) stock was prepared as described before [17] . all assays were carried out using fully confluent llc-mk2 (1.5 × 10 4 cells per well) cultured for 48 h on 96-well plates (tpp, switzerland). cells were infected with virus at tcid 50 (50% tissue culture infectious dose) of 400 per ml (moi = 0.05) in 0% dmem (dulbecco's modified eagle's medium (thermo scientific, poland) deprived of fbs, supplemented with penicillin (100 u/ml), streptomycin (100 μg/ml), gentamicin (50 μg/ml), amphotericin (2.5 μg/ml) and trypsin (1 μg/ml) (sigma-aldrich, poland)). virus titers were assessed as described by reed and muench [28, 29] . cell viability was evaluated using xtt cell viability assay kit (biological industries, usa) according to the manufacturer's instructions. cells were incubated with tested polymers for 6 days at 37˚c in atmosphere containing 5% co 2 . after incubation, the medium was discarded, cells were rinsed with phosphate-buffered saline (pbs) and 100 μl of fresh medium was added to each well. next, 50 μl of the activated 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2h)-tetrazolium-5-carboxanilide (xtt) solution was added and samples were incubated for 2 h at 37˚c. the absorbance (λ = 450 nm) was measured using spectra max 250 spectrophotometer (molecular devices, usa). virus yield was determined by reverse transcription (rt) quantitative real-time pcr (qpcr). to ensure that polymers do not affect the isolation and/or amplification the template, supernatants were diluted 1000 times and total rna was isolated with viral dna/rna kit (a&a biotechnology, poland) in accordance with the manufacturer's instructions. reverse transcription reaction was carried out with a high-capacity cdna reverse transcription kit (thermo scientific, poland) according to the manufacturer's instructions. qpcr was performed with rt hs-pcr mix probe (a&a biotechnology, poland), as described before [28, 30] . the number of virus copies was determined using a series of 10-fold diluted dna standards containing a known number of template copies. the reaction was carried out as follows: 2 min at 50˚c, 10 min at 92˚c, followed by 40 cycles of 15 s at 92˚c and 1 min at 60˚c. for each virus a standard curve was prepared separately. the lower limit of detection was 10 3 viral rna copies per milliliter. to study antiviral properties of the compounds, cells were infected with hmpv or inoculated with the mock sample in the presence or absence of polymer. after 2 h infection at 37˚c, unabsorbed hmpv virions were removed by rinsing cells thrice with pbs. subsequently, media supplemented with inhibitors were added and cultures were incubated for 6 days at 37˚c. after incubation, culture supernatants were analyzed by titration and by rt-qpcr, as described above. to study the mechanism of action of tested polymers, a battery of functional assays were conducted as described before [26] , with some modifications. the assay shows the influence of tested compounds on the viral particle; polymers at a final concentration of 1000 μg/ml were mixed with virus stock and incubated for 1 h at room temperature while mixing at 1000 rpm. hmpv stock incubated in the absence of the polymer was treated as a control. after incubation, samples were diluted 200 times to ensure that the polymer concentration is below the lower limit of the effective range. samples were titrated as described by reed and muench [29] . the assay shows the effect of polymers on the cell; a confluent culture of llc-mk2 cells was inoculated with 100 μl of polymer solution (1000 μg/ml) per well. samples were incubated at 37˚c for 2 h, media were removed, and 100 μl virus or mock solution (moi = 0.05) was added to each well. after 2 h incubation at 37˚c media were discarded and cells were rinsed thrice with pbs to remove residual virus particles. then, 100 μl of fresh medium was added to each well and plate was incubated for 6 days at 37˚c. the assay allows to examine the effect of the compound on the virus-receptor interaction; a confluent culture of llc-mk2 cells was pre-cooled to 4˚c and overlaid with 100 μl of media supplemented with tested polymer (1000 μg/ml) and virus (moi = 0.05) or mock control. the samples were incubated at 4˚c for 1 h to allow virus attachment but to block virus internalization [31] . after incubation, cells were rinsed thrice with ice-cold pbs to remove residual virus particles. then, 100 μl of fresh medium was added and cells were incubated for 6 days at 37˚c. the assay allows to evaluate virus entry into the susceptible cell. pre-cooled (4˚c) confluent cells were infected with hmpv at moi of 0.05 or treated with mock. the plate was incubated for 2 h at 4˚c to allow virus attachment, but not internalization. supernatants were discarded and cells were washed thrice with ice-cold pbs to remove unbound virus particles. next, 100 μl of media supplemented with polymers (1000 μg/ml) was added and cells were incubated for 2 h at 37˚c to allow virus internalization. finally, the supernatants were discarded, cells were washed with an acidic buffer (ph 3.0, 0.1 m glycine, 0.1 m sodium chloride) to inactivate particles that did not enter the cell. cultures were incubated for 6 days at 37˚c in medium deprived of polymers. the assay eevaluates virus replication and production of infectious progeny. confluent cells were incubated with 100 μl of the hmpv at moi of 0.05. the infection was carried out for 2 h at 37˚c. subsequently, medium was discarded and cultures were rinsed thrice with pbs. cells were overlaid with media supplemented with polymers or control samples and incubated for 6 days at 37˚c. in this test, both supernatants and cell lysates were analyzed. lysates were prepared by 3 fast cycles of freezing and thawing at -80˚c in r9f buffer (a&a biotechnology, poland). for confocal microscopy analysis, replication inhibition assay was performed on cells seeded on coverslips in 6-well plates (tpp, switzerland all experiments were conducted at least in triplicate. the results are expressed as means ± standard deviations (sd). the 50% inhibitory concentration (ic 50 ) values were calculated using the graph pad prism function. to determine the significance of the results obtained, student t-test was used and p values < 0.05 were considered significant. virus yields and virus titers are presented as normalized data, i.e., log reduction values (lrvs) [26] for easier comparison of the inhibitory effect between assays. lrv was calculated according to the following formula: lrv = log (c i /c 0 ) where c i is the number of viral rna copies per milliliter in the sample treated with inhibitor and c 0 is the number of viral rna copies per milliliter in control sample (untreated cells). raw research data (number of copies per milliliter or tcid 50 values) are presented in supporting information file. the cells were incubated for 6 days in the presence or absence of evaluated compound, and the cell viability was assessed using an xtt assay. while some cytotoxicity was observed for ι-car and nspah-65-96 at 5000 μg/ml (~80% of the control), at lower concentrations the viability of cells was not affected (fig 1) . obtained results are consistent with previously published data [26] . in the initial experiment, polymers were tested for their ability to hamper hmpv replication in llc-mk2 cells. cells were overlaid with media supplemented with polymers or control (fig 2a; s1 fig) . to test if infectivity of progeny virus is also diminished, cell culture supernatants were titrated. as visible in fig 2b, nspah-15 -94 exhibits the best antiviral properties. confocal imaging confirmed this observation, as in the presence of the polymers the number of infected cells was lower compared to the control (fig 3) . to ensure broad specificity of nspah towards different hmpv strains, the study was performed also using different hmpv strain (clade a1). similar antiviral effect was observed (fig 4; s8 fig) . in the next step, we aimed to elucidate the mechanism of action for described polymers. at 1000 μg/ml no inhibition of early steps of virus infection was noted for carrageenans or nspahs (first four functional assays, s4-s7 figs), but we observed strong inhibition of progeny virus production in assay 5, wherein the inhibitors were added 2 h after the inoculation. all tested compounds inhibited hmpv progeny virus production, as illustrated by decreased virus yields and titers (p < 0.05). rt-qpcr analysis indicated that the inhibition is the most obtained results suggest that tested polymers hamper late stages of virus replication cycle, after the virus enters the cell. to further investigate the mechanism, supernatants from infected cultures were discarded and cells were lysed by three freeze and thaw cycles (titration) or with lysis buffer (rna isolation). surprisingly, rt-qpcr analysis revealed only minor inhibition (p < 0.05) of viral rna replication by carrageenans and no inhibitory activity for nspahs (fig 6, black bars; s3 fig) . this is consistent with our previous results on the influenza virus [26] . in the case of titration, we observed slight but significant increase in the infectivity of the samples in case of ι-car and nspah-65-96. nspah-15-94 also increase the infectivity of cell lysates, however, this increase was statistically non-significant (fig 6, grey bars; s2 fig) . this indicates that hmpv virions correctly progress to the budding stage, but in the presence of the polymers they remain cell-associated. altogether, obtained data indicate that nspahs do not affect virus replication per se, but lock them on cells and limit their spread to the neighboring cells. while some previous reports suggest inhibition of early stages of viral replication by carrageenans [19] , we were not able to replicate these results. it is, however, possible that the mechanism of action may very depending on the origin of these natural polymers [15, 20, 21, 26] . to study whether the observed antiviral effect is virus-specific, the same cell line was infected with an unrelated virus, i.e., human coronavirus nl63 (hcov-nl63). this virus efficiently replicates in llc-mk2 cells but is released from the cell in a different mechanism. in contrast to hmpv and influenza a viruses, which bud from the cell surface, hcov-nl63 buds from the golgi apparatus and mature infectious viral particles are transported via exocytosis to the cell surface [32] . none of the tested polymers inhibited hcov-nl63 (fig 7; s9 fig) . this observation suggests that the polymers block virus release from the cell surface, e.g., by modification of membrane plasticity or anchoring the progeny virions to the cell. one may also consider that the difference in nspahs activity between hmpv and hcov-nl63 results from different requirements for the hspgs presence, but it would not explain strong inhibitory effect of nspahs against the influenza virus. moreover, hcov-nl63 was also shown to employ hspgs during the entry to the cell [33] . to sum up, we demonstrated a dose-dependent inhibition of hmpv replication by sulfonated poly(allylamine)-based polymers. among tested polymers, ι-carrageenan and nspah-15-94 show the strongest antiviral properties against hmpv. studies on the mechanism of antinovel hmpv inhibitors hmpv properties of tested polymers imply that the decrease of samples' infectivity is due to blocking virus release from the cellular membrane. developed synthetic polymers are not toxic and offer similar antiviral properties as carrageenans but show better physicochemical properties. in our previous work we reported that nspahs hamper influenza virus replication in vitro and in vivo and we believe that they may be considered as broad-spectrum drug candidates competitive to marketed compounds. the role of human metapneumovirus in upper respiratory tract infections in children: a 20-year experience human metapneumovirus infections in hospitalized children. emerging infectious diseases a 1-year experience with human metapneumovirus in children aged human metapneumovirus in lung transplant recipients and comparison to respiratory syncytial virus a newly discovered human pneumovirus isolated from young children with respiratory tract disease prevalence of human metapneumovirus among hospitalized children younger than 1 year in catalonia, spain epidemiology and genetic variability of human metapneumovirus during a 4-year-long study in southeastern brazil seroprevalence of human metapneumovirus in japan human metapneumovirus infection in the united states: clinical manifestations associated with a newly emerging respiratory infection in children burden of human metapneumovirus infection in young children human metapneumovirus (hmpv) binding and infection are mediated by interactions between the hmpv fusion protein and heparan sulfate protective effect of a natural carrageenan on genital herpes simplex virus infection in mice differential inhibition of dengue virus infection in mammalian and mosquito cells by iota-carrageenan sulfated polyanions prevent hiv infection of lymphocytes by disruption of the cd4-gp120 interaction, but do not inhibit monocyte infection carrageenan is a potent inhibitor of papillomavirus infection antiviral activity of lambda-carrageenan prepared from red seaweed (gigartina skottsbergii) against bohv-1 and suhv-1 novel polymeric inhibitors of hcov-nl63 structure and anti-metapneumovirus activity of sulfated galactans from the red seaweed cryptonemia seminervis inhibition of human metapneumovirus binding to heparan sulfate blocks infection in human lung cells and airway tissues iota-carrageenan is a potent inhibitor of influenza a virus infection iota-carrageenan is a potent inhibitor of rhinovirus infection λ-carrageenan p32 is a potent inhibitor of rabies virus infection antiviral activity of carrageenan on hepatitis a virus replication in cell culture efficacy and safety of iotacarrageenan nasal spray versus placebo in early treatment of the common cold in adults: the icicc trial lessons learned from a double-blind randomised placebo-controlled study with a iota-carrageenan nasal spray as medical device in children with acute symptoms of common cold novel polyanions inhibiting replication of influenza viruses carrageenan: a review porphyromonas gingivalis enzymes enhance infection with human metapneumovirus in vitro a simple method of estimating fifty per cent endpoints entry of human coronavirus nl63 into the cell virucidal activity of polysaccharide extracts from four algal species against herpes simplex virus morphogenesis of coronavirus hcov-nl63 in cell culture : a transmis-sion electron microscopic study human coronavirus nl63 utilizes heparan sulfate proteoglycans for attachment to target cells key: cord-001387-2g9dc5z4 authors: mcintyre, k. marie; setzkorn, christian; hepworth, philip j.; morand, serge; morse, andrew p.; baylis, matthew title: a quantitative prioritisation of human and domestic animal pathogens in europe date: 2014-08-19 journal: plos one doi: 10.1371/journal.pone.0103529 sha: doc_id: 1387 cord_uid: 2g9dc5z4 disease or pathogen risk prioritisations aid understanding of infectious agent impact within surveillance or mitigation and biosecurity work, but take significant development. previous work has shown the h-(hirsch-)index as an alternative proxy. we present a weighted risk analysis describing infectious pathogen impact for human health (human pathogens) and well-being (domestic animal pathogens) using an objective, evidence-based, repeatable approach; the h-index. this study established the highest h-index european pathogens. commonalities amongst pathogens not included in previous surveillance or risk analyses were examined. differences between host types (humans/animals/zoonotic) in pathogen h-indices were explored as a one health impact indicator. finally, the acceptability of the h-index proxy for animal pathogen impact was examined by comparison with other measures. 57 pathogens appeared solely in the top 100 highest h-indices (1) human or (2) animal pathogens list, and 43 occurred in both. of human pathogens, 66 were zoonotic and 67 were emerging, compared to 67 and 57 for animals. there were statistically significant differences between h-indices for host types (humans, animal, zoonotic), and there was limited evidence that h-indices are a reasonable proxy for animal pathogen impact. this work addresses measures outlined by the european commission to strengthen climate change resilience and biosecurity for infectious diseases. the results include a quantitative evaluation of infectious pathogen impact, and suggest greater impacts of human-only compared to zoonotic pathogens or scientific under-representation of zoonoses. the outputs separate high and low impact pathogens, and should be combined with other risk assessment methods relying on expert opinion or qualitative data for priority setting, or could be used to prioritise diseases for which formal risk assessments are not possible because of data gaps. disease or pathogen risk prioritisation exercises are used by organisations charged with providing surveillance and mitigation measures including disease management and control, and biosecurity measures. qualitative, semi-quantitative or quantitative approaches can be used, but most take significant time to develop, so their use is limited, and when research involving the study of multiple diseases or pathogens is planned, agents are rarely systematically selected. quantitative measures for risk prioritisation include the calculation of epidemiological parameters such as disease incidence, prevalence, mortality and morbidity rates, costs of prevention, treatment or control, and for human disease, years lived with disability (yld) and disability-adjusted-life-year estimates (daly). additional measures for animals include losses to production. for many diseases, robust estimates of these measures do not exist. semi-quantitative and qualitative risk assessments are less demanding of data than quantitative approaches. nevertheless, they require significant time and physical resources (for example, to obtain parameters and effect sizes from the scientific literature), need updating regularly, and they usually require expert-opinion, adding subjectivity [1, 2, 3, 4, 5] . the h-index is an alternative approach to disease prioritisation. it objectively and rapidly provides a quantitative proxy of human disease or pathogen impact [6] , (mcintyre, unpublished) . the hindex captures scientific interest in a disease by deriving a metric from the number of papers published and how many citations each receives. combining scientific impact (citations) with technical productivity (papers published) is useful as, individually, total papers does not account for the quality of publications, while citation count may be influenced by a small number of seminal papers or if a disease becomes 'fashionable' briefly. the h-index method is significantly correlated with more comprehensive measures of human infectious disease impact, including dalys [6] , and deaths from disease (mcintyre, unpublished data). it can be rapidly obtained at low cost, attained automatically, and repeated regularly to reflect changes in impact, serving as a generic tool to assess the relative bearing of diseases or pathogens, in an easier, timelier manner than traditional risk assessments. while the h-index method undoubtedly has limitations, these tend to be different to those of other approaches; its use could be a step forward in separating high and low priority diseases or pathogens, in combination with other risk assessment methods. the enhanced infectious diseases (eid2) database integrates published data sources on pathogens, their hosts (including vectors) and geographic ranges [7] . by coupling the h-index method with the eid2, the primary aim of this study was to establish priority lists of human and domestic animal pathogens (including zoonoses) present in europe. we then consider reasons for the omission of some pathogens in our lists from those of other disease prioritisations: the 2010 global burden of disease (gbd) estimates [8] , communicable human diseases reportable in the european community [9] , the oie list of notifiable animal diseases, infections and infestations [10] , and the eu fp7 discon-tools project [11] . the gbd 2010 study was a large collaborative five year project which used all relevant published and unpublished evidence to create the strongest evidence-based epidemiological assessment of people's infectious and non-infectious health problems around the world [8] . the discon-tools project, funded by the european commission over five years, investigated the impacts of 52 domestic animal diseases, to focus and prioritise future research [11] . as the zoonotic and emerging status of pathogens as well as their taxonomic division could affect the likelihood of their inclusion in surveillance and impact quantification work, these factors were also investigated as reasons for omission from the other disease prioritisations. the h-index can be obtained in the same way for both human and animal pathogens. it therefore has potential as a single metric for prioritising across both host groups. its potential as a quantitative one health indicator (i.e. a single measure applicable to both human and animal diseases) was investigated by comparing scores for human-only, zoonotic, and animal-only pathogen groups, including emerging status as this would likely drive research impact. previous work has shown that the h-index is a proxy for human disease impact [6] , (mcintyre, unpublished) . we investigated its value as a proxy for animal disease impact by comparing domestic animal pathogen h-indices with other measures of impact including presence on the oie list [10] , and inclusion in discontools [11] . the eid2 database collates data on human and domestic animal pathogens: where, when, and in which hosts there is evidence of their occurrence. the database is built largely using automated procedures to interrogate publicly available databases. an eid2 background has been described previously [7] ; here, we used similar criteria to define pathogens, including pathogenic status (frequently pathogenic: a pathogen which frequently causes a clinically pathogenic effect -morbidity or mortality -in humans or domestic animals; non-pathogenic: an organism which causes no clinical signs within any of its hosts; unknown pathogenicity: an organism for which there is insufficient evidence to decide), evidence of pathogens affecting hosts ('host-pathogen interactions': evidence from at least one piece of meta-data uploaded with dna or rna sequence information to [12] , which describes where, when and from which host the pathogen came, or specific scientific publications [13]), and evidence of pathogens occurring within countries (evidence from at least one piece of meta-data [12] , or at least five publications in [13] where pathogen name and a country mesh-term [14] co-occurred in the title/abstract). information on host-pathogen interactions was collated when there was evidence of a pathogen occurring in at least one host of interest to the study (including humans and european domestic animals; see table 1 ). further information about each organism, such as their taxonomic division for pathogens (bacteriaincluding rickettsia, fungi -including algal pathogens, helminths -including thorny-headed worms and pentastomids, protozoa, and viruses -including prion agents) or their taxonomic rank (genus, species, etc.) is stored using a series of statements. previously, we examined characteristics of pathogen species [7] ; here, we include sub-species, to account for important strains e.g. escherichia coli o157:h7. information on whether pathogens were zoonotic, non-zoonotic, emerging and not emerging was examined based upon previously published information [15, 16] . if not included in earlier work or if their status had changed due to more recent scientific evidence, updated pathogen information was based upon the previous definitions. zoonotic pathogens were classified as those naturally transmitted between vertebrate (non-human) animals and humans (as the definitive host), not including species which have recently evolved from animal pathogens but are no longer transmitted between animals and humans [15, 17] . emerging pathogens are those that have appeared in a host population for the first time (including newly-evolved strains), or have occurred previously but are increasing in incidence or expanding into areas where they had not previously been reported [15, 17] . pathogens needed to have emerged in several geographically distinct areas to be 'emerging'. information sources. h-index searches were undertaken in january 2012 using web of science (wos) [18] . previous work established that results of h-index searches for pathogens undertaken using different bibliographic sources (e.g. wos, scopus, google scholar) are not identical but are highly correlated [6] . eligibility criteria. searches were restricted to the years 1900 to 2010, inclusive. english is used in wos, however searches also include foreign-language publication title translations. all literature in the wos database has been published. searches. searches were undertaken using search phrases specified in quotation marks (''''), the 'topic' search field and with no lemmatization. phrases were compiled including pathogen scientific name, alternative names, synonyms and alternative spellings according to ncbi taxonomy [19] . h-indices for clinical diseases used clinical terms as well as pathogen phrases for the main pathogens of disease. virus searches also included synonyms and acronyms from the ncbi taxonomy database and international committee on taxonomy of viruses [19, 20] , and the term 'virus', and excluded other entities (viral or non-viral) which shared acronyms. the boolean operators 'and', 'or', and 'not' linked multiple search phrases. example of a search phrase. (''mycobacterium tuberculosis'' or ''bacillus tuberculosis'' or ''bacterium tuberculosis'' or ''mycobacterium tuberculosis typus humanus'' or ''mycobacterium tuberculosis var. hominis''). a full list of human and domestic animal pathogens frequently causing pathogenic effects and for which there was evidence of european occurrence was created using eid2 information [7] , and defined criteria, see figure 1 . the relative impact of pathogens in this full list was assessed by calculating h-indices using the specified search protocol; high impact pathogens had the highest h-indices. the list was split according to host-pathogen interaction information, into two directories, one including pathogens with evidence of their occurrence in humans, and the second including domestic animal occurrence; zoonotic pathogens appeared in both lists. information was manually obtained on whether these pathogens cause diseases featuring in other prioritisation lists [8, 10, 11] , by examining pathogens listed under each disease's details in the ncbi mesh library [14] ; specific lists of diseases had been provided in other work [9, 11] . these additional pieces of information are included in the results (tables 2 and 3 ). finally, information on the pathogenic status of each pathogen, whether they frequently occurred in the relevant hosts and in europe was verified by the study authors using manual literature searches of the scientific literature, for the pathogens with the highest h-indices. h-indices and previous prioritisations. pearson's chisquared tests with yates' continuity correction and fisher's exact tests (fet) were used to test for differences in counts of pathogens included in previous work [9, 10, 11] , according to outcomes including their taxonomic division, zoonotic and emerging status. where appropriate, odds ratios (or) and 95% confidence intervals (ci) are presented. h-indices for one health. differences in h-indices for human-only, zoonotic, and animal-only pathogens were examined using a two-way analysis of variance (anova) with log 10transformation of the response, including emerging status as an explanatory covariate. post-hoc tukey multiple comparisons of treatments were undertaken using the hsd.test [21] . h-indices for domestic animal pathogens. -oie list. homogeneity of variances of h-indices for animal-only (and not zoonotic) pathogens included or not within the oie list of notifiable animal diseases was examined using the fligner-killeen (median) test. one-way anova thereafter established differences in the (log 10 -transformed) h-indices of pathogens included or not in the oie list. -discontools. h-indices and discon-tools scores were compared using spearman's rank correlations. if more than one pathogen had been included within disease information for the discontools rankings (for campylobacter, leishmaniasis, and salmonellosis), the higher h-index score was used for analyses. table 1 . animal species including humans for which pathogens have been studied, including domestic animals we eat or companion animals we keep as pets, and exotic animals also used as food sources or as pets. all analyses were undertaken using the statistical software package r [22] , with statistical significance determined by a pvalue of less than 0.05. two lists each including the top 100 human ( table 2 ) and domestic animal pathogens (table 3 ) which cause significant clinical disease and which therefore need consideration from a health and well-being perspective were short-listed using the hindex prioritisation method (for alternative names and synonyms see [19] ). when combined, 114 (72.6%) pathogens appeared solely in the human or animal list, and 43 (27.4%) were in both lists. of the top 100 human pathogens, 66 were classed as zoonotic and 67 were emerging, compared to 67 and 57 for domestic animal pathogens, respectively. of the top 100 human pathogens identified, 42 were either included in the gbd [8] , or are reportable to the ec [9] , or both. reasons for failure to include pathogens may be that pathogenic agents cause rarely diagnosed disease (e.g. human t-lymphotropic virus 1, lymphocytic choriomeningitis virus, and moraxella catarrhalis), or because disease agents are diverse, e.g. pneumonia or other lung infections (aspergillus niger, chlamydophila pneumonia, cryptococcus neoformans, klebsiella pneumonia, and mycoplasma pneumoniae) and gastro-intestinal (gi) symptoms or gi-tract infections (aeromonas hydrophila, bacillus cereus, bacteroides fragilis, clostridium species, vibrio parahaemolyticus, and yersinia enterocolitica). the impact of chronic disease or diseases causing low morbidity may be difficult to quantify or seen as less important (bartonella henselae, borrelia burgdorferi, human enterovirus c, human herpesvirus group, human papillomavirus, human parvovirus b19, mycobacterium bovis, mycobacterium avium, and mycoplasma genitalium). in addition, some pathogens may generally be commensals or natural biota (aggregatibacter actinomycetemcomitans, candida species, enterobacter, enterococcus, staphylococcus species, candida tropicalis, helicobacter pylori, and porphyromonas gingivalis) or species existing in the environment (acinetobacter baumannii, burkholderia cepacia, candida glabrata, entamoeba histolytica, fusarium oxysporum, gibberella moniliformis, proteus mirabilis, pseudomonas species, rhizopus oryzae, serratia marcescens, staphylococcus aureus, and stenotro-phomonas maltophilia) causing opportunistic infections in immunecompromised individuals (including those young, old or pregnant); their impact upon the general population may not be quantified. of the top 100 domestic animal pathogens described, 76 were either notifiable according to the oie [10] , or included in discontools [11] , or both. reasons for failure to include may be similar to for human pathogens (only pathogens not previously mentioned are cited: multiple disease symptoms or lack of diagnosis -ascaris suum, feline immunodeficiency virus, feline leukemia virus, gallid herpesvirus 2, haemonchus contortus, and yersinia pseudotuberculosis; causes of specific disease being diverse, for respiratory infection -feline calicivirus, and gi symptoms -campylobacter fetus, cryptosporidium parvum, and listeria monocytogenes,; and existing in the environment and opportunistic -pseudomonas aeruginosa). in addition, some omitted pathogens may be production issues with impact difficult to quantify (streptococcus agalactiae causing mastitis in cattle and neospora caninum causing abortion in cattle and dogs) and some may be issues of pets (canine parvovirus, neospora caninum, and parainfluenza virus 5). for human pathogens in our list (table 2) , fungi and helminths are particularly under-represented in gbd assessments (percentage included in gbd: bacteria, 25%; fungi, 0%; helminths, 0%; protozoa, 60%; viruses, 42.3%; fet, p = 0?046). there was no difference between taxonomic divisions in the percentage reportable to the ec (fet, p = 0.109). human pathogens classed as emerging (compared to not emerging) were statistically more likely to have gbd estimates (fet, p,0.001, or = 10.27, ci = 2.28-95.77) and be ec reportable (fet, p,0.001, or = 16.00, ci = 3.49-144.92), but not more likely to have gbd estimates or be ec reportable if they were zoonotic compared to nonzoonotic (pearson's x 2 , p = 0.219 and fet, p = 0.745, respectively). for domestic animal pathogens in our list (table 3) , fungi were particularly under-represented in the oie list (percentage included in oie: bacteria, 20%; fungi, 0%; helminths, 33.3%, protozoa, 33.3%, viruses, 52.5%; fet, p = 0.014). by contrast, viruses are particularly under-represented in discontools (percentage included in discontools: bacteria, 55.6%; fungi, 100%; helminths, 33.3%; protozoa, 33.3%; viruses, 25%; fet, p = 0.008). animal pathogens classed as zoonotic (compared to non-zoonotic) were (of borderline statistical significance) less likely to be included in the oie list (pearson's x 2 , p = 0.055, effect size(ã¸) = 0.22, or = 0.39) but there was no difference in the percentage included in discontools, (pearson's x 2 , p = 0.309) nor any effect of being emerging (versus not emerging) there was a statistically significant difference between the hindices of zoonotic, human-only or animal-only pathogens (twoway anova, f 2,152 = 24?40, p,0.001); h-indices were significantly higher for human-only (untransformed mean = 132.3966.14 and lower for animal-only pathogens (68.1166.06) compared to zoonotic (100.8369.93). h-indices were higher (with borderline statistical significance) for emerging (106.61610.19) compared to not emerging (86.9168.14) pathogens (two-way anova, f 1,152 = 3.78, p = 0.054). the interaction between zoonotic and emerging factors was not significant (p = 0.25). -discontools. there were significant correlations between h-indices and discontools estimates of public (human) health (zoonotic and animal pathogens) and impact on wider society (animal-only pathogens), and a further relationship of borderline significance between h-indices and the discon-tools overall result; no other correlations were significant (table 4 ). the european commission has outlined measures to strengthen coordinated approaches to health security at eu level, including monitoring, early warning and combating specific threats of a cross-border nature. these measures could be for climate change resilience [23] or for biosecurity, particularly for infectious diseases including communicable diseases, antimicrobial resistant and healthcare-associated infections related to communicable diseases, and biotoxins or other biological agents [24] . in this study, we implement a number of previously defined actions [25] , including presenting a quantitative evaluation for the impact of infectious pathogens affecting human health and well-being (via effects upon domestic animals) [24] . the work is unique, starting with all known infectious pathogens, and then objectively and systematically deciding which occur in relevant hosts in europe using a transparent process. the study establishes priority lists of human and domestic animal pathogens (including zoonoses) present in europe, using the h-index as a proxy measure for impact. previous work suggests that higher h-indices indicate higher impact for a pathogen relative to lower h-indices [6] , (mcintyre, unpublished material). the h-index method has both strengths and weaknesses. the strengths include that it is much more evidence-based and objective than semi-quantitative and qualitative approaches, and the results provide an easily understood quantitative estimate of impact. h-indices estimates can be simply and rapidly calculated, and they can therefore be repeatedly obtained to reflect changes in status, with the potential for automation of this process. the results are available for all pathogens at a global scale, and the scores reflect the wider scientific interest that would be expected to follow from a pathogen being either zoonotic or emerging [6] . most importantly, within a study of 27 human diseases, h-indices were correlated with daly estimates [6] , (mcintyre, unpublished material). dalys are an accepted measure of true disease burden in humans which accounts for the years of healthy life lost as a result of poor health or disability as well as the potential years of life lost due to premature death [26] . in further work, h-indices were also correlated with the number of human deaths (mcintyre, unpublished material). the weaknesses of the h-index method include that calculations need some manual oversight, as false positives can occur for instance when pathogens are used as model organisms; biases in results may happen because of trends in interest in specific pathogens, diseases or research fields or in certain regions; and estimates are subject to biases in funding (mcintyre, unpublished material) and research publication. h-indices are likely to underestimate the contribution of scientific literature published in non-english languages, although after translation some publications are included in wos and consequently in our calculations of h-indices. the literature searching method also doesn't account for the quality of publications in which pathogen names appear and the typical number of citations within different fields, and all bibliographic software packages incorporate newly published literature from different literature sources into their databases at different rates. finally, h-indices are only a proxy for impact, with the results susceptible to a lag in time-to-publication, and newly emerging pathogens likely to be under-represented. as the strengths and weaknesses of using the h-index method are different to those of other prioritisation methods, it is probably best used in combination with other approaches, for example, to shortlist a set of pathogens for more detailed risk assessment relying on expert opinion or qualitative data. it may also be used to prioritise diseases for which formal risk assessments are not possible because of data gaps. our priority lists of pathogens enabled investigation of why infectious pathogens are omitted from disease surveillance and impact quantification work [8, 9, 10, 11] . we considered several reasons for exclusion, including lack of diagnosis or misdiagnosis [27] , because the impact of particularly chronic infections is difficult to quantify or they are seen as less important, and because some pathogens are commensals or natural biota causing opportunistic infections in immune-compromised individuals; their pathogens include those which are zoonotic (z), non-zoonotic (nz), emerging (e) and not emerging (ne) [15, 16] , or given a new status (ns) in this work. pathogens also included in the list of top 100 animal pathogens are noted (a). the major pathogens causing diseases included within the 2012 global burden of disease (gbd) report are noted [8, 31] , as are those reportable in the ec (ec) [9] . doi:10.1371/journal.pone.0103529.t002 table 3 . top 100 domestic animal pathogens in europe, prioritised according to the h-index methodology [6] with the same emerging and zoonotic definitions as for table 2 . [6] ; for some animal pathogens, this is the first time that emerging status has been examined. methods to assess disease impacts use metrics capturing either human or animal host effects; they neither measure the magnitude in all hosts nor take account of scientific knowledge and tools for control. it is hard to prioritise human and animal diseases, because of the different metrics used (health or societal impacts versus welfare or economic impacts). significant differences between hindices mean values for human-only, zoonotic, and animal-only pathogens provide evidence that this single measure may have some use as a one health metric accounting for such factors. for example values for zoonotic pathogens were higher than for animal-only, suggesting that they account for human as well as animal-impact. higher values for human-only compared to other pathogen groups suggests that zoonoses may be under-represented due to underestimation of their global burden [28, 29] , or research impact [6] , or because of biases in research impact and funding for chronic human pathogens [29] . in addition, lower animal-only hindices may be due to funding biases. finally, there was limited evidence that the h-index method is a reasonable proxy for the impact of animal pathogens; animal pathogen h-indices were significantly positively correlated with subsections of discontools [11] , including impact on public (human) health and overall results (borderline significance). if animal-only (not zoonotic) diseases were included, there was a significant positive relationship with impact on wider society. as the more animal-focussed subsections (disease knowledge, impact on animal health and welfare, impact on trade, and available control tools) were not correlated with h-indices, and h-indices were not affected by inclusion in the oie list [10] , this suggests a human-centric bias in h-indices; for example, a pathogen causing little impact in animals may nevertheless have a high h-index if zoonotic. the priority lists presented in this work should be used by agencies and research organisations in combination with other risk assessment methods to identify gaps in working for priority setting. it has been suggested that zoonoses must be dealt with at the interface of human and animal health using all available information [30] ; this work, combining the eid2 and h-index technique, demonstrates such 'big-data' approaches. pathogens also included in the list of top 100 human pathogens are noted (h). the major pathogens causing diseases included within the oie list of notifiable terrestrial and aquatic animal diseases (oie) are noted [10] , as are those included in the discontools project (disc) [11] . doi:10.1371/journal.pone.0103529.t003 table 4 . results of spearman's rank correlations between h-indices and the discontools prioritisation of major animal diseases [11] . fungi ascaris suum, z, ne, ns 65 helminths yersinia enterocolitica, z, e, h 126 bacteria borna disease virus, z, e, ns, disc 65 viruses bacillus anthracis evidence-based semiquantitative methodology for prioritization of foodborne zoonoses global change: impact, management, risk approach and health measures -the case of establishing priorities for national communicable disease surveillance prioritizing emerging zoonoses in the netherlands establishing priorities for european collaboration in communicable disease surveillance the h-index as a quantitative indicator of the relative impact of human diseases using open-access taxonomic and spatial information to create a comprehensive database for the study of mammalian and avian livestock and pet infections disability-adjusted life years (dalys) for 291 diseases and injuries in 21 regions, 1990-2010: a systematic analysis for the global burden of disease study commission implementing decision of 8 august 2012 amending decision 2002/253/ec laying down case definitions for reporting communicable diseases to the community network under decision no. 2119/98/ec of the european parliament and of the council oie-listed diseases, infections and infestations in force the ncbi nucleotide database homepage the ncbi medical subject headings (mesh) database homepage risk factors for human disease emergence host range and emerging and reemerging pathogens global trends in emerging infectious diseases overview -web of science the ncbi taxonomy database homepage agricolae package cran 1.1-6 ed r: a language and environment for statistical computing adapting to climate change: towards a european framework for action decision no 1082/2013/eu of the european parliament and of the council of 22 october 2013 on serious cross-border threats to health and repealing decision no 2119/98/ec monitoring eu emerging infectious disease risk due to climate change quantifying the burden of disease -the technicial basis for diability-adjusted life years emerging infectious diseases: vulnerabilities, contributing factors and approaches neglected and endemic zoonoses the socioeconomic burden of parasitic zoonoses: global trends emerging zoonoses: the challenge for public health and biodefense years lived with disability (ylds) for 1160 sequelae of 289 diseases and injuries 1990-2010: a systematic analysis for the global burden of disease study thanks to helen roberts (uk department for environment, food and rural affairs) and john stephenson (institute of infection and global health, university of liverpool) for input during study development. key: cord-001071-bjx5td52 authors: vanhems, philippe; barrat, alain; cattuto, ciro; pinton, jean-françois; khanafer, nagham; régis, corinne; kim, byeul-a; comte, brigitte; voirin, nicolas title: estimating potential infection transmission routes in hospital wards using wearable proximity sensors date: 2013-09-11 journal: plos one doi: 10.1371/journal.pone.0073970 sha: doc_id: 1071 cord_uid: bjx5td52 background: contacts between patients, patients and health care workers (hcws) and among hcws represent one of the important routes of transmission of hospital-acquired infections (hai). a detailed description and quantification of contacts in hospitals provides key information for hais epidemiology and for the design and validation of control measures. methods and findings: we used wearable sensors to detect close-range interactions (“contacts”) between individuals in the geriatric unit of a university hospital. contact events were measured with a spatial resolution of about 1.5 meters and a temporal resolution of 20 seconds. the study included 46 hcws and 29 patients and lasted for 4 days and 4 nights. 14,037 contacts were recorded overall, 94.1% of which during daytime. the number and duration of contacts varied between mornings, afternoons and nights, and contact matrices describing the mixing patterns between hcw and patients were built for each time period. contact patterns were qualitatively similar from one day to the next. 38% of the contacts occurred between pairs of hcws and 6 hcws accounted for 42% of all the contacts including at least one patient, suggesting a population of individuals who could potentially act as super-spreaders. conclusions: wearable sensors represent a novel tool for the measurement of contact patterns in hospitals. the collected data can provide information on important aspects that impact the spreading patterns of infectious diseases, such as the strong heterogeneity of contact numbers and durations across individuals, the variability in the number of contacts during a day, and the fraction of repeated contacts across days. this variability is however associated with a marked statistical stability of contact and mixing patterns across days. our results highlight the need for such measurement efforts in order to correctly inform mathematical models of hais and use them to inform the design and evaluation of prevention strategies. the control of hospital-acquired infections (hai) is largely based on preventive procedures derived from the best available knowledge of potential transmission routes. the accurate description of contact patterns between individuals is crucial to this end, as it can help to understand the possible transmission dynamics and the design principles for appropriate control measures. in particular, the mutual exposures between patients and health-care workers (hcws) have been documented for bacterial and viral transmission since decades [1, 2, 3] . transmission might be the result of effective contact, as in the cases of s. aureus [4, 5] , k. pneumoniae [6] or rotavirus [7] , of exposure to contaminated aerosols, as for m. tuberculosis [8] , or the result of exposure to droplets, as for influenza [9] . some pathogens such as influenza can also be transmitted by different routes. although close-range proximity and contacts between individuals are strong determinants for potential transmissions, obtaining reliable data on these behaviors remains a challenge [10] . data on contacts between individuals in specific settings or in the general population are most often obtained from diaries and surveys [11, 12, 13, 14] and from time-use records [15] . these approaches provide essential information to describe contacts patterns and inform models of infectious disease spread. the gathered data, however, often lack the longitudinal dimension [10, 12, 16] and the high spatial and temporal resolution needed to accurately characterize the interactions among individuals in specific environments such as hospitals. moreover, they are subject to potential biases due to behavioral modifications due to the presence of observers, to short periods of observation, and especially to missing information and recall biases. evaluating biases and understanding the accuracy of the collected data is therefore a difficult task [16] . in this context, the use of electronic devices has recently emerged as an interesting complement to more traditional methods [10] . in particular, wearable sensors based on active radio-frequency identification (rfid) technology have been used to measure face-to-face proximity relations between individuals with a high spatio-temporal resolution in various contexts [17] that include social gatherings [18, 19] , schools [20, 21] and hospitals [22, 23] . the amount of available data, however, is still very limited, high-resolution contact data relevant for the epidemiology of infectious diseases are scarce, and the longitudinal aspects of contact patterns have not been investigated in detail, prompting further investigation. in this paper we report on the use of wearable proximity sensors [17] to measure the numbers and durations of contacts between individuals in an acute care geriatric unit of a university hospital. we investigate the variability of contact patterns as a function of time, as well as the differences in contact patterns between individuals with different roles in the ward. we document the presence of individuals with a high number of contacts, who could be considered as potential super-spreaders of infections. some implications of our results regarding prevention and control of hospital-acquired infections are discussed. the measurement system, developed by the sociopatterns collaboration [24] , is based on small active rfid devices (''tags'') that are embedded in unobtrusive wearable badges and exchange ultra-low-power radio packets [17, 18, 21, 23] . the power level is tuned so that devices can exchange packets only when located within 1-1.5 meters of one another, i.e., package exchange is used as a proxy for distance (the tags do not directly measure distances). individuals were asked to wear the devices on their chests using lanyards, ensuring that the rfid devices of two individuals can only exchange radio packets when the persons are facing each other, as the human body acts as a rf shield at the frequency used for communication. in summary the system is tuned so that it detects and records close-range encounters during which a communicable disease infection could be transmitted, for example, by cough, sneeze or hand contact. the information on face-to-face proximity events detected by the wearable sensors is relayed to radio receivers installed throughout the hospital ward (bedrooms, offices and hall). the system was tuned so that whenever two individuals wearing the rfid tags were in face-to-face proximity the probability to detect such a proximity event over an time interval of 20 seconds was larger than 99%. we therefore define two individuals to be in ''contact'' during a 20-second interval if and only if their sensors exchanged at least one packet during that interval. a contact is therefore symmetric by definition, and in case of contacts involving three or more individuals in the same 20-second interval, all the contact pairs were considered. after the contact is established, it is considered ongoing as long as the devices continue to exchange at least one packet for every subsequent 20 s interval. conversely, a contact is considered broken if a 20-second interval elapses with no exchange of packets. we emphasize that this is an operational definition of the human proximity behavior that we choose to quantify, and that all the results we present correspond to this precise and specific definition of ''contact''. we make the raw data we collected available to the public as datasets s1-s5 in file s1 and on the website of the sociopatterns collaboration (www. sociopatterns.org). data were collected in a short stay geriatric unit (19 beds) of a university hospital of almost 1000 beds [3] in lyon, france, from monday, december 6, 2010 at 1:00 pm to friday, december 10, 2010 at 2:00 pm. during that time, 50 professional staff worked in the unit and 31 patients were admitted. we collected data on the contacts between 46 staff members (92% participation rate) and 29 patients (94% participation rate). the participating staff members were 27 nurses or nurses' aides, 11 medical doctors and 8 administrative staff. in the ward, all rooms but 2 were single-bed rooms. each day 2 teams of 2 nurses and 3 nurses' aides worked in the ward: one of the teams was present from 7:00 am to 1:30 pm and the other from 1:30 pm to 8:00 pm. an additional nurse and an additional nurse' aid were moreover present from 9:00 am to 5:00 pm. two nurses were present during the nights from 8:00 pm to 7:00 am. in addition, a physiotherapist and a nutritionist were present each day at various points in time, with no fixed schedule, and a social counselor and a physical therapist visited on demand (in our analysis they are considered as nurses). two physicians and 2 interns were present from 8:00 am to 17:00 pm each day. visits were allowed from 12:00 am to 8:00 pm but visitors were not included in the study. in advance of the study, staff members and patients were informed on the details and aims of the study. a signed informed consent was obtained for each participating patient and staff member. all participants were given an rfid tag and asked to wear it properly at all times. no personal information was associated with the tag: only the professional category of each hcw and the age of the patients were collected. the study was approved by the french national bodies responsible for ethics and privacy, the ''commission nationale de l'informatique et des libertés'' (cnil, http://www.cnil.fr) and the ''comité de protection des personnes'' (http://www.cppsudest2.com/) of the hospital. individuals were categorized in four classes according to their activity in the ward: patients (pat), medical doctors (physicians and interns, med), paramedical staff (nurses and nurses' aides, nur) and administrative staff (adm). med and nur professionals form a group named hcw. the contact patterns were analyzed using both the numbers and the durations of contacts between individuals. for each individual we measured the number of other distinct individuals with whom she/he had been in contact, as well as the total number of contact events she/he was involved in, and the total time spent in contact with other individuals. these quantities were aggregated for each class and for each pair of role classes in order to define contact matrices that describe the mixing patterns between classes of individuals. the longitudinal evolution of the contact patterns was studied by considering, in addition to the entire study duration, several shorter time intervals: we divided the study duration into 5 daytime periods (7:00 am to 8:00 pm) and 4 nights (8:00 pm to 7:00 am); daytime periods were divided in morning (7:00 am to 1:30 pm) and afternoon (1:30 pm to 8:00 pm) shifts, and we also considered data aggregated on a 1-hour timescale. we finally considered the similarity of contact patterns between successive days, by measuring the fraction of contacts that were repeated from one day to the next, as such information is particularly relevant when modeling spreading phenomena [18, 25, 26] . overall, 14,037 contacts occurred during the study, with a cumulative duration of 648,480 s (approx. 10,808 minutes or 180 hours). 10,616 contacts (75.6%) included at least one nur, 4,003 (28.5%) included at least one med, and 3,849 (27.4%) at least one patient. table 1 reports the average number and duration of contacts of individuals in each class over the whole study duration. most contacts involve at least one nur and/or one med, and nurs and meds have on average the largest number of contacts, as well as the largest cumulative duration in contact. large standard deviations are however observed: the distributions of the contact durations and of the numbers and cumulative durations of contacts are broad, as also observed in many other contexts [20, 21, 23, 27] . important variations are observed even within each role class. in particular, contacts of much larger duration than the average are observed with a non-negligible frequency. the total number of contacts between individuals belonging to specific classes is reported in table 2 and the corresponding contact matrices are shown in figure 1 . we report contact matrices giving the total numbers and cumulative durations of contacts between individuals of given classes, as well as contact matrices taking into account the different numbers of individuals in each class. contacts were most frequent between two nurs (5,310 contacts, 38%), followed by nur-par contacts (2,951 contacts, 21%), and by contacts between two meds (2,136 contacts, 15%). very few contacts between pats or between members of the adm group were observed. as reported in table 3 , among the 14,037 contacts detected, 13,206 (94.1%) occurred during daytime, for a total duration of 612,900 s (approx. 10,215 min or 170 h). 831 contacts (5.9%) occurred during nights (lasting 35,580 s, approx. 593 min or 10 h). on average we recorded 2,265 contacts per morning, 1,041 per afternoon, and 207 per night. the evolution of the number of contacts at the more detailed resolution of one-hour time windows is reported in figure 2 . the number of contacts varied strongly over the course of a day, but the evolution was similar from one day to another (for day 1 and day 5, contacts were recorded after 1:00 pm and before 2:00 pm respectively, see methods), with very few contacts at night and a maximum around 10-12 am. the number of contacts between individuals of specific classes also depends on the period of the day. contacts between nurs, and between nurs and pats, were predominant in the morning while contacts between meds remained similar between mornings and afternoons. overall, 63.3% of contacts between nurs and pats occurred on the morning, 25.5% on the afternoon and 9.2% during the night. figure 3 reports the contact matrices giving the numbers of contacts between individuals of specific classes for each morning, afternoon and night. the absolute numbers of contacts varied from one morning (resp., afternoon or night) to the next, but the mixing patterns remained very similar. differences were observed between morning, afternoon and night patterns. the main difference between morning and afternoon periods came from larger numbers of contacts involving nurs in the morning. almost only contacts involving nurs and pats were observed at night. although the aggregated observables reported above are very similar from day to day, the precise structure of the daily contact network varied strongly: the fraction of common neighbors of an individual between two different days is on average just of 48%. this value is smaller than the one observed in a school [21] , but much larger than the one measured for the attendees of a conference [18] . the cumulative number and duration of contacts of each individual, as identified by his/her badge identification number, are reported in figure 4 and table 4 . a small number of hcws accounted for most of the contacts observed between hcws and pats, both in terms of number and cumulative duration. for instance, 6 nurs (representing 16% of all hcws) accounted for 42.1% of the number of contacts and 44.3% of the cumulated duration of the contacts with pats (number of contacts and cumulative duration of contacts of a given individual are strongly correlated, r = 0.98). the number of distinct individuals contacted by a given individual was also correlated with the total number of contacts of the same individual (r = 0.69). these 6 hcws had a much larger number and duration of contacts than average, as shown in table 4 . the objective of the present study was to describe in detail the contacts between individuals in a healthcare setting. such data can help to accurately inform computational models of the propagation of infectious diseases and, as a consequence, to improve the design and implementation of prevention or control measures based on the frequency and duration of contacts. numbers and duration of contacts were characterized for each class of individuals and for individuals belonging to given class pairs, yielding contact matrices that represent important inputs for realistic computational models of nosocomial infections. as also measured in other contexts [17, 20, 21, 23, 27] , the numbers and durations of contacts display large variations even across individuals of the same class: the resulting distributions were broad, with no characteristic time scale. as a consequence, even though the average durations of contacts were rather short, contacts of much longer durations than average occured with nonnegligible frequency. contacts involving either two nurs or between nurs and pats accounted for the majority of contacts, both in terms of numbers and of global durations. very few contacts occurred between pats: this might be a specificity of wards with mostly single rooms, and other wards in which patients are not alone in a room or in which they move around more might yield more numerous contacts between pats. these results are consistent with previous studies [23, 28] carried out in pediatrics, surgery and intensive care units, and provide additional evidence that nurses and assistants may be the most essential target group for prevention measures [27, 28] . the detailed information about the number and duration of contacts also allowed us to highlight the presence of a limited number of ''super-contactors'' among hcws who account for a large part of all contacts. a large number of contacts could correspond to different situations, namely to contacts with many different patients, or to many contacts with few patients. our results show that the cumulated number of contacts and the number of distinct persons contacted are correlated; this indicates that in the hospital context under study the super-contactors have contacts with many different patients. they could therefore potentially play the role of super-spreaders, whose importance in the spread of infectious agents has been highlighted both theoretically [29, 30] and empirically [31] . this suggests that their role class should be targeted for prevention measures. these results are in concordance with the central role of hcws in hospital wards, as repeated contacts with patients are often necessary for the quality of healthcare. however, since outbreaks of measles and influenza involving this population have been observed [32, 33] , the possibility for hcws to be super-contactors emphasizes the need to reduce their exposure to infection and to limit the risk of transmission to patients. this should stimulate the strict implementation of preventive measures including hand washing, vaccination, or wearing of masks [34] . in addition, hcws could be warned against the risk brought forth by unnecessary large numbers or long durations of contacts, especially with patients. limiting the contacts of hcws (either with pats or with other hcws) might however not be feasible without altering the quality of care. in this respect, the investigation of the temporal evolution of the numbers of contacts may help envision and discuss changes in the organization of care during epidemic or pandemic periods. the numbers of contacts varied indeed greatly along the course of each day, clearly highlighting the periods of the day (here, the mornings) during which transmission could occur with higher probability. the high numbers of contacts during mornings may indicate a potential overexposure to infection for pats and nurs, and one may imagine a different organization toward a smoothing of the number of contacts throughout mornings and afternoons. this would decrease the density of contacts, in particular between nurs, at each specific moment, while maintaining the daily number and duration of contacts between nurs and pats, and overall tend to limit their overexposure [35] . the potential efficacy of such or other changes in the healthcare organization should of course be tested through numerical simulations of spreading phenomena, and their feasibility would moreover need to be asserted through discussions with the staff. the measurement of contact patterns by means of wearable sensors presents strengths and limitations that are worth discussing. strong advantages are the versatility of the sensing strategy (i.e., the unobtrusiveness of wearable sensors and the prompt deployability of receivers) and the fact that it does neither require the constant presence of external observers nor interfere with the delivery of care in the ward. another strength lies in the high spatial and temporal resolution: behavioral differences across role classes can be detected, and longitudinal studies are possible. high participation ratios are also crucial: similarly to a previous study in another hospital [23] , the rate of acceptance among hcws and patients turned out to be very high (92%). the information meetings held before the study, providing a clear exposition of the scientific objectives and of the privacy aspects, most probably played an important role in achieving such a high participation rate. the versatility of a system based on wearable sensors and easily deployable data receivers makes it possible to repeat similar studies in different environments and to compare results across contexts [19] . in particular, several of the reported findings are very similar to those described in [23] in a different hospital, situated in a different country, and in a different type of ward (paediatric): large variability in the cumulative duration of contacts, small number of contacts between patients, and large numbers and durations of contacts between nurs. repeating measurements in the same ward and in other wards represents an important step towards understanding the similarities and differences of contact patterns in hospital settings, and allows to generalize the observations to more correctly inform models. the measurement approach we used here has also several limitations. contacts were defined as face-to-face proximity, without any information on physical contact between individuals. therefore, the assumption that the number of contacts reflects disease exposure can be appropriate for respiratory infections such as influenza, or for similar diseases that can be transmitted by various routes at a distance of 1 meter around an index case [36] . the use of close-range proximity as a proxy for the transmission of bacterial infection acquired by cross transmission, such as s aureus or enterobacteriacea, is more questionable. other factors related to specific attributes of individuals (e.g., vaccination or immunosuppression), of the microbial agent (e.g., resistance or virulence) or of the environment (e.g., specialty of ward) may also alter the relationship between contact frequency/duration and transmission. in this respect, a validation with simultaneous direct observation and human annotation of the contacts would be of particular interest. finally, it is difficult to assess whether individuals modified their behavior in response to wearing rfid badges, but direct observation indicated that hcws were focusing on their daily activities and most probably were not influenced by the presence of the badges. badges were not proposed to visitors and this potential external source of infection was not studied. this study complements previous work [22, 23, 27, 28, 30, 34] and provides data that can be used to explore the spread of infection in confined settings through mathematical and computational modeling. models of transmission within hospitals might be based on contact matrices such as those presented here, and used to better understand the epidemiology of different types of microbial agents, to assess the impact of control measures, and to help improve the delivery of care during emergency epidemic situations. in our study, specific mixing patterns were observed between different classes of individuals, showing a clear departure from homogeneous mixing, as it is expected in a hospital setting, and highlighting the relevance of correctly informed contact matrices. moreover, although an important turnover between the persons in contact with a given individual was observed across different days, and although the average contact durations between different classes of individuals varied between mornings, afternoons and nights, the contact patterns remained statistically very similar across successive days. these results suggest that, in order to correctly inform computational models, data collected over just a few hours might be insufficient, but that measures lasting 48 hours would be sufficient to evaluate the statistical properties of contact patterns as well as the mixing patterns between individual classes, and to estimate the similarity between the contacts of an individual across days. the statistical features of the gathered data could then be used to model contact patterns over longer time scales. the scarcity of contact data [10, 37] calls for further measurement campaigns to validate and consolidate the results across other hospital units, other contexts, and over longer periods of time. additional data sets would also be useful to build and test proxies that could replace systematic detailed measurement of contact patterns, such as the ones put forward in [15, 38, 39] . in order to explore the relationship between complex contacts network and the spreading of infections, it would be particularly interesting to collect simultaneously high-resolution contact data and microbiological data describing the infection status of participating individuals. combining these heterogeneous sources of information within appropriate statistical models would allow elucidating the relation between the risk of disease transmission and contacts patterns, in order to disentangle transmission likelihood from contact frequency. finally, feedback of the results to hcws could be an innovative pedagogical tool in health care settings. file s1 dataset s1. time-resolved contact network for day 1, in gexf format. each node corresponds to one rfid tag and has an attribute ''role'' that indicates the role of the individual wearing the tag: patient (pat), medical doctor (med), paramedical staff (nur) or administrative staff (adm). each edge has 3 attributes: ''ncontacts'', the number of contact events between the corresponding rfid tags; ''cumulativeduration'', the total duration of these contacts, and ''list_contacts'', the explicit list of time intervals during which the individuals were in contact. dataset s2. timeresolved contact network for day 2, in gexf format. each node corresponds to one rfid tag and has an attribute ''role'' that indicates the role of the individual wearing the tag: patient (pat), medical doctor (med), paramedical staff (nur) or administrative staff (adm). each edge has 3 attributes: ''ncontacts'', the number of contact events between the corresponding rfid tags; ''cumulativeduration'', the total duration of these contacts, and ''list_contacts'', the explicit list of time intervals during which the individuals were in contact. dataset s3. time-resolved contact network for day 3, in gexf format. each node corresponds to one rfid tag and has an attribute ''role'' that indicates the role of the individual wearing the tag: patient (pat), medical doctor (med), paramedical staff (nur) or administrative staff (adm). each edge has 3 attributes: ''ncontacts'', the number of contact events between the corresponding rfid tags; ''cumulativeduration'', the total duration of these contacts, and ''list_contacts'', the explicit list of time intervals during which the individuals were in contact. dataset s4. time-resolved contact network for day 4, in gexf format. each node corresponds to one rfid tag and has an attribute ''role'' that indicates the role of the individual wearing the tag: patient (pat), medical doctor (med), paramedical staff (nur) or administrative staff (adm). each edge has 3 attributes: ''ncontacts'', the number of contact events between the corresponding rfid tags; ''cumulativeduration'', the total duration of these contacts, and ''list_contacts'', the explicit list of time intervals during which the individuals were in contact. dataset s5. timeresolved contact network for day 5, in gexf format. each node corresponds to one rfid tag and has an attribute ''role'' that indicates the role of the individual wearing the tag: patient (pat), medical doctor (med), paramedical staff (nur) or administrative staff (adm). each edge has 3 attributes: ''ncontacts'', the number of contact events between the corresponding rfid tags; ''cumulativeduration'', the total duration of these contacts, and ''list_contacts'', the explicit list of time intervals during which the individuals were in contact. (zip) health-care workers: source, vector, or victim of mrsa? tuberculosis exposure of patients and staff in an outpatient hemodialysis unit risk of influenza-like illness in an acute health care setting during community influenza epidemics in modeling the spread of methicillin-resistant staphylococcus aureus in nursing homes for elderly community and nosocomial transmission of panton-valentine leucocidinpositive community-associated meticillin-resistant staphylococcus aureus: implications for healthcare klebsiella pneumoniae bloodstream infections among neonates in a high-risk nursery in cali, colombia outbreak of rotavirus gastroenteritis in a nursing home transmission of drug-susceptible and drug-resistant tuberculosis and the critical importance of airborne infection control in the era of hiv infection and highly active antiretroviral therapy rollouts transmission of pandemic a/h1n1 2009 influenza on passenger aircraft: retrospective cohort study close encounters of the infectious kind: methods to measure social mixing behaviour social mixing patterns for transmission models of close contact infections: exploring self-evaluation and diary-based data collection through a web-based interface comparison of three methods for ascertainment of contact information relevant to respiratory pathogen transmission in encounter networks social contacts of school children and the transmission of respiratory-spread pathogens social contacts and mixing patterns relevant to the spread of infectious diseases using time-use data to parameterize models for the spread of close-contact infectious diseases collecting closecontact social mixing data with contact diaries: reporting errors and biases dynamics of person-to-person interactions from distributed rfid sensor networks simulation of an seir infectious disease model on the dynamic contact network of conference attendees what's in a crowd? analysis of face-to-face behavioral networks a highresolution human contact network for infectious disease transmission high-resolution measurements of face-to-face contact patterns in a primary school using sensor networks to study the effect of peripatetic healthcare workers on the spread of hospital-associated infections close encounters in a pediatric ward: measuring face-to-face proximity and mixing patterns with wearable sensors modelling disease spread through random and regular contacts in clustered populations models of epidemics: when contact repetition and clustering should be included prioritizing healthcare worker vaccinations on the basis of social network analysis nurses' contacts and potential for infectious disease transmission superspreading and the effect of individual variation on disease emergence peripatetic health-care workers as potential superspreaders severe acute respiratory syndrome-singapore nosocomial transmission of measles: an updated review hospital-acquired influenza: a synthesis using the outbreak reports and intervention studies of nosocomial infection (orion) statement monitoring hand hygiene via human observers: how should we be sampling? global perspectives for prevention of infectious diseases associated with mass gatherings cough-generated aerosols of mycobacterium tuberculosis: a new method to study infectiousness invited commentary: challenges of using contact data to understand acute respiratory disease transmission modeling and estimating the spatial distribution of healthcare workers a low-cost method to assess the epidemiological importance of individuals in controlling infectious disease outbreaks we are particularly grateful to all patients and the hospital staff who volunteered to participate in the data collection. key: cord-000008-3dgjv0x1 authors: vali, bahareh; yue, feng yun; jones, r. brad; sheth, prameet m.; kaul, rupert; betts, michael r.; wong, david; kovacs, colin; loutfy, mona; common, andrew; halpenny, roberta; ostrowski, mario a. title: hiv-specific t-cells accumulate in the liver in hcv/hiv co-infection date: 2008-10-20 journal: plos one doi: 10.1371/journal.pone.0003454 sha: doc_id: 8 cord_uid: 3dgjv0x1 background and aims: hepatitis c virus (hcv)-related liver disease progresses more rapidly in individuals co-infected with human immunodeficiency virus-1 (hiv), although the underlying immunologic mechanisms are unknown. we examined whether hiv-specific t-cells are identified in the liver of hcv/hiv co-infected individuals and promote liver inflammation through bystander immune responses. methods: ex-vivo intra-hepatic lymphocytes from hcv mono-infected and hcv/hiv co-infected individuals were assessed for immune responses to hiv and hcv antigens by polychromatic flow cytometry. results: hcv/hiv liver biopsies had similar frequencies of lymphocytes but lower percentages of cd4(+) t-cells compared to hcv biopsies. in co-infection, intra-hepatic hiv-specific cd8(+) and cd4(+) t-cells producing ifn-γ and tnf-α were detected and were comparable in frequency to those that were hcv-specific. in co-infected individuals, viral-specific cd8(+) t-cells produced more of the fibrogenic cytokine, tnf-α. in both monoand co-infected individuals, intra-hepatic hcv-specific t-cells were poorly functional compared to hiv-specific t-cells. in co-infection, haart was not associated with a reconstitution of intra-hepatic cd4(+) t-cells and was associated with reduction in both hiv and hcv-specific intra-hepatic cytokine responses. conclusion: the accumulation of functional hiv-specific t-cells in the liver during hcv/hiv co-infection may represent a bystander role for hiv in inducing faster progression of liver disease. approximately 25% of human immunodeficiency virus-1 (hiv) infected individuals are also infected with hepatitis c virus (hcv) [1] . hiv adversely affects each stage of the natural history of hcv infection. fewer individuals recover spontaneously from hcv infection when also infected with hiv [2] . among those with persistent hcv infection, hiv co-infection is associated with higher hcv viremia and more rapid progression to cirrhosis and hepatocellular carcinoma [3] . a recent meta-analysis showed that hiv co-infection increased the risk of histological hepatic cirrhosis by two-fold and clinically decompensated liver disease by six-fold [4] . in addition, hcv co-infection is associated with increased incidence of haart (highly active antiretroviral therapy) related liver injury [5] . the mechanisms for hepatic damage in hcv/hiv co-infection are poorly defined. although intra-hepatic t-cell immune responses are necessary for hcv clearance, they have also been shown to play a central role in mediating hepatocellular injury by direct cytotoxicity or indirectly by releasing cytokines. in this regard, ifn-c has been shown to be anti-fibrogenic, whereas, tnf-a activates hepatic stellate cells, which induce fibrosis, and likely contributes to progression to cirrhosis [6, 7] . potent and broad cd4 + and cd8 + t-cell immunity are important for virologic control in both hcv and hiv viral infections. ex-vivo hcv-specific cd8 + t-cell responses in peripheral blood mono-nuclear cells (pbmcs) from mono-infected individuals are generally weak [8] . although, peripheral hcvspecific cd4 + and cd8 + t-cell responses are somewhat weaker in hcv/hiv co-infected individuals [9] , similar frequencies of intra-hepatic hcv-specific responses appear to be obtained in hcv versus hcv/hiv co-infection [10, 11] . however, ex-vivo hiv-specific cd8 + t-cell responses in pbmcs from hiv monoinfected individuals are about one log higher than ex-vivo hcvspecific responses in hcv mono-infection. in addition, impairment in cellular immune responses to hcv compared to hiv has been shown in hcv/hiv co-infection [12] . hiv-specific cd8 + t-cells are easily detectable in blood of untreated hiv infected individuals [13] . such high frequencies of hiv-specific t-cells circulating in peripheral blood led us to question whether these cells could also migrate to the liver in hcv/hiv co-infection and through bystander responses add to the inflammation induced by hcv-specific t-cells. hcv mono-infected and hcv/hiv co-infected individuals who required liver biopsies for work up of liver disease were recruited for the study (see results and table 1 ). all study participants provided informed, written consent and the study protocol was approved by the research ethics board at the university of toronto and st. michael's hospital. both blood and liver biopsy samples were received from each participant. liver biopsy samples were washed in rpmi-1640 to remove contaminating blood lymphocytes, manually homogenized with a plastic plunger, and treated with dnase (0.002%, sigma) and collagenase iv (0.02%, sigma) for 30 minutes, stirring at 37uc. the digested cell suspension was filtered through a 70 mm strainer, washed and re-suspended in r-10 medium (10% fetal calf serum). in order to identify candidate epitope-specific responses to be detected in ex-vivo liver samples, we first mapped antigen-specific t-cell responses in blood against the entire hiv-1 clade-b and hcv-1a proteome using the matrix approach by ifn-c eli-spot assay as described previously [14] . mapped peptides were then pooled to evaluate hepatic responses. in order to address the possibility that differing epitopes were only targeted in the liver, we also used four peptide pools that previously were shown to target a majority of responses. these pools spanned hiv-gag and hcv-ns3, hcv-ns4 and hcv-core protein (2 mg/peptide/ml, from national institute of health reagent program). of the hcv pools, the pool that gave the strongest elispot response in pbmcs was used for hepatic cell stimulation (see below). all the extracted cells from each liver biopsy were split in three wells and stimulated on the same day as pbmcs. 1610 6 pbmcs and liver-isolated cells were stimulated with either dmso, hiv or hcv peptide pools as described previously [14] . hiv pools consisted of peptides that were screened by the matrix approach in that individual plus the hiv-gag pool. likewise, hcv pools consisted of mapped peptides plus an hcv pool that gave the strongest response in pbmcs. cd107a antibody (pe-cy5, bd pharmingen) was added at the time of stimulation. the following antibodies were used for staining: cd8-pe texas-red (beckman coulter), cd4-pacific blue (e-bioscience), cd3-apccy7, ifnc-fitc, tnfa-pecy7, il2-apc, mip-1b-pe (bd bioscience), pd-1 fitc (biolegend) and dead cell stain aqua (invitrogen, molecular probes). cells were analyzed on a multi-color facsaria flow cytometer (bd biosciences). for blood samples between 500,000 to 1,000,000 total events and for liver biopsy samples between 50,000 to 200,000 total events were collected. data analysis was performed using flowjo version 8.6 (treestar inc., san carlos, ca). polychromatic flowjo data were analyzed with pestle software, and pie-chart graphs were generated using spice software (obtained from m. roederer, national institutes of health, bethesda md). multi-parameter analysis of hiv-gag: 77-85 (slyntvatl: sl9) specific cd8 + t-cells was conducted in both blood and liver of co-infected individuals initially identified with a positive elispot response to the 15-mer hiv peptide including sl9 epitope, using the corresponding tetramer (itag mhc class-i tetramer, beckman coulter). tetramer staining was performed prior to peptide stimulation, at room temperature for 20 minutes. tetramer stained cells were then washed and stimulated with 10 mg/ml of sl9 peptide followed by ics staining as mentioned above. additional hiv, hcv and cmv-specific pentamer staining (pro5 mhc class i pentamers -proimmune) was conducted followed by pd-1 staining. data were analyzed by performing two-tailed non-parametric mann-whitney test using graphpad prism version 4.00. p-values#0.05 were considered significant. three groups of individuals were studied as depicted in table 1 ; hcv mono-infected (n = 6), hcv/hiv co-infected who were not receiving haart (n = 8) and hcv/hiv co-infected who were receiving haart for greater than one year at the time of evaluation (n = 12). all individuals never received prior treatment for hcv and underwent liver biopsies for staging and evaluation for pegylated-interferon/ribavirin treatment. hcv/hiv co-infected individuals had higher hcv viral loads. on average, cd4 t-cell counts of hiv infected individuals were .400/ml in both groups. of note, the mean hepatic fibrosis scores were higher in the haart treated and mono-infected groups in this cohort, indicating that individuals in these groups had more advanced disease at the time of biopsy in this study. although similar frequencies of intra-hepatic lymphocytes were obtained in dual versus mono-infection, haart-treated individuals showed a trend towards greater percentages of lymphocytes in their biopsies ( figure 1a ). the percentage of intra-hepatic cd4 + t-cells was significantly reduced in dual infection [31.6%613.8 for hcv vs 6.5%62.9, for hcv/hiv therapy naïve, p,0.01], and was not associated with any improvement in haart-treated individuals, as previously shown in the gut [15] (figure 1b) . however, compared to hcv mono-infected individuals the percentage of intra-hepatic cd8 + t-cells was higher in both co-infected groups [33.8%65.5% for hcv vs 67.3%615.5% for hcv/hiv therapy naïve vs 59.5615.1% for hcv/hiv on haart, p,0.01] (figure 1b ). to determine the presence of intra-hepatic viral specific t-cell responses, liver isolated cells were stimulated with hcv and hiv peptide pools. summary data of viral specific responses are depicted in figure 2 . in response to stimulation with hiv peptide pool, untreated co-infected individuals showed significantly higher frequencies of intra-hepatic cd4 + t-cells producing ifn-c, compared to hcv mono-infected [0.1660.05% vs 0.0260.01%, p,0.05], and haart-treated co-infected individuals [0.1660.05% vs 0.0360.05%, p,0.05] (figure 2a ). untreated co-infected individuals showed a trend towards lower frequencies of intra-hepatic ifn-c producing cd4 + t-cells in response to hcv peptides. surprisingly, haart-treated co-infected individuals had significantly reduced hcv-specific ifn-c producing cd4 + t-cells when compared to untreated co-infected individuals [0.0260.01% vs 0.4660.11%, respectively, p,0.01] (figure 2a). therapy naïve co-infected subjects had greater ifn-c producing cd8 + t-cells in response to hiv peptides compared to hcv mono-infected individuals [1.3960.37% vs 0.0260.0%, p,0.05], and haart was associated with a significant reduction in the frequencies of these cells [1.3960.37% vs 0.3060.26%, p,0.05] (figure 2b). although there was a trend for enhanced intra-hepatic cd8 + t-cells producing ifn-c in response to hcv peptides in therapy-naive co-infection compared to hcv mono-infection, this was not found to be statistically significant. haart on the other hand, was associated with a significant reduction in hcv-specific, intra-hepatic cd8 + t-cells producing ifn-c [1.360.37% vs 0.0360.01%, p,0.05] (figure 2b). similarly, co-infected individuals had significantly greater intrahepatic tnf-a expressing cd4 + t-cells after hiv peptide stimulation compared to hcv mono-infected [0.260.05 vs 0.0260.01, p,0.01], although haart had no significant effect on their frequencies (figure 2c ). hcv mono-infected individuals showed significantly higher frequencies of hcv-specific tnf-a producing cd4 + t-cells compared to haart-treated co-infected individuals [0.9160.25% for hcv vs 0.2360.20 for hcv/hiv on haart, p,0.01] (figure 2c), but did not show significant differences with the untreated co-infected group. the therapy-naïve co-infected group showed significantly higher frequencies of intra-hepatic tnf-a producing cd8 + tcells in response to both hiv-1 and hcv antigens. both types of to study the functional profile of virus-specific t-cells in hcv/ hiv co-infection, simultaneous expression of 5 distinct cd8 + t-cell markers were analyzed in 3 individuals within each cohort using a previously developed multicolor flow cytometry method [16] . expression levels of degranulation marker cd107a and cytokines ifn-c, tnf-a and il-2, as well as the chemokine mip-1b were simultaneously measured in response to hcv or hiv peptides in both blood and liver of each individual. figure 3 depicts a representative multi-parameter analysis of cd8 + t-cell responses in liver and blood of a therapy-naïve, co-infected subject in response to hiv and hcv peptide pools. these data indicate that both hcv and hiv specific cd8 + t-cells expressing one or more functions are detectable in the liver and blood. compared to blood, the frequency of hiv-specific t-cells producing cd107a and il-2 was shown to be significantly higher in the liver of therapy-naïve, co-infected individuals. consistent with previously reported data [10] , hcv-specific responses were compartmentalized to the liver and stronger than peripheral hcv-specific responses (figure 3c ). recognition of functional ctl, specific for the hla-a0201-restricted hiv-sl9 epitope in hcv/hiv co-infected liver to determine if t-cells specific for an hiv immuno-dominant epitope are present in hcv/hiv co-infected liver, we quantified cd8 + t-cells specific for hla-a*0201-restricted slyntvatl (sl9) epitope in the liver of individuals with positive sl9 responses in their blood. we identified 3 co-infected hla-a*0201 individuals, among them one showed a response to the sl9 epitope of hiv-gag antigen. figure 4 shows the multi-parameter analysis of tetramer positive cd8 + t-cells in blood and liver of this therapy-naïve, co-infected individual. the tetramer cytokine response pattern was shown to be different in the liver compared to blood of the same individual, with diminished intra-hepatic tetramer-specific ifn-c responses and an increase in both cd107a and tnf-a responses, with the majority of sl9 tetramer positive cells expressing these two markers. we also included cmv as a non-hepatotropic control virus in our liver analysis. using a pool of hla-a*0201-and hla-a*2402-restricted, cmv-specific pentamers, we did not detect any cmv-specific cd8 + t-cells in hcv/hiv co-infected liver, although we could readily detect them in blood (figure 4c ). using the panel of markers tnf-a, ifn-c, mip-1b, il-2, and cd107a, we characterized the ability of cd8 + t-cells to simultaneously exert these 'functions' in response to both hcv and hiv peptides. figure 5 depicts a representative functional profile of virus specific cd8 + t-cells in blood and liver during hcv/hiv co-infection (fig. 5a) and hcv mono-infection (fig. 5b) . analysis of co-infected subjects demonstrates a very limited functional hierarchy of hcv-specific t cells in the blood, with majority of t-cells producing one function. hiv-specific tcells from blood had a more expanded functional hierarchy. in accordance with previous reports on hiv mono-infected individuals [16] , cells expressing all 5 functions were absent in the blood of co-infected subjects, mainly due to lack of il-2 production. in co-infection, intra-hepatic cd8 + t-cells responding to hcv peptides were within the single-responding and 2+ populations. intra-hepatic cd8 + t-cell responses to hiv peptides produced a larger spectrum of responses. cd107a responding cells were represented in nearly all of the different hiv-specific populations in the liver of co-infected individuals. as expected, hcv-specific responses in the blood of hcv mono-infected subjects were mainly single functional. the profile of hcv-specific responses in the liver of hcv mono-infected individuals showed the appearance of a very small population of 4+ responding cells in the liver. we and others have considered a cutoff point of more than 2 simultaneously expressed markers to demonstrate poly-functional characteristic of responding t-cells [16, 17] . figure 5c shows a comparison between average frequency of intra-hepatic viralspecific responses within the pool of cd8 + t-cell populations expressing 2 markers or less, and those within the pool of populations expressing more than 2 markers simultaneously. for both hcv and hiv-specific cd8 + t-cells the majority of responses had two or less functions. however, intra-hepatic hiv-specific responses demonstrated more poly-functionality, compared to hcv-specific responses either within co-infected or mono-infected individuals [0.0560.01 vs 0.00760.00, p,0.05; hiv-specific responses vs hcv-specific responses in hcv/hiv co-infected group]; [0.0560.01 vs 0.0160.00, p,0.05; hivspecific responses in hcv/hiv co-infected group vs hcvspecific responses in hcv mono-infected group]. in summary, although viral-specific t-cells, simultaneously expressing all 5 measured markers were rarely found in the liver, intra-hepatic hiv-specific t-cells showed greater functional capacity when compared to those being hcv-specific. based on the recently highlighted role of pd-1 contributing to the dysfunction of t-cells in chronic viral infections, we also determined whether hiv and hcv-specific intra-hepatic t-cells differ in the degree of pd-1 expression. in an hcv/hiv coinfected liver, we found that 100% of intra-hepatic hcv-specific cd8 + t-cells were pd-1 positive, compared to 48.8% of those cells that were hiv-specific (figure 5d ). this is the first study to demonstrate the presence of hivspecific t-cells within the liver of hcv/hiv co-infected individuals. the finding of hiv-specific t-cells within liver of co-infected individuals may not altogether be surprising, given the high frequencies of hiv-specific cd8 + t-cells found in the peripheral blood in untreated hiv infection. nevertheless, it is surprising to find functional t-cells of such viral specificities to be accumulating in liver. in contrast, we could not detect cmvspecific t-cells in co-infected liver despite their abundance in blood indicating that different viruses target t-cells to the liver. recent studies have demonstrated that systemic viral infections may recruit viral-specific t-cells to the liver. the significance of non-hepatotropic viral-specific t-cells that are found in liver is unclear. it has been postulated that the liver can non-selectively trap activated t-cells during any infection, and thus act as a 'sink' or 'graveyard' [18] , however it is unclear whether these cells are rendered anergic while traveling in the liver or contribute to inflammation and damage as a result of bystander activation. of note, is that hepatitis has been observed in measles [19] , sars [20] and in 20% of individuals with acute hiv infection [21] . polakos et. al. [22] found that some individuals infected with influenza-a develop transaminitis and showed in a murine influenza model that influenza-specific cd8 + t-cells migrate to figure 3 . polychromatic facs analysis of viral-specific t cells in hcv/hiv co-infection. shown are representative facs data of the hiv and hcv specific multi-parameter cd8 + t-cell responses from (a) liver and (b) blood of subject om 405, a therapy-naïve hcv/hiv co-infected individual, after in vitro stimulation using pool of hiv and hcv peptides. initial gating on forward scatter area (fsc-a) versus height (fsc-h) was used to remove doublets. the events were further gated on forward scatter (fsc) versus the dead cell marker to remove dead cells. lymphocytes were gated on the remaining live cells on a fsc versus ssc plot. gates on cd3 + /cd8 + cells were then generated. all responses are gated on a cd3 + /cd8 + population and presented against tnf-a on the x-axis. figure (c) shows a comparison of the frequency of hiv and hcv-specific cd8 + t-cells in the liver and blood of therapy-naïve, co-infected individuals. all intra-hepatic hcv-specific responses are significantly stronger than peripheral hcv-specific responses. doi:10.1371/journal.pone.0003454.g003 non-hepatotropic viruses such as hiv, cmv and ebv, in general do not induce chronic hepatitis, thus, it is possible that the coexistence of hepatotropic viruses may alter the hepatic environment to allow recruitment of activated t-cells non-specifically. this could be due to an up-regulation of integrins such as icam-1 and vcam-1 in hepatic sinusoids as previously shown during hcv infection [23] that could enhance t-cell recruitment. in this regard, spangenberg et. al. [24] demonstrated the presence of influenza-specific t-cells in about 50% of liver biopsies from hcv mono-infected individuals. there are several lines of evidence demonstrating that the liver efficiently clears many foreign pathogens, including rna viruses. it is shown that liver is a major organ for clearing simian immunodeficiency virus in rhesus monkeys [25] . there is also evidence for the detection of hiv rna in the liver of hiv infected individuals [26] . these findings support the identification of hiv-specific t-cells in the liver. in hcv/hiv co-infection, it is possible that intra-hepatic hcv-specific cd4 + t-cells become infected with hiv and recruit hiv-specific immune responses to this site. evidence for these potential mechanisms will need further analysis on liver biopsies of co-infected individuals. our analysis of liver biopsies from hcv/hiv co-infected individuals not only demonstrate that hiv-specific t-cells producing ifn-c and tnf-a are detected in the liver, but also exhibit comparable frequencies of responses to those that are hcv-specific. this observation may explain the added contribution of hiv-specific immune responses to the ongoing intrahepatic damage induced by hcv-specific t-cell responses that are inefficient in clearing the virus. therapy naïve co-infected individuals demonstrated a higher frequency of intra-hepatic cd8 + t-cells that produce tnf-a in response to both hcv and hiv antigen stimulation compared to hcv mono-infected individuals. in addition, we identified cd8 + t-cells specific for an immunodominant hiv epitope in co-infected liver, demonstrating high frequency of tnf-a expression. intra-hepatic tnf-a has been previously associated with liver fibrosis, and the accumulation of cells expressing this marker may explain in part the faster rate of liver disease progression found in hcv/hiv coinfection. further comparisons of tnf-a responses between immunodominant hcv and other hiv epitopes in a larger cohort of individuals are warranted. contrary to our expectation, viral-specific, intra-hepatic levels of ifn-c were also higher in the therapy-naïve co-infected group, which would be against the expected protective role of ifn-c. however, we interpret this observation as a potential effect of the fibrogenic tnf-a to mask ifn-c protection. on the other hand, viral-specific t-cells are composed of several major populations with unique functional patterns. therefore, measurement of only one or two t-cell functions may not provide a comprehensive picture of the quality of t-cell responses. recent lines of evidence demonstrate the importance of the qualitative rather than quantitative characteristics of cd8 + t-cell responses to efficient viral control [13, 27] . the significance of tcell populations simultaneously representing 5 different functions has been discussed as a hierarchical functional model in viral infections such as cmv and ebv which are effectively controlled by respective cd8 + t-cells [16] . hcv-specific cd8 + t-cells were not poly-functional which is consistent with the notion that although hcv-specific t-cells are found in hepatic tissue, their loss of poly-functionality may be associated with inefficient control of hcv replication. hiv-specific t-cells in the liver of co-infected individuals however, simultaneously could express 4 and 5 of the measured markers. recently, t-cell exhaustion has been related to signaling pathways through pd-1 [28, 29] . our analysis of pd-1 levels of antigen-specific cd8 + t-cells from co-infected liver demonstrates higher expression of pd-1 on hcv-specific t-cells, compared to those specific for hiv, supporting the notion that the former are less functional. the observed poly-functionality of intra-hepatic hiv-specific t-cells, should have little effect on hcv replication but would further enhance the cytokine milieu induced from bystander activation, and contribute to liver damage during co-infection with hcv. in this regard, we found that the degranulation marker cd107a dominates the hiv-specific cd8 + t-cell responses in the liver, with the majority of the responding cells expressing cd107a, a surrogate marker for the cytotoxic function of cd8 + t-cells. activated hiv-specific cd8 + t-cells with the potential to degranulate could induce bystander damage. in addition, the release of chemokines such as mip-1b by the same cells could also attract further lymphocytes without hcv specificity to the liver. bystander function of these non-specific t-cells could expand the tissue damage triggered by hcv infection and ultimately activate fibrogenesis. we found that the frequency of cd4 + t-cells within livers of coinfected individuals was reduced compared to hcv monoinfection. surprisingly, haart did not appear to reconstitute the cd4 + t-cell population within liver. despite this defect of cd4 + t-cell help, comparable frequencies of hcv-specific-cd8 + t-cells were found in co-infected livers. haart-treated biopsies showed further reduced frequencies of hcv-specific responses. these data support previous findings that show haart induces cd4 + t-cell recovery but not any restoration of hcv-specific tcell responses peripherally [30] . further investigation is needed to clarify the role of cd4 + t-cell help in affecting the frequencies of hcv-specific cd8 + t-cells in hcv/hiv-1 co-infection. haart was also associated with a reduction in frequencies of hiv-specific t-cell responses within liver, indicating that removing the hiv antigenic load may also reduce the opportunity for such cells to accumulate within hepatic tissue. here, we propose a novel mechanism for enhanced hcvrelated liver disease progression in hiv co-infection; that of bystander activation and induced inflammation from hiv-specific t-cells accumulated in the liver. our data however are limited in the cross-sectional nature of our cohort, the low number of analyzed liver biopsies and the narrow range of cd4 + t-cell counts among the studied individuals. we should also acknowledge that ex-vivo functional t-cell capacity may not exactly reflect the situation in-vivo. further studies, particularly, those which are prospective are warranted in order to understand the role that hiv-specific t-cells play in contributing to fibrosis and in particular how haart modulates these responses. frequency of cd8 + t-cells specific for hcv, hiv and cmv in hcv/hiv co-infected liver (om 385). liver isolated mononuclear cells were stained with pools of hla-a*0201 and hla-a*2402-restricted pentamers (pro5 mhc class i pentamers, proimmune), followed by staining for cell surface markers cd3 and cd8. the following pentamers were used for each group: hcv pentamers: ns3-cingvcwtv and ns3-klvalginav; hiv pentamers: pol-ilkepvhgv and gag p24-tlnawvkvv; cmv pentamers: pp65-nlvpmvatv and pp65-qydpvaalf. no cd8 + t-cells specific for cmv were detected in this co-infected liver sample. similar findings were found in another individual (data not shown). doi:10.1371/journal.pone.0003454.g004 background is shown to become extremely low when examining combinations of functions, nearly reaching 0 events for multiple functions simultaneously. this permits a very low threshold for detection of positive responses from multiple combinations. consequently, for multi-parameter analysis, the results were thresholded based on a minimum criterion of positivity, as calculated by spice software and presented as the 90 th percentile of negative values for each analysis. each pie chart generated by spice software, represents the hierarchy of responses to either hcv or hiv antigen stimulation. for simplicity, responses are grouped by number of functions and matched to the colored bars, with black bars representing the percentage of responding cells to hiv peptides and gray bars representing the percentage of responses to hcv peptide stimulation. in all pie charts, color red represents the 5+ responding population and the colors blue, green, turquoise, and yellow representing the 4+, 3+, 2+, and 1+ populations respectively. color-coded arcs represent the dominant marker within each pie slice, with color blue representing tnf-a, red for cd107-a, green for ifn-c and black for mip-1b. although il-2 is included in the presentation and demonstrated by bar graphs, the software would not allow for arc colors for more than 4 responses. as a result there is no arc representative for il-2. figure (c) represents the average frequency of intra-hepatic viral specific responses within the pool of cd8 + t-cell populations simultaneously expressing 2 functions or less, compared with those within the pool of populations expressing more than 2 functions simultaneously; as analyzed in 3 subjects within each cohort of hcv mono and hcv/hiv co-infected individuals. the cutoff point of simultaneous expression of more than 2 measured markers is considered to show cd8 + t-cell poly-functionality. effect of human immunodeficiency virus on hepatitis c virus infection among injecting drug users impaired hepatitis c virus-specific t cell responses and recurrent hepatitis c virus in hiv coinfection retrospective analysis of the impact of hiv infection and alcohol use on chronic hepatitis c in a large cohort of drug users influence of human immunodeficiency virus infection on the course of hepatitis c virus infection: a meta-analysis hepatotoxicity associated with antiretroviral therapy in adults infected with human immunodeficiency virus and the role of hepatitis c or b virus infection pathogenic roles of tumor necrosis factor receptor p55-mediated signals in dimethylnitrosamine-induced murine liver fibrosis the role of tumor necrosis factor-alpha in liver toxicity, inflammation, and fibrosis induced by carbon tetrachloride impaired effector function of hepatitis c virus-specific cd8+ t cells in chronic hepatitis c virus infection hiv longterm non-progressors maintain brisk cd8 t cell responses to other viral antigens cd8+ cell responses to hepatitis c virus (hcv) in the liver of persons with hcv-hiv coinfection versus hcv monoinfection comparison of hcv-specific intrahepatic cd4+ t cells in hiv/hcv versus hcv human immunodeficiency virus type 1-hepatitis c virus coinfection: intraindividual comparison of cellular immune responses against two persistent viruses analysis of total human immunodeficiency virus (hiv)-specific cd4(+) and cd8(+) t-cell responses: relationship to viral load in untreated hiv infection hiv-specific cd8+ lymphocytes in semen are not associated with reduced hiv shedding severe cd4+ t-cell depletion in gut lymphoid tissue during primary human immunodeficiency virus type 1 infection and substantial delay in restoration following highly active antiretroviral therapy hiv nonprogressors preferentially maintain highly functional hiv-specific cd8+ t cells polyfunctional analysis of human t cell responses: importance in vaccine immunogenicity and natural infection the liver as a site of t-cell apoptosis: graveyard, or killing field? measles associated hepatobiliary disease: an overview clinical significance of hepatic derangement in severe acute respiratory syndrome acute human immunodeficiency virus type 1 infection kupffer cell-dependent hepatitis occurs during influenza infection inflammatory markers in chronic hepatitis c intrahepatic cd8+ t-cell failure during chronic hepatitis c virus infection the liver is a major organ for clearing simian immunodeficiency virus in rhesus monkeys identification and quantitation of hiv-1 in the liver of patients with maintenance of large numbers of virus-specific cd8+ t cells in hivinfected progressors and long-term nonprogressors pd-1 expression in acute hepatitis c virus (hcv) infection is associated with hcvspecific cd8 exhaustion pd-1 expression on hiv-specific t cells is associated with t-cell exhaustion and disease progression differences in hcv-specific t cell responses between chronic hcv infection and hiv/hcv co-infection we are grateful to the patients who contributed their time and effort to this study. we would also like to thank ms. dionne white for her expertise and assistance in flow cytometry and dr. george makedonas for spice and pestle software training. we specially thank dr. mario roederer for providing us with the mentioned software. key: cord-001007-645zegcv authors: kim, hak; kim, kisoon; kim, dae-won; jung, hee-dong; min cheong, hyang; kim, ki hwan; soo kim, dong; kim, you-jin title: identification of recombinant human rhinovirus a and c in circulating strains from upper and lower respiratory infections date: 2013-06-27 journal: plos one doi: 10.1371/journal.pone.0068081 sha: doc_id: 1007 cord_uid: 645zegcv human rhinoviruses (hrvs), in the enterovirus genus within the family picornaviridae, are a highly prevalent cause of acute respiratory infection (ari). enteroviruses are genetically highly variable, and recombination between serotypes is known to be a major contribution to their diversity. recently it was reported that recombination events in hrvs cause the diversity of hrv-c. this study analyzed parts of the viral genes spanning the 5′ noncoding region (ncr) through to the viral protein (vp) encoding sequences of 105 hrv field isolates from 51 outpatient cases of acute respiratory infectious network (arinet) and 54 inpatient cases of severe lower respiratory infection (slri) surveillance, in order to identify recombination in field samples. when analyzing parts of the 5′ncr and vp4/vp2 encoding sequences, we found intraand interspecies recombinants in field strains of hrv-a and -c. nineteen cases of recombination events (18.1%) were found among 105 field strains. for hrv-a, there were five cases (4.8%) of intraspecies recombination events and three cases (2.8%) of interspecies recombination events. for hrv-c, there were four cases (3.8%) of intraspecies recombination events and seven cases (6.7%) of interspecies recombination events. recombination events were significantly more frequently observed in the arinet samples (18 cases) than in the slri samples (1 case; p< 0.0001). the recombination breakpoints were located in nucleotides (nt) 472–554, which comprise stem-loop 5 in the internal ribosomal entry site (ires), based on the hrv-b 35 sequence (accession no. fj445187). our findings regarding genomic recombination in circulating hrv-a and -c strains suggest that recombination might play a role in hrv fitness and could be a possible determinant of disease severity caused by various hrv infections in patients with ari. human rhinoviruses (hrvs), first discovered in 1953, are nonenveloped, positive single-strand rna viruses of the genus enterovirus in the family picornaviridae [1, 2] . hrvs are the major cause of upper and lower respiratory tract infections in humans [3] . in particular, hrvs are the second main cause of bronchiolitis and wheezing illnesses in infancy, which are strongly associated with a high risk of developing asthma in childhood [4] . it is recognized that 50-85% of most asthma exacerbations are caused by hrv infections [5, 6, 7, 8] . hrvs are transmitted commonly by the respiratory-salivary route, both by contact and airborne transmission [9] . hrvs have a genome of approximately 7,200 base pairs (bp) containing a single reading frame that encodes four viral capsid proteins (vp1, vp2, vp3 and vp4) and seven nonstructural proteins (2a pro , 2b, 2c, 3a, 3b, 3c pro and 3d pol ) [8, 10] . hrvs have a 5′ non-coding region (ncr) of about 650 bp that consists of a cloverleaf-like (cl) motif and an internal ribosome entry site (ires) at the 5′ end of the genome, with roles in viral replication and translation initiation, respectively. the ires of hrvs contains five secondary rna structures called stem-loops (sls) 2-6 and a polypyrimidine tract (ppt) located between sl5 and sl6 [11] . currently, 153 proposed types of hrvs have been identified and classified into three species (a, b and c) based on the nucleotide sequences that encode the vp1 protein, as hrv-c species are difficult to isolate by in vitro culture and to serotype [9, 12, 13, 14, 15, 16, 17, 18, 19] . hrvs share many features of their genome organization and structure with other picornaviruses. the genus enterovirus in the family picornaviridae has undergone much evolutionary genetic diversification through recombination events [20, 21, 22, 23] . hrvs have also developed genetic diversity by recombination near the 5′ ncr/p1, p1/p2 and p2/p3 boundaries [8, 10, 24, 25] . recombination events between the 5′ ncr and the vp4 encoding sequences have mainly been observed in hrv-c, and the recombination breakpoints have been identified at sl5 and the ppt region [10] . in addition, some hrv-c that are closely related to hrv-a based on sequence analysis of the 5′ ncr have been designated "hrv ca", and these hrv ca subspecies have been suggested to arise from interspecies recombination [10, 25, 26] . however, the inter-or intraspecies recombination events of field hrv-a and intraspecies recombination events of field hrv-c have not been studied until recently. after finding of hrv-c, numerous studies on epidemiological and clinical manifestations have been conducted to elucidate the pathogenicity of hrv-c infection associated with disease severity. however the correlation between disease severity and hrv-c is still controversial [27, 28, 29, 30, 31] . in addition, hrvs are genetically heterogeneous and recombination events between or within species could cause complicate the identification and typing of hrvs, as well as a differentiation of clinical consequences. this study aimed to understand and characterize the various recombination events between 5' ncr and vp4/vp2 region of field strains of hrv including species a, b and c, using 105 hrvs identified from two distinctive laboratory surveillance systems, the acute respiratory infectious network (arinet) and severe lower respiratory tract infections (slri) surveillances undertaken from october 2008 to march 2009. we investigated the occurrence and location of recombination events in these field strains of hrvs by phylogenetic analysis and by applying the recombination detection program and described comparative analysis of unbiased recombination events in two surveillance systems with different disease severity. for specimens from arinet, this study was approved by the institutional review board of korea centers for disease control and prevention (kcdc; 2012-09con-03-4c) as it involved de-identified remaining respiratory tract samples which were not related to human gene study and collected for the respiratory virus diagnosis with written informed consent from patients, their parents or legal guardian. de-identification was performed except for each subject's age, gender, reported diagnosis, time of collection and virus detection results. in the case of specimens from slri, ethical clearance was obtained from yonsei university health system institutional review board, seoul, korea (4-2008-0649). target population was the total population of children less than 5 years needed to be admitted for their lower respiratory infections. patients who had their parents or legal guardians' written consent to participate in surveillance were enrolled. we obtained their nasopharyngeal aspirate specimens and their clinical information without personal ones. nasal aspirate specimens from patients with ari (n = 3082) and nasopharyngeal aspirate specimens from patients with slri (n = 381) were collected in the arinet and slri surveillances and marked as ka and kl in sample name respectively, in south korea from october 2008 to march 2009 [32] . among the hrv-positive samples-827 from arinet and 85 from slri-51 and 54 samples, respectively, were selected by random sampling method. the viral rnas of collected specimens were extracted using the qiaamp viral rna mini kits (qiagen, hilden, germany) according to the manufacturer's instructions and stored at -70 °c until used for experiments. the extracted rna was applied to one-step reverse transcription-polymerase chain reaction (rt-pcr) reagents and the labopass™ rv detection kit (cosmo genetech, seoul, south korea) for detection of hrv. the kit was developed by the division of influenza and respiratory virus in kcdc with cosmo genetech [32] . [33] . from the analysis of 131 genomes of reference hrvs from genbank and modification of previously reported primer sequences, a new primer set was designed covering the vp4/vp2 sequences (nt 447-1083). for the amplification of target genes, a 20 µl master mixture containing 2 µl of cdna, 1 µl of each of the 10 pm target primers, 12 µl of depc-treated ddh 2 o, 1 µl of 2.5 mm dntp mix (cosmo genetech), 2 µl of 10x sp-taq buffer (cosmo the hrv sequences were aligned with those of 53 reference hrvs and previously published hrv strains: ny-074 from new york (genbank accession number dq875932); cl170085 from geneva (genbank accession number eu840952); qpm from australia (genbank accession number ef186077); c subtype 35 from sweden (genbank accession number jf436925); n10, n36 and n46 from shanghai (genbank accession numbers gq223228, gq213131 and gq213134, respectively); lz268, lzy79, lz508 and lz101 from beijing (genbank accession numbers jf317013, jf317014, jf317015 and jf317017, respectively); and a21_p1177_sr3307, c36_p1075_s3911 and c43_p1154_sr1124 from wisconsin (genbank accession numbers jn837693, jn541267 andjx074056, respectively). the clustal w method of megalign (ver. 8.0.2(13.4) 2) in the lasergene 8 program suite (dnastar, madison, wi, usa) was used [34] . the 5′ncr and vp4/vp2 sequence analyses were based on nt 193-470 and nt 623-1053, respectively (genbank accession number fj445187) [13, 25] . the phylogenetic trees of the 5′ ncr and vp4/vp2 were predicted using mega 4 (ver. 4.0.2) by the neighbor-joining method [35] . bootstrap analysis was performed using 1000 replicates. in addition, each part of the 5′ ncr and vp4/vp2 sequences was identified using megablast. the sequences, covering nt 193-1053, were applied to splitstree 4 (http://www.splitstree.org) [36] and rdp 3 (http:// darwin.uvigo.es/rdp/rdp.html) for predicting recombination events [37, 38] . the 19 genome sequences of recombinant viruses described in this study have been deposited in genbank under accession numbers jx177615-jx177617, jx177619-jx177633 and jx177643. we obtained samples from the arinet and slri surveillances as described in the materials and methods. the arinet surveillance for outpatients with acute respiratory illness covers about 100 hospitals located all over korea and includes all ages. the slri surveillance for inpatients covers four general hospitals in metropolitan areas and includes infants and children of less than 5 years of age [32] . these arinet and slri surveillances represented mild and severe disease respectively, depending on clinical symptoms. among hrv-positive samples, 51 arinet samples and 54 slri samples collected during the 2008-2009 winter season were selected for further analysis. even though the age distributions of the arinet and slri patients were not comparable directly (because the object of arinet were patients from all ages but the slri from patients less than 5 years old), the majority of samples from both sets were from patients who were 1 year old or younger: 30/51 from arinet (59%), and 43/54 from slri (80%), as shown in table 2 . there were no differences in the gender ratio between the arinet (28 female and 23 male) and slri (28 female and 26 male) samples. patients from the arinet surveillance were diagnosed with pharyngitis, bronchitis, common cold, otitis media, pneumonia and sinusitis by the hospitals involved. patients in the slri surveillance were diagnosed with bronchiolitis, pneumonia, croup or asthma, as shown in table 3 . the 5′ ncr and vp4/vp2 sequences were applied to phylogenetic analysis using the mega 4 program with 53 reference hrvs and 14 previously isolated hrv strains, as described in the materials and methods. in this study, we only analyzed the sequences from 5' ncr to vp2 region, therefore the vp4/vp2 sequences were used to define the hrv types. the phylogenetic trees predicted by the vp4/vp2 sequences were divided into hrv-a, -b and -c clusters, as shown in figure s1a . the frequencies of presumed hrv-a, -b and -c were, respectively, 31 (60.8%), 1 (2.0%) and 19 (37.6%) in the 51 arinet samples, and 21 (38.9%), 4 (7.4%) and 29 (53.7%) in the 54 slri samples. the ratios of these species differed slightly between the slri and arinet samples, but the difference was not significant. in analysis using the 5′ncr regions, the phylogenetic trees ( figure s1b ) showed branches and cluster compositions differing from the vp4/vp2 sequence-based tree for both hrv-c and a. fourteen hrv-c reference strains (qpm, qce, nat001, ny-074, c 24, c 25, c 26, n36, n46, c-43 p1154, cl170085, lz269, lzy79 and lzy101), which were previously reported as ca subspecies having a hrv-a 5′ncr, also clustered together with hrv-a reference strains. in addition, seven field strains classified as hrv-c by the vp4/vp2 tree were also clustered with hrv-a clusters in the 5′ ncr-based tree. in contrast, three field strains classed as hrv-a by the vp4/vp2 tree were classified as hrv-c in the 5′ncr-based tree. furthermore, we found that nine field strains of hrv-a and -c that were categorized as the same species but related to a different type at the 5′ncr and vp4/vp2 sequences had possible intraspecies recombination between the same species. to further study these inconsistencies, all 5′ncr and vp4/vp2 sequences from these 19/105 (18.1%) strains were identified with the megablast program, and showed high identities with different types in the same or different species, depending on the region analyzed. however, some reference sequences with the highest identities did not cover the entire region from the 5′ncr to the vp4/vp2 sequences. in these cases, the reference or previously published sequences having full coverage but showing slightly less identity were searched and selected as parent genomes for detecting recombination (table 4 ). to summarize, only the 19 selected sequences in table 4 were re-applied to the mega 4 program (figure 1) , and the phylogenetic trees showed the same results as in figure s1 . these results suggested the possibility of recombination events between the 5′ ncr and the vp4/vp2 sequences. traditional bifurcating phylogenetic trees do not properly display the evolutionary history of different field strains, because one strain might be linked to more than one ancestral sequence. to confirm the possibility of inter-and intraspecies recombination, the 19 sequences were tested with the split decomposition network method and rdp3 using the regions from nt 193-1053 (from the 5′ncr to the vp2 sequence). each sequence with a parent sequence selected from megablast was applied to the splitstree 4 method [36] . all 19 selected field strains showed an interconnected relationship in the network, supporting recombination history between them, as shown in figure s2 . in addition, from the analysis using six methods in rdp, the 19 samples were predicted as being recombinant, and the recombination breakpoints were also suggested as expected from the results of phylogenetic analysis (figure 2 and table 5 ). the breakpoint indicated by at least three methods was identified as the breakpoint of each recombination strain, as shown in figure 3 . to summarize, 105 field strains were characterized. among these, five cases (4.8%) of intraspecies recombination strains and three cases (2.8%) of interspecies recombination strains were found among hrv-a viruses, and four cases (3.8%) of intraspecies recombination strains and seven cases (6.7%) of interspecies recombination strains were identified among hrv-c viruses. recombination events were significantly more frequent in the arinet samples (18/54; 33%) than in the slri samples (1/51; 2%; p< 0.0001). in our study, 52 strains of hrv-a (49.5%) and 48 strains of hrv-c (45.7%) were identified in 105 field samples, whereas only five hrv-b viruses (4.8%) were found. there was no clustering difference in the distribution of arinet and slri strains representing acute, mild and severe illness. coinfection with another respiratory virus-mainly rsv-was found in 10 cases of hrv-a, 15 cases of hrv-c and four cases of hrv-b. hrvs have remarkable genetic and antigenic variability, 102 known serotypes of hrv-a and b, and new types of hrv-c are being discovered continually. it is known that recombination events in the hrv genome have increased the diversity of each virus in the family picornaviridae [20, 21, 22, 23] . in earlier studies, lee et al. (2007) analyzed the 5′ncr of 103 hrvs from wisconsin and confirmed nine novel field strains [13, 25] . in 2009, huang et al. referred to these results and studied recombinant hrv-c among 66 hrvs from cases of ari. in that study, 14 of 34 strains of hrv-c were found to be related closely to hrv-a by analysis of the 5′ ncr sequences, and these strains of hrv-c were designated as "hrv ca". the authors suggested that this strain had arisen from interspecies recombination, and that the cc subspecies containing the hrv-c 5′ncr and vp4/vp2 sequences had not experienced a recombination event [10, 25] . palmenberg et al. identified intraspecies recombination of the hrv-a strain in three hrv-a reference viruses and interspecies recombination events in hrv-c viruses by phylogenetic and recombinant detection analysis [8, 10, 24, 25] . in the present study, intraspecies recombination events of hrv-a and interspecies recombination events of hrv-c were also detected using similar methods. surprisingly, we also detected three cases (2.8%) of interspecies recombination in hrv-a and four cases (3.8%) of intraspecies recombination in hrv-c viruses. the incidence of recombination events was found to be similarly distributed in both hrv-c (11/48 cases) and hrv-a (8/52 cases) strains (p>0.05) by testing for any difference between the two proportions [39] . we also confirmed that interspecies recombination events had occurred in 14 reference strains of hrv-c reference strains: qpm, qce, nat001, ny-074, c 24, c 25, c 26, n36, n46, c-43 p1154, cl170085, lz269, lzy79 and lzy101. interestingly, ka08-3539 and ka08-4189 have the same nucleotides in the 5′ncr regions which clustered with the c isolate lz508, but have the different vp4/vp2 sequences. thus, ka08-3539 (interspecies recombination in hrv-a) and ka08-4189 (intraspecies recombination in hrv-c) were related to hrv-a 42 and to the c isolate lzy101, respectively. ka08-4374 (interspecies recombination in hrv-a) and ka09-756 (intraspecies recombination in hrv-c) also had the same 5′ ncr and differences in the vp4/vp2 sequences. we assume that recombination events in the 5′ncr between vp4 and vp2 could have led to this diversity of hrvs. the recombinant breakpoint was located at the ires in sl5 at nt 472-554, as shown in table 5 and figure 3 . mcintyre et al. have identified the recombination breakpoints, which were at sl5 (nt 484-548, based on accession number fj445187) and ppt-sl6 (nt 560-581), of an interspecies recombinant rhinovirus of hrv-c [10] . in our study, although the breakpoint was not identified at ppt, the breakpoints in sl5 were similar to mcintyre's results. in addition, most of the breakpoints were scattered at nt 515-554 (the conserved nucleotide region in sl5), as shown in figure 3 . the 5′ncr including the ires plays an important role during viral replication, transcription and translation through the construction of a secondary rna structure. the sl5 region of the 5′ ncr forms an rna-protein complex with ptb, the cellular translation initiation protein, and the amplified sequences of vp4/vp2 and 5′ncr were identified by megablast program. 11 strains of hrv-c recombination events were identified compared to 8 strains of hrv-a recombination events. interestingly, the recombinant viruses were mainly found in arinet samples (94.7%: 18 of 19 strains). a identity b. these reference strains clustered with hrv-a in the 5′ncr phylogenetic tree in this study and previous report [10, 25] . c the parent genomes, that contained 5′ncr and vp4/vp2 region, of low identity for confirming recombination events it is known that the efficiency of ptb in stimulating ires activity is affected by variations in ires structure in polioviruses [40] . recently, artificial 5′ ncr interspecies strains produced by recombination between enteroviruses and rhinoviruses were investigated for studying the efficiency of the 5′ ncr in translation and replication in vitro. this study showed that the genome of the field virus was more efficient with translation and replication than the artificial recombination genomes [41] . accordingly, we assume that the translation and replication efficiencies are generally decreased by recombination events during evolution, except for a few favorable combinations and the recombinant viruses may have a different optimal temperature resulting to upper and lower respiratory tract infections. although genetic and immunological predispositions of patients are primary contributor for determination of disease severity, current results presented here also support that hypothesis, with 18 cases of recombinant viruses in the arinet isolates but only one case in the slri isolates. another suggestion from the current hypothesis is that sequence information of 5' ncr and recombination characteristics may lead us to identify a closer relationship between viral diversity and disease severity rather than single criteria of hrvs classification based on vp region sequences. in conclusion, this study is the first report describing intraand interspecies genomic recombination in circulating hrv-a and -c isolated from patients with acute or severe respiratory illness and these results will assist in investigating the causes of the diversity and evolution of hrvs arising through recombination events. further study should be required on the correlation between recombination at sl5 and the assignment of virulence factor(s) in recombinant viruses to elucidate the public health impact of hrv diversity. results are shown for all recombination events with 6 analysis methods by rdp program. a : position referred by genbank accession no. fj445187 the isolation of a new virus associated with respiratory clinical disease in humans a cytopathogenic agent isolated from naval recruits with mild respiratory illnesses human rhinoviruses: the cold wars resume wheezing rhinovirus illnesses in early life predict asthma development in high-risk children 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lower respiratory tract infections in korean infants and young children rhinoviruses replicate effectively at lower airway temperatures clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice the neighbor-joining method: a new method for reconstructing phylogenetic trees application of phylogenetic networks in evolutionary studies the fine-scale structure of recombination rate variation in the human genome rdp3: a flexible and fast computer program for analyzing recombination a note on effective sample size for constructing confidence intervals for the difference of two proportions polypyrimidine tract-binding protein stimulates the poliovirus ires by modulating eif4g binding experimental human rhinovirus and enterovirus interspecies recombination prospects for inferring very large phylogenies by using the neighbor-joining method key: cord-000237-mticfoic authors: guan, xuhua; silk, benjamin j.; li, wenkai; fleischauer, aaron t.; xing, xuesen; jiang, xiaoqing; yu, hongjie; olsen, sonja j.; cohen, adam l. title: pneumonia incidence and mortality in mainland china: systematic review of chinese and english literature, 1985–2008 date: 2010-07-23 journal: plos one doi: 10.1371/journal.pone.0011721 sha: doc_id: 237 cord_uid: mticfoic background: pneumonia is a leading infectious disease killer worldwide, yet the burden in china is not well understood as much of the data is published in the non-english literature. methodology/principal findings: we systematically reviewed the chineseand english-language literature for studies with primary data on pneumonia incidence and mortality in mainland china. between 1985 and 2008, 37 studies met the inclusion criteria. the quality of the studies was highly variable. for children <5 years, incidence ranged from 0.06–0.27 episodes per person-year and mortality ranged from 184–1,223 deaths per 100,000 population. overall incidence and mortality were stable or decreased over the study period and were higher in rural compared to urban areas. conclusions/significance: pneumonia continues to be a major public health challenge in young children in china, and estimates of pneumonia incidence and mortality vary widely. reliable surveillance data and new prevention efforts may be needed to achieve and document additional declines, especially in areas with higher incidence and mortality such as rural settings. despite the availability of safe and effective antibiotics and vaccines for treatment and prevention, pneumonia is a leading cause of death worldwide and the leading infectious disease killer [1, 2] . pneumonia is the single leading cause of death globally among children under 5 years of age accounting for approximately 2 million deaths annually [2, 3] . children in developing countries have an estimated 0.29 episodes of pneumonia per person-year, compared with 0.05 episodes per person-year in developed countries [3] . pneumonia is one of the leading causes of death in adults and children in china [4] . in urban areas, pneumonia is the fourth leading cause of death, and in rural areas pneumonia is the leading cause of death [5, 6] . a recent article in the chinese literature estimated that each year in china there are 2.5 million patients with pneumonia and that 125,000 (5%) of these patients die of pneumonia-related illness [5] . a 2008 global review by rudan and colleagues estimated that there were 21.1 million new cases of clinical pneumonia annually in china in children under 5 years of age (0.22 episodes/person-year), which is second only to india in burden (43.0 million new cases, 0.37 episodes/person-year) [3] . available estimates of the burden of childhood pneumonia in china vary widely, and pneumonia accounts for an estimated 17% of all child deaths in china and 67% of all childhood pneumonia deaths in the western pacific region [3, 6, 7] . although the global community recognizes that pneumonia is an important public health issue in china, the disease burden is not well studied nor necessarily reported in the english language and these data have not been systematically reviewed for english-or chinese-language readers [8] . complicating an assessment of the pneumonia incidence and mortality in china is the lack of ongoing and systematic surveillance. with the exception of human avian influenza and severe acute respiratory syndrome (sars), pneumonia and other respiratory diseases are not included in the 39 nationally notifiable infectious diseases in china [9] . however, in the wake of the 2003 sars outbreak, enhanced surveillance using pneumonia of unknown etiology for early detection of suspected sars was initiated in 2004 (case definition given in table s1 ). we conducted a systematic review of the chinese-and englishlanguage literature in order to describe pneumonia incidence and mortality in china, evaluate the quality of published studies, and identify gaps in the literature that can be addressed through surveillance and epidemiologic research projects in the future. no ethical review was required since all results are from the published literature. using pubmed, we searched the national library of medicine database for manuscripts published before october 31, 2008. the reference terms``pneumonia,''``acute respiratory infection,'' and`l ower respiratory tract infection'' were each combined with`c hina'' and``mainland china.'' using equivalent terms, we performed additional searches for publications in the chinese medical literature using the wanfang (http://www.wanfangdata. com/) and chongqingvip (www.cqvip.com) databases [10, 11] . in these databases, publications were available since 1982 and 1989, respectively. the english search terms were translated into chinese (by author x.g.) for use in the chinese-language searches [º, %'|8só, |8só (p-ý-ý' f)]. in addition, journal articles cited in the identified manuscripts were collected and added to the review. clinical and community-based studies with primary data collection in humans were identified through a literature search conducted in november 2008 ( figure 1 ). studies conducted exclusively in hong kong special administrative region (sar), macao sar or taiwan, china, were excluded. we also excluded studies focusing exclusively on sars and avian influenza, outbreak reports, diagnostic studies of pneumonia etiologies, and studies that did not include population-based estimates of incidence or mortality. two coauthors who read chinese as a first language (x.g., w. l.) abstracted from these references the year of publication; province, prefecture, and city; study population (e.g., age group); study design; site of case detection (i.e., inpatient, outpatient, or both); pneumonia case definition; and estimates of pneumoniarelated incidence and mortality. english data abstraction was each checked by a native english and mandarin speaker. data abstractions were validated during direct discussions with english-speaking epidemiologists (b.j.s., a.t.f., s.j.o., a.l.c.). incidence is reported as the annual number of pneumonia episodes per year (i.e., person-years). for manuscripts presenting incidence per 100,000 population, data were converted to personyears to be comparable. for example, if there were 200 cases in a year in a population of 10,000, the incidence would be converted from 2,000 per 100,000 per year to 0.02 person-years. when incidence was reported on an annual basis during a multi-year study period within a single study, a simple mean was calculated to report overall incidence during the study period. for mortality measures, when possible, we calculated case fatality rate (i.e., the percentage of pneumonia cases that died), mortality per 100,000 population, and mortality per 1,000 live births annually. we evaluated trends in incidence and mortality and made comparisons across studies by using studies with similar study methods and case definitions when possible. there were three pneumonia case definitions used in the studies: i. the world health organization (who) case definition of pneumonia for integrated management of childhood illness [12] , ii. chinese medical association guidelines (iia: communityacquired pneumonia (cap) [13] or iib: hospital-acquired pneumonia (hap) [14] ), and iii. physician assessment (iiia: acute lower respiratory infection; iiib: newborn pneumonia; or iiic: pneumonia as a cause of death in children under 5 years of age). the specific signs and symptoms for each case definition are detailed in table s1 . for each study that we identified through the systematic review, we critically reviewed the quality of each manuscript. based on published recommendations for measuring quality of epidemiologic studies of pneumonia [15] , we assessed quality using the following six criteria: (1) geographic location was reported, (2) study was conducted for a period of at least one year or multiples of one year to account for seasonal factors, (3) site of case detection or surveillance location was reported, (4) age and population size of cohort of at least 50 cases were reported, (5) quality assurance and monitoring methods were employed to assure that data was complete and high quality, and (6) a clearly defined case definition (e.g., not based solely on clinical diagnosis) was used and reported. these six criteria were selected to represent basic and essential indicators of epidemiologic study quality. each criteria was dichotomous (1 = reported and 0 = not reported); the sum of all reported criteria yielded the manuscript's overall quality criteria score (0 to 6; tables s2 and s3). at least one coauthor who read chinese as a first language (x.g., w.l.) reviewed each of the studies to evaluate these six criteria; evaluations were then validated during direct discussions with an english-speaking epidemiologists (b.j.s., a.t.f., s.j.o., a.l.c.). we included all studies in this review to report the differences in study quality. thirty-seven published studies met the inclusion criteria ( figure 1 ); 14 publications included data on pneumonia incidence and 28 publications reported pneumonia mortality estimates. at least three studies were conducted in beijing, guangxi ar, hubei, jiangsu, shanghai, shanxi, and sichuan each ( figure 2 ). five (36%) of the 14 incidence articles and 3 (11%) of the 28 mortality articles were identified using pubmed [16, 17, 18, 19, 20, 21, 22, 23] . the studies were each evaluated on six quality indicator criteria (tables s2 and s3) . first, all studies reported the geographic location of the study, but few, if any, reported more specific information on setting such as altitude and annual rainfall, prevalence of malnutrition and aids, or immunization coverage against pneumonia vaccines and access to health care [15] . second, all but 2 studies (35 [95%] of 37) reported at least one year of data. third, all studies reported the site of case detection and the age of the cases. fourth, the population size was not given for 15 (54%) of the 28 mortality studies; when population was reported, at least 50 cases were reported. fifth, four (22%) of 18 studies of incidence and 27 (97%) of 28 studies of mortality reported quality assurance and monitoring. the sixth quality criteria evaluated the case definition used. of the 14 studies that reported incidence, 5 (36%) used the who case definition for pneumonia and 4 (26%) used the chinese medical association guidelines case definition; 5 (36%) used physician diagnosis, which was considered less reliable than the other case definitions. the incidence estimates using the who case definition were generally higher than those using a physician diagnosis, so we report incidence estimates separately by case definition; there was no clear trend in mortality estimates based on case definition, so we did not separate these estimates by case definition. five (33%) of 15 incidence studies and only 1 (4%) mortality study included chest x-ray in their case definition. most of the mortality data relied on physician diagnosis (23 [82%] of 28 studies). one quarter (7 of 28) of the mortality studies were reports of deaths for children under 5 years of age collected through the national death surveillance program. in summary, all of the studies met at least four of the six quality criteria. only 5 (18%) of the 28 mortality studies and none of the incidence studies met all six quality criteria. age. based on 6 studies, the age-specific incidence of pneumonia in children ,1 year of age ranged from 0.01±0.68 episodes per person-year using either clinical or who case definitions [16, 17, 18, 19, 24, 25] . based on 9 studies of children ,5 years, the incidence ranged from 0.06±0.27 episodes per personyear using clinical case definition and from 0.14±0.66 using the who case definition (table s2) [16, 18, 19, 21, 24, 25, 26, 27, 28] . incidence was lower in older children [16, 25] , and one study presented a pneumonia incidence for adults: 0.037 episodes per person-year for people $65 years of age [20] . time trends. a study conducted in two provinces in the west found that pneumonia incidence in children ,5 years of age decreased from 1997 to 2000 by 64% in yunnan province (0.083 in 1997 versus 0.030 episodes per person-year in 2000) and by 47% in qinghai province (0.017 in 1997 versus 0.009 in 2000) [21] . rural vs. urban areas. seven incidence studies were performed in urban areas (50%), five in rural areas (36%), and two (14%) in both urban and rural areas. pneumonia incidence rates were generally higher in rural areas compared with urban areas (table s2) . for example, a study in guangdong that compared pneumonia rates in both rural and urban settings found that rates in rural areas were more than four times higher than urban rates in children ,1 year of age (0.91 vs. 0.19 episodes per person-year, respectively) and more than six times higher in children ,5 years of age (0.79 vs. 0.12 episodes per person-year, respectively) [24] . regions. most (83.3%) studies on pneumonia incidence were conducted in northeast and southeast china. the incidence of pneumonia in children ,5 years of age in northeastern china (range: 0.06±0.27 episodes per person-year) [16, 18, 19, 26] , was lower than southern china (range: 0.32±0.66 episodes per personyear), which includes the southeast, south central, and southwest regions (table s2) [24, 27, 28] . there were no studies exclusively in the north central or northwest regions. one multi-site study showed that pneumonia incidence among children ,5 years of age was higher in southern than in northern provinces (0.057 versus 0.013 episodes per person-year in southern yunnan and northern qinghai, respectively) [21] . a multi-site study in 1986 reported that pneumonia incidence in children ,14 years of age in eastern china (range 0.041±0.057 episodes per person-year) was higher than in western and central china (range 0.032±0.035 episodes per person-year) [25] . age. twelve (43%) of 28 studies that evaluated pneumoniarelated mortality presented estimates for children ,1 year of age; 23 (82%) presented estimates for children aged ,5 years; most (20 studies, 71%) presented mortality as deaths per 1,000 live births per year (table s3) . mortality rates ranged from 485 to 890 deaths from pneumonia per 100,000 population for children ,1 year of age [25, 29, 30] , and from 184 to 1223 deaths from pneumonia per 100,000 population for children ,5 years of age [25, 26, 27, 28, 29] . when mortality was measured per 1,000 live births, the estimates ranged from 0.66 to 12.8 deaths for children ,1 year of age [23, 31, 32, 33, 34, 35, 36, 37, 38] and from 0.71 to 16 for children ,5 years of age [21, 23, 26, 31, 33, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] (table s3 ). case fatality rates were higher in children ,1 year of age (4.67±4.88%) than in children ,5 years of age (0.52± 1.94%). one study in adults, which included older adults up to age 94 years, found a case fatality rate of 9.98% [22] . time trends. nine (50%) of 18 studies that examined multiple years of data reported decreasing mortality trends during their study periods [23, 27, 29, 30, 35, 36, 37, 38, 47] . for example, a seven-year study in shandong province found that the case fatality rate for pneumonia decreased from 1.94% to 0.50% from 1995 to 2001, mortality decreased from 421 to 119 deaths from pneumonia per 100,000 population, and mortality per 1000 live births decreased from 2.50 to 1.07 [26] . however, another nine studies did not show a clear declining trend [21, 33, 40, 41, 42, 43, 44, 45, 46] . for example, a separate study in shandong province found that while the mortality rate was lowest in the most recent year of the 11-year study (0.14 deaths per 1000 live births in children ,5 years in 2006), there was no clear decrease in mortality over the previous decade [31] . no studies showed increasing mortality. rural vs. urban areas. nineteen (68%) of 28 mortality studies were conducted in both urban and rural areas, while 6 (21%) were conducted in rural areas and 2 (7%) were conducted in urban areas only. as with incidence, pneumonia mortality in rural areas was generally higher than in urban areas. in one study conducted in zhejiang from 1991 to 1993, mortality for infants ,1 year old ranged from 10.63 to 15.06 cases per 1,000 live births in rural areas compared with 2.29 to 2.54 cases per 1,000 live births in urban areas; for children ,5 years, pneumonia mortality ranged from 10.75 to 15.13 in rural areas compared with 2.60 to 2.79 in urban areas [46] . a study in jiangsu province found 4.18 and 1.12 deaths per 1000 live births in rural and urban areas, respectively [35] . regions. studies on pneumonia mortality were more geographically representative than studies of pneumonia incidence, and there was at least one study of mortality from each of the six regions (table s3 ). estimates of mortality from pneumonia in infants ,1 year old were consistent in four of the six studies conducted in southern china (3.04±3.73 deaths from pneumonia per 1,000 live births) [32, 33, 35, 36] ; only one study in northern china reported mortality rates from pneumonia in infants (0.66 deaths from pneumonia per 1,000 live births) [31] . in children aged ,5 years, the highest mortality rates were reported by four studies that were each conducted in multiple regions throughout mainland china (9.55±14.40 deaths from pneumonia per 1,000 live births; table s3 ) [21, 23, 38, 48] . relatively high mortality rates in this age group were also reported in the northwest [42, 43, 44] and the southwest [37, 47] (3.91±7.51 and 7.7±12.08 deaths from pneumonia per 1,000 live births, respectively). three studies evaluated hospital-acquired pneumonia [49, 50, 51] . in shanghai where the studies were performed, the hospital-acquired pneumonia rate ranged from 1.6% to 2.4% of hospitalized patients. given the population size of china (1.3 billion persons) [6] , the varied health utilization and economic development, the diverse climate (tropical to subarctic), and the range of population densities, the burden of pneumonia in china would be expected to be large and highly variable across regions. despite overall trends from these studies suggesting that pneumonia incidence and mortality are stable or decreasing, pneumonia continues to be a major public health concern in china [7] . the studies in this review found incidence of pneumonia in children ,5 years of age that were as low as what has been estimated for the developed world globally (0.05 episodes per person-year) and that were as high as what has been estimated for the developing world (0.29 episodes per person-year) [52] . the studies included in this review reported pneumonia incidence for children ,5 years of age (0.06± 0.27 episodes per person-year from 1985 to 2008) that was similar or less than what has been estimated for china (0.22 episodes per person-year in 2008) [3] . although the studies reported a wide range of pneumonia mortality estimates (184±1,223 deaths per 100,000 population), these are consistent with pneumonia remaining the leading cause of childhood mortality in china [7] . while the data summarized here provide insights into pneumonia in china, they also serve as a reminder that reliable and high quality national and regional data on pneumonia incidence and etiology are needed to adequately direct prevention and control efforts. perhaps the largest limitation to this study is that study comparisons of morbidity and mortality rates were constrained by the wide variation and quality of the study designs. this is particularly evident in the wide range of mortality estimates among the different studies. in general, the incidence estimates were higher when the more standardized who case definition was used compared with estimates obtained from physician diagnosis; however, there was significant variability even among studies that used similar case definitions. the use of a standard pneumonia case definition that is designed for surveillance and epidemiologic research would improve generalizability and could allow for direct comparisons of incidence and mortality estimates in china and elsewhere [15, 53] . most studies spanned multiple years, which would account for differences in seasonality of pneumonia, but a few were conducted for one year or less. although a few of the studies reported large surveillance populations, many calculated incidence based on relatively small populations or did not report the population under surveillance. most of these studies were conducted in large, urban centers primarily serving residents of densely-populated areas; few included adults or populations from rural western china. although many of the studies were conducted prospectively, none calculated incidence using active, population-based surveillance and population denominators were not reliably measured in each study. until standardized case definitions and appropriate surveillance methodology are applied, pneumo-nia incidence and mortality estimates should be interpreted cautiously. in regions for which we identified published data, pneumonia incidence in china appears to be declining and mortality is stable or declining from the 1980s to the 2000s. there are several factors that could have contributed to these changes over time. china experienced substantial economic growth during these years, a trend that was more pronounced in the coastal (eastern) areas. from the beginning of economic reforms in 1978 to 2006, china's gross domestic product (gdp) increased 5,719% from 362.4 billion rmb to 21,087.1 billion rmb [54] . the income of the average chinese person also improved during this time period; gdp per capita rose by 4,144% from 379 rmb to 16,084 rmb [54] . economic development may have led to improvements in healthcare quality and access to health services. in addition to economic development, china is undergoing dramatic healthcare reform, including government-sponsored healthcare in rural areas [55, 56] . declines in the incidence of pneumonia are likely attributable to the implementation of pneumonia intervention measures, such as technical training for village doctors, health education to parents, improved pneumonia surveillance and case management, and the use of vaccines against pneumonia in the routine immunization program (namely measles and pertussis). the improved detection and recognition of pneumonias following the sars, avian influenza and 2009 influenza h1n1 epidemics could lead to more cases of pneumonia being promptly identified and treated. large scale programs to introduce less polluting cookstoves in china have led to decreases in lung cancer and chronic obstructive pulmonary disease [57, 58] ; studies from other countries suggest that reductions in exposure to indoor air pollution from solid fuels used for cooking can also lead to fewer cases of pneumonia [59] . other strategies, including better access to care, improved hygiene, and better nutrition may need strengthening to effectively reduce the incidence of pneumonia in china [60] . for many chinese, adequate healthcare remains difficult to access; this review revealed a disparate incidence and mortality of pneumonia across different regions of china [55, 61] , some of which is likely due to inequalities in health care. for example, respirators and ventilators for children are not currently available in many county hospitals. eastern china is more developed, whereas western china is more rural. according to a report on national maternal and child health endorsed by the china ministry of health, unicef, and who, pneumonia is the leading cause of death in children under 5 years of age in some rural areas, and comprises a larger proportion of deaths in children under 5 years of age as the area becomes more rural [62] . this report also demonstrated the decreasing trends in pneumonia mortality across china, especially in areas where few clinical studies have been completed. better access to proven public health interventions, including vaccines, is needed in the public sector in china. vaccines against haemophilus influenzae type b (hib), streptococcus pneumoniae, and influenza are not part of routine childhood vaccination programs in many countries worldwide [63, 64] ; none of these vaccines are included in the routine childhood immunization schedule in mainland china. however, hib and influenza vaccines are commonly available in many parts of china through vaccination clinics, and hong kong sar is the first region in china, as well as asia, where pneumococcal conjugate vaccine will be included in their routine childhood immunization program starting september 2009 [65] . in addition, hong kong sar recommends seasonal influenza vaccine use in high risk groups. vaccine clinical trials in other countries have estimated that 21% of radiologically confirmed pneumonia is caused by hib [66] and 36% by pneumococcus [66, 67] ; over 10% of hospitalized pneumonia in children in nearby thailand are due to influenza [68] . studies within china have suggested that hib and pneumococcus are common causes of pneumonia in children [69, 70, 71] , suggesting that widespread use of these two vaccines, as well as influenza vaccine, could reduce the incidence and mortality of pneumonia in china. this paper has several strengths, particularly the inclusion of papers published in both the english and chinese literature. a recent global review on childhood pneumonia incidence included only two of the 14 articles presented here, suggesting that articles not published in english are usually overlooked and difficult to obtain [3, 16, 18] . we did not search three of the five major chinese-language literature databases (the china national knowledge infrastructure china academic journals full-text database, chinese biomedical literature database, and chinese medical current content), so we may not have captured all relevant manuscripts; however, the two databases that we did search include some of the greatest number of journals and articles of the five major databases [10, 11, 72] . in addition, this review includes information on all ages, although only two studies included adults. while global pneumonia prevention efforts often focus on children, the burden of pneumonia in adults and the elderly is also substantial. importantly, interventions aimed at children may have underappreciated benefits on adults. for example, the introduction of universal childhood pneumococcal vaccination in the united states in 2000 resulted in significant declines in pneumococcal incidence in both children and adults. in 2003, the indirect effect of preventing invasive pneumococcal disease in adults was over twice the direct effect of preventing cases in children [73] . comprehensive data on 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(20tob°?ºñå ê{¡þ~ae). health vocational education key: cord-000478-88wo4xen authors: gowen, brian b.; ennis, jane; russell, andrew; sefing, eric j.; wong, min-hui; turner, jeffrey title: use of recombinant adenovirus vectored consensus ifn-α to avert severe arenavirus infection date: 2011-10-24 journal: plos one doi: 10.1371/journal.pone.0026072 sha: doc_id: 478 cord_uid: 88wo4xen several arenaviruses can cause viral hemorrhagic fever, a severe disease with case-fatality rates in hospitalized individuals ranging from 15-30%. because of limited prophylaxis and treatment options, new medical countermeasures are needed for these viruses classified by the national institutes of allergy and infectious diseases (niaid) as top priority biodefense category a pathogens. recombinant consensus interferon alpha (cifn-α) is a licensed protein with broad clinical appeal. however, while cifn-α has great therapeutic value, its utility for biodefense applications is hindered by its short in vivo half-life, mode and frequency of administration, and costly production. to address these limitations, we describe the use of def201, a replication-deficient adenovirus vector that drives the expression of cifn-α, for preand post-exposure prophylaxis of acute arenaviral infection modeled in hamsters. intranasal administration of def201 24 h prior to challenge with pichindé virus (picv) was highly effective at protecting animals from mortality and preventing viral replication and liver-associated disease. a significant protective effect was still observed with a single dosing of def201 given two weeks prior to picv challenge. def201 was also efficacious when administered as a treatment 24 to 48 h post-virus exposure. the protective effect of def201 was largely attributed to the expression of cifn-α, as dosing with a control empty vector adenovirus did not protect hamsters from lethal picv challenge. effective countermeasures that are highly stable, easily administered, and elicit long lasting protective immunity are much needed for arena and other viral infections. the def201 technology has the potential to address all of these issues and may serve as a broad-spectrum antiviral to enhance host defense against a number of viral pathogens. the arenaviridae family of viruses has several members that can cause viral hemorrhagic fever, an acute, often-fatal, viral syndrome characterized by intense fever, malaise, and less frequently, bleeding and neurologic manifestations. case fatality rates of hospitalized patients suffering from arenaviral hemorrhagic fever (ahf) range from 15-30% [1, 2, 3, 4] . arenaviruses known to cause ahf include junín, machupo, guanarito, sabiá, and chapare in the south american continent, and lassa and lujo in west and southern africa, respectively. primary transmission of the arenaviruses from respective rodent reservoir hosts to humans occurs via exposure to contaminated excreta [5] . person-to-person transmission can occur through contact with blood or other body fluids during the care and management of infected individuals [1, 6] . notably, these viruses are considered a threat to national security and are classified as highest priority pathogens by the niaid [7] . at present, the treatment of ahf is limited to ribavirin and immune plasma [8, 9] . the latter has only been proven to be effective in treating cases of argentine hemorrhagic fever (junín virus infection) within 8 days of disease onset. off-label usage of ribavirin has been shown to be effective in treating lassa fever when therapy was initiated within 6 days of the development of clinical symptoms. however, there are toxicities associated with ribavirin therapy at dosages required for efficacious use, which may contribute to the observed poor patient compliance in completing prescribed treatment regimens [10, 11] . very limited case data using ribavirin to treat other ahfs supports the use of emergency protocols [1, 12, 13] , however the utility of ribavirin therapy remains to be seen. interferon alpha (ifn-a) is an effective part of the host innate immune response, which can be manufactured as a recombinant human protein with broad clinical appeal [14] . consensus (c)ifna, also known as ifn alfacon-1 and infergen, is a licensed, second generation ifn-a engineered to contain the most frequently occurring amino acids among the nonallelic ifn-a subtypes. previously, we have demonstrated that cifn-a can be used effectively alone, or in combination with ribavirin, to treat pichindé virus (picv) infection in hamsters [15, 16] , an experimental model of acute arenaviral disease [17] . however, while cifn-a has clinical value, its usefulness is hindered by its short half-life and cost to manufacture. there is an initial distributive half-life of 7 minutes and a beta half-life of 2 to 5 hours [14] . the rapid systemic clearance requires frequent dosing to achieve desired therapeutic levels. consequently, treatment can result in well-documented toxicities which include headache, depression, hair loss, fever, and malaise. in order to combat the rapid degradation, pegylated forms of recombinant ifn-a have been introduced with half-lives that are on the order of days instead of hours, thus reducing the number of injections to once per week [18] . however, the cost to manufacture peg-ifn-a is exceedingly high, and the pegylation process has been shown to reduce the activity of ifn-a, thereby further increasing the production costs. to circumvent the fast decay of cifn-a, a replicationincompetent, recombinant adenovirus type 5 (rad5) gene delivery platform was designed to drive constitutive expression of the cifna gene from transduced nasal epithelial target cells. this rad5 cifn-a virus, called def201, was first developed in mice and recently shown to be active against yellow fever virus (yfv) infection in hamsters [19, 20] . the intranasal (i.n.) inoculation used in the yfv study prevents the host immune system from recognizing the ad5 vector, thereby bypassing any possible preexisting immunity [21] . in the present study, we evaluated the use of def201 administered i.n. for the prevention and treatment of picv infection in hamsters. all animal procedures complied with usda guidelines and were conducted at the aaalac-accredited laboratory animal research center at utah state university under protocol 1229, approved by the utah state university institutional animal care and use committee. female golden syrian hamsters were obtained from charles river laboratories (wilmington, ma) and acclimated for a minimum of 6 days prior to experimentation. they were fed standard hamster chow and tap water ad libitum. animals were approximately 7-9 weeks old at the time of virus challenge. picv, strain an 4763, was provided by dr. david gangemi (clemson university, clemson, south carolina). the virus was passaged once through hamsters. virus stocks were prepared from pooled livers harvested from infected hamsters. virus dilutions were made in minimal essential medium (mem), and infectious inoculum was given bilaterally in two intraperitoneal (i.p.) injections of 0.1 ml each. the recombinant adenovirus vectored cifn-a (rad5-huifn-a; def201) and the rad5 empty vector (rad ev) control virus were provided by defyrus, inc. (toronto, on, canada) at a concentration of 6610 9 and 2610 11 plaque-forming units (pfu)/ml, respectively. both viruses were prepared in pbs for i.n. instillation in a 200 ml volume. virus titers were assayed using an infectious cell culture assay as previously described [22] . briefly, a specific volume of liver or spleen homogenate or serum was serially diluted and added to triplicate wells of vero (african green monkey kidney; american type culture collection, manassas, va) cell monolayers in 96well microplates. the viral cytopathic effect (cpe) was determined 7 to 8 days post-virus inoculation, and the 50% endpoints were calculated as described [23] . the assay detection ranges were 2.8 to 9.5 log 10 50% cell culture infectious doses (ccid 50 )/g of liver or spleen and 1.8 to 8.5 log 10 ccid 50 /ml of serum. in samples presenting with undetectable liver or spleen virus, a value of ,2.8 was assigned (,1.8 for serum). conversely, in cases wherein virus exceeded the detection range, a value of.9.5 (.8.5 for serum) was assigned. for statistical analysis, values of 2.8 or 9.5 log 10 (1.8 or 8.5 for serum) were assigned as needed for samples with undetectable or saturated virus levels, respectively. detection of alt in serum is an indirect method for evaluating liver disease. serum alt levels were measured using the alt (sgpt) reagent set purchased from pointe scientific, inc. (lincoln park, mi) per the manufacturer's recommendations. the reagent volumes were adjusted for analysis on 96-well microplates. experimental design def201 dose range titration experiment. hamsters were weighed on the morning prior to the day of infection and grouped (n = 15 for drug treatment groups, 26 for the placebo group) so that the average hamster weight per group across the entire experiment varied by less than 5 grams. varying pfu amounts of def201, the rad ev control virus, or saline placebo treatments were administered in a single i.n. dose 24 h prior to challenge with ,5 pfu of picv. five animals from each group were sacrificed on day 7 of infection. serum was collected for assaying alt activity, and virus titers were determined for liver, spleen, and serum samples as described above. the remaining 10 animals (21 for the placebo group) were observed 21 days for mortality and weighed individually every 3 days starting on day 0. sham-infected normal controls (n = 3) were included for comparison. extended pre-exposure prophylaxis experiment. the design was similar to the def201 titration experiment with the following differences. hamsters were weighed on the morning of initial pretreatment (day 214 relative to the infection) and grouped (n = 15 per group). groups were treated once i.n. with 10 8 pfu of def201, rad ev control virus, or saline placebo. treatments were given 14 or 7 days prior to challenge with ,5 pfu of picv. animals were observed for 28 days post-challenge for mortality. post-exposure prophylaxis experiment. the design was similar to the def201 pre-exposure prophylaxis experiment with the following differences. single dose i.n. treatments with 10 8 pfu of def201 or rad ev were administered 24 h prior to, or 6, 24, or 48 h after challenge with ,5 pfu of picv. on day 28 postinfection, the surviving animals (including 6 naïve sham-infected controls) were re-challenged. morbidity and mortality were observed out to 58 days after the initial challenge. kaplan-meier survival plots and all statistical evaluations were done using prism (graphpad software, ca). the log-rank test was used for survival analysis. for analyzing differences in viral titers, alt levels, and weight change, a one-way analysis of variance (anova) with newman-keuls post test or the kruskal-wallis (two-tailed) test with the dunn's post test was performed based on gaussian distribution of the data. in the initial trial, hamsters were treated with 10 6 to 10 8 pfu of def201 one day prior to challenge with a lethal dose of picv. pretreatment with the highest dose of 10 8 pfu of def201 resulted in 100% survival, and 10 7 and 10 6 pfu doses also significantly protected 90% and 60% of hamsters, respectively, from mortality ( figure 1a) . moreover, the hamster that succumbed in the 10 7 group, survived 19 days. importantly, only one out of ten hamsters treated with 10 8 pfu of the control rad ev virus survived the infection; however, there did appear to be a slight delay in the time to death in the hamsters that received the control virus treatment. the weights of the hamsters were measured every 3 days to assess weight gain over the course of the experiment as a marker of well being ( figure 1b) . notably, from day 3 to day 6, a time before weight loss due to illness from picv infection would have been expected, hamster weights decreased as the dose of def201 increased. this would suggest that the higher treatment doses may have resulted in some loss of appetite, probably due to mild illness due to expression of consensus ifn since no overt effects were noticeable when handling the animals. the hamsters that received the 10 6 pfu dose of def201 gained weight through day 6 similarly to the animals treated with saline placebo and the normal controls (sham-infected, untreated) ( figure 1b) . the high-dose of rad ev control virus also resulted in a slight reduction in weight compared to the controls, suggesting that the immune response to the adenoviral vector alone may have caused some malaise in the animals. there was no elevation in serum alt levels on day 7 of infection in samples collected from parallel treated and infected hamsters receiving def201 (figure 2a ). eighty percent of the rad ev group and 100% of the pbs placebo group had elevated levels of alt, reflective of liver disease. interestingly, the 10 7 and 10 8 pfu def201 groups presented with little to no day-7 virus burden in the serum, liver, or spleen, while the 10 6 group developed viral titers that were comparable to the rad ev and placebo controls ( figure 2b-d) . a delay in the development of liver disease in the 10 6 pfu def201treated animals may explain the reduced alt levels. alternatively, saturation of liver virus titers in the low-dose def201, rad ev, and placebo groups may have masked a substantial difference between the former and the viral vector and vehicle control groups. we next evaluated the prophylactic window of protection against picv infection using the 10 8 pfu dose of def201. animals were treated one or two weeks prior to challenge with a lethal dose of picv. consistent with the trend observed in initial dose titration study, hamsters treated with the 10 8 pfu dose of def201 had significantly reduced weights compared to those that received the rad ev and placebo control treatments ( figure 3a) . nevertheless, the pretreatment with def201 seven days before infection was highly protective (90% survival rate; figure 3b ). notably, the single hamster that failed to survive the challenge succumbed on day 5, which was several days before the mean time to death measured in both the placebo and rad ev groups. an autopsy to determine the cause of death was not performed. in hamsters treated two-weeks prior to picv challenge, def201 significantly reduced mortality (50% survival) and extended the time of death in the animals that succumbed ( figure 3c ). in contrast, uniform lethality was seen with animals that received the rad ev and placebo treatments. of the 5 surviving animals pre-treated with def201, one was anorexic at the conclusion of the study on day 28 post-infection. this was reflected by a 27% weight loss compared to the animals starting weight. it is possible that this hamster, which appeared ill and lethargic, was not able to completely prevent the infection. it was unclear whether it would have ultimately recovered if the observation period had been extended. on both the 7-day ( figure 4a , c, e, g) and 14-day ( figure 4b , d, f, h) pretreatments, def201 significantly reduced day-7 viral loads and liver disease (alt) compared to the controls. the absence of elevated alt levels in the def201-treated hamsters may be explained by the 2-3 log 10 reduction in liver virus burden ( figure 4e , f) and a delay in the development of liver disease. although tissue titers were slightly lower when def201 was given 7 days prior to challenge compared to the 14 day pretreatment, this was not evident with serum viral burden. because most animals had measurable replicating picv ( figure 4c-h) , it is likely that survivors would have been immunized and protected from subsequent challenge. this may not be the case with hamsters treated with def201 24 h prior to challenge since most had no detectable virus titers in spleen, liver, or serum on day 7 of picv infection ( figure 2b-d) . having observed dramatic protection when administered up to 2 weeks prior to challenge, a final experiment was conducted to determine the therapeutic value of def201 in the hamster picv infection model. when def201 was administered 6 or 24 h after challenge, highly significant protection was observed ( figure 5 ). efficacy waned when def201 treatment was delayed to 48 h postinfection. as anticipated, the treatment given 24 pre-challenge verified previous activity, with all animals surviving challenge. interestingly, there was higher than expected survival with the control rad ev treatments initiated 24 and 48 h post-challenge, suggestive of a slight antiviral effect as the time of treatment was further delayed ( figure 5) . the surviving hamsters from this experiment were re-challenged with picv to assess the ability of def201 to enhance longer-term protection via acquired immunity. with the exception of 4 animals in the 24 h def201 pretreatment group, and a single animal in the rad ev 48 h group, all animals that were challenged with picv on day 0 of the experiment survived a second challenge figure 3 . def201 extended pre-exposure prophylaxis protects hamsters from lethal picv challenge. animals were treated i.n. with a single dose 10 8 pfu of def201, the rad ev control virus, or pbs placebo 7 or 14 days prior to picv infection. animal weights were measured two weeks prior to, and at the time of, picv challenge. the effect of 7-day and 14-day pretreatments on a) weight change over the two-week period prior to picv challenge and the extended picv prophylaxis efficacy data for the b) 7-day and c) 14-day pretreatments are shown. ***p,0.001 compared to respective placebo-treated animals. b p,0.01, c p,0.001 compared to respective rad ev-treated animals. doi:10.1371/journal.pone.0026072.g003 on day 28 ( figure 5 ). all six naïve animals that were initially shaminfected succumbed as expected. in animals that were sacrificed on day 7 relative to the first infection, reductions in alt and viral titers were most evident in the groups that received def201 within 24 h of infection ( figure 6) . notably, in the animals treated with the control rad ev, there was an interesting trend that developed with the 6 h post-infection group having the greatest alt levels and viral titers, followed by the 24, 48, and 224 h groups. this trend may suggest a low-level immune stimulation in the hamsters relative to the time at which the rad ev was given. the resulting lack of measurable viral replication in the 224 h def201 group ( figure 6b-d) is likely insufficient to elicit immunological memory. it is unclear as to why one of the first infection survivors from the 48 h rad ev group ultimately succumbed to the second infection. in the present study, our findings demonstrate that expression of cifn-a following a single i.n. administration of def201 offers a strong protective effect in hamsters against challenge with picv that included limiting liver disease and inducing an antiviral state that inhibited systemic and tissue viral replication. the lack of significant antiviral activity elicited by the rad ev control virus suggests that the enhanced antiviral response produced by def201 is largely due to the expression of the cifn-a gene. the weak stimulatory effect seen in 1 of the 3 experiments was not surprising considering the number of host systems that play a role in sensing the adenovirus vector [24] ; however, the effect was short-lived. in contrast, the enhancement of the host antiviral defenses by def201 was long-lasting with a 14-day pre-picv challenge prophylactic window. moreover, def201 was effective when given 1-2 days post-picv infection. these data also suggest that sufficient viral replication may be necessary to elicit an adaptive immune response that confers lasting protective immunity, as, for the most part, only re-challenged animals from the 24 h def201 pretreatment group succumbed to a second challenge with a lethal picv inoculum. presumably, the robust innate immunity and antiviral state induced by the def201 pretreatment rapidly controlled the ,5 pfu challenge dose obviating the development of the adaptive immune response and immunological memory. the pathogenic arenaviruses have evolved strategies to suppress and evade the host immune response [25, 26, 27, 28] , resulting in uncontrolled replication and broad dissemination. however, they appear to be unable to block the induction of ifn stimulated genes via exogenous type i ifn [29] , which may, in part, explain the success of def201 and cifn-a treatments [15] . also essential to the success of def201 was early intervention prior to significant viral replication and engagement of innate immune suppressive functions. indeed, early induction of a strong type i ifn response is associated with favorable disease outcome in nonhuman primates challenged with lassa virus [30] . early post-exposure prophylaxis was also required with exogenous cifn-a protein administered by the i.p. route [15, 16] . with the multiple strategies that arena and other pathogenic viruses have in place to subdue the ifn-mediated host antiviral response [31] , the utility of def201, recombinant ifn proteins, and ifn inducing agents will depend upon the nature of the ifn pathway blockade and require early administration to be effective post-exposure. notably, with daily cifn-a protein injections of up to 40 mg/kg, significant protection was observed; however, survival rates did not exceed 80% in those studies employing the same picv hamster model system and virus stock [15, 16] . in contrast, def201 consistently elicited greater protection (90-100%). the improved efficacy observed with def201 may be explained by a combination of factors that includes constitutive expression of fully glycosylated protein and reduced animal stress levels by avoiding daily injections for 7-10 days. we hypothesize that with the appropriate dose of def201, therapeutic levels of consensus ifn-a can be maintained, effectively eliminating the daily bolus effect produced by i.p. injections. in addition, because cifn-a is produced in genetically engineered escherichia coli, the native glycosylation pattern is lost. conceivably, enhanced immunotherapeutic activity results from fully glycosylated cifn-a expressed from cells transduced with def201. previous studies in mice with a related def201 virus expressing mouse ifn-a (mdef201) have shown the utility of adenovirusbased system to counter viral infections [20, 32, 33] . more recently, in a different hamster model of viral hemorrhagic fever, several of us reported on efficacy of def201 in mitigating yfv infection and disease [19] . yfv infection appears to be more sensitive to the effects of def201, as a lower dose was able to provide complete protection. taken together with the results of the present study, the experimental animal data support the broad use of def201 for extended pre-exposure and early post-exposure prophylaxis applications. further investigations using advanced arenavirus models based on challenge of nonhuman primates with pathogenic arenaviruses [17] are needed to better evaluate the potential of def201 to prevent severe disease in humans. nonhuman primate models should allow the full spectrum of cifn-a activity not possible in hamsters or guinea pigs. the familiarity of the fda with adenovirus gene delivery technology and approved cifn-a protein support the development of def201 for clinical use. an important step in the development process is the safety/toxicology testing in rodents, which is presently underway. in our studies, the highest dose of 10 8 pfu of def201 administered by the i.n. route appeared to be well-tolerated in hamsters despite evidence of weight loss. they did not appear visibly ill, but clearly the treatment was having some effect that possibly led to reduced food and water consumption consistent with mild toxicity seen with ifn-a therapy. the i.n. delivery route is designed to circumvent pre-existing immunity to adenovirus type 5 in humans [21, 34] , and may limit systemic inflammation that could occur by parenteral administration of large numbers of adenovirus particles. ultimately, the production of a shelf-stable, powdered formulation of def201 for easy i.n. administration and long-term storage would be ideal for stock-piling in the event of the need for mass distribution due to intentional release or (re)emerging disease outbreaks of arena or other viral etiology. nosocomial outbreak of novel arenavirus infection, southern africa treatment of argentine hemorrhagic fever new opportunities for field research on the pathogenesis and treatment of lassa fever venezuelan hemorrhagic fever: clinical and epidemiological studies of 165 cases arenaviridae: the viruses and their replication the counter-bioterrorism research agenda of the national institute of allergy and infectious diseases (niaid) for cdc category a agents efficacy of immune plasma in treatment of argentine haemorrhagic fever and association between treatment and a late neurological syndrome lassa fever. effective therapy with ribavirin review of the literature and proposed guidelines for the use of oral ribavirin as postexposure prophylaxis for lassa fever ribavirin for lassa fever postexposure prophylaxis brief report: treatment of a laboratory-acquired sabia virus infection animals were treated as described in figure 5 and sacrificed on day 7 of infection for analysis of serum (a) alt and (b) virus titer, and (c) liver and (d) spleen viral titers. *p,0.05, ***p,0.001 compared to rad ev-treated animals treatment of bolivian hemorrhagic fever with intravenous ribavirin interferon-alpha as an immunotherapeutic protein interferon alfacon-1 protects hamsters from lethal pichinde virus infection combinatorial ribavirin and interferon alfacon-1 therapy of acute arenaviral disease in hamsters animal models of highly pathogenic rna viral infections: hemorrhagic fever viruses enhanced circulating half-life and antitumor activity of a site-specific pegylated interferonalpha protein therapeutic treatment of yellow fever virus with an adenovirus-vectored interferon, def201, in a hamster model pre-and post-exposure protection against western equine encephalitis virus after single inoculation with adenovirus vector expressing interferon alpha nasal delivery of an adenovirus-based vaccine bypasses pre-existing immunity to the vaccine carrier and improves the immune response in mice vitro and in vivo activities of t-705 against arenavirus and bunyavirus infections a simple method of estimating fifty percent endpoints recognition of virus infection and innate host responses to viral gene therapy vectors human macrophages, but not dendritic cells, are activated and produce alpha/beta interferons in response to mopeia virus infection differential inhibition of type i interferon induction by arenavirus nucleoproteins short double-stranded rnas with an overhanging 59 ppp-nucleotide, as found in arenavirus genomes, act as rig-i decoys z proteins of new world arenaviruses bind rig-i and interfere with type i interferon induction inhibition of the type i interferon response by the nucleoprotein of the prototypic arenavirus lymphocytic choriomeningitis virus early and strong immune responses are associated with control of viral replication and recovery in lassa virus-infected cynomolgus monkeys type 1 interferons and the virus-host relationship: a lesson in detente singledose intranasal administration with mdef201 (adenovirus vectored mouse interferon-alpha) confers protection from mortality in a lethal sars-cov balb/c mouse model alpha interferon as an adenovirus-vectored vaccine adjuvant and antiviral in venezuelan equine encephalitis virus infection comparative seroprevalence and immunogenicity of six rare serotype recombinant adenovirus vaccine vectors from subgroups b and d key: cord-001083-vy1nxax2 authors: malagnac, fabienne; fabret, céline; prigent, magali; rousset, jean-pierre; namy, olivier; silar, philippe title: rab-gdi complex dissociation factor expressed through translational frameshifting in filamentous ascomycetes date: 2013-09-19 journal: plos one doi: 10.1371/journal.pone.0073772 sha: doc_id: 1083 cord_uid: vy1nxax2 in the model fungus podospora anserina, the payip3 gene encoding the orthologue of the saccharomyces cerevisiae yip3 rab-gdi complex dissociation factor expresses two polypeptides, one of which, the long form, is produced through a programmed translation frameshift. inactivation of payip3 results in slightly delayed growth associated with modification in repartition of fruiting body on the thallus, along with reduced ascospore production on wood. long and short forms of payip3 are expressed in the mycelium, while only the short form appears expressed in the maturing fruiting body (perithecium). the frameshift has been conserved over the evolution of the pezizomycotina, lasting for over 400 million years, suggesting that it has an important role in the wild. all living cells have to accurately convert gene information into proteins in a complex multistep process. translation is suspected to be the key step where errors may occur. one of the main issues is to keep translating the correct reading frame from the start codon to the stop codon. the reading frame is defined by the start codon, as the genetic code is unpunctuated; any shift leads to an aberrant protein. spontaneous frameshift errors occur at low levels in comparison to missense errors. the ribosome has developed several mechanisms to allow proper translocation of the trnas in the different sites. signals are present on some mrnas and induce an alternative reading of the genetic code by diverting the standard rules. these elements are very efficient at disrupting ribosome accuracy, increasing error rate from background (,5610 25 ) to 50% [1] . such programmed events are known as recoding. among the recoding sites identified, programmed frameshifting (prf) are the most frequently found signals. +1 frameshifting motifs are widespread and regulate several important cellular functions in both eukaryotes and prokaryotes [2, 3] whereas 21 frameshifting events are currently mainly limited to viral genomes [4] , with very few exceptions such as dnax in prokaryotes or the edr/peg10 and ma3 genes in eukaryotes [5, 6, 7, 8] . 21 frameshifting motifs are minimally formed by a slippery sequence of the general structure x xxy yyz (the initial reading frame is indicated) where trnas shift reading frame, and a stimulatory element (mainly a secondary structure) [9,10] located 5-9 nucleotides (nt) downstream of the slippery sequence. in the filamentous fungus podospora anserina, it was hypothesized about 30 years ago that translation accuracy changes during cell differentiation allowing the production of key regulatory proteins required for the development of this model fungus [11] . indeed, tampering with accuracy of the p. anserina cytosolic translation apparatus results in impaired development [12, 13] . especially, decreasing the error rate triggers abnormal ascospore formation [14, 15] and the development of an epigenetic cell degeneration mechanism known as crippled growth [16] . the regulatory proteins, whose diminished expression could be responsible for both alterations as hypothesized by picard-bennoun, are still unknown. moreover, altering translation accuracy results in altered lifespan [17, 18] , through presently unknown mechanism(s) [15, 17, 19, 20] . while sequencing the p. anserina genome, we identified potential candidates regulated by recoding that could explain the importance of maintaining a certain level of translational error [21] . among these candidate genes, a 21 frameshifting site located in the p. anserina orthologue of the saccharomyces cerevisiae yip3/pra1 protein appears conserved in pezizomycotina. yip3 is a rab-gdi displacement factor involved in endoplasmic reticulum to golgi transport. it is conserved in animals [22] and plants [23] . we present here a functional analysis of this factor in p. anserina showing that its production is indeed regulated by a programmed translational 21 frameshift, which appears widely conserved in filamentous ascomycetes. all the p. anserina strains used in this study derived from the ''s'' (uppercase s) wild-type strain that was used for sequencing [21, 24] . the latest genome sequence and est derived from the s strain are available at http://podospora.igmors.u-psud.fr. construction of the dmus51::su8-1 strain lacking the mus-51 subunit of the complex involved in end-joining of broken dna fragments was described previously [25] . dna integration in this strain proceeds almost exclusively by homologous recombination. standard culture conditions, media and genetic methods for p. anserina have been described [26] and the most recent protocols can be accessed at http://podospora.igmors.u-psud.fr/more.php; the m2 minimal medium is a medium in which carbon is supplied as dextrin, and nitrogen as urea. the methods used for nucleic acid extraction and manipulation have been described [27, 28] . transformation of p. anserina protoplasts was carried out as described previously [29] . for the frameshifting measurement, the fy1619 s. cerevisiae strain was used (mata ura3-52 trp1d63 his3d200 leu2d1). the reporter plasmid for frameshifting measurement was the pac vector [30] . the podospora target sequences, corresponding to either the 21 frameshift sequence (podo-1) (59-gcttttt ccggggaggtggtctaggtggtcggccacgagctcc or the in-frame control sequence (podo0) (obtained by addition of a g at the 59 end) were inserted at the msci restriction site located at the junction between bgalactosidase and the firefly luciferase coding sequences. hybrid sequences consisting of the ibv (infectious bronchitis virus) slippery site with either the ibv spacer sequence (ibv-podo-1 sp6) (59-tatttaaacgggtacgggaggtggtcaaggtggtc ggccacgagctcccaaattttcgcccc-39) or the podospora spacer sequence (ibv-podo-1 sp2) (59-tatttaaaccgg ggaggtggtctaggtggtcggccacgagctcccaaat tttcgcccca-39) followed by the podospora pseudoknot were also cloned into the pac msci site, as well as a sequence consisting of the ibv slippery site out-of frame with the ibv spacer sequence and the p. anserina pseudoknot (ibv-podo0) (59-ttatttaa acgggtacgggaggtggtcaaggtggtcggccacgag ctcccaaattttcgccccat-39). the b-galactosidase and firefly luciferase activities were quantified in the same crude extract as described previously [31] for standard growth conditions. quantifications were the mean of six independent measurements. the efficiency, defined as the ratio of firefly luciferase activity to b-galactosidase activity, is expressed as percentage, and calculated by dividing the firefly luciferase/b-galactosidase ratio obtained from the 21 frameshift construct by the same ratio obtained with the in-frame control construct [30] . statistical significance was determined using the mann-whitney test (xl stat 2007 software). payip3 was inactivated by replacing the payip3 cds with a hygromycin b-resistance marker. the flanking regions of the payip3 gene were amplified by pcr, using the primers 8470gf (59-aagcttccggatccgagaataacc-39) and 8470gr (59-tctagacggtttggagaaggaaaagg-39) for the upstream sequence, and 8470df (59-ctcgagaccattaacgcccgttgttt-39) and 8470dr (59-aagcttcttcgcgaccttctcaa-39) for the downstream sequence. two primers contain sites for restriction enzymes to help in cloning. the pcr products were digested with xbai/hindiii for the upstream region and with hindiii/xhoi for the downstream region and cloned into the pbchygro vector [13] digested with xbai and xhoi. the plasmid recovered was then linearized with hindiii and introduced into the p. anserina dmus51::su8-1 strain by transformation. numerous transformants were obtained. selected transformants were crossed with the wild-type strain to remove the dmus51::su8-1 marker and obtain mat+ and mat2 strains carrying the deletion. southern blot analysis confirmed that for two transformants the payip3 gene was correctly deleted ( figure s1 ). one such mutant was selected for further phenotypic analyses. to complement the payip3 d mutant, the wild-type gene was amplified by pcr using the phusionh high-fidelity dna polymerase (fermentas), p. anserina wild-type genomic dna and primers 8470df and 8470gr. the product was cloned into the pbc-phleo vector [13] , to yield ppayip3 + . sequencing of the insert proved that no mutation occurred in payip3 during amplification and cloning. the ppayip3 + plasmid was then introduced by transformation into the payip3 d mutant. twentysix transformants, all with a wild-type phenotype, were recovered. two were selected for further studies and crossed with wild-type. the phleomycin-resistant payip3 d f1 progeny carrying the payip3 + transgene had a wild-type phenotype, demonstrating that restoration of the phenotype was due to the introduction of a wild-type copy of the payip3 gene. the corrected version of the payip3 cds was pcr amplified using the phusionh high-fidelity dna polymerase, p. anserina payip3 c -gfp genomic dna (see below) and primers 8470df and 8470rl (59-agcggccgcttaactgacacaaacgacaatcg-39). because the payip3 c allele was amplified from a gfp-tagged version it has no stop codon. therefore the pcr product was cloned into the pbcphleo vector [13] along with the rib2 terminator harboring a taa codon in its 5 prime end, to yield ppayip3 c . sequencing the insert proved that no mutation occurred in payip3 c during amplification and cloning. the ppayip3 c plasmid was then introduced by transformation into the payip3 d mutant. 19 transformants, all with a payip3 d mutant phenotype, were recovered. two were selected for further studies and crossed with wild-type. analysis was performed on f1 progeny with the payip3 c payip3 d genotype. the truncated version of payip3 cds was pcr amplified using the phusionh high-fidelity dna polymerase, p. anserina wild-type genomic dna and primers 8470df and 8470rc (59-atctagactagaccacctccccggaaaaagcc-39). the pcr product was cloned into the pbc-phleo vector [13] , along with the rib2 terminator to generate the payip3 t plasmid. sequencing the insert proved that no mutation occurred in payip3 t during amplification and cloning. the ppayip3 t plasmid was then introduced by transformation into the payip3 d mutant. thirteen transformants, showing a range of phenotype intermediates between the wild-type and the payip3 d mutant, were recovered. three of them were selected for further studies and crossed with wild-type. analysis was performed on the f1 progeny with the payip3 t payip3 d genotype. to construct a c-terminal gfp fusion of the frameshifted protein, the 535 bp 39-end fragment of the 39 orf of the payip3 gene was pcr-amplified using the orf39w-bgl2 (59-gaa-gatctggctctagtgcatccaggacac-39) and orf39c-sma1 (59-tttcccgggtcgctttccttctagcagtacc-39) oligonucleotides, containing, respectively, the bglii and smai sites (underlined in the sequence). after enzymatic digestion, the fragment was cloned into the bglii and smai sites of the pegfphygro vector, resulting in the pyip3 + -gfp vector. pegfp-hygro contains the hygromycin b resistance gene from pbc-hygro inserted into the noti site of the pegpf-1 vector from clontech. a c-terminal gfp fusion with the frameshifted protein was artificially obtained using the pyip3 c -gfp vector, which was constructed by cloning a 2 kb fragment of the payip3 gene, in which the frameshift was eliminated. to this end, two pcrfragments corresponding to the 59 orf (860 bp) and the 59-end of the 39 orf (1208 bp) were amplified, respectively, with the orf59w-sal1 (59-acgtgtcgaccggttcagccgttctttc ggagc-39) (sali site underlined) and phase-c (59-cctcc ccggaaaaagccctcatcgataggcttg-39) oligonucleotides, and with the phase-w (59-caagcctatcgatgaggg ctttttccggggagg-39) and orf39c-sma1 oligonucleotides. phase-w and phase-c oligonucleotides are complementary and contain the slippery sequence (indicated in italic) of the 21 frameshifting site. they also carry a supplemental nucleotide (in bold) compared to the genomic sequence in order to place the 39 orf in frame with the 59 orf. both fragments were pcr-joined, saliand smai-digested and cloned into the correspondingly digested pegfp-hygro vector to yield the pyip3 c -gfp vector. a last c-terminal gfp fusion was constructed with only the 59 orf. a 869 bp fragment of the the 59 orf was pcr-amplified using the orf59w-sal1 and the orf59c-sma1 (59-agacccgggc-cacctccccggaaaaagcctc-39) (smai site underlined) oligonucleotides. after digestion with both the sali and smai enzymes, and cloning into the sali-smai-digested pegfp-hygro, the pyip3 t -gfp vector was obtained. all constructs were verified by sequencing and then transformed into the dmus51::su8-1 strain. dna was extracted from selected transformants, pcr-amplified so as to sequence the inserted chimaeric genes. for each of the three plasmids (pyip3 + -gfp, pyip3 c -gfp and pyip3 t -gfp), two transformants, in which a correct integration of the chimaeric gfp transgenes had occurred, were selected and crossed with wild-type. in the progeny, mat+ and mat2 strains carrying the gfp chimaeric genes and devoid of the dmus51::su8-1 were selected for observation. gfp localization was identical in the two transformants of all three constructs. the anti-gfp monoclonal antibodies from roche applied science (catalog number 11814460001) were used for the detection of gfp-fusion proteins by western blot analysis. proteins were extracted as described [32] . pictures were taken with a leica dmire 2 microscope coupled with a 10-mhz cool snap hq charge-coupled device camera (roper instruments). they were analyzed with imagej. the gfp filter was the gfp-3035b from semrock (excitation: 472 nm/30, dichroïc: 495 nm, emission: 520 nm/35). the trees were constructed using phyml [33] with the default parameters [34] and 100 bootstrapped data sets. trees were visualized with the itol server [35] . the structure of the -1 translational frameshifting motif of payip3 as seen in figure 1a , the mrna of the p. anserina payip3 gene (cds number pa_1_8470) overlaps two open reading frames (orfs). the small one located at the 59 end of the messenger is similar to the s. cerevisiae yip3 gene (ynl044w), while the larger one at the 39 end is not present in this yeast (fig. 1b) . this second orf has no known functional domain and is only found in the genomes of pezizomycotina, a large group of ascomycota fungi (see phylogenetic analysis below). translation from the first 59 orf should produce a 19.5 kda protein similar to yip3. it is possible to generate a larger protein of 61.6 kda in p. anserina that would encompass both the small 59 and large 39 orfs by hypothesizing a -1 frameshift. analysis of 12 independent cdnas obtained during the p. anserina genome sequencing project showed that the mrna sequence was identical to that of the gene except for a 173 bp intron located in the 59 untranslated region [21] . this indicates that the putative frameshift is not corrected by either rna editing or splicing and thus it had to occur during translation. we could not find a canonical frameshift signal; however, examination of the payip3 sequence suggested that frameshifting could happen at codon nu 170, since there is at this position the sequence ''u uuu ucc'', a potential slippery sequence [10] . despite the fact that this slippery sequence does not match the consensus x xxy yyz motif, this sequence would still allow trna tandem slippage by pairing of the trna ser (iga) to the -1 codon (uuc) since g-u base pairing between codon and anticodon is possible. moreover, there is a sequence capable of forming a stem-loop and possibly a pseudoknot two nt downstream of the potential slippery sequence (fig. 1c ), a feature that should increase frameshifting frequency [9, 10] . such close vicinity between the slippery sequence and the stimulatory element is rather unusual for a 21 frameshifting stimulatory element. however the g-c stretch at the base of stem 1 is reminiscent of the very well-studied ibv pseudoknot [36] . despite these unusual features we were able to detect the long frameshifted protein fused with gfp, demonstrating that this 21 frameshifting motif is indeed functional (see below). in our previous study [21] , we observed that the yip3 -1 frameshifting motif is conserved during evolution, at least in pezizomycotina. the availability of additional fungal genome sequences now makes it possible to refine the phylogenetic analysis. to this end, the yip3 orthologues from representative species, whose genome sequences were available in public databases and covering the entire diversity of the eumycota, were manually annotated. the sequences of the 59 short and 39 large orfs of the pezizomycotina proteins, along with those of other fungi, were aligned and phylogenetic trees were constructed ( figures s2 and s3 ). both trees were compatible with the known evolution of the eumycota and pezizomycotina (fig. 2) . in most fungi, yip3 is encoded by a single gene (fig. 2) . the presence of a c-terminal extension was detected in all species of pezizomycotina, except for ascosphaera apis and arthrobotrys oligospora (fig. 2) . examination of the structure downstream the frameshift sites showed that it was highly conserved in most pezizomycotina and compensatory changes could be detected in the pseudoknot structure ( fig. 3a and 3b) . otherwise, changes were restricted to the predicted loops (fig. 3b) . however, in the case of tuber melanosporum, the adopted structure may not be a pseudoknot, but rather a simple stem-loop. interestingly, in two clades, the capnodiales (eight species investigated) and the ophiostomatales (one species investigated), the c-terminal extension was not in the 21 frame but in the +1 frame ( fig. 2 and fig. 3c ). the uuuuu slippery sequence and the pseudoknot were conserved in grosmania clavigera, the ophiostomatales, while the uuuuu sequence was changed to uuucu and only a small stem loop could be predicted in the case of mycosphaerella graminicola, a capnodiales (fig. 3c ). this could indicate that a single trna slippage would occur in this sequence instead of the classical tandem trna slippage. deletion of payip3 uncovers a growth phenotype but evidences no role in ascospore formation and crippled growth development to define the role of payip3 in the physiology of p. anserina, the gene was inactivated by replacing its entire coding sequence by a hygromycin b resistance marker, i.e., the deleted gene is unable to produce either the short or the long forms of payip3 (see materials and methods). the payip3 d mutant strain was investigated during the entire life cycle of the fungus and no difference was detected during the reproductive phase, i.e., kinetics and modalities of fruiting body development, ascospore genesis and germination. on the contrary, mycelium defects appeared. first, the mycelium of payip3 d grew slightly more slowly than wild-type (6+0.2 mm/d instead of 7+0.2 mm/d). however, growth resumed at the same speed as wild-type after 3 to 4 days of incubation. this was accompanied by an altered pattern in the repartition of the fruiting bodies (perithecia; fig. 4 ) when grown on m2 minimal medium. indeed, on this medium the wild-type produced perithecia mainly as a 1 cm-thick ring with an internal diameter of 1 cm, whereas in the mutant the ring was 0.7 cm thick the presence (species in black) and absence of the 39 orfs (species in grey) of yip3 was mapped on the most probable phylogenetic tree of the eumycota [43, 46] . the most parsimonious scenario of evolution is a single appearance of the 39 orf after the split of the orbiliomycetes from the other pezizomycotina (slanted arrow) and a secondary loss in a. apis (cross). * denotes the two groups for which a+1 frameshift is hypothesized and 62 the species in which a duplication of yip3 has occurred. doi:10.1371/journal.pone.0073772.g002 and its internal diameter was 0.7 cm. moreover, although the kinetics of perithecium development was identical after fertilization, perithecia from mat+/mat2 heterokaryotic mutant cultures yielded ascospores half a day before wild-type cultures, suggesting that fertilization occurred earlier in the mutant, in line with the smaller diameter of the ring of fruiting bodies. finally, when grown on a medium with wood shavings as sole carbon source, fertility of the mutant was diminished and fewer ascospores were produced, while growth with whatman paper (i.e. pure cellulose) as carbon source was only marginally modified (fig. 4) . significantly, crippled growth and life span were not modified in the mutant (data not shown). to assess whether the ring and wood shaving phenotypes were due to deletion of payip3, a wild-type copy of the payip3 gene was introduced by transformation into the payip3 d mutant. as seen in figure 5 , complete restoration of the wild-type phenotype was observed in transformants carrying an ectopic copy of payip3 + , whereas the complemented strains formed a wild-type ring of perithecia and produced as much ascospores as wild-type on wood shaving medium, demonstrating that inactivation of payip3 was responsible for all the phenotypes observed (compare payip3 d with payip3 + ). the mutant was also transformed with truncated and corrected alleles of the payip3 gene. the payip3 t truncated allele was obtained by removing the 39 orf and produced only the 19.5 kda polypeptide. the payip3 c corrected allele was created by inserting a c in codon nu168, which is just upstream of the frameshift site, and produced the 61.6 kda polypeptide resulting from the fusion of the proteins produced by the 39 and 59 orfs. introduction of payip3 c did not result in the restoration of a wildtype phenotype: ring and ascospore production on wood shaving medium was identical to that observed with the payip3 d mutant (fig. 5) . a heterogeneous restoration depending on the transformants was observed with the payip3 t allele. recovery ranged from complete (e.g., the t1 transformant had a wild-type phenotype) to inexistent (e.g., the t2 transformant has a payip3 d mutant phenotype, fig. 5) , while others presented a modified repartition of perithecia and diminished fertility on wood shaving medium (e.g., the t3 transformant had a more diffuse ring and produced reduced amount of ascospores on wood shaving medium). integrative transformation in p. anserina results mostly in non-homologous insertion. variability in the complementation observed with the truncated allele could be explained by influence of the insertion point on the expression of the transgene. to check whether expression of both the truncated and corrected form in the same strain could rescue a complete wild-type phenotype, we constructed by genetic crosses strains carrying both the corrected and truncated alleles of payip3. as seen in figure 5 , these strains exhibited the same phenotypes as those expressing the truncated allele alone, indicating that co-expression of both forms is not sufficient to recover a wild-type phenotype. to determine the in vivo expression level and localization of the short and long forms of yip3, we constructed plasmids carrying chimaeric versions of the wild-type, truncated and corrected alleles by adding in frame the egfp cds at the 39 end of payip3 (see materials and methods). transformation of the three plasmids into p. anserina resulted in their integrations at the payip3 chromosomal locus by a single crossing-over, generating the modified loci depicted in fig. 6a . in the case of the wild-type payip3 + -gfp transgene only a small ,500 pb truncated payip3 cds was present downstream of the chimaeric gene, while for the two other constructs a complete cds was retained. however, these copies had a 59-untranslated region truncated just before the intron (fig. 1) , and thus lacked the promoter region as well as 350 pb of the 59-utr of the payip3 mrna. to ensure that the recovered gfp-transgenes were expressed, we monitored by western blotting with an anti-gfp antibody the production of the proteins from payip3 + -gfp, payip3 t -gfp and payip3 c -gfp (fig. 6b) . a doublet of bands, likely due to different levels of posttranslational modification observed for proteins with reticulum/golgi localization, was detected at ,45 kda for payip3 t -gfp, as well as a band of about 88 kda for payip3 + -gfp and payip3 c -gfp. these sizes are expected for the fusion proteins, showing that they were actually produced in p. anserina. although present in low amounts, the detection of a band of high molecular weight with the payip3 + -gfp construct demonstrated that the 61.6 kda-long form of the protein is naturally produced by the wild-type allele. when analyzed on m2, the strains carrying the payip3 + -gfp and payip3 t -gfp transgenes grew and presented a ring of perithecia as did the wild-type strain, while those carrying payip3 c -gfp were similar to the payip3 d mutant (fig. 7) . to test whether the phenotype of payip3 c -gfp resulted from lack of activity of the remaining downstream wild-type cds or to a dominant negative effect of the payip3 c -gfp copy, we constructed by genetic crossings balanced heterokaryon with the following genotype: payip3 c -gfp lys2-1/payip3 + leu1-1. these had a wildtype phenotype (fig. 7) , showing that the payip3 c -gfp phenotype was recessive. the same expected result was observed with a payip3 d -gfp lys2-1/payip3 + leu1-1 heterokaryon used as control. therefore, the payip3 c -gfp protein displayed no dominant negative effect. hence as observed for the ectopic payip3 c protein, payip3 c -gfp is not functional and the downstream copy microscopic examination revealed a clear expression of the wild-type fusion payip3 + -gfp protein in the hyphae confirming that the recoded form is efficiently expressed in vivo in the host. this longer protein seems to display the same localization as the smaller form (payip t -gfp; fig. 8 ). the corrected in-frame form (payip3 c -gfp) is more difficult to observe in the hyphae, as expected if it is present in lower amounts. in all three strains, the fluorescence was concentrated in foci compatible with a reticulum/golgi localization as observed for the orthologous yip3/ pra1/pra2 protein [37, 38] . intriguingly, while we did not detect fluorescence in the centrum of fruiting bodies from the payip3 + -gfp and payip3 c -gfp strains, a clear staining was observed with the payip3 t -gfp protein, suggesting that only this short form of the protein is present in this tissue. unfortunately it is not possible to evaluate frameshifting efficiency by western-blot as the small peptide is not tagged with gfp in the payip3 + -gfp construct. moreover western-blot results from payip3 c -gfp suggest that the longer form could be unstable, due to the weak intensity of the band (fig. 6) , which corresponds to 100% of corrected protein. to determine if the identified payip3 recoding sequence was able to promote 21 frameshifting, it was inserted between the b-galactosidase and firefly luciferase open reading frames of a yeast s. cerevisiae reporter plasmid [30] . the 21 frameshifting efficiency was quantified as described in materials and methods. the level was estimated to be 1%, which in s. cerevisiae indicated a low but true -1 frameshift event. we also replaced the slippery sequence by the very well characterized slippery sequence from the ibv 21 frameshift (t tta aac) in frame or out-of-frame. positioning the ibv slippery sequence out-of-frame results in a significant drop in frameshifting efficiency (0.3%), indicating that the 1% obtained with the payip3 sequence is significant. however, despite having tested several spacer distances between the ibv slippery sequence and the payip3 pseudoknot, it is clear that this pseudoknot is unable to stimulate frameshifting in the ibv sequence to the same extent as does the ibv pseudoknot does ( figure s4 ). this confirms the unusual nature of the frameshifting event that occurs on this sequence. we present evidence that the p. anserina gene orthologous to s. cerevisiae yip3, payip3, is expressed in a complex fashion, whereby two proteins are produced with a single mrna through a translational -1 frameshifting event. the mrna produced by this gene encompasses two orfs, the 59 orf is highly conserved and is similar to the cds of yip3, while the 39 orf is less conserved and present only in pezizomycotina. the two orfs can be expressed in p. anserina as a single polypeptide if a 21 frameshift occurs during translation. we were able to reveal in vivo a fusion protein corresponding to the translation of the two orfs. we know from the analysis of est that the mrna does not undergo any modification (splicing, editing); therefore, only a translational event can explain the fusion protein. several translational recoding events such as ribosome hopping or frameshifting could explain how the fusion protein is synthesized. however, two characteristic features reminiscent of frameshifting motifs are found in the overlapping sequence between the two orfs: i) a pseudoknot that is an mrna structure commonly stimulating 21 frameshifting [9, 10] ; ii) a slippery sequence. this sequence does not match the 21 frameshifting slippery sequence consensus; nevertheless, the slippage of the trna ser (iga), which base pairs to the ucc codon in the initial frame, can occur on the -1 codon (uuc) since g-u base pairing between codon and anticodon is possible. another unconventional feature of this new frameshifting site is the proximity of the slippery sequence to the stimulatory pseudoknot. indeed the slippery sequence is located only 2 nt upstream of the pseudoknot whereas the spacer region is usually around 5 to 9 nt. to gain further details about this new frameshifting site we tested this motif in the yeast s. cerevisiae with a dual reporter system. frameshifting efficiency was estimated as 1%, which is low but significant since frameshifting efficiency dropped to as low as 0.3% when the reading frame in which the ribosome encounters this slippery sequence was changed. moreover our western-blot results suggest that frameshifting efficiency could be higher in the natural host. to better characterize the frameshifting mechanism the slippery site was replaced by a canonical slippery sequence from the ibv pseudoknot. despite efforts to position the ibv slippery sequence either at 6 nt from the pseudoknot (the size of the ibv spacer region), or at 2 nt (the size of the payip3 pseudoknot) the ibv frameshifting efficiency in yeast (10-15%) was not reached and only a moderate increase with a 2 nt spacer was observed. this indicates that the payip3 pseudoknot is unable to stimulate frameshifting as does the ibv pseudoknot with a 6 nt spacer. however we cannot rule out that the slight increase observed with figure 7 ). (b) cellular extracts from transformants with the indicated genotypes were separated on a 10% acrylamide 1 mm-thick gel and probed with an anti-gfp antibody. the left part results from a short exposition time that reveals the control and the payip3 t peptides. the right part is a longer exposition time to reveal the proteins produced from payip3 + and payip3 c transgenes. brackets and arrow, respectively, indicate the short and the longer proteins specifically revealed by the anri-gfp antibody. controls: 59 kda fusion protein of su12 ribosomal protein in frame with gfp (su12-gfp, [48] ). doi:10.1371/journal.pone.0073772.g006 the 2 nt spacer is significant. this tentatively suggests that the payip3 pseudoknot may slightly improve basal frameshifting of the ibv slippery sequence when positioned 2 nt downstream. it is always difficult to extrapolate the functional role from a predicted structure, as the pseudoknot can fold in an inactive form [39] . however one plausible explanation comes from the lack of conservation of the potential slippery sequence between the two organisms, suggesting that the frameshifting event does not occur by a dual trna slippage as for ibv but more probably by a single p-trna slippage, similarly to frameshifting found in bacterial transposable elements [40] . this would also explain why the payip3 pseudoknot is unable to fully stimulate dual trna slippage on the ibv sequence. this is also in accordance with the fact that this pseudoknot displays several unusual features. it is positioned very close to the slippery sequence (only 2 nt), and there is a highly conserved bulge of 3 nt in stem 1 of the pseudoknot. such a 3 nt bulge has already been described for the edr frameshifter pseudoknot [6] but remains very unusual. mutagenesis of edr frameshifting supports a tandem trna slippage model and the bulge found in edr pseudoknot does not play any role in frameshifting. the situation could be very different for payip3 frameshifting as a trans-acting factor could bind this pseudoknot to stimulate frameshifting in the host. this frameshifting is not conserved in s. cerevisiae so this unknown factor would be absent in this species explaining the inability of the pseudoknot to stimulate frameshifting at the ibv slippery site. whatever the mechanism, this frameshifting site is functional in p. anserina. it is worth mentioning that up to now the only two eukaryotic genes (ma3 and edr/peg10), that use a -1 frameshift to extend the size of a protein, are derived from domesticated retroviruses. no such signature is found in payip3 suggesting that this will be the first cellular gene using a -1 frameshifting event to extend the protein length and not related to a retrovirus. in s. cerevisiae, yip3 facilitates the dissociation of endosomal rab-gdi complexes [41] . however, yip3 may have multiple functions in the cell, which are as yet not well characterized [37] . in p. anserina, deletion of payip3 triggers three phenotypes. indeed, on m2 standard medium, the mutants initiate their growth slightly more slowly than wild-type and differentiate a smaller ring of fruiting bodies that produce ascospores half a day earlier. they are also impaired in their ability to produce abundant ascospores on medium with wood shavings as sole carbon source. at the present time, the molecular defects underlying these phenotypes are unknown, but defects in protein routing may account for them. in particular, degradation of wood shavings relies on the secretion of many enzymes that act synergistically, and impairment in secretion can alter the ability to degrade lignocellulose. remarkably, the mutants are not affected in their ability to complete maturation of ascospores (although their yield is reduced) and to undergo crippled growth, the two main developmental phenotypes triggered by increasing translation accuracy. likewise, their lifespan is not modified, as typically seen in accuracy mutants. therefore, payip3 is not the factor produced by a recoding event and hypothesized to regulate either one of these phenomena [11, 15, 16, 17] . some of the other p. anserina genes found to be potentially regulated by recoding [21] could be involved. the possible role of payip3 frameshifting in p. anserina is not clear at the present time. indeed, expression of the 19.5 kda alone can restore a wild-type phenotype, when present at the payip3 chromosomal location (fig. 5) . however, when present at an ectopic location, complementation may not be complete, likely due to abnormal expression of the transgenes integrated at an ectopic location (fig. 5) , unlike what is observed for the wild-type allele with frameshift. moreover, expression of the 61.6 kda polypeptide alone is unable to restore a wild-type phenotype, indicating that it is inactive. it does not appear to act as a dominant negative form (fig. 5) . finally, expressing both the 19.5 kda and 61.6 kda forms within the same cells from different transgenes does not improve phenotypic rescue. several explanations can be proposed. possibly, the laboratory conditions tested are not appropriate to detect an effect of the lack of frameshift. redundancy from another factor along with payip3 may be masking an effect of the frameshift product. the 19.5 kd and 61.6 kd forms may need to be present in defined ratio to observe an effect. another possibility would be that doing an error during translation rather than producing two different polypeptides is important. this could allow for spatial or temporal regulation of payip3. interestingly, while expression of the 19.5 kda form is detected in the perithecium centrum, expression of the 61.6 kda form was not detected either from the wild-type allele or the corrected allele. possibly, the c-terminus of the 61.6 kda polypeptide acts as a degradation signal in the centrum. this situation is reminiscent of the s. cerevisiae pde2 gene. indeed this gene undergoes translational stop codon readthrough producing a short and a long protein. the longer protein is destabilized and quickly degraded by the proteasome [42] . in the case of payip3 this degradation signal is tissue specific adding a supplementary layer of regulation. the reason for the lack of the 61.6 kda in the centrum of fruiting bodies is not clear, as we detected no change in the ascospore maturation and ejection processes in the mutants. possibly, a protein degradation mechanism exists in the centrum so as to ensure that only certain proteins are transmitted to the progeny. the 61.6 kda protein may be recognized and specifically degraded, without causing any harmful effect. whatever the role of the recoding, it seems important from an evolutionary point of view as it is conserved in all the pezizomycotina that we have investigated except for a. oligospora and a. apis. a. apis is a fungus that infects beehives and may present features associated with parasitism, such as simplified metabolism. more interesting is the fact that a. oligospora belongs to the orbiliomycetes, a class of pezizomycotina, which is believed to be the first to have diverged during evolution [43] . if correct, the most parsimonious explanation of the phylogenetic pattern of the c-terminal extension would be its origin after the split of the orbiliomycetes from the other pezizomycotina and its removal from the ancestors of a. apis due to their parasitic lifestyle (fig. 7) . this split likely occurred more than 400 million years ago [44] . during this time, the 21 frameshift was conserved except in two orders for which a+1 frameshift may occur. in the ophiostomatales, the frameshift site is well conserved, i.e., a slippery sequence and a pseudoknot are present, yet only a+1 frameshit can join the 59 and 39 orfs. in the capnodiales, the slippery sequence and the pseudoknot are not conserved, but the two orfs are conserved in a+1 frameshift configuration. this confirms that recoding during translation is important for the regulation of yip3 in filamentous ascomycetes. a recent report [45] shows that recodings are involved in metabolic control in a wide range of fungi through the production of distinct polypeptides in the targeting of enzymes to different compartments. here, we show that a gene involved in routing proteins is also subjected to translation recoding control, possibly adding a level of complexity to the metabolic network. reprogrammed genetic decoding in cellular gene expression autoregulatory frameshifting in decoding mammalian ornithine decarboxylase antizyme expression of peptide chain release factor 2 requires high-efficiency frameshift non-canonical translation in rna viruses a functional -1 ribosomal frameshift signal in the human paraneoplastic ma3 gene characterization of the frameshift signal of edr, a mammalian example of programmed -1 ribosomal frameshifting programmed ribosomal frameshifting generates the escherichia coli dna polymerase iii gamma subunit from within the tau subunit reading frame 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mitochondrial dna of podospora is under the control of nuclear genes deletion and dosage modulation of the eef1a gene in podospora anserina: effect on the life cycle informational suppressor alleles of the eef1a gene, fertility and cell degeneration in podospora anserina the genome sequence of the model ascomycete fungus podospora anserina targeting rab gtpases to distinct membrane compartments the pra1 gene family in arabidopsis les phénomènes de barrage chez podospora anserina. i. analyse génétique des barrages entre souches s and s the crucial role during ascospore germination of the pls1 tetraspanin in podospora anserina provides an example of the convergent evolution of morphogenetic processes in fungal plant pathogens and saprobes contribution à l'étude génétique d'un ascomycète tétrasporé: podospora anserina (ces.) rapid methods for nucleic acids extraction from petri dish grown mycelia transformation by integration in podospora anserina.i. methodology and phenomenology nonsense-mediated decay mutants do not affect programmed -1 frameshifting versatile vectors to study recoding: conservation of rules between yeast and mammalian cells a mitotically inheritable unit containing a map kinase module a simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood phylogeny.fr: robust phylogenetic analysis for the non-specialist interactive tree of life (itol): an online tool for phylogenetic tree display and annotation characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot saccharomyces cerevisiae rab-gdi displacement factor ortholog yip3p forms distinct complexes with the ypt1 rab gtpase and the reticulon rtn1p pra isoforms are targeted to distinct membrane compartments an equilibrium-dependent retroviral mrna switch regulates translational recoding programmed translational -1 frameshifting on hexanucleotide motifs and the wobble properties of trnas yip3 catalyses the dissociation of endosomal rab-gdi complexes translational readthrough of the pde2 stop codon modulates camp levels in saccharomyces cerevisiae orbilia ultrastructure, character evolution and phylogeny of pezizomycotina dating divergences in the fungal tree of life: review and new analyses cryptic peroxisomal targeting via alternative splicing and stop codon read-through in fungi a multigene phylogeny of olpidium and its implications for early fungal evolution freiburg rna tools: a web server integrating intarna, exparna and locarna in vivo labelling of functional ribosomes reveals spatial regulation during starvation in podospora anserina we thank sylvie françois for her expert technical assistance and anne-lise haenni for correcting the manuscript. key: cord-001065-j4hvyyoi authors: boncristiani, humberto f.; evans, jay d.; chen, yanping; pettis, jeff; murphy, charles; lopez, dawn l.; simone-finstrom, michael; strand, micheline; tarpy, david r.; rueppell, olav title: in vitro infection of pupae with israeli acute paralysis virus suggests disturbance of transcriptional homeostasis in honey bees (apis mellifera) date: 2013-09-05 journal: plos one doi: 10.1371/journal.pone.0073429 sha: doc_id: 1065 cord_uid: j4hvyyoi the ongoing decline of honey bee health worldwide is a serious economic and ecological concern. one major contributor to the decline are pathogens, including several honey bee viruses. however, information is limited on the biology of bee viruses and molecular interactions with their hosts. an experimental protocol to test these systems was developed, using injections of israeli acute paralysis virus (iapv) into honey bee pupae reared ex-situ under laboratory conditions. the infected pupae developed pronounced but variable patterns of disease. symptoms varied from complete cessation of development with no visual evidence of disease to rapid darkening of a part or the entire body. considerable differences in iapv titer dynamics were observed, suggesting significant variation in resistance to iapv among and possibly within honey bee colonies. thus, selective breeding for virus resistance should be possible. gene expression analyses of three separate experiments suggest iapv disruption of transcriptional homeostasis of several fundamental cellular functions, including an up-regulation of the ribosomal biogenesis pathway. these results provide first insights into the mechanisms of iapv pathogenicity. they mirror a transcriptional survey of honey bees afflicted with colony collapse disorder and thus support the hypothesis that viruses play a critical role in declining honey bee health. over the last years, the declining health of the honey bee (apis mellifera) and other pollinators has caused concern all over the world. particularly over the last six years, honey bee health has shown alarming rates of deterioration [1, 2, 3] , questioning the sustainability of our food production system. there are many possible threats to honey bees health, including pesticides, malnutrition, management stress, and pathogens [3, 4, 5, 6, 7] . numerous studies suggest that novel or emerging pathogens play a role in honey bee health declines [1, 2, 3, 7, 8, 9] . however, insufficient knowledge on honey bee pathogens compromises our ability to assess their importance and to develop control measures. this is particularly true for honey bee viruses although their importance for honey bee losses has become evident in recent years [6, 7, 8, 10, 11, 12] . specifically in combination with the ectoparasitic bee mite varroa destructor [13, 14, 15 ] that serves as a vector, several viruses appear to become more virulent [16, 17, 18] . viruses may cause covert infections [19] and were considered mostly harmless until varroa mites were introduced to a. mellifera populations almost 30 years ago [20, 21, 22] . the increased virulence of viruses has been confirmed experimentally by direct inoculation of bees with viruses [23, 24, 25, 26, 27] , opening an important research field to explore. approximately twenty honey bee viruses have been described so far [4, 16, 28, 29] , affecting the morphology, physiology, and behavior of bees. most belong to the families dicistroviridae [30] and iflaviridae in the order picornavirales. viruses in these families have a positive sense rna genome, covered by an icosahedral, pseudo t = 3 structure symmetry capsid (around 30 nm) that is responsible for rna protection, host specificity, and tissue infection. picornaviruses are well known for their capacity to shut off the translational system of their host cells, by cleavage of translation factors leading to a decrease in cap-dependent host translation, a conserved replication strategy among all members studied to date [31, 32] . picornavirus infection also triggers hostimmune responses (i.e., pkr) that result in decreased capdependent (host) translation. picornaviruses circumvent this immune response by encoding an internal ribosomal entry site (ires) which is recognized and translated by the host machinery (non-canonical translation) [30, 32] . over time, the accumulation of produced virus particles and repression of the synthesis of essential cell components lead to cell death in most cases [32] . little is known about the specific biology of the viruses in these families that infect honey bees, although they contain important bee pathogens, such as deformed wing virus (dwv) and israeli acute paralysis virus (iapv). iapv has previously been associated with the unusual honey bee disappearance syndrome called colony collapse disorder (ccd) [8] and is frequently seen in many honey bee pathogen surveys [6, 7] . despite the importance of iapv and the feasibility to work with iapv in the laboratory [25, 33] , little is known about iapv's interactions with its host and resulting pathogenesis. in general, the lack of adequate tools for honey bee virus research has hampered our understanding of basic biology of the relevant viruses and little is known about the molecular bases of honey bee viruses replication and pathogenesis [18, 28] . therefore, many assumptions regarding their replication are made based on other picornaviruses (e.g., cricket paralysis virus [34, 35] and human poliovirus [32] ), highlighting the need of specific, mechanistic studies on honey bee viruses [36] . focusing on iapv, we report here the development of an inoculation method of invitro reared honey bee worker pupae that provides the basis for mechanistic, in-depth studies of honey bee viruses. we report acute but variable disease symptoms, compare viral replication among pupae of two colonies and patrilines within these colonies, and report on measures of gene expression in response to viral infection that indicate major disruption of cellular homeostasis. initially, approximately 20 adult bees from a heavily iapvinfected colony were frozen in liquid nitrogen, ground to a fine powder, and homogenized in 10 ml extraction buffer (0.1 m potassium phosphate buffer, ph 7.5, 0.2% diethyldithiocarbamate, 1/5 volume of diethyl ether). emulsification ensued by adding 5 ml carbon tetrachloride and centrifuging at 10,000 rpm for at 4uc for 10 minutes (rotor: sorvall rc-5b) and collecting the supernatant. the supernatant containing viruses was filtered through a 0.2micron filter (milex-gs, millipore, #slgs033ss) to remove small tissue debris, fungi, and bacteria. the filtrate was then centrifuged at 30,000 rpm (61,740 rcf) in a beckman lb-70m ultracentrifuge with a 70.1/ti rotor for six hours at 4uc to pellet picornavirus particles. the pellet was resuspended in 0.2 ml of pbs and centrifuged against a cscl gradient (0.44 g/ml) at 52,000 rpm (185,000 rfc) overnight (beckman lb-70m ultracentrifuge, 70.1/ti rotor). the fractions containing virus particles were dialyzed using ''slide-a-lyzer dialysis cassettes'' against cold (10uc) 0.2 ml of pbs overnight. rt-pcr was conducted to test for the presence of acute bee paralysis virus (abpv), israel acute paralysis virus (iapv), kashmir bee virus (kbv), sacbrood virus (sbv), chronic bee paralysis virus (cbpv), black queen cell virus (bqcv), and deformed wing virus (dwv). iapv, and small amounts of abpv, dwv, and bqcv were detected in the purified viral solution (positive amplification with pcr primers). viral quantification was performed by absolute quantification using the standard curve method as described previously [37] [38] . 5.0 ml of viral solution was examined for the presence of virus particles and their phenomenological characterization by electron microscopy. virus particles were negatively stained with 2% uranyl acetate on a formvar-coated ni grid and viewed in a hitachi h-7000 electron microscope at 150,000x to 200,000x. iapv replicates readily in pupae [25] . therefore, white-eyed pupae were inoculated for virus propagation, using 1.0 ml of the viral suspension per pupa. after 4 days of incubation, with disease symptoms apparent (figure 1 ), viruses were purified using the approach outlined above. qrt-pcr showed ,10 5 more iapv genomes than the second most detected virus, dwv after a single round of virus injection pupal amplification, and isolation. this procedure was repeated twice using pupae from very strong hives to further reduce contaminating viruses and increase the amounts of iapv. the high concentration of iapv over other honey bee viruses in these purifications allowed us to strongly dilute the inoculum, decreasing the chances of cross inoculation with another virus. in the experiments described below, we injected 10 4 iapv genome equivalents keeping the probability of cross contamination at negligible levels. pupae were individually homogenized and submitted to total rna extraction, using trizolh (invitrogen, carlsbad ca) following the manufacturer's protocol. the resultant rna pellets were resuspended in diethyl pyrocarbonate-treated water in the presence of rnase inhibitor (invitrogen, carlsbad ca) and treated with dnase i (invitrogen, carlsbad ca) to remove any contaminating dna. the resulting rna was quantified on a nanodrop spectrophotometer (thermo scientific, wilmington, de). first-strand cdna was then synthesized by incubating 2mg of total rna per sample in a 96-well plate with master mix containing 50 u superscript ii (invitrogen, carlsbad, ca), 2 nmol dntp mix, 2 nmol poly(dt)18, and 0.1 nmol poly (dt) (12) (13) (14) (15) (16) (17) (18) at 42uc for 50 minutes followed by 15 minutes at 70uc as described previously [39] . the cdna was diluted 1:5 with molecular-grade water. the primers used in this study were validated for relative quantification of the target genes and are commonly used in honey bees [39, 40, 41] . reactions to amplify the cdna products were conducted in 96-well plates using the applied biosystems step one real time pcr machine (applied biosystems, carlsbad, california). one microliter of diluted cdna from each sample was used as a template for rt-qpcr reactions using sybr green tm (applied biosystems, carlsbad, california) following the manufacturer's protocols. the reactions were conducted under a fixed thermal protocol consisting of 3 minutes at 95uc, followed by 40 cycles of a three-step protocol of 95uc for 20 sec, 60uc for 30 sec, 72uc for 1 minute. fluorescence measurements were taken at each cycle during the last 72uc step. this procedure was followed by a melt-curve dissociation analysis to confirm the specificity of the reactions. based on the results of the preliminary experiments 1 and 2 (preliminary experiments s1), a more extensive iapv inoculation experiment was designed to study the time line of infection and associated gene expression patterns, and to assess bees for variability in iapv susceptibility. one microliter of the inoculant (pbs as control or virus solution containing 10 4 genome equivalents of iapv) was injected using a nanojet tm syringe pump (chemix, usa) with an infusion flow rate of 0.1ml/sec, following manufacturer's parameters. the needle was inserted in the lateral abdomen between the fourth and fifth tergite of young, white-eye honey bee pupae ( figure 2a ). two strong, iapv-free hives were selected from the uncg research apiary, representing two distinct sources of bees for the experiment. from each hive, 200 white-eye pupae were collected for each of the following treatment groups: without inoculation (w/o), pbs inoculated (pbs), and iapv inoculated (iapv). from each treatment group and hive, 50 bees were frozen at 0 h, 5 h, 20 h and 48 h after inoculation and a subset of these samples was individually analyzed for viral titers and gene expression patterns. the first time point directly after inoculation was used as a control of the initial states of the bees in the experimental and control groups. the time point of five hours post-infection was chosen to measure the virus impact before completion of the replication cycle, based on the assumption that iapv follows the picornavirus family average timing for a replication cycle, of 7-12 hours [32, 34, 35] . any gene expression changes at this time point represent the bees' response to inoculation without complications from virus-related tissue damage. the time point of 20 h postinfection was considered representative of events after one complete cycle of virus replication, and the 48 h time point represents the established diseased state, characterized by visual symptoms. based on the results of the preliminary experiments (preliminary experiments s1), we tested the effect of iapv injection on gene expression of six commonly used reference genes that have been reported to be constantly expressed across different experimental conditions [4, 7, 41] . we studied the transcription of actin, ribosomal 28s rna, ribosomal 18s rna, ribosomal protein rps5, mgst1, and histone h2a, under iapv infection. histone h2a is not common used in honey bees, but it was added to our experiment because its expression is constitutive and cell-cycle independent, and it is commonly used on other models [42] . the sequences of utilized h2a primers are: 59-aaaggaaattacg-cagaacga-39 (h2a forward) and 59-cggctaaatattc-cataacgg-39 (h2a reverse). in addition, the titers of iapv and dwv were quantified in these samples. one hind leg was removed from each pupa and stored at -20uc before dna extraction to determine the subfamily (patriline) for each individual. dna was extracted from each leg using a standard chelex 100h method [43] . briefly, each sample was incubated for 60 min at 55uc, 15 min at 99uc, 1 min at 37uc, and 15 min at 99uc in 150 ml of a 5% chelex 100h solution with 5 ml 0.35 mg/ml proteinase k. subfamily identification for each sample was determined using microsatellite alleles following previously described methods [44] . this genomic paternity analysis was conducted using two multiplex pcr reactions (plex 1 and plex 2) with 10 ml reaction volumes containing approximately 100 ng sample dna, 16pcr buffer (takarah without mgcl 2 ), 1 mg/ml bsa, 1.5 u taq polymerase, 300 mm dntps, and either 1.5 mm (plex 1) or 1.1 mm (plex 2) mgcl 2 . following [45] , primer sets in plex 1 included 2.0-2.5 pmol am061, am052, am010, and am553, and primer sets in plex 2 included 2.5-3.5 pm am043, am098, am125, and am059. all reactions were performed using a thermoh px2 thermocycler with 7 min at 95uc followed by 30 cycles of 30 sec at 95uc, 30 sec at 55uc (plex 1) or 54uc (plex 2), and 30 sec at 72uc, then a final 10 min at 72uc. the pcr products were run on an abi 3730h dna analyzer at the genomic sciences laboratory at ncsu. data was acquired with genemapper 4.0 (abi) to score microsatellite fragment sizes. loci with poor amplification were excluded from analyses and only samples for which more than half of the loci could be scored were used for analysis. the data were analyzed with the computer package colony 1.2 to assign subfamily membership to each sample [46] . all experiments revealed that the absolute quantities (ct values) of the standard reference genes were affected by the iapv treatment (see results) and that they did not fulfill the criterion of expression stability. therefore, a larger set of potential reference genes was evaluated in the main experiment (raw data s1). however in the absence of an internal control, the transcript level of these genes and iapv could not be normalized by the customary dct or ddct methods [47, 48] . instead, transcripts were evaluated by ct values, based on the assumption that the amount of template after quantification and appropriate dilution did not differ systematically among treatment groups. to ensure consistency, a fixed fluorescence threshold for each gene and experiment was determined manually to avoid inter rt-qpcr runs inconsistencies. tests of technical error indicated a high replicability for several genes, with variation between replicate ct values of 1.3% on average (minimum: 0.002%, maximum: 2.6%). all statistical analyses of this study were done using the r stats package, version 2.15.0, http://www.r-project.org/ or with spss 20.0 (ibm). heat maps were generated by heatmap.plus r package version 1.3. patterns of gene expression were analyzed with parametric linear models, using time and treatment as fixed effects. bonferroni and scheffe's post-hoc tests were performed and did not differ in their results. in the experiment, the virus titers of inoculated individuals were compared among colonies and patrilines within colonies. patrilines that were represented by only one individual were omitted from the patrline but not the colony analysis. thus, separate anovas were used instead of one nested anova. attempts to isolate pure iapv directly from naturally infected adult bees were unsuccessful due to co-infection of the bees with other honey bee viruses. pcr tests resulted repeatedly in positive amplification of multiple viruses, such as bqcv, abpv, and dwv. co-infection seems to be the rule rather than the exception and it is generally rare to find bees infected with a single virus [49] . however, our artificial inoculation of pupae led to selective increases of iapv, relative to the other viruses. after three rounds of pupae inoculation and subsequent virus purification, the amount of iapv was at least 10 5 genome copies higher than all other common honey bee viruses found in our initial inoculum (bqcv, abpv, dwv). after serial dilutions, iapv was the only virus that could be detected by pcr. electron microscopy analysis of this sample showed uniform viral particles around 27 nm ( figure 2b ), consistent with picornavirus particles. sequence analysis verified these particles to be iapv. the site for injection of the virus inoculum into the honey bee was chosen based on the ability of the pupae to complete development to become an adult after sham injections. when the junction between the last abdominal sternites (figure 2a ) was selected more than 95% of bees were able to complete development after pbs inoculation. this region is very soft, enabling smooth penetration of the needle with little physical damage to the pupae. in addition, varroa destructor nymphs were often observed in this same region when pupae were prepared for inoculations, suggesting that this area is an attractive feeding site. in the standardization process both controls, without inoculation (w/o) and pbs buffer injected bees (pbs), developed normally ( figure 1a) , culminating in full development after 144 hours ( figure 1c ). iapv-inoculated bees showed strong but variable symptomatology over time ( figures 1a and 1b) , inhibited metamorphosis, and ultimately death. symptoms ranged from a complete cessation of development with no visual evidence of disease ( figure 1b-1) , rapid darkening of body parts ( figure 1b-3 and 1b-4) , simultaneous darkening and hindered development ( figure 1b-5) , to apparently normal development ( figure 1b-2) with eventual sudden death. iapv titer increased in all inoculated bees but no correlation between symptomatology and virus titer determined by rt-qpcr at the end of the experiment was observed. the experiment investigated iapv infections in pupae from two unrelated colonies to compare these two colonies and patrilines within the colonies. rt-qpcr analyses showed no initial evidence of iapv infection in either experimental colony and even the initial inoculum was below our detection limit (figure 3) . a twofactorial anova indicated that the two colonies differed significantly in the build-up of virus titers (f colony (1,104) = 5.3, p = 0.023; f time (3,104) = 69.7, p,0.001; f interaction (3,104) = 8.0, p,0.001). specifically, a significant difference between the colonies was found at 20 hours (f colony (1,42) = 39.2, p,0.001; figure 3 ). post-hoc tests also revealed a significant difference among all time points, except between 0 and 5 hours. within colonies, some patriline differences were suggestive ( figure s1 ) but not significant after bonferroni correction (colony 1: symptomatic differences were also observed between the two colonies (table 1) . generally, pupae from colony 1 showed some evidence of developmental completion, as evidenced by the presence of brown eye pigmentation and darkened abdomens. pupae from colony 2 showed higher development debilitation: few individuals developed eye pigmentation or darkened abdomens. no correlations between virus titer and symptomatology were found. three-factorial anova revealed a significant treatment effect on the expression on all six genes ( table 2 ). in general, gene expression also differed among time points but the differences for actin were not significant. in contrast, the two colonies only differed in actin expression (table 2) . post-hoc tests of main treatment effects showed significantly higher gene expression in the iapv-inoculated bees compared to the two control groups for actin, 28s rrna, and mgst1. conversely, 18s rrna was significantly less expressed in iapv-inoculated bees than in both control groups. for histone h2a, significantly lower expression was found in the untreated group than in the pbs and virus injected group, and all treatment groups differed significantly in the rps5 expression in the following order: ''control group'',''pbs-injected'',''iapv-injected''. post-hoc test results for time effects were more complex (results s1). the anova models also revealed many significant interaction terms (results s1), indicating time-specific and colony-specific treatment effects (figure 4) . the entire anova model explained most of the gene expression variation for rps5 (65.8%), followed by actin (62.1%), mgst (40.7%), h2a (38.0%), 18s rrna (35.0%), and 28s rrna (18.9%). the additionally performed ancovas revealed that all associations between iapv and transcripts were also significant, independently of treatment or timing ( table 2) . no correlations between gene expression and symptomatology were found. honey bee viruses play an important role in the recent declines in honey bee health [8, 9, 16, 17, 50, 51] but very little is known about how virus infections damage honey bees. the developed study model is a crucial step to much-needed mechanistic studies of honey bee viruses. on the one hand, honey bee pupae do not require feeding and can be easily maintained under laboratory conditions until full development into adult. they are highly relevant in the pathology of several viruses [16] . on the other hand, iapv has proven an excellent choice because its preferential replication in pupae [25] enables the production of inoculum that is virtually free of contaminating viruses. in addition, iapv is relevant for bee health [8] but it is not ubiquitous in the bee population, which makes it possible to set up experiments with iapv-free bees. our experiment showed significant differences in iapv replication between the two studied colonies, and also suggested patrilineal variation, although small sample sizes per patriline precluded significance after correction for multiple testing. environmental factors are not to be disregarded and can include colony propolis [52] and pesticide [53] levels, or larval nutrition [54] . colony 01 showed a more abrupt increase in virus titers, while iapv increased more gradually in colony 02. however, the relative resistance of bees from colony 02 only delayed iapv build-up and the iapv titers were invariably high in pupae after 48 hours. the identification of genotypic variation in virus susceptibility would improve the prospect for selective breeding to improve honey bee health. in any case, our study demonstrates significant heterogeneity in virus amplification and gene responses (see below), highlighting the importance of standardization in honey bee health studies. distinct symptomology patterns were observed between the two colonies (table 01) . it is not clear whether virus-induced tissue damage and necrosis or melanization as part of the immune response is responsible for the observed darkening. melanization is key component of insect immune response and is active in antiviral immunity [55, 56, 57] . melanization would be predicted to correlate with resistance to iapv, contrary to our observations of the darkening of larvae. therefore, necrosis or other forms of cell death are a more likely explanation for the tissue darkening [58] . quantifying gene expression responses to iapv infection in the honey bee pupa according to standard protocols was complicated because iapv infection significantly affected all investigated reference genes in all experiments (preliminary experiments s1 and figure 4 ), precluding their meaningful incorporation into dct or ddct analyses [48] . even though relative quantification has considerable advantages and is used almost universally, it depends on appropriate references [59] , which were not available for our study. therefore, we relied on absolute quantification after standardizing the amount of rna in each experiment. the ct values were converted to another measure of absolute quantification (copy number) for iapv by comparison to a standard curve. variation in cdna synthesis or other inequalities among samples might have contributed some experimental error. however, it is highly unlikely that technical errors are responsible for the observed significant differences among our experimental treatments, particularly given the consistency among our three separate experiments. thus, we conclude that ct values are most appropriate for our study and absolute quantification is necessary in studies of the transcriptional response to virus infections in honey bees. caution needs to be exerted in general when interpreting relative gene expression patterns with respect to virus infections in honey bees and other organisms [60, 61] . the investigation of multiple reference genes confirmed the earlier conclusions that basic cellular pathways were significantly being affected by iapv infection (preliminary experiments s1). interestingly, the transcription of many genes in the pbs-injected bees was intermediate between the negative control and the iapv group, demonstrating an effect of wounding itself. overall, the expression of all genes was affected by time, although for actin this effect was non-significant in the full factorial model. in contrast, actin was the only gene that exhibited an overall expression difference between the two colonies. furthermore, all genes were significantly associated with iapv titers, independently of the treatment effects. in sum, all analyzed genes failed to fulfill the criteria for a reliable reference gene and instead indicated a profound disruption of fundamental cellular processes by iapv. in addition to treatment effects, the expression of the putative reference genes also changed over time or was dependent on genotype. this transcriptional instability of putative reference genes might present a general disadvantage of the pupa as study system because the ongoing metamorphosis presumably affects numerous genes, independently of any treatment effects [62] . the biological interpretation of the main effects of host colony, time, and treatment is complicated by numerous significant interaction effects observed. for actin all interactions among the three factors were significant and for the 28s rrna no significant interactions were observed. the other four genes all showed a significant 3-way interaction and one or two 2-way interactions. interactions between time and treatment are not surprising for any transcript because the treatment effects only appear at the later stages of the experiment. however, interactions between colony and treatment confirm the finding that source colony significantly affects the interaction between iapv and its host. bees of the more resistant colony 02 showed a down-regulation of the 18s rrna by iapv injection. in contrast, the transcript was increased by iapv injection in bees from the more susceptible colony 01. similarly, for most other transcripts, the strongest up-regulation by iapv occurred after 20 hours in colony 01 but after 48 hours in colony 02. further experiments are needed to determine the causal relationships among host genotype and environment, gene expression patterns, and iapv replication. the observed gene expression patterns could be due to viral manipulation of the cells to increase virus replication or present cell compensatory responses to iapv infection. typically, picornaviruses express a protease that cleaves the scaffold eif4g initiator factor. this process inhibits the 59 cap mediated translation of cellular peptides and redirects the cell translational machinery to viral mrnas that depend on internal ribosomal entry sites (ires)-mediated translation [30, 31] . the proteasemediated shut-down of cellular translation is widespread [63, 64] and homologs of the protease gene have been identified in all members of the dicistroviruses so far [30] . however, direct evidence for a translational inhibition that increases transcriptional activities via feedback loops is so far missing for all honey bee viruses and insect picornaviruses in general. rps5 is a key component for ires recognition in the dicistrovirus family [65, 66, 67] . the up-regulation of this gene benefits virus replication directly, suggesting that rps5's strong and consistent upregulation may be directly induced by iapv. however, the widespread transcriptional response to iapv also suggests that the cell may respond to the lack of certain cell components by increasing their transcription. the up-regulation of actin, mgst1, and the histone h2a in most experimental groups suggests a far-reaching, although variable, response in a range of basic cellular processes in addition to a disturbance of the ribosomal biogenesis pathway discussed below. more research is needed to understand these processes and it variability among environments and genotypes. the three components of the ribosomal biogenesis pathway studied exhibited different responses to iapv injection. while 28s rrna, and rps5 transcripts were invariably increased after iapv replication (20 and 48 hours post-injection), 18s rrna transcripts were decreased in colony 02 at both time points and were only increased in the more susceptible colony 01 at the first time point. the reason for these disparities is unclear, particularly because the 18s rrna and 28s rrna transcripts are derived from a polycistronic precursor mrna [68] . however, the regulated balance between small and large ribosomal subunits [69] is controlled by independent maturation pathways [70] and iapv presumably affects these pathways differently. the differences between colonies may indicate that the more resistant individuals may have either resisted transcriptional manipulation by iapv or dedicated more cellular resources to immediate immune functions instead of ribosomal biogenesis. consistent with this interpretation, gene expression patterns converged between the two colonies at the later time point. ribosome biogenesis is a highly complex and energetically costly pathway that is essential for all eukaryotic cells [70] . it is highly regulated and integrated with other cell functions, such as p53 signaling, and deregulation of ribosomal biosynthesis has been associated with oncogenesis [71] and apoptosis [72] . apoptosis is a widespread cellular response to virus infection [73] and could explain some of the observed differences in iapv symptomology. on the other hand, viruses can also directly interfere with the ribosomal biogenesis pathway by either up-or down-regulation [74, 75] . in any case, our result of a disturbance of the ribosomal biogenesis corroborates an important microarray survey of transcripts in the honey bee intestine that has linked picornaviruses and 28s rrna transcript abundance to colony collapse disorder [9] . the injection of pbs served as an experimental control to account for the effect of wounding during iapv inoculation. for all genes, the observed transcription patterns of the pbs-injected bees were intermediate between the iapv-injected and the negative control group. this observation may suggest that a similar disruption of basic cellular functions occurs in response to wounding and cellular trauma, resulting in profound changes at the transcriptome level [76] . however, our data show also an increase of dwv titers in the pbs-injected pupae over time and relative to both other treatment groups. increased dwv titers in response to wounding have been observed before [77] . the increase of another picorna-like virus may have triggered responses in the pbs-injected bees that were similar to the iapv injection, supporting a similar gene expression pattern observed between pbs group and iapv injected bees compared to the negative control groups. in contrast to the pbs-injected bees, the iapv-injected bees did not show an increase in dwv titers, suggesting that iapv or cellular responses to iapv interfere with dwv replication [78] . in summary, this study introduces an important model system to advance mechanistic studies on virus-host interactions in insects. it is particularly valuable to study honey bee viruses and their role in compromising honey bee health. the results demonstrate significant variability and indicate sources for this variability. the transcriptional analyses show profound, correlated perturbations of basic cellular functions and call into question the use of typical reference genes in this system. the investigated responses to iapv inoculation in honey bees seem typical for picornavirus infections and provide a first step towards understanding the basic biology of this important honey bee virus. more detailed studies need to follow to manipulate virus and host and assess host responses to iapv at the systemic level. figure s1 patriline differences of iapv titers within colonies suggest a genetic basis for virus resistance in honey bees. preliminary experiments s1 two experimental inoculations of a limited number of bees with iapv suggested that viral infection causes broad-scale alterations of transcript patterns, including immune and reference genes. raw data s1 raw ct values for all qpcrs run in the main experiment. (pdf) results s1 the expression of genes evaluated by full, 3factorial anovas indicated that developmental time is a significant factor, that was as important as treatment but for most genes interacted with treatment. 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(apis mellifera) resin collection and social immunity in honey bees pesticide exposure in honey bees results in increased levels of the gut pathogen nosema nutrigenomics in honey bees: digital gene expression analysis of pollen's nutritive effects on healthy and varroa-parasitized bees the host defense of drosophila melanogaster antiviral melanization reaction of heliothis virescens hemolymph against dna and rna viruses in-vitro plasma phenoloxidase of the larval tobacco budworm, heliothis virescens, is virucidal baculoviruses and apoptosis: a diversity of genes and responses the miqe guidelines: minimum information for publication of quantitative real-time pcr experiments reference gene selection for quantitative real-time pcr analysis in virus infected cells: sars corona virus, yellow fever virus, human herpesvirus-6, camelpox virus and cytomegalovirus infections identification and validation of reference genes for normalization of transcripts from virus-infected arabidopsis thaliana the developmental transcriptome of drosophila melanogaster identification of the cleavage site and determinants required for poliovirus 3cpro-catalyzed cleavage of human tata-binding transcription factor tbp poliovirus proteinase-3c converts an active form of transcription factor-iiic to an inactive form -a mechanism for inhibition of host-cell polymerase-iii transcription by poliovirus structural basis for ribosome recruitment and manipulation by a viral ires rna eukaryotic ribosomal protein rps25 interacts with the conserved loop region in a dicistroviral intergenic internal ribosome entry site cryo-em visualization of a viral internal ribosome entry site bound to human ribosomes: the ires functions as an rna-based translation factor characteristics of the nuclear (18s, 5.8s, 28s and 5s) and mitochondrial (12s and 16s) rrna genes of apis mellifera (insecta : hymenoptera): structure, organization, and retrotransposable elements autoregulation of ribosome biosynthesis by a translational response in fission yeast ribosome biogenesis: of knobs and rna processing changes in ribosome biogenesis may induce cancer by down-regulating the cell tumor suppressor potential ribosomal stress induces l11-and p53-dependent apoptosis in mouse pluripotent stem cells apoptosis in viral pathogenesis genome-wide expression profiling shows transcriptional reprogramming in fusarium graminearum by fusarium graminearum virus 1-dk21 infection cardiovirus 2a protein associates with 40s but not 80s ribosome subunits during infection epigenetic reprogramming during wound healing: loss of polycomb-mediated silencing may enable upregulation of repair genes expression levels of immune-genes in developing workers of apis mellifera in response to reproductive timing and infestation level by the parasitic mite varroa destructor competition-colonization dynamics in an rna virus we would like to thank the usda-beltsville bee research laboratory members: dr. miguel corona, for the histone h2a primer design and mr. andy ulsamer and ms. michele hamilton for their technical support. in addition, we would like to thank all members of the uncg social insect group for their suggestions and support. key: cord-000833-m6abyuvx authors: sekiguchi, satoshi; kimura, kiminori; chiyo, tomoko; ohtsuki, takahiro; tobita, yoshimi; tokunaga, yuko; yasui, fumihiko; tsukiyama-kohara, kyoko; wakita, takaji; tanaka, toshiyuki; miyasaka, masayuki; mizuno, kyosuke; hayashi, yukiko; hishima, tsunekazu; matsushima, kouji; kohara, michinori title: immunization with a recombinant vaccinia virus that encodes nonstructural proteins of the hepatitis c virus suppresses viral protein levels in mouse liver date: 2012-12-17 journal: plos one doi: 10.1371/journal.pone.0051656 sha: doc_id: 833 cord_uid: m6abyuvx chronic hepatitis c, which is caused by infection with the hepatitis c virus (hcv), is a global health problem. using a mouse model of hepatitis c, we examined the therapeutic effects of a recombinant vaccinia virus (rvv) that encodes an hcv protein. we generated immunocompetent mice that each expressed multiple hcv proteins via a cre/loxp switching system and established several distinct attenuated rvv strains. the hcv core protein was expressed consistently in the liver after polyinosinic acid–polycytidylic acid injection, and these mice showed chronic hepatitis c-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. immunization with one rvv strain (rvv-n25), which encoded nonstructural hcv proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis c within 7 days after injection. furthermore, hcv protein levels in liver tissue also decreased in a cd4 and cd8 t-cell-dependent manner. consistent with these results, we showed that rvv-n25 immunization induced a robust cd8 t-cell immune response that was specific to the hcv nonstructural protein 2. we also demonstrated that the onset of chronic hepatitis in cn2-29((+/−))/mxcre((+/−)) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) tnf-α and (interleukin) il-6. thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of hcv proteins because these mice had not developed immune tolerance to the hcv antigen. in addition, we propose that rvv-n25 could be developed as an effective therapeutic vaccine. hepatitis c virus (hcv) is a major public health problem; approximately 170 million people are infected with hcv worldwide [1] . hcv causes persistent infections that can lead to chronic liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (hcc) [2] . antiviral drugs are not highly effective in individuals with a chronic infection; furthermore, an effective vaccine against hvc has not been developed. a convenient animal model of hcv infection will greatly facilitate the development of an effective hcv vaccine. transgenic mice that express hcv proteins have been generated to study hcv expression [3, 4] ; however, in each of these cases, the relevant transgenes is expressed during embryonic development; therefore, the transgenic mice become immunotolerant to the transgenic products, and consequently, the adult mice are not useful for investigations of the pathogenesis of chronic hepatitis c. to address this problem, we developed a system that can drive conditional expression of an hcv transgene; our system involves the cre/loxp system and a recombinant adenovirus capable of expressing cre recombinase [5, 6] . concerns have been expressed that an adenovirus and transient expression of hcv proteins could induce immune responses [5] and, therefore, obscure any evidence of the effect of the host immune responses on chronic liver pathology. therefore, here, we used a cre/loxp switching system to generate an immunocompetent mouse model of hcv protein expression; with this system, we could study the host immune responses against hcv proteins. folgori et al. (2006) reported effective vaccination of chimpanzees with an adenoviral vector and plasmid dna encoding the hcv nonstructural region. this technique protected the liver tissues from acute hepatitis, which results when whole animals are challenged with virus [7] . however, this vaccine has not yet been shown to be effective against chronic hcv infection. here, we aimed to address how hcv expression causes chronic liver diseases and to provide new options for hcv vaccine development. using lc16m8, a highly attenuated strain of vaccinia virus (vv), we generated three recombinant vaccinia viruses (rvvs) that each encoded one of three different hcv proteins and found that one recombinant virus (rvv-n25), which encoded nonstructural hcv proteins, resolved pathological chronic hepatitis c symptoms in the liver. we also found that immunization with rvv-n25 suppressed hcv core protein levels in the livers of transgenic mice; moreover, this suppression was mediated by cd4 and cd8 t cells, as has been previously reported [8] . to produce adult mice that express an hcv transgene, we bred cn2-29 transgenic mice, which carry an hcv transgene, [5, 6, 9] with mx1-cre transgenic mice [10] , which express cre recombinase in response to interferon (ifn)-a or a chemical inducer of ifn-a, poly(i:c) ( figure 1a ). following poly(i:c) injection, the hcv transgene was rearranged, and hcv sequences were expressed in the livers of f1 progeny (cn2-29 (+/2) /mxcre (+/2) mice) within 7 days after poly(i:c) injection ( figure 1b) . to evaluate the characteristic features of these cn2-29 (+/2) / mxcre (+/2) mice, we analyzed serum alanine aminotransferase (alt) and liver hcv core protein levels after poly(i:c) injection. as illustrated in figure 1c , serum alt levels increased and reached a peak at 24 h after the first poly(i:c) injection; this elevation appeared to be a direct result of the poly(i:c) treatment, which causes liver injury [11] . after this peak, serum alt levels dropped continuously until day 4, and then alt levels began to increase, as did hcv core protein levels. thereafter, the hcv core protein was expressed consistently for at least 600 days. histological analysis showed hcv core protein expression in most hepatocytes of the transgenic mice; these mice showed evidence of lymphocytic infiltration that was caused by the hcv core proteins ( figure 1d and e). these observations, in addition to the modified histology activity index (hai) scores, indicated that expression of hcv proteins caused chronic hepatitis in the cn2-29 (+/2) /mxcre (+/2) mice because a weak, though persistent, immune response followed an initial bout of acute hepatitis ( figure s1 ). moreover, we observed a number of other pathological changes in these mice -including swelling of hepatocytes, abnormal architecture of liver-cell cords, abnormal accumulation of glycogen, steatosis, fibrosis, and hcc (figures 1e and f, table s1 ). steatosis was mild in the younger mice (day 21) and became increasingly severe over time (days 120 and 180; figure s2 ). importantly, none of the pathological changes were observed in the cn2-29 (+/2) /mxcre (2/2) mice after poly(i:c) injection ( figure 1f ). to determine whether activation of the host immune response caused the reduction with hcv protein levels in the livers of cn2-29 (+/2) /mxcre (+/2) mice, we used a highly attenuated vv strain, lc16m8, to generate three rvvs [12] . each rvv encoded a different hcv protein; rvv-cn2 encoded mainly structural proteins, rvv-n25 encoded nonstructural proteins, and rvv-cn5 encoded the entire hcv protein region (figure 2a ). because rvvs can express a variety of proteins and induce strong and long-term immunity, they have been evaluated as potential prophylactic vaccines [13] . we used western blots to confirm that each hcv protein was expressed in cell lines. each of seven proteins -the core, e1, e2, ns3-4a, ns4b, ns5a, and ns5b -was recognized and labeled by a separate cognate antibody directed ( figure s3 ). to induce effective immune responses against hcv proteins in transgenic mice, we injected an rvv-hcv (rvv-cn2, rvv-cn5, or rvv-n25) or lc16m8 (as the control) intradermally into cn2-29 (+/2) / mxcre (+/2) mice 90 days after poly(i:c) injection ( figure 2b ). analysis of liver sections 7 days after immunization with rvv-n25 revealed dramatic improvement in a variety of pathological findings associated with chronic hepatitis -including piecemeal necrosis, hepatocyte swelling, abnormal architecture of liver-cell cords, abnormal accumulation of glycogen, and steatosis ( figures 2c-e) . collectively, these results demonstrated that only the rvv-n25 treatment resulted in histological changes indicative of improvement in the chronic hepatitis suffered by the transgenic mice. to determine whether rvv-n25 treatment induced the same effect in other strains of hcv transgenic mice, we analyzed rzcn5-15 (+/2) /mxcre (+/2) mice, which express all hcv proteins; in these mice, chronic hepatitis was resolved within 28 days of immunization with rvv-n25. taken together, these findings indicated that rvv-n25 had a dramatic therapeutic effect on both types of hcv transgenic mice ( figure s4 ). to assess in detail the effects of rvv-hcv immunization on hcv protein clearance from the livers of cn2-29 (+/2) /mxcre (+/ 2) mice, we monitored the levels of hcv core protein in liver samples via elisa. we found that within 28 days after immunization the hcv core protein levels were significantly lower in livers of rvv-n25-treated mice than in those of control mice ( figure 3a ). immunohistochemical analysis indicated that, within 28 days after immunization, levels of hcv core protein were substantially lower in the livers of cn2-29 (+/2) /mxcre (+/2) mice than in those of control mice ( figure 3b ). importantly, neither resolution of chronic hepatitis nor reduction in the hcv protein levels was observed in the mice treated with lc16m8, rvv-cn2, or rvv-cn5. these results indicated that hcv nonstructural proteins might be important for effects of therapeutic vaccines. in contrast, rvv-cn5 which encoded hcv structural and non-structural proteins did not show any significant effects. these results indicated that hcv structural proteins might have inhibited the therapeutic effects of the non-structural proteins. therefore, it may be important to exclude the hcv structural proteins (aa 1-541) as antigenic proteins when developing therapeutic vaccines against chronic hepatitis c. in addition, we measured serum alt levels in cn2-29 (+/2) / mxcre (+/2) mice from all four treatment groups 28 days after rvv-hcv immunization. serum alt levels were not significant(+/2) and the cremediated activation of the transgene unit. r6cn2 hcv cdna was cloned downstream of the cag promoter, neomycin-resistant gene (neo), and poly a (pa) signal flanked by two loxp sequences. this cdna contains the core, e1, e2, and ns2 regions. (b) cre-mediated genomic dna recombination. after poly(i:c) injection, genomic dna was extracted from liver tissues and analyzed by quantitative rtd-pcr for cre-mediated transgenic recombination. the transgene was almost fully recombined in transgenic mouse livers 7 days after the injection. in all cases, n = 3 mice per group. (c) hcv core protein expression was sustained for at least 600 days after poly(i:c) injection. (d) immunohistochemical analysis revealed that most hepatocytes expressed the hcv core protein within 6 days after injection. (e) liver sections from cn2-29 (+/2) /mxcre (+/2) mice after the poly(i:c) injection. infiltrating lymphocytes (arrows) were observed on days 6 and 180; hepatocellular carcinoma (hcc) was observed on day 360. in contrast, these pathological changes were not observed in cn2-29 (+/2) /mxcre (2/2) mice after the injection. the inset image shows abnormal mitosis in a tumor cell. (f) hepatocyte swelling and abnormal architecture of liver-cell cords (silver staining), as well as abnormal glycogen accumulation (pas staining) were observed on day 90 in cn2-29 (+/2) /mxcre (+/2) mice. we observed steatosis (oil-red-o staining) on day 180 and, subsequently, fibrosis (azan staining) on day 480. the scale bars indicate 50 mm. doi:10.1371/journal.pone.0051656.g001 ly different in the rvv-n25-treated mice and control mice ( figure 3c ); this finding indicated that rvv-n25 treatment did not cause liver injury and that the antiviral effect was independent of hepatocyte destruction. we hypothesized that the reduction in the levels of hcv core protein in rvv-hcv-treated mice was not caused by cytolytic elimination of hepatocytes that expressed hcv proteins. to investigate this hypothesis, we conducted an rtd-pcr analysis of genomic dna from liver samples of cn2-29 (+/2) /mxcre (+/2) mice. the recombined transgene was similar in rvv-n25-treated and control mice 28 days after immunization ( figure 3d ). we also measured the expression of hcv mrna in lc16m8-treated cn2-29 (+/2) /mxcre (+/2) mice with that in rvv-n25-treated cn2-29 (+/2) /mxcre (+/2) mice 28 days after immunization; the hcv mrna levels did not differ between rvv-n25-treated cn2-29 (+/2) /mxcre (+/2) and control mice ( figure 3e ). these results indicated that rvv-n25-induced suppression of hcv core protein expression could be controlled at a posttranscriptional level. viral clearance is usually associated with cd4 and cd8 t-cell activity that is regulated by cytolytic or noncytolytic antiviral mechanism [14] . to determine whether cd4 or cd8 t-cell activity was required for the reduction in hcv core protein levels in the livers of transgenic mice, we analyzed the core protein levels in cn2-29 (+/2) /mxcre (+/2) mice immunized with rvv-n25 in the absence of cd4 or cd8 t cells ( figure 4a ). as expected, the mice lacking cd4 or cd8 t cells failed to show a reduction in hcv core protein levels ( figure 4b ). however, in mice lacking either cd4 or cd8 t-cells, the pathological changes associated with chronic hepatitis were resolved following rvv-n25 immunization, and the steatosis score of rvv-n25-treated mice was significantly lower than that of control mice (figures 4c-e) . these results indicated that cd4 and cd8 t cells were not responsible for the rvv-n25-induced amelioration of histological findings and that other inflammatory cell types may play an as-yet-unidentified role in the resolution of the pathological changes in these mice. because we found that hcv protein reduction in the liver required cd8 t cells, we tested whether hcv-specific cd8 t cells were present in splenocytes 28 days after immunization. to determine the functional reactivity of hcv-specific cd8 + t cells, we performed a cd107a mobilization assay and intracellular ifnc staining. cn2-29 transgenic mice expressed the hcv structural protein and the ns2 region. however, rvv-n25 comprised only isolated splenocytes from immunized mice were co-cultured with el-4cn2 or el-4ns2 cell lines for 2 weeks and analyzed. cytolytic cell activation can be measured using cd107a, a marker of degranulation [15] . the ratio of cd8 + cd107a + cells to all cd8 t cells significantly increased in rvv-n25-treated splenocytes after co-culture with el-4cn2 or el-4ns2 (p,0.05), whereas splenocytes that had been treated with any other rvv were not detected ( figure 5a, b and c) . these results indicated that rvv-n25 treatment increased the frequency of hcv ns2specific activated cd8 t cells. consistent with these results, the ratio of cd8 + ifn-c + cells to all cd8 t cells for rvv-n25-treated mice was also significantly higher than that for mice treated with any other rvv (p,0.05). taken together, these findings indicated that rvv-n25 induced an effective cd8 t-cell immune response and that ns2 is an important epitope for cd8 t cells. to determine whether rvv-n25 treatment affected inflammatory cytokine production, we measured serum levels of inflammatory cytokines after rvv immunization. the serum levels of these inflammatory cytokines increased in the cn2-29 (+/2) / mxcre (+/2) mice ( figure 6a, figure s5 ). immunization with rvv-n25 affected serum levels of inflammatory cytokines in cn2-29 (+/ 2) /mxcre (+/2) mice and caused a return to the cytokine levels observed in wild-type untreated mice ( figure 6a ). in wild-type mice, the cytokine levels remained unchanged after immunization ( figure 6a ). these results indicated that inflammatory cytokines were responsible for liver pathogenesis in the transgenic mice. to test the hypothesis that inflammatory cytokines were responsible for liver pathogenesis in cn2-29 (+/2) /mxcre (+/2) mice, we administered transgenic mouse serum intravenously into nontransgenic mice. we observed the development of chronic hepatitis in the nontransgenic mice within 7 days after the serum transfer ( figures 6b and c) . this finding was consistent with the 28 days after immunization with lc16m8 or rvv-n25. the scale bars indicate 100 mm (c) and 50 mm (d). (e) histological evaluation of steatosis in liver samples from cd4-depleted or cd8-depleted cn2-29 (+/2) /mxcre (+/2) mice 28 days after immunization with lc16m8 or rvv-n25. significant relationships are indicated by a p-value. doi:10.1371/journal.pone.0051656.g004 hypothesis that inflammatory mediators played a key role in inducing hepatitis. furthermore, to investigate whether tnf-a and il-6 played particularly critical roles in the pathogenesis of chronic hepatitis in the transgenic mice, we neutralized tnf-a and blocked the il-6 receptor in the livers of these mice. as expected, chronic hepatitis did not develop in these mice. (figure 6d and e) . next, to determine which cell population(s) produced tnf-a, il-6, or both during continuous hcv expression in cn2-29 (+/2) / mxcre (+/2) mice, we isolated intrahepatic lymphocytes (ihls) and labeled the macrophages (the f4/80 + cells) with anti-tnf-a and anti-il-6 antibodies using an intracellular cytokine detection method. macrophages in cn2-29 (+/2) /mxcre (2/2) mice produced small amounts of tnf-a and il-6, while those in cn2-29 (+/2) /mxcre (+/2) mice produced much larger amounts of these cytokines ( figure 6f ). finally, we evaluated whether rvv-n25 treatment affected the number of macrophages, cytokine production by macrophages, or both; specifically, we isolated ihls from cn2-29 (+/2) /mxcre (+/2) mice 7 days after immunization with rvv-n25 or with lc16m8. the percentage of macrophages (cd11b + f4/80 + ) among ihls and il-6 production from these macrophages were significantly lower in rvv-n25-treated mice than in control mice ( figure 6g ). though the percentage of tnf-a-producing macrophages was not significantly different in rvv-n25-treated and control mice (p = 0.099), rvv-n25 treatment appeared to suppress these macrophages. these results demonstrated that rvv-n25 had a suppressive effect on activated macrophages, and they indicated that this suppression ameliorated the histological indicators of chronic hepatitis. various hcv transgenic mouse models have been developed and used to examine immune response to hcv expression and the effects of pathogenic hcv protein on hepatocytes [4, 16, 17] . however, these transgenic mice develop tolerance to the hcv protein; therefore, examining immune response to hcv protein has been difficult. to overcome the problem of immune tolerance in mouse models of hcv expression, we developed an hcv model in mice that relies on conditional expression of the core, e1, e2, and ns2 proteins and the cre/loxp switching system [5, 6] ; we showed that the injection of an ad-cre vector enhanced the frequency of hcv-specific activated cd8 t cells in the liver of these mice and caused liver injury. however, the ad-cre adenovirus vector alone causes acute hepatitis in wild-type mice. nevertheless, the transgenic model was useful for evaluating interactions between the host immune system and viral protein (serum alt level over 2,000 iu/l) [5] ; hcv core protein levels were reduced and expression of this protein was transient (about 2 weeks). therefore, this ad-cre-dependent model cannot be used to effectively investigate immune responses to chronic hcv hepatitis. here, we used poly (i:c)-induced expression of cre recombinase to generate hcv transgenic mice in order to study the effect of hcv protein and confirmed that these mice developed chronic active hepatitis-including steatosis, lipid deposition, and hepatocellular carcinoma. these pathological findings in the transgenic mice were very similar to those in humans with chronic hepatitis c; therefore, this mouse model of hcv may be useful for analyzing the immune response to chronic hepatitis. however, experimental results obtained with this mouse model may not directly translate to clinical findings from patients with hcv infection because the expression of hcv proteins was not liver specific in these mice. furthermore, poly(i:c) injection can activate innate immune responses and, consequently, might induce temporary liver injury [18] . additionally, poly(i:c) injection has an adjuvant effect; specifically, it stimulates tlr3 signaling [19] . to evaluate whether poly(i:c) injection caused hepatitis in cn2-29 (+/2) /mxcre (2/2) mice, we examined serum alt levels and liver histology following poly(i:c) injection. we found that, following poly(i:c) injection, serum alt levels in cn2-29 (+/2) / mxcre (2/2) mice increased, reached a peak one day after injection, declined from day 1 to day 6, and were not elevated thereafter; this time-course indicated that poly(i:c) injection alone did not induced continuous liver injury ( figure s6 ). based on these findings, we believe that the effects of poly(i:c) injection in these mice did not confound our analysis of chronic hepatitis. immunization with rvv-n25 suppressed hcv protein levels in the liver, and this suppression was associated with ameliorated pathological chronic hepatitis findings (see figure 3 ). importantly, rvv-n25 treatment did not cause liver injury based on the serum alt levels; therefore, this treatment was unlikely to have cytopathic effects on infected hepatocytes. these findings provided strong evidence that rvv-n25 treatment effectively halted the progression of chronic hepatitis. immunization with plasmid dna or with recombinant vaccinia virus can effectively induce cellular and humoral immune responses and exert a protective effect against challenge with hcv infection [20, 21] . however, findings from these previous studies revealed hcv immunization of both uninfected, naïve animals and immune-tolerant animals induced a hcv-specific immune response. in the model describe here; the animals were immune competent for hcv; therefore, our findings provided further important evidence that rvv-n25 was effective in the treatment of chronic hepatitis. in addition, we demonstrated that rvv-n25 treatment in the absence of cd4 and cd8 t cells had no effect on hcv clearance. this important observation indicated that rvv-n25-induced hcv clearance was mediated by cd4 and cd8 t cells. many studies have shown that spontaneous viral clearance during acute hcv infection is characterized by a vigorous, broadly reactive cd4 and cd8 t-cell response. [8, 22] hcv clearance and hepatocellular cytotoxicity are both mediated by cd8 antigenspecific (cytotoxic t lymphocyte) ctls [23] . consistent with these observations, rvv-n25 treatment effectively induced the accumulation of ns2-specific cd8 t cells, which express high levels of figure 6 . immunization with rvv-n25 suppresses serum inflammatory cytokine levels. (a) daily cytokine levels in the serum of cn2-29 (+/ 2) /mxcre (+/2) mice during the week following immunization with lc16m8, rvv-cn2, rvv-n25, or rvv-cn5. values represent means 6 sd (n = 3) and reflect the concentrations relative to those measured on day 0. the broken lines indicate the baseline data from wild-type mice. in all cases, n = 6 mice per group. (b) liver sections from cn2-29 (+/2) /mxcre (+/2) and cn2-29 (+/2) /mxcre (2/2) mice. (c) histology activity index (hai) scores of liver samples taken from cn2-29 (+/2) /mxcre (+/2), or cn2-29 (+/2) /mxcre (2/2) mice. (d) liver sections from cn2-29 (+/2) /mxcre (+/2) mice in which tnf-a was neutralized and the il-6 receptor was blocked. the scale bars indicate 50 mm. (e) hai scores of liver samples taken from cn2-29 (+/2) /mxcre (+/2) in which tnf-a was neutralized and the il-6 receptor was blocked. tg and non-tg indicate cn2-29 (+/2) /mxcre (+/2) and cn2-29 (+/2) /mxcre (2/2) , respectively. (f) macrophages were the main producers of tnf-a and il-6 in cn2-29 (+/2) /mxcre (+/2) mice following poly(i:c) injection. (g) immunization with rvv-n25 reduced the number of macrophages in liver samples from cn2-29 (+/2) /mxcre (+/2) mice and suppressed tnf-a and il-6 production from macrophages ( figure 6g ). significant relationships are indicated by a p-value. doi:10.1371/journal.pone.0051656.g006 cd107a and ifn-cin the spleen. notably, even with rvv-n25 immunization, the frequency of activated cd8 t cells was very low, and a minimum of 2-weeks incubation was required to distinguish the difference between rvv treatments. even if a small population of specific cd8+ t cells played a relevant role in the reduction of core protein, it is difficult to assert that the only ns2specific cd8+ t cells were important to this reduction. however, based on the results presented in figure 4b , we are able to conclude that at least cd8+ and/or cd4+ t cells were important to the reduction in hcv core protein. therefore, to elucidate the mechanism of hcv protein clearance, further investigation of not only the other t cell epitopes but also other immunocompetent cells is required. interestingly, rvv-n25 treatment-but not the rvv-cn2 or rvv-cn5 treatment-efficiently induced a hcv-specific activated cd8 t cells response; this difference in efficacy could have one or more possible causes. the hcv structural proteins (core, e1, and e2 proteins) in the rvv-cn construct may cause the difference; saito et al. reported that injection with plasmid constructs encoding the core protein induced a specific ctl response in balb/c mice [24] . reportedly, ctl activity against core or envelope protein is completely absent from transgenic mice immunized with a plasmid encoding the hcv structural proteins, but core-specific ctl activity is present in transgenic mice that were immunized with a plasmid encoding the hcv core [21] . in contrast, when recombinant vaccinia virus expressing different regions of the hcv polyprotein were injected into balb/c mice, only the hcv core protein markedly suppressed vaccinia-specific ctl responses [25] . thus, the hcv core protein may have an immunomodulatory function [26] . based on these reports and our results, we hypothesize that the causes underlying the effectiveness of rvv-n25 treatment were as follows: 1) this rvv construct included the core and envelope proteins and 2) the core protein had an immune-suppressive effect on ctl induction. therefore, we suggest that exclusion of the core and envelope antigen as immunogen is one important factor in hcv vaccine design. interestingly, immunization with rvv-n25 rapidly suppressed the inflammatory response; however, immunization with either of the other rvvs did not (see figure 6a ). this result indicated that rvv-n25 may modulate inflammation via innate immunity, as well as via acquired immunity. reportedly, toll-like receptor (tlr)-dependent recognition pathways play a role in the recognition of poxviruses [27] . tlr2 and tlr9 have also been implicated in the recognition of the vaccinia virus [28, 29] . these findings indicate that tlr on dendritic cells may modulate the immunosuppressive effect of rvv-n25 in our model of hcv infection; however, further examination of this hypothesis is required. the finding that pathological symptoms in the hcv transgenic mice were completely blocked by intravenous injection of tnf-a and il-6 neutralizing antibodies indicated that the progression of chronic hepatitis depended on inflammatory cytokines in serum, rather than the hcv protein levels in hepatocytes. lymphocytes, macrophages, hepatocytes, and adipocytes each produce tnf-a and il-6 [30, 31] , and hcv-infected patients have elevated levels of tnf-a and il-6 [32, 33] . both cytokines also contribute to the maintenance of hepatosteatosis in mice fed a high-fat diet [34] , and production of tnf-a and il-6 is elevated in obese mice due to the low grade inflammatory response that is caused by lipid accumulation [35] . these findings indicate that both cytokines are responsible for hcv-triggered hepatosteatosis, and anti-cytokine neutralization is a potential treatment for chronic hepatitis if antiviral therapy is not successful. the reduction of macrophages in number might be due to the induction of apoptosis by vaccinia virus in vitro infection as previous reported [36] . to understand the mechanisms responsible for the reduction of the number of macrophage, we performed another experiment to confirm whether the macrophages were infected with vaccinia virus inoculation. however, based on pcr analyses; vaccinia virus dna was not present in liver tissue that contained macrophages ( figure s7 ). furthermore, apoptosis of macrophages was not detected in liver samples (data not shown). based on these results, it is unlikely that the reduction in the number of macrophages was due to apoptosis induced by vaccinia virus infection. although rvv-n25 reduced the number of macrophage, precise mechanism is still unknown. further examination to elucidate the mechanism is required. in conclusion, our findings demonstrated that rvv-n25 is a promising candidate for an hcv vaccine therapy. additionally, the findings of this study indicate that rvv-n25 immunization can be used for prevention of hcv infection and as an antiviral therapy against ongoing hcv infection. r6cn2 hcv cdna (nt 294-3435) [37] and full genomic hcv cdna (nt 1-9611) [38, 39] were cloned from a blood sample taken from a patient (#r6) with chronic active hepatitis (text s1). the infectious titer of this blood sample has been previously reported [40] . r6cn2hcv and r6cn5hcv transgenic mice were bred with mx1-cre transgenic mice (purchased from jackson laboratory) to produce r6cn2hcv-mxcre and r6cn5hcv-mxcre transgenic mice, which were designated cn2-29 (+/2) /mxcre (+/2) and rzcn5-15 (+/2) /mxcre (+/2) mice, respectively. cre expression in the livers of these mice was induced by intraperitoneal injection of polyinosinic acid-polycytidylic acid [poly(i:c)] (ge healthcare uk ltd., buckinghamshire, england); 300 ml of a poly(i:c) solution (1 mg/ml in phosphate-buffered saline [pbs]) was injected three times at 48-h intervals. all animal care and experimental procedures were performed according to the guidelines established by the tokyo metropolitan institute of medical science subcommittee on laboratory animal care. tissue samples were fixed in 4% paraformaldehyde in pbs, embedded in paraffin, sectioned (4-mm thickness), and stained with hematoxylin and eosin (h&e). staining with periodic acid-schiff stain, azan stain, silver, or oil-red-o was also performed to visualize glycogen degeneration, fibrillization, reticular fiber degeneration, or lipid degeneration, respectively. for immunohistochemical staining, unfixed frozen liver sections were fixed in 4% paraformaldehyde for 10 min and then incubated with blocking buffer (1% bovine serum albumin in pbs) for 30 min at room temperature. subsequently, the sections were incubated with biotinylated mouse anti-hcv core mono-clonal antibody (5e3) for 2 h at room temperature. after being washed with pbs, the sections were incubated with streptavidin-alexa fluor 488 (invitrogen). the nuclei were stained with 4',6diamidino-2-phenylindole (dapi). fluorescence was observed using a confocal laser microscope (laser scanning microscope 510, carl zeiss). the pbr322-based plasmid vector pbmsf7c contained the ati/p7.5 hybrid promoter within the hemagglutinin gene region of the vaccinia virus, which was reconstructed from the psfj1-10 plasmid and pbm vector [41, 42] . separate full-length cdnas encoding either the hcv structural protein, nonstructural protein, or all hcv proteins were cloned from hcv r6 strain (genotype 1b) rna by rt-pcr. each cdna was inserted into a separate pbmsf7c vector downstream of the pbmsf7c ati/p7.5 hybrid promoter; the final designation of each recombinant plasmid was pbmsf7c-cn2, pbmsf7c-n25, or pbmsf-cn5 (figure 2 ). they were then transfected into primary rabbit kidney cells infected with lc16m8 (multiplicity of infection = 10). the viruscell mixture was harvested 24 h after the initial transfection by scrapping; the mixture was then frozen at 280uc until use. the hemagglutinin-negative recombinant viruses were cloned as previously described [42] and named rvv-cn2, rvv-n25, or rvv-cn5. insertion of the hcv protein genes into the lc16m8 genome was confirmed by direct pcr, and expression of each protein from the recombinant viruses was confirmed by western blot analysis. the titers of rvv-cn2, rvv-n25, and rvv-cn5 were determined using a standard plaque assay and rk13 cells. data are shown as mean 6 sd. data were analyzed using the nonparametric mann-whitney or kruskal-wallis tests or an-ova as appropriate; graphpad prism 5 for macintosh (graph-pad) was used for all analyses. p values ,0.05 were considered statistically significant. table s1 incidence of hepatocellular carcinoma in male and female transgenic mice at 360, 480, and 600 days after poly(i:c) injection. text s1 supporting information including material and methods, and references. (docx) hepatitis c virus infection epidemiology of hepatitis c in the west transgenic expression of hepatitis c virus structural proteins in the mouse the core protein of hepatitis c virus induces hepatocellular carcinoma in transgenic mice possible role of cytotoxic t cells in acute liver injury in hepatitis c virus cdna transgenic mice mediated by cre/loxp system efficient conditional transgene expression in hepatitis c virus cdna transgenic mice mediated by the cre/loxp system a t-cell hcv vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees hepatitis b virus immunopathology inhibition of cytochrome c release in fas-mediated signaling pathway in transgenic mice induced to express hepatitis c viral proteins inducible gene targeting in mice distinct poly(i-c) and virus-activated signaling pathways leading to interferon-beta production in hepatocytes characteristics of an attenuated vaccinia virus strain, lc16m0, and its recombinant virus vaccines evidence for protection against chronic hepatitis c virus infection in chimpanzees by immunization with replicating recombinant vaccinia virus viral clearance without destruction of infected cells during acute hbv infection a novel flow cytometric assay for evaluating cell-mediated cytotoxicity hepatitis c virus core and e2 protein expression in transgenic mice steatosis and liver cancer in transgenic mice expressing the structural and nonstructural proteins of hepatitis c virus immunoprivileged status of the liver is controlled by toll-like receptor 3 signaling ampligen: a potential toll-like 3 receptor adjuvant for immunotherapy of cancer immunization with hepatitis c virus-like particles results in control of hepatitis c virus infection in chimpanzees genetic immunization of wild-type and hepatitis c virus transgenic mice reveals a hierarchy of cellular immune response and tolerance induction against hepatitis c virus structural proteins the liver as a lymphoid organ unscrambling hepatitis c virus-host interactions plasmid dna-based immunization for hepatitis c virus structural proteins: immune responses in mice suppression of host immune response by the core protein of hepatitis c virus: possible implications for hepatitis c virus persistence flying under the radar: the immunobiology of hepatitis c a46r and a52r from vaccinia virus are antagonists of host il-1 and toll-like receptor signaling innate immunity against vaccinia virus is mediated by tlr2 and requires tlr-independent production of ifnbeta survival of lethal poxvirus infection in mice depends on tlr9, and therapeutic vaccination provides protection hepatitis c virus infection: molecular pathways to metabolic syndrome gut, inflammation and osteoporosis: basic and clinical concepts serum interleukin 6 concentrations in chronic hepatitis c patients before and after interferon-alpha treatment tumor necrosis factor alpha gene expression and the response to interferon in chronic hepatitis c dietary and genetic obesity promote liver inflammation and tumorigenesis by enhancing il-6 and tnf expression inflammatory mechanisms in obesity vaccinia virus induces apoptosis of infected macrophages isolation of a cdna clone derived from a blood-borne non-a, non-b viral hepatitis genome activation of the cki-cdk-rb-e2f pathway in full genome hepatitis c virusexpressing cells hepatitis c virus impairs p53 via persistent overexpression of 3beta-hydroxysterol delta24-reductase correlation between the infectivity of hepatitis c virus in vivo and its infectivity in vitro prior immunization with severe acute respiratory syndrome (sars)-associated coronavirus (sars-cov) nucleocapsid protein causes severe pneumonia in mice infected with sars-cov sars-cov spike protein-expressing recombinant vaccinia virus efficiently induces neutralizing antibodies in rabbits pre-immunized with vaccinia virus we thank dr. fukashi murai for supporting this study. we also thank dr. keiji tanaka for providing the mxcre mice, dr. shigeo koyasu for providing the gk1.5 (anti-cd4) and 53-6.72 (anti-cd8) monoclonal antibodies, and dr takashi tokuhisa for helpful discussions. key: cord-000742-0r4z1zea authors: vittecoq, marion; grandhomme, viviane; champagnon, jocelyn; guillemain, matthieu; crescenzo-chaigne, bernadette; renaud, françois; thomas, frédéric; gauthier-clerc, michel; van der werf, sylvie title: high influenza a virus infection rates in mallards bred for hunting in the camargue, south of france date: 2012-08-27 journal: plos one doi: 10.1371/journal.pone.0043974 sha: doc_id: 742 cord_uid: 0r4z1zea during the last decade, the role of wildlife in emerging pathogen transmission to domestic animals has often been pointed out. conversely, far less attention has been paid to pathogen transmission from domestic animals to wildlife. here, we focus on the case of game restocking, which implies the release of millions of animals worldwide each year. we conducted a 2-year study in the camargue (southern france) to investigate the influence of hand-reared mallard releases on avian influenza virus dynamics in surrounding wildlife. we sampled mallards (cloacal swabs) from several game duck facilities in 2009 and 2010 before their release. a very high (99%) infection rate caused by an h10n7 strain was detected in the game bird facility we sampled in 2009. we did not detect this strain in shot ducks we sampled, neither during the 2008/2009 nor the 2009/2010 hunting seasons. in 2010 infection rates ranged from 0 to 24% in hand-reared ducks. the 2009 h10n7 strain was fully sequenced. it results from multiple reassortment events between eurasian low pathogenic strains. interestingly, h10n7 strains had previously caused human infections in egypt and australia. the h10 and n7 segments we sequenced were clearly distinct from the australian ones but they belonged to the same large cluster as the egyptian ones. we did not observe any mutation linked to increased virulence, transmission to mammals, or antiviral resistance in the h10n7 strain we identified. our results indicate that the potential role of hand-reared mallards in influenza virus epizootics must be taken into account given the likely risk of viral exchange between game bird facilities and wild habitats, owing to duck rearing conditions. measures implemented to limit transmission from wildlife to domestic animals as well as measures to control transmission from domestic animals to wild ones need to be equally reinforced. during the last decade, awareness concerning the intimate links between human and animal health has rapidly increased in the context of disease emergence [1] . indeed, approximately 80% of the infectious diseases that recently emerged were zoonotic [2] . the role of wildlife in emerging pathogen transmission to humans and domestic animals has in many cases been pointed out [3±5] . conversely, pathogen transmission from domestic animals to wildlife has received far less attention, although the importance of this issue was often mentioned [6, 7] . indeed, contacts between wildlife and livestock or their environment sometimes result in wildlife diseases with conservation issues [8, 9] . besides, handreared animal releases into the wild for either conservation or exploitation purposes represent a particular case in which handreared individuals eventually share natural habitats with their wild congeners. in both cases such releases can dramatically influence disease dynamics in the surrounding wild animal populations [ 10± 14] . in the present study we focused on the case of game restocking, which implies the release of millions of individuals worldwide each year [15] . birds are the most frequently involved, with millions of individuals being released annually in europe only. for example more than 3 millions red-legged partridges are released annually in spain [15] , and ca. 1.4 million mallards are being so in france [16] . the camargue region, a complex network of wetlands situated in the rhone delta, is a major duck winter quarter [17] and a central place for wildfowl hunting in france [18] . hunting is also among the most important economic activities in the area, which is one of the reasons for the massive mallard releases in the camargue [19] . at least 30 000 hand-reared individuals are released annually in the region [20] . maximum mallard numbers in the wild are reached in september after the beginning of the hunting season, with 56 500 individuals on average over the last seven years (gauthier-clerc, unpubl. data), these numbers certainly include a mixing of wild and released mallards. given its central position on the flyway of many european migratory species, the camargue is also a potential hotspot for the introduction and transmission of bird-borne pathogens [21] . for this reason, avian influenza viruses (aiv) have been studied since 2004 in the area. these negative-sense single stranded rna viruses belonging to the orthomyxoviridae family are commonly characterized by the combination of their surface proteins: hemagglutinin (ha) and neuraminidase (na) [22, 23] . aivs are highly variable and undergo continuous genetic evolution via two mechanisms: i) accumulation of point mutations at each replication cycle, ii) reassortment involving gene segment exchanges that occur when a cell is co-infected by different viruses [24] . these mechanisms contribute to the emergence of new variants with the ability to transmit to new hosts and/or with epidemic or even pandemic potential. aquatic birds, particularly anseriforms (ducks, geese and swans) and charadriiforms (gulls, terns and shorebirds) constitute their major natural reservoir [23, 25] . the aiv circulating in wild birds are usually low pathogenic ones (lpaiv). lpaiv generally have little impact on their host [26, 27] , although some studies have reported a possible influence on migration capacities [28] . besides, when lpaiv of h5 or h7 subtypes are transmitted from wild birds to domestic ones reared in artificial environments, their virulence can evolve to high pathogenicity [29, 30] . highly pathogenic avian influenza viruses (hpaiv), such as hp h5n1 strains currently circulating in asia and africa, are still of great economic concern, notably due to the cost of preventive actions including vaccination and massive birds culling [31] . moreover, hpaiv infections represent a threat for human health since 603 hpaiv h5n1 human infections including 356 fatal cases have been reported worldwide since 2003 [32] . relatively high prevalence of aiv was regularly detected in the wintering mallard population of the camargue (e.g. 5.4% prevalence during the 2006±2007 hunting season) [33] . moreover a seasonal infection pattern was identified in mallards during autumn and winter, with higher infection rates in early fall [33] . mallards hence represent a focal study species in aiv research. indeed wild mallards are one of the main low pathogenic aiv natural reservoir host [34] , and have proven to be healthy carriers of some of the h5n1 hpaiv strains [35] . however, to our knowledge no study ever aimed at investigating the potential role of hand-reared mallards released for hunting in the epidemiology of aiv, despite the very large number of ducks being released in the wild annually. to clear this gap we conducted a 2-year study in the camargue to investigate the potential influence of hand-reared mallard releases on aiv dynamics in surrounding wildlife. we first hypothesized that, owing to high density rearing conditions and to their genetic uniformity, hand-reared mallards should be highly susceptible to aiv infections and could play an amplification role in aiv dynamics. this phenomenon has already been pointed out in red-legged partridge (alectoris rufa) reared for hunting in spain, where escherichia coli prevalence was much higher in hand-reared populations before their release than in the wild ones [36] . to test this assumption we collected cloacal swabs from mallards reared for hunting in several game bird facilities (gbf) in 2009 and 2010, and analysed these samples to measure aiv prevalence. second, we hypothesized that aiv exchange occurs between wild and hand-reared mallards, potentially leading either to the circulation of new strains in wild populations or to the amplification and dispersal of wild strains. indeed, no barrier prevents aiv exchange between wild birds and hand-reared ducks in the gbf since water flows exist between pens and ponds used by wild birds. moreover, as the gbf roofs are made of nets wild birds can deposit feces in the pens. finally, hand-reared ducks are in direct contact with wild ones after their release. to investigate these issues, we tested shot waterfowl before and after handreared mallards were sampled. noteworthy, genetic analyses suggest that 76% of hunted mallards in the camargue have a captive origin [37] . considering the low annual survival of released mallards (0.8±15.9% depending on the release site) [37] , the individuals we tested from the camargue hunting bags certainly included a large proportion of ducks released some months before being shot. these released ducks cannot be differentiated morphologically from the wild ones [38] . here we hence analyzed shot ducks as a whole since they are a representative sample of the mallard population wintering in the camargue, which is composed of individuals of both wild and captive origin that share habitats and can thus be considered as a single epidemiological unit. our third hypothesis was that any aiv strain potentially found in captive reared mallards might present genetic characteristics linked to its circulation in domestic populations. we therefore performed a molecular study of the identified strains in order to look for such characteristics, and in particular to test for the presence of mutations known to be associated with increased virulence, since it has been highlighted that artificial environments are favorable to the appearance of such mutations in birds [30] . we also searched for mutations linked with transmission to other species including humans, since some studies proved that they can be acquired during their transmission among birds [39] . thirdly, we tested for the presence of mutations conferring resistance to common antiviral drugs, since such resistance has recently been recorded in wild birds, notably in sweden [40] . lastly, full sequence analysis of some strains was performed, so as to get insight into their geographic origin through a phylogenetic study, and to determine their relatedness with strains, which have caused human infections in the past. the infection rates in the different gbf were determined by real-time rt-pcr targeting the conserved m gene of aiv (table 1) . a very high infection rate (99%) was observed in the single gbf sampled in 2009. in 2010 the infection rates ranged between 0 and 24%, being of 8% in the farm infected in 2009. initial subtyping, searching for h5, h7, h9, n1, and n7 by realtime rt-pcr performed on positive samples led to the identification of an h10n7 strain in the gbf in 2009 and further testing for h10 by real-time rt-pcr showed that the outbreak involved a single h10n7 virus (named h10n7 camargue below). the viral strains involved in the infections observed in 2010 were lpaiv. h5, h7, h9, h10, and n7 subtypes were searched among them but none was detected. in shot ducks, mean aiv infection rates were 15% and 5% during the 2008/2009 and the 2009/2010 hunting seasons, respectively ( table 2) . during these two periods the maximum monthly infection rates (respectively 20% and 16%) were observed in september (table 2 ). all the involved strains were lpaiv but an important proportion of these were of the h5 subtype (2008/ 2009:17%; 2009/2010:30%). no h7 or h10n7 subtype was detected in aiv found in shot ducks. phylogeny. the h10n7 camargue strain was isolated in embryonated chicken eggs and amplified. determination of the sequence of the whole genome was performed in order to gain insights into its phylogeny and molecular characteristics. the phylogenetic analysis of four h10n7 camargue isolates confirmed that they were very closely related to each other and formed a cluster (figures 1 and s1 ). analysis of each of the sequences of the 8 viral segments highlighted some shared characteristics (figures 1 and s1). first, the sequences of all segments were clearly distinct from those of lpaiv strains from the north-american lineage. second, none were closely related to highly pathogenic viruses (h5n1 or h7n7) that circulated in europe and asia. however, the pb2 segment was more closely related to the asian hp h5n1 cluster than to most of the european lpaiv strains we included in our phylogenetic analysis. this finding suggests an ancient asian origin of this segment. third, the sequences of all segments were closely related to eurasian strains. interestingly, the most closelyrelated eurasian strains differed between segments. as an example the lpaiv strain a/mute swan/hungary/5973/2007 (h7n7) was very closely related to the h10n7 camargue virus for the n7 segment, but less so for the m segment and belonged to a distant phylogenetic group for the pb2 segment. in the same way, the lpaiv strain a/mallard/netherlands/9/2005 (h7n7) was closely related to the h10n7 camargue virus for the m segment, whereas its pb2 segment belonged to a distinct phylogenetic group. african strains that we included in our analysis were part of eurasian clusters and clearly distinct from american strains. for one of them, a/pekin duck/south africa/ai1642/2009 (h10n7) the ns, n7 and h10 sequences were closely related to those of the h10n7 camargue virus but available sequences of two other segments (np and m) were clearly distinct. finally, it is important to note that based on the analysis of partial sequences of the h10 (data not shown) the h10n7 strains that caused human infections in australia (genebank accession numbers adg58106 and adg58107; ratnamohan,v.m. and dwyer, d.e. unpublished) are clearly distinct from the h10n7 camargue strain. on the contrary egyptian h10n7 strains that circulated in birds in 2004, i.e. at the time of the detection of two human infections [41] , are relatively closely related to the h10n7 camargue strain. however, this relatedness is difficult to interpret since the involved nodes are not strongly supported statistically (figure 1) . study of the extremities of the viral segments. we also determined the sequences of the extremities including the full noncoding sequences of all segments of the h10n7 camargue strain, which to our knowledge was not performed before for any h10n7 strain. the extremities of the n7 segment revealed two characteristics that confirmed the phylogenetic distinction between the h10n7 camargue strain and those of the american lineage. first, in the non-coding region of the 59 extremity, an a was found at position 1440, as commonly seen in eurasian strains, while strains of the american lineage possess a t at this position. second, at position 145±147 of the 39 extremity that belongs to the coding region the camargue strain, like other eurasian ones, has a supplementary amino acid compared to strains of the american lineage. molecular characterization. the molecular analysis of the h10n7 camargue strain is detailed in table 3 . the focus strain is a low pathogenic one since the ha segment possesses a monobasic cleavage site. ha, na and pb2 sequences all presented some typical avian characteristics (see table 3 ). no known determinants of adaptation to mammals (humans or mice) were detected. in addition, we did not identify any mutation known to be linked with increased virulence in mammals. yet, we detected the v149a mutation in pb2, which is associated with high virulence in chickens. however, 149a was also observed in low pathogenic h7 strains sampled from wild and domestic ducks in china. this suggests that this mutation alone does not increase the virulence in ducks [42] . furthermore, no mutation was detected in the h10n7 camargue strain at positions known to be linked with reduced susceptibility or resistance to neuraminidase inhibitors or m2blockers. this study confirms our first hypothesis that hand-reared mallards are highly sensitive to aiv infections. indeed, we detected high infection rates in the gbf we sampled in 2009 (99%) and in 2010 (up to 24%), whereas the prevalence usually observed in duck populations very rarely exceeds 20% in the wild [33, 43, 44] . this shows that massive outbreaks can occasionally affect gbf, potentially impacting on aiv dynamics in the wild populations with which hand-reared mallards are in contact before and after their release into the wild. this pattern has already been highlighted in spain where rabbit (oryctolagus cuniculus) restocking caused sarcoptic mange dispersion in the wild rabbit population, leading to massive decrease in numbers [11] . the possible outcomes of gbf infection onto wild duck populations should here depend on the origin of the strain involved. indeed, low pathogenic aiv strains that naturally circulate in wild ducks have little impact on their health [26] . our data did not permit to determine the origin of the strain we identified in 2009. indeed, we did not detect the h10n7 camargue strain among the samples collected on shot mallards, neither during the hunting season that preceded the release of the infected individuals, nor during the following one. our sample size for hunted mallards was reasonably large (n = 299 in 2008/2009 and 555 in 2009/2010). moreover, the sites where we sampled hand-reared and shot mallards were close to each other (see figure 2 ; for instance one of the hunting estates we sampled was within 4 km of the gbf where we detected an aiv outbreak). nevertheless, our sampling effort was limited in time to the hunting seasons: we do not have any data from february to august 2009. we therefore cannot exclude that the h10n7 strain originated from wild birds. population density during the breeding season is low in wild mallard [45] , and thus, even if the h10n7 camargue virus originated from wild birds, hand-reared mallards would then have played an amplifying role. in addition, commercial trade of mallard chicks is common and most of the juveniles sampled in this study traveled 600 km from the producer when one-day old. it is also possible that the h10n7 camargue strain originated from the birthplace of the chicks. if so, domestic ducks would have both played an amplifying role and potentially a dispersal one, since they could have spread a new strain in wild duck populations. the lack of samples during the weeks that directly followed the release of the positive mallards (because the hunting season had not yet started) could explain why we did not detect the h10n7 camargue virus in wild individuals. furthermore, the lack of large-scale dispersal of that h10n7 strain could be due to different parameters. first, we sampled the hand-reared ducks of the gbf1 21 days before their release. it is possible that only few birds were still excreting viruses when released into the wild, since excretion time in birds can be as short as 2 days (although it can also last up to 30 days in some cases) [46±48]. second, a parallel capturerecapture demographic study ran in the camargue has shown that hand-reared mallards exhibit low monthly survival before the hunting season (only 44% survive from release until the onset of the hunting period on average) [49] . moreover, their dispersal capacities appear to be very low [49] . the same pattern has been observed for red-legged partridges released in spain [13] . although intestinal parasites were much more numerous in the individuals living in the hunting estates where domestic birds were released, this was interestingly not observed in the neighboring estates [13] . it is possible that the poor survival and low dispersal of the hand-reared mallards after release limited the potential spread of the h10n7 camargue virus within the mallard population in the wild. besides, we did not detect any h5 subtype circulating in the gbf whereas 23% of all the aiv we isolated from samples collected on shot mallards in the camargue belonged to this subtype. again we could not highlight any aiv exchange between ducks living in captivity and in the wild. yet, hand-reared ducks are exposed to wild bird feces and gbf are connected to wild bird habitats through common use of water. thus, aiv exchanges are very likely to exist. our results highlighted that: i) gbf represent an epidemiological compartment into which important aiv outbreaks can occur; ii) a significant proportion of mallards wintering in the camargue are infected by lpaiv, including h5 strains that are known to be able to evolve to hpaiv in domestic birds [30] . knowing that rearing conditions in the gbf may favor aiv exchanges between wild and hand-reared mallards, these findings outline the need for influenza surveillance in gbf to prevent hpaiv outbreaks in both wild and domestic birds in the region. we used the phylogenetical analysis of whole-genome sequence of the h10n7 camargue strain as a supplementary tool to get insights into its evolutionary and geographical origins. the four isolates studied clearly belonged to a cluster that did not include other strains. the infections we detected were therefore certainly due to a single introduction event. all the viral segments of the h10n7 camargue strain belong to the eurasiatic lineage also including african strains. the sequences of the extremities of the n7 segment, that exhibit features characteristic of the eurasian lineage viruses, confirmed this finding. the eurasian lineage is clearly distinct from the american lineage and our observations further support the fact that inter-hemispheric aiv exchanges are rare. the camargue strain was also distinct from the h10n7 viruses that previously caused human infections in australia, but belonged to the same large cluster as the egyptian h10n7 strains that circulated in birds when h10n7 human infections occurred in this country. these egyptian strains did not cause severe disease. nevertheless, this information raises concern about a possible transmission to humans of strains such as the camargue one, in particular to the gbf owners and hunters who live in close contact with ducks. interestingly, the strains that are genetically closest relatives differ for each segment of the h10n7 camargue strain. the camargue strain thus seems to result from multiple reassortments between different eurasiatic strains, a common phenomenon which has been illustrated by many studies [50, 51] . regrettably, we could not determine if the h10n7 camargue strain was more closely related to strains observed in the wild or in gbf. indeed, the data associated with viral sequences from aiv infected mallards in genbank are often too scarce to determine if the sample was taken on a wild or a domestic individual. this underlines the need for more detailed information on aiv sequences included in common databases. the molecular analysis of the h10n7 camargue strain allowed us to question whether it may exhibit some characteristics related to its circulation in an artificial environment, namely some mutations linked to increased virulence, resistance to antiviral drugs or transmission to humans. our results do not support this assumption. the sequences of the studied segments presented typical avian characteristics. no mutation known to contribute to transmission to humans was detected. moreover, the camargue strain is a low pathogenic one like all viruses isolated during our research program in the camargue since 2005 [33, 52] . no mutation conferring antiviral resistance was detected. this analysis therefore failed to highlight any risk factor linked to this particular strain. yet, one must keep in mind that we only looked for mutations previously described in the literature, while many others may remain undiscovered so far. it is also important to stress that most experimental studies focusing on mutations modifying host specificity and virulence are carried out on chicken, although it has been demonstrated that phenotypic outcomes of a given mutation can greatly differ from one host species to another [42] . in conclusion, our results point out the important role that could be played by hand-reared ducks released for hunting in aiv dynamics. as we did not detect similar aiv strains in shot and hand-reared ducks we could not prove that aiv exchanges exist between these two epidemiological compartments. yet, due to rearing conditions in gbf, aiv exchange risks seem to be high enough to urge for sanitary control of hand-reared animals prior to their release into the wild, which appears to be highly insufficient so far. such surveillance would also prevent hpaiv circulation that may arise from the evolution in gbf of h5 lpaiv that proved to be commonly infecting free-living ducks in the camargue. the world organization for animal health (oie) stresses that surveillance of aiv infection should be applied to all domesticated birds including those used``for restocking supplies of game'' [53] , but control measures generally appear to be poor in game bird rearing estates. this surveillance gap has recently been highlighted in the usa, where game bird holders reported very variable sanitary practices [54] . the problem appears similar in europe, including france. additionally, according to french law, game birds should be ringed to allow for the differentiation of wild and released individuals [55] . most of gbf owners ignore this obligation. if released birds were clearly identified the specific role they may have in aiv dynamics could be addressed both before and after their release. our study illustrates the reality of the epidemiological consequences that can result from surveillance gaps and the knowledge that could be gained if released individuals were recognizable. sharing knowledge and strictly controlling viral exchanges between wild birds and all kinds of domestic ones represent major steps to anticipate and face hpaiv epizootics. this study has been submitted for approval and the results reviewed by the scientific council of the tour du valat foundation. birds were handled, ringed and sampled under the supervision of a veterinarian (michel gauthier-clerc) and a registered duck ringer of the « museum national d'histoire naturelle » of paris (matthieu guillemain). all procedures were carried out in accordance with the permit delivered by the prefect of paris to the « office national de la chasse et de la faune sauvage » (permit n 2009±014). (table 1) . samples were stored in 3 ml of universal transport medium (biolys kit) and frozen at 280uc until molecular analysis was performed. rna isolation from 140 ml of each sample was carried out using a macherey-nagel nucleospin 96 virus system with rna elution into a final volume of 60 ml. influenza a virus was detected by real-time rt-pcr targeting the conserved matrix as describe previously [56] . all real-time rt-pcr assays were performed on a lightcycler 480 (roche diagnostic) in a final volume of 10 ml with 2.5 ml rna, 0.5 mm of each primer, 0.2 mm probe and 0.4 ml enzyme mix using a superscript iii platinum one-step quantitative rt-pcr system (invitrogen). the reaction was carried out with the following temperature profile: 15 min at 45uc, 3 min at 95 c, 50 cycles of 10 s at 95uc, 10 s at 55uc, 20 s at 72uc, and finally 30 s at 40uc. positive samples were further tested for h5, h7, h9, n1 and n7 subtypes using the same real-time rt-pcr technique (primers available upon request). for 10 rt-pcr positive samples, 200 ml of specimen was inoculated in the allantoic cavity of 10-days-old embryonated hen's eggs. the allantoic fluid was harvested after 72 h at 37uc and influenza a virus was detected by hemagglutination assay with hen erythrocytes. viral rna was purified from allantoic fluid using a qiaamp viral rna mini kit (qiagen) following manufacturer's instructions. ha subtype of virus isolates was determined by rt-pcr and sequencing of the ha 0 cleavage site region using a universal set of primers as described previously [57] . ha sequence was analyzed with the basic local alignment search tool available from ncbi (data not shown) and confirmed by sequencing of the whole ha gene using a set of h10-specific primers (primer sequences available upon request). the na subtype was deduced from this analysis and confirmed by rt-pcr and sequencing of 172 nt of the na using a set of h10specific primers (primer sequences available upon request). amplification of viral rna extracted from 4 virus isolates was carried out using a superscript platinum one-step rt-pcr system (invitrogen) and primers specific of each segment. sequencing was done using a big dye terminator v1.1 kit and a sequencer abi dna analyzer 3730xl (applied biosystems). sequences of all primers are available upon request. sequences of the whole viral genome were analyzed with clc main workbench 5.6.1. we performed alignments for the 8 segments using sequences available from the influenza sequence database with clc main workbench 5.6.1. phylogenetic trees were constructed using maximum parsimony (mp) methods with the dnapars program of the phylip 3.68 package and the maximum likelihood (ml) with the software phyml 2.4.4. evolutionary model was selected using model generator 0.85 [58] . nodal supports were assessed with 100 bootstrap replicates generated for each method. determination of 39 and 59 end non coding sequences of the 8 segments of the h10n7 virus viral genomic rna was extracted using the qiaamp viral rna mini kit (qiagen) from 140 ml of allantoic fluid according to the manufacturer's recommendations. the rna was eluted in 60 ml of rnase-free water and the 39 and 59 nc regions were amplified as previously described [59] using an anchored (dt) 14 oligonucleotide and primers specific for the coding sequences of both segments for reverse transcription and amplification. after purification, the pcr products were sequenced with internal oligonucleotides using a big dye terminator sequencing kit and an automated sequencer (perkin elmer). all sequences of the primers are available from the authors upon request. one-step real-time rt-pcr assays were developed to be specific of the virus identified. all influenza a positive samples detected between august 2008 and july 2010 on wild and domestic mallards were tested for h10 and n7 subtypes respectively. detection was performed in the same conditions as described above except annealing temperature was decreased 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adamantane derivatives we are grateful to game bird facility owners, hunting managers and hunters who authorized us to collect samples from mallards. we also thank two anonymous referees for their valuable comments. key: cord-000642-mkwpuav6 authors: moreira, rebeca; balseiro, pablo; planas, josep v.; fuste, berta; beltran, sergi; novoa, beatriz; figueras, antonio title: transcriptomics of in vitro immune-stimulated hemocytes from the manila clam ruditapes philippinarum using high-throughput sequencing date: 2012-04-19 journal: plos one doi: 10.1371/journal.pone.0035009 sha: doc_id: 642 cord_uid: mkwpuav6 background: the manila clam (ruditapes philippinarum) is a worldwide cultured bivalve species with important commercial value. diseases affecting this species can result in large economic losses. because knowledge of the molecular mechanisms of the immune response in bivalves, especially clams, is scarce and fragmentary, we sequenced rna from immune-stimulated r. philippinarum hemocytes by 454-pyrosequencing to identify genes involved in their immune defense against infectious diseases. methodology and principal findings: high-throughput deep sequencing of r. philippinarum using 454 pyrosequencing technology yielded 974,976 high-quality reads with an average read length of 250 bp. the reads were assembled into 51,265 contigs and the 44.7% of the translated nucleotide sequences into protein were annotated successfully. the 35 most frequently found contigs included a large number of immune-related genes, and a more detailed analysis showed the presence of putative members of several immune pathways and processes like the apoptosis, the toll like signaling pathway and the complement cascade. we have found sequences from molecules never described in bivalves before, especially in the complement pathway where almost all the components are present. conclusions: this study represents the first transcriptome analysis using 454-pyrosequencing conducted on r. philippinarum focused on its immune system. our results will provide a rich source of data to discover and identify new genes, which will serve as a basis for microarray construction and the study of gene expression as well as for the identification of genetic markers. the discovery of new immune sequences was very productive and resulted in a large variety of contigs that may play a role in the defense mechanisms of ruditapes philippinarum. the manila clam (ruditapes philippinarum) is a cultured bivalve species with important commercial value in europe and asia, and its culture has expanded in recent years. nevertheless, diseases produced by a wide range of microorganisms, from viruses to metazoan parasites, can result in large economical losses. among clam diseases, the majority of pathologies are associated with the vibrio and perkinsus genera [1] [2] [3] . although molluscs lack a specific immune system, the innate response involving circulating hemocytes and a large variety of molecular effectors seems to be an efficient defense method to respond to external aggressions by detecting the molecular signatures of infection [4] [5] [6] [7] [8] ; however, not many immune pathways have been identified in these animals. although knowledge of bivalve immune-related genes has increased in the last few years, the available information is still scarce and fragmentary. most of the data concern mussels and eastern and pacific oysters [9] [10] [11] [12] [13] [14] , and very limited information is available on the expressed immune genes of r. philippinarum. recently, the expression of 13 immune-related genes of ruditapes philippinarum and ruditapes decussatus were characterized in response to a vibrio alginolyticus challenge [15] . also, a recent 454 pyrosequencing study was carried out by milan et al. [16] , who sequenced two normalized cdna libraries representing a mixture of adult tissues and larvae from r. philippinarum. even more recently ghiselli et al. [17] , have de novo assembled the r. philippinarum gonad transcriptome with the illumina technology. moreover, a few transcripts encoded by genes putatively involved in the clam immune response against perkinsus olseni have been reported by cdna library sequencing [18] . currently (19/12/ 2011) , there are 5,662 ests belonging to r. philippinarum in the genbank database. the european marine genomics network has increased the number of ests for marine mollusc species particularly for ecologically and commercially important groups that are less studied, such as mussels and clams [19] . unfortunately, most of the available resources are not annotated or well described, limiting the identification of important genes and genetic markers for future aquaculture applications. the use of 454-pyrosequencing is a fast and efficient approach for gene discovery and enrichment of transcriptomes in non-model organisms [20] . this relatively low-cost technology facilitates the rapid production of a large volume of data, which is its main advantage over conventional sequencing methods [21] . in the present work, we undertook an important effort to significantly increase the number of r. philippinarum ests in the public databases. specially, the aim of this work was to discover new immune-related genes using pyrosequencing on the 454 gs flx (roche-454 life sciences) platform with the titanium reagents. to achieve this goal, we sequenced the transcriptome of r. philippinarum hemocytes previously stimulated with different pathogen-associated molecular patterns (pamps) to obtain the greatest number of immune-related transcripts as possible. the raw data are accessible in the ncbi short read archive (accession number: sra046855.1). the r. philippinarum normalized cdna library was sequenced with 454 gs flx technology as shown in figure 1 . sequencing and assembly statistics are summarized in table 1 . briefly, a total of 975,190 raw nucleotide reads averaging 284.1 bp in length were obtained. of these, 974,976 exceeded our minimum quality standards and were used in the mira assembly. a total of 842,917 quality reads were assembled into 51,265 contigs, corresponding to 29.9 megabases (mb). the length of the contigs varied from 40 to 5565 bp, with an average length of 582.4 bp and an average coverage of 5.7 reads. singletons were discarded, resulting in 37,093 contigs formed by at least 2 ests, and 26,675 of these contigs were longer than 500 bp. clustering the contigs resulted in 1,689 clusters with more than one contig. the distribution of contig length and the number of ests per contig, as well as the contig distribution by cluster are all shown in figure 2 . even though the knowledge of expressed genes in bivalves has increased in the last few years, it is still limited. indeed, only 41,598 nucleotide sequences, 362,149 ests, 24,139 proteins and 704 genes from the class bivalvia have been deposited in the genbank public database (19/12/11) , and the top entries are for the mytilus and crassostrea genera. for ruditapes philippinarum, these numbers are reduced to 5,662 ests, 612 proteins and 12 genes. this evidences the lack of information which prompted the recent efforts to increase the number of annotated sequences of bivalves in the databases. for non-model species, functional and comparative genomics is possible after obtaining good est databases. these studies seem to be the best resource for deciphering the putative function of novel genes, which would otherwise remain ''unknown''. ncbi swissprot, ncbi metazoan refseq, the ncbi nonredundant and the uniprotkb/trembl protein databases were chosen to annotate the contigs that were at least 100 bp long (49, 847) . the percentage of contigs annotated with a cut off evalue of 10e-3 was 44.7%. contig sequences and annotations are included in table s1 . of these contigs, 3.26% matched sequences from bivalve species and the remaining matched to non-bivalvia mollusc classes (4.13%), other animals (81.38%), plants (2.58%), fungi (1.78%), protozoa (1.50%), bacteria (4.95%), archaea (0.20%), viruses (0.21%) and undefined sequences (0.01%). as shown in figure 3a , the species with the most sequence matches was homo sapiens with 3,106 occurrences. the first mollusc in the top 35 list was lymnaea stagnalis at position 11. the first bivalve, meretrix lusoria, appeared at position 17. r. philippinarum was at position 25 with 124 occurrences. notably, a high percentage of the sequences had homology with chordates, arthropods and gastropods ( figure 3b and c), and only 343 contigs matched with sequences from the veneroida order ( figure 3d ). these values can be explained by the higher representation of those groups in the databases as compared to bivalves and the quality of the annotation in the databases, which has been reported in another bivalve transcriptomic study [22] . the data shown highlight, once again, the necessity of enriching the databases with bivalve sequences. a detailed classification of predicted protein function is shown for the top 35 blastx hits ( figure 4a ). the list is headed by actin with 903 occurrences, followed by ferritin, an angiopoietin-like protein and lysozyme. an abundance of proteins directly involved in the immune response was predicted for this 454 run; ferritin, lysozyme, c1q domain containing protein, galectin-3 and hemagglutinin/amebocyte aggregation factor precursor are immune-related proteins present on the top 35 list. ferritin has an important role in the immune response. it captures circulating iron to overcome an infection and also functions as a proinflammatory cytokine via the iron-independent nuclear factor kappa b (nf-kb) pathway [23] . lysozyme is a key protein in the innate immune responses of invertebrates against gram-negative bacterial infections and could also have antifungal properties. in addition, it provides nutrition through its digestive properties as it is a hydrolytic protein that can break the glycosidic union of the peptidoglycans of the bacteria cell wall [24] . the c1q domain containing proteins are a family of proteins that form part of the complement system. the c1q superfamily members have been found to be involved in pathogen recognition, inflammation, apoptosis, autoimmunity and cell differentiation. in fact, c1q can be produced in response to infection and it can promote cell survival through the nf-kb pathway [25] . galectin-3 is a central regulator of acute and chronic inflammatory responses through its effects on cell activation, cell migration, and the regulation of apoptosis in immune cells [26] . the hemagglutinin/amebocyte aggregation factor is a single chain polypeptide involved in blood coagulation and adhesion processes such as self-nonself recognition, agglutination and aggregation processes. the hemagglutinin/ amebocyte aggregation factor and lectins play important roles in defense, specifically in the recognition and destruction of invading microorganisms [27] . other proteins that are not specifically related to the immune response but could play a role in defense mechanisms include the following: angiopoietin-like proteins, apolipoprotein d and the integral membrane protein 2b. in other animals, angiopoietin-like proteins (angptl) potently regulate angiogenesis, but a subset also function in energy metabolism. specifically, angptl2, the most represented angptl, promotes vascular inflammation rather than angiogenesis in skin and adipose tissues. inflammation occurs via the a5b1 integrin/rac1/nf-kb pathway, which is evidenced by an increase in leukocyte infiltration, blood vessel permeability and the expression of inflammatory cytokines (tumor necrosis factor-a, interleukin-6 and interleukin-1b) [28] . apolipoprotein d (apod) has been associated with inflammation. pathological and stressful situations involving inflammation or growth arrest have the capacity to increase its expression. this effect seems to be triggered by lps, interleukin-1, interleukin-6 and glucocorticoids and is likely mediated by the nf-kb pathway, as there are several conserved nf-kb binding sites in the apod promoter (apre-3 and ap-1 binding sites are also present). the highest affinity ligand for apod is arachidonic acid, which apod traps when it is released from the cellular membrane after inflammatory stimuli and, thus, prevents its subsequent conversion in pro-inflammatory eicosanoids. within the cell, apod could modulate signal transduction pathways and nuclear processes such as transcription activation, cell cycling and apoptosis. in summary, apod induction is specific to ongoing cellular stress and could be part of the protective components of mild inflammation [29] [30] [31] . finally, the short form of the integral membrane protein 2b (itm2bs) can induce apoptosis via a caspase-dependent mitochondrial pathway [32] . to avoid redundancy, the longest contig of each cluster was used for gene ontology terms assignment. a total of 23.05% of the representative clusters matched with at least one go term. concerning cellular components ( figure 4b ), the highest percentage of go terms were in the groups of cell and cell part with 25.9% in each; organelle and organelle part represented 19.67% and 11.38%, respectively. within the molecular function classification ( figure 4c ), the most represented group was binding with 49.25% of the terms, which was followed by catalytic activity (29.12%) and structural molecular activity (4.60%). with regard to biological process ( figure 4d ), cellular and metabolic processes were the highest represented groups with 16.78% and 12.43% of the terms, respectively, which was followed by biological regulation (10.18%). similarities between the r. philippinarum transcriptome and another four bivalve species sequences were analyzed by comparative genomics (crassostrea gigas of the family ostreidae, bathymodiolus azoricus and mytilus galloprovincialis of the family mytilidae and laternula elliptica of the family laternulidae). this analysis could identify specific transcripts that are conserved in these five species. a venn diagram was constructed using unique sequences from these databases according to the gene identifier (gi id number) of each sequence in its respective database: 207,764 from c. gigas, 76,055 from b. azoricus, 121,318 from m. galloprovincialis and 1,034,379 from l. elliptica. c. gigas was chosen because is the most represented bivalve species in the public databases. the other three species are bivalves that have been studied in transcriptomic assays. figure 5 shows that of the total 29,679 clusters, 72% were found exclusively in the r. philippinarum group, while only 7.59% shared significant similarity with all five species. the number of coincidences among other groups was very low (4.14% to 0.31% of sequences), suggesting that 21,454 new sequences were discovered within the bivalve group. the percentage of new sequences is very high compared to previous transcriptomic studies [33] [34] , in which the fraction of new transcripts was approximately 45%. one possible explanation for this discrepancy is the low number of nucleotide and est sequences currently available in public databases for r. philippinarum, but these transcripts could also be regions in which homology is not reached, such as 59 and 39 untranslated regions or genes with a high mutation rate. on the other hand, a comparison between our 454 results and the milan et al. [16] transcriptome using a blastn approach is summarized in table 2 immune-related sequences r. philippinarum hemocytes were subjected to immune stimulation using several different pamps to enrich the est collection with immune-related sequences. the objective was to obtain a more complete view of clam responses to pathogens. a keyword list and go immune-related terms were used to find proteins putatively involved in the immune system. after this selection step, we found that more than 10% of the proteins predicted from the contig sequences had a possible immune function. some sequences were found to be clustered in common, well-recognized immune pathways, such as the complement, apoptosis and toll-like receptors pathways, indicating conserved ancient mechanisms in bivalves ( figures 6, 7, 8 ). the complement system is composed of over 30 plasma proteins that collaborate to distinguish and eliminate pathogens. c3 is the central component in this system. in vertebrates, it is proteolytically activated by a c3 convertase through both the classic, lectininduced and alternative routes [35] . although the complement pathway has not been extensively described in bivalves, there is evidence that supports the presence of this defense mechanism. ests with homology to the c1q domain have been detected in the american oyster, c. virginica [36] , the tropical clam codakia orbicularis [37] , the zhikong scallop chlamys farreri [38] and the mussel m. galloprovincialis [39] [40] . more recently, a novel c1q adiponectin-like, a c3 and a factor b-like proteins have been identified in the carpet shell clam r. decussatus [41] [42] . these data support the putative presence of the complement system in bivalves. our pyrosequencing results, using the blastx similarity approach, showed that the complement pathway in r. philippinarum was almost complete as compared to the kegg reference pathway ( figure 6 ). only the complement components c1r, c1s, c6, c7 and c8 were not detected. i. lectins. lectins are a family of carbohydrate-recognition proteins that play crucial self-and non-self-recognition roles in innate immunity and can be found in soluble or membraneassociated forms. they may initiate effector mechanisms against pathogens, such as agglutination, immobilization and complement -mediated opsonization and lysis [43] . several types of lectins have been cloned or purified from the manila clam, r. philippinarum [44] [45] [46] , and their function and expression were also studied [18, 47] . also, a manila clam tandemrepeat galectin, which is induced upon infection with perkinsus olseni, has been characterized [46] . lectin sequences have been found in the stimulated hemocytes studied in our work: 23 of the contigs are homologous to c-type lectins (calcium-dependent carbohydrate-binding lectins that have characteristic carbohydrate-recognition domains), 115 are homologous to galectins (characterized by a conserved sequence motif in their carbohydrate recognition domain and a specific affinity for bgalactosides), 4 contigs have homology with ficolin a and b (a group of oligomeric lectins with subunits consisting of both collagen-like and fibrinogen-like domains) and 34 contigs have homology with other groups of lectins such as lactose-, mannoseor sialic acid-binding lectins. ii. b-glucan recognition proteins. b-glucan recognition proteins are involved in the recognition of invading fungal organisms. they bind specifically to b-1,3-glucan stimulating short-term immune responses. although these receptors have been partially sequenced in several bivalves, there is only one complete description of them in the scallop chlamys farreri [48] . two contigs with homology to the beta-1,3-glucan-binding protein were found in our study. iii. peptidoglycan recognition proteins. peptidoglycan recognition proteins (pgrps) specifically bind peptidoglycans, which is a major component of the bacterial cell wall. this family of proteins influences host-pathogen interactions through their pro-and anti-inflammatory properties that are independent of their hydrolytic and antibacterial activities. in bivalves, they were first identified in the scallops c. farreri and a. irradians [49, 50] and the pacific oyster c. gigas, and from the latter four different types of pgrps were identified [51] . peptidoglycan-recognition proteins and a peptidoglycan-binding domain containing protein have been found for the first time in r. philippinarum in our results and were present 4 and 1 times, respectively. iv. toll-like receptors. toll-like receptors (tlrs) are an ancient family of pattern recognition receptors that play key roles in detecting non-self substances and activating the immune system. the unique bivalve tlr was identified and characterized in the zhikong scallop, c. farreri [52] . tlr 2, 6 and 13 were present among the pyrosequencing results. tlr2 and tlr6 form a heterodimer, which senses and recognizes various components from bacteria, mycoplasma, fungi and viruses [53] . tlr13 is a novel and poorly characterized member of the toll-like receptor family. although the exact role of tlr13 is currently unknown, phylogenic analysis indicates that tlr13 is a member of the tlr11 subfamily [54] suggesting that it could recognize urinary pathogenic e. coli [55] . it has been demonstrated that tlr13 colocalizes and interacts with unc93b1, a molecule located in the endoplasmic reticulum, which strongly suggests that tlr13 might be found inside cells and might play a role in recognizing viral infections [56] . figure 7 summarizes the tlr signaling pathway with the corresponding molecules found in the r. philippinarum transcriptome. pathogen proteases are important virulence factors that facilitate infection, diminish the activity of lysozymes and quench the agglutination capacity of hemocytes. because protease inhibitors play important roles in invertebrate immunity by protecting hosts through the direct inactivation of pathogen proteases, many bivalves have developed protease inhibitors to regulate the activities of pathogen proteases [1] . some genes encoding protease inhibitors were identified in c. gigas [57] , a. irradians [58] , c. farreri [59] and c. virginica; in the latter a novel family of serine protease inhibitors was also characterized [60] [61] [62] . a total of 23 contigs with homology to serine, cystein, kunitzand kazal-type protease inhibitors and metalloprotease inhibitors were found among our results. lysozyme was one of the most represented groups of immune genes in this transcriptome study with 208 contigs present. it is an antibacterial molecule present in numerous animals including bivalves. although lysozyme activity was first reported in molluscs over 30 years ago, complete sequences were published only recently including those of r. philippinarum [24] . antimicrobial peptides (amps) are small, gene-encoded, cationic peptides that constitute important innate immune effectors from organisms spanning most of the phylogenetic spectrum. amps alter the permeability of the pathogen membrane and cause cellular lysis [63] . in bivalves, they were first purified from mussel hemocyte granules [64, 65] . in mussels, the amp myticin c was found to have a high polymorphic variability as well as chemotactic and immunoregulatory roles [66, 67] . in clams, two amps with similarity to mussel myticin and mytilin [68] and a big defensin [69] are known. we were able to detect 36 contigs with homology to different defensins: defensin-1 (american oyster defensin), defensin mgd-1 (mediterranean mussel defensin) and the big defensin previously mentioned. four contigs were similar to an unpublished defensin sequence from venerupis ( = ruditapes) philippinarum. the primary role of heat shock proteins (hsps) is to function as molecular chaperones. their up-regulation also represents an important mechanism in the stress response [70] , and their activity is closely linked to the innate immune system. hsps mediate the mitochondrial apoptosis pathway and affect the regulation of nf-kb [71] . hsps are well studied in bivalves. for r. philippinarum, several assays have been developed to better understand the hsps profile in response to heavy metals and pathogen stresses [72] [73] [74] . the most important and well-studied groups of hsps were present in our r. philippinarum transcriptome (hsp27, hsp40/ dnaj, hsp70 and hsp90), but other, less common hsps were also represented (hsp10, hsp22, hsp83 and some members from the hsp90 family). recently, several genes related to the inflammatory response against lps stimulation have been detected in bivalves. such is the case of the lps-induced tnf-a factor (litaf), which is a novel transcription factor that critically regulates the expression of tnfa and various inflammatory cytokines in response to lps stimulation. it has been described in three bivalve species: pinctada fucata [75] , c. gigas [76] and c. farreri [77] . other tnf-related genes have been identified in the zhikong scallop, such as a tnfr homologue [78] and a tumor necrosis factor receptor-associated factor 6 (traf6), which is a key signaling adaptor molecule common to the tnfr superfamily and to the il-1r/tlr family [79] . figure 7 shows that several components of the tlr signaling pathway that are present in our transcriptomic sequences (myd88, irak4, traf-3 and -6, tram, btk, rac-1, pi3k, akt, btk and tank). a total of 1,918 contigs, 8.43% of those annotated, had homology with the main groups of putatively pathogenic organisms such as viruses (47 hits), bacteria (1,126 hits), protozoa (341 hits) and fungi (404 hits). figure 9 displays the taxonomic classification of these sequences and table 3 summarizes a list of the known bivalve pathogens found in our results. bacteria constitute the main group found among the sequences not belonging to the clam. as filter-feeding animals, bivalves can concentrate a large amount of bacteria and it could be one of their sources of food [24] . because vibrio spp. are ubiquitous in aquatic ecosystems, it was expected that the vibrionales order, with 141 hits, would be the most predominant. several species of the vibrio genus are among the main causes of disease in bivalves specifically causing bacillary necrosis in larval stages [80] . is noticeable that sequences belonging to the causative agent of brown ring disease in adults of manila clam, vibrio tapetis, have not been found. perkinsus marinus, with 2 matches, is the only bivalve pathogen found within the protozoa (alveolata) group. perkinsosis is produced by species from the genus perkinsus. both p. marinus and p. olseni have been associated with mortalities in populations of various groups of molluscs around the world and are catalogued as notifiable pathogens by the oie. viruses were the least represented among pathogens. the baculoviridae family was the most predominant with 21 matches, but the corresponding sequences were inhibitors of apoptosis (iaps) [81] that could also be part of the clam's transcriptome. five viral families were found in our transcriptome study: iridoviridae, herpesviridae, malacoherpesviridae, picornaviridae and retroviridae. a well-known bivalve pathogen was also identified, the ostreid herpesvirus 1, which has been previously been found to infect clams [82] . fungi had 404 matches in our results. it is known that bivalves are sensitive to fungal diseases, which can degrade the shell or affect the larval bivalve stages [83, 84] . this study represents the first r. philippinarum transcriptome analysis focused on its immune system using a 454-pyrosequencing approach and complements the recent pyrosequencing assay carried out by milan et al. [16] . the discovery of new immune sequences was effective, resulting in an enormous variety of contigs corresponding to molecules that could play a role in the defense mechanisms. more than 10% of our results had relationship with immunity. this new resource is now gathered in the ncbi short read archive with the accession number: sra046855.1. our results will provide a rich source of data to discover and identify new genes, which will serve as a basis for microarray construction and gene expression studies as well as for the identification of genetic markers for various applications including the selection of families in the aquaculture sector. we have found sequences from molecules never described in bivalves before like c2, c4, c5, c9, aif, bax, akt, tlr6 and tlr13, among others. as a part of this work, three immune pathways in r. philippinarum have been characterized, the apoptosis, the toll like signaling pathway and the complement cascade, which could help us to better understand the resistance mechanisms of this economically important aquaculture clam species. animal sampling and in vitro stimulation of hemocytes r. philippinarum clams were obtained from a commercial shellfish farm (vigo, galicia, spain). clams were maintained in open circuit filtered sea water tanks at 15uc with aeration and were fed a total of 100 clams were notched in the shell in the area adjacent to the anterior adductor muscle. a sample of 500 ul of hemolymph was withdrawn from the adductor muscle of each clam with an insulin syringe, pooled and then distributed in 6-well plates, 7 ml per well, in a total of 7 wells, one for each treatment. hemocytes were allowed to settle to the base of the wells for 30 min at 15uc in the darkness. then, the hemocytes were stimulated with 50 mg/ml of polyinosinic:polycytidylic acid (poly i:c), peptidoglycans, ã�-glucan, vibrio anguillarum dna (cpg), lipopolysaccharide (lps), lipoteichoic acid (lta) or 1610 6 ufc/ml of heat-inactivated vibrio anguillarum (one stimulus per well) for 3 h at 15uc. all stimuli were purchased from sigma. pyrosequencing. after stimulation, hemolymph was centrifuged at 1700 g at 4uc for 5 minutes, the pellet was resuspended in 1 ml of trizol (invitrogen) and rna was extracted following the manufacturer's protocol. after rna extraction, samples were treated with turbo dnase free (ambion) to eliminate dna. next, the concentration and purity of the rna samples were measured using a nanodrop nd1000 spectrophotometer. the rna quality was assessed in a bioanalyzer 2010 (agilent technologies). from each sample, 1 mg of rna was pooled and used for the production of normalized cdna for 454 sequencing in the unitat de genã²mica (sct-ub, barcelona, spain). full-length-enriched double stranded cdna was synthesized from 1,5 mg of pooled total rna using mint cdna synthesis kit (evrogen, moscow, russia) according to manufacturer's protocol, and was subsequently purified using the qiaquick pcr purification kit (qiagen usa, valencia, ca). the amplified cdna was normalized using trimmer kit (evrogen, moscow, russia) to minimize differences in representation of transcripts. the method involves denaturation-reassociation of cdna, followed by a digestion with a duplex-specific nuclease (dsn) enzyme [85, 86] . the enzymatic degradation occurs primarily on the highly abundant cdna fraction. the single-stranded cdna fraction was then amplified twice by sequential pcr reactions according to the manufacturer's protocol. normalized cdna was purified using the qiaquick pcr purification kit (qiagen usa, valencia, ca). to generate the 454 library, 500 ng of normalized cdna were used. cdna was fractionated into small, 300-to 800-basepair fragments and the specific a and b adaptors were ligated to both the 39 and 59 ends of the fragments. the a and b adaptors were domain; pkc: protein kinase c; pten: phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase pten; raidd: caspase and rip adapter with death domain; tnf r1: tumor necrosis factor receptor 1; tnf-a: tumor necrosis factor alpha; tradd: tnf receptor type 1-associated death domain protein; traf2: tnf receptor-associated factor 2; trail: tnf-related apoptosis-inducing ligand; trail decoy: decoy trail receptor without death domain; trail-r: trail receptor. doi:10.1371/journal.pone.0035009.g008 used for purification, amplification, and sequencing steps. one sequencing run was performed on the gs-flx using titanium chemistry. 454 sequencing is based on sequencing-by-synthesis, addition of one nucleotide, or more, complementary to the template strand results in a chemiluminescent signal recorded by the ccd camera within the instrument. the signal strength is proportional to the number of nucleotides incorporated in a single nucleotide flow. all reagents and protocols used were from roche 454 life sciences, usa. pyrosequencing raw data, comprised of 975,190 reads, were processed with the roche quality control pipeline using the default settings. seqclean (http://compbio.dfci.harvard.edu/tgi/software/) software was used to screen for and remove normalization adaptor sequences, homopolymers and reads shorter than 40 bp prior to assembly. a total of 974,973 quality reads were subjected to mira, version 3.2.0 [87] , to assemble the transcriptome. by default, mira takes into account only contigs with at least 2 reads. the other reads go into debris, which might include singletons, repeats, low complexity sequences and sequences shorter than 40 bp. ncbi blastclust was used to group similar contigs into clusters (groups of transcripts from the same gene). two sequences were grouped if at least 60% of the positions had at least 95% identity. the 51,265 contigs were grouped into a total of 29,679 clusters. an iterative blast workflow was used to annotate the r. philippinarum contigs with at least 100 bp (49,847 contigs out of 51,265). then, blastx [88] with a cut off value of 10e-3, was used to compare the r. philippinarum contigs with the ncbi swissprot, the ncbi metazoan refseq, the ncbi nr and the uniprotkb/trembl protein databases. after annotation, blast2go software [89] was used to assign gene ontology terms [90] to the largest contig of a representative cluster (minimum of 100 bp). this strategy was used to avoid redundant results. default values in blast2go were used to perform the analysis and ontology level 2 was selected to construct the level pie charts. to make a comparison between r. philippinarum and other bivalve species, the nucleotide sequences and ests from c. gigas, m. galloprovincialis, l. elliptica and b. azoricus were obtained from genbank and from dedicated databases, when available. [93] . unique sequences from these databases (based on gi number) were used from each of the databases. these sequences were compared by blastn against the longest contig from each of 29,679 r. philippinarum clusters with a cut off e-value of 10e-05. hits to r. philippinarum sequences were represented in a venn diagram. the comparison between our 454 results, the longest contig from each of 29,679 clusters, and the milan et al. [16] transcriptome, contigs downloaded from ruphibase (http:// compgen.bio.unipd.it/ruphibase/query/), was made by blastn with a cut off e-value of 10e-05. another analysis was carried out to compare just the longest contig from each of 2,005 clusters identified as immune-related and the milan et al. contigs as well. the results were summarized in a table ( table 2 ). the percentage of coverage is the average % of query coverage by the best blast hit and the percentage of hits is the % of query with at least one hit in database, in parenthesis were added the total number of hits. identification of immune-related genes all the contig annotations were revised based on an immunity and inflammation-related keyword list (i.e. apoptosis, bactericidal, c3, lectin, socsâ�¦) developed in our laboratory to select the candidate sequences putatively involved in immune response. the presence or absence of these words in the blastx hit descriptions was checked to identify putative immune-related contigs. the remaining non-selected contigs were revised using the go terms at level 2, 3 and 4 assigned to each sequence after the annotation step that had a direct relationship with immunity. selected contigs were checked again to eliminate non-immune ones and distributed into functional categories. immune-related genes were grouped in three reference immune pathways (complement cascade, tlr signaling pathway and apoptosis) to describe each route indicated by our pyrosequencing results. to identify and classify the groups of organisms that had high similarity with our clam sequences, the uniprot taxonomy [94] was used except for the protozoa group. because protozoa are a highly complex group, a specific taxonomy [95] was followed. briefly, after the blastx annotation step all the hit descriptions included the species name (i.e. homo sapiens) or a code (i.e. human) meaning that protein has been previously identified as belonging to that species. with such information sequences were classified in taxonomical groups and represented in pie charts. table s1 list of contigs (e-value,10-3) of ruditapes philippinarum including sequence, length, description (hit description), accession number of description (hit acc), e-value obtained and database used for annotation (blast). study of diseases and the immune system of bivalves using molecular biology and genomics bacterial disease in marine bivalves, review of recent studies. trends and evolution perkinsosis in molluscs: a review bacteria-hemocyte interactions and phagocytosis in bivalves role of lectins (c-reactive protein) in defense of marine bivalves against bacteria modulation of the chemiluminescence response of mediterranean mussel (mytilus galloprovincialis) haemocytes immune parameters in carpet shell clams naturally infected with perkinsus atlanticus nitric oxide production by carpet shell clam (ruditapes decussatus) hemocytes generation and analysis of a 29,745 unique expressed sequence tags from the pacific oyster (crassostrea gigas) assembled into a publicly accessible database, the gigasdatabase immune gene discovery by expressed sequence tags generated from hemocytes of the bacteria-challenged oyster, crassostrea gigas sequence variability of myticins identified in haemocytes from mussels suggests ancient host-pathogen interactions mytibase, a knowledgebase of mussel (m. galloprovincialis) transcribed sequences insights into the innate immunity of the mediterranean mussel mytilus galloprovincialis development of expressed sequence tags from the pearl oyster, pinctada martensii dunker gene expression analysis of clams ruditapes philippinarum and ruditapes decussatus following bacterial infection yields molecular insights into pathogen resistance and immunity transcriptome sequencing and microarray development for the manila clam, ruditapes philippinarum: genomic tools for environmental monitoring de novo assembly of the manila clam ruditapes philippinarum transcriptome provides new insights into expression bias, mitochondrial doubly uniparental inheritance and sex determination analysis of est and lectin expression in hemocytes of manila clams (ruditapes phylippinarum) (bivalvia, mollusca) infected with perkinsus olseni increasing genomic information in bivalves through new est collections in four species, development of new genetic markers for environmental studies and genome evolution rapid transcriptome characterization for a nonmodel organism using 454 pyrosequencing sequencing technologies -the next generation transcriptomic analysis of the clam meretrix meretrix on different larval stages ferritin functions as a proinflammatory cytokine via iron-independent protein kinase c zeta/nuclear factor kappab-regulated signaling in rat hepatic stellate cells cloning and characterization of an invertebrate type lysozyme from venerupis philippinarum c1q and tumor necrosis factor superfamily: modularity and versatility the regulation of inflammation by galectin-3 isolation, cdna cloning, and characterization of an 18-kda hemagglutinin and amebocyte aggregation factor from limulus polyphemus angiopoietin-like proteins: emerging targets for treatment of obesity and related metabolic diseases modulation of apolipoprotein d expression and translocation under specific stress conditions neuroprotective effect of apolipoprotein d against human coronavirus oc43-induced encephalitis in mice apolipoprotein d itm2bs regulates apoptosis by inducing loss of mitochondrial membrane potential transcriptomic signatures of ash (fraxinus spp.) phloem transcriptomics of the bed bug (cimex lectularius) complement and its role in innate and adaptive immune responses potential indicators of stress response identified by expressed sequence tag analysis of hemocytes and embryos from the american oyster, crassostrea virginica analysis of a cdna-derived sequence of a novel mannose-binding lectin, codakine, from the tropical clam codakia orbicularis a novel c1q-domaincontaining protein from zhikong scallop chlamys farreri with lipopolysaccharide binding activity the c1q domain containing proteins of the mediterranean mussel mytilus galloprovincialis: a widespread and diverse family of immune-related molecules mgc1q, a novel c1q-domain-containing protein involved in the immune response of mytilus galloprovincialis differentially expressed genes of the carpet shell clam ruditapes decussatus against perkinsus olseni characterization of a c3 and a factor b-like in the carpet-shell clam, ruditapes decussatus structural and functional diversity of lectin repertoires in invertebrates, protochordates and ectothermic vertebrates purification and characterisation of a lectin isolated from the manila clam ruditapes philippinarum in korea characterization, tissue expression, and immunohistochemical localization of mcl3, a c-type lectin produced by perkinsus olseni-infected manila clams (ruditapes philippinarum) noble tandem-repeat galectin of manila clam ruditapes philippinarum is induced upon infection with the protozoan parasite perkinsus olseni lectin from the manila clam ruditapes philippinarum is induced upon infection with the protozoan parasite perkinsus olseni cdna cloning and mrna expression of the lipopolysaccharide-and beta-1,3-glucan-binding protein gene from scallop chlamys farreri molecular cloning and characterization of a short type peptidoglycan recognition protein (cfpgrp-s1) cdna from zhikong scallop chlamys farreri molecular cloning and mrna expression of peptidoglycan recognition protein (pgrp) gene in bay scallop (argopecten irradians, lamarck 1819) distribution of multiple peptidoglycan recognition proteins in the tissues of pacific oyster, crassostrea gigas molecular cloning and expression of a toll receptor gene homologue from zhikong scallop, chlamys farreri pattern recognition receptors and inflammation the evolution of vertebrate toll-like receptors a tolllike receptor that prevents infection by uropathogenic bacteria unc93b1 delivers nucleotide-sensing toll-like receptors to endolysosomes cg-timp, an inducible tissue inhibitor of metalloproteinase from the pacific oyster crassostrea gigas with a potential role in wound healing and defense mechanisms molecular cloning, characterization and expression of a novel serine proteinase inhibitor gene in bay scallops (argopecten irradians, lamarck 1819) molecular cloning and expression of a novel kazal-type serine proteinase inhibitor gene from zhikong scallop chlamys farreri, and the inhibitory activity of its recombinant domain a novel slowtight binding serine protease inhibitor from eastern oyster (crassostrea virginica) plasma inhibits perkinsin, the major extracellular protease of the oyster protozoan parasite perkinsus marinus evidence indicating the existence of a novel family of serine protease inhibitors that may be involved in marine invertebrate immunity serine protease inhibitor cvsi-1 potential role in the eastern oyster host defense against the protozoan parasite perkinsus marinus antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? innate immunity. isolation of several cysteine-rich antimicrobial peptides from the blood of a mollusc, mytilus edulis a member of the arthropod defensin family from edible mediterranean mussels (mytilus galloprovincialis) evidence of high individual diversity on myticin c in mussel (mytilus galloprovincialis) mytilus galloprovincialis myticin c: a chemotactic molecule with antiviral activity and immunoregulatory properties analysis of differentially expressed genes in response to bacterial stimulation in hemocytes of the carpetshell clam ruditapes decussatus: identification of new antimicrobial peptides molecular characterization of a novel big defensin from clam venerupis philippinarum heat shock proteins: facts, thoughts, and dreams heat shock proteins, cellular chaperones that modulate mitochondrial cell death pathways djla, a membrane-anchored dnaj-like protein, is required for cytotoxicity of clam pathogen vibrio tapetis to hemocytes alternation of venerupis philippinarum hsp40 gene expression in response to pathogen challenge and heavy metal exposure identification of two small heat shock proteins with different response profile to cadmium and pathogen stresses in venerupis philippinarum molecular characterization and expression analysis of a putative lps-induced tnf-alpha factor (litaf) from pearl oyster pinctada fucata cloning, characterization and expression analysis of the gene for a putative lipopolysaccharide-induced tnf-alpha factor of the pacific oyster molecular cloning and characterization of a putative lipopolysaccharide-induced tnf-alpha factor (litaf) gene homologue from zhikong scallop chlamys farreri first molluscan tnfr homologue in zhikong scallop: molecular characterization and expression analysis identification and expression of traf6 (tnf receptor-associated factor 6) gene in zhikong scallop chlamys farreri diversity and pathogenecity of vibrio species in cultured bivalve molluscs an apoptosis-inhibiting baculovirus gene with a zinc finger-like motif detection of ostreid herpesvirus 1 dna by pcr in bivalve molluscs: a critical review synopsis of infectious diseases and parasites of commercially exploited shellfish a fungus disease in clam and oyster larvae a novel method for snp detection using a new duplex-specific nuclease from crab hepatopancreas simple cdna normalization using kamchatka crab duplex-specific nuclease using the miraest assembler for reliable and automated mrna transcript assembly and snp detection in sequenced ests basic local alignment search tool blast2go, a universal tool for annotation, visualization and analysis in functional genomics research gene ontology, tool for the unification of biology. the gene ontology consortium pyrosequencing of mytilus galloprovincialis cdnas: tissue-specific expression patterns insights into shell deposition in the antarctic bivalve laternula elliptica: gene discovery in the mantle transcriptome using 454 pyrosequencing highthroughput sequencing and analysis of the gill tissue transcriptome from the deep-sea hydrothermal vent mussel bathymodiolus azoricus newt, a new taxonomy portal the new higher level classification of eukaryotes with emphasis on the taxonomy of protists key: cord-000666-je9t4i6q authors: verbist, katherine c.; rose, david l.; cole, charles j.; field, mary b.; klonowski, kimberly d. title: il-15 participates in the respiratory innate immune response to influenza virus infection date: 2012-05-18 journal: plos one doi: 10.1371/journal.pone.0037539 sha: doc_id: 666 cord_uid: je9t4i6q following influenza infection, natural killer (nk) cells function as interim effectors by suppressing viral replication until cd8 t cells are activated, proliferate, and are mobilized within the respiratory tract. thus, nk cells are an important first line of defense against influenza virus. here, in a murine model of influenza, we show that virally-induced il-15 facilitates the trafficking of nk cells into the lung airways. blocking il-15 delays nk cell entry to the site of infection and results in a disregulated control of early viral replication. by the same principle, viral control by nk cells can be therapeutically enhanced via intranasal administration of exogenous il-15 in the early days post influenza infection. in addition to controlling early viral replication, this il-15-induced mobilization of nk cells to the lung airways has important downstream consequences on adaptive responses. primarily, depletion of responding nk1.1+ nk cells is associated with reduced immigration of influenza-specific cd8 t cells to the site of infection. together this work suggests that local deposits of il-15 in the lung airways regulate the coordinated innate and adaptive immune responses to influenza infection and may represent an important point of immune intervention. influenza virus is a major human pathogen that causes substantial morbidity and mortality-approximately 36,000 deaths annually in the united states alone [1] . combined with the severe economic burden imposed from seasonal influenza outbreaks and growing concerns over potential imminent influenza pandemics, there is considerable need for a firm understanding of the disease pathology, prevention strategies, and mechanisms of host defense against the virus [2] . influenza virus is primarily transmitted via inhaled aerosols and results in an infection localized to the upper respiratory tract, with viral replication largely limited to epithelial cells [3] . mechanisms by which the immune system eliminates influenza have been well studied and are known to involve the coordinated actions of the innate and adaptive immune systems. namely, the cytolytic action of influenza-specific cd8 t cells has been shown to be the primary mediator of complete viral clearance, but important roles have also been described for cd4 t cells [4, 5, 6] . in addition to t cells, a crucial role has also been established for innate immune effectors including natural killer (nk) cells, which provide short-term control of viral replication prior to t cell activation [7] . nk cells become activated following the loss of inhibitory signals coupled with positive activating signals resulting in direct (via release of cytotoxic granules and interferon c) or indirect (via activation of macrophages and dendritic cells) target cell lysis [8] . nk cells are vital in limiting influenza viral replication as depletion of nk cells dramatically increases morbidity and mortality in hamsters and mice [9] , and in humans severe infections with the 2009 pandemic h1n1 virus positively correlated with reduced numbers of nk cells in the lungs [10] . studies have indicated that the natural cytotoxicity receptors nkp44 and nkp46, which recognize hemagglutinin proteins of several different influenza strains [11, 12] is one mechanism used by nk cells to protect against lethal viral challenge [13] . secondarily, nk cells also aid in viral clearance indirectly through the production and secretion of cytokines which both amplifies local inflammation and recruits antigen-specific cd8 t cells to sites of inflammation [14] . implicit in both of these functions is the ability of nk cells to accumulate within the respiratory tract to contact infected cells and provide a source of chemotactic signals to recruit recently activated cd8 t cells. type i ifns expressed within hours after viral infection have been documented to induce expression of the chemokines cxcl9 and 10 which function to recruit cxcr3 expressing nk cells to sites of infection [15] . however, type i ifns also modulate the expression of the common gamma chain cytokine interleukin 15 [16, 17, 18] , which we recently reported to be temporally and locally increased following influenza infection [19] . this expression of il-15 in the respiratory tract facilitates the recruitment of antigen-specific cd8 t cells to the respiratory tract. however, it is unclear whether the chemotactic properties of il-15 uniquely affect migratory cd8 t cells or could be extended to other il-15-sensitive immune cells. nk and nkt cells are nearly absent in il-15 2/2 animals [20] , highlighting the important role of il-15 on nk cell development and homeostasis in the steady-state. following viral infections, de novo production of il-15 by dendritic cells results in the activation and proliferation of nk cells [21, 22] , and transient systemic stimulation of nk cells with soluble il-15/il-15ra complexes also results in an accumulation of phenotypically and functionally mature nk cells [23, 24] . in addition to these roles, il-15 also can stimulate the migration of nk cells in vitro and enhances their adhesion to cultured endothelial cells [25] . we therefore hypothesized that virally induced il-15 functionally assists in the migration of nk cells into the lung airways. we show here that an il-15 deficiency results in a site-specific reduction in nk cells from the lung airway and an exacerbation of viral load at early time points post influenza infection. additionally, exogenous il-15 induces the specific migration of nk cells in vitro and in vivo. this il-15dependent enhanced mobilization of nk cells to the lung airways correlates with decreased viral loads. importantly, in the absence of nk cells, antigen-specific cd8 t cells fail to accumulate at the site of infection, providing a possible link between il-15-mediated migratory effects of both the innate and adaptive immune responses to influenza infection and suggest therapeutic possibilities regarding the use of il-15 to simultaneously regulate both arms of the immune system for improved responses to viral infection. all animals were handled in strict accordance with good animal practice as defined by the american association for accreditation of laboratory animal care as well as federal and state agencies. all animal work presented here was approved by institutional animal care and use committee of university of georgia (aup no. a2009-6-114). mice, viruses, il-15 blocking, and nk cell depletion c57bl/6 mice were purchased from charles river (wilmington, ma) through the nci program. influenza a/hk-x31 (x31, h3n2) was generously donated by dr. s. mark tompkins (university of georgia, athens, ga). animals were infected intranasally (i.n.) with 10 3 pfu hkx31 diluted in 50 ml sterile pbs. il-15 was blocked using 25 mg anti-il-15 mab (clone aio3) (ebioscience, san diego, ca) administered daily via intraperitoneal injection (i.p.) in 200 ml sterile pbs. il-15 depletion was confirmed by reductions in frequencies of both nk and cd44 hi cd8 + t cell in antibody-treated animals compared to untreated animals 7 days after the initiation of mab treatment. nk1.1+ cells were depleted via intravenous (i.v.) injections of 200 ul pbs containing 200 mg anti-nk1.1/mouse (clone pk136) every other day (ucsf monoclonal antibody core facility, san francisco, ca), and depletion of nk cells was verified using an anti-nkp46 mab (clone 21a9.4, ebioscience) throughout the experiment and 2 days after the last injection of the nk1.1 depleting mab. lung airway-resident cells were harvested by bronchioalveolar lavage (bal) with 3 consecutive washes of 1 ml pbs. to isolate cells from the lung parenchyma, lungs were perfused with ,25 ml pbs/heparin sodium solution, harvested, minced and incubated at 37uc for 30 minutes in 1.25 mm edta. the tissue was subsequently incubated in collagenase diluted in rpmi (6 mg/ml) at 37uc for 1 hour and passed through a 5 mm cell strainer. isolated cells were subjected to separation via density gradient centrifugation by resuspending cells in 47% percoll underlain with 67% percoll. the gradients were then centrifuged at 2800 rpm for 20 minutes, and lymphocytes at the interface were collected. spleen and lymph nodes were harvested from animals, homogenized, and then passed through a cell strainer. spleen homogenate was depleted of erythrocytes by incubation in tris-buffered ammonium chloride. single cell suspensions were stained with combinations of cocktails containing anti-cd3, nk1.1, nkp46, cd11c, cd11b, cd122, and cd132 (ebioscience, san diego, ca) as indicated for 20 minutes at 4uc. where indicated, cells were concurrently stained with or without biotinylated anti-il-15ra (r&d systems, minneapolis, mn) followed by 20 minute incubation with apcconjugated streptavidin at 4uc. influenza nuclear protein (np) mhc class i tetramer [h2-d b /asnenmetm] were generated by the national institute of allergy and infectious diseases tetramer facility (emory university, atlanta, ga). tetramer staining was conducted at room temperature for 1 hour concurrently with anti-cd3, nk1.1, nkp46, cd8, and cd44 (ebioscience, san diego, ca). stained cells were analyzed using a bd lsrii digital flow cytometer (bd biosciences, san jose, ca) and either bd facsdiva or flowjo software (tree star, inc., ashland, or). il-15 complexes (il-15c) were generated on the day of use by incubating 1.5 mg recombinant mil-15 with 7 mg il-15ra fcchimera (r&d systems, minneapolis, mn) at 37uc for 20 minutes followed by 4uc for at least 10 minutes. for ra only controls, 7 mg il-15ra fc-chimera (r&d systems, minneapolis, mn) was incubated similarly to complexes without addition of the cytokine. complexes or ra alone were administered via passive inhalation into both nostrils using a micropipette delivering 36.25 ml (for daily treatments) or 72.5 ml (for one time treatments) of complexes in sterile pbs. for assessment of cell proliferation, animals received 2 mg of brdu (sigma-aldrich, st. louis, mo) administered i.p. in a 200 ml volume of pbs. cells were isolated from these animals 12 hours after treatment and stained with 20 ml aminoactinomycin d (7-aad; bd pharmingen, san jose, ca) for 20 minutes at 4uc. cells were surface stained as previously described and stained intracellularly with fitc-labeled anti-ki-67 and apc-labeled anti-brdu monoclonal antibodies (bd pharmingen, san jose, ca) according to manufacturer's instructions. in vitro migration assays were performed by placing bulk populations of lymphocytes containing predetermined numbers of nk cells (verified by facs analysis) from the indicated tissues on the top insert of a 5 mm chemotaxis transwell (fisher scientific, waltham, ma) in which the bottom well contained warm media alone or supplemented with 100 ng/ml il-15c. il-15c was generated by incubating 100 ng of il-15 with 500 ng il-15ra fcchimeric protein at 37u for 20 minutes and 4u for 10 minutes. plates containing transwells were then incubated at 37uc with co 2 exchange, and 90 minutes after plating, cells were harvested from bottom chambers and the percent migration of nk cells was calculated as the ratio of the number of nk cells in the bottom chamber compared to the number of nk cells determined in the input sample. plaque assays were preformed as previously described [26] . briefly, lungs from hk-x31 infected animals were collected and homogenized using a tissue lyser (qiagen, hilden, germany). monolayers of madin-darby kidney cells were incubated with 10fold serial dilutions of 10% homogenate in dilution media (16mem, 1 mg/ml tpck-treated trypsin) for 1 hour at 37uc. cells were washed with 16 sterile pbs and overlaid with mem containing 1.2% avicel microcrystalline cellulose (fmc biopoly-mer, philadelphia, pa), 0.04 m hepes, 0.02 mm l-glutamine, 0.15% nahco 3 (w/v), and 1 mg/ml tpck-treated trypsin. after 72 hours, the overlay was removed, and the cells were washed with 16 sterile pbs, fixed by incubation with cold methanol/acteone (60:40%), and stained with crystal violet. statistical significance was determined by student's t test using prism 5 software (graphpad software). significance was determined to be any p-value where p,0.05. we and others have shown that following influenza infection, il-15 message and protein is increased in the lung airways [19, 27] . because this il-15 expression was rapidly induced by influenza infection and reached significant levels as early as day 3 post infection (p.i.) [19] , we hypothesized that influenza-induced il-15 expression may be an important mediator of nk cell responses to influenza infection. we therefore first sought to determine whether nk cells responding to influenza infection were capable of receiving signals from this locally produced il-15. to this end, lymphocytes were isolated from the lung airways of influenzainfected animals via bal, and cd3 2 , nk1.1 + nk cells were analyzed for the expression of il-15 receptor components by flow cytometry. the il-15 receptor is a heterotrimer, consisting of the common gamma chain (cd132), the shared il-2/il-15rb chain (cd122), and the specific il-15ra chain [28] . to date the majority of biological effects of il-15 on nk cells, however, are mediated through the paired co-expression of cd122 and cd132 [29, 30, 31] , while il-15ra is only required by accessory cells which present il-15 to respondent cells, a mechanism referred to as trans-presentation [32] . however, some groups have suggested that il-15ra expression alone contains some signaling moieties which may participate in distinct biological functions [27] . therefore it is important to establish the kinetics of il-15 receptor component expression and il-15 signaling potential on nk cells in our model. nk cells are known to respond rapidly to influenza infection and continue accumulating in the lung airways through day 5 p.i. ( [13] and data not shown). since we wished to specifically evaluate recent nk cell immigrants responding to the airway inflammation resulting from influenza infection, we restricted our analyses of nk cell kinetics to before and up to day 4 p.i.. nk cells were first detected in the bal at day 2 post influenza infection (albeit at a low frequency, ,0.1-0.6% of lymphocytes, figure 1a and figure 2a ) but were completely absent in control mock-infected animals (data not shown). despite the low frequency of nk cells in the lung airway at this early time point, one fifth consistently expressed il-15ra. additionally, 30-40% of them expressed cd122 and cd132 ( figure 1b ). by day 3 p.i., when nk cells represented a much more discernible population (,2-3% of lymphocytes, figure 1a & 2a), greater than 90% of these gated cells expressed cd122 and cd132, and expression levels of these receptors on a per-cell basis increased over time as indicated by a higher median fluorescence intensity by day 4 p.i. ( figure 1b ) and consistent with evidence that expression of il-15r components is induced by activating stimuli [33] . expression of il-15ra however, was variable but consistently much lower that the expression levels of cd122 and cd132. this biased expression of cd122/132 receptor chains over il-15ra and enhanced il-15 levels [19] following influenza infection, indicate that nk cells accumulating at the site of influenza infection are capable of responding to locally produced il-15 via the trans-presentation pathway. since co-expression of cd122 and cd132 render nk cells responsive to il-15 signals, we next wished to determine whether virally induced il-15 and subsequent signaling through these receptors affected the accumulation of these cells in the respiratory tract. because il-15 2/2 mice exhibit a severe developmental defect in both nk and nkt cell lineages and are nearly devoid of these cell populations [20] , we chose to use an anti-il-15 blocking antibody to selectively deplete il-15 concurrent with infection. therefore, we monitored the influx of nk cells in animals receiving either pbs or anti-il-15 mab administered i.p. daily from day 0 through day 4 post influenza infection. in order to more accurately define bona fide nk cells, particularly in the lung airways harboring few nk cells at very early time points post influenza infection, we included nkp46 reactivity in our staining protocol and henceforth define nk cells as cd3 2 lymphocytes positive for both nk1.1 and nkp46. while nk cells accumulated in the lung airways of untreated mice as expected, by day 2 p.i. the overall frequency of nk cells in il-15 blocked animals was reduced by half and remained this low through day 4 p.i. (figure 2a) . concordantly, total numbers of nk cells in il-15blocked mice were partially reduced at day 2 p.i., and substantially (figure 2a ). in fact, whereas the numbers of nk cells continued to accumulate in the lung airways of control-treated animals through day 4 p.i., numbers of nk cells in the lung airways of treated animals plateaued by day 3 p.i. (figure 2a ). in the lung parenchyma of control-treated animals, nk cells also accumulated over time post infection, similar to those in the lung airways, but the frequencies of nk cells in this site remained unchanged in il-15-blocked mice. numbers of nk cells in the lung parenchyma of anti-il-15 treated animals also remained similar to control animals with only a slight reduction at day 2 p.i. ( figure 2b ). importantly, while the numbers of nk cells were significantly reduced at the site of infection as a result of an il-15 deficiency, anti-il-15 treatment had little effect on the frequency or number of nk cells found in anatomical sites distal to the site of infection such as the spleen ( figure 2c ). in order to determine whether an absence of il-15 in the lung airways resulted in the reduced frequencies and numbers of nk cells specifically, numbers of other populations of innate cells in the airways at these early time points post infection were analyzed. cd11c 2 cd11b + cells (granulocytes) or cd11c + cd11b 2 (dendritic cells) in the airways following infection were mostly unaffected by the il-15 deficiency, with only a small reduction observed at day 4 p.i. (figure 2d ). in contrast, nk1.1 + cd3 + (nkt cells) were markedly reduced in the lung airways of il-15blocked mice ( figure 2d ), but overall, these cells represented a low proportion of the innate cells responding to influenza infection at these early time points following infection ( figure 1a ). together, these data indicated that short term blockade of il-15 did not result in global defects in nk cell homeostasis or survival in peripheral tissues and the effects of il-15 were largely specific to nk cells as blocking il-15 selectively resulted in a significant loss of nk cells recovered from the site of infection. to test whether this local reduction in nk cells impairs the control of viral replication, we performed plaque assays on control-and anti-il-15 treated mice to quantify viral load in the lungs of animals with intact or diminished il-15 and nk cell responses. viral load was quantified on days 1-5 and 7-8 p.i. to specifically look at control during the time frame of nk cell entry and accumulation in the lung airways and the kinetics of subsequent viral clearance. in il-15 blocked animals, differences in viral load were apparent as early as d2 p.i. where viral titers were about 36 higher through day 3 p.i. ( figure 2e) ; however, these animals seemed to regain control of viral replication by day 4 p.i., which perhaps corresponds with the early entry of cells of the adaptive immune response as anti-influenza specific cd8 t cells are first detectable in the lung airways by d6 post infection by flow cytometry ( [34] and data not shown). thus, while viral elimination is not ultimately dependent on il-15, early control of the virus is impaired in the absence of il-15, which correlates with the arrival of a significant number of nk cells in the lung airways. we thus hypothesized that il-15 was important for the migration of nk cells in the lung airways following influenza infection. we observed significant reductions in the numbers of nk cells in the lung airways of influenza-infected animals in which il-15 was blocked at time points associated with their arrival at the site of infection, and failure of these cell populations to accumulate had implications in early viral control (figure 2) . in order to determine whether il-15 might be an important signal for nk cells in the migration to and/or the proliferation within the site of infection, we chose to provide exogenous il-15 in an attempt to enhance any il-15-dependent nk cell migration and/or in situ proliferation within the lung airways of influenza-infected animals. to this end, either pbs or recombinant il-15/il-15ra fusion protein complexes (il-15c) were administered intranasally to mice three days following influenza infection. to ensure that any biological effects of the il-15c could be attributed to activity of the cytokine (which is merely stabilized by complexing to il-15ra), a control group of mice received the il-15ra only. concurrent with treatment, mice received an i.p. pulse of the thymidine analog brdu to identify proliferating cells. twelve hours post treatment, the frequency and total number of nk cells in the bal was quantified as well as the percentage of these cells incorporating brdu. isolated cells were simultaneously stained with 7-aad as an indicator of cell viability, as only cells with disrupted membranes stain positive for this fluorescent dye. importantly, neither the il-15ra alone nor the il-15c affected cell viability, as cells isolated from animals receiving these treatments had similar percentages of 7-aad + nk cells as those from pbs-treated mice (data not shown). upon introduction of il-15c to the lung airways, the overall frequency of nk cells isolated from the bal was significantly increased ( figure 3a and b) , and the total number of nk cells isolated from this site was nearly three times that of pbs-treated control animals ( figure 3b ). interestingly, the percentage of nk cells expressing cd122, the il-2/15 rb chain, was reduced in il-15c-treated animals ( figure 3b ), perhaps indicative of increased signaling through and subsequent internalization of this receptor complex by il-15 responsive cells. unlike t cells, which require large clonal bursts of proliferation to achieve effector status, the effector function of nk cells is more related to activation and mobilization to the site of inflammation [35] . nevertheless, we wished to test whether the large increases in nk cell number in the lung airways following il-15c administration could be attributed to il-15-induced proliferation of nk cells at the site. to assess the potential role of proliferation in this observed increase in nk cell frequency and number in the bal of treated mice, these nk cells were analyzed for brdu incorporation. concomitantly, cells isolated from the bal were assessed for expression of the cellcycle-specific protein ki-67. since brdu is incorporated into the dna of only cells in s phase whereas ki-67 is expressed by cells in any stage of the cell cycle, it was unsurprising that brdu + cells were only a fraction of ki-67 + cells ( figure 3a) . therefore, we considered only cells positive for both markers as cells undergoing proliferation at the time of treatment. although the percentage of brdu-incorporating cells was modestly increased in il-15c-treated animals, the overall frequency of proliferating cells was low in untreated (,10%) and treated (,12%) animals ( figure 3b ). these data suggest that il-15c may trigger the proliferation of nk cells, but proliferation of this cell population in the lung airways is, in general, low and not likely to be solely responsible for the total number of cells extracted from the lung airways. il-15ra alone had no significant impact on the accumulation or proliferation of this cell population ( figure 3a and b) . finally, differences in cell of cd11c 2 cd11b + cells, cd11c + cd11b 2 cells, and cd3 + nk1.1 + cells from bal are shown 6 sem in pbs control (solid lines) or ail-15-treated (dashed lines) mice (n = 3 mice/group). (e) at the indicated times p.i. with 10 3 pfu hkx31, lung viral titers from pbs and ail-15-treated mice were determined by plaque assay. mean viral titer is plotted 6 sem (n = 2 mice/group on days 1 and 5 p.i. or 3 mice/group for remaining time points; day 2 p = 0.0503). data are representative of two independent experiments. doi:10.1371/journal.pone.0037539.g002 frequencies, numbers, cd122 expression, and brdu incorporation were specific to the lung airways (the sight of treatment), as nk cells isolated from spleens were similar in control and il-15ctreated animals ( figure 3c and data not shown). together, these data demonstrate that exogenous il-15 results in increased numbers of nk cells in the lung airways. since this increase appeared to be independent of il-15-mediated effects on cell survival, and proliferation was low, we hypothesized that il-15 may be responsible for a substantial amount of migration of nk cells into the lung airways following influenza infection similar to its effects on cd8 t cells [19] . intranasal administration of il-15c resulted in increased numbers of nk cells in the lung airways that appeared to be due to increased migration into that site. previous studies have indicated that il-15 is indeed chemotactic for nk cells. in vitro checkerboard assays revealed that freshly isolated nk cells migrated to il-15 gradients, and il-15 stimulation increased lfa-1-dependent binding of nk cells to cultured endothelial cells [25] . il-15 has also been shown to play a central role in the recruitment of cd16 2 human nk cells into the endometrium following ovulation [36] . to test the direct chemotactic potential of il-15c for nk cells in our own system, we employed an in vitro chemotaxis transwell assay. bulk lymphocytes (or purified splenic nk cells, data not shown) from the bal, lung, and spleen of mice collected three days after infection with influenza were placed in the top chamber of a transwell filter support with either media alone or media supplemented with il-15c in the bottom chamber. after 90 minutes of incubation, il-15c significantly enriched nk cells isolated from the lung and spleen ( figure 4a ). consistent with our findings with cd8 t cells [19] , nk cells from the lung airways did not migrate to il-15c, perhaps because this site represents the terminal destination for these cell populations. unlike cd8 t cells, which lose expression of cd122 upon residence in the lung airways following influenza infection [19] [37] , nearly 100% of the nk cells residing in the bal of influenza-infected mice express cd122 ( figure 1a and b) . nonetheless, these data indicate that nk cells in the lung parenchyma or the general circulation (as represented by the spleen) migrate to il-15 in vitro. intranasal administration of il-15c during the innate phase enhances early viral control a temporal cessation in il-15 bioavailability reduced the numbers of nk cells (figure 2a ) in the respiratory tract resulting in increased viral titers ( figure 2e) , presumably due to impaired nk cell responses. conversely, exogenous il-15c promoted the migration of nk cells ( figure 4 ) and resulted in increased numbers of nk cells in the lung airways ( figure 3b ). we therefore hypothesized that intranasal administration of il-15c early after infection could be used to enhance the early innate immune response to influenza and augment viral control. to this end, influenza-infected animals received either pbs or il-15c intranasally on days 1-4 p.i., a time frame corresponding to the migration of nk cells into the lung airways and limiting any confounding effects il-15c might have on adaptive immune cells entering the lung airways at later time points. every other day, from day 2-8 p.i., whole lungs were collected and viral titers were quantified via plaque assay ( figure 5a ). early (d2) p.i., we observed no difference in viral load between pbs and il-15c-treated mice, but as viral replication reached more significant levels at day 4 p.i., animals receiving the il-15c had 2.46 less viral load than animals receiving only pbs ( figure 5b and c) . although viral titers dropped 10 fold in both groups of animals by day 6 p.i., as expected, surprisingly, there remained a greater than 2 fold significant difference in viral load ( figure 5b and d) . although both groups of animals cleared the influenza virus completely by day 8 p.i. (figure 5b ), our data suggest administration of il-15c on days 1-4 p.i. can enhance early control of influenza virus by cells of the innate immune system. because we have previously reported that il-15 is important for the migration of influenza-specific cd8 t cells into the lung airways [19] , it was possible that those observed effects were secondary to the recruitment of nk cells to the lung. to test whether the accumulation of nk cells in the lung was required for the subsequent immigration of influenza-specific cd8 t cells to the respiratory tract, influenza-infected animals were assayed for the accumulation of anti-influenza specific cd8 t cells in nk deficient animals, generated by administration of the ank1.1 depleting mab pk136 every other day ( figure 6a ). flow cytometric analyses of nkp46 expression of day 4 p.i. lymphocytes isolated from the lung, bal, spleen, and mediastinal lymph nodes (mdln) of ank1.1-treated animals revealed robust depletion of nk cells as the number of cd3 2 nkp46 + cells were reduced to less than one third of those observed in pbs-treated control animals ( figure 6b and data not shown). on days 6 and 8 p.i., the numbers of influenza-specific cd8 t cells in these same tissues were quantified through the identification of cells staining positive for a tetrameric reagent loaded with the immunodominant peptide derived from the influenza nucleoprotein (np). tetramer positive cd8 t cells could first be detected in the lung airways at day 6 p.i., and although numbers were low, they were somewhat reduced in the bal of pk136-treated mice ( figure 6c ). no reduction in influenza-specific cd8 t cell numbers could be observed in any other tissue. in fact, cd8 t cells seemed to accumulate in other tissues of animals depleted of nk1.1expressing cells. by day 8 p.i., the frequency of influenza-specific cd8 t cells in the bal of pk136-injected animals was less than half than that of control animals, and the total numbers were reduced nearly threefold ( figure 6d ). again, this effect was specific to the lung airways, since frequencies and numbers of nptetramer + cd8 t cells were unchanged in other tissues examined ( figure 6d ). therefore, the largely tissue-specific nature of the dependence of influenza-specific cd8 t cells on the presence of nk1.1-expressing cells in the lung airways partially indicates that lung-resident nk and possibly nkt cells are requisite for the subsequent migration of influenza-specific cd8 t cells into this site. these data suggest that il-15-mediated migration of cd8 t cells to the lung airways may be, at least partially, an indirect effect of nk1.1 + cells moving into the lung airway space in response to il-15 and that subsequent tissue remodeling or production of an intermediate factor is responsible for the subsequent recruitment of the cd8 t cells. to our knowledge, these data for the first time describe a role for il-15 in linking the innate and adaptive responses to influenza infection necessitating the further inquiry of il-15 as a potential vaccine adjuvant. here, we have demonstrated that nk cells, which accumulate in the lung airways early after influenza infection, are dependent on il-15 for this accumulation and subsequent ability to control viral load. nk cells in the lung airways express high levels of the common gamma chain (cd132) and cd122, receptors responsible for imparting il-15 responsiveness. since il-15 is known to be produced in the lung airways following influenza infection, we investigated the role of il-15 in nk cell responses to influenza. in the absence of il-15, nk cell frequencies and numbers were significantly reduced in the bal, resulting in impaired early control of influenza virus. although numbers of cd3 + nk1.1 + cells were also substantially reduced following anti il-15 treatment, it is unclear whether this cell population plays a relevant role in controlling primary infections with influenza. while nk cells are thought to be very important participants in the control of influenza replication, evidence for nkt cells playing a similar role is more controversial [38] . models of nkt cell activation using agal-cer revealed enhanced innate responses to influenza and improved disease outcome [39] , but challenge of cd1d 2/2 mice with influenza led to increased survival, implying an immuno-regulatory role for nkt cells in this model [40] . whether or not these cells are critically involved in viral clearance, in our model, they represent a very small percentage (,1%) of the lymphocytes in the lung airways in the first five days following infection. therefore, we believe that an abrogation of virallyinduced il-15 in the lung airways most dramatically affects cd3 2 , nk1.1, nkp46 double positive nk cells responding rapidly to infection. by the same principle, exogenous il-15 could be used therapeutically to increase nk cell populations in the bal-primarily as a result of il-15-induced migration-to enhance viral control. therefore, these combined data indicate that influenza-induced il-15 is an important signal for the migration of nk cells to the lung airways where they help limit viral replication. il-15 is produced by a variety of cell types within and outside of the immune system, including dendritic cells, monocytes and macrophages, stromal cells, endothelial cells, and epithelial cells [41] , and pathogenic stimuli are known to induce this expression above constitutive levels [17, 42, 43, 44] . although dendritic cells are known to be an important source of il-15 at day 6 post influenza infection [27] , it is unclear whether dcs or other cell types produce il-15 to facilitate specific immune responses to influenza. for example, lung epithelial cells constitutively express il-15 and il-15ra [45] , and neutrophils and macrophages can be a major source of il-15 that is produced during a variety of lung inflammatory diseases including sarcoidosis, tuberculosis, bronchitis, and asthma [46] . following influenza infection, the il-15producing cell type(s) is/are still unknown, but influenza-induced expression of il-15 is clearly an important regulator of the nk and cd8 t cell responses to this virus [19] . not only might there be different cellular sources of il-15 at different times after infection, but il-15 signaling in nk cells could also be regulated by the context in which il-15 is presented. at least one isoform of il-15 is known to enter the cell secretory pathway and could therefore be released as a soluble molecule [47, 48] . these data lend the idea that soluble il-15 could bind heterotrimeric receptors on nk cells. however, il-15 is also known to be transpresented to nk cells, and indeed, this mode of signaling appears to be most important for il-15-mediated nk cell survival [49] . while il-15ra is dispensable for nk cell survival, il-15ra is thought to be an important component in signaling the migration of cd16 2 human nk cells into the endometrium, since this particular nk cell population expresses higher levels of this receptor component than cd16 + nk cells that do not migrate to il-15 [36] . in our studies, il-15ra was only expressed on a relatively low proportion of nk cells in the lung airways of influenza-infected animals ( figure 1 ). in contrast, cd122 signaling appears to be important for nk cell migration to il-15 as this receptor chain is down regulated on nk cells exposed to il-15c in vivo ( figure 3 ). thus, we consider it likely that signaling through il-15ra is not essential to il-15-mediated of migration of nk cells to the lung airways. therefore, future work is needed to identify the il-15-producing cell population(s) and the mechanism of migration to this cytokine in order to target this response for eventual applications in influenza vaccines and treatments. a significant finding of our work is that nk cells immigrating into the lung airways are partially required for substantial trafficking of influenza-specific effector cd8 t cells to this location. we found that nk cells are necessary for the optimal accumulation of antigen-specific cd8 t cells, important effectors of eventual viral clearance [4, 5] , at the site of infection, since depletion of nk1.1-expressing cells resulted in a significant decrease in the number of influenza-specific cd8 t cells in the bal. a subpopulation of cd8 t cells has been shown to express nk1.1 upon activation [50] , and while these cells were detected in the lung parenchyma of influenza-infected c57bl/6 mice, they comprised less than 1% of the np-tetramer+ cd8 t cells at this site alone and were never detected in the lung airways. this and the fact that the reduction of activated, influenza-specific cd8 t cells was observed only in the lung airways, and only at later time points post infection lead us to believe that direct depletion of nk1.1-expressing cd8 t cells cannot account for this reduction in treated mice. these data strongly suggest that nk1.1 + cells in the lung airways are requisite for the subsequent accumulation of influenza-specific cd8 t cells in the bal and implicate il-15mediated nk cell migration into the lung airways as a link between the innate and adaptive responses to influenza infection. we currently favor a model in which nk cells migrate directly to influenza-induced il-15 in the lung airways, and, cd8 t cell trafficking, in turn, occurs to both il-15 directly and to chemotactic factors produced by nk cells already present at the site. a positive feedback loop in which nk cells responding to il-15 induce further expression of il-15 by dendritic cells for the stimulation of cd8 t cells is an additional possibility [51] . moreover, il-15 has also been shown to modulate chemokine and chemokine receptor expression by nk cells and t cells [52, 53, 54] . as il-15 deficiencies also cause reductions in cd8 t cell accumulation in the bal [19] , chemotactic potential of il-15 for nk cells presented here provides a possible link between il-15-mediated effects of both the innate and adaptive immune responses to influenza infection. future work will attempt to tease out the roles of direct and indirect migration of both nk cells and cd8 t cells to il-15 following influenza infection. regardless of mechanism, il-15-induced trafficking of lymphocytes is an important part of our overall understanding of influenza immunobiology. the studies presented here emphasize the importance of il-15 in mediating the innate response to influenza via the trafficking of nk cells, as viral titers were not as efficiently controlled early after infection in il-15-blocked animals. these and previous studies also suggest that exogenous il-15c could be used to modulate the immune response at both the innate and adaptive phases. overall, we believe that il-15 may be in important point of eventual immune intervention for treatment of primary infection and for adjuvanting vaccines. newer respiratory virus infections: human metapneumovirus, avian influenza virus, and human coronaviruses influenza pandemics: can we prepare for the unpredictable? host responses to respiratory virus infection and immunization effector cd4+ and cd8+ t-cell mechanisms in the control of respiratory virus infections cellular events in the lymph node and lung of mice with influenza. consequences of depleting cd4+ t cells cell-mediated immunity to respiratory virus infections role of natural killer cells in the generation of influenza virus-specific cytotoxic t cells functions of natural killer cells in vivo treatment of mice and hamsters with antibodies to asialo gm1 increases morbidity and mortality to pulmonary influenza infection reduction of natural killer but not effector cd8 t lymphocytes in three consecutive cases of severe/lethal h1n1/09 influenza a virus infection recognition of viral hemagglutinins by nkp44 but not by nkp30 recognition of haemagglutinins on virus-infected cells by nkp46 activates lysis by human nk cells lethal influenza infection in the absence of the natural killer cell receptor gene ncr1 immune regulation: a critical link between nk cells and ctls cxcr3 ligands: redundant, collaborative and antagonistic functions viral activation of interleukin-15 (il-15): characterization of a virus-inducible element in the il-15 promoter region interferon-alpha/beta upregulate il-15 expression in vitro and in vivo: analysis in human hepatocellular carcinoma cell lines and in chronic hepatitis c patients during interferon-alpha/beta treatment il-15 is expressed by dendritic cells in response to type i ifn, double-stranded rna, or lipopolysaccharide and promotes dendritic cell activation a role for il-15 in the migration of effector cd8 t cells to the lung airways following influenza infection reversible defects in natural killer and memory cd8 t cell lineages in interleukin 15-deficient mice dendritic cells support the in vivo development and maintenance of nk cells via il-15 trans-presentation natural killer cell-dendritic cell crosstalk in the initiation of immune responses combined il-15/il-15ralpha immunotherapy maximizes il-15 activity in vivo mature natural killer cells with phenotypic and functional alterations accumulate upon sustained stimulation with il-15/il-15ralpha complexes il-15 is chemotactic for natural killer cells and stimulates their adhesion to vascular endothelium new low-viscosity overlay medium for viral plaque assays il-15 trans-presentation by pulmonary dendritic cells promotes effector cd8 t cell survival during influenza virus infection il-15/il-15 receptor biology: a guided tour through an expanding universe il-15 trans-presentation promotes human nk cell development and differentiation in vivo thymic and peripheral microenvironments differentially mediate development and maturation of inkt cells by il-15 transpresentation role of trans-cellular il-15 presentation in the activation of nk cell-mediated killing, which leads to enhanced tumor immunosurveillance il-15ralpha recycles and presents il-15 in trans to neighboring cells distribution of il-15 receptor alpha-chains on human peripheral blood mononuclear cells and effect of immunosuppressive drugs on receptor expression activated antigen-specific cd8+ t cells persist in the lungs following recovery from respiratory virus infections nk cell development, homeostasis and function: parallels with cd8 t cells central role of interleukin-15 in postovulatory recruitment of peripheral blood cd16(2) natural killer cells into human endometrium loss of il-7r and il-15r expression is associated with disappearance of memory t cells in respiratory tract following influenza infection the invariant nkt cell subset in anti-viral defenses: a dark horse in anti-influenza immunity? activation of invariant nkt cells enhances the innate immune response and improves the disease course in influenza a virus infection heterosubtypic immunity to influenza a virus in mice lacking iga, all ig, nkt cells, or gamma delta t cells cloning of a t cell growth factor that interacts with the beta chain of the interleukin-2 receptor herpes simplex virus-1 up-regulates il-15 gene expression in monocytic cells through the activation of protein tyrosine kinase and pkc zeta/lambda signaling pathways induction and regulation of il-15 expression in murine macrophages longitudinal analysis of cd8(+) t-cell phenotype and il-7, il-15 and il-16 mrna expression in different tissues during primary simian immunodeficiency virus infection subcellular expression pattern and role of il-15 in pneumococci induced lung epithelial apoptosis expression of il-15 in inflammatory pulmonary diseases expression of two interleukin-15 mrna isoforms in human tumors does not correlate with secretion: role of different signal peptides regulation of il-15 secretion via the leader peptide of two il-15 isoforms interleukin (il)-15r[alpha]-deficient natural killer cells survive in normal but not il-15r[alpha]-deficient mice cd8+ t cells rapidly acquire nk1.1 and nk cell-associated molecules upon stimulation in vitro and in vivo nk cells provide helper signal for cd8+ t cells by inducing the expression of membranebound il-15 on dcs differential cytokine and chemokine gene expression by human nk cells following activation with il-18 or il-15 in combination with il-12: implications for the innate immune response il-15 induces the expression of chemokines and their receptors in t lymphocytes interleukin (il)-15 and il-2 reciprocally regulate expression of the chemokine receptor cx3cr1 through selective nfat1-and nfat2-dependent mechanisms we would like to thank dr. r. tripp and jamie barber for use of the lsrii and guidance using the equipment. key: cord-001909-yy9xp5ms authors: buß, o.; jager, s.; dold, s. -m.; zimmermann, s.; hamacher, k.; schmitz, k.; rudat, j. title: statistical evaluation of hts assays for enzymatic hydrolysis of β-keto esters date: 2016-01-05 journal: plos one doi: 10.1371/journal.pone.0146104 sha: doc_id: 1909 cord_uid: yy9xp5ms β-keto esters are used as precursors for the synthesis of β-amino acids, which are building blocks for some classes of pharmaceuticals. here we describe the comparison of screening procedures for hydrolases to be used for the hydrolysis of β-keto esters, the first step in the preparation of β-amino acids. two of the tested high throughput screening (hts) assays depend on coupled enzymatic reactions which detect the alcohol released during ester hydrolysis by luminescence or absorption. the third assay detects the ph shift due to acid formation using an indicator dye. to choose the most efficient approach for screening, we assessed these assays with different statistical methods—namely, the classical z’-factor, standardized mean difference (ssmd), the kolmogorov-smirnov-test, and t-statistics. this revealed that all three assays are suitable for hts, the ph assay performing best. based on our data we discuss the explanatory power of different statistical measures. finally, we successfully employed the ph assay to identify a very fast hydrolase in an enzyme-substrate screening. introduction β-amino acids are intermediates in the synthesis of a great variety of pharmaceutically important compounds [1, 2] . the objective of this study is to select an hts assay to screen for one enzyme for a two-step reaction cascade for the synthesis of β-amino acids. these occur in a number of biologically active compounds such as paclitaxel, bleomycin and the lipopeptide ym-170320 [3, 4] . paclitaxel is used as a drug in the treatment of certain types of cancers [5] . as the correct configuration of the stereocenters is essential for biological activity, synthesis strategies with high enantioselectivity are desirable [2] . a variety of synthesis strategies have been established for the production of chiral β-amino acids. however, the limitations of these strategies are unfavourable for industrial production [6, 7] . in addition to the chemical approaches, enzymatic strategies have been established to produce chiral β-amino acids. since all enzymatic approaches established in a industrial scale are based on kinetic resolutions, their theoretical yield is limited to 50% at most [8] . both the enzymatic and chemical synthesis strategies are still a subject of research for the production of β-amino acids [9] . in order to achieve higher yields than 50%, β-amino acids can also be obtained with high enantiomeric excess by enzymatic conversion of β-keto acids using transaminase [9] . however, the substrates of this synthesis strategy, the β-keto acids, are not stable and decarboxylate. to avoid this side reaction β-keto acids can be generated in situ by hydrolysis of a β-keto ester catalyzed by a hydrolase. in the synthesis of natural and non-natural substrates hydrolases are a beneficial and commonly used enzyme class [10] and a number of lipases and esterases are commercially available [11] . many enzymes are well characterized, but often there is no perfect match between the requirements of an efficient catalysis reaction and the properties of the biocatalyst. besides being able to acquire hydrolases commercially, there is the possibility to test hydrolases from different sources, such as biomolecular libraries containing purified enzymes, microorganisms from the environment or hydrolases variants from directed evolution and from in silico gene data basis. the limiting factor is the fast and reliable identification of the best fitting enzyme. while investment in both, experimental time and costs, can increase sample throughput in large screenings with standard analytical hardware [12, 13] , this route can quickly become prohibitively expensive. high throughput assays permit simultaneous measurement of samples in 96-to 1536-well plates so that 10 5 to 10 7 samples may be screended per day [14] [15] [16] . this way, large biomolecular libraries can be screened for optimal enzymes [16] . a wide variety of assays has been established for the detection of efficent hydrolases in vivo or in vitro [17, 18] . hts assays for lipases and esterases have been extensively reviewed [19, 20] . hts assays may directly detect the reaction products or convert these products for indirect detection. for example a simple indirect hts-assay detects the ph-shift during a hydrolysis reaction by a ph-indicator molecule [21] . enzyme activity can be detected by a number of parameters such as microbial growth or changes in spectral properties, temperature or electrochemical potential as well as by luminescence [17] . chromatography methods are well suited for medium-throughput screenings [13] . the assessment of temperature change due to exothermic reactions has been reported, as a technically sophisticated measure requiring specialized infrared cameras, which are no standard laboratory equipment [22] . in direct hts assays product formation is monitored by a change in a physical quantity associated with the product. for this purpose, chromogenic and fluorogenic substrates are frequently applied in these assays [18] . additional analytical reactions are also commonly used [18] . these mostly synthetic substrates allow for an easy readout by changes in absorbance, fluorescence or by chemiluminescence [12] . however, when a substrate analog with an artificial chromophore or chromogenic group is employed in the the optimization process instead of the substrate of interest this may lead to an enzyme optimized processing the analog but not the substrate of interest ("you get what you screen for" you and arnold et al. 1996) [13, 19, 23, 24] . a variety of hydrolase screening assays is based on non-natural substrates with chromophoric groups. well-known examples are umbelliferyl-, 4-nitrobenzofurazane-and 4-nitrophenylassays [25] [26] [27] . the disadvantage of this assays is caused by the difference between the properties of the substrate to the non-natural substrate. the most active enzymes in this kind of screening do not have to be the most active enzyme for the natural substrate. as an example, for screening hydrolases, 4-nitrophenyl esters are popular, because standard photometric plate readers can easily monitor the reaction by detection of the colored product at 348 to 405 nm [13] . however, 4-nitrophenyl esters per se are no substrate with industrial pertinence [28, 29] . furthermore, the absorption of the liberated 4-nitrophenol at 405 nm depends on the phvalue, so that the ph value needs to be controlled. alternatively, absorbance may be measured at the isosbestic point of 4-nitrophenol at 348 nm [30, 31] . moreover, 4-nitrophenyl esters are more readily hydrolyzed than esters comprising aliphatic alcohol residues like methanol or ethanol. when 4-nitrophenyl esters are used to in hydrolase screening, the hits may therefore exhibit lower activity towards the actual substrate [19, 32] . in 2008, j. córdova et al. [33] reported enzymatic activity of bovine serum albumin (bsa) showing high hydrolysis rates for 4-nitrophenyl esters at 80-160°c. under these conditions, a spontaneous hydrolysis of 4-nitrophenyl esters is likely, so that the observed hydrolysis may not be necessarily due to an enzymatic activity of bsa. another argument against the use of 4-nitrophenyl esters is the potential reaction with nucleophilic amino acid side chains as it was shown for the reaction of 4-nitrophenyl acetate with l-tyrosine esters by b.s. hartley et al. in 1953 and the acetylation of insulin in the reaction with 4-nitrophenyl acetate [34] . modification of the enzyme by acyl-group transfer may lead to artifacts during screening. taken together, 4-nitrophenyl esters are are often not suitable as substrate analogues for screening. if possible, alternatives to the fluorophoric and chromophoric non-natural substrates should be used. if it is impossible to directly detect the conversion of the native substrate, the alternative approach is to further convert the products for indirect detection. a simple approach are colorimetric ph-assays, which can be employed if one of the products is an acid that subsequently deprotonates or a base that lowers the proton concentration in the medium. this change in ph value can be monitored by an indicator dye [35, 36] . enzymatically coupled systems that transform one of the products yielding a detectable compound are more complex. as more reaction steps and compounds required for quantification more parameters need to be optimized and the readout is more prone to errors. one of the first examples for a convenient indirect assay was the nadh-dependent, coupled enzyme assay for urease by kaltwasser et al.(1966) [37] . for the hydrolysis of β-keto esters, we compared three different indirect assays for the activity of hydrolases. one assay relies on the change of the ph-value, the second is based on enzymatic oxidation of the released ethanol to acetic acid and the third, which we expected to be most sensitive, is based on the oxidation of ethanol to ethanal and hydrogen peroxide which is then converted by horseradish peroxidase (hrp) in a luminescence reaction. the first assay is the simplest one, because only a buffer-ph indicator system is needed and many examples are established for enzyme screening with ph-indicators [21, 35, 38, 39] . the second assay is also well-known and established for measurements of alcohol in food [40] . this assay was miniaturized to microtiter plates. the third assay is a modification of a chromogenic alcohol-oxidase (aox)/peroxidase (hrp) ethanol assay for determination of ethanol in beverages, which normally based on 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (abts) as chromogenic substrate [41] . this assay was modified to a luminometric assay by adding luminol instead of a chromogenic substrate. the luminometric measurements should be more sensitive due to photons are released by the detection reaction. for the first time the system was established in a flow system with separated bioreactors by marschall and gibson [42] . for the quantification of ethanol, we adapted the luminol-aox-hrp system to microtiter plates by using design of experiments (doe). the aim was to optimize the system for endpoint measurements of enzymatic hydrolysis reactions. therefore, for the first time a luminometric aox-hrp-system was tested for quantification of ethanol in 96-well plates. in this study all assays were tested for hts compatibility. to compare the quality of high-throughput assays the z'factor is frequently employed [14, 43] . in addition, the assessment of measurement procedures can be based on traditional statistic parameters like signal-to-noise-ratio(s/n), signal to background ratio (s/b), and coefficient of variation (cv) ( table 1) . cv is the ratio of mean to variance and often used as quality control for assays [44] . the s/b-ratio is a criterion that indicates whether the level of the signal is sufficiently high above the background. the rule of thumb for a good hts assay is s/b >3. however, fluctuations in both the signal and the background are not considered. in contrast, the s/ n-ratio takes into account the standard deviation of the background. the higher the s/n ratio, the less do background fluctuation influence the desired signal. both measures indicate whether a sample could be distinguished from the background, however they don't quantify to what extent the positive and negative controls can be distinguished. therefore methods have been explicitly developed to evaluate high-throughput screenings, like z'-factor and the strictly standardized mean difference (ssmd) [45] . j.h. zhang (1999) defined the z'-factor based on the normal distribution and the 3-sigma rule of thumb [43, 46] , which implies that 99.7% of all samples lie within less than three standard deviations distance from the mean. the z'-factor has become a common metric for hts quality control as it allows to decide whether an assay is suitable to just distinguish positive samples from negative ones (z'>0,5) or whether it can distinguish well performing samples from poor ones (z'>0,8) [14, 43, 47] . the strictly standardized mean difference (ssmd) takes the mean difference of negatives and positives in proportion to the standard deviation. ssmd gained recognition in screening e.g. for antiviral drugs [48] . in 2007, x.d. zhang derived the ssmd under the condition of independence of both distributions through maximum likelihood estimation (mle) and method of moment (mm). finally, for increased robustness against outliers, the median of absolute deviation (ssmd r ) can be used. in this work, we applied for the first time these different ssmds for evaluation of a biocatalyst screening assay. ssmd have already been used in rnai screening and cell-based systems table 1 . overview of statistical measures for evaluation of hts assays. σ = standard deviation; μ = mean; m = median; pos = data set of positive controls; neg = data set of negative controls; n = sample size; f k = cumulative density function for data set k; max j = maximum distance between two distributions; ssmd = strictly standardized mean difference; x i = ith value of the (ordered) data set x. definition or for quantification of enzyme inhibition [49, 50] but never for assays of enzyme catalysis. we compared these measures to the z'factor. we also chose non-parametric methods like the ks-test and t-statistic. non-parametric test statistic differs from parametric statistic in that no explicit distribution is given, but the procedure is solely based on the data without the need for any model. the t-statistic describes the difference of two sample distributions measured in standard errors of both means. this is suitable for small sample sizes, especially when the underlying distribution is unknown or not derivable [51] . in contrast to all other methods, the kolmogorow-smirnow-test (ks-test) [52] calculates the maximum distance between two cumulative density functions (cdf). the cdf can be computed for finite number of data points as a cumulative sum of the frequency of occurrence of the (sorted) data. the unique benefit of this kind of calculation is that no previous knowledge about distribution or models is needed. we include the ks test here to contrast it with the other measures that do not rely on any cdf. all enzymes and chemicals used in the assays were purchased from sigma-aldrich (st. louis, usa) and carl roth (karlsruhe, germany). reagents were dissolved in 40 mm potassium phosphate buffer (ph 7.2) unless stated otherwise. for all buffers and solutions deionized water was used. expression and purification of para-nitrobenzyl-esterase 13 (pnb-est 13) expression was performed in e. coli bl 21 codonplus using a pet-22b vector system with a pelb-leader sequence for export to the periplasm of e.coli. after transformation cells were cultivated at 180 rpm and 30°c in shaking flasks in 400 ml lb-medium. after the od600 had using the standard conditions of the aox-hrp-abts system (described in the protocol of sigma-aldrich [54] ) the luminescence signal was very weak for ethanol (2.0 mm) [55, 56] . to gain a much longer and more intensive luminescence reaction a design of experiments with a statistic based optimization program (modde 10.1 software (umetrics, sweden)) was carried out (s4 fig and s5 fig) . for measuring luminescence, white 96-well plates (greiner, austria) were used. the final reaction volume per well was 200 μl. reaction mixtures were prepared on a tecan freedom evo 200 liquid handling station (lhs). the lhs is equipped with one eightport liquid handling arm (liha), a standard robotic plate handling arm (roma) equipped with a centric gripper, a 96-channel liquid handling arm (multichannelarmtm (mca 96), tecan) with an eccentric gripper, and an integrated spectrophotometer (infinite m200 pro, tecan). pipetting precision and accuracy of aqueous solutions was determined by pipetting on an analytical balance as described by oelmeier et al., 2010 [57] . a variation coefficient of less than 1.6% was determined for volumes between 20 and 1,000 μl. the concentrations of luminol, hrp, aox and ethanol were varied in doe to search for the maximum of luminescence intensity (slope). for quantification of ethanol in samples different dilutions of ethanol (0 to 2 mm) were added to the assay mixture. finally a 1:8330 dilution of aox (10-40 u/mg, sigma-aldrich) containing of 2 μg of hrp (150 u/mg, sigma-aldrich) were added to the assay mixture, containing 0.5 mm luminol (sigma-aldrich) dissolved in 5% (v/v) dmso (carl roth) and 95% 40 mm sodium phosphate buffer, ph 8.0. for statistical evaluation, 2.0 mm ethanol was used as a positive control without hydrolase and ester substrate (see also subsection statistical analysis). all ingredients, except hrp, were mixed at room temperature and then incubated under shaking at 150 rpm at 30°c for 15 min. the reaction was started by adding 15 μl of hrp solution (total activity 0.3 u) to the reaction mixture and mixing with a 96-tip automatic pipette of the evo workstation. afterwards the 96-well plates were placed in the infinite m200 pro reader to measure luminescence. for each measure point luminescence intensity was integrated over 350 ms. each well was repeatedly measured for up to 1 h. the working temperature was 30°c +/-2°c. the luminescence raw data was processed either as mean or as numeric integral over the duration of the experiment. to determine the ethanol concentration the commercial ethanol kit from r-biopharm ag (germany) was used. according to the manufacturer, this assay is carried out in a total volume of 3 ml measured in cuvettes at 340 nm in an ordinary spectrophotometer. the manufacturer's instructions were adapted to the 96-well plate format using 100 μl per well maintaining the ratio of compounds. the assay was started by addition of a 1:10 dilution of aldehyde dehydrogenase solution. the plate was briefly centrifuged to eliminate bubbles. the ethanol concentration was determined by an ethanol calibration curve in the range from 0 to 3 mm (s3 fig). according to the other assays a positive control for the evaluation test contained only 2.0 mm ethanol without ester and hydrolase (see also subsection statistical analysis). the protocol from moris-varas et al. was adapted [38] for endpoint measurements. a weak 2.5 sodium phosphate buffer (pk a2 = 7.2) was adjusted to ph 7.0. bromothymol blue (pk a = 7.1) [58, 59] was dissolved under heating and stirring in this buffer to yield a 0.54 mm stock solution. the assay system contained 10% (v/v) indicator stock solution. according to the other assays a positive control for the evaluation contained only 2.0 mm hcl without ester and hydrolase (see also subsection statistical analysis). absorbance was measured at the absorption maxima of bromothymol blue at 440 nm and 620 nm in transparent 96-well plates in a conventional plate reader (epoch, biotek). the hydrolytic activity was also determined based on β-keto acid formation as an indicator of product formation using the ph indicator bromothymol blue. for the determination of activity of hydrolases substrate concentration was 2.0 mm. the calibrations were done by adding different concentrations of hcl (0 to 2 mm) to the buffer in the presence of each tested ester to determine a calibration line (s3 fig) [39] . during the experiment the ph range was between ph 6.0 to ph 7.0. in this range, the 3-oxo-3-phenylpropanoic acid (β-keto acid) is almost completely dissociated. the calculated pk a for 3-oxo-3-phenylpropanoic acid is 3.56 [60] . for comparison of hydrolases equal masses of protein from 10 mg/ml stock solutions were used. the enzyme concentration of the stock solution was tested by bradford assay. for each enzyme the blank was determined in buffer with bromothymol blue and without substrate. the incubation temperature was 30°c and 2 mm (concentration in situ) ethyl benzoyl acetate was added to the reaction mixture containing hyrolase (see table 2 ). the specific activity [mol/(min á mg)] was calculated by the slope at the beginning of the reaction at 620 nm. the specific activity was converted by the number of active sites into the turnover number [1/s]. to evaluate the quality of all proposed assays, we used the equations shown in table 1 . for the ph-assay and spectroscopic ethanol-assay μ pos was the mean absorbance of the positive controls and μ neg that of the negative controls. in case of the oxidative luminescence assay μ pos was the mean intensity or integral of the positive controls and μ neg of the negative controls. for the ph indicator assay the positive control contained 2 mm hcl in 5 mm sodium phosphate buffer (ph 7.2), and the negative control was made from plain buffer. for the two different ethanol based assays, the positive control consisted of assay buffer and 2 mm ethanol, while plain assay buffer was used for the negative controls. between 44 and 48 positives and negatives were measured for each assay. the measurements took place in 96-well plates. ssmd, student t-statistic as well as the ks-statistic and other metrics were computed in r [69] for the t-statistic we used the t.test function with welch correction and for the ks test the ks.test function in r. all other metrics were implemented in r. all plots were created using the ggplot2 library [70] . for tstatistics the welch correction has been designed to account for unequal variances of both groups (positive and negative) [71] . the criteria for evaluation of the assays based on the statistic measures are listed in table 3 . r is an environment for interactive analysis of statistical data in bioinformatics offering many additional software packages. we combined all approaches and implemented the assay-toolbox package in r to make the applied methods accessible to a wide community. software link: http://www.cbs.tu-darmstadt.de/htsassay.zip upon hydrolysis of β-keto ethylesters, ethanol is released and a β-keto acid is formed. afterwards the acids can decarboxylate to carbon dioxide and acetophenone. a small amount of table 2 . enzymes for screening. for comparison of enzyme activity equal protein concentrations (mg/ml) were used. the concentrations of all hydrolase solutions were determined by bradford assay. stock solutions of hydrolases were 10 mg/ml. all enzymes but pnb-est13 were were purchased comerially. pnb-est13 was hetrologous expressed in e. coli. hydrolase carbon dioxide forms carbonic acid in water, which lowers the ph-value as well [72] (fig 1) . we used the ph shift (fig 1(b) ) visualized by the indicator bromothymol blue as a measure of product formation. we choose this indicator due to its expected ph value of the enzyme coupled system consisting of lipase and transaminase. in addition we created a new luminometric assay (fig 1(c) ) for use in 96-well plates. this assays detects the formation of the product ethanol by an enzymatic reaction. ethanol was oxidized by alcohol-oxidase to yield acetaldehyde and hydrogen peroxide, which is used to oxidize luminol to 3-aminophthalate by horseradish peroxidase (fig 1(c) ). this well-known chemiluminescent reaction is frequently used in immunoassays such as western blot or elisa [73] . for the luminescence-based assay we expected a higher sensitivity as the emitted light can be accumulated (high signal) and there is no stray light from excitation (low background) [74] . other alcohols can also be oxidized by alcohol oxidase [75] so that this assay would be applicable to the hydrolysis of different types of esters. for comparison we used the commercial ethanol assay from r-biopharm, based on the oxidation of ethanol to acetic acid in a two-step oxidation by alcohol dehydrogenase and aldehyde dehydrogenase [40] . in this reaction cascade, two equivalents of nadh are generated, which can be detected by uv/vis spectroscopy at 340 nm. the aim was to identify the best assay out of these three, i.e. the one with the highest dynamic range and most stable readout. the system also has to be sensitive enough to operate reliably with small quantities of substrate and enzymes to save costs in high-throughput screening. the first step was to optimize all assays by testing different enzyme and compound concentrations without hydrolases, with hcl or ethanol as signal inducing molecules, mostly for the luminescence assay. for comparison of the three hts assays, endpoint measurements were done. the two step oxidative luminescence assay was optimized by design of experiments (doe) to maximize the duration of luminescence and the sensitivity for endpoint measurements. the isochronic induction of luminescence in all wells was very important for the reliable quantification of ethanol. the assay mixtures were automatically pipetted by the tecan evo pipetting robot that can simultaneously pipette 96 wells. it turned out that a pre-incubation time of 15 min without peroxidase was necessary. without pre-incubation, the resulting luminescence signal was too weak for the quantification of ethanol. luminescence enhancers like 4-iodophenol [76] , were added in order to maximize the luminescence output, however no sufficient signal amplification was observed so that these additives were omitted. due to the relatively weak signal the luminescence assay was only suitable for endpoint determinations. for quantification, we calculated both the numeric integral and the mean of luminescence over the measured time. a clear luminescence signal was detectable for about 50 min. likewise, the commercial spectrometric ethanol assay was only suitable for endpoint determinations when carried out in 96-well plates. the assay was tested for kinetic measurements with the result that no correlation between substrate concentration and reaction rate of hydrolase was observed. the pre-assembled commercial assay might not be suitable for kinetics, because the concentration of nad and alcohol-dehydrogenase can not separately be varied. in contrast to these two assays, the ph indicator assay has no additional enzymes. the ph indicator, bromothymol blue, has two absorption maxima in the uv-vis spectrum at 440 and 620 nm and a pk a at the desired ph value for the cascade reaction of a hydrolase in combination with a ω-transaminase. the extinction coefficient at both wavelengths depends on the phvalue so that both were used for evaluation measurements. to evaluate these assays, we performed simple tests with positive and negative controls without the real substrate and hydrolases. one-half of each plate was filled with the positive control and the other half with the negative control. for the ph-assay, we used 2 mm hcl as positive control, which corresponds to the maximum product concentration, if all substrate was converted. for evaluation of the oxidative luminescence and spectrometric ethanol assay, we added 2.0 mm ethanol as positive control for the tested enzyme coupled assays. reaction buffer without substrate and hydrolase was used as the negative control for all assays. in fig 2 the distribution of negative and positive controls for each assay is shown. for evaluation we used the criteria for evaluation listed in table 3 . the ks-statistic value was almost equal for all assays, because all distributions seem to have a maximal distance. ks-statistic shows that all positives and negatives have a clearly separated distribution, but it cannot answer the question whether the degree of separation is acceptable for hts. consequently ks-statistic test is not necessarily helpful to compare the performance of these types of assays. z'-factor, t-statistic and ssmd allowed for a more detailed evaluation: due to the low performance in all statistical tests the lum(int) and ph-based assay at 440 nm were considered unsuitable and were rejected. the ph-based assay at 620 nm performed best, because it has the largest dynamic range (fig 2(b) ) and thus is a reliable test for the applicability of our statistical measures. ssmd yielded similar results as the z'-factor and student t-test, showing that this measure, that was developed for rnai screens, can be employed for the evaluation of enzymatic reactions (fig 3) . robust ssmd r differs from all metrics for the ph-based assay at 440 nm which indicates that outliers exist for this assay. the negative consequence of outliers can be avoided, if at least triplicates are measured. in contrast to all other measures ssmd r is suitable for selection of sensitive assays with more outliers. on the one hand, additional metrics like the statistical based screening for β-keto esters degrading enzymes mentioned ssmd r are particular useful in case of assays with outliers, because in some screening approaches they have a minor influence on the evaluation. however on the other hand statistics depend on explanatory power of the experimental data. as a conclusion of this, it should be carefully considered which metrics as well as processed raw data are suitable for a certain hts evaluation. as the ph indicator assay at 620 nm obtained the highest statistical scores it was chosen for a subsequent small scale screening of a set of different hydrolases with different derivates of ethyl benzoyl acetate (eba; fig 4) . first of all, we determined for each ester a calibration curve with hcl concentrations between 0 and 2 mm at 620 nm (s3 fig). the strong acid hcl (pk a = -6) nearly dissociates to 100% in a wide range of the ph-scale. in contrast the weak β-keto acid (pk a = 3.56) can only completely dissociate, when the ph is clearly above the pk a . a weak sodium phosphate buffer (ph 7.0) was very important to of the ph change during the reaction. the range of ph-values during the experiment was between ph 6 and ph 7. this was the prerequisite for the calculation of the enzyme activity at the beginning of an activity test by the absorbance over the time. the concentrations were calculated by the different calibration curves of each substrate. we tested each hydrolase in combination with each ester to determine which hydrolases are the most active ones and which substrates have the highest accessibility (fig 4) . when adding the enzymes to the reaction buffer containing bromothymol blue we observed a color change for amano lipase. however, upon substrate addition we detected no change in ph-value, so we assume that interactions between the enzyme and the ph-indicator, a polyaromatic molecule, may have interfered with the reaction or its colorimetric detection. therefore, it was not possible to screen the activity of amano lipase of pseudomonas fluorescens(alfp). such interfering effects of indicator molecules and enzymes are well known, see for instance banyai [12, 77] . an alternative option in ph-indicator screening might be the use of 4-nitrophenol (pk a = 7.16) as an indicator molecule. it has only one aromatic ring and is therefore less hydrophobic [77, 78] . none of the other hydrolases showed any change in absorbance with only the ph indicator in the absence of substrate. the experiment revealed that every substrate was converted by hydrolases. the lipase from rhizomucor miehei(rml) showed the highest activity of the tested enzymes while the porcine pancreas lipase mix (ppl) showed the lowest hydrolysis activity towards aromatic β-keto esters. the only esterase in the screening (pnb-est13), showed the fourth highest activity for statistical based screening for β-keto esters degrading enzymes all substrates. this shows that esterases may be considered as alternatives to lipases. this is of interest as the activity of lipases depends on hydrophobic surface interactions so that the substrate spectrum is limited. esterases could help to broaden the substrate range for enzymatic βketo acid and β-amino acid synthesis towards more hydrophilic compounds. taken together, the assay results confirm, that the ph-assay at 620 nm can indeed be used for substrate-hydrolase screenings, albeit other indicator dyes may have to be tested to reduce non-specific interactions. to explain the high activity of rml against all substrates, we consider the previous work of rehm et al. [79] . thus we compared the active site volumes and structural conformations like the opened as well as closed state of rml,candida rugosa lipase (crl) and tll [79] . for example, in water, the lipase active site is covered by a mobile element, the lid, which opens upon substrate binding due to the hydrophobic interface [63] . in contrast to rml and tll, crl possesses a large and complex lid (residues 66-92), consisting of a short and a long αhelix. a comparison of the closed and open crystal structure of crl revealed that the lid has to refold partially (s1b fig). as expected for this reason, a slower opening and closing when compared to rml and tll could be shown using molecular dynamics [79] . in contrast to crl, a fast rigid body movement of the lid was suggested for rml and tll. this opening event is suggested to be the rate limiting step during catalysis [64] . in addition several studies proposed that conformational rearrangement during the catalysis could have a much greater influence on the activity than binding energy inside the substrate pocket [80] [81] [82] . the discussion on the influence of the dynamic on the activity of the enzyme is still on going [83] . although crl possesses a even bigger lid than rml and tll, the same approximated active site volume was calculated for all three lipases (rml: 59.5 nm 3 ; crl 60.3 nm 3 ; tll 57.3 nm 3 , s1 fig) beside this, the substrate properties of the ethyl benzoyl acetate substituents might have an influence on the activity caused by polarization and steric differences. we compared three different assays based on a set of positive and negative controls by end point measurements. for this purpose we applied for the first time an alcohol oxidase-peroxidase-luminescence assay for ethanol quantification in 96-well format. additionally, we evaluated a ph-indicator and a commercial ethanol assay for hts. we applied several statistical measures for biocatalysis assay evaluation and found that classical z'-factor, ssmd and t-statistic can indicate whether an assay is suitable for hts. the ph-indicator assay based on color change of bromothymol blue at 620 nm upon acid formation performed best. however, strictly considered is the most accurate way to evaluate hts assays to test the complete coupled assays (consisting of: enzyme(s) of interest, substrate(s) and assay compounds), then all necessary assay compounds, substrates and catalysts have to be tested in combination with each other. otherwise possible cross-interactions of all assay substances can not be excluded when not the complete system is tested. but this approach is not suitable for a screening with the variation of more than one compound, caused by the exponentially growing number of experiments when two or more different compounds are tested. a further obstacle for evaluation of hts assays by the whole testing system is that the hydrolases have to be previously inactivated in presence of the substrate for an end-point measurement. moreover, the limiting factors for an evaluation are often enzymes as well as substrates availability (e.g. environmental samples). therefore, a robust reduced evaluation approach might be suitable and weighed against potential benefits of a complete coupled system. in addition the non-coupled evaluation system could be also adapted for more complex screenings e.g. in case for enzymes from microbial lysates. to accomplish this a standard lysate from the expression host could be included into the evaluation setup. beside this, using the ph assay in a screen of a panel of mostly commercially available lipases we identified the rml as the most efficient enzyme for the hydrolysis of the tested set of aromatic β-keto ethyl esters. we were able to explain those findings by structural models of the enzymes' binding pocket and lid as well as by comparison of our data with descriptive literature on lipases dynamics, including md. furthermore, we demonstrated that the esterase pnb-est-13 is also suitable for aromatic βketo ester hydrolysis. this may help to broaden the substrate spectrum since esterases may accept more hydrophilic substrates than lipases. the ph-assay is a very useful method in the search for efficient hydrolases for a cascade reaction as it is robust, inexpensive and allows to record reaction kinetics. our best-performing hydrolase, rml, could be used in combination with ω-transaminase, in cascade reactions for the synthesis of β-amino acids. all tested β-keto esters could be utilized for cascade reaction with hydrolase and ω-transaminase [84] . the newly proposed enzymatic oxidative luminescence assay, still requires further optimization. however, it bears the potential for a more sensitive assay, due to the cumulative measurement and the lower background compared to fluorescence and absorption measurements [85] . beside this, we want to extend our modelling efforts to validate our hypothesis for the activity of the rml and to make predictions for the 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by the yeast, pichia pastoris. purification and properties of the alcohol oxidase phenols as enhancers of the chemiluminescent horseradish peroxidase-luminol-hydrogen peroxide reaction: application in luminescence-monitored statistical based screening for β-keto esters degrading enzymes plos one | quantitative screening of hydrolase libraries using ph indicators: identifying active and enantioselective hydrolases solvent-induced lid opening in lipases: a molecular dynamics study. protein science conformational flexibility in glutamate dehydrogenase: role of water in substrate recognition and catalysis intrinsic dynamics of an enzyme underlies catalysis enzyme dynamics during catalysis are there dynamical effects in enzyme catalysis? some thoughts concerning the enzymatic chemical step biochemical properties and crystal structure of a β-phenylalanine aminotransferase from variovorax paradoxus a comparison of assay performance measures in screening assays: signal window, z' factor, and assay variability ratio novel analytic criteria and effective plate designs for quality control in genome-scale rnai screens the authors would like to thank for the german federal ministry of education and research (bmbf) for funding within the molecular interaction engineering (mie) project (funding code 031a095b). in addition, we gratefully acknowledge christoph syldatk at karlsruhe institute of technology (kit), technical biology, for the provision of laboratory materials. key: cord-000079-533xlisc authors: huszthy, peter c.; giroglou, tsanan; tsinkalovsky, oleg; euskirchen, philipp; skaftnesmo, kai ove; bjerkvig, rolf; von laer, dorothee; miletic, hrvoje title: remission of invasive, cancer stem-like glioblastoma xenografts using lentiviral vector-mediated suicide gene therapy date: 2009-07-20 journal: plos one doi: 10.1371/journal.pone.0006314 sha: doc_id: 79 cord_uid: 533xlisc background: glioblastoma is the most frequent and most malignant primary brain tumor with a poor prognosis. the translation of therapeutic strategies for glioblastoma from the experimental phase into the clinic has been limited by insufficient animal models, which lack important features of human tumors. lentiviral gene therapy is an attractive therapeutic option for human glioblastoma, which we validated in a clinically relevant animal model. methodology/principal findings: we used a rodent xenograft model that recapitulates the invasive and angiogenic features of human glioblastoma to analyze the transduction pattern and therapeutic efficacy of lentiviral pseudotyped vectors. both, lymphocytic choriomeningitis virus glycoprotein (lcmv-gp) and vesicular stomatitis virus glycoprotein (vsv-g) pseudotyped lentiviral vectors very efficiently transduced human glioblastoma cells in vitro and in vivo. in contrast, pseudotyped gammaretroviral vectors, similar to those evaluated for clinical therapy of glioblastoma, showed inefficient gene transfer in vitro and in vivo. both pseudotyped lentiviral vectors transduced cancer stem-like cells characterized by their cd133-, nestinand sox2-expression, the ability to form spheroids in neural stem cell medium and to express astrocytic and neuronal differentiation markers under serum conditions. in a therapeutic approach using the suicide gene herpes simplex virus thymidine kinase (hsv-1-tk) fused to egfp, both lentiviral vectors mediated a complete remission of solid tumors as seen on mri resulting in a highly significant survival benefit (p<0.001) compared to control groups. in all recurrent tumors, surviving egfp-positive tumor cells were found, advocating prodrug application for several cycles to even enhance and prolong the therapeutic effect. conclusions/significance: in conclusion, lentiviral pseudotyped vectors are promising candidates for gene therapy of glioma in patients. the inefficient gene delivery by gammaretroviral vectors is in line with the results obtained in clinical therapy for gbm and thus confirms the high reproducibility of the invasive glioma animal model for translational research. glioblastoma is the most frequent and most malignant primary brain tumor. despite advances in neurosurgery, radiation and chemotherapy, the prognosis of patients remains poor with a median survival of 14 months [1] . a major drawback in translational brain cancer research has been the lack of suitable animal models. syngeneic-or xenograft tumors based on glioblastoma cell lines cultured as monolayers grow as circumscribed and highly angiogenic lesions in vivo [2] , lacking the invasive tumor cells, which represent an important feature of human glioblastoma. the invasive cells migrate away from the initial tumor mass and can cause recurrent tumors in different regions of the brain. thus, these cells represent a major therapeutic target. a recently established model in which glioblastoma biopsybased spheroids are serially passaged in the brains of nude rats shows highly invasive and angiogenic features [3] . therefore, this model is well suited for the study of new therapeutic strategies. still, reports using this or other clinically relevant models for experimental therapy are scarce. recently, we analyzed the therapeutic potential of the hsv-1-based oncolytic herpes vector g207 in the biopsy spheroid-based gbm model. the tumor volume in treated animals was reduced compared to control groups, but there was no significant survival advantage [4] . in contrast, the same therapy was more effective in a cell line-based animal model [5] and as a result is currently investigated in a phase i/ii clinical study [6] . in the present investigation we used the invasive xenograft model to evaluate transduction and therapeutic efficacy of lentiviral pseudotyped vectors. gammaretroviral vectors derived from the moloney murine leukemia virus (mmlv) have been the most frequently used retroviruses for gene therapy of brain tumors [7] [8] [9] [10] . however, despite promising results in animal models, clinical trials using retroviral vector supernatants or retroviral packaging cells have failed [11] [12] [13] . one major drawback of gammaretroviral vectors is the exclusive transduction of dividing cells, since in human gliomas, the majority of tumor cells do not divide within a given treatment window. therefore, lentiviral vectors with their ability to also transduce non-dividing cells are attractive candidates for the treatment of brain cancer. in previous studies, we have developed gammaretroviral and lentiviral vectors pseudotyped with the glycoproteins (gp) of the lymphocytic choriomeningitis virus (lcmv) [14, 15] . these vectors have a broad host range and can be concentrated by ultracentrifugation for in vivo applications. in addition, lcmv-gp is not cytotoxic, and stable recombinant packaging cell lines can be established [16, 17] . recently, we showed that lentiviral lcmv-gp pseudotypes efficiently delivered transgenes to rat glioma cells in vivo, while resident neurons were not transduced [18] . furthermore, we showed a significant therapeutic effect of lcmv-gp pseudotyped lentiviral vectors in the cell-line based 9l rat glioma model using the suicide gene hsv-1-tk. vsv-g lentiviral pseudotypes also showed a significant efficacy, similar to that of lcmv pseudotypes, which was mainly mediated by a bystander effect of transduced normal brain cells [19] . in the presented work, we showed that both, vsv-g and lcmv-gp pseudotyped lentiviruses efficiently transduced human glioma cells in vitro and in vivo, whereas gammaretroviral transduction was inefficient. the gene transfer to glioma cells was efficient for both lentiviral pseudotyped vector types. however, it was more specific using lcmv-gp pseudotyped vectors, as vsv-g pseudotypes also transduced host brain cells in invasive areas. analysis of transduced tumor cells revealed that both lentiviral vectors targeted cd133-positive as well as cd133negative cancer cells. furthermore, transduced glioblastoma cells expressed the stem cell markers nestin and sox2. importantly, when evaluated for therapeutic application using hsv-1-tk as a transgene, both lentiviral vectors mediated complete tumor regression on mri, resulting in a highly significant survival benefit (p,0.001) compared to the control groups. the collection of human biopsy tissue was approved by the regional ethical committee. the handling of the animals and the surgical procedures were done in accordance with the norwegian animal act and the local ethical committee approved the protocol. the human embryonic kidney cell line 293t (atcc number crl-11268) and the te671 cell line were obtained from the american type culture collection (atcc, manassas, va) and maintained in dulbbeco's modified eagle medium (dmem) supplemented with 10% fetal calf serum (fcs) and 1% glutamine. all cell lines were grown at 37uc in a humidified atmosphere of 5% co 2 . tumor fragments from glioblastoma multiforme patients were obtained during surgery. tissue specimens were taken from viable tumor areas that corresponded to regions with contrast enhancement on preoperative mri-scans. the specimens were transferred to test tubes containing complete growth medium, and spheroids were prepared as previously described [20] . the same method was applied for tumor material passaged in nude rats. briefly, tissue samples were minced into ,0.5 mm fragments and placed into 80 cm 2 tissue culture flasks (nunc, roskilde, denmark) base-coated with 0.75% agar (difco, detroit, mi). the spheroids were maintained in a standard tissue culture incubator with 5% co 2 and 100% relative humidity at 37uc. the medium was changed once a week. spheroids with diameters between 400 and 600 mm were selected for in vitro experiments and for intracerebral implantation. tumors were dissociated using the neuronal dissociation kit (miltenyi, bergisch-gladbach, germany) according to the manufacturer's protocol. cells were analyzed and sorted on a moflo cell sorter (beckman coulter, usa; former cytomation, usa), equipped with a coherent enterprise 621 argon-ion laser tuned to 488 nm (used at 180 mw), and 635 nm diode (25 mw). two mg/ml propidium iodide -pi (molecular probes) were added to the samples before flow sorting to facilitate dead cell discrimination. the gfp and pi were excited at 488 nm and fluorescence was measured through 530/40 bp and 613/20 bp optical filters (all filters from omega optical, brattleboro, vt, usa), respectively. doublets were discriminated using a forward light scatter (fsc) versus pulse width. fl3 channel (in logarithmic mode) with fsc were used to display and gate out pi positive/dead cells. fsc and side light scatter (ssc) signals were detected and shown in linear mode. gfp+ cells were defined on ssc versus fl1 (in logarithmic mode) dot plot after exclusion of dead cells and debris as described above. for analysis of cd133 expression cells were stained with allophycocyanin (apc) conjugated monoclonal cd133/1 (ac133) antibodies (miltenyi, bergisch-gladbach, germany), according to the manufacturer's general protocol for immunofluorescent staining (for 10 min in the dark at 4uc). cd133-apc was excited at 635 nm, the fluorescence was measured through 670/ 30 bp optical filter, and alive cd133+ cells were defined on ssc versus fl6 (in logarithmic mode) dot plot. non-stained cell suspension was used as a control. gfp+ tumor cells were sorted in ''purify 1'' mode into polypropylene round-bottom falcon tubes (becton dickinson labware europe, france) containing culture media, that were placed on ice. aliquots from some samples at the end of the sorting were removed and reanalyzed for control of the sort purity that was greater than 98%. sorted cells were either cultured in neurobasal medium (invitrogen, carlsbad, ca) with b27 supplement (20 ml/ml; invitrogen), glutamax (10 ml/ml; invitrogen), fibroblast growth factor 2 (20 ng/ml; peprotech, rocky hill, nj), epidermal growth factor (20 ng ml; peprotech) or transferred to dmem supplemented with 10% fetal calf serum (fcs) and 1% glutamine and grown on cover slips in 24 well plates. immunofluorescence staining of spheroids/adherent cells spheroids/adherent cells were stained with human-specific mouse-anti-nestin antibodies (millipore, billerica, ma), goat-anti-sox2 antibodies (r&d), mouse-anti-b2tubuliniii (millipore) antibodies and mouse-anti-gfap antibodies (millipore). primary antibodies were incubated overnight at 4uc. alexa-fluor647-goatanti-mouse und alexa-fluor647-donkey-anti-goat antibodies (dianova, hamburg, germany) were used as secondary antibodies over night at 4uc (for spheroids) or for 2h at room temperature (for adherent cells). spheroids were examined under a fluorescence microscope (nikon, tokyo, japan) and adherent cells were analyzed by confocal scanning laser microscopy (zeiss, jena, germany). the lentiviral vector plasmid prrl.sincmvegfppre was published by naldini et al. [21] . the construction of the lentiviral vector prrl.sincmv-tk/egfppre has been described previously. the retroviral vector pmp71-egfp-pre has been described previously [22] . the 293t cell line was used for transient lentiviral vector production. the lentiviral vector plasmid prrl.sincmv-tk/ egfppre (5 mg) or prrl.sincmvegfppre (5 mg), the hiv gagpol-rev expression plasmid pcmv-dr8.91(12.5 mg) [21] and 2 mg of the envelope expression plasmid phcmv-lcmv-gp [14] or pcmv-g [23] were cotransfected into 293t cells and concentrated as described previously [18] . for the production of retroviral vectors, 293t cells were transfected with 7.5 mg of pmp71-egfp-pre, 12.5 mg of psv-mo-mlvgagpol, and 2 mg of the envelope expression plasmid phcmv-lcmv-gp [14] or pcmv-g [23] . vectors were harvested and concentrated as described previously [24] . vectors were titered on te671 cells as described previously [18] . intracranial implantation of glioblastoma spheroids was done as described previously [25] . three weeks to one month after implantation, the animals were anesthetized and prepared for vector injection. the skin was withdrawn to reveal the location of the craniotomy. 2 times 10 ml of vector stocks were delivered into the centre of the tumors using a glass syringe (model 701, hamilton, bonaduz, switzerland) secured in a microprocessor-controlled infusion pump (ump 2-1, world precision instruments, aston, stevenage, uk). the injection coordinates were estimated after analyzing mri images for each individual lesion. vector infusion was done by convection enhanced delivery in the course of 25 min (200 nl/min for 10 min, followed by 400 nl/min for 10 min, and finally 800 nl/ min for 5 min). after infusion, the needle was left in place for 5 min to avoid vector reflux. the needle was slowly retracted and the skinfolds were closed with polyamide surgical thread. following surgery, rats were allowed to recover in an incubator set at 35uc before returning them to the cages. rats bearing glioblastoma xenografts were treated by daily i.p. injections of 50 mg/kg ganciclovir (gcv, roche, basel, switzerland). animals were euthanized and perfused with sterile saline and thereafter with 4% paraformaldehyde. brains were removed, suspended in 30% sucrose for three days, and then snap frozen in isopentane chilled with dry ice. coronal sections (12 mm) were prepared on a cryostat. for immunofluorescence analysis, sections were stained with human-specific anti-nestin antibodies (millipore) for human glioblastoma cells, mouse-anti-neun (millipore) antibodies for neurons, rat specific mouse-anti-nestin antibodies (millipore) for astrocytes and progenitor cells. primary antibodies (dilution 1:200) were incubated overnight at 4uc. biotinylated goat-anti-mouse and goat-anti-rabbit (vector laboratories, burlinghame, ca) were used as secondary antibodies (dilution 1:100) for 2 h at room temperature. sections were incubated with extravidin-cy3 (sigma, st. louis, mo) as fluorochrome (dilution 1:200) for 1 h at room temperature. the sections were examined under a fluorescence microscope (nikon) and analyzed by confocal scanning laser microscopy (zeiss, jena, germany). for analysis of transduction efficacy, consecutive sections (every 20.-30.) throughout the tumors were examined under a fluorescence microscope (nikon) with an automated stage using 106magnification. the transduction volume was calculated using nikon lucia imaging software. paraffin embedded formalin-fixed tissue sections from rat brain and patient material were placed in xylene bath for 263 minutes, absolute ethanol 263 minutes, 96% ethanol 262 minutes and finally in distilled water for 30 seconds for removal of paraffin and rehydration. epitope retrieval was performed by heating the sections at 99uc for 20 minutes in 10 mm citrate buffer at ph 6.0. the sections were incubated with a monoclonal human-specific anti-nestin antibody 1:200 in tbs/1%bsa over night at 4uc. a biotinylated goat-anti-mouse antibody (vector laboratories) was used as secondary antibody (dilution 1:100) for 1 h at room temperature followed by abc-complex incubation for 30 min. sections were developed with with 393-diaminobenzidine (dako cytomation), following the manufacturer's instructions. using a bruker pharmascan 7 tesla mr scanner (bruker biospin, billerica, ma), axial t2-weighted rare sequences were acquired (repetition time, 4,200 ms; echo time, 36 ms; slice thickness, 1 mm; field of view, 3.2 cm; matrix size, 2566256; 20 slices). during scanning, the animals were kept under anesthesia with 1.5% isofluorane (schering-plough, kenilworth, nj) mixed with 50% air and 50% o2. survival was analyzed by a log-rank test based on the kaplan-meier test using spss software. differences between pairs of groups were determined by the student's t-test. p values,0.05 were considered significant. human glioblastoma spheroids derived either directly from the patient (low generation) or from serial in vivo passages in the brains of nude rats (high generation), were infected with lentiviral lcmv-gp (5610 4 in 10 ml) or vsv-g pseudotyped lentiviral vectors (both 5610 4 particles in 10 ml) or with retroviral mlv-based vectors pseudotyped with lcmv-gp (1610 5 particles in 10 ml). the vectors were prepared in the same way for in vitro and in vivo experiments (see materials and methods). both lentiviral vectors transduced patient spheroids and high generation spheroids very efficiently ( figure 1 ). in contrast, retroviral vectors transduced only a few single cells in high generation spheroids ( figure 1 ) and failed to transduce patient spheroids (data not shown). in conclusion, both lentiviral vectors are much more efficient in transducing human glioblastoma spheroids in vitro than retroviral vectors. to compare the transduction efficiency of lentiviral and gammaretroviral vectors in vivo, we used a xenograft model that reflects the angiogenic and invasive features of human glioblastoma in situ. the xenograft also expresses the neural progenitor marker nestin and closely recapitulates the histology of the patient tumor ( figure 2 ). the vectors were injected into the center of progressively growing lesions using microprocessor-controlled stereotactic infusion. the injection coordinates were estimated after analyzing mri images for each individual lesion. the injection volume applied was 2610 ml and the vector titre 1610 7 / ml for all vectors. transduction efficiency was evaluated 7 days after vector injection. both lentiviral pseudotyped vectors showed very efficient transduction of the tumors ( figure 3a,d) . when analyzed at higher magnification, both lcmv-gp and vsv-g pseudotyped lentiviral vectors showed efficient transgene delivery to nestin-positive tumor cells in solid ( figure 3b ,e) and invasive tumor areas ( figure 3c ,f). in contrast, the retroviral vector only transduced a few scattered tumor cells near the injection site (figure 3 g,h) . for a quantitative comparison of transduction efficiency between the two lentiviral pseudotyped vectors, the gfp-positive areas were measured on histological slides (see material and methods). the total volume of transduced tumor tissue was 7.0563.51 mm 3 for lcmv-gp pseudotyped vectors and 4.0562.04 mm 3 for vsv-g pseudotyped vectors ( figure 3i) . although there was a difference in the mean, it was not statistically significant (p = 0.269) due to high interindividual differences (standard deviations). to analyze transduction specificity, histological sections of invasive tumor areas were stained for rat specific markers neun (for neurons) and nestin (for astrocytes and progenitor cells). lcmv-gp pseudotyped lentiviral vectors exclusively transduced tumor cells in all invasive areas ( figure 4a ), while normal brain cells were not transduced ( figure 4b,c) . also vsv-g pseudotyped vectors showed specific transduction of tumor cells in some invasive areas ( figure 4d,e) , however, they also transduced a few host brain cells at other sites ( figure 4g,h) . to analyze the potential of both lentiviral vectors to infect cancer stem-like cells, transduced tumors were enzymatically dissociated and cd133 expression was measured by flow cytometry. both vectors transduced cd133-positive and cd133-negative cells ( figure 5a ). although there were high interindividual differences in the fraction of total cd133-positive tumor cells in the different xenografts, both vectors showed similar efficiency in transducing cd133-positive cells, which was slightly higher than the overall fraction of cd133-positive cells (table 1) . the gfp-positive cells from tumors transduced with lcmv-gp or vsv-g pseudotyped lentiviral vectors were sorted and cultured in neural basal medium supplemented with egf and bfgf. transduced tumor cells from both vectors were able to form spheroids ( figure 5b,e) . spheroids expressed the stem cell markers nestin and sox2 ( figure 5c,d,f,g) . sorted cells were also plated under serum conditions. the cells continued to show significant expression of the stem cell markers nestin and sox2, but also of the differentiation markers gfap and b-tubuliniii ( figure 5h-o) . to evaluate the therapeutic efficacy of both lentiviral pseudotyoped vectors in the invasive xenograft model, vectors expressing the suicide gene hsv1-tk fused to egfp were injected into established tumors when visible on mri using the same method as described for the in vivo tropism study. the animals were treated daily with 50 mg/kg ganciclovir for 30 days starting 7 days post vector injection. tumor growth was measured every 7-14 days by mri. after 4 weeks of treatment, 7 out of 8 animals in both the lcmv-and the vsv-pseudotype treated groups had a complete remission on mri ( figure 6 ). one animal in each group had a stable disease until the end of gc treatment. all animals in the control groups developed large tumors during the treatment period of 30 days ( figure 6 ). both, lcmv-and vsv-pseudotype treated animals had a highly significant survival advantage (p,0.001) compared to the control groups ( figure 7a ). there was no statistically significant difference in survival between the two treatment groups. upon cessation of prodrug administration, all animals developed recurrent tumors, which could be classified into three different groups ( figure 7b-e, table 2) . the animals showed either 1) local recurrences or 2) local and/or contralateral recurrences or 3) recurrences in other distant brain areas. there was no clear difference in the recurrence pattern between the two vector types, but lcmv-pseudotyped vector-treated animals had more contralateral recurrences, whereas vsv-g pseudotyped vector-treated animals had more local recurrences ( figure 7b -e, table 2). histopathological and confocal microscopic analysis of the lesions revealed gfp-positive cells in all recurrent tumors demonstrating that not all transduced glioma cells were killed by ganciclovir treatment (figure 7f-m) . in vsv-g pseudotype-treated animals, the gfp-positive surviving cells were found in invasive areas ( figure 7f ,g), the corpus callosum region ( figure 7h ) and also in some regions of distant recurrences. one animal also showed a focus of transduced normal brain cells at the tumor border that survived gc treatment ( figure 7i ). in the lcmv group, most gfp-positive cells were found in the ipsilateral hemisphere, in solid and invasive tumor areas ( figure 7j ,k,l), with only a few cells seen in the contralateral hemisphere ( figure 7m ). future success of glioma gene therapy will depend on more potent vector systems that show higher transduction efficiency than the systems that are available today. in addition, the application of representative animal models that recapitulate both, the invasive and angiogenic features of patient tumors, is vital in order to minimize the huge discrepancies between the experimental results and clinical outcomes previously observed for gene therapeutic strategies for brain cancer. to this end, we applied one of the most clinically relevant animal models for glioblastoma known. this model was established several years ago [20] and its growth pattern as well as geno-and phenotypic similarity to glioblastoma in patients has been extensively characterized [3] . a striking difference of our model compared to other cell-line based models is the highly invasive behaviour of the lesions, similar to glioblastoma in patients. our model is based on spheroids derived from patient biopsies that are passaged serially in the brains of nude rats. first generation tumors are highly invasive and grow without signs of angiogenesis. late generation tumors show an angiogenic phenotype, but are still invasive. our in vitro experiments revealed that both lentiviral vectors transduced spheroids derived from both low and high generation tumors very efficiently. in contrast, retroviral vectors transduced only high generation spheroids and displayed a much lower efficiency of gene transfer than both lentiviral vectors. this difference can only be attributed to the vector backbone, as the glycoprotein which is responsible for virus entry into the cell was the same for the retroviral and one of the lentivral vectors applied (lcmv-gp). the most important feature that distinguishes lentiviral from retroviral vectors is their ability to infect non-dividing cells. it is known that glioma spheroids, especially primary biopsy spheroids, contain a significant fraction of non-dividing cells, which cannot be transduced by retroviral vectors. it has previously been shown that the cultured biopsy spheroids show a similar cell proliferation as seen in glioblastoma patients [20] . previous studies have demonstrated that retroviral vectors can very efficiently transduce highly proliferative monolayer cultures of glioma cell lines [14] . however, monolayer cultures change their geno-and phenotypic characteristics under long term culture and thus are not a suitable model to answer clinically relevant questions [26] . for in vivo experiments, we selected a high generation xenograft that showed both angiogenic and invasive features and recapitulated the histology of the patient lesion. furthermore, this xenograft showed a high level of nestin expression similar to the patient material. in translational research it is crucial to assure that the experimental tumors truly reflect the corresponding patient's tumor properties to avoid using non-relevant animal models. however, this strategy is not common practice yet, based on the simplicity of using established cell lines for in vivo experiments. we showed a high transduction efficiency of lentiviral vectors for glioma cells in vivo, whereas retroviral vectors transduced a few scattered tumor cells near the injection track. this is in contrast to in vivo studies by others where retroviral vectors were very efficient, but as mentioned above, the models applied were non-invasive, based on cell lines cultured as monolayers [27] . of note, the results of clinical studies using retroviral vectors showed the same low transduction efficiency as observed in our model system [12] . this finding provides further evidence that the glioma model used here has a higher predictive value for the performance of a novel therapeutic approach in the clinic than previous animal models. the tropism for glioma cells was more specific with lcmv-gp lentiviral pseudotyped vectors, as vsv-g pseudotyped lentiviral vectors also transduced few normal brain cells in invasive areas. in previous studies using a rat glioma model, we also showed a more specific transduction of glioma cells by lcmv-gp pseudotyped vectors compared to vsv-g pseudotyped vectors [18, 19] . in fact, in these studies, the vsv-g pseudotyped vectors transduced normal brain cells at a much higher frequency than in this study. this can be explained by the mode of vector delivery, because in the previous studies, we injected the vectors both into the tumor core as well as into tumor border areas. in the present study, we injected the vectors into the tumor core only using convection enhanced delivery. we used this method because it results in a high distribution volume of drug and vector and is currently used as a delivery method in clinical studies [28] . in addition, the tumor model we apply here is highly invasive and lacks a sharply demarcated brain tumor/normal brain interface, present in the rat glioma model. another explanation could be the species difference as we used human glioma cells in this study in contrast to rat glioma cells in the previous work. vsv-g pseudotyped vectors might have a higher tropism for human glioma cells than for rat normal host cells. however, as the receptor for vsv-g is unknown [29] , this remains a hypothesis. the targeting of cancer stem cells or cancer stem-like cells in human tumors including glioblastoma has recently evolved as a major aim in cancer therapy. these stem cells are suggested to initiate cancer and might be resistant to therapy, thus being responsible for tumor recurrence [30] [31] [32] . yet, recent studies have initiated a controversial discussion whether cancer stem cells really exist [33, 34] . therefore, we use the term ''cancer stem-like cells'' in our study to designate cells which have certain stem-like properties described previously. we showed that both lentiviral vectors transduced cd133-positive and cd133-negative cells. cd133-positive cells have been identified as cancer initiating cells and cancer stem cells in many different cancers including glioblastoma [30, 35] . however, more recent reports questioned these findings showing that cd133-negative popopulations can include cancer initiating cells as well [36, 37] . the efficient targeting of cd133-positive and cd133-negative glioma cells has also been described for adenoviral vectors [38, 39] . we further demonstrated that sorted cells from tumors transduced either by lentiviral lcmv-gp or vsv-g pseudotyped lentiviral vectors had the ability to form spheroids upon culturing in neural basal medium supplemented with egf and bfgf. spheroids from both sorted cell populations expressed the neural stem cell markers nestin and sox2 and showed the ability to express the differentiation markers gfap and b-tubuliniii under serum conditions. these properties have been described for neural progenitor cells [40] as well as for cancer stem-like cells in human glioblastoma [26] . thus, the cell populations transduced by lcmv or vsv pseudotyped lentiviral vectors showed features of cancer stem-like cells which might be an important target for therapy. in a therapeutic approach using the suicide gene hsv-1-tk fused to egfp, we demonstrated a highly significant therapeutic effect for both lentiviral vectors compared to control groups. using mri to follow tumor growth, we detected complete remission in 7 out of 8 animals for lcmv-gp and vsv-g pseudotyped vectors after 30 days of ganciclovir treatment. hsv-1-tk has been reported to be an effective therapeutic gene in previous studies [19, 41] . the limited success in clinical studies has been a result of inefficient gene delivery systems rather than lack of efficacy of the suicide mechanism [11] . however, there is still space for improvement of the prodrug delivery such as application length, treatment intervals and route of delivery. tai et al. demonstrated that multiple cycles of prodrug application are superior over one cycle of prodrug [42] . in our study, we still detected gfp-positive tumor cells after one cycle (30 days) of ganciclovir administration in treated animals indicating that application in cycles might also improve the therapeutic effect in this setting. further, these results demonstrate that vector-transduced tumor cells retain the ability to invade brain tissue and migrate even to distant brain regions. in conclusion, the present study demonstrates an efficient transduction and therapy of experimental human glioblastoma by lentiviral vectors. the inefficient gene transfer by gammaretroviral vectors is in line with the results obtained in clinical trials and thus confirms the high relevance of the spheroid-based glioma animal model for translational research. radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma expression of extracellular matrix components in a highly infiltrative in vivo glioma model angiogenesis-independent tumor growth mediated by stem-like cancer cells oncolytic herpes simplex virus type-1 therapy in a highly infiltrative animal model of human glioblastoma attenuated multi-mutated herpes simplex virus-1 for the treatment of malignant gliomas phase ib trial of mutant herpes simplex virus g207 inoculated pre-and post-tumor resection for recurrent gbm eradication of rat malignant gliomas by retroviral-mediated, in vivo delivery of the interleukin 4 gene inhibition of glioma angiogenesis and growth in vivo by systemic treatment with a monoclonal antibody against vascular endothelial growth factor receptor-2 eradication of murine brain tumors by direct inoculation of concentrated high titer-recombinant retrovirus harboring the herpes simplex virus thymidine kinase gene highly efficient and tumorrestricted gene transfer to malignant gliomas by replication-competent retroviral vectors clinical trials with retrovirus mediated gene therapywhat have we learned? a phase iii clinical evaluation of herpes simplex virus type 1 thymidine kinase and ganciclovir gene therapy as an adjuvant to surgical resection and radiation in adults with previously untreated glioblastoma multiforme thymidine kinase gene therapy for human malignant glioma, using replication-deficient retroviruses or adenoviruses oncoretrovirus and lentivirus vectors pseudotyped with lymphocytic choriomeningitis virus glycoprotein: generation, concentration, and broad host range retroviral vectors pseudotyped with lymphocytic choriomeningitis virus recombinant expression of lymphocytic choriomeningitis virus strain we glycoproteins: a single amino acid makes the difference a retroviral packaging cell line for pseudotype vectors based on glioma-infiltrating progenitor cells selective transduction of malignant glioma by lentiviral vectors pseudotyped with lymphocytic choriomeningitis virus glycoproteins normal brain cells contribute to the bystander effect in suicide gene therapy of malignant glioma multicellular tumor spheroids from human gliomas maintained in organ culture in vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector context dependence of different modules for posttranscriptional enhancement of gene expression from retroviral vectors a general method for the generation of high-titer, pantropic retroviral vectors: highly efficient infection of primary hepatocytes retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus s protein widespread dispersion of adeno-associated virus serotype 1 and adenoassociated virus serotype 6 vectors in the rat central nervous system and in human glioblastoma multiforme xenografts tumor stem cells derived from glioblastomas cultured in bfgf and egf more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines vesicular stomatitis virus g pseudotyped retrovector mediates effective in vivo suicide gene delivery in experimental brain cancer convection enhanced drug delivery of novel therapeutic agents to malignant brain tumors phosphatidylserine is not the cell surface receptor for vesicular stomatitis virus identification of human brain tumour initiating cells isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma glioma stem cells promote radioresistance by preferential activation of the dna damage response tumor growth need not be driven by rare cancer stem cells efficient tumour formation by single human melanoma cells identification of a cancer stem cell in human brain tumors cd133(+) and cd133(2) glioblastoma-derived cancer stem cells show differential growth characteristics and molecular profiles cd133 negative glioma cells form tumors in nude rats and give rise to cd133 positive cells lowdose radiation enhances survivin-mediated virotherapy against malignant glioma stem cells adenoviruses 16 and cv23 efficiently transduce human low-passage brain tumor and cancer stem cells isolation and characterization of neural progenitor cells from post-mortem human cortex bystander killing of malignant glioma by bone marrow-derived tumorinfiltrating progenitor cells expressing a suicide gene single-shot, multicycle suicide gene therapy by replication-competent retrovirus vectors achieves long-term survival benefit in experimental glioma we thank mariana carstov, narve brekka, roswitha seyd, hege dale and endy spriet for expert technical assistance. key: cord-000540-bbjmcdo5 authors: hellard, eléonore; pontier, dominique; sauvage, frank; poulet, hervé; fouchet, david title: true versus false parasite interactions: a robust method to take risk factors into account and its application to feline viruses date: 2012-01-03 journal: plos one doi: 10.1371/journal.pone.0029618 sha: doc_id: 540 cord_uid: bbjmcdo5 background: multiple infections are common in natural host populations and interspecific parasite interactions are therefore likely within a host individual. as they may seriously impact the circulation of certain parasites and the emergence and management of infectious diseases, their study is essential. in the field, detecting parasite interactions is rendered difficult by the fact that a large number of co-infected individuals may also be observed when two parasites share common risk factors. to correct for these “false interactions”, methods accounting for parasite risk factors must be used. methodology/principal findings: in the present paper we propose such a method for presence-absence data (i.e., serology). our method enables the calculation of the expected frequencies of single and double infected individuals under the independence hypothesis, before comparing them to the observed ones using the chi-square statistic. the method is termed “the corrected chi-square.” its robustness was compared to a pre-existing method based on logistic regression and the corrected chi-square proved to be much more robust for small sample sizes. since the logistic regression approach is easier to implement, we propose as a rule of thumb to use the latter when the ratio between the sample size and the number of parameters is above ten. applied to serological data for four viruses infecting cats, the approach revealed pairwise interactions between the feline herpesvirus, parvovirus and calicivirus, whereas the infection by fiv, the feline equivalent of hiv, did not modify the risk of infection by any of these viruses. conclusions/significance: this work therefore points out possible interactions that can be further investigated in experimental conditions and, by providing a user-friendly r program and a tutorial example, offers new opportunities for animal and human epidemiologists to detect interactions of interest in the field, a crucial step in the challenge of multiple infections. numerous parasites species circulate simultaneously in natural populations. many of them are able to infect a same host species and a host individual can therefore be infected by several parasites at the same time. these multiple infections are not only common in nature but usually more frequently encountered than infections by a single parasite [1] . within a host individual, parasites can thus interact, either in a synergistic manner (parasite a favours infection by parasite b or worsens the symptoms caused by b) or in an antagonistic manner (parasite a decreases the infection risk by parasite b or reduces the symptoms caused by b) [2] . as these interactions can have important epidemiological, biological and clinical consequences (e.g., [3] [4] [5] [6] [7] ), detecting, understanding and evaluating them is essential to understand the phenomena and to control and manage infectious diseases. in recent years, the question of polyparasitism has attracted considerable attention [4, 8, 9] , although in reality the subject has a long history of experimental investigation under laboratory conditions [10, 11] . many epidemiological studies have also been conducted on the main human pathogens, motivation for the study of polyparasitism being in particular driven by the urgency to understand the epidemiological and clinical consequences of infection by parasites potentially interacting with hiv and other emerging diseases [12] and the mechanisms of their interactions. a large amount of work indeed revealed interactions between hiv and tuberculosis, malaria, sexually transmitted diseases, and helminths (e.g., [6, [13] [14] [15] [16] [17] [18] [19] [20] ); as well as interactions between plasmodia parasites and helminths (e.g., [21] [22] [23] [24] ). studies on animal hosts also revealed interactions between their parasites, with many studies on helminth communities (mammals: [4, 25, 26] , birds: [27] , fish: [28] ), and fewer on protozoan species (e.g., [29] ) or viruses (e.g., [30] ). many diseases have been revealed to be affected by the presence of other disease-causing agents, altering the rates of species co-occurrence, levels of infection and disease severity. parasite interactions have also been shown to affect the success of parasite vaccination strategies [31] and could be involved in disease (re)emergence [32] , reinforcing the interest of these studies. if laboratory experiments have clearly demonstrated that interspecific parasite interactions occur, often mediated by host immune responses [1, [33] [34] [35] , attempts to detect such effects in natural populations have generally been less successful. indeed, detecting their existence on the field is not easy, due to complex networks of indirect effects making it difficult to infer underlying processes. field studies are however essential as experimental systems are oversimplified and require an existing suspicion of interaction between the studied parasites. in addition, only studies in natural populations can give access to infection and co-infection probabilities. in other words, before studying their mechanisms in the lab, interactions of interest must be identified in the field. main difficulties encountered in field studies are methodological. many confounding factors can create statistical associations between parasites even if there is no true biological interaction between them, which may alter conclusions about the importance of interspecific interactions [36] [37] [38] [39] [40] . a similar transmission mode, for example, can alone increase the risk of co-infection. the excess of positive associations found in strongylid communities in domestic horses, ruminants and macropod marsupials is in particular likely to be due to the common habit of these hosts feeding on pastures contaminated with the larvae of a number of nematode species [41] [42] [43] . in addition, environmental, behavioural or host-specific factors can be associated with both types of infection and influence epidemiological and geographic patterns of infection and disease. among such common risk factors, some have long been recognised, such as sexual behaviours for sexually transmitted diseases (e.g., [44] ), socio-economic status for infections particularly prevalent in poor regions such as helminth infection and malaria [45] , or age for many diseases (e.g., [46] ). as apparent associations between two infections may be due to common risk factors, they are crucial to identify and to take into account in the analysis. however, such confounding factors are difficult to control and few methods enable to take them into account. a variety of analytical approaches have been suggested to detect associations in parasite communities, primarily focusing on macroparasite (parasitic helminth) communities (e.g., [4, 39, 47, 48] ). however, they implicitly assume that the direction and strength of an observed association between parasite species reflects an underlying biological interaction, and their reliability to detect interactions has been recently questioned [49] . the adoption of a generalized linear mixed modelling (glmm)-based approach has been rather suggested by fenton et al. [49] (see also [50] ). apparently more robust to detect interactions between macroparasites, this method has the advantage of offering the opportunity of taking into account the variance caused by other factors. nevertheless, field data, particularly relating to microparasites, are most of the time serological (i.e. presence-absence data). indeed, viral excretion is usually too short to make antigen detection an efficient tool to follow microparasites in natural populations, as host capture and sampling would have to be done exactly during the excretion period, especially during nonepidemic phases. most field data are thus limited to observed frequencies of seronegative, seropositive and doubly seropositive individuals. in this context, the search for potential interactions between pairs of microparasites is traditionally done by calculating odds ratios in stratified data or by a pearson's chi-square test of independence (e.g., [29, 51, 52] ). the latter compares the observed frequencies to the frequencies expected if parasites are independent, under the null hypothesis that the joint distribution of the cell counts in a 2-dimensional contingency table is the product of the row and column marginals. however, such methods ignore confounding factors and/or the possible simultaneous action or interaction of several of them. significant associations detected in this manner can therefore be either true biological interactions or statistical associations, with no means of distinguishing the two. alternative methods have been therefore proposed to determine the expected frequencies in a modified chi-square analysis. some are based on the estimation of ''pre-interactive'' species prevalences [53] , which requires previous knowledge of dominance relationships between parasites species. some others are based on log-linear models [e.g., [54] [55] [56] . in addition, another way to take risk factors into account is to include them in a logistic regression analysis and to determine whether parasite b status is still a predictor of parasite a status [52, 57] . however, the main drawback of methods based on log-linear or logistic regression models is that they are based on an asymptotic approximation of the deviance, which might not be relevant for small sample size data. in the present paper, we propose another method (termed the ''corrected chi-square'') to detect microparasite interactions from serological data, based on an adaptation of the pearson's chisquare test. by combining logistic regressions and chi-square tests, we are able to calculate the expected frequencies of co-infected individuals if parasites are independent considering their risk factors, and to compare them to the observed ones. in a first step, we perform a theoretical comparison of the robustness of the corrected chi-square and the logistic regression approaches. in a second step, both approaches are applied to serological data obtained in natural populations of domestic cats to search for potential interactions between four feline viruses. the domestic cat is indeed an appropriate model to investigate such questions as its main viruses are well known and rather easy to survey on the field, and its natural populations, although very flexible in their social and spatial organisation, have been extensively studied [32, [58] [59] [60] [61] [62] . 1.1. logistic regression analysis. a first way to test the interaction between two pathogens is to test the effect of the serological status to one virus on the probability of being seropositive to the other. a logistic regression was used for that purpose. the approach allows correcting for common risk factors by adding known or suspected risk factors as correction variables. the logistic regression model reads: where f k denotes the k-th risk factor, p 1 is the probability of seropositivity to pathogen 1 and s 2 the serological status to pathogen 2. the coefficients a k (k = 0…k) and b are the coefficients of the logistic regression. the interaction between the two pathogens was tested using a likelihood-ratio test (lrt) testing h0: b = 0 vs h1: b=0. the asymptotic chi-square approximation was used to derive the pvalue of the test of independence between the two viruses [63] . 1.2. corrected pearson's chi-square tests. the corrected chi-square approach is based on the idea that the coefficients of the logistic regression of the two viruses can be used to estimate the number of seronegative, single-and double-seropositive individuals expected if the two pathogens are independent. as the classical chi-square, the corrected chi-square compares the observed (o i,j ) and theoretical (e i,j ) numbers of individuals with different combinations of status (seropositive or seronegative) for the two pathogens using the chi-square statistic: where i is the status to pathogen 1 (0 for seronegative and 1 for seropositive) and j is the status to pathogen 2. to calculate the e i,j , for each pathogen taken separately, a logistic regression including k risk factors (see previous section) is run to estimate the probability of being seropositive for each individual (termedp p p,x for individual x and pathogen p, p[ 1,2 f g): whereâ a p,k denotes the estimation of the regression coefficients for pathogen p and f k,x the value of the k-th risk factor in individual x. the theoretical contingency table is then deduced from these probabilities: for each pair of viruses, the distribution of the corrected chisquare was determined by a parametric bootstrap run as follows: step 1: estimated seropositivity probabilities (p p p,x ) are used to generate in silico serological data for both pathogens independently. step 2: the corrected chi-square is calculated for this in silico dataset. steps 1 and 2 were repeated 1000 times, leading to 1000 independent realisations of the corrected chi-square statistic under the null hypothesis of independence between the two pathogens. two ways of calculating the p-value were derived from this procedure. p-value1 was estimated assuming that the corrected chi-square is proportional to a chi-square with one degree of freedom, the coefficient of over-(or under-) dispersion (ĉ) being defined by the mean of the bootstrapped corrected chi-square. p-value2 was given by the proportion of bootstrapped corrected chisquares which were smaller than the observed value. in principle, p-value2 is better (no assumption on the distribution of the likelihood ratio test, lrt, is made), but requires running enough simulations, which may be long in some cases. p-value1 allows working with smaller numbers of simulations when simulation times are too long. the r program is available as supplementary file (file s2) and can be applied to any presence-absence data to calculate the corrected chi-square and the associated p-values. a tutorial example (file s3) illustrates its use step-by-step using an example dataset (file s4). the main criticism that could be made to the logistic regression approach is that it is based on the asymptotic distribution of the lrt. in practice, the chi-square approximation is true only for large datasets. in the present paper we investigated the robustness of the logistic regression to different sample sizes and numbers of correction risk factors. we also aimed to compare how robustness is affected by the type of risk factors considered (qualitative or quantitative). the same investigations were performed with the corrected chi-square test to compare the robustness of the two approaches. for that purpose, random seroprevalence datasets were generated, assuming independent viruses. random data were always generated assuming that all individuals had an independent 0.5 probability of being seropositive for each pathogen. n f randomly generated risk factors were considered in the logistic regression for the two pathogens. by construction these factors have no effect (they are chosen independently of the serological status of the individuals) but from a theoretical point of view it is interesting to measure how their inclusion in the model can introduce biases depending on the approach. randomly generated factors could be either qualitative or quantitative. for simplicity, qualitative factors had only two modalities, individuals having a 0.5 probability of being in each one. quantitative factors were chosen for each individual randomly according to a standard normal distribution. to investigate how the nature of risk factors affects robustness, three scenarios were tested: i) all factors are qualitative; ii) all factors are quantitative and iii) half of the factors are quantitative while the other half are qualitative factors (mixed scenario). our objective now was to understand how data characteristics (the number of individuals, n, the number of factors, n f and their type, scenario i, ii or iii) would affect the probability of wrongly concluding that there is an interaction between the two pathogens (type i error). for a given combination of these characteristics, a thousand random seroprevalence datasets were generated and we estimated the type i error associated to each approach as the proportion of random datasets for which the p-value was below 5%. 3. application to cat data 3.1. ethics statement. the field work has been made by qualified people according to the french legislation. accreditation has been granted to the umr-cnrs 5558 (accreditation number 692660703) for the program. 3.2. the feline viruses. the feline immunodeficiency virus (fiv), is a major non-traumatic cause of death in adult cats, and is associated with immunosuppression causing secondary infections [64] . this retrovirus can infect other felids, most of which are threatened or endangered species e.g., the european wildcat (f. s. silvestris) [64] [65] [66] . it is mainly transmitted by bites, through a direct horizontal mode [67] , principally during aggressive or sexual contacts [64, 68] . the feline herpesvirus (fhv) and the feline calicivirus (fcv) are responsible of upper respiratory tract disease, of concern in veterinary medicine [69, 70] . both viruses are transmitted through 'amicable' contacts, by oral, nasal and ocular secretions during close interactions [71, 72] . fhv infected cats become asymptomatic carriers, but the latent infection can be reactivated by a stress (i.e., change of habitat, lactation or fights between males; [73] ). the feline parvovirus (fpv) infects all felids, as well as other carnivores [74] , and fpv infection may be fatal especially in kittens [75] . the virus is transiently excreted in feces, urine, saliva and vomiting and its high resistance in the environment (still infectious after 13 months at 4-25uc; [76] ) makes indirect transmission through feces and contaminated areas largely predominant [77, 78] . 3.3. serological data. the serological statuses for fiv, fhv, fcv and fpv were obtained in 2007 in 15 natural rural populations of domestic cats in north-eastern france [62, 79] . cats were captured using baited traps or directly caught by the owner, anaesthetized, measured, and blood samples were taken from the jugular vein. fiv-antibodies were immediately searched for with a commercial kit using the elisa method (snap combo +, idexx), whereas specific antibodies against fhv, fcv or fpv were measured by a specific blocking elisa [80] . none of the cats was vaccinated. all six pairs of viruses were tested for potential association. between 467 and 474 cats were tested for each virus and 465 to 469 were double-tested (depending on the virus pair). previous analyses using logistic regression models with the same dataset revealed the combination of risk factors that were supported by our data [62] . five factors were initially investigated: age (age), sex (sex), way of life (owned or unowned, wol), orange phenotype (orange or non orange, pheno) and body mass (mass) and one correction factor (the population of origin, pop) was considered. for each virus, the most appropriate model was selected using the akaike information criterion adjusted for small sample size (aicc, [81] ). ideally, all factors potentially creating apparent associations should be included in the model. but to limit the number of correction risk factors, the minimal model containing the identified risk factors for the two viruses was retained as a compromise for each pair (table 1) . the corrected chi-square was robust for all tested sample sizes and numbers of parameters, whatever the nature of the factors (scenarios i, ii, iii) and the method used to calculate the p-value (p-value1, p-value2, see file s1 and fig. s3 for more details). the type i error of this method remained indeed very close to 5% (fig. 1) . on the contrary, the robustness of the logistic regression approach decreased with the n f /n ratio (number of factors/sample size). in scenarios i (only qualitative factors) and ii (only quantitative factors), the type i error was around 5% for a ratio of 0.005, around 8% for a ratio of 0.15 and around 20% for a ratio of 0.35. it became significantly different from 5% for ratios larger than 0.12 (type i error = 6.7%, z = 2.47, p = 0.019) and 0.08 (type i error = 7.9%, z = 4.21, p = 5.7610 25 ) for scenarios i and ii, respectively. in the mixed scenario (iii), the type i error became significantly different to 5% for all n f /n ratio larger than 0.075 (type i error = 7.1%, z = 3.047, p = 0.0038). more details are available in file s1 and fig. s2 . taken together, these results show that, as a rule of thumb, the logistic regression approach is robust for n f /n ratios below 0.1 for all types of factors. the two approaches (corrected chi-square and logistic regression) were used for the analysis of the interactions between four cat viruses ( table 2) . results showed that the interaction was not significant for pairs involving fiv. all other pairs (fhv-fcv, fhv-fpv and fcv-fpv) were found to interact, i.e., the number of individuals coinfected by two viruses could not be explained by shared risk factors. the three significant associations were all positive, meaning that there were always more co-infected individuals than expected considering shared risk factors (table 3) . pairwise interactions between fhv, fcv and fpv could have come from the fact that one virus was a common risk factor for the two others. this possibility was tested (see the three last lines of table 2 ) by adding the serological status to one virus as a common risk factor for the two others. results led to reject this hypothesis, meaning that the observed associations cannot be solely explained by the fact that one virus interacts with the two others. the two p-values obtained for the corrected chi-squares are coherent. as for the p-values obtained for the logistic regression approach, they are usually slightly lower than those of the corrected chi-squares, probably because of the over-predictive trend of logistic regressions. in addition, as with simulated data, the logistic regression approach was less robust to small sample sizes than the corrected chi-square (table s1 ). this was tested by randomly sampling smaller subsets of the cat data in order to increase the n f /n ratio. finally, to emphasise the need to consider risk factors in the analysis of interactions, we also calculated the classical independence pearson's chi-square. this approach, which does not integrate risk factors, predicted an association between five of the six tested pairs. in the case of the fiv-fcv and fiv-fhv pairs, it would lead to wrongly conclude on the existence of an interaction, whereas the two approaches have shown that these apparent interactions were in fact explicable by shared factors. common risk factors can create statistical associations. this work confirmed that ignoring them would lead to wrong conclusions. ignoring them would indeed result in an over-estimation of the number of interactions as any association, biological or statistical, would be put in one basket. the loss of significance after controlling for other factors was illustrated in this paper with feline viruses data, and was previously found by behnke et al. [39] for helminth parasites of the wood mice. the next step was to identify an appropriate way to take those risk factors into account. two approaches to take risk factors into account with serological data (i.e., presence-absence) were proposed and examined. those are the use of logistic regression models as table 1 . risk factors models used to test for potential association between pairs of feline viruses. previously done by some authors [52, 57] , or an adaptation of the chi-square test for independence presented for the first time in this paper. to determine which method should be used under which circumstances, we need to make the following considerations. first, the corrected chi-square involves 2n+2 estimations of the logistic regression coefficients, n being the number of bootstraps. in comparison, only two models must be parameterized in the logistic regression. as a consequence, the logistic regression approach is much faster to run (less than a second versus 2.5 minutes for the corrected chi-square for a model with 6 factors in full interaction and 300 individuals, for 1000 bootstraps, using a desktop computer with an intel(r) core(tm)2 quad cpu q6600 processor). second, the corrected chi-square is more robust than the logistic regression, especially for small sample size. a first solution would be to use the corrected chi-square as soon as simulation times are acceptable. for a 5% rejection threshold, a more straightforward alternative is to use the corrected chi-square by default as soon as the ratio between the sample size and the number of parameters is below 10 and the logistic regression in the opposite case. however, we did not test all potential situations and further analyses are needed to determine the limit of robustness of the logistic regression approach (in particular in situations where the probability of infection is not 50% and can be affected by risk factors). two p-values have been proposed for the corrected chi-square. the first one relies on the assumption that the corrected chi-square is proportional to a chi-square with one degree of freedom; the second one simply counts the proportion of in silico datasets for which the value of the corrected chi-square is above the observed value. both p-values led to consistent results using a 5% rejection threshold, consistently with the fact that for all tested pairs the corrected chi-square fitted well with an under-dispersed chi-square with one degree of freedom (fig. s1, fig. s2 ). which one should be used in practice actually depends on the simulation time. if simulations are fast enough and if running 1000 bootstrap is acceptable, p-value2 should be preferred. in the opposite case, a good option is to run much less bootstraps (typically 30) and to use p-value1. even if other alternative methods allow taking covariates into account, we only compared the corrected chi-square to the logistic regression approach. we could have compared it as well to loglinear models, which model the probability of infection with single and multiple parasite species from contingency tables and allow including known risk factors. however, in this approach the independence between parasites is tested using likelihood ratio tests, which are based on an asymptotic approximation of the deviance as in the logistic regression approach. they should therefore have the same limitations than logistic regressions and their robustness should be similarly influenced by the n f /n ratio. in addition, continuous variables are usually discretized in loglinear models, whereas the corrected chi-square allows working with continuous data. after correction by the known risk factors of the viruses, three pairs of feline viruses out of six appeared to be significantly associated. the n f /n ratio being 0.04 to 0.06, the logistic regression approach can be considered robust, at least for a 5% rejection threshold. first, it is worth noting that age is a crucial covariate. the infection probability of all viruses increases with host' age [62] , thus age must strongly participate in the generation of false interactions. this age-dependence is due to both a biological effect (i.e., behaviors and immune defenses may evolve with age, [82, 83] ) and a mechanical effect (i.e., older individuals are more likely to be seropositive because of a longer exposure time). disentangling both effects would require the use of susceptible-infected-recovered (sir) models, but was not necessary here. indeed, to the type i error of the corrected chi-square tests represented here is based on p-value2 but similar results were observed with p-value1 (fig. s3) . note that for the logistic regression approach, points resulting from a given sample size were linked to see the effect of the n f /n ratio for different sample sizes (solid line: n = 100, dashed line: n = 200, dotted line: n = 300). the dashed horizontal line represents a type i error of 5%. doi:10.1371/journal.pone.0029618.g001 correct for age in the study of interactions, the important is to model the evolution of the probability of infection with age. correcting for all risk factors, no pair of viruses involving the feline immunodeficiency virus (fiv-fhv, fiv-fcv, fiv-fpv) was significantly associated. this result is at first surprising because, as in humans infected by hiv, feline aids is characterised by a chronic immunodeficiency, allowing subsequent opportunistic infections (review in [84] ). indeed, although fiv positive cats can mount immune responses to administered antigens other than during the terminal phase of infection, their primary immune responses may be delayed or diminished [85, 86] . experimental studies also revealed that cats co-infected by fiv and fcv or fhv had more severe disease signs than non-fiv infected cats [87, 88] . in addition, the presence of fhv was shown to accelerate fiv transcription through the activation of the fiv long terminal repeat [89] , a phenomenon that was also shown in vitro for the human versions of the viruses, hsv2 and hiv [90] [91] [92] [93] . those laboratory experiments show that fiv infection may increase the severity of fhv or fcv-induced clinical signs but do not address the question of the effect of fiv on the sensitivity to fhv or fcv infection. furthermore, the few epidemiological studies interested in the question did not demonstrate any epidemiological association between fiv and fhv [94] . in other words, if experimental investigations suggest a synergy between fiv and fhv and between fiv and fcv towards a more severe disease, our sero-epidemiological study suggests that the identified risk factors explain by themselves the apparent increase of double sero-positive individuals. as for the fiv-fpv pair, this study is to our knowledge the first to search for a potential association. whether risk factors were taken into account or not, we did not find any significant association between the two viruses. again, this could be at first surprising as both viruses are supposed to be immunosuppressive [84, 95, 96] . in experimental conditions, fpv infection is more severe in fiv-infected cats [97] . consequently, a positive association could have been expected if infections had facilitated each other (leading to numerous co-infections) or a negative association if the co-infection had led to a strong host mortality (leading to few co-infections). however, the fpv-induced decrease in the immune response is transient and more likely to occur in young kittens, whereas fiv infection is more frequent in adult cats. the persistence of fpv-antibodies can be longer than 7 years [98] , and consequently, double seropositivity against fpv and fiv is not synonymous of co-infection. it is likely that co-infections by the two viruses are not frequent and mainly occur in adult animals which are less sensitive to fpv. as no association was evidenced for these three pairs of viruses, the fiv infection does not seem to modify the risk of infection by another virus. however, our results do not exclude the occurrence of an interaction once both parasites are in contact within the host (e.g., directly through competition or indirectly via the host immune system), as suggested by several experimental co-infection studies. in addition, the fiv seropositivity status may encompass different stages of the infection with various degrees of immunodeficiency. the results of this study do not exclude the possibility that late stage fiv infection may increase the sensitivity to the other feline viruses. on the contrary, the three other pairs (fhv-fcv, fhv-fpv and fcv-fpv) were significantly associated after correction by their known risk factors. it is to our knowledge the first evidence of a possible interaction between those viruses. as more double seropositive cats than expected under the independence hypothesis were observed, possible synergies are suggested. after an acute infection, fhv is known to persist life-long in a latent form, which can be reactivated in stressful conditions [73] . infection with fpv or fcv could thus be responsible for the reactivation of fhv in latently infected animals, resulting in seroconversion against both fhv and the new infecting virus. this could explain the fhv-fcv and fhv-fpv associations. in addition, since fpv is more immunosuppressive than fcv, the interaction between fpv and fhv is expected to be stronger than that between fcv and fhv, which is consistent with our results. the immunosuppressive effect of fpv could also explain the association with fcv. in that case however, contrary to fhv, it would require that the fcvinfection occurs at the time of the immunosuppression occuring within the two weeks post-fpv infection. interestingly, a similar association between fpv and fcv antibodies was described in free-ranging lions in east africa [99] . this work pointed out new probable synergies between feline viruses that can now be further investigated in laboratory conditions. however, the associations could also result from the existence of an unknown confounding factor common to fhv, fcv and fpv. the feline parvovirus is immunosuppressive, as a result of the strong leukopenia occurring within the two weeks post-infection [95, 96] . this virus could therefore be a confounding factor to the fhv-fcv pair if fpv-seropositive cats are more susceptible to fhv and fcv at the same time. however, as shown in this paper, the fhv-fcv interaction remained significant after correction by fpv (table 2) . if fpv is not a confounding factor, we cannot exclude the existence of another one, such as a greater susceptibility of certain individuals to infections whatever the parasite involved. numerous studies have shown that an extensive inter-individual variability exists in response to certain pathogens, such as hiv (review in [100] ), trypanosomiasis (review in [101] ), or human and bovine tuberculosis (reviews in [102, 103] ), including variations in susceptibility to the parasite, its transmission, and/or the course of disease progression. it has been attributed to host determinants and variability in multiple genes that regulate virus cell entry, acquired and innate immunity (e.g., macrophages, molecular and cellular actors of the inflammatory reaction), and others that influence the outcome of the infection. hosts with a diminished or delayed innate immune response may in fact be more susceptible to any infection, with physiological parameters, such as hormonal profiles (e.g., [104] ), possibly playing a role in the modulation of transmission efficiency and/or in the immune response intensity. a weaker physical condition could also lead to a higher sensitivity to infectious agents (lower dose-effect, different intra-host dynamic) (e.g., [105] ). more generally, individuals' personality may as well be involved [61, 106] . a better understanding of genetic, physiological and immunological basis of such inter-individual variability would therefore be of particular interest in the context of polyparasitism. another perspective of this work is the development of new methods able to distinguish pairwise interactions from those due to common confounding factors shared by the three viruses. such methods could use the proportion of infected individuals that are in reality triply infected. while the study of macroparasites usually uses quantitative data (i.e., parasite load per individual host), the study of microparasites on the field is most of the time limited to presence-absence data (i.e., serology), making the detection of associations between parasites more complicated from a methodological point of view. the corrected chi-square proposed in this study is, with the logistic regression approach, currently one of the rare ways to search for interaction between parasites from presence-absence data. this work provides evidence of the efficiency of such methods to reduce the bias introduced by common risk factors and encourages their use. however it also points out the low robustness of the likelihood ratio test for certain data characteristics. the corrected chi-square test must indeed be preferred for small sample size. those methods can be applied to any epidemiological study based on serology, within human or animal host populations. applied here to feline viruses, they revealed significant associations between three pairs of feline viruses. if they still do not allow us to decide whether such associations are really true interactions or whether they reveal the existence of ''over-susceptible'' hosts, we believe it is an important step forward as it offers the possibility to point out parasites associations that should be further investigated in experimental conditions. the understanding of parasites interactions and of their consequences on diseases evolution, emergence and management is indeed a crucial challenge for human and animal epidemiologists of our time. figure s2 issue of the conformity tests of the type i error to 5% according to the n f /n ratio for the logistic regression approach. the issue was coded 1 when the test was significant, 0 when not and the resulting logistic regression was drawn (dark line). three scenarios are considered: i) all factors are qualitative (a); ii) all factors are quantitative (b) and iii) half of the factors are quantitative and the other half are qualitative (mixed scenario, c). (eps) figure s3 type i error (%) of the corrected chi-square tests according to the n f /n ratio and the type of p-value used for the corrected chi-square: p-value1 (blue empty points) or p-value2 (red full points). three scenarios are considered: i) all factors are qualitative (a); ii) all factors are quantitative (b) and iii) a half of the factors is quantitative and the other half is qualitative (mixed scenario, c). the dashed horizontal line represents a type i error of 5%. table s1 corrected chi-square tests and logistic regressions to search for feline viruses' interactions using subsets randomly sampled in cat data such that the n f / n ratio takes various values. file s1 robustness of the logistic regression approach and of the corrected chi-square test. (1) conformity tests of the type i error to 5%, (2) influence of the way to calculate the pvalue of the corrected chi-square test on the robustness of the study. (doc) file s2 ''chi2corr'', an r program for the application of the corrected chi-square test to any presence-absence data: test statistic, observed and expected frequencies, estimated dispersion coefficient (parametric bootstrap), p-values and distribution of the bootstrapped corrected chi-square. file s3 a step-by-step example of application of the corrected chi-square test to search for interaction between two parasites, using a provided dataset (''da-ta_example.txt'', file s4) and the provided r program (''chi2corr.r'', file s2). concomitant infections, parasites and immune responses préliminaires d'une étude historique des maladies susceptibility to trypanosoma cruzi is modified by a previous non-related infection competition and mutualism among the gut helminths of a mammalian host malaria-filaria coinfection in mice makes malarial disease more severe unless filarial infection achieves patency dual infection with hiv and malaria fuels the spread of both diseases in sub-saharan 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syndrome concept at the population level we thank guillaume leblanc for data collection on the field and béatrice tarin, vincent badol and christine coupier for laboratory analyses. we are grateful to two anonymous reviewers for fruitful suggestions. analyzed the data: eh. contributed reagents/materials/analysis tools: hp. wrote the paper: eh df dp fs hp. designed the method: df eh. designed the r program and the tutorial example: fs eh. specialists of the cat-feline viruses system: dp hp. supervised the work: df. key: cord-002890-g7aje88u authors: wood, paul l.; steinman, margaret; erol, erdal; carter, craig; christmann, undine; verma, ashutosh title: lipidomic analysis of immune activation in equine leptospirosis and leptospira-vaccinated horses date: 2018-02-23 journal: plos one doi: 10.1371/journal.pone.0193424 sha: doc_id: 2890 cord_uid: g7aje88u currently available diagnostic assays for leptospirosis cannot differentiate vaccine from infection serum antibody. several leptospiral proteins that are upregulated during infection have been described, but their utility as a diagnostic marker is still unclear. in this study, we undertook a lipidomics approach to determine if there are any differences in the serum lipid profiles of horses naturally infected with pathogenic leptospira spp. and horses vaccinated against a commercially available bacterin. utilizing a high-resolution mass spectrometry serum lipidomics analytical platform, we demonstrate that cyclic phosphatidic acids, diacylglycerols, and hydroperoxide oxidation products of choline plasmalogens are elevated in the serum of naturally infected as well as vaccinated horses. other lipids of interest were triacylglycerols that were only elevated in the serum of infected horses and sphingomyelins that were increased only in the serum of vaccinated horses. this is the first report looking at the equine serum lipidome during leptospiral infection and vaccination. leptospirosis is a worldwide zoonotic disease that affects horses and many other mammalian species, including man [1] . leptospira interrogans serovar pomona is commonly associated with clinical leptospirosis in horses in the united states [2, 3] the disease in horses is mainly characterized by spontaneous abortions and recurrent uveitis, with leptospiral abortions occurring late in gestation, typically without any prior clinical signs [4] . infected mares shed leptospires in the urine for up to 14 weeks and can potentially be a source of infection to other animals. recurrent uveitis is an important sequela to leptospiral infection and a major cause of blindness in horses [5] . a leptospiral serosurveillance conducted in 2012, reported a prevalence of 45% among horse population in 29 states of the united states and a canadian province [6] . a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the microscopic agglutination test (mat) is the gold standard in serodiagnosis of leptospirosis. the mat is performed by mixing serial dilutions of patient serum with a battery of live leptospira serovars, and the presence of leptospiral antibodies in the serum is detected by dark-field microscopic examination for agglutination [7] . among several obvious limitations to the mat is the test's inability to distinguish between leptospiral antibodies generated as a result of natural infection from that by vaccination. vaccinated horses have antibodies to leptospiral bacterin and give positive agglutination reactions in mat. a test that overcomes the technical limitations of the mat and distinguishes between infected and vaccinated horses would improve the diagnosis of equine leptospirosis. recent advances in leptospiral research has resulted in identification of a number of immunogenic leptospiral proteins that are either exclusively expressed or significantly upregulated during infection in horses, but their usefulness in differentiating infected and vaccinated horses is still under investigation [8, 9, 10] . as a result, there currently are no diagnostic tests to differentiate these two immune responses. alterations in lipid metabolism due to pathogen-induced immune activation have previously been reported [11, 12, 13, 14, 15] . in this study, we asked if differences in host's responses to live, multiplying leptospira versus killed leptospires, present in the vaccine, are reflected in the serum lipidome of these two groups of horses. to that end, we used a non-targeted lipidomics approach to compare serum lipidome of horses with leptospiral infection and horses vaccinated with a commercially available bacterin. fifteen serum samples each from these three groups of horses were used in the study: (1) unvaccinated, naturally infected (microscopic agglutination test (mat)-positive) horses, (2) horses vaccinated with lepto eq innovator (zoetis inc., kalamazoo, mi), and (3) unvaccinated, unexposed (mat-negative) horses (table 1) . initial screening was performed by mat, following oie protocol (http://www.oie.int/fileadmin/home/eng/health_standards/tahm/2. 01.12_lepto.pdf). naturally infected horses were never vaccinated and had a mat titer of (table 1) . horses in the vaccinated group did not have a history of prior exposure to leptospira spp. but ruling out any prior exposure is not possible. the control group horses were never vaccinated and had no known history of a prior exposure. all vaccinated and six of the fifteen samples in the infected group were sent to the ukvdl for mat titers. remaining nine samples in the infected group were collected from two different farms in virginia and kentucky. five milliliters of blood was obtained from the jugular vein of horses using a vacutainer needle (20g, 1.5"), a sleeve, and a 10 ml dry blood collection tube (red top). clotted blood samples were centrifuged at 2,000 x g for 15 minutes. serum was separated, stored frozen at -20˚c, and when required, shipped on dry ice. none of the samples were thawed more than 2 times before the lipidomic analyses were done. the samples used in this study were left-over aliquots of either clinical diagnostic samples (university of kentucky veterinary diagnostic laboratory) or blood samples collected in a phlebotomy teaching lab. the phlebotomy lab protocol was approved by the lincoln memorial university's institutional animal care and use committee. for the lipid extraction, 100 μl of serum were vortexed with 1 ml of methanol containing stable isotope internal standards ( [16] [17] [18] . next 1 ml of water and 2 ml of methyl-tert-butyl ether were added and the tubes were vigorously shaken at room temperature for 30 min. the tubes were next centrifuged at 5,000 xg for 15 min at room temperature and 1 ml of the upper organic extracts was dried by centrifugal vacuum evaporation and dissolved in isopropanol: methanol: chloroform (4:2:1) containing 7 mm ammonium acetate. constant infusion lipidomics were performed utilizing high-resolution (140,000 at 200 amu) data acquisition, with sub-millimass accuracy on an orbitrap mass spectrometer (thermo q exactive) with successive switching between polarity modes. in negative ion esi, the anions of ethanolamine plasmalogens (plse), phosphatidylethanolamines (ptde), lysophosphoethanolamines (lpe), phosphatidylglycerols (pg), phosphatidic acids (pa), lysophosphatidic acids (lpa), cyclic phosphatidic acids (cpa), phosphatidylinositols (pi), ceramides, phosphatidylserines (ps) were quantitated and lipid identities validated by ms/ms. in positive ion esi, the cations of choline plasmalogens (plsc), hydroperoxy plscs, phosphatidylcholines (ptdc), lysophosphocholines (lpc), sphingomyelins (sm), monoacylglycerols (mg), and acylcarnitines (acar), and the ammonium adducts of diacylglycerols (dg), triacylglycerols (tg), and cholesterol esters (ce) were quantitated and lipid identities validated by ms/ms. in the case of hydroperoxy plscs, structural identities were validated by the loss of h 2 o 2 with ms 2 and generation of a major fragment for choline phosphate. the cations and anions of bromocriptine were used to monitor for potential mass axis drift. between injections, the transfer line was washed with successive 500 μl washes of methanol and hexane/ethyl acetate/chloroform (3:2:1) to minimize potential ghost effects. r values (ratio of endogenous lipid peak area to the peak area of an appropriate internal standard) per 100 μl of serum were calculated. data are presented as mean ± sem. data were analyzed by anova followed by the dunnets test to compare the vaccinated and infected groups to the controls. individual data values are available in the supplementary information (s1 table) results cpa 16:0 (fig 1) was elevated in the serum of both the vaccinated and infected equine groups. tandem mass spectrometry validated the structure of cpa 16:0, with the anions for 16:0 and glycerophosphate (-h 2 o) being monitored with 1.2 and 0.98 ppm mass errors, respectively ( table 2 ). other cyclic phosphatidic acids also were elevated in the serum of both the vaccinated and infected groups. these included cpa 18:0, cpa 18:1, and cpa 18:2 (fig 2) . the structures were validated by ms 2 with the product anions for glycerophosphate (-h 2 o) monitored for all 3 cpas and the respective fatty acid constituents 18:0, 18:1, and 18:2 monitored (table 2) . these data clearly demonstrate that the th1-type immune responses initiated by leptospirosis [19] or by vaccination [20, 21] result in the generation of cpas and that these may be important as indicators of immune activation. of particular interest, horse lv7, while vaccinated, did not demonstrate a mat titer (table 1 ). in contrast cpa changes similar to other vaccinated horses were detected, indicating that this lipid is a more sensitive indicator of immune activation. cyclic phosphatidic acids are generated by phospholipase d-dependent transphosphatidylation of lysophosphatidylcholines [22] , which are generated by phospholipase a2 hydrolysis of phosphatidylcholines [23] (fig 3) . in parallel with augmented cpas, we monitored increased serum levels of the associated lysophosphatidylcholine (lpc) precursors (fig 3) , albeit, the increases in lpc levels were greater in vaccinated compared to infected horses (p < 0.01). diacylglycerols (dag) also were elevated in the serum of both vaccinated and infected horses. these included dag 34:1, dag 34:2, dag 34:3, dag 36:1, dag 36:2 (fig 4) , dag 36:3, dag 36:4, and dag 36:5. of particular interest, horse lv7, while vaccinated, did not demonstrate a mat titer (table 1 ). in contrast dag changes similar to other vaccinated horses were detected, indicating that this lipid is a more sensitive indicator of immune activation. in contrast, triacylglycerols were only increased in the serum of infected horses. these included tag 48:1, tag 48:2, tag 48:3, tag 50:1, tag 50:2, tag 50:3, tag 50:4 (fig 4) , tag 50:5, tag 52:1, tag 52:2, tag 52:3, and tag 52:4. from evaluations of sphingolipids we noted that sphingomyelins were selectively elevated in the serum of vaccinated horses. these included sm d18:1/18:3, sm d18:1/20:0, sm d18:1/22:1, sm d18:1/22:3, sm d18:1/24:0 (fig 5) , sm d18:1 /24:1, sm d18:1/24:2 (fig 5) , and sm d18:1/ 24:3. in contrast sm changes similar to other vaccinated horses were detected, indicating that this lipid is a more sensitive indicator of immune activation. while infections generally result in the induction of serine palmitoyltransferase and thereby augmentation of sphingomyelin synthesis [11] , this is not universally noted. the hydroperoxy oxidation products of a number of choline plasmalogens possessing unsaturated fatty acid substituents were detected in infected and vaccinated horses but were greater in the vaccination group (fig 6) . the identities of these oxidation products were validated via tandem mass spectrometry. using this approach we monitored the loss of both h 2 o and h 2 o 2 ( table 3 ) and the generation of choline phosphate (184.0739; < 1 ppm mass error), hallmark features for this class of oxidized lipids [24, 25] . there is an ever increasing knowledge base regarding the biochemistry of the immune response during infections and inflammatory diseases. a shift in the th1 and th2 responses generally results in up-regulation of th2-type pro-inflammatory cytokines with bacterial infections [26, 27] , viral infections [28, 29] , and parasitic invasions [30] . in addition local immune responses, such as in the lung [31] , brain [32] , and the intestine in inflammatory bowel disease (crohn's disease and ulcerative colitis) [33] elicit alterations in the th1 and th2 cytokine responses. pro-inflammatory cytokines act to induce indoleamine-2,3-dioxygenase-1 thereby acting to deplete tryptophan and generate kynurenine. as a result of this activated pathway, the kyneurenine/tryptophan ratio is often used as a surrogate biomarker of immune activation [34, 35] . lipid metabolism is also altered by the acute phase reactant response. for example, elevated levels of circulating triglycerides have been observed experimentally with cytokine and lipopolysaccharide injections [11, 12, 36] , as a result of augmented hepatic lipogenesis. elevated triglycerides have also consistently been reported with bacterial [11, 12, 13] and parasitic infections [14, 15] . altered sphingomyelin metabolism has also been reported, with increases in pneumonia patients [37] and decreases in hiv patients [38] . to further evaluate alterations in the serum lipidome during immune activation we took advantage of the opportunity to compare the serum lipidome of horses with active leptospirosis infection [1, 4, 39] and horses vaccinated with a commercial bacterin [40, 41] . our results show that serum levels of cyclic phosphatidic acids (cpa), diacylglycerols, and hydroperoxide oxidation products of choline plasmalogens were elevated in both vaccinated and naturally infected horses. perhaps more importantly, we observed that triacylglycerols were only elevated in the serum of infected horses and sphingomyelins were increased only in the serum of vaccinated horses. in previous and ongoing studies in our lab we have demonstrated increased levels of cpas in airway surfactant of horses with asthma [42] . phospholipase a2 [43, 44] and pld [45, 46] are both augmented during the early phase of infections suggesting that our cpa biomarkers may be simple biochemical readouts of the induction of these enzymes by immune activation (see fig 3) . of particular note is that our data is the first to demonstrate that vaccination can activate the same enzyme systems. these data suggest that cpas may be useful as global biomarkers of immune activation during various infections in horses and possibly other animal species. considering the breadth of bioactivities of this class of lipids [47] , their contributions to immune responses may be diverse, particularly since they modulate nuclear function [48, 49] . in this regard, pharmacological studies have shown that cpa analogs potently reverse experimental osteoarthritis [50] , block immune-induced demyelination [51] , and inhibit the growth of cancer cells [52] . plasmalogens are essential membrane lipids, particularly in lipid rafts [23] . alterations in these structural glycerophospholipids, induced by lipid oxidation, may play a role in the host's immune response, particularly in the development of immunity as evidenced by the dramatic increases in circulating hydroperoxy choline plasmalogens in the leptospira-vaccinated animals. the roles of singlet oxygen-oxidation, free radicals and/or oxygenases in the production of these lipids with vaccination remain to be defined. in this study we demonstrated that triacylglycerols (tags) are elevated only in the serum of naturally infected horses. previous reports also have consistently demonstrated elevated serum levels of tags in both experimental and clinically unresolved immune activation [12] [13] [14] [15] 36] , including human leptospirosis [13] . the mechanism involved in immune-dependent hypertriglyceridemia is thought to involve cytokine activation of triglyceride synthesis in the liver [11] . in contrast, dags were elevated in both infected and vaccinated cohorts, suggesting that the synthesis or metabolism of these neutral lipids is altered in both resolved and unresolved immune activation. this is the first report of these changes in dag levels with immune activation. in this regard, the reports of increased expression of pld [45, 46] during the early phase of infections suggest that this immune-activated mechanism may be involved in the generation of increased levels of dags from glycerophospholipids since pld metabolizes glycerophospholipids to phosphatidic acids, the direct precursors of dags [23] . in our study, sphingomyelins were increased only in the serum of vaccinated horses. sphingomyelin levels have previously been shown to be elevated in patients with pneumonia [37] , and melioidosis [53] while they are unaltered in several bacteremic conditions [53] and decreased in aids patients [38] . our data indicate that the immune response induced by vaccination has a more dramatic effect than leptospiral infection on sphingomyelin synthesis in horses. the role of these lipids in the immune response remains to be more clearly defined. in summary, our results provide important information about differences in serum lipidome of naturally infected and leptospira-vaccinated horses. since this study utilized diagnostic and clinical samples, a more controlled, time-matched study is required to further ascertain usefulness of the candidate lipids in differentiating vaccine from infection responses to leptospira spp. supporting information s1 table. (xlsx) leptospira and leptospirosis a unique genotype of leptospira interrogans serovar pomona type kennewicki is associated with equine abortion prevalence and serovars of leptospira involved in equine abortions in central kentucky during the 1991-1993 foaling seasons leptospirosis in horses leptospiral uveitis-there is more to it than meets the eye! zoonoses public health seroepidemiology of equine leptospirosis utilizing diagnostic laboratory specimens from 29 states (us) and one canadian province. proceedings of 55th annual aavld meet improved microtechnique for the leptospiral microscopic agglutination test host-inducible immunogenic sphyngomyelinase-like protein, lk73.5, of leptospira interrogans lfha, a novel factor h binding protein of leptospira interrogans cloning and molecular characterization of an immunogenic liga protein of leptospira interrogans effects of infection and inflammation on lipid and lipoprotein metabolism: mechanisms and consequences to the host tumor necrosis factor, cytokines, and the hyperlipidemia of infection leptospirosis is associated with markedly increased triglycerides and small dense low-density lipoprotein and decreased high-density lipoprotein visceral leishmaniasis is associated with marked changes in serum lipid profile discovery of infection associated metabolic markers in human african trypanosomiasis lipidomics of equine sperm and seminal serum: identification of amphiphilic (o-acyl)-ω-hydroxy-fatty acids non-targeted lipidomics of csf and frontal cortex grey and white matter in control, mild cognitive impairment, and alzheimer's disease subjects non-targeted lipidomics utilizing constant infusion high resolution esi mass spectrometry proinflammatory and immunomodulatory cytokine mrna time course profiles in hamsters infected with a virulent variant of leptospira interrogans protective killed leptospira borgpetersenii vaccine induces potent th1 immunity comprising responses by cd4 and gammadelta t lymphocytes a leptospira borgpetersenii serovar hardjo vaccine induces a th1 response, activates nk cells, and reduces renal colonization phospholipase d2-dependent inhibition of the nuclear hormone receptor ppargamma by cyclicphosphatidic acid lipidomics of alzheimer's disease: current status hydrophilic interaction liquid chromatography-electrospray ionization-tandem mass spectrometry of a complex mixture of native and oxidized phospholipids determination of phosphatidylcholine hydroperoxide (pcooh) as a marker of membrane lipid peroxidation pro-inflammatory immune responses are associated with clinical signs and symptoms of human anaplasmosis infection and inflammation-induced proatherogenic changes of lipoproteins cd4/cd8 ratio and kt ratio predict yellow fever vaccine immunogenicity in hiv-infected patients immunological and virological characterization of hiv-1 viremia controllers in the north region of brazil th2 and th1 responses: clear and hidden sides of immunity against intestinal helminths inflammatory, anti-inflammatory and regulatory cytokines in relatively healthy lung tissue as an essential part of the local immune system roles of cns macrophages in neurodegeneration crohn's disease and ulcerative colitis show unique cytokine profiles microenvironment tumor metabolic interactions highlighted by qmsi: application to the tryptophan-kynurenine pathway in immuno-oncology kynurenine pathway metabolism and neuroinflammatory disease differential regulation of il-1 alpha and tnf alpha release from immortalized murine microglia (bv-2) lipid metabolites as potential diagnostic and prognostic biomarkers for acute community acquired pneumonia serum metabolomics biosignature according to hiv stage of infection, pace of disease progression, viremia level and immunological response to treatment preliminary evaluation of a dual antigen elisa to distinguish vaccinated from leptospira infected horses clinical, serological and echocardiographic examination of healthy field dogs before and after vaccination with a commercial tetravalent leptospirosis vaccine levels of cyclic phosphatidic acid are increased in surfactant from asthmatic horses exposed to hay secretory phospholipase a2 in the pathogenesis of acute dengue infection critical role of phospholipase a2 group iid in age-related susceptibility to severe acute respiratory syndrome-cov infection targeting phospholipase d in cancer, infection and neurodegenerative disorders phospholipase d promotes arcanobacterium haemolyticum adhesion via lipid raft remodeling and host cell death following bacterial invasion cyclic phosphatidic acid-a unique bioactive phospholipid ppar γ networks in cell signaling: update and impact of cyclic phosphatidic acid cyclic phosphatidic acid inhibits alkyl-glycerophosphate-induced downregulation of histone deacetylase 2 expression and suppresses the inflammatory response in human coronary artery endothelial cells cyclic phosphatidic acid relieves osteoarthritis symptoms cyclic phosphatidic acid treatment suppress cuprizone-induced demyelination and motor dysfunction in mice cyclic phosphatidic acid stimulates camp production and inhibits growth in human colon cancer cells metabolomic profiling of serum from melioidosis patients using uhplc-qtof ms reveals novel biomarkers for diagnosis funds for this work were provided by lmu-college of veterinary medicine's internal grant program. we thank brittney beigel, dan kish, joey morgan, charles faulkner, john dascanio for technical assistance or helpful comments on the manuscript. key: cord-001280-skavefji authors: choi, sang-ho; hong, sang-bum; hong, hyo-lim; kim, sung-han; huh, jin won; sung, heungsup; lee, sang-oh; kim, mi-na; jeong, jin-yong; lim, chae-man; kim, yang soo; woo, jun hee; koh, younsuck title: usefulness of cellular analysis of bronchoalveolar lavage fluid for predicting the etiology of pneumonia in critically ill patients date: 2014-05-13 journal: plos one doi: 10.1371/journal.pone.0097346 sha: doc_id: 1280 cord_uid: skavefji background: the usefulness of bronchoalveolar lavage (bal) fluid cellular analysis in pneumonia has not been adequately evaluated. this study investigated the ability of cellular analysis of bal fluid to differentially diagnose bacterial pneumonia from viral pneumonia in adult patients who are admitted to intensive care unit. methods: bal fluid cellular analysis was evaluated in 47 adult patients who underwent bronchoscopic bal following less than 24 hours of antimicrobial agent exposure. the abilities of bal fluid total white blood cell (wbc) counts and differential cell counts to differentiate between bacterial and viral pneumonia were evaluated using receiver operating characteristic (roc) curve analysis. results: bacterial pneumonia (n = 24) and viral pneumonia (n = 23) were frequently associated with neutrophilic pleocytosis in bal fluid. bal fluid median total wbc count (2,815/µl vs. 300/µl, p<0.001) and percentage of neutrophils (80.5% vs. 54.0%, p = 0.02) were significantly higher in the bacterial pneumonia group than in the viral pneumonia group. in roc curve analysis, bal fluid total wbc count showed the best discrimination, with an area under the curve of 0.855 (95% ci, 0.750–0.960). bal fluid total wbc count ≥510/µl had a sensitivity of 83.3%, specificity of 78.3%, positive likelihood ratio (plr) of 3.83, and negative likelihood ratio (nlr) of 0.21. when analyzed in combination with serum procalcitonin or c-reactive protein, sensitivity was 95.8%, specificity was 95.7%, plr was 8.63, and nlr was 0.07. bal fluid total wbc count ≥510/µl was an independent predictor of bacterial pneumonia with an adjusted odds ratio of 13.5 in multiple logistic regression analysis. conclusions: cellular analysis of bal fluid can aid early differential diagnosis of bacterial pneumonia from viral pneumonia in critically ill patients. severe pneumonia requiring intensive care unit (icu) admission is associated with high rates of morbidity and mortality. delays in the provision of adequate antimicrobial therapy have been reported to be associated with excess mortality [1] [2] [3] ; therefore, rapid and accurate etiologic diagnosis of severe pneumonia is essential for successful treatment. in recent years, bronchoscopic bronchoalveolar lavage (bal) has been established as a useful technique for collecting lower respiratory tract specimens from the alveolar level, and can thus be used to accurately define the causative organisms of pneumonia [4] [5] [6] . however, a conventional culture usually takes at least a few days, and microbiological yield is often compromised by prior empirical usage of antimicrobial agents. in addition, identification of viruses and atypical organisms requires a separate etiologic work-up. cellular analysis of bal fluid, including total and differential cell counts and the cd4+:cd8+ t-lymphocyte ratio, is useful for the diagnosis of various interstitial lung diseases [7] [8] [9] . under an appropriate clinical setting, bal fluid analysis can provide highly suggestive or even diagnostic information for specific interstitial lung diseases in the absence of a lung biopsy [10] . however, only a few previous studies with limited patient populations [11] [12] [13] have evaluated the role of cellular analysis of bal fluid in patients with suspected pneumonia. most of these studies focused on the differential diagnosis of pneumonia from non-infectious pulmonary diseases, not on the prediction of pneumonia etiology. bal fluid analysis can be performed within several hours. therefore, such analysis would be useful for guiding early treatment if it could predict the etiology of pneumonia, similar to the role of cerebrospinal fluid cellular analysis, which can reliably differentiate among meningitis etiologies. therefore, this study investigated whether analysis of the cellular profile of bal fluid can predict the etiology of pneumonia in critically ill patients admitted to the medical icu. this study was based on data from a prospective observational cohort study conducted from march 2010 to may 2013. all patients admitted to the medical icu of asan medical center, a 2,700-bed tertiary care university-affiliated hospital in seoul, republic of korea, with suspected severe pneumonia were prospectively identified and monitored until discharged [14] . the data collected included patient demographics, underlying diseases or conditions, illness severity scores including acute physiological and chronic health evaluation (apache) ii and sequential organ failure assessment (sofa), type of pneumonia, laboratory data including microbiological tests, length of icu stay, and outcome. the prospectively collected data were retrospectively analyzed. this study was approved by the institutional review board of asan medical center and the requirement for informed consent was waived because of the observational nature of the study. all patients information was anonymized and deidentified prior to analysis. inclusion criteria were as follows: (1) patients aged $18 years with a clinical diagnosis of pneumonia (see below for definition), and (2) patients who underwent bronchoscopic bal for etiologic diagnosis of pneumonia. exclusion criteria were as follows: (1) patients in whom the pathogen was not identified, (2) patients in whom bal fluid analysis was impossible (due to severe neutropenia or clotting of specimen) or not performed, (3) patients with a mixed infection (identification of bacteria and virus), (4) patients who were treated with antimicrobial agents for more than 24 hours before bronchoscopic bal, (5) patients with invasive pulmonary aspergillosis, (6) patients with mycobacterial infection, and (7) patients with pneumocystis jirovecii pneumonia. pneumonia was defined as the presence of an acute infiltrate on a chest radiograph and at least one of the following: fever (temperature $38.0uc) or hypothermia (temperature ,35.0uc), cough, pleuritic chest pain, dyspnea, and altered breath sounds on auscultation [15] . pneumonia was categorized as communityacquired pneumonia (cap), healthcare-associated pneumonia (hcap), or hospital-acquired pneumonia (hap), as defined previously [16, 17] . fiberoptic bronchoscopy with bal was performed following a standardized protocol as previously described [14] . briefly, bal was performed by instillation of three consecutive aliquots of sterile saline solution (20-30-30 ml) into the bronchial tree at the area that was most abnormal on the chest radiography. the right middle lobe or lingual segment was chosen in patients with bilateral diffuse infiltration. bal fluid that was first retrieved was discarded, and bal fluid that was subsequently retrieved was collected. the total cell count was determined using a hemocytometer. the corresponding amount of bal fluid for 10 3 cells was centrifuged onto a microscope slide using a thermo shandon cytospin (thermo fisher scientific inc., waltham, ma, usa), at 500 rpm for 5 minutes at room temperature. the slide was airdried and stained with wright-giemsa stain. differential cell counts that included percentages of neutrophils, lymphocytes, alveolar macrophages, and eosinophils were determined. bacterial, fungal, and mycobacterial cultures of endotracheal aspirates and bal fluid were performed. respiratory viruses were tested by a multiplex reverse-transcription polymerase chain reaction (pcr) assay using a seeplex rv15 ace detection kit (seegene inc., seoul, korea) and/or shell vial culture. pcr to detect mycoplasma pneumoniae, chlamydophila pneumoniae, and legionella pneumophila, and a urinary antigen test to detect streptococcus pneumoniae and l. pneumophilia serogroup 1 species were also performed. data were expressed as mean 6 standard deviation or median and 25-75% interquartile range according to data distribution. categorical variables were compared using the chi-square test or fisher's exact test as appropriate. receiver operating characteristic (roc) curves were constructed to determine the performances of bal fluid cellular components, serum procalcitonin concentration, and c-reactive protein concentration for predicting bacterial pneumonia. youden's index (sensitivity + specificity-1) [18] was used to select the optimal cutoff points of the roc curve. area under the curve (auc), sensitivity, specificity, positive likelihood ratio and negative likelihood ratio were calculated. for positiveand negative predictive values, the prevalence of bacterial pneumonia in severe pneumonia patients admitted to the medical icu was assumed to be 35.9%, based on our previous study [14] . multivariable logistic regression analysis was used to identify independent predictors of bacterial pneumonia. variables with p values less than 0.2 in the univariate analysis were included in the multivariate analysis. the correlation between bal fluid white blood cell (wbc) count and apache ii score was determined by calculating pearson's correlation coefficient. significance was accepted at p # 0.05. all tests were performed using spss (version 18.0; spss, inc.) and graphpad prism (version 5; graphpad, inc.) software. pneumonia underwent bronchoscopic bal (67 with cap, 159 with hcap, and 133 with hap). of these patients, 100 were excluded because the pathogen was not identified, 42 were excluded because bal fluid analysis was not possible (due to severe neutropenia or specimen clotting) or not performed, 52 were excluded because two or more types of pathogens were identified, and 100 were excluded because they received antimicrobial therapy for more than 24 hours before bronchoscopic bal. ten patients with pneumocystis jirovecii pneumonia, 5 patients with invasive pulmonary aspergillosis, and 3 patients with mycobacterial pneumonia were also excluded. finally, 47 patients (24 with bacterial pneumonia and 23 with viral pneumonia) were included. the characteristics of the 47 patients are shown in table 1 . thirty-two patients (68.1%) were men and the mean age was 62.1 years. structural lung disease was the most common underlying disease (29.8%), followed by diabetes mellitus (19.0%), and hematologic malignancy/solid cancer (both 12.8%). sixteen patients (34.0%) had cap, 25 (53.2%) had hcap, and 6 (12.8%) had hap. most baseline characteristics did not significantly differ between the bacterial pneumonia and viral pneumonia groups. by contrast, mean apache ii (27.066.8 vs. 20.865.3, p = .002) and sofa (11.363.7 vs. 7.663.2, p = .001) scores were significantly higher in the bacterial pneumonia group than in the viral pneumonia group. however, mortality rates, including 28-day mortality, did not significantly differ between the groups. pathogens that were identified in 47 patients are summarized in table 2 . twenty-eight bacterial pathogens were identified in 24 patients. in 4 patients, two different bacteria were identified. staphylococcus aureus (n = 5) was the most common bacteria, followed by legionella pneumophila (n = 4), and streptococcus pneumoniae (n = 3). bacteria were identified from bal fluid cultures or pcrs in 15 patients, from endotracheal aspirates or sputum cultures in 15 patients, from blood cultures in 4 patients, and from urinary antigen tests in 4 patients (two patients with pneumococcal antigens and two patients with legionella antigens). eleven patients had two or more positive tests. twenty-six viruses were identified in 23 patients. in three patients, two different viruses were identified. rhinovirus was the most common virus (n = 11), followed by influenza virus (n = 6), and respiratory syncytial virus (n = 3). viruses were identified from bal fluid specimens in 18 patients and from nasopharyngeal specimens in 13 patients. viruses were detected in both in bal fluid and nasopharyngeal samples in 8 patients. cellular bal fluid profiles and distributions of bal fluid cell counts in the two groups are shown in table 3 and figure 2 . detailed data of each patient are summarized in table s1 . the median total wbc count (2,815/ml vs. 300/ml, p,0.001), percentage of neutrophils (80.5% vs. 54.0%, p = 0.02), and absolute neutrophil count (2,661/ml vs. 204/ml, p,0.001) of bal fluid were significantly higher in the bacterial pneumonia group than in the viral pneumonia group. the median serum procalcitonin concentration was also higher in the bacterial pneumonia group than in the viral pneumonia group (1.9 ng/ml vs. 0.3 ng/ml, p = 0.02), and the c-reactive protein concentration tended to be higher in the bacterial pneumonia group than in the viral pneumonia group (20.3 mg/dl vs. 14.9 mg/dl, p = 0.09). of the 100 pathogen-identified patients who had received antimicrobial agent for more than 24 hours prior to bronchoscopic bal (figure 1) , 44 had bacterial pneumonia, 54 had viral pneumonia, and 2 had invasive pulmonary aspergillosis. of the 98 patients with bacterial pneumonia or viral pneumonia, the median duration of antimicrobial therapy before bronchoscopic bal was 5 days (interquartile range, 3-9 days). the median total wbc count (395/ml vs. 200/ml, p = 0.52) and percentage of neutrophils (69.0% vs. 67.0%, p = 0.26) were not significantly different between these two groups. figure s1 shows the changes recorded in the bal fluid total wbc count and percentage of neutrophils according to the duration of exposure to antimicrobial agents. diagnostic performances of bal fluid cellular components, serum procalcitonin concentration, and serum c-reactive protein concentration for the prediction of bacterial pneumonia the ability of bal fluid cellular analysis to distinguish between bacterial pneumonia and viral pneumonia was assessed using roc analysis (table 3 last the sensitivities, specificities, positive predictive values, negative predictive values, positive likelihood ratios, and negative likelihood ratios are summarized in table 4 . when the cutoff value of bal fluid total wbc count was $510/ml, which was selected using youden's index, sensitivity was 83.3% (95% ci; 67.9-93.2), specificity was 78.3% (95% ci; 62.2-88.6), positive predictive value was 68.2% (95% ci; 49.2-82.6), negative predictive value was 89.3% (95% ci; 77.0-95.5), positive likelihood ratio was 3.83 (95% ci; 1.80-8.17), and negative likelihood ratio was 0.21 (95% ci; 0.08-0.52). a combination of bal fluid total wbc count $ 510/ml or serum procalcitonin concentration $0.71 ng/ml had a sensitivity of 95.8% (95% ci; 81.6-99.8) and a negative likelihood ratio of 0.07 (95% ci; 0.003-0.40), whereas bal fluid total wbc count $510/ml and serum c-reactive protein concentration $26.1 mg/dl had specificity of 95.7% (95% ci, 81.6-99.8) and a positive likelihood ratio of 8.63 (95% ci, 1.31-180.96). when a cutoff value of bal fluid total wbc count $510/ml was applied to 100 pathogen-identified patients who had received antimicrobial agent for more than 24 hours, sensitivity was 36.8% (95% ci; 21.8-54.0), specificity was 71.4% (95% ci; 56.7-83.4), positive predictive value was 41.9% (95% ci; 28.2-57.0), negative predictive value was 66.9% (95% ci; 59.9-73.2), positive likelihood ratio was 1.29 (95% ci; 0.70-2.37), and negative likelihood ratio was 0.89 (95% ci; 0.66-1.19). multiple logistic regression analysis revealed that bal fluid total wbc count $510/ml was an independent predictor of bacterial pneumonia with an adjusted odds ratio of 13.5 (95% ci; 2.3-80.4) ( table 5 ). there was a modest but significant positive correlation between the degree of bal leukocytosis and the apache ii score (r = 0.419, p = 0.05) (figure 4) . table 3 . bronchoalveolar lavage total and differential cell counts (%) in patients with pneumonia. this study analyzed the usefulness of cellular analysis of bal fluid for predicting the etiology of pneumonia in critically ill adult patients. neutrophilic pleocytosis in bal fluid was frequently found in patients with bacterial-and viral pneumonia. the degree of pleocytosis, which was higher in the bacterial pneumonia, was useful for differential diagnosis of bacterial pneumonia. total wbc count had the best diagnostic accuracy for predicting bacterial pneumonia, and its diagnostic performances was better than those of serum procalcitonin and c-reactive protein concentrations. combinations of bal fluid total wbc count, serum procalcitonin concentration, and serum c-reactive protein concentration provided the best diagnostic yields. the data suggest that cellular analysis of bal fluid is a rapid and useful technique for differentiating bacterial pneumonia from viral pneumonia, and can be used to direct early appropriate treatment. information about the role of cellular profiles of bal fluid for differential diagnosis of bacterial pneumonia in adult patients is limited. stolz et al [11] . evaluated potential markers of bacterial infection in a cohort of immunocompromised patients with pulmonary complications. they reported that the percentage of neutrophils in bal fluid and the serum procalcitonin concentration are independent predictors of bacterial infection. they suggested that the optimal cutoff value of the percentage of neutrophils in bal fluid is 15% (sensitivity 84%; specificity 77%), which is much lower than the cutoff value in the current study. sternberg et al. [13] investigated the usefulness of bal in assessing pneumonia in renal transplant patients, and suggested that the optimal cutoff value of the percentage of neutrophils in bal fluid is .20% for predicting bacterial pneumonia. however, neither of these previous studies included patients with severe pneumonia caused by respiratory viruses alone, and both compared bal findings between patients with bacterial pneumonia and those with non-infectious diseases. in the current study, patients with viral pneumonia were included by using the newly developed multiplex respiratory virus rt-pcr. this showed that cases of viral pneumonia were frequently associated with neutrophilia in bal fluid (median 54.0%). we speculate that this underlies why the optimal cutoff value of percentage of neutrophils in bal fluid for predicting bacterial pneumonia is much higher in the current study (64%, table 4 ) than in previous studies. several authors of the current study previously investigated the diagnostic utility of soluble triggering receptor expressed on myeloid cells-1 (strem-1) in bal fluid of various patient populations with bilateral lung infiltrates. a cutoff value of $60% neutrophils in bal fluid is useful for differential diagnosis of bacterial or fungal pneumonia from other causes of pneumonia or non-infectious diseases (auc = 0.77, 95% ci; 0.54-0.84, p = 0.001) [12] . in comparison to this previous study, the current study did not include patients with non-infectious diseases or fungal pneumonia, included much more cases of severe viral pneumonia, and analyzed the counts of various cell types. among the currently available inflammatory markers, serum procalcitonin is one of the best indicators of bacterial infections, including lower respiratory tract infections [19] . the usefulness of serum procalcitonin measurements has been validated in the diagnosis, severity assessment, and follow-up of patients with lower respiratory tract infections [20] [21] [22] . in the current study, the auc of serum procalcitonin concentration for predicting bacterial pneumonia (auc = 0.705) was smaller than those of total wbc (auc = 0.855) and neutrophil (auc = 0.837) counts. the combination of bal fluid wbc counts and serum procalcitonin concentration tended to improve the diagnostic accuracy of the roc model. this indicates that combinations of these markers can be useful to rule-out (bal fluid total wbc count $510/ml or serum procalcitonin concentration $0.71 ng/ml with a sensitivity of 95.8% and negative likelihood ratio of 0.068) or rule-in (bal fluid total wbc count $510/ml and serum c-reactive protein concentration $26.1 mg/dl with a specificity of 95.7% and positive likelihood ratio of 8.63) bacterial pneumonia. diagnostic accuracy could be further improved if bal fluid cellular profiles are interpreted alongside clinical presentations, radiographic studies, and other relevant test results. using this approach, it might be possible to identify patients who can be managed without antibacterial agents or those who require antiviral agents. although not included in the current study, strem-1 in bal fluid is another notable biomarker for the diagnosis of pneumonia. strem-1 is reportedly a potent discriminator of bacterial pneumonia from non-infectious lung infiltrations [12, [23] [24] [25] [26] [27] . however, the proposed cutoff values of strem-1 concentration vary widely (5-900 pg/ml) and some studies have questioned the reliability of bal fluid strem-1 [28] [29] [30] . studies on bal fluid strem-1 have mainly been confined to patients with ventilator-associated pneumonia. therefore, the usefulness of strem-1 for etiologic diagnosis of pneumonia, especially differential diagnosis of viral pneumonia, has not been elucidated yet. the current study directly compared patients with bacterial and viral pneumonia, and therefore differs from previous studies of strem-1. bal fluid strem-1 concentration in combination with the bal fluid cellular profile might exhibit better diagnostic performance, although this warrants further studies. a strength of the current study is the relatively strict enrollment criteria used. to minimize bias associated with antimicrobial therapy, all patients who received antimicrobial therapy for more than 24 hours were excluded, regardless of the adequacy of prior antimicrobial therapy. by using strict enrollment criteria, however, only a small proportion of pneumonia patients who underwent bronchoscopic bal was finally included (figure 1 ), which might have influenced on the results. however, positive likelihood and negative likelihood ratio, which are not influenced by disease prevalence, were good, which supports the validity of the data. in clinical practice, the results might be applied to patients who have received antimicrobial therapy more than 24 hours. that is, if bal fluid cellular analysis shows evident pleocytosis even after antimicrobial therapy for more than 24 hours, it would be a strong suggestion for bacterial etiology. the study has several limitations. first, the small sample size of the select critically ill patient population analyzed limits the general applicability of our findings. moreover, since our study included critically ill patients with acute respiratory failure secondary to pneumonia who were not receiving antimicrobial therapy, our results may not be applicable to the majority of severe pneumonia patients in clinical practice. second, the impact of antimicrobial therapy on the both bal fluid cellular profiles and other inflammation markers such as procalcitonin, remains to be further elucidated. third, cases of invasive pulmonary aspergillosis, pneumocystis jirovecii pneumonia, and mycobacterial pneumonia, were not included, mainly because few patients had these types of pneumonia. fourth, patients with non-infectious causes of pulmonary infiltrates that can often mimic infectious causes, such as acute respiratory distress syndrome, cryptogenic organizing pneumonia, eosinophilic pneumonia, and drug-induced pneumonitis, were also excluded from our analyses. the inclusion of those cases may have caused a marked decrease in the specificity of our bal fluid criteria. finally, all the pathogens were not directly identified from bal fluid. some patients were included in whom pathogens were identified by other means, such as blood culture, endotracheal aspirates culture, urinary pneumococcal antigen test, and pcr from nasopharyngeal samples, as long as clinically and radiographically compatible and no other etiology was demonstrated. therefore, patients with coincidental upper respiratory infections or colonization may have been included. in conclusion, the data indicate that cellular analysis of bal fluid, alone or in combination with serum procalcitonin and creactive protein concentrations, may rapidly provide valuable diagnostic information for the early differential diagnosis of pneumonia in critically ill adult patients. timing of antibiotic administration and outcomes for medicare patients hospitalized with community-acquired pneumonia inadequate antimicrobial treatment of infections: a risk factor for hospital mortality among critically ill patients prognostic factors of non-hiv immunocompromised patients with pulmonary infiltrates quantitative culture of bronchoalveolar lavage fluid for the diagnosis of bacterial pneumonia diagnostic accuracy of bronchoalveolar lavage samples in immunosuppressed patients with suspected pneumonia: analysis of a protocol bronchoalveolar lavage for diagnosing acute bacterial pneumonia interstitial lung disease guideline: the british thoracic society in collaboration with the thoracic society of australia and new zealand and the irish thoracic society an official american thoracic society clinical practice guideline: the clinical utility of bronchoalveolar lavage cellular analysis in interstitial lung disease bronchoalveolar lavage cell populations in the diagnosis of sarcoidosis bronchoalveolar lavage: a forgotten tool bal neutrophils, serum procalcitonin, and c-reactive protein to predict bacterial infection in the immunocompromised host diagnostic utility of the soluble triggering receptor expressed on myeloid cells-1 in bronchoalveolar lavage fluid from patients with bilateral lung infiltrates utility of bronchoalveolar lavage in assessing pneumonia in immunosuppressed renal transplant recipients viral infection in patients with severe pneumonia requiring intensive care unit admission health care-associated pneumonia requiring hospital admission: epidemiology, antibiotic therapy, and clinical outcomes infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia optimal cut-point and its corresponding youden index to discriminate individuals using pooled blood samples procalcitonin in bacterial infections-hype, hope, more or less usefulness of procalcitonin levels in community-acquired pneumonia according to the patients outcome research team pneumonia severity index effect of procalcitonin-guided treatment on antibiotic use and outcome in lower respiratory tract infections: cluster-randomised, single-blinded intervention trial pneumonitis-associated hyperprocalcitoninemia soluble triggering receptor expressed on myeloid cells and the diagnosis of pneumonia combined measurement of procalcitonin and soluble trem-1 in the diagnosis of nosocomial sepsis triggering receptors expressed on myeloid cells in pulmonary aspiration syndromes diagnostic implications of soluble triggering receptor expressed on myeloid cells-1 in patients with acute respiratory distress syndrome and abdominal diseases: a preliminary observational study diagnostic implications of soluble triggering receptor expressed on myeloid cells-1 in bal fluid of patients with pulmonary infiltrates in the icu soluble triggering receptor expressed on myeloid cells-1 in bronchoalveolar lavage fluid is not predictive for ventilator-associated pneumonia soluble triggering receptor expressed on myeloid cells-1 (strem-1) as a diagnostic marker of ventilator-associated pneumonia soluble triggering receptor expressed on myeloid cell-1 is increased in patients with ventilator-associated pneumonia: a preliminary report key: cord-000425-isw6jeir authors: flori, laurence; gao, yu; laloë, denis; lemonnier, gaëtan; leplat, jean-jacques; teillaud, angélique; cossalter, anne-marie; laffitte, joëlle; pinton, philippe; de vaureix, christiane; bouffaud, marcel; mercat, marie-josé; lefèvre, françois; oswald, isabelle p.; bidanel, jean-pierre; rogel-gaillard, claire title: immunity traits in pigs: substantial genetic variation and limited covariation date: 2011-07-29 journal: plos one doi: 10.1371/journal.pone.0022717 sha: doc_id: 425 cord_uid: isw6jeir background: increasing robustness via improvement of resistance to pathogens is a major selection objective in livestock breeding. as resistance traits are difficult or impossible to measure directly, potential indirect criteria are measures of immune traits (its). our underlying hypothesis is that levels of its with no focus on specific pathogens define an individual's immunocompetence and thus predict response to pathogens in general. since variation in its depends on genetic, environmental and probably epigenetic factors, our aim was to estimate the relative importance of genetics. in this report, we present a large genetic survey of innate and adaptive its in pig families bred in the same environment. methodology/principal findings: fifty four its were studied on 443 large white pigs vaccinated against mycoplasma hyopneumoniae and analyzed by combining a principal component analysis (pca) and genetic parameter estimation. its include specific and non specific antibodies, seric inflammatory proteins, cell subsets by hemogram and flow cytometry, ex vivo production of cytokines (ifnα, tnfα, il6, il8, il12, ifnγ, il2, il4, il10), phagocytosis and lymphocyte proliferation. while six its had heritabilities that were weak or not significantly different from zero, 18 and 30 its had moderate (0.1<h2≤0.4) or high (h2>0.4) heritability values, respectively. phenotypic and genetic correlations between its were weak except for a few traits that mostly include cell subsets. pca revealed no cluster of innate or adaptive its. conclusions/significance: our results demonstrate that variation in many innate and adaptive its is genetically controlled in swine, as already reported for a smaller number of traits by other laboratories. a limited redundancy of the traits was also observed confirming the high degree of complementarity between innate and adaptive its. our data provide a genetic framework for choosing its to be included as selection criteria in multitrait selection programmes that aim to improve both production and health traits. increasing robustness by improving resistance/tolerance to pathogens is an important selection objective in most livestock species, particularly in pigs. in the past 30 years, selection for growth, carcass leanness, meat quality and prolificacy, combined with stringent sanitary rules, vaccination and use of antibiotics, has been highly effective in pigs [1] . since the early 2000's, prophylactic use of antibiotics as growth promoters has been forbidden by european legislation. as a result, the health status of numerous farms has deteriorated, leading to an increase in the therapeutic use of antibiotics. indeed, animals highly selected for production traits may be more susceptible to pathogens or less able to maintain performance after infection. deterioration of the global health status may also be due to environmental trends. in this context, including health traits in existing breeding schemes using direct and/or indirect strategies is an emerging trend in pig breeding. direct strategies target animal resistance/tolerance to specific pathogens but may result in increased susceptibility to other diseases [2, 3] . alternatively, an indirect and putatively more global approach focuses on immune traits (its) providing a measure of immune capacity (i.e. immunocompetence) and hopefully predicting the responses to pathogens in general [4] . the choice of relevant its is further based on knowledge of the immune system. this highly interactive and cooperative system is classically separated into two arms referred to as innate and adaptive, which produce a combined response. innate immunity is the first line of defence. its activation is non pathogen-specific and depends on the recognition of evolutionarily conserved pathogenassociated molecular patterns such as lipopolysaccharides constituting bacterial cell walls [5] . innate immunity involves physical barriers, innate immune cells such as dendritic cells (dcs), monocytes, natural killers (nk cells) or cd t lymphocytes, and inflammatory cytokines such as il1b, il6 and tnf. adaptive immunity is antigen-specific and requires the recognition of specific ''non-self'' antigens via a process of antigen presentation and results in an immunological memory. adaptive immunity is divided into cell-and humoral-mediated immunity with different effector functions [6] . in order to include its in a breeding plan to improve pig immunocompetence, the genetic and phenotypic parameters of the different its need first to be estimated. several studies in swine, mice, poultry and cattle demonstrated the possibility of selecting animals with high or low immune response (ir) as characterized by one or a few its [2, 7, 8, 9, 10] . a study on yorkshire pigs selected for eight generations for high and low adaptive ir (hir and lir, respectively) on an index combining four standardized measures of specific antibodies and cellmediated ir, after stimulation with specific antigens (bacillus calmette-guérin and hen egg white lysozyme), has revealed that hir and lir animals differ in response to immunization and infection [2, 11, 12, 13, 14] . other studies have also shown that various innate and adaptive its are genetically controlled. for example, variation in innate its, such as nk cells, monocytes, interferon a (ifna) production or phagocytosis [15, 16, 17] is heritable and several adaptive its have moderate to high heritability values including total white blood cells (wbc), cd4 + t lymphocyte, cd8a + t lymphocyte and b lymphocyte subsets [15, 16, 17] , delayed-type hypersensitivity reaction [15, 18] , lymphocyte proliferative response [15] , interleukin-2 (il2) production by lymphocytes [15] and antibody response [12, 15, 18, 19] . clapperton and colleagues have also reported that variation in acute phase protein levels is heritable [16, 17] . finally, several significant qtls for total leukocyte count ( [20, 21] ; animal-qtldb, http://www.animalgenome.org/cgi-bin/qtldb/index), mitogen-induced proliferation [20] , antibody response [20, 22] , cytokine production (il10 and ifnc) [23] , complement activity [22] , and acute phase protein serum concentration [22] have been detected and mapped to different pig chromosomes. taken together these data demonstrate that variation in some its is under genetic control. however, most of the results reported so far have targeted a limited number of traits and very few studies have combined innate and adaptive its. our global goal is to identify immunocompetence traits for inclusion in selection schemes aiming to improve both zootechnical performances and health traits in pigs. for this purpose, we have launched a genetic and genomic study of numerous its covering innate and adaptive ir [24] . in this report, we present the results of a global genetic study, combining principal component analysis (pca), and genetic parameter estimation applied to a large number of innate and adaptive its in a pig population vaccinated against mycoplasma hyopneumoniae (m. hyopneumoniae). a set of 54 its was measured on a population of 443 pigs three weeks after vaccination against m. hyopneumoniae (tables 1 and 2; table s1 ; figure 1 ). these its comprise either traits related to ir (phagocytosis, lymphocyte proliferation, cytokine production after in vitro stimulations, levels of total and specific antibodies, levels of acute phase proteins) or traits related to total leukocyte and leukocyte subpopulation counts. the various characteristics and descriptive statistics of the traits measured on each animal of the studied population (n = 383 to 442) are summarized in tables 1 and 2 . among the cell-mediated its evaluated after diverse stimulations, higher responses in cytokine levels were observed after phorbol myristate acetate (pma)-ionomycin (pmaiono) stimulation compared to lipopolysaccharide (lps) and concanavalin a (cona) stimulations, except for il2 production. conversely, a higher lymphocyte proliferation was detected after cona and pmaiono stimulations than after lps stimulation. ample phenotypic variation was observed for most traits. the coefficient of variation (cv) was equal to 0.8 on average and ranged from 0.07 to 3.9 (tables 1 and 2 ). limited dispersion (cv#0.9) was observed for traits derived from hemograms, cell subsets characterized by fluorescence-activated cell sorting (facs), phagocytosis capacity and non-specific immunoglobulins. moderate dispersion (0.9,cv#1.5) was observed for most cytokines produced in vitro except for tumour necrosis factor a (tnfa) and mitogen proliferation-related traits. finally, the cv for seric c reactive protein (crp) and haptoglobin (hapt) levels were close to 1.6 and 2, respectively. the highest cv (3.9) was obtained for the specific iggs directed against m. hyopneumoniae. these data clearly indicate that the seric inflammatory protein levels and the specific iggs had the greatest phenotypic variance in our study. in order to analyse the factors causing the variation, we performed a normed pca with 32 traits (tables 1 and 2 ). for cellmediated adaptive ir, we included only those traits related to cytokine production and lymphocyte proliferation after pmaionomycin stimulation. for innate its, we included 10 facscharacterized cell subtypes including the percentages of b lymphocytes (igm + ), cd t lymphocytes (tcrcd + ), three subsets of ab t lymphocytes (cd4 + cd8 + , cd4 -cd8 + and cd4 + cd8 -), nk cells (cd16 + cd2 + ) and three monocyte subsets (cd16 + cd172a + , cd16 + mhcii + , mhcii + cd172a + ). we excluded from the analysis four haematological traits not directly involved in immunity: red blood cell count (rbc), hematocrit (ht), red blood cell distribution width (rdw) and platelet count (plt). the percentage of variance (inertia) explained by the first five components was over 50% (figure 2a ). each of these five components explained more than 5% of the total variance and the first two components accounted for 16.4 and 10.8% of the total variance, respectively. taking into account the 32 components from pca, multivariate normal mixture modelling and modelbased clustering (see materials and methods) were used to identify clusters of its. the highest bayesian information criterion (bic) was obtained using the diagonal model with variable shape and variable variance (vvi in pink on figure 2b ) and k = 3 (first factorial plan on figure 2c ). no parameter was located near the correlation circle indicating that the phenotypic correlations between its are globally weak. a first cluster (k1 in blue on figure 2c ) groups together four hemogram-derived cell counts: white blood cell count (wbc), lymphocyte count (lym), monocyte count (mon) and neutrophil count (neu). this cluster is representative of total cell number traits despite the eosinophil count (eos) not being included. a second cluster (k2 in green on figure 2c ) groups together all the traits related to facscharacterized leukocyte subpopulations (expressed as the percentage of cells with one or two surface antigens) except cd t lymphocytes (tcrcd + ), with a cell response parameter (il10-pmaiono) and the seric level of haptoglobin (hapt). this cluster can be considered as representative of the leukocyte subsets. a third cluster (k3 in red on figure 2c ) includes all other cell response traits, one hemogram-derived cell count (eos) and one facs-characterized leukocyte subpopulation. note that k1 and k2 related traits, which mainly correspond to cell subsets and explain around 25% of the phenotypic variance, show clear clustering ( figure 2c ). these traits are grouped on the first pca axis and separated on the second axis. traits belonging to k3, representative of cell activity (cytokine production, phagocytosis and antibody production), are more spread out on the other axes (data not shown). interestingly, cluster analysis did not highlight any cluster of innate or adaptive its. the estimation of phenotypic correlations (r r p ) with wombat confirmed that the its are weakly correlated, except i) among a few cell count traits (wbc, lym, mon, neu), and ii) between cell count traits and a few leukocyte subsets (wbc, lym, cd16 + cd2 + and cd4 -cd8 + ) for whichr r p greater than 0.4 were estimated (table s2; figure s1 ). weakr r p were mainly positive (330) with 166 negative. no strongly negativer r p (#20.4) were found. taken together, pca and estimations of phenotypic correlations showed that the level of redundancy between the different immune parameters was limited. heritability estimates of the 54 analyzed its was equal to 0.45 on average (se = 0.20; table 1 for the large set of adaptive its, the mean heritability was 0.48 (se = 0.21). no significant difference in average heritability values was detected either between group of its qualifying the innate and adaptive immunity or the humoral and cellular adaptive immunity. in addition, an equivalent proportion of innate and adaptive its had significant heritability values. indeed, 40% (10/25), 40% (2/5) and among the traits involved in cell-mediated immunity, variation in ab t lymphocyte (cd4 -cd8 + , cd4 + cd8 + and cd4 + cd8cells) counts was highly heritable. heritability estimates of the three cytokine (il4, il10, ifng) levels were moderate to high after pmaiono and cona stimulations and weak to moderate after lps stimulation. for those cytokines induced by cona or lps stimulation, confidence interval (95ci) for the heritabilities overlapped zero, except for il4-cona. il2 production after pmaiono, cona and lps stimulations gave high estimates of heritability significantly different from zero for il2-pmaiono and il2-lps. proliferation measurements after various stimulations (prolif-cona, prolif-pmaiono, prolif-lps) provided moderate estimates of heritability not significantly different from zero. among the traits involved in humoralmediated adaptive immunity, heritabilities for total igg and iga antibody levels were higher than for total igm and specific antibodies, and weak h 2 values were obtained for b lymphocyte count (igm + cells). heritability for innate its such as i) total cell number (eos and neu), ii) leukocyte subsets (cd16 + cd2 + cells, cd16 + cd172acells, cd16 + mhcii + cells and tcrcd + lymphocytes), iii) cytokine production (ifna and il12), and iv) phagocytosis were high and significantly different from zero. in addition, several innate its showed weak to moderate heritability, including proinflammatory cytokines (il1b, il8, tnf and il6), mon, cd16 -cd2 + cells, cd16 + cd2cells, mhcii -cd172a + cells, mhcii + cd172acells, cd16 -cd172a + cells and cd16 + cd172a + cells. variation in acute phase proteins was moderately (crp) to highly (hapt) heritable. among the four traits, which measured the total number (mon) or proportions (mhcii + cd172a + , cd16 + cd172a + and cd16 + mhcii + ) of monocytes, moderate to high h 2 , but not significantly different from zero, were estimated, except for cd16 + mhcii + cells. other haematological traits (rbc, ht, rdw and plt) gave high h 2 estimates, of which ht and plt were significant. pairwise genetic correlations are presented in table s2 and illustrated in figure 3 . genetic correlation estimates among most its were generally weak but a few high genetic correlations were observed. the number of positive genetic correlations (310) was higher than that of negative correlations (183), as already observed for phenotypic correlations. positive genetic correlations were higher (in absolute values) than negative ones (table s2, figures 3 and s1). only 28 (2.7% of the total number of correlations) and five r ĝ (0.4% of the total number of correlation estimates) were higher than 0.4 or lower than -0.4, respectively. the unsupervised hierarchical clustering distinguished two main clusters of traits ( figure 3 ). the first cluster of 14 traits (cluster a) could be divided into two groups: i) a group of four traits including one innate immunity cytokine (ifna), two antibody levels (total igg and specific igg-mh), and one facscharacterized leukocyte subpopulation (cd16 + cd172a -) and ii) a group of 10 traits with nine hemogram-based cell counts or facscharacterized leukocyte subpopulations (wbc, lym, mon, neu, eos, cd16 + mhcii + , cd16 + cd2 + , cd4 -cd8 + cells) and two cell activity traits (igm and il10-pmaiono). the second cluster of 18 traits (cluster b) is also subdivided into two groups of traits: i) a group of three different cell response traits (il6, iga and ifng-pmaiono) and one facs-characterized leukocyte subpopulation (igm + cells), and ii) a group of nine cell response traits (il12, il4-pmaiono, il2-pmaiono, tnf, prolif-pma, hapt, crp, il1b, il8 and phag) and four facs-characterized leukocyte subpopulations (cd4 + cd8 -, cd4 + cd8 -, tcrcd + and mhcii + cd172a + cells). in cluster a, the first group of 10 traits showed moderately to highly positive genetic correlations with each other. indeed, r ĝ values greater than 0.4 were estimated between wbc, lym, mon, neu, eos, cd4 -cd8 + and cd16 + cd2 + (nk) cells ( figure 3 , table s2 ). in cluster b, r ĝ values greater than 0.4 were estimated between i) tnf, il8 and phag, and ii) crp and hapt ( figure 3 , table s2 ). strong negative r ĝ values (,20.4) were found between a few traits from both clusters: i) tnf and three cell number traits (wbc, lym, mon), ii) il6 and cd16 + mhcii + , and iii) crp and iga. the measured its globally cover innate and adaptive immunity the large-scale study reported here allowed us to estimate the genetic and phenotypic parameters of numerous its measured on pigs bred in the same environment. innate immunity is represented by 25 traits, humoral-mediated immunity by five traits and cell-mediated adaptive immunity by 18 traits. we have also considered the total number of white blood cells and lymphocytes, and four other haematological traits (rbc, ht, rdw and plt). within cell-mediated immunity, we explored both th1 and th2 responses by measuring cytokine production. figure 1 summarizes the traits that we selected to cover immunity globally. these traits include in vivo measures on blood such as quantification of cell populations by hemogram, dosage of circulating immunoglobulins and acute phase proteins, as well as ex vivo measures obtained after in vitro tests such as lymphocyte proliferation, phagocytosis capacity and cytokine production after blood stimulation. all these its have been widely studied in humans [25, 26] . the trait typology we have used (table 1 ) follows a model based on a clear distinction between innate and adaptive immunity, which may be over-simplistic since both immune systems are closely interconnected [27, 28] . monocytes are involved in innate immunity but are also antigen-presenting cells required for adaptive immunity, and cytokines such as il12 are at the interface between innate and adaptive immunity. similarly, the cell-mediated and humoral adaptive immunity subdivision is artificial. for example, il4 is a cytokine produced by th2 lymphocytes that is usually classified as part of adaptive cellmediated immunity, whereas it is also involved in antibody production and thus adaptive humoral immunity. in addition, the conventional paradigm that cd4 + ab t lymphocytes differentiate into th1 and th2 lineages expressing specific cytokines is collapsing. indeed, recent studies have revealed that cytokine production by the different cd4 + t cell subsets (th1, th2, th, th17 and itreg) is highly flexible, providing new insight into the th cell plasticity [29] . nevertheless, although schematic, the approach used in our report provides a comprehensive overview of genetic variation and co-variation across the entire immune spectrum in pigs (figure 1 ). table 1 illustrates various ranges of phenotypic variation in the measured immune traits, with most having a cv over 0.5. such variability has already been reported in large panels of healthy humans [26] , and in previous studies on pigs [2, 16, 17, 30] . for instance, we substantiated the high level of variation of cytokine production previously reported for il2 production and virusinduced ifna production in a swedish yorkshire population [20] . for innate immunity-related cytokines and il12, stimulation was performed with a mixture of lps, pma and ionomycin. il8 was the cytokine produced with the highest levels followed by il1b, tnf, il12 and lastly il6. these four cytokines are not expected to be expressed at similar levels at all time points after stimulation and a kinetic study would help to improve comparison of cytokine production levels. weaker levels of adaptive ir cytokines are observed after lps stimulation compared to cona or pmaiono. these differences could be related to the distinct modes of action of these molecules. indeed, pma, a plant-derived functional analog of diacylglycerol, in conjunction with ionomycin, a calcium ionophore produced by streptomyces conglobatus, and cona, a lectin originally extracted from the jack-bean canavalia ensiformis, are known to be potent mitogens of blood lymphocytes [31, 32] . lps, a major structural component of the outer membrane of gram-negative bacteria, which binds the cd14/ the five first components, which explain more than 50% of the total variance, are in red. b. plot of the bayesian information criterion (bic) calculated with different models according to number of clusters. six models are compared: eii (spherical with equal volume and equal shape), vii (spherical with variable volume and equal shape), eei (diagonal with equal shape and equal volume), vei (diagonal with variable shape and equal volume), evi (diagonal with equal shape and variable volume), vvi (diagonal with variable shape and variable volume). c. first factorial plan (1: first component, 2: second component) with three clusters identified by multivariate normal mixture modelling and model-based clustering taking into account the 32 components (clusters k1, k2 and k3 are in blue, green and red, respectively). doi:10.1371/journal.pone.0022717.g002 tlr4/md2 receptor complex and promotes the secretion of proinflammatory cytokines, has been extensively used to study innate immune response [33] . we have already shown that transcriptome modifications in peripheral blood mononuclear cells (pbmcs) differ between pmaiono and lps stimulation and that pmaiono and lps target different cells and cellular pathways [34] . the combination of pma and ionomycin induces a stronger stimulation that may be related to a higher production of cytokines as detected in the present study. in addition, the lymphocyte proliferation induced by lps is weaker than that observed with other stimulants, as expected. our study provides the first heritability estimates for innate and adaptive cytokine production and for lymphocyte proliferation after pma-ionomycin and lps stimulations in pig. pro-inflammatory cytokines appear to show less heritable variation than adaptive system-related cytokines. among the adaptive system-related cytokines, estimated heritability was weakest for cytokines produced after lps stimulation, except for il2 production. heritability estimates for lymphocyte proliferation after cona, pma and lps stimulations were moderate and that of lymphocyte proliferation after cona stimulation was comparable to the value obtained by edfors-lilja and colleagues [15] . moderate to high heritability estimates for cell count traits from hemogram or facs also confirmed those previously obtained for wbc, total lymphocytes, neutrophils, eosinophils and some leukocyte subsets (for cd4 + and cd8 + t lymphocytes, cd t lymphocytes, cd11r1 + , cd11r1 + cd8a -, cd11r1 + cd8a + and cd16 + mhcii + ) [15, 16, 17, 19] . likewise, our results confirmed the high heritability estimate for phagocytosis [15] . conversly, a lower heritability estimate for crp than previously reported was observed [15, 16] . overall, the heritability estimates for these traits appear robust regardless of populations, environments and protocols. some discrepancies exist between our heritability estimates and previous results for humoral-mediated adaptive its and some innate its. indeed, in our study, b lymphocyte levels (igm + cells) are not significantly heritable contrary to other results [16, 17] and specific iggs (igg-mh) have lower heritability estimates (0.12, se = 0.19) than previously reported (range from 0.27 to 0.45) for specific antibodies directed against other antigens [12] . our estimated heritability for total igg is higher (0.92, se = 0.20) than in published reports [3, 15, 18, 19] . in addition, our estimated h 2 for ifna production is moderate to high (0.60, se = 0.23), contrary to previous results (range from 0 to 0.08) [15] , and for haptoglobin (0.55, se = 0.21) is higher than previously published (range from 0.14 to 0.23) [17, 19] . these discrepancies could be due to differences in the pig breeds and in environment factors but also to the absence of common standardised protocols between laboratories. in order to better qualify the phenotypes, protocol standardisation is needed. overall, we show that variation in both innate and adaptive its is under substantial genetic control (figure 1 ; tables 1 and 2) . similar heritability estimates for innate and adaptive its and also between cell number and cell response parameters were observed. further, heritability estimates do not differ consistently between in vivo and ex vivo measures with no apparent bias due to phenotyping methods. these data also suggest candidate its for qtl mapping. indeed, mapping studies have already started for total leukocyte count ( [20, 21] ; animalqtldb, http://www.animalgenome.org/cgi-bin/qtldb/index), mitogen-induced proliferation [20] , antibody response [20, 22] , cytokine production (il2, il10 and ifnc) [23, 35] , complement activity [22] , and acute phase protein serum concentration [22] . compared to the previously limited data on genetic correlations between its in pigs, our study provides a large-scale estimation of phenotypic and genetic correlations among 32 its. pca results and correlation estimations highlight the weak phenotypic and genetic correlations between the different its, except mainly for cell subsets. no cluster of innate and adaptive its is revealed. these results illustrate that many of the its included in our study provide more or less independent potential clues for selecting for improved immunocompetence. such complementarity is expected since innate immunity is in place or ready for activation prior to infection or antigenic stimulation and collaborates with adaptive immunity, which is induced by infection or antigenic stimulation. nevertheless, a few highly positive genetic correlations have been detected between total number of white blood cells and some leukocytes subsets, and between some leukocyte subsets such as total number of lymphocytes, cd4 -cd8 + lymphocytes (which contains ab cd4 -cd8 + lymphocytes and nk), and nk cells (cd16 + cd2 + cells). phagocytosis, production of il8 and tnf, two pro-inflammatory cytokines produced by monocytes and macrophages, were positively correlated with acute inflammatory phase proteins produced by hepatocytes i.e. crp and hapt. a high correlation was also found between crp and hapt. clapperton and colleagues [17] have shown that phenotypic correlations between leukocyte subsets and acute phase proteins are weak (,0.2) and not significantly different from zero in agreement with our results. in contrast to our study, clapperton and collaborators have not detected any significant genetic and phenotypic correlations between different leukocyte subsets except when one subset was nested in another [16] . our results provide a framework for including its in multitrait selection for immunocompetence in pigs. criteria for inclusion should take into account heritability, biological relevance, biological sensitivity and feasibility of measurement [25] . the weak genetic correlations between most its suggest that it will be difficult to choose only a few its to select for immunocompetence, and that a combination of many traits may be required [26] . the moderate to high heritabilities estimated for many traits together with the selection study on pigs carried out by wilkie and colleagues a decade ago support the feasibility of selecting for immunocompetence [2, 13] . chickens have also been successfully divergently selected for carbon clearance (phagocytic activity), high antibody response to newcastle disease virus three weeks after vaccination (adaptive humoral ir) and wing web response to pha (high cell-mediated immune response) for more than twelve generations [9, 36] . in order to include immunocompetence in selection for improved health, a major challenge will be to correlate variation in heritable its in healthy animals with inter-individual variability in response to various pathogens. testing this hypothesis will be a key point for further use of its as indirect selection criteria in multitrait selection to improve resistance to disease. some results on genetic and phenotypic relationships between immunocompetence and susceptibility to specific pathogens have already been reported in the literature for pigs. among pigs selected for eight generations for high (hir) or low (lir) response based on an index of four cell and humoral-mediated immunity traits, an increased specific antibody response and lower polyserositis were observed in the hir pigs compared to the lir pigs after challenge with a novel pathogen, mycoplasma hyorhinis [12, 37] . thus, animals with a high ir level to unrelated challenges, as defined by wilkie and colleagues [12, 37] , have a better response to infection with mycoplasma hyorhinis. however, hir pigs develop more severe arthritis than lir pigs. indeed, the levels of humoral and cell-mediated adaptive its included in the wilkie et al index induce the formation of immune complexes and/or the development of inflammatory responses, central to the pathogenesis of mycoplasma hyorhinis-induced arthritis. other correlation tests between its and response to various infections are needed. however, it is important to remain cautious with high responder animals, which could develop autoimmune pathologies or pathological iummne responses. all the studies on immunocompetence and resistance to disease will have to be completed by estimation of genetic correlations with economically important traits already under selection. negative genetic correlations have been reported between some its (monocytes, cd11r1 + cells) and average daily gain [16] . however a larger correlation study considering a higher number of its and pig performances is needed and is ongoing in our population. in the future, a more sustainable production system may require a compromise with a slight decrease in performance traded off for a gain in animal robustness. more studies are required to better understand the correlations between its and production traits and it is not established which levels of its would be good predictors for resistance to pathogens if any. in conclusion, our results show that variation in many its is under significant genetic control in pigs and these findings may provide insights in other species. moreover, based on heritability and correlation estimations, some of the its that we have studied might be incorporated into selection schemes, provided they are associated with improved global health and do not exhibit strong antagonisms with other economically important traits. our experiment was conducted in accordance with the french national regulations for humane care and use of animals in research. no ethics approval was required for the vaccination and the collection of blood samples under the then current regulations. experiments were performed under the individual license numbers 77-01 assigned to marcel bouffaud who was responsible for experiments in the test farm, and 78-16 assigned to a veterinarian, dr silvia vincent-naulleau. the experimentation agreement number for the test farm at le rheu was a35-240-7. a total of 443 large white pigs (castrated males, dam line) tested for performance traits in a pig test station (ue450, inra, le rheu, france) was included in the study. the pigs were distributed in seven contemporary groups and belonged to 307 nuclear families obtained from 106 boars, with an average of 4.1 (+/2 1.7) piglets per boar. animals were born and weaned in 16 different selection herds and arrived in the test station at five weeks of age with no prior vaccination. they were placed into pens of 30 piglets in a post weaning unit and vaccinated against m. hyopneumoniae (stellamune, pfizer, one injection) one day after their arrival in the test station, when 36.3 days old in average. all pigs were apparently healthy with no clinical sign of infection. all animals were sampled three weeks after vaccination. blood samples were collected via the external jugular vein into tubes with or without anti-coagulants, according to further use. blood collected in 10 ml tubes with no anti-coagulant was centrifuged at 3200 g for 15 min at 4uc. the serum was collected and stored at 220uc until use. plasma was collected from blood sampled in heparinised tubes and stored at 220uc before use. hemograms were measured with an ms4-5 counter (eli-techgroup, france) with blood sampled in edta tubes. among a set of 18 traits, nine were included in the genetic analyses: total number of leukocytes, lymphocytes, monocytes, neutrophils, eosinophils, erythrocytes, platelets and hematocrit (tables 1 and 2 ). total concentrations of immunoglobulin subsets were measured by elisa as previously described [38] . plasma samples were diluted 1:6000, 1:4000 and 1:60,000 to detect igm, iga and igg, respectively, in tris-buffered saline and added to plates coated with immunoglobulin class specific pig antibody (bethyl laboratories inc., interchim, france). the different subsets were detected with the appropriate peroxidase anti-pig igm, iga or igg (bethyl laboratories inc.) and were quantified by reference to standard curves constructed with known amounts of pig immunoglobulin subsets. anti-m. hyopneumoniae igg titers were also measured by elisa using a commercial kit (elisa id screenh m. hyopneumoniae indirect, idvet, france). absorbance was read at 450 nm using an elisa plate reader (spectra thermo, tecan, nc, usa) and the biolise 2.0 data management software. haptoglobin and c reactive protein levels were measured in pig serum by colorimetric tests (phase haptoglobin assay, abcys biologie, france) and elisa assays (porcine c reactive protein assay, abcys biologie, france), respectively. absorbance was read at 450 nm using an elisa plate reader (mrx revelation, dynex). pbmcs were purified by density gradient centrifugation. a volume of 13 ml heparinised blood was added to leucosept tubes (greiner bio-one, france) pre-filled with 17 ml ficoll (lymphocytes separation medium, eurobio, france) and centrifuged at 1200 rpm for 35 min. pbmcs were collected at the ficoll interface and washed in 50 ml d-pbs without mgcl 2 and cacl 2 (gibco, invitrogen, france). cells were then incubated in 2 ml bd pharmlyse 1x (bd biosciences, france) at room temperature. purified pbmcs were washed in 50 ml d-pbs without mgcl 2 and cacl 2 , incubated with 2 ml pig serum at 4uc for 20 min, washed again in 50 ml d-pbs without mgcl 2 and cacl 2 and then washed in 50 ml s/w buffer (1 g/l nan 3 , 10 g/l bovine serum albumin in pbs, ph 7.3) at a final concentration of 5.10 6 cells/ml. 10 6 cells were used for each antibody labelling. single, double or triple staining was performed using monoclonal antibodies (mabs) directed against i) cd2 (msa4, isotype igg2a, vmrd) and cd16 (mca1971, isotype igg1, serotec), ii) cd4 (pt90a, isotype igg2a, vmrd) and cd8a (pt81b, isotype igg2b, vmrd) iii) tcrcd (mac320, isotype igg2a, bd biosciences pharmingen), iv) igm (pig45a, isotype igg2b, vmrd) and v) mhcii (msa3, isotype igg2a, vmrd), cd16 (mca1971, isotype igg1, serotec) and cd172a (74-22-15a, isotype igg2b, bd biosciences pharmingen). briefly, pbmcs were stained with primary mabs for 25 min at 4uc, washed in s/w buffer and stained with allophycocyanin-conjugated anti-mouse igg1 (bd biosciences, france), phycoerythrin-conjugated antimouse igg2a (southern biotech, france), fitc-conjugated antimouse igg2b (southern biotech, france), apc-conjugated antimouse igg1 (bd biosciences, france), phycoerythrin-conjugated anti-rat igg2a (bd pharmingen, france), or phycoerythrinconjugated anti-mouse igg2b (southern biotech, france). after washing in s/w buffer, cells were fixed in bd cellfix solution (becton dickinson, germany). data acquisition and analysis were carried out with the facscan and cellquest software (becton dickinson, uk). synthesis of ifna by leukocytes of pigs was tested in vitro by incubating diluted total blood in the presence of pseudorabies virus (prv, suid herpesvirus 1)-infected, glutaraldehyde-fixed, pk15 cell monolayers, according to a protocol previously described for transmissible gastroenteritis virus [39] . confluent pk15 cell monolayers grown in 24 well plates were infected by prv at a multiplicity of infection of 20, fixed with 0.05% glutaraldehyde 8 h post-infection and washed with d-pbs and rpmi1640 before the addition of blood samples. for each animal, monolayers were incubated with diluted heparinized blood (270 ml of blood diluted 1:5 in dmem supplemented with antibiotics) for 18 h at 37uc. plates were then centrifuged at 450 x g for 20 min at 4uc, and supernatants were collected and stored at -20uc. ifna was assayed in the supernatants using a classical sandwich elisa test as previously described [40] . production and dosage of il1b, il6, il8, tnfa and il12 heparinized blood samples (400 ml) were fivefold diluted in 24well plates in 1.6 ml rpmi 1640 medium (biowhittaker, belgium) supplemented with 10% heat-inactivated fetal bovine serum (qb perbio, uk), 2 mmol/l l-glutamine, 100 u/ml penicillin and 100 mg/ml streptomycin. for stimulation, a mixture of 10 ng/ml pma (sigma, france), 1 mg/ml ionomycin (sigma, france) and 1 mg/ml lps from escherichia coli o111:b4 (sigma, france) was added to the diluted blood. for mock stimulation, a volume of pbs equal to the volume of stimulation reagents was added to the diluted blood. after incubation at 37uc for 24 h, culture supernatants were collected by centrifugation at 450 g for 20 min and stored at 220uc before use. the cytokines il1b, il6, il8, tnf and il12 were quantified using commercial elisa tests (duoset elisa development kits, r&d systems, usa). for quantification of basal levels of cytokines in supernatants from mock-stimulated cells, the samples were not diluted for quantification. supernatants collected from stimulated cells were diluted (1:22 for il1b and il8, 1:1 for il6, 1:10 for tnf and 1:2 for il12). all samples were tested in duplicates. absorbance was read at 450 nm using an elisa plate reader (mrx revelation, dynex). results were expressed as pg of cytokine/ml. heparinized blood diluted 1:5 in complete culture medium consisting of dmem (dulbecco's modified eagle medium, eurobio, france) supplemented with 5% fetal calf serum (hyclone, perbio, france), 2 mm l-glutamine, 100 u/ml penicillin and 50 mg/ml streptomycin (eurobio, france) was stimulated with 10 mg/ml cona (sigma, france), or with 50 ng/ml of pma (sigma, france) and 1 mg/ml of ionomycin (sigma, france) or 1 mg/ml lps from escherichia coli (sigma, france). cytokine content was measured in supernatants using elisa tests as already described [41] . briefly, purified fractions of anti-swine il-2, il-4, ifnc (clones a150d 3f1, a155b 16f2 and a151d 5b8 respectively, biosource, france) and il10 (clone 148801, r and d system, france) were used as capture antibodies, in conjunction with the biotinylated anti-swine il-2, il-4, il10 and ifnc monoclonal antibodies (clones a150d 8h10, a155b 15c6 and a151d 13c5, respectively, biosource, clinisciences, france) or anti-swine polyclonal antibody (goat anti-porcine il-10, r and d system, france). streptavidin-horseradish peroxidase (biosource) and tmb (fermentas, md, usa) were used for detection. absorbance was read at 450 nm using an elisa plate reader (spectra thermo, tecan, nc, usa) and the biolise 2.0 data management software. recombinant pig il-2, il-4, il10 and ifnc were used as standards. the detection limits were 700 pg/ ml, 60 pg/ml, 90 pg/ml and 100 pg/ml for il-2, il-4, il10 and ifnc, respectively. results were expressed as pg of cytokine/ ml. lymphocyte proliferation was performed in 96 well plates as already described [42] . briefly, heparinized blood samples were diluted 1:15 in complete culture medium consisting of dmem (dulbecco's modified eagle medium, eurobio, france) supplemented with 5% fetal calf serum (hyclone, perbio, france), 2 mm l-glutamine, 100 u/ml penicillin and 50 mg/ml streptomycin (eurobio, france). for detection of unspecific lymphocyte proliferation, the diluted blood samples were seeded in 96 well plates (200 ml/well) and mock-stimulated for 48 h by incubation in culture medium (control wells), or stimulated for 48 h by incubation with the culture medium supplemented with either 10 mg/ml cona (sigma, france), or 50 ng/ml of pma (sigma, france) and 1 mg/ml of ionomycin (sigma, france), or 1 mg/ml lps (sigma, france). control wells remained unstimulated. after 48 h of incubation at 39uc, 0.5 mci of 3 h-methylthymidine (icn, france) was added to each well. after another 24 h incubation period, the cells were harvested through glassfiber filters (whatman, united kingdom) by means of an automatic harvester (titerteck-skatron, molecular devices, france). incorporation of tritiated thymidine was measured with a liquid scintillation beta counter (kontron instruments, france). results were expressed as a stimulation index of lymphocyte proliferation calculated as mean counts per min (cpm) of the triplicate cultures in stimulated culture/mean cpm in control non-stimulated culture. preliminary statistical analyses were performed using r software [43] . in order to test if trait distributions deviated from gaussian, a d'agostino normality test was used (p = 0.05). since most traits were not sampled from a gaussian distribution, they were all normalized using a box-cox transformation except for phenotypes reaching the value zero, which were normalized using ln(1+x) transformation. significant effects of age at the time of vaccination, of time of vaccination, of breeding unit and of time of experiment were detected for most traits by variance analysis taking into account these effects. normed principal component analysis (pca, [44] , dudi.pca function, ade4 package [45] , r software [43] ) on a subset of its adjusted for age at the time of vaccination, time of vaccination, breeding unit and experiment (table 1) were performed using a linear model. clusters of its were detected using the r package mclust for normal mixture modelling and model-based clustering [46] . it combines model-based agglomerative hierarchical classification, based on the classification likelihood, and the expectation-maximization (e-m) algorithm for maximum likelihood estimation of multivariate mixture models. variance components, genetic parameters and their standard errors were estimated by the restricted maximum likelihood (reml) method [48] , using the wombat software [49] . this is the reference method to estimate genetic parameters with a mixed model. univariate and bivariate mixed linear animal models were employed to estimate heritability and genetic correlations, respectively. for the univariate analyses, the fixed part of the model included experiment time, age at the time of vaccination, vaccination time and herd of origin effects and the random part included a common litter environmental effect and direct genetic effects. in matrix notation y~x b zw a azw c cze where y = the vector of observations; x b , w a and w c are known incidence matrix relating observations to fixed and random effects; ß = a vector of fixed effects and covariates; a = the vector of direct genetic effects; c = the vector of common litter environmental effects; and e = the vector of random residual effects. all random effects were assumed to follow a normal distribution with zero mean. for bivariate analyses, the same effects as for univariate analyses were taken into account in the fixed part of the model and a direct genetic effect was included in the random part of the model. 95% confidence intervals (95ci) were calculated for heritability (h 2 ) estimates (h 2 ). heritability estimates have been classified: high (h 2 .0.4), moderate (0.1,h 2 ,0.4) or weak (h 2 #0.1). a graphical representation of the genetic correlations combined with a hierarchical clustering (euclidian distance, average link) was obtained with the heatmap function from the bioconductor software [50] . comparisons of heritability average between subsets of traits were tested using the wilcoxon mann-whitney test (significance threshold p-value = 0.05). figure s1 heatmap of the phenotypic correlations between 32 its. the correspondence between colour scale and genetic correlation levels are presented on the right-hand side of the heatmap. (tif) estimation of genetic trends in french large white pigs from 1977 to 1998 for growth and carcass traits using frozen semen selection for high immune response: an alternative approach to animal health maintenance? genetic aspects of health and disease resistance in pigs relationships between genetic change and infectious disease in domestic livestock toll-like receptors and innate immunity kuby immunology toward genetic dissection of high and low antibody responsiveness in biozzi mice genetic parameters for antibody response of chickens to sheep red blood cells based on a selection experiment correlated effects of selection for immunity in white leghorn chicken lines on natural antibodies and specific antibody responses to klh and m. butyricum evaluation of immune responses of cattle as a means to identify high or low responders and use of a human microarray to differentiate gene expression use of estimated breeding values in a selection index to breed yorkshire pigs for high and low immune and innate resistance factors immune responsiveness in swine: eight generations of selection for high and low immune response in yorkshire pigs multi-trait selection for immune response; a possible alternative strategy for enhanced livestock health and productivity cytokines in mycoplasma hyorhinis-induced arthritis in pigs bred selectively for high and low immune responses genetic variation in parameters reflecting immune competence of swine pig peripheral blood mononuclear leucocyte subsets are heritable and genetically correlated with performance traits associated with innate and adaptive immunity in pigs: heritability and associations with performance under different health status conditions an evaluation of immune competence in different swine breeds immunological traits have the potential to improve selection of pigs for resistance to clinical and subclinical disease mapping quantitative trait loci for immune capacity in the pig confirmation of qtl on porcine 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aged adults characterization of the response of human thymocytes and blood lymphocytes to the synergistic mitogenicity of 12-otetradecanoylphorbol-13-acetate (tpa)-ionomycin lps/tlr4 signal transduction pathway transcriptome analysis of porcine pbmcs after in vitro stimulation by lps or pma/ionomycin using an expression array targeting the pig immune response mapping quantitative trait loci for innate immune response in the pig immune modulation: the genetic approach mycoplasma hyorhinis infection of pigs selectively bred for high and low immune response ingestion of deoxynivalenol (don) contaminated feed alters the pig vaccinal immune responses induction of alpha interferon by transmissible gastroenteritis coronavirus: role of transmembrane glycoprotein e1 a sensitive immunoassay for porcine interferon-alpha fumonisin b1 alters cell cycle progression and interleukin-2 synthesis in swine peripheral blood mononuclear cells ingestion of low doses of deoxynivalenol does not affect hematological, biochemical, or immune responses of piglets a language and environment for statistical computing. vienna: r foundation for statistical computing on lines and planes of closest fit to systems of points in space the ade4 package. i. one-table methods model-based methods of classification: using the mclust software in chemometrics the significance of the difference between two means when the population variances are unequal recovery of interblock information when block sizes are unequal wombat: a tool for mixed model analyses in quantitative genetics by restricted maximum likelihood (reml) bioconductor: open software development for computational biology and bioinformatics sincere thanks are due to mathieu gautier (inra, umr gabi, jouy-en-josas, france) for his computational help and to thierry tribout (inra, umr gabi, jouy-en-josas, france) for providing the pig genealogy. we thank fabrice andreoletti and stephan bouet (inra, umr gabi, jouyen-josas, france) for their contribution to animal experimentation. we are also grateful to hélène hayes (inra, umr gabi, jouy-en-josas, france) for helping in preparing the manuscript. we warmly thank christopher moran (faculty of veterinary science, university of sydney) for helpful comments and suggestions for the final submission of the manuscript. key: cord-001620-yy5gq0ki authors: woo, hye-min; lee, jin-moo; yim, sanggyu; jeong, yong-joo title: isolation of single-stranded dna aptamers that distinguish influenza virus hemagglutinin subtype h1 from h5 date: 2015-04-22 journal: plos one doi: 10.1371/journal.pone.0125060 sha: doc_id: 1620 cord_uid: yy5gq0ki surface protein hemagglutinin (ha) mediates the binding of influenza virus to host cell receptors containing sialic acid, facilitating the entry of the virus into host cells. therefore, the ha protein is regarded as a suitable target for the development of influenza virus detection devices. in this study, we isolated single-stranded dna (ssdna) aptamers binding to the ha1 subunit of subtype h1 (h1-ha1), but not to the ha1 subunit of subtype h5 (h5-ha1), using a counter-systematic evolution of ligands by exponential enrichment (counter-selex) procedure. enzyme-linked immunosorbent assay and surface plasmon resonance studies showed that the selected aptamers bind tightly to h1-ha1 with dissociation constants in the nanomolar range. western blot analysis demonstrated that the aptamers were binding to h1-ha1 in a concentration-dependent manner, yet were not binding to h5-ha1. interestingly, the selected aptamers contained g-rich sequences in the central random nucleotides region. further biophysical analysis showed that the g-rich sequences formed a g-quadruplex structure, which is a distinctive structure compared to the starting ssdna library. using flow cytometry analysis, we found that the aptamers did not bind to the receptor-binding site of h1-ha1. these results indicate that the selected aptamers that distinguish h1-ha1 from h5-ha1 can be developed as unique probes for the detection of the h1 subtype of influenza virus. influenza viruses are responsible for serious respiratory diseases and are deemed to be one of the biggest threats to human health. belonging to the family orthomyxoviridae, influenza virus is an enveloped virus with single-stranded negative-sense rna consisting of eight segments [1] . its two major surface glycoproteins, hemagglutinin (ha) and neuraminidase (na) [2] , are highly expressed during viral infection and serve as targets for immune detection. thus far, 18 ha (h1-h18) and 11 na (n1-n11) have been identified [3] , and influenza is classified on the basis of the subtypes of ha and na proteins. subtype h5 is known as highly pathogenic in protein (gst-h5-ha1) was incubated with 100 μl of glutathione agarose beads in 100 μl of binding buffer (50 mm tris/hcl; ph 8.0, 150 mm nacl, 1.5 mm mgcl 2 , 2 mm dtt, and 1% [w/v] bsa) for 30 min at room temperature with occasional shaking. the synthetic ssdna library was denatured by heating at 95°c for 10 min and immediately annealed on ice for 10 min. second, 2 μg of the dna library was incubated with the opponent protein bound to glutathione agarose beads for 30 min at room temperature. bead/opponent protein-bound dna was precipitated and discarded. the pre-cleared supernatant was collected and incubated with 2 μg target protein (gst-h1-ha1) in 100 μl binding buffer for 30 min at room temperature. glutathione agarose beads (100 μl) were added, the reaction mixture was incubated for another 30 min, centrifuged to remove unbound dna molecules, and the pellets were washed five times with 500 μl binding buffer. the gst-h1-ha1-ssdna pool complexes were dissociated from the glutathione agarose beads with elution buffer (binding buffer plus 10 mm glutathione). ssdna pool bound to gst-h1-ha1 were recovered from the supernatant by phenol-chloroform extraction and ethanol precipitation. using this procedure, a total of 14 sequential selection rounds were performed, applying more stringent conditions from round 8 onwards, by increasing the opponent protein concentration (4 μg: rounds 8-9, 8 μg: rounds 10-11, 16 μg: rounds 12-13, and 32 μg: round 14) and reducing the target protein concentration (1 μg: rounds 8-9, 0.5 μg: rounds 10-11, 0.25 μg: rounds 12-13, and 0.125 μg: round 14). after the 14 th round, ssdna was amplified by pcr, cloned into a linearized pgem-t vector (promega, madison, wi), and dna sequences were determined as previously described [23] . sequence alignment of selected aptamers was performed by clustalw2 [25] , and secondary structures were predicted using mfold [26] . ha1 protein-ssdna aptamer binding analysis by elisa aptamers were 5 0 -biotinylated by asymmetric pcr using the forward primer 5 0 -biotin-gcaatgtacggtacttcc-3 0 followed by lambda exonuclease digestion, as previously described [27, 28] . the 5 0 -biotinylated ssdna aptamers (100 nm) were heated at 90°c for 10 min, immediately placed on ice, added to the wells of a streptavidin-coated plate (pierce biotechnology, rockford, il), and incubated for 1 h at room temperature while shaking at 100 rpm. the wells were washed four times with pbst (0.1% tween 20 in pbs; ph 7.4), blocked with 5% bsa in pbst at room temperature for 1 h, re-washed four times, and incubated with various concentrations of purified gst-h1-ha1 in pbs at room temperature for 1 h. after washing four times with pbst, incubation with gst antibody-conjugated horseradish peroxidase (hrp; 1:1,000 in pbst, santa cruz biotechnology, dallas, tx) at room temperature for 1 h, and four additional washes, bound gst-tagged ha1 protein was detected by adding 3,3 0 ,5,5 0 -tetramethylbenzidine (tmb) solution (merck, darmstadt, germany) and terminating with 0.5 n h 2 so 4 . the absorbance of each well was measured at 450 nm by using a triad microplate reader (dynex technologies, chantilly, va). gst-h5-ha1 and gst served as negative controls. for binding affinity measurements, the surface plasmon resonance (spr) technology-based proteon xpr36 (bio-rad, hercules, ca) was used with pbs (ph 7.4) at 25°c. biotinylated ssdna aptamers (0.25 μg/μl) in pbs were immobilized on a neutravidin (nlc) sensor chip (bio-rad) in vertical orientation at a flow rate of 30 μl/min. concentrations of 0.625, 1.25, 2.5, 5, and 10 μm h1-ha1 protein in pbs were run across the surface in horizontal orientation at a flow rate of 100 μl/min for 60 s with a dissociation time of 600 s. data were analyzed with the proteon manager software, and binding constants were determined using a simple 1:1 langmuir model. equilibrium dissociation constants (k d ) were calculated from association and dissociation rate constants (k d = k d /k a ). purified h1-ha1 protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) and electrophoretically transferred to a polyvinylidene fluoride membrane. the membrane was washed, blocked with 5% bsa in pbst at room temperature for 1 h, and incubated with 5 0 -biotinylated ssdna aptamer in pbst (500 ng/ml) for 1 h. after four washes, the membrane was incubated with streptavidin-hrp for 1 h, washed again, and bands were visualized using an enhanced chemiluminescence (ecl) system and imagequant las 4000 (ge healthcare bio-sciences, piscataway, nj). for specificity analysis, h5-ha1 and gst proteins were treated accordingly and served as controls. circular dichroism (cd) spectroscopy and prediction of g-quadruplex structures cd spectra were collected as previously described [28] , using a chirascan-plus cd spectrometer (applied photophysics, leatherhead, surrey, uk). aptamers (25 μm) were resuspended in 10 mm tris/hcl (ph 7.5) buffer containing 100 mm kcl. cd spectra data were obtained from 200~320 nm at a step size of 1 nm, a 0.2 s time-per-point, and a bandwidth of 1 nm. each spectrum was an average of three scans at room temperature and was buffer baseline corrected. the presence of g-quadruplex structures in aptamers was predicted by quadruplex-forming g-rich sequences (qgrs) mapper [29, 30] . cells of the human embryonic kidney epithelial cell line hek293t (atcc, usa) were cultured in dulbecco's modified minimal essential medium supplemented with 100 u penicillin and 10% fetal bovine serum at 37°c and 5% co 2 . to confirm the selectivity of ssdna aptamers, hek293t cells were harvested with trypsin-edta, washed three times with 1 ml of pbs, added to a pre-incubated protein-aptamer complex of 100 μg gst-h1-ha1 protein and 8 μg fitc-labeled aptamer in pbs, and incubated at 4°c for 30 min. the cells were washed, blocked with 1% bsa in pbs for 30 min, incubated with a phycoerythrin (pe)-conjugated anti-gst antibody (abcam, cambridge, uk) in pbs at 4°c for 30 min, washed, fixed in 4% paraformaldehyde solution, and suspended in 500 μl pbs with 10% fetal calf serum and 1% nan 3 . fluorescence was determined with a guava easycyte flow cytometer (millipore, billerica, ma) by counting 10,000 events. gst-h5-ha1 was used as a negative control. the ha1 genes were cloned in the pgex-4t-1 expression vector (s1a fig), transformed into rosetta 2(de3) cells, and gst-tagged proteins h1-ha1 and h5-ha1 were purified by glutathione-agarose affinity chromatography and sephadex g-100 gel-filtration chromatography, as described in supporting information. purified ha1 protein was identified as a band of the expected mass of~64 kda on 10% sds-page gels stained with coomassie brilliant blue and on western blots using anti-gst antibody (s1b fig). blast (basic local alignment search tool) was used to compare the amino acid sequence similarity of h1-ha1 and h5-ha1. since h1 and h5 belong to the same group (group 1) of hemagglutinins, their amino acid sequences were expected to be highly similar. the blast result showed that they have 55% identity and 73% similarity (s1c fig). to determine whether the purified ha1 proteins have biological activity, hemagglutination assay was carried out with chicken red blood cells (rbcs). s2 fig shows that both ha1 proteins were able to efficiently agglutinate the erythrocytes, and subsequent selex procedure was performed. in vitro selection of ssdna aptamers specific for h1-ha1 rather than h5-ha1, and determination of binding affinity to select specific ssdna aptamers that can distinguish h1-ha1 from h5-ha1, counter-selex was performed with an ssdna library of 88-mers containing a randomized sequence region of 45 nucleotides in the center, followed by lambda exonuclease digestion, as shown in fig 1a. enrichment of selected ssdna aptamers specific for h1-ha1 protein was assessed by elisa on the basis of the interaction between h1-ha1 protein and biotinylated ssdna aptamers. a total of 14 cycles of selection were performed, and ssdna pools isolated after rounds 8, 10, 12, and 14 were tested for binding affinity. as shown in fig 1b, the absorbance at 450 nm increased significantly from round 10 onwards. after 14 cycles of selection, we obtained 22 independent ssdna aptamers and measured their binding affinity by elisa and spr, as described in materials and methods. binding curves were fitted to a michaelis-menten equation where abs is the absorbance, a is the amplitude, [h1-ha1] is the concentration of h1-ha1, and k d is the dissociation constant), and the amplitude and k d value of each ssdna aptamer were obtained. out of 22 candidates, three aptamers, named api, apii, and apiii, were found to have high binding affinities to h1-ha1, but schematic representation of the counter-selex procedure. after removing ssdna species binding nonspecifically to glutathione agarose beads, the ssdna pool was incubated with h5-ha1 (negative control) and centrifuged to remove h5-ha1-binding ssdnas, after which the unbound ssdnas were incubated with h1-ha1 (target protein), and the h1-ha1-bound dnas were extracted using phenol-chloroform and amplified by pcr. after 14 rounds of selection, the enriched dna was pcr-amplified, cloned, and sequenced. (b) specific binding activity as measured by elisa after 8, 10, 12, and 14 rounds of selection. gst-tagged h1-ha1 (100 nm) incubated on selected dna-coated plates and analyzed using anti-gst antibody-hrp with tmb color detection. (fig 2a) . h5-ha1 and gst served as negative controls to which the selected aptamers did not bind ( fig 2b) . binding affinities of the three selected aptamers were also measured by spr technology (fig 3a) . the k d values were calculated from the resonance unit as 96.6 nm (api), 1.09 μm (apii), and 293 nm (apiii), whereas the selected aptamers did not bind to gst-h5-ha1 or gst used as negative controls (fig 3b) . western blotting was performed to determine whether the selected ssdna aptamers could be used as molecular probes for h1 protein recognition. various amounts of h1-ha1 protein (0, 1, 5, and 10 μg) were analyzed by western blot using ssdna aptamers (500 ng/ml). indicating that the selected aptamers were bound to the h1-ha1 protein in a dose-dependent manner. control experiments with gst-h5-ha1 and gst showed that the three aptamers did not bind to these proteins, corresponding to the results of elisa and spr, as described above. to rule out the non-specific binding of aptamers to cells, another western blot analysis was carried out using whole cell lysates. s3 fig shows that non-specific binding was not detectable. the secondary structure of the selected aptamers was predicted using zuker's mfold program (fig 5a) , showing that the 45-nucleotide random sequence region (shaded) forms a major loop (api) or a major loop with a small hairpin (apii and apiii). bioinformatic analysis using clus-talw2 (fig 5b) revealed that the selected aptamers comprise of characteristic clusters of nucleotides in the 45-nucleotide random sequence region (indicated by asterisks) and contain seven nucleotides (gggtggg) followed by (g(n)gggggtgg), (ggt), and (ggg(n)t(n)g). we also found that the g-rich sequences were located in the large major loop (api, apii, and apiii) as well as in the hairpin regions (apii and apiii) (solid line in fig 5a) . although the exact binding structure is still unclear, the g-rich sequence might be involved in the interaction with h1-ha1. interestingly, we also found that each aptamer was likely to have two plausible g-quadruplex structures in the 45-nucleotide random sequence region. therefore, we used qgrs mapper (representing underlined dots and asterisks in fig 6a) and circular dichroism to determine whether each aptamer has a g-quadruplex structure. in fig 6b, a positive peak at 280 nm was obtained with the starting ssdna library, indicating a normal dna population [31] . on the other hand, the selected aptamers were found to have parallel g-quadruplex structures, which were identified by a positive maximum peak at 266 nm and a negative minimum peak at 244 nm [32] . to confirm that the selected aptamers were bound to the sialic acid-binding region of ha1, we performed a flow cytometry analysis (fig 7) . we pre-incubated fitc-labeled aptamers with h1-ha1 or h5-ha1 and added the complexes to hek293t cells harboring sialic acid on the surface. as shown in fig 7, the shifts of fitc and pe labels with h1-ha1 and h5-ha1 indicate that aptamer-h1-ha1 complexes were bound to sialic acid on the hek293t cell surface, whereas h5-ha1 was bound to sialic acid with no aptamer attached. therefore, the selected aptamers were found to bind selectively to h1-ha1 and not to h5-ha1, while they did not interfere with the sialic acid-binding ability of ha1 regardless of the subtype, suggesting that the aptamers were not binding to the sialic acid-binding region of ha1. upon influenza virus infection, surface glycoprotein ha recognizes and binds to sialic acid of the host cell membrane, leading to membrane fusion between the virus envelope and the endosome [33] . the identification of the type of ha present on the viral surface could aid in the determination of the species that can be infected and the type of sialic acid that is necessary (siaα2-6gal or siaα2-3gal) for viral infection [34] . therefore, an efficient and accurate method to detect the several types of ha is important for the determination of the influenza subtype in infected species. when ha1 sequences of subtypes h1 and h5 were aligned by blast, the two proteins were found to have 55% identity and 73% similarity. therefore, the development of an ssdna aptamer that can distinguish between h1 and h5 was assumed to be useful for the subtle differentiation between two influenza subtypes. we constructed an ssdna pool of 88-mers containing a randomized sequence region of 45 nucleotides in the center, which was screened using counter-selex in order to isolate ssdna aptamers binding specifically to h1-ha1 protein, but not to h5-ha1. after 14 cycles of selection, we were able to select three ssdna aptamers and measure their binding affinities to h1-ha1 by elisa and spr. both methods produced similar results in a range of nanomolar k d values. in addition to determining k d values, elisa and spr experiments demonstrated that the fixed aptamers on the surface of a plate or chip were able to bind to the h1-ha1 protein. this indicates that appropriate protein-specific ssdna aptamers would make good probes for the development of biosensors. further analysis by western blotting demonstrated that the selected aptamers could be used as substitutes for commercial anti-h1-ha1 protein antibodies (s4 fig). based on the fact that aptamers are called "chemical antibodies" for their high affinity to target molecules, the isolation of good aptamers should be very attractive for the development of immunological reagents and diagnostic systems [35, 36] . when we aligned the sequences of the selected aptamers using clustalw2, we found that the aptamers have g-rich sequence clusters in the 45-random nucleotide region. based on the fact that a characteristic g-rich sequence of nucleotides, (gg(n) x gg) or (ggg(n) x ggg), forms a g-quadruplex structure [37] , we predicted those by qgrs mapper [29, 30] , because grich sequences of the selected aptamers could fold into a g-quadruplex structure [38] . in addition to prediction, the structure of aptamers was analyzed by cd, showing that the selected aptamers have two g-quadruplex structures. according to a previous study, a stable g-quadruplex structure of selected aptamers has a strong binding capacity to a target protein [39] . therefore, it is likely that a g-quadruplex structure plays an important role in the binding to h1-ha1, although the exact binding mode is still unclear [40] . in fact, there have been several attempts to discover nucleic acid aptamers against ha protein as a target. the arnon group discovered a dna aptamer that can prevent viral infection by blocking the receptor-binding region of ha (amino acid 91-261) regardless of the subtype (h1n1, h2n2, and h3n2) [41] . using counter-selex, the kumar group selected an rna aptamer that can distinguish h3 subtypes (h3n2) of different strains [42] , while they recently isolated an aptamer binding to h5n1 and h7n7 viruses and inhibiting ha-glycan interactions [43] . the kim group isolated rna aptamers that bind to h5-ha1 (h5n2) and suppress viral infection [44, 45] . in most previous studies, the isolated aptamers were binding to the receptorbinding region of ha proteins. to determine whether our selected aptamers were also binding to the receptor-binding region of ha, thereby inhibiting the interaction with the receptor of the host cell, flow cytometry analysis was performed. interestingly, our selected aptamers did not block the interaction between h1-ha1 protein and the receptor of the host cell, indicating that the aptamers bind to h1-ha1 in a region other than the receptor-binding region. therefore, our selected aptamers are not expected to inhibit ha-mediated membrane fusion. considering the vital role of the ha protein in influenza infection, it is important to determine the ha subtype present in infected species for the purpose of diagnosis and prophylaxis. therefore, methods for rapid and reliable detection of various subtypes of ha need to be established. in the present study, we isolated new ssdna aptamers that specifically bind to h1-ha1 protein and distinguish it from subtype h5-ha1. we anticipate that our selected aptamers can be used as sensitive probes for the development of new detection devices. in the future research, the selected aptamers will be modified to have functional groups for immobilization on the appropriate surface including gold nanoparticles or magnetic beads [46, 47] . combination of probe development with up-to-date chemical and physical methodology would expand the application of current aptamer-based system. orthomyxoviridae: the viruses and their replication influenza hemagglutinin and neuraminidase membrane glycoproteins new world bats harbor diverse influenza a viruses the epidemiology and control of avian influenza and newcastle disease emergence and pandemic potential of swine-origin h1n1 influenza virus the structure and function of the hemagglutinin membrane glycoprotein of influenza virus the mechanisms of lipid-protein rearrangements 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influenza viruses and inhibits haemagglutinin-mediated membrane fusion an aptamer that binds efficiently to the hemagglutinins of highly pathogenic avian influenza viruses (h5n1 and h7n7) and inhibits hemagglutinin-glycan interactions an rna aptamer that specifically binds to the glycosylated hemagglutinin of avian influenza virus and suppresses viral infection in cells selection of an antiviral rna aptamer against hemagglutinin of the subtype h5 avian influenza virus conceived and designed the experiments: yjj. performed the experiments: hmw jml. analyzed the data: hmw jml. contributed reagents/materials/analysis tools: sy. wrote the paper: yjj. key: cord-001571-drcfdv9z authors: alvarez, julio; sarradell, javier; morrison, robert; perez, andres title: impact of porcine epidemic diarrhea on performance of growing pigs date: 2015-03-13 journal: plos one doi: 10.1371/journal.pone.0120532 sha: doc_id: 1571 cord_uid: drcfdv9z the impact of porcine epidemic diarrhea virus (pedv) infection on the us pork industry has mainly been attributed to the mortality that it causes in suckling piglets, and, consequently, much effort has been invested in the quantification of its effect in sow farms. however, no information on the performance of surviving pigs that were exposed to the pedv as piglets is available. here, a retrospective cohort study to evaluate the impact of porcine epidemic diarrhea virus (pedv) infection on growing pigs’ performance, as indicated by mortality, average daily gain (adg), average daily feed intake (adfi), and feed conversion ratio (fcr) was performed using production records from weaned pigs in nursery and wean-to-finish sites from sow farms that became pedv-infected between may 2013 and june 2014. production records from the first batch of growing pigs weaned in infected flows after the pedv outbreak (“infected batches”) were compared with those from pigs weaned within the previous 14 to 120 days (“control batches”). performance records from infected and control batches, paired by flow, were compared using non-parametric paired tests. mortality, adg and fcr were significantly different in pedv-positive (infected) compared with pedv-negative (control) batches, with a mean increase of mortality and fcr of 11% and 0.5, respectively, and a decrease of adg of 0.16 lb/day. our results demonstrate a poorer performance of growing pigs weaned after a pedv outbreak compared with those weaned within the previous 14-120 days, suggesting that in addition to the mortality induced by pedv in suckling pigs, the disease also impairs the performance of surviving pig. these findings help to quantify the impact of pedv infection in the us and, ultimately, contribute to efforts to quantify the cost-effectiveness of disease prevention and control measures. since its first detection in may 2013 [1] porcine epidemic diarrhea virus (pedv) has caused a major impact on the us pork industry. pork production is expected to decline around 6-7% in 2014 due to a decrease in the number of hogs that may range from 3 to 11%. [2, 3] the devastating effect of pedv is mainly due to the acute watery diarrhea induced in infected pigs, which may lead to a high (50-80%) mortality, particularly in neonatal piglets. [4] in many sow farms, it may take at least 12 weeks to recover to the baseline piglet production level. [5] however, if piglets survive the first seven days after infection, their chance of survival increase. [6] the impact of pedv infection in subsequent production stages (i.e., post-weaning/ growing and finishing pigs) of pigs surviving the first contact with pedv has not been reported. performance indicators routinely used to measure pigs' productivity include average daily gain (adg), average daily feed intake (adfi), and feed conversion ratio (fcr). [7] in addition, increased mortality in growing pigs is also related with decreased profitability in swine operations. [8] here, those four indicators were used to quantify the impact of pedv infection in growing pigs. these results will help to quantify the impact that the disease has on the swine industry and to estimate the cost-effectiveness of alternative prevention and control measures. data from all batches of weaned pigs were collected from one large swine system between 1 st of march 2013 (before pedv had been reported in the usa) and 1 st of june 2014. the database included all batches of animals produced in all farms from the company, located in the midwest. all sows and boars were cross bred, commercial genetics. all pigs were vaccinated for mycoplasma hyopneumoniae, porcine circovirus (pcv) and porcine reproductive and respiratory syndrome (prrs). pcv was present in all farms while prrs virus (prrsv) was occasionally detected in a proportion of them (see below). collected data included information on the site in which pigs were located, site production type (nursery or wean to finish (wf)), pig source (sow farm from which pigs originated), start and close period on each site, number of pigs per batch, mortality (defined by percentage of total pigs started), average daily gain (adg), average daily feed intake (adfi), feed conversion (fcr), and status of the sow farm from which pigs were weaned for prrs and ped. because weaned pigs from different sow farms were sometimes commingled in the subsequent growing stages, the term "flow" is used to refer to the origin of a given pig batch (and may include one or more single sow farms). only flows in which ped was detected (based on diagnostic investigation including clinical signs and pcr detection of pedv) during the period of study were selected. the identity of the data provider cannot be disclosed due to legal restrictions, but all relevant information underlying the analyses presented here are contained in the text and s1 dataset. to evaluate the effect of pedv infection on mortality and performance, a retrospective cohort study was carried out in which the exposed group was the first ped-positive batch from a given flow (infected batch) and the unexposed group (control batch) was defined as the batch that had left the sow farm immediately before the exposed group. records were screened to identify ped-positive flows, and, for each ped-positive flow, the first ped positive batch (infected group) and the preceding batch (control group), that had to precede the infected group by more than 14 but less than 120 days. for flows with more than one sow farm, only batches with the same combination of sow farms were paired. candidate control batches leaving sow farms within the first two weeks before the ped-infected batch were excluded from the analysis in an effort to have high specificity for the "control batches". target parameters (mortality, adg, adfi, and fcr) of infected and control groups were compared using the wilcoxon test, and differences observed in nursery and wf pairs were compared using a mann-whitney test. eighteen flows fulfilled the inclusion criteria, i.e., were positive to ped at some stage during the study period and had a control batch leaving the sow farms within the previous 14-120 days to the first ped-positive batch (s1 dataset). of those 18 flows, 4 sent pigs to nurseries ("nursery flows") and 14 to wf sites ("wf flows"). only one of the four nursery batches originated from a single sow farm, whereas the other three originated from up to five different sow farms and were commingled in the nursery ("mixed flows"). in contrast, all 14 wf flows originated from one single sow farm (table 1) . during the four months before the pedv outbreak, nursery flows produced 23 batches of weaned pigs, with a mean time between batches of 19 days (median = 13 days, range = 3-34 days), whereas 82 batches were produced by the 14 wf flows (between-batch mean and median time of 14 and 15 days, range = 4-63). median average days on feed was 64 days (interquartile range = 58-69 days) for nursery batches and 66 days for wf batches (iqr = 55-72). pigs from only 3 of the 82 wf batches weaned before the ped outbreak were on feed for >150 days, with records from the remaining pigs coming from the first stage of a double stocking practice. those 3 batches were not considered eligible as control/infected batches due to the effect of the longer period on feed on the performance of the pigs. within the four previous months to the ped outbreak, prrsv was detected in four of the 18 flows (a, c, e, h) but batches selected as controls from all but one flow (flow c) were already negative. four case batches were positive for prrs (flows o, p and q, in addition to flow c). before the pedv outbreak overall mean monthly mortality and fcr ranged between 4.3-4.8% and 1.71-1.89 respectively (fig. 1) , whereas adg and adfi values were between 0.75-0.85 and 1.29-1.62 (data not shown). analysis of the mortality of the first ped-positive batches on each flow revealed an increase in the mortality up to 14.9% in nursery and 15.5% in wf (fig. 1) . adg was lower in wf batches compared with the previous four months, while this effect was not so apparent in nursery pigs, and adfi observed in the outbreak batches did not increase in wf batches and increased moderately in nursery pigs compared with the values observed in the previous months. as a consequence, a strong increase in the fcr recorded in the infected batches was observed (fig. 1) . mortality was significantly (p<0.001) higher in ped-positive batches compared with the control pigs, with an overall mean difference of 12.5% (95% confidence interval = 6.4-18.4) (fig. 2) . difference observed in wf pairs was not significantly different from that found in nursery pairs. the difference in mortality between control and infected batches increased for those cases in which control batches left the sow farms in the previous 14-60 days compared with 61-120 days (fig. 2) . adg and fcr were significantly worse in case batches, with a 0.16 lb mean decrease of the former (95% ci = 0.07-0.26) (fig. 3a) and a 0.55 increase in the latter (95% ci = 0.30-0.79) (fig. 3b) . adfi remained largely unaffected (p = 0.9). again, differences observed in wf batches were larger than those observed in nursery pigs, but differences were not significant (p>0.3). the devastating effect of pedv in suckling piglets caused a decrease in all production indicators in us sow farms since its first detection in may 2013. however, although mortality in piglets may be as high as 80% in the first days of the outbreak, sow farms may return to their baseline production within 13 weeks, although reaching 100% of the baseline production may take longer [5] . in addition, pedv infection may have an effect in surviving piglets that may impair their future performance and, therefore, increase the economic burden caused by pedv infection; still, to the authors' knowledge, this hypothesis has not been tested yet. in the present study we compared the performance of pigs produced in 18 different flows before and just after diagnosis of ped in the sow farms in a first attempt to detect the impact of pedv infection in growing pigs. comparison of the mean mortality rates observed in the batches produced in the four months before the pedv reveals a rather stable trend with point estimates usually below 6%, and with 95% confidence intervals always including expected values for pigs of this age. [9] even though there was a certain degree of variation in the mortalities observed in that period, overall mean mortality for both nursery and wf pigs was below 5%, and in more than 95% of the batches the mortality was below 10%. the impact of ped on the mortality of the first positive weaned batches was evident, since more than half of the 18 case batches having mortalities above 10% (table 1 ). use of mortality as an indicator of performance of growing pigs is impaired by the limited number of studies focused on defining baseline levels in grow-finish pigs. [10] indeed, the variation observed in the batches produced before the pedv outbreak in the flows under study likely reflects the impact of other variables (such as coinfection with other pathogens, or certain stressors or management practices) not captured in the analysis. still, the absence of a visible trend in the mortality in a four month period supports the suitability of the selection of the control pair among batches produced within those 120 days, as all were considered to belong to the same population. even with the limited sample size analyzed here, differences in mortality evidenced by the comparison of the paired values were enough to reach statistical significance, with a mean increase of at least 10% and 13 of 18 pairs displaying a difference above 5% (table 1 , fig. 1 ). three of the four pairs in which the control batch was weaned between 60-120 days before the infected batch displayed smaller differences than the rest of the data points (fig. 1) . two of these flows had weaned other pigs between the infected and control batches that had been commingled with pigs from other sow farms, with mortality rates in these "intermediate batches" above 10% in all batches. however they could not be included in this study due to the lack of a suitable control pair for them (animals from the same sow farms that were ped negative within the previous 14-120 days). therefore, our results would suggest that mortality in batches that were weaned several weeks after pedv infected the sow farm was more similar to pre-pedv mortality, and that the increase due to pedv in the first pigs weaned after the outbreak may be even higher than estimated here. use of paired batches with the same origin allowed for control of certain factors known to affect mortality in growing pigs that occur at an early stage of their lives (such as mean weaning age). such analytical procedure increased the homogeneity of the compared infected-and control-populations, in terms of breed, management and disease prevention procedures at the sow farm, thus accounting for their effect as possible confounders affecting mortality. [10, 11] data from wf and nursery batches were analyzed separately, but the fact that the period on feed for most of the wf pigs (only three ped-and one ped+ batch stayed >150 days) indicated that the study period in which performance was measured was very similar regardless of productive type of the site in which pigs were placed. still, data were presented separately for both sites due to other management related factors that may differentiate wf and nursery sites, such as size of barns, floor, feeders, and that could have an impact, as suggested by the differences observed in the pre-pedv batches). infection with other pathogens could also increase mortality, and prrsv was detected in four of the 18 flows before the ped outbreak, and in four case batches compared with one control batch. still, among those three pairs in which only the case batch was positive (flows o, p and q), difference in case-control mortality was less than 3% in two cases ( table 1 ), suggesting that prrs was not a significant factor in the overall trend observed across flows. moreover, the mean difference between the mortality recorded in case and control batches from the four flows affected with prrs within the four months before the ped outbreak was lower than that observed in flows in which prrsv was not detected throughout the whole study (8.7% and 14.2%, respectively). the site where pigs were placed was not taken into account in the analysis due to the extensive variability found in the study population (that did not allow for any adjustment there). the location in which the pigs stayed during the period in which mortality was measured would undoubtedly influence this parameter. however, provided that weaned pigs were shipped to the different sites depending on the production needs and capacities and not depending on the ped status of the sow farm at that time, the "site effect" likely affected infectedand control-batches similarly, and for that reason, the authors do not think that such effect has affected the results reported here. as with any other observational study, causality cannot be demonstrated and for that reason, it is unclear if the higher mortality in ped+ batches was due to pedv infection alone. however, we are not aware of any epidemiological factor that could increase mortality and that was significantly more prevalent in ped+ batches compared with ped-batches. for this reason, we believe that the most biologically sound explanation for the significantly poorer performance observed in ped+ batches was the infection with the virus. mortality can be directly recorded by swine barn managers and is related with profitability of swine operations. [10] this makes it an attractive target to evaluate the importance of pedv in growing pigs. however, adg. adfi and fcr are also basic indicators of performance usually recorded in growing pigs. [7] our data reveal that adg was significantly lower in ped-positive compared with ped-negative batches, whereas adfi remained unaltered (table 1) , thus yielding a mean increase in the fcr of 0.55. assuming that the feed costs associated to produce a 40 lb feeder pig is 11.47$/head (mean estimate for december 2013-june 2014, [12] ), our results would suggest a mean increase of the cost of each lb. of pig of around 16 cents due to the increased feed costs only. variability in these parameters within the four months before the outbreak was larger than in the case of mortality, as shown for fcr in fig. 1 , therefore suggesting that these results should be interpreted carefully given the limited sample size. still, comparison of paired values revealed a consistent trend towards a poorer performance of pigs from case batches ( fig. 3a and 3b ). this suggests that while clinical signs of diarrhea can be mild in growing pigs, the negative consequence on performance is important. in summary, our results demonstrate that mortality, adg and fcr recorded in growing pigs (mean ages 23-90 days) were significantly poorer from those observed in pigs weaned 14 to 120 days before the introduction of ped. this is the first reported description of the effect of pedv infection in performance of growing pigs that survive the infection at the sow farm. supporting information s1 dataset. pig batches produced by the 18 flows that broke with ped during the study period (march 2013-june 2014) in the 120 days before the first ped+ batch. (xls) emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences virus killing 5 million pigs spurs hog-price rally: commodities. bloomberg news this little piggy cried p-e-d-v all the way home porcine epidemic diarrhea, diagnosis and elimination production impact study update. swine health monitoring project 08/01/2014 emergence of porcine epidemic diarrhoea in north america management of growing-finishing pigs swine costs and production. agri-practice finishing weights rise; mortality drops. feedstuffs a retrospective study of mortality in grow-finish pigs in a multi-site production system an analysis of mortality in the grower/finisher phase of swine production in the united states estimated livestock returns-wean to feeder key: cord-002601-d8908t93 authors: arellano-llamas, rocío; alfaro-ruiz, luis; arriaga canon, cristian; imaz rosshandler, ivan; cruz-lagunas, alfredo; zúñiga, joaquín; rebollar vega, rosa; wong, christopher w.; maurer-stroh, sebastian; romero córdoba, sandra; liu, edison t.; hidalgo-miranda, alfredo; vázquez-pérez, joel a. title: molecular features of influenza a (h1n1)pdm09 prevalent in mexico during winter seasons 2012-2014 date: 2017-07-10 journal: plos one doi: 10.1371/journal.pone.0180419 sha: doc_id: 2601 cord_uid: d8908t93 since the emergence of the pandemic h1n1pdm09 virus in mexico and california, biannual increases in the number of cases have been detected in mexico. as observed in previous seasons, pandemic a/h1n1 09 virus was detected in severe cases during the 2011–2012 winter season and finally, during the 2013–2014 winter season it became the most prevalent influenza virus. molecular and phylogenetic analyses of the whole viral genome are necessary to determine the antigenic and pathogenic characteristics of influenza viruses that cause severe outcomes of the disease. in this paper, we analyzed the evolution, antigenic and genetic drift of mexican isolates from 2009, at the beginning of the pandemic, to 2014. we found a clear variation of the virus in mexico from the 2011–2014 season due to different markers and in accordance with previous reports. in this study, we identified 13 novel substitutions with important biological effects, including virulence, t cell epitope presented by mhc and host specificity shift and some others substitutions might have more than one biological function. the systematic monitoring of mutations on whole genome of influenza a ph1n1 (2009) virus circulating at iner in mexico city might provide valuable information to predict the emergence of new pathogenic influenza virus since the emergence of the pandemic h1n1pdm09 virus in mexico and california, biannual increases in the number of cases have been detected in mexico [1, 2] the first outbreak began from late march to july 2009, followed by a second wave from late august 2009 to march 2010 [1] . the 2010-2011 winter season was characterized by low cases of pandemic virus and a predominance of influenza a h3n2 and influenza b [3] . as viral genome sequencing 50 clinical samples were positive for ah1n1pdm09 and we obtained the whole viral genome sequence in 23 of these samples. 13 samples were sequenced using high-density oligonuclotides microarrays and 10 samples were sequenced using massive parallel sequencing with the illumina platform. 4 samples were sequenced using both technologies. additionally, there were 4 samples where we could not achieve whole genome sequences and we only obtained sequences from specific segments (inmegen-iner 1 the whole genome sequences from clinical samples were obtained using high-density oligonuclotides microarrays for re-sequencing, (gis h1n1 flu biosurveillance array nimblechip 132k) as previously described [10] retro transcription (rt) of the influenza ah1n1pdm09 viral genome was carried out from rna extracted from nasal swabs of clinical samples positive to this viral type. we used the invitrogen super script iii high fidelity enzymes kit (superscript iii first strand synthesis system for rt-pcr, cat no. 18080-051) and primers from the biosurveillance resequencing oligo kit (aitbiotech, aitbh1n1m-50), for rt-pcr. later we used 2 ul of cdna from the rt for the 8 segments amplification by pcr with platinum taq dna polymerase (platinum taq dna polymerase, thermo scientific, foster city, ca, usa) and cocktail of primers included in the resequencing oligo kit. pcr conditions were 94˚c, 2min; 40 cycles (94˚c, 30 s; 66˚c, 3min); 72˚c, 5min. pcr fragments from each sample were pooled and labeled with cy3 (nimbelgen onecolor dna labeling kit, ref 05 223 555 001) independently and 800 ng of the labeled reaction were used for array hybridization, following the manufacturer´s instructions (nimblegen hybridization kit, ref 05 583 683 001). microarrays were scanned using a genepix 4000b scanner and processed with the nimble-scan and evolstar softwares to translate the fluorescence intensity into nucleotides and obtain a sequence fasta file. in total, the array contains 8,236 control probes and 121,928 h1n1(2009) probes, which provides 2x coverage of the entire h1n1(2009) genome, and up to 8x coverage of the regions comprising the 36 mutation hotspots and 10 drug-binding sites [11] . another set of 10 clinical samples were sequenced using massive paralleled sequencing (mps) on an illumina miseq (illumina, ca, usa), as described below. four samples were sequenced in parallel by microarrays and (mps). massive parallel sequencing. the 8 viral segments were amplified simultaneously and directly from clinical samples, using mbtuni12 and mbtuni13 primers, as described elsewhere [12] . amplification products for 10 samples were gel-purified (qiaquick gel extraction kit; qiagen, valencia, ca). barcoded libraries for ngs were produced using nextera xt dna library preparation kit and paired-end sequencing (2x250 cycles) was performed using the miseq platform (illumina). the reads were mapped to a/california/07/2009(h1n1)/2009 (h1n1), data was obtained from the niaid influenza research database (ird) [13] (access number of this sequence is presented in s1 table) using smalt v.0.7.6. assembly was performed using velvet package (velvet v1.2.10) and visualized with tablet (v 1.15.09.01). a 100% coverage was achieved for each virus, with at least 90 depth for ha, ns, na, m and np segments (mean coverage: 10). the pa, pb1 and pb2 segments had 80% coverage at >10x. the sequences from this study were deposited at the niaid influenza research database (ird, http://www.fludb.org) [13] and are available in genbank (accesion numbers can be found in s1 table) mutation analysis the sequences were analyzed using a/california/07/2009(h1n1)/2009 amino acid sequence as reference and compared to global sequences at flusurver [14] from 2009 to 2016, this for each of the eight viral segments (access number in s1 table) , in order to identify novel substitutions and determine how many times reported substitutions have been previously reported. sequences were aligned using the clustal w method in molecular evolutionary genetics analysis (mega 6.0) software [15] . [16] and edited by mega 6.0. a maximum likelihood tree was constructed for each influenza segment using mega 6.0. the tajima-nei model was selected with 5-parameter gamma distributed rates and 1,000 bootstrap replicates. editing of trees was made using tree (http://tree.bio.ed. ac.uk/software/figtree/). clinical and demographic characteristics of the studied individuals are summarized in table 1 . the mean age of the patients was 45.4 ± 20.8 years and 76.4% of these individuals were male. fatal outcome (%) 5.8 data are means ± standard deviation (sd), or number and percentage. *2 patients had asthma and 1 copd. the mean bmi (kg/m 2 ) was 29.2, and 23.5% required mechanical ventilation for 15.2 (mean) days. patients had 22 days of hospitalization stay, 17.6% of them had co-morbidities such as asthma and chronic obstructive pulmonary disease (copd), and 5.8% of these patients died. patients who required mechanical ventilation displayed a kirby index (pao 2 /fio 2 ) (mean: 104.7). the main signs and symptoms at the start of the illness included fever, myalgia, cough, and headaches, while chest pain, dyspnea, and cyanosis occurred after the third day. critically ill patients received oseltamivir (150 mg/day) during the period while they were under mechanical ventilation while non-critically ill patients received 150mg/day of oseltamivir for 5 days. substitutions detected by whole genome sequencing of the ah1n1 09pdm influenza virus 13 of the substitutions we identified were novel, and their potential biological role has not been defined. substitutions that have been previously reported are involved in host specificity shift, viral oligomerization interfaces (voi), binding small ligands (bsl), ab's recognition, binding host proteins (bhp), binding nucleic acids (bna) and antigenic drift. voi is calculated automatically from known structures and captures sites both in viral oligomerization sites as well as crystal contacts. bhp includes mostly interaction with human immune response proteins. bsl is derived from small ligands seen in known crystal structures and, in case of influenza surface proteins, often signifies interaction with small glycans which can be in or near a glycosylation site. fig 1) . these mexican sequences clustered next to ha sequences of strains from new york (cy189313, cy189361, bootstrap value 66). these sequences share two substitutions in signal peptide of the immature ha at amino acid 7 and 15 (-11 v/i and -3 a/t). group 2 (kr271559 and kr1571583) sequences clustered separately and shared a novel substitution in ha h138q. group 3 (kr271599 and kr271567) clustered with strains isolated in new york, washington and helsinki. one isolate (kr271567) shared mutation t474m with sequences from helsinki and finland principally. we mapped the amino acid substitutions occurring at the nodes of h1n1/2009 ha phylogeny, revealing amino acid changes at k163q and a256t for 2013-2014 sequences as reported previously. additionally, a cluster of 2 mexican sequences was marked by substitution h138q. regarding na, we observed 3 substitutions that marked sequences from 2013-2014; i34v, i321v and k432e. to determine whether individual sites were under positive selection, we used the mixed effects model of evolution (meme) method. only i34v was statistically significant (p = 0.05). additionally we found substitutions that distinguished the three groups mentioned above with ha analyses. we observed substitution q308l in group 1, while t452i was found in group 2 and the substitutions n449k, n386k and s82p were present on sequence kr271569 in group 3. for ns1 we detected the substitution l36i in group 1, the n127s in group 2 and finally the substitution k131e in group 3. regarding pa we found the substitution i13v as a signature of the group 1 (statistically significant with p = 0.05), while in group 3 we detected two substitutions f35l and i459t. in pb1 we associated the substitution a374t with group 2 and m646i with group 3. in pb2 t676i was found in group 2 [17] . other substitutions red dots at nodes show branches with 50% bootstrap support leading to the 2014 sequences described in this work. trees for the rest of the viral genome segments can be found in s1-s7 figs (s1 fig na, s2 fig pb2, s3 fig pb2, s4 fig pa, s5 fig m, s6 fig np, s7 fig ns) . were found in m1, m2, ns2 and np but were similar to those reported in other countries ( table 2 ). influenza a viruses cause acute respiratory tract infections and represent a significant public health threat [18] . the outbreak strain of swine-origin influenza a/h1n1 virus infection in 2009 is still circulating during the winter season in many countries, and may cause severe pulmonary illness in susceptible individuals [4, 19] . therefore continuous analysis of the entire genome of these viruses will provide comprehensive information of its molecular evolution in order to maintain effective prevention measures for public health. it has been extensively demonstrated that pandemic a/h1n1 influenza virus contains a mixture of segments, including ha, np, pb1, pb2, pa and ns from a triple reassortant virus isolated in north america and segments na and m from the eurasian swine influenza virus [20, 21] . due to the differential mutation rates of each viral segment, molecular epidemiology surveillance is important to detect antigenic variations among circulating influenza strains, which can modify the pathogenicity and antiviral resistance patterns of these viral strains. in this study we sequenced the entire genome of pandemic a/h1n1 strains isolated from patients in a reference hospital in mexico city (iner) in different years and we compared these sequences with consensus sequences in order to detect mutations that might be associated with viral evolution or might influence the antigenicity of the virus. it is important to mention that the sequences were obtained directly from clinical samples, in order to avoid in vitro artificial selection. we found a clear variation of the virus in mexico from the 2011-2014 season due to different markers and in accordance with previous reports [22, 23] . mutations v344m and i354l of pb2 and n321k of pa, i397m, i435t of pb1, s498n of np, n44s, v241l, n369k of na v80i of m1, l90i of ns1 and s185t, s203t, e374k and s451n of ha appeared together during the evolution of influenza virus in mexico. however we found other unique substitutions in pb2 g644r and t676i, pb1 a374t, ha h138q, na q308l, and l36i in ns1 of some mexican strains that could indicate geographical divergence. these findings should be confirmed with more sequences obtained in other regions of the country. recently a very extensive analysis of sequences of influenza virus in a post-pandemic era showed that different geographical regions generated local epidemic peaks and might act as a potential seed of local virus. further studies would be required in order to determine which of them are fixed in the next generation of influenza virus in mexico. it is known that influenza viruses undergo positive selection during the process of cross-species transmission and during the initial stages of a human outbreak, followed by purifying (negative) selection, when viruses adapted to the new human host in late epidemic [24] [25] [26] . in this context we still observed positive selection of substitutions of na i34v and pa i13v in winter season 2013-2014. our data indicate that the polymerase complex appears to have different evolutionary dynamics; probably due to differences in the evolutionary rate among the viral segments. this might also be a reflection of differences in selective pressure once the virus is in the infected host; regarding the rest of the viral genome segments, all the mexican isolates from season 2013-2014 clustered with sequences from new york and helsinki. mexican sequences from 2013-2014 belonged to clade 6b characterized by d97n, k163q, s185t, k283e and a256t substitutions (fig 1) . however a different branch was observed with 2 sequences presenting specific substitutions in all segments, including the substitution h138q which lied in antigenic site in ha, these results suggest that strains could appear temporarily, with different molecular characteristics and potentially with antigenic or pathogenic implications. a recently significant change occurred in virus from 2013 to 2014, the substitution k163q were predominant from these years until now and has been associated with binding prevention of antibodies (abs) elicited in a larger number of middle-aged humans all over the world [27] . in this study we identified 13 novel substitutions with important biological effects, including virulence, t cell epitope presented by mhc and potential roles in host specificity shifts and it should be noted that although these mutations have not been particularly characterized, they are found to be close to or belong to regions of biological importance previously ( table 2) . substitution pb2-r591p is on a position where r has been implicated in a more efficient replication of pandemic h1n1 viruses in mammals [28] while the effect of proline is not known. pb1-s704t is part of the h-2k b epitope and might affect the efficiency of antigen processing during cytotoxic t lymphocyte (ctl) response [29, 30] . e16a is in the n-terminal ectodomain of m2 at the outside of the viral membrane near to the disulfide bridges connecting the m2 tetramer 18235503] but its specific effect has not been particularly characterized yet [31] . other novel substitutions in this report are involved in voi, such as pa at position t162p [32, 33] ; bsl in pb2 at v640m [34] and in pa at l109f [32, 33] ; voi/bna in np at g356r [35] and bsl/voi in ns1 at q63k [36] . regarding previously observed substitutions, some have been computationally predicted to be linked to host specificity, such as pb2 v613a [37] . other roles of residues include voi, i13v in pa [38] , s482n in np [35] ; g18s and a33t in m1; a122t in ns1 [39] [40] . bsl, f35l in pa [41] . it was observed before that some substitutions might have more than one biological function, such as ha h25r and e373g, which are implicated in ab's recognition, bsl, voi, and bhp [42, 43] . na d199n is also associated with antigenic drift [44, 45] ; bsl/voi in pb2 g644r [46] ; pa t151n and e623d [32, 33, 47] ; na, v177i [45]; ns1, l36i, q63k, n127s [40, 48] . bhp in na t452i involved in bsl [49] and, in ns1 s74t [40, 48] , related to bsl and voi. bna in np g356r [35] and ns1, g45 related with voi [50]. all of these sites have been described by crystalographic or computational analysis ( table 2 ). in ha we observe changes that could affect immunogenicity of the influenza virus; sequences from 2015 to 2016 had additional mutations (s162n) in antigenic site (sa) and together with the substitution i276t are defining a new clade 6b.1 [21, 22] . importantly s162n may confer an additional glycosylation site in ha that could affect even more the immunogenicity of influenza virus in the future. our results indicate that the pandemic influenza virus changes through the seasons and show how the use of whole genome sequences are able to provide a deeper understanding of virus evolution through time. supporting information s1 table. accession numbers of sequence used as reference and accession numbers of sequences from this study. recrudescent wave of pandemic a/h1n1 influenza in mexico, winter 2011-2012: age shift and severity. plos currents influenza molecular features tracking the evolution of the sars coronavirus resequencing arrays large-scale evolutionary surveillance of the 2009 h1n1 influenza a virus using resequencing arrays single-reaction genomic amplification accelerates sequencing and vaccine production for classical and swine origin human influenza a viruses single-reaction genomic amplification accelerates sequencing and vaccine production for classical and swine origin influenza research database: an integrated bioinformatics resource for influenza research and surveillance. influenza and other respiratory viruses mega6: molecular evolutionary genetics analysis version 6.0 muscle: a multiple sequence alignment method with reduced time and space complexity single mutation at the amino acid position 627 of pb2 that leads to increased virulence of an h5n1 avian influenza virus during adaptation in mice can be compensated by multiple mutations at other sites of pb2 epidemic viral pneumonia. current opinion infectious desease clinical aspects of pandemic origin of the 2009 mexico influenza virus: a comparative phylogenetic analysis of the principal external antigens and matrix protein accumulation of human-adapting mutations during circulation of a(h1n1)pdm09 influenza virus in humans in the united kingdom comparative analysis of whole-genome sequences of influenza a(h1n1)pdm09 viruses isolated from hospitalized and nonhospitalized patients identifies missense mutations that might be associated with patient hospital admissions in finland during molecular evolution of the sars coronavirus during the course of the sars epidemic in china cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human. proceedings of the national academy of sciences of the united states of america adaptive evolution of the spike gene of sars coronavirus: changes in positively selected sites in different epidemic groups characterization of an influenza a virus in mexican swine that is related to the a/ h1n1/2009 pandemic clade biological and structural characterization of a host-adapting amino acid in influenza virus diversity of epitope and cytokine profiles for primary and secondary influenza a virus-specific cd8+ t cell responses crystal structures of murine mhc class i h-2 d b and k b molecules in complex with ctl epitopes from influenza a virus: implications for tcr repertoire selection and immunodominance structure and mechanism of the m2 proton channel of influenza a virus crystal structure of an avian influenza polymerase pa(n) reveals an endonuclease active site structural analysis of specific metal chelating inhibitor binding to the endonuclease domain of influenza ph1n1 (2009) polymerase fine mapping of the subunit binding sites of influenza virus rna polymerase the mechanism by which influenza a virus nucleoprotein forms oligomers and binds rna conserved surface features form the double-stranded rna binding site of non-structural protein 1 (ns1) from influenza a and b viruses identification of human-to-human transmissibility factors in pb2 proteins of influenza a by large-scale mutual information analysis nucleoside monophosphate complex structures of the endonuclease domain from the influenza virus polymerase pa subunit reveal the substrate binding site inside the catalytic center structural basis for suppression of a host antiviral response by influenza a virus a transient homotypic interaction model for the influenza a virus ns1 protein effector domain synergistic adaptive mutations in the hemagglutinin and polymerase acidic protein lead to increased virulence of pandemic 2009 h1n1 influenza a virus in mice key: cord-001599-pfdnmzx2 authors: wee, yin shen; weis, janis j.; gahring, lorise c.; rogers, scott w.; weis, john h. title: age-related onset of obesity corresponds with metabolic dysregulation and altered microglia morphology in mice deficient for ifitm proteins date: 2015-04-09 journal: plos one doi: 10.1371/journal.pone.0123218 sha: doc_id: 1599 cord_uid: pfdnmzx2 the ifitmdel mouse lacks all five of the ifitm genes via loxp deletion. this animal breeds normally with no obvious defect in development. the ifitmdel animals exhibit a steady and significantly enhanced weight gain relative to wild-type controls beginning about three months of age and under normal feeding conditions. the increased weight corresponds with elevated fat mass, and in tolerance tests they are hyporesponsive to insulin but respond normally to glucose. both young (4 mo) and older (12 mo) ifitmdel mice have enhanced levels of serum leptin suggesting a defect in leptin/leptin receptor signaling. analysis of the gene expression profiles in the hypothalamus of ifitmdel animals, compared to wt, demonstrated an altered ratio of pomc and npy neuropeptide expression, which likely impairs the satiation response of the ifitmdel animal leading to an increased eating behavior. also elevated in hypothalamus of ifitmdel mice were pro-inflammatory cytokine expression and reduced il-10. anatomical analysis of the hypothalamus using immunohistochemistry revealed that microglia exhibit an abnormal morphology in ifitmdel animals and respond abnormally to poly:ic challenge. these abnormalities extend the phenotype of the ifitmdel mouse beyond abnormal responses to viral challenge to include a metabolic phenotype and weight gain. further, this novel phenotype for the ifitmdel mouse could be related to abnormal neuropeptide production, inflammatory status and microglia status in the hypothalamus. the interferon-induced transmembrane gene family (ifitm) consists of four genes in humans and five in the mouse that encode very similar proteins of 40-60 residues. each ifitm protein consists of a unique extracellular n-terminus, a highly conserved transmembrane domain, and equally well-conserved hydrophilic (cytoplasmic) domain followed by a much more diverse transmembrane-like domain [1] . although the ifitm proteins can be detected with sequences on the outside of the plasma membrane, an alternative prediction is that they are embedded on the cytoplasmic side of the membrane [2] . the proteins possess a number of functional sites including cysteine residues that are palmitoylated, lysine residues that are ubiquitinylated and serine/threonine residues that are phosphorylated following cellular activation ( [1, 2] ,unpublished data). ifitm cell location includes on the surface as well as inside the cell where they are associated with endosomal and golgi membranes [3] [4] [5] . as a class, the ifitm proteins have a proclivity to bind to multi-spanning/tetraspanin proteins such as cd81 and cd9 [6, 7] . of the ifitm gene family members, ifitm3 is the best characterized and it shows the greatest transcriptional response to type i and type ii interferon induction [8] . ifitm3 was shown in a broad sirna screen to be essential for the interferon-induced cellular resistance to viruses that infect from the endosomal compartment to the cytoplasm such as influenza and dengue [4, 5, 9, 10] . a defective human ifitm3 allele has been linked to increased severity of human infections to influenza virus [11] and we have recently shown this same allele is linked to coronary heart damage associated with kawasaki disease, an immune inflammation of unknown initiation [12] . a number of models have been proposed to describe the function of ifitm3 in providing resistance to cellular infections including building a protein lattice in the membrane to block endosomal exit, blocking fusion pores during virus-endosome hemifusion, enhancing the deposition of cholesterol to also block virus exit or by blocking virus entry by enhancing the stability of the clathrin/vatpase complexes on the endosomal membrane [13] [14] [15] . the mouse ifitm gene family encompasses about 65,000bp on mouse chromosome 7. this section of the chromosome has been removed by loxp mediated deletion to create the ifitmdel animal which lacks all five of the ifitm genes [16] . no other coding sequences or functional noncoding rna's are included within this section of the genome. the ifitmdel animal was originally created to test the necessity of the ifitm proteins for germinal cell speciation [17] [18] [19] and embryo generation [20] . ifitmdel animals are generated in normal mendelian numbers and have few if any obvious defects in development and survival [16] . we have made extensive use of these animals to study the roles that the ifitm proteins have in immune signaling pathways. as we maintain these animals as homozygous deletion lines, over time we have observed a pronounced enhanced weight gain and an obesity phenotype (e.g., [21] [22] [23] ) in older ifitmdel mice compared to c57bl/6 controls. in this report we quantify the obesity phenotype and link this to altered leptin/neuropeptide signaling, and demonstrate abnormal microglia morphology in the ifitmdel animal. the mice were housed and used for this study in accordance with protocols approved in advance by the institutional animal care and use committee at the university of utah (protocol number (09-07003). in all cases animals were maintained in according to the guide for the care and use of laboratory animals of the national institutes of health. ifitmdel mice were backcrossed for greater than 10 generations to the c57bl/6 strain. background-and age-matched littermates were used as wt controls. these mice were fed ad libitum with normal chow. for food intake studies, mice were kept individually and a similar amount of normal chow was given to each mouse. average three-day consumption of food was measured for 21 days. for insulin and glucose tolerance tests, mice fasted 5 hours followed by intraperitoneal injection of human recombinant insulin (1u/kg, novalin r) or glucose (1.5g/kg, sigma) respectively. blood levels were measured at indicated time points by freestyle lite blood glucose monitoring system. blood samples were obtained from tail bleeds. for blood tests of fasting and fed animals, including blood glucose and leptin, blood was obtained at 10 am and 10 pm (12h) for analysis. energy expenditure and locomotor activity were analyzed by indirect colorimetry using clams metabolic cages, hsc cores research facility at university of utah. body composition was determined by nuclear magnetic resonance (nmr bruker minispec). for rna isolation, the hypothalamus was dissected, total rna of the hypothalamus extracted by using qaizol reagent kit (qaigen), and reverse transcribed with maloney murine leukemia virus reverse transcriptase (m-mlv rt, invitrogen). for qrt-pcr, 1x faststart universal sybr green master (roche) was used to analyze gene expression on lightcycler 480 system (roche). the accession numbers of genes ifitm1 (nm_026820. , f4/80 (nm_010130) and actin (m12866.1). primer sequences were as follows: ifitm1, forward: 5'-cttcaaaagccgagagatg-3', reverse: 5'-ccaccatcttcctgtcccta-3'; ifitm2, forward: 5'-ccatcctccagacggggcgattg-3', reverse: 5'-tattcaggcacttggcagtg-3'; ifitm3, forward: 5'-ctttgctccgcac catgaacca-3', reverse: 5'-aggcacttagcagtggaggcgt-3'; ifitm6, forward: gagg gatcctgactcagc-3', reverse: 5'-agcatgggattgggccccagtc-3'; pomc, forward: ctgcttcagacctccatagatgtg-3', reverse: 5'-cagcgagaggtcgagtttgc-3'; npy, forward: 5'-tactccgctctgcgacacta-3', reverse: 5'-gatgagggtggaaac ttgga-3'; actin, forward: 5'-gtaacaatgccatgttcaat-3', reverse: 5'-ctccatcg tgggccgctctag-3'; ifnβ, forward: 5'-caagaaaggacgaacattcg-3', reverse: 5'-agacattctggagcatctct-3'; ifit1, forward: 5'-atgggagagaatgctgatggtg, reverse: 5'-tgtcaaggaactggacctgctc-3'; tnfα and il-1β [24] , il-10, [25] f4/80 [26] and inos [27] can be found in indicated citations. the epididymal fat pads were dissected from animal and fixed immediately in 4% paraformaldeyde in pbs overnight at 4°c. next day, tissues were dehydrated with serial alcohol solutions (50%, 70% and 100%) at room temperature for 2 hours each. tissues were then infiltrated in immuno-bed resin (polyscience, inc.) at room temperature overnight. tissues were moved to fresh immuno-bed resin with catalyst (polyscience, inc.) at 1:25 ratio and polymerized in the mold at room temperature overnight. tissues were carefully removed from the mold and sectioned to 3 microns with a rotary microtome (thermo scientific, microm hm310). tissue sections were adhered to microscope slides and proceed to h&e staining according to manufacturer's instructions (vwr). stained slides were mounted with cytoseal mounting medium (fisher scientific) before imaging. pictures were acquired with zeiss axiovert 100 microscope equipped with a microfire ccd camera (optimetrix). brain sections were prepared as before [28] [29] [30] . briefly, mice were lethally anesthetized with tribromoethanol (tbe) by intraperitoneal injection. for perfusion, the right ventricle of the heart was punctured and 10ml of saline was perfused into the left ventricle, followed by 20ml of 3% paraformaldehyde (pfa, ems) plus 5% sucrose in pbs. the brains were post-fixed with 3% paraformaldehyde plus 5% sucrose solution and subsequently infiltrated with 15% and then 30% sucrose. brain tissues were embedded in 2% gelatin (sigma-aldrich) and sectioned at 10μm with the thermo scientific hm 550 cryostat (thermofisher scientific). sections were permeabilized with 0.02% triton (sigma) and stained with iba1 (abcam), f4/80 (ebioscience), cd11b (ebioscience), gfap (abcam), o2a (abcam) and map-2(abcam) followed by secondary antibody fitc-or pe-conjugated anti-rabbit antibodies or alexa fluor 546 goat anti-rat igg (invitrogen) as appropriate. bmdm were isolated and cultured using standard protocols (14) . cells were fixed with 4% paraformaldehyde and permeabilized with 0.02% triton (sigma) and stained with iba1. the ifitmdel animals demonstrate enhanced adiposity the ifitmdel strain possesses a defined and engineered (via cre-lox) chromosomal deletion of all five of the ifitm genes on a mouse genetic background of c57bl/6 [16] . no coding sequences or regulatory sequences other than those associated with the ifitm genes are known to be lost in this deletion. mice lacking these ifitm genes are fertile and thrive in colonies except for the welldescribed sensitivity to viral infections. in maintaining such mice in our colony we noted that the older ifitmdel animals were generally larger than their wt (c57bl/6) age and sex matched counterparts. to quantify these differences, body weights were taken from male mice maintained in the colony on regular mouse chow. fig 1a, shows the slow but significant enhanced weight gain associated with the ifitmdel animals compared to wt. this body weight increase corresponded with an increased total fat mass as the animals aged ( fig 1b) . measurement of 3 day food intake averages for a period of 21 days shows that the ifitmdel mice consume more chow than the wt age-matched controls ( fig 1c) . also increased levels of epididymal fat deposits were present in these ( fig 1d) . histological examination suggested the adipocyte size was increased compared to wt adipose tissue ( fig 1e) and this was confirmed by quantitation as shown in fig 1f. the same trends were observed for female mice (not shown). we next determined if the enhanced body weight of the ifitmdel animals was associated with altered physiological stasis. blood glucose levels of fasting 2 month and 9-month-old male ifitmdel animals were measured and observed to be consistently higher than wt (fig 2a) . however, this difference was not observed under normal feeding (fed) schedules. this difference in fasting blood glucose was clear in the 2 month old animals. two standard tests to measure blood glucose levels are the insulin tolerance test (itt) ( fig 2b) where blood glucose levels are quantified in fasted mice following a single injection of insulin and the glucose tolerance test (gtt; fig 2c) in which blood glucose levels are quantified following a single injection glucose [31] . as shown, the ifitmdel animals responded poorly in the itt, maintaining higher levels of blood glucose than the wt animals. the ifitmdel and wt animals were indistinguishable in their response in the gtt. the same trends were observed with female ifitmdel animals (not shown). these results suggest that as the ifitmdel animals age, they enter into a metabolic syndrome with a more moderate phenotype than animals displaying morbid obesity [23] . the ifitmdel animals were also analyzed using metabolic chambers that quantify oxygen consumption (v02), heat production, and activity (movement). as shown in fig 2d the ifitm-del animals have reduced metabolism compared to the wt animals as reflected by lower oxygen consumption and decreased heat production in both the light and dark cycles. the activity of the ifitmdel animal in the cage trended towards less than the wt in the 12 month age group, but these differences were not statistically significant. mammals with a metabolic syndrome phenotype often have altered levels of leptin in the blood stream [32] . the leptin cytokine is critical in regulating appropriate food uptake [22, 33, 34] and we have shown (fig 1c) that the ifitmdel mice have increased food intake. this level of food intake, however, is significantly less than observed with the ob/ob or db/db mice. to assess leptin levels in the ifitmdel and control mice, two sets of male animals (4 and 12 months) were analyzed ( fig 3a) . in order to correct for the differences in body weight, serum leptin levels are shown as ng per μl per gram of lean body mass. as shown in fig 3a, the blood leptin levels were dramatically elevated in the ifitmdel animal compared to wt. when blood leptin levels in mice of the same weight (e.g., ifitmdel vs wt weighing 35 g), but differing age, were compared the ifitmdel animals still consistently have higher levels of blood leptin compared to wt controls (not shown). leptin is primarily produced by fat cells and serves, via the leptin receptor expressed in the hypothalamus, to regulate food intake [23, 34, 35] . animals lacking leptin (ob/ob) or a functional leptin receptor (db/db) become morbidly obese due to their lack of control over food intake [34] . leptin's actions are largely through interaction with leptin receptors expressed in the hypothalamus, which makes this brain region a target for the interaction between leptin and ifitm. to confirm that ifitm genes are expressed by cells of the hypothalamus, the expression of ifitm1, ifitm2, ifitm3 and ifitm6 was quantified in the wt hypothalamus samples (ifitm5 is only expressed by osteoblasts). as shown in fig 3b, ifitm2, ifitm3 and ifitm6 are all expressed in wt tissue, with ifitm2 and ifitm3 showing dramatically elevated expression in older animals. therefore, the correlation between the deletion of ifitm genes and leptin levels occurs in both young and older mice, the older ifitmdel mice have the added impact of not having the normal increase in these proteins seen during the aging process. the leptin receptor, expressed by neurons in the hypothalamus, signals through a stat3 dependent pathway, controlling the expression of two contrasting neuropeptides [34] . these are the proopiomelanocortin (pomc) gene whose expression is increased upon leptin signaling while the transcription of the neuropeptide y (npy) gene is depressed [36] [37] [38] [39] . bio-active peptides from the pomc protein serve to dampen eating while those of npy have the opposite effect and promote food intake. the expression of pomc and npy gene transcripts were analyzed from hypothalami obtained from wt and ifitmdel animals of various ages from 1 to 12 months of age. as shown in fig 3c, ifitmdel animals have significantly reduced pomc transcripts than wt suggesting that the ifitmdel animals lack appropriate signals to depress eating. further, the ifitmdel samples have trending higher levels of npy transcripts compared to wt samples suggesting elevated npy peptides may contribute to the positive signal to maintain feeding. ifitmdel mice demonstrated altered responses to chronic type i interferon induction chronic poly i:c treatment, via activation of tlr3 and the rig-i-like receptors, results in the production of il-6 and ifnγ that can, when provided chronically as a model for cachexia, lead to a progressive weight loss [40] . when administered in vivo, the major cell types responding to poly i:c include macrophages, dendritic cells (dc) and microglia. additionally responding dc's can undergo necroptosis that can exacerbate the inflammatory response. metabolic dysfunction and neurological disorders have also been linked to cachexia, especially as the outcome of chronic infections and cancer metastasis that can lead to the chronic release of inflammatory cytokines. based upon the previously described findings that the absence of the ifitm proteins can alter cellular induction pathways following type i interferon treatment, we tested whether or not the ifitmdel animals would have an altered response, compared to wt, to chronic poly i:c treatment. as shown in fig 4a, 8 week old wt animals treated with poly i: c over a time course of 28 days demonstrated the expected progressive weight loss, but age and sex matched ifitmdel animals were much more resistant to the cachexic effects of the poly i:c treatment. while the ifitmdel animals treated with poly i:c did lose weight, especially compared to their pbs-treated counterparts, the degree of weight loss was much less dramatic. to determine if the altered response to poly i:c in the ifitmdel animals was also mirrored in altered cytokine responses, animals were treated with poly i:c for 10 days (treatment every two days) weighed and analyzed. the hypothalamus was removed by dissection, total rna isolated and cytokine expression by quantitative rt-pcr was measured. as a positive control, the ifninduced protein with tetratricopeptide repeats (ifit1), which is genetically and functionally distinct from the ifitm proteins, was measured in response to type i interferon stimulated by poly i:c injection. as shown in fig 4b, wt and ifitmdel mice have equivalent levels of ifit1 rna following poly i:c treatment indicating these two strains were equally responsive. however, the ifitmdel and wt animals displayed significant differences in tnfα and il-1β mrna levels with the ifitmdel animal showing significantly elevated levels of these inflammatory cytokines (fig 4c and 4d) . pbs mock activated mice of the wt and ifitmdel did not induce elevated cytokine levels in the hypothalamus (not shown). the expression of the ifnγ and inos genes was also elevated in the ifitmdel animals (fig 4e and 4f) with differences trending toward significance. interestingly the expression of the anti-inflammatory cytokine il-10 showed lower levels of expression in the ifitmdel animal samples (fig 4g) . finally the expression of f4/80 (emr1), a macrophage/microglial marker, was compared between the wt and ifitmdel poly i: c treated animals (fig 4h) . the increased level of f4/80 rna in the hypothalamus of the poly i:c treated ifitmdel animals could be for a number of reasons including enhanced recruitment of blood monocytes/macrophages to that anatomic site compared to similarly activated wt animals. similar analyses were performed with rna obtained from abdominal fat pads (not shown) which showed the same trend of inflammatory cytokine signature as that obtained from the hypothalamus samples. in total these cytokine profiles suggest the response to interferon induced by the poly i:c treatment is intact but much more polarized towards a proinflammatory signature in the ifitmdel animal than wt. this is the case even though the weight loss in these animals upon poly i:c treatment is less severe. the rna signaling results (fig 4) led us to examine the morphology of the hypothalamus which to our knowledge has not been examined in depth in the ifitmdel mice. we first assessed the hypothalamus in unstimulated mice using markers for microglia/macrophages (iba1), astrocytes (gfap), oligodendrocytes (o2a) and neurons (map2). coronal serial sections from the brains of saline perfused and paraformaldehyde-fixed tissue of ifitmdel and control animals (methods) were prepared from equivalent anatomical locations, stained for cell specific markers and photographed for visual analysis. overall no gross abnormalities in brain anatomy were detected (data not shown), which is consistent with earlier reports [16] that the ifitmdel animals exhibited no developmental defects (although cns morphology was not specifically characterized). the regional distribution, appearance and staining of neurons, astrocytes or oligodendrocytes was also equivalent between animals of both genotypes (methods). however, microglia revealed by immunostaining for the expression of the iba1 antigen exhibited a markedly altered morphology between wt and ifitmdel mice (fig 5) . among the most notable morphology differences was the occurrence of dramatically elongated processes that were often observed to extend well over 150 microns (fig 5b, dotted line) . these processes were almost exclusively unipolar, and they exhibited infrequent branching and few varicosities. the cell bodies of these microglia also tended to appear as smaller and more poorly defined relative to controls in the hypothalamus (fig 5a-5d ). the altered microglia morphology of the ifitmdel mice was evident in other brain regions including the cortex and hippocampus (data not shown). microglia are mesoderm/mesenchymal derivatives that share a bone-marrow macrophage lineage [41] . to determine if the morphological differences observed in the ifitmdel microglia are intrinsic to this cell type, bone marrow macrophages (bmdm) from controls and ifitmdel mice were prepared from culture. after 10 days, cells were gently rinsed with pbs, cells fixed with paraformaldehyde and immunostained to reveal iba1 expression. as shown in fig 5e and 5f , the morphology of the double arrow identifies a cell with an elongated cell morphology that also common to cultures prepared from either genotype. both are typical to both preparations. the arrow head identifies a cell with morphology unique to the ifitmdel culture that is characterized by exceptionally long thin processes (dots) that resemble the microglia morphology seen in the brain. bar = 20 microns. doi:10.1371/journal.pone.0123218.g005 a subpopulation of ifitmdel bmdms exhibited strikingly similar features to those seen in the microglia. in particular, these bmdms from ifitmdel bm produced very thin extensions that extended for long distances in the cultures. these processes were rare or not detected with the control cells (fig 5e) . also evident is the impression that ifitmdel bmdms were more compact and in general looked smaller than cells of similar morphology in the wt preparation. this is also evident in cells producing fan-shaped lamella which are more compact in ifitmdel bmdms. thus the curious morphology of the ifitmdel microglia was recapitulated in cultured bone marrow derived macrophages possessing the same genetic defect suggesting that common cellular abnormalities may be associated with the ifitmdel in vulnerable cells. having found that microglia exhibit altered morphology in ifitmdel animal and the hypothalamic transcriptional response to poly i:c is also altered in this mouse (fig 4) , we examined by immunohistochemistry changes in the cns induced by poly i:c. wt and ifitmdel animals were activated with poly i:c as above, sacrificed and perfused. a notable difference in the response of the wt compared to the poly i:c treated mice was evident in choroid plexus structures within ventricles. macrophages mobilize to the cns upon inflammation and this is often evident by increased cellularity of the choroid plexus. in fig 6 we show that poly i:c treatment of wt and ifitmdel animals stimulates an increase in the accumulation of iba1/cd11b positive cells (fig 6c and 6d; i.e., macrophages) in the choroid plexus. further, these cells are also iba1/ f4-80 positive (fig 6g and 6h) , indicative of activation of macrophage lineage cells. a striking difference between wt and ifitmdel animals is the appearance of masses of these cells (fig 6d fig 6 and 6h) that are not evident in the pbs animals (fig 6a and 6b ), poly i:c treated wt animals (fig 6e and 6f ) or the pbs-treated ifitmdel animal (fig 6c and 6d) . similar data were also obtained from stained sections of the median eminence of the ventral hypothalamic region (fig 7) , again showing enhanced macrophage/microglia staining in the poly i:c treated ifitmdel animal compared to controls or sham activated ifitmdel animals. collectively these findings suggest that the enhanced numbers of transcripts encoding for f4/80 in the hypothalamus of poly i:c treated ifitmdel animals (see figs 6h and 7h) may be due to increased recruitment and/or accumulation of inflammatory macrophages to the brain. further, the activated macrophages in these regions at the site of blood-brain interfaces suggests that perhaps the macrophages do not enter the brain but may become entangled in these regions during normal migration. this report describes novel peripheral and central alterations associated with the lack of the ifitm proteins. the ifitmdel animals lack all five of the ifitm genes via an engineered genetic deletion without impact on other coding or control sequences that reside within the gene family locus. when bred as heterozygotes, the homozygous ifitmdel progeny are produced in the vivarium at normal mendelian ratios and exhibit normal losses of the adult animals compared to wt c57bl/6 animals. what is clear, however, is that the longer the ifitmdel animals are maintained on normal chow diet, the more obese they become. this obesity is due to an enhanced accumulation of white adipose mass. we have not examined changes in brown adipose tissue in these animals. while there are a variety of pathways that can lead to obesity, perhaps the best characterized are the aberrant feeding behaviors associated with alterations in the leptin/leptin receptor pathway [34] . animals deficient in either the ligand or receptor become morbidly obese due to the uncontrolled feeding behavior of the animals. leptin, produced by fat cells, binds to leptin receptor-bearing cells in the hypothalamus and engages the stat3 signaling pathway to influence the expression of genes encoding neuropeptides [42] . these include genes that impact upon feeding behavior such as an increase in pomc that suppresses feeding and npy that promotes this activity. hence, normally low leptin levels allow pomc expression to drop and npy to increase which leads to enhanced feeding. however, ifitmdel animals, even at an early age (1 month) with normal weight, despite increased leptin levels, exhibit depressed expression of pomc that corresponds to the increased feeding behavior of these animals. this reveals a novel disparity in leptin signaling through its receptor in the hypothalamus. our preliminary analyses of the stat3 signaling pathways in the ifitmdel animals revealed no difference with wt animals (data not shown). further, other signaling systems that require stat3 activation such as the il-6 receptor is similar in ifitmdel splenocytes to that of wt-responses (data not shown). however, the elevated expression of certain inflammatory cytokines such as tnfα, which is often associated with cachexia and weight loss, require further exploration in the ifitmdel mice. thus, a future line investigation will be to detail the mechanism(s) through which the ifitm proteins contribute to this unique metabolic phenotype. because we are unaware of any reports of brain structural anomalies for the ifitmdel animal, we performed a survey of brain using a histological approach to evaluate whether the altered pomc expression regulation could be due to structural deficiencies in the hypothalamus. overall, we observed no abnormal anatomical defects in any brain regions when compared to the c57bl/6 controls. additional analyses of these sections stained with cell type specific immunofluorescent markers also failed to reveal any overall gross inconsistencies in the overall distribution or numbers of neurons, oligodendrocytes and astrocytes. microglia, however, were different. the overall numbers appeared to be similar between the ifitmdel and control mice. it is worth noting that in occasional ifitmdel animals there appeared to be a substantial decrease in microglia in the cortex (not shown). what did consistently differ was a striking and common microglial cell morphology throughout the brain tissues of the ifitmdel animal. most notable was the reduced elaboration of microglia morphology accompanied by extremely long and usually mono-polar processes that extended from the cell body with few bifurcations. also, the cell bodies appeared smaller and less distinct than their control counterparts. how altered microglia cell morphology could specifically affect leptin signaling and the production of neuropeptides such as pomc or npy is not known. however, it is possible that ifitmdel microglia are unable to produce normal interactions with other cells types including neurons and astrocytes such as trophic interactions or clearance of debris thereby leading to altered and potentially toxic microenvironments. the function(s) of the ifitm proteins in development and maintenance of the central nervous system is a newly developing field. elevated expression of ifitm family members has been noted in the brains of schizophrenic patients, patients with autism, bipolar disorders and alzheimer's disease [43] [44] [45] [46] . besides being reported in neurodegenerative diseases, ifitm3 also has been shown to respond to poly i:c as an inducer of type i interferons, by increasing gene expression in astrocytes [47] . the ifitm3 protein is found in the endosomes of astrocytes and knockdown of ifitm3 expression inhibits clathrin dependent uptake in such cells [47] , similar to our description for cells obtained from the ifitmdel animal [14] . it appears that the expression of ifitm proteins is crucial to function in astrocytes. however, our observations failed to reveal any gross morphological abnormalities in astrocytes of ifitmdel animals (stained by gfap, data not shown). this will require further evaluation to assure astrocytes are not functionally compromised despite the overall appearance of normal morphology. the dysregulation of the signaling pathways in regulating energy homeostasis in the hypothalamus leading to metabolic disorders are well-known. this includes neuronal dysfunctions or inflammation in certain brain regions may also be linked to metabolic disorders [48] [49] [50] . activated microglia are capable of secreting pro-inflammatory cytokines that serve to recruit even more microglia to the site of inflammation that can further influence the metabolic response in the brain [51] [52] [53] [54] . in neuronal dysfunctions such as schizophrenia, autism, bipolar disorders and alzheimer's disease [43] [44] [45] [46] , the activation of microglia in addition to astrocytes can worsen the disease progression by secreting excessive inflammatory cytokines. one byproduct of these activation-dependent cytokines is the development of metabolic syndrome phenotypes characterized by weight gain and/or leptin resistance [52, 55, 56] . while there is a growing body of evidence suggesting the ifitm proteins may influence neuronal function through poorly defined mechanism, our study suggests they actually play a role in normal brain cellular architecture and interaction. a final point is that the ifitmdel animal is lacking all five of the ifitm genes. as shown in fig 4c, the ifitm1, 2, 3 and 6 genes are expressed in the hypothalamus and the expression of ifitm2 and ifitm3 normally increases with age. as the ifitmdel animal does not produce this agerelated alteration in expression, there is the intriguing possibility that these increases produce normal compensatory functions towards control of the age-related increases in obesity and altered leptin modulation of metabolic homeostasis. since an obesity phenotype has not been described for single ifitm gene deletions (ifitm3 deficient or ifitm1 deficient) [16, 53] nor have any brain anomalies such as the deficiency in microglia as shown in this report been described for any of these single ifitm gene deletion strains, this will require further investigation. for example, ifitm6 is primarily expressed in osteoclasts and macrophage lineages (of which microglia are related [6, 57] ). but whether or not the microglia deficiency seen in the ifitmdel animal is due to the lack of ifitm6 during microglial cell development remains to be evaluated. also other possibilities such as the indirect regulation of other modulators of adipose cell signaling by ifitm (e.g., regulation of leptin signaling through carbonic anhydrase activation [58] ) will need to be investigated. what does appear important is that ifitm genes are implicated in modulating important endocrine functions. in the framework of the microglia and hypothalamic interactions, our data also suggest that the phenotype could vary depending upon exposure to ifitm-specific pathogens. the broad-spectrum antiviral functions of ifit and ifitm proteins s-palmitoylation and ubiquitination differentially regulate interferon-induced transmembrane protein 3 (ifitm3)-mediated resistance to influenza virus the cd225 domain of ifitm3 is required for both ifitm protein association and inhibition of influenza a virus and dengue virus replication distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus ifitm3 inhibits influenza a virus infection by preventing cytosolic entry expression of the mouse fragilis gene products in immune cells and association with receptor signaling complexes functional dissection of the cd21/cd19/tapa-1/leu-13 complex of b lymphocytes ifn-gamma and ifn-alpha induce the expression and synthesis of leu 13 antigen by cultured human endothelial cells ifitm proteins restrict antibody-dependent enhancement of dengue virus infection identification of five interferon-induced cellular proteins that inhibit west nile virus and dengue virus infections ifitm3 restricts the morbidity and mortality associated with influenza kawasaki disease patients homozygous for the rs12252-c variant of interferon-induced transmembrane protein-3 are significantly more likely to develop coronary artery lesions the antiviral effector ifitm3 disrupts intracellular cholesterol homeostasis to block viral entry interferon-inducible transmembrane proteins of the innate immune response act as membrane organizers by influencing clathrin and v-atpase localization and function ifitm3 restricts influenza a virus entry by blocking the formation of fusion pores following virus-endosome hemifusion normal germ line establishment in mice carrying a deletion of the ifitm/fragilis gene family cluster developmentally regulated expression of mil-1 and mil-2, mouse interferoninduced transmembrane protein like genes, during formation and differentiation of primordial germ cells regulation of expression of mouse interferon-induced transmembrane protein like gene-3, ifitm3 (mil-1, fragilis), in germ cells ifitm/mil/fragilis family proteins ifitm1 and ifitm3 play distinct roles in mouse primordial germ cell homing and repulsion the fragilis interferon-inducible gene family of transmembrane proteins is associated with germ cell specification in mice carbonic anhydrase inhibitors as emerging drugs for the treatment of obesity leptin signaling and obesity: cardiovascular consequences positional cloning of the mouse obese gene and its human homologue gene expression profiling reveals unique pathways associated with differential severity of lyme arthritis a critical role for type i ifn in arthritis development following borrelia burgdorferi infection of mice adenoviral delivery of interleukin-10 fails to attenuate experimental lyme disease outer surface lipoproteins of borrelia burgdorferi stimulate nitric oxide production by the cytokine-inducible pathway mouse strain-specific nicotinic acetylcholine receptor expression by inhibitory interneurons and astrocytes in the dorsal hippocampus nicotinic acetylcholine receptor expression in the hippocampus of 27 mouse strains reveals novel inhibitory circuitry mouse chromosome 11 harbors genetic determinants of hippocampal strain-specific nicotinic receptor expression ptp1b antisense oligonucleotide lowers ptp1b protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice leptin levels in human and rodent: measurement of plasma leptin and ob rna in obese and weight-reduced subjects direct leptin action on pomc neurons regulates glucose homeostasis and hepatic insulin sensitivity in mice hormonal regulation of food intake identification and expression cloning of a leptin receptor leptin differentially regulates npy and pomc neurons projecting to the lateral hypothalamic area coexpression of agrp and npy in fasting-activated hypothalamic neurons specificity of leptin action on elevated blood glucose levels and hypothalamic neuropeptide y gene expression in ob/ob mice the role of neuropeptide y in the antiobesity action of the obese gene product homeostatic myd88-dependent signals cause lethal inflammation in the absence of a20 fate mapping analysis reveals that adult microglia derive from primitive macrophages socs3 deficiency in the brain elevates leptin sensitivity and confers resistance to diet-induced obesity molecular markers distinguishing supragranular and infragranular layers in the human prefrontal cortex immune transcriptome alterations in the temporal cortex of subjects with autism molecular characterization of bipolar disorder by comparing gene expression profiles of postmortem brains of major mental disorders microarray analysis in alzheimer's disease and normal aging astroglial ifitm3 mediates neuronal impairments following neonatal immune challenge in mice consumption of a fatrich diet activates a proinflammatory response and induces insulin resistance in the hypothalamus lipid-induced insulin resistance mediated by the proinflammatory receptor tlr4 requires saturated fatty acid-induced ceramide biosynthesis in mice hypothalamic proinflammatory lipid accumulation, inflammation, and insulin resistance in rats fed a high-fat diet differential regulation of trophic and proinflammatory microglial effectors is dependent on severity of neuronal injury molecular dissection of reactive astrogliosis and glial scar formation in vivo functional requirement of the mouse ifitm1 gene for germ cell development, interferon mediated immune response and somitogenesis the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus lower counts of astroglia and activated microglia in patients with alzheimer's disease with regular use of non-steroidal anti-inflammatory drugs inflammation, microglia, and alzheimer's disease overexpression of rankl implicates ifn-beta-mediated elimination of b-cell precursors in the osteopetrotic bone of microphthalmic mice the effect of carbonic anhydrase inhibition on leptin secretion by rat adipose tissue the authors would like to thank the university of utah core facilities (facs, metabolic and transgenic and knockout mouse), dr. donald mcclain for his assistance in measuring serum leptin and the weis labs for their critique of this work and many useful suggestions. key: cord-001734-bbeznd3r authors: gupta, garvita; lim, liangzhong; song, jianxing title: nmr and md studies reveal that the isolated dengue ns3 protease is an intrinsically disordered chymotrypsin fold which absolutely requests ns2b for correct folding and functional dynamics date: 2015-08-10 journal: plos one doi: 10.1371/journal.pone.0134823 sha: doc_id: 1734 cord_uid: bbeznd3r dengue genome encodes a two component protease complex (ns2b-ns3pro) essential for the viral maturation/infectivity, thus representing a key drug target. previously, due to its “complete insolubility”, the isolated ns3pro could not be experimentally studied and it remains elusive what structure it adopts without ns2b and why ns2b is indispensable. here as facilitated by our previous discovery, the isolated ns3pro has been surprisingly deciphered by nmr to be the first intrinsically-disordered chymotrypsin-like fold, which exists in a loosely-packed state with non-native long-range interactions as revealed by paramagnetic relaxation enhancement (pre). the disordered ns3pro appears to be needed for binding a human host factor to trigger the membrane remodeling. moreover, we have in vitro refolded the ns3pro in complex with either ns2b (48–100) or the full-length ns2b (1–130) anchored into the lmpc micelle, and the two complexes have similar activities but different dynamics. we also performed molecular dynamics (md) simulations and the results revealed that ns2b shows the highest structural fluctuations in the complex, thus providing the dynamic basis for the observation on its conformational exchange between open and closed states. remarkably, the ns2b cofactor plays a central role in maintaining the correlated motion network required for the catalysis as we previously decoded for the sars 3cl protease. indeed, a truncated ns2b (48–100;δ77–84) with the flexible loop deleted is able to trap the ns2b-ns3pro complex in a highly dynamic and catalytically-impotent state. taken together, our study implies potential strategies to perturb the ns2b-ns3pro interface for design of inhibitors for treating dengue infection. dengue is the most prevalent mosquito-borne viral disease with over 500 million human infections annually and 2.5 billion people at risk, particularly in tropical and subtropical regions [1, 2] . the disease is caused by dengue virus (denv) belonging to the flaviviridae family, which also includes several other human pathogens such as the west nile virus, japanese encephalitis virus, and yellow fever virus [1] [2] [3] [4] . so far, four antigenically distinct denv serotypes have been identified: denv-1 to -4, all of which cause dengue fever, dengue haemorrhagic fever and dengue shock syndrome. despite intense studies, currently there are neither vaccines nor other treatments available to treat this disease [5, 6] . the denv genome is composed of an 11-kb single-stranded positive sense rna. upon infection, the rna genome is translated into a large polyprotein by the host-cell translation machinery, that is subsequently processed into 10 proteins, including three structural proteins (capsid, membrane, and envelope) and seven nonstructural proteins (ns1, ns2a/b, ns3, ns4a/b, and ns5). the structural proteins form the viral particle while the nonstructural proteins participate in the replication of the rna genome, virion assembly, and attenuation of the host antiviral response. the cleavage of the polyprotein is carried out by host cell proteases including furin and signalaseas, as well as a virus-encoded serine protease ns2b-ns3pro, which has been established as a valuable target of therapeutic interest [5, 6] . the protease domain ns3pro consisting of the n-terminal part of ns3 adopts a chymotrypsin-like fold with two β-barrels, each composed of six β-strands, with the catalytic triad (his51-asp75-ser135) located at the cleft between the two β-barrels [7, 8] . amazingly, unlike other proteases with a chymotrypsin-like fold, the flavivirus proteases including dengue protease, requires a stretch of approximately 40 amino acids from the cytosolic domain of ns2b for catalytic activity, thus called two-component protease [6] [7] [8] . intriguingly, while the protease domains adopt highly similar structures in all crystal structures determined to date, the ns2b cofactors have been found to assume two distinctive structures, namely open (inactive) and closed (active) conformations by x-ray crystallography and nmr spectroscopy [7] [8] [9] [10] [11] [12] . to understand why the ns2b cofactor is indispensable for activating the dengue ns3 protease is not only of fundamental interest for enzymology, but also bears considerable implications for design of molecules with high affinity and specificity to inhibit the protease [13, 14] . however, due to its "complete insolubility", it has been previously impossible to carry out any experimental studies on the isolated ns3pro domain. in 2005, we discovered that previously-thought "insoluble proteins" in fact could be solubilized in water with minimized salt ions [15] [16] [17] [18] , and therefore we have used this to study various previously-thought insoluble proteins including tdp-43 n-terminus [19] . here with this approach, we have successfully characterized the solution conformations and dynamics of the isolated protease domain by cd, nmr and paramagnetic relaxation enhancement (pre). the results reveal that surprisingly the isolated ns3pro domain with the native sequence is intrinsically disordered without any stable secondary and tertiary structures, as well as has no detectable activity. nevertheless, upon meeting the ns2b cofactor, the disordered ns3pro spontaneously folds into the well-structured and active enzyme highly similar to those co-expressed [10, 11] . we have also successfully refolded the ns3pro complexed with the full-length ns2b anchored into the lmpc micelle. to further decipher the roles of the ns2b cofactor in protein dynamics, we performed molecular dynamics (md) simulations which is very powerful in pinpointing the role of protein dynamics in the catalysis of proteases such as the sars 3c-like protease [20, 21] . the correlation analysis reveals that the ns2b residues play a central role in coordinating the correlated motion network in the ns2b-ns3pro complex. indeed, a truncated ns2b is able to form a buffer-soluble complex with ns3pro, but this complex is highly dynamic and catalytically-impotent. our results imply that the discovery/ design of molecules to block the correct folding of the ns2b-ns3pro complex or/and to decouple its correlated motion network might represent promising strategies to inhibit the dengue protease, thus holding the considerable potential to treat dengue infections. plasmid construction dna sequences encoding the ns3 protease and ns2b from dengue virus type 2 strain tsv01 (genbank accession number ay037116) were optimized and synthesized by genscript (piscataway, nj). with designed primers, the optimized dna by genscript were used as templates for amplifying the dna fragments encoding residues 14-185 of ns3pro and residues 48-100 of ns2b, which have the same starting and ending residues as the constructs used in the previous nmr study [10] , as well as the full-length ns2b over residues 1-130 including the n-and c-terminal transmembrane domains. amplified dna fragments for ns3 (14-185), ns2b (1-130) and were subsequently cloned into pet28a vector with n-terminal his-tag (novagen) using ncoi and xhoi restriction sites, while fragments for ns2b (48-100) and ns2b (48-100; δ77-84) with thr77-met84 replaced by three gly residues were cloned into pgex-4t1 vector with gst-tag (ge healthcare) using bamhi and xhoi restriction sites. dna sequences of cloned constructs were verified by automated dna sequencing. competent escherichia coli bl21 (de3) star cells were transformed with pet28a-ns3pro or pgex-4t1-ns2b plasmids. transformed single colony was inoculated overnight in 10 ml luria-bertani broth containing 25 μg/ml kanamycin for pet28a and 100 μg/ml ampicillin for pgex-4t1. then cells were transferred to 1 lit lb media containing respective antibiotics and grown at 37°c with shaking until the a 600 reached to 0.6. cultures were induced with 0.5 mm isopropyl β-d-thiogalactopyranoside (iptg) for 16 h at 20°c. cells were harvested and resuspended in cold phosphate buffered saline (pbs) ph 7.4 buffer for lysis by sonication. after centrifugation at 40000g, gst-tagged ns2b (48-100) protein was purified from supernatant using affinity chromatography. the gst-ns2b (48-100) protein was cleaved with thrombin to remove the gst tag, and the released ns2b peptide was further purified by rp-hplc on a vydac c 8 column. the ns2b (1-130) and ns3pro proteins were completely insoluble and all found in inclusion body, which were solubilized with pbs buffer (ph7.4) containing 8 m urea. cell debris was removed by centrifugation at 40000g, and supernatant containing his-tagged ns2b (1-130) and ns3pro were purified by ni-nta affinity chromatography under denaturing condition. eluted protein was further purified with rp-hplc on a vydac c 8 column. ( 15 nh 4 ) 2 so 4 , [ 13 c6]-glucose and d 2 o were purchased from cambridge isotope laboratories (andover, ma). the generation of the isotope-labeled proteins for nmr studies followed a similar procedure except that the bacteria were grown in m9 medium with the addition of ( 15 nh 4 ) 2 so 4 for 15 n labeling and ( 15 nh 4 ) 2 so 4 /[ 13 c]-glucose for 15 n-/ 13 c-double labelling as previously described [22] . the purity of the recombinant proteins was checked by sds-page gels and their molecular weights were verified by esi-ms and voyager str matrix-assisted laser desorption ionization time-of-flight-mass spectrometer (applied biosystems). the concentration of protein samples was determined by the uv spectroscopic method in the presence of 8 m urea [23] . was measured in the assay buffer containing 50 mm tris-hcl (ph 7.5), 0.001% triton x-100, 0.5 mm egta, or 100 mm sodium acetate (ph 4.0), 0.001% triton x-100, 0.5 mm egta. briefly, in the 100 μl reaction mixtures containing 0.3 μm protease, 20 μm protease specific fluorophore-tagged substrate benzoyl-nle-lys-arg-arg-aminomethylcoumarin (bz-nkrr-amc) was added and reaction mixtures were incubated at 37°c, and the liberated coumarin fluorophore was continuously monitored at λ ex of 380 nm and λ em of 450 nm on infinite m200 pro tecan microplate reader. for steady state kinetics, 0.3 μm refolded ns3-ns2b protease was incubated with various concentrations of bz-nkrr-amc substrate in assay buffer containing 50 mm tris-hcl (ph 7.5), 0.001% triton x-100, 0.5 mm egta at 37°c. progression of enzymatic reaction was monitored as an increase in fluorescence at λ ex of 380 nm and λ em of 450 nm. initial fluorescence velocities (relative fluorescence units/sec) were calculated and curves were fitted to the michaelis-menten equation by nonlinear regression using graph-pad prism. steady-state kinetic constants were determined from triplicate measurements and reported as mean standard error. as the ns3pro contains no free cys residue, three single-cys mutants were prepared: q27c, e86c, s158c by use of the quikchange site-directed mutagenesis kit (stratagene, la jolla, ca, usa) as previously described [18] . the mutated plasmids were confirmed by dna sequencing and their recombinant proteins were subsequently expressed and purified by the same procedures described above. 1 h-15 n heteronuclear single quantum coherence spectroscopy (hsqc) experiments were performed on each mutant to validate that these mutations did not significantly perturb the conformation of the native ns3pro sequence. the recombinant proteins of three single-cysteine mutants were cys-modified following the previous procedure [18] , by the thiol-reactive nitroxide free radical probe, mtssl (1-oxyl-2,2,5,5-tetramethyl-δ3-pyrroline-3-methyl) methanethiosulfonate (toronto research chemicals inc.). briefly, the hplc-purified recombinant protein of the each mutant was dissolved in the buffer containing 8 m urea, 20 mm phosphate (ph 8.0), which was pre-degassed with nitrogen gas for 20 minutes. subsequently, the mtssl reagent was added from 3.8 mm stock solution in acetonitrile to reach a six-fold molar concentration of the protein, followed by incubation at room temperature with constant stirring for 5 hours. to ensure a complete labeling, another dose of mtssl was added to a six-fold molar concentration of the protein for an overnight incubation. the mtssl-labeled protein was purified by reverse-phase hplc on a c8 column and lyophilized. based on the verification by the time-of-flight-mass spectrometer, the purity of the mtssl-modified proteins of all mutants was > 99% after the hplc purification. all circular dichroism (cd) experiments were performed on a jasco j-810 spectropolarimeter equipped with a thermal controller using 1-mm path length cuvettes. data from five independent scans were added and averaged [18] . the ns2b, ns3pro and ns2b-ns3pro samples were prepared at a protein concentration of 20 μm in either milli-q water (ph 4.0) and 1 mm phosphate (ph 7.5) respectively. secondary structure contents of different samples were obtained by decovoluting cd spectra with continll program (http://lamar.colostate.edu/~sreeram/ cdpro/main.html). all nmr experiments were acquired on an 800 mhz bruker avance spectrometer equipped with pulse field gradient units as described previously [18] . for characterizing the conformation of the isolated ns3pro in water, a pair of triple-resonance experiments hncacb, cbca (co)nh as well as 15 n-edited hsqc-tocsy and hsqc-noesy were collected for the sequential assignment on a 15 n-/ 13 c-double labelled or 15 n-labelled sample at a protein concentration of 500 μm in 90% h 2 o/10% d 2 o (ph 4.0). for the refolded ns2b-ns3pro complexes, hsqc spectra were collected for 15 n-labeled samples in either acetate buffer (ph 4.0), or phosphate buffer (ph 7.5) in the absence and in the presence of inhibitor p-nitrophenyl-pguanidino benzoate [11] . for assessing the backbone dynamics on the ps-ns time scale, { 1 h}-15 n steady-state noes were obtained by recording spectra on the 15 n-labeled ns3pro domain at 500 μm in either milli-q water (ph 4.0), with and without 1 h presaturation with duration of 3 s plus a relaxation delay of 6 s at 800 mhz. all nmr data were processed with nmrpipe [24] and analysed with nmrview [25] . nh, n, cα and cβ chemical shifts of the isolated ns3pro in water at ph 4.0 were further analyzed by both delta2d [26] to derive the secondary structure population. for each spin-labeled single-cysteine mutant, a pair of 2d 1 h-15 n hsqc spectra were acquired at a protein concentration of 150 μm in milli-q water (ph 4.0): one for the spin-labeled sample in the paramagnetic form, and another after adding ascorbic acid (to 10 mm) to the sample to reduce the nitroxide, yielding the diamagnetic sample. we also acquired hsqc spectra for 3 corresponding cysteine mutants without spin-labelling at the same conditions and only several hsqc peaks slightly shifted after spin-labeling, indicating that the spin-labeling would not significantly change the conformation. the spectra were subsequently analyzed to obtain intensity ratios of hsqc peaks in the paramagnetic and diamagnetic forms using the programs nmrpipe [24] . protein structures were displayed by pymol molecular graphics system (w. l. delano, delano scientific llc, san carlos, ca). the crystal structure of the ns2b-ns3pro complex (pdb code: 2fom) with an open conformation [7] was selected for molecular dynamics simulations. as ns2b residues thr77-met84 are missing in the crystal structure, those residues were added and the obtained structure was post-processed as previously described [20, 21] . the simulation cell is a periodic cubic box with a minimum distance of 10 å between the protein and the box walls to ensure the protein would not directly interact with its own periodic images given the cutoff. the water molecules, described using the tip3p model, were filled in the periodic cubic box for the all atom simulation. 6 na + ions were randomly placed to neutralize the charge in md system. three independent 20-ns md simulations for either ns2b-ns3pro complex or ns3pro alone were performed with the program gromacs [27] with the amber-03 [28] all-atom force field. the long-range electrostatic interactions were treated using the fast particle-mesh ewald summation method [29] , with the real space cutoff of 9 å and a cutoff of 14 å was used for the calculation of van der waals interactions. the temperature during the simulations was kept constant at 300 k by berendsen's coupling. the pressure was held at 1 bar. the isothermal compressibility was 4.5 ã 10 −5 bar -1 . the time step was set as 2 fs. all bond lengths including hydrogen atoms were constrained by the lincs algorithm [29] . prior to md simulations, all the initial structures were relaxed by 500 steps of energy minimization using the steepest descent algorithm, followed by 100 ps equilibration with a harmonic restraint potential applied to all the heavy atoms of the protease. as the 20-ns simulations cannot reproduce the folding-unfolding event, so here we attempted to capture the low-frequency correlation motions by a recently established approach called mutinf [30] . mutinf represents an entropy-based approach to analyze ensembles of protein conformers, such as those from molecular dynamics simulations by using internal coordinates and focusing on dihedral angles. in particular, this approach is even applicable for detecting conformational changes which are subtle in the short md simulations because the coupling is mostly entropic in nature [30] . briefly, this approach utilizes second-order terms from the configurational entropy expansion, called the mutual information, to identify pairs of residues with correlated conformations, or correlated motions, in an equilibrium ensemble [30] . in the present study, the normalized matrix values were used, and 0.3 was set up to be the threshold value to determine the pairs of highly correlated residues. the residue pairs with correlated values ! 0.3 make up the top 1% of the entire matrix. dna fragments encoding ns2b (48-100) and ns3pro (14-185) were sub-cloned into the expression vectors pgex-4t1 and pet-28 respectively and subsequently expressed in e. coli bl21 (de3) star cells. the recombinant ns3pro protein was found to be completely insoluble and all in inclusion body as previously reported [7, 11] . as a consequence, the ns3pro proteins were first purified by ni 2+ -affinity column under denaturing condition in the presence of 8 m urea, followed by purification with reverse-phase (rp) hplc. on the other hand, the gst-fused ns2b (48-100) cofactor was found in supernatant and thereby purified under native condition, followed by the thrombin cleavage to release the cofactor which was also purified by rp-hplc. the lyophilized powder of the ns2b (48-100) was soluble both in mill-q water and in buffers, while the ns3pro protein was highly soluble at protein concentration of 1 mm without any detectable aggregation for at least half a year in mill-q water. the ns3pro protein also could be quickly diluted into 5 mm phosphate buffer with a final protein concentration of 100 μm and ph of 7.0 without visible aggregates for 1 hr, but formed aggregates in nmr tube after 4 hr, which has been commonly observed on a variety of "completely insoluble" proteins previously reported by us and other groups [15] [16] [17] [18] . as shown in fig 1a, ns3pro has very similar far-uv cd spectra in milli-q water (ph 4.0) and 5 mm phosphate buffer (ph 7.0), with the maximal negative signal at 200 nm and no positive signal below 200 nm, which is typical of a predominantly disordered protein without any stable secondary structure. this indicates that the isolated ns3pro domain is highly disordered in aqueous solution. indeed, the deconvolution of its cd spectrum reveals that the isolated ns3pro consists of 53% random coil, 14% turn, 29% extended strand and 4% helix secondary structures. moreover, it has a hsqc spectrum with very narrow spectral dispersions at both 1 h (~0.9 ppm) and 15 n (~19 ppm) dimensions (fig 1b) , further indicating the absence of tight tertiary packing. interestingly, on the other hand, ns2b (48-100) has a cd spectrum with the maximal negative signal at 206 nm and positive signal at 190 nm, as well as an additional negative signal at 222 nm. further deconvolution shows that it contains 32% random coil, 24% turn, 7% extended strand and 37% helix secondary structures. previously ns2b has been shown to have slightly different secondary structures in the open [7] and closed [8] states of the ns2b-ns3pro complexes. in the open (inactive) state (pdb id: 2fom), ns2b has a short helix over residues glu62-gly69 while in the closed (active) state (pdb id: 3u1i), the ns2b only has β-strands but no helical segment. therefore, it is possible that upon losing tertiary contacts with the ns3pro domain, the free-state ns2b has more helical conformations populated over residues glu62-gly69, or/and even has helical conformations populated over residues which adopt βstrands in the ns2b-ns3pro complexes. indeed, we previously observed that upon disrupting the all β-barrel native fold, the mutants of a sh3 domain became highly helical even over the residues which adopt β-strands in the native fold [31] . unfortunately, hsqc peaks of the ns2b (48-100) are much more broadened than those of ns3pro, implying that the isolated ns2b (48-100) has dynamic aggregation or/and conformational exchanges on the μs-ms time scale, thus preventing from further high-resolution nmr studies. despite its narrow spectral dispersions, we have successfully assigned nmr resonances of almost all non-proline residues of the 172-residue ns3pro (14-185) by analyzing nmr spectra including hn(co)cacb/cbca(co)hn and hsqc-tocsy/hsqc-noesy. fig 1c pres ents the obtained (δcα-δcβ) chemical shifts, which is a sensitive indicator of the residual secondary structures in disordered proteins [32] . previously, by analyzing nmr chemical shifts, the ns2b (48-100)-ns3pro (14-185) complex was characterized to adopt the same structure in solution and crystal [10] . here we downloaded the chemical shifts of ns3pro in complex with ns2b (bmrb id of 19080) and included them (grey bars in fig 1c) in parallel to those of the isolated ns3pro (red bars in fig 1c) for comparison. the ns3pro domain in complex with ns2b has very large (δcα-δcβ) deviations characteristic of a well-folded protein. by contrast, the isolated ns3pro has dramatically decreased (δcα-δcβ) over the whole sequence ( fig 1c) . for example, many ns3pro residues in the complex have the absolute values of (δcα-δcβ) > 4 ppm, while all isolated ns3pro residues have the absolute values of (δcα-δcβ) < 2 ppm. interestingly, many residues of the isolated ns3pro still have the (δcα-δcβ) patterns similar to those in the complex, implying that similar secondary structures might be weakly populated over these residues. indeed, we further analysed the nh, 15 n, cα and cβ chemical shifts by delta2d program [26] and the results indicate that the isolated ns3pro adopts highly-populated random coil conformations over the whole sequence. nevertheless, over some short segments, the extended strand conformation is also populated to some degree but the helix conformation is almost lacking (fig 1d) , completely consistent with the deconvolution result of the cd spectrum. furthermore, as seen in fig 1e, only sequential noes d nn(i,i+1) and d αn(i,i+1) manifest over the majority of the sequence, clearly indicating that the isolated ns3pro has no stable secondary structures and its nmr conformation represents an average of an ensemble of different structures. taken together, cd and nmr results define the 172-residue ns3pro domain to be an intrinsically disordered protein which is lacking of both stable secondary and tertiary structures in the absence of the ns2b cofactor [22, [31] [32] [33] [34] [35] [36] . to pinpoint the backbone flexibility, we measured the { 1 h}-15 n heteronuclear steady-state noe (hnoe) of the isolated ns3pro, which is a measure of the backbone motions on the psns time scale [18, 19, 31, 32, 37] . previously, all except for several c-terminal residues of the complexed ns3pro were shown to have positive hnoe values, with many even close to 1, clearly indicating that the backbone of the ns3pro in the complex is very rigid [10] . by contrast, residues of the isolated ns3pro have small or even negative hnoes (fig 2a) , with an average hnoe of only 0.08. more specifically, all residues have hnoe values < 0.4, while many residues even have negative hnoe, such as n-/c-termini, ser34-gly39, arg54-arg64, gln110-ile139 and arg157-gly159 (fig 2a) . this strongly suggests that the isolated ns3pro has largely unrestricted backbone motions on the ps-ns time scale, consistent with its absence of any stable secondary and tertiary structures. nevertheless, residues over ile65-leu100 have relatively large hnoe, implying that this region might have transit tertiary packing to a certain degree. previously many intrinsically disordered proteins such as α-synuclein have been shown to have transient long-range interactions by measurements of paramagnetic relaxation enhancement (pre), which is a powerful tool in detecting transiently existing contacts in highly disordered proteins with distances up to~25 å [18, 32, 38] . here, by site-directed mutagenesis, we introduced cys residue into ns3pro one by one at three locations: gln27, glu86 and ser158 (s1 fig). three single cys mutants were subsequently labelled with the nitroxide free radical probe, mtssl whose pre were measured by hsqc (s1 fig) as we previously described [18] . fig 2b shows the intensity ratios of hsqc peaks from the oxidized (paramagnetic) and reduced (diamagnetic) spectra of q27c mutant. interestingly, the residues which were significant affected (ratio < 0.85) are all located in the first β-barrel of the chymotrypsin fold adopted by the ns3pro domain in the native ns2b-ns3pro complex (fig 2c) . this suggests that these affected residues have long-range contacts with cys27 with distances < 25 å. interestingly, although residues thr48-glu66 assuming a short helix and β-sheet have very close contacts with cys27 in the native structure, they were not significantly perturbed by the spin-label at cys27. this strongly implies that the tertiary packing is non-native in the isolated ns3pro domain. the spin-label at the position 86 significantly affected the residues on both β-barrels (fig 2d and 2e) , suggesting that these residues have long-range contacts to cys86 with distances < 25 å. again, many residues have very close contacts with cys86 in the native fold but were not significantly perturbed by the spin-label at cys88. the residues affected by the spin-label at the position 158 are mostly located on second β-barrel, the loop connecting two βbarrels, and the last β-sheet over residues ser68-gly71 (fig 2f and 2g) . the pre measurements revealed that despite lacking of stable secondary and tight tertiary structures, the isolated ns3pro domain is not completely extended, but instead has a loose tertiary packing. however, this packing is highly dynamic and non-native, in which the residues over the middle region of the ns3pro sequence are slightly less dynamic than the n-and c-terminal ones, completely consistent with hnoe results (fig 2a) . the isolated ns3pro (14-185) and ns2b (48-100) are largely disordered in solution (fig 3a) and the ns3pro alone showed no detectable catalytic activity even with a protease concentration up to 5 μm in buffers at ph 7.5 ( fig 3b) . however, upon mixing them at an equal molar ratio in milli-q water at ph 4.0, the mixture has a cd spectrum for a protein with a substantial amount of secondary structures, which has a large positive signal at 190 nm and the maximal negative signal at 208 nm ( fig 3a) . the deconvolution shows that this refolded complex between ns2b (48-100) and ns3pro in water at ph 4.0 contains a large portion of β-strand and βturn structures: 34% random coil, 22% turn, 37% extended strand and 7% helix secondary structures, which is consistent with its three-dimensional structure (fig 2) . this sample was subsequently split into two: one at the same condition at ph 4.0, and another with buffer added to reach ph 7.5. as shown in fig 3a, the cd spectrum of the mixture in buffer at ph 7.5 has slight changes: the maximal positive signal shifted to 192 nm as well as became larger; and the maximal negative signal shifted from 208 to 217 nm, almost identical to that previously reported on the co-expressed ns2b-ns3pro complex [10] . this change appears to result from the slight increase of β-strand and reduction of random coil conformations. the slight variations in cd spectra of the refolded ns2b-ns3pro complex at ph 4.0 and 7.5 suggest that the complex may have some conformational differences at two ph values. indeed, the complex at ph 4.0 has a hsqc spectrum with many hsqc peaks too broad to be detected (fig 3c) , indicating that the complex undergoes significant dynamic aggregation, or/and conformational exchanges on the μs-ms time scale. by contrast, the refolded ns2b-ns3pro complex at ph 7.5 has a hsqc spectrum typical of a well-folded protein, which has very large spectral dispersions at both 1 h (~3.6 ppm) and 15 n (~28 ppm) dimensions (fig 3d) . in particular, although in the present study the ns2b peptide is unlabelled, a large portion of the hsqc peaks of the present ns3pro domain are almost superimposable to those of the previous complex with both ns2b and ns3pro 15 n-labelled and co-expressed [10, 11] , as exemplified by the characteristic hsqc peaks (green cycled), furthermore, the addition of the protease inhibitor p-nitrophenyl-p-guanidino benzoate previously used [11] triggered dramatic shifts of many hsqc peaks, indicating the refolded ns2b-ns3pro is active in binding the inhibitor (fig 3e) . we measured the enzymatic activity of the complex refolded at ph 4.0 in two buffers: one at ph 4.0 and another at ph 7.5. interestingly, it has no detectable activity in the acetate buffer at ph 4.0 even with the protein concentration reaching 5 μm. nevertheless, if the sample refolded at ph 4.0 was measured in the buffer at ph 7.5, it is highly active with no detectable difference from that refolded in the buffer at ph 7.5 (fig 3b) . the enzymatic parameters of the refolded ns2b-ns3pro complex in the buffer at ph 7.5 were obtained with km = 92.39 ± 9.94 μm and kcat = 0.15 ± 0.01 s -1 , which are very similar to the previous results with a co-expressed ns2b-ns3pro complex [10] . the structure and enzymatic activity revealed that once ns2b and ns3pro meet, they will bind to each other and initiate the folding process to form the two-component complex. however, it seems that if at ph 4.0, the ns2b-ns3pro complex exists as an intermediate which already has substantial secondary structures and tertiary packing. however, the tight tertiary packing is not completely achieved and consequently the complex undergoes μs-ms conformational exchanges, very similar to what we previously observed on a molten globule formed by a small protein at ph 4.0 [39, 40] . the intermediate of the ns2b-ns3pro complex at ph 4.0 is enzymatically inactive, but nevertheless, once it is transferred into the buffer at ph 7.5, the tightly-packed structure will immediately form and the complex can reach the fully active state. to study the structure and activity of the ns3pro in complex with the full-length ns2b (1-130), which is expected to be anchored into the membranes by forming the transmembrane helices at both n-and c-termini, the dna fragment encoding the full-length ns2b (1-130) was cloned into pet28a vector and the ns2b (1-130) protein was purified under denaturing condition. the full-length ns2b (1-130) was reconstituted in the lmpc micelle at a molar ratio of 1:200 (ns2b:lmpc) in buffer (ph 7.5). furthermore, ns3pro and ns2b (1-130) were also refolded together at the equimolar concentrations in presence of lmpc in buffer (ph 7.5). interestingly, the far-uv cd spectrum of the full-length ns2b (1-130) reconstituted in the lmpc micelle have the maximal negative signal at 209 nm and positive signal at 192 nm, as well as an additional negative signal at 222 nm ( fig 4a) . the deconvolution reveals that the ns2b (1-130) reconstituted in the lmpc micelle contains 28% random coil, 23% turn, 17% extended strand and 32% helix secondary structures. the large portion of the helix conformation is anticipated to result from the formation of the transmembrane helices at both of n-and c-terminal termini. interestingly, the ns2b (1-130) in the lmpc micelle has~10% higher β-strand conformation than the ns2b (48-100) in buffer, implying that they only have a slight difference in secondary structures if considering the error in cd deconvolution. on the other hand, only a small set of broad peaks could be detected in its hsqc spectrum (fig 4b) , indicating that the ns3b (1-130) in the lmpc micelle undergoes significant conformational exchanges on μs-ms time scale, or/and dynamic aggregation, which thus prevents from further high-resolution nmr studies. the observed line-broadening cannot mainly result from the increased molecular weight in the lmpc micelle as much more narrow peaks for almost all residues could be detected for the helical superoxide dismutase 1 (sod1) with 153 residues in the dpc micelle [41] . we also refolded the ns3pro with the full-length ns2b (1-130) in the lmpc micelle. as shown in fig 4a, the far-uv cd spectrum of the complex between ns2b (1-130) and ns3pro in the lmpc micelle has the maximal negative signal at 206 nm and positive signal at 190 nm, as well as an additional negative signal at 222 nm (fig 4a) . the deconvolution shows that its secondary structure contents are very similar to those of the complex between ns2b (48-100) and ns3pro in buffer, only with~3% increase of β-strand conformation and~1.6% reduction of the helix conformation. however, very different from the complex between ns2b (48-100) and ns3pro in buffer (fig 3d) , for the hsqc spectrum of the complex between ns2b (1-130) and ns3pro in the lmpc micelle (fig 4c) , only a small portion of the hsqc resonance peaks could be observed, which are also much more broad. this is partly due to the increased molecular weight of the complex upon being anchored into the lmpc micelle. a closer examination of the spectra revealed that a small set of the ns3pro hsqc peaks of the ns2b (1-130)-ns3pro complex in the lmpc micelle is almost superimposable to those of the ns2b (40-100)-ns3pro complex. these residues were identified to be the c-terminal residues glu173-lys185 of ns3pro, which were not visible in crystal structures of the ns2b-ns3pro complexes, thus suggesting that they are similarly flexible in both complexes ns2b (40-100)-ns3pro in buffer and ns2b (1-130)-ns3pro in the lmpc micelle). on the other hand, some hsqc peaks of the ns3pro domain complexed with the ns2b (1-130) in the lmpc micelle are not superimposable to those in the ns2b (40-100)-ns3pro complex. this implies that these ns3pro residues may have different structures, or/and dynamics, or/and chemical environments in the ns2b (40-100)-ns3pro and ns2b (1-130)-ns3pro in the lmpc micelle. furthermore the fact that most well-dispersed hsqc peaks disappeared (fig 4c) implies that the ns3pro may undergoes significant conformational exchanges on μs-ms time scale upon being anchored in the lmpc micelle. nevertheless, the ns2b (1-130)-ns3pro in the lmpc micelle is similarly active, with km = 101.1 ± 3.163 μm and kcat = 0.2469 ± 0.003 s -1 , which are very similar to the activity of the ns3pro in complex with the ns2b (48-100) with transmembrane regions deleted. molecular dynamics simulation is a powerful tool to gain insights into protein dynamics and folding/unfolding that underlies protein functions. although it remains extremely challenging to simulate the folding/unfolding for large proteins such as ns3pro, which usually occurs on the ms-s time scale, short md simulations in conjunction with the correlation analysis are able to capture the low-frequency correlated motions, which have been recently found to be essential for the catalysis of the sars 3c-like protease [20, 21] . therefore, to understand the role of the cofactor in the dynamics of the dengue ns3 protease, we conducted 20-ns md simulations for both ns2b-ns3pro complex and isolated ns3pro respectively. here we used the crystal structure of the ns2b-ns3pro complex (pdb code: 2fom) with an open conformation [7] for md simulations, because this complex has sequences almost identical to what we studied here, with only one and two conserved residue variations respectively as compared to our ns3pro and ns2b constructs. furthermore, in this structure, the ns2b cofactor assumes an open conformation, thus has a minimal contact surface with the ns3pro domain as compared to other structures with the closed conformation. this would facilitate the identification of the most important residues of the ns2b in mediating the dynamics of the protease complex. fig 5a and 5b present the structure snapshots in the first md simulations for the ns3pro alone and the ns2b-ns3pro complex, showing that within 20 ns, both isolated ns3pro domain and that in the complex remains dynamically stable, in particular over the regions with regular secondary structures. by contrast, the ns2b cofactor has large structural fluctuations, particularly over the residues thr77-met84 which are invisible in all previous crystal structures with an open conformation. fig 5c-5e show the root-mean-square deviations (rmsd) of cα atoms (from their positions in the energy minimized structures) for three independent simulations of the isolated ns3pro, the ns2b and ns3pro in the context of the complex respectively, and fig fig 5. overall dynamic behaviors in the md simulations. structure snapshots (one structure for 2-ns interval) of the first md simulations respectively for the isolated ns3pro (a) and ns2b-ns3pro complex (b). root-mean-square deviations (rmsd) of the cα atoms (from their positions in the energy minimized structures) for three independent md simulations of the isolated ns3pro (c), the ns3pro (d) and ns2b (e) in the context of the ns2b-ns3pro complex. (f) rmsd trajectories averaged over three independent md simulations of the isolated ns3pro (blue), the ns3pro (red) and ns2b (cyan) in the context of the ns2b-ns3pro complex. 5f shows their averaged rmsd trajectories. the averaged rmsd values are 1.85 ± 0.23, 1.56 ± 0.22 and 4.40 ± 0.71 å respectively for the isolated ns3pro, the ns3pro and ns2b in the context of the complex. this clearly indicates that upon removing ns2b, the isolated ns3pro domain will have higher conformational dynamics even within the 20-ns simulations. as such, it is expected that the isolated ns3pro domain may become unfolded if the simulations could reach ms-s time scale. on the other hand, even in the context of the complex, the ns2b cofactor has much higher structural fluctuations than ns3pro, implying that the bound ns2b cofactor is still capable to sample a large ensemble of conformations. the high flexibility of ns2b uncovered by md simulations here is completely consistent with the invisibility of many cofactor residues in crystal structures and also provides a dynamic basis for the conformational exchange of ns2b between open and closed conformations as previously observed [8] [9] [10] [11] [12] . noticeably, similar dynamic behaviours are also reflected by the root-mean-square fluctuations (rmsf) of the cα atoms in the md simulations (fig 6a and 6b) . fig 6c presents the averaged rmsf of three trajectories for the isolated ns3pro and ns2b-ns3pro complex. interestingly, only three regions of the isolated ns3pro have slightly higher fluctuations than the corresponding ones in the complex. the highest structural fluctuations are observed on the ns2b residues. more specifically, the ns2b residues with rmsf > the average are over n-/cterminal residues g43-ala49 and leu95-gly96, as well as ser75-glu89 which include the missing residues thr77-met84 in all crystal structures of the open form [7] . strikingly, the ns3pro residues with rmsf > the average are not only over loop/turn residues and c-terminal residues which include g29-leu31, glu43-thr45, his60-gly62, leu85-val95, thr118-gly121, gly144-val146 and arg157-gly159; but also over the short helix val72-lys74, and β-strands gly32-gln35, lys63-ile65, asp75-ile77 hosting the catalytic triad residue asp75, thr122-val126 and gly159-val162 (fig 6d and 6e) . recently, we have deciphered that a global correlation motion network exists in the sars 3c-like protease [20, 21] . remarkably, a mutation n214a on the extra domain which is far away from the active site is sufficient to decouple the correlated motions and consequently leads to the inactivation of the enzymatic catalysis. here, the correlation analysis of the md simulation trajectories revealed that like the sars 3c-like protease, a global correlation network does exist in the ns2b-ns3pro complex. most strikingly, in this network, the majority of the significant correlated motions are established between the cofactor and ns3pro residues ( fig 7a) . surprisingly, although the cofactor residues ser44-glu63, ser75-thr94 are highly dynamic in the md simulations, and thr77-met84 are even missing in all crystal structures of the open form, they were revealed to play a key role in coordinating the correlated motions with the ns3pro residues asp20-gln28, gly39-gly44, met59-glu66, ly84-val95, pro106-gln110, and lys145-gly148, which cover not only the residues having direct contacts with the cofactor, but also residues located far away from the cofactor (fig 7b and 7c ). furthermore, the global correlation network is largely eliminated as uncovered by the correlation analysis of the md trajectories of the isolated ns3pro with the ns2b cofactor removed (fig 7d) , which is similar to what was observed on the inactivated n214a mutant of the sars 3c-like protease [19, 20] . therefore, as implied by our previous results with the sars 3c-like protease, slight manipulations of the ns2b-ns3pro interface may be sufficient to decouple the correlation network to inactivate the activity of the dengue protease. the conformation and activity of the refolded ns3pro in complex with ns2b (48-100;δ77-84) although residues thr77-met84 are missing in all crystal structures of the open form, they were revealed by md simulations to play a key role in coordinating the correlated motions with the ns3pro residues. therefore, we generated a truncated ns2b (48-100;δ77-84) in which the residues thr77-met84 were replaced by three gly residues and subsequently conducted the refolding of ns2b (48-100;δ77-84) with ns3pro using the same protocol for refolding of ns2b . interestingly, although the isolated ns3pro is completely insoluble in buffer, the ns3pro became highly soluble in buffer at ph 7.5 in the presence of ns2b (48-100;δ77-84). this suggests that ns3pro forms a complex with ns2b (48-100;δ77-84) and the complex is soluble in buffer. indeed, as shown in, the decovolution analysis of the far-uv cd spectrum (fig 8a) reveals that the ns2b (48-100;δ77-84)-ns3pro complex contains 19% helix, much higher than those of the isolated ns3pro (4%), and ns2b (48-100)-ns3pro complex (8%). on the other hand, the ns2b (48-100;δ77-84)-ns3pro complex contains much lower strand (8%) but higher (36%) turn conformations than the isolated ns3pro and ns2b (48-100)-ns3pro complex. interestingly, the ns2b (48-100;δ77-84)-ns3pro complex has ~37% unstructured conformation, slightly higher than that of the ns2b (48-100)-ns3pro complex (31%), but much lower than that of the isolated ns3pro (53%). these results indicate that the ns2b (48-100;δ77-84)-ns3pro complex has a very different structure from either isolated ns3pro or ns2b (48-100)-ns3pro complex. indeed, very different from the hsqc spectra of the isolated ns3pro ( fig 1b) or ns2b (48-100)-ns3pro complex (fig 4d) , only a small set of hsqc peaks could be detected for the 15 nlabeled ns3pro in complex with ns2b (48-100;δ77-84) ( fig 8b) . furthermore, a large portion of detectable hsqc peaks are superimposable to those of the ns2b (48-100)-ns3pro complex (fig 8b) , implying that some residues of the ns2b (48-100;δ77-84)-ns3pro complex have conformations similar to those of the ns2b (48-100)-ns3pro complex. however, hsqc peaks of the majority of residues were not detected, indicating that the ns2b (48-100;δ77-84)-ns3pro complex undergoes conformational exchanges on μs-ms, or/and dynamic oligomerization, thus retarding further nmr characterization of its high-resolution conformation. most strikingly, the ns2b (48-100;δ77-84)-ns3pro complex had no detectable catalytic activity even with a concentration up to 10 μm in buffers at ph 7.5, suggesting that this complex is trapped in a highly-dynamic and catalytically impotent state. as virus-encoded proteases have been shown to be essential for the replication and infectivity of many viruses, consequently they become important targets for design of anti-viral drugs [42, 43] . indeed, drugs have been successfully developed to treat hiv and hcv infections by targeting their proteases [44, 45] . the dengue ns3 protease has its catalytic machinery hosted by a chymotrypsin fold, which has been extensively shared by a variety of proteases. on the other hand, unlike most other chymotrypsin-like proteases, only flavivirus proteases including dengue ns3 protease need an additional cofactor ns2b to form the enzymatically active complex. noticeably, even within the flavivirus proteases, the ns2b cofactors are very divergent, only with sequence identity of~19% among different members [6] . so the ns2b-ns3pro interface may represent an attractive target for developing molecules which specifically inhibit flavivirus proteases. however, due to its "complete insolubility", the isolated ns3pro domain has never been experimentally characterized so far. in the present study, as facilitated by our discovery in 2005 [15] [16] [17] [18] [19] , for the first time, the isolated ns3pro domain has been extensively characterized in solution by cd, nmr and pre. the results decipher an unexpected fact that despite owning a high-complexity sequence which is very different from classic iups [31] [32] [33] [34] [35] [36] , the ns3pro with the native sequence is intrinsically disordered without the ns2b cofactor, with no stable secondary structures and tight tertiary packing. this indicates that the isolated ns3pro becomes completely insoluble in buffers by following the same mechanism as we previously established for other "completely insoluble" proteins [15] [16] [17] [18] [19] . nevertheless, in the presence of the ns2b cofactor, the disordered ns3pro domain folds into the well-structured chymotrypsin-like fold hosting the active catalytic machinery by the "binding-coupled folding" mechanism [32] [33] [34] [35] [36] . previously, the chymotrypsin fold has been found to be an autonomous folding unit even in the context of the sars 3clike protease with an extra domain [46] . therefore, to the best of our knowledge, the ns3pro domain represents the first intrinsically-disordered chymotrypsin-like fold which absolutely requests the additional cofactor to achieve its correct folding. so an interesting question is whether being intrinsically disordered for the isolated ns3pro bears any in vivo relevance? interestingly, a recent study has revealed that the dengue ns3pro domain without ns2b was capable of binding human fatty acid synthase (fasn) to trigger the membrane remodelling [47] . as such, our biophysical results imply that this interaction needs the dengue ns3pro to be largely disordered. indeed, we found that the well-folded ns2b-ns3pro complex only had weak binding to the fasn domain. furthermore, denv and other members of the flaviviridae family are dependent on the host er to translate, replicate and package their genome, and their infection has been found to induce significant rearrangements of intracellular membranes [47] [48] [49] [50] . in particular it has been recently revealed that the rearrangement and expansion of the er appear to be driven by viral but not host protein synthesis early after denv infection, independently of the upr or srebp2 pathways [50] . therefore, on a speculative note, we propose that the highly disordered ns3pro itself might also play a role in triggering the early rearrangement of the er before activating specific pathways [50] . previously, we have shown that the als-causing p56s mutation of the er-anchored vapb protein rendered the well-structured β-barrel fold of its msp domain to be highly disordered and also become "completely insoluble" in buffer [51] , similar to what we found here on the isolated ns3pro. remarkably, the p56s-vapb suddenly gained the novel capacity to remodel the er structure which was not observed for the wild-type vapb [52] . therefore, the disordered human p56s-vapb and dengue ns3pro may use similar mechanisms to remodel the er structure. in fact, the degue ns3 protein itself is also anchored into the er membrane before ns3 forms the catalytically-active complex with ns2b to cleave itself from the polyprotein. in this study, with our previous discovery that "insoluble proteins could be solubilized in water with minimized salt ions, we also successfully refolded the ns3pro protease with the fulllength ns2b (1-130) anchored into the lmpc micelle. interestingly, although nmr characterization deciphers that the ns3pro domains have different dynamics on the μs-ms time scale in the contexts of being complexed with ns (48-100) in buffer and with ns2b (1-130) in the lmpc micelle, they have very similar enzymatic activities. this membrane-anchored complex may provide a platform for screening the inhibitors for the dengue protease. in this regard, despite challenging, it is of both fundamental and therapeutic interest in the future to explore how the different structures and dynamics upon being anchored into membranes affect the affinity and specificity of the dengue ns3pro in binding substrates and inhibitors. further md simulations provide critical insights into the indispensable role of the ns2b cofactor. even within 20-ns simulations, the isolated ns3pro domain already showed higher dynamic instability than the ns3pro domain in the ns2b-ns3pro complex (fig 5f) . it is thus expected that on the long time scale, the isolated ns3pro would become unfolded as experimentally demonstrated here. strikingly, the ns2b residues show much higher structural dynamics than ns3pro even in the complex, suggesting that the cofactor is capable of sampling a large ensemble of conformations, thus providing the dynamic mechanism for the observed conformational exchange of ns2b between open and closed conformations [8] [9] [10] [11] [12] . the correlated motions in proteins have been extensively recognized to be critical for their diverse functions [20, 21, 30, 53] . in particular, we have recently revealed that a global correlated motion network was essential for the catalysis of the sars 3c-like protease [20, 21] , which also utilizes the chymotrypsin fold to harbour its catalytic machinery. more specifically, without altering the three-dimensional structure of the enzyme, the n214a mutation on the extra domain is sufficient to inactivate the catalytic machinery by globally decoupling the correlation network, while the sti/a mutations also on the extra domain enhance the catalytic machinery by altering the correlation network pattern [20, 21] . in the present study, a similar scenario has been observed for the dengue ns2b-ns3pro complex. a global correlation network does exist in the ns2b-ns3 complex. most intriguingly, this global correlation network is mostly coordinated by the ns2b residues which have very high structural dynamics. as such, the md results not only rationalizes the central role of the ns2b cofactor in maintaining the dynamic stability of the ns3pro domain, but further implies that a slight perturbation of the ns2b-ns3pro interface may be sufficient to decouple the correlation network to inactive the catalytic machinery of the dengue ns2b-ns3pro protease, as we found on the n214a mutation of the sars 3clike protease [20, 21] . indeed, by deleting the ns2b residues highly flexible in md simulations, we obtained a truncated ns2b (48-100;δ77-84), which is able to trap the ns2b-ns3pro complex in a catalytically-impotent state with significant μs-ms dynamics. in the further, it would be of significant interest to test whether this truncated ns2b can act as a specific inhibitor of the dengue protease in vivo. despite the success in treating viral infections with inhibitors to target the active sites of hiv and hcv proteases [41] [42] [43] , many challenges/difficulties still remain for this approach. for example, the structural architecture and catalytic mechanism of viral proteases are also largely shared by many human proteases. this makes it extremely challenging to design inhibitors which only specifically bind to viral proteases but not to human ones. moreover, there is an addition challenge associated with the dengue ns3pro protease. it has been proposed that the flat and charged nature of its active site may at least partly contribute to the current failure in developing effective inhibitors [7, 45] . to overcome these difficulties, alternative strategies are requested to targets sites other than the active sites of the viral proteases, such as to inhibit the dimerization required for activity [46] , to trigger allosteric inhibition [45] , or even to block the folding of the protease [54] . in this regard, our present study successfully implies potential strategies to perturb the ns2b-ns3pro interface for future developing effective and specific inhibitors for the dengue protease. more specifically, inhibitory molecules might be developed to: 1) trap the ns2b-mediated folding into an inactive intermediate as exemplified by the truncated ns2b; and 2) decouple the global correlation network of the ns2b-ns3pro complex. furthermore, our success in in vitro refolding of the active ns3pro in complex with both ns2b (48-100) in buffer and ns2b in the micelle provides experimental platforms for implementing the above-proposed approaches of inhibitor design as well as to decode the underlying mechanism for inhibitors obtained by high through-put screening. for example, recently the dengue protease inhibitors were identified by high through-put screening, which were implied to target the ns2b-ns3pro interface by in silico prediction or mutagenesis [55, 56] . it would be of significant interest to delineate how these molecules achieve the inhibitory effect: by trapping the folding or modulating the dynamics of the protease complex, if they directly target the ns2b-ns3pro interface. in summary, our study decrypted that the dengue ns3pro domain is the first intrinsicallydisordered chymotrypsin-like fold which absolutely requests the ns2b cofactor to coordinate the correct folding as well as correlated motions of the ns2b-ns3pro complex to achieve its catalytic function. in light of a recent study [47] , the disordered ns3pro is needed for interacting with the human host factor to initiate the membrane remodeling. furthermore, our results also imply potential strategies to manipulate the ns2b-ns3pro interface for design of molecules in the future, which may effectively and specifically inhibit the protease activity for treating the dengue infection. best practices in dengue surveillance: a report from the asia-pacific and americas dengue prevention boards the global distribution and burden of dengue the dengue viruses dengue: a continuing global threat structural proteomics of dengue virus structural biology of dengue virus enzymes: towards rational design of therapeutics structural basis for the activation of flaviviral ns3 proteases from dengue and west nile virus ligand-bound structures of the dengue virus protease reveal the active conformation binding of low molecular weight 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replication factories early dengue virus protein synthesis induces extensive rearrangement of the endoplasmic reticulum independent of the upr and srebp-2 pathway elimination of the native structure and solubility of the hvapb msp domain by the pro56ser mutation that causes amyotrophic lateral sclerosis a vapb mutant linked to amyotrophic lateral sclerosis generates a novel form of organized smooth endoplasmic reticulum structured crowding and its effects on enzyme catalysis hiv-1 protease folding and the design of drugs which do not create resistance novel dengue virus-specific ns2b/ns3 protease inhibitor, bp2109, discovered by a high-throughput screening assay. antimicrob agents chemother a small compound targeting the interaction between nonstructural proteins 2b and 3 inhibits dengue virus replication key: cord-000725-rafwlw0t authors: hindinger, claudia; bergmann, cornelia c.; hinton, david r.; phares, timothy w.; parra, gabriel i.; hussain, shabbir; savarin, carine; atkinson, roscoe d.; stohlman, stephen a. title: ifn-γ signaling to astrocytes protects from autoimmune mediated neurological disability date: 2012-07-27 journal: plos one doi: 10.1371/journal.pone.0042088 sha: doc_id: 725 cord_uid: rafwlw0t demyelination and axonal degeneration are determinants of progressive neurological disability in patients with multiple sclerosis (ms). cells resident within the central nervous system (cns) are active participants in development, progression and subsequent control of autoimmune disease; however, their individual contributions are not well understood. astrocytes, the most abundant cns cell type, are highly sensitive to environmental cues and are implicated in both detrimental and protective outcomes during autoimmune demyelination. experimental autoimmune encephalomyelitis (eae) was induced in transgenic mice expressing signaling defective dominant-negative interferon gamma (ifn-γ) receptors on astrocytes to determine the influence of inflammation on astrocyte activity. inhibition of ifn-γ signaling to astrocytes did not influence disease incidence, onset, initial progression of symptoms, blood brain barrier (bbb) integrity or the composition of the acute cns inflammatory response. nevertheless, increased demyelination at peak acute disease in the absence of ifn-γ signaling to astrocytes correlated with sustained clinical symptoms. following peak disease, diminished clinical remission, increased mortality and sustained astrocyte activation within the gray matter demonstrate a critical role of ifn-γ signaling to astrocytes in neuroprotection. diminished disease remission was associated with escalating demyelination, axonal degeneration and sustained inflammation. the cns infiltrating leukocyte composition was not altered; however, decreased il-10 and il-27 correlated with sustained disease. these data indicate that astrocytes play a critical role in limiting cns autoimmune disease dependent upon a neuroprotective signaling pathway mediated by engagement of ifn-γ receptors. cns resident cells are targets of autoimmune mediated damage but also active participants in disease development, progression and control [1, 2] . however, their contributions to neuroprotection and regulation by inflammatory mediators are not well defined. cns insults, including autoimmune disease, initiate rapid astrocyte activation characterized by cellular hypertrophy and increased intermediate filament glial fibrillary acidic protein (gfap) expression [3] [4] [5] . astrocytes form a physical barrier surrounding areas of inflammation initially limiting bystander tissue damage [6, 7] . however, this barrier subsequently impedes axonal regeneration contributing to sustained disability [3, 5, 8] . innate and adaptive pro-inflammatory astrocyte functions include production of pro-inflammatory cytokines, reactive oxygen species, chemokines, and matrix metalloproteinases [3] [4] [5] . by contrast, secretion of anti-inflammatory cytokines and scavengers of reactive oxygen species, as well as inhibition of both microglial activation and tumor necrosis factor (tnf) secretion, all support an astrocyte mediated anti-inflammatory function [3] [4] [5] . therefore, astrocyte activation constitutes a ubiquitous, yet heteroge-neous response associated with both promoting and inhibiting cns repair [3] [4] [5] . ms and eae are both associated with t cells secreting ifn-c (th1 cells) and il-17 (th17 cells) which play complex, not fully understood roles in disease initiation and progression [2, 9] . in vivo and in vitro evidence indicates that suppression of encephalitogenic t cell proliferation within the cns during eae and activation of anti-inflammatory programs are in part mediated via astrocytes [4, 5] . similar to astrocytes, ifn-c mediates both proand anti-inflammatory functions during autoimmune disease [10] . early ifn-c induced effects are pro-inflammatory; ifn-c facilitates inflammatory cell access, shapes their composition, increases major histocompatibility complex (mhc) expression, contributes to macrophage and microglia activation, and initiates oligodendrocyte death [11] . similarly, ifn-c mediated protection during eae is also multifaceted [12] [13] [14] [15] . it acts as a negative regulator of neutrophil accumulation, th17 cell activation, il-1r signaling, protease secretion, and chemokine activity. it also inhibits proinflammatory cytokine secretion via induction of suppressor of cytokine secretion (socs) proteins, facilitates t cell apoptosis and protects oligodendrocytes via inducing endoplasmic reticulum (er) stress responses [10, 11, 16, 17] . this highlights the critical role of a single mediator in both promoting disease but also limiting inflammatory mediated damage required to initiate repair cascades. based on the gatekeeper functions of astrocytes and the diverse biological effects of ifn-c, we set out to determine how ifn-c signaling specifically to astrocytes influences cns autoimmune disease. the results demonstrate that ifn-c signaling to astrocytes had no profound effects on initial disease progression, but played an essential protective role during the transition from acute to chronic disease. clinical remission induced by ifn-c signaling to astrocytes coincided with reduced demyelination, axonal degeneration, and astrocyte activation. the ifn-c receptor is expressed ubiquitously; however, these data reveal astrocytes as the primary target and mediator of the well established antiinflammatory activity of ifn-c within the cns. to understand the role of ifn-c signaling to astrocytes during the pathogenesis of cns autoimmune disease, eae was induced in transgenic mice expressing a signaling deficient dominant negative ifn-c receptor 1 specifically on astrocytes (gfapcr1d mice) [18] . peripheral activation of self reactive t cells in gfapcr1d mice was similar to wt mice (data not shown). this is consistent with both the cns restricted transgene expression in the gfapcr1d mice as well as the similar t cell activation following peripheral immunization with a non-self antigen [18] . following immunization the incidence of disease, initiation of clinical symptoms, and initial symptom progression were unaltered by the inability of astrocytes to respond to ifn-c ( fig. 1a ; table 1 ). in addition, neither immunized group exhibited clinical symptoms of atypical eae associated with the absence of ifn-c [19] . however, clinical symptoms in gfapcr1d mice began to diverge from wt mice at , day 12 post immunization (p.i.) prior to the peak of clinical disease. in both groups clinical disease peaked at , day 14 p.i., but severity was increased from a score of 3.1 in wt mice to 4.0 in the gfapcr1d group ( fig. 1a ; table 1 ). gfapcr1d and wt mice were compared at the peak of acute disease to determine if ifn-c signaling altered astrocyte activation or cns inflammation. astrocyte hypertrophy and gfap expression ( fig. 1b) were similar in both groups, indicating no overt effects of ifn-c on initial astrocyte reactivity. furthermore, neither the extent of inflammation nor the anatomic distribution of inflammatory cells was altered (fig. 1b) . flow cytometry confirmed no difference in the overall extent of cd45 hi inflammatory cells recruited into the cns ( fig. 2a) . furthermore, percentages of cd4 + t cells ( fig. 2a) , cd8 + t cells and macrophages within the infiltrates were also similar (fig. 3) . in contrast to the association between increased eae severity and neutrophil accumulation in the global absence of ifn-c [14, 15] , only a small percentage of neutrophils were identified in the cns of both groups by flow cytometry (fig. 3) ; their low presence was confirmed by the inability to identify cells with the characteristic morphology of neutrophils in the brain by histopathology (fig. 1b) . the absence of increased neutrophils in the cns of gfapcr1d mice during acute disease suggested that clinical disease was aggravated by a mechanism distinct from global ifn-c deficiency. in addition to the similar frequency of cd4 + t cells in the brains of the two groups ( fig. 2a) , myelin oligodendrocyte glycoprotein (mog) reactive cd4 + t cells secreting ifn-c were also similar ( fig. 2b ). by contrast, mog specific cd4 + t cells secreting il-17 ( fig. 2b ) and foxp3 + regulatory cd4 + t cells (tregs) were decreased in gfapcr1d mice compared to wt mice (fig. 2c) . reduced th17 and tregs may be attributed to increased ifn-c [10, 16] . alternatively, as ifn-c is protective in eae [12] [13] [14] [15] , increased disease severity may reflect reduced bioavailable ifn-c due to sequestration of ifn-c binding to the dominant negative receptor. however, measurement of ifn-c in cell free supernatants derived from dissociated brains at the peak of acute disease demonstrated protein levels of 2.160.2 ng/brain in wt mice versus 3.660.5 ng/brain in gfapcr1d mice (n = 3; p,0.05). as overall frequencies of mog reactive cd4 + t cells secreting ifn-c were similar, increased ifn-c in the brains of gfapcr1d mice suggested enhanced secretion at the cellular level. although a contribution of nk or cd8 t cells could not be excluded, these potential sources of ifn-c were unlikely due to their low frequencies (nk ,5% and cd8 + t cells , 5%, see fig. 3 ) and their equivalent frequencies in the wt and gfapcr1d mice. furthermore, reduced th17 cell and treg frequencies were consistent with suppression of these populations due to elevated ifn-c [10, 16, 20] . inflammation in spinal cords from the gfapcr1d mice was also similar to wt controls at the peak of clinical symptoms (fig. 4) . although numbers and distribution of cd4 + t cells were also similar (4.661.3/mm 2 in gfapcr1d mice vs. 3.860.1/ mm 2 in wt mice; fig. s1 ), spinal cords of gfapcr1d mice exhibited a ,2-fold increase in demyelination (fig. 4) . areas of demyelination encompassed 7.361.0% of spinal cord white matter in gfapcr1d mice versus 3.860.9% in wt mice (p#0.01). furthermore, the increase in demyelination was associated with a prominent loss of axons in gfapcr1d mice compared to controls (fig. 4) . despite elevated demyelination and axonal loss in the absence of ifn-c signaling to astrocytes, spinal cords showed no evidence of differential astrocyte activation by either immunohistochemistry (fig. 4 ), or differences in gfap mrna expression during the peak of acute disease (fig. 5 ). although ccl5, il-1 and tnf mrna expression were increased in the spinal cords of the gfapcr1d mice, no differences in ifn-c, inos, cxcl10 or il-6 mrna were consistent with similar inflammation (fig. 5) . these data suggest that the initial disease pathogenesis, reflected by an increased demyelination in spinal cords, but not brain, during ascending paralysis is dampened by ifn-c signaling to astrocytes. subsequent to peak disease severity the clinical symptoms in wt mice began a modest decline by day 16 p.i. (fig. 1a ). by contrast, gfapcr1d mice not only exhibited increased severity of clinical symptoms following day 14 p.i., but the continued disease escalation was associated with increased mortality ( fig. 1a ; table 1 ). sustained morbidity and significant mortality in gfapcr1d mice after day 30 p.i. implied a critical role for ifn-c induced astrocyte signaling in neuroprotection and limiting disability. a similar absence of clinical remission was found in a preliminary experiment comparing eae sjl mice carrying the gfapcr1d gene (data not shown). these data suggest that astrocyte responses to ifn-c are protective, irrespective of genetic background. escalating disease in gfapcr1d mice coincided with focal areas of intense inflammation in spinal cords, which contrasted with the more diffuse inflammation in wt mice (fig. 6 ). demyelination was also increased with myelin loss encompassing 5.761.8% of spinal cord area in gfapcr1d mice versus 2.361.4% in wt mice (p#0.05) at day 35 p.i. (fig. 6 ). the demyelinated areas further exhibited enhanced axonal damage in gfapcr1d mice (fig. 6) , supporting a correlation between enhanced tissue damage, sustained morbidity and increased mortality ( fig. 1a ; table 1 ). astrocyte activation associated with areas of myelin loss is a prominent finding in the cns of both patients with ms and rodents with eae. although demyelination was increased in the cns of gfapcr1d mice, the extent of astrocyte activation associated with spinal cord white matter lesions was similar in both groups ( fig. 6 ; ,60 gfap + cells/mm 2 ). by contrast, the frequency of activated astrocytes in spinal cord grey matter areas that were not associated with demyelinated lesions, was increased in gfapcr1d mice with 101.5623.0 gfap + activated astrocytes/mm 2 versus 25.5615.5 in wt mice (p,0.001) (fig. 7) . a similar increase in astrocyte activation within grey matter distal to white matter lesions was also detected in gfapcr1d sjl mice during chronic eae (data not shown). flow cytometric analysis during chronic disease revealed an ,4fold increase in cd45 hi inflammatory cells confirming increased inflammation in the absence of ifn-c signaling to astrocytes (fig. 8a ). similar to the acute disease, relative percentages of neutrophils, macrophages, cd4 + and cd8 + t cells were all similar (fig. 3) . furthermore, no differences in expression of activation markers on cns derived cd4 + t cells, including cd69, cd122, cd127, fas, fasl, icos and cd152 were evident between the groups (data not shown). the percentages of mog reactive th1 cells were also not altered in the cns of gfapcr1d relative to wt mice (fig. 8b ) and the decreased percentage of potentially destructive th17 cells identified during acute disease (fig 2) , was also sustained during chronic disease (fig. 8b ). equivalent expression of il-7, il-23 and tgf-b mrna (fig. 8c ) suggested that the decrease in th17 cells was dependent upon an increase in ifn-c and not related to a defect in activation or maintenance signals [20] . increased inflammation and sustained mhc class ii expression on microglia (fig. 8a ) further suggested that ifn-c signaling to astrocytes down regulates inflammation universally without altering the composition of the cns infiltrates. this concept is supported by sustained composition of cns infiltrates during inflammation induced astrocyte apoptosis [21] . the inability to restrain ongoing inflammation during eae in gfapcr1d mice was evident at multiple levels. astrocyte activation was sustained consistent with increased expression of gfap mrna. ccl2, ccl5 and cxcl10 mrna levels were increased (fig. 8c ). the expression of mrna encoding potentially destructive immune mediators including il-1, il-6, inos, and tnf were all increased (fig. 8c ). ifn-c was ,2-fold higher in the cns of gfapcr1d mice (fig. 9) . lastly, the level of the antiinflammatory cytokine il-10 was reduced ( fig. 9 ), suggesting limited availability of il-10 may be a key in prolonging astrocyte activation and inflammation [22] . a possible link between reduced il-10 and the inability of ifn-c signaling to astrocytes is provided by the capacity of ifn-c to induce il-27 in astrocytes [23] , thereby promoting il-10 production. indeed il-27 in the cns of the gfapcr1d mice was significantly reduced compared to wt mice during chronic disease (fig. 9 ). by contrast, il-12 ( fig. 9) , an indirect suppressor of cns inflammation by promoting ifn-c production [24] , was similar in the cns of both gfapcr1d and wt mice. astrocytes derived from gfapcr1d and wt mice were stimulated with ifn-c to confirm ifn-c dependent il-27 secretion by astrocytes [25] . while ifn-c induced il-27 secretion by astrocytes from wt mice, il-27 secretion was significantly reduced in cultures derived from gfapcr1d mice (fig. 10) . these data support the possibility that ifn-c mediated il-27 secretion by astrocytes regulates eae effector t cell function, inflammation and tissue destruction via induction of il-10; however, this does not exclude the possibility that the inability of astrocytes to respond to ifn-c could directly or indirectly dysregulate a variety of other immunomodulatory functions [4, 5, 17] . in the eae model of ms, ifn-c functions as a proinflammatory cytokine in the early stages of disease [10, 11, 26 ], yet it also assumes a prominent anti-inflammatory role during the transition to remission [10, [12] [13] [14] [15] . protection has been attributed to a variety of potentially interrelated mechanisms including limiting neutrophil accumulation, th17 cell activation, il-1r signaling, matrix metalloproteinase secretion, pro-inflammatory cytokine secretion and chemokine activity; in addition ifn-c facilitates t cell apoptosis and protects oligodendrocytes from death via an er stress response [10, 11, 17] . the activities of astrocytes during autoimmunity also range from pro-to antiinflammatory [3] [4] [5] . however, to what extent a direct action of ifn-c on astrocytes contributes to inhibitory mechanism is unclear. the data herein demonstrate that among the multiple early functions of astrocytes imposed by innate responses are largely pro-inflammatory during eae [3, 6, 7, 26] . for example, astrocytes contribute to the loss of bbb integrity via secretion of reactive oxygen species, chemokines and pro-inflammatory cytokines [3] [4] [5] . this pro-inflammatory milieu is associated with initial axonal damage prior to accumulation of self reactive t cells [27] , which further promotes immune mediated damage. blocking the pro-inflammatory transcription factor nf-kb in astrocytes improves recovery during chronic eae [28, 29] . supporting an initial pro-inflammatory role of astrocytes dependent on innate, not ifn-c responsiveness, inhibition of ifn-c signaling to astrocytes did not influence eae onset or incidence, initial disease progression, astrocyte activation, or bbb integrity as indicated by similar entry of inflammatory cells into the brain. by contrast, an inflammation dampening effect of ifn-c became evident during the onset of disease remission. astrocytes limit ongoing inflammation and pathogenic processes at several levels. they not only facilitate repair by inhibiting inflammatory cell entry into the cns parenchyma [6, 7, 30] , but also down regulate t cell effector function and proliferation [4, 5, 21] . for example, eae in gfap deficient mice results in more severe and widespread inflammation [31] . in addition, t cell-astrocyte interactions as well as astrocyte secretion of an unidentified soluble product, distinct from nitric oxide, prostaglandins, or tryptophan metabolism, suppress t cell proliferation [4, 5] . t cell proliferation was also not inhibited via defective il-2 release, despite the suggestion that t cell-astrocyte interactions facilitate secretion of anti-inflammatory cytokines [4, 5] . lastly, astrocytes may limit inflammation by triggering apoptosis in t cells [32] . nevertheless, the role of ifn-c signaling in these potentially anti-inflammatory, protective mechanisms is not clear. elevated ifn-c in the cns of gfapcr1d mice during both acute and chronic disease excluded limited ifn-c due to sequestration by the transgenic receptor as a mediator of increased clinical severity and mortality. indeed, enhanced ifn-c production may reflect an attempt to compensate for the loss of ifn-c dependent astrocyte mediated control of inflammation [33] . sustained expression of pro-inflammatory cytokines, particularly il-6 and tnf, thus represents a primary mechanism underlying ongoing tissue destruction in gfapcr1d mice. il-6 is known to mediate neurological dysfunction [34] and astrocytes are the predominant source of il-6 during cns autoimmune disease. furthermore, its secretion is down regulated by ifn-c induced socs activity [17] . on the other hand, sustained tnf implicated dysregulated activation of microglia or macrophages via increased ifn-c or il-6, as tnf is predominantly secreted by activated cns macrophages and microglia during eae [35, 36] . however, the down regulation of microglia activation, including tnf secretion, following interaction with activated astrocytes [37] questions this notion. while both il-6 and tnf may thus contribute to sustained pathological changes, the source of tnf remains unclear. similarly, astrocytes are a potential source of the ifn-c induced chemokine cxcl10 during eae, one of the chemokines controlling t cell recruitment [38] . however, the increased expression of cxcl10 coupled with the inability of the astrocytes in the gfapcr1d mice to respond to ifn-c suggests altered microglia activation and secretion of cxcl10 [38] or activation of cxcl10 transcription via an independent signaling pathway mediated by tnf or type 1 interferons [39, 40] . importantly, pathology further correlated with decreases in both tregs and il-10, but not with increased antigen specific th17 cells, although astrocytes are critical targets of il-17 [41] . although it is possible that the increased ifn-c in the cns influenced peripheral activation of antigen specific th17 cells, previous data demonstrated no evidence for expression of the transgene in peripheral organs, including lymphoid organs [18] . il-10, secreted by a variety of cells types including cd4 + t cells, cd8 + t cells and tregs [42] , inhibits both acute and chronic eae [43, 44] . the decrease in this anti-inflammatory cytokine suggests that the induction of il-27 secretion is a critical aspect of astrocyte responses to ifn-c, thus promoting il-10 secretion by t cells [23, 25] . however, our data is unable to distinguish the contribution of paracrine versus autocrine ifn-c induced signals to astrocytes on il-10 production, as il-10 also regulates astrocyte activation [22] , and can itself be secreted by astrocytes to exert autologous functions [45] . in addition to mediating an imbalance of protective versus detrimental cytokines, our results demonstrate that ifn-c signaling to astrocytes limits the extent of astrocyte activation during chronic eae, specifically in grey matter. activated astrocytes, especially within and adjacent to areas of demyelination are a prominent feature of white matter plaques associated with both chronic ms and eae [3] [4] [5] . astrocytes protect neurons and oligodendroglia, but also form glial scars which inhibit regeneration after neuronal injury [3, 5, 8, 20] . the absence of reactive astrocytes increases axonal regeneration after injury [46] , consistent with the concept that limiting astrogliosis is critical for axonal regeneration after neuronal injury. while the significance of sustained astrocyte activation in grey matter tracks is unclear in our model, it is consistent with increased axonal damage, and suggests possible damage to axons outside the lesions, which may not be manifested by histological analysis. limiting inflammation following either infection or during an autoimmune attack is a prerequisite for the initiation of repair. this is critically important within the cns which has limited regenerative capacity. in summary, our data identify astrocytes as prominent targets underlying ifn-c mediated suppression of chronic cns inflammation [19] . the data further provide a link between sustained inflammatory responses, enhanced demyelination and axonal degeneration associated with loss of neurological function during eae and chronic progressive ms. although the inability of astrocytes to respond to ifn-c did not alter disease in the brain, engagement of the ifn-c receptor on astrocytes in spinal cord limits demyelination and functions in a neuroprotective capacity. current therapies for ms are primarily focused on antiinflammatory and immunomodulatory approaches and have been partially successful in treating acute episodes. the identification of astrocytes as critical responders and mediators of ifn-c signaling in limiting cns autoimmune disease may provide insights into new approaches to limit long term progression to disability. brains and spinal cords from perfused mice were homogenized separately as previously described [47, 48] . homogenates were centrifuged at 4506g for 7 min at 4uc. supernatants were stored at -80uc for cytokine determination (see below). cells were resuspended in 30% percoll (amersham biosciences, piscataway, nj) and isolated by centrifugation (8006g for 30 min at 4uc) onto 70% percoll cushions. non-specific binding was inhibited by incubation with anti-cd16/cd32 (2.4g2; bd biosciences, san diego, ca) and a 10% mixture of normal goat, human, mouse and rat serum for 10 min on ice. fitc, pe, percp, and apc conjugates with monoclonal antibodies (mab) used to identify and quantify microglia and inflammatory cells were: cd4 (gk1.5), cd8a (53-6.7), cd45 (30-f11), mhc class ii (2g9), ly6g (1a8) (bd biosciences), and f4/80 (serotec, raleigh, nc). microglia and inflammatory cells were distinguished based on differential cd45 expression. cd4 + t cells were identified as cd45 hi cd4 + , cd8 + t cells as cd45 hi cd8 + , macrophages as cd45 hi f4/80 + and neutrophils as cd45 hi ly6g + mhc class ii -. mog specific induction of cytokines was determined by stimulation of 1610 6 cns cells with 20 mg/ml peptide for 6 h at 37uc with golgistop (bd biosciences) added for the last 4 h. intracellular cytokines were detected with fitc-anti-ifn-c (clone xmg1.2; bd biosciences) and pe-anti-il-17 (clone tc11-18h10; bd biosciences). intracellular foxp3 was detected by staining for cell surface markers, followed by permeabilization with fixation/ permeabilization reagent (ebioscience, san diego, ca) and incubation with pe-labeled anti-foxp3 (fjk-16s; ebioscience). cells were analyzed on a facscalibur flow cytometer (bd biosciences) using cellquest pro software (bd biosciences). data was analyzed using flowjo (7.6.1) software (tree star inc., ashland, or). cytokines were determined by elisa using antibody pairs and recombinant cytokine standards from bd bioscience. il-27 was measured using quantikine mouse il-27p28 immunoassays (r&d systems inc., minneapolis, mn). following anesthesia, mice were perfused with pbs (ph 7.2). brains and spinal cords were fixed with clark's solution (75% ethanol and 25% glacial acetic acid), and embedded in paraffin. spinal cords were divided into 6 sections prior to embedding, corresponding to cervical, thoracic and lumbar levels. cross sections (6 mm), were stained with either hematoxylin and eosin (h&e) or luxol fast blue (lfb). immunoperoxidase staining was used to identify activated astrocytes with anti-gfap (abcam, cambridge, ma) and axonal integrity with smi-31 and smi-32 mab (sternberger monoclonals inc., lutherville, md) followed by visualization using vectastain abc kit (vector laboratories, burlingame, ca) and 3,39-diaminobenzidine (sigma-aldrich). sections from at least 3 separate experiments containing at least 3 mice per group were reviewed in a blinded manner. numbers of gfap + cells in spinal cord were determined in 15 non-overlapping 406 fields (0.2 mm 2 ) in white matter and gray matter. stained spinal cord sections of all 6 levels on individual glass slides were scanned (406) and digitally imaged at high resolution with an aperio scanscope (vista, ca). aperio software was used to quantify areas of demyelination within the white matter tracks of each of the 6 sections per individual mouse. for analysis of cd4 + t cells spinal cords were embedded in tissue-tek o.c.t. (andwin scientific, tryon, nc), flash frozen in liquid nitrogen and stored at 280uc. blocks were warmed to 220uc prior to cutting 10 mm sections by cryostat. following fixation with acetone for 2 min at 4uc, non-specific antibody binding was blocked with cyto q background buster (innovex biosciences, richmond, ca) for 15 min. sections were stained with anti-cd4 (l3t4) antibody (vector laboratories) diluted in cyto q immuno diluent (innovex biosciences) followed by biotinylated rabbit anti-rat, peroxidase abc reagent and visualized with novared substrate (all from vector laboratories). sections were counter stained with hematoxylin to visualize the nuclei. spinal cord sections were scanned (406) and digitally imaged at high resolution with an aperio scanscope. mixed glial cultures (,70% astrocytes) were established from neonatal gfapcr1d and wt mice as previously described [47] . il-27 secretion was determined 48 h after rifn-c (100 ng/ml) stimulation. frozen tissues were homogenized in trizol (invitrogen, carlsbad, ca) and cdna prepared as described [47, 48] . quantitative real-time pcr was performed using 4 ml of cdna and sybr green master mix (applied biosystems, foster city, ca) in duplicate on a 7500 fast real-time pcr system (applied biosystems). expression levels were normalized to ubiquitin or gapdh using the following formula: statistical significance was determined by two-tailed student's t test. a value of p,0.05 was considered statistically significant. figure s1 cd4 + t cell recruitment into the spinal cord during acute eae. cd4 + t cells were visualized in 10 mm frozen sections of spinal cords from wt and gfapcr1d tg mice at day 18 p.i. immunoperoxidase stain (novared chromogen with hematoxylin counterstain). scale bars = 50 microns. (tif) multiple sclerosis: a complicated picture of autoimmunity the immunology of multiple sclerosis astrocytes: biology and pathology astrocytes in the tempest of multiple sclerosis astrocytes-friends or foes in multiple sclerosis leukocyte infiltration, neuronal degeneration, and neurite outgrowth after ablation of scar-forming, reactive astrocytes in adult transgenic mice reactive astrocytes form scar-like perivascular barriers to leukocytes during adaptive immune inflammation of the cns regeneration beyond the glial scar autoimmune t cell responses in the central nervous system how interferon-gamma keeps autoimmune diseases in check the integrated stress response prevents demyelination by protecting oligodendrocytes against immune-mediated damage intrathecal delivery of ifn-gamma protects c57bl/6 mice from chronicprogressive experimental autoimmune encephalomyelitis by increasing apoptosis of central nervous system-infiltrating lymphocytes interferon-gamma confers resistance to experimental allergic encephalomyelitis mice with a disrupted ifn-gamma gene are susceptible to the induction of experimental autoimmune encephalomyelitis (eae) ifn-gamma plays a critical down-regulatory role in the induction and effector phase of myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis cross-regulation of signaling pathways by interferongamma: implications for immune responses and autoimmune diseases socs1 and socs3 in the control of cns immunity astrocyte expression of a dominant-negative interferon-gamma receptor regional cns responses to ifn-gamma determine lesion localization patterns during eae pathogenesis empowering t helper 17 cells in autoimmunity gp130-dependent astrocyte survival is critical for the control of autoimmune central nervous system inflammation attenuation of astroglial reactivity by interleukin-10 suppressive effect of il-27 on encephalitogenic th17 cells and the effector phase of experimental autoimmune encephalomyelitis early administration of il-12 suppresses eae through induction of interferon-gamma suppression of autoimmune inflammation of the central nervous system by interleukin 10 secreted by interleukin 27-stimulated t cells astrocytes are active players in cerebral innate immunity astrocyte-associated axonal damage in pre-onset stages of experimental autoimmune encephalomyelitis inhibition of transcription factor nf-kappab in the central nervous system ameliorates autoimmune encephalomyelitis in mice transgenic inhibition of astroglial nf-kappa b improves functional outcome in experimental autoimmune encephalomyelitis by suppressing chronic central nervous system inflammation reactive astrocytes protect tissue and preserve function after spinal cord injury experimental autoimmune encephalomyelitis in mice lacking glial fibrillary acidic protein is characterized by a more severe clinical course and an infiltrative central nervous system lesion apoptosis of inflammatory cells in immune control of the nervous system: role of glia ifn-gamma shapes immune invasion of the central nervous system via regulation of chemokines neurologic disease induced in transgenic mice by cerebral overexpression of interleukin 6 increased production of interferon gamma and tumor necrosis factor precedes clinical manifestation in multiple sclerosis: do cytokines trigger off exacerbations? tnfr1 signalling is critical for the development of demyelination and the limitation of t-cell responses during immune-mediated cns disease il-12 production by central nervous system microglia is inhibited by astrocytes chemokines and glial cells: a complex network in the central nervous system tnfa and ifnc synergistically enhance transcriptional activation of cxcl10 in human airway smooth muscle cells via stat-1, nf-kb, and the transcriptional coactivator creb-binding protein concomitant detection of ifna signature and activated monocyte/dendritic cell precursors in the peripheral blood of ifna-treated subjects at early times after repeated local cytokine treatments astrocyterestricted ablation of interleukin-17-induced act1-mediated signaling ameliorates autoimmune encephalomyelitis regulation and functions of the il-10 family of cytokines in inflammation and disease il-10 is critical in the regulation of autoimmune encephalomyelitis as demonstrated by studies of il-10-and il-4-deficient and transgenic mice central nervous system expression of il-10 inhibits autoimmune encephalomyelitis multiple sclerosis: cytokine receptors on oligodendrocytes predict innate regulation axonal plasticity and functional recovery after spinal cord injury in mice deficient in both glial fibrillary acidic protein and vimentin genes inhibition of interferon-gamma signaling in oligodendroglia delays coronavirus clearance without altering demyelination distinct regulation of mhc molecule expression on astrocytes and microglia during viral encephalomyelitis we thank bruce trapp for helpful discussions, wenqiang wei, eric barron and ernesto barron for assistance with the histopathology and jeffrey tarcy, kate stenson and maria ramirez for assistance with mouse husbandry and immunological assays. key: cord-000580-dcid9emx authors: sällman almén, markus; bringeland, nathalie; fredriksson, robert; schiöth, helgi b. title: the dispanins: a novel gene family of ancient origin that contains 14 human members date: 2012-02-20 journal: plos one doi: 10.1371/journal.pone.0031961 sha: doc_id: 580 cord_uid: dcid9emx the interferon induced transmembrane proteins (ifitm) are a family of transmembrane proteins that is known to inhibit cell invasion of viruses such as hiv-1 and influenza. we show that the ifitm genes are a subfamily in a larger family of transmembrane (tm) proteins that we call dispanins, which refers to a common 2tm structure. we mined the dispanins in 36 eukaryotic species, covering all major eukaryotic groups, and investigated their evolutionary history using bayesian and maximum likelihood approaches to infer a phylogenetic tree. we identified ten human genes that together with the known ifitm genes form the dispanin family. we show that the dispanins first emerged in eukaryotes in a common ancestor of choanoflagellates and metazoa, and that the family later expanded in vertebrates where it forms four subfamilies (a–d). interestingly, we also find that the family is found in several different phyla of bacteria and propose that it was horizontally transferred to eukaryotes from bacteria in the common ancestor of choanoflagellates and metazoa. the bacterial and eukaryotic sequences have a considerably conserved protein structure. in conclusion, we introduce a novel family, the dispanins, together with a nomenclature based on the evolutionary origin. membrane proteins are essential for the ability of all cellular organisms to respond and interact with their environment. therefore they have attained large research interest and are one of the major groups of drug targets [1] . we have previously estimated that 27% of the human genes codes for alpha-helical membrane proteins and provided a comprehensive classification based on their function and evolutionary origin [2] . however, the identification and annotation of many membrane bound protein families is still being revised. we have during recent years worked on the annotation of both g protein-coupled receptors [3, 4] and solute carriers [5] and most of the genes of these large superfamilies now have a clear identity and annotation. there is however still large work to be done to clarify the identity, annotation and the evolutionary history of several families of membrane bound proteins. establishing a rigid nomenclature based on evolutionary information and structural features of the predicted proteins facilitates prediction of the functional role of these genes that often have only have been studied in large gene or transcription consortia. in previous studies we have found that membrane proteins with few transmembrane (tm) helices are less studied than other. this is particularly true for 2tm proteins where more than 70% of the about 700 proteins remained unclassified. interestingly, we found evidence for several uncharacterized homologues to a small group of genes known as the interferon-induced transmembrane proteins (ifitm) family. the ifitms constitute a group with four human members (ifitm1-3, 5) that are found in a consecutive order on chromosome 11, having two transmembrane (2tm) helices. the ifitm4 gene is not present in human, but is located in proximity to the other four genes in the mouse genome. the ifitm1-3 proteins were identified 25 years ago as being upregulated by interferons (ifn) [6] . recently they received considerable attentions as ifitm1-3 were found to prevent infection of a growing list of viruses such as hiv-1, sars influenza a h1n1, west nile and dengue fever viruses [7, 8, 9, 10] . hence, proteins of the ifitm family mediate part of the antiviral response orchestrated by ifns. however, the ifitm family is also involved in other processes such as oncogenesis, bone mineralization (ifitm5) and germ cell development (ifitm1 and 3) and ifitm5 has not been identified as interferon-inducible [11, 12, 13, 14] . although the biological roles of the ifitm genes are emerging, no thorough evolutionary analysis has been performed on this group. in this study, we sought to infer the evolutionary history of the human ifitm genes and identify potential homologues. we mined 36 eukaryotic species, covering all major eukaryotic groups, and found that the ifitms form a subfamily in a larger novel family that has ten human members in addition to the four ifitm genes. we propose dispanins as a novel name for this family, which refers to their common 2tm structure. further, we find that the eukaryotic dispanins first appeared before the radiation of metazoa and that they branch out into four subfamilies (a-d). more surprisingly, we also discover that the dispanins are found in a large range of bacteria and in brown alga. in total, we collected 87 eukaryotic ifitm homologues from h. sapiens (14 genes) m. musculus (17 genes), g. gallus (6 genes), x. tropicalis (13 genes), d. rerio (7 genes), p. marinus (1 gene), c. intestinalis (1 gene), b.floridae (12 genes), s. manosoni (1 gene), s. purpuratus (9 genes), n. vectensis (4 genes) and m. brevicollis (1 gene). no ifitm genes could be detected in any of the remaining 24 analyzed proteomes, which covers all other major eukaryotic groups. the search of the nr database with hmmer did not get any hits outside metazoa except bacteria, choanoflagellates and the brown alga ectocarpus siliculosus (2 genes). nine genes were deemed as pseudogenes based on annotation and sequence analysis and removed from further analysis. in addition to the four previously identified human ifitm genes, ten novel human homologous genes were detected. these ten genes together with the four ifitm genes form a human gene family that we choose to call dispanins based of their common 2tm structure. in uniprot we identified 65 annotated ifitm homologues from full bacteria proteome sets spread over seven different phyla (see table s1): acidobacteria (2 genes), actinobacteria (43 genes), cyanobacteria (3 genes), tg1 (1 gene), bacteroidetes (2 genes), firmicutes (1 gene) and proteobacteria (13 genes). out of these, 46 bacterial sequences from 32 species were included for further analysis. no viral or archaean genes were annotated as ifitm homologues in uniprot. the phylogeny of the vertebrate dispanins ( figure 1 ) allows the division of the dispanins into four subfamilies a-d that are supported by strong confidence with respect to posterior probabilities (pp) or bootstraps (pp.0.75 and bs.90% for all nodes). we propose a common nomenclature for the dispanins that are based on their subclass and a number (dspa1 etc). the proposed names together with previous gene symbols and accession number can be found in table s1 . the finding of two dispanin homologs in the brown alga e. siliculosus, which is evolutionary distant to metazoa, and the single dispanin in the close metazoan relative m.brevicollis are the two only non-metazoan eukaryotic dispanins. a blast search gives that the e. siliculosus proteins have a higher similarity to metazoan family members (best hit e-value,10 210 ) than bacterial dispanins and m. brevicollis. the m. brevicollis dispanin share a conserved splice site with all metazoan family members, which suggest that the eukaryotic dispanins first emerged in a common ancestor of m. brevicollis and the metazoan lineage. within the metazoan lineage the dispanins have been lost in at least two separate occasions, i.e. t. adhaerens and in the ecdysozoan lineage (d. melanogaster and c. elegans). the vertebrate dispanins sort into subfamilies a-d ( figure 1 ). the dspa subfamily has six human genes (dspa1, dspa2a-d and dspa3) of which the dspa2a-c corresponds to ifitm1-3 and dspa1 to ifitm5. dspa2d (ac023157) and dspa3 (ac068580) are two novel identified genes, closely related to the ifitm family. the phylogenetic tree indicates that the dspa2 genes have undergone an independent duplication in h. sapiens and m. musculus and these were given a species specific nomenclature, e.g. dspa2a-d in human. the dspa4, dspa5, dspa6 genes in the phylogenetic tree do not have any clear human orthologs. the dspb subfamily is only found in tetrapoda and contains three human genes called dspb1 (tusc5), dspb2 (tmem233) and dspb3 (prrt2) whereas dspb4 is only present in g. gallus. dspc1 (tmem90a), dspc2 (tmem90b) and dspc3 (tmem91) make up the human dspc subfamily, which is represented in all investigated vertebrates. the dspd1 (pprt1) gene is found in all the vertebrate species except the basal organism p. marinus whereas dspd2 (al160276) is mammalian specific. five vertebrate genes and all invertebrate were excluded from the phylogenetic analysis and instead classified into subfamilies by using a blast approach (table s1) . some genes could not unambiguously be classified into the vertebrate subfamilies: c. intestinalis (1 gene), s. mansoni (1 gene), b. floridae (2 genes), s. purpuratus (3 genes), n. vectensis (1 gene) and m. brevicollis (1 gene). by combining the results of the phylogenetic analysis and blast classification, we created a schematic overview of the organisms' gene repertoire and a schematic picture of the dispanin family's evolutionary history, which suggests that the invertebrate dispanins share more similarity towards the dspc and d subfamilies than dspa and b ( figure 2 ). all the members of the human dspa are located on chromosome 11 except for dspa2d which resides on chromosome 12. the genes of the other subfamilies are not enriched on any chromosome. several features are common to all the eukaryotic dispanin proteins ( figure 3 ). they comprise two transmembrane helices that are predicted between 20 and 30 amino acids in length with the second helices often being slightly longer. the dispanins are rich in both glycosylation-and phosphorylation sites that predominantly are found on the nterminus. the n-terminus is often long (.100 amino acids) compared to the c-terminal (,10 amino acids) and both are always oriented towards the outside of the cell. the dispanins contain several conserved motifs ( figure 3 and 4), which are found both among eukaryotes and bacteria. the most conserved pattern is the g-d motif and the a-x(6)-a motif, both situated in the intracellular loop between the transmembrane helices that also is frequently rich in positive amino acids (k and r). the first helix is the most conserved with an alanine (a) residue and double cysteine c-c (c-f-c in the dspb family) motif whereas a glycine (g) residue is the most conserved in the second helix. another highly conserved motif, though only amongst the eukaryotes, is the single aspartic acid (d) on the n-terminus, flanking the first helix. analysis of the exon structure in the protein sequences was made for all eukaryotic dispanins except e. siliculosus where no such information was found. the eukaryotic dispanins have a conserved splice site in the intracellular loop that separates the two transmembrane helices into different exons ( figure 4 ). this site is only missing in the s. mansoni dispanin and the mouse dspa2f genes. the vertebrate proteins of the dspa and dspb subfamilies only have these two exons whereas the whole dspc subfamily and the dspd1 proteins have an additional exon that codes for their n-terminus. the dspd2 proteins that only are found in mammals seem to have lost their n-termini exon. all the classified b. floridae and s. purpuratus sequences has the corresponding splice site in the n-terminus, whereas the n. vectensis and m. brevicollis proteins has 3-6 and 7 exons respectively. we provide evidence that the four ifitm genes together with ten additional human genes, known as tusc5, tmem233, prrt2, tmem90a, dspc2, tmem90b, tmem91, ac023157, al160276 and ac068580, form a novel gene family that we call the dispanins, which refers to the 2tm membrane topology that is common to all identified members. this family is the second largest 2tm family in the human genome, superseded only by the inwardly rectifying potassium channel family that has 15 members [2] . except for the 2tm memebrane topology the dispanins are not homologous or share domains with any other 2tm proteins in the human genome and constitute a distinct gene family. we have discovered that this family is found in metazoan, the choanoflagellate m. brevicollis and the brown alga e. siliculosus, but not in other eukaryotes. surprisingly it is widely present in bacteria where it is found in several different phyla such as actinobacteridae, acidobacteria, cyanobacteria, bacteriodetes, firmicutes and proteobacteria. the highest number of bacterial dispanins is detected in actinobacteria and proteobacteria, which diverged around three billion years ago [15] . we find that dispanins in eukaryotes and bacteria have high sequence similarities and share several conserved sequence motifs (figure 4) , which is strong evidence for a common evolutionary origin and possibly a functional relationship. as the family is found in several bacterial phyla we suggest that it first emerged in bacteria to later be introduced in eukaryotes through a horizontal gene transfer event. however, we were not able to construct a stable phylogenetic tree including bacterial and eukaryotic dispanins. as the eukaryotic dispanins only is widespread in metazoa it was unexpected to find the family in the evolutionary distant brown alga e. siliculosus (figure 2 ). our sequence analysis supports that all metazoan dispanins have their origin in the common ancestor of m. brevicollis and metazoa as the choanoflagellate share a conserved splice site in the intracellular loop with nearly all metazoan dispanins (figure 4) . however, the finding of the family in e. siliculosus suggests that the family has undergone two horizontal gene transfer events. as the e. siliculosus dispanins are more similar to metazoan family members than m. brevicollis and bacteria we propose that the first horizontal gene transfer event was from bacteria to a common ancestor of choanoflagellates and metazoa followed by a second transfer between metazoa and brown alga. during the course of metazoan evolution the dispanins have expanded and diverged into four distinct subfamilies. however, it has also been lost in the basal metazoa t. adhaerens and the ecdysozoan lineage, which show that it is not essential for all metazoan life. although we were unable to create a stable phylogenetic tree that include both vertebrate and invertebrate sequences blast searches suggest that the dspc and d subfamilies are the oldest of the vertebrate subfamilies as the invertebrate sequences has higher resemblance to these two subfamilies ( figure 2) . moreover, the dspc and d subfamilies forms a separate cluster from dspa-b (figure 1) . hence, the phylogenetic analysis suggests that the dspa and b subfamilies have their origin close to the radiation of teleost, although dspb have been lost in d. rerio (figure 1 and 4) . the dspc family is found in two to three copies in all vertebrates and is the most widespread family as the blast classification suggest that it is present in two invertebrate species and e. siliculosus. in mouse, the family members are expressed predominantly in brain tissues (dspc1/tmem90a, dspc2/tmem90b) or ubiquitously (dspc3/ tmem91). dspc1 (tmem90a) has been proposed to have a role in striatial functioning and the pathophysiology of huntington's disease and is localized to the golgi apparatus [16] . the dspd family has been lost at several occasions, both in vertebrates and invertebrates ( figure 2 ). the mouse dspd1 (prrt1) gene is ubiquitously expressed with the highest expression in b-cells according to biogps [17] . however, no previous studies have been performed on the dspd subfamily. the dspb subfamily is found in tetrapoda and has three members in human and mouse. mice expression profiles from biogps shows that dspb3 (prrt2) is exclusively expressed in brain tissues and that dspb1 (tusc5) is expressed in dorsal root ganglia and adipose tissues. in agreement with this expression data, dspb1 (tusc5) has been suggested to be involved in neural regulation of adipocyte differentiation and is regulated by pparc [18, 19] . the dspa/ifitm subfamily is the most numerous and the mouse and human genes are all clustered in a consecutive manner on chromosome six and eleven, respectively. these regions share a conserved synteny (http:// cinteny.cchmc.org/) and are flanked by the athl1 and b4galnt4 genes on each side, which is strong evidence for the genes to have their origin in common evolutionary gene duplications. this is supported by the phylogenetic analysis ( figure 1 ) for dspa1 (ifitm5) and dspa3 (ac068580), which have orthologs in all tetrapoda. however, for the dspa2 group (dspa2a-d/ifitm1-3 and ac068580) the phylogeny suggests that m. musculus and h. sapiens have undergone independent expansions of the group. rather than being created by independent gene duplications in the two species, it is possible that these genes are subject to concerted evolution, where paralogous genes within a species are more conserved towards each other than towards orthologs in other species. this phenomenon is most common in tandemly repeated genes, such as the dspa2 group, and is believed to primarily be the result of recombination mechanisms [20] . interestingly, also the dspa4a-f genes of x. tropicalis seem to have undergone and independent expansion. however, the phylogeny is not strong enough to prove that the dspa4a-f genes are orthologous to the mammalian dspa2 genes (figure 1 ). the dspa/ifitm subfamily is the most well studied and is a multifunctional family of which its antiviral properties are best understood [8] . the family is expressed in many mouse tissues with the highest expression in mast cells, macrophages and osteoblasts according to biogps. we add two novel human members to this subfamily: dspa2d, which is closely related to dspa2c (ifitm3) and dspa3, which forms a distinct cluster (figure 1 ). both these genes are poorly characterized. microarray data from array express (www.ebi.ac.uk/ arrayexpress/) shows that dspa3 is upregulated by interferon after exposure of macrophages to interferon-gamma in a study (e-geod-5099) where dspa2a-c (ifitm1-3) also were induced [21] . the mouse dspa3 gene is like the other genes of this subfamily situated on chromosome seven, but is 1.5 mbp away from the dspa (ifitm) cluster where the other genes reside. hence, dspa3 could explain the mild phenotypes and be responsible for the suggested functional redundancy that was found when deleting the whole dspa (ifitm) loci [22] . the dispanin family has several conserved motifs across subfamilies that are also detected in bacteria (figure 4) . one of the most prominent is the double cysteine motif (c-c) in the first transmembrane helix. this motif has recently been shown to undergo post-translational modification through s-palmitoylation in dspa2c (ifitm3), which increases hydrophobicity [23] . further, yount and colleagues shows that the antiviral activity of dspa2c (ifitm3) is dependent on this modification, which induces clustering of the proteins. as this motif is highly conserved, it is likely that s-palmitoylation is an important regulatory mechanism also among the other subfamilies. intriguingly, this motif is also found in the bacterial dispanins even though bacterial proteins do not undergo s-palmitoylation. hence, the cysteine motif of the may have other means of structural and functional importance apart of from the s-palmitoylation. in this study, we introduce the dispanin family, of which the ifitm genes constitute a subfamily. in addition to the ifitm genes we identify 10 novel human dispanins and investigate the family's evolutionary history and suggest that the eukaryotic members are descending from bacteria through a horizontal gene transfer. thus, the expansion and diversification of dispanins in vertebrates may reflect the evolution of a larger functional repertoire, which is a supported by the distinct expression profiles figure 3 . the protein features and topology of the dispanin subfamilies. the picture shows the membrane topology and sequences features of a representative human member of each subfamily. conserved motifs and residues are shown and those which have a sequence identity of more than 90% are framed in black and those with 80-90% sequence similarity are framed in blue. predicted phosphorylation (green) and glycosylation (orange) sites are shown. doi:10.1371/journal.pone.0031961.g003 of the subfamilies. by identifying homologs to the ifitm genes and establishing the dispanins as a family together with a solid detailed and evolutionary based nomenclature for the vertebrate genes, we provide a fundament for future functional characterization these genes. the whole proteome dataset for the following eukaryotic species was included in the analysis: homo sapiens, mus musculus, gallus gallus, xenopus tropicalis, danio rerio, petromyzon marinus, drosophila melanogaster, caenorhabditis elegans, saccharomyces cerviciae, schistosoma mansoni, apis mellifera, anopheles gambiae, pediculus humanus, ixodes scapularis, daphnia pulex, oryza sativa, pristionchus pacificus, acyrthosiphon pisum, trypanosoma brucei, leishmania braziliensis and ciona instestinalis were downloaded from ensembl; strongylocentrous purpuratus was downloaded from spbase (www.spabase.org); branchiostoma floridae, nematostella vectensis, trichoplax adhaerens, phytophtera soyae, thalassiosira pseudonana, naegleria gruberi and monosiga brevicollis were downloaded from the joint genome institute; dictyostelium discoideum was downloaded from dictybase (www.dictybase.org); arabidopsis thaliana was downloaded from tair (http://www.arabidopsis.org/); entamoeba histolytica was downloaded from amoebadb (http://amoebadb.org); paramecium tetraurelia was downloaded from ncbi; tetrahymena thermophila was downloaded from uniprot; trichomonas vaginalis was downloaded from trichdb (http://trichdb.org); giardia lamblia was downloaded fromgiardiadb (http://giardiadb.org). all proteomes were searched against a local installation of the pfam database (v.23) [24] using hmmer3 [25] and the script pfam_scan.pl, which was obtained from the pfam ftp-site (ftp://ftp. sanger.ac.uk/pub/databases/pfam/tools/), with pfam's default settings. in pfam, the ifitm family is represented by a specific hidden markov model [pfam: p04505]. all the proteins that were assigned to this model in the pfam-search were considered to be homologous to the ifitm family and were therefore included for further analysis. the script pfam_scan.pl uses the homology criterion set by the pfam database, which is based on a manually curated gathering threshold for each model. the gathering threshold for pf04505 is a score of 20.6. the sequence datasets were controlled for annotated pseudogenes and transcript variants from the same gene. in the case of multiple transcript variants, the longest sequence was kept. the resulting non-redundant datasets were used for the analysis. the bacterial sequences were obtained by querying uniprot (www.uniprot.org) for the pfam id [pfam: pf04505]. thereafter, the sequence set was downloaded by browsing by taxonomy and restricting it to species with a full proteome set. to assure that no lineages were missing in the selection of proteomes the nr protein dataset from ncbi was downloaded. the nr datasets contained 15,322,545 (20-09-11) protein sequences from a wide range of organisms. the dataset was searched against the pf04505 pfam model using hmmer3 with default settings and sequences with a score above the pfam gathering threshold (20.6) were deemed as homologous to ifitm. mafft-einsi was used, with default settings, to create a multiple sequence alignment (msa) for the vertebrate protein sequences [26] . the msas were thereafter examined and refined in jalview 2.5.1 [27] , i.e. the sequences were trimmed and well conserved and aligned regions were kept, which included 82 aligned amino acid columns. phylogenetic analysis was performed with a bayesian approach implemented in mrbayes [28] . the following settings for the eukaryote proteins were adjusted: the analysis was run using a gamma shaped model for the variation of evolutionary rates across sites (rates = gamma) and the mixed option (aamodelpr = mixed) was used to estimate the best amino acid substitution model. we generated 5 000 000 trees and the markov chain monte carlo analysis reached well below a standard deviation of split frequencies of 0.01. each hundred tree was sampled from the mcmc run and the first 25% of the sampled trees were discarded (burnin = 0.25) to reassure a good sample from the posterior probability distribution. a consensus tree was built from the remaining 37 500 trees with the mrbayes sumt command using the 50% majority rule method. the sump command was used to assure that an adequate sample of the posterior probability distribution was reached during the mcmc procedure. to validate the phylogenetic inference with mrbayes a maximum likelihood method implmemented in raxml was used [29] . the combined rapid bootstrapping and search for the bestscoring ml tree option (-f a) in raxml was used to create 1000 bootstraps (-# 1000) using a gamma model of evolutionary rates and the jtt substitution model (-m protgammajtt). the jtt substitution model was identified as the most suitable model in the bayesian analysis and therefore selected for the maximum likelihood phylogeny. the consensus phylogenetic tree found with mrbayes was drawn in dendroscope 3.0 and the bootstrap support values from raxml were annotated on the corresponding nodes [30] . the phylogenetic tree was used to determine subfamilies by identifying clusters with a high posterior probability and bootstrap support. invertebrate sequences were excluded from the phylogenetic analysis as they induced highly unstable topologies together with 1 m. musculus, 2 d. rerio and 3 x. tropicalis sequences. these excluded sequences were categorized into their respective subfamilies by using a blast search towards the categorized sequences. the top five hits were examined to classify the invertebrate and excluded sequences into subfamilies. a sequence was assigned to the subfamily if four out of the five top hits are from the same subfamily. several resources were used to identify the protein sequence features of the dispanins. netphos 2.0 [31] identified potential phosphorylation sites. netnglyc 1.0 and netoglyc 3.1 [32] were used to find possible n-and o-glycosylation sites respectively. transmembrane helix prediction was made using tmhmm 2.0 [33] . motifs were found manually through jalview 2.5.1 and the server meme 4.4.0. finally, emboss:cons was used to create consensus sequences of the different dispanin families that emerged from the phylogenetic analysis and the bacterial sequences. the consensus sequences were aligned together with the m. brevicollis and the invertebrate sequences. the resulting msa was viewed and trimmed in jalview and the conserved region around the tm helices was kept. splice sites were detected in the eukaryotic dispanins by studying their annotation in the respective databases and align them to their genome using blat at the ucsc genome browser website (http://genome.ucsc.edu). table s1 this is a record of all identified dispanins together with their accession numbers, nomenclature and species belonging. (xls) trends in the exploitation of novel drug targets mapping the human membrane proteome: a majority of the human membrane proteins can be classified according to function and evolutionary origin the g-proteincoupled receptors in the human genome form five main families. phylogenetic analysis, paralogon groups, and fingerprints structural diversity of g protein-coupled receptors and significance for drug discovery the solute carrier (slc) complement of the human genome: phylogenetic classification reveals four major families transcriptional and posttranscriptional regulation of interferon-induced gene expression in human cells the ifitm proteins inhibit hiv-1 infection the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus identification of five interferon-induced cellular proteins that inhibit west nile virus and dengue virus infections distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus identification of the ifitm family as a new molecular marker in human colorectal tumors characterization of the osteoblast-specific transmembrane protein ifitm5 and analysis of ifitm5-deficient mice ifitm/mil/ fragilis family proteins ifitm1 and ifitm3 play distinct roles in mouse primordial germ cell homing and repulsion bril: a novel bone-specific modulator of mineralization timetree: a public knowledge-base of divergence times among organisms capucin: a novel striatal marker down-regulated in rodent models of huntington disease biogps: an extensible and customizable portal for querying and organizing gene annotation resources characterization of tusc5, an adipocyte gene co-expressed in peripheral neurons molecular characterization of the tumor suppressor candidate 5 gene: regulation by ppargamma and identification of tusc5 coding variants in lean and obese humans concerted evolution: molecular mechanism and biological implications transcriptional profiling of the human monocyte-to-macrophage differentiation and polarization: new molecules and patterns of gene expression normal germ line establishment in mice carrying a deletion of the ifitm/fragilis gene family cluster palmitoylome profiling reveals s-palmitoylation-dependent antiviral activity of ifitm3 the pfam protein families database a new generation of homology search tools based on probabilistic inference recent developments in the mafft multiple sequence alignment program jalview version 2-a multiple sequence alignment editor and analysis workbench mrbayes: bayesian inference of phylogenetic trees raxml-vi-hpc: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models dendroscope: an interactive viewer for large phylogenetic trees sequence and structure-based prediction of eukaryotic protein phosphorylation sites prediction, conservation analysis, and structural characterization of mammalian mucin-type oglycosylation sites a hidden markov model for predicting transmembrane helices in protein sequences key: cord-000554-p4ufea6x authors: gao, wei; sun, wenkui; qu, bingqian; cardona, carol j.; powell, kira; wegner, marta; shi, yi; xing, zheng title: distinct regulation of host responses by erk and jnk map kinases in swine macrophages infected with pandemic (h1n1) 2009 influenza virus date: 2012-01-18 journal: plos one doi: 10.1371/journal.pone.0030328 sha: doc_id: 554 cord_uid: p4ufea6x swine influenza is an acute respiratory disease in pigs caused by swine influenza virus (siv). highly virulent siv strains cause mortality of up to 10%. importantly, pigs have long been considered “mixing vessels” that generate novel influenza viruses with pandemic potential, a constant threat to public health. since its emergence in 2009 and subsequent pandemic spread, the pandemic (h1n1) 2009 (h1n1pdm) has been detected in pig farms, creating the risk of generating new reassortants and their possible infection of humans. pathogenesis in siv or h1n1pdm-infected pigs remains poorly characterized. proinflammatory and antiviral cytokine responses are considered correlated with the intensity of clinical signs, and swine macrophages are found to be indispensible in effective clearance of siv from pig lungs. in this study, we report a unique pattern of cytokine responses in swine macrophages infected with h1n1pdm. the roles of mitogen-activated protein (map) kinases in the regulation of the host responses were examined. we found that proinflammatory cytokines il-6, il-8, il-10, and tnf-α were significantly induced and their induction was erk1/2-dependent. ifn-β and ifn-inducible antiviral mx and 2′5′-oas were sharply induced, but the inductions were effectively abolished when erk1/2 was inhibited. induction of ccl5 (rantes) was completely inhibited by inhibitors of erk1/2 and jnk1/2, which appeared also to regulate fasl and tnf-α, critical for apoptosis in pig macrophages. we found that nfκb was activated in h1n1pdm-infected cells, but the activation was suppressed when erk1/2 was inhibited, indicating there is cross-talk between map kinase and nfκb responses in pig macrophages. our data suggest that map kinase may activate nfκb through the induction of rig-1, which leads to the induction of ifn-β in swine macrophages. understanding host responses and their underlying mechanisms may help identify venues for effective control of siv and assist in prevention of future influenza pandemics. swine influenza is an acute respiratory disease caused by swine influenza viruses (siv). the symptoms and signs generally include fever, sneezing, nasal rattles, and respiratory distress in pigs. pigs recover within a few days, but severe signs can develop and mortality can reach up to 10% when highly virulent strains are involved [1] or pigs are infected at young ages [2, 3] . pigs have long been considered to be the intermediate host of various subtype viruses and ''mixing vessels'' for the evolution and genesis of influenza viruses with pandemic potential because of their susceptibility to swine, avian, and human influenza viruses [4, 5, 6] . this broad susceptibility is due to the presence of both sialic acid (sa)2,3 gal-and sa2,6-gal receptors present in the respiratory epithelium. three major siv subtypes are prevalent: h1n1 (classical swine h1n1 and avian-like h1n1), h3n2 (triple reassortant h3n2 and human-like h3n2), and h1n2 [2, 7, 8, 9, 10, 11] . pigs are also susceptible to and show clinical signs when infected with pandemic (h1n1) 2009 virus (referred to hereafter as h1n1pdm) [12] , which emerged in april 2009 in north america [13] , arising at least in part from contemporaneous siv. to date h1n1pdm has been found in a few swine farms [12, 14, 15] , which further demonstrates a two-way process of both gene and virus trafficking between humans and pigs. though h1n1pdm has remained antigenically and genetically stable in humans since its emergence, a novel reassortant siv containing a h1n1pdm-like na and seven other genes from triple-reassortant h1n2 and european ''avian-like'' h1n1 viruses was identified in early 2010 [16] , and that same year h1n1pdm was shown to be evolving genetically at a faster pace in pigs than it was in humans [12, 15, 17] . effective control of circulating influenza viruses in swine populations is key to reducing consequent genesis of novel pandemic strains that threaten the health of both humans and animals. studies have been conducted to identify proinflammatory cytokines including tnf-a, il-6, il-12, and ifn-a or ifn-c, which are upregulated in lung or bronchoalveolar secretions in siv-infected pigs [18, 19, 20, 21] and may be correlated with clinical manifestations. in an alveoli macrophage-depleted pig model, macrophages appeared to be indispensible to effective clearance of siv from lungs. a higher frequency of cytotoxic t, cd t, and treg cells were also detected in infected pig lungs [18] , which together with the induction of cytokines, contribute to pathogenesis of influenza infection in pigs. exploring the mechanism of regulation of host responses is crucial for understanding the pathogenesis of siv and for controlling swine influenza in pigs. macrophages residing beneath the respiratory epithelium and surrounding alveoli are part of the first line defenses against influenza viruses. during influenza viral replication in bronchial epithelial cells, macrophages are one of the earliest targets to be infected. together with dendritic cells, macrophages coordinate innate immune responses, which subsequently lead to adaptive immunity by initiating antigen presentation and lymphocyte activation. macrophages are indispensable in alveolar host defense and controlling influenza virus in pulmonary organs in pigs [22] . while protective in launching host antiviral responses and restricting virus spread, induced proinflammatory cytokines and chemokines are also the cause of pathogenicity for the host and may lead to acute respiratory failure (arf), a major cause of death in highly pathogenic h5n1 or h1n1pdm-infected humans [23] . needless to say, the roles of macrophages are critical to pathogenicity as well as host protection in siv-infected pigs. however, little is known about the mechanisms of how host responses are regulated in pigs or their macrophages. considering the critical role macrophages play in siv infections, and the threat that h1n1pdm could further evolve higher virulence in pigs and subsequently infect humans, we were interested in profiling host responses of swine macrophages to h1n1pdm, and more importantly, in exploring the underlying mechanism of host response regulation including antiviral, proinflammatory responses, and apoptosis in pigs. in this report, we will demonstrate that swine macrophages are susceptible to infection by h1n1pdm. we will show a unique pattern of proinflammatory cytokine responses to the infection, which are distinctly regulated by swine mitogen-activated protein (map) kinases. we have also observed cross-talk between map kinase and nfkb pathways, and our data indicate that map kinase erk1/2 and jnk1/2 may impact the activation of nfkb through the induction of rig-1, leading to ifn-b induction in h1n1pdm-infected swine macrophages. the 3d/4 cells used in our study are a spontaneouslytransformed line of swine macrophages purchased from atcc (manassas, va) and grown in rpmi 1640 medium (invitrogen) containing 10% fetal bovine serum (fbs). mouse anti-erk and anti-jnk antibodies as well as rabbit anti-phospho erk and antiphospho jnk antibodies (cell signaling), anti-cytochrome c, anti-influenza ns1, and alkaline phosphatase (ap)-conjugated anti-rabbit and anti-mouse igg antibodies (santa cruz biotechnology) were obtained from their respective providers. anticleaved caspase antibody was obtained from cell signaling technology, and anti-bak antibody was obtained from emd chemicals. the chemicals purchased from emd chemicals also included inhibitors for map kinases, u0126 (erk1/2), sb203580 (p38), and insolution jnk inhibitor ii (jnk1/2), and the inhibitors for nfkb and ikk (6-amino-4-(4-phenoxyphenylethylamino) quinazoline (cat. 481406) and wedelolactone (cat. 401474), respectively). a/nanjing/108/2009 (h1n1), a pandemic (h1n1) 2009 virus, was isolated from a swab sample of an outpatient febrile child at the nanjing children's hospital during the pandemic in 2009, nanjing, china. the sampling procedure was performed in accordance with the rules set by the institutional review board at the hospital. the eight genomic segments of this virus have been fully sequenced and the raw data are deposited at genbank under accession numbers jq173100 through jq173107. the virus was grown in 9-day-old embryonating chicken eggs; virus allantoic fluid (vaf) was harvested 48 hrs after inoculation, then titrated with standard haemagglutination tests (ha) and plaque assays in mdck cells for ha and infectious viral titers, respectively [24] . for viral infection, the 3d/4 cells were trypsinized, resuspended in rpmi 1640 medium containing 10% fbs, and plated on 6-cm tissue culture plates at 5610 6 cells per plate 12 hrs before infection. the cells were infected with h1n1pdm inocula in vaf at a multiplicity of infection (moi) of 1. after 1 hr of adsorption, the virus inocula were discarded and 3 ml of serum-free rpmi 1640 medium containing tpck-trypsin (1 mg/ml, sigma) was added. the cells were incubated at 37uc and 5% co 2 for various time points before cell lysates or total rna extraction were prepared. mrna transcript levels of ifn-b, il-1b, il-6, il-8, ccr5, ip-10, tnf-a, fasl, trail, mx, 2959-oas, retinoic acid-inducible gene i (rig-1), melanoma differentiation-associated antigen 5 (mda-5), and glyceraldehyde-3-phosphate dehydrogenase (gapdh) genes were analyzed by a two-step real-time rt-pcr assay as described previously [25] . 1 mg of total rna, prepared from the cells using the rneasy kit (qiagen), was reverse transcribed with the quantitect reverse transcription kit (qiagen) following the manufacturer's instructions. the sequences of primers used in the study are listed in table 1 . the rt reaction was carried out with the rna after treatment with dnase i at 42uc for 2 min. real-time pcr was conducted with 1 ml of cdna in a total volume of 25 ml with the iq sybr green supermix (bio-rad) following the manufacturer's instructions. relative expression values were normalized using an internal gapdh control. the fold change of relative gene expression levels was calculated following the formula: 2 (dct of gene2dct of gapdh) [25, 26] . for each reaction, melting curves were analyzed to determine the specificity of each amplicon. to determine the viral rna level, the total rna from infected cells was reverse transcribed and cdna used for taqman-based real-time pcr (applied biosystems) to measure viral m gene transcripts in the infected cells [27] . cell lysates were prepared by lysing uninfected and infected 3d/4 cells in 1% np-40 lysis buffer containing 1 mm pmsf, 1% aprotinin, 20 mg leupeptin ul 21 and 1 mm sodium vanadate (sigma) as described previously [10] . cell lystes were clarified by low speed centrifugation (1000 g, 5 min at 4uc) and subjected to sds-page (10 to 12%). proteins were transferred to the immuno-blot pvdf membrane (bio-rad), and western blot analysis was performed following standard protocols [28] using rabbit or mouse anti-map kinase or phosphor-map kinase antibodies (1:500) in tbst containing 5% fat-free milk powder for 90 mins incubation at rt. after washes, incubation with apconjugated anti-rabbit or anti-mouse igg antibody for another 90 mins followed. after incubation and thorough washes, bcip/ nbt reagents (sigma) were used for the development of colorimetric signals on the membrane. the membrane was also blotted with a monoclonal anti-actin antibody (santa cruz biotechnology) for input control. for statistical analysis, a two-tailed student's t-test was used to evaluate realtime rt-pcr data. an x 2 analysis was used to evaluate significant differences of the data in two and more groups. the 0.05 level of probability (p,0.05) was considered statistically significant. to examine the susceptibility of pig macrophages to h1n1pdm originating from a human host, we infected 3d/4 cells with the a/ nanjing/108/2009 (h1n1). typical cytopathic effect (cpe) appeared 16 hrs post infection and the cell monolayer was destroyed 32 hrs post infection (fig. 1a) . this result demonstrated that h1n1pdm retains the ability to infect and replicate in swine macrophages, and can reach 1.8610 4 pfu/ml as shown in a replicative curve (fig. 1b) . apoptosis occurred and proceeded through the course of the infection, as we observed cleaved/ activated caspase-9 as well as the emergence of downstream executioner caspases-6, -7, and -3, which eventually destroyed the infected swine macrophages (fig. 1c) . clearly, cytochrome c was released into the cytosol (fig. 1e) , which activated mitochondriamediated intrinsic apoptosis as early as 3 hrs post infection. bak, a pro-apoptotic bcl-2 family member, was upregulated as detected in the infected cells (fig. 1d) , and may be involved in the release of cytochrome c from mitochondria in swine macrophages. to elucidate the pathogenesis of h1n1pdm in pigs, we examined the pattern of cytokine responses in ph1n1-infected swine macrophages. total rna from infected and uninfected 3d/ 4 cells collected at different time points post infection (p.i.) were prepared and used for realtime rt-pcr analyses with specific primers to swine cytokines. we found that the levels of proinflammatory cytokines il-6 and il-8 were upregulated up to 51-and 38-fold at 16 hrs, respectively, and the level of il-8 continued to rise up to 142-fold at 36 hrs p.i.. however, the level of il-1b remained unchanged throughout the infection ( fig. 2a) , indicating that il-6 and il-8, as well as tnf-a (fig. 2b ) as described later, were the main proinflammatory cytokines upregulated. we observed a robust induction of antiviral ifn-b, which rose up to 620-and 5,100-fold at 16 and 36 hrs p.i., respectively (fig. 2c ). ifn-inducible antiviral proteins mx and 295.-oas were induced accordingly up to 910-and 12,510-fold, respectively, at 36 hrs p.i. (fig. 2c) . tnf family members were also induced in response to h1n1pdm infection, which may be attributable to cell death. we found that in pig macrophages the levels of fasl and tnf-a remained undetectable, while tnf-related apoptosis-inducing ligand (trail) seemed to be most abundant before infection, based on ct values from realtime rt-pcr (data not shown). fasl and tnf-a were induced most robustly, but trail was only mildly induced in response to infection (fig. 2b) . among the induced, the level of tnf-a, critical in both cell death and inflammation, was sharply upregulated up to 14-and 162-fold, and fasl up to 43-and 22-fold at 16 and 36 hrs p.i., respectively. fasl and tnf-a may play a major role in h1n1pdm-triggered extrinsic apoptosis. to understand the mechanism of proinflammatory cytokine and tnf family ligand induction in h1n1pdm-infected swine macrophages, we investigated how map kinases were activated and whether their signaling pathways were involved in the regulation of various cytokines and tnf family ligands in pig immune cells. 3d/4 cells were infected with h1n1pdm, and cell lysates were prepared at various time points for sds-page and western blot analyses with specific anti-erk1/2 and anti-jnk1/2 antibodies. activated forms of erk and jnk (phospho-erk1/2 and phosphor-jnk1/2) were detected by anti-phospho-erk1/2 and anti-phospho-jnk antibodies. as shown in figure 3a , erk1/2 was basally phosphorylated at a low level before infection, but further phosphorylated between 9 and 18 hrs and thereafter p.i.. phosphorylation and activation of jnk1/2 appeared at 9 hrs and increased to the peak around 18 hrs p.i. (fig. 3b) . although both erk1/2 and jnk1/2 were activated in response to h1n1pdm infection in swine macrophages, erk1/2 remained active at basal level even before infection, so did jnk1/2 as shown in some of our experiments (fig. 4b) . however, our data showed that basal level phosphorylation of both erk1/2 and jnk1/2 remained unchanged in uninfected 3d/4 cells through the period of our infection. in addition to erk1/2 and jnk1/2, we have also observed the phosphorylation and activation of p38 map kinase in h1n1pdm-infected cells (data not shown). to evaluate the role of map kinases in the regulation of proinflammatory cytokine responses in h1n1pdm-infected swine macrophages, we pre-treated 3d/4 cells with specific inhibitors for erk1/2, p38, and jnk1/2 1 hr prior to infection. we then infected the cells with the virus and observed how infectioninduced activation of map kinases was affected by inhibition of the respective map kinases. as shown in figure 4a , 3d/4 cells were pre-treated with inhibitors of erk1/2 (u0126), p38 (sb230058), and jnk1/2 (jnk insolution), at concentrations of 10 mm, 5 mm, and 50 mm, respectively. while the phosphorylation of erk1/2 was unaffected by treatment with the p38 and jnk inhibitors, it was completely abolished at both 18 and 30 hrs p.i. (lines 4-5) by the erk1/2 inhibitor u0126 (fig. 4a) . we noted that the basal level phosphorylation of erk1/2 diminished in the presence of u0126. on the other hand, in light of the p38 and jnk inhibition with their specific inhibitors, the phosphorylation of erk1/2 appeared to be enhanced (fig. 4a, lines 6-9 ), indicating that a compensatory mechanism may exist among map kinases. we observed a similar response in which a complete suppression of jnk1/2 phosphorylation was observed (lines 8-9) when the cells were pre-treated with the jnk1/2 inhibitor (fig. 4b) . however, the phosphorylation of jnk1/2 was not suppressed at all by the inhibitors of erk1/2 and p38. we noted that there were double bands for jnk1, and a lower band of jnk1 usually appeared at a later stage of infection (30 hrs p.i.). this band was detected mainly by anti-jnk1/2, but not by anti-phospho-jnk1/ 2, indicating that jnk activation was transient and dephosphorylation of jnk occurred at later stages of infection, probably by an uncharacterized map kinase phosphatase (mkp) present in pigs. a basal level phosphorylation of nfkb was also observed in 3d/4 pig macrophages, and was further enhanced upon h1n1pdm infection, indicating that the nfkb pathway was activated as well in infected pig macrophages (fig. 4c) . when the cells were pre-treated with specific inhibitors of nfkb (10 nm) or ikk (10 mm), the phosphorylation/activation of nfkb was effectively decreased or diminished. map kinases and nfkb pathways were activated in h1n1pdm infected pig macrophages, which could be reversed or inhibited by their specific inhibitors. we used these inhibitors to study the regulation of host responses, which may be controlled by these pathways. to determine how cytokine responses are regulated by individual map kinases, we pre-treated the cells with erk1/2 and jnk1/2 inhibitors, respectively, and measured the induction of the cytokines after infection with realtime rt-pcr. we observed that il-1b was barely detected and not induced during h1n1pdm infection. interestingly, we noticed that il-1b was upregulated in the presence of the jnk inhibitor, although no change was observed after the treatment by the erk inhibitor, indicating that il-1b could have been induced in swine macrophages infected with h1n1pdm, but was virtually suppressed by jnk1/2 (fig. 5a) . we observed that the induction of il-6, il-8, and il-10 was completely suppressed in the presence of the erk1/2 inhibitor, which indicates that il-6, il-8, and il-10 inductions are all dependent on the erk signaling pathway (fig. 5b-d) . it is interesting to note that jnk1/2 may play different roles in the induction of il-6, il-8, and il-10 based on their responses in the presence of the jnk inhibitor. jnk1/2 may have moderate effects in the induction of il-6 ( fig. 5b ), but may be not relevant at all to the induction of either il-8 or il-10 ( fig. 5c-d) . we also noted that ccl5 (rantes) was strongly regulated by erk1/2 and jnk1/2 in swine immune cells. as shown in figure 5e , induction of ccl5 was efficiently blocked in the presence of either erk1/2 or jnk1/2 inhibitors, indicating that ccl5 is induced by h1n1pdm infection through erk and jnk signaling pathways. as for antiviral ifn-b, which was robustly induced with h1n1pdm infection in swine macrophages, erk1/2 appeared to be essential since the induction of its mrna transcripts was virtually abolished in the presence of the erk inhibitor (fig. 6a ). jnk1/2 may also play a role in ifn-b induction because of its significant decrease at the earlier stage of infection (16 hrs p.i.) when 3d/4 cells were pre-treated with the jnk inhibitor. however, erk1/2 seemed to be the primary pathway in the ifn-b induction in swine macrophages. the distinct contributions to the induction of ifn-b by erk1/2 and jnk1/2 were also reflected in the decreased mrna transcript levels of ifn-inducible antiviral proteins, mx and 2959-oas, in the presence of erk and jnk inhibitors, respectively (fig. 6b-c) , which is in accordance with the suppression of the ifn-b induction by these same compounds in the infected cells. both mx and 2959-oas were suppressed significantly by the erk inhibitor, but only the in contrast to the abundance of trail transcripts, mrna levels of fasl and tnf-a were barely detectable by realtime rt-pcr in swine macrophages (data not shown). however, both fasl and tnf-a were induced profoundly in response to ph1n1 infection ( fig. 2b and 7a-c) , while the change of trail was mild. by using inhibitors, we concluded that the induction of fasl and tnf-a are mainly controlled by the erk1/2 and jnk1/2 pathways in pig macrophages. the nfkb pathway could also be critical in host responses, as has been shown in humans and mice infected with influenza a virus. nfkb can be phosphorylated and activated in swine macrophages in response to h1n1pdm infection ( fig. 8a and 4c ), albeit at a later stage. interestingly, when the cells were pre-treated with erk1/2 or jnk1/2 inhibitors, the phosphorylation of nfkb was also suppressed. however, when the cells were pre-treated with the p38 inhibitor, nfkb phosphorylation decreased much less than with erk1/2 or jnk1/2 inhibitors (fig. 8a) . this result suggests that a cross-talk may exist between map kinase and nfkb pathways, and that among the map kinases, erk1/2 and jnk1/2 are mainly involved. figure 5 . regulation of swine proinflammatory cytokine gene transcripts by map kinases. 3d/4 cells were pretreated with u0126 and insolution jnk inhibitor, which are inhibitors of erk1/2 and jnk1/2, respectively, 1 hr before h1n1pdm infection. total rna was prepared at 24 and 36 hrs post infection for reverse transcription. cdna was used for realtime pcr with specific primers to measure fold changes of cytokine transcripts at different time points. each assay was repeated at least twice. a-e. regulation of il-1b, il-6, il-8, il-10, and ccl5, respectively, by erk1/2 and jnk1/ 2 inhibitors. data show mean fold changes plus standard deviation of two or three independent assays. *p,0.05, student's t-test. doi:10.1371/journal.pone.0030328.g005 we next examined the expression levels of rig-1 and mda-5, the rlr family members and cytosolic sensors for rna viruses. we found that rig-1 in particular was significantly induced up to 1280-fold, while mda-5 was also upregulated up to 42-fold in infected pig macrophages (fig. 8b) . we further examined the induction of rig-1 and mda-5 and their relevance to map kinases. to do this, we pre-treated the cells with inhibitors of map kinases. as shown in figure 8c , the induction of rig-1 was completely abolished by the inhibition of erk1/2 or jnk1/2 inhibitors, and to a much lesser extent, by the p38 inhibitor, suggesting that the induction of rig-1 was dependent on erk1/ 2 and jnk1/2, but not as much on p38. this differentially regulated pattern of rig-1 induction by erk1/2, p38, and jnk1/2 was similar to the suppression of nfkb phosphorylation/activation by map kinases (fig. 8a) , suggesting that the induction of rig-1 was associated with erk1/2 or jnk1/2 activation, but to a much lesser extent with p38. since nfkb could be downstream activated by rig-1/ips-1 [29, 30] , we postulate that erk1/2 or jnk1/2 may activate nfkb through the activation of rig-1/ips-1 during h1n1pdm infection in pig macrophages. a similar, albeit less dramatic, induction and suppression of mda-5 expression was also observed (fig. 8d) , which indicated that mda-5 might also be an intermediate adaptor bridging the map kinases erk1/2 and jnk1/2 to the nfkb pathway activation. the cells were pretreated with u0126, sb230058, and insolution jnk inhibitor, 1 hr before h1n1pdm infection. total rna was prepared from infected cells for reverse transcription. cdna was used for realtime pcr with rig-1 and mda-5 primers to measure fold changes of rig-1 and mda-5 transcripts in treated swine macrophages. each assay was repeated at least twice. data show mean fold change plus standard deviation of two or three independent assays. *p,0.05, student's t-test. doi:10.1371/journal.pone.0030328.g008 in the present study, we have demonstrated a pattern of host responses in swine macrophages to h1n1pdm infection. strong proinflammtory and antiviral cytokine responses including il-6, il-8, tnf-a, as well as ifn-b, were observed. in contrast, il-1b was not induced, and was barely detectable in pig macrophages. this pattern differs from that in bronchoalveolar secretions of siv-infected pigs in which il-1b was induced but il-8 was not [19, 20, 21, 22, 25] . the different cell types involved (macrophages and epithelial cells) may account for the difference. it has previously been reported that in human immune cells and patients a weak innate immune response, evidenced by a poor induction of proinflammatory and antiviral cytokines including ifn-b and tnf-a, has been observed in human monocyte-derived dcs and macrophages infected with h1n1pdm, compared to seasonal h1n1 infection [31] . highly pathogenic h5n1 viral infection in human macrophages induced higher expression of il-6 and ccl5 (rantes) than ph1n1 [32] , which may explain generally mild clinical disease among h1n1pdm-infected patients. in human macrophages, similar to our findings, il-1b was not detected. map kinase signaling pathways and their roles in the regulation of cytokines and viral replications have not been characterized in influenza-infected pig immune cells. in this study, we found that erk1/2 and jnk1/2 could both be activated in swine macrophages. we noted that erk1/2 was phosphorylated and active at a low level constitutively, which may be important for the rapid physiological responses required upon infection. to elucidate the mechanism that regulates swine host responses, we used specific inhibitors of map kinases to pre-treat macrophages before infection. we determined that the induction of ifn-b, il-6, il-8, and il-10 were regulated by erk1/2, while jnk1/2 may only play a minor or no role in the regulation of these cytokines. as described earlier, il-1b was not induced in response to the ph1n1 infection, which could be explained by our data indicating that its induction was in fact efficiently suppressed by jnk1/2 in swine macrophages. this may be the first time that jnk1/2 inhibitory effects on the induction of proinflammatory cytokines have been demonstrated. previous studies found that ifn induction was dependent on the jnk1/2 signaling pathway in epithelial cells infected with influenza virus infection [33] . however, our data clearly demonstrate that erk1/2 plays a major role in the regulation of ifn-b in pig macrophages, which may indicate that the regulation of ifn differs in different cell types. we noted that basal level activities of both erk1/2 and jnk1/2 were constitutively present in non-infected 3d/4 cells, which may be important in the induction of proinflammatory and antiviral cytokines at the early stages of infection. our data indicate that the induction of il-6, il-8, il-10, ccl-5, as well as ifn-b, were apparent at the earliest stages of viral infection even before erk1/2 was further activated. we realized that a transformed monocytic cell line, instead of primary cells, was used in the study, which may compromise the significance of our data. basal level phosphorylation of both erk1/ 2 and jnk1/2, which may affect certain cytokine production, would be minimal in primary monocytes. however, specific inhibitors used in the study completely wiped out phosphorylation of both erk1/2 and jnk1/2 (fig. 4) . the effect of map kinase phosphorylation and activation on the regulation of affected cytokines as observed in our study with the inhibitors is, therefore, valid, even though the cells were not primary cultures. macrophages appear to die inevitably of apoptosis when infected with influenza virus [26] . the fas-mediated extrinsic apoptotic pathway is apparently triggered by tnf family ligands. while both fasl and tnf-a were induced vigorously upon the viral infection, induction of trail was rather mild in h1n1pdminfected swine macrophages. we knew previously that fasl and tnf-a were barely detectable, while the level of trail remained high prior to the infection based on our realtime rt-pcr data (ct) (xing et al., unpublished data). we can therefore presume that h1n1pdm-induced apoptosis may be mainly attributed to fasl and tnf-a, while pig macrophages could be resistant to trail, since the cells remained intact despite the presence of a high level of trail before infection. furthermore, we were also able to determine that both erk1/2 and jnk1/2 were involved in the induction of fasl, tnf-a, and trail. fasl is also regulated by erk1 in chicken macrophages infected with an h9n2 avian influenza virus [34] . both toll-like receptors (tlr) and rna helicases, such as rig-1 and mda-5, are critical to antiviral innate immunity [35, 36] . as a cytosolic sensor, rig-1 binds to dsrna and viral ssrna that contain a 59-triphosphate not present in host rna, and then is recruited to mitochondrial protein ips via the card domain, leading to activation of nfkb, irf-3/-7, and induction of ifn [37, 38, 39] . rig-1 can be induced by viral infection [40] . in this study, we observed a robust induction of rig-1 and mda-5 in h1n1pdm-infected swine macrophages, which appeared to be suppressed completely by inhibitors of erk1/2 or jnk1/2, but to be a much lesser extent, by the inhibitor of p38. this indicates that the induction of rig-1 or mda-5 depends on the activation of erk1/2 and jnk1/2 in pig macrophages. we postulate a mechanism, therefore, that the cross-talk between map kinase and nfkb pathways is through the regulation of rig-1 and maybe mda-5, and that erk1/2 controls the activation of nfkb, leading to the induction of ifn in swine macrophages. characterization of an influenza a virus isolated from pigs during an outbreak of respiratory disease in swine and 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influenza a (h1n1) flu replication and plaque assay of influenza virus in an established line of canine kidney cells immune-related gene expression in response to h11n9 low pathogenic avian influenza virus infection in chicken and pekin duck peripheral blood mononuclear cells modulation of the immune responses in chickens by low-pathogenicity avian influenza virus h9n2 genetic and phenotypic characterization of a low-pathogenicity avian influenza h11n9 virus host immune and apoptotic responses to avian influenza virus h9n2 in human tracheobronchial epithelial cells rna recognition and signal transduction by rig-i-like receptors rig-i-like receptors: sensing and responding to rna virus infection pandemic h1n1 2009 influenza a virus induces weak cytokine responses in human macrophages and dendritic cells and is highly sensitive to the antiviral actions of interferons cytokine profiles induced by the novel swine-origin influenza a/h1n1 virus: implications for treatment strategies influenza virus-induced ap-1-dependent gene expression requires activation of the jnk signaling pathway roles of the erk mapk in the regulation of proinflammatory and apoptotic responses in chicken macrophages infected with h9n2 avian influenza virus toll-like receptors and rna helicases: two parallel ways to trigger antiviral responses intracellular pattern recognition receptors in the host response 59-triphosphate rna is the ligand for rig-i recognition of rna virus by rig-i results in activation of card9 and inflammasome signaling for interleukin 1 beta production rig-imediated antiviral responses to single-stranded rna bearing 59-phosphates an rna helicase, rhiv -1, induced by porcine reproductive and respiratory syndrome virus (prrsv) is mapped on porcine chromosome 10q13 we thank members of xing laboratory for their technical assistance in the study and helpful discussions for manuscript preparation. we appreciate excellent editing work performed by sandy shanks. key: cord-000501-qz68gtd4 authors: greatorex, jane s.; digard, paul; curran, martin d.; moynihan, robert; wensley, harrison; wreghitt, tim; varsani, harsha; garcia, fayna; enstone, joanne; nguyen-van-tam, jonathan s. title: survival of influenza a(h1n1) on materials found in households: implications for infection control date: 2011-11-22 journal: plos one doi: 10.1371/journal.pone.0027932 sha: doc_id: 501 cord_uid: qz68gtd4 background: the majority of influenza transmission occurs in homes, schools and workplaces, where many frequently touched communal items are situated. however the importance of transmission via fomites is unclear since few data exist on the survival of virus on commonly touched surfaces. we therefore measured the viability over time of two h1n1 influenza strains applied to a variety of materials commonly found in households and workplaces. methodology and principal findings: influenza a/puertorico/8/34 (pr8) or a/cambridge/aho4/2009 (pandemic h1n1) viruses were inoculated onto a wide range of surfaces used in home and work environments, then sampled at set times following incubation at stabilised temperature and humidity. virus genome was measured by rt-pcr; plaque assay (for pr8) or fluorescent focus formation (for pandemic h1n1) was used to assess the survival of viable virus. conclusions/significance: the genome of either virus could be detected on most surfaces 24 h after application with relatively little drop in copy number, with the exception of unsealed wood surfaces. in contrast, virus viability dropped much more rapidly. live virus was recovered from most surfaces tested four hours after application and from some non-porous materials after nine hours, but had fallen below the level of detection from all surfaces at 24 h. we conclude that influenza a transmission via fomites is possible but unlikely to occur for long periods after surface contamination (unless re-inoculation occurs). in situations involving a high probability of influenza transmission, our data suggest a hierarchy of priorities for surface decontamination in the multi-surface environments of home and hospitals. influenza transmission is well documented in households and other residential settings [1] [2] [3] [4] . yet the underlying mechanisms of transmission remain poorly understood and hotly debated [5, 6] . although transmission by aerosols (particles typically ,5 mm in diameter), larger droplets and contact transmission (direct and via fomites) probably all play some role, the relative importance of each is uncertain, which has led to difficulties regarding the provision of evidence-based infection control advice for both pandemic and seasonal influenza [7] . if virus can survive for meaningful periods on surfaces and objects, or alternatively, if surfaces are frequently re-inoculated (e.g. by toddlers), then it is feasible that transmission via fomites might occur. the potential for transmission of influenza by indirect contact (i.e. via fomites) is linked to the ability of virus to survive in transmissible titres on commonly touched surfaces; however few data exist on this subject. parker et al (1944) demonstrated improved survival of influenza viruses in the presence of human mucus [8] ; and in 1962, buckland demonstrated experimentally that influenza virus was inactivated relatively quickly on glass, probably through desiccation [9] . in 1982, widely cited work by bean et al showed that both influenza a and b, directly applied to stainless steel surfaces or hard plastic, could survive for 24-48 hours, and be transferred, from there to hands, for 24 hours; survival was much shorter on porous materials such as paper and cotton (8-12 hours) , with transferability to hands for only 15 minutes [10] . in contrast, thomas et al, recently demonstrated survival of human seasonal a (h1n1) and a (h3n2) on swiss banknotes for up to three days, increasing to up to eight days when applied with nasopharyngeal secretions from children (17 days if applied at very high concentration). although viable virus was recovered at each of these time points, it was noted that virus load declined sharply after the first few days; no other materials were tested [11] . other studies have detected influenza virus on fomites in homes and health and childcare facilities, using rt-pcr to establish the presence of the viral genome [12] [13] [14] . however, data obtained using this technique (even quantitatively) do not distinguish adequately between viable and non-viable virus and are therefore problematic to interpret in the context of practical infection control guidance. in another recent study, virus was detected by pcr on commonly touched household surfaces, but only one sample proved culture positive [15] . however, the time from deposition to recovery was not known, nor the extent of any cleaning undertaken. we evaluate the survival of influenza a (h1n1) viruses deliberately applied to a range of commonly touched household and workplace surfaces, using rt-pcr for genome detection and culture methods to determine viability. we conclude that rt-pcr is only useful to demonstrate the absence of virus and that on most surfaces, virus viability drops rapidly. nevertheless, on certain non-porous surfaces, viable virus persists for several hours, rendering fomite transmission possible without re-inoculation. to test the surface survival of influenza virus, we used a variety of materials commonly encountered in the home and workplace, including a hospital setting ( table 1) ; choice of surfaces to be tested was discussed with the department of health, england to ensure relevance to public health policy. these included fibrous materials such as the ubiquitous j-clothh (associated brands) widely used for cleaning, a silver impregnated fabric with known bacteriostatic properties (toray textiles europe ltd.) of the type sometimes encountered in hospital staff clothing to combat nosocomial bacterial infections, as well as fabric from a child's soft toy. the latter fabric was made of non-absorbent polyester and, although a porous item overall, individual fibres might perform as a nonporous surface. a variety of non-porous plastic surfaces representing objects highly likely to be touched by multiple individuals such as light switch, telephone and keyboard plastics were also tested, as well as porous and non-porous 'background' materials such as various wood surfaces, glass, perspex/plexiglass (poly (methyl methacrylate) -a thermoplastic often used as a light or shatterresistant alternative to glass) and metals. as a control surface, we used standard laboratory polystyrene culture dishes. as viruses, we used two human h1n1 strains: the laboratory adapted a/puerto rico/8/34 (pr8) strain because of ready availability and robust, convenient assay systems with a wide dynamic range, and an isolate of the current 2009 pandemic virus a/cambridge/ah04/ 2009 (ah04), as a low passage history representative of a virus likely to be encountered in the current environment. the source and disinfection method used to clean the various surfaces before testing are listed in table 1 . human influenza a virus pr8 (cambridge lineage) was grown in embryonated hens' eggs and harvested at a titre of 9610 8 pfu/ ml. for inoculation of the surfaces, the virus was diluted 1:10 in 1% bsa and serum free media (dulbecco modified eagles medium, dmem, gibco, uk). this represented a viral titre approximating 1.5610 8 tcid50/ml, just above the upper end of titres reported for human shedding [10, 11] . preliminary experiments established that virus survival was improved by the addition of extra protein to the suspension. we tested 0.5% or 1% bsa as well as four preparations of artificial mucus produced from pig stomach mucosa (nbs biologicals), pig stomach mucin types ii or iii or bovine sub maxillary glands mucin, type i-s (all from sigma aldrich). 1% bsa had the largest effect on titre and duration of survival, followed by the bovine mucin (data not shown). in the interests of simplicity and reproducibility, 1% bsa was therefore used in all subsequent experiments. to test a 2009 pandemic influenza a (h1n1) virus strain on selected surfaces, a clinical isolate designated influenza a/cambridge/aho4/2009 (ah04) was passaged once in mdck cells and then grown in caco-2 cells (colorectal adenocarcinoma cells, atcc htb-37 tm ). the virus is a recent isolate from an received sterile in packaging from manufacturer. fumigation in a cliii room using a laycock fumigator (tolbest ltd). doi:10.1371/journal.pone.0027932.t001 immunocompetent patient who was hospitalised briefly at the start of their illness, but recovered. this virus does not form discrete plaques in mdck cells and could therefore not be titred by this method. instead, virus stocks were quantified by qpcr for segment 7 [12] . although this method scores viable and non-viable virus particles alike, preparations of wild type influenza a viruses generally have similar particle:pfu ratios and quantitative comparison of rt-pcr and other titration methods have shown good agreement [13, 14] . the ah04 stock contained 6.5610 8 genome copies/ml and was used at a 1:10 dilution as for pr8. for comparison, the pr8 stock had a genome titre of 1.6610 11 genome copies/ml. mouse monoclonal aa5h (abcam) was used to detect influenza np by immunofluorescence. surfaces were cut into 2 cm 2 pieces and sterilised by a variety of means depending on the surface to be tested (e.g. autoclaving, fumigation etc). sterile surfaces were glued into sterile 6-well tissue culture dishes using cyanoacrylate adhesive (henkel, uk). preliminary experiments (data not shown) demonstrated that dried adhesive alone was non-inhibitory to influenza virus. under the same conditions of temperature and humidity (ranges 17-21uc and 23-24% respectively), 10 ml volumes of virus were applied to six samples of each surface at the same time. sampling was conducted immediately -time zero -to demonstrate recoverability. a cotton swab was moistened by dipping in 3 ml of virus transport medium (vtm, remel, uk) and then wiped carefully in 6 different directions for 1 minute across the top of the surface. keeping everything on ice, the swab was placed into the tube containing the residual (3 ml) volume of vtm and vortexed for 1 minute. after this, the sample was split directly into 6 eppendorf tubes and stored on dry ice prior to freezing at 270uc. the remaining samples in the plate were kept in a plastic, lidded box at constant temperature and humidity. at 4, 9, 24, 48 and 72 hrs, further samples were taken and stored. after initial experiments it was clear that the virus did not survive in detectable amounts for more than 24 hrs, therefore for the majority of the experiments only the first 4 time points (0, 4, 9, and 24 hrs) were taken. initial experiments with pr8 virus also showed that loss of virus on the swab was not a major factor, with recovery of virus at time zero from polypropylene surfaces approaching 50% of initial titre (data not shown). the qrt-pcr assay used has been described previously [12] . in brief, primers and probes to the matrix gene of influenza a were used to detect the presence of the virus on the surfaces. samples from all time points were stored and then extracted. virus genome was amplified to check that the quantity of virus deposited on the different surfaces was consistent and to determine whether any of the surfaces affected the genome over time. plaque assays were performed as previously described in mdck cells using avicell overlays [15, 16] , in duplicate or where possible in triplicate. to detect ah04 virus by fluorescent focus assay, infectious material from swabs was first allowed to amplify by inoculation into 1610 6 mdck cells and incubation for 48 h. supernatant virus was then diluted 1:10 in serum free dmem and 250 ml used to inoculate 1.5610 5 mdck cells in a 24 well tissue culture plate. after virus absorption, the cells were overlaid with 1 ml serum free dmem media containing 1 mg/ml worthington's trypsin and 0.14% bsa and incubated overnight at 37uc. the following day they were fixed with 4% formaldehyde in pbs, permeabilised by the addition of 0.2% triton 6100 in pbs for 5 minutes at rt and fluorescently stained with anti-np monoclonal antibody and counterstained for dna with 4,6-diamino-2-phenylindole (dapi) as previously described [17] . cells were examined blind by two people and scored semi quantitatively for the presence of infected cells using a standardised schema (2 no fluorescence seen; +/2 some fluorescence seen (,5% cells infected); +5-10% cells infected; ++ .10% cells infected). the literature indicates immunofluorescence to be at least as sensitive in general as plaque assay [13, 18, 19] . confirming this, tests using serial dilutions of known quantities of pr8 virus, our method reliably detected 20 pfu of virus in the original sample prior to amplification and 50% of the time detected 2 pfu (data not shown). to test the surface survival of the virus genome, replicate samples of the various materials were inoculated with 10 ml samples containing 1610 6 pfu of virus and incubated for defined periods of time before sample recovery was attempted by swabbing. it was noted that the liquid was absorbed by the wooden surfaces within 5 minutes whereas a droplet could be seen on non-porous surfaces for considerably longer, although in all cases, surfaces had dried by 7 hours. material eluted from the swabs was then titred for virus genome by quantitative rt-pcr. for both pr8 (fig. 1a , table 2 ) and ah04 (table 3) viruses the results were unambiguous. on most surfaces, the viral genome persisted well, with only around a 10-100 fold drop from the initially recoverable titre after 24 h. the exceptions were unsealed wood surfaces, where both viruses lost genome titre rapidly and on pine surfaces in particular, became undetectable after a few hours. thus in general, viral rna survives well for at least 24 h and few surfaces had any significant 'contact effect' in immediately reducing genome titre. when pr8 surface viability was assessed by plaque assay, virus inoculated onto a control surface of a tissue culture dish could be recovered efficiently at t0, but thereafter infectivity fell away rapidly with no live virus recovered at 24 h (table 4 ). fitting the data to a one-phase exponential decay model (fig. 1b ) estimated the t 1/2 of the virus under these conditions to be around 1.5 h. a similar pattern of rapid loss of infectivity was seen when the household surface samples were tested, with the difference that greater initial losses of infectivity ranging between 20-fold (telephone handset) to nearly 4000-fold (unsealed pine) were seen (table 4 ). nevertheless, viable virus was recovered at 4 h (but not later) from the silver-impregnated cloth, soft toy fabric and in trace quantities, from light switch material. the only material (other than the control tissue culture dish) for which even low amounts of viable virus could be detected at 9 h was stainless steel. thus despite the persistence of the viral genome on a wide variety of household surfaces, pr8 infectivity decayed sharply, with evidence of significant contact effects from some materials; most notably unsealed pine, but also a wide variety of other porous and nonporous surfaces. to test whether these findings could be extrapolated to a currently circulating virus, we next tested the survival of ah04 virus, a 2009 pandemic isolate, on a subset of the materials. unlike pr8, as a recent clinical isolate this virus does not grow to high titres in the laboratory and nor was a workable plaque assay available. we therefore used a fluorescent focus assay in which live virus is detected by immunofluorescent detection of the viral nucleoprotein in infected cells. to boost the sensitivity with which viable virus could be detected, infectious virus present in the swabs was first amplified by growth in mdck cells before subsequent assay. the assay therefore provides a highly sensitive but semi quantitative measure of virus infectivity, ideally suited to working with low titre samples [13, 19] . by this measure, the ah04 virus persisted for at least 24 h on the control tissue culture dish material, although titres were evidently lower at 9 and 24 h ( table 5) . consistent with the results obtained with pr8 virus, all household surfaces tested showed lower persistence of infectious virus, with none providing recoverable titre at 24 h and the majority failing to produce live material at 9 h. once again the pine surface showed very rapid inactivation of viability, with no infectivity recovered at 4 h. thus both an historic virus isolate and an example of the recent pandemic strain fail to survive in high titres for long periods of time on a variety of household surfaces, but with significant survival over shorter time spans on certain materials. prior to the influenza a(h1n1) pandemic of 2009-10, few data were available with regard to virus survival on different household surfaces. with a few notable exceptions [10, 11] , the majority of studies had been carried out based on rt-pcr to detect the presence of the genome [20] [21] [22] ; these shed no light on the presence or absence of viable virus. in this study we sought to provide contemporary data about virus survival on a wider range of materials found in or on household surfaces than previously described in the literature; these exemplars were chosen after discussion with uk pandemic policy makers. however, one limitation is that our study was confined to h1n1 influenza a viruses (pr8 and the 2009 pandemic virus) due to resource issues. however, we know of no evidence to suggest there are substantial differences in survival between human influenza viruses. moreover, when we compared the survival of pr8 virus with two seasonal isolates of influenza a (a/solomon islands/12/5/08 (h1n1) and influenza a/brisbane/12/5/08 (h3n2), obtained from professor alan hay of the national institute of medical research, mill hill), we saw no significant differences, with all three viruses losing plaque titre on a plastic surface with a t1/2 of around 90 minutes (data not shown). we therefore think it is reasonable to generalise from the findings here to other human strains of influenza a. further studies, especially of influenza b are warranted however. we applied concentrations of virus (,1610 6 tcid 50 ), which were within the range of those reported in the respiratory secretions of naturally infected individuals [5, 10] . in addition, we suspended virus in 1% bovine serum albumin (bsa), reflecting our (unpublished) finding that bsa improved virus survival, and similar findings from thomas et al [11] using mucus obtained from children. our experiments were conducted within a narrow range of humidity and temperature conditions consistent with normal human indoor living conditions in temperate zones, and all survival assays were performed in duplicate and where possible triplicate. we used plaque assay techniques and immunofluorescence techniques for pr8 and pandemic viruses, respectively. the differing methodologies used to detect the two strains of h1n1 virus (lower titre inoculum of the pandemic ah04 virus but higher sensitivity detection method) make it difficult to directly compare the survival of the two strains, but we see little to suggest any major difference. our data on the survival of the laboratory adapted pr8 virus indicated that viable virus was no longer recoverable in detectable amounts from 9 of 14 (64%) surfaces four hours after deposition; however, contrary to the findings of bean et al., non-porous surfaces were not consistently more conducive to virus survival than porous ones [10] . nevertheless, no test surfaces supported detectable virus survival beyond nine hours. broadly similar outcomes in which infectivity tended to be lost after 4-9 hours were obtained with the recent pandemic isolate ah04. overall, our results indicate that influenza virus does not remain viable in large quantities on most surfaces in indoor domestic conditions for more than a few hours. our data are consistent with recent findings from a study of environmental deposition of pandemic h1n1 virus in the homes of infected patients, involving our laboratory, when almost 10% of tested surfaces yielded viable virus [15] . however, in this and similar studies in community settings where environmental samples are taken relatively infrequently and the infectious source remains present, it is not possible to establish the time elapsed since virus deposition [15, 23] . with regard to the testing of specific materials, we examined survival on a range of porous items: a children's soft toy, a silver impregnated fabric with known bacteriostatic properties of the type sometimes encountered in hospital staff clothing to combat nosocomial bacterial infections, and a branded cleaning cloth (j clothh, associated brands). we hypothesised that the inclusion of an antimicrobial agent, microbanh (microban international ltd) in the j cloth might inhibit viral growth. microbanh is based on triclosan and has been demonstrated to have anti-bacterial and anti-fungal activity; it has not however, been demonstrated or claimed to be anti-viral. notwithstanding, in our laboratory setting, some constituent or quality of the j clothh appeared to limit virus survival to under 4 hours. the result for the silver impregnated fabric also deserves further comment. whilst silver has been demonstrated to have bacteriostatic properties, it has not been documented to show antiviral activity. our data would tend to suggest that it is not significantly inhibitory to influenza a. surfaces that allowed pr8 virus to survive longest (between four and nine hours) included light switch material (polyvinyl chloride) and a computer keyboard. interestingly these are likely to be the materials from which the most frequently touched communal household objects are made. both pr8 and pandemic viruses survived less than four hours on all of the wood surfaces tested. this may have been due to a number of factors including porosity of the surface, oils in the wood or a potentially virucidal 'contact effect' of varnish finishes. pine oil in particular has been demonstrated to have virucidal activity against respiratory viruses [24] . our findings suggest they are not hospitable environments for enveloped viruses. as observed in other studies, we found that stainless steel supported the viability of influenza viruses longer than other tested metals. metals have been demonstrated to have low levels of anti viral activity [25] [26] [27] ; and stainless steel has previously been demonstrated to support influenza virus viability for longer than that of copper [28] . confirmation of these results raises questions about the use of stainless steel in healthcare and daycare settings in particular. in conclusion, testing two h1n1 strains of influenza a (one of which was a 2009 pandemic virus) demonstrates that in an environment that is consistent with indoor domestic settings in temperate zones, virus deposited onto the touched environment is likely to survive up to a few hours, though rarely more than nine hours, on the vast majority of surfaces. metallic and non-metallic non-porous materials pose the greatest risk and should be targeted for frequent cleaning if situated in close proximity to patients infected with influenza virus; fortunately the latter are also more conducive to surface cleaning with a wide variety of simple cleaning agents [12] . whilst our data suggest that the risk of virus transmission might last several hours after deposition, we generated very little data suggesting that appreciable amounts of virus survived much beyond nine hours. this probably means that frequently touched environments such as classrooms, offices and living rooms, which are then left unoccupied overnight, will not contain much viable virus on surfaces by the next morning. nevertheless, the data still support frequent cleaning of commonly touched items and surfaces throughout the working day, particularly when symptomatic persons are present, for example in physician waiting rooms. in terms of cleaning regimens, one critically important consideration is that survival of virus in high titres for prolonged periods is not necessary for fomite transmission if surfaces are frequently re-inoculated (e.g. by toddlers). however the contribution of such indirect transmission relative to respiratory droplets directly from one person to another or relative to aerosol transmission remains unknown. estimating household and community transmission parameters for influenza statistical procedures for estimating the community probability of illness in family studies: rhinovirus and influenza simulation studies of influenza epidemics: assessment of parameter estimation and sensitivity using data on social contacts to estimate age-specific transmission parameters for respiratory-spread infectious agents review of aerosol transmission of influenza a virus transmission of influenza a in human beings influenza and related infection control issues resistance of the melbourne strain of influenza virus to desiccation loss of infectivity on drying various viruses survival of influenza-viruses on environmental surfaces survival of influenza virus on banknotes effectiveness of common household cleaning agents in reducing the viability of human influenza a(h1n1) biophysical characterization of influenza virus subpopulations using field flow fractionation and multiangle light scattering: correlation of particle counts, size distribution and infectivity genome packaging in influenza a virus virus shedding and environmental deposition of novel a(h1n1) pandemic influenza virus: interim findings mutational analysis of cis-acting rna signals in segment 7 of influenza a virus interaction of the influenza virus nucleoprotein with the cellular crm1-mediated nuclear export pathway comparison of enzyme-linked immunosorbent assay, indirect immunofluorescence assay and virus isolation for detection of respiratory viruses in nasopharyngeal secretions enhanced detection of infectious airborne influenza virus the occurrence of influenza a virus on household and day center fomites respiratory viral rna on toys in pediatric office waiting rooms occurrence of bacteria and viruses on elementary classroom surfaces and the potential role of classroom hygiene in the spread of infectious diseases influenza virus contamination of common household surfaces during the 2009 influenza a (h1n1) pandemic in bangkok, thailand: implications for contact transmission the antiviral action of common household disinfectants and antiseptics against murine hepatitis virus, a potential surrogate for sars coronavirus neutralizing viruses in suspensions by copper oxide-based filters inactivation and morphological changes of avian influenza virus by copper ions 2-oximinocarboxylates of certain metals and their antiviral activity inactivation of influenza a virus on copper versus stainless steel surfaces we thank penny powell, roberto vivancos and darren sexton at the university of east anglia for their contributions to early discussions; helen wise and amanda stuart for help with the virus work. some materials were kindly supplied free of charge by brushwood ltd uk (kitchen work surfaces) and toray textiles europe ltd (silver-containing fabric); the remaining items were sourced commercially or as shown in table 1 . key: cord-000723-wo20st5w authors: xu, zhenqiang; shen, fangxia; li, xiaoguang; wu, yan; chen, qi; jie, xu; yao, maosheng title: molecular and microscopic analysis of bacteria and viruses in exhaled breath collected using a simple impaction and condensing method date: 2012-07-25 journal: plos one doi: 10.1371/journal.pone.0041137 sha: doc_id: 723 cord_uid: wo20st5w exhaled breath condensate (ebc) is increasingly being used as a non-invasive method for disease diagnosis and environmental exposure assessment. by using hydrophobic surface, ice, and droplet scavenging, a simple impaction and condensing based collection method is reported here. human subjects were recruited to exhale toward the device for 1, 2, 3, and 4 min. the exhaled breath quickly formed into tiny droplets on the hydrophobic surface, which were subsequently scavenged into a 10 µl rolling deionized water droplet. the collected ebc was further analyzed using culturing, dna stain, scanning electron microscope (sem), polymerase chain reaction (pcr) and colorimetry (vitek 2) for bacteria and viruses. experimental data revealed that bacteria and viruses in ebc can be rapidly collected using the method developed here, with an observed efficiency of 100 µl ebc within 1 min. culturing, dna stain, sem, and qpcr methods all detected high bacterial concentrations up to 7000 cfu/m(3) in exhaled breath, including both viable and dead cells of various types. sphingomonas paucimobilis and kocuria variants were found dominant in ebc samples using vitek 2 system. sem images revealed that most bacteria in exhaled breath are detected in the size range of 0.5–1.0 µm, which is able to enable them to remain airborne for a longer time, thus presenting a risk for airborne transmission of potential diseases. using qpcr, influenza a h3n2 viruses were also detected in one ebc sample. different from other devices restricted solely to condensation, the developed method can be easily achieved both by impaction and condensation in a laboratory and could impact current practice of ebc collection. nonetheless, the reported work is a proof-of-concept demonstration, and its performance in non-invasive disease diagnosis such as bacterimia and virus infections needs to be further validated including effects of its influencing matrix. bioaerosols are present virtually anywhere in the environment, and their exposure is shown to cause numerous adverse health effects [1] [2] . in addition, there is also a possible release of biowarfare agents in a man-made bio-terror event. a number of studies demonstrated that the respiratory tract can be colonized with disease organisms [3] [4] [5] . through talking, coughing, sneezing or singing, the potential virulent organisms can be exhaled and spread into the ambient environment [6] , which accordingly causes air contamination. for example, sars in 2003 and h1n1 in 2009 outbreaks were shown to be attributed to the airborne route of disease transmission [7] [8] [9] [10] . among many other diseases, respiratory infection accounts for 23.3-42.1% of the total hospital infections [11] , and is listed as the third leading killer [12] . however, present diagnosis procedures using nasal swabs, bronchoalveolar lavages, nasopharyngeal aspirates or sputum samples, appear to cause unpleasant experiences in addition to long detection time. during flu outbreaks, body temperature or isolation procedures are often used to control and prevent further spread, however such methods are lacking scientific evidence and not always effective with those patients infected but in latent period. on another front, exhaled breath condensate (ebc) as a simple and noninvasive method is increasingly being utilized in early disease screening and infectious aerosols measurements, e.g., lung cancer [13, 14] , asthma [15, 16] , and other respiratory problems [17, 18] . in previous studies, human influenza a viruses were detected in exhaled breath using ebc [19, 20] as well as filter [21] , mask [22, 23] and a liquid sampler [24] . in another study, foot-and-mouth disease viruses were also found in the exhaled air from experimentally infected cattle [25] . in addition, high levels of bacterial concentrations in ebc were also observed in other studies [26] [27] [28] [29] . it was recently shown that exhaled breath could be also analyzed for fungal infection by relevant biomarker, e.g., 2-pentyl furan (2pf) for aspergillosis [30] . overall, ebc has demonstrated great potential and advantages in early disease screening and diagnosis [31] , opening a new arena for studying airway inflammation and chemistry [32] . recently, vereb et al (2011) suggested that exhaled breath can be also used for assessing a variety of environmental exposures [33] . for ebc related studies, the first key step is the collection of exhaled breath. over the years, a variety of devices (table s1 , supporting information) were developed including rtube collection system (respiratory research, inc, charlottesville, va) and ecoscreenh condenser (erich jaeger gmbh, wurzbur, germany). typically, these devices would be able to collect 1000 ml of ebc samples within about 10 min, however the collection often comes with a lengthy procedure and a higher cost. for example, use of the ecoscreen involves 7 steps: 1) turn on to cool, 2) clean collection tube, 3) clean condensation chamber insert, 4) retrieve cooling sleeve from freezer, 5) sample collection, 6) sample storage and transport, 7) removal of sample (respiratory research, inc, charlottesville, va). the rtube eliminates the first 3 steps, but each collection still requires 10 min and costs $23.25 (respiratory research, inc, charlottesville, va) compared to 31 min and $47.17 per collection for the ecoscreen. these collection devices are generally expensive, e.g., the ecoscreen costs around $9000. a recent study compared the sampling efficiency of the rtube (widely used ebc collection device) with that of throat swab method, showing detection rates of 7% and 46.8% for the rtube and the throat swab method, respectively [20] . it was suggested that the rtube is not applicable for viral detection in exhaled breath [20] . in addition, condenser coatings [34] , sampling temperature [35] and sampling times [36] were shown to affect physical collection efficiencies of available ebc collectors. among others, the noted problems with these available ebc collection devices are the device availability, reusability and possible cross contamination [37] , which would negatively impact their wide applications. in addition, ebc collection is strictly limited to the method condensation only in most studies [38] . to fully utilize ebc in early disease screening, diagnosis and environmental exposure assessment, simple yet efficient ebc collection device using different methods and biological characterization of the ebc sample are needed. in this study, a novel ebc collection method was developed by using hydrophobic surface, a layer of ice, and a droplet scavenging procedure. the physical collection efficiency (amount of ebc collected per unit of time) of the device was evaluated. in addition, biological analysis and characterization of ebc samples collected from human subjects were conducted using culturing, dna stain, sem, qpcr and species identification tool vitek 2. this work contributes to the effort in applying ebc together with molecular tools as a non-invasive method in rapid disease diagnosis. the collection method and device developed and experiential set up for collecting ebc are shown in figure 1 and figure 2 , respectively. as observed in figure 1 , a simple ebc collection device was developed here. the ebc collection device is composed of four major parts as shown in figure 1 : collection device cover, collection device base, a layer of ice, and a hydrophobic film (treated by ultralow temperature 270uc). the collection device cover and base were made of teflon tm polytetrafluoroethylene (ptfe) material, and a parafilm (parafilm co. menasha, wi) used as the hydrophobic surface. the dimensions of the collection device are measured as 80640640 (mm) (length6width6height). in the collection device cover, there is a hole with a diameter of 6 mm as the exhaled breath inlet. the thickness of the collection device cover and base was about 3 mm, and the whole collection device weighs around 105 g. the layer of ice is used to keep the treated hydrophobic film cool. for ebc collection, sterile water (dna and rna free) was first added into the collection base of the device up to a depth of 5 mm as observed in figure 2 . and then, the device base together with the cover was placed in an ultralow temperature (270uc) refrigerator (thermo fisher scientific co. marietta, oh) to form a layer of ice. following this step, a sterile hydrophobic parafilm measured as 8064060.3 (mm)(length6width6thickness) was placed onto the surface of the ice suited in the collection base. to collect ebc samples, a disposable sterile straw with a diameter of 5 mm (16 cm long) is inserted through the exhaled breath inlet shown in figure 2 , with its end 2 mm above the hydrophobic parafilm. the human subjects are then advised to mouth-breathe without wearing a nose clip through the exhaled breath inlet shown in figure 2 toward the hydrophobic film for a selected time (1-4 min). due to the low temperature and hydrophobic nature of the parafilm surface, exhaled breath quickly condenses into tiny liquid droplets on the hydrophobic surface. assuming an average breathing rate of 12 l/min for an adult, the particle speed from the exhaled breath would be around 10 m/s given the size of the straw (5 mm in diameter). therefore, during the exhaled breath collection, the bacteria or virus particles would impact onto the hydrophobic surface at a speed of 10 m/s. in addition to condensing used for other ebc collection procedures, the method developed here also rely on the impaction to collect the bacterial and viral particles. given such a speed, there might be possible particle bounce problems, however the bacterial or viral particles in the exhaled breath usually come with water droplets, which thus minimizes the potential particle bounce problem. after the collection, about 10 ml of dna and rna free di water was pipetted onto the hydrophobic film as observed in figure 1 . to collect breath samples on the hydrophobic film, one only needs to use the pipette to touch the di water droplet, and then drag the di water droplet to scroll over the entire surface. the di water droplet would move with the pipette without an extra step. the materials collected on the surface would be subsequently scavenged into the water droplet. after this operation, the collected ebc samples in the form of bigger liquid droplet as shown in figure 1d were transferred to a sterile tube by a pipette for subsequent analysis. the samples collected without the exhaled breath from human subjects are used as the negative controls. the ebc collection efficiency and biological analysis of collected samples were performed as outlined in the experimental procedure shown in figure s1 (supporting information). to investigate the amount of variability in ebc collected by the method developed, six student volunteers were recruited to exhale through the device for 1, 2, 3 and 4 min. the volume of collected ebc was measured by a calibrated pipette (eppendorf, hauppauge, ny). the amount of ebc per unit time collected by the device was determined using averages of ebc samples obtained by the volunteers under each of specific collection time tested. for each ebc collection, a different hydrophobic film and a different exhalation straw were used. in addition, the particle size distributions in the exhaled breath through mouth-breathing were also measured in a particle free bio-safety hood using an optical particle counter (opc) (grimm co. ltd., ainring, germany) at a flow rate of 1.2 l/min. to ensure air stream balance, the opc was connected to a two-way tubing, which connects to clean air (biological safetyhood) and the breathing straw, respectively. in this work, seven patients with onset flu symptoms (their medical information is listed in table s2 , supporting information) were also recruited from the respiratory clinic of peking university third hospital in beijing. about 40 ml of exhaled breath condensate collected from each of 7 patients was diluted by 10 times and then plated on trypticase soy agar (tsa) (becton, dickson and company, sparks, md) plates at 26uc for 2-3 days, and colony forming units (cfus) were manually counted. the total culturable bacterial aerosol concentration was calculated as cfu/m 3 (exhaled breath) by considering the collection time and an average breathing rate of 12 l/min for an adult. besides, the culturable bacterial species were identified using vitekh 2 (biomérieux, inc,100 rodolphe street, durham, nc). in addition, molecular detection of bacteria and virus using qpcr and rt-qpcr, respectively, were performed according to the procedures described in supporting information s1. to further confirm the bacterial presence dna stain of ebc sample by acridine orange (ao) was also conducted. the differences in collected ebc volumes and culturable bacterial aerosol concentrations obtained by the ebc collection device were analyzed by analysis of variance (anova). a pvalue of less than 0.05 indicates a statistically significant difference at a confidence level of 95%. collection of ebc from human subjects was approved by peking university ethnics committee. here, a novel ebc collection method and device was developed and evaluated in collecting ebc samples from human subjects using culturing and molecular methods. compared to those currently available devices shown in table s1 , our device is lightweight with simplicity, reusability, and lower cost. the developed collection device itself costs less than $10, with about $0.5 for consumables (straw and hydrophobic film) per collection. the time needed for 100 ml ebc including sample collection and removal was around 2 min. the physical collection efficiency of the device is shown in figure 3 . the data points shown in the figure were averages of the ebc samples collected from six volunteers under each of the collection times (1, 2, 3 and 4 min) tested. in general, the amount of ebc sample collected was observed to increase with increasing collection time were observed among subjects. as also observed in figure 3 , the method has a good reproducibility (small variations). anova analysis indicated that the collection time had a statistically significant effect on the amount of ebc sample collected per unit of time (p-valtable s2 ; f and m indicate female and male, respectively, 1-7 indicate the subject id corresponding to those listed in table s2 ; ebc collection time was 3 min; data points represent averages and standard deviations from at least three replicates. doi:10.1371/journal.pone.0041137.g005 ue = 0.0026). for the 4 min collection, the volume of collected ebc (168.7 ml) was 1.8 times of that (60.0 ml in average) by 1 min. in our study, when no ebc was collected about 1 ml of liquid was obtained from the hydrophobic surface in an environment with a temperature of 17.9-19.3uc and a relative humidity level of 46-52%. in addition, during the breath sample collection, the collection device had a higher air pressure due to the exhaling, thus it is less likely that environmental air would come into the device. this suggests that environmental water vapor had limited impact on the collection method given the total amount of ebc collected. a recent study indicated that the minimum required volume of ebc was 50 ml for follow-up biological and chemical analysis, such as multiplexed cytokine analysis [35] . this on the other hand implies that the ebc device developed in this study can provide adequate amount of ebc sample for rapid analysis. here, only one type of hydrophobic surface (parafilm) was tested, and in the future different table s2 ; di water was used as the negative control. table s2 ; bacillus subtilis species was used as the positive control and di water was used as the negative control; the curves shown here include two duplicates for each ebc sample. doi:10.1371/journal.pone.0041137.g007 hydrophobic materials should be also explored to improve the overall efficiency. as listed in table s1 , currently available ebc collection devices, e.g., the rtube and the ecoscreen, are comparable to ours with respect to rate of ebc collection. however, our ebc device has advantages in size, weight, and simplicity. in our study, we used a 16 cm long straw for exhaling toward to the super hydrophobic surface without any control of saliva for the possible contamination. however, our collection time was only 1-4 min, and during such short sampling period the sample contamination by saliva is very limited given the length of the straw. another advantage of our developed device is the one time use of the hydrophobic parafilm (disposable) and exhalation straw with an easy collection of ebc, which thus prevents the possible cross contamination and facilitates the collection of ebc samples from a large number of subjects. this is particularly useful during an influenza outbreak or a man-made bio-terrorism attack in which a rapid screening of exposed persons needs to be conducted immediately. here, the ebc samples collected by the developed device from seven human subjects recruited from a respiratory unit of peking university third hospital in beijing were studied using culturing, dna stain, sem and molecular methods. in this study, the particle size distributions trends in a typical exhaled breath were also measured and are shown in figure 4 . as observed in the figure, the number concentration decreased with increasing particle diameter. for bacterial size ranges (0.65-2.2 mm), a concentration level of 329 to 25819 particles/l was observed, while for larger particles of 2.2-4 mm a concentration level of 60 to 400 particles/l was obtained. in previous studies, similar particle size distribution trend in exhaled breath was also found using the opc, although the droplet concentrations for respective size ranges were slightly different [21, 39] . nonetheless, due to its rapid evaporation water droplet itself or those adsorbing on bacterial particles in the exhale breath will certainly affect the results obtained here. the results from opc indicated that particles of larger than 2.5 mm only accounted for 0.4% of the total particles exhaled. according to icrp (1994), the total lung deposition efficiency for particles larger than 2 mm is more than 80%, while for smaller particles of less than 1 mm, the deposition efficiency is less than 40%, i.e., 60% exhaled out [44] . in addition, larger particles could stick to the straw wall. therefore, in the exhaled breath as well as those collected into di water droplet smaller particles would dominate. figure 5 shows the concentrations of culturable bacterial aerosols in ebc samples collected from seven human subjects. as shown in the figure, bacterial concentration levels ranged from 693 to 6,293 cfu/m 3 . anova tests indicated that there were statistically significant differences in culturable bacterial aerosol concentrations for ebc samples collected from different subjects (p-value = .0001). in a recent study, human occupants are also identified as the significant contributors for indoor bacteria, i.e., the emission rate is about 37 million gene copies per person per hour, and a distinct indoor air signature of bacteria was demonstrated to be associated with human skin, hair, and nostrils [40] . during human breathing, the bacterial particles from environmental air are continuously inhaled, some of which, i.e., smaller ones, can be exhaled out again by the lung and reside with nostrils. here, bacterial species sphingomonas paucimobilis and kocuria rosea were detected using vitek2 in six ebc samples as shown in table s2 . because of limitation of vitek 2, certain bacterial species were not identified in our study. among the subjects, subject #6 had substantially higher culturable bacterial concentrations than other subjects. from his medical conditions shown in table s2 , it was likely that his fever was caused by the bacterial infections. in his ebc sample, we found kocuria variants which were thought to cause catheter-related bacteremia [41] . for other human subjects, the culturable bacterial aerosol concentration levels ranged from 700 to 3000 cfu/m 3 and sphingomonas paucimobilis, a non-fermenting gram-negative bacillus, were detected. in a previous study, s. paucimobilis was found to cause nosocomia bacteremia outbreak [42] . for negative control samples, we did not observe the bacterial growth, indicating no contamination during the ebc collection. ideally, bacterial particles in ebc should be collected using a suitable size-selective sampling tool to investigate the bacterial counts for different size range. however, such device is currently not available yet. compared to the environmental culturable bioaerosol concentrations, those in ebc samples collected had relatively higher levels, thus representing an important source of bioaerosols particularly in a high human occupancy environment. in addition to viruses, rhodococcus equi, a bacterium causing pyogranulomatous bronchopneumonia, were detected in the exhaled air from foals in a recent study [43] . when pathogenic bacteria are breathed out, they could pose a serious public health threat. figure 6 shows the qpcr amplification plot from ebc samples collected from seven human subjects in a respiratory clinic. as observed from the figure, bacterial samples were successfully amplified (ct values were [16] [17] [18] [19] , while the positive sample (b. subtilis) had a ct value of 15 and the negative control had a value of 28. based on the dna standards used, the concentrations of bacterial dna in the ebc samples (sample 1-7) were in the range of 0.32 mg/ml-3.15 mg/ml. detection of the bacterial dna in ebc samples was also confirmed by the melting curve of qpcr amplification as shown in figure 7 . as observed in the figure, most ebc samples had a peak at 68uc, the same as that of the positive control b. subtilis. for a few different peaks observed, they might be the possible primer dimer (pd) from the pcr non-specific amplification process. in addition to the qpcr amplification of bacteria in ebc samples collected, dna stain (ao method) was also performed and the results are shown in figure s2 . as observed in the figure, both viable (green) and dead (yellow) were found in the ebc samples collected and the positive control b. subtilis samples, while no cells were detected in the negative control. sem images with different resolutions and agar plate culturing shown in figure 8 also indicated that ebc samples (cultured) had various types of bacteria based on their morphologies and colony color. from sem images, it can be estimated that most bacteria are in the range of 0.5-1.0 mm. according to total particle deposition curve developed by icrp (1994) [44] , more than 60% of bacterial particles of below 1 mm could be exhaled out. these smaller bacterial particles could remain airborne for a prolonged time period, thus playing an important role in airborne transmission of potential diseases. results shown in figures 5, 6 , 7, and 8 indicate that high levels of bacterial aerosols were detected in the ebc samples collected, and the results on the other hand also implied that the developed device was efficient in collecting bacterial particles in the exhaled breath. these experimental data further confirm that exhaled breath is an important source of bacterial aerosols in the built environments. in this study, qpcr was also applied to detecting influenza a h3n2 viruses in ebc samples collected by the device. as observed in figure s3 , h3n2 viruses were detected in the ebc sample collected from subject #3 with a ct value of 28, while those for subject #1, #2 were shown below the detection limits. in addition, spiking viruses into the samples in general enhanced the overall qpcr signal as observed in figure s3 . this on the other hand suggests no inhibition or amplification occurred when amplifying h3n2 viruses in ebc samples using qpcr. according to information shown in table s2 , subject #3 had a fever, but no other information was available at the time of the experiment. in a previous study, it was indicated that use of the rtube for ebc collection had a very low viral detection rate (7%) compared to nasal swabs (46.8%) [20] . recently, a mask-like sampler was also tested and proved to be useful in detecting viruses using pcr in exhaled breath [23] . it was indicated that airborne virus detection is difficult due to their low concentration and the presence of a wide range of inhibitors, thus optimized molecular biology should be performed to enhance their detection [45] . although the number of the subjects tested is limited here, the developed method, i.e., ebc collection and qpcr application, was demonstrated successful in detecting viruses from human exhaled breath. this would offer a non-invasive method for diagnosis of respiratory infections by using ebc. in the future, more patients should be tested with the ebc collection device developed here for viral detections. exhaled breath holds great promise for monitoring human health and for the diagnosis of various lung and systemic diseases, but analysis challenges remain due to the complex matrix of the breath [46, 47] . in this study, different from available devices restricted solely to condensation a simple and low cost ebc collection method using impaction and condensing was developed here for collecting bacteria and virus particles. an important advantage is the reusability of the collection device with a disposable hydrophobic film and an exhalation straw yet with a rapid ebc collection. this would offer the opportunity to collect ebc samples from a large number of subjects, especially during an influenza outbreak or a man-made bioterrorism event, within a shorter time frame. the developed ebc collection method was shown highly successful in detecting bacteria in ebc samples in a clinical setting. the developed ebc collection method was also shown applicable in detecting influenza viruses too. experimental data here also suggest that exhaled breath, which was shown to contain smaller bacterial particles, could play an important role in airborne transmission of potential diseases. the collection efficiency of other substances including bio-markers (no,co, 8isoprostane, hydrogen peroxide, nitrite, volatile organic compounds) using the developed method here is subject to further investigations. in addition, different exhalation modes should be also investigated with the method in collecting ebc. besides, the dynamics of the air flow, mixing, and effects of temperatures and humidity, condensation, evaporation, growth of particles during the collection as well as the optimal straw length should be also investigated for improving the developed technique. overall, our developed method here could be easily made available to a laboratory, and have impacts on current practice of ebc collection. nonetheless, the reported work is a proof-of-concept demonstration, and its performance in non-invasive disease diagnosis such as bacterimia and virus infections needs to be further validated including effects of those influencing factors described. figure s1 experimental procedures used in this study include physical characterization and molecular analysis of the ebc collection efficiencies of the device and its pilot application in a respiratory clinic. (tif) figure s2 optical images of ebc samples stained by acridine orange (ao): bacillus subtilis species were used as the positive control and di water was used as the negative control. (tif) figure s3 detection of h3n2 influenza viruses in ebc samples collected from three human subjects with id: 1, 2, 3 corresponding to those listed in table s2 ; in addition, spiked h3n2 virus samples were also amplified; h3n2 viruses were used as the positive control and di water was used as the negative control. 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breath and the airborne environment exhaled breath analysis: the new frontier in medical testing the great challenge for exhaled breath analysis: embracing complexity, delivering simplicity key: cord-002398-0a3okta0 authors: myllykoski, matti; kursula, petri title: structural aspects of nucleotide ligand binding by a bacterial 2h phosphoesterase date: 2017-01-31 journal: plos one doi: 10.1371/journal.pone.0170355 sha: doc_id: 2398 cord_uid: 0a3okta0 the 2h phosphoesterase family contains enzymes with two his-x-ser/thr motifs in the active site. 2h enzymes are found in all kingdoms of life, sharing little sequence identity despite the conserved overall fold and active site. for many 2h enzymes, the physiological function is unknown. here, we studied the structure of the 2h family member ligt from escherichia coli both in the apo form and complexed with different active-site ligands, including atp, 2′-amp, 3′-amp, phosphate, and nadp(+). comparisons to the well-characterized vertebrate myelin enzyme 2′,3′-cyclic nucleotide 3′-phosphodiesterase (cnpase) highlight specific features of the catalytic cycle and substrate recognition in both enzymes. the role played by the helix α7, unique to cnpases within the 2h family, is apparently taken over by arg130 in the bacterial enzyme. other residues and loops lining the active site groove are likely to be important for rna substrate binding. we visualized conformational changes related to ligand binding, as well as the position of the nucleophilic water molecule. we also present a low-resolution model of e. coli ligt bound to trna in solution, and provide a model for rna binding by ligt, involving flexible loops lining the active site cavity. taken together, our results both aid in understanding the common features of 2h family enzymes and help highlight the distinct features in the 2h family members, which must result in different reaction mechanisms. unique aspects in different 2h family members can be observed in ligand recognition and binding, and in the coordination of the nucleophilic water molecule and the reactive phosphate moiety. the 2h phosphoesterase superfamily is an ancient group of proteins and protein domains characterized by a common fold and a few conserved active site residues [1, 2] . various catalytic activities have been assigned for the 2h enzymes: hydrolysis of a 2 0 ,3 0 -cyclic phosphate, in either nucleotides or 3 0 -ends of rna molecules, into 2 0 -phosphate [3] [4] [5] , hydrolysis of adpribose 1 00 ,2 00 -cyclic phosphates into adp-ribose 1 00 -phosphate [6] [7] [8] , generation and/or cleavage of 2 0 -5 0 -linkages between nucleotides or rna molecules, such as trna halves [3, [9] [10] [11] [12] [13] , and 3 0 -5 0 exonucleolytic removal of terminal uridine nucleotides from snrna with the simultaneous generation of 2 0 ,3 0 -cyclic phosphates at the terminus [14] . the structurally best-a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 characterized 2h enzyme is the mammalian myelin enzyme 2 0 ,3 0 -cyclic nucleotide 3 0 -phosphodiesterase (cnpase) [15] [16] [17] [18] [19] , but even for this enzyme, the biological function remains enigmatic [5] . e. coli ligt is a 20-kda protein that exhibits 2 0 ,3 0 -cyclic nucleotide 3 0 -phosphodiesterase and 2 0 -5 0 -ligase/phosphodiesterase activities [3, 9] , but the biological function of the enzyme is unknown. it is potentially a source of 2 0 -5 0 oligoadenylates (2-5a) and similar compunds with 2 0 -5 0 -linkage detected in e. coli [20] . a genomic knockout of ligt in e. coli did not obviously affect cellular growth or viability in laboratory conditions, while ligt overexpression produced a phenotype sensitive to elevated temperature [9] . a ligt orthologue in deinococcus radiodurans was massively upregulated after acute irradiation, and it was speculated to function in the handling of damaged rna species [21] . in a recent study, e. coli ligt was crystallized, and its structure was refined in complex with the in vitro reaction product 2 0 -amp [22] . we extend the previous study here, providing highresolution crystallographic data and different active-site ligand complexes. comparisons to the apo form of the enzyme determined in the current work allow the highlighting of conformational changes and binding determinants of the reactive species. based on further comparisons to mouse cnpase, we also modelled the substrate complex of ligt and discuss unique features of each protein. an extended binding surface for rna substrates is also identified, which possibly involves flexible loops of the 2h family. all in all, we provide novel data on the structurefunction relationships in a bacterial 2h enzyme, which can be used to understand the common and divergent properties of enzymes in the entire 2h superfamily. overall structure the structure of the putative trna ligase ligt from e. coli was determined by x-ray crystallography in the apo form. in addition, a number of ligt complex structures with active-site ligands were solved, hence significantly extending the earlier results on the ligt structure, which was only available as a complex with the reaction product 2 0 -amp [22] . intriguingly, a total of five different crystal forms were observed in this study, which also highlights the flexible properties of the enzyme. the ligt structure presents a typical 2h phosphoesterase fold, in which the catalytic residues reside at the beginning of strands β2 and β6 ( fig 1a) . as in other 2h family members, the active site has 2-fold symmetry; this symmetry also includes the 4 water molecules at the bottom of the active site, connecting the active-site β strands through water-mediated hydrogen bonds ( fig 1b) . these water molecules coordinate the substrate/product throughout the reaction in cnpase [17, 18] , and they are likely to play a similar role in ligt and other 2h phosphoesterases. the strict conservation of the active-site water structure between 2h enzymes is remarkable, taking into account the amino acid sequence identity of slightly above 10% between cnpase and ligt. for an insight into the active-site properties and catalytic mechanism, we co-crystallized ligt with different nucleotide compounds in the active site (table 1, fig 2) , including substrates, products, and other compounds. in addition to the presumed obvious substrates and products, we also used other nucleotide compounds in an attempt to screen ligt crystallographically for nucleotide-like ligand-binding properties. this was considered of interest, as open questions remain related to the physiological function of both ligt and many 2h family enzymes in general. all structures had more than one protein chain in the asymmetric unit, and in general, the one with best-defined electron density for ligands was used in the analyses below. previously, ligt has been crystallized with the reaction product 2 0 -amp [22] . we also observed such a complex after cocrystallization with 2 0 ,3 0 -camp (fig 2a) . the binding mode is similar to that seen before, and the 2 0 -amp ligand is well defined in one of the two ligt monomers, while the occupancy in the other monomer is low. binding involves aromatic stacking of the nucleotide base against phe48 and a c-h. . .π interaction between the ribose ring and phe8. in addition to the catalytic residues, arg130 plays a key role in coordinating the phospho moiety of the product. considering reaction geometry, a water molecule coordinated by his125 in the apo structure can now be designated as the nucleophilic water (fig 2a) , analogously to cnpase. the complex also proves that ligt has cnpase activity towards 2 0 ,3 0 -cyclic mononucleotides, since the reaction product is bound in the crystals grown in the presence of the substrate. a possible role for 2 0 ,3 0 -cyclic nucleotides in mammalian systems has been lately discovered [23] , but their importance in prokaryotic systems is not known. however, 2 0 ,3 0 -cyclic cytidine and uridine monophosphate were recently detected in the extracts of pseudomonas fluorescens [24] , and several bacterial phosphodiesterases that cleave 2 0 ,3 0 -cyclic nucleotides have been described over the years [25, 26] . whether ligt activity plays a role in the metabolism of such compounds in vivo, remains to be studied. another product analogue of the reaction catalysed by ligt, nadp + , was also trapped in the active site, being well defined in electron density (fig 2b) . in the nadp + complex, there are 4 monomers in the asymmetric unit, and the nicotinamide end of the ligand binds to each of them differently, having different conformations or being disordered-depending on crystal contacts. the adenosine 2 0 ,5 0 -bisphosphate moiety in each monomer binds identically, however, and in a mode highly similar to that seen in 2 0 -amp. the 5 0 -phosphate mimics the next phosphodiester in an rna molecule, and it can be used to deduce further binding determinants for rna substrates. this phosphate moiety is bound by arg6 from the n-terminal strand β1 in the crystal structure, and it is likely that this residue plays a direct role in rna a. overall structure of ligt. secondary structure elements and the n and c termini are labeled, and the two active site hxt motif side chains are also shown. b. stereo view of the organization and conservation of the active site between ligt (white) and mouse cnpase (blue) [18] . the four water molecules between strands 2 and 6 are conserved (ligt, red; cnpase, blue). the nucleophilic water molecule in ligt (green) is coordinated by his125 and arg130, while the corresponding water molecule in cnpase (magenta) interacts with the n terminus of helix 7 (right) and the catalytic histidine. substrate binding also. an additional nadp + fragment is seen stacked on top of trp82 in one monomer. we also cocrystallized the enzyme with atp and 3 0 -amp, which are neither substrates nor products. the fact that they bind the active site can imply that they may be weak inhibitors, and it can be taken as evidence of a general propensity to bind nucleotides in the active site. the ligt orthologue protein pf0027 from pyrococcus furiosus was shown to require a gtp cofactor for the rna ligation reaction [3] , while for the e. coli enzyme, such a cofactor has not been reported. although we attempted crystallization in the presence of gtp as with atp, no corresponding gtp electron density was found in the resulting crystals (data not shown). two similar datasets were collected with atp, one of which has one atp bound to only one of the 2 monomers in the asymmetric unit. this atp molecule probably has a partial occupancy and/or some degree of flexibility, as evidenced by residual difference electron density. the major conformation was built in the structure. the other complex has three atp molecules for two monomers; the space group remains the same. the reason for this fortuituous ambiguity is unknown. in addition to the ligand in both active sites (fig 2b) , another atp in the latter crystal form is bound in the vicinity of it in one monomer; this atp is also involved in crystal contacts. the binding of this second atp is a strong indication of a propensity for binding further nucleotides in the active site vicinity by e. coli ligt. residues interacting with the second atp include phe159 and arg6. again, the α-5 0 -phosphate of the active-site atp is in the same location as the corresponding phosphate in nadp + , and it is similarly coordinated by arg6, highlighting putative rna recognition features extending from the active site. as far as the 3 0 -amp experiment is concerned, one of the 4 monomers in the asymmetric unit has a bound 3 0 -amp molecule, with the phosphate group in the same position as in the 2 0 -amp product complex. binding of the ligand to the remaining monomers is apparently prevented by crystal contacts. the conformation of 3 0 -amp differs slightly from that observed for 2 0 -amp, but the essential recognition features remain the same; stacking of the base against phe48, binding of the phosphate to the two hxtx motifs in the active site, and c-h. . .π interaction of the sugar ring against phe8 ( fig 2c) . as incubation with 2 0 ,3 0 -camp resulted in 2 0 -amp, the 3 0 -amp complex is irrelevant for the reaction mechanism. to generate 3 0 -amp in the ligt reaction, the nucleophilic water should attack from the opposite side. in addition, attempts to cocrystallize ligt with trna resulted in a structure with apparent phosphate ions bound to the active site; these probably originated as impurities in the trna preparation. two distinct po 4 -binding sites are observed in the active site, on both sides of arg130 (fig 2d) . their locations could correspond to binding sites for phosphomoieties in a bound rna substrate. one of the phosphates lies in the active site, while the second is in a nearby cavity, close to arg35. the density for the second phosphate molecule was strongest in monomer d, and it was not built into the other three monomers in the model, as the electron density suggested only partial occupancy. the crystal structure of ligt with 2 0 -amp was superimposed on the corresponding complex of cnpase, in order to distinguish common and divergent ligand binding determinants in 2h enzymes. the binding mode of the reaction product is very similar in both enzymes, and the base and sugar moieties make similar interactions. while in cnpase, the n terminus of helix α7 is important in coordinating the reaction product [17] , arg130 is an important residue for binding the corresponding phosphate group in ligt ( fig 3a) . as the crystals obtained in the presence of substrate resulted in the structure of a product complex, we also modeled the likely substrate complex of ligt and 2 0 ,3 0 -camp, based on our earlier liganded complexes of cnpase [16] [17] [18] , coupled to manual docking and energy minimization. the predicted binding mode and interactions are very similar to those seen in cnpase. compared to the product binding mode, his125 is not directly interacting with the substrate, but coordinates the nucleophilic water instead (fig 3b) . within the active site region, the presence of helix α7 in cnpase is a major difference (fig 4a) . the cnpase-specific helix α7 is missing in ligt, as predicted based on sequence analysis and a structure-based alignment (fig 4b) . cnpase is the only known 2h phosphoesterase family protein with helix α7 and also the only one, for which the catalytic mechanism has been structurally characterized in detail [5, [15] [16] [17] 19 ]. in light of the x-ray crystallographic results, indicating propensity for binding of different nucleotide compunds, we probed ligand compounds also for binding to ligt in solution, in order to clarify whether the observed ligand complexes were possibly artifacts of the crystallization environment. itc was carried out for 2 0 -and 3 0 -amp, atp, and nadp + (s1 fig). the dissociation constants observed for these compounds were 140, 180, 70, and 40 μm, respectively. in light of the k m values of mouse cnpase towards 2 0 ,3 0 -cyclic nadp + of around 500 μm [27] , the k d values determined for ligt are similar and indicate binding of the studied nucleotide compounds to ligt also in solution, in addition to the crystal lattice. the active site is surrounded by several loops, which might provide flexibility in substrate binding. the α5-β6 loop, which emerges from helix α5 and leads to the strand β6 that contains one half of the catalytic residues, is disordered in many structures, but can be resolved in selected monomers in the different crystal forms. in mouse cnpase, this loop corresponds to the mobile loop α6-β5, which may play a role in cnpase interaction with larger substrates. other flexible loops close to the ligt active site include the c-terminal hairpin loop β8-β9 and the loop connecting strands β4 and β5. the ligt ligand complexes highlight mobility of the ligt loops and possible rna-interacting residues close to the active site. in different crystal forms, the active-site loops can be observed in slightly different conformations (fig 5a) , and especially residues arg6 and arg35 take different conformations depending on the bound ligand, further implying their role in phosphomoiety recognition. in a ligand-free active site, also phe48 is seen to take a conformation distinct from that seen in ligand complexes. hence, as opposed to cnpase, in which the active site is to a large extent pre-organized for substrate binding [16, 17] , ligt shows more flexibility. to get further information on the flexibility of the loops, we performed a search for the closest 2h structural homologues (fig 5b) . the salami structural homology search detected 11 homologous proteins from the pdb, the best hit representing the previous ligt structure. vertebrate cnpases were not included in this list, which had 2h proteins from bacteria, viruses, and plants; many of these are annotated as 2 0 -5 0 rna ligases or phosphoesterases. superposition of the structures indicated highly similar folding, despite sequence identities as low as 7%, and the largest differences were in the loops mentioned above. this result further highlights the flexibility of the active-site vicinal loops and suggests they may be important in rna substrate recognition in the entire 2h enzyme family. visualization of the temperature factors in the ligt crystal structure is in line with this loop flexibility (fig 5c) . the loop corresponding to the α5-β6 loop is also very flexible in mouse cnpase (fig 5d) . the dynamics of ligt were also studied using md simulations. analysis of the root mean square fluctuations (rmsf) during the simulation (fig 5e) indicates that the most dynamic segments of the protein correspond to the loops described above, surrounding the active-site cavity. the most dynamic structure appears to be the β hairpin formed between the two c-terminal β strands; interestingly, this hairpin loop is not present in the mammalian cnpase structure. some loops are more rigid in the simulation than predicted by the crystallographic b factors; this observation may be related to the fact that the reaction product 2 0 -amp was present in the active site in the simulation run. the results further highlight the flexible nature of the loops, likely to play roles in ligt substrate binding. to obtain an insight into larger ligand binding, we carried out small-angle x-ray scattering (saxs) experiments with samples of ligt and yeast trna (fig 6, table 2 ). ligt was monomeric in solution, and the obtained 3d shape corresponded closely to that seen in the crystal structure (chi 2 = 0.8 between experimental saxs data and calculated data from a ligt monomer). an exception was the highest concentration (>15 mg/ml), which fitted well to a dimeric species; the relevance of this dimerization is not known, and we believe it was an artifact of the very high concentration. trna is slightly larger and more elongated than ligt, as expected. a multiphase ab initio modeling approach, taking advantage of the different x-ray scattering properties of rna and protein, based on saxs data from the complex and both components alone was employed, and an elongated complex (fig 6c) fit the experimental data well. the result is a clear indication of the ability of ligt to bind rna molecules, in this case specifically trna. furthermore, the good fit of the 1:1 complex to the experimental data and the volume of the corresponding ab initio model both imply that the sample was mostly in complex form, with free ligt and trna not interfering with saxs analysis. whether trna would be a true biological substrate or ligand for ligt, is unclear at present, and remains a subject for future work. the fact that trna was not observed in the crystal structure can be explained by the commercial yeast trna preparation being a mixture of different trnas, not homogeneous enough to support crystallization of the complex. some viral and eukaryotic 2h enzymes cleave the 2 0 -5 0 -phosphodiester bond of 2 0 -5 0 -polyadenylates [11] [12] [13] . structural data [29] from these enzymes incidate that the 2 0 ,5 0 -adenosine bisphophate substrate binds along the opposite side of the active site, compared to ligt and cnpase (fig 7a) . in cnpase, the side used for 2 0 ,5 0 -adenosine bisphophate in these enzymes is blocked by the α7 helix, which coordinates the nucleophilic water. in ligt, this opposite side is open, and there would be room for a larger nucleophile than water, even though recent data suggest that short rna molecules would not act as nucleophiles in ligt [22] . the central phosphate moiety sits nearly identically on top of the hxtx motifs, however. combining both binding modes by superposition, interesting conservation can be observed (fig 7a) . for example, ligt trp82 is in a perfect position to stack against a base, and arg120 is conserved. in the nadp + complex, we actually observe unidentified electron density of a stacking interaction, modeled as a fragment of nadp + , on top of trp82, which could reflect low-affinity binding of some moiety in nadp + . these observations highlight several possible sites for interaction between phosphate, sugar, and base moieties in rna with residues lining the active-site groove in ligt. ligt has been characterized as an enzyme that can ligate trna fragments cleaved by yeast endonuclease. the ligation joins the 2 0 ,3 0 -cyclic phosphate and 5 0 -hydroxyl termini in a 2 0 -5 0 phosphodiester linkage, where the linking phosphate group is derived from the cyclic phosphate moiety [10] . therefore, ligt should bind rna in the close vicinity of its active site. we looked at the surface properties of ligt to better understand this process. earlier, we identified a possible rna-binding groove in mouse cnpase, which additionally has a polynucleotide kinase-like domain [17] . the surface analysis of ligt, specifically looking at electrostatics, aromatic surface residues, and basic residues, indicates that a similar surface extends away from the ligt active site. the active site is formed at the bottom of a groove lined with basic and aromatic residues, and this groove has a very high positive electrostatic potential (fig 7b and 7c ). these characteristics fit well to the hypothesis of rna binding. the substrate of ligt in an rna ligation reaction is an rna molecule, for which the 3 0 -terminal residue, with a 2 0 ,3 0 -cyclic phosphate group, sits in the active site. the orientation of the reaction products 2 0 -amp and nadp + in the active site indicates the direction, into which an rna molecule would continue, considering the last residue sits in the active site. likely participants in rna binding include several aromatic and arg residues lining the active-site groove. as the different structures predicted an rna-binding surface on ligt, we further modeled a 3-residue rna molecule with a terminal 2 0 -phosphate into the active site. possible rna recognition features can be deduced from this model (fig 7d) , which fit to the binding determinants observed for different ligands crystallographically. the predicted binding mode includes both electrostatic interactions between arg residues and the phospho groups, stacking of bases against aromatic residues, and c-h. . .π interactions of the ribose rings. the 2 0 -5 0 phosphodiester linkage between rna nucleotides is apparently rare, but is nevertheless found all across biology. in animals, 2-5as synthesized by 2 0 -5 0 -oligoadenylate synthases 2h enzyme active site structure have functions in innate immunity, whereby they specifically activate rnase l [30] . rnase l then cleaves singe-stranded viral or cellular rnas. 2-5as can also have antibacterial activity [31] . some viruses produce an enzyme that degrades 2 0 -5 0 -linked oligoadenylates and prevents the activation of rnase l [13, 32] . 2 0 -5 0 oligoadenylates have also been detected in e. coli. the level of these oligoadenylates increased as a response to phage infection, similarly to animals [20] . bacterial oligoadenylates are apparently adequate to activate rnase l, since the overexpression of recombinant mammalian nuclease resulted in rna degradation and cell growth inhibition [33] . ligt might function in the metabolism of bacterial 2-5as [20] . the original paper describing ligt identified it as having enzymatic activity resembling trna ligases [10] . these activities included the 3 0 -phosphodiesterase activity towards the 2 0 ,3 0cyclic phosphate present in the 3 0 -terminus of the 5 0 -half of the cleaved trna molecule, and the subsequent ligation of the 3 0 and 5 0 halves with a 2 0 -5 0 -phosphodiester bond between the 2 0 -phosphate group formed in the previous reaction and the 5 0 -hydroxyl group of the 3 0 -half of the cleaved trna molecule [10] . the ligation was later found to be reversible, as the enzyme additionally functions as a 2 0 -5 0 -phosphodiesterase [3, 9] . recently, however, concomitant with the publication of the first e. coli ligt structure, this view was challenged, as ligt did not appear to ligate short 10-nucleotide rna oligomers with 2 0 ,3 0 -cyclic phosphate and 5 0hydroxyl ends, but only acted on the cyclic phosphate [22] . thus, ligt was claimed to be a cnpase rather that rna ligase. it should be noted that the experimental conditions in the different studies varied, and it is hard to draw a definite conclusion at this point. our structural data do highlight close structural similarities of bacterial ligt to rna ligases and clear differences with respect to the vertebrate cnpase active site. despite the current lack of identity of the physiological activity of ligt, the crystal structures presented here further highlight the versatility of the active-site architectures in 2h phosphoesterases. the best-characterized 2h enzyme is mammalian myelin cnpase, for which a central role in the reaction mechanism is played by the n terminus of helix α7 [17] . this helix is missing in most 2h family members, including ligt, and therefore, the reaction mechanisms must also be different across the enzyme family. the α7 helix blocks access of larger nucleophiles than water into the cnpase active site, and it could be argued that its absence in ligt and most other 2h enzymes hints towards larger molecules, most likely rna, as potential nucleophiles. genomic dna from the bl21(de3) strain of e. coli was purified and used as a template for pcr (s1 table) . the initial primers for the first pcr added a tev cleavage site to the n terminus of the coded protein sequence. a second pcr reaction added attb cloning sites to both ends of the insert. the product from the latter reaction was subcloned into the pdonr221 vector (invitrogen) and further subcloned into the pth27 expression vector [34] , which adds an n-terminal his 6 tag to the expression product. clones were verified by dna sequencing and found to be identical with the ligt database entry (genbank id. am946981.2). ligt was overexpressed in the bl21(de3) strain using zym-5052 autoinduction medium [35] , supplemented with 100 μg/ml ampicillin and 0.01% antifoam 204 (sigma). the expression culture was incubated at +37˚c for 24 h. overexpression did not lead to any obvious toxic effects. the dry cell weight was around 12 g for 1 liter of culture. cells were harvested by centrifugation and resuspended in lysis buffer containing 50 mm na-hepes (ph 7.5), 500 mm nacl, 20 mm imidazole, 0.5 mm tcep, and 1x edta-free protease inhibitor (roche). the suspension was flash-frozen in liquid nitrogen and stored at -70˚c until use. the cell suspension was supplemented with 0.1 mg/ml lysozyme and sonicated. the lysate was clarified by a 30-min centrifugation at 27000 g. the clarified lysate was then mixed with ni-nta (qiagen) matrix. the matrix was washed with lysis buffer, and the protein was eluted from the matrix with elution buffer containing 500 mm imidazole. fractions were analysed using sds-page. his-tagged tev protease [36] was added to the eluted fractions containing ligt, in order to remove the his 6 tag, and this mixture was dialyzed overnight against lysis buffer devoid of imidazole. the dialyzed sample was passed through the ni-nta matrix to remove tev protease, uncleaved ligt, and any other ni-nta binding contaminants. the eluted fractions were analyzed using sds-page. fractions with cleaved ligt were concentrated and applied to a superdex 75 16/60 (ge healthcare) size exclusion chromatography column equilibrated with 10 mm na-hepes (ph 7.5), 100 mm nacl, and 0.5 mm tcep. fractions were analysed using sds-page, and the fractions containing pure ligt were pooled. approximately 25 mg of pure protein was obtained from one liter of expression culture. pure ligt was flash-frozen in small batches with liquid nitrogen and stored at -70˚c until use. for crystallization, ligt was in the gel filtration buffer at 10 mg/ml. sitting drop crystallization experiments were prepared with 2:1, 1:1, and 1:2 drop ratios. the crystallization conditions were composed of 0.1 m tris-hcl at ph 7.4-7.5, 0.2 m mgcl 2 , and peg 8000 at 12-20% (w/ v). for obtaining liganded complexes, ligt was mixed with putative active-site ligands prior to crystallization at 5 mm ligand concentration; these compounds included atp, nadp + , 2 0 ,3 0 -camp, and 3 0 -amp. crystals were cryoprotected by soaking them for a few minutes in the well solution supplemented with the ligand (if present) and 15% (v/v) peg 200. x-ray diffraction data were collected using synchrotron radiation at 100 k. data were collected on beamlines p11 and p13 [37] at petra iii/desy (hamburg, germany), as well as on beamline i911-2 at max-lab (lund, sweden). all data were processed using xds [38] . at the time of these experiments, the bacterial ligt structure had not been solved. the structure was solved here by molecular replacement with phaser [39] using as a search model the ligt orthologue structure from thermus thermophilus [40] (pdb entry 1iuh, sequence identity 28%), modified with phenix.sculptor [41] to resemble more closely the target sequence. refinement was carried out in phenix.refine [42] and rebuilding in coot [43] . final structures were validated using molprobity [44] . coordinates and structure factors were deposited at the pdb under accession codes 5ldi (apo), 5ldj (phosphate complex), 5ldk (atp complex), 5ldm (2 0 -amp complex), 5ldo (3 0 -amp complex), 5ldp (atp complex 2), and 5ldq (nadp + complex). structural sequence alignments were done with swiss pdb viewer [45] and espript [46] . for structure analyses, pymol and ucsf chimera [47] were used. superpositions were done with the ssm algorithm [48] , and structural homologues were searched using salami [49] . modeling of the substrate complex and a complex with a 3-base rna oligonucleotide was done in yasara [50] . electrostatic surfaces were calculated using pdb2pqr and abps [51] . the complex of ligt with the bound product 2'-amp was subjected to md simulations with the program yasara [50] , version 16.2.21. the structure was placed in a cubic box filled with water, the ph was kept at 7.4, and nacl was added to keep the salt concentration at the physiological 0.9% (w/w). after initial energy minimization, an md run of 260 ns was carried out at 298 k. the default yasara settings were used, saving snapshots every 250 ps, and employing the amber14 force field. yasara scripts were further used for simulation data analysis. small-angle x-ray scattering saxs data were collected in batch mode on the embl/desy synchrotron beamline p12 [52] , using standard procedures [53] . in addition to ligt alone, yeast trna (sigma) and a 1:1 molar mixture of ligt and trna were analyzed. data were processed with the atsas package [54] . distance distribution functions were analyzed using gnom [55] . for modelling ligt alone, gasbor [56] was used. for modelling the protein-rna complex, we used the program monsa [57] with the protein and rna in different phases. the trna alone was modelled using dammin [57] . molecular weight was estimated through a comparison of the forward scattering intensity of the sample to that of a fresh sample of monomeric bovine serum albumin. itc was carried out using an itc200 instrument (microcal). in brief, 0.2-0.4 mm ligt was titrated with 5-10 mm ligand (2 0 -amp, 3 0 -amp, atp, nadp + ); both the protein and ligand were in a buffer consisting of 10 mm hepes (ph 7.5), 0.2 m nacl, and 0.1 mm tcep. the titration was carried out at +25˚c, and the data were analysed using microcal origin. detection of novel members, structure-function analysis and evolutionary classification of the 2h phosphoesterase superfamily structural and functional evolution of 2',3'-cyclic nucleotide 3'-phosphodiesterase characterization of a heat-stable enzyme possessing gtp-dependent rna ligase activity from a hyperthermophilic archaeon, pyrococcus furiosus saccharomyces cerevisiae trna ligase. purification of the protein and isolation of the structural gene the myelin membrane-associated enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase: on a highway to structure and function trna splicing in yeast and wheat germ. a cyclic phosphodiesterase implicated in the metabolism of adp-ribose 1",2"-cyclic phosphate cloning and characterization of the arabidopsis cyclic phosphodiesterase which hydrolyzes adp-ribose 1'',2''-cyclic phosphate and nucleoside 2',3'-cyclic phosphates characterization of the saccharomyces cerevisiae cyclic nucleotide phosphodiesterase involved in the metabolism of adp-ribose 1",2"-cyclic phosphate the 2'-5' rna ligase of escherichia coli. purification, cloning, and genomic disruption rna ligase in bacteria: formation of a 2',5' linkage by an e. coli extract murine akap7 has a 2',5'-phosphodiesterase domain that can complement an inactive murine coronavirus ns2 gene homologous 2',5'-phosphodiesterases from disparate rna viruses antagonize antiviral innate immunity antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns2 protein is required for virus replication and liver pathology aberrant 3' oligoadenylation of spliceosomal u6 small nuclear rna in poikiloderma with neutropenia identification of essential residues in 2',3'-cyclic nucleotide 3'-phosphodiesterase. chemical modification and site-directed mutagenesis to investigate the role of cysteine and histidine residues in enzymatic activity myelin 2',3'-cyclic nucleotide 3'-phosphodiesterase: active-site ligand binding and molecular conformation crystallographic analysis of the reaction cycle of 2',3'-cyclic nucleotide 3'-phosphodiesterase, a unique member of the 2h phosphoesterase family determinants of ligand binding and catalytic activity in the myelin enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase crystal structure of the catalytic fragment of human brain 2',3'-cyclic-nucleotide 3'-phosphodiesterase the occurrence of 2'-5' oligoadenylates in escherichia coli transcriptome dynamics of deinococcus radiodurans recovering from ionizing radiation structure and mechanism of e. coli rna 2',3'-cyclic phosphodiesterase discovery and roles of 2',3'-camp in biological systems identification of cytidine 2',3'-cyclic monophosphate and uridine 2',3'-cyclic monophosphate in pseudomonas fluorescens pfo-1 culture isolation and sequence analysis of the gene (cpdb) encoding periplasmic 2',3'-cyclic phosphodiesterase the hd domain of the escherichia coli trna nucleotidyltransferase has 2',3'-cyclic phosphodiesterase, 2'-nucleotidase, and phosphatase activities expression, purification, and initial characterization of different domains of recombinant mouse 2',3'-cyclic nucleotide 3'-phosphodiesterase, an enigmatic enzyme from the myelin sheath restrained refinement of two crystalline forms of yeast aspartic acid and phenylalanine transfer rna crystals structural basis for 2'-5'-oligoadenylate binding and enzyme activity of a viral rnase l antagonist rnase l and the nlrp3-inflammasome: an old merchant in a new trade an essential role for the antiviral endoribonuclease, rnase-l, in antibacterial immunity middle east respiratory syndrome coronavirus ns4b protein inhibits host rnase l activation expression of interferon-inducible recombinant human rnase l causes rna degradation and inhibition of cell growth in escherichia coli effect of n-terminal solubility enhancing fusion proteins on yield of purified target protein protein production by auto-induction in high density shaking cultures improved solubility of tev protease by directed evolution p13, the embl macromolecular crystallography beamline at the low-emittance petra iii ring for high-and low-energy phasing with variable beam focusing automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants phaser crystallographic software crystal structure of the 2'-5' rna ligase from thermus thermophilus hb8 improvement of molecular-replacement models with sculptor towards automated crystallographic structure refinement with phenix.refine coot: model-building tools for molecular graphics molprobity: all-atom structure validation for macromolecular crystallography swiss-model and the swiss-pdbviewer: an environment for comparative protein modeling espript: analysis of multiple sequence alignments in post-script ucsf chimera-a visualization system for exploratory research and analysis secondary-structure matching (ssm), a new tool for fast protein structure alignment in three dimensions the salami protein structure search server yasara view-molecular graphics for all devices-from smartphones to workstations web servers and services for electrostatics calculations with apbs and pdb2pqr versatile sample environments and automation for biological solution x-ray scattering experiments at the p12 beamline (petra iii, desy) interaction between the c-terminal region of human myelin basic protein and calmodulin: analysis of complex formation and solution structure new developments in the program package for small-angle scattering data analysis determination of the regularization parameter in indirect-transform methods using perceptual criteria determination of domain structure of proteins from x-ray solution scattering restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing we would like to thank beamline staff for excellent support at embl/desy and max-lab. the use of the facilities and expertise of the biocenter oulu crystallization core facility, a member of biocenter finland and instruct-fi, is also gratefully acknowledged. key: cord-002901-u4ybz8ds authors: yu, chanki; yang, sejung; kim, wonoh; jung, jinwoong; chung, kee-yang; lee, sang wook; oh, byungho title: acral melanoma detection using a convolutional neural network for dermoscopy images date: 2018-03-07 journal: plos one doi: 10.1371/journal.pone.0193321 sha: doc_id: 2901 cord_uid: u4ybz8ds background/purpose: acral melanoma is the most common type of melanoma in asians, and usually results in a poor prognosis due to late diagnosis. we applied a convolutional neural network to dermoscopy images of acral melanoma and benign nevi on the hands and feet and evaluated its usefulness for the early diagnosis of these conditions. methods: a total of 724 dermoscopy images comprising acral melanoma (350 images from 81 patients) and benign nevi (374 images from 194 patients), and confirmed by histopathological examination, were analyzed in this study. to perform the 2-fold cross validation, we split them into two mutually exclusive subsets: half of the total image dataset was selected for training and the rest for testing, and we calculated the accuracy of diagnosis comparing it with the dermatologist’s and non-expert’s evaluation. results: the accuracy (percentage of true positive and true negative from all images) of the convolutional neural network was 83.51% and 80.23%, which was higher than the non-expert’s evaluation (67.84%, 62.71%) and close to that of the expert (81.08%, 81.64%). moreover, the convolutional neural network showed area-under-the-curve values like 0.8, 0.84 and youden’s index like 0.6795, 0.6073, which were similar score with the expert. conclusion: although further data analysis is necessary to improve their accuracy, convolutional neural networks would be helpful to detect acral melanoma from dermoscopy images of the hands and feet. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 in asians, melanoma is rare, compared to its prevalence in caucasians, and usually occurs in acral areas such as the hands and feet. it can be misrecognized as benign nevi (bn), is occasionally hidden by calluses, and eventually results in late diagnosis at an advanced stage, with a poor prognosis [1] [2] [3] . since effective anti-cancer agents for treating melanoma have not yet been developed, early detection and wide excision of the skin lesion is more crucial to the cure for melanoma. recently, to aid the early diagnosis of melanoma and the reduction of unnecessary skin biopsy, dermoscopy has been widely used [4, 5] . moreover, because it is difficult for nonexperts to use [6] , artificial intelligence and deep-learning models have been applied to help physicians who are untrained to handle a digital dermoscope [7] ; its use is expected to increase in the field of teledermatology. a convolutional neural network (cnn) is one of the representative models among the various deep-learning models. it has already shown potential for general and highly variable tasks across many fine-grained object categories [8] [9] [10] [11] [12] and has been shown to exceed human performance in object recognition [9] . recently, it was applied to detect skin cancers in images, including from dermoscopy, and successfully demonstrated artificial intelligence capable of classifying skin cancer with a competence level comparable to that of dermatologists [13] . for the success of cnn models, a large amount of training data labeled with class types to produce a rich feature hierarchy is necessary, and therefore, its usefulness in the diagnosis of rare diseases with insufficient data has not been fully established. in this study, we applied an end-to-end cnn framework to detect a rare disease in asians, acral melanoma (am), from the dermoscopy images of pigmentation on the hands and feet. to overcome the insufficiency of the datasets, we adopted a transfer learning technique to leverage learned features from a cnn model pre-trained on a large-scale natural image dataset [14] . moreover, we also applied a half-training and half-trial method to validate its clinical usefulness for the early diagnosis of patients compared with the dermatologist's and non-expert's evaluation. a total of 724 dermoscopy images were collected from january 2013 to march 2014 at the severance hospital in the yonsei university health system, seoul, korea, and from march 2015 to april 2016 at the dongsan hospital in the keimyung university health system, daegu, korea. among them, 350 dermoscopy images were from 81 patients with am and 374 images were from 194 patients with bn of the acral area (fig 1) . a total of 632 dermoscopy images were captured by the dermlite cam (3gen inc., usa), and 92 images were captured by the dermlite hybrid ii (3 gen inc., usa), connected to a digital camera (nikon coolpix p6000, japan). all diagnoses were histopathologically confirmed and multiple images were captured in cases of large lesions. we provide a strobe checklist for the study of diagnostic efficacy as supporting information (s1 table) . dermoscopy images of bn were divided into nine types, and am images into three types according to the reference [15] , by two dermatologists. this study protocol was approved by the institutional review board of yonsei university, severance hospital and keimyung university, dongsan hospital and was conducted according to the declaration of helsinki principles. patient records/information was anonymized and deidentified prior to analysis. we have described the cnn architecture we adopted in section 2. 1 and presented the training and inference methods for detecting melanoma in section 2. 2. 2.1 convolutional neural network. cnns are composed of several convolutional layers, each involving linear and nonlinear operators, as well as fully connected layers. the architecture for the state-of-the-art cnn has many parameters; for example, the vgg-16 model has 138 million parameters, where the parameters are learned from the imagenet dataset containing 1.2 million general object images of 1,000 different object categories for training [16] . deep neural networks are difficult to train using small datasets (i.e., a few hundred images). to circumvent this problem, we used the fine-tuning technique, which is one of the regularization techniques. we fine-tuned a modified vgg model with 16 layers (13 convolutional and three fully connected layers), which uses the convolution filters of the same size (i.e., 3 × 3) for all convolution layers, as seen in table 1 . our network configuration is depicted in fig 2 and table 1 . each layer and feature map in the cnn is represented by a three-dimensional array of table 1 ; "conv" represents a convolutional layer and "fc" represents a fully connected layer). the input with a fixed-size, 224 × 224, was passed through a stack of convolutional layers, where each followed a rectified linear unit (relu) activation function, and max-pooling was performed over a 2 × 2 pixel window with a stride of 2. a series of convolutional layers (conv1, conv2, conv3, conv4, and conv5) were followed by three fully connected layers: the first 2 fully connected layers (fc6 and fc7) had 4,096 channels each, where each followed a relu activation function, while the last fully connected layer (fc8) had 2 channels since our problem was a two-way classification problem (melanoma and non-melanoma class). it should be noted that the number of channels of the last fully connected layer was the same as the number of classes. hence, we replaced the original fully connected layer (fc8: fc with 1000 channels) with a fully connected layer with two channels. the last layer had the soft-max activation function and predicted whether the input patch was a melanoma or non-melanoma lesion. moreover, the vgg-16 model pre-trained on the imagenet database are used to perform transfer learning, and the weights of the last convolutional layers (the last two layers of conv5) and three fully convolutional layers (fc6, fc7, and fc8) are initialized using xavier weight initialization [17] . in order to perform fine-tuning, we froze the weights of conv1, conv2, conv3, conv4, and the first layer of conv5 on pre-trained imagenet, and trained the initialized weights on our dermoscopy image dataset. the above procedure is performed to prevent the large gradient caused by randomly initialized weights from ruining the pre-trained weights. after several training epochs, we trained the all weights of our network without freezing any layer. our dataset consisted of 724 images and associated labels, which were split into two mutually exclusive subsets (group a and b); half of the total image dataset was selected for training and the rest for testing. the scale and location of a skin lesion in a captured image were changed according to the capture conditions. to resolve this issue, we adopted a sliding window strategy and used the cropped patches instead of the full image at the training and inference time. at the inference time, we extracted about 12 image patches from each test image on a regularly spaced grid with a partial overlap between neighboring patches and then each patch was rescaled to the size of 224 × 224 pixels, as seen in fig 3. in addition, to increase the robustness of the variation of geometric transformation in our cnn model, the training dataset was artificially augmented at training. additional augmented data were formed by rotating and flipping images from the original training set. we generated 216 image patches from a single image using rotations by 0˚, 45˚, 90˚, and 135˚, as well as leftright and top-bottom reflections. in addition, the patches that did not contain any melanoma lesions among the melanoma training images were manually removed and the patches that did not contain any skin lesions among the non-melanoma training images were assigned to the non-melanoma class at training time. we randomly selected 30% of the training dataset as a validation set and the rest as a training set at the onset of training. the validation data were used to prevent the overfitting of the training data and to provide guidance on when to stop training the network. the training of our cnn was stopped when the validation error on the validated dataset stopped decreasing. we trained the network using an adaptive stochastic sub-gradient method where the batch size is set to 50, and the momentum parameter, learning rate, and weight decay are set to 0.9, 0.0001, and 0.0005, respectively. some of the filters learned from our melanoma dataset may be seen in fig 4. fig 4(a) shows 64 learned filters at the 1 st convolutional layer, where each represents a learned filter with a 3 × 3 kernel size. the input of the first layer is an rgb image with 224x224 pixel size, and it is convolved with 64 learned filters with 3x3 kernel size as shown in fig 3(a) and 64 feature maps with 224x224 size are generated. in addition, the output feature maps are used as the input of the next layer. fig 4(b) -4(m) shows 100 filters among the learned filters from the 2 nd to the 13 th convolution layer, respectively, where each represents a learned filter with a 3 × 3 kernel size. at the time of inference, we interpreted 12 image patches per test image, and when one or more images were predicted as containing melanoma, the corresponding test image was interpreted as containing melanoma. each input of the network was an rgb image subtracted from the average image and calculated over the entire training image dataset. we implemented our method using matconvnet, a matlab-based cnn framework for computer vision applications [18] . moreover, we fine-tuned a vgg model with 16 layers downloaded from http:// www.vlfeat.org/matconvnet/pretrained). to assess the clinical usefulness of the cnn, we compared its diagnostic rate with those of two dermatologists who had five or more years of clinical experience in dermoscopy (expert group) and two non-trained general physicians (non-expert group). all images on the computer screen were evaluated simultaneously. if there was a dissensus between two physicians, they reached a conclusion under the agreement. since 724 images were randomly and equally the agreement between the pathologic result and each rater's diagnosis was measured using the calculation of cohen's kappa coefficient. all statistical analyses were performed with medcalc software version 17.9. (po = accuracy, pe = hypothetical probability of a chance agreement) among 724 dermoscopy images, 71 images were from the hands and fingers, and the others were from the feet and toes. a total of 350 am images included homogenous diffuse irregular pigmented, parallel ridge, and multicomponent patterns, while 374 bn images included parallel furrow, fibrillar, lattice-like, reticular, globular, and homogenous patterns (s1 table) . in the group a results obtained by the training of group b images, cnn showed 92.57% sensitivity and 75.39% specificity, which were similar to those of the expert (94.88% and 68.72%, respectively). however, the non-expert showed lower sensitivity (41.71%) and relatively higher specificity (91.28%, table 2 ). for diagnostic accuracy, both the cnn and expert group showed similar scores (83.51% and 81.08%, respectively), which were higher than that of the non-expert (67.84%, fig 5) . in the result of group b by the training of group a images, cnn also showed a higher diagnostic accuracy (80.23%) than that of the non-expert (62.71%) but was similar to that of the expert (81.64%). for validating diagnostic reliability, both the cnn and expert showed an auc above 0.8 in group a and b (fig 5) . however, the non-expert regarding the concordance rate between the cnn and expert group, 73 cases (73/362, 20.17%) in group a (am: 14 cases, bn: 59 cases) were discordant. of these, 41 cases (56.16%) of the cnn and 32 cases (43.84%) of the expert were identical with the pathologic results. however, in the concordant cases between them, 29 cases (29/362, 8.01%) differed from the pathology reports. in group b, 57 cases (am: 12 cases, bn: 45 cases) showed discordance between the cnn and expert, and 26 cases (45.61%) of the cnn and 31 cases (54.39%) of the expert were identical with the pathologic results. among the concordant cases in group b, 39 cases (39/362, 10.77%) differed from the pathology results. cohen's kappa between cnn and expert, cnn and non-expert, expert and non-expert is shown in table 3 . to verify the performance of cnn architecture for the discrimination of acral melanoma, we perform the deep learning architecture, inception-v3, in [13] , the state-of-the-art publication for the classification of skin cancer. in [13] , a single image was used for learning. meanwhile, we applied multiple images for learning. thus, we compared inception-v3 with a single image and inception-v3 with multiple images to cnn with multiple images. the results are shown in table 4 . although non-invasive and automated diagnostic techniques have been introduced for the early detection of melanoma, they are still not easy to apply in the acral type [7, 19] . this may be due to the overall low occurrence rate of melanoma in asians, depending on the ethnic differences, which need a longer time to provide a sufficient dataset to improve diagnostic accuracy. to overcome the problem of an insufficient dataset, we adopt a 2-fold cross validation method, for the training and test groups. in addition, capturing images at different places for one lesion helps to construct a robust cnn. similarly, data augmentation generating virtual images using rotation, translation, different angle positioning from one image also helps for a robust cnn. these procedures are necessary to construct an automated diagnosis system from small datasets due to the low occurrence rate of acral melanoma. for the effective screening of melanoma, higher sensitivity is required. thus, if there is a small compartment corresponding to the melanoma in one image, our system considers it as melanoma. also, our system recognizes one image as one patient. from the results, the accuracy of the cnn was above 80%, which was similar in both groups and was close to that of the expert. the cnn and expert also showed auc values above 0.8, indicating good discrimination. generally, higher auc values are considered to demonstrate better discriminatory abilities as follows: excellent discrimination, auc of !0.90; good discrimination, 0.80 auc < 0.90; fair discrimination, 0.70 auc < 0.80; and poor discrimination, auc of <0.70 [20] . since the auc of the non-expert was lower than 0.7, cnn can be a useful tool for the early detection of am by the physicians who are not familiar with the dermoscopic images. moreover, additional datasets of am images can improve the diagnostic accuracy of cnn [21] , making it a more reliable tool for the evaluation of the need for skin biopsy for hand and feet pigmentation. there were several auto-classification methods independent of the size of training data using dermatologists' checklist, such as the abcd rule and 7-point scale [22] [23] [24] [25] . this method used particular features such as color, shape, size, the boundary of the skin lesion, and statistical features of wavelength, which showed 91.26% of accuracy and 0.937 auc value [25] . however, these cannot be directly applied to acral melanoma due to the different morphologic features such as ridge or furrow patterns. although there was a new dermoscopic algorithm reflecting these characteristics for diagnosing acral melanoma: braaff [26] , it has not yet been applied to the automated diagnosis. in addition, although there is a state-of-art automated classification method for acral melanoma, these methods cannot be generalized and only work well for a particular pattern of acral melanoma, which is a ridge-and-furrow pattern [27] . automated diagnosis methods using particular features are able to reflect experts' perception and the speed of performance is fast. however, it is not easy to catch experts' perception, although we are trying to reach the goal with significant features. on the other hand, deep learning does not require specific features as inputs. it automatically finds the most correlated features with expert's perception by learning. thus the accuracy is higher than feature-based methods. however, a large database is critical for the successful completion of deep learning. recently, the melanoma classification performance of cnn using 1,010 dermoscopy images was reported as having an auc of 0.94 [13] , which was higher than noted in our results (0.84, 0.8). our inferior results may be due to the characteristics of am; it occurs on the pressure area, thick skin, callus, etc., which can hinder and transform the classic pigmented lesion into an atypical case. because of this, experts in our experiment also showed an auc of 0.82. therefore, if the datasets are analyzed separately considering these anatomic characters, cnn may perform a more precise discrimination. furthermore, if combined with images from noninvasive devices for melanoma diagnosis, which may overcome the problems presented by a thick skin, the accuracy of cnn can be markedly improved. several non-invasive devices such as confocal and photon microscopy are being introduced to provide convenient ways to diagnose melanoma early [28] . however, they require much effort and time for a physician to gain expertise. an automated diagnostic system using a cnn, even with a small dataset, may alleviate the difficulty of learning how to use these newly developed devices. in conclusion, a half-training and half-trial method were useful for creating a comparatively accurate deep-learning model from a relatively small dataset. although further data analysis is necessary to improve its accuracy, cnn would be helpful for the early detection of am, which is usually associated with delayed diagnosis and poor prognosis. supporting information s1 conceptualization: sejung yang, byungho oh. treatment and outcomes of melanoma in acral location in korean patients epub 2010/05/26 plantar malignant melanoma-a challenge for early recognition improvement in survival rate of patients with acral melanoma observed in the past 22 years in sendai biopsy of the pigmented lesion-when and how dermoscopy of pigmented skin lesions: results of a consensus meeting via the internet diagnostic accuracy of dermoscopy systematic review of dermoscopy and digital dermoscopy/ artificial intelligence for the diagnosis of melanoma deep learning imagenet large scale visual recognition challenge imagenet classification with deep convolutional neural networks. advances in neural information processing systems rethinking the inception architecture for computer vision deep residual learning for image recognition dermatologist-level classification of skin cancer with deep neural networks convolutional neural networks for medical image analysis: full training or fine tuning? an atlas of dermoscopy very deep convolutional networks for large-scale image recognition understanding the difficulty of training deep feedforward neural networks matconvnet: convolutional neural networks for matlab. proceedings of the 23rd acm international conference on multimedia non-invasive tools for the diagnosis of cutaneous melanoma can routine laboratory tests discriminate between severe acute respiratory syndrome and other causes of community-acquired pneumonia? revisiting unreasonable effectiveness of data in deep learning era computer image analysis in the diagnosis of melanoma reliability of computer image analysis of pigmented skin lesions of australian adolescents combination of features from skin pattern and abcd analysis for lesion classification computer-aided diagnosis of melanoma using border and waveletbased texture analysis the braaff checklist: a new dermoscopic algorithm for diagnosing acral melanoma. the british journal of dermatology ridge and furrow pattern classification for acral lentiginous melanoma using dermoscopic images innovations and developments in dermatologic non-invasive optical imaging and potential clinical applications key: cord-001088-dugsh7mp authors: kim, so-hee; kim, joo young; choi, youngjoo; nguyen, huan h.; song, man ki; chang, jun title: mucosal vaccination with recombinant adenovirus encoding nucleoprotein provides potent protection against influenza virus infection date: 2013-09-25 journal: plos one doi: 10.1371/journal.pone.0075460 sha: doc_id: 1088 cord_uid: dugsh7mp influenza vaccines that target the highly variable surface glycoproteins hemagglutinin and neuraminidase cause inconvenience of having vaccination every year. for this reason, development of universal vaccines targeting conserved viral components is needed. in this study, we generated recombinant adenovirus (rad) vaccine encoding nucleoprotein (np) of a/pr/8/34 influenza virus, designated rad/np. balb/c mice were immunized intranasally or sublingually with rad/np vaccine and subsequently challenged with lethal doses of heterologous as well as homologous influenza viruses. we found that intranasal immunization of rad/np elicited strong mucosal iga responses as well as stronger cd8 t-cell responses toward immunodominant k(d)-restricted np(147-155) epitope than sublingual immunization. importantly, only single intranasal but not sublingual immunization of rad/np provides potent protection against both homologous and heterologous influenza virus challenges. these results suggest that recombinant rad/np could be a universal vaccine candidate for mucosal administration against influenza virus. influenza virus is an important respiratory pathogen accounting for 3-5 million infections and responsible for 250,000-500,000 deaths annually worldwide [1] . recently, newly emerging influenza virus subtypes have infected humans and caused significant public health concerns. for example, since several human cases of highly pathogenic h5n1 avian influenza virus infection have been first reported in hong kong in the late 1990s, hundreds of additional confirmed cases of human infection by h5n1 virus have been reported with a lethal outcome in over 50% of the documented cases [2, 3, 4] . also, in 2009, a new swine/human/avian-origin h1n1 influenza virus emerged in mexico and resulted in a worldwide pandemic [5] . moreover, recent outbreak of h7n9 avian influenza virus in china has claimed multiple human lives, while the numbers of reported human cases are growing continually [4] . hence, these examples underscore the necessity for better preparedness against potential influenza virus pandemic caused by different influenza virus strains. vaccination is the most cost-effective way to control and/or prevent influenza outbreaks. however, live-attenuated and inactivated influenza vaccines that are currently licensed for human use are designed to induce strain-specific humoral immunity and cannot offer cross-protection against different strains of influenza virus expressing sequentially and/or conformationally related, but unique, viral surface proteins generated by random antigenic changes that influenza virus frequently undergo. thus, development of vaccines that offer broad-range protection against multiple strains of influenza virus can be immensely beneficial for public health. for development of such influenza vaccines, it is important to consider that the immune response elicited by the vaccination targets viral antigens that are highly conserved among multiple influenza virus strains. influenza virus nucleoprotein (np) contains a conserved immunodominant cd8 t-cell epitope which is associated with the induction of cross-protective immunity against heterologous and heterosubtypic influenza virus infections [6, 7, 8] . it has been previously shown that immunization with recombinant adenovirus (rad) vaccines encoding conserved influenza antigens such as np and m2e generated cross-reactive immune responses, which can provide protection from lethal virus challenge in mice [9, 10, 11] . accordingly, in our present study, we generated a recombinant adenovirus expressing fulllength np (rad/np) derived from influenza virus a/puerto rico/ 8/1934 (pr8) and evaluated its potential as a mucosal vaccine candidate that could offer broad-range cross-protection against multiple strains of influenza virus. we focused on the advantages of adopting mucosal vaccination strategy, which has been shown to effectively target both systemic and mucosal immunity, over parenteral vaccination strategy [12, 13, 14] . additionally, we compared the vaccination-induced immune responses generated following administration of our candidate vaccine virus via two different, intranasal and sublingual, mucosal routes. female balb/c mice (5 week-old) were obtained from orient bio (seoul, korea). all of the mice were maintained under specific pathogen-free conditions in the experimental facility at the ewha womans university. the viral rna from pr8 virus was acquired by using qiaamp minelute virus spin kit (qiagen, valencia, ca) in accordance with the manufacturer's instructions. the cdna corresponding to np gene was generated by rt-pcr using forward primer (5'-gggtaccgccaccatggcgtcccaaggcacc-3') which contains a kpni restriction enzyme site and the kozak sequence to enhance translation and the reverse primer (5'-ttctagattaattgtcgtactcctc-3') that contains a stop codon and an xbai restriction enzyme site at 3' terminus. the whole open reading frame was then digested with kpn i/xba i double digestion and inserted into the pshuttle-cmv vector. for generation of replication-defective adenovirus (serotype 5), the np sequence was first inserted into adenovirus genome through homologous recombination as described previously [15] . subsequently, recombinant adenoviral dna containing the np gene was transfected into hek293 cells to generated rad/np virus. mock adenovirus (rad/mock) was generated by the same method using the vacant pshuttle-cmv vector. the recombinant adenoviruses were amplified on hek293 cells and purified by double cscl 2 density-gradient ultracentrifugation. the expression of np protein by rad/np was confirmed by infecting hek293 cells at multiplicity of infection (moi) of 10 and immunoblotting using mouse polyclonal pr8-specific antiserum and horseradish peroxidase (hrp)-conjugated goat anti-mouse igg (invitrogen, eugene, or) as a secondary antibody. to extract cellular proteins from the infected-hek293 cells, cell lysates were prepared by resuspending cell pellets with a buffer containing 50 mm tris-hcl, 150 mm sodium chloride, 1% np-40, 0.5% sodium deoxycholate and 0.1% sds, and clear supernatants and pellets were separated by centrifugation. female balb/c mice were kept under specific pathogen-free conditions. for vaccination, 5 week-old mice were inoculated with varying doses of rad/np vaccine through intranasal (i.n.) or sublingual (s.l.) route. for i.n. immunization, mice were lightly anesthetized by isoflurane (ifran®; hana pharm, kyonggi-do, korea), and 1 × 10 7 to 1 × 10 8 plaque forming unit (pfu) of rad/np or 1 × 10 8 pfu rad/mock in a volume of 50 µl of phosphate-buffered saline (pbs) was applied to the both nostrils. for s.l. immunization, mice were anesthetized by intraperitoneal injection of 100 mg/kg of body weight ketamine (yuhan co., seoul, korea) and 10 mg/kg of body weight xylazine hydrochloride (bayer, kyonggi-do, korea), as described elsewhere [16, 17] . and then forceps were placed under the tongue of the mouse and its mouth was stretched open. the total volume of vaccines were kept to <5 µl to avoid swallowing effects. the s.l. groups were secondarily immunized by the same procedure two weeks after primary immunization. three weeks after last immunization, mice were lightly anesthetized by isoflurane and challenged i.n. with 10 ld 50 of pr8, ca04, a/philippines or a/vietnam. all animal studies were performed according to the guidelines of ewha womans university institutional animal care and use committee (iacuc, approval no. 2011-01-032). at five days post challenge, subsets of mice were sacrificed and tracheotomy was executed. the lung airways were washed with 1 ml of pbs. the collected bronchoalveolar lavage (bal) fluid was centrifuged and supernatants were used for measuring secretory iga titers. blood was acquired from the retro-orbital plexus by a heparinized capillary tube, centrifuged and serum collected was stored at -70°c. antibody titers from immunized mice were measured by a direct enzyme-linked immunosorbent assay (elisa). briefly, for coating antigens, 1.8 × 10 7 pfu of pr8 virus in allantoic fluid was disrupted with 0.5% triton x-100. next, 96-well plates were coated with 100 μl/well of split influenza virus diluted in pbs (1 : 500) and incubated overnight at 4°c, and then blocked with pbs containing 1% non-fat milk and 0.05% tween 20 for 2 h at rt. samples of sera or bal fluids were added in serial dilutions and incubated for 2 h. after a washing step with pbs containing 0.05% tween 20, hrp-conjugated goat anti-mouse igg (invitrogen) or hrp-conjugated goat anti-mouse iga (zymed laboratories, san francisco, ca) as a secondary antibody to measure np-specific igg in the sera or np-specific iga in the bal fluids, respectively. for color development, 3,3', 5,5'-tetramethylbenzidine (kpl, gaithersburg, md) was added and stopped by adding 1 m h 3 po 4 . the color development was analyzed at 450 nm by a thermo multiskan® ex (vantaa, finland). five days after influenza virus challenge, a subset of each group was euthanized and the influenza vial titers in the lungs were measured as described elsewhere [18] . briefly, the lung tissues were removed into pbs and processed through a 70µm cell strainer (bd labware, franklin lakes, nj) with 3 ml mem. the supernatants were collected by centrifugation, and virus titers in the supernatants were analyzed by standard plaque assay on subconfluent mdck cells. the data are expressed as the pfu per gram of lung tissue. the lungs perfused with 5 ml pbs including 10 u/ml heparin (sigma, st. louis, mo) using a syringe with 25-gauge needle through the right ventricle were dissected and collected. to obtain single-cell suspensions, the tissues were homogenized with 3 ml iscove's modified dulbecco's medium (imdm) through 70-µm cell strainers. following centrifugation, lymphocytes were resuspended in fresh imdm and erythrocytes were removed by red blood cell lysing buffer (sigma). after washing with imdm, cells were washed two times with facs buffer (0.5% fbs, 0.09% nan 3 in pbs) and were blocked with purified rat anti-mouse cd16/cd32 (bd pharmingen, san diego, ca) and 5 µg/ml streptavidin (invitrogen). then, cells were stained with anti-mouse cd8a-apc (clone 53-6.7; biolegend, san diego, ca), anti-mouse cd44-fitc (clone im7; biolegend) and k d / np 147-155 (tyqrtralv)-tetramer-pe. after staining, the cells were fixed in pbs-2% (wt/vol) paraformaldehyde and analyzed using facscalibur flow cytometer (bd biosciences, san diego, ca) and flowjo software (treestar inc., ashland, or). all data were plotted as mean±standard error (n=5) and the difference comparison was conducted by using an unpaired, two-tailored student t-test. the difference was considered statistically significant when p values were ≤ 0.05. the full length coding region of np gene from pr8 under the control of cmv promoter followed by a kozak sequence, which enhance the translation of gene inserts, and polyadenylation stop signal (polya) was inserted into early region 1 (e1) of the adenovirus genome by homologous recombination, resulting in the generation of recombinant replication-defective adenovirus expressing pr8-dervied np (rad/np) ( figure 1a ). the proper expression of np in our recombinant adenovirus was evaluated by infecting hek293 cells with rad/np and conducting immunoblot analysis with the infected cell lysates using pr8specific polyclonal antibody. we detected a robust, single band at approximate molecular weight of 56 kda representing np in rad/np-infected hek293 cell lysates. however, no such band was detected in uninfected hek293 cell lysates or rad/mockinfected hek293 cell lysates that were used as negative controls ( figure 1b) . in order to determine the optimal route and dose of rad/np vaccination to elicit appropriate influenza virus np-specific immune responses, balb/c mice were inoculated intranasally or sublingually with either 1×10 7 or 1×10 8 pfu of rad/np. also, mice were immunized intranasally with 1×10 8 pfu of rad/mock to be used as negative control. in our preliminary studies, a single s.l. immunization with rad/np was insufficient to produce detectable immunogen-specific antibody levels in balb/c mice (data not shown). therefore, in order to enhance the efficacy of immunogen-specific antibody responses for the s.l. vaccination, sublingually primed mice were boosted two weeks after their first immunization using the same vaccination scheme used during their priming. however, all mice immunized intranasally received a single immunization with the indicated dose of rad/np or rad/mock. subsequently, three weeks after their respective final immunization, sera were harvested from all immunized mice, and the levels of pr8-specific igg in the immune sera were determined via elisa using detergentdisrupted pr8 virus as the coating antigen. all i.n. and s.l. vaccination groups that received rad/np elicited significant levels of pr8-specific serum igg compared to control mice given rad/mock (figure 2a) . interestingly, while mice immunized via s.l. route generated comparable levels of pr8specific serum igg titers regardless of the administered vaccine dose, mice immunized via i.n. route showed significant dose-dependent differences in serum igg titers between the two vaccination doses used. further, when pr8-specific serum igg levels were determined in the sera harvested from pr8infected mice at day 5 post-challenge, we observed significant increase in the pr8-specific igg titers in mice vaccinated intranasally with either dose of rad/np. however, such increase in pr8-specific serum igg titers following pr8challenge was not observed in sublingually vaccinated mice. in summary, these data indicate that both i.n. and s.l. immunizations with rad/np elicit considerable levels of pr8 np-specific serum iggs and that pr8-challenge following rad/np immunization significantly increases pr8-specific serum igg levels, compared to that of pre-challenge levels, in intranasally immunized mice, but not in sublingually immunized mice. additionally, when the levels of np-specific mucosal iga in bal collected at day 5 post-pr8-challenge were evaluated, we observed that extensive levels of pr8-specific iga titers were detected in mice vaccinated intranasally with rad/np regardless of the administered vaccine dose. we also observed that pr8-challenge following 1×10 8 pfu of rad/np immunization increases pr8-specific mucosal iga levels, compared to that of pre-challenge levels, in intranasally immunized mice. as expected, bal fluid collected from mice immunized with rad/mock contained no detectable levels of pr8-specific mucosal iga. interestingly, however, we did not detect pr8-specific mucosal igas in bal collected from mice that were given rad/np via s.l. route ( figure 2b ). taken together, these results suggest that rad vector-based vaccine expressing the np gene of pr8 virus is capable of eliciting npspecific immune responses and that a single immunization with rad/np via i.n. route sufficiently induces strong systemic as well as mucosal immunity to pr8 virus as represented by increased serum igg and mucosal iga levels, respectively. previous studies have reported that influenza virus np contains a k d -restricted immunodominant cd8 t-cell epitope between amino acids from 147 to 155, against which dominant cd8 t-cell responses are elicited during influenza virus infection. in order to determine the ability of rad/np vaccination to induce cd8 t-cell responses against the mentioned epitope, the levels of k d /np 147-155 tetramer-specific cd8 t-cell recruitment in the lungs following rad/np vaccination and subsequent pr8-challenge were evaluated. briefly, mice were immunized i.n. once with either 1×10 7 or 1×10 8 pfu of rad/np or immunized s.l. with rad/np using the same prime-and-boost vaccination regimen as aforementioned. again, a group of mice immunized i.n. with rad/mock was used as our negative control. all immunized mice were then challenged with a lethal dose of pr8. at day 5 post-challenge, mice lungs were harvested and k d /np 147-155 tetramer-specific cd8 t-cell frequency was determined via flow cytometry. as expected, we observed significant increase in the percentage (of total lung cd8 t cells) of k d /np 147-155 tetramer-specific cd8 t cells in the lungs of mice that received rad/np via i.n. route compare to the lungs of control mice that received rad/mock (figure 3) . moreover, this increase in np-specific cd8 t-cell recruitment was dose-dependent, as higher percentage (of total lung cd8 t cells) of np-specific cd8 t cells were detected in mice that received 1×10 8 pfu dose compared to those that received 1×10 7 pfu (figure 3 ). however, we did not observed any significant differences in the levels of k d /np 147-155 tetramerspecific cd8 t cells in any of the s.l. immunized groups compared to rad/mock immunized control group, notwithstanding the dose of rad/np vaccine administered ( figure 3 ). such phenomena should be underscored as similar trend was observed in the pr8-specific serum igg and mucosal iga levels between the animals that received differential doses of rad/np vaccine via i.n. route. in order to determine whether rad/np immunization confer protection against homologous influenza virus infection, mice were challenge with 10 ld 50 of live pr8 virus, three weeks after their last respective immunization regimens. our data show that all groups that received i.n. immunization of rad/np, regardless of the administered vaccination dose, survived the lethal pr8 challenge and demonstrated considerable resistance to weight loss ( figure 4a and b) . however, all mice that received either dose of rad/np via s.l. route as well as control mice that received rad/mock or un-immunized naïve mice succumbed to pr8 infection by day 9 post-challenge ( figure 4a and b) . interestingly, however, lung virus titers at day 5 post-challenge were detected at similar levels in all immunization groups ( figure 4c ). it is probable that rad/np may induce long-lasting innate immunity that contributes complementarily with other specific immune arms to the control of the disease by uncharacterized mechanisms [19] . as a result, the protection may not necessarily correlate with virus titers detected in the lungs upon lethal challenge. overall, these results indicate that i.n. immunization of rad/np can confer complete protection against the lethal homologous virus challenge while allowing competent virus replication to perpetuate even to day 5 post-challenge. in order to determine whether immunization with rad/np can offer cross-protection against heterologous and/or heterosubtypic influenza infections [20] , immune mice were challenged at 3 weeks after their last respective immunization regimen. first, to investigate the protective efficacy of rad/np immunization against heterologous influenza virus infection but with the same h1n1 subtype, mice were challenged with a lethal dose of ca04 and monitored daily for body weight change and mortality. the group of mice that received i.n. immunization of 1×10 8 pfu of rad/np experienced some weight loss, but 100% of the animals survived the lethal challenge. the group that received i.n. immunization of 1×10 7 pfu of rad/np experienced somewhat heavier weight loss compared to the group that received the higher dose, and 60% of the animals survived the challenge ( figure 5 ). however, all animals that received s.l. immunization experienced substantial weight loss. interestingly, we observed that 20% of the animals that received 1×10 7 pfu of the vaccine virus via s.l. route survived the lethal challenge whereas all animals that received 1×10 8 pfu of the vaccine virus via the same route succumbed to the challenge. next, in order to evaluate heterosubtypic protection offered by rad/np vaccination, mice were challenged with a lethal dose of influenza a/philippines (h3n2) or a/vietnam (h5n1) and monitored daily for body weight change and mortality. all mice immunized with rad/np via s.l. route, notwithstanding the administered vaccine dose, suffered considerable weight loss before succumbing to death within 8 days following a/ philippines influenza virus challenge ( figure 5 ). in contrast, both groups of i.n. immunized animals that received either 1×10 7 or 1×10 8 pfu of the vaccine were completely protected and experienced minimal weight loss following a/philippines challenge ( figure 5) . overall, influenza a/vietnam challenge produced steeper weight loss in animals compared to other influenza virus challenges. both s.l. immunized groups that received either 1×10 7 or 1×10 8 pfu of the vaccine suffered quick and severe weight loss upon h5n1 challenge and, thereafter, succumbed to death within 6 days ( figure 5 ). the group that received 1×10 7 pfu of rad/np via i.n. route also experienced severe weight loss comparable to s.l. immunized groups and were not protected from the lethal challenge. interestingly, however, i.n. immunization with 1×10 8 pfu of rad/np provided 40% of protection against the challenge with the same dose of h5n1 virus ( figure 5b ). taken together, our data demonstrate that rad/np immunization via i.n. route, but not s.l. route, provides complete protection against heterologous h1n1 and heterosubtypic h3n2 virus challenges and suggest that a high dose intranasal immunization may be required to confer protection against h5n1 virus challenge. complete protection offered by i.n. immunization of rad/np during heterologous h1n1 or heterosubtypic h3n2 virus challenge rendered us to investigate whether the observed cross-protection correlates with the magnitude np-specific cd8 t-cell responses, given that influenza np contains a conserved immunodominant cd8 t-cell epitope as indicated previously. in order to determine the possible presence of such correlation, blood lymphocytes of mice challenged with 10 ld 50 dose of influenza a/philippines were analyzed by k d /np 147-155 -tetramer staining from day 0 to day 12 post-challenge. as expected, all animals that received control rad/mock immunization or rad/np immunization via s.l. route succumbed to death by day 7 postchallenge ( figure 5 ). however, i.n. immunization with rad/np, notwithstanding the administered dose, conferred complete protection ( figure 5) . moreover, at day 6 post-challenge, significant increases in the percentage of np tetramer-positive blood cd8 t lymphocytes were observed in mice immunized via i.n. route, and greater proportion of np-specific blood cd8 t lymphocytes was observed in mice that received i.n. immunization of 1×10 8 pfu dose of rad/np than in mice that received 1×10 7 pfu dose ( figure 6 ). taken together, our results strongly suggest that np-specific cd8 t cells, primed by i.n. rad/np immunization, can be successfully recalled during a subsequent heterosubtypic influenza virus infection and can be ascribed to cross-protection observed in previous figures. recent abrupt outbreak of influenza pandemic caused by a new swine/human/avian-origin influenza a (h1n1) virus raised a significant concern for global public health and awareness for the necessity of better preparedness against potential recurrence of influenza pandemic. currently, inactivated and live-attenuated influenza vaccines are widely used for vaccination in humans. however, the efficacy of these vaccines largely depends on the antigenic relatedness of the vaccine virus to the circulating influenza virus strains. given that influenza virus is a highly dynamic virus that readily undergoes genetic shifts and drifts, development of vaccines that offer previous studies have identified influenza a virus np as a major target antigen for cross-reactive influenza virus-specific cd8 t cells and suggested that strategy to induce np-specific cd8 t cells should be considered for the development of broadly protective influenza virus vaccines [21, 22, 23, 24] . accordingly, we engineered a recombinant adenovirus expressing full-length np derived from pr8 influenza virus as a potential vaccine candidate in order to investigate whether priming for np-specific immune responses could offer crossprotection against different strains and subtypes of influenza virus. however, the use of adenovirus-based vaccine raises an important concern regarding development of vector-specific immunity in vaccine recipients as pre-existing vector immunity could interfere with the vaccine efficacy in scenarios requiring multiple booster vaccinations [25] . to address this challenge, we chose to administer of our recombinant adenovirus vaccine via mucosal route as previous studies have shown that a single immunization via nasal route can bypass the development of vector-specific immunity to adenovirus vector in human while eliciting potent immune responses specific for the vaccine antigen [26, 27, 28] . moreover, the airway mucosa is the main entry point for various invading pathogens, functioning as the first line of defense against respiratory infections [29] . the mucosal immune system is distinct from the systemic immune system as it possesses distinctly organized immunological tissues of its own which function to maintain the homeostasis within the mucosa [30] . current method of delivering influenza vaccines via parenteral route relies on the systemic induction of virus-specific iggs for protection. however, previous studies have reported that influenza vaccination efficacy is also closely associated with the immune responses induced within the respiratory mucosa [31, 32, 33] . given that parenteral influenza vaccines presently in use are inefficient in stimulating immune responses in mucosal tissues [34] , our study examined the potential application of mucosal immunization strategy which has been shown to effectively target both systemic and mucosal immunity [12, 13, 14] . however, there is a safety issue concerning the redirection of antigens to the central nervous system in i.n. immunization [35] , while s.l. route is thought to be relatively safe [36] . therefore, in our present study, we assessed i.n. and s.l. immunizations as an approach to evaluate the protective efficacy generated by mucosal vaccination of our rad-based influenza vaccine, as these two routes of immunization have been shown to promote induction of protective immune responses characterized by the localized responses in the respiratory mucosa [37, 38] as wells as systemic induction of vaccine antigen-specific responses [39] . additional aspects of our study focused on the comparison of humoral and cell-mediated immune responses generated following administration of our vaccine virus via these two different mucosal routes. further, we wanted to determine the specific branches of immunity, primed by mucosal rad/np immunization, which correlate to the establishment crossprotection against different influenza virus strains. in our present study, we demonstrated that rad/np immunization increases the frequency of np-specific cd8 t cells recruited to the lungs of i.n. immunized mice following homologous challenge with pr8 virus. accordingly, complete protection against pr8 challenge was observed only in the groups that received the vaccine virus via i.n. route, indicating that np-specific ctl response may be directly correlated to protection against homologous influenza virus infection. moreover, protection against heterosubtypic influenza virus infection may also be correlated to np-specific ctl response as np-specific blood cd8 t lymphocyte levels considerably increased (starting from day 4 post-challenge) in mice that survived the lethal challenge with heterosubtypic h3n2 virus. however, i.n. immunization failed to confer complete protection against h5n1 infection as only 40% of mice in the group that were given 1×10 8 pfu dose of rad/np survive this lethal challenge. our explanation for such lack of protection against h5n1 virus is that, although the immunodominant cd8 t-cell epitope within in np (np 147-155 ) are fully conserved among all influenza a subtypes, the magnitude and characteristic of ctl response elicited during differential influenza virus infection may be distinct for each influenza virus strain causing the concurrent infection. hence, even subtle differences in the ctl responses may affect the degree of protection offered during influenza infection caused by different influenza virus strains [40, 41] . mucosal immunization of mice with rad/np also dramatically increases np-specific igg levels in the serum independent of the immunization route. however, we observed that subsequent challenge with the homologous influenza virus additionally increases the np-specific serum igg levels in i.n. immunized mice, but not in s.l. immunized mice. further, substantial levels of np-specific respiratory mucosal igas were detected in i.n. immunized mice, whereas no such igas were detected in s.l. immunized mice. thus, it is possible that the presence of influenza np-specific iggs and igas in the respiratory mucosa may also be involved in the protection against the lethal influenza challenges [42, 43] , even though the exact mechanisms remain to be determined further. the immunization with adenovirus vector encoding np induced both cellular and antibody responses. it has been shown recently that influenza virus-infected cells can be eliminated by anti-m2e igg-mediated cellular cytotoxicity or phagocytosis since these cells express m2 on their surface after infection [44] . similarly, the np-specific antibodies may interact with the viral np expressed on cell surface of infected cells and mediate cell lysis by antibody-dependent cellular cytotoxicity. overall, our study demonstrates that a single i.n. administration of rad/np confer cross-protection against lethal challenge by different influenza virus strains, while s.l. administration of the same vaccine failed to confer protection, and we ascribe the high levels of np-specific ctls and antibodies found in intranasally immunized mice to the observed cross-protection. given that influenza np contains a conserved immunodominant cd8 t-cell epitope shared among all influenza a virus and that mucosal immunization can stimulate both mucosal and systemic immune responses, we believe that i.n. immunization with rad/np can induce protective immunity against different strains of influenza virus by priming for cross-reactive np-specific ctl response and possibly by local and systemic induction of np-specific antibodies. as such, our vaccination approach, examined in the present study, could be further explored as a next-generation influenza vaccination strategy which could generate broadly protective immunity 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protection we thank members of immunology laboratory for technical support and helpful suggestions. conceived and designed the experiments: jc. performed the experiments: shk jyk. analyzed the data: jyk yc hhn mks. wrote the manuscript: yc. key: cord-001603-vlv8x8l8 authors: ul-haq, zaheer; saeed, maria; halim, sobia ahsan; khan, waqasuddin title: 3d structure prediction of human β1-adrenergic receptor via threading-based homology modeling for implications in structure-based drug designing date: 2015-04-10 journal: plos one doi: 10.1371/journal.pone.0122223 sha: doc_id: 1603 cord_uid: vlv8x8l8 dilated cardiomyopathy is a disease of left ventricular dysfunction accompanied by impairment of the β(1)-adrenergic receptor (β(1)-ar) signal cascade. the disturbed β(1)-ar function may be based on an elevated sympathetic tone observed in patients with heart failure. prolonged adrenergic stimulation may induce metabolic and electrophysiological disturbances in the myocardium, resulting in tachyarrhythmia that leads to the development of heart failure in human and sudden death. hence, β(1)-ar is considered as a promising drug target but attempts to develop effective and specific drug against this tempting pharmaceutical target is slowed down due to the lack of 3d structure of homo sapiens β(1)-ar (hsβadr1). this study encompasses elucidation of 3d structural and physicochemical properties of hsβadr1 via threading-based homology modeling. furthermore, the docking performance of several docking programs including surflex-dock, fred, and gold were validated by re-docking and cross-docking experiments. gold and surflex-dock performed best in re-docking and cross docking experiments, respectively. consequently, surflex-dock was used to predict the binding modes of four hsβadr1 agonists. this study provides clear understanding of hsβadr1 structure and its binding mechanism, thus help in providing the remedial solutions of cardiovascular, effective treatment of asthma and other diseases caused by malfunctioning of the target protein. g-protein coupled receptor (gpcr) superfamily constitutes the largest family of receptors in cell responsible for mediating the effects of over 50% of drugs in the market now-a-days [1] [2] [3] [4] [5] [6] [7] [8] . gpcrs are involved in the transmission of a variety of signals to the interior of the cell and can be activated by a diverse range of small molecules including nucleotides, amino acids, peptides, proteins and odorants. activation of gpcrs results in a conformational change followed by a signal cascade that passes information to the inside of the cell by interacting with a protein known as heterotrimeric g-proteins. there are three main classes of gpcrs (a, b and c) depending on their sequence similarity to rhodopsin (rho) (class a). class a gpcrs is the largest group and encompasses a wide range of receptors including receptors for odorants, adenosine, β-adrenergic and rhodopsin [1] [2] [3] [4] [5] [6] [7] [8] . the β-adrenergic receptors (β-ars) are g s protein-coupled receptors that play important roles in cardiovascular function and disease, through serving as receptors for the neurohormones: norepinephrine and epinephrine. norepinephrine released from cardiac sympathetic nerves activates myocyte β 1 -ars, which activates adenylyl cyclase via stimulatory g-protein (g s ). the rise in the intracellular [camp] level causes the phosphorylation of several intracellular proteins by means of camp-dependent protein kinase a. such type of activated β 1 -ar results in an increased cardiac inotropy, lusitropy, and chronotropy and the secretion of rennin, all of which contribute to regulate the cardiac functions and blood pressure [9] [10] . β 1 -ar predominates in the heart, representing about 80% of the myocardial β-ars; thus, they tend to be viewed as the most significant β-ars with respect to the cardiovascular system. β 1 -and β 2 -ars in kidneys stimulate the release of renin, thereby playing a role in the activation of renin-angiotensin-aldosterone system [9] [10] . the role of β-ars in cardiovascular function and disease is also highlighted by the significant roles of drugs whose actions are based on binding to the β-ars blockers (β-blockers). βblockers represent first line therapy for the management of chronic heart failure, hypertension, acute and post myocardial infarction patients, chronic stable angina, and unstable angina [11] . they are also commonly used to control the symptoms of atrial fibrillation and other arrhythmias [11] . there are no cardiovascular drugs that have a wider range of indications than βblockers, making them a critical drug class for the management of cardiovascular disease. the availability of uses for β-blockers also suggests that the activation of β-ars, or the sympathetic nervous system (sns), plays an essential function in most cardiovascular diseases. the fact that β 1 -ar selective antagonists are equivalent to non-selective blockers in essentially all situations provides additional evidence that β 1 -ars are the more important β-receptors with respect to cardiovascular disease. the development of a large number of rational inhibitors that have the ability to modulate the activity of such receptors has been a major goal for the pharmaceutical industries to improve the clinical treatment of various disease including hypertension, heart failure and asthma [12] . however, finding specific drug against a particular β-ars drug target is a slow and laborious process. furthermore, the lack of 3d structure of hsadrb1 is an obstacle in the identification of specific drug like molecules. on the other hand, the development of computational approaches for drug designing can be effectively carried out with low cost [13] [14] . the use of computational techniques in drug discovery and development process is rapidly gaining popularity, implementation and appreciation. there will be an intensifying effort to apply computational power to combine biological and chemical space in order to rationalize the drug discovery, designing and optimization phenomena. today, computer aided drug design (cadd) is based on the knowledge of structure, either of the receptor, or that of the ligand. the former is described as structure-based while the later as ligand-based drug designing. because it is difficult and time-consuming to obtain experimental structures from methods such as x-ray crystallography and protein nmr for every protein of interest, homology modeling is a widely used in silico technique providing the useful structural models for generating hypotheses about a protein's function and directing further experimental work [15] . the main objective of this study is to employ "in silico" homology modeling technique to construct the 3d structure of hsadrb1 that will be used to identify and characterize new inhibitors for hsadrb1 by structure-based computational approaches. this model serves as a starting point to gain knowledge of protein-ligand interactions and the structural requirements of active site of protein. computational studies were performed on intel xeon quad core (2.33 ghz processor) server installed with linux os (opensuse version 12.0). multiple sequence alignment was carried out by clustalw of the closest homologue identified by ncbi p-blast to find out the identity, similarity and gap region between the target and template [16] . homology modeling was accomplished by orchestrar [17] implemented in biopolymer module of sybyl7.3 [18] . an online server, i-tasser [19] , was used for modeling a region absent in template structure. the finally selected model of hsβadr1was minimized by amber (version10.0) [20]. stereochemical properties of modeled protein structure were validated by procheck [21] , veri-fy3d [22] and errat [23] . molecular docking experiments were conducted by surflex-dock implemented in sybyl (version 7.3) [24] , fred (version 2.2.5) [25] and gold (version 2.5) [26] . ucsf chimera [27] [28] and moe [29] were used for visualization purpose. the flowchart of work plan is illustrated in (fig 1) . search the closest homologue. top-ranked template sequences as determined by blast were subjected for multiple sequence alignment on the basis of optimized e-value of the specified target sequence (table 1) . however, meleagris gallopavo β 1 -ar (mgβadr1, pdb id: 2y00) retrieved as the closest homologue, was manually edited for optimal alignment along with the target sequence. best alignment was selected based on alignment score and the reciprocal position of the conserved amino acid residues across the members of class a gpcr superfamily. the confined ballesteros and weinstein numbering scheme [32] was used to identify the transmembrane (tm) segments relative to the conserved position of amino acids in tm helices assigned as locant.50 shareing the common features in all class a gpcr superfamily. this is followed by the representation of amino acids tm helix numbers. the immediately preceding and following the .50 residue are numbered .49 and .51, respectively. orchestrar is specifically designed for homology or comparative protein modeling that identifies structurally conserved regions (scrs), models loops using model-based and ab-initio methods, models side chains, and combine them all to prepare a final model. initially, a homology model was generated by orchestrar that lacks a region of 45 amino acid residues (209-254) of the cytoplasmic loop of tm5 located within the target sequence but absent in the template structure. this region was modeled by i-tasser, an integrated platform for automated protein structure and function whose prediction is based on sequence-to-structureto-function paradigm as per multiple threading alignments by lomets [33] . the model generated by i-tasser was named as sub-model 1. five sub-models were evaluated by replica-exchange monte carlo simulations with low free-energy states, spatial restrains and alignments tm regions [34] to identify the best structural alignment almost closed to the structural analogs on the basis of structural similarity. any further steric clashes were removed to refine the coordinates, and the final results of all sub-models were based on sequence-structurefunction paradigm obtained from the consensus of structural similarity and confidence score (c-score) of i-tasser server. c-score value is the quality for the predicted sub-model on the basis of threading method. stereochemical properties of each sub-model were evaluated and the best selected sub-model was incorporated to the homology model of hsβadr1, generated previously by orchestrar and after insertion of the model the finalized modelled is subjected for optimization. homology model of hsβadr1 generated by orchestrar was minimized by sybyl using conjugate gradient and steepest descent method with 10,000 iterations each. the selected submodel generated by i-tasser was also individually minimized to 10,000 cycles by amber10, followed by the insertion of sub-model into the homology model of hsβadr1 by chain joining option in sybyl. the finally generated model is minimized further to 30,000 cycles using ff99sb force field by amber10. selection of complexes for re-docking and cross-docking validation. to identify a suitable docking program for the docking of hsβadr1 agonists, re-docking and cross-docking experiments were performed by surflex-dock, fred, and gold. six βadr1-ligand complexes, three βadr2-ligand complexes and two rhodopsin-ligand complexes were retrieved from pdb. the details of the protein-ligand complexes used in this study are summarized in table 2 and s1 fig. selection of complexes was based on following criteria: availability of the protein-ligand complexes, the crystallographic resolution of protein-ligand complexes should be 3 ǻ, the binding interaction of the protein-ligand complexes should be known. cross-docking experiments conducted in using multiple docking methods with their scoring function are utilized in this study mentioned in s2, s3 and s4 tables. the details of docking methodology are discussed in supporting informations. the re-docking results were analyzed to check the ability of docking programs to correctly identify the bound conformation of co-crystallized ligand in the top-ranked solution. rmsds were calculated between the corresponding co-crystallized ligand against its predicted docked pose. cross-docking experiments were conducted to identify which docking program exactly identified its cognate ligand among the diverse set of ligands within the top-ranked solution. for cross-docking, 11 complexes were extracted from pdb in which eight proteins are homodimers (chain a and chain b) while the rest of three are monomers (chain a). for those proteins that are present as homodimers, ligands were docked into both chains. overall, 19 complexes were evaluated for cross-docking. the results were quantified as best (1-3 position), moderate (4-5 position) and worst when the cognate ligand ranks position lowers than 5 within its cognate protein, respectively. blast results and multiple sequence alignments blast predicted mgβadr1 (pdb id: 2y00) [35] as the best match for hsβadr1 with 68% identity and 75% positivity (with an e-value of 3×10 -165 ). 2r4r, 3kj6, 3p0g and 2rh1 have 79% while 2r4s and 3sn6 have 74% query coverage, more sequence coverage than observed for 2y00 (73%). since 2r4r, 3kj6 and 24rs are available as apo form with fewer scores, identity and positive values, these structures were not used in this study. similarly, the complexes 3p0g, 2rh1 and 3sn6 were not used for the modeling of hsβadr1 structure due to their lower scores. hence, 2y00 is used according to the blast results but to establish more confidence on the top-ranked search, we opted for two sorts of multiple sequence alignments: raw multiple sequence alignment and manually-edited multiple sequence alignment. for raw alignment, the ten top-ranked templates sequences (2y00, 2vt4, 2r4r, 3kj6, 2r4s, 3sn6, 4gbr, 3p0g, 2rh1 and 3pds) were aligned against the target sequence illustrated in s2 fig and s1 table. for manually-edited alignment, both the target and template (2y00) sequences were truncated. the first 50 residues from n-terminus and 84 residues (393-477) from c-terminus were omitted from the target sequence due to the absence of corresponding homologous sequence in the template and has no important residue which is necessary to be in helical segments. the template sequence has 483 amino acid residues whereas the structure of 33-368 residues has been resolved (total 315 residues as some residues are missing). the first 3 residues (33-36) from n-terminus and 14 residues (337-351) from c-terminus were omitted. finally, 342 residues of target sequence was aligned with ten top-ranked blast search, 2y00 (297 residues), 2vt4, 2r4r, 3kj6, 2r4s, 3sn6, 4gbr, 3p0g, 2rh1 and 3pds the average alignment score for manually edited multiple sequence alignment is better (76.47) than the score obtained by raw multiple sequence alignment (74.89). overall, there are 15 instances where alignments are improved, 7 alignments are improved when the target sequence is aligned with the rest of the sequences and 8 times the alignments have better quality when the template sequence is aligned with the remaining sequences. the generalized ballesteros and weinstein numbering scheme is beneficial for the understanding, recognition and structural alignments of gpcrs family [32] . the ballesteros and weinstein numbering is illustrated in (fig 2) and the conserved amino acid residues of class a gpcrs is tabulated in table 3 . ballesteros and weinstein numbering is useful for the understanding of integrating methods for the construction of 3d models and computational probing of structure-function relations in gpcrs. these criteria pertain to the selection of correct inputs for the a1ignment programs and to structural considerations applicable to checking and refining the sequence alignments generated by alignment programs. this selection criterion depends on the information that is determined by the extent of homology among the compared sequences. alignment of sequences with intermediate homologies (i.e., 30-70%) can identify continuous patterns of conservation distributed over the entire sequence. such patterns provide structural inferences based on conservation. the hsβadr1 model is selected after structural comparison, superimposition and procheck results ( fig 3a) . orchestrar generated homology model using template 2y00 was incomplete since the structure of residues 209-254 was missing. orchestrar fills the gap but not more than1-12 residues long, therefore, an ab-initio based threading method is used to predict the structure of missing region (s3 fig). subsequently, five sub-models were generated. each sub-model is further analyzed by ramachandran plot (table 4 ). among them, sub-model 1 is selected on the basis of highest c-score (-2.43) and stereochemical properties. the c-score value being lower than -1.5 likely indicates a lack of an appropriate template within the i-tasser library. the selected sub-model 1 was subsequently inserted into the homology model of hsβadr1 by sybyl. the c-terminus val208 and the n-terminus lys254 of the homology model is connected with the n-terminus val209 and the c-terminus thr255 of submodel 1, respectively ( fig 3b) . according to the ramachandran plot,~85%, 13.5% and 1.3% residues are located within the favorable, allowed and the generously allowed regions, respectively while only one residue (ile208) is found to be in the disallowed region. the visual inspection revealed that ile208 is far away from the active site region and do not lie within 5å of active site. additionally, stereochemical properties of the model were validated by verify3d web server. verify3d evaluated the local environment and inter-residue contacts in the model. ideally, the 3d-1d profile for each of the 20 amino acids should be in range of 0-0.2. values less than zero are considered as inaccurate for the homology model. the verify3d plot of hsβadr1 model shows that the average score of all amino acid residues is 0.16 which is relatively closed to 0.20. moreover, errat, a protein structure verification web server was used to verify the model on the basis of model building and refinement, and is extremely useful in making decisions about reliability of the homology model. errat results showed that the overall quality factor for the hsβadr1 model is 73.35%., suggesting that the generated model is robust and can be use for virtual screening purpose in future. the 3d model of hsβadr1 revealed an excellent agreement with the experimentally determined 3d structure of mgβadr1. (fig 4) shows the superimposed view of hsβadr1 model and mgβadr1 structure. the calculated polypeptide backbone (cα, these pdbs have comparable sequence similarities, identities and source as that of the template but some conformational changes has been observed for helix6 [36] . however, we found no observable conformational changes especially for those amino acid residues that are involved in molecular interactions with high-affinity antagonists i32, p32 and cau located within h6 and cl-3. finally, the hsβadr1 model is subjected to the sequence manipulation suite ident and sim [37] to observe the similarity and identity of the model with respect to the template structure. the results are better but afterwards much improved after manual editing of the target and template sequences, similarity and identity ratios are increased from 73% to 75.4% and 67% to 68.4%, respectively. the overall topology and secondary structural elements particular for the class a gpcr family remain quite conserved in the model of hsβadr1, that is, an extracellular n-terminus domain, seven 7-tm domains linked by three intracellular cytoplasmic loops (cl-1, cl-2 and cl-3), three extracellular loops (el-1, el-2 and el-3), and a cytoplasmic c-terminus domain. the nterminus domain comprises of nine amino acids residues (1-9) that are located outside the membrane. the tm-1-tm-7 helices spans from 10-34, 44-67, 80-104, 125-146, 198-173, 297-274 and 308-326 amino acid residues, respectively, while the c-terminus domain comprises of amino acid residues ranging from 327 to 342 at the inner face of membrane. the cytoplasmic loops, (cl-1, cl-2 and cl-3) comprise of residues 35-43, 105-124 and 199-273, respectively. the cytoplasmic loops cl-2 and cl-3 are believed to be important in the binding, selectivity or specificity and activation of g-proteins [38] . the extracellular loops, (el-1, el-2 and el-3) comprising 68-79, 147-172 and 298-307 residues, respectively. two conserved disulfide bridges which are important for cell surface expression, ligand binding, receptor activation and maintenance of the secondary structure are located in el-2 and el-3 regions at positions cys81-cys166 and cys159-cys165, respectively (table 5 ). conserved motifs of hsβadr1 homology model dry motif also known as ionic lock switch [39] is observed at position asp105(3.49), arg106 (3.50) and tyr107 (3.51) in helix 3 of hsβadr1 model. the conserved asp in dry motif at the cytoplasmic end of helix 3 believes to regulate the transition state of active state, while the adjacent arg is crucial for g-protein activation [40] . another conserved penta-peptide npxxy motif known as tyrosine toggle switch (where x usually represents a hydrophobic residue and n is rarely exchanged against d) located at the c terminus of tm-7 which contributes to gpcr internalization and signal transduction. several site-directed mutagenesis studies revealed the importance of this motif in signaling [41] . the npxxy motif is present at position arg323(7.49), pro324(7.50), ileu325(7.51), ileu326(7.52) and tyr327(7.53) in the [44] . these domains help anchor tm to the cytoskeleton and hold together signaling complexes. pdz domain have many functions, from regulating the trafficking and targeting of proteins to assembling signaling complexes, and networks designed for efficient and specific signal transduction [45] . the amphipathic amino acid residues present in helix 8 are conserved among all human gpcrs (residues 327-341), located between the tm7 bound with helix 7. the palmitoylation occurs at n-terminus while the biosynthesis of receptor and the proper regulation of surface expression occur at c-terminus of hsβadr1. the side chain of two crucial residues of helix8, asp332(8.49) and arg334 (8.51) , are projected within the hydrophilic interface involved in stimulatory g-protein (g s ) activation while the residue phe333(8.50) and phe337(8.54) are buried in the hydrophobic core of the helix [46] . salt bridges play important roles in protein structure and function. disruption and the introduction of a salt bridge reduce and increase the stability of the protein, respectively [47] . in membrane proteins, one expects salt bridges to be particularly important because of a smaller dehydration penalty (loss of favorable contacts with water) on salt bridge formation [48] . charged groups become largely dehydrated when inserted into membranes, and therefore, experience a smaller change in hydration between non-salt-bridging and salt-bridging states. there should also be a smaller effect because of solvent screening, strengthening salt-bridge interactions [48] . multiple salt bridges are observed in the homology model of hsβadr1; asp154:arg157, asp209:arg213, asp332:lys335, glu155:arg158, glu200:lys203 and glu212: arg213. in addition, salt bridges can also serve as key interactions in much the same way as disulfide bonds (s6 fig) . re-docking analysis. the success of docking is usually scrutinized by its accurate pose prediction ability [49] [50] , hence prior to the docking of βadr1agonists into the homology model of hsβadr1, the reliability of three docking programs including surflex-dock, fred, and gold was assessed. the re-docking results were quantified on the basis of rmsd between the top-ranked docked conformation and the co-crystallized (termed as ˈreferenceˈ) ligand and visual analysis. the prediction is termed as good when rmsd >1 or < 2 å and the docked pose is superimposed on the ligand's co-crystallized position, fair when rmsd > 2 and < 3 å and the docked pose is in active site but not superimposed on its native conformation, and poor or inaccurate when rmsd >3 å and the docked pose is inverted or far away from the active site. the re-docking results showed that gold outperformed surflex-dock and fred (fig 5) . among the 19 complexes used, gold, surflex-dock, and fred generated 100%, 74%, and 68% good solutions in the top-ranked position, respectively. surflex-dock and fred identified 5% and 10% fair poses, respectively in the top-ranked docked poses. while both the programs generated 21% inaccurate solutions in the top-ranked docked pose. the results are summarized in (table 6) . furthermore, docking methods utilized in cross-docking is illustrated in (table 7) , was conducted to find out which program utilized in correctly ranks 19 ligands into their cognate binding site. the prediction was quantified on the basis of ligand's ranking (s2, s3 and s4) tables. the cross-docking results indicates that surflex-dock is superior with 47% best results in ranking the ligand in top 1-3 position in their cognate receptors. gold and fred are returned with 42% and 44% best results, respectively (fig 5) . the position and the interaction of each ligand within the cognate receptor are visually analyzed. the results showed that the conformation of each ligand generated by surflex-dock is much better than the docked conformations generated by gold and fred. most of the interactions generated by surflex-dock are similar to the interactions present in the x-ray conformation. hence, surflex-dock was found to be more appropriate for the docking of gpcr's ligands and it is further used in this study to explore the binding mode of hsβadr1 agonists into the active site of hsβadr1 model. table 8 and table 9 . binding mode of y00, whj, 5fw, 68h the docked pose of y00 reveals that multiple hydrogen bonding interactions are formed between the surrounding amino acid residues that stabilize y00 in the catechol binding pocket. the −oh group at the phenol moiety is involved in hydrogen bonding with the γ carboxylate side chain of asp167 (1.83 å). the substituted −oh group at meta and para positions of ring b shows hydrogen bonding interactions with the side chains γ −oh of thr170 (1.93 å) and ser178 (1.80 å), respectively. furthermore, the side chain phenyl ring of phe168 and the carboxylate of asp168 provide cation-π stacking interactions to the phenolic moiety of y00 that further helps to stabilize the orientation of agonist. (fig 6a) displays the binding mode of compound y00. the binding mode of whj demonstrates that the amino group of whj mediates hydrogen bond with the side chain carboxylate of asp88 at a distance of 1.86 ǻ. similarly, thr170 γ -oh group probes hydrogen bonding interactions with multiple ligand atoms including n atom and o atom at a distance of 2.03 ǻ and 2.64 ǻ, respectively. the same thr170 is also involved in forming hydrogen bond at a distance of 1.76 ǻ, the most significant hydrogen bonding interaction for whj. phe168 forms cation-π interaction with one of the fused aromatic ring of whj. the binding orientation of compound whj is shown in (fig 6b) . the binding mode of 5fw shows that the para −oh moiety of 5fw establishes hydrogen bonding interaction with the side chain carboxylate of ser179 at a distance of 2.87 ǻ. additionally; ser178 forms bi-dentate hydrogen bonding with the para and meta −oh groups at distances of 2.22 ǻ and 2.17 ǻ, respectively. the main chain carbonyl moiety of phe168 mediate hydrogen bond with the amino group of 5fw (2.66 ǻ). the docked binding mode of compound 5fw is depicted in (fig 6c) . as revealed in (fig 6d) , the -oh of 68h shows similar interactions as observed for compound 5fw. the para substituted −oh group of 68h mediates bi-dentate hydrogen bonds with the side chain −oh groups of ser178 and ser179 at distances of 2.22 ǻ and 2.44 å, respectively. furthermore, asp88 mediates bi-dentate interaction with the linear chain amino and −oh groups of 68h at a distance of 1.91 ǻ and 1.90 ǻ, respectively. the binding mode analysis of agonists y00, 5fw, 68h displays that the ser178 plays crucial role in stabilizing the agonists within the catechol binding pocket of hsβadr1 homology model. the docking results reveals that ser178 and phe168 are crucial residues in ligand binding by providing h-bonding, and π-π interactions, respectively, thus helps in the activation of hsβadr1. we intend to incorporate molecular dynamic simulation studies in order to investigate the dynamic behavior of protein-inhibitor complex formation in the near future; and the role of most important residues will be determined. the study will be helpful to pursue structure based drug design of hsβadr1 blockers. human βadr1 is found to be involved in several cardiovascular diseases. the lack of crystal structure of hsβadr1 provoked us to apply in silico techniques to initiate the drug discovery process for hsβadr1. hence, to understand the characteristics structural features of hsβadr1 and to execute the structure-based drug design strategy for hsβadr1, threading-based homology modeling of mammalian origin were applied in this study. the model possesses acceptable structural profiles. furthermore, the binding modes of four hsβadr1 agonist were determined via molecular docking simulation. h-3, h-5, and el-2 regions were found to be important in ligand binding. several residues including trp84, asp88, val89, asp167, phe168, thr170, ser178, and ser179 are involved in direct interactions with the ligand. among all, ser178, and phe168 provides h-bonding, π-π interactions, respectively, hence found to be crucial residue in ligand binding and for the activation of hsβadr1. we are also investigating the dynamic behaviour of the apo and ligand bound forms of hsβadr1 that will be published in future. note: the coordinate file of hsadrb1 is submitted to the publicly accessible protein model database (pmdb) [51] ; www.caspur.it/pmdb). the pmdb id of hsadrb1 is pm0079652 respectively. table 8 and (fig 6) ). (tif) s1 table. alignment scores (a) alignment scores obtained from the alignment scores raw multiple sequence alignment (b) alignment scores obtained from the manually edited multiple sequence alignment (c) alignment scores obtained from the raw target and template pair wise sequence alignment. (doc) s2 table. cross-docking results of surflex-dock analyzed the basis of ranking of the cognate ligand in their respective receptor. criteria for ranking: 1-3 position is best (green cell), 4-5 is moderate (blue cell) and >5 is inaccurate (red cell). (doc) s3 table. cross-docking results of fred analyzed on the basis of ranking of the cognate ligand in their respective receptor. criteria for ranking: 1-3 position is best, 4-5 is moderate and >5 is inaccurate. (doc) s4 table. cross-docking results of gold analyzed on the basis of ranking of the cognate ligand in their respective receptor. 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predicting molecular interactions in silico: i. a guide to pharmacophore identification and its applications to drug design virtual screening for sars-cov protease based on kz7088 pharmacophore points structure-based 3d-qsar models and dynamics analysis of novel n-benzyl pyridinone as p38α map kinase inhibitors for anticytokine activity clustalw and clus-talx version 2.0 comparison of composer and orchestrar sybyl molecular modeling software version 7.3, tripos associates i-tasser: a unified platform for automated protein structure and function prediction procheck: a program to check the stereochemical quality of protein structures verify3d: assessment of protein models with three-dimensional profiles verification of protein structures: patterns of nonbonded atomic interactions surflex: fully automatic flexible molecular docking using a molecular similarity-based search engine fred pose prediction and virtual screening accuracy gold version 3.0. cambridge crytallographic data center chimera: an extensible molecular modeling application constructed using standard components molecular operating environment (moe), 2012.10 chemical computing group inc. 1010 sherbooke st. west, suite #910 uniprot website basic local alignment search tool integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in g protein-coupled receptors i-tasser: fully automated protein structure prediction in casp8 tm-align: a protein structure alignment algorithm based on the tm-score the structural basis for agonist and partial agonist action on a beta 1-adrenergic receptor two distinct conformations of helix 6 observed in antagonist-bound structures of a β1-adrenergic receptor the sequence manipulation suite: javascript programs for analyzing and formatting protein and dna sequences structure of a beta1-adrenergic g-protein-coupled receptor the significance of g protein-coupled receptor crystallography for drug discovery the effect of mutations in the dry motif on the constitutive activity and structural instability of the histamine h2 receptor mutation of tyrosine in the conserved npxxy sequence leads to constitutive phosphorylation and internalization, but not signaling, of the human b2 bradykinin receptor hallucinogen actions on 5-ht receptors reveal distinct mechanisms of activation and signaling by g protein-coupled receptors homology modeling of g-protein-coupled receptors and implications in drug design pdz domains and their binding partners: structure, specificity, and modification evolutionary expansion and specialization of the pdz domains characterization of the residues in helix 8 of the human β1-adrenergic receptor that are involved in coupling the receptor to g proteins esbri: a web server for evaluating salt bridges in proteins salt bridges: geometrically specific, designable interactions. proteins structure-based design, synthesis and biological evaluation of β-glucuronidase inhibitors identification of novel interleukin-2 inhibitors through computational approaches the pmdb protein model database structure of bovine rhodopsin in a trigonal crystal form advances in determination of a highresolution three-dimensional structure of rhodopsin, a model of g-protein-coupled receptors (gpcrs) high resolution crystal structure of an engineered human β2-adrenergic g protein coupled receptor a specific cholesterol binding site is established by the 2.8 å structure of the human β2-adrenergic receptor conserved binding mode of human β2 adrenergic receptor inverse agonists and antagonist revealed by x-ray crystallography we are thankful to prof. bernd m. rode (university of innsbruck) for the computational software support during this research work. financial support required to conduct this scientific work from higher education commission (hec), is highly acknowledged. supporting information s1 fig. 2d representation of the bound ligands of 11 gpcrs complexes utilized in this study (see also table 2 ). key: cord-000180-howix091 authors: macleod, iain j.; minson, tony title: binding of herpes simplex virus type-1 virions leads to the induction of intracellular signalling in the absence of virus entry date: 2010-03-05 journal: plos one doi: 10.1371/journal.pone.0009560 sha: doc_id: 180 cord_uid: howix091 the envelope of hsv-1 contains a number of glycoproteins, four of which are essential for virus entry. virus particles lacking gb, gd, gh or gl are entry-defective, although these viruses retain the ability to bind to the plasma membrane via the remaining glycoproteins. soluble forms of gd have been shown to trigger the nuclear translocation of the nf-κb transcriptional complex in addition to stimulating the production of type i interferon. by taking advantage of the entry-defective phenotype of glycoprotein-deficient hsv-1 virus particles, the results presented here show that binding of virions to cellular receptors on the plasma membrane is sufficient to stimulate a change in cellular gene expression. preliminary microarray studies, validated by quantitative real-time pcr, identified the differential expression of cellular genes associated with the nf-κb, pi3k/akt, jak/stat and related jak/src pathways by virions lacking gb or gh but not gd. gene induction occurred at a few particles per cell, corresponding to physiological conditions during primary infection. reporter assay studies determined that nf-κb transcriptional activity is stimulated within an hour of hsv-1 binding, peaks between two and three hours post-binding and declines to background levels by five hours after induction. the immediate, transient nature of these signalling events suggests that hsv-1 glycoproteins, particularly gd, may alter the cellular environment pre-entry so as to condition the cell for viral replication. subjugation of the intracellular environment by viruses is essential to ensure the effective expression and replication of the viral genome to allow production of progeny virions. one such viral strategy involves hijacking signalling pathways that ultimately control host gene transcription. interactions between intracellular viral proteins and cellular kinases responsible for signal transduction are a means by which to achieve this. however, there is growing evidence to support the premise that glycoproteins on the surface of virus particles may trigger intracellular signalling pathways by interacting with their cognate receptors on the host cell membrane [1] . binding of hsv-1 to permissive cells occurs through viral glycoproteins on the viral envelope interacting with specific receptors on the cell surface, triggering fusion of the plasma membrane with the outer envelope. five of these glycoproteins are known to be involved in virion binding to the cell surface: gb, gc, gd and the heterodimer gh-l. of the five, only gc is dispensable for allowing productive infection as deletion of gb, gd or gh-l results in an entry-defective phenotype [2] [3] . gd is known to bind independently to hvem, nectin-1 and nectin-2, whereas gh interacts with the avb3 integrin, with the paired immunoglobulin-like receptor, pilra, acting as a receptor for gb [2] [4] [5] . one of the first studies to examine whether hsv-1 glycoproteins play a role in the induction of signalling found that gd was able to block fas-mediated apoptosis [6] . u937 monocytoid cells were rendered resistant to apoptosis after infection with uvinactivated hsv virions, when co-cultured with a stably transfected hsv-1 gd-expressing cell line or after treatment with soluble gd. inhibition of nf-kb signalling by the introduction of a dominant-negative nf-kb repressor abolished protection from gd-induced fas-mediated apoptosis. more specifically, the interaction of gd with one of the gd receptors, hvem, was involved in preventing apoptosis induction [1] . there are reports showing that hsv-1 triggered the translocation of nf-kb by six hours post-infection [7] . however, it has come to light that hsv-1 may induce two distinct phases of nf-kb activity. the initial phase is transient, lasting only two hours, and occurred shortly after viral adsorption by both wild-type and uv-inactivated virus particles. this correlates with evidence showing that supernatant taken from gd-expressing cells may also cause an increase in nf-kb binding activity in monocytoid cells by 30 minutes post-treatment [6] . a second phase relied on de novo viral protein synthesis as it was stimulated only by replication-competent hsv-1 and not by uv-inactivated virions [8] . during this second phase, nf-kb complexes were shown to have associated with their consensus sequence found in the promoter region that drives icp0 expression [8] . in comparison to wild-type gd, a mutated form of gd that was unable to bind to hvem did not stimulate nf-kb activity in cocultured monocytoid cells [9] . hep-2 cells, which do not express hvem, lacked detectable nf-kb signalling after infection with uv-inactivated hsv-1, implying that gd-mediated nf-kb stimulation may be dependent on expression of hvem [9] . soluble glycoproteins from beta-and gammaherpesviruses can also stimulate intracellular signalling pathways; gb of cmv has been shown to stimulate the differential expression of cellular transcription factors and interferon-stimulated genes (isgs), and soluble gp350 of ebv can trigger nf-kb activation [10] [11] [12] . the treatment of cells with soluble glycoproteins or uvinactivated virus has provided strong evidence that hsv-1 binding to the plasma membrane triggers signal transduction in the host cell as a prelude to infection, but interpretation of these experiments is not straightforward. the use of soluble glycoproteins does not mimic the binding of individual virus particles and the physiological significance of the results is difficult to assess. the use of uv-inactivated particles in signalling studies simulates infection but these particles are able to enter cells and deliver virion proteins to the cytoplasm. experiments with uv-inactivated particles do not therefore distinguish between events arising from virus binding from those resulting from virus entry. the main focus in these studies was to examine signalling events using virus particles that are capable of binding to the cell surface but are incapable of entry due to the absence of one of the three essential glycoproteins, gb, gd or gh [13] [14] [15] [16] , and to find whether signalling occurs at particle concentrations that would mirror physiological conditions. all cells other than human foreskin fibroblasts (hff) (atcc cell line crl-2522) were grown in glasgow modified eagles medium supplemented with 10% foetal bovine serum, 4 mm glutamine, 100 units/ml penicillin and 100 ug/ml streptomycin. hff cells were grown in dulbecco modified eagles medium supplemented as described above but with the addition of 1x mem non-essential amino acids. working stocks of hsv-1 sc16 were grown on hacat cells and assayed on vero cells [17] . mutant viruses lacking functional genes for gh (scghz) [14] , gb (hfemdul27-lacz) [18] or gd (sc16gddelz) [18] were grown and assayed on helper cell lines expressing the corresponding glycoprotein: cr1 cells, expressing gh [19] ; d6 cells expressing gb [13] ; and vd60 cells expressing gd [18] . all stocks were grown using an moi of 0.1. purified preparations of wt virus, or of virions lacking individual glycoproteins, were produced as previously described [20] . purified preparations were assayed for particle numbers using electron microscopy [21] and for infectivity by plaque assay on vero cells or, in the case of glycoprotein-deficient virions, on the relevant helper cell line. preparations of wt virions had a particle to infectivity ratio of 3610 1 . preparations of ghnegative, gd-negative and gb negative virions had particle to infectivity ratios of 2610 10 , 5610 6 and 3610 6 respectively. the maximum virion dose of entry-incompetent virions used in the experiments described in this paper was 1000 particles per cell and it therefore follows that in the maximum infectivity of a preparation of an entry-defective virus (i.e. using gb-negative virions) would be less than one cell per thousand. virus preparations were deglycosylated with 500 u of pngase f for 18 hrs at 37uc in the absence of denaturing buffer [22] . removal of n-linked sugars was confirmed by a shift in the electrophoretic mobility of gd on a protein denaturing gel. human foreskin fibroblast cells (hff) were seeded at 1.5610 6 in a 175 2 cm flask and grown to 90% confluence then growtharrested in medium supplemented with only 100 u/ml penicillin-g and 100 mg/ml streptomycin for five days. real-time pcr experiments were conducted with growth-arrested hffs that were inoculated in triplicate with 1000 particles/cell of entry-defective hsv-1 in warm, serum-free medium, with parallel mock-infected cells that received an equivalent volume of pbs in warm serumfree medium. inoculations took place at 37uc. at the indicated times after the addition of virus, the medium was removed, the cells were lysed with tri reagent (sigma) and total rna was immediately purified. for each entry-defective hsv-1 mutant, three independent biological replicates were carried out. microarray experiments were conducted under the same conditions using wild-type hsv-1 (sc16) at an moi of 20. total rna was isolated from tri reagent lysates as per the manufacturer's instructions. contaminating genomic dna was digested with 2 u dnase i (invitrogen) and rna was re-purified with tri reagent. cdna was derived from 30 mg total rna using anchored oligo (dt)20 primers (invitrogen) as described in the manufacturer's instructions. for each entry-defective hsv-1 mutant, the cdna from three independent infections were used in technical triplicates for real-time pcr reactions. microarray analysis was carried out using the signal transduction pathwayfinder cdna array as per the manufacturer's instructions (superarray). nylon cdna microarrays membranes were pre-hybridised with supplied sheared salmon sperm dna at 60uc for one to two hours. the pre-hybridisation solution was replaced with the biotin-dutp labelled cdna probe, which was diluted in sheared salmon sperm dna. the solution was left to hybridise at 60uc overnight. the following morning the cdna probe was discarded and the membrane was washed twice then blocked in solution q, as provided by the manufacturer. after 40 minutes, the blocking solution was discarded and the membrane was incubated with a binding buffer, solution f. after washing, the chemiluminescent substrate was incubated with the membrane for two to five minutes at room temperature. the microarray membrane was then exposed to x-ray film. analysis was performed using gearray expression analysis suite as provided by the manufacturer (superarray). all the data sets used in the microarray analysis are available in table s1 . cdna derived from cells that were mock-infected or inoculated with 1000 particles/cell of entry-defective dgb, dgd and dgh virions was used for real-time pcr analysis. primers were designed, using the primer3 software [23] , to anneal at 60uc, with a minimum product melting temperature of 80uc (table s2) . reactions were set-up with cdna corresponding to 100 ng total rna, primers (70 nm), and 10 ml sybr green pcr master mix (abgene) made to a total volume of 20 ml with nuclease-free dh 2 o. a corbett rotorgene 3000 was used to determine the levels of sybr green fluorescence over 40 cycles. samples were denatured for 10 minutes at 95uc, annealed at 60uc for 30 seconds, with extension of the primer product at 72uc for 40 seconds then a final 80uc step for 20 seconds that removes background fluorescence from primer-dimers. a melt curve analysis was produced at the end of the cycling to ensure the specificity of pcr product amplification and associated sybr green fluorescence. relative gene expression levels between mockinoculated and inoculated cells were calculated using the pfaffl method with potential variations in cdna quantity between samples normalised to the transcript for ribosomal protein l13a (rpl13a) [24] . serial dilutions of pcr product across 8 logs were used to determine the efficiency of pcr amplification for each primer set under the above conditions so that the relative quantitation could be adjusted as defined by the pfaffl method. a relative change in expression of two-fold was set as a threshold for determining whether differential expression of a gene had occurred. the p values associated with the fold-change in expression were calculated using a students t-test on the realtime pcr c t values comparing triplicate mock-inoculated and hsv inoculated cells. an nf-kb reporter assay system was designed using a pgl4-nfkb construct (promega). quadruplicate repeats of the nf-kb binding consensus sequence 59-gggaatttcc-39 were excised from a p-nf-kb-luc (gift from dr. heike laman, university of cambridge) vector using kpni and hindiii and ligated into the pgl4.20 vector multiple cloning site using the same restriction sites. pgl4.20 was chosen as it is optimized for more efficient expression in mammalian cells, compared to previous generations of luciferase constructs, and contains fewer transcription factor consensus sequences such that background luciferase expression is reduced. hffs were electroporated in batches of 6610 5 cells per cuvet (amaxa) with 2 mg of pgl4-nf-kb, pooled together and aliquoted at 10 5 cells per well of a 24-well plate. after 48-hours recovery in supplemented media, the transfected hffs were serum-starved for five days. hffs were inoculated with 1000 particles/cell of dgh hsv-1 and were lysed at the indicated time points in 100 ml passive lysis buffer (promega) and 20 ml of lysate were examined for luciferase activity using 50 ml assay buffer (promega). western blotting gd and the major tegument protein, vp16, were detected by immunoblotting using the monoclonal antibodies lp2 and lp1, respectively [25] [26] . sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and immunoblotting were performed as described previously [27] . signalling-specific microarrays were chosen as an initial route to identifying signalling pathways that may be stimulated early in hsv-1 infection, and more precisely, by virion binding. the signal transduction pathwayfinder cdna array from superarray incorporates 96 targets covering 19 associated signalling pathways, in addition to two negative controls corresponding to puc18 plasmid dna and a 'blank' spot, as well as four positive controls representing the housekeeping genes for gapdh, beta actin, cyclophilin a and ribosomal protein l13a. serum-starved hffs were inoculated with 1000 particles/cell of dgb, dgd or dgh entry-defective hsv-1 virions. total rna was isolated immediately after inoculation, corresponding to 0 hpi, and 6 hpi. changes in gene expression associated with the activation of signalling pathways were identified by comparison to the transcript abundance at 0 hpi. the results from duplicate experiments indicated that downstream gene targets of a number of signalling pathways are differentially expressed above the twofold threshold after virion binding (table 1) . these preliminary results were then used as the basis of quantitative pcr studies. using the same methodology, serum-starved hff cells were exposed to 1000 particles per cell of virions lacking either gd, gb or gh and rna was extracted for cdna synthesis immediately (0 h) or after 6 h. table 2 gives the results of three independent experiments for those genes that were differentially expressed between 0 h and 6 h post treatment and is compared to the initial microarray results in table 3 . it is striking that in virtually every instance where gene expression is modified by virion binding, gd appears to be the key effector. virions lacking gh or gb had were capable of altering cellular gene expression while those lacking gd had no significant effect. the results can be summarised as follows. the jak/stat and related jak/src pathway show a similar response to that seen for nf-kb. of the jak/stat-responsive genes, only nos2a is regulated by more than one pathway, with dgb and dgh virions inducing their transcription and dgd particles having no significant effect ( table 2) . although the jak/ src pathway has only two targets on the signalling microarraysupon which the real-time pcr experiments were based -bcl2l1 is unique to the pathway, with bcl2 expression regulated by multiple signalling routes. interestingly, while dgb and dgh virions up-regulated bcl2 and bcl2l1, dgd virus particles appeared to down-regulate both targets by a significant amount. induction of this pathway may be responsible for the upregulation of ccnd1, myc, bcl2 and mmp7 after inoculation with dgb and dgh but not dgd hsv-1 ( table 2 ). differential expression of ccnd1 and myc can be regulated by the wnt and pi3k signalling pathways, with myc also being controlled by protein kinase c. the up-regulation of mmp7, which is the only target unique to the pi3k pathway that is differentially expressed, highlights the potential crosstalk between signalling pathways and cannot exclude the involvement of pi3 kinase signalling as a pathway leading to transcription of all four cellular genes. both dgb and dgh virions are capable of stimulating an nf-kb response (table 2) , a result that correlates with the preliminary microarray data (table 1) . transcripts for both birc-2 and birc-3 are increased above the two-fold threshold after inoculation with dgb and dgh hsv-1 but not dgd. there appears to be no change in birc-1 expression after inoculation with any of the three entry-defective mutants. components of the nf-kb transcriptional complex, nfkb1 and rel, showed similar levels of increased transcription after binding by dgb and dgh virions, a response that was absent in cells stimulated with gddeficient virions. two markers of inflammation, ccl2 and pecam1, also showed a significant change in gene expression at 6 hpi with entry-defective hsv-1 lacking gb or gh but not gd. the downstream signalling appears specific as not all nf-kb targets were up-regulated. the nf-kb repressors, nfkbia and nfkbib, were only up-regulated by dgb virion binding ( table 2) . to confirm that virion binding induces nf-kb and to examine the kinetics, an nf-kb promoter-driven luciferase construct was transfected into hff cells and cells were treated with dgh virions (1000 particles per cell) or were mock-treated. cells were harvested every hour until 9 hr post treatment, lysed and assayed for luciferase activity in triplicate. figure 1(a) shows the fold-change in luciferase activity of those hffs inoculated with dgh in comparison to mock-inoculated cells at the same time point. biological duplicates of the inoculations were performed with the lysates used in triplicate reporter assays. at 1 hpi, there is an average of 2.4 fold change in luciferase activity from the nf-kb reporter, rising to 3.5 at 2 hpi and is maintained through to 3 hpi whereby it drops to 1.8 at 4 hpi, which correlates with the half-life of luciferase [28] . by 5 hpi, and until 9 hpi, luciferase activity drops to baseline levels at, or near, 1-fold change. error bars represent the standard error across the six readings taken from technical triplicates on biological duplicates. as induction of the nf-kb reporter construct occurred within one hour of inoculation with dgh virions and peaked at around two-and-a-half hours post-inoculation, then the transcripts previhffs were stimulated with 1000 particles/cell of dgb, dgd or dgh hsv-1 for six hours and a cdna microarray corresponding to targets of 19 signalling pathways was used to detect changes in cellular gene expression when compared to mock-infected. infections and mock infections were carried out in duplicate. a number of signalling pathways were shown to be stimulated by one or more of the entry-defective mutants. these data were used to independently verify changes in expression using real-time pcr. doi:10.1371/journal.pone.0009560.t001 ously shown in table 2 to be up-regulated as a result of dgh virions binding were examined for changes in transcript levels at two-and-a-half hours after inoculation with dgh. figure 1 (b) compares expression levels at two-and-a-half and six hours postinoculation. transcripts for the nf-kb-responsive chemokine ccl2 were up-regulated to a greater degree at two-and-a-half hours than at six hours post-inoculation. of the nf-kb-responsive genes, rel transcription was induced to a higher level whereas pecam1 transcript abundance was reduced in comparison to six hours. although jak/stat-responsive targets were not differentially expressed by two-and-a-half hours, the jak/src targets bcl2 and bcl2l1 were up-regulated above the two-fold arbitrary threshold to similar transcript levels that were seen at six hours post-inoculation. the initial microarray screen was uninformative with respect to interferon signalling, but this was of interest because soluble gd has been reported to induce ifn [29] , while, in contrast, isg induction is reported to require virion entry but not de novo protein synthesis [30] . primers were designed to type i interferons, a (ifna1) and h (ifnb1), and the newly designated type iii interferon l (il29). human fibroblasts do not produce interferon gamma. additionally, the cdna preparation was used to identify any change in the abundance of isgs. both dgb and dgh virions were able to stimulate the up-regulation of ifn-a expression 2.81 fold and 2.34 fold, respectively, with a high degree of statistical significance (p#0.05) ( table 4 ). hsv-1 particles lacking gd were unable to do so, with a fold change of 21.11 and a p = 0.15 (table 4) . ifn-h transcript levels were up-regulated 2.22 fold by dgb virions and 2.06 fold (p = 0.01) by dgd virions, again with a high degree of statistical significance. inoculation with virus particles lacking gh led to a 21.07 fold change (p = 0.44) in ifn-h expression when compared to mock-infected cultures. all three entry-defective mutants were unable to modulate the expression of ifn-l above the arbitrary two-fold threshold. the glycosylation status of ifn-inducing viral glycoproteins is a potential determinant of induction efficacy [22] [31] . hsv-1 gd has three potential n-glycosylation sites but lacks o-linked glycosylation. dgh virions, that retain expression of gd, were treated with the endoglycosidase, pngase f, which removes nlinked sugars. a long incubation was required as the denaturing buffer was excluded from the reaction to avoid conformational changes that might abrogate virion binding. as previously shown, wild-type virions treated in a similar manner retained infectivity ( figure s1 ) [22] . hff were inoculated as above with dgh virions that had been incubated for 18 hrs in the presence or absence of pngase. glycosylated and deglycosylated dgh virions stimulated a 2.23 and 2.28 fold increase in ifn-a transcription, respectively. these results suggest that glycosylation has no impact on the interferogenic properties of dgh virions, unlike the glycoproteins of mhv and tgev, which appear to stimulate type i ifn through there was a degree of correlation between the change in expression determined by microarray studies and those confirmed by real-time pcr, particularly for genes under the control of nf-kb. note: rel and nfkbib were not present on the original signalling-specific microarray, but were included in the real-time analysis, as they are known to be up-regulated as part of the nf-kb feedback mechanism. doi:10.1371/journal.pone.0009560.t003 a lectin-like action, hsv-1 gd may trigger ifn up-regulation through a different mechanism, possibly analogous to that used to stimulate other signalling pathways. five interferon-stimulated genes were chosen to examine whether they were differentially expressed by virion binding. ifn-induced protein 54 (isg54) transcripts are routinely used as a marker of isg induction. binding by dgb, dgd or dgh virions was insufficient to cause changes in the transcript abundance of interferon regulatory factor-1, -3, -7, -9 or isg54 (table 4) . to ensure that these observations could not result from a background of infection by competent virus, parallel cultures were infected with wild type virus at an moi of 0.01, a level of infection some tenfold higher than would result from the 'worst case' scenario using preparations of entry defective virions (see methods). these cultures exhibited no induction of ifn or isgs ( table 4 ). all the experimental work described above was performed using treatments with 1000 virus particles per cell, a condition that would be unlikely to pertain in natural primary infections. to establish whether much lower virion numbers would be effective, hffs were serum-starved for five days then mock-inoculated or inoculated with 1, 10, 100 or 1000 particles per cell of dgh hsv-1 for six hours. infections were carried out in duplicate with mockinoculated controls that received an equivalent volume of pbs. total rna was purified and reverse transcribed, with the cdna from each duplicate inoculation used in triplicate for real-time pcr. the nf-kb responsive genes nfkb1, rel and ccl2, in addition to the jak/stat target nos2a, were used as they had previously been shown to be differentially expressed after inoculation with 1000 particles/cell of dgh hsv-1. recalling that the more abundant a transcript the fewer number of cycles it takes to cross a threshold set in the exponential phase of pcr product accumulation (the c t value), figure 2 plots the dc t value on the y-axis, (i.e. the c t value for the gene of interest after normalisation against the rpl13) against the multiplicity of infection on the x-axis. as the multiplicity of dgh increases the c t value decreases, indicating an increase in the abundance of transcripts for all three nf-kb targets (figure 2(a) ). a logarithmic regression analysis across three logs of multiplicity show a high correlation between the four c t values for each transcript with r 2 .0.98. similarly for the jak/stat responsive target nos2a there are almost two cycles of a difference between the level of transcripts after infection with 1 particle/cell compared to 1000 particles/cell (figure 2(b) ). this larger response than those for nf-kb responsive genes is indicative of the different level of induction shown with previous real-time pcr analysis of these transcripts ( table 2) . arrows in figure 2 indicate the c t value for each of the four targets examined. inoculation with 1 particle cell gave no detectable increase in transcript abundance but an increase was observed using 10 particles per cell and the dose response curves strongly suggest that lower doses have an effect. interpreting these data in the context of infection is not straightforward as particle:infectivity ratios for herpes simplex virus are, at best, 20 or more. on this basis a single infectious unit per cell is clearly sufficient to increase transcript abundance, indicating that signalling occurs at 'physiological' multiplicities of infection. evidence from previous studies using soluble hsv-1 glycoprotein and uv-inactivated virus suggested that binding of hsv-1 virions to the cell surface might be sufficient to stimulate intracellular signalling pathways. we undertook preliminary microarray studies with entry-defective hsv-1 virions and identified a number of pathways that were stimulated at early time points during infection. microarray experiments are inevitably vulnerable to false positives and negatives due to non-specific binding of labelled cdna probes, necessitating a more robust follow-up with highly sensitive methods. due to such confounders, these data were used exclusively as a guide to select, for real-time pcr, the transcriptional targets of intracellular signalling pathways stimulated after inoculation with entry-defective hsv-1. gene targets associated with the nf-kb, jak/stat/src, and pi3k/akt pathways were shown to be differentially expressed after inoculation with glycoprotein-deficient virions, with the majority of signalling events being associated with the presence of gd on the envelope. nineteen other signalling pathways present on the preliminary microarray experiments, and confirmed by real-time pcr, were not stimulated (data not shown). these results are compatible with our model, shown in fig. 3 . real-time pcr confirmed that changes in transcription associated with the nf-kb, jak/stat, jak/src and pi3k pathways were modulated as a result of virion binding, all of which required gd on the envelope surface to demonstrate that signalling occurred at physiologically relevant multiplicities of infection, hffs were inoculated with either 1000, 100, 10 or 1 particles per cell of entry-defective hsv-1. changes in gene transcription occurred in a dose-dependent manner and were detectable at 10 virus particles per cell. given the particle:infectivity ratios usually quoted for hsv1, we argue that this corresponds to physiological conditions (i.e. less than one infectious unit per cell), but it is impossible to know the circumstances that pertain in vivo when a single cell becomes infected. what seems almost certain is that an infected cell will normally present an uninfected neighbouring cell with 10 or more progeny particles. hsv-1, as well as beta-and gammaherpesviruses, are capable of stimulating the nf-kb pathway in a bi-phasic manner, with our data supporting that an early, transient induction is reliant on virions expressing gd [8] [32] [33] . suppression of nf-kb activity is via negative feedback up-regulation of the inhibitor ikba (nfkbia), which was also stimulated by the binding of entry-defective hsv-1 virions. the triggering of early nf-kb transcriptional activity was most likely through the coupling of gd on entry-defective virions to the tnf superfamily receptor hvem [1] . in doing so, not only does the initial activation of this pathway allow for the subsequent sequestration of the nf-kb p65 subunit to the icp0 promoter, but is crucial for immediate-early gene transcription and subsequent hsv-1 replication [8] . intracellular signalling induced by soluble gd can protect against fas-mediated apoptosis with inhibition of nf-kb signalling leading to a loss of this protection [6] . infection with uv-inactivated virions also led to an increase in the expression of the anti-apoptotic protein c-iap2 (birc3), which we have demonstrated to be up-regulated after inoculation with entry-defective virions containing gd. additional studies have supported the anti-apoptotic role for nf-kb during hsv-1 infection yet there are conflicting data that demonstrate possible pro-apoptotic activity [34] [35] . this inconsistency may be due to differing cell types used in those studies. primary human foreskin fibroblasts have been shown to be resistant to apoptosis after infection with recombinant hsv-1 that is unable to express icp4 or icp27 whereas infection with either virus has been shown to cause apoptosis in transformed cell lines [36] . aspects of the hsv-1 life cycle, such as stimulating the progression of the cell cycle in the absence of serum, may be sufficient to induce a stress response and trigger apoptosis. both bcl2 (bcl-2) and bcl2l1 (bcl-xl) belong to the bcl-2 family of apoptosis regulators that provide cellular protection from a range of harmful stimuli such as cytokine deprivation, uv-and cirradiation [37] . bcl-2 and bcl-xl are found in the outer mitochondrial membrane and are thought to suppress apoptosis by blocking mitochondrial outer-membrane permeabilisation through the sequestration of pro-apoptotic bcl2 family members [38] . given the up-regulation of four anti-apoptotic genes, birc2, birc3, bcl2 and bcl2l1, through the activation of multiple signalling pathways by entry-defective hsv-1, this establishes a role for gd binding in shifting the intracellular environment towards a more anti-apoptotic stance. . model for glycoprotein-receptor interactions in the induction of intracellular signalling pathways by hsv-1. glycoprotein d acts as the main signalling molecule on the surface of the hsv-1 envelope. gh interacts with a v b 3 integrins to potentially trigger the production of ifn-b, which is known to involve irf-3 and 7 [48] . binding by gd to hvem may lead to the activation of traf molecules, which in turn stimulate the nf-kb signaling cascade. this pathway up-regulates a number of cellular genes in addition to augmenting early viral gene expression. nf-kbresponsive genes, birc2 and birc3, have an anti-apoptotic role, but paradoxically, inflammatory mediators such as ccl2 are also up-regulated. gdinduced signalling of the jak/stat and jak/src pathways also results in the differential expression of genes associated with anti-apoptosis and inflammation. the up-regulation of c-myc could lead to a corresponding increase in cdk2, which has a role in promoting dna replication and gene transcription during infection. it should be noted that most signalling cascades have been elucidated in non-fibroblast cells lines, so the role of specific kinases may vary in hffs. doi:10.1371/journal.pone.0009560.g003 it is less apparent as to the biological relevance of an innate immune response stimulated through hsv-1 binding. it may be that there is a ''cost'' associated with altering the intracellular environment, which leads to the differential expression of cytokines, such as ccl2, that are under similar transcriptional regulation as those host factors that are favourable for virus replication. signalling by secreted type i ifns occurs through the jak/stat pathway results in the expression of various isgs; a response that is also triggered by virus entry [39] . nevertheless, productive infection with hsv-1 can down-regulate the triggered isg response, allowing viral replication to continue unhindered [40] . despite our evidence that gd binding by entry-defective virions can induce ifn-a mrna expression, independent of gd glycosylation status, these data also fit with published observations that binding by hsv-1 is insufficient to cause the up-regulation of interferon-stimulated genes [30] . the up-regulation of ifn-b, albeit it through a different mechanism, is suggestive of a previously unidentified role for gh in eliciting a change in host gene expression. an entry-defective hsv-1 mutant lacking gb that also contains an rge rather than the integrin binding rgd motif of gh has been constructed and future studies may further elicit the role of gh in interferon stimulation. the methodology used here required the serum-starvation of primary human fibroblasts for five days. in the absence of serum, primary fibroblasts rapidly enter a quiescent state. as a dna virus that requires host nuclear factors to replicate its genome, it is therefore not surprising that hsv-1 would stimulate cells from a g0 state into one that would favour dna replication and possibly promote the transcription of viral genes. quiescent cells in vitro have very low levels expression of the transcription factor c-myc. its up-regulation is rapidly induced after mitogenic stimulation or the introduction of serum and increased expression of c-myc is consistent with the advancement of cellular proliferation [41] . control of myc transcription can be influenced by a number of pathways, including pi3k/akt signalling, which was shown here to occur as a result of binding by gd. a central role for c-myc in promoting cell-cycle progression is evident from the genes that it can up-regulate such as eif2 [42] . progression of the cell cycle depends on the additional activity of cyclin-dependent kinases. by interacting with the promoters for genes encoding cyclins and cyclin-dependent kinases, c-myc can influence the advancement on the cell cycle into the g1/s phase [43] . cyclin-dependent kinase 2 (cdk2) is one such downstream target of c-myc activity, as well as the androgen pathway, highlighting the signalling cross-talk that may occur [44] . cdk2 is involved in the progression of the cell cycle from g1 through to s phase. transient activation of cdk2 was shown to occur early in hsv-2 infection at two hours post-infection, and is crucial in early hsv-1 infection [45] [46] . kinase action by the cyclin a/cdk2 complex liberates the bound transcription factor e2f from rb, a transcription factor that has previously been shown to be active during hsv-1 infection [47] . epithelial cells at the initial site of hsv-1 infection in vivo are likely to be in a resting state, necessitating the virus to evolve a preentry signalling mechanism by which to stimulate the cell to provide host factors that are necessary for viral replication. we have demonstrated that signalling induced by hsv-1 glycoproteins, primarily gd, has the potential to: activate cellular transcription factors that augment viral gene transcription, differentially express a number of cellular genes so as to condition the cell for optimal replication or, alternatively, signal transduction may occur as a secondary effect to the appropriation of cellular receptors to achieve viral entry. involvement of gd/hvem interaction in nf-kb-dependent inhibition of apoptosis by hsv-1 gd entry of herpesviruses into mammalian cells the role of glycoprotein h in herpesvirus membrane fusion herpes simplex virus type 1 glycoprotein h binds to alphavbeta3 integrins pilralpha is a herpes simplex virus-1 entry coreceptor that associates with glycoprotein b protection by herpes simplex virus glycoprotein d against fas-mediated apoptosis: role of nuclear factor {kappa}b herpes simplex virus type 1 induction of persistent nf-kb nuclear translocation increases the efficiency of virus replication*1 herpes simplex virus disrupts nf-{kappa}b regulation by blocking its recruitment on the i{kappa}b{alpha} promoter and directing the factor on viral genes signaling pathway used by hsv-1 to induce nf-kb activation global modulation of cellular transcription by human cytomegalovirus is initiated by viral glycoprotein b binding of the epsteinbarr virus major envelope glycoprotein gp350 results in the upregulation of the tnf-a gene expression in monocytic cells via nf-kb involving pkc, pi3-k and tyrosine kinases1 epstein-barr virus and its glycoprotein-350 upregulate il-6 in human b-lymphocytes via cd21, involving activation of nf-kb and different signaling pathways1 role of glycoprotein b of herpes simplex virus type 1 in viral entry and cell fusion construction and properties of a mutant of herpes simplex virus type 1 with glycoprotein h coding sequences deleted a herpes simplex virus mutant in which glycoprotein d sequences are replaced by beta-galactosidase sequences binds to but is unable to penetrate into cells a mutant herpes simplex virus type 1 unable to express glycoprotein l cannot enter cells, and its particles lack glycoprotein h herpes simplex virus tegument protein vp16 is a component of primary enveloped virions a study of primary neuronal infection by mutants of herpes simplex virus type 1 lacking dispensable and nondispensable glycoproteins a genetically inactivated herpes simplex virus type 2 (hsv-2) vaccine provides effective protection against primary and recurrent hsv-2 disease assembly and organization of glycoproteins b, c, d, and h in herpes simplex virus type 1 particles lacking individual glycoproteins: no evidence for the formation of a complex of these molecules electron microscopic particle counts on herpes virus using the phosphotungstate negative staining technique virus-cell interactions regulating induction of tumor necrosis factor alpha production in macrophages infected with herpes simplex virus primer3 on the www for general users and for biologist programmers a new mathematical model for relative quantification in realtime rt-pcr an analysis of the biological properties of monoclonal antibodies against glycoprotein d of herpes simplex virus and identification of amino acid substitutions that confer resistance to neutralization monoclonal antibodies to three non-glycosylated antigens of herpes simplex virus type 2 an endoplasmic reticulumretained herpes simplex virus glycoprotein h is absent from secreted virions: evidence for reenvelopment during egress modulation of firefly luciferase stability and impact on studies of gene regulation induction of interferona by glycoprotein d of herpes simplex virus: a possible role of chemokine receptors activation of interferon response factor-3 in human cells infected with herpes simplex virus type 1 or human cytomegalovirus single amino acid changes in the viral glycoprotein m affect induction of alpha interferon by the coronavirus transmissible gastroenteritis virus human cytomegalovirus up-regulates the phosphatidylinositol 3-kinase (pi3-k) pathway: inhibition of pi3-k activity inhibits viral replication and virus-induced signaling binding of the epsteinbarr virus major envelope glycoprotein gp350 results in the upregulation of the tnf-[alpha] gene expression in monocytic cells via nf-[kappa]b involving pkc, pi3-k and tyrosine kinases efficient replication by herpes simplex virus type 1 involves activation of the ikappab kinase-ikappab-p65 pathway the patterns of accumulation of cellular rnas in cells infected with a wild-type and a mutant herpes simplex virus 1 lacking the virion host shutoff gene viral oncoapoptosis of human tumor cells bcl-2, bcl-xl and adenovirus protein e1b19kd are functionally equivalent in their ability to inhibit cell death the bcl2 family: regulators of the cellular life-ordeath switch innate cellular response to virus particle entry requires irf3 but not virus replication identification of a novel pathway essential for the immediate-early, interferon-independent antiviral response to enveloped virions increased expression of eukaryotic translation initiation factors eif-4e and eif-2 alpha in response to growth induction by c-myc expression analysis with oligonucleotide microarrays reveals that myc regulates genes involved in growth, cell cycle, signaling, and adhesion induction of cyclin e-cdk2 kinase activity, e2f-dependent transcription and cell growth by myc are genetically separable events analysis of cyclin-dependent kinase activity after herpes simplex virus type 2 infection requirement for cellular cyclin-dependent kinases in herpes simplex virus replication and transcription e2f proteins are posttranslationally modified concomitantly with a reduction in nuclear binding activity in cells infected with herpes simplex virus 1 irfs: master regulators of signalling by toll-like receptors and cytosolic pattern-recognition receptors we thank susanne bell for invaluable technical assistance and helena browne for helpful discussions and critical reading of the manuscript. conceived and designed the experiments: ijm tm. performed the experiments: ijm. analyzed the data: ijm. contributed reagents/ materials/analysis tools: ijm. wrote the paper: ijm tm. key: cord-001275-a9o2dvke authors: chen, xue; liu, hongyang; zhang, ting; liu, yanchun; xie, xixiu; wang, zhirong; xu, xuemei title: a vaccine of l2 epitope repeats fused with a modified igg1 fc induced cross-neutralizing antibodies and protective immunity against divergent human papillomavirus types date: 2014-05-06 journal: plos one doi: 10.1371/journal.pone.0095448 sha: doc_id: 1275 cord_uid: a9o2dvke current human papillomavirus (hpv) major capsid protein l1 virus-like particles (vlps)-based vaccines in clinic induce strong hpv type-specific neutralizing antibody responses. to develop pan-hpv vaccines, here, we show that the fusion protein e3r4 consisting of three repeats of hpv16 l2 aa 17–36 epitope (e3) and a modified human igg1 fc scaffold (r4) induces cross-neutralizing antibodies and protective immunity against divergent hpv types. e3r4 was expressed as a secreted protein in baculovirus expression system and could be simply purified by one step protein a affinity chromatography with the purity above 90%. vaccination of e3r4 formulated with freunds adjuvant not only induced cross-neutralizing antibodies against hpv pseudovirus types 16, 18, 45, 52, 58, 6, 11 and 5 in mice, but also protected mice against vaginal challenges with hpv pseudovirus types 16, 45, 52, 58, 11 and 5 for at least eleven months after the first immunization. moreover, vaccination of e3r4 formulated with fda approved adjuvant alum plus monophosphoryl lipid a also induced cross-neutralizing antibodies against hpv types 16, 18 and 6 in rabbits. thus, our results demonstrate that delivery of l2 antigen as a modified fc-fusion protein may facilitate pan-hpv vaccine development. more than 150 hpv genotypes have been identified with different epithelial tropisms [1] . infection with cutaneous hpv types (such as hpv1, 2, 3, 5) causes benign cutaneous warts or epidermodysplasia verruciformis (hpv5). mucosal hpv types infect the upper part of the respiratory tract (hpv6, 11), oral cavity (hpv13), and the epithelium of the anogenital region. the low-risk anogenital hpvs (such as hpv6, 11, 42) cause genital warts whereas the high-risk hpvs (such as hpv16, 18, 31, 45, 52, 58) are associated with progression to carcinoma of the cervix, vulva, vagina, penis, anus and oropharynx [2] . cervical cancer is the third most common cancer worldwide, and about 70% of cervical cancers are caused by infections with hpv types 16 and 18. currently there are two licensed hpv major capsid protein l1 virus-like particle (vlp)-based vaccines, cervarix, a bivalent hpv16/18 vaccine, and gardasil, a quadrivalent hpv16/18/ 6/11 vaccine. although some cross-reactivity has been observed between closely related hpv genotypes, the protection provided upon vaccination with hpv l1 vlp vaccine is largely hpv typespecific, indicating that vaccination provides very little cross-protection to the hpv types not covered by the vaccines [3, 4] . the limited cross-protective capacity of l1-based vaccines makes it necessary to develop a pan-hpv vaccine. vaccination with recombinant minor capsid protein, l2, or peptides derived from l2 results in the production of crossneutralizing antibodies that are protective in animal models [5, 6] . in the context on native virions, l2 is poorly immunogenic. neither natural infection nor immunization with hpv l1/l2 vlps elicits anti-l2 antibody responses [7] . studies showed that l2 is poorly exposed on the surface of virions. it is generally accepted that after hpv virus binds to heparin sulfate moieties on the basement membrane, the capsid undergoes a conformational change that exposes the amino terminus of l2 [8] . the exposed nterminus of l2 is susceptible to protease cleavage, thus exposing l2 epitopes near the n-terminus of the protein [9] . several regions in the n-terminus of l2 can be targeted by neutralizing antibodies [10, 11, 12, 13, 14] , which prevent viruses from transferring from basement membrane to unidentified receptor on epithelial cells. a major cross-neutralizing epitope located in amino acid 17 to 36 represents an attractive candidate antigen for broadly protective vaccination [15] . the neutralizing titers produced by l2 vaccination are considerably lower than that induced by l1 vlp vaccination, particularly against heterologous hpv types [16] . therefore, it is likely that an l2 vaccine will only be effective if its immunogenicity is enhanced. b-cell activation is initiated following engagement of the b-cell receptor (bcr) by a specific antigen. large antigens, such as immune complexes and viruses, can be presented to b cells more efficiently than small soluble molecules [17, 18, 19] . unlike tcell receptor (tcr) which recognizes processed epitopes in the context of major histocompatibility complex molecules, bcr may recognize unprocessed antigens presented on the surface of antigen presenting cells (apcs) [20, 21, 22] . displaying multivalent l2 epitope in exposed regions on vlps derived from papillomavirus [23, 24, 25] , bacteriophage [26, 27] and adeno-associated virus [28] , or in the surface region of bacterial thioredoxin [10] has shown to induce enhanced epitope-directed antibody responses and broadly protective immunity. the fc receptors for igg (fccrs), expressed on dendritic cells (dcs) and apcs, can bind and internalize antigen-igg immune complexes via the interaction with the igg, resultsing in enrichment of exogenous antigens in dcs, which facilitates dc maturation and antigen-specific t cell responses and humoral responses. recombinant antigen-immunoglobin fc-fusion proteins were shown to increase the immunogenicity of the fused antigens and elicit neutralizing antibody responses to hiv [29, 30] and protective immunity to virulent herpes simplex virus [31] , influenza viruses [32] and ebola viruses [33] . in this study, we showed for the first time that fusing hpv16 l2 aa 17-36 epitope repeats to a recombinant ligand for fccrs (designated l2r4, see figure 1a -b) could significantly increase the immunogenicity of the l2 peptide and induce cross-neutralizing antibodies and protective immunity against a range of phylogenetically distant hpv types. a recombinant ligand for fccrs, r4, is an fc-like fusion protein that contains a tandem repeat composed of 4 copies of the hinge and ch2 regions (hch2) of human igg1 for each chain [34] , and the hch2 region of igg encompasses the sequences that bind fccrs [34, 35, 36, 37] . to express l2 peptide as an fc-like fusion protein, the l2 antigen gene encoding three repeats or twelve repeats of hpv16 l2 aa 17-36 epitope with a three amino acid linker (gly-gly-pro) in between was fused in-frame to the upstream of r4 ( figure 1a) , and the resulting fusion protein was designated as e3r4 or e12r4 ( figure 1b ). to facilitate purification, the signal peptide sequence of gp67 of baculovirus was added to the upstream of each l2r4 fusion gene. the l2r4 genes were codon optimized based on the codon usage bias in sf9 cells (sangon, shanghai), subcloned into pfastbac1 vector and expressed as secreted proteins in baculovirus expression system as described previously [7, 38, 39] . e3r4 and e12r4 proteins were purified from the culture supernatant by rprotein a sepharose affinity chromatography according to the manufacturer's instructions (ge healthcare). r4 protein was also expressed and purified as a scaffold control. the expression and analysis of the recombinant proteins were evaluated by sds-page with coomassie blue staining and western blot with 1:5,000 dilution of rg-1, a cross-neutralizing and protective monoclonal antibody that recognizes residues 17-36 of hpv16 l2 (generously provided by richard roden) [15] as previous reported [38] . the purity of purified proteins was determined by sf9 host cell protein (hcp) elisa as described previously [39] . four-to six-week-old female balb/c mice and new zealand white rabbits were purchased from the institute of laboratory animal science, chinese academy of medical sciences, and kept in the animal facility of the institute of basic medical sciences, chinese academy of medical sciences. all animal work was done in accordance with the guidelines of the institutional animal care and use committee of the institute of laboratory animal science, chinese academy of medical sciences, and all experimental protocols were approved by the institutional animal care and use committee. groups of 5 mice were vaccinated on weeks 0, 2, 4 and 6 with 1.7 nmol of e3r4, e12r4, r4, e3 peptide (lifetin, beijing) or phosphate-buffered saline (pbs) formulated with complete freunds adjuvant (cfa) for priming dose and with incomplete freunds adjuvant (ifa) for booster immunizations. groups of 2 rabbits were vaccinated subcutaneously on weeks 0, 2, 4 and 6 with 4 nmol of e3r4 alone or with alum (sigma-aldrich) and monophosphoryl lipid a (sigma-aldrich) (alum-mpl) adjuvant. sera were collected at weeks 6 and 8 and stored at -20uc. pseudoviruses of hpv16, 18, 45, 58, 6, 11 and 5 (psv16, psv18, psv45, psv58, psv6, psv11 and psv5, respectively) with encapsidated reporter plasmid plucf which encoding both luciferase and green fluorescence protein (gfp), or plasmid pegfp-n1 (clonetech) which encoding gfp were produced in 293tt cells as previously described [38, 40, 41] with minor modifications. for psv52 preparation, 16 mg dna (8 mg of shell plasmid and 8 mg of reporter plasmid) was mixed with 56 ml of turbofect (thermo fisher) and 1.6 ml of unsupplemented dmem, then added to 6.5610 6 293tt cells as suggested by prof. reinhard kirnbauer. the titer of psv (infectious units per ml, iu/ml) was determined by gfp expression in 293tt cells as described in http://home.ccr.cancer.gov/lco/pseudovirusproduc tion.htm. l1/l2 expression plasmids, plucf plasmid, were generously provided by john schiller, susana pang, chris buck, martin müller and tadahito kanda. to assess the binding of l2r4 proteins to rg-1, elisa plates were coated with 100 ng of each protein (e3r4, e12r4, r4), or 5 ng (equivalent to the amount of the recombinant proteins on a mole basis) of e3 peptide at 4uc overnight. elisa was performed as previously described [38, 39] . briefly, wells were blocked with 5% bovine serum albumen (bsa) in pbst at room temperature for 2 hours. rg-1 monoclonal antibody (1:3,000 dilution) was added to each well and incubated at room temperature for 2 hours. after wash, wells were incubated with horseradish peroxidase (hrp)-conjugated goat anti-mouse igg (1:5,000 dilution) at room temperature for 1 hour. plates were developed by the addition of 0.4 g/ml of o-phenylenediamine diluted in phosphate-citrate buffer, ph 5.0, containing 0.045% (v/v) hydrogen peroxide. the enzymatic reaction was stopped by the addition of 50 ml of a solution containing 2 m h 2 so4. plates were read at 490 nm, and antibody titer was determined as the reciprocal of the highest serum dilution with an od 490 greater than 0.2 and 2-fold higher than control sera at the same dilution. to measure cross-reactive antibody responses to hpv capsids in antisera, elisa plates were coated with 1.5610 4 to 2.0610 4 iu of hpv psvs (depending on the psv stock) encoding gfp. sera collected at week 8 were assessed by end-point dilution elisa as described above. binding titer was determined as the reciprocal of the highest serum dilution with an od 490 greater than 0.2 and 2-fold higher than control sera at the same dilution. statistical significance was determined by one-way anova and bonferroni multiple comparison test (graphpad prism 5). p value ,0.05 was considered to be statistically significant. hpv psv-based neutralization assays were performed as previous reports [38, 39, 41] . pseudovirus (encoding gfp) diluents and serially diluted sera were mixed and added to the cell culture plate, after incubation, the 293tt cells were digested with trypsin and transferred to cell sorting tube. the fluorescent cells were detected by fluorescence activated cell sorting. the endpoint titer was calculated as the reciprocal of the highest serum dilution with percent infection inhibition higher than 50%, and a titer ,50 was considered as nonsignificant [42, 43] . every sample was detected in duplicate. statistical significance was determined by one-way anova and bonferroni multiple comparison test. mice were treated with 3 mg of progesterone subcutaneously four days before psv challenge. the immunized mice were intravaginally pretreated with 50 ml of 4% nonoxynol-9 (n9, igepal, sigma) at six hours prior to psv challenge, r4 and pbs injected female mice were used as controls. 20 ml of psv preparation containing 2.5610 5 to 1.0610 7 iu of psvs (encapsi-dated reporter plasmid plucf) and 1% carboxymethyl cellulose (cmc, sigma) was intravaginally instilled using a positivedisplacement pipette. forty-eight hours post-psv challenge, mice were vaginally instilled with 0.4 mg of 59-f-luciferin (cellcyto life sciences). three minutes later, luciferase signals were acquired for 5 min with a biofluorescence imaging (bfi) system of the lb 983 nightowl ii (berthold technologies), and analyzed with indigo 2 software (berthold technologies). statistical significance was determined by one-tailed unpaired ttest. we first examined the expression of l2r4 proteins in the culture medium by sds-page since they are expressed as secreted proteins. we observed e3r4 (120 kda) or e12r4 (130 kda) band by coomassie blue staining ( figure 1c) , which was further confirmed by western blot ( figure 1d ). the recombinant proteins were highly expressed, with approximately 3 mg of e3r4 or 2.5 mg of e12r4 obtained per 100 ml culture medium. the proteins were purified with rprotein a affinity chromatography, and the purity was analyzed by sds-page with coomassie blue staining ( figure 1e ) and sf9 hcp elisa [39] . when 10 mg of each protein was loaded, a single band was observed in e3r4 sample, indicating that e3r4 was stable, but two additional small bands were also observed in e12r4 sample, indicating that e12r4 was partially degraded as these small bands were all reactive with rg-1 antibody by western blot (not shown). by hcp elisa, we found that the purity of purified e3r4 and e12r4 was above 90%. to further characterize the stability of e3r4, we kept the purified protein at room temperature for 5 days and found a single band in e3r4 sample by sds-page with coomassie blue staining (not shown). taken together, we conclude that e3r4 fusion protein can be highly expressed and simply purified with stability in vitro. to assess the immunogenicity of l2r4 proteins, we first showed that rg-1 strongly bound to e3r4, e12r4 and e3 peptide, but not to r4 scaffold control (figure 2) , indicating that l2 epitopes in l2r4 proteins are well exposed to be recognized by rg-1 antibody. we next asked whether l2r4 proteins were able to induce cross-reactive antibody responses. as our goal was to develop a vaccine inducing the broadest cross-reactivity against divergent hpv types, a panel of hpv psvs, including hpv16, 18, 45, 52, 58 (five common oncogenic types), hpv6, 11 (the most common types found in benign genital warts) and hpv5 (causes epidermodysplasia verruciformis) were used to coat elisa plates. sera were collected at 2 weeks after four immunizations from mice, and the hpv capsid-reactive antibody titer was measured by endpoint dilution elisa. the binding titers against each psv type in e3r4 and e12r4 antisera were significantly higher than e3 antisera (p,0.001) (figure 3) , suggesting that the r4 scaffold can enhance the immunogenicity of fused antigens. we also observed stronger antibody responses induced by e3r4 than e12r4 (p, 0.001), which may be due to the instability of e12r4 ( figure 1e ). thus, these data show that e3r4 fusion protein is highly immunogenic and induces broad cross-reactive antibodies against divergent hpv types. hpv capsid-based elisa assay detects both non-neutralizing and neutralizing antibodies, and the neutralizing antibodies are thought to be the primary immune mechanism of protection by hpv vaccination. thus, we further examined the cross-neutral-izing antibody responses induced by l2r4 proteins using psvbased in vitro neutralization assay. as proof-of-concept, a panel of psvs encoding gfp from divergent hpvs, including hpv16, 52, 58 (alpha-9), hpv6, 11 (alpha-10), hpv18, 45 (alpha-7) and hpv5 (beta-1), were used in neutralization assays. although the cross-neutralizing antibody levels in the antisera after the third immunization of e3r4 or e12r4 were very low (titer, ,50, not shown), we did observed broad cross-neutralizing antibody responses induced by e3r4 after the fourth immunization ( figure 4) . the mean neutralizing antibody titers of the e3r4 antisera against hpv16, 52, 58, 6, 11, 18, 45 and 5 were 1040, 320, 360, 110, 110, 400, 420 and 380, respectively, which were significantly higher than e12r4 or e3 antisera (p,0.05). e12r4 only induced weak anti-hpv16 neutralizing antibody response (mean titer, 80) and the neutralizing antibody titers against heterologous psvs in e12r4 antisera were all below 50. although e12r4 elicited significantly higher cross-reactive antibody responses than e3 measured by hpv capsid-based elisa assay (figure 3 ), there were no significant differences between the crossneutralizing antibody titers in e12r4 and e3 antisera (p.0.05), suggesting that the majority of serum antibodies induced by e12r4 were not neutralizing antibodies. we didn't detect any neutralizing antibodies in r4 scaffold control antisera (not shown). collectively, we show that freunds adjuvanted e3r4 vaccine induces broad cross-neutralizing antibody responses in mice. given the broad cross-neutralizing antibody responses elicited by freunds adjuvanted e3r4 protein in mice, we next examined the in vivo protection against multiple hpv types using the vaginal challenge assay. mice were immunized with e3r4 or r4 formulated with freunds adjuvant for four times with 2-week intervals. eleven months after the first immunization, mice were vaginally challenged with divergent psvs (hpv16, 45, 52, 58, 11, 5) encoding luciferase. consistent to relatively high-levels of crossneutralizing antibodies in antisera (figure 4 ), mice immunized with e3r4 were completely protected from challenge of psv16 ( figure 5a , p,0.05), psv45 ( figure 5b , p,0.05), psv52 ( figure 5c , p,0.05), psv58 ( figure 5d , p,0.01), psv11 ( figure 5e , p,0.01) and psv5 ( figure 5f , p,0.05), whereas the mean luminescence signal in mice immunized with r4 scaffold was comparable to that in pbs control mice. thus, our data demonstrate that e3r4 vaccine could provide substantial protection against diverse types of hpv psvs. to further evaluate the potency and potential clinical application of e3r4 fusion protein, we examined the immune responses induced by e3r4 formulated with fda approved adjuvant alum-mpl in another animal model, the new zealand white rabbit. two rabbits were vaccinated with either e3r4 alone or formulated with alum-mpl according to previous studies [24, 44] . sera collected after the fourth immunization were analyzed by psv-based in vitro neutralization assay. the results were shown in table 1 . while the neutralizing antibody titers against psv16 and other psv types were all below 25 in e3r4 alone antisera, we observed neutralizing antibody responses against hpv16, 18 and 6 in antisera from rabbits immunized with e3r4 formulated with alum-mpl. the neutralizing antibody titers of the two rabbits were 400 and 50 against hpv16, 50 and 25 against hpv18, 50 and less than 25 against hpv6. the neutralizing antibody titers against other psv types were all below 25. we did notice that the cross-neutralizing antibody responses by e3r4 in rabbits were weaker than in mice, which may be due to the fact that alum-mpl adjuvant used in rabbits is less effective than freunds adjuvant used in mice. in addition, reported antigen doses used in rabbits were usually 5fold-higher or more than in mice [24, 43, 45] , but the e3r4 dose in rabbits is only 2.35-fold-higher than that in mice. thus, the relatively lower dose injected in rabbits may also cause the weaker cross-neutralizing antibody responses than in mice. nevertheless, our accumulative data show that e3r4 induces cross-neutralizing antibodies in two different animal models when formulated with alum-mpl or freunds adjuvants. neutralizing antibodies binding to linear epitopes in hpv16 l2 aa 17-36, 65-81 and 108-120 have been described [11, 15, 23, 42] . passive transfer of hpv16 l2 aa 17-36 antiserum or rg-1 antibody has shown to protect the mice from hpv16 psv infection, suggesting that the neutralizing antibodies induced by l2 epitope is sufficient for in vivo protection [15, 42] . it was reported that sera from mice immunized with a tandem repeat of l2 aa 17-36 derived from 22 different hpv types with gpi-0100 adjuvant could neutralize hpv psv types 16, 18, 45, but not types 6, 58 [43] . in this study, we fused three repeats of hpv16 l2 aa 17-36 with a modified igg1 fc to generate e3r4 fusion protein, which can be highly expressed, simply purified with high purity and is relatively stable in vitro. importantly, immunization of e3r4 with freunds adjuvant elicited broad neutralizing antibody responses against hpv types 16, 18, 45, 52, 58, 6, 11 and 5 in mice. induction of long-term and broadly protective immunity against all clinically relevant hpv types is the goal of pan hpv vaccine development. similar to the rg1-vlps (presenting rg-1 epitope on the surface of hpv16 l1 vlps) which were shown to induce enduring protective antibody against hpv58 for 12 month after vaccination [25] , our results also showed that the broadly protective immunity induced by e3r4 with freunds adjuvant sustained at least 11 months after the first immunization, highlighting that l2 epitope-based protein vaccine is able to induce long-lasting protective immunity when the epitope is properly delivered. in the current study, we used r4, a modified igg1 fc, as a scaffold to display l2 epitopes and observed strong effects of r4 scaffold on enhancing the immune responses induced by l2 epitopes. fc of igg is considered as an important fusion tag for coexpressing several viral proteins to promote correct folding of the fusion proteins, facilitate purification, enhance the binding to apcs expressing fccr and improve the immunogenicity of fused proteins [30, 32, 33, 46, 47] . previous studies showed that recombinant proteins by fusing r4 to antigen proteins, such as human serum albumin domain 1 and the clostridal botulinum neurotoxin, could be effectively targeted to all classes of fccrs on apcs and elicit enhanced antigen-reactive antibody responses [34, 35] . thus, we think the enhanced immunity induced by e3r4 may result from targeted antigen delivery to apcs. we do not exclude other mechanisms contributing to the enhanced immune responses, such as prolonged serum half-life of fc-fusion protein [48] . in addition, as r4 is derived from human igg1, we speculate that administration of e3r4 which containing a modified human igg1 fc in humans may result in better immune responses than in mice because human fc can bind to human fccr on immune cells, such as macrophages and dcs, more efficiently, thus contributing to an enhanced immunity. previous studies have shown that the immunogenicity of hpv16 l2 aa17-36 (rg-1 epitope) is relatively low [28, 43] . in our study, we failed to detect neutralizing antibodies in the antisera from the mice immunized with e3r4 alone for four times (not shown), which may be mainly due to the weak immunogenicity of rg-1 epitope. our results were consistent with the previous report on a chimeric adeno-associated virus-like particle bearing l2 epitopes from hpv16 and 31, which also failed to induce neutralizing antibodies when it was immunized alone [28] . a concatenated multi-type l2 fusion protein was shown to elicit more broad neutralizing antibody responses than recombinant l2 derived from a single hpv type [43] , and a thioredoxin fusion protein with multiple copies of l2 peptide induced stronger immunogenicity than with single copy of l2 peptide [10] . in this study, we also generated e12r4 protein which contains 12 copies of rg-1 epitope. unfortunately, due to the poor protein stability, e12r4 only induced lower-level of anti-hpv16 neutralizing antibodies and non-detectable cross-neutralizing antibodies against other hpv types. consistent with our observations, jagu et al found that hpv16 l2 aa 13-88, an unstable peptide, produced much weaker cross-neutralizing antibody responses than hpv16 l2 aa 1-88, a stable peptide containing the same amount of epitopes [43] , suggesting an essential role of the stability of antigen protein in vaccine efficacy. we speculate that the vaccine potency can be further enhanced if a stable l2r4 protein containing much more l2 epitopes from multiple hpv types is developed. it is worthy to note that unlike freunds adjuvanted e3r4 immunization in mice, alum-mpl adjuvanted e3r4 immunization in rabbits only induced low-titers of neutralizing antibodies against hpv16, 18 and 6. adjuvant [24, 49, 50, 51] , antigen dose, animal model and vaccine scaffold are all contributing to the final antibody responses. for example, the rg1-bpv1 vlps (with bovine papillomavirus l1 vlp as a scaffold) formulated with freunds adjuvant (cfa/ifa) induced much higher neutralizing antibody titers against hpv types 16, 18, 45, 58 and 5 than formulated with alum-mpl [24] . furthermore, even using the same adjuvant alum-mpl, the neutralizing antibody titers against hpv types 16 and 18 were much higher in mice receiving 10 mg dose of rg1-bpv1 vlps than those in rabbits receiving 50 mg dose (5-fold-higher dose than in mice) [24] . in our study, the adjuvant used in rabbits (alum-mpl) is less effective than that used in mice (freunds adjuvant), and we only used 2.35-foldhigher dose (4 nmol) in rabbits than that in mice (1.7 nmol). we also noticed that the cross-neutralization capacity of alum-mpl adjuvanted e3r4 in rabbits in this study is lower than the alum-mpl adjuvanted rg1-vlp, which uses papillomavirus vlp as a platform to display the rg-1epitope [25] . papillomavirus vlps themselves are good ''adjuvant'' because of their highly immunofigure 3 . cross-reactive antibody titers in sera from mice vaccinated with l2r4 proteins. mice were immunized on weeks 0, 2, 4 and 6 with each protein (e3r4, e12r4 and r4) or e3 peptide formulated with freunds adjuvant, and sera were collected at week 8. dilutions of each of hpv l1/l2 pseudovirus, including psv16, 18, 45, 52, 58, 6, 11 and 5, were coated in elisa plates and used to determine the cross-reactive antibody titers of the antiserum. the binding titer was determined as the reciprocal of the highest serum dilution with an od 490 greater than 0.2 and 2-fold higher than control sera at the same dilution. the experiment was repeated twice. the horizontal bars represent the geometric mean antibody titers. the statistically significant differences (using one-way anova and bonferroni multiple comparison test) were indicated by: ***, p,0.001. doi:10.1371/journal.pone.0095448.g003 figure 4 . neutralizing antibody titers in sera of mice vaccinated with l2r4 proteins. mice were immunized and sera were collected (see figure 3 legend for detail). antisera were tested for in vitro neutralization titers against hpv16 (a), hpv18 (b), hpv45 (c), hpv52 (d), hpv58 (e), hpv6 (f), hpv11 (g) and hpv5 (h) pseudovirus. the horizontal bars represent the geometric mean antibody titers. the statistically significant differences (using one-way anova and bonferroni multiple comparison test) were indicated by: *, p,0.05; **, p,0.01; ***, p,0.001. nd, not detectable. doi:10.1371/journal.pone.0095448.g004 figure 5 . broad-spectrum protection against in vivo genital hpv psv challenges. mice were immunized four times with e3r4, r4 or phosphate-buffered saline (pbs) with cfa/ifa adjuvant. at eleven months after the first immunization, mice were intravaginally challenged with hpv16 (a), hpv45 (b), hpv52 (c), hpv58 (d), hpv11 (e), or hpv5 (f) pseudovirus. luciferase signals were acquired for 5 min with a lb 983 nightowl ii imager 48 h after psv infection. infection is measured as bioluminescence. the statistically significant differences (using one-tailed unpaired t-test) were indicated by: *, p,0.05; **, p,0.01. doi:10.1371/journal.pone.0095448.g005 l2 epitope-based vaccine induced broad immunity plos one | www.plosone.org genic surface characteristics [52, 53] . moreover, theoretically, there are up to 360 copies of rg-1 epitope are surface exposed per chimeric rg1-vlp, while only 6 copies of rg-1 epitope are surface exposed per e3r4 protein. thus, we reason that the differential broadness is likely resulted from the usage of different scaffolds to display the rg-1 epitope. taken together, our data demonstrate that a modified human igg1 fc can be used as a scaffold to display l2 antigen to induce cross-neutralizing antibodies and protective immunity against divergent human papillomavirus types. this type of fusion protein can be expressed with high yield and easily purified with high purity. therefore, delivery of l2 antigen by a modified fc scaffold opens a new avenue for pan-hpv vaccine development. groups of 2 rabbits were immunized four times with e3r4 alone or with alum-mpl adjuvant, and sera were collected at two weeks after the fourth immunization. antisera of two rabbits (nos. 1 and 2) were tested for cross-neutralization of eight hpv pseudovirus types. neutralization titers were determined as previously described [38, 39, 41] . abbreviations: alum-mpl, alum and monophosphoryl lipid a; hpv, human papillomavirus. doi:10.1371/journal.pone.0095448.t001 the biology and life-cycle of human papillomaviruses hpv prophylactic vaccines and the potential prevention of noncervical cancers in both men and women the impact of quadrivalent human papillomavirus (hpv; types 6, 11, 16, and 18) l1 virus-like particle vaccine on infection and disease due to oncogenic nonvaccine hpv types in sexually active women aged 16-26 years hpv16/ 18 l1 vlp vaccine induces cross-neutralizing antibodies that may mediate cross-protection protection against heterologous human papillomavirus challenge by a synthetic lipopeptide vaccine containing a broadly cross-neutralizing epitope of l2 protection of rabbits against challenge with rabbit papillomaviruses by immunization with the n terminus of human papillomavirus type 16 minor capsid antigen l2 type-specific and cross-reactive antibodies induced by human papillomavirus 31 l1/l2 virus-like particles mechanisms of human papillomavirus type 16 neutralization by l2 cross-neutralizing and l1 type-specific antibodies cleavage of the papillomavirus minor capsid protein, l2, at a furin consensus site is necessary for infection potent anti-hpv immune responses induced by tandem repeats of the hpv16 l2 (20 -38) peptide displayed on bacterial thioredoxin the n-terminal region of the human papillomavirus l2 protein contains overlapping binding sites for neutralizing, cross-neutralizing and non-neutralizing antibodies neutralization of hpv16, 18, 31, and 58 pseudovirions with antisera induced by immunizing rabbits with synthetic peptides representing segments of the hpv16 minor capsid protein l2 surface region monoclonal antibodies recognizing cross-neutralization epitopes in human papillomavirus 16 minor capsid protein l2 crossneutralization potential of native human papillomavirus n-terminal l2 epitopes a protective and broadly cross-neutralizing epitope of human papillomavirus l2 minor capsid protein of human genital papillomaviruses contains subdominant, crossneutralizing epitopes subcapsular encounter and complement-dependent transport of immune complexes by lymph node b cells b cells acquire particulate antigen in a macrophage-rich area at the boundary between the follicle and the subcapsular sinus of the lymph node subcapsular sinus macrophages in lymph nodes clear lymph-borne viruses and present them to antiviral b cells transport of immune complexes from the subcapsular sinus to lymph node follicles on the surface of nonphagocytic cells, including cells with dendritic morphology b cell recognition of membrane-bound antigen: an exquisite way of sensing ligands taking dendritic cells into medicine a papillomavirus-like particle (vlp) vaccine displaying hpv16 l2 epitopes induces cross-neutralizing antibodies to hpv11 chimeric l1-l2 virus-like particles as potential broad-spectrum human papillomavirus vaccines efficacy of rg1-vlp vaccination against infections with genital and cutaneous human papillomaviruses vlps displaying a single l2 epitope induce broadly cross-neutralizing antibodies against human papillomavirus a universal virus-like particle-based vaccine for human papillomavirus: longevity of protection and role of endogenous and exogenous adjuvants development of aavlp(hpv16/31l2) particles as broadly protective hpv vaccine candidate a recombinant mimetics of the hiv-1 gp41 prehairpin fusion intermediate fused with human igg fc fragment elicits neutralizing antibody response in the vaccinated mice hiv-1 and influenza antigens synthetically linked to igg2a fc elicit superior humoral responses compared to unmodified antigens in mice efficient mucosal vaccination mediated by the neonatal fc receptor a recombinant vaccine of h5n1 ha1 fused with foldon and human igg fc induced complete cross-clade protection against divergent h5n1 viruses ebola virus glycoprotein fc fusion protein confers protection against lethal challenge in vaccinated mice a novel fc gamma receptor ligand augments humoral responses by targeting antigen to fc gamma receptors rapid immune responses to a botulinum neurotoxin hc subunit vaccine through in vivo targeting to antigen-presenting cells localization of the binding site for the human high-affinity fc receptor on igg human fc gamma ri and fc gamma rii interact with distinct but overlapping sites on human igg trivalent human papillomavirus (hpv) vlp vaccine covering hpv type 58 can elicit high level of humoral immunity but also induce immune interference among component types human papillomavirus type 58 l1 virus-like particles purified by two-step chromatography elicit high levels of long-lasting neutralizing antibodies efficient intracellular assembly of papillomaviral vectors generation of hpv pseudovirions using transfection and their use in neutralization assays optimization of multimeric human papillomavirus l2 vaccines concatenated multitype l2 fusion proteins as candidate prophylactic panhuman papillomavirus vaccines immunization with potato plants expressing vp60 protein protects against rabbit hemorrhagic disease virus differential antibody responses to a distinct region of human papillomavirus minor capsid proteins receptor-binding domain of sars-cov spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine surface display of igg fc on baculovirus vectors enhances binding to antigen-presenting cells and cell lines expressing fc receptors crossreactive hiv-1-neutralizing activity of serum igg from a rabbit immunized with gp41 fused to igg1 fc: possible role of the prolonged half-life of the immunogen immunization with the haemophilus ducreyi hemoglobin receptor hgba with adjuvant monophosphoryl lipid a protects swine from a homologous but not a heterologous challenge evaluation of several adjuvants as alternatives to the use of freunds-adjuvant in rabbits comparison of antibody-response by use of synthetic adjuvant system and freund complete adjuvant in rabbits the coming of age of virus-like particle vaccines understanding and learning from the success of prophylactic human papillomavirus vaccines key: cord-000261-ip32y0j5 authors: becker, pablo d.; legrand, nicolas; van geelen, caroline m. m.; noerder, miriam; huntington, nicholas d.; lim, annick; yasuda, etsuko; diehl, sean a.; scheeren, ferenc a.; ott, michael; weijer, kees; wedemeyer, heiner; di santo, james p.; beaumont, tim; guzman, carlos a.; spits, hergen title: generation of human antigen-specific monoclonal igm antibodies using vaccinated “human immune system” mice date: 2010-10-04 journal: plos one doi: 10.1371/journal.pone.0013137 sha: doc_id: 261 cord_uid: ip32y0j5 background: passive transfer of antibodies not only provides immediate short-term protection against disease, but also can be exploited as a therapeutic tool. however, the ‘humanization’ of murine monoclonal antibodies (mabs) is a time-consuming and expensive process that has the inherent drawback of potentially altering antigenic specificity and/or affinity. the immortalization of human b cells represents an alternative for obtaining human mabs, but relies on the availability of biological samples from vaccinated individuals or convalescent patients. in this work we describe a novel approach to generate fully human mabs by combining a humanized mouse model with a new b cell immortalization technique. methodology/principal findings: after transplantation with cd34(+)cd38(−) human hematopoietic progenitor cells, balb/c rag2(−/−)il-2rγc(−/−) mice acquire a human immune system and harbor b cells with a diverse igm repertoire. “human immune system” mice were then immunized with two commercial vaccine antigens, tetanus toxoid and hepatitis b surface antigen. sorted human cd19(+)cd27(+) b cells were retrovirally transduced with the human b cell lymphoma (bcl)-6 and bcl-xl genes, and subsequently cultured in the presence of cd40-ligand and il-21. this procedure allows generating stable b cell receptor-positive b cells that secrete immunoglobulins. we recovered stable b cell clones that produced igm specific for tetanus toxoid and the hepatitis b surface antigen, respectively. conclusion/significance: this work provides the proof-of-concept for the usefulness of this novel method based on the immunization of humanized mice for the rapid generation of human mabs against a wide range of antigens. hyper-immune sera containing polyclonal immunoglobulins (igs) have been widely used in both therapeutic and prophylactic clinical settings [1] . however, the use of polyclonal sera was associated with several problems, such as the stimulation of allergic reactions, low reproducibility between clinical batches and high off-label use, which finally caused a decline in their use [2] . the advent of technologies to make monoclonal antibodies (mabs) derived from animals, especially mice, has overcome many of the problems associated with the use of polyclonal sera. the technology to make monoclonal cell lines of antibody-producing cells by fusing antibody producing plasma cells with myeloma cells was described for the first time in 1975 by milstein and kohler [3] . the therapeutic potential of mabs was immediately recognized and in 1980 the first mab, okt3, was approved for therapeutic applications. this antibody inactivates t cells, thereby preventing rejections of organ transplants [4] . however, because of the animal origin of the first generation of mabs that were used in clinical trials, human subjects treated with these antibodies developed vigorous immune reactions against the animal proteins, which were thereby eliminated preventing their therapeutic actions [5] . to overcome these problems technologies were developed to diminish the immunogenicity of mouse antibodies by replacing part or the complete mouse antibody backbone by its human equivalent, first generating chimeric, and subsequently fully humanized antibodies [6] . in a parallel approach transgenic mice bearing the human ig region were created to obtain fully human antibodies following immunization. the use of these mice obviates the elaborate molecular engineering of antibodies that is needed to humanize antibodies generated in wild-type mice, however, the maturation process of the mouse b cells expressing human igs is different from that of fully human b cells [7] . immortalization of b cells from immune humans seems to be the logical strategy to avoid these problems. however, the methods to achieve this goal have showed low efficiencies, although some progress has recently been reported [8, 9] . nevertheless, the major disadvantage of human b cells immortalization is the need for cells from either vaccinated individuals or patients who had recovered from an infection. thus, to fully exploit the ig repertoire of human b cells in an in vivo setting, we explored the possibility to raise mabs following de novo induction of human b cell responses in mice carrying elements of the human immune system (his). his mice are generated by engrafting immunodeficient mice with human hematopoietic stem cells (hsc) with or without human lymphoid tissues from fetal origin [10, 11, 12] . in particular, mice deficient for the recombinase activating gene-2 (rag2) and the common gamma chain of the il-2 receptor (il2rg) on a balb/c or a non-obese diabetic (nod) background are permissive for human hsc xenografts. inoculation of newborn mice from these strains with human hsc of fetal or umbilical cord blood origin gives rise to robust engraftment of a number of immune cells, including t, b, nk and dendritic cells. in this work, we describe a convenient approach to generate fully human mabs based on the immunization of balb/c rag2 2/2 il-2rcc 2/2 engrafted with human cd34 + cd38 2 hsc [13, 14] . to this end, his mice were immunized with commercial vaccines against hepatitis b virus (hbv) and tetanus. following immunization, human cd19 + b cells were sorted based on surface cd27 expression, as a marker of memory phenotype, and the isotype of surface igs. the sorted b cell populations were immortalized in vitro by retroviral transduction with human b cell lymphoma (bcl)-6 and bcl-xl genes and antigen-specific b cell clones were established and characterized. the obtained results provided the proof-of-concept for the usefulness of this generic approach based on his mice combined with immortalization of human b cells for the rapid and inexpensive development of human mabs against a wide range of antigens. the use of fetal liver tissue obtained from elective abortions with gestational age ranging from 14 to 20 weeks was approved by the medical ethical committee of the amc-uva and was contingent on informed written consent. balb/c rag2 2/2 il-2rcc 2/2 mice were bred and maintained in individual ventilated cages, and fed with autoclaved food and water. his mice were generated as previously described [13, 14, 15, 16] , with the approval of the animal ethical committee of the amc-uva (permit number dhl-100970). in brief, human fetal livers were obtained from elective abortions with gestational age ranging from 14 to 20 weeks. magnetic enrichment of cd34 + cells (.98% pure) was performed by using the cd34 progenitor cell isolation kit (miltenyi biotech), after preparation of single cell suspensions and isolation of mononuclear cells by density gradient centrifugation over lymphoprep (axis shield). finally, newborn (,5 days old) sub-lethally irradiated (3.5 gy) balb/c rag2 2/2 il-2rcc 2/2 mice were injected via intra-hepatic route with 5-10610 4 sorted cd34 + cd38 2 human fetal liver hematopoietic stem cells in 30 ml. all manipulations of his mice were performed under laminar flow. cell suspensions were prepared in rpmi medium supplemented with 2% fetal calf serum (fcs). twelve to sixteen weeks after cd34 + cd38 2 hsc engraftment, his mice were killed and single cell suspensions of splenocytes were prepared. red cells lysis was performed in 1 ml of red cell lysis buffer (sigma) for 10 min. splenocytes were washed, resuspended in 600 ml of rlt lysis buffer (qiagen) and homogenized by passing through a 21-gauge needle several times using rnase free syringes. rna was prepared using rneasy mini kits (qiagen) according to manufactures instructions. bcr v h immunoscope was performed as previously described [17] . briefly, cdna was prepared and real-time pcr performed by combining primers for the different v h chains (v h 1-7) and specific fluorochrome-labeled probes against the different constant regions (c h m, c h a and c h c). an additional four pcr cycles 'run-off reactions' were then performed on the pcr products using fluorescent primers specific for the constant regions (fcm, fca and fcc). products were gel separated to determine cdr3 lengths. analysis of six individual his-mice containing greater than 30% human chimerism in the spleen was performed. the number of human cd19 + b cells in chimeric spleens ranged from 5-12610 6 . eight weeks after hsc transplantation, blood was taken from his mice to verify the level of engraftment by flow cytometry, as described elsewhere [18] . his mice with a good level of human reconstitution (.20% hcd45 + cells) were immunized by intramuscular route (biceps femoris) using a 29g needle, three times on weeks 14, 16 and 18 with either 100 ml of the hbv vaccine (engerix-b, glaxosmithkline) or 50 ml of tetanus toxoid (tt) containing vaccine (tetanus vaccine, the netherlands vaccine institute). these amounts correspond to 1/10 of the normal human dose. negative controls received the same volume of pbs buffer. two weeks after the last immunization, his mice were exsanguinated under isofluran/oxygen narcosis. spleens and mln were removed aseptically and cellular suspensions were prepared. the bm cells were isolated from the femur and tibia. from beckman coulter; cd3 (sk7), cd4 (sk3), cd8 (sk1), cd19 (hib19), cd38 (hit2), cd45 (2d1 and hi30), cd45ra (hi100), cd138 (mi15), igm (g20-127), igd (ia6-2), igg (g18-145) and ccr7 (3d12) from bd biosciences; cd27 (lt27) from abd-serotec; cd27 (lg.7f9) from ebioscience. tt-specific b cells were also occasionally stained with pe-coupled tt, kindly provided by dr. andreas radbruch (german rheumatism research center, berlin, germany). dead cells were excluded based on dapi incorporation. all washings and reagent dilutions were done with pbs containing 2% fcs and 0.02% nan 3 . stained cells were analyzed with an lsr-ii interfaced to a facs-diva software system (bd biosciences). cell sorting of b cell subsets were performed on his mouse bm and spleens using a facs-aria cell sorter interfaced to a facs-diva software system (bd biosciences). for these experiments, all washings and reagent dilutions were done with 2% fcs supplemented pbs without nan 3 . the human bcl6 [19, 20] and bcl-xl [21] encoding cdnas were further cloned in a lzrs retroviral expression vector, around a t2a cleavage-promoting peptide sequence and upstream a cassette containing an internal ribosome entry site (ires) and the gene encoding gfp. we therefore obtained a lzrs vector in the following configuration: bcl6-t2a-bclxl-ires-gfp [9] . transfection of phoenix-galv packaging cells and virus production were performed as previously described [22] . before retroviral transduction, memory b cells were activated on c-irradiated (50 gy) mouse l cell fibroblasts stably expressing cd40l (cd40l-l cells) in the presence of 25-50 ng/ml recombinant mouse interleukin-21 (rmil-21, r&d systems) for 36 h [19] . the b cells were washed, mixed with retroviral supernatants in retronectin-coated plates (takara), centrifuged at room temperature for 60 min at 360 g, and subsequently incubated with the retroviruses at 37uc, 5% co 2 for 6-8 h. transduced b cells were maintained in co-cultures using cd40l-l cells (10 5 cells/ml) and in standard imdm (gibco) culture medium supplemented with 8% fetal bovine serum (fbs; hyclone), penicillin/streptomycin (roche) and 25 ng/ml rmil-21. the analysis of human ig-v h sequences was performed as follows. total rna was isolated from approximately 5610 5 monoclonal b cells with trizol (invitrogen). the cdna was generated and subjected to pcr with primers specific to the different v h family members. pcr products were sequenced to determine the cdr3 region of the different clones. sequence analysis was performed using bigdye terminator chemistry (applied biosystems inc.) and codoncode aligner software. the plasma harvested from his mice (7 days after the first and second immunization; 10 days after the third immunization) and b cell clone culture supernatants were screened by elisa for the presence of total human igm, total human igg and antigenspecific antibodies. measurement of total igm and igg was performed by coating 96-well plates either with affinipure f(ab') 2 fragment goat anti-human igm (fc5m-specific, jackson immu-noresearch) or affinipure goat anti-human igg (fcc fragmentspecific; jackson immunoresearch). control human serum protein calibrator (dako) with known igm (0.8 mg/ml) and igg (10.4 mg/ml) concentrations was used as a standard to be compared to the samples. for the detection of antigen-specific antibodies, 96-well plates were coated either with tetanus vaccine (nederlands vaccin instituut) or engerix b (glaxosmithkline) (106 diluted in pbs) for 1 h at 37uc or overnight at 4uc. alternatively, ridascreen tetanus igg elisa plates (biopharm) were also used to screen for tt-specific antibodies. after coating, the plates were washed in elisa wash buffer (pbs, 0.5% tween-20). a pbs solution containing 4% of milk was used as a blocking agent, before adding serial dilution of his mouse plasma (starting at a dilution of 1:5) or cell culture supernatants (starting at a dilution of 1:2). enzyme-conjugated detection antibodies were added at a dilution of 1:2500 for hrp-conjugated anti-igg and a dilution of 1:5000 for hrp-conjugated anti-igm (both from jackson immunoresearch). then, tmb substrate/stop solution (biosource) was used for the development of the elisa assay. statistical analyses were performed using graphpad prism version 5.02 for windows (graphpad software). data were subjected to two-tailed unpaired student t test analysis. the obtained p values were considered significant when p,0.05. we have generated his mice by transplanting human hsc into alymphoid balb/c rag2 2/2 il-2rcc 2/2 newborn mice ( figure 1a ). as reported previously, multilineage human hematopoietic reconstitution is observed in his mice, which demonstrate human thymopoiesis, b cell differentiation, nk cell and plasmacytoid dendritic cell development, and myelopoiesis [10, 13, 14, 15, 16] . human immune cells accumulate in lymphoid tissues, and several b cell subsets are observed in his mice ( figure 1b) . we analyzed the human b cell repertoire present in naive his mice by using b cell receptor (bcr) immunoscope analysis based on quantitative pcr of ig variable (v h ) and constant (c h ) region gene segments [17] . due to the lack of human spleen samples, the cells isolated from his mouse spleens, which contained sufficient numbers of human b cells to perform the immunoscope analysis, were compared to control human peripheral blood mononuclear cells (pbmc) samples, which were considered acceptable for the purpose of the performed comparison. we observed that igm-expressing b cells as well as ig isotype-switched b cells are found in naive his mice ( figure 1c) . the vast majority of b cells of his mice expressed an igm (97.961.0%), whereas igg (1.861.0%) and iga (0.0760.04%) expressing b cells represented minor populations. only the frequency of iga-expressing b cells was found significantly higher in control human pbmc samples (p,0.0001). at 10-14 weeks post-transplantation (i.e. in steady state conditions), the human ig concentrations in the blood were 12268 mg/ml (igm) and 143612 mg/ml (igg) ( figure 1d ), as previously reported [8, 16] . in comparison, the normal range for ig concentration in healthy humans is 400-3100 mg igm/ml and 7200-14700 mg igg/ml. in brief, despite a low frequency of igg-expressing cells, both human igm and igg accumulated in the plasma of ,3 month-old his mice to levels representing around 10% and 1% of adult human igm and igg concentrations, respectively. we further examined the antigen receptor repertoire diversity in his mice, by determining the length of cdr3 hypervariable regions for each ig-v h gene family. the analysis of cdr3 length distribution of individual his mouse splenocytes showed that igm repertoires are undistinguishable from normal human pbmc igm repertoires, as measured by the bcr immunoscope analysis ( figure 1e ). this observation suggests that his mice contain a broad variety of naive igm + b cell clones. the v h -family usage was large and similar to control human pbmc ( table 1) . the bcr immunoscope analysis was also performed for igg and iga repertoires and we observed more restricted repertoires, as expected from b cells undergoing clonal selection and ig class switch recombination ( figure s1 ). immunization of his mice with hbv and tetanus vaccines results in the generation of antigen-specific antibody responses since his mice contained broad naïve b cell repertoires, we analyzed the induction of human antigen-specific b cell responses after immunization with commercially available human vaccines. we designed a vaccination protocol based on repeated intramuscular immunizations (3 injections with 2-week intervals) of 10-14-week old his mice with vaccines containing hepatitis b surface antigen (hbsag) or tt. seven days after the last immunization mice were sacrificed, the blood and the lymphoid organs were harvested, and the phenotype and function of human cells was analyzed. all his mice showed human reconstitution (.20% hcd45 + cells) in the blood before starting the immunization protocol, which correlated with human engraftment in lymphoid organs. overall, 42% of hbsag-vaccinated (8 out of 19 vaccinated animals) and 40% of tt-vaccinated (6 out of 15) his mice showed significant production of antigen-specific igm antibodies, as detected by elisa (figure 2a) . we performed a kinetic monitoring of antigen-specific plasma ig levels in individual hbsag-vaccinated responder his mice and we observed that after the first immunization antigen-specific igs were rarely detected. in contrast, after the second immunization antigen-specific igm was detected, which steadily increased after the third immunization with approximately 25-40% of responder mice also showing an antigen-specific igg response (figure 2a) . this suggests that repeated vaccination leads to enhanced antigen-specific antibody production. the responder mice exhibited higher total igm (173641 mg/ml) and total igg (4596140 mg/ml) concentrations in their plasma, as compared to pbs-injected (igm: 37612 mg/ml; igg: 191658 mg/ml) and non-responder vaccinated (igm: 44611 mg/ml; igg: 192667 mg/ml) animals ( figure 2b) . at the end of the immunization protocol, vaccinated animals showed significantly higher numbers of hcd45 + cells in all organs (i.e. spleen, bone marrow (bm) and mesenteric lymph nodes (mln)) in comparison to mock-injected control mice. responder his mice exhibited higher numbers of human t and b cells in the spleen, as well as t cells in the bm ( table 2; table s1 ), suggesting that the vaccination protocol had a positive impact on the accumulation of human b and t cells. moreover, the mln isolated from vaccinated his mice contained 4 to 5-fold more hcd45 + cells than those of control animals ( figure 2c ), suggesting that the mln structure might play a role in eliciting an immune response in the his mice. in humans, the cd27 + memory b cell population contains the majority of antigen-experienced b cells [23, 24] , and we reasoned that the same should be true in vaccinated his mice. we therefore cell sorted several different cd19 + cd27 + b cell subsets from individual his mice. we used two strategies to isolate the following human b cell (cd45 + cd19 + ) subsets from bm and spleens of vaccinated his mice: (i) cd27 hi cd38 hi , (ii) cd27 + cd38 lo/int igd + , and (iii) cd27 + cd38 lo/int igd 2 on the one hand ( figure 3 , although the number of cells was increased for each of these subpopulations in the vaccinated animals, as expected from the enhanced number of total b cells ( table 2) . we only observed a significant increase in the frequency of igg + b cells within the cd27 hi cd38 hi plasmablast population of vaccinated responder his mice, as compared to pbs-injected animals (13.262.7% and 4.361.8% of cd27 hi cd38 hi b cells, respectively; p = 0.0311). in order to identify, isolate and immortalize the antigen-specific antibody-producing b cells, the aforementioned b cell subsets were transduced immediately after cell sorting with a retroviral vector encoding both human bcl6 and bcl-xl [9, 19] . by ectopically expressing bcl6 and bcl-xl in splenic or peripheral blood memory b cells and culturing them with factors produced by follicular helper t cells (cd40l and il-21), we generated highly proliferative, bcr positive b cell lines that secrete igs. since these cells express bcl6, the differentiation of memory b cells to terminal plasma cells is blocked [28, 29, 30] . therefore, the resulting b cells can expand extensively in vitro for long periods of time in presence of cd40l and il-21, and provide a tool to generate antigen-specific human bcr-positive, antibody-secreting b cell lines. the number of isolated cells from spleen and antigen-specific b cell clones that were generated with the bcl6/bcl-xl transduction approach is provided in the table s2 . since the frequency of antigen-specific b cell clones was unknown, we started with microcell cultures ranging from 0.6 to 640 cells per well. the wells containing antigen-specific b cells -as determined by hbsagspecific or tt-specific elisa -were subsequently cultured by limiting dilution to obtain monoclonal b cell lines. overall, we generated 15 anti-hbsag igm + b cell clones from 3 his mice vaccinated with hbsag, and 18 anti-tt igm + b cell clones from 3 his mice vaccinated with tt ( table s2 ). the estimated frequency of hbsag-specific b cells (clones) in the his mice after vaccination was 1/350. the igm secretion level of the b cell clones were in the range of 1 mg per 10 5 cells over 3 days in culture, which was in a similar range of secretion (0.6-5 mg/10 5 cells/3 days) to what was previously reported for b cell clones generated from human blood [9] . the igm v h regions of the bcr of the antigen-specific igm + b cell clones were sequenced. overall, the bcr of hbsag-specific and tt-specific b cell clones exhibited a v h sequence close to the germ-line sequence, although limited frequencies of somatic hyper-mutations were observed (table s3 and table s4 ). somatic hyper-mutations were occasionally detected in all framework regions (fr) and complementary determining regions (cdr), and most of the bcr diversity was the result of nadditions in the cdr3 region. based on the bcr sequence, we observed that 12 out the 15 anti-hbsag igm + b cell clones were unique, as well as 5 out the 18 anti-tt igm + b cell clones ( table s2, table s3 and table s4 ). the supernatants of tt-specific b cell clones were further tested for their capacity to recognize different antigens by elisa. we observed that igm mabs did not cross-react with unrelated antigens (i.e., hbsag and respiratory syncytial virus (rsv) antigens) ( figure 4a) . the tt-specific b cell clones were also screened by flow cytometry for direct binding of the tt antigen labeled with a fluorochrome ( figure 4b) . interestingly, three types of clones that produced antibodies that gave a similar signal in elisa were detected, with high, intermediate and low binding of the fluorescent tt antigen. in the present work we established a new approach to generate fully human mabs. we immortalized b cells from vaccinated his mice by transduction with bcl6 and bcl-xl followed by expansion in presence of il-21 and cd40l. antigen-specific b cell clones were obtained that expressed the bcr on their cell surface and secreted antigen-specific antibodies. similarly to methods based on the immortalization of human memory b cells from individuals that were either vaccinated or exposed to pathogens, our strategy exploits the antibody repertoire of human b cells which is likely to be different from that of b cells of mice expressing human ig gene segments. naïve his mice display an extensive human igm-expressing b cell repertoire. based on the analysis of the length of the cdr3 regions, this igm b cell repertoire is similar to the repertoire of healthy individuals. thus, his mice have no obvious limitations for the generation of human igm mabs against any possible antigen. upon intramuscular vaccinations with either tt or hbsag, approximately 40% of the his mice were able to mount an antigen-specific antibody response. human igm-producing b cell lines against both antigens were obtained after isolation of memory b cells followed by ex vivo differentiation into plasmablastlike cells. it is important to highlight that the selection of the antigen-specific human b cell clones relied on relevant bioassays (e.g., elisa or neutralization test). in contrast to ebv-based approaches, human b cell immortalization using transduction with bcl-6 and bcl-xl preserves the expression of the bcr at the surface and antigen-specific b cell clones can also be selected by binding of labeled antigen to the bcr of immortalized memory b cells (e.g. by using a labeled antigen). even when igg was used as a selection criterion, we were unable to establish antigen-specific igg + human b cell clones. the reason for this might be that t cell help in this system is suboptimal as indicated by the absence of antigen-specific t cell responses after vaccination (not shown). we also observed that the bcr of the b cell clones had a close to germ-line sequence, suggesting that also the induction of somatic hyper-mutation is sub-optimal in his mice. in our hands the great majority of the vaccinated his mice showed a defective formation of germinal centers [14, 16] , which further explains the absence of antigenspecific ig-class switched b cells. so far, humanized mouse models based on the transplantation of human hsc only -i.e. without additional human tissues -share these limitations, and immunization strategies result in the limited generation of class-switched antigen-specific b cell responses [14, 31, 32] . similar patterns are observed in human hsc-transplanted immunodeficient mice infected with lymphotropic pathogens, such as hiv [33] or ebv [34] , although dengue virus infection in his mice was reported to induce an igg response in a majority of the responder animals [35] . it is not clear why igg antigen-specific responses are limited while serum igg can accumulate efficiently, considering the low frequency of igg + b cells in his mice. it remains to be determined whether this apparent discrepancy might be explained by the conjunction of particularly effective igg production on a cell basis by igg + b cells (which might occur in a t cell independent manner, such as in the case of the igg3 subclass), long-term stability of human igg in the his mouse serum as compared to human igm, and/or defective survival of igg + b cells under specific conditions (e.g. after antigen-specific triggering of the bcr). although igm mabs might already be useful for some specific applications or could be modified by ig class swapping to obtain igg mabs [36] , optimized humanized mouse models with improved b cell function are highly desirable. one reason for the suboptimal interaction of t and b cells may be the poor survival resulting in a high turnover of human t cells (discussed in [10, 37] ), making it very likely that procedures leading to improved accumulation of human t cells may promote b cell responses and isotype switching. it was already shown that human b cells undergoing isotype switching can be obtained in humanized mice, provided that a human environment supporting this process is present, e.g. in scid mice transplanted with human fetal bones, thymus and lymph nodes [38] . consistent with this notion, enhanced human peripheral t cell accumulation was observed in nod/scid mice transplanted with human bone marrow hsc, fetal liver and fetal thymus tissues (referred to as blt mice), as compared to conventional humanized mouse systems [39] . interestingly, blt mice consistently generated an antigen-specific igg response after hiv-infection [40] . although it is yet unknown whether the isotype switch observed in blt mice is truly t cell dependent, those data might support the idea that improved t cell homeostasis has a positive impact on b cell responses. to obtain humanized mouse models with improved b and t cell homeostasis, alternative strategies not relying on the transplantation of human fetal tissues -which are not necessarily easy to access to, for ethical, legal or practical reasons -will likely be favored in the future. the replacement of mouse genes involved in the hematopoietic system by their human equivalent is a valuable strategy to improve development, maintenance and/or function of several hematopoietic cell subsets in humanized mouse models, as shown with cytokines, such as il-7 and il-15 [15, 21, 41, 42] , or mhc molecules (n.d.h and j.p.d., manuscript submitted) [43, 44] . the fact that the human cd47 was shown to be unable to properly interact with the mouse sirpa indicates that reintroducing a functional phagocyte inhibition mechanism via the cd47/sirpa signaling axis is another strategy of potential interest [45] . in conclusion, our results show using two standard vaccine antigens the general applicability of an innovative b cell immortalization method in combination with the his mouse model to generate human mabs. similarly to methods based on the immortalization of human memory b cells from vaccinated or convalescent individuals [9] , our approach exploits the broad antibody repertoire of human b cells, overcoming the potential limitations of conventional humanized murine mabs such as laboriousness or impaired biological properties, synthetic antibody libraries that require a known target antigen, and transgenic mice bearing the human ig locus that have limited b cell repertoires. in addition, our method enables to exploit experimental infection models and immunization regimes that would be unethical or untenable in humans. considering the upcoming advances in his mice models [37] , this new approach will provide a powerful tool to generate human mabs for either diagnostic or therapeutic purposes. figure s1 igg/iga b cell repertoire in naïve his mice. similarly to figure 1e , the naive igg (a) and iga (b) b cell repertoires of his (balb-rag/c) mice were evaluated on splenocytes by performing a bcr immunoscope for each v h family. the profiles obtained with control human pbmc are also shown. found at: doi:10.1371/journal.pone.0013137.s001 (1.61 mb tif) figure 3 . limited dilutions of b cells transduced with bcl-6 and bcl-xl were performed with 6.4 and 0.64 cells/well. after sub-cloning of the positive wells, we generated 15 igm + anti-hbsag mabs, of which 13 are unique (as determined by ig-v h sequence, see table s3 ), and 18 igm + anti-tt mabs, of which 5 are unique (see table s4 ). in the case of hbsag vaccination, the number of screened b cells was ((192*6.4)+(96*0.64))*3 = 3870, which eventually suggests that the frequency of hbsag-specific b cells is at least 1/350 b cells. found at: doi:10.1371/journal.pone.0013137.s003 (0.13 mb doc) igm v h amino-acid sequence of generated hbsagspecific b cell clones. the germ-line sequence is given for each v h family, with indication of framework regions (fr) and complementary determining regions (cdr). highlighted amino-acids correspond to n-additions (in the cdr3 region) and somatic hyper-mutation events, whether it results in a silent mutation (green) or not (red). clones with identical bcr sequences are grouped together. found at: doi:10.1371/journal.pone.0013137.s004 (0.02 mb pdf) the mechanism of diphtheria immunity and tetanus immunity in animals immunogenicity of therapeutic monoclonal antibodies continuous cultures of fused cells secreting antibody of predefined specificity upping the ante on antibodies mice with a human touch molecular engineering and design of therapeutic antibodies from xenomouse technology to panitumumab, the first fully human antibody product from transgenic mice an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus generation of stable monoclonal antibody-producing b cell receptorpositive human memory b cells by genetic programming experimental models to study development and function of the human immune system in vivo human-hemato-lymphoid-system mice: opportunities and challenges humanized mice in translational biomedical research monitoring the effect of gene silencing by rna interference in human cd34+ cells injected into newborn rag2-/-gammac-/-mice: functional inactivation of p53 in developing t cells development of a human adaptive immune system in cord blood celltransplanted mice il-15 trans-presentation promotes human nk cell development and differentiation in vivo t cellindependent development and induction of somatic hypermutation in human igm+ igd+ cd27+ b cells many human peripheral vh5-expressing igm+ b cells display a unique heavy-chain rearrangement experimental model for the study of the human immune system: production and monitoring of ''human immune system'' rag2-/-gamma c-/-mice stat3-mediated up-regulation of blimp1 is coordinated with bcl6 down-regulation to control human plasma cell differentiation a senescence rescue screen identifies bcl6 as an inhibitor of anti-proliferative p19(arf)-p53 signaling il-7 enhances thymic human t cell development in ''human immune system'' rag22/2il-2rgammac2/2 mice without affecting peripheral t cell homeostasis stat5 regulates the self-renewal capacity and differentiation of human memory b cells and controls bcl-6 expression cd27: a memory bcell marker human b cell subsets rapid cloning of high-affinity human monoclonal antibodies against influenza virus human immunoglobulin m memory b cells controlling streptococcus pneumoniae infections are generated in the spleen human blood igm ''memory'' b cells are circulating splenic marginal zone b cells harboring a prediversified immunoglobulin repertoire control of inflammation, cytokine expression, and germinal center formation by bcl-6 bcl-6 represses genes that function in lymphocyte differentiation, inflammation, and cell cycle control the bcl-6 protooncogene controls germinal-centre formation and th2-type inflammation antigen-specific antibody production of human b cells in nog mice reconstituted with the human immune system antigen-specific human t-cell responses and t cell-dependent production of human antibodies in a humanized mouse model disseminated and sustained hiv infection in cd34+ cord blood cell-transplanted rag2-/-gamma c-/-mice a new humanized mouse model of epstein-barr virus infection that reproduces persistent infection, lymphoproliferative disorder, and cell-mediated and humoral immune responses dengue virus infection and immune response in humanized rag2(2/2)gamma(c)(2/2) (rag-hu) mice potent antibody therapeutics by design humanized mice for modeling human infectious disease: challenges, progress, and outlook generation of primary antigen-specific human t-and b-cell responses in immunocompetent scid-hu mice humanized mice mount specific adaptive and innate immune responses to ebv and tsst-1 intrarectal transmission, systemic infection, and cd4+ t cell depletion in humanized mice infected with hiv-1 lentiviral vector delivery of human interleukin-7 (hil-7) to human immune system (his) mice expands t lymphocyte populations human lymphoid and myeloid cell development in nod/ltsz-scid il2r gamma null mice engrafted with mobilized human hemopoietic stem cells generation of functional human t-cell subsets with hla-restricted immune responses in hla class i expressing nod/scid/il2r gamma(null) humanized mice priming of protective t cell responses against virus-induced tumors in mice with human immune system components polymorphism in sirpa modulates engraftment of human hematopoietic stem cells we thank berend hooibrink for expert cell sorting and maintenance of the amc-uva flow cytometry facility. we acknowledge the bloemenhove clinic (heemstede, the netherlands) for providing fetal tissues and the staff of the animal research institute amsterdam for animal care. access to technologies available at the centre d'immunologie humaine of the institut pasteur was greatly appreciated. key: cord-001145-hc9ssruz authors: akazawa, yuko; isomoto, hajime; matsushima, kayoko; kanda, tsutomu; minami, hitomi; yamaghchi, naoyuki; taura, naota; shiozawa, ken; ohnita, ken; takeshima, fuminao; nakano, masayuki; moss, joel; hirayama, toshiya; nakao, kazuhiko title: endoplasmic reticulum stress contributes to helicobacter pylori vaca-induced apoptosis date: 2013-12-13 journal: plos one doi: 10.1371/journal.pone.0082322 sha: doc_id: 1145 cord_uid: hc9ssruz vacuolating cytotoxin a (vaca) is one of the important virulence factors produced by h. pylori. vaca induces apoptotic cell death, which is potentiated by ammonia. vaca also causes cell death by mitochondrial damage, via signaling pathways that are not fully defined. our aim was to determine whether endoplasmic reticulum (er) stress is associated with vaca-induced mitochondrial dysfunction and apoptosis. we found that c/ebp homologous protein (chop), a key signaling protein of er stress-induced apoptosis, was transcriptionally up-regulated following incubation of gastric epithelial cells with vaca. the effect of vaca on chop induction was significantly enhanced by co-incubation with ammonium chloride. phosphorylation of eukaryotic initiation factor 2 (eif2)-alpha, which is known to occur downstream of the er stress sensor pkr-like er-localized eif2-alpha kinase (perk) and to regulate chop expression, was also observed following incubation with vaca in the presence of ammonium chloride. knockdown of chop by sirna resulted in inhibition of vaca-induced apoptosis. further studies showed that silencing of the perk gene with sirna attenuated vaca-mediated phosphorylation of eif2-alpha, chop induction, expression of bh3-only protein bim and bax activation, and cell death induced by vaca with ammonium chloride, indicating that er stress may lead to mitochondrial dysfunction during vaca-induced toxicity. activation of er stress and up-regulation of bh3-only proteins were also observed in human h. pylori-infected gastric mucosa. collectively, this study reveals a possible association between vaca-induced apoptosis in gastric epithelial cells, and activation of er stress in h. pylori-positive gastric mucosa. infection with helicobactor pylori (h. pylori) may result in chronic gastritis, gastric ulcer, and gastric cancer [1] [2] [3] [4] . vacuolating cytotoxin a (vaca) is one of the major toxins produced by h. pylori that may trigger molecular changes in gastric epithelial cells. [3] [4] [5] [6] [7] [8] [9] [10] . secreted by the bacteria as a ,88-kda single polypeptide [11] , vaca contains amino-terminal 33.4-kda (p33) and carboxyterminal 54.8-kda (p55) domains [11] [12] [13] . it is believed that p33 is responsible for the assembly of vaca into stable hexamers that form an ion channel, which is required for vaca-induced toxicity, while p55 is responsible for vaca binding to cells [10, 12, 14, 15] . following acid activation, a p33-dependent, anion-selective channel is formed, leading to vaca internalization and association with endosomal membranes [13] . it has been proposed that internalized vaca incorporated into channels accelerates the turnover of endosomal v-atpases by augmenting the permeability of the endosomal membrane to anions, leading to the accumulation of osmotically active species such as nh 4 + [13, 16] . this event is believed to induce an osmotic imbalance involving late endosomes that provokes vacuolation. in this regard, vacainduced vacuolation is inhibited by the v-atpase activity inhibitor, bafliomycin a1 [17] . in contrast, weak bases including nh 4 cl that can be produced by the high urease activity of h. pylori significantly potentiates vaca-mediated vacuole formation in cultured cells [5, 18, 19] . vaca is also known to cause apoptosis in gastric epithelial cells. it is now accepted that vaca targets mitochondria to mediate cell death [6, 10, 12, 20, 21] . the unresolved question has been whether vaca induces cytochrome c release by directly or indirectly targeting mitochondria. domanska et al. showed that vaca forms ion channels on mitochondria in a p33-dependent manner [12] , whereas yamasaki and others suggested that vaca causes cytochrome c release indirectly by activating the pro-apoptotic bcl-2 family protein bax [20] . the mechanism by which vaca induces bax activation is not fully understood. notably, both vaca-induced vacuolation and mitochondrial dysfunction were significantly enhanced by nh 4 cl, while ammonia per se did not induce significant cell injury [5, 20, 22] . according to these studies, nh 4 cl is likely not necessary for vaca to initiate apoptosis but it significantly increases vaca-induced mitochondrial dysfunction and cytotoxicity [5] . enhancement of both vacuolation and mitochondrial dysfunction by nh 4 cl is inhibited by ion channel blockers, suggesting that membrane channel formation is required for both activities [17] . on the other hand, a study employing az-521 cells and mkn 28 cells demonstrated that apoptosis by neither vaca alone nor vaca in combination with nh 4 cl was attenuated by bafilomycin a1 [5, 20] . thus at least in selected cell lines, nh 4 cl may potentiate vaca-mediated apoptosis via an unknown mechanism that is independent of vacuolation. endoplasmic reticulum (er) plays a role in critical cellular functions by controlling protein folding and trafficking [23, 24] . failure of the er's capacity to resolve stress results in induction of the unfolded protein response (upr), which interacts with other stress signaling pathways including those involved in inflammation and cell death [24, 25] . the er stress transducers in mammalian cells are pkr-like er-localized eukaryotic initiation factor 2 (eif2)-a kinase (perk), inositol-requiring enzyme 1(ire-1), and activating transcription factor 6 (atf6) [23, 26, 27] . in unstressed cells, these proteins are retained in an inactive conformation via their association with the er-resident chaperone protein, glucoseregulated protein 78/immunoglobulin-heavy-chain-binding protein (grp78) [23, 26] . when unfolded proteins increase in the er, grp78 is released from perk, atg-6 and ire-1, thereby activating the three er stress sensors [23, 24] . er stress, especially activation of perk, leads to induction of nuclear c/ebphomologous protein (chop) via phosphorylation of eukaryotic initiation factor (eif2)-a [28, 29] . chop has been implicated as a key mediator of er stress-induced cell death in diverse pathological conditions including gastric epithelial cell damage [30] [31] [32] [33] [34] , and is known to activate proteins that mediate mitochondrial dysfunction [25, 32, 34, 35] . of note, chop has been reported to activate pro-apoptotic bh3-only proteins including bcl-2 interacting mediator of cell death (bim) and p53 up-regulated modulator of apoptosis (puma) [35] . these bh3-only proteins usually monitor cellular wellbeing but they participate in promoting bax activation to initiate mitochondrial cell death when activated by cytotoxic signals [36] . however, contribution of er stress and its downstream effectors during vaca-induced cell injury remains to be defined. to gain further mechanistic insight into vaca-induced mitochondrial dysfunction and cell death stimulated by ammonia, we investigated the effects of vaca and ammonia on the perk-and chop-signaling pathway and its potential role in the activation of bax, parp cleavage, and cell death. a human gastric cancer cell line, az-521 (culture collection of health science resource bank, japan health science foundation, tokyo, japan), was used in the study. cells were grown in eagle's minimal essential medium (sigma) containing 10% fetal calf serum (invitrogen, carlsbad, ca) under a 5% co 2 atmosphere at 37uc. the toxin-producing h. pylori strain atcc 49503 was the source of vaca, with purification by a modification of our published procedure [37] . in brief, after growth of h. pylori in brucella broth containing 0.1% b-cyclodextrin at 37uc for 3-4 days with vigorous shaking in a controlled microaerobic atmosphere of 10% o 2 and 10% co 2 , vaca was precipitated from the culture supernatant with 50% saturated ammonium sulfate. precipitated proteins were then dialyzed against rx buffer (10 mm kcl, 0.3 mm nacl, 0.35 mm mgcl 2 , and 0.125 mm egta in 1 mm hepes, ph 7.3) and applied to an anti-vacaspecific immunoglobulin g (igg) antibody column equilibrated with rx buffer. after washing the column with rx buffer, vaca was subsequently eluted with 50 mm glycine-hcl buffer (ph 1.0), which was promptly neutralized with 1 m tris-hcl (ph 10). after gel filtration on superose 6hr 10/30 equilibrated with tbs buffer (60 mm tris-hcl buffer, ph 7.7, containing 0.1 m nacl), purified vaca was concentrated and stored at 220uc (200 mg/ ml). vaca concentration was measured using a bead enzymelinked immunosorbent assay method. to activate vaca, 0.2 v/v 300 mm hcl was added to vaca preparations, which were then incubated for 10 min at room temperature, and then neutralized with the same volume of 300 mm naoh. the cells were treated with 120 nm of vaca [20] . in selected experiments, cells were treated with vaca in the presence of 5 mm nh 4 cl, which was similar to the concentrations observed in human h. pyloriassociated gastritis [38] . neutral red uptake into vacuoles was quantified as previously described [37] . cells were incubated with 50 ml of 0.05% neutral red in pbs containing 0.3% bovine serum albumin. cells were then washed three times with 0.1 ml of pbs containing 0.3% bovine serum albumin. after addition of 0.1 ml of 70% ethanol in water containing 0.4% hcl, absorbance at 540 nm (a 540 ) was measured. cells were stained with 5 mg/ml of 49,6-diamidine-29-phenylindole dihydrochloride (dapi) for 30 min at 37uc and visualized under fluorescence microscopy (nikon eclipse te200; nikon, tokyo, japan). apoptotic cells were quantified by counting 100 random cells per study. cells with the characteristic nuclear changes of chromatin condensation and nuclear fragmentation were considered apoptotic. in addition, apoptosis was also confirmed biochemically by immunobloting for cleaved parp (catalog # 9541, cell signaling, beverly, ma). whole cell lysates were directly lysed for 15 min. on ice using a commercially available cell lysis reagent from thermo scientific (rockford, il). for chop protein analysis, nuclear extracts from whole cell lysates were obtained using nuclear extraction reagent (thermo scientific, rockford, il) following manufacturer's instructions. samples containing 50 mg of protein were resolved by 4-15% sds-page, transferred to nitrocellulose membranes, followed by incubation overnight with primary antibodies at a dilution of 1:1000. the primary antibodies used are as follows; mouse anti-bax (catalog # sc-7480), rabbit anti-bad (catalog # sc-7869), rabbit anti-bik (catalog # sc-10770), mouse anti-chop (catalog # sc-7351), and anti-lamin b (catalog # sc-6216) antibodies were from santa cruz (santa cruz, ca). goat anti-bid antibodies were from r and d systems (catalog # baf860), rabbit anti-puma antibodies (catalog # 23404) were from rockland (gilbertsville, pa), and rabbit anti-bak antibodies(catalog # 06-536) were from upstate (billerica, ma). rabbit anti-bim, rabbit anti-perk (catalog # 3129s), rabbit anti-cleaved parp (catalog# 5625s) rabbit anti-eif2-a (catalog # 9722), and rabbit anti-phospho-eif2-a (catalog # 9721) antibodies were from cell signaling technology (beverly, ma). on the following day, membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (biosource international, camarillo, ca) at a dilution of 1:3000 for 2 hr. bound antibody was incubated with chemiluminescent substrate (supersignalh west pico chemiluminescent, thermo scientific, rockford, il) for 5 min and was visualized with a chemiluminescent imaging system (fluorchemh fc2, alpha innotech, san leandro, ca). in selected experiments, density of bands was analyzed using image j software (national institutes of health, bethesda, md) in order to quantify the results. biopsy specimens were placed immediately into 1 ml of rna later (applied biosystems, foster city, ca), followed by extraction of total rna using a commercially available kit (genelute tm mammalian total rna miniprep kits, sigma-aldrich, munich, germany). total rna was extracted from the cells and tissue small interfering (si) rna for perk and chop small interfering rna (sirna) was employed to knockdown chop and perk. chop targeting nucleotides, which included 4 different sequences, were obtained from dharmacon (lafayette, co, chop sigenome smartpool, catalog # m-004819-03-0005). perk targeting nucleotides were obtained from sigma and ambion. the target sequences were (perk sirna: 59-cacaaacuguauaacgguuua-39), and (perk sirna #2: 59-gugacgaaauggaacaaga-39), respectively. briefly, cells were grown in 6-well plates and transiently transfected with sirna using lipofectamin rnaimax (invitrogen grand island, ny). cells were used for experiments 48 hr after transfection. knock down of chop was confirmed by real-time pcr and knock down of perk was assessed by immunoblotting. immunocytochemistry of activated bax was performed using mouse monoclonal anti-bax (clone 6a7, 1:400 dilution, exalpha biologicals, watertown, ma) as previously described in detail [39] . cells were imaged by confocal microscopy with excitation and emission wavelengths of 488 and 507 nm, respectively. patients who underwent upper gastrointestinal endoscopy from june 2007 to may 2011 were enrolled in the study. upper gastrointestinal endoscopy was performed in nagasaki university hospital. endoscopies were performed for medically indicated reasons. endoscopies were not performed only for research. written informed consent of the patient was obtained prior to enrollment, in agreement with the helsinki declaration. the study was reviewed and approved by an ethics committee of nagasaki university hospital (office of human subjects protection registration number iorg0007678). exclusion criteria were as follows: age ,18 or .80 years, pregnancy, body mass index .30 kg/m 2 , diabetes mellitus, malignancies, renal impairment, systemic infection, liver diseases, drug addiction, alcohol abuse, use of medications effective against h. pylori during the preceding 3 months, and chronic corticosteroid or nonsteroidal anti-inflammatory drug use. during endoscopic examination of japanese patients, 2 biopsy specimens were obtained from the gastric antrum along the lesser curvature. one of the samples was subjected to rna isolation. the other specimen was fixed in 10% formalin and embedded in paraffin for histopathological examination. h. pylori status was assessed by the rapid urease test (helicocheck, otsuka pharmaceutical, tokushima, japan) and histology with giemsa staining. patients were considered positive for h. pylori infection when at least one of these examinations yielded positive results. patients were defined as h. pylori-negative if all test results were negative. immunohistochemistry for grp78 was performed with the streptavidin-biotin-peroxidase-complex method (histofine sab-poh kits, nichirei co., tokyo, japan). paraffin-embedded biopsy specimens were sliced into 5-mm-thick sections, deparaffinized, and rehydrated. after inhibition of endogenous peroxidase activity for 30 min with methanol containing 0.3% h 2 o 2 , the sections were reacted for 20 min with 5% albumin. samples were subsequently incubated overnight at 4uc with anti-grp78 antibody (sc-1050, santa cruz, ca), at a dilution of 1:1000. on the following day, sections were washed in 0.01 m phosphatebuffered saline (pbs) and incubated for 20 min with 10 mg/ml biotinylated antiserum. after washing in pbs, the sections were incubated for 20 min with 100 mg/ml horseradish peroxidaseconjugated streptavidin and stained with 0.02% 3,39-diaminobenzidine tetrahydrochloride in 0.05 m tris-hcl buffer containing 0.03% h 2 o 2 . the sections were then washed in pbs and counterstained with hematoxylin. all data represent at least three independent experiments and are expressed as means 6 se unless otherwise indicated. differences between groups were compared by using either an unpaired two-tailed t-test, one-way anova followed by post hoc test (bonferroni method), or two-way anova followed by post hoc test (bonferroni method), as indicated in figure legends. p values ,0.05 were considered statistically significant. we initially tested whether nh 4 cl increases vacuolation and apoptosis in az-521 cells. as previously reported, vaca alone triggered vacuolation in az-521 cells ( figure 1a ) [18] , which was increased in the presence of 5 mm nh 4 cl ( figure 1a ). vaca induced a mild increase in the number of apoptotic cells as confirmed by dapi staining, whereas cell death was significantly enhanced when cells were incubated with vaca plus nh 4 cl ( figure 1b) . next, we assessed whether vaca increases expression of chop, which has a pivotal role in er-induced apoptosis. we incubated the cells with nh 4 cl alone, vaca alone, or vaca plus nh 4 cl and examined the chop protein by immunoblot analysis. chop protein was not present in untreated cells or nh 4 cltreated cells, whereas intoxication with vaca alone triggered weak expression of chop protein (figure 2a ). of note, chop protein expression was augmented in cells treated with vaca plus nh 4 cl (figure 2a) . expression of chop mrna was also increased by 13 fold in cells treated with vaca plus nh 4 cl compared to untreated cells ( figure 2b ), implying that chop was transcriptionally activated by apoptotic stimuli following er stress. in fact, eif2-a, which can cause transcriptional induction of chop downstream of er-stress sensor perk, was phosphorylated by incubation of cells with vaca plus nh 4 cl ( figure 2c ). these results suggested that induction of chop resulted from perk activation via phosphorylation of eif2-a. expression of er resident chaperone protein grp78 mrna was also significantly elevated in vacatreated cells (figure 3 ). splicing of xbp-1 mrna occurs downstream of the ire-1 arm of er stress; xbp-1 mrna existed in both spliced and un-spliced form in untreated cells and this splicing pattern was not altered by vaca treatment ( figure s1 ). up-regulation of atf-6 mrna was also not observed following vaca treatment (data not shown). collectively, these results indicate that vaca in the presence of nh 4 cl is capable of inducing er stress, especially involving the downstream targets of perk. based on the above findings, we next examined whether activation of er stress, particularly chop, is associated with vaca-induced apoptosis. az-521 cells were transfected with differences between groups were compared by using an unpaired two-tailed t-test. *p,0.05, nonspecific sirna-transfected cells vs chop sirna-transfected cells. the data represent the mean 6 sem for n = 3 studies. (b) cells were treated with either vehicle (control), 5 mm nh 4 cl, 120 nm vaca, or 5 mm nh 4 cl plus 120 nm vaca for 24 hr. apoptosis was assessed by morphological changes after 30 min of dapi staining. the data represent the mean 6 sem for n = 3 studies each performed in triplicate. data was assessed by two-way anova followed by bonferroni method. *p,0.05, non-specific sirna-transfected cells vs chop sirna-transfected cells in vaca-treated cells. **p,0.01, non-specific sirna-transfected cells vs chop sirna-transfected cells in nh 4 cl plus vaca-treated cells. (c) cells were treated as indicated above. cleaved parp was assessed by immunoblotting. (d) after incubation with 5 mm nh 4 cl plus 120 nm vaca for 24 hr, cells were collected and cleaved parp was assessed by immunoblotting. densitometry was performed and differences between groups were compared by using an unpaired two-tailed t-test.*p,0.05, non-specific sirna vs chop sirna transfected cells. the data represent the mean 6 sem for n = 3 studies. doi:10.1371/journal.pone.0082322.g004 sirna targeted to chop and inhibition of transcriptional expression was assessed by real-time pcr ( figure 4a ). in response to treatment with both vaca alone and vaca plus nh 4 cl, cells with knockdown of chop showed significant suppression of apoptosis ( figure 4b ) and parp cleavage ( figure 4c and d) , indicating that chop participates in vaca-induced apoptosis. in subsequent studies, we tested whether induction of chop and its downstream apoptotic signaling was mediated by perk. indeed, perk knockdown attenuated vaca-mediated phosphorylation of eif2-a ( figure 5a and b) and chop mrna expression ( figure 5c ). more importantly, perk knockdown decreased apoptosis ( figure 5d ) and production of cleaved parp caused by vaca treatment (figure 5e and f) . these results were further confirmed by employing an additional sirna targeting perk ( figure s2a , b, c, d). next, we tested whether activation of er stress occurs upstream or downstream of mitochondrial dysfunction. if er stress occurs upstream of mitochondrial damage, knockdown of perk should inhibit vaca-induced bax activation. in fact, perk sirna attenuated bax activation induced by vaca plus nh 4 cl treatment ( figure 5g ). these data suggest that upon vaca treatment, especially in the presence of nh 4 cl, er stress leads to perk-mediated chop induction followed by mitochondrial dysfunction, leading to apoptotic cell death. 4 cl, 120 nm vaca, or 5 mm nh 4 cl plus 120 nm vaca for 8 hr, cells were lysed and were subjected to immunoblot analysis with anti-phospho-eif2-a and anti-total eif2-a antibodies. (c) after incubation with 5 mm nh 4 cl plus 120 nm vaca for 8 hr, cells were collected and real-time pcr was performed to assess chop mrna expression. the data represent the mean 6 sem for n = 3 studies. differences between groups were compared by using an unpaired two-tailed t-test. *p,0.05 non-specific sirna vs perk sirna. (d) cells were treated with either vehicle (control), 5 mm nh 4 cl, 120 nm vaca, or 5 mm nh 4 cl plus 120 nm vaca for 24 hr. apoptosis was assessed by morphological changes after 30 min of dapi staining. data was assessed by two-way anova followed by bonferroni method. *p,0.01, non-specific sirna-transfected cells vs perk sirna-transfected cells. the data represent the mean 6 sem for n = 3 studies. (e) cells were treated as indicated above. cleaved parp was assessed by immunoblotting. (f) after incubation with 5 mm nh 4 cl plus 120 nm vaca for 24 hr, expression of cleaved parp was assessed by immunoblotting followed by densitometry. differences between groups were compared by using an unpaired two-tailed t-test. *p,0.05, non-specific sirna vs perk sirna transfected cells. the data represent the mean 6 sem for n = 3 studies. (g) cells were examined by immunofluorescence microscopy for bax following the treatment with vehicle (control) or 5 mm nh 4 cl plus 120 nm vaca for 16 hr. the primary antibody used for the study recognizes the n-terminal region of bax, which is exposed upon activation. green fluorescence shows activated bax, whereas blue fluorescence indicates dapi staining of nucleus. data represent the results of 3 independent experiments. doi:10.1371/journal.pone.0082322.g005 since er stress-mediated apoptosis can be executed via activation of bh3-only proteins [25, 40] , we investigated whether bh3-only proteins are also up-regulated by vaca. we found that bim protein was up-regulated in response to vaca treatment ( figure 6a and b) , whereas other bh3-only proteins, such as puma, noxa, bad, bik, and pro-apoptotic multidomain bcl-2 proteins (bak, bax) were unchanged. of note, hrk and bid were not detected in az-521 cells. we found that bim mrna was also up-regulated in cells treated with vaca plus nh 4 cl ( figure 6c ). furthermore, vaca-induced expression of bim protein ( figure 6d and e, figure s2e and 2f) and mrna ( figure 6f ) were significantly attenuated by perk sirna, suggesting that, upon vaca treatment, bim may be up-regulated downstream of er stress pathways. to examine the above findings in h. pylori-infected human mucosa, we investigated the expression of er stress markers. we found that expression of er chaperone protein grp78 mrna was significantly elevated in h. pylori-infected samples compared to h. pylori-negative samples ( figure 7a ), indicating activation of er stress in h. pylori-infected mucosa. immunohistochemistry for grp78 showed that the protein was strongly positive in the epithelium in h. pylori-positive biopsy specimens, suggesting that activation of er stress occurred in gastric epithelium ( figure 7b ). in contrast, in the h. pylori-negative biopsy specimens, grp78 was detected primarily in the crypts near the basement membrane and not in the upper layers of epithelium ( figure 7b ). expression of chop mrna was unaltered in h. pylori-infected gastric mucosa (figures3). next, mrna expression of bh3-only proteins in h. pylori-infected and uninfected human mucosa was compared. we 4 cl plus vaca-treated cells compared to control. data was assessed by one-way anova followed by bonferroni method.*p,0.05, control vs vaca plus nh 4 cl-treated cells. the data represent the mean 6 sem for n = 3 studies. (c) cells were treated with either vehicle (control), 5 mm nh 4 cl, 120 nm vaca, or 5 mm nh 4 cl plus 120 nm vaca for 8 hr. bim mrna expression was assessed by real-time pcr. data was assessed by one-way anova followed by bonferroni method. *p,0.05, control vs nh 4 cl plus vaca-treated cells. the data represent the mean 6 sem for n = 3 studies. (d) after incubation with perk sirna or nonspecific sirna for 48 hr, cells were treated as above and were subjected for immunoblot analysis for bim and actin. (e) after incubation with perk sirna or non-specific sirna for 48 hr, cells were incubated with 5 mm nh 4 cl plus 120 nm vaca for 8 hr. expression of bim was assessed by immunoblotting followed by densitometry. differences between groups were compared by using an unpaired two-tailed t-test.*p,0.05, non-specific sirna-transfected cells vs perk sirna-transfected cells. the data represent the mean 6 sem for n = 3 studies. (f) cells were treated with either nonspecific sirna or perk sirna for 48 hr. after incubation with 5 mm nh 4 cl plus 120 nm vaca for 8 hr, cells were collected and real-time pcr was performed to assess bim mrna expression. the data represent the mean 6 sem for n = 3 studies. differences between groups were compared by using an unpaired two-tailed t-test. *p,0.05 non-specific sirna vs perk sirna. data represent the results of 3 independent experiments. doi:10.1371/journal.pone.0082322.g006 found that bim and puma mrnas were significantly elevated in h. pylori-infected mucosa compared to uninfected controls ( figure 7c ). collectively, these results suggest that a group of er stress markers and bh3-only proteins are up-regulated in h. pylori infected-gastric mucosa. the principal findings of this study indicate the following: (i) vaca induces er stress in gastric epithelial cells, especially in the presence of nh 4 cl, (ii) vaca-induced apoptosis is at least in part, dependent on activation of chop-mediated intracellular signaling, (iii) er stress is responsible for induction of pro-apoptotic bh3-only protein bim as well as bax activation, and (iv) er stress markers and bh3-only proteins are up-regulated in h. pylori infected-human gastric mucosa. apoptosis in response to h. pylori infection may play a role not only in gastric injury, but also in development of gastric atrophy and cancer. vaca, one of the most important virulence factors of h. pylori, induces vacuolation and apoptosis in gastric epithelial cells. mitochondria serve as a target of vaca during apoptosis. the mechanism(s) underlying vaca effects on the mitochondria are unclear. it has been proposed that mitochondrial membrane potential is lost because of bax induction, although the pathway leading to bax activation has not been defined [13, 20, 41] . others have suggested that loss of the membrane potential results from translocation of vaca to the mitochondria [12, 42] . the current study indicates that vaca induces er stress in az-521 cells, consistent with previous reports demonstrating that other bacterial virulence factors including shigella toxins and lipopolysaccharide provoke er stress [43, 44] . of note, some viruses and bacterial virulence factors trigger chop activation [44] [45] [46] , implying that chop may participate in the pathogenesis of a number of infectious diseases. the significance of chop in facilitating er stress-induced cell death has been well documented, for instance, macrophages from chop knockout mice are more resistant to apoptosis induced by thapsigargin, an activator of er stress [31] ; further, indomethacin-induced apoptosis is attenuated in cultured guinea-pig gastric mucosal cells expressing a dominant-negative form of chop [30] . fibroblasts from bax 2/2 bak 2/2 mice are resistant to thapsigargin-induced apoptosis, indicating a pivotal role for these pro-apoptotic bcl-2 family proteins in er stressmediated cell death [47] . consistent with these reports, our study showed that vaca-mediated activation of chop, at least in part, contributes to apoptosis in gastric epithelial cells. studies have demonstrated that nh 4 cl enhances vacainduced bax activation and cell death [8, [18] [19] [20] . in agreement, this study demonstrated that addition of 5 mm nh 4 cl to incubation medium, a concentration often found in gastric juice of h. pylori-infected patients, significantly augmented vacamediated chop induction and apoptosis. furthermore, chop was transcriptionally activated by perk via phosphorylation of eif2-a, which was also augmented by nh 4 cl. although it is currently unclear how nh 4 cl increases vaca-induced er stress, it is believed that vaca enters cells independent of ammonia [19] , therefore, the effect of nh 4 cl on apoptosis probably does not result from increased vaca entry into cells. we also note that the effect of nh 4 cl on enhancing vaca-mediated er stress and apoptosis may not be directly linked to enhanced vacuolation since chop induction upon incubation with vaca plus nh 4 cl was not inhibited by bafilomycin a1 (data not shown). these data support the prior conclusions that cytochrome c release and vacuolation are independent cellular outcomes of vaca treatment while both of these actions can be potentiated by the presence of nh 4 cl in the culture medium [17, 20] . our data showed that some er stress markers including atf-6 up-regulation and enhanced xbp-1 splicing were not altered by vaca treatment, suggesting that vaca in combination with nh 4 cl does not broadly activate er stress signals but rather specifically enhances perk-chop signaling pathways. interestingly, we did not observe increased expression of chop in human h. pylori-infected samples. the following possible scenarios are considered: persistent sub-lethal er stress in human chronic gastritis caused by h. pylori might allow the cells to adapt for survival by retaining a minimal level of chop in vivo. in this respect, we observed up-regulation of cytoprotective grp78 in vaca-treated cells as well as in human gastric mucosa infected by h. pylori. this er-chaperone protein plays a critical role in a upr-protective response and its up-regulation is commonly used as a sentinel marker of er stress in various pathologic conditions [48] . therefore, gastric cells may try to adapt to a toxic environment and upr by increasing the content of chaperone proteins including grp78. if er homeostasis is not restored, apoptotic signals can be executed by activation of the perk-chop pathway. additional studies are necessary to test this hypothesis. er stress-mediated signaling pathways are coupled to activation of several death signaling molecules, which promote mitochondrial dysfunction [25] . functionally, chop is known to up-regulate the bh3-only proteins bim and puma and induce bax activation [34, 35] . the role of bh3-only proteins in vaca-mediated apoptosis has not been explored, but vaca was reported to reduce expression of anti-apoptotic bcl-2 proteins that antagonize the activation of bh3-only proteins [49] , and wei et al. have shown that bh3-only proteins are up-regulated in h. pyloriinfected gastric epithelial cells [50] . in the current study, we found elevation of bim and puma mrna in human h. pylori-positive gastric mucosa, as well as transcriptional up-regulation of bim in az-521 cells treated with vaca plus nh 4 cl. since knockdown of perk decreased vaca-mediated bim mrna expression, activation of bim likely occurred downstream of er stress. although further investigations are required to identify precisely how vacainduced er stress leads to activation of bh3-only proteins and bax, it was recently reported that chop potentially cooperates with foxo3a in neuronal cells to regulate er stress-induced bim expression [35] . thus, it would be intriguing to investigate if foxo3a also modulates vaca mediated-apoptosis in gastric epithelial cells. in summary, the present study demonstrates that perkmediated chop up-regulation contributes to vaca-triggered bax activation and apoptosis in gastric epithelial cells. future research involving mouse models may lead to a better understanding of the role of er stress in the pathogenesis of h. pylori-induced gastric mucosal injury. figure s1 cells were treated with either vehicle (control), 5 mm nh 4 cl, 120 nm vaca, or 5 mm nh 4 cl plus 120 nm vaca for 8 hr. cells were lysed and mrna was collected. xbp-1 cdna was amplified by pcr, following 3 hr incubation with restriction enzyme pst-1. 4 cl, 120 nm vaca, or 5 mm nh 4 cl plus 120 nm vaca for 24 hr. apoptosis was assessed by morphological changes after 30 min of dapi staining. data was assessed by two-way anova followed by bonferroni method. *p,0.01, non-specific sirna-transfected cells vs perk sirna-transfected cells. the data represent the mean 6 sem of n = 3 studies. (c) cells were treated as indicated above. cleaved parp was assessed by immunoblotting. (d) cells were treated with 5 mm nh 4 cl plus 120 nm vaca for 24 hr. cleaved parp was assessed by immunoblotting followed by densitometry. *p,0.05, non-specific sirna vs perk sirna#2-transfected cells. differences between groups were compared by using an unpaired two-tailed t-test. the data represent the mean 6 sem of n = 3 studies. (e) cells were treated with either vehicle (control), 5 mm nh 4 cl, 120 nm vaca, or 5 mm nh 4 cl plus 120 nm vaca for 16 hr. expression of bim was assessed by immunoblotting. (f) cells were treated with 5 mm nh 4 cl plus 120 nm vaca for 8 hr. expression of bim was assessed by immunoblotting followed by densitometry. differences between groups were compared by using an unpaired two-tailed t-test. *p,0.05, nonspecific sirna vs perk sirna#2-transfected cells. (tif) figure s3 (a) mrna was extracted from biopsy specimens of h. pylori-negative and -positive gastric mucosa. real-time pcr for chop mrna expression was performed on the samples. (tif) helicobacter pylori in health and disease inflammation, immunity, and vaccines for helicobacter helicobacter pylori virulence factors in gastric carcinogenesis life in the human stomach: persistence strategies of the bacterial pathogen helicobacter pylori relationship between vac a toxin and ammonia in helicobacter pylori-induced apoptosis in human gastric epithelial cells helicobacter pylori vacuolating cytotoxin a (vaca) engages the mitochondrial fission machinery to induce host cell death remodeling the host environment: modulation of the gastric epithelium by the helicobacter pylori vacuolating toxin (vaca) induction of gastric epithelial cell apoptosis by helicobacter pylori vacuolating cytotoxin lowdensity lipoprotein receptor-related protein-1 (lrp1) mediates autophagy and apoptosis caused by helicobacter pylori vaca intoxication strategy of helicobacter pylori vaca toxin carboxy-terminal proteolytic processing of helicobacter pylori vacuolating toxin helicobacter pylori vaca toxin/subunit p34: targeting of an anion channel to the inner mitochondrial membrane helicobacter pylori vaca: a new perspective on an invasive chloride channel the nterminal 34 kda fragment of helicobacter pylori vacuolating cytotoxin targets mitochondria and induces cytochrome c release helicobacter pylori vaca, a paradigm for toxin multifunctionality gastric cell apoptosis and h. pylori: has the main function of vaca finally been identified? cellular vacuolation and mitochondrial cytochrome c release are independent outcomes of helicobacter pylori vacuolating cytotoxin activity that are each dependent on membrane channel formation potentiation of helicobacter pylori vacuolating toxin activity by nicotine and other weak bases persistence of helicobacter pylori vaca toxin and vacuolating potential in cultured gastric epithelial cells helicobacter pylori vacuolating cytotoxin induces activation of the proapoptotic proteins bax and bak, leading to cytochrome c release and cell death, independent of vacuolation vacuolating cytotoxin a (vaca), a key toxin for helicobacter pylori pathogenesis formation of anion-selective channels in the cell plasma membrane by the toxin vaca of helicobacter pylori is required for its biological activity endoplasmic reticulum stress in nonalcoholic fatty liver disease endoplasmic reticulum stress and the inflammatory basis of metabolic disease er stress triggers apoptosis by activating bh3-only protein bim mediators of endoplasmic reticulum stress-induced apoptosis molecular mechanisms of lipotoxicity in nonalcoholic fatty liver disease feedback inhibition of the unfolded protein response by gadd34-mediated dephosphorylation of eif2alpha perk is required at the er-mitochondrial contact sites to convey apoptosis after rosbased er stress endoplasmic reticulum stress response is involved in nonsteroidal antiinflammatory drug-induced apoptosis chop is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum initiation and execution of lipotoxic er stress in pancreatic beta-cells he b (2006) viruses, endoplasmic reticulum stress, and interferon responses chop and ap-1 cooperatively mediate puma expression during lipoapoptosis chop potentially cooperates with foxo3a in neuronal cells to regulate puma and bim expression in response to er stress life in the balance: how bh3-only proteins induce apoptosis essential domain of receptor tyrosine phosphatase beta (rptpbeta) for interaction with helicobacter pylori vacuolating cytotoxin detection of helicobacter pylori infection of the gastric mucosa by measurement of gastric aspirate ammonium and urea concentrations jnk1-dependent puma expression contributes to hepatocyte lipoapoptosis palmitoleate attenuates palmitate-induced bim and puma up-regulation and hepatocyte lipoapoptosis bax translocation and mitochondrial fragmentation induced by helicobacter pylori helicobacter pylori vacuolating cytotoxin enters cells, localizes to the mitochondria, and induces mitochondrial membrane permeability changes correlated to toxin channel activity molecular mechanisms of the lps-induced non-apoptotic er stress-chop pathway signaling through c/ebp homologous protein and death receptor 5 and calpain activation differentially regulate thp-1 cell maturation-dependent apoptosis induced by shiga toxin type 1 upregulation of chop/gadd153 during coronavirus infectious bronchitis virus infection modulates apoptosis by restricting activation of the extracellular signal-regulated kinase pathway hepatitis c virus envelope proteins regulate chop via induction of the unfolded protein response proapoptotic bax and bak: a requisite gateway to mitochondrial dysfunction and death critical role of the stress chaperone grp78/bip in tumor proliferation, survival, and tumor angiogenesis in transgene-induced mammary tumor development helicobacter pylori vaca reduces the cellular expression of stat3 and prosurvival bcl-2 family proteins, bcl-2 and bcl-xl, leading to apoptosis in gastric epithelial cells interaction of helicobacter pylori with gastric epithelial cells is mediated by the p53 protein family we thank yoko kido and kayoko maeda for research assistance. we thank justin mott, biochemistry and molecular biology, university of nebraska medical center, for advice in statistical analysis. joel moss was supported by the intramural research program, national institutes of health, national heart, lung, and blood institute. key: cord-001982-arczqdza authors: khajah, maitham a.; fateel, maryam m.; ananthalakshmi, kethireddy v.; luqmani, yunus a. title: anti-inflammatory action of angiotensin 1-7 in experimental colitis date: 2016-03-10 journal: plos one doi: 10.1371/journal.pone.0150861 sha: doc_id: 1982 cord_uid: arczqdza background: there is evidence to support a role for angiotensin (ang) 1–7 in reducing the activity of inflammatory signaling molecules such as mapk, pkc and src. enhanced angiotensin converting enzyme 2 (ace2) expression has been observed in patients with inflammatory bowel disease (ibd) suggesting a role in its pathogenesis, prompting this study. methods: the colonic expression/activity profile of ace2, ang 1–7, mas1-receptor (mas1-r), mapk family and akt were determined by western blot and immunofluorescence. the effect of either exogenous administration of ang 1–7 or pharmacological inhibition of its function (by a779 treatment) was determined using the mouse dextran sulfate sodium model. results: enhanced colonic expression of ace2, ang1-7 and mas1-r was observed post-colitis induction. daily ang 1–7 treatment (0.01–0.06 mg/kg) resulted in significant amelioration of dss-induced colitis. in contrast, daily administration of a779 significantly worsened features of colitis. colitis-associated phosphorylation of p38, erk1/2 and akt was reduced by ang 1–7 treatment. conclusion: our results indicate important anti-inflammatory actions of ang 1–7 in the pathogenesis of ibd, which may provide a future therapeutic strategy to control the disease progression. the renin-angiotensin-aldosterone system (raas) plays an important role in the homeostatic control of cardiovascular, renal and adrenal functions. this peptide-based hormonal system is comprised of two main pathways; angiotensin converting enzyme/angiotensin ii/angiotensin type 1/2 receptor (ace/ang ii/at1/2r; which is the classical raas), and ace2/ang 1-7/ mas-1 receptor (mas1-r) (a recently described version of raas) [1, 2] . the proteolytic enzyme renin is released by the juxtaglomerular cells in the kidneys to cleave the n-terminal region of angiotensinogen to form ang i (or decapeptide). ang i is further degraded to ang ii by the removal of two c-terminal amino acids by proteases such as ace and chymas-1. ang ii binds to two main receptors; at1r and at2r. whereas at1r plays an important role in blood pressure control and cardiac cell remodeling, at2r antagonizes the signaling associated with at1r stimulation [1] . furthermore, ang ii can be converted to smaller peptide products with various biological activities. one of these, ang 1-7, can be synthesized not only from ang ii (via postproline carboxypeptidase) but also from ang i (via tissue endopeptidases; neprilysin, prolyl endopeptidase, and thimet oligopeptidase), or directly from ang 1-9 following its formation from ang 1-10 by ace2, bypassing the synthesis of ang ii [3] . ang 1-7 induces its biological effects through the only known receptor for this peptide; the mas-1 r oncogene gprotein coupled receptor [4] . the physiological roles of ang 1-7 on renal function, fluid homeostasis, vascular tone and cardiac contractility and remodeling are well documented, and most of these actions are directly opposed to ang ii-mediated effects [5] [6] [7] [8] . it is thought that the beneficial effects of ace-inhibitors (aceis) and angiotensin receptor blockers (arbs) on blood pressure control and in delaying/inhibiting the cardiac remodeling process is through increasing serum levels of ang1-7 [9] [10] [11] [12] . several studies have suggested an important role for ang ii in the pathogenesis of inflammatory bowel disease (ibd), a chronic disorder of the gastrointestinal tract which comprises two conditions: crohn's disease and ulcerative colitis [13, 14] . when the production and/or the activity of ang ii is reduced, colitis severity is ameliorated in various animal models of ibd through various mechanisms. treatment with aceis or arbs resulted in significant reduction in colitis severity in 2, 4, 6-trinitrobenzene sulphonic acid (tnbs) and dextran sulfate sodium (dss) induced models of experimental colitis [15] [16] [17] [18] [19] [20] [21] . furthermore, colitis severity can also be ameliorated by making mice homozygous for targeted disruption of the angiotensin gene [21] or atr1a deficiency [22] . these protective effects are considered to be due in part to inhibition of ang ii mediated enhancement of nfκb phosphorylation, adhesion molecule expression in the gut (the mucosal addressin; madcam-1) and serum pro-inflammatory cytokine release (tnfα, ifnγ, il-1β). furthermore, acei or arb treatment in mice results in reduced colonic epithelial cell apoptosis and enhanced expression of the anti-inflammatory cytokine il-10. interestingly, ace gene polymorphism was detected in ibd patients and is associated with reduced ace serum levels; this might be associated with the disease pathogenesis and its extraintestinal side effects [23] [24] [25] . it is thought that chronic treatment with raas blockers (acei or arbs) may lead to increased ang 1-7, which could then oppose the harmful effects of ang ii in the cardiac and renal system, and possibly in the gut as well. anti-inflammatory effects of ang 1-7 have been reported in atherosclerosis plaque [26] and in an arthritic model [27] which was mediated through inhibition of nfκb activity and reduction of cytokines and chemokines such as tnfα, il-1β, mcp-1 and cxcl1. several reports [28] [29] [30] [31] have suggested that ang 1-7 treatment reduces the activity of intracellular signaling molecules such as mapk family (p38, erk1/2 and jnk), protein kinase c (pkc) and c-src kinase, which play an important role in intensifying the inflammatory response. ang 1-7 has been found to reduce the severity of allergic inflammation in mice by suppressing the activity of erk1/2 and nfκb pathways [32] . of particular interest, ace2 (the main enzyme responsible for ang 1-7 generation) expression has been observed in epithelial and sub-mucosal cells throughout the gut, with significant expression in the ileum and the colon [33] [34] [35] . in the present study, we used the mouse dss colitis model to examine the ability of ang 1-7 to influence colitis severity. we show for the first time that the expression of ace2/ang 1-7/ mas-1 r are modulated post colitis induction. blockade of the endogenous function of ang 1-7 (by the mas-1 r antagonist a779) exacerbated colitis severity, while daily administration of ang 1-7 ameliorated it. the anti-inflammatory effects of ang 1-7 were associated with reduction in the expression level of ang ii and the phosphorylation of p38 mapk, erk1/2 and akt in the colon. male and female balb/c mice (1:1 ratio, 6-10 weeks old, mean weight 20 g.) were supplied by the animal resource center of the health sciences center at kuwait university. all animals were kept under standard conditions including controlled temperature (25°c), a 12-h lightdark cycle and had free access to food and drinking water ad libitum. all experimentations were approved by the animal care committee at kuwait university health sciences center and conformed to their rules and regulations. during this study, the general health and wellbeing of the mice was continuously monitored and overseen by a veterinary physician in the animal house. all mice were monitored for their eating/drinking habits, activity, or other severe signs of hunched or lateral recumbency or starry fur or lethargy. there were no signs of illness or mortality during the treatment time points that required sacrificing the animals. the number of animals in each control and treated group for each set of experiments is indicated in the respective figure/table legends. colitis was induced in mice by delivering dss polymers (3.5% w/v, m.wt 40 kd; mp biomedicals, france) in autoclaved drinking water and provided ad libitum for 4-7 days [36] . control (untreated; ut) mice received autoclaved tap water only. the dss solution was replaced every 2 days for the duration of the experiment. there was no difference in the amount of water consumption by mice between the groups. decrease in body weight provides indirect indication of colitis severity. mice were sacrificed by cervical dislocation at 4-7 days post-colitis induction. blood samples were collected by cardiac puncture using a heparinized syringe (for total and differential wbc count). colons were resected and fixed in 10% formalin solution. treatment protocols. angiotensin fragment 1-7 acetate salt hydrate (ang 1-7; m.wt 899; sigma-aldrich, st louis, usa) was dissolved in 0.9% saline (vehicle) at 1 mg/ml and stored at -80°c. various doses (0.01, 0.06, 0.1, 0.3 and 1 mg/kg) were freshly prepared from the stock each day of the experiment, and administered to mice by daily intra-peritoneal (i.p) injections in a volume of 500 μl per injection, either before (prophylactic approach) or after (treatment approach) dss treatment. a779 (mas-1 r antagonist; m.wt 873; genscript, usa) was similarly dissolved in distilled water at 1 mg/ml and stored at -80°c. a freshly prepared dose of 1 mg/kg was administered to a second group of mice by daily i.p injections in a volume of 500 μl daily (for 4 days) along with colitis induction (prophylactic approach). a third group of mice received dss containing water and daily i.p injections of 0.9% saline (vehicle). the fourth group received dss containing water along with daily i.p injections with dexamethasone (dex) at doses of 0.01-1.0 mg/kg or its vehicle (0.9% saline) (prophylactic approach). gross (macroscopic) assessment of colitis severity. using sterile forceps and scissors, the entire colon of each mouse was removed by a ventral midline incision and opened longitudinally. its length and maximal bowel thickness was measured (in mm) with calipers. several other macroscopical parameters were used to assess the colitis severity including stool consistency, blood in stool, adhesion, erythema, edema and ano-rectal bleeding [36] . the data is presented as the percentage of mice in each group showing these features. histological (microscopic) assessment of colitis severity. the colon was cleaned from stools and blood with a few drops of sterile 0.9% saline and 'swiss-rolled' from the descending to the ascending part. samples were fixed in 10% neutral buffered formalin and placed in tissue processing and embedding cassettes in pbs for a few minutes and then overnight in a leica asp 3005 tissue processing machine. tissues were rinsed in two changes of formalin, then dehydrated in several changes of graded alcohol (70%, 90% and 100%). three changes of xylene were used for tissue condensation and clearing. using an slee-mps embedding machine, processed tissues were embedded in paraffin wax (24 h at room temperature) and stored at 4°c before trimming and sectioning using an leica rm 2235 microtome. sections (6 μm thick) were floated in a water bath and then placed on uncoated slides at 37°c overnight. after de-paraffinisation in three changes of xylene (5 min each) and rehydration by serial immersion for 2-3 min in each of absolute, 90% and 70% alcohol, sections were washed briefly with distilled water and stained in meyer's alum haematoxylin solution for 7 min followed by thorough rinsing with running tap water. before counter-staining sections in eosin solution for 2 min, slides were dehydrated in graded alcohol. a clearing step was performed by rinsing in three changes of xylene (2 min each) followed by mounting with dpx. the stained sections were (blindly) scored by 3 observers using a standard semi-quantitative histology scoring system [36] [37] [38] which graded the following features: extent of destruction of normal mucosal architecture (0, normal; 1, 2, and 3, mild, moderate, and extensive damage respectively), presence and degree of cellular infiltration (0, normal; 1, 2, and 3, mild, moderate, and transmural infiltration respectively), extent of muscle thickening (0, normal; 1, 2, and 3, mild, moderate, and extensive thickening respectively), presence or absence of crypt abscesses (0, absent; 1, present) and the presence or absence of goblet cell depletion (0, absent; 1, present). the scores for each feature were summed with a maximum possible score of 11. the extent of ulceration was determined on each section along the muscularis mucosa and expressed as percentage ulcerated mucosa [38] . differential white blood cell counts. during dissection, blood was collected by cardiac puncture using a 1 ml heparinized syringe. to determine differential white blood cell count (wbc), a blood smear was made on a glass slide by placing a drop of blood on one slide surface and using a second glass slide to push the blood forward from the edge of the drop, and left to dry for 24h. siemens, diff-quik™ stain set was used for rapid differential staining. slides were dipped 5 times in a methanolic fixative to stabilize cellular components, then dipped 10 times in a buffered solution of eosin y and 7 times in a buffered solution of thiazine dye (methylene blue and azure a). finally, slides were rinsed in distilled water, dried and stored at room temperature. wbcs were identified as monocytes, neutrophils, basophils, eosinophils and lymphocytes. a total of 100 cells were randomly counted in each slide and the data are presented as % of the presence of each cell type. colon tissue samples (descending part) were cut and homogenized (with teflon glass homogenizer) in 1 ml of buffer composed of 1.2g of 50 mm hepes, 0.3g of 50 mm nacl, 1ml of 0.5m edta, 1ml of 1% triton x-100 and 98ml of deionized water. a protease inhibitor cocktail (10μg/ml aprotinin, 10μg/ml leupeptin and 100μm pmsf) was added separately. homogenates were centrifuged at 1,800 rpm for 10 min at 4°c, and the supernatant collected. protein concentration was determined by the bradford assay (bio-rad, hercules, ca). samples containing 50 μg protein were dissolved in an equal volume of 2 x lammeli sample buffer and β-mercaptoethanol, heated at 90°c for 10 min and loaded onto a 12.5% sds-polyacrylamide gel and electrophoresed at 125 v for 1 h. proteins were transferred (at 100 v for 1 h) onto a pvdf membrane (millipore, ireland) and then blocked with 4% bsa for 90 min before overnight incubation at 4°c with primary antibodies for actin (cell signaling technology, boston, ma, usa; 1/1000 dilution), mas-1 r (m 13, santa cruz, tx, usa; 1/100 dilution), ang ii (novus biological; 1/500 dilution), and ace2 (santa cruz, tx, usa; 1/50 dilution). membranes were washed 3 times for 1 h with 1x tbs-t buffer and incubated with appropriate horseradish peroxidase (hrp)-labeled secondary antibodies [anti-rabbit igg, hrp linked antibody (cell signaling technology, boston, ma, usa; 1/1000 dilution), and donkey anti-goat igg-hrp linked antibody (santa cruz, tx, usa; 1/100 dilution)]. bands were visualized using super signal ecl substrate (thermo scientific, rockford, usa) and kodak x-ray film. colon sections (5μm) were deparaffinized, rehydrated through a series of washes in graded ethanol and water, followed by an antigen retrieval step (by boiling the sections in 10 mm sodium citrate buffer, ph 6.0 for 20 min). sections were then incubated in blocking solution (5% bovine serum albumin (bsa) + 0.3% triton x-100 in pbs) for 1 h, followed by incubation overnight at 4°c with primary antibodies [p-erk1/2, p-akt (1:50 dilution), p-p38 mapk and mas-1r (1:100 dilution); cell signaling, usa, and ang ii (1/50 dilution); novus biological] diluted in 1% blocking solution. on the following day, sections were washed and incubated with secondary antibody conjugated to alexa fluor 555 (goat anti rabbit sfx kit; life technologies,usa, 1:400 dilution) for 2 h at room temperature) in the dark. after washes in pbs sections were stained with 4',6 diamidino-2-phenylindole and mounted. images were captured on a ziess lsm 700 confocal microscope and fluorescence intensity estimated in defined fields using image j software package. colon tissue (100 mg/ml) was homogenized as described above and sonicated for 1 min for further disruption of the cell membrane. homogenates were centrifuged for 15 min at 5,000 rpm and the supernatants collected for subsequent analysis. blood was collected by cardiac puncture, centrifuged at 5,000 rpm for 10 min and the plasma stored at-80°c. ang 1-7 was measured using mouse angiotensin 1-7 elisa kit (mybiosource # mbs700453). following the manufacturer's protocol: 50 μl of standards, samples and blank (pbs) were pipetted into the pre-coated wells and mixed with 5 μl of the balance solution. enzyme conjugate (50 μl) was added to samples (except blank) and kept for 1 h at 37°c in the dark. after removal of suspension, wells were washed five times with 1 x wash solution prior to addition of hrp substrates a and b and 15 min later, of stop solution. intensity of the yellow complex was measured at 450 nm and concentration of ang 1-7 interpolated from a standard curve. data were analyzed using graphpad prism version 5.0 for windows (graphpad software, calfornia, usa). differences between groups were assessed using unpaired students' t test or oneway anova, with p 0.05 being regarded as significant. a seven fold decrease in the plasma level of ang 1-7 was demonstrated in dss treated mice compared to untreated (ut) group at day 7 post colitis induction (fig 1a) . on the other hand, a significant increase in ang 1-7 was observed in colon homogenates of dss treated mice at day 7 (0.09 ng/ml) compared to ut mice (0.04 ng/ml) as shown in fig 1b . ang ii expression determined by immunoblotting was increased at days 4 and 6 post-colitis induction compared to ut mice (fig 2a) , as was ace2. an elevation was seen at day 6 in the mas-1 r (fig 2b) . immunofluorescent staining showed presence of ace2 and mas-1 r in the mucosal layers of colon sections (fig 2c) observed at days 4 and 6 post-colitis induction, whereas very little reaction was detectable in colon tissue of untreated control mice. the effect of ang 1-7 on modulating colitis severity in mice was tested by daily i.p injections of various doses of the peptide. dss administration resulted in approximately 20% drop in body weight from day 5 onwards (in contrast with mice receiving tap water only). ang 1-7 at doses of 0.01 and 0.06 mg/kg (but not at higher doses of 0.1-1 mg/kg; data not shown) reduced this drop in body weight by 5-10% compared with the dss/saline i.p treated group (fig 3a) . dss treatment in the dss/saline i.p group resulted in increased circulating neutrophils; this was prevented by ang 1-7 treatment at doses of 0.06-1 mg/kg (fig 3b) . no significant changes in circulating lymphocytes were observed between control and treatment groups. the decrease in colon length and thickness seen after dss treatment was prevented by ang 1-7 treatment at doses of 0.01-0.06 mg/kg but not at higher doses (fig 3c and 3d ). as indicated in table 1 , at the gross level, dss administration for 7 days with daily i.p injections of saline (vehicle) resulted in diarrhea, blood in stool, adhesion, erythema, edema and ano-rectal bleeding, none of which was seen in the ut group. daily administration of ang 1-7 at doses of 0.01-0.06 mg/kg significantly reduced most of these effects. the presence of erythema was reduced by 40-60%, incidence of diarrhea by 30-60%, ano-rectal bleeding by 6-50%, blood in stool by 30%, and edema by 20-30% relative to dss/saline i.p group. ang 1-7 at 0.1 mg/kg dose reduced ano-rectal bleeding, blood in stool and erythema by 15-40% but at the groups indicated. panel g represent plasma levels (ng/ml) of ang 1-7 in mice treated with dss (for 7 days) plus either daily i.p saline or ang 1-7 (0.06 mg/kg). histobars represent means ± sem for 4-18 mice in each group (8 for ut, 18 for dss/i.p saline, 6 for ang 0.01 mg/kg, 5 for ang 0.06 mg/kg, 9 for ang 0.1 mg/kg, 4 for ang 0.3 mg/kg and 9 for ang 1.0 mg/kg). asterisks denote significant difference from ut mice with p<0.05 (*) and p<0.001 (**). # denotes significant difference from dss/i.p saline treated mice with p<0.05. . panels e and f represent quantitatively the histological assessment of colitis severity and the % of ulceration in the whole colon section for each group as indicated. histobars represent means ± sem for 5-18 mice 0.3-1.0 mg/kg, this protective effect was lost. the histological score of colitis correlated well with the macroscopic score (as shown in fig 3e) ; a significant reduction in colitis severity was seen with ang1-7 treatment only at the lower doses of 0.01-0.06 mg/kg. similarly, the % of ulceration (fig 3f) was significantly reduced (40-55%) but again only with the lower dose of 0.01-0.06 mg/kg ang 1-7. furthermore, daily i.p injections of ang 1-7 at 0.06 mg/kg dose significantly increased the plasma levels of ang 1-7 compared to mice receiving daily i.p saline at day 7 post-colitis induction (fig 3g) . the histological features of the resected tissues are illustrated in fig 4. compared to the normal architecture in untreated (ut) controls, dss/i.p saline treatment for 7 days resulted in significant mucosal destruction and infiltration by immune cells (fig 4b) . injection of dss treated animals with 0.01-0.06 mg/kg doses of ang 1-7 improved mucosal integrity and reduced the degree of immune cell recruitment to the colon (fig 4c and 4d ). this anti-inflammatory effect was lost with higher ang 1-7 doses (fig 4e, 4f and 4g). to determine whether ang 1-7 can reduce the severity of colitis once it is established, mice were given daily i.p injections of ang 1-7 (for 3 days) 4 days after dss administration (when effects are less severe). by 7 days, at both doses of 0.01 or 0.06 mg/kg, ang 1-7 reversed the loss in body weight induced by dss (fig 5a) . in addition, the elevation in % circulating neutrophils seen with dss treatment was also reversed by ang 1-7 back to levels seen in the ut group ( fig 5b) . the small increase in colon thickness observed in the dss group was reversed by ang 1-7 though this effect did not reach statistical significance (fig 5d) . histological examination ( fig 5g-5i and quantified in fig 5e and 5f ) showed that ang 1-7 could reverse some of the dss induced mucosal damage and ulceration, though in the case of the latter it did not reach statistical significance. at the macroscopic level, ang 1-7 was seen to reduce the dss induced increases in edema and erythema but not in adhesion ( table 2) . we compared the effects of ang 1-7 with the classic anti-inflammatory agent dexamethasone (dex) using the same treatment approach. the dss induced loss in body weight was partly in each group (9 for ut, 18 for dss/i.p saline and 5 for ang 0.01 mg/kg and ang 0.06 mg/kg). in panels a asterisks denote significant difference from ut mice with p<0.05 (*) and p<0.001 (**). # denotes significant difference from dss/ i.p saline treated mice with p<0.05. panel g shows colon section taken from a mouse treated with dss for 4 days followed by 3 days of i.p saline (vehicle) indicating the extent of mucosal destruction and immune cells recruitment. panels h and i show colon sections taken from dss mice subsequently treated with ang1-7. significant improvement in the mucosal integrity and reduction in immune cell recruitment can be seen. bars represent 150μm. reversed after 7 days of dex treatment at 0.01 mg/kg dose ( fig 6a) . as observed with ang 1-7, this effect was lost at higher doses. dex did not reverse the increase in neutrophils (data not shown). whereas there was no difference in the colon thickness, colon length was significantly increased by dex treatment at 0.01-0.1 mg/kg doses (but not higher) compared to dss/i.p saline group (fig 6b) . both histological score of colitis and extent of ulceration (fig 6d and 6e ) were reduced at low (but not higher) doses of dex. with respect to the macroscopic parameters, dex treatment specifically at 0.01 mg/kg dose significantly reduced diarrhea, blood in stool, ano-rectal bleeding and erythema, but did not have any effect on either the edema or adhesion ( table 3) . to determine the effect of inhibiting the action of endogenous ang 1-7, the mas-1 r antagonist a779 was administered (for 4 days) with daily i.p injections at 1 mg/kg dose to dss treated mice. dss/i.p saline group did not show weight reduction since dss treatment was given for 4 days only ( fig 7a) . a779 treatment exacerbated the weight loss but did not affect the changes in lymphocytes or neutrophils induced by dss (fig 7a and 7b ). there was also no effect on colon length or thickness but histologically, there was significant additional deterioration of the mucosal structures and increased ulceration as compared to the dss/i.p saline group as shown by the examples in fig 7 panels g and h and quantitatively in panels e and f. at the macroscopic level, a779 treatment resulted in reduction of the dss induced edema but worsened the erythema, with no effect on the other parameters (table 4 ). prolonged administration of dss plus a779 to mice (for 5-7 days) resulted in death (data not shown), highlighting the intensified colitis severity as a result of prolonged inhibition of the endogenous levels of ang 1-7. the level of phosphorylated forms of three key signaling intermediates, erk1/2 (fig 8) , p38 mapk (fig 9) and akt (fig 10) , were measured by immunofluorescence in sections from resected colon tissue of untreated mice or mice treated with dss (for 7 days) plus daily ang 1-7 or saline (vehicle) treatment. in each case, expression was enhanced by dss and reduced by ang 1-7 back to the basal levels observed in the ut group. in the case of erk1/2, ang 1-7 at 0.06 mg/kg dose suppressed phosphorylation even below basal levels ( fig 8e) . . panels e and f represent histological assessment of colitis severity and the % of ulceration in the whole colon section respectively in the groups indicated. histobars represent means ± sem for 5-9 mice in each group (9 for ut and 5 for dss/i.p saline and dss/a779). asterisks denote significant difference from ut mice with p<0.05 and # denotes significant difference from dss/ i.p saline treated group with p<0.05. panels g and h show representative colon sections from dss/i.p saline or dss/a779 treated mice. the expression level of ang ii was examined by immunofluorescence in sections from resected colon tissue of untreated mice (ut) or mice treated with dss (for 7 days) plus daily ang 1-7 or saline (vehicle) treatment. ang ii expression was enhanced by dss administration (fig 11b) compared to ut mice ( fig 11a) . ang ii expression was reduced by daily administration of ang 1-7 at doses of 0.01-0.1 mg/kg (fig 11c-11e ). in fact, daily administration of ang 1-7 at 0.06 mg/kg dose ( fig 11d) reduced ang ii to the basal levels observed in the ut group. daily administration of a high dose of ang 1-7 (1.0 mg/kg, fig 11f) resulted in increased ang ii levels similar to levels seen in dss/i.p saline group. in this study, we have presented data indicating a novel role of ang 1-7 in reducing colitis severity in the murine dss model. daily i.p injections (at doses of 0.01-0.06 mg/kg) ameliorated colitis severity when given either prophylactically or after colitis induction. blockade of the mas-1 r (by a779) aggravated colitis severity, highlighting the involvement of the ang 1-7/mas-1 r axis in the pathogenesis of ibd. this protective effect was in part modulated through the expression/activity of several signaling molecules such as ang ii, p38 mapk, erk1/2, and akt. it has been demonstrated that ace2 is expressed in the epithelial, sub-mucosal and muscular layers of the colon in both humans and mice [33] [34] [35] , and its expression levels are increased in the plasma of ibd patients [39] . we observed increased expression of ace2 and mas-1 r proteins in the colonic tissues of mice after dss challenge (fig 2) , and higher ang 1-7 levels in the inflamed colon homogenates (fig 1b) . plasma levels of ang 1-7 were significantly reduced at 7 days post dss treatment (0.002 ± 0.006 ng/ml) when compared to healthy controls (ut; 0.232 ± 0.134 ng/ml). partial restoration was achieved with daily i.p injections of ang 1-7 at 0.06 mg/kg dose (0.061 ± 0.009 ng/ml; fig 3g) . the enhanced expression of the ace2/ang 1-7/mas-1 r axis post dss challenge might reflect a compensatory protective mechanism to reduce the effect of the inflammatory response. daily ang 1-7 administration significantly reduced colitis severity observable at both gross (table 1 ) and histological levels (fig 3e and 3f ). in addition, it also opposed the dss-induced drop in body weight (fig 3a) , and restored the changes in colon length and thickness post colitis induction (fig 3c and 3d) . the protective effect of ang 1-7 was lost with higher doses in vivo (fig 3) . one explanation is that higher concentrations of ang-1-7 attenuate the mas-1 r responsiveness through receptor desensitization [40] [41] [42] . another report also showed that ang 1-7 treated hek 293t cells at high concentrations redistributed the mas-1 r to intracellular vesicles and co-localized ang 1-7 with rab5 and the adaptor protein complex 2, suggesting regulation by a clathrin-mediated pathway [43] . enhanced activity of members of the mapk family, p38 mapk and erk1/2 as well as akt (the downstream target of pi3k), which we observed in colonic sections post dss treatment (figs 8-10 ) has previously been reported in colonic biopsies from ibd patients [44] [45] [46] [47] . treatment with inhibitors of these signaling molecules ameliorated colitis severity in mice and reduced the disease activity index in patients, underlining their role in ibd pathogenesis [44] [45] [46] [47] . in this study, we showed that the anti-inflammatory effects of ang 1-7 treatment were associated with a reduction in the phosphorylated forms of p38 mapk, erk1/2 and akt post dss induction (figs 8-10) , consistent with previous reports showing reduced activity of these molecules post ang 1-7 treatment in the anterior pituitary [31] , proximal tubular cells [29] , and lung tissues in an animal model of lung inflammation [32] . using immunofluorescence analysis, we observed enhanced colonic expression of ang ii at day 7 post-dss treatment (fig 11) , confirming western blotting data presented in fig 2a. ang ii expression was reduced by daily ang 1-7 treatment (at doses of 0.01-0.1 mg/kg, fig 11c-11e ). this suggest that the antiinflammatory properties of ang 1-7 are in part mediated through reduction of ang ii levels. regarding the influence of the ace2/ang 1-7/mas-1 r axis in modulating colitis severity, one report demonstrated that administration to mice of gl1001 (as a chemical inhibitor of ace2) reduced colitis severity [48] . however, it should be noted that the specificity of this inhibitor is questionable, since ace2 inhibition may increase the levels of other substrates such as ang i/ii, ghrelin, dynorphin, bradykinin, neurotensin and apelin [49] . other studies (in the murine tnbs model) have demonstrated that ace2 deficiency results in increased likelihood of developing intestinal inflammation due to the disruption of dietary amino acid homeostasis, innate immunity and gut microbial ecology [50] , as well as increased leukocyte recruitment and pro-inflammatory cytokine release in the colonic tissues. these data are consistent with a protective role of the ace2/ang1-7/mas-1 r axis observed in this study. in regard to the effect of dex on modulating colitis severity, targeted delivery of dex to the colon was shown to reduce colitis severity in rodents [51] [52] [53] . however, daily i.p administration of dex was shown to either increase [54] , or decrease colitis severity [55, 56] . in our study, we showed that daily i.p injection of dex at doses 0.01-0.06 mg/kg significantly reduced colitis severity at macroscopic (table 3 ) and histological level (fig 6d-6e) . in conclusion, this study is the first demonstration (to our knowledge) that the ang 1-7/ mas-1 r axis plays a role in modulating colitis severity, in part through down-regulation of the levels of ang ii and the phosphorylation of key signaling molecules involved in the inflammatory process such as erk1/2, p38 mapk and akt. blockade of the mas-1 r inhibited the protective effects of endogenous ang 1-7 and 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infarction angiotensin (1-7) induces mas receptor internalization inhibition of stressactivated map kinases induces clinical improvement in moderate to severe crohn's disease. gastroenterology anti-inflammatory and anti-oxidative activities of paeonol and its metabolites through blocking mapk/erk/p38 signaling pathway. inflammation inhibition of phosphoinositide 3-kinase ameliorates dextran sodium sulfate-induced colitis in mice pi3k/akt signaling pathway is involved in the pathogenesis of ulcerative colitis effects of the ace2 inhibitor gl1001 on acute dextran sodium sulfate-induced colitis in mice hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase ace2 links amino acid malnutrition to microbial ecology and intestinal inflammation investigation of the colon-targeting, improvement on the side-effects and therapy on the experimental colitis in mouse of a resin microcapsule loading dexamethasone sodium phosphate immunomodulation of rheum tanguticum polysaccharide (rtp) on the immunosuppressive effects of dexamethasone (dex) on the treatment of colitis in rats induced by 2,4,6-trinitrobenzene sulfonic acid dexamethasone 21-sulfate improves the therapeutic properties of dexamethasone against experimental rat colitis by specifically delivering the steroid to the large intestine the effect of dexamethasone treatment on murine colitis bioluminescence imaging for il-1beta expression in experimental colitis expression of na-h exchanger-8 isoform is suppressed in experimental colitis in adult rat: lack of reversibility by dexamethasone this work was supported by kuwait university research sector grant pt02/12. parts of this work were supported by grant srul02/13 to the research unit for genomics, proteomics and cellomics studies (omics), kuwait university. key: cord-002094-7tewne3a authors: tago, damian; hammitt, james k.; thomas, alban; raboisson, didier title: the impact of farmers’ strategic behavior on the spread of animal infectious diseases date: 2016-06-14 journal: plos one doi: 10.1371/journal.pone.0157450 sha: doc_id: 2094 cord_uid: 7tewne3a one of the main strategies to control the spread of infectious animal diseases is the implementation of movement restrictions. this paper shows a loss in efficiency of the movement restriction policy (mrp) when behavioral responses of farmers are taken into account. incorporating the strategic behavior of farmers in an epidemiologic model reveals that the mrp can trigger premature animal sales by farms at high risk of becoming infected that significantly reduce the efficacy of the policy. the results are validated in a parameterized network via monte carlo simulations and measures to mitigate the loss of efficiency of the mrp are discussed. financial aid to farmers can be justified by public health concerns, not only for equity. this paper contributes to developing an interdisciplinary analytical framework regarding the expansion of infectious diseases combining economic and epidemiologic dimensions. movement restriction policies (mrps) are one of the most popular strategies implemented within and between countries to fight the spread of infectious animal diseases in europe and worldwide [1] . their efficiency has been demonstrated in the field, often in combination with other measures. in europe, following the council directive 91/119/eec, whenever an infectious disease is detected three zones are delimited over which different restrictions on animal movements are implemented. scale-free networks [2] , which characterize the structure of many real-world networks, provide a theoretical basis for designing strategies for controlling infectious diseases in cattle and other livestock. applications have been developed recently for animal [3] and human diseases [4] and for viral computer infections [5] . the main characteristic of scale-free networks is the existence of highly connected nodes, i.e. with a degree that greatly exceeds the average such that the degree distribution (the frequency distribution of the number of connections of each node) is fat-tailed. these nodes are called hubs and their high connectivity is the main reason why a disease, technology, or fashion can spread much more quickly through a scale-free network than through a random network [6] . the idea behind control strategies such as the mrp and vaccination [7] is that removing infected nodes or immunizing susceptible ones are efficient mechanisms to fight the spread of a disease. it has been found that in fat-tailed degree distribution networks, random immunization (for example vaccinating a proportion of nodes chosen randomly) is not effective in stopping the spread of a disease since for many diseases immunization of 80%-100% of the population is required to halt epidemics [8] . on the other hand, targeted immunization of the most highly connected nodes (hubs) is very effective, but requires global information on the network [9] . nevertheless, strategies that use only partial information have been found to efficiently reduce the spread of diseases [10] . new efforts regarding the traceability of infected animals, such as the construction of databases recording every movement of cattle in a country [11] , make implementation of the mrp more efficient since they allow tracking the origin of an infection and improving the efficiency of emergency procedures. the removal or isolation of infected nodes will prevent the disease from continuing to spread but this strategy is effective only under demanding conditions. for example, surveillance protocols must be sufficiently efficient that infections are detected almost immediately, or farmers must detect and quickly report the presence of an infectious disease. the time between contagiousness of an animal and its detection depends on the disease, and early detection may not occur when clinical signs are not easily visible. even active-surveillance programs may not detect contagious animals quickly enough when tests are imperfect. moreover, farmers may be slow to report possible infection, in particular when immobilization of their animals can be associated with very large costs [12] . a great advantage of the mrp is that it can be implemented to control an infectious disease as soon as the disease is detected, whereas strategies based on vaccination require time to develop, produce, distribute, and administer the vaccine to large populations. the efficiency of the mrp also depends on the disease to be controlled. even if detection and isolation of infected farms are perfectly efficient, if the disease is transmitted by insects or other vectors the mrp cannot by itself assure that the epidemic will be halted, as observed during the 2008 btv-1 epizootic [13] . studies of human-disease epidemics have recently begun to incorporate human reactions [14] . most of these studies point to the existence of preventive responses that can reduce the spread of the infection, such as the voluntary use of vaccines or facemasks that reduce the risk of being infected [15] . in the field of animal diseases, the human factor has received less attention. however, the role of biosecurity measures on the spread of animal diseases has been analyzed taking into account the externalities and strategic behavior of farmers [16, 17] . among english and welsh cattle farmers, it has been shown that behavioral changes have an impact on the implementation of disease-control programs [18] . in asian countries, misbehavior by farmers (lack of perfect compliance with regulation) and the emergence of underground poultry markets have been recognized as possible consequences of implementing control strategies [19] , but their impacts have not been quantified. in the model we propose, changes in farmers' behavior induced by implementation of the mrp can make the policy ineffective under certain conditions. this is due to a mismatch between the time that the mrp is announced in a country (when an infectious disease is detected) and the time that immobilization is implemented in a specific region of that country. this result holds even under the assumption of perfect compliance by farmers (no misbehavior). it can be explained by reasoning similar to that of the so-called "green paradox", which states that some environmental policies oriented to slow global warming act as an announced expropriation of fossil fuels, inducing the owners of these resources to accelerate extraction, which accelerates global warming [20] . hence possible induced changes in farmers' behavior should be considered when designing control strategies for infectious diseases. the remainder of the paper is organized as follows. first, a susceptible-infected epidemiological model at the farm level is described to analyze diseases that can be transmitted through both the cattle trade network and the geographical network. second, a two-period economic decision model is presented to understand how the mrp can change the strategic behavior of farmers located close to the restriction zone. the conditions that trigger anticipatory sales are established and the effect of the mrp on the spread of an infectious disease is analyzed, with and without anticipation effects. the results are presented, the implications for anticipatory sales are discussed, and a corrective mechanism is proposed. finally the conclusions are presented. the epidemiological model national cattle trade networks have been characterized by previous studies, which find that they have a fat-tailed degree distribution [21, 22] . the degree of a node represents the number of connections (edges) that this node has with the rest of the network. in networks with fattailed degree distributions, highly connected nodes (hubs) are more frequent than in random networks; the distribution of degrees is heavy-tailed with power-law behavior of the form p(k)~k −φ with 2 φ 3, where p(k) is the density function evaluated for degree "k". to study the spread of an infectious disease under different scenarios a fat-tailed degree distribution network is constructed following the characteristics of the french cattle trade network described by rautureau et al. [21] . the trade network was constructed using the complex networks package [23] with a power-law degree exponent φ = 2.15 [21] . to mimic the french cattle network (table 1) , the nodes were ranked by degree and the top 0.04% were classified as markets, the next 0.54% as dealers, and the remaining 99.42% as farms. each group (farms, dealers, and markets) is characterized by a probability of selling derived from the french cattle network and modeled as a random variable with a uniform distribution in order to capture some of the heterogeneity (table 2) . a node is connected to the trade network in each period when it sells animals, which is simulated as the outcome of a bernoulli trial using the corresponding probability of selling. rewiring of the network takes place at each time period. we extend the standard susceptible-infected (si) epidemiological model to incorporate features that are essential for our analysis: the efficiency with which infectious disease is detected and the existence of multiple channels of infection. in our framework, each node represents an agent involved in the cattle trade (farm, dealer, or market) and each agent can be in one of two states: susceptible (s) or infected (i). if detection is imperfect, there are two types of infected nodes: detected (i d ) and non-detected (i nd ), so the total number of infected nodes is i = i d + i nd . to adapt the model to vector-borne diseases an additional transmission channel must be considered. with vector-borne diseases, the infection can propagate through (a) commercial flows and (b) geographical proximity to infected nodes. the incorporation of both channels has been highlighted as a desirable feature for realistic epidemiologic models [24] . an additional geographic network capturing the risk of vector-transmitted infection of nodes located close to infected ones has to be taken into account. an overview of the baseline model for a susceptible node exposed to infection is illustrated in fig 1. at each time period there are 4 stages. in the first stage the trade of cattle takes place. at this stage all the market decisions are taken, animals are traded, and susceptible nodes can be exposed to infection through the arrival of infected animals. second, there is the infection stage in which susceptible nodes exposed to infection (i.e. directly connected to an infectious node) can change from the s-state to the i-state with probability λ. the probability of a node becoming infected depends on the number of infectious neighbors and can be computed as 1-(1-λ) n , where n is the number of infectious neighbors. after the infection stage the disease is detected with probability γ, which is equal to 1 if detection is perfect. finally the control stage takes place, in which movement-restriction policies are implemented in two steps: first, the i dnodes are isolated with probability α; second, direct neighbors at the geographic network of infected and controlled nodes are isolated. table 3 summarizes the main parameters involved in the transition between states. the transmission rate λ depends on the disease and may also depend on other factors such as cattle density [25] and environmental factors [13] , which affect vector capacity (ability of vectors to acquire and transmit pathogens). the detection rate γ depends on two factors: the characteristics of the disease and the efficiency of the detection tools. the former is associated with the length of the incubation period or the rate of subclinical cases while the latter may depend on the sensitivity of available tests. the parameter α reflects the efficiency of the authorities in implementing the mrp. notice that the mrp isolates the nodes detected as infected as well as their geographic neighbors, which are considered to be those nodes directly linked through the geographic network. this simulates establishing a protection zone around the infected perimeter in which no infected animals have been detected but movements are restricted. the geographic network is simplified to a square lattice in which each node is connected to its immediate neighbors, so there are no hubs in the network. both networks have the same number of nodes and are associated through a one-to-one mapping that randomly assigns the at the infection stage, the node changes to infected status with probability λ; otherwise it retains the s-status during the whole period. at the detection stage, an infected node is detected with probability γ and adopts the i d -status; otherwise it takes the i nd -status. when disease is detected the node is removed from the network with probability α. nodes with i nd -status and those with i d -status that are not removed (i dnc ) remain in the network until the next time period when they are again at risk of being detected or removed. nodes can be removed from the network if they are infected, detected, and effectively controlled (i dc at control stage i), or if they enter a protection zone triggered when any direct neighbor in the geographic network is infected and controlled (c at control stage ii). node's location, which remains fixed throughout the simulation. the infection is introduced by infecting one random node in the first period and the spread of the infection is summarized by the number of infected nodes up to 300 time periods. since the probability of selling was derived for weekly cattle trade networks, a time period can be interpreted as a week in this analysis. for each scenario explored, 300 simulations were performed using matlab (mathworks). the immobilization of animals may impose costs on producers whose profits rely on the movement of their animals [26, 27] . to illustrate the implications of the mrp on the behavior of farmers, we consider a simple two-period economic model where a seller of cattle has to choose when to sell his animals. consider a risk neutral agent who has to choose between selling or not selling in each period (t = {1,2}). at any time period t, if the farmer decides to sell he receives: where: y t is the farmer's income, p t is the market price of cattle, and w t is the total weight of the animals that the farmer sells in period t. if the farmer decides to keep his animals and wait for the second period, y 1 = -c, where c represents the costs associated with keeping the animals for an additional period such as feeding and labor costs. to simplify the problem, assume there is no market risk or price trend such that p 1 = p 2 = p, and the animals gain weight according to the growth parameter d (i.e. w 2 = w 1 ã (1 + d), where d represents the one-period weight gain percentage). in case the farmer decides not to sell at t = 1 nor at t = 2, he receives an option value v, which is smaller than the revenue he would receive by selling at t = 2 (i.e. v < p ã w 1 ã (1 + d)). in the context of beef weaned calves (young animals that are sold to fattening units), if animals become too heavy/old, farmers may face a lower price, which can be represented by a penalty p>0 on the option value (v = p ã w 1 ã (1 + d) ã (1 − p)). this penalty does not represent fluctuations of market prices (which are fixed in our model) but the fact that if a farmer does not sell at the right moment, the animal losses some of the characteristics valued by buyers. for example, the cost of transporting beef weaned calves increases as the animals become heavier, which makes fattening units less prone to buy these animals. the present study focuses on the most interesting case, where market conditions are such that the gain from waiting one period is larger than the cost of keeping the animals (i.e. c < p ã w 1 ã d), making the strategy of waiting at t = 1 and selling at t = 2 strictly dominant. usually, beef weaned calves producers face this type of situation and feeding costs play an important role in their decision. if an infectious disease is detected at t = 1, a farmer sufficiently close to the infected zone will face the risk that the restricted zone (rz) will expand to include his location by the next period (with probability q). if at t = 2 the farmer is located in the rz he will not be able to sell. hence a farmer that does not expect any compensation in case of falling into the rz will decide to anticipate and sell prematurely if: solving for q yields if the gain from trading in the first period is larger than the payoff from keeping the animals and being unable to sell them in the second period (i.e., [v − c] < p ã w 1 ) the existence of q 2 (0,1) is assured. to incorporate the anticipatory behavior into the si model, define nr it as farm i's set of neighbors in the geographic network that are located in the rz at period t, and b q p ã w 1 ã dàc p ã w 1 ð1þdþàv as the threshold for q above which anticipatory selling is an optimal choice. we assume 8i such that nr it 6 ¼ ;; i.e. farms geographically adjacent to a farm in the rz perceive the probability of being in the rz in the next period to be high enough such that it is optimal to sell in anticipation (fig 2) . therefore, anticipatory sales affect the rewiring process, which modifies the spread of the disease. a monetary transfer to farmers in the rz is a simple mechanism to avoid (or reduce) the anticipatory behavior of farmers at risk of entering the rz. the size of the monetary transfer (denoted by t) that makes farmers with probability q it of entering the rz in the next period indifferent between anticipating and waiting can be derived from: therefore: the size of the monetary transfer was calibrated taking information on prices, weight evolution of animals, and production costs, using data corresponding to the resurgence of the french btv-8 epidemic in 2007. the monetary transfer is expressed as a function of the subjective probability of being immobilized in the next week. the main parameters of the economic model are summarized in s1 table, including the values used to derive the monetary transfer required to avoid anticipatory sales. the efficiency of the mrp was quantified by an index defined as the proportional increase in the time-weighted number of uninfected nodes associated with a policy, compared with no policy. this represents the proportional increase in the area above the curve of accumulated infected nodes due to the policy (s1 fig). the index ranges from 0 to infinity, where 0 is associated with a policy with no benefits in terms of slowing the disease spreading. a rank-correlation analysis was performed to explore how the characteristics and location of the initial case of infection contribute to variability in the results [28] . in this type of analysis, the degree of statistical dependence between two variables that follow different distributions is quantified and used to identify which factors are the most relevant to the spreading of a disease. the correlations explored are between the accumulated number of infected nodes at a specific time period (the period was chosen such that the average fraction of nodes infected was 50%) and three different variables that characterize the early periods of infection: the degree (number of edges) and closeness (defined as the inverse of farness, which is the sum of the distances from the selected node to the rest of the nodes in the network) of the initially infected node and the time period at which the first infection of a hub (dealer or market) takes place. results focus on the accumulated number of infected nodes over time after the introduction of a disease. in the absence of control strategies, a 5% infection rate leads to 100% of nodes being infected within approximately 100 weeks (fig 3, red line) . in the case of non-vector-borne diseases, i.e. when the transmission channel is restricted to the trade network, the mrp is an effective control strategy. if the detection tools are perfect it can immediately stop the spread of the disease (fig 3, green line) . in cases where the disease can be transmitted through both geographic and trade networks (as in the case of vector-borne diseases), the outbreak cannot be contained by movement restrictions alone even if the detection and control rates are 100% since the geographic channel cannot be closed by the mrp (fig 3, blue line) . however, the mrp helps to slow the disease spreading. when a disease is difficult to detect (γ close to 0) the benefits of the mrp are severely dampened (s2 fig). when both the geographic and trade networks are considered, a disease with a 5% infection rate infects half of the nodes around periods 275, 185, and 125 for detection rates equal to 100%, 50%, and 20%, respectively. when the strategic behavior of agents is incorporated the outcome of the mrp changes significantly. the anticipation effect increases the speed of infection, and for a low detection rate, γ = 20%, the benefits of the mrp are severely reduced (fig 4) . as the detection rate increases, the anticipation effects become less relevant since infected farms are removed from the network before they can sell. although there exists some overlap in the results of the simulations under different scenarios, the mean trajectory of infected nodes under the mrp with anticipation effects is significantly higher than in the case without anticipation. such overlapping disappears when the 15% most extreme outcomes are omitted from the analysis (s3 fig). the effect of anticipation anticipatory behavior-connectivity of a susceptible node. a susceptible node can be located in the rz or the unscathed zone (uz). if located in the rz, the node is unable to sell (probability of selling = pr = 0) and has no effect on the dissemination of the disease. a node located in the uz can face two situations: a) the node is not connected to any node located in the rz so it will decide to sell or not according to pr = p s , which is associated with its type (farm, dealer, market; see table 2 ); b) the node is connected to a node located in the rz so q i;t > b q, which triggers anticipatory sales (pr = 1). doi:10.1371/journal.pone.0157450.g002 can be assessed using the efficiency index, which decreases from 1.06 without anticipation effects to 0.41 when anticipation effects are included. the rank correlation coefficients, whose range is [-1,1], are small and positive for the degree and closeness of the initially infected node (between 0.02 and 0.18), and large and negative for the time at which the first hub is infected (between -0.53 and -0.88, see fig 5) . finally, the size of the monetary transfer required to prevent anticipatory selling is derived for two option values that correspond to different penalties (10% and 30%) on the value of the animal, considering that too old/heavy animals the efficiency of the mrp is significantly reduced when facing a vector-borne disease because the policy cannot control the spread by vectors. however, the mrp restricts the transmission of the disease to the geographical channel. because the geographic network has no hubs, the rate of spreading is significantly slower than through the commercial network. geographic networks can be important even for diseases that are not spread by vectors. for example, it has been found that in the case of diseases transmitted through close animal-to-animal contact, such as foot-and-mouth disease, the plumes of the virus can be dispersed over long distances [29] . therefore, considering both the geographic and trade networks can be important for some non-vector-borne diseases. factors that limit the speed of detecting infections are an obstacle for implementation of the mrp. the mrp becomes significantly less efficient when infected nodes that have not been detected spread the disease through both trade and geographic networks. the distribution of the time between the infection and control events has previously been highlighted as a crucial ingredient to identify for effectively managing disease spreading [30] . factors that can limit the rate of detection include a high rate of subclinical cases, long incubation periods, and poor detection tools. the detection period can also be lengthened by the absence of active surveillance activities or lack of declaration in cases of clinical suspicion. for cow-calf systems or for dry dairy cows, farmers may easily miss some moderate clinical signs on some animals and not identify cases until clinical signs have become severe. note that in the present study, changes in the efficiency of implementation of the control policy (α) have analogous results to changes in the detection rate (γ), since the effective removal of the infected nodes depends on the product αã γ. the introduction of strategic behavior of farmers into the analysis significantly reduces the efficiency of the mrp. according to our efficiency index, the anticipatory behavior of farmers facilitates spreading of the disease and reduces the efficiency of the policy by 61%. when the mrp is implemented, farmers that perceive a high risk of entering the restriction zone will prefer to sell prematurely to avoid the immobilization costs. this increase in the connectivity of the network accelerates the dissemination of disease. in the case of vector-borne diseases, the holdings located close to the rz have a higher risk of becoming infected. if the rate of subclinical cases is high or the incubation period is long, infection at farms outside the rz may be unnoticed and may be transmitted through premature selling of undetected but infected animals. although the mrp by itself is unable to stop the spreading of a vector-borne disease, it can be very useful for reducing the speed of spreading, especially when waiting for the availability of vaccines or for the seasonal inactivity of vectors. the fat-tailed degree distribution of the cattle trade network plays an important role in accelerating the rate of disease spreading. anticipatory sales can infect hubs, which can quickly infect many other nodes. if the trade network were homogenous, hubs would not exist and the effect of anticipatory sales on the spread of diseases would be smaller. the assumption that all farmers having a neighbor (through the geographic network) in the rz perceive that the risk of becoming immobilized in the next period is sufficiently high to induce them to sell their animals (eq (4)) is strong. if this assumption is relaxed, there would be fewer anticipatory sales and the disease would spread more slowly. moreover, when an infected node is detected and isolated, its neighboring nodes are isolated too. therefore, the way the restriction zone is defined has an impact on the results. if the mrp were modified to include not only direct neighbors but the neighbors of neighbors as well (2nd degree connections), the set of nodes that sell prematurely would change. in particular, at early stages of an epidemic, the number of nodes engaging in anticipatory selling (i.e. those at the frontier of the rz) is increasing in the size of the rz and decreases as the entire territory becomes rz. but if there is a correlation between the risk of infection and the geographic proximity to infected nodes (as is the case of vector-borne diseases), by increasing the rz, the nodes located at the frontier are less likely to be infected so they have a smaller effect on disease spreading even if they sell prematurely. the rank-correlation analysis shows that the longer the hubs remain uninfected, the slower the epidemic will spread (fig 5) . this result is consistent with the recommendation of immunizing hubs as quickly as possible to avoid the expansion of a disease [31] . this relationship is stronger when no policy control takes place (r = -0.88) than when the mrp is implemented (r = -0.66 with anticipation, r = -0.53 without anticipation). on the one hand, the role of hubs as amplifiers of disease spreading is reduced through the mrp, but on the other hand, complementary strategies to control the spread of infection focused on the immunization of hubs will be less efficient compared to the situation without mrp. this trade-off between the benefits of implementing the mrp and the decrease in efficiency of complementary strategies (focused on the immunization of hubs) should be taken into account when designing an integrated control strategy. the risk imposed by the anticipatory behavior is not a consequence of farmers violating the rules; anticipation effects can be important even in contexts where farmers report new infections promptly and comply with the movement restrictions. the key element in this model is that detection is imperfect (because the tools are limited or the latent or subclinical period of the disease is long) so a farm could sell infected animals without knowing it. a monetary transfer conditional on entering the rz is proposed as a simple mechanism to avoid the change in behavior of farmers affected by the policy. this monetary transfer must be credible and announced in advance so that farmers know of it when considering anticipatory selling. in practice, the monetary transfer can be directed to a specific type of farmer since not all farmers have incentives to sell prematurely. for example, the mrp affects farms specialized in dairy products to a lesser extent than farms specialized in the production of young beef weaned calves, which are sold to fattening units as intermediate products. recognizing this fact, during the 2006-2008 bluetongue outbreak the french government provided financial aid only to farmers who generated at least 50% of their revenue by the sale of livestock (c-dgpei/ sdepa/c2007-4027) and to the producers of young beef weaned calves (ns-dgpei/sdepa/ n2008-4019). the monetary transfer to farmers located in the rz reduces the costs associated with being immobilized and therefore discourages premature sales. the size of the monetary transfer for risk-neutral farmers is given by eq (6) . when the risk of falling into the rz exceeds a threshold, (q>b q), the monetary transfer that should be offered in the case of getting into the rz is positive (it can be shown that the transfer is increasing and concave in q). in eq (6), the first element of the monetary transfer pãw 1 q it à ðv à cþ h i represents the gain from selling prematurely adjusted by the risk of falling into the rz compared with waiting and being unable to sell in the second period. the second element ( 1àq it q it ã ½p ã w 1 ã ð1 þ dþ à c) represents the discount due to the uncertainty of the event. this discount is given by the odds of not getting into the rz in the next period ( 1àq it q it ) that multiplies the gain from selling in the discount goes to zero for farms with a high risk of getting in the rz (q it !1). in this case the government should offer full compensation if the farmer gets in the rz to avoid the anticipation. the rationale for this discount is that the government benefits from the incentives of the seller to wait and sell in the second period (which is the dominant strategy) and therefore does not need to offer a transfer equal to the full cost of immobilization. in the past the rationale for providing financial aid to farmers affected by the mrp was compensatory. when anticipation effects are a risk factor for spreading the disease, giving financial aid to farmers facing movement restrictions becomes an issue of public health and the arguments in favor of this measure are stronger. additional measures that help in the control of infectious diseases can be classified in three categories: measures oriented to reduce the transmission rate such as vaccination or the use of insecticides in the case of vector-borne diseases; measures oriented to increase the detection rate such as the adoption of new technologies (e.g., thermal scanners) and the implementation of preventive protocols; and measures oriented to detect high-risk zones to target surveillance. the third type of measure is possible only with the collection of accurate data and the construction of reliable models. the european effort of constructing national databases to register all the cattle movements is a significant advance and could be extended to other species. network analysis of the spread of disease that incorporates human responses to policy provides new insight into the efficacy of alternative control measures. extensions of this analysis to risk-averse agents and multiple periods should be useful. the current study considers both geographic and commercial networks as potential channels of disease transmission and highlights the relevance of incorporating economic and behavioral elements to evaluate control strategies. for simplicity, we assumed the same transmission probability in both networks. in reality, the transmission probability can differ between transmission paths (geographic or trading) and can change over time. efforts to identify temporal and spatial risk factors of disease spreading associated with the landscape and meteorological conditions should be included in the analysis. the mrp is the main strategy to control the spread of infectious diseases when vaccination is not available (as is the case for newly discovered serotypes of diseases). arguments for implementing mrp are weaker for vector-borne than other diseases because this additional transmission channel reduces the mrp's efficiency. however, it must be taken into consideration that even in the worst-case scenario (vector-borne disease with high rate of subclinical cases) the mrp helps to slow the spreading. this can be very useful in some contexts, such as when the period of inactivity of the vectors (cold months) is approaching, when a vaccine is in the process of development, or even when it is necessary to wait for the vaccines to be dispatched, injected, or boosted (when two injections are needed). for each disease, the costs and the benefits of the mrp should be analyzed in order to take the best possible decision. the strategic behavior of agents can be an important element to consider when analyzing the efficiency of control strategies. the mrp produces incentives that may yield behavioral responses that reduce its effectiveness. we have shown how the strategic behavior of farmers can reduce the efficiency of mrp in controlling the spread of infection. this loss in efficiency should be considered in order to produce more-accurate estimates of the expected benefits of the mrp. the inclusion of complementary measures among emergency protocols related to infectious diseases can increase the efficacy of these policies. by including in the emergency protocols the financial aid for farmers in the rz, farmers know in advance that they will be compensated in case of entering the rz and the anticipatory behavior may be avoided. the monetary transfer required to avoid anticipatory sales by farmers is a function of the subjective probability of being in the rz in the next period (q) for different option values (v = pãw 1 ã(1+d)ã(1-p), where p is the penalty associated with less desirable animals). the model is calibrated using french data for august 30 th , 2007 and estimates for the weight evolution of a charolais calf: p = 2.56 eur/kglwt; w 1 = 350 kg; d = 10/350; c = 8.73 eur. (tif) s1 table. parameters for the economic model. (docx) the effects of the bse outbreak in the united states on the beef and cattle industry emergence of scaling in random networks where diseases and networks collide: lessons to be learnt from a study of the 2001 foot-and-mouth disease epidemic spread of a novel influenza a (h1n1) virus via global airline transportation epidemic spreading in scale-free networks velocity and hierarchical spread of epidemic outbreaks in scale-free networks immunization of complex networks infectious diseases of humans: dynamics and control network robustness and fragility: percolation on random graphs efficient immunization strategies for computer networks and populations identification and registration of animals in the european union risk factors of poultry outbreaks and human cases of h5n1 avian influenza virus infection in west java province, indonesia why did bluetongue spread the way it did? environmental factors influencing the velocity of bluetongue virus serotype 8 epizootic wave in france capturing human behaviour on the existence of a threshold for preventive behavioral responses to suppress epidemic spreading strategic behavior and spread of animal infectious diseases plos one | behavioral incentives, equilibrium endemic disease, and health management policy for farmed animals biosecurity and spread of an infectious animal disease perceptions, circumstances and motivators that influence implementation of zoonotic control programs on cattle farms wet markets-a continuing source of severe acute respiratory syndrome and influenza? the lancet public policies against global warming: a supply side approach. int tax and public finance vulnerability of animal trade networks to the spread of infectious diseases: a methodological approach applied to evaluation and emergency control strategies in cattle network analysis of italian cattle trade patterns and evaluation of risks for potential disease spread available: www.levmuchnick.net/ content/networks/complexnetworkspackage.html modelling bluetongue virus transmission between farms using animal and vector movements did vaccination slow the spread of bluetongue in france? minimization of the impact of aujeszky's disease outbreaks in the netherlands: a conceptual framework cost assessment of the movement restriction policy in france during the 2006 bluetongue virus episode (btv-8) spearman rank correlation forecasting the airborne spread of foot-and-mouth disease the foot-and-mouth epidemic in great britain: pattern of spread and impact of interventions epidemic spreading in a variety of scale free networks conceived and designed the experiments: dt. analyzed the data: dt jkh at dr. wrote the paper: dt jkh at dr. key: cord-000176-z76vjkxg authors: nguyen, jack t.; hoopes, justin d.; le, minh h.; smee, donald f.; patick, amy k.; faix, dennis j.; blair, patrick j.; de jong, menno d.; prichard, mark n.; went, gregory t. title: triple combination of amantadine, ribavirin, and oseltamivir is highly active and synergistic against drug resistant influenza virus strains in vitro date: 2010-02-22 journal: plos one doi: 10.1371/journal.pone.0009332 sha: doc_id: 176 cord_uid: z76vjkxg the rapid emergence and subsequent spread of the novel 2009 influenza a/h1n1 virus (2009 h1n1) has prompted the world health organization to declare the first pandemic of the 21(st) century, highlighting the threat of influenza to public health and healthcare systems. widespread resistance to both classes of influenza antivirals (adamantanes and neuraminidase inhibitors) occurs in both pandemic and seasonal viruses, rendering these drugs to be of marginal utility in the treatment modality. worldwide, virtually all 2009 h1n1 and seasonal h3n2 strains are resistant to the adamantanes (rimantadine and amantadine), and the majority of seasonal h1n1 strains are resistant to oseltamivir, the most widely prescribed neuraminidase inhibitor (nai). to address the need for more effective therapy, we evaluated the in vitro activity of a triple combination antiviral drug (tcad) regimen composed of drugs with different mechanisms of action against drug-resistant seasonal and 2009 h1n1 influenza viruses. amantadine, ribavirin, and oseltamivir, alone and in combination, were tested against amantadineand oseltamivir-resistant influenza a viruses using an in vitro infection model in mdck cells. our data show that the triple combination was highly synergistic against drug-resistant viruses, and the synergy of the triple combination was significantly greater than the synergy of any double combination tested (p<0.05), including the combination of two nais. surprisingly, amantadine and oseltamivir contributed to the antiviral activity of the tcad regimen against amantadineand oseltamivir-resistant viruses, respectively, at concentrations where they had no activity as single agents, and at concentrations that were clinically achievable. our data demonstrate that the tcad regimen composed of amantadine, ribavirin, and oseltamivir is highly synergistic against resistant viruses, including 2009 h1n1. the tcad regimen overcomes baseline drug resistance to both classes of approved influenza antivirals, and thus may represent a highly active antiviral therapy for seasonal and pandemic influenza. globally, influenza viruses cause substantial morbidity and mortality in humans and economic dislocation in afflicted nations. each year in the united states, seasonal influenza virus infection result in an estimated 36,000 deaths and 200,000 hospitalizations [1] . antiviral drugs are an important means to mitigate the impact of the yearly influenza epidemics and potential pandemics. currently, two classes of antiviral drugs have been approved for the prevention and treatment of influenza infection -the m2 channel inhibitors (aminoadamantanes; amantadine and rimantadine) and the neuraminidase inhibitors (nais; oseltamivir and zanamivir). however, the prevalence of drug-resistant strains, which has been reported for both classes of antiviral drugs for seasonal influenza [2, 3] , could undermine their clinical benefit when utilized as monotherapy. indeed, in 2009, the centers for disease control and prevention (cdc) reported that 100% of the seasonal h3n2 virus isolate tested were resistant to the adamantanes, and 99.6% of the seasonal h1n1 viruses tested were resistant to oseltamivir [4] . in april, 2009, a novel h1n1 virus of swine-origin capable of human-to-human transmission likely emerged in mexico and was first isolated from patients enrolled in two separate surveillance activities in southern california [5] . the emergence and spread of 2009 h1n1 to over 168 countries has led u.s. officials [6] and the world health organization (who) [7] to declare a public health emergency; on june 11, 2009 the who raised the influenza pandemic alert from phase 5 to phase 6, the official declaration of a pandemic [8] . early published results from the cdc showed that 2009 h1n1 bears the amantadine-resistance associated s31n mutation in the m2 ion channel, but remains susceptible to oseltamivir and zanamivir [9] . more recently, however, the cdc has reported that ten 2009 h1n1 isolates tested in the united states have been found to be resistant to oseltamivir, raising the concern that dually resistant viruses may become prevalent [4] . in an earlier study, we explored the in vitro antiviral activity and synergy of single, double, and triple combinations of amantadine, ribavirin and oseltamivir against a panel of influenza a viruses that were susceptible to these drugs [10] . our hypothesis was that a triple combination antiviral drug (tcad) regimen composed of drugs with different mechanisms of action, and which act at different stages in the viral life cycle, could result in synergistic antiviral activity. our results showed that these drugs did indeed act synergistically, with the triple combination showing significantly greater synergy than any of the double combinations evaluated. furthermore, we found that the synergy of the tcad regimen was maintained across multiple seasonal and avian influenza a strains, including the three major subtypes -a/h1n1, a/h3n2, and the avian a/h5n1 -that currently cause significant morbidity and mortality in humans. in this study, we sought to evaluate the activity and synergy of the tcad regimen against influenza viruses which were resistant to amantadine or oseltamivir. our data showed that against amantadine-resistant viruses -both seasonal and 2009 h1n1and oseltamivir-resistant seasonal viruses, the tcad regimen was strongly synergistic, and the synergy of the tcad regimen was greater than the synergy of any double combination. surprisingly, we found that amantadine and oseltamivir contributed to the synergy of the tcad regimen at concentrations where they had no activity as single agents, and at concentrations that were clinically achievable. our findings highlight the utility of the tcad regimen as a potential approach to address the current limitations of antiviral potency and drug resistance, and as a viable broad-spectrum therapeutic option for serious influenza virus infection. oseltamivir carboxylate, the active metabolite of oseltamivir, was obtained from charnwood molecular (loughborough, u.k.) through synthesis via the nboc-protected acid from oseltamivir phosphate. amantadine was obtained from moehs catalana (barcelona, spain). ribavirin and rimantadine were purchased from sigma-aldrich (st. louis, mo). peramivir was obtained from jubilant chemsys ltd (uttar pradesh, india) as free base through nboc synthesis, and purity was confirmed using nmr and chiral hplc. zanamivir was obtained from haorui pharma-chem, inc. (edison, nj). cells were passaged in minimal essential medium containing 5% fetal bovine serum (hyclone laboratories, logan, ut). during antiviral evaluations, the serum was removed and the medium was supplemented with gentamicin (50 mg/ml), porcine trypsin (10 units/ml) and edta (1 mg/ml). to obtain single agent concentration-response curves, individual drugs were added to mdck cells in 96-well microplates (8610 4 cells/well) using three wells for each concentration used. the compounds were added at the following concentrations: oseltamivir carboxylate, zanamivir, and peramivir at 0, 0.000032, 0.0001, 0.00032, 0.001, 0.0032, 0.01, 0.032, 0.1, 1.0, 10.0 and 100 mg/ml; amantadine, rimantadine, and ribavirin at 0, 0.001, 0.0032, 0.01, 0.032, 0.1, 0.32, 1, 3.2, 10, 32 and 100 mg/ml. untreated wells of infected cells (virus controls), uninfected cells (cell controls), and drug cytotoxicity controls (cells and drugs only, using the same dilution range for each drug as the test wells) were included on each test plate. at three days after infection, the virus control wells exhibited 100% cytopathology. the 50% effective concentration (ec 50 ) and 50% cytotoxic concentration (tc 50 ) was determined for each drug as outlined below. for double and triple combination studies, each drug was tested in triplicate at five or six concentrations (including no drug), in which the high concentration for each drug was set to approximate the ec 50 of the drug as a single agent. the concentrations of each drug used in double and triple combinations against each virus are provided in table s1 . the cytotoxicity of the double and triple drug combinations were determined using the same experimental format in three separate experiments, and using the same concentration ranges as outlined in table s1 . the extent of viral cytopathology in each well was determined by staining with neutral red (nr) as detailed elsewhere [11] . briefly, the cells were stained with 0.011% nr diluted in mem to determine cell viability. two hours later the plates were processed for quantification of nr uptake into viable cells. the amount of nr taken up by cells was determined spectrophotometrically. ec 50 , tc 50 , and synergy calculations ec 50 , tc 50 , and synergy calculations were done as described previously [10] . briefly, ec 50 and tc 50 calculations for single agents were made by normalizing the nr data for each well against the cell and virus controls, which was assumed to represent 100% cell viability and 0% cell viability, respectively. normalized data were plotted as percent cell viability versus compound concentration. the data points were then fitted using fourparameter curve fitting in graphpad prism (graphpad software, la jolla, ca) to derive the ec 50 and tc 50 . statistical comparisons between best-fit ec 50 values for any two curves were performed in prism using the extra sum-of-squares f test; differences in ec 50 values between two curves with a p-value of ,0.05 were considered significant. synergy was calculated using the macsynergy ii software developed by prichard and shipman, which was modified to accommodate a three-drug combination [12] and is similar to that reported previously describing this approach [13] . the theoretical additive interactions were calculated from the concentrationresponse curves of each drug as a single agent. this calculated additive surface was then subtracted from the observed, experimental surface to reveal regions that deviate from the calculated additive effects. purely additive interactions are represented graphically as areas in grey, indicating that they do not differ from the calculated additive effects. synergistic interactions result in greater inhibition than the calculated inhibition, and are represented as areas in blue. conversely, antagonism is represented as areas in red. synergy plots are shown as the percent inhibition above or below expected (calculated additive inhibition), and are presented as the mean of three replicates at a level of 95% confidence, which eliminates insignificant deviations from the additive surface. synergy volume for each double and triple combination was also calculated, which represents the sum of the synergy or antagonism across all concentrations of a combination. synergy volumes are presented as a quantitative measure of the overall interaction of the drugs within a combination. as determined by cytopathic effect (neutral red assay), synergy volumes .100 mg/ ml 2 % for double combinations or .100 mg/ml 3 % for triple combinations are considered to be strongly synergistic. conversely, combinations with synergy volumes ,2100 mg/ml 2 % or mg/ ml 3 % are considered to be strongly antagonistic. the synergy of the cytotoxic effects of double and triple drug combinations were calculated in a similar manner. between three and nine independent experiments were conducted using identical dosing ranges for amantadine, ribavirin, and oseltamivir carboxylate against each virus. the experiments used a 3-replicate plate format, for a total of 9 to 27 observations for each condition. the data from the independent experiments were merged and subjected to statistical analysis using the random effects model. the raw data were imported into the program r and were normalized to the virus and cell controls as described above, and synergy (percent inhibition above calculated) is calculated as above. for synergy volume estimates, random effects models were applied to the data to account for variation between replicate measures within experiments. therefore, the models took the form as given by: where y ij is the measure of the j th replicate in experiment i; b 0 is the grand mean; u 0i ,n(0,t 2 ); and e ij ,n(0,s 2 ). as the observations in the data are not independent, this model allowed for proper estimation of standard errors. the standard errors were then used to determine the statistical significance of synergy volume. the inhibitory quotient (iq) is defined herein as the ratio of the expected average total (free and protein-bound) plasma concentration (c ave ) of each drug at recommended dosage to the ec 50 (c ave /ec 50 ). thus, an iq of 1 or greater means that the achievable total plasma concentration of the drug is equal to or greater than the in vitro 50% effective concentration, and the higher the iq translates to a greater predicted efficacy. the c ave of each drug was determined by pharmacokinetic modeling using a non-compartmental model in the progam winnonlin. the recommended doses, along with the pharmacokinetic parameters, used for modeling were obtained from the package inserts for amantadine [14] , oseltamivir [15] , and ribavirin [16] , and the references therein. for ribavirin, since the plasma concentration does not achieve steady-state until ,4 weeks after the start of dosing, the c ave was determined for the first 10-day window. based on these parameters, the expected c ave for amantadine was determined to be 0.43 mg/ml based on the recommended dosage of 100 mg twice daily for the treatment of influenza infection; the expected c ave for oseltamivir was determined to be 0.3 mg/ml based on the recommended dosage of 75 mg twice daily for the treatment of influenza infection; and the expected c ave of for ribavirin was determined to be 1.3 mg/ml after 10 days of treatment based on the recommended dosage of 600 mg twice daily for the treatment of hepatitis c infection. to determine the iq of the triple combination, amantadine, ribavirin, and oseltamivir carboxylate were tested as a fixed ratio combination, wherein the ratio of the three drugs was kept constant even as the total concentration of drugs varied. the ratio of drugs in the tcad regimen was based on the expected c ave of each drug. a dilution curve of tcad regimen was created by first preparing a solution of all three drugs at 100-fold the c ave of each drug (43 mg/ ml amantadine, 30 mg/ml oseltamivir carboxylate, and 130 mg/ ml ribavirin), and then serially diluting this solution in 0.5-log 10 increments. in this manner, the ec 50 of tcad regimen as a fixed dose combination was determined as ratio of the c ave and expressed in units of fold-c ave . the susceptibility of three 2009 h1n1 influenza strains -a/ california/04/09 (ca04), a/california/05/09 (ca05) and a/ california/10/09 (ca10) -to each of six antiviral drugs (amantadine, rimantadine, oseltamivir carboxylate, zanamivir, peramivir, and ribavirin) as single agents was determined by measuring the inhibition of virus-induced cpe in mdck cells. against the three strains, oseltamivir carboxylate, zanamivir, peramivir, and ribavirin produced concentration-dependent inhibition of cytopathic effect (data not shown). amantadine was active only at higher concentrations (ec 50 of 16-20 mg/ml; 85-106 mm) (table s2) , which represents a .250-fold reduction in susceptibility compared to the published values for a wild-type virus [17] . rimantadine did not produce inhibition up to the 50% cytotoxic concentration (ec 50 .12 mg/ml; .55 mm). the ec 50 values for the six drugs as single agents against all three strains are summarized in table s2 , and confirm that the three virus strains remain susceptible to oseltamivir carboxylate, zanamivir, peramivir, and ribavirin. these results are consistent with the results previously published that demonstrated that 2009 h1n1 contained a mutation (s31n) in the m2 channel that has been associated with resistance to adamantanes, but remained susceptible to nais [18] . we next assessed the synergistic activity of double and triple combinations of amantadine, ribavirin, and oseltamivir carboxylate over a range of concentrations of each drug against each 2009 h1n1 isolate. a quantitative measure of the total synergy (or antagonism) of a drug combination can be expressed in terms of synergy volumes, which represents the cumulative synergy and antagonism across all concentrations for all the drugs in a combination. based on the empirically determined criterion of synergy volume .100 mg/ml 2 % using the lower confidence interval, all three double combinations were additive against the 2009 h1n1viruses (table 1) . by contrast, the tcad regimen was synergistic against all three viruses over multiple concentrations of all three drugs, with the synergy occurring at 0.1 mg/ml and above for amantadine, 0.32 mg/ml and above for ribavirin, and 0.0032 mg/ml and above for oseltamivir carboxylate (table 1) . these data show that the synergy of the tcad regimen occurred at clinically achievable concentrations for all three drugs, given that the expected average plasma concentrations based on the recommended doses are 0.43 mg/ml for amantadine, 1.3 mg/ml for ribavirin, and 0.3 mg/ml for oseltamivir carboxylate. furthermore, the synergy of the tcad regimen was greater than the synergy of any double combination tested for all three 2009 h1n1 strains. figure 1b -1d shows the synergy of the tcad regiment as a function of increasing concentrations of amantadine, ribavirin, or oseltamivir carboxylate as the third drug, representing the contribution of each drug to the synergy of the double combination without the third drug. these data reveal that the addition of each drug as the third drug to the double combinations resulted in a concentration-dependent increase in synergy, indicative that each drug contributed to the synergy of the tcad regimen. importantly, despite the fact that amantadine had no significant antiviral activity as a single agent below 3.2 mg/ml (data not shown), we find that the amantadine contributed to the activity of the tcad regimen at clinically achievable concentrations (0.43 mg/ml). statistical analysis of the variability across all replicates from the six experiments for each virus revealed that amantadine made a significant contribution to the synergy of the tcad regimen at concentrations 0.1 mg/ml and 0.32 mg/ml and above against ca05 and ca10, respectively, compared to the double combination of ribavirin/oseltamivir carboxylate without amantadine ( figure 1b ). for ca04, while only the 3.2 mg/ml amantadine concentration had statistically significant greater synergy volume than the double combination without amantadine, there was a trend toward increasing synergy volume starting at 0.32 mg/ml. thus, amantadine contributed to the activity of the tcad regimen at concentrations where it was inactive as a single agent, and at concentrations that were clinically achievable. synergy plots, which reveal the extent of synergy at each concentration of each drug in the combinations, are shown in figures s1 for ca05. the data are presented as contour plots, in which regions where inhibition is greater (synergy) or less (antagonism) than expected are identified by subtracting the theoretical additive inhibition from the observed inhibition. synergy plots for tcad regimen showed a concentrationdependent increase in synergy with respect to amantadine ( figure s1b , top plane). at the highest concentration tested (3.2 mg/ml), synergy was observed over a wide range of concentrations of ribavirin and oseltamivir carboxylate tested. at lower concentrations of amantadine, synergy occurred at 1 mg/ml ribavirin and higher, and at 0.0032 mg/ml oseltamivir carboxylate and higher. no significant antagonism was observed at any dose of any drug in the double combinations or the tcad regimen. similar patterns of synergy were observed with double and triple combinations of these antiviral drugs against ca04 and ca10 (data not shown). one notable consequence of synergy is that the antiviral activities of each drug in the combination is enhanced compared to the activities of the drugs as single agents. to demonstrate this, we compared the antiviral activity of each of the three drugsamantadine, ribavirin, and oseltamivir carboxylate -as single agents and in the presence of fixed concentrations of the second and third drugs against ca10 replication. for each drug, the ec 50 was reduced in triple combination compared to the ec 50 as a single agent, indicative that each drug was active at lower concentrations (greater potency). for example, the ec 50 for amantadine as a single agent was reduced by 3.2-fold in combination with 1 mg/ml ribavirin and 0.0032 mg/ml oseltamivir carboxylate; the ec 50 for ribavirin as a single agent was reduced by 2.7-fold in combination with 0.0032 mg/ml oseltamivir carboxylate and 1 mg/ml amantadine; and the ec 50 for oseltamivir carboxylate as a single agent was reduced 16.2-fold in combination with 1 mg/ml ribavirin and 1 mg/ml amantadine ( table 2) . for all three drugs, the reduction in ec 50 values observed in the triple combination compared to the single agent was statistically significant (p,0.05), indicative that each drug had greater antiviral potency and was effective at lower concentrations. in addition, the ec 50 of all three drugs in triple combination were reduced 1.5-to 6.5-fold compared to the ec 50 in double combinations, indicative of that the antiviral activity of the drugs in triple combination were enhanced compared to double combinations. importantly, the data presented here do not represent the maximum reductions in ec 50 values for the three drugs. due to the dynamic range of the assay, we were only able to obtain precise dose-response curves for each drug at fixed concentrations of the second and third drugs which were well below their ec 50 values and well below concentrations where maximum synergy occurred. at higher concentrations, the antiviral activity of the second and third drug contributed significantly to the inhibition, which decreased the linear range of the assay and reduced the accuracy of the curve-fitting (data not shown). a comprehensive assessment of the interaction of two or three drugs in combination requires the evaluation of multiple concentrations of each drug in order to quantify synergy over the entire dosing range, as was done in the section above. we also evaluated the interactions of double combinations of neuraminidase inhibitors (nais) against 2009 h1n1. as shown in table 1 , the synergy volumes for the zanamivir/oseltamivir carboxylate were 2246116 mg/ml 2 %, 2155689 mg/ml 2 %, and 2197698 mg/ml 2 % against ca04, ca05, and ca10, respectively. these values suggest that the zanamivir/oseltamivir carboxylate combination was additive against these viruses. for the zanamivir/peramivir combination, synergy volumes were 2356112 mg/ml 2 %, 21976108 mg/ml 2 % and 2239693 mg/ ml 2 % against ca04, ca05, and ca10, respectively, indicative of additivity to moderate antagonism. consistent with these values, the synergy plots for the nai double combinations against ca05 revealed regions of antagonism (red areas), which occurred at higher concentrations of zanamivir (0.01-0.1 mg/ml) and at variable concentrations of the second nai (oseltamivir or peramivir, figure s2 ). similar results were found with the other two 2009 h1n1 strains. furthermore, evaluation of the ec 50 of each nai in combination with a fixed concentration of a second nai revealed that the antiviral activity of each drug was not enhanced in combination with a second drug (data not shown). the observation that double combinations of neuraminidase inhibitors were not synergistic is consistent with the fact that all three drugs are known to target the same enzyme, and all bind in the same substrate binding pocket in a similar manner [19] . as an extension of these studies we assessed the contribution of amantadine to the synergy of the tcad regimen against . as a single agent, amantadine had no activity against these viruses at concentrations up to the highest concentration tested (32 mg/ml; data not shown). as shown in figure 2 , against nc v27a and wi s31n, amantadine contributed to the synergy of the tcad regimen in a concentration-dependent manner. the synergy of the tcad regimen in the presence of amantadine was greater than the synergy of the ribavirin/oseltamivir carboxylate double combination without amantadine, and the increase in the synergy of the tcad regimen was observed at 0.32 mg/ml amantadine for nc v27a and 1 mg/ml amantadine for wi s31n, when significance was evaluated at the level of ,0.05. against dk a30t, however, amantadine did not make a contribution to the synergy of the tcad regimen within the concentration range tested (0.1-3.2 mg/ml), and the synergy of the tcad regimen was not greater than the synergy of the ribavirin/oseltamivir carboxylate double combination. whether the lack of contribution from amantadine against the dk a30t virus was due to the specific subtype (h5n1), the m2 mutation (a30t), or the combination of both remain to be determined. however, it should be noted that while amantadine made no contribution to the activity of the tcad regimen against dk a30t, both ribavirin and oseltamivir remain active and their interactions were additive, indicative that two out of three drugs contribute to the activity of the tcad regimen against this virus (data not shown). given that a large percentage of seasonal influenza circulating viruses are resistant to oseltamivir, we next assessed the activity and synergy of the tcad regimen against oseltamivir-resistant viruses to evaluate the spectrum of activity of the tcad regimen, and to determine whether oseltamivir contributed to the activity of the tcad regimen against oseltamivir-resistant viruses. two h1n1 oseltamivir-resistant viruses were used, both of which bear the h274y substitution in na which has been demonstrated to confer resistance to oseltamivir [20] : a/mississippi/3/01 (ms h274y) and a/hawaii/21/07 (hi h274y). as a single agent, oseltamivir carboxylate had no antiviral activity against either virus below 1 mg/ml (data not shown). the synergy volumes of double and triple combinations of amantadine, ribavirin, and oseltamivir were determined against both viruses, and the data are presented in figure 3 . as double combinations, amantadine/ oseltamivir carboxylate, amantadine/ribavirin, and ribavirin/ oseltamivir carboxylate were all additive ( figure 3a) . as was seen with the 2009 h1n1 viruses, the tcad regimen was synergistic, and the synergy volume was greater than the synergy volume of any double combination, with each drug making a contribution to the synergy of the tcad regimen against the oseltamivir-resistant viruses ( figure 3b-d) . importantly, oseltamivir carboxylate contributed to the synergy of the tcad regimen starting at 0.1 mg/ml against ms h274y (p,0.05) and at 0.32 mg/ml against hi h274y (p,0.01). at these concentrations, which are achievable clinically, oseltamivir carboxylate is not active as a single agent against these resistant strains. synergy plots for the tcad regimen against ms h274y and hi h274y are provided in figure s3 , and reveal increasing synergy with increasing concentrations of oseltamivir carboxylate. at the highest concentration of oseltamivir carboxylate tested (3.2 mg/ml), synergy occurred over wide concentrations of ribavirin and amantadine. at lower concentrations of oseltamivir carboxylate, synergy occurred at higher concentrations of amantadine (0.1 mg/ml or higher), and at wide concentrations of ribavirin. no significant antagonism was detected at any concentrations of the three drugs. we also determined the ec 50 of oseltamivir carboxylate as a single agent and in double and triple combinations against both oseltamivir-resistant viruses. as summarized in table 3 , the ec 50 of oseltamivir carboxylate as a single agent was 74 mg/ml against ms h274y and 15 mg/ml against hi h274y. these represent 1480-and 300-fold reductions in susceptibility compared to the published values for a wild-type virus [11] . the ec 50 of oseltamivir carboxylate was not reduced in double combination with 1mg/ml ribavirin or 0.032 mg/ml amantadine against ms h274y, and was reduced by only 1.7-and 1.4-fold, respectively, against hi h274y. however, in triple combination with ribavirin and amantadine at the same concentrations used in double combination, the ec 50 of oseltamivir carboxylate was reduced by 21-fold against ms h274y and by 5.8-fold against hi h274y. the tc 50 of amantadine as a single agent was 37-40 mg/ml, whereas the tc 50 of ribavirin and oseltamivir carboxylate as single agents were .100 mg/ml (table s2) . these values are more than 10-fold higher than the highest concentration of each drug used in the combination experiments (table s1 ). furthermore, synergy analysis of the double and triple combinations revealed no synergistic cytotoxicity for any double combination or the tcad regimen within the concentration ranges tested (data not shown). for example, cells treated with the tcad regimen at the highest concentrations tested for all three drugs used in the combination experiments (3.2 mg/ml amantadine, 10 mg/ml ribavirin, and 3.2 mg/ml oseltamivir carboxylate) exhibited 97% viability, which was not statistically different than the cell control (p = 0.47 as determined by student's t-test, figure s4 ). one indicator of the expected clinical antiviral activity is the inhibitory quotient (iq), defined herein as the ratio between the average total plasma concentration (c ave ) and the 50% effective inhibitory concentration (ec 50 ). in order to predict the effectiveness of the tcad regimen against the circulating influenza strains in the clinical setting, we calculated and compared the iqs for amantadine, ribavirin, and oseltamivir carboxylate as single agents and the tcad regimen against susceptible and resistant influenza viral strains (including 2009 h1n1). to determine the iq of the tcad regimen, amantadine, ribavirin, and oseltamivir carboxylate was tested as a fixed ratio combination, wherein the ratio of the three drugs was kept constant even as the total concentration of drugs varied. a dilution curve of the tcad regimen was created by first preparing a solution of all three drugs at 100-fold the c ave of each drug (43 mg/ml amantadine, 130 mg/ml ribavirin, 30 mg/ml oseltamivir carboxylate), and then serially diluting this solution in half-log 10 increments. in this manner, the ec 50 of the tcad regimen was determined as a ratio of the c ave and expressed in units of fold change from c ave . as representatives of the current circulating strains, we used a/ new caledonia/20/99 (h1n1) (nc20) as the susceptible virus (susceptible to amantadine, ribavirin, and oseltamivir carboxylate), ca10 as the amantadine-resistant virus, and ms h274y as the oseltamivir-resistant virus. concentration-response curves for amantadine, ribavirin, oseltamivir carboxylate, and tcad were generated against all three viruses, and the ec 50 s and iqs are summarized in table 4 . amantadine was effective at inhibiting nc20 and ms h274y in vitro, resulting in iqs of 1.95 and 7.17, respectively. however, the iq for amantadine was reduced to 0.02 when tested against ca10, indicative that an in vitro concentration representing the achievable plasma concentration at the recommended dose was not adequate to inhibit the amantadineresistant virus in vitro. an iq of 0.02 represents a 100-fold reduction compared to nc20 and a 350-fold reduction compared to ms h274y. similarly, oseltamivir carboxylate was effective at inhibiting nc20 and ca10 in vitro resulting in iqs of 1.5 and 5.0, respectively, but not ms h274y (iq = 0.004). similar to amantadine against the amantadine-resistant virus, the iq for oseltamivir was reduced 350-to 2300-fold against the oseltamivirresistant virus compared to susceptible viruses. the iqs for ribavirin were uniformly low and below 1 for all three viruses (0.23 to 0.42). on the other hand, the iqs for the tcad regimen as a fixed dose combination were consistently high against all three viruses (8.33 to 17.24), varying by no more than 2-fold between susceptible and resistant viruses. these data suggest that the tcad regimen may have broad utility against all circulating influenza strains, including strains that are resistant to either amantadine or oseltamivir. given that virtually all seasonal h3n2 and 2009 h1n1 strains are resistant to amantadine, and virtually all currently circulating seasonal h1n1 strains are resistant to oseltamivir, the pharmacologic rationale for the development of a triple combination antiviral drug (tcad) composed of amantadine, ribavirin, oseltamivir is that at least two, and possibly three drugs, in the tcad regimen will be active against all of these viruses. a number of studies have evaluated double combinations of antivirals [21] [22] [23] [24] [25] [26] [27] against influenza a infection in vitro, and hayden et al. have tested a triple combination of two antivirals with human interferon a [28] . however, there have been few reports on the effects of drug combinations on resistant influenza viruses [26, 27, 29] . recently, smee et al. evaluated the effects of double combinations of amantadine, ribavirin, and oseltamivir against the same amantadine-resistant h5n1 virus used in this study (a/duck/mn/1525/81) [26] . the authors found that the presence of amantadine in double combinations did not provide added benefit over the second drug alone, either in cell culture or in mouse models. in the present study, we examined the efficacy and synergy of the tcad regimen against viruses which were resistant to oseltamivir or amantadine, including 2009 h1n1. consistent with the previous findings [9] , we found that the 2009 h1n1 strains were susceptible to nais (oseltamivir carboxylate, zanamivir, and peramivir) and ribavirin, but were resistant to adamantanes (amantadine and rimantadine). surprisingly, we found that amantadine as a single agent retained partial activity against these viruses (table s2) , albeit the activity was reduced by 100-fold compared to a susceptible virus, whereas rimantadine had no activity below the 50% cytotoxic concentration. this observation suggests that phenotypic testing, in addition to determination of the genotype, may be necessary in order to fully understand the susceptibility profile of a novel virus and may have important implications in guiding the choice of antivirals for use in combinations. against the 2009 h1n1 strains, the interactions of oseltamivir carboxylate, peramivir, and zanamivir in double combinations ranged from additive to moderately antagonistic, indicative that the activity of these drugs was not enhanced in combination compared to their activity as single agents. these results suggest that double combinations of nais may not provide any added benefit over the drugs as single agents. given that all nais bind in the same substrate binding pocket in na, the use of these drugs in combination in the absence of enhanced activity raises the risk of selecting for a single mutation that could confer resistance to both neuraminidase inhibitors simultaneously. indeed, cross-resistance to oseltamivir and zanamivir resulting from a single amino acid change has been documented for seasonal influenza a and b viruses [30] . if this were to occur in the 2009 h1n1 background, the resulting virus would be resistant to all approved anti-influenza drugs. in total, we tested the activity and synergy of the tcad regimen against six amantadine-resistant viruses, including three strains of 2009 h1n1, and two oseltamivir-resistant viruses. the viruses tested in this study come from the three subtypes that cause significant morbidity and mortality in humans (h1n1, h3n2, and h5n1), and include seasonal, avian, and pandemic strains. the double combinations of amantadine/oseltamivir carboxylate, amantadine/ribavirin, and ribavirin/oseltamivir carboxylate were additive against 2009 h1n1, and ranged from additive to moderately synergistic against the other viruses (data not shown). in contrast, with the exception of the duck h5n1 virus, we found that the tcad regimen was synergistic at clinically achievable concentrations of all three drugs, and that the synergy of the tcad regimen was greater than that of any double antiviral drug combination. these data suggest that the tcad regimen may have broad-spectrum antiviral activity against circulating influenza a viruses, including strains that are resistant to either classes of antivirals. to date, most influenza a strains in circulation (,99%) are resistant to either the adamantanes or oseltamivir, and not to both [31] , and thus are expected to be susceptible to the tcad regimen. currently, rapid diagnostic tests are not available to determine the susceptibility profile of influenza viruses in real time, and thus clinicians do not often have the necessary information with which to guide appropriate antiviral use. the availability of a broad-spectrum antiviral therapy that would be effective against the majority of circulating strains regardless of the susceptibility would be of high clinical utility. importantly, we found that amantadine and oseltamivir contributed to the synergy of the tcad regimen against amantadine-resistant and oseltamivir-resistant viruses. the contributions from both drugs to the synergy of the tcad regimen were significant at clinically achievable concentrations where they had little or no antiviral activity as a single agent. for instance, a comparison of the synergy volume of the tcad regimen at 0.32 mg/ml amantadine to the synergy volume of the ribavirin/ oseltamivir carboxylate double combination (no amantadine) revealed that amantadine contributed 39%, 24%, and 44% to the total synergy of the tcad regimen against ca04, ca05, and ca10, respectively. similarly, against the oseltamivir-resistant viruses, oseltamivir carboxylate at 0.32 mg/ml contributed 76% and 83% to the total synergy of the tcad regimen against ms h274y and hi h274y, respectively. thus, all three drugs contributed to the synergy and activity of the tcad regimen against amantadine-and oseltamivir-resistant viruses, and the activities of amantadine and oseltamivir were restored in the context of the tcad regimen against influenza strains that were resistant to these drugs, thereby maximizing the clinical utility of these drugs. the mechanism(s) by which amantadine and oseltamivir carboxylate contribute to the synergy of the tcad regimen against resistant strains is unclear. the interactions between m2, ha, and na on the surface of the influenza particle are complex and not well understood, and a number of studies have demonstrated that ha-m2 and ha-na interactions can affect the susceptibility to amantadine and oseltamivir, respectively [32, 33] . furthermore, amantadine has been demonstrated to exert antiviral activity via interactions with ha at higher concentrations [34, 35] . it is conceivable that, as the result of protein-protein interactions between m2, ha, and na, the binding of a drug at one site may affect the conformation and therefore affinity for another drug at another site. the mechanism by which ribavirin contributes to the synergy of the tcad regimen is also unclear. ribavirin has been documented to act through multiple mechanisms affecting both virus replication and host immune response [36] [37] [38] , and it remains to be elucidated which of these mechanisms are responsible for the synergy with amantadine and oseltamivir. finally, we evaluated the activity and inhibitory quotient (iq) of tcad against susceptible and resistant viruses representing the currently circulating strains. while the correlation between iq and clinical efficacy has not been demonstrated for influenza, it is valuable to construct a relative ranking of the iq of different antiviral regimens against susceptible and resistant viruses in order to assess the spectrum of their activity. when tested against a seasonal susceptible h1n1 virus, an amantadine-resistant 2009 h1n1, and a seasonal oseltamivir-resistant h1n1 virus, tcad was uniformly active against all three viruses with significantly high iqs (8.33 to 17.24; table 4 ). this suggests that tcad may have broad antiviral activity against all currently circulating influenza strains and may have good efficacy in the clinical setting against these strains. our data suggests that a triple combination antiviral drug (tcad) composed of amantadine, ribavirin, and oseltamivir may be an effective and viable therapeutic option for the treatment of pandemic and seasonal influenza infection. the body of data presented in this report validates the tcad hypothesis, which states that for any given susceptible or resistant circulating influenza virus, at least two, and in some cases all three, drugs in tcad will be active. furthermore, the tcad regimen appears to overcome baseline drug resistance and thus may represent a highly active antiviral therapy for seasonal and pandemic influenza. the safety, pharmacokinetics, distribution, and metabolism of amantadine, ribavirin, and oseltamivir as single agents are well understood, and it is not expected that co-administration of the three drugs will result in substantially increased risk to patients compared to the administration of the individual drugs. in addition, all three double combinations have been tested in humans without adverse effects, including amantadine plus oseltamivir [39] , amantadine plus ribavirin [40] , and ribavirin plus oseltamivir [41] . clinical trials to assess the efficacy and safety of tcad for the treatment of influenza have been initiated, and will provide important data on the use of tcad against both pandemic and seasonal influenza. table s1 concentration ranges (mg/ml) of each drug tested in double and triple combinations against different influenza a viruses. drugs were titrated at half log 10 figure s1 synergy plot of double and triple combinations of amantadine, ribavirin, and oseltamivir carboxylate against 2009 h1n1 a/california/05/09 (ca05) replication as determined by neutral red assay in mdck cells. calculated additive interactions were subtracted from the experimentally determined inhibition to reveal regions of synergy (inhibition above expected) or antagonism (inhibition below expected). values were derived from mean triplicate data and presented at 95% confidence. this experiment was repeated a total of six times with similar results. blue areas indicate concentrations of each drug that are synergistic, gray areas indicate concentrations that are additive, and red areas indicate concentrations that are antagonistic. the intensity of the color (blue or red) corresponds to percent inhibition above or below expected. figure s4 viability of mdck cells treated with the tcad regimen. mdck cells were incubated with the tcad regimen at the highest concentrations of all three drugs used in the synergy experiments (3.2 mg/ml amantadine, 10 mg/ml ribavirin, and 3.2 mg/ml oseltamivir carboxylate), and cell viability was determined by neutral red assay after 72 hours. values are the mean of nine replicates from three experiments, with standard deviations. the difference in viability between the tcad treated cells and the cell controls was not statistically significant (p = 0.47, student's t-test). found at: doi:10.1371/journal.pone.0009332.s006 (1.00 mb tif) the impact of influenza epidemics on mortality: introducing a severity index surveillance of resistance to adamantanes among influenza a(h3n2) and a(h1n1) viruses isolated worldwide surveillance for neuraminidase inhibitor resistance among human influenza a and b viruses circulating worldwide from swine influenza a (h1n1) infection in two children-southern california acting secretary of the u.s. department of health and human services (2009) determination that a public health emergency exists director-general of the world health organization (2009) swine influenza world now at the start of 2009 influenza pandemic update: drug susceptibility of swine-origin influenza a (h1n1) viruses triple combination of oseltamivir, amantadine, and ribavirin displays synergistic activity against multiple influenza virus strains in vitro cyclopentane neuraminidase inhibitors with potent in vitro anti-influenza virus activities strategic design and threedimensional analysis of antiviral drug combinations a three-dimensional model to analyze drug-drug interactions schering corporation generation and characterization of recombinant influenza a (h1n1) viruses harboring amantadine resistance mutations update: drug susceptibility of swine-origin influenza a (h1n1) viruses current and future antiviral therapy of severe seasonal and avian influenza oseltamivir resistance-disabling our influenza defenses neuraminidase inhibitorrimantadine combinations exert additive and synergistic anti-influenza virus effects in mdck cells enhancement of activity against influenza viruses by combinations of antiviral agents combination chemotherapy, a potential strategy for reducing the emergence of drug-resistant influenza a variants in vitro inhibitory effects of combinations of anti-influenza agents combination treatment of influenza a virus infections in cell culture and in mice with the cyclopentane neuraminidase inhibitor rwj-270201 and ribavirin effects of double combinations of amantadine, oseltamivir, and ribavirin on influenza a (h5n1) virus infections in cell culture and in mice low dose oral combination chemoprophylaxis with oseltamivir and amantadine for influenza a virus infections in mice combined interferonalpha 2, rimantadine hydrochloride, and ribavirin inhibition of influenza virus replication in vitro oseltamivir-ribavirin combination therapy for highly pathogenic h5n1 influenza virus infection in mice susceptibilities of antiviralresistant influenza viruses to novel neuraminidase inhibitors fluview: 2009-2010 influenza season: week 50 ending contribution of h7 haemagglutinin to amantadine resistance and infectivity of influenza virus overexpression of the alpha-2,6-sialyltransferase in mdck cells increases influenza virus sensitivity to neuraminidase inhibitors fusion mutants of the influenza virus hemagglutinin glycoprotein amantadine selection of a mutant influenza virus containing an acid-stable hemagglutinin glycoprotein: evidence for virus-specific regulation of the ph of glycoprotein transport vesicles mechanism and specificity of action of ribavirin the mechanism of action of ribavirin: lethal mutagenesis of rna virus genomes mediated by the viral rna-dependent rna polymerase the broadspectrum antiviral ribonucleoside ribavirin is an rna virus mutagen a randomized, crossover study to evaluate the pharmacokinetics of amantadine and oseltamivir administered alone and in combination effects of alpha interferon induction plus ribavirin with or without amantadine in the treatment of interferon non-responsive chronic hepatitis c: a randomised trial identification of severe acute respiratory syndrome in canada we thank brett hurst, benjamin christensen, and christopher myers for technical support, kristin porter for statistical analysis, and linda wuestehube for editorial assistance. key: cord-000013-pr9i9swk authors: croyle, maria a.; patel, ami; tran, kaylie n.; gray, michael; zhang, yi; strong, james e.; feldmann, heinz; kobinger, gary p. title: nasal delivery of an adenovirus-based vaccine bypasses pre-existing immunity to the vaccine carrier and improves the immune response in mice date: 2008-10-29 journal: plos one doi: 10.1371/journal.pone.0003548 sha: doc_id: 13 cord_uid: pr9i9swk pre-existing immunity to human adenovirus serotype 5 (ad5) is common in the general population. bypassing pre-existing immunity could maximize ad5 vaccine efficacy. vaccination by the intramuscular (i.m.), nasal (i.n.) or oral (p.o.) route with ad5 expressing ebola zaire glycoprotein (ad5-zgp) fully protected naïve mice against lethal challenge with ebola. in the presence of pre-existing immunity, only mice vaccinated i.n. survived. the frequency of ifn-γ+ cd8+ t cells was reduced by 80% and by 15% in animals vaccinated by the i.m. and p.o. routes respectively. neutralizing antibodies could not be detected in serum from either treatment group. pre-existing immunity did not compromise the frequency of ifn-γ+ cd8+ t cells (3.9±1% naïve vs. 3.6±1% pre-existing immunity, pei) nor anti-ebola neutralizing antibody (nab, 40±10 reciprocal dilution, both groups). the number of inf-γ+ cd8+ cells detected in bronchioalveolar lavage fluid (bal) after i.n. immunization was not compromised by pre-existing immunity to ad5 (146±14, naïve vs. 120±16 sfc/million mncs, pei). however, pre-existing immunity reduced nab levels in bal by ∼25% in this group. to improve the immune response after oral vaccination, the ad5-based vaccine was pegylated. mice given the modified vaccine did not survive challenge and had reduced levels of ifn-γ+ cd8+ t cells 10 days after administration (0.3±0.3% peg vs. 1.7±0.5% unmodified). pegylation did increase nab levels 2-fold. these results provide some insight about the degree of t and b cell mediated immunity necessary for protection against ebola virus and suggest that modification of the virus capsid can influence the type of immune response elicited by an ad5-based vaccine. the ability of human adenoviruses to induce strong innate and adaptive immune responses makes them powerful adjuvants that facilitate the immune response against an encoded antigen. recombinant adenoviruses have been shown to elicit significant immune responses to bacterial (anthrax, plague), viral (hepatitis c, rabies, sars) and tumour-associated antigens [1] [2] [3] . while these results are encouraging, immunity eventually develops against virus capsid proteins. this severely reduces the immunogenicity of adenovirus-based vaccines in mice, [4] [5] [6] [7] [8] , primates [9] and humans [10] . this problem is also significant since a large portion of the western world has marked levels of anti-adenovirus serotype 5 (ad5) antibodies and is also prominent in regions of sub-saharan africa and southeast asia, where many of these vaccines are needed [11, 12] . thus, assessment of the impact of pre-existing immunity on immune protection and alternative vaccination strategies may be needed for successful use of many adenovirusbased vaccines. several strategies have been developed to address the prevalence of pre-existing immunity to ad5 in the general population. increasing the vector dose or adopting a prime-boost regimen in order to overcome pre-existing immunity to the virus is a common approach [2] . there is mixed enthusiasm for this plan, however, due to the documented toxicity associated with high doses of adenovirus and the length of time required for primeboost regimens when only a prime could be sufficient [13, 14] . 'seroswitching', using recombinant adenoviruses constructed from chimpanzees or rare human serotypes with limited exposure rates such as adenoviruses 35 and 11 can elicit potent immune responses that are minimally affected by pre-existing immunity [7, 8, [15] [16] [17] [18] [19] [20] [21] . hexon-chimeric adenoviruses can also avoid neutralization [22, 23] . both approaches offer promise in the context of addressing pre-existing immunity, but require further investigation in response to concerns regarding safety and feasibility of largescale production. covalent attachment of polyethylene glycol or incorporation of the virus into polymer matricies can also effectively protect ad5 from neutralization [24] [25] [26] [27] [28] [29] [30] [31] . delivery of ad5-based vaccines by mucosal routes can also circumvent the effect of pre-existing immunity and induce a significant immune response against an encoded antigen [32] . recombinant adenoviruses are one of the few well-studied vectors currently under development for vaccination against ebola virus infection. the first protocol for an ebola vaccine employed a prime-boost regimen consisting of naked dna expressing either ebola glycoprotein (gp) or nucleoprotein (np) and recombinant ad5 expressing ebola gp to successfully protect non-human primates against a lethal challenge of ebola [33] . this has since led to several phase i clinical trials [34, 35] in which each component of the vaccine is administered by intramuscular injection. to date, there have been only two reports describing mucosal administration of an ebola vaccine [36, 37] . nasal administration of recombinant human parainfluenza virus type 3 (hpiv3) vectors expressing ebola gp and/or np to guinea pigs and rhesus monkeys conferred complete protection against a lethal challenge with ebola. we have previously found that a single dose of a recombinant adenovirus expressing ebola zaire gp given by either the oral or the nasal route is capable of affording protection against lethal challenge in naïve mice and that mucosal immunization can stimulate a broad, prolonged t cell-mediated immune response in both the systemic and mucosal compartments [37] . the primary objective of this study was to test the hypothesis that administration of an ad5-based ebola vaccine by either the nasal or oral route can circumvent pre-existing immunity and confer full protection upon challenge. systemic and mucosal t and b cell responses to ebola gp were assessed in both naïve mice and those with pre-existing immunity. the influence of pegylation of the vaccine carrier on the immune response after oral immunization is also described. the e1/e3-deleted adenovirus vector expressing the ebola zaire glycoprotein was created by cloning the open reading frame (orf) sequence of the glycoprotein in the plasmid pshuttle (adeno-x expression system i, bd clonetech, palo alto, ca) for subsequent insertion in the e1 region of the human adenovirus serotype 5 genome. the human cytomegalovirus (cmv) promoter included in the adeno-x expression system was used to drive the expression of the ebola zaire glycoprotein in the final recombinant adenovirus serotype 5 construct. authenticity of the final product was confirmed by sequencing of the recombinant virus rescued by transfecting the linearized dna into 293 cells. virus was sequentially amplified to large-scale infections (5610 8 cells) and purified on an affinity column (adeno-x virus purification mega kit, bd clonetech, mountain view, ca) according to the manufacturer's instructions. genome structures of vectors were analyzed by restriction digestion of isolated viral dna and compared with those of the original molecular clones. particle number and infectivity of vectors were determined by standard optical density reading and immunodetection of the hexon protein, respectively, following infection of 293 cells with limiting dilutions of each vector preparation according to the recommendations by the manufacturer (adeno-x rapid titer kit, clontech, mountain view, ca). purified virus was administered in sterile phosphate buffered saline (ph 7.4) and had particle to plaque forming unit (pfu) ratios of 100:1 or less. first generation recombinant adenovirus expressing ebola zaire glycoprotein was prepared and purified as described above. the protein content of the virus preparation was determined using biorad dc protein assay reagents (biorad, hercules, ca) and bovine serum albumin as a standard in a microplate format. according to established protocols, 10 mg of monomethoxypoly(ethylene) glycol, activated by tresyl chloride (sigma aldrich, st. louis, mo), was added for each microgram of protein present [38] . the coupling reaction was performed at 25uc with gentle agitation. the reaction was stopped by the addition of l-lysine, in a 10-fold excess with respect to the amount of peg added. unreacted peg, excess l-lysine, and reaction byproducts were removed by buffer exchange over a second econo-pac 10dg disposable chromatography column equilibrated with 100 mm potassium phosphate-buffered saline (ph 7.4). pegylated preparations were administered in sterile potassium phosphate buffered saline (ph 7.4) and had particle to plaque forming unit (pfu) ratios of 100:1 or less as determined by the adeno-x rapid titer kit. characterization of these preparations revealed significant changes in biophysical properties of the virus once the reaction was complete such as the peg-dextran partition coefficient and peak elution times during capillary electrophoresis (as described previously [30, 38] ). approximately 13,000 peg molecules were associated with each virus particle in the studies outlined here as determined by a peg-biotin assay [30] . b10.br mice were immunized with 1610 10 particles of recombinant virus per mouse either by intramuscular injection (50 ml) in the right hindlimb, or by oral gavage (100 ml) using oral feeding needles (18g, 2.25 mm dia., popper & sons, inc, new hyde park, ny). for nasal immunization, mice were anesthetized with isoflurane. once anesthesia was achieved, 1610 10 particles of virus slowly delivered as a bolus into the nostrils using a standard micropipette (gilson, middleton, wi) as previously described [39] . pre-existing immunity to adenovirus serotype 5 was established by injecting 5610 10 particles of adenovirus expressing betagalactosidase (adlacz) by intramuscular injection in the right hindlimb 30 days prior to vaccination with ad5-zgp. this protocol has been documented to activate t and b cells against virus capsid proteins and elicit humoral immunity [8, 11] . at the time of vaccination, mice had an average anti-adenovirus circulating nab titer of 1:320, which falls within lower range of average values reported in humans after natural infection [11] . mice were challenged by intraperitoneal injection of 2006ld 50 of mouse-adapted ebola virus, zaire strain (ma-zebov) in 200 ml sterile saline [40] . after challenge, the animals were weighed daily for 13 days and monitored for clinical signs of ebola infection using an approved scoring sheet. all procedures and the scoring method were approved by the institutional animal care committee at the national microbiology laboratory (nml) of the public health agency of canada (phac) according to the guidelines of the canadian council on animal care. all infectious work was performed in the 'biosafety level 4' (bsl4) facility at nml, phac. a) anti-ebola neutralizing antibody (serum). sera collected from immunized mice were inactivated at 56uc for 45 minutes. serial dilutions of each sample (1:10, 1:20, 1:40, etc, in 50 ml of dmem) were mixed with equal volumes of recombinant ebola zaire expressing the enhanced green fluorescent protein (egfp) reporter gene (zebov-egfp, 100 transducing units/well, according to egfp expression) and incubated at 37uc for 90 minutes [41] . the mixture was then added to subconfluent veroe6 cells in 96-well flat-bottomed plates and incubated for 5-10 minutes at room temperature. control wells were infected with equal amounts of either zebo-egfp with media without serum or that containing non-immune serum. 100 ml of dmem supplemented with 20% fbs was then added to each well and plates were incubated at 37uc in 5% co 2 for 48 hr. cells were subsequently fixed with 10% buffered formalin for 24 h and examined under a fluorescent microscope. sample dilutions which showed .50% reduction in the number of green cells compared to controls scored positive for neutralizing antibody. b) anti-ebola neutralizing antibody (mucosal). for the evaluation of the specific levels of igg and iga antibodies, bronchoalveolar lavage (bal) fluid was collected in situ with a 20gauge catheter inserted into the proximal trachea, flushing the lower airways three times with 1 milliliter of l15 media (sigma). bal from each animal was incubated at 56uc for 45 minutes. two-fold serial dilutions were added to 96 well plates pre-coated overnight with 30 ng of zebov-like particles per well and incubated at 37uc for 1 hr. goat anti-mouse secondary antibody conjugated to horseradish peroxidase (hrp) was then added and the plate was incubated for one additional hour at 37uc. the abts peroxidase substrate system (kpl) was used for detection and data collected using an asys uvm 340 elisa plate reader (isogen life science) at od405. all infectious in vitro work was performed in the bsl4 laboratory at the nml, phac. c) anti-adenovirus serotype 5 neutralizing antibody (serum). pre-existing immunity against recombinant adenovirus 5 was assessed by determining the amount of neutralizing antibody present in serum according to established methods [30] . in brief, serum was incubated at 56uc for 30 minutes and then diluted in dmem in twofold increments starting from a 1:20 dilution. each dilution (100 ml) was mixed with an aliquot of a standard stock of adenovirus type 5 expressing e. coli beta-galactosidase (10 6 pfu), incubated for 1 hour at 37uc, and applied to hela cells in 96-well plates (2610 4 cells/well). one hundred microliters of dmem supplemented with 20% fbs was then added to each well. cells were incubated at 37uc for 24 hours. neutralizing antibody titers were calculated as the highest dilution at which 50% of the cells stained blue by visual inspection. for the evaluation of inf-c positive cd8+ t cells 10 days postimmunization, splenocytes were harvested and cultured (1610 6 / sample) for 5 hours at 37uc in 96-well round bottom microtiter plates in dmem supplemented with 10% fbs, 2-beta-mercaptoethanol (10 26 m) and golgistop (1 ml/ml, bd pharmingen, san diego, ca). the telrtfsi peptide which carries the ebola zaire gp immunodominant mhc class i epitope for mice of the h-2 k haplotype (b10.br) was used for stimulation at a concentration of 1 mg/ml [42] . control cells were treated with either an unrelated peptide or no peptide. after washing, cells were stained with 100 ml of a fitc-anti mouse cd8a antibody (1:100 dilution, pharmingen) at 4uc for 30 minutes. cells were washed again, permeabilized in 16cytofix/cytoperm (pharmingen) for 20 minutes at 4uc, washed with 16perm/wash (pharmingen) and stained with 100 ml of a peanti mouse ifn-c antibody (1:100 dilution, pharmingen) in the same buffer at 4uc for 30 minutes. quantitation of inf-c positive cd8+ t cells isolated from splenocytes or mononuclear cells from bronchioalveolar lavage (bal), mesenteric lymph nodes (mln) and peyer's patches (pp) 45 days after vaccination was performed using an elispot assay (elispot mouse set, bd pharmingen, san diego, ca) according to the manufacturer's instructions. briefly, a 96-well elispot plate was coated with 5 mg/ml anti-mouse ifn-c capture antibody. cells pooled from 4 b10.br mice per experimental group were added to microwells along with the telrtfsi peptide (2 mg/well). control cells were incubated either without peptide or with the non-specific stimulator, seb (200 ng/ml). after incubation with a biotinylated anti-mouse ifnc detection antibody and streptavidin-horseradish peroxidase antibody, wells were counted using an elispot reader (aid elispot reader system, cell technology, colombia, md). data were analyzed for statistical significance by performing unpaired t tests (two-tailed p value) or one-way analysis of variance (anova) when appropriate. the differences in the mean or raw values among treatment groups were considered significant when p,0.05. in an effort to correlate markers of immunity with protection against ebola infection after mucosal immunization, t and b cell specific immune responses against ebola glycoprotein were analyzed in mice in the presence or absence of pre-existing immunity (pei) to adenovirus 10 days after vaccination with a first generation adenovirus serotype 5 vector expressing the zaire ebola glycoprotein (ad5-zgp). intracellular staining and flow cytometry (facs) revealed that ebola glycoprotein peptide-specific activation of cd8+ t cells, as measured by production of ifn-c, occurred in naïve animals immunized by the nasal route at a frequency of 3.961% (i.n., figure 1a ). pre-existing immunity was induced by intramuscular administration of recombinant ad5 expressing a nonrelevant antigen, beta-galactosidase (adlacz), 30 days prior to vaccination with ad5-zgp. at the time of vaccination, mice had an average anti-adenovirus circulating nab titer of 1:320. pre-existing immunity did not significantly alter activation of cd8+ t cells when the vaccine was given intranasally (i.n.+pei, 3.661%, p = 0.07). oral immunization lowered the response against ebola glycoprotein (p.o., 260.5%). samples obtained from animals with pre-existing immunity and vaccinated in the same manner were barely above the positive threshold (three times the average background level obtained from animals treated with the irrelevant virus, adlacz). samples obtained from naïve animals immunized by the intramuscular route (i.m.) contained the largest population of ifn-c positive cd8+ t cells (1062%). animals treated with the adlacz vector alone (adlacz-control) served as negative controls and produced 0.5% cd8+, ifn-c+ t cells in response to the ebola glycoproteinspecific peptide. the b cell response against ebola glycoprotein achieved by administration of the vaccine was determined by incubating a recombinant ebola virus (zaire strain) expressing green fluorescent protein (zebov-egfp) with serum collected 25 days after vaccination [8, 37] . neutralizing antibody (nab) levels equivalent to 40610 reciprocal dilution were detected in both naïve animals and those with pre-existing immunity after intranasal immunization ( figure 1b) . samples from naïve animals immunized by the oral route contained nab levels equivalent to 2065 reciprocal dilution whereas anti-ebola nab could not be detected in samples obtained from animals immunized by the same route with preexisting immunity (p.o.+pei). nab levels of 80610 were detected in samples obtained from naïve mice immunized by the intramuscular route. anti-ebola nab could not be detected in samples from mice given the adlacz vector alone (adlacz control). since the mucosa is often the primary sight of exposure, longterm, localized immune responses against ebola virus are desirable for providing optimal protection against infection. in this context, t cell mediated immune responses were assessed by elispot from various tissue compartments specific to the route of immunization 45 days after vaccination. the number of activated ifn-c secreting mononuclear cells harvested from splenocytes was significantly reduced by 38 and 59% in mice vaccinated nasally or orally when compared to those immunized by intramuscular injection (p#0.05, figure 2a ). in contrast, samples obtained from the spleen of animals with pre-existing immunity that were immunized by the i.n. route contained the highest number of activated ifn-c secreting cells (8156190 spot-forming cells (sfc)/ million mononuclear cells (mncs), figure 2c ) although this was approximately 30% lower than that seen in naive animals (1,3406146 sfc/million mncs, figure 2a ). pre-existing immunity also significantly reduced the number of these cells produced by animals immunized by the i.m. (210668 sfc/million mncs) and p.o. routes (71612 sfc/million mncs) with respect to those seen in naïve animals (2,145612 (i.m.) and 8856168 (p.o.)), p#0.05, figure 2a) . a very limited number of activated ifn-c secreting mononuclear cells were detected in splenocytes of naïve animals immunized with the adlacz vector (50620 sfc/million mncs, figure 2a ) and those with pre-existing immunity to the same virus (1261, adlacz, figure 2c ). significant levels of inf-c positive cells were detected in bronchioalveolar lavage fluid (bal) after intranasal immunization (146614 sfc/million mncs, p#0.05, figure 2b ). this was not significantly compromised by pre-existing immunity to adenovirus (120616 sfc/million mncs, p#0.06, figure 2d) . a limited number of inf-c secreting cells were detected in bal from naïve animals immunized by the i.m. or p.o. routes (462 and 1165 sfc/million mncs respectively, figure 2b ) and were similar to that seen in samples obtained from animals given the adlacz vector (1261, negative control). pre-existing immunity to adenovirus reduced these cell populations further to values that were not statistically significant (161 (i.m.), 562 (p.o.) and 261 (adlacz) sfc/million mncs (p = 0.08, figure 2d) ). inf-c positive cells were found in mesenteric lymph nodes (mln) and peyer's patches (pp) (146611 and 2965 sfc/million mncs, respectively) only after oral vaccination with ad-zgp ( figure 2b ). pre-existing immunity, however, reduced production of these cells to 18612 sfc/million mncs (mln) and 1662 sfc/million mncs (pp) ( figure 2d ). the nab response was also monitored from bronchioalveolar lavage fluid 45 days post-vaccination. nab to zebov-egfp was undetectable in samples obtained from control (adlacz) or i.m. immunized mice, whereas levels of 40610 and 1065 reciprocal dilution were detected in the bal of nasally and orally vaccinated animals, respectively ( figure 3 ). those with pre-existing immunity and immunized by the nasal route had nab levels of 30610. nab was not detected in mice with pre-existing immunity to ad5 and immunized by either the intramuscular or oral route. further characterization of ebola glycoprotein-specific immunoglobulin isotypes obtained from bronchioalveolar lavage fluid revealed that pre-existing immunity correlated with a marked decrease in the production of both igg and iga antibodies in mice immunized by intramuscular injection (figure 4a and 4b ). igg and iga levels were the lowest in that were vaccinated orally regardless of whether they had pre-existing immunity ( figure 4a and 4b). immunization by the nasal route induced a strong igg response that was reduced by an average of 25% in the presence of pre-existing immunity. the strongest iga response was detected in samples obtained from animals given the vaccine by the nasal route. iga levels, however, were also reduced by 25% in mice with pre-existing immunity to ad5. the most direct means of evaluating vaccine efficacy in mice is to assess protection by monitoring weight loss and death rates after a lethal challenge of ebola [43] . therefore, mice were immunized with 1610 10 pre-existing immunity. at the time of vaccination, animals had an average anti-adenovirus neutralizing antibody titer of 1:320 reciprocal dilution. naïve mice vaccinated by intramuscular injection with saline (vehicle) served as controls for complete lethality following challenge with mouse-adapted ebola virus. twenty-eight days after vaccination, animals were challenged with 200 ld 50 of mouse-adapted (ma)-zebov. only mice immunized by the nasal route survived the lethal challenge ( figure 5a ). this was evident as early as 5 days after challenge when controls and mice immunized by the other routes began to lose weight ( figure 5b ). all eventually expired 7 days post-challenge ( figure 5a , some data not shown for clarity). in contrast, naïve mice given the vaccine survived challenge regardless of the route of administration. the studies outlined above strongly suggest that only intranasal immunization can successfully afford protection against ebola virus in the absence and presence of pre-existing immunity. it was also clear that additional strategies were needed to improve vaccination by either the oral or intramuscular route in the presence of preexisting immunity. covalent attachment of activated monomethoxypolyethylene glycol to the protein capsid of the ad5-zgp vector was evaluated as a potential way to improve the immune response after oral vaccination. based upon previous observations, we hypothesized that this modification would protect the virus from neutralization by immune sera and improve survival in gastrointestinal tract [26, 27, 30, 44] . the frequency of ebola glycoprotein peptide-specific activation of ifn-c positive cd8+ t cells was not significant in naïve animals given the pegylated virus (0.360.3%) with respect to those given the unmodified virus (2.060.5%, p = 0.09, figure 6a ). similar results were also seen in the presence of pre-existing immunity in either treatment group (0.460.2% pegylated vaccine, 1.760.5%, unmodified vaccine). in contrast, nab levels equivalent to 30610 reciprocal dilution were detected in naïve animals given the pegylated vaccine ( figure 6b ). samples obtained from animals given the unmodified virus had levels of 2065 reciprocal dilution. animals with preexisting immunity against adenovirus and treated with the pegylated preparation were also able to produce detectable levels of nab (1065 reciprocal dilution) whereas nab was not found in sera from mice given the unmodified vector orally. further characterization of ebola glycoprotein-specific ig isotypes revealed that the pegylated vector could possibly stimulate production of igg in the presence or absence of pre-existing immunity with respect to levels found in animals given the unmodified vector ( figure 6c ). modification of the virus induced a slight increase of iga in the presence of pre-existing immunity with respect to that seen in naïve animals given unmodified virus (vaccine, figure 6d ) and those with pre-existing immunity (vaccine+pei). despite this, animals with pre-existing immunity to adenovirus and immunized with the pegylated vaccine orally did not survive after challenge with 200 ld 50 of (ma)-zebov. with mortality rates as high as 95%, ebola infection occurs largely by direct contact with blood, tissues or skin of patients, and through mucosal exposure [45, 46] . to date, few efforts have been made to focus on the mucosa as the primary sight of exposure to ebola virus and priming it for participation in the host defense by vaccination [47] . this is surprising given that the pulmonary, nasal and oral immune systems which comprise the mucosal-associated lymphoid tissues (malt) are responsible for the production of approximately 80% of all immunocytes [48, 49] and mucosal immunity is often the first line of defense against pathogens coming in contact with susceptible hosts. many studies in rodents indicate that systemic immunization produces strong anti-viral systemic responses while mucosal vaccination can stimulate both the mucosal and systemic immune systems and can confer long-term immunological memory against a given pathogen despite the fact that the magnitude of these responses are often reported to be somewhat reduced [37, 48, 50] . we have found this to be the case in the studies outlined here since the systemic cellular response and neutralizing antibody levels against ebola zaire gp were consistently lower following nasal and oral vaccination. mucosal vaccination did, however, generate significant cellular and antibody responses in the periphery (bal, mln or peyer's patches) and a single intranasal immunization with ad5-zgp conferred 100% protection even in the presence of pre-existing immunity. administration of recombinant adenovirus-based vaccines to the mucosa has also conferred sufficient protection against challenge with a variety of pathogens in the presence of preexisting immunity to the vaccine carrier in mice and other preclinical models of disease [5, 19, 32, 51, 52] . this served as a basis for the present study. a single intranasal dose of a recombinant ad5 vaccine expressing the zaire ebola glycoprotein conferred 100% protection in both naïve mice and those with pre-existing immunity despite the fact that the strength of the immune response generated by this route of administration was quantitatively lower than that seen in animals vaccinated by intramuscular injection. it is also important to note that pre-existing immunity induced by intramuscular injection did not severely compromise the t cell-mediated response at either the systemic or mucosal levels in these animals. the level of anti-ebola gp antibodies in the circulation and in the lung was also not significantly compromised by pre-existing immunity to adenovirus. while these results suggest that intranasal vaccination with an ad-based vaccine is indeed a promising strategy to overcome pre-existing immunity, one might wonder how accurately our results translate to natural exposure to the wild type virus via the respiratory tract. this is somewhat difficult to establish in the mouse model since the ability of the wild-type adenovirus to replicate is limited [53] . in addition, the amount of neutralizing antibody present in the nasal cavity of those in the general population with pre-existing immunity to adenovirus 5 has not been assessed to the degree that serum neutralizing antibodies have, making it difficult to set a relative parameter for one to target in pre-clinical animal models. lack of this data may be due to the invasive nature of the technique for acquiring samples and/or the fact that antibody levels in the mucosa of individuals with established pre-existing immunity to adenovirus type 5 are quite low and transient in contrast to systemic levels of anti-adenovirus neutralizing antibodies which are quite robust and persist over time (unreported observations). thus, for these studies, we decided to induce pre-existing immunity against adenovirus by intramuscular injection at a dose that has been shown to induce production of systemic neutralizing antibodies at 1:320, a level mostly below what was reported in humans with documented preexisting immunity [8, 11, 12] . additional studies designed to assess the amount of neutralizing antibody to adhu5 in the lung over time after intranasal administration of varying doses of adhu5 are currently underway in an effort to further define stringent conditions under which pre-existing immunity can be established for experimental testing. data obtained from animals vaccinated by the oral route provided several insights about the immunological requirements for protection against ebola in a mouse model. immunization by this route induced quantitatively lower t and b cell mediated immune responses against ebola glycoprotein with respect to that achieved by either intramuscular or intranasal immunization. despite this, every naïve animal immunized with a single oral dose of ad-zgp survived challenge, giving better precision on the minimal threshold of immunity required to achieve protection. as seen with intranasal immunization, the t cell-mediated response was not compromised by pre-existing immunity 10 days after oral vaccination. the number of ifn-c secreting t cells in both mucosal and systemic compartments of these animals at day 45, however, was significantly reduced by pre-existing immunity. it is possible that the peak t cell response at day 10 does not reflect the extent by which pre-existing immunity decreases the ad5-zgp-induced t cell response. alternatively, it is also possible that the elispot assay could detect subtle variations that flow cytometery could not monitor accurately due to differences in the sensitivity of each assay. neutralizing antibody was not detected in the serum of animals with pre-existing immunity given a single oral dose of the vaccine. more importantly, none of these animals survived challenge with mouse adapted ebola. we attempted to improve the immunogenicity of the adenovirus-based vaccine by protecting it from the harsh environment of the gastrointestinal tract and from neutralization by antiadenovirus antibodies by pegylation. interestingly, the t-cell mediated immune response was significantly reduced and antibody levels increased in naïve animals given a single oral dose of the pegylated vaccine with respect to that seen in animals given the same dose of unmodified ad5-zgp. it has been shown that modification of virus capsids by pegylation can significantly dampen the t-cell mediated immune response against the virus and stimulate the antibody response against a secreted antigen [26, 27, 30, 54] . taken together, these data support the notion that the exposition of the virus capsid proteins facilitates the immune response against the encoded antigen. optimization of pegylation chemistries and/or densities on adenovirus-based vaccine that promote and strengthen protective immune responses following oral immunization is currently underway. delivery of recombinant adenoviral vaccines to either the nasal or intestinal mucosa is an attractive vaccination strategy for many reasons. vaccines administered in this manner will offer improved safety with respect to disease transmission and needle-stick injuries among health care workers, significant issues of concern in developing countries where the demand for many vaccines is high [55] . mucosal administration of vaccines reduces the pain associated with vaccination, eliminates the need for specialized training programs for large vaccination campaigns and makes selfadministration of the vaccine possible. this route of administration may also significantly reduce systemic toxicity associated with recombinant adenovirus despite the fact that it has been shown that nasal immunization with recombinant adenovirus-based vaccines can facilitate translocation of the virus to the central nervous system [52] . even though testing of other virus-based vaccines such as influenza have reported similar findings and are currently used in the clinic [56] , studies designed to fully assess the toxicological profile of adenovirus vaccine after nasal administration are also currently underway. we have shown that nasal immunization with an ad5-based vaccine can induce a long-term protective immune response against ebola virus in a mouse model which is not impeded by preexisting immunity to adenovirus serotype 5. while these results are extremely encouraging, further characterization of the immune response against both the encoded antigen and the adenovirus vector in larger, clinically relevant animal models is vital for both understanding the biology of ad vaccines and for the development of an effective ebola vaccine suitable to populations with different requirements [57, 58] . the issue of pre-existing immunity must also be adequately addressed in order to develop efficient recombinant adenovirus-based vaccines. while the majority of the literature suggests that pre-existing immunity significantly hamper the effective use of adhu5 vaccine carriers, other investigators have reported that pre-existing immunity did not interfere with the potency of recombinant ad5-based vaccines in both pre-clinical models of disease and in humans [59, 60] . thus, additional studies identifying clinically relevant conditions under which to test ad-based vaccine candidates are necessary to assess the full impact of pre-existing immunity on vaccine potency, including in different compartments. better define the role of preexisting immunity on vaccine-induced immunity will further the understanding of how individuals previously exposed to adenovirus will respond to these immunization regimens. adenovirus-based genetic vaccines for biodefense adenovirus as vehicle for anticancer genetic immunotherapy use of viral vectors for the development of vaccines the effect of pre-existing adenovirusspecific immunity on immune responses induced by recombinant adenovirus expressing glycoprotein d of bovine herpesvirus type 1 oral vaccination of mice with adenoviral vectors is not impaired by preexisting immunity to the vaccine carrier oral vaccination with pegylated adenovirus improves the b-cell mediated but not the t-cell mediated immune response against ebola glycoprotein. naïve mice and those with pre-existing immunity were vaccinated with 1610 10 particles of either unmodified (vaccine) or pegylated (peg-vaccine) ad5-zgp by oral gavage. pre-existing immunity (pei) was induced by i.m. injection with 5610 10 particles of adenovirus expressing beta-galactosidase (adlacz). (a) frequency analysis of ifn-c secreting cd8+ t cells neutralizing antibody (nab) levels against zebov-egfp were evaluated 25 days post-vaccination (n = 10/group). (c) profile of anti-ebola-specific igg antibodies. (d) profile of anti-ebola-specific iga antibodies. in all panels, error bars represent the standard deviation of the data overcoming immunity to a viral vaccine by dna priming before vector boosting immunogenicity of recombinant adenovirus serotype 35 vaccine in the presence of pre-existing anti-ad5 immunity chimpanzee adenovirus vaccine protects against zaire ebola virus comparative immunogenicity in rhesus monkeys of dna plasmid, recombinant vaccinia virus, and replication-defective adenovirus vectors expressing a human immunodeficiency virus type 1 gag gene clinical trials yield promising results from two adenovirusbased vaccines prevalence of neutralizing antibodies to adenoviral serotypes 5 and 35 in the adult populations of the gambia, south africa, and the united states comparative seroprevalence and immunogenicity of six rare serotype recombinant adenovirus vaccine vectors from subgroups b and d adenovirus vector induced innate immune responses: impact upon efficacy and toxicity in gene therapy and vaccine applications viral vectors: a wide range of choices and high levels of service mucosally delivered e1-deleted adenoviral vaccine carriers induce transgene product-specific antibody responses in neonatal mice replication-deficient human adenovirus type 35 vectors for gene transfer and vaccination: efficient human cell infection and bypass of preexisting adenovirus immunity adenovirus types 5 and 35 seroprevalence in aids risk groups supports type 35 as a vaccine vector novel replication-incompetent vector derived from adenovirus type 11 (ad11) for vaccination and gene therapy: low seroprevalence and non-crossreactivity with ad5 induction of protective immunity to anthrax lethal toxin with a nonhuman primate adenovirus-based vaccine in the presence of preexisting anti-human adenovirus immunity immunogenicity of heterologous prime-boost regimens involving recombinant adenovirus serotype 11 (ad11) and ad35 vaccine vectors in the presence of anti-ad5 immunity efficacy of severe acute respiratory syndrome vaccine based on a nonhuman primate adenovirus in the presence of immunity against human adenovirus hexon gene switch strategy for the generation of chimeric recombinant adenovirus hexon-chimaeric adenovirus serotype 5 vectors circumvent pre-existing antivector immunity poly (lactic-glycolic) acid copolymer encapsulation of recombinant adenovirus reduces immunogenicity in vivo pegylation of adenovirus with retention of infectivity and protection from neutralizing antibody in vitro and in vivo stealth'' adenoviruses blunt cell-mediated and humoral immune responses against the virus and allow for significant gene expression upon readministration in the lung pegylation of e1-deleted adenovirus vectors allows significant gene expression on readministration to liver encapsulation of recombinant adenovirus into alginate microspheres circumvents vector-specific immune response microsphere-liposome complexes protect adenoviral vectors from neutralising antibody without losses in transfection efficiency, in-vitro pegylated helper-dependent adenoviral vectors: highly efficient vectors with an enhanced safety profile pegylated adenovirus vectors containing rgd peptides on the tip of peg show high transduction efficiency and antibody evasion ability adenoviral vectors for mucosal vaccination against infectious diseases development of a preventive vaccine for ebola virus infection in primates a dna vaccine for ebola virus is safe and immunogenic in a phase i clinical trial immune protection of nonhuman primates against ebola virus with single lowdose adenovirus vectors encoding modified gps a single intranasal inoculation with a paramyxovirus-vectored vaccine protects guinea pigs against a lethal-dose ebola virus challenge mucosal delivery of adenovirus-based vaccine protects against ebola virus infection in mice development of a rapid method for the pegylation of adenoviruses with enhanced transduction and improved stability under harsh storage conditions immunogenicity of novel consensus-based dna vaccines against avian influenza a mouse model for evaluation of prophylaxis and therapy of ebola hemorrhagic fever infection of naive target cells with virus-like particles: implications for the function of ebola virus vp24 cytotoxic t lymphocytes to ebola zaire virus are induced in mice by immunization with liposomes containing lipid a pathogenesis of experimental ebola zaire virus infection in balb/c mice pegylated adenoviruses for gene delivery to the intestinal epithelium by the oral route lethal experimental infections of rhesus monkeys by aerosolized ebola virus lethal experimental infection of rhesus monkeys with ebola-zaire (mayinga) virus by the oral and conjunctival route of exposure how ebola and marburg viruses battle the immune system nalt-versus peyer's-patch-mediated mucosal immunity intestinal and pulmonary mucosal t cells: local heroes fight to maintain the status quo mucosal vaccines: the promise and the challenge immunization with recombinant adenovirus synthesizing the secretory form of japanese encephalitis virus envelope protein protects adenovirus-exposed mice against lethal encephalitis a chimpanzee-origin adenovirus vector expressing the rabies virus glycoprotein as an oral vaccine against inhalation infection with rabies virus infection of mouse liver by human adenovirus type 5 fully detargeted polyethylene glycol-coated adenovirus vectors are potent genetic vaccines and escape from pre-existing anti-adenovirus antibodies estimation of the global burden of disease attributable to contaminated sharps injuries among health-care workers productive infection in the murine central nervous system with avian influenza virus a (h5n1) after intranasal inoculation use of animal models in the development of human vaccines status and challenges of filovirus vaccines protective immunity against botulism provided by a single dose vaccination with an adenovirus-vectored vaccine safety and immunogenicity of adenovirus-vectored nasal and epicutaneous influenza vaccines in humans key: cord-000413-h2e6h076 authors: zhang, jingyu; ma, zhenni; dong, ningzheng; liu, fang; su, jian; zhao, yiming; shen, fei; wang, anyou; ruan, changgeng title: a conformation-sensitive monoclonal antibody against the a2 domain of von willebrand factor reduces its proteolysis by adamts13 date: 2011-07-11 journal: plos one doi: 10.1371/journal.pone.0022157 sha: doc_id: 413 cord_uid: h2e6h076 the size of von willebrand factor (vwf), controlled by adamts13-dependent proteolysis, is associated with its hemostatic activity. many factors regulate adamts13-dependent vwf proteolysis through their interaction with vwf. these include coagulation factor viii, platelet glycoprotein 1bα, and heparin sulfate, which accelerate the cleavage of vwf. conversely, thrombospondin-1 decreases the rate of vwf proteolysis by adamts13 by competing with adamts13 for the a3 domain of vwf. to investigate whether murine monoclonal antibodies (mabs) against human vwf affect the susceptibility of vwf to proteolysis by adamts13 in vitro, eight mabs to different domains of human vwf were used to evaluate the effects on vwf cleavage by adamts13 under fluid shear stress and static/denaturing conditions. additionally, the epitope of anti-vwf mab (sz34) was mapped using recombinant proteins in combination with enzyme-linked immunosorbent assay and western blot analysis. the results indicate that mab sz34 inhibited proteolytic cleavage of vwf by adamts13 in a concentration-dependent manner under fluid shear stress, but not under static/denaturing conditions. the binding epitope of sz34 mab is located between a1555 and g1595 in the central a2 domain of vwf. these data show that an anti-vwf mab against the vwf-a2 domain (a1555-g1595) reduces the proteolytic cleavage of vwf by adamts13 under shear stress, suggesting the role of this region in interaction with adamts13. plasma von willebrand factor (vwf) is a large multimeric glycoprotein that interacts with platelet surface receptors and is crucial for normal hemostasis. the adhesive activity of vwf is positively correlated with the size of the multimers in plasma [1, 2] . vwf multimer size is regulated by metalloproteinase adamts13, which cleaves the central a2 domain of vwf at the tyr1605-met1606 bond [3] [4] . the importance of vwf proteolysis by adamts13 is demonstrated in two syndromes, i.e., thrombotic thrombocytopenic purpura and von willebrand disease type 2a. the former is associated with the deficiency of plasma adamts13 activity, either due to congenital mutations or acquired autoantibodies [5] [6] [7] . the latter is mostly caused by mutations in the a2 domain of vwf that lead to the increased proteolysis of vwf multimers by adamts13 [8, 9] . many factors modulate the proteolysis of vwf by ada-mts13. these ligands that bind the a1 domain of vwf such as platelet glycoprotein iba, heparin and ristocetin promote vwf proteolysis by adamts13 [10] . in addition, platelets also significantly enhance the cleavage of vwf multimers by adamts13 under fluid shear stress [11, 12] . on the contrary, thrombospondin-1, an extracellular matrix adhesion protein, may compete with adamts13 for binding to the a3 domain of vwf, which reduces the rate of vwf proteolysis by adamts13 [13, 14] . in this study, we investigated the effects of eight murine monoclonal antibodies (mabs) against various domains of vwf on its proteolysis by adamts13 under physiologically relevant conditions. among those, mab sz34 dramatically decreased the susceptibility of vwf to adamts13 under shear stress, but not under static/denaturing conditions. the epitope of sz34 was mapped to the amino acid residues between a1555 and g1595 in the central a2 domain of vwf. our findings highlight the critical role of this region for adamts13-mediated proteolysis under shear stress. anti-vwf mab sz34 decreases the susceptibility of vwf to proteolysis by adamts13 under shear stress in this study, we used radamts13 and pvwf as the sources of enzyme and substrate to determine the effect of sz34 on vwf proteolysis under shear stress. the cleavage product was determined by sds-page under non-reducing conditions followed by western blot. the results showed that the 350 kda cleavage product of vwf under shear stress was reduced in the presence of sz34 in a concentration-dependent manner ( figure 1 ). the half maximal (50%) inhibitory concentration (ic 50 ) of the sz34 was estimated to be approximately 50 mg/ml ( figure 1 ). in contrast, other anti-vwf mabs including 1c1e7 (an anti-vwf d'd3 mab), sz129 (an anti-vwf a1 mab), sz123 (an anti-vwf a3 mab) and so on had no effects on the proteolytic cleavage of vwf by adamts13 (figure 1 and table 1 ). the control experiments established the lack of detectable proteolysis in the absence of adamts13 or in the addition of 20 mm edta to reaction mixtures ( figure 1 ). the proteolysis of vwf by adamts13 in the absence and presence of anti-vwf mabs was also determined by agarose gel electrophoresis visualizing vwf multimers. we showed that the decreased amount of the high and intermediate molecular weight multimers were dramatically reduced by mab sz34 in a concentration-dependent manner under shear stress (figure 2 ), further confirming the role of sz34 in decreasing the susceptibility of vwf to proteolytic cleavage by adamts13 under physiologically relevant conditions. sz34 had no effects on vwf proteolysis by adamts13 under denaturing/static condition when pvwf was pre-denatured with guanidine-hcl before incubation with sz34 and radamts13, no obvious difference in the 350 kda cleavage products was detected ( figure 3 and purified pvwf (150 nm) was preincubated with sz34 (0-200 mg/ml) for 30 min at 37uc, and then incubated with 50 nm radamts13. after 5 min of vortexing at 2,500 rpm on a mini vortexer, the 350 kda cleavage products were visualized by 5% sds-page under non-reducing conditions and western blot analysis. 1c1e7 (an anti-vwf d'd3 mab), sz129 (an anti-vwf a1 mab) and sz123 (an anti-vwf a3 mab) were used as negative controls. (b) changes in the cleavage products detected relative to that observed in the absence of mabs were determined under shear stress by densitometry. the extent of cleavage was analyzed by detection of the intensity of the 350 kda cleavage products. results represent the mean 6 standard deviation of four independent experiments. doi:10.1371/journal.pone.0022157.g001 table 1 ). thus, sz34 had no effect on the digestion of unfolded vwf by adamts13. in the meantime, anti-vwf mabs including 1c1e7, sz129, sz123, etc, exhibited no effects on adamts13-mediated proteolysis of the unfolded vwf under static conditions either ( figure 3 and table 1 ). to exclude the influence of denaturing condition on interaction between vwf and antibodies, we constructed and expressed vwf-r1597w mutant and investigated whether sz34 inhibited its proteolysis by adamts13. r1597w is the most frequent mutation in von willebrand disease type 2a and the mutation site is adjacent to tyr1605-met1606 bond. this mutant vwf can be cleaved by adamts13 under static condition and in the absence of denaturants such as urea and guanidine [8] . the results showed that with or without sz34 r1597w vwf multimers were equally effectively cleaved by adamts13 under static conditions (figure 4 ), which suggested that sz34 had no effects on the proteolysis of this vwf mutant by adamts13. preliminary experiments have shown that sz34 binds to vwf-r1597w multimers by an elisa method and the dissociation constants (kd) is 0.6760.11 nm. to determine the binding epitope of sz-34, we prepared a series of recombinant vwf fragments, including a1a2a3, a1, a2, a3 and d'd3 ( figure 5a ), and five gst fusion vwf-a2 fragments, i.e. a2-1, a2-2, a2-3 (vwf73), a2-12 and a2-23 (vwf114) ( figure 5b ). we measured the binding capacities (dissociation constants, kd) of sz34 to native and guanidinedenatured vwf and various vwf fragments by elisa method. sz34 bound to native and pre-denatured full-length vwf, a1a2a3, and a2, but only native a2-12 and a2-23 (table 2) . furthermore, the binding of sz34 to the pre-denatured vwf and vwf fragments was much weaker than the native counterparts ( table 2 ). these data suggest that the epitope of sz34 is conformationally sensitive. the binding site was mapped to the central a2 domain of vwf. because sz34 bound to both native a2-12 and native a2-23, the epitope of sz34 appeared to be located within the a2-2 (a1555-g1595 region). to further to testify that sz34 is a conformational mab against vwf, a western blot in combination with elisa based on polystyrene microspheres was performed to compare the binding activities of sz34 to native pvwf and denatured pvwf. compared with native pvwf, the binding activity of sz34 to heated pvwf or pvwf treated with 1.5 m guanidine-hcl was significantly reduced (figure 6 ), further confirming that sz34 is a conformation-sensitive mab to vwf. we found that sz34, a mab against the a2 domain of human vwf, reduces the susceptibility of vwf to proteolysis by adamts13 under fluid shear stress. however, this effect was not observed under static and chemically denaturing conditions. seven other mabs against vwf including sz129 and sz130 (two mabs against vwf a1), sz29 (another mab against vwf a2), sz123 and sz125 (two mabs against vwf a3) and 1c1e7 and 75h4b12 (two mabs against vwf d'd3) exhibited no effects on adamts13-mediated vwf proteolysis under either shear stress or denaturing conditions. the epitope mapping shows that the epitope of sz-34 is located within the a1555-g1595 region of the a2 domain of native vwf, suggesting that this region may directly interact with adamts13. furthermore, the mab sz-34 is a conformation-sensitive mab because it preferentially binds to native vwf. vwf-d'd3 no effect no effect 75h4b12 25 vwf-d'd3 no effect no effect sz129 26 vwf-a1 no effect no effect sz130 26 vwf-a1 no effect no effect sz29 28 vwf-a2 no effect no effect sz34 27,28 vwf-a2 inhibition no effect sz123 27 vwf-a3 no effect no effect sz125 27 vwf-a3 no effect no effect doi:10.1371/journal.pone.0022157.t001 only two reports have been published showing the inhibitory effects of vwf antibodies on the proteolysis of vwf by adamts13, but the mechanism of inhibition by sz34 may be different from that of the two other mabs vp-1 and ru8 reported by tsai et al [15] and zanardeli et al [16] , respectively. the mab vp-1, which was directly against residues 1591-1605 (inside vwf a2 domain) of vwf polypeptide, inhibited the susceptibility of the recombinant type 2a vwf mutants r1606w and r1606q to proteolytic cleavage by adamts13 under the static condition. but they did not examine whether vp-1 inhibited native pvwf or wild-type recombinant vwf proteolysis by adamts13 [15] . because vp-1 was raised against the synthetic vwf peptide (residues 1591-1605) and reacted poorly with the intact subunit (250 kda fragment) of vwf in reduced sds-page followed by immunoblotting [3, 15] , we speculated that vp-1 could not bind to native vwf and could not affect its proteolysis by adamts13 without denaturization or under shear stress. in addition, zanardelli et al [16] reported the inhibition of vwf proteolysis by adamts13 under shear by using ru8, a mab directed against the vwf d4 domain. they suggested that a binding site in the cterminal region (d4ck) of vwf is constitutively exposed and participates at the initial step of a multi-step interaction, ultimately leading to proteolysis of vwf by adamts13. thus, ru8 may inhibit vwf proteolysis by inhibiting the initial binding of the distal domains of adamts13 to the c-terminal region of vwf. we found that the mab, sz34, bound to native vwf better than pre-denatured vwf at the central a2 domain, and decreased the cleavage of native vwf under shear stress, but not pre-denatured vwf under static conditions. these findings suggest that sz34 binds native vwf-a2 domain, which may block the conformational change of vwf or block the access of adamts13 to the ancillary binding site and cleavage bond located at the central a2 domain under fluid shear stress. the adamts13 cleavage site (the y1605-m1606 bond) in the vwf a2 domain is buried within the native, folded structure of vwf multimers and is not accessible to cleavage by the metalloprotease [3] [4] [5] . although a report found that recombinant vwf-a2 peptide (a.a.1481-1668) was sensitive to proteolysis by adamts-13 under physiological ph and non-denaturing conditions [17] , most of studies have shown that even for the isolated a2 domain, unfolding by high fluid shear stress or chemical denaturants such as urea and guanidine-hcl is required for proteolysis by adamts13 [10, 18] . thus, for vwf with its multi-domain structure, not only the inter-domain but also the intra-domain a2 structural changes have regulatory roles in adamts13-mediated cleavage of vwf. our data demonstrate that the epitope of sz34, a mab raised against native vwf multimers, is located within the a1555-g1595 region, which comprises the a2-helix and a3-helix according to the crystal structure of vwf a2 domain [19] . it is possible that part or all of the a2-helix and a3-helix structure of a2 domain is constitutively exposed on the native vwf multimers and is involved in the regulation of vwf proteolysis by adamts13. previous studies, however, considered that the a2 domain of vwf is normally sandwiched between the much larger a1 and a3 domains [19, 20] , and the d1596-r1668 region in vwf a2 domain (the so called vwf73) is the minimal substrate for adamts13 [21] . we conclude that sz34 is a conformationally sensitive anti-vwf mab which can modulate proteolytic cleavage of vwf by adamts13 under physiologically relevant conditions. this monoclonal anti-vwf antibody may be a useful tool for further investigating biological function of vwf in vivo. plasma-derived human vwf (pvwf) was purified from commercial vwf/fviii concentrate by gel filtration with a sepharose 4b-cl column (amersham pharmacia biotech ab, uppsala, sweden) and heparin-sepharose 6ff (pharmacia) affinity chromatography, as reported [22] . vwf antigen (vwf:ag) concentration was determined by an enzyme-linked immunosorbent assay (elisa) kit from dako (glostrup, denmark) and vwf multimers analysis was performed as reported [23] . plasmid adamts13 containing human full-length cdna sequence was generously provided by dr. jingfei dong (baylor college of medicine, houston, tx, usa) [24] . recombinant adamts13 (radamts13) with the c-terminal his-tag was expressed in a stably transfected hela cell line. expression medium was concentrated and purified using a ni-nta agarose column (qiagen gmbh, hilden, germany). eight murine mabs against human vwf were used. the anti-vwf d'd3 domain mabs 1c1e7 and 75h4b12 were kind gifts from dr. deckmyn (laboratory for thrombosis research, ku leuven campus kortrijk, kortrijk, belgium) [25] . others were all previously produced in our laboratory. sz129 and sz130 recognize vwf a1 domain [26] , whereas sz123 and sz125 interact with vwf a3 domain [27] . sz29 and sz34 are mabs to vwf, whose epitopes are indefinite because of being directly raised against purified plasma-derived full-length human native vwf [27, 28] . horse-radish peroxidase (hrp)-conjugated polyclonal rabbit antihuman vwf igg was purchased from dako (glostrup, denmark). purified vwf (1.5 mm) was pre-denatured with 1.5 m guanidine-hcl in 20 mm tris-hcl (ph 8.0) at 37uc for 2 h. after a 1:10 dilution with 20 mm tris-hcl (ph 8.0), the denatured vwf was incubated with anti-vwf mabs (0-200 mg/ml) at 37uc for 30 min and then treated with 25 nm radamts13 that had been activated by incubation with 5 mm cacl 2 at 37uc for 1 h. after incubation at 37uc for 1.5 h, the reaction was stopped by the addition of 20 mm edta. expression of vwf-r1597w mutant and its cleavage by adamts13 in the presence of mab sz34 under static/ nondenaturing conditions the plasmid psvhvwf1, which harbors a full-length cdna insert of human vwf (kindly provided by je sadler, washington university school of medicine, st louis, usa) [29] , was used to construct the r1597w mutant by pcr-based single-nucleotide mutagenesis. recombinant vwf-r1597w mutant was expressed in transiently transfected hela cell line using serum-free opti-mem i (invitrogen) for 48 h. the media were collected and added into edta-free 16 proteinase inhibitor cocktail (roche), followed by concentration using amicon ultra-15 centrifugal filter (millipore). vwf antigen (vwf:ag) concentration was determined by an elisa kit (dako). recombinant vwf-r1597w (150 nm) was incubated with 0 or 100 mg/ml sz34 at 37uc for 30 min and then for 18 h with 25 nm radamts13 at 37uc. the cleavage reactions were measured by the distribution of proteolytic fragments (350 kda) on a 5% sds-page under nonreducing condition or by vwf multimeric size distribution on a 1.5% agarose gel electrophoresis, and then analyzed by western blotting with hrp-conjugated anti-vwf igg (dako) and visualized by chemiluminescence as reported [23, 30] . the intensity of the bands was analyzed using image j software. plasmids encoding different vwf fragments were all generated from psvhvwf1 [29] . recombinant vwf d'd3, a1a2a3, a1, a2 and a3 all contained a 66his tag (novagen, san diego, ca, usa) ( figure 5a ). the preparation of vwf a1, a2 and a3 was described previously [26, 27, 31] . plasmid construction, expression and purification of vwf a1a2a3 were similar to vwf a3 [27] . two primers were used for amplifying vwf a1a2a3: 59-gga tcc gca gga gcc ggg agg c-39 and 59-ctc gag tcc aga gca cag ttt gtg-39 (lowercase letters for bamhi and xhoi sites). four recombinant vwf domains including a1a2a3, a1, a2 and a3 were all expressed in inclusion bodies. after purification, protein refolding was achieved using an 8 to 0 m urea linear gradient. plasmids encoding a2-12, a2-23 (vwf114), a2-1, a2-2 and a2-3 (vwf73) ( figure 5b ) were constructed similarly in the gst fusion vector pgex-6p-1 (amersham biosciences, piscataway, nj). these recombinant plasmids were prepared by use of primers as follows: a2-12 (59-cgg gat cc gac ctt gcc cct gaa gcc cct c-39 and 59-cgg aat tc tca gtg atg gtg atg gtg atg acc ctg gct gac caa gaa gct g-39), a2-23 (59-cgg gat cc gca cag tcc aaa ggg gac atc c-39 and 59-cgg aat tc tca gtg atg gtg atg gtg atg cct ctg cag cac cag gtc agg a-39), a2-1 (59-cgg gat cc gac ctt gcc cct gaa gcc cct c-39 and 59-cgg aat tc tca gtg atg gtg atg gtg atg ctc gct gaa ggg gta ctc cac ag-39), a2-2 (59-cgg gat cc gca cag tcc aaa ggg gac atc c-39 and 59-cgg aat tc tca gtg atg gtg atg gtg atg acc ctg gct gac caa gaa gct g-39) and a2-3 (59-cgg gat cc gac cgg gag cag gcg ccc aac c-39 and 9-cgg aat tc tca gtg atg gtg atg gtg atg cct ctg cag cac cag gtc agg a-39). lowercase letters indicate added restriction enzyme sites (bamhi and hindiii) and the underlined sequence was the inserted c-terminal 66his-tag. the gst fusion proteins were expressed and purified by chromatography on ni-nta agarose (qiagen gmbh, hilden, germany) as described [21] . all five recombinant gst fusion proteins were mainly expressed in soluble fractions at low temperatures. heat treatment and denaturization treatment with guanidine-hcl were used to unfold recombinant vwf fragments and pvwf. purified pvwf was denatured by heating at 80uc for 20 min in a thermo-block heater (thermostat plus, eppendorf) as reported [32] . recombinant vwf fragments and pvwf were denatured with guanidine-hcl [30] as following. purified pvwf or various vwf fragments (10 mm) were incubated with 1.5 m guanidine-hcl at 37uc for 2 h. then, guanidine-hcl treated vwf fragments and pvwf were diluted serially with 20 mm tris-hcl (ph 8.0), 0.15 m nacl. the binding affinity of denatured vwf and sz34 was determined by an elisa. because the initial concentrations of pvwf and vwf fragments were high, the final concentration of guanidine-hcl in solution was ,20 mm, which did not interfere with the binding activity of sz34 (data not shown), consistent with what was reported by tsai [30] in which he showed that guanidine-hcl at 24 mm did not interfere with the platelet aggregation. affinity measurements of sz34 with full-length vwf and various vwf fragments using elisa the native or denatured vwf fragments and pvwf at various concentrations were first incubated in solution with the antibody (sz34 or sz29) at constant concentration until equilibrium was reached, respectively. the denatured vwf fragments and pvwf were obtained by denaturization treatment with guanidine-hcl as above. the concentration of free antibody was then determined by an elisa. the equilibrium dissociation constants (kd) of antigen/antibody complexes in solution were obtained according to the equation [33] : where a 0 and a are the absorbances measured for the antibody in the absence and presence of antigen, respectively, and a 0 and i 0 respectively the total concentrations of antigen and antibody. increasing concentrations (0.5-100 nm) of the vwf fragments or pvwf with or without being denatured were mixed with a constant amount of sz34 (0.3 nm), in 1% (w/v) bsa-pbs, respectively, until equilibrium were reached (15 h incubation at 22uc). then the proportion of sz34 which remained unsaturated at each concentration of various vwf fragments and pvwf was measured by an elisa method as following. the antigen/ antibody complexes were added to the microtiter plates coated with 100 ml of pvwf (7.5 mg/ml) and incubated at 37uc for 2 h. after being washed, the wells were incubated at 37uc for 2 h with hrp-labeled goat anti-mouse igg (sigma) in 1% (w/v) bsa-pbs. the plates were washed 5 times and tetramethyl benzidine (tmb) solution was added for color development. absorbance was read at 450 nm and a 0 and a were then obtained, respectively. binding of sz34 to native and unfolded vwf by western blot/elisa based on polystyrene microspheres to distinguish the linear from the conformational epitope of sz34, we used an in-house western blot/elisa (western blot in combination with elisa) based on polystyrene microspheres. sz129 (a mab to vwf a1 domain) was used as a control. briefly, 20 mg/ml of sz34 or sz129 in 0.05 m sodium carbonate buffer (ph 9.6) was mixed with 1:10 volume of 50 mg/ml polystyrene microsphere suspension (sdl37; takeda chemical industries, co., ltd., osaka, japan). the mixtures were incubated at room temperature for 2 h with shaking. the microspheres were then centrifuged and resuspended in a two-fold dilution of the starting volume of 2.5% (w/v) bsa-pbs to block the free binding sites. this solution was incubated at room temperature for 2 h with constant shaking. after washing three times with pbs, the microspheres were incubated with 100 nm of pvwf with or without denaturing at room temperature for 1 h with gentle agitation. the microspheres were washed three times with pbs, added in sample buffer (0.0625 m tris-hcl buffer, ph 6.8, 10% (v/v) glycerol, 2% (w/v) sds, 5% (v/v) b-mercaptoethanol, 0.001% (w/v) bromophenol blue, 4 mm edta), and then heated at 99uc for 5 min. after centrifugation at 10,000 g for 3 min, the supernatants were subjected to 6% sds-page. the proteins were transferred to membranes and blotted with hrp-conjugated anti-vwf igg (dako). structure and function of von willebrand factor: relationship to von willebrand's disease binding of von willebrand factor to glycoproteins ib and iib/iiia complex: affinity is related to multimeric size identification of a cleavage site directing the immunochemical detection of molecular abnormalities in type iia von willebrand factor partial purification of a protease from human plasma cleaving von willebrand factor to fragments produced by in vivo proteolysis mutations in a member of the adamts gene family cause thrombotic thrombocytopenic purpura antibodies to von willebrand factor-cleaving protease in acute thrombotic thrombocytopenic purpura von willebrand factor, adamts13, and thrombotic thrombocytopenic purpura impact of mutations in the von willebrand factor a2 domain on adamts13-dependent proteolysis effect of von willebrand disease type 2b and type 2m mutations on the susceptibility of von willebrand factor to adamts-13 binding of platelet glycoprotein iba to von willebrand factor domain a1 stimulates the cleavage of the adjacent domain a2 by adamts13 factor viii and platelets synergistically accelerate cleavage of von willebrand factor by adamts13 under fluid shear stress platelet-vwf complexes are preferred substrates of adamts13 under fluid shear stress role of thrombospondin-1 in control of von willebrand factor multimer size in mice thrombospondin-1 controls vascular platelet recruitment and thrombus adherence in mice by protecting (sub)endothelial vwf from cleavage by adamts13 proteolytic cleavage of recombinant type 2a von willebrand factor mutants r834w and r834q: inhibition by doxycycline and by monoclonal antibody vp-1 a novel binding site for adamts13 constitutively exposed on the surface of globular vwf evaluation of adamts-13 activity in plasma using recombinant von willebrand factor a2 domain polypeptide as substrate mechanoenzymatic cleavage of the ultralarge vascular protein von willebrand factor structural specializations of a2, a force-sensing domain in the ultralarge vascular protein von willebrand factor cleavage of ultra-large von willebrand factor by adamts-13 flow conditions vwf73, a region from d1596 to r1668 of von willebrand factor, provides a minimal substrate for adamts-13 n-linked glycosylation of vwf modulates its interaction with adamts13 factor viii accelerates proteolytic cleavage of von willebrand factor by adamts13 recombinant cub-1 domain polypeptide inhibits the cleavage of ulvwf strings by adamts13 under flow conditions anti-vwf antibodies induce gpibalpha and fcgammarii mediated platelet aggregation only at low shear forces preparation and characterization of monoclonal antibodies against human von willebrand factor a1 domain two novel monoclonal antibodies to vwfa3 inhibit vwf-collagen and vwf-platelet interactions studies on monoclonal antibodies to human von willebrand factor the mutation arg (53)-trp causes von willebrand disease normandy by abolishing binding to factor viii. studies with recombinant von willebrand factor physiologic cleavage of von willebrand factor by a plasma protease is dependent on its conformation and requires calcium ion a2 domain of human von willebrand factor expressed in e. coli and its biological activity comprehensive antibody epitope mapping of the nucleocapsid protein of severe acute respiratory syndrome (sars) coronavirus: insight into the humoral immunity of sars measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay we thank dr. j. evan sadler for providing the psvhvwf1 plasmid, dr. jing-fei dong for providing the adamts13 plasmid, and dr. hans deckmyn for providing the recombinant vwf d'd3 protein and two murine anti-vwf d'd3 mabs (1c1e7 and 75h4b12). the authors especially thank dr. x. long zheng at the children's hospital of philadelphia, philadelphia, pa, usa, for his critical reading and comments. we also thank dr. lijun xia (cardiovascular biology research program, oklahoma medical research foundation, university of oklahoma health sciences center, oklahoma city, ok, usa) and dr. yi wu (cyrus tang hematology center, soochow university, suzhou, china) for giving valuable advice during the preparation of this manuscript. key: cord-000715-zl1s82yi authors: shulman, lester m.; hindiyeh, musa; muhsen, khitam; cohen, dani; mendelson, ella; sofer, danit title: evaluation of four different systems for extraction of rna from stool suspensions using ms-2 coliphage as an exogenous control for rt-pcr inhibition date: 2012-07-16 journal: plos one doi: 10.1371/journal.pone.0039455 sha: doc_id: 715 cord_uid: zl1s82yi knowing when, and to what extent co-extracted inhibitors interfere with molecular rna diagnostic assays is of utmost importance. the qiaamp viral rna mini kit (a); magna pure lc2.0 automatic extractor (b); kingfisher (c); and nuclisens easymag (d) rna extraction systems were evaluated for extraction efficiency and co-purification of inhibitors from stool suspensions. real-time reverse transcriptase polymerase chain reaction (rrt-pcr) of ms-2 coliphage spiked into each system’s lysis buffer served as an external control for both. cycle thresholds (cts) of the ms2 were determined for rna extracted from stool suspensions containing unknown (n = 93) or varying amounts of inhibitors (n = 92). stool suspensions from the latter group were also used to determine whether ms-2 and enterovirus rrt-pcr inhibitions were correlated. specifically 23 rna extracts from stool suspensions were spiked with enterovirus rna after extraction and 13 of these stool suspension were spiked with intact enterovirus before extraction. ms2 rrt-pcr inhibition varied for rnas extracted by the different systems. inhibition was noted in 12 (13.0%), 26 (28.3%), 7 (7.6%), and 7 (7.6%) of the first 93 rna extracts, and 58 (63.0%), 55 (59.8%), 37 (40.2%) and 30 (32.6%) of the second 92 extracts for a, b, c, and d, respectively. furthermore, enterovirus rrt-pcr inhibition correlated with ms2 rrt-pcr inhibition for added enterovirus rna or virus particles. in conclusion, rrt-pcr for ms-2 rna is a good predictor of inhibition of enterovirus rna extracted from stool suspensions. easymag performed the best, however all four extraction methods were suitable provided that external controls identified problematic samples. diagnosis of enteric viral infections by real time reverse transcription -polymerase chain reaction (rrt-pcr) of rna extracted from stool suspensions is complementing and increasingly replacing diagnosis based on viral isolation and characterization in tissue culture [1, 2] . however, interpretation of results is not always straightforward. the sensitivity of the rrt-pcr is negatively impacted, by compounds present in the clinical sample that may partially or completely inhibit the rt and/or pcr chemistries [3, 4, 5, 6, 7] . potential inhibitors that might be incompletely removed from stool suspensions during rna extraction include hemoglobin, immunoglobulins, bilirubin, triglycerides, complex polysaccharides, organic and phenolic compounds, glycogen, fats, and metabolic products especially those from pathological conditions, bacteria, vegetables, medications, anticoagulants, and drugs or alcohol [4, 6, 8, 9, 10, 11] . adding to the list of endogenous rrt-pcr inhibitors are exogenous inhibitors from extraction protocols such as detergents, chelating compounds and guanidinium hcl [9] . the presence of inhibitory compounds in the extracted rna and the extent of inhibition can be determined by semiquantitative rrt-pcr of an rna template that is present in the sample, added to it prior to extraction, or introduced into the rrt-pcr mix. natural or modified, encapsulated coliphage ms2 rna is one source of protected rna that has been used as a noncompetitive external rna template control [5, 12, 13, 14] . the efficiency of removing inhibitors in patient samples may be related to the intrinsic properties of the method used to extract the rna [15] . in this study we compared four commercial rna extraction systems efficiencies for rna isolation and removal of inhibitors in stool samples. the extraction systems were: qiaamp viral rna mini kit: manual extraction using silica columns (qiagen inc, valencia, ca, usa); magna pure lc2.0 automatic extractor with magna pure lc total nucleic acid isolation kit-high performance: automatic rna extraction using magnetic beads (roche diagnostics, in, usa); kingfisher (thermo electron corporation, waltham, ma, usa) semi-automatic extraction using magnetic beads of ambion magmax viral rna isolation kit (ambion, inc, austin, tx, usa); nuclisens easymag (biomerieux, marcy l'etoile, france): semi-automatic extractor using magnetic beads of easymag extraction kit. in addition, the effectiveness of ms2 as an exogenous template control for measuring inhibitors co-extracted with rna from stool suspen-sions and its relevance for validation of enterovirus diagnostic rrt-pcr suspensions is discussed. ninety-three stool suspensions were extracted by the four extraction systems after spiking the lysis buffer of each with ms2 that yielded similar final ms2 concentrations. any contribution from endogenous ms2 rna was ruled out since no ms2 rna was amplified from any of these 93 stool suspensions upon extraction with protocol d when exogenous ms2 was omitted from the lysis buffer. results for each extraction procedure by assigned levels of inhibition are shown in table 1 . individual values are shown in fig. s1 . the proportion of samples with inhibitors varied among the extraction methods. of the 93 rna extracts, 12 (13.0%), 26 (28.3%), 7 (7.6%), and 7 (7.6%) extracted by protocols a, b, c, and d, respectively, contained inhibitors of ms2 rrt-pcr. median interquartile range (iqr) reduction in cts for protocols a, b, c and d were 2.4 (0 to 34), 1.9 (0 to 33), 0.58 (0 to 29), and 0.26 (0 to 7), respectively. finally, the number of samples with an inhibition .6 ct were 6 (6.5%), 3 (6.4%), 2 (2.2%), and 1 (1.1%) for extraction protocols a through d, respectively. the friedman test indicated that there was a statistically significant difference in co-extraction of inhibitors of ms2 rrt-pcr among the extraction protocols used, p,0.001. in order to further challenge the performance of the 4 extraction systems to exclude inhibitors of rrt-pcr, 92 stool samples with levels of inhibition ranging from a single cycle to complete inhibition were evaluated. these samples were selected from among archived stool samples previously tested for enterovirus and ms2 after extraction by qiaamp viral rna mini kit. a sufficient number of samples with high, intermediate, and low levels of inhibitors were chosen for re-analysis to enable comparison between extraction procedures at each of these levels of inhibition. the results of ms2 inhibition for each sample by extraction protocol is shown in fig. 1 and summarized by levels of inhibition in table 1 , experiment 2. the number of samples with inhibitors differed among the four protocols and for some individual samples, the amount of inhibition varied depending on the extraction protocol. specifically, inhibitors were present in 58 (63.0%), 55 (59.8%), 37 (40.2%) and 30 (32.6%) of the samples prepared by protocols a, b, c, and d, respectively. median iqr reduction in ct levels for protocols a, b, c and d were 11.7 (0 to 34), 7.0 (0 to 30), 4.2 (0 to 29), and 1.3 (0 to 30), respectively. more importantly the number and proportion of samples where inhibition was .6 ct also varied among the protocols; 44 (47.8%), 29 (31.5%), 14 (15.2%), and 1 (1.1%), for protocols a, b, c, and d, respectively. the friedman test indicated that there was a statistically significant difference in co-extraction of inhibitors of ms2 rrt-pcr among the extraction protocols used, p,0.001. to differentiate between rrt-pcr inhibition due to the presence of inhibitors in the rna after extraction and variation due to differences in efficiency of extraction, 23 rna extracts from samples with inhibitors were spiked with two concentrations of purified coxb3 enterovirus rna (fig. 2 ). the first concentration was equivalent to that of ms2 (r 1 , equivalent) and the second contained 64 fold (6 cts) more enterovirus rna (r 2 , high). no significant difference between inhibition of ms2 rrt-pcr and enterovirus rrt-pcr was noted when equivalent amounts of enterovirus rna was added to extracts prepared by protocols a, b and c (fig. 2. part 2) . however, when the higher amount of enterovirus rna was added, there was significantly less inhibition of enterovirus rrt-pcr than ms2 rrt-pcr extracts prepared using protocols b, c, and d, but not a. thus, the decrease in ms2 rrt-pcr can serve as a measure of the amount of inhibitors in the rna extracted by each of the extraction protocol. analysis of variance, indicated that there were no significant differences between inhibition of cts for ms2 and enterovirus rna for protocols a, b, and c, but not d, when the amount of enterovirus rna was equivalent to that of ms2 (p.0.5), whereas there was significantly less inhibition when the 64-fold higher amount of enteroviral rna was tested for rna extracted by protocols b, c, and d (p,0.05), but not a (p.0.05). thirteen stool suspensions with varying amounts of inhibitors were spiked with intact coxb3 virus at a concentration adjusted to yield a ct equivalent to that of the ms2. these spiked suspensions were re-extracted in parallel by all four methods. with few exceptions, the extent of inhibition of enterovirus rrt-pcr was similar to that for inhibition of ms2 rrt-pcr (fig. 3) . the number of samples with $6 ct inhibition in ms2 rrt-pcr, that also had .6 ct inhibition of enterovirus rrt-pcr, was 10/10 (100%), 9/9 (100%), 6/7 (85.7%), and 4/4 (100%) for protocols a, b, c, and d, respectively. analysis of variance ( fig. 3 , part 2), indicated that there was no significant difference (paired t-test, p.0.05) between the inhibition of rrt-pcr of the ms2 external control and the added enterovirus (p.0.05) for protocols a, c, and d. in protocol b the inhibition of the rrt-pcr for enterovirus was actually significantly higher than that for ms2 (p = 0.045). recovery of enterovirus rna by the four different protocols was equivalent. one of the trends in pathogen identification is the increasing reliance on molecular methods to achieve high throughput, high sensitivity and specificity of results in clinically relevant times [2, 16] . however, low pathogen copy number, inefficient extraction and the incomplete removal of inhibitors, reduce the sensitivity and specificity of the molecular assays. among clinical samples, stool suspensions contain a wide range of materials that have the potential to inhibit rt and/or pcr chemistries [4, 6, 8, 9, 10, 11] . thus, it is important to develop a reliable method to determine whether inhibitors are present in nucleic acid prepared directly from stool suspensions. an appropriate internal control does not exist because of wide differences in eukaryotic and prokaryotic composition in fecal material between patients. four protocols for extracting rna from stool samples were compared in this study. these four commercial extraction methods are among the most commonly used in the diagnostic laboratories. none of samples 1 to 93 contained ms2 rna. similarly nonove et al (14) did not report any unusual amounts of ms2 rna that would have indicated prior presence of ms2 in their samples when they added ms2 to 106 stool suspensions at concentrations two to eight-fold less than used in this study. ms2 coliphage was thus used as a non-competitive external control because of its absence from human clinical samples. ms2 is a better external control for rrt-pcr than plasmid or phage dna since it also measures the efficiency of the rt reaction [5, 12, 13, 14, 17, 18] . here, the ms2 was added to the lysis buffers to minimize sample manipulation and increase the uniformity between manual and semi-automatic extraction protocols. the presence of rrt-pcr inhibitors was evaluated by extracting equal volumes of stool suspension and eluting with equal volumes of elution buffer. extractions were performed in parallel to eliminate storage related differences, and rna was assayed on the same rrt-pcr run to minimize analytical differences [19] . absence of ms2 in the stool samples 1-93 was confirmed by ms2 rrt-pcr of rna extracted with the easymag rna extraction system using lysis buffer without exogenous ms2. this extraction protocol, the system that had the least amount of samples with inhibitors, was chosen to increase the chance of finding any endogenous ms2 rna in the stools. the number of stool suspension rna extracts with inhibitors of ms2 rrt-pcr varied between rna extraction protocols for randomly chosen stool suspensions. the amount of inhibitors in rna extracts for individual stool suspensions also varied according to extraction procedures. this suggests that more than one type of inhibitor may be present and that different procedures do have different proficiencies in excluding them. stool samples extracted by the kingfisher and the nuclisens easymag had fewer extracts with inhibitors than the qiaamp [18, 20, 21, 22] found that procedures similar to that used by easymag outperformed manual extraction procedures similar to qiagen, and that magna pure and kingfisher-like procedures were intermediate. in contrast fewer did not find major differences between some of these systems [23, 24] . results presented here further confirm that rna extracted with magnetic bead-based systems contained fewer inhibitors than column-based systems [21] . the number of samples with inhibitors was low in the first group of samples (samples 1-93) that were used to compare rna extraction procedures. in order to better compare the four extraction procedures, we selected from among previously tested samples, sufficient numbers of archived stool suspensions that had high, intermediate, and low levels of inhibitors after extraction by qiaamp viral rna mini kit (samples 101-192). the stool suspensions were re-extracted in parallel by all four protocols and evaluated for inhibition of ms2 rrt-pcr. as before, kingfisher and nuclisens easymag semi were better than the qiaamp viral rna mini kit and magna pure lc2.0 automatic extractor. differences in extraction efficiency might be at least as important as inhibitors, as hata et al. [25] showed by adding separate controls for both extraction and amplification. we performed a similar analysis by adding enterovirus rna to rna samples after extraction and intact enterovirus to suspensions before extraction. the inhibition of enterovirus rrt-pcr correlated with ms2 rrt-pcr inhibition. it is important to note that all four extraction protocols yielded equivalent amounts of enterovirus rna from enterovirus added to suspensions where there was no inhibition of ms2 rna rrt-pcr in the samples for equal starting and elution volumes. the pattern and amount of enterovirus rrt-pcr inhibition was similar for specific suspensions regardless of whether enterovirus was added before extraction or viral rna added to the reaction mix after extraction. this implies that the apparent decrease in the amount of ms2 rna in an rna extract where ms2 rna was added to the lysis buffer was primarily due to failure to remove inhibitors present in particular stool suspensions. the standard maximum volumes that could be extracted differ for the procedures: 140 ml for qiagen; 100 ml for magna pure; 50 ml for kingfisher; and, 200 ml for easymag. the effect of using these manufacturers' recommended volumes for stool suspensions was not evaluated in the present study, although to have done so would have meant that depending on the procedure used, there would have been a 2 to 4 fold increase in the amount of endogenous inhibitors in the starting volume without a concomitant increase in the extraction, washing and elution buffer volumes. others have shown that increasing the starting volume increased the co-extraction of inhibitors [21, 25, 26] and that the final rrt-pcr outcome is a balance between specific template and coextracted inhibitors [27] . the majority of rrt-pcr assays have a lower limit of quantitation of 10 target sequences per reaction with a ct of approximately 35 cycles. a reaction with a three-fold shift upwards in ct due the presence of inhibitors would still be positive, i.e. a specific signal would still be detectable below the 40 cycle cap recommended for the majority of rrt-pcr assays. a two ct difference is within the statistical variation that may occur between repeated analyses of the same rna. in contrast, a positive signal would no longer be detectible if there were a six-fold shift in ct, which would produce a false negative outcome. in conclusion, inhibition is a complex process. all four extraction methods were suitable provided that an external control was used to identify problematic samples. rrt-pcr of ms2 rna recovered from ms2 coliphage added to the lysis buffers of rna extraction systems is a good predictor of inhibition of enteroviral rna extracted from stool suspensions. more than one inhibitor may be present in the stool suspensions or added during extraction and their efficiency of removal differs between the extraction protocols. the correlation between the extent of ms2 rrt-pcr inhibition and enteroviral rrt-pcr inhibition increases inversely in relation to the amount of enteroviral rna in the sample. in agreement with dreier et al [5] , ms2 rrt-pcr inhibition should be tested for each rna sample from a stool suspensions each time it is tested since there is no way to predict in advance whether inhibitors have been efficiently removed during extraction or remain active after cycles of frozen storage. viral rrt-pcr results should not be considered as quantitative results when ms2 rrt-pcr is inhibited by more than 3 ct. finally, we recommend that any negative viral rrt-pcr result from a sample with an inhibition of .6 ct for ms2 rrt-pcr should be considered invalid and alternative methods used to re-assay or re-extract the sample. if the rna is diluted and re-tested only samples with positive results for enterovirus rrt-pcr should be considered as valid. the ethical review board of the sheba medical center, tel hashomer approved this study (smc-8859). the samples and results were stripped of all links to personal details pertaining to, or which could be used to identify individual patients. all data were analyzed anonymously. the ethical review board exempted this study from a requirement for obtaining informed consent. stool suspensions (n = 185) prepared for routine analysis of clinical stool samples sent to the central virology laboratory (cvl) at chaim sheba medical center in israel were used to evaluate the efficiency of four different rna extraction systems in excluding inhibitors of rrt-pcr. ninety-three of the stool samples contained unknown amounts of rrt-pcr inhibitors (table 1; fig. s1 ). the remaining ninety-two stools suspensions were selected from among 860 stool suspensions archived between 2009 and 2011 that had been sent for routine enterovirus analysis (table 1 , figs. 1, 2, 3 and s2 ). the rna from these archived suspensions had been extracted manually with the qiaamp viral rna mini kit and ms2 rrt-pcr and inhibition levels were known. ms2 rrt-pcr inhibitors .5 ct were found in 93 (10.8%) of these samples. a non-random subset of 92 of these 860 samples (samples 101 to 192) with high, intermediate, and low levels of inhibitors were chosen for re-analysis so that would be a sufficient number of samples for comparison at each of these levels of inhibition. small portions of fecal matter were vortexed for 15 seconds in stool suspension buffer, 2 ml 0.9% saline with glass beads (samples 1 to 93) or 5 ml of m199 containing 15.6 mg of dihydrostreptomycin, 15,625 u of penicillin g, and 156 u of mycostatin and 0.1 volume (v/v) of chloroform (samples 101-192). suspensions were clarified by centrifugation at 2,5006g for 10 min and stored at 220uc until use. a natural e. coli rna ms2 coliphage [28] (ms2, atcc 15597-b1) stock was prepared on e. coli top 10f in nzycm broth as described by dreier et al [5] . aliquots of the stock were frozen at 270uc. the concentrated stock was thawed, serially diluted in dilution buffer [100 mm nacl, 8 mm mgso 4 , 50 mm tris ph 7.5 and 0.01% (w/v) gelatin] and added to the respective extraction lysis buffers used in each of the extraction procedures before use. the amount of external control ms2 template added to the lysis buffers was adjusted to give a ct of approximately 27 (,10,000 copies/ml) in the rrt-pcr mix upon addition of 5 ml of rna. rna was extracted from clarified fecal suspensions using four different commercial protocols. samples were extracted in parallel to eliminate storage related differences [19] . extractions were performed according to manufacturers' instructions except that rna was extracted from 50 ml of stool suspension for all four protocols with addition of respective suspension buffer to reach the recommended aqueous volumes listed above. the rna was stored at 270uc pending analysis and between analyses (see fig. s2 ). samples 1 to 93 were also extracted by protocol d as above, except that exogenous ms2 was omitted from the lysis buffer. the abi prism 7500 sequence detection system (applied biosystems, foster city, ca, usa) was used for the amplification and detection of the ms2 and enterovirus rna by taqman technology as previously described [5, 29, 30] . briefly, for ms2 rrt-pcr, 5 ml of rna was added to the agpath mastermix (ambion, applied biosystems inc, foster city, california), which contained the published concentrations of primers and probes and 5-carboxy-x-rhodamine succinimidyl ester (rox) as an internal reference dye, whereas 8 ml of rna was added for all enterovirus rrt-pcr assays. rrt-pcr was performed under the following conditions: 30 min at 48uc, 10 min at 95uc, and 60 cycles of 15 s at 95uc and 1 min at 60uc. rnas extracted from the same stool suspension by the four procedures were assayed on the same rrt-pcr run to minimize analytical differences [19] . data were managed and analyzed using excel (microsoft) and spss (ver. 15) software. for each extraction protocol, the presence and relative amount of rrt-pcr inhibitor(s) was determined by comparing ms2 rrt-pcr ct results obtained in the absence or presence of stool suspensions. measured ms2 cts results were assigned to one of five levels of inhibition: no inhibition, inhibition of 1 to 3 cts (2 to 8 fold), inhibition of 4 to 6 cts (16 to 64 fold), inhibition of 7 to 9 cts (128 to 512 fold) and inhibition of $10 cts ($1024 fold). similarly, the presence and relative amount of inhibitors of enterovirus rna rrt-pcr was determined by comparing enterovirus rrt-pcr ct results obtained in the absence or presence of stool suspensions for enterovirus added before extraction or enterovirus rna added after extraction. the inhibition for suspensions that gave cts for ms2 rrt-pcr below that for ms2 in suspension buffer alone was set to 0. the upper limit for rrt-pcr inhibition was capped at 29 ct. the non-parametric friedman test was used to determine to determine whether there were significant differences between results among the four different extraction protocols. post-hoc analysis of pairwise differences between protocols was performed using a wilcoxon signed-rank test (spss) with a bonferroni correction that set the significance level for the pairwise comparisons to p,0.008. comparison of ms2 rrt-pcr and enteroviral rrt-pcr cts was by repeated measurements, analysis of variants. figure s1 ms2 rrt-pcr inhibition in rna extracted from stool suspensions using four different rna extraction protocols. equal amounts of stool suspensions chosen randomly from among samples sent to the laboratory were extracted by four protocols: (a) qiagen, (b) magna pure, (c) kingfisher, and (d) easymag as described in methods. ms2 coliphage calculated to give 27 ct by rrt-pcr was added to the extraction buffer. rrt-pcr values for ms2 in rna extracted from buffer controls were subtracted from the values for ms2 in rna extracted from stool suspensions. this difference, the number of cts of inhibition, is shown in the box to the right of the sample numbers. negative values were set to 0 and the maximum values for inhibition ''c'' were capped at 29 cts. samples with inhibition .10, 7 to 9, 4 to 6, and 1 to 3 ct are indicated by the colors of the boxes: black, red, tan, and light yellow, respectively. blank white boxes indicate no inhibition. the numbers of samples in each category and the significance in differences are shown in table 1 , experiment 1. (pdf) figure s2 ms2 rrt-pcr inhibition for rna extracted from stool after re-freezing and thawing by extraction protocol. 1. three repeated measurements of ms2 rrt-pcr were performed for the 23 samples by protocols: (a) qiagen, (b) magna pure, (c) kingfisher, and (d) easymag as described in methods. the rna was stored at 270uc between tests. ms2 coliphage was added to the extraction buffer. rrt-pcr values for ms2 in rna extracted from buffer alone were subtracted from the values for ms2 in rna extracted from stool suspensions. this difference, the number of cts of inhibition, is shown in the boxes to the right of the sample numbers. negative values were set to 0 and the maximum values for inhibition ''c'' were capped at 29 cts. samples with inhibition $10, 7 to 9, 4 to 6, and 1 to 3 ct are indicated by the colors of the boxes: black, red, tan, and light yellow, respectively. blank white boxes indicate that the samples were not inhibited. samples were stored at 270uc between measurements. 2. the means for each of the three repeat tests the square of the means (m sq), the standard deviation of each run (s.d.), and the significance of differences between the means of the repeated measurements by extraction protocol are presented. there were no significant differences between the repeat measurements for each extraction protocol. 3. friedman test values for comparison among the 4 extraction methods. there were significant differences among the extraction protocols (shaded yellow boxes). 4. the wilcoxon singed-rank test (spss) with bonferroni correction (significance if p,0.008) for the pairwise comparison of the means of the three measurements of the rrt-pcr results from the different extraction protocols. shaded yellow boxes indicate where pairwise differences between extraction protocols were significant. (pdf) real-time pcr in virology real-time pcr in clinical microbiology: applications for routine laboratory testing quality control in nucleic acid testing-where do we stand? inhibition and facilitation of nucleic acid amplification use of bacteriophage ms2 as an internal control in viral reverse transcription-pcr assays removal of realtime reverse transcription polymerase chain reaction (rt-pcr) inhibitors associated with cloacal swab samples and tissues for improved diagnosis of avian influenza virus by rt-pcr inhibition of legionella pneumophila pcr in respiratory samples: a quantitative approach validation of laboratory-developed molecular assays for infectious diseases complex polysaccharides as pcr inhibitors in feces: helicobacter pylori model identification and characterization of immunoglobulin g in blood as a major inhibitor of diagnostic pcr pcr inhibition in stool samples in relation to age of infants preparation of his-tagged armored rna phage particles as a control for real-time reverse transcription-pcr detection of severe acute respiratory syndrome coronavirus duplex realtime reverse transcriptase pcr assays for rapid detection and identification of pandemic (h1n1) 2009 and seasonal influenza a/h1, a/h3, and b viruses rna and dna bacteriophages as molecular diagnosis controls in clinical virology: a comprehensive study of more than 45,000 routine pcr tests evaluation of preanalytical variables in the quantification of dengue virus by real-time polymerase chain reaction molecular detection of human viral pathogns enhanced reverse transcription-pcr assay for detection of norovirus genogroup i comparison of automated nucleic acid extraction methods with manual extraction comparative evaluation of in-house manual, and commercial semi-automated and automated dna extraction platforms in the sample preparation of human stool specimens for a salmonella enterica 59-nuclease assay clinical evaluation of nuclisens magnetic extraction and nuclisens analyte-specific reagents for real-time detection of human metapneumovirus in pediatric respiratory specimens multicenter comparison of nucleic acid extraction methods for detection of severe acute respiratory syndrome coronavirus rna in stool specimens evaluation of nuclisens easymag for automated nucleic acid extraction from various clinical specimens comparison of the nuclisens easymag and qiagen biorobot 9604 nucleic acid extraction systems for detection of rna and dna respiratory viruses in nasopharyngeal aspirate samples comparison of automated and manual nucleic acid extraction methods for detection of enterovirus rna validation of internal controls for extraction and amplification of nucleic acids from enteric viruses in water samples rapid one-step quantitative reverse transcriptase pcr assay with competitive internal positive control for detection of enteroviruses in environmental samples comparison of four rna extraction methods for the detection of small round structured viruses in faecal specimens complete nucleotide sequence of bacteriophage ms2 rna: primary and secondary structure of the replicase gene molecular detection of human viral pathogns evaluation of a rapid realtime rt-pcr assay for detection of enterovirus rna in cerebrospinal fluid specimens we thank prof. david steinberg (tel aviv university) for the statistical analysis of the data. we thank virginia levy, roberto azar, ilana silberstein, batia abramovitz, victoria indenbaum, and merav weil of the central virology laboratory, sheba medical center (tel hashomer, israel) for their excellent technical assistance. key: cord-000809-9voqa1oy authors: archer, brett n.; tempia, stefano; white, laura f.; pagano, marcello; cohen, cheryl title: reproductive number and serial interval of the first wave of influenza a(h1n1)pdm09 virus in south africa date: 2012-11-16 journal: plos one doi: 10.1371/journal.pone.0049482 sha: doc_id: 809 cord_uid: 9voqa1oy background/objective: describing transmissibility parameters of past pandemics from diverse geographic sites remains critical to planning responses to future outbreaks. we characterize the transmissibility of influenza a(h1n1)pdm09 (hereafter ph1n1) in south africa during 2009 by estimating the serial interval (si), the initial effective reproductive number (initial r(t)) and the temporal variation of r(t). methods: we make use of data from a central registry of all ph1n1 laboratory-confirmed cases detected throughout south africa. whenever date of symptom onset is missing, we estimate it from the date of specimen collection using a multiple imputation approach repeated 100 times for each missing value. we apply a likelihood-based method (method 1) for simultaneous estimation of initial r(t) and the si; estimate initial r(t) from si distributions established from prior field studies (method 2); and the wallinga and teunis method (method 3) to model the temporal variation of r(t). results: 12,360 confirmed ph1n1 cases were reported in the central registry. during the period of exponential growth of the epidemic (june 21 to august 3, 2009), we simultaneously estimate a mean r(t) of 1.47 (95% ci: 1.30–1.72) and mean si of 2.78 days (95% ci: 1.80–3.75) (method 1). field studies found a mean si of 2.3 days between primary cases and laboratory-confirmed secondary cases, and 2.7 days when considering both suspected and confirmed secondary cases. incorporating the si estimate from field studies using laboratory-confirmed cases, we found an initial r(t) of 1.43 (95% ci: 1.38–1.49) (method 2). the mean r(t) peaked at 2.91 (95% ci: 0.85–2.91) on june 21, as the epidemic commenced, and r(t)>1 was sustained until august 22 (method 3). conclusions: transmissibility characteristics of ph1n1 in south africa are similar to estimates reported by countries outside of africa. estimations using the likelihood-based method are in agreement with field findings. during 2009, the emergence and worldwide spread of influenza a(h1n1)pdm09 (ph1n1) was observed [1] . while a rapid and timely estimation of the transmission parameters of this novel virus played an important role in informing transmission potential and mitigation interventions during the 2009 pandemic period, the post-pandemic documentation of these parameters is equally important as many previous estimates were established from analyses conducted during the early stages of epidemics and often from preliminary data [2, 3] . additionally enhancing our knowledge of past pandemics assists in providing greater insight to prepare and respond in future outbreaks. four key measures are typically used to describe the transmissibility of an infectious disease. first, the serial interval (si) describes the mean time between illness onset of two successive cases in the chain of transmission. second, the secondary attack rate (sar) describes the proportion of susceptible contacts that acquire infection from an infectious person. third, the basic reproductive number (r 0 ) is defined as the average number of secondary cases per primary case in an idealised entirely susceptible population in the absence of control measures. finally, the effective reproductive number (r t ) at any given time point represents the actual average number of secondary cases per primary case observed in a population. r t reflects the impact of control measures and the depletion of susceptible persons over time. the initial r t may approximate r 0 in pandemic situations. [2] [3] [4] [5] . previously published estimates of ph1n1 transmission parameters vary by study setting and methods employed. the majority of studies found the mean si of ph1n1 to range from 2.5-3.3 days [2, [6] [7] [8] [9] [10] [11] ; however, canada and texas reported a longer si of 4-5 days, respectively [12, 13] . estimates of the r 0 of pandemic influenza from the usa range from 1.3-2.3 [2, 9, 11] . estimates from mexico range from 1.4-2.9 [2, 14, 15] . outside of north america, r 0 estimates include: australia (mean 2.4) [16] , canada (mean 2.62) [12] , thailand (mean 2.07) [17] , peru (range 1.2-1.7) [18] and new zealand (mean 1.96) [19] . finally, japan revised their mean r 0 estimates from 2.3 to 1.28 after repeating analyses later in the pandemic [20] ; thus demonstrating a need to revisit revised and more complete datasets. a variation in r t with progression of the pandemic was observed in mexico, averaging at 1.47 (based on a negative binomial model) [14] , but peaking between 2.1-4.0 depending on the generation interval chosen [21] . in a previous work, we estimated the sar and si of ph1n1 among the first 100 cases detected in south africa by prospectively examining virus transmission between household contacts [22] . we found a sar of 10% and a mean si of 2.3 days (sd 61.3, range 1-5) between successive laboratory-confirmed cases in the transmission chain. when additionally including suspected secondary cases into the analysis, the sar increased to 17% and the si to 2.7 days (sd 61.5, range 1-6). in this work we incorporate data collected on all laboratory-confirmed cases detected during the 2009 ph1n1 epidemic in south africa with the aim of describing the transmissibility characteristics (initial r t and temporal variation of r t ) of the epidemic in the country and compare its dynamics with those observed in other countries in the same year. during 2009, the national institute for communicable diseases (nicd), of the national health laboratory service (nhls), south africa, maintained a central registry of all ph1n1 laboratory-confirmed cases detected throughout the country. the methodology of collating this data has previously been described in detail [23] . briefly, we collated individual case-based data from all laboratories offering ph1n1 testing throughout south africa, which included patient age, sex, dates of illness onset and specimen collection, and the administrative location (province) of the healthcare facility where the patient presented. testing was performed by accredited laboratories, including: the national influenza centre (nicd-nhls), nhls public-sector laboratories or private-sector laboratories. all testing laboratories performed detection and characterisation of ph1n1 virus by real-time pcr by either the protocol developed by the who collaborating centre for influenza, u.s. centers for disease control and prevention [24] , or using commercially available kits. wherever the date of symptom onset was missing, we estimated it from the date of specimen collection using a multiple imputation approach. firstly, we modelled the lag time from date of symptoms onset to date of specimens collection from cases with complete data via a poisson regression model using predictors significant at p,0.05. the covariates assessed in the model were patient age, gender, province, date of specimen collection, and collection of a specimen on a weekend day (i.e. saturday or sunday). secondly we obtained an estimated lag-time for each observation with missing date of symptoms onset using a random sampling process from a poisson distribution centred on the predicted value from the poisson regression model. a poisson distribution was selected to model count data. thirdly we imputed missing dates of symptoms onset by subtracting the estimated lag-time from the date of specimen collection. the imputation process was repeated 100 times for each missing value, creating 100 datasets with information on the onset date (imputed or observed) for 12,630 laboratory-confirmed cases. we based the estimation of initial r t and temporal variation of r t on date of symptoms onset (observed and imputed). in all analyses we modelled the si via a multinomial distribution. when estimating initial r t , we focus our analysis on the exponential growth phase of the epidemic in south africa (i.e. the period from the first occurrence of five consecutive days with confirmed cases reported to the epidemic peak). the parameters were estimated using three methods: method 1. we make use of the likelihood-based method for the simultaneous estimation of initial r t and the si described by [25] . this method is well suited for estimation of initial r t and si in real-time with observed aggregated daily counts of new cases, denoted by n = (n 0 , n 1 …,n t ) where t is the last day of observation and n 0 are the initial number of seed cases that begin the outbreak. the n i are assumed to be composed of a mixture of cases that were generated by the previous k days, where k is the maximal value of the serial interval. we denote these as x j , the number of cases that appear on day i that were infected by individuals with onset of symptoms on day j. we assume that the number of infectees generated by infectors with symptoms on day j follows a poisson distribution with parameter r t n j . additionally, x j = (x j,j+1 , x j,j+2 …,x j,j+k+1 ), the vector of cases infected by the n j individuals, follows a multinomial distribution with parameters p, k and x j . here p is a vector of probabilities that denotes the serial interval distribution. using these assumptions, the following likelihood is obtained: where m i~rt ( p k j~1 p j n i{j ). parameter estimates are obtained using maximum likelihood methods. for this method we used 6 days as the maximal value of the si (k), which is consistent with the length of the si observed in field investigations in south africa [22] . in addition we implemented a sensitivity analysis to assess the variation of the initial r t estimates vis-à-vis k values of 4 days and 8 days, respectively. method 2. we assume a known distribution of the si in south africa and we estimate the initial r t using the maximum likelihood estimator for known si described by [9, 25] . the estimator of initial r t in this case is a modification of method 1 and is given by: for this analysis we use the two si distributions observed from investigations of the first 100 ph1n1 cases in south africa [22] : (1) the si distribution between primary cases and laboratoryconfirmed secondary cases only (39%, 24%, 14%, 17%, 3% and 3% for day 1 to 6 respectively), and (2) the si distribution between primary cases and suspected plus laboratory-confirmed secondary cases (30%, 17%, 20%, 23%, 7% and 3% for day 1 to 6 respectively). we consider suspected secondary cases, individuals that developed ili symptoms within 14 days from the symptom onset of a confirmed index case within the same household. method 3. we make use of the wallinga and teunis' method for estimation of r t from the imputed data [26] . this method uses the daily case counts of cases and assumes the serial interval is known. we make the same assumptions for the serial interval as in method 2. the method calculates the relative probability a case on day i infects a case on day j as: table 1 . observed lag-time between date of symptom onset and date of specimen collection, incidence rate ratio (irr) and significance value of the covariates significant in the poisson regression model. where p k is the probability of a serial interval of length k. then the estimate for the reproductive number for case i, is: this method requires that we make use of the entire epidemic curve. we calculate r t as the average of the r i when i is in the epidemic period, as previously defined. estimates are reported as the means across the 100 imputations. for all estimates, we calculate bootstrap confidence intervals as has been described previously [9, 26] . we combine the results from all 100 imputations to obtain a confidence interval that incorporates both imputation error, as well as random error [27] . all analyses were performed using r version 2.14. 12,630 laboratory-confirmed ph1n1 cases were captured by the south african central registry during 2009. the overall demographic, spatial and temporal distribution of these cases has been previously described [23] . data on date of symptom onset was available for 758 (6%) cases and date of specimen collection for 12,500 (99%) cases. the first case reported illness onset of june 12, 2009 the lag-time between symptom onset and specimen collection was significantly associated with the provincial location of specimen collection, as well as the collection of a specimen on a weekend day (table 1) . we used these two covariates in the multiple-imputation to predict the date of symptom onset where missing for all cases (figure 1 ). other available variables, including date of specimen collection (period during the epidemic), patient age and sex were not significantly associated with the lag-time between symptom onset and specimen collection and, therefore, not included in the final model. analyses to simultaneously estimate initial r t and serial interval, and estimate initial r t given a known serial interval, were performed over the exponential growth phase of the epidemic from june 21 to august 3, 2009. using the likelihood-based method to simultaneously estimate initial r t and the si across 100 imputations of the dataset (method 1), we estimated ar r t of 1.47 (95% ci: 1.30-1.72) and a mean si of 2.78 days (95% ci: 1.80-3.75) (figure 2 ).r r t estimates ranged from 1.31 (95% ci: 1.21-1.48) to 1.54 (95% ci: 1.37-2.03) when the maximal value of the si ranged from 4 to 8 days. we first utilised the si established from the aforementioned field investigations of the initial 100 cases in estimating r t , as described in method 2. when performing the analysis using the si distribution observed for laboratory-confirmed ph1n1 secondary cases only (mean 2.3 days, sd 61.3, range 1-5) [22] , we found an initialr r t of 1.43 (95% ci: 1.38-1.49) ( figure 3a) . when performing the analysis using the si distribution observed for both confirmed and suspected secondary cases (mean 2.7 days, sd 61.5, range 1-6) [22] , we found an initialr r t of 1.49 (95% ci: 1.44-1.55) ( figure 3b ). figure 4 shows the variation inr r t with the progression of the outbreak over time. we observed relatively highr r t values following the introduction of ph1n1 virus into south africa, corresponding to high rates of transmission and exponential growth of the local epidemic during this period.r r t peaked on the first day of the epidemic growth period (june 21) at 2.91 (95% ci: 0.85-3.99).r r t began to drop from july 27 onward and remained consistently below one after august 22. this corresponds with the decline in the daily incidence of new cases detected. averaging the r t values obtained during the epidemic growth period (june 21 to august 3, 2009), we estimate initial r t to be 1.42 (95% ci: 1.20-1.71). utilising temporal data on illness onset and specimen collection, and the epidemic curve derived from these data, we provide estimates of the transmissibility parameters of ph1n1 during the first wave experienced in south africa. our results focus primarily on the use of analytical techniques to estimate initial r t and si without incorporating contact tracing or household transmission studies. however, when parameters from field studies are available, we show that these can be incorporated to provide robust estimates of transmission parameters. we found that initial r t estimates established using the likelihood-based method for the simultaneous estimation of r t and si (method 1: initialr r t : 1.47, si: 2.78 days) are in agreement with those obtained using si observed in field investigations [22] (method 2: initialr r t : 1.43 and 1.49 using observed si for laboratory confirmed or laboratory confirmed and suspected cases respectively). in addition, the mean si estimate obtained with method 1 (2.78 days) is in agreement with field findings (si: 2.3-2.7 days using observed si for laboratory confirmed or laboratory confirmed and suspected cases respectively). previous estimates of initial r t and the mean si for ph1n1 have ranged between 1.3-2.9 and 2.5-3.3 days, respectively [2, [6] [7] [8] [9] [10] [11] [14] [15] [16] [17] [18] [19] . our estimates are consistent with these findings, regardless of the method used for the analysis and despite difference in climate, demography and health systems across these countries. it appears that once established, the transmission characteristics of ph1n1 are very consistent. differences in transmission rates may occur within smaller subgroups of the overall population; however, this has not been well-studied. previous estimates of the epidemiological parameters of seasonal influenza epidemics found a si = 2-4 days [28] [29] [30] , and a r t a little over 1 with slight variation between climates; r t = 1.03 in brazil [31] versus r t = 1.1-1.3 in more temperate climates [32] . a number of studies have retrospectively estimated the transmissibility of influenza pandemics. during the 1918 spanish influenza a(h1n1) pandemic, when assuming a si = 4 days, r 0 estimates range from 2.0-4.3 in community settings [33, 34] , and even higher values (r 0 = 2.6-10.6) in confined settings such as ships and prisons [34] . a separate analysis predicted a slightly lower si of 3.3 in community settings and a si of 3.81 in confined settings during the 1918 pandemic, and subsequently estimated r 0 values of 1.34-3.21 and 4.97 in these respective settings [35] . r 0 estimates from the 1957 asian influenza a(h2n2) pandemic range from 1.65-1.68 [36, 37] . during the first wave of the 1968-1969 hong kong influenza a(h3n2) pandemic, estimates of r 0 range from 1.06-2.06 and increased to 1.21-3.58 during the second wave [38] . given our findings, the overall transmissibility of ph1n1 in south african during 2009 was more similar to that of seasonal influenza strains than the 1918 pandemic, and comparable to lower end estimates of the latter pandemics. however, by showing variation in transmissibility with time, we demonstrate that shortly after introduction of ph1n1 into the country, transmission of the virus reached anr r t of 2.9, resulting in exponential growth of the local epidemic and widespread illness. nonetheless, we show that after a period of less than 2 months of heightened transmission,r r t dropped below 1, corresponding to a decline in the incidence of new cases; likely a result of a combination of herd immunity, there are several limitations in this analysis which merit discussion. first, we assume that all cases are known and reported. it has been shown previously that, if cases are not reported, this may bias estimates generated using this method [39] . if the proportion of cases reported remains consistent over the study, then the estimates of transmissibility will not be biased; however, if the reporting fraction varies through time, then biased estimates of the reproductive number and serial interval may result. likewise, variation in case ascertainment with time may bias our estimates of the temporal variation of r t . generally higher reporting rates may be anticipated in the early phase, with reporting fatigue later becoming a factor. secondly, data for this study are derived from laboratory-based surveillance data from several regions across south africa; a large and diverse country. our findings do not incorporate heterogeneities (such as spatial and demographic differences) that likely exist in transmission patterns, or assess the degree to which these impact aggregate measures of initial r t . methodologies that incorporate heterogeneities inherent in public health data warrant further study. despite these limitations, the post-pandemic estimates presented here add to the body of knowledge of ph1n1 transmissibility parameters, which were previously dominated by estimates from developed nations and often based on preliminary data. it remains important that revised parameters, from complete datasets and diverse geographies, are incorporated into planning mitigation strategies for future pandemics. nonetheless, the methods used in this study would be adaptable to generating real-time estimates during future epidemics. as we continue to build epidemiological capacity in developing nations, including south africa, we must keep in mind the need for rapid assessments of transmissibility of novel pathogens, in addition to disease severity, to better inform public health interventions. pandemic (h1n1) 2009 -update 112 the transmissibility and control of pandemic influenza a (h1n1) virus transmission parameters of the a/h1n1 (2009) influenza virus pandemic: a review mathematical models of infectious disease transmission comparative estimation of the reproduction number for pandemic influenza from daily case notification data household transmission of 2009 pandemic influenza a (h1n1) virus in the united states outbreak of 2009 pandemic influenza a (h1n1) at a new york city school assessment of secondary attack rate and effectiveness of antiviral prophylaxis among household contacts in an influenza a(h1n1)v outbreak in kobe estimation of the reproductive number and the serial interval in early phase of the 2009 influenza a/h1n1 pandemic in the usa comparative epidemiology of pandemic and seasonal influenza a in households serial intervals and the temporal distribution of secondary infections within households of 2009 pandemic influenza a (h1n1): implications for influenza control recommendations estimated epidemiologic parameters and morbidity associated with pandemic h1n1 influenza household transmission of pandemic (h1n1) pandemic potential of a strain of influenza a (h1n1): early findings initial human transmission dynamics of the pandemic (h1n1) 2009 virus in north america early transmission characteristics of influenza a(h1n1)v in australia: victorian state a preliminary analysis of the epidemiology of influenza a(h1n1)v virus infection in thailand from early outbreak data epidemiological and transmissibility analysis of influenza a(h1n1)v in a southern hemisphere setting: peru estimating the reproduction number of the novel influenza a virus (h1n1) in a southern hemisphere setting: preliminary estimate in new zealand pros and cons of estimating the reproduction number from early epidemic growth rate of influenza a (h1n1) a preliminary estimation of the reproduction ratio for new influenza a(h1n1) from the outbreak in mexico introduction of 2009 pandemic influenza a(h1n1) into south africa: clinical presentation, epidemiology and transmissibility of the first 100 cases interim report on pandemic h1n1 influenza virus infections in south africa cdc protocol of realtime rtpcr for swine influenza a(h1n1), revision 1 a likelihood-based method for real-time estimation of the serial interval and reproductive number of an epidemic different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures statistical analysis with missing data estimation of the serial interval of influenza strategies for containing an emerging influenza pandemic in southeast asia risk factors of influenza transmission in households the reproduction number of seasonal influenza epidemics in brazil seasonal influenza in the united states, france, and australia: transmission and prospects for control transmissibility of 1918 pandemic influenza estimates of the reproduction numbers of spanish influenza using morbidity data transmissibility of the influenza virus in the 1918 pandemic containing pandemic influenza with antiviral agents potential impact of antiviral drug use during influenza pandemic estimates of the transmissibility of the 1968 (hong kong) influenza pandemic: evidence of increased transmissibility between successive waves reporting errors in infectious disease outbreaks, with an application to pandemic influenza a/h1n1 we would like to acknowledge and thank the many individuals and organisations who contributed to the investigation and response to pandemic influenza in south africa, including the public-and privatesector laboratories that contributed data to the national central registry of confirmed cases. we also acknowledge prof. marc lipsitch, harvard school of public health, who contributed to the conceptualization of this analysis. key: cord-000158-d08buwtu authors: corti, davide; langedijk, johannes p. m.; hinz, andreas; seaman, michael s.; vanzetta, fabrizia; fernandez-rodriguez, blanca m.; silacci, chiara; pinna, debora; jarrossay, david; balla-jhagjhoorsingh, sunita; willems, betty; zekveld, maria j.; dreja, hanna; o'sullivan, eithne; pade, corinna; orkin, chloe; jeffs, simon a.; montefiori, david c.; davis, david; weissenhorn, winfried; mcknight, áine; heeney, jonathan l.; sallusto, federica; sattentau, quentin j.; weiss, robin a.; lanzavecchia, antonio title: analysis of memory b cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from hiv-1-infected individuals date: 2010-01-20 journal: plos one doi: 10.1371/journal.pone.0008805 sha: doc_id: 158 cord_uid: d08buwtu background: the isolation of human monoclonal antibodies (mabs) that neutralize a broad spectrum of primary hiv-1 isolates and the characterization of the human neutralizing antibody b cell response to hiv-1 infection are important goals that are central to the design of an effective antibody-based vaccine. methods and findings: we immortalized igg(+) memory b cells from individuals infected with diverse clades of hiv-1 and selected on the basis of plasma neutralization profiles that were cross-clade and relatively potent. culture supernatants were screened using various recombinant forms of the envelope glycoproteins (env) in multiple parallel assays. we isolated 58 mabs that were mapped to different env surfaces, most of which showed neutralizing activity. one mab in particular (hj16) specific for a novel epitope proximal to the cd4 binding site on gp120 selectively neutralized a multi-clade panel of tier-2 hiv-1 pseudoviruses, and demonstrated reactivity that was comparable in breadth, but distinct in neutralization specificity, to that of the other cd4 binding site-specific neutralizing mab b12. a second mab (hgn194) bound a conserved epitope in the v3 crown and neutralized all tier-1 and a proportion of tier-2 pseudoviruses tested, irrespective of clade. a third mab (hk20) with broad neutralizing activity, particularly as a fab fragment, recognized a highly conserved epitope in the hr-1 region of gp41, but showed striking assay-dependent selectivity in its activity. conclusions: this study reveals that by using appropriate screening methods, a large proportion of memory b cells can be isolated that produce mabs with hiv-1 neutralizing activity. three of these mabs show unusual breadth of neutralization and therefore add to the current panel of hiv-1 neutralizing antibodies with potential for passive protection and template-based vaccine design. neutralizing antibodies provide one arm of the adaptive immune response against the human immunodeficiency virus type 1 (hiv-1). several reports demonstrated that the neutralizing antibody response exerts selective pressure during hiv-1 replication in vivo, which accounts in part for the extensive variation in the env gene observed soon after primary infection [1, 2] . furthermore, selective pressure imposed by neutralizing antibodies has been demonstrated in a human trial where three neutralizing monoclonal antibodies (mabs) administered during haart treatment-interruption led to a reduction in viremia followed by selection of escape mutants [3, 4] . passive transfer studies in macaques showed that the administration of hiv-1 neutralizing mabs protects against vaginal or intravenous challenge with siv-hiv-1 chimeric viruses (shiv) [5, 6, 7, 8, 9] . in some models protection depended not only on viral neutralization but also on fc-mediated antibody effector functions [10, 11] . given the predicted low-titer inoculum driving hiv-1 sexual transmission, a vaccine capable of eliciting antibodies that neutralize a broad spectrum of viral strains could potentially reduce or prevent infection. it has been anticipated that the identification of broadly neutralizing mabs from hiv-1 infected individuals, and the characterization of their cognate epitopes will be instrumental in the design of immunogens capable of eliciting such a broad neutralizing response [12] . this idea has led to a major international cooperative effort within consortia of laboratories with complementary expertise in human immunology, structural biology and vaccine design [13, 14] . hiv-1 is characterized by an extraordinary genetic diversity, reflected by the presence of several clades (subtypes), a fact that represents a significant impediment to vaccine development. env is the most variable hiv-1 gene, with up to 35% sequence diversity among clades, 20% diversity within clades, and 10% diversity in a single infected individual [15, 16, 17] . several conserved epitopes have been defined by a small panel of neutralizing mabs isolated using different experimental approaches. one epitope that appears to be relatively conserved and overlaps with the cd4 binding site (cd4bs) on the surface env glycoprotein gp120 is recognized by mab b12, which is the most potent and broadly-reactive mab of such specificity [18, 19, 20] . this site was recently shown to be a significant target of neutralizing antibodies present in the sera of selected patients [21, 22] . however, b12 was derived from a phage library in which heavy and light chains have been randomly reassorted, thus its relevance to naturally-occurring b cell responses in hiv-1 infection is unclear. a second epitope in a carbohydraterich region on the outer domain of gp120 is composed of glycans recognized by mab 2g12, which shows an unusual interlocked vh domain-swapped dimer generating an extended and monovalent binding surface [23, 24, 25, 26] . the 2g12 epitope is not present in the majority of clade c isolates [27] , but, of more concern, no 2g12-like activity has been detected in the sera of hiv-1 infected individuals [21, 22] , suggesting that this type of neutralizing antibody may not be generally amenable to elicitation by b cells. a third target of the broadly-neutralizing mab 4e10, and relatively broad mabs 2f5 and z13, is located on the membrane-proximal external region (mper) of the transmembrane glycoprotein gp41 [28, 29, 30, 31] . as with 2g12, mperspecific neutralizing antibodies are infrequently encountered in the sera of hiv-1-infected individuals [21, 32, 33] , potentially reducing their relevance to vaccine development strategies. moreover, mper-specific mabs have features that suggest they are products of autoreactive b cells, casting doubts over the possibility of inducing such antibodies by vaccination [34] . a fourth target of neutralizing mabs is the v3 loop, which is immunogenic but also a highly variable region involved in env-coreceptor binding. antibodies to the v3 loop, such as mabs 447-52d and f425-b4e8, are mostly effective against neutralization sensitive (tier-1) viruses and have generally a limited breadth of reactivity almost exclusively specific for clade b viruses, reflecting their provenance from clade b-infected individuals [11, 35, 36, 37] . a cluster of human v3 mabs raised from b cells derived from non-b cladeinfected individuals showed variable neutralizing activity for two ag and two c clade primary isolates, although these were particularly neutralization sensitive [38] . finally, the cd4-induced (cd4i) site has been described as a neutralization epitope primarily on tier-1 isolates with limited accessibility on tier-2 isolates [39] . very recently, the isolation and partial characteriza-tion of two related mabs from a clade a-infected donor that appear to have very broad and potent neutralization profiles was reported [40] . these mabs recognize a discontinuous epitope only expressed on the native env trimer, made up of segments of the cd4i, v2 and v3 loops. this publication is the first to provide new broadly neutralizing reagents from a non-clade b donor and provides proof-of-principle that new neutralization specificities remain to be discovered on hiv-1 env. since the number of existing neutralizing mabs with a relatively broad neutralization profile is very limited, and those described to date, with the exception of the walker et al mabs were isolated from clade b virus-infected donors, we undertook the task of isolating new human mabs from hiv-1-infected donors. our primary aims were: i) to isolate novel mabs with cross-clade neutralizing activity from non-b clade donors; ii) to analyze the memory b cell repertoire in a representative panel of predominantly non-b clade-infected individuals. by using an improved memory b cell immortalization method [41] , combined with highthroughput parallel screening with a panel of recombinant envbased antigens, we isolated a panel of 58 human mabs which we have characterized with regard to epitope specificity and breadth of neutralization. we have more fully characterized three mabs from this panel that demonstrated complementary and relatively broad cross-clade neutralizing profiles that target the gp120 cd4bs and the v3 crown, and the gp41 heptad repeat 1 (hr-1). the study was approved by the ethical committee at institute of tropical medicine and queen mary university. all participants gave written informed consent. tzm-bl and hos.cd4-r5 cells were obtained from the nih-aids research and reference reagent program (arrrp). 293t/17 cells were obtained from the atcc. mabs 2g12 [29] , 2f5, 4e10 [42] , b12 [18] , 3d6 [43] and 5f3 [29] were obtained from polymun scientific gmbh (austria), as part of the collaboration for aids vaccine discovery program. the clade b and c hiv-1 reference panels of env clones [44, 45] and the sf162 clone were obtained through the nih-arrrp. other non-clade b isolates were provided by the comprehensive antibody vaccine immune monitoring consortium (ca-vimc). hiv-1 subtype b clone jrfl was provided by dennis burton (scripps institute, la jolla, us). pseudoviruses were produced by co-transfecting hek293t/17 cells with the envexpressing plasmids and the complementing viral-genome reporter gene vector, pnl4-3.luc + .e 2 r + (kindly provided by john r. mascola, vrc, niaid, nih, us). recombinant gp140 from hiv-1 isolates 92ug37, ca18, cn54, k530, ug21 and br29 were provided by simon jeffs (imperial college london, uk). recombinant gp120 from hiv-1 isolates cm235, 93th975, bal and sf162 were obtained through the nih-arrrp, while gp160 from isolates mn and lai were purchased by prospec-tany technogene ltd (israel). recombinant yu2 gp120 and the two mutants d368r and i420r were provided by john r. mascola and richard wyatt (vrc, niaid, nih, us). recombinant soluble cd4 and recombinant gp120 from hiv-1 iiib and cn54 were obtained from the cfar, nibsc (uk). the recombinant ectodomain of gp41 from isolate hxb2 (amino acids 541-682) was purchased by vybion inc. (us). hr-1 is a modified version of gp41 [46] exposing residues hllqltvwgikqlqarilave (hxb2). 5hb was produced as described [47] . hr-1-fp comprises gp41-residues 512-594 (hxb2) including the fusion peptide and hr-1. hr-2 (hxb2) comprises gp41 residues 591-693 (hxb2) including the cys-loop region, hr-2 and mper flanked by two coiled coil regions. patients were selected for inclusion into the study on the basis of the clade of their infecting virus (predominantly non-b clade) and on the ability of their plasma to neutralize a panel of hiv-1 primary isolates using two differerent assays. at the institute for tropical medicine a panel of 4 primary a strains (vi191, 92ug37, vi820, vi 1031), 4 primary c strains (vi829, vi882, vi1144, vi1358) and 6 primary crf02 strains (vi1090, vi2680, ci20, ca18, vi1380, vi2727) was used to select patients with .80% neutralization was measured at a 1/20 dilution of plasma. the plasma dilution was mixed with virus for 24 hours and absorbed to pha/il-2 stimulated freshly isolated pbmc for 1 hour. virus-antibody mixture was then removed by extensive washing and p24 elisa was performed after 14 days of culture to determine neutralization. at queen mary university of london, the tzmbl-based recombinant pseudovirus assay was employed with a selected panel of tier 2-type clade a, b, c, crf02_ag, _ae, f and bf viruses to measure .50% neutralization at a 1/20 dilution. mabs were isolated using a previously described improved ebv immortalization method [41] . culture supernatants were harvested 14 days after immortalization and assayed in parallel for binding to trimeric gp140 proteins (ug37, clade a and cn54, clade crf07_bc) [48] , monomeric gp120 (cn54, crf07_bc and iiib, clade b) and gp41 recombinant ectodomain (hxb2, clade b). those that were positive were then subcloned and grown up on a large scale for supernatant purification. the purity of all mab preparations was assessed using a chromogenic lal endotoxin assay (genscript) and endotoxin levels were always ,0.05 eu/ml. a standard elisa was used to determine binding of mabs to the panel of hiv-1 envs. briefly, elisa plates were coated with each env antigen, blocked with 10% fcs in pbs, incubated with human mabs and washed. bound mabs were detected by incubation with ap-conjugated goat anti-human igg (southern biotech). plates were then washed, substrate (p-npp, sigma) was added and plates were read at 405 nm. the relative affinities of mabs binding to respective coated antigens were determined with elisa by measuring the concentration of each mab required to achieve 50% maximal binding at saturation (k 50 ). the ability of mabs to inhibit binding of scd4 to gp120 or gp140 was evaluated by elisa. serial dilutions of mabs were pre-incubated with gp120 (or gp140) and added to plates pre-coated with scd4. after 1 h plates were washed and incubated with sheep polyclonal antibody d7324 (aalto bio-reagents) followed by washing, incubation with ap-conjugated rabbit anti-sheep igg antibody (abcam, cambridge, uk), extensive washing and detection as above. mabs were purified on protein g columns (ge healthcare) and biotinylated using the ez-link nhs-peo solid phase biotinylation kit (pierce). the competition between unlabeled and biotinylated mabs for binding to immobilized env antigens was measured by elisa. briefly, unlabelled competitor mabs were added at different concentrations. after 1 h biotinylated mabs were added at a concentration corresponding to the 70-80% of the maximal od level. after incubation for 1 h, plates were washed and bound biotinylated mab was detected using aplabeled streptavidin (jackson immunoresearch). the percentage of inhibition was tested in triplicates and calculated as follow: (12[(odsample-odneg ctr)/(odpos ctr -odneg ctr)])6100. overlapping linear 15-mer and cyclized 15-mer peptides based on gp160 of hiv-1 ug037 and 93mw965 and the overlapping 15-mer peptides of gp41 strain sf162 were synthesized on polypropylene support (minicards), and were tested for reactivity with mabs as described [49, 50] . pepscan peptide binding analysis was carried out by an elisa-based format in which the colored substrate was quantified with a charge-coupled device (ccd)camera and an image processing system. the values mostly ranged from 0 to 3000, a log scale similar to 1 to 3 of a standard 96-well plate elisa-reader. a single-cycle infectivity assay was used to measure the neutralization of luciferase-encoding virions pseudotyped with the desired hiv-1 env proteins, as previously described [51] . briefly, appropriate dilutions of the virion-containing culture supernatants were pre-incubated at 37uc for 1 h with mabs at various concentrations. the virus-mab mixtures were added to hos-cd4-ccr5 cells and incubated for 3 days at 37uc. a similar protocol was used for supernatants screening using tzm-bl cells [45] . the cells were then lysed with britelite reagent (perkin-elmer) and the relative light units in the cell lysates were determined on a luminometer microplate reader (veritas, turner biosystems). the 50% inhibitory dose (ic50) was defined as the sample concentration at which relative luminescence units were reduced 50% compared to virus control wells. some of the neutralization data for the reference mabs b12, 2g12, 2f5 and 4e10 are taken from previous analyses [41, 42] carried out under identical standardized conditions to those used for the new mabs reported here. a multi-cycle virus replication assay was used to measure the neutralization of replication competent luciferaseencoding viruses in 5.25.egfp.luc.m7 cells [52] . this cell line is a genetically engineered clone of cemx174 that expresses multiple siv and hiv-1 entry receptors (cd4, ccr5, cxcr4, gpr15/bob) [53] . for the neutralization assay, 5000 tissue culture infectious dose 50 (tcid 50 ) of virus was incubated with multiple dilutions of test sample in triplicate for 1 hr at 37uc in a total volume of 150 ml in 96-well flat-bottom culture plates. a 100 ml suspension of cells (5610 5 cells/ml of growth medium containing 25 mg deae dextran/ml) was added to each well. plates were incubated until approximately 10% of cells in virus control wells were positive for gfp expression by fluorescence microscopy (approximately 3 days). at this time, 100 ml of cell suspension was transferred to a 96-well white solid plate (costar) for measurements of luminescence using the britelite luminescence reporter gene assay system (perkinelmer life sciences). plasma samples were obtained from patients entered into the antwerp and london cohorts who were infected with a variety of hiv-1 clades. patients were selected for mab preparation on the basis of the clade of their infecting virus (predominantly non-b clade), and on the ability of their plasma to neutralize, to .80% at a 1/20 dilution, members of a selected panel of tier 2-type clade a, b, c, crf02_ag, ae, f and bf viruses in both a pbmcbased infectious primary isolate virus assay [54] and in the tzmbl-based recombinant pseudovirus assay [55] . in some cases only a restricted plasma neutralization analysis was possible due to limiting amounts of patient plasma. twelve and nine infected donors from the london and antwerp cohorts respectively were selected for b cell immortalization. summarized clinical and virological data including hiv-1 clade, viremia, cd4 + t cell counts, therapy and breadth of neutralization are shown in table s1 . igg + memory b cells were isolated from the selected donors and immortalized with ebv in the presence of cpg as described [41] . the immortalization efficiency of memory b cells from hiv-1 patients was significantly lower than that of non-hiv-1-infected donors (3% versus 20%, n = 21, p,0.001). this finding may be explained by the recent report that hiv-specific b cells are present within a population of ''exhausted'' memory b cells, characterized by the expression of inhibitory receptors and low levels of cd21 [56, 57] . to maximize detection of antibodies recognizing conserved features, cell culture supernatants were screened by parallel highthroughput elisas using five recombinant hiv-1 env proteins, including trimeric gp140 [48] , monomeric gp120 and gp41 recombinant ectodomains. from the 21 donors interrogated we selected 58 b cell clones producing mabs that bound to at least one of the screening antigens. the mabs were purified and further characterized for binding specificity and neutralizing activity using an extended panel of recombinant env proteins and pseudoviruses representative of several hiv-1 clades with diverse coreceptor usage, geographic origin and conformation. the binding data for each mab are displayed in figure s1 and expressed as half-maximal binding concentrations at equilibrium (k 50 ), which approximate to the equilibrium dissociation constants [58] . of the 58 mabs, 37 bound to gp120 and 21 to gp41. several gp120-specific and gp41-specific mabs showed a broad pattern of reactivity with most recombinant proteins, although usually within a broad range of k 50 values. overall, there was no relationship between the donor's hiv-1 clade and the clade specificity of the isolated mabs. the neutralizing activity of the mabs was initially measured using 20 pseudotyped hiv-1 primary isolate envs characterized by different sensitivity to neutralization [44, 45] . the mabs were tested at a fixed concentration (100 mg/ml) in a luciferase-based neutralization assay using hos-cd4.r5 as target cells. out of the 46 mabs tested against the whole virus panel, 37 showed neutralizing activity on at least one isolate ( figure 1a) . three mabs, hj16, hgn194 and hk20, stood out for their breadth of neutralizing activity, neutralizing 10, 11 and 17 out of the 20 pseudoviruses, respectively ( figure 1a) . these mabs were further characterized in the same assay for their potency ( figure 1b) . hj16 showed high neutralizing activity, while hgn194 and hk20 were less potent. in addition, while most antibodies preferentially neutralized tier-1 isolates, hj16 preferentially neutralized tier-2 isolates ( figure 1a-b) . since it has been reported that hiv-1 neutralization is target cell type-sensitive [59, 60] , we next used tzm-bl as target cells and titrated the mabs against a larger panel (n = 46) of predominantly (38/46) tier-2 pseudoviruses (table s2) . generally the results obtained with the extended panel confirmed the previous results and showed that several mabs had potent neutralizing activity, with ic 50 values ,20 ng/ml. a striking exception was hk20, which neutralized 17/20 pseudoviruses in the hos-based assay, but only 3/46 pseudoviruses in the tzm-bl based assay. we established the breadth of neutralization of these three novel mabs by comparing them in the tzmbl assay with the five mabs currently considered to be the most broadly reactive, namely b12, 2g12, 2f5, 4e10 and 447-52d. we used a large multi-clade panel comprising 10 tier-1 and 82 tier-2 isolates including clade b early-transmitted viruses [61] . the three new mabs showed a pattern that was clearly distinct from that of previously described mabs (figure 2 ). in particular, hgn194 neutralized with high potency all tier-1 viruses from clade a, b and c and 11% of tier-2 viruses. by contrast hj16 neutralized only 1 out of 10 tier-1 viruses, but neutralized 39% of tier-2 viruses. thus hj16 is comparable to b12 and 2f5 in terms of percentage of tier-2 isolate neutralization and is superior to 2g12 and 447-52d. 4e10, as previously reported [27, 41] showed an extremely broad pattern of reactivity. taken together the above results indicate that novel neutralizing mabs can be isolated from memory b cells of nonclade b hiv-1-infected individuals, some of which display relatively broad neutralizing activity. since one of our primary aims was to isolate and characterize novel mabs with unusually broad and potent neutralization activity, we focused our attention on hj16, hk20 and hgn194. mab hj16, derived from a donor infected with clade c, has a unique neutralization profile with potent and selective neutralization of multiple tier-2 pseudoviruses ( figure 1b and figure 2 ). when compared with the cd4bs-specific mab b12 for gp120 binding, hj16 showed similar binding curves ( figure 3a ) and inhibited to a comparable extent the binding of gp120 to solid-phase scd4 ( figure 3b , ic 50 values of 1.57 and 1.16 mg/ml, respectively). however, cross-competition analysis of hj16 and b12 for binding to gp120 revealed incomplete heterologous inhibition, with plateau values of approximately 80% ( figure 3c-d) . these results suggest that hj16 and b12 recognize related but non-overlapping cd4bsproximal epitopes. to further characterize hj16 specificity we measured its binding to two yu2 gp120 mutants: the d368r mutant, which is not bound by cd4 or cd4bs-specific mabs [21] and the i420r mutant, which is not recognized by cd4i-specific mabs [62] . as already reported, b12 bound the i420r cd4i mutant, but failed to recognize the d368r cd4bs mutant. by contrast, hj16 bound both mutants and indeed bound better to the d368r cd4bs mutant compared to the wild-type molecule ( figure 3e-f) . attempts to map the epitope by pepscan analysis using overlapping linear peptides were unsuccessful (not shown), consistent with hj16 recognition of a discontinuous epitope. hj16 and b12 neutralized 1/10 and 8/10 tier-1 and 32/82 and 35/82 tier-2 pseudoviruses, respectively ( figure 2) . interestingly, 22/32 tier-2 isolates neutralized by hj16 were not neutralized by b12, and reciprocally 24/35 tier-2 isolates neutralized by b12 were not neutralized by hj16. another interesting finding is that hj16 does not discriminate between clades as much as b12, 2g12 and 2f5 (e.g. b12 and 2g12 rarely neutralize clade a isolates while 2f5 and 2g12 rarely neutralize clade c isolates, table s3 ). these results reveal a largely nonoverlapping pattern of reactivity of hj16 and b12 and suggest that the combination of these two mabs could be effective against a dominant fraction of hiv-1 isolates (57/82, i.e. 69%). taken together these results are consistent with hj16 recognition of a surface proximal to the cd4bs domain within gp120, but with an epitope completely distinct from that recognized by b12. mab hgn194, isolated from a donor infected with crf02_ag clade, neutralized 11/20 pseudoviruses in the hos-based assay and 19/92 pseudoviruses in the tzm-bl-based assay. of note, hgn194 neutralized all tier-1 isolates tested (10/10 neutralized) across clades a, b, c and recombinant ag and bc. using pepscan analysis with linear and cyclic peptide libraries of gp120 the epitope recognized by hgn194 was mapped to the sequence rrsvrigpgqtf in the crown of the v3-loop ( figure 4a) . similarly, the epitope of 7 additional gp120specific mabs was mapped to the same v3 region using different peptide libraries generated from the sequence of the isolate to which each mab was mostly reactive ( figure 4a) . the minimal sequence recognized by these mabs ranged from 7 to 17 amino acids and, with a single exception, comprised the g(a)pgr/q/k sequence, which is modeled to interact with ccr5 or cxcr4 during the viral entry process [63] . however, in contrast to hgn194 which neutralizes with high potency all tier-1 isolates tested, the other v3-specific mabs neutralized only a few tier-1 isolates ( figure 1a -b and figure 2 ), similar to the activity of other recently characterized v3 loop-specific mabs derived from non-b clade infected individuals [38] . we confirmed the neutralization activity of hgn194 in an m7 cell-based assay challenged with replication-competent hiv-1 viruses carrying a luc reporter [52] and we observed that 3/5 clade b viruses were neutralized (table s5) . to better characterize the epitope recognized by hgn194 we performed a replacement scanning of each position with the 18 complementary amino acids. this analysis revealed only 3 positions (rrsvrigpgqtf) where amino acid substitutions abrogated binding. remarkably, only one mutation out of the 21 found in viral isolates (i to m in position 6) affected binding of hgn194 ( figure 4b-c) . however, we observed that several hiv-1 isolates that were not neutralized by hgn194 encoded the same amino acid sequence shared by other hiv-1 isolates that were neutralized ( figure 4b ). for instance, the epitope rksvrigpgqtf on 93mw965.26, which is strongly neutralized by hgn194 (i.e. ic50 ,0.02 mg/ml), is shared with du422.1, zm197m.pb7 and du172.17, isolates not neutralized by hgn194. this implies that the hgn194 v3 epitope is unavailable for antibody recognition on the assembled trimer of these non-neutralized isolates. taken together these results indicate that hgn194 has unusual potency and breadth for a v3-specific monoclonal antibody. of note, hgn194 appears to have a broader reactivity than 447-52d since it neutralizes all tier-1 isolates and 11% of tier-2 isolates, while 447-52d neutralizes 88% of tier-1 and 4% of tier-2 isolates (figure 2 and table s3 ). another v3 mab with unusual breadth of activity is f425-b4e8 [37] . we have not compared hgn194 with f425 here, but f425-b4e8 appears somewhat broader than 447-52d, neutralizing 1 clade c and 2 clade d pseudoviruses [37] . further comparison between f425-b4e8 and hgn194 is difficult as different viruses and assays were used to determine neutralization breadth and potency. hk20, an hr-1 specific mab with target cell-specific neutralizing activity hk20 was initially characterized as a gp41-specific mab with broad neutralizing activity in the hos-based assay but lacking robust activity in the tzm-bl assay. to map the epitope, we tested hk20 against all overlapping 15-mer peptides of the extracellular region of hiv-1 gp41. hk20 bound peptide qqhllqltvwgikql that overlaps the hydrophobic pocket sequence of hr-1 ( figure 5a ). the specificity of hk20 was confirmed by immunoprecipitation of the 5-helix bundle (5hb) construct [47] and of a trimeric hr-1 construct that includes the gp41 fusion peptide, indicating that the fusion peptide does not interfere with hk20 binding ( figure 5b ). in elisa, hk20 bound to 5hb and hr-1 gp41 constructs with k 50 values of 210 and 95 ng/ml, respectively and also to the gp41 ectodomain, although with lower avidity (1.27 mg/ml) ( figure 5c ), a finding that may be due to the partial unfolding of solid phase-bound gp41. taken together, the above results indicate that hk20 recognizes a highly conserved site within the hr-1 region of gp41. since hr-1 occupies a restricted surface only transiently exposed during the hiv-1 entry process, we hypothesized that accessibility of the hk20 target epitope might be limited by the size of an igg molecule. we therefore compared intact igg and fab fragments of hk20 for their capacity to neutralize a panel of 22 pseudoviruses in the hos-based assay. the hk20 fab fragments showed markedly increased breadth and potency compared to igg, being able to neutralize 21/22 isolates with ic 50 values ranging from 0.01 to 5.7 mg/ml ( figure 5d -e and table s4 ). by contrast, hk20 igg neutralized 19/22 isolates with ic 50 values ranging from 1.46 to 84 mg/ml. in addition, when the hk20 fab fragment was tested in the tzm-bl assay it neutralized 10 out of 33 isolates with ic 50 values ranging between 0.4 and 8.8 mg/ml (table s2) , thus suggesting that the lack of activity observed with hk20 igg was possibly associated to a different susceptibility of this cell line to this type of entry inhibitors. overall, these findings suggest that the reduced size of the fab molecule allows increased access to the hr-1 region and imply that hr-1 accessibility is largely dependent on the target cell used. this is similar in concept to the enhanced neutralization profiles of fab and scfv specific for the cd4i surface [39] . further studies using m7 cells [52] challenged with replication-competent hiv-1 viruses carrying a luc reporter showed that hk20 exhibits broad neutralization consistent with the results from the hos cell assay but unlike the tzm-bl assay, being able to neutralize 4 out 5 viruses tested (table s5) . the epitopes recognized by the 55 remaining mabs were mapped using three experimental approaches: i) cross-competition against mabs of known specificity or soluble cd4; ii) binding to gp120 mutants or gp41 constructs representing different conformational intermediates; iii) pepscan analysis. the results of the analysis performed on the gp120-specific and gp41-specific mabs are presented in table 1 and table 2 , and the data are summarized in a pie chart in figure 6 . thirty-seven mabs bound to gp120 targeting primarily the cd4bs and the v3 loop and to a lesser extent the cd4i site ( table 1) . a first group of mabs was assigned to the v3 loop based on cross-competition with two v3 loop specific mabs (hr10 and hga9). this group comprised 8 mabs, which were already mapped to the v3 loop using the pepscan analysis approach, and 7 additional mabs that were not analyzed by peptide scanning (i.e. hgf9, hgt4, hgp21, hgp51, hgw48, hgd129 and hgp27). four mabs (hx44, hgp105, hgw7, hgy38) reacted with wt and cd4bs yu2 mutants but failed to bind the cd4i mutant, and therefore were assigned to the cd4i cluster. notably, one mab (hgp68) was mapped by pepscan analysis to a novel epitope in the v2 loop (tvyalfyrldivp) and neutralized 4 tier-1 isolates (table s2) . fifteen other mabs were assigned tentatively or conclusively to the cd4bs based on their capacity to inhibit gp120-scd4 binding. most of these mabs competed other cd4bs-specific mabs, such as b12 and hj16, while in some cases this assay could not be performed due to lack of epitope expression on the gp120 proteins used. furthermore, this group of mabs showed variable reactivities with the yu2 mutants. while most of them did not bind the d368r mutant similar to b12, others including hj16, bound this mutant avidly. in addition, most of these new cd4bs-specific mabs also showed decreased binding to the cd4i mutant, suggesting that they may span a broader, as yet undescribed region that includes elements of the cd4bs and cd4i sites. finally, for 2 mabs these analyses did not provide any relevant information. twenty-one mabs bound to gp41 by targeting primarily the immunodominant c-c region, the hr-1 and the region recognized by 5f3 mab [64] (table 2) . mabs hgk129, hgn146 and hgn35 were assigned to the c-c loop since they competed with 3d6 [43] , reacted with an hr2 construct and recognized synthetic peptides within this region. mabs hgw17 and hgy25 were provisionally assigned to the c-c region since they competed with 3d6. these data are consistent with the immunodominance of the c-c region and with the notion that the antibody response against this region overlaps with the 3d6 epitope. several mabs competed with 5f3 and hk20, and bound both the hr-1 and 5hb constructs, suggesting that they may bind between the fusion peptide and the hr-1 region. hgb33 was provisionally assigned to the hr-1 region according to competition with hk20 and binding to the 5hb construct. other mabs bound specifically the 5hb construct but did not compete with any of the mabs tested, thus indicating that the complete hr-1 coiled coil region exposed in 5hb harbors antibody epitopes available for b cell recognition in the gp41 pre-hairpin conformation [65, 66] . the concomitant binding to recombinant gp41 suggests that the latter may partially resemble the gp41 prefusion state. of note, three gp41 binding mabs (hgp40, hgp48 and hgy50) neither competed with any of the mabs tested, nor bound to any constructs representing pre-hairpin conformations or native gp140 ( figure s1 ), indicating that they recognize as yet uncharacterized regions that are only available in the recombinant gp41 protein. interestingly, mab hgf24 was assigned by pepscan analysis to an epitope in the c-terminal region of hr-2 (tnliytlieesqn), proximal to the 2f5 epitope, and neutralized 4 tier-2 isolates (table s2) . this mab competed with hk20 for gp41 binding, in spite of their distinct cognate specificities (hr-2 and hr-1, respectively). this finding would be consistent with the proximity of hr-1 and hr2 in the six-helix bundle structure. finally, although the gp41 protein used in the primary screening includes the 2f5 and 4e10 epitopes, no mper specific mabs were isolated, reinforcing the idea that this portion of gp41 is poorly immunogenic in humans [21, 22] in conclusion, regardless of their limited breadth of neutralization, this extended panel of human mabs may represent a useful tool for understanding the molecular basis of env recognition in humans. using an improved ebv immortalization method combined with a broad screening strategy we isolated from memory b cells of hiv-1 infected donors 58 mabs that cover an extensive antigenic surface of env. of these, 37 neutralized at least one of the isolates tested and 3 mabs in particular bound to the cd4bs, v3 crown and hr-1, showing considerable neutralizing breadth against a panel of hiv-1 pseudoviruses of different clades and spanning tier-1 and tier-2 isolates. several studies have questioned the existence of individual broadly neutralizing antibody specificities as part of a normal immune response to hiv-1 infection. in this respect, b12 was isolated from a phage library, while 2f5, 4e10 and 2g12, although isolated from memory b cells, do not appear to have a counterpart in human sera [22] . in a recent study, nussenzweig and coworkers used trimeric gp140 to isolate antigen-binding memory b cells from which 500 antibody sequences were retrieved by single-cell pcr [62] . surprisingly, although the b cell donors had broadly neutralizing serum activity, none of the mabs isolated had this property. this raised the possibility that broad serum neutralizing activity results from multiple clonal responses each of unique epitope specificity but restricted breadth of viral strain recognition. however, an alternative interpretation might be that since available recombinant trimeric antigens vary in structure, the 'bait' used for b cell isolation was not optimal for enriching those secreting neutralizing antibodies. our results indicate that monoclonal antibodies with a limited spectrum of neutralizing activity can be easily isolated, while those with a broad pattern of cross-clade reactivity are rare, in line with the study of walker et al [40] and consistent with inferences from serum neutralization specificity mapping analyses [21] . in this respect our study demonstrates the feasibility of the ebv immortalization method, which is limited only by the size of the blood samples obtained (20-50 ml of peripheral blood). the characterization of the specificity and neutralizing activity of this new panel of human mabs reveal some interesting features of the human antibody response to hiv-1 within a population of hiv-1-infected donors. first, most of the gp120-specific mabs showed neutralizing activity (37 out of 58), indicating that they bind to sites available on the functional env trimer. the selection of a highpercentage of trimer binding specificities may be a consequence our use of trimeric env antigens during the screening of the evb-b cell supernatants. the second aspect relates to gp41-specific antibodies, which, with a few remarkable exceptions, were not neutralizing. interestingly the non-neutralizing antibodies bound gp41 in the context of the recombinant trimer, whereas the most effective neutralizing mabs bound to gp41 fusion intermediates. the three new neutralizing mabs hj16, hgn194 and hk20 show some interesting features. hj16 showed a breadth of neutralizing activity comparable to, and generally complementary to b12, which aside from pg9 and pg16 [40] is currently the most potent of the relatively broad neutralizing antibodies available. hj16 also showed selective neutralization of multiple tier-2 isolates, making it particularly relevant as a template for vaccine design. hgn194 binds to an extremely conserved epitope in the v3 crown, and neutralizes all tier-1, but only 11% of tier-2 isolates tested. hgn194 showed broader activity than the well-characterized v3-specific mab 447-52d against a panel of 92 pseudoviruses. the preference for tier-1 isolates characteristic of v3-specific mabs is consistent with the idea that the v3 loop is displayed to varying degrees in the context of the native trimeric env protein of individual isolates [67, 68] and with the recent observation that scd4 broadens neutralization of v3 mabs [69] . the results obtained with hk20 are particularly intriguing, since this mab has a broad pattern of neutralization observed with hos but not tzmbl target cells. this mab recognizes a highly conserved epitope in the hr-1 trimer and it is more effective as an fab fragment as compared to intact igg, a fact that is may be explained by the limited accessibility of the epitope, which is likely to be exposed only transiently on the cell surface during the viral entry process [70] . intriguingly, a recent study indicates that in tzm-bl cells hiv-1 pseudoviruses penetrate the cytoplasm from within endosomes rather than from the cell surface [71] , a finding that might help explain the lack of activity of hk20 in the tzmbl assay if binding of this mab is affected by the endosomal environment. further studies on primary cells will be required to address the relative importance of these two entry pathways and to establish whether hk20 may be capable of exerting a broad and potent neutralizing activity in vivo. preliminary studies using m7 cells [52] challenged with replication-competent hiv-1 viruses cross competition with biotinylated mabs of known specificity: +, 90-100% inhibition, +/2, 50-90% inhibition, 2, ,50% inhibition. binding to different gp41 constructs by elisa. minimal linear epitopes using the pepscan analysis and specificity assignment. c-c, c-c loop; 5hb, 5-helix bundle; fp, fusion peptideshown is also the number of isolates neutralized in the hos-based and tzm-bl based assays. doi:10.1371/journal.pone.0008805.t002 carrying a luc reporter showed that hk20 exhibits reasonably broad neutralization consistent with the results from the hos cell assay, and similar to fab hk20 in the tzm-bl cell assay. whereas the tzm-bl assay is sensitive for certain antibodies such as cd4bs mabs, some mabs such as 2g12 to other epitopes are less sensitive to neutralization in this assay [72] , perhaps owing to the unnaturally high level of ccr5 expression on the tzm-bl cells [60] . in this respect, low coreceptor expression levels have been associated with delayed fusion kinetics and hence enhanced virus sensitivity to hr-1 binding inhibitors, such as t-20 [73] . therefore, despite the advantages of high throughput with the tzm-bl assay, alternative neutralization assays might have closer relevance to the in vivo situation. in conclusion, we have shown that in hiv-1-infected individuals a high proportion of hiv-specific b cells produce neutralizing antibodies but only a few possess neutralizing activity with relatively broad neutralization coverage. in the short term, some of these reagents will be tested for their ability, singly or in combination, to prevent hiv-1 or siv transmission in a macaque challenge model as done previously with other mabs [5, 6, 7, 8, 9] . in the longer term, and combined with an atomic-level analysis of epitope specificity, some of these mabs may facilitate the templatebased design of immunogens for the development of a vaccine able to induce neutralizing antibodies against the wide range of hiv-1 strains present in the global pandemic. figure s1 binding of gp120 and gp41-specific mabs to a panel of 15 recombinant env proteins from different clades. found at: doi:10.1371/journal.pone.0008805.s001 (0.47 mb pdf) figure 6 . epitope specificity and neutralizing activity of the mab panel. the pie chart shows the specificity of the 58 mabs isolated from the 21 interrogated individuals. the fraction of antibodies with neutralizing activity against at least one isolate is indicated in parentheses. partial cd4bs, incomplete inhibition of cd4 binding; hr-1/ fp, epitope located in between hr-1 and the fusion peptide as determined by 5f3 competition; hr-1/5hb, recognition of trimeric hr-1 and 5-helix bundle; 5hb, 5 helix bundle; c-c region, gp41 immunodominant region; hr-2, heptad-repeat 2; nd, not defined epitopes within gp120 or gp41. doi:10.1371/journal.pone.0008805.g006 antibody neutralization and escape by hiv-1 rapid evolution of the neutralizing antibody response to hiv type 1 infection in vivo and in vitro escape from neutralizing antibodies 2g12, 2f5, and 4e10 delay of hiv-1 rebound after cessation of antiretroviral therapy through passive transfer of human neutralizing antibodies protection of macaques against pathogenic simian/human immunodeficiency virus 89.6pd by passive transfer of neutralizing antibodies protection of macaques against vaginal transmission of a pathogenic hiv-1/siv chimeric virus by passive infusion of neutralizing antibodies antibody protects macaques against vaginal challenge with a pathogenic r5 simian/ human immunodeficiency virus at serum levels giving complete neutralization in vitro human neutralizing monoclonal antibodies of the igg1 subtype protect against mucosal simian-human immunodeficiency virus infection complete protection of neonatal rhesus macaques against oral exposure to pathogenic simian-human immunodeficiency virus by human anti-hiv monoclonal antibodies fc receptor but not complement binding is important in antibody protection against hiv neutralization of primary human immunodeficiency virus type 1 isolates by the broadly reactive anti-v3 monoclonal antibody antibodies, viruses and vaccines hiv vaccine design and the neutralizing antibody problem medicine. the need for a global hiv vaccine enterprise consistent viral evolutionary changes associated with the progression of human immunodeficiency virus type 1 infection regulation of human natural killer cell migration and proliferation by the exodus subfamily of cc chemokines human immunodeficiency virus type 1 subtype distribution in the worldwide epidemic: pathogenetic and therapeutic implications a large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals efficient neutralization of primary isolates of hiv-1 by a recombinant human monoclonal antibody recognition properties of a panel of human recombinant fab fragments to the cd4 binding site of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1 broad hiv-1 neutralization mediated by cd4-binding site antibodies profiling the specificity of neutralizing antibodies in a large panel of plasmas from patients chronically infected with human immunodeficiency virus type 1 subtypes b and c dissection of the carbohydrate specificity of the broadly neutralizing anti-hiv-1 antibody 2g12 antibody domain exchange is an immunological solution to carbohydrate cluster recognition molecular characterization of five neutralizing anti-hiv type 1 antibodies: identification of nonconventional d segments in the human monoclonal antibodies 2g12 and 2f5 the mannose-dependent epitope for neutralizing antibody 2g12 on human immunodeficiency virus type 1 glycoprotein gp120 comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency virus type 1 monoclonal antibodies fine definition of the epitope on the gp41 glycoprotein of human immunodeficiency virus type 1 for the neutralizing monoclonal antibody 2f5 generation of human monoclonal antibodies against hiv-1 proteins; electrofusion and epstein-barr virus transformation for peripheral blood lymphocyte immortalization anti-human immunodeficiency virus type 1 (hiv-1) antibodies 2f5 and 4e10 require surprisingly few crucial residues in the membrane-proximal external region of glycoprotein gp41 to neutralize hiv-1 broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41 analysis of neutralization specificities in polyclonal sera derived from human immunodeficiency virus type 1-infected individuals factors associated with the development of cross-reactive neutralizing antibodies during human immunodeficiency virus type 1 infection cardiolipin polyspecific autoreactivity in two broadly neutralizing hiv-1 antibodies the v3 loop is accessible on the surface of most human immunodeficiency virus type 1 primary isolates and serves as a neutralization epitope crossclade neutralizing activity of human anti-v3 monoclonal antibodies derived from the cells of individuals infected with non-b clades of human immunodeficiency virus type 1 analysis of the neutralization breadth of the anti-v3 antibody f425-b4e8 and re-assessment of its epitope fine specificity by scanning mutagenesis cross-clade neutralizing activity of human anti-v3 monoclonal antibodies derived from the cells of individuals infected with non-b clades of human immunodeficiency virus type 1 access of antibody molecules to the conserved coreceptor binding site on glycoprotein gp120 is sterically restricted on primary human immunodeficiency virus type 1 broad and potent neutralizing antibodies from an african donor reveal a new hiv-1 vaccine target an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus a potent cross-clade neutralizing human monoclonal antibody against a novel epitope on gp41 of human immunodeficiency virus type 1 characterization of monoclonal antibodies to human immunodeficiency virus type 1 gp41 by hiv-1 polypeptides expressed in escherichia coli human immunodeficiency virus type 1 env clones from acute and early subtype b infections for standardized assessments of vaccine-elicited neutralizing antibodies genetic and neutralization properties of subtype c human immunodeficiency virus type 1 molecular env clones from acute and early heterosexually acquired infections in southern africa design and characterization of an engineered gp41 protein from human immunodeficiency virus-1 as a tool for drug discovery protein design of an hiv-1 entry inhibitor expression and characterisation of recombinant oligomeric envelope glycoproteins derived from primary isolates of hiv-1 antigenic structure of the central conserved region of protein g of bovine respiratory syncytial virus rapid and quantitative cyclization of multiple peptide loops onto synthetic scaffolds for structural mimicry of protein surfaces human monoclonal antibodies specific for conformation-sensitive epitopes of v3 neutralize human immunodeficiency virus type 1 primary isolates from various clades evaluating neutralizing antibodies against hiv, siv and shiv in luciferase reporter gene assays association of chemokine-mediated block to hiv entry with coreceptor internalization neutralization kinetics of sensitive and resistant subtype b primary human immunodeficiency virus type 1 isolates recommendations for the design and use of standard virus panels to assess neutralizing antibody responses elicited by candidate human immunodeficiency virus type 1 vaccines b cells in hiv infection and disease evidence for hiv-associated b cell exhaustion in a dysfunctional memory b cell compartment in hiv-infected viremic individuals identification and characterization of a new cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody international network for comparison of hiv neutralization assays: the neutnet report increased efficacy of hiv-1 neutralization by antibodies at low ccr5 surface concentration identification and characterization of transmitted and early founder virus envelopes in primary hiv-1 infection broad diversity of neutralizing antibodies isolated from memory b cells in hivinfected individuals structure of a v3-containing hiv-1 gp120 core generation of human monoclonal antibodies against hiv-1 proteins; electrofusion and epstein-barr virus transformation for peripheral blood lymphocyte immortalization atomic structure of the ectodomain from hiv-1 gp41 core structure of gp41 from the hiv envelope glycoprotein cryptic nature of a conserved, cd4-inducible v3 loop neutralization epitope in the native envelope glycoprotein oligomer of ccr5-restricted, but not cxcr4-using, primary human immunodeficiency virus type 1 strains antibodies that are cross-reactive for human immunodeficiency virus type 1 clade a and clade b v3 domains are common in patient sera from cameroon, but their neutralization activity is usually restricted by epitope masking soluble cd4 broadens neutralization of v3-directed monoclonal antibodies and guinea pig vaccine sera against hiv-1 subtype b and c reference viruses hiv infection does not require endocytosis of its receptor hiv enters cells via endocytosis and dynamin-dependent fusion with endosomes hiv sensitivity to neutralization is determined by target and virus producer cell properties sensitivity of hiv-1 to entry inhibitors correlates with envelope/coreceptor affinity, receptor density, and fusion kinetics we thank john mascola and richard wyatt for generously providing yu2 mutant molecules, marlen aasa-chapman for experimental support, jane anderson and werner leber who managed the follow up of patients and provided blood samples; we are also thankful to all patients who participated in this study. we would like to express our thanks to vasia dekou who coordinated reagent supply and the work within our consortium. key: cord-000006-104sqoxz authors: bray, daniel p.; bennett, malcolm; stockley, paula; hurst, jane l.; kipar, anja title: composition and function of haemolymphatic tissues in the european common shrew date: 2008-10-15 journal: plos one doi: 10.1371/journal.pone.0003413 sha: doc_id: 6 cord_uid: 104sqoxz background: studies of wild animals responding to their native parasites are essential if we are to understand how the immune system functions in the natural environment. while immune defence may bring increased survival, this may come at a resource cost to other physiological traits, including reproduction. here, we tested the hypothesis that wild common shrews (sorex araneus), which produce large numbers of offspring during the one breeding season of their short life span, forgo investment in immunity and immune system maintenance, as increased longevity is unlikely to bring further opportunities for mating. in particular, we predicted that adult shrews, with shorter expected lifespans, would not respond as effectively as young animals to infection. methodology/principal findings: we examined haemolymphatic tissues from wild-caught common shrews using light and transmission electron microscopy, applied in conjunction with immunohistology. we compared composition and function of these tissues in shrews of different ages, and the extent and type of inflammatory reactions observed in response to natural parasitic infections. all ages seemed able to mount systemic, specific immune responses, but adult shrews showed some signs of lymphatic tissue exhaustion: lymphatic follicles in adults (n = 21) were both smaller than those in sub-adults (n = 18; wald = 11.1, p<0.05) and exhibited greater levels of depletion (wald = 13.3, p<0.05). conclusions/significance: contrary to our expectations, shrews respond effectively to their natural parasites, and show little indication of immunosenescence as adults. the pancreas of aselli, a unique lymphoid organ, may aid in providing efficient immune responses through the storage of large numbers of plasma cells. this may allow older animals to react effectively to previously encountered parasites, but infection by novel agents, and eventual depletion of plasma cell reserves, could both still be factors in the near-synchronous mortality of adult shrews observed shortly after breeding. the immune system is the primary mechanism through which animals defend against parasites and pathogenic organisms. immunity is believed to be a major factor in regulating host survival, as under natural conditions, even a mild parasitic infection may weaken an animal sufficiently to increase the chances of mortality through starvation or predation [1] . however, maintenance and up-regulation of the immune system requires energetic and nutritional resources [1] , resulting in a trade-off between investment in immunity and other physiological processes, including growth and reproduction [2] [3] [4] . an appreciation of the extent to which hosts invest in immunity is therefore critical to understanding the strategies through which animals maximise fitness, and how trade-offs are mediated between current offspring production, longevity and future reproductive success [3, 4] . while there have been a number of studies of ecological immunology in birds and insects [4] [5] [6] , there has been little effort to understand immune function of small mammals in this context outside of the laboratory. here, we examined the unique haemolymphatic system of the european common shrew (sorex araneus) to investigate the capacity of this short-lived mammal, restricted by a fast metabolism and extremely limited fat reserves, to defend against its unusually diverse parasite fauna, both as a young animal and an adult. common shrews have attracted considerable attention from both ecologists and parasitologists, and have a life history strategy characterized by a high investment in reproduction and a short life span [7] [8] [9] . their life cycle takes 14 to 16 months to complete, with the first young born in mid-may [10] [11] [12] , and only one breeding season in the spring of the second year of life [9, 12, 13] . both sexes can mate with multiple partners, and females are extremely promiscuous [14, 15] . females can produce up to three litters of around seven offspring [10, 11, 16] , with energy intake during lactation increasing to around three times the non-reproductive level [8] . both males and females die shortly after breeding, such that there is little overlap between generations [12, 13, [17] [18] [19] . s. araneus has an unusually diverse parasite fauna, which includes ectoparasites [20] [21] [22] , bartonella and trypanosome infections [23] [24] [25] [26] [27] , anaplasma phagocytophilum [27, 28] , and pneumocystis carinii [29] , as well as over 20 helminth species [30] . the extent to which shrews are able to mount immune responses to these parasites is unknown: commons shrews have a fast metabolic rate even for their small body size [31] but store very little energy as fat [32] . lack of resources may therefore limit the capacity of s. araneus to mount immunological responses, particularly as reproductive adults, and parasitism has been suggested as one of the causes of mortality of adults after breeding [12, 13, 18] . common shrews possess a large lymphatic organ, known as the pancreas of aselli, which may function in defence against parasites, though its exact role is unknown, and remains the subject of discussion [33] [34] [35] . to date, there have been no studies of spleen or bone marrow function in s. araneus. we hypothesised that common shrews, which are not expected to survive beyond the first breeding season, would gain little benefit from investing their limited resources in immunity and immune system maintenance, at the expense of reproduction. instead, we predicted that wild shrews would demonstrate only limited responses to parasites, and that their immune system would show signs of deterioration with age. the aim of the study was therefore to evaluate the capacity of sub-adult and adult shrews to mount immunological responses. we examined and compared the structure, composition and function of relevant haemolymphatic tissues including the pancreas of aselli, in wild-caught common shrews of different ages pre and post maturation, and the extent and type of inflammatory reactions produced in response to naturally occurring parasitic infections. light and electron microscopy were applied in conjunction with immunohistological characterisation of leukocyte populations. contrary to our predictions, our results indicated that shrews are capable of mounting immune/inflammatory responses throughout their entire life span. while some degree of lymphatic exhaustion was obvious in adult animals (perhaps as a result of age-related changes, or reduced investment in immunity as a consequence of breeding effort), there was also evidence of some degree of compensation, in the form of storage of plasma cells particularly in the pancreas of aselli, possibly as a defence against previously encountered parasites. forty-three common shrews (19 male, 24 female) were live-caught in cheshire, england between september 2001 and june 2003. the work was performed with approval of and under a licence from english nature (licence number 20030767) held by ps. shrews were classified into three age categories: 18 sub-adults, showing no sign of sexual development (11 female, 7 male), 3 animals undergoing sexual maturation (2 female, 1 male) and 22 sexually mature animals (11 female and 11 male) caught during or after the breeding season. adult females all exhibited signs of mating, pregnancy and/or lactation. all animals appeared healthy when captured, and were killed humanely by overdose of inhalation anaesthetic (fluothane, schering-plough animal health, uk). animals were inspected for ectoparasites (data not presented) before full necropsy was performed and body mass (minus gastrointestinal tract) recorded. bladder and oesophagus were removed and dissected in hanks saline (406 magnification), with nematode and digenean parasites in both tissues counted and identified using keys [30] . stomachs and guts were removed, weighed, stored in 10% formalin and later dissected in hanks saline (406 magnification). recovered helminths were identified as nematodes, cestodes or digeneans [30] and counted. tissue samples from all major organs from all animals were fixed in 4% buffered paraformaldehyde for 24-48 h prior to routine embedding in paraffin wax. sections (5 mm) were stained with haematoxylin-eosin for histological evaluation. sections from haemolymphatic tissues (spleen, pancreas of aselli, bone marrow (sternum) and in selected cases mesenteric or mediastinal lymph nodes and thymus) were prepared for immunohistological examinations and the tunel method. samples from the pancreas of aselli of one sub-adult common shrew were fixed in 2.5% glutaraldehyde and 4% paraformaldehyde in cacodylate buffer (ph 7.4) and subsequently embedded in epoxy resin. semi-thin (1 mm) and thin sections were prepared and the latter examined using transmission electron microscopy. labelling of leukocytes, proliferating and apoptotic cells by immunohistology and the tunel method leukocytes, proliferating and apoptotic cells in haemolymphatic tissue samples from 21 common shrews were identified using immunohistology and the tunel (terminal deoxynucleotidyl transferase-mediated dutp-biotin nick end labelling of dna fragmentation sites) method respectively. for immunohistology, monoclonal and polyclonal antibodies (cross) reacting in other species were used in conjunction with peroxidase anti-peroxidase and avidin biotin peroxidase complex methods as previously described [36] [37] [38] [39] . antibodies, their sources and detection methods are listed in table 1 . cellular turnover in haemolymphatic tissues was assessed by counting proliferating and apoptotic cells. proliferating cells were identified by their expression of the proliferating cell nuclear antigen (pcna; [38] [39] [40] ), while apoptotic cells were demonstrated in situ by the tunel method ( [39, 41] ) using a commercially available kit according to the manufacturer's instructions (apoptag tm in situ apoptosis detection kit; chemicon, california, usa). consecutive tissue sections, incubated with normal rabbit or rat serum or a non-reacting mouse monoclonal antibody, were used as negative controls for polyclonal and monoclonal antibodies respectively. for tunel, terminal deoxynucleotidyl transferase (tdt) was replaced by distilled water on negative control slides. all antibodies used cross-reacted with shrew leukocytes. the b cell markers cd45r and cd79a were expressed by b cells, cd45r being strongly expressed in follicular germinal centres, but relatively faintly in well-differentiated b cells within follicular mantle zones, whereas cd79a was mainly expressed by mature b cells (weak expression in germinal centres (dark zone), strong expression in the periphery of secondary follicles, positive reaction of all cells in primary follicles). plasma cells were negative or exhibited faint staining for both b cell markers. cd3 acted as a pan t cell marker, being expressed by entire t cell zones. both the myeloid/histiocyte antigen and lysozyme were markers for mature monocytes/macrophages and their precursors, myelomonocytic cells, as they were expressed by monocytes and macrophages and a high percentage of cells in the bone marrow. however, they seemed not to be a marker for all neutrophils, as in both the splenic red pulp and sinuses of the pancreas of aselli, a proportion of cells with the morphology of neutrophils were negative for both antigens. pcna-positive, proliferating cells were present in follicle germinal centres, t cell zones, bone marrow (including megakaryocytes) and splenic red pulp, the sites in the haemolymphatic tissue expected to contain proliferating cells. the tunel method identified cells with the morphology of apoptotic cells [39, 41] as well as apoptotic bodies (both free and within tingible body macrophages), located predominantly in follicular germinal centres. assessment of lymphatic follicle and bone marrow activity, and the severity of inflammatory infiltration in the liver spleen and lymph node exhibited a composition very similar to laboratory mice, which allowed direct comparison for evaluation. lymphatic follicles in the spleen and lymph nodes of all animals were classified as primary and/or secondary follicles and as small, medium or large. the presence and degree of follicle depletion (none, mild, moderate or severe) was assessed on the basis of the cellularity of the germinal centres. large, undepleted secondary follicles were interpreted as evidence of high activity, whereas small, depleted primary follicles indicated lowest activity. similarly, bone marrow activity was classified as low, moderate or high based on the ratio of haematopoietic cells to adipose tissue in a cross section of the marrow in the sternum. the degree of inflammatory infiltration in the liver was assessed semi-quantitatively as mild, moderate or severe, based on the number of cells and cell layers in the portal areas or between hepatic cords. statistical analyses were restricted to sub-adult and adult animals as only 3 pubescent shrews were caught. ordinal logistic regression examined whether bone marrow activity, lymphatic follicle activity (primary/secondary follicles, presence and degree of follicular depletion) in the spleen and pancreas of aselli and severity of inflammatory infiltration in the liver (mild, moderate or severe) varied with age class or sex. sex and age category were entered simultaneously as independent variables into models for each dependent variable listed above, and the significance of both terms assessed using wald tests. mann-whitney u tests were used to test for differences in helminth abundances between sub-adult and adult shrews. the spleen in adult shrews exhibits lesser activity in the white pulp but higher cellularity in the red pulp compared to the spleen in sub-adults red and white pulp of shrews of all age groups were examined for their composition and cellular turnover. the white pulp (lymphatic follicles and t cell zones) was generally confined to the organ's centre, where follicles were arranged singly or in groups ( fig. 1 ). germinal centres exhibited numerous proliferating, pcna-positive cells (up to 50%) and few (#5%) apoptotic cells. t cell zones were arranged around medium-sized arteries, forming periarterial lymphatic sheaths similar in size and cell density in all animals, exhibiting up to 10% proliferating, pcna-positive cells and generally few apoptotic cells. the generally cell-rich red pulp contained neutrophils, erythrocytes and smaller numbers of lymphocytes (each ,5% up to 30% t cells and mature b cells) and macrophages as well as numerous evenly distributed megakaryocytes (fig. 1b) ; the red pulp exhibited some degree of cellular turnover, with approximately 10% proliferating, pcnapositive cells and scattered apoptotic cells. follicles and follicle groups were often delineated by a variably distinct rim of macrophages and neutrophils (fig. 1c) , together with variable numbers of mature, strongly cd79a-positive b cells, t cells and erythrocytes. in general, 30-50% of cells in the red pulp were positive for myeloid/histiocyte antigen (fig. 1d ) and lysozyme and had the morphology of monocytes/macrophages or neutrophils. age groups differed in both the composition and functional state of the spleen. within the red pulp, the amount of neutrophils and table 1 . antibodies and detection methods used to identify leukocytes and proliferating cells in the common shrew with references to suppliers and use in other species. [36, 53] . f [37] . g [53] . h [38] [39] [40] . megakaryocytes often appeared greater in adult animals than in sub-adults. the white pulp of sub-adults was exclusively comprised of secondary follicles, which often appeared interconnected and formed large groups (fig. 1a) . these follicles were for the most part large and without signs of depletion and included numerous apoptotic cells and tingible body macrophages, as well as several mitotic cells. in contrast, the majority of adults exhibited fewer follicles which were a mixture of primary and secondary follicles (figs. 1b, 2) and generally smaller than those of sub-adults (n = 17, wald = 17.17, p,0.05, figs. 1a, 2). follicles in adults also differed from those in sub-adults, in that they were only partially surrounded by distinct perifollicular rims. where present, germinal centres in adults contained both mitotic as well as apoptotic cells. follicle centres in four adults exhibited collagen deposition, and while there was a tendency for greater follicle depletion in older animals (fig. 2) , the difference between adults and sub-adults was not statistically significant (wald = 2.10, not significant (ns)). the white pulp of the three pubescent animals exhibited primary and/ or secondary follicles, the latter with features similar to those found in sub-adult shrews. males (n = 18) and females (n = 21) did not differ with respect to either size (wald = 0.67, ns) or depletion (wald = 0.13, ns) of follicles in the spleen. lymph node composition is similar in all age groups, with a relatively high proportion of plasma cells, particularly in adults mesenteric or mediastinal lymph nodes were examined from selected sub-adult and adult animals. all features normally associated with mammalian lymph nodes were represented: a cortex containing primary and secondary follicles, paracortex, lymphatic cords, medulla and both marginal and medullary sinuses. compared to lymph nodes in other mammalian species [39, 42] , the medulla often appeared to contain a high number of plasma cells, particularly in adults. no other differences were observed between the age groups. the pancreas of aselli represents a specialised abdominal lymph node that appears to function as a plasma cell store, in particular in adult shrews in the past, there has been some controversy as to the composition and function of the pancreas of aselli (lymph nodelike or equivalent to the avian bursa of fabricius [35] ). the aim of this study was therefore to clarify this matter with up-to-date methodology. in general, the composition of the pancreas of aselli was very similar to that of a lymph node. beneath the capsule were marginal sinuses of variable width, containing disseminated lymphocytes (mostly t and b cells in equal proportions), macrophages and neutrophils, the latter either disseminated or as small accumulations. in four adult shrews, the marginal sinuses exhibited focal to extensive fibrosis. beneath the sinuses lay a cortex containing exclusively secondary follicles ( fig. 3a-c) , with the exception of one adult male where both primary and secondary follicles were present (fig. 4) . germinal centres generally exhibited a high cellular turnover, with variable but high numbers of pcna-positive, proliferating cells (often more than 50% of cells; fig. 3e ) and often numerous apoptotic cells (fig. 3f ). t cell zones formed a paracortex located immediately beneath the follicles (fig. 3d ) and were generally similar in size and cell density in animals of all age groups. the organ's centre (medulla) contained loosely arranged sinuses with only low numbers of macrophages. the remainder of the medulla was made up almost entirely of plasma cells (fig. 3g, h) . in sub-adult animals the cortex generally appeared tightly packed with large follicles that exhibited no, mild or moderate depletion (figs. 3a, 4) . in adult shrews, the cortex often contained only a small number of follicles (9/20 animals; 45%), frequently with large areas of cortex devoid of follicles (5/20; 25%; fig. 3b ). follicles occasionally seemed to extend outwards into marginal sinuses and in two animals exhibited central collagen deposition. follicles in adults (n = 21) were both smaller than those in sub-adults (n = 18; wald = 11.06, p,0.05, fig. 4 ) and exhibited greater levels of depletion (wald = 13.28, p,0.05, fig. 4) . no difference was found between males (n = 17) and females (n = 22) with respect to follicle size (wald = 0.30, ns) or depletion (wald = 0.42, ns). cortical areas devoid of follicles were also devoid of t cell zones. as a consequence, overall numbers of t cells in the pancreas of aselli often seemed lower in adult than sub-adult animals. where the cortex was devoid of follicles, plasma cells extended from the medulla to the marginal sinuses or beyond to the capsule, such that the medulla often occupied most of the organ in adult animals (fig. 3b) . accordingly, in adult animals, the whole organ frequently appeared as an accumulation of plasma cells, surrounded by a fragmentary cortex and paracortex. extramedullary haematopoiesis was observed in 9/21 (43%) adult animals, as represented by scattered megakaryocytes within the outer medulla. this was not seen in sub-adult animals. thymic tissue was recovered from seven animals across all age groups. this generally consisted of a variable number of lymphocyte layers which were arranged around blood vessels and encased by a thin capsule of fibrous connective tissue. taking the histological features of a mouse thymus in account, the thymus appeared to exhibit a variable, but generally low degree of involution in all examined shrews, regardless of age. the bone marrow generally exhibits moderate to high activity, regardless of age the bone marrow generally exhibited moderate to high activity. all haematopoietic cell types known from other mammalian species were present. approximately 10% of cells were identified as t cells, 10% as b cells (cd79a-positive, cd45r-negative; interpreted as circulating mature b cells) and 30% to 50% were myeloid/histiocyte antigen-and/or lysozyme-positive cells. at least 30% to 40% of all cells were pcna-positive, representing relatively high levels of proliferation. no difference in bone marrow activity was found between sub-adult (n = 15) and adult (n = 16) shrews (wald = 1.91, ns) or between males (n = 12) and females (n = 19; wald = 0.74, ns). all animals harboured helminths ( table 2 ). the abundance of infection was greater in adults than sub-adults (table 3) . nematodes (liniscus incrassatus; roots, 1992) recovered from the lumen and between epithelial cells of the urinary bladder were associated with mild to moderate acute to chronic lymphocytedominated cystitis and/or mild degeneration and sloughing of epithelial cells. infestation of the gall bladder by the digenean dicrocoelium soricis resulted in only a mild lymphocytic submucosal infiltration in one of two animals infected. neither helminths within the gastrointestinal tract nor porrocaecum sp. larvae within interscapular adipose tissue were associated with inflammatory reactions or other histological changes. a high proportion of shrews (14/18 sub-adults; 78%, 2/3 pubescent animals; 67%, 12/22 adults; 55%) exhibited protozoan parasites within vessel walls that could not be further identified ( table 2 ). these occurred predominantly in kidneys (21/43 shrews; 49%) and myocardium (12/43 shrews; 28%), but also occasionally in the liver, the splenic red pulp, the medulla of the pancreas of aselli and the wall of the urinary bladder. in general, these cysts did not induce any alterations apart from an occasional slight thickening of the affected vessel wall, or a mild granulomatous inflammatory infiltration. protozoan cysts with features of sarcocystis sp. were found within skeletal muscle myocytes of one adult female, without any associated reaction. the liver of all animals exhibited a variable degree of mixed cellular (neutrophils, lymphocytes and macrophages) portal inflammatory infiltration, where t cells and b cells were present in equal amounts. no difference in the severity of the inflammatory infiltration was apparent between sub-adults (n = 18) and adults (n = 22; wald = 0.03, ns), or between males (n = 22) and females (n = 18; wald,0.01, ns). infiltrates often occurred together with follicle-like accumulations of lymphocytes, which occasionally exhibited germinal centres. additional findings in the liver included a granuloma with central necrosis in one sub-adult, focal necrosuppurative hepatitis in two adults and variably intense (multi)focal hepatic necrosis with haemorrhage and pyogranulomatous inflammation in another three adults. in one pubescent shrew helminth parasites were observed within and outside multifocal suppurative hepatitis and haemorrhage. five of the 43 animals exhibited granulomas within the pancreas of aselli, with a central area of necrosis and/or mineralization (four shrews) or an embedded nematode (one animal). focal pyogranulomatous (two adults) or suppurative inflammation (two adults), the latter in one case surrounding a nematode, were also observed. the marginal sinuses of one subadult shrew contained extensive focal accumulations of neutrophils, occasionally surrounding areas of necrosis. none of the animals exhibited other major gross or histological changes. in particular there were no findings suggestive of a systemic non-infectious disease and/or a neoplastic disease. while studies of laboratory animals allow aspects of immunity to be studied in controlled, repeatable environments, they may not reflect how wild animals, constrained by limited resources and at threat from a variety of infectious agents, respond to parasites and disease. we predicted that common shrews, which are short lived, store little energy as fat, and invest heavily in reproduction, would show limited responses to parasites, and their haemolymphatic tissues would deteriorate with age as expected survival decreased. our study is the first to examine haemolymphatic tissue structure, compoosition and function in shrews using modern techniques, and one of only a small number to explore immune responses of wild animals in their natural environment. with regards to both morphology and composition, the spleen, lymph nodes, thymus and bone marrow in s. araneus were found to be very similar to their equivalents in other mammalian species [39, 42] . in common with a number of species, including the musk shrew suncus murinus, both bone marrow and spleen were identified as sites of haematopoiesis in s. araneus [43] . the results of our study also confirm that the pancreas of aselli, which is specific to shrews, can be considered as a large, specialised lymph node [33] . the presence of a cortex with both follicles and paracortical t cell zones renders previous controversial assumptions regarding the organ's function incorrect: the pancreas of aselli is neither a specific site of exclusive b cell production nor a functional analogue of the bursa of fabricius in birds [35] . however, it differs from normal lymph nodes in that the centre (medulla) contains a very high proportion of plasma cells. in adulthood, the number of plasma cells and the relative size of the medulla seem to increase, until almost the entire organ is composed of plasma cells. such a feature has not been described under physiological circumstances in any other species, and suggests that the pancreas of aselli in s. araneus functions as a storage site for plasma cells, particularly in older animals. lymph nodes in s. araneus were also found to contain a higher number of plasma cells than normally observed in other species [39, 42] , which may emphasise a general tendency towards progressive plasma cell storage in common shrews. in comparing the lymphatic tissues of sub-adult and adult shrews, young animals were generally found to have an activated immune system, as represented by a predominance of large, active, secondary follicles in the spleen and pancreas of aselli. this suggests sub-adults were responding effectively to a diverse array of infectious agents, including the helminth and protozoan parasites detected in a number of tissues. post-reproductive animals, however, exhibited characteristics indicative of immune system exhaustion: follicles were generally smaller and were often depleted, with a smaller proportion of secondary follicles, particularly in the spleen. this could indicate decreased follicular activity in adult animals, with impaired germinal centre reactions resulting in reduced b cell production [44] . impairment of germinal centre reactions is a known feature of immunosenescence in vertebrates and has been studied extensively: in humans it has been shown to be a product of defective t cell-dependent b cell activation [44, 45] . reduced lymphocyte production as a consequence of follicular and t cell impairment could explain why significantly lower numbers of white blood cells, and specifically lymphocytes, have been reported in old common shrews [46] . differences between sub-adults and adults were also evident in the so-called ''marginal'' or ''intermediate zone'' of the spleen, the variably distinct rim of macrophages, lymphocytes, neutrophils and erythrocytes surrounding follicles and follicle groups seen in s. araneus and previously described in other species, including the musk shrew [43, 47] . this zone is considered to be the site of most intensive blood filtration in the spleen [43, 47] , and its loss of integrity/intensity in older animals may indicate a reduction in filtration capacity. this might however be counterbalanced by an increase in the capacity of the peripheral phagocytic response, as represented by an increase in neutrophil numbers within both the spleen (as observed here) and the peripheral blood [46] . both in bone marrow and splenic red pulp, the degree of haematopoiesis was similar in animals from all age groups. this concurs with similar findings in the musk shrew [43] , where both the splenic red pulp and bone marrow have been identified as physiological sites of erythropoiesis, leukocytopoiesis and platelet production over the animal's lifespan. in this aspect, shrews are similar to some reptiles, whereas in other mammals the haematopoietic capacity of the spleen seems to cease after birth [43] . we also found evidence of haematopoiesis in the pancreas of aselli in adult s. araneus, as demonstrated by the presence of megakaryocytes in the medulla of some individuals. interestingly, we found no evidence of major thymic involution in s. araneus, even in older animals. the rate of thymic involution is known to vary between species and breeds and with intraspecific factors such as sex and diet [48, 49] . perhaps in shrews thymic involution is delayed to maintain production of t cells into adulthood. it has been suggested that short-lived species should limit their investment in immunity to immediate, innate responses, as the energetic costs associated with mounting specific immune reactions are unlikely to be outweighed by the benefits of increased long-term survival [1] . the dependence on innate responses may be greater for species with limited energetic reserves (such as s. araneus), as even a mild immune challenge is likely to result in starvation if allowed to persist for more than a short time [1] . here, however, the presence of numerous active secondary follicles in the spleen and pancreas of aselli, the development of small lymphatic follicles in portal areas in the liver and the generally high number of plasma cells in the pancreas of aselli all indicate that common shrews remain consistently able to mount systemic, specific immune responses. we also observed macrophage-dominated (granulomatous) inflammatory reactions with lymphocyte involvement in both sub-adult and adult shrews, which included reactions to helminths in tissues. the increasing number of plasma cells in the medulla of the pancreas of aselli and in lymph nodes with advancing age might even suggest a 'refocusing' of the immune system, from reacting to novel antigens in follicles as a young animal, to combating previously experienced parasites or pathogens with appropriate antibody responses as an adult. plasma cells are long-lived and can survive for weeks after immunisation, particularly when not too tightly packed [50] ; perhaps young common shrews invest in long term immunity by producing and storing plasma cells in the pancreas of aselli, which can then be used to mount efficient responses against previously encountered parasites in adulthood, when reproduction places greater demands on internal resources [8] . while this strategy may allow older animals to react effectively to previously encountered parasites, infection by novel agents or eventual depletion of plasma cell reserves, could still be factors in 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survival surface phenotype and migratory capability of peyer's patch germinal center cells cd79: a review immunohistochemical characterization of inflammatory cells in brains of dogs with granulomatous meningoencephalitis the occurrence of centrorhynchus (acanthocephala) in shrews (sorex araneus and sorex minutus) in the united kingdom the morphology of hymenolepidid and dilepidid cestodes from common and pygmy shrews (soricidae) in southeast england the authors are grateful to mrs. a. griffiths, for excellent technical assistance, and to mr. g. ross, for preparation of ultrastructural samples. all shrews were trapped under licence from english nature. key: cord-000410-av8b8g8c authors: radoshitzky, sheli r.; longobardi, lindsay e.; kuhn, jens h.; retterer, cary; dong, lian; clester, jeremiah c.; kota, krishna; carra, john; bavari, sina title: machupo virus glycoprotein determinants for human transferrin receptor 1 binding and cell entry date: 2011-07-07 journal: plos one doi: 10.1371/journal.pone.0021398 sha: doc_id: 410 cord_uid: av8b8g8c machupo virus (macv) is a highly pathogenic new world arenavirus that causes hemorrhagic fever in humans. macv, as well as other pathogenic new world arenaviruses, enter cells after their gp1 attachment glycoprotein binds to their cellular receptor, transferrin receptor 1 (tfr1). tfr1 residues essential for this interaction have been described, and a co-crystal of macv gp1 bound to tfr1 suggests gp1 residues important for this association. we created macv gp1 variants and tested their effect on tfr1 binding and virus entry to evaluate the functional significance of some of these and additional residues in human and simian cells. we found residues r111, d123, y122, and f226 to be essential, d155, and p160 important, and d114, s116, d140, and k169 expendable for the gp1-tfr1 interaction and macv entry. several macv gp1 residues that are critical for the interaction with tfr1 are conserved among other new world arenaviruses, indicating a common basis of receptor interaction. our findings also open avenues for the rational development of viral entry inhibitors. arenaviruses have single-stranded, bisegmented ambisense rna genomes and form enveloped virions [1] . seven arenaviruses cause viral hemorrhagic fever in humans: the old world arenaviruses lassa and 'lujo,' and the new world clade b arenaviruses machupo (macv), junín (junv), guanarito (gtov), sabiá (sabv), and chapare (chpv) [2, 3, 4] . all of these viruses are us select agents and risk group 4 pathogens [5, 6] . macv, junv, and gtov are also classified as us national institute of allergy and infectious disease category a priority pathogens [7] . macv causes human disease outbreak with high case-fatality rates. to date, at least 1,200 cases with <200 fatalities have been recorded [8, 9] . arenaviral genomes encode at least four proteins from two segments (large and small). the large segment encodes a matrix protein (z) and an rna-dependent rna polymerase (l); the small segment encodes a nucleoprotein (np) and the glycoprotein precursor gpc [2] . gpc is cleaved by a cellular protease to a stable signal peptide (ssp), and two subunits, gp1 and gp2. the three cleavage products form a stable complex and mediate virus attachment to host cells and fusion of the arenavirion envelope with that of the cell [10, 11, 12, 13, 14, 15] . gp1 mediates the binding of the virion to a cell-surface receptor, whereas the class i fusion protein gp2 mediates membrane fusion after internalization of the virion into an acidified endosome [16, 17] . interrupting the interaction of gp1 with its cell-surface receptor is a current focus of antiviral development since it would prevent the first and pivotal step of arenavirus host-cell infection. transferrin receptor 1 (tfr1) is the principle cell-surface receptor of macv, junv, gtov, and sabv [17, 18] , and a major determinant of host adaptation. however, studies on receptor use and cellular tropism suggest that the non-pathogenic clade b viruses amapari (ampv) and tacaribe (tcrv) can enter human cells in a human tfr1-independent manner [18, 19, 20] . nevertheless, ampv and tcrv use tfr1 orthologs of their principal host animals to infect nonhuman cells [21] . these studies reveal a complex pattern of receptor use for new world arenaviruses, suggesting the existence of additional receptor molecules, and a possible relationship between receptor use and disease potential. the gp1-binding site on human tfr1 (htfr1) has been pinpointed to a prominent loop within the apical domain (residues 201-212) [22] . conversely, until recently the tfr1-binding site on arenavirus gp1 has been poorly characterized. one study showed that the 20 n-terminal amino acids of macv gp1 are dispensable for tfr1-binding as a macv gp1 variant lacking these residues (macv gp1d, residues 79-258) binds htfr1 even more efficiently than wild-type gp1 [17] . a second study demonstrated that the central region of gtov gp1 (residues 85-221) interacts with htfr1 and that residues 159-221 are essential for this interaction [23] . a recently published crystal structure of a truncated macv gp1 (region 87-239) [24] , as well as a co-crystal structure of the macv gp1:tfr1 complex [25] provided structural insight of gp1 residues that contact tfr1. the latter study identified five interaction motifs in the interface between macv gp1 and the apical domain of tfr1 involving some relatively conserved gp1 residues, such as d114 (motif 2), d123 (motif 4), and f226 (motif 3) (colored red, fig. 1 ), some less conserved residues, such as y122 (motif 4) and k169 (motif 4) (colored blue, fig. 1 ), as well as some nonconserved residues, such as r111 (motif 1),and s116 (motif 2) (colored green, fig. 1 ) [25] . to evaluate experimentally the functional significance of these residues and other conserved solvent-accessible macv gp1 residues in tfr1-mediated entry, we created a panel of macv gp1 variants replacing the mentioned amino acids with alanine, and tested the variants' ability to bind htfr1 and mediate macv entry in simian and human cells experimental systems. we aligned the gp1 sequences of macv (residues 59-258) with those of junv, gtov, and sabv to identify conserved residues mapping to the surface of the determined macv gp1 crystal structure (dnastar lasergene software). we identified ten residues of interest that did not contact htfr1 in the co-crystal structure yet are conserved in at least three of these viruses: d140, w147, d155, d159, p160, k167, n178, and k211 (colored red, fig. 1 ). we followed a strategy previously employed for virus receptor identification to evaluate the importance for tfr1 binding of these conserved residues, as well as gp1 residues recently indicated in the published macv gp1:tfr1 crystal structure [17, 26] . briefly, we designed codon-optimized open reading frames (orfs) of macv gp1d in which one or more of the chosen residues were mutated to alanine, and generated them by gene synthesis (dna2.0). these orfs were cloned into a pcdm8-like expression vector [17, 26] with the signal sequence of human cd5 upstream and the fc region of human immunoglobulin g1 downstream of the orf. fc alone and wild-type macv gp1d-fc, which was described previously [17] , served as controls. proteins were purified as described [17, 26] : the individual plasmids were transfected into hek 293t/17 cells (atcc) using the calcium-phosphate method and grown in 293 sfm ii medium (gibco-invitrogen). media were harvested and clarified, and fc fusion proteins precipitated with protein a-sepharose fast flow beads (ge healthcare). fc fusion proteins were eluted with 3 m mgcl 2 , dialyzed in pbs, and concentrated. purified proteins were assayed by sds-page followed by bio-safe coomassie staining (bio-rad), and measured using the quant-it protein assay kit (invitrogen). all mutant plasmids expressed proteins ( fig. 2a) . we evaluated the ability of the individual mutants to bind to htfr1 using co-immunoprecipitation (co-ip) assays as described [17, 22, 26] . briefly, hek 293t/17 cells were transfected with plasmid encoding htfr1 [17, 22] and lysed 48 h post transfection with ripa lysis and extraction buffer (thermo scientific pierce). cleared lysates were added to equimolar amounts (200 nm) of fc, macv gp1d, or variants thereof, and bound proteins were immunoprecipitated with protein a-sepharose fast flow beads. htfr1 in the cell lysate immunoprecipitates was analyzed by sds-page and western blot using the westernbreeze chromogenic kit (invitrogen) and a murine monoclonal anti-tfr1 antibody (invitrogen). four mutants, d114a, d140a, k167a, and k169a, precipitated htfr1 as efficiently as wild-type (wt) macv gp1d or at slightly lower levels. reduction in gp1 interaction with htfr1 was observed with mutants s116a, k211a, and d114a/s116a (motif 2 in [1] ), whereas mutants r111a, y122a, d123a, w147a, d155a, d159a, p160a, n178a, f226a, and the triple mutant y122a/d123a/k169a effectively inhibited macv receptor interactions (fig. 2b) . we performed cell-binding assays as described [17, 26] to determine the ability of these mutants to bind to the surface of macv-permissive cells. briefly, fc constructs were added to 5610 5 vero cells (atcc) to a final concentration of 200 nm. cells with bound proteins were incubated with a 1:40 dilution of fitc-conjugated anti-human fc antibody (sigma-aldrich). cell-surface binding of constructs was detected by flow cytometry. baseline fluorescence was determined by measuring cells treated only with the secondary antibody, which was then subtracted from binding values of the tested constructs and control proteins. overall, cell-binding of mutants supported the co-ip results as mutants that precipitated htfr1 exhibited high cell-surface binding and vice versa (fig. 2c) . exceptions were y122, s116a and d114a/s116a (motif 2), which bound the cell-surface of vero cells as or almost as efficiently as wt macv gp1d, yet exhibited lower htfr1 levels in the co-ip assays. the k167a mutant exhibited an opposite trend, as it bound less efficiently to the cell-surface than wt macv gp1d whereas it immunoprecipitated htfr1 as effectively. we next tested whether gross misfolding was the reason for the inability of some of the mutants to precipitate htfr1 and to bind to the cell-surface of macv-permissive vero cells. we performed far-uv circular dichroism (cd) of the wt gp1d and selected mutants (fig. 2d ). far-uv cd spectra were taken in pbs at 5uc with a jasco-810 spectropolarimeter and a 1 mm pathlength rectangular quartz cell. protein solutions used were from 0.05 to 0.08 mg/ml. ten scans were accumulated and averaged, without smoothing. the spectra all revealed a single broad minimum in the range of 108 to 216 nm. these results are consistent with similar folding of the wt and mutant proteins, although small changes in the conformation of the mutants cannot be ruled out (the fc portion of the protein as well as gp1d contributed to the measured signal). we then cloned orfs encoding mutant gp1s into plasmids expressing full-length macv gpc. retroviral pseudotypes were created as described [17, 26] : hek 293t/17 cells were transfected by the calcium-phosphate method with plasmid encoding 1) macv gpc and variants thereof, together with 2) the pqcxix vector (clonetech) expressing enhanced green fluorescent protein (egfp) flanked by the moloney murine leukemia virus (momlv) long terminal repeats, and 3) plasmid containing the momlv gag/pol genes. cell supernatants were cleared and measured using the retro-x tm qrt-pcr titration kit (clonetech). equivalent amounts of pseudotypes were adsorbed on macv-permissive cells for 2 h. after 48 h, cells were stained with hoechst 33342 (invitrogen) and the percentage of egfp-positive cells was determined by high-content quantitative image-based analysis using an opera confocal imager (perkin elmer). cells exposed to mock pseudotypes (media of cells transfected with the pqcxix vector and the mlv gag/pol plasmid) were used as negative controls. western blot analysis of macv gp2 expression in the producer cells (data not shown) and in the various momlv pseudotype preparations ( fig. 3 lower panel) was performed using an antibody against macv gp2 (new england peptide) to examine expression, processing, and incorporation levels of wt macv gpc and variants thereof. in agreement with the co-ip and cell-binding data, momlv carrying gpc mutants d114a, s116a, d140a, and k169a had no effect on entry into human (hela) and nonhuman primate (vero) cells (fig. 3) . mutants d123a, d155a, p160a, f226a, k167a, and k211a had an intermediate effect on macv entry, with the first four amino acids playing a more prominent role. interestingly, macv gpc mutant f226a had a much more pronounced effect on hela cell entry (.80% reduction) than on vero cell entry (.40% reduction). d155a, p160a, and k211a mutants also exhibited decreased incorporation levels into momlv pseudotypes. however, as the cell-entry assay results are in agreement with our results using purified proteins (fig. 2b and 2c) , we suggest that the observed phenotype is not due to decreased incorporation levels but due to the mutations themselves. macv gpc mutants n178a and d159a were not incorporated into momlv pseudotypes and therefore were unable to transduce either cell line. macv gpc mutant w147a behaved unexpectedly in that it had no effect on momlv transduction efficiency, despite its decreased precipitation of htfr1 and attachment to the cell surface of macvpermissive cells (fig. 2) . we hypothesize that w147a might be misfolded in the context of macv gp1d but folded correctly in the context of full-length gpc. the two mutants with the most significant effect on cell entry were the r111a mutant and the triple mutant y122a/d123a/k169a, exhibiting more than 70% reduction in momlv transduction efficiency when compared to wt gpc. finally, the y122a mutant, while minimally affecting entry into vero cells, decreased momlv entry into hela cells. this is in agreement with our assays using purified proteins (fig. 2) , in which the y122a mutant bound efficiently to the cell surface of vero cells (fig. 2c) yet was unable to immunoprecipitate htfr1. in summary, our results demonstrate that gp1 residues r111, d123, y122, and f226 are important for htfr1 binding and cellentry of macv (figs. 2 and 3) . these results are in accordance with a recent study demonstrating that the central region of gtov gp1 (residues 85-221) interacts with htfr1 and that residues 159-221 are essential for this interaction [17] . these results are also in agreement with the recently published structure of macv gp1 bound to htfr1 [25] . macv gp1 r111, which is not conserved in any of the other new world arenaviruses, is part of interaction motif 1 and makes prominent contacts with htfr1 bii-2 strand, namely v210 (fig. 1, top panel, and fig. 4) . f226, unique to macv (w in other new world arenaviruses), is part of interaction motif 3 and has a hydrophobic interaction with htfr1 v210, as well as a van der waals contact with htfr1 i201 and l212 (fig. 1, top panel, and fig. 4 ). d123 and y122 are part of interaction motif 4 and are conserved between macv, junv, and sabv. d123 forms a hydrogen bond with htfr1 k344, whereas y122 forms one with htfr1 e343 and in addition contacts htfr1 residues a293, e294, and a340 (fig. 1, top into hela cells (fig. 3) . in accordance, this mutant bound efficiently to the cell surface of vero cells (fig. 2c ) yet was unable to immunoprecipitate htfr1 (fig. 2b) . these results suggest that y122 might be important for binding htfr1 but dispensable for binding simian tfr1, or that additional receptors/attachment factors are facilitating binding and entry of macv into vero cells. whereas tfr1 residues e343, a293, and a340 are conserved among human and simian tfr1, glutamate 294 is an aspartate in simian tfr1 and the proximal asparagine 292 is a lysine. macv gpc residues d114, s116, d140, k169, and most likely w147 are dispensable for cell attachment and entry of macv. according to the crystal structure, d114 and s116 form interaction motif 2, which contacts htfr1 residues n348, s370, and k371. k169, a part of interaction motif 4, contacts k344, and e294 of htfr1. in our functional assays, using both purified proteins and momlv pseudotypes, mutating these residues had no or minimal effect on htfr1 binding and cell-entry (figs. 2 and 3), suggesting that although they might contact htfr1, they by themselves play no significant role in htfr1 usage by macv. residues d155, p160, and k211 of macv gpc had an intermediate effect on htfr1 binding and cell-entry, with the first two amino acids being more predominant. these residues were not found to contact htfr1 residues in the crystal structure and could exert their effect on cell-surface binding and entry by affecting overall or local folding of macv gp1 and possibly by interacting with a yet unknown attachment or entry factor. p160 is conserved among all clade b new world arenaviruses (fig. 1, top panel) , which use tfr1 as a receptor [1, 21] . however, d155 is conserved only among clade b new world arenaviruses that cause hemorrhagic fevers in humans and bind htfr1. amapari (amav) and tacaribe (tcrv) viruses, which are considered non-pathogenic for humans [2] and which do not bind htfr1 but bind tfr1 of their mammalian hosts [21] , do not have an aspartate residue in this position (fig. 1, top panel) . finally, it is difficult to establish the role of macv gpc residues n178 and d159 in cell binding and entry of the virus. in the context of purified gp1d, mutation of these amino acids did not affect overall folding (fig. 2d) , but in the context of gpc these mutants failed to be incorporated into momlv pseudotypes. these results suggest that mutagenesis of n178 and d159 might lead to small changes in folding of gp1 (not being detected by cd) that might affect accessibility of htfr1-binding residues in their vicinity. alternatively, trafficking, folding, or binding of gp1 to ssp or gp2 might be defective only in the context of full-length gpc. the macv gp1-htfr1 crystal structure revealed seven nlinked glycans on asn178 [25] . it is plausible that these glycans play an important role in macv glycoprotein folding, trafficking, gp complex formation, or entry. n178 and d159 (with the exception of chpv) are conserved among all clade b new world arenaviruses (fig. 1, top panel) , which use tfr1 as a receptor [1, 21] . we have used purified macv gp1 and momlv pseudotyped with the macv glycoproteins in all experiments. several studies suggest that retroviral vectors pseudotyped with the glycoproteins of arenaviruses adopt the receptor-binding characteristics of the corresponding viruses [17, 20, 27] . therefore, we believe this surrogate system to be valuable for initial studies of viral cell binding and entry. however, it is important that the system is simplistic and uses only a few viral proteins in the absence of other viral-encoded proteins or genomic elements. therefore, any results obtained with this system should be verified with infectious virus. unfortunately, no reverse genetics system has been developed for macv to date, making it impossible to evaluate the effect of the mutated residues on entry of infectious macv. currently there is no fda-approved treatment to stop the spread of macv once humans are infected. defining interactions between the viral glycoprotein and a cellular receptor may allow for drug development that could limit viral spread in the infected individual and reduce infection during an outbreak. our mutagenesis analysis of macv gp1 experimentally elucidates the structure and functional interaction motifs of macv gp1 and its receptor htfr1. these studies therefore provide a solid platform for the future design and development of small molecule inhibitors and antivirals specifically targeting viral entry. family arenaviridae ) fields virology genetic detection and characterization of lujo virus, a new hemorrhagic feverassociated arenavirus from southern africa national institutes of health (nih) (2007) biosafety in microbiological and biomedical laboratories (bmbl) niaid category priority pathogens hemorrhagic fever in cochabamba reemergence of bolivian hemorrhagic fever site-specific antibodies define a cleavage site conserved among arenavirus gp-c glycoproteins mechanisms for lymphocytic choriomeningitis virus glycoprotein cleavage, transport, and incorporation into virions the lassa virus glycoprotein precursor gp-c is proteolytically processed by subtilase ski-1/ s1p bitopic membrane topology of the stable signal peptide in the tripartite junin virus gp-c envelope glycoprotein complex endoproteolytic processing of the lymphocytic choriomeningitis virus glycoprotein by the subtilase ski-1/s1p identification of lassa virus glycoprotein signal peptide as a trans-acting maturation factor identification of an n-terminal trimeric coiled-coil core within arenavirus glycoprotein 2 permits assignment to class i viral fusion proteins transferrin receptor 1 is a cellular receptor for new world haemorrhagic fever arenaviruses new world clade b arenaviruses can use transferrin receptor 1 (tfr1)-dependent and -independent entry pathways, and glycoproteins from human pathogenic strains are associated with the use of tfr1 differences in tropism and ph dependence for glycoproteins from the clade b1 arenaviruses: implications for receptor usage and pathogenicity receptor use by pathogenic arenaviruses host-species transferrin receptor 1 orthologs are cellular receptors for nonpathogenic new world clade b arenaviruses investigation of clade b new world arenavirus tropism by using chimeric gp1 proteins unusual molecular architecture of the machupo virus attachment glycoprotein structural basis for receptor recognition by new world hemorrhagic fever arenaviruses angiotensinconverting enzyme 2 is a functional receptor for the sars coronavirus characterization of the cellular receptors for the south american hemorrhagic fever viruses junin, guanarito, and machupo opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the us army, the us department of health and human services or of the institutions and companies affiliated with the authors. key: cord-001537-i34vmfpp authors: lima, francisco esmaile de sales; cibulski, samuel paulo; dos santos, helton fernandes; teixeira, thais fumaco; varela, ana paula muterle; roehe, paulo michel; delwart, eric; franco, ana cláudia title: genomic characterization of novel circular ssdna viruses from insectivorous bats in southern brazil date: 2015-02-17 journal: plos one doi: 10.1371/journal.pone.0118070 sha: doc_id: 1537 cord_uid: i34vmfpp circoviruses are highly prevalent porcine and avian pathogens. in recent years, novel circular ssdna genomes have recently been detected in a variety of fecal and environmental samples using deep sequencing approaches. in this study the identification of genomes of novel circoviruses and cycloviruses in feces of insectivorous bats is reported. pan-reactive primers were used targeting the conserved rep region of circoviruses and cycloviruses to screen dna bat fecal samples. using this approach, partial rep sequences were detected which formed five phylogenetic groups distributed among the circovirus and the recently proposed cyclovirus genera of the circoviridae. further analysis using inverse pcr and sanger sequencing led to the characterization of four new putative members of the family circoviridae with genome size ranging from 1,608 to 1,790 nt, two inversely arranged orfs, and canonical nonamer sequences atop a stem loop. viruses of the circoviridae family are known to infect a wide range of vertebrates. the virions consist of naked nucleocapsids of about 20 nm in diameter, with a circular single stranded dna (ssdna) genome of approximately 2.0 kb [1] . they have an ambisense genome organization containing two major open reading frames (orfs) inversely arranged, responsible for encoding the replicase (rep) and capsid (cap) proteins, and are separated by a 3' intergenic region (igr) between the stop codons and a 5' igr between the start codons [2] . some circoviruses are major pathogens of pigs [3] [4] [5] , e.g. porcine circovirus 2 (pcv2) which causes either asymptomatic infections or clearly apparent disease which may be responsible for significant economic losses [6] [7] [8] [9] [10] . in birds, avian circoviruses, within the genus gyrovirus, have been identified in a broad range of avian species; one of them, chicken anemia virus (cav), is a major cause of disease, associated to lymphoid depletion, immunosuppression and developmental abnormalities [11] [12] [13] [14] [15] . according to the document 2014.006a-gv from ictv, there is a proposal of the gyrovirus genus removal from circoviridae to anelloviridae family due to recent metagenomic studies on gyroviruses showing a very high sequence divergence when compared to other circoviruses members. recent metagenomic approaches, high-throughput sequencing techniques and degenerate pcrs have led to the identification of small circular dna genomes in fecal samples of wild mammals, in insects as well as from environmental samples [2, [16] [17] [18] . some of the newly described circular genomes are similar to those of circoviruses, but phylogenetically different from the previously known avian and porcine circoviruses [18] . their distinct nucleotide/ amino acid composition and genome organization allowed authors to propose the creation of a new genus within the circoviridae, which was named cyclovirus. in comparison to members of the genus circovirus, both rep and cap cycloviruses genes are smaller, with shorter or no 3' igr between the stop codons of the two major orfs and a longer 5' igr between the start codons of the two major orfs [2] . sequences related to circoviruses have been identified based on the detection of the conserved rep region involved in rolling circle replication (rcr) [19] . cyclovirus genomes were detected in wild animal's samples, human feces and cerebrospinal fluids; muscular tissues of farm animals such as chickens, cows, sheep, goats, and camels [20, 21] . currently, eight different species of cycloviruses have been detected in winged-insect populations highlighting they circulate in a wide host range possessing a high genetic diversity, as well [18] [19] [20] [22] [23] [24] . so far, classification for the genus circovirus considers circoviruses sharing >75% genomewide nucleotide identity and >70% amino acid identity in the capsid (cap) protein to the same species. although, there are no species demarcation criteria for the genus cyclovirus, the taxonomic classification for the family circoviridae considers viruses sharing >60% in their cap amino acid identity level as belonging to distinct genera [19] . in the present article, the detection of ssdna genomes from bat fecal samples is reported. genome segments were amplified by consensus/degenerate pcr. whole genome sequencing and phylogenetic analyses of the sequences obtained revealed that four of the sequences represent viral genomes of new members of the family circoviridae. permission for this work on protected bats was granted by health monitoring (cevs-centro estadual de vigilância em saúde) of the brazilian federal state of rio grande do sul. the study did not involve any direct manipulations of bats and relied entirely on collection of fecal samples from the attic floor. all experiments were performed in compliance with the european convention for the protection of vertebrate animals used for experimental and other scientific purposes (european treaty series-no. 170 revised 2005) and the procedures of the brazilian college of animal experimentation (cobea). it must be highlighted that we had the owner's permission to access the attic for the purposes of this study. in case of future surveys in porto alegre, the health monitoring (cevs) will be contacted to obtain the permissions. a maternity roost of bats was identified in the summer of 2012 in the attic of a private residence in the central area of the municipality of porto alegre, rio grande do sul, southern brazil. the colony was estimated to harbor about 500 bat specimens of insectivorous bats of two species, velvety free tailed bats (molossus molossus) and brazilian free tailed bats (tadarida brasiliensis) [25] . speciation was confirmed by dna extraction from fecal pellets, amplification and sequencing of the mitochondrial cytochrome b (cytb) gene as described [26] . one hundred fecal samples were collected from the attic floor as follows: a plastic film was spread on the ground of the attic compartment and fresh droppings were collected with clean disposable forks in the following night. each sample consisted of pool of 5 fecal droppings, which were immediately sent to the laboratory and stored at -80°c. the samples were then thawed, resuspended and in 1 ml of hank's balanced salt solution (hbss), vortexed and centrifuged at 10 .000 x g for 5 min. the supernatants were then transferred to fresh tubes, filtered through 0.45 μm pore-size syringe filters (fisher scientific, pittsburgh, pa) and submitted to dna extraction. total fecal dna was extracted from 400 μl of the supernatants (above) with phenol-chloroform (invitrogen) [27] . the extracted dna was eluted in 50 μl of te (tris-hydrochloride buffer, ph 8.0, containing 1.0 mm edta), treated with 20 μg/ml of rnase a (invitrogen) and stored at -80°c. subsequently, samples were submitted to amplification in a nested-pcr targeting the rep gene of circoviruses/cycloviruses with the following degenerate primers: cv-f1 (5´-ggiayiccicayyticargg-3´), cv-r1 (5`-awccaiccrtaraartcrtc-3`), cv-f2 (5´-ggiayiccicayyticarggitt-3´), and cv-r2 (5´-tgytgytcrtaiccrtccc acca-3´) [2] . briefly, the nested pcr was performed as follows: the first reaction was performed in a 25 μl volume containing 20 to 50 ng of sample dna 1 mm mgcl 2 , 0.2 μm of each primer (cv-f1 and cv-r1), 1.5 u taq dna polymerase (invitrogen), 10% pcr buffer and 0.6 mm dntps. the cycling conditions were: 5 min at 95°c; 40 cycles of 1 min at 95°c, 1 min at 52°c, 1 min at 72°c and a final incubation at 72°c for 10 min. for the second (nested) reaction, the 25 μl mix components were: 1 μl of the 1 st reaction product, 1 mm mgcl 2 , 0.2 μm of each primer (cv-f2 and cv-r2), 1.5 u taq dna polymerase (invitrogen), 10% pcr buffer and 0.6 mm dntps. the cycling conditions were: 5 min at 95°c; 40 cycles of 1 min at 95°c, 1 min at 56°c, 1 min at 72°c, and a final incubation at 72°c for 10 min. products with a size of approximately 400 bp were purified and directly sequenced using primer cv-r2. to confirm the sequences, each product was sequenced three times. samples were sequenced with the big dye terminator cycle sequencing ready reaction (applied biosystems, uk) in an abi-prism 3100 genetic analyzer (abi, foster city, ca), according to the protocol of the manufacturer. sequences similar to the rep gene sequences of circovirus-like-genomes were aligned for designing of new sets of primers to perform the inverse pcr (ipcr). the ipcr were carried out in a 25 μl reaction mixture optimized with platinum taq hi-fi (invitrogen™) (cycling conditions can be informed upon request) and the primer sequences as follows: . standard precautions were taken to avoid contamination and negative controls were added to each batch of reactions. five microliters of the pcr products were electrophoresed in 0.7% agarose gels and the products visualized on uv light after staining with ethidium bromide. the amplicons corresponding to the sizes ranging from 1-2 kb were purified and cloned into pcr 2.1-topo cloning kit (invitrogen™). three insert-containing plasmids of each clone were sequenced with m13 forward and reverse primers as described above. the full-length sequence of genomes was constructed by "genome walking" using the geneious software (version 7.1.3). identification of putative orfs was made with aid of orf finder (ncbi; http://www.ncbi. nlm.nih.gov/gorf/gorf.html). sequence analyses were performed with the blastx software (http://www.ncbi.nlm.nih.gov/blast/). nucleotide sequences were aligned and compared to sequences of human, animal and sewage-associated members of the circoviridae available at genbank database using clustalw [28] . the alignments were optimized with the bioedit sequence alignment editor program version 7.0.9 [29] . the hairpin and stem-loop structures were identified in mfold [30] . phylogenetic analysis was carried out in mega5 [31] . the confidence of each branch in the phylogeny was estimated with bootstrap values calculated from 2000 replicates. for the purpose of this work, the samples were named bat circovirus porto alegre (batcv poa), followed by the cluster number to which each one was assigned. amplicons with the expected size (about 400 bp) were obtained from 24 out of the 100 (24%) fecal samples screened. the amplified dna was direct sequenced. the nucleotide sequences corresponding to part of the rep gene were determined and submitted to genbank (km401658-km401681). blastx analysis showed that these partial rep sequences have an amino acid identity of 10-76% with those of known circoviruses and 87-100% among themselves. a phylogenetic tree was constructed based on the alignment of the deduced amino acid sequences herein detected with those of the representative circovirus and cyclovirus sequences (fig. 1) . as shown in the tree, it was observed the arrangement of five main groups with clusters ii (4 sequences), vi (3 sequences) and vii (2 sequences) falling into the clade of cycloviruses, in contrast to clusters i (13 sequences) and v (2 sequences) that formed distinct and distant groups from those formed by circoviruses and cycloviruses. the arbitrary division of these sequences in clusters was carried out to analyze their genomic features, assuming that according to the criteria used for circovirus diversity analysis, distinct species comprising more than >20% sequence divergence are considered to be classified as an individual viral [32] . according to this, we could infer the detection of five potential new species from bat samples (3 cycloviruses and 2 circoviruses). the impossibility to achieve the complete sequencing of virus dna from clusters i and v was probably due to the high gc-rich content present in the 3´igr gc region, even though attempts on pcr amplification before sequencing were made without much success by varying the concentrations of dmso and/or in the presence of 50% 7-deaza-gtp and 50% dgtp (new england biolabs), as performed by rijsewijk et al. [33] . the predicted two orfs, rep and cap, are present and inversely arranged in all sequences as shown in fig. 2 . the predicted cap protein sequences consist of 197-231 amino acids and share an amino acid identity of 24-76% with the known cycloviruses/circoviruses and 15.5-88.8% among themselves (tables 1 and 2) . the predicted rep protein sequences ranged from 232 to 280 amino acid and have an amino acid identity ranging from 9.2-44.4% among themselves (tables 1 and 2) . stem-loop structures were found in all 4 bat circular genomes. they have a conserved nonanucleotide motif located at the 5' igr (nantattac) and are considered to be responsible for initiating the rolling-cycle replication of circoviruses [18, 34] . as shown in table 3 , all four batcv poa also contain a conserved nonamer sequence in the loop region of the 5' igr, different from the conserved cyclovirus and circovirus nonanucleotide motif sequence, but similar to the loop motif of cycloviruses found on bat, human and chimpanzee feces (batcv poa ii, v, vi) and slightly modified from those of cyclovirus and circovirus (batcv poa i) [2, 17, 18, 20] . the predicted protein sequences encoded by orf2 (cap) and orf1 (rep) of batcv i-vi genomes were used for phylogenetic analysis with representative and recently discovered circoviruses/cycloviruses; pepper golden mosaic virus was used as outgroup, as they are somewhat related to other members in the circoviridae family (fig. 3a, 3b and 3c ). as shown in the trees, batcv poa/2012/ii and vi fell into the cyclovirus clade already identified in chickens, chimps, bats, goats, humans and dragonflies [2, 17, 18, 20, 22] . when analyzing the cap-encoding region (fig. 3a) , batcv poa/2012/ii was related to a cyclovirus detected in muscle tissues of a goat from pakistan through degenerate/consensus pcr [20] , and batcv poa/2012/vi was more related to dragonfly cyclovirus detected through viral metagenomics [22] . however, when analyzing both genomes according to the conserved rep-encoding region, it was observed that they formed a monophyletic clade (fig. 3b) . on the other hand, batcv poa/2012/i and v fell outside the circovirus and cyclovirus clades, not yet related to any genus of circoviridae family along with bat circovirus-like virus tm6 and batcv-sc703 [17, 18] . this situation was confirmed based on the alignments of the whole genomes, producing a similar tree topology (see fig. 3c ). these sequences are closer to sequences detected in guano and fecal samples collected from bats in the united states and china through metagenomic approaches, suggesting that these viruses have the same host origin, likely from bats [17, 18] . however, currently, no classification has been fully considered to these sequences. in this work we report the discovery of 4 novel circular ssdna genomic sequences from insectivorous bats feces from brazil. in the recent years, many genomes of circoviruses, cycloviruses and rep-containing circular dna viruses have been characterized in mammals, birds, insects and environmental samples [19] bringing to light a high level of genetic diversity among these viruses [19, 35] . according to our results, two genomes belong to genus cyclovirus (batcv poa ii and vi). these genomes are organized and contain two major orfs in opposite directions, presenting in their 5' igr of the rep orf the cyclovirus-conserved nonanucleotide motif (5'-taatactat-3') in their loop region (table 3) . batcv poa i and v present their cap located in the positive strand and the larger rep located on the minus strand, as expected for circoviruses, but this pattern was not present in batcv poa ii and vi, as shown in table 1 . the phylogenetic analysis constructed based on the alignments of the complete rep and cap protein confirms that batcv poa/ii and vi cluster into the genus cyclovirus along with the chinese cycloviruses sequences clade detected in bat feces [18] and sharing less than 65% of identity at the cap/rep amino acid level. batcv poa i and v had a low amino acid identity with cap (<20%) and rep (<10%) sequences of two other sequences detected in bat feces in this study with known circoviruses/cycloviruses (table 2) . consequently, they formed a distinct clade along with other bat-sourced sequences, expanding the view of diversity in these new ssdna viruses that are divergent enough at the sequence level that they could very likely be part of a different genus. in our study, we detected cyclovirus and circovirus related sequences at a frequency of 24% in the examined samples. however, due to methodological limitations, restriction in location and variety of bat species, we were not able to extrapolate our results to epidemiological data (such as incidence and prevalence) or to which bat species the ssdna positive samples belonged. as performed by ge et al. in china [36] , further investigation is needed to determine the prevalence of circoviruses in other brazilian bat species. nevertheless, it becomes clear that such study is worthy to understand the great diversity of circoviruses found worldwide. our study was based on the phylogenetic analysis and comparison to the sequences recovered. the finding of known insect viruses in bat feces simply reflects the diet of these insectivorous bats, which play an important role on predating insects. viral dna detection in bat feces does not allow one to differentiate between viral replication in bats or simple passage through the digestive track from ingested food [20, 35] . to date, few members of the circovirus genera can be related to severe clinical conditions in animals, with the exception of pcv2 and some of the avian circoviruses [5] . even with the recent discovery of many cycloviruses, circoviruses-like or rep-like sequences in a variety of mammals tissues and feces, including humans fecal samples [20, 36, 37] , there is no syndrome yet associated with these viruses. nevertheless, a recent identification of a new cyclovirus from vietnamese and malawi patients with acute central nervous system infection of unknown etiology raises the possibility of disease association, yet to be proven [38, 39] , although possibly with limited geographic distribution [38] . in this work, two more circular dna genomes were characterized which did not fall within the circo/cycloviruses clade grouping instead distantly with tm6 and batcv-sc703 [17, 18] both also from bat feces. these new genomes have in common the presence in the rep n-terminus of the same motifs associated with rolling circle replication (ftlnn, tphlqgy) and dntp-binding (gxgks), as well as the conserved identified in the carboxy half of rep amino acid motifs associated with 2c helicase function (wwdgy and dryp) [19] . the n-terminal regions related to cap proteins of batcv poa i and v are highly basic and arginine-rich, as is typical for circoviruses capsid proteins with arginine residues ranging from 36%-42% (genome i and v, respectively) along the first 50 aa, in contrast to tm6 (28%) and sc703 (26%). they are also distinguishable based on their cap and rep sizes (data not shown), as well as on the low amino acid level for both proteins, as the percentage of amino acid identity of batcv poa i and v shows a rep identity <45% and <35% for cap identity in relation to tm6 and sc703. based on these genomes characteristics, even though they are clustered in a separate clade, not yet characterized, they are new viral species. upon the discovery of other sequences grouping along with these genomes, it will be of interest to propose the creation of a new genus within circoviridae by the international committee on taxonomy of viruses (ictv). here we report the detection of four novel circular ssdnas from bat feces after whole-genome characterization within the family circoviridae. so far, it is not clear if these new ssdna detected have some important role on pathogenesis. in addition to bioinformatics analysis, future investigations must include attempts in virus isolation to confirm host origin, which will give some light to better understand the relationships between these circular dna viruses and bats. conceived and designed the experiments: fesl spc pmr. performed the experiments: fesl spc hfs tft apmv. analyzed the data: spc ed. contributed reagents/materials/analysis tools: pmr acf. wrote the paper: fesl spc pmr acf ed. virus taxonomy: classification and nomenclature of viruses multiple diverse circoviruses infect farm animals and are commonly found in human and chimpanzee feces pathogenesis of postweaning multisystemic wasting syndrome caused by porcine circovirus 2: an immune riddle isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome insights into the evolutionary history of an emerging livestock pathogen: porcine circovirus 2 porcine circoviruses: a review quantification of porcine circovirus type 2 (pcv2) dna in serum and tonsillar, nasal, tracheo-bronchial, urinary and faecal swabs of pigs with and without postweaning multisystemic wasting syndrome (pmws) a review of porcine circovirus 2-associated syndromes and diseases porcine circovirus type 2 associated disease: update on current terminology, clinical manifestations, pathogenesis, diagnosis, and intervention strategies recent advances in the epidemiology, diagnosis and control of diseases caused by porcine circovirus type 2 psittacine beak and feather disease virus nucleotide sequence analysis and its relationship to porcine circovirus, plant circoviruses, and chicken anaemia virus cloning and sequencing of duck circovirus (ducv) genome sequence determinations and analyses of novel circoviruses from goose and pigeon circoviruses: immunosuppressive threats to avian species: a review identification of a novel circovirus in australian ravens (corvus coronoides) with feather disease frequent detection of highly diverse variants of cardiovirus, cosavirus, bocavirus, and circovirus in sewage samples collected in the united states bat guano virome: predominance of dietary viruses from insects and plants plus novel mammalian viruses genetic diversity of novel circular ssdna viruses in bats in china a field guide to eukaryotic circular single-stranded dna viruses: insights gained from metagenomics possible cross-species transmission of circoviruses and cycloviruses among farm animals identification of a new cyclovirus in cerebrospinal fluid of patients with acute central nervous system infections dragonfly cyclovirus, a novel single-stranded dna virus discovered in dragonflies (odonata: anisoptera) novel cyclovirus discovered in the florida woods cockroach eurycotis floridana (walker) high global diversity of cycloviruses amongst dragonflies detection of alphacoronavirus in velvety free-tailed bats (molossus molossus) and brazilian free-tailed bats (tadarida brasiliensis) from urban area of southern brazil genomic characterization of severe acute respiratory syndrome-related coronavirus in european bats and classification of coronaviruses based on partial rna-dependent rna polymerase gene sequences molecular cloning: a laboratory manual: cold spring harbor laboratory press clustal w and clustal x version 2.0 bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt; 1999 well-determined" regions in rna secondary structure prediction: analysis of small subunit ribosomal rna mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods virus taxonomy, ixth report of the international committee for the taxonomy of viruses discovery of a genome of a distant relative of chicken anemia virus reveals a new member of the genus gyrovirus rolling-circle replication of an animal circovirus genome in a theta-replicating bacterial plasmid in escherichia coli rapidly expanding genetic diversity and host range of the circoviridae viral family and other rep encoding small circular ssdna genomes evaluation of the in vivo radiosensitizing activity of etanidazole using tumor-bearing chick embryo host effect on the genetic diversification of beet necrotic yellow vein virus single-plant populations limited geographic distribution of the novel cyclovirus novel cyclovirus in human cerebrospinal fluid key: cord-000765-r7y1cqou authors: chang, yu-ming; chen, cammy k. -m.; chang, yuan-chih; jeng, wen-yih; hou, ming-hon; wang, andrew h. -j. title: functional studies of ssdna binding ability of marr family protein tcar from staphylococcus epidermidis date: 2012-09-21 journal: plos one doi: 10.1371/journal.pone.0045665 sha: doc_id: 765 cord_uid: r7y1cqou the negative transcription regulator of the ica locus, tcar, regulates proteins involved in the biosynthesis of poly-n-acetylglucosamine (pnag). absence of tcar increases pnag production and promotes biofilm formation in staphylococci. previously, the 3d structure of tcar in its apo form and its complex structure with several antibiotics have been analyzed. however, the detailed mechanism of multiple antibiotic resistance regulator (marr) family proteins such as tcar is unclear and only restricted on the binding ability of double-strand dna (dsdna). here we show by electrophoretic mobility shift assay (emsa), electron microscopy (em), circular dichroism (cd), and biacore analysis that tcar can interact strongly with single-stranded dna (ssdna), thereby identifying a new role in marr family proteins. moreover, we show that tcar preferentially binds 33-mer ssdna over double-stranded dna and inhibits viral ssdna replication. in contrast, such ssdna binding properties were not observed for other marr family protein and tetr family protein, suggesting that the results from our studies are not an artifact due to simple charge interactions between tcar and ssdna. overall, these results suggest a novel role for tcar in regulation of dna replication. we anticipate that the results of this work will extend our understanding of marr family protein and broaden the development of new therapeutic strategies for staphylococci. staphylococci are among the most common causes of bacterial infection in the community and pose a major danger to human health. s. aureus is the most well-known of the species which produce hospital-and community-acquired infections, with methicillin-resistant s. aureus presenting a serious public health threat [1] . s. epidermidis is the sister species of s. aureus which often causes infection in immunocompromised individuals or those following damage to the epithelium. both of them produce biofilm to protect themselves from host immune system and enhance their resistance to antibiotic chemotherapy [2] . the key component of the biofilm extracellular matrix in staphylococci is polysaccharide intercellular adhesin (pia) [3] , an essential factor in biofilm formation composed of homopolymer of b-1,6-linked n-acetylglucosamine (glcnac). the production of pia depends on the expression of the icaadbc operon, and tcar and icar are a weak and a strong negative regulator of transcription of the ica locus, respectively [4] . the transcription regulator tcar is a member of the marr family, and is involved in teicoplanin and methicillin resistance in staphylococci [5] . the marr family proteins function as regulators of protein expression and these regulated proteins confer resistance to multiple antibiotics, household disinfectants, organic solvent virulence factors, and oxidative stress agents [6, 7, 8, 9, 10, 11] . the crystal structures of a number of marr family proteins have been reported, including marr from escherichia coli [12] , ohrr from bacillis subtilis [13] , mexr from pseudomonas aeruginosa [14] , marr from xanthomonas campestris [15] , slya from salmonella typhimurium [16] , adcr from streptococcus pneumonia [10] and tcar, which is studied in this work, from s. epidermidis [17] . these structures revealed that marr family proteins are all homodimers. the overall structure of each monomer could be divided into two functional domains, one is the dimerization domain and the other is the winged helix-turn-helix (whth) dna binding domain. the n and the c-terminal a-helices (a1, 5, 6) of one monomer interdigitate with those of the other monomer to produce dimerization interaction. in addition, the whth dna binding domain is composed of a2-a3-a4-ba-w1-bb which adopts the winged-helix-fold, and the amino acid sequences of this domain are highly conserved. as the marr-type proteins can act as positive, negative, or bifunctional regulators, tcar also acts as a multi-functional regulator. it is not only as a regulatory factor to affect the transcription of icaadbc [4] , the first regulator reported for cell wall-anchored proteins (spa and sasf), but also as the regulator of sars [18, 19] . we previously described the 3d structures of tcar in its apo form and in complex with salicylate as well as several aminoglycoside and b-lactam antibiotics [17] . in this research, comparison of the native tcar structures and those in complexes indicated that the regulation mechanism involves a large conformational shift in the dna binding lobe. several antimicrobial compounds inhibited tcar-dna interaction and further induced biofilm formation in s. epidermidis. in the present study, we found that tcar could interact with ssdna and the result demonstrated that tcar shows a stronger preference toward gcrich ssdna than to dsdna by using emsa, cd, and biacore experiments. however, the detailed mechanism of the interaction between tcar and ssdna still remains to be elucidated. in order to investigate the regulation mechanism of the ssdna binding ability of tcar, we applied electron microscopy (em) technique to reveal tcar-ssdna complex. furthermore, we clarified the role of tcar-ssdna interaction by in vitro replication assay and in vivo plaque assay. taken together, these results provide an in-depth investigation on the multiple functions of tcar in s. epidermidis. strong tcar binding to ssdna oligomers revealed by emsa tcar is known to bind and regulate the ica promoter [4] . we previously identified that tcar most strongly interacts with icar dna1 (a 33-mer pseudo-palindromic sequence containing consensus sequence ttnnaa) compared with other designed icar dna fragments [17] . however, when using the sense strand of icar dna1 (icar dna1s) and the antisense strand of icar dna1 (icar dna1a) ( figure 1a ) as controls in electrophoretic mobility shift assays (emsa), the result demonstrated that tcar shows a stronger preferences toward ssdna fragments (icar dna1s and dna1a) ( figure 1b) . to determine the type and length of the tcar-binding site on ssdna, a series of gc-rich and at-rich ssdna segments were designed ( figure 1a ) [20, 21] . their tcar binding ability was tested using emsa. as shown in figure 1c , tcar does not significantly interact with 17-mer gcrich (gc17) and at-rich (at17) ssdna oligomers, but shows strong interaction with 33-mer gc-rich (gc33) and at-rich (at33) ssdna sequences with a preference toward the 33-mer ssdna sequence with a molar ratio of 1:1. thus, we suggest that tcar prefers binding to the 33-mer ssdna. in order to evaluate the minimal dna binding length of tcar, gc-rich fragments of different lengths were designed. as seen in figure 1d , gc-rich fragments with 33, 29, and 25 bases showed similar binding strength to tcar; with tcar forming a large, apparently multimeric complex with gc33, a small complex with gc25, and both small and large complexes with gc29 in emsa. these results indicated that the minimal observed ssdna fragment size to allow tcar binding ranges between 17 to 25mer; providing useful information for the design of a dna fragment with precise length suitable for crystal packing. up to now, only three marr family protein complex structures have been reported, and the first one is complexed with dsdna [13, 16, 22] . the second one is complexed with salicylate [12, 17, 22, 23] and we discovered the third case which is complexed with antibiotics [17] . we have already obtained tcar-ssdna crystals and collected x-ray diffraction data to 3.6 å resolution at spring-8 (hyogo, japan), beamline bl12b2. however, the phase problem is still the main challenge and the works are currently under progress. moreover, to investigate whether tcar preferentially binds to ssdna or dsdna, the ability of ssdna to compete with the tcar-dsdna complex was evaluated. for the competition assay, the icar dna1 probe was preincubated with tcar (dimer) protein to allow formation of the dsdna-tcar complex prior to mixing with increasing amounts of single-stranded gc33 dna. it has been known that ssdna products have lower migration velocity compared to its dsdna counterparts in polyacrylamide electrophoresis [24, 25] . as shown in figure 1e , ssdna, as a competitor, interfered the binding of tcar to the dsdna, suggesting a binding preference for ssdna. to further confirm this result, icar dna1 and gc33 ssdna oligomers were mixed, and their interaction strengths with tcar were compared using emsa ( figure 1f ). findings indicated that increasing the concentration of tcar produces a ssdna band shift greater than that for dsdna, confirming a stronger interaction between tcar and ssdna. moreover, to investigate possible ph effect of ssdna binding activity of tcar, a series of buffers with increasing ph were tested for their potential interfere in tcar-ssdna binding. as shown in figure 1g , the emsa results showed that tcar had a strongest affinity for gc33 at ph 8.0 and the affinity was reduced by decreasing ph. consequently, the result indicates that the ssdna binding activity of tcar is ph-dependent. to clarify whether the ssdna binding site of tcar is close, or identical, to the dsdna binding site, a tcar quadruple mutant (4 positively charged amino acids responsible for dna binding mutated to alanines to produce r71a/k73a/r93a/k95a) [17] was designed and its ssdna and dsdna binding ability tested. as seen in figure 1h , the mutant failed to interact with either dsdna or ssdna. this indicated that these amino acids are essential for binding in both ssdna and dsdna. the marr protein of e. coli is a multidrug binding transcription regulator. a wide range of compounds, including 2,4-dinitrophenol, plumbagin, menadione, and salicylate, attenuate and inhibit its association with cognate dna [26] . in our previous study, salicylate and multiple antibiotics interfered with the transcriptional repressor activity of tcar [17] . these findings prompted the current investigation into the possible effects of antibiotics on the ssdna binding ability of tcar. here, to investigate the possible effect of some drugs on tcar, four compounds were tested for their potential inhibition on tcar-ssdna interaction. these include one beta-lactam antibiotics (ampicillin) that contain a b-lactam nucleus in their molecular structure and act by inhibiting the synthesis of the peptidoglycan layer of bacterial cell walls, two aminoglycoside antibiotics (kanamycin and gentamicin) that composed of several sugar groups and amino groups, and bacteriostatic antimicrobial (chloramphenicol) which is considered as a prototypical broad-spectrum antibiotic. as shown in figure 1i , kanamycin, chloramphenicol and gentamycin interfered with the ssdna (gc33) binding activity of tcar at a concentration of 2 mm and this effect was more pronounced at a higher concentration, suggesting that antibiotics inhibit formation of the tcar-ssdna complex. results indicated that ampicillin also antagonized the ssdna binding activity of tcar at a concentration of 20 mm. this result is consistent with the observation seen in spr, as discussed below. taken together, we believe that diverse kinds of antibiotics may interact with tcar to regulate its ssdna binding ability. the binding affinity of gc33 and tcar was determined quantitatively using surface plasmon resonance. increasing concentrations of tcar were passed across a flow cell coated with ssdna gc33 and the binding response was recorded as changes in response units (ru) after subtraction of the binding response for the reference flow cell. figure 2a shows a representative sensorgram. analysis of the sensorgram data indicates k a for the interaction of tcar with the ssdna gc33 is 8.8610 5 m 21 s 21 ; k d for the interaction of tcar with the ssdna gc33 is 9.5610 23 s 21 (table 1) . to investigate whether tcar binds to different types and different lengths of dna molecules, biacore experiment was used to test the binding of tcar to dna fragments of biotin-labeled ssdna and hairpin dna duplex (icar dna1). consistent with previous observations in fig. 1b , tcar binds to ssdna gc33 and ssdna at33 with the higher affinity and the association rates of tcar to icar dna1, and ssdna gc17, ranging from 4.3610 5 m 21 s 21 to 1.2610 5 m 21 s 21 , are much lower (shown in figure 2b ). however, when look at a wider range of dna sizes, tcar showed no detectable binding ability to ssdna 8-mer gc8 but the highest binding ability to ssdna gc99. the association rate with ssdna gc99 is 36-fold higher than with ssdna gc33, along with a 20-fold higher off-rate, suggested that cooperative binding of tcar may contribute significantly in its ssdna binding activity (table 1) . interestingly, the association rate of gc33 ssdna is two-times higher than icar dna1, but the dissociation rate of icar dna1 is the lowest compared to the dna fragments tested in this study, suggesting different modes of interaction occurs in ssdna-tcar and dsdna-tcar complex ( figure 2b ). furthermore, in order to determine whether other marr family and tetr family proteins such as sar2349 from s. aureus and . icar dna1 probe duplex of 1 mm was pre-incubated with 2 mm tcar (dimer) at room temperature for 15 min before mixing with increasing concentration of gc33 ssdna, followed by the same procedure as described in the legend to figure 1b . (f) competition experiment was carried out to compare the binding strength of tcar to ssdna and to dsdna. in the emsa analysis, 1 mm icar dna1 probe duplex was pre-incubated with 1 mm gc33 ssdna fragment for 15 min at room temperature before mixing with tcar protein of increasing concentration. (g) the effects of ph to the tcar binding to ssdna gc33 in emsa experiment. molar ratio of gc33:tcar was 1:2. (h) emsa analysis of mutant tcar protein binding to ssdna (gc33) and dsdna (icar dna1) fragments with different protein-dna ratio. (i) emsa analysis of the binding of tcar protein to ssdna(gc33) in the present of different types of antibiotics. gc33 ssdna probe of 1 mm was preincubated with 2 mm tcar (dimer) at room temperature for 15 min before mixing with 2 mm or 20 mm antibiotics, followed by the same procedure as described in the legend to figure 1b icar from s. epidermidis have the ssdna binding ability, we conducted a series of spr experiments to analyze the binding ability of sar2349 and icar proteins to gc33 ssdna ( figure 2c ). the result shows that not all marr family proteins have this ssdna binding ability, thus pointing to the specific ssdna-binding feature of tcar. in addition, we have previously demonstrated that antibiotics appear to antagonize the ssdna binding activity of tcar ( figure 1i ). therefore, a measurement for the effect of kanamycin and ampicillin to the gc33 ssdna binding affinity of tcar is conducted using surface plasmon resonance to confirm the result. as seen in figure 2d , the affinity between tcar and gc33 ssdna is shown by a decrease in ru values in the presence of antibiotics. this was especially apparent with kanamycin, which yield the lower binding capacity. taken together, we demonstrate that tcar shows a higher binding affinity to ssdna than to dsdna, and several antibiotics could regulate the ssdna binding activity of tcar. conformational changes of tcar in response to ssdna were monitored using cd spectroscopy [27] . as shown in figure 3 , the cd spectra of tcar protein were scanned from 200 to 250 nm in the presence of gc33. with increasing concentrations of gc33 ssdna, the cd spectrum shows a concomitant decrease in the intensity of the negative peak at 222 and 210 nm, revealing the conformational change of tcar. furthermore, in order to examine whether tcar shows a binding ability towards much longer ssdna fragments such as viral wx174-ssdna, the interaction between them was also examined with increasing concentration (0, 2.5, 10 mm) of viral in order to further confirm our finding that tcar forms complex with viral ssdna, the m13 and wx174 phage ssdna circles were used as probes in emsa to evaluate tcar binding. as shown in figure 4a , tcar reduced the mobility of the m13 and wx174 ssdna, but s. epidermidis icar and s. aureus marr family protein sar2349 had no specific interactions with ssdna. this indicated that tcar has strong viral ssdna-binding ability. it is also worth noting that other marr family protein and tetr family protein do not have such ssdna binding properties with high affinity, suggesting that the results from tcar studies are not an artifact due to simple charge interactions between tcar and ssdna. furthermore, since the attempt to obtain the tcar-ssdna complex structure was not successful, we resorted to em analysis to image tcar-wx174 complex and to pursue its 3-d reconstruction. after staining for 4 min with 2.5% uranyl acetate, em analysis was performed with a tecnai tm g 2 spirit bio twin (fei co., the netherlands) using 120kv acceleration voltage. as seen in figure 4b -d, em imaging revealed that no complex was found in the negative control sections, whereas tcar form a nucleoprotein filament with a circular viral wx174-ssdna fully covered with proteins, suggesting strong and cooperatively interaction between viral ssdna and tcar. this is consistent with the emsa results we observed. we are now testing another em method as described by lurz r et al. [28] to confirm the cooperative binding between the tcar and viral wx174-ssdna. a distinct group of dna-binding proteins called the ssdnabinding proteins (sbp) could specifically bind ssdna and be used in processes where the double helix is separated, including dna replication, transcription, and recombination. because tcar is known as a marr family transcription regulator that binds to specific dsdna sequence with the winged helix-turn-helix (whth) dna binding domain, the ssdna binding ability of tcar may not be involved in transcription. in order to clarify the role of tcar-viral ssdna interaction, our approach is to examine it with in vitro replication. we used single-primed m13 replication assay to measure the ability of purified tcar protein to convert a primed single-strand m13 template to the duplex form in a manner that requires processive dna synthesis. as seen in figure 5a , m13-based in vitro dna replication assay showed that the addition of tcar protein to the reaction mixture, and incubation for up to 30 min, resulted in almost no dna replication activity compared to controls. this indicated that tcar markedly inhibits dna replication and that the mechanism of inhibition, at least in part, involves interaction with viral ssdna. these results suggest a possible role for tcar in bacteriophage resistance. since 1980, investigators have developed an increasing number of bacteriophage therapies for the treatment, or prophylaxis, of bacterial infectious diseases [29, 30, 31] . reports have described that appropriately administered phages can treat lethal infectious diseases caused by gram-negative and gram-positive bacteria, such as pseudomonas aeruginae, klebsiella pneumoniae, enterococcus faecium, and s. aureus [32, 33, 34, 35] . antibiotic resistance has become a global public health concern; thus investigators are extensively reevalu-ating phage therapies to fully exploit their antimicrobial potential [36, 37] . however, phages encounter a variety of different antiviral mechanisms during their infection of bacterial cells, such as prevention of phage adsorption and dna entry, cutting of phage nucleic acids, and abortive infection systems [38] . most reported antiphage systems have been shown to be relevant to the dsdna phage, but not ssdna, ssrna, or dsrna phages. to further confirm and clarify the first relationship between the tcar protein and ssdna phage resistance, the standard plaque assay was performed in e. coli as a model system since little is known about the ssdna phage infecting staphylococci. as seen in figure 5b , induction of the tcar protein in e. coli conferred increased host resistance to ssdna phage (m13 and wx174) infection. however, a tcar-expressing strain did not demonstrate reduced sensitivity to dsdna phage lambda (l) infection, suggesting that the phage resistance was caused by tcar-viral ssdna complex. the observed biological differences point to a remarkable plasticity of tcar. these findings, thus, may support a hypothesis that tcar might interfere with viral ssdna replication and establish a link between tcar and ssdna phage resistance. the marr family transcriptional regulators serve as sensors of changing environments, allowing pathogenic bacteria to survive and persist in a dynamic environment [39] . however, up to now, the knowledge of marr family protein-nucleic acid interaction has been focused on dsdna and the marr family protein-ssdna interaction ability as well as their contribution to the multiple functions of tcar is yet to be discovered. better understanding of these interactions not only will benefit the understanding of many biological mechanisms but also is expected to provide a concept for designing a new therapy for streptococci. in this work, we present the first attempt to investigate the tcar-ssdna interaction. the information of tcar-ssdna binding mode and the minimal binding length that we obtained from emsa analysis and biacore will be helpful for us to obtain tcar-ssdna complexed structure successfully. moreover, we used in vitro replication assay and plaque assay to elucidate the specific biological role of the ssdna binding ability of tcar. such observations may help us understand the mechanism of antibiotic resistance in the marr family regulators. the icar and tcar proteins were expressed in e. coli bl21 (de3) and purified as already described [17, 40] . the marr homologous gene, sar2349, was amplified directly from the s. aureus mrsa252 genome by polymerase chain reaction (pcr) and subsequently cloned into expression vector pet-32. this construct was transferred into e. coli of arctic express tm (de3) ril strain. the his 6 -tagged wild-type protein was over-expressed in difco luria-bertani (lb) broth containing 50 mg/l ampicillin to an optical density at 600 nm of 0.5-0.6 and then induced with 0.5 mm iptg (isopropyl-b-d-thiogalactopyranoside). cells were grown for 2 days at 16uc. the cells were then harvested by centrifugation at 12,000 g for 30 min and disrupted by constant cell disruption system (constant system ltd, uk) with lysis buffer containing 20 mm tris-hcl (ph 8.0), 500 mm nacl, and 20 mm imidazole. the homogenate was centrifuged at 27,000g for 30 min and the cell-free extract was loaded onto a ni 2+ -nta column, which had been previously equilibrated with lysis buffer. the column was washed with lysis buffer, and the his 6 -tagged sar2349 was subsequently eluted by a linear gradient of imidazole from 10 mm to 500 mm. his-tagged sar2349 eluted was dialyzed twice against 5 liters of buffer (20 mm tris-hcl, ph 8.0, and 500 mm nacl) and then subjected to thrombin digestion to remove the tag. the mixture was then passed through another ni 2+ -nta column, and subsequently untagged sar2349 protein was dialyzed twice against 3 liters of buffer (20 mm tris-hcl, ph 8.0) and then passed through a q-sepharose anion-exchange column for further purification, and subsequently sar2349 was eluted by a linear gradient of 10 mm to 500 mm nacl-containing buffer and then dialyzed twice against 5 liters of buffer (20 mm tris-hcl, ph 8.0, 150 mm nacl, and 2 mm dtt) for storage. the purified sar2349 protein was finally concentrated by 3 kda cut-off size membrane of amicon ultra-15 centrifugal filter units (millipore, ma, usa) for storage at -80uc. the six oligonucleotide probes used in emsa experiments were purchased from mdbio inc. (taiwan) ( figure 1a ). the viron wx174 and m13 ssdna were purchased from new england biolabd (usa). for the preparation of double-stranded icar dna1, equimolar amounts (100 mm each) of complementary oligonucleotides were mixed, fully denatured by heating at 95uc for 5 min in 10 mm tris-hcl ph 8.0, 20 mm nacl and allowed to cool gradually to room temperature. gel shift assays were performed by incubating 1 mm of ssdna or dsdna with 1-4 mm purified recombinant proteins under binding conditions (20 mm tris-hcl, ph 8.0, 150 mm kcl, 0.1 mm mgcl 2 , 0.05 mm edta, 12.5% glycerol, 0.1 mm dtt and 1 mg/ml bsa) for 15 min at room temperature with gentle vortex. after incubation, 15 ml of the reaction solution was mixed with 3 ml of the sample loading dye and subsequently electrophoresed on 6% preequilibrated polyacrylamide gels in 1/2 tris/acetate/edta (tae) at 100 v for 30 min and visualized using sybr green i nucleic acid gel stain (invitrogen). for competition assay, icar dna1 probe duplex of 1 mm was pre-incubated with 2 mm tcar (dimer) at room temperature for 15 min, then mixed with increasing concentration of gc33 ssdna. in the assay for analyzing the effect of different antibiotics on the interaction between tcar and ssdna, 1 mm gc33 probe was pre-incubated with 2 mm tcar (dimer) at room temperature for 15 min before mixing with 2 mm or 20 mm antibiotics. the affinity, association and dissociation between the drug and the dna duplexes were measured using a biacore 3000a surface plasmon resonance (spr) instrument (pharmacia, uppsala, sweden) with a sensorchip sa5 from pharmacia by monitoring the refractive index change of the sensor chip surface [41, 42, 43] . these changes are proportional to the amount of bound analyte. the spr angle change is reported as resonance units (ru). five fragments of 59 biotinylated oligonucleotides probes purified by page were purchased from mdbio inc. (taiwan). activation buffer (100 mm nacl, 50 mm naoh) was injected for 1 min (20 ml) to remove any unbound streptavidin from the sensor chip. to control the amount of the dna bound to the sa chip surface, 200 nm of the biotinated oligonucleotides were immobilized manually onto the surface of a streptavidin chip until 120 ru was reached in the first cell. the chip surface was then washed with 10 ml of 10 mm hcl to eliminate non-specific binding. the second flow cell was unmodified and served as a control. different concentration of tcar, icar, and sar2349 proteins were injected at a flow rate of 30 ml/min in 50 mm tris, 150 mm nacl, ph 7.5 for 170 s to reach equilibrium. blank buffer solution was then passed over the chip to initiate the dissociation reaction. at the end of each cycle, the surface was recovered with two 30 s injections of 0.025% sds. spr-binding constant is analyzed as described previously [44] . sensorgrams for the interactions between dna and tcar were analyzed using bia evaluation software to determine the association and dissociation rate constant (k a /k d ). in the assay analyzing the effect of antibiotics on the interaction between tcar and ssdna, tcar protein with 640 nm kanamycin or ampicillin in 50 mm tris, 150 mm nacl, ph 7.5 was injected on to the sensor chip. circular dichroism (cd) spectra were obtained using a jasco-815 cd spectropolarimeter. temperature was controlled by circulating water at the desired temperature in the cell jacket. tcar and dna samples were prepared under the conditions identical to those prepared for emsa assay. the cd spectra were collected between 250 and 200 nm with 1 nm bandwidth at 1 nm intervals. all spectra were obtained from an average of five scans. the photomultiplier absorbance did not exceed 600 v during the analysis. cd spectra were normalized by subtraction of the background scan with buffer alone. the mean residue ellipticity, [h], was calculated based on the equation, [h] = mrw6hl/ 106l6c, where mrw is the mean residue weight, hl is the measured ellipticity in milidegrees at wavelength l, l is the cuvette pathlength (0.1 cm), and c is the protein concentration in g/ml. the tcar proteins (0.3 mm) were first incubated at 30uc for 15 min in reaction buffer [20 mm tris-hcl, ph 8.0, 150 mm kcl, 0.1 mm mgcl 2 , 0.05 mm edta, 12.5% glycerol, 10 mm dtt, 12 mm circular viron wx174 ssdna (5386 nucleotides in length), 0.2 m ammonium acetate], and then chilled on ice to stop the reaction. the reaction product was diluted 100-fold with em sample dilution buffer (2 mm mgcl 2 , 0.5 mm dtt, 10 mm hepes ph 7.0). a droplet (4 ml) was placed for 1 min at room temperature on a copper grid (300 mesh, pelco, usa) coated with fresh carbon. the excess buffer was then carefully blotted away from the edge of the grid with whatman #1 filter paper (whatman inc., usa). after staining for 1 min with 2% uranyl acetate, excess liquid was removed and samples were dried at room temperature. bio-transmission em was performed with a tecnai f20 bio twin (fei co., netherlands) using an acceleration voltage of 200 kv. images were recorded with a slow scan ccd camera (gatan multiscan tm 600, usa) at a resolution of at least 4k64k pixels. for m13 replication assay, 250 mm of single primed m13mp18 ssdna was incubated with/without 2 mm tcar protein in the reaction mixture (20 ml) containing 25 unit klenow fragment, 25 mm nacl, 7 mm mgcl 2 , 1 mm edta, and 0.5 mm dtt for 3 min at 30uc to allow replication complexes to assemble at the primer template junction [45, 46] . replication was allowed to proceed by addition of 60 mm dntp. after incubation at 30uc for 30 min, the reactions were terminated by addition of 10 mm tris-hcl, 5 mm edta, 0.5% sds, and 50 mg of proteinase k (with total volume of 20 ml) and incubated at 50uc for 1 h. conventional electrophoresis was then performed to verify the result of dna replication (20-cm 0.8% agarose gel, 15 ma, 0.5x tbe buffer). the bands are visualized using sybr green i nucleic acid gel stain (invitrogen) and quantified by quantify one (bio-rad, usa). host sensitivity to phages was tested using a virulent variant of phage (m13, wx174 and c) and e. coli bl21 (de3) ril transformed with engineered pet-16b-tcar plasmid containing laci gene and lac operator as host [47, 48, 49] . cells were grown in lb media until the optical density (od 600 ) reached 0.6. tcar protein was then induced by adding a final concentration of 0.1 mm iptg and used in plaque assays as previously described [50, 51] . plaque assays were performed in triplicate. plates and topagar contained lb and above mentioned concentrations of inducers. the sensitivity of the host to phage infection was calculated as the efficiency of plaquing, which is the plaque count ratio of a non-iptg set to the iptg set [52] . error-bars were calculated as one standard deviation. the atomic coordinates and structure factors for the tcar-rna complex have been deposited in the wwpdb with accession numbers of 4ejt. ceftobiprole: an extended-spectrum antimethicillin-resistant staphylococcus aureus cephalosporin antibiotic resistance of bacteria in biofilms polysaccharide intercellular adhesin (pia) protects staphylococcus epidermidis against major components of the human innate immune system the teicoplaninassociated locus regulator (tcar) and the intercellular adhesin locus regulator (icar) are transcriptional inhibitors of the ica locus in staphylococcus aureus inactivation of a novel three-cistronic operon tcar-tcaa-tcab increases teicoplanin resistance in staphylococcus aureus the many faces of the helix-turn-helix domain: transcription regulation and beyond the mar regulon: multiple resistance to antibiotics and other toxic chemicals overlaps and parallels in the regulation of intrinsic multiple-antibiotic resistance in escherichia coli structural insights into the redox-switch mechanism of the marr/duf24-type regulator hypr crystal structure of the zincdependent marr family transcriptional regulator adcr in the zn(ii)-bound state lactococcus lactis zitr is a zinc-responsive repressor active in the presence of low, nontoxic zinc concentrations in vivo the crystal structure of marr, a regulator of multiple antibiotic resistance, at 2.3 a resolution structure of an ohrr-ohra operator complex reveals the dna binding mechanism of the marr family crystal structure of the mexr repressor of the mexrab-oprm multidrug efflux operon of pseudomonas aeruginosa the crystal structure of xc1739: a putative multiple antibiotic-resistance repressor (marr) from xanthomonas campestris at 1.8 a resolution crystal structures of slya protein, a master virulence regulator of salmonella, in free and dna-bound states structural study of tcar and its complexes with multiple antibiotics from staphylococcus epidermidis tcar, a putative marr-like regulator of sars expression identification and characterization of sarh1, a new global regulator of virulence gene expression in staphylococcus aureus the hsv-1 icp27 rgg box specifically binds flexible, gc-rich sequences but not gquartet structures molecular mechanism of upregulation of survivin transcription by the at-rich dna-binding ligand, hoechst33342: evidence for survivin involvement in drug resistance st1710-dna complex crystal structure reveals the dna binding mechanism of the marr family of regulators structural insight on the mechanism of regulation of the marr family of proteins: high-resolution crystal structure of a transcriptional repressor from methanobacterium thermoautotrophicum promotion of homologous recombination and genomic stability by rad51ap1 via rad51 recombinase enhancement the n-terminal domain of twinkle contributes to single-stranded dna binding and dna helicase activities alteration of the repressor activity of marr, the negative regulator of the escherichia coli marrab locus, by multiple chemicals in vitro conformational changes in dna upon ligand binding monitored by circular dichroism electron microscopic study of dna complexes with proteins from the archaebacterium sulfolobus acidocaldarius the lancet (1983) phage therapy bacteriophage therapy and prophylaxis: rediscovery and renewed assessment of potential phage therapy-advantages over antibiotics? phage therapy: past history and future prospects bacteriophage therapy the prospect for bacteriophage therapy in western medicine population and evolutionary dynamics of phage therapy bacteriophage therapy-cooked goose or phoenix rising? bacteriophage and their lysins for elimination of infectious bacteria bacteriophage resistance mechanisms ligand-responsive transcriptional regulation by members of the marr family of winged helix proteins crystal structure of icar, a repressor of the tetr family implicated in biofilm formation in staphylococcus epidermidis studies of sequence-specific dna binding, dna cleavage, and topoisomerase i inhibition by the dimeric chromomycin a3 complexed with fe(ii) elucidation of the stability and functional regions of the human coronavirus oc43 nucleocapsid protein effects of polyamines on the dna-reactive properties of dimeric mithramycin complexed with cobalt(ii): implications for anticancer therapy effects of polyamines on the thermal stability and formation kinetics of dna duplexes with abnormal structure methods for analysis of poxvirus dna replication parvovirus initiator protein ns1 and rpa coordinate replication fork progression in a reconstituted dna replication system the phage abortive infection system, toxin, functions as a protein-rna toxinantitoxin pair small crispr rnas guide antiviral defense in prokaryotes crispr provides acquired resistance against viruses in prokaryotes inhibition of bacteriophage m13 replication with esterified milk proteins generating a synthetic genome by whole genome assembly: phix174 bacteriophage from synthetic oligonucleotides phage response to crispr-encoded resistance in streptococcus thermophilus we, the authors, acknowledge the support of grants from the institute of biological chemistry at academia sinica and national research program for biopharmaceuticals. we are also grateful for the use of the tecnai f20 in the cryo-em core facility, scientific instrument center at academia sinica. key: cord-000574-7eflwyxk authors: liu, yanli; huangfu, jie; qi, feng; kaleem, imdad; e, wenwen; li, chun title: effects of a non-conservative sequence on the properties of β-glucuronidase from aspergillus terreus li-20 date: 2012-02-07 journal: plos one doi: 10.1371/journal.pone.0030998 sha: doc_id: 574 cord_uid: 7eflwyxk we cloned the β-glucuronidase gene (atgus) from aspergillus terreus li-20 encoding 657 amino acids (aa), which can transform glycyrrhizin into glycyrrhetinic acid monoglucuronide (gamg) and glycyrrhetinic acid (ga). based on sequence alignment, the c-terminal non-conservative sequence showed low identity with those of other species; thus, the partial sequence atgus(-3t) (1–592 aa) was amplified to determine the effects of the non-conservative sequence on the enzymatic properties. atgus and atgus(-3t) were expressed in e. coli bl21, producing atgus-e and atgus(-3t)-e, respectively. at the similar optimum temperature (55°c) and ph (atgus-e, 6.6; atgus(-3t)-e, 7.0) conditions, the thermal stability of atgus(-3t)-e was enhanced at 65°c, and the metal ions co(2+), ca(2+) and ni(2+) showed opposite effects on atgus-e and atgus(-3t)-e, respectively. furthermore, km of atgus(-3t)-e (1.95 mm) was just nearly one-seventh that of atgus-e (12.9 mm), whereas the catalytic efficiency of atgus(-3t)-e was 3.2 fold higher than that of atgus-e (7.16 vs. 2.24 mm s(−1)), revealing that the truncation of non-conservative sequence can significantly improve the catalytic efficiency of atgus. conformational analysis illustrated significant difference in the secondary structure between atgus-e and atgus(-3t)-e by circular dichroism (cd). the results showed that the truncation of the non-conservative sequence could preferably alter and influence the stability and catalytic efficiency of enzyme. glycyrrhizin (gl), the main constituent of licorice extract (glycyrrhiza glabra), is a natural edulcorant as well as an important ingredient of traditional chinese medicine [1, 2, 3] . by hydrolyzing one or two distal glucuronides, gl can be transformed into glycyrrhetinic acid monoglucuronide (gamg) or glycyrrhetinic acid (ga) ( figure 1 ). as an important derivative of gl, gamg displayed stronger physiological functions than gl such as antiviral, anti-inflammatory, anti-tumor functions, and so on; and it is also 1000-fold sweeter than saccharose [4] . on the other hand, ga is the bioactive substance of gl well known for its pharmacological features [4, 5, 6] . the research on gl biotransformation catalyzed by b-glucuronidase (gus, ec 3.2.1.31) was reported mainly in animal tissues such as duck [7] and human [8] , whereas studies on gl biotransformation in fungal species are few [9] . in our previous work, a fungal strain, aspergillus terreus li-20 was screened, which can use gl as a carbon source and produce gamg and ga after catalysis by b-glucuronidase (atgus). the main disadvantages of atgus were low enzyme productivity, low catalytic efficiency, and pathogenicity, which rendered it unsafe for use in the food and medical industries. to overcome disadvantages of a natural enzyme, many methods was applied to obtain artificial evolution enzymes, and this approach is not only faster than natural evolution but also provides a deeper understanding of enzyme evolution. several methods of designing new enzymes are available, and gene sequence truncation is also investigated for its effects on enzymatic properties. the non-conservative n-terminal domain of the protein phosphatase1 (pp1), with 1-8 residues deleted, showed higher sensitivity to three substrates and influenced the structure and properties of pp1 [10] , whereas the truncation of the cterminal region improved the thermal stability of endo-bglucanase from bacillus subtilis ja18 [11] . however, the loss of the c-terminal regulatory domain resulted in a loss of the ability to catalyze the aldol reaction [12] . with development of molecular biology and bioinformatics characterization, an increasing number of sequence data have been cloned and applied in the biotransformation industry. bioinformatics characterization from the national center for biotechnology information (ncbi) showed that most b-glucuronidases belong to the glycoside hydrolase family (ghf) 2, and all of them consist of sugar-binding, immunoglobulin-like b-sandwich, and tim barrel domains (triosephosphate isomerase, tim) [13, 14, 15] . the tim barrel domain, which is one of the most common catalytic domains, is adopted by about 10% of the enzymes; thus, sequence modification inside or outside the domain to improve the enzymatic property and determine the catalytic mechanism was reported in many studies. the site-directed mutagenesis of seven amino acids (aa) in the tim barrel domain was performed to investigate the importance of the residue in the catalysis of an exo-b-d-glucosaminidase from trichodema reesei [16] . heparanase is an endo-b-d-glucuronidase, and its c-terminal region, which is not an integral part of the tim barrel domain, is essential for the enzymatic activity and secretion of heparanase [17] . although b-glucuronidases from many species have been registered in genbank, only a few genes have been published for gl biotransformation [18] . three fungal strains, namely, a. terreus li-20, p. purpurogenum li-3, and a. ustus li-62, were screened in our previous studies and represented three modes of gl biotransformation: (1) glrga+gamg; (2) glrgamg; and (3) glrga [9] . the three b-glucuronidase genes were cloned in our laboratory, and the aa sequence alignment showed that the bglucuronidase from a. terreus li-20 (atgus) was quite different from the other two b-glucuronidases(pgus and augus) in cterminal non-conservative sequence. atgus can hydrolyze gl into two products; thus, the different modes of gl biotransformation of the b-glucuronidases from the other two fungi may be related to the natural evolution in the sequence. in the present research, atgus and the partial sequence [atgus(-3t)] without cterminal non-conservative sequence behind the tim barrel domain were amplified in order to investigate effects of nonconservative sequence on enzymatic property. no specific permits were required for the described field studies. no specific permissions were required for these locations/ activities. no location is privately-owned or protected in any way. the field studies did not involve endangered or protected species. in our previous work, a. terreus li-20 was isolated and screened from a g. glabra planting field in shihizi, xinjiang. it was incubated in 100 ml liquid czapek's medium in a 500 ml erlenmeyer flask at 30uc and placed in a shaker incubator at 170 rpm. escerichia coli dh5a and e. coli bl21 were used as hosts for plasmid amplification and expression, respectively. the plasmids pmd19-t (takara, japan) and pet28a (+) (invitrogen, u.s.) were used as vectors. the recombinant cells were inoculated in a lysogeny broth (lb) medium with kanamycin (50 mm) and operated at 37uc for 3 h. the recombinant protein was induced by adding 0.4 mm isopropyl-b-d-thiogalactopyranoside (iptg). gl was purchased from xinjiang tianshan pharmaceutical co. (china). ga was purchased from sigma chemical co. (u.s.), whereas gamg was obtained from the nanjing university of technology, china. methanol was of chromatographic grade. all other chemicals used were of analytical grade. the dl2000 marker and the protein low weight marker were purchased from takara, japan. an intron in an atgus genomic sequence was removed via three-step polymerase chain reaction (pcr) to express the gene in e. coli bl21 (figure 2a ). according to the database of a. terreus nih2624, a primer set containing p1(59-ccgtacgtaatgct-gaagccccgacaaacacctt-39) and p2(59-catgcgg-ccgcttaagcgccaaataggaagtatagt-39) was designed to obtain the sequence with an intron from the a. terreus li-20 genome under the following conditions: 94uc for 10 min, 30 cycles of 94uc for 1 min, 58uc for 1 min, 72uc for 2 min, and a final extension at 72uc for 10 min with ex tag (takara, japan). after ligated into pmd19-t, a primer set containing p3(59-cactccaccgtgttttcaatgtatgagctgcagc-39) and p4(59-ccggcttcgcagctatgtgtcttgagcatc-39) was used for the second pcr, and the reaction was performed by pfu polymerase (shenggong, china) under the following conditions: 94uc for 10 min, 30 cycles of 94uc for 1 min, 55uc for 1 min, and 72uc for 5 min. the fragment amplified in the second pcr was ligated by t4 dna ligase after a terminal phosphation with t4 polynucleotide kinase (takara, japan), and the positive clones were screened in an lb plate with 100 mg/ml ampicillin. the primer set containing p1 and p5(catgcg-gccgcttaactccaccgtgttttcaatgtatg-39) was used for atgus(-3t) under the same conditions as those of p3 and p4. after iptg introduction and the ultrasonication of the recombinant e. coli bl21 cells, supernatant was brought to 70% saturation with (nh4) 2 so 4 and stored overnight at 4uc, and then again centrifuged. the enzymes expressed by pet28a(+) vector were fused to an n-terminal six-histidine tag and purified via nickel chelate affinity chromatography (ge, u.s.), which was eluted with 150 mm imidazol. the quality of the purified protein was evaluated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and coomassie blue staining. hplc for analysis of gl, gamg, and ga gl, gamg, and ga concentrations were measured via reverse-phase high performance liquid chromatography (hplc) on a c18 column (4.6 mm6250 mm, 5 mm particle size, kromasil) at 40uc. the sample (injection volume, 10 ml) was separated with a mobile phase consisting of 6% acetic acid/ methanol (19:81 v/v), and the elution was monitored via ultraviolet detection at 254 nm. the gl, gamg, and ga amounts were calculated from the standard curve of the peak area and concentration. determination of ph and temperature profiles the activity of b-glucuronidase was determined using gl as the substrate. the reaction mixture consisted of the enzyme and substrate (2 g/l gl) at a 1:4 (v/v) ratio. 50 mm na 2 hpo 4 -citric acid buffer at ph 4.0-8.0 was used for determine the ph effects of the enzyme. the catalytic activity of the enzyme was examined at 30 to 70uc in 50 mm na 2 hpo 4citric acid buffer (ph 7.0). the enzyme activity under the optimal temperature and ph was defined as 100%. the temperature stability of the enzyme was determined by incubating the enzyme samples at different temperatures (45, 55, 65, and 75uc) for 15, 30, 45, 60, and 120 min at optimum ph without the substrate gl, and the residual activity was determined at the optimum temperature. the effect of several metal ions on the activity of atgus(-3t)-e and atgus-e was investigated. the enzyme activity was determined in the reaction mixture consisting of k + (kcl), na + (nacl), mg 2+ (mgcl 2 ), mn 2+ (mncl 2 ), co 2+ (cocl 2 ), ca 2+ (-cacl 2 ), ni 2+ (niso 4 ), cu 2+ (cuso 4 ), and al 3+ (alcl 3 ) ions at final concentrations of 1 and 5 mm. the enzyme activity was subsequently determined at the optimum temperature after incubation for 30 min. different concentrations of the substrate gl, ranging from 0.375 to 4 mm, were prepared to determine the kinetic constants. the catalytic reactions were continuously monitored, and the initial velocities were fitted to the michaelis-menten equation using the origin 7.5 software (originlab). the values of the michaelis-menten constant (km), maximal velocity (vmax), catalytic turnover rate (kcat), and catalytic efficiency (kcat/km) were evaluated. far-uv circular dichroism (cd) spectra were recorded at 25uc in the range from 190 to 260 nm with a spectral resolution of 0.2 nm using a jasco j-715 spectropolarimeter. the scan speed was 100 nm/min and the response time was 0.125 s with a bandwidth of 1 nm. quartz cells with an optical path of 0.1 cm were used. typically, scans were accumulated and subsequently averaged. the spectra were corrected for the corresponding protein-free control. the protein three-dimensional structural was modeled by modeler 9v7 to analyze three domains of the protein. the 2,193 bp product was amplified and sequenced using a genomic template ( figure 2b) , and its 219 bp intron was analyzed by ncbi. after a three-step pcr, the full encoding sequence was cloned. the results show that the open reading frame of this gene was 1,974 bp ( figure 2b) , which encodes for 657 aa. the conserved domain database (cdd) was performed to analyze domains of atgus, and there were sugar-binding domain, immunoglobulin-like beta-sandwich domain, and tim barrel domains in it which all belonged to glycoside hydrolase family (ghf) 2. ghf 2 comprised b-galactosidase (ec 3.2.1.23), bmannosidase (ec 3.2.1.25), and b-glucuronidase (ec 3.2.1.31), so the phylogenetic tree was constructed according to it (figure 3) . it showed that the gene cloned was a b-glucuronidase gene named atgus (genbank accession no. jf894133), which was found very similar to pgus(genbank accession no. eu095019) from p. purpurogenum li-3 and augus (genbank accession no. jn247805) from a. ustus li-62, especially in the sugar-binding, immunoglobulin-like beta-sandwich, and tim barrel domain ( table 1 ). the obvious difference among them lied in the non-conservative sequence of the c-terminal behind the tim barrel domain which may result in the difference enzymatic properties. therefore, the 1-1,776 bp segment, named atgus(-3t), was amplified in the both pet28a(+)-atgus and pet28a(+)-atgus(-3t) were constructed and transformed into the e. coli bl21 strain, and the recombinant proteins atgus-e and atgus(-3t)-e were successfully expressed ( figure 4) . the induction condition for the optimum production of the two recombinant proteins was 20uc with 0.4 mm iptg. both atgus-e and atgus(-3t)-e were purified through ni-nta sepharose (figure 4) . the target protein was eluted with 150 mm imidazole. furthermore, the concentrations of the soluble purified proteins of atgus-e and atgus(-3t)-e were determined as ,7 and ,12 mg/l, respectively. both purified enzymes could hydrolyze gl into gamg and ga. we investigated the enzymatic properties to determine the effect of the non-conserved sequence on the enzyme. the optimal ph for the bioconversion reaction by atgus-e was 6.6, whereas that for atgus(-3t)-e was 7.0 ( figure 5a) .the optimal temperatures for atgus-e and atgus(-3t)-e were both 55uc ( figure 5b) . enzyme thermal stability experiments showed that the enzymes remained more than 80% residual activity at 45uc and 55uc for 120 min heat treatment, respectively ( figure 5c and 5d) . at 65uc, the residual activity of atgus(-3t)-e remained almost 60% of enzymatic activity after 30 min heat treatment, which was comparatively higher than that of atgus-e with less than 5% residual activity after the same treatment. at a higher temperature (75uc), both enzyme residual activity rapidly vanished, and within 15 min heat treatment, almost all enzymatic activity was lost. the effect of various metal ions with different concentration gradients (from 1 to 5 mm final concentration) on the activities of atgus(-3t)-e and atgus-e was evaluated, and the results are presented in table 2 . the enzymatic activity assayed in the absence of metal ions was taken as 100%. the effect of monovalent cations on the two enzymes was similar: the 1 and 5 mm k + and 1 mm na + exhibited no obviously affecting effects on the activity of atgus(-3t)-e and atgus-e, while the 5 mm na + inhibited the enzymatic activity. the divalent cations mg 2+ and mn 2+ discretely promoted the activities of atgus-e and atgus(-3t)-e. with increasing concentration of co 2+ , atgus-e was firstly activated and then inhibited while atgus(-3t)-e showed an inverse effect. ca 2+ and ni 2+ also exhibited opposite effects on the two enzymes. ca 2+ at 5 mm final concentration enhanced the activity of atgus-e by 107% but inhibited atgus(-3t)-e activity by 61%. in the presence of 5 mm ni 2+ buffer, atgus(-3t)-e was increased by 78%, whereas atgus-e was decreased by 40%. cu 2+ distinctively inhibited the enzyme activity, while al 3+ showed activation at 1 mm concentration and inhabitation at 5 mm concentration to both enzymes. these results reveal that k + , na + , mg 2+ , mn 2+ , cu 2+ , and al 3+ exhibited nearly similar effects on the activity of atgus-e and atgus(-3t)-e; however, co 2+ , ca 2+ , and ni 2+ , showed opposite effects on both enzymes, respectively. the data reported here have been taken from three replicate samples from three independent experiments. the reaction kinetics of atgus-e and atgus(-3t)-e were determined. the vmax of the atgus-e and atgus(-3t)-e enzymes toward gl were calculated using lineweaver-burk plots ( table 3 ) and were determined as 1.84 and 0.97 mmolmin 21 mg 21 , respectively. the km of the recombinant atgus(-3t)-e was 1.95 mm, which was approximately one-seventh that of atgus-e (12.9 mm), indicating that a higher affinity of atgus(-3t)-e for gl than atgus-e. in addition, the catalytic efficiency (kcat/km) of atgus(-3t)-e (7.16 mm s 21 ) was 3.2 folds higher than that of atgus-e (2.24 mm s 21 ). the enzymatic activities were determined at different concentrations of the substrate gl from three independent experiments. to determine the impact of the sequence truncation on the structure of the protein, a circular dichroism (cd) spectra was amplified. the far-uv spectra for atgus-e and atgus(-3t)-e have been presented in figure 6 . it illustrated that the curves exhibited significant difference between the two proteins. these results suggest that the secondary structure of atgus-e has changed after deletion of non-conservative sequence. the b-glucuronidase (gus) gene was first cloned in 1987 [19] , and in subsequent years, many gus genes were cloned and registered in the genbank. however, this gene has never been cloned for the hydrolysis research of gl into gamg or/and ga, with more valuable merits. based on cdd analysis, the three domains of the enzyme atgus were well investigated, and the main aim of the present study is to modify the non-conservative sequence of atgus and try to obtain an artificial evolution enzyme with better enzymatic properties. the tim barrel domain is a canonical (b/a) 8 -barrel composed of eight units, each of which consists of a b-strand and an a-helix [20] . there was a non-conservative segment behind the catalytic domain (tim barrel domain) of atgus which showed low identity with pgus and augus after the primary sequence alignment. a model of the three-dimensional structure of atgus was presented in the current research ( figure 7) . the deleted sequence exhibited no involvement in the tim barrel domain, locating near the ''stability face'' rather than the ''catalytic face'' [21] . atgus-e and atgus(-3t)-e were very similar with each other at some enzymatic properties, such as optimal ph and optimal temperature. it was reported in previous studies that many modified enzymes maintained some original enzymatic properties even though some sequence has been modulated [10] . furthermore, we could also speculate that the non-conservative sequence lied outside of the catalytic face of the tim barrel domain which may not affect the catalytically active residues and the gl biotransformation mode. interestingly, the stability of atgus(-3t)-e was slightly higher than that of atgus-e at 65uc. similar results have been reported that the modification of the c-terminal region could improve the thermal stability of endo-b-glucanase from bacillus subtilis ja18 [11] . in addition, previous study showed that ab-loops in ''stability face'' are important for the stability [22] . the truncation of the non-conservative sequence lies near ab-loops of the stability face, therefore, we can predicted that the deletion of the c-terminal region outside the tim barrel domain has influence on thermal stability of atgus. the effect of nine metal ions with different concentration gradients on the activities of atgus(-3t)-e and atgus-e was evaluated, and co 2+ , ca 2+ , and ni 2+ showed opposite effects on the two enzymes, respectively. in addition, atgus(-3t)-e showed higher affinity and catalytic efficiency than atgus(-3t)-e. both of the result might suggest that the spatial structural rearrangement, and the speculation has been proved by cd spectra, which showed great difference between the secondary structure of the two enzymes. it has been reported that the loops above the catalytic face was very important for substrate hydrolysis [23] , so we can conclude that the truncation of the non-conservative domain firstly changed the secondary structure of the enzyme and then influenced the substrate affinity, catalytic efficiency and metal ions effects. moreover, the crystal structure of human bglucuronidase was firstly reported in 1996 [24] , and the structure of bacterial b-glucuronidase has also been published recently [25] . both b-glucuronidase structures were tetramers. the deleted region of the atgus non-conservative sequence lies outside the main three domains, so it was predicted that the non-conservative might change the combination pattern of each monomer. different methods have been applied in creating new enzyme such as error-prone pcr [26] , dna shuffling [27] and staggered extension process (step) [28] . some efforts have been made to modify the enzyme by directed screening but high ratio of negative mutated forms of enzyme in the initial screening is a big hurdle and requires further screening for positive mutated forms of enzyme. every method has its own advantages and disadvantages that determines the feasibility of a particular method so as sequence truncation [10, 11, 12] . based on the same hydrolyzing mode, relatively higher thermal stability, and especially the enhanced affinity and catalytic efficiency for gl, deletion of the non-conservative sequence behind the tim barrel domain was a successful evolution of atgus. the truncation of non-conservative region based on sequence alignment could be an effective way of artificial enzyme evolution as it can alter and influence the stability and catalytic efficiency of enzyme, and 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marker in higher plants catalytic versatility, stability, and evolution of the (beta/alpha) 8 -barrel enzyme fold stability, catalytic versatility and evolution of the (beta/alpha)8-barrel fold protein sequence randomization: efficient estimation of protein stability using knowledge-based potentials the aglycone specificitydetermining sites are different in 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (dimboa)-glucosidase (maize beta-glucosidase) and dhurrinase (sorghum beta-glucosidase) structure of human beta-glucuronidase reveals candidate lysosomal targeting and active-site motifs alleviating cancer drug toxicity by inhibiting a bacterial enzyme singlestep assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides molecular evolution by staggered extension process (step) in vitro recombination key: cord-000895-z5rdf0mi authors: belalov, ilya s.; lukashev, alexander n. title: causes and implications of codon usage bias in rna viruses date: 2013-02-25 journal: plos one doi: 10.1371/journal.pone.0056642 sha: doc_id: 895 cord_uid: z5rdf0mi choice of synonymous codons depends on nucleotide/dinucleotide composition of the genome (termed mutational pressure) and relative abundance of trnas in a cell (translational pressure). mutational pressure is commonly simplified to genomic gc content; however mononucleotide and dinucleotide frequencies in different genomes or mrnas may vary significantly, especially in rna viruses. a series of in silico shuffling algorithms were developed to account for these features and analyze the relative impact of mutational pressure components on codon usage bias in rna viruses. total gc content was a poor descriptor of viral genome composition and causes of codon usage bias. genomic nucleotide content was the single most important factor of synonymous codon usage. moreover, the choice between compatible amino acids (e.g., leucine and isoleucine) was strongly affected by genomic nucleotide composition. dinucleotide composition at codon positions 2-3 had additional effect on codon usage. together with mononucleotide composition bias, it could explain almost the entire codon usage bias in rna viruses. on the other hand, strong dinucleotide content bias at codon position 3-1 found in some viruses had very little effect on codon usage. a hypothetical innate immunity sensor for cpg in rna could partially explain the codon usage bias, but due to dependence of virus translation upon biased host translation machinery, experimental studies are required to further explore the source of dinucleotide bias in rna viruses. amino acid sequence of proteins is encoded by nucleotide triplets. the majority of organisms use the standard genetic code, with 61 sense codons translated into 20 amino acids. therefore, most amino acids are encoded by several synonymous codons, which are not used evenly. this codon usage bias (cub) can have distinct causes and consequences in different organisms. two major factors are implicated to explain cub, termed (somewhat ambiguously) translational (selection) and mutational pressure [1] . translational pressure means preference towards codons that are most suitable for translation in a given context. evidence for translational pressure towards common codons has been found in certain highly expressed bacterial genes [2] [3] [4] .use of rare codons was hypothesized to slow down the translation rate to ensure optimal levels of protein expression and proper folding [5] , however regulation of translation rate in bacteria might rather rely on specific sequence fragments than simply on rare codons [6] . moreover, it was shown that a synonymous substitution can result in a protein with different folding and different function, probably by causing ribosome stalling [7] . there are also reports of translational pressure in eukaryotes [8, 9] . while the implications of translational pressure are ubiquitous (reviewed in [10, 11] ), a growing body of evidence suggests that it is not the main driver of synonymous codon preference. mutational pressure is produced by distinct probability of different substitution types. a predominant factor driving codon usage is believed to be gc content, i.e. the summed relative abundance of g and c nucleotides [12, 13] . additional factors that might contribute to mutational pressure are deoxycytidine methylation in cpg (c-phosphate-g) dinucleotide context and subsequent deamination that results in c-t substitution [14] . nucleotide content bias in viral rna genomes could also be produced by rna editing by cellular transaminases (e.g. adar and apobec); however, the extent of such editing of cellular mrna is still a matter of controversy [15, 16] . although rna editing has been speculated to complement to variability of viral genomes [17] , no mechanistic proof has been presented, and its role in the evolution of viruses remains to be proven. several additional factors of dna/rna variation can be classified as selection, but not translational pressure. firstly, there may be a selection against a sequence pattern that triggers innate immunity, e.g. toll-like receptor 9 (tlr9) by cpg rich bacterial dna [18] , or against a target of immunity effectors. the latter case is exemplified by upa dinucleotide, targeted by rnase l [19] , which presumably resulted in reduced upa content in, e.g. hepatitis c virus [20] . another factor that might constrain synonymous codon usage, especially in rna viruses, is the secondary rna structure, either nonspecific rna folding, presumably to protect it from intracellular defense mechanisms [21, 22] , or regulatory structural rna elements commonly found in viruses within the coding sequence [23, 24] . these forces are hard to distinguish from the mutational pressure because they act above the translation level. investigation of cub source is most commonly performed in silico and relies on several approaches, which can be generally classified as host-dependent and host-independent. host-dependent methods rely upon either total codon usage statistics for an organism and compare it to codon usage in specific genes, or upon trna abundance, which can be measured experimentally or extrapolated from the number of different trna genes in the genome [25] [26] [27] . these approaches have limited value in virology because trna statistics are available only for a few model organisms. also, the native host of a virus is often not known, and virus replication commonly takes place only in a specific type of cell, which may have relative trna concentrations and codon usage distinct from other tissues [28] . in addition, viral infection can result in inhibition or activation of rna polymerase iii and thus affect transcription of multiple genes, including trnas, in an infected cell [29, 30] . the host-independent approach accounts only for uneven frequency of synonymous codons and is commonly expressed as effective number of codons, enc [31] . this single number theoretically ranges from 61 (all codons used equally) to 20 (only one codon used per amino acid). enc below 40 is commonly treated as evidence of a strong cub. it is a robust and organism-independent method that is universally used to evaluate cub. a statistical correction termed nc' was subsequently developed to account for genomic gc content [32] . while a notable improvement over the original method, nc' has its faults and limitations, especially in the case of short analyzed sequences [33] . in addition, nc' does not explicitly account for mononucleotide and dinucleotide composition bias that may be especially high in viral genomes. most rna viruses display low to moderate cub, which was attributed mainly to gc and dinucleotide content [34] . combined gc content was the first measure of genome composition, implemented even before the genetic code was deciphered [35] . while this simple criterion covers the majority of genome composition bias, it does not account for different mononucleotide frequencies. this work investigates the impact of different types of mutation pressure on cub in rna viruses and provides an analysis of cub in common rna viruses. coding sequences of 29 animal rna viruses representing 12 families (table 1) were analyzed for codon usage and dinucleotide bias. reference genbank records and sequences of prototype strains were preferred. concatemers of open reading frames (orfs) were used for viruses with segmented genomes. for viruses with a complex coding strategy concatemers of orfs, excluding regions with overlapping frames (as described in the corresponding genbank records), were used. the exact genes used in the study and virus name acronyms are provided in table 1 . additionally, five representative datasets of common rna viruses were created. the sequences were chosen from all available complete coding sequences by automatically omitting identical or very similar sequences. the cutoff for dropping similar sequences was selected individually for each virus to produce datasets of a manageable size. the datasets were 69 out of 1525 available complete coding sequences of hepatitis c virus (hcv), all of 39 of hepatitis a virus (hav) sequences, 74 out of 1283 dengue 1 sequences, 68 out of 154 human enterovirus c sequences, and 79 out of 12327 segment 2 coding sequences of influenza a virus (pb1 polymerase fragment, not excluding pb1-f2 orf). simulated random sequences were created by first generating random values of third-position nucleotide frequencies. the randomization algorithm was designed to generate extreme values to cover all theoretically possible range of values in a reasonably sized dataset. then 1000 random amino acid sequences were generated using these third-position nucleotide frequencies for synonymous codon choice. the effective number of codons, enc [31] , was calculated with the ''chips'' module in the emboss package (http://emboss. sourceforge.net/). enc of 20 corresponds to extreme bias (only one synonymous codon per amino acid), enc of 61 indicates absence of bias. python 2.6 scripts were developed for dinucleotide content analysis (dint-stat), sequence randomization and shuffling (cub-stat) (available at http://www.poliomielit.ru/images/doc/ enc-stat.rar). the difference between these two approaches is that shuffling moves sequence fragments (e.g. dinucleotides) from one place in a sequence to another, while randomization first counts sequence statistics, and then generates a random sequence aiming to reach these values. as generation of sequences is random, they never have precisely the same composition as the original sequence. a total of 1000 scrambled replicates were analyzed for each virus and each algorithm. the algorithms of these scripts are summarized below. n 3 correction n 3 correction shuffled third positions of synonymous codons, thus precisely preserving genomic mononucleotide frequencies at the third codon position (fig. 1) . technically, all synonymous third position nucleotides were first extracted from the genome to a pool, and then randomly distributed back among synonymous positions whilst preserving the amino acid sequence. (gc) 3 correction (gc) 3 correction shuffled third positions of synonymous codons like n 3 correction, but prior to distribution of the third-position nucleotides from the pool back to sequence g content was set equal to c, and a equal to t, therefore preserving combined gc content, but not individual nucleotide content. dn 23 and dn 31 corrections dn 23 and dn 31 corrections similarly shuffled dinucleotides between positions 2-3 (or 3-1, respectively) of synonymous codons whilst preserving amino acid sequence; 6-fold degenerate codons were treated as independent 2-and 4-fold degenerate. as a result, first or second codon position was intact and only the third position was changed. use of dn 23 dinucleotide shuffling ensured precise preservation of position 2-3, but not position 3-1 dinucleotide content, while the dn 31 had an opposite effect. dn 231 correction dn 231 correction swapped compatible trinucleotides between codon positions 2-3-1 whilst preserving amino acid sequence (fig. 1) ; 6-fold degenerate codons were treated as independent 2and 4-fold degenerate. as a result, codon position 2 and position 1 of the following codon were identical before and after shuffling, and distinct genomic dinucleotide content at both codon positions 2-3 and 3-1 was the same as in the original sequence (fig. 1 ). relative dinucleotide bias (rdb) was calculated as the ratio of observed to expected dinucleotide frequency (the product of genomic nucleotide frequencies at the corresponding codon positions, e.g. £ [c 2 ]6£ [g 3 ] for cpg 23 dinucleotide) to exclude the effect of mononucleotide composition on apparent dinucleotides ratio. statistical analysis was carried out with student's t-test as implemented in microsoft excel. spearman correlation was calculated with graphpad prism 4.0 (graphpad software inc.). variance of nucleotide frequencies was calculated as , where n is the number of states (2 for gc content, 4 for mononucleotides, 16 for dinucleotides) and x is the frequency of a (di)nucleotide. a well-known feature of genomes in general, and rna viruses in particular, is dinucleotide bias [36] , which also complements to cub [34] . there are three dinucleotide positions in a codon. variation of dinucleotide 1-2 is strongly restricted by amino acid sequence. dinucleotides 2-3 and 3-1 may seem equally evolutionarily flexible because they both include the variable third codon position. usually, a correction for either position 2-3 or 3-1 dinucleotide content is used to explore origins of cub [37] . however, dinucleotide content varies between codon positions 2-3 and 3-1. to illustrate this, the frequency of the cpg dinucleotide at these codon positions was calculated in rna viruses. as reported previously [36] , cpg dinucleotide was suppressed in all rna viruses, albeit to a different extent. relative frequency of cpg dinucleotide was lower at codon positions 2-3 compared to 3-1 in 26 out of 29 rna viruses (fig. 1a) . to evaluate the significance of this observation, extended datasets were tested for five viruses (fig. 1b) . in four viruses lower cpg frequency at codon position 2-3 was statistically significant with p-values below 10 27 (student's t-test). in influenza a virus there was an insignificant excess of cpg at codon positions 2-3 (p.0.05), which could be explained, for example, by different selection pressures in the mammalian and bird hosts [38] . statistically significant difference of rdb between codon positions could also be observed for other dinucleotides in many rna viruses (data not shown), but without an obvious common pattern among the codon positions, as seen for the cpg dinucleotide. we then evaluated how accounting for the complex dinucleotide composition patterns could improve analysis of codon usage bias. a general rationale for analyzing impact of (di)nucleotide bias on enc is randomizing or shuffling synonymous codons in the original genome sequence whilst preserving the factor under study [37] . when shuffled sequences have an enc close to the original sequence, the constraint (e.g. the nucleotide content) fully explains the cub (or, in simple words, at that (di)nucleotide composition enc cannot possibly be higher than it is). on the other hand, a difference between enc in the original and simulated sequences shows the extent of cub that could not be explained by the kind of pressure (nucleotide or dinucleotide) that was used as a constraint for the shuffling algorithm. shuffling was preferred to randomization because it precisely preserved the positional dinucleotide content, produced sequences with more uniform enc values, evident as lower standard deviation (sd) in sequences that were shuffled vs. randomized under the same constraint, and slightly lower enc values than upon randomization (data not shown). unfortunately, common statistical tests were poorly applicable to this approach, because all factors that affect codon preference (e.g. structural rna elements) could not be accounted for, and therefore even a negligible difference in enc between the original and generated sequences (e.g. 0.2 sd) passed a formal significance test upon increasing the number of replicates. as the standard deviation of enc in scrambled sequences of a virus was on average 0.29 upon (gc) 3 correction, 0.27 upon n 3 correction,0.22 upon dn 23 correction and 0.21 upon dn 231 correction), and maximum sd in any virus/algorithm combination was 0.62, enc difference above 1 was considered as significant. consistent with previous studies [34] , enc in rna viruses ranged from 38.2 to 58.3. in sequences shuffled with preserved third codon position gc content, enc was below 61 in all viruses ( fig. 1) , indicating the effect of gc content on cub. this finding was compatible with enc values in rna viruses that were observed upon nc' correction, which is also based on gc content [34] . however, the enc in (gc) 3 shuffled sequences was higher than the original enc on average by 3.5, but by up to 8.9, indicating that gc content imparity cannot explain all cub observed in rna viruses. viruses often have uneven mononucleotide content, thus compromising gc correction approaches. for example, aichi picornavirus has 54% c and only 16% g content at the third codon position [39] . shuffling of the third-position nucleotide (n 3 correction) produced enc values lower than (gc) 3 in some instances the difference between (gc) 3 and n 3 randomization was marginal, but in 8 out of 29 viruses it was above 1 and up to 4 in, e.g. htlv1 and hev. overall, n 3 shuffling, although an improvement over gc randomization, produced enc values that were higher than the original enc by more than one in all viruses except for rubella, indicating residual unexplained bias. the impact of dinucleotide content on cub was next tested. a notable disparity of dinucleotide frequencies at codon positions 2-3 and 3-1 (fig. 2) justified treating them independently in shuffling algorithms. in 22 out of 29 viruses, shuffling of codon position 2-3 dinucleotide (dn 23 ) produced enc values that were lower by more than 1 than enc upon n 3 correction (fig. 1) . in 15 viruses, dinucleotide content-corrected enc values were lower than n 3 corrected values by more than 2, indicating a strong impact of position 2-3 dinucleotide content on cub. shuffling of dinucleotide position 3-1, on the contrary, did not result in a decrease of enc, which differed from n 3 shuffled sequences by less than 0.3 sd in all viruses. to evaluate the impact of distinct dinucleotide content in codon positions 2-3 and 3-1 simultaneously, the codon position 2-3-1 triplet was shuffled between compatible codon pairs (dn 231 correction, fig. 1 ). consistent with results of dn 23 and dn 31 shuffling, enc upon dn 231 shuffling did not differ significantly from dn 23 shuffling, supporting negligible impact of dinucleotide content at codon positions 3-1 on synonymous codon preference. shuffling synonymous codons is a practical way to approach origins of cub. another way to evaluate the effect of rna composition on cub is plotting enc against gc content in studied sequences and comparing this to the theoretically expected enc at various (gc) 3 content [31] . in rna viruses, actual enc was always lower than the expected enc at that gc content, implying that gc content does not fully explain cub [34] . unfortunately, the formula suggested by wright to predict enc values as a function of gc content [31] is not applicable to distinct frequencies of four nucleotides. to study limitations of classic gc content correction methods and develop a better measure of genome composition, a data set of one thousand 10002 nucleotide (3334 codon) long simulated coding sequences with highly variable third-position nucleotide content was generated. firstly, relation of enc and gc content in simulated sequences was compared to viral genomes and to the theoretical prediction of the wright's curve [31] . while the theoretical estimation properly described maximum possible enc as a function of gc content (fig. 3a) , it was not informative of a possible range of enc at a given gc content in simulated sequences with extreme mononucleotide biases (fig. 3a, grey dots) . in fact, enc values close to 30 could be achieved at any gc content. enc in actual viruses was always below the wright's curve (fig. 3a, black dots) ; however the result for simulated sequences indicated that this is not necessarily an evidence of translational bias. importantly, the limitations of gc content as the sole genome composition measure equally apply to eukaryotic genomes, as mrnas may also have uneven mononucleotide composition [40] . next, enc in the simulated sequences was plotted against the variance of sequence content characteristics (fig. 3b, 3c, 3d ). consistent with figure 2a , gc content variance was a poor measure of sequence composition because it described only maximum enc in simulated sequences, but poorly reflected possible enc range at a given variance (fig. 3b) . variance of mononucleotide and dinucleotide frequencies in simulated sequences provided a single measure of genome composition bias with a narrow corresponding enc range (fig. 3c, 3d , grey dots). actual rna virus sequences processed similarly (fig. 3c, 3d , black dots) had notably lower enc values than random sequences with the same variance of the thirdposition mononucleotide frequencies (fig. 3c) . this was consistent with results of mononucleotide content shuffling that only partially explained cub (fig. 1) . when enc in rna viruses was plotted against the variance of dinucleotide frequencies at codon position 2-3 (fig. 3d) , the values overlaid with enc values of simulated sequences, implying that dinucleotide bias can explain almost all apparent cub in rna viruses. also, consistent with shuffling studies, in few viruses enc was slightly lower than that of simulated sequences with the same variance of nucleotide content, indicating additional sources of cub. therefore, variance of dinucleotide frequencies is a good descriptor of mutational bias and can be used to predict a range of enc for a given genome composition. this and previous studies identified mutational pressure, and the third-position nucleotide content in particular, as the main driver of synonymous codon preference. it has also been reported that gc content affects all codon positions in plant viruses [41] and can influence amino acid usage in hiv [42] . a plot of e.g. c content at codon positions 1+2 against position 3 in studied viruses (fig. 4a ) exemplifies that nucleotide content is generally consistent at different codon positions. a similar observation has been reported for vertebrate dna viruses and plant viruses [41, 43] . a further analysis showed that genomic nucleotide content could significantly affect choice between compatible amino acids. the ratio of isoleucine (encoded by auh triplets) to leucine (encoded by cun and uur triplets) in the viral protein sequence correlated with a content at the synonymous third codon position (fig. 4b) . similar but weaker correlation could be observed between the third codon position g content and valine (gun codons) to leucine (cun and uur codons) ratio (data not shown), but not for the ratio between the third-position g content and poorly compatible arginine (cgn and agr codons) and glycine (ggn codons) content in the encoded protein (fig. 4c) . previously, it has been shown that gc content in genomes of higher organisms can affect amino acid composition [44] [45] [46] . our results indicate that this effect is maintained in rna viruses. therefore, genomic nucleotide content is not only the key driver of synonymous codon choice, but also a significant factor that defines choice between compatible amino acids. the concept of synonymous and non-synonymous genetic variation is central to evolutionary studies. synonymous, however, does not equal identical, because many factors can affect choice of synonymous codons. in rna viruses ''71% and 88% of cub'' was attributed to mutational pressure on nucleotide and dinucleotide content, respectively [34] , which indicated the key role of mutational bias, but left a notable unexplained bias. a method that considered the impact of each of four nucleotides was superior to classical gc content correction (figs. 1, 3 ), yet it failed to completely explain cub in all but one virus. dinucleotide bias at codon positions 2-3, but not 3-1, complemented notably to cub in most viruses in our dataset. overall, in 25 of 29 of the rna viruses, mononucleotide and codon position 2-3 dinucleotide bias explained almost the entire apparent cub. there is, however, a fundamental problem that complicates attributing dinucleotide bias to translational or mutational pressure. on one hand, in mammalian genomes there is cpg and upa dinucleotide bias, and, consequently, ncg and nua codons are relatively less frequent [47] . therefore, translational bias towards common mammalian codons could produce secondary dinucleotide bias at codon positions 2-3. on the other hand, in rna viruses there is also dinucleotide bias at codon positions 3-1 (fig. 2) , which implies additional types of pressure against, at least, cpg dinucleotide. dinucleotide shuffling at distinct codon positions presented here allows distinguishing individual factors in a complex interplay of translational and mutational pressures on codon preference. negligible effect of position 3-1 dinucleotide content on enc (fig. 1 ) may be explained by the fact that only few dinucleotides are usually highly biased, and e.g. pressure against the cpg dinucleotide at codon position 2-3 would strongly affect the four ncg codons and produce a strong bias. at positions 3-1 cpg bias would affect sixteen nnc codons, and only when they precede gnn codons. therefore, the apparent effect of dinucleotide bias at codon positions 3-1 on cub may be many times weaker that at codon positions 2-3, and below the detection limit of our method. results of dinucleotide shuffling at codon positions 2-3 could also reflect the effect of translational bias, which could have produced secondary dinucleotide bias and increased dinucleotide content bias at codon position 2-3 compared 3-1 in most rna viruses (fig. 2) . however, these are not the only possible explanations for the shuffling results. viruses are a unique form of life, and their life cycle that is largely distinct from host genome replication makes them both a valuable tool for exploring sources of cub, and a source of confusion. in the mammalian host genome the cpg dinucleotide is underrepresented [48] , because in this context c is methylated and then deaminated, producing c-t transition [14] . this creates an obvious mutational pressure on codon usage and results in underrepresentation of ncg codons. such a feature of an organism's own genome allows recognition of non-self bacterial dna by tlr9, an innate immunity sensor for cpg-rich dna [18] . in mitochondria, cpg is also underrepresented [49] , probably to avoid triggering tlr9. a similar sensor for cpg in rna was predicted [38] , but is not yet known. this hypothetical sensor or effector could explain cpg 31 bias (fig. 2) and also the dramatic effect of introduction of cpg dinucleotide at codon positions 3-1 on poliovirus viability [50] . if such a sensor was linked to translation, as, for example, translation-coupled endonucleases involved in no-go rna decay [51] , it could produce codon position-specific pressure on cpg dinucleotide content. unfortunately, due to the host cub driven by host mutational pressure, it is fundamentally impossible to unambiguously distinguish translational and mutational pressure on cub in mammalian rna viruses using only a computational approach. overall, dn 23 and dn 231 shuffling produced enc values that differed from the original sequence by less than one in 25 of 29 viruses, indicating that we explored almost all types of pressure that affect overall codon usage. in three viruses (hiv, bunyavirus, rotavirus, and thogotovirus) enc values upon any correction were still higher than enc of the original sequence by more than 1. therefore, additional factors are required to explain this residual cub. in hiv, editing of cdna by apobec3 could introduce additional genome composition bias [52] . one additional unaccounted factor could be the influence of (di)nucleotide bias on 6fold degenerate codons. unfortunately, it is not possible to prudently evaluate such an effect. randomization of 6-fold degenerate codons would inevitably rely on inadequate estimates in genome composition, either nucleotide content at codon positions 1 and 2, which is dependent on amino acid sequence, or the third-position statistics, which would produce changes in positional (di)nucleotide content. shuffling dinucleotides between different synonymous codon positions, on the other hand, would ruin the positional (di)nucleotide frequencies in the genome. the latter approach to 6-fold degenerate codons was not superior over the algorithms used here, moreover it could introduce additional bias evident as enc values higher than upon dn 231 correction or lower than in the original sequence (data not shown). the algorithms presented here provide a substantial improvement in dissecting origins of codon usage bias in rna viruses. it is obvious that corrections based on gc content are inadequate not only in viruses, but in any application, as conventional dsdna genomes may have different genome composition biases for different genome strands [40] . as for dinucleotide composition, different codon positions should always be analyzed alongside, because they are affected by different kinds of pressure. considering only dinucleotide content at positions 2-3 or 3-1, as well as combining different codon positions for analysis, can lead to false conclusions. altogether, different shuffling algorithms allowed almost complete exploration of the correlation of nucleotide content and enc in all but a few rna viruses, but led to a conclusion that it is not possible to definitively discover forces affecting dinucleotide bias and cub by only computational means. a number of additional questions remain unanswered in the field of synonymous variation in the genomes. firstly, the origin of nucleotide bias that is strong enough not only to drive synonymous codon preference, but also to affect amino acid usage, remains largely unknown. second, the source of primary dinucleotide content bias in rna viruses remains obscure. third, a single virus genome might not contain enough information to discover fine factors affecting cub. scanning window approaches working with an alignment of multiple genomes, akin to bootscanning, and the corresponding significance criteria, are required to analyze local cub, while algorithms that properly address the synonymous variation in 6-fold degenerate codons and account for rna secondary structure might further clarify the role of translation pressure. such statistical methods, coupled with experimental analysis, can also elucidate fundamental steps of virus-cell interaction. coevolution of codon usage and transfer rna abundance codon usage in highly expressed genes of haemophillus influenzae and mycobacterium tuberculosis: translational selection versus mutational bias preference for guanosine at first codon position in highly expressed escherichia coli genes. a relationship with translational efficiency codon usage in bacteria: correlation with gene expressivity ribosome-mediated translational pause and protein domain organization the anti-shine-dalgarno sequence drives translational pausing and codon choice in bacteria synonymous mutations and ribosome stalling can lead to altered folding pathways and distinct minima evidence for translational selection in codon usage in echinococcus spp similar rates but different modes of 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in a gene accounting for background nucleotide composition when measuring codon usage bias accounting for background nucleotide composition when measuring codon usage bias: brilliant idea, difficult in practice the extent of codon usage bias in human rna viruses and its evolutionary origin comparison of the base composition of nucleic acids of nuclei and cytoplasm of different mammalian tissues why is cpg suppressed in the genomes of virtually all small eukaryotic viruses but not in those of large eukaryotic viruses? patterns of evolution and host gene mimicry in influenza and other rna viruses patterns of oligonucleotide sequences in viral and host cell rna identify mediators of the host innate immune system genetic variation and recombination in aichi virus mutational and selective pressures on codon and amino acid usage in buchnera, endosymbiotic bacteria of aphids codon usage bias amongst plant viruses codon and amino acid usage in retroviral genomes is consistent with virus-specific nucleotide pressure evolutionary basis of codon usage and nucleotide composition bias in vertebrate dna viruses heuristic approach to deriving models for gene finding nucleotide composition bias affects amino acid content in proteins coded by animal mitochondria nucleotide bias causes a genomewide bias in the amino acid composition of proteins codon usage tabulated from the international dna sequence databases: status for the year 2000 initial sequencing and analysis of the human genome skew of mononucleotide frequencies, relative abundance of dinucleotides, and dna strand asymmetry introduction of cpg and upa dinucleotides within and across synonymous capsid region codons differentially decreases replicative fitness endonucleolytic cleavage of eukaryotic mrnas with stalls in translation elongation apobec3f properties and hypermutation preferences indicate activity against hiv-1 in vivo authors are grateful to alexey nesterenko and petr mamonov for support with python code and to an anonymous reviewer for helpful suggestions. conceived and designed the experiments: anl. performed the experiments: isb. analyzed the data: anl isb. wrote the paper: anl. key: cord-001011-vjxmrmfc authors: lei, daoxiong; li, faqian; su, huabo; liu, jinbao; wei, ning; wang, xuejun title: hepatic deficiency of cop9 signalosome subunit 8 induces ubiquitin-proteasome system impairment and bim-mediated apoptosis in murine livers date: 2013-07-01 journal: plos one doi: 10.1371/journal.pone.0067793 sha: doc_id: 1011 cord_uid: vjxmrmfc the cop9 signalosome (csn), an evolutionally highly conserved protein complex composed of 8 unique subunits (csn1 through csn8) in higher eukaryotes, is purported to modulate protein degradation mediated by the ubiquitin-proteasome system (ups) but this has not been demonstrated in a critical mitotic parenchymal organ of vertebrates. hepatocyte-specific knockout of the cops8 gene (hs-csn8ko) was shown to cause massive hepatocyte apoptosis and liver malfunction but the underlying mechanism remains unclear. here, we report that csn8/csn exerts profound impacts on hepatic ups function and is critical to the stability of the pro-apoptotic protein bim. significant decreases in cis (cytokine-inducible src homology 2 domain-containing protein), a bim receptor of a cullin2-based ubiquitin ligase, were found to co-exist with a marked increase of bim proteins. csn8 deficiency also significantly decreased 19s proteasome subunit rpt5 and markedly increased high molecular weight neddylated and ubiquitinated proteins. the use of a surrogate ups substrate further reveals severe impairment of ups-mediated proteolysis in hs-csn8ko livers. inclusion body-like materials were accumulated in csn8 deficient hepatocytes. in addition to bim, massive hepatocyte apoptosis in hs-csn8ko livers is also associated with elevated expression of other members of the bcl2 family, including pro-apoptotic bax as well as anti-apoptotic bcl2 and bcl-xl. increased interaction between bcl2 and bim, but not between bcl2 and bax, was detected. hence, it is concluded that hepatic csn8 deficiency impairs the ups in the liver and the resultant bim upregulation likely plays an important role in triggering hepatocyte apoptosis via sequestering bcl2 away from bax. the ubiquitin-proteasome system (ups) constitutes a major degradation pathway for intracellular proteins and plays important roles in virtually all cellular processes. ups-mediated proteolysis involves two essential steps: ubiquitination and proteasomal degradation. ubiquitination tags the target proteins with a chain of the small protein molecule ubiquitin and the ubiquitin chain serves as a signal of the target proteins for degradation by the 26s proteasome [1] . the 26s proteasome is a multi-unit protein proteolytic machine composed of a cylinder-shaped core particle (i.e., the 20s proteasome) and the 19s regulatory particle (i.e., the 19s proteasome) at one or both ends of the 20s. proteasomal proteolytic enzymes reside, and peptide cleavage takes place, in the 20s proteasome. the 19s serves to regulate protein degradation by the 20s proteasome. the 19s proteasome consists of two distinct substructures known as the "lid" and the "base". the base of the 19s directly interacts with the α (outer) ring of the 20s, responsible for unfolding the target protein and regulating the gating of the 20s. the lid plays a critical role in recognizing and binding polyubiquitinated target proteins and the removal of ubiquitin from the target proteins for ubiquitin recycling [2] . besides the 19s, the 11s proteasome which is formed by heteropolymerization of pa28α and pa28β or by homopolymerization of pa28γ, can also associate with the 20s to form hybrid proteasomes (11s-20s-19s) or 11s-associated 20s proteasome complexes [3] . upregulation of 11s proteasomes by pa28α overexpression can facilitate the degradation of misfolded proteins by the ups [4, 5] . ups involvement in the pathogenesis of many forms of liver disease is well documented [6, 7] . accordingly, the ups has been suggested as a therapeutic target for many liver diseases [8, 9] . hence, it is important to achieve a better understanding on the regulation of the ups and the consequence of ups dysfunction in the liver. the cop9 signalosome (csn) consists of 8 unique protein subunits (csn1 through csn8) in mammalian cells. it is an evolutionarily conserved multifunctional protein complex that is essential in plants and animals [10] . the csn has an intrinsic metalloproteinase that removes the ubiquitin-like protein nedd8 from cullins via a process known as deneddylation [11, 12] . csn-mediated deneddylation is purported to regulate the assembly and catalytic dynamics of various cullin-ring ubiquitin ligases (crls) [13] [14] [15] , a large family of ubiquitin e3's that control the ubiquitination and degradation of many proteins. csn subunit 8 (csn8) is the smallest and the least conserved subunit of the csn complex. conditional knockout of the csn8 gene in mice has helped determine the essential role of csn8/csn in antigen-induced initiation of t cell proliferation [16] and in the postnatal cardiac development and heart function [17, 18] . csn8 deficiency was shown to cause ups impairment in the heart but this has not been examined in other organs [17] . we have previously reported that csn8/csn is essential to hepatocyte survival and effective proliferation in mice [19] . in livers deficient of csn8, hepatocytes showed massive apoptosis, resulting in marked proliferation of the oval cells and cells of the biliary lineage, and extensive interstitial fibrosis. the liver pathology induced by hepatocyte-specific csn8 knockout (hs-csn8ko) recapitulates the sequalea of chronic hepatic injury such as viral hepatitis [19] . hence, dissecting the molecular links between csn8 deficiency and the resultant hepatocyte apoptosis will shine light on the pathogenesis of a large group of liver diseases. hepatocyte apoptosis is a common mechanism of liver injury including hepatitis, liver fibrosis and cirrhosis. it appears that hepatocyte apoptosis can be induced via both the extrinsic and the intrinsic pathways [20] . in the intrinsic pathway, mitochondria release several apoptosis-promoting factors in response to diverse death signals via sensor molecules, such as bh3-only proteins. activation of pro-apoptotic proteins, such as bax, can trigger a series of events that lead to the activation of caspases [21] , resulting in dna fragmentation and cellular morphologic changes characteristic of apoptosis. notably, the bcl2 family proteins constitute a critical intracellular checkpoint in the mitochondrial pathway of apoptosis [22] . the bcl2 family consists of both anti-apoptotic and pro-apoptotic proteins. the anti-apoptotic members, including bcl2, bcl-xl and mcl1, display sequence conservation through four bcl2 homology domains (bh1 through bh4), whereas the pro-apoptotic members are composed of more fully conserved 'multidomain' proteins (e.g., bax and bak) possessing homology in bh1~bh3, as well as the bh3-only proteins (e.g., bid, bim, bad) which harbor only the bh3 domain [22, 23] . the bh3-only proteins can be further categorized into the 'activator' (e.g., bim, tbid) and the "sensitizer" (e.g., bad, noxa) subgroups [24, 25] . in viable cells, the anti-apoptotic proteins, bcl2 and bcl-xl, bind to and repress the multidomain pro-apoptotic proteins bax and bak, and keep them in inactive monomers. in response to cellular stresses, bh3-only proteins bim and tbid can bind to bcl2 or bcl-xl, thereby neutralize their antiapoptotic effects, and relieve bax or bak from bcl2-bound complexes [23] ; or alternatively bim and tbid directly activate bax and bak [25, 26] , thereby initiating caspase activation and cell death. therefore, the interaction and the functional balance between pro-apoptotic and anti-apoptotic bcl2 family proteins in a cell determine the fate of the cell [21] . using a cre-loxp-mediated hs-csn8ko mouse model, we have uncovered in the present study that csn8 deficiency leads to ups derangement and functional impairment, which are associated with massive apoptosis. we also presented the first evidence that the upregulation of pro-apoptotic protein bim and its interaction with bcl2 may have mediated the bax-dependent activation of the intrinsic pathway of hepatocyte apoptosis in csn8 deficient livers. hs-csn8ko mice were generated with the use of the cre-loxp system as previously described [19] . briefly, csn8 flox/flox mice were cross-bred with the cre transgenic mice (alb-cre) in which the cre expression is controlled by an albumin promoter [27] . ultimately, littermate mice with a genotype of either csn8 flox /flox ::alb-cre(+) or csn8 flox /flox ::alb-cre(-) were used in the present study as the hs-csn8ko or the control (ctl) mice, respectively. the generation and characterization of transgenic mice with ubiquitous overexpression of a surrogate ups substrate gfpdgn were previously described [28] . gfpdgn is an enhanced green fluorescence protein (gfp) modified by carboxyl fusion of degron cl1 [28] . using a cross-breeding strategy similar to what we previously reported [17] , the gfpdgn transgene was introduced into the hs-csn8ko and ctl background via cross-breeding, yielding the hr-csn8ko-gfpdgn group and the ctl-gfpdgn group used in the present study. the care and use of animals in this study was approved by the institutional animal care and use committee (iacuc) of the university of south dakota. transferred to pvdf membranes. western blots were performed with following antibodies: rpn2, rpn8, rpn10, rpt5, pa28α, pa28β, pa28γ, vhl (von hippel-lindau), hif1α (hypoxia inducible factor 1α), and csn8 (biomol), bim and bcl-xl (chemicon), bax and bcl2 (bd pharmingen tech.), nedd8 (alexis), β-tubulin and ubiquitin (sigma), cis (cytokineinducible src homology 2 domain-containing protein) and gfp (santa cruz biotechnology), and 20s subunit β5 (custom made). horseradish peroxidase-conjugated anti-mouse,rabbit, or -rat secondary antibodies (santa cruz) were used to probe the bound primary antibodies and detected using ecl advanced detection reagents (amersham pharmacia biotech, piscataway, nj). a versadoc model 3000 imaging system (bio-rad laboratories, hercules, ca) was used to visualize and digitalize the western blot images. the densitometry of the western blots was performed with the quantity-one software (bio-rad). for co-immunoprecipitation, the proteins (400µg) solubilized in ripa lysis buffer were incubated respectively with primary antibody against bim, bcl2, or bax (2µg/ml) at 4°c for 3 hours and precipitated with protein-a/g-sepharose (santa cruz) at 4°c for 2 hours. after centrifugation, the pellets were washed with ripa buffer for four times. the immunoprecipitates dissolved in sds-sample buffer were analyzed by western blotting. the terminal deoxynucleotidyl transferase-mediated dutp nick-end labeling (tunel) assay was performed using the tunel cell death detection kit (roche diagnostics, mannheim, germany), as previously described [19] . the label solution only (without terminal transferase) instead of tunel reaction mixture was used as negative control. at least 300 nuclei were counted from three different random fields in each liver, and data from 3 livers per group are presented. tunel index was expressed as a ratio of the number of tunel positive nuclei and the total number of nuclei stained with dapi. gfpdgn direct fluorescence of cryosections from paraformaldehyde fixed mouse livers, and the tunel or dapi labeled fluorescence in cryosections were visualized using a zeiss axiovert 200m epi-fluorescence microscope (carl zeiss microimaging gmbh, germany). tissue samples were collected from the right lobe of the liver, fixed in 10% formalin, and processed for paraffinembedding. serial 5-μm paraffin sections were used for csn8 immunohistochemistry as previously described [19] . briefly, paraffin-embedded sections were de-paraffinized and rehydrated. the slides were further incubated in 1n hcl for 8 minutes at 65°c and blocked with 0.3% h 2 o 2 for 20 minutes, washed in pbs, and blocked with 1% (w/v) bovine serum albumin (bsa) in pbs. the bsa-blocked slides were incubated with rabbit anti-csn8 antibody for 1 hour at room temperature. biotinylated anti-rabbit secondary antibody was used at a 1:200 dilution and visualized with peroxidase reaction using the abc vectastain kit (vector laboratories, burlingame, ca). this was performed essentially as previously described [18] . mice were anesthetized with isoflurane and the tissue samples from the left and right lobes of livers were fixed in 3.5% glutaraldehyde in 100 mm cacodylate buffer (ph 7.3). at least 3 tissue samples from each lobe of the liver were chosen randomly for ultrastructural analysis. two mice for each genotype were examined. toluidine (tol) blue stain of the semi-thin sections of resin-embedded tissue was performed and observed using bright-field light microscope to help orientate the tissue sample and select areas of interest for ultra-thin sectioning. ultrathin sections were picked up on nickel grids, dried and etched with a saturated solution of sodium m-periodate and 0.1n hcl. thin sections were counterstained with uranyl acetate and lead citrate. the sections were viewed in a zeiss, omega 912 electron microscope at 100 kv. all quantitative data were presented as mean ± sd unless otherwise indicated and analyzed using student t-test for unpaired two group comparison. a p-value <0.05 is considered statistically significant. to dissect the molecular link between csn8 deficiency and the activation of apoptotic pathways, we examined changes in the ups in hs-csn8ko livers. significantly increases in the mono-neddylated form of cullins have been reported in hs-csn8ko livers [19] , confirming that csn-mediated cullin deneddylation in hepatocytes requires csn8. csn-mediated cullin deneddylation is purported to prevent self-ubiquitination and destabilization of crls. consistent with this postulate, we detected in hs-csn8ko livers significant decreases in cis (cytokine-inducible src homology 2 domain-containing protein) and vhl (p<0.01, figure 1a ). cis and vhl serve as the receptor protein for bim-el and hif1α in the elongin b/c-cullin2-cis and the elongin b/c-cullin2-vhl ligases that target bim-el and hif1α for degradation, respectively [29, 30] . in agreement with the decrease of cis, the extra-long form of bim (bim-el) protein which is a known substrate of cis [29] , was significantly increased in hs-csn8ko livers compared with the ctl (p<0.01). by contrast, the protein level of hif1α, a substrate of vhl [31] , showed no discernible increase (p>0.05, figure 1a ). we previously observed marked increases in the mononeddylated form of cullins in hs-csn8ko livers [19] ; here we report that hmw nedd8 conjugates were significantly higher in hs-csn8ko liver than the ctl at 4 weeks of age (p<0.01, figure 1b, 1c) . interestingly, the abundance of hmw ubiquitin conjugates were also remarkably greater in hs-csn8ko livers than in the ctl (p<0.01, figure 1d ). the csn holo-complex shares morphology and subunit domain structure similarities with the lid of the 19s proteasome; hence, it has been proposed that the csn may serve as an alternative "lid" for the 19s proteasome to compete with the lid of the 19s proteasome in binding the base of the 19s. csn8 deficiency is known to reduce the abundance of csn holocomplex in mice [16, 17] . therefore, we examined the subunit protein levels of the 19s proteasome in hs-csn8ko mouse livers. csn8 deficiency show no discernible effect on the protein levels of rpn2, rpn8, and rpn10, three 19s lid subunits. interestingly, the level of a 19s base subunit rpt5 was significantly lower in hs-csn8ko liver than the ctl (p<0.01, figure 2a , 2b). hs-csn8ko showed no discernible effect on the protein level of 20s subunit β5. strikingly, all three subunits (pa28-α, -β, -γ) of the 11s proteasome showed greater protein expression in hs-csn8ko livers than in the ctl (p<0.05, 0.01; figure 2c , 2d). proteolysis via the ups plays important roles in a variety of basic cellular processes. the csn may act as a ups regulator by cullin neddylation of crls and potential association with the 19s proteasome [32, 33] . to investigate ups proteolytic function in intact animals, we previously established a transgenic mouse model that ubiquitously overexpresses gfpdgn, a modified green fluorescence protein (gfp) with its carboxyl fusion of degron cl1. gfpdgn, which is similar to gfp u [34] , has proven to be a surrogate substrate of the ups [28] . gfpdgn protein levels inversely correlate to ups proteolytic function in the cell [35, 36] . therefore, we introduced gfpdgn into the ctl and hs-csn8ko mice via crossbreeding, to explore the in vivo impact of hepatic csn8 deficiency on ups-mediated proteolysis in the liver. hs-csn8ko increased significantly hepatic gfpdgn protein levels (p<0.01; figure 3a , 3b). the accumulation of gfpdgn was further confirmed by the increased intensity of gfp direct fluorescence in hs-csn8ko mouse livers ( figure 3c ). these results compellingly demonstrate that csn8 deficiency in liver leads to severe impairment of ups-mediated proteolysis. meanwhile, the quantitative western blot analyses confirmed a dramatic decline in the protein level of subunit rpt5 of the 19s proteasome in hs-csn8ko-gfpdgn livers ( figure 3d, 3e) , indicating that ups malfunction resulted from csn8 deficiency in the liver is closely associated with the down regulation of a key subunit of the base of 19s proteasomes. consistent with protein accumulation resulting from impaired ups function in hs-csn8ko livers, immunohistochemistry examination revealed that csn8-negative hepatocytes displayed striking enlargement in cell size and nuclear size, compared with those in the littermate ctl at 6 weeks of age ( figure 4a ). toluidine blue stain of the semi-thin sections of resin-embedded liver tissue samples of 4-week-old mice revealed a large number of greenish granules or puncta in the cytoplasm of hepatocytes of hs-csn8ko but not ctl livers ( figure 4b ). transmission electron microscopy (tem) of the liver tissue from 4-week-old ctl mice revealed that hepatocytes were enriched with mitochondria which were frequently associated with rough endoplasmic reticulum. the hepatocyte in ctl livers usually contained a centrally located sphere-shaped nucleus which displayed a smooth outline profile and a diameter of approximately 2µm. electron-lucent or -dense vacuoles and autophagosomes were rare in the hepatocytes of ctl livers ( figure 5a , 5c). in hs-csn8ko mice, however, the nucleus of hepatocytes was markedly enlarged and lost its normal spherical shape. the outline of the nuclear envelop became rather rough and cytoplasmic invaginations resulting cytoplasmic inclusions were sometimes seen in the nuclear territory ( figure 5b ). in hs-csn8ko livers, a prominent feature is the presence of a large number of electron-lucent vacuoles of various sizes (diameter ranging from 0.2µm to 0.8µm) in the majority of hepatocytes. electron dense vacuoles with a diameter of approximately 0.2µm were also more frequently observed in hs-csn8ko than in ctl hepatocytes. large inclusion body-like (ibl) structures full of homogenous dense fine granules were present in a subpopulation of hepatocytes in hs-csn8ko livers. some of the ibl structures appeared to be membrane-bound ( figure 5d ) but others were not ( figure 5e) ; both often showed a clear boundary from their surroundings. in addition, multi-layered myelin-like structures ( figure 5b ) and autophagosome-like structures of various stages ( figure 5f ) were also observed in hs-csn8ko hepatocytes. tem feature of apoptosis was also observed in hs-csn8ko livers (data not shown). we have previously reported massive apoptosis in the liver of hs-csn8ko mice at 4 weeks of age [19] . our previous characterization of this hs-csn8ko mouse model has revealed that the depletion of csn8 protein starts in between postnatal week 1 and 2 and a significant decrease of csn8 protein is evidenced at week 2 [19] . hence, we characterized in the present study the time course of hepatocyte apoptosis in hs-csn8ko mice during the first 6 weeks after birth. we observed that hepatocyte apoptosis, as revealed tunel assays, was significantly increased at week 2, peaked at week 3, and remained at a significantly increased level at 4 and 6 weeks notably, we have previously observed cardiomyocyte massive necrosis in cardiomyocyte-restricted csn8ko mice produced using the same csn8-floxed allele [17] ; however, the same in vivo evans blue dye permeability assay as we used in cardiac csn8ko mice [17] , showed no discernible increases in hepatocyte membrane permeability in the hs-csn8ko mice at in the regulation of apoptosis, the bcl2 family proteins play a critical role in response to specific stress signals either to promote or to inhibit cell death. thus, we examined the expression of pro-apoptotic bcl2 family proteins in hs-csn8ko livers. we found that the protein levels of bh3-only protein bim-el and bim-l were significantly upregulated ( figure 7a , 7b) and the multi-domain pro-apoptotic protein bax was also expressed at a higher level compared with that in the ctl group ( figure 7a, 7c) . likewise, the protein abundance of antiapoptotic family members bcl2 and bcl-xl was elevated in hs-csn8ko livers ( figure 7a, 7d) . these findings imply that disequilibrium or altered interaction between the pro-apoptotic and anti-apoptotic proteins of the bcl2 family might be involved in csn8 deficiency-induced hepatocyte apoptosis, prompting us to examine their interactions (see below, figure 8 ). activation and polymerization of bax and/or bad constitute a critical step in the mitochondrial pathway of apoptosis. the mechanism by which bh3-only protein bim activates bax remains a matter of active debate. one possibility is the neutralization of anti-apoptotic bcl2 family members that releases bax from the anti-apoptotic complex and the other is for bim to bind directly and activate bax [23] [24] [25] . to gain insight into the mechanism of bh3-only protein bim-induced apoptosis and its interactions with other bcl2 family members, we performed reciprocal immunoprecipitation experiments to determine whether bim directly or indirectly activates bax. first, we used the bim antibody to pull down bim proteins from the crude protein extracts of hs-csn8ko livers. the immunoprecipitated bim-containing protein complexes were then probed with antibodies against bcl2, bcl-xl, or bax by western blotting, respectively. as shown in figure 8a and 8b, anti-apoptotic protein bcl2 but not bcl-xl was coimmunoprecipitated with bim, indicating that bim preferentially binds to bcl2 in hs-csn8ko hepatocytes. no bax was detected in the bim-precipitated complexes, which indicates that bim does not bind directly to bax in this case. second, the antibody against bcl2 was used to pull down the respective protein complexes, which were then immunobloted for bim and bax ( figure 8c and 8d) . the results showed that bim-el not bax was co-immunoprecipitated with bcl2, which further confirmed that bim most likely activates indirectly bax via binding and engaging anti-apoptotic protein bcl2. lastly, we also employed bax antibodies for immunoprecipitation. bim was nearly undetectable in bax-containing protein complexes from csn8ko livers ( figure 8e ). thus, our results do not support a direct interaction between bim and bax, but rather suggest the exclusive binding of bim with bcl2 for the indirect activation of pro-apoptotic protein bax to trigger apoptosis in hs-csn8ko livers. a growing body of evidence indicates that the csn is involved in the regulation of a variety of cellular processes including development [37] [38] [39] , cell cycle progression [40, 41] , dna damage checkpoint [42] , and immune responses [16, 43, 44] . the most studied functions of the csn are its roles in the regulation of ubiquitination via its deneddylation activity toward crls. previous studies with cardiomyocyte-or hepatocyte-restricted csn8ko in mice have revealed that csn8 is essential for the cell survival and the functioning of the heart and livers [17] [18] [19] . csn8 is required for ups function in the perinatal heart (a post-mitotic organ) but this has not been examined in a mitotic organ. in the present study, we investigated the effect of hs-csn8ko on ups function in livers and explored the potential molecular pathway through which hepatocyte apoptosis is activated by csn8 deficiency. our results reveal that the conditional targeting of the csn8 gene in hepatocytes perturbs the ups and leads to the impairment of ups proteolytic function. our data further suggest that the ups impairment is associated with increased hepatocyte apoptosis and the latter is likely activated via a bim-mediated proapoptotic signaling pathway. liver csn8 knockout previous studies have demonstrated that csn8 is required for the formation of the csn holocomplex and the bona fide cullin deneddylation activity of the csn in mammals [16, 17, 19] . csn regulation of crls has long been believed to depend on csn deneddylase activity but additional mechanisms independent of deneddylation were also suggested by recent reports [14, 45] . consistent with a role of the csn in preventing self-destruction of the components of crls, we observed in hs-csn8ko livers significant decreases in cis and vhl, two socs (suppressor of cytokine signaling)-box-containing substrate receptors of crls. the cis down-regulation was accompanied by increases of its substrate bim-el but decreased vhl was not associated with an increase of hif1α, a known substrate of vhl ( figure 1a) . interestingly, some other examined substrate receptor proteins (e.g., β-trcp, skp2) did not show significant changes although their bona fide substrates (e.g., β-catenin for β+-trcp and p27 for skp2) were significantly increased (data not shown). these in vivo data support the notion that the csn differentially regulates the stability and activity of crls. recent data demonstrate that not only cullins but also a number of other proteins, such as p53, epidermal growth factor (egf) receptor, ribosomal protein l11, etc., can also be modified by nedd8 [46] . in the present study, our examination of nedd8 conjugates reveals a remarkable increase of hmw nedd8-conjugated proteins in hs-csn8ko livers ( figure 1b, 1c) . these increased hmw nedd8 conjugates are likely neddylated forms of non-cullin proteins because immunoblotting shows that they are negative for cullin1 through cullin4 that we have examined. these new findings imply that the csn may also be responsible for deneddylation of target proteins other than cullins. it will be interesting to identify the hmw neddylated proteins accumulated in hs-csn8ko mouse livers. crls are a large family of ubiquitin ligases and are by estimate responsible for ~20% of ubiquitination in the cell [47] . it is reasonable to expect that defects in the csn impair crls and thereby lead to an appreciable decrease in global ubiquitination. similar to what was observed in the heart with cardiomyocyte-restricted csn8ko, we detected remarkable increases in the hmw ubiquitin conjugates in hs-csn8ko livers ( figure 1d ). this suggests that the removal of ubiquitinated proteins by the 26s proteasome is likely also impaired by csn8 deficiency. consistent with decreased proteasomal function, the surrogate ups substrate gfpdgn, when overexpressed in the hs-csn8ko, was also significantly accumulated ( figure 3a ~ 3c) . the marked reduction of the protein levels of rpt5 (a critical subunit of the 19s base) in hs-csn8ko livers (figures 2a, 3d, 3e ) may have contributed to the proteasomal impairment because rpt5 has been elegantly shown to play an indispensable role in 26s proteasome assembly [48, 49] . the mechanism for rpt5 down-regulation is unclear and does not appear to be attributable to an increased caspase activity in hs-csn8ko livers. activated caspase 3 can cleave rpt5, rpn2, and rpn10 of the 19s proteasome during apoptosis [50] . indeed, hepatocyte apoptosis was immediately increased upon the depletion of csn8 protein in the hs-csn8ko livers and remained significantly increased during the first 6 weeks of age that were examined ( figure 6 ) and we have previous reported that activated (the cleaved form) caspase 3 was significantly increased in csn8 deficient livers [19] . however, no discernible decreases of rpn2 and rpn10 were evidenced in the same hs-csn8ko livers where rpt5 decreases were observed, which stands against caspase activation as the main cause of rpt5 down-regulation. regardless of the cause of rpt5 downregulation, the depletion of this key component of the19s proteasome is at least in part responsible for the impaired proteasomal function in hs-csn8ko livers. supporting this postulate, it has been reported that myocardial ischemic preconditioning preserves ups function in ischemic myocardium via diminishing oxidative damage to rpt5 [51] . the csn has been proposed by some as an alternative and competing "lid" for the 19s proteasome [33, 52] ; however, as described above, none of the examined 19s lid subunits (rpn2, rpn8) or linker subunit (rpn10) showed discernible changes in protein abundance upon csn8 deficiency ( figure 2a) . interestingly, the expression of all three 11s proteasome subunits (pa28α, β, γ) was significantly increased in hs-csn8ko livers ( figure 2c, 2d) . this is likely a compensatory response to proteasome functional impairment because upregulation of 11s proteasomes by pa28α overexpression has been demonstrated to enhance proteasome-mediated degradation of misfolded proteins [4, 5] . apparently, as revealed by the increases in gfpdgn proteins, this compensatory response was inadequate. consistent with impairment of upsmediated protein degradation, striking accumulation of ibl structures and increases in the nuclear and cell size of hepatocytes as shown by histological characterizations are prominent morphological features (figures 4, 5) accompanying severe liver malfunction that was previous described for liver with hs-csn8ko [19] . blocking neddylation (including cullin neddylation) by, for example, a nedd8 activating enzyme (nae) inhibitor (e.g., mln4924) can effectively induce cell death in cancer cells and is being clinically tested for treating malignancy [47] . here we show that blocking cullin deneddylation by hs-csn8ko can also cause massive apoptosis in the liver ( figure 6) . a common mediating mechanism for the two opposing approaches to cause apoptosis resides likely in the inhibition of crls and the resultant defects in the degradation of critical regulators of cell cycle and cell death. it is in active investigation regarding how inhibition of crls causes apoptosis. inactivation of cullin neddylation by mln4924 in cancer cells can accumulate multiple crl substrates, including (1) cell cycle inhibitors such as p21, p27, and wee1, resulting in cell cycle arrest [53] ; (2) dna replication licensing factors cdt1 and orc1, leading to dna re-replication stress and the dna damage response [47, 54] ; and (3) iκbα, inhibiting nfκb activity [55] . these factors may all contribute to apoptosis induced by nae inhibition. in the present study, we collected compelling evidence that csn8 deficiency reduces a crl substrate receptor protein cis and increases its substrate bim (figures 1, 7) . bim is a pro-apoptotic bh3-only protein of the bcl2 family, acting in the upstream of mitochondria-mediated apoptosis [22] [23] [24] 56] . bim has at least 18 different splicing variants, among which bimel (extra-long), biml (long), and bims (short) are the three major isoforms. bimel and biml expression are tightly regulated at both the transcriptional and posttranslational levels [29] . in healthy cells, bim is sequestered to the microtubule-associated dynein motor complex by binding to dynein light chain lc8, and is thereby unable to promote cell death. upon death stimulation, bim is released from the microtubules, promoting bax activation and apoptosis [57, 58] . in hr-csn8ko mouse livers, both the pro-apoptotic members (bim, bax) and the anti-apoptotic members (bcl2 and bcl-xl) of the bcl2 family were significantly increased ( figure 7) , suggesting that the intrinsic apoptotic pathway has likely mediated the increased hepatocyte apoptosis induced by csn8 deficiency. the interaction and the functional balance between pro-apoptotic and anti-apoptotic bcl2 family proteins in a cell determine the fate of the cell [21] . anti-apoptotic bcl2 family members usually inhibit the pro-apoptotic activity of bax in healthy cells. there are two competing theories for the mechanism by which the pro-apoptotic bh3-only proteins, such as bim, promote the activation of bax: one proposes that bim binds to bax and thereby activates bax directly [26, 59] ; the other postulates that bim binds and sequesters the antiapoptotic bcl2 family proteins, which relieves bax from being bound by bcl2 and allows polymerization and activation of bax [24] . our data favor the latter as the mechanism taken by the increased bim to promote hepatocyte apoptosis in hs-csn8ko mouse livers. this is because our co-immunoprecipitation reveals that increased bimel and bcl2 existed in the same complex, while increased bim did not interact with bax and the increased bcl2 did not appear to interact with the increased bax in hs-csn8ko livers (figure 8 ). the present study demonstrates that csn8 deficiency compromises protein deneddylation, differentially downregulates substrate receptor proteins of crls, and selectively accumulates crl substrates such as pro-apoptotic protein bim, and reduces 19s proteasome subunit rpt5, leading to severe ups functional impairment and massive apoptosis in hepatocytes of intact mice. increased bim expression and its interaction with bcl2 may play an important role in mediating hepatocyte apoptosis in hs-csn8ko mice. posttranslational modification and quality control ubiquitin receptors and protein quality control properties of the hybrid form of the 26s proteasome containing both 19s and pa28 complexes enhancement of proteasome function by pa28α overexpression protects against oxidative stress enhancement of proteasomal function protects against cardiac proteinopathy and ischemia/reperfusion injury in mice hepatocellular carcinoma and the ubiquitinproteasome system the ubiquitin-proteasome system plays an important role during various stages of the coronavirus infection cycle the proteasome inhibitor velcade enhances rather than reduces disease in mouse hepatitis coronavirus-infected mice proteasome inhibitor treatment in alcoholic liver disease the cop9 signalosome cop9 signalosome: a multifunctional regulator of scf and other cullin-based ubiquitin ligases promotion of nedd-cul1 conjugate cleavage by cop9 signalosome the cop9 signalosome: more than a protease deconjugation of nedd8 from cul1 is directly regulated by skp1-f-box and substrate, and the cop9 signalosome inhibits deneddylated scf by a noncatalytic mechanism characterization of the role of cop9 signalosome in regulating cullin e3 ubiquitin ligase activity cop9 signalosome subunit 8 is essential for peripheral t cell homeostasis and antigen receptor-induced entry into the cell cycle from quiescence perturbation of cullin deneddylation via conditional csn8 ablation impairs the ubiquitinproteasome system and causes cardiomyocyte necrosis and dilated cardiomyopathy in mice cop9 signalosome regulates autophagosome maturation cop9 signalosome subunit 8 is required for postnatal hepatocyte survival and effective proliferation hepatocyte death: a clear and present danger traveling bax and forth from mitochondria to control apoptosis cell death: critical control points the bcl2 family: regulators of the cellular life-or-death switch apoptosis initiated when bh3 ligands engage multiple bcl-2 homologs, not bax or bak hierarchical regulation of mitochondrion-dependent apoptosis by bcl-2 subfamilies bax activation is initiated at a novel interaction site dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knockouts using cre recombinase a novel transgenic mouse model reveals deregulation of the ubiquitinproteasome system in the heart by doxorubicin rack1 and cis mediate the degradation of bimel in cancer cells rbx1, a component of the vhl tumor suppressor complex and scf ubiquitin ligase substrate-mediated regulation of cullin neddylation the cop9 signalosome: an assembly and maintenance platform for cullin ubiquitin ligases? the cop9 signalosome: an alternative lid for the 26s proteasome? impairment of the ubiquitinproteasome system by protein aggregation intrasarcoplasmic amyloidosis impairs proteolytic function of proteasomes in cardiomyocytes by compromising substrate uptake impairment of the ubiquitin-proteasome system in desminopathy mouse hearts targeted inactivation of the cop9 signalosome impairs multiple stages of t cell development cop9 signalosome subunit 8 (csn8) is essential for drosophila development unified nomenclature for the cop9 signalosome and its subunits: an essential regulator of development the cop9 signalosome inhibits p27(kip1) degradation and impedes g1-s phase progression via deneddylation of scf cul1 degradation of the cyclindependent-kinase inhibitor p27kip1 is instigated by jab1 the ubiquitin ligase activity in the ddb2 and csa complexes is differentially regulated by the cop9 signalosome in response to dna damage cop9 signalosome subunit 5 (csn5/jab1) regulates the development of the drosophila immune system: effects on cactus, dorsal and hematopoiesis role of scf ubiquitin-ligase and the cop9 signalosome in the n genemediated resistance response to tobacco mosaic virus structural basis for a reciprocal regulation between scf and csn protein modifications: beyond the usual suspects' review series an inhibitor of nedd8-activating enzyme as a new approach to treat cancer c termini of proteasomal atpases play nonequivalent roles in cellular assembly of mammalian 26 s proteasome atp binding by proteasomal atpases regulates cellular assembly and substrateinduced functions of the 26 s proteasome caspase activation inhibits proteasome function during apoptosis myocardial ischemic preconditioning preserves postischemic function of the 26s proteasome through diminished oxidative damage to 19s regulatory particle subunits consequences of cop9 signalosome and 26s proteasome interaction induction of p21-dependent senescence by an nae inhibitor, mln4924, as a mechanism of growth suppression nedd8-targeting drug mln4924 elicits dna rereplication by stabilizing cdt1 in s phase, triggering checkpoint activation, apoptosis, and senescence in cancer cells mln4924, a nedd8-activating enzyme inhibitor, is active in diffuse large b-cell lymphoma models: rationale for treatment of nf-{kappa}b-dependent lymphoma the bcl-2 protein family: opposing activities that mediate cell death bim: a novel member of the bcl-2 family that promotes apoptosis the proapoptotic activity of the bcl-2 family member bim is regulated by interaction with the dynein motor complex identification of novel isoforms of the bh3 domain protein bim which directly activate bax to trigger apoptosis key: cord-000984-64p3wpav authors: huang, shang-hui; zhao, li-xiang; hong, chao; duo, cui-cui; guo, bing-nan; zhang, li-juan; gong, zheng; xiong, si-dong; gong, fang-yuan; gao, xiao-ming title: self-oligomerization is essential for enhanced immunological activities of soluble recombinant calreticulin date: 2013-06-10 journal: plos one doi: 10.1371/journal.pone.0064951 sha: doc_id: 984 cord_uid: 64p3wpav we have recently reported that calreticulin (crt), a luminal resident protein, can be found in the sera of patients with rheumatoid arthritis and also that recombinant crt (rcrt) exhibits extraordinarily strong immunological activities. we herein further demonstrate that rcrt fragments 18–412 (rcrt/18-412), rcrt/39-272, rcrt/120-308 and rcrt/120-250 can self-oligomerize in solution and are 50–100 fold more potent than native crt (ncrt, isolated from mouse livers) in activating macrophages in vitro. we narrowed down the active site of crt to residues 150–230, the activity of which also depends on dimerization. by contrast, rcrt/18-197 is almost completely inactive. when rcrt/18-412 is fractionated into oligomers and monomers by gel filtration, the oligomers maintain most of their immunological activities in terms of activating macrophages in vitro and inducing specific antibodies in vivo, while the monomers were much less active by comparison. additionally, rcrt/18-412 oligomers are much better than monomers in binding to, and uptake by, macrophages. inhibition of macrophage endocytosis partially blocks the stimulatory effect of rcrt/18-412. we conclude that the immunologically active site of crt maps between residues 198–230 and that soluble crt could acquire potent immuno-pathological activities in microenvironments favoring its oligomerization. calreticulin (crt) is a 46 kda ca 2+ -binding glycoprotein in the endoplasmic reticulum of eukaryotic cells [1] [2] . it folds into 3 domains including a lectin-like globular n domain (amino acid residues , a proline-rich p domain (residues 198-308) and a ca 2+ -binding c domain (residues 309-412) [3] [4] [5] . crt can also appear at the surface of various types of cells exhibiting multiple biological functions [6] [7] [8] [9] [10] [11] [12] . recently it has been shown that soluble crt is present in the sera of patients with rheumatoid arthritis and with sle [13] [14] and that natural crt (ncrt), isolated from human or mouse tissues, can directly activate macrophages in vitro [15] . additionally, rcrt/39-272, a prokaryotically-expressed murine crt fragment covering amino acid residues 39-272 fused with an n-terminal his-tag, was extraordinarily potent in activating b cells and macrophages in vitro and also in eliciting specific ab production in mice [16] . this recombinant polypeptide also exhibited potent adjuvanticity, effectively assisting igg production against conjugated target ags with or without t cell help [16] [17] . however, molecular mechanisms underlying this phenomenon are far from clear. recent x-ray crystographical studies by kozlov [18, 19] . the sequence of rcrt/39-272 encompasses most of the globular n domain (aa residues , and we have previously shown that it possess lectin-like activity (selective binding with polysaccharides including carrageenan, alginic acids, and hyaluronic acids in elisas) [16] , implying that the prokaryotically expressed recombinant polypeptide retained the lectin activity of crt. it is of interest to determine if destroying the carbohydrate binding and/or peptide-binding sites (by deleting first half of the n domain sequence) would also abolish the potent immunological activities observed in rcrt/ 39-272. additionally, the fact that rcrt/39-272 is substantially more potent than ncrt in activating macrophages(see below) raised concerns regarding the possibility of lps contamination in the e. coli.-expressed recombinant product. the ''lps contamination'' hypothesis suggests tight binding between bacterial lps and rcrt and also that, due to a synergistic effect, the lps-crt complex is a more potent immune activator than free lps and rcrt alone. based upon the observation that interaction between the n and c domains of crt influences its structural stability as well as functional activity [20] , a ''c-domain deletion'' hypothesis has also been postulated, suggesting that deletion of the c-domain (as in rcrt/39-272) leads to exposure of an immunologically active site (ias) in the n and/or p domains of crt. the present study compares the biochemical characteristics of ncrt and a series of truncated rcrt polypeptides and investigates the molecular mechanisms underlying the potent immunological activities of soluble rcrt. the results arising from this study have important implications for our understanding of the potential role of soluble crt in immunopathological conditions. native crt was extracted from mouse livers by (nh 4 ) 2 so 4 precipitation followed by ion exchange chromatography on a deae-a50 column using a linear gradient of 280-500 mm nacl for elution. samples of the eluted fractions were assayed by sds-page (fig. 1a) , which showed that the eluent between 360-380 mm nacl contained protein bands of the expected molecular weight for ncrt (55 kda), which showed approximately 90% homogeneity judging by density of the major band in coomassie blue (cbb)-stained gel (fig. 1c ). recombinant murine crt fragment with an n-terminal his-tag and without c-terminal kdel) was expressed in e. coli and affinity purified using a ni 2+ -column. the resultant rcrt/18-412 product contained 3 major protein bands at 60, 46 and 32 kda (figs. 1b & 1c); the two larger bands (namely rcrt-60 kda and rcrt-46 kda), but not the smaller one (cp32), were recognized by polyclonal rabbit-anti-crt antisera (crt-abs) in western blot (wb) (fig. 1d ). purified ncrt, but not bsa or recombinant enhanced green fluorescence protein (regfp, 28 kda with a his-tag), was positively recognized by crt-abs. as evidenced by native page analysis, a substantial amount of rcrt/18-412 formed higher-molecular-weight oligomers, whilst ncrt existed mostly in monomeric form (figs. 1e & 1f). in our previous study, rcrt/39-272 could effectively activate mouse macrophages in vitro [16] . interestingly, rcrt/18-412 was as effective as rcrt/39-272 in inducing no 2 2 production by [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] were analyzed by a sds-page 12% gel followed by cbb staining. fractions 6-10 were combined, dialyzed against pbs, and then adjusted to 1 mg/ml for use as ncrt. (b) a ni-column was employed for purification of rcrt/18-412 from a lysate of iptg-induced e. coli harboring the expression vector for rcrt/18-412. samples of e. coli before and after iptg induction were loaded in lanes, flow through (binding buffer), wash through (20 mm imidazole), resultant rcrt/18-412 (300 mm imidazole), and stripping through were loaded, in the order of 1-6 into a sds-page 12% gel followed by cbb staining. samples of ncrt, rcrt/18-412, bsa and regfp were compared in cbb-stained sds-page 12% gel (c) and native-page (e), followed by wb using rabbit anti-crt polyclonal antibody for detection (d, f). the secondary ab was hrplabeled goat-anti-rabbit igg, with opd as substrate. doi:10.1371/journal.pone.0064951.g001 mouse peritoneal macrophages in vitro, indicating that the presence, or deletion, of the c-domain did not affect the immunological activity of crt ( fig. 2a ). purified ncrt was also able to activate macrophages in vitro, but with a 50-100-fold lower potency than rcrt/18-412 ( fig. 2a) . although rcrt/18-412 and rcrt/39-272 preparations had been repeatedly treated with polymyxin b (an efficient lps inhibitor) prior to functional assays, the possibility that bacterial lps might form a tight complex with rcrt, thereby resisting pmb treatment, was always a concern. figs. 2b & 2c demonstrate that ncrt was not able to bind lps in elisas, implying that formation of tight lps-crt complexes in the expressing e. coli cells is perhaps an unlikely event. moreover, lps and ncrt at sub-optimal concentrations did not show any synergistic effect in activating macrophages (fig. 2d) , providing circumstantial evidence against the ''lps contamination'' hypothesis. contaminating rpl2 does not contribute to rcrt activity when preparing rcrt/18-412, cp32 was a relatively persistent contaminant protein (figs. 1 & 3) . to characterize this protein, the cp32 band was sliced out of sds-page gels and then (i) used to immunize c57/bl6 mice for preparation of specific antisera; and (ii) subjected to q-tof mass spectrometry analysis for sequence identification. the resultant antisera (cp32-abs) were able to recognize the immunizing protein band, but not the rcrt-60 kda and rcrt-46 kda bands, in wb (fig. 3b) . the ms result identified cp32 as bacterial 50s ribosomal protein l2 (rpl2), which was confirmed by positive recognition of recombinant rpl2 (rrpl2, commercially available) by cp32-abs (fig. 3b) . it is noteworthy that rrpl2 was unable to activate macrophages, as evidenced in no 2 2 production assays (fig. 3c ), and also that rrpl2 was not recognized by crt-abs (figs. 4c & 4d). these results exclude the possibility that the contaminant cp32 could make a substantial contribution to the immunological activities of rcrt. 2 in the culture supernatant was then determined using griess reagent and the results are expressed as mean no 2 2 concentration (mm) 6 sd. lps-based elisas were performed for detection of lps binding with crt (b) or lactoferrin (c). lf or ncrt (2 mg/ml) were added to wells in polyvinyl plates pre-coated with lps (10 mg/ml), with bsa as a negative control. combination of polyclonal rabbit abs against crt, or lactoferrin, and hrp-labeled goat-anti-rabbit igg was used for detection with opd as substrate. the results are expressed as absorbance at od492 nm6sd. for sinergy analysis (d), freshly isolated mouse peritoneal macrophages were stimulated with ncrt (0.3-30 mg/ml) in the presence, or absence, of lps (0.1 ng/ml) for 24 h. cells in medium alone (medium) or stimulated with lps (0.1 ng/ml) alone (lps) were included as controls. tnf-a in the culture supernatant was then quantitated using an elisa kit and the results are expressed as mean concentration (pg/ml)6sd. these are representatives of 3 independent experiments. doi:10.1371/journal.pone.0064951.g002 since rcrt and ncrt differ in their ability to form oligomers in solution, we next asked if the potent immunological activities of rcrt polypeptides were the result of self-oligomerization. a sephadex g-75 column was employed for fractionation of rcrt/ 18-412 oligomers and monomers. fig. 4a shows that rcrt/18-412 was successfully separated into 3 peaks, designated sequentially as orcrt (higher-molecular-weight rcrt/18-412 oligomers, major peak), mrcrt-60 kda (monomeric rcrt/18-412, 60 kda) and mrcrt-46 kda (monomeric rcrt-46 kda, aa18-386, see below). guided by the sds-page results ( , revealed their molecular mass as 46.78 kda and 43.57 kda, respectively. it can therefore be calculated that rcrt-46 kda is a degradation product of rcrt-60 kda less the c-terminal 26 amino acid residues. as shown in fig. 5a , orcrts were modestly more effective than unfractionated rcrt/18-412 in eliciting tnf-a production by murine macrophages in vitro, while mrcrt-60, mrcrt-46 and ncrt were 50-100-fold less active than orcrts by comparison. a similar conclusion was also drawn when no 2 2 production was taken as a measure for macrophage activation (fig. 5b) . moreover, s.c. immunization of balb/c mice with orcrts or unfractionated rcrt/18-412 (in the absence of adjuvant) elicited high titer serum igg capable of recognizing both orcrts and mrcrts in elisas (figs. 5c-5f). by contrast, mrcrt-60 kda (monomeric rcrt/18-412) and mrcrt-46 kda (monomeric rcrt/18-386) were almost non-immunogenic in parallel experiments. in order to assess whether the ias is located in the n or p domain, the following fragments were designed: rcrt-n (residues 18-197, full length n domain), rcrt/120-250 (partial n, half p domains), rcrt/150-230 (partial n, one third p domains), rcrt/120-308 (partial n, full length p domain), and rcrt/ 198-308 (full length p domain). all, but rcrt/198-308 (very low yield and poor solubility), were successfully expressed in e. coli and affinity-purified. both rcrt/120-250 and rcrt-n formed homodimers as well as higher-molecular-weight species (fig. 6a) . rcrt/120-250 was as potent as rcrt/39-272 and rcrt/18-412 in terms of eliciting no 2 2 production by murine macrophages, while rcrt-n was almost completely inactive (fig. 6b ). similar to rcrt/120-250, rcrt/120-308 also formed homodimers and higher-molecular-weight oligomers and showed strong macrophage-stimulatory activity in vitro (table 1) . rcrt/150-230, which contains a single cysteine residue (cys163) and could only form homodimers (fig. 6a) , was approximately 10 fold less effective than rcrt/39-272 and rcrt/120-250 but nevertheless substantially more potent than mrcrts and ncrt (fig. 6b) . a mutant form of rcrt/150-230 (namely rcrt/150-230-c163a) was prepared by substituting the cysteine residue at position 163 with alanine. the c163a mutant was neither able to form homodimers/oligomers in solution nor activate macrophages in vitro (figs. 6c-6e). these results map the ias of crt to residues 150-230 and suggest that the n-domain probably does not contribute to the immunological activity of the molecule. moreover, the importance of oligomerization for its immunological activities is further confirmed. in order to investigate the mechanisms for the relationship between rcrt oligomerization and its potent immunological activities, fractionated orcrts and mrcrt-60 kda (monomeric rcrt/18-412) were conjugated with fitc and then compared for ability to stain macrophages. after 30 min incubation at 4uc, fitc-orcrts showed stronger binding to murine macrophages than fitc-mrcrts, as evidenced by flow cytometric analysis and confocal laser scanning microscopy (fig. 7a&b) . substantially more fitc-orcrts than fitc-mrcrts were endocytosed by macrophages after 30 min incubation at 37uc (fig. 7b) . moreover, monodansylcadaverine (mdc), an endocytosis inhibitor [21] , partially suppressed no 2 2 production by macrophages under stimulation with orcrts, while lps-triggered macrophage activation was unaffected by the same treatment (fig. 7c ). in this study, we have examined different hypotheses (i.e. cdomain deletion hypothesis, lps contamination hypothesis, rpl2 hypothesis and oligomerization hypothesis) for explanation of the much stronger immunogenicity and immunostimulatory activities of rcrt than ncrt. our data strongly suggests that selfoligomerization of the rcrt polypeptides is a key factor for their strong immunological activities. as summarized in table 1 , all four rcrt fragments containing residues 120-250 (including rcrt/18-412, rcrt/39-272, rcrt/120-250 and rcrt/120-308) self-oligomerized and exhibited potent macrophage activating ability in vitro and strong immunogenicity in vivo, while ncrt, which existed mainly in monomeric form, showed only modest stimulatory activities towards macrophages and was non-immunogenic in mice (figs. 1 & 3) . moreover, fractionated rcrt/18-412 oligomers were 50-100 folds more active than mrcrts in activating macrophages in vitro (fig. 5) . the n domain polypep-tide (rcrt/18-197) also formed higher-molecular-weight oligomers (fig. 6a ), but it is almost completely inactive in terms of stimulating macrophages in vitro (fig. 6b ) and inducing ab responses in vivo (table 1) . clearly, oligomerization is necessary but not sufficient to arm the rcrt polypeptides with potent immunological activities. in general, oligomerized (aggregated) proteins are more immunogenic than their monomeric counterparts. however, the immunogenicity of orcrts is by far the most impressive and not comparable by other protein aggregates. even in the absence of any adjuvant, minute amount (1 ng/mouse) rcrt/18-412 or rcrt/39-272 can elicit strong igg responses in mice ( fig. 2; ref. 16 ). most, if not all, other protein antigens are unable to induce igg production in t-cell-deficient nude mice, yet rcrt/39-272 and rcrt/18-412 could do so relatively efficiently [16, 22] . moreover, the potent adjuvanticity of the rcrt polypeptides is also quite phenomenal. for instance, rcrt/39-272 (mostly in oligomeric forms) is able to assist the production of igg abs against fused target proteins or conjugated polysaccharides in healthy mice or t-cell-deficient nude mice [17, 22] . dimerization/oligomerization of soluble crt was also observed by previous investigators. for example, jorgensen et al documented that shielding of the free cys163 in the n domain is the main reason that ncrt exists mainly in monomeric form under physiological conditions. under partial unfolding conditions such as high temperature or low ph, however, the free cys could be exposed and subsequently help crt oligomerization [23] . ncrt (isolated from human placenta) formed homodimers and higher-molecular-weight species through disulfide bonding as well as non-covalent association, and that oligomerized ncrt showed higher binding affinity to peptides and denatured proteins [23] . in the case of prokaryotically expressed rcrt polypeptides (the folding of which may differ from ncrt), all cys residues in sequence could contribute to its olimerization, although it might not be absolutely necessary that all 3 cys residues have to be available for inter-molecular cross-linking at the same time. mancino and colleagues illustrated that self-oligomerized rcrt could better assist hla folding in vitro [24] . there are 3 conserved cysteine residues in the amino acid sequence of crt. cys105 and cys137 form intramolecular disulfide bonds, while cys163 is free [25] [26] . it is likely that rcrt polypeptides are unable to form appropriate intra-molecular disulfide bonds like in ncrt, thereby allowing all 3 cysteine residues to participate in self-oligomerization, although formation of higher-molecularweight oligomers could also occur through non-covalent association of crt [23, 27] . crt is considered one of the heat shock proteins (hsps) that share many immunological and biochemical activities [28] [29] . interestingly, self-oligomerization also occurs to other hsps such as grp94 and hsp90, which is likely associated with their chaperone function [30] [31] [32] . koslov et al and chouquet et al have recently solved the crystal structure of the lectin site as well as a peptide-binding site in the crt n domain [18, 19] , which apparently play important roles in the physiological function of crt. however, our data maps the ias of crt to a region of 80 residues between aa150-230 (fig. 6 ). as rcrt-n was almost completely inactive in functional assays (fig. 6, table 1 ), the ias may be narrowed down further to aa198-230 in the p domain, although a series of truncated synthetic peptides covering this region would be needed for a concrete conclusion. interestingly, this sequence of 30 amino acid residues contains the ra shared epitope (se)-binding site (residues 217-224) of crt recently mapped by ling et al. [33] . such coinciding results from 2 independent groups further emphasize the importance of the aa198-230 region of the p domain to the immunological activities of crt. it has been documented that the crt p domain adopts a hairpin-shaped structure, its sequence is consisted of 3 copies of a repeat motif (type 1: ixdpxxxk-pedwd) followed by 3 copies of another repeat motif (type 2) [34] [35] [36] . interestingly, the se-binding site almost completely overlaps the type 1 motif [33] . it is reasonable to suggest that the type 1 motif might also represent the core of ias of crt, responsible for direct biding with activation receptors on the surface of immune cells. functional comparison data showed that rcrt/120-308 and rcrt/120-250 (possessing 3 type 1 repeats, the latter without type 2 repeats) are 10 times more active than rcrt/120-230 (with 2 type 1 repeats, no type 2 repeats) in activating macrophages (table 1, fig. 6b ), implying that (i) type 2 repeats do not contribute to the immunological activity of the molecule; and (ii) the presence of 3 copies of the type 1 motif is of crucial importance for the potent immunological function of orcrts. it can be envisaged that oligomerization of crt multiplies its binding avidity to immune cells with receptors for the type 1 motif, thereby enabling it to deliver stimulatory signals to the cells in a highly efficient manner. we further predict that the high-avidity binding of orcrts to macrophages may easily trigger their uptake process. indeed, orcrts can activate macrophages in an endocytosis-dependent pathway (fig. 7) . perhaps orcrts could use certain intracellular sensors to deliver immunostimulatory activity to the responding immune cells. an example of an intracellular sensor for endocytosed polymeric proteins is the nod-like receptor (nlr) protein nlrp3, a key component of the nlrp3 inflammasome and an important intracellular sensor for microbial ligands and endogenous danger signals [37] . masters and colleagues demonstrated that soluble oligomers of islet amyloid polypeptide (iapp), a protein that forms amyloid deposits in the pancreas during type 2 diabetes, could be endocytosed and trigger the nlrp3 inflammasome and generate mature il-1b [38] . finally, the region of aa150-230 of crt has a 98% homology between mouse and human. it would be of interest to examine if there is a functional relationship between the se binding site and the ias of crt. irrespective of such a relationship, oligomerization might occur to extracellular crt released by tissue cells thereby converting crt into a highly active form, which may play important roles in the development and pathogenesis of autoimmune disorders in humans. purification of ncrt ncrt was purified from mouse livers using a modification of a previously described methods [39, 40] . briefly, fresh mouse liver cells (erythrocytes depleted) were collected and centrifuged at 1200 rpm for 5 min. the cell pellet was lysed in 3 volumes of lysis buffer (1% triton-x 100, 0.2 mm pmsf in pbs) for 30 min on ice, followed by centrifugation at 35,000 g for 60 minutes. the supernatant was then precipitated using (nh 4 ) 2 so 4 and the final precipitate dissolved in binding buffer (150 mm nacl, 20 mm tris, ph7.4) followed by dialysis against this buffer. the sample was applied to a deae sephadex a50 column (1062 cm, ge healthcare, us) which was then sequentially washed with binding buffer and washing buffer (280 mm nacl, 20 mm tris, ph7.4) at 1 ml/min to remove contaminating proteins. the fractions were eluted with a linear salt (280-500 mm nacl) gradient. the preparation of rcrt/39-272 and regfp was as previously described [16] and the other rcrt polypeptides used in this study were prepared using the same prokaryotic system. specific primers were as follows: 59-cccaagcttctaatctgtggggtcatc-gatcttg-39. the rcrt/150-230-c163a mutant was constructed using takara mutan best kit (takara biotechnology co. ltd) following manufacturer's instruction. the mutant primer was as follows: sense: 59-attcacacacctataca-cactgatt-39, antisense: 59-tcatcatccttagcccgga-tatcct-39. all proteins were desalted by passing through pd10 columns (pierce, rockford, il, usa). protein concentration was determined using coomassie protein assay reagent (pierce, rockford, il, usa). all recombinant proteins were used at over 90% purity as judged by cbb-stained sds-page gels. the separated protein bands in sds-page gels were electroblotted onto pvdf membranes, at a constant current of 250 ma in transbuffer (50 mm tris, ph 8.0, containing 0.192 m glycine and 20% methanol), using a bio-rad trans-blot cell. the strips were incubated for 1 hr at room temperature in blocking buffer (tbs containing 5% nonfat milk), followed by a overnight incubation at 4uc with constant agitation in indicated antibody diluted in blocking buffer. after 3 washes with tbs containing 0.05% tween 20, strips were incubated for 1 hr with hrpconjugated secondary antibody (southern biotechnology associates inc., usa) and visualized using the ecl detection system as recommended by the manufacturer (applygen technologies inc., beijing, china). lps-based and crt-based elisas were as previously described [41] . briefly, elisa plates were coated at 4uc overnight with rcrt or lps and subsequently incubated with blocking solution (1% bsa in pbs) for 2 hrs at 37uc. the wells were washed five times with pbs containing 0.05% tween 20 (pbs-t) prior to incubation at 37uc with 100 ml of diluted mouse sera or with indicated protein (ncrt and lactoferrin) followed by corresponding antibody in triplicate. after 5 washes with pbs-t, the plates were further incubated with hrp-labeled goat-antimouse or goat-anti-rabbit igg abs (southern biotechnology associates inc., al., usa) for 1 hr at 37uc. the reaction was developed with 100 ml of o-phenylenediamine (opd, sigma) for 5 min and stopped with 100 ml 2 m h 2 so 4 . optical density (od) was measured at 492 nm in an elisa spectrophotometer (titertek multiscan plus mk ii; icn flow laboratories, irvine, uk). all cells were cultured in complete r10 medium: rpmi-1640 supplemented with 10% (v/v) fetal bovine serum (hyclone, usa), penicillin/streptomycin (100 u/ml), l-glutamine (2 mm), and b-me (5610 25 m). for preparation of mouse peritoneal macrophages, mice were injected i.p. with 3% thioglycollate (1 ml/ mouse) and the macrophages retrieved from the peritoneum 3 days later using a syringe. the resultant cells were.90% positive for f4/80 marker, as determined by facs analysis. relative macrophage stimulation activity (rmsa) of crt is estimated using ncrt as reference, which is able to induce tnf-a production by macrophages in vitro at a concentration of 10 mg/ml or above (see fig. 5a ). the listed results are based on several batches of independent experiments including fig. 5a . b) ability to elicit specific igg responses in healthy balb/c mice after s.c. immunization, in the absence of adjuvant, with 100 mg protein and a booster immunization with 50 mg protein a fortnight later. the mice were monitored for up to 28 days after the second immunization. nd: not detected; -: not immunogenic; +: strong humoral response; 2/+: weak response. freshly prepared c57/bl6 mouse peritoneal macrophages (1.5610 5 cells/well) were stimulated with rcrt fragments, or ncrt, or lps, in r10 medium in 96-well tissue culture plates for 24 hrs in a 5% co 2 incubator at 37uc. the concentration of tnf-a in the culture supernatant was determined using elisa kits (biolegend, san diego, usa) following the manufacturer's instructions. the concentration of no 2 2 in the supernatant was determined by griess reagent. standard curves were established using nano 2. female c57bl/6 mice between the age of 6-8 weeks were purchased from the model animal research center, nanjing, china. all animals were maintained under specified-pathogen-free (spf) conditions and animal usage was conducted according to protocols approved by the soochow university institutional animal care and use committee. for immunization with rcrt, mice were immunized s.c. at the base of the tail with 100 mg protein in total 100 ml pbs. when booster immunization was needed, 50 mg of protein in 200 ml pbs was injected intraperitoneally (i.p.). for immunization with page gel slices containing cp32, the band was cut out with a razorblade and then frozen in liquid nitrogen and emulsioned with cfa, which was then injected s.c. into female c57/bl6 mice. serum samples were collected by tail bleeding, aliquoted and kept at 220uc until use. a sephadex g-75 (ge healthcare, us) column of 8062 cm was employed. 5 ml of rcrt/18-412 at 10 mg/ml was loaded into the column, followed by elution with 0.9% nacl at 20 ml/h and collected every 2 ml. protein was labeled by fitc using fluorotag tm fitc conjugation kit (sigma, us) according to the manufacturer's instructions. in brief, 1 mg of protein was dialyzed against 0.1 m na 2 co 3 , ph 9.5 at 5 mg/ml. fluoresceine isothiocyanate (fitc, sigma) was dissolved in the same buffer with dmso at 1 mg/ml and 50 ml of this solution was added to the protein. the sample was gently mixed for 2 h at rt. separation of labeled protein from unbound fitc was performed on a sephadex g-25 column. flow cytometric analysis 10 6 freshly isolated peritoneal macrophages were stained with apc-anti-f4/80 and then incubated with 15 mg/ml of fitc-orcrt, fitc-mrcrt or fitc-ova for 30 min at 4uc. the binding of crt by macrophages was determined by flow cytometry (bd facs canto ii, us). macrophages (1.5610 5 /well) were plated onto poly-l-lysine coated glass slides and allowed to adhere. the cells were then incubated in a total volume of 200 ml 0.5% bsa in pbs with 15 mg/ml fitc-orcrt, fitc-mrcrt or fitc-ova for 30 min at 4uc or 37uc. after washing to remove unbound protein, macrophages were fixed in 1% paraformaldehyde and stored at 4uc until microscopic analysis. finally, 1% of glycerol was added and slides were counterstained with 1 mg/ml dapi. the cells were imaged with a nikon confocal microscope system a1. all experiments were repeated at least 3 times and the results are expressed as mean6standard deviation of the mean (sd). statistical analysis was performed using the independent-samples t test or two-side paired t test between groups using the spss 14.0 program (spss, chicago, il). differences were considered statistically significant at p,0.05. calreticulin: one protein, one gene, many functions definition of the lectin-like properties of the molecular chaperone, calreticulin, and demonstration of its copurification with endomannosidase from rat liver golgi delineation of the lectin site of the molecular chaperone calreticulin oligosaccharide binding characteristics of the molecular chaperones calnexin and calreticulin calreticulin is expressed on the cell surface of activated human peripheral blood t lymphocytes in association with major histocompatibility complex class i molecules cell surface calreticulin is a putative mannoside lectin which triggers mouse melanoma cell spreading thrombospondin mediates focal adhesion disassembly through interactions with cell surface calreticulin activation of human monocyte cell line u937 via cell surface calreticulin cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of lrp on the phagocyte calreticulin in tlymphocytes. identification of calreticulin in t-lymphocytes and demonstration that activation of t cells correlates with increased levels of calreticulin mrna and protein regulation of peripheral t cell activation by calreticulin candidate autoantigens identified by mass spectrometry in early rheumatoid arthritis are chaperones and citrullinated glycolytic enzymes calreticulin is released from activated neutrophils and binds to c1q and mannan-binding protein cd91-dependent programming of t-helper cell responses following heat shock protein immunization functional analysis of recombinant calreticulin fragment 39-272: implications for immunobiological activities of calreticulin in health and disease ajuvanticity of recombinant calreticulin calreticulin as a hydrophilic chimeric molecular adjuvant enhances igg responses to the spike protein of sars coronavirus structural basis of carbohydrate recognition by calreticulin x-ray structure of the human calreticulin globular domain reveals a peptide-binding area and suggests a multi-molecular mechanism calreticulin is a thermostable protein with distinct structural responses to different divalent cation environments amantadine and dansylcadaverine inhibit vesicular stomatitis virus uptake and receptor-mediated endocytosis of alpha 2-macroglobulin adjuvanticity of recombinant calreticulin fragment 39-272 in assisting anti-b-glucan igg responses in t celldeficient mice dimerization and oligomerization of the chaperone calreticulin calreticulin recognizes misfolded hla-a2 heavy chains calreticulin, a ca2 + -binding chaperone of the endoplasmic reticulum human placental calreticulin characterization of domain structure and post-translational modifications the metal ion binding properties of calreticulin modulate its conformational flexibility and thermal stability heatshock proteins as activators of the innate immune system heat shock proteins as regulators of the immune response heat-induced chaperone activity of hsp90 endoplasmic reticulum chaperone grp94 subunit assembly is regulated through a defined oligomerization domain substrate-binding characteristics of proteins in the 90 kda heat shock protein family identification of the rheumatoid arthritis shared epitope binding site on calreticulin trosy-nmr reveals interaction between erp57 and the tip of the calreticulin pdomain nmr structure of the calreticulin p-domain nmr structures of 36 and 73-residue fragments of the calreticulin p-domain nlrp3: an immune sensor of cellular stress and infection activation of the nlrp3 inflammasome by islet amyloid polypeptide provides a mechanism for enhanced il-1beta in type 2 diabetes human placental calreticulin: purification, characterization and association with other proteins a single purification procedure for the major resident proteins of the er lumen: endoplasmin, bip, calreticulin and protein disulfide isomerase heat shock protein 60: specific binding of lipopolysaccharide key: cord-000736-6f8vyziv authors: pripuzova, natalia; wang, richard; tsai, shien; li, bingjie; hung, guo-chiuan; ptak, roger g.; lo, shyh-ching title: development of real-time pcr array for simultaneous detection of eight human blood-borne viral pathogens date: 2012-08-17 journal: plos one doi: 10.1371/journal.pone.0043246 sha: doc_id: 736 cord_uid: 6f8vyziv background: real-time pcr array for rapid detection of multiple viral pathogens should be highly useful in cases where the sample volume and the time of testing are limited, i.e. in the eligibility testing of tissue and organ donors. findings: we developed a real-time pcr array capable of simultaneously detecting eight human viral pathogens: human immunodeficiency virus types 1 and 2 (hiv-1 and -2), hepatitis b virus (hbv), hepatitis c virus (hcv), human t-cell leukemia virus-1 and -2 (htlv-1 and -2), vaccinia virus (vacv) and west nile virus (wnv). one hundred twenty (120) primers were designed using a combination of bioinformatics approaches, and, after experimental testing, 24 primer sets targeting eight viral pathogens were selected to set up the array with sybr green chemistry. the specificity and sensitivity of the virus-specific primer sets selected for the array were evaluated using analytical panels with known amounts of viruses spiked into human plasma. the array detected: 10 genome equivalents (geq)/ml of hiv-2 and hcv, 50 geq of hiv-1 (subtype b), hbv (genotype a) and wnv. it detected 100–1,000 geq/ml of plasma of hiv-1 subtypes (a – g), group n and crf (ae and ag) isolates. further evaluation with a panel consisting of 28 hiv-1 and hiv-2 clinical isolates revealed no cross-reactivity of hiv-1 or hiv-2 specific primers with another type of hiv. all 28 viral isolates were identified with specific primer sets targeting the most conserved genome areas. the pcr array correctly identified viral infections in a panel of 17 previously quantified clinical plasma samples positive for hiv-1, hcv or hbv at as low as several geq per pcr reaction. conclusions: the viral array described here demonstrated adequate performance in the testing of donors’ clinical samples. further improvement in its sensitivity for the broad spectrum of hiv-1 subtypes is under development. rapid progress and improvement in molecular technologies have allowed researchers to switch from the traditional approaches of virus detection in clinical samples to multiplexing for simultaneous detection of multiple pathogens in a single assay. a number of different pcr based assays for detection and discovery of multiple pathogens have been developed [1] [2] [3] [4] . detection microarrays are proven to be useful in the identification and discovery of viruses homologous to known species. they have been used to guide the selection of samples for further analysis by sequencing [1] [2] [3] 5] . however, microarrays based on nucleic acid hybridization are too complex in design and performance for the routine donors testing, and exhibit a comparatively low sensitivity of detection, usually around 10021,000 genome copies of target virus per analyzed sample [1] [2] . several pcr based assays coupled with oligonucleotide microarray technology (so called resequencing arrays) have been designed to allow simultaneous detection or genotyping of a target group of viruses, such as some critical blood-borne pathogens (3 viruses) [6] , respiratory viruses (16-21 viruses) [7] [8] , and respiratory adenoviruses (6 different serotypes) [9] . such pcr based approach allows increasing the sensitivity of detection down to 10-100 copies of the target rna or dna in a sample. pcr multiplexing should be highly useful when both the volume of the samples and the time of testing are critical, as in the donor eligibility (de) testing for tissue or organ transplantation [10] . current regulation requires that de testing be performed using assays approved and licensed by the u.s. food and drug administration (fda). however, the automated assay systems that are designed to screen large numbers of samples, without the strict limitation of sample volumes, may not be completely suitable or ideal for the needs of de testing for tissue or organ transplantation. the main goal of the study presented here was to evaluate the feasibility of developing a sensitive and specific assay for rapid detection and identification of a group of target viral pathogens. the following viral pathogens were included in our array: human immunodeficiency virus types 1 and 2 (hiv-1 and hiv-2), human t-cell leukemia virus-1 and -2 (htlv-1 and htlv-2), hepatitis c virus (hcv) and west nile virus (wnv), all with singlestranded rna genome; vaccinia virus (vacv) and hepatitis b virus (hbv), both with double-stranded dna genome, hbv also has single-stranded rna stage. some of the listed viruses are included to the required de testing for tissue transplantation. besides, historically, some of the targeted viruses have been found to be allograft-transmitted to recipients [11] [12] . in the present study, a real-time pcr array with sybr-green chemistry targeting these eight viral pathogens listed above was developed and evaluated with analytical and clinical panels. the array demonstrated acceptable performance in the testing with both analytical panels and donors' clinical samples. the research study conducted at fda using previously frozen blood samples was reviewed by department of health and human services, food and drug administration, research involving human subjects committee (rishc protocol #10-008b entitled ''detection of infectious agents in previously frozen blood samples from patients with various illnesses and healthy blood donors''). the 17 clinical plasma samples positive for hbv, hcv or hiv-1 used in this study were existing clinical diagnostic samples kept in nih blood bank. information of these left over samples had been recorded in such a manner that subjects can not be identified, directly or through identifiers. the written informed consent from the participants was waived under 45 cfr 46.101 (b) (4) . the six plasma samples positive for hiv-1 were estimated to contain 50 to ,90,000 genome copy numbers per ml; six plasma samples positive for hcv were estimated to contain 780 to ,123,000 genome copy number/ml and five plasma samples positive for hbv were estimated to contain ,150 to 16,000 copy number/ml. the copy numbers of viruses in these samples were provided by the nih blood bank. no information about the subtypes or genotypes of these viruses was available. the amount of each clinical sample was sufficient to be tested only once by the pcr array in the study. all positive pcr products obtained in the testing using the pcr array were confirmed for validity by sequencing in the facility for biotechnology resources of fda/ cber. we used the ''insignia'' program (http://insignia.cbcb.umd. edu/query.php), a bioinformatics on line tool developed in the center for bioinformatics and computational biology, university of maryland [13] to choose a specific dna or rna ''signature'' (a sequence, with customized length and g/c content) for targeted viruses. comparative sequence analysis of the complete genomes was performed using mvista (http://genome.lbl.gov/vista/ mvista/submit.shtml). multiple nucleotide sequence alignments (nsas) were then created to visualize the most conserved genome areas using mega4 (http://www.megasoftware.net). specific criteria for the primers and amplicon selection for the sybr green based pcr array were: 1) the same range of annealing temperature (t) -57-60uc -for all primers, 2) high g/c content for primers, allowing higher specificity of annealing, and 3) an amplicon size in the range of 100-200 b.p. in order to have a high pcr amplification efficiency and to sufficiently distinguish the products from primer dimers based on melting t peak (tm). all primers were checked for potential dimer formation using ''primer express'' software (version 3.0, applied biosystems). after design, all primers were again checked using the national center for biotechnology information (ncbi) basic local alignment search tool (blast) (http://blast.ncbi.nlm.nih.gov/blast.cgi) to avoid any cross-reactivity with other species. newly designed and previously published primer sets adapted for the final version of the real-time pcr array are listed in table 1 . in addition, primers specific for the human beta-globin gene were included in the array as an internal control for the quality of dna/rna preparation as well as for estimation of viral copy number per host cell if needed (the last row of table 1 ). total cellular dna of the chronically infected cell cultures (for hiv-1, hiv-2; htlv-1, htlv-2 and vacv), viral genome cdna copy spiked into human dna (for hcv and wnv), and dna isolated from human plasma of the infected individual (for hbv) were used as the positive templates in the initial testing and are listed in the last column of table s1 . dna or rna panels were created by cloning of specific synthetic templates for each virus into the pgem-t-easy vector (promega) by ta cloning, following by in vitro transcription to obtain rna standards for hcv and wnv. all created plasmids are listed in table s1 . nucleotide numbers in table s1 refer to the location of the partial viral genome cloned into pgem-t-easy vector according to the following complete genome sequences available in genbank: htlv-1 -l03562.2, htlv-2 -m10060.1, hiv-1 -k02083.1, hiv-2 -j03654.1, hbv -af462041.1, hcv -af271632.1, vacv -ay243312.1, wnv -hq596519.1. to establish the real-time pcr standard curve the copy number was calculated for each plasmid carrying one copy of the specific viral gene. the size of each plasmid (x b.p.) was used to determine the molecular weight in daltons (g/mol): w (g/mol) = x b.p. (330 da 6 2). the copy number of the target viral gene (molecules/ml) was determined from the plasmid concentration (c dna ) and the molecular weight of each plasmid molecule: copy number = c dna (ng/ml) 6 6.02 6 10 23 (avogadro's number)/w. knowing the number of plasmid molecules with the target viral gene in a ml, a series of dilutions was made to generate a pcr standard curve. the developed analytical standards were used to calculate the intra and inter-assay reproducibility of quantification for each virus-specific primer set. mean c(t) values, standard deviation (sd) and coefficient of variation (cv) were calculated from the data obtained in three replicates of each standard dilution for the intraassay reproducibility, and in three real-time pcr assays consisted of three replicates each (nine total) for the inter-assay reproducibility. cv was calculated as sd/mean c (t) * 100%. preparation of viral rna/dna for pcr array analysis 0.5-1 ml of human plasma was used for the total viral rna/ dna extraction using ''qiaamp viral rna mini-kit'' (qiagen) and trna (sigma) was used as a carrier rna during the preparation. after the final elution step rna/dna in 160 ml of buffer ave was precipitated with 100% ethanol and 3 mm nacl at 220uc overnight. an rna/dna pellet was washed with 70% ethanol, dissolved in 10 ml of depc-treated water and then immediately used for cdna synthesis with superscript ii rt (invitrogen) and random hexamers (invitrogen) in a total reaction volume of 20 ml. the volume of cdna/dna sample was then adjusted up to 30 ml with depc-treated water and the whole pcr was performed using ''bio-rad cfx96 real-time system'' with ''power sybr green pcr master mix'' (applied biosystems). one reaction (25 ml total) contained: 12.5 ml of pcr master mix, 0.5 mm of each primer and 1.25 ml of dna/cdna template. in the single virus testing (sections 3 and 4 of the ''results'') we used 2.5 ml of dna/cdna template from 20 ml of sample after cdna synthesis. ''universal'' pcr conditions for all primer sets included to the array were: 95uc for 8 min (one cycle), then 50 cycles of: denaturation at 95uc for 15 s, annealing and extension at 60uc for 1 min, followed by melting curve read from 65uc to 95uc with increment 0.2uc for 5 s. real-time pcr data were downloaded in 96-well plate format from ''bio-rad cfx manager 2.1'' to ms excel and analyzed manually. two types of samples served as the background control for determination of the c(t) cut off. the 1 st type of negative control was 50 ng of human cellular dna. the 2 nd type of negative control was negative donors' plasma. data were collected in separate experiments from 8 human cellular dna controls and 3 negative donors' plasma. to standardized the c(t) cut off for all primers the threshold was set at the pcr machine default setting. based on a false positive rate of less than 5% the following method [14] was used to estimate the c(t) cut off from the range of c(t) obtained with negative samples for all 24 primer sets in the array: 1. calculate the margin of error of the confidence interval (ci), w = t* 6 sd/!n, where: n -number of obtained c(t) values, sd-standard deviation, df = n21, and t* (for 95% confidence) is a ''critical value of the t distribution'' [14] . 2. one side ci covers this range: m-w, where m is sample mean. the c(t) cut off calculated from the range (n = 50) of the 1 st type of negative control data was c(t)#41.03. the c(t) cut off calculated from the range (n = 50) of the 2 nd type of negative control data was c(t)#42.7. even some overlap between the c(t) measurements of truly positive and truly negative samples was detected in pcr array data, the tm parameter was used to define if the obtained pcr product is specific by comparison to the expected tm peak range. the analytical sensitivity of each primer set was determined in the single virus testing using fda/cber panels (kindly provided by dr. stephen kerby, fda/cber) consisting of various amounts of the viruses (0-1,000 genome copies/ml) spiked into the ''normal'' human plasma. panels for the following viruses were used in testing: hiv-1 (three different panels), hiv-2, hbv (based on genotype a), hcv (genotype 1b), and wnv (based on strain hu2002). these panels have been used for testing of commercially licensed nat assays and their development was described previously [15] . three separate hiv-1 rna panels were used for testing. the first consisted of various amounts of an hiv-1 group m subtype b isolate: 0, 5, 10, 25, 50, 100, and 500 copies/ml. the second consisted of 25 samples representing various concentrations of hiv-1 groups o and n, and group m subtypes a, c, d, e, f and g: 10, 100 and 1,000 copies/ml for each virus. the third panel consisted of hiv-1 circulating recombinant forms (crfs) ae and ag: 100, 1,000, and 10,000 copies/ml for each crf. np24 caacttcatccacgtttcacc a -to simplify the process of evaluation we used our primer names with sequential numbers, however some of the primers have been designed previously with their original names in the articles listed in the last column of this [25] [26] . hiv-1 groups and subtypes are based on designations reported by the nih arrrp. low passage virus stocks were prepared and median tissue culture infective dose (tcid 50 )/ml of virus containing supernatant determined in fresh human pbmcs isolated as previously described [27] . infectious unit (iu) of the virus stocks in tcid 50 s were titrated for each isolate and the number of iu used for viral rna isolation and pcr was calculated based on the dilution factor (1:100) and ranged from ,10 to 2,500 or 1 to 3.4 log 10 tcid 50 per pcr reaction. pcr approach based on sybr green chemistry, allowing simultaneous detection of multiple targets, was chosen to be applied for the array performance. virus-specific primer sets targeting at least three different genomic sites for each viral pathogen were designed for the real-time pcr array. we used the ''insignia'' program, a bioinformatics tool that helps to choose a specific dna or rna ''signature'' for different bacteria and viral pathogens that are included in the pre-built ''insignia'' database [13] . sequences of the highly conserved regions, such as coding viral polymerase or structural proteins were selected to design the candidate primers. in addition, we performed nucleotide sequence alignments (nsa) of the complete viral genomes and of the most conserved genome areas for different subtypes/genotypes or different isolates of all targeted viruses. some previously published primers designed for pcr detection of the target viruses were also adapted and evaluated. overall, a total of 120 primers were initially designed using specific criteria for the current real-time pcr array (see materials and methods) to cover the eight targeted viruses: hiv-1, hiv-2, hbv, hcv, htlv-1, htlv -2, wnv, and vacv. the primers were designed and tested for their effectiveness and specificity of amplifying the respective target under uniform pcr conditions (using the same annealing temperature for all primers). each candidate primer pair was first tested for its specificity and sensitivity of pcr amplification. this was assessed in a real-time pcr cross-testing against human pbmc dna (50 ng/pcr reaction) from a healthy donor, dna from human cell cultures infected by various target viruses, or human dna spiked with a known amount of genome copies of various viruses. it was important to ensure that the selected primers targeting a specific virus would not non-specifically amplify any dna in a sample. the melting temperature (tm) peak of the product amplified from the target virus should be clearly differentiable from the tm peak of primer dimers or any non-specific products produced in pcr. dna or rna panels were created from the cloned synthetic templates (listed in table s1 ) by spiking of 2-10 4 genome copies of each virus into 50 ng of human background dna. the sensitivity of each candidate primer set for each target virus was assessed using these panels. example of the experimental testing of hiv-1 specific primer set targeting gag gene (np3/4) for its sensitivity with dna analytical standards is shown in figure s1 . serial dilutions of a plasmid dna corresponding to 5-10 4 hiv-1 genome copies spiked into 50 ng of normal human dna, hiv-1 infected cells (h9/iiib) (''positive'' control dna) and uninfected human pbmc (''negative'' control dna) are used in the experiment. only a single melting peak tm = 72.5uc corresponding to hiv-1 specific product was observed and no unspecific amplification was registered. figure s1b revealed a standard curve showing the correlation between copy number of the target gene and ct values with a slope = 23.77. the limit of sensitivity was determined in this assay to be 10 viral genome copies/pcr. after evaluation, a total of 24 primer pairs targeting the eight different viruses were chosen for the real-time pcr array based on their specificity and sensitivity (table 1) . among them, five of the primer sets were previously published and the other 19 primer sets were originally designed in the current study. table 1 shows the sequences of the previously published primer sets selected for the real-time pcr array with the reference to the original source. analytical sensitivity expressed in genome copy/pcr for each primer set (table 1 ) was estimated using dna/rna analytical panels, as described above. coverage of variants (i.e., different subtypes or genotypes) for each virus (table 1 ) was estimated using the nsas. degenerative nucleotides were introduced into some of the primer sets based on the nsas performed in-house to obtain a broader coverage. the tm peak range of the product stated in table 1 for each primer set was established during further testing of selected primers with analytical and clinical panels. in addition, the intra and inter-assay reproducibility of quantification for all primer sets was evaluated using three replicates of each standard dilution (of dna or rna analytical standards) in each of three real-time pcr assay runs. the coefficient of variation (cv) for the c (t) values was #3.3% and #6.7% for intra-and inter-assay, respectively. all the data depicting mean c (t), standard deviation (sd), and cv for each primer set selected for the real-time pcr array with each standard concentration are shown in table s2 . to further evaluate the sensitivity of the selected primers, we used fda/cber panels (kindly provided by dr. stephen kerby, fda), consisting of various amounts of viruses spiked into ''normal'' human plasma, that are specifically developed and used for the evaluation of commercially licensed nat assays. table 2 summarizes the testing results for the selected primers against the target viruses. one out of four primer sets targeting hiv-1, np3/4, could detect the subtype b hiv-1 rna at the concentration of 50 copies/ml of human plasma. two other hiv-1 specific primer sets (np51/52 and np170/171) detected the hiv-1 rna at 100 copies/ml of plasma. the 4th primer set, np175/174, (targeting the conserved pol region and containing degenerative nucleotides to support broader variant coverage) could detect the virus only at 500 viral genome copies/ml of plasma. hiv-2 rna was detected with primer set np86/87 at the concentration of 10 copies/ml and with the primer set np76/77 at the concentration of 50 copies/ml. another primer set (np84/ 85) detected hiv-2 at 100 copies/ml. hbv (genotype a) dna was detected with the primer set np11/97 at 50 copies/ml and with np94/100 at 100 copies/ml. the third hbv specific primer set (np11/97-mod) detected hbv at 500 copies/ml of plasma. both hcv specific primer sets targeting the viral 59ntr detected hcv rna at the concentration of 10 copies/ml of plasma. two of the wnv-specific primer sets (np21/22, targeting e protein gene, and np176/177, targeting ns5) detected wnv at 50 copies/ml and the third primer set (np178/179, also targeting ns5) gave a positive signal at 100 copies/ml of plasma. the four hiv-1 specific primer sets were further evaluated by testing them against another fda/cber analytical panel containing a broad spectrum of hiv-1 subtypes. as summarized in table 3 , the hiv-1 subtype f (group m) and group o isolates in table 2 . the results of sensitivity testing of the real-time pcr array primer sets specific for hiv-1, hiv-2, hbv, hcv, and wnv the with fda/cber analytical plasma panels. table 3 . sensitivity of four hiv-1 specific primer sets selected for the real-time pcr array in testing with fda/cber analytical hiv-1 broad spectrum panel. detection limit (copy number/ml of plasma) was evaluated using fda/cber analytical panel, containing pre-set copy number of hiv-1 spiked into 1 ml of ''normal'' human plasma. rna from 1 ml of plasma was converted to cdna and divided into eight pcr reactions; two pcr repeats were performed for each primer set. the estimated copy number per pcr reaction is shown in parentheses. the panel could not be successfully amplified by any of the four selected primer sets (detection limit is .1,000 rna copy/ml of plasma). all other subtypes and crfs of group m, and the group n isolate could be amplified with at least one out of the four primer sets at 50 to 1,000 rna copies/ml of plasma (table 3) . the tm peaks of the pcr products amplified from different subtypes of hiv-1 varied within 1.5uc ( table 3 ). all of the four selected hiv-1 specific primer sets amplified hiv-1 subtype b (group m) with the highest sensitivity (50-500 copies/ml of plasma). to examine the specificity and the ability of the array to detect different isolates within subtypes of hiv-1 and different isolates of hiv-2 by the array, we additionally tested our primers with another panel, developed by the southern research institute. this panel contained three different isolates from each subtype (a to g) of hiv-1 group m, three isolates from hiv-1 group o, one isolate from hiv-1 group n and three different isolates of hiv-2. the infectious dose of each hiv isolate in the panel was determined by tcid 50 (median tissue culture infective dose) titration and the dose of virus used in each pcr reaction was calculated. the results of testing of hiv-1 specific primers are shown in figure 1 . in this experiment we evaluated the coverage of hiv-1 variants and estimated the relative sensitivity of the four hiv-1 specific primer sets based on cycle threshold (c(t)) values obtained with each isolate tested. two primer sets targeting the conserved pol regions (np170/171 and np175/174) were able to detect most of the hiv-1 subtypes with a high sensitivity (c(t) = 15-25). only one primer set (np175/174) detected both the group n isolate and all three isolates of group o with ct = 20-35. we did not detect crossreactivity of hiv-1 or hiv-2 specific primers with the other type of hiv. all three hiv-2 isolates studied (1-3 log 10 tcid 50 /pcr input) were detected with all hiv-2 specific primer sets with a low ct value: 12-20 (data not shown). after completion of sensitivity testing of primer sets with analytical panels we finalized the expected tm peaks range for each amplicon and arranged a working array in 96-well plate format ( figure s2 ). to evaluate the specificity and possible crossreactivity of primers in the array, 20-100 genome copies of each virus were spiked into 50 ng of human dna to be used as positive templates and the same amount of human dna was used as a negative control in each experiment. the array was tested with all targeted viruses using dna standards (listed in table s1 ). each positive template was tested in duplicate; one human dna negative control and one no-template control (ntc) were included in every testing. in these experiments the definition of ''positive'' signal was set up as the threshold cycle cut-off of c(t)#41 (see materials and methods) and all tm peaks are within the expected range ( figure s2) . no cross-reactivity was detected for any primer sets in the array using dna standards with this setting (data not shown). seventeen (17) clinical plasma samples (obtained from nih blood bank) from donors who tested positive for hiv-1, hbv, or hcv were used to evaluate the array sensitivity and specificity. the genome copy of each virus in virus-positive plasma samples was previously determined by the nih blood bank using commercial assays approved for donor screening. representative results from the testing of three hbv-positive plasma samples (pt.#13-pt.#15) using our array are shown on figure 2 . the threshold cycle cut off of the assay performed with plasma samples was set up as c(t)#43 (see materials and methods). the wells with c(t) lower than the cut-off and the obtained tm peaks within the expected tm range indicate positive amplification of hbv target genes and are highlighted with blue circles (figure 2 , green color code for hbv). for samples #13 and #14 tm peaks of the products, obtained with only two hbv specific primer sets (wells g2, g3-pt.#13 and wells g5, g6-pt.#14), were within the expected range, with c(t) values of 36.7-42.9 and 36.5-36.6 respectively. for sample #15, all three hbv specific primer sets gave the tm peaks within the expected range (wells g7, g8 and g9), and the c(t) values range was 33.8-34.2. the genome copy number of hbv in plasma samples #13-15 was 151-518 copies/ ml, corresponding to 6-21 copies/pcr. the wells (white color code, red circles) with the human beta-globin gene specific primer set, serving as the internal control, produced positive signals from all three plasma samples tested, with c(t) values of 25.5-26.3 (wells h3, h6 and h9 figure 2 ), which shows that the quality of the rna/dna sample preparation was equally good for the plasma of these three patients. the final tm range for each primer set was adjusted after testing of this clinical panel. based on the results from the testing of clinical samples and according to nsa performed during the design, two primer sets targeting the s gene region of hbv (np11/97 and np11/97-mod) have two different tm ranges depending on the hbv genotype ( figure 2 -wells g1 and g3). the lower tm range (74.5-75.5uc) was detected for genotype a and the higher tm range (76.8-77.4uc) -for genotypes b, c, and d. the difference in tm ranges occurred due to single nucleotide polymorphism (snp) in the amplicon sequence leading to a difference in the g/c nucleotide content of the products. table 4 summarizes the results from testing the 17 virus-positive clinical samples using the developed array. at least two different specific primer sets could detect a target virus in all cases, with one exception: one hiv-1 positive sample (#4) with ,4 viral genome copies per pcr reaction was amplified with only one primer set (np175/174). hiv-1 positive clinical samples (#1-#3) with 51-80 viral genome copies/ml of plasma or 2-3 copies/pcr reaction were detected with at least two primer sets. all six hcv-positive samples (#7 to #12) with 16-2,570 viral genome copies/ml of plasma tested positive with both hcv-specific primer sets. three hbv-positive samples (#15, #16, #17) at 21-66 copies/pcr tested positive by the three hbv-specific primer sets and two hbv-positive samples (#13, #14) with 6-10 copies/pcr tested positive with two out of three hbv-specific primer sets. it is important to note that none of the primer sets showed any crossreactivity with other viruses in the panel of clinical samples tested. in the study presented here we applied a real-time pcr array approach in a 96-well plate platform for detection of a group of target viral pathogens. in contrast to taqman pcr, which is commonly used for viral diagnostics, this platform based on pcr with sybr green chemistry supports simultaneous detection and identification of 24 different targets corresponding to eight different viruses. pcr based on sybr green staining of the double stranded dna is economically affordable and allows for the detection of mutants with snps within the amplicon sequence [34] . the strategy of primer selection for the array included the sequential use of different bioinformatics programs to identify highly-specific primer sets with a maximal variants' coverage of the targeted viruses, while working under ''universal'' pcr conditions. experimental selection process using a panel of dna or rna analytical standards allowed choosing two to four most sensitive and specific primer sets for each targeted virus. the sensitivity (in copy number per pcr reaction) and the intra and inter-assay reproducibility of quantification were characterized for each primer set selected for the array. fda/cber analytical plasma panels were used to assess the detection sensitivity of the primer sets selected for the working array. the hiv-2 and hcv rna was detected at as low as 10 genome copies/ml of human plasma by one out of three and two out two primer sets, respectively. similarly, wnv rna and hbv dna were detected at 50 genome copies/ml of plasma by two out of three and one out of three selected primer sets respectively. all four of the selected hiv-1 specific primer sets detected group m, subtype b, the most common subtype of hiv-1 in the americas and western europe [35] , at 50-00 genome copies/ml of plasma. however, the array was able to amplify all other subtypes, excluding subtype f, of hiv-1 group m with only one primer set (np170/171), targeting the conserved pol region, at 100-1,000 genome copies/ml of plasma. in addition, the array amplified the group n isolate at 1,000 copies/ml with another primer set (np175/174) targeting the conserved pol region. all the selected hiv-1 specific primer sets of the array failed to amplify the group o isolate (detection limit is .1,000 copies/ml of plasma). in comparison, the limit of detection (lod) of recently improved commercial multiplex nat assays for the broad spectrum of hiv-1 group m isolates is 40 copies/ml, and the lod for group o is 200 copies/ml [36] [37] [38] . thus, specific primer sets targeting group o isolates of hiv-1, as well as subtype f (group m) will need to be included in the array to increase the coverage of all existing subtypes/groups of the hiv-1. no crossreactivity was shown for hiv-1 or hiv-2 specific primers with the other type of hiv in a testing with medically relevant levels of viruses in a diversity panel containing 25 different hiv-1 isolates and three natural hiv-2 isolates collected worldwide. there is presently no us food and drug administration (fda) approved pcr-based nat testing for blood donors' screening for htlv-1 and htlv-2. there is no official fda (or world health organization (who)) viral panel released for these two viruses. it is also difficult to obtain htlv-1 or htlv-2 positive blood donors' samples in the united states. in the absence of the analytical panels and clinical samples we tested htlv-1, htlv-2 and vacv specific primers only with cell culture derived dna and with dna standards. the minimum detection limit of htlv-1 and htlv-2 specific primer sets estimated with analytical dna standards was 5-10 genome copies/pcr reaction, which is in the same range as for other primers included to the array. the coverage of the viral variants for the primers targeting these viruses was estimated in silico using multiple sequence alignments performed with complete genome sequences available in gen bank. the working array arranged in 96-well plate format was subsequently tested for specificity and potential cross-reactivity with human dna and with each of the targeted viruses. none of the primer sets selected for a particular target virus in the array produced non-specific cross-reaction toward the other viruses. comparative performance of the array was also evaluated through the testing of 17 clinical specimens from the united states patient population. all 17 samples were correctly identified in our pcr array with a high sensitivity to contain hiv-1, hcv, or hbv. we found that a combination of several primer sets targeting each virus in the array allows for the detection of different variants of the virus; however, it makes the absolute quantification with uniform dna/rna standards a challenge. quantification by the assay is not always possible when the genetic group of the viral isolate being tested is different than the assay standards. this is one of the reasons why in certain cases the commercial assays underestimate viral loads by up to 1-10 log 10 copies per ml [37] [38] [39] . nevertheless, relative quantification can be done using all primer sets selected for the array, and the absolute quantification can be performed with dna/rna standards using the primers targeting the most conserved genome areas. there are several commercial qualitative multiplex nat assays now available on the market simultaneously targeting three most important blood-borne viral pathogens (hiv-1, hcv and hbv) [40] . one of them was recently approved by u.s. fda for screening of blood and organ donors (http://www.fda.gov/biologicsblood vaccines/bloodbloodproducts/approvedproducts/licensedproduc tsblas/blooddonorscreening/infectiousdisease/ucm306073.htm). we compared the lod of these multiplex nat assays [40] with the results of our working pcr array in sensitivity of testing against these important viruses. the lod for hbv are 38.1-195 geq/ml by ''procleix ultrio tigris'' and 9.2-37. other pcr-coupled techniques have been developed previously for highly-sensitive pathogens' detection that could reach the sensitivity of the assay up to 1 genome copy per pcr reaction. for example the bioactive amplification with probing (bap), utilizing a nested pcr and magnetic bead-based hybridization with the specific probe, has been developed for the detection of bovine and avian viruses [41] [42] [43] . in spite of the exceeding sensitivity of such assays targeting a single virus, it may be difficult to adapt the approach or method to meet the main objective of simultaneous detection of multiple target viral pathogens by an array using universal pcr conditions. it is important to note that this array was developed to be adapted by any laboratory. comparison of experimentally obtained tm peaks to the range of expected specific tm peaks allows rapid identification or exclusion of the viral pathogen in a sample. this is an initial study to examine the suitability of using pcr arrays for the detection of a group of target viruses. the current array was developed utilizing five previously published and table 4 . tm and c(t) values obtained with primer sets specific for hiv-1, hcv, or hbv in testing of 17 human clinical samples in the format of pcr array targeting eight different viruses. 19 originally designed primers sets. however, it can be expanded to a larger number of targets for the same virus. targeting of several genome areas increases the detection sensitivity of the target virus and provides an intra-assay confirmation of positive signals. additionally, any new virus of interest can be added to the list of targeted pathogens. efforts are underway to test the utility of this real-time pcr array using samples with more diverse biological origin and pathogen content. panmicrobial oligonucleotide array for diagnosis of infectious diseases testing and validation of high density resequencing microarray for broad range biothreat agents' detection crossspecies transmission of a novel adenovirus associated with a fulminant pneumonia outbreak in a new world monkey colony identification of pathogenic vibrio species by multilocus pcr-electrospray ionization mass spectrometry and its application to aquatic environments of the former soviet republic of georgia pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity an oligonucleotide microarray for multiplex real-time pcr identification of hiv-1, hbv, and hcv a low density oligonucleotide microarray for the detection of viral and atypical bacterial respiratory pathogens application of taqman low density arrays for simultaneous detection of multiple respiratory pathogens use of oligonucleotide microarrays for rapid detection and serotyping of acute respiratory disease-associated adenoviruses eligibility determination for donors of human cells, tissues, and cellular and tissue-based products; final rule and notice. 21 cfr part 1271 infections and human tissue transplants: review of fda medwatch reports reported infections after human tissue transplantation before and after new food and drug administration (fda) regulations, united states insignia: a dna signature search web server for diagnostic assay development intuitive biostatistics development and evaluation of hiv-1 subtype rna panels for the standardization of hiv-1 nat assays genetic variation of hiv type 1 in four world health organization-sponsored vaccine evaluation sites: generation of functional envelope (glycoprotein 160) clones representative of sequence subtypes a, b, c, and e. who network for hiv isolation and characterization human immunodeficiency virus type 1 t-cell tropism is determined by events prior to provirus formation dual infection of the central nervous system by aids viruses with distinct cellular tropisms biologic and genetic characterization of a panel of 60 human immunodeficiency virus type 1 isolates, representing clades a, b, c, d, crf01_ae, and crf02_ag, for the development and assessment of candidate vaccines subgroup g hiv type 1 isolates from nigeria variability of human immunodeficiency virus type 1 group o strains isolated from cameroonian patients living in france identification of a new human immunodeficiency virus type 1 distinct from group m and group o biological and molecular variability of human immunodeficiency virus type 2 isolates from the gambia genetically divergent strains of human immunodeficiency virus type 2 use multiple coreceptors for viral entry aids verursacht durch hiv-2 preliminary characterization of an hiv-2 isolate derived from a german virus carrier development of hexadecyloxypropyl tenofovir (cmx157) for treatment of infection caused by wild-type and nucleoside/nucleotide-resistant hiv dna amplification for direct detection of hiv-1 in dna of peripheral blood mononuclear cells quantitation of hepatitis b virus dna in plasma using a sensitive cost-effective ''in-house'' realtime pcr assay a genotypeindependent real-time pcr assay for quantification of hepatitis b virus dna rapid detection of west nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a taqman reverse transcriptase-pcr assay hpv dna detection and typing in cervical scrapes complete coding sequences of the rabbit pox virus genome sybr green-based real-time quantitative pcr assay for detection of west nile virus circumvents falsenegative results due to strain variability human immunodeficiency virus type 1 subtype distribution in the worldwide epidemic: pathogenetic and therapeutic implications evaluation of the roche cobas taqman and abbott realtime extraction-quantification systems for hiv-1 subtypes comparative rna quantification of hiv-1 group m and non-m with the roche cobas ampli prep/cobas taqman hiv-1 v2.0 and abbott real-time hiv-1 pcr assays a new real-time quantitative pcr for the diagnosis and monitoring of hiv-1 group o infection performance characteristics and comparison of abbott and artus real-time systems for hepatitis b virus dna quantification comparison of the analytical and operational performance of two viral nucleic acid test blood screening systems: procleix tigris and cobas s 201 development of a reliable assay protocol for identification of diseases (rapid)-bioactive amplification with probing (bap) for detection of bovine ephemeral fever virus development of a reliable assay protocol for identification of diseases (rapid)-bioactive amplification with probing for detection of avian reovirus development of a reliable assay protocol for identification of diseases (rapid)-bioactive amplification with probing (bap) for detection of newcastle disease virus key: cord-001251-forh7lw4 authors: jank, johanna m.; maier, esther m.; reiß, dunja d.; haslbeck, martin; kemter, kristina f.; truger, marietta s.; sommerhoff, christian p.; ferdinandusse, sacha; wanders, ronald j.; gersting, søren w.; muntau, ania c. title: the domain-specific and temperature-dependent protein misfolding phenotype of variant medium-chain acyl-coa dehydrogenase date: 2014-04-09 journal: plos one doi: 10.1371/journal.pone.0093852 sha: doc_id: 1251 cord_uid: forh7lw4 the implementation of expanded newborn screening programs reduced mortality and morbidity in medium-chain acyl-coa dehydrogenase deficiency (mcadd) caused by mutations in the acadm gene. however, the disease is still potentially fatal. missense induced mcadd is a protein misfolding disease with a molecular loss-of-function phenotype. here we established a comprehensive experimental setup to analyze the structural consequences of eight acadm missense mutations (p.ala52val, p.tyr67his, p.tyr158his, p.arg206cys, p.asp266gly, p.lys329glu, p.arg334lys, p.arg413ser) identified after newborn screening and linked the corresponding protein misfolding phenotype to the site of side-chain replacement with respect to the domain. with fever being the crucial risk factor for metabolic decompensation of patients with mcadd, special emphasis was put on the analysis of structural and functional derangements related to thermal stress. based on protein conformation, thermal stability and kinetic stability, the molecular phenotype in mcadd depends on the structural region that is affected by missense-induced conformational changes with the central β-domain being particularly prone to structural derangement and destabilization. since systematic classification of conformational derangements induced by acadm mutations may be a helpful tool in assessing the clinical risk of patients, we scored the misfolding phenotype of the variants in comparison to p.lys329glu (k304e), the classical severe mutation, and p.tyr67his (y42h), discussed to be mild. experiments assessing the impact of thermal stress revealed that mutations in the acadm gene lower the temperature threshold at which mcad loss-of-function occurs. consequently, increased temperature as it occurs during intercurrent infections, significantly increases the risk of further conformational derangement and loss of function of the mcad enzyme explaining the life-threatening clinical courses observed during fever episodes. early and aggressive antipyretic treatment thus may be life-saving in patients suffering from mcadd. medium-chain acyl-coa dehydrogenase deficiency (mcadd; mim #201450) is the most common fatty acid disorder arising from mutations in the acadm gene (mim #607008). in the absence of catabolic stress individuals with mcadd are mostly asymptomatic, but intercurrent illness or prolonged fasting may lead to episodes of coma with hypoketotic hypoglycemia and hepatic dysfunction [1, 2] . in the absence of newborn screening (nbs) for mcadd, premature death or serious disability occurs in 20% to 25% of children with the disorder [3] . if mcadd is identified by nbs, families are instructed to avoid prolonged fasting and to have their children hospitalized for glucose infusion during acute illness. these measures reduce the frequency of sudden death in mcadd, but unfortunately, some patients die in spite of early diagnosis and implementation of emergency procedures [4] . still occurring fatal courses may be due to the incomplete understanding of the underlying molecular phenotype linked to the mutations found in these patients. eighty percent of symptomatic patients diagnosed before the establishment of expanded nbs programs in europe and in the us are homozygous for the mutation p.lys329glu (c.985a.g), 18% carry this allelic variant in the heterozygous state [5, 6] with an allele frequency of 90% in this group. thus, this mutation is tightly linked to the risk of clinical disease manifestation. mcadd patients identified by expanded nbs show a considerably wider genetic heterogeneity with the p.lys329glu mutation present only on 47% to 63% of defective alleles [3, [7] [8] [9] [10] . p.tyr67his (c.199c.t) is the second most prevalent mutation identified in newborns with mcadd and is discussed to be less severe [7, [9] [10] [11] [12] . in addition, numerous novel mutations have been identified in the nbs cohort, leading to the current number of 81 different acadm mutations with 80% of these being missense or small insertions/deletions (www.hgmd.org). the clinical relevance of the majority of these mutations is so far unknown. notably, novel genotypes identified in nbs are associated with risk of sudden death [4] . clinical observation may support the hypothesis that the two most common mutations in the acadm gene causing the amino acid substitutions p.lys329glu, the predominant variant in symptomatic patients, and p.tyr67his, a biochemically mild variant solely found in presymptomatic infants in nbs, form the two ends of a phenotypic spectrum in mcadd. experimental evidence led to the current view that missense induced mcadd is a protein misfolding disease with a loss-offunction molecular phenotype [13] [14] [15] [16] . in spite of differences in severity both p.lys329glu and p.tyr67his caused protein misfolding [16, 17] . in a previous study, we provided first experimental evidence for the impact of eight additional acadm mutations identified in nbs on mcad structure and function and all of these were clearly associated with conformational derangement and decreased protein stability [16] . high rates of aggregation and degradation upon recombinant expression indicated a severe protein misfolding molecular phenotype for two further mutations from bavarian nbs, namely p.asp223gly and p.ile331thr, and precluded these variants from further experimental exploration [16] . the analysis of the structural consequences of side-chain replacements in the mcad protein may contribute to a better understanding of the mechanisms underlying mutation-induced protein misfolding and loss of enzyme function. mcad is a homotetrameric protein with each subunit consisting of three structural domains, the n-terminal a-domain (ad n ), the middle bdomain (bd), and the c-terminal a-domain (ad c ). within the family of fatty acid acyl-coa dehydrogenases the mcad enzyme shows a comparatively broad spectrum of substrate utilization [18] with the highest affinity for octanoyl-coa, and contains a fad moiety as catalytic redox factor. substrate and cofactor bind to neighboring but structurally distinct grooves. substrate binding is confined to the individual subunits, whereas parts of the cofactor molecules adopt an inter-subunit orientation. in an attempt to classify novel mutations found in nbs we established a comprehensive experimental setup which includes circular dichroism (cd) spectroscopy, differential scanning fluorimetry (dsf) monitoring 8-anilino-1-naphtalenesulfonic acid (ans) and flavin adenine dinucleotide (fad) fluorescence, right angle light scattering, and kinetics of thermal inactivation. this allowed for detailed analysis of the conformation of wild-type and variant mcad proteins focusing on a domain-related view of folding and mutation-induced unfolding events in the mcad protein. with fever being a distinctive clinical risk factor for patients with mcadd, special emphasis was put on structural and functional derangements related to thermal stress. moreover, we summarized and quantified the structural data and visualized the molecular protein misfolding phenotype of the variant mcad proteins in a 3d plot comparing them to the wild-type, to p.lys329glu, the classical severe mutation, and to p.tyr67his, discussed to be a mild mutation. in a previous study [16] we analyzed oligomerization, enzyme kinetics, and overall stability of the mcad wild-type protein and 10 variant mcad proteins derived from acadm missense mutations identified in bavarian nbs. in this study, we provide detailed conformational analysis of wild-type and eight variant mcad proteins, p.ala52val, p.tyr67his, p.tyr158his, p.arg206cys, p.asp266gly, p.lys329glu, p.arg334lys, and p.arg413ser that were amenable to recombinant expression and purification. mutations were grouped according to the localization of the respective amino acid side-chain replacement within the three structural domains of the mcad protein: i) n-terminal adomain (ad n ), p.ala52val (c.155c.t), p.tyr67his (c.199t.c), ii) middle b-domain (bd), p.tyr158his (c.472t.c), p.arg206cys (c.616c.t), p.asp266gly (c.797a.g), and iii) c-terminal adomain (ad c ), p.arg334lys (c.1001g.a), p.arg413ser (c.1237c.a). a color code linked to the individual protein domains (green, ad n ; blue, bd; red, ad c ) was used throughout. mutations are summarized in table 1 . genebank nm_000016.4 was used as cdna reference sequence. the recommendations of the nomenclature working group [19] were followed. nucleotide +1 is the a of the atgtranslation initiation codon. amino acid numbering starts with the translation initiator methionine as +1. additionally, the traditional nomenclature system of the mcad protein is shown in a separate column in table 1 . in this system, amino acid numbering starts at codon 26 with the first 25 amino acids forming a mitochondrial targeting peptide and being cleaved off to produce the mature mcad monomer [20] . the cdna of the human acadm gene (acadm-encoding pkk223 plasmid obtained as a gift from jerry vockley, pittsburgh, usa) was cloned into the prokaryotic expression vector pmalc2x (neb) encoding an n-terminal mbp (maltosebinding protein) and a factor xa cleavage site. acadm mutations were constructed by use of the quikchange site-directed mutagenesis kit (stratagene) and verified by dna sequencing. transient expression of wild-type and variant mcad in cos-7 cells cos-7 cells were maintained in rpmi 1640 medium with stable glutamine supplemented with 10% fetal bovine serum and 1% antibiotics (paa). for transient expression of wild-type and variant mcad proteins, wild-type and mutant acadm cdna was subcloned in pef-dest51. after transfection of 2 million cells with 2 mg dna using the amaxa electroporation system (lonza), cells were cultured for 48 h in basic medium. culture medium was changed every 24 h. cells were harvested by scraping and lysed by three freeze-thaw cycles in a lysis buffer (20 mm hepes ph 7.0, 200 mm nacl containing 1% triton x-100, 100 mg/ml digitonin and proteinase inhibitors), followed by 20 min centrifugation at 14,000 rpm at 4uc. supernatants were kept at -80uc until being used for the activity assay. the mcad enzyme activity was determined essentially as described elsewhere [21] using a reaction medium with the following composition: 200 mm tris-hcl, 0.2 mm phenylpropionyl-coa, 1 mm ferrocenium hexafluorophosphate, ph 8.0. final protein concentration in the assay was 0.02 mg protein/ml. separation of substrate and product was performed by uhplc-uv. expression and purification were essentially performed as previously described [16] . in brief, the mcad wild-type pmalc2x expression plasmid was used to transform the e. coli strain bl21-codonplus (stratagene), whereas the e. coli strain dh5a (invitrogen) was co-transformed with expression plasmids of variant human acadm and pgroesl [22] . bacteria transformed with wild-type mcad were grown in 2 l of dyt medium supplemented with 100 mg/ml ampicillin, while variant proteins were grown in the presence of 100 mg/ml ampicillin and 50 mg/ ml chloramphenicol. protein overexpression was induced with 0.01 mm isopropylthio-b-d-galactoside (iptg) at a post induction temperature of 28 uc for 20 hours. recombinant proteins were purified at 4 uc using ä ktapurifier and ä ktaprime systems (ge healthcare). after loading the crude extract on a mbptrap affinity chromatography column (ge healthcare), equilibrated with column buffer (20 mm tris-hcl ph 7.4, 200 mm nacl, 1 mm edta, 1 mm dtt), unbound proteins were washed out and mbp fusion proteins were eluted with the same buffer supplemented with 10 mm maltose, followed by sizeexclusion chromatography using a hiload 16/600 superdex 200 column (ge healthcare) equilibrated with size-exclusion buffer (20 mm hepes ph 7.0, 200 mm nacl). the isolated tetrameric fusion proteins were pooled and incubated with factor xa (novagen) for 12 hours at 4 uc to cleave off the mbp tag using a protease to protein ratio of 1:20 (u mg) followed by final rechromatography (hiload 16/600 superdex 200 column, ge healthcare) in size-exclusion buffer. the concentration of the isolated tetrameric fraction was determined using the fluorescent dye binding quant-it assay (invitrogen). all purification steps of wild-type mcad are shown in figure s1a and wild-type and variant mcad proteins were purified to homogeneity ( figure s1b ). far-uv cd spectra were recorded using a jasco j-715 spectropolarimeter (jasco, gross-umstadt, germany) equipped with a ptc 343 peltier element. experiments were performed in quartz cuvettes with 0.1 cm path length at a protein concentration of 0.15 mg/ml and far-uv spectra were recorded from 190 to 260 nm in 10 mm potassium phosphate, ph 8.0 at 20 uc. 16 spectra were accumulated for each protein analyzed and subsequently baseline corrected. the composition of secondary structure elements was calculated using the cd spectra deconvolution software cdnn [23] . thermal transition experiments were monitored at 208 nm with a heating rate of 20 uc per hour. data were normalized by setting the signal of the native protein to 1. fluorescence measurements with the wild-type and variant mcad enzymes (0.5 mg/ml) diluted in 20 mm hepes buffer, ph 7.0 and 200 mm nacl were performed on a cary eclipse fluorescence spectrophotometer equipped with a temperaturecontrolled peltier multicell holder (varian). intrinsic fad fluorescence and binding of the hydrophobic dye 8-anilino-1naphtalenesulfonic acid (ans, sigma-aldrich) to surface exposed hydrophobic groups was co-monitored (multiwavelength program) during heat-induced denaturation experiments in the temperature range from 25 uc to 65 uc at a heating rate of 1 uc/min. changes in ans fluorescence emission were detected at 450 nm (excitation at 395 nm, 5.0/10.0 nm slit widths), whereas intrinsic fad emission was monitored simultaneously at 530 nm (excitation at 450 nm, 5.0/10.0 nm slit widths). we ensured that no fluorescence resonance energy transfer occurred at the wavelengths used. transition midpoints (t m1/2 ) were determined graphically with t m1/2 being the temperature at which half denaturation occurred. significances between wild-type and variant proteins were calculated by one-way anova followed by a dunnett's post test (graphpad prism 5.0). right angle light scattering (rals) experiments were performed on a cary eclipse fluorescence spectrophotometer equipped with a temperature-controlled peltier multicell holder (varian). samples contained 0.08 mg/ml protein diluted in 20 mm hepes buffer, ph 7.0 and 200 mm nacl. the increase in turbidity was monitored in the temperature range from 25 uc to 65 uc at a heating rate of 1 uc/min (excitation at 330 nm, emission 335 nm; 5.0 nm slit widths). turbidity curves were illustrated with graphpad prism 5.0. thermal inactivation experiments were performed using a ferrocenium-based spectrophotometric assay [24] . mcad protein diluted in 100 mm hepes, ph 7.6 supplemented with 0.1 mm edta was incubated at five different temperatures in the range from 32 uc to 44 uc for the indicated time intervals. mcad enzyme activity was subsequently measured by addition of 10 mm octanoyl-coa. residual enzyme activities of n = 5 to n = 7 independent experiments were normalized to initial enzyme activity prior to incubation (25 uc) . the kinetic constants of inactivation k were calculated by plotting the residual activity against the measurement time. arrhenius plots result from plotting the logarithm of the five kinetic constants against the respective inverse temperature. activation energy (e a ) was determined from the slope of the arrhenius plot and is given as single value without measures of variation. to visualize results from experiments investigating thermal stability, kinetic stability, and conformation of mcad wild-type and variants, we combined data and developed relative scores assessing the severity of mutation-induced changes. the parameter thermal stability (y-axis) reflects the thermal unfolding behavior. values were based on the means of transition midpoints of thermal denaturation as determined by cd spectroscopy, ans-dsf or fad-dsf. the parameter kinetic stability (x-axis) represents the activation energy (e a ) as a measure of the threshold from the active to the non-active protein upon application of thermal stress. the parameter conformation (z-axis) arises from the results of cd spectroscopy, ans-fluorescence or intrinsic fad-fluorescence reflecting secondary structure, tertiary structure, and structural integrity with no stress applied, i. e. at room temperature. a numerical score was assigned to each of the experimental data with values ranging from 9 (native state) to 1 (non-native state). the mutations analyzed in this study affect side chains distributed over the entire mcad protein. side chains ala27 and tyr133 of the mature peptide lacking the 25 amino acid signal sequence are located at the core of the mcad monomer, lys304 and arg309 are positioned at the subunit interface inside the tetramer ( figure 1a ), whereas all other side chains (tyr42, arg181, asp241, arg388) are surface-exposed ( figure 1b and c). both substrate and cofactor are oriented towards the catalytic core, however, they differentially interact with the mcad protein ( figure 1d ). only the acyl-moiety of the substrate is buried in the substrate-binding cleft while the coenzyme-a moiety remains solvent-exposed allowing for rapid exchange within catalytic cycles. by contrast, the fad cofactor is integral to the mcad 3d structure and diffusion is impeded in the natively folded tetrameric state. residual activity of all variant mcad proteins expressed in cos-7 cells was determined in order to analyze loss of function in living cells. the variants p.ala52val, p.tyr67his, and p.arg334lys revealed high residual activity in the range from 45 to 91% as compared to the wild-type, whereas p.arg206cys, p.asp266gly, and p.arg413ser showed activities between 21 and 34%. only p.tyr158his displayed low activity of 5%. when compared to the specific enzyme activity of recombinantly expressed and purified mcad variants (maier et al 2009), p.arg206cys and p.lys329glu displayed a significant loss of protein function in the eukaryotic environment. note that p.arg413ser is a kinetic variant with a substrate affinity reduced by two orders of magnitude resulting in a catalytic efficiency (v max /k m ) of 1.7 compared to that of the wild-type of , 100. first, we analyzed missense mutation-induced alterations of variant mcad conformations with respect to secondary structure, hydrophobicity, and fad binding in comparison to the wild-type. the relative composition of secondary structure elements was monitored by cd scans recorded in far-uv light between 190 and 260 nm. the cd spectra showed typical secondary structure characteristics of proteins with predominating a-helical structures and a lower amount of b-sheets represented by minima at 208 nm and 222 nm, and a maximum at 193 nm, respectively ( figure 2a) . when compared to the wild-type, spectra of variant mcad proteins showed only slight differences. this was confirmed by quantitative analysis of secondary structure elements by means of cd spectra deconvolution ( table 2 ). for p.tyr67his and p.lys329glu, a decrease in the relative amount of a-helical structures resulted in a higher share of b-strands. although to a lesser extent, the two other mutations mapping to ad c (p.arg334lys, p.arg413ser) showed the same phenomenon, whereas mutations in bd did not induce marked changes in the secondary protein structure. global conformational changes were determined probing the accessibility of a fluorescent dye (ans) to hydrophobic groups within the protein. the two variants mapping to ad n , p.ala52val and p.tyr67his, showed similar ground-state fluorescence at ambient temperature as the wild-type ( figure 2b ). by contrast, all mutations affecting bd (p.tyr158his, p.arg206cys, and p.as-p266gly) displayed elevated ground-state fluorescence indicating partial unfolding at 25 uc. p.tyr158his showed the most pronounced changes with an increase by 15-fold, while the ground-state fluorescence of p.arg206cys and p.asp266gly was increased by 2-and 4-fold, respectively. two of the three mutations mapping to ad c , p.lys329glu and p.arg413ser, produced a 2-fold elevation of the ground-state fluorescence, whereas hydrophobicity of p.arg334lys was similar to that of the wild-type. intrinsic fluorescence of the fad cofactor allows for tracking structural integrity of the mcad protein as fad is integral to the natively folded tetrameric state. to investigate whether mutations alter fad binding capacity, we analyzed fad fluorescence of mcad variants at 530 nm after 15 min incubation at room temperature. thermal denaturation assays demonstrated that fad is released from the protein in the course of the unfolding process ( figure s2 ). all mutations induced elevated ground-state fad fluorescence signals as compared to wild-type mcad indicating partial loss of fad binding capacity due to alteration of the native state conformation ( figure 2c ). the variant p.tyr158his showed the most pronounced alterations with an increase in fad fluorescence by 3-fold. three mutations, each mapping to one of the three protein domains, were associated with an increase in intensity by 2-fold (p.tyr67his, p.arg206cys, p.arg413ser) while the impact of the remaining mutations including p.lys329glu was less pronounced. taken together, all mutations with the exception of p.ala52val showed conformational alterations with individual patterns of table 1 ). side chains, fad cofactor (yellow) and the substrate analogue 3-thiaoctanoyl-coa (orange) are shown as stick models. (b, c) structural localization of affected side-chains at the surface of subunit a (gray) relative to the other subunits (b, green; c pink; d, blue) in the mcad tetramer. (d) surface view of the mcad tetramer with 3-thiaoctanoyl-coa (orange) bound to the substrate binding site and the positioning of fad (yellow) relative to subunits a (gray), b (green), and d (blue). (e) relative enzyme activity of wild-type and variant mcad proteins. specific activity was determined using purified recombinant protein [16] and residual activity was measured upon expression in cos-7 cells. data are given as means and sd of three replicates. doi:10.1371/journal.pone.0093852.g001 table 2 . secondary structure analysis (cd spectroscopy). derangement as to different structural aspects. p.tyr67his and p.lys329glu had a particular impact on the secondary protein structure, partial unfolding probing global hydrophobicity was most pronounced for mutations mapping to bd, and variants of all domains showed disturbed fad binding. mutations mapping to bd are associated with accelerated heat-induced unfolding among protein misfolding diseases with a loss-of-function molecular phenotype, the mcadd clinical phenotype has proven to be particularly vulnerable to fever [15, 25, 26] . in order to get structural insight into this disease mechanism, we investigated heat-induced unfolding of variant mcad proteins by means of cd-spectroscopy, as well as ans-dsf and fad-dsf. we monitored denaturation of a-helical secondary structures by means of cd at 208 nm in order to determine transition midpoints between folded and unfolded states (t m1/2 ) ( figure 3a , table 3 ). in comparison to wild-type mcad (t m1/2 56.5 uc), p.tyr158his was by far the most instable variant with a marked shift of the curve to lower temperatures and a t m1/2 deviation of 12 uc (t m1/2 44.5 uc). p.arg206cys also displayed markedly reduced stability with a decrease in t m1/2 by 6.5 uc. all other variants exhibited a reduced stability of the secondary structure, though to a lesser degree with t m1/2 -deviations of 2.5 to 4 uc. in this group, p.arg413ser and p.ala52val showed the most pronounced shift (t m1/2 52.0 uc), the variants p.tyr67his, p.arg334lys, and p.asp266gly were in the medium range, whereas p.lys329glu was the most stable variant (t m1/2 54.0 uc). to analyze heat-induced loss of native-state conformation, we monitored fad fluorescence emission at increasing temperatures. the mcad wild-type protein showed a t m1/2 of 58.3 uc denoting the midpoint of transition from the native cofactorbound state to the unfolded state with complete cofactor release (table 3, figure s2 ). none of the variants showed marked changes in the shape of the denaturation curves. however, most curves were shifted to lower temperatures indicating early fad release upon thermal stress ( figure 3b) . surprisingly, the variant p.lys329glu was associated with a shift of the fad fluorescence curve to higher release temperatures (t m1/2 59.0 uc). for p.arg334lys we observed a curve virtually mapping to that of wild-type mcad and p.asp266gly, p.arg413ser, and p.tyr67-his displayed only moderately left-shifted curves. three variants (p.ala52val, p.arg206cys, p.tyr158his) mapping to ad n and bd were associated with structural rearrangements at the fad binding site leading to fad release at lower temperatures. again, the variant p.tyr158his showed the most pronounced alteration with a dt m1/2 of 12 uc (table 3) correlating well with the shift in t m1/2 observed in cd-spectroscopy experiments. interestingly, the amino acid residue affected by this mutation is an essential part of the active site directly interacting with the fad cofactor [27] . ans-dsf is an excellent means to monitor global thermal unfolding events [16, [28] [29] [30] and transition midpoints for mutations analyzed in this study have been determined before [16] . however, application of varying scan rates as a function of temperature in our previous work did not allow for fine-mapping of unfolding profiles. to enable this, we performed ans-dsf experiments at a fixed scan rate of 1 uc/min. under these conditions, the values for t m1/2 were well comparable to those obtained by cd-spectroscopy ( table 3) . analysis of the pattern of thermal denaturation curves revealed a subtle biphasic transition with a different slope in the low temperature range between 45 uc and 53 uc for the wild-type (t m1/2 56.2 uc) ( figure 3c ). biphasic unfolding transitions have previously been reported for the mcad protein [14] and other disease-related multi-domain proteins and different transition phases were associated with unfolding of distinct structural domains [31] [32] [33] . variants with an amino acid side-chain substitution in ad n displayed straight curves with a minor shift to lower temperatures indicating moderately decreased stability of the enzyme equally affecting the conformation of all domains. unfolding patterns of bd variants were severely altered. the p.tyr158his variant revealed a marked shift in transition and a steeper slope than all other mutations, reflecting the most pronounced destabilization of the protein (t m1/2 44.9 uc). accordingly, t m1/2 was changed to a similar extent as observed by the above methods. p.arg206cys and p.asp266gly disclosed a marked biphasic transition with a left-shift of the whole curve and an even more pronounced shift of the low-temperature transition. thus, mutations mapping to bd particularly affected the low-temperature unfolding transition. these observations may indicate that unfolding of bd correlates with the low-temperature transition. mutations mapping to ad c , in particular p.lys329glu and p.arg334lys, showed unfolding curves very similar to that of the wild-type protein indicating good thermal stability. the p.arg413ser variant displayed a reduced mean unfolding transition (t m1/2 51.9 uc) and a clear shift of the low-temperature transition but this was less pronounced than for variants p.arg206cys and p.asp266gly that also showed biphasic transitions. in conclusion, considering the localization of side chain replacements in different domains, mutations mapping to bd displayed the most pronounced alterations affecting all parameters tested. in addition to bd variants, p.ala52val of ad n was associated with early loss of fad binding and the ad c variant p.arg413ser displayed a marked left-shift of the low-temperature range unfolding transition. to investigate whether thermal stress leads to accelerated protein aggregation of mcad variants we carried out rals experiments probing the formation of insoluble aggregates. based on hydrophobic self-association of the purified proteins, the intensity of the scattered light at 335 nm was detected in right angle to the excitation light of 330 nm and plotted as a function of temperature applied to the sample (figure 4 ). mutations mapping to ad n induced a slight shift of the onset of aggregation to lower temperatures resulting in curves indicative for a similar aggregation behavior as the wild-type protein. also in this set of experiments mcad variants of bd displayed the most distinct alterations. aggregation curves for all three variants (p.tyr158his, p.arg206cys, p.asp266gly) were markedly shifted indicating an earlier onset of aggregation than observed for the wild-type and all other variants analyzed here. for p.tyr158his we observed the most pronounced left-shift with the steepest slope. the pattern of aggregation for mutations mapping to ad c was moderately leftshifted and comparable to that observed in ad n . in summary, missense mutations in the acadm gene mapping to ad n or ad c only showed minor acceleration in the formation of insoluble aggregates upon thermal stress, whereas variants of bd were distinctly aggregation prone and induced aggregation already at lower temperatures. following the analysis of the impact of increasing temperatures on protein structure we investigated loss of mcad enzyme function upon thermal stress. thermal inactivation of recombinant mcad wild-type and variants was determined over time at temperatures between 32 uc and 44 uc ( figure 5a ). velocity constants (k) were calculated from the resulting thermal inactivation profiles and plots of the logarithm of the kinetic constants (lnk) against the inverse temperature revealed a linear arrhenius relationship ( figure 5b ). differences in the slope of arrhenius plots reflect distinct activation energies (e a ) for conversion of mcad variants from an active conformational state into an inactivated and potentially non-native state ( figure 5c ). p.ala52val and p.tyr67-his of ad n as well as p.arg334lys of ad c showed e a at wild-type level. all other variants mapping to bd and ad c displayed reduced kinetic stability. the lowest e a of all variants was observed for p.tyr158his. in summary, kinetics of thermal inactivation allowed to gain insight into unfolding dynamics at the active center of the mcad protein. variants located in ad n were completely unaffected, possibly due to the remote position to the active site. mutations mapping to bd and ad c induced decreased kinetic stability as to both global and local unfolding processes. notably, a side chain replacement remote to the active site structure, p.arg334lys, did not alter dynamics of heat-induced unfolding. to provide an integrated view of the structural consequences of acadm mutations we classified mcad variants based on conformation, kinetic stability, and thermal stability and visualized the results as a 3d plot ( figure 6 ). all mutations analyzed here induced reduced thermal stability when compared to the wild-type. the deleterious structural effects of the p.tyr158his missense mutation demonstrated by the several experimental approaches applied in this study resulted in opposite positioning of this variant to wild-type in the 3d plot. the other two variants located in the bd, namely p.arg206cys and p.asp266gly, together with p.lys329glu and p.arg413ser of ad c , built a cluster of relative instability with respect to nativestate conformation and thermodynamic stability. the variants p.ala52val and p.arg334lys were assigned an intermediate position between p.tyr67his and wild-type with respect to native-state conformation. p.ala52val showed a slightly reduced thermal stability and both variants presented with equal or higher thermal stability as compared to p.tyr67his. thus, p.lys329glu, the classical severe mutation, and p.tyr67his, discussed to be a particularly mild mutation, did not constitute the edges of the plot. to summarize, mutations mapping to bd showed the most severe structural alterations with some of the variants displaying more unfavorable structural properties than p.lys329glu and others being even less affected than p.tyr67his. the introduction of expanded nbs programs including early detection of mcadd raised uncertainties about the clinical relevance of novel acadm mutations identified in individuals showing biochemical abnormalities in the first days of life. before the screening era p.lys329glu was by far the most prevalent mutation, preferentially identified in symptomatic patients presenting with a metabolic crisis [34, 35] . later, p.tyr67his was identified as the second most prevalent mutation. to date, it has only been detected in babies identified by nbs and there are no reports of patients presenting clinically with this mutation [7, 11, 15] . on the basis of mcad enzyme activity determinations, one study came to the conclusion that this variant is of no clinical relevance [36] . the assessment of pathogenicity of mutations identified after positive nbs is of major clinical relevance since the disclosure of a genetic disease is associated with a high burden for the patients and their families [37, 38] . however, data describing the consequences of these mutations at the molecular level and the availability of experimental tools for this assessment, are scarce. at the clinical level, the natural course of the disease in individuals carrying these novel mutations is unlikely to be unraveled as detected asymptomatic babies are protected from metabolic decompensation by the implementation of emergency protocols. at the biochemical level, markers such as blood acylcarnitine concentrations do not correlate well with the risk of metabolic decompensation [39, 40] . a recent study established risk stratification based on residual mcad enzyme activity obtained from patient blood cells and showed that an enzyme activity of less than 1% is associated with an increased risk of fatal neonatal presentation and enzyme activities below 10% enhance the risk of hypoglycemia [41] . however, reported enzyme activities linked to single mutations vary over a wide range and can differ substantially when residual enzyme activity is analyzed in patient cells or after overexpression of variant proteins [7, 16, 36, 42, 43] . furthermore, in all cohorts evaluating the pathogenicity of acadm genotypes [11, 36, 42] mutations identified in nbs cohorts are mostly observed in compound heterozygosity with p.lys329-glu or p.tyr67his. only few mcad variants (5% in [41] and 20% in [11] ) were found in other combinations, and even less in the homozygous state. prediction tools or 3d-structural analyses are often used to estimate the pathogenic impact of acadm mutations. however, this approach often is not straight-forward and may lead to misassumptions. based on visual analysis of side-chain replacements in the mcad 3d structure, one would judge mutations p.tyr67his, p.arg206cys, p.asp266gly, and p.arg413ser, all affecting protein surface regions, as not damaging. in the case of p.arg413ser (mapping to arg388 in the mature protein) the interaction with the substrate, however, would suggest a loss-offunction phenotype. at the experimental level, we showed that p.arg413ser, p.asp266gly (asp241), and p.arg206cys (arg181) displayed a severe structural phenotype, whereas only tyr67his (tyr42) showed a mild structural phenotype in accordance with the mild clinical phenotype associated to this mutation. the polyphen-2 polymorphism phenotyping tool (www.genetics.bwh. harvard.edu/pph2/) predicts the possible impact of an amino acid substitution on structure and function of a human protein. a query for p.tyr67his resulted in a score of 0.077 and the side chain replacement was classified as benign. mutations p.arg206cys and p.arg413ser were scored 0.999 and classified as probably damaging. however, a score of 0.000 for p.asp266gly suggested a benign structural phenotype. this in silico prediction did not match our observation of a severe structural phenotype for p.asp266gly resembling that of p.arg206cys and p.arg413ser. thus, elucidating the molecular consequences of mutations on variant proteins in model systems, which generate data specific to individual mcad variants, is considered to be a useful tool to gain insight into their pathogenicity. we previously provided first evidence that acadm mutations identified in newborn screening induce considerable structural alterations [16] . in the work presented here we significantly expanded the experimental program focusing on the effect of thermal stress on conformational integrity as well as on enzymatic function. in a domain-related view, we tracked mutation-induced structural derangements with respect to ad n , bd, and ad c . a 3d plot summarizing and quantifying the structural phenotype of the variants in comparison to the wild-type on one hand and to the two benchmark mutations p.tyr67his and p.lys329glu on the other, allowed for visualization of the severity of conformational derangement induced by acadm mutations identified by newborn screening. notably, the mild p.tyr67his and the severe p.lys329glu did not constitute the phenotypic extremes of the panel of mutations analyzed. the classification applied here rather resulted in two groups of variant mcad proteins. the group associated with mild structural deteriorations comprised the wildtype protein and p.tyr67his as well as p.ala52val and p.arg334lys. the other group presenting moderate to severe structural deteriorations comprised p.lys329glu as well as p.arg206cys, p.asp266gly, p.arg413ser and at the extreme, p.tyr158his. interestingly, mutations identified in nbs either induced structural alterations that were less severe than those observed for p.tyr67his or more severe than those seen in the presence of p.lys329glu. moreover, mutations p.tyr67his and p.lys329glu displayed similar behavior with respect to various parameters of protein conformation and stability. for both variants only moderate changes were observed with respect to conformation and thermal unfolding or aggregation. furthermore, both variants displayed a similar shift in the relative distribution of secondary structure elements towards b-strands and b-turns. however, p.tyr67his and p.lys329glu were clearly separated by kinetic stability. this parameter was the best discriminator for both groups. by contrast, thermal stability as determined by dsf assays did not discriminate these two groups. based on the data from the present study, all mutations mapping to bd can be categorized as prone to misfolding, unfolding, and functional inactivation. in addition, two out of three mutations affecting residues in ad c can be assigned to the group of severely affected variant mcad proteins. variants arising from mutations located in bd displayed more severe structural alterations in all aspects as compared to p.lys329glu located in ad c . the mutation p.tyr158his mapping to the core of the mcad monomer induced the most profound alterations. mutations p.arg206cys, p.asp266gly, and p.arg413ser showed molecular phenotypes comparable to those induced by p.lys329-glu and may therefore be classified as just as severe. the latter group of mutations displayed substantial reduction in residual enzyme activity when compared to the respective specific activity, a fact that may underscore the impact of structural derangement on protein function in vivo. in the mild group, p.ala52val induced alterations comparable to p.tyr67his and the structural phenotype of p.arg334lys was almost unaffected. it seems tempting to assume that the mutations assigned to the mild group or mutations mapping to ad n in general bear a lower risk of metabolic decompensation. nevertheless, eight mutations mapping to this domain (c.145c.g, q24e; c.157c.t, r28c; c.233t.c, p.i78t; c.250c.t, p.l84f; c.253g.t, p.gly85cys; c.320t.c, p.leu107ser; c.346t.g, p.cys116gly; c.443g.a, p.arg148lys) have previously been associated with intermediate to severe clinical phenotypes including sudden death [7, 11, 44] . moreover, the mild structural effects determined for p.ala52val (ala27 in the mature protein) are in apparent conflict with the loss in enzymatic function observed. the side chain 27 is not in close proximity to the active site but ala27 maps to the conjunction of two a-helices and its replacement by valine is likely to displace the far n-termini that exhibit mutual side chain interactions between subunits across the dimer interface. in line with our observation that p.ala52val displayed the third lowest melting point of fad-dsf, proper positioning of n-termini may be of fundamental importance for mcad structural integrity and consequently for mcad function. notably, the classification of the two groups with respect to kinetic stability, i. e. thermal stress-induced loss of enzymatic function, correlated with residual activity determined in eukaryotic cells. residual enzyme activity ranged from 45% to 91% in the mildly affected group and from 5% to 34% in the group of severely affected mcad variants. in conclusion, classification of mutations constituting the mild group either as pathogenic or as clinically non-relevant that would exclude patients from clinical follow-up and emergency procedures is still challenging. however, data derived from structural and functional analyses of variant mcad proteins support the notion that mutations identified in nbs induce significant alterations and may thus be of clinical relevance. in particular, mutations constituting the severe group, p.tyr158his, p.arg206cys, p.asp266gly, and p.arg413ser may be associated with a significant clinical risk comparable to that associated with the classic acadm mutation p.lys329glu. in the absence of adequate biochemical or clinical criteria, the degree of conformational alterations could be a valuable tool to estimate the risk of metabolic decompensation. this may hold particularly true, when data from protein structure analysis are considered in conjunction with functional parameters. on the structural level, the molecular phenotype resulting from acadm mutations identified in nbs presented here depends on the intramolecular site of structural alterations with the central b-domain being particularly prone to conformational derangement and destabilization. on the functional level it is of critical importance whether and to what extent thermal stress affects the function of the mcad enzyme. the clinical observation of metabolic decompensation being considerably aggravated with fever has led to the assumption that the patient's temperature has impact on the molecular phenotype [4, 7, 10, 14, 39, 41, 45] . we showed here that mutations in the acadm gene lower the temperature threshold at which mcad loss-of-function occurs. consequently, increased temperature as it happens during intercurrent infections, significantly enhances the risk of further conformational derangement and loss of function of the mcad enzyme explaining the life-threatening clinical courses observed during fever episodes in patients suffering from mcadd. two possible strategies may help to address this issue. first, at short notice, the necessity of early and aggressive antipyretic treatment to avoid further loss in mcad enzyme activity should be emphasized towards caregivers. secondly, in the medium term, the development of pharmacological chaperones that bind to the variant mcad protein and induce its conformational stabilization and functional rescue would provide a specific treatment strategy protecting mcadd patients from metabolic decompensation, morbidity, and early death. this view is strongly supported by positive experiences with two approved drugs acting as pharmacological chaperones in genetic diseases, sapropterin dihydrochloride for phenylketonuria [46, 47] and tafamidis for familial amyloid polyneuropathy [48] . morbidity and mortality in medium chain acyl coenzyme a dehydrogenase deficiency medium-chain acyl-coenzyme a dehydrogenase deficiency: clinical course in 120 affected children the epidemiology of medium chain acyl-coa dehydrogenase deficiency: an update sudden death in medium chain acyl-coenzyme a dehydrogenase deficiency (mcadd) despite newborn screening molecular basis of medium chain acyl-coenzyme a dehydrogenase deficiency. an a to g transition at position 985 that causes a lysine-304 to glutamate substitution in the mature protein is the single prevalent mutation prevalent mutations in fatty acid oxidation disorders: diagnostic considerations medium-chain acyl-coa dehydrogenase (mcad) mutations identified 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know, what we do not know, and how we will know it medicine. newborn screening: gaps in the evidence mediumchain acyl-coa dehydrogenase deficiency: genotype-biochemical phenotype correlations lack of genotype-phenotype correlations and outcome in mcad deficiency diagnosed by newborn screening in new york state risk stratification by residual enzyme activity after newborn screening for mediumchain acyl-coa dehyrogenase deficiency: data from a cohort study in vitro and in vivo consequences of variant medium-chain acyl-coa dehydrogenase genotypes a novel tandem mass spectrometry method for rapid confirmation of mediumand very long-chain acyl-coa dehydrogenase deficiency in newborns a novel mutation of the acadm gene (c.145c.g) associated with the common c.985a.g mutation on the other acadm allele causes mild mcad deficiency: a case report vulnerability to oxidative stress in vitro in pathophysiology of mitochondrial short-chain acyl-coa dehydrogenase deficiency: response to antioxidants tetrahydrobiopterin as an alternative treatment for mild phenylketonuria efficacy of sapropterin dihydrochloride in increasing phenylalanine tolerance in children with phenylketonuria: a phase iii, randomized, double-blind, placebocontrolled study tafamidis, a potent and selective transthyretin kinetic stabilizer that inhibits the amyloid cascade we are grateful to our patients and their families. we would like to thank anja schultze, heike preisler, anna heckel-pompey, jos ruiter, mirjam doolaard and petra mooyer for excellent technical assistance. we thank jerry vockley for providing the acadm-encoding plasmid. key: cord-002602-2qvyhjlp authors: roy, amrita; lim, liangzhong; srivastava, shagun; lu, yimei; song, jianxing title: solution conformations of zika ns2b-ns3pro and its inhibition by natural products from edible plants date: 2017-07-10 journal: plos one doi: 10.1371/journal.pone.0180632 sha: doc_id: 2602 cord_uid: 2qvyhjlp the recent zika viral (zikv) epidemic has been associated with severe neurological pathologies such as neonatal microcephaly and guillain-barre syndrome but unfortunately no vaccine or medication is effectively available yet. zika ns2b-ns3pro is essential for the proteolysis of the viral polyprotein and thereby viral replication. thus ns2b-ns3pro represents an attractive target for anti-zika drug discovery/design. here, we have characterized the solution conformations and catalytic parameters of both linked and unlinked zika ns2b-ns3pro complexes and found that the unlinked complex manifested well-dispersed nmr spectra. subsequently with selective isotope-labeling using nmr spectroscopy, we demonstrated that c-terminal residues (r73-k100) of ns2b is highly disordered without any stable tertiary and secondary structures in the zika ns2b-ns3pro complex in the free state. upon binding to the well-characterized serine protease inhibitor, bovine pancreatic trypsin inhibitor (bpti), only the extreme c-terminal residues (l86-k100) remain disordered. additionally, we have identified five flavonoids and one natural phenol rich in edible plants including fruits and vegetables, which inhibit zika ns2b-ns3pro in a non-competitive mode, with ki ranging from 770 nm for myricetin to 34.02 μm for apigenin. molecular docking showed that they all bind to a pocket on the back of the active site and their structure-activity relationship was elucidated. our study provides valuable insights into the solution conformation of zika ns2b-ns3pro and further deciphers its susceptibility towards allosteric inhibition by natural products. as these natural product inhibitors fundamentally differ from the currently-known active site inhibitors in terms of both inhibitory mode and chemical scaffold, our finding might open a new avenue for development of better allosteric inhibitors to fight zikv infection. zika virus (zikv) was a neglected, mosquito-borne flavivirus because of its assumed small geographical spread and mild clinical symptoms [1] such as fever, headache, rashes and etc [2] . the first biological zikv sample was isolated from a sentinel rhesus monkey in the zika plos one | https://doi.org/10.1371/journal.pone.0180632 july 10, 2017 1 / 22 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 forest of uganda in 1947 [3] , and it was later found zikv is transmitted to humans by aedes mosquitoes. however since 2007, large epidemics of asian genotype zikv have been reported around the world [4, 5] . recently it is estimated that one-third of the world population might be at risk of infection [6] . the rapid rise in zika infection is compounded by the ease of vertical [7] and sexual human-to-human transmissions [8] . recent studies have associated zikv infection with other diseases: guillain-barré syndrome and microcephaly in newborn infants of mothers infected with zikv during pregnancy [7, [9] [10] [11] , thrombocytopenia [12] , multipleorgan failures [13] , and possibly male infertility [14] . consequently who has declared a public health emergency for zikv infection [15] . zikv represents a significant challenge to the public health of the whole world but unfortunately there is no available effective vaccine or therapy so far. zikv has a single-stranded positive sense rna genome of 10.7 kb, belongs to the flavivirus genus which also contains viruses causing dengue, yellow fever, west nile, japanese encephalitis and tick-borne encephalitis [6, 16] . zikv shares a high degree of sequence and structural homology with other flaviviruses particularly dengue virus, thus resulting in immunological cross-reactivity [17] . current zika outbreaks are largely localized within dengue-endemic areas, it is thus proposed that preexisting dengue-induced antibodies may enhance zika infection by antibody-dependent enhancement (ade), a factor that makes the vaccine approaches extremely challenging [17] . while several recent studies focused on the possibility of developing neutralizing antibodies against zikv [18] [19] [20] , it may take quite a while before such neutralizing antibodies for zikv enter clinical trials. zikv genome is translated into a single~3,500-residue polyprotein, which is further cleaved into 3 structural proteins and 7 non-structural proteins [16] . the correct processing of the polyprotein is essential for replication of all flaviviruses, which requires both host proteases and a viral ns2b-ns3 protease (ns2b-ns3pro) and this property makes the flaviviral ns2b-ns3pro a well-established target for developing antiviral drugs [16, [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] . while there are studies testing the inhibition effects of certain drugs on ns2b-ns3pro such as bromocriptine [33] , boronate [34] , or small compounds inhibitors [35] , we are interested in new chemical scaffolds that are found in the edible foods. our focus here is on whether natural edible products contain small molecules that have some inhibitory effects on the enzymatic activities of zikv ns2b-ns3pro. thus in the present study, we have characterized the solution conformations and catalysis of two enzymatically active zika ns2b-ns3pro: one with ns2b and ns3pro linked by extensively used (gly) 4 -ser-(gly) 4 sequence; and another with ns2b and ns3pro unlinked. subsequently, we identified several small molecular inhibitors from edible plants: five flavonoids and one natural phenol which were shown to inhibit zika ns2b-ns3pro in a non-competitive mode. further molecular docking suggests that these molecules inhibit zika ns2b-ns3pro by binding to a pocket on the back of the active site and allosterically affect the structure-activity property of zika ns2b-ns3pro. as such, identification of these natural product inhibitors here night provide new avenues for development of allosteric inhibitors against zikv infection. based on the sequence alignment with ns2b and ns3pro of the dengue serotype 2 we previously characterized [21] , the corresponding zika sequences were identified for the ns2b (s1a we also constructed a zika protease with ns2b and ns3pro linked by a (gly) 4 -ser-(gly) 4 sequence which was extensively used for functional and structural characterization of flaviviral ns2b-ns3pro complexes [23] [24] [25] [26] [27] . the linked ns2b-ns3pro protein was detected in the pellet of e. coli cells with induction of 1 mm isopropyl β-d-thiogalactopyranoside (iptg) for four hours at 37˚c, while a portion of recombinant protein was found to be in supernatant with induction of 0.2 mm iptg overnight at 18˚c. consequently, we purified the linked ns2b-ns3pro by ni 2+ -affinity column under two conditions: the soluble form directly from the supernatant under native condition, but the insoluble form from inclusion body under denaturing condition, which was easily refolded by overnight dialysis against pbs buffer (ph 7.4) with 10 mm β-mercaptoethanol (column 2 of s1c fig) . the linked complexes without his-tag were successfully obtained by cleavage with thrombin covalently linked to beads, followed by fplc purifications on a gel filtration column (hiload 16/60 superdex 200) (column 3 of s1c fig) . nevertheless, the linked zika ns2b-ns3pro complexes purified directly from supernatant and from the refolding were indistinguishable as judged from both enzymatic activity and biophysical characterizations by cd, fluorescence and nmr. on the other hand, the wild-type ns2b and ns3pro domain are not covalently linked. furthermore, it has been previously demonstrated that only the unlinked dengue ns2b-ns3pro manifested well-dispersed nmr spectra [30, 31] . therefore, we went on further to express the isolated zika ns2b and ns3pro. while the ns3pro protein was found to be in inclusion body, the ns2b was detected in supernatant. so we purified them by ni 2+ -affinity column under denaturing condition for ns3pro and under native condition for ns2b. we first attempted to refold ns3pro alone without ns2b by dialyzing ns3pro overnight against pbs buffer (ph 7.4) with 10 mm β-mercaptoethanol, but all ns3pro protein precipitated during dialysis and no enzymatic activity could be detected, suggesting that zika ns3pro domain also needs ns2b to fold correctly, as previously observed on all other flaviviral ns2b-ns3pro [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] . however, using the same protocol, the mixture of ns2b and ns3pro was easily refolded into the soluble complex (column 2 of s1d fig) , was subjected to further cleavage of his-tag and the final fplc purification (column 3 of s1d fig). as small peptides diffuse significantly and thus usually cannot be seen in the sds-page system we used here, we checked the presence of the ns2b peptide in the finally purified unlinked ns2b-ns3pro complex by the reverse-phase (rp) high pressure liquid chromatography (hplc) with an analytic c8 column. the hplc profile clearly showed that two peaks exist: one with the shorter retention time is for ns2b while another with the longer retention time is for ns3pro (s1f fig) . first we acquired 1 h nmr one-dimensional spectra for both linked and unlinked ns2b-ns3pro ( fig 1a) . both spectra have similar up-field peaks, which can only be observed on a well-folded protein with the tight tertiary packing and will disappear even upon a slight disruption to its tight tertiary packing [36] . fig 1a clearly indicates both linked and unlinked complexes are well folded. an interesting note here is the peaks of linked complex are broader than those of the unlinked complex, which implies the linkage between ns2b and ns3pro introduced additional μs-ms conformational dynamics; this phenomenon was observed for the linked dengue ns2b-ns3pro [21, 30, 31] . while this linkage significantly facilitated the crystallization of the linked flavi-viral ns2b-ns3pro complexes [27] [28] [29] , this linkage significantly broadened nmr signals of linked ns2b-ns3pro complexes [21, 30, 31] . thus, high-resolution nmr can be done on the unlinked form of dengue ns2b-ns3pro which was found to manifest well-dispersed nmr spectra [30, 31] . similarly here, the linked zika ns2b-ns3pro also had broad nmr signals and consequently only a very small portion of hsqc peaks could be detected ( fig 1b) . by contrast, the unlinked complex had sharper 1d up-field peaks ( fig 1a) and a well-dispersed hsqc spectrum (fig 1b) . and unlinked (red) zika ns2b-ns3pro, as well as ns2b (48-74)-ns3pro (green) complexes at a protein concentration of 30 μm. cyan arrows are used to indicate the very up-field peaks. (b) superimposition of 1 h-15 n hsqc spectra of 15 n-labeled unlinked (blue) and linked (red) ns2b-ns3pro complexes at a protein concentration of 30 μm. nmr spectra were acquired at 25˚c in 10 mm sodium phosphate buffer at ph 6.5. pink arrows are used to indicate five hsqc peaks for ns2b-trp61, as well as ns3pro-trp50, ns3pro-trp69, ns3pro-trp83 and ns3pro-trp89 side chains only observed for the unlinked complex. (c) far-uv cd spectra of linked (blue) and unlinked (red) zika ns2b-ns3pro, as well as ns2b (48-74)-ns3pro (green) complexes at a protein concentration of 10 μm, together with that of dengue-2 ns2b-ns3pro complex previously obtained (black) (ref. we further characterized zika ns2b-ns3pro complexes with one being labeled and the other unlabeled. as seen in s2a fig, the [21, 30, 31] which unambiguously indicates dengue ns2b has a tight tertiary packing unlike the zika one. therefore, our results suggest that in the zika ns2b-ns3pro complex, ns2b has a portion of residues undergo μs-ms dynamics which made their nmr peaks too broad to be detectable; while the rest of ns2b is highly disordered and lacks tight tertiary packing, which results in a narrowly-dispersed hsqc spectrum (s2b fig) . both linked and unlinked zika ns2b-ns3pro complexes have far-uv cd spectra with the maximal negative signal at the wavelength of~201 nm and lacks any positive signal below 200 nm, which is different from that of the unlinked dengue complex of the same length [21] which has the maximal negative signal at 217 nm and large positive signal at 192 nm ( fig 1c) . cd studies provide another piece of evidence that zika ns2b-ns3pro complexes contain more disordered regions than the dengue one. we have also collected spectra of intrinsic uv fluorescence from five trp residues in both linked and unlinked ns2b-ns3pro complexes ( fig 1d) , and both complexes have similar spectra with the emission maxima ranging from 344 to 348 nm, very similar to what were observed on the linked ns2b-ns3pro complexes of all four dengue serotypes (348 nm) [25] , which suggests that in zika ns2b-ns3pro, all five trp residues are similarly buried as dengue ones, as trp residue in the unfolded proteins has an emission maximum wavelength > 352 nm [37] . to screen inhibitors from natural products which are largely insoluble in aqueous buffers, we introduced organic solvents such as dimethyl sulfoxide (dmso) and glycerol into the activity assay buffers. we assessed their effects on the conformations of zika ns2b-ns3pro by monitoring intrinsic uv fluorescence. the results indicate that dmso induced significant changes in the intrinsic uv fluorescence spectra of zikv's ns2b-ns3pro (fig 1e) , while glycerol has no significant effect even with concentration up to 20% (fig 1f) . as the isolated zika ns2b (48-100) was soluble in buffers, we acquired triple-resonance experiments hncacb, cbca(co)nh on a double labeled sample, and achieved the sequential assignment for all residues of ns2b (48-100) except for pro72, pro92 and pro93 which have no amide protons. fig 2a presents its (δcα-δcβ) chemical shifts, which represent a sensitive indicator of the residual secondary structures in disordered proteins [37, 38] . the small absolute values of (δcα-δcβ) chemical shifts over the whole sequence clearly indicate that the isolated zika ns2b (48-100) lacks stable secondary structure. we used ssp program [39] to gain quantitative insights into the populations of different secondary structures and found residues arg73-lys100 all have small but negative ssp score ( fig 2b) which implies these residues are in extended conformations that are weakly populated [37, 39] . strikingly, superimposing the hsqc spectra of the 15 n-labeled ns2b in the isolated state against its complex form with unlabeled ns3pro revealed that c-terminal residues arg73-lys100 were the detectable hsqc peaks of ns2b in complexed form, while n-terminal residues ser48-ser71 were the disappeared hsqc peaks (fig 2c) . furthermore, we titrated the 15 n-labeled ns2b in complex with unlabeled ns3pro by adding unlabeled bpti, which is a tight inhibitor and converts open conformation of flaviviral ns2b-ns3pro complexes to the closed form by both nmr [31] and crystallography [40] . here, upon adding bpti, the hsqc peaks of arg73-ser85 disappeared and only very weak hsqc peaks of leu86-lys100 were detectable ( fig 2d) . previously, we have characterized many binding interactions by nmr and found that the binding-induced disappearance of hsqc peaks was due to intermediate binding affinity (with kd values between nm and μm), or/and enhancement of μs-ms dynamics triggered by binding and binding interactions between two well-folded proteins [41] and between disordered peptides and a well-folded protein [42] . our nmr results here indicated that only c-terminal residues of zikv ns2b remained disordered both in the complex form (arg73-lys100; fig 2e) and in presence of bpti (leu86-lys100; fig 2f) , and the conformation of these c-terminal residues in zikv ns2b in these two forms are similar to the uncomplexed form as their hsqc peaks are superimposable ( fig 2c) . our observations are also supported secondary structure score obtained by analyzing chemical shifts with the ssp program. a score of +1 is for the well-formed helix while a score of -1 for the well-formed extended strand. (c) superimposition of 1 h-15 n hsqc spectra of 15 n-labeled ns2b alone (blue) and 15 n-labeled ns2b in complex with unlabeled ns3pro (red). the green is used to highlight two residues gly57 and gly69, which could only be detected in the hsqc spectrum of ns2b in the free state. (d) superimposition of 15 n-labeled ns2b alone (blue) and 1 h-15 n hsqc spectra of 15 n-labeled ns2b in complex with unlabeled ns3pro in the presence of unlabelled bovine pancreatic trypsin inhibitor (bpti) at a molar ratio of 1:2 (red). nmr spectra were acquired at 25˚c in 10 mm sodium phosphate buffer at ph 6.5. a proposed diagram showing the conformations of ns2b in the open conformation (e) and in the close conformation (f) triggered by complexing with bpti. blue arrows are used for indicating β-strands formed over ns2b, while purple dashed lines are for flexible regions of ns2b, whose hsqc peaks could be detected. https://doi.org/10.1371/journal.pone.0180632.g002 by two independent observations: the c-terminal residues of ns2b beyond val87 are completely invisible in the crystal structure of zika ns2b-ns3pro in the closed conformation [34] ; a report on the apo/open-form of zika ns2b-ns3pro [43] stated ". . . c-terminus of ns2b (residues 69-87) is largely unseen, highlighting its high intrinsic flexibility." in sharp contrast, the crystal structure of apo denv ns2b-ns3pro complex showed the c-terminal loop of ns2b adopts a defined conformation despite some discontinuous electron densities beyond residue 76 [27] . so why do zika and dengue ns2b have such radical differences in conformations and dynamics? examination of ns2b sequences reveals that the zika ns2b region corresponding to the dengue ns2b glu91-thr96 shows no sequence homology at all (s3 fig) , thus implying sequence variations over this region may at least partly account for the loss of the contacts between the zika ns2b and ns3pro corresponding to the dengue ns2b gln93-leu95 and ns3pro leu31-ser34, thus leading to the high dynamics of zika ns2b c-half. thus we cloned and expressed a his-tagged zika ns2b (48-74) with the c-half deleted. despite its low expression level and insolubility, we managed to purify a sufficient amount of ns2b (48-74) for refolding with ns3pro with the same protocol described above. interestingly, ns2b (48-74) is sufficient to form a soluble complex with ns3pro (s1e fig) . however, its nmr peaks are broader than those of the linked ns2b-ns3pro (fig 1a) , which is likely due to μs-ms conformational dynamics or/and dynamic aggregation. on the other hand, its intrinsic uv fluorescence spectrum indicates that its four trp residues are similarly buried as linked and unlinked zika complexes with the full-length soluble domain of ns2b ( fig 1d) . most interestingly, this complex with the c-half of ns2b deleted contains less amount of disordered region than both linked and unlinked zika complexes with the full-length ns2b, as seen by its cd spectrum having maximal negative signal shifted to 207 nm and has positive elliptical signal at 191 nm ( fig 1c) , despite being less disordered, this complex (with the later c-half of ns2b deleted) showed no detectable enzymatic activity even with the protease concentration up to 20 μm, which suggests this complex is enzymatically inactive. previously, we generated a truncated dengue ns2b with the same c-half deleted but the shorter ns2b is unable to form a soluble complex with its ns3pro [21] . however, we also generated another truncated dengue ns2b with only residues 77-84 deleted, which was designated as ns2b (48-100; δ77-84). interestingly, ns2b (48-100; δ77-84) was able to form a soluble but inactive complex with its ns3pro, which appeared to be highly disordered as reflected by its cd spectrum, and highly dynamic as judged by its nmr spectrum [21] . together with recent reports on the crystal structures of zika ns2b-ns3pro complexes in both open and closed conformations [34, 43] , our current results reveal that in solution the ns2b residues over arg73-lys100 are highly disordered in the open conformation. however, upon conversion into closed conformation such as triggered by bpti binding, the ns2b residues arg73-ser85 become further bound to the ns3pro domain. on the other hand, our results suggest that despite being intrinsically disordered [44] , the c-half of zika ns2b is absolutely required for implementing the catalytic actions, thus implying that the closed conformation might be enzymatically-active, which was also previously speculated [27] [28] [29] [30] 34, 40] . additionally, being disordered for the ns2b c-half might have other functional advantages offered by intrinsically disordered proteins [44] , such as to allow the formation of dynamic replication complex observed in hcv replication [45, 46] . we have extensively characterized the catalytic properties of both linked and unlinked zika ns2b-ns3pro complexes in different buffer conditions. to allow comparison with the previously published results on the (gly) 4 -ser-(gly) 4 linked ns2b-ns3 proteases of all four dengue serotypes [26] , we have selected the same three substrates. these substrates, namely bz-nkrr-amc, boc-grr-amc and boc-gkr-amc, were extensively used for characterization of other flaviviral ns2b-ns3pro. moreover, we have used the same buffer as previously published [26] , for the measurement of proteolytic activities. furthermore, the effects of salts and organic solvents on the zika protease activity were assessed. interestingly, as shown in s4a fig, the zika ns2b-ns3pro efficiently cleaved bz-nkrr-amc, but unexpectedly showed weak activity on other two substrates. these results imply that besides requiring dibasic residues at the p1 and p2 sites (which is characteristic of the flaviviral ns3 proteases), zika ns2b-ns3pro appears to have a higher need for a basic residue at p3 site than dengue complexes [26] ; as our current focus was not on profiling substrate specificity, we did not test this hypothesis on more substrates but instead measured all kinetic constants of zika complexes with bz-nkrr-amc. we found enzymatic activities of both linked and unlinked zikv complexes showed similar dependence on the ph values with the optimal ph at~9.5 (s4b fig), which were similar to other flaviviral ns2b-ns3pro linked by (gly 4 )-ser-(gly 4 ) [23] [24] [25] [26] , different from dengue-2 ns2b-ns3pro, for which the optimal ph significantly switched from basic ph to neutral ph upon separating ns2b from ns3pro [30] . secondly, like other flaviviral ns2b-ns3pro, the catalytic activity of zika ns2b-ns3pro also reduced significantly upon increasing the concentrations of nacl (s4c fig). in the presence of 150 mm nacl, the catalytic activity is only 13.6% and 8.2% of the linked and unlinked enzymes respectively in 50 mm tris buffer at ph 8.5. thirdly, glycerol concentrations also largely affect the enzymatic activity. the linked zika complex showed the highest activity in the presence of 30% glycerol while the unlinked had the highest activity in the presence of 20% glycerol (s4d fig) . hence, we measured the kinetic parameters for the unlinked complex in the same buffers but with varying concentrations of organic solvents (s4e fig) and the results were presented in table 1 . the linked and unlinked zika complexes only have slightly differences for their kinetic constants in the buffer without any organic solvent. recently, the kinetic constants were just published for a zika ns2b-ns3pro which has ns2b (49-95) and ns3 (1-170) also linked by the same (gly) 4 -ser-(gly) 4 linker [34] . it has a km of 18.3 μm and kcat of 44.6 s -1 respectively. while the kcat values are almost identical, the km value is~2.4-fold less than our current one. this small difference is likely due to: 1) the difference of substrate as bz-nkkr-amc was used in the study [34] ; or/and 2) the slight differences of ns2b and ns3pro lengths, 3) or/and the difference of the buffers: our buffer is 50 mm tris ph 8.5 while the buffer in the publication [34] is only 10 mm tris ph 8.5 with 20% glycerol and 1 mm chaps. it is extensively demonstrated that high salt concentrations has decreased, while glycerol at low concentrations has increased the activity of the flaviviral proteases [21] [22] [23] [24] [25] [26] [27] . indeed, we measured the activity of our linked zika protease in 10 mm tris ph 8.5, and the catalytic activity is 1.36-time higher. due to the urgency to fight zikv infection, we attempted to screen the inhibitors of zika ns2b-ns3pro from natural products rich in edible plants. to better reflect the situation in vivo, here we selected the unlinked zika ns2b-ns3pro complex for screening. however as most natural products are largely insoluble in aqueous buffers, we have assessed effects of dmso and glycerol on the conformations (fig 1e and 1f ) as well as on catalytic parameters (table 1) . consequently, 50 mm tris buffer at ph 8.5 + 20% glycerol was utilized as the screening buffer as it could dissolve all natural products in the present study but has no significant effect on the conformation of zika ns2b-ns3pro (fig 1f) . remarkably, we have identified six compounds to have significant inhibitory effects, which belong to flavonoid and natural phenol ( fig 3a) . subsequently, we have determined their values of ic 50 (s5 fig and table 2 ); and inhibitory constant ki (fig 3c; table 2 ). noticeably, despite sharing the same scaffold, five flavonoids have very distinctive inhibitory effects, with myricetin being the highest (ic50 of 1.26 μm and ki of 0.77 μm) and apigenin the weakest (ic50 of 56.32 μm and ki of 34.02 μm). on the contrary, no inhibitory activity was detected even at a compound concentration up to 500 μm for daidzein, an isoflavone; as well as catechine that has a chemical structure identical to that of quercetin, except that the c-ring of catechin is a dihydropyran heterocycle (fig 3b) . noticeably, isorhamnetin, also called 3'-methylquercetin, has a much weaker inhibitory activity (ic50 of 15.46 μm and ki of 6.22 μm) than quercetin (ic50 of 2.42 μm and ki of 1.12 μm), although isorhamnetin is a derivative of quercetin only with proton of the hydroxyl group at 3' position of phenyl ring replaced by a methyl group (fig 3a) . by contrast, quercetin and luteolin have almost the same inhibitory activity (table 2) , although a hydroxyl group at 3 position of benzopyran ring is absent in luteolin (fig 3a) . furthermore, a significant inhibitory effect (ic50 of 3.45 μm and ki of 2.61 μm) was detected for curcumin, a natural phenol with two aromatic rings linked by heptadiene group (fig 3a) , while no inhibitory effect was detected for resveratrol, also a natural phenol with two aromatic rings but linked by unsaturated ethene group (fig 3b) . these results suggest that these natural products inhibit zika ns2b-ns3pro specifically. another significant finding is that the six compounds inhibit zika ns2b-ns3pro by changing vmax but not km (fig 3c) . this indicates that these compounds inhibit zika ns2b-ns3pro in a non-competitive mode. in other words, six compounds are most likely to allosterically inhibit zika ns2b-ns3pro with their binding sites having no overlap with that for the substrate. indeed, previously myricetin and quercetin have been characterized to allosterically inhibit dengue-2 ns2b-ns3pro with ki values of 4.7 and 20.7 μm respectively, which, however, are much weaker than those for zika ns2b-ns3pro here. unfortunately, nmr spectroscopy cannot be utilized to investigate the interaction between those compounds and zika ns2b-ns3pro as the presence of 20% glycerol significantly increased the rotational tumbling time of the protein which made nmr peaks too broad for detection. to facilitate a better understanding of the experimental results and elucidate structure-activity relationship of the compounds, we used autodock software [47] to dock six small molecules to the crystal structure (5lc0) [34] of zikv ns2b-ns3pro with the substrate-derived inhibitor cn-716 removed. strikingly, all six compounds bind to the pockets on the back of the active site of zika ns2b-ns3pro (fig 4a) , similar to flavonoids binding to dengue-2 ns2b-ns3pro such as myricetin and quercetin [32] . interestingly in the complexes, the short β-sheet formed by ns2b residue leu74-leu78 and asp83-leu86 has direct contacts with the active site inhibitor cn-716 on one side, and with the six compounds on another side (fig 4b and 4c ). as such the pocket for binding six compounds is constituted by the surfaces provided by both zika conformations and inhibition of zika ns2b-ns3pro ns2b and ns3pro (fig 4d and 4e) , whose inner surfaces are relatively polar and negatively charged. interestingly, five flavonoids are highly superimposable in the pocket with the phenyl ring contacting the inner wall formed by ns2b, and with the benzopyran ring located in a pocket provided by ns3pro (fig 4e) . amazingly, although one phenyl ring of curcumin also occupies the same pocket like flavonoids, another phenyl ring has established the binding to a new pocket of ns3pro, which is not observed for five flavonoids (fig 4f and 4g) . previously a similar pocket has been extensively identified on the dengue-2 ns2b-ns3pro structure 3u1i [40] to bind flavonoids including myricetin and quercetin [32, 48] . however, the binding affinities of myricetin and quercetin to bind dengue ns2b-ns3pro are much weaker than those for zika one. as seen in s6 fig, although the overall rmsd of all heavy atoms is only 0.62 å between the crystal structures of zika [34] and dengue-2 [32] ns2b-ns3pro complexes, which are both bound with active-site inhibitors, the binding pockets have slightly-different local geometries but very distinctive electrostatic properties: the zika pocket is more polar and negatively charged, while the dengue-2 pocket is less polar and positively charged (s6 fig). we further analyzed the hydrogen bond networks formed between zika ns2b-ns3pro and six compounds. interestingly, myricetin, the strongest inhibitor, establishes six hydrogen bonds with four zika ns3pro residues (fig 5a) . protons of 3', 4'-hydroxyl groups on phenyl ring form two hydrogen bonds with the backbone oxygen atoms of lys73 of the zika ns3pro domain, while oxygen atoms of 4', 5'-hydroxyl groups on phenyl ring establishes two hydrogen bonds with the side chain nh atom of asn152. furthermore, proton of 3-hydroxyl group on the benzopyran ring forms a hydrogen bond with the side chain oxygen atom of gln74, while proton of 7-hydroxyl group on benzopyran ring forms a hydrogen bond with the backbone nitrogen atom of gly124. due to the absence of 5'-hydroxyl group on phenyl ring in quercetin, one hydrogen bond is missing between oxygen atom of 5'-hydroxyl groups and the side chain nh atom of asn152 (fig 5b) , which may at least partly account for its slightly weak affinity than that of myricetin. strikingly, due to the replacement of proton of 3'-hydroxyl group of quercetin by a methyl group in isorhamnetin, the hydrogen bond between proton of 3'-hydroxyl group and backbone oxygen of lys73 is lost (fig 5c) . this hydrogen bond appears to significantly contribute to the inhibitory activity as the inhibitory constant ki of isorhamnetin increases by~6 times as compared to quercetin (table 2) . on the other hand, luteolin establishes the same hydrogen bond network with zika ns2b-ns3pro residues as quercetin except for the loss of the hydrogen bond with gln74 due to the absence of 3-hydroxyl group in luteolin (fig 5d) . however, this hydrogen bond appears to be not very important as only a very slight reduction of the inhibitory activity was observed for luteolin as compared to quercetin (table 2 ). however, the further absence of 3'-hydroxyl group on phenyl ring in apigenin results in loss of a hydrogen bond between 3'-hydroxyl group on phenyl ring and backbone oxygen atom of lys73 (fig 5e) . this hydrogen bond appears to be critical for its inhibitory activity as apigenin has a ki increased by~25 times as compared to luteolin (table 2) . amazingly, resveratrol has no detectable inhibitory activity but curcumin shows strong inhibitory activity comparable to quercetin. in fact, resveratrol and curcumin have similar structures but the linker between two phenyl rings of resveratrol is 5-carbon shorter than that of curcumin. the complex model between zika ns2b-ns3pro and curcumin (fig 5f) provides an explanation to the experimental result. with a longer linker, one phenyl ring of curcumin occupies the pocket identical to five flavonoids with the formation of hydrogen bonds with gln74 and gly124, while another phenyl ring has additional contacts with a new pocket with unique hydrogen bonds with asp122 and ile165. as such, although curcumin and isorhamnetin have the same 3'-methoxy and 4'-hydroxyl groups on the phenyl rings, likely by having bivalent binding sites, curcumin gains an inhibitory affinity which is much higher than that of isorhamnetin (table 2) . a high affinity, which is achieved by establishing bivalent or multivalent binding sites, has been extensively found, such as on bivalent thrombin-inhibitor interactions [49] . knowledge of catalysis, structures and dynamics of all structural states is beneficial for design of inhibitors with high affinity and specificity towards enzymes including viral proteases [49] [50] [51] [52] [53] . this knowledge is particularly relevant to the flaviviral ns2b-ns3pro complexes as it is proposed that their catalytic activities require a transition from the open (inactive) to closed (active) conformation [21, 28, 29, 40] . interestingly, regardless of being in the open or closed conformations, ns3pro domains of different flaviviral ns2b-ns3pro complexes universally adopt the same chymotrypsin fold. by contrast, while the n-half of ns2b assumes a similar βstrand packed to the ns3pro domain in both open and closed conformations, the c-half of ns2b shows a significant structural diversity in different structures determined so far. in the closed conformation, ns2b structures of flaviviral ns2b-ns3pro complexes show a similar a short β-hairpin formation over the c-half of ns2b and is tightly bound to the ns3pro chymotrypsin fold (fig 4a) . however in the open conformation, the structural properties of the ns2b c-half have been shown to be quite diverse. for the well-studied dengue-2 ns2b-ns3pro in the open conformation, most ns2b residues are tightly packed with the ns3pro domain as revealed by the crystal structure [27] , and evident from its well-dispersed hsqc spectrum (s2c fig) reconstructed from a previous report [30] . in the present study, we first constructed and characterized the zika ns2b-ns3pro complex with ns2b and ns3pro linked by an artificial (gly) 4 -(ser)-(gly) 4 sequence which has been found to dramatically facilitate the crystallization of flaviviral ns2b-ns3pro complexes [27, 34, 40, 43] . despite slight differences in sequence length, the catalytic parameters (table 1) of our linked zika ns2b-ns3pro complex have no significant difference from those recently published [34] . unfortunately, as previously observed on dengue-2 ns2b-ns3pro complexes [21, 30, 43] , our linked zika complex also underwent significant μs-ms dynamics, thus making its nmr signals too broad to be detected (fig 1a and 1b) . as a consequence, we devoted efforts to generate and characterize an unlinked zika ns2b-ns3pro complex by using a protocol we previously established for the dengue-2 ns2b-ns3pro complex [21] . this approach is also required for the selective isotope-labeling of zika ns2b or ns3pro for high-resolution nmr studies. indeed, despite showing no significant difference of catalytic properties from the linked one (table 1) , the unlinked zika ns2b-ns3pro complex suddenly manifested a well-dispersed hsqc spectrum of the 15 n-labeled ns3pro domain in complex with unlabeled ns2b with sharper nmr peaks (fig 1a and 1b) , which are consistent with previous nmr results on the unlinked dengue complexes [21, 30, 31] . most importantly, this allowed us to selectively study the 15 n-labeled ns2b in complex with unlabeled ns3pro. the results revealed that the zika ns2b-ns3pro complex, the c-terminal residues arg73-lys100 of ns2b remain highly disordered unlike the dengue-2 ns2b-ns3pro complex in the open conformation. binding to bpti appeared to trigger the conversion of zika ns2b-ns3pro complex into the closed conformation, in which the ns2b c-terminal residues arg73-ser85 become further bound to the ns3pro domain. the intrinsic dynamics of the zika ns2b c-half might be due to the significant sequence variations over ns2b residues 91-96 (s3 fig). strikingly, this unique property for zika ns2b-ns3pro is not only observed in solution by our nmr investigation, but has been recently shown by the crystal structure of the apo/open-form of zika ns2b-ns3pro [43] . in the future, it is of significant interest to explore what is the functional consequence of this unique property. one possibility might be that with the intrinsically disordered ns2b c-half [44] , the zika ns2b-ns3pro is more susceptible to the allosteric regulation [50] [51] [52] . although many adults infected with zikv will have only mild or even no detectable symptoms, the zikv can be transmitted from a pregnant woman to her fetus, thus leading to birth defects such as microcephaly. this imposed a great challenge and urgency to fight zikv. therefore we attempted to screen inhibitors from natural products rich in edible plants for the unlinked zika ns2b-ns3pro, which represents a more realistic form in vivo. remarkably, we identified five flavonoids and one natural phenol to specifically inhibit zika ns2b-ns3pro with the strongest myricertin having a ki of 770 nm. recently, lim et. al. has reported a ki value of 8.9 um for myricitin [54] , the difference might be due to 1) the linked zika ns2b-ns3pro which they have used for their enzyme inhibition assay whereas we have used an unlinked enzyme and 2) the ph of the assay buffer they used is ph 7 whereas we have used ph 8.5. furthermore, analysis of structures and inhibitory activities reveals that for flavonoids, the presence of benzopyran ring and the position to connect to phenyl ring are critical for the inhibitory activity, as evident from the results that daidzein and catechin were inactive on inhibiting zika ns2b-ns3pro. furthermore, the number of hydroxyl groups on phenyl ring is a key determinant for the inhibitory activity. similar observation has been reported in a recent study where a number of polyphenols has been studied for inhibitory activity against linked zika ns2b-ns3pro [54] . interestingly, our current molecular docking reveals that these compounds bind to a pocket on the back of the active site of the zika ns2b-ns3pro complex. while five flavonoids bind very similarly to the pocket constituted by both ns2b and ns3pro, curcumin can have an additional binding site outside of this pocket. amazingly, the pocket we observed on the zika ns2b-ns3pro to bind five flavonoids is very similar to that previously observed on the dengue ns2b-ns3pro [32, 48] , but the additional binding site for curcumin represents a novel one. therefore, zika ns2b-ns3pro appears to be highly susceptible to allosteric inhibitions, as previously found on other flaviviral ns2b-ns3pro complexes [24, 32, 48, 53] . noticeably, probably due to slight variations of the local geometry or/and significant difference in electrostatic potentials of the allosteric pockets of zika and dengue complexes, the same flavonoids inhibit the zika ns2b-ns3pro complex with higher activity [32] . five flavonoids and curcumin are rich in various edible plants including caper, tea, red onion, celery, broccoli, green pepper and turmeric. additionally, they have been previously demonstrated to have beneficial effects on human health and thus extensively used as supplements [32, [55] [56] [57] . as such, our finding might help the public to fight against zikv infection immediately. in the further, it is also of both fundamental and therapeutic interests to characterize the allosteric mechanisms by which these natural products inhibit zika ns2b-ns3pro by both experimental and computational approaches [53] [54] [55] [56] [57] [58] [59] [60] . as these natural product inhibitors fundamentally differ from the currently-known active site inhibitors in both inhibitory mode and chemical scaffold [34, 43] , our finding may also provide a promising foundation for further development of novel allosteric inhibitors of higher affinity and specificity to fight zikv infection. the identified genes encoding ns3pro (1-185) and ns2b (1-130) from the asian zika strain 8375 (genbank id: ku501217.1) were optimized and synthesized by genscript (piscataway, nj). with designed primers, the genes by genscript were used as templates for amplifying dna fragments encoding the isolated ns3 (14-185) and ns2b (48-100) with the transmembrane regions removed, as well as ns2b with the c-region further deleted. furthermore, dna fragments were designed for linking ns2b (48-100) to ns3 (14-185) by (gly) 4 -ser-(gly) 4 using overlap pcr as previously described (14) . amplified dna fragments were subsequently cloned into his-tagged pet28a vector (novagen). dna sequences of all constructs were verified by automated dna sequencing. all pet28a vectors containing different zika genes were transformed into escherichia coli bl21 (de3) star cell (thermo fisher scientific), which was cultured in luria-bertani broth containing 25 μg/ml kanamycin at 37˚c until the a600 reached 0.6. subsequently, protein expressions of different constructs were induced with different conditions. to obtain soluble form of the linked ns2b-ns3pro complex, protein expression was induced with 0.2 mm isopropyl β-d-thiogalactopyranoside (iptg) overnight at 18˚c. however, the expression level of the soluble form was low and consequently the linked complex was also prompted into inclusion body with induction of 1 mm iptg for 4 hours at 37˚c, which yielded to a much higher expression level. for isolated ns2b and ns3, protein expressions were induced with 1 mm iptg for 4 hours at 37˚c. for the linked ns2b-ns3pro complex, the soluble form was purified by ni 2+ -affinity chromatography under native condition. subsequently, the his-tag was removed by the cleavage with thrombin-agarose beads from thrombin cleancleave™ kit (sigma-aldrich, st. louis, mo), followed by binding to an excess amount of ni-nta beads to remove his-tag and uncleaved fusion protein, as well as a final fplc purification on a gel filtration column (hiload 16/60 superdex 200). to obtain the linked complex in inclusion body, the cell pellets were re-suspended in cold pbs buffer at ph 7.4, containing 10 mm β-mercaptoethanol and 8 m urea and the supernatant containing the recombinant proteins were purified by ni-nta affinity column under denaturing condition. eluted fractions were subjected to dialysis against pbs buffer (ph 7.4) with 10 mm β-mercaptoethanol at 4˚c overnight to allow the refolding of the complex. the refolded complex was subjected to the cleavage of his-tag and final fplc purification as described above. the isolated ns2b (48-74) and ns3 were purified by ni-nta affinity column under denaturing condition, while ns2b (48-100) was purified under native condition. subsequently, the purified ns2b (48-100)/ns2b and ns3 were mixed up with an excess amount of ns3. the mixture was subjected to refolding by dialysis against pbs buffer (ph 7.4) with 10 mm β-mercaptoethanol at 4˚c overnight as we previously established for dengue ns2b-ns3pro complex (12) . the refolded complex was subjected to the cleavage of his-tag and further fplc purification as described above. the generation of the isotope-labeled proteins for nmr studies followed a similar procedure except that the bacteria were grown in m9 medium with the addition of ( 15 nh 4 ) 2 so 4 for 15 n labeling and ( 15 nh 4 ) 2 so 4 /[ 13 c]-glucose for double labeling [21] . all recombinant protease samples were checked by tris-glycine sds page. however, for this sds-page system, small peptides diffuse significantly and thus usually cannot be seen. therefore, reverse-phase (rp) high pressure liquid chromatography (hplc) with an analytic c8 column was used to check the presence of the isolated ns2b peptides with a molecular weights less than 6 kda. molecular weight verification and protein sequencing were conducted with time-of-flightmass spectrometer (applied biosystems). protein concentration was determined by the uv spectroscopic method with 8 m urea [21] . intrinsic uv fluorescence spectra were measured with a cary eclipse fluorescence spectrophotometer as we previously described [37] with the excitation wavelength at 280 nm. circular dichroism (cd) experiments were performed on a jasco j-1500 spectropolarimeter and data from five independent scans were added and averaged [21] . to assess the effects of dmso and glycerol on the conformation of zika ns2b-ns3pro, we monitored the change of its intrinsic uv fluorescence instead of circular dichroism (cd), because organic solvents were found to provoke very high non-specific noises. all nmr experiments were acquired on an 800 mhz bruker avance spectrometer equipped with pulse field gradient units as described previously [21] . to achieve sequential assignment, 15 n-/ 13 c-double labeled zika ns2b sample was prepared at a protein concentration of 200 μm in 10 mm phosphate buffer. a pair of triple-resonance experiments hncacb, cbca(co) nh were acquired [21] . to investigate the binding interaction between zika ns2b-ns3pro and bpti, hsqc spectra of zika ns2b-ns3pro only with ns2b 15 n-labeled were acquired in the absence and in the presence of bpti (sigma-aldrich) at different ratios. to allow comparison with the ns2b-ns3pro complexes of four dengue serotypes (17), we selected three fluorophore-tagged substrates previously used (17) : namely bz-nle-lys-arg-arg-amc, boc-gly-arg-arg-amc and boc-gly-lys-arg-amc (bachem ag, bubendorf), which were dissolved in dimethyl sulfoxide for preparing stock solutions (100 mm). all enzymatic experiments were performed in triplicate and data are presented as mean ± sd, while ic 50 , km and ki were obtained by fitting with graphpad prism 7.0 [61] . the ph dependence was measured with a protease concentration of 50 nm and substrate (bz-nkrr-amc) concentration of 250 μm at 0.5 ph intervals using the following buffers: 50 mm citrate-phosphate buffer for ph 4-5, 50 mm phosphate buffer for ph 5.5-8, 50 mm tris-hcl buffer for ph 8.5-9.5, and 50 mm na-bicarbonate buffer for ph 10-10.5 . for steady state kinetics, we used the exactly the same buffer as a previous one on profiling substrate specificity for the ns2b-ns3pro complexes of all four dengue serotypes 17): 50 mm tris-hcl at ph 8.5. to screen natural product inhibitors of zika ns2b-ns3pro, we also measured the km values of zika ns2b-ns3pro in the presence of dmso and glycerol which allow the solubilization of these compounds in the assay buffer. briefly, zika protease at 50 nm was incubated with substrates ranging from 10 to 1000 μm in 100 μl assay buffer at 37˚c. progression of enzymatic reaction was monitored as an increase in fluorescence at λ ex of 380 nm and λ em of 450 nm. fluorescence intensity is reported in arbitrary units. initial fluorescence or absorbance velocities (relative fluorescence units per minute or relative absorbance units per minute) were converted to ms -1 from a standard acmc calibration curve. subsequently, the curves were fitted to the michaelis-menten equation by nonlinear regression. all natural products were purchased from sigma-aldrich, which are all hplc purified. after optimization of buffer conditions, here, we selected 50 mm tris-hcl buffer at ph 8.5 in the presence of 20% glycerol which could dissolve all natural products. briefly, for the initial screening, the zika protease at 50 nm was preincubated for 30 min with different compounds at final concentrations of 5 and 500 μm dissolved in 1 μl dmso, followed by adding bz-nkrr-amc to 250 μm to initiate reaction. only the compounds showing significant inhibitions at both concentrations were subjected to further determination of ic 50 and ki. for ic 50 determination, the zika protease at 50 nm was preincubated at 37˚c for 30 min with natural products at various final concentrations in 1 μl dmso; and subsequently the reaction was initiated by adding bz-nkrr-amc to 250 μm. for ki determination, the assay was performed with different final concentrations of the inhibitors and substrate. briefly, the zika protease at 50 nm was preincubated with the inhibitor at different concentrations for 30 min at 37˚c. subsequently, the reaction was initiated by addition of the corresponding concentration series of the substrate. all measurements were performed in triplicate and data are presented as mean ± sd. the ki was obtained by fitting in the non-competitive inhibition mode with graphpad prism 7.0, with an equation: vmaxinh = vmax/(1+i/ki), while i is the concentration of inhibitor [61] . to gain insight into structural details of the binding pocket, we docked all six active natural products to the crystal structure (pdb code of 5lc0) of zika ns2b-ns3pro in complex with an active site inhibitor cn-716 [34] . the chemical structures of the compounds were downloaded from zinc (http://zinc.docking.org) and chemicalbook database (http://www. chemicalbook.com) respectively. subsequently the structures were geometrically optimized with avogadro [62] . the partial charges of all atoms in small compounds and zika ns2b-ns3pro were assigned with gasteiger-marsili charges, and non-polar hydrogen atoms were merged into the appropriate heavy atoms with autodocktools [47] . autodock software (version 4.2) was utilized to dock six compounds to the crystal structure of zika ns2b-ns3pro. the grid box was set with 74 × 70 × 66 (x,y,z axis) with the default 0.375å spacing. the initial population size was set to 300, and the number of energy evaluations was set to 25,000,000, and number of docking runs was set as 150. the results were clustered with each cluster having a tolerance of 2 å. the complexes with the lowest energy were selected for analysis and display. zika fever and congenital zika syndrome: an unexpected emerging arboviral disease zika virus: an update on epidemiology, pathology, molecular biology, and animal model isolations and serological specificity zika virus: history of a newly emerging arbovirus complete coding sequence of zika virus from a french polynesia outbreak in 2013 potential for zika virus introduction and transmission in resource-limited countries in africa and the asia-pacific region: a modelling study zika virus associated with microcephaly evidence of sexual transmission of zika virus zika virus disrupts neural progenitor development and leads to microcephaly in mice the brazilian zika virus strain causes birth defects in experimental models experts fear zika's effects may be even worse than thought zika virus infection associated with severe thrombocytopenia the emergence of zika virus as a global health security threat: a review and a consensus statement of the indusem joint working group (jwg) zika virus infection in dexamethasone-immunosuppressed mice demonstrating disseminated infection with multi-organ involvement including orchitis effectively treated by recombinant type i interferons ihr 2005) emergency committee on zikv and observed increase in neurological disorders and neonatal malformations predicting zika virus structural biology: challenges and opportunities for intervention dengue virus sero-cross-reactivity drives antibody-dependent enhancement of infection with zika virus neutralizing human antibodies prevent zika virus replication and fetal disease in mice structures of the zika virus envelope protein and its complex with a flavivirus broadly protective antibody a human antibody against zika virus crosslinks the e protein to prevent infection nmr and md studies reveal that the isolated dengue ns3 protease is an intrinsically disordered chymotrypsin fold which absolutely requests ns2b for correct folding and functional dynamics flavivirus genome organization, expression, and replication activity of recombinant dengue 2 virus ns3 protease in the presence of a truncated ns2b co-factor, small peptide substrates, and inhibitors solution conformations of a linked construct of the zika virus ns2b-ns3 protease a comparative biochemical analysis of the ns2b (h)-ns3pro protease complex from four dengue virus serotypes functional profiling of recombinant ns3 proteases from all four serotypes of dengue virus using tetrapeptide and octapeptide substrate libraries structural basis for the activation of flaviviral ns3 proteases from dengue and west nile virus structural biology of dengue virus enzymes: towards rational design of therapeutics the flavivirus ns2b-ns3 protease-helicase as a target for antiviral drug development nmr analysis of a novel enzymatically active 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structures of the dengue virus protease reveal the active conformation structural characterization of the epha4-ephrin-b2 complex reveals new features enabling eph-ephrin binding promiscuity structural insight into the binding diversity between the tyr-phosphorylated human ephrinbs and nck2 sh2 domain mechanisms of activation and inhibition of zika virus ns2b-ns3 protease classification of intrinsically disordered regions and proteins intrinsically unstructured domain 3 of hepatitis c virus ns5a forms a "fuzzy complex" with vapb-msp domain which carries als-causing mutations the intrinsic disorder status of the human hepatitis c virus proteome autodock4 and auto-docktools4: automated docking with selective receptor flexibility docking of noncompetitive inhibitors into dengue virus type 2 protease: understanding the interactions with allosteric binding sites nmr for the design of functional mimetics of protein-protein interactions: one key is in the building of bridges structured crowding and its effects on enzyme catalysis. dynamics in enzyme catalysis protein dynamics: conformational footprints the role of protein loops and linkers in conformational dynamics and allostery allosteric inhibition of the ns2b-ns3 protease from dengue virus inhibitory effect of flavonoids against ns2b-ns3 protease of zika virus and their structure activity relationship health effects and bioavailability of dietary flavonols flavonoid intake in european adults (18 to 64 years) curcumin: a review of anti-cancer properties and therapeutic activity in head and neck squamous cell carcinoma dynamic principle for designing antagonistic/agonistic molecules for epha4 receptor, the only known als modifier dynamically-driven inactivation of the catalytic machinery of the sars 3c-like protease by the n214a mutation on the extra domain dissection study on the severe acute respiratory syndrome 3c-like protease reveals the critical role of the extra domain in dimerization of the enzyme defining the extra domain as a new target for design of highly specific protease inhibitors fitting models to biological data using linear and nonlinear regression: a practical guide to curve fitting avogadro: an advanced semantic chemical editor, visualization, and analysis platform key: cord-000082-jy7j8sh0 authors: huang, ting; wang, wei; bessaud, mael; ren, peijun; sheng, jun; yan, huajie; zhang, jing; lin, xin; wang, yongjin; delpeyroux, francis; deubel, vincent title: evidence of recombination and genetic diversity in human rhinoviruses in children with acute respiratory infection date: 2009-07-27 journal: plos one doi: 10.1371/journal.pone.0006355 sha: doc_id: 82 cord_uid: jy7j8sh0 background: human rhinoviruses (hrvs) are a highly prevalent cause of acute respiratory infection in children. they are classified into at least three species, hrv-a, hrv-b and hrv-c, which are characterized by sequencing the 5′ untranslated region (utr) or the vp4/vp2 region of the genome. given the increased interest for novel hrv strain identification and their worldwide distribution, we have carried out clinical and molecular diagnosis of hrv strains in a 2-year study of children with acute respiratory infection visiting one district hospital in shanghai. methodology/findings: we cloned and sequenced a 924-nt fragment that covered part of the 5′utr and the vp4/vp2 capsid genes. sixty-four hrv-infected outpatients were diagnosed amongst 827 children with acute low respiratory tract infection. two samples were co-infected with hrv-a and hrv-b or hrv-c. by comparative analysis of the vp4/vp2 sequences of the 66 hrvs, we showed a high diversity of strains in hrv-a and hrv-b species, and a prevalence of 51.5% of strains that belonged to the recently identified hrv-c species. when analyzing a fragment of the 5′ utr, we characterized at least two subspecies of hrv-c: hrv-cc, which clustered differently from hrv-a and hrv-b, and hrv-ca, which resulted from previous recombination in this region with sequences related to hrv-a. the full-length sequence of one strain of each hrv-ca and hrv-cc subspecies was obtained for comparative analysis. we confirmed the close relationship of their structural proteins but showed apparent additional recombination events in the 2a gene and 3′utr of the hrv-ca strain. double or triple infections with hrv-c and respiratory syncytial virus and/or bocavirus were diagnosed in 33.3% of the hrv-infected patients, but no correlation with severity of clinical outcome was observed. conclusion: our study showed a high diversity of hrv strains that cause bronchitis and pneumonia in children. a predominance of hrv-c over hrv-a and hrv-b was observed, and two subspecies of hrv-c were identified, the diversity of which seemed to be related to recombination with former hrv-a strains. none of the hrv-c strains appeared to have a higher clinical impact than hrv-a or hrv-b on respiratory compromise. human rhinoviruses (hrvs) are a highly p revalent cause of the acute respiratory infection (ari) defined as the common cold [1, 2, 3] , which is frequently associated in children with bronchitis, bronchiolitis, wheezing, pneumonia, asthma and otitis [4, 5, 6, 7, 8, 9] . hrvs are classified in genus enterovirus (hevs) in family picornaviridae [10] . hrvs are non-enveloped, single-stranded, positive-sense rna viruses of approximately 7200 nt, composed of a 59 untranslated region (utr), followed by a long open reading frame coding for capsid proteins vp4, vp2, vp3 and vp1, and seven non-structural proteins 2a, 2b, 2c, 3a, 3b, 3c and 3d, and terminated by a short 39utr and poly a tract. more than 100 serotypes of hrv are known, which have been classified into two species, hrv-a and hrv-b, according to comparative alignment of nucleotide fragments of vp1 [11, 12] , vp4/vp2 [13] and 59utr [14, 15] , and more recently, on complete genome nucleotide sequences [16] . moreover, some genomic sequences have been found not to cluster with hrv-a and hrv-b species, which suggests the existence of other species (hrv-c and hrv-d) [16] . a new species of hrv-c was recently identified worldwide by comparative analysis of vp4 or vp4/vp2 genes [7, 17, 18, 19, 20, 21] and 59utr [14, 15] . however, discrepancies have appeared in the classification of some of the new hrv-a or hrv-c strains, depending on the size and location of the nucleotide sequence in the viral genome and on the phylogenetic methods used for direct analysis of hrv sequences [3, 7, 14, 15, 17, 18, 20, 21, 22, 23] . these recent data underline the lack of knowledge about the biodiversity of hrv strains and their worldwide distribution [14, 17] . moreover, little is known about the characteristics and diversity of hrvs circulating in a given area in a short period of time. in the present study, we looked for hrvs in a 2-year collection of nasopharyngeal swabs (npss) of children with ari visiting a district hospital in shanghai, and compared sequences in two regions previously defined for genetic classification of hrv serotypes [13, 15] . our study showed a high diversity of hrv species and genotypes, and a prevalence of the novel hrv-c species in npss of children with bronchitis and pneumonia. this biodiversity appeared to result partly from recombination events in the 59utr, between hrv-c strains and those close or similar to hrv-a species, which led to the suggested classification of hrv-c into at least two subspecies. eight hundred and twenty-seven samples were collected from a group of children consulting the shanghai nanxiang hospital during a 2-year period, and tested for 17 respiratory viruses using a multiplex rt-pcr (mrt-pcr). sixty-four samples (7.7%) were positive for hrv, according to the length of the amplified fragment in the vp4/vp2 region visualized on agarose gel (data not shown). a larger fragment of 924 nt, including part of the 59utr (starting at nt 163) and the vp4/vp2 genes (ending at nt 1086), was amplified and cloned into plasmid vectors for genetic analysis (table 1) . only one sample, n1, could not be amplified and was amplified in two steps in the 59utr (nt 163-552) and in the vp4/vp2 region (nt 528-1086), respectively. analyses of different clones for each sample allowed the identification of multiple infections: sample n16 was shown to contain one hrv-a and one hrv-b (n16a and n16b, respectively), while n58 contained one hrv-a and one hrv-c (n58a and n58c, respectively) (see below). in order to further characterize the 66 hrvs identified in the 64 samples, the nucleotide sequences located in the 59utr (285 nt) and the vp4/vp2 genes (420 nt) were chosen to allow comparative alignment with sequences of reference serotypes and field strains available in genbank. we first compared and classified shanghai strains according to their vp4/vp2 sequences. the pairwise nucleotide divergence in the vp4/vp2 region ranged from 0 to 72.2%. twenty-seven hrvs (40.9%) showed .81% nucleotide identity with the closest hrv-a clusters, and five hrvs (7.6%) showed .88.8% nucleotide identity with hrv-b clusters ( table 2 ). the remaining 34 strains (51.5%) diverged from hrv-a and hrv-b species by .47.3% in their vp4/vp2 nucleotide sequence ( table 2) . these strains showed from 68.3 to 100% nucleotide identity with each other and were related to the recently described hrv-c strains, nat001 and nat045, isolated in california, usa [18] , c024, c025 and c026 in hong kong [20] , and qpm in australia [7, 24] ( fig. 1 ). strains n34, n35 and n68 were closely related to the recently identified strains c025 and nta001 with .95.9% nucleotide identity, whereas the 31 remaining hrv-c strains showed only 74.4-86.4% nucleotide identity with six other recent strains (qpm, nat001, nat045, c024, c025 and c026; table 2 ), which were classified tentatively as hrv-c species [7, 18, 20, 24] . classification of the strains into three different species was also demonstrated by construction of a phylogenetic tree using aligned vp4/vp2 sequences (fig. 1) . to characterize and classify further the chinese hrv strains, 59utr sequences were considered (table 3 , fig. 2 ). they were compared to the 59 utr of all 101 reference hrvs, to those of 26 new strains identified in children with respiratory illness in wisconsin (indicated as w) [15] , and to those of other recently identified hrv-c strains [14] . pairwise nucleotide divergence between the three hrv species was 0.7-64.3%, and a limit of ,9% divergence between genotype pairs was chosen for similar genotype assignment in one species [15] . new genotypes were identified when they had 9-30% pairwise nucleotide divergence from the nearest serotype in the same species (table 3) . fifty-five hrvs shared .94.4% nucleotide identity with strains already identified, and 11 hrvs showed 9.5-20.9% nucleotide divergence with the nearest known hrvs. they may represent newly discovered genotypes. these strains clustered with hrv-a (n6) or hrv-c species (n4, n8, n21, n62, n63, n67, n24, n25, n28 and n32) ( table 3) . most surprisingly, 20 of the 34 strains classified as hrv-c by comparative analysis of vp4/p2 sequences ( table 2) were related more closely to hrv-a strains when their 59utrs were analyzed, and showed incongruent clustering in phylogenetic trees (figs. 1 and 2). the nucleotide sequences in the 59utr of these strains were related closely to those of formerly identified qpm, nat001, nat045, c024, c025 and c026 hrv-c strains, and to some of the w strains recently identified as hrv-a [15] (fig. 2) . however, our 20 strains clustered together with these six strains in two major branches in the phylogenetic tree constituted a subspecies of hrv-c called hrv-ca (table 3 , figs. 2 and 3) . the fourteen other hrv-c strains formed another unique branch of hrv-c subspecies, called hrv-cc, which clustered differently from other species of hrv-a and hrv-b, and from subspecies hrv-ca in the phylogenetic tree based on 59utr sequences (table 3 ; figs. 1 and 2). these strains were related closely to some w strains of hrv that were classified as hrv-c [15] (fig. 2 , table 3 ) in order to characterize more precisely the differences observed in the 59utr between hrv-ca and hrv-cc subspecies, and to localize possible recombination sites in the 59utr of the genome of hrv-ca subspecies, bootscanning and similarity plot analyses were conducted in the gene fragment of 868 nt that included the 59utr and adjacent capsid genes. hrv-ca nucleotide sequences were scanned against sequences of n10, r16 and r52, which are considered as representative strains of hrv cc subspecies and hrv-a and hrv-b species, respectively. stretches of nucleotide sequences that were closer to hrv-a (r16) than to hrv-cc (n10), flanked by sequences related to hrv-c could be detected in the 59utr of hrv-ca strains, as exemplified with hrv-ca n25 strain ( fig. 3a and b) . these hrv-a-related nucleotide stretches were thus flanked by putative recombination sites. these sites were located differently among the hrv-ca strains, delimiting hrv-a-related stretches that ranged from 150 to 400 nt in length (fig. 3c ). while variable among the strains, the identified recombination sites were all located inside the 59 utr and none of them was identified in the downstream vp4/vp2 coding sequence. hrv-a-related nucleotide sequences and putative recombination sites were also found in the 59utr of the previously described hrv-c strains c024, c025, c026, nat001, nat045 and qpm (fig. 3) . the results corroborated the clustering observed in the phylogenetic tree based on 59utr sequences (fig. 2) , since strains gathered in the same hrv-ca subcluster (for example n24, n25, n28 and n32, or n4, n7, n8, n21, n36 and n46) displayed the same recombination pattern. these subclusters revealed different recombinant lineages, each of which originated from independent recombination events. in order to further characterize the genome of the hrv-cc subspecies, for which no full-length sequence was yet available, we sequenced the remaining genes that covered the whole coding sequence and 39utr of n10 strain, which was chosen as the representative of this subspecies (tables 2 and 3 ). the full-length n10 genome sequence was compared to those of the hrv-ca subspecies strains c024, c025 and c026, and to that of n4 strain, which was sequenced as the representative of the hrv-ca subspecies. the genome sequences were also compared to those of the hrv-a strains n13 and r44, and to the hrv-b strains r14 and r52 (table 4 ). the full-length nucleotide sequence of n10 strain contained 7111 nt, excluding the poly(a) tract, which was shorter than sequences from hrv-a and hrv-b strains, but similar to those of hrv-ca strain n4 and other related strains (c024, c025 and c026). the 2144 aa lengths of the polyprotein and of each of the individual proteins of n10 were slightly different from those of hrv-a and hrv-b species, but similar to those of other hrv-c strains. the most divergent amino acid length between hrv was observed for the vp1 protein that was shorter in hrv-c species ( table 4 ). the unique putative cleavage (m/s) site between vp4 and vp2 protein identified previously for qpm, c024, c025 and c026 strains [20] was also observed for n10 and n4 strains. it was different from those of the hrv-a strains n13 and r44 (q/s), and from those of the hrv-b strains r14 and r52 (n/s) (data not shown). alignment of the vp1 amino acid sequence of hrv-cc strain n10 with those of other hrv-a and hrv-b species and hrv-ca subspecies, designated in table 4 , showed structural features typical of hrv-c species [16, 20, 25] (data not shown). in particular, footprints including deletions in the bc, de and hi loops and conserved amino acids potentially involved in inter-cellular adhesion molecule 1 (icam-1) receptor binding [7, 11, 20, 24] were conserved within the hrv-c species (data not shown). bootscanning and similarity plot analysis conducted on the genomic sequences of n4 (hrv-ca), n10 (hrv-cc), r16 (hrv-a) and r52 (hrv-b) confirmed that n4 featured a 59utr sequence that was related to the r16 sequence (stretch i), followed by a capsidic sequence related to the n10 sequence. n4 nonstructural sequence (2a to 39utr) was related more closely to n10 than to r16 and r52 sequences. however, in stretch ii (nt 3300-3500 according to n4 numbering), n4 strain (hrv-ca) was closer to r16 (hrv-a) than to r52 (hrv-b) or n10 (hrv-cc), which resulted in high bootstrap values between n4 and r16 2a sequences (fig. 4) . this may have been the result of a recombination event that occurred in the 39 part of the 2a-encoding sequence of the parental strain n4, and which involved an hrv-a strain. nevertheless, the hrv-a parental strain or ancestral strain could not be identified since the closest hrv-a 2a nucleotide sequence available was ,80% identical to that of n4 in this region. in contrast from nt 6,550 to the 39 end (stretch iii in fig. 4) , the n10 strain genome was found to be closely related to that of n4, with nucleotide identity .98%. this result is corroborated in figure 5 , which shows a phylogenetic analysis of the 39utr sequences of n10 and n4 compared to those of hrv-ca subspecies and hrv-a and hrv-b species. this suggests that n4 and n10 strains share a common recent ancestor through recombination. 1 and 2 ) and on local recombination in 59utr (see fig. 3 ). b strains closely related with more than 93% identity. c these strains clustered differently when based on vp4/vp2 sequences (see table 2 and figs. 1 and 2) . doi:10.1371/journal.pone.0006355.t003 clinical outcome from hrv strains isolated from pediatric outpatients among the pediatric patients, 46 were males and 18 females, and their age ranged from 5 months to 14 years. the majority of hrv infections were diagnosed between 2 and 6 years of age (84.6%). bronchitis (73.4%) and pneumonia (26.6%) were highly prevalent in children with comparable incidence in hrv-a and hrv-c infections (table 5) . moreover, the ratio of pneumonia over bronchitis (36.2%) was comparable to that in the whole cohort of 827 children (40.7%). only one child among the 64 hrv-positive patients had asthma and was co-infected with hrv-c, influenza a virus (iav) and respiratory syncytial virus (rsv) ( table 5) , whereas 100 of the 827 patient were diagnosed with asthma. hrvs were isolated throughout the 2 years, with a predominance of hrv-c viruses in the cold season (table 5) . interestingly, different hrv genotypes were detected within the same period (for example, n1 and n4, n9 and n11/n12, n44/n48 and n51, and n55/n56 and n62/n67), with a larger diversity and distribution of individual or paired hrv-a genotypes compared to hrv-c strains, which clustered in closely-related genotypes (fig. 2, tables 2 and 3) . conversely, n4 and n21 strains of samples collected at 10 months interval showed 99.8% identity (fig. 2) . single hrv infection was diagnosed in 42 children and coinfections were identified in 22 patients (table 5) , with 17 double and five triple infections. the viruses most often identified in hrv co-infection were rsv (six cases) and human bocavirus (hbov; four cases), and two patients were co-infected with hrv, hbov and rsv (table 5 ). there was no difference between hrv-ca or hrv-cc subspecies and any of the clinical or epidemiological data (data not shown). in this report, we looked for hrvs in a 2-year collection of npss from children with ari visiting a district hospital in shanghai, and found a high diversity of hrv strains that belonged to different species and genotypes. we characterized by rt-pcr and sequenced 66 hrvs, among them 27 hrv-a, five hrv-b, and 34 hrv-c strains. when sequencing the vp4/vp2 region of the hrv genome, several recent studies have identified new strains of viruses from children and adults with ari, asthma, or otitis, which are clustered differently from hrv-a and hrv-b, and have been classified into a novel hrv-c species [7, 8, 17, 18, 19, 20, 21, 25, 26, 27, 28] . other groups have also identified novel hrv-c strains by sequencing the vp1 gene [29] or the 59utr [14, 15] . the different sizes and locations of the regions amplified in the hrv genomes renders difficult comparative genetic analysis. recently, palmenberg et al. (2009) have finalized the full-length genome sequences of all hrv-a and hrv-b reference strains, and identified structural features of these two species and the novel hrv-c species [16] . in our study, we identified 34 hrvs (51.5%) that clustered differently from hrv-a and hrv-b in a phylogenetic tree that was established on the basis of vp4/vp2 sequences, which were related to recent strains classified in the novel hrv-c species (fig. 1, table 2 ). fourteen hrv-c strains (41.2%) segregated from the other 20 strains (58.8%) that were closely related to hrv-a in their 59utr sequence (fig. 2) . this led us to propose a classification of two hrv-c subspecies, hrv-cc and hrv-ca. in previous studies [15] . these strains clustered with our field strains within the hrv-cc subspecies. moreover, 17 strains that clustered with hrv-a, and had 12-35% pairwise nucleotide divergence from the nearest reference serotype [15] , clustered within the two major branches of hrv-a and hrv-ca strains (fig. 3) . therefore, we cannot ensure that some of the 17 strains were hrv-a or hrv-ca strains. kiang et al. (2008) have identified five novel hrvs out of 24 clinical samples (20.8%), which segregated from hrv-a and hrv-b, and were classified as hrv-c, and three additional strains (12.5%) that also clustered with qpm, c024, c025, c026, nat001 and nat045 [14] (fig. 2) . however, the field hrv strains of these previous studies were sequenced using a 59utr that did not match fully our sequence and that of lee et al. (2007) [15] , and could not be included in the present study for comparative analysis. interestingly, the five strains identified in california in 2007 [14] and n42 and n45 from our study were closely related to strain w37 isolated in wisconsin in the late 1990s [15] , and to nat001 isolated in the winter of 2004 in california [18] , which confirms that similar genotypes of hrv-ca are widespread [17] . the strains of hrv-c species identified in the present study were characterized by analyzing the 59utr, vp4, and part of vp2 (fig. 3) . this approach showed the advantages of covering only 59ncr, vp4/vp2, vp1 or 3d genome fragments. analyzing sequences that covered the 59utr and the downstream vp4/ vp2 capsid region allowed identification of co-infections when several clones were sequenced, and helped to locate the recombination sites in strains of the hrv-ca subspecies. thus, this region of the genome may be useful for building a database of the novel strains that are circulating worldwide. the genome of hevs is subject to frequent recombination [30, 31, 32, 33, 34, 35, 36] , with interspecies exchanges observed in the 59utr [37] . palmenberg et al. (2009) have observed intraspecies recombination in three hrv-a, with structural characteristics and phylogenetic evidence that suggests a novel clade d classification [16] . tapparel et al. (2009) observed phylogenetic incongruities in 59 utr, vp1 and 3cd sequences of two clinical isolates of hrv-a related to recombination [38] . we observed incongruent clustering of n12, n44 and n48 strains of hrv-a species in the phylogenetic trees based on the 59utr or the vp4/vp2 regions of their genomes (figs. 1 and 2, table 3 ), which suggests intraspecies recombination in the 59utr. we observed one co-infection with hrv-a and hrv-b (n16a and n16b), one with hrv-a and hrv-c (n58a and n58c), and three co-infections of hrv-a and hrv-b with hevs that may favor recombination events. previous comparison of genome sequences between 34 hrvs showed only limited recombination events and a pattern of genetic diversity lower than that observed with other picornaviruses [25] . the presence in hrv-c subspecies of sequences that share 90.5-98.6% identity with hrv-a strains (table 3) suggests that recombination events occurred between hrv-c and hrv-a. bootscanning of the 59utr of hrv-c strains also showed different sites and lengths of recombination (fig. 3c) , which suggested that there were several independent events that led to several groups of hrv-ca genotypes, which formed clusters in the phylogenetic tree (fig. 2) . comparative analysis of the full-length nucleotide sequences of two field strains of different hrv-c subspecies (n4 and n10) with those of other hrv species suggested that multiple interspecies recombination events occurred in the 59utr and in the ns2a protein gene, and that recombination also occurred in the 39utr between n4 and a strain close to n10. these findings are in agreement with those observed for other hevs, for which recombination events in the capsid-encoding sequence are very rare, probably because of structural constraints that restrict the functioning of chimeric capsids [31] . this result appeals for the full-length genome sequencing of the major representatives of the hrv-c species, in order to establish a clear understanding of the evolution and classification of the novel virus into subspecies. comparison of the coding sequences of n10 hrv-cc with other strains of hrv-ca subspecies [20] , including our field strain n4, showed high similarities in the lengths of the 11 proteins, their cleavage sites, and the structural features of vp1. these characteristics and the absence of growth in cell culture, noted in our laboratory and by others (data not shown), support the classification of the novel strains into a unique hrv-c subspecies. our clinical specimens all originated from npss from pediatric outpatients. the remarkable outcome of the study is the large diversity of genotypes that has circulated in a relatively small group of people in a district of shanghai during a 2-year observation. although some clusters of similar genotypes in a limited period of time were observed, co-circulation of different genotypes and hrv species and subspecies, and co-infections with two hrv species were observed. the prevalence of the novel hrv-c in our specimens (4.1%) differed from previous studies that associated the prevalence of the novel variant with severe disease outcomes, which ranged from influenza-like illness or infection of the low respiratory tract [17, 28] to asthma exacerbation, bronchiolitis, and febrile wheeze [7, 8, 15, 18, 20, 21, 29, 39] . all our patients showed bronchitis or pneumonia, with no etiological correlation with any of the species or subspecies of hrv. only one patient co-infected with hrv-c, iav and rsv was diagnosed with asthma among the hrv-positive patients (1.6%), whereas 100 of the 827 children had asthma (12%). the difference observed with previous studies, 44.6% [8] and 12% [29] asthma in hrv-positive patients, may be related to the criteria for enrolment. moreover, none of the patients in our study were hospitalized, which makes comparison with hospitalized children difficult [20, 24] . another criterion to consider in the trend to correlate clinical symptoms with hrv infection is the presence of co-infecting pathogens. in our study, four strains of hbov and six strains of rsv (17.6%) were identified in association with hrv-c (11.7%). hbov and rsv are common viruses diagnosed in ari, which are often associated with hrv [40, 41] , and hbov was identified in .50% of children co-infected with hrv [20] . nevertheless, the incidence of hbov in ari and in severe outcomes remains elusive [42] . more studies need to be carried out on large numbers of samples from severe and mild diseases, to identify any obvious role of hrv sequence diversity and association with other pathogens in disease severity. since a large diversity of recombination in hrvs has become obvious, we must be aware of the occurrence of novel hrvs that may become highly virulent. this study was approved by the ethical committee of shanghai nanxiang hospital and written informed consent was obtained from the parents of the children. clinical specimens (n = 827) from npss were collected from children under 14 years old, who experienced a lower respiratory tract infection, and who were consulting the pediatric department of shanghai nanxiang hospital during the period october 2006 to october 2008. total rna was extracted from nps specimens using qiaamp viral rna mini kit (qiagen, hilden, germany), and stored at 280uc. rna was amplified using the qiagen one step rt-pcr kit. a five-tube mrt-pcr was used for virus identification as previously described [43, 44] . tube 1 targeted iav, influenza b virus, rsv, and human metapneumovirus; tube 2, parainfluenza viruses 1 to 4; tube 3, hrv and influenza c virus; tube 4, human coronaviruses (hcovs) 229e-hcov, oc43-hcov, nl63-hcov and hku1-hcov; and tube 5, adenovirus and hbov. amplified products were analyzed in 0.5 mg/ml ethidium bromide/2% agarose gel. samples that showed positive results for hrv were amplified again using specific primers p1-1f and vp4/2r, located in the 59utr and vp2 gene, respectively (table 1) . one strain of hrv-c (n1) could not be amplified using the p1 and vp2 extreme primers and was amplified using primers in 59utr and vp4/ vp2, respectively (table 1 ). in brief, 2.5 ml of extracted rna was mixed with 56 buffer and 0.4 mm dntps, 0.2 mm of each of the primers, and 1 ml of enzyme mix, and diethylpyrocarbonatetreated ultrapure water was added to a final volume of 25 ml. amplification programs included reverse transcription at 50uc for 30 min, inactivation at 95uc for 15 min, followed by 40 cycles at 94uc for 30 s, 50uc for 30 s, 72uc for 70 s, and final extension at 72uc for 10 min. the amplified dna products were detected by ethidium bromide-agarose gel electrophoresis. the lengths of p1-vp2, vp4-vp2 and p1-p3 amplicons were 924, 559 and 390 nt, respectively. dna products were extracted from agarose gels by using qiaquick gel extraction kit (qiagen), and were ligated into pmd20-t vector (takara biotechnology, dalian, china), and at least two recombinant plasmids were sequenced in biosune sequence company and life biotechnology in shanghai, china. sequences of different clones of n16 and n58 showed identities for either hrv-a or hrv-b strains. more plasmids were sequenced for these strains to confirm that the two patients were originally coinfected with two different hrv species. sequences of three complete genomes of hrv were obtained for strains n4 (reference r3061207002 collected on december 7, 2006) , n10 (r3070614001 collected on june 14, 2007) and n13 (r3070719007 collected on july 19 2007). primers used for the amplification of viral genomes were designed after multiple alignments of sequences from the genomes of different hrvs available in genbank (table 1) . overlapping amplified dna products were obtained after pcr of cdna that was obtained using oligodt and a transcriptor high fidelity cdna synthesis kit (roche, mannheim, germany), following the manufacturer's protocols. briefly, 10.4 ml viral rna was mixed with 1 ml oligodt and heated at 65uc for 10 min, and then kept on ice for 2 min. after addition of 4 ml 56buffer, 0.5 ml protector rnase inhibitor, 2 ml dntps, 1 ml dtt, and 1 ml rt enzyme, the reaction was incubated at 50uc for 1 h, inactivated at 85uc for 5 min, and stored at 220uc. amplification of a 3d region of n4, n10 and n13 hrv strains was carried out by nested-pcr using takara extaq (takara biotechnology) and specific primers (table 1) , for 35 cycles of 30 s at 94uc, 30 s at 55uc, and 70 s at 72uc. to amplify vp1 (upstream of 2a) sequences of n4 and n13 strains, nested pcr was carried out using takara lataq with gc buffer i(takara biotechnology), specific primers (table 1) , and incubation for 35 cycles of 30 s at 94uc, 30 s at 60uc, and 4 min at 68uc. the fragment vp2-3d of n10 hrv strain was obtained by seminested pcr and specific primers vp2 f, and 3d inner and outer reverse primers (table 1) , using takara lataq with gc buffer ii, for 35 cycles of 30 s at 94uc, 30 s at 60uc, and 6 min at 68uc. the terminal part of the whole genome was obtained by rapid amplification of cdna ends using 59/39 rapid amplification of the cdna kit, following the manufacturer's protocol (roche). to perform 59 terminal race, 4 ml of 56cdna buffer, 2 ml dntps, 1.25 ml specific primer 1 (10 mm), 9.2 ml rna, 1 ml control primer neo1/rev (12.5 mm), 1 ml control rna, 1 ml rt enzyme, and 0.6 ml rnase inhibitor (roche) were mixed and incubated for 55uc for 60 min, followed by inactivation at 85uc for 5 min, and stored on ice. the product was purified using the qiagen pcr purification kit and eluted with 30 ml deionized distilled water. a polya tail was added to the cdna, by mixing 9.5 ml dna with 1.25 ml 106 reaction buffer, 1.25 ml (2 mm) datp, and after incubation at 95uc for 3 min, the reaction was chilled on ice for 2 min. after addition of 0.5 ml terminal transferase, the reaction was incubated at 37uc for 30 min, inactivated at 70uc for 10 min, and kept on ice. nested pcr was performed by using the expend high fidelity pcr kit (roche). a mixture of 2.5 ml poly-da-tailed cdna, 0.5 ml oligodt-anchor primer 37.5 mm, 0.62 ml sp2 primers (10 mm) (table 1) , 0.5 ml control neo2/rev primer (12.5 mm), 0.5 ml dntp, 0.35 ml enzyme, 2.5 ml 106 buffer, and 18 ml ddh 2 o was incubated for 40 cycles of 30 s at 94uc, 30 s at 60uc, and 30 s at 72uc. to perform 39 terminal race, the method was similar to normal two-step rt-pcr using 3d inner and outer f primers (table 1) . sequence alignment, phylogenetic analyses and recombination analysis dna sequences used for p1-p2 gene analysis were based on hrv-16 nt 178-462 and those used for vp4/vp2 gene analysis were based on hrv-16 nt 626-1045. multiple sequences were aligned using clustal x [45] . the multiple-sequence alignment was subjected to phylogenetic analyses using programs in the phylip package (v3.6). bootstrap analysis was performed using seqboot, with a replicate number of 1000. then, dnadist and neighbor were used to obtain distance matrices with the f84 parameter, and a transition/transversion ratio of 4. consensus trees were computed by consense, and then re-rooted with retree. the final tree was visualized and edited with mega version 4 [46] . recombination analysis was carried out by using recombination detection program v.3.22. manual bootscanning was performed by using the juke-cantor algorithm and the neighbor-joining method [47] , with a window size of 200 nt, a step size of 20 nt and 100 replicates. pairwise identities between sequences were determined with simplot software method [48] ,with a window size of 200 nt and a step size of 20 nt. pairwise homology matrices were obtained by using clc combined workbench 3.0 software (clc bio, aarhus, denmark). the original p1-vp2 sequences described in this study were deposited in genbank under accession nos. gq223119 to gq223136. the vp4-vp2 sequences were deposited under the nos gq223137 to gq223181, and the p1-p2 sequences under the nos gq223182 to gq223226. the full length genomes sequences 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genomic features of intertypic recombinant sabin poliovirus strains excreted by primary vaccinees recombination in circulating enteroviruses improved molecular identification of enteroviruses by rt-pcr and amplicon sequencing co-circulation and evolution of polioviruses and species c enteroviruses in a district of madagascar evidence of recombination among enteroviruses frequency and dynamics of recombination within different species of human enteroviruses genome analysis of circulating picornavirus reveals rhinovirus recombination and a new enterovirus serotype. xv european study group on the molecular biology of picornaviruses meeting enterovirus surveillance reveals proposed new serotypes and provides new insight into enterovirus 59-untranslated region evolution new respiratory enterovirus and recombinant rhinoviruses among circulating picornaviruses molecular characterization of a variant rhinovirus from an outbreak associated with uncommonly high mortality association of rhinovirus infection with increased disease severity in acute bronchiolitis the incidence of human bocavirus infection among children admitted to hospital in singapore human bocavirus: clinical significance and implications development of three multiplex rt-pcr assays for the detection of 12 respiratory rna viruses detection of human bocavirus in hospitalised children clustal w and clustal x version 2.0 mega4: molecular evolutionary genetics analysis (mega) software version 4.0 a modified bootscan algorithm for automated identification of recombinant sequences and recombination breakpoints full-length human immunodeficiency virus type 1 genomes from subtype cinfected seroconverters in india, with evidence of intersubtype recombination key: cord-000979-cav9n18w authors: hoppe, sebastian; bier, frank f.; nickisch-rosenegk, markus v. title: rapid identification of novel immunodominant proteins and characterization of a specific linear epitope of campylobacter jejuni date: 2013-05-29 journal: plos one doi: 10.1371/journal.pone.0065837 sha: doc_id: 979 cord_uid: cav9n18w campylobacter jejuni remains one of the major gut pathogens of our time. its zoonotic nature and wide-spread distribution in industrialized countries calls for a quick and reliable diagnostic tool. antibody-based detection presents a suitable means to identify pathogenic bacteria. however, the knowledge about immunodominant targets is limited. thus, an approach is presented, which allows for the rapid screening of numerous cdna derived expression clones to identify novel antigens. the deeper understanding of immunodominant proteins assists in the design of diagnostic tools and furthers the insight into the bacterium’s pathogenicity as well as revealing potential candidates for vaccination. we have successfully screened 1536 clones of an expression library to identify 22 proteins that have not been described as immunodominant before. after subcloning the corresponding 22 genes and expression of full-length proteins, we investigated the immunodominant character by microarrays and elisa. subsequently, seven proteins were selected for epitope mapping. for cj0669 and cj0920c linear epitopes were identified. for cj0669, specificity assays revealed a specific linear epitope site. consequently, an eleven amino acid residue sequence tlikelkrlgi was analyzed via alanine scan, which revealed the glycine residue to be significant for binding of the antibody. the innovative approach presented herein of generating cdnas of prokaryotes in combination with a microarray platform rendering time-consuming purification steps obsolete has helped to illuminate novel immunodominant proteins of c.jejuni. the findings of a specific linear epitope pave the way for a plethora of future research and the potential use in diagnostic applications such as serological screenings. moreover, the current approach is easily adaptable to other highly relevant bacteria making it a formidable tool for the future discovery of antigens and potential biomarkers. consequently, it is desirable to simplify the identification of structural epitopes, as this would extend the spectrum of novel epitopes to be detected. c. jejuni is a gram-negative, microaerophilic bacterium possessing a helical-shaped morphology [1] . in industrialized countries, c. jejuni has been one of the primary causal agents of gastroenteritis. in 2012, in germany alone 62626 cases have been reported [2] . campylobacteriosis predominantly induces mild, self-limiting diarrhoea, however severe cases have been reported [3] . several studies have shown the potential contribution of campylobacteriosis in the development of neuropathies such as the guillain-barré syndrome [4, 5] . the prominent route of infection is the improper handling and insufficient cooking of poultry [6] . the broad distribution of this pathogen in combination with a high clinical relevance necessitates fast and reliable diagnosis. although several genomic typing methods exist [7, 8] , these are often timeconsuming and inappropriate for a point-of-care application. instead, a direct approach detecting the whole bacterium is beneficial. this can be achieved by using specific antibodies to membrane-associated antigens similar to the latex-agglutination-test that is already available for several bacterial pathogens including campylobacter [9] . in order to achieve this, copious knowledge of potentially suitable targets, i.e. immunodominant proteins, is indispensable. in the past, screening for immunogenic proteins has been carried out on nitrocellulose membranes [10] or using microarray library screening with extensive protein purification [11] . however, these methods have some major drawbacks as the former is prone to non-specific binding and cross-reactivity when using polyclonal sera for screening of bacterial libraries [12] , while the latter method is time-consuming and laborious due to the purification steps needed prior to microarray printing. as we have shown elsewhere, an approach using halotagh and specifically coated halolink tm slides is better suited to detect immunodominant proteins while reducing cross-reactivity to a minimum [13] . the halotagh provides several advantages to other commonly used tags as it enhances the amount of soluble proteins expressed [14] reducing the formation of inclusion bodies. in addition, the interaction of tag and its specific ligand is based on covalent binding [15] . this negates the need for additional purification steps as the crude lysate can simply be spotted onto coated microarrays. only the target proteins presented as fusion constructs bearing a halotagh will bind to the surface, whereas the remaining proteins are washed off. combining the described screening method with expression libraries derived from c. jejuni cdna allows for the fast analysis of hundreds of different proteins. thus, suitable immunodominant proteins can be detected, isolated and identified via sequencing the encoding cdna sequence. the generation of a cdna derived expression library offers advantages in contrast to genomic libraries. the latter demand excessive screenings as the genetic information is mostly truncated or of little relevance representing areas within the genome that do not encode for proteins, whereas the former focuses on the genes transcribed [16] . this reduces the amount of clones to be screened. nevertheless, for effective cdna library screening normalization is needed, as rrna is mainly overrepresented due to its extreme abundance within a total rna extraction prior to reverse transcription [17] . bacteria only posses a poly(a)-tail on their mrna in rare cases [18, 19] . although methods exist to isolate mrna from bacteria [20, 21] , it is generally considered to be more challenging as compared to eukaryotic rna, where oligo(dt) primers are sufficient [22] . therefore, we refrained from isolating the mrna prior to reverse transcription. instead, the generated cdna was normalized, i.e. trimmed down, afterwards by the use of a duplex-specific nuclease [23] . this approach has been shown to effectively reduce the amount of rrna-derived molecules, thereby altering the overall composition in favour of the mrnaderived cdna without including a bias [24] . further optimization of library construction was achieved by using a ligationindependent cloning as well as electroporation, which have been shown to enhance overall cloning efficiency [25, 26] . using this approach, a relatively small number of clones can be screened to illuminate immunodominant proteins effectively. the identification of previously unknown or incompletely described antigens offers several benefits and potential applications. first, these proteins might serve in a diagnostic tool to rapidly identify c. jejuni in biological samples, e.g. in food manufacturing industry or hospitals. secondly, proteins eliciting an immune response might be suitable candidates for vaccination. finally, gaining insight into the structure and function of novel immunodominant proteins might improve the overall understanding of a bacterium's pathogenicity as it could accelerate the elucidation of novel virulence-associated factors. in this paper, we show the successful screening of an expression library of c. jejuni identifying several potentially immunodominant proteins. after further investigations, we selected a subset of these proteins for epitope mapping and succeeded in identifying linear epitopes for two proteins, namely cj0669 and cj0920c not described before. furthermore, assays were performed to assess specificity of the binding as well as investigating the relevance of the amino acid residues involved via alanine scanning. additionally, the structure and antigenicity of the proteins and epitopes were modelled to analyze the suitability of the identified sequences for future applications like diagnostic tools or vaccine development. the rin values for the c. jejuni rnas isolated were above 8.5 in all cases (see s1: rin). after polyadenylation of the rna, it was reverse transcribed and subsequently normalized using a duplexspecific nuclease (dsn). assessing the performance of normalization, we sequenced a sample set of 96 individual clones after transformation with trimming and without trimming. without dsn treatment 28% of clones contained 23s rrna derived cdna and 8% other rrna derived cdna (16s and 5s). in comparison, after incubation with dsn only one clone in 96 showed a 23s rrna derived cdna. a total number of 1536 different clones were screened using halolink tm slides. we used hisj (uniprot/swiss-prot: q46125), cjaa (q0p9s0) and peb1a (q0p9x8) as positive reference markers, as they have been described previously to be immunodominant [27] [28] [29] . in contrast, argc (q9pis0) and pyrc (q0pbp6) were used as negative reference proteins. after comparing the median value and the standard deviation of each sample to the values of the positive references, a selection of 192 clones were picked to be sequenced. generally, we grouped clones into three categories after screening. group i included samples showing a higher median contrast value than all the positive references used, group ii encompassed the samples with median contrast values in between those of different positive references and group iii the remaining samples which albeit below the lowest positive reference were still above all the negative reference signals. after screening 1536 clones, 32% fell into group i, 15% encompassed group ii and 4% made up group iii while the remaining 50% were below the negative controls. the sequenced clones were ''blasted'' against the genome of c. jejuni nctc 11168 (refseq: nc_002163) to identify the corresponding genes and proteins. for further experiments, known antigens of c. jejuni found within the sequenced clones were discarded and we focused solely on 22 novel potentially immunogenic proteins. several of the identified clones carried only truncated fragments of the corresponding genes. in addition, some of the sequenced inserts possessed a frame shift. in order to overcome these limitations fulllength inserts were prepared and subcloned to express the fulllength proteins. the 22 genes and their corresponding proteins are summarized in table 1 including the length as well as the size of the protein without the attached halotagh. recombinant expression of the full-length fusion proteins was assessed by sds-page using a halotagh 488 alexa ligand, see figure 1 . the correct-sized proteins are highlighted as they are expressed fused to the 34 kda halotagh. bands at roughly 34 kda in size are visible throughout, likely representing early-termination transcripts comprised only of the halotagh. evaluating immunodominance of these proteins, we performed 32 microarray and elisa analyses using three different primary antibodies against c. jejuni. summing up these results, table 1 shows the respective mean q values and errors (n = 25) for each of the proteins identified. the highest scores were attained by cj0926 and cj0927 with 1.7760.75 and 2.2360.58 respectively, indicating a potentially stronger immunogenicity as compared to the known immunodominant proteins hisj and cjaa. however, we restrained from further characterizing the former two proteins via epitope mapping as both are highly conserved and show multiple homologies in cellular organisms (cj0927) or epsilonproteobacteria (cj0926) according to the ncbi protein cluster database [30] . the same rationale was applied for cj1576, which is conserved in cellular organisms and peaks at 1.5560.43. instead, we focused on cj0623, cj1575 and cj0669, which are highly conserved in campylobacter only as well as cj0920c and cj1380, which in turn are conserved in campylobacteraceae. cj1380, cj0623 and . for almost all proteins, bands were visible with the respective sizes of their molecular weight and the halotag tm . the halotag tm alone at 34 kda is visible throughout. the proteins were as follows: 1 -cj0476, 2 -cj1723, 3 -cj1208, 4 -cj1619, 5 -cj0920c, 6 -cj1380, 7 -cj1381, 8 -cj0669, 9 -cj1624, 10 -cj1320, 11 -cj1486, 12 -cj0130, 13 -cj1366, 14 -cj0016, 15 -cj1575, 16 -1576, 17 -cj1729, 18 -cj0571, 19 -cj0926, 20 -cj0927, 21 -cj0623 and 22 -cj1138. the pictures of the two gels as well as the righthand marker were fused by coreldraw to minimize space. for all proteins except cj0920c (5) and cj1320 (10) the overlapping peptide sequences corresponding to each protein were mapped with three different primary antibodies to c. jejuni. each peptide contains 15 amino acid residues with an overlap of 11 to each adjacent peptide. cj0669, an abc-transporter atp-binding protein, showed significant intensities in two adjacent peptide spots as shown in figure 2 . the consensus sequence derived from these spots is tlikelkrlgi, which is highly conserved in c. jejuni (see s2: tlikelkrlgi is conserved in c. jejuni). next, we assessed specificity of these signals by applying antibodies reactive to other bacteria then c. jejuni in the epitope mapping to assess whether the binding occurs via the paratope of the antibody or in a nonspecific manner. figure 3 shows the results of these investigations. the mean rfis (n = 12) of peptide 44 and 45 are at 8000 and 6000 a.u. respectively after incubation with polyclonal antibody to c. jejuni, whereas these intensities drop below 50 a.u. if an e.coli antibody is used. the remaining proteins painted a different picture. for cj1575 (s3:epitope mapping of cj1575) -a fragment with only 75 residues -no linear epitope was identified. the same was true for cj0623 (s4:epitope mapping of cj0623), cj0476 (s5:epitope mapping of cj0476) as well as cj1723 (s6:epitope mapping of cj1723) which harboured no linear epitopes. for the protein cj1380 (s7:epitope mapping of cj1380) only one peptide showed signal intensities above the positive control. another abc-transporter protein, cj0920c, possesses several regions, where signal intensities were above those of the positive controls, namely peptides 6 to 8 (aa 21 -43), 17 to 19 (aa 65 -87) as well as 56 and 57 (aa 221 -239), see s8: epitope mapping of cj0920c. the three potential antigenic regions were further assessed using transmembrane prediction tool tmhmm2.0 and the antigenic prediction tool by emboss. here, peptides 6 -8 showed a consensus sequence, namely spfavwkfldal, which ought to be presented extracellular as well as being antigenic according to the prediction tools. for the other two sites, either no antigenicity was predicted or most of the amino acids lie within a transmembrane region. this is true for amino acids 47 -69, 90 -112 and 214 -236. for a summary of the predicted characteristics, see s9: transmembrane and antigenic potential of three potential epitope sites for cj0920c.further, specificity control assays revealed that these signals do not drop significantly when using antibodies to salmonella enterica indicative of nonspecific binding to occur, see s10: specific vs. non-specific binding to potential linear epitopes of cj0920c. the influence of each amino acid within the consensus sequence tlikelkrlgi was assessed by performing an alanine scan. as figure 4 shows, the majority of amino acids in the sequence do not confer a change in binding intensity if replaced by alanine. however, in case of glycine a substantial drop in rfi values to less than 10% of the original value is observed. this indicates an important role of the glycine residue in the binding of the antibody. further, these results were highly reproducible in a second set of experiments and the specificity of the binding was evaluated using antibodies reactive to helicobacter pylori as summarized in figure 5 . the green boxes on the left represent the values of original sequence tlikelkrlgi and altered sequence tlikelkrlai after incubation with polyclonal antibodies to c. jejuni. as was observed in the previous figure, the drop in intensity is significant from a mean value of approximately 38000 a.u. to less than 7900 equal to roughly 80% decrease in the signal. in comparison, the adjacent boxes represent the data of the original sequence as well as tlikelkrlai after incubation with antibodies reactive to helicobacter pylori (orange).here, no significant difference between unaltered and altered sequence could be observed. furthermore, the mean intensities were below 800 a.u. thus, binding of an anti-helicobacter pylori antibody to the sequence is approximately 2% as compared to the original binding of polyclonal antibodies to campylobacter jejuni against this linear epitope. the same trend was observed when using antibodies reactive to s. enterica (s11: binding specificity assay of tli-kelkrlgi with anti-salmonella antibodies). this indicates low non-specific binding. modelling of cj0669's structure was performed by swiss-model using automated mode. the template chosen during automatic identification was pdb1ji0a from the rcsb protein data bank [35] , which referred to the crystal structure analysis of the abc transporter from thermotoga maritima [36] . the model spans amino acids 3 to 236 of the molecule incorporating more than 90% of the residues. figure 6 shows a graphical display of the structure with the distinct secondary structures highlighted. in addition, the epitope sequence tli-kelkrlgi is marked in orange. the first nine residues comprise parts of an alpha helix, whereas glycine and isoleucine form a loop to attach the alpha helix with a beta strand further down the primary sequence of the protein. the modelling was supported by statistical analyses including determination of a z-value [37] based on published data in the protein data bank. figure 7 shows the accuracy of the model based on the z-value calculations. we analyzed the consensus peptide sequence tlikelkrlgi by geneious pro 5.6.5. notably, when predicting secondary structure and antigenic regions by emboss garnier and antigenic tool respectively, the results matched the previous observations. an alpha helix precedes a loop that is connected to a beta-sheet. the only difference to the structural modelling by swiss model is the number of residues within the loop as emboss only includes the glycine residue in the loop. for antigenic potential, emboss antigenic predicts all amino acids within the consensus sequence to be part of antigenic sites except for lysine. the development of a cdna derived expression library and subsequent screening of the expressed fusion proteins has revealed several novel immunodominant proteins. optimization during library construction by reducing the amount of rrna molecules via dsn treatment has proven to greatly reduce the amount of rrna clones as was shown for other applications elsewhere [38, 39] . this enhances the number of clones carrying mrnaderived cdna, thus increasing the number of clones potentially expressing proteins of interest. for screening purposes antibodies were used, which were generated by immunizing rabbits with whole and partly lysed cells of c. jejuni. therefore, both membrane-associated as well as soluble proteins were available during immune response. thus, it was to be expected that the antibodies used in screening target both membrane and cytosolic proteins, which could be observed in the proteins identified. the screening not only detected the 22 proteins listed here but included other already known immunogenic proteins. these were excluded from further analyses as the main goal of this work was to identify novel proteins and their linear epitopes. the screening and analysis of immunodominant behaviour by microarrays was highly reproducible. some of the proteins showing the strongest signals during screening and microarray experiments were discarded, as they possess strong homologies within a wide spectrum of organisms. however, we focused on proteins specific for campylobacter due to the potential application in a diagnostic tool. thus, linear epitope mapping was performed on a subset of seven proteins. this revealed linear epitopes for two proteins, cj0669 and cj0920c, both abc-type transporters, which were expected to be immunogenic [40] . the identified region tlikelkrlgi of cj0669 proved to be specific showing little to none non-specific interactions with other antibodies. most notably, h. pylori antibodies did not reveal any binding to the linear epitope. in the wake of the other antibodies tested, i.e. anti-e.coli and anti-s. enterica, this strongly suggests non-specific binding to be minute. furthermore, the applicability of tlikelkrlgi as a specific target for campylobacter identification, antibody or vaccine production seems plausible. alanine scan has further identified the glycine residue to be of utmost importance for the antibody binding as replacing it by alanine reduces the intensity by at least 80%. this has been somewhat surprising as glycine is a non-charged, achiral residue. however, the presence of glycine might cause the protein to turn more easily provided by the glycine's flexibility and consequently leading to a better accessibility. thus, immunogenic ability might be rendered by the presence of glycine. in fact, epitopes containing glycine as an important residue have been reported before [41] [42] [43] . next, we modelled the 3-dimensional structure of cj0669 in order to evaluate the accessibility of the potential epitope. this is an important feature when bearing a diagnostic application in mind. from the model, we could conclude that the glycine residue and the linear epitope are most likely presented as part of a loop structure adjacent to the end of an alpha helix. these are located on the outer rim of the protein, hence easily accessible for antibody binding to occur. although, the structural information is merely based on modelling, the z-values of this model fall within close proximity to zero (-0.88) commonly associated with x-ray crystallographic results [37] . consequently, the probability of this model to match the real structure is high. these findings are supported by further calculations performed using the emboss garnier and emboss antigenic algorithms. these results underline the antigenic potential of the consensus sequence tlikelkrlgi as well as predicting almost identical secondary figure 5 . binding specificity assay of epitope sequence in alanine scan. box-whisker-plot (n = 12) of the eleven residue long consensus peptide sequence tlikelkrlgi and its derivative tlikelkrlai of cj0669 after incubation with antibodies reactive to c. jejuni (green) and h. pylori (blue). each box represents 50% of the values, while 98% fall within the whiskers. the median is represented by a horizontal line within each box and the small rectangle corresponds to the mean for each sample. for references the rfi values of the positive control, rabbit igg, and negative control, mbp, are indicated on the right for both antibody incubations. for the original sequence a mean value of 38000 a.u. can be observed after incubation with antibody to c. jejuni. this dropped to less than 8000 a.u. after alanine substitution. this represents a drop of roughly 80% in overall intensity. in contrast, neither original nor substituted peptide showed any significant mean values after incubation with the anti-h. pylori antibodies indicating specific binding to the peptide by the anti-campylobacter antibody. doi:10.1371/journal.pone.0065837.g005 structures as the automated swiss model. moreover, the secondary structure prediction changes when glycine is replaced by alanine, which further underlines the important role glycine most likely plays in antibody binding due to its ability to create a loop structure which would otherwise be absent with high probability. in conclusion, tlikelkrlgi seems a suitable candidate to be studied further and shows high potential for applications in a diagnostic tool or vaccination due to its good accessibility and antigenicity. this gains further momentum as tlikelkrlgi is highly conserved in c. jejuni, whereas other species of campylobacter, e.g. c. coli, c. upsaliensis or c. lari possess other amino acid residues within this region. for helicobacter pylori, this effect is even more pronounced. specifically, the glycine residue at the tenth position that was revealed paramount for the binding is not present in helicobacter pylori, see s2. thus, tlikelkrgli seems a suitable candidate for specific diagnostic applications targeting c. jejuni. the analysis of the remaining six proteins revealed linear epitopes only in the case of cj0920c, another abc-type transporter. the region encompassing amino acids 27 -38 spfavwkfldal seems to be the most promising as its rfi values are high in microarray experiments. furthermore, the prediction tools determined this sequence to be antigenic and located extracellularly, thus easily accessible. this is not true for the other two regions detected from cj0920c, which are either lacking antigenic potential or are located within the cell or in transmembrane regions. still, the results indicate non-specific binding to contribute mainly to the positive signal. this exempts spfavwkfldal from a suitable diagnostic application; however, further analysis might be helpful to investigate the full potential of this sequence as a specific epitope. although the analyses of full-length proteins on microarrays hint at the presence of antigenic sites within each protein, a lack of linear epitopes was observed. however, this was expected, as most naturally occurring epitopes are conformational and not linear [44] [45] [46] [47] . the current method used for epitope mapping cannot detect conformational epitopes. still, the presence of linear epitopes and their detection are important features in serological applications. this is particularly true for the high-throughput analysis of sera on microarrays as described elsewhere [48, 49] . the present work has accomplished two main goals. first, we have been able to identify a previously unknown antigenic site tlikelkrlgi of cj0669 from c. jejuni and were able to determine the important residue involved in antibody binding as well as modelling the epitope's accessibility within the full-length protein. for c. jejuni the generation of monoclonal antibodies against cj0669 is mandatory to further investigate the affinity and kinetics of the observed binding event by biacore. this might grant further insight whether or not this sequence is a suitable candidate for specific detection in a biosensor or if cj0669 is a suitable vaccine candidate. mutagenesis of cj0669 might help to illuminate the function of this protein and its potential role if any in pathogenicity. once this is determined, a monoclonal antibody might be used to cocrystallize the antigen for x-ray crystallography to assign a measured structure to the predicted model. on top of that, antibody validation by elisa with a wide array of c. jejuni isolates is needed to evaluate the applicability of the derived antibody for future clinical point-of-care devices. additionally, next-generation sequencing of transcriptomes of a broad spectrum of c. jejuni strains ought to be beneficial in analyzing the presence and expression of the protein. this might provide further insight into the suitability of the protein for clinical applications and vaccine development. consequently, it could help to identify potential sequence homologies or discrepancies, which as of now are limited to already published datasets. thus, nextgeneration sequencing might be an attractive procedure to complement the approach presented herein. secondly, we have established and applied a quick and easy method for the screening of cdna expression libraries in order to rapidly detect novel immunodominant proteins of bacteria, see figure 8 for a summary. this approach is easily transferable to other bacterial pathogens such as methicillin-resistent staphylococcus aureus, klebsiella pneumonia, neisseria gonorrhoea, pseudomonas aeruginosa, salmonella enterica and others. the information needed prior to analysis is minute, yet using fully sequenced strains is advantageous as it speeds up the identification of genes and proteins. still, even unsequenced strains such as clinical isolates can easily be analyzed and should not pose any hindrance, as homologous proteins ought to be identified via blast. the more important prerequisite for the screening is the availability of polyclonal antibodies or patient sera. in the context of a diagnostic tool to be used in hospitals, patient sera are beneficial, as they resemble the desired immune response better, thus narrowing down the results to the clinically most relevant proteins. finally, suitable references are useful, yet in most cases, some immunodominant antigens have already been described. regardless, even without a suitable known antigen, this method could identify novel immunoreactive proteins and their linear epitopes with some minor adjustments to the overall calculations. in conclusion, we have successfully shown the application of this method while gaining insight into some novel immunodominant proteins of an important gut pathogen, c. jejuni. the current research emphasizes the need for future investigations in two distinct areas. first, more insight into the newly identified proteins of c. jejuni might foster the understanding of this enigmatic pathogen and help to illuminate its pathogenicity while providing suitable means for rapid detection and combating its spreading. second, transferring this method to other bacterial pathogens will help to discover other immunodominant proteins, potentially leading to a broad spectrum of clinical applications and serological assays. additionally, it might be preferable to simplify the identification of conformational epitopes as this could greatly enhance the retrieval rate of suitable antigenic sites. the state-of-the-art technique involves the cocrystallization of an antigen with a monoclonal antibody [50, 51] . besides, mutation analysis [52] , mass-spectrometry [53] or clips technology [54] are other methods to identify structural epitopes. however, the existing methods are extremely costly, time-consuming and demand high material inputs. therefore, advances to simplify structural epitope detection are essential and would be ideally suited to be combined with our current approach. nevertheless, linear epitopes provide an important tool for clinical applications. while their low abundance poses a problem, their simplicity grants major benefits. they are rapidly synthesized and modified. this enables them to be used in a broad spectrum of assays. in a clinical context where antibody titer determination of patient sera is necessary, short peptides are extremely valuable. production costs using chemical synthesis are relatively low and easier than recombinant expression of full-length proteins. furthermore, specific peptides of pathogenic bacteria remove the need to use the entire pathogen, thus reducing the associated risk. although prediction-based strategies for antigens and linear epitopes have been published [55] [56] [57] [58] , their accuracy is often lacking [59] . in contrast, our approach offers an attractive dual procedure as it rests on experimental data that are complemented by a widespread support of bioanalytical tools. thus, such a thorough approach facilitates to focus on relevant epitopes quickly, while rapidly evaluating their suitability for prospective diagnostic applications as compared to prediction-based or experimental methods alone. consequently, we believe our present findings to be of outstanding interest for diagnostic applications and to pave the way for future implementation. moreover, the ability to quickly generate cdna libraries and identify novel immunodominant figure 8 . schematic summary of the methods involved for library construction, screening and characterization. a bacterial pathogen is selected and its rna isolated. after cdna generation, normalization is performed to minimize the number of rrna derived clones. next, screening is performed using polyclonal antibodies and potentially immunogenic proteins are selected. the corresponding clones are sequenced and the genes and proteins identified, either by exact match (sequenced strain) or homology (clinical isolate). a set of candidate proteins is selected focusing only on previously unknown antigens, while known antigens are discarded. immunodominant nature of each full-length protein is assessed by elisa and microarray analyses to further narrow down the number of proteins to be tested via epitope mapping, if desired. linear epitope mapping reveals potential antigenic sites, which are tested for specificity and in alanine scan. finally, bioinformatic tools are used to model the 3-dimensional structure, accessibility and antigenicity of each protein. afterwards, the most promising candidates can be used in future applications including but not limited to monoclonal antibody generation, kinetic measurements, structural and functional analyses and diagnostic applications. doi:10.1371/journal.pone.0065837.g008 proteins independent of the bacterium used should lay the foundations for future research with highly relevant pathogens. creating a method to extend the detection to structural epitopes would add another tier to the current approach and greatly enhance the knowledge about antigenic sites. further, we are expanding our focus to other pathogens to help elucidate novel antigens within a wide array of clinically relevant bacteria. the strain c. jejuni nctc 11168 was grown on solid karmali media for 48 h at 37 uc under microaerophilic conditions (85% n 2 , 5% o 2 and 10% co 2 ) within a hypoxic workstation (coylabs). for rna isolation, a liquid culture was prepared by inoculating 10 ml of brain-heart-infusion broth (bhi) with a single colony and incubated overnight at 37uc, 140 rpm under microaerophilic conditions. this overnight culture was used to inoculate a flask containing 100 ml fresh bhi medium and incubated for 16 h prior to harvesting. for initial screening, a rabbit polyclonal igg antibody to c. jejuni (acris ap24002pu-n) was used. for further microarray analyses of a subset of candidate proteins, elisa, epitope mapping and alanine scanning this antibody was used as well as two other rabbit polyclonal igg antibody to c. jejuni (abcam ab22542 and abd serotec 1744-9035). the immunogen used for generation of antibodies to c. jejuni was c. jejuni atcc 29428. for specificity assays rabbit polyclonal igg antibody to e.coli bl21 (micromol #322), s.enterica (abcam ab35156), s. aureus (fitzgerald 20c-cr1274rp) and h. pylori (abcam ab20459) were used respectively. detection was achieved by usage of secondary antibodies. a goat polyclonal to rabbit igg conjugated with chromeo tm -546 (abcam ab60317) for fluorescent and conjugated with horseradish peroxidase (abcam ab6721) for a colorimetric readout was applied. the cells were harvested by centrifugation (2000 x g, 10 min). the supernatant was discarded and the pellets resuspended in fresh bhi medium. subsequently, 0.5 ml of the bacterial suspension were added to 1 ml of rnaprotect bacteria reagent (qiagen) in a 2 ml tube, vigorously vortexed for 5 s and incubated for 5 min at room temperature. after centrifugation (5000 x g, 10 min) the pellets were resuspended in 200 ml of lysis buffer (30 mm tris-cl, 1 mm edta, 15 mg/ml lysozyme, .12 mau proteinase k) by pipetting and vortexing for 10 s. the solution was incubated for 10 min using a thriller thermomixer (peqlab) at room temperature and 1000 rpm. after addition of 700 ml buffer rlt and 500 ml 96% ethanol, the lysate was applied to rneasy bacteria mini kit spin columns (qiagen) for rna isolation following the manufacturer's instructions. after loading of the lysate, an on-column dnase digest was performed using rnasefree dnase i solution (qiagen) according to manufacturer's instructions. the isolated total rna was eluted in 50 ml of rnasefree water and its concentration and purity analyzed by nanodrop (peqlab) measurements. the quality of isolated rna was assessed using the rna 6000 pico kit and bioanalyzer 2100 (agilent). the total rna was diluted to a working concentration of 200 -500 pg/ml. the analysis was performed following manufacturer's instructions and the rna integrity number (rin) calculated by the 2100 expert software (agilent). the rin is defined to fall into a range of 0 to 10, with a higher score indicating an intact rna, whereas lower numbers are associated with degraded rna molecules [60] . in order to use bacterial mrna as a substrate in cdna synthesis, polyadenylation was mandatory. the tailing was achieved using the poly(a) polymerase tailing kit (epicentre) following the alternate protocol offered by the manufacturer. briefly, up to 10 mg of total rna were combined with 2 ml poly(a) polymerase reaction buffer, 2 ml 10 mm atp, 0.5 ml riboguard rnase inhibitor and 1 ml poly(a) polymerase (4 u) in a total reaction volume of 20 ml. the reaction was incubated for 20 min at 37uc, terminated by the addition of 1 ml 0.5 m edta and purified by rneasy mini kit (qiagen) following manufacturer's instructions. yield and purity were determined by nanodrop measurements. for cdna synthesis, the in-fusionh smarter tm directional cdna library construction kit (clontech) was used according to manufacturer's instructions with slight modifications. 3.5 ml total, polyadenylated rna were mixed with 1 ml of 39 in-fusion smarter cds primer, heated first for 3 min at 72uc and then incubated for additional 2 min at 42uc. after addition of 5.5 ml mastermix (2 ml 5x first strand buffer, 0.25 ml 100 mm dtt, 1 ml 10 mm dntps, 1 ml 12 mm smarter v oligonucleotide, 0.25 ml rnase inhibitor and 1 ml smartscribe tm reverse transcriptase) the tubes were incubated for 90 min at 42uc. the reaction was terminated at 68uc for 10 min. for second strand cdna synthesis two 2 ml aliquots of first strand reaction were used in long distance pcr using phusion polymerase (finnzymes). each pcr reaction was comprised as follows: 2 ml first-strand reaction, 70 ml rnase-free water, 20 ml 5x phusion hf buffer and 2 ml each of dntp mix (10 mm), 59 pcr primer ii a (12 mm), 39 in-fusion smarter pcr primer (12 mm) and phusion polymerase with a total reaction volume of 100 ml. the pcr reactions were subjected to the cycling program with 98uc for 1 min as initial denaturation followed by 15 cycles of 10 s denaturation at 98uc, 30 s of primer annealing at 65uc and 6 min extension at 72uc. for improved pcr results optimization was performed as follows; 30 ml of the 15 cycle experimental tube were transferred to a separate pcr tube, cycling commenced and aliquots of 5 ml each were collected after 15, 18, 21, 24 and 27 cycles total. the different cycles were compared by gel electrophoresis and the experimental tubes subjected to additional cycles if necessary. finally, pcr reactions were purified using the qiaquick pcr purification kit (qiagen). the purity and yield of each reaction were analyzed by nanodrop measurements. normalization of double-stranded cdna was achieved with the trimmer-2 cdna normalization kit (evrogen) to reduce the number of cdna molecules derived from rrnas. briefly, 12 ml of cdna (approx. 100 ng/ml) were mixed with 4 ml of 4x hybridization buffer. for the trimming reaction 4 ml of this mixture were distributed to four different pcr tubes and overlaid with a drop of pcr-grade mineral oil. after centrifugation (13000 x g, 2 min), the tubes were incubated for 2 min at 98uc followed by 5 h at 68uc. next, pre-heated (68uc) duplex-specific nuclease (dsn) master buffer was added to each tube and incubation prolonged for 10 min. dsn was added to the first three tubes in decreasing concentrations -1 u/ml, 0.5 u/ml and 0.25 u/ml -with the fourth tube receiving dsn storage buffer and no enzyme as a control reaction. the incubation was continued for 25 min at 68uc. after addition of 5 ml dsn stop solution and subsequent incubation for 5 min at 68uc, the tubes were placed on ice. the chilled reaction was diluted by addition of 25 ml sterile, rnase-free water. for amplification of normalized cdna, 1 ml of each reaction was used as template in pcr. each pcr reaction contained 1 ml of template from the normalization reaction, 33 ml nuclease-free water, 10 ml 5x phusion hf buffer, 1 ml 10 mm dntp mix (neb), 2 ml of each primer 59 pcr primer ii a (12 mm), 39 in-fusion smarter pcr primer (12 mm) and 1 ml phusion polymerase. the pcr was performed with initial denaturation at 98uc for 1 min and seven cycles of denaturation at 98uc for 10 s, primer annealing at 65uc for 30 s and extension at 72uc for 3 min, respectively. for optimization, the control tube was subjected to 7, 9, 11, and 13 cycles with 12 ml aliquots taken every two cycles. the optimization samples were analyzed by gel electrophoresis (1% agarose, tae, 100 v) and the optimal cycle number determined. the remaining three tubes were subjected to 9 + x cycles with x being the differential of the optimized cycles to the originally performed seven cycles. after the second pcr, the experimental reactions were compared to the optimal control reaction using gel electrophoresis as above. reactions showing a successful normalization were combined and used in a third pcr reaction. after the final pcr, the reactions were purified by qiaquick pcr purification kit. for cloning pfn18a (promega) was used as a vector, as it features a n-terminal encoded halotagh fusion protein, which allows for specific and covalent binding to a unique ligand, thus reducing background and minimizing cross-reactivity in immunoassays with halolink tm microarrays harbouring the ligand on its surface. first, the vector needed to be linearized to be used with the in-fusion cloning technology. this was achieved by reverse pcr using ifs 18a for (59 ttgataccactgcttttc-catggcgatcgcgttatc 39) and ifs 18a rev (59 tctcatcgtaccccgtgtttaaacgaattcgggctcg 39) . each reaction contained 2 ml each of 1:10 diluted pfn18a (10 ng/ml) and the two primers, 10 ml 5x phusion hf buffer, 1 ml 10 mm dntps, 0.5 ml phusion polymerase and 32.5 ml nucleasefree water to reach a total reaction volume of 50 ml. the pcr was run using a 25 cycle two-step program with 98uc denaturation for 10 s and 4 min extension at 72uc. after completion, 2 ml of dpni (20 u/ml) were added to the reaction and incubated at 37uc for 1 h. the presence of a single band was checked by gel electrophoresis and the remaining reaction purified by qiaquick pcr purification kit. cloning of normalized cdna and linearized pfn18a vector was performed following the manufacturer's instructions within the in-fusion smarter directional cdna library construction kit (clontech). electroporation 2 ml of the cloning reaction were mixed with 25 ml of electrocompetent acella tm e.coli cells (mobitec), a bl21 derivative, and electroporated in 1 mm cuvettes using the easyject plus electroporator (peqlab). conditions for electroporation were as follows: voltage = 1400 v, capacity = 25 mf, resistance = 200 v and pulse duration of 5 ms. the electroporated cells were added to 970 ml of super optimal broth with catabolite expression (soc) and incubated at 37uc for 1 h with shaking at 250 rpm. afterwards, 150 ml of the transformation reaction were plated on lysogeny broth (lb) agar containing ampicillin. for each reaction, at least two plates were prepared and incubated at 37uc for 16 h. a total number of 1536 clones were selected and transferred to 1.3 ml u96 deepwell tm plates (nunc) containing 0.8 ml lbamp. the plates were incubated overnight at 37uc, 130 rpm. on the next day, the deepwell tm plates were centrifuged, the supernatant discarded and the pellets resuspended in 370 ml of lbamp. a new set of u96 deepwell tm plates was prepared with 850 ml of fresh lb-amp and inoculated with 100 ml each from the resuspended overnight cultures. the remaining 270 ml of resuspended overnight culture were mixed with 30 ml of sterile-filtered dmso and stored at -80uc. the newly inoculated plates were incubated for 6 h at 37uc, 130 rpm. afterwards, the temperature was reduced to 20uc, incubation continued for 1 h and protein expression induced by addition of 2 ml of 0.5 m b-d-1thiogalactopyranoside (iptg). incubation persisted overnight at 20uc, 130 rpm. the cells were harvested by centrifugation (2500 x g, 10 min), the supernatant discarded and the pellets frozen at -20uc. after 15 min the pellets were resuspended in 180 ml of easylyse tm bacterial protein extraction solution (epicentre) and incubated for 5 min at room temperature. dnase i was mixed with dnase reaction buffer (10 mm tris-hcl, 2.5 mm mgcl 2 , 10 mm cacl 2 ), added to the reaction and incubation was carried on for 10 min at 37uc. the plates were centrifuged to collect cell debris for 3 min at 2500 x g. for each sample 10 ml of lysate were transferred to 384 microtiter plates (genetixx), which were used as reservoirs for the spotting procedure. the samples were spotted onto halolink tm slides (promega) using the qarray2 microarray spotter (molecular devices). 384 different samples were spotted per slide with three replicate slides per screening. in total 1536 samples were screened on 12 slides (n = 3). each sample was spotted as quadruplicates with controls in two identical sets of eighteen 10610 subarrays each (total number of spots per slide 3600). the controls used included ht-hisj, ht-cjaa and ht-peb1a as positive reference proteins as these have been described as immunodominant before. as specificity controls ht-argc and ht-pyrc were used, representing proteins without known immunodominant behaviour, thus binding of the polyclonal antibodies is not expected. in addition, two different e.coli strains -acella tm electrocompetent cells and krx single-step competent cells (promega) -were spotted as further controls. as those two lack proteins expressed with a halotagh, they are used as negative controls. after spotting of the samples, the slides were incubated for 1 h at room temperature in a humidity chamber. next, slides were washed with pbs + 0.05% igepalh ca-630 (pbsi, sigma aldrich) and dried by nitrogen flow. the 2 well proplate tm module (grace biolabs) was attached to each slide. the top chamber was filled with 1.5 ml of rabbit-polyclonal antibody to c. jejuni (acris, 2 mg/ml) in pbs. the bottom chamber was incubated with pbs only. after 2 h of incubation at room temperature with gentle rocking, both chambers of each slide were washed three times with 2 ml of pbsi. secondary antibody (goat-polyclonal to rabbit igg conjugated with chromeo tm -546, abcam, 5 mg/ml) was subjected to each chamber in pbs and the slides were incubated at room temperature for 2 h in the dark under gentle rocking. finally, slides were washed three times with pbsi, the proplate tm modules removed and the slides dried by nitrogen flow. the slides were scanned on an axon genepix 4200a laser scanner (molecular devices) with the following settings: 532 nm laser, pmt gain 400, 40% laser power, lines to average 1, 10 mm resolution and standard green emission filter at 575 nm. the raw data sets of all the microarray analyses in this publication have been deposited in ncbi's gene expression omnibus [61] and are accessible through geo series accession number gse44717. the median fluorescence intensity of each spot corrected by the local background (median f532 -b532) was used. further, relative fluorescence intensity (rfi) was calculated by subtracting the signals of the bottom chamber from the raw data signals of the top chamber to account for non-specific binding of secondary antibodies. for screening of expression libraries we used the contrast method with either argc or pyrc as specificity control to determine the contrast via the formula: with rf f i control the median of all rfis of the control used: clones harbouring strong signals in microarray screening were selected to be sequenced. sequencing was performed externally by lgc genomics using ht7f (59 acatcggcccgggtct-gaatc 39) and flxr (59 cttcctttcgggctttgttag 39) primers. after sequencing and identification of potentially immunodominant proteins, primers were designed to generate full-length clones for each identified gene, see s12 for a list of the primers used. cloning was performed as mentioned above with slight modifications. the pfn18a vector was linearized using the following primer set; 18a if linear for (59 gtttaaac-gaattcgggctc 39) and 18a if linear rev (59 ggcgatcgcgttatcgctctg 39) with pcr conditions as mentioned before. protein expression, lysis and spotting of fulllength proteins were performed as described above. the slides were incubated for 1 h at room temperature in a humidity chamber. for incubation with antibodies 3 well or 16 well proplate tm modules (grace biolabs) were attached to the halolink tm slides. processing of the slides was done similar to the original screening, however several different antibodies were used, see section antibodies. for testing of immunodominant characteristics with elisa, the crude lysate was first purified using halolink tm magnetic beads (promega) following the manufacturer's instructions. the proteins of interest were subsequently cleaved off by digestion with protev protease (promega) and concentration was determined by nanodrop measurements. the samples were diluted to a total protein content of 20 mg/ml in pbs and 50 ml of each sample was added to maxisorb plates (nunc). each sample was analyzed at least in triplicate. the elisa plate was covered with a lid and incubated overnight at 4uc in a humidity chamber. after five washing steps each with pbs + 0.05% tween-20 (pbst), the plates were blocked using 200 ml 5% non fat dried milk in pbs per well for 2 h. afterwards, plates were washed three times with pbst. 100 ml of primary antibody solution (c = 4 mg/ml) in pbs containing 1% non fat dried milk were applied to each well using the respective desired antibody or pbs for controls. the plates were incubated for 2 h at room temperature and washed four times with pbst. next, 100 ml of conjugated secondary antibody (goat polyclonal to rabbit igg conjugated with horseradish peroxidase, abcam ab6721, c = 20 ng/ml) were added to each well and incubation carried on for 1 h. finally, plates were washed once again four times with pbst and 100 ml 3,39,5,59-tetramethylbenzidine (tmb, sigma-aldrich) was added to each well for detection. after 30 min of incubation at room temperature in the dark, the reaction was stopped by applying 100 ml of 2 m h 2 so 4 to each well. the optical density of each well was measured using the omega fluostar (bmg labtech) at a wavelength of 450 nm. primers were designed using primer3 [62] within geneious pro 5.6.5 [63] . the sequenced inserts were identified by blast [64] .peptide sequence secondary structures were predicted using the emboss garnier [65] algorithm and the transmembrane regions predicted by tmhmm2.0 [66, 67] . antigenic sites were predicted by emboss antigenic [68, 69] . data evaluation was performed by originpro 8 g (originlab) and microsoft excel. 3dimensional structure predictions were performed using the swiss model automated mode [70] [71] [72] [73] [74] and pdb files were visualized and analyzed by the ucsf chimera package [75] . chimera is developed by the resource for biocomputing, visualization, and informatics at the university of california, san francisco (supported by nigms p41-gm103311). analysis of full-length proteins was achieved by combining the results from elisa and microarray data. hence, the rfi of each sample was calculated. next, a normalized rfi was generated by dividing the rfi of each sample by the median rfi of all the samples within an area of interest, i.e. incubation compartment. from these normalized rfis a median and standard deviation was calculated. if the median normalized rfi of the positive control was below the median normalized rfi of any of the negative references whilst considering the standard deviations, the test was rendered invalid. if the test passed the above criterion, the q values were calculated as follows: with rf f i sample the median of normalized rfis of the sample and rf f i pos:control the median of the normalized rfis of the positive control hisj respectively cjaa. the resulting error was calculated by error propagation according to gauss. finally, incorporating all valid tests, the mean q value was determined along with its resulting error following error propagation by gauss, see table 1 . several proteins were chosen for epitope mapping. these were the proteins encoded by cj0467, cj1723, cj0669, cj1380, cj0920, cj1575 and cj0623. the proteins were divided into 15-mer oligopeptides with an overlap of 11 amino acids in silico. the synthesis and coupling to microarray slides was performed externally by jpt peptide technologies gmbh. each peptide sequence was applied 9 times to one slide. the slides were used with proplate 3-well chamber system (grace) allowing for incubation with different antibodies. first, the slides were blocked with superblock blocking buffer (thermo fischer) for 2 h, washed five times with pbs + 0.05% tween-20, primary antibodies applied, incubated overnight at 4uc with mild rocking, washed again, secondary antibodies applied for 2 h in the dark and after a final washing procedure, dried and scanned as above. three different primary antibodies to c.jejuni were tested. the bottom chamber was always used as a control chamber, incubated only with secondary antibody. the peptide tlikelkrlgi and 11 derivatives thereof substituting one amino acid for alanine were synthesized and coupled to microarray slides by jpt peptide technologies gmbh. each peptide was applied 9 times to one slide. incubation procedure was performed as described above for epitope mapping. the expression of the desired halotagh fusion proteins was checked by sds-page. after lysis of cells, 2 ml of each protein extract was mixed with 1 ml of 10 mm halotagh alexa 488 ligand. after addition of 7 ml 1x tbs (100 mm tris, 150 mm nacl, ph 7.6) the reaction was incubated at room temperature for 30 minutes. 2 ml of each reaction were removed, mixed with 8 ml of 5x loading buffer (fermentas) and 1 ml dtt and heated for 5 min at 70uc. the separation was performed on a mini-protean tgx gel (biorad, any kd, 15 wells) in a protean ii xi cell chamber (biorad) for 30 min at 200 v. as a size reference benchmark fluorescent protein standard (life technologies) was used. fluorescence was measured in a fla-5100 (fujifilm) with excitation at 473 nm. figure s1 rin. electropherogramm and rna integrity number (rin) for sample 5, a total rna isolated from c. jejuni nctc 11168, after analysis using the rna 6000 pico kit and the agilent bioanalyzer 2100. the rin equals 9 and the ratio of 23s to 16s rrna is 1.8. on the right hand, a virtual gel picture is presented as calculated by the agilent expert 2100 software. (tif) figure s2 likelkrlgi is conserved in c. jejuni. the protein sequence of cj0669 was blasted and subsequently aligned according to a blosum62 matrix. as a reference sequence tlikelkrlgi of q0pak5, the protein encoded by cj0669 from c. jejuni nctc 11168 was used. the dots indicate an agreement to the reference, while differences are given by the one letter amino acid code. the first 13 sequences including the references are 100% identical and are all derived from c. jejuni proteins. for other species of campylobacter such as c. coli or c. upsaliensis several of the residues are replaced. for other organisms, especially helicobacter pylori the degree of replaced residues increases. the positions showing the most conservation throughout are residues 3, 6, 9 and 11. however, residue 10, the glycine which was revealed to be paramount for the binding of the antibody is not found in helicobacter pylori proteins. (pdf) figure s10 specific vs. non-specific binding to potential linear epitopes of cj0920c. the bars represent the mean values (n = 15) of rfi values after incubation with polyclonal antibody to c. jejuni (green) and salmonella enterica (orange). the mean values for each peptide fall within the same range or possess overlapping standard deviations. thus, no specific interaction of the antibody to the epitope is present; rather a non-specific binding seems likely. (tif) figure s11 binding specificity assay of tlikelkrlgi with anti-salmonella antibodies. the different sequences tested in alanine scanning are shown in the box-whisker-plot (n = 15) with each box representing 50% of the values. the whiskers encompass 98% of the values, the median is indicated by a horizontal line and the mean represented by a small rectangle. the 12 boxes in green on the left represent the results after incubation with polyclonal antibody to s. enterica. for comparison, the two red boxes show the original signals from fig. 4 for the sequence tlikelkrlgi as well as tlikelkrlai, after incubation with polyclonal antibodies to c. jejuni. all the green boxes fall into the same range as the altered sequence tlikelkrlai, where alanine replaced the glycine residue, which possessed only 10% intensity of the original sequence. thus, no specific interaction of the antibody to the epitope is present; rather a non-specific binding seems likely. (tif) figure s12 primers used in this study. the name of each primer, the corresponding target gene or vector and its sequence in 59 to 39 direction is given. for each gene, f represents the forward primer and r the reverse. the primers were used for cloning in the in-fusion smarter tm directional cdna library construction kit. (xls) proposal for a new family epidemiologisches bulletin 03 campylobacter jejuni and related species campylobacter and guillain-barré syndrome genetic basis for the variation in the lipooligosaccharide outer core of campylobacter jejuni and possible association of glycotransferase genes with post-infectious neuropathies risk factors for sporadic campylobacter infection in the united states. a case-control study in foodnet sites typing of campylobacter jejuni and campylobacter coli isolated from live broilers and retail broiler meat by flaa-rflp, mlst, pfge and rep-pcr rapid pulsed-field gel electrophoresis protocol for subtyping of campylobacter jejuni evaluation of three commercial latex agglutination tests for identification of campylobacter spp molecular cloning: a laboratory manual, third edition severe acute respiratory syndrome diagnostics using a coronavirus protein microarray immunogenic cross-reaction among outer membrane proteins of gram-negative bacteria microarray-based 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quality of individual protein structure models construction and evaluation of normalized cdna libraries enriched with full-length sequences for rapid discovery of new genes from sisal (agava sisalana perr.) different development stages construction of normalized rna-seq libraries for next-generation sequencing using the crab duplex-specific nuclease atp-binding cassette transporters are targets for the development of antibacterial vaccines and therapies a glycine-rich bovine herpesvirus 5 (bhv-5) ge-specific epitope within the ectodomain is important for bhv-5 neurovirulence identical igm antibodies recognizing a glycine-alanine epitope are induced during acute infection with epstein-barr virus and cytomegalovirus glycine-rich cell wall proteins act as specific antigen targets in autoimmune and food allergic disorders epitope mapping x-ray crystallography of antibodies b-cell epitopes: fact and fiction antigenic diversity among helicobacter pylori vacuolating toxins functional peptide microarrays for specific and sensitive antibody diagnostics serodiagnosis of echinococcus spp. infection: explorative selection of diagnostic antigens by peptide microarray three-dimensional structure of an antibody-antigen-complex epitope mapping using the x-ray crystallographic structure of complement receptor type 2 (cr2)/cd21: identification of a highly inhibitory monoclonal antibody that directly recognizes the cr2-c3d interface high-resolution epitope mapping of hghreceptor interactions by alanine-scanning mutations characterization of an anti-borrelia burgdorferi ospa conformational epitope by limited proteolysis of monoclonal antibody-bound antigen and mass spectrometric peptide mapping functional reconstruction and synthetic mimicry of a conformational epitope using clips technology diagnostic peptide discovery: prioritization of pathogen diagnostic markers using multiple features best: improved prediction of b-cell epitopes from antigen sequences an introduction to epitope prediction methods and software immunoinformatics and the in silico prediction of immunogenicity. an introduction benchmarking b cell epitope prediction: underperformance of existing methods the rin: an rna integrity number for assigning integrity values to rna measurements gene expression omnibus: ncbi gene expression and hybridization array data repository primer3 on the www for general users and for biologist programmers geneious v5 basic local alignment search tool analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins predicting transmembrane protein topology with a hidden markov model: application to complete genomes a hidden markov model for predicting transmembrane helices in protein sequences a semi-empirical method for prediction of antigenic determinants on protein antigens new hydrophilicity scale derived from high-performance liquid chromatography peptide retention data: correlation of predicted surface residues with antigenicity and x-ray derived accessible sites the swiss-model workspace: a web-based environment for protein structure homology modeling the swiss-model repository and associated resources swiss-model: an automated protein homology-modeling server swiss-model and the swiss-pdbviewer: an environment for comparative protein modeling protein modeling by e-mail ucsf chimera -a visualization system for exploratory research and analysis the c. jejuni strain nctc 11168 was a kind gift of the group of s. bereswill (department of microbiology and hygiene, charité -university medicine berlin, berlin, germany). sh is greatly indebted to martina obry for her assistance during expression library construction and clone isolation. the authors would like to thank simone aubele for technical assistance. we also gratefully acknowledge michaela schellhase for microarray printing. conceived and designed the experiments: sh mvnr. performed the experiments: sh. analyzed the data: sh ffb mvnr. contributed reagents/materials/analysis tools: sh. wrote the paper: sh ffb mvnr. key: cord-001983-zo9yngfc authors: napp, s.; allepuz, a.; purse, b. v.; casal, j.; garcía-bocanegra, i.; burgin, l. e.; searle, k. r. title: understanding spatio-temporal variability in the reproduction ratio of the bluetongue (btv-1) epidemic in southern spain (andalusia) in 2007 using epidemic trees date: 2016-03-10 journal: plos one doi: 10.1371/journal.pone.0151151 sha: doc_id: 1983 cord_uid: zo9yngfc andalusia (southern spain) is considered one of the main routes of introduction of bluetongue virus (btv) into europe, evidenced by a devastating epidemic caused by btv-1 in 2007. understanding the pattern and the drivers of btv-1 spread in andalusia is critical for effective detection and control of future epidemics. a long-standing metric for quantifying the behaviour of infectious diseases is the case-reproduction ratio (r(t)), defined as the average number of secondary cases arising from a single infected case at time t (for t>0). here we apply a method using epidemic trees to estimate the between-herd case reproduction ratio directly from epidemic data allowing the spatial and temporal variability in transmission to be described. we then relate this variability to predictors describing the hosts, vectors and the environment to better understand why the epidemic spread more quickly in some regions or periods. the r(t) value for the btv-1 epidemic in andalusia peaked in july at 4.6, at the start of the epidemic, then decreased to 2.2 by august, dropped below 1 by september (0.8), and by october it had decreased to 0.02. btv spread was the consequence of both local transmission within established disease foci and btv expansion to distant new areas (i.e. new foci), which resulted in a high variability in btv transmission, not only among different areas, but particularly through time, which suggests that general control measures applied at broad spatial scales are unlikely to be effective. this high variability through time was probably due to the impact of temperature on btv transmission, as evidenced by a reduction in the value of r(t) by 0.0041 for every unit increase (day) in the extrinsic incubation period (eip), which is itself directly dependent on temperature. moreover, within the range of values at which btv-1 transmission occurred in andalusia (20.6°c to 29.5°c) there was a positive correlation between temperature and r(t) values, although the relationship was not linear, probably as a result of the complex relationship between temperature and the different parameters affecting btv transmission. r(t) values for btv-1 in andalusia fell below the threshold of 1 when temperatures dropped below 21°c, a much higher threshold than that reported in other btv outbreaks, such as the btv-8 epidemic in northern europe. this divergence may be explained by differences in the adaptation to temperature of the main vectors of the btv-1 epidemic in andalusia (culicoides imicola) compared those of the btv-8 epidemic in northern europe (culicoides obsoletus). importantly, we found that btv transmission (r(t) value) increased significantly in areas with higher densities of sheep. our analysis also established that control of btv-1 in andalusia was complicated by the simultaneous establishment of several distant foci at the start of the epidemic, which may have been caused by several independent introductions of infected vectors from the north of africa. we discuss the implications of these findings for btv surveillance and control in this region of europe. the basic reproduction ratio (r 0 ) is the most widely used parameter in epidemic theory and is a key tool for understanding the behaviour of infectious diseases [1] . it is defined as the average number of secondary cases produced when a single infected individual is introduced into a fully susceptible population [2] . as the epidemic progresses, as a result of the depletion of susceptible animals or the application of control measures, the basic reproduction ratio changes to the case reproduction ratio (r t ), i.e. the average number of secondary cases arising from a single infected case at time t (for t>0). knowledge of r t is very relevant for the control of the epidemic [3] . if r t <1, each case will, on average, produce less than one secondary case, and the epidemic will tend to die out, even if no further measures are applied. however, if r t >1, each case will, on average, produce more than one secondary case, and extra measures will be needed to control the epidemic. the proportion of the population that would need to be protected to achieve the eradication of the disease (p) can be estimated as: p>1-(1/r t ) [2] . therefore, the higher the value of r t , the more difficult it will be to control the epidemic. both r 0 and r t are usually derived from explicit deterministic susceptible-infectious-recovered (sir) models, and estimated by fitting a equations to epidemic (case or seroprevalence) data [4, 5] . however, there are two problems associated with this approach. first, many assumptions need to be made, for example about the size of the susceptible population, which in the context of an epidemic is continuously expanding. second, r 0 and r t are estimated as mean values, without consideration of how these parameters vary over both space and time, thereby excluding spatio-temporal information of considerable epidemiological importance [6] . to overcome these difficulties, haydon and collaborators [4] developed a method based on the construction and analysis of epidemic trees, which has the advantage that the case-reproduction ratio can be estimated directly from epidemic data [7] . bluetongue is a viral disease caused by bluetongue virus (btv), which belongs to the genus orbivirus within the family reoviridae. traditionally, 24 different serotypes have been classified, but in recent years, two new serotypes btv-25 and btv-26 have been identified [8] , reflecting the dynamic nature of this disease. although bluetongue affects all ruminant species, severe disease is mainly restricted to certain breeds of sheep [9] . btv is transmitted between hosts almost exclusively by the bites of certain species of culicoides biting midges (diptera: ceratopogonidae). in 2007, andalusia, the southernmost region of spain, was affected by a devastating epidemic caused by bluetongue virus serotype 1 (btv-1) resulting in more than four thousand infected farms [10] . besides btv-1, andalusia has been affected by several other btv serotypes: btv-10 was introduced in 1956, btv-4 in 2004 and btv-8 in 2008 [11] . except in the case of btv-8, the other serotypes were likely to have been introduced through wind transportation of infected culicoides from the north of africa [11] . introduction of bluetongue from northern africa into southern spain is considered to be one of the main routes of introduction of new serotypes of bluetongue into europe [12] . therefore, understanding the pattern of btv spread in andalusia is critical for facilitating effective surveillance and control of future epidemics. the objective of this study was to calculate the between-herd case-reproduction ratio (r t ) of the btv-1 epidemic in andalusia in 2007 using epidemic trees, and to describe the main factors that determined spatial and temporal variation in its value. andalusia is a region located in the south of spain and has an area of 87597 km 2 . the ruminant population comprises approximately 2.6 million sheep, 1.2 million goats and 0.6 million cattle, divided into 13346 sheep flocks, 10789 goat flocks, 8745 cattle herds and 4339 mixed farms. epidemiological data on the 2007 btv outbreak was collected by the veterinary services of the autonomous government of andalusia. a case was defined as a farm where btv-1 was confirmed by the national reference laboratory as positive in at least one animal (sheep, goat or cattle) by means of rt-pcr analysis according to the oie guidelines. as detection relied on previous clinical detection, only seven cattle herds affected by btv-1 were detected in the epidemic [10] , and therefore they were excluded from our analysis. a total of 4421 infected farms, 3699 in small ruminant flocks and 722 in mixed farms (sheep, goat and/or cattle in the same farm) were included in the analysis. 2. determination of the date of infection and the date of onset of infectiousness of the affected farms (step 1 in s1 fig & s2 fig) for the first 974 farms affected during the epidemic, questionnaires were conducted so that the date when clinical symptoms were first observed, the date of btv notification to the authorities and the date of btv confirmation at the farms were recorded. that information allowed the average delay between the detection of clinical symptoms at the farm and btv confirmation to be calculated (s2 fig). then, for the remaining farms, the approximate date of development of clinical symptoms could be estimated based on its date of btv confirmation. in sheep, the average incubation period is between 6 and 8 days [13] , so a conservative delay of twice that period (14 days) was considered between the infection of the farm and the detection of the clinical signs by the farmer. by subtracting these 14 days from the estimated date of development of clinical symptoms, the approximate date of infection of each of the 4421 farms was estimated. from the date of infection of the farm, the date of onset of infectiousness on that farm may be calculated, taking into account [14] : the eip is defined as the time between the infection of the vector and when it first becomes capable of transmitting the virus, and was calculated as the reciprocal of the virogenesis rate. the virogenesis rate (v) depends on the temperature (t), and may be estimated as [16] : where t min is the threshold temperature for virus replication, which for btv-1 in c. imicola (the main vector of btv in andalusia) was estimated as 12.7°c; and α represents the mean rate of replication above the threshold temperature, which for btv-1 in c. imicola was estimated as 0.016 per degree-day. for the estimation of the eip, the mean daily temperatures at the point location of each farm, for 50 days after the date of infection of the farm were obtained. temperature data was taken from the uk met office's numerical weather prediction (nwp) model, the unified model [17] . for the time period of this study data is available from the european domain of the model at a horizontal resolution of 12km. hourly values of temperature at the surface were extracted to the point location of each farm and from these daily mean temperatures were calculated. the virogenesis rate on a given day (i.e. for a given mean temperature) represented the proportion of the eip completed that day [14] . the duration of the eip on that farm was given by the number of days required for the summation of these proportions to reach one (i.e. for the eip to be completed). for each infected farm, the day a farm got infected, plus the titv, plus the number of days needed to complete the eip, determines the day from which a given farm became infectious. because the animals within a farm are infected (and then become infectious) progressively over time, once a farm became infectious, it was assumed to remain infectious until november (when the last infections of the epidemic occurred). for each of the infected farms (daughter farms), the source of its infection (parent farm) was selected using an algorithm. the majority of the results presented in this paper were based on an algorithm in which the parent farm was assigned to the closest infectious farm (algorithm 1), as in haydon and collaborators [4] , however we also considered two other types of algorithms: 1. one in which all infectious farms within a given distance (5 km) of the daughter farm were assumed to have the same probability of having been the parent farm (algorithm 2). the choice of the distance was based on sedda and collaborators [18] , who found that in the btv-8 epidemic in northern europe, the majority of infections occurred over distances of 5 km or less. 2. another in which all infectious farms within 5 km of the daughter farm were considered as potential parent farms, but their probabilities were inversely proportional to their distance (algorithm 3). one advantage of the method developed by haydon and collaborators [4] is that it allows the case-reproduction ratios to be estimated for different areas and periods. similarly, the btv-1 epidemic in andalusia can be viewed as sets of clades within which transmission was local. the difficulty lies in differentiating local transmission from a new focus (i.e. how far from the closest infectious farm a new infection can still be considered as local transmission, versus when it should be considered as the onset of a new focus). in order to define this distance, we estimated for each affected farm, the distance to the nearest infectious farm, and used the 99 th percentile of these distances, (52.1 kilometers) to set as the spatial limit to differentiate local transmission from a new focus. once the parent farm (i.e. the farm responsible for the infection) had been assigned to each infected farm by means of one of the algorithms, we calculated the number of farms infected by each of the 4421 farms affected throughout the epidemic: n i (step 3 in s1 fig) . then, the case-reproduction ratio (r t ) for a given focus (x) and time period (t) was calculated as the average value of n i of the farms infected in focus x during the period t (step 4 in s1 fig) . the algorithms were programmed in r software [19] . maps were generated using qgis [20] . the relative influence of different factors on the value of r t was assessed by means of a statistical model. for each time step, a buffer of 5 km around the farms that belonged to each focus was created. these buffers were used to extract the mean values of environmental variables from different raster layers. variables assessed included (s1 table) : 1. variables related to the host species: density of domestic hosts, i.e. cattle, sheep and goats per km 2 , and in relation to wild hosts: red deer habitat suitability [21] and roe deer habitat suitability [22] . two new variables, total domestic ruminants (cattle, sheep and goats) and total wild ruminants (red deer and roe deer) were also created. data on the population of domestic ruminants in a raster format was provided by the autonomous government of andalusia. 2. variables related to the vector species: the median and the 95% upper limit of the credible interval of predicted seasonal maximum abundance of c. imicola, c. obsoletus and c. pulicaris (the three main bluetongue vector species in andalusia) within the buffer area. two new variables, median and 95% upper limit of the ci of culicoides captures (sum of the 3 species) were also created. models for the maximum abundance of culicoides vector species were developed from national surveillance datasets of weekly ovi light trap catches at approximately 300 sites across the uk and spain covering a five year period (2005-2010) [23] . briefly, at 404 site by year combinations, five culicoides vector species or species complexes were identified morphologically and counted (c. imicola, c. newsteadi, c. impunctatus, c. obsoletus complex, and c. pulicaris complex). for each site and year the maximum size of weekly female catch per species per year was determined. only sites that had intense seasonal coverage of trapping, defined as no more than 2 weeks missing per year, were used in the analysis. the analysis involved fitting poisson generalised linear mixed models (glmm) using integrated nested laplacian approximation (inla) to identify the best-fitting models using a range of environmental covariates, and random effects for site and year. all possible combinations of explanatory variables were tested over 20 splits in the dataset into testing (75%) and training (25%) subsamples. an out of sample predictive r 2 was calculated for each of the 20 splits in the data for each combination of explanatory variables for each species or species complex. these 20 out of sample predictive r 2 values per combination were then averaged to give a final out of sample statistic for each possible combination of explanatory variables. finally, all possible combinations of variables were ranked according to their out of sample predictive ability and the top 20 best models were selected for each species to make predictions at new sites across western europe [23] . predictions for the maximum annual abundance from each of the top 20 models (based on out of sample prediction performance) were averaged for each species, and weighted by their relative support in the data. predictions were only made for pixels that were environmentally similar to the training data set, defined as those with a malhalanobis distance (md) falling within the 0-80% cumulative distribution of mds from the complete training data set (404 site by year combinations from the uk and spain). uncertainty in the estimation of model parameters was properly accounted for in the final predictions by using multiple samples from the posterior distribution of each model parameter and averaging across multiple predictions to generate a final averaged prediction. all predictions included a 95% credible interval [23] . median eips in the affected farms, were also obtained. eip represents a measure of the favourability of temperatures for btv transmission. intuitively, r t within a specific focus is likely to decrease as the time since the initiation of that focus progresses. therefore, a variable to account for the time since the start of each focus was created, defined as the days elapsed since the earliest date of infection amongst the farms in the focus. as the epidemic expands and progresses, some foci grow and end up overlapping others. in these overlapping areas r t values are likely to be lower as the farms that get the infection are shared by different foci. therefore, a variable to account for the proportion of a given focus area at a given time period, overlapped by other foci was also created. land cover, elevation and slope were also included as variables to be assessed. elevation was obtained from srtm data [24] at 85.7m. using the terrain function of the raster package in r [19] , slope and aspect were calculated from the elevation layer. furthermore, a terrain ruggedness index (tri), defined as the mean of the absolute differences between the value of a cell and the value of its eight surrounding cells, was calculated for both slope and elevation with higher values of these indices indicating a wider variability in slope or elevation in the surrounding area, which we hypothesised might make it more difficult for infected midges to move around the landscape. land cover data were obtained from corine land cover 2006 at a 100m resolution and used to create an index of the suitability of landcover for movement of infected midges around the landscape. landcover categories that were wholly unsuitable for farms or midges were assigned a high cost of movement value (2) while agricultural or grassland/shrubland categories with a high percentage of farms were assigned the lowest cost of zero. the intermediate cost of movement category, assigned a value of one, contained agricultural or grassland/shrubland categories which had a lower percentage of farms. s2 table includes the cost category assigned to each individual corine land cover class. having reclassified the corine land cover map according to these cost categories, the cost of movement were averaged at a 1km grid cell level. as stated above, these landscape factors were extracted for a 5km buffer zone around the farms in each focus at each time step. statistical analysis. due to the hierarchical nature of the data, a mixed-effects model was used within the nlme library [25] implemented in r [19] . because the different foci were observed through time, a nested random factor where focus was nested within time was included in the model. the spearman rank correlation coefficient was used to assess the correlation between the different environmental predictors included in the analysis. in those cases where the correlation was higher than 0.9 the variable with the higher biological significance was retained. the model was run with all the predictors, and full subset model selection was performed using reml comparing all possible subsets of environmental predictors. models were compared using aicc [26] . the mean temperatures at the farms infected by btv was calculated for each week since the start of the epidemic, and the relationship between those temperatures and the weekly r t values for the whole epidemic (i.e., for the whole of andalusia), was evaluated using the pearson correlation coefficient. first we evaluated how the case-reproduction ratio (r t ) of the btv-1 epidemic for the whole of andalusia varied through the epidemic. considering the natural months between july and november, and using the algorithm for the closest farm (algorithm 1), the r t value reached its maximum value (4.6) in july, at the start of the epidemic, then decreased to 2.2 by august, and dropped below 1 by september (0.8). by october it had decreased to 0.02 and remained close to 0 in november. there were no significant differences in r t values when algorithm 2 (equally proportional within a distance) was applied, except that values for july and august were a bit lower (3.3 and 1.9, respectively) and for september and october values were a bit higher (0.9 and 0.2, respectively). similarly, r t values when algorithm 3 (proportional to distance) was applied were analogous to those obtained using algorithm 1, but values for july were a bit lower (3.3) and for august were a bit higher (2.4) . in contrast to the monthly values of r t , the number of infected farms reported per month went from 113 in july, to 1010 in august and reached a maximum in september (2089 infected farms). in october, despite the low value of r t , there was a significant number of infected farms reported (1192), which rapidly decreased to only 15 farms infected in november. if we use a smaller time step (i.e. one week) to evaluate the variation of the case-reproduction ratio for the whole of andalusia, we observe a much higher heterogeneity in the values of r t , particularly at the start of the epidemic. fig 1 shows how the first week of the epidemic r t reached a very high value (20) , decreased to 2.6 by week 2, increased to 6.3 in the third week (mid-july), and then there was a slow progressive decrease, so that it was not until week 12 (mid-september) that r t fell below 1. looking at this smaller time-step (r t values per week) it is easier to appreciate that the temporal pattern of the for the whole btv-1 epidemic in andalusia is the result of both the intensity of local btv transmission within established foci and btv expansion to new areas (i.e. new foci). if we look at the weekly estimations of r t for the whole of andalusia, the high value obtained in week 1 is likely to be the result of the r t value in focus 4 (where the epidemic starts), while estimations for weeks 2 and 3 are probably linked to introduction and spread within focus 1, and the maintenance of high r t values between week 4 (22 nd of july) and week 7 (12 th of august) are highly dependent on the introduction and spread within focus 20 (the largest focus in the epidemic) (fig 1 and fig 2) . while in some of the foci (e.g. focus 4 in fig 1) , there was a delay of 1 or 2 weeks between the primary infection (as a result of btv introduction) and the first secondary cases arising as a result of local spread, in other cases (e.g. focus 1 and 20 in fig 1) , there was no such a delay. in general, the foci initiated earlier in the epidemic tended to be larger (i.e. more farms infected) (fig 3) , and of longer duration than the foci initiated later. the results show that there was a great variation in the effectiveness of btv spread among the 58 foci. in some foci, btv-1 seemed to fail to establish a continuous transmission within the new area, and none or a few secondary cases were produced (e.g. foci 10 and 11 in fig 2) . in fact, of the 58 foci, 21 had 4 infected farms or less. however, in some other cases, btv easily spread in the new area, and transmission was maintained for many weeks with hundreds of secondary cases, for example focus 4 (791 cases in 18 weeks) and focus 1 (748 cases in 18 weeks) (fig 2) . in some cases as a consequence of btv local spread, foci ended up overlapping with each other (for example foci 2, 3, 7 and 8 in fig 2) . between weeks 3 and 9 (end of august), the slow progressive decrease of r t values, coincided with an even slower progressive increase of the time needed for the completion of the eip in the affected farms (fig 4) . between weeks 10 and 16 (mid-october), increase of eips becomes steeper, going from a mean of 5.7 days between weeks 3 and 9 to mean of 9.3 days between weeks 10 and 16, which results in a rapid decrease of r t values, that after week 13 are close to 0 (fig 2 and fig 4) . after week 16 (mid-october), the eips of infected farms were frequently above 20 days, and in many cases temperatures did not allow the completion of the eip. fig 5 shows the relationship between the weekly r t values for the whole epidemic and the mean weekly temperatures at the farms affected by the epidemic. for the first half of the epidemic (between the 1 st of july and the 2 nd of september) temperatures were more or less constant at approximately 25°c, while r t values showed a decreasing trend. in contrast, for the second half of the epidemic (between the 9 th of september and the 4 th of november) decreasing mean temperatures coincided with a further decrease of r t values, which fell below the threshold of 1 when temperatures dropped below 21°c. we found a positive correlation between temperature and the r t values (r = 0.44), which was very close to significant (p = 0.06). correlation analysis of the different environmental predictors potentially related to r t showed that the estimated median maximum abundance of c. imicola was correlated with the 95% upper limit of the credible interval of estimated maximum abundance of c. imicola, as well as with the median and 95% upper limit of the credible interval for total culicoides (as c. imicola represented more than 99% of the captures in the affected areas of andalusia). therefore, only median estimated maximum abundance of c. imicola was retained for further analysis. there was also a positive correlation between slope and elevation (spearman's rank correlation = 0.99), therefore only slope was retained. considering the relationship between r t and the predictors, support in the data for the best model was split amongst several models; with 16 models receiving approximately equal support in the data (delta aicc value of <2.0; s1 table) . of these top 16 models, all models included the proportion of area overlapped and sheep density, 14 models included eip, 8 models included red deer habitat suitability, and 7 models included slope (s3 table) . the best fitting model included these five predictors, of which proportion of area overlapped, eip and sheep were strongly significant predictors of r t , and red deer suitability was close to significant (table 1) . for each day of increase in the duration of the eip of the farms within a given focus, the value of r t was reduced by 0.0041. for each increase of 1% in the area overlapped, the value of r t was reduced by 2.64. for each unit increase in the number of sheep per km 2 , the value of r t was increased by 0.14. red deer suitability showed a weak negative correlation with r t values, although it was not statistically significant. the random effects components of the model indicated that residual spatial variability between foci not accounted for by fixed effects was much lower (sd: 0.00017; table 1 ) than residual temporal variability through time (weeks) within the different foci (sd: 4.38; table 1 ). overall residual variation not attributed to spatial variation between foci or temporal variation within foci was 0.029 (table 1) . based on our estimations, the infection of the first farm occurred in the south-east of the province of cadiz on the 25 th of june (fig 6) . the second farm was infected 5 days later (30 th of june), but it was located at 128 km from the first infected farm, in the south-east of the province of huelva. the third farm was infected on the 1 st of july, in the south of huelva, 26.8 km away from the previous farm. on the 2 nd of july, a fourth farm was infected, also in huelva, but in the north-east of the province, 71.3 km away from the closest infected farm at that time. the fifth farm was infected on the 4 th of july in tarifa, in the southernmost area of the province of cadiz, but 46 km away from the first infected farm. however, taking into account the temperatures on those dates on those farms, which determine the time needed for the completion of the eip, it seems that none of those farms became infectious before the 5 th of july, and therefore were not epidemiologically linked. the abovementioned case in tarifa (fifth infected farm) was actually the first case of btv-1 confirmed by the spanish ministry of agriculture (on the 25 th of july 2007). by the 26 th of july movement restrictions were imposed in cadiz, the majority of malaga and seville, and the eastern area of huelva [27] (fig 7) . however, by that date, btv-1 had spread to other areas of andalusia, including 3 farms outside restricted areas (fig 7) . despite movement restrictions, 35 of the 58 foci identified throughout the whole epidemic, were initiated during the 10 days that followed the implementation of those measures (fig 3 and fig 7) , with appearance of new foci in the western area of huelva and the previously unaffected provinces of cordoba and almeria period. although the majority of farms infected throughout the epidemic were located in western andalusia, there were foci in the 8 provinces of andalusia. this study represents the first time that spatial and temporal variability in the case-reproduction ratio has been estimated for a midge-borne disease and related to the variability in key host, climate and landscape factors across the invaded region. here, we discuss the implications of these findings for management of future disease incursions, both in andalusia, a critical point of entry for bluetongue virus strains into europe, and further afield. the evaluation of the evolution of the monthly case-reproduction ratio of the btv-1 epidemic for the whole of andalusia evidenced that r t declined progressively: from 4.6 in july, to 2.2 in august, such that by september r t fell below the key threshold of 1 (value of 0.8), and by october the value was close to 0. de koeijer and collaborators [28] estimated the temporal pattern of the basic reproduction number between herds for the btv-8 epidemic in northern europe, and they obtained a value of around 4 for the second half of the summer, which progressively declined, falling below 1 by autumn. values in andalusia were therefore very similar to those obtained in northern europe despite differences in the epidemiology of btv between the two areas, including the fact that the main vector in andalusia is culicoides imicola, while in northern europe is culicoides obsoletus. in contrast to the gradual decrease of r t , the number of infected farms progressively increased, from only 113 farms infected in july to a maximum of 2089 in september, and remained high in october (1192) despite the low value of r t that month. this apparent discrepancy between r t and the number of infected farms may be explained by the fact that, as the epidemic advances, more and more farms become infectious, which results in an increase of the number of farms infected, but also an increase of the number of potential parents for those farms, and therefore a decrease of the values of r t . there were no significant differences between the r t values obtained by the three different algorithms used for the determination of parent farms on the basis of the distance. regardless of the algorithm, r t values for a given focus area and period are averaged across that area and period, and therefore whether the parenthood is assigned to a given farm or its neighbour does not have a great effect on the estimate of r t for that area and period. therefore, we can be fairly confident in the reliability of the casereproduction ratio estimated for the different areas and periods. however, a relevant difference is that when algorithm 1 (closest infectious farm) was applied, the number of potential parent farms was much lower as compared to when algorithm 2 (equal probability within a distance) was applied, as in that case, the probability of infection was shared among all the infectious farms within 5 kilometers. with algorithm 1, those particular farms, especially the farms that initiate the new foci, tended to be responsible for the infection of a great number of farms (i.e. they act as super-spreaders). in fact, with algorithm 1, the 20% most infective farms infected 87% of the total farms affected throughout the whole epidemic, close to the 20/80 rule (i.e. 20% of the population contributes to 80% of the net transmission) which applies in many disease systems [6] . that may be relevant in relation to the control of the disease, if we were able to achieve early identification of those farms, control measures would be much more effective [6] . in fact, with algorithm 1, 67% of the farms did not infect any farm, a value which is also very similar to the 70% of "end-point" farms obtained by sedda and collaborators [18] for the btv-8 epidemic in northern europe. when algorithm 2 was applied, the 20% most infective farms infected only 56% of the total farms affected throughout the whole epidemic, and only 10% of the farms were considered "end-point" farms, i.e. they have a zero probability of infecting other farms. however, for the majority of the remaining 90% farms, the probabilities of infecting other farms were very low. finally, the values obtained with algorithm 3 (proportional to distance) were similar to those obtained with algorithm 1, as the 20% most infective farms were responsible for the infection of 88% of the total farms affected, and 62% of the farms did not contribute to any further infections. when we used a smaller time-step (i.e. one week) to evaluate the temporal pattern of the r t values for the whole btv-1 epidemic in andalusia, we observed an increase in the variability of the case-reproduction ratio, which went from 20 in the first week, to 2.6 in the second and then to 6.3 in the third week. that heterogeneity can be more easily understood if we look at the epidemic not as a whole, but as a set of different foci, and realise that those global r t values are simply the result of the complex combination of both of local btv transmission within established foci and btv spread to new (distant) areas, i.e. new foci, where btv may spread more or less efficiently depending on different factors. the mechanism by which btv-1 jumped to those new distant areas in andalusia to establish new foci, which implied travelling distances over 52.1 kilometers, remains uncertain. in the 2006 btv-8 epidemic in northwest europe, 2% of infections seemed to have occurred at distances over 31 kilometers, probably related to wind dispersal of culicoides, although other mechanisms such as human transport of midges or virus, or movement of domestic or wild animals, could not be ruled out [18] . unless we are able to clarify those mechanisms of long-distance dispersal, the prediction of disease spread and the establishment of effective control measures will be hampered. the heterogeneity of btv spread through time and space means that control measures applied on the basis of global parameters (e.g. vaccination of a proportion of the population calculated from the r t value for the whole epidemic) would probably result in measures more exhaustive than needed in some areas (with the subsequent waste of resources), while in other areas the same measures will be insufficient to control the disease. this highlights the need for more detailed understanding of spatio-temporal variation in transmission as epidemics progress. the delay observed in many foci between the initial infection and the secondary cases arising as a result of local spread may have been due to a lack of suitable conditions for disease spread in that period or a failure of detection of those secondary cases. in addition, if the new focus resulted from the introduction of a recently infected host (rather than infected vectors), before local transmission could occur, the infected host needs to reach the viraemic stage and then the local vectors need to complete the eip before onwards transmission can occur. the fact that the duration of the eips increased progressively throughout the epidemic, seems to indicate that the decrease in temperature may have played a more relevant role in stopping btv-1 spread than vaccination (which mainly was applied from november onwards). however, vaccination was probably effective at preventing the persistence of the disease in the following year: only 10 cases of btv-1 were reported in andalusia in 2008, and they occurred by mid-october, when btv-1 was widespread in other regions of spain. within the range of values at which btv-1 transmission occurred in andalusia (i.e., between 20.6°c and 29.5°c) there was a general positive correlation between temperature and r t values (i.e., the higher the temperature, the higher the r t values). however, our results indicated that the relationship was not linear, but had a more complicated pattern. this pattern is probably the result of the complex relationship between temperature and different parameters affecting btv transmission. while higher temperatures increase the virogenesis rate (i.e., reduce the eip) and the biting rate, and therefore favor btv transmission, they also increase the adult vector mortality rate (i.e., reduce the culicoides lifespan), which hampers the transmission of btv (as reviewed in gubbins and collaborators [29] ). as well as influencing parameters that determine btv transmission, temperature is also one of the main determinants of culicoides abundance, though the effect of temperature may interact with moisture availability [30] . r t values for btv-1 in andalusia fell below the threshold of 1 when temperatures dropped below 21°c. in contrast, in the btv-8 epidemic in northern europe, the reproduction ratio between herds was estimated to drop below 1 only when temperatures fell below 15°c [31] . this divergence may be explained by differences in the adaptation to temperature of the main vectors of the btv-1 epidemic in andalusia (culicoides imicola) and the btv-8 epidemic in northern europe (culicoides obsoletus). the distribution and abundance of c. imicola seems to be constrained by their relatively poor tolerance of lower temperatures [32] . on the other hand, c. imicola kieffer infected with btv-1 showed no apparent transmission potential at 15°c [33] . the results of the mixed effects model indicated that variability through time (weeks) within the different foci was significantly higher than among different spatial foci. this is likely to be related to the importance of temperature on btv transmission, which is consistent with the fact that the duration of eip was found to be significantly associated with the rate of spread. the significance of overlapping foci indicates the relevance of this phenomenon in btv transmission in andalusia. overlapping of disease foci may result in the underestimation of the r t value for a given focus because some of the farms within its area may be infected by farms of another (overlapping) focus, which needs to be taken into account when interpreting the r t values. finally, the significant increase of r t with sheep density is an indication that this species was the key host in the btv-1 epidemic in andalusia. the negative correlation between red deer suitability and r t values is in contradiction with previous studies [10, 34] . however, this result should be interpreted with care as the correlation was weak and not statistically significant, and negativity may have been associated to the fact that many of the areas with the highest suitability are located in areas that were invaded later in the epidemic, when the conditions for btv transmission were less favourable. based on the estimated dates of infection, it seems that the first five farms affected in the epidemic were infected between the 25 th of june and the 4 th of july. however, taking into account the temperatures on those dates on those farms, which determine the time needed for the completion of the eip, it seems that none of those farms became infectious before the 5 th of july. this almost simultaneous infection and the fact that these farms were located far away from each other, suggests that the btv-1 epidemic in andalusia in 2007 may have not been the result of a single introduction, but several almost simultaneous introductions at distant locations. in the summer of 2007 btv-1 was circulating in morocco, and culicoides carried on the wind were considered as the most likely explanation for the introduction of btv-1 into spain, as neither bovines nor small ruminants were reported to have been imported from morocco [10] . therefore, btv-1 infected culicoides from morocco may have been transported to these distant locations, where local transmission resulted in the initiation of different disease foci. if btv-1 (and previously other serotypes) were believed to be introduced into andalusia through wind transportation of infected vectors from the north of africa, it would be reasonable to think that that phenomenon occurred not only at a single point at the start of the epidemic, but at several times and different locations throughout the epidemic. whether wind transportation of infected vectors from the north of africa actually occurs, and if it does, its frequency deserves further investigation, because that would have a great influence on how btv spreads and therefore on the effectiveness of control measures. even though movement restrictions were imposed the day after the confirmation of the first outbreak in an extensive area around the first confirmed focus, by that time, btv-1 had spread to other areas of andalusia, including some regions outside restricted areas. therefore, it seems clear that the movement restrictions imposed in andalusia were not able to stop the spread of the disease. for example, 35 out of the 58 foci of the epidemic were reported during the 10 days that followed those restrictions. long distance transmission, which results in new foci, may be the result of the introduction of infected vectors into the new area (e.g. transported on the wind) or of infected hosts, either domestic or wild. further studies would be needed to clarify the relative importance of the different mechanisms of long distance spread in andalusia, which would allow the implementation of more effective measures for the control of the disease. failure of movement restrictions to stop btv spread is in accordance with previous btv epidemics, such as the btv-8 epidemic in northern europe [35] . however, it is reasonable to think that if movement of animals had not been forbidden, the spread of disease would have been greater. some of the limitations of the study are related to the fact that the detection relied on clinical suspicion. some lack of accuracy in the dates of infection of the farms should be expected and that may influence the results. besides, some underreporting would be expected, although we assumed that veterinary inspection was homogeneous over the territory and therefore the different areas would be equally affected by underreporting. this assumption is supported by previous studies, for example méroc and collaborators [36] , concluded that the surveillance system based on clinical detection underestimated the real impact of the btv epidemic, but described accurately the spatial distribution of the virus. another problem was that as neither clinical signs nor mortality were observed in cattle, cattle herds had to be excluded from the analysis. this limitation probably did not have a great influence on the spatial distribution of the epidemic, as sheep are widely distributed and btv-1 spread to all the provinces in andalusia. in contrast, underreporting in cattle may have biased the estimation of the intensity of transmission in some areas, although the analysis showed that the density of cattle did not have a significant effect on the level of spread, in contrast to the density of sheep. moreover, the cattle population represents only a 13.6% (0.6 out of 4.4 million) of the total ruminant population in andalusia, and of the 4421 affected farms, 722 were mixed farms (sheep, goat and/or cattle in the same farm), which were included in the analysis. in order to assess the effect of vectors we used estimates of the median annual maximum abundance derived from an analysis of national surveillance weekly trapping data. however, using a static estimate of vector abundance ignores how seasonal variation in culicoides numbers throughout the year may have affected transmission. in fact a drop off in vector numbers in autumn may be coincident with the lengthening of the extrinsic incubation period found to significantly reduce r t in this period. moreover, the large differences in culicoides catches between neighbouring farms [37] indicate the limitations of extrapolating the results of entomological surveillance systems to describe the abundance of culicoides in nearby areas. this may be why we detected no significant effect of vector abundance on the spread of btv in the epidemic. other estimates of r 0 value for btv [29, 38, 39, 40] have largely considered animals as units, rather than farms (i.e. average number of secondary cases arising from the introduction of a single infected individual into a totally susceptible population) and therefore assume that animals are homogeneously distributed, rather than clustered in farms. the models by gubbins and collaborators [29] , hartemink and collaborators [38] and guis and collaborators [39] are theoretical models using the density of hosts and vectors, plus several assumptions about transmission parameters. since european culicoides vectors are diverse, taxonomically cryptic and difficult to colonise the range of variability of many of these transmission parameters is poorly described [41] . by contrast santman-berends and collaborators [40] , estimated r 0 using serological field data from the btv-8 epidemic in the netherlands, but these data are expensive to collect and highly dependent on how well the sites selected for surveillance represent relevant environmental drivers. while hartemink and collaborators [38] considered variability of r 0 across space and seasons, the final predictions are highly dependent on input geographical data for densities of animal hosts and vectors with their associated uncertainties. we argue that estimating the between-herd reproduction ratio for bluetongue directly from empirical epidemic data provides the best opportunity for understanding spatial and temporally variability in transmission and for linking this variability to environmental conditions. this is because relatively few simplifying assumptions and entomological parameters are required and a wide spectrum of environmental conditions that may impact btv is potentially encompassed within farms across the whole epidemic region. btv-1 epidemic in andalusia resulted from a combination of local transmission and long distance jumps with the establishment of new foci that produced large variability in transmission, not only through time, but also among the different geographical areas. this heterogeneity means that area-wide control measures are unlikely to be effective. transmission was favoured by the increase in the density of sheep and by the reduction of the eip (which is dependent on the temperature). there seemed to be a general positive correlation between temperature and r t values, although the relationship was not linear, probably as a result of the complex relationship between temperature and the different parameters affecting btv transmission. r t values for btv-1 in andalusia fell below the threshold of 1 when temperatures dropped below 21°c, while for btv-8 epidemic in northern europe, that occurred at 15°c. this divergence may be explained by differences in the adaptation to temperature of the main vectors of the btv-1 epidemic in andalusia (culicoides imicola) and the btv-8 epidemic in northern europe (culicoides obsoletus). control of btv-1 in andalusia was further complicated by the simultaneous establishment of the disease at several distant foci, which may have been caused by the repeated introductions of infected vectors at distant locations. therefore, whenever orbiviral diseases are circulating in the north of africa, surveillance in andalusia should be intensified (and extended beyond the closer areas), as the south of spain is one of the main entry point for these diseases into europe. individual-based perspectives on r 0 infectious diseases of humans: dynamics and control different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures the construction and analysis of epidemic trees with reference to the 2001 uk foot-and-mouth outbreak modeling infectious diseases in humans and animals heterogeneities in the transmission of infectious agents: implications for the design of control programs new approaches to quantifying the spread of infection complete genome characterisation of a novel 26th bluetongue virus serotype from kuwait the pathology and pathogenesis of bluetongue monitoring bluetongue disease (btv-1) epidemic in southern spain during bluetongue in spain: from the first outbreak to 2012 bluetongue in europe: past, present and future bluetongue in northern europe quantitative assessment of the probability of bluetongue virus overwintering by horizontal transmission: application to germany duration of bluetongue viraemia and serological responses in experimentally infected european breeds of sheep and goats temperature dependence of the extrinsic incubation period of orbiviruses in culicoides biting midges a new dynamical core for the met office's global and regional modelling of the atmosphere a new algorithm quantifies the roles of wind and midge flight activity in the bluetongue epizootic in northwest europe r: a language and environment for statistical computing. r foundation for statistical computing qgis geographic information system a first attempt at modelling red deer (cervus elaphus) distributions over europe a first attempt at modelling roe deer (capreolus capreolus) distributions over europe ecology and eco-epidemiology of culicoides-borne diseases hole-filled srtm for the globe version nlme: linear and nonlinear mixed effects models model selection and multimodel inference: a practical information-theoretic approach quantitative analysis of transmission parameters for bluetongue virus serotype 8 in western europe in 2006 assessing the risk of bluetongue to uk livestock: uncertainty and sensitivity analyses of a temperature-dependent model for the basic reproduction number incriminating bluetongue virus vectors with climate envelope models confirmation of spatial patterns and temperature effects in bluetongue virus serotype-8 transmission in nw-europe from the 2007 reported case data thermal limits of two biting midges, culicoides imicola kieffer and c. bolitinos meiswinkel (diptera: ceratopogonidae). parasit vectors vector competence of south african culicoides species for bluetongue virus serotype 1 (btv-1) with special reference to the effect of temperature on the rate of virus replication in c. imicola and c. bolitinos role of wild ruminants in the epidemiology of bluetongue virus serotypes 1, 4 and 8 in spain impact of human interventions on the spread of bluetongue virus serotype 8 during the 2006 epidemic in north-western europe establishing the spread of bluetongue virus at the end of the 2006 epidemic in belgium modelling the spatial distribution of culicoides biting midges at the local scale mapping the basic reproduction number (r 0 ) for vector-borne diseases: a case study on bluetongue virus modelling the effects of past and future climate on the risk of bluetongue emergence in europe estimation of the reproduction ratio (r 0 ) of bluetongue based on serological field data and comparison with other btv transmission models bionomics of temperate and tropical culicoides midges: knowledge gaps and consequences for transmission of culicoides-borne viruses this work was supported by the edenext fp7-261504 eu project. additional support was provided for ks and bp from the nerc centre for ecology and hydrology national capability allocation. conceived and designed the experiments: sn. analyzed the data: sn aa krs. contributed reagents/materials/analysis tools: igb lb. wrote the paper: sn aa bvp jc igb lb krs. key: cord-003024-17f1evh3 authors: nunes, márcio roberto teixeira; de souza, william marciel; acrani, gustavo olszanski; cardoso, jedson ferreira; da silva, sandro patroca; badra, soraya jabur; figueiredo, luiz tadeu moraes; vasconcelos, pedro fernando da costa title: revalidation and genetic characterization of new members of group c (orthobunyavirus genus, peribunyaviridae family) isolated in the americas date: 2018-05-24 journal: plos one doi: 10.1371/journal.pone.0197294 sha: doc_id: 3024 cord_uid: 17f1evh3 group c serogroup includes members of the orthobunyavirus genus (family peribunyaviridae) and comprises 15 arboviruses that can be associated with febrile illness in humans. although previous studies described the genome characterization of group c orthobunyavirus, there is a gap in genomic information about the other viruses in this group. therefore, in this study, complete genomes of members of group c serogroup were sequenced or re-sequenced and used for genetic characterization, as well as to understand their phylogenetic and evolutionary aspects. thus, our study reported the genomes of three new members in group c virus (apeu strain bean848, itaqui strain bean12797 and nepuyo strain bean10709), as well as re-sequencing of original strains of five members: caraparu (strain bean3994), madrid (strain bt4075), murucutu (strain bean974), oriboca (strain bean17), and marituba (strain bean15). these viruses presented a typical genomic organization related to members of the orthobunyavirus genus. interestingly, all viruses of this serogroup showed an open reading frame (orf) that encodes the putative nonstructural nss protein that precedes the nucleoprotein orf, an unprecedented fact in group c virus. also, we confirmed the presence of natural reassortment events. this study expands the genomic information of group c viruses, as well as revalidates the genomic organization of viruses that were previously reported. introduction group c viruses are antigenically characterized into the genus orthobunyavirus, family peribunyaviridae, order bunyavirales [1] . this name is historically based on their serological characteristics, which makes them distinct from members of group a (alphaviruses genus of the family togaviridae) and group b (flavivirus genus of the family flaviviridae) antigenic groups [2] . currently, the group c serogroup is composed of 15 distinct viruses isolated from humans, wild animals (mainly rodents, monkeys, marsupials, and bats), and mosquitoes. these viruses are present in tropical and subtropical areas of the americas, including the united states, mexico, panama, honduras, guatemala, trinidad, brazil, peru, ecuador, venezuela, and french guiana [1, 3, 4] . clinically, the human infections caused by group c viruses are asymptomatic or characterized by unspecific febrile illness [1, 5, 6] . the percentage of asymptomatic group c viruses infections are unknown, and apparently, the prevalence of antibodies against group c viruses is directly related to people living nearby or maintaining close contact to forest or ecological niches in tropical areas [5] . the genomes of members of group c viruses presents the typical organization of other orthobunyaviruses, which are a tri-segmented negative-sense rna named small (srna), medium (mrna) and large (lrna) segments. the srna encodes a nucleocapsid protein (n protein) and a non-structural protein (nss), while the mrna encodes a polyprotein precursor that after a post-cleavage process gives rise to two envelope glycoproteins (gc and gn) and a non-structural protein (nsm). lrna segment encodes a large rna-dependent rna polymerase (rdrp) [7] . so far, previous studies have described the genomic characteristics s and m rna segments of members of group c viruses, but many sequences generated by sanger sequencing approach were divergent, despite using the same strains of group c viruses [1, [7] [8] [9] . therefore, in this study, we combined high-throughput sequencing (hts), rapid amplifica viruses strains used in this study were propagated into vero cells (atcc 1 ccl-81™), as previously described [10] . the infected cells were incubated for 3 to 5 days until visualization of viral cytopathic effect. table 1 provides the names, strains, year, sources and local of isolation, as well as the genbank accession numbers. viral rnas were extracted from the supernatant of infected vero cells using the purelink viral rna mini kit (invitrogen, usa) following the manufactures instructions. then, the strands of the rna synthesis were performed using the kit cdna synthesis system and 400 μm roche "random" primer according to the manufacturer's instructions. the cdnas were prepared for hts on a gs flx+ pyrosequencer (roche, 454 life sciences) at the center for technological innovation at the evandro chagas institute, ministry of health, brazil. the de novo assembling strategy applied to obtain the genomes was used with program newbler v. 3.0 [11] . additionally, sequences for terminal untranslated regions (utrs) were determined by 5'/3' rapid amplification of cdna ends (race) sequencing (s1 table) [12] . virus genomes were evaluated regarding it sizes, annotations of putative open reading frame (orf), 5' and 3' non-coding regions (ncrs) and conserved motifs with geneious 9.1.2 (biomatters, new zealand), for identification of transmembrane regions and signal peptide we used the topcons web server [13] and for identification of n-glycosylation sites we used netnglyc 1.0 server (http://www.cbs.dtu.dk/services/netnglyc/). the annotations of protein domains were performed with interpro 60.0 [14] . the presence of potential characteristic motifs for orthobunyaviruses were identified on multiple sequence alignments (msa) based on amino acids sequences, which were carried out using muscle v.3.7 [15] , and visualized in geneious 9.1.2 (biomatters, new zealand). maximum likelihood (ml) phylogenetic trees were constructed using nucleotide and amino acids sequences of viruses reported in our study and additional sequences of members of group c viruses with complete coding sequences (s, m, and l) available in the genbank database (http://www.ncbi.nlm.nih.gov/) until 10 th of april of 2018. the msas were carried out using muscle v.3.7 [15] . the phylogenies were inferred by iq-tree version 1.4.3 software using the best-fit model based on bayesian information criterion, and the gtr+i+g4 nucleotide substitution model was used to all rna segments, and lg+g4, lg+i+g4, and lg+g4 amino acids substitution model to s, m, and l, respectively. statistical supports for individual nodes were estimated using the bootstrap value using 1,000 replicates [16] . the phylogenetic trees were visualized using the figtree software v.1.4.2. in addition, the nucleotides and amino acid distances among viruses of group c were estimated with segment s, m, and l using the p-distance values. standard error estimations were calculated by bootstrapping method (1,000 replicates) using the mega v.6 program [17] . potential reassortment events were analyzed by distinct phylogenetic topologies based on the depicted trees at the nucleotide level. in addition, all genes were concatenated in a single sequence, and an msa was performed using the program muscle 3.7 [15] . potential reassortment events were then analyzed using the rdp, geneconv, bootscan, maxchi, chimaera, siscan and 3seq methods implemented in rdp4 [18] . common program settings for all methods were used to perceive sequences as linear, to require phylogenetic evidence, to refine breakpoints and to check alignment consistency. the highest acceptable p-value was set as 0.05, after considering bonferroni correction for multiple comparisons. all method-specific program settings remained at their default values. our results showed that the complete srna segments of group c viruses ranged from 1,003 to 1,111 nucleotides (nt) and presents the open reading frame (orf) of the nucleocapsid (n) protein, with a conserved size of 235 amino acids (aa) and 26.72 to 27.03 kda for all viruses (fig 1) . the second orf in the s segment in these viruses encodes a putative non-structural protein (nss). interestingly, the orf to nss gene starts at an atg codon -38 nucleotides downstream of the n orf start codon, which potentially encodes an nss protein of 318 nt (105 aa) with a molecular mass of~11.8 kda (fig 1) . the nss protein is common in most orthobunyaviruses that have been isolated from vertebrates, such as the members of group c viruses [19] . previously studies have been reported that caraparu and madrid from group c viruses encode an nss protein with 83 amino acids, and also a 62 aa nss, predicted to be truncated in the n-terminus of marituba and oriboca viruses, and consequently, could not be expressed [6, 7, 9] . indeed, our analysis demonstrates that possibly the members of group c viruses present a pre-n coding strategy, as recently described for the brazoran and enseada orthobunyaviruses [20, 21] . therefore, some s segment sequences previously reported for group c viruses are possibly incomplete, as shown in fig 2. whereas that nss protein has been demonstrated to play crucial roles in virulence and pathogenesis of orhobunyaviruses [19] , further studies using reverse genetics may elucidate the biological importance of nss protein found in viruses of group c. the complete mrna segments ranged from 4,534 to 4,613 nt in length, which encodes the glycoprotein precursor (gpc), ranging from 1,428 to 1,435 aa in length (159.94 to 161.87 kda) with a similar topology to other members of the orthobunyavirus genus (fig 1) . the gpc is proteolytically cleaved to yield two structural glycoproteins, gn (204 to 211 aa and 34.01 to 35.13 kda) and gc (942 to 947 aa and 100.45 to 102.8 kda), and a nonstructural protein (nsm) with 281 or 282 nt (24.99 to 26.27 kda) (fig 1) . the gpc contains the specific n-terminal signal peptide that is common to all members of the genus, which is responsible for delivering the nascent polypeptide to the endoplasmic reticulum [22] . the conserved zinc finger region (amino acids position 257 to 295) and fusion peptide (amino acids position 1068 to 1091) in the gpc protein were also identified, which is an important rna binding site and is probably involved in viral assembly [23] . furthermore, the gpc was predicted to contain five transmembrane regions (tmds): a single tmd in gn (close to the c-terminus); three tmds in nsm; and a single one, close to the c-terminus in gc (fig 1) . the tmds in gn and gc have been shown to play crucial roles in membrane fusion, assembly, and morphogenesis [24] . the complete lrna segments ranged from 6,907 to 6,979 nt in length, with an orf that encodes an rna-dependent rna polymerase (rdrp) of 2,248 aa, with a predicted molecular weight of 255.64 to 262.13 kda. the unique exception is nepuyo virus strain bean 10709 with a rdrp of 2,249 aa. this protein contains the conserved polymerase activity domains consisting of pre-motif a and motifs a through e in position 951 to 1230 aa, as well as the n-terminal endonuclease motifs h, pd, and dxk (fig 1) . these domains are highly conserved in negative sense rna viral polymerases as well as other members of the bunyavirales order and are directly involved in the polymerase function activity [25] . in addition, we observed two mismatches in the 8th and 9th nucleotide of the s segments, and another mismatch in the 9th nucleotide of the l segments, but we did not found mismatches in the utr of m segments (s2 fig). murv strain bean974, oriv strain bean17, mtbv strain bean15, carv strain bean3994 and madv strain bt4075 viruses present 99.96 to 100% nucleotide identities with the corresponding partial genomic sequences that were previously reported (fig 3d-3f and parts d-f of s1 fig) [4, 7, 8 ]. on the other hand, the new viruses that we have been sequenced the complete coding sequences shared higher nucleotide identify with partial genomes, such as apeu strain bean 848, which shared 99% nucleotide identify in all segments with partial sequences of same strains previously described [9] . in addition, the amino acid substitutions were observed as follow: carv strain bean3994 (s344p and k605n) on gpc. mtbv strain bean15 (q335r); (l728q); (q1073k) and (k1345r) on rdrp and (g456r and d626n) in gpc. murv strain bean974 (f949s) in rdrp. however, we did not observe any amino acid substitutions in the nucleoprotein sequence of the viruses that were submitted to the resequencing protocol. on the other hand, the identified amino acids substitutions observed in gpc and rdrp of our results compared with previously sequences reported to same strains. therefore, we suspected that this fact is probably due to serial passages in cultured cells [26] . collectively, our results are consistent with the same viral strains sequenced previously [4, [7] [8] [9] , but the results of segment s suggested that some complete coding sequences previously reported for group c viruses are possibly are incomplete. to better understand the genetic relationships among group c viruses, we conducted the ml trees based on the complete coding sequences in nucleotide and amino acids level for all segments (fig 3 and s1 fig) . these viruses were clustered in a unique monophyletic clade with different topologies for each segment. the s and l segments present two major clades, namely of clade a and b (fig 3a-3c ). the clade a was subdivided in three: subclade ia, composed by murv, oriv, and restan (resv); subclade iia, with mtbv and apeuv; subclade iiia comprised by nepv, gumbo limbo (glv). the clade b was split into two subclades: subclade ib, represented by carv strains, itqv, itaya (ityv) strain iqt9646 and fsl2923 and madv and the subclade iiib with bruconha virus (bruv) (fig 3a-3c) . the topology of the ml tree for the m segment reveals four phylogenetic groups that were well supported (bootstrap > 80%). these groups were named following previous serological classifications based on complement fixation, neutralization, and hemagglutination inhibition tests: marituba, caraparu, madrid and oriboca complexes (fig 3b) [1, 27] . the caraparu complex includes carv, apeuv and bruv, madrid complex comprises madv and ityv, marituba complex comprises mtbv, murv, resv, nepv and glv, and the oriboca complex was composed by oriv and itqv [1] . unfortunately, there are no complete sequences available to vinces and ossa viruses, but probably these viruses will be clustered into caraparu complex as previously showed by serological assays [28] . possibly, the concordance observed between the phylogeny of the m segment and the serological classification occurs because the m segment encodes the glycoproteins, which are the primary antigenic determinants recognized by the neutralization and hemagglutination inhibition tests [1, 29] . reassortment is an important evolutionary mechanism of segmented rna viruses in which co-infection of a host cell with multiple viruses may result in the shuffling of genomic segments [30] . this fact has been shown to be involved in virus emergence and interspecies transmission, which include schmallenberg and oropouche virus [31] [32] [33] . in group c viruses, a classic and informative study using standard antigenic tests showed cross-reactivity in marituba and murutucu by hemagglutination-inhibition and neutralization, murutucu and oriboca by complement-fixation, oriboca and itaqui by hemagglutination-inhibition and neutralization, itaqui and caraparu by complement-fixation, caraparu and apeu by hemagglutination-inhibition and neutralization and apeu and marituba by complement-fixation [27] . however, some these viruses cross-reacted completely by one test, but not by another test and still are related. thus, these and other data indicated that the group c viruses include many natural reassortants [34] . to elucidate this point, we compared the topology of both s and l trees with the phylogenetic tree generated using the m segment sequences and observed some discrepancies, which were supported by higher bootstrap values and significantly different likelihood scores, which suggested natural reassortment. also, after combining these branching inconsistencies in the phylogenetic trees with rdp4 analyses of concatenated segments, we confirmed four reassortment events with different genome segment organization than previously described [1, 7] . the first reassortment event was identified in apeuv strain bean848 contains s and l segments from mtbv strain bean15 and an m segment that is probably unique. also, itqv strain bean 12797 has s and l segments from carv strain bean3994 and a possibly unique m segment. interestingly, based on the classification of international committee on taxonomy of viruses (ictv), the reassortment events described in this study occurs between different viral species, as the apeu virus that is classified into caraparu orthobunyavirus, but the s and l this virus is from marituba orthobunyavirus. also, the itaqui and oriboca viruses are classified into oriboca orthobunyavirus, but the s and l segments of both viruses are from viruses of caraparu orthobunyavirus. despite, previous studies based on serological tests and partial genome sequences indicated that apeuv and itqv are potential reassortments, the first complete coding sequences for three segments were reported in this study [1, 7, 9, 34] . the other reassortment event was observed in oriv strain bean17, which possesses the s and l segments from murv strain bean974, and a unique m segment (fig 4) . interestingly, all reassortments observed in this study have been reported cross-reactions only by the complement-fixation method [27] . this fact, suggests that cross-reactivity observed by complement-fixation test was determined by s segment because serologic makers identified by the l segment remains unidentified, as well as hemagglutination-inhibition, and neutralization assay that is determined by m segment, therefore, these viruses probably are unique [34] . also, we confirmed the reassortments in ityv identified in amazon region of peru in 1999, both strains of this virus have s and l segments from caraparu virus and a unique m segment [4] . on the other hand, murv and resv were previously reported to be reassortments, but we did not find any evidence to support this phenomenon [1] . additional studies, as well as other strains of group c viruses, may help to clarify this point. in summary, our study provides a better understanding and clarifies the genome sequences, genomic characterization and reassortment events, as well as the evolutionary relationship of group c serogroup. thus, our results may be helpful to better understand the evolution and diversity of these viruses, as well as can be used in further molecular epidemiological, evolutionary studies, and development of molecular methods to diagnostic of infection by group c viruses. nucleotide sequence accession number. the nucleotide sequence determined in this study have been deposited in genbank under the accession number mg029269 to 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diseases bunyamwera orthobunyavirus glycoprotein precursor is processed by cellular signal peptidase and signal peptide peptidase mutagenesis of the la crosse virus glycoprotein supports a role for gc (1066-1087) as the fusion peptide. virology role of the cytoplasmic tail domains of bunyamwera orthobunyavirus glycoproteins gn and gc in virus assembly and morphogenesis bunyaviridae rna polymerases (l-protein) have an n-terminal, influenza-like endonuclease domain, essential for viral cap-dependent transcription acquisition of cell-cell fusion activity by amino acid substitutions in spike protein determines the infectivity of a coronavirus in cultured cells further studies on the serological relationships of group c arthropod-borne viruses and the application of these relationships to rapid identification of types. the american journal of tropical medicine and hygiene identification of hitherto unrecognized arboviruses from ecuador: members of serogroups b, c, bunyamwera, patois, and minatitlan. the american journal of tropical medicine and hygiene characterization of the candiru antigenic complex (bunyaviridae: phlebovirus), a highly diverse and reassorting group of viruses affecting humans in tropical america rna virus reassortment: an evolutionary mechanism for host jumps and immune evasion genetic reassortment between sathuperi and shamonda viruses of the genus orthobunyavirus in nature: implications for their genetic relationship to schmallenberg virus oropouche virus: clinical, epidemiological, and molecular aspects of a neglected orthobunyavirus. the american journal of tropical medicine and hygiene iquitos virus: a novel reassortant orthobunyavirus associated with human illness in peru viruses of the family bunyaviridae: are all available isolates reassortants? key: cord-000321-ee7q7jhv authors: powell, michael l.; leigh, kendra e.; pöyry, tuija a. a.; jackson, richard j.; brown, t. david k.; brierley, ian title: further characterisation of the translational termination-reinitiation signal of the influenza b virus segment 7 rna date: 2011-02-08 journal: plos one doi: 10.1371/journal.pone.0016822 sha: doc_id: 321 cord_uid: ee7q7jhv termination-dependent reinitiation is used to co-ordinately regulate expression of the m1 and bm2 open-reading frames (orfs) of the dicistronic influenza b segment 7 rna. the start codon of the bm2 orf overlaps the stop codon of the m1 orf in the pentanucleotide uaa ug and ∼10% of ribosomes terminating at the m1 stop codon reinitiate translation at the overlapping aug. bm2 synthesis requires the presence of, and translation through, 45 nt of rna immediately upstream of the uaa ug, known as the ‘termination upstream ribosome binding site’ (turbs). this region may tether ribosomal 40s subunits to the mrna following termination and a short region of the turbs, motif 1, with complementarity to helix 26 of 18s rrna has been implicated in this process. here, we provide further evidence for a direct interaction between mrna and rrna using antisense oligonucleotide targeting and functional analysis in yeast cells. the turbs also binds initiation factor eif3 and we show here that this protein stimulates reinitiation from both wild-type and defective turbs when added exogenously, perhaps by stabilising ribosome-mrna interactions. further, we show that the position of the turbs with respect to the uaa ug overlap is crucial, and that termination too far downstream of the 18s complementary sequence inhibits the process, probably due to reduced 40s tethering. however, in reporter mrnas where the restart codon alone is moved downstream, termination-reinitiation is inhibited but not abolished, thus the site of reinitiation is somewhat flexible. reinitiation on distant augs is not inhibited in eif4g-depleted rrl, suggesting that the tethered 40s subunit can move some distance without a requirement for linear scanning. eukaryotic viruses have evolved a variety of translational control strategies to facilitate expression of downstream open reading frames (orfs) on polycistronic mrnas and examples have been described at all three steps of protein synthesisinitiation, elongation and termination [1] . these include leaky scanning of 40s ribosomal subunits past the most 59 aug [2] , the de novo recruitment of ribosomes to intercistronic internal ribosomal entry sites (iress; [3] ); programmed ribosomal frameshifting [4, 5] and the circumvention of normal termination by programmed stop codon readthrough [6, 7] . generally, these processes allow the expression of two (or more) proteins from a single mrna and may also permit a level of control over their relative quantities. another way of accessing a downstream orf in viral mrnas is by termination-dependent reinitiation of translation (termination-reinitiation), a phenomenon first described in the expression of the influenza b virus bm2 protein [8] . the dicistronic mrna that is derived from genomic segment 7 of this virus has two orfs encoding matrix protein 1 (m1) and bm2, with the termination codon of m1 in close proximity to the start codon of the bm2 orf (uaaug; stop codon of m1 in bold, start codon of bm2 underlined) [8] [9] [10] . following translation of m1, some 10-20% of ribosomes terminating at the m1 stop codon go on to reinitiate translation at the immediately adjacent bm2 start codon [8, 11] . this capacity to reinitiate protein synthesis following translation of a long upstream orf was unexpected. during the elongation phase, initiation factors are likely to be rapidly lost, thus reinitiation of translation following termination was believed to be restricted to cases where the upstream orf (uorf) is very short [12] [13] [14] . our knowledge of the mrna signals that allow efficient reinitiation following translation of a long upstream orf has largely been obtained from studies of caliciviruses, namely in the expression of the vp2 protein of feline calicivirus (fcv; [15] [16] [17] ) and the vp10 protein of rabbit haemorrhagic disease virus (rhdv; [18, 19] ). here, expression of the downstream orf by termination-reinitiation requires a stretch of mrna (between 69 and 87 nt in length) upstream of the stop codon of the first orf (termed the termination upstream ribosome binding site or turbs) and the close proximity of the stop and start codons of the two orfs [15, 17, 19] . within the turbs, two essential sequence motifs (motifs 1 and 2) have been described. motif 1 is highly conserved amongst caliciviruses and is composed of a short stretch of 6-9 nt (with a conserved core of uggga) with complementarity to the apical loop of helix 26 of 18s rrna. this motif, which lies towards the 59 boundary of the turbs, likely acts to tether the 40s subunit to the mrna posttermination, facilitating the recruitment of the initiation factors necessary for reinitiation [15, 19] . motif 2, which lies some 15-20 nt upstream of the termination-reinitiation window (auga in fcv), is a second essential stretch of 4-8 nt but shows little sequence conservation amongst caliciviruses. recent work has revealed that this motif in fact forms the 39 arm of a stem-loop structure whose formation is necessary for efficient terminationreinitiation [16] . the features identified in caliciviral turbs suggest a model for termination-reinitiation in which post-termination 40s subunits are tethered to the mrna through interactions between the mrna (through motif 1) and 18s rrna, initiation factors are recruited and the aug restart codon located, processes which may require precise rna folding (involving motif 2) within the turbs. the turbs is not highly active as an ires and 40s subunit recruitment probably requires high local 40s subunit concentrations [17, 18] . an important question, therefore, is how the interaction between turbs motif 1 and 18s rrna helix 26 is facilitated. analysis of primary and secondary structural features of the bm2 signal has revealed that it contains a short turbs (of 45 nt) which is largely single-stranded, with motif 1 likely to be located in the apical loop of a metastable stem-loop structure when the ribosome is positioned at the termination codon of the upstream orf [11] . we have suggested that motif 1 may thus be ''presented'' to the solvent-accessible apical loop of helix 26, promoting the interaction and subsequent 40s tethering. however, this hypothesis remains contentious. the paucity of stable rna secondary structure within the bm2 turbs [11] , the turbs of fcv [i. brierley, unpublished observations] and the calicivirus murine norovirus (mnv; [20] ), limits the predictive power of rna folding programs in the assessment of the likely rna folds present before and after ribosomal transit through the turbs. it is also known that eukaryotic initiation factor 3 (eif3) plays a role in the termination-reinitiation process, perhaps interacting with the turbs to provide another link between mrna and the 40s subunit [17] . in this paper, we describe further analysis of the bm2 turbs. in contrast to the caliciviral turbs, which contain stretches of non-essential bases between motifs 1 and 2, all of the bm2 turbs appears to be required for function perhaps due to its shorter length, with sequence-specific and sequence-independent elements. evidence is provided, from oligonucleotide targeting and from expression studies in yeast cells, to support the hypothesis that bm2 motif 1 interacts directly with helix 26 of 18s rrna, consistent with a tethering role. we also provide evidence that a close spacing between the m1 stop and bm2 start codons is not critical; rather, there is dependence upon the distance between the terminating ribosome and the turbs. indeed, the ribosome is able to locate start codons placed some distance downstream of the wild-type position if termination occurs at the normal distance relative to the turbs. the role of rna secondary structure in turbs function was also investigated by mutagenesis, but the experiments gave only limited support for the predicted secondary structures. however, the capacity of eif3 to stimulate terminationreinitiation, particularly from a defective turbs, was confirmed, including a turbs rendered defective through a point mutation in motif 1. together, these data support the view that efficient termination-reinitiation requires both mrna-rrna interactions and the participation of eif3. the p2luc-bm2 plasmid series was prepared by subcloning sequences encompassing the influenza b virus terminationreinitiation signal (prepared by rt-pcr) into the dual-luciferase reporter plasmid p2luc [21] . most of these plasmids have been described previously [11] . the p2luc-bm218s-ag, 18s-at and 18s-ac plasmids were generated by site directed mutagenesis of p2luc-bm2wt or p2luc-bm2-204 (see below). the crpv-p2luc-bm2 plasmid series was generated by digestion of p2luc-bm2wt with psti and nhei, and insertion of a pcr fragment comprising the cricket paralysis virus (crpv) ires downstream of a bacteriophage t7 promoter. the t7-crpv fragment was generated by pcr from plasmid crpv/+8norm [14] . the resulting plasmid contains a t7 promoter located 25 nt upstream of the crpv ires. translation starts on an alanine codon within the inserted crpv fragment, resulting in an nterminal extension to rluc of 10 amino acids relative to that of the p2luc-bm2wt. derivative plasmids were generated by site-directed mutagenesis using primers described previously [11] . termination-reinitiation in yeast cells was studied using the yeast dual-reporter plasmid pac99 [22, 23] . the wild-type termination-reinitiation signal of bm2 was prepared by pcr as an ecorv fragment that was subsequently cloned into mscidigested pac99. pac99-bm2 derivative plasmids were generated by site directed mutagenesis as described below. sequences were confirmed by commercial dideoxy sequencing (using the facility at the department of biochemistry, university of cambridge). site-directed mutagenesis was performed using the quikchange ii site-directed mutagenesis kit (stratagene) according to manufacturer's instructions. mutagenesis to introduce insertions longer than 6 bp was performed in two steps [24] , by first subjecting the mutagenesis reactions (containing either the sense or antisense primer) to three cycles of pcr, then mixing the reactions and performing a further 18 cycles as previously described [11] . in vitro transcription and translation p2luc-bm2 reporter plasmids were linearised with hpai and capped run-off transcripts generated using t7 rna polymerase as described previously [25] . messenger rnas were recovered by a single extraction with phenol/chloroform (1:1 v/v) followed by ethanol precipitation. remaining unincorporated nucleotides were removed by gel filtration through a nucaway spin column (ambion). the eluate was concentrated by ethanol precipitation, the mrna resuspended in water, checked for integrity by agarose gel electrophoresis and quantified by spectrophotometry. unless otherwise stated, mrnas were translated in flexih rabbit reticulocyte lysate (flexihrrl, promega) programmed with template mrna at 50 mg/ml. typical reactions were of 10 ml and composed of 60% (v/v) flexihrrl, 20 mm amino acids (lacking methionine), 500 mm mgoac, 2 mm dtt, 5u rnase inhibitor (rnaguard, ge healthcare life sciences), 130 mm-160 mm kcl (optimised for each batch of flexihrrl) and 0.2 mbq [ 35 s]methionine. reactions were incubated for 1 h at 30uc and stopped by the addition of an equal volume of 10 mm edta, 100 mg/ml rnase a followed by incubation at room temperature for 20 minutes. samples were prepared for sds-page by the addition of 4 volumes of 4x laemmli's sample buffer, boiled for 3 minutes and resolved on 12% sds-page gels. the relative abundance of products on the gels was determined by direct measurement of [ 35 s]-methionine incorporation using a packard instant imager 2024. eif4g-depleted rrl was prepared and used as described previously [26] . purified eif3 was a kind gift of dr. chris fraser (department of molecular and cell biology and howard hughes medical institute, university of california). dominant-negative eif4a-r362q was prepared as described [14] . reporter gene assay in yeast cells pac99 reporter plasmids were transformed into yeast strain y349 using the lioac/ssdna/peg method [27] . for each experiment three transformants cultivated under the same conditions were assayed. cells were disrupted by vortexing with acid washed glass beads (sigma) at 4uc for 30 minutes. cell debris was removed by centrifugation and reporter enzyme assays carried out as described [23] . termination-reinitiation efficiency was calculated as the ratio of firefly luciferase activity to b-galactosidase activity relative to that of constructs in which the open reading frames were fused in frame. the bm2 turbs is streamlined into 45nt of rna essential for termination-reinitiation, containing sequence-specific and sequence-independent elements in rhdv and fcv, turbs motifs 1 and 2 are separated by some 30 nt, much of which appears to be non-essential [15] [16] [17] 19] . however, given that the minimal sequence requirement for termination-reinitiation of bm2 (,45 nt; [11] ) is shorter than that documented in the caliciviruses (69 and 87 nt; [15, 17, 19] ), we wished to test whether any of the residues were non-essential. to do this, we introduced 6 nt deletions throughout the length of the sequence upstream of the m1 termination codon in the context of the bm2 termination-reinitiation reporter plasmid, p2luc-bm2-204 ( figure 1a ; [11] ). as expected, translation of the bm2-204 reporter mrna generated products corresponding to the capdependent upstream product (rlucm1-204 ,36 kda) and the termination-reinitiation product (bm2fluc ,62 kda). the latter protein was not observed in translations of the negative control reporter mrna, p2luc-bm2ps (rluc'm1 ,33 kda, [11] ), which contains an in-frame stop codon immediately upstream of the turbs. significantly, deletion of any part of the minimal required region led to a strong inhibition of bm2fluc synthesis, suggesting that the entire turbs is required for efficient termination-reinitiation on the segment 7 rna ( figure 1a ). alternatively, it was possible that the defect in bm2 synthesis in this deletion series may be a consequence of the altered spacing between the m1 stop codon and upstream features required for termination-reinitiation. to test this, we selected three of the 6 nt deletion mutants surrounding motif 1 (mutants 6.1, 6.4 and 6.6) and replaced the deleted regions with two different random 6 nt sequences ( figure 1b ). no restoration of bm2 synthesis was observed with either of the 6.1 nucleotide replacement mutants ( figure 1b) . replacement of nucleotides into the 6.4 deletion mutant had no effect with the first of the mutants (6.4r1), but some recovery was observed in the second of these mutants (6.4r2), albeit the frequency of termination-reinitiation was diminished compared to that of the wild-type mrna ( figure 1b ). in contrast, insertion of 6 nt back into the 6.6 deletion mutant fully restored bm2 synthesis in both cases ( figure 1b) . these results suggest that the minimal 45 nt turbs region is split into both sequencespecific and sequence-independent elements, where the sequence towards the 59 end of the minimal region is important in basespecific interactions, whereas the 39 end of the turbs may act solely as a spacer, important in placing motif 1 relative to the terminating ribosome. targeting the 18s rrna:mrna interaction using antisense oligonucleotides termination-reinitiation on the bm2 orf is likely to be dependent on interactions between the influenza b segment 7 rna and helix 26 of the 18s rrna [11] . however, other work has revealed that motif 1 may also be important in eif3 binding [17] . to investigate the mrna-18s rrna interaction further, antisense 2-o-methyl oligonucleotides (aons) were synthesised that would target either the loop of helix 26 of 18s rrna ( figure 2a ) or motif 1 of the bm2 turbs ( figure 2b ). control oligonucleotides were also prepared to target sequences both upstream and downstream of motif 1 to control for potential nonspecific effects on translation ( figure 2b ). an aon designed to target helix 26 of the 18s rrna had no effect on bm2 synthesis ( figure 2a ), although high concentrations (320-fold to 2960-fold molar excess of the aon to the mrna) inhibited global translation ( figure 2a and data not shown). conversely, an aon targeting motif 1 specifically inhibited bm2 synthesis, with little effect on overall translation ( figure 2b ). importantly, the upstream and downstream control aons had little effect on bm2 synthesis or global translation within the range tested for the bm2 motif 1 complementary aon ( figure 2b ). it should be noted that the 80s ribosome will strip annealed aons from the mrna as it translates through the turbs. however, both the upstream and motif 1 complementary aons will be able to re-anneal once the ribosome reaches the uaaug. however, it is possible that any effect of the downstream oligo is masked due to it being unable to reanneal in the presence of the terminating ribosome. nevertheless, these data indicate that the effect of the bm2 motif 1 complementary aon is specific and not an effect on ribosome processivity, and provide further evidence in support of an interaction between motif 1 and 18s rrna. however, we cannot rule out that these effects may also be due to perturbations of rna secondary structure within the turbs or due to interactions of motif 1 with an unspecified molecule (see later). the dependence of bm2 synthesis on interactions between the mrna and 18s rrna raises the possibility that increasing 18s rrna complementarity within motif 1 would lead to increased synthesis of the bm2fluc polypeptide. to test this, the bases adjacent to motif 1 were substituted such that the complementarity to 18s rrna was increased to the 59, to the 39 or in both directions ( figure 3a ). in all cases, however, the substitution mutations reduced the frequency of termination-reinitiation events, albeit to a lesser extent with the 39 complementary extension mutant ( figure 3a ). we went on to examine the possibility that multiple copies of motif 1 would stimulate termination-reinitiation, through the provision of an increased number of tethering sites. as a precedent for this, the murine gtx mrna (also known as nkx 6-2) contains a short 9 nt ires element that is known to act more efficiently when present in multiple copies. similarly to motif 1, ribosomes are recruited through interactions between the gtx ires and helix 26 of the 18s rrna [28] [29] [30] , although with the arm of the helix [31] rather than the apical loop ( figure 3c , left panel). two sets of constructs were prepared. in the first, one or two additional copies of the 6 nt (auggga) core of motif 1 were introduced alongside the original ( figure 3b ). control constructs were also generated in which one or two copies of the hexanucleotide augcga were looped in as a negative control, since this motif 1 mutation was shown previously to be unable to support termination-reinitiation, presumably due to the abolition of mrna:rrna base pairing [11] . in the second set of constructs, the bases downstream of the wild-type auggga sequence were substituted to generate one or two extra copies of the auggga motif ( figure 3b ). in vitro translations revealed, however, that none of the mutations had a stimulatory effect on reinitiation on the bm2 orf. looping in a single extra copy of the auggga (or augcga) hexanucleotide had very little effect on the process, whilst insertion of two copies led to inhibition in both contexts ( figure 3b ). in constructs where motif 1 was duplicated by virtue of nucleotide substitutions, termination-reinitiation was inhibited to a similar extent in constructs containing either one or two copies of the 18s complementary motif ( figure 3b ). these data indicate that there is no additive effect on termination-reinitiation of adding extra copies of the 18s complementary region. in fact, the inhibitory effects seen most likely reflect the importance of the precise spacing of the ribosome with respect to motif 1 (in the loop-in mutants) or, alternatively, some effect on turbs rna secondary structure. as discussed above, the gtx mrna is thought to be able to recruit ribosomes by virtue of interactions between helix 26 of the 18s rrna and the mrna [28] [29] [30] ). it was therefore of interest to determine whether the minimal 7 nt gtx motif could functionally replace motif 1. two constructs were generated, one in which the inserted gtx element was complementary to murine 18s rrna (which would result in a mismatch with helix 26 of rabbit 18s rrna) and one with full complementarity to the rabbit 18s rrna, such that efficient tethering would be expected in rabbit reticulocyte lysates (rrl; figure 3c , top-right panel). termination-reinitiation was found to be inhibited in either mutant and to a similar extent as mutants in which a single nucleotide substitution was present to abolish 18s rrna:mrna base pairing ( figure 3c ; the single nucleotide mutants have been described previously [11] ). these results suggest that motif 1 and the gtx ires element cannot functionally replace each other. termination-reinitiation in yeast cells: further evidence for mrna:18s rrna interactions in ribosome tethering it is known that nucleotide substitutions within motif 1 that are predicted to destabilise the motif 1:18s rrna interaction are inhibitory to termination-reinitiation, but it is possible that these mutations may affect turbs structure or interaction with other translational components, like eif3 [17] . in an attempt to resolve this issue, we assayed termination-reinitiation in yeast cells, whose 18s rrna helix 26 equivalent has a somewhat different primary sequence. this experiment was carried out in the context of the yeast dual reporter vector pac99, into which we had cloned the relevant bm2 information appropriately framed between bgalactosidase and firefly luciferase reporter genes. in pac99-bm2wt, b-galactosidase is synthesised by cap-dependent translation, whereas firefly luciferase should only be synthesised following reinitiation on the bm2fluc orf ( figure 4a ). to confirm that firefly luciferase was being synthesised as a result of a genuine termination-reinitiation event (as opposed to internal ribosome entry, for example), we generated a premature stop (ps) mutant (pac99-bm2-ps) in which a stop codon was inserted into the m1 sequence 233 nt upstream of the start codon of bm2fluc. in pac99-bm2-yeast, motif 1 was changed to give full complementarity to the yeast 18s rrna partner ( figure 4b ). the plasmids were transformed into yeast strain y349, and b-galactosidase and luciferase assays were carried out. termination-reinitiation frequency was determined in comparison to the reporter gene activities observed in a vector in which the reporter orfs were fused in-frame (pac99-bm2ifc). as expected, termination reinitiation was significantly (p,0.01) reduced in pac99-yeastps, although there was still some background synthesis of fluc ( figure 4c ). importantly, mutation of the motif 1 homologue such that it was fully complementary to yeast 18s rrna led to a significant (p,0.01) increase in bm2fluc synthesis relative to the wild-type bm2 reporter ( figure 4c ), supporting the view that mrna:rrna base pairing is a key determinant in bm2 orf expression. it has been suggested previously that termination-reinitiation is dependent on the stop codon of the uorf and the start codon of the downstream orf lying in close proximity [8, 11, 15, [17] [18] [19] 32, 33] . indeed, movement of the m1 stop codon more than 24 nt downstream of its original context in the terminationreinitiation 'window' of the bm2 signal results in inhibition of reinitiation on the bm2 orf [11] . however, in all previous investigations into proximity effects of the start and stop codons, the termination site was moved downstream of the start codon of the upstream orf [8, 11, 15, [17] [18] [19] 32] ). given that terminationreinitiation almost certainly depends on interactions between the terminating ribosome and the turbs, it is conceivable that these experiments have overestimated the importance of the relative proximity of the stop and start codons and that the distance between the terminating ribosome and the turbs is the crucial issue. to investigate this, a series of mrnas were prepared in which the uaaug overlap was moved downstream en masse such that the distance between the stop and start codons was conserved but the ribosome would terminate at varying distances relative to the turbs ( figure 5b) . a series of control mrnas were also examined in which the stop codon alone was shifted downstream of the bm2 start codon ( figure 5a ; details in [11] ). in all constructs, nucleotides at -3 and +4 relative to the bm2fluc aug were maintained to control for context effects of the start codon. as observed previously in the control series [11] , reinitiation occurred efficiently when the stop codon was moved up to 24 nt downstream of its original placement but was abolished when the stop was moved further ( figure 5a ). importantly, bm2 translation the 44 kda product produced from this mrna corresponds to the rlucm1 product, the 62 kda product is the bm2fluc reinitiation product. the molar excess of aon (oligo) in relation to the mrna is shown above the autoradiograph. (b) the sequence upstream of the termination-reinitiation site (uaaug) is shown, with the a residue at the start of the turbs underlined. also shown is the core sequence of motif 1 in bold. the regions targeted followed the same pattern in mrnas where the uaaug motif was moved as a unit, with ablation of synthesis observed when this sequence was moved any further than 24 nt downstream of its original site ( figure 5b) . these experiments reveal that the placement of the 40s subunit relative to the turbs, posttermination, is quite critical. as reinitiation is not absolutely dependent on the close proximity of stop and start codons ( figure 5a) , it seemed conceivable that termination-reinitiation could still occur efficiently if the start codon of the bm2fluc orf was moved downstream of the stop codon of rlucm1. a series of mrnas were prepared in which the original bm2fluc start codon was mutated to agc and new aug restart codons inserted independently at +12, +24, +36, +48 and +63 nt, again preserving the wild-type kozak consensus. as can be seen in figure 6a , movement of the start codon to +12 resulted in 50% inhibition of fluc synthesis, but there was no further reduction in product yield when the restart codon was displaced further downstream. however, the electrophoretic mobility of this reinitiation product did not show the clear increase that would be expected given that the restart product would become progressively smaller with increased displacement of the restart codon. rather, the reinitiation product band seemed less sharp and more diffuse with the +48 and +63 mrnas. this suggested the possibility that reinitiation might be occurring at two sites: one in the region of the wild-type reinitiation site (and so necessarily at a non-aug codon), and the other at the displaced aug restart codon. the most obvious potential non-aug codon for the first type of reinitiation event is the aua codon located immediately upstream (-1 position) of the wild-type reinitiation site at +1 ( figure 6a ). we therefore tested whether reinitiation can indeed occur at this -1 position, by generating an aug in this position and removing the wild-type +1 aug. the results showed that the efficiency of reinitiation at an aug in the -1 position was similar to when it was in the normal (+1) position ( figure 6b , compare lanes 8 and 10), in agreement with the finding that reinitiation efficiency in the rhdv system was only slightly compromised if the restart codon was moved one codon upstream [19] . given that quite efficient reinitiation still occurs in fcv and bm2 rnas when the wild-type aug is mutated to non-aug codons [11, 15, 17] , this observation suggests that when the wild-type +1 aug has been mutated, there is a strong possibility of some reinitiation occurring at the -1 aua codon. as for reinitiation at the displaced aug codon, the data of figure 6b (lanes 1-6) demonstrate that reinitiation certainly occurs at these downstream sites, because on this higher resolution gel the product size clearly decreases as the displacement is increased, although the efficiency is only 20-25% of the reinitiation frequency in the wild-type mrna (lane 1). in fact, in this particular experiment there is much more downstream cistron product from the displaced aug than product from the putative -1 aua reinitiation site, which was only just visible on the autoradiogram (highlighted by the asterisk). in reviewing the results of many experiments of this type we conclude that the relative use of the two potential reinitiation sites shows quite a large degree of variability, which seems related to the use of different batches of reticulocyte lysate and so may possibly be due to small batch to batch differences in the endogenous ionic conditions. the variability seems to mainly affect the yield of putative -1 aua reinitiation product, whereas the yield of product from the displaced aug codon is relatively invariant at ,20% of the wild-type mrna yield. the two extremes of the variability range are well illustrated by figure 6b (very minimal use of the -1 aua site) and figure 6c , where there is actually more putative reinitiation at the -1 aua than at the displaced aug codon. the fact that reinitiation, albeit at reduced efficiency, can occur when the first aug is moved 63 nt downstream raises the question of whether the reinitiating 40s subunits access this site by linear scanning from the turbs. we examined this by exploiting the dependence of ribosomal scanning on eif4g [34] . the bm2wt and bm2start +63 mrnas were modified such that translation of the upstream m1 orf was driven by the cricket paralysis virus (crpv) ires, which does not require eif4g for function. these mrnas (crpv-bm2wt, crpv-bm2start+63) were then translated alongside capped brome mosaic virus rnas (bmv, promega) and bm2wt rnas (as a control for the efficacy of eif4g depletion) in mock-and eif4g-depleted rabbit reticulocyte lysates ( figure 6c ). translations were also performed in the absence or presence of dominant-negative eif4a (eif4a r362q [35] ) to inhibit the activity of any residual eif4g that had escaped depletion. as expected, the translation of the capped wildtype rna (bm2wt) and bmv rnas were inhibited in the eif4gdepleted rrl, but the crpv ires-containing mrnas were translated efficiently in both control and depleted rrl ( figure 6c ). importantly, depletion of eif4g had no effect on the reinitiation efficiency in either the crpv-bm2wt or crpv-bm2start +63 mrnas, suggesting that the reinitiation site can be located in a scanning-independent manner. when two aug codons were present, one at +63 and the other at either the +1 (wild-type) or -1 position, the upstream site took complete precedence over the +63 site ( figure 6b, lanes 8 and 10) , and this precedence was maintained even when the stop codon was moved 12 codons downstream ( figure 6b, lanes 11-13) . thus the strongly preferred site for reinitiation is an aug codon just downstream of the turbs. if there is no aug in this region, the reinitiation mechanism apparently seeks alternatives: either an acceptable non-aug codon in this same region or an aug codon further downstream. reinitiation in the later case does not involve eif4g/4a-dependent linear scanning, and so it is more likely to involve a direct transfer of the 40s subunit from the turbs to the aug, a transfer which might be facilitated by looping out the mrna between the tethered 40s ribosomal subunit and the aug. studies of the fcv signal have indicated a role for eif3 in termination-reinitiation as, for example, addition of supplementary eif3 to rrl is able to specifically stimulate synthesis of the orf3 reinitiation product [17] . furthermore, greater stimulation is observed with mrnas that show a partial defect in reinitiation in the absence of eif3, suggesting that the increase in eif3 concentration may rescue the negative phenotype, perhaps by allowing increased binding of eif3 to the mrna [17] . to analyse whether the same is true of reinitiation on the bm2 orf, we examined the effect of exogenous eif3 on reinitiation on mrnas containing a fully functional signal (bm2wt, bm2-204), or mutants by aons are shown above the sequence, colour coded to match the relevant aon sequence. the bm2wt rna was translated as above in the presence of increasing aon concentrations as indicated above each autoradiograph. doi:10.1371/journal.pone.0016822.g002 defective by virtue of a deletion (bm2-207, bm2-210, described in [11] ) or a substitution in motif 1 (bm2-ag, bm2-ac). to focus specifically on the effect of eif3 on termination-reinitiation rather than 59-dependent initiation, the bm2 sequences were sub-cloned such that the rlucm1 orf would initiate on an alanine codon provided as part of the crpv ires. as can be seen in figure 7 , exogenous eif3 specifically stimulated synthesis of the bm2fluc reinitiation product with all of the mrnas tested, including those mutants that were essentially inactive (bm2-210 and bm2-ac; most evident in long exposures, see right panel, figure 7) . importantly, the addition of an unrelated, similarly purified protein had no effect on reinitiation in any of the rnas described above (figure s1 ), further highlighting the specificity of eif39s effect on reinitiation. given the likely importance of the interaction between motif 1 and 18s rrna, we previously carried out rna structure mapping and minimal free energy predictions to assess whether motif 1 was in basepaired or single stranded conformation in the native mrna. this work implicated two potential structures, mfold 1 and mfold 2 ( figure 8a ; [11] ). in mfold 1 the 18s complementary region is sequestered in a base-paired region of stem 2. we previously hypothesised that translation through the segment 7 mrna and termination of ribosomes at the m1 stop codon would prevent these interactions, creating a structure similar to mfold 2 ( figure 8b ). in this structure, the 18s complementary region would then be presented to helix 26 of the 18s rrna on the apical loop of a metastable stem-loop structure. given that translation through the turbs up to the termination-reinitiation window is a prerequisite for efficient reinitiation [11] , one can speculate that mfold 2 is the biologically relevant fold, and that destabilisation of the central stem of mfold 1 (stem 2) would have little effect on the reinitiation process. to test this hypothesis we created destabilising mutations within either arm of stem 2 (chosen to avoid disruption of the 18s rrna complementary region) in the context of the p2luc-bm2-204 parental plasmid (which encodes the minimal required region for termination-reinitiation [11] ). the mutations created were of either 2 or 3 nt to control for context effects on the bm2 aug, creating the arm 1 mutants p2luc-bm2a1-xnt, and the arm 2 mutants p2luc-bm2a2-xnt (where x = the number of nucleotides mutated). we also prepared double mutants (a1/2-xnt) to create a pseudowild-type structure where base-pairing between the two arms is restored. in the context of both the 2 nt and 3 nt mutations, substitution of bases in arm 1 abolished terminationreinitiation, however substitution of the bases at the bottom of arm 2 had little effect on bm2fluc synthesis relative to the bm2-204 control. the double pseudowild-type mutation also demonstrated abolition of bm2fluc expression ( figure 8a ). taken together these results suggest that the structure presented in mfold 1 is unlikely to play a role in termination-reinitiation as disruption of arm 2 of stem 2 has little effect on bm2 translation, and no restoration of bm2 synthesis was observed in the pseudowild-type construct. as opposed to mfold 1, the structure presented for mfold 2 would still be able to form when the ribosome has translated through the upstream orf and is in the process of termination. we tested this structure in the same way, by introducing destabilising mutations independently in both arms at the base of stem 29 (creating mutants p2luc-bm2a1' and p2luc-bm2a29) and preparing a double mutant that would yield a pseudowild-type structure, which would be expected to exhibit bm2fluc synthesis (p2luc-bm2a19/29). it should be noted that the a19 mutation is very similar to that of the a1-2/ 3 nt mutant, given that this arm base pairs to form both stem 2 and stem 29 in the two putative folds. as expected, this mutation dramatically inhibited synthesis of bm2fluc similarly to that observed with the a1 mutation ( figure 8b ). however, as before, no inhibition of bm2 synthesis was observed when stem 2 was destabilised in the 39 arm ( figure 8b ). nevertheless, the partial restoration of bm2 expression (to around 50% of wild-type) seen with the a19/29 double mutant, indicates that base-pairing in this region may play some role in termination-reinitiation. the simplest conclusion from these experiments, however, is that neither mfold 1 nor mfold 2 represent the sole active confirmation, although it is possible that these mutants may form another structure that can present the turbs motif 1 to helix 26 of 18s rrna. previous work has revealed that termination-dependent reinitiation on the bm2 orf of segment 7 of influenza b virus is dependent on a relatively short (45 nt), largely unstructured, turbs containing a typical motif 1 element towards the 59 end [11] . in the present study, we investigated features of the turbs required for efficient reinitiation, focusing primarily on the proposed interaction between turbs motif 1 and helix 26 of 18s rrna. whilst the effects of point mutations within motif 1 are consistent with such an interaction, direct evidence is lacking in the bm2 system. we began by attempting to block the interaction by targeting the binding partners with antisense oligonucleotides. such an approach was successfully employed in studies of the ires-like properties of the gtx leader (which recruits ribosomes de novo by virtue of interactions between the 18s rrna and the mrna) and revealed that the ires activity could be blocked by aons that bind either the mrna or the rrna [36] . whilst we were able to observe specific inhibitory effects with an oligonucleotide that targeted motif 1, no effect was observed with an oligonucleotide targeting the ribosomal rna ( figure 2 ). our failure to observe an effect of the aon complementary to the apical loop of helix 26 may be due to a failure of the aon to access the target. the gtx mrna:rrna interaction occurs at a in the p2luc-bm2gtx mutants, the 7 nt core of motif 1 (bold italics in p2luc-bm2wt) was substituted for nucleotides complementary to the region of 18s rrna where the gtx ires is believed to bind. note the p2luc-bm2gtx rabbit sequence is altered from that of the p2luc-bm2gtx mouse reporter such that 100% complementarity with the different rabbit 18s rrna could be achieved. nucleotides differing between gtx mouse and gtx rabbit are underlined. rnas were translated and analysed as in the legend to figure 1 . a series of previously described motif 1 mutants [11] were also translated as controls. doi:10.1371/journal.pone.0016822.g003 different region of helix 26 than the putative segment 7 mrna:rrna interaction, and this may be more accessible. indeed, termination-reinitiation could not be reconstituted in reporter mrnas in which motif 1 was replaced by the gtx 18s rrna binding motif ( figure 3c) , and thus different responses to oligonucleotides targeting the rrna may be expected. it may also be that the 18s rrna-targeting oligonucleotide may need to adopt a similar structure to the mrna if it is to effectively bind to the 18s rrna and block reinitiation. the inhibition of termination-reinitiation by the aon that targeted motif 1, in contrast, is highly consistent with its proposed role in binding to helix 26, although effects via rna conformation or binding to an unknown molecule cannot be ruled out. however, the weight of evidence from mutational analysis, the yeast data described below and the aon titrations strongly suggest that the inhibitory effect of aons directed against motif 1 are likely due to their blocking of the interaction between mrna and 18s rrna. subsequently, we sought to confirm the interaction by investigating termination-reinitiation in yeast cells, exploiting the fact that the helix 26 equivalent in yeast 18s rrna has a primary sequence distinct from that of the rabbit. using a dicistronic reporter mrna with variant motif 1 sequences, we found that increasing the complementarity of the motif to that of yeast 18s rrna stimulated termination-reinitiation, supporting strongly the view that intermolecular interactions are important in reinitiation on the bm2 orf. however, two aspects of this experiment require comment. first, a high background activity was observed in control assays (figure 4) , the origin of which is uncertain secondly, in yeast cells, the bm2wt reporter mrna, whose motif 1 is not fully complementary to the helix 26 target, showed an efficiency of termination-reinitiation only two-fold lower than the bm2yeast mrna. in rrl, a single base mismatch between mrna and rrna is sufficient to inhibit bm2 synthesis 10-20 fold [11] . one possible explanation for this difference is that the higher concentrations of mrna and rrna in an intact cell can act to stabilise the (presumably) weakened mrna:rrna interaction. during preparation of this manuscript, luttermann and meyers (2009) published a similar, more sophisticated, investigation of fcv motif 1:18s rrna interactions using a yeast expression system in which both mrna and rrna partners could be the rlucm1/bm2fluc overlap window was changed from uaaug to caaug such that termination occurs at the next in-frame stop codon, some 63 nt downstream of the original stop site (+63). new stop codons were then inserted, preserving the stop codon context, at +12, +24, +36 and +48 nt downstream of the natural termination site. messenger rnas derived from these plasmids and a control mrna (ps) were translated and analysed as detailed in the legend to figure 1 . this experiment is a repeat of that found in [11] and is shown here as a control. (b) the rlucm1/bm2fluc overlap window was changed from uaaug to caacu, and the uaaug sequence reintroduced at +12, +24, +36, +48 or +63 nt downstream preserving both the initiation codon and stop codon context. messenger rnas derived from these plasmids were translated and analysed as above. doi:10.1371/journal.pone.0016822.g005 figure 6 . effect on termination-reinitiation of altering the bm2 start codon position. (a) the termination-reinitiation overlap was mutated from auaaug to auaagc and the bm2fluc start codon reintroduced at +12,+24, +36, +48 or +63 nt, preserving the start codon context at the -3 and +4 positions. the codon immediately 59 of the native reinitiation site is denoted -1, the reinitiation site is shown as +1. messenger rnas derived from these plasmids were translated and analysed as above. (b) left panel: translations were performed as in figure 6a and the samples were separated by sds-page and autoradiographed. the putative aua reinitiation product is marked with an asterisk. in addition, the three right hand modified. this work provided strong evidence in support of a requirement for mrna:rrna interactions in termination-reinitiation in the expression of fcv vp2 [16] . since the reinitiation mechanism shows a fairly strong preference for an aug codon, it seems a certainty that initiator met-trna is involved. this, in turn, implies that the ribosome lanes of the left panel show translations of mutant mrnas in which the codon -1 of the aug start codon and the start codon (+1) were mutated to the indicated codons in the context of the bm2start +63 rna. lane numbers are shown below the figure. right panel: translations of rnas containing stop and start codons at the indicated positions. c) capped bmv (bmv) rnas, bm2 wt reporters (cap-wt), crpv-ires driven bm2wt (crpv-wt) and bm2start +63 (crpv+63) rnas were translated in control (c) and eif4g-depleted (d) rrls in the absence or presence of ,1 mm dominant-negative eifa (4a-r362q). rnas were translated as described in [26] and analysed as above. doi:10.1371/journal.pone.0016822.g006 which has just terminated at the stop codon must dissociate into subunits prior to reinitiation, in order to allow eif2/gtp/met-trna i ternary complex binding to the 40s subunit. so it is a 40s subunit rather than an 80s ribosome that interacts with turbs motif 1. the fact that reinitiation efficiency decreases quite abruptly with increasing distance of displacement of the stop [11] ) is shown with motif 1 shown in red, the termination-reinitiation overlap shown in cyan and the sequences likely to be occupied by a ribosome terminating at the m1 orf are 39 of the purple line. mutations were introduced at the bottom of stem 2, with both the 2 nt and 3 nt substitution mutations shown next to arm 1 (a1) or arm 2 (a2). messenger rnas derived from these plasmids and control mrna (204, ps) were translated and analysed as detailed in the legend to figure 1 . rrf denotes the relative reinitiation frequency adjusted for relative methionine content as compared to the wt (set as 100%). (b) the mfold 2 structure (from [11] ) is shown with motif 1, the termination-reinitiation overlap, and the ribosome protected region marked as above. mutations were introduced at the bottom of stem 29 and are shown next to arm 1 (a19) or arm 2 (a29). messenger rnas derived from these plasmids and a control mrna (204) were translated and analysed as above. rrf denotes the relative reinitiation frequency adjusted for relative methionine content as compared to the wt (set as 100%). doi:10.1371/journal.pone.0016822.g008 codon further downstream ( figure 5 ) favours a model in which this 40s subunit is transferred directly from the termination site to the turbs, rather than dissociating from the mrna after termination followed by reassociation with the turbs. recent work has implicated eif3 in the termination-reinitiation process [17] , which is of interest because eif3 plays an important role in disassembly of ribosomes following the termination event which leaves the 80s ribosome with bound deacylated trna and still associated with the mrna [37, 38] . at low (sub-optimal) mg 2+ , this disassembly requires eif1 and eif3, but no other protein factor. first, eif3 binds to the solvent face of the 40s subunit, promoting dissociation of the 60s subunit, and leaving the 40s subunit (with bound deacylated trna) still associated with the mrna. then eif1 ejects the deacylated trna, while the eif3j subunit promotes dissociation of the 40s/eif1/eif3 complex from the mrna [37] . at higher, more physiologically relevant mg 2+ , as would pertain in our translation assays, there is also a requirement for abce1 and atp [38] , which catalyses the first step of dissociation of the ribosomal subunits, ejecting the 60s subunit and again leaving the 40s subunit (plus bound deacylated trna) still associated with the mrna. then eif3 binds to the solvent face of the 40s subunit, thereby preventing any ribosomal subunit reassociation, and events thereafter are exactly as at low mg 2+ [37, 38] . initiation factor eif3 has been shown to crosslink to the fcv turbs [17] . more important, supplementary eif3 has been shown to stimulate reinitiation at the wild-type fcv turbs, but more especially at turbs derivatives which are partially defective due to deletions or point mutations [17] , just as has been observed here (figure 7) . these results, particularly the stimulation of the partially defective turbs, have suggested a model in which it is specifically the eif3 associated with the turbs that binds those 40s subunits which are destined to reinitiate translation [17] . there is no conflict between this hypothesis and the model in which tethering the 40s subunit to the turbs is due to base-pairing between turbs motif 1 and 18s rrna. eif3 binds predominantly to the back or solvent face of the 40s ribosomal subunit, and is known to make direct contacts with the 18s rrna component of the small ribosomal subunit [39, 40] . a bridging interaction in which eif3 binds simultaneously to the 40s subunit and to the turbs could help stabilise the binding of the 40s subunit to the turbs for sufficient time as is required to acquire an eif2/gtp/met-trna i ternary complex, and other necessary initiation factors. after all, the analogous shine-dalgarno interaction of prokaryotic 30s subunits is generally considered to be very transitory unless it is accompanied and stabilised by a p-site fmet-trna base-pairing with an aug (or gug) at an appropriate distance further downstream. an important question that remains to be resolved is whether there is a role for turbs rna secondary structure. we previously proposed that the turbs may fold into two main configurations, in the first of which (mfold 1) motif 1 is sequestered in a base-paired region [11] , which may explain why it does not have significant ires activity (as is seen with gtx [28] [29] [30] ). translation through and termination at the m1 orf would remodel mfold 1 to a structure similar to that shown in mfold 2 ( figure 8b ) such that motif 1 would then be presented to the 18s rrna on the apical loop of a stem-loop structure. the experiments described in figure 1 agree well with dependence on a structure similar to that presented in mfold 2, in that the substituted nucleotides in the 6.1 and 6.4 replacement mutants would act to disrupt the stem, whereas substitution of nucleotides in the 6.6 replacement mutants would be expected to have no effect as they would lie within the mrna channel of the terminating ribosome. however, we also carried out a mutational analysis of the main stem regions present in mfold 1 and mfold 2 but the data did not corroborate our structural predictions. whilst disruption of either of the putative stems in one arm inhibited reinitiation, little effect was observed when stem formation was disrupted in arm 2 of either stem-loop structure, although some restoration of bm2 synthesis was observed in an mrna containing a pseudowild-type mfold 2 structure. it should be noted that rna structure mapping of the turbs of bm2 [11] , fcv (brierley et al. unpublished observations) and mnv [20] turbs reveals that they are largely single-stranded, and probably metastable. it may be that turbs are able to adopt a variety of conformations, some of which are able to facilitate terminationreinitiation, for example the 6 nt replacement mutant 6.4r2 ( figure 1b) . alternatively, the requirement for translation through the turbs may not be due to a dependence on translational remodelling and 'unzipping' of motif 1 from paired regions, but rather just to place the ribosome in proximity to motif 1 (similar to the case for reinitiation in bacteriophage where the ribosome is placed close to the vestigial shine-dalgarno [sd] motif [41] ). it is clear that further work is required to understand the putative role of rna secondary structure in termination-reinitiation. previous studies on the termination-reinitiation process have suggested the importance of the close proximity of the stop codon of the upstream orf and the start codon of the downstream orf [8, 11, 15, 17, 18, 32] . this is believed to reflect a restricted mobility of the tethered ribosome; that is, it may be able to undergo only limited movement following termination. however, we show here that the distance between the terminating ribosome and the turbs is more critical to reinitiation efficiency ( figure 5 ) and that when the start codon of the bm2 orf is placed downstream of the stop codon, reinitiation is detectable even when the start codon is moved up to 63nt downstream of its original position ( figure 6 ). this suggests that it is not solely the distance between the start and stop codons per se that affects reinitiation efficiency but rather how well the ribosome can be tethered (by virtue of the distance between where the ribosome terminates and the turbs). however, whilst efficient reinitiation requires an aug at, or very near, the wild-type site ( figure 6a and 6b) , other reinitiation sites can be used when the native reinitiation codon is absent, albeit at lower efficiency. under the latter circumstance, ribosomes can reinitiate at a downstream aug codon ( figure 6b ), or at a near-cognate initiation codon (such as the -1 aua codon, figure 6 ) close to the native reinitiation site. however, if given a choice of a wild-type aug and one located downstream, the ribosome always selects the wild-type aug, even if the ribosome artificially terminates downstream of the reinitiation window ( figure 6b) , suggesting that the turbs may cause the tethered 40s subunit to 'snap back' to the proper site of reinitiation. importantly, we show that there is no requirement for eif4g in location of either wild-type or downstream reinitiation sites. as such, location of downstream augs is likely due to the 40s subunit being transferred directly to the aug whilst tethered to the turbs (perhaps in complex with eif3 and other factors) by an rna-looping mechanism. figure s1 recombinant 4ebp1 has no significant effect on reinitiation on the bm2 orf. motif 1 and turbs deletion mutants were translated in the absence or presence of 250nm 4ebp1 (details as in figure 7) . the fold-stimulation over the reinitiation levels observed in the absence of 4ebp1 are shown below the autoradiographs. (eps) translational control of viral gene expression in eukaryotes regulation of expression of human immunodeficiency virus naturally occurring dicistronic cricket paralysis virus rna is regulated by two internal ribosome entry sites recoding in bacteriophages and bacterial is elements programmed ribosomal frameshifting in hiv-1 and the sars-cov endless possibilities: translation termination and stop codon recognition translational control in positive strand rna plant viruses eukaryotic coupled translation of tandem cistrons: identification of the influenza b virus bm2 polypeptide sequence of rna segment 7 of the influenza b virus genome: partial amino acid homology between the membrane proteins (m1) of influenza a and b viruses and conservation of a second open reading frame nucleotide sequence of rna segment 7 of influenza b/singapore/222/79: maintenance of a second large open reading frame characterization of the termination-reinitiation strategy employed in the expression of influenza b virus bm2 protein constraints on reinitiation of translation in mammals efficiency of reinitiation of translation on human immunodeficiency virus type 1 mrnas is determined by the length of the upstream open reading frame and by intercistronic distance what determines whether mammalian ribosomes resume scanning after translation of a short upstream open reading frame? a bipartite sequence motif induces translation reinitiation in feline calicivirus rna the importance of inter-and intramolecular base pairing for translation reinitiation on a eukaryotic bicistronic mrna the mechanism of an exceptional case of reinitiation after translation of a long orf reveals why such events do not generally occur in mammalian mrna translation translation of the minor capsid protein of a calicivirus is initiated by a novel termination-dependent reinitiation mechanism characterization of the sequence element directing translation reinitiation in rna of the calicivirus rabbit hemorrhagic disease virus expression of the vp2 protein of murine norovirus by a translation terminationreinitiation strategy a dualluciferase reporter system for studying recoding signals translational readthrough of the pde2 stop codon modulates camp levels in saccharomyces cerevisiae versatile vectors to study recoding: conservation of rules between yeast and mammalian cells two-stage pcr protocol allowing introduction of multiple mutations, deletions and insertions using quikchange site-directed mutagenesis structure-function analysis of the ribosomal frameshifting signal of two human immunodeficiency virus type 1 isolates with increased resistance to viral protease inhibitors truncated initiation factor eif4g lacking an eif4e binding site can support capped mrna translation transformation of yeast by lithium acetate/singlestranded carrier dna/polyethylene glycol method a 9-nt segment of a cellular mrna can function as an internal ribosome entry site (ires) and when present in linked multiple copies greatly enhances ires activity biochemical and functional analysis of a 9-nt rna sequence that affects translation efficiency in eukaryotic cells an mrna-rrna basepairing mechanism for translation initiation in eukaryotes rrna-complementarity in the 59 untranslated region of mrna specifying the gtx homeodomain protein: evidence that base-pairing to 18s rrna affects translational efficiency expression of the orf-2 protein of the human respiratory syncytial virus m2 gene is initiated by a ribosomal termination-dependent reinitiation mechanism translation termination reinitiation between open reading frame 1 (orf1) and orf2 enables capsid expression in a bovine norovirus without the need for production of viral subgenomic rna the roles of individual eukaryotic translation initiation factors in ribosomal scanning and initiation codon selection dominant negative mutants of mammalian translation initiation factor eif-4a define a critical role for eif-4f in cap-dependent and cap-independent initiation of translation antisense masking reveals contributions of mrna-rrna base pairing to translation of gtx and fgf2 mrnas recycling of eukaryotic posttermination ribosomal complexes the role of abce1 in eukaryotic posttermination ribosomal recycling specific interaction of one subunit of eukaryotic initiation factor eif-3 with 18s ribosomal rna within the binary complex, eif-3 small ribosomal subunit, as shown by cross-linking experiments the yeast eif3 subunits tif32/a, nip1/c, and eif5 make critical connections with the 40s ribosome in vivo scanning model for translational reinitiation in eubacteria purified eif3 was a kind gift of dr. chris fraser (department of molecular and cell biology and howard hughes medical institute, university of california, berkeley). key: cord-000359-y0m1utug authors: walpita, pramila; barr, jennifer; sherman, michael; basler, christopher f.; wang, linfa title: vaccine potential of nipah virus-like particles date: 2011-04-06 journal: plos one doi: 10.1371/journal.pone.0018437 sha: doc_id: 359 cord_uid: y0m1utug nipah virus (niv) was first recognized in 1998 in a zoonotic disease outbreak associated with highly lethal febrile encephalitis in humans and a predominantly respiratory disease in pigs. periodic deadly outbreaks, documentation of person-to-person transmission, and the potential of this virus as an agent of agroterror reinforce the need for effective means of therapy and prevention. in this report, we describe the vaccine potential of niv virus-like particles (niv vlps) composed of three niv proteins g, f and m. co-expression of these proteins under optimized conditions resulted in quantifiable amounts of vlps with many virus-like/vaccine desirable properties including some not previously described for vlps of any paramyxovirus: the particles were fusogenic, inducing syncytia formation; pcr array analysis showed niv vlp-induced activation of innate immune defense pathways; the surface structure of niv vlps imaged by cryoelectron microscopy was dense, ordered, and repetitive, and consistent with similarly derived structure of paramyxovirus measles virus. the vlps were composed of all the three viral proteins as designed, and their intracellular processing also appeared similar to niv virions. the size, morphology and surface composition of the vlps were consistent with the parental virus, and importantly, they retained their antigenic potential. finally, these particles, formulated without adjuvant, were able to induce neutralizing antibody response in balb/c mice. these findings indicate vaccine potential of these particles and will be the basis for undertaking future protective efficacy studies in animal models of niv disease. since it was first recognized in 1998, nipah virus (niv) has caused several outbreaks in humans of encephalitic disease associated with high lethality. in the first outbreak, which was in malaysia and singapore, 265 humans became sick and some ,40% of them died. epidemiological links pointed to human contact with sick pigs in commercial piggeries, and the outbreak was brought under control through culling of approximately ,1.1 million pigs [1, 2, 3, 4] . since then, the virus has re-emerged in bangladesh and neighboring india, starting in 2001, and between then and now, has caused several smaller but even deadlier disease outbreaks with case fatality rates ranging between 60 and 90% [5, 6, 7, 8] . unlike the malaysian outbreak, the route of transmission in these outbreaks was considered to be bat-to-human via food contaminated with bat saliva [9] . in some cases, nosocomial transmissibility and person-to-person spread was also noted [5, 10, 11, 12] . an additional concern is that niv is also potentially an agent of agro-terror since the rate of transmission of this virus in the pig population is close to 100% [13] . effective vaccine and therapies are needed to combat the threats posed by niv. niv is a member of the genus henipavirus in the subfamily paramyxovirinae, family paramyxoviridae. it has several distinctive genetic and biologic features [14, 15, 16, 17, 18] although its morphology and genome organization is similar to that of other members of the subfamily. niv has six genes arranged in tandem, 39-n, p, m, f, g and l-59 [15, 16] . the n, p and l are required for reconstituting viral rna polymerase activity, the matrix protein m is required for particle formation and budding, and the two surface glycoproteins g and f are required for attachment and entry into the host cell [19, 20] . ephrinb2 and b3 have been identified as the niv entry receptors [21, 22, 23] . after fusion of the virus and the cell membrane, the viral ribonucleoprotein is released in to the cell cytoplasm. following transcription and replication, the viral components migrate to the plasma membrane for assembly and budding of progeny particles [24, 25] . two vaccination strategies for niv disease prevention have already been explored experimentally: a canarypox virus-based vaccine vector approach was effective as veterinary vaccine [26] , and it is in the process of further development. the same approach for human use vaccines is undergoing extensive evaluation, largely for hiv and aids [27] . a soluble niv g protein approach has also shown promise [28, 29] . however, subunit approaches are in general less effective than particulate immunogens, and can suffer from suboptimal presentation to the immune system [30, 31, 32] . immunogenicity in mice to niv glycoproteins has been reported recently using two vectored approaches for gene delivery; one using venezuelan equine encephalitis virus replicons [33] and the other involving inoculation of a mix of two complementing defective vesicular stomatitis virus (vsvdg) vectors, one for expressing each of the two niv glycoproteins [34] . the latter approach is new and seems promising but its regulatory approval as human vaccine might be problematic [34, 35] . in this study we have explored the potential of niv virus-like particles (vlps) as a vaccine. plasmid-mediated expression of selected viral proteins results in the spontaneous assembly and release of vlps. these particles make highly effective immunogens because they possess several features of the authentic virus such as their surface structure and dimensions [31, 36] . they are also safe because they do not contain any viral genetic material. vlps, where one or more of the constituent proteins serve as immunogens (native vlps), are particularly effective as vaccines for infectious disease. the fact that two such vaccines [gardasil (merck & co) for human papillomavirus (hpv), and sci-b-vac (scigen) and bio-hepb (glaxosimthkline) for hepatitis b virus (hbv)] have already been approved for human use, and many, for non-enveloped and enveloped viruses [31, 32, 37, 38, 39, 40, 41, 42] are at various stages of development, attests to the desirability of this approach for vaccine development. the budding capacity of virus proteins as vlps, the proteinprotein interactions that facilitate this process, and the central role of m protein in vlp assembly and release has been described for several paramyxoviruses such as sendai virus (sev), newcastle disease virus (ndv), respiratory syncytial virus (rsv), paramyxovirus simian virus 5 (piv-5) and human parainfluenza virus type 1 (hpiv1) [20, 43, 44, 45, 46, 47] : the efficiency of vlp formation in virtually all these studies was based on m protein release in the supernatant. niv virus-like particles have also been described [48] ; the results of this study showed 1) that niv g and f proteins individually retained some budding capacity although it was far less efficient than that of the m protein and 2) niv n, m, f and gcontaining vlps resembled the virus in some respects but differed significantly from it with respect to ratio of vlp-incorporated f protein; most of it was present in precursor f 0 form. recently, the vaccine potential of native vlps of ndv [49] has been described: these particles, composed of hn, f, m and np proteins, had several virus-like properties. however, since the f protein in this formulation was modified by design to ablate the cleavage site, it remained in its precursor form; consequently, the ndv vlps were non-fusogenic, and therefore incapable of inducing syncytia formation. here we describe niv vlps composed of the two surface glycoproteins g, and f, and the matrix protein m. the g and f proteins were included because they mediate attachment and entry into the host cell [50, 51, 52] , both are major targets of neutralizing antibodies, and both are major players in vaccine induced protection [52, 53, 54] . niv g and f together are also the most effective as immunogens; this was elucidated in a canary pox virus vector-based experimental protective efficacy study [26] . the m protein was included in our formulation because it is required for particle formation and release [20, 25, 45] . under optimized conditions, we were able to make substantial, quantifiable amounts of niv vlps composed of these three niv proteins. this has allowed us to characterize their properties in detail to show that they possessed many virus-like/vaccine desirable properties in vitro. it has also allowed us to test for immunogenicity in vivo in balb/c mice; note that although niv does not cause disease in these animals, niv proteins injected in them are known to induce robust neutralizing antibody response [29, 33, 34] . importantly, nivspecific mouse monoclonal antibodies are protective in the hamster model of niv disease [55] . in this study, careful assessment of immunogenicity has shown for the first time, that these niv vlps are able to induce neutralizing antibody response. we have also provided a detailed methodology to optimize production of the vlps for research purposes. beyond this, we have provided the first cryoem study of niv vlps and thus provide a careful assessment of their morphology. we further demonstrate that niv vlps can trigger ''fusion from without'' upon addition to cells. to our knowledge this is a first for an enveloped vlp. finally, we have shown that niv vlps activate innate immune signaling in ''infected'' cells and provide a transcriptional profile of this response. based on all these attributes, niv m, f and g-protein-containing vlps show promise as vaccine and will be the basis for undertaking future protective efficacy studies in animal models of niv disease. niv expression plasmids pcaggs-g, f, and m are all under the control of chicken beta actin promoter [56] , and they were constructed in the laboratory of one of the co-authors of this study (cb) as described previously [20] . human embryonic kidney 293 cells (atcc, crl-1573) and 293t cells (atcc, crl-11268) were grown in dulbecco's minimum essential medium supplemented with10% fetal bovine serum (fbs) and penicillin and streptomycin, and maintained in the same medium containing 2% fbs. the minigenome that was used for optimizing vlp formation has been described previously [57] . all the initial minigenome-based optimization steps were done in bhk-t7 cells (a gift from dr. n. ito). the same conditions were applicable to produce vlps in 293t cells and they were used throughout to generate the vlps used for the work described in this study. 293t cells were grown in dulbecco's complete medium to achieve semi-confluent (80-90% density) cell monolayers. the cells were transiently transfected with the plasmids constructs using the lipid reagent lipofectamine 2000 according to the general guidelines provided by the manufacturers' instructions (invitrogen inc). at 48 hrs post-transfection, the vlp-containing cell supernatants (sup) were harvested for concentration and purification of the vlps. because of the fusogenic property of our vlps, there was widespread syncytia formation at this time point although the cells were still adherent. vlps released in the transfected-cell sup were harvested and clarified by centrifugation at 3,500 rpm for 30 minutes at 4uc and concentrated by sucrose density gradient centrifugation based on previous descriptions [44, 45, 58] . briefly, the clarified sups were concentrated by ultracentrifugation through 20% sucrose cushion in tn buffer (0.1 m nacl; 0.05 m tris-hcl, ph 7.4) at 200,0006 g for 8 hours at 4uc. the resulting vlp pellet in ,0.5 ml volume was purified on a discontinuous sucrose gradient formed by layering 80%, 65%, 50% and 10% sucrose in tn buffer. after centrifugation at 186,0006 g for 8 hours, the top ,1.5 ml of the gradient (which included the vlp-containing band at the interface between the 10% and 50% sucrose layers) was resuspended in 20% sucrose buffer and centrifuged once more at 160,0006 g for one hour. the resulting pellet was resuspended in 20% sucrose solution in endotoxin-free tn bufffer and stored at 4uc for subsequent analysis. supernatant of 293t cells transfected with empty pcaggs plasmid and processed similarly (referred to as ''mock'' particles) served as negative control when needed. since the ratio of the protein expression plasmids used at transfection and the time of harvest may have a bearing on the level of vlp formation, a minigenome-based vlp infectivity assay, similar to those described previously [59, 60] was used to determine the relative concentrations of the constituent plasmids, and to determine the kinetics of vlp formation for optimal production. this assay provides only a comparative assessment of vlp formation since it only accounts for vlps that are able to incorporate and passage minigenomes. however, based on the assumption that the ratio of empty and minigenome-containing vlps will be equivalent in each reaction, the method provides an indirect means to determine the optimal set of conditions for vlp production as determined by vlp-incorporated minigenomeencoded cat enzyme activity. briefly, the steps involved in the vlp infectivity assay were 1) transfection of niv minigenome construct and co-transfection with full complement of the niv protein expression plasmids, n, p, l, m, f and g, using lipofectamine 2000. 2) following replication (48 hours posttransfection), passage of equal volume of vlp-containing transfected cell sup on to fresh cells previously transfected with n, p and l plasmids and 3), determination of cat activity in the vlp infected cells 48 hours later. replication of the vlpincorporated incoming mingenomes based on reporter gene activity indicates the level of particle formation and release, vlp infectivity, and successful minigenome packaging. fast cat assay kit (molecular probes) was used according the manufacturer's instructions and allowed accurate quantification of cat enzyme levels over a wide linear range. vlps were purified as described. the particles were adsorbed on formvar carbon coated copper grid by floating it on a drop of vlp suspension for 15 minutes, the grids were blotted, and then negatively stained with 2% aqueous uranyl acetate for viewing by transmission electron microscopy. the vlps were vitrified as reported previously [61] on holey carbon film grids (c-flat tm , protochips, raleigh, north carolina). vlps were imaged at 40,000x indicated magnification using a 4k64k slow-scan ccd camera (ultrascan 895, gatan, inc., pleasanton, ca) using a low-dose imaging procedure. unfixed vlps were used for immunogold labeling to limit antibody reactivity to the cell surface proteins. the particles were adsorbed on formvar coated nickel grids, stained with niv specific primary antibody (hyper immune mouse ascites fluid, hmaf, obtained from dr. p. rollin, cdc) diluted in buffer (1% bsa in 0.05 m tris buffer) rinsed in wash buffer (0.1% bsa in 0.05 m tris buffer), stained with colloidal gold labeled goat anti-mouse secondary antibody (jackson immunoresearch laboratories), washed, and then negatively stained with 2% uranyl acetate for viewing by em. the total protein concentration of the purified vlp preparations was measured by the bca (bicinchoninic acid) method (thermo scientific laboratories). vlp composition was determined by western blot analysis. briefly, purified vlps resuspended in endotoxin free pbs were lysed by resuspending them in equal amount of 2x sds proteinloading buffer and loaded into a 12% sds-polyacrylamide gel with a 4% stacking gel. 293t cell lysates processed similarly were run in parallel as negative cell control. following electrophoresis to resolve the protein bands, and transfer to membrane, the blot was incubated with niv-specific hmaf primary antibody at a dilution of 1:1000 dilution, overnight at 4uc, and hrp-conjugated antimouse secondary antibody (from ge healthcare) at a 1: 20,000 dilution for one hour at room temperature. the proteins were revealed using western blot detection reagents according to instructions provided by the manufacturer (ge healthcare). these studies were undertaken with the approval of the institutional biosafety committee (protocol# #01/08-2010-1) and the institutional animal care and use (iacuc) committee (protocol # 0904028). five to six week old female balb/c mice (harlan laboratories) were housed in microisolater cage for 4 days in the animal resource center at the university of texas medical branch before beginning the immunization protocol. mice in groups of five were immunized by subcutaneous inoculation of four different concentrations of vlps (1.75, 3.5, 7 or 14 mg/ mouse, referred to subsequently as treatment groups a through d respectively) prepared just prior to use in sterile endotoxin free pbs. no adjuvant was used. a group of five mice inoculated with sterile endotoxin free pbs served as negative control group. mice in the four treatment groups (a through d) were boosted (6 mg/ mouse) on days 15 and 29; the negative control group received pbs. blood was collected from the submandibular vein of the animals on days 21, 14, 21, 28 and 35; they were euthanized on day 35. plaque reduction neutralization test (prnt). two-fold dilutions of test sera were made in 50 ml cell culture medium. under biohazard level 4 conditions, each of the diluted sera were mixed with 50 ml of niv diluted to generate ,30 plaque forming units and incubated for 30 min at 37uc. the pre-incubated virusantibody mix was added to vero cell monolayers grown in 96 well plates and incubated for 30 min at 37uc when the inoculum was removed and replaced 150 ml of cell media. after incubation at 37uc for 24 h, the cells fixed in 100% ice-cold methanol and staining by indirect immunofluorescence assay as follows: the wells in the plate were blocked with bsa/pbs and stained with rabbit sera raised against the g protein of hev, and goat antirabbit alexa fluor 488 conjugate (invitrogen) diluted 1:1000 in blocking buffer. viral plaques were visualized and counted, and neutralizing antibody titers were reported based on reduction in plaque count by 50% relative to the untreated control (prnt 50 ). antibody levels measured by immunofluorescence assay (ifa). for ifa, niv-specific total antibody levels were measured by using niv g, f and m expressing 293t cells as target antigen. thirty six hours post-transfection, the cells were harvested, fixed in paraformaldehyde, cytospun (cytocentrifuge, thermoscientific) on glass slides to obtain monolayered preparations and then stored at 4uc, and used as antigen within three weeks of preparation. on the day of use, the slides were washed in pbs, permeabilized with triton-x-100 and blocked with bsa/pbs. after incubation with two fold dilutions of the test sera, the cell monolayers were washed and stained with alexa fluor 488-conjugated goat anti-mouse antibody according the manufacturer's (molecular probes) instructions. negative and positive controls were run in parallel with each batch. gene expression profile by real-time pcr vlp-mediated transcriptional activation was tested for eighty four genes involved in toll-like receptor (tlr)-mediated signal transduction using rt 2 profiler pcr array (sabiosciences). the 96 well array format included mediators of tlr signaling including adaptors and proteins that interact with the tlrs, and members of nfkb, jnkp38, nf/il6 and irf signaling pathways downstream of tlr signaling. briefly, 293 cells grown overnight in 60 mm dishes were exposed to 10 mg of purified vlps suspended in 1 ml of opti-mem (invitrogen). ''mock particles'' (see methods, vlp harvest and purification) resuspended similarly and exposed to 293 cells served as negative control. the inoculums were adsorbed on the cell monolayers for 3 hours at 37uc when additional 1.5 ml of opti-mem was added and the dishes further incubated. twenty fours post vlp exposure, total cell rna was extracted according to the manufacturer's (sabiosciences) instructions. the integrity of the rna was verified by agarose gel electrophoresis and the same concentration of total cell rna from the vlp-stimulated and ''mock'' stimulated cells were used for gene expression profiling by real-time pcr using eppendorf mastercycler unit. the array plate included positive and negative controls for quality assurance, and three sets of housekeeping genes for normalization for data analysis. the fold-change in gene expression in the vlp stimulated 293 cells relative to the ''mock'' stimulated 293 cells was calculated by the ddct method according to the manufacturer's instructions. in preliminary studies it was found that co-expression of niv g, f and m proteins in 293t cells resulted in the formation of vlps that bud out into the transfected cell sup and that they can be harvested, concentrated and purified as described under methods. however, the vlp yield was low. to improve the efficiency of vlp formation we proceeded to optimize the ratio of the three expression plasmids used at transfection. we speculated that this would be important based on the fact that a), during replication, paramyxoviruses form a transcription gradient where the 39 proximal genes are transcribed more abundantly than the successive downstream genes [52] and b), the stoichiometry of interaction of the viral proteins has proved to be critical in plasmid-driven minigenome and full-length rescue systems [62] . the importance of protein ratios for vlp formation was alluded to in a previous ndv study where the expression plasmids were co-transfected at ''pre-determined concentrations'' to produce vlp-incorporated protein ratios analogous to those in virus infected cells [49] . in a study by patch el al [48] , equivalent amounts of niv n, m, f and g were initially used to produce the vlps. in that study, vlps were subsequently also made by adjusting niv expression plasmid concentrations by experimental variations similarly to that in the ndv study [49] . the efficiency of particle formation and budding in both these, and many other paramyxovirus vlp formation systems was based on m protein release [43, 44, 45, 46, 48] . we have chosen a minigenome-based functional assay, the vlp infectivity assay (described under materials and methods), to determine optimal expression plasmid ratios for efficient vlp formation based on reporter gene readouts. briefly, 293t cells were transfected with plasmids as shown in figure 1 . for titrating niv g, f and m plasmids, increasing concentrations of either g, or f or m expression plasmids were, in turn, co-transfected with fixed concentrations of the other two plasmids. the minigenome and n, p and l plasmids were transfected using a predetermined ratio [57] . the vlp-containing cell sup was harvested 48 hours post-transfection, clarified by centrifugation, and equal volume from each was passaged onto fresh cell monolayers (vlp-infected cells) previously transfected with the core proteins required to support the incoming packaged minigenomes. the vlp infected cells were harvested 48 hours later and tested for optimal particle production based on incoming minigenome-encoded cat activity. this time point was chosen because maximal vlp formation was also found to be time dependent and optimal at 48 hours post-passage (data not shown). the reproducibility of the results was verified in an independent repeat experiment. results presented in figure 1 show that within the given range, and based on the levels of minigenome-encoded cat activity, varying the concentrations of g, f and m plasmids had a bearing on vlp formation. cat activity in the vlp infected cells appeared optimal in the boxed lanes 7 and 8 but further analysis to ensure reporter activity in the linear range (data not shown) indicated that the largest amount of minigenome-containing niv vlps were produced when the cells were transfected with the niv m, f and g plasmid ratios of 3:1:1 as in lane 7. this ratio was used for making all our vlp preparations. the optimized conditions were applied to transfect g, f and m expression plasmids in 293t cells grown in 10 cm dishes. the vlp-containing culture sups were harvested 48 hours later, and concentrated and purified as described. briefly, the clarified sups were concentrated by ultracentrifugation through 20% sucrose cushion, and then purified on a discontinuous sucrose gradient. the vlp pellet was resuspended in tn buffer and viewed by em after negative staining. the result presented in figure 2a shows a vlp-containing band in the sucrose gradient. viewing of the negatively stained purified particles by transmission electron microscopy ( figure 2b) showed numerous virus-like particles. the size variation of these vlps was consistent with the parental virus: niv is a pleomorphic virus ranging in size from 40-1900 nm [63, 64] ; the sizes of the vlps ranged from ,40-500 nm. the particles also resembled authentic niv morphologically, and this is seen more clearly in the magnified images presented in figure 2c ; here, the fringe of the glycoproteins is clearly visible on the vlp surface. an occasional vlp had what appeared to be a double fringe (shown with an arrow), a feature more frequently associated with hendra rather than niv virus particles [64] . the image in figure 2d is a cryoelectron micrograph of one of our vlps; the overall surface appearance is virus-like, which is described as dense, ordered and repetitive [31] , and it shows the surface glycoproteins and their spatial arrangement even more definitively. to verify whether the niv proteins were incorporated into the vlps as designed, purified particles were analyzed by western blotting using niv-specific mouse antibody, and hrp-conjugated anti-mouse secondary antibody as described under methods. the right hand panel in the figure 3 shows vlp-incorporated proteins in two different preparations of niv vlps. the protein bands are consistent in size to niv proteins g, f 0 , f 1 and m proteins [17, 48] . the relative amounts of the vlp-incorporated g and m proteins appeared to be similar to that reported in niv virions also [17] . this was in spite of the fact that the viral proteins in that study were revealed using rabbit sera raised against bacterially expressed hendra virus proteins. however, the ratio of the vlpincorporated f 1 to f 0 was different from that in the virions. this difference is more likely to be a reflection of timing and protein turnover rather than the reagents used to reveal them since in a previous study, pulse chase experiments have shown that similarly to the vlp-incorporated f 1 to f 0 , intracellular cleavage of the precursor niv fusion protein by cathepsin l results in near equal mix of mature fusogenic, and the precursor forms [65] . absence of the niv-specific bands in two different 293t cell lysate preparations processed similarly (and shown in the left hand panel in figure 3 ) confirms specificity of the vlp-incorporated proteins. the immunoreactivity of the vlp surface glycoproteins was verified by staining purified unfixed vlps by the immunogold labeling technique using niv-specific mouse antiserum and 6 nm colloidal gold particle-conjugated goat anti-mouse secondary antibody (jackson immunoresearch laboratories inc). the particles were viewed by em after negative staining. the use of unfixed particles assured that only the surface-exposed antigens would be reactive. numerous vlps with the gold particles decorating their surface were seen; figure 2e shows two such vlps. syncytium formation is a classical feature of niv and other paramyxovirus-induced cytopathology that can be blocked by virus-specific neutralizing antibody. a similar observation was made when 293 cells were ''infected'' with the niv vlps. briefly, the vlps were pre-incubated with niv-specific antibody, junin virus (jv)-specific antibody, and with opti-mem i (invitrogen inc) medium only for one hour at 37uc before inoculating onto near confluent 293 cell monolayers grown overnight in 60 mm dishes. the inoculum was removed after incubation for 3 hours at 37uc, replaced with opti-mem i, and the plates were further incubated overnight at 37uc overnight. the monolayers were then viewed for the formation of syncytia after staining with crystal violet. the results in figure 4 show that 293 cells exposed to niv vlps induced syncytium formation and that this process was neutralized by niv-specific antibodies; prior incubation with the unrelated jv antibodies failed to block this process. mice in groups of five were inoculated subcutaneously with four different concentrations of purified vlps and boosted as described under methods. the negative control group of five mice were inoculated with sterile endotoxin free pbs for each inoculation. the mice were bled from the submandibular vein on the day before primary inoculation, and then on days 14, 21, 28 and 35. for initial evaluation, sera from each treatment group were pooled, and the ifa method used to determine levels of niv-specific antibodies. the results in figure 5a show that titers (reciprocal of the highest serum dilution showing reactivity) increased with time post primary inoculation, i.e., the highest titers (1:2560) were seen on day 35. titers also increased with vlp dosage although by day 35, the three higher treatment groups seemed to produce similar titers. as expected, the mice in the negative control group remained nonresponsive. all sera were tested individually by plaque reduction neutralization method by doubling dilution of each sample (1:5 to 1:80) as figure 5b ) showed distinct association between vlp dosage and the ability to mount a neutralizing antibody response. mice inoculated with the two highest vlp doses (treatment groups c and d) were each able to induce neutralizing antibodies by day 35. when samples from mice receiving the two lower concentrations of vlps (3.5 ug/dose and 1.75 ug/dose, corresponding to treatment groups b and a respectively) were similarly tested, 3 of 5 and 1 of 5 mice respectively induced neutralizing antibody response; the titers ranged from 1:5 to .1:80. as expected, the control mice did not induce neutralizing response. niv vlp-induced activation of genes involved in signaling innate immune response a pcr array format (sabiosciences) was used to investigate modulation in transcription profile of 84 genes involved in innate immune responses to include tlr signaling family and members of the downstream signaling pathways, nfkb, nf/il6, irf and jnkp38. these genes represent key sensors of non-self that signal, and ultimately shape the nature of innate immune response that modulates the type and duration of adaptive immune responses [66, 67] . the differential expression of genes in vlp-exposed 293 cells relative to the ''mock'' infected 293 cells was measured by real-time pcr. same concentration of total cell rna from the vlp-stimulated and the ''mock'' stimulated control cells were used for first strand synthesis and sybr green pcr amplification of the relevant genes as described under methods. the integrity of rna in each sample was confirmed by gel electrophoresis (figure 6a ). data representing the differential transcription profile of vlp exposed vs. ''mock'' stimulated cells is shown as a heat map ( figure 6b) . a 4-fold cutoff threshold was used to determine modulation in gene expression. we noted significant vlp-stimulated up-regulation (89 fold and 7 fold) in the expression of nfkb2 and tbk1 genes respectively. close to four fold (3.9 fold) up-regulation was noted also in il-8 and mapk8 genes. nfkb2 and il-8 are target genes in the downstream nfkb pathway, and tbk1 which are in the irf and jnk/p38 pathways respectively. using a minigenome-based functional assay, we have established conditions (described under results and shown in figure 1 ) that have allowed us to produce substantial quantities of niv vlps to be able to undertake the studies described in this manuscript. we have shown that these particles are functionally assembled, biologically active and are able to induce innate immune responses, and a neutralizing antibody response. native vlps have been used to study various aspects of the virus lifecycle, as carriers to deliver heterologous proteins for vaccination, and to deliver small molecules for gene therapy purposes. particularly importantly, they have been used highly effectively as vaccines in their native form [31, 32, 39, 40, 41] . no vaccine for niv disease has been developed so far that would be both safe and protective for humans. the two vaccination strategies that have already been explored are the canary pox-based vector approach [26] and soluble subunit approach [28, 29] . niv vaccine by the former method is undergoing development as a veterinary vaccine [26] . the same approach is being evaluated for human use vaccines, mainly for the prevention of hiv and aids [27] . the subunit approach has limitations as already mentioned above [28, 29, 30, 31, 32] . one particular challenge revealed by studies that tested a soluble niv g protein-based subunit vaccine formulated with adjuvant is the potential difficulty of eradicating infection in the central nervous system. in that study [28] , live virus was present in the brain of one cat, and viral rna was present throughout the 21 day postchallenge period in the brains of the remaining challenged animals. a recently reported vaccination strategy [34] requires simultaneous inoculation of two vsvdg vectors, one expressing niv g, and the other expressing niv f proteins. it was of interest to note that supernatants of cells co-infected with these two defective viruses were infectious and could be passaged indefinitely in the absence of vsv g trans-complementation. this vaccination approach seems promising since self-propagated stock of these two viruses induced robust neutralizing antibody response in mice. however, potential pathogenicity of vsv-based vaccine vectors remains a concern [34, 35] . the potential of a recombination event resulting in a single vsv vector virus expressing both these niv proteins is unlikely, but it may still be problematic for a human use vaccine. native vlps like the ones we have produced allow the viral proteins to be presented to the immune system in the same conformation as in the virion for effective b and t cell response [31] . vlps are particularly effective in producing a protective antibody response because of their virus-like size range, their particulate nature, and their virus-like dense, repetitive and ordered surface structure [31, 36] . the spacing of the antigenic epitopes on the vlp is also optimal for b cell activation [31] : em analysis showed that our particles resembled the real virus in terms of size and surface structure [63, 64] . the image in figure 2d is the first elucidation of vlp structure of any paramyxovirus imaged by cryoem, and it provides a careful assessment of their morphology; it alludes to a surface similar to that revealed for measles virus by the same imaging technique (dr. elizabeth wright, emory university). the proteins on the vlp surface are clearly visible here; the average distance between the spikes was 9.13 nm and standard deviation was 1.72 nm. this is of interest given that epitopes spaced between 5 and 10 nm are known to be sufficient to drive optimal b cell activation [36] . niv m, f, g and n protein-containing vlps consistent in size and morphology to the parental virus have also been reported in a previous study which evaluated protein-protein interaction that facilitate vlp formation [48] . however, in that study, most of the particle-incorporated niv f protein was predominantly in the uncleaved precursor form. this finding is clearly distinct from ours since our vlps contained substantial amounts of cleaved f protein, and this may have been related to ratios of the interacting proteins expressed in 293t transfected cells. in a recent study of ndv vlps [49] , the particle-incorporated proteins were reported to have virus-like protein ratios, but the f protein remained in its precursor form because the cleavage site required to produce the fusion competent form was mutated by design. what effect a vlp-incorporated non-fusogenic f protein may have, relative to the fusogenic form, on the level and quality of vlp-induced immune response is not clear at present since difference in immunogenicity between fusion-competent and fusion-defective vlps has not been experimentally evaluated so far. however, a recent report suggests that viral fusogenic membrane glycoproteins may enhance vaccine potency [68] . immunogold labeling of our unfixed niv vlps confirmed that the surface proteins in our vlps were functionally assembled and they were biologically active ( figure 2e ). we could deduce the presence of biologically active g and f proteins on the vlp surface by the fact that they were able to induce the formation of syncytia in 293 cells ( figure 4 ); this is a process that requires the interaction of both the surface glycoproteins, the attachment protein g, and the fusion competent f protein, when they come in contact with the cognate receptor-bearing cells. formation of syncytia or multinucleated cells in replication competent enveloped viruses, especially paramyxoviruses, is induced by a process that is described as ''fusion from within'', and it can be blocked or neutralized by prior treatment of the virus with specific antisera. in contrast, ''fusion from without'' is induced by non-replicating viruses at high multiplicities of infection, and it too can be blocked by pretreatment with virus-specific antibodies ( [69] , and references therein; [70] ). our non-replicating particles likewise induced syncytia formation in 293 cells that could be neutralized with nivspecific antibodies (figure 4 ). to our knowledge, this is the first study describing fusion from without induced by vlps of any paramyxovirus, or any other enveloped viruses, although it has been described for the vlps of the non-enveloped rotavirus [71] . the mechanism(s) of fusion from without is not clear but two models have been proposed [72, 73] ; one proposes that particles connecting adjacent cells effectively promote fusion between them, and the other is that when particles decorated with the surface glycoproteins fuse with the target cell membrane, the glycoprotein complexes diffuse freely in the lipid bilayer, and mimic fusion from within. the type of vlp-induced syncytia formation and eventual cell death is also not known. we are in the process of investigating it. neutralizing antibody response is the critical correlate of protection mediated by prophylactic vaccines [53, 54] and native vlps promise to be highly effective prophylactic vaccines for paramyxoviruses like niv, and others like ndv and measles where neutralizing immune response is known to play a pivotal role in protection against disease [53, 54, 55, 74] . our vlps were highly effective immunogens, and all, especially in the three higher treatment groups produced high levels of response by day 35 ( figures 5a) . importantly, niv vlps were able to induce neutralizing antibodies. this response was clearly dose-dependent ( figure 5b ). all ten mice receiving a primary inoculation of 7 or 14 mg vlps (subgroup c and d) were able to produce such response; but even of those animals that received a first dose of only 3.5 or 1.75 ug/mouse (treatment group b and a respectively), 3 of 5 and 1 of 5 produced neutralizing antibodies. neutralization antibody response was first seen on day 28, and increasing titers were seen in some animals within a week of it; we believe that this response, induced by our non-replicating and potentially safe particles, formulated without adjuvant, compares favorably with the levels of such response induced at an equivalent time point by some replication competent pseudotype viruses [34] . immunogenicity to native vlps has been reported previously for one other paramyxovirus namely ndv [49] . in that report, immune response to ndv vlps was evaluated by primary inoculation of mice intraperitoneally with vlp concentrations ranging between 10 and 40 mg, and a booster dose of 10 mg, without adjuvant. ndv-specific titers by elisa were high in each mouse in each treatment group. neutralizing antibody response to 20 and 40 mg of these particles was also detected. the nature of innate immune response dictates the type and duration of adaptive immune response [67, 75] . the mechanism by which niv vlps are recognized by host cells and trigger the induction of innate immune response, and how this translates into effective adaptive immunity is not known. here we have taken the first step ( figure 6 ) towards understanding this process. with the experimental conditions as described, we observed vlp-induced activation of some of the genes that are known to be involved in the induction of an effective innate immune response [75] . results presented in the heat map in figure 6 show that relative to the ''mock'' treated cells, nfkb2 gene (in the nfkb pathway) was up-regulated 89 fold as a result of vlp exposure, and tbk1 (in the irf pathway) was 7 fold higher. in the light of these findings, we are testing pcr array expression profiles of the same set of 84 genes in 293 and other cells at earlier and later time points to identify their upstream effectors, and niv vlp-responsive signaling networks. in this respect, the murine system, with the many available immunological reagents and knockout strains may provide the best system to identify these host sensors. currently there is minimal information on live niv infection-responsive cellsignaling changes [76] and there is none on array-based transcriptional alterations for comparative analysis. likewise, it has not been possible to compare the niv vlp-induced transcription modulation with those induced by other paramyxovirus vlps since to our knowledge, such studies have not been undertaken so far. lastly, a growing number of reports point to viral surface glycolproteins as relevant in host cell signaling and triggering of innate immune response. we believe that particles like niv vlps, with many virus-like properties (including their surface glycoproteins organized to resemble the parental virus, figure 2d ) would induce an effective innate immune response for the promotion of the desired adaptive immunity [67, 76] . finally, as described above, our vlps were highly effective as immunogens, able to induce neutralizing antibody response in all animals with primary inoculation of as little as 7 mg vlp protein each. fusogenic property of our vlps may be critically relevant in this regard in the light of recent findings, and would need to be experimentally verified by comparing the potency of fusioncompetent and fusion-defective vlps as vaccine [68] . in conclusion, we have been successful in producing substantial quantities of niv vlps needed to characterize niv vlps, we have demonstrated their many virus-like properties, and their effectiveness as immunogens in balb/c mice. these findings are the basis on which we will be undertaking future challenge studies in the hamster model of niv disease [77] . a survey of nipah virus infection among various risk groups in singapore fatal encephalitis due to nipah virus among pig-farmers in malaysia nipah virus: a recently emergent deadly paramyxovirus nipah virus outbreak in malaysia nipah virusassociated encephalitis outbreak nipah 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characterization of the antiviral and inflammatory responses against nipah virus in endothelial cells and neurons a golden hamster model for human acute nipah virus infection we thank dr. v. popov and julie wen for their help and advice with electron microscopy. vishal naik provided much valued technical assistance. joshua lisinicchia, provided essential help at various stages of the project. key: cord-002180-gsdk5x3e authors: davies, colin; ward, vernon k. title: expression of the ns5 (vpg) protein of murine norovirus induces a g1/s phase arrest date: 2016-08-24 journal: plos one doi: 10.1371/journal.pone.0161582 sha: doc_id: 2180 cord_uid: gsdk5x3e murine norovirus-1 (mnv-1) is known to subvert host cell division inducing an accumulation of cells in the g(0)/g(1) phase, creating conditions where viral replication is favored. this study identified that ns5 (vpg), is capable of inducing cell cycle arrest in the absence of viral replication or other viral proteins in an analogous manner to mnv-1 infection. ns5 expression induced an accumulation of cells in the g(0)/g(1) phase in an asynchronous population by inhibiting progression at the g(1)/s restriction point. furthermore, ns5 expression resulted in a down-regulation of cyclin a expression in asynchronous cells and inhibited cyclin a expression in cells progressing from g(1) to s phase. the activity of ns5 on the host cell cycle occurs through an uncharacterized function. amino acid substitutions of ns5(y26a) and ns5(f123a) that inhibit the ability for ns5 to attach to rna and recruit host eukaryotic translation initiation factors, respectively, retained the ability to induce an accumulation of cells in the g(0)/g(1) phase as identified for wild-type ns5. to the best of our knowledge, this is the first report of a vpg protein manipulating the host cell cycle. noroviruses are non-enveloped viruses from the caliciviridae family that cause gastroenteritis in a variety of mammals including humans [1] [2] [3] [4] [5] . human norovirus (hunov) infections account for significant mortality in the developing world, and in the developed world norovirus outbreaks come with a substantial financial burden [6] . hunov research has been hampered by the lack of a reproducible animal or cell culture system that supports viral replication. using mnv-1 as a model allows norovirus replication and host cell interactions to be studied in cell culture and in small animals [7] . mnv-1 is a positive-sense rna virus of approximately 7.4 kb, containing four open reading frames (orf). orf1 encodes for 6 non-structural proteins (ns1-2, ns3, ns4, ns5, ns6, and ns7) while orf2 and orf3 encode the major and minor structural proteins respectively [8] . orf4 encodes for virulence factor 1, a non-essential protein involved in interactions with host apoptotic pathways [9] . the mnv ns5 (vpg; virus protein, genome linked), is a~16 kda protein that is covalently linked to the 5 0 end of the genomic and subgenomic rna [8] . linkage to the genome is thought to prevent detection by host pathogen recognition receptors such as rig-1 and protein kinase r that detect uncapped 5 0 triphosphorylated rna, leading to an antiviral response. ns5 additionally has a role in genome replication, acting in place of an rna 5 0 cap to provide a free hydroxyl that can be extended by the virally encoded rna-dependent rna polymerase (ns7) [10] . the ns5 protein also acts to aid viral translation, recruiting host eukaryotic translation initiation factors to initiate translation of viral proteins [11] . the ns5 protein also contains regions of predicted disorder that are often associated with multiple functions [12, 13] . as more viruses are characterized, it is becoming increasingly common to observe interactions between viral replication and the host cell cycle. each phase of the cell cycle presents distinctive biological conditions that have a significant impact on viral replication. many viruses can subvert the host cell division in order to create an environment where viral propagation is preferred. several rna viruses, including murine norovirus 1 (mnv-1) have been characterized to manipulate cell cycle progression at the g 1 /s restriction point, often creating favorable conditions for viral replication [14] [15] [16] [17] [18] [19] [20] [21] . cell cycle progression is a complex process that is tightly controlled by multiple pathways. the g 1 /s checkpoint controls progression from the first gap phase (g 1 ), a period of substantial cell growth, into the synthesis phase (s) where the host dna is replicated. progression through g 1 /s is predominantly controlled by the phosphorylation status of the retinoblastoma protein (prb), which is in turn controlled by the activities of cyclins and cyclin-dependent kinases (cdk) (reviewed in [22] ). cyclins are expressed at various stages of cell division and bind to their corresponding cdk and phosphorylate numerous targets including prb. in early g 1 phase, cyclin d family members bind to cdk4/6 and phosphorylate prb, driving g 1 phase progression and expression of e and a cyclins. cyclin e forms a complex with cdk2, which further phosphorylates prb to release an e2f transcription factor, driving s phase entry [23] . cyclin a levels continue to increase during s phase and help drive cell cycle progression through the later stages of the cell cycle, through to the initiation of prophase during mitosis [24, 25] . recently, we have shown that mnv-1 is able to manipulate the host cell division in murine macrophages, inducing an accumulation of cells in the g 0 /g 1 phase due to an arrest at the g 1 /s restriction point [20] . additionally, this g 1 /s arrest created conditions where mnv-1 replication was favored compared to other stages of the cell cycle. in this study, we show that expression of viral ns5 protein in cell culture induces an accumulation of cells in the g 0 /g 1 phase through a g 1 /s arrest in an analogous manner to mnv-1 infection. furthermore, the effects of ns5 on the host cell cycle are independent of the known replication and translation activities attributed to ns5 (vpg). raw-blue cells, a mouse leukemic monocyte macrophage cell line (obtained from invivogen, san diego, ca) were maintained in dulbecco's modified eagle's medium (dmem) (life technologies, gaithersburg, md) containing 10% heat-inactivated fetal calf serum (thermo fisher scientific), penicillin (100 u/ml), streptomycin (0.1 mg/ml), normocin (100 μg/ml) and zeocin (200 μg/ml) (life technologies). cells were maintained at 37°c in a 5% co 2 humidified atmosphere and passaged every 48 h. mnv-1 ns1-2 and ns5 sequences were amplified from the mnv-1 infectious clone pmnv ã , derived from mnv strain cw1 (genbank accession dq285629) as described in ward et al [26] , and the pcr products ligated into puc8 and the sequence verified. primers used for ns1-2 amplification were 5 0 gaaattaatacgactcactatagtgaaatgaggatggc aacgccatc 3 0 (forward) and 5 0 agcaaggtcgaagggttattcggc 3 0 (reverse) and for ns5 5 0 gaaattaatacgactcactatagggagaatgggaaagaagggcaaga`3 0 (reverse). each forward primer added a gaaat motif and a t7 promoter (underlined) to the 5 0 end of the insert for t7 rna polymerase binding as recommended for mmessage machine in vitro rna transcript production (ambion). bold sequences indicate start (atg) or stop (tta) codons. the expression constructs matched the mnv-1 ns1-2 and ns5 sequences except for the addition of a methionine codon at the 5 0 end of the ns5 coding sequence to facilitate translation and stop codons in the ns1-2 and ns5 constructs to terminate translation. the resulting plasmids were transformed into xl1-blue mrf 0 escherichia coli cells and used as templates for in vitro rna synthesis. cloning of ns5(y26a) and ns5(f123a). constructs for the expression of ns5 variants ns5(y26a) and ns5(f123a) were designed with the requirements for rna synthesis and translation and were synthesized by genscript and cloned into a puc57-simple vector (gen-script, piscataway, nj, usa) [27] . the ns5 sequences (genbank accession dq285629) were flanked at the 5 0 end by a bamhi restriction site, a t7 promoter sequence (underlined), a kozak sequence for optimal rna translation and a methionine codon (bold) (ggatccgaa attaatacgactcactatagggagaatg). flanking the 3 0 end of the ns5 sequence was a stop codon (bold) and a hindiii restriction site (tgaaagctt). the ns5 variants sequences had alanine substitutions inserted at the tyrosine 26 and phenylalanine 123, named ns5 (y26a) and ns5(y26a) respectively. the resulting plasmids were transformed into xl1-blue mrf 0 e. coli cells and used as templates for in vitro rna synthesis. in vitro rna synthesis. plasmids were linearized at the 3 0 end of viral genes with ecori (for ns1-2), avai (for ns5) or hindiii (for ns5(y26a) and ns5(f123a)). subsequently, messenger rna (mrna) transcripts were synthesized from the linearized plasmids using the mmessage mmachine transcription kit (ambion) and purified using megaclear transcription clean-up kit (ambion). electroporation. transfection of rna transcripts was performed using a neon transfection system (life technologies), following manufacturer's instructions. briefly, raw-blue cells were suspended in resuspension buffer and approximately 1 × 10 6 cells transfected with 4-6 μg of rna using 1 pulse at 1730 v and 20 ma. transfected cells were added to 2 ml of pre-warmed medium in a 6-well plate and incubated for the times indicated. western blot analysis. transfected cells were collected posttransfection and washed twice in dulbecco's phosphate buffered saline (dpbs). cells were lysed directly in 25 μl dpbs and 25 μl sample buffer (120 mm tris-hcl [ph 6.8], 5% sds, 10% 2-mercaptoethanol, 20% glycerol, 0.01% bromophenol blue), boiled for 10 minutes and separated by sds-page electrophoresis. proteins were transferred to nitrocellulose membranes (amersham hybond-c extra; ge healthcare) and detected with the corresponding primary and secondary antibodies. the following primary antibodies were used; cyclin a (h-432) (sc-751; santa cruz), actin (i-19) (sc-1616; santa cruz), gfp (ab6556; abcam), mnv-1 anti-ns1-2 [13] and mnv-1 anti-ns5 [13] . secondary antibodies used were 680rd donkey anti-goat igg (926-68074; li-cor) and 800cw donkey anti-rabbit igg (926-32213; li-cor). synchronization of cells. to synchronize cells to the g 1 phase, approximately 2 × 10 6 cells were seeded in 25-cm 2 flasks and treated with 3 mm sodium butyrate (n-butyrate) (b5887; sigma) for 20 h [28, 29] . cell cycle analysis by flow cytometry. nuclear dna content was measured by propidium iodide staining and fluorescence-activated cell sorting (facs) as previously described [20] . briefly, cells were scraped, washed in dpbs and fixed in 3 ml of cold 70% absolute ethanol overnight. fixed cells were washed in dpbs and stained in 50 μg/ml propidium iodide (p4170; sigma) and 0.1 mg/ml rnase a (r4875; sigma) for 45 min at 37°c in 5% co 2 . stained cells were washed and analyzed using fluorescence-activated cell sorting (facs); data was analyzed with modfit lt 3.0 software (verity software house). statistical and densitometric analyses. data is presented as means and standard deviations (sd). results were analyzed with either a student's t-test or a one-way anova with the appropriate post-test as stated. p values of 0.05 were considered statistically significant. western blots are shown for one of three independent experiments. band analysis for each protein was quantified using image studio lite software. each protein quantification was first normalized against actin loading before comparisons for changes (recorded as a percentage of mocktransfected) were made. expression of ns5 induces a g 0 /g 1 phase arrest mnv-1 infection of raw-blue cells induces an arrest at the g 1 /s restriction point, increasing the g 0 /g 1 population in order to change the internal cellular environment to favor viral replication [20] . we sought to determine if a viral encoded protein was responsible by analysis of the non-structural proteins of mnv-1 for effects to the host cell cycle. the ns5 protein from caliciviruses is essential to viral rna transcription and translation and is essential for calicivirus replication. the ns5 protein has been shown to bind host eukaryotic translation initiation factors, recruiting these proteins for preferential viral translation and potentially inhibiting host protein expression [11, 30] . we hypothesized that an inhibition of host protein expression may contribute to a cell cycle arrest. the effect of ns5 on the host cell cycle was therefore determined by transfection of raw-blue cells with rna transcripts, encoding individual viral genes, ns1-2 from mnv-1 was included as a negative control (fig 1a) . ns1-2 and ns5 were detected by their corresponding antibodies 18 h posttransfection (fig 1c) . expression of ns5 increased the population of cells in the g 0 /g 1 phase by 28% and decreased the s phase by 27% when compared to the mock-transfected population ( fig 1b) . furthermore, ns5 expression decreased cyclin a expression by 68% when compared to the mock-transfected control ( fig 1d) . the cyclin a protein governs s phase entry and progression, so a decrease in expression would imply a decrease in s phase entry, further indicating ns5 as the protein responsible for the mnv-1 induced cell cycle manipulation. ns1-2 had no significant effects on the host cell cycle or cyclin a expression. if the ns5 protein is responsible for the cell cycle arrest, then it should induce its cell cycle arrest at the g 1 /s restriction point, as observed during mnv-1 infections [20] . the transition through g 1 /s is a highly regulated checkpoint during cell division and is often targeted by viruses to induce changes to the host cell cycle [14, 18, 31, 32] . the transition of cells through the g 1 /s restriction point was examined in cells expressing ns5. cells were synchronized to the g 1 phase through n-butyrate treatment, released from the arrest and transfected with ns5, ns1-2 and gfp coding rna. ns1-2 was used as a viral control protein that did not affect the cell cycle and gfp as a non-viral rna negative control. n-butyrate was also added to released populations and used as a positive control. cells were then analyzed for their transition from g 1 into s phase by facs analysis of the host cell cycle. gfp, ns1-2 and ns5 rna transcripts were translated into protein and detected from 15 to 24 hours post-g 1 release (fig 2a) . the nbutyrate treated cells remained predominantly in g 1 phase post-release while the mock-transfected cells came out of the g 1 arrest, indicated by a decrease in the g 0 /g 1 population and an increase in the s phase cells post-release (fig 2c) . transfection of gfp and ns1-2 rna had no effect on the transition of cells from g 1 into s phase compared to the mock-transfected population, with both gfp and ns1-2 transfected populations progressing into s phase, with a substantial decrease in the g 0 /g 1 population observed at 21 hours post-release, similar to the mock-transfected population (fig 2c) . in contrast, transfection of ns5 rna induced a g 1 /s phase arrest, as indicated by cells remaining in the g 1 phase post-release, with no increase in the s phase population. at 24 hours post-release in ns5 transfected populations, 73% of cells remained in the g 0 /g 1 phase compared to 46% in the mock-transfected population (fig 2c) . because cyclin a expression is inhibited by mnv-1 infection in cells transitioning through the g 1 /s restriction point [20] , cells were analyzed post-g 1 release for cyclin a expression by western blot analysis. expression of ns5 inhibited the accumulation of cyclin a in populations progressing from g 1 to s phase. in a g 1 arrested population expression of cyclin a is low, indicated by undetectable levels of cyclin a in n-butyrate treated cells (fig 2d) . in mock-transfected, gfp and ns1-2 transfected populations, cyclin a expression increased from 18 to 24 hours post-release as cells entered s phase. in ns5 transfected cells, cyclin a levels remained below detectable limits post-release, as transfected cells remained in g 0 /g 1 phase and did not progress into s phase. these results indicate that ns5 is responsible the cell cycle arrest induced by mnv-1. not only does ns5 expression increase the g 0 /g 1 population, but it also inhibits progression at the g 1 /s restriction point and inhibits cyclin a expression. a well-characterized function of ns5 in viral replication is to recruit host eukaryotic initiation factors for preferential translation of viral proteins [30, 33] . it was hypothesized that the manipulation of the host cell cycle by ns5 could be driven by its association with host eukaryotic initiation factor eif4g, leading to inhibition of host translation, as seen with plant vpg proteins [34] , and thus potentially inducing a cell cycle arrest. the ability of mnv ns5 to bind to eif4g can be abolished through the introduction of a phenylalanine to alanine substitution at position 123 (ns5(f123a)) [30] . not only does the ns5(f123a) substitution inhibit binding to scaffold protein eif4g, it abolishes viral replication. we predicted that the introduction of the ns5(f123a) substitution could inhibit its cell cycle control. rna transcripts encoding wt ns5, ns5(f123a) and ns1-2 were generated, transfected into an asynchronous cell population and their cell cycle effects analyzed by flow cytometry. both ns5 and ns5(f123a) could be detected by the α-ns5 antibody (fig 3c) . expression of viral ns1-2 had no effect on the host cell cycle while expression of both ns5 and the ns5(f123a) variant increased the g 0 /g 1 population by~22% and decreased the s phase population proportionally when compared to the mock-transfected population (fig 3b) . furthermore, the ns5(f123a) variant decreased cyclin a protein expression by 67% when compared to the mocktransfected population in a synonymous manner to ns5, strongly implying that the host eukaryotic initiation factor binding domain of ns5 does not play a role in its cell cycle manipulation (fig 3d) . the ns5 protein from mnv is covalently attached at the 5 0 terminus of viral rna, acting as a cap to prime rna synthesis [8] . attachment of ns5 to viral rna occurs via the tyrosine residue at position 26 (y26) in mnv, lying within a highly conserved eyde motif in caliciviruses [10, 35, 36] . substitution of the y26 residue with an alanine residue ns5(y26a) prevents the formation of ns5-viral rna [10, 37] . the nucleotidylation of ns5 at y26 is likely contingent upon viral rna polymerase (ns7), which is not present in the expression system used in this study. to further confirm that y26 residue is not involved in the cell cycle arrest through an unidentified mechanism, an alanine substitution at y26 was introduced (ns5 (y26)). although the viral genome and ns7 protein is absent in the transfection, we could not exclude that ns5 could be attaching via the y26 residue to host rna. rna encoding wt ns5, ns5(y26a) and ns1-2 were synthesized, transfected into an asynchronous cell population and their cell cycle effects analyzed by flow cytometry. both ns5 and ns5(y26a) could be detected by the α-ns5 antibody (fig 4c) . expression of viral ns1-2 had no effect on the host cell cycle while both ns5 and ns5(y26a) expression increased the g 0 /g 1 population by~20% and decreased the s phase population proportionally when compared to the mock-transfected population ( fig 4b) . furthermore, wt ns5 and ns5(y26a) decreased cyclin a protein expression by 69% and 73% respectively when compared to the mock-transfected population (fig 4d) . these results, combined with the absence of viral rna polymerase, imply that nucleotidylation to rna via the y26 residue has no role in ns5 ability to manipulate the host cell cycle. we recently identified that mnv-1 was able to manipulate the host cell cycle, causing an arrest and accumulation in an early cell growth phase leading to enhanced viral replication [20] . this study identifies that the ns5 protein from mnv-1 is able to induce a cell cycle arrest analogous to that of mnv-1 infection in a mouse macrophage cell line, in the absence of other viral factors. changes to the host cell cycle post ns5 expression was nearly identical to that observed under mnv-1 infection with the g 0 /g 1 population increasing by 24% and 28% and the corresponding s phase population decreasing by 26% and 27% respectively. expression of ns5 alone was sufficient to induce an accumulation of cells in the g 0 /g 1 phase and reduce cyclin a expression in an asynchronous population. further confirmation of ns5 as the cell cycle regulator comes from analyzing the ability of ns5 to inhibit the g 1 /s transition and prevent cyclin a accumulating in a population progressing from g 1 into s phase. these effects on the host cell cycle are consistent with the observed effects of mnv-1 infection, showing ns5 to be a causative agent of this cell cycle arrest. it was initially hypothesized that the cell cycle arrest induced by ns5 was due to its known interaction with host eif4g. the ns5 protein from several caliciviruses has been documented to aid in viral protein translation, through binding to several host eukaryotic initiation factors and recruiting ribosomes to the site of viral replication [11, 30, [38] [39] [40] . the concentration of host eukaryotic initiation factors by vpg proteins is predicted to impair host translation [36] . shut-off of host protein translation has been shown to induce cell cycle arrests in several cell phases including g 1 and g 2 /m [41, 42] . we analyzed the impact on the host cell cycle of ns5 binding to host eukaryotic initiation factors through the introduction of an amino acid substitution that was known to impede binding to eif4g [30] . the ns5(f123a) substitution had no effect on the ability of ns5 to induce a g 1 arrest indicating an alternative mechanism is responsible for the cell cycle manipulation. another well characterized function of ns5 is in priming of rna synthesis via attachment to viral genomic and subgenomic rna. while nucleotidylation of the ns5 y26 residue is dunnett's post-test. *, p 0.05; **, p 0.01; ***, p 0.001. (c) expression of ns1-2, wt ns5, ns5(f123a) and cyclin a was determined by western blot analysis, actin was used as a loading control. (d) cyclin a levels from three experiments were quantified with image studio lite (li-cor) and normalised against the results for actin and results presented as means and sd from three independent experiments. statistical significance was determined for comparisons between transfected populations and the mock-transfected population using a one-sample t-test. *, p 0.05; **, p 0.01. unlikely in the absence of viral rna polymerase (ns7) we wanted to confirm that y26 had no role in cell cycle arrest. attachment to host rna could result in changes in protein expression, leading to a cell cycle arrest. the ns5(y26a) protein is unable to undergo the nucleotidylation reaction that attaches ns5 to viral rna but still induced cell cycle changes in an analogous manner to wt ns5. this indicates that attachment to rna via the y26 residue is not the cause of cell cycle affects. mnv ns5 is indicated to be located in the perinuclear region, so an interaction with host dna is unlikely [43] . because 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structures of the compact helical core domains of feline calicivirus and murine norovirus vpg proteins calicivirus translation initiation requires an interaction between vpg and eif 4 e sapovirus translation requires an interaction between vpg and the cap binding protein eif4e the genome-linked protein vpg of the norwalk virus binds eif3, suggesting its role in translation initiation complex recruitment rapamycin stimulates viral protein synthesis and augments the shutoff of host protein synthesis upon picornavirus infection avian reovirus nonstructural protein p17-induced g(2)/ m cell cycle arrest and host cellular protein translation shutoff involve activation of p53-dependent pathways subcellular localization of the mnv-1 orf1 proteins and their potential roles in the formation of the mnv-1 replication complex special thanks to dr estelle baker and joanna ward for generation of the wild-type ns1-2 and ns5 plasmids. key: cord-001186-jkg7qkj6 authors: skowronski, danuta m.; hamelin, marie-eve; de serres, gaston; janjua, naveed z.; li, guiyun; sabaiduc, suzana; bouhy, xavier; couture, christian; leung, anders; kobasa, darwyn; embury-hyatt, carissa; de bruin, erwin; balshaw, robert; lavigne, sophie; petric, martin; koopmans, marion; boivin, guy title: randomized controlled ferret study to assess the direct impact of 2008–09 trivalent inactivated influenza vaccine on a(h1n1)pdm09 disease risk date: 2014-01-27 journal: plos one doi: 10.1371/journal.pone.0086555 sha: doc_id: 1186 cord_uid: jkg7qkj6 during spring-summer 2009, several observational studies from canada showed increased risk of medically-attended, laboratory-confirmed a(h1n1)pdm09 illness among prior recipients of 2008–09 trivalent inactivated influenza vaccine (tiv). explanatory hypotheses included direct and indirect vaccine effects. in a randomized placebo-controlled ferret study, we tested whether prior receipt of 2008–09 tiv may have directly influenced a(h1n1)pdm09 illness. thirty-two ferrets (16/group) received 0.5 ml intra-muscular injections of the canadian-manufactured, commercially-available, non-adjuvanted, split 2008–09 fluviral or pbs placebo on days 0 and 28. on day 49 all animals were challenged (ch0) with a(h1n1)pdm09. four ferrets per group were randomly selected for sacrifice at day 5 post-challenge (ch+5) and the rest followed until ch+14. sera were tested for antibody to vaccine antigens and a(h1n1)pdm09 by hemagglutination inhibition (hi), microneutralization (mn), nucleoprotein-based elisa and ha1-based microarray assays. clinical characteristics and nasal virus titers were recorded pre-challenge then post-challenge until sacrifice when lung virus titers, cytokines and inflammatory scores were determined. baseline characteristics were similar between the two groups of influenza-naïve animals. antibody rise to vaccine antigens was evident by elisa and ha1-based microarray but not by hi or mn assays; virus challenge raised antibody to a(h1n1)pdm09 by all assays in both groups. beginning at ch+2, vaccinated animals experienced greater loss of appetite and weight than placebo animals, reaching the greatest between-group difference in weight loss relative to baseline at ch+5 (7.4% vs. 5.2%; p = 0.01). at ch+5 vaccinated animals had higher lung virus titers (log-mean 4.96 vs. 4.23pfu/ml, respectively; p = 0.01), lung inflammatory scores (5.8 vs. 2.1, respectively; p = 0.051) and cytokine levels (p>0.05). at ch+14, both groups had recovered. findings in influenza-naïve, systematically-infected ferrets may not replicate the human experience. while they cannot be considered conclusive to explain human observations, these ferret findings are consistent with direct, adverse effect of prior 2008–09 tiv receipt on a(h1n1)pdm09 illness. as such, they warrant further in-depth investigation and search for possible mechanistic explanations. during spring-summer 2009, several observational studies from canada reported that prior receipt of the 2008-09 trivalent inactivated influenza vaccine (tiv) was associated with increased risk of medically-attended, laboratory-confirmed a(h1n1)pdm09 illness, with estimated risk or odds ratios of 1.4-2.5 compared to those unvaccinated [1] . this increased risk was not apparent among vaccinated people when comparing hospitalized to community cases [1] , and observational studies in other settings showed contradictory results, including increased [2] [3] [4] [5] , null [6] [7] [8] [9] or protective [10, 11] effects from vaccination. hypotheses to explain findings from canada initially focused on methodologic (observational designs) or product-specific (domestically-manufactured vaccine) considerations. however, a randomized-controlled trial (rct) in hong kong spanning november 2008 to october 2009 also showed significantly increased relative risk (2.58) among children who had received a different manufacturer's 2008-09 tiv product (vaxigrip, sanofi pasteur, lyon, france) [12, 13] . previous ferret studies have also shown mixed results although none have demonstrated 2008-09 tiv to have been protective against a(h1n1)pdm09 [14] [15] [16] [17] [18] [19] . two small ferret studies reported no tiv effect on virus replication in nasal or lung specimens [14, 15] but, where clinical outcomes have been assessed, several studies have shown consistent albeit non-significant trend toward greater weight loss and worsening of severity indicators in vaccinated ferrets [16] [17] [18] [19] . all of these ferret studies to date, however, have suffered from small sample size, typically comparing #5 animals per group in total. mechanistic hypotheses to explain increased a(h1n1)pdm09 risk among prior tiv recipients have included both direct and indirect vaccine effects [1] . the direct effect hypothesis postulates that seasonal vaccine may directly influence host resistance to pandemic virus infection and/or replication whereas the indirect hypothesis proposes that seasonal vaccine may block the more robust, complex and cross-protective immunity otherwise afforded by seasonal virus infection thereby indirectly increasing the risk of pandemic illness. here we report on a randomized, blinded, placebo-controlled ferret study to test whether the commerciallyavailable tiv predominantly used in canada in 2008-09 may have directly influenced a(h1n1)pdm09 disease risk. overview experimental procedures were conducted at the laval university animal care facility in québec between april 27 and july 4, 2011 ( figure 1 ). animals were housed two per cage in the same room and permitted food and water ad libitum. the primary outcome of this study was weight loss. sample size was assigned to test differences in proportionate weight loss from baseline following infection with pandemic h1n1 virus. based on 80% power and 2-sided alpha of 0.05 to detect mean difference in weight loss relative to baseline of 5% in the placebo group and 10% in the vaccine group, and given standard deviation (sd) of 4-5%, 12-17 ferrets per group would be required. we used 16 ferrets per group. thirty-two male ferrets were randomly assigned to receive 0.5 ml intra-muscular injection of either 2008-09 tiv (''vaccine'') or pbs (''placebo'') on days 0 and 28. study personnel and investigators remained blinded to vaccine/placebo assignment throughout experimental procedures and assays. glaxosmithkline (gsk fluviral; manufactured in laval, québec, canada) and sanofi pasteur (vaxigrip; manufactured in lyon, france) supplied approximately 75% and 25%, respectively, of the seasonal split tiv distributed in canada during the 2008-09 season. in this experiment we therefore used the dominant canadian manufactured, commercially-available, non-adjuvanted gsk sodium deoxycholate split-antigen fluviral containing the three whothe potency of all three vaccine strains of the post-expiry but cold-chain maintained 2008-09 fluviral lot that was used was confirmed by single radial immuno-diffusion (srid) testing by the center for biologics evaluation and research, united states (us) food and drug administration (fda) [21] . us specifications require $27 mg/ml hemagglutinin (ha) for each antigen (dose 0.5 ml) based on the mean of three tests, with additional requirements around the standard deviation (sd). as last assessed in february 2012, for each antigen, mean ha content for the tiv lot used remained $30 mg/ml with sds within required specifications. on day 49, animals were lightly anesthetized and challenged (ch0) intra-nasally with 250 ml (4.5logtcid50/ml) [22, 23] (table s1) , administered half-volume per nostril. four randomly-selected ferrets per group were sacrificed on day 54 (i.e. 5 days post-challenge: ch+5) and the remainder sacrificed on day 63 (i.e. 14 days post-challenge: ch+14). pre-shipment sera collected between march 14 and 24, 2011 and serum samples from anesthetized animals collected on days 0 (may 2, 2011), 28, 49 (ch0), 54 (ch+5) and 63 (ch+14) were assessed for antibody to tiv components and a(h1n1)pdm09 by haemagglutination inhibition (hi), microneutralization (mn), nucleoprotein (np)-based elisa and ha1-based protein microarray assays. hemagglutination inhibition and microneutralizationantigens and analyses. reference viruses used in the hi and mn assays were obtained from canada's influenza reference laboratory, the national microbiology laboratory (nml), winnipeg, and passaged in mdck cells two to three times (table s1) . mdck-passaged viruses were tested by both hi and mn against reference ferret anti-serum provided by the nml, confirming antigenic integrity was maintained relative to the who-recommended reference viruses. viral hemagglutinin (ha) and neuraminidase (na) were also sequenced to assess amino acid (aa) identity relative to who reference viruses and the actual vaccine components selected by the manufacturer (table s1 and table s2 ). compared to respective vaccine strains, passaged assay viruses showed complete ha and na identity for seasonal h1n1 and $98% for h3n2. compared to challenge virus, passaged a(h1n1)pdm09 assay virus also showed $98% ha and na identity. percent identity was reduced as expected when comparing seasonal to pandemic h1 strains (72-73% in the ha1 protein). hi and mn assays were conducted in duplicate according to established protocols [24] as detailed below and the individual result assigned as the geometric mean titer (gmt) of duplicate values. summary hi and mn serologic statistics were compared including the number meeting threshold titers of 10 and of 40. changes in titers from baseline (day 0) to days 28, 49 (ch0), and 54 (ch+5) or 63 (ch+14) were assessed through sero-conversion, group gmts with 95% confidence intervals (ci) and group gmt ratios (gmtr). haemagglutination inhibition methods. in preparation for the hi assay, sera were treated with receptor destroying enzyme (accurate chemical & scientific, ny) to remove nonspecific inhibitors of agglutination, and further hemadsorbed with 50% turkey erythrocytes (lampire biologic laboratories, pennsylvania). sera were serially diluted beginning at 1:10 with phosphate buffered saline and 25 ml of each serum dilution was reacted with 25 ml of antigen (infected mdck cell lysate supernatant) containing 4 ha units of virus for 30 minutes. to each mixture 50 ml of 0.5% turkey erythrocytes were added, and after mixing, the preparations were incubated for 30 minutes. results were recorded by photography. the hi titer was designated as the inverse of the highest dilution at which detectable hi activity was still present. influenza b virus was used in the hi assay in its ether-treated, inactivated form. briefly, the clarified influenza b virus cell lysates were each mixed by agitation with an equal volume of diethyl ether. the mixture was kept at 4uc for 15 minutes to allow for the phases to separate. the top ether layer was aspirated and nitrogen gas bubbled through the virus preparation to remove residual ether, and the preparation was then used in the hi assay. microneutralization methods. for mn assay, 50% tissue culture infectious dose (tcid50) viral titers were determined on mdck cells. the sera were treated with receptor destroying enzyme (accurate chemical & scientific, ny) and serially diluted in serum-free medium (megavir, hyclone, utah) beginning at 1:10. to each 50 ml dilution, 100 infectious units of virus were added. the plates were incubated for 2 hours at 37uc to allow for virus antibody interaction. the contents of each well were then transferred onto microtiter plates with confluent monolayers of mdck cells. after 3 hours of further incubation at 37uc, the medium in each well was removed and replaced with fresh megavir medium containing 2 mg/ml l-1-tosylamido-2-phenylethyl chloromethyl ketone (tpck)-treated trypsin. the plates were further incubated at 37uc and monitored for cytopathic effects on days 3 and 5. the mn titer was defined as the inverse of the serum dilution immediately preceding the wells with cytopathic effects. and ha1-based protein microarray methods. competitive elisa was conducted on sera using a commercially-available np-based influenza a antibody test kit from idexx laboratories, inc. (switzerland) [25] . competitive elisa antibody values were calculated as sample optical density (od) divided by negative control od with ratios ,0.60 considered positive and mean values with 95%ci displayed; smaller elisa ratios denote higher antibody levels. an ha1-based protein microarray serological assay was conducted for study and non-study antigens listed in table s3 , performed as previously described [26] with adaptation for detection of ferret antibodies. serum samples were tested in a single 1:10 dilution in blotto containing 0.1% surfact ampt (both thermo fisher scientific inc., rockford, usa). all incubation steps were one hour at 37uc. recombinant ha1 proteins were spotted in duplicate and incubated with 70 ml blotto followed by 70 ml of diluted ferret serum and then two-step conjugation by incubation with 70 ml mouse anti-mustelid igg (antibodiesonline, aachen, germany) followed by 70 ml dylight 649conjugated goat anti-mouse igg (jackson immunoresearch, baltimore pike, usa) both in blotto containing 0.1% surfact ampt. after washing and drying, the slides were scanned using a powerscanner (tecan, mä nnedorf, switzerland) and signals quantified using a scanarray scanner (perkin elmer, waltham, usa). individual spot signals were valued with correction by subtraction for per spot background fluorescence, truncated at zero where signal was below background. final individual per ferret values were assigned as the average of duplicates, log 10transformed after imputing a value of 0.1 for any zero values and compared for each antigen by study group and day. activity, rectal temperature, appetite and weight were recorded daily from days 42 through 46 and then from challenge (day 49; ch0) until sacrifice. baseline reference weight per ferret was computed as the average of day 42 to 46 and ch0 weights. activity was scored 1-5: one being the most alert and playful; two, alert but playful only if induced; three, alert but not playful (stays in hiding place); four, neither alert nor playful and five was the humane endpoint. appetite was scored as usual, diminished or no appetite. a(h1n1)pdm09 virus titers were assessed in nasal wash prechallenge day 46 and daily post-challenge (ch+1) until sacrifice and in whole right lung homogenates at each scheduled sacrifice. virus titers were determined by standard plaque assays using st6gali-expressing mdck cells [27, 28] , expressed as log 10transformed plaque-forming units (pfu) per ml. paraffin tissue sections were prepared from whole left lung of ferrets for histo-pathology assessment at each scheduled sacrifice. inflammation was graded on six indicators (bronchial/endobronchial, peribronchial, perivascular, interstitial, pleural and intraalveolar), each scored as: 0 (normal), 1 (mild), 2 (moderate) or 3 (marked) for a maximum combined score of 18 [28] . vascular congestion and pulmonary edema were similarly scored. lung immuno-histochemistry was undertaken to identify cells with virus antigen. paraffin tissue sections were quenched for 10 minutes in aqueous 3% h 2 o 2 then pre-treated with proteinase k for 15 minutes. a 1:10,000 dilution of mouse monoclonal antibody to influenza a nucleoprotein (f26np9; in-house) was applied for one hour. sections were visualized using horseradish peroxidaselabelled polymer, envisionh+system (anti-mouse) (dako, usa), reacted with the chromogen, diaminobenzidine and counterstained with gill's hematoxylin. change in lung cytokine mrna gene expression was assessed by relative quantitative pcr (qpcr) with rna extracted from 140 ml of right lung homogenate using the qiaamp viral rna mini-kit. reverse transcription was performed using the high capacity rna-to-cdna kit (applied biosystems) on 0.5 mg of the total rna following manufacturer's protocol. qpcr was performed using the taqmanh gene expression master mix (abi) with primers designed to target published cytokine sequences [29] [30] [31] from mustela putorius furo mrna sequences at a final concentration of 0.25 mm. assays were run on the stepone plus (abi) with the following conditions: 50uc-2 minutes; 95uc-10 minutes; followed by 40 cycles of 95uc-15 seconds; and 60uc-1 minute. fold-change was calculated using the delta-delta ct method [32] with uninfected ferrets as reference and gapdh as endogenous control. chi-square or fisher's exact test were used to compare categorical variables and t-test or wilcoxon methods for continuous variables. weight, nasal virus titers, and ha1 protein microarray values were analysed via contrasts in a mixed-effects linear model (group, visit and their interaction, with repeated measurements on each animal). for protein microarray values, analyses were repeated after excluding statistical outliers without substantively affecting main conclusions. inflammatory scores by individual ferret are presented and combined inflammatory scores and cytokine values at ch+5 versus ch+14 were compared via contrasts in a two-way anova model (group, scheduled sacrifice day and their interaction). observed p,0.05 are described as significant; no attempt is made to correct for multiple inference. individual hi, mn and elisa assay results are displayed concurrently for pre-shipment, day 0, 28, 49 and 54/63 time points, for the four vaccinated and four placebo animals sacrificed at ch+5 in table s4 , and for the 12 animals per group sacrificed at day 63 in table s5 for vaccinated animals and table s6 for placebo animals. summary elisa results are shown in table 1 and summary microarray results for study antigens in figure 2 with study and non-study antigen cross-reactive responses shown in figure s1 . summary hi and mn statistics are shown in table s7 and table s8 , respectively. all animals were confirmed by np-based elisa to be influenza a naïve at pre-shipment and day 0. by day 49 there was significant elisa antibody rise following immunization in the vaccine group, with further antibody rise evident by day 54 (ch+5) after a(h1n1)pdm09 challenge in vaccinated animals but not until day 63 (ch+14) in placebo ferrets ( table 1, table s4, table s5 , table s6 ). ultimately, total influenza a elisa antibody was significantly higher among vaccine compared to placebo animals at both scheduled sacrifices ( table 1) . protein microarray results were consistent with elisa but in addition showed vaccine-induced ha1 antibody to the seasonal h1 antigen, for which values were significantly higher in vaccinated animals relative to pre-immunization and compared to placebo from day 28, most pronounced from day 49 after the first tiv dose (i.e. three weeks after two-dose vaccine series completion) ( figure 2 ). there was significant rise in antibody relative to pre-immunization for the h3n2 vaccine component at day 49, but only relative to the placebo group at day 63 (i.e. five weeks after vaccine series completion). slight but significant decrease in a(h1n1)pdm09 antibody was evident at day 28 postimmunization compared to baseline but not thereafter or relative to placebo, calling into question its clinical relevance. significant rise in antibody to a(h1n1)pdm09 relative to pre-immunization was evident at day 63 in both the vaccine and placebo groups. of interest, infection with a(h1n1)pdm09 induced antibody by day 63 not only to itself but also to closely related 1918 h1 antigen in both study groups (significantly higher for the latter in the vaccinated) ( figure s1 ). furthermore, at day 63 among vaccinated but not among placebo animals, a(h1n1)pdm09 challenge induced significantly greater cross-reactivity to nonstudy (1977, 1999 ) h1 variants to which the animals had never been exposed, evident relative to pre-immunization, to the placebo group and to pre-challenge at day 49 ( figure s1 ). increase following a(h1n1)pdm09 challenge among the vaccinated was evident at day 63 for other non-h1, non-vaccine antigens (h2, h3) relative to pre-immunization but not relative to placebo. all animals were also shown by hi and mn assays to be influenza naïve at pre-shipment (table s4, table s5, table s6 ). more variability in hi than mn antibody titers was evident thereafter. overall, however, the significant rise in vaccineinduced antibody shown by elisa and ha1 protein microarray by day 49 was not evident by hi or mn assays except among a few of the vaccinated ferrets ( table s4, table s5 ). conversely, ferrets in both groups showed substantial neutralizing antibody to a(h1n1)pdm09 at day 63 (table s5, table s6 ) with very high gmts and gmtrs relative to baseline, slightly (but nonsignificantly) higher in the placebo compared to the vaccine group by both hi and mn (table s7, table s8 ). average baseline weight was comparable between the vaccine (0.96 kg) and placebo (0.97 kg) groups (p = 0.87). beginning at ch+2, more vaccinated animals showed diminished or no appetite compared to placebo recipients (7/16 versus 3/16; p = 0.25) with the greatest between-group difference at ch+5 (12/16 versus 6/ 16; p = 0.07), that resolved by ch+10 (1/12 vs. 0/12; p = 1.0). the greatest between-group difference for no appetite was at ch+6 (7/ 12 vs. 1/12; p = 0.03). weight loss from baseline was greater in the vaccine than placebo group, evident beginning at ch+2 and marginally significant across the full study period (% weight loss p = 0.048; absolute loss p = 0.06) ( figure 3a) . the greatest between-group difference in percentage weight loss from baseline was at ch+5 (7.4% vs. 5.2%; p = 0.01) ( figure 3a ) on which day 4/16 (25%) vaccinated versus 1/16 (6%) placebo animals had lost 10% or more of their body weight compared to baseline (p = 0.3) ( table s4, table s5, table s6 ). consistent with random selection, the same pattern was evident regardless of sacrifice day, with mean percentage weight loss from baseline at ch+5 of 6.9% for the vaccine group and 4.6% for the placebo group among animals selected for sacrifice at ch+5 and 7.6% and 5.3%, respectively, among animals sacrificed instead at ch+14. animals had comparable scores of one for alertness/playfulness at all time points except from ch+2-8 for which activity levels were marginally worse (scored as two) in all animals in both groups. temperature patterns did not differ between vaccine and placebo groups with a peak in mean/median temperatures at ch+2 of 40.0uc and 40.1uc, respectively. nasal a(h1n1)pdm09 titers did not differ significantly between groups over time (p = 0.37). nasal virus titers rose more steeply between ch+1-2 in the vaccine versus placebo group (mean difference in log-titres 1.14 versus 0.88 pfu/ml; p = 0.25) and then fell more steeply at ch+2-3 (mean difference in log-titres 0.80 versus 0.46 pfu/ml; p = 0.01) ( figure 3b ). lung a(h1n1)pdm09 titers at ch+5 were significantly higher in the vaccine versus placebo group (log-mean 4.96 versus 4.23 pfu/ ml; p = 0.01) ( figure 3c ). neither group had detectable virus in the lung at ch+14. at ch+5, animals sacrificed from the vaccine group had higher combined mean/median lung inflammatory scores than placebo animals (5.8/5.5 versus 2.1/2.0), a difference that did not reach statistical significance (p = 0.051) ( table s9) . two of four vaccinated animals showed a combined lung inflammatory score of $10 at ch+5 compared to none of the four placebo animals (maximum score = 4) ( table s9 ). salient histologic features are shown in figure 4 for animals of both groups with the highest and lowest combined ch+5 inflammatory scores, illustrating the severe bronchopneumonia that was evident in half of the vaccinated but none of the placebo animals. the increased micrometric scale in panel d reflects that the pathologic changes are marked and diffuse, best rendered at low magnification, whereas the mild to moderate and more focal changes in panels a to c necessitate photomicrographs at higher magnification. compared to ch+5, combined mean lung inflammatory score was significantly lower in the vaccine group among animals sacrificed at ch+14 (5.8 vs. 1.8; p = 0.01) whereas this showed little change over time in the placebo group (2.1 vs. 2.4; p = 0.84) with significant interaction between scheduled sacrifice day and group (p = 0.048). combined mean/median lung inflammatory scores were not significantly different between the vaccine versus placebo group at ch+14 (1.8/1.2 versus 2.4/1.5). in both the vaccine and placebo groups at ch+5, influenza antigen was detected by immuno-staining in bronchial and bronchiolar epithelium. within alveolar walls, most of the positive cells were identified as pneumocytes using double immunolabelling [not shown]. occasional cells had the morphology of macrophages. no viral antigen was detected in animals at ch+14. ifn gamma was below the limits of detection in both groups at both scheduled sacrifices. other lung cytokines were consistently but non-significantly higher in the vaccine versus placebo group at ch+5. all cytokine values were lower in both groups at ch+14, consistently but non-significantly lower in the vaccine animals. the ch+5 versus ch+14 difference was statistically significant for all vaccine group cytokines (all p#0.02) except ifn alpha (p.0.05) whereas differences were not significant in the placebo group, except for il17 (p = 0.046). overall, the interaction between scheduled sacrifice day and study group was not significant for any cytokine (figures 5a and 5b ). during spring-summer 2009, several observational studies from canada reported that prior recipients of 2008-09 tiv experienced approximately two-fold increased risk of medically-attended, laboratory-confirmed a(h1n1)pdm09 illness [1] . recognizing that all observational designs are susceptible to methodological bias, gold standard rct analysis would typically be considered essential in clarifying such unexpected findings. in hong kong, an rct of the 2008-09 tiv (vaxigrip) already underway had shown similar association among vaccinated children with significant relative risk of 2.58 (increased to 2.74 with adjustment for seasonal infection) [12, 13] ; however, during follow-up rct using 2009-10 vaxigrip and spanning august 2009 to december 2010, the same investigators instead reported significant protective effects [33] . both rcts lacked sufficient power for analysis based on virologically-confirmed infection so that conclusions were drawn instead from less reliable serologically-defined outcomes [12, 33] . further rct test of the association in humans has now become practically impossible given that, since fall 2010, all seasonal tiv routinely contains protective, homologous a(h1n1)pdm09 antigen [20] . we therefore undertook rct evaluation of the possible direct effects of prior heterologous tiv receipt on a(h1n1)pdm09 disease risk in ferrets as the ideal alternate model of human influenza infection [34] . although none of the animals became moribund or so severely ill as to require euthanasia, ferrets immunized with two doses of 2008-09 tiv did show significantly worse clinical, virologic and pathological features following pandemic h1n1 infection compared to placebo recipients. as originally powered to show, vaccinated animals experienced significantly greater weight loss relative to baseline following infection, maximally different from placebo at ch+5. nasal wash titers did not differ, but vaccinated animals showed significantly higher lung virus titers. consistent with lung virus findings, lung inflammation was also increased more than 2.5-fold in vaccinated compared to placebo animals at ch+5 although with fewer animals sacrificed on that day the difference fell just short of statistical significance. inflammatory indicators were, however, significantly higher at ch+5 compared to ch+14 in the vaccinated animals and showed no change across that period in placebo recipients. lung cytokines showed a similar pattern. although illness activity levels appeared similar between groups based on categories of induced playfulness, these may have been of insufficient resolution to reflect clinical differences in lung disease, manifest otherwise through significant loss of appetite and weight. by day 14 post-challenge animals in both groups had recovered. in human studies, a doubling of the risk of medically-attended pandemic h1n1 illness was observed among 2008-09 tiv recipients, but an increase in the risk of hospitalization was not shown [1] . this was broadly interpreted to suggest increased risk of acquiring infection per se whereas here we report increased disease severity among influenza-naïve, systematically-infected ferrets. as such, our findings in ferrets may not replicate the experience in humans. it is worth noting, however, that the source population in the human observational studies was patients seeking medical care [1] . although illness severity among outpatient visits was not specifically compared between vaccinated and unvaccinated participants, subjects had experienced influenza-like illness severe enough to prompt medical consultation within one week of illness onset, and that outcome was significantly increased among the vaccinated. in that regard, the pattern of acute worsening of a(h1n1)pdm09 illness during the first week of infection in vaccinated ferrets, followed by subsequent recovery by day 14, is consistent with increased outpatient but not hospitalization risk observed in vaccinated humans. as such, this ferret rct suggests that earlier findings from observational studies in humans cannot be dismissed on the basis of methodological bias alone and that direct mechanistic explanations should be sought. whether these findings may be product-specific or may also apply to other tiv products has yet to be separately assessed in follow-up studies. to date, hypotheses about biological mechanisms to explain increased a(h1n1)pdm09 risk among prior tiv recipients have included both direct and indirect vaccine effects [1] . indirect mechanisms include the infection block hypothesis whereby effective seasonal vaccine may prevent the more robust and complex cross-protective immunity against heterologous viruses afforded by seasonal infection, such as through cell-mediated responses to conserved internal virus components [1, 35, 36] . other epidemiological investigators have favoured this hypothesis, or related variations (such as temporary immunity hypothesis) [33, 37] , but in appendix g of our original publication [1] we demonstrated these indirect hypotheses to be insufficient and implausible to fully explain a doubling of risk, requiring as they do unreasonably high estimates of infection attack rates, infectioninduced cross-protection, and tiv effectiveness [1, 13] . to test whether increased risk in vaccinees may have occurred without invoking infection block mechanisms we specifically designed the current ferret experiment without the intermediary of heterologous seasonal influenza infection. as such, we cannot rule out an additive role for indirect vaccine effects mediated through infection block mechanisms, but show that direct vaccine effects are likely to have at least contributed to our previous findings. possible direct vaccine effects include antibody-dependent enhancement (ade) whereby virus uptake by cells is enhanced in the presence of low-level, cross-reactive, non-neutralizing antibodies, best described for dengue [1, 38, 39] . a possible role for cross-reactive antibodies in explaining severe a(h1n1)pdm09 manifestations in otherwise healthy adults, and in archived lung sections from fatal adult cases during the 1957 h2 pandemic has previously been suggested [40] . another recent study has reported an association between higher ratios of cross-reactive elisa versus neutralizing antibody titers early during a(h1n1)pdm09 infection and more severe illness [41] . enhanced respiratory disease in vaccinated swine has also been reported following challenge with a(h1n1)pdm09 or other heterologous, homosubtypic h1 viruses that do not share cross-reactive neutralizing antibodies [42] [43] [44] [45] . as in our ferret experiment, clinical worsening in vaccinated swine was evident at two to five days post-challenge [43, 44] and was correlated with elevated pro-inflammatory cytokine responses in the lung [44] . unlike the current ferret or prior human studies, however, these swine studies used adjuvanted whole virion vaccine that was additionally heterosubtypic for the neuraminidase surface protein (i.e. n2 versus n1), the relevance of which is uncertain. ade is classically associated with enhanced virus uptake in macrophages or other fc-receptor-bearing cells, demonstrated in vitro for influenza [46] [47] [48] [49] and more recently also specifically for a(h1n1)pdm09 in the presence of heterologous human anti-sera [50] . in our vaccinated ferrets, higher lung virus titers were observed, but immuno-histochemistry could not distinguish affected cells of the lung in vaccine versus placebo animals, and macrophages were not predominant in either group. more recently in swine, however, heterologous antibody has been shown to enhance a(h1n1)pdm09 infection of other mammalian (mdck) cells, described in the context of fusion-enhancing crossreactive anti-ha2 stalk antibodies and absent neutralizing antibodies targeting the ha1 globular head [51, 52] . our experiment was unable to further elucidate these putative immunologic mechanisms. we identified significant vaccineinduced influenza a antibody rise by elisa and confirmed this to include anti-ha1 antibody response to the seasonal h1n1 tiv component. there was also rapid and significant increase in influenza a elisa antibody following a(h1n1)pdm09 challenge among vaccinated but not placebo ferrets sacrificed at ch+5 but ha1-based and neutralizing antibodies to a(h1n1)pdm09 were not evident until ch+14 in either group. lower neutralizing antibody to a(h1n1)pdm09 even at ch+14 among vaccinated versus placebo ferrets, although not statistically significant, is consistent with human immunogenicity trials showing blunting of pandemic h1n1 vaccine-induced responses in association with prior seasonal vaccine receipt [53] [54] [55] [56] . conversely, pandemic h1n1 immunization has been observed to boost pre-existing heterosubtypic antibody which may also be consistent with our ch+14 findings for the h3n2 tiv component and the broad boosting of cross-reactive antibodies to other antigenically-distant h1 variants observed by protein microarray in vaccinated but not placebo animals [57] . although protein microarray did not show cross-reactive a(h1n1)pdm09 antibody prior to ch+14, it was not designed to detect the sort of anti-ha2 stalk antibodies highlighted above in association with severe disease in vaccinated swine [51] . ultimately, therefore, we are unable to discern whether the rise in ch+5 elisa antibody in vaccinated animals suggests early crossreactive, non-neutralizing antibody to a(h1n1)pdm09 or further antibody increase to tiv antigens 4 weeks after their second dose (or both) although microarray indicates the latter certainly contributed. t-cell hypo-responsiveness may be an alternate explanation compatible with a hypothesis of direct vaccine effect. this phenomenon has been reported in same-season influenza vaccine booster-dose studies [58] with parallels also in the allergy literature suggesting peptide-induced t-cell hypo-responsiveness beginning at 2-8 weeks and lasting up to 40 weeks [59] . however, while ferret #81 (placebo; inflammatory score 0.5) indicating very mild/minimal peri-bronchial inflammation; (b) ferret #63 (vaccinated; inflammatory score 0.5) indicating very mild/minimal peri-vascular inflammation; (c) ferret #58 (placebo; inflammatory score 4.0) indicating moderate bronchial and mild peri-bronchial/peri-vascular inflammation; (d) ferret #69 (vaccinated; inflammatory score 11.5) indicating severe bronchopneumonia. the increased micrometric scale in panel d reflects that the pathologic changes are marked and diffuse, best rendered at low magnification, whereas the mild to moderate and more focal changes in panels a to c necessitate photomicrographs at higher magnification. corresponding histopathology scores are shown in table s9 . doi:10.1371/journal.pone.0086555.g004 interferon-gamma was below detectable limits in both groups, lung cytokines in vaccinated ferrets were otherwise consistently (but non-significantly) higher compared to placebo animals at ch+5, notably including the th2 il4 [60] , pro-inflammatory il17 [61, 62] and regulatory il10 [63] cytokines. il17 has been implicated as super-inducer of neutrophil infiltration and acute lung immuno-pathology following influenza infection [62] , with counteractive dampening interactions by il10 [63] . in an earlier canadian ferret experiment in which animals administered a single dose of 2008-09 tiv also experienced worse a(h1n1)pdm09 illness, il6 in nasal wash was substantially raised in the fluviral group and il10 significantly in the flumist group, with disease enhancement suggested in both vaccine groups compared to controls [16] . we did not assess nasal wash cytokines or flumist and were not statistically powered to explore cytokine differences, but lung il6 and il10 were also both non-significantly raised at ch+5 in our fluviral versus placebo ferrets. all cytokine values were then lower at ch+14 in the absence of lung pathology. however, none of the between-group cytokine differences at either time point were statistically significant. there are limitations to this study. although ferrets are considered the ideal animal model for human influenza infection, there are anticipated differences in immunologic and clinical aspects of immunization, infection and illness responses (timing, dosing and intensity) across species. overall patterns may be compared but ferret studies do not support precise quantification of actual risk in humans. the greater likelihood of more severe disease based on several clinical indicators (weight loss, lung virus titers) among vaccinated compared to unvaccinated ferrets may not replicate the greater likelihood of medically-attended a(h1n1)pdm09 illness we previously reported in vaccinated humans. in using influenza-naïve, systematically infected ferrets there are clear differences from the human experience with respect to pre-conditions (influenza exposure history and immunologic context), process (infection acquisition), and other relevant parameters (vaccine immunogenicity, clinical outcomes and monitoring). clinical relevance of the differences we report between vaccinated and placebo ferrets is ultimately best interpreted in the context of our study objectives assigned in follow up to the prior human observations we reported. the main objective of the ferret study was to assess through randomized, controlled design whether prior receipt of 2008-09 tiv may have had direct, adverse effects on a(h1n1)pdm09 illness, specifically powered related to weight loss. although we cannot more precisely elucidate the underlying mechanisms involved, the current ferret study supports the hypothesis of direct vaccine effect. taken together with prior human and swine studies, these findings represent a signal that warrant further investigation and better understanding though they cannot be considered conclusive. the most prominent concern in this ferret study may relate to our failure to show neutralizing antibody response to vaccine, an issue we therefore consider in detail. we observed some greater albeit low-level variability in antibody titers by hi, a nonfunctional assay, than by microneutralization or np-based elisa. intra-and inter-laboratory variability in antibody assay results is well-recognized [64, 65] and we thus interpret findings in the context of combined hi, mn, and elisa results, overall indicating our animals were naïve at pre-shipment and baseline. vaccine-induced hi and mn responses were not evident thereafter but significant vaccine-induced antibody rise was shown by both np-based elisa and ha1-based protein microarray assays, the latter also shown only at high serum/low antibody concentrations (i.e. testing dilution of 1:10). although srid testing of the expired 2008-09 vaccine lot that we used still met standard ha potency requirements for annual commercial vaccine approval by the fda [21] , we cannot rule out other unrecognized vaccine changes with time that may have been influential. mdck-passaged viruses used in our hi and mn assays were antigenically equivalent to reference strains and amino acid sequencing showed they were also identical in their ha and na to the 2008-09 h1n1 vaccine component. this argues against such linear differences to explain the suboptimal neutralizing responses we measured; however, we cannot rule out other conformational changes to protein structure in the original vaccine comprised of (sodium-deoxycholate) disrupted and inactivated virus. the h3n2 virus used in our assays was also antigenically equivalent to the who-recommended reference virus with which it shared $98% ha antigenic site amino acid identity. however, we did not conduct antigenic testing in relation to the actual vaccine component used by manufacturers and cannot rule out differences between assay and vaccine viruses in the suboptimal vaccine responses we measured (table s1 and s2). protein micro-array also showed variable low-level response to the h3n2 component among immunized ferrets at day 49, significantly greater than placebo and compared to pre-immunization only at day 63 (i.e. five weeks after the second vaccine dose). whether the latter was due to further antibody rise with time following immunization or cross-reactive response following a(h1n1)pdm09 challenge is unknown; however, similar effect was not observed relative to placebo for other non-vaccine h3 subtype viruses included in the microarray, suggesting h3n2 vaccine response. thresholds for defining antibody response are also anticipated to vary across species and assays; these have not been crosscorrelated/2validated in ferrets for the various antigens and assays we used. in fact, sero-protective thresholds have not yet been established for ferrets by any assay. however, failure to induce a robust hi or mn antibody response to inactivated influenza vaccine in naïve ferrets has long been recognized, after single or several doses, in the absence of prior infection or adjuvant for seasonal, novel or pandemic vaccines [66] [67] [68] [69] [70] [71] [72] [73] . in order to replicate more closely the observations in humans, we used a commercially available, non-adjuvanted 2008-09 fluviral lot that had been administered in canada. it is of note that in human studies conducted with the same product in 2008-09 (pediatric) [74] , 2009-10 (elderly) [75] and in a mouse study conducted in 2010-11 [21] , fluviral also induced suboptimal hi and/or mn responses to the same seasonal a/brisbane/59/2007-like h1n1 vaccine antigen. for example, in the pediatric trial including infants and toddlers 6-23 months of age similarly naïve to influenza as were our ferrets, the same schedule of two 0.5 ml doses of a thimerosal-free version of the 2008-09 fluviral induced significantly lower h1n1 antibody responses than even half that volume (0.25 ml) per dose of vaxigrip [74] . [74] . a similar pattern, though less pronounced, was also observed with the h3n2 component [74] . the reasons for diminished immunogenicity of the canadian vaccine are unknown although authors of the pediatric trial proposed more complete clearance of intact virus among other possible explanations. in human observational studies, the 2008-09 tiv was still shown to be protective overall against homologous seasonal influenza [1] . spring-summer 2009 observations of increased risk of heterologous a(h1n1)09 illness were identified six or more months after tiv receipt. in that regard, lower vaccine-induced antibody titers in ferrets at our three-week post-immunization a(h1n1)pdm09 challenge time point may better replicate end-ofseason antibody conditions when vaccinated humans were exposed to a(h1n1)pdm09 virus. a prior ferret study to assess the same 2008-09 fluviral also suggested disease enhancement but was able to induce homologous hi antibody response to the h1n1 component with mean antibody titre exceeding 100 within two weeks of a single 0.5 ml vaccine dose [16] , higher even than induced in the pediatric study population cited above. such variability in serologic responses may reflect lot-to-lot or laboratory differences. not knowing the precise mechanisms involved in vaccine-associated enhanced respiratory disease, and unable to exactly know or replicate the human immunologic context in spring-summer 2009, we did not adjust the human vaccine formulation, dose or schedule to force higher ferret vaccine responses. instead we focused on clinical parameters, recording the observed effects according to standard immunization practice. in general, ferret studies to date have suffered from small sample size and insufficient power [14] [15] [16] [17] [18] [19] 76] . our study was powered for clinical (percentage weight loss) comparison and follow-up to 14 days post-challenge. failure to reach statistical significance for other consistent indicators should not prompt their dismissal but should stimulate further investigation. it may be argued that lung findings at ch+5 were chance occurrences among few ferrets poorly-representative of the full group experience. however, animals in both groups were randomly and blindly selected for ch+5 sacrifice, the comparison of baseline and ch+5 characteristics showed no significant within-group differences according to scheduled endpoint, and ch+5 lung findings in vaccinated animals (higher virus titers and lung inflammation) were consistent with overall clinical patterns (greater loss of appetite and weight). nevertheless, future experiments should be powered with more animals to specifically examine these early acute clinical, immunologic, and pathologic findings and explore their possible mechanisms in greater detail. we assessed only influenza-naïve animals whereas most humans, other than young children, will have prior potentially cross-attenuating influenza infection history. the use of influenzanaïve animals in previous swine [42] [43] [44] [45] 51, 52] and the current ferret studies may be relevant to the increased severity highlighted in vaccinated animals but to a lesser extent noted with the association in people. further experiments are needed to explore nuances related to infection and/or immunization history which additionally and variously complicate the human experience. in a recent publication, disease enhancement was included among possible hypotheses to explain greater 2009 pandemic h1 morbidity in the americas compared to australia, new zealand or europe, with reference to findings in vaccinated swine interpreted ecologically in the context of regional differences in prior heterologous seasonal h1n1 virus circulation; given findings in vaccinated ferrets and swine, however, regional differences in prior heterologous seasonal h1n1 vaccine (i.e. tiv) coverage may also be relevant to consider [77] . of note, mechanisms such as ade, if explanatory, require a precise balance of low-level, crossreactive, non-neutralizing antibody to be manifest [1, 38, 39] , a particular but sliding immunologic scale that may not have been captured in all animals or humans at the time of a(h1n1)pdm09 exposure. a spectrum of illness is anticipated with any infection process and a greater likelihood of severity does not require that all exposed individuals experience that outcome. however, this additional immunologic complexity related to ade, if involved, may have contributed to the variability in clinical outcomes we observed among vaccinated ferrets and to the variability in reporting the association in humans. our experiment assessed the unique context of heterologous but homosubtypic pandemic h1n1 challenge. it has been suggested that original antigenic sin as an aspect of the cross-reactive, non-neutralizing antibody required for ade applies when antigenic differences of less than 33-42% exist across related but distinct prime-boost strains [38] ; amino acid differences in the ha1 between the 2008-09 seasonal and 2009 pandemic h1 antigens were within this range (72-73% similarity; table s2 ) with much closer homology (92%) across the ha2 (18 amino acid differences across 222 residues). however, without better understanding of the underlying mechanisms or specific virologic interactions we cannot speculate whether the same association could apply to other emerging heterologous or hetero-subtypic variants; the antigenic distance and other criteria required to define or forecast that likelihood remain unknown. in summary, although these ferret findings cannot be considered conclusive in explaining earlier human observations from canada, they support the hypothesis that prior receipt of 2008-09 tiv may have had direct, adverse effects on a(h1n1)pdm09 illness. both human and ferret findings from canada are consistent with observations elsewhere of enhanced disease following heterologous influenza challenge in vaccinated swine. given the potential implications for informing influenza immuno-epidemiology and public health response to other emerging viruses, these signals warrant further in-depth evaluation and a search for possible mechanistic explanations. figure s1 ha1 microarray values for study and nonstudy antigens by group and study day. table s1 amino acid sequence substitutions in hemagglutinin (ha) and neuraminidase (na) of mdck-passaged assay and challenge viruses. (pdf) table s2 pairwise identity (% (number of amino acid mutations)) in influenza a hemagglutinin 1 (ha1) peptide and antigenic sites across viruses used in antibody assays and a(h1n1)pdm09 challenge. (pdf) association between the 2008-09 seasonal influenza vaccine and pandemic h1n1 illness during spring-summer 2009: four observational studies from canada seasonal influenza vaccine and increased risk of pandemic a/h1n1-related illness: first detection of the association in british columbia clinical and epidemiologic characteristics of an outbreak of novel h1n1 (swine origin) influenza a virus among united states military beneficiaries association between seasonal influenza vaccination in 2008-2009 and pandemic influenza a (h1n1) 2009 infection among school students from kobe effectiveness of pandemic h1n1 vaccine against influenza-related hospitalization in children effectiveness of 2008-09 trivalent influenza vaccine against 2009 pandemic influenza a (h1n1) -united states notes from the field: outbreak of 2009 pandemic influenza a (h1n1) virus at a large public university in delaware pandemic influenza h1n1 2009 infection in victoria, australia: no evidence for harm or benefit following receipt of seasonal influenza vaccine in 2009 no association between 2008-09 influenza vaccine and influenza a(h1n1)pdm09 virus infection partial protection of seasonal trivalent inactivated vaccine against novel pandemic influenza a/h1n1 2009: case control study in mexico city infection and deaths from influenza a h1n1 virus in mexico: a retrospective analysis protective efficacy of seasonal influenza vaccination against seasonal and pandemic influenza virus infection during 2009 in hong kong mechanism for seasonal vaccine effect on pandemic h1n1 risk remains uncertain comparison of a live attenuated 2009 h1n1 vaccine with seasonal influenza vaccines against 2009 pandemic h1n1 virus infection in mice and ferrets seasonal influenza vaccine provides priming for a/h1n1 immunization assessment of the efficacy of commercially available and candidate vaccines against a pandemic h1n1 2009 virus impact of prior seasonal influenza vaccination and infection on pandemic a(h1n1) 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of humoral immune responses to influenza viruses by using protein microarray enhanced expression of an alpha2,6-linked sialic acid on mdck cells improves isolation of human influenza viruses and evaluation of their sensitivity to a neuraminidase inhibitor enhanced lung disease and th2 response following human metapneumovirus infection in mice immunized with the inactivated virus early cytokine mrna expression profiles predict morbillivirus disease outcome in ferrets molecular cloning and phylogenetic analysis of inflammatory cytokines of the ferret (mustela putorius furo) early gene expression events in ferrets in response to sars coronavirus infection versus direct interferon-alpha2b stimulation a new mathematical model for relative quantification in realtime rt-pcr protective efficacy against pandemic influenza of seasonal influenza vaccination in children in hong kong: a randomized controlled trial the ferret as a model organism to study influenza a virus infection vaccination 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precede the induction of antigen-specific hyporesponsiveness in atopic allergic asthmatic subjects type 1/type 2 immunity in infectious diseases il-17 boosts proinflammatory outcomes of antiviral response in human cells critical role of il-17ra in immunopathology of influenza infection il-10 deficiency unleashes an influenza-specific th17 response and enhances survival against high-dose challenge haemagglutination-inhibiting antibody to influenza virus serologic assays for influenza surveillance, diagnosis and vaccine evaluation immunity to influenza in ferrets. ii. influence of adjuvants on immunization immunity to influenza in ferrets. v. immunization with inactivated virus in adjuvant 65 immunity to influenza in ferrets. i. response to live and killed virus immunity to influenza in ferrets. vii. effect of previous infection with heterotypic and heterologous influenza viruses on the response of ferrets to inactivated influenza virus vaccines immunization of ferrets against influenza: a comparison of killed ferret grown and egg grown virus immunity to influenza in ferrets x. intranasal immunization of ferrets with inactivated influenza a virus vaccines multiple infections with seasonal influenza a virus induce cross-protective immunity against a(h1n1) pandemic influenza virus in a ferret model control of pandemic (h1n1) 2009 influenza virus infection of ferret lungs by nonadjuvant-containing pandemic and seasonal vaccines immunogenicity and safety of 2 dose levels of a thimerosal-free trivalent seasonal influenza vaccine in children aged 6-35 months: a randomized, controlled trial seasonal trivalent inactivated influenza vaccine does not protect against newly emerging variants of influenza a (h3n2v) virus in ferrets global mortality estimates for the 2009 influenza pandemic from the glamor project: a modeling study authors wish to acknowledge ms. lisan kwindt for assistance with literature retrieval, manuscript preparation and formatting, and ms. catharine chambers for assistance with literature review. we also wish to thank dr. yan li of the national microbiology laboratory for providing the reference viruses and anti-sera used in the serologic assays, as well as the authors and originating and submitting laboratories of the reference who and vaccine virus sequences obtained from gisaid's epiflu database. contributed reagents/materials/analysis tools: ss xb cc al ceh mk edb rb. wrote the paper: dms meh gds nzj gl ss xb cc al dk ceh sl mp gb mk edb rb. key: cord-002141-9mxi4dzi authors: memczak, henry; lauster, daniel; kar, parimal; di lella, santiago; volkmer, rudolf; knecht, volker; herrmann, andreas; ehrentreich-förster, eva; bier, frank f.; stöcklein, walter f. m. title: anti-hemagglutinin antibody derived lead peptides for inhibitors of influenza virus binding date: 2016-07-14 journal: plos one doi: 10.1371/journal.pone.0159074 sha: doc_id: 2141 cord_uid: 9mxi4dzi antibodies against spike proteins of influenza are used as a tool for characterization of viruses and therapeutic approaches. however, development, production and quality control of antibodies is expensive and time consuming. to circumvent these difficulties, three peptides were derived from complementarity determining regions of an antibody heavy chain against influenza a spike glycoprotein. their binding properties were studied experimentally, and by molecular dynamics simulations. two peptide candidates showed binding to influenza a/aichi/2/68 h3n2. one of them, termed peb, with the highest affinity prevented binding to and infection of target cells in the micromolar region without any cytotoxic effect. peb matches best the conserved receptor binding site of hemagglutinin. peb bound also to other medical relevant influenza strains, such as human-pathogenic a/california/7/2009 h1n1, and avian-pathogenic a/mute swan/rostock/r901/2006 h7n1. strategies to improve the affinity and to adapt specificity are discussed and exemplified by a double amino acid substituted peptide, obtained by substitutional analysis. the peptides and their derivatives are of great potential for drug development as well as biosensing. influenza a virus is an enveloped virus belonging to the orthomyxoviridae family. it can cause annual epidemics and infrequent pandemics [1] . the spanish flu pandemic of 1918 as well as the asian flu of 1957 and the hongkong flu in 1968 pandemics caused the death of millions of people [2] . in 2009 the pandemic swine origin influenza a h1n1 virus as well as the outbreak of h7n9 in china in 2013 has reminded the world of the threat of pandemic influenza [3] [4] [5] [6] . the genome of influenza virus consists of eight segmented negative rna strands. the envelope bilayer harbors the two spike glycoproteins hemagglutinin (ha) and neuraminidase (na), and the m2 proton channel. the homotrimeric ha is the most abundant protein on the viral surface. it mediates attachment to the host cell surface via binding to sialic acid (sa) residues of cellular receptors, and upon endocytic virus uptake it triggers fusion of the envelope with the endosomal membrane releasing the viral genome into the cytoplasm. na cleaves glycosidic bonds with terminal sa facilitating the release of budding virions from the cell. in diagnostics, antibodies against spike proteins are the preferred tool for identification and serotyping of viruses. development of therapeutic antibodies against influenza is a challenge, as the high viral mutation rate (antigenic drift) and genetic reassortment of the virus genome (antigenic shift) continuously lead to new strains escaping from neutralization by antibodies [7, 8] . this goes along with adaptation to small molecule inhibitors (e.g. oseltamivir) [9] . vaccines can only temporarily control the recurring epidemics of influenza, because antigenic changes are typical for ha and na. 16 avian and 2 bat serotypes of influenza a virus ha (h1-h18) are known, but only three (h1, h2, and h3) have been adapted to humans. antibodies binding to regions of hemagglutinin conserved among serotypes have been developed which demonstrated broad specificity and neutralization potency [10] [11] [12] [13] [14] [15] . however, development, production and quality control of antibodies is expensive and time consuming. as an alternative, short peptides binding specifically to the spike proteins can be produced in automated high-throughput synthesis at low costs. ha-binding peptides have been recently obtained by phage display, lead structure optimization of natural products and specific toxins, bioinformatics tools and discovery from side effects of known anti-inflammatory peptides [16] [17] [18] [19] [20] [21] [22] [23] . some of them showed antiviral activity [17, [19] [20] [21] [22] [23] . a more epitope-oriented accession to binding peptides is the search for paratope-derived peptides from variable regions of specific antibodies [24] . antibodies against ha have been described, and at least 6 antigenic sites (a-f) on the ha-trimer have been identified, localized either at the receptor binding site, the interface of the three ha-monomers, or at other sites like the stalk [8, 11, 25] . several structures of ha-antibody complexes have been published deposited in the protein data bank (pdb) [11] [12] [13] [14] . indeed, an antibody was described, whose ha binding is mediated mainly by one cdr, namely hcdr3 [12] . inspired by this finding, we chose linear peptides corresponding to the cdrs of vh of monoclonal antibody hc19, having the majority of contacts with the ha1 domain of the strain a/aichi/2/1968 [26, 27] . the antibody and the derived peptides bind to ha at the sa binding site, in particular to the 130-loop and the 190-helix, which belong to the antigenic sites a and b, respectively. this binding site is conserved among several ha serotypes providing a basis for a peptide with broader specificity [28] . we used complementary experimental and theoretical approaches to select ha binding vh-cdr peptides and to improve their potential to inhibit binding, and finally, infection of cells by influenza a virus. the inhibitory potential of the most efficient cdr-peptide was improved by microarray-based site-directed substitutions of amino acids. we could demonstrate a broader specificity of the selected peptides as they bound to ha of human and avian pathogenic influenza strains. influenza strain a/aichi/2/68 h3n2 x31 (aichi h3n2), reassorted with a/puertorico/8/1934 h1n1, and low pathogenic a/mute swan/rostock/r901/2006 h7n1 k3141 (rostock h7n1) were harvested from allantoic fluid of hen eggs. virus isolates were clarified upon low speed centrifugation (300 x g, 10 min) and concentrated by ultracentrifugation (100 000 x g, 1 h). for safety reasons, viruses prepared for spr experiments were inactivated by 5 min irradiation with uv-light on ice. infectivity of inactivated virus was precluded using mdck ii based cellassays, remaining binding ability of ha was proven using standard hemagglutination assay (ha) with human red blood cells [29] . for for infection and cytotoxicity assays madin-darby canine kidney epithelial cells (mdck ii, nbl-2, ccl-34) were used (atcc). cells were cultivated under standard cell culture conditions with dmem (supplemented with 10% fetal calf serum (fcs), 2 mm l-glutamine) in humid atmosphere at 37°c and 5% co 2 . hemagglutination inhibition assays (hai) were performed using either human erythrocytes (α-2,6'-sialosugars) for aichi h3n2, or turkey erythrocytes (α-2,3'-sialosugars) for rostock h7n1 [29] . peptides were synthesized with a linker for immobilization or without linker for in vitro inhibition assays. for spr based binding experiments, antibody derived peptides were extended with an n-terminal lysine linker: kkkk-sgfllisn-amide (pea-lys), kkkk-fydydvfy-amide (peb-lys), kk-ßaßa-lgviwaggntny-amide (pec-lys). in the spr based screen for virus binding, single and double mutated variants of peb-lys were used. all lysine variants were purchased from biosyntan or genecust with >95% purity. these peptides were dissolved in water and diluted in appropriate buffers for immobilization on spr sensor chips. spr based binding inhibition assays were performed with the full length peptides sgfllisngvhwv-amide (pea), ardfydydvfyyamd-amide (peb), and its double mutant ardfygydvffyamd-amide (peb gf ). for the biological assays peb, peb gf and the control peptide ardfydpdvfyyamdamide (peb p ) were applied. these peptides, and the peptides eb, s2(1-5), phage 1 (p1) h5n1 and phage 1 (l-p1) h9n2, mucroporin m-1were synthesized by rudolf volkmer (charité, berlin). hplc purification and analysis were achieved using a linear solvent gradient (a: 0.05% tfa in water; b: 0.05% tfa in acetonitrile; gradient: 5-60% b over 30 min; uv detector at 214 nm; rp-18 column). the identities of the peptides were validated by mass spectrometry using mal di-tof (microflex lt, bruker daltonik), and esi (q-tof micro, micromass). lyophilized peptides could be stored for at least one year at -20°c as controlled by hplc/ms analysis. stock solutions of peb, peb gf and peb p were prepared by initial dissolving in dmso, followed by dilution in pbs (8 mm peptide with 10% dmso v/v). solvation was improved by sonication. stock solutions could be stored for several weeks at 4°c. circular dichroism (cd) spectroscopy for determination of secondary structure and melting temperature was performed (s1 fig) using a jasco j-715 cd spectrometer. cd spectra of 100 μg ml -1 peptide solutions were recorded at 1 nm wavelength steps. the primary signal in millidegrees (mdeg) was recorded over 4 s, and then the molar ellipticity (θmrw, [deg cm 2 dmol -1 10 −3 ]) was calculated. measurements were performed on a biacore™ t200 (ge-healthcare bio-sciences ab) using cm5 chips, and running buffer hbsp (10 mm hepes, 150 mm nacl, 0.05% tween 20, ph 7.4) at 25°c. peptides and proteins were immobilized via amine coupling with edc/nhs at a flow rate of 10 μl min -1 , according to the manufacturer's instructions (ge healthcare). optimum preconcentration was evaluated before, using acetate and phosphate buffers (10 mm) of ph 4 to 7, and ligand concentrations of 10 μg ml -1 . for the immobilization of biotinylated sialyllactose (lectinity), neutravidin (thermo fisher scientific) (100 μg ml -1 ) was immobilized by amine coupling at ph 5.5 enabling high ligand density. biotinylated sialyllactose was then injected at a concentration of 10 μg ml -1 in hbsp for 3 min. several concentrations of analyte (ha, virus) were applied at least onto two channels with generally 500 s association and a 600 s dissociation time, followed by a 60 s regeneration step with 50 mm naoh. a flow rate of 10 μl min -1 was used. quantitative data were obtained by extracting the amount of bound material 10 s after injection was terminated (mean value over 5 s). reverse peptide binding experiments were run, with immobilized ha or aichi h3n2, under the same conditions. double referencing of binding curves was performed, using a control flow cell without ligand, and using control injections of buffer, in order to eliminate bulk refractive index effects and unspecific binding to the sensor surface. the dissociation constant (k d ) was calculated from a plot of steady state binding levels against analyte concentration, using a biacore t200 evaluation software. a constant concentration of 50 or 100 μg ml -1 aichi h3n2 virus was used as analyte, while the concentration of the inhibitors was varied. the suspensions of virus and inhibitor were mixed and subsequently incubated on a thriller1 thermoshaker at 500 rpm and 37°c for at least 15 min to reach equilibrium before injection on the spr surface. the inhibitors used were peptides or α-2,3'and α-2,6'-sialyllactose (carbosynth). the suspensions of virus and inhibitor were mixed and subsequently incubated for at least 15 min to reach equilibrium before injection on the spr surface. all data were referenced against buffer or inhibitor injection of identical concentration. the decrease of binding capacity of the chip over time was considered by referencing against multiple virus injections at various time points during the experiment. inhibition was calculated against the mean value of at least five binding events with the virus alone, using the sigmoidal four parameter logistic fit (origin). microarray-based substitutional analysis of peptide peb was performed using a pepstar 1 peptide library spotted on glass slides by jpt peptide technologies. the slides were used without additional treatment. for the labeling of proteins with a fluorescent dye, dyomics dy-634 (λ ex = 635 nm, λ em = 654 nm, fluorospin 634 kit (emp biotech) was used, according to the manufacturer´s instructions. the following materials were labeled: newyork h3n2, victoria h3n2, aichi h3n2, and california h1n1. labeled analytes were incubated several hours or overnight at indicated concentrations using femtotip buffer (ftp) (20 mm tris, 30% glycerol, 3% polyvinylpyrrolidon 90, 0.1% tween 20, ph 8.4) for dilution. the slides were washed twice in ftp and twice in ultrapure water and subsequently dried under a stream of nitrogen. fluorescence measurements were performed using an axon 4200a laser scanner (molecular devices). fluorescence intensity was evaluated using genepix pro 6.0 software. for evaluation of binding efficiency the contrast (c) was calculated from the assay was performed according to potier et al. [30] . aichi h3n2 or rostock h7n1 viruses were incubated with human or turkey erythrocytes, respectively, to yield agglutination (in the absence of an inhibitor) and concentration-dependent inhibition of agglutination (in the presence of an inhibitor). inhibitors were twofold serially diluted in pbs. then, 2 hemagglutination units (hau) containing 2á10 7 virus particles were added to all wells. viral particle concentration was estimated as described by desselberger et al. [31] . after 30 min incubation at room temperature, 50 μl of a 1% erythrocyte solution (~2á10 6 cells μl -1 ) was added, gently mixed and incubated for 60 min at room temperature. for aichi h3n2 human erythrocytes (α-2,6'-sialosugars), for rostock h7n1 turkey erythrocytes (α-2,3'-sialosugars) have been used. the inhibitor constant k i (hai), reflects the lowest inhibitor concentration, which is necessary to achieve complete inhibition of hemagglutination caused by the influenza virus. to check for full hemagglutination inhibition, the microtiter plate was tilted by 60°to cause droplet formation from the red blood cell pellet [29] . the experiment has been assessed using an mts reagent (promega) according to the manufacturer's protocol: 15,000 mdck ii cells were seeded the day before infection. aichi h3n2 or rostock h7n1 were pretreated with peptides in a twofold dilution series for 30 min at room temperature under slight agitation. cells were washed once with pbs (supplemented with 0.49 mm mg 2+ , 0.90 mm ca 2+ ), before pretreated virus (moi 0.05) was added to the cells and maintained for 1 h at room temperature to allow binding. unbound virus was removed by washing once with infection medium (dmem, 2 mm l-glutamine, 0.1% fcs, 0.1% bovine serum albumin (bsa), 2.5 μg ml -1 l-(tosylamido-2-phenyl) ethyl chloromethyl ketone (tpck) treated trypsin, 100 u ml -1 penicillin and 100 μg ml -1 streptomycin), and subsequently incubated for 24 h at 37°c. then, 20 μl mts solution was added to each well followed by 2 hours incubation at 37°c. finally, absorbance at 490 nm was recorded and data were normalized as follows: experiments have been performed for both virus strains in at least triplicate experiments. the microneutralization assay was performed according to the protocol of klimov et al. [32] with minor modifications. briefly, peptide dilutions were mixed with either 200 tcid 50 aichi h3n2 or 50 tcid 50 rostock h7n1 in infection medium for 30 min at room temperature. subsequently, 30,000 mdck ii cells were added to each well, followed by incubation for 18 h at 37°c. next, the medium was removed, washed with pbs and fixed with ice cold acetone (80% v/v). after removal of the fixative, plates were dried and undergone immunostaining. cells were stained with igg primary antibody against the influenza a nucleoprotein (millipore, cat. # mab8257, mouse monoclonal, 1:1000 in antibody diluent: pbs, 0.3% tween 20 (v/v) and 5% (w/v) nonfat, dry milk) for 1 h at room temperature. following, cells were washed with washing buffer (pbs, 0.3% tween 20), before a secondary antibody (1:1000 goat anti-mouse igg, kpl, cat. # 074-1802 in antibody diluent) conjugated to horseradish peroxidase (hrp) was added for another hour at room temperature. next, the antibody was removed and the cells were washed with washing buffer. finally, σ-phenylenediamine dihydrochloride (opd) in citrate (0.05 m phosphate-citrate, 0.03% sodium perborate, ph 5.0 at 25°c), was added for approximately 30 minutes until the supernatant turned yellow. the reaction was stopped with 0.5 m sulfuric acid and absorbance of the reaction product in the microwell plate was recorded at 490 nm. after subtraction of the background of non-infected cells, neutralization was calculated in ratio to untreated, infected cells. one day before treatment 15,000 mdck ii cells were seeded. on the following day media was removed and replaced with a twofold serial dilution of either peb, peb gf or peb p in supplemented dmem (see above). next, cells have been incubated in the presence of inhibitor for 24 h at 37°c. then, 20 μl mts solution was added to each well. finally, absorbance at 490 nm was measured and data were normalized to untreated cells as follows: cell viability % ð þ ¼ ðtreated cells à medium backgroundþ ðuntreated cells à medium backgroundþ x 100 an ensemble of configurations for the complexes ha-pea, ha-peb, ha-pec and ha-peb gf was built using the amber14 package of programs [33] . original coordinates for all atoms were taken from crystal structure pdbid = 2vir. in each case, the antibody moiety excepting the sequence of the peptide under study was erased. in the case of peb gf , in silico d6g and y11f mutations were performed. systems were then solvated using the tip3p water molecule model [34] , extending at least 10 å from the complex. nearly 18250 water molecules were added to solvate the complex and the resulting truncated octahedral box size was nearly 108 å x 108 å x 108 å. an appropriate number of chloride ions were added to keep the total system charge neutral. all bond lengths involving hydrogen atoms were constrained using the shake algorithm allowing the usage of a 2 fs time-step [35] . the temperature was fixed at 300 k using a langevin thermostat with a collision frequency of 2 ps -1 . the electrostatic interactions were treated using the particle-mesh ewald (pme) scheme with a fourth-order b-spline interpolation and a tolerance of 10 -5 [36] . the non-bonded cut-off was 8 å and the non-bonded pair list was updated every 50 fs. simulations were carried out according to the same protocol that has been used in our previous studies [37] [38] [39] [40] [41] , with the amber ff99sb force field [42] . briefly, each complex configuration was first optimized by 1000 steps of steepest descent followed by another 1000 steps of conjugate gradient minimization, keeping all atoms of the complex restrained to their initial position with a weak harmonic potential. each system was then simulated for 50 ps at constant volume with a 2 kcal mol -1 å -2 restraint on the complex, in order to equilibrate the solvent at 300 k without undesirable drifts of the structure. subsequently, a 50 ps md simulation with a 2 kcal mol -1 å -2 restraint on each complex at a pressure of 101,325 kpa was conducted to relax the density using berendsen's barostat. after 1 ns without restraint equilibration phase, a 10 ns simulation at constant pressure was carried out and the coordinates were stored every 10 ps, resulting in 1000 configurations for each simulation. subsequently, molecular mechanics-poisson-boltzmann surface area (mm-pbsa) calculations were performed. in the mm-pbsa method, the binding free energy of the receptor-ligand complex, δg bind , is determined from where g com , g rec , and g lig denote the absolute free energies of the complex, receptor and the ligand, respectively. this method has been discussed elsewhere [37] [38] [39] [40] [41] . the free energy g for each species is estimated from here, e mm is the molecular mechanical energy in the gas phase, g solv the solvation free energy, and -ts mm the contribution from the conformational entropy. the term e mm is comprised of the internal (bond, angle, dihedral) (e int ), electrostatic (e elec ), and van der waals energies (e vdw ), according to to incorporate all possible nonbonded interactions, the term e mm was estimated for each snapshot with no cut-offs. the solvation free energy, g solv , is approximated as the sum of the polar (g pol ) and the nonpolar contribution (g np ) using a continuum representation of the solvent according to here, γ = 0.00378 kcal mol -1 å -2 and b = -0.5692 kcal mol -1 . the popular linear poisson-boltzmann method was used to estimate the polar component of the solvation free energy. here, sasa is the solvent accessible surface area estimated by the linear combination of pairwise overlap (lcpo) algorithm using a probe radius of 1.4 å [43] . the electrostatic contribution to the solvation free energy was estimated from the poisson-boltzmann (pb) approach using the pbsa solver implemented in amber. in order to solve the pb equation the grid spacing was set to 0.5 å in all dimensions and the relative dielectric constants in the protein and in the water were chosen to be 1 and 80, respectively. the ionic strength was set to 0.15 m. the ratio between the longest dimension of the rectangular finitedifference grid and that of the solute was chosen to be 4.0. the linear pb equation was solved with a maximum of 1000 iterations. in order to understand the inhibitor-residue interaction in more detail, the interaction energy was further decomposed into the contributions from each residue of the peptide by using the theory of free energy decomposition [44] . the peptide-residue interaction is approximated by where δe vdw and δe elec are the contributions from the van der waals and electrostatic interactions between the inhibitor and each residue in the gas phase. the polar solvation free energy, δg pb , was estimated using the pbsa module of amber. the 3d structure of the fab-hemagglutinin complex of the antibody hc19 (pdb: 2vir; complex of immunoglobulin igg1 with ha of aichi h3n2) was analyzed to identify peptide sequences of the fab fragment at the ha contact area [26] . essentially, three hypervariable complementarity determining regions (cdr) of the heavy chain variable domain (vh) of fab interact with ha ( fig 1a, 1b and 1c ). from these three loop-like organized sequences interacting with the sa binding pocket of ha, peptides pea, peb and pec ( fig 1d) were derived containing as a core the cdr sequences (table 1) . in order to predict and compare binding affinities to ha of the three peptides, corresponding peptide-ha complexes were simulated in explicit water model for 10 ns using md. as deduced from the root-mean-square deviation for peb already after three ns a steady state for binding was observed (fig 2a) . in contrast, peptide pea dissociated from ha within the simulation time of 10 ns. gibbs free energies of peptide binding to ha were -20.4 and -5.3 kcal mol -1 for peb and pec, respectively (table 1) . even though absolute values for free energy changes derived from md simulation have been proven not to correlate exactly with experimental values, a comparison between md derived values implicates a stronger binding of peb to ha. also, as shown in fig 1b and 1c , peb fits best the conserved sa binding site of aichi h3n2 ha. based on this and experimental results (see below) we selected peb as a potential inhibitor of virus binding to host cells. as expected for its short primary sequence, and revealed experimentally by cd spectroscopy, peb displayed no secondary structure elements in solution at physiological ph (s1a and s1b fig) . to unravel the molecular basis of peb binding to ha, an analysis of energetic contribution was conducted by a combined md/mm-pbsa approach. molecular configurations obtained from md simulations of the complexes in explicit water were used for calculation of binding free energies using an implicit solvation scheme. the binding free energy was decomposed into contributions of the individual peptide residues (fig 2b, peb) . residues within and close to the loop (y5 to y12) provide the largest contributions to the binding free energy. significant attractive contributions to the binding free energy change arise from f4 (-0.6 kcal mol -1 ), y7 (-3.9 kcal mol -1 ), and f10 (-3.0 kcal mol -1 ). however, d8 and d6 contribute repulsively by 2.4 kcal mol -1 and 3.7 kcal mol -1 , respectively, to the binding free energy. hence, exchanging d8 and d6 to other amino acids would predict to reduce the repulsive interactions of these residues and, thus, to increase the affinity of the modified peb to ha. the detailed analysis of electrostatic, van der waals and polar contributions to the free energy change of each residue is presented in s3 fig. we have also analyzed the contributions of the individual residues of ha to this complex. attractive contributions mainly originate from residues s136 (-3.0 kcal mol -1 ), n137 (-1.8 kcal mol -1 ), and l194 (-1.0 kcal mol -1 ), whereas the most significant repulsive contribution is caused by e190 (1.1 kcal mol -1 ) (numbering according to pdb 2vir) (s4 fig). the attractive contributions from the polar residues s136 and n137 arise mainly from the intermolecular electrostatic interaction energy, whereas the hydrophobic residue l194 derives most of its contribution from van der waals interactions. binding of aichi h3n2 virus to surface immobilized peptides was measured using spr. for this purpose, peptides pea-lys, peb-lys, and pec-lys, containing lysine residues at the amino terminus, acting as spacer were used. as deduced from md simulations, the n-terminus of peb does not significantly contribute to binding of the peptide to ha, as the contribution of these residues to the total free energy change is close to 0 kcal mol -1 (fig 2b, peb) . binding significant virus binding was observed on peb-and pec-, but not on pea-modified surfaces consistent with our md simulations. the minimum detection level, corresponding to the lowest virus concentration, and showing binding 3 times above the spr response of a buffer control, was 0.19 μg and 6.25 μg aichi h3n2 virus protein ml -1 for immobilized peb and pec, respectively. the higher sensitivity of peb in the viral binding assay confirms the selection of this cdr derived peptide for further experiments, as mentioned above. to confirm specificity three peb variants containing single amino acid residue exchanges, which were immobilized with the same ligand density, were investigated. no binding was observed with the three variants, suggesting that the binding of virus is indeed specific to the cdr sequence ( fig 4a) . the results also exclude the possibility that binding is mediated by the oligolysine terminus. furthermore, several well-known proteins were tested for unspecific binding to the peptide modified chip surface ( fig 4b) . all proteins were injected at the same mass concentration. only for core-streptavidin and lysozyme minor binding to peb was found showing that surface inertness is not impaired. very likely, viruses bind to the peptide-covered surfaces in a multivalent manner (see discussion). as it is not possible to calculate reliable binding constants from the multivalent binding, the inverse experiment using surface immobilized ha from aichi h3n2 was performed. from steady-state affinity calculation for peb a binding constant k d of 56.8 μm (r max = 35.6 ru; chi 2 = 0.088), was obtained (fig 5a and 5b ). to assess whether peb binds to and thus blocks the sialic binding pocket of aichi h3n2 neuraminidase, an enzyme activity assay with the substrate munana was performed (for details see material and methods). while the neuraminidase inhibitor dana (n-acetyl-2,3-dehydro-2-deoxyneuraminic acid), serving as a positive control, was able to inhibit neuraminidase activity (ic 50 to further support specificity of binding, inhibition of binding of aichi h3n2 to surface immobilized fetuin by peb was studied. fetuin contains α-linked 2,3'-and 2,6'-sialic acids, table) . all proteins were diluted to a concentration of 10 μg ml -1 . columns represent the mean value of duplicate experiments. the error bars show the sem. provide evidence that peb competes successfully for the sa binding site and, thus, may serve as a virus binding inhibitor. our md simulations have shown that the main contributions of peb binding to ha originate from residues 4 to 12 of the peptide. in search for peptides with higher binding affinity, a full substitutional analysis using microarrays was performed. thus, 152 peb mutant variants were generated. in each of them, the full-length sequence was preserved except for only one amino acid from the eight loop-forming amino acids of the original peb peptide (ardfydydv-fyyamd) which was substituted with the 19 remaining natural amino acids (fig 6b) . to assess binding, aichi h3n2 was labeled with a protein reacting fluorophore. most notably, substitution of d6 generally leads to an increased binding signal. this is in agreement with our md simulations, predicting that this residue causes repulsive forces (+3.7 kcal mol -1 ) by interacting with ha. furthermore, most substitutions (out of d, g, t) of f10, which was predicted to provide high attractive contribution (-3.0 kcal mol -1 ), reduced the obtained fluorescence signal. although the results are essentially consistent with the theoretical predictions, we noticed a deviation: following the microarray results, aspartic acid d8 seems to be essential for binding while the md simulation assigned it to be repulsive. notably, for some substitutions of y11 we observed also an enhanced binding of virus. the microarray approach was applied to other influenza virus strains which are of higher medical relevance (s7 fig): new york h3n2, victoria h3n2 and california h1n1. similar as for aichi h3n2, most substitutions of d6 increased binding of california h1n1 and-although rather marginal-of new york h3n2. however, for the latter as well as for victoria h3n2 substitutions of other residues caused significantly enhanced binding. for both strains all substitutions of y11 lead to enhanced affinity. virus binding of selected peptide variants was evaluated by spr to identify variants with improved binding. particularly, we were interested in variants showing binding to various influenza virus strains. to this end, several doubly substituted variants were probed. based on the results for monosubstituted variants (see above), we have chosen d6 and y11 for substitution. fig 6c shows the results for the binding of three virus strains to peb-lys and 10 monoand double-substituted variants. the best variant was peb gf with the sequence ardfygydvffyamd. the repulsive amino acid d6 (+0.8 kcal mol -1 ) and the only slightly attractive residue y11 (-0.1 kcal mol -1 ) (see md simulation above) were replaced by g and f, respectively. contribution to the total binding free energy change of every amino acid of peb gf binding to ha of aichi h3n2 obtained by md-simulations is shown in fig 2b. d6g and y11f mutations decreased the contribution of free energy changes of these residues (s4 fig and discussion) . y11f alters only moderately its interaction with ha, but it induces a favorable contribution change in the interaction of its neighbor residue phenylalanine 10 with ha. the side chain of residue 11 is oriented towards the solvent in peb, due to its polar property. on the other hand, the absence of a -oh group in the residue side chain in peb gf induces a slight change in the orientation of the loop, tilting now the phenylalanine side chain towards a hydrophobic region of the ha αhelix, located between residues 160-163. the amount of hemagglutinin bound to peb gf compared to peb was 2-, 4-and 20-fold for aichi h3n2, new york h3n2 and california h1n1, respectively. specific binding of peb gf to the receptor binding site of ha was evidenced by spr ( fig 6a, table 2 ). compared to peb, peb gf showed a 6-fold lower ic 50 (13 μm vs 75 μm) for inhibition of aichi h3n2 binding to a fetuin-immobilized spr surface. for peb we observed a minor tendency to bind to immobilized fetuin, which becomes obvious from calculated inhibition values higher than 100%. to characterize the potential of antibody derived peptides to prevent virus replication, we studied virus binding and infection of host cells in the presence of peb, peb gf and peb p . the latter is assumed to be a structurally distinct variant due to the presence of a proline residue, used as a negative control. both peb and peb gf efficiently prevented aichi h3n2 mediated aggregation of red blood cells (rbc) as assessed by hai (fig 7a) . however, peb gf was much more efficient in comparison to peb. the inhibitory constant k i (hai) was 235 μm and 31.9 μm for peb and peb gf , respectively. furthermore, we could show binding inhibition of rostock h7n1 table 2 . mean values of ic 50 [μm] from inhibition experiments using spr (left column), together with its sem (n!2). spr binding of virus aichi h3n2 to fetuin is shown in fig 6a. inhibitor constant ki(hai) [μm] from hemagglutination inhibition experiments with its sem (n!3). data is plotted in fig 7. mean values of ic 50 [μm] from infection inhibition (fig 7b and 7c ) and microneutralization assays (fig 7d and 7e ) are presented together with its sem (n!3). (table 2) . hemagglutination by both viral strains was not inhibited by peb p . both peptides also protected efficiently mdck ii cell from infection (moi 0.05) by aichi h3n2 (fig 7b) and rostock h7n1 (fig 7c) . peb p did not compromise the infection at all. importantly, all three peptides did not impair cell viability within 24 h treatment with peptides up to the maximum concentration (400 μm) studied (s8 fig). similar results were obtained from a microneutralization assay, in which the peptide inhibitors are present in solution throughout the whole 24 h incubation (fig 7d and 7e and table 2 ). here, viral nucleoprotein expression was detected by immunostaining of infected cells, and the results were compared to untreated, infected cells. the ic 50 values of rostock h7n1 were similar to that obtained by the mts based infection inhibition assay. however, the ic 50 values for aichi h3n2 were somewhat lower (3-5 fold) with respect to the mts assay. although micromolar ic 50 values seem to be rather high concentrations from the point of view of medical applications, the results are very promising for developing multivalent inhibitors (see discussion). to evaluate the inhibitory potential of the antibody derived peptides in relation to that of already published ha binding peptides, we compared these peptides in the hai assay (fig 8) [ 16-18, 20, 45] . we normalized k i (hai) values to that of 2,6-sialyllactose as the natural receptor for h3. with reduction of k i (hai) by 200 and 1600 fold, peb respectively peb gf showed superior potential compared to other published peptides, with the exception of the "entry blocker" eb. as in previous experiments, peptide peb gf showed an higher inhibitory potency than peb as observed by its 8-fold lower k i (hai). additionally both peptides showed lower k i (hai)-values than peptides obtained by phage display against h3n2 (s2(1-5)), h5n1 and h9n2. only the entry blocker eb showed superior hemagglutination inhibition, revealed by its 20-fold lower k i (hai) as compared to peb gf . however, due to its amphiphilic character, eb forms micelles and thus may act in a multivalent manner [45] . therefore, it inhibits aichi h3n2 hemagglutination more efficiently than other monovalent peptides. for the sake of completeness we mention that mucroporin m-1 showed strong hemolytic effects and strong interference with gold surfaces, which made it impossible to obtain reliable data [20] . our aim was to obtain peptide inhibitors that recognize the conserved region of the sialic acid binding pocket of ha with a broader specificity to cover several influenza virus strains. the three cdrs of the vh-chain of antibody hc19 against hemagglutinin of influenza virus aichi h3n2 were used as templates to design peptides being a potential inhibitor of virus binding to host cells. we used complementary experimental approaches as spr and peptide microarrays, and md to select appropriate peptides and to enhance their affinity to ha by site directed substitutions of amino acids. this was validated by assessing their potential to prevent virus binding to cell surfaces and, eventually, infection of host cells. three peptides-pea, peb and pec-have been derived from the three cdr sequences of the hc19 antibody's heavy chain. in agreement with predictions from our md calculations of peptide-ha complexes, we found by spr experiments that binding of surface immobilized peptide peb to ha from aichi h3n2 was superior to pec (fig 3) . therefore, and as peb matches the conserved sialic acid binding site better than pec (pdb 2vir) [46] , subsequent studies were performed with peb and its derivatives. inhibition of virus binding to surface immobilized fetuin and 2,6'-sialyllactose by peb confirmed the specific binding of the peptides to the sialic acid binding pocket of ha. the ic 50 values are in the range of 75-120 μm, depending on the assay, which is about one order of magnitude lower than the ic 50 of α-2,6-sialyllactose. an important observation which may be also of interest for upcoming studies is that we found specific binding of viruses to surface immobilized peptides consisting only of amino acids representing the peb core sequence, and a four lysine long linker (fig 3) . nevertheless, peb and its derivatives harboring only those amino acids which are essential for the recognition of sa are presumably not efficient binders in their monomeric form. the affinities of the peptides are in the micromolar range, whereas the reported k d value of antibody hc19 binding to hemagglutinin from aichi h3n2 is 28 ± 8 nm [47] . however, weak binding interactions for such shorter monomeric peptides could be enhanced by a multivalent interaction as it occurs very likely in the spr based experiments, where binding of even 6.5á10 8 viruses ml -1 to immobilized peb could be detected (fig 3) . extensions of the c-and n-termini of the cdr sequences could be important for (i) the formation of a stable peptide structure, which exposes the cdr loop region in its native arrangement or (ii) direct involvement in binding and foster interaction with ha. according to the md simulations peb adopts a strand-loop-strand conformation with the loop consisting of residues d6 to y11. as shown in fig 2b, residues within and adjacent to the loop (y5 to y12) include the residues yielding the largest contributions to the binding free energy. most of these contributions are attractive (negative free energies changes) but some are also repulsive (positive free energies changes). the contributions of each amino acid to the free energy calculated by mm-pbsa were largely consistent with the results of microarray-based substitutional analysis, despite the described discrepancy for d8. possibly, substitution of d8 changes the secondary structure leading to a lower affinity. in the light of improving the affinity of peb and to enable a broader specificity we also probed the binding of the peptide to ha of other virus strains by microarray-based substitutional analysis. we identified that y11 is also an important residue for improving binding. based on that, we investigated the affinity of selected variants of peb having two substitutions. in this screen, we identified peb gf as the best binder. the two substitutions, d6g and y11f, altered the ligand moiety and its binding contacts, resulting in favorable changes to the contribution to the free energy change of residues 6, 8 and 10 ( fig 2b) . the mutation of residue 6 (d6g), replacing a charged amino acid by a neutral one, resulted in two major changes. firstly, it induces a decrease of its own electrostatic contribution to the binding free energy, but such a change is largely overcome by an increase in the contribution to the polar solvation free energy change (s3 fig). on the other hand, one should notice that residues 6 and 8 are located in close contact to each other in the loop region. as a consequence for peb, this involves repulsive interaction between two negatively charged residues (d6 and d8). the replacement of d6 by an uncharged residue in peb gf probably induces a slight reorganization in the loop structure, favoring van der waals and polar solvation interaction contributions between this residue and the ha moieties (s3 fig). due to its orientation towards the sa binding region of ha, cdr3 sequences of antibody hc19 [26, 27] against ha of aichi h3n2 are promising candidates for getting variants with broad influenza strain specificities. indeed, both peptides peb and peb gf were found to bind to ha of other strains (rostock h7n1, new york h3n2, and california h1n1). it is worth to note that all has of different influenza subtypes, which could be bound and inhibited by peptides peb or peb gf reveal strong structural similarities, especially for those amino acids involved in peptide binding (s2 table) . this close relation supports the wide binding ability and inhibitory potency of peb for a variety of influenza a strains. such broader specificity is also supported by the similarity between the vh-cdr3 of the mouse antibodies against aichi h3n2 hemagglutinin in pdb 1ken (antibody hc63, amino acids 97-109: aafyydydfffdy) and pdb 2vir, antibody hc19, amino acids 97-109: rdfydydvfyyam), and also a human antibody against h3: (egdydiltgyyyyfdy), respectively [26, 48, 49] . notably, there is enrichment of aromatic amino acids tyr and phe, and of asp. in line with these findings, a recently published human antibody against h1 shows a higher proportion of aromatic amino acids in the hcdr3 domain, too [50] . the crucial role of an asp residue and hydrophobic amino acids in the ha binding of many receptor mimicking antibodies is highlighted by lee et al [13] . as shown exemplarily for aichi h3n2 and rostock h7n1 inhibition of virus binding by peptides leads to a strong protection from virus infection of host cells. in the mts based infection inhibition assay peb gf was found to be superior with respect to peb, too. a similar observation was made for rostock h7n1 when using the microneutralization assay, which provides a direct measure for viral replication. however, for aichi h3n2 we did not find a preference for peb gf indicating strain dependent differences of the antiviral potential of the peptides. the inhibitory potential of our antibody derived peptides demonstrated a remarkable performance compared to other published peptides binding to ha (fig 8) . as summarized in table 2 , peb and peb gf provides better inhibition against aichi h3n2 compared to rostock h7n1, with the exception of the results from the hai assay. however, we should take into consideration that the performance of a peptide as inhibitor depends on many factors. as a consequence, the hemagglutination inhibition efficiency may not necessarily be stronger against aichi h3n2, even though the peptide was derived from an antibody binding to this influenza strain. one can speculate that rostock h7n1 is more prone to neutralization by blocking this binding site, while for aichi h3n2 peb and peb gf can be displaced more easily in a binding competition experiment with erythrocytes. this trend could also be explained by the use of erythrocytes derived from different species (see material and methods). similar strain-dependent efficiencies have been obtained by lópez-martinez et al. [22] . although a peptide was designed against broadly conserved epitopes, the antiviral efficiency was not the same against all tested serotypes (three different h1n1 strains and h5n2). short oligomeric biomolecules with sufficient affinity are of high interest for diagnostics and drug design, as development, synthesis and further chemical modifications are easy to implement. linear short cdr derived peptides as used here can be expected to differ from the loop structure they adopt within the antibody, leading to lower affinity. however, there are promising ways to largely improve them. cyclisation of cdr peptides using a d-pro-l-pro template has been shown to force them into canonical conformations [51] . in this report, the l3 loop of the hc19 antibody adopted a hairpin structure and an aromatic t-stacking as in the antibody crystal structure. this and other strategies to generate peptidomimetics by stabilizing or mimicking turns, ß-sheets and helices, have been recently reviewed [52] . whereas the gain in affinity by cyclization of a peptide is limited by the affinity of the native structure, larger improvements can be achieved by multivalency, especially when a virus with a high target protein density on its surface has to be bound [53] [54] [55] [56] . for example, sialyl-containing oligosaccharides constituting the natural receptors of hemagglutinin on mammalian epithelial cells, exhibit a millimolar 1:1 affinity, but due to multivalent virus-cell attachment the binding is strong enough to enable infection. effective inhibition of influenza virus infection by multivalent sialic-acid-functionalized gold nanoparticles and other glycoarchitectures has been described [57, 58] . more recently, a trivalent glycopeptide mimetic containing three sialyl residues matching the three sialyl binding sites in a ha trimer, was reported to bind to h5 hemagglutinin 4000 fold better than the monomer [59] . recently, we demonstrated that the antiviral effectivity of acylated peb gf can be increased, upon presentation in a multivalent fashion by 10-fold against rostock h7n1 and by 20-fold against aichi h3n2 [60] . further, multivalent presentation of the short s2 peptide (arlpr) with a dendrimer resulted in sub-micromolar inhibitory activities [61] . the antibody derived peptide peb and its mutant variant peb gf are promising hemagglutinin binding peptides, which both could have potential for application in viral diagnostics and therapeutics. infection inhibition could be achieved at micromolar concentrations. strategies to improve the peptide structure and to apply the peptides for developing multivalent binders with high affinities are discussed. finally, the workflow presented involving complementary experimental and theoretical approaches could be a template for the development of antiviral agents, which could be also applied for influenza detecting biochips. in a coevolutionary approach the peptide's primary sequence may also be adapted to seasonal upcoming mutations of the influenza virus with minor additional efforts, costs and in short time without the additional development of novel monoclonal antibodies. contributions of individual residues of peb (black) and peb gf (grey) to the binding free energies of the corresponding peb-ha or peb gf -ha complexes. a) electrostatic, b) van der waals and c) polar contributions to the total binding free energy change. the mutation of residue 6 (d6g) replacing a charged amino acid by a neutral one, decreases the electrostatic contribution, but such a change is largely overcome by an increase in the contribution to the solvation free energy change, as calculated. mutation of amino acid 11 (y11f) appears not to alter directly the contribution of the interaction between this residue and the ha, but it induces a favorable free energy change contribution in the interaction between residue 10 and the ha, essentially by a more favorable van der waals interaction energy change between uncharged residues. (tif) labeled influenza california h1n1 (left), new york h3n2 (middle) and victoria h3n2 (right) were used as analytes. numbers represent mean value of the contrast relative to contrast for positive control fetuin. false-colors are used to illuminate fluorescence intensities, color changes from blue (lower) to yellow (higher intensity than peb). data just represent qualitative relation between peb mutants within single influenza strains. quantitative comparison between different strains is not valid due to the varying types of samples, while precise quantitation fails due to unknown ligand density (most likely varying per peptide). table. alignment of ha sequences of used influenza viruses. amino acids involved in binding of peb as obtained from md-simulations are highlighted in gray. pdb2vir: sequence obtained from protein database; a/mute/swan/r901/06-h7n1: sequence obtained from prof. harder (friedrich-loeffler-institut, riems, germany); all other sequences were obtained from influenza virus resource (ivr) as indicated by their accession numbers [62] . while l219 is identical for all subtypes, s159 and e215 are replaced in some cases by functional closely related t or d, respectively. the sequence differ mostly in n160, which is replaced by s, a or v. the n160 substitutions could be the main reason for differences in the observed binding ability. (docx) influenza neuraminidase: a druggable target for natural products influenza: the mother of all pandemics unraveling the mystery of swine influenza virus human infection with a novel avian-origin influenza a (h7n9) virus avian-origin influenza a(h7n9) infection in influenza a(h7n9)-affected areas of china: a serological study emergence of a novel swine-origin influenza a (h1n1) virus in humans variation and infectivity neutralization in influenza structural basis of immune recognition of influenza virus hemagglutinin influenza virus resistance to antiviral therapy a neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza a hemagglutinins a highly conserved neutralizing epitope on group 2 influenza a viruses cross-neutralization of influenza a viruses mediated by a single antibody loop heterosubtypic antibody recognition of the influenza virus hemagglutinin receptor binding site enhanced by avidity structural insights into key sites of vulnerability on hiv-1 env and influenza ha a common solution to group 2 influenza virus neutralization identification and characterisation of a novel anti-viral peptide against avian influenza virus h9n2 sialic acid-mimic peptides as hemagglutinin inhibitors for anti-influenza therapy phage displayed peptides to avian h5n1 virus distinguished the virus from other viruses bovine lactoferrin-derived peptides as novel broad-spectrum inhibitors of influenza virus virucidal activity of a scorpion venom peptide variant mucroporin-m1 against measles, sars-cov and influenza h5n1 viruses a novel family of peptides with potent activity against influenza a viruses inhibition of influenza a virus infection in vitro by peptides designed in silico influenza a virus entry inhibitors targeting the hemagglutinin systematic exploration of the antigen binding activity of synthetic peptides isolated from the variable regions of immunoglobulins structural identification of the antibody-binding sites of hong kong influenza haemagglutinin and their involvement in antigenic variation antigen distortion allows influenza virus to escape neutralization refined three-dimensional structure of the fab fragment of a murine iggl,lambda antibody structurally conserved binding sites of hemagglutinin as targets for influenza drug and vaccine development hemagglutination inhibition assays. seminars in avian and exotic pet medicine fluorometric assay of neuraminidase with a sodium (4-methylumbelliferyl-alpha-d-n-acetylneuraminate) substrate relation of virus particle counts to the hemagglutinating activity of influenza virus suspensions measured by the ha pattern test and by use of the photometric hcu method influenza virus titration, antigenic characterization, and serological methods for antibody detection comparison of simple potential functions for simulating liquid water numerical-integration of cartesian equations of motion of a system with constraints-molecular-dynamics of n-alkanes particle mesh ewald-an n.log(n) method for ewald sums in large systems importance of polar solvation for cross-reactivity of antibody and its variants with steroids energetic basis for drug resistance of hiv-1 protease mutants against amprenavir origin of decrease in potency of darunavir and two related antiviral inhibitors against hiv-2 compared to hiv-1 protease energetics of mutation-induced changes in potency of lersivirine against hiv-1 reverse transcriptase mutation-induced loop opening and energetics for binding of tamiflu to influenza n8 neuraminidase comparison of multiple amber force fields and development of improved protein backbone parameters approximate solvent-accessible surface areas from tetrahedrally directed neighbor densities insights into protein-protein binding by binding free energy calculation and free energy decomposition for the ras-raf and ras-raigds complexes identification of the minimal active sequence of an anti-influenza virus peptide receptor binding and membrane fusion in virus entry: the influenza hemagglutinin a complex of influenza hemagglutinin with a neutralizing antibody that binds outside the virus receptor binding site an antibody that prevents the hemagglutinin low ph fusogenic transition broad neutralizing human monoclonal antibodies against influenza virus from vaccinated healthy donors broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza virus hemagglutinin structural mimicry of canonical conformations in antibody hypervariable loops using cyclic peptides containing a heterochiral diproline template structure-based design of inhibitors of protein-protein interactions: mimicking peptide binding epitopes monomeric inhibitors of influenza neuraminidase enhance the hemagglutination inhibition activities of polyacrylamides presenting multiple c-sialoside groups polyvalent interactions in biological systems: implications for design and use of multivalent ligands and inhibitors effective inhibitors of hemagglutination by influenza virus synthesized from polymers having active ester groups. insight into mechanism of inhibition multivalency as a chemical organization and action principle inhibition of influenza virus infection by multivalent sialic-acid-functionalized gold nanoparticles inhibition of influenza virus activity by multivalent glycoarchitectures with matched sizes a nanomolar multivalent ligand as entry inhibitor of the hemagglutinin of avian influenza potential of acylated peptides to target the influenza a virus synthesis and influenza virus inhibitory activities of carbosilane dendrimers peripherally functionalized with hemagglutinin-binding peptide influenza virus resource the authors thank ines kretzschmar (charité, berlin) for help with peptide synthesis, prof. harder (friedrich-loeffler-institut, riems, germany) for providing virus rostock h7n1, dr. zierenberg (gsk dresden) for providing the influenza monobulks a/victoria/210/2009 h3n2 and a/newyork/55/2004 h3n2, and dr. thaa (fu berlin) for providing the turkey erythrocytes. for support with cd spectroscopy measurements we thank dr. jahnke (university of potsdam). conceived and designed the experiments: ffb ah eef ws. performed the experiments: hm dl pk sdl. analyzed the data: hm dl pk sdl vk ws. contributed reagents/materials/analysis tools: rv. wrote the paper: ws ah dl. key: cord-001263-hqxiyxfj authors: kam, yiu-wing; lee, wendy w. l.; simarmata, diane; le grand, roger; tolou, hugues; merits, andres; roques, pierre; ng, lisa f. p. title: unique epitopes recognized by antibodies induced in chikungunya virus-infected non-human primates: implications for the study of immunopathology and vaccine development date: 2014-04-22 journal: plos one doi: 10.1371/journal.pone.0095647 sha: doc_id: 1263 cord_uid: hqxiyxfj chikungunya virus (chikv) is an alphavirus that causes chronic and incapacitating arthralgia in humans. although patient cohort studies have shown the production of chikv specific antibodies, the fine specificity of the antibody response against chikv is not completely defined. the macaque model of chikv infection was established due to limitations of clinical specimens. more importantly, its close relation to humans will allow the study of chronic infection and further identify important chikv targets. in this study, serum samples from chikv-infected macaques collected at different time-points post infection were used to characterize the antibody production pattern and kinetics. results revealed that anti-chikv antibodies were neutralizing and the e2 glycoprotein, capsid, nsp1, nsp3 and nsp4 proteins were targets of the anti-chikv antibody response in macaques. furthermore, linear b-cell epitopes recognized by these anti-chikv antibodies were identified, and mapped to their structural localization. this characterizes the specificity of anti-chikv antibody response in macaques and further demonstrates the importance of the different regions in chikv-encoded proteins in the adaptive immune response. information from this study provides critical knowledge that will aid in the understanding of chikv infection and immunity, vaccine design, and pre-clinical efficacy studies. chikungunya virus (chikv) was first described during an epidemic in 1952 in tanzania, east africa as the causative agent of chikungunya fever (chikf) [1, 2] . chikv belongs to the genus alphavirus of the family togaviridae and is an enveloped virus with a single-stranded positive-sense rna genome [3] . the 12kb rna genome is capped at the 59 end and polyadenylated at the 39 end and consists of two open reading frames coding for four nonstructural proteins (nsp1-4), three major structural proteins (capsid, e1, and e2) and two small cleavage products (e3 and 6k) [3, 4] . the e1 and e2 glycoproteins form heterodimers that associate as trimeric spikes on the virion surface while e3 and 6k were demonstrated to act as helper proteins in the budding and maturation process of the virion envelope [5] [6] [7] . in the last decade, multiple chikf epidemics have occurred in east africa, the indian ocean islands, and many parts of south east asia [8] [9] [10] [11] [12] [13] . more recently, new episodes of chikf have been reported in the americas, further broadening the geographical spread of the disease [14] . the aedes species of mosquito has been the major arthropod vector associated with chikv transmission to humans [15] . chikv infection usually leads to the development of chikf and is characterized by an abrupt onset of fever, headache, fatigue, nausea, vomiting, rash, myalgia, and severe arthralgia. similar to other arthralgia-causing arbovirus infections, a fraction of patients developed chronic symptoms lasting from several weeks to months [1, 2, 15] . currently, there are no licensed vaccines or antiviral drugs against chikv infection for human use. therapy for chikv infection is often limited to supportive care [4] . despite the development of several animal models, few have met the requirement to be used in pre-clinical study to assess potential therapeutics. recent epidemiological data showed the increasing importance of antibody-mediated protection against chikv [16] [17] [18] [19] , highlighting the feasibility of using anti-chikv antibodies as therapeutics or as a prophylactic treatment [20] . however, information about the exact target of the adaptive immune response either in human or in animal models remains limited, although b-cell epitopes have been identified within the e1/e2 glycoproteins [17, 21] . due to the close lineage relationship between humans and macaques, macaque models of chikv infection have been developed [22] [23] [24] . these models allow comparison of the adaptive immunity between humans and macaques. furthermore, information obtained from macaque studies will be valuable for the design of future therapeutics. in this study, we aimed to investigate the kinetics and specificity of anti-chikv antibodies induced after experimental infection in cynomolgus macaques (macaca fascicularis). in addition to the anti-e2 glycoprotein responses, we also identified new linear b-cell epitopes that were recognized by anti-chikv antibodies from chikv-infected macaques. these epitopes can potentially be used for future development of vaccine candidates. all animals were handled in strict accordance with good animal practice as defined by the european directive 63/2010/eu and in accordance with recommendations of the weatherall report. four to six-year-old cynomolgus macaques (macaca fascicularis) were imported from the international accredited breeding facilities from mauritius (negative for siv, stlv, herpes b virus, filoviruses, srv-1, srv-2, measles, dengue virus and chikv) and were housed in a bsl3 facility (permit number a 92-032-02), in accordance with office for laboratory animal welfare (olaw, usa; #a5826-01) standards at the cea in accordance with the french national regulation under the number b-92-032-02 for animal use, under the number 2005-69 for macaque breeding. animals are fed daily and monitored closely by caretakers reporting directly to the veterinarians in charge of the animal facilities. all studies were reviewed and approved by the regional animal care and use committee in accordance with european directive 63/2010/eu: ''comité regional d'éthique pour l'expérimentation animale ile-de-france sud'', fontenay aux roses, decision #07_012. cell lines originally purchased from american tissue culture collection (atcc), such as human embryonic kidney (hek 293t, atcc crl-3216) and baby hamster kidney (bhk21, atcc ccl-10) cells, were adhered to recommended ethics approvals and standards. hek 293t cells were cultured in dulbecco's modified eagle medium (dmem) supplemented with 10% fetal bovine serum (fbs) (gibco, invitrogen). chikv isolates (lr2006 opy-1, imt and sgp11) used in this study were originally isolated from two french patients returning from reunion island during the 2006 chikf outbreak [25, 26] , and from a singaporean admitted to the national university hospital in 2008 [27] respectively. chikv lr2006 opy-1 clone was rescued from corresponding infectious cdna clone as previously described [28] . virus stocks (imt and sgp11) used for in vitro studies were prepared via numerous passages in vero-e6 cultures, titered, washed and precleared by centrifugation before storage at 280uc [29] . lr2006-opy1 was isolated from patient serum in marseille and passaged three times in vero-e6 culture. virus stocks were produced following a single passage in bhk21 cells for experimental infection of macaques. the animals were first sedated with ketamine chlorhydrate (10 mg/kg; rhone-mérieux) and were inoculated with 10 5 up to 10 8 pfu of chikv via the saphenous vein [22] . clinical examinations were carried out as described previously [22] , and temperature and weight of the macaques were recorded 15 minutes after sedation. sera were collected by bleeding from the femoral vein before the first chikv inoculation and on a daily basis after the infection until 16 days post-infection (dpi). chikvinfected macaques were kept for a maximum of up to 180 dpi. neutralizing activity of antibodies from chikv-infected macaque samples were tested in triplicates and analyzed by immunofluorescence-based cell infection assay in hek 293t cells. amount of chikv virions corresponding to moi 10 were mixed with heat-inactivated macaque serum (1:100-1:800 dilutions), and incubated for 2 hours at 37uc with gentle agitation in a thermomixer. virus-antibody mixtures were then added to hek 293t cells seeded in 96-well plates and incubated for 1.5 hours at 37uc. medium was removed, and cells were replenished with dmem medium supplied with 5% fbs and incubated for 6 hours at 37uc before fixation with 4% paraformaldehyde followed by immunofluorescence quantification using the cellomics arrayscan v. percentage of infectivity was calculated according to the equation: % infectivity = 1006 (% responder from sero-neutralization group/% responder from virus infection group). proteins from chikv virions were detected using modified techniques previously described [29, 30] . for reducing samples preparation, sample containing imt and sgp11 virions were boiled for 5 minutes at 100uc in laemmli buffer supplemented with 2% sds and 1 mm dtt. non-reducing samples were prepared in laemmli buffer supplemented with 2% sds but without dtt and were not boiled. sds migration buffer was used for electrophoresis. proteins from chikv virions were separated by 10% sds-page respectively, and transferred to nitrocellulose membrane at 180 ma for 45 min in transfer buffer (24 mm tris, 77 mm glycine, 20% methanol) using semi-dry transfer method. the membranes were blocked overnight in blocking buffer (tbst supplemented with 5% dry milk and 3% fbs), followed by 1 hour incubation at room temperature with either antigen-specific rabbit serum (1:2000), or macaque serum samples diluted (1:2,000) in blocking buffer. antigen-specific sera against nsp1, nsp2, nsp3, nsp4, capsid, e2 and e1 proteins were raised in rabbits by passive immunization. the appropriate hrp-conjugated anti-rabbit igg or anti-monkey igg secondary antibodies were then added and incubated for 1 hour, followed by chemiluminescence detection using ecl plus detection reagents (amersham biosciences). blots were exposed to films (pierce, thermo scientific) and developed. polystyrene 96-well microtiter plates (maxisorp, nunc) were coated with purified chikv (20,000 infectious virions per ml in pbs; 50 ml per well). wells were blocked with pbs containing 0.05% tween-20 and 5% non-fat milk (pbst-milk), and plates were incubated for 1.5 hours at 37uc. serum samples were then diluted 1:2,000 in pbst-milk and incubated 1 hour at 37uc. hrp-conjugated rabbit anti-monkey igg (alpha diagnostic) antibodies were used to detect macaque antibodies bound to virus-coated wells respectively. reactions were developed using tmb substrate (sigma-aldrich) and terminated by stop reagent (sigma-aldrich). absorbance was measured at 450 nm. noninfected macaque serum was used as negative controls. elisa readings were done in duplicates and the values were plotted as mean 6 standard error mean (sem). biotinylated peptide library consisting of 18-mer overlapping peptides (mimotopes) were generated from sequence alignments of different chikv amino acid sequences as previously described [17, 29] . peptides were dissolved in dimethyl sulphoxide (dmso) to obtain a stock concentration of approximately 15 mg/ml. all the peptides were screened in triplicates. briefly, streptavidincoated plates (pierce) were first blocked with 1% sodium caseinate (sigma-aldrich) diluted in 0.1% pbst (0.1% tween-20 in pbs), before coating with peptides diluted at 1:1,000 in 0.1% pbst and incubated at room temperature for 1 hour on a rotating platform. plates were then rinsed with 0.1% pbst before incubation with chikv-infected macaque serum samples (1:2,000) diluted with 0.1% pbst for 1 hour. plates were rinsed and then followed by incubation with the respective anti-monkey igg antibodies conjugated to hrp diluted in 0.1% blocking buffer for 1 hour at room temperature to detect for peptide bound antibodies. reaction was detected with tmb substrate solution (sigma-aldrich) and terminated with sulphuric acid (sigma-aldrich). absorbance was read at 450 nm in a microplate autoreader (tecan). peptides are considered positive if absorbance values are higher than the mean 63 standard deviation (sd) values of noninfected macaque serum controls. data are presented as mean 6 sem. structural data of the e2 glycoproteins were retrieved from pdb (id: 3n44) and visualized using the ucsf chimera software [31] . solvent excluded molecular surfaces were generated with the help of msms package [32] . structures of capsid, nsp1, nsp3 and nsp4 sequences were predicted separately using individual i-tasser queries, and visualized using ucsf chimera software [33, 34] . all data are presented as mean 6 sem or sd. differences in responses among groups at various time points and between groups and controls were analyzed using appropriate tests (mann-whitney u tests). a two-sided p value of less than 0.05 was considered to be statistically significant. in order to characterize the chikv antibody response, we obtained serum samples taken at two post-infection time-points from chikv (lr2006 opy-1)-infected macaques. the total igg present in the serum was quantified by a virion-based elisa assay using chikv isolate from la reunion (imt) [25, 29] . high levels of chikv-specific igg antibody responses were detected at 16 dpi ( figure 1a ) confirming previous observations [17] . even though the igg levels decreased over time, they were still detectable at 180 dpi ( figure 1a ). to determine if the anti-chikv antibodies were neutralizing, in vitro infection of hek 293t cells with different chikv isolates (imt and sgp11) was carried out in the presence of sera from chikv-infected macaques ( figure 1b and 1c ) [29] . serum samples from chikv-infected macaques contained antibodies that displayed neutralizing activity when assessed in sero-neutralization assays [16, 17] with the highest neutralizing efficiency for samples taken at 16 dpi ( figure 1c ). interestingly, a stronger neutralizing activity against imt was observed when compared to sgp11 for samples at 16 dpi, at a dilution of 1:800 ( figure 1c ). this is not unexpected as these macaques were experimentally infected with the lr2006 opy-1 isolate that is more closely related to the imt isolate. the presence of neutralizing antibodies in the sera of chikv-infected macaques was sustained till 180 dpi, although less efficiently than sera at 16 dpi ( figure 1c ). to better define the antigenic recognition profile of the antibodies from chikv-infected macaques, we used chikvinfected cell lysates to determine which chikv antigens were recognized by the macaque antibodies ( figure 2a) . none of the non-structural proteins (nsps) were recognized by antibodies in the serum samples taken at 16 or 180 dpi ( figure 2b ). the serum contained antibodies against e2 glycoprotein and capsid protein ( figure 2b and s1). anti-e2 and anti-capsid antibodies persisted till 180 dpi. in addition, chikv virions were purified from supernatant of cells infected with chikv isolates from reunion island (imt) [25] , or from singapore (sgp11) [27] . protein extracts were prepared under reducing or non-reducing conditions as described [29, 30] , and were then assessed by immunoblot assays using serum samples from chikv-infected macaques ( figure 2c ). this first screen revealed that these serum samples contained antibodies recognizing multiple proteins from two isolates of chikv. a stronger reactivity against proteins prepared under non-reducing conditions compared to proteins prepared under reducing conditions indicated that the serum samples contained antibodies recognizing both linear and disulfide bonds dependent conformational epitopes ( figure 2c ) [35] . similar reactivity was observed between two closely related chikv isolates (imt and sgp11) that have more than 99% amino acid sequence similarity [36] , suggesting that the two isolates present epitopes that are common or similar in part and are recognized by a fraction of the figure 1 . antibody profiles of sera from chikv-infected macaques. a, virus-specific igg and igm antibody titers in serum samples, at a dilution of 1:2,000 were determined by elisa using purified chik virions. serum samples from chikv-infected macaques (n = 1-3) were collected at 16 and 180 dpi and subjected to virion-based elisa, using 96-well plates pre-coated with purified chikv (imt) virions. sera from non-infected macaques were used as negative controls. data are presented as mean 6 sd and representative of 2 independent experiments with similar results. b, visualization of chikv by immunofluorescence in chikv-infected cultures after seroneutralization. virus samples were pre-incubated with heatinactivated sera from chikv-infected macaques collected at 16 and 180 dpi, before being added to hek 293t cells. infection without pre-incubation with sera (no sera) was used as a control. analysis was performed at 6 hours post-infection (hpi). scale bar in white: 50 mm. representative microscopic images for each treatment condition (macaque sera dilution at 1:800) are shown. c, in vitro neutralizing activity of sera from chikvinfected macaques. samples (healthy macaque sera, 16 and 180 dpi) were tested against imt or sgp11 viruses, in triplicates at a dilution 1:800 for healthy macaque sera; between 1:100 and 1:800 for chikv-infected macaque sera. results are representative of 3 independent experiments, presented as mean 6 sd, and expressed as percentage of control infection. *p,0.05, mann-whitney u test. doi:10.1371/journal.pone.0095647.g001 antibodies from macaques infected with the third closely related isolate of chikv. elisa assays were performed using a biotinylated peptide library (mimotopes) with minimized non-specific binding. the library consisted of 18-mer overlapping peptides covering the whole chikv proteome. pooled peptides were screened with pooled serum samples taken at two different time-points [17, 29] . several peptide pools were identified that contained linear chikv b-cell epitopes recognized by the serum antibodies ( figure 3 , peptide pools that positively bound serum antibodies are marked with *). results showed that there were two such peptide pools for the capsid protein ( figure 3a) , five for the e2 glycoprotein ( figure 3b) , one for the nsp1 protein ( figure 3c) , three for the nsp3 protein ( figure 3d ) and one for the nsp4 protein ( figure 3e ). the recognition pattern of antibodies from the two different time-points was not identical. no peptide pools containing linear b-cell epitopes were detected for the remaining proteins (nsp2, e3, 6k and e1) (data not shown). to study the kinetics of the establishment of the adaptive immunity in chikv-infected macaques, serum samples from macaques infected intravenously with the reunion island strain figure 2 . anti-chikv antibodies demonstrate polymorphic epitope recognition against different chikv isolates. a, schematic representation of the coding regions of chikv genome. b, total cell lysates were prepared from chikv-infected 293t cells and mock-infected 293t cells. lysates were subjected to sds-page gel electrophoresis (left lane -mock-infected lysates, right lane -chikv-infected lysates) and probed with sera from chikv-infected macaques at a dilution of 1:2,000, followed by hrp-conjugated anti-monkey igg secondary antibodies. the arrows at the top panel indicate the chikv antigens (capsid and e2) detected by antibodies in the sera of chikv-infected macaques. arrows at the middle panel (non-structural proteins) and the lower panel (structural proteins) indicate chikv proteins detected by corresponding chikv antigen-specific rabbit sera performed at a dilution of 1:2,000 followed by hrp-conjugated anti-rabbit igg secondary antibodies. housekeeping protein gapdh was probed with a specific anti-gapdh antibody (biolegend) as an indicator for loading control. sizes of molecular weight markers are indicated in the left part of the diagram. c, purified chikv virions (imt and sgp11) were prepared under reducing (100uc, 5 min+dtt) or non-reducing conditions and subjected to sds-page gel electrophoresis, then probed with sera from chikv-infected macaques at a dilution of 1:2,000, followed by secondary hrpconjugated anti-monkey igg. sizes of molecular weight markers are indicated on the left part of the diagram. non-annotated arrows represent the presence of disulfide bonds dependent conformational epitopes recognized by antibodies from the sera of chikv-infected macaques. doi:10.1371/journal.pone.0095647.g002 figure 3 . mapping of chikv b-cell epitopes within chikv proteome. sera from chikv-infected macaques (16 and 180 dpi) were diluted 1:2,000 and subjected to peptide-based elisa with a peptide library covering the chikv proteome, using pooled peptides (named p1, p2, etc) from the structural (a, capsid, b, e2) and non-structural (c, nsp1, d, nsp3 and e, nsp4) proteins. sera from non-infected macaques were used as negative controls. *pooled peptides were considered to contain positive linear b-cell epitopes when od values obtained with sera from chikv-infected macaques were above mean +3 sd of the od values obtained with sera from non-infected macaques. data are presented as od values obtained using sera from infected macaques, minus the od values obtained using sera from non-infected macaques, for the corresponding pooled peptides. data represent an average of two independent experiments (mean 6 sd). doi:10.1371/journal.pone.0095647.g003 (lr2006 opy-1) were collected over a 6-month period [22] . anti-chikv antibody profiles were followed longitudinally through different post-infection phases ( figure 4 ). all the macaques presented with fever, rash and lymphopenia, and exhibited signs of inflammation as indicated by cytokine patterns and biochemistry assays, from 1 to 6-8 dpi [22] . the 9 dpi time point in the acute phase was the earliest time point when specific anti-chikv antibodies were detected (figure 4 ). at 9 dpi, 90% of the total signal came from igm that was no longer detectable at 16 dpi, in the early convalescent phase. furthermore, at 16 dpi, the first detectable levels of igg were also observed. importantly, the later time-points of 100 and 180 dpi in the recovery phase allowed the plateau and persistence of the igg specific response to be assessed, while levels of igm were no longer detectable (data not shown). next, to define the location of the linear b-cell epitopes, the complete set of single peptides from each of the antibody-binding peptide pools identified in figure 3 , was screened with serum samples. the positions of the epitope sequences within the nonstructural and structural proteins are illustrated in the schematic diagrams ( figure 4a ). the two recognized regions for the capsid protein are located at the n-and c-terminus of the protein while the epitopes for the e2 glycoprotein are distributed along the entire protein. on the other hand, antibodies from the macaque serum samples failed to recognize any regions in the 6k protein and e1 glycoprotein. comparison of the reactivity between the two time-points showed that antibodies recognized the linear epitopes in capsid, and in the immunodominant e2 region in a concerted manner ( figure 4b ). taken together, results from the peptide-based elisa assay were consistent with the immunoblot assays, confirming the recognized regions in the e2 glycoprotein, and the capsid protein. epitope-containing sequences were mapped onto the available three-dimensional (3d) crystal structure of the e2 glycoprotein (pdb number 3n44), or predicted 3d structures of the capsid, nsp1, nsp3 and nsp4 proteins as described previously ( figure 5 ) [29] . one of the epitope-containing regions of the capsid protein (amino acids 2561-2586) was located on the surface of the protein, while the other was concealed in the folded protein ( figure 5a) . furthermore, the two recognized regions of the nsp3 protein were located on the surface of the protein ( figure 5a ). similar analyzes for the e2 glycoprotein revealed that one out of four recognized regions mapped onto the surface of the protein ( figure 5b ). majority of the epitopes clustered in the middle of the protein and were embedded in the e2 glycoprotein. defining an ideal animal model for chikv studies remains a constant challenge. in order to understand the immunology and pathophysiology of chikv infections, an animal model with disease presentations closely similar to humans is preferred. in order to study the temporal pattern of chikv b-cell epitopes in detail, experimental infections were performed in macaques with serum samples collected over 6 months. linear determinants were targeted since they are more easily identifiable in a medium-throughput approach. although conformational epitopes could also contribute to the anti-chikv antibody response, these determinants are not explored due to the limitation of detection by the linear peptides screen described here. results showed two structural proteins (capsid protein and e2 glycoprotein) and three nonstructural proteins (nsp1, nsp3 and nsp4 proteins) contained linear epitopes that were recognized by macaque anti-chikv antibodies. experimental infection of macaques induced antibodies not only against the structural proteins, but also against several non-structural proteins. the important role of antibodies against structural proteins has been previously demonstrated in sero-neutralization and protection assays [17, 18, 29] . whether antibodies targeted against the nonstructural proteins also offer protection during chikv infection will require further investigation. studies on antibody responses against various chikv proteins using plasma from patients obtained at different post-infection time-points have shown that the n-terminal region of e2 glycoprotein provides long-lasting anti-chikv antibody response during the whole course of disease [17, 29] . specifically, high levels of antibodies from chikv-infected patients were targeted against a linear b-cell epitope called e2ep3 during the acute phase of chikv infection [17] . here, e2ep3 is one of the major b-cell linear epitopes recognized by antibodies from the sera of all chikv-infected macaques during the early phase of disease (amino acids 2800-2818, table 1 ). this verifies that the presence of anti-e2ep3 antibodies is a common marker for early chikv infection in both humans and macaques [17] . previous findings that the e2 and e3 glycoproteins, capsid and nsp3 proteins were specifically detected during the convalescent and recovery phase of human disease [29] suggest that the pattern of b-cell epitope recognition by anti-chikv antibodies alters as the disease progresses. the acid-sensitive region (asr) of e2 glycoprotein has been suggested to play a role in regulating chikv particle generation and virulence in vivo [37, 38] . moreover, the functional role of the asr in regulating e1/e2 glycoprotein conformational changes suggests that the asr could be a target for neutralizing antibodies [39] . our previous results showed that natural chikv infection induced antibodies against only one asr epitope (amino acids 3025-3058, table 1 ). these antibodies were detected during the recovery phase (2 to 3 months post illness onset) [29] . however, anti-chikv antibodies against two asr epitopes (amino acids 2961-2978, and 3025-3066, table 1 ) were detected in the sera of experimentally infected macaques. furthermore, these two asr epitopes were immunodominant from the early convalescent to recovery phase, contrary to the immunodominance of the e2ep3 epitope observed in natural human chikv infection [17, 29] . the observation that one of the asr epitopes (amino acids 3025-3058) is important in both natural human infection and experimental macaque infection, suggests that this epitope could be useful for vaccine development. in addition, our results showed that antibody recognition of the e2 glycoprotein changes throughout the course of disease in the experimentally infected macaques. this could be due to the spatial positions of the b-cell epitopes on the native form of the e1/e2 glycoprotein complex. differential induction of neutralizing antibodies against exposed or hidden b-cell epitopes could contribute to antibody-mediated clearance during the entire course of disease [40] . this is important in chikv vaccinology since it underscores the importance of eliciting a broad antibody response targeting exposed and hidden b-cell epitopes that would be sufficient to cover most of the important antigens for virus neutralization. it has been established that patient antibodies from a singapore cohort have stronger binding capacity to the chikv sgp11 isolate than the imt isolate due to different epitope sequences between the two isolates, which influences epitope-antibody binding capacity [29] . here, we further demonstrated this phenomenon in the non-human primate model. infection of macaques with the lr2006-opy1 isolate, which encodes k 252 in the e2 glycoprotein, strongly induced anti-chikv antibodies against a particular linear b-cell epitope (amino acids 3025-3066) at 16 dpi. in line with this, we observed significantly stronger neutralizing activity against the imt isolate (encoding k 252 in e2) compared to the sgp11 isolate (encoding q 252 in e2) ( figure 1c) . these data complement a separate study that showed stronger neutralizing antibody response in animals infected with the ecsa strain (dhs-4263) compared to animals infected with the west african strain (37997) [24] . paradoxically, the difference in neutralizing capacity observed here was lost in macaque sera taken at 180 dpi. incidentally, sera taken at this time point also had relatively low levels of anti-chikv antibodies against the same b-cell epitope (amino acids 3025-3066). these observations highlight the importance of incorporating various components in antigen preparation for chikv vaccine development. epitope sequences covering all the relevant isolates from various geographical regions will be favored in order to offer complete coverage against chikv infections globally. only four common linear b-cell epitopes in the e2 glycoprotein could be identified by antibodies obtained from patients and chikv-infected mice [18] , while two epitopes were common between patients and chikv-infected macaques (table 1, underlined sequences) . recent studies have also demonstrated that chikv-infected patients reacted sero-positively to the e2 glycoprotein [17, 29, 41] . these observations were further supported by other studies with b-cell epitopes identified along the e2 glycoprotein by mouse and human monoclonal antibodies [21, [42] [43] [44] . contrastingly, linear epitopes along the e1 glycoprotein were detected only by antibodies from chikv-infected mice. comparatively, antibodies obtained from experimentally-infected mouse models in other alphaviruses have also identified epitopes from both the e1 and e2 glycoproteins [18, 40, [45] [46] [47] [48] . divergence in epitope recognition in the various animal models suggests the existence of species-related differences in the humoral response upon virus infection. only a few vaccine candidates against chikv have been tested in macaques [49, 50] . all other studies were assessed in mouse models [51] [52] [53] [54] . in these models, the protective effect was closely associated to the generation of neutralizing antibodies, but only few have reported on the exact epitopes involved [50, 52] . our previous work in chikv-infected macaques showed that natural infection led to viral persistence in all macaques tested up to 90 dpi [22] despite a robust innate immune response that was protective in vitro. as such, a comprehensive study on the exact targets of infection-versus vaccination-induced antibodies will provide more information about antibody-mediated protection against the chronic chikv infection observed in macaques. figure s1 . 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prediction structure-function relationships of hiv-1 envelope sequence-variable regions refocus vaccine design comparative analysis of the genome sequences and replication profiles of chikungunya virus isolates within the east, central and south african (ecsa) lineage a specific domain of the chikungunya virus e2 protein regulates particle formation in human cells: implications for alphavirus vaccine design deliberate attenuation of chikungunya virus by adaptation to heparan sulfatedependent infectivity: a model for rational arboviral vaccine design host alternation of chikungunya virus increases fitness while restricting population diversity and adaptability to novel selective pressures an epitope of the semliki forest virus fusion protein exposed during virus-membrane fusion analysis of antibody response (igm, igg, igg3) to chikungunya virus using panel of peptides derived from envelope protein for serodiagnosis production and characterization of mouse monoclonal antibodies reactive to chikungunya envelope e2 glycoprotein characterisation of mouse monoclonal antibodies targeting linear epitopes on chikungunya virus e2 glycoprotein a neutralizing monoclonal antibody targeting the acid-sensitive region in chikungunya virus e2 protects from disease monoclonal antibodies that cross-react with the e1 glycoprotein of different alphavirus serogroups: characterization including passive protection in vivo sindbis virus conformational changes induced by a neutralizing anti-e1 monoclonal antibody characterization of epitopes for virus-neutralizing monoclonal antibodies to ross river virus e2 using phagedisplayed random peptide libraries analysis of murine b-cell epitopes on eastern equine encephalitis virus glycoprotein e2 a viruslike particle vaccine for epidemic chikungunya virus protects nonhuman primates against infection a dna vaccine against chikungunya virus is protective in mice and induces neutralizing antibodies in mice and nonhuman primates evaluation of recombinant e2 protein-based and whole-virus inactivated candidate vaccines against chikungunya virus effective chikungunya virus-like particle vaccine produced in insect cells a recombinant measles vaccine expressing chikungunya virus-like particles is strongly immunogenic and protects mice from lethal challenge with chikungunya virus dna vaccine initiates replication of live attenuated chikungunya virus in vitro and elicits protective immune response in mice we are grateful to laurent rénia for critical discussions of this study and to kai er eng from sign, a*star for manuscript editing. we wish to thank benoit delache and christophe joubert from cea for technical assistance, and marc grandadam for providing us with the virus isolate imt from la réunion. key: cord-001092-pkps5j8w authors: coleman, ryan g.; carchia, michael; sterling, teague; irwin, john j.; shoichet, brian k. title: ligand pose and orientational sampling in molecular docking date: 2013-10-01 journal: plos one doi: 10.1371/journal.pone.0075992 sha: doc_id: 1092 cord_uid: pkps5j8w molecular docking remains an important tool for structure-based screening to find new ligands and chemical probes. as docking ambitions grow to include new scoring function terms, and to address ever more targets, the reliability and extendability of the orientation sampling, and the throughput of the method, become pressing. here we explore sampling techniques that eliminate stochastic behavior in dock3.6, allowing us to optimize the method for regularly variable sampling of orientations. this also enabled a focused effort to optimize the code for efficiency, with a three-fold increase in the speed of the program. this, in turn, facilitated extensive testing of the method on the 102 targets, 22,805 ligands and 1,411,214 decoys of the directory of useful decoys enhanced (dud-e) benchmarking set, at multiple levels of sampling. encouragingly, we observe that as sampling increases from 50 to 500 to 2000 to 5000 to 20000 molecular orientations in the binding site (and so from about 1×10(10) to 4×10(10) to 1×10(11) to 2×10(11) to 5×10(11) mean atoms scored per target, since multiple conformations are sampled per orientation), the enrichment of ligands over decoys monotonically increases for most dud-e targets. meanwhile, including internal electrostatics in the evaluation ligand conformational energies, and restricting aromatic hydroxyls to low energy rotamers, further improved enrichment values. several of the strategies used here to improve the efficiency of the code are broadly applicable in the field. molecular docking is widely used to predict protein-ligand complexes [1, 2] and to screen large libraries for molecules that will modulate the activity of a biological receptor. though it suffers from well-known liabilities, it has predicted new ligands for over 50 targets in the last five years alone . in prospective, comparative studies with experimental high-throughput screening (hts), it has enriched hit-rates by over 1000-fold [58] . while hts has illuminated docking false negatives [56] ; docking has correspondingly illuminated false negatives from hts [3] . ever more frequently, docking predictions are tested by subsequent xray crystallographic structures, often confirming the predicted geometries of the docked complex [7, 14, [59] [60] [61] [62] [63] [64] [65] . notwithstanding these successes, docking retains crucial liabilities. as it is used to screen increasingly large compound libraries for new candidate ligands, the speed of the docking calculations has remained a goal for optimization. the need for efficient docking programs has become more pressing as the size of the accessible compound libraries has risen. whereas docking campaigns in the early 1990s addressed libraries like the fine chemical directory (mdl) of about 60,000 molecules, and the available chemicals directory of about 250,000 molecules in the early 2000s, the advent of zinc and related databases [66, 67] increased the number of purchasable molecules for screening to over 700,000 in 2005 and to almost 20,000,000 molecules of molecular mass less than 500 daltons today [68] . more crucial still is the need for sufficient sampling of ligand and protein states in docking, and of accurate evaluation of the binding energies of potential protein-ligand complexes. conformational space grows exponentially with ligand size, and sampling this space remains challenging. a key issue is whether docking is sampling sufficiently, and how increased sampling relates to improved scoring and outcomes. this includes sampling the internal degrees of freedom within the ligand as well as sampling ligand poses between the ligand and the protein receptor. several widely-used docking methods have been introduced to address these problems, and to exploit the opportunities that large compound libraries present for the discovery of new ligands. the program fred [69] exhaustively samples geometries defined by a regular latice, filters using pharmacophores, and then evaluates the remaining poses with an energy function. icm [70] uses multiple stochastic runs to sample poses to be scored with an energy function, while gold [71] uses a genetic algorithm to sample poses and includes a variety of scoring functions. glide sp [72] uses several levels of sampling and scoring, ending with a modified version of chemscore with ten scoring terms [73] , and glide xp [74] uses eighty parameters for scoring and is trained to reproduce binding affinity data for known complexes. autodock 4 [75] and autodock vina [76] are different versions of the same grid-based energy approach with a genetic algorithm to sample poses. the dock series of programs have typically focused on physics-based scoring functions with relatively few terms and sampling by graph-matching between ligand atoms and receptor ''hot-spots''-points of likely complementarity for a particular ligand atom. there are two main branches of dock, the dock 6.x [77] and dock 3.x families, of which the former has focused more on accurate prediction of ligand geometries and adopted a wider range of scoring functions. meanwhile, the dock 3.x programs have cleaved more tightly to physics-based scoring functions with fewer terms, and have focused on optimizing for the speed necessary to tackle large library screens. it is the latter program that has been most extensively tested by experiment for new ligand discovery, and is among the docking programs most thoroughly tested by direct comparison to prospective hts, and crystallographic confirmation, at least in the literature. dock3.5.54 managed a relatively rapid screening of chemical libraries by efficient sampling of possible orientations and by use of a flexibase [78] of pre-calculated ligand conformations [79, 80] . the former relied on an implementation of dock's traditional hotspot-based graph matching [81, 82] which focused the search for complementary ligand orientations to the protein likely to lead to favorable fits, while the latter eliminated the need to build ligand conformations on the fly, especially useful when docking the same ligand to multiple proteins as the time is saved for additional screens beyond the first. as we tried to optimize the program further, however, we found that the sampling of orientations behaved erratically as parameters were varied. when using histograms to limit the sampling, the orientations sampled were always a subset of what was possible at any given distance tolerance. changing the histogram parameters always returned different possible graph clique matches, but did not return subsets or supersets of the possible orientations made by other histogram parameters, leading to confusion when trying to explore and optimize orientational sampling. similarly we were concerned about the sampling of ligand conformations in the flexibase. the main issue was with the recombination of different conformations generated by omega [83] into new conformations, which had the potential to create internal steric clashes. these conformations were often present in dock3.5.54 [79, 80] library screens. a scheme to filter for conformations without internal clashes in dock3.6 [84] was not entirely satisfactory, as these strained geometries were still generated and the filters were not entirely successful, leading to effectively good scoring decoy conformations. additionally, problems with conformations in the flexibase to be docked, such as the sampling of aromatic hydroxyls out of plane, introduced further erors. here we explore new algorithms and engineering strategies to address these problems. we adapt an exhaustive graph-matching technique [85, 86 ] that ensures we sample all possible matching graph cliques. by graph cliques we mean superpositions of sets of ligand atoms on sets of receptor hot-spots ( figure 1a) . now, as we increase the amount of matches, ligand orientation sampling grows regularly, predictably and nonstochastically. this allows us to explore how, and if, increased ligand sampling leads to better docking performance, as judged by energies and enrichment of known ligands over matched decoys. this is crucial to understanding whether our core challenges in docking are sampling or scoring. we further explore whether physically improved calculations of ligand geometry, using an electrostatic term in the ligand conformation generation, as well as more realistic sampling of aromatic hydroxyls, leads to better docking performance. alert to the need for efficiency in a method that seeks to rank order the protein complementarity of 20,000,000 unrelated molecules, we also explored software engineering for efficient docking, which ultimately improved the raw speed of the method. what results is a docking method whose sampling increases regularly and predictably while retaining its physics-based energy calculation and speed: on common 2.66 ghz cores, it can reliably dock the 1,400,000 compound library used in dud-e [87] in as little as 1000 cpu hours. given the ready accessibility of multi-core clusters, this speed allows us to test dock stringently using different parameters, on the dud-e library of 102 diverse protein targets with a total of 22,805 ligands and 1,411,214 property-matched decoys. several of the methods here may find wide application. the new program is called dock3.7 [88] and incorporates updates to the dock source code, the flexibase generation program mol2db2 (an update of mol2 db [79, 80] ), blastermaster (an updated dock blaster [89] ) and other accessory scripts. dock remains available as a free download, with source for all our programs, for academics and nonprofit research institutions. a web-based implementation for those interested in using it for ligand discovery without investing in a local installation is also available [90] . our first goal was to make the sampling of ligand orientations in dock3.7 regular, non-stochastic, and smoothly variable. the dock programs have used a graph matching strategy since the program's first inception [81] , mapping ligand atoms onto receptor hot-spots, regions where ligand atoms are likely to bind. in this scheme, hot-spots and atoms are matched based on internal distances. in the dock3.x program series, an effort to speed and focus this matching had led to irregular sampling, which made performance hard to anticipate as variables were changed and made it impossible to smoothly increase orientation sampling, reproduce results or optimize docking performance. to overcome this problem, the algorithm was upgraded to use full graph matching instead of using histogram binning to reduce the number of orientational matches found [82] (figure 1 ). full distance matrices are now used in place of histograms that reduced the number of potential matches and, therefore, orientations. in this way, we use a single parameter to control how many orientational matches are desired, much like that used in dock4.0 [86, 91] . we do not specify a minimum internal distance parameter; it made sense when entire small molecules were being matched, but the current dock architecture matches only rigid rings [78] [79] [80] , allowing the rest of the molecule encoded in the flexibase hierarchy to move with respect to that rigid ring positioned in the binding site. we use an adaptive system where a desired number of matches is specified, as well as a minimum, maximum and increment of the distance tolerance. the latter three parameters are unchanged throughout all these tests and chosen to start very low (0.05å ) and grow slowly with each iteration, allowing the first parameter of desired number of orientational matches to control the docking run. in this way, the orientational matches found with a match goal of 1000 orientations include all the matches found with a lesser match goal. increasing the sampling by changing this desired number of match goals parameter will always find all of the old poses as well as additional poses. correspondingly, increasing the match goal always maintains or improves the score of any docked molecule ( figure 2) . also, the orientations tested are always identical-the algorithm is deterministic and non-random at all parts of the sampling. we wanted to know how actual docking performancegeometry prediction and enrichment over decoys-varied with sampling; just because we can guarantee sampling has increased, does not translate to improved docking performance. though every docked molecule will find a better scoring pose with a higher level of sampling, a situation where decoys find better poses relative to the ligands would decrease the performance in terms of enrichment. for 13 pdb [92] structures of glutamate receptor ionotropic kainate 1 (grik1) (1vso, 2f34, 2f35, 2pbw, 2qs1, 2qs2, 2qs3, 2wky, 3c31, 3gba, 3gbb, 3s2v, 4dld), the ligands were extracted and compared to their docked poses based on the heavy atom root mean square deviation (rmsd) using the kuhn-munkres algorithm [77] to account for symmetry. with these 13 ligands, as sampling was increased from 50 through 20000 match goals, the mean rmsd went from 3.1 å to 2.7 å to 2.6 å to 2.9 å to 3.0å . by this criterion, although docking scores always improved with sampling, rmsd to crystallographic poses did not. however, when we quantified the correctness of the docked poses by defining a set of critical contact atoms, judged to be important in the ligand binding to the protein by being present in most, if not all, of the ligands, matters improved. three atoms; two carboxylate oxygens and an amide nitrogen, were chosen; the carboxylate atoms interact with arg95 and thr90, the amide nitrogen interacts with thr90, pro88 and glu190 of grik1 ( figure 3a ). for the docked dud-e ligands of grik1 the median critical contact rmsd drops from 3.9 å to 2.8 å to 2.2 å to 2.1 å to 1.9 å as orientational sampling increases from 50 to 20000. 86 of the 99 docked known ligands showed a decreased critical contact rmsd as orientational sampling increased ( figure 3 ). the dock3.x series of programs [79, 80, 84, 89, 93] has had as its major focus the screening of large compound libraries for new ligand discovery. to support this effort, and those of others, we have introduced benchmarking sets that measure the ability of a docking program to enrich known ligands from decoy molecules that are matched to the ligand in physical properties such as molecular weight, charge, and hydrophobicity, among other terms. perhaps the most ambitious of these sets is the dud-e collection of 102 targets, with 22,805 literature-annotated ligands (a mean of 223.6 per target), and 1,411,214 property-matched decoys [87] . to investigate the performance of the new matching method, we screened the full 102 dud-e targets against their corresponding ligands and decoys. in 77 of the 102 dud-e systems, increased sampling improves enrichment ( figure 4 ). this may be judged by the area under a receiver operator characteristic (roc) curve, or by enrichment of ligands over decoys at the 1% point of the docking ranked list (often referred to as the ef1 statistic), or, as we prefer, by an adjusted logauc [93] . adjusted logauc is the area under the roc curve, where the x-axis is logarithmic to favor early ligand enrichment, combining the strengths of the auc and ef1 metrics. the area under a random curve is subtracted from the logauc so that an adjusted logauc of zero represents random enrichment, while positive numbers represent enrichment of ligands over physically-matched decoys. as orientational sampling is increased from 50 to 500 to 2000 to 5000 to 20000 poses, the mean adjusted logauc for all dud-e systems rises from 13.1 to 14.7 to 16.0 to 16.6 to 17.4 (the same monotonic trends are apparent for linear auc and for ef1, tables s3 and s4). thus, the mean over 102 protein targets with more thorough sampling of ligand orientions leads to better docking performance, as judged by enrichment of many known ligands over many more property matched decoys. this was not something we could previously investigate in a regularly variable manner. admittedly, though mean performance over all systems improved with greater sampling, this was not always the case for every system; there were 25 targets where increased sampling had little effect on enrichment ( figures 4a and c) , or even reduced it ( figures 4b and d) . in cases where the effect of increased sampling on enrichment is negative, it is possible that 1) decoys are finding . dock score effects with varying degrees of orientational sampling. a) the crystal ligand from pdb code 1vso. the critical contacts are defined as 3 atoms from the ligand crystal structure making key polar contacts with the protein, highlighted with spheres. 4 poses of zinc00013260 are shown in b through e, with increasing sampling going from left to right, better dock scores and lower critical contact rmsd (with the exception of the critical contact rmsd rising from match goal of 50 to 500). protein is shown in gray, crystal ligand shown in purple and representative docked pose shown in green, with hydrogen bonds drawn according to ucsf chimera defaults. an additional molecule, zinc00374553, is similarly shown in subfigures f through i, with a similar trend of increasing dock energies and decreasing critical contact rmsd. doi:10.1371/journal.pone.0075992.g003 unrealistic poses, 2) the scoring function has not properly captured some aspect of the system, 3) some decoys may actually be ligands, or 4) a combination of these possibilities. one example is map kinase-activated protein kinase 2 (mapk2), where the enrichment gets worse with more orientational sampling ( figure 5c ). when examined, the ligands find better energies ( figure 5a ) and the poses get better, as judged by critical contact rmsd, but the decoys improve even more in energy than do the ligands. for any given target, the level of sampling should be carefully checked against enrichment and other indicators of success before prospective screening is undertaken. the focus on screening multi-million compound libraries has motivated rapid calculations in the dock3.x programs, but as ambitions increasingly turn to docking across families of proteins or even the proteome [89] the need for further optimization remains pressing. whereas previously we have investigated methods to optimize efficiency in sampling orientations [82] or conformations [79, 80, 94] or in their scoring [95] , here we investigated compiler-level optimization and code efficiencies to increase raw speed. whereas this may seem inelegant, it has the virtue of being applicable to most other programs. we investigated systematic optimization of compiler flags for the code. we found that four flags led to measurable increases in performance, with several increasing speed by between 2% and 26% (table 1) ; overall, compiler optimization improved speed by 41%. a second major improvement came from optimizing energy scoring grids. these grids pre-compute receptor energy potential functions, such as van der waals and poisson-boltzmann-derived electrostatic potentials. these grid potentials, combined with an atomic property like atom type or charge, are multipled into energies. in this way, each atom is scored for each of the energy functions desired. to look up grid potentials for ligand atoms that do not fall precisely on a lattice point, trilinear interpolation is used between neighboring points for atoms that fall in-between lattice points, as most do. such interpolation at run time turns out to be costly, but we found that we could pre-compute large portions of the calculation before docking began, saving substantial calculation time when scoring, at the cost of memory usage. further time was saved aligning continuously in memory all 8 precomputed values associated with every trilinear interpolation cube for the cache controller. for instance, with the optimized interpolation we can look up over 1 million atom grid scores per second in the grik1 system, whereas before optimization only 300,000 grid lookups per second were performed ( table 2 ). as this step was rate determining for many systems, it contributed substantially to the 2.5 to 3-fold overall speed-up realized for the optimized code over the dock3.5.54 code (table 2) . because most docking programs [72, 75, 76, 81, 85, [95] [96] [97] [98] [99] use a grid-based approach for at least initial scoring, such a stratagem may be widely applicable. what results is a program that is suitable for large-scale library screens. as described above, ligand enrichment against decoys across the dud-e systems already achieves an adjusted logauc of 13, or an ef1 of 11.6, at a match goal of 50 orientations. at an orientation goal of 500, the adjusted logauc and ef1 values improve to 14.8 and 12.8, respectively, and further rises as matches rise further. a point of diminishing returns is eventually reached, undoubtedly because the higher orientation numbers are achieved by lower stringency matching of atoms to receptor hotspots, leading to poorer correspondence between the ligand atoms and the receptor hot-spots. if we take 500 ligand orientations as a sensible level of sampling, screening 1.5 million molecules in the lead-like available-now set in zinc [68] would require about 2700 hours on a single core (about one ligand every 5 seconds). if this remains a substantial investment, it is less than two days on a small cluster of 100 cores, and an afternoon on a cluster of 1000 cores, a size that is increasingly common. if one wanted to take on a larger number of systems, it is a simple matter to reduce the orientation sampling goal for the program-a goal of 50 orientations, which still performs relatively well against the dud-e set, would only demand 1.5 hours to screen 6.6 million lead-like compounds against a single representative target for a 1000 core cluster. as an aside, we note that calculation time did not scale linearly with the orientation goal. thus, for the 50/500/2000/5000/ 20000 matches, the mean calculation time over the 102 dud-e targets was 15.9/34.7/104.0/243.7/810.3 core hours (table s2 ). this likely reflects the greater likelihood of finding productive poses using higher-stringency of the lower matching goal; the more productive poses that are calculated, the more time that must be spent in scoring them, as clashing poses are quickly discarded. mean times across all match goals across all systems in dud-e are shown figure 6 , with the three different performance metrics also shown. if the challenges of efficient sampling of conformations and orientations are widely appreciated in molecular docking, those of ensuring that ligand conformations are low energy are sometimes overlooked. they are, however, important, as high energy conformations are essentially decoys that can obscure the presence of more favorable poses. among the features that can contribute to such high-energy ligand conformations is the neglect of electrostatic energies in ligand conformations, which are often ignored owing to concerns about overweighting the term in an effectively low dielectric calculation [100] (figure 7) . indeed, conformations that neglected electrostatics have been previously used for all molecules in the zinc [101, 102] dud [103] and dud-e [87] ligands databases (since in the dock3.x programs ligand conformations are pre-calculated and docked as a flexibase, relatively expensive ligand calculations are affordable). to explore the effect of the electrostatic term on docking, we re-built all ligands and decoys for all dud-e systems with both the mmff94s [104] forcefield in omega [83, 105] and with the mmff94s_noestat forcefield, where electrostatics are turned off. to our knowledge, this is the first time this has been attempted on such a large scale (over 1.4 million ligands and decoys), and the first time it has been judged by docking all the resulting conformations and computing enrichments. with electrostatics turned on for compound conformation generation, enrichment of ligands over decoys improved in 60% of dud-e targets (62 out of 102), shown in figure 8e . however, in the 40% of targets where enrichment diminished, it diminished further than it had increased in the 60% of the systems where it improved. the mean enrichment over dud-e was essentially zero ( figure 8e , green bars). on inspection, many of the systems where performance declined on adding electrostatics to the ligand conformational energy calculation had charged ligands leading to relatively high-magnitude contributions to the conformational energy from the electrostatic term. this is important because we only calculate conformations within a certain energy window, which is arbitrarily set [100] , but as the absolute magnitude of the energies of the conformations rises, so will the difference between them, and so too should the energy window (figure 7) . these effects were explored in a subset of 17 dud-e targets (ace, aces, andr, comt, dpp4, dyr, fa10, fa7, fabp4, fpps, grik1, pyrd, thrb, try1, tryb1, urok, xiap). increasing the energy window from 12.5 to 15 kcal/mol for conformations built with internal electrostatics increased enrichment by a mean of 1.0 logauc units. increasing the energy window further still, to 30 kcal/mol, improved mean logauc by 3 units. overall, of these 17 dud-e targets, 12 were rescued by the increase in the energy window during ligand the ligand building procedure ( figure 8e, black bars) . taken together, these results support the use of ligand internal electrostatics in conformation calculation, which has, moreover, the added benefit of being physically more realistic. a related challenge emerged in the placement of ligand aromatic hydroxyls. in previous versions of dock3.x [79, 80, 93, 106] and zinc [101, 102] , aromatic hydroxyl protons were usually placed in high-energy, incorrect conformations, out of the plane of the ring. this is inconsistent with high-resolution small molecule structures in the cambridge structural database [107] , which reveals a strong preference for aromatic hydroxyls to be in the ring plane ( figure 9 ). to investigate the impact of such hydroxyl sampling on docking enrichment, 29 dud-e targets that had a substantial number of hydroxyl-bearing ligands were investigated (ada, adrb1, adrb2, andr, bace1, braf, comt, def, drd3, esr1, esr2, fpps, gcr, glcm, gria2, grik1, hivint, hivpr, hmdh, hs90a, inha, kith, mk01, nram, pnph, pur2, sahh, thb, wee1). ligands were built using the mmff94s force-field with hydroxyls placed as in the earlier version, or with a new method that only places them in the ring plane. the latter improved the adjusted logauc by 0.78 to 0.88 across the 29 dud-e targets, depending on number of orientations sampled; five systems showed much higher improvement ( figure 10 ). in these systems, improvement typically reflected better interactions made by the in-plane ligand hydroxyl than could be made by its out-of-plane counterpart. several other changes made the docking output more extendable and widely usable. full atom type and bond information is now encoded in the ''flexibase'' (i.e., the precalculated molecular conformations in the library). previously, to save disk space, the molecular library had been represented in a format that sacrificed ligand topology, among other features, for a highly compressed format that could support the millions of molecules and billions of conformations in a typical lead-like or drug-like docking library from zinc [79, 80] . with the increased disk capacity of modern computer systems this is no longer necessary. by storing full atom and bond information, the flexibase may now be directly understood and interrogated, and the docked output has lost none of the information of the original mol2 file and may be readily converted to other formats [108] . correspondingly, fields have been added to the output to support anticipated new directions in scoring and docking information, such as weighting ligands by internal energies that we are now beginning to calculate. additionally, the scoring breakdown for each atom and for each scoring term can be written to the output file, enabling further analysis and visualization. finally, multiple topscoring poses per molecule may now be written out, not only the very top-scoring pose as was previously the case. this will often enable a much richer investigation of the docking results, and the consideration of ensemble energies; it is limited only by memory and disk space. a bump filter using simple distance cutoffs embedded into a grid has been a part of the dock line of programs for a long time. the bump grid itself was removed in this version and replaced by a calculation of the van der waals energy. if the repulsive term for any portion of the ligand is very high, it is certain that further exploring that orientation and/or conformation is unlikely to yield a good energy score. even if it does yield an overall total good score due to a very favorable electrostatic interaction caused by placing oppositely charged atoms near each other, it is not a physically realistic pose. we ran each dud-e target with 500 orientational samples through a bump limit of +10 kcal/mol, 20, 50 or no limit (not using the repulsive filter). favorable energies are negative. the mean difference in logauc was 0.3; only 8 dud-e targets had a logauc difference higher than one. of these, one extreme example of a logauc change is shown in figure 11 . in these targets that are sensitive to the bump limit, there was a charged ligand that needed to be placed slightly inside the repulsive radius of the protein atom to obtain a favorable electrostatic and overall energy. however, the time is essentially unaffected by the bump limit, showing only a small improvement with lower limits ( figure 11c) . importantly, the speed of the docking procedure is roughly doubled simply from using a bump limit at all. for all other tests in this study a bump limit of 50 is used, though the default could likely be as low as ten for many systems. a further analysis of the thyroid hormone receptor beta-1 (thb) target shown in figure 11 was done on poses of the ligands found. importantly, many poses of the known ligands were not found with the low bump limits, only 26 of the possible 103 ligands had a pose identified at all. as the bump limit was raised to 20 kcal/mol, 50 or no limit this number rose to 41, 67 and finally 102. again, this is the extreme case, as the other 94 dud-e targets did not show this dependence. three observations emerge from this work that may be broadly useful to other investigators in the field. first, once we regularize our sampling of orientations, it becomes apparent that increased energies for ligands and decoys for that target. b & d) protein farnesyltransferase/geranylgeranyltransferase type i alpha subunit (fnta), where the logauc does not change significantly with increased orientational sampling, again the difference between the ligand and decoy energies is shown above in the upper right. e), grik1 is shown, where the logauc goes up with increased orientational sampling. doi:10.1371/journal.pone.0075992.g005 sampling of poses improves docking performance. this is supported both by enrichment of ligands over decoys and by capturing canonical interactions. second, this algorithmic change was accompanied by code optimization-using techniques that may be widely applicable-that increased the speed of the program three-fold; this will be important as the field takes on ever-more ambitious library screening campaigns. third, improving our physical treatment of ligand conformations-including internal electrostatics energies when building them, and insisting on low-energy rotamers for aromatic hydroxyl hydrogens-not only improved overall performance, but did so in a way that establishes a foundation for further development of physics-based scoring in molecular docking. a longstanding concern in not only ligand docking but in other modeling techniques, such as protein comparative modeling, is whether increased sampling will necessarily improve performance. after all, if the scoring functions have serious liabilities it could well be that increased sampling will simply exploit these, leading to better scoring but worse enrichment, owing to decoy molecules scoring even better than the true ligands. broadly this was not what we observed: despite the well-known liabilities in docking scoring functions, in three quarters of the 102 dud-e targets enrichment rose with greater sampling. this suggests that the scoring function, with all of its gaps, approximations, and errors, is capturing important aspects of ligand recognition, even in the face of a benchmarking set where every ligand is matched with 50 property-matched decoys. admittedly, performance remains far from perfect: in 23 dud-e targets enrichment did not rise with greater orientation sampling, and in almost all of the 102 targets improvements in enrichment plateaued after about 2000 orientations had been sampled. this reflects the limitations on our current scoring function. a key to exploring the variation of enrichment with sampling was a code base optimized for speed. whereas it may seem easy to optimize compiler flags and grid interpolation, as opposed to fundamental algorithmic efforts, the result is a program that is three-fold faster. this not only enabled extensive testing on a large and challenging benchmark, the dud-e set, but also ambitious prospective campaigns against multiple targets. for instance, a longstanding goal in chemical biology has been to screen the world's chemistry against all of the pharmacologically relevant targets. this remains infeasible for empirical screening, but it is feasible, even today, for docking. to dock the 2 million zinc lead-like available-now compounds against the 3000 or so pharmacologically interesting targets for which structures are available, with 50 orientations sampled per ligand, would demand about seven months for a 1000 core cluster, which is no longer considered a large cluster in the field. if it's true that the limited improvement of ligand enrichment with increasing sampling reflects limitations in our current scoring, the fact that it does improve monotonically, in most systems, supports the idea that there is room to build upon the current physics-based scoring. whereas scoring optimization was not the major focus of this work, building ligands with internal electrostatics, and with low-energy aromatic hydroxyl rotamers, ultimately improved enrichment, supporting this idea. perhaps more important still, including electrostatics and, more broadly, ligand internal energies [109] , provides a foundation for including crucial physical terms now missing from the scoring function. exploring such terms is enabled by the method developments and optimization described here. given that the main purpose of dock 3.7 will be prospective screening of fragment [110] and lead-like [111] subsets of zinc, the problems with electrostatics in screening drug-like [112] ligands can be considered less important. most of the problems appear isolated to very large, peptide-like and drug-like ligands, not usually attempted during prospective virtual screening campaigns. when targets with ligands in the lead-like or fragment-like range were examined, no problems with building ligands with the full mmff94s energy function were encountered. this work has explored several ligand sampling parameters used in docking across the 102 dud-e test targets [87] . dock3.7 served as an ideal platform for these tests, as many sources of error were removed, allowing ''apples to apples'' tests. additional orientation sampling, while expensive, improves most dud-e systems, and prospective users of docking software should sample as much as they can reasonably afford given the resources available. using a bump limit based on van der waals score to prune out bad, repulsive conformations can double the speed of the docking procedure, similar to the ideas of dead-end elimination or a* searching from computer science [113] . these lessons will guide future users of dock3.7 but could be applied to all docking software and systems. in building ligands, we concluded that careful attention should be paid to hydroxyl positions. even though they may seem small, they can matter a great deal for some targets in terms of enrichment. the final lesson for building ligands is that the full mmff94s force field can and should be used, as it improves most systems. these lessons for building ligands will be applied in future releases of zinc [102] , but are also relevant for anyone building large collections of ligands for future docking. since the steps of ligand library generation and docking are separated in the docking pipeline presented here, it could also be used to test alternate methods for building ligands, including replacements for omega [83, 105] like dg-ammos [114] or frog2 [115] . other procedures for computing partial charges of ligand atoms and ligand desolvation terms besides amsol [116] , like qequil [117] , pdb2pqr [118] or am1bcc [119] , could be tested. also, since building the ligands for these systems is a timeconsuming process, once built many docking parameters and code changes can be explored and examined on a fast basis. the code can dock all ligands and decoys to their dud-e targets in as few as 11 hours, but longer and more extensive tests take thousands of hours. on a cluster of 500 computers, a full set of tests at high levels of orientational sampling take as little as 8 hours; as computers get faster and clusters grow, this time will only come down. one expansive area of future research is to incorporate changes to the dock scoring function presented here. since all ligands and decoys are pre-built, changes to the scoring function are very fast to test. dock3.7 has many features to make further energy function modifications easier. these are primarily 1) many output poses can be saved, though this uses additional disk space 2) atomic breakdowns of each part of the overall score are saved in the output file and 3) integration of the output file in mol2 format with the viewdock module in ucsf chimera [120] . the 102 dud-e targets were prepared here in a completely automatic fashion from pdb codes [89] , and the automatic docking procedure was improved and will also be an area of future improvement. testing and using molecular docking systems remains an important area of future research, and using a very fast system allows full parameter explorations and guides future large database builds and prospective screens. target preparation for docking proceeded in a new protocol derivative of the dock blaster pipeline [89] . the be-blasti routine of dock blaster was used to download the pdb codes for each dud-e target [87] , the list of ligands, cofactors and ions was modified to correctly account for all targets in dud-e without intervention. previous methods for protonation of sidechains have been replaced with reduce [121] as it was the most adaptable program, capable of protonating only sidechains that are requested and to not move heavy atoms during the protonation procedure. chemgrid [122] is used to make the van der waals grid using an amber forcefield [123] for the receptor; solv-map [93] is used to calculate a ligand desolvation grid. proteinligand electrostatics is calculated using qnifft [124, 125] , a version of delphi [126] . qnifft improved performance on dud [103] slightly as compared to preparations with delphi, likely due to the increased default grid size of 193 from 179. sphgen and related programs are used to place the receptor spheres used in the matching routine [81] . the general pipeline for placing matching spheres is to use the crystallographic ligand heavy atoms as the first set of spheres and to add nearby spheres generated by sphgen until a set number is reached, for further details see [89] . cofactor parameters were taken from previous versions of dud [103] and dud-e [87] as is, except for the parameters for the comt cofactor s-adenosylmethionine, which were modified to properly protonate the sulfur moeity. all parts of the new protein target preparation script are written in python. there are many steps in preparing the many conformations of a ligand for flexible ligand docking to a receptor. many of the steps here were identical to the steps in the zinc processing pipeline [101, 102] . corina [127] is used to produce an initial 3d conformation from input smiles, amsol is used to compute partial charges and ligand desolvation terms [116] , and omega [83, 105] is used to enumerate multiple 3d conformations. the biggest changes are to the procedure for collecting flexible ligands for use during docking. previously, mol2db [79, 80] , an implementation of the flexibase concept [78] , was used to collect ligand conformations for docking. the flexibase concept, in short, uses a collection of conformations built around a single rigid component, often a ring, to represent ligand flexibility. in mol2db, each part of the molecule stemming from the rigid component was independent, which represented a speedup in terms of time, but many conformations found during with dock 3.5.54 [79, 80] were energetically unrealistic, often internally clashing. dock 3.6 [84, 93, 106] avoided this problem by implementing runtime clash checking to find a slightly worse pose, as judged by docking score, but one that did not have an internal clash. however, this procedure was limited to simple distance checks and often still produced bad poses. to improve this system, mol2db2 was written, with a new hierarchy format derived from the original flexibase concept [78] . the new hierarchy format tracks the input conformations, and only docks complete input conformations instead of ones that have been pieced together from different input conformations. in early testing on the directory of useful decoys (dud) [103] , we found that some targets needed additional input conformations represented in order to appropriately sample the target. for this reason, the new pipeline uses a lower rmsd cutoff in openeye omega [83, 105] (0.4 å now as opposed to 0.8å ) and more output conformations (2000 versus 600) than before. hydroxyls are reset and rotated inside mol2db2 to be in plane when connected to sp2 carbons or at 3 equiangular positions when connected to sp3 carbons. except for hydroxyl resets and rotations, all input conformations are stored for later scoring during docking. the rigid component is typically a ring structure that serves as the basis for which the other atoms in the ligand move relative to. heavy atoms in the rigid component are used for orientation matching during docking. as before, partial charges and ligand desolvation energies are calculated for only one input conformation per set of conformations for a given molecule. as opposed to the previous method [80] , mol2db2 preserves atom typing and bond information according to the tripos mol2 format (http://www.tripos.com/mol2/atom_types.html) for each ligand. the.db2 file format used as output from mol2db2 and input for dock3.7 is documented on the shoichet lab wiki: http://wiki. bkslab.org/index.php/mol2db2_format_2 including sample lines for reading/writing for various programming languages. this is also supplied as supplementary information s1 of this paper. the goal by distributing this format freely is to encourage others to write code that can read/write or modify the files for their own docking programs or other purposes. future versions of zinc [102] will contain and distribute pre-built db2 files for many purchasable ligands as well as active molecules from chembl [128] . code for dock 3.7[88] is based on dock 3.6 [84, 89, 93, 106] with extensive modifications. mechanically, the code has been rewritten in fortran95 with 2003 extensions. as dock 3.6 before it, dock 3.7 uses the libfgz c library to read and write gzipped files. many algorithmic details have been updated in this process. the new db2 file format is used as input to dock 3.7, preserving input conformations, atom typing and bond information. this allows valid mol2 files to be generated as output. the histogram binning steps of the matching algorithm [82] have been removed in favor of a complete matching algorithm in the style of dock4 [86] . orientations of the ligand into the receptor can be generated using a single parameter of distance tolerance-the difference between the distances between the matched pair of points. a more tolerant parameter leads to more matches (and therefore more orientations of the ligand in the receptor) but always includes the matches found with a lower distance threshold. ligand matching spheres are heavy atom positions of atoms in the rigid component. receptor matching spheres can be specified by any positions within the binding site. during automated docking preparation [89] , receptor spheres are placed at the crystallographic ligand heavy atom coordinates and at other nearby ''hotspots'' identified by sphgen [81] . as before, limits can be placed on how many distances must match before an orientation is generated. throughout this work, four was the minimum and maximum number necessary, requiring that all 6 distances specified by the four points at the corners of tetrahedra are within the distance tolerance. additionally, the ability to save multiple top poses of a ligand is now implemented, with almost no speed penalty up to 100 poses. above that disk access times can begin slow down the docking, but the option to save any number of top poses remains available. the top poses are kept using an insertion sort by score, which incurs a small speed and memory penalty. for speed and memory usage, grids can be trimmed to the bare minimum necessary for docking. all grids are allocated dynamically so that only the input files must be changed; code does not need to be recompiled to run different grid sizes. this strategy uses much less memory than using larger grids. each docking job completed here used less than 250 megabytes of memory. dock3.7 output consists of a file containing information about each small molecule as well a file containing the tripos mol2 (http://www.tripos.com/mol2/atom_types.html) conformation of each small molecule relative to the protein receptor. multiple poses and per-atom scoring breakdowns can also be included in this output file, at the cost of disk space and time required. ucsf chimera remains the preferred visualization tool to use with dock, with the built-in viewdock tool [120] , though visualization with pymol is also possible [129] and the mol2 output files are likely usable with other molecular graphics programs. though it may only be of special interest, some readers may find additional technical details of the optimizations made useful. the optimization techniques generally fell within four categories: 1. precomputation 2. attention to data structure layout to accommodate the underlying memory architecture of the computer 3. changes to the underlying operating system to better accommodate the needs of dock and 4. trial and error with various heuristic based compiler switches. one optimization was to precompute and store as much information about the trilinear interpolations as possible before docking is executed. these computations take less than a few seconds but come with a significant time savings later. the only cost is additional memory usage as eight times the memory is necessary. furthermore, special attention was paid to the memory layout and access patterns of this precomputed data so as to maximize availability in local cpu cache. this amounted to reorganizing the 3 dimensional grid of data and various access loops to maximize spatial and temporal locality. next, given the size of these 3 dimensional data structures, and the inevitable need to access data representing the periphery of the binding cleft, a sizable amount of execution time was discovered to be spent handling virtual memory page table exceptions. this was minimized by changing the translation lookaside buffer (tlb) page size within the linux kernel from 4 kb to 2 mb. lastly, we optimized the build procedure to include only compiler optimizations known to benefit execution time as not all compiler heuristics benefit every application. for those interested in the complete compiler optimizations we applied, they are: ''-cbyteswapio -mallocatable = 03 -tp px-64 -gopt -o3 -fastsse -minline -mipa = fast,inline:10,libinline,libopt,vestigial -munroll = c:8,m:4,n:8 -mfprelaxed -mvect = sse,assoc,altcode,short -mcache_align -msmartalloc = huge''. these were used with the portland group fortran compiler, as it produced the fastest compiled code [130] . despite the sophistication of the portland group v9.0.4 vectorizing compiler, development time was also invested inspecting the assembly code of critical loops and modifying the fortran code to ensure vector instruction invocation. for the purposes here, we examined the crystallographic ligand and found the critical contacts made with the protein. these few atoms are used with an rmsd algorithm where any element of the same type can match to compute a critical contact rmsd [131, 132] . this rmsd comparison uses the munkres-kuhn or hungarian algorithm, as used by dock6 to evaluate poses [77] . when comparing these critical contact atoms to the list of atoms contained in the docked ligands, some small number of ligands will not have these critical atoms so their rmsd is undefined. for this reason, medians are reported instead of means. this avoids the arbitrariness of bias induced from single proteinligand crystal structure rmsd; though there is still bias in this form of analysis, it should be ameliorated over many docked ligand poses. as before, all code for dock3.7 including the updated receptor preparation routines and mol2db2 is available for free for academic and non-profit use with complete source from the dock website [88] . commercial licenses remain available with a license fee. tools utilized during the ligand building procedure figure 11 . effects of changing the bump limit on docking performance and time. a & b) differences for varying the bump limit at a match goal of 500 are shown here for one dud-e target, thyroid hormone receptor beta-1 (thb). this is one of the few cases where ligands and even some decoys find scores with a higher bump limit than they do with a lower bump limit in kcal/mol. c) the timings for this run and the mean over all 102 dud-e targets is shown. the bump limit itself does not have a large effect on the time, but using a high bump limit instead of none roughly doubles the speed of docking. doi:10.1371/journal.pone.0075992.g011 which are not available for re-distribution must be acquired from the appropriate sources. dock3.7 documentation is available at https://sites.google.com/site/dock37wiki/ under the creative commons attribution-sharealike 3.0 unported license. the shoichet lab cluster of over 800 cpus was used for all processing. cpu times should be taken with a caveat as the cluster is heterogeneous, containing 32 bit and 64 bit nodes, with varying levels of processing speed. challenges and advances in computational docking: 2009 in review structure-based virtual screening for drug discovery: a problem-centric review comprehensive mechanistic analysis of hits from high-throughput and docking screens against b-lactamase docking study yields four novel inhibitors of the protooncogene pim-1 kinase a virtual screen for diverse ligands: discovery of selective g protein-coupled receptor antagonists discovery of novel human histamine h4 receptor ligands by large-scale structure-based virtual screening rescoring docking hit lists for model cavity sites: predictions and experimental testing discovery of novel agonists and antagonists of the free fatty acid receptor 1 (ffar1) using virtual screening receptor-based virtual ligand screening for the identification of novel cdc25 phosphatase inhibitors novel ppargamma agonists identified from a natural product library: a virtual screening, induced-fit docking and biological assay study structure-based tailoring of compound libraries for high-throughput screening: discovery of novel ephb4 kinase inhibitors discovery of novel chemotypes to a g-protein-coupled receptor through ligand-steered homology modeling and structure-based virtual screening discovery of novel alpha-glucosidase inhibitors based on the virtual screening with the homologymodeled protein structure predicting ligand binding affinity with alchemical free energy methods in a polar model binding site ligand discovery from a dopamine d3 receptor homology model and crystal structure structurebased discovery of a2a adenosine receptor ligands structure-based drug screening for gprotein-coupled receptors structure-based ligand discovery for the protein-protein interface of chemokine receptor cxcr4 structurebased discovery of prescription drugs that interact with the norepinephrine transporter high selectivity of the gamma-aminobutyric acid (gaba) transporter 2 (gat-2, slc6a13) revealed by structure-based approach crystal structure-based virtual screening for fragment-like ligands of the human histamine h1 receptor selective structure-based virtual screening for full and partial agonists of the beta2 adrenergic receptor discovery of diverse human dihydroorotate dehydrogenase inhibitors as immunosuppressive agents by structure-based virtual screening identification of anti-malarial compounds as novel antagonists to chemokine receptor cxcr4 in pancreatic cancer cells conformation guides molecular efficacy in docking screens of activated beta-2 adrenergic g protein coupled receptor structure-based discovery of beta2-adrenergic receptor ligands novel inhibitor discovery through virtual screening against multiple protein conformations generated via ligand-directed modeling: a maternal embryonic leucine zipper kinase example identification by virtual screening and in vitro testing of human dopa decarboxylase inhibitors structure-based virtual screening of src kinase inhibitors structure-guided fragment-based in silico drug design of dengue protease inhibitors virtual screening identification of novel severe acute respiratory syndrome 3c-like protease inhibitors and in vitro confirmation structure-based virtual screening and identification of a novel androgen receptor antagonist novel non-peptide beta-secretase inhibitors derived from structure-based virtual screening and bioassay identification of a sub-micromolar, nonpeptide inhibitor of beta-secretase with low neural cytotoxicity through in silico screening structure-based virtual screening approach to identify novel classes of cdc25b phosphatase inhibitors discovery of novel pim-1 kinase inhibitors by a hierarchical multistage virtual screening approach based on svm model, pharmacophore, and molecular docking identification of novel malarial cysteine protease inhibitors using structure-based virtual screening of a focused cysteine protease inhibitor library identification of novel adenosine a2a receptor antagonists by virtual screening one scaffold, three binding modes: novel and selective pteridine reductase 1 inhibitors derived from fragment hits discovered by virtual screening novel inhibitors of dengue virus methyltransferase: discovery by in vitro-driven virtual screening on a desktop computer grid discovery of novel fibroblast growth factor receptor 1 kinase inhibitors by structure-based virtual screening virtual screening identification of nonfolate compounds, including a cns drug, as antiparasitic agents inhibiting pteridine reductase combination of virtual screening and high throughput gene profiling for identification of novel liver x receptor modulators discovery of selective inhibitors against ebna1 via high throughput in silico virtual screening tyrosine kinase syk non-enzymatic inhibitors and potential anti-allergic drug-like compounds discovered by virtual and in vitro screening novel human mpges-1 inhibitors identified through structure-based virtual screening nf-oe#b inducing kinase (nik) inhibitors: identification of new scaffolds using virtual screening discovery of non-peptide inhibitors of plasmepsin ii by structure-based virtual screening identification of novel human dipeptidyl peptidase-iv inhibitors of natural origin (part ii): in silico prediction in antidiabetic extracts identification of novel inhibitors for a low molecular weight protein tyrosine phosphatase via virtual screening structure-based virtual screening approach to the discovery of novel ptpmt1 phosphatase inhibitors discovery of novel seca inhibitors of candidatus liberibacter asiaticus by structure based design an integrative in silico methodology for the identification of modulators of macrophage migration inhibitory factor (mif) tautomerase activity discovery of novel checkpoint kinase 1 inhibitors by virtual screening based on multiple crystal structures discovery of novel human acrosin inhibitors by virtual screening complementarity between a docking and a high-throughput screen in discovering new cruzain inhibitors structure-based discovery of antagonists of nuclear receptor lrh-1 molecular docking and high-throughput screening for novel inhibitors of protein tyrosine phosphatase-1b docking for fragment inhibitors of ampc b-lactamase comprehensive mechanistic analysis of hits from high-throughput and docking screens against beta-lactamase molecular docking and ligand specificity in fragment-based inhibitor discovery roles for ordered and bulk solvent in ligand recognition and docking in two related cavities probing molecular docking in a charged model binding site blind prediction of charged ligand binding affinities in a model binding site crystal structures of trna-guanine transglycosylase (tgt) in complex with novel and potent inhibitors unravel pronounced induced-fit adaptations and suggest dimer formation upon substrate binding zinc-a free database of commercially available compounds for virtual screening ligand-info, searching for similar small compounds using index profiles zinc: a free tool to discover chemistry for biology pose prediction and virtual screening accuracy docking and scoring with icm: the benchmarking results and strategies for improvement improved protein-ligand docking using gold glide: a new approach for rapid, accurate docking and scoring. 1. method and assessment of docking accuracy empirical scoring functions: i. the development of a fast empirical scoring function to estimate the binding affinity of ligands in receptor complexes extra precision glide:'ä â docking and scoring incorporating a model of hydrophobic enclosure for protein'àíligand complexes autodock4 and autodocktools4: automated docking with selective receptor flexibility autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading evaluation of dock 6 as a pose generation and database enrichment tool flexibases: a way to enhance the use of molecular docking methods flexible ligand docking using conformational ensembles hierarchical docking of databases of multiple ligand conformations a geometric approach to macromolecule-ligand interactions molecular docking using shape descriptors conformer generation with omega: learning from the dataset and the analysis of failures dock 4.0: search strategies for automated molecular docking of flexible molecule databases critical evaluation of search algorithms for automated molecular docking and database screening directory of useful decoys, enhanced (dud-e): better ligands and decoys for better benchmarking automated docking screens: a feasibility study dock 4.0: search strategies for automated molecular docking of flexible molecule databases the protein data bank rapid context-dependent ligand desolvation in molecular docking a model binding site for testing scoring functions in molecular docking automated docking with grid-based energy evaluation grid-based molecular footprint comparison method for docking and de novo design: application to hivgp41 icm-disco docking by global energy optimization with fully flexible side-chains detailed analysis of grid-based molecular docking: a case study of cdocker-a charmmbased md docking algorithm automated docking of flexible ligands: applications of autodock comparative performance assessment of the conformational model generators omega and catalyst:' a large-scale survey on the retrieval of protein-bound ligand conformations zinc -a free database of commercially available compounds for virtual screening zinc-a free tool to discover chemistry for biology benchmarking sets for molecular docking merck molecular force field. i. basis, form, scope, parameterization, and performance of mmff94 conformer generation with omega: algorithm and validation using high quality structures from the protein databank and cambridge structural database the cambridge structural database: a quarter of a million crystal structures and rising open babel: an open chemical toolbox contribution of conformer focusing to the uncertainty in predicting free energies for protein-ligand binding fragment-based lead discovery: leads by design the design of leadlike combinatorial libraries experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings a formal basis for the heuristic determination of minimum cost paths. systems science and cybernetics dg-ammos: a new tool to generate 3d conformation of small molecules using distance geometry and automated molecular mechanics optimization for in silico screening frog2: efficient 3d conformation ensemble generator for small compounds atomic charge parameters for the finite difference poisson-boltzmann method using electronegativity neutralization pdb2pqr: expanding and upgrading automated preparation of biomolecular structures for molecular simulations fast, efficient generation of high-quality atomic charges. am1-bcc model: ii. parameterization and validation ucsf chimera-a visualization system for exploratory research and analysis asparagine and glutamine: using hydrogen atom contacts in the choice of side-chain amide orientation automated docking with grid-based energy evaluation polyelectrolyte electrostatics: salt dependence, entropic, and enthalpic contributions to free energy in the nonlinear poisson-boltzmann model electrostatic contributions to heat capacity changes of dna-ligand binding protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons virtual computational chemistry laboratory, design and description chembl: a large-scale bioactivity database for drug discovery the pymol molecular graphics system pgi workstation 9 an extension of the munkres algorithm for the assignment problem to rectangular matrices algorithms for the assignment and transportation problems an experimental approach to mapping the binding surfaces of crystalline proteins we thank kim sharp for his contribution of the qnifft program to the docking pipeline and assistance in integration, and therese demers and pascal wassam for maintaining the lab cluster and installing software. we thank dahlia weiss, henry lin and joel karpiak for reading this manuscript, while suggestions on the input/output formats was provide by many shoichet laboratory members past and present. we are grateful to openeye scientific software (santa fe, n.m.) for licenses to omega and oechem. we thank molecular networks (germany) for corina, and the university of minnesota for amsol. key: cord-001748-7e8px4vx authors: nobach, daniel; bourg, manon; herzog, sibylle; lange-herbst, hildburg; encarnação, jorge a.; eickmann, markus; herden, christiane title: shedding of infectious borna disease virus-1 in living bicolored white-toothed shrews date: 2015-08-27 journal: plos one doi: 10.1371/journal.pone.0137018 sha: doc_id: 1748 cord_uid: 7e8px4vx background: many rna viruses arise from animal reservoirs, namely bats, rodents and insectivores but mechanisms of virus maintenance and transmission still need to be addressed. the bicolored white-toothed shrew (crocidura leucodon) has recently been identified as reservoir of the neurotropic borna disease virus 1 (bodv-1). principal findings: six out of eleven wild living bicoloured white-toothed shrews were trapped and revealed to be naturally infected with bodv-1. all shrews were monitored in captivity in a long-term study over a time period up to 600 days that differed between the individual shrews. interestingly, all six animals showed an asymptomatic course of infection despite virus shedding via various routes indicating a highly adapted host-pathogen interaction. infectious virus and viral rna were demonstrated in saliva, urine, skin swabs, lacrimal fluid and faeces, both during the first 8 weeks of the investigation period and for long time shedding after more than 250 days in captivity. conclusions: the various ways of shedding ensure successful virus maintenance in the reservoir population but also transmission to accidental hosts such as horses and sheep. naturally bodv-1-infected living shrews serve as excellent tool to unravel host and pathogen factors responsible for persistent viral co-existence in reservoir species while maintaining their physiological integrity despite high viral load in many organ systems. most of emerging viruses that are continuously detected belong to the rna viruses and are often zoonotic in nature with epidemic or epizootic potential in case of transmission to livestock or humans [1] [2] [3] . interestingly, approximately 50% of the highly pathogenic diseases caused by these agents affect the central nervous system [4] [5] [6] . emerging viruses and also viruses highly pathogenic for animal species often arise from animal reservoirs, namely bats, rodents and insectivores. thus, reliable animal models for the in vivo analysis of host-pathogen interactions in respective reservoir species and the mechanisms that drive crossing of species barriers are urgently needed. this could also allow characterization of transmission routes and maintenance in reservoir populations of these viruses. the order mononegavirales comprises non segmented negative stranded rna viruses with a considerable number of highly pathogenic viruses which reside inconspicuously in natural reservoirs, e.g. lyssaviruses, paramyxoviruses and henipaviruses in bats. in case of transmission to susceptible animals or humans they cause fatal disease [7, 8] . borna disease virus-1 (bodv-1) also belongs to the order mononegavirales and was classified within an own and currently growing family named bornaviridae. a new classification of this family with subdivision into 5 species has been proposed with the classical borna disease virus-1 as part of the species mammalian 1 bornavirus [9] . recently, a variegated squirrel-derived bornavirus (vsbv-1) was found in association with the death of three people indicating the zoonotic potential for this newly discovered bornavirus [10] . comparably to other reservoir-bound viruses of the order mononegavirales, bodv-1 infection can lead to a lethal neurological disorder in accidental hosts such as horses and sheep due to a severe immune mediated non purulent meningoencephalitis [11] . the strictly endemic course of borna disease with seasonal appearance in spring and early summer, the varying incidence between years with peaks every three to five years as well as the highly conserved viral genome pointed to a natural reservoir for bodv-1 already for a long time [12] . however, many studies in wild rodents did not reveal any signs of bodv-1 infection in these species [13] . first evidence of natural bodv-1 infection in small mammals was provided by the detection of bodv-1 antigen and rna in bicolored white-toothed shrews (crocidura leucodon) originating from an endemic area in switzerland [14, 15] . this was substantiated by a study based on a geographic information system analysis which connects the prevalence of borna disease and the distribution of c. leucodon [16] . recently, similar occurrence of bodv-1 infection in c. leucodon in endemic areas in bavaria and in saxony-anhalt [17, 18] further underlines the role of this shrew species as bodv-1 reservoir. overlapping feature of all bodv-1-infected shrewsregardless of their endemic origin-is the widespread virus distribution not only in the central nervous system (cns) but also in peripheral organs capable of shedding virus in secretions and excretions [15, 17, 18] . experimental bodv-1 infection of neonatal immune incompetent rats leads to a quite comparable mode of virus distribution [19] . in these animals, persistent infection is achieved by immune tolerance. obvious neurological signs are lacking but behavioural deficiencies have been noted. in contrast, adult lewis rats exhibit a severe neurological biphasic disease due to a non purulent meningoencephalitis closely resembling the accidental host situation. certain mice strains develop a fatal neurological disease only after intracerebral infection of newborns [20, 21] . thus, outcome of experimental bodv-1 infection in rodents such as mice and rats depend on the species and even the particular strain and, the age at time point of infection. the latter is most likely explainable by the status of the immune system. this leads to significant differences in virus-host interactions resulting in variable clinical outcome and fatality of disease, reaction pattern of the immune system, virus distribution and shedding. whether natural bodv-1 infection of c. leucodon may fit to any of the known experimental courses or even run a different and so far unknown way of infection remains unknown. thus, clinical outcome, routes of virus shedding including demonstration of infectivity was characterized in bodv-1-infected c. leucodon. this contributes to understand not only bodv-1 pathogenesis but also serve as in vivo model for the analysis of general mechanisms of viral coexistence of reservoir-bound neurotropic viruses in physiologically normal appearing hosts. to further characterize viral maintenance in reservoir species, bicolored white-toothed shrews were caught alive. trapping was performed at two sites in the administrative district of swabia (permission no. 55.1-8646-2/75), a known endemic area for bodv-1 infections and clinical apparent disease (borna disease) in horses. after trapping, shrews were transported separately and put in husbandry. the animals were kept isolated from each other in single cages. they were kept in adapted standard cages type 4 and with respect to the natural requirements they were fed with a mixture of chicken heart muscle, chicken liver and insects. after an adaption period of 4 weeks and the initial veterinary care the animals stayed in husbandry for breeding. with respect to the high vulnerability to stress of wild-born animals, a health monitoring was installed. once a day a visual examination of body condition, haired skin and behaviour was carried out and food intake was measured. once a week body mass was recorded. the animals were monitored for any sign of direct abnormal behaviour or any indirect evidence like altered food intake, altered skin care or loss of body weight. in case of suffering, an early humane endpoint scheme adopted from laboratory rodents could be applied including euthanasia by anaesthesia conducted with co 2 and decapitating. a postmortem examination scheme with evaluation of gross and histologic lesions could be performed to evaluate the cause of death. for comparative analysis, tissues from three naturally bodv-1-infected dead bicolored white-toothed shrews from pest control (#2001 and #5017, [17] and another animal #5072 from the same stable as #5017) were used. at trapping, infection status of the animals was unknown therefore high hygiene standards were applied to avoid accidental transmission. to detect naturally bodv-1-infected animals, first samples of skin surface were taken directly on first day in husbandry and screened for the presence of bodv-1 rna as described below. non-infected animals were sampled in the same way. in animals caught in 2013 (group 1: female #2, male #5, female #6), after an adaption phase of one month, samples of saliva, lacrimal fluid, skin surface, urine and excrements from the bodv-1-infected shrews were taken weekly over a period of 4 weeks as necessary veterinary care. initial veterinary care could be reduced in animals caught in 2014 (#9, #10, #12) and only an initial sampling was performed. as health monitoring, possibility of long lasting virus shedding after at least more than 250 days in the husbandry was investigated and infected shrews (group 2: female #2, male #9, male #10, male #12) were sampled again. quantitative amplification of bodv-1 rna was carried out by real time rt-pcr as described elsewhere [22] by using commercially available kits for rna extraction and real-time reverse transcription polymerase chain reaction (qiasymphony rna kit, onestep rt-pcr kit, qiagen). qualitative isolation of infectious virus was performed on rabbit embryonic brain cells (reb cells) according to herzog et al., 1980 [23] . briefly, cells were incubated with diluted samples from shrews no #2, #5, #6 and virus replication in reb cells was visualized by indirect immunofluorescence test [24] . viral rna was extracted from reb cells persistently infected with the isolated bodv-1 by using commercially available kits for rna-extraction (rneasy mini kit, qiagen) and was sequenced according to previous protocols [25] . the nucleotide sequences were submitted to genbank database. phylogenetic studies were performed as described elsewhere [25] using the phylogeny inference program package, phylip [26] . representative sequences of all five regional bodv-1 subclusters were obtained from genbank (group 1a: l27077, ay374524; group 1b: ay374551, ay374550; group 2: ay374521, ay374531; group 3: ay374519, ay374534; group 4: u04608, ay374522; borna disease virus-2 aj311524). firstly, seqboot program was used for testing stability of the trees by bootstrap resampling analysis of 100 replicates. secondly, genetic distances between each pair of sequences were calculated based on the kimura two-parameter model, transition/transversion ratio of 2, computed with the dnadist program. thirdly, using the neighbour-joining method of the neighbor program a phylogenetic tree was generated and printed out as a consensus tree by the consense program. finally, the phylogenetic tree was displayed using seaview [27] . immunohistochemistry (ihc) was carried out using the monoclonal anti-bodv-1 nucleoprotein (bodv-1-n) antibody bo18 as described elsewhere [28, 29] . in-situ hybridization (ish) to detect genomic rna and respective mrna sequences of the bodv-1-n gene was performed additionally applying established protocols [29] . all statistical analyses were performed using statistica 10 software package (statsoft, tulsa, oklahoma, usa). female shrew #3 was excluded from the statistical analyses of relative body mass trend as it was used for breeding during the observation period and could therefore show changes of body mass due to pregnancy. relative body mass trend was calculated by ratio of body mass of an individual at time point x to the body mass at day 1 of husbandry. normality of data was assessed using kolmogorov-smirnov and lilliefors tests. we used a kruskal-wallis test to assess significant differences in weekly body mass trend between individuals. we used mann-whitney-u test to test for significant differences relative in body mass trend between non-infected and infected shrews within the same week in husbandry. the criterion to accept statistical significance was p < 0.05. animal husbandry and health management were performed in accordance with the german law and were declared to the animal welfare officer of the university, additional ethical waiver of an ethical animal care and use committee was not required. prior to animals capture, capture protocol and gathering of animals were approved and permitted by the administrative district of swabia (permission no. 55.1-8646-2/75) for establishing an insectivore animal model. additional approval by an animal ethics committee for capture of wild animals was not required. capture of wild animals was performed by skilled veterinarians according to the "guidelines for the capture, handling and care of mammals as approved by the american society of mammalogists" of the animal care use committee [30] . animals were kept in an animal facility of the philipps-university in marburg, animal housing was licenced (az lrv fd 83.4.1-19c 20/21) by the administrative district of marburg-biedenkopf according to the law (animal welfare act = "tierschutzgesetz", §11) and to the guidelines of the veterinarian association for animal welfare (= "tierärztliche vereinigung für tierschutz e.v."). only noninvasive diagnostic sampling procedures during routine veterinary care were applied that did not need to be additionally approved by an animal ethics committee. rabbit embryonic brain cells were generated in the early 1990s by s. herzog and frozen until usage. generation of these primary cells was licenced (gi 23-1/89) by the administrative district of giessen. eleven bicolored white-toothed shrews were caught (4 females [#2, #3, #6, #8], 7 males [#1, #5, #7, #9, #10, #12, #13] ). an overview about the different shrews is given in s1 table. as animals were integrated into husbandry at different time points, the observation period varied between the animals. in totally six out of eleven shrews (female #2, male #5, female #6, male #9, male #10, male #12) natural bodv-1-infection was confirmed by detection of viral rna, in three out of eleven shrews (female #2, male #5, female #6) additionally by detection of infectious virus. two of the naturally infected shrews (male #5, female #6) died about 9 weeks after the start of the observation period (see below). the five other shrews did not exhibit any evidence for bodv-1-infection, neither infectious virus nor viral rna was detected at any time point investigated. during the whole observation period up to 600 days, activity and behaviour during day or night light regime and food intake did not differ between infected and noninfected animals. furthermore, there was no significant difference of relative body mass trend between infected and non-infected individuals (mann-whitney u-test, p > 0.06 for each comparison) (fig 1) . there was also no significant difference of relative body mass trend between different weeks in husbandry of non-infected animals (kruskal-wallis-test: h(10;42) = 4.3123; p = 0.9322) and between different weeks in husbandry of infected animals (kruskal-wallis-test: h(10;50) = 6,8237; p = 0.7420). body mass of the individual shrews are shown in s1 fig. six shrews (#2, #7, #9 , #10, #12, #13), both infected and non-infected ones, developed focal alopecia after 4 to 5 months. two animals of group 1 (#5, #6) were found dead without previous symptoms shortly after the initial health monitoring. post mortem examination revealed intestinal invagination as cause of death in one case and hepatitis and pneumonia without known etiology in the other case. infected animals caught in 2013 received an intensive initial health monitoring including shedding of the virus for 4 weeks. in these three naturally infected shrews (#2, #5, #6), viral rna was present in saliva, lacrimal fluid, skin swabs, urine and faeces as well as in the ground substrate from their lairs (table 1) . during the observation period viral rna was consistently present in swabs from saliva and skin, however detection varied in urine, lacrimal fluid and was solely sporadically possible in faeces. ct-values were lowest in samples of saliva. for the investigation of long lasting virus shedding bodv-1 infected animals were sampled again after at least more than 250 days in the husbandry. in these four naturally infected shrews(#2, #9, #10, #12), viral rna was present in swabs from saliva, lacrimal fluid, skin and urine, but was not detectable in faeces (table 2) . ct-values varied between different animals and between the samples but were lowest in saliva in two animals. furthermore, infectious virus was successfully isolated on reb cells from all of the bodv-1-positive shrews caught in 2013 (#2, #5, #6) in samples from saliva (#2, #6), skin/sebum (#2, #6) and urine (#5, #6) (fig 2) . viral rna of isolates from saliva of #2 and saliva of #6 was sequenced (genbank accession no. km349818, km 349819). in a 2150 nucleotide stretch (nt 17 to 2161 covering the n, x, p, half of m-protein-encoding regions) sequences of both isolates revealed a homology of 99% compared to a recent bodv-1-sequence (genbank accession no. kf275185) obtained from a shrew of the same endemic area and to an equine bodv-1-sequence from a horse housed in the same region (genbank accession no. kf275184) [17] . both isolates are part of the regional bodv-1 subcluster 1a. (fig 3) the results obtained from the living shrews were compared to the organ distribution of viral antigen and bodv-1-rna in three naturally bodv-1-infected c. leucodon from pest control (#2001 and #5017, [17] and another animal #5072 from the same stable as #5017) and in the two deceased shrews. detailed information about organ distribution is given in s3 fig. in all of these animals, mrna, genomic rna and/or viral antigen were detected in the nervous system and widespread in peripheral organs (e.g. epithelial cells of the parotid gland, lacrimal gland, sebaceous glands, bronchi, kidney tubules, esophagus and epidermal keratocytes) [17] (fig 4) . interestingly, viral antigen was also present in the uterus in one shrew. thus, detection of viral rna and infectious virus from secretions and excretions in the living shrews (saliva, lacrimal fluid, skin swabs, urine and faeces) fits well with the morphological demonstration of viral antigen and rna in the respective organ systems and further confirms successful viral replication in peripheral organs. beside virus shedding via secretions and excretions shedding of bodv-1 seems also to be possible via scaling of epidermal epithelial cells. reservoir-bound rna viruses reside typically inconspicuously in animal reservoirs such as bats, rodents and insectivores. however, transmission routes, host-pathogen interactions necessary for viral maintenance in the respective animal population and factors needed to cross the species barrier are still rudimentarily known. thus, reliable animal models are urgently needed. the order mononegavirales comprises many viruses with high zoonotic and pathogenic properties, e.g. filoviruses, henipaviruses, paramyxoviruses and lyssaviruses which reside in bat reservoirs [7, 31] . in their biological behaviour, bornaviruses, as known from the mammalian borna disease virus-1 (bodv-1), are unique [10, 32] , but in several aspects pretty comparable to other neurotropic mononegavirales. the recently found zoonotic variegated squirrel 1 bornavirus (vsbv-1) clearly differs in its homology to the classical mammalian bodv-1 but provides evidence for its zoonotic capacities [10] . as the current knowledge is sparse, it is not known if vsbv-1 share features with bodv-1 behaviour. however, detection of vsbv-1 in several organs including cns and peripheral organs like lung and kidney of the squirrel [10] also indicate a widespread virus distribution comparable to the bodv-1 infected bicolored white-toothed shrew. typically shrews rear up to four litters from march to september and winter resource shortage is the most important source for mortality [33] . trapping took place during summer and autumn, therefore caught shrews were likely born in the same year and the age at time of trapping could be estimated between 1 to 6 months. as most of the offspring settles locally [33] kinship between the individuals and joint rearing cannot be excluded. during trapping, infection status of the individuals was unknown. previous studies showed different infection prevalence of shrews that also differed between the trapping sites in the study. hilbe et al [14] found only infected shrews (100%), puorger et al. [15] detected 2/6 infected shrews (33%), bourg et al. [17] showed 1/1 infected shrews (100%) at one site und 1/ 19 infected shrews (5%) at the other site whereas dürrwald et al. [18] found an amount of 9/17 infected shrews (53%) at one site with a variance between the years from 25% to 100%. these differences can be due to the small number of animals in the respective population or represent the natural variation within the shrew population between sites and years. since examination of larger cohorts has not been carried out so far, the percentage of naturally infected shrews among the trapped animals could not be predicted in detail. six naturally infected shrews out of eleven shrews implies a percentage of 55% of infected shrews with variations between the sites and years from 50% (site a, year 2013 3/6, year 2014 1/2) to 66% (site b, year 2014 2/3). as all non-infected animals did not show any shedding during the whole observation period, transmission of the virus in the husbandry could be successfully prevented in captivity. current data from living shrews provide reliable evidence that natural bodv-1-infection in these animals is indeed clinically inconspicuous over a long time period as already previously assumed [15, 18] despite persistent infection with shedding of infectious virus via various sites. during the observation period of up to 600 days, only two naturally infected animals were lost due to an intestinal invagination in one case and hepatitis/pneumonia in the other case which did not seem to be directly related to bodv-1 infection. in the bronchial epithelium of the animal suffering from hepatitis/pneumonia only few cells harboured bodv-1 nucleoprotein, bodv-1 mrna and genomic rna without associated distribution to the pneumonia and in the liver only genomic rna was detected in very few cells. interestingly, shedding of viral rna was continuously present.as shrews were naturally infected before trapping, the time between the infection and first virus release remain unknown. however, low ct-values were found in samples taken at time points at least more than 4 to 8 weeks after infection and at time points at least more than 200 days after infection. this indicates a persistent bodv-1 infection as known from other animals [11, 13] with long lasting and continuous virus release. there was certain variability in the amount of viral rna, sites of shedding, between individual animals and for the time points of sampling. some of these variations can be due to variations in sample size, as gathering of samples had to be performed non-invasive on non-anaesthesized animals. however, several shrews exhibited lowest ct-values in saliva and lacrimal fluid regardless of time point of sampling. whether this might have a role for virus transmission, e.g. combating, needs to be further investigated. the simultaneous presence of viral antigen, viral mrna and genomic rna in cns and peripheral tissues points to many sites of viral replication thereby enhancing probability of successful virus transmission to other animals [17] . horizontal transmission of bodv-1 in shrews might be either achieved via direct contact with secretions or excretions or even via contaminated environment. since shrews are known to behave territorially, infection by infected saliva during combating for a habitat might also occur. vertical transmission of bodv-1 in shrews cannot be excluded as viral antigen has been detected in the uterus. however, the route of entry in the reservoir still remains unknown. offspring might already be infected early by their mothers due to the various sites of viral shedding even from the skin. the underlying viral mechanisms of maintenance in the reservoir are still incompletely understood but might include adjusted viral life cycle possibly with attenuated pathogenicity, differences in viral entry and circumvention of the antiviral host immune system [4, 34, 35] . the latter could be achieved best in specific situations of the host immune system. infection of animals in an immune-incompetent stage can lead to persistent, immune-tolerant virus infections, often associated with shedding of high doses of infectious virus and without any severe clinical signs and notable inflammatory lesions. to date it remains unknown whether disseminated bodv-1 infection of shrews is only possible when infected in an immune incompetent state as known for rats [19] . however, the clinical inconspicuous course could point to an immune tolerant infection and a highly adapted viral-host interaction. neonatally bodv-1 infected rats display no neurological signs but increased motor activity, learning deficits and subtle changes in social behaviour and memory [36, 37] . moreover, experimental bodv-1 infection of the prosimian tree shrew (tupaia glis) leads to a persistent infection and transient mild encephalitis, resulting in a disorder characterized primarily by hyperactivity and pronounced disturbances in social and breeding behavior rather than neurological signs [38] . in the neonatally bodv-1 infected rat the behavioral changes were attributed to lesions in the hippocampus and cerebellum and in the tree shrew to alterations of the limbic system. whether naturally bodv-1 infected shrews also display subtle deficits in learning, memory and/or social behavior, especially mating, needs to be addressed in further behavioral and breeding experiments. as known so far, c. leucodon did not exhibit any morphological changes in cerebellum, hippocampus or elsewhere in the brain as noted for the neonatally infected rat. however, any behavioral changes might contribute to higher contact frequency or increased aggressive and territorial behavior thereby facilitating viral transmission and maintenance in the reservoir. characteristics of the shrew population correspond well to the epidemiologic pattern of borna disease. the distribution of c. leucodon in bavaria and the prevalence of borna disease seem to be connected [16] . the yearly varying peaks of borna disease in accidental hosts and the decline of borna disease within the last decades could be related to population dynamics of the shrews between the years and the restriction of habitats indirectly caused by modern agriculture [18] . inbreeding and low dispersal distance of the offspring correlates with the limited distribution of bodv-1 within endemic territories [18] . moreover, the continuous secretion and excretion of infectious bodv-1 and the detection of viral rna in the lair substantiates the hypothesis that "infectious dust" is responsible for bodv-1 transmission to accidental hosts through the intranasal route as known for hantavirus infections [39] . in this scenario, the bodv-1 infection of horses and sheep might rather represent an accidental occasion. to date it still remains to be solved whether and which factors are responsible for successful crossing the species barrier. amount of infectious virus, virulence, immune status and age of reservoir and accidental host as well as their genetic makeup might function as essential co-factors. taken together, shedding of bodv-1 in the bicolored white-toothed shrew is achieved via various routes which enable successful viral maintenance in the reservoir population and even fatal transmission to susceptible accidental hosts such as horses and sheep. moreover, these animals serve as suitable model to investigate host and pathogen factors that enable persistent viral co-existence in apparently healthy carriers. public health threat of new, reemerging and neglected zoonoses in the industrialized world global trends in emerging infectious diseases emergence and re-emergence of viral disease of the central nervous system the evolving epidemiology of viral encephalitis emerging viral infections of the central nervous system bats: important reservoir hosts of emerging viruses bats host major mammalian paramyxoviruses taxonomic reorganization of the family bornaviridae a variegated squirrel bornavirus associated with fatal human encephalitis bornaviridae epidemiological pattern of classical borna disease and regional genetic clustering of borna disease virus point towards the existence of to-date unknown endemic reservoir host populations persistence of borna disease virus in naturally infected sheep shrews as reservoir hosts of borna disease virus distribution of borna disease virus antigen and rna in tissues of naturally infected bicolored white-toothed shrews, crocidura leucodon, supporting their role as reservoir host species landscape features and reservoir occurrence affecting the risk for equine infection with borna disease virus bicolored white-toothed shrews as reservoir for borna disease virus the bicolored white-toothed shrew crocidura leucodon is an indigenous host of mammalian borna disease virus replication of borna disease virus in rats: age dependent differences in tissue distribution borna disease virus-induced neurological disorder in mice: infection of neonates results in immunopathology tnf-overexpression in borna disease virus-infected mouse brains triggers inflammatory reaction and epileptic seizures reverse transcription real-time pcr assays for detection and quantification of borna disease virus in diseased hosts replication of borna disease virus in indirect immunofluorescence assay for intra vitam diagnosis of avian bornavirus infection in psittacine birds genetic clustering of borna disease virus natural animal isolates, laboratory and vaccine strains strongly reflects their regional geographical origin phylip (phylogeny inference package), version 3.6. distributed by the author seaview version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building distribution of borna disease virus in the brain of rats infected with an obesity-inducing virus strain restricted expression of borna disease virus glycoprotein in brains of experimentally infected lewis rats animal care use committee (1998) guidelines for the capture, handling and care of mammals as approved by the bats as a continuing source of emerging infections in humans reverse-genetic approaches to the study of borna disease inbreeding in the greater white-toothed shrew cell surface expression of 27c7 by neonatal rat olfactory ensheathing cells in situ and in vitro is independent of axonal contact two ways to survive infection: what resistance and tolerance can teach us about treating infectious diseases early and persistent abnormalities in rats with neonatally acquired borna disease virus infection borna disease virusinduced hippocampal dentate gyrus damage is associated with spatial learning and memory deficits behavior alterations in tree shrews (tupaia glis) induced by borna disease virus risk factors for human infection with puumala virus, southwestern germany we thank franz schmid (veterinäramt günzburg) for his helpful support and cooperation. h. lange-herbst is supported by the margarete ammon-stiftung. conceived and designed the experiments: dn mb sh hlh jae me ch. performed the experiments: dn mb sh hlh me. analyzed the data: dn mb sh hlh jae me ch. contributed reagents/materials/analysis tools: sh jae me ch. wrote the paper: dn mb sh hlh jae me ch. key: cord-001219-517gka4h authors: timpka, toomas; spreco, armin; gursky, elin; eriksson, olle; dahlström, örjan; strömgren, magnus; ekberg, joakim; pilemalm, sofie; karlsson, david; hinkula, jorma; holm, einar title: intentions to perform non-pharmaceutical protective behaviors during influenza outbreaks in sweden: a cross-sectional study following a mass vaccination campaign date: 2014-03-07 journal: plos one doi: 10.1371/journal.pone.0091060 sha: doc_id: 1219 cord_uid: 517gka4h failure to incorporate the beliefs and attitudes of the public into theoretical models of preparedness has been identified as a weakness in strategies to mitigate infectious disease outbreaks. we administered a cross-sectional telephone survey to a representative sample (n = 443) of the swedish adult population to examine whether self-reported intentions to improve personal hygiene and increase social distancing during influenza outbreaks could be explained by trust in official information, self-reported health (sf-8), sociodemographic factors, and determinants postulated in protection motivation theory, namely threat appraisal and coping appraisal. the interviewees were asked to make their appraisals for two scenarios: a) an influenza with low case fatality and mild lifestyle impact; b) severe influenza with high case fatality and serious disturbances of societal functions. every second respondent (50.0%) reported high trust in official information about influenza. the proportion that reported intentions to take deliberate actions to improve personal hygiene during outbreaks ranged between 45–85%, while less than 25% said that they intended to increase social distancing. multiple logistic regression models with coping appraisal as the explanatory factor most frequently contributing to the explanation of the variance in intentions showed strong discriminatory performance for staying home while not ill (mild outbreaks: area under the curve [auc] 0.85 (95% confidence interval 0.82;0.89), severe outbreaks auc 0.82 (95% ci 0.77;0.85)) and acceptable performance with regard to avoiding public transportation (auc 0.78 (0.74;0.82), auc 0.77 (0.72;0.82)), using handwash products (auc 0.70 (0.65;0.75), auc 0.76 (0.71;0.80)), and frequently washing hands (auc 0.71 (0.66;0.76), auc 0.75 (0.71;0.80)). we conclude that coping appraisal was the explanatory factor most frequently included in statistical models explaining self-reported intentions to carry out non-pharmaceutical health actions in the swedish outlined context, and that variations in threat appraisal played a smaller role in these models despite scientific uncertainties surrounding a recent mass vaccination campaign. although encouraging the public to undertake specific protective behaviors has proved useful in containing outbreaks of infectious disease [1] , more research has been called for examining the social, demographic, and cultural factors that influence these efforts [2] . this is particularly important to understanding people's hesitations to heed official advice, particularly in the absence of clear scientific evidence regarding the disease outbreak [3] . the as03-adjuvanted pandemrixh was the most commonly used vaccine in response to the influenza a(h1n1)pdm09 outbreak in europe [4] ; finland and sweden recommended this vaccine to their entire populations. in august 2010 reports of a possible association between exposure to the vaccine and occurrence of narcolepsy in children and adolescents emerged in both the latter countries, which led to a review of the vaccine by the european medicines agency (ema). subsequently, increased narcolepsy diagnoses associated with the start of the campaign have been confirmed [5] . in sweden, scientific uncertainty regarding the safety of this mass vaccination was both publicly discussed [6] and questioned by researchers [7] . beliefs that the interventions suggested are effective and safe [8] , that the illness has severe consequences [9] , and that there is a high likelihood of exposure [10] have been associated with compliance with behavioral recommendations. it has also been pointed out that behavioral research in epidemics should not only identify determinants of individual and population behavioral responses, but also clarify the mechanisms underpinning these [11] . protection motivation theory (pmt) [12, 13] posits that an intention to perform protective activities is determined by perceptions of threat and the ability to cope. in addition to intentions and preceptions, a recent review concluded that protective behavior needs to be investigated with regard to sociodemiograpic characteristics in order to identify the ''contagious'' effect and contextual nature of perceptions and mediating mechanisms [14] . for instance, coping appraisals are made in interaction with environmental resources, which vary in availability across population subgroups. protective behavior associated with influenza outbreaks has also been investigated with regard to general estimates of health status [15] , but few studies have used validated measures of self-rated health as a means for the sub categorization. at present, several such measures are available for use in population-based research [16] . to provide a snapshot of intended self-protective behaviors during a period when scientific uncertainty pervaded public discussions addressing infectious disease control, we carried out a cross-sectional telephone survey of a demographically representative sample of the swedish population. the specific aim was to examine to what extent self-reported intentions to improve personal hygiene and increase social distancing during influenza outbreaks can be explained by perceptions of threat and the ability to cope as outlined in pmt, self-reported assessments of health, trust in official information, and sociodemiographic factors. the study used a cross-sectional design to analyze associations between intended protective behaviors during influenza outbreaks and items in a theoretical model of explanatory factors [14, 17] . a random sample of 1,011 persons ranging between 20-90 years of age was drawn from the swedish national population register. a combined telephone and questionnaire survey was carried out during the first quarter of 2012. the study was conducted according to the world medical association's declaration of helsinki from 1964 regarding ethical principles for medical research involving human subjects, revised in 2008. potential study sample participants were informed about the study by letter via postal mail and invited to participate in a telephone survey on protective behaviors during influenza outbreaks. those agreeing to participate returned their consent in writing. all collected data were managed confidentially and analyzed anonymously. the study design was approved by the institutional (ethics) review board at umeå university (dnr 2011-314-31ö ). a hypothetical explanatory model was constructed to inform the analysis of the main research question; i.e. to what extent selfreported intentions to perform protective behaviors during influenza outbreaks can be explained by perceptions of threat and the ability to cope as outlined in the pmt, self-assessments of health status, trust in official information, and sociodemiographic factors. in this model, protective behaviors during outbreaks are restricted to two categories: increased personal hygiene (use of disinfectants and other handwash products; frequent washing of hands when having touched common objects, such as door knobs) and social distancing (staying home from work or school; avoiding use of public transportation). the intentions to carry out a protective behavior are assessed by asking whether the respondent would try to perform the behavior during a mild and severe influenza outbreak, respectively. both outbreak scenarios described personal risk of infection as high (i.e., 1 in 3 people infected). the mild influenza description details moderate health consequences (less than 1 in 1000 infected people dying) and a minor lifestyle impact (services mainly operating normally). the severe scenario describes serious health consequences (1 in 50-100 infected people dying) and services no longer being able to operate normally. the first set of explanatory factors concerned perceptions of threat and the ability to cope. based on the notion of subjective expected utility [18] , which postulates that people's choices are a product of assessments of probability and utility of options, healthrelated methodologies such as the pmt and the health belief model [19] have included formally quantified models of subjective health risk perceptions, i.e., as the likelihood of contracting a disease multiplied by disease severity. together with different types of cost-benefit valuations and self-efficacy expectations, these perceptions of risk are presumed to determine health-protective behaviors. in the present study, the collection and analysis of data on protection motivation in relation to influenza outbreaks are structured according to the pmt. this theory suggests that threat appraisal will generate an intention to act, while coping appraisal determines the type of action. threat appraisal is in this study characterized in its three dimensions [11, [20] [21] : -perceived relative risk of catching influenza; measured by one item assessing personal likelihood of infection, if no preventative action was taken, -anxiety about catching mild and severe influenza; measured by one item for each influenza type, and -perceived severity of the consequences of catching mild and severe influenza; measured by one item for each influenza type. coping appraisal is also represented in its three dimensions: -response efficacy; assessed by one item asking about protecting oneself from influenza by employing enhanced personal hygiene and one item asking about social distancing, -self-efficacy; measured by two items asking whether the respondent felt it is possible to carry out protective behaviors by social distancing and increased personal hygiene, respectively, and whether they were confident they could carry out these actions if they so desired [22] , and -response costs; defined as the estimated efforts needed to overcome perceived barriers on carrying out protective actions. for social distancing, this dimension was assessed by asking for 'work concerns', i.e. guilt and anxiety about not completing work. response costs for increased personal hygiene were assessed through items asking for concerns associated with acquiring adequate soaps and disinfectants (handwash products) and learning the correct techniques to use them. self-reported health assessments have in epidemiological studies been found to be valid indicators of health status as measured by prediction of future physician contacts and all-cause mortality [23] . in this study, self-reported health is measured by the sf-8 tm 24-hour recall questionnaire in order to examine associations with intentions to carry out protective behaviors. this general selfreported health instrument contains eight health-related questions that, in turn, can be summarized in two overall measures of physical and mental health: physical component summary (pcs) and mental component summary (mcs), respectively [24] . it is derived from the sf-36 for the purposes of yielding comparable scores for the 8 health dimensions and 2 summary measures of the sf-36 with minimal respondent burden. trust in government information during influenza outbreaks has in previous studies been found to be associated with greater self-efficacy and personal hygiene [25] . trust in official information was therefore included in the explanatory model, asking for agreement with a single statement about trust in government information during outbreaks. the sociodemiographic factors included in the model were marriage status, number of children living at home, formal education, employment status, and ethnicity. prior to the telephone call, the subjects were asked to complete a paper-based survey, querying for sociodemiographic data and data elements from the sf-8 tm . the remaining data were collected in the telephone interview. to catalyze their considerations about the research topics, each subject was presented with brief scenarios of mild and severe influenza outbreaks. interview data were derived from open statements, and the respondents were asked to score their agreement along a seven-point scale from 1 (strongly disagree) to 7 (strongly agree). the collection of data on perceptions associated with precautionary behaviors was structured in accordance with the pmt (text s1). to assess trust in official information in this study, the single statement ''for information during influenza outbreaks i do rely on government sources'' was used. we conducted a drop-out analysis based on the demographic variables available for the entire sample, i.e. gender, age and place of residence. all collected data were first subjected to descriptive statistics, i.e. mean, median and standard deviation for continuous data and frequency and proportions (%) for categorical data. the primary end points for the ensuing analyses were intentions to increase social distancing (staying home while not ill; avoid public transportation), and enhance personal hygiene (use of handwash; frequent washing of hands after touching common objects) during mild and severe influenza outbreaks, respectively. the theoretical model of potential explanatory factors was used as the basis for the analysis. for each endpoint, logistic regression analyses were applied using the items in the model as explanatory variables. these included trust in official information; variables corresponding to pmt items (the threat appraisal items of perceived personal risk, emotional response (worry), perceived severity; and the coping appraisal items of general response efficacy, self-efficacy, and response costs); variables representing the sf-8 summary items (pcs and mcs); and sociodemographic characteristics (age, gender, educational level, living with partner, living with child, and employment). when used as response variables, ordinal variables were dichotomized (agree/do not agree). to contrast expected perceptions against other perceptions, the variables were converted with the agreement scores in the expected extreme as one category. for threat appraisal, agreement scores in the low extreme were contrasted against other opinions, except for the estimates of the severity of the consequences of getting infected where the scores in the high extreme were contrasted against the other opinions. regarding coping appraisal, the personal hygiene scores in the high extreme were contrasted against other opinions for response efficacy and self-efficacy and in the low extreme for response costs. for social distancing, agreement scores in the low extreme were contrasted against other opinions for response efficacy and in the high extreme for self-efficacy and response costs. the area under the roc curve (auc) was used as model performance indicator and nagelkerke r 2 to estimate the determination level for each model. the limits for interpreting the auc (or c-statistic) were set to 0.90, 0.80, and 0.70, denoting very strong (outstanding), strong (excellent), and acceptable discriminatory performance, respectively [26] . all tests were twosided and p,0.05 was regarded as statistically significant. all calculations were done using spss version 18 or higher. two-hundred and fifty-four persons in the total population sample (n = 1,011) could not be reached by a telephone call. of the 757 persons reached, 443 provided a complete response, leading to a 59% response rate to the telephone survey and a 44% participation rate with regard to the total sample. the age category 65-90 years was slightly over-represented (54% response rate) among the study participants when compared to the total population sample (p = 0.039). however, the effect size of this difference in participation was small (cramer's v = 0.08). thus, while elderly individuals were overrepresented in our data, the impact of this deviation from the reference population was of a small magnitude. in terms of place of residence, those living in small labor market regions (with a total population of less than 100,000 inhabitants) exhibited the highest participation rates: 51%, compared to 41% in large regions (with a population greater than 1 000,000 inhabitants). the basic sociodemiographic characteristics of the final study participants are displayed in table 1 . the general level of health in the study population as measured by sf-8 scores was above the reference values for all items except for physical functioning and vitality (lower scores) and general health (equal scores) ( table 2 ). there was no statistically significant difference between men and women regarding the mean scores of any sf-8 item or summary component. every second respondent (50.0%) reported high or very high trust (scores 6-7) in information about influenza provided by official sources (mean score 5.3; median 5.5; standard deviation (sd) 1.7)). neither age, education, employment nor any component of self-rated health was associated with trust in official information about influenza. however, the level of trust was associated with gender, with men reporting lower trust levels than women (p = 0.018; odds ratio (or) 0.60 (95% confidence interval (ci) 0.40;0.91)). regarding social distancing measures, 9% of the respondents scored strong (strong or very strong) agreement with the stated intention to stay home when not ill during mild influenza outbreaks, and 11% of the respondents scored strong agreement with this intent during severe outbreaks. more than twice as many respondents (23%) scored strong agreement with avoiding use of public transportation during a mild outbreak, while 29% of the respondents scored strong agreement with this intention during a severe outbreak. regarding measures related to personal hygiene, 77% of the respondents scored strong agreement with the stated intention to use handwash products during mild outbreaks, while 85% of the respondents scored strong agreement with this intention during severe outbreaks. regarding the intention to frequently engage in handwashing, 46% reported strong agreement in association to mild influenza outbreaks and 60% in association to severe outbreaks. a model describing the intention to stay home without being ill during a mild influenza outbreak included eight significant variables and displayed a strong discriminative performance (auc 0.85 (95% ci 0.82;0.89)) ( table 3 ). this self-reported intention was strongly associated with coping appraisal; low perceived response costs associated with staying home and selfefficacy with regard to social distancing; and, interestingly, to a disbelief in the general efficacy of social distancing as an infectious disease control measure. planning to stay home was also strongly associated with male gender and, with a weaker association, to being unemployed and living with a partner. the intention was also associated with threat appraisal, although with a weaker strength; with worry about getting infected and high perceived severity of the influenza threat. in comparison, the intention to stay home without being ill during a severe outbreak was represented by a model including only four significant variables, but that also displayed a strong discriminative performance (auc 0.82 ((95% ci 0.77;0.85)). as for the mild outbreak scenario, this intention was strongly associated with coping appraisal; to response costs and perceived self-efficacy with regard to social distancing. however, staying home during a severe outbreak was also strongly associated with threat appraisal related to concerns about getting infected. regarding sociodemographic factors, this intention was only associated with not having employment. the intention to avoid using public transportation during a mild influenza outbreak was represented by a model including six significant variables and an acceptable discriminative performance (auc 0.78 (95% ci 0.74;0.82)) ( table 4 ). this self-reported intention was, also, strongly associated with coping appraisal; to perceived response costs associated with avoiding public transportation and to self-efficacy with regard to social distancing. the intention was also strongly associated with threat appraisal in terms of worry about getting infected. in addition, avoiding use of public transportation was associated with a lower level of formal education, living with a partner, and high trust in official information. in contrast, the intention to avoid public transportation during a severe influenza outbreak was described by a model including four significant variables and an acceptable discriminative performance (auc 0.77 (95% ci 0.72;0.82)). as for the mild outbreak scenario, avoiding public transportation during severe outbreaks was strongly associated with coping appraisal; to response costs; and, with weaker strength, to perceived self-efficacy with regard to personal social distancing. with regard to threat appraisal, avoiding public transportation during a severe outbreak was associated with worry about getting infected and a high perceived severity of the influenza threat. planning to use handwash products during a mild influenza outbreak was described by a model including three significant variables and an acceptable discriminative performance (auc 0.70 (95% ci 0.65;0.75)) ( table 5 ). planning to use handwash was strongly associated with female gender. this intention was, for mild outbreaks, also explained by self-efficacy with regard to personal hygiene and trust in official information. for the severe outbreak scenario, planning to use handwash products was represented by a model including four significant variables and an acceptable discriminative performance (auc 0.76 (95% ci 0.71;0.80)). this intention was, too, strongly associated with female gender. in addition, it was strongly associated with coping appraisal; to a belief in the general efficacy of increased personal hygiene; and low response costs associated with acquiring of suitable products. contrary to any of the other intended behaviors studied, the intention to use handwash products during severe outbreaks was associated with low self-rated physical health. an intention to frequently engage in handwashing after having touched common objects during a mild influenza outbreak was represented by a model including four significant variables and an acceptable discriminative performance (auc 0.71 (95% ci 0.66;0.76)) ( table 6 ). the intention was strongly associated with coping appraisal in terms of self-efficacy with regard to personal hygiene. it was also associated with female gender, higher age, and lower education. in comparison, planning to frequently wash hands during a severe outbreak was represented by a model including three significant variables and an acceptable discriminative performance (auc 0.75 (95% ci 0.71;0.80)). similar to the mild influenza scenario, it was strongly associated with coping appraisal in terms of a high self-efficacy with regard to personal hygiene. the intention was also associated with female gender and being born in the country. despite the fact that the safety of the mass vaccination during the a(h1n1)pdm09 outbreak had been questioned by national mass media in a campaign-like manner, two years after the outbreak every second respondent in a representative sample of the swedish adult population reported high trust in official information about influenza. while the proportion of persons reporting intentions to improve personal hygiene during influenza outbreaks ranged between 45-85%, the proportion reporting intentions to increase social distancing did not exceed 25%. this pattern can generally be explained by the notion that the initial behavioral changes during an influenza outbreak are more likely to resemble familiar reactions and well-known routines [27] , such as increasing personal hygiene, rather than changes that require deductive planning, such as increasing social distancing. the explanatory models developed in this study showed statistical associations ranging from strong (staying home without being ill) to acceptable (avoiding public transportation and increasing personal hygiene). among the explanatory factors considered, coping appraisal was the factor most frequently showing associations (as displayed by odds ratios) with the reported intentions. in a validation analysis (data not shown), we fitted each model fully (including all terms in the five explanatory factors categories trust of information, threat appraisal, coping appraisal, sociodemographic factors, and self-rated health) and calculated the proportion of correctly classified cases for these full models for all eight scenarios. then we left the terms from one of the five categories out separately, and calculated the proportion of correctly classified cases for each of these subset models. we found that the proportion of correctly classified cases without coping appraisal was lower than the corresponding proportion for all full models and lower or equal to the corresponding proportion for 28 of the 32 models excluding one of the other four categories. we interpret these observations combined as indicative evidence that of the explanatory factors considered, coping appraisal was the factor strongest associated with the reported intentions. analogous to our results, a recent british web-based survey of university employees found that coping appraisal was the principal predictor of variability in protective intentions during pandemics [21] , and response costs have been reported as the largest predictor for emergency nurses not reporting to work during an influenza pandemic [28] . a contributing influence to the lesser relative importance of threat appraisal suggested by our results may be a scandinavian tendency to perceive risks lower than in other countries [29] [30] [31] . one of the explanations for this tendency is that the media in scandinavia appear to report more about risks abroad with less attention to risk inside the country [29] . in contrast to our results, self-efficacy during the a(h1n1)pdm09 outbreak in hong kong was found to be only weakly associated with social distancing [25] . however, hong kong residents are limited in their ability to avoid crowds, and the relatively mild impact of the outbreak could have led to the notion that people saw no reason to jeopardize their economic well-being and curtail other social activities. a socio-geographic theory of protective behaviors during infectious disease outbreaks suggested that efficacy beliefs of chinese living in the uk and the netherlands were comparable to those of native uk and dutch residents during the sars outbreak in 2003 [32] , indicating that country of residence is more important than ethnicity or country and culture of origin in determination of protective behaviors. however, with coordinated regional disease control efforts and increasing influence from social media, this may change. gender was the sociodemiographic characteristic that showed the strongest association with the observed variation in reported intentions. as also found in a norwegian study from the same time table 4 . simple and multiple logistic regression models of explanatory factors for the intention to avoid using public transport displayed by influenza outbreak scenario. period [33] , the swedish women in this study were more disposed to enhance their protective behaviors related to personal hygiene than were men. one explanation of this finding could be an interaction with concerns about the consequences of getting infected. a recent study from the u.s. reported that women were more worried than men about getting seriously ill or even dying during a severe influenza outbreak [34] . however, no gender differences with regard to threat appraisal were reported from the norwegian study [33] . originally, we did not include interaction terms in our statistical analyses. a secondary analysis (data not shown) did not reveal any statistically significant interaction between gender and any threat or coping appraisal item such that omitting the interaction from the model would disturb the estimation of the main effects. therefore, an alternative explanation of our findings is that the female respondents were more disposed to enhance their protective behaviors related to personal hygiene than the male respondents because swedish women purchase and use hygiene products more often than men [35] , and, in consequence, were more confident about the practical handling of handwash and liquid soap. conversely, men were more inclined to stay home without being ill during influenza outbreaks. this could be explained by the fact that fewer of the employed swedish men (12%) than women (46%) were at the time of the study working in caring or educational occupations that require physical presence at the workplace, such as nursing, child care, and teaching [36] . in other words, a larger proportion of men could consider the possibility of staying home while continuing to work during an ongoing influenza outbreak, which was not an option for many women. these findings indicate that more research is needed to understand gender-related differences in protective behavior during influenza outbreaks. the main strengths of this study are its foundation on a current theoretical model [14] and a relatively large representative sample of the swedish population. however, the study has also important limitations that must be taken into consideration when interpreting the results. the demographic characteristics available may not be the most important factors biasing the results. for instance, it is possible that individuals with low trust in official information about influenza were under-represented, and anxious individuals worrying about disease risks were over-represented, among the participants. moreover, interpreting cross-sectional data on protective behaviors is difficult because they confound the motivation and accuracy-associated aspects regarding the causaltemporal relationship between perception and behavior [37] . the motivational hypothesis assumes that high perceived risk leads people's intention to adopt protective behaviors, while the accuracy hypothesis suggests that people who act in a more risky way should also feel more at risk. as an example, individuals having physical contact with many people through their occupation may have been aware of that daily routines are associated with a higher risk for getting infected. accordingly, a negative correlation may indicate accurate relative risk perceptions, i.e. that people are aware of their risk status [20, 37] . further longitudinal studies of protective behaviors during influenza outbreaks are thereby warranted [38] . another limitation is that we assessed self-reported intentions rather than objectively measured behavior. nevertheless, intentions are a well-validated proxy for behavior predicting a moderate amount (30-42%) of the variance in actual behavior across a wide range of contexts [39, 40] . moreover, proponents of dual-process health behavior models have suggested that analytic central and emotional-heuristic processes work in concert to select decisions [14] , and under certain circumstances emotions may even be the dominant force [41] . while the pmt used in this study does include an emotional component, it still represents a cognitive appraisal model in assuming that cognitive risk assessment determines experience of fear. such a model is naturally applicable for the study of behaviors aimed at fending off long-term disease, where fear is likely to be less imminent and therefore secondary to more rational reflections about gains and losses related to protective behavior. however, in an acute threat situation, like a severe influenza outbreak, emotional aspects might gain more immediate importance. this would even be more likely during periods of scientific uncertainty, when fewer facts are available. it is in this context interesting to note that coping appraisal in this study was found to be the motivation factor that contributed most to the discriminatory performance despite the fact that threataffect was included in the general model, although indirectly through cognitive assessment. however, what role affect-or emotion-based judgments play in interaction with threat and coping appraisals is still an issue in need of clarification. finally, it should be noted that there were relatively small differences between the reported intended behaviors associated to the mild and severe scenarios, respectively. one explanation of this observation can be the fact that the a(h1n1)pdm09 outbreak was relatively mild in sweden, and that the respondents, wrongfully, related the severe scenario to their recent personal experience rather than the scenario description. however, the lack table 6 . simple and multiple logistic regression models of explanatory factors for the intention to wash hands after touching common objects displayed by influenza outbreak scenario. of difference can also be seen as a sign of its own, i.e. that the swedish population may not be fully aware of the seriousness of a full influenza pandemic. failure to monitor the beliefs and attitudes of the public has recently been identified as a weakness in preparedness strategies against infectious disease outbreaks [42] . we examined how items in a general explanatory model of intended health behavior were associated with personal hygiene and social distancing practices following a questioned mass vaccination campaign against influenza in the swedish population. we observed a relatively high trust in official recommendations and a higher proportion of intentions to improve personal hygiene than those used to increase social distancing. among the explanatory factors considered, coping appraisal was the factor most frequently included in models explaining self-reported intentions. variations in threat appraisal played a smaller role in these models despite the uncertainties surrounding the mass vaccination during the a(h1n1)pdm09 outbreak. the results also show that not just from a third world perspective [43] it is necessary to consider that not all population sub groups have the same predispositions to enact specific behaviors to protect their health. for instance, they suggest that further studies are needed of gender differences in protective behaviors during influenza outbreaks. we conclude that developing interventions that support the general population's efforts to perform self-protective behaviors during influenza outbreaks and longitudinal studies of such interventions across several influenza seasons are warranted also in european countries. text s1 interview guideline for collection of data on perceptions associated with precautionary behaviors. 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netherlands, compared to the general population in these countries influenza-like illness in norway: clinical course, attitudes towards vaccination and preventive measures during the 2009 pandemic deriving behavior model parameters from survey data: self-protective behavior adoption during the 2009-2010 influenza a(h1n1) pandemic energy consumption by gender in some european countries statistics sweden (2012) women and men in sweden. facts and figures 2012. stockholm: statistics sweden use of correlational data to examine effects of risk perceptions on precautionary who takes precautionary action in the face of the new h1n1 influenza? prediction of who collects a free hand sanitizer using a health behavior model how well do the theory of reasoned action and theory of planned behaviour predict intentions and attendance at screening programmes? a metaanalysis does changing behavioral intentions engender behavior change? a meta-analysis of the experimental evidence responses to information about psychosocial consequences of genetic testing for breast cancer susceptibility: influences of cancer worry and risk perceptions developing pandemic preparedness in europe in the 21st century: experience, evolution and next steps delivering interventions to reduce the global burden of stillbirths: improving service supply and community demand item specific for intention to wash hands after touching common objects. 1 formal education past high school/secondary school. doi:10.1371/journal.pone.0091060.t006 key: cord-001605-8p06bpt1 authors: sapmak, ariya; boyce, kylie j.; andrianopoulos, alex; vanittanakom, nongnuch title: the pbrb gene encodes a laccase required for dhn-melanin synthesis in conidia of talaromyces (penicillium) marneffei date: 2015-04-13 journal: plos one doi: 10.1371/journal.pone.0122728 sha: doc_id: 1605 cord_uid: 8p06bpt1 talaromyces marneffei (basionym: penicillium marneffei) is a significant opportunistic fungal pathogen in patients infected with human immunodeficiency virus in southeast asia. t. marneffei cells have been shown to become melanized in vivo. melanins are pigment biopolymers which act as a non-specific protectant against various stressors and which play an important role during virulence in fungi. the synthesis of the two most commonly found melanins in fungi, the eumelanin dopa-melanin and the allomelanin dhn-melanin, requires the action of laccase enzymes. the t. marneffei genome encodes a number of laccases and this study describes the characterization of one of these, pbrb, during growth and development. a strain carrying a pbrb-gfp fusion shows that pbrb is expressed at high levels during asexual development (conidiation) but not in cells growing vegetatively. the pbrb gene is required for the synthesis of dhn-melanin in conidia and when deleted results in brown pigmented conidia, in contrast to the green conidia of the wild type. talaromyces marneffei (basionym: penicillium marneffei) is an opportunistic human fungal pathogen endemic to southeast asia and southern china [1] [2] [3] . most cases of t. marneffei infection occur in immune-deficient hosts, especially those infected with human immunodeficiency virus (hiv), however, infections have also been reported in non-hiv children with the underlying immune defects (e.g. severe combined immunodeficiency (scid), congenital lymphopenia, hyper-igm syndrome, and hyper-ige syndrome). failure to treat infections is fatal, especially in children with primary immunodeficiency [2, 3] . t. marneffei is a dimorphic fungus with a thermally regulated dimorphic switch. as such, t. marneffei is capable of undergoing a transition from the saprophytic filamentous multicellular hyphae found in the environment (or in vitro at 25°c) to a unicellular yeast growth form in vitro at 37°c and during infection. during growth at 25°c, hyphae can also undergo asexual development (conidiation) to produce conidia, the infectious propagules. hyphae differentiate by sequential production of an aerial stalk followed by the budding of the metula and phialide cell types from the stalk tip plos one | doi: 10 all transformants were produced from a uracil auxotrophic strain (g526, δpkua pyrg -) derived from talaromyces marneffei frr2161 (cbs 334.59, atcc18224) [26, 27] . strain g526 lacks the pkua gene encoding the ku70 protein which functions in non-homologous dna end joining repair [28] . therefore, genetic transformation is mediated via homologous recombination only. g526 harbors a spontaneous, loss-of-function mutation in pyrg (orotidine-5'-phosphate decarboxylase encoding gene) selected by growth in the presence of 5-fluoroorotic acid [27] . all pyrg + transformants and the t. marneffei g681 (δpkua::pyrg + ) strain used as a control, were maintained on anm medium containing 1% (w/v) glucose, 10 mm ammonium sulfate [26] . the t. marneffei f4 strain isolated from an aids patient (cbs 119456) [29] was maintained on malt extract (me) agar. conidial suspensions of transformants and the g526 strain were harvested from conidiating colonies growing on anm solid medium at 28°c for 1 week. colonies on petri dishes were flooded with sterilized phosphate buffer saline, gently scraped, filtered through miracloth (calbiochem), and recovered by centrifugation. conidia were harvested from t. marneffei f4 colonies cultured on me agar at 28°c for 1 week. a suspension of 10 6 conidia was spread on me agar with and without 30 μg/ml tricyclazole in 6-well plate. the plate was incubated at 28°c for 1 week. the expression of pbrb was investigated by rt-pcr. rna samples were extracted from t. marneffei f4 cultured in brain heart infusion (bhi) broth at 28°c and 37°c for 3 days. rna was extracted and used as a template to produce cdna using omniscript reverse transcription kit (qiagen). the pbrb cdna was then amplified using specific primers pmlac25-f and l25re. the 18s rrna was amplified as a control using pm1 and pm2 primers [30] . primers used in this study are listed in table 1 . talaromyces (penicillium) marneffei pbrb gene to construct the δpbrb plasmid, pmlac25u and pmlac25l primers were used to amplify the pbrb gene containing approximately 1.4 kb of upstream and downstream untranslated regions (utrs) from t. marneffei strain g681. purified pcr product (4,747 bp) was ligated into pgem-t easy (promega) to produce the pbrb plasmid (plac25). this plasmid was digested with bglii and stui to remove the pbrb coding region (138 bp before atg to 155 bp before stop codon). the bglii/stui pbrb plasmid was ligated to a bamhi/ecorv fragment containing the pyrg blaster selectable marker cassette (pab4342 [27] ) to generate pplilac25. the linearized insert from pplilac25 was excised by noti and purified before transformation. for the complementation construct, p25n9, the noti fragment from plac25 was ligated into a noti-digested/ dephosphorylated pab4342 vector. in order to generate the pbrb(p)::pbrb::gfp strain, plac25 was amplified by inverse pcr using ill25ec1/iul25ec1 primers. the pcr product was digested with ecorv and acli and then ligated with ecorv/clai fragment containing egfp to produce plac25gfp. the egfp coding region was inserted at the 5' end of pbrb, 87 bases after the start codon. the egfp coding region was amplified from the pegfp-c1 vector (kindly provided by dr. amornrat o'brien [31] ) using fpegfpc1/rpegfpc1 primers. the whole pbrb(p)::pbrb::gfpfragment was excised from plac25gfp by noti digestion and cloned into noti-digested/dephosphorelated pab4342 to generate pgfplac4n. t. marneffei strain g526 was used to generate the δpbrb::pyrg + mutant and pbrb(p)::pbrb:: gfp strain. dna-mediated transformation was performed using peg-mediated transformation protocol described previously [27] . the pyrg + transformants were selected on medium without uracil. to generate a δpbrb pyrgstrain, the δpbrb::pyrg + strain was inoculated onto anm agar containing 1 mg/ml 5-fluoroorotic acid to select for loss of the pyrg cassette via homologous recombination between the inverted cat repeats that flank pyrg in the construct [32] . to generate the δpbrb pbrb + complementation strain, the δpbrb pyrgstrain was transformed with p25n9 and pyrg + transformants were selected. genetically modified t. marneffei transformants were examined by pcr and/or southern blot analysis [26] . the pbrb amplification was performed using pmlac25f/l25re primers, while fpegfpc1/l25re primers were for egfp-pbrb amplification. the t. marneffei fetc gene (pmaa_057450) was amplified as an internal control of pcr using pmlac4f/pmlac4r primers. the hybridization probe for southern blot analysis was the pbrb gene amplified by pcr using pmlac25u/pmlac25l primers. for germination assays, conidia (10 6 ) of each strain were inoculated in a six well microtitre plate containing bhi broth and the plate was incubated at 28°c or 37°c for 24 hours. aliquots of the broth culture were placed on slides to count germinating conidia. three replicates were performed counting at least 100 cells each time. for growth rate assays, approximately 10 conidia were spread onto 3 plates of anm agar and incubated at 28°c for 3 days. the diameter of 5 individual colonies was measured daily over a period of 7 days. mean and standard error of the mean (sem) values were calculated and analyzed for statistical significance using graph-pad software (http://www.graphpad.com/). conidia (10 6 ) of the pbrb(p)::pbrb::gfp strain were inoculated into two flasks containing 50 ml of bhi broth. each flask was incubated in a shaking incubator at 28°c or 37°c for 3 days. mycelia from the 28°c culture were poured onto miracloth and rinsed with cold pbs before microscopic examination. fungal cells from the 37°c culture were harvested by centrifugation, washed with cold pbs, placed on a microscope slide and examined using an olympus provis ax70 fluorescence microscope. to examine conidiating fungal cells, a piece of anm agar block (about 10 x 10 x 5 mm) was placed on a sterile glass slide. conidia from the pbrb(p)::pbrb::gfp strain were inoculated on each side of agar block. a cover glass was placed on the top of the agar block. the slide culture was incubated in a humidified chamber at 28°c for 10 days. the agar block was removed and fungal cells that remained attached to the slide were fixed with absolute ethanol before air drying in a biosafety cabinet. fungal cells on the slide were incubated with 5 mg/ml trichoderma harzianum cell wall lysing enzyme (sigma-aldrich, uk) dissolved in osmotic buffer [27] at 37°c for 30 minutes and then the solution was poured off. fungal cells were permeabilized by the addition of 0.2% triton x-100 in pbs for 5 minutes. slides were washed with cold pbs solution. immunofluorescence staining was performed as previously described [20] . briefly, slide cultures were blocked with superblock blocking buffer (pierce, usa) at 37°c for 2 hours. the slides were incubated with 10 μg/ml of anti-melanin igm antibody, kindly provided by dr. sirida youngchim [20] , at 37°c for 1.5 hours. slides were washed with pbs and then incubated with a dilution 1:100 of rhodamine-labeled goat anti-mouse igm antibody (jackson immunoresearch laboratories, usa) at 37°c for 1.5 hours. after washing, cells were observed under a fluorescence microscope. the negative control samples were produced by omitting the antimelanin igm binding step in order to assess the background caused by non-specific binding of secondary antibody. the superblock blocking buffer used to dilute anti-melanin igm was added, instead of using anti-melanin igm, before incubating with secondary antibody. to examine chitin deposition, calcofluor white staining was performed as previously described [33] . three-day-old fungal cells were harvested from bhi broth culture as described above and the cells were washed with pbs before staining. the green pigment synthesized during conidiation in t. marneffei is dihydroxynaphthalene melanin to examine if the green coloration of conidia was attributed to dhn-melanin, t. marneffei conidia were inoculated onto medium with or without the inhibitor tricyclazole. tricyclazole inhibits the two hydroxynaphthalene reductases functioning in dhn-melanin synthetic pathway ( fig 1a) [23] [24] [25] . in the absence of tricyclazole, t. marneffei conidia appeared green ( fig 1b) . in contrast, in a presence of tricyclazole, the t. marneffei conidia appeared yellow (fig 1b) . this suggests that the synthesis of dhn-melanin contributes to the green coloration of conidia during asexual development. the t. marneffei genome encodes ten multicopper oxidases laccases are members of the multicopper oxidase (mco) family, which also includes ferroxidases and ascorbate oxidases. to identify laccase encoding genes in t. marneffei the c. neoformans lac1 and a. fumigatus abr2 (fig 2a) encoding genes were used as a query sequence in blast searches of the t. marneffei atcc 18224 genome sequence (genbank, ncbi). this identified two highly homologous genes (pmaa_072680 and pmaa_085520) (cnlac1 homology of 88% and 78%, respectively) that were then also used for additional blast searches to retrieve related sequences in the t. marneffei genome. these homology searches identified 10 genes encoding putative multicopper oxidases (pmaa_008350, pmaa_050860, pmaa_055370, pmaa_057450, pmaa_062880, pmaa_072680, pmaa_082010, pmaa_082060, pmaa_085520, and pmaa_100410). generally, laccases contain four copper atoms including type1 cu, type 2 cu, and a pair of type 3 cu. the patterns of conserved amino acids coordinated with each type of copper are hxhg, hxh, hchxxxhxxxm/f/l, and hxxhxh, which occupy the l1-l4 signature sequences starting from the n-terminus [34] [35] [36] . we found that 9 of the t. marneffei mcos (excluding pmaa_062880) have these conserved patterns (see s1 fig) . previous phylogenetic studies reveal that mco sequences are often clustered with other genes according to function, fungal division, and source organism [10, 11, 13] . to predict which t. marneffei mco participates in conidial dhn-melanin synthesis, we combined 55 fungal mco sequences and performed alignments using clustalw (http://www.genome.jp/ tools/clustalw/). tree construction was conducted in mega version 6 [37] using the neighbor--joining method. the 55 fungal mco sequences were separated into 5 clades with wellsupported branches (>90% bootstrap support) (fig 2a) . each clade was highlighted and named according to sequences with known functions and fungal divisions. the ascomycete laccase lineage can be divided into 2 clades supported with very high bootstrap values (99% and 100%). focusing on the second clade containing t. marneffei pbrb, this clade comprises of characterized laccases functioning in conidial dhn-melanin synthesis. t. marneffei pbrb is more closely related to a. fumigatus abr2 than a. nidulans ya [16] [17] [18] . pbrb is expressed during asexual development and localizes to all cell types of the conidiophore the expression of pbrb was investigated by rt-pcr. rna samples were extracted from t. marneffei f4 grown in bhi broth at 28°c and 37°c for 3 days. a transcript was not detected under these conditions suggesting there is little or no pbrb expression in vegetative hyphal or yeast cells (data not shown). to analyze pbrb expression further, a strain was generated that expresses a fusion construct in which pbrb, expressed from the native promoter, is fused to gfp (pbrb(p)::pbrb::gfp). conidia of the pbrb(p)::pbrb::gfp strain were inoculated into bhi broth and cultured at 28°c and 37°c for 3 days. in support of the rt pcr analysis, gfp fluorescence was not detected in vegetative hyphal cells at 28°c or vegetative yeast cells at 37°c nlm.nih.gov/protein/) and used to build sequence alignments in clustalw. the alignment was then used to construct a relatedness tree using mega 6 software. phylogenetic relationships of the 55 mcos were inferred using the neighbor-joining method and bootstrap tested (1000 replicates). branch lengths of the tree under standard conditions (fig 3a and 3b ). in addition, vegetative cells were grown at 28°c and 37°c in a variety of other types of medium (anm, synthetic dextrose and malt extract) and in the presence of copper with low glucose (0.2%) or under acidic condition ph 5.0, which are known laccase inducing conditions [38] . no signal from pbrb::gfp could be detected under any of these conditions after 3, 5 and 7 days of incubation (data not shown). a slide culture of the pbrb(p)::pbrb::gfp strain was prepared to observe expression of pbrb during conidiation. compared to the negative control (fig 3c) , strong gfp fluorescence was observed in conidiophores of the pbrb(p)::pbrb::gfp strain, suggesting that pbrb is expressed during asexual development (fig 3d) . the pbrb::gfp protein was localized as distinct spots in metulae and phialides (fig 3d) . to investigate if pbrb co-localizes with melanin, melanin was detected in the pbrb(p)::pbrb:: gfp strained by immunolabeling with an anti-melanin antibody. immunostaining detected strong fluorescence in phialide cells with some weaker staining in the stalk, metulae and conidial cell types. there was also some punctate melanin staining in the various cell types. this staining are drawn to scale and bootstrap support indicating at the branch sites. fungal mcos that share a common ancestry with more than 60% bootstrap value are shaded in gray. functions are defined at each clade based on characterized function of mco members. (b) a partial alignment representing the conserved motifs of copper binding sites and l1-l4 signature sequences found in t. marneffei pbrb (pmaa_082060) and its orthologs a. fumigatus abr2 (afua_2g17530) and a. nidulans ya (an6635). the numbers in front of each sequence indicate the amino acid position of a particular protein. cu binding motifs are identified above the sequences, numbers including 1, 2, and 3 are type 1 cu, type 2 cu, and type 3 cu. the asterisk indicates the potential proton donor for the reaction intermediates. was highly co-localised with the fluorescence form the pbrb::gfp fusion protein (fig 4) . this supports the hypothesis that pbrb is likely to be involved in melanin biosynthesis during conidiation. to characterize the role of pbrb in t. marneffei, δpbrb mutants were generated. two independent mutants were characterized with respect to conidiation, germination and growth. in order to examine morphogenesis during conidiation, δpbrb conidia were grown on anm plates. in contrast to the wild type control, which exhibits green colored conidiation at 28°c after 14 days, conidiation of the δpbrb mutants appeared a light brown color (fig 5) . reintroduction of a wild type copy of pbrb at the native locus (materials and methods) in the δpbrb mutant complemented the conidiation phenotype (fig 5) . slide cultures of the δpbrb and δpbrb pbrb + complemented strains growing on anm agar at 28°c for 7 days were prepared to examine the morphology of conidiophores. deletion of pbrb did not result in any morphological defects in any of the conidiophore cell types (fig 6c and 6d ). the change in phenotype indicates that pbrb has a unique function that cannot be compensated by other t. marneffei laccases. to assess if deletion of pbrb and the consequent change in pigmentation of the conidia affected germination, 10 6 conidia of the δpbrb and δpbrb pbrb + strains were inoculated in bhi broth and incubated at 28°c or 37°c for 24 hours then the number of germinated conidia were counted. there was no difference in the germination of the δpbrb and δpbrb pbrb + strains at 28°c (fig 6a and 6b ) or 37°c (data not shown). hyphal cells from the δpbrb and δpbrb pbrb + strains were grown at 28°c for 3 days and examined microscopically. staining of hyphae with 1 μg/μl calcofluor white showed no defects in chitin deposition nor was any morphological difference detected (data not shown). growth rates of the δpbrb and δpbrb pbrb + strains were determined by measuring colony diameters daily over the course of 7 days on anm medium at 28°c. mean and standard error of the mean (sem) were calculated and statistical differences were assessed using the t-test. the differences in growth rates between δpbrb and δpbrb pbrb + strains at 6 and 7 days were found to be statistical significant (p = 0.002 and 0.015, respectively) (fig 7) . deletion of pbrb results in brown conidiation color, in contrast to the green coloration of wild type. this suggests that like a. fumigatus and a. nidulans, pbrb in t. marneffei is required for the synthesis of dhn-melanin. as melanins protect fungal cells against environmental assaults, the susceptibility of the δpbrb mutant to a variety of stresses was determined. ten-fold dilutions of δpbrb and δpbrb pbrb + conidial suspensions were dropped on anm and bhi agar containing 2% h 2 o 2 (oxidative stress), 20 μg/ml sds (cell wall stress), 3 μm congo red (cell wall stress), 1 m sorbitol (osmotic stress), 0.6 m nacl (salt stress), 5 mm nano 2 (nitrosative stress) or antifungals (0.15 μg/ml amphotericin b, 0.1 μg/ml clotrimazole, 40 μg/ml fluconazole, and 0.04 μg/ml itraconazole) and incubated at either 28°c and 37°c for 7 days. in addition, susceptibility to antifungal activity of macrophage (j774) was examined. however, no differences in stress susceptibility were observed (data not shown). talaromyces (penicillium) marneffei pbrb gene cytoplasmic protein extracts from the wild type and δpbrb mutant cultured in brain heart infusion broth at 37°c for 3 days were capable of catalyzing l-dopa (data not shown). in addition, δpbrb cells growing in bhi medium at 37°c for 5 days showed equivalent staining as the wild type using the anti-melanin antibody. these results suggest that melanization of vegetative cell types is not dependent on pbrb and this is consistent with the results from the susceptibility tests using the various stress agents. the multicopper oxidase (mco) family comprises enzymes that typically contain four copper atoms classified into three types (type 1 cu, type 2 cu and type 3 cu). members of mco family include laccases, ferroxidases, ascorbate oxidases, bilirubin oxidases, cueo, and ceruloplasmin [10, 11, 34, 35] . among mco members, laccases can be found in various organisms (e.g. plants, fungi, bacteria and insects), but not in humans. a large number of laccases are produced in many basidiomycete and ascomycete fungal species and a variety of physiological roles have been reported including morphogenesis, stress defense, fungal pathogen/host interaction and delignification [7, 12, 36] . in pathogenic fungi, laccases have attracted attention due to their involvement in melanization and the correlation with host invasion and protection against host immunity. laccases contribute to the melanization pathways producing the most commonly isolated fungal melanins; dopa-and dhn-melanin [7, 9] . generally, melanins are deposited in the cell wall of conidia and fungal cells [6, 15] . this study has determined the role of a developmentally regulated laccase, encoded by pbrb, during dhn-melanin synthesis in conidia. neither the pbrb transcript nor the gfp-tagged protein in a pbrb(p)::pbrb::gfp strain could be detected during vegetative hyphal or yeast growth. however, gfp fluorescence was observed in conidiophores of the pbrb(p)::pbrb::gfp strain, suggesting that pbrb is only expressed during asexual development in t. marneffei. likewise in a. fumigatus and a. nidulans, expression of the orthologous abr2 and ya genes characteristically occurs during conidiophore development but not during vegetative growth [16, 18] . phylogenetic examination showed that t. marneffei pbrb is more closed to a. fumigatus abr2 than a. nidulans ya (fig 2a) . moreover, t. marneffei δpbrb colonies displayed brown-pigmented conidia that resemble those of the δabr2 strain [17, 18] . our data suggests that t. marneffei pbrb is polymerizing polymers of 1,8-dhn to form dhn-melanin, as has been shown in a. fumigatus [39] . t. marneffei pbrb is located within a genomic cluster of genes with homology to those required for conidial pigment biosynthesis in other systems and includes pmaa_082010 (conidial biosynthesis oxidase abra), pmaa_082020 (conidial biosynthesis protein ayga), pmaa_082030 (1,3,6,8-tetrahydroxynaphthalene reductase arpa), pmaa_082040 (conidial pigment biosynthesis scytalone dehydrogenase arpa) and pmaa_082120 (conidial pigment polyketide synthase alba) (see s2a fig) . this cluster is conserved in a. fumigatus (s2b fig). the biochemical pathway for dhn-melanin production was first described by wheeler and bell (1988) and has been well characterized both biochemically and genetically in many ascomycetes. the genus aspergillus comprises many species and pigmented conidia appear in various colors among species. effects of inhibitors on dhn-melanin synthesis (using tricyclazole and phthalide) and dopa-melanin synthesis (using kojic acid and tropolone) in a range of aspergillus species demonstrate that differences in the amounts and types of pigments and melanins synthesized by related species are likely to be a common theme [40, 41] . among aspergillus and penicillium species, most exhibit green to bluish green conidial pigments and the use of inhibitors of melanization pathways has revealed that most of these pigments are made from pentaketide metabolites [41] . the starting carbon units are acetate [25, 42] , malonyl coa and/or acetyl coa [6, 15, 43] and are catalyzed by a polyketide synthase (pks) in the first step before subsequent processing through enzymatic steps to produce dhn-melanin [6, 9, 12, 23] . mutation of the gene encoding the pks in a. nidulans (wa), a. fumigatus (pksp) and t. marneffei (wa) similarly results in white, non-melanized conidia [44, 45, 22] . while this early part of the pathway seems to be conserved amongst these fungi, differing results have been shown when tests are conducted with the dhn-melanin synthesis inhibitor tricyclazole. tricyclazole specifically affects the reductases that reduce 1,3,6,8-tetrahydroxynaphthalene to scytalone and 1,3,8-trihydroxynaphthalene to vermelone [23] [24] [25] 46] . tricyclazole alters the conidial color phenotype of a. fumigatus but it does not affect conidial coloration of a. nidulans, a. flavus, and a. parasiticus [41, 47] . further studies showed that the conidial pigment biosynthesis in a. fumigatus and a. nidulans possess some steps that are distinct from the general model of dhn-melanin biosynthetic pathway (fig 8) [6, 15] . instead of producing tetrahydroxynaphthalene (thn) from the starter units, a. nidulans and a. fumigatus utilize malonyl coa and acetyl coa to produce heptaketide naphthopyrone ywa1 by heptaketide synthase (hks) [44, 48] . although ywa1 is a precursor for green conidial pigmentation in both a. nidulans and a. fumigatus, the downstream metabolic steps are different in the two organisms. in a. nidulans, ya laccase catalyzes ywa1 to produce the green pigment of conidia. in contrast, a. fumigatus ywa1 is catalyzed through a hydrolytic polyketide-based shortening step to produce 1,3,6,8-thn by ayg1 [49] . a homolog of a. fumigatus ayg1 is also presents in t. marneffei genome (see s2 fig) . as the addition of tricyclazole affected conidial color of t. marneffei, it suggests that hydroxynaphthalene reductases function in dhnmelanin synthetic pathway (fig 8) . similar to a. fumigatus, ywa1 should be converted to 1,3,6,8-thn in t. marneffei. then 1,3,6,8-thn is processed through additional steps to produce 1,8-dhn. finally, 1,8-dhn is polymerized by pbrb laccase to form dhn-melanin, which appears as the green color of t. marneffei conidia (fig 8) . melanization of t. marneffei has been described previously, however the type of melanin was not determined [20] . in this study, we found that pbrb encodes a laccase enzyme expressed during conidiation. pbrb is required for the synthesis of dhn-melanin in conidia and when deleted results in brown conidiation, in contrast to the green conidiation of wild type. the existence of additional uncharacterized multicopper oxidase-encoding genes in the t. marneffei genome suggests that in addition to dhn-melanin, t. marneffei may also have the capacity to produce dopa-melanin depending on growth conditions and supply of precursors. [15, 39, 50] . processing steps of the well-known dhn pathway are presented with dark arrows. pks, polyketide synthase; 4hnr, tetrahydroxynaphthalene reductase; sd, scytalone dehydratase; 3hnr, trihydroxynaphthalene reductase; vd, vermelone dehydratase; 1,3,6,8-thn, tetrahydroxynaphthalene; 1,3,8-thn, trihydroxynapthalene; 1,8-dhn, dihydroxynaphthalene. tc, tricyclazole, can inhibit both reductases (4hnr and 3hnr) presented in a model pathway. distinctive steps described in a. nidulans (an), a. fumigatus (af), t. marneffei (tm) are shown with dashed arrows. asterisks, * and **, refer to data from previous study [22] and this study, respectively. doi:10.1371/journal.pone.0122728.g008 phylogeny and nomenclature of the genus talaromyces and taxa accommodated in penicillium subgenus biverticillium penicilliosis in children without hiv infectionare they immunodeficient? insights into the pathogenicity of penicillium marneffei penicillium marneffei infection and recent advances in the epidemiology and molecular biology aspects control of morphogenesis in the human fungal pathogen penicillium marneffei melanin synthesis in microorganisms-biotechnological and medical aspects fungal laccases-occurrence and properties color me bad: microbial pigments as virulence factors synthesis and assembly of fungal melanin function and molecular evolution of multicopper blue proteins phylogenetic comparison and classification of laccase and related multicopper oxidase protein sequences laccase: microbial sources, production, purification, and potential biotechnological applications multiple multi-copper oxidase gene families in basidiomycetes-what for? curr genomics effect of the laccase gene, cnlac1, on virulence of cryptococcus neoformans biosynthesis of fungal melanins and their importance for human pathogenic fungi molecular characterization of the aspergillus nidulans ya locus a developmentally regulated gene cluster involved in conidial pigment biosynthesis in aspergillus fumigatus characterisation of the laccase-encoding gene abr2 of the dihydroxynaphthalene-like melanin gene cluster of aspergillus fumigatus cryptococcus neoformans laccase catalyses melanin synthesis from both d-and l-dopa. microbiol melanization of penicillium marneffei in vitro and in vivo. microbiology detection of dopa-melanin in the dimorphic fungal pathogen penicillium marneffei and its effect on macrophage phagocytosis in vitro strategies for the molecular genetic manipulation and visualization of the human fungal pathogen penicillium marneffei melanins and their importance in pathogenic fungi melanin biosynthesis inhibitors (mbis) for control of rice blast the second naphthol reductase of fungal melanin biosynthesis in magnaporthe grisea: tetrahydroxynaphthalene reductase area controls nitrogen source utilisation during both growth programs of the dimorphic fungus penicillium marneffei an ste12 homolog from the asexual, dimorphic fungus penicillium marneffei complements the defect in sexual development of an aspergillus nidulans stea mutant new tools for the genetic manipulation of filamentous fungi phagocytosis and killing of human pathogenic, penicillium marneffei and non-pathogenic, penicillium citrinum by mouse macrophage j774.1 cells rapid identification of penicillium marneffei by pcr-based detection of specific sequences on the rrna gene membrane topology of murine coronavirus replicase nonstructural protein 3 a basic helix-loop-helix protein with similarity to the fungal morphological regulators, phd1p, efg1p and stua, controls conidiation but not dimorphic growth in penicillium marneffei the fungal type ii myosin in penicillium marneffei, myob, is essential for chitin deposition at nascent septation sites but not actin localization combined sequence and structure analysis of the fungal laccase family basic and applied features of multicopper oxidases, cueo, bilirubin oxidase, and laccase laccases: a never-ending story mega6: molecular evolutionary genetics analysis version 6.0 induction and transcriptional regulation of laccases in fungi melanin is an essential component for the integrity of the cell wall of aspergillus fumigatus conidia dopa and dhn pathway orchestrate melanin synthesis in aspergillus species the effects of tricyclazole, pyroquilon, phthalide, and related fungicides on the production of conidial wall pigments by penicillium and aspergillus species the melanin biosynthesis gene of alternaria alternate can restore pathogenicity of the melanin-deficient mutants of magnaporthe grisea 1,8-dihydroxynaphthalene (dhn)-melanin biosynthesis inhibitors increase erythritol production in torula corallina, and dhn-melanin inhibits erythrose reductase isolation and molecular characterization of the aspergillus nidulans wa gene identification of a polyketide synthase gene (pksp) of aspergillus fumigatus involved in conidial pigment biosynthesis and virulence site of inhibition by tricyclazole in the melanin biosynthetic pathway of verticillium dahliae characterization of melanin pigment produced by aspergillus nidulans the developmentally regulated alb1 gene of aspergillus fumigatus: its role in modulation of conidial morphology and virulence pentaketide melanin biosynthesis in aspergillus fumigatus requires chain-length shortening of a heptaketide precursor hydrolytic polyketide shortening by ayg1p, a novel enzyme involved in fungal melanin biosynthesis the authors acknowledge members of the andrianopoulos lab, department of genetics, university of melbourne, and vanittanakom lab, department of microbiology, chiang mai university for helpful discussions and providing the necessary facilities and research training. competing interests: the authors have declared that no competing interests exist.supporting information s1 fig. patterns of copper binding sites in t. marneffei mcos. sequence alignments showing the general copper signature sequences (l1-l4) found in fungal laccases [34, 35] . site of the first amino acid of each sequence is indicated next to pmaa number. the copper binding residues are highlighted and note the type of copper (1, 2, and 3). asterisk is a potential proton donor. type 1 copper ligand (m/l/f) superscripted with i/ii/ii refers to redox potential class from low to high [35] . genomic region in t. marneffei (tm) from gene pmaa_082000 to pmaa_08120, which encompasses a cluster of genes, predicted to be required for conidial pigment synthesis. genes required for conidial pigment biosynthesis are colored brown (pmaa_082010 oxidase, abra), blue (pmaa_082020 ayga), orange (pmaa_082030 1,3,6,8-tetrahydroxynaphthalene reductase, arpb), aqua (pmaa_082040 scytalone dehydratase, arpa), red (pmaa_080260 laccase, pbrb) and green (pmaa_080260 polyketide synthase, wa). (b) a. fumigatus gene cluster involved in dhn melanin synthesis. this gene cluster is conserved in a. fumigatus (af) (afug_2g17530, abr2; afug_2g17540, abr1; afug_2g17550 ayg1; afug_2g17560 arp2; afug_2g17580 arp1 and afug_2g17600 alb1).(tif) key: cord-000609-dpcgl6ig authors: raju, sammeta v.; wang, guoshun title: suppression of adenosine-activated chloride transport by ethanol in airway epithelia date: 2012-03-19 journal: plos one doi: 10.1371/journal.pone.0032112 sha: doc_id: 609 cord_uid: dpcgl6ig alcohol abuse is associated with increased lung infections. molecular understanding of the underlying mechanisms is not complete. airway epithelial ion transport regulates the homeostasis of airway surface liquid, essential for airway mucosal immunity and lung host defense. here, air-liquid interface cultures of calu-3 epithelial cells were basolaterally exposed to physiologically relevant concentrations of ethanol (0, 25, 50 and 100 mm) for 24 hours and adenosine-stimulated ion transport was measured by ussing chamber. the ethanol exposure reduced the epithelial short-circuit currents (i(sc)) in a dose-dependent manner. the ion currents activated by adenosine were chloride conductance mediated by cystic fibrosis transmembrane conductance regulator (cftr), a camp-activated chloride channel. alloxazine, a specific inhibitor for a(2b) adenosine receptor (a(2b)ar), largely abolished the adenosine-stimulated chloride transport, suggesting that a(2b)ar is a major receptor responsible for regulating the chloride transport of the cells. ethanol significantly reduced intracellular camp production upon adenosine stimulation. moreover, ethanol-suppression of the chloride secretion was able to be restored by camp analogs or by inhibitors to block camp degradation. these results imply that ethanol exposure dysregulates cftr-mediated chloride transport in airways by suppression of adenosine-a(2b)ar-camp signaling pathway, which might contribute to alcohol-associated lung infections. alcohol abuse is a risk factor for pulmonary infections. it is not fully understood how alcohol exposure compromises the lung host defense. previous studies suggest that multiple pathophysiological mechanisms may be involved [1, 2, 3] . airway mucosal immunity and mucociliary clearance are the two primary host defense mechanisms, which take place in a thin layer of liquid on the top of airway epithelia, known as airway surface liquid (asl). asl, composed of a gel-like mucus layer and a sol-like periciliary liquid layer [4, 5, 6] , is the ''battlefield'' for pulmonary infection and immunity. the viscous mucous blanket traps inhaled microorganisms and particles to restrict their spreading in the lung. in contrast, the watery periciliary liquid (pcl) underneath pools antimicrobial substances, antibodies, cytokines, chemokines and other immune modulators [7, 8, 9] . more importantly, pcl provides the milieu for innate and adaptive immune cells including neutrophils and macrophages to home and function. moreover, pcl prevents cilia from being entrapped in viscous mucus and bathes them for mechanical movement for mucociliary clearance [5, 10] . asl composition and volume are collectively regulated by epithelial chloride secretion, sodium absorption and secondarily water secretion and absorption [6, 11] . mounting evidence indicates that paracrine/autocrine purinergic signaling is critical to airway epithelial ion transport and asl hydration [12] #. adenosine has been shown to be a potent regulator in the process, which can be directly released by local epithelial cells and immune cells [13] or from extracellular metabolism of atp [12] #. it is known that atp is constitutively released by epithelia due to various stimuli including mechanical stretch and shear stress due to respiration [14] . the released atp is then converted to adenosine by extracellular ectonucleotidases [15] . thus, asl has relatively high levels of adenosine. further studies demonstrate that adenosine largely regulates epithelial cftr channel function by acting on a 2b ar [16, 17, 18] . thus, the adenosine-a 2b ar signaling pathway is a crucial element in lung host defense [19, 20] . previous alcohol studies have documented that ethanol exposure decreases camp signaling and protein kinase a (pka) activation [3, 21] . ethanol also up-regulates phosphodiesterase 4 (pde4), which increases camp degradation [22] #. in spite of the clear link between alcohol exposure and alteration of adenosine signaling, no published data are currently available concerning alcohol effects on airway ion transport through this signaling pathway. the current report directly measured the adenosineinduced chloride secretion of airway epithelia under the exposure of physiologically relevant concentrations of alcohol and found that ethanol attenuates epithelial cftr-mediated chloride transport by modulating cellular camp levels. no human subjects or animals were used in this study. calu-3 cells, a human airway epithelial cell line (atcc, manassas, va), were seeded on collagen-coated millicellh-pcf membrane inserts (millipore, billerica, ma) at a density of 1610 6 cells per insert of 0.6 cm 2 surface area. two days after the initial submerged culture, the apical media were aspirated off and the cells cultured at an air-liquid interface according to previously published protocol [23] . regardless of submerged culture or air-liquid interface culture, the media used were the same, consisting of advanced-mem (gibco, carlsbad, ca) containing 10% fetal bovine serum, 1% l-glutamine, 100 u/ml penicillin, 100 mg/ml streptomycin and 0.25 mg/ml amphotericin b. after 2 weeks at 37uc in presence of 5% co 2 , the epithelia established a dry apical surface and had a transepithelial electrical resistance greater than 1000 v/cm 2 . the cystic fibrosis (cf) airway epithelial cells cfbe41o[24] were similarly cultured. the fully differentiated cf epithelia after 2 weeks exhibited a transepithelial resistance greater than 700 v/cm 2 . air-liquid interface cultures were basolaterally exposed to different concentrations of ethanol (200 proof; aaper alcohol and chemical co., shelbyville, ky), as indicated in individual experiments. all the cultures were kept at 37uc, 5% co 2 in incubators that had been pre-saturated with specified concentrations of ethanol. the teer of the airway epithelial cell cultures was measured by using a ''chop stick'' epithelial ohmmeter (world precision instruments, sarasota, fl), as described previously [25, 26] . calu-3 cells, cultured at the air-liquid interface for 2 weeks, were exposed to different concentrations of ethanol for 24 hours and mounted on an ussing chamber apparatus (world precision instruments, sarasota, fl). the cells were bathed with an apical low chloride buffer (135 mm sodium gluconate, 5.0 mm hepes, 1.2 mm mgcl 2 , 0.6 mm kh 2 po 4 , 4 mm cacl 2 , 2.4 mm k 2 hpo 4 3h 2 o, and 10 mm dextrose, ph 7.4) and with a basal high chloride buffer (135 mm nacl, 5.0 mm hepes, 1.2 mm mgcl 2 , 0.6 mm kh 2 po 4 , 1.2 mm cacl 2 , 2.4 mm, k 2 hpo 4 3h 2 o, and 10 mm dextrose, ph 7.4). both buffers were continuously stirred, gassed with 95% o 2 and 5% co 2 and maintained at 37uc. these buffers were to maintain a chloride gradient across the epithelial monolayer. the teer was measured with an open circuit by applying an electrical pulse across the epithelial monolayer. short circuit currents (i sc ) were measured by applying an epithelial voltage clamp. for all the experiments, 100 mm of amiloride was added to the apical side to block sodium channels. anion currents were induced by apical addition of 100 mm adenosine. the a 2b ar blockade was achieved by apical application of 50 mm alloxazine. to differentiate between chloride and bicarbonate currents, 100 mm bumetanide was used basally to block chloride transport. acetazolamide (20 mm) or dnds (4,49dinitro stilbene-2,29-disulfonate) (100 mm) was employed apically to block bicarbonate transport. further, to inhibit epithelial phosphodiesterases, 100 mm of ibmx (3-isobutyl-1-methylxanthine) or 50 mm papaverine was added apically. the air-liquid interface cultures of calu-3 cells were exposed to either 0 mm or 100 mm of ethanol at the basolateral side for 24 hours. on the apical surface the cells were stimulated with 100 mm of adenosine. the cells were washed with pbs containing 100 mm ibmx to inhibit phosphodiesterases that might degrade camp. the cells were lysed and camp was measured by camp immunoassay (r&d systems, minneapolis, mn). ibmx (100 mm) was also included in the lysis buffer. the samples were read at 450 nm using a spectrophotometer (biotek, winooski, vt). all the treatments were carried out in quadruplicates. data presented represent mean of multiple experiments and error bars indicate standard deviation from the mean. where indicated, the data points were analyzed by student's t-test or one-way anova test. the p values smaller than 0.05 were considered statistically significant. to explore if ethanol affects adenosine-activated ion transport function of airway epithelium, we employed air-liquid interface cultures of calu-3 cells, a system widely used to investigate airway epithelial electrophysiological properties [27, 28] . the cultured epithelia were exposed basolaterally for 24 hours to different concentrations of ethanol (0, 25, 50 and 100 mm) and adenosineinduced transepithelial ion transport was assessed by measuring i sc with an ussing chamber apparatus. two buffers with asymmetric chloride were applied: apical low chloride (10.4 mm) and basolateral high chloride (139.8 mm). after voltage clamp, sodium channels were blocked by apical amiloride (100 mm) and i sc was stimulated by apical addition of adenosine (100 mm). as shown (fig. 1) , the ethanol exposure decreased adenosine-activated i sc in a dose-dependent manner. significant differences were detected by figure 1 . effect of ethanol pre-exposure on adenosine-induced epithelial ion transport. calu-3 cells were cultured at an air-liquid interface on membrane filters and basolaterally exposed to 0, 25, 50 and 100 mm of ethanol for 24 hours. these calu-3 epithelia were placed in an ussing chamber with asymmetrical buffers of chloride (apical 10.4 mm and basolateral 139.8 mm). following voltage clamp, stable short circuit baselines were attained in 15 to 20 min. i sc was measured after blocking na + channels with 100 mm of apical amiloride and stimulated with 100 mm of apical adenosine. alterations in ion transport are expressed as difference in i sc from their baseline. ethanol preexposure decreased 100 mm adenosine mediated epithelial ion transport in a dose-dependent manner. asterisks indicate significant differences between groups by one-way anova test (p,0.05, n = 5 for each condition). doi:10.1371/journal.pone.0032112.g001 one-way anova test between the control and the alcoholexposed (50 mm and 100 mm) groups (p,0.05, n = 5). it was previously documented that adenosine-stimulated anion secretion in airway epithelia is largely mediated by chloride transport [17, 29] . to explore if the alcohol-suppressed i sc , as identified above, is actually a chloride current, we first confirmed chloride transport by calu-3 epithelia in our experimental setting. to this end, the cultured calu-3 epithelia without any alcohol exposure were subjected to i sc measurement in the presence of various inhibitors to block different ion channels. bumetanide (100 mm), a specific inhibitor for the na + -k + -2cl 2 cotransporter, was applied to the basolateral side of the calu-3 epithelia after adenosine stimulation. this drug decreased i sc by ,58%, while acetazolamide, a carbonic anhydrase inhibitor, and dnds, an inhibitor for na + /hco 3 2 cotransporters and cl 2 /hco 3 2 exchangers, had no effect on i sc (fig. 2a) . these data suggest that the adenosine-stimulated anion current that is suppressed by alcohol is the chloride channel conductance. the result was further validated by measuring the adenosine-stimulated i sc with identical apical and basal chloride buffers. without chloride gradient (fig. 2b) , adenosine-induced i sc was dropped significantly by student's t-test (p,0.01, n = 5). the i sc was only ,8% of that measured with the asymmetric chloride buffers. the adenosine-stimulated chloride transport is mediated through the cftr channel cftr has been found to be a major chloride channel in the airway epithelium responsible for adenosine-induced ion transport [30] #. to validate if cftr is responsible for the observed adenosine-stimulated chloride transport in the calu-3 epithelia, we applied cftr channel inhibitor cftr inh 172 to the apical side of calu-3 epithelia for 30 minutes, followed by adenosine stimulation. the chloride conductance was decreased by ,76% when cftr was inhibited (fig. 3) , which is significantly lower than that of the no drug control (p,0.01, n = 5). to seek a second approach to confirm the result, cfbe41o cells, an airway epithelial cell line derived from a cystic fibrosis (cf) patient with homozygous df508 mutations in cftr, were used [24] . strikingly, adenosine failed to elicit any chloride currents across the cf epithelia (p,0.01, n = 4). thus, the currents under our experimental condition and drug profile are cftr-mediated chloride conductance. these data altogether confirmed that in airway epithelial cells ethanolsuppression of chloride transport is mediated through cftr, a camp-activated chloride channel. the aforementioned data suggest that the negative modulation of epithelial chloride secretion by ethanol is likely through the adenosine-adenosine receptor signaling pathway. up to date, four different adenosine receptors have been identified: a1, a 2a , a 2b and a3 receptors [31] . in airway epithelial cells, a 2b ar predominantly regulate cftr function [18] . here, we wanted to examine if the same signaling pathway is involved in the alcohol-induced inhibition of cftr-mediated chloride transport. to this end, alloxazine (50 mm), a commonly used a 2b ar specific blocker, was applied to the apical buffer after adenosine stimulation. the data ( figure 4) demonstrate that ,75% adenosine-stimulated epithelial i sc was reduced by alloxazine, indicating that a 2b ar was largely responsible for the adenosinemediated chloride secretion. figure 2 . adenosine-stimulated short circuit current is chloride conductance. a) i sc of calu-3 epithelia was measured by using the asymmetric chloride buffers. after stimulated with 100 mm of apical adenosine, the cells gave rise to i sc of ,75 ma/cm 2 . the i sc was blocked by ,58% by basolateral addition of bumetanide (100 mm), but not by acetazolamide (20 mm) or dnds (100 mm). b) i sc of calu-3 epithelia was measured with either asymmetric chloride buffers or symmetric chloride buffers. the adenosine-stimulated i sc with no chloride-gradient buffers was ,92% lower than that with chloride-gradient buffers. significance of the difference was determined by student's t-test (p,0.01, n = 5). doi:10.1371/journal.pone.0032112.g002 figure 3 . adenosine-stimulated transepithelial i sc was mediated by cftr channel. calu-3 epithelia were stimulated with 100 mm of apical adenosine, resulting in a significant increase in i sc above baseline. such an adenosine-induced i sc was mostly absent in cfbe41o cells in which cftr channel was dysfunctional or was significantly inhibited with 25 mm of cftr inhibitor cftr inh 172. student's t-test was performed to determine the statistic significance (p,0.05, n = 5). doi:10.1371/journal.pone.0032112.g003 ethanol exposure affects cftr-mediated chloride secretion through modulation of cellular camp level upon adenosine binding, the a 2b ar signals through g s protein to activate adenylyl cyclase and raises intracellular camp [13, 32] . because cftr is a camp-activated chloride channel, we hypothesized that ethanol inhibits adenosine-stimulated camp production to cause a reduced cftr channel activity. to test this hypothesis, air-liquid interface cultures of calu-3 cells were exposed basolaterally to either 0 mm or 100 mm of ethanol for 24 hours. these cells were apically stimulated with 100 mm of adenosine as described above for 10 minutes, the time point at which the epithelial i sc was at its peak levels. then, the cells were lysed and the levels of cellular camp assayed. the results demonstrate that ethanol pre-treatment significantly decreased adenosine-stimulated camp levels (p,0.05, n = 5) (fig. 5a ). in addition, to test whether the decrease in camp was in fact causing the suppression of adenosine-stimulated chloride transport by ethanol, sp-camps, a cell permeable and phosphodiesteraseresistant camp analogue, was used to directly activate pka which then activates the cftr channel. as shown in figure 5b , the alcohol-treated calu-3 cells in the presence of 10 mm sp-camps obliterated the ethanol suppressive effect on adenosine-induced chloride secretion (p,0.05, n = 6). thus, we conclude that ethanol inhibits cftr-mediated chloride secretion by directly affecting the cellular camp level instead of the downstream pka enzyme. based on the data that ethanol impairs the adenosine-activated chloride secretion by reducing the cellular camp level, we chose to block endogenous camp degradation pharmacologically to counteract the alcohol-suppressive effect on chloride secretion. air-liquid interface cultures of calu-3 cells were similarly exposed to ethanol for 24 hours and treated with 100 mm of non-specific phosphodiesterase inhibitor ibmx along with adenosine stimulation. the data in figure 6 indicate that ibmx almost completely restored the ethanol suppression of calu-3 chloride secretion (p,0.05, n = 4). papaverine is a clinically used phosphodiesterase inhibitor. this drug also overcame the inhibitory effect of ethanol on adenosine-induced transepithelial chloride conductance (p,0.05, n = 4) (fig. 6) . these results not only confirm that ethanol modulates adenosine-camp signaling but also suggest that phosphodiesterase inhibitors may be useful as the potential therapeutic agents for improving the airway epithelial ion transport and mucociliary clearance in alcoholic patients. airway epithelial cells not only constitute the physical barrier that separates airway lumen from interstitial compartments, but also actively participate in innate and adaptive immunity to protect the host from pulmonary infections [33] . multiple defense systems have been evolved in airways. first, the polarized airway epithelia have a mechanical clearance mechanism. goblet cells or submucosal glands secrete mucus that entraps airborne particles and inhaled infectious agents. synchronized ciliary movement sweeps inhaled particulate matter toward the mouth to be expectorated or swallowed [34] #. second, airway epithelia secrete antimicrobial factors, such as lysozyme and b-defensins [35] . third, airways and alveoli are patrolled by phagocytic cells, most notably the alveolar macrophage, which can engulf microbes [36] . fourth, airways can recruit neutrophils and monocytes to sites of inflammation [37] . fifth, the acquired immune system, including antigen-stimulated t and b lymphocytes, provides cellular and humoral defenses for the airways [38, 39] . it is noteworthy that all the above-mentioned pulmonary defense mechanisms are executed in asl, the thin film of liquid on top of the airway epithelia. therefore, asl homeostasis is pivotal to lung host defense. here we provide the first evidence suggesting that ethanol exposure suppresses adenosine-stimulated chloride secretion that regulates asl. our data demonstrate that ethanol affects adenosine-stimulated chloride secretion through cftr. on epithelial apical surface, adenosine binds to a 2b ar, a predominant adenosine receptor present in airway epithelia [16] . this receptor is coupled to g s protein to activate adenylyl cyclase, which elevates intracellular camp and consequently activates pka. then, cftr is phosphorylated by pka and the channel opens to permeate chloride [40] #. more chloride in asl will cause less na + absorption and more water retention, thus increasing asl height and volume [15, 20] . it is even speculated that adenosine is a sensor for asl homeostasis. when asl falls, adenosine levels increase in airways beyond their basal levels and activate a 2b ar and cftr channels [16, 29] . our data have linked ethanol-suppression of camp levels to attenuation of chloride secretion by airway epithelia. previous studies have documented that ethanol decreases ciliary beating and epithelial cell migration during wound repair which are associated with compromised camp signaling and pka activation [21, 41] . moreover, alcohol is reported to upregulate the pde4 enzyme expression as well as the enzymatic activity in epithelia [22] #, which results in accelerated camp degradation. thus, our data are consistent with the data reported, indicating that alcohol modulates various cellular pathways by reducing cellular camp levels. pulmonary mucociliary clearance is largely affected by three factors: 1) mucus production, 2) ciliary sweeping and, 3) asl state. studies on human bronchial epithelial cells have revealed that a 24-hour exposure with 100 mm of alcohol caused an 8-fold increase in trachea-bronchial mucin gene expression [42] . interestingly, experiments using bovine bronchial epithelial cells have established that alcohol exposure at 100 mm beyond 6 hours decreases ciliary beating frequency [22, 41] . a recent publication by allen-gipson and colleagues reports that purinergic stimulation of cbf requires a 2b ar activation [43] . similarly, in rats chronic alcohol administration decreased ciliary beating frequency and enhanced lung colonization of nasally administered s. pneumoniae [44] #. our current results demonstrate that 24-hour ethanol exposure reduces chloride secretion, which consequently alters asl composition and volume. thus, alcohol affects all three mucociliary clearance components. pharmacologically, blocking camp degradation has the potential of restoring ethanol-suppressed cellular camp levels and therefore epithelial functions. this finding is of clinical implications. phosphodiesterase inhibitors such as papaverine may improve mucociliary clearance in alcoholics by enhancing airway epithelial ion secretion and ciliary beating. moreover, enhancement of airway epithelial ion secretion alters the composition of asl. our laboratory has reported that extracellular chloride levels affect neutrophil microbial killing ability [45] . thus, increasing asl chloride level by phosphodiesterase inhibitors may also improve phagocytic innate immunity in airways of alcoholics. in summary, ethanol exposure compromises chloride ion secretion which is pivotal to maintain asl volume and composition. such an effect may alter the properties of airway host defenses predisposing ethanol abusers to an increased risk of infection in the lung. restoration of cellular camp level with phosphodiesterase inhibitors could potentially ameliorate mucociliary clearance by improving epithelial ion secretion and hence lung host defense. figure 6 . phosphodiesterase inhibitors restore the ethanol suppression of adenosine-induced transepithelial chloride conductance. the air-liquid interface cultures of calu-3 cells were exposed to 0 and 100 mm of ethanol for 24 hours. adenosinestimulated i sc was measured, showing that ethanol-exposed calu-3 epithelia had significantly lower adenosine-stimulated i sc than the no ethanol control. however, phosphodiesterase inhibitors, ibmx (100 mm) or papaverine (50 mm), fully restored the ethanol-suppressed adenosine-stimulated i sc . asterisks indicate statistically significant differences between groups (p,0.05, n = 4 for each group). doi:10.1371/journal.pone.0032112.g006 alcohol, immunosuppression, and the lung alcohol abuse, 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structure and function of the cftr chloride channel chronic ethanol downregulates pka activation and ciliary beating in bovine bronchial epithelial cells transcriptional regulation of tracheo-bronchial mucin (tbm) gene by ethanol adenosine activation of a(2b) receptor(s) is essential for stimulated epithelial ciliary motility and clearance smoke exposure exacerbates an ethanol-induced defect in mucociliary clearance of streptococcus pneumoniae the role of chloride anion and cftr in killing of pseudomonas aeruginosa by normal and cf neutrophils the authors would like to thank drs. richard painter, nicholas lanson, elisa ledet for their helpful discussion of the experiments and critical reading of the manuscript. conceived and designed the experiments: gw. performed the experiments: svr. analyzed the data: gw svr. wrote the paper: gw svr. key: cord-000813-gagakqw4 authors: xue, mei; shi, xingming; zhang, jing; zhao, yan; cui, hongyu; hu, shunlei; gao, hongbo; cui, xianlan; wang, yun-feng title: identification of a conserved b-cell epitope on reticuloendotheliosis virus envelope protein by screening a phage-displayed random peptide library date: 2012-11-21 journal: plos one doi: 10.1371/journal.pone.0049842 sha: doc_id: 813 cord_uid: gagakqw4 background: the gp90 protein of avian reticuloendotheliosis-associated virus (rev-a) is an important envelope glycoprotein, which is responsible for inducing protective antibody immune responses in animals. b-cell epitopes on the gp90 protein of rev have not been well studied and reported. methods and results: this study describes the identification of a linear b-cell epitope on the gp90 protein by screening a phage-displayed 12-mer random peptide library with the neutralizing monoclonal antibody (mab) a9e8 directed against the gp90. the mab a9e8 recognized phages displaying peptides with the consensus motif svqyhpl. amino acid sequence of the motif exactly matched (213)svqyhpl(219) of the gp90. further identification of the displayed b cell epitope was conducted using a set of truncated peptides expressed as gst fusion proteins and the western blot results indicated that (213)svqyhpl(219) was the minimal determinant of the linear b cell epitope recognized by the mab a9e8. moreover, an eight amino acid peptide svqyhpla was proven to be the minimal unit of the epitope with the maximal binding activity to mab a9e8. the rev-a-positive chicken serum reacted with the minimal linear epitopes in western blot, revealing the importance of the eight amino acids of the epitope in antibody-epitope binding activity. furthermore, we found that the epitope is a common motif shared among rev-a and other members of rev group. conclusions and significance: we identified (213)svqyhpl(219) as a gp90-specific linear b-cell epitope recognized by the neutralizing mab a9e8. the results in this study may have potential applications in development of diagnostic techniques and epitope-based marker vaccines against rev-a and other viruses of the rev group. reticuloendotheliosis viruses (revs) are a group of viruses in the family retroviridae, specifically gammaretroviruses in the same genus as mammalian c-type retroviruses [1] . the rev group includes defective rev-t [2, 3] , non-defective rev-a [4, 5] , chick syncytial virus (csv) [6] , duck infectious anemia virus [7] , and spleen necrosis virus (snv) [8] . except for the defective rev-t, all isolated rev strains belong to a single serotype [5] and their genetic sequences show little variation [9] . rev genome consists of three structural genes (gag, pol and env) flanked by long-terminal repeats (ltrs) [10] . the major mature env gene products of revs are the surface glycoprotein (gp90) and the transmembrane protein (gp20) [11, 12] . the gp90 protein containing both continuous and discontinuous epitopes functions as the immunodominant protein [13] and is responsible for eliciting rev antibodies. previous studies indicated that the cterminal epitope of gp90 was exposed on the outer surface of the rev-a-infected cell [12] . however, the epitope identified in rev gp90 protein has not been finely mapped, and the core sequence of the epitope needs to be determined. detailed analysis of epitopes is important for the understanding of immunological events, and the development of epitopebased marker vaccines and diagnostic tools for various diseases [14, 15] . in this study, we prepared a neutralizing monoclonal antibody (mab) against gp90 protein from the rev-a strain hlj07i, and used it to screen a phage-displayed random 12mer peptide library for the linear b-cell epitope. this study describes the first identification of the precise location of the epitope on gp90 protein. the information provided in this study will facilitate the development of specific serological diagnosis of rev infection, and will contribute to the rational design of vaccines by further understanding of the antigenic structure of gp90. care of laboratory animals and animal experimentation were performed in accordance with animal ethics guidelines and approved protocols. all animal studies were approved by the animal ethics committee of harbin veterinary research institute of the chinese academy of agricultural sciences (syxk (h) 2006-032). rev-a strain hlj07i (genbank accession no. gq375848) was isolated from heilongjiang province in china in 2007. chicken embryo fibroblasts (cefs) were prepared as primary cultures from 10-day-old chicken embryos as previously described [16] and were grown in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal bovine serum plus antibiotics. viruses were grown in cefs and incubated at 37uc with 5% co 2 for 5 days. the suspension was frozen and thawed three times to disrupt cells and release virus, and then clarified by two centrifugation steps (2000 g for 15 min, and 10,000 g for 60 min). virus present in the upper phase was precipitated with 10% (w/v) polyethylene glycol 6000 (peg 6000) for 4 hours at 4uc. precipitates were collected by centrifugation at 9,000 g for 30 minutes and resuspended in tne buffer (50 mm tris-hc1, ph 7.5; 0.1 m nac1, 10 mm edta). finally, they were centrifuged through a 30% (w/v) sucrose cushion for 90 minutes at 200,000 g and resuspended in tne buffer. the purified virus was analyzed in sds-page. six-week-old female balb/c mice were subcutaneously immunized with 100 mg of the purified recombinant gp90 protein emulsified with an equal volume of freund's complete adjuvant (sigma, st. louis, mo, usa). two boosters of the freund's incomplete adjuvant (sigma, st. louis, mo, usa) emulsified antigen were given at two week interval. two weeks after the third immunization, the mice were intraperitoneally boosted with 100 mg antigen alone. three days later, the spleen cells from immunized mice were fused with myeloma cells sp2/0 (sp2/0-agl4; atcc crl 1581) [17] , using 50% (wt/vol) polyethylene glycol and 10% dimethyl sulfoxide (dmso) (vol/vol) (sigma, st louis, mo, usa). hybridomas were screened by indirect enzyme-linked immunosorbent assay (elisa) and indirect immunofluorescence assay (ifa). the hybridomas producing mabs were cloned three times by limiting dilution of the cells. antibody subtype identification was performed using sba clonotyping tm system/hrp kit (southern biotech, birmingham, al, usa). plates were coated with 100 ml/well of purified rev gp90 antigen diluted in carbonate-bicarbonate buffer (ph 9.6) for incubation overnight at 4uc. following 4 washes with 200 ml/ well of pbs/0.05% tween-20, the plates were blocked with 200 ml/well of blocking buffer (pbs containing 5% skim milk) for 1 h at 37uc. the supernatant of hybridoma culture (100 ml/well) was added in duplicate and the plates were incubated for 1 h at 37uc. after washing three times with pbs, 100 ml of horseradish peroxidase (hrp)-conjugated goat anti-mouse immunoglobulin g (igg, 1:5,000 dilution,sigma, st louis, mo, usa) was added to each well and incubated for 1 h at 37uc. plates were washed three times with pbs and incubated with 100 ml/well of o-phenylenediamine dihydrochloride (opd, sigma, st louis, mo, usa) containing 0.3% h 2 o 2 for 5 minutes at room temperature in the dark. the reaction was stopped with 50 ml/well of 2 m h 2 so 4 and the absorbance measured at 492 nm. about 70-80% confluent cef cells in 96-well plates were infected with rev-a hlj07i at a moi of 0.2. at 5 days postinfection, the infected cells were fixed with icy cold ethanol absolute for 15 min at 4uc, and air dried. the fixed cells were incubated with mab a9e8, rev-a-positive chicken serum, antiporcine ifn-c mab (sigma, st louis, mo, usa), or rev-anegative chicken serum for 1 h at 37uc. after washing three times with pbs, 50 ml/well of fitc-conjugated goat anti-mouse igg or fitc-conjugated rabbit anti-chicken igg (sigma, st louis, mo, usa) at 1:100 dilutions were added and incubated for 1 h at 37uc. the cells were rinsed three times with pbs and once with deionized water, and mounted in 50 ml of 90% glycerol in pbs, and then observed under the nikon eclipse ti-e microscope equipped with nis-elements ar software. the micro-neutralization assay was modified from a previously described procedure [18] . the ascitic fluid was heat inactivated for 30 min at 56uc, and two fold serial dilutions were incubated with 2610 3 tissue culture infective doses 50% (tcid 50 /ml ) of rev-a in a 96-well micro-plate. four uninfected control wells were included on each plate as control wells. after 2 h incubation at 4uc, 100 ml of cef cells at 1.5610 5 cells/ml was added to each well. the plates were incubated for 5 days at 37uc and 5% co 2 . the monolayers were washed with pbs and fixed in icy cold ethanol for 15 minutes. the presence of viral gp90 protein was detected by elisa with the mab a9e8. the absorbance was measured at 492 nm with an elisa microplate reader. the average a492 was determined for quadruplicate wells of virusinfected and uninfected control wells, and a neutralizing endpoint was determined by using a 50% specific signal calculation. the endpoint titer was expressed as the reciprocal of the highest dilution of ascitic fluid with a492 value less than x, where 6= [(average a492 of infected wells) 2 (average a492 of control wells)]/2+ (average a492 of control wells). the ph.d.-12 tm phage display peptide library kit was purchased from new england biolabs inc. the dodecapeptide library consisted of 2.7610 9 electroporated sequences (1.5610 13 pfu/ml). the mab was purified from the ascites uid of mice inoculated with the hybridma cells secreting a9e8 by affinity chromatography using rprotein g agorose (invitrogen, carlsbad, ca,usa) according to the manufacturer's instructions. the concentration of the purified protein was determined using the bradford protein assay kit (beyotime, shanghai, china). three successive rounds of biopanning were carried out according to the manufacturer's instruction manual. briey, one well of a 96well microtiter plate was coated with 10 mg/ml of mab a9e8 in coating buffer (0.1 m nahco 3 , ph 8.6) overnight at 4uc, followed by blocking with blocking buffer (0.1 m nahco 3 , ph 8.6, 0.02% nan3, and 5 mg/ml bsa) for 2 h at 4uc. the phage library (1.5610 11 phages/100 ml) was added to the blocked wells and the plate incubated for 1 h at room temperature. the unbound phages were removed by successive washings with tbs buffer (50 mm tris-hcl, ph 7.5, 150 mm nacl) containing gradually increased concentrations (0.1%, 0.3%, and 0.5%) of tween-20, and the bound phages were eluted by 0.2 m glycine-hcl containing 1 mg/ml bsa (ph 2.2) and immediately neutralized with 1 m tris-hcl (ph 9.1). the eluted phages were amplified by infecting e. coli (er2738), and were titered on lb/iptg/xgal plates for the subsequent rounds of selection. the output to input ratio was calculated as follows: (titer of the amplified eluent phages/titer of the input phages (1.5610 11 ))6100%. after three rounds of biopanning, eight individual phage clones were selected for target binding in elisa as described in the manufacturer's instructions. briey, 96-well plates were coated with 100 ng of purified mab a9e8, or anti-porcine ifn-c mab (sigma, st louis, mo, usa) as negative controls overnight at 4uc. the coated wells were blocked for 2 h at room temperature and then the phages (10 10 pfu/100 ml/well) diluted in blocking solution were added. the plates were incubated for 1 h at room temperature followed by washing ten times with tbst. bound phages were subjected to reaction with horseradish peroxidase (hrp)-conjugated sheep anti-m13 antibody (pharmacia, piscataway, ny, usa), followed by color development with substrate solution containing o-phenylenediamine (opd). the positive phage clones identified by phage elisa were sequenced with the 296 giii sequencing primer 59-tga gcg gat aac aat ttc ac-39 as described in the manufacturer's instructions. a series of complementary oligonucleotides (table 1) coding for wild-type and truncated motif svqyhpl were synthesized, annealed, and cloned into the bamhi/xhoi sites of the prokaryotic expression vector pgex-6p-1 (pharmacia, piscataway, ny, usa), producing a group of recombinant plasmids. all the resulting recombinant plasmids were validated by restriction analysis and dna sequencing. expression plasmids were transformed into bl21 (de3) competent cells, followed by the addition of 1 mm isopropyl-d-thioga-lactopyranoside (iptg; ge healthcare, usa) for induction. approximately equivalent amount of each gst fusion protein was subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% sds-page). the gel was either stained with commassie blue staining solution or electrophoretically transferred to nitrocellulose membrane. after being blocked with 5% skim milk in pbs overnight at 4uc, the membrane was incubated with mab a9e8 (diluted 1:2,000 in pbs) or rev-a-positive chicken serum (diluted 1:100 in pbs) at 37uc for 1 h. after being washed three times with pbst, the membrane was probed with a 1:5,000 dilution of hrp-conjugated goat anti-mouse igg to investigate the conservation of the epitope among rev viruses, sequence alignment of the epitope and the corresponding regions on gp90 proteins of 32 rev-a strains, one rev-t strain, four snv strains and one csv strain was performed using the dnastar lasergene program (windows version; dnastar inc., madison, wi, usa). purified gp90 protein was used to immunize balb/c mice. after cell fusion and screening, several hybridoma cell lines were generated, which produced gp90-reactive mabs. one monoclonal antibody produced by the line designated as a9e8 was selected for strong reactivity with recombinant gp90 protein in western blot ( figure 1a ) and in an indirect elisa (data not shown). it also showed strong reactivity with purified whole virus in western blot ( figure 1b) and could be used to detect rev-a antigen by an indirect immunofluorescence assay (ifa; figure 1c ). the mab a9e8 was compose of an igg2b heavy chain paired with a k-type light chain, as determined using the sba clonotyping tm system/ hrp kit. the titers of antibody in hybridoma cell culture supernatants and in ascites were measured by indirect elisa and determined to be 1:3,200 and 1:128,000, respectively. the neutralizing activities of the mab a9e8 were then determined by a micro-neutralization assay on cef cells using rev-a hlj07i. the mab a9e8 neutralized the virus with a neutralization titer (nt 50 ) of 100. to determine the epitope recognized by mab a9e8, biopanning of a phage displayed 12-mer random peptide library was performed using the affinity purified mab a9e8. after three rounds of biopanning, an enrichment of phages bound to the mab a9e8 was obtained. the output to input ratios of the three rounds of biopanning were 0.00008%,0.038% and 0.79%. eight phage clones were selected for reactivity with the mab a9e8 after three rounds of biopanning and enrichment of the phages binding to the mab a9e8. these selected clones were further evaluated by phage elisa for reactivity with the mab a9e8 and a negative control mab (anti-porcine ifn-c). as shown in figure 2 , all the selected eight phage clones (a1-a8) showed specific reactivity with a9e8 (od492 nm .1.10), but not with anti-porcine ifn-c mab (od492 nm ,0.15). the eight phage clones were sequenced, and were shown to display a consensus sequence svqyhpl, which was identical to the motif 213 svqyhpl 219 at the c-terminus of the gp90 protein of rev-a strain hlj07i (table 2) . to verify whether the identified motif represented an epitope recognized by the mab a9e8, a dna fragment coding for the motif svqyhpl was expressed as a gst fusion protein (gst-h7wt) in e. coli. western blot analysis showed that the fusion protein was recognized by the mab a9e8 ( figure 3a ) and rev-a infected chicken antiserum ( figure 3b ), indicating that the motif represented a linear b-cell epitope. to define the epitope precisely, four mutants with deletions at c-and n-termini of the motif svqyhpl (table 1) were constructed to express the gst fusions gst-h7ds, gst-h7dl, gst-h7dsv, and gst-h7dpl representing -vqyhpl, svqyhp-, -qyhpl and svqyh-(deletions were shown as dashes) in e. coli, respectively. we found that only the full-length svqyhpl polypeptide (gst-h7wt) was recognized by the mab a9e8 ( figure 3a ). removal of one or more amino acids at either the amino or carboxyl terminus of the peptide abolished antibody binding, indicating that the peptide svqyhpl represented the minimal requirement for the reactivity of the epitope with a9e8. minimal unit of the epitope with the maximal binding activity to mab a9e8 to investigate minimal unit of the epitope with the maximal binding activity to mab a9e8, a series of gst-fusion proteins were expressed with extended amino acid residues at both n and c termini of the motif svqyhpl (table 1) . these gst-fusion proteins were subjected to sds-page and testing for reactivity with mab a9e8 in western blot. fusion proteins gst-r1 (svqyhpla), gst-r2 (svqyhplal) and gst-r3 (svqyh-plalp) reacted strongly with mab a9e8 in western blot ( figure 4) . the gst-r2 and gst-r3 showed similar binding activity to the gst-r1, indicating that alanine alone significantly increased binding activity of the core epitope to mab a9e8. in contrast, gst-fusion proteins with extended amino acid residues at the n terminus of the motif svqyhpl showed no increased binding activity compared with gst-h7wt in western blot (data not shown). taken together, these results showed that svqyh-pla was the minimal unit of the epitope with the maximal binding activity to mab a9e8. to investigate the conservation of the svqyhpl epitope, we aligned the epitope identified in this study with revs gp90 coding regions available in genbank. the alignment results showed that all amino acids in the motif were identical among all rev strains ( figure 5 ), indicating that the motif represented a conserved epitope on the gp90 protein of revs. figure 2 . detection of the selected phages for antibody binding by phage elisa. eight phage clones selected after three rounds of biopanning were added to the microplate wells (10 10 pfu/100 ml/well) coated with the mab a9e8 or anti-porcine ifn-c mab (negative control) (100 ng/well ), and incubated for 1 h at room temperature. bound phages were subjected to reaction with horseradish peroxidase (hrp)-conjugated anti-m13 antibody, followed by color development with substrate solution containing o-phenylenediamine (opd). three independent assays were performed for each selected phage. doi:10.1371/journal.pone.0049842.g002 table 2 . sequence comparison of random peptide inserts displayed on the positive phages. amino acid sequence of the insert a conservative amino acid motifs are bold and underlined. doi:10.1371/journal.pone.0049842.t002 the gp90 protein of rev is an important antigenic protein and is associated with virus neutralization, which is the major candidate antigen for vaccine development and disease serological diagnosis [12, 13] . studies showed that recombinant gp90 protein expressed in pichia pastoris induced a protective immune response against rev in chickens [19] . precise mapping of epitopes in gp90 is important for understanding antibody-mediated protection and developing epitope-based marker vaccines and diagnostic tools. cui et al. [20] reported the generation and partial characterization of a panel of 11 mabs against the nondefective rev strain t, and showed that the epitope was on the viral envelope glycoprotein. however, they only identified the relative regions in rev envelope glycoprotein recognized by the mabs, and did not map the fine locations of the epitopes. to our knowledge, there has been no report on linear epitope mapping of the gp90 of rev. mapping epitopes using monoclonal antibodies has become a powerful tool to study protein structure and has been used to diagnose diseases and design marker vaccines [21, 22, 23] . in this study, we described the generation and epitope mapping of a gp90 protein specific mab, and demonstrated that the epitope was conserved among the rev group. precise analysis of rev-a gp90 protein epitope will provide the fundamental information for development of epitope-based vaccines and diagnostic tools for rev-a and/or other rev group infection. phage display is an in vitro selection technique in which a peptide or protein is genetically fused to a coat protein of bacteriophage and the fused peptide or protein is displayed on the exterior surface of the phage virion. the phage displayed random peptide library is a powerful and high throughput tool for rapid mapping of epitopes [24] . in this study, we generated a gp90-specific mab a9e8 using recombinant gp90 protein expressed in e. coli. the mab a9e8 showed strong reactivity against purified whole virus in western blot and could be used to detect rev-a antigen by an indirect immunofluorescence assay. the linear epitope recognized by the mab a9e8 was defined as svqyhpl by screening a random phage display peptide library. this peptide sequence was identical to 213 svqyhpl 219 of the gp90 protein of rev-a. n-or cterminal deletions of amino acids of this epitope demonstrated that 213 svqyhpl 219 is the minimal requirement for recognition by a9e8. fusion proteins gst-r1 with extended amino acid residues at the c terminus of the motif svqyhpl showed increased binding activity compared with that of gst-h7wt in western blot, indicating that alanine alone significantly increased binding activity of the core epitope to mab a9e8. thus, the peptide svqyhpla was determined to be the minimal unit of the epitope with the maximal binding activity to mab a9e8. the peptide was also recognized by rev-a-positive chicken serum, revealing the importance of the eight amino acids of the epitope in antibody-epitope binding reactivity. sequence alignments of rev-a strains, rev-t strain and five other rev strains demonstrated that the motif was highly conserved among rev viruses, indicating that it is a broad group-specific epitope. since a9e8 was identified as a neutralizing mab, the epitope identified with a9e8 in this study was a neutralizing epitope. many neutralizing epitopes have been mapped in the variable regions of the proteins of viruses, including infectious bursal disease virus [25] , infectious bronchitis virus [26] , hepatitis c virus [27] , and hiv [28] . some neutralizing epitopes, however, are highly conserved across most of the viruses in the same group [29, 30] . a novel epitope was mapped within the highly conserved flavivirus fusion loop peptide 98 drxw 101 by phage-display biopanning and structure modeling using mab 2a10g6 that had broad cross-reactivity with dengue virus (denv) 1-4, yellow fever virus (yfv), west nile virus (wnv), and japanese encephalitis virus (jev) viruses. this mab potently neutralized denv 1-4, yfv, and wnv and conferred protection against lethal challenge with denv 1-4 and wnv in murine model. further functional studies revealed that 2a10g6 blocked infection at a step after viral attachment. these results show that the broad cross-reactivity epitope recognized by neutralizing mab 2a10g6 is highly conserved among denv 1-4, yfv and wnv [29] . an epitope recognized by mab 51 belonging to isotype igm was mapped to 215 kqekd 219 of the vp1 capsid protein of enterovirus 71 (ev71), which possessed neutralizing activity in vitro and provided 100% in vivo passive protection against lethal challenge with ev71 strain hfm 41. blast analyses of the neutralizing epitope revealed that it was highly conserved among all ev71 strains, but not coxsachievirus 16 [30] . in this study, the epitope recognized by neutralizing mab a9e8 was mapped to a highly conserved region of the gp90 protein among revs, which would be useful for development of rev marker vaccines and diagnostic techniques. in summary, a highly conserved neutralizing linear b-cell epitope on the gp90 protein of rev-a was identified in this study. the identified conserved epitope may have potential for development of rev specific diagnostic assays and epitope-based marker vaccines. identification of an epitope on rev gp90 protein plos one | www.plosone.org retrovirus restriction revealed transformation by reticuloendotheliosis virus: development of a focus assay and isolation of a nontransforming virus hematopoietic cell transformation by reticuloendotheliosis virus: characterization of the genetic defect tolerance, viral shedding, and neoplasia in chickens infected with non-defective reticuloendotheliosis viruses serologic differences among nondefective reticuloendotheliosis viruses cultivation of a filterable agent associated with marek's disease duck infectious anemia virus associated with plasmodium lophurae a new virus of ducks interfering with development of malaria parasite (plasmodium lophurae) phylogenetic analyses indicate little variation among reticuloendotheliosis viruses infecting avian species, including the endangered attwater's prairie chicken full genome sequence and some biological properties of reticuloendotheliosis virus strain apc-566 isolated from endangered attwater's prairie chickens biosynthesis and chemical and immunological characterization of avian reticuloendotheliosis virus env geneencoded proteins site-directed cytotoxic antibody against the cterminal segment of the surface glycoprotein gp90 of avian reticuloendotheliosis virus the immunodominant proteins of reticuloendotheliosis virus identification of immunodominant b-and t-cell combined epitopes in outer membrane lipoproteins lipl32 and lipl21 of leptospira interrogans identification of a conserved linear b-cell epitope at the n-terminus of the e2 glycoprotein of classical swine fever virus by phage-displayed random peptide library plasma membrane proteins of normal and rous sarcoma virus-transformed chick-embryo fibroblasts a better cell line for making hybridomas secreting specific antibodies detection of antibody to avian influenza a (h5n1) virus in human serum by using a combination of serologic assays recombinant gp90 protein expressed in pichia pastoris induces a protective immune response against reticuloendotheliosis virus in chickens monoclonal antibodies against avian reticuloendotheliosis virus: identification of strain-specific and straincommon epitopes structure and function analysis of therapeutic monoclonal antibodies against dengue virus type 2 identifying diagnostic peptides for lyme disease through epitope discovery first peptide vaccine providing protection against viral infection in the target animal: studies of canine parvovirus in dogs phage display for epitope determination: a paradigm for identifying receptor-ligand interactions sequence analysis and expression of the host-protective immunogen vp2 of a variant strain of infectious bursal disease virus which can circumvent vaccination with standard type i strains amino acids within hypervariable region 1 of avian coronavirus ibv (massachusetts serotype) spike glycoprotein are associated with neutralization epitopes in vivo and in vitro evidence that cross-reactive antibodies to c-terminus of hypervariable region 1 do not neutralize heterologous hepatitis c virus broadly neutralizing antibodies elicited by the hypervariable neutralizing determinant of hiv-1 a broadly flavivirus cross-neutralizing monoclonal antibody that recognizes a novel epitope within the fusion loop of e protein characterization of an isotype-dependent monoclonal antibody against linear neutralizing epitope effective for prophylaxis of enterovirus 71 infection key: cord-000326-a18rch1f authors: zhou, jun-wei; tsui, stephen k. w.; ng, maggie c. y.; geng, hua; li, sai-kam; so, wing-yee; ma, ronald c.; wang, ying; tao, qian; chen, zhen-yu; chan, juliana c. n.; ho, yuan-yuan title: apolipoprotein m gene (apom) polymorphism modifies metabolic and disease traits in type 2 diabetes date: 2011-02-24 journal: plos one doi: 10.1371/journal.pone.0017324 sha: doc_id: 326 cord_uid: a18rch1f this study aimed at substantiating the associations of the apolipoproein m gene (apom) with type 2 diabetes (t2d) as well as with metabolic traits in hong kong chinese. in addition, apom gene function was further characterized to elucidate its activity in cholesterol metabolism. seventeen apom snps documented in the ncbi database were genotyped. five snps were confirmed in our study cohort of 1234 t2d and 606 control participants. three of the five snps rs707921(c+1871a), rs707922(g+1837t) and rs805264(g+203a) were in linkage disequilibrium (ld). we chose rs707922 to tag this ld region for down stream association analyses and characterized the function of this snp at molecular level. no association between apom and t2d susceptibility was detected in our hong kong chinese cohort. interestingly, the c allele of rs805297 was significantly associated with t2d duration of longer than 10 years (or = 1.245, p = 0.015). the rs707922 tt genotype was significantly associated with elevated plasma totaland ldlcholesterol levels (p = 0.006 and p = 0.009, respectively) in t2d patients. molecular analyses of rs707922 lead to the discoveries of a novel transcript apom5 as well as the cryptic nature of exon 5 of the gene. ectopic expression of apom5 transcript confirmed rs707922 allele-dependent activity of the transcript in modifying cholesterol homeostasis in vitro. in conclusion, the results here did not support apom as a t2d susceptibility gene in hong kong chinese. however, in t2d patients, a subset of apom snps was associated with disease duration and metabolic traits. further molecular analysis proved the functional activity of rs707922 in apom expression and in regulation of cellular cholesterol content. the human apolipoprotein m gene (apom, gene id: 55937) is located on chromosome 6p21.33 and contains six exons spanning a region of 2.3 kb in length with gene structure conserved across species [1, 2] . in human and mice, apom mrna is highly expressed in liver and kidney [2] . the human apom protein (mim 606907) of 188 amino acids is mainly associated with hdl and to a minor degree with ldl, very low density lipoprotein, and chylomicrons [2] . plasma apom has been positively associated with plasma total cholesterol (tc), ldl cholesterol (ldl-c), and hdl cholesterol (hdl-c) [3] . apom knockdown in mice by sirna revealed its anti-atherosclerotic effect by participating in pre-b hdl formation and reverse cholesterol transport [4] . kruit et al., recently reported the effect of cellular cholesterol accumulation on beta cell dysfunction in type 2 diabetes [5] . such finding implies that factors (i.e., apom) affecting the balance of cellular cholesterol content are likely to modify beta cell function and thus the susceptibility to or progression of type 2 diabetes. several additional lines of evidence also indicated the possible involvement of apom in the development of diabetes and metabolic disturbances: 1) the human apom gene is located within a high susceptibility region (6q21-q23) to type 2 diabetes (t2d) in genome-wide linkage analyses [6] . 2) snp rs805296 (t-778c) in apom promoter has been associated with the levels of plasma total cholesterol (tc) and fasting plasma glucose (fpg) in non-diabetic participants, 3) snp rs805296 has also been associated with the susceptibility to t2d and coronary artery disease among the northern chinese [7, 8] . in 2010, china became the country with the largest diabetic population in the world. the northern and southern chinese populations are distinct in genetic marker analyses [9] , meaning disease markers identified in northen populations may not be shared by the southern populations. the primary aim of the current study is to establish the association between apom and t2d susceptibility in a southern chinese cohort in hong kong. by assuming the same effect size (or = 1.934) and disease allele frequency as observed in the studies of northern chinese [8] , the power of the current case-control study is over 95% with 1234 cases and 606 controls. the secondary aims are to examine for association between apom and component metabolic traits as well as to further assess the function of the gene. the pilot cohort consisted of 103 male and 95 female controls (average age = 43 yrs). they were hong kong chinese adults recruited from a community health screening program of cardiovascular risk factors with normal response at a 75 g oral glucose tolerance test [10] . the study cohort had 1234 unrelated t2d patients and 606 controls. all participants gave written informed consent at the time of blood sampling. ethics approval was obtained from the clinical research ethics committee of chinese university of hong kong, shatin, nt, hong kong. all t2d participants were selected from the hong kong diabetes registry. control participants were recruited in a community health screening program for cardiovascular risk factors and some were hospital staff (3.1%, n = 19). no subdemographic differences were detected in control participants. all control participants had no known history of diabetes and had fasting plasma glucose (fpg) , 6.1 mmol/l. clinical assessments of participants had been described elsewhere [11] . body mass index (bmi), blood pressure (bp) as well as fasting blood biochemical and metabolic profiles were measured. among the 1234 t2d patients, 9.8% (n = 121) were on diet treatment only, 41.3% (n = 510) were on oral anti-diabetic drugs only, 12.5% (n = 154) were on insulin only, 9.5% (n = 117) on both oral anti-diabetic drugs and insulin, and 7.3% (n = 90) were treated for dyslipidemia. 2.1. snp selection and genotyping analyses. genomic dna was prepared from whole blood as previously described [12] . seventeen apom snps including rs6921907, rs1266078, rs9267528, rs805297, rs4947251, rs9404941, rs805296, rs805264, rs3117581, rs34490746, rs11462733, rs2273612, rs707922, rs707921, rs28432254, rs3132449, rs3178094 enlisted in the ncbi database [13] were selected for genotyoping in the pilot cohort of 198 controls by multiplex reactions using the mass array system (sequenom, san diego, ca, usa) at the genome quebec innovation centre, mcgill university (montréal, quebec, canada). six of the seventeen snps were confirmed in the pilot cohort: rs1266078(t-1628g), rs805297(c-1065a), rs9404941(t-855c), rs805264(g+203a), rs707922(g+1837t) and rs707921(c+1871a). these six snps were further genotyped in the study cohort of 1840 participants (1234 cases and 606 controls). case and control dna samples were genotyped in parallel on the same plates. two hundred ninety one duplicate samples (15.8%) were used to assess intra-plate and inter-plate genotype quality. no genotyping discrepancies were detected. the overall call rate was 98.0%. five out of the six snps (except for rs1266078) were successfully genotyped in the study cohort. 2.2. plasma lipids and apom levels. plasma apom concentration was estimated by dot-blot analysis using monoclonal mouse anti-human apom antibody (abnova, taipei, taiwan) following previously established protocols [14, 15] . recombinant human apom (abnova, taipei, taiwan) was used as protein standard after serial dilution. the mean signal densities of each specimen and protein standards in triplicate measures were determined by imagej 1.42q software (http://rsbweb.nih.gov/ij/). apom concentration was derived from the standard curves developed using the recombinant apom protein. ectopic apom1 and apom5 expression. wrl-68 and hepg2 hepatic cell lines were purchased from american type culture collection (rockville, md, usa) and maintained in rpmi-1640 medium supplemented with 10% fbs and 100 units/ ml penicillin and 100 mg/ml streptomycin in a humidified atmosphere containing 5% co 2 at 37uc. prior to the expreiments measuring cellular and medium cholesterol content, cells were switched to serum free and phenol red free rpmi medium (invitrogen, carlsbad, ca, usa). ectopic expression of apom was achieved by transient transfection of apom1 or apom5 cdna (cloned into the pcmv-myc vectors) into cultured cells at 70% confluence using lipofectaminetm 2000 reagent (invitrogen, carlsbad, ca, usa) following the manufactorer's protocols [16] . cellular lipids were extracted as previously described [17] . total cellular cholesterol was measured using the infinitytm cholesterol liquid stable reagent (thermo fisher scientific inc., middletown, va, usa) following the manufacturer's instructions. the measured amount of total cholesterol was normalized by cell protein concentration. 3.1. comparative genomic and protein sequence analyses. apom transcript and gene sequences were obtained from the ncbi human genome browser [18] , the ensembl genome browser [19] and the human expressed sequence tags (est) databases. the evolutionary conserved regions browser [20] and the ensembl genome browser were used to identify sequence conservation. 3.2. rapid amplification of cdna ends (race). the human liver firstchoice race-ready cdna kit (ambion, austin, texas, usa) was used to amplify the 59 and 39 ends of novel apom transcripts (supplementary figure s1 , and supplementary table s1). 3.3. semi-quantitative rt-pcr analysis. human normal adult tissue rna samples were purchased commercially (stratagene, la jolla, ca, usa or millipore chemicon, billerica, ma, usa). cdna was synthesized using the geneamp rna pcr kit (applied biosystems, foster city, ca, usa) in combination with rnase inhibitor (roche applied science, indianapolis, in, usa) and m-mulv reverse transcriptase. the subsequent pcr amplification of cdna was performed using the amplitaq gold dna polymerase (applied biosystems, foster city, ca, usa) following standardized protocols [21, 22] . the vulgate transcript apom1 (ensembl: enst00000375916) and the novel transcript apom5 were amplified using primer rt-pcr-yy12 paired with rt-pcr-yy13 and rt-pcr-yy12 paired with rt-pcr-yy14, respectively (supplementary table s4 ). gapdh was amplified as a house-keeping gene control for rna integrity and equal loading using the gapdh primers, rt-pcr-tao1 and rt-pcr-tao2 [23] (supplementary table s1 ). continuous variables were compared using student's t test or one-way analysis of variance (anova) for traits with normal distribution. plasma triglycerides (tg) were skewed and logarithmically transformed. association tests between genotypes and quantitative traits were performed in t2d patients and nondiabetic controls separately. categorical variables, including genotype distributions were compared by x 2 tests. genotype distributions were tested for hardy-weinberg equilibrium using goodness-of-fit test (1 df). informative missingness was checked by coding successful genotypes into one group and failed genotypes into another group followed by 262 chi-square test for t2d and t-test for the quantitative traits of interest. one snp (rs805264) with significant result (p,0.001) indicative of informative missingness (im) was excluded for further t2d association analyses (supplelmentary table s2 ). pairwise ld of d' and r 2 analyses were performed using haploview (broad institute of mit and harvard, usa, version 4.0). 262 contingency tables were used for comparing the differences of allele frequencies and 263 contingency tables were used for detecting the differences of genotype frequencies between cases and controls. allelic, dominant, recessive and additive genetic models were used to test the association between each snps and t2d. multivariate logistic regression analysis was used to assess the significance of covariates and adjusted for confounders in the association of genetic factors with t2d. the independent contributions of all traits, covariates, snps, and haplotypes were determined by multiple regression analysis. association between haplotypes and t2d or metabolic traits were tested by haploview (version 4.0, broad institute of mit and harvard, usa) and phase software (version 2.1, uw tech-transfer digital ventures, university of washington, seattle, wa, usa) [24] . when using phase software, the probability thresholds were set at 90% for haplotype inference to deal with ambiguous haplotypes. it was only used to infer the haplotype of each individual and thus the case-control permutation test was not conducted. the phase-imputed haplotypes were counted 20 times using different seed numbers. no difference between runs was detected. to account for multiple testing, we used the bonferroni correction and a statistical significance was considered only when an snp association with t2d/metabolic traits was p,0.017 (equivelent to 0.05/3), and a haplotype association with t2d/ metabolic traits was p,0.0125 (equivelent to 0.05/4). the human plasma apom concentrations determined by dot-blot assays were compared by a nonparametric kruskal-wallis h test. a value of p,0.05 was considered significant. results from functional analyses were analyzed by student's ttest for two-group comparison, and one way anova for multiple group comparisons. a statistical significance is considered at p,0.05 level. all statistical analyses were performed using the spss program (spss version 15.0, chicago, il, usa) unless otherwise specified. seventeen apom snps enlisted in the public databases were selected for genotyping in a pilot cohort of 198 control participants. six snps were confirmed polymorphic in this pilot cohort of hong kong chinese. these six snps were further genotyped in the full study cohort of 1840 participants. five of the variants were successfully genotyped: rs805297(c-1065a), rs9404941(t-855c), rs805264(g+203a), rs707922(g+1837t), and rs707921(c+1871a) with genotype distributions fitting hardy-weinberg equilibrium. table 1 summarized the allele and genotype frequencies of snps in non-diabetic controls and t2d patients. snp rs805264 was removed from further association analysis for t2d due to informative missingness (supplementary table s2 ). 2.1. apom snps and t2d susceptibility. no significant association was detected between individual snps and t2d (table 1) . further multiple logistic regression analysis adjusting for age, bmi, sbp (systolic blood pressure), dbp (diastolic blood pressure), tc and tg again detected no significant association between individual snps and t2d (data not shown). 2.2. apom snps and t2d duration. we next examine the association between apom snps and t2d disease duration. t2d patients were subgrouped into disease duration of # 10 years (n = 583) and disease duration of .10 years (n = 586). as shown in table 2 , the c allele of rs805297 was associated with t2d duration of longer than 10 years (odds ratio = 1.245, p = 0.015). 2.3. apom snps and metabolic traits. we next analyzed the association between snps and metabolic variables in patients and controls separately. snp rs805297(c-1065a) was not associated with metabolic traits in either patients or controls. since rs707922(g+1837t) was in near perfect ld with rs805264(g+203a) and rs707921(c+1871a) (supplementary figure s2b) , similar association results were expected and observed. table 3 showed the representative results using rs707922 as the marker snp. under recessive model, homozygous minor allele tt of rs707922 was associated with significantly higher tc (p = 0.006), ldl-c (p = 0.009) in t2d patients. in controls, no association between snps and metabolic traits was detected. when plasma apom concentration was measured in t2d patients and controls subgrouped by their rs707922 genotype, the tt genotype was found associated with significantly higher apom level as compared to the gt (and gg) genotype(s) (p = 0.002). as mentioned above, rs805264, rs707922, and rs707921 are located within the same ld block. therefore, in the subsequent haplotype analysis, rs707922 was used to 'tag' the three snps. haplotype construction was conducted for rs707922(g+1837t) with other independent snps rs805297(c-1065a) and rs9404941(t-855c). four haplotypes (a-t-g, c-c-g, c-t-g, and c-t-t) accounting for 99.9% of all possible haplotypes were detected in our hong kong chinese population (supplementary table s3 ). no significant association was found between these haplotypes with t2d. homozygous c-t-t was significantly associatiated with elevated tc, ldl-c and hba1c in t2d patients (p,0.0125, supplementary table s4 ). figure s3) . snps rs707922 (g+1837t) and rs707921 (c+1871a) which associated with plasma levels of tc, ldl-c and apom in t2d patients fell within the evolutionary conserved region. figure 1 (top panel) illustrated the cross-species sequence conservation of the rs707922-and rs707921-flanking region. blast search using this conserved sequence as the template returned unique human est clones bi757556 (human brain) and aa975560.1 (human kidney) which are likely other apom transcripts. the ensembl browser also displayed three apom transcripts (apom1: ensembl-enst00000375916, apom2: ensembl-enst00000375920 and apom3: ensembl-enst00000375918). 4.2. molecular cloning of apom transcripts. since the sequences of est clones bi757556 and aa975560.1 were different than the known apom transcripts, we proceeded with cloning alternative transcripts of apom. a novel transcript, designated apom5, was identified by 59 race and 39 race. as shown in figure 1 , the 39 end of apom5 was identical to the 39 end of apom3. the 59 end, however, was similar to that of the vulgate apom transcript (designated apom1) except that the transcription start site of apom5 was 21 nucleotides downstream that of the apom1. the full-length sequence of apom5 is provided in supplementary figure s4 . it is important to note that the two snps rs707922(g+1837t) and rs707921(c+1871a) associated with metabolic traits in t2d are located to the exon 5 of apom5. on the contrary, when reference to the vulgate apom1 transcript, rs707921 and rs707922 are located to intron 5. these observations support the cryptic nature of exon 5 of the gene. the results of this study did not support an assoiation between apom and t2d suseptibility in hong kong chinese. for a subset of snps, we presented evidence of association between apom and disease duration as well as metabolic traits in t2d patients. further characterization of rs707922, one of the metabolic traitassociated snp at molecular level lead to the discoveries of a novel transcript apom5 and its snp-dependent effect on cellular cholesterol content. the ld block formed among rs805264, rs707922, and rs707921 in our cohort agreed with the ld structure reported in the northern chinese [25] . it is currently unknown whether this subset of snps is also associated with metabolic traits in northern chinese with t2d. among the four common haplotypes constructed from rs805297, rs9404941, and rs707922, only homozygous haplotype c-t-t was significantly associated with higher tc, ldl-c and hba1c levels in t2d patients. given the established association between rs7070922 and plasma tc and ldl-c levels, these association results did not support additional effects of the haplotypes on serum cholesterol levels. interestingly, the association between the homozygous haplotype c-t-t with hba1c indicated the interaction among the three alleles to control systemic glucose level in t2d patients. it would have been ideal if the previously reported association between snp rs805296(t-778c) and t2d in northern chinese were reproduced in this hong kong chinese population. unfortunately, genotyping of this snp failed to produce results in this study, precluding it being used for discussions attempting to reconcile the current findings with prior results. although rs805296 is physically close to rs9404941, the existing information/data does not allow the relationship between snp rs805296 and t2d/t2d metabolic traits to be predicted in our cohort. it is noteworthy that the case-control study design adopted by the current and other studies tend to be limited by the heterogeneity of the prevalent cases with regards to t2d ascertainment, i.e., both those have developed t2d and those have survived in the setting of t2d were included as cases. therefore, those 'susceptible to' the disease were not distinguished from those 'survived' the disease'. one possibility to circumvent such issue is to examine for similar duration of diabetes across studies being compared and test for difference in duration of t2d by snp. interestingly, while our results did not support an association between apom and t2d susceptibility, stratification of our cases by disease duration allowed us to detect an association between rs805297(c-1065a) and t2d duration. this result implied the possibility that relative to the rs805297-a carriers, the rs805297-c carriers better survived the diabetic condition over the long term and such possibility can be further tested. interestingly, zhao et al., recently reported a positive association between rs805297-a and the risk of stroke in norhtern chinese (or = 1.38, p = 0.002) after adjusting for other risk factors including history of diabetes [26] . whether such association is present among the southern chinese requires further investigation. nevertheless, losing rs805297-a carriers with t2d to stroke over time provides a plausible explanation for the observed higher frequency of rs805297-c allele in the t2d duration .10 years subgroup (relative to t2d duration #10 years subgroup). previous studies attempting to correlate plasma apom and cholesterol levels have generated inconsistent results [3, 27] . in this study, rs707922 homozygous minor allele (tt) was associated with elevated tc and ldl-c as well as plasma apom levels in diabetic cases (average bmi of 25.26). these observations are consistent with previously reported positive association between plasma apom and plasma tc and ldl-c in overweight-obese individuals [3] . results presented by han et al. from the study of a northern chinese cohort showed significant association between rs707922 t allele and increased risk of cerebral infraction (or = 1.78, p = 0.000). in parallel they also confirmed hypercholesterolemia as an independent risk factor for cerebral infraction [25] . these results implied the possibility that rs707922 is also a modifier of serum cholesterol in northern chinese. the association between rs707922 tt genotype and elevated serum total-/ldl-cholesterol levels in type 2 diabetes found in the current report deserves to be further substantiated in strict replicate studies. the mechanism underlying the effects of rs707922 on plasma tc and ldl-c levels in diabetes remains elusive. richter et al., reported hnf-1 alpha being a potent transcription activator of apom [28] . the decreased serum apom level in maturity-onset diabetes of the young subjects as compared to the controls could be explained by the hnf-1 alpha mutations in these patients [28] . given the association between apom and metabolic traits found in this study, one may speculate that snp rs707922 (g+1837t), in the capacity of an intronic snp (reference to the apom1 transcript), may modify apom expression through snp-specific recruitment of transcription factors (i.e., pax 6 showed an allelespecific interaction with rs707922 t by computer prediction as presented in supplementary figure s5 ) and subsequently affect cellular cholesterol homeostasis in liver and possibly other tissues. more interestingly, we found that rs707922 can also assume the capacity as an exonic snp (i.e., reference to the apom5 transcript). while the function of apom5 requires further elucidation, the high renal and hepatic expression levels of apom1 and apom5 indicated the possibility of these transcripts coordinate to regulate cholesterol homeostasis in these tissues. such possibility is further supported by the results showing the activities of ectopically expressed apom5 in modifying hepatic cell cholesterol content. with regards to systematic cholesterol homeostasis, we observed that homozygous rs707922-t allele associated with elevated total-and ldl-cholesterol levels. one possible mechanism of such elevation is through reducd hepatic and/or pheripheral clearance of circulating cholesterol. consistent with this notion, our in vitro data showed that hepatic cells overexpressing apom5-t transcript had lower cholesterol content relative to cells expressing the apom5-g counterpart. in conclusion, the apom snp frequencies and the ld structure reported in this study of hong kong chinese population will facilitate future population genetics studies. while our results did not support an association between apom and t2d susceptibility in hong kong chinese, subgroup analyses found snp as well as haplotype associations between apom and metabolic traits in t2d. bioinformatics/molecular analyses revealed the cryptic nature of exon 5 responsible for the expression of a novel transcript apom5, predominantly in liver and kidney. the activity of apom5 on modifying cellular cholesterol content revealed another layer of regulation underlying the expression and function of apom. (mus musculus; chr17) , rat (rattus norvegicus; chr20), cow (bos taurus; chr23) and dog (canis familiaris; chr12) genes are shown. conserved sequences were defined as coding exons (blue), the evolutionary conserved regions (ecrs) were indicated by pink lines (on top of the panel for each species) with a default value of 70%. the human apom was depicted as a horizontal blue line above the graph, with strand/transcriptional orientation indicated by arrows. apom coding exons were shown as blue boxes along the line, while untranslated regions (utr) were indicated as yellow boxes. peaks within the conservation profile which corresponded to these five exons of apom were similarly coloured within the plot. peaks within the conservation profile that did not correspond to transcribed sequences were highlighted in red colour. regions of transposable elements and simple repeats were highlighted in green color. figure s5 computer-predicted transcription factor interaction sites in nucleotide sequences spanning snps rs707922(g+1837t) and rs707921(c+1871a). this figure is generated by the match program. top panel: the transcription factors predicted to interact with the nucleotide sequences spanning the major allele of snps rs707922 (g allele) and rs707921 (the c allele). bottom panel: the transcription factors predicted to interact with the nucleotide sequences spanning the minor allele of snps rs707922 (the t allele) and rs707921 (the a allele). the predicted transcription factors are marked by blue text with scores of matrix match indicated in parentheses. the locations and orientations of the binding sites for these predicted transcription factors are marked by black horizontal dashed lines with arrows. highlighted in pink boxes are allele-specific transcription factors (pax6 and areb6 for rs707922-t; hnf4 and oct1 for rs707921-a) and their corresponding binding sites. the vertical dashed lines indicate the locations of snps rs707922 and rs707921. the precise nucleotide positions of snp rs707922 and rs707921 are also highlighted with grey boxes in the dna sequences represented by red colored text. (tiff) table s1 sequences of primers used in this study. (pdf) table s2 summary of data quality of the five successfully genotyped apom snps in the full cohort (n = 1840). the clinical traits tested for im include t2d, tg, hba 1c , fpg, tc, hdl-c, and ldl-c. (pdf) table s3 frequencies of common haplotypes constructed by apom snps rs805297, rs904941, and rs707922. (pdf) table s4 haplotype c-t-t formed from snps rs805297(c-1065a), rs9404941(t-855c), and rs707922 (g+1837t) and clinical characteristics of t2d patients. values are either number of subjects, mean 6 sd, or geometric mean (95% confidence interval). p values here represent the comparisons between subgroup of homozygotes of c-t-t haplotype vs. subgroup with one c-t-t haplotype and without c-t-t haplotype (a recessive model). p values are adjusted for age, sex, bmi and disease duration in t2d. in non-diabetic controls, the p values without parentheses are adjusted for age, sex and bmi. ''+/+'' represent the homozygote of haplotype c-t-t, ''+/2'' represent the heterozygote of haplotype c-t-t, ''2/2'' represent the subgroup who do not have the haplotype of c-t-t. individuals on lipid lowering medications (n = 90) were excluded for association analysis with lipid traits. * statistical significance (p,0.0125). 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haploinsufficiency is associated with reduced serum apolipoprotein m levels we thank dr. tilla worgall, department of pathology and dr. richard, deckelbaum, institute of human nutrition, columbia university, new york for their generous technical support and scientific input. key: cord-001363-irysq6pf authors: liu, zhenjiang; yuan, zhengwei; zhao, qun title: seldi-tof-ms proteomic profiling of serum, urine, and amniotic fluid in neural tube defects date: 2014-07-23 journal: plos one doi: 10.1371/journal.pone.0103276 sha: doc_id: 1363 cord_uid: irysq6pf neural tube defects (ntds) are common birth defects, whose specific biomarkers are needed. the purpose of this pilot study is to determine whether protein profiling in ntd-mothers differ from normal controls using seldi-tof-ms. proteinchip biomarker system was used to evaluate 82 maternal serum samples, 78 urine samples and 76 amniotic fluid samples. the validity of classification tree was then challenged with a blind test set including another 20 ntd-mothers and 18 controls in serum samples, and another 19 ntd-mothers and 17 controls in urine samples, and another 20 ntd-mothers and 17 controls in amniotic fluid samples. eight proteins detected in serum samples were up-regulated and four proteins were down-regulated in the ntd group. four proteins detected in urine samples were up-regulated and one protein was down-regulated in the ntd group. six proteins detected in amniotic fluid samples were up-regulated and one protein was down-regulated in the ntd group. the classification tree for serum samples separated ntds from healthy individuals, achieving a sensitivity of 91% and a specificity of 97% in the training set, and achieving a sensitivity of 90% and a specificity of 97% and a positive predictive value of 95% in the test set. the classification tree for urine samples separated ntds from controls, achieving a sensitivity of 95% and a specificity of 94% in the training set, and achieving a sensitivity of 89% and a specificity of 82% and a positive predictive value of 85% in the test set. the classification tree for amniotic fluid samples separated ntds from controls, achieving a sensitivity of 93% and a specificity of 89% in the training set, and achieving a sensitivity of 90% and a specificity of 88% and a positive predictive value of 90% in the test set. these suggest that seldi-tof-ms is an additional method for ntds pregnancies detection. the prevalence of neural tube defects (ntds) is known to vary significantly based upon geography and ethnicity, with ranges from 0.5 to 6 in 1,000 newborns [1] . the mothers of an ntdaffected child are 10-fold more likely to give birth to a second child with an ntd, suggesting the involvement of both environmental and genetic factors in their etiology. there are multiple types of isolated ntds including spina bifida and anencephaly [2] . current prenatal screening efforts are based on two complementary methods, maternal serum alpha-fetoprotein (msafp) and ultrasound screening. it has been determined that in a fetus with an ntd, exposed membranes allow afp to leak into the amniotic fluid and then into maternal serum, at a level of roughly in proportion to the size of the exposed area. however, the level of msafp is not a specific indicator of an ntd, since it is also increased in ventral wall defects (omphalocele or gastroschisis), abnormal glomerular diseases such as nephrotic syndrome, defective placental membranes (fetal hydrops), and fetal blood contamination due to a traumatic amniocentesis, as well as other pregnancy-related problems [1] . although a matter of some controversy, when the spina bifida lesion is covered with healthy skin, msafp and amniotic fluid afp (afafp) concentrations are generally found to be normal. therefore, due to the low specificity of msafp or afafp levels, its use as a screening tool, has limited diagnostic value. norem and coworkers found that among the 102 ntds cases who had received msafp testing, 25 cases (25%) had negative maternal serum screening results, including 15 (38%) of the 40 spina bifida cases tested, 6 (67%) of the 9 encephalocele cases tested, and 4 (8%) of the 53 anencephaly cases [3] . at present, there does not appear to be a more specific marker of ntds that has been identified in maternal serum [1] . kooper et al. found that 27 out of 6,188 pregnancies (0.4%) without any increased ntd risk had afafp levels .2.5 mom (multiples of the median), two of which were associated with ntds; two out of 258 pregnancies (0.8%) with an increased ntd risk had elevated afafp levels and were associated with affected pregnancies; and 44 of 55 pregnancies (80%) with clinically diagnosed fetal ntds had an increased afafp levels [2] . biochemical diagnosis of ntds is based on the electrophoresis of amniotic fluid cholinesterases [4] . cholinesterases includes butyrylcholinesterase, normally present in the serum and amniotic fluid, and acetylcholinesterase, which is specific to neural tissue but is normally absent from amniotic fluid. when the fetus has an ntd, acetylcholinesterase is present as a rapidly-migrating eletrophoretic band, in addition to butyrylcholinesterase. however, amniocentesis is an invasive procedure, and it has not been used routinely in the clinical practice. surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (seldi-tof-ms) is a breakthrough in clinical proteomics, and can detect different protein expression patterns of body fluid and tissue specimens between patients and healthy subjects, and its rapid development provides an alternative tool to search for biomarkers. seldi-tof-ms detects proteins selectively adsorbed onto the surface of a protein chip array after nonspecifically bound proteins are washed off by stringent buffers, and has been shown to be sensitive, rapid and reliable. this technique has been successfully applied in the discovery of serum biomarkers for many cancers [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] , nervous system diseases [19] [20] [21] [22] and renal diseases [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] . furthermore, construction of a classification tree using ''artificial intelligence'' algorithms to process seldi data improves the accuracy to differentiate cancer patients from non-cancer groups [33] [34] . our hypothesis is that protein expression profiles of ntds may be considered as a potential diagnostic approach using seldi-tof-ms. the purpose of this pilot study was to preliminarily explore the differential protein expression pattern between ntd case mothers and normal control mothers using seldi-tof-ms protein profiling and classification and regression tree (cart) analysis, in order to differentiate pregnancies complicated by the presence of an ntd-affected fetus from healthy controls. experimental protocol was approved by china medical university institutional review board. written informed consent was obtained from all mothers with ntd-affected fetuses and healthy volunteers for this study. 120 maternal serum samples (64 mothers with ntd-affected fetuses and 56 normal control mothers), 114 maternal urine samples (61 mothers with ntdaffected fetuses and 53 normal control mothers) and 113 maternal amniotic fluid samples (61 mothers with ntd-affected fetuses and 52 normal control mothers) were collected between gestational weeks 15.0 and 34.0. gestational age was determined from the date of the last menstrual period (lmp) or by an ultrasonography examination when the lmp was uncertain. healthy volunteers receiving prenatal medical examination in the shengjing hospital were recruited into the control group. all of the normal control mothers, which had no pregnancy-related problems, such as diabetes or hypertension, had or were carrying fetuses lacking any congenital defects. all of the normal control mothers had normal biochemistry detection of serum and urine. control amniotic fluid samples were collected from pregnant women with a normal fetal karyotype. karyotypes of all fetuses were evaluated following amniocentesis. prenatal diagnoses of ntd-affected and normal fetuses were confirmed using ultrasonography. ntds in our study were classified into two different clinical types: spina bifida and anencephaly. two ml of whole blood was collected in the morning after fasting overnight and stored at 4uc for 1 hour. blood samples were then centrifuged at 4,000 rpm, 4uc for 10 min and 100 ml aliquots were stored at 280uc until analyzed. two ml of urine was collected in the morning after fasting overnight. urine were immediately centrifuged at 2,500 rpm, 4uc for 5 min, and 100 ml aliquots were stored at 280uc until analyzed. amniotic fluid were obtained from mothers carrying ntds fetuses during transabdominal amniocentesis before induced labor, and mothers suspected of having a fetus with trisomy 21 or 18 that were ultimately confirmed as a normal karyotype during the prenatal examination. amniotic fluid samples were collected during an aseptic procedure through the abdominal wall and by direct ultrasound guidance using a 21-gauge puncture needle. two ml of amniotic fluid were collected, immediately centrifuged at 2,500 rpm, 4uc for 5 min, distributed into 100 ml aliquots and stored at 280uc until analyzed. the efficacies of both strong anion exchange and weak cation exchange (wcx) protein chips (ciphergen biosystems, inc., fremont, ca, usa) for serum protein profiling were tested in this study. results showed that the weak cationic exchange proteinchip, cm10, had a higher capture ability and signal-tonoise ratio (s/n). therefore, cm10 was selected for all subsequent experiments. binding of proteins to the proteinchip cm10 array can also be affected by ph dependent of the buffer and changing the ionic strength of the buffer. serum, urine or amniotic fluid samples were thawed on ice and centrifuged at 10,000 rpm for 2 min at 4uc, at which time 20 ml u9 buffer (9 mol/l urea, 2% 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate, 50 mmol/l tris-hcl, ph 9.0, and dithiothreitol) was added into 10 ml supernatant, and vortexed for 30 min at 4uc. at this point, 360 ml binding buffer (50 mmol/l naac, ph 4.0) was added into serum sample, 250 ml of binding buffer was added into urine sample, 170 ml of binding buffer was figure 1 . seldi profile showed a peak with average mass of 2750 da that was up-regulated in ntds compared with control serum. serum mass spectra of 2 mothers with ntd-affected fetuses were compared with those of 2 control mothers and the mass-tochange ratio (m/z) ranged between 2700-3100 da. x-axis showed molecular weight of peaks and y-axis showed intensity of peaks. doi:10.1371/journal.pone.0103276.g001 figure 2 . seldi profile showed a peak with average mass of 5497 da that was down-regulated in ntds compared with control serum. serum mass spectra of 2 mothers with ntd-affected fetuses were compared with those of 2 control mothers and the massto-change ratio (m/z) ranged between 5300-5600 da. x-axis showed molecular weight of peaks and y-axis showed intensity of peaks. doi:10.1371/journal.pone.0103276.g002 added into amniotic fluid sample, and immediately mixed. to equilibrate the chip, 200 ml binding buffer was applied to each chip spot, shaking at 250 rpm for 5 min and excess buffer was removed without contacting the active surface. the same procedure was repeated once. 100 ml prepared serum sample, or urine sample, or amniotic fluid sample, was then added into each bioprocessor well, and chip was incubated in a humidity chamber for 1 h, shaking at 250 rpm at room temperature. excess buffer was removed. 200 ml binding buffer was added into each well, shaking at room temperature for 5 min. the above procedure was repeated once. each spot was washed with 200 ml deionized water. then 0.5 ml saturated sinnapinic acid was applied to each spot and the chips were allowed to air dry. chips were re-incubated with sinnapinic acid and air dried again. captured proteins by the arrays were detected on a pbs-ii c reader (ciphergen biosystems). the accuracy of mass spectrum was calibrated using the all-inone peptide molecular mass standard (ciphergen biosystems, inc., fremont, ca, usa) at the day of experiment. the high cutoff molecular weight was set as 50,000 daltons (da), with an optimized range of 1,000 to 30,000 da, laser intensity of 225 and detection sensitivity of 9. each sample was activated 130 times. error range of molecular weight was controlled at ,0.1%. intrachip and interchip quality controls were included. the coefficient of variation (cv) was controlled at ,10% for peak amplitude. all spectra were analyzed using biomarker wizard software (ciphergen biosystems, inc.). qualified mass peaks (s/n .5) with m/z between 1000 and 30000 da were detected automatically and majority of resolved protein/peptides were within this range. molecular masses from 0-1000 da were excluded from analysis since they were mainly noises from the energy-absorbing molecule (eam). peak intensities were normalized to the total ionic current of peaks with m/z between 1000 and 30000 da using proteinchip software version 3.1.1 (ciphergen biosystems, inc.). in a validation experiment, protein peaks should present in more than 10% samples and deviation of peak amplitude should be smaller than 0.3% in each sample. parameters of protein peaks were presented as mean value plus or minus standard deviation and spectral data of the training set were further analyzed using biomarker pattern software (bps) (version 5.0; ciphergen biosystems) to establish a classification tree. classification and regression tree (cart) analysis decision tree classification pattern was generated using bps version 5.0, a statistical tool developed by breiman et al. [35] to implement cart. bps uses peak information generated from the sample training set to build the classification tree. cart consisted of tree construction and tree pruning [36, 37] . the tree construction process primarily splits spectral data of the training set into two nodes by questionnaire criteria. the splitting decision is based on the intensity of a peak. the answer to ''does mass a have intensity less than or equal to x'' splits the data set into two nodes, a left node for yes and a right node for no. the splitting process was repeated until no further gain in classification and terminal nodes were reached. further classification of terminal nodes is based on the clinic grouping of most samples (i.e., ntd and control) that represents the majority of samples in that node. in the tree pruning step, the classification tree is cut down to a desired size using tree-cost complexity pruning [33] . calculation of sensitivity, specificity, positive predictive value and negative predictive value were performed using bps version 5.0 (ciphergen biosystems, inc.). comparison of parameters between ntd group and control group were performed using the t-test. p,0.05 was considered statistically significant. total of 55 qualified mass peaks (s/n .5) were detected. seldi was particularly effective in resolving low molecular weight (,10 kda) proteins and polypeptides. the intensities of 12 of 55 mass peaks between ntds group and control group were significantly different, among which 8 proteins with m/z of 4105, 4297, 4188, 6650, 8583, 3282, 2750 (fig. 1) and 3327 da were up-regulated, while 4 peaks with m/z of 5497 (fig. 2) , 28078, 9155 and 9434da were down-regulated in ntds group compared to normal control group (table 1) . total of 39 qualified mass peaks (s/n .5) were detected. the intensities of 5 of 39 mass peaks were significantly different between ntds group and normal control group, among which 4 proteins with m/z of 8320, 8209, 9099 (fig. 3) and 10567 da were up-regulated, while 1 peak with m/z of 3458 da was downregulated in ntds group compared to control group (table 2) . total of 35 qualified mass peaks (s/n .5) were detected. 7 of 35 mass peaks in ntds group have significantly different intensity to that of normal control group, among which 6 proteins with m/z of 14700, 7995, 15891, 16027, 13776 (fig. 4) and 11040 da were up-regulated, while 1 peak with m/z of 23417 da was downregulated in ntds group compared to control group (table 3) . no single peak was identified alone, indicating that no peak could completely differentiate the ntd group from the normal control group for serum, urine and amniotic fluid samples, respectively. a decision tree classification algorithm was built using all 55 protein peaks and 2 protein peaks at 4105 and 7788 da were automatically selected as splitters. the 4105 da peak was used as the root node in the classification tree to split all samples into two groups (fig. 5) : the left node (terminal node 1) included peaks with intensity #53.002 and the right node (node 2) contained the remaining peaks with intensity .53.002. each branch node was then further classified into next layer using the same method with 7788 da as the cutoff. finally, all samples were classified into 3 terminal nodes and a classification tree was obtained (fig. 5) . this pattern analysis process yielded a sensitivity of 91%, specificity of 97%, positive predictive value of 98% and negative predictive value of 90% in the training set ( table 4 ). the validity and accuracy of the classification tree algorithm were then evaluated by challenging to classify blinded objects correctly in the test set, which consisted of 20 samples of serum from mothers with ntd-affected fetuses, 18 analysis of urine samples from mothers of ntd-affected fetuses and normal control mothers revealed two major, differentially expressed proteins at 9096 and 8244 da used in the classification figure 3 . seldi profile showed a peak with average mass of 9099 da that was up-regulated in ntds compared with control urine. urine mass spectra of 2 mothers with ntd-affected fetuses were compared with those of 2 control mothers and the mass-to-change ratio (m/z) ranged between 8800-9400 da. x-axis showed molecular weight of peaks and y-axis showed intensity of peaks. doi:10.1371/journal.pone.0103276.g003 figure 4 . seldi profile showed a peak with average mass of 13776 da that was up-regulated in ntds compared with control amniotic fluid. amniotic fluid mass spectra of 2 mothers with ntd-affected fetuses were compared with those of 2 control mothers and the mass-to-change ratio (m/z) ranged between 12000-18000 da. x-axis showed molecular weight of peaks and y-axis showed intensity of peaks. doi:10.1371/journal.pone.0103276.g004 pattern to generate 3 terminal nodes (fig. 6 ). our results yielded sensitivity of 95%, specificity of 89%, positive predictive value of 91% and negative predictive value of 94% (table 5 ). the validity of the classification tree was then challenged with a blind test set including another 19 samples of urine from mothers with ntd-affected fetuses and 17 samples of urine from healthy controls. the algorithm correctly classified 86% ( analysis of amniotic fluid samples from mothers of ntdaffected fetuses and normal control mothers revealed two major, differentially expressed proteins at 14700 and 13776 da used in the classification pattern to generate 3 terminal nodes (fig. 7) . our results yielded sensitivity of 93%, specificity of 89%, positive predictive value of 90% and negative predictive value of 91% (table. 6 comparing among serum, urine and amniotic fluid seldi profile, four differentially expressed masses with the same m/z of 4188, 6451, 11744 and 23425 da, coexists in the serum, urine and amniotic fluid (table 7 ). msafp, afafp, and amniotic fluid cholinesterases are the primary protein biomarkers used in the prenatal diagnosis for ntds [1] . msafp screening and ultrasonography are both used for prenatally detecting ntd-affected fetuses. although the american college of obstetricians and gynecologists recommended that all pregnant women should be offered msafp screening in the second trimester of pregnancy, the detection rate for ntds using elevated msafp level as a screening tool was only 75% to 80% [3] . causes for elevated msafp levels other than ntds include underestimated gestational age, congenital skin defects, pilonidal cysts, abdominal wall defects, gastrointestinal defects, obstruction, liver necrosis, cloacal exstrophy, cystic hygroma, sacrococcygeal teratoma, renal anomalies, urinary obstruction, polycystic kidney, absent kidney, congenital nephrosis, osteogenesis imperfecta, low birth weight, oligohydramnios, multiple gestation and maternal underweight [38] . szajkowski et al. suggested that msafp screening had a low sensitivity for fetal hydrocephalus and was rarely elevated in isolated cases [39] . screening for ntd is now a routine prenatal test, mainly due to its association with second-trimester maternal serum screening for down syndrome determined by combination of low afp value and high hcg value. afafp can also be used as an indicator for ntds, but its specificity is not satisfactory [1] , and sensitivity is 80% [2] . therefore, the suggestion of performing routine afafp test in early second-trimester genetic amniocentesis by widlund et al. [40] to rule out the possibility of an open fetal ntd, does not seem to be justified given its limited clinical diagnostic value. electrophoresis of amniotic fluid cholinesterases can also be used to biochemical diagnose of ntds, which has good sensitivity, specificity, positive predictive value and negative predictive value [4] . however, as an invasive technique, amniocentesis might cause abortion, infection, injury to mother and fetus, which must all be weighed against the value of performing the test. proteomic analysis provides a unique tool for the identification of diagnostic biomarkers, evaluation of disease progression and development of drugs [41] . seldi-tof-ms has been used to resolve proteins in biological specimens through binding to biochemically distinct proteinchips [34, 42] , and has many other advantages compared with traditional approaches: 1) it is much faster to perform; 2) it has high-throughput capability; 3) it requires only small amount of protein sample; 4) it has relatively high sensitivity to detect proteins at picomole to attamole range; 5) it can effectively resolve low mass proteins (2-20 kda) and 6) it is directly applicable for development of clinical assays [26] . since ntds are multifactorial traits, it is likely that a combination of several biomarkers will become a signature which is necessary to effectively differentiate ntds from controls, such as the combination of peaks at 4105 and 7788 da in the serum samples, peaks at 9096 and 8244 da in the urine samples, and peaks at 14700 and 13776 da in the amniotic fluid samples. in our study, profiling of multiple proteins was performed using seldi technology, and biomarker patterns software was used to analyze the large volume of generated data. proteins/peptides derived from both the fetus and the mother could be used in this classification system only if they produce a relatively accurate diagnosis, that is, these biomarkers can be detected by seldi and accurately selected by the classifier. our results further confirmed the suitability of seldi-tof-ms for protein profiling of serum, urine and amniotic fluid samples, which yielded relatively high sensitivity and specificity for the diagnosis of ntds. there were a greater number of different proteins in the serum samples than the amniotic fluid samples, when the amniotic fluid is more directly measuring fetal proteins than maternal serum. however, when we analyzed the data, we found that only four protein/peptide biomarkers, 4188, 6451, 11744 and 23425 da, were detected in all serum, urine and amniotic fluid samples. one possible explanation is that these four protein/peptide biomarkers might be related to the abnormal protein expression of ntds. since identification of these biomarkers is essential for understanding their biological roles in ntds, we are currently making efforts to characterize these protein/peptide biomarkers. the ultimate clinical application of protein profiling is for the early detection of ntds from the maternal body fluid, such as serum or urine. detection of larger number of peaks in serum samples than those of urine and amniotic fluid samples suggested that the identification of candidate biomarkers from maternal serum samples may be productive. it was noted that not all differentially expressed protein peaks were used in the cart analysis. wadsworth et al. suggested that cart analysis should examine all possible protein peak combinations in input spectral data to generate the best classification tree, in which any statistically insignificant protein peak included was also important for the classification algorithm after stratification [43] . although peak of 7788 da shown in figure 5 had no statistical difference between the two groups of serum, it was crucial for the classification tree to delineate subsets of groups that had been stratified by the significant peak of 4105 da. the use of peak of 8244 da in the urine sample cart shown in figure 6 was similar with that of peak of 7788 da in the serum sample cart. according to literature, another research group has used two-dimensional gel electrophoresis (2-de)/mass spectrometry (ms) to characterize differentially expressed proteins in amniotic-fluid samples (afss) of embryonic day (e) 17.5 rat fetuses with spina bifida aperta induced by retinoic acid (ra) [44] . they identified five proteins differentially expressed in afss of spina bifida aperta, including three upregulated proteins (transferrin, alpha-1 antiproteinase and signal recognition particle receptor, b subunit [srprb] 55 kda), two downregulated proteins. specifically, they found 11 alpha-1 fetoprotein (afp) fragments that were downregulated and 35 afp fragments that were upregulated in afss from embryos with spina bifida aperta. the comparative proteomic study of afss from rat fetuses with spina bifida aperta may provide new insights in neural tube defects and contribute to the prenatal screening. this study suggests that ntds-specific proteomic signatures are likely to present in serum, urine, and amniotic fluid of mothers carrying a ntds fetus. it is reported that at present although afp screening detects 88% of affected fetuses with a false positive rate of 3% in the second trimester, ultrasound has 100% specificity and 98% sensitivity for ntd at 18-20 week [45, 46] . currently, it is still controversial whether or not it is sufficient to rely only on ultrasound to detect ntd. recent literature has even suggested only ultrasound be used. new techniques in ultrasound have high specificity and sensitivity [47] . in the paper, there is a recommendation to abolish msap and rely only on ultrasound (which approaches 100% specificity and sensitivity). however, on the other hand, some recent studies also demonstrated the limitation of ultrasound approach alone to detect ntd. for example, one study showed that ultrasound biparietal diameter alone would detect 50% of cases for a 5% false-positive rate or 63% for 10%; adding afp increases detection by 2%; and a combined test with ultrasound biparietal diameter, afp, and free b-hcg detects 58% for 5% or 70% for 10% [48] . another study indicated that there were 20 perinatal deaths from ntd using ultrasound approach alone that could potentially have been prevented through the use of pre-conceptual folate [49] . therefore, in new zealand, new guidelines were implemented in 2010 that required all eligible pregnant women to be offered a nuchal translucency scan combined with a blood test to improve ntd detection. in our study, we could only take samples from those pregnant women who have already been diagnosed with ntd-affected pregnancies by prenatal ultrasound. if ultrasound is not sufficient enough for ntd in some cases, we think, seldi-tof-ms can at least serve as an additional method for ntds pregnancy detection because of its relatively high sensitivity and specificity. our pilot study demonstrated the application of seldi protein profiling in the detection of mothers with ntd-affected fetuses. we have preliminarily established a relatively specific procedure to detect ntd-affected pregnancies using a combination of proteinchip technology and classification and regression tree analysis. conceived and designed the experiments: zl qz. performed the experiments: zl zy qz. analyzed the data: zl qz. wrote the paper: zl qz. prenatal biochemical screening for neural tube defects fetal anomaly scan potentially will replace routine afafp assays for the detection of neural tube defects routine ultrasonography compared with maternal serum alpha-fetoprotein for neural tube defect screening amniotic fluid acetylcholinesterase measurement in the prenatal diagnosis of open neural tube defects. second report of the collaborative acetylcholinesterase study differential expression of proteomics models of colorectal cancer, colorectal benign disease and healthy controls identification of serum proteins as prognostic and predictive 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abnormalities? an audit of early pregnancy care in new zealand key: cord-001253-3jnkki5z authors: mohammad, fahim; theisen-toupal, jesse c.; arnaout, ramy title: advantages and limitations of anticipating laboratory test results from regressionand tree-based rules derived from electronic health-record data date: 2014-04-14 journal: plos one doi: 10.1371/journal.pone.0092199 sha: doc_id: 1253 cord_uid: 3jnkki5z laboratory testing is the single highest-volume medical activity, making it useful to ask how well one can anticipate whether a given test result will be high, low, or within the reference interval (“normal”). we analyzed 10 years of electronic health records—a total of 69.4 million blood tests—to see how well standard rule-mining techniques can anticipate test results based on patient age and gender, recent diagnoses, and recent laboratory test results. we evaluated rules according to their positive and negative predictive value (ppv and npv) and area under the receiver-operator characteristic curve (roc aucs). using a stringent cutoff of ppv and/or npv≥0.95, standard techniques yield few rules for sendout tests but several for in-house tests, mostly for repeat laboratory tests that are part of the complete blood count and basic metabolic panel. most rules were clinically and pathophysiologically plausible, and several seemed clinically useful for informing pre-test probability of a given result. but overall, rules were unlikely to be able to function as a general substitute for actually ordering a test. improving laboratory utilization will likely require different input data and/or alternative methods. laboratory testing is the single highest-volume medical activity [1] . its main role is to help adjust the level of clinical suspicion of a diagnosis to help rule it in or out; it is also used for disease monitoring. in practice, the level of clinical suspicion and the probability of a given test result can be correlated: the higher the suspicion, the more likely it is that the result will confirm the diagnosis. information that feeds into the clinical suspicionincluding the age and gender of the patient, prior diagnoses, and prior laboratory results-thus may also influence the test result. in principle, this relationship can be used to improve laboratory testing by making it possible to estimate the pre-test probability of getting a given test result before ordering the test, and, in the limit, to reduce test utilization without adversely affecting patient outcomes. indeed, ordering fewer tests, where warranted, might benefit outcomes by saving the patient the burden of following up false positives (or negatives) [2] [3] [4] . conceptually, the relationship between clinical suspicion and pre-test probability is used routinely to help set guidelines regarding when and when not to order a given test. for example, the pre-test probability of lyme serology being positive given a targetoid rash is high enough that, given the test's sensitivity and specificity, ordering the test is contraindicated [5] . because of the large number of tests and clinical scenarios that exist, and in light of evidence from across medicine that utilization of laboratory testing can be improved [1, 6] , it is of interest to understand whether analyzing large clinical databases using the robust application of standard statistical techniques can turn this relationship into actionable decision-support rules-or whether progress toward better laboratory utilization might instead lie elsewhere. we sought to test the limits of rule-mining for this purpose. to what extent can laboratory results be anticipated computationally based on data available to the clinician, or a clinical decision support system, at the time of the order? we addressed this question using generalized linear modeling (glm), a generalized form of linear regression [7] , and, for comparison, classification trees (ct) [8, 9] . we used four types of input-age, gender, diagnoses (three-digit icd-9 codes), and results of laboratory tests on blood samples added to the record in the seven days before a given test was ordered-to build simple, robust models for whether the result of a test would be within the reference interval (''normal'') or outside of it in a given direction (''abnormal''), treating high and low results separately. we based our study on 10 years of records from the beth israel deaconess medical center (bidmc), a 585-bed tertiary care center in boston, ma. we first anonymized records and reconciled test names (work approved by bidmc committee on clinical investigation's institutional review board for research involving human subjects, protocol 2012-p-000229/ 01). informed consent was not obtained because patient records/ information was anonymized prior to analysis. each blood test (the test of interest), over 69.4 million in all, was marked as an in-house test (performed at the hospital) or a sendout (performed off-site). for each test, we compiled a list of all instances in which the test was ordered and performed. for each instance, we recorded the patient's age, gender, and any diagnoses or other blood-test test results from the seven days prior to the result of interest. when a test was ordered multiple times within a seven-day period, we considered only the most recent one (i.e., the one closest in time to the sendout order) as input data. for relevance, we considered only those tests that were ordered at least 1,000 times over the entire 10-year period, for an average of at least twice a week. we randomly divided the resulting instances into a training set and a test set (see below for details). all tests had either two (reference vs. abnormal) or three (low, normal, or high) possible response values. for tests with three values, we performed two separate rule searches: one for high vs. not high-i.e., grouping normal and low-and one for low vs. not low. we sought to identify simple, robust subsets of our input data to evaluate as linear predictors (''rules'') for whether a test result would be normal or abnormal. to do this, we used glm twice: first to find rules based on a particular training set and a second time to find rules based on just those items that were common to rules found from a number of different training sets (to avoid overfitting any one training set). we did this as follows, for each test of interest (the response variable or ''response''). we first excluded those input variables (''features'') that appeared with fewer than 5 percent of the response. we then temporarily set aside the most common features (those of the complete blood count and basic metabolic panel) as well as age and gender, and searched the remaining items for frequent featuresets (using the apriori algorithm [10, 11] ). we then added back to each resulting featureset the common features, age, and gender (which are frequent items by definition, since they appear in all instances) with a support threshold of 0.60 (i.e., itemsets for which all items were present with at least 60 percent of instances of the response variable). this set-aside/add-back approach sped the search for featuresets without loss of comprehensiveness. we used each featureset to create a model for the test of interest using r's glm function (with the family argument set to ''binomial''). we used backward feature elimination to remove non-significant features one at a time from the featureset (using a significance threshold p-value of 1610 25 ; see below) until the only features that remained were all significantly correlated with the response. we also removed features that are used to calculate the result for the test of interest-e.g., cd4 and cd8 count for t-cell count, which is the sum of cd4 and cd8-for all but proof-ofprinciple runs. the significance threshold was corrected for multiple comparisons by dividing the traditional threshold of p = 0.05 by the product of the total number of tests considered and the average number of rules generated for each test. the combined total number of features (in-house tests plus sendout tests plus diagnoses) was 170+81+434 = 685. the average number of rules after application of glm for the first time for each test is 6. thus our threshold p-value was 0.05/(6*685) = 1.2610 25 , which we rounded to 1610 25 . we constructed a model for the result by running glm a second time on a training set (see below) based on this reduced featureset. of note, there was no guarantee that any feature would be significantly correlated (p#1610 25 ) or that there would be enough instances (glm's threshold was 200) of the test appearing with all features of even the reduced featureset for glm to produce a model. when feature elimination resulted in no significant features or too few instances, no model was constructed. we scored models using ppv, npv, and roc auc. we were interested only in models that were robust to the size and choice of training set. therefore we repeated the above process for a range of training set-test set splits (80-20, 70-30, 60-40, 50-50, 40-60, 30-70, and 20-80 percent). for each split, we ran the above process 10 times and found the number of rules with auc$0.75. we decided on using a 60-40 split for downstream analyses as this split generated a total number of rules comparable to 70-30 and 80-20 splits but with less training data (fig. 1) . finally, for each test of interest, we selected features that appeared in a strict majority of rules for that test and reran glm using only those features. this made rules both simpler and more robust by removing features whose presence was contingent on a particular choice of training or test set. for each of the inhouse and sendout tests we used cart, implemented as rpart in r (rpart v3.1-50; cran.r-project.org/ package = rpart), to predict the response from all input features, using 60:40 training:test-set splits. we fixed some of the metrics (see below) that were used in building the final tree. the cart grows classification tree in two stages. in stage one, a tree is grown by finding a feature which best splits the data into two groups. splitting is done only if the overall ''impurity,'' the number of outcomes different from the majority (e.g., a ''low'' response alongside many ''normal'' responses), decreases, above some threshold (the ''complexity parameter;'' 0.01). then, in top-down fashion, these two subgroups are further divided in a recursive manner until the subgroups reach a minimum size (minsplit = 20 records) or until no further improvement can be made. the resulting tree may overfit the training data. to avoid this, crossvalidation (xval = 10; 10-fold cross-validation) was used in the second stage by pruning the tree. we fixed the maximum depth (maxdepth) of the tree, i.e., the maximum number of branchings from stem to leaf, to be 20. the final models were tested on the test data and performance statistics are found. we repeated modelbuilding 10 times for each test and summarized the statistics. data-processing was performed in python (enthought canopy python version 2.7.3. r (version 2.15.3) was used for statistical analysis and reports generation. to determine how well sendout and in-house test results can be anticipated based on basic information available in the medical record, we used two independent methods-generalized linear modeling (glm) and classification and regression trees (cart)to build simple, robust test-result predictors and then evaluated the performance of these predictors according to the standard clinical metrics of positive predictive value (ppv) and negative predictive value (npv), as well as sensitivity and specificity via the receiveroperator curve (roc) area under the curve (auc). as proof of principle for glm, we first tested it on the anion gap, a result calculated by subtracting the serum concentrations of the anions chloride and bicarbonate from those of the cations sodium and potassium, and confirmed that our methods found a rule for elevated anion gap based on these four items. we next applied glm to 81 sendout tests ordered regularly at our hospital. glm generated rules for just 11 of these tests. for the remaining tests, either no recent diagnosis or in-house test result (or age or gender) was sufficiently correlated with the sendout test result, or there were not enough instances in which correlated items appeared with the result, to generate a rule. only two tests-for high corticotropin (acth) and for low ceruloplasmin-had npv$0.95. of these, ceruloplasmin had a ppv$0.94. the mean auc for all rules was 0.69, with models for only three tests having an average auc$0.75 over 10 repeat runs. removal of features that did not appear in a majority of rules had essentially no effect on these aucs (difference in mean auc#0.02). cart generated rules for 60 tests. however, the auc for most of these rules was low, with only five tests having auc$0.75: free t3, alpha-macroglobulin, ca27-29, hyaluronic acid, and alpha fetoprotein (auc 0.75-0.79). we next applied glm to in-house tests. a total of 170 in-house tests were analyzed. a number of rules exhibited a high ppv (the probability of seeing an abnormal value given a prediction of an abnormal value by the rule) or npv (the probability of seeing a normal value given prediction of a normal value). these were mostly components of the complete blood count (cbc) and metabolic panels. interestingly, the predictive power of these rules was almost exclusively based on a previous measurement of the test in question: in other words, the best rules were for repeat tests, and the best predictor of a result being normal or abnormal was whether it had been normal or abnormal within the previous seven days. for example, the npv for a low red blood cell count was 0.95 (with ppv = 0.75), with a rule that depended most on the previous red blood cell count also having been low, and the ppv for high total calcium was 0.98 (npv = 0.76) and based exclusively on the previous total calcium having been high. for comparison, we applied cart to in-house tests, again including in the input data the most recent result for that test if performed within a week of the order. again, a number of rules exhibited a high ppv ($0.95), and again these were often tests of the cbc and metabolic panels, with rules based almost exclusively on a previous abnormal value. examples included low white blood cell count (wbc; ppv = 0.97, npv = 0.79), platelet count (0.95, 0.88), and serum sodium (0.96, 0.65), and high total calcium (0.99, 0.67), mean corpuscular volume (0.98, 0.84), and iron (0.97, 0.56) all of which were determined almost exclusively from the previous value being low or high (table 1) . overall, there was good agreement in ppv between glm and cart for tests for which both methods found rules, but cart outperformed glm noticeably in npv (fig. 2 ). the growing availability of large clinical databases has raised interest in the possibility of using systematic rule-mining for clinical decision support [12] [13] [14] [15] . one popular and well characterized approach has been logistic regression [16] [17] [18] , a special case of generalized linear modeling (glm). researchers have applied these approaches for diverse health-related purposes including prediction of cardiovascular risk [19] , mortality in head trauma [18] , texture analysis of magnetic resonance images [16] , and many other applications [17, 20, 21] . however, we note that glm does not easily incorporate missing values, as it removes records with missing features; a feature will be ''missing'' for any record in which that test (the feature) was not performed. other methods, such as classification and regression trees (cart) and artificial neural networks [18] , have also been applied. most of these studies were limited in scope to predicting risk of a particular diagnosis. harper [22] compared four classification techniques (regression, cart, artificial neural networks, and discriminant analysis) on four different datasets and concluded that there was no obvious best choice for their data; while cart performed best, regression was fastest and nearly as good. similar comparative studies on coronary artery disease [20] and alzheimer disease [23] indicated that newer algorithms such as ann and random forests [24] have little advantage over simpler, more traditional approaches. also, the utility and limitations of these approaches for predicting laboratory results (as opposed to diagnoses) are unclear. however, while cart is both a top performer and overcomes glm's problem with missing values, it is also more computationally intensive and potentially less sensitive to simple algebraic relationships among features (e.g., among sodium, chloride and bicarbonate and the anion gap). therefore we chose glm as a well-understood approach with strong performance and excellent speed, and cart as the bestperforming complementary approach for purposes of comparison. given the importance of laboratory testing, we asked how much information regression-or classification tree-based rules could provide in assessing the pre-test probability of a test result being abnormal for 251 commonly ordered in-house and sendout tests at our hospital. data-mining can sometimes find spurious correlations, artifacts of the particular partitioning of the data into training and test set. to avoid such artifacts, we repeated our regression on multiple independent partitions of the data and kept only items that appeared in a majority of the resulting rules. this safeguard also had the effect of simplifying rules by making each rule dependent on a smaller number of items. as expected, the effect on performance was negligible and dependence on the resulting items was more often clinically and pathophysiologically plausible than rules derived from each run. when data-mining it is also important to consider the setting. the rules we found do not exist in a vacuum but are ''contingent'' in the sense that they depend on current clinical practice. certain tests and panels are ordered in patterns. in a sense, contingency is a form of selection bias: there may well be other diagnoses or test result results that correlate with the result for the test of interest that are not routinely measured according to current best practices. however, as long as the setting in which such rules would be applied is the substantially similar to that in which they were found, selection bias would have little if any effect on finding rules. as long as one is clear that one is looking for relationships in a current practice process, and not among all things that could possibly be measured, any rules that are discovered will by construction be setting-appropriate. but while our rules appear to be plausible and settingappropriate, the motivating question behind this study is whether the rules we found could be useful clinically. one way to approach this question is by considering the positive and negative predictive value of each rule (ppv and npv). these metrics are in contrast to sensitivity and specificity, by which rules are often measured but which do not incorporate disease prevalence in spite of its importance to clinical decision-making. a ppv of 0.95 means that when a rule suggests that the test result will be abnormal, the result actually will be abnormal 95 percent of the time. a npv of 0.95 means that when a rule suggests that the test result will be normal, the result actually will be normal 95 percent of the time. we found rules with ppv and/or npv$0.95 (by glm) for only two tests that are sendouts at our hospital-one of which is ceruloplasmin, which we have previously suggested is overordered via chart review [25] . in contrast, for in-house tests we found over a dozen such rules. interestingly, the main determinant for rules for in-house tests was a normal or abnormal result for the same test within the previous seven days. although in this study we did not set out explicitly to make a statement about repeat laboratory testing, the appropriateness of which has been investigated elsewhere [4] , these results suggest that repeat laboratory testing within one week does not always add information that could not have been anticipated from the previous result. refining this observation using the same unbiased approach we have followed here is potentially an area for future investigation. our results should not be taken as a categorical criticism of repeat testing. first, while the ppv was $0.95 in several cases, the npv was more typically 0.70-0.85. thus, while prediction that a result will be abnormal may be correct 95 percent of the time, which may be good enough to discourage repeat ordering, prediction that a result will be normal may not be so dependable. therefore use of a rule depends on the subtle distinction of whether the clinical question is ''will the result be abnormal'' vs. ''will the result be normal.'' second, we note that no rules with such strong performance were found for the majority of our sendout or in-house tests by either of our two complementary approaches. thus while the rules we found can inform clinical decision-makers, the information they provide rarely replaces the information obtained from actually performing these tests. it is interesting to note that on average, our simple rules yielded a ppv of 0.84 and an npv of 0.75. this means that on average, rules will correctly predict an abnormal laboratory result 5 times out of 6 (5/6<0.84) and correctly predict a normal result 3 times out of 4. while not good enough to replace testing (especially for rules that depend on previous test results), these observations raise the question of how much better prediction can get. integration of information not considered in the present study, including vital signs, chief complaints, and physical findings, may improve prediction by these methods. the ulysses syndrome the dangers of false-positive and false-negative test results: false-positive results as a function of pretest probability the landscape of inappropriate laboratory testing: a 15-year systematic review and meta-analysis laboratory evaluation in the diagnosis of lyme disease elementary, my dear doctor watson generalized linear models electronic health record surveillance algorithms facilitate the detection of transfusion-related pulmonary complications using classification trees to assess low birth weight outcomes fast algorithms for mining association rules fast discovery of association rules data mining and clinical data repositories: insights from a 667,000 patient data set mining association rules from clinical databases: an intelligent diagnostic process in healthcare machine learning for personalized medicine: predicting primary myocardial infarction from electronic health records predictive data mining in clinical medicine: current issues and guidelines textural analysis of contrast-enhanced mr images of the breast establishing a clinical decision rule of severe acute respiratory syndrome at the emergency department comparison of artificial neural network and logistic regression models for prediction of mortality in head trauma based on initial clinical data improved cardiovascular risk prediction using nonparametric regression and electronic health record data comparing performances of logistic regression, classification and regression tree, and neural networks for predicting coronary artery disease predicting improvement in urinary and bowel incontinence for home health patients using electronic health record data a review and comparison of classification algorithms for medical decision making application and comparison of classification algorithms for recognition of alzheimer's disease in electrical brain activity (eeg) random forests the overuse of serum ceruloplasmin measurement key: cord-001045-jm60nxc2 authors: delisle, sylvain; kim, bernard; deepak, janaki; siddiqui, tariq; gundlapalli, adi; samore, matthew; d'avolio, leonard title: using the electronic medical record to identify community-acquired pneumonia: toward a replicable automated strategy date: 2013-08-13 journal: plos one doi: 10.1371/journal.pone.0070944 sha: doc_id: 1045 cord_uid: jm60nxc2 background: timely information about disease severity can be central to the detection and management of outbreaks of acute respiratory infections (ari), including influenza. we asked if two resources: 1) free text, and 2) structured data from an electronic medical record (emr) could complement each other to identify patients with pneumonia, an ari severity landmark. methods: a manual emr review of 2747 outpatient ari visits with associated chest imaging identified x-ray reports that could support the diagnosis of pneumonia (kappa score = 0.88 (95% ci 0.82∶0.93)), along with attendant cases with possible pneumonia (adds either cough, sputum, fever/chills/night sweats, dyspnea or pleuritic chest pain) or with pneumonia-in-plan (adds pneumonia stated as a likely diagnosis by the provider). the x-ray reports served as a reference to develop a text classifier using machine-learning software that did not require custom coding. to identify pneumonia cases, the classifier was combined with emr-based structured data and with text analyses aimed at ari symptoms in clinical notes. results: 370 reference cases with possible pneumonia and 250 with pneumonia-in-plan were identified. the x-ray report text classifier increased the positive predictive value of otherwise identical emr-based case-detection algorithms by 20–70%, while retaining sensitivities of 58–75%. these performance gains were independent of the case definitions and of whether patients were admitted to the hospital or sent home. text analyses seeking ari symptoms in clinical notes did not add further value. conclusion: specialized software development is not required for automated text analyses to help identify pneumonia patients. these results begin to map an efficient, replicable strategy through which emr data can be used to stratify ari severity. effective responses to epidemics of infectious diseases hinge not only on early outbreak detection, but also on an ongoing assessment of disease severity. indeed, the proportion of infected patients who develop severe illness often governs public perception and is a key factor in deciding whether or not to trigger interventions that can cause harm and exact significant social and financial costs. for surveillance systems aimed at epidemics of acute respiratory infections (ari), the rationale for incorporating information about disease severity is particularly compelling: 1) doing so could help discover outbreaks that involve only a small number of very sick patients, such as what initially occurred with sars [1] or what could be anticipated shortly after a criminal release of plague [2] or tularemia [3] ; 2) such systems could help adjust ongoing responses to seasonal or pandemic influenza, where severity can vary by orders of magnitude between epidemics [4] or even between waves of the same epidemic [5, 6] . to be useful, information about ari severity needs to be both timely and specific [7, 8] . current methods of monitoring influenza-related hospitalizations or deaths fall short of meeting these requirements [9] . electronic medical records (emr) are fast becoming commonplace, and form a rich source of information that could be secondarily used for surveillance purposes. in the past, we initiated a project to unravel how emr data could be combined to identify outpatients with ari [10] . in this work, we sought to develop casedetection algorithms (cda) aimed at pneumonia, a key landmark in the severity spectrum of ari. in particular, we asked how information retrieved from the free-text of chest imaging reports and clinical notes could complement structured data to uncover pneumonia cases. this study was approved by the institutional review boards at the university of maryland and the va maryland health care system. research-related risks were limited to maintaining the confidentiality of data generated during routine patient care. a waiver of consent was granted because the research-related risks were minimal and did not adversely affect the rights and welfare of the participants, and because the work would not have otherwise been feasible, given the large number of participants. we applied a previously validated ari case-detection algorithm (cda) [10] to emr-derived information related to outpatient visits at the veterans administration maryland health care system, from january 1, 2004 through december 31, 2006 . this ari cda was chosen as a screening tool because it identifies 99% of outpatients that satisfied a broad definition of ari: positive respiratory virus culture/antigen or any two of the following symptoms, of no more than 7 days duration: a) cough; b) fever or chills or night sweats; c) pleuritic chest pain; d) myalgia; e) sore throat; f) headache and illness not attributable to a non-infectious etiology [10] . the ari cda flagged an outpatient visit if the provider assigned it an ari-related international disease classification, 9 th revision, clinical modification (icd-9) diagnostic code or issued a prescription for a cough remedy or documented at least two symptoms from the above ari case definition in his/her clinical note, as retrieved by computerized text analysis [10] . visits flagged by the ari cda were included if chest imaging was obtained within 24 hours of clinic registration time. participants were sampled only once, at their first eligible visit during the study period. the methods to validate the performance of selected pneumonia cda on a separate population are described in the next section. reference chest imaging report review. a pulmonary disease physician read all eligible chest imaging reports (n = 2,861 in 2747 unique patients). reports were labeled ''negative'' if they did not support the diagnosis of pneumonia. this category included all images within normal limits or showing no evidence of active pulmonary disease. reports with comments on shrapnel or bullet fragments, pleural plaques or other abnormalities outside the lung parenchyma, calcified granulomas, old nodules, scars or chronic emphysematous changes were put in this category. reports were labeled ''non-negative'' if they could possibly support the diagnosis of pneumonia. these reports described a wide range of abnormalities, from ill-defined densities where the diagnosis of pneumonia could not be excluded, to frank infiltrates characteristic of pneumonia. all ''non-negative'' reports and a 10% sample of the ''negative'' reports were blindly reviewed by a second pulmonary physician (n = 537). kappa score between the two independent reviewers was 0.88 (95% ci 0.82:0.93). ''nonnegative'' reports containing wording typically used to describe abnormalities indicative of pneumonia were also flagged and used as an alternative training set in the development of the automated imaging report classifier (see below). reference clinical record review. reference cases with pneumonia were identified by manually reviewing all emr entries made during the calendar day of index visits that corresponded to the reference, manually reviewed, ''non-negative'' chest imaging reports outlined above. symptoms and diagnostic impressions were abstracted by a pulmonary physician, entered into a data collection instrument (ms access, microsoft corp., redmond wa) and recombined into two case definitions: 1) ''possible pneumonia'': non-negative chest imaging report and at least one of the following symptoms, new or changed within the last 7 days: a) cough; b) sputum; c) fever or chills or night sweats; d) dyspnea; e) pleuritic chest pain and illness not clearly attributable to a noninfectious etiology; 2) ''pneumonia-in-plan'': a non-negative chest imaging report and pneumonia listed as one of the top two diagnostic possibilities in a physician's or nurse practitioner's note. cases with possible pneumonia or pneumonia-in-plan were labeled ''admitted'' if they gained admission to the hospital within 48 hours of index visit registration. otherwise, they were labeled ''outpatient''. development of chest imaging report classifier. we used open-source automated software that couples a clinical nlp pipeline (clinical text analysis and knowledge extraction system (ctakes) [11] ) with an implementation of a conditional random fields probabilistic classifier [12] to develop the text analyses that could separate non-negative from negative chest imaging reports (automated retrieval console (arc) software, v.2.0 [13, 14] ). in a preliminary effort to improve the performance of the classifier, the reference imaging reports were presented for machine-learning as four alternative training sets where: a) the text of the reports was fed either whole or scrubbed from the characters preceding the string ''impression'' when the latter was found; b) targeted reports were either all of the non-negative reports (n = 450) or only those that described abnormalities typical for a pneumonia (n = 316). text classification models with the highest f-measure were retained for each training set. the four retained models were then separately combined with other emr-derived data and performance of the resulting cdas at identifying patients that fitted our case definition compared (see next paragraph). the text classification models trained with reports that contained typical pneumonia descriptions and whose text was restricted to the ''impression'' field led to the best performing pneumonia cdas, and were those used for this report. candidate components for cdas included those previously found useful to identify patients with ari: ari-related icd-9 codes (labeled as ''ari icd-9 codes''), cough remedies [10] , and clinical notes identified as positive for ari symptoms by text analysis [10] (''text of clinical notes''). we also considered the following cda components, when related to the index outpatient visit: 1) a subset of the ari icd-9 codes whose narrative included the string ''pneumonia'' (''pneumonia icd-9 codes'': 480-483, 485-487); 2) a new prescription for antibiotics of a class of commonly used to treat pneumonia (cephalosporins, fluoroquinolones, macrolides, penicillins); 3) admission to the hospital, for any reason, within 48 hours of the index outpatient visit (''(not) admitted to hospital''); 4) chest imaging performed (''imaging obtained''); 5) whether at least one chest imaging report related to the index visit was labeled ''non-negative'' by the automated text classifier described above (''text of imaging reports''). performance measures. the performance of the pneumonia cdas was summarized with standard test descriptors (sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and f-measure (2 * ppv * sensitivity/(ppv + sensitivity)). denominators used to calculate these tests were either the whole study population (n = 2747), those patients who were hospitalized for any reason following their index visit (n = 602) or those who were not (n = 2145). validation of selected cdas. the ari cda and imaging report classifier were applied to emr-derived databases for a 5year period anterior to the original study period i.e. 1/1/2007-12/31/2011. a random, 50% sample of the visits flagged by the [ari cda and text of imaging reports] query were manually reviewed. cases identified served as the reference to validate the ppv of selected pneumonia cdas. the ari cda flagged 22,960 first visits from unique patients during the algorithm development phase of the study period. of these, 2,747 were associated with at least one report for chest imaging performed within 24 hours of check-in time. the study population was 93% male, older (61615 years old, mean 6 standard deviation) and 52.6% african american (table 1) . a manual review of emr entries on the day of the 2,747 index visits identified 380 cases that satisfied at least one pneumonia case definition, 370 with possible pneumonia and 250 with pneumoniain-plan. most patients with a pneumonia-in-plan also had possible pneumonia (240/250), including nearly all (124/127) patients admitted to the hospital. patients who satisfied either case definitions were therefore merged into a common target group for the development of the ''admitted pneumonia'' cdas. ninety percent of all index visits occurred in urgent/same day care settings. patients with possible pneumonia and pneumonia-in-plan had similar demographics (table 1 ) and symptoms and signs (table 2) , with the possible exception that the latter population had more febrile symptoms. compared with their outpatient counterparts, admitted pneumonia patients were overrepresented in the older age groups (71-90 years old, table 1 ) and appeared to have more dyspnea, fever-related symptoms, and clinical signs of lung consolidation ( table 2) . the composition and performance of illustrative cdas for cases with possible pneumonia or pneumonia-in-plan are shown for all locations of care in table 3 , and for those cases that remained outpatients or were admitted (tables 4 and 5, respectively). structured emr information ipso facto included as components of the relevant cdas included: 1) that chest imaging was obtained (''imaging obtained'', tables 3-5); 2) whether or not a case was admitted to the hospital (''(not) admitted'', tables 4-5 ). an icd-9 code set for pneumonia diagnoses (''pneumonia icd-9 codes'', tables 3-5) helped identify pneumonia with ppvs of 52.8-55.3% but had limited sensitivity (28.5-56%, cdas 1, 8, 15, 22 , and 29, tables 3-5), even when providers had indicated that pneumonia was a likely diagnosis in their clinical notes i.e. in pneumonia-in-plan or admitted pneumonia cases (cdas 8, 22 and 29, tables 3-5). a broadly inclusive ari icd-9 code set (''ari icd-9 codes'', tables 3-5) increased detection sensitivity to 86-97%, but degraded ppv (6.3-23.5%) and overall performance, as reflected by lower f-measures (compare cda 3 to 1, 10 to 8 in table 3 , 17 to 15, 24 to 22 in table 4 , and 31 to 29 in table 5 ). cdas that did not include icd-9 diagnostic codes were not among the most successful (data not shown). prescriptions for medications aimed at ari symptoms and various groupings of antibiotics that could be used to treat bacterial pneumonias did not add value (data not shown). we retrieved information from free-text emr entries according to two different strategies. in the first strategy, text analysis routines were used to search for ari symptoms in the providers' clinical notes (''text of clinical notes'', . coupling positive results of text of clinical notes analyses to ari icd-9 codes using an or logical operand increased detection sensitivity over otherwise comparable cdas. however, specificity and ppv decreased and overall performance either did not improve or worsened (compare cda 6 to 3 and 13 to 10, table 3 ; cda 20 to 17 and 27 to 24, table 4 ; cda 34 to 31 and 35 to 33, table 5 ). coupling the text of clinical notes analysis to ari icd-9 codes using an and logical operand further increased ppv, but severely reduced sensitivities and overall performance (cda 4, 11, 18, 25 and 32, tables 3-5). in the second strategy, text analysis was used to flag chest imaging reports that could support the diagnosis of pneumonia (''and text of imaging reports'' component, . adding this component increased the ppv of otherwise identical cdas by 23-70 absolute percentage points (compare cda 2 to 1, 5 to 3, 7 to 6 and so on, tables 3-5). despite attendant losses in sensitivity, results from the ''text of imaging reports'' classifier increased the f-measure of all cdas that included the broad ari icd-9 code set. with the possible exception is cda 7, whose fmeasure was the highest achieved in this study, the or text of clinical notes component did not add further value to cdas that already included analyses of the chest imaging reports (compare cda 7 to 5 and 14 to 12, table 3 ; cda 21 to 19 and 28 to 26, table 4 ; cda 35 to 33, table 5 ). table 4 ) and was in large part due to flagging of follow-up rather than initial pneumonia visits (data not shown). ppvs actually increased for patients admitted to the hospital (cda 33, 35, table 5 ). discussion automated text analyses of chest imaging reports improved the performance of emr-based cdas that included structured data elements and free-text search for ari symptoms. this contribution persisted across pneumonia case definitions, applied to outpatients and hospitalized patients alike, and helped cdas reach precisions of 64-86% while maintaining sensitivities of 58-75%. these data support our working hypothesis that selected free text analyses can supplement structured emr data to assess the severity of ari outbreaks. this work benefits from prior efforts to combine emr data to identify patients with ari. the ari cda used as an initial screen for the current study had been developed and validated against a population-based sample of over 15,000 emr records, where it recognized 99% of cases that satisfied a broad definition of ari [10] . this screening algorithm forms a practical starting point for an emr data flow intent on monitoring the incidence and severity of aris, and is likely to have flagged most symptomatic pneumonia patients. pneumonia is seldom a definitive diagnosis, even when histological information is available [15] . absent a standard, we sought clinically acceptable case definitions that could be reliably abstracted from clinical records. as is both customary and recommended by treatment guidelines [16] [17] [18] [19] , our case definitions required supportive chest imaging. to this common imaging requirement, the possible-pneumonia definition added clinical symptoms whereas pneumonia-in-plan relied solely on the provider's final diagnostic assessment. despite these differences, more than 95% of patients with pneumonia-in-plan also satisfied the more permissive possible pneumonia definition in both our development and validation reference populations, indicating that the two definitions addressed related clinical conditions. given that independent emr abstractors could identify respiratory symptoms [10] , pneumonia diagnostic impressions and supportive chest imaging with a high degree of agreement, our data suggest that the possible pneumonia and the pneumonia-in-plan case definitions can serve as useful tools to reproducibly retrieve pneumonia-related information from an emr. prior attempts to automatically identify pneumonia patients through medical records have concentrated on diagnostic codes assigned after hospital discharge. discharge codes have been found to be good markers for hospitalized pneumonia patients, whether benchmarked against retrospective record reviews [20] [21] [22] or prospective data acquired for clinical trials [23] [24] [25] [26] . discharge codes, however, are of limited value in epidemic surveillance because they are untimely and do not distinguish between community-and hospital-acquired pneumonia [22] . in this study, we evaluated diagnostic codes assigned by providers at the conclusion of outpatient visits, as is practiced at the veterans administration health system. we found these codes to represent a key component of pneumonia detection, even if they proved less accurate at finding pneumonia patients who were sent home rather than hospitalized [27] . while the utility of diagnostic codes vary when they are assigned by third parties or have reimbursement repercussions, our results nevertheless provide an impetus for diagnostic codes to be made available as soon as possible following outpatient services, so that they can be used for surveillance, decision support and quality control. the chest imaging report has long been recognized as a fruitful context in which to mine for evidence of pneumonia. over the last 20 years, various combinations of approaches, including natural language processing [28] [29] [30] [31] , expert rules [32, 33] , bayesian [32, 34] or neural networks [35] and machine-learning [33] , have held their own compared to physicians for their ability to find pneumonia-related concepts in report narratives. imaging report analyses have been compared to discharge diagnostic codes [36, 37] , but have seldom been evaluated for their added value against a broader reference standard for clinical pneumonia [38] [39] [40] . to our knowledge, only one previous publication used imaging report analyses to detect outpatients with communityacquired pneumonia [40] . besides bolstering the evidence for the utility of these text analyses, our data illustrate the importance of targeting them properly: in the course of this study, classifying 26,581 imaging reports did more to improve detection performance than extracting ari symptoms from almost 14 million clinical notes. although an assessment of the significance of the performance gained through imaging report text analysis must await purpose-specific evaluations, our data nevertheless support the notion that a generalized machine learning approach can perform well across information retrieval tasks [13, 14] . also significant, in our view, is the ease with which we could develop the classifier. clinical users focused on the document-level classification needed to create the reference training set. once the latter was fed to the arc software, model development required little further user interaction, and there was no need for custom programming. such an efficient workflow makes it possible to quickly rebuild the classifier elsewhere, should it proves less robust than our validation exercise suggests. our study is subject to limitations beyond those already mentioned. first, we did not evaluate cda components that have been associated with pneumonia in the past such as abnormalities in vital signs [41] , white blood cell count [42] or oxygenation [41] , and microbiological results. while these data elements could be missing in some patients [43] , they could provide an opportunity to further improve detection performance. second, our work was performed in a health system whose population and health care practices may not be generalizable. even if diffusion of our approaches was initially restricted to va institutions, at least some automated pneumonia surveillance could nevertheless be deployed across all 50 states. third, sampling was not random but instead based on a screening algorithm. while this algorithm has been validated using a random, population-based sample, our study sample remains subject to verification bias [44] such as the systematic exclusion of pneumonia patients for whom chest imaging was not obtained [45] . fourth, the retrospective nature of the record review coupled with shortcomings of clinical acumen and chest imaging [46] imply that we may have missed pneumonia patients whose symptoms, signs or imaging abnormalities were absent [46, 47] , missed, atypical, inadequately documented or miscoded [23] . despite these potential failings, our results do reflect information committed to a real-world emr, and thus represent a realistic environment in which to compare the relative performance of alternative detection approaches. in summary, our results indicate that an emr-based approach that couples queries of structured data with text analysis of imaging reports can be used to assess disease severity in outpatients with ari. by identifying high-performing yet parsimonious cdas that could be replicated without creating customized software, our results begin to map an efficient strategy by which pneumonia surveillance could be more widely implemented. severe 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reports that support pneumonia the role of domain knowledge in automating medical text report classification bayesian modeling for linking causally related observations in chest x-ray reports automatic identification of patients eligible for a pneumonia guideline: comparing the diagnostic accuracy of two decision support models using discordance to improve classification in narrative clinical databases: an application to community-acquired pneumonia detection of pneumonia using free-text radiology reports in the biosense system use of computerized surveillance to detect nosocomial pneumonia in neonatal intensive care unit patients extracting information on pneumonia in infants using natural language processing of radiology reports combining decision support methodologies to diagnose pneumonia vitalsign abnormalities as predictors of pneumonia in adults with acute cough illness laboratory tests for pneumonia in general practice: the diagnostic values depend on the duration of illness processes and outcomes of care for patients with community-acquired pneumonia: results from the pneumonia patient outcomes research team (port) cohort study assessment of diagnostic tests when disease verification is subject to selection bias testing strategies in the initial management of patients with community-acquired pneumonia high-resolution computed tomography for the diagnosis of community-acquired pneumonia influence of age on symptoms at presentation in patients with community-acquired pneumonia the authors would like to thank robert sawyer md for technical help with the institutional databases. conceived and designed the experiments: sd ag ms ld. performed the experiments: bk jd ts. analyzed the data: sd ts. contributed reagents/materials/analysis tools: sd ld. wrote the paper: sd ag ms ld. key: cord-002957-gw2cow0d authors: gray, darren w.; welsh, michael d.; mansoor, fawad; doherty, simon; chevallier, olivier p.; elliott, christopher t.; mooney, mark h. title: diva metabolomics: differentiating vaccination status following viral challenge using metabolomic profiles date: 2018-04-05 journal: plos one doi: 10.1371/journal.pone.0194488 sha: doc_id: 2957 cord_uid: gw2cow0d bovine respiratory disease (brd) is a major source of economic loss within the agricultural industry. vaccination against brd-associated viruses does not offer complete immune protection and vaccine failure animals present potential routes for disease spread. serological differentiation of infected from vaccinated animals (diva) is possible using antigen-deleted vaccines, but during virus outbreaks diva responses are masked by wild-type virus preventing accurate serodiagnosis. previous work by the authors has established the potential for metabolomic profiling to reveal metabolites associated with systemic immune responses to vaccination. the current study builds on this work by demonstrating for the first time the potential to use plasma metabolite profiling to differentiate between vaccinated and non-vaccinated animals following infection-challenge. male holstein friesian calves were intranasally vaccinated (pfizer rispoval(®)pi3+rsv) and subsequently challenged with bovine parainfluenza virus type-3 (bpi3v) via nasal inoculation. metabolomic plasma profiling revealed that viral challenge led to a shift in acquired plasma metabolite profiles from day 2 to 20 p.i., with 26 metabolites identified whose peak intensities were significantly different following viral challenge depending on vaccination status. elevated levels of biliverdin and bilirubin and decreased 3-indolepropionic acid in non-vaccinated animals at day 6 p.i. may be associated with increased oxidative stress and reactive oxygen scavenging at periods of peak virus titre. during latter stages of infection, increased levels of n-[(3α,5β,12α)-3,12-dihydroxy-7,24-dioxocholan-24-yl]glycine and lysophosphatidycholine and decreased enterolactone in non-vaccinated animals may reflect suppression of innate immune response mechanisms and progression to adaptive immune responses. levels of hexahydrohippurate were also shown to be significantly elevated in non-vaccinated animals from days 6 to 20 p.i. these findings demonstrate the potential of metabolomic profiling to identify plasma markers that can be employed in disease diagnostic applications to both differentially identify infected non-vaccinated animals during disease outbreaks and provide greater information on the health status of infected animals. introduction bovine respiratory disease (brd) is a multifactorial disease characteristic of a viral-bacterial synergistic infection with predisposition from environmental stressors [1] . the disease constitutes a major source of economic loss through mortality, clinical disease and the associated treatments and long lasting reduced growth performance of infected young stock [2, 3] . the annual cost of brd is estimated at $1billion in the usa, with preventative measures contributing a further $3billion [4, 5] . vaccines are commonly used for controlling brd viral pathogens [6] , but despite seasonal vaccination, animals can become infected with each new outbreak [7] , maintaining the infection within the population. the viral pathogens associated with brd [bovine parainfluenza virus type-3 (bpi3v), bovine respiratory syncytial virus, bovine viral diarrhoea virus and bovine herpes virus-1] impair immune responses in infected animals and damage the respiratory tract allowing the establishment of secondary infections, that may develop further into bacterial pneumonia [8] . however, vaccinated animals can successfully clear viral infections faster than non-vaccinated animals through immune memory response, reducing the associated viral tissue damage or impairment of immune functions preventing the establishment of secondary bacterial and mycoplasma infections [6] . during disease outbreaks, identification of unvaccinated animals at the early stages of infection could provide a window for effective treatment and facilitate the removal of animals that pose a greater risk of becoming infected and transmitting the infection to more susceptible juvenile stock. furthermore, halting viral disease progression to more severe and costly secondary bacterial infections through the identification of vaccine failure animals during infection outbreaks would reduce the level of antibiotic use in the agricultural industry. the only definitive method for successfully identifying vaccinated animals in the presence of an active viral infection is to determine the rate of viral shedding by virus isolation, cytokine/interleukin profiling or virus neutralization assay [9] . these types of analysis require repeated sampling, a period for seroconversion and are expensive compared to serology based elisa, and are therefore not routinely employed during endemic viral infection outbreaks. differentiating infected from vaccinated animals (diva) marker vaccines (e.g. a modified wild type virus with a gene deletion resulting in the absence of a particular diagnostic antigen) can be employed to differentiate vaccine antibody responses from that of wild type virus. companion serology based tests rely on seroconversion, and upon exposure to wild type virus the antibody response to diva vaccines will be masked by that of the wild type virus. vaccine diva functionality is often limited to large viruses with increased potential for gene deletion and removal of redundant expressed antigens. therefore, for viruses with small genomes such as paramyxoviruses (e.g. bpi3v and bovine respiratory syncytial virus of the brd complex) where gene deletion of neutralizing antigens may reduce vaccine efficacy, alternative approaches are required to provide diva functionality. one approach is to design molecular diva vaccines that contain a marker nucleotide sequence differing from the wild type virus that can be employed in combination with pcr-based molecular diagnostics to differentiate between vaccine and wild virus strains [10, 11] . successful differentiation of vaccinated from non-vaccinated animals using this technique requires concurrent vaccination and infection [12, 13] , with a narrow diagnostic window post-infection for detection of diva vaccine and viral genetic material. furthermore, detection of vaccine genetic material only demonstrates exposure to the vaccine and not the successful generation of immune protection, limiting functionality in assessment of herd level immunity. consequently, there is a clear need for alternative diagnostic methods that can assess efficacy of vaccines and vaccination status of animals exposed to brd viral pathogens at the early stages of infection prior to seroconversion and which do not require repeated sampling. additionally, the lower initial exposure rates to viral infections in field settings combined with variation in strain nucleotide sequences and short periods of virus secretion highlights the requirement for a diva approach with a long diagnostic window which is not strain specific. a potential approach that can meet these needs is based on the application of metabolomics to identify metabolites or 'small molecules' in biological samples that are signatures that correlate or provide some evidence of immune protection. these metabolites are often the end stage products of biological processes and therefore provide an accurate representation of an organism's homeostatic status at time of sampling [14, 15] . metabolomic analysis of bio-fluids has provided new insights to the understanding of the patho-physiological processes involved in disease establishment, development and diagnosis [16] [17] [18] [19] . whilst metabolomics has had limited application in the field of veterinary research, several studies have demonstrated the potential of this technique in the prediction of brd disease outcome [20] , differentiation of stress from viral infection responses [21] , and characteristic of immune responses following vaccination [22] . this study focuses specifically on bpi3v due to its endemnicity within cattle populations and absence of clinical symptoms which still predispose animals to more severe bacterial infections [23] . due to its small genome and absence of non-redundant proteins suitable for removal in diva vaccines, bpi3v is an excellent model for assessing the potential of metabolomics to establish vaccination status in infected animals. the aims of the current study were therefore to assess the performance of reverse phase (rp) and hydrophobic interaction liquid chromatography (hilic) separation methods for ultra performance liquid chromatography-mass spectrometry (uplc-ms) metabolomic profiling of bovine plasma and identify plasma metabolomic markers capable of differentiating between vaccinated and nonvaccinated calves following intranasal challenge with bpi3v. this work for the first time reports the metabolomic responses following challenge with bpi3v and demonstrates how the application of metabolomic profiling may help overcome current limitations in diva diagnostics by identifying markers capable of differentiating between vaccinated and non-vaccinated animals, and importantly allow the development of better tools to assess the performance of vaccines. assessment of clinical findings of animals post bpi3v vaccination and challenge have been reported in detail previously [22] . briefly, animals were healthy throughout the duration of the study with no clinical signs of disease in vaccinated or non-vaccinated study groups. calves were sourced from respiratory disease free farms with no history of vaccination against brd. prior to the commencement of vaccination all calves tested seropositive for anti-bpi3v igg. these residual levels of maternally derived anti-bpi3v immunoglobulin are in keeping with other studies employing calves of the same age [24] . at the commencement of vaccination there was no significant difference in anti-bpi3v igg observed between treatment groups. vaccination with rispoval 1 pi3+rsv resulted in a significant increase in anti-bpi3v igg with no significant increase in non-vaccinated controls. following bpi3v challenge vaccinated calves had significantly higher plasma anti-bpi3v igg relative to non-vaccinated animals. post-bpi3v challenge, anti-bpi3v igg remained elevated in vaccinated animals. although no significant differences in anti-bpi3v igg levels were observed in non-vaccinated animals at days 12-20 post-infection (p.i.), igg was elevated in 2/3 animals relative to day 0 levels, with the remaining animal demonstrating elevated levels from days 14 to day 20 p.i. there was a significant increase in lymphocyte counts in non-vaccinated animals from day 0 to 5 p.i., and a significant decrease from days 5 to 12, with no significant variations observed in vaccinated animals. there were no significant differences in neutrophil counts between study groups at sampling points post-infection, however significant (p < 0.05) temporal variations were observed with a decrease from day 0 to 5 in non-vaccinated animals, and an increase from day 5 to 12 in both study groups. at day 6 p.i., 3 animals per group were prepared for gross postmortem analysis. two of 3 animals in the non-vaccinated group showed gross inflammatory lesions, with some peri-vascular cuffing consistent with pneumonia. no significant gross / histological abnormalities were detected in 3/3 of the vaccinated calves at 6 day p.i. at postmortem. preliminary metabolomic analysis was performed to select a suitable method for the comprehensive profiling of metabolites within bovine plasma. rp and hilic chromatographic methods were compared using day 6 p.i. samples (n = 6), which corresponds to peak viral titre [25] . despite improved resolution of poorly retained hydrophobic compounds eluting between 1-2min by hilic separation, increased chromatographic peaks corresponding to plasma derived compounds (relative to blank injections) were observed in rp acquired profiles (fig 1a and 1b) relative to hilic profiles (fig 1c and 1d ). observable differences between plasma samples from vaccinated and non-vaccinated animals within chromatogram profiles were only present using rp (s1 fig) . furthermore, upon data extraction and processing (metabolites with cv > 50% in qc pools), increased numbers of accurate mass retention time pairs (amrtps) were observed following rp (n = 1165) relative to hilic (n = 538) separation of plasma. unsupervised pca analysis of plasma profiles acquired by rp-uplc-ms resulted in clear separation between study groups when assessing principle components (pc) 1 and 2 (57.4% of systemic (r2x) variation within the dataset) as illustrated in fig 2a. in contrast, hilic-uplc-ms metabolomic profiles facilitated only partial separation between study groups based on the pca scores plot (pc1 and pc2, 35.6% of systemic (r2x) variation) ( fig 2b) . due to the small number of chromatographic peaks, fewer amrtps and poor unsupervised pca separation of plasma profiles acquired following hilic-uplc-ms chromatography, rp-uplc-ms was selected as the method of choice for metabolomic profiling of remaining study plasma samples. plasma samples from days 0, 2, 14 and 20 p.i. were extracted, analysed and combined with day 6 p.i. data for multivariate analysis. mass accuracy and retention time deviation of reference compounds between analysis runs was excellent (s1 table) and within marker lynx extraction parameters (0.02da and 0.2min respectively) ensuring accurate peak matching. amrtps (n = 1593) with %cv less than 50% in inter-run quality control plasma pools were used to construct of pca models (pre-filtered from 7690 amrtps extracted from raw data). the effect of bpi3v challenge on the metabolite profile of all animals irrespective of vaccination status, was illustrated in un-supervised pca scores plot ( fig 3a) by separation in pre-(day 0 p.i.) and post-bpi3v challenge samples (days 2-20 p.i.) when observing pc2 and pc3 (14.1% r2x). the greatest separation was observed between day 0 pre-and day 2 p.i. post-challenge stages in vaccinated and non-vaccinated animals. differentiation of vaccinated from non-vaccinated animals based on metabolite profiles following challenge with bpi3v was investigated using un-supervised (pca) and supervised (opls-da) multivariate analysis. prior to bpi3v challenge metabolite profile variation between vaccinated and non-vaccinated animals was low, evidenced by no separation at day 0 ( fig 3b) when observing all pcs. with study progression to post-challenge stages, the variation between vaccinated and non-vaccinated animal metabolite profiles increased from partial separation at day 2 p.i. (fig 3c, 9 .45% r2x with pcs 5 and 7) to clear separation at days 6, 14 and 20, with 19% (fig 2a) , 32.1% ( fig 3d) and 51.1% ( fig 3e) variation (r2x) respectively in pcs 2 and 3. opls-da analysis was employed for the selection of marker amrtps that could discriminate between vaccinated and non-vaccinated animals post-infection. fig 4a-4d illustrates opls-da score (inset) and s-plots for supervised discriminate analysis of plasma metabolite profiles from bpi3v infected vaccinated and non-vaccinated animals at days 2, 6, 14 and 20 p. i. respectively. the amount of variation responsible for differentiation of animals of different vaccination status (r2x) was 7.02%, 8.09%,12.7% and 20.4% in models generated for days 2, 6, 14 and 20 p.i. respectively, with excellent fit (r2y > 98.5%) and good cross-validated prediction (q2 > 95%). as opls-da is known to over-fit, particularly in megavariate datasets where the number of variables are higher than the sample size we employed permutation and false discovery rate testing to reduce the chances of selecting false positives. leave-one-out cross validation was performed to assess the performance of the models generated. briefly, all technical replicates from a single biological sample (test sample) were removed and the remaining dataset employed to generate a predictive opls-da model (vaccinated vs non-vaccinated). this predictive opls-da model was employed to predict the vaccination treatment group of the test sample. this cross-validation was permeated until all biological samples had been assessed for treatment classification. the results (s2 table) indicated that all biological replicates were accurately classified to their respective treatment groups upon cross validation prediction model testing. amrtps which contributed to class discrimination were selected on a criterion of a variable importance score (v.i.p.) score > 1 from opls-da discriminate analysis. amrtps were further filtered to select only those with a fold change (fc) >1.5 and significant difference (anova with bonferroni post-hoc test) p < 0.05 between study groups. final selection of amrtps was performed by assessment of raw mass spectrometric data to determine those with good peak shape and consistent peak intensity (height) in replicate injections. 383 unique potential markers combined from all opls-da analysis (44 at day 2 p.i., 152 at day 6 p.i., 128 at day 14 p.i., and 91 at day 20 p.i.) were selected for further refinement to remove fragments and adducts for parent ion identification. the selected panel of 383 unique amrtps (s3 table) differentiating animals of different vaccination status at various time-points post-bpi3v challenge were deconvoluted to identify parent ion mass, adducts and low energy fragments using low and high energy data (function 1 and 2 respectively), yielding 26 parent ions for elemental composition determination. nonparametric mann whitney was also employed on the selected panel of metabolites from day 0, 2 and 6 p.i. time points, with all markers attaining significance levels within thresholds comparative with anova. the false discovery rate was calculated from the cv (50%) filtered dataset via the benjamini-hochberg procedure with a threshold of 20%. all selected markers passed this threshold and met further selection criteria (v.i.p. score > 1 and fc > 1.5) were selected as potential markers. retention times and accurate masses of potential markers post-bpi3v challenge (days 2, 6, 14 and 20 p.i.) are shown in table 1 this is the first study to report the potential to differentiate between infected animals with differing vaccination status through the use of metabolomic profiling techniques (diva metabolomics). previous work [22] by the authors has demonstrated that metabolomics can identify metabolites associated with immune responses to vaccination, and the current study has advanced this concept by applying metabolomics analysis of plasma to identify unique metabolite marker profiles that are capable of distinguishing between vaccinated and non-vaccinated animals following infection. calves vaccinated (rispoval 1 pi3+rsv) and infection-challenged (bpi3v) demonstrated a significantly stimulated anti-bpi3v igg post-vaccination response which remained elevated post-challenge. lung histology and haematology confirmed that the bpi3v inoculum employed for viral challenge was sufficient to induce some evident respiratory pathology in non-vaccinated animals, with an absence in vaccinated animals. the subsequent challenge in primed vaccinated animals also resulted in maintenance of pre-challenge elevated anti-bpi3v igg (indicating response to inoculum), with absence of respiratory pathology. following preliminary analysis of a subset of samples (day 6 p.i.), rp-uplc-ms analysis demonstrated increased capacity to profile bovine plasma metabolites relative to hilic-based chromatographic separation and was subsequently used for extensive metabolomic plasma profiling. unsupervised pca analysis of acquired metabolomic profiles of preand post-challenge plasma revealed clear and distinguishable variation in plasma metabolites between vaccinated and non-vaccinated animals. considering that elevated anti-bpi3v igg typically occurs at 2 weeks post-challenge [6, 26] , variation in plasma metabolite profiles as early as day 2 p.i. illustrates the capacity of metabolomic profiling methods to detect changes stimulated by immune responses at early post-infection phases. supervised multivariate discriminant analysis of plasma metabolomic profiles yielded 383 potential metabolites (amrtps) that were significantly different in the plasma of vaccinated compared to non-vaccinated animals following bpi3v challenge. following de-convolution (with removal of adducts and fragment ions), 26 parent metabolite ions were identified and shown to be present at different levels within plasma from vaccinated and non-vaccinated animals from days 6-20 p.i. despite no significant differences in the parent ion intensity between treatment groups at day 2 p.i. plasma metabolite profiles differed between vaccinated and nonvaccinated animals through supervised opsl-da. identities of 17 of these 26 parent metabolites were revealed via database searching, in silco fragmentation and spectral matching. divergent plasma metabolite profiles have previously [22] been reported following vaccination, with altered metabolites shown to be associated with primary or secondary immune responses to vaccination. the metabolomic profiling performed here in this study on post-bpi3v challenge acquired samples, has identified a unique panel of plasma metabolites which differ between vaccinated and non-vaccinated animals, and significantly are involved in recognised immune response mechanisms. at day 6 p.i., increased biliverdin (fc = 2.86), bilirubin (fc = 3.25) and decreased 3-indolepropionic acid (fc = -2.40) levels were observed in plasma of non-vaccinated animals. biliverdin is degraded from heme by heme-oxygenases, and is further reduced to bilirubin by bilirubin reductase [27] . heme-oxygenase-1 exerts anti-inflammatory effects as demonstrated by reduced tumour necrosis factor alpha release in lipopolysaccharide-stimulated macrophages [28] . 3-indolepropionic acid is a reactive oxygen species scavenger [29] , produced in the microbiome [30] . phagocyte activation by rna viruses results in both increased reactive oxygen species release and the production of pro-oxidant cytokines (tumour necrosis factor alpha and interleukin-1) which promote iron uptake by the mononuclear phagocyte system [31, 32] . decreasing plasma 3-indolepropionic acid levels in concert with elevated biliverdin and bilirubin levels (via heme-oxygenase-1 action) in non-vaccinated animals at day 6 p.i. maybe indicative of 3-indolepropionic acid scavenging of reactive oxygen species produced by phagocytes, or increased downstream ros production from cytokine signalling. fluctuations in the levels of a number of bile acids (3-oxocholic acid, cholic acid and ndgca) were observed in plasma of animals at various stages in the study. 3-oxocholic acid and ndgca were found to be significantly increased (fc = 3.4 and 1.74 respectively) in the plasma of non-vaccinated animals at day 6 and 14 p.i respectively, whereas cholic acid was significantly higher in vaccinated animals (fc = -2.72) at day 20 p.i. altered plasma bile acid profiles at distinct study phases suggest that bile acid metabolism and conjugation is associated with different immune response mechanisms. bile acid driven farnesoid x receptor activation (expressed in pulmonary endothelial cells [33, 34] ) enables a regulatory immune response as demonstrated by dendritic cell modulation [35] , natural killer t-cell inhibition, reduced proinflammatory osteopontin production [36] and reduced lung permeability, suppressing leukocyte movement to sites of tissue inflammation [37] . when transported into cells ndgca is a potent farnesoid x receptor activator [38] and elevated ndgca at day 14 in non-vaccinated animals suggests an association with the switching and/or suppression of innate inflammatory responses towards adaptive immune responses. cholic acid, the primary metabolite of cholesterol, was found to significantly increase from day 0 to 20 p.i. in vaccinated animals. cholesterol, a component of lymphocyte lipid rafts, supports b-and t-cell receptor signalling [39] . reverse cholesterol transport is associated with immunosuppression via reduction in b-and t-cell receptor signalling, lymphocyte activation and proliferation [40, 41] , and elevated cholic acid may indicate increased cholesterol metabolism via reverse cholesterol transport in proliferating lymphocytes, down-regulating the secondary immune response to viral challenge during later stages of infection (day 20 p.i.). lysophosphatidylcholine produced from phosphatidylcholine [42] [43] [44] stimulates dendritic cell maturation through the action of a g-protein-coupled receptor with further ability to stimulate interleukin-2 and interferon-γ production in t cells [45] indicating a role in innate-toadaptive immune response progression. plasma levels of six lysophosphatidylcholine derivatives were significantly up-regulated (fc > 2.23) in non-vaccinated animals at day 14 p.i. significantly higher plasma levels suggest involvement in systemic immune responses to primary bpi3v antigen stimulation with dendritic cell recruitment to lymph nodes and subsequent dendritic cell driven t-cell activation. decreased plasma peak intensity of enterolactone (fc = -2.35), a lignan metabolite formed in ruminants through the action of colonic bacteria on secoisolariciresinol diglucoside [46] , was observed in non-vaccinated animals at days 14 and 20 p.i. enterolactone crosses the intestinal barrier and exerts anti-inflammatory functions by suppressing nuclear factor-κb signalling, tumour necrosis factor alpha production [47] and interleukin-1β release [48] . as enterolactone is dietary derived it cannot be readily replenished or up-regulated during periods of high demand, therefore decreased enterolactone in non-vaccinated animals during the latter stages of bpi3v infection (days 14 and 20 post-challenge) may reflect its metabolism to induce anti-inflammatory effects associated with the transition of acute to adaptive immune responses. from a diagnostic perspective, those plasma metabolite markers found to be altered at early stages of infection prior to sero-conversion and which persist to the later stages of infection are of particular interest. hexahydrohippurate, despite not showing significantly altered plasma levels until day 6 p.i., remained elevated in vaccinated animals (fc > -2.05) for the duration of the study. significantly lower levels of n-methylhippuric acid (fc = 2.46) and higher n-(cyclohex-1-en-1-ylcarbonyl)glycine (fc = -5.52) were also observed in vaccinated calves at day 6 p.i. n-(cyclohex-1-en-1-ylcarbonyl)glycine, n-methylhippuric acid and hexahydrohippurate are formed through the action of glycine n-acyltransferase on cyclohexanecarboxy-coa, cyclohexene-1-carboxyl-coa or benzoyl-coa, produced during shikimate and phenylalanine metabolism [30, [49] [50] [51] [52] . with no significant variation in the plasma levels of phenylalanine (which would attribute post-bpi3v challenge acyl-glycine conjugate to dietary variations), elevated hexahydrohippurate and n-(cyclohex-1-en-1-ylcarbonyl)glycine levels may be a consequence of an increased demand for coa in the liver due to the need to free coa otherwise sequestered in cyclohexanecarboxy-coa or cyclohexene-1-carboxyl-coa. increased coa demand may therefore be associated with increased rate of b-and t-cell proliferation and antibody production following an adaptive memory response to bpi3v challenge (as illustrated previously by post-parental immunization [22] ). assessment of hexahydrohippurate concentrations in plasma could allow for differentiation of vaccination status in infected calves with a wider diagnostic window to that observable currently through monitoring differences in anti-bpi3v igg levels. hexahydrohippurate occurs at high abundance in plasma offering potential for use as a realistic marker that could be measured using on-site based testing methods. in conclusion, this study highlights the potential of untargeted uplc-ms metabolomics to differentiate the vaccination statuses of virus challenged animals (i.e. diva metabolomics). the differential pre-challenge immune status of vaccinated and non-vaccinated animals resulted in divergent plasma metabolite profiles following bpi3v challenge, evident as early as 2 days p.i., with increasing variation from time post challenge. the metabolites identified were associated with immune cell regulation mechanisms, including t/b-cell proliferation and phagocyte activation and maturation. the wide diagnostic window for hexahydrohippurate combined with metabolite markers altered at distinct time periods associated with specific immune response mechanism (e.g. lysopc, biliverdin, bilirubin or 3-indolepropionic acid), could find application in staging of infection. the effectiveness of these efforts is reflected in the large magnitude fold-change and low intra-group variation of statistically significantly metabolites identified at days 14 and 20 p.i. similar to cytokine profiling, metabolomic based diagnostics are unlikely to match the pathogen specificity of molecular and serological testing but instead provide greater information on physiological health status, with future disease diagnostics potentially employing multiple methods to improve disease management decisions. a limitation of this study, as with many other biomarker discovery investigations involving large bovine animals, is the small cohort size (i.e. n = 6 per group; n = 3 at days 14 and 21 p.i.) which can be incorporated effectively into experimental groups. this potentially may impact on how findings from resulting analysis can be accurately translated to that observed within the wider more variable herd population. to mitigate against such issues, animals were extensively screened with regards to health and maternal antibody status prior to study group inclusion, and post-metabolomics profiling, stringent metabolite selection criteria (fc > 1.5; p < 0.05; v.i.p. score > 1; 20% false discovery rate) were applied to select only the most robust metabolites as marker candidates. as such, further investigation using a larger, more comprehensive sample set (differing sexes, breeds and ages of animals) with alternative routes of vaccination, vaccination failure (degradation or immunosuppression) and challenge (with multiple pathogens) may determine a panel or 'fingerprint' of metabolites with greater diagnostic specificity. a number of unidentified markers showed promising expression profiles and as metabolomics is still a relatively new field, lacking the level of database curation compared to genomics and proteomics for marker identification, these markers may be identified in the future. further studies are required to expand upon this initial proof-of-concept to determine if the observed diva metabolite markers are robust. importantly these studies have identified potential markers that would also be of benefit in screening and assessing new vaccine formulations and allow identification of trails indicative of protective immune responses. hplc grade acetone and analytical standards were purchased from sigma aldrich (dorset, uk). lc-ms grade acetonitrile, water, methanol and chloroform were purchased from fisher scientific (loughborough, uk). all animal studies were carried out in accordance with the uk animals (scientific procedures) act 1986 and with the approval of the agri-food and biosciences institute northern ireland ethical review committee. calves were vaccinated with rispoval 1 pi3+rsv as previously reported [22] . briefly, 12 male holstein friesian calves aged between 20 and 25 weeks were sourced commercially from farms with no history of prior respiratory disease outbreaks. the calves had no prior vaccination for brd and were clinically examined and declared fit for the study on day 0 by the named veterinary surgeon. claves were divided into two study groups (n = 6) and assigned as non-vaccinated and vaccinated calves. vaccinated calves were treated with pfizer rispo-val 1 pi3+rsv intranasal vaccine (designated vaccinated animals) as per manufacturer's instructions, and non-vaccinated calves treated with empty poly-(lactic-co-glycolic) acid nanoparticles (designated non-vaccinated) prepared using standard double emulsification solvent evaporation technique (w/o/w) [53] . calves received two dosages of vaccine formulation at 70 and 35 days prior to intranasal bpi3v challenge (post-infection = p.i.) (inoculation with 2 ml of virus suspension (tcid 50 of 10 6.78 /ml) per nostril). calves were screened weekly following vaccination and at days 0, 5, 12, 14 and 20 post-bpi3v infection (p.i.) for the presence of bpi3v igg in blood serum using svanovir-pi3v-ab kit (boehringer ingelheim svanovir, uppsala, sweden) as per manufacturer's instructions. at day 6 p.i. 3 animals per group were sacrificed for viral isolation and histology after sampling. blood samples drawn via jugular venepuncture at days 0, 2, 6, 14 and 20 p.i. into 6ml plastic k3 edta vacuette tubes (greiner bio-one, stroudwater, uk) were processed at random to platelet poor plasma via a double centrifugation method [54] optimized for metabolomic analysis within 2 hours of initial blood drawing and plasma stored at -80˚c prior to use. samples processing order was randomized to negate processing bias on sample metabolite profiles. 400 μl of plasma was added to 1.6 ml of ice cold acetone, vortexed for 30 sec and placed on ice for 15 min. the sample was then deproteinated by centrifugation at 15,000g at 4˚c for 15 min. 1.6 ml of supernatant was removed and dried under nitrogen for 45 min at 40˚c using turbovap lv (caliper life sciences, hopkinton, usa). resulting residue was reconstituted in 500 μl of ultra-pure h 2 o and liquid/liquid extraction of lipids performed by addition of 500 μl of ice-cold methanol:chloroform (1:1 v/v) and vortexing for 30 sec followed by centrifugation at 16,000g at 4˚c for 15 min. liquid/liquid extraction was repeated and after centrifugation 900 μl of the aqueous layer was removed and dried under nitrogen. the residue was reconstituted in 150 μl ultra-pure h 2 o and filtered by centrifugation at 10,000g, 4˚c using 0.22μm costar spin-x 1 centrifuge tube filter for 5 mins. 100 μl of plasma was added to a well in an ostro protein precipitation and phospholipid removal plate (waters corporation, milford, ma, usa). 100μl of 1% formic acid/acetonitrile (v/v) was added to the sample well, the plate shaken for 30 sec and extracted metabolites drawn through under vacuum into a 96 well 2 ml collection plate. rp-uplc-ms analysis was performed using an acquity uplc system coupled to a xevo g2 q-tof (waters corporation, milford, ma, usa). a test mix of acetominophen, sulfaguanidine, sulfadimethoxine, val-tyr-val, verapmil, terfenadine, leucine-enkephalin, reserpine and erythromycin was injected to ensure calibration of mass spectrometer mass accuracy and uplc performance prior to analysis. pooled samples (comprising a 10μl aliquot from all study samples) were injected 5 times before the start of each run for column conditioning [55] and intermittently throughout the run to validate instrument performance. the run-order of samples entering the mass spectrometer was constructed using a randomized sample list comprising 5 technical replicates per biological sample. no other samples were injected during the analysis run. 8 μl of prepared sample extracts were injected onto an acquity uplc hss-t3 column (100 mm x 2.1 mm i.d., 1.8 μm; waters corporation, milford, ma, usa). column and autosampler temperature were maintained at 50˚c and 10˚c respectively. chromatographic separation was carried out at a flow rate of 600 μl/min with mobile phase consisting of 99.9% h2o/0.1% formic acid (a) and 99.9% acetonitrile/0.1% formic acid (b). the elution gradient was as follows: 0-2 min isocratic at 1% of b, 2-14.5 min linear gradient form 1-100% of b, 14.5-17.5 min isocratic at 100% of b, and finally 17.5-20 min linear gradient at 100-1% of b. mass spectrometry was performed in positive-ion mode (esi+) with the capillary voltage set to 1500 v and the sampling cone voltage 30v. the desolvation and cone gas flows were set at 750 l/h and 100 l/h respectively. source and desolvation temperatures were 120˚c and 400˚c respectively. leucine enkephalin ([m+h] + = 278.1141 da, and [m+h] + = 556.2771 da) was used for accurate lockmass calibration during data acquisition. lockmass acquisition setting were: 0.5 sec scan time, 30 sec interval, 3 scan average, mass window +/-0.5da. centroid data were acquired in positive mode using resolution mode. collision energy was only applied on function 2, with ramping between 15 ev and 30 ev. for hilic-uplc-ms/ms analysis the test mix was cytosine, o-acetyl-l-carnitine and lvaline. pooled samples (comprising a 10μl aliquot from all study samples) were injected 5 times before the start of each run for column conditioning [55] and intermittently throughout the run to validate instrument performance. the run-order of samples entering the mass spectrometer was constructed using a randomized sample list comprising 5 technical replicates per biological sample. no other samples were injected during the analysis run. 1 μl of prepared sample extracts were injected onto an acquity uplc beh hilic column (2.1 mm x 100 mm i.d., 1.7 μm; waters corporation, milford, ma, usa). column and autosampler temperature were maintained at 45˚c and 6˚c respectively. chromatographic separation was carried out at a flow rate of 600 μl/min with mobile phase consisting of 10 mm ammonium formate ph 3.3 (a) and acetonitrile with 0.2% formic acid (b). the elution gradient was as follows: 0-2 min isocratic at 5% of a, 2-10 min linear gradient form 5-30% of a, 10-11 min linear gradient from 30-90% of a, 11-12 min isocratic at 90% of a, 12-12.5 min linear gradient from 90-5% of a, 12.5-16 min isocratic at 5% of a. mass spectrometry was performed using a waters xevo g2 q-tof (milford, ma) operating in positive-ion mode (esi+) with the capillary voltage set to 300v and the sampling cone voltage 20v. the desolvation and cone gas flows were set at 700l/h and 5l/h respectively. source and desolvation temperatures were 120˚c and 450˚c respectively. leucine enkephalin was used for accurate lockmass calibration during data acquisition, with acquisition settings the same as with rp-uplc-ms analysis. centroid data were acquired in positive mode using resolution mode. collision energy was only applied on function 2, with ramping between 20v and 35v. total ion count (tic) chromatograms and spectra were acquired with masslynx version 4.1 (waters corporation, milford, ma, usa) in centroid format and metabolite data was processed using the markerlynx software. the markerlynx method for data extraction and deconvolution was as follows. ions were extracted from function 1 data using peak detection analysis of retention time window 0.30-14.50min, with a mass range of 100da to 1200da. the xic window for data collection was 0.2da and apex peak tracking parameters were set to automatic with no smoothing. data collection parameters consisted of an intensity threshold (counts) of 500, a mass window of 0.02da with a retention time window of 0.20min. a noise elimination level of 6 was applied and isotopes were removed. peak heights for extracted ions were normalized against the total peak height of all extracted ions and standardized to a total ion count of 10,000. the results were exported in .csv format as a two dimensional data table in which rows and columns respectively represented analysed samples and the relative normalized peak heights of each detected mass spectrometric signal, i.e. as an accurate mass (m/z) and retention time (min) pair (amrtp). extracted and processed data submitted for downstream multivariate and statistical analysis for metabolite marker selection can be found in supplementary data (s4 table) . temporal changes in bpi3v antibody titre were analysed using two-tailed paired t-test, and significant differences between treatment groups at sampling stages was assessed using two-tailed heteroscedastic t-test. simca-p+ version 13.0 (umetrics, sweden) was used for multivariate metabolite marker selection. the dataset was pre-filtered to exclude amrtps with coefficient of variation greater than 50% in inter-run quality control pools (generated from equal aliquots of all extracted samples and injected intermittently throughout the analysis run). all centroid data were pareto scaled and analysed by unsupervised principle component analysis (pca) and supervised discriminatory analysis by orthogonal projections of latent structures-discriminant analysis (opls-da). unsupervised pca models were generated at each sampling day to reveal potential relationships between treatment groups. supervised analysis by opls-da was performed to reveal potential markers of response to treatment in vaccinated calves compared to non-vaccinated calves at each sampling day. robustness of final opls-da discriminative models was assessed by setting a predictive model of each case in which 2/3 of the data (known treatment) was used to predict the remaining 1/3 (unknown treatment). significance of the identified markers at all time points was determined using anova with bonferroni post-hoc test and non-parametric mann whitney for day 14 and 21 p.i. time points. the elemental composition of selected parent compounds was determined in masslynx using both positive and negative mode data. mass uncertainty was set to 3 mda, odd and even electron state, carbon isotope filter of +/-5% and elements included were c, h, o, n, p and s. where applicable na and k adduct elemental composition were determined with the respective element included in the analysis parameters. elemental compositions were searched against pubchem and chemspider online databases, and where possible function 2 fragments were matched against metlin, hmdb or massbank databases. where fragmentation spectra for the analyte in question was not available, in silico fragmentation was performed using metfrag and function 2 fragmentation data was validated against potential in silico fragments. identified compounds were validated using mass spectrum and retention time relative to authentic analytical standards analysed under identical experimental conditions (s4 fig). pooled plasma samples and individual analytical standards (1 μm) were analysed under identical uplc and mass spectrometric run conditions as utilized previously [22] , and metabolite identities confirmed by matching retention time and function 1 and function 2 spectra (including low and high energy fragments and adducts). putative annotations were acquired for compounds with spectral similarity to public spectral libraries (metlin, hmcb or massbank). table. cross-validation of opls-da models. leave-one-out (loo) cross validation was performed to assess the performance of the models generated. all technical replicated from a single biological sample were removed and an opls-da model containing the remaining biological and technical replicates employed to predict class representation. (xlsx) s3 table. selected amrtps of plasma metabolomic markers of response bpi3v challenge in vaccinated and non-vaccinated animals. combined list of 383 amrtps selected using opls-da at days 2, 6, 14 and 20 post-bpi3v infection filtered to exclude those with p < 0.05, fc < 1.5 and v.i.p. score < 1. (xlsx) s4 table. processed raw data employed for downstream metabolomic analysis. markerlynx was employed for data extraction and processing. amrtp levels are reported as peak height. the medicine and epidemiology of bovine respiratory disease in feedlots factors of respiratory disease: review of management factors. bovine respiratory disease: total health management use of treatment records and lung lesion scoring to estimate the effect of respiratory disease on growth during early and late finishing periods in south african feedlot cattle economic impact associated with respiratory disease in beef cattle. veterinary clinics of north america-food animal practice a dynamic range compression and three-dimensional peptide fractionation analysis platform expands proteome coverage and the diagnostic potential of whole saliva duration of immunity of a quadrivalent vaccine against respiratory diseases caused by bhv-1, pi3v, bvdv, and brsv in experimentally infected calves iowa: iowa state university press pathogenesis and pathology of bovine pneumonia. the veterinary clinics of north america food animal practice comparison of cytokine measurements using elisa, elispot and semi-quantitative rt-pcr differentiation of c-strain "riems" or cp7_e2alf vaccinated animals from animals infected by classical swine fever virus field strains using real-time rt-pcr escape of classical swine fever cstrain vaccine virus from detection by c-strain specific real-time rt-pcr caused by a point mutation in the primer-binding site laboratory test descriptions for bovine respiratory disease diagnosis and their strengths and weaknesses: gold standards for diagnosis, do they exist? canadian veterinary journal-revue veterinaire canadienne a comparison of diagnostic methods for the detection of bovine respiratory syncytial virus in experimental clinical specimens. canadian journal of veterinary research-revue canadienne de recherche veterinaire towards quantitative metabolomics of mammalian cells: development of a metabolite extraction protocol metabolic profiling of serum using ultra performance liquid chromatography and the ltq-orbitrap mass spectrometry system identification of potential biomarkers for ovarian cancer by urinary metabolomic profiling high performance liquid chromatographymass spectrometry for metabonomics: potential biomarkers for acute deterioration of liver function in chronic hepatitis b investigation of the human brain metabolome to identify potential markers for early diagnosis and therapeutic targets of alzheimer's disease lc-ms based serum metabolomics for identification of hepatocellular carcinoma biomarkers in egyptian cohort biomarkers for prediction of bovine respiratory disease outcome comparative approaches to the investigation of responses to stress and viral infection in cattle identification of systemic immune response markers through metabolomic profiling of plasma from calves given an intra-nasally delivered respiratory vaccine infectious bovine rhinotracheitis, parainfluenza-3, and respiratory coronavirus. veterinary clinics of north america-food animal practice comparison of levels and duration of detection of antibodies to bovine viral diarrhea virus 1, bovine viral diarrhea virus 2, bovine respiratory syncytial virus, bovine herpesvirus 1, and bovine parainfluenza virus 3 in calves fed maternal colostrum or a colostrum-replacement product efficacy of a quadrivalent vaccine against respiratory diseases caused by bhv-1pi(3)v, bvdv and brsv in experimentally infected calves effect of parainfluenza-3 virus challenge on cell-mediated immune function in parainfluenza-3 vaccinated and non-vaccinated calves heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway indole-3-propionic acid attenuates neuronal damage and oxidative stress in the ischemic hippocampus metabolomics analysis reveals large effects of gut microflora on mammalian blood metabolites human leukocyte pyrogen induces release of specific granule contents from human neurtophils oxidative stress during viral infection: a review. free radical biology and medicine downregulation of endothelin-1 by farnesoid x receptor in vascular endothelial cells fxr-mediated regulation of enos expression in vascular endothelial cells ursodeoxycholic acid suppresses eosinophilic airway inflammation by inhibiting the function of dendritic cells through the nuclear farnesoid x receptor the bile acid sensor farnesoid x receptor is a modulator of liver immunity in a rodent model of acute hepatitis fxr protects lung from lipopolysaccharide-induced acute injury endogenous bile acids are ligands for the nuclear receptor fxr bar lipid rafts and signal transduction high-density lipoproteins and the immune system hdl in innate and adaptive immunity hydrolysis of phosphatidylcholine during ldl oxidation is mediated by platelet activating factor acetylhydrolase serum lysophosphatidic acid is produced through diverse phospholipase pathways differential hydrolysis of molecular species of lipoprotein phosphatidylcholine by groups iia, v and x secretory phospholipases a mature dendritic cell generation promoted by lysophosphatidylcholine weekly excretion of the mammalian lignan enterolactone in milk of dairy cows fed flaxseed meal enterodiol and enterolactone modulate the immune response by acting on nuclear factor-kappa b (nf-kappa b) signaling flaxseed, and the lignan enterolactone increase stroma-and cancer cell-derived il-1ra and decrease tumor angiogenesis in estrogen-dependent breast cancer metabolism of cyclohexanecarboxylate in rat glycine conjugation activity of benzoic-acid and its acinar locaalization in the perfused-rat-liver benzoic acid glycine conjugation in the isolated perfused rat-kidney. drug metabolism and disposition the origin of urinary aromatic compounds excreted by ruminants. 1. the metabolism of quinic, cyclohexanecarboxylic and non-phenolic aromatic acids to benzoic acid the preparation and characterization of poly(lactide-co-glycolide) microparticles. 2. the entrapment of a model protein using a (water-in-oil)-in-water emulsion solvent evaporation technique peptidomic analysis of human blood specimens: comparison between plasma specimens and serum by differential peptide display global metabolic profiling procedures for urine using uplc-ms we acknowledge the staff at afbi animal services unit vsd (stormont) for their help in the animal study and the staff at the advanced asset centre igfs (qub) for their excellent technical assistance. key: cord-001761-yvd1n42f authors: yoshimura, takeo; suzuki, takamasa; mineki, shigeru; ohuchi, shokichi title: controlled microwave heating accelerates rolling circle amplification date: 2015-09-08 journal: plos one doi: 10.1371/journal.pone.0136532 sha: doc_id: 1761 cord_uid: yvd1n42f rolling circle amplification (rca) generates single-stranded dnas or rna, and the diverse applications of this isothermal technique range from the sensitive detection of nucleic acids to analysis of single nucleotide polymorphisms. microwave chemistry is widely applied to increase reaction rate as well as product yield and purity. the objectives of the present research were to apply microwave heating to rca and indicate factors that contribute to the microwave selective heating effect. the microwave reaction temperature was strictly controlled using a microwave applicator optimized for enzymatic-scale reactions. here, we showed that microwave-assisted rca reactions catalyzed by either of the four thermostable dna polymerases were accelerated over 4-folds compared with conventional rca. furthermore, the temperatures of the individual buffer components were specifically influenced by microwave heating. we concluded that microwave heating accelerated isothermal rca of dna because of the differential heating mechanisms of microwaves on the temperatures of reaction components, although the overall reaction temperatures were the same. rolling circle amplification (rca), which is based on the mechanism of the replication of viral genomes, is an isothermal nucleic acid amplification technique that uses two primers, a circularized template and a dna polymerase with strand displacement activity [1] [2] [3] [4] [5] . rca efficiently synthesizes many copies of repeated sequences from circular dna and rna templates (fig 1) . moreover, it detects single nucleotide polymorphisms using a circularized dna probe called a padlock probe as well as efficiently amplifying bacterial genomes by a hyper-branch method [6] [7] [8] [9] [10] . isothermal dna polymerization has various application possibilities [11] [12] [13] [14] . microwave heating technology is applied to organic and inorganic chemistry using a microwave generator to produce useful effects such as rapid heating, decreased reaction times, and improved product yield and purity [15] [16] [17] [18] . the microwave effect is debated to be the "thermal-effect," "non-thermal effect," and "specific microwave effect (superheating or selectivity heating)" [19, 20] . one of the mechanisms of microwave heating in organic chemistry involves heating a low molecular weight compound with a dipole polarization and an ionic conduction. enzymatic reactions (in the fields of proteomics, pretreatment, degradation, chemical synthesis, and optical resolution) are performed using microwave heating [21] [22] [23] [24] [25] . published studies on pcr using microwave heating (microwave-assisted pcr, mw-pcr) show that amplicon synthesis is inefficient, large reaction volumes are required, and temperature is difficult to control or measure [26] . we reasoned that these problems may be attributed to the use of three temperatures (for annealing, elongation, and denaturation) in the mw-prc method that are not precisely controlled by microwave heating. temperature measurement under microwave irradiation is a matter of importance [27, 28] . for strict temperature control by microwave irradiation, a gene amplification study using microwave was reported in milliliter-scale pcr [29] . microwave device with several frequencies for pcr is developed to accurately control temperature [30, 31] . in contrast, rca does not require complex temperature control compared with conventional pcr methods, and isothermal conditions are appropriate for controlling microwave heating. in our previous study, we reported the microwave-assisted rca for the first time using a multi-mode microwave generator [32] , but the reproducibility was difficult. we developed a novel single mode resonant cavity microwave generator with fiber-optic probe to improve accurate temperature measurement and reproducibility. further, this applicator can be used by a conventional pcr-scale (50-100 μl). mw-rca was performed using a circular dna template and four thermostable dna polymerases with strand displacement activity. we focused on microwave selectivity heating, which is one of the features of microwave heating. microwave selectivity heating is thought to alter molecular motion state compared with conventional heating when the reaction temperature is the same. to examine the components of rca responsible for microwave selectivity heating, we measured the temperature of rca components under conventional and microwave heating conditions at the same temperature. mw-rca, which increased the components of selectivity heating, was performed. here we show that microwave heating facilitated the synthesis of repetitive dna through rca using the four dna polymerases. analysis of the temperature profiles of each rca component subjected to microwave heating revealed the selectivity heating of buffer components compared with primers, template dna, dntp, and rnase-free water. we show further that the components of rca responsible for microwave selectivity heating related to the acceleration of mw-rca. the rca template and primers were designed to amplify sequences encoding a zinc finger protein for the expression of an artificial repetitive protein. a circular rca template was prepared according to previous published study [32] . template oligonucleotide (0.1 nmol) was phosphorylated using t4 polynucleotide kinase (10 u) in 100 μl of solution at 37°c for 60 min. after heating at 72°c for 10 min, the phosphorylated template nucleotide (1 μl) was diluted with 36 μl of lk buffer (50 mm tris-hcl, 10 mm mgcl 2 , 10 mm dithiothreitol, 1 mm datp, and 25 μg/ml bovine serum albumin) containing 0.1 nmol of primer (primer-2). the primer-template mixture was annealed and incubated at 16°c for 1 h with t4 dna ligase (1050 u) in 950 μl of lk buffer. the circularized primertemplate was precipitated from the phenol-chloroform-isoamyl alcohol extraction mixture and suspended in rnase-free water (takara). the dna concentration of the final sample used for rca was determined using a nanodrop 2000c (thermo scientific) and was diluted to a final concentration of 60 ng/μl. rca reactions were performed to amplify the repetitive sequences using dna polymerases with strand-displacement activity. the four thermostable dna polymerases were as follows: bst dna polymerase large fragment (bst-lf) (new england biolabs, neb), bst dna polymerase, csa dna polymerase, and 96-7 dna polymerase (nippon gene). all rca reactions were performed in a final volume of 50 μl at the optimum temperature of each enzyme in a 0.2-ml polypropylene tube (qsp) using a thermal-cycler (takara). reactions were initiated after raising the temperature from 13°c to each initial temperature (60°c or 55°c) for 2 min. the control rca reaction contained a dntp mixture (each 2.5 mm), 10 pmol each of two primers, buffer specific for each dna polymerase, eights units of each dna polymerase, and 60 ng of each circular template-primer complex. bst-lf was incubated in thermopol buffer (20 mm tris-hcl, 10 mm (nh 4 ) 2 so 4 , 10 mm kcl, 2 mm mgso 4 , and 0.1% triton x-100; neb) at 60°c for 20-60 min. the bst dna polymerase (nippon gene) was incubated in the bst reaction buffer (8 mm mg 2+ ) at 60°c for 20-60 min. the csa dna polymerase (nippon gene) was incubated in the csa reaction buffer (8 mm mg 2+ ) at 60°c for 10-60 min. the 96-7 dna polymerase (nippon gene) was incubated in the 96-7 reaction buffer (9.5 mm mg 2 + ) at 55°c for 10-180 min. a thermal-cycler was used to inactivate each dna polymerase. microwave-assisted heating experiments were performed using a microwave applicator in tm 010 mode (saida fds), equipped with a fiber-optic probe (neoptix) for internal online temperature control (fig 2) . this resonant cavity-type microwave applicator (2.45 ghz) more precisely controls output compared with the magnetron power generated by a solid-state device (maximum 10 w). all microwave reactions were performed using a 0.2-ml polypropylene tube (qsp). this instrument was operated in a cold room (5°c) for the continuous generation of microwave power. this cavity resonator allows users to control the temperature of a 50 μl volume reaction mixture. the temperature of the mw-rca reaction mixture contained in a pcr tube was directly measured and was controlled using the fiber-optic probe ( fig 2b) . all mw-rcas were performed in a cold room. to prevent exceeding the target temperature, the temperature of the mw-rca reaction mixture was raised from 13°c to 60°c in 2 min, and the reactions were incubated for 30 min (s1 and s5 figs) because the microwave applicator was unable to maintain the reaction volume at 60°c over 30 min. the average microwave output at 60°c during the 30-min incubation was <2 w (s2 and s6 figs). after terminating the mw-rca reaction, dna polymerases that were used in microwave heating were denatured by placing the tubes in a heating block at 90°c. conventional heating using a 60°c heating block and microwave heating were measured using a fiber-optic probe in the cold room for 10 min beginning at 13°c. the data used to plot the temperatures represent the average of three measurements. electrophoresis rca products were electrophoresed in 1.5% agarose gels and visualized using ethidium bromide. the electrophoresis buffer contained 222 mm tris-borate buffer and 5 mm edta. the samples (5 μl) and a dna marker (onestep marker 5, nippon gene) were electrophoresed for 30 min at 100 v. gels were analyzed using an las-3000 imaging system (fujifilm) equipped with an ultraviolet filter (605df40, fujifilm). the intensity of sybr green i fluorescence emission was measured using a fluorescence plate reader (fluorocount, packard) with a 96-well white microwell plate (thermo scientific). comparison of mw-rca with conventional rca using four dna polymerases to determine the effect of microwave heating, mw-rca was performed using four thermostable dna polymerases that had strand displacement activity. temperature, power, and frequency profiles of mw-rca using bst-lf at 30°c for 30 min is shown in fig 3. a power profile of microwave is a value obtained by subtracting reflected power from incident power. temperature profile data proved that mw-rca was performed at precise temperatures. conventional rca and mw-rca reactions were sampled at intervals from 10 to 60 min and 10 to 30 min, respectively, and analyzed using agarose gel electrophoresis and fluorescence emission (fig 4) . the use of the microwave applicator ended in 30 min under high temperature conditions. a comparison of mw-rca with conventional rca using the bst-lf revealed that the reaction product of mw-rca was increased by a factor of four compared with conventional rca (fig 4a) . fluorescence intensity showed a 4-fold increase than conventional rca in 30 min. repetitive dna sequences repeated four times (300 bp) and eight times (600 bp) were accurately replicated using mw-rca using bst-lf under microwave irradiation. the results of each assay showed that the yields of dna produced by mw-rca using the bst dna polymerase (60°c) were markedly greater compared with those by conventional rca during the first 30 min ( fig 4b) . the fluorescence intensities of the mw-rca reaction sampled at 20 and 30 min and those of the conventional rca reaction sampled at 60 min showed that the former plateaued at 20 min ( fig 4b) . the bst dna polymerase was the most effective enzyme for mw-rca. in the same way, mw-rca using the csa dna polymerase was accelerated at 60°c compared with conventional rca, and polymerization was increased at 20 min. in contrast, dna synthesis was not detectable in the conventional rca reaction at 30 min ( fig 4c) . the fluorescence intensity in mw-rca using csa was higher by a factor of 30 at 30 min compared with that of the conventional rca, and it was almost equal to that of conventional rca at 60 and 90 min ( fig 4c) . reactions using the 96-7 dna polymerase were incubated at 55°c, its optimum temperature, and showed an increase in discrete bands at 120 min, representing repeated sequences that were detected in the conventional rca reaction. in contrast, mw-rca using the 96-7 dna polymerase showed diffuse migration of high molecular weight dna molecules at 20 min, although dna synthesis using conventional rca was undetectable after 60 min. the fluorescence intensity of the conventional rca reaction products at 90 min was twice as that of mw-rca at 30 min (fig 4d) . temperature and power profiles of four dna polymerases under microwave heating are indicated in the supporting information (s1 file). comparison of temperature profiles of rca components using the microwave applicator and the heating block to determine the component of rca by microwave selectivity heating, we measured the temperatures of the five components (circularized template with primers, dntps, thermopol buffer, bst-lf, and rnase-free water) of the rca and mw-rca mixtures for 10 min from 13°c to 60°c. a fiber-optic probe was used to measure the temperature of the conventional heating block. all six samples (rca and its five components) attained 60°c using a 60°c heating block (fig 5a) . by microwave heating the rca mixture including all components reached 60°c in 2 min. moreover, the thermopol buffer with 45 μl of rnase-free water reached 60°c by microwave heating 10 s after the addition of the rca mixture. four samples (dntps with 46 μl of rnase-free water, circularized template with primers in 50 μl of rnase-free water, 8 u of the bst dna polymerase in 50 μl of rnase-free water, and 50 μl of rnase-free water) reached a maximum temperature of 42°c by microwave heating in 10 min (fig 5b) . effect of microwave irradiation on the temperatures of higher concentrations of buffer components added to rca reaction mixtures the results shown in fig 5 suggest that the thermopol buffer was a primary factor leading to an increase in temperature under microwave irradiation. to determine whether the thermo-pol buffer contained a specific thermal component that was selectivity affected by microwave irradiation, we measured the temperatures of each of the four thermopol buffer components (fig 6a) . the microwave applicator heated the four components (20 mm tris-hcl, 10 mm (nh 4 ) 2 so 4 , 10 mm kcl, and 2 mm mgso 4 ) from 13°c to 60°c for 10 min. the four components did not reach 60°c (fig 6a) , i.e., the temperature increase of 10 mm (nh 4 ) 2 so 4 was the highest (47°c). in contrast, 10 mm kcl reached 44°c and the temperatures of 20 mm tris-hcl and 2 mm mgso 4 (42°c) were nearly identical to the temperature of the rnase-free water. further, to examine the mw-rca increased component of microwave selectivity heating, we measured the temperatures of 4-fold excess concentrations of buffer components under microwave irradiation (fig 6b) . the temperature of 40 mm (nh 4 ) 2 so 4 reached 60°c after 195 seconds and that of 40 mm kcl plateaued at 55°c; the maximum temperature of 80 mm tris-hcl was 46°c. increasing the concentration of mgso 4 from 2 mm to 8 mm increased its maximum temperature from 42°c to 44°c. the quadruple density of all four samples produced an to reveal the effect of the selectivity heating in mw-rca, we compared the efficiency of dna amplification in the rca and mw-rca reactions mixtures containing a 4-fold excess concentration of each rca component (dntp, template-primers, bst-lf, tris-hcl, kcl, (nh 4 ) 2 so 4 , and mgso 4 ). all reactions were performed for 30 min at 60°c using a thermal cycler with the microwave applicator, and the reaction products were analyzed using agarose gel electrophoresis and sybr green assay. profiles of temperature and power in microwave heating are indicated in the supporting information (s3 file). mw-rca of the control and all seven types of mw-rca reactions, each containing a 4-fold excess concentration of each rca component, effectively amplified 75-bp circular templates compared with each control rca (fig 7) . rca reactions containing a 4-fold excess concentration of dntps incubated under conventional or microwave heating conditions did not amplify repetitive dna (fig 7a) . moreover, rca reactions containing a 4-fold excess concentration of the bst-lf were accelerated by conventional as well as microwave heating. rca and mw-rca reaction mixtures each containing template-primers at a 4-fold excess concentration were most effectively replicated by the other seven sample pairs (fig 7a) . the 4-fold increases in the concentrations of tris-hcl and kcl accelerated rca and mw-rca reactions compared with controls. in contrast, mw-rca reaction mixtures containing a 4-fold excess concentration of (nh 4 ) 2 so 4 were accelerated to a greater extent compared with the control mw-rca reaction, whereas the microwave heating accelerates rca yield of the rca reaction mixture containing a 4-fold excess concentration of (nh 4 ) 2 so 4 was equal to that of the control conventional rca reaction mixture. similarly, mw-rca using a 4-fold excess concentration of mgso 4 equivalently accelerated to an mw-rca reaction containing a 4-fold excess concentration of (nh 4 ) 2 so 4 , although the conventional rca reaction mixture containing a 4-fold excess concentration of mgso 4 yielded a slight increase of dna. our research focused on the acceleration of rca by microwave heating. to accomplish our objective, we developed a microwave applicator, which avoided the problem of temperature measurement under microwave irradiation and allowed us to implement new reaction conditions that enhanced conventional rca. we considered the possibility that microwave-assisted enzymatic reactions are more effective at higher temperatures compared with those at typical physiological conditions. using four thermostable dna polymerases with strand displacement activity, we compared the products of mw-rca and conventional rca and found that microwave heating accelerated the reactions catalyzed by the four dna polymerases compared with conventional heating at the same temperature (fig 4) . in a previous study, dna amplification of mw-pcr, which was controlled at the precise temperature using the three steps, was reported [29] . in this study, rca using four thermostable dna polymerases was accelerated by microwave heating. a rca reaction is equivalent to an elongation reaction of three steps (annealing, elongation, and denaturation) of pcr. these results of mw-rca suggest that the reason of acceleration in mw-pcr was dna polymerization by microwave heating. why was rca with four heat-stable dna polymerases accelerated by microwave heating, although the overall reaction temperatures were the same? in another report of microwaveassisted enzymatic reactions, the authors hypothesized that microwave effects on enzymatic digestion are due to the increased dipole moments of α-helices of proteins [33] . a microwave study using immobilized thermostable lipase b suggested that the effect of microwave irradiation of enzymes was due to superheating of the water layer near the enzyme surface [34] . a study using thermostable β-glucosidase under microwave irradiation at frequencies of 2.45 and 5.8 ghz suggested, with respect to the heating mechanism, that 5.8 ghz irradiation affected only the water molecules in the buffer solution, whereas 2.45 ghz acted on both water molecules and buffering ions [35] . to elucidate the mechanism of this microwave heating effect in rca, we compared the temperature increase of rca components under conventional or microwave heating (see fig 5) . the rca components of the bst dna polymerase-lf reaction mixture were used for this purpose, because only the thermopol buffer of this polymerase is a nonproprietary reagent. the temperature increases for all rca components, which were placed in a 60°c heating block, were the same. microwave heating to 60°c increased the temperature of the rca mixture as well as that of the thermopol buffer to 60°c. in contrast, the temperature of the four rca components (bst dna polymerase-lf, dntp, template-primer, and water) was 42°c. these data indicate that the thermopol buffer in mw-rca was selectively heated by microwave irradiation. thermopol buffer, which contains a high concentration of ionic molecules, is considered to be heated by conduction loss of microwave irradiation [19, 20] . we postulated that the acceleration of mw-rca is caused by the components of thermopol buffer on selective microwave heating. the approach to the microwave heating effect is expected to change the amount of the thermopol buffer, which was selectively heated by microwave irradiation. however, a substantial change in concentration of the thermopol buffer is impossible because enzymatic reactions have an optimum concentration. we focused on the four components of thermopol buffer. we hypothesized that the microwave selective heating could be observed with heating of only one of each buffer component under microwave irradiation. we accordingly prepared four samples of one-fold thermopol buffer components [tris-hcl, kcl, (nh 4 ) 2 so 4 , and mgso 4 ] and four samples of thermopol buffer components in four-fold excess concentrations, and measured their temperatures under microwave heating ( fig 6) . temperature measurements of thermopol buffer components at four-fold excess concentrations showed an increase in temperature compared with one-fold concentrations. the microwave heating method correlated with the increase in concentrations of buffer components. we performed mw-rca reactions containing a four-fold higher concentration of each rca component [dntp, template-primers, bst dna polymerase-lf, tris-hcl, kcl, (nh 4 ) 2 so 4 , and mgso 4 ] to identify a link between microwave selective heating and dna amplification. there was no influence of ph change by the additional buffer component in rca (s4 file). all results of microwave heating were accelerated compared with conventional heating (fig 7) . two notable results were observed. mw-rca with 40 mm of (nh 4 ) 2 so 4 was accelerated relative to the control mw-rca, whereas rca with 40 mm of (nh 4 the effect of tris-hcl and kcl on mw-rca was unclear, given that the conventional results of rca containing four-fold excess concentrations of tris-hcl (80 mm) and kcl (40 mm) were effectively amplified relative to control rca (fig 7b) . control rca may improve the reaction by buffer composition of tris-hcl and kcl. the complex dielectric constant of protein in water or buffer solution has been described in [35, 36] . we need to measure the complex dielectric constant of rca components to understand the microwave heating effect of mw-rca. in conclusion, we report here the development of a microwave applicator for enzymatic reactions and demonstrate the effect of microwave heating on rca of dna. the temperature profiles of rca components by microwave heating suggest that the ionic components of the thermopol buffer are selectively heated and that the rate of temperature increase induced by microwave heating depends on the components of a molecule and the concentrations of buffer components. these findings indicate that (nh 4 ) 2 so 4 and mgso 4 play important roles in the mechanism of mw-rca. the shortened reaction time and higher product yields indicate that microwave heating systems can be widely used to enhance enzymatic reactions. studies are being conducted to further improve the maximum incubation time at temperatures higher than 60°c as well as to enhance the visualization of the heating process. preliminary results indicate that mw-rca can be accelerated at 37°c. thermopol-buffer and enzyme component was measured (ph 8.71). the ph of fictitious rca mixtures of one 4-fold constituent of thermopol-buffer were measured (4-fold tris-hcl: 8.73, 4-fold kcl; 8.69, 4-fold (nh4) 2 so 4 : 8.37, 4-fold mgso 4 : 8.67). thus, to assess the effect of ph, rca using the thermopol-buffer prepared by us (ph 8.80 and 8.34) was performed and was compared with a control rca using neb thermopol-buffer. as a result, no effect of rca products by ph alteration of this range was confirmed by electrophoresis and fluorescence analysis. (docx) rolling replication of short dna circles rolling circle dna synthesis: small circular oligonucleotides as efficient templates for dna polymerases a replication cycle for viroids and other small infectious rna's replication of avocado sunblotch viroid: evidence for a symmetric pathway with two rolling circles and hammerhead ribozyme processing padlock probes: circularizing oligonucleotides for localized dna detection amplification of padlock probes for dna diagnostics by cascade rolling circle amplification or the polymerase chain reaction in situ genotyping individual dna molecules by target-primed rolling-circle amplification of padlock probes cell-free cloning using phi29 dna polymerase rapid and sensitive detection of severe acute respiratory syndrome coronavirus by rolling circle amplification homogeneous and label-free fluorescence detection of singlenucleotide polymorphism using target-primed branched rolling circle amplification rolling circle amplification in a prokaryotic translation system using small circular rna rolling circle amplification-templated dna nanotubes show increased stability and cell penetration ability rapid identification of bio-molecules applied for detection of biosecurity agents using rolling circle amplification selective aluminum passivation for targeted immobilization of single dna polymerase molecules in zero-mode waveguide nanostructures microwave assisted organic synthesis-a review controlled microwave heating in modern organic synthesis: highlights from the 2004-2008 literature microwave chemistry for inorganic nanomaterials synthesis microwave-assisted synthesis of colloidal inorganic nanocrystals microwaves in organic and medicinal chemistry microwaves in organic synthesis recent developments in microwave-assisted protein chemistries-can this be integrated into the drug discovery and validation process? drug discovery today microwave energy potential for biodiesel production microwave-assisted synthesis using ionic liquids evaluating the potential nonthermal microwave effects of microwave-assisted proteolytic reactions lipase catalyzed kinetic resolution of (±)-1-(1-naphthyl) ethanol under microwave irradiation microwave-assisted high-speed pcr how to measure reaction temperature in microwave-heated transformations measurements of accurate temperatures in the microwave reactors an efficient method to perform milliliter-scale pcr utilizing highly controlled microwave thermocycling localized microwave heating in microwells for parallel dna amplification applications rapid pcr amplification using a microfluidic device with integrated microwave heating and air impingement cooling microwave assisted rolling circle amplification microwave energy: a versatile tool for the biosciences effect of ionic liquid properties on lipase stabilization under microwave irradiation efficiency of 2.45 and 5.80 ghz microwave irradiation for a hydrolysis reaction by thermostable beta-glucosidase ht1 hydration study of proteins in solution by microwave dielectric analysis we thank dr. s. maezawa and k. kajimoto from the tokyo university of science for their valuable advice. furthermore, we thank n. ohneda, h. odajima and saida fds for the microwave applicator. the authors would like to thank enago for the english language review. key: cord-000255-73nlxqgk authors: hosseini, parviez; sokolow, susanne h.; vandegrift, kurt j.; kilpatrick, a. marm; daszak, peter title: predictive power of air travel and socio-economic data for early pandemic spread date: 2010-09-15 journal: plos one doi: 10.1371/journal.pone.0012763 sha: doc_id: 255 cord_uid: 73nlxqgk background: controlling the pandemic spread of newly emerging diseases requires rapid, targeted allocation of limited resources among nations. critical, early control steps would be greatly enhanced if the key risk factors can be identified that accurately predict early disease spread immediately after emergence. methodology/principal findings: here, we examine the role of travel, trade, and national healthcare resources in predicting the emergence and initial spread of 2009 a/h1n1 influenza. we find that incorporating national healthcare resource data into our analyses allowed a much greater capacity to predict the international spread of this virus. in countries with lower healthcare resources, the reporting of 2009 a/h1n1 cases was significantly delayed, likely reflecting a lower capacity for testing and reporting, as well as other socio-political issues. we also report substantial international trade in live swine and poultry in the decade preceding the pandemic which may have contributed to the emergence and mixed genotype of this pandemic strain. however, the lack of knowledge of recent evolution of each h1n1 viral gene segment precludes the use of this approach to determine viral origins. conclusions/significance: we conclude that strategies to prevent pandemic influenza virus emergence and spread in the future should include: 1) enhanced surveillance for strains resulting from reassortment in traded livestock; 2) rapid deployment of control measures in the initial spreading phase to countries where travel data predict the pathogen will reach and to countries where lower healthcare resources will likely cause delays in reporting. our results highlight the benefits, for all parties, when higher income countries provide additional healthcare resources for lower income countries, particularly those that have high air traffic volumes. in particular, international authorities should prioritize aid to those poorest countries where both the risk of emerging infectious diseases and air traffic volume is highest. this strategy will result in earlier detection of pathogens and a reduction in the impact of future pandemics. predicting the origin and emergence of new diseases is critical to preventing and controlling them [1, 2] . in particular, if the early spread of a newly emerging pathogen can be predicted and curtailed before it becomes pandemic, its impact on public health and global economies may be much reduced [3, 4, 5, 6] . in march and april of 2009, a novel h1n1 influenza a virus (2009 a/ h1n1) with gene segments from humans, swine, and birds led to the first pandemic of influenza in forty years [7, 8, 9, 10] . current evidence points to a mexican origin for the initial human-tohuman transmission of this virus, although preliminary genetic analyses suggest the virus has an older and highly-mixed lineage [8] . the virus' lineage and rapid spread suggest that global trade and travel may have played an important role in its early emergence [7, 8] . here, we attempt to elucidate how these factors may relate to the emergence and spread of this newly detected virus. one unresolved question is to what degree does a country's development affects its ability to detect and respond to an emerging disease in a timely manner? development may affect spending on healthcare infrastructure, and particularly, spending on the high cost, intensive public health surveillance needed during the early stages of a pandemic [11, 12, 13] . socioeconomic factors will also likely affect individuals' abilities or desire to seek diagnosis or treatment, and a country's capacity to test and identify pathogens. here, we analyze socio-economic and travel data to understand the initial spread of this virus. we focus on the early stages of the epidemic, when travel from mexico was likely to be the dominant mode of viral spread. finally, we examine poultry and swine trade data prior to the 2009 a/h1n1 pandemic to add to our understanding the processes that led to the emergence of this virus. as of may 8 th 2009, only two weeks after it was first reported, the 2009 a/h1n1 influenza strain had spread to 24 countries, 40 u.s. states (plus the district of columbia) in the us, and 9 provinces in canada ( figure 1 ). this rapid spread resulted, in part, from the tight connectivity of the globe through air travel ( figure 2) . a log-logistic survival analysis regression model was used to predict the time-to-reporting of the first confirmed 2009 a/h1n1 case to each country. of all the models evaluated, a multivariate model with three predictors, (1) total country-level healthcare spending per capita, (2) estimated passenger volume arriving from mexico via direct flights (direct flight capacity), and (3) passenger volume from mexico via indirect, or two-leg, flights (indirect flight capacity), provided the best fit to the data using aic, as detailed under methods (table 1 , daic = 0, overall x 2 = 54.33 on 5 degrees of freedom, p-value,0.0001). the correlation between total country-level healthcare spending and the flight data was low (r,0.4). although the correlation between direct and indirect flight data was high for countries with direct flights (r.0.9), the indirect flight information provided critical additional information for areas without direct flights. the aic scores demonstrated this, as the model that included only direct flight information and healthcare spending did not explain the data as well as the best fit model (daic = 9.044). alternate socio-economic measures, even those directly related to healthcare, such as the number of physicians per capita, gdp, or population density were much less predictive than total healthcare spending per capita. notably, out of univariate analyses, the model with healthcare spending per capita as the sole predictor fit better than models with flight information alone (table 1) , demonstrating just how informative this data is in predicting the date of reporting. in the best fitting multivariate model, indirect flight capacity had the largest effect size, but including healthcare spending per capita substantially increased the fit to the data (tables 1, 2 ). for canadian provinces and american states, we conducted an analysis with just the flight data (table 3 overall x 2 = 22.89 on 2 degrees of freedom, p-value ,0.001). while the direct flight information does not have a statistically significant effect, the indirect does, most likely because only a few key hubs had direct flights, and these hubs also have a large volume of indirect connections. for the country-level analysis, we compared the predicted reporting dates with the actual reporting dates, for countries where the disease arrived by may 8 th , 2009 ( figure 3 , supplemental online figure s1 ). we validated the model by determining how well a model fit to data up until may 8th predicted reporting dates for fourteen countries where the disease was detected between may 9 th and may 19 th (supplemental online figure s2 ). the correlation between forward predicted and observed dates was 0.62, and the observed reporting date fell within the 95% confidence interval for all countries. many of the actual reporting dates are earlier than predicted, which is expected due to the nonlinear nature a of log-log survival analysis regression. in particular, countries that had not reported disease by the cut-off date were included in the analysis by designating these as locations that ''survived'' the entire study period without acquiring the disease (i.e, censoring). this appropriately extends the predicted reporting dates by including information on both countries that had reported disease by the cut-off date as well as countries that had not. using this methodology, we also estimated the reporting date of the disease in the remaining 103 countries and the 95% confidence intervals ranged from april 17 th to may 29 th , 2009 (supplemental online figure s3 ). to elucidate the potential origins of this novel viral strain, and to shed light on targets for future surveillance and prevention programs, we analyzed global trade in live poultry and swine during the decade preceding the current pandemic [14] . we estimate the trade in live swine between canada, the united states and mexico to be over 1.75 million animals over the last decade, previous studies suggest that data on air travel can be used to predict the spread of newly emerged human pathogens and better target public health measures [15, 16, 17, 18] . our analyses support this, but demonstrate that the ability of a country to rapidly detect, diagnose, and report the new infection is a critical element that enhances our predictive power and control capacity. other studies suggest that analysis of the underlying drivers of disease emergence (e.g. agricultural intensification, land-use change) can be used to predict the geographic origins of new emerging diseases [2] . the currently circulating pandemic influenza strain is a triple reassortment virus with closest known relatives from europe, asia, and north america, but there is uncertainty regarding its origin due to the large temporal separation between this pandemic 2009 a/h1n1 strain and the nearest ancestors (10-15 years) [7] . our analyses of swine and poultry trade demonstrate an enormous potential for intercontinental mixing of potentially zoonotic pathogens, including influenza a viruses. although artificial insemination is the predominant strategy for interbreeding of commercial swine, live swine are still routinely traded for breeding purposes [19] . large numbers of poultry are also traded globally, and low pathogenicity influenza viruses are likely to spread unnoticed among poultry until they reassort or mutate to highly pathogenic forms, such as the a/h1n1v strain. this strain notably was the results of reassortment of several relatively low pathogenic influenza strains, as explained by garten et al. [8] . in addition, as the recent cases of workers exposing a herd of pigs to the 2009 a/h1n1 virus makes clear [20, 21] , even dramatic reductions in the international live animal trade may not prevent the exposure of local livestock to novel viral types from distant locations [9, 10] . although extensive trade of poultry and swine between continents and within the north american countries almost certainly contributed to the emergence of this virus, surveillance of influenza strains circulating among traded animals is poor [10] , so that it is impossible to designate any single country, trade connection or market as the key point at which the new strain evolved. expanded surveillance for influenza in livestock populations may allow more of the markers of transmissibility and virulence to be identified, or factors driving higher virus transmission to be determined [9, 22] . in particular, we need to analyze all influenza strains, including the non-and low pathogenic influenzas, in addition to the highly pathogenic ones, with greater regularity. only by this thorough surveillance can we begin to understand what differentiates the strains that cause pathogenesis in humans from those that do not. such that eventually we may be able to predict viral emergence and develop vaccines against pandemic influenza viruses in advance of their spread. in order to develop such capability, we need to do more surveillance of livestock and wild influenza strains now. the speed at which 2009 a/h1n1 spread during the early phases of this pandemic is striking. it was detected in four continents within three weeks after mexican authorities first reported it. in contrast, the 1918 spanish flu took 3 years to circle the globe [23] . our analyses of air-travel data support the who's decision to recommend against closing all air travel from mexico, since the virus most likely had already spread to several other countries by the time it was first reported to be widespread in mexico on april 29 th . in particular, cases had already been detected in the united states, which is a major hub for connecting flights [24] . our current report is the first published analysis of h1n1 spread to include indirect flight data, and this significantly increased the predictive power of our model. our analysis suggests that airports serving as major hubs could be targets for disease surveillance, and could become facilities that train people and stockpile medicines in preparation for pandemics. this approach differs from previous reports that focus on the role of travel restrictions at hubs [6, 17] . our results further suggest a critical role for health care spending in determining a country's probability of detecting, confirming and reporting influenza cases in the early phases of a pandemic. the negative relationship between healthcare spending and detection of 2009 a/h1n1 influenza may be due to a delay in testing or in the collecting of specimens from individuals in countries lower healthcare resources. these countries likely have lower rates of health insurance, less healthcare infrastructure, lower self-reporting, and lower numbers of doctors per capita. one consequence of lower health care resources is that the threshold for detection (i.e., the number of cases that need to occur before a case is detected, tested and confirmed by medical authorities) is likely higher in lower-income countries that cannot afford to invest as much in public health and healthcare infrastructure. similar socioeconomic factors have been shown to play an important role in determining spatiotemporal patterns of diseases such as tuberculosis, schistosomiasis, west nile virus, and hiv/aids [12, 13, 25, 26] . we found that incorporating data on healthcare spending per capita significantly increased our power to predict the time of reporting of 2009 a/h1n1. this suggests important strategies for future disease control. during the early stages of a pandemic, countries with moderate to high air travel from a pandemic origin, but relatively low healthcare spending, are likely to significantly under-report cases. it is therefore in the best global health interest for intergovernmental and other aid agencies to specifically target these nations for assistance to test and report cases early in a new pandemic. we propose that subsidies for outbreak response to these nations with high connectivity and low resources would be the most effective strategy to reduce the spread and impact of a pandemic. efforts to better target pandemics would be more effective in reducing disease spread if they were set up in advance of a pandemic [5, 6, 16] , as there is a very small window of opportunity in which to act once a new emerging disease is detected. such efforts could be strategically positioned to target emerging disease 'hotspots' [2] that are also hubs of trade and travel for surveillance and prevention [16, 27] . for influenza viruses, any future identification of a spillover of a novel strain from poultry or swine to farm workers should be rapidly followed by analyses of the travel routes out of the country where the index case was discovered. at that point, intergovernmental agencies such as who could best target limited resources to the poorer countries that are most likely to receive high numbers of airline travelers from the pandemic origin. these are the countries where reporting is likely to be poorest, and where a significant, undetected caseload is likely to exist by the time resources are allocated. these at-risk countries are also the least capable of affording control measures. on the whole, this h1n1 strain appears to be relatively mild, although it is still inflicting additional morbidity and mortality. however, if a strain with a higher mortality rate, such as that observed with the h5n1 avian influenza subtype, were to spread in a similar fashion, the outcome would be catastrophic both in terms of human suffering and economic damage. for example, the impact of an h5n1 avian influenza outbreak, should the virus become easily transmissible between humans, on the united states economy has been estimated to be $71.3-$166.5 billion [28] . the measures we have proposed are likely to have economic benefits that far outweigh their costs. we compiled the data on international air travel from the iata database, supplied by diio, llc through their apgdat service [29] . similar to prior analyses [15, 16, 17, 18] , we used direct connection information with regards to aircraft type and passenger capacity to calculate the connectivity of mexico with all airports included in the database, and summarized this information (as direct flight capacity) at the country level. additionally, we estimated the number of connecting passengers (indirect flight capacity) by calculating the number of passengers (p i,j ) arriving at airport j from airport i, and then estimating the number of passengers (p j,k ) going from airport j to airport k, based on all flights reported in the database. we limited the potential connections (trip jrk) to flights that departed no sooner than one hour after the first trip (irj), and no later than six hours after the arrival of the first trip. we also disallowed return of passengers to mexico once they left the country, and the return of passengers to north america once they left that region. we thus obtained a quantity, x i,j,k , that estimates the total potential connections to airport k available to passengers from the first trip (irj). setting constant the fraction of all passengers that connect (x), we obtained an estimate of the number of passengers with two leg itineraries for each potential destination (irk; eq. 1): we summarized these connections at the country scale, thereby estimating connectivity for nearly every country on the globe with mexico through either direct or indirect flights; the only countries excluded would require an overnight stay in a hub airport, or three or more connecting flights. we validated our algorithm (eq. 1) for connections within the contintental u.s.a. (the only data on actual itineraries, including connecting flight information, to which we had access). we randomly chose 50 connecting itineraries within the u.s.a. and compared our predictions to the actual routes. our predictions were statistically significant, using a simple proportional model with log-normal errors, and explained over 60% of the variance in actual routes (f = 83.71, p,0.001 on 1, 49 d.f, adjusted r 2 = 0.6232). we determined the date a country reported its first whoconfirmed 2009 a/h1n1 case through may 8 th , 2009. we chose this date in order to limit the analysis, as much as possible, to initial spread from mexico, because it served as a natural breakpoint in the distributions of reporting dates, as well as being the date our initial analysis. we performed a survival analysis using r [30] , and used an accelerated life time model using a log-logistic distribution. we also examined using a scale-free exponential distribution, as opposed to a log-logistic distribution, which requires a scale parameter, but these models did not fit nearly as well, as measured by aic. we followed burnham and anderson [31] , in using akaike information criterion (aic) to choose the model that best explains the data (i.e., the one with the lowest aic, or equivalently daic, score). additionally we provided the akaike weights, which estimate the likelihood that a specific model is the true model, assuming that the true model is in the set of examined models [14] . using this methodology, we choose to evaluate 22 models that made mechanistic sense including a null model for a reference. we did not include any models with only the indirect flight data, and without the direct flight data, because we feel that this does not make mechanistic sense. to reduce multicollinearity we included at most two socio-economic indicators. we evaluated four independent predictors for the date of first confirmed 2009 a/h1n1 case: the volume of (1) direct and (2) indirect passengers on international flights, (3) the country-specific gross domestic product and (4) healthcare spending per capita, by both private and public entities, from 2006 (the most recent year with all data available) from world bank estimates [32] . we also examined alternate socio-economic metrics as compiled by the world bank [32] , such as the number of physicians, and average population density. however models including these predictors did not perform as well (as measured by aic) and often had many more missing values if limited to most recent information. for all analyses, dates were transformed to julian day since february 15 th , and all predictor variables were standardized (mean subtracted, then divided by standard deviation) in order make possible the direct comparison of coefficients. this standardization has the added advantage of canceling out the x factor in equation 1 for the statistical analysis; thus, our analyses do not require any assumptions about the number of passengers who make connecting flights. these statistical models were used to predict the expected time of detection for all countries in our database that had gdp, population density, healthcare, and flight data. confidence intervals were constructed from the best model fit based on the variance of the data, using the ''predict'' functions in r [30] . we obtained united nations food and agriculture organization data on trade in live swine (commodity code hs96:s0103) and live poultry (s0105) from the u.n. comtrade data portal [14] . we analyzed data from the last ten years (the approximate time since 2009 a/h1n1 diverged from the nearest sampled virus) [7] , and focused on trade to north america (mexico, canada and united states) from outside this region, as well as trade to mexico within the north american region. figure s1 model predictions compared with actual case arrival dates. dates of case arrivals (black diamonds) for cases that were reported before our cut off of may 8th. grey whisker plots represent 95% confidence intervals for predicted arrival date, with interior grey bar as expected (mean) date of arrival from survival analysis. found at: doi:10.1371/journal.pone.0012763.s001 (0.02 mb pdf) figure s2 forward prediction of future case arrival dates. dates of case arrivals (black diamonds) for cases that were reported after our cut off of may 8th, but before may 19th. grey whisker plots represent 95% confidence intervals for predicted arrival date, with interior grey bar as expected (mean) date of arrival from survival analysis. found at: doi:10.1371/journal.pone.0012763.s002 (0.02 mb pdf) figure s3 forward prediction of future case arrival dates. grey whisker plots represent 95% confidence intervals for predicted arrival date, with interior grey bar as expected (mean) date of arrival from survival analysis. found at: doi:10.1371/journal.pone.0012763.s003 (0.03 mb pdf) microbial threats to health: emergence, detection, and response global trends in emerging 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database we acknowledge aleksei a. chmura, jon epstein, alonso a. aguirre, barry nickel, and evan girvetz for assistance. conceived and designed the experiments: prh pd. analyzed the data: prh shs kjv amk. contributed reagents/materials/analysis tools: amk. wrote the paper: prh shs amk pd. key: cord-000837-rdpsxb4n authors: perez-pepe, marcelo; slomiansky, victoria; loschi, mariela; luchelli, luciana; neme, maximiliano; thomas, maría gabriela; boccaccio, graciela lidia title: buho: a matlab script for the study of stress granules and processing bodies by high-throughput image analysis date: 2012-12-20 journal: plos one doi: 10.1371/journal.pone.0051495 sha: doc_id: 837 cord_uid: rdpsxb4n the spontaneous and reversible formation of foci and filaments that contain proteins involved in different metabolic processes is common in both the nucleus and the cytoplasm. stress granules (sgs) and processing bodies (pbs) belong to a novel family of cellular structures collectively known as mrna silencing foci that harbour repressed mrnas and their associated proteins. sgs and pbs are highly dynamic and they form upon stress and dissolve thus releasing the repressed mrnas according to changes in cell physiology. in addition, aggregates containing abnormal proteins are frequent in neurodegenerative disorders. in spite of the growing relevance of these supramolecular aggregates to diverse cellular functions a reliable automated tool for their systematic analysis is lacking. here we report a matlab script termed buho for the high-throughput image analysis of cellular foci. we used buho to assess the number, size and distribution of distinct objects with minimal deviation from manually obtained parameters. buho successfully addressed the induction of both sgs and pbs in mammalian and insect cells exposed to different stress stimuli. we also used buho to assess the dynamics of specific mrna-silencing foci termed smaug 1 foci (s-foci) in primary neurons upon synaptic stimulation. finally, we used buho to analyze the role of candidate genes on sg formation in an rnai-based experiment. we found that fak56d, gcn2 and pp1 govern sg formation. the role of pp1 is conserved in mammalian cells as judged by the effect of the pp1 inhibitor salubrinal, and involves dephosphorylation of the translation factor eif2α. all these experiments were analyzed manually and by buho and the results differed in less than 5% of the average value. the automated analysis by this user-friendly method will allow high-throughput image processing in short times by providing a robust, flexible and reliable alternative to the laborious and sometimes unfeasible visual scrutiny. an emerging concept in cell biology is the formation of microscopically visible supramolecular assemblies involved in very distinct cellular processes, ranging from metabolism to rna silencing. noxious insults that activate the cellular stress response trigger the transient accumulation of stress granules (sgs) in the cytoplasm. sgs belong to a novel family of cellular structures collectively known as mrna silencing foci that harbour repressed mrnas and their associated proteins [1] . sgs assemble as a consequence of the global translation silencing provoked by the inactivation of the translation initiation factor 2a (eif2a) by specific kinases that are activated upon cell stress. processing bodies (pbs) are related foci and are as well induced upon stress. there is controversy on whether sgs and pbs are cause or consequence of mrna repression. sgs and pbs from different organisms appear to differ on composition and function and a continuous spectrum of entities exits. nevertheless, sgs and pbs are highly dynamic and they may dissolve thus releasing the repressed mrnas to allow translation according to cellular needs [2, 3, 4, 5, 6] . sgs contribute to the cell survival response by regulation of specific signalling pathways [7] , and the cellular mechanisms that control their assembly and disassembly are incipiently described (reviewed in [1] ). cell damage induces the assembly of additional supramolecular complexes in both the nucleus and in the cytoplasm. the endoribonuclease inositol-requiring enzyme 1 (ire-1) and uvdamaged rna concentrate in cytoplasmic ire-1 foci or uv-bodies, respectively. dna replication factories form foci associated to damaged dna [8, 9, 10, 11, 12, 13] . translational silencing is frequently linked to the formation of distinct aggregates. for instance, local translation regulation is relevant to synaptic activity and involves the dynamic formation of a plethora of mrna silencing foci located at the synapse [14, 15] . the eukaryotic initiation factor 2b (eif2b) bodies are another example linked to translation regulation and their integrity and dynamics are crucial for eif2 recycling [16, 17, 18] . finally, a number of dynamic supramolecular factories were reported to occur in several cell types. these include purinosomes and glutamine synthetase foci, which concentrate specific biosynthetic enzymes and dissolve when the levels of the end-product metabolite increase [19, 20, 21] . enzymes may also reversibly aggregate for storage in an inactive state, as is the case of the ctp synthetase in yeast cells, drosophila embryo and mammalian axons [22] . in addition to all these structures, a large variety of cytoplasmic or nuclear supramolecular complexes such as nuclear stress bodies, eisosomes [23] , ubodies, splicing speckles and cajal bodies, among others, assemble and disassemble dynamically, depending on changes in cell physiology (reviewed in [1, 24] ). besides its relevance in mrna silencing and decay, sgs and pbs are relevant to the pathophysiology of several conditions. sg and pb dynamics are affected by virus infections (reviewed in [25, 26, 27] ). more recently, sgs have been linked to abnormal protein aggregates involved in neurodegenerative diseases [28, 29, 30, 31] . thus, there is an increasing interest in identifying pharmacological and genetic enhancers and inhibitors of sg and pb formation. a pioneer work for identifying regulators of sgs and pbs involved the analysis of thousands of micrographs [32] . these high-throughput studies would be facilitated by automated methods. here we report a matlab script that we named buho for the analysis of supramolecular aggregates in mammalian and drosophila cells. briefly, objects were identified by their similarity to a number of prototypes, which were of different size, shape and intensity. buho is easy to use and simple to adjust for the analysis of a variety of cellular components. we used it to analyze sg and pb induction in mammalian and drosophila cells exposed to stress. with minor adjustments, the script was also useful to study the dynamics of specific neuronal mrna silencing foci termed s-foci. finally, we used buho to analyze the effect of candidate genes on sg formation in an rnai-based experiment in drosophila s2r+ cells. we found that focal adhesion kinase 56d (fak56d), general control non-derepresible 2 (gcn2), and protein phosphatase 1 (pp1) govern sg assembly. pp1 mediates sg dissolution and its role is conserved in mammalian cells, as judged by the enhancement of sg formation provoked by the pp1 inhibitor salubrinal. all these experiments were evaluated manually and by buho, and in all cases the differences were lower than 5% of the average value. the automated analysis of diverse cellular components by this user-friendly method allows massive image processing in short times as well as minimizes variations between different operators by providing a robust, flexible and reliable high-throughput alternative to the laborious and sometimes unfeasible visual scrutiny. the accurate identification and characterization of cellular components using high-throughput microscopy, which retrieves a large amount of data, requires an automated methodology. to develop a matlab script for the computerized analysis of supramolecular aggregates we used sgs as a model system. these mrna silencing foci are an ideal model since their size and number vary depending on the cellular physiology. drosophila s2r+ cells were exposed or not to arsenite, a known inductor of oxidative stress that promotes sg formation [1, 33] . to visualize sgs and cell nuclei we co-stained the cells with oligodt-cy3 and dapi as indicated in materials and methods and 636 confocal images of 5126512 pixels were taken. as expected, in addition to sgs the fish for polyadenylated rna stained also the nucleus as well as faintly the cytoplasm ( figure 1a ). six images of stressed cells and three images of untreated cells were then manually analyzed. as previously reported [33] , sgs formed in half of the cells (table 1) . these images were used as a training set to adjust a matlab script to identify cells and the sgs inside them. matlab imported the cy3 and dapi images as 5126512 matrices where each matrix element has values from 0 to 65535 corresponding to the intensity of each pixel in the16-bit images. then, matlab operations were adjusted iteratively to minimize deviation from the manual analysis (table 1) , as described below. the first step was to identify the cells present in the micrographs. cell segmentation was performed using the matlab watershed transform algorithm (watershed function). first, high-intensity noise was eliminated by setting a convenient threshold for each channel. average dapi nuclear intensity was 200 in the training set of images (available at https://sourceforge.net/projects/buho/ ) and less than 0.1% of dapi-pixels reached values above 255. cy3 intensities in sgs and cytoplasm were lower than 50 and less than 0.01% of the cy3-pixels had values above 255. thus, intensity was clipped to 255 in both the dapi and the cy3 channels. the contrast of the dapi signal was adjusted with the imadjust function and the dapi signal was used to create a nuclear mask image. all pixels with intensities lower than 60% of maximal values were converted to 0 and points above this threshold were converted to 1 with the image to black and white (im2bw) function. we combined the nuclear mask and the negative counterpart of the oligodt-cy3 matlab matrices to generate a single image where all dapi-positive pixels were converted to 0 and cells are brightly stained. cells were clearly recognized in the image corresponding to this operation ( figure 1b ). next, to identify the cells by matlab, we calculated the watershed lines according to the topographic distances method. with this operation, the cell area corresponds to a catchment basin and the nuclei to its local minima or bottom of the basin. in summary, the nucleus acted as a marker for cell position and the inverted cy3 intensity gradient in the cytoplasm allowed the identification of the cell border ( figure 1b , c). previous to these operations, to compensate the faint overall cytoplasmic oligodt-cy3 staining, values in the matrix corresponding to the cy3 signal were multiplied by a factor of two, and additive noise was reduced using the wiener 2-d adaptive noiseremoval filtering method (wiener2 function). these adjustments allowed for a better identification of the cell border, and were not required when the cytoplasm is strongly stained, as in the case of sg detection by immunofluorescence against eukaryotic initiation factor 3g (eif3g) shown below (see figure 2) . we observed that a reduced number of cells were irregular and smaller than average, with an area smaller than 30 mm 2 .these include partially attached cells, due to the semi-adherence of the s2r+ cell line, as well as a small number of unhealthy cells, and were not included in subsequent analysis. the next step was to identify the sgs present in the micrographs of the training set. this was done using a normalized 2-d cross-correlation between the matrix that correspond to the oligodt-cy3 signal and several prototype sgs. then, a set of 8 prototypes were selected among 50 different example sgs taken from the confocal images that represent the most frequent patterns of sizes, shapes and intensities: round, elliptical, with uniform intensity or with a brighter core ( figure 1d ). among other factors, we found that the inclusion of a margin of cytoplasm surrounding table 1 ) were used to investigate the effect of varying the similarity threshold (st) and of increasing numbers of prototypes. e, the proportion of false positives relative to the number of seeds and the proportion of positive hits relative to the manual-counted number of sgs was measured at different st for the prototype iv, depicted in d. an st of 0.85 allowed no false positives. sg recognition dropped from 80% to 20% at this st value, and similar values were observed for the other prototypes (not shown). f, the number of positive hits that resulted by correlation with the indicated prototypes subsets was measured. all the sgs present in the micrographs were identified with the six prototypes depicted in d. size bar, 5 mm. g, a representative sg from the cell in the first row showing the seeds (red) created by the script. doi:10.1371/journal.pone.0051495.g001 nine images from wells treated or not with arsenite (training set) were manually analyzed. cells were considered positive when they contain two or more sgs [33] . each prototype granule greatly improved their performance in identifying the sgs present in the original images. a preliminary analysis using gaussian filters did not perform better than the prototypes selected from the micrographs. for each combination of prototype and micrograph, a matrix was obtained containing correlation coefficients with values from 21 to 1, according to the similarity between the local pixel pattern and the prototype granule. next, the points with correlation coefficients lower than a threshold termed similarity threshold (st), which we empirically adjusted for each prototype (see below and figure 1e ) were eliminated using the image to black and white (im2bw) function, and a value of 0 was assigned to them. points with correlation coefficients above the st were assigned a value of 1. we called these points ''seeds''. as expected, this operation generated a significant number of seeds inside the nucleus, as the abundant nuclear polyadenylated rna is recognized by the cy3labelled probe ( figure 1a ). seeds inside the nucleus were eliminated, together with a small amount of seeds generated in extracellular regions. we observed that a reduced number (7.5%) of sgs were weakly stained and were not recognized by the above operations. this was compensated by the application of the imfilter function with the fspecial: unsharp filter thus allowing faint or blurry sgs to be detected. these newly acquired sgs were added to the sgs identified in the unfiltered image, so for each micrograph, 16 correlations (8 prototypes, 2 cy3-micrographs with or without filter) were assessed. for each prototype, we investigated the effect of varying the st ( figure 1e and data not shown). by analyzing the 6 representative images of the training set with a total of 282 sgs, we empirically determined that a st of 0.89 for prototype i; 0.88 for prototype ii; 0.8 for prototype iii; 0.88 for prototype iv; 0.86 for prototype v; 0.86 for prototype vi; 0.92 for prototype vii; and 0.85 for prototype viii; eliminated all false positives in each case. as expected, these strict st values reduced the number of positive hits ( figure 1e and data not shown). this was compensated by the use of several prototypes. we found that with the above indicated st, each prototype identified a fraction between 2% and 32% of the sgs present in the images. we found that with the 8 proposed prototypes ( figure 1d ) the script recognized all the sgs present in the original micrographs of the training set ( figure 1f ). however, as expected, a single sg was frequently recognized by more than one prototype ( figure 1g and table 2 ). the whole process of nuclei, cell and sg identification is depicted in figure s1 . sgs frequently display anisotropic staining and the selected prototypes are examples of anisotropic sgs. then, we analyzed sg recognition by rotated versions of the prototypes depicted in fig. 1d . twenty four new prototypes were generated by three successive 90u clockwise rotations of the prototypes i to viii, and their performance in sg identification was assessed at the same st that the respective non-rotated prototypes. as expected, a variable fraction of sgs were identified by these 24 rotated prototypes (ranging from 2 to 32%). more important, we found that in all cases, the sgs identified by the rotated prototypes are redundantly identified by non-rotated prototypes ( figure s2 ). thus, without losing sensitivity and to minimize the number of cross-correlations to be performed, rotated versions were not included in the proposed collection of prototypes. the redundant recognition of a single sg leads to the assignment of more than one seed per sg. as a result, the sg will be counted more than once. this was corrected as follows: we applied a 363 dilation with the imdilate function to each seed to create a centered 363 square, which is the size of a small sg at this magnification and resolution (636, 5126512 pixels), so that all the squares surrounding seeds in the same sg overlapped and became a single object. we confirmed that with this operation close sgs were identified as different objects. finally, every object was reduced to a point with the function bwmorph-shrink, and the number of these points represented an accurate measurement of the number of sgs present in the original image. a combination of this matrix with that obtained by the watershed transform algorithm allowed calculating the number of sgs in each cell. as expected, the number of pixels in the above objects is a good estimation of the sg size in the original micrographs. however, we found that object size was better measured when it was above 6 pixels in width, thus higher resolution and/or higher magnification images are required for accurate measurements of granule size (see figure 3 below). however, the object size in the micrograph is largely affected by the point spread function of the objective and this may represent a serious limitation to size measurements by confocal microscopy. in summary, buho informs the cell number, the proportion of cells with sgs, the number of sgs per cell and their size with minimal deviation from manual-counted values (table 1 ). in addition, the algorithm calculates the distances between equal or distinct objects stained simultaneously using the bwdist function. to assess the suitability of buho for the analysis of additional cell structures, we investigated several examples of cytoplasmic foci stained with different strategies, including sgs and pbs in mammalian and insect cells, and synapses and smaug 1-mrna silencing foci (s-foci) in primary neurons. finally, we used buho for the analysis of a pilot experiment aimed to identify new genes regulators of sg formation. in all cases, we compared the buho output with values obtained manually and found that differences were lower than 5% of the values. example i: time-course of sg formation analyzed by buho sgs form transiently [1] and we analyzed the time-course of sg formation in drosophila cells upon induction of oxidative stress. sgs in drosophila s2r+ cells were visualized by fish as described in material and methods (figure 2a) , the lsm images (636, 102461024 pixels) were imported to matlab and scaled-down to 512 pixels using the imresize function. then, cells and sgs were identified as described above using the following sts: 0.84, 0.88, 0.7, 0.8., 0.83, 0.8, 0.92, and 0.85 for prototypes i-viii respectively. we compared the number of cells with sgs calculated by buho with the values obtained by manual analysis. maximal formation was detected by both methods at 2 hs after the oxidative stress stimulus, with half of the cells showing sgs (figure 2a) . at all the time points analyzed buho and manual values diverge in less than 5% relative to the average values. next, we extended this study to the analysis of sgs induced in a different experimental system. we used mammalian u2os cells exposed to thapsigargin, a known inductor of er-stress, as indicated in materials and methods. to challenge our automated method with different data input, sgs were visualized by immunofluorescence against eif3g, an accepted sg marker ( figure 2b ). images were taken at 636, 102461024 pixels and scaled down to 512 pixels previous to analysis with the prototypes i-viii as above. as before, buho allowed us to assess transient sg formation with high precision. deviation from manuallycounted values was less than 7% and both analysis showed a maximal response at 1 hour, when 93% of the cells had sgs. we concluded that buho perform successfully in the identification of sgs in cells stained by fish against polyadenylated rna or immunofluorescence against eif3g. moreover, the same 8 prototypes are suitable for sg identification in mammalian or drosophila cells exposed to different insults. example ii: analysis of pb induction upon stress pb size increases upon cellular stress [1] , and we used this as a model system to test the buho suitability to assess changes in foci size. drosophila s2r+ or mammalian u2os cells were exposed to oxidative or er-stress as indicated in materials and methods, and pbs were visualized by immunostaining of the specific marker dcp1a ( figure 3a ). drosophila pbs were analyzed at 636, 102461024 pixels, a resolution that we found suitable for size determination of objects with these dimensions (0.2-1 mm 2 /11-56 square pixels). low intensity noise was removed with the im2bw function and prototypes i-viii were compared at st 0.8. mammalian pbs were analyzed at 406, 204862048 pixels. in this case, low intensity noise was removed and prototypes i-viii were compared at sts 0.8, 0.88, 0.8, 0.8, 0.86, 0.86, 0.8, and 0.85 respectively. adjustment of these parameters was performed by analyzing 123 representative s2r+ cells and 38 representative u2os cells. next, pb size was determined using buho and the manual method in parallel. as expected, both methods indicated that pbs were significantly larger after exposing the cells to oxidative stress ( figure 3a, b) . drosophila pbs enlarged by a factor of 3.5 and mammalian pb size by a factor 1.5 relative to their normal values. the difference of these values from manual parameters was less than 4% for basal pbs and 10% for induced pbs in drosophila cells, and less than 7% for basal and 12% for induced pbs in mammalian cells. the total number of pbs in the micrographs was also measured, and we found that the buho-generated values (drosophila basal, 350; drosophila stress, 102; u2os basal, 185; u2os stress, 148) were comparable with the manual values (drosophila basal, 309; drosophila stress, 122; u2os basal, 167; u2os stress, 129). we concluded that buho successfully addresses changes in foci size and number. moreover, as expected given that sgs and pbs are morphologically similar, we found that prototype sgs were useful to identify pbs. to test the performance of buho in analyzing distances between objects, we focused on the presence of synaptic mrna silencing foci at the synapse surroundings. briefly, neurons contain a plethora of mrna-silencing foci collectively termed synaptic activity-regulated mrna silencing foci (syas). different synaptic stimuli enhance or reduce the presence of syas and there is an increasing interest in understanding syas dynamics [14, 15] . for instance, we recently found that a specific kind of synaptic foci named s-foci dissolve upon n-methyl-d-aspartate (nmda) stimulation [14] . we studied the suitability of buho to assess this effect and compared it with the manual analysis. primary neurons were exposed to nmda as previously described [14] and s-foci and synapses were identified by immunostaining with specific antibodies as indicated in materials and methods. using 636 micrographs, 102461024 pixels, we first evaluated s-foci size. the s-foci are morphologically similar to pbs ( [14] figure 4 ) and we used prototypes i-viii to analyze them. as we did for pbs, low intensity noise was removed and prototypes were compared at st 0.8. as previously reported, s-foci size was reduced to 71% of basal levels when obtained manually and to 61% when evaluated with the matlab script, which are comparable values [14] . we recently reported that in addition to this general reduction in size, synaptic stimulation by nmda provokes the complete dissolution of the s-foci located at the synapse [14] . to evaluate this important local response we counted the number of synapses with s-foci in their surroundings. synapses were stained with a specific antibody and identified by correlation with the prototypes i to viii. optimal st was determined to be 0.7 in a preliminary analysis. we used the bwdist function to measure the distances between each synapse and the closer s-foci, and calculated the percentage of synapses with s-foci at a distance lower than 0.5 mm, which is physiologically relevant [14] (figure 4 ). we found that the presence of s-foci at the synapse surroundings was reduced to 50% of basal values. when evaluated manually, they were affected in a similar proportion (41%, figure 4 ). we concluded that buho is a reliable bioinformatics tool to identify synapses and to assess the vicinity between different cellular structures. finally, to simulate a high-throughput rnai screen, we used the buho script for the analysis of a pilot experiment aimed to identify sg regulators. we used drosophila s2r+ cells in 384 mw plates with robotized manipulation for cell plating, stress induction, staining and imaging as indicated in materials and methods. we analyzed three candidate genes predicted to affect sg formation. previous to the induction of oxidative stress, we treated the cells with dsrna against fak56d, gcn2, or pp1a-96a, or with a non-relevant dsrna against lacz. fak56d is homologous to mammalian fak, a kinase involved in sg disassembly [34] . gcn2 is an eif2a kinase that mediates sg formation upon oxidative stress in drosophila [35] . pp1a-96a is the main phosphatase of eif2a and we speculated that this molecule affects sg stability. triplicate plates including three well with a dsrna for each gene, 20 wells with a dsrna against lacz and 68 wells with no dsrna were analyzed. seven images of each well were taken under the same conditions as those from the training set used for the script adjustment and analyzed manually and by buho using the prototypes i-viii, as in figure 2a . as expected, the values for the number of cells and sgs, and the proportion of cells with sgs were equivalent with the two methods (table 3) when this test set of images was analyzed. we defined a score as the ratio of the percentage of cells with sgs upon dsrnatreatment relative to the average percentage of cells with sgs in the controls without dsrna present in the same plate. as expected, we found that the dsrna against lacz had no effect on sg formation ( figure 5a ). as previously reported [35] , gcn2 knockdown impaired sg formation. fak56d knockdown had a stimulatory effect, as reported before for the mammalian counterpart [34] . in addition, we found that the knockdown of pp1a-96a enhanced sg formation in s2r+ cells. to investigate whether the role of this phosphatase is conserved in mammalian cells, we followed a pharmacological approach by using the pp1a inhibitor salubrinal. we exposed u2os cells to a er-stress insult in the presence or absence of salubrinal [36] , which was added 1 hour after the stress-stimulus as indicated in materials and methods and investigated sg formation by immunostaining of eif3g at different time points. sgs were identified by buho as in figure 2b . we found that paralleling the effect of pp1a-96a knockdown in drosophila cells the pharmacological inhibition of mammalian pp1a impaired sg disassembly in u2os cells ( figure 5b ). all these experiments were simultaneously assessed by manual analysis by an independent operator, and deviation from buho-obtained values was less than 5%. pp1 is the main eif2a phosphatase and we evaluated phosphorylated eif2a (peif2a) levels upon exposure to er-stress in the presence or absence of salubrinal. as previously reported [1, 33, 37] we found that eif2a phosphorylation increased 13 fold relative to basal values one hour after stimulation, when sg formation is maximal (fig. 5b and data not shown) . at this time, salubrinal was added to inhibit dephosphorylation and peif2a levels were monitored three hours after stress induction. paralleling the prolonged occurrence of sgs in the presence of salubrinal, we found that peif2a levels were 50% higher in the presence of the pp1 inhibitor (fig. 5c, p#0 .01). we speculate that dephosphorylation of eif2a is key in regulating sg dissolution and this opens new lines of research. the success of buho in this pilot rnai-based screen documented by automated microscopy underscores its potential in high-throughput imaging analysis. in this work, we developed an original algorithm for the automated analysis of sgs and related cytoplasmic foci, and used it to identify pp1 as a novel regulator of sg formation. this matlab script, termed buho is easily commanded using windows operating system and is based in the recognition by similarity to a collection of prototypes. the analysis of a pilot highthroughput experiment including 164304 images at 5126512 pixels was completed in 120 hours in a relatively low power computer (perez-pepe et al, unpublished). the script was optimized for the analysis of sgs in drosophila cells, and is easy to adjust for the study of a wide variety of supramolecular aggregates in different cell types. with minor modifications, we have successfully applied it for processing images of different magnification and resolution of synapses, sgs, pbs and similar foci in mammalian and drosophila cell lines as well as in primary cultured neurons. remarkably, the deviation between buho and manual values was less than 5% of the average values. by using a different set of prototypes taken exclusively from confocal images or in combination with prototypes generated with gaussian filters, we anticipate that buho will be also a valuable tool for the analysis of additional cellular structures and supramolecular aggregates that are emerging in the literature [17, 19, 20, 22, 23, 24] . to further validate the multiple uses of this bioinformatics method, we utilized buho in the analysis of a pilot experiment aimed to investigate the effect of candidate genes on sg dynamics. as expected, we found that this script is a reliable tool to assess changes in sg formation. as reported before for the mammalian fak56d homologue, we found that the knockdown of this kinase in drosophila cells enhances sg formation, whereas downregulation of gcn2 impairs their assembly [34] . in addition, we found that the catalytic subunit of pp1 mediates sg dissolution. the role of this main phosphatase appeared conserved in flies and mammals, and furthermore, we found that pp1 affected the disassembly of sgs induced by different stress stimuli. phosphorylation of eif2a provokes translation inhibition and concomitant sg formation, and dephosphorylation of eif2a correlates with figure 5 . pp1a governs sg disassembly. a, drosophila s2r+ cells were exposed to the indicated dsrna and the effect on sg formation was evaluated. triplicate wells of the indicated rnai treatments were analyzed both manually and by buho. 204 control wells were analyzed by buho, and a subset of 6 wells (see table 3 ) was analyzed manually. the score is the ratio of the percentage cells with sgs in each well relative to the average percentage of cells with sgs in the control wells in the same plate. gcn2 knockdown impaired sg formation, and the kd of fak56d or pp1a-96a facilitated their assembly. scores determined manually differ in less than 7% from those calculated by buho. b and c, mammalian u2os cells were exposed to er-stress in the presence (sal translation recovery after acute stress [1] . accordingly, our results indicate that pp1 mediates eif2a dephosphorylation, and we speculate that this plays an important role in sg stabilization upon pp1 kd or inhibition by salubrinal. the relevance of additional pp1 targets involved in sg dissolution is unknown. salubrinal is a relatively new drug and its potential as therapeutic agent is expanding [36] . the contribution of sg regulation to the beneficial effect of this inhibitor remains to be investigated. we anticipate that the high-throughput screening of natural or synthetic compounds will benefit from the automated analysis of sg formation described here. sgs always form when cells are exposed to stress insults and their formation will be indicative of cell toxicity, which is an important effect to assess when analyzing novel chemicals. the use of buho for the automated analysis of other mrna silencing foci, protein aggregates and cytoplasmic structures in addition to sgs, pbs, synapses and s-foci will require only minor modifications. the spontaneous formation of microscopically visible aggregates that concentrate molecules involved in distinct pathways is common in many different cell types. a fraction of a yeast library including thousands of genes with cterminal gfp-fusions was visually screened to reveal that numerous proteins form filaments and foci [20, 22] . the usefulness of these libraries will be greatly potentiated by high-throughput image analysis like the one described here. purinosomes, glutamine synthetase foci and other supramolecular factories optimize the biosynthetic pathway by channeling substrates, minimizing diffusion to the cytosol and protecting labile intermediates [19, 20] . conversely, a number of enzyme aggregates appears to serve as depots [22] . in addition to enzyme aggregates, uv-rna granules, ire-1 foci, splicing speckles, nuclear stress bodies, cajal bodies, eif2b bodies, and other cytoplasmic or nuclear structures assemble and disassemble dynamically depending on cellular needs. the script here described will result a suitable tool for the high-throughput analysis of these different supramolecular aggregates in diverse experimental settings. among other outputs, buho addresses the number, size and distance between objects, and categorize them by similarity in shape, size and intensity to a variable number of prototypes isolated from original images. by adjusting the correct parameters buho can be adapted to the automated analysis of many different cellular components. this user-friendly script allows the analysis of high-throughput imaging data in short times and eliminates variations associated to tedious manual scrutiny that frequently involves more than one operator. this novel bioinformatics tool is a step forward in the expanding field of cellular supramolecular aggregates. drosophila s2r+ cells were grown in 384 mw plates (figure 1 and 5, table 1 , 2, and 3) or in 10 mm glass coverslips (figure 2 and 3), as previously described [33] . oxidative stress was induced by exposure to 500 mm arsenite during 2 hs unless otherwise indicated. when required, cells in 384mw plates were treated with 0.25 mg dsrna during 3 days, as is routine at the drosophila rnai screening center (drsc), harvard medical school. we used the following dsrnas, designed by the drsc: drsc16678 against gcn2, drsc16795 against pp1a-96a and drsc07426 against fak56d. all manipulations, including plating, treatment and staining in 384 mw plates were performed in a robotized system at the drsc. u2os cells from the american type culture collection (atcc) were grown on 10 mm glass coverslips in dmem (sigma) as before [33, 37] . when indicated, 100 nm thapsigargin (sigma) was added to conditioned media, and 25 mm salubrinal (calbiochem emd bioscience, san diego, ca) in dmso or dmso alone as control were added one hour afterwards. mild oxidative stress was triggered with 250 mm arsenite plus 100 mg/ ml puromycin and strong oxidative stress with 500 mm arsenite plus 250 mg/ml puromycin during one hour, as previously described [38] . primary hippocampal neurons were prepared and treated with 30 mm nmda (sigma) during 5 minutes as previously described [14] . sgs in drosophila s2r+ cells were visualized by fish for polyadenylated rna using oligodt-cy3 (sigma), as previously indicated [33] and with automated processing for the 384 mw plates. immunofluorescence of drosophila and mammalian cells was performed after fixation, permeabilization and blocking as previously done [14, 33, 39] . primary antibodies were used as follows: goat anti-eif3g (santa cruz); 1:200; mouse anti-dcp1a (abnova), 1:500; rabbit anti-peif2a (cell signaling), 1:100; antimammalian smaug1, 1:500 [40] ; igg1 anti-synapsin (synaptic systems), 1:250. the secondary antibodies used were: anti-goat cy5 (jackson immuno research laboratories), 1:200 and antimouse alexa 488 (molecular probes), 1:500, anti-rabbit cy3 (jackson immuno research laboratories), 1:200. nuclei were visualized by dapi staining. cells grown in glass coverslips were mounted in mowiol 4-88 (calbiochem, emd bioscience, san diego, ca). cells in 384 mw plates were analyzed in an opera autoscope at the drsc and 636 micrographs were captured at 5126512 pixels. cells in glass coverslips were analyzed after mounting. images were acquired in a lsm510 meta confocal microscope (carl zeiss, oberkochen, germany) using zeiss lsm software at 25uc and ec 'plan-neofluor' 406/1.30 oil or a plan-apochromat 636/1.4 oil. equipment adjustment was assessed using 1 mm focalcheck fluorescent microspheres (molecular probes). magnification and resolution were as follows: figures 2a, 2b , 3a, 4 and 5b: 636, 102461024 pixels; figure 3b : 406, 204862048 pixels; figure 1a and 5a: 636, 5126512 pixels. manual and buho analysis were carried out independently by two different operators. cells and particles were manually analyzed by image j (nih). signal intensity was analyzed by image j. lsm or tiff format images were used for the analysis with matlab (matrix laboratory, the mathworks inc.), version r2008a. the bwdist; bwmorph; imadjust; imcrop; imdilate; imfill; imfilter; imread; regionprops; watershed and wiener2 functions from the matlab image processing toolbox were used. monochromatic images correspond to a 5126512, 102461024 or 204862048 matlab matrix, where the intensity of each pixel is a value ranging from 0 to 255 for 8-bit images (figures 2, 3, 4 and 5b) , or from 0 to 65535 for 16-bit images (figures 1 and 5a ). when indicated, resolution was scaled down to 512 pixels. the matlab script developed here, termed buho and the confocal images are available at https://sourceforge.net/projects/buho/. the proposed collection of prototypes is available at proto-type.rar at the same url. figure s1 buho and manual analysis of cells and sgs. a representative region from the training set of images (see table 1 ) is depicted through the whole process of nucleus, cell and sg identification. a, the dapi staining is converted to a nuclear mask using the im2bw matlab function (image to black and white). the oligodt-cy3 staining was negatively converted with the imcomplement function (image complement). the two matrices were combined to generate an image with black nuclei and bright cytoplasm. then, the watershed transform algorithm (watershed function) was used to delimitate the cell borders. b, the oligodt-cy3 image was used to detect sgs similar to prototype i. the pixel pattern of each pixel of the cy3 image is compared with the pixel pattern of prototype 1 by a normalized 2-d cross-correlation (normxcorr2), which generate an image where each pixel has a value from 21 to 1, with 1 indicating the higher similarity. then, a similarity threshold empirically adjusted to avoid false positives (see text) is applied and pixels with values higher than st = 0.89 are assigned a value of 1. these operations generate seeds in each hit. finally, the imdilate function convert the seeds into larger objects, the identified sgs. c, identification of sgs by prototypes i to viii. a normalized 2-d cross-correlation was performed for each prototype. the prototype i identifies 6 different sgs (yellow arrows). the prototype ii identifies two additional sgs (blue arrows). the prototype iii identifies seven sgs (red arrows), one of them previously detected by prototype ii (left upper corner). the prototype iv identifies five sgs (orange arrows), only one of them previously undetected (second cell from right). the prototype v identifies three sgs (green arrows) previously detected by prototypes i, ii and iii, respectively. the prototype vi identifies two new sgs and two sgs already detected by prototype iii (pink arrows). the prototypes vii and viii don't match any sg in this image, according to their low identification rate ( figure 1f and table 2) . see text and buho_process at https://sourceforge.net/projects/ buho/ for more detail. (tif) figure s2 redundant identification by rotated prototypes. prototypes i-viii depicted in figure 1f were rotated 90, 180 or 270 degrees clockwise using the imrotate function and used as probes to detect sgs in the training set of images. the subsets of sgs recognized by each rotated prototype was then tested against non-rotated prototypes i to viii in an additive manner (against prototype i only; against prototype i and ii, et cetera). in all cases 100% of the sgs identified by rotated prototypes were redundantly recognized by the collection of non-rotated prototypes. (tif) rna granules: the good, the bad and the ugly movement of eukaryotic mrnas between polysomes and cytoplasmic processing bodies stress-specific composition, assembly and kinetics of stress granules in saccharomyces cerevisiae analyzing p-bodies and stress granules in saccharomyces cerevisiae the dynamics of mammalian p body transport, assembly, and disassembly in vivo real-time and quantitative imaging of mammalian stress granules and processing bodies transient sequestration of torc1 into stress granules during heat stress messenger rna targeting to endoplasmic reticulum stress signalling sites a novel class of mrna-containing cytoplasmic granules are produced in response to uv-irradiation sequential assembly of translesion dna polymerases at uv-induced dna damage sites dynamics of dna replication factories in living cells dynamic targeting of the replication machinery to sites of dna damage differentially regulates dna replication and dna-repair-associated processes after uv irradiation smaug1 mrna-silencing foci respond to nmda and modulate synapse formation synaptic activity regulated mrna-silencing foci for the fine tuning of local protein synthesis at the synapse localization of the translational guanine nucleotide exchange factor eif2b: a common theme for gefs? dynamic cycling of eif2 through a large eif2b-containing cytoplasmic body: implications for translation control fusel alcohols regulate translation initiation by inhibiting eif2b to reduce ternary complex in a mechanism that may involve altering the integrity and dynamics of the eif2b body reversible compartmentalization of de novo purine biosynthetic complexes in living cells widespread reorganization of metabolic enzymes into reversible assemblies upon nutrient starvation microtubuleassisted mechanism for functional metabolic macromolecular complex formation identification of novel filament-forming proteins in saccharomyces cerevisiae and drosophila melanogaster eisosomes mark static sites of endocytosis biogenesis and function of nuclear bodies regulation of stress granules in virus systems p bodies, stress granules, and viral life cycles mouse hepatitis coronavirus replication induces host translational shutoff and mrna decay, with concomitant formation of stress granules and processing bodies tdp-43 is recruited to stress granules in conditions of oxidative insult alsassociated fused in sarcoma (fus) mutations disrupt transportin-mediated nuclear import survival motor neuron protein facilitates assembly of stress granules trapping of messenger rna by fragile x mental retardation protein into cytoplasmic granules induces translation repression a functional rnai screen links o-glcnac modification of ribosomal proteins to stress granule and processing body assembly dynein and kinesin regulate stress-granule and p-body dynamics regulation of stress granule dynamics by grb7 and fak signalling pathway visibly stressed: the role of eif2, tia-1, and stress granules in protein translation sustained translational repression by eif2[agr]-p mediates prion neurodegeneration mammalian staufen 1 is recruited to stress granules and impairs their assembly a monoclonal antibody against p53 cross-reacts with processing bodies staufen recruitment into stress granules does not affect early mrna transport in oligodendrocytes mammalian smaug is a translational repressor that forms cytoplasmic foci similar to stress granules we are grateful to stephanie mohr from the drsc for advice and to lorena b. benseñ or for critical reading of the manuscript. key: cord-000166-36bfeoqv authors: tracht, samantha m.; del valle, sara y.; hyman, james m. title: mathematical modeling of the effectiveness of facemasks in reducing the spread of novel influenza a (h1n1) date: 2010-02-10 journal: plos one doi: 10.1371/journal.pone.0009018 sha: doc_id: 166 cord_uid: 36bfeoqv on june 11, 2009, the world health organization declared the outbreak of novel influenza a (h1n1) a pandemic. with limited supplies of antivirals and vaccines, countries and individuals are looking at other ways to reduce the spread of pandemic (h1n1) 2009, particularly options that are cost effective and relatively easy to implement. recent experiences with the 2003 sars and 2009 h1n1 epidemics have shown that people are willing to wear facemasks to protect themselves against infection; however, little research has been done to quantify the impact of using facemasks in reducing the spread of disease. we construct and analyze a mathematical model for a population in which some people wear facemasks during the pandemic and quantify impact of these masks on the spread of influenza. to estimate the parameter values used for the effectiveness of facemasks, we used available data from studies on n95 respirators and surgical facemasks. the results show that if n95 respirators are only 20% effective in reducing susceptibility and infectivity, only 10% of the population would have to wear them to reduce the number of influenza a (h1n1) cases by 20%. we can conclude from our model that, if worn properly, facemasks are an effective intervention strategy in reducing the spread of pandemic (h1n1) 2009. novel influenza a (h1n1) (hereafter referred to as pandemic (h1n1) 2009 in keeping with the world health organization (who) nomenclature) is a new flu virus of swine, avian, and human origin that was first identified in mid-april 2009 in mexico and the united states [1] . the virus soon spread to the rest of the world and on june 11, 2009 the who declared novel influenza a (h1n1) a pandemic. the virus continues to spread, with most countries reporting cases of pandemic (h1n1) 2009 [1] . even though the who's declaration of a phase six pandemic alert level does not explicitly refer to the severity of the disease, as many people contracting the virus recover without medical treatment, the number of deaths continues to rise [1] . the rapid spread of influenza, due to its short incubation period and lack of strainspecific vaccine, pose a challenge to the implementation of effective mitigation strategies during the expected reemergence of pandemic (h1n1) 2009 in the fall/winter flu season. every year approximately 36,000 people die from seasonal influenza or flurelated causes in the u.s. [2] . however, the number of casualties may increase with a new and more virulent strains of influenza, such as the pandemic (h1n1) 2009. the emergence of an unexpected or new strain of influenza means there are no prepared vaccines and the existing antivirals may be ineffective in combating the spread of infection. vaccination is typically the first line of defense against influenza viruses [3] . the entire vaccine production process takes at least six months to complete [4] and although a pandemic (h1n1) 2009 vaccine became available in the u.s. in october 2009, there are severe shortages in the amount of vaccines available. another concern is that the currently circulating h1n1 strain could mutate, making the vaccine ineffective or less effective. in the recent pandemic (h1n1) 2009 outbreak, non-pharmaceutical interventions such as school closings and thermal screenings at airports were implemented to slow the spread of disease [5, 6] . other common non-pharmecuetical interventions include quarantine, isolation, travel restrictions, closing of public places, fear-based self quarantine, and cancellation of events. these interventions all have economic costs to individuals and society related to lost work, increased school absenteeism, and decreased business revenues. another non-pharmaceutical option is the use of facemasks. in the 2003 sars outbreak many individuals used facemasks to reduce their chances of contracting infection. in hong kong 76% of the residents reported using masks during the 2003 sars epidemic [7] . even though individuals have taken upon themselves to wear facemasks during disease outbreaks, little research has been done to quantify the impact of the use of facemasks during an epidemic. mathematical models of the spread of infectious disease can be useful in assessing the impact of facemasks on reducing the spread of a disease, specifically pandemic (h1n1) 2009. pandemic (h1n1) 2009 spreads through person-to-person contact, airborne particles, coughing and sneezing, and by fomites [1] , therefore, the use of facemasks is a logical line of defense. the centers for disease control and prevention (cdc) have interim recommendations on the use of facemasks and respirators for the current pandemic (h1n1) 2009 virus. the cdc defines the term facemask as a disposable mask cleared by the u.s. food and drug administration (fda) for use as a medical device, such as surgical masks. surgical masks are designed to help stop droplets from being spread by the person wearing the mask, not to protect against breathing in very small particle aerosols that may contain viruses [8] . we will use of the term 'respirator' for an n95 or higher filtering facepiece respirator certified by the cdc/national institute for occupational safety and health (niosh); a respirator is designed to protect the person wearing the mask against breathing in very small particles that may contain viruses [8] . the cdc states that the effectiveness of the use of facemasks and respirators in various settings is unknown and do not generally recommend the use of facemasks or respirators in home or community settings nor in non-medical occupational settings [8] . in certain circumstances the cdc recommends the use of masks for individuals who are at high risk of infection and cannot avoid situations with potential exposure to the disease [8] . there have been a handful of studies that have analyzed the effectiveness of facemasks against nanoparticles in the size range of viruses using manikin-based protocol in which the masks were sealed on the manikin's face so that no leakage would occur [9] [10] [11] . all three studies show similar results in penetration percentage for the n95 respirator. the high fit n95 respirator had penetration percentages from about 0.5% to 2.5% at 30 l/ min and from about 0.5% to 5% at 85 l/min [9] [10] [11] . the low fit n95 respirator had penetration percentages from about 1.5% to 3.5% at 30 l/min and from about 1.5% to 6% at 85 l/min [9] [10] [11] . the surgical masks tested in balazy et al.'s [10] study show a much greater penetration percentage. at 30 l/min one model of surgical mask (sm1) allowed 20-80% of particles to penetrate the mask, while another model (sm2) allowed 2-15% [10] . at 85 l/min sm1 allowed penetration of 30-85% of particles while sm2 allowed 5-21% [10] . the n95 respirator in a sealed manikin test seems to be fairly effective against nanoparticles, almost holding up to its 95% certification. the surgical masks are not as effective, allowing a much greater percentage of particles to pass through to the wearer even when sealed tightly to a manikin. unfortunately, this type of testing does not provide an accurate estimate of the level of protection for everyday use of a mask by a person. while these studies provide data on the actual protection of masks against nanoparticles in a perfect setting, it does not take into consideration that a mask will not be completely sealed on an individual nor will it fit perfectly. furthermore, one must consider that an individual will not always be wearing the mask, for example, a mask will be taken off to eat and sleep, or possibly because it becomes uncomfortable to wear. lee et al. [12] performed a study on n95 respirators and surgical masks using human subjects. the challenge aerosol used was nacl, with particles in the size range of bacteria and viruses (.04-1.3mm). they tested four models of n95 respirators: 1) high protection level, 2) medium protection level, 3) exhalation valve, and 4) exhalation without valve and three models of surgical masks: 1) high protection level, 2) medium protection level, and 3) low protection level. the results from the study showed that the lowest protection offered from n95 respirators is when particles are in the size range of 0.08-0.2mm and for surgical masks when particles are in the size range of 0.04-0.32mm. the size range of influenza virus is in the range of 0.08-0.12mm, which falls into both masks most penetrating particle size range. the n95 respirator was found to be 21.5% effective and the surgical mask was 2.4% effective in protecting against nanoparticles. the n95 respirator provides approximately nine times greater protection than a surgical mask and is clearly a better option in protecting against infection. a university of michigan school of public health study led by dr. allison aiello [13] is evaluating the effectiveness of handwashing and facemasks in preventing influenza from spreading. the study, called m-flu, conducted a randomized cluster intervention trial among students living in dorm housing. the students were randomly separated into two intervention groups, one wearing masks and practicing hand hygiene, one just wearing masks, and also in a control group. the study was carried out over the 2006-2007 influenza season, which was a mild season. the study found that facemasks and hand hygiene were correlated with a 35-51% reduction in influenza-like illness [13] . there are many factors that influence people's willingness to wear a mask. in a study by tang and wong [14] a total of 1,329 . the arrows that connect the boxed groups represent the movement of individuals from one group to an adjacent one. nonmask wearing susceptible individuals (s) can either become exposed (e) or susceptible wearing a mask (s m ). non-mask wearing exposed individuals (e) can either become infectious non-mask wearing (i) or mask wearing exposed (e m ). non-mask wearing infectious individuals (i) can either recover (r), die (d), or become infectious wearing a mask (i m ). mask wearing susceptible individual (s m ) can either become an exposed mask wearer (e m ) or a non-mask wearing susceptible (s). mask wearing exposed individuals (e m ) can either become an infectious mask wearer (i m ) or a non-mask wearing exposed individual (e). a mask wearing infectious individual (i m ) can either recover (r), die (d), or stop wearing the mask while they are still infectious (i). doi:10.1371/journal.pone.0009018.g001 adult chinese residing in hong kong were surveyed on their use of facemasks during the 2003 sars epidemic. overall 61.2% of the respondents reported the consistent use of facemasks to prevent contracting the disease. the study found that women in the age group 50-59 and married respondents were more likely to wear facemasks, suggesting that the aesthetics of wearing a facemask may be a concern. also, the study found that individuals who had a university education or earned more than us$5,000 per month were more likely to wear a mask. tang and wong also showed that perceived susceptibility, cues to action, and perceived benefits, were significant predictors in whether or not an individual consistently wore a mask. following the approached developed in [15] , the population is divided into two subgroups: a mask wearing group (subscript m) and a non-mask wearing group. people move back and forth between the mask and non-mask groups based on the number of individuals infected with pandemic (h1n1) 2009. individuals in each activity group are characterized by their epidemiological status: susceptible, denoted by s and s m , exposed, denoted by e and e m (i.e., people who are infected but not yet fully contagious), and infectious individuals, i and i m . definitions of the eight epidemiological classes are summarized in table 1 and the transfers are shown diagrammatically in figure 1 . because we are evaluating the effectiveness of masks in a single influenza period, we use a closed system with no migration in or out, and births and natural deaths are not included in the model. as seen in figure 1 , the transfer rates of people from the exposed classes, e and e m , to the infectious classes, i and i m , are ve and ve m . infectious individuals can move to group d, at rate mi and mi m , when they die from infection or to group r, at rate di and di m , upon recovery. the mean times in the infectious classes, i and i m , are 1=(dzm). hence, the infectious fraction d=(mzd) recovers and the infectious fraction m=(mzd) dies as a consequence of this disease. we assume that there is homogeneous mixing between groups and that contact activity levels remain normal throughout the epidemic. we define t 0 as the beginning of the epidemic. movement of individuals between mask and non-mask groups depends upon the number of pandemic (h1n1) 2009 cases in the population. a specified percentage of the population starts wearing masks as the number of infected people increases. we define q sm s, q em e, and q im i to be the transfer rates from the s, e, and i classes to the s m , e m , and i m classes, respectively, similarly q s s m , q e e m , and q i i m are the transfer rates from the s m , e m , and i m classes to the s, e, and i classes, respectively. the rate coefficients are modeled by step-functions of the number of infectious individuals: for i = s, e, i, s m , e m , and i m . here the parameters a and b are positive constants that determine the rate of movement and t is the number of pandemic (h1n1) 2009 cases that determines when masks are implemented. for i = s, e, and i, b i is set at 0.1 or 10% of the population. using the transfer diagrams in figure 1 we obtain the following system of differential equations: here l (non-mask group) and l m (mask group) are the forces of infection and ls and l m s m are the transfer rates from the susceptible classes, s and s m , to the exposed classes, e and e m . the infection rates, l and l m , incorporate the probability of transmission per contact, b, the reduced infectiousness due to incubation, a, the reduced number of contacts because of symptomatic infection, h, and 1{g j , (j = s or i), which accounts for the effectiveness of the mask in reducing either susceptibility (g s ) or infectivity (g i ). the transmissibility, b, is defined as the effective reproduction number < con for n95 respirators. notice that < con decreases as a higher percentage of people wear masks as well as when masks are more effective. < con is greatly reduced when 50% of the population wears masks and masks are 50% effective. doi:10.1371/journal.pone.0009018.t005 susceptibility of the population multiplied by the infectivity of the disease multiplied by the average number of contacts an individual has per day. the definitions of the parameters are summarized in table 2 . the forces of infection for the non-mask group and mask group are shown by: where r~n{(1{h)(izi m ) and n is the total population the effective reproduction number, < eff , is the average number of secondary cases produced by a typical infectious individual effective reproduction number, < con , for surgical masks. notice that < con decreases as a higher percentage of people wear masks as well as when masks are more effective. however, < con is not greatly reduced even when 50% of the population wears masks and masks are 50% effective. doi:10.1371/journal.pone.0009018.t006 during the infectious period [16, 17] . the effectiveness of intervention strategies are often measured by their ability to reduce the spread of a disease in a given population. in an epidemic model the magnitude of the effective reproduction number, < eff , determines whether or not an epidemic occurs and its severity [15] . when < eff w1, the number of infections grow and an epidemic occurs, however when < eff v1, the number of infections does not increase and there is no epidemic outbreak [15] . without any interventions the model has an initial effective reproduction number (uncontrolled) < unc given by: this < unc is the product of the average number of people infected per unit time b and the weighted sum of the average infectious period 1=(mzd) plus the average incubation period 1=v. the 'next-generation operator' approach [17] is used to find an expression for the effective reproduction number (controlled) < con for our epidemic model when masks are used as an intervention strategy. the computation is done by linearizing the system of equations (2) around the disease-free equilibrium (dfe). the dfe has e, e m , i, and i m equal to zero with s 0 , s 0 m , and r 0 positive. since there is no immunity from previous infection or vaccination r 0 is also equal to zero. the resulting fourdimensional linearized system is of the form dx dt~( the effective reproduction number < con is the largest eigenvalue of the matrix fv {1 [17] . hence < con is the only non-zero eigenvalue of the matrix fv {1 and is given by the expression: figure 2 with respirators is also seen here: as the masks effectiveness is higher the number of cumulative cases decreases and the number of cases also decreases if a higher percentage of people wear masks. however, the difference in the number of cumulative cases is not nearly as large when surgical masks are worn; this is due to their lower effectiveness. doi:10.1371/journal.pone.0009018.g003 where c 1~qem zv, c 2~qe zv, c 3~qim zdzm, c 4~qi zdzm, and s~s 0 zs 0 m . we use equations 4 and 7 to define the effective reproduction number for the model as: where t is the threshold number of infected individuals at which masks start to be used. the epidemiology of pandemic (h1n1) 2009 is not accurately known since it continues to spread across the world. the parameter values shown in table 2 were chosen based on the best available data. the incubation period for pandemic (h1n1) 2009 has been reported to be 2-10 days with a mean of 6 days [18] . the mean time in the exposed classes e and e m corresponding to the incubation period has been assumed to be 6 days, making the transfer rate to the infectious classes, i and i m , constant at v = 1/6. the infectious period is believed to be between four and seven days, with an average of five days [19, 20] . thus, the baseline value for the recovery rate is constant at d = 1/5. the fatality rate of the pandemic (h1n1) 2009 is thought to be in the range of 0.3%-1.5%, with a mean of 0.46% [21] [22] [23] . the case fatality rate for our model is m=(dzm), setting this equal to 0.0046 results in m~0:0046d=0:9954~0:001. the current estimates on the transmission of pandemic (h1n1) 2009 are that one infected person may typically infects one to two people [24] [25] [26] . the transmissibility, b, is the product of the susceptibility of the population, the infectivity of the disease, and the number of contacts an individual has in a day [27, 28] . the susceptibility of the population is set to one, as it is believed few people are immune to pandemic (h1n1) 2009, and the number of contacts an individual has per day is assumed to be 16 [29] . the infectivity is found by 1:8=(16( a v z h mzd )), so that r 0 = 1.8 in a completely susceptible population and the infectivity is .0141. so b~0:23 gives the transmission rate, the fraction of contacts per day that is sufficient for the transmission of pandemic (h1n1) 2009. the baseline population size n for the model is set at one million people and all are initially in the susceptible class s. the initial infected fraction, i/n, is set at 0.00001 so that when n = 1000000, i = 10. the model scales linearly so that the initial population size n and the initial number of infected individuals i are both scaled up or down by the same factor. we assume that individuals will start wearing masks after 100 people are infected, figure 4 . sensitivity to < unc < unc . the number of cumulative cases is very sensitive to the value of the uncontrolled effective reproduction number (< unc ). higher values of < unc result in a larger number of cumulative cases. a large difference in the number of cases is seen when the < unc is equal to 1.83 and when < unc is equal to 1.7; for such a slight difference in < unc the difference in the number of cases is quite large. doi:10.1371/journal.pone.0009018.g004 (6) once there is enough number of cases in a community to convince people to start wearing masks. we analyzed the impact of masks when 10%, 25%, and 50% of the population wear them. using the studies published on the effectiveness of masks we determined the baseline values for the effectiveness of n95 respirators to be g s = 0.2 and g i = 0.5 and for the surgical masks g s = 0.02 and g i = 0.05 [12] . the effectiveness of masks in decreasing the infectivity of a sick individual is greater because the mask contains the virus particles, preventing them from becoming airborne, and therefore preventing the contamination of surrounding surfaces as well as people [30] . although it is possible that some sick individuals may change their behavior due to the symptoms [15] , we assume that sick individuals will not change their behavior and continue to have the same number of daily contacts as a healthy individual. therefore, we set the baseline value for the reduced number of contacts due to illness h at 1, as people usually do not greatly alter their daily behavior during the incubation period. individuals in the exposed classes, e and e m , are thought to be 50% less infectious due to incubation than those in the infected classes, i and i m , so we set a = 0.5 [19, 31] . we analyzed two scenarios: one in which the n95 respirator is worn and one in which surgical masks are worn; for both types of masks we considered three different variations in mask effectiveness. each case is evaluated with 10%, 25%, and 50% of susceptible and exposed individuals wearing masks, while in each case the fraction of infectious individuals wearing masks is slightly larger. when 10%, 25%, and 50% of susceptible and exposed individuals are wearing masks the fraction of infectious individuals wearing masks is 30%, 40%, and 50%, respectively. all simulations assume that in a population of one million there are initially 10 infected individuals reported and everyone else is susceptible. mask start being used when there have been 100 reported cases of pandemic (h1n1) 2009. the numerical results for the percentage of pandemic (h1n1) 2009 cases are shown in table 3 for the n95 respirator and in table 4 for surgical masks. the effective reproduction numbers for each case are shown in table 5 for n95 respirators and in table 6 for surgical masks. the cumulative number of pandemic (h1n1) 2009 cases can be seen graphically for the varying mask effectiveness and the different fractions of individuals wearing masks in figure 2 and in figure 3 for n95 respirators and surgical masks, respectively. table 3 and table 4 show that when masks are not used, then the total percentage of the population who will be infected is 74.61% in a population of 1 million people. with the implementation of n95 respirators table 3 exhibits a reduction in the cumulative number of cases of almost 200,000, or a 19% decrease, when 10% of the population wears masks and they are 20% effective. table 5 shows the implementation of the n95 respirators' impact on the effective reproduction number < con ; it is reduced from 1.83 to 1.66 when masks are 20% effective in reducing both susceptibility and infectivity and 10% of the population is wearing masks. when effectiveness is increased to 50% < con is reduced even further to 1.4. as the fraction of the population wearing n95 respirators increases, < con is reduced table 4 shows that surgical masks do not have as large of an impact in reducing the cumulative number of cases as does the n95 respirator. table 6 displays the effective reproduction number < con when surgical masks are implemented. the lowest value surgical masks reduce < con to is 1.77. in figure 2 the effectiveness of the n95 respirator in reducing the spread of pandemic (h1n1) 2009 is significant. as the percentage of the population wearing masks increases the number of cumulative cases decreases and when the mask effectiveness is greater, the number of cases is also greatly reduced. the impact of surgical masks is not as large as seen graphically in figure 3 , the reduction in the cumulative number of cases is relatively small compared to that of the n95 respirator. if mask effectiveness is 5% and 50% of the population wears surgical masks the reduction in the number of cumulative cases is 6%. even though the parameter values were estimated from epidemiological data, there is still some uncertainty in their values. since pandemic (h1n1) 2009 is a new virus, there is a wide range of estimated values for the parameters. in our model we chose the averages for our baseline parameters, here we look at a range of parameters and how changing a specific one effects the outcome of the model. this sensitivity analysis examines the effects of changes in the reproduction number (< unc ), mask effectiveness (g s and g i ), index cases (i=n), fraction of population wearing masks (q i ), number of initially infected at which masks are implemented (t), as well as the effect of which epidemiological group wears masks (s or i). unless otherwise stated the other parameters are fixed at their baselines values found in table 2 . effective reproduction number. the effective reproduction number < unc determines the average number of secondary cases resulting from one typical infectious individual during the infectious period without the implementation of facemasks. since there is a delay in the implementation of facemasks the initial growth of the epidemic is affected by < unc . the estimates of < unc for pandemic (h1n1) 2009 vary widely, the common range is assumed to be between 1.2 and 2.2. as the value of < unc increases the number of pandemic (h1n1) 2009 cases increases significantly as shown graphically in figure 4 . mask effectiveness. the effectiveness of the mask greatly affects the number of cumulative cases. the higher the effectiveness the fewer number of cases (shown in the results section). the effectiveness of the masks not only depends upon the type of mask and quality but also proper usage. index cases. the number of initially infected individuals can have a major impact on the size of the epidemic. in figure 5 we vary the number of initially infected individuals in the population. fraction of population wearing masks. we consider variations in the percentage of the population that wears masks. we look at the effect of 10%, 25% and 50% of the population wearing masks. the model shows that the higher the percentage of the population wearing masks the fewer the number of cumulative cases, this is shown in figure 6 . implementation of masks. the epidemic is sensitive to the delay in the implementation of masks as seen in figure 7 . we look at the cumulative number of pandemic (h1n1) 2009 cases for the n95 respirator when 10% of the population is wearing masks. figure 7 shows that the earlier masks are implemented, the bigger the reduction in the cumulative number of cases. who wears masks. the model is sensitive to who wears masks. here we look at the effect if only infected individuals wear masks and if only susceptible and exposed individuals would wear masks. figure 8 shows that it is important for both infected, as well as susceptible and exposed individuals, to wear masks. the standard mitigation strategies used for influenza viruses are vaccines and antivirals. however, in the case of a novel virus these may not be readily available and other mitigation strategies will be needed. as seen during the 2003 sars outbreak and the current pandemic (h1n1) 2009 people are willing to wear facemasks to reduce the spread of disease. we used a mathematical model to examine the possible impact of n95 respirators and surgical masks on reducing the spread of pandemic (h1n1) 2009. when modeled with a low mask effectiveness and a small fraction of the population wearing masks, the implementation of facemasks still has a relatively large impact on the size of the pandemic (h1n1) 2009. the numerical simulation results in the results section show that without any interventions, we predict that a large percentage of the population will be infected with pandemic (h1n1) 2009 influenza strain. this result is not surprising as the population is 100% susceptible and the effective reproduction number < unc is 1.83, which is higher than that of typical seasonal influenza. in reality, the r unc may be lower due to heterogeneous mixing patterns, pre-existing immunity, and other interventions in place. with 10% of the population wearing n95 respirators with effectiveness at 20% in reducing both susceptibility and infectivity there is a 19% reduction in the cumulative number of cases. with the same mask effectiveness but 25% of the population wearing n95 respirators, the total number of pandemic (h1n1) 2009 cases is reduced by almost 30% and with 50% of the population wearing masks, it results in over a 36% reduction in the number of cases. the effectiveness of surgical masks is low, therefore the impact of wearing them during an epidemic is not significant. even at 50% effectiveness in reducing both susceptibility and infectivity and with 50% of the population wearing surgical masks only a 6% reduction in the number of cumulative cases is seen. the sooner an epidemic is recognized and masks are implemented, the bigger the reduction in the number of cases will be. as seen in the results section the epidemic is sensitive to the delay in implementing masks. the difference in the total number of pandemic (h1n1) 2009 cases when masks are implemented at 100 infected individuals and 1,000 infected individuals is over 7%. the implementation of neither n95 respirators nor surgical masks lowered the effective reproduction number < unc below one. however, n95 respirators greatly decreased < unc , in some scenarios very close to one. while facemasks will not stop the pandemic (h1n1) 2009, they could greatly reduce its severity and allow for more time to develop effective vaccines and antivirals. there are currently more trials being conducted on the effectiveness of surgical masks and n95 respirators [32] , which will allow us to refine the assumptions made in the model. however, it must be noted that in order for masks to be effective they must be: (1) available, (2) affordable, (3) worn properly, (4) replaced or sanitized daily, and (5) n95 respirators should be fit-tested. only 10% of the population would have to wear masks in order to reduce the percentage of cases by 20%. facemasks are inexpensive, relatively easy to implement, and would not cause a large economic burden to society. masks are a powerful tool and can be used by countries with limited supplies of antiviral drugs and vaccines. in addition, economically feasible preventative global mitigations will benefit the world as a whole. we can conclude from our model that n95 respirators if worn properly are an effective intervention strategy in reducing the spread of the pandemic (h1n1) 2009. figure 8 . sensitivity to who wears masks. in order to achieve the greatest possible reduction in the cumulative number of cases both infectious individuals and susceptible and exposed individuals should wear masks. if only infectious individuals wear masks the number of cases is not significantly reduced. doi:10.1371/journal.pone.0009018.g008 center for disease control and prevention website. 2. (2009) questions and answers regarding estimating deaths from influenza in the united states mitigation strategies for pandemic influenza in the united states how do they make influenza vaccine? 5. 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of behavioral changes in a smallpox attack model the mathematics of infectious diseases reproduction numbers and subthreshold endemic equilibria for compartmental models of disease transmission interim guidance for clinicians on identifying and caring for patients with swine-origin influenza a (h1n1) virus infection local and systemic cytokine response during experimental human influenza a virus infection. relation to symptom formation and host defense duration of influenza a virus shedding in hospitalized patients and implications for infection control influenza a (h1n1)-update 44. world health organization website: global alert and response pandemic potential of a strain of influenza a (h1n1): early findings the effect of public health measures on the 1918 influenza pandemic in u.s. cities outbreak of swine-origin influenza a (h1n1) virus infection -mexico update: infections with a swine-origin influenza a virus -united states and other countries, april. center for disease control and prevention website episims los angeles case study transmission dynamics of the great influenza pandemic of 1918 in geneva, switzerland: aassessing the effects of hypothetical interventions mixing patterns between age groups in social networks a schlieren optical study of the human cough with and without wearing masks for aerosol control quantifying the routes of transmission for pandemic influenza surgical mask vs n95 respirator for preventing influenza among health care workers transmission potential of the new influenza a (h1n1) virus and its age-specificity in japan pandemic (h1n1) 2009. world health organization website: global alert and response hhs pandemic influenza plan. us department of health and human services website we would like to thank carlos castillo-chavez and gerardo chowell for their helpful comments. conceived and designed the experiments: smt sdv jmh. performed the experiments: smt. analyzed the data: smt sdv jmh. contributed reagents/materials/analysis tools: smt sdv jmh. wrote the paper: smt sdv jmh. key: cord-000374-gt2pwc9b authors: yang, albert c.; tsai, shih-jen; hong, chen-jee; wang, cynthia; chen, tai-jui; liou, ying-jay; peng, chung-kang title: clustering heart rate dynamics is associated with β-adrenergic receptor polymorphisms: analysis by information-based similarity index date: 2011-05-04 journal: plos one doi: 10.1371/journal.pone.0019232 sha: doc_id: 374 cord_uid: gt2pwc9b background: genetic polymorphisms in the gene encoding the β-adrenergic receptors (β-ar) have a pivotal role in the functions of the autonomic nervous system. using heart rate variability (hrv) as an indicator of autonomic function, we present a bottom-up genotype–phenotype analysis to investigate the association between β-ar gene polymorphisms and heart rate dynamics. methods: a total of 221 healthy han chinese adults (59 males and 162 females, aged 33.6±10.8 years, range 19 to 63 years) were recruited and genotyped for three common β-ar polymorphisms: β(1)-ar ser49gly, β(2)-ar arg16gly and β(2)-ar gln27glu. each subject underwent two hours of electrocardiogram monitoring at rest. we applied an information-based similarity (ibs) index to measure the pairwise dissimilarity of heart rate dynamics among study subjects. results: with the aid of agglomerative hierarchical cluster analysis, we categorized subjects into major clusters, which were found to have significantly different distributions of β(2)-ar arg16gly genotype. furthermore, the non-randomness index, a nonlinear hrv measure derived from the ibs method, was significantly lower in arg16 homozygotes than in gly16 carriers. the non-randomness index was negatively correlated with parasympathetic-related hrv variables and positively correlated with those hrv indices reflecting a sympathovagal shift toward sympathetic activity. conclusions: we demonstrate a bottom-up categorization approach combining the ibs method and hierarchical cluster analysis to detect subgroups of subjects with hrv phenotypes associated with β-ar polymorphisms. our results provide evidence that β(2)-ar polymorphisms are significantly associated with the acceleration/deceleration pattern of heart rate oscillation, reflecting the underlying mode of autonomic nervous system control. instantaneous heart rate in response to physiological perturbations often exhibits remarkable oscillations at multiple time scales. these oscillations, known as heart rate variability (hrv), are mainly mediated by the autonomic nervous system via parasympathetic and sympathetic innervations. analysis of hrv has been suggested to reveal subtle patterns of heart rate dynamics that are relevant to the underlying physiological state and autonomic nervous system function [1] . prior studies have shown that hrv measures are highly heritable traits that can be used to support genetic association and linkage studies [2, 3, 4, 5] . family and twin studies have shown a significant genetic influence on a variety of hrv measures [6, 7] . genetic polymorphisms related to cardiovascular functions have been associated with altered hrv [8, 9, 10, 11] . recently, we have also made progress in identifying variations in two genes related to neuropsychiatric function that are associated with altered heart rate dynamics in samples of healthy adult and elderly subjects: those encoding brain-derived neurotrophic factor (bdnf) [12] and apolipoprotein e [13] , respectively. despite increasing focus on investigating the genetic influence on autonomic functions, current approaches to genotype-hrv associations have largely been characterized by a top-down approach involving a direct comparison of continuous hrv variables among pre-defined groups of subjects (i.e., healthy vs. ill or groups of known genotypes), yet it is unclear how a particular genetic polymorphism may determine a similar pattern of autonomic heart rate control from one subject to another. specifically, heart rate dynamics is a phenotypic ''expression'' of the autonomic nervous system, so comparing similar heart rate oscillation phenotypes among individuals may reveal a global profile of autonomic function relevant to genetic variants. with these considerations in mind, in the present study, we introduce a bottom-up genotype-phenotype analysis to investigate the association between genetic polymorphisms and autonomic control of heart rate dynamics, using three common polymorphisms in genes encoding b-adrenergic receptor (b-ar) as an example. b-ar has a pivotal role in the functions of the cardiac autonomic nervous system. activation of b 1 -ar provides strong stimulus to increase the frequency and contractility of the heart, whereas the activation of b 2 -ar results in smooth muscle relaxation and increased cardiac output with less extent compared to b 1 -ar. thus, the connection between b-ar and hrv is plausible and warrants evaluations. limited evidences suggest that variations in genes coding subtypes of b-ar may be associated with heart rate or hrv. for example, ser49gly polymorphism in b 1 -ar gene has been found to be associated with resting heart rate [14, 15] , and an association of b 2 -ar gene polymorphisms with spectral components of hrv measures has been reported in a relatively small healthy adult male sample [11] . we applied an information-based similarity index (ibs) [16, 17] to measure the pairwise dissimilarity of interbeat interval time series among a sample of healthy adult volunteers. the ibs method is based on rank-order frequency analysis of acceleration/ deceleration patterns of heart rate fluctuation. because stimulus of b-ar results in acceleration of heart rate, the functional changes in genetic polymorphisms of b-ar may affect acceleration/deceleration patterns of heart rate, which can be detected by the ibs method. the analyses of the present study were two-fold: 1) a nonrandomness index [17] derived from the ibs method was applied to quantify the nonlinear aspect of hrv according to b-ar genotype and to test the correlation of this index with standard hrv indices; and 2) using agglomerative hierarchical cluster analysis, we unsupervisedly categorized these subjects into clusters based on pairwise dissimilarity among heart rate dynamics, and then we investigated the association of these clustering patterns with b-ar gene polymorphisms. we show that this bottom-up, categorization approach combining the ibs method and hierarchical cluster analysis can detect subgroups of subjects based on phenotypes that are associated with b-ar gene polymorphisms. two hundred forty-seven healthy han chinese adult volunteers were recruited from two medical centers: taipei veterans general hospital and kaohsiung e-da hospital, taiwan. subjects were recruited by advertisement among medical employees, research laboratory staff working at both hospitals, and their relatives. all subjects gave informed consent before commencement of the study. the protocol was approved by the institutional review boards of the taipei veterans general hospital (taipei, taiwan) and e-da hospital (kaohsiung, taiwan). each subject was given an interview using a standard questionnaire to carefully review the history of medical disease, psychiatric illness, and medication use. subjects included in the study did not have a personal history of medical conditions (e.g., malignant tumors, heart failure, or diabetes mellitus), pregnancy, psychiatric illnesses or substance abuse/dependence. none of the subjects was taking any medication. the collected demographic data included age, sex, body mass index, and smoking. of note, most volunteers were hospital colleagues, and the rate of smoking was low (n = 2, 0.9%). of these subjects, 228 were successfully contacted for ambulatory electrocardiogram (ecg) monitoring. holter recordings (myecg e3-80 portable recorder, microstar inc., taipei, taiwan) were used to obtain two hours of ecg signals. the e3-80 device continuously recorded three channels of ecg signals at a sampling rate of 250 hz. all ecg monitoring took place in the daytime, and participants were asked to avoid smoking or drinking alcoholic beverages and to stay in a resting state while being monitored. valid dna samples were obtained in 221 subjects by drawing blood or by buccal swabs. the final study sample therefore consisted of 221 healthy adult subjects (59 males and 162 females, aged 33.6610.8 years, range 19-63 years). among the present study sample, 211 subjects have been included elsewhere in a previous report on the altered sympathovagal balance associated with val66met polymorphisms of the bdnf gene [12] . each subject was genotyped for three polymorphisms (rs1801252, rs1042713 and rs1042714), and genomic dna was isolated using the puregene dna purification system (gentra systems, minneapolis, mn, usa). the genotypes of rs1042713 were determined using polymerase chain reaction and restriction fragment length polymorphism analysis. briefly, primers and probes were designed with spectrodesigner software (sequenom, san diego, ca, usa). pcr was then performed, and unincorporated double-stranded nucleotide triphosphate bases (dntps) were dephosphorylated with shrimp alkaline phosphatase (hoffman-laroche, basel, switzerland) followed by primer extension. the purified primer extension reaction product was spotted on to a 384-element silicon chip (spectrochip, sequenom) and analyzed in a bruker biflex iii maldi-tof spectro-reader mass spectrometer (sequenom). the resulting spectra were then processed with spectrotyper (sequenom). all samples were genotyped for eight unrelated snps for dna quality examination. the samples were diluted onto 96-well plates, and only the plates on which each of the eight unrelated snps had a successful genotyping rate greater than 95% were used for further study. all experiments were performed by investigators who were blind to phenotype. failure in genotyping for rs1801252 polymorphism was noted in 6 cases. the ecg signals were automatically processed and analyzed by open-source hrv algorithms [18] . the standard hrv analysis has been well reviewed [19] . briefly, time domain measures of hrv include the mean heart rate and standard deviation of the normal interbeat intervals (sdnn), the root mean square successive difference between adjacent normal interbeat intervals (rmssd), and the percentage of adjacent intervals that varied by greater than 50 ms (pnn50) [20] . the sdnn assesses the overall variability of interbeat intervals. the rmssd and pnn50 measure the short-term variation of interbeat intervals, which is mainly modulated by parasympathetic innervation [21] . standard spectral hrv measures [19] include high-frequency power (hf; 0.15-0.40 hz), low-frequency power (lf; 0.04-0.15 hz), and very low-frequency power (vlf; 0.003-0.04 hz). lf power is suggested to be modulated by both sympathetic and parasympathetic activities, whereas hf power is mainly modulated by parasympathetic activity [22, 23] . the lf/hf ratio is considered a measure of the shift of sympathovagal balance toward sympathetic activity [19, 24] . the physiological mechanism underlying vlf power is disputed but has been suggested to be mediated partly by the renin-angiotensin-aldosterone system or parasympathetic modulation [19, 25, 26] . in addition, we incorporated two nonlinear hrv indices: detrended fluctuation analysis (dfa) [27, 28] and multiscale entropy (mse) [29] . dfa quantifies the presence of long-range (fractal) correlations whereas mse measures the entropy over multiple time scales inherent in physiologic signals and is therefore a complexity measure. both methods are available at physionet (http://physionet.org), a research resource for complex physiologic signals [18] . in the dfa method, the root-mean-square fluctuation of integrated and detrended time series is measured at different observation windows and plotted against the size of the observation window on a log-log scale. the scaling exponent a is then derived from the slope of line fitting to the obtained log-log plot. the short-term exponent a 1 (4 to 11 heartbeats) and the longterm scaling exponents a 2 (.11 heartbeats) were also calculated [27, 30, 31] . low-exponent values represent reduced fractal propertiy of heart rate dynamics and have been implicated in the risk of fatal cardiac arrhythmia, increased mortality, or poor prognosis in cardiovascular diseases [32, 33, 34, 35] . mse has been proposed as a biologically meaningful complexity measure by quantifying the entropy over multiple time scales inherent in physiologic signals. the procedure and calculation of the mse is summerized as following three steps: 1) construction of coarse-grained time series, 2) quantification of the sample entropy of each coarse-grained time series, and 3) summation of the sample entropy values over a range of scales. in the present study, sample entropy was calculated using a pattern length (m) of 2 and a similarity factor (r) of 0.15. the sum of sample entropy over all scale factors from 1 to 20 was computed to represent the overall mse measure. in addition, the sum of sample entropy over scale factors from 1-5 and 6-20 was calculated to represent short-term and long-term mse measures, respectively [36] . several methods of symbolic dynamic analysis of hrv have been proposed previously [37, 38, 39, 40, 41] . the ibs method was developed to effectively categorize symbolic sequences according to their information content. the method has been fully described and validated [16] , with applications to heart rate time series, literary texts, and genetic sequences [17, 42, 43] . an interbeat interval time series (or heart rate time series) is mapped to a symbolic sequence, according to a mapping rule that accelerated heart rate in consecutive heartbeats is designated as 0 and a deceleration of heart rate is designated as 1 ( figure 1a ). this way of mapping captures the essential dynamics of the autonomic nervous system's control of heart rate and is less sensitive to noisy fluctuations in interbeat interval time series commonly caused by ectopic heartbeats [17] . a binary, symbolic ''word'' is then defined as a n-tuple sequence derived from n+1 consecutive interbeat intervals. we determined the frequencies of each pattern of n-tuple sequences by applying a sliding window (moving one interbeat interval/step) across the entire interbeat interval time series and then ranked each n-tuple sequence according to its frequency in descending order. to compare the similarity between symbolic sequences, we plotted the rank number of each n-tuple sequence in the first sequence against that of the second sequence ( figure 1b ). if two sequences are similar in their rank order of n-tuples, the scattered points will be located near the diagonal line (e.g., comparison between healthy subjects) [17] . therefore, the average deviation of these scattered points away from the diagonal line is a measure of the dissimilarity index between these two sequences. we defined the distance (d n ) using n-tuples between two sequences, s 1 and s 2 , as here r 1 (w k ) and r 2 (w k ) represent the rank of a specific n-tuple, w k , in sequences s 1 and s 2 , respectively. n = 2 n is the number of different n-tuple sequences (or patterns). the absolute difference of the mapping procedure for a n-tuple binary symbolic sequence (here n = 6 for illustrative purposes) from part of an interbeat interval time series. (b) rank-order comparison of two interbeat interval time series from the two healthy subjects, using 6tuple binary symbolic mapping. in this case, frequencies of 2 6 = 64 6tuple symbolic patterns were determined and ranked accordingly. for each 6-tuple symbolic sequence (black dot), its rank in subject a is plotted against its rank in subject b. the dashed diagonal line indicates the case where the rank order of 6-tuple symbolic sequence for both subjects is identical. doi:10.1371/journal.pone.0019232.g001 ranks, r 1 (w k ){r 2 (w k ) j j , is weighted by summing shannon's entropy h for w k in sequences s 1 and s 2 [44] . shannon's entropy measures the information richness of each n-tuple in both sequences. thus, the more frequently used n-tuples contribute more to measuring similarity among symbolic sequences. of note, the ibs is an empirical index which does not necessarily obey the triangular inequality criterion of a distance measure. therefore, the triangular inequality test is required before generating a cluster [16, 17] . the ibs algorithm was available at physionet (http:// www.physionet.org/physiotools/ibs/). the applications of the ibs method in the present study were two-fold: 1) the pairwise distance of heart rate dynamics among individuals was calculated by an average of ibs distance using ntuple symbolic sequences (n = 2-10) and was used in subsequent hierarchical cluster analysis. 2) to quantify the nonlinear aspect of heart rate time series, a non-randomness index was defined to measure the symbolic patterns of heart rate dynamics away from complete randomness (i.e., absence of ordered control of the autonomic nervous system) [17] . the non-randomness index is independent of conventional entropic measures (i.e., sample entropy or approximate entropy) and has been evaluated in other studies [17, 45, 46, 47] . the calculation of the non-randomness index is based on estimating the average n-tuple distance between raw interbeat interval time series and its randomly shuffled surrogates using the ibs method, where the number of surrogates was 100 in the present study. the averaged non-randomness index derived from each n-tuple non-randomness index (n = 2-10) was then used in this study for comparisons among b-ar genotypes. we calculated allele and genotype frequencies and performed hardy-weinberg equilibrium tests for each b-ar genotype. the spectral hrv indices were log-transformed to produce normalized distributions. to control for the effects of non-genetic factors, differences in hrv variables were compared for individual genotypes using a general linear model (glm) followed by the bonferroni post hoc test for corrections of multiple between-group comparisons., using age, gender, and body mass index as covariates. effect sizes were calculated with cohen d, defined as the difference between the means of two groups, divided by the pooled standard deviation of these groups. partial correlation analysis was applied, controlling for age and bmi, to determine the associations between standard hrv indices and nonrandomness index derived from the ibs method. a p value of less than 0.05 (two-tailed) was required for all statistical comparisons. a complete-linkage, hierarchical clustering tree was estimated using the generalized association plot (gap) (http://gap.stat. sinica.edu.tw/). gap is implemented here as an unsupervised clustering algorithm to visually categorize all subjects based on the dissimilarity matrix, which is derived from calculating the pairwise dissimilarity of heart rate dynamics among subjects using the ibs method. cluster-specific association was analyzed by the chisquare test to assess the frequency of b-ar polymorphisms and by the glm model to test the differences in standard hrv indices among resulting clusters. to validate cluster-specific associations, we tested the differences in hrv and b-ar genotypes in estimated clusters based on first-and second-half ecg data. demographic and clinical data for subjects with each b-ar genotype are presented in table s1 and s2. the subgroups in three genotypes did not differ in age, gender ratio, smoking status, or body mass index. there was no detectable deviation from hardy-weinberg equilibrium in b 1 -ar ser49gly genotype (x 2 = 0.169, p = 0.681), b 2 -ar arg16gly genotype (x 2 = 0.824, p = 0.364) or b 2 -ar gln27glu genotype (x 2 = 2.188, p = 0.139). tables 1, 2, 3 summarize the association of b-ar genotypes with hrv indices determined by a glm model using age, gender, and bmi as covariates. there was no significant effect of interaction on hrv indices detected between b-ar genotypes and demographic variables. b 1 -ar ser49gly genotype was associated with mean heart rate and sdnn with borderline significance (table 1 ; p = 0.087 and p = 0.064, respectively). no statistical association was found between b 2 -ar gln27glu genotype and hrv indices ( table 2) . for the b 2 -ar arg16gly genotype (table 3) , a significant codominant association was found with lf% (f = 3.636, p = 0.028) and the non-randomness index derived from the ibs method (f = 5.642, p = 0.004). multiple comparisons showed that lf% was significantly lower in homozygous arg16 carriers compared to subjects with the arg16/gly16 genotype (p = 0.016), but not to homozygous gly16 carriers (p = 0.985). likewise, a significantly lower non-randomness index was found in homozygous carriers of the arg16 allele (p = 0.016) compared to subjects heterozygous for arg16/gly16 (p = 0.001), and a borderline significance was seen in the comparison to homozygous gly16 carriers (p = 0.057). there was no significant difference in hrv indices between heterozygotes arg16/gly16 and homozygous gly16 carriers, correlations between non-randomness index and standard heart rate variability to ascertain the relationship of non-randomness index with autonomic function, we estimated the partial correlation between non-randomness index and standard hrv variables, controlling for age and bmi (table s3) . a weak but significant positive correlation of the non-randomness index with the lf/hf ratio was found (r = 0.234, p = 0.001), whereas negative correlations were found with rmssd (r = 20.274, p,0.001) and pnn50 (r = 20.279, p,0.001). association of heart rate dynamic clusters with b-ar gene polymorphisms next, we investigated the b-ar genotype distributions in an unsupervised cluster tree based on the dissimilarity (distance) measure of heart rate dynamics. we first estimated the pairwise distance between interbeat interval time series of all subjects using the ibs method. no violation of the triangular inequality was observed. we then applied a hierarchical clustering algorithm to cluster these subjects based on the obtained distance matrix. two major clusters were identified in the resulting dendrogram ( figure 2 ). there was no significant difference in age, gender, or other demographic variables between the two clusters (data not shown). table 4 shows b-ar genotypes and standard hrv characteristics according to two major clusters. a significant deviation in the distribution of homozygous b 2 -ar arg16 carriers was detected between the two clusters (p = 0.011), whereas the other genotypes showed no deviation in genotype distribution. in terms of standard hrv characteristics, cluster 1 (with a higher rate of homozygous b 2 -ar arg16 carriers) showed significantly reduced lf power (p,0.001) and significantly increased hf power (p = 0.001). the clusters did not differ in other hrv indices. we verified the genotype distribution with clusters based on first-and second-half ecg data, and the results were consistent (table s4 ). the data presented in this study demonstrate a significant association of a common b 2 -ar polymorphism, arg16gly, with the non-randomness index, a nonlinear hrv measure derived from the ibs method. moreover, we illustrate that a bottom-up approach using the ibs method was able to measure the dissimilarity of heart rate dynamics among individuals, and we show that the resulting clusters were associated with b 2 -ar arg16gly genotype, indicating an impact of this b 2 -ar polymorphism on acceleration/deceleration patterns of heart rate oscillations. our study offers several advantages for studying genetic associations with physiological parameters and might be generalizable to other genotype-phenotype studies based on different types of time series (e.g., brain electroactivity or time series derived from functional magnetic resonance imaging). first, our method enables us to cluster heart rate dynamics by an ibs method based on their acceleration/deceleration patterns of heartbeat sequence. the ibs method was not exclusively developed for the analysis of heartbeat sequence but also for other generic datasets consisting of repetitive elements [42, 43] . with an appropriate mapping rule that is meaningful to a target dataset, the ibs method can be applied to other types of phenotypic data derived from the time series [16] . second, we employed a visually aided hierarchical analysis by a software tool called a generalized association map, which enables a bottom-up approach to unsupervisedly identify heart rate clusters to study genotype-phenotype associations. this bottom-up approach may help reduce the false-positivity commonly seen in conventional association studies. b 1 -ar is the predominant b-ar subtype in the heart. compared to activation of b 2 -ar, stimulation in b 1 -ar can result in more figure 2 . unsupervised, hierarchical cluster tree of subjects according to the pairwise dissimilarity matrix among heart rate dynamics using an information-based similarity index method. dissimilarity matrix data were visualized and clustered by a generalized association plot algorithm [58] . the bars on the left indicate the b-adrenergic receptor (b-ar) gene polymorphisms rs1801252 (green: homozygous ser49 carriers; blue: gly16 allele carriers; red: unknown genotype), rs1042713 (green: homozygous arg16 carriers; blue: gly16 allele carriers) and rs1042714 (green: homozygous gln27 carriers; blue: glu27 allele carriers). doi:10.1371/journal.pone.0019232.g002 significantly increased frequency and contractility of heart. although substantial efforts have been made to link b 1 -ar polymorphisms with cardiovascular function, studies have been mixed in this regard. for example, b 1 -ar ser49gly polymorphism was found to be associated with increased blood pressure and heart rate [14, 15] but data are inconsistent in our findings and other reports [48, 49] . because we studied only one polymorphism in b 1 -ar, further research is needed to identify other possible b 1 -ar polymorphisms associated with hrv. this study found that b 2 -ar arg16gly genotype was associated with clusters based on acceleration/deceleration patterns of heart rate. although b 2 -ar is expressed in the heart at lower concentrations than is the b 1 -ar subtype, it also expresses on the presynaptic sympathetic nerve terminals and expresses abundantly in vascular and bronchial smooth muscle. therefore, changes in b 2 -ar function may not only alter sympathetic activity [50] , but also respiration and vascular responses [51, 52] . it can be reasonable to speculate that these factors have influences in hrv. of note, the association between b 2 -ar arg16gly polymorphism and heart rate fluctuation patterns may simply reflect a functional genetic component nearby due to linkage disequilibrium and warrants future genetic research targeting on this region. the non-randomness index was developed initially as a nonlinear hrv index [17] and its connection with other traditional hrv indices needs to be explored. though the nonrandomness index was weakly associated with hrv indices (rsquare,0.1; table s3 ), it did correlate negatively with hf power and positively with the lf/hf ratio, suggesting that the non-randomness index is a marker related to sympathovagal balance. our findings of lower non-randomness index and lf% seen in homozygous b 2 -ar arg16 carriers are in line with prior studies showing that the b 2 -ar arg16 polymorphism is associated with lower sympathetic activity, manifested as lower blood pressure [53, 54] , lower plasma norepinephrine [54, 55] , and enhanced agonist-mediated desensitization in the vasculature [56] . strength of this work include a larger sample size, matched gender distribution, and long recording of ecg signals using a holter recorder, compared to prior genetic association study of hrv. several limitations influence the interpretation of the findings presented in this study. first, we are unable to repeat findings of spectral hrv indices seen in a smaller male sample [11] . this may be due to different gender distributions [57] and means of ecg recording. twenty-four hour holter recording could validate and reinforce the association of b-ar genetic polymorphism with hrv seen in this study. second, we were unable to evaluate the correlation of the non-randomness index with other commonly used sympathetic indicators, such as blood pressure or plasma catecholamine levels. third, our findings are based on a healthy population and may not be generalizable to medically ill patients, such as those with cardiovascular diseases. the cross-sectional nature of our study also limited us examining the age modification of the effect of b-ar receptor polymorphism on autonomic function. in conclusion, this study shows that a bottom-up, categorization approach combining the ibs method and hierarchical cluster analysis can detect subgroups of subjects based on phenotypes that are associated with b 2 -ar gene polymorphisms. further research should aim to identify the physiological mechanisms underlying these findings. fractal dynamics in physiology: alterations with disease and aging heritability of heart rate variability: the framingham heart study heritability of ambulatory heart rate variability ethnic differences and 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analysis of two randomised studies arg16gly polymorphism of the beta2-adrenergic receptor is associated with differences in cardiovascular function at rest and during exercise in humans beta2-adrenoceptor polymorphisms relate to insulin resistance and sympathetic overactivity as early markers of metabolic disease in nonobese, normotensive individuals beta2-and beta3-adrenergic receptor polymorphisms are related to the onset of weight gain and blood pressure elevation over 5 years the effect of common polymorphisms of the beta2-adrenergic receptor on agonist-mediated vascular desensitization influence of age, gender, body mass index, and functional capacity on heart rate variability in a cohort of subjects without heart disease methods for simultaneously identifying coherent local clusters with smooth global patterns in gene expression profiles key: cord-001432-d4zavkcn authors: nishida, yoriko; aida, kaoru; kihara, makoto; kobayashi, tetsuro title: antibody-validated proteins in inflamed islets of fulminant type 1 diabetes profiled by laser-capture microdissection followed by mass spectrometry date: 2014-10-16 journal: plos one doi: 10.1371/journal.pone.0107664 sha: doc_id: 1432 cord_uid: d4zavkcn background: there are no reports of proteomic analyses of inflamed islets in type 1 diabetes. procedures: proteins expressed in the islets of enterovirus-associated fulminant type 1 diabetes (ft1dm) with extensive insulitis were identified by laser-capture microdissection mass spectrometry using formalin-fixed paraffin-embedded pancreatic tissues. results: thirty-eight proteins were identified solely in ft1dm islets, most of which have not been previously linked to type 1 diabetes. five protein-protein interacting clusters were identified, and the cellular localization of selected proteins was validated immunohistochemically. migratory activity-related proteins, including plastin-2 (lcp1), moesin (msn), lamin-b1 (lmnb1), ras gtpase-activating-like protein (iqgap1) and others, were identified in cd8(+) t cells and cd68(+) macrophages infiltrated to inflamed ft1dm islets. proteins involved in successive signaling in innate/adaptive immunity were identified, including sam domain and hd domain-containing protein 1 (samhd1), ras gtpase-activating-like protein (iqgap1), proteasome activator complex subunit 1 (psme1), hla class i histocompatibility antigen (hla-c), and signal transducer and activator of transcription 1-alpha/beta (stat1). angiogenic (thymidine phosphorylase (tymp)) and anti-angiogenic (tryptophan-trna ligase (wars)) factors were identified in migrating cd8(+) t cells and cd68(+) macrophages. proteins related to virus replication and cell proliferation, including probable atp-dependent rna helicase dead box helicase 5 (ddx5) and heterogeneous nuclear ribonucleoprotein h (hnrnph1), were identified. the anti-apoptotic protein t-complex protein 1 subunit epsilon (cct5), the anti-oxidative enzyme 6-phosphogluconate dehydrogenase (pdg), and the anti-viral and anti-apoptotic proteins serpin b6 (serpinb6) and heat shock 70 kda protein1-like (hspa1l), were identified in ft1dm-affected islet cells. conclusion: the identified ft1dm-characterizing proteins include those involved in aggressive beta cell destruction through massive immune cell migration and proteins involved in angiogenesis and islet vasculature bleeding, cell repair, and anti-inflammatory processes. several target proteins for future type 1 diabetes interventions were identified. many cascades related to viral infections and innate and adaptive immunity and beta cell responses are postulated to lead to beta cell dysfunctions in human type 1 diabetes and type 1 diabetic rodent models [1, 2, 3] . proteins involved in beta cell destruction have been identified based on in vivo animal model studies of type 1 diabetes [1, 3, 4, 5] . however, the proteins and mechanisms associated with the destruction or defense of beta cells in human type 1 diabetes have yet to be elucidated. furthermore, to date there have been no reports of in situ protein profiling in human inflamed islets affected by type 1 diabetes (insulitis). laser-capture microdissection (lmd) coupled with liquid chromatography (lc)-tandem mass spectrometry (ms) (lmd-lc-ms) is an emerging method useful for profiling proteins in situ [6] . we performed a proteomic analysis of formalin-fixed and paraffin-embedded pancreatic islet tissues of enterovirus-associated fulminant type 1 diabetes (ft1dm), a representative subtype of virus-related human type 1 diabetes in which most of the islets are affected by insulitis [7] [8] [9] [10] [11] . furthermore, we immunohistochemically validated the localization of the identified proteins in type 1 diabetic islets. an important feature of our findings was how selectively many proteins function in affected islets, including remaining islet cells and infiltrating immune cells. the catalog of profiled proteins and the protein-protein network model presented here provide new insights that will both enhance our understanding of the pathogenesis of virus-induced type 1 diabetes and facilitate development of future interventions. in addition, our lmd-lc-ms method is applicable to routine analysis of pathological specimens of formalin-fixed and paraffin-embedded tissues. three pancreatic specimens were obtained at autopsy and fixed with 5% formaldehyde and paraffin-embedded. more than 95% of the islets had extensive mononuclear cell (mnc) infiltration (insulitis) [10, 11] . clinical profiles of the three autopsied patients with ft1dm were reported elsewhere [10] . briefly, case 1 was a 14-year-old boy who died from diabetic ketoacidosis following onset of flu-like symptoms 5 days earlier. case 2 was a 25-year-old man who died from diabetic ketoacidosis following sudden onset of nausea and symptoms of epigastric pain 2 days earlier. case 3 was a 29-year-old man who died from diabetic ketoacidosis following onset of slight fever, nausea, and vomiting 2 days earlier. pancreatic tissues from five autopsied non-diabetic men (2264 years of age, mean 6 sd, range: 18-28 years of age) were used as non-diabetic controls. methods for immunohistochemical analyses were reported previously [10, 11] . the primary antibodies used in this study are listed in table s1 . serial 10-mm-thick sections were prepared from pancreas blocks and attached onto director tm slides (expression pathology, rockville, md). every fifth section was stained with anti-insulin antibody (dako, carpinteria, ca) as described elsewhere [10, 11] . for lmd, the sections were de-paraffinized twice with xylene for 5 min, rehydrated with graded ethanol solutions and distilled water and then stained with hematoxylin alone. stained uncovered slides were air-dried and 6.4,6.8 mm 2 islets area (1,700,2,700 islets) were collected in a 0.2-ml low-binding plastic tube using a leica lmd7000 (leica microsystems gmbh, ernst-leitz-strasse, wetzlar, germany). protein extraction was performed using a liquid tissue ms protein prep kit (expression pathology) according to the manufacturer's protocol. because tissues from ft1dm cases were scarce, islets obtained from three ft1dm pancreata by lmd were combined and analyzed using mass spectrometry. lc-ms analysis of digested samples was carried out essentially as described previously [6] . we used a reversed-phase liquid chromatography (rp-lc) system interfaced with an ltq-orbitrap hybrid mass spectrometer (thermo fisher scientific, bremen, germany) equipped with a nano-electrospray source (amr, tokyo, japan). the rp-lc system (paradigm ms4, michrom bioresources, auburn, ca) consisted of an l-column micro trap (0.365.0 mm) and a capillary separation column (0.16150 mm lcolumn micro packed with reversed-phase l-c18 gel particles of 3 mm in diameter and 12-nm pore size [ceri, tokyo, japan]) fitted with an emitter tip (fortistip, 20-mm id and 150-mm od with a perfluoropolymer-coated blunt end, omniseparo-tj, hyogo, japan). an autosampler (htc-pal, ctc analytics, zwingen, switzerland) was used to load aliquots of samples onto the trap, which then was washed with solvent a (98% distilled water with 2% acetonitrile and 0.1% formic acid) for concentrating peptides on the trap and desalting. subsequently, the trap was connected in series to the analytical column and the columns were eluted for 100 min at a flow rate of 0.3 ml/min with a linear gradient from 5% solvent b (10% distilled water and 90% acetonitrile containing 0.1% formic acid) to 45% b, then from 45% b to 90% b over 5 min, maintaining at 90% b for 5 min, then from 90% b to 5% b over 0.01 min, followed by reequilibration with 5% b for 9 min. the ltq was operated in the data-dependent ms/ms mode to automatically acquire up to three successive ms/ms scans in the centroid mode. the three most intense precursor ions for these ms/ms scans could be selected from a high-resolution ms spectrum (survey scan) previously acquired by the orbitrap during a predefined short time window in the profile mode at a resolution of 30,000 in the m/z range 450 to 1,800. the sets of acquired highresolution ms and ms/ms peptide spectra were converted to single data files and merged into mascot generic format files for database searching. database searching and semi-quantification with spectral counting all ms/ms data were searched against the uniprot/swiss-prot (release 2012_03) database using mascot (version 2.2.06, matrix science, london, uk), in which the peptide and fragment mass tolerances were 10 ppm and 0.8 da, respectively, and up to two missed cleavages were allowed for errors in trypsin specificity. for variable peptide modifications, methionine oxidation and formylation of lysine, arginine, and n-terminal amino acids were taken into account. reported results were obtained from triplicate lc-ms runs for each sample with all peptide hits included. unique peptides and proteins were identified by the following proteomics guidelines. mascot search results were processed through scaffold software (version 3.3.3, proteome software, portland, or) for gene ontology analyses and validation of ms/ ms-based peptide and protein identifications. peptide identifications were accepted if they could be established at a scaffold peptide probability of .95%. protein identifications were accepted if they could be established at a scaffold protein probability of .99% and contained at least two identified peptides. identified proteins were also analyzed in terms of putative functional association networks using the string 9.01 server (http://www.string-db.org). the ethics committee of the university of yamanashi approved all of the procedures performed in this study. witten informed consent was obtained from the next of kin or parents/ guardians on behalf of the children or autopsied cases. the informed consent was written on the form and kept in the medical records. the ethics committee of the university of yamanashi approved the consent procedures. fisher's exact test was used to compare the frequencies of specific immunostaining results between ft1dm-affected and non-diabetic control pancreata. proteins identified by lmd-lc-ms and the protein profile of inflamed ft1dm pancreas tissue we identified a total of 300 different proteins. a total of 193 proteins were identified in the islet mixture from the three ft1dm patients, and 262 proteins were identified in the islets of at least one of the control patients (table 1 and tables s2 and s3) . overall, 38 of the 300 proteins identified (12.7%) ( table 1) were found only in the ft1dm islets, 107 proteins (35.7%) (table s3) were found only in the control islets, and 155 proteins (51.7%) ( table s2) were found in both control and ft1dm-affected islets. most proteins have not been previously implicated as being involved in type 1 diabetes ( table 1 ). the proteome of the control islets included insulin, glucagon, and proteins associated with glycolysis/gluconeogenesis, oxidative phosphorylation, ribosomes, and secretory granules (tables s2 and s3 ). glucagon was identified in ft1dm-affected islets but insulin was not, which was in agreement with our previous pathological studies [10, 11] . in ft1dm, enterovirus infection induces innate immune responses, activates the cxcl10-cxcr3 axis, and induces massive infiltration of autoreactive t cells, dendritic cells, and macrophages, resulting in subsequent severe and rapid beta cell destruction [10, 11] . we therefore sought to identify proteins that promote beta cell destruction in situ using lmd-lc-ms, and we immunohistochemically validated the presence and cellular localization of some of the candidate proteins, as described below. a protein-protein interaction network consisting of 38 proteins unique to ft1dm, and proteins previously identified immunohistochemically in inflamed ft1dm [10, 11] , was constructed for ft1dm-affected pancreas tissue. as shown in figure 1 , there appear to be five protein-protein interacting clusters as determined by string analyses. figure 2 shows the cellular locations of the selected proteins in the islets, and figure 3 summarizes the putative functions and locations of the proteins in ft1dmaffected islets. cluster 1 (lcp1, actr3, iqgap1, msn, flna, lmnb1, uso1/p115) cluster 1 includes proteins that interact with the mnc cytoskeleton and are involved in activation, polarization and infiltration of these cells to the islets. plastin-2 (lcp1) was highly expressed on cd8 + t cells and cd68 + macrophages that aggressively infiltrated to or around the islets in ft1dm tissue (figure 2a -d). lcp1 has been implicated in cell migration [12, 13] . chemokine binding to the receptor results in phosphorylation of lcp1, which induces movement of the f-actin cytoskeleton to the leading edge of the t cell and subsequent activation of cellular polarization and migration of the chemokinestimulated t lymphocytes [12, 13] . thus, aggressive homing of activated cd8 + t cells to affected islets in response to cxcl10 chemokine stimulation in ft1dm can be explained by the increased expression of lcp1. actin-related protein 3 (actr3) was over-expressed in the cytoplasm and nucleus of mncs infiltrated to the islets in ft1dm-affected pancreas tissue. actr3 is a major constituent of the actin-related protein (arp) 2/3 complex and is postulated to be located at the cell surface and to play an essential role in cell motility [14] . ras gtpase-activating-like protein (iqgap1) was highly expressed in infiltrating mncs, islet beta cells, and islet non-beta cells ( figure 2e ). in non-diabetic controls, iqgap1 was weakly expressed in the islet cells ( figure 2f ). iqgap1 regulates cell morphology and motility through interaction with components of the cytoskeleton (i.e., actr3 in cluster 1) as well as cell adhesion molecules and several signaling molecules [15, 16] . similar to lcp1, iqgap1 expression on mncs contributes to the aggressive translocation of mncs to beta cells in ft1dm. moesin (msn) was hyper-expressed in the cytoplasm of infiltrating cd8 + t cells and cd68 + macrophages. moesin plays crucial roles in mediating cell-cell contact between lymphocytes and movement of cells to the target site [14, 17] . this effect on cellcell contact and movement is mediated by the t cell receptor and may play a role in the aggressive infiltration of cells that is characteristically observed in islets affected by ft1dm [7] [8] [9] [10] [11] . filamin a (flna), an actin filament cross-linking protein similar to lcp-1 and initially identified in macrophages, plays a central role in mechanotransduction [18] and is also involved in aggressive t cell/macrophage translocation and infiltration into islets. lamin b-1 (lmnb1) is expressed in the nuclei of mncs that have infiltrated into islets in ft1dm-affected pancreas tissue. the lamins play important roles in providing mechanical support and shape to the nucleus and in regulating many nuclear functions, including dna replication, rna polymerase ii transcription, dna repair, mitotic spindle formation, response to oxidative stress and chromosome positioning [19] . general vesicular transporter factor p115 (uso1, p115) was densely stained in alpha-cells in ft1dm-affected pancreas tissue. uso1 associates with gamma-tubulin and plays a role in golgi structure and mitosis progression [20] . cluster 2 (ddx5, hnrnph1, cct5) cluster 2 included several proteins involved in virus replication. probable atp-dependent rna helicase dead box helicase 5 (ddx5) was over-expressed in the nucleus and cytoplasm of all subsets of islet cells affected by ft1dm ( figure 2g ). in the nucleus of non-diabetic islet cells, staining of ddx5 was faint ( figure 2h ). ddx5 is a member of the asp-glu-ala-asp (dead) box family of rna helicases and interacts directly with viral proteins and is involved in virus production [21, 22] . these findings are in agreement with our previous finding that enteroviruses are capable of infecting and replicating in all subsets of endocrine cells [10, 11] . heterogeneous nuclear ribonucleoprotein h (hnrnph1) was over-expressed in the nucleus of ft1dm-affected islet cells. hnrnph1 is also involved in virus replication [23] . t-complex protein 1 subunit epsilon (cct5) chaperone protein was also over-expressed in the islet cells of ft1dm-affected pancreas. cct5 is a chaperone protein known to play a role in the proper folding of actin and tubulin [24] . cluster 3 included several ribosomal proteins. ribosomal proteins are vital for cell proliferation, apoptosis and survival processes [25] . 40s ribosomal protein s24 (rps24) and 60s ribosomal protein l15 (rpl15) were identified as part of cluster 3. rpl15 also plays a role in innate immune responses stimulated by type i ifn [26] . cluster 4 included several proteins related to viral infection, innate/adaptive immunity, and successive signal transduction. thymidine phosphorylase (tymp) was highly expressed in cd68 + macrophages and cd8 + t cells infiltrating to ft1dmaffected islets ( figure 2i , j, k, l). tymp is a well-known mediator induced by pro-inflammatory and pro-angiogenic conditions [27] [28] [29] . however, there are no reports regarding the involvement of tymp in islet inflammation and beta cell destruction. in many types of cancers, tymp is expressed in the cancer cells and by infiltrating macrophages [27] . tymp is assumed to contribute to angiogenesis by accelerating the migration of endothelial cells. expression of tymp is induced by many inflammatory mediators including ifn-gamma, ifn-alpha, tnf-alpha, and il-1 beta [27, 28] . the jak-stat signaling pathway, probably through stat1 (see below), mediates ifn-gamma-induced activation of macrophages/t cells and exhibits angiogenic effects, such as migration to islet cell clusters and vascular neogenesis with bleeding, which are characteristic pathological feature of ft1dm. this in turn leads to aggressive macrophages/t cell infiltration to the islets and massive destruction of beta cells. tymp is a target of manipulation in oncology, suggesting that it could serve as a target for interventional applications of anti-cancer agents [29] to the treatment of type 1 diabetes. sam domain and hd domain-containing protein 1 (samhd1) was highly expressed in the nucleus and cytoplasm of islet beta cells, non-beta cells, and infiltrating mncs in ft1dm-affected islets ( figure 2m, n) . samhd1 was initially isolated as an ifngamma-induced factor in dendritic cells, and viral infection induces its expression [30] . mutations in samhd1 are associated with aicardi-goutieres syndrome, which resembles a congenital viral infection [31] . serpin b6 (serpinb6) was strongly expressed both in islet cells and exocrine cells in ft1dm-affected tissues but was only weakly expressed in non-diabetic control tissues. serpinb6 is a member of the serpin (serine protease inhibitor) superfamily. serpins are structurally related proteins that inhibit the activity of serine proteases involved in a diverse array of processes, such as inflammation, apoptosis, coagulation, and tumorigenesis [32] . serpinb6 also inhibits microbial and viral proteases [33] . serpinb6 expression is induced by virus infection-associated ifn-gamma production and confers resistance to cytotoxic t cells and nk cells [33] . in the islets of non-obese diabetic (nod) mice, the expression of mrnas of several serpin family members was figure 1 . protein-protein interaction networks involving 38 islet proteins unique to ft1dm-affected tissue that were identified by lmd-lc-ms in this study (yellow labeled) and proteins previously identified immunohistochemically in inflamed ft1dm-affected islets [10, 11] and classified using string (http://www.string-db.org). the five clusters are indicated by numerals. the width of the edges depends on the confidence score for each protein association as determined by string analysis. doi:10.1371/journal.pone.0107664.g001 no expression of tymp was observed in non-diabetic control pancreas tissue (j). triple immunostaining of ft1dm pancreatic tissue for tymp (green), cd68 + (red), and insulin (blue). merged image (k) shows that tymp is expressed on cd68 + macrophages and appears yellow (arrowheads). triple immunostaining of ft1dm-affected pancreatic tissue for tymp (green), cd8 + (red), and insulin (blue). merged image (l) shows shown to be highly upregulated. treatment with the serpin family member alpha1-antitrypsin reduces insulitis and prevents diabetes in nod mice [34] . clinical trials evaluating alpha-antitrypsin are underway (http://clinicaltrials.gov/ct2/show/nct01319331). 6-phosphogluconate dehydrogenase, decarboxylating (pgd), a key enzyme of the pentose phosphate pathway that is responsive to increased oxidative stress [35] , was over-expressed in islet-cells and non-islet cells ( figure 2o ). pgd over-expression is indicative of compensatory responses to increased oxidant activity in the inflamed milieu of ft1dm. signal transducer and activator of transcription 1-alpha/beta (stat1) was highly expressed in islet endocrine cells and mncs in ft1dm tissue ( figure 2q, r) . stat1 is a key transcription factor of antiviral responses and inflammation. stat1 is activated upon phosphorylation by various ligands (i.e., ifn-gamma, ifnalpha). many functions of ifn-gamma have been ascribed to direct stat1-mediated induction of a number of effector genes, including those encoding various immune-modulatory proteins (i.e., mhc class i and mhc class ii), antiviral proteins (i.e., double-stranded rna-activated protein kinase), antimicrobial proteins (i.e., inos), apoptosis-inducing proteins (i.e., irf-1, that tymp is localized on cd8 + t cells (arrowheads). (m)-(n), expression of sam domain and hd domain-containing protein 1 (samhd1) in ft1dmaffected pancreas (m) and control pancreas (n) tissues. hyper-expression of samhd1 (brown) in the nucleus and cytoplasm of islet beta cells (red), alpha cells (green), and infiltrating mncs is shown. no expression of samhd1 was observed in non-diabetic control pancreas tissue (n). (o)-(p), 6-phosphogluconate dehydrogenase, decarboxylating (pgd) expression in ft1dm-affected pancreas tissue (o). pdg (brown) was over-expressed in the cytoplasm of islet-cells, and non-islet cells (arrowheads). pdg was only faintly expressed in the cytoplasm of non-diabetic islet cells (p). (q)-(r), signal transducer and activator of transcription-1 alpha/beta (stat1) expression in ft1dm-affected pancreas (q) and control pancreas (r) tissues. stat1 was over-expressed in the cytoplasm and nucleus of islet beta cells (red), alpha cells (green), and mncs (arrowheads). no staining for stat1 was observed in control islet tissue (r). (s)-(t), proteasome activator complex subunit 1 (psme1, pa28a) expression in ft1dm-affected pancreas (s) and non-diabetic pancreas (t) tissues. psme1 (brown) was over-expressed in the nucleus and cytoplasm of beta cells (red), alpha cells (green), and infiltrating mncs (arrowheads) (s). psme1 was not expressed in non-diabetic control pancreas tissue (t). (u)-(v), tryptophanyl-trna synthetase (wars) expression in the islets of ft1dm-affected (u) and non-diabetic control (v) pancreas tissues. merged image shows expression of wars (brown) in beta cells (red) and mncs (arrowheads) in ft1dm-affected tissue (u). no staining of wars was observed in non-diabetic control pancreas tissue (v). (w)-(x), heat shock protein 70 kda protein 1-like (hspa1l) expression in the islets of ft1dm-affected (w) and non-diabetic pancreas (x) tissues. strong expression of hspa1l (brown) was observed in beta cells (red), alpha cells (green), and other subsets of endocrine cells (w). weak expression of hspa1l was observed in non-diabetic control islets (x). positive staining for each identified protein was significantly more frequent (p = 0.048) in ft1dm-affected islets than control islets. doi:10.1371/journal.pone.0107664.g002 figure 3 . the location and putative function of proteins unique to ft1dm as identified by lmd-lc-ms in this study (red characters) and proteins previously identified by immunohistochemistry (green characters) in ft1dm-affected pancreas tissue [10, 11] . doi:10.1371/journal.pone.0107664.g003 fas/fas ligand), and cytokines (i.e., mig, cxcl10, icam-1, mcp-1) [36] . hla class i histocompatibility antigen-c (hla-c) interacts with cytotoxic t cells to activate them. in enterovirus-induced ft1dm, hla class i molecules are over-expressed in islet cells, and beta cells secrete cxcl10 [10] . cxcl10 expression may recruit islet-reactive t cells [37] . it was also reported that peripheral t cells from some ft1dm patients are reactive against self-antigens such as gad and insulin-b-9-23 [38, 39] . these findings suggest a crucial role for hla-c in both rapid beta cell destruction and the regulation of virus exclusion. proteasome activator complex subunit 1 (psme1, pa28a) and proteasome activator complex subunit 2 (psme2, pa28b) were over-expressed in the nucleus and cytoplasm of islet beta cells, non-beta cells, and infiltrating mncs in ft1dm-affected islets ( figure 2s ). proteasomes are constitutively expressed in almost all cells and function in cleaving peptides in an atp/ubiquitindependent process in a non-lysosomal pathway. psme 1 and 2 are constituents of the immunoproteasome, which is an alternative type of proteasome that plays a central role at the interface between the innate and adaptive immune responses. immunoproteasomes are constitutively expressed in immunity-related cells and somatic cells, including islet beta cells, after stimulation with ifn-gamma and ifn-beta [40] . the immunoproteasome degrades peptides in a ubiquitin-independent manner and is exclusively connected with the adaptive immune response and increased mhc class i antigen presentation. in addition, the immunoproteasome maintains protein homeostasis against ifninduced oxidative stress [40] . we previously reported strong expression of ifn-gamma and ifn-beta in ft1dm-affected islet cells [11] . in response to ifn-gamma-induced oxidative stress, psme1 and 2 will act to maintain cell homeostasis and mhc class i expression. tryptophanyl-trna synthetase (wars) was strongly expressed in the beta cells and in mncs infiltrating the islets ( figure 2u ) in ft1dm-affected tissue. while wars catalyzes the aminoacylation of trnas with tryptophan, they also play roles in splicing, apoptosis, viral assembly, and in regulating transcription and translation [41] . wars expression is strongly induced by ifngamma. wars is secreted by various cell types, and this protein exhibits potent anti-angiogenic activity through inhibition of the expression in endothelial cells of genes encoding proteins associated with angiogenesis, such as akt and no synthetase [42] . negative regulation of angiogenesis induced by viral infection is important in reducing the inflammatory angiogenic response in ft1dm. heat shock 70 kda protein1-like (hspa1l or hsp70t) was over-expressed in the cytoplasm of all islet cell subsets ( figure 2w ). hspa1l is an isoform of 70 kda heat shock protein. in conjunction with other heat shock proteins, hspa1l is also annotated as a protein involved in type 1 diabetes [43] . hspa1l exhibits chaperone activity in the remaining islet cells in ft1dmaffected pancreas, which is in agreement with findings indicating that all subsets of islet endocrine cell are involved in the inflamed milieu and are affected by the degenerative processes in ft1dm [10, 11] . eosinophil peroxidase (epx) is an enzyme that is thought to be involved in host defense in inflammation. leucine aminopeptidase 3 (lap3) was over-expressed in both mncs and the cytoplasm of ft1dm-affected islet cells. lap3 expression is induced by ifn-gamma. lap3 cleaves a single residue from the amino terminus of a peptide to promote antigen presentation by mhc class i molecules. cluster 5 (hist1h213k, hist1h1e, hist1h1b) histone h2b type 1-k (hist1h213k), histone h1.4 (hist1h1e), and histone h1.5 (hist1h1b) are core components of the nucleosome. foxp3 interacts with hist1h1b to alter its binding to target genes in order to modulate their expression and to program the regulatory t cell (treg). apolipoprotein l2 (apol2) was over-expressed in islet endocrine cells. apol2 is stimulated by ifn-gamma and exhibits anti-apoptotic activity in ifn-gamma-induced cytotoxicity [44] . the proteins we profiled in ft1dm-affected islets are primarily expressed in islet endocrine cells and/or cd8 + t cells and cd68 + macrophages infiltrating the islets. we detected expression in islet cells of viral replication-associated proteins typically induced by virus infection and proteins for which expression is induced by ifn-gamma. these proteins exert their major effects through the jak-stat1 signaling cascade, which leads to activation of innate/adaptive immunity, up-regulation of angiogenesis and antiangiogenesis factors, and initiation of cell repair mechanisms through chaperone activity. in cd8 + t cells and cd68 + macrophages in the islets, proteins involved in cell motility and migration to target islets are over-expressed. we hypothesize that through immunological mechanisms and angiogenesis with bleeding in the pancreas, some of the identified proteins play crucial roles in the aggressive beta cell destruction that is a characteristic pathological feature of ft1dm. other molecules we identified have antiviral, cell repair, and anti-inflammatory actions. several proteins that may prove useful in future vaccination or chemical interventions to treat type 1 diabetes were also identified. we could not identify some proteins that were previously detected immunohistochemically, including enterovirus capsid protein (vp1), rig-i, and mda5 [10, 11] . the very low amount of protein present in samples from ft1dm-affected islets may explain our inability to identify these proteins in the present study. proteins present in crude samples at the femtomole (10 215 ) level can be detected using lcm-lc-ms, whereas immunohistochemical staining enables detection of molecules present at the attomole (10 218 ) level, depending on the antisera used. the role of inflammation in insulitis and beta-cell loss in type 1 diabetes progression of autoimmune diabetes driven by avidity maturation of a t-cell population animal models of human type 1 diabetes presumed guilty: natural killer t cell defects and human disease immune cell crosstalk in type 1 diabetes proteomic analysis of laser-microdissected paraffin-embedded tissues: (1) stagerelated protein candidates upon non-metastatic lung adenocarcinoma immunology and immunogenetics of type 1 diabetes in japan a novel subtype of type 1 diabetes mellitus characterized by a rapid onset and an absence of diabetes-related antibodies a novel subtype of type 1 diabetes mellitus enterovirus infection, cxc chemokine ligand 10 (cxcl10), and cxcr3 circuit: a mechanism of accelerated beta-cell failure in fulminant type 1 diabetes rig-iand mda5-initiated innate immunity linked with adaptive immunity accelerates beta-cell death in fulminant type 1 diabetes l-plastin regulates polarization and migration in chemokine-stimulated human t lymphocytes plastins: versatile modulators of actin organization in (patho)physiological cellular processes actin cytoskeletal dynamics in t lymphocyte activation and migration cortactin localization to sites of actin assembly in lamellipodia requires interactions with f-actin and the arp2/3 complex get to grips: steering local actin dynamics with iqgaps moesin interacts with the cytoplasmic region of intercellular adhesion molecule-3 and is redistributed to the uropod of t lymphocytes during cell polarization filgap and its close relatives: a mediator of rho-rac antagonism that regultes cell morphology and migration nuclear lamins is a general vesicular transport factor related to the yeast endoplasmic reticulum to golgi transport factor uso1p host cell interactome of hiv-1 rev includes rna helicases involved in multiple facets of virus production interaction between sars-cov helicase and a multifunctional cellular protein (ddx5) revealed by yeast and mammalian cell two-hybrid systems identification of hnrnph1, nf45, and c14orf166 as novel host interacting partners of the mature hepatitis c virus core protein cellular chaperonin cctc contributes to rabies virus replication during infection ribosomal proteins in cell proliferation and apoptosis a novel interaction between interferon-inducible protein p56 and ribosomal protein l15 in gastric cancer cells interferon gamma-dependent induction of thymidine phosphorylase/platelet-derived endothelial growth factor through gamma-activated sequence-like element in human macrophages interferons upregulate thymidine phosphorylase expression via jak-stat-dependent transcriptional activation and mrna stabilization in human glioblastoma cells thymidine phosphorylase (platelet-derived endothelial-cell growth factor) in cancer biology and treatment inhibition of gip3 expression found in the differential display study on respiratory syncytial virus infection mutations involved in aicardi-goutieres syndrome implicate samhd1 as regulator of the innate immune response serpin structure, mechanism, and function antiviral cytokines induce hepatic expression of the granzyme b inhibitors, proteinase inhibitor 9 and serine proteinase inhibitor 6 alpha1-antitrypsin gene therapy modulates cellular immunity and efficiently prevents type 1 diabetes in nonobese diabetic mice the activity of the pentose phosphate pathway is increased in response to oxidative stress in alzheimer's disease interferon-c: an overview of signals, mechanisms and functions beta cells are responsible for cxcr3-mediated t-cell infiltration in insulitis t-cellmediated autoimmunity may be involved in fulminant type 1 diabetes t lymphocyte response against pancreatic beta cell antigens in fulminant type 1 diabetes immunoproteasomes at the interface of innate and adaptive immune responses: two faces of one enzyme differential regulation of the human, interferon inducible tryptophanyl-trna synthetase by various cytokines in cell lines biologically active fragment of a human trna synthetase inhibits fluid shear stress-activated responses of endothelial cells heat-shock proteins induce tcell regulation of chronic inflammation a novel antiapoptotic role for apolipoprotein l2 in ifn-c-induced cytotoxicity in human bronchial epithelial cells we thank prof. a. nakano, department of immunology, university of yamanashi, for critical reading of the manuscript and prof. r. kato, department of human pathology, university of yamanashi, for help in recruiting age-matched control pancreas donors. we thank dr. taro maruyama for generous help in material sampling, s. osada, c. imai, and k. hosaka for editorial assistance, and h. imai, y. kanemaru, and drs. t. key: cord-001014-8yrpcl94 authors: kitagawa, hiroshi; kawano, mitsuo; yamanaka, keiichi; kakeda, masato; tsuda, kenshiro; inada, hiroyasu; yoneda, misao; sakaguchi, tadashi; nigi, akina; nishimura, koumei; komada, hiroshi; tsurudome, masato; yasutomi, yasuhiro; nosaka, tetsuya; mizutani, hitoshi title: intranasally administered antigen 85b gene vaccine in non-replicating human parainfluenza type 2 virus vector ameliorates mouse atopic dermatitis date: 2013-07-03 journal: plos one doi: 10.1371/journal.pone.0066614 sha: doc_id: 1014 cord_uid: 8yrpcl94 atopic dermatitis (ad) is a refractory and recurrent inflammatory skin disease. various factors including heredity, environmental agent, innate and acquired immunity, and skin barrier function participate in the pathogenesis of ad. t -helper (th) 2-dominant immunological milieu has been suggested in the acute phase of ad. antigen 85b (ag85b) is a 30-kda secretory protein well conserved in mycobacterium species. ag85b has strong th1-type cytokine inducing activity, and is expected to ameliorate th2 condition in allergic disease. to perform ag85b function in vivo, effective and less invasive vaccination method is required. recently, we have established a novel functional virus vector; recombinant human parainfluenza type 2 virus vector (rhpiv2): highly expressive, replication-deficient, and very low-pathogenic vector. in this study, we investigated the efficacy of rhpiv2 engineered to express ag85b (rhpiv2/ag85b) in a mouse ad model induced by repeated oxazolone (ox) challenge. ear swelling, dermal cell infiltrations and serum ige level were significantly suppressed in the rhpiv2/ag85b treated mouse group accompanied with elevated ifn-γ and il-10 mrna expressions, and suppressed il-4, tnf-α and mip-2 mrna expressions. the treated mice showed no clinical symptom of croup or systemic adverse reactions. the respiratory tract epithelium captured rhpiv2 effectively without remarkable cytotoxic effects. these results suggested that rhpiv2/ag85b might be a potent therapeutic tool to control allergic disorders. atopic dermatitis (ad) is a refractory and recurrent inflammatory skin disease. heredity, environmental agent, immunity, and skin barrier function participate in the pathogenesis of ad. ad symptoms are triggered by various non-specific or specific allergic reactions. the cytokine pattern of ad, especially in the acute phase skin lesion is th2-type cytokine dominant [1] . the barrier disrupted skin in ad is easily permitted the percutaneous entry of environmental allergens that strongly promotes th2 immunological responses [2] . th2 cells as well as t regulatory cell (treg) subsets play key roles in development of ad. patients with ad have significantly increased numbers of peripheral blood treg compared with healthy controls, which is correlated with disease activity in ad [3, 4] . this suggests involvement of some self regulation system in immune responses in ad [5] . repeated elicitation with hapten such as oxazolone (ox) on the ear of balb/c mice develops immediate type responses with late phase reactions followed by delayed type hypersensitivity responses. this accompanied with balance shift of cytokines in the lesional skin from th1 to th2 type [6] , and has been utilized as mouse ad. ag85b is 30-kda major secretory protein well conserved in mycobacterium species [7] . the studies for the tuberculosis vaccine revealed strong activities of ag85b in priming naïve t cells for th1 effector cells under the appropriate conditions, and induction of strong th1-type immune responses in mice as well as in humans [8, 9] . recently we reported that plasmid dna vaccination encoding ag85b derived from m. kansasii inhibits immediate-type hypersensitivity responses with treg induction in skin [10] , and a combined vaccination with heat-killed bcg followed by ag85b also suppressed skin eczematous reactions in ad model mice by inducing treg [11] . human parainfluenza type 2 virus (hpiv2) is one of the human respiratory pathogens and a member of the genus rubulavirus of the family paramyxoviridae in the order mononegavirales, possessing a non-segmented and negative-stranded rna genome of 15,654 nucleotides. the genome of hpiv2 encodes 7 mrnas [12] [13] [14] and has about 60-nt leader sequence at 39 end and about 20-nt noncoding trailer sequence. the gene order is 39 (leader)-np-v/p-m-f-hn-l-(trailer)-59. the coding proteins are the nucleocapsid (np), the v (v) and phospho (p), the matrix (m), the fusion (f), the haemagglutinin-neuraminidase (hn), and the polymerase protein (l). the genomic rna of the virus: viral rna (vrna) is encapsidated with the np proteins, and the nucleocapsids are associated with the p and l proteins to form the figure 1 . schematic diagram of constructs and strategy used in this study. a. the constructs of recombinant hpiv2/egfp and hpiv2/ag85b. the egfp or ag85b gene open reading frame was engineered to be flanked by hpiv2-specific gene end of np gene (r2), intergenic sequence (ig), and gene start (r1) transcriptional signal of v/p gene. it was inserted into a cloned cdna of the hpiv2 antigenome at a not i site that had been engineered to be at 59-noncoding region of np gene. a genomic nucleotide length divisible by six (the rule of six) was maintained. for generating of replicationdeficient virus, two stop codons (.) were introduced on the m gene. b. schedule for the development of a hapten-induced atopic dermatitis model and vaccination of rhpiv2/ag85b. mice were initially sensitized with 20 ml of 0.5% ox solution to their right ear 7 days prior to the first challenge (day -7) and then 20 ml of 0.5% ox solution was repeatedly applied on the right ear 3 times per week from day 0 until day 21. mice were inoculated intranasally with 20 ml (5610 6 tcid 50 ) of rhpiv2/ag85b or rhpiv2 on day 4. rhpiv2 vector or phosphate buffered saline (pbs) were also applied as controls. on day 16, mice were vaccinated again intranasally or subcutaneously with pbs, rhpiv2 or rhpiv2/ag85b. c. summarized schedule of the experimental groups. doi:10.1371/journal.pone.0066614.g001 ribonucleoprotein complex. in paramyxovirus particles, vrna is enclosed by the viral envelope composed of a cellular lipid bilayer and two envelope glycoproteins, hn and f, which are integral transmembrane proteins mediating virus attachment and cell fusion, respectively [15] . m protein underlies the lipid bilayer to ensure the structural integrity of the viral particles and is essential for interactions between the viral envelope and the rnp complex [15] . this association leads to the budding and release of viral particles from the cell surface [15] . recently, as technology advances in reverse genetics [16] , hpivs offer several advantages as a vaccine vector. hpivs efficiently infect the respiratory tract but don't spread far beyond it, which is an important safety factor. hpiv-based vectors have proven the effect in inducing local and systemic immunity against a number of foreign antigens [17] . hpivs infect to various cell types and cause little cytopathic effects. moreover, they replicate exclusively in the cytoplasm of infected cells, don't have a dna phase during their life cycle and can thus avoid the possibility of integration of foreign genes into the host dna genome [18] . in the present study, we utilized newly engineered rhpiv2: replication-deficient rhpiv2 vector. rhpiv2 lacks m gene that is an essential gene for virus particle formation by insertion of two stop codons. this alteration might support much safer application to animals than original proliferating virus vector. we first figure 2 . expression of egfp from rhpiv2/egfp. a. hacat cells were infected with rhpiv2/egfp at an moi of 0.5. three days after, egfp was clearly visualized using a fluorescence microscopy (x100). b. the rhpiv2/egfp (5610 6 tcid 50 ) were administered to a wild type balb/c mice intranasally egfp was visualized clearly in the airway epithelial cells 4 days after administration (x200, upper right box, x400). doi:10.1371/journal.pone.0066614.g002 animals balb/c 6-week old male mice were purchased from japan slc co. (shizuoka, japan) and used at 7-week. animal care was performed according to ethical guidelines, and approved by the institutional board committee for animal care and use of mie university. construction of rhpiv2/ag85b and rhpiv2/egfp rhpiv2/ag85b and rhpiv2/egfp was constructed according to the method reported previously, except for methods of the supply of t7 and hpiv2 rna polymerases (np, p, l). in brief, to generate replication-deficient rhpiv2 vector, two nucleotides change [atg to tag (position of 89aa) and aag to tag (259aa)] were introduced into the m frame of the plasmid ppiv2, a full-length cdna copy of hpiv2 anti-genome [19] (fig. 1a) . consequently, the 6 n length cdna of ag85b or egfp, followed by transcriptional end sequence of np gene (r2), intergenic sequence (ig), and transcriptional start signal of v/p gene (r1) ( [20] was synthesized by pcr using appropriate primers), was inserted into a not i site of the plasmid dna encoding the replication-deficient rhpiv2 genome described above. then, the viruses (rhpiv2/ag85b and rhpiv2/egfp) were recovered by co-transfection of each anti-genomic plasmid and plasmids expressing the np, p, m and l, each cloned in a mammalian gene expression vector (pcaggs) [21] into bsr7/5 cells expressing t7 rna polymerase [22] . the cells were harvested, and then co-cultured with fresh vero cells every 48 hr. approximately 90% of the cells showed syncytia formation in the 10 th cocultured cells, and its state was maintained in further co-culture. furthermore, for virus propagation, cos7 cells were transfected with the plasmid expressing m and co-cultured with abovementioned 10 th cells. the supernatant was centrifuged at 9,000 g for 12 h at 4uc. the virus pellet was suspended in opti-mem (invitrogen, carisbad, ca, usa). the virus titers were determined by cpe method using vero cells, and were expressed as 50% tissue culture infectious dose (tcid 50 ). hacat cells (cell line service, eppelheim, germany) were cultured in dulbecco's mem supplemented with 5% (v/v) fbs, 2.0 mm l-glutamine, 100 u/ml penicillin, and 100 mg/ml streptomycin. hacat cells were seeded one day before the transduction at 1610 6 cells/ml (1 ml/well) in 6-well culture plates (costar, ny, usa). the cells were incubated for 8 hours at 37uc in a 5% co 2 atmosphere. the next day, the media was removed and 1 ml of the rhpiv2/egfp viruses were added to the cells to be adjusted to 1610 6 tcid 50 . two hours after infection, the media was removed and fresh culture media was supplemented to the cells. after 3 days culture, each well was observed by fluorescence microscopy. at the next step, 20 ml of concentrated rhpiv2/egfp (5610 6 tcid 50 ) were administered into the cavity of the nose of the mice. four days after infection, the respiratory tract and lung were sampled, embedded in tissue-tek oct compound (miles, elkhart, usa), frozen in liquid nitrogen, and cut into 7 mm-thick sections. sections were examined and recorded by fluorescence microscopy. repeated hapten sensitization and challenge system was introduced in this experiment. ox (sigma, st. louis, mo) was dissolved in acetone/olive oil (1:1). as shown in fig. 1b , mice were initially sensitized by pasting 20 ml of 0.5% ox solution to their right ear 7 days prior to the first challenge (day -7) and then 20 ml of 0.5% ox solution was repeatedly applied on the right ear 3 times per week from day 0 until day 21. repeated application of ox causes delayed type hypersensitivity followed by immediatetype and late phase reaction. for vaccination, mice were infected intranasally under general anesthesia with 5610 6 tcid 50 of the virus in a 20 ml inoculum or phosphate-buffered saline (pbs) on day 4. on day 16, mice were vaccinated again with pbs, rhpiv2/ ag85b or control rhpiv2 vector intranasally or subcutaneously to the pinna skin (fig. 1b,c) . ear swelling was measured with thickness gauge calipers before and 6 hours after last ox challenge on day 21. blood and pinna skins were also sampled. the ear skins were sampled at six hours after last ox challenge on day 21. samples were fixed in 10% neutral buffered formaldehyde and embedded in paraffin. histological sections were of 6 mm thickness and stained with hematoxylin & eosin (h&e). the mrna was extracted from the mouse ear using isogen (nippon gene, tokyo, japan) according to the manufacturer's instructions: one ml of homogenate was mixed with 200 ml of chloroform, and then centrifuged. the aqueous phase was separated and mixed with 0.5 ml of 2-propanol (nacalai tesque, kyoto, japan) to precipitate rna. after centrifuging, the precipitate was washed with 70% ethanol (nacalai tesque) and the rna was suspended in 40 ml of rnase-free water. the concentration of rna was measured at 260 nm absorbent, and the quality was confirmed by electrophoresis. cdna was synthesized from 2 mg of mrna using an archive kit (applied biosystems, foster city, ca, usa) according to the manufacturer's protocol. real time quantitative reverse transcriptionpolymerase chain reaction (rt-pcr) was performed to measure transcriptional activity in skin lesions. a 25 ml reaction mixture containing 1 mg of cdna, 900 nm of each primer, and 250 nm of taqman probe was mixed with 12.5 ml of taqman master mix (ab). quantitative rt-pcr for cytokine transcripts was performed using prequalified primers and probes for il-2, il-4, il-10, il-17a, mip2, tnf-a, tgf-b, ifn-c and gapdh (ab). the on the panel) revealed marked inflammatory reactions with acanthosis and ulceration in epidermis, and marked edema with cellular infiltration including mononuclear cells and neutrophils in the dermis. the skin infiltration of inflammatory cells and epidermal thickness were decreased in rhpiv2/ag85b treated group (ag85b ear and ag85b nasal on the panel). original magnification 6100. d. plasma ige levels on day 21. plasma ige level was decreased in rhpiv2/ag85b treated groups (ag ear and ag nasal). *p,0.05, **p,0.01. doi:10.1371/journal.pone.0066614.g003 dct method was used to standardize the transcripts to gapdh, and the ratio to that of control mice was calculated. the ear skins sampled on day 21 were snap-frozen, and the frozen sections prepared at 7 mm thickness were subjected to a blocking procedure with 5% normal goat serum (vector laboratories, burlingame, ca). sections were then incubated with fitcconjugated rat anti-mouse cd4 antibody (beckman coulter) and pe conjugated anti-mouse foxp3 antibody (biolegend), and examined under fluoview fv1000 laser scanning confocal microscopy (olympus, tokyo, japan). skin infiltrating cd4 + t cells and foxp3 + cd4 + t cells were counted at x100 field, and the numbers in 10 randomly chosen fields of five samples were evaluated. serum ige level was determined by a sandwich enzyme-linked immunosorbent assay (yamasa, tokyo, japan) according to the manufacturer's instructions. optical density of each well was determined by using a microplate reader (multiscan jx, thermo electron, yokohama, japan). statistical analysis was performed using mann-whitney u-test. p,0.05 was considered as significant. to investigate expression levels of the inserted gene in rhpiv2 in vitro, hacat cells were infected with rhpiv2/egfp at an moi of 0.5 and were examined directly using a fluorescence microscopy. the egfp from rhpiv2/egfp was highly expressed in hacat cells, and remarkable fluorescence extended to nearly all the cells in spite of low moi ( fig. 2a) . then, to evaluate the gene expression in vivo, mice were intra-nasally inoculated with rhpiv2/egfp (5610 6 tcid 50 ), and the intense egfp expression was revealed in the lung epithelium of the mice (fig. 2b ). to evaluate the clinically relevant therapies, mice were treated following the strategy shown in fig. 1b . ear lobes of the rhpiv2 (vector alone) or pbs-treated mice developed severe edematous erythema with exudation and erosion at 6 hours after ox challenge on day 21. however, rhpiv2/ag85b-treatment reduced dermatitis in both of the intra-nasal and subcutaneous application groups (fig. 3a) . ear swelling was dramatically suppressed in both of the rhpiv2/ag85b-treated mice compared to pbs or rhpiv2 treated mice (fig. 3b ). pbs or rhpiv2-treated mice showed marked inflammatory reactions with acanthosis and ulceration in epidermis, and marked edema with cellular infiltration including mononuclear cells and neutrophils in the dermis. both of the intra-nasal and subcutane-ous rhpiv2/ag85b application successfully reduced inflammatory cell infiltration and epidermal thickness (fig. 3c ). high levels of ige were detected in sera from pbs or rhpiv2treated mice. on the other hand, the ige levels in the sera from ag85b-treated mice by two ways were suppressed significantly (fig. 3d ). expression of il-4 mrna was significantly decreased in the ear skin of intra-nasally rhpiv2/ag85b treatment group compared to that of pbs treated mice (fig. 4a) . in clear contrast, ifn-c mrna expression was significantly increased in rhpiv2/ag85b intranasally treated group (fig. 4b ). as expected, il-10 levels were significantly increased in intra-nasally treated with rhpiv2/ag85b and rhpiv2-vector groups (fig. 4c) . surprisingly, tgf-b expression is remarkably elevated in the intra-nasal rhpiv2/ag85b group (fig. 4d) . mrna expressions of tnf-a and mip-2 were significantly decreased in both of intra-nasally and subcutaneously rhpiv2/ag85b treated groups compared with pbs or vector treated group (fig. 4e, f) . the expression of il-2 mrna was also significantly elevated in both of the rhpiv2/ag85b intra-nasally and subcutaneously treated groups (fig. 4g) . no obvious suppression in il-17 mrna expression was detected in rhpiv2/ag85b group (fig. 4h ). as shown in fig. 5a , cd4 + t cells are displayed with green fluorescence, and foxp3 + t cells are with red. merged yellow color means foxp3 + cd4 + t cells. the skin infiltrating cd4 + t cells are significantly decreased in ag nasal group and ag ear group compared to that of pbs-treated group. although it does not reach the significance, the cd4 + t cells number is less in ag nasal group compared to ag ear group (fig. 5b) . the foxp3 + cd4 + t cells are significantly increased in both of ag nasal and ag ear groups (fig. 5c ). immune system is finely controlled on the balance of four main subsets of th1, th2, th17, and treg [5] cells. ad, especially in its acute phase, is a disease that th2 cells are dominantly involved in the pathogenesis. in fact pbmcs from patients with ad have increased production of il-4, il-5, and il-13 with limited capacity in production of ifn-c [23] [24] [25] . repeated elicitation of ox on mice ear shifts the cutaneous th1 cytokine milieu to th2, which represents the characteristic immunological features of ad. immunotherapy for ad has some different options to correct the imbalance of the shifted cytokine profile. in the present study, we investigated effects of vaccination using replication-deficient rhpiv2 vector expressing ag85b gene to mouse ad model. bcg is known as a strong th1 response modifier; however, it has a risk for granuloma formation. to avoid granuloma formation, non-wax protein antigen is required. ag85b is a conserved protein in mycobacterial species and can elicit a strong th1-type immune expressions of il-4, tnf-a and mip2-a mrna were significantly decreased in the ear skin treated with intranasally rhpiv2/ag85b treated group compared to those of control groups. meanwhile, the expression levels of mrna of ifn-c, il-10, tgf-b and il-2 were significantly elevated in rhpiv2/ag85b intra-nasally treated group compared to those of control groups. *p,0.05, **p,0.01, ***p,0.001. doi:10.1371/journal.pone.0066614.g004 figure 5 . immunostaining for tregs in the inflamed ear skin lesions. a. cd4 + t cells are displayed with green fluorescence (1), and foxp3 + t cells are with red (2) . merged yellow color means foxp3 + cd4 + t cells (3) (x100). b. the number of skin infiltrating cd4 + t cells is less in ag nasal group and ag ear group compared to that of pbs-treated group. although it does not reach statistical significance, cd4 + t cell number is less in ag nasal group compared to that of ag ear group. c. the number of foxp3 + cd4 + t cells in the inflamed ear skin is significantly increased in both of the intranasal and ear-subcutaneous rhpiv2/ag85b application groups. doi:10.1371/journal.pone.0066614.g005 response [8] . therefore, ag85b has been used as immunemodulator to control acute ad lesions or asthma. ag85b dna vaccine suppressed airway inflammation in a murine model of asthma [26] . furthermore, resent studies suggested that ag85b vaccination promotes th1-type immune responses as well as treg responses. administration of ag85b showed therapeutic effects to th2-type cytokine mediated acute phase ad models by inducing regulatory t cells [10] . selection of the vector and its application pathway has importance in successful dna vaccination therapy. we selected hpiv2 as a potential vector for ag85b vaccination. hpivs are human respiratory pathogens, and the most distinctive clinical feature of infection of hpivs is croup (i.e., laryngotracheobronchitis or swelling around the vocal chords and other parts of the upper and middle airway). among hpivs, hpiv1 and hpiv3 are the major cause of croup in children, whereas hpiv2 is rarely identified as a clinical pathogen. therefore, hpiv2 has been suspected as less virulent and cytotoxic virus. hpiv2 enters the cell by cell fusion at the plasma membrane, and replicates exclusively in the cytoplasm, and buds at the plasma membrane. therefore, hpiv2 has no risk for integration in the host genome, not like retrovirus. in addition, since hpiv2 has a non-segmented and negative-stranded rna genome, there is no antigenic shift among rna segments, not like influenza viruses. using technology of advanced reverse genetics [20] , we constituted replicationdeficient hpiv2 vector with additional advantage as a highly safe virus vector. to confirm the target and effective infection of rhpiv2 vector, we inoculated rhpiv2/egfp to hacat cells. hacat cells successfully expressed egfp up to 7 days postinfection (pi). also, balb/c mice intranasally single-administrated with rhpiv2/egfp showed intense egfp expression in the airway epithelial cells. these results strongly support activities of long-term high-level expression of the exogenous gene and efficiency of rhpiv2 in vivo. in the present study, ad symptoms including ear swelling at late phase reaction were significantly suppressed in rhpiv2/ag85b treated groups in both of intra-nasal and subcutaneous administration. inflammatory cell infiltration including mast cells and eosinophils in the lesional skin was also suppressed. in the cytokine profile, mrna expression of ifn-c and il-2 in the ear skins was significantly increased. interestingly, il-4 mrna was significantly reduced in intranasal rhpiv2/ag85b treated groups. in il-4 suppression and ifn-c induction, intra-nasal application showed stronger effects compared with subcutaneous application. hpiv2 is a virus infectious to the respiratory tract mucosa, and therefore more effective capture of rhpiv2/ag85b by respiratory epithelium compared with that of skin resident cells is reasonable. in addition, the skin derived anti-infectious molecule, horny layer proteases and epithelial skin barrier might decrease efficiency of rhpiv2. treg induction in the effects of rhpiv2/ag85b therapy has importance. present study unveiled augmentation of tgf-b and il-10 expression by intranasal rhpiv2/ag85b. tgf-b and il-10 have been described as critical regulatory cytokines produced by treg. in fact in the current experiment, the numbers of skin infiltrating cd4 + t cells are decreased in the nasal application and ear skin application groups accompanied with increased foxp3 + treg population. a heat-killed mycobacterium vaccae (m. vaccae) gives rise to allergen specific regulatory t cells that produce il-10 and tgf-b, which confer the protection against airway inflammation [27] . recently tgf-b was proved to suppress gata-3 function through sox4 signal, and tgf-b controls th2 cellmediated inflammation [28] . in addition, it is crucial that piv2 itself has some effects in induction of treg without obvious effects in clinical manifestation and th1/th2 balance. in conclusion, the respiratory tract epithelium captured rhpiv2 effectively without remarkable cytotoxic effects. the treatment with rhpiv2/ag85b especially by trans-nasal mucosa approach ameliorates ox-induced ad model by altering th2/th1 cytokine balance with induction of regulatory cytokines induction. thus, nasal rhpiv2/ag85b vaccination is a novel, less invasive and useful therapeutic approach for ad and related allergic disorder. a role for th1 and th2 cells in the immunopathogenesis of atopic dermatitis percutaneous sensitization with allergens through barrier-disrupted skin elicits a th2-dominant cytokine response t regulatory cells in atopic dermatitis and subversion of their activity by superantigens expansion of foxp3-positive cd4+cd25+ t cells associated with disease activity in atopic dermatitis the role of cytokines/chemokines in the pathogenesis of atopic dermatitis repeated elicitation of contact hypersensitivity induces a shift in cutaneous cytokine milieu from a t helper cell type 1 to a t helper cell type 2 profile isolation and partial characterization of major protein antigens in the culture fluid of mycobacterium tuberculosis the immunogenic peptide for th1 development naive human t cells develop into th1 effectors after stimulation with mycobacterium tuberculosisinfected macrophages or recombinant ag85 proteins administration of ag85b showed therapeutic effects to th2-type cytokinemediated acute phase atopic dermatitis by inducing regulatory t cells heat-killed bacillus calmette-guerin and mycobacterium kansasii antigen 85b combined vaccination ameliorates dermatitis in a mouse model of atopic dermatitis by inducing regulatory t cells complete nucleotide sequence of the matrix gene of human parainfluenza type 2 virus and expression of the m protein in bacteria sequence of the fusion protein gene of human parainfluenza type 2 virus and its 39 intergenic region: lack of small hydrophobic (sh) gene sequence analysis of p gene of human parainfluenza type 2 virus: p and cysteinerich proteins are translated by two mrnas that differ by two nontemplated g residues paramyxoviridae: the viruses and their replicatio infectious rabies viruses from cloned cdna mucosal immunisation of african green monkeys (cercopithecus aethiops) with an attenuated parainfluenza virus expressing the sars coronavirus spike protein for the prevention of sars recombinant parainfluenza virus 5 (piv5) expressing the influenza a virus hemagglutinin provides immunity in mice to influenza a virus challenge recovery of infectious human parainfluenza type 2 virus from cdna clones and properties of the defective virus without v-specific cysteine-rich domain characterizations of the human parainfluenza type 2 virus gene encoding the l protein and the intergenic sequences efficient selection for high-expression transfectants with a novel eukaryotic vector generation of bovine respiratory syncytial virus (brsv) from cdna: brsv ns2 is not essential for virus replication in tissue culture, and the human rsv leader region acts as a functional brsv genome promoter decreased frequency of interferon-gamma-and interleukin-2-producing cells in patients with atopic diseases measured at the single cell level the quantitative and qualitative defect of cd4+ cd45ro+ memory-type t cells are involved in the abnormality of th1 immunity in atopic dermatitis patients evidence that defective interferon-gamma production in atopic dermatitis patients is due to intrinsic abnormalities ag85b dna vaccine suppresses airway inflammation in a murine model of asthma suppression of airway eosinophilia by killed mycobacterium vaccae-induced allergen-specific regulatory t-cells the transcription factor sox4 is a downstream target of signaling by the cytokine tgf-beta and suppresses t(h)2 differentiation we thank dr. k.k. conzelmann for bsrt7/5 cells. key: cord-003244-abs3tc3r authors: chong, ka chun; hu, pei; lau, steven; jia, katherine min; liang, wenjia; wang, maggie haitian; zee, benny chung ying; sun, riyang; zheng, huizhen title: monitoring the age-specificity of measles transmissions during 2009-2016 in southern china date: 2018-10-08 journal: plos one doi: 10.1371/journal.pone.0205339 sha: doc_id: 3244 cord_uid: abs3tc3r background: despite several immunization efforts, china saw a resurgence of measles in 2012. monitoring of transmissions of individuals from different age groups could offer information that would be valuable for planning adequate disease control strategies. we compared the age-specific effective reproductive numbers (r) of measles during 2009–2016 in guangdong, china. methods: we estimated the age-specific r values for 7 age groups: 0–8 months, 9–18 months, 19 months to 6 years, 7–15 years, 16–25 years, 26–45 years, and ≥46 years adapting the contact matrix of china. the daily numbers of laboratory and clinically confirmed cases reported to the center for disease control and prevention of guangdong were used. results: the peak r values of the entire population were above unity from 2012 to 2016, indicating the persistence of measles in the population. in general, children aged 0–6 years and adults aged 26–45 years had larger values of r when comparing with other age groups after 2012. while the peaks of r values for children aged 0–6 years dropped steadily after 2013, the peaks of r values for adults aged 26–45 years kept at a high range every year. conclusions: although the provincial supplementary immunization activities (sias) conducted in 2009 and 2010 were able to reduce the transmissions from 2009 to 2011, larger values of r for children aged 0–6 years were observed after 2012, indicating that the benefits of the sias were short-lived. in addition, the transmissions from adults aged between 26 and 45 years increased over time. disease control strategies should target children and adult groups that carry high potential for measles transmission. increased over time. disease control strategies should target children and adult groups that carry high potential for measles transmission. measles is a highly contagious acute viral disease. throughout the world, and most countries have set goals for its elimination. in 1978, the national expanded program on immunization (epi) in china started to implement a standard schedule for the routine administration of one dose of measles-containing vaccine (mcv1) among children between 8 and 24 months of age. subsequently, the mean annual measles incidence decreased from 355 per 100,000 in 1970-1979 to 53 per 100,000 in 1980-1989 [1] . in 1986, a two-dose routine measles immunization program was implemented for children aged between 8 months and 7 years. the age schedule for the second dose of mcv (mcv2) was shifted to 18-24 months in 2005. during 2000-2009, the number of measles cases showed a remarkable decrease but remained around 6.8 per 100,000 on average [2] . in 2006, the government of china set a goal to eliminate measles by 2012, for which purpose a series of programs was implemented, including strengthening routine immunization surveillance, supplementary immunization activities (sias), and casebased surveillance [2] . an sia is defined as the administration of a supplementary dose of a vaccine to a specific age population in a certain area during a short period, regardless of the recipients' previous vaccination histories. sias enhance routine immunization programs, including catch-up campaigns, follow-up campaigns, and outbreak-response immunization. the estimated coverage rate of routine immunization with mcv1 increased from 80.4% in 2000 to 91.1% in 2009, whereas the estimated coverage rate for mcv2 was <80% before 2005 and 84.3% in 2009 [1] . in september 2010, china conducted a synchronized, nationwide sia that targeted children aged 8 months to 14 years, covering 102 million children with a reported coverage rate of 97.5% [1] [2] [3] . although the annual measles incidence had dropped to 0.46 per 100,000 in 2012, it resurged to more than 1.96 per 100,000 in 2013 [3] . despite the implementation of two-dose routine vaccines since 2005, frequent outbreaks have occurred over the past years [4] [5] [6] . guangdong, a highly populated province with 108 million population in 2015, is located in the southernmost part of mainland china (fig 1) . the incidence of measles in guangdong dropped to a remarkable low in 2011 (0.30/100,000) after a series of sias targeting children aged 8 months to 14 years during 2009 and 2010. however, guangdong had the highest number of reported cases in china in 2012 and 2013, with incidences of 1.84 and 6.64 per 100,000, respectively [7] , even though the reported coverage of mcvs was kept above 98% every year after 2009. to this end, the guangdong government implemented some mop-up vaccination programs after 2012 targeting children aged 8 months to 6 years (fig 1) . monitoring the effectiveness of the measles control policy is done by surveillance. in china, measles is a category b infectious disease which indicates it is highly contagious and must be reported to the surveillance system within 24 hours after confirming the laboratory samples [8] . since 2004, china has a direct network reporting system and automatic warning information system for infectious diseases. the system focusses on the number of reported cases, but does not evaluate the transmissibility of measles. the effective reproduction number, r, is a key epidemiologic variable that summarizes the transmissibility of infectious diseases. it is defined as the expected average number of secondary cases produced by an infectious individual in a population in which not all the individuals are susceptible [9] . when r is larger than 1, an infectious individual is expected to infect more than one secondary case. when r is less than 1, an infectious individual tends to infect less than one secondary case, and the incidence will decrease. nevertheless, some infectious diseases have been shown to be strongly age-specific, for example, measles. age-specific r, defined as an average total number of secondary cases from all age groups generated by a single case with respect to his age group was recommended to study the differences in transmission potential taking account of social mixing [10] [11] [12] [13] [14] [15] . although a usual interpretation of age-specific r for gauging the control measures required to eliminate an infection is inappropriate [16, 17] , age-specific rs provide valuable information on the underlying heterogeneous transmission between and within different groups of individuals. for example, glass et al. estimated the rs of pandemic influenza a(h1n1) for children and adults and identified children had a higher transmission then adult cases. in china, the demography of measles infections has changed over time. while infants aged between 9 months and 18 months and young adults aged 16 to 25 years were the primary population of measles infections, children aged 0 to 8 months and adults aged 26-45 years became the primary sources after the national sia. apparently, the age specificity in measles transmissions could be affected by vaccination policies. chong et al. [18] showed that even though substantial decreases in the numbers of cases were observed after mass vaccination campaign, measles could still persist in a population given a high value of r. the majority of the relevant epidemiological studies conducted in china have been based on reported cases and have aimed to describe the incidence and characteristics of population distribution [19, 20] . however, the age specificity in measles transmissions had hardly been studied. in the present study, we compared the age-specific r of measles infections between different age groups by using laboratory and clinically confirmed data collected from 2009 to 2016. daily notifications of measles cases from january 1, 2009 to december 31, 2016 were collected from the national infectious disease monitoring information system (nidmis), as complied by the center for disease control and prevention (cdc) of guangdong province. for some of the cases with typical clinical symptoms, case notifications were sent to the person in charge of reporting by outpatient or resident doctors, and the cases were then recorded as "clinically confirmed". blood samples from these cases were sent to cdc clinical laboratories for confirmation, if laboratory capacity allowed. other cases with atypical clinical symptoms were recorded as "suspected cases," and the blood samples of these cases were subsequently transferred to a diagnostic laboratory to obtain a confirmed diagnosis. the test results were returned to the patients' doctors. if the test results were positive, the cases were relabeled as "laboratory-confirmed cases," and the person in charge of reporting was notified. if the results were negative, the cases were relabeled according to the specific disease that had been detected before handing over to the reporting personnel. for the (clinically confirmed or suspected) cases without laboratory confirmation, epidemiological investigations were conducted to determine whether the patients' infections had any linkage to other confirmed cases within 7-21 days before the onset of any symptom. the epidemiological investigations were performed through direct contacts in the relevant village, community, or school, or through direct contacts for mass gathering events. the clinically confirmed and laboratory-confirmed cases were both regarded as cases, and reporting personnel were required to report such cases to the nidmis within 6 hours. we divided the population into 7 age groups according to the age of onset: 0-8 months (pre-vaccination age), 9-18 months (received the first dose of the measles containing vaccine, mcv-1), 19 months to 6 years (received the second dose of the measles containing vaccine, mcv-2), 7-15 years (primary and secondary school students), 16-25 years (high school and college students/young adults), 26-45 years (mature adults), and !46 years (aged adults). this study was reviewed and approved by the medical ethics committee of the guangdong cdc. the application of the data in this study has been authorized by the guangdong cdc. all data were fully anonymized prior to access by any of the authors and does not involve patients' privacy prior collection. informed consents were exempt from the ethics committee in accordance to the cdc policy of continuing public health investigations of notifiable infectious diseases, in which the patient names, addresses, medical histories with infectious diseases, and their family information will not be disclosed to the public by guangdong cdc in any form. the data are available without restrictions (the link will be provided after the acceptance of the paper). the method for estimating the age-specific effective reproduction numbers suggested by white et al. [10] , which is a modification of the wallinga and teunis approach [21] , was adopted. let p d denote the probability of a serial interval of length d, (d = 1,2,. . .,d where d be the maximum serial interval length), n t;g i denote the frequency of symptom onset in age group g i (i = 1,2,. . .,g where g is the total number of age groups) on day t (t = 1,2,. . .,t where t is the length of the study period), r g i !g j denote the contact rate between two individuals from age group g i and age group g j (j = 1,2,. . .,g). the effective reproduction number of age group g i on day t, r t;g i , can be calculated by summing the expected number of individuals in each age group from t+1 to t+d infected by an individual in age group g i whose symptom onset was on day t: denote the relative probability that an individual in group g j on day t+d was infected by an individual in group g i on day t. in this study, there were 7 age groups (i.e., g = 7) and the maximum serial interval length d was set at 20. p d was generated from a gamma distribution with a mean of 7 days and standard deviation of 3 days [14] . r g i !g j was estimated by using the contact matrix of china, projected by the bayesian hierarchical model in prem et al [22] . the estimation formulas were implemented in microsoft excel. we extended the probabilistic method described in white et al. to generate the statistical uncertainty [15] . a parametric bootstrapping approach was employed to generate 1,000 realizations of fr t;g i g. in each iteration, we generated a new dataset by first simulating the total number of individuals in group g j (j = 1, 2, . . ., 7) infected by those in group g i (i = 1, 2, . . ., 7) with symptom onset of day t (t = 1, 2, . . ., t-1) from a poisson distribution with mean = n t;g i â x minðd;tà tþ d¼1 n tþd;g j â pði t;g i ! i tþd;g j þ, which can be interpreted as the estimated total number of individuals in group g j infected by all the individuals in group g i with symptom onset on day t, where n t;g i and pði t;g i ! i tþd;g j þ were directly obtained and calculated from the original dataset respectively. the simulated number was then distributed within the serial interval t + 1 and t + 20 according to a gamma distribution with a mean of 7 days and standard deviation of 3 days. the above procedure was repeated for all i,j, and t. the resulting data were used to calculate a realization of fr t;g i g and further averaged by months. the 95% credible intervals (ci) of the monthly estimates were obtained from the 2.5 th and 97.5 th percentiles over the 1,000 realizations. apart from china's contact matrix, contact matrixes from 8 other countries were employed to test the sensitivity of our results [23] . we also tried evaluating the estimates using 12 days and 3 days as the mean and standard deviation of the gamma distribution of the serial interval [24] . the r values estimated for children aged 7-15 years were low across the study period in general, even though the values also increased since 2012, indicating that primary and secondary school students had a limited contribution to measles transmissions. similarly, the results for the adults aged !46 years were extremely low across the study period, indicating that these persons were unlikely to infect more than a case on average. the for adults aged between 26 and 45 years, a clear seasonal pattern of r values was observed after 2012, which showed a similar trend to that observed in children. in 2014 and 2015, the estimated annual peaks of r were 1.24 (95% ci: 1.03 to 1.33) and 1.20 (95% ci: 1.01 to 1.39) respectively. given that the r peak of the entire population was significantly above unity in 2016, adults aged 26-45 years had the largest contribution to measles transmissions. s1 fig shows the sensitivity of the results to the use of contact matrices from other countries [23] . in general, the major findings were robust with the variation of contact patterns, for example, children aged 0-6 years still had a large contribution in measles transmissions after 2012. nevertheless, due to a difference in contact frequency, larger estimates were observed for children aged 0-8 months and 9-19 months when using the contact matrix of germany. moreover, while using poland's contact matrix drew a lower estimates for children aged 0-8 we also investigated using a different set of parameters for the distribution of the serial interval, and found that the results were generally consistent with the main analysis (s2 fig). the r values of children groups were slightly increased, whereas the r values of adult groups were slightly decreased. monitoring the age specificity of measles transmissions could provide information that would be valuable to officials who seek to develop adequate disease control strategies. for example, it could help to select appropriate age groups for supplementary vaccination. in this study, we compared the age-specific r of measles infections between different age groups, using laboratory and clinically confirmed data from 2009 to 2016 for guangdong province. according to the results, measles transmissions varied across most age groups before and after 2012 and the large values of r from the entire population indicated a persistence of measles in the population from 2012 to 2016. in general, children aged 0-6 years and adults aged 26-45 years had higher contributions in measles transmissions when comparing with other age groups after 2012. after 2013, while the peaks of r values for children aged 0-6 years dropped steadily by years, the peaks of r values for adults aged 26-45 years remained unchanged and kept at a high range every year, demonstrating the highest contributions in measles transmissions. the findings suggest that disease control strategies should target children and adult groups that carry a high potential for measles transmission. as has been previously noted, we found that children aged 0-6 years had r values that increased after 2012, even though sias targeted this population in 2009 and 2010. the increasing r values could have resulted from low mcv coverage in this cohort, for which the official reported coverage was usually over-estimated [25, 26] . an in-house survey of a similar cohort of children aged 24-47 months showed that mcv1 and mcv2 coverage rates were only 83% and 75%, respectively [25] , results that were inconsistent with the generally reported figure of >98% in china [27] . the geographic heterogeneity of vaccine coverage in china could be another explanation [28, 29] . a chinese study indicated that the measles antibody levels of children aged 2-10 years old were significantly lower for residents of rural areas than for residents of urban areas [28] . the primary reasons why rural children had missed their mcvs were because they were living far from the clinics and because they were unable to access vaccination information [30] . the incomplete immunization records of rural children also made it more difficult for public health officials to track them in order to administer the vaccine. we observed elevated transmissions in infants aged 0-8 months, which may primarily be attributed to the design of the immunization system, which regarded them as too young to be vaccinated by either routine immunization or sias. a longitudinal study of maternal measles antibody titers in infants in guangzhou (the provincial capital of guangdong province) showed that titers among infants decreased rapidly after 3 months of age, and were generally undetectable at 7 months of age [31] . several other studies reported similar results [32, 33] . hence, there was a remarkable immunity gap among children under 8 months old. some studies showed that only around 2.7% to 6.8% of infants are seropositive for measles at 6 months of age [34, 35] . nevertheless, even though infants aged 0-8 months were identified as a high transmissibility group, reducing the minimum age for receiving mcv-1 to 6 months is controversial. we also found that the transmissions from children aged 7-15 years were comparatively low, which was expected given that given that they were the main target of previous sias. moreover, many primary schools implemented screening of children's vaccination certificates and administered supplementary doses of the measles vaccine to fill immunity gaps before the annual entrance [36] . the values of age-specific r for adults aged between 26 and 45 years kept at a high range from 2013 to 2016 and it could be attributed to the lower efficacy of measles vaccines, the low vaccination coverages during 1980s and earlier, and the reduced chance of natural infections. persons aged 26-45 years at the time of the present study were thought to be the first recipients of the vaccination after the approval of routine immunization. liquid vaccines were used for immunization at that time. they had a lower effective dosage and may have resulted in the lower level or shorter protective duration of antibodies among the population. in addition, a functional cold chain, transportation, and communication system for the measles vaccine had not been established at that time; hence, the quality and efficacy of the vaccines could not be guaranteed. secondly, several parents knew nothing about the measles vaccine and underestimated the severity of measles, thereby resulting in a low vaccination rate and a high rate of unsure inoculation history. thirdly, secondary vaccine failure (i.e. measles onset after vaccination and successful seroconversion) due to waning immunity might have occurred among vaccinated adults. although our study could not identify secondary vaccine failure from other cases as serological evidence of previous successful vaccination were lacked, it has been concluded by who that waning immunity has not played a major role in the transmission of measles compared to the absence of initial immunity [37] . the proportion of cases attributable to secondary vaccine failure varied greatly across outbreaks [38] . in a cohort study (n = 2882) in zhuji county of zhejiang province, around 11-13% of those given with single doses of domestic vaccines would become sero-negative (measured by haemagglutination-inhibition tests) after 14 years, yet clinical measles cases rarely happened among them who had humoral immunity waned as they were still protected by secondary immune response [37, 39] . finally, the subsequent sias did not cover these persons; thus, the immunity gaps among people aged 26 to 45 years increased. on the other hand, the transmissions from individuals aged !46 years were the lowest among the age groups studied, even though there was almost no vaccination history in this group. we believe that the majority of these individuals acquired antibodies through natural infection, owing to the highly contagious nature of measles when they were young. moreover, many studies have shown that seropositivity after natural infection persists longer and generates a stronger response than the immunity acquired from vaccination [39] [40] [41] . given the increasing values of r for the entire population observed after 2012, some mopup vaccination campaigns in 2012 and 2013 appear to have had limited effectiveness, even though they aimed to control measles transmission. one reason for this is that rural families usually have a lower level of education and do not fully understand information regarding mop-up campaigns, which results in a lack of initiative to get the vaccine. moreover, some of the susceptibles were migrants, and officials reported difficulties tracking their vaccination histories. although door-to-door notifications, text messages, and telephone notifications have been used to inform migrant families to join mop-up campaigns [42] , it is often difficult to contact these families because of changes to their addresses or phone numbers. from a policy-making perspective, these results imply that for a successful measles control campaign, the public health department should carry out control measures for appropriate age groups. for children between 9 and 18 months old, it is necessary to take measures to improve vaccination coverage, including providing more publicity to improve parents' awareness about vaccination against measles, creating integrated multichannel notifications to inform parents of the vaccination, and strengthening the supervision of kindergartens. for adjustments to the immunization strategy, adult-specific vaccination programs should be considered to fill the immunity gaps among adults, especially for those aged between 26 and 45 years. one of the major limitations in this study is the quality of notification data. indeed, a proportion (~30%) of the notification data in the early phase study (2009) (2010) (2011) was only clinically confirmed which may lead to some misdiagnosis as well as an underestimate of the age-specific rs. nevertheless, more than 95% of cases were laboratory confirmed after 2011. particularly, the reliance on clinically confirmation was more in rural hospitals in which doctors may lack sufficient knowledge on measles diagnosis. the positive predictive value of a clinical definition would also be changed over time as measles has become rarer by time. to minimize the chance of misdiagnosis, the clinically confirmed cases were not only identified by clinical symptoms, but were also investigated with any potential epidemiological association with other laboratory-confirmed cases. moreover, the completeness of the data could be affected by underreporting as some of the parents might have regarded measles as a kind of skin disease or might have confused it with other diseases that involve skin rashes. apart from that, age-specific rs are common to be used as a metric to identify appropriate age groups most responsible for transmission as for a target of interventions [10, 12, 13] . however, when determining the effort required to eliminate an infection, the interpretation of an age-specific r is different from that of an overall r in a heterogeneous population as using the original threshold of unity could lead to an underestimation of target population for interventions [16, 17] . alternatively, roberts & heesterbeek [17] suggested a type-reproduction number which can single out particular subgroup rather than averaging over all subgroups. further works such as generalizability and statistical inference [16] on the alternative measures worth being investigated. in summary, we compared the age specificity in measles transmissions from 2009 to 2016 in guangdong province. although the provincial sias conducted in 2009 and 2010 were able to reduce the transmission rates from 2009 to 2011, larger effective reproductive numbers for children aged 0-6 years were observed after 2012, which indicates that the benefits of the sias were short-lived. in addition, the transmissions from adults aged between 26 and 45 years 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measles cases during the 1997 são paulo epidemic duration of immunity following immunization with live measles vaccine: 15 years of observation in zhejiang province decreasing seroprevalence of measles antibodies after vaccination-possible gap in measles protection in adults in the czech republic immunoglobulin response in serum and secretions after immunization with live and inactivated poliovaccine and natural infection the influence of children's immunization leak replant measures on improving immunization vaccination rate we thank guangdong cdc for providing the data for analysis. we thank the two anonymous reviewers whose comments helped improve and clarify this manuscript. key: cord-001639-p9mbmfaq authors: alfonso-morales, abdulahi; rios, liliam; martínez-pérez, orlando; dolz, roser; valle, rosa; perera, carmen l.; bertran, kateri; frías, maria t.; ganges, llilianne; díaz de arce, heidy; majó, natàlia; núñez, josé i.; pérez, lester j. title: evaluation of a phylogenetic marker based on genomic segment b of infectious bursal disease virus: facilitating a feasible incorporation of this segment to the molecular epidemiology studies for this viral agent date: 2015-05-06 journal: plos one doi: 10.1371/journal.pone.0125853 sha: doc_id: 1639 cord_uid: p9mbmfaq background: infectious bursal disease (ibd) is a highly contagious and acute viral disease, which has caused high mortality rates in birds and considerable economic losses in different parts of the world for more than two decades and it still represents a considerable threat to poultry. the current study was designed to rigorously measure the reliability of a phylogenetic marker included into segment b. this marker can facilitate molecular epidemiology studies, incorporating this segment of the viral genome, to better explain the links between emergence, spreading and maintenance of the very virulent ibd virus (vvibdv) strains worldwide. methodology/principal findings: sequences of the segment b gene from ibdv strains isolated from diverse geographic locations were obtained from the genbank database; cuban sequences were obtained in the current work. a phylogenetic marker named b-marker was assessed by different phylogenetic principles such as saturation of substitution, phylogenetic noise and high consistency. this last parameter is based on the ability of b-marker to reconstruct the same topology as the complete segment b of the viral genome. from the results obtained from b-marker, demographic history for both main lineages of ibdv regarding segment b was performed by bayesian skyline plot analysis. phylogenetic analysis for both segments of ibdv genome was also performed, revealing the presence of a natural reassortant strain with segment a from vvibdv strains and segment b from non-vvibdv strains within cuban ibdv population. conclusions/significance: this study contributes to a better understanding of the emergence of vvibdv strains, describing molecular epidemiology of ibdv using the state-of-the-art methodology concerning phylogenetic reconstruction. this study also revealed the presence of a novel natural reassorted strain as possible manifest of change in the genetic structure and stability of the vvibdv strains. therefore, it highlights the need to obtain information about both genome segments of ibdv for molecular epidemiology studies. infectious bursal disease (ibd), or gumboro disease, is a viral infection that was described for the first time in the 60's in gumboro, delaware, united states [1] and now occurs worldwide. the most important lesions observed in affected animals are lymphoid tissue damage found in the bursa of fabricius [2] . ibd is caused by the ibd virus (ibdv), a non-enveloped virus belonging to the birnaviridae family with a genome consisting of two segments of double-stranded rna (segments a and b) [3] . segment a (3.2 kbp) encodes a precursor polyprotein in a major open reading frame (orf) that is cleaved by auto-proteolysis to yield the mature vp2 (outer capsid), vp4 (protease) and vp3 (inner capsid) proteins [4] . segment b (2.8 kbp) encodes the virus rna-dependent rna polymerase (rdrp) vp1 [5] which exists in the virus particle both as a free protein and as a genome-linked protein; it interacts with the viral genome and the carboxy-terminal region of vp3 [6, 7] . two different serotypes of ibdv have been reported, which can be differentiated by virus neutralisation test [8] . all pathogenic isolates belong to serotype-1 strains [9, 10] . additionally, the serotype-1 strains, based on in vivo studies, molecular and phylogenetic analyses, have also been classified as: attenuated ibdv (atibdv), classical virulent ibdv (cvibdv), antigenic variant ibdv (avibdv) and very virulent ibdv (vvibdv) [11] . in particular, vvibdv strains appeared in belgium during the early 1980s associated with high mortality in young chickens [12, 13] . since vvibdv strains emerged, they have been the source of great economic losses in the poultry industry in many countries. whereby the scientific community has focused special attention related to the emergence and expansion of vvibdv strains [14, 15, 16] . on the one hand, phylogenetic analyses have revealed independent evolutionary histories for the two genome segments (a and b), suggesting that a reassortment event may have played a role in the emergence of vvibdv [14] . on the other hand, analyses based on reverse genetic have evidenced the fact that both genome segments influence in vvibdv's pathogenicity [17] . hence, to conduct molecular epidemiology studies, including sequence analysis for both genomic segments, is an important step to explain the links between emergence, spreading and maintenance of the vvibdv strains around the world. most of the phylogenetic studies based on segment a use the hyper-variable region of vp2 (hvr-vp2) as phylogenetic marker [18, 19, 20] , whereas those few studies including segment b use the complete segment [21, 22] . however, sequencing the full genome of ibdv is still expensive, from both computational and laboratory perspectives. moreover, for many computationally intensive analyses, utilizing the full genome is unfeasible. it would be, therefore, beneficial to use only those genomic regions that contain the highest phylogenetic signal to reduce cost without losing valuable information [23, 24] . in the current study, the reliability of a phylogenetic marker included into segment b (bmarker) was assessed. this b-marker could be applied in feasible molecular epidemiology studies involving both genome segments of ibdv. in addition, based on the reliability of the bmarker results and using phylogenetic inference based on hvr-vp2, the presence of a novel ibdv natural reassortant between segments a and b was reported. the present work also highlights the need to obtain information about the genetic structure of both genome segments of ibdv, to elucidate the causes of the emergence and spreading not only of the vvibdv strains but also of the novel ibdv natural reassortant (between segments a and b) strains. novel ibdv natural reassortant strains have been described in few countries [25, 26] and their effect on the epidemiological behavior of ibdv around the world remains unknown. ethics statement. international standards for animal welfare were used for all animal samples collected, following the regulations for animal sampling of the institute of veterinary medicine (imv), ministry of agriculture (minagri) of the republic of cuba. the protocol was approved by the committee on the ethics of the minagri of the republic of cuba and all efforts were made to minimize suffering of the animals. birds were euthanized using cervical dislocation to collect the samples. the samples were sent directly from the imv to the animal virology laboratory at censa. the imv is the official regulatory body of the republic of cuba; therefore, additional permits were not required. collection, selection and processing of samples. forty-one bursa of fabricius samples previously confirmed ibdv positive, which had been selected for molecular studies [16] were used in the current study. additionally, the atibdv strain used as vaccine was also included in the current work: the commercial vaccine strain "gumboro" (labiofam, s.a., cuba) (http:// www.labiofam.cu/productos/vacuna-gumboro.html) which is currently applied in the vaccination program conducted by cuban veterinary services (reviewed in [27] ). the 41 bursal samples and the vaccine strain were printed on fta cards, suited for the preservation of genetic material and adequate transportation [28] . the fta cards were sent to centre de recerca en sanitat animal, barcelona, spain (cresa) where the laboratory procedures were conducted. the rna of all 42 samples was extracted from fta cards using the method described by moscoso et al. [282] with modifications. briefly: one gram from the spotted areas in the fta cards was cut by a disposable scalpel blade and placed in 1.5 ml microcentrifuge tubes. for each fta paper portion, 200 μl of nuclease free water were added, vortexed and incubated for 10 min at room temperature. the final suspensions were centrifuged at 7500g for 5 min at 4°c. rna was extracted from 150 μl of supernatant recovered using nucleospin rna virus kit (macherey-nagel, düren, germany) following the manufacturer's instructions. rna was eluted in a final volume of 60 μl of nuclease free water. the rna extracted from fta cards was used to amplify a region of the segment b using the primer pair gb2f:5´-gaccaggagtacttcccaaar-3´/gb2r:5´-gtccacttgat gacttgaggt-3´, previously described by lojkic et al. [29] . within this region, the b-marker of 430 bp of length was selected, which is framed between n-terminal and f domains (s1 fig) . both domains have a functional role in vp1 more than structural constrains [30] . the amplification products were visualized by electrophoresis on 1.8% agarose gels stained with ethidium bromide and were cleaned by qiaquick pcr purification kit (qiagen inc., valencia, ca) following manufacturer's instructions. the resulting products were submitted to bi-directional dna sequencing using a bigdye terminator v3.1 cycle sequencing kit following the manufacturer's directions (applied biosystems). sequencing products were read on an abi prism-3100 genetic analyzer (applied biosystems). the sense and antisense sequences obtained from each amplicon were assembled, and a consensus sequence for each gene was generated using the chromaspro v1.5 program (technelysium pvt. ltd., 2009). nucleotide blast analysis (http://www.ncbi.nlm.nih.gov/ blast/blast.cgi) was initially used to verify the identity of each fragment sequence obtained. the sequences were submitted to the genbank database under accession numbers (lk022327-lk022343). different sequence datasets were organized: i) to assess the reliability of the b-marker, a set of 70 complete segment b sequences of ibdv available at genbank database (http://www.ncbi. nlm.nih.gov/) were used (table 1 , sequences denoted). from this sequences dataset two subsets were prepared: one containing the alignment of the entire b segments and the other one containing only the b-marker region. ii) to conduct the classification study, the cuban sequences obtained were used together with reference strain sequences selected (table 1 , sequences denoted). for this study, sequences from the hypervariable region of segment a (hvr-vp2) were also used (s1 table) . finally, iii) to estimate rates of nucleotide substitution per site, per year and the time to the most recent common ancestors (tmrca) of specific groups, only sequences with a known year of collection were included. in all cases, the alignments of the sequences were performed using the algorithm clustalw method included in the program bioedit sequence alignment editor [31] . the software jmodeltest 2.0 [32] was used to estimate the best-fit model using the akaike and bayesian information criteria (aic and bic). the best-fit models for the complete segment b and phylogenetic marker were selected and used for phylogenetic analysis. to remove sequences with a possible recombinant event from the alignment of all sequence datasets, searches for recombinant sequences and crossover regions were performed using geneconv [33] , rdp [34] , maxchi [35, 36] , chimera [35] , bootscan [37] , siscan [38] , 3seq [39] and lard [40] , all implemented in rdp3 beta 4.1 [41] . programs were executed with modified parameter settings determined according to the guidelines in the rdp3 manual for the analysis of divergent sequences (available upon request). recombinant sequences were tested with a highest acceptable p value of 0.05, and bonferroni's multiple comparison correction was used. analyses were conducted twice to ensure the repeatability of results. phylogenetic relationships of the ibdv strains were analyzed using the bayesian inference (bi) and maximum likelihood (ml) methodologies. bayesian inference analyses were performed with the software mrbayes 3.1 [42, 43] . the markov chain monte carlo (mcmc) searches were run with four chains for 10 million generations, with sampling of the markov chain every 100 generations. at the end of the run, the convergence of the chains was inspected through the average standard deviation of split frequencies and the first 25% of the trees were discarded. after discarding the burn-in, the four mcmc chains were combined and summarized on a majority rule consensus tree. the convergence was again assessed on the basis of the effective sampling size (ess) using tracer software version 1.4 (http://tree.bio.ed.ac.uk/ software/tracer/). only a log-likelihood with ess's of > 250 was accepted. a tree with clade credibility was constructed using the posterior probability distribution. the trees yielded from segment b and b-marker were unrooted. the tree yielded from segment a analysis was rooted using the sequence of ibdv serotype 2 with accession number at genbank database m66722. ml trees were computed using the phyml v3.0 [44] , and confidence levels were estimated by 1000 bootstrap replicates. all topologies were tested by the kishino and hasegawa test (k-h) [45] and the shimodaira-hasegawa test (s-h) [46] , which computed the log-likelihoods per site for each tree and compared the total log-likelihoods for each proposed topology, using the pamlv4.3 program [47] . ten thousand replicates were performed using the k-h and s-h topologies tests by re-sampling the estimated log-likelihoods for each site (rell model) [48] . finally, the trees selected were visualized by figtree v1.1.2 [49] . evaluation of the substitution saturation. the loss of phylogenetic information due to substitution saturation was evaluated comparing the complete segment b and the phylogenetic marker. the level of saturation was studied by plotting the pairwise number of observed transitions and transversions versus genetic distance. in addition, substitution saturation was evaluated with xia's test [23] . all these studies were performed using the dambe program [50] . likelihood mapping. the phylogenetic signal of each sequence dataset was investigated by the likelihood mapping analysis of 100,000 random quartets generated using treepuzzle [51] . in this strategy, if more than 30% of the dots fall in the center of the triangle, the data are considered unreliable for phylogenetic inference purposes. evaluation of the phylogenetic-relationship reconstruction. to assess if the b-marker selected contains the adequate phylogenetic signal to reduce cost without losing valuable information, topologies using complete segment b and the phylogenetic marker were constructed following the methodology described above. all topologies obtained using both datasets were compared as described above the dataset of 31 sequences previously selected, was used to generate the beast input file by beauti within the beast package v1.8.1 [52] (freely available at http://beast.bio.ed.ac.uk). rates of nucleotide substitution per site, per year and the tmrca were estimated employing a bayesian mcmc approach. the model selection was performed by estimating model marginal log-likelihood through the path sampling (ps) and stepping-stone (ss) sampling methods described by baele et al. [53] . the estimation of model marginal log-likelihood through the ps and ss for the twelve coalescent demographic models including parametric models (constant population size, exponential growth and logistic growth) and nonparametric models (bayesian skyline plot, bsp) with strict, uncorrelated lognormal distribution (ucld) and uncorrelated exponential distribution (uced) relaxed molecular clocks were calculated (s2 table) . rates of nucleotide substitution per site, per year and the tmrca were also estimated. in addition, a bayesian skyline plot to infer the population dynamics in terms of changing levels of relative genetic diversity (neτ) through time was performed. for bsp analysis data were collected and plotted using graphpad prism software 5.0 (1992-2007, graphpad prism software inc.). an unpaired t test with whelch's correction was performed for statistical analysis of comparison of neτ between groups. in all cases the mcmc chains were run for 100 million generations, in order to obtain an ess > 250, and the first 10% trees were discarded as ''burn-in", as recommended by the beast package manual [54] (freely available at http://beast.bio.ed.ac.uk). convergence was assessed by estimating the effective sampling size (ess) after a 10% burn-in, using tracer software version 1.5 (http://tree.bio.ed.ac.uk/software/tracer/). the tree with maximum log clade credibility was selected and visualized by figtree v1.1.2 [48] . saturation effects were investigated plotting the absolute number of transitions and transversions versus genetic distance for complete segment b and b-marker (fig 1) . the number of observed transversions relative to that of transitions gradually increased with growing divergence, and both datasets resembled a line, indicating that transitions and transversions were not saturated. moreover xia's test did not support saturation for complete segment b and the b-marker (iss < iss.c, p < 0.0001) (fig 1) . the phylogenetic noise in each data set was investigated by means of likelihood mapping. the percentage of dots falling in the central area of the triangles ranged from 6.2% for the complete segment b to 11.7% for the b-marker (fig 1) . none of the datasets showed more than 30% noise, which enabled the use of the b-marker to deduce the phylogenetic signal. the phylogenetic relationships among the ibdv strains were reconstructed based on complete segment b and b-marker sequences by means of ml and bi analyses. both algorithms yielded congruent results showing the same topologies, which was supported by moderate to high confidence values given by the bootstrap percentage and the posterior probability (fig 2) . even though the ml tree yielded from complete segment b was the best, the statistical support for this tree was not significantly different from the bi tree from the complete segment b or the ml/bi trees from b-marker (s3 table) . thus, all topologies obtained from both datasets yielded two highly divergent lineages, corresponding to both recognized vvibdv and non-vvibdv clades (fig 2) . each lineage was strongly supported by the maximum posterior probability value of 1.00 and the highest bootstrap value of 100% (fig 2) . the phylogenetic relationships among the ibdv strains were reconstructed based on b-marker sequences using ml analysis. moderate to high confidence values given by the bootstrap percentage supported the topology yielded (fig 3, left panel) . the ml tree that estimated the phylogenetic relationships between the cuban ibdv sequences and other reference ibdv strains is shown in fig 3 (left panel) . all cuban ibdv field strains, excepting the cuban vaccine strain and the 117/96pir strain, were grouped in the defined lineage corresponding to the vvibdv strains (fig 3, left panel) . therefore, the cuban vaccine strain and the 117/96pir strain were grouped in the defined lineage corresponding to the non-vvibdv strains (fig 3, left panel) . at the same time, the topology yielded from hvr-vp2 showed that the cuban vaccine strain was grouped within the defined cluster corresponding to atibdv strains (fig 3, right panel) . whereas, the remaining cuban field strains were grouped within the defined cluster corresponding to vvibdv strains (fig 3, right panel) . these cuban field strains included the 117/96pir ibdv, which had been grouped within the non-vvibdv strain lineages for segment b (fig 3) . thus, the cuban 117/96pir ibdv strain can be classified as natural segment-reassortant vvibdv-like segment a and non-vvibdv-like segment b. nucleotide and amino acid deduced comparisons were carried out among the sequences of the ibdv cuban field strains obtained in the current study. the nucleotide sequence identities of the b-marker sequences among the 17 ibdv cuban field strains ranged 87.2-100% and the deduced amino acid identities ranged 95.8-100% (table 2) . ps and ss analyses based on b-marker showed an exponential coalescent and an exponential, uncorrelated clock best fitted to our data (s2 table) . the estimated mean (95% hpd) for the substitution rate of the segment b of all populations assessed of ibdv was 7.09x10 -4 (2.56x10 -4 -1.37x10 -3 ) substitutions/site/year (table 3) . nevertheless, both lineages showed different substitution rates for segment b, the estimated mean (95% hpd) of the substitution rate for non-vvibdv lineage was 1.88x10 -4 (1.11x10 -6 -5.18x10 -4 ) substitutions/site/year, and the mean of (table 3) . thus, the substitution rate of segment b for vvibdv lineage was approximately 40 times higher than the substitution rate of segment b for non-vvibdv lineage. the date bayesian phylogenetic tree obtained for the global ibdv strains was characterised by a clear temporal structure; the oldest samples tended to fall closer to the root of the tree, while the most recent samples were located at the most distal tips. the mean tmrca for diversification of both lineages for segment b of ibdv was framed in different dates. the diversification of the non-vvibdv lineage was located at approximately 1917 (95% hpd from 1810 to 1965), while the mean of tmrca for vvibdv lineage was 1981 (95% hpd from 1965-1987) (fig 4) . in this context, the ancestor for the cluster in which cuban strain 117/96pir96 was located was framed around the year 1976, whereas the ancestors for the remaining cuban strains were defined between the years 1985 and 1986 (fig 4) . demographic inference using the bsp model is summarised in fig 5, which essentially plots neτ as a function of time. neτ can be considered a measure of relative genetic diversity that reflects the number of effective infections established by the virus. the bsp for both ibdv lineages showed different patterns for neτ, indicating different epidemiological behaviours for both viral populations (fig 5) . for non-vvibdv lineage, a decrease in neτ from its emergence to early '80s was observed, with a subsequent maintenance in the neτ, suggesting stability in the diversity of this population. on the contrary, an abrupt increase in neτ from the emergence of vvibdv lineage (approximately in 1986) to 1993 was observed (fig 5) , which proves an epidemic behaviour of this viral population during this period. subsequently, a mild increase in neτ was observed until year 2001 followed by a maintenance until 2011, suggesting a stability in diversity of this population. despite of the stability of neτ for vvibdv lineage, the genetic diversity was statistically higher for this lineage than for the non-vvibdv lineage (fig 5) . the sudden and dramatic emergence of vvibdv strains, which have caused high mortality rates in birds and considerable economic losses in different parts of the world since more than two decades [57] , still represents a considerable threat to the poultry industry. the polygenic nature of ibdv pathogenicity, in particular the clear role of both segments (a and b) in vvibdv's pathogenesis [17, 58] , has shown that the characterization of ibdv strains based on both genome segments is essential to understand the epidemiological behavior of this viral agent. nonetheless, the complete sequencing of both segments is impractical in routine practice in diagnostic laboratories. in addition, utilizing the full genome for molecular epidemiology studies would result in costly computational analyses, which would be also unfeasible [59] . evaluation of a phylogenetic marker based on genomic segment b of ibdv therefore, the use of phylogenetic markers for molecular epidemiology studies of ibdv is a useful and needful approach. in the present study, a novel phylogenetic marker (b-marker) included into segment b of ibdv's genome is proposed. the initial selection of b-marker was based on two main aspects: length of the fragment and location. being the b-marker less than 500bp (430bp) of length, this fragment can be easily obtained and sequenced in laboratories with limited resources. likewise, the size of the b-marker facilitates the computational analyses, avoiding delayed results generated by large size fragments (reviewed in: [24] ). in addition, b-marker is framed between nterminal domain and partial f domain of vp1 [30] . the n-terminal domain of ibdv vp1 folds into a mixed α/β structure. this domain has been associated with the protein priming process and interacts with the fingers and thumb domains [60] . the f domain has been linked to the nucleotide recognition and binding process as well as to the rna template binding mechanism [30] . other domains such as domain d, starting from residue 252 (end of b-marker) are involved in structural integrity, and c-terminal domain is highly conserved not only among ibdv strains but also for all members of birnaviridae family [30] . thus, the structural domains in which b-marker is framed have a functional role in vp1 more than structural constrains; therefore, this region could be more influenced by evolutionary changes than possible negative selective restrictions. the reliability of this b-marker to conduct feasible molecular epidemiology studies involving both genome segments of ibdv was also evaluated. furthermore, the global phylodynamic of the ibdv strains was studied based on the consistency of the results obtained for this bmarker. in this context, the genetic diversity of the cuban ibdv strains was also analyzed. besides, the current work revealed the presence of a novel ibdv natural reassortant between segment a from vvibdv strains and segment b from non-vvibdv strains in cuban ibdv population. reliable reconstruction of phylogenies using molecular data can be affected by several factors; one of these critical factors is the saturation of substitutions [61] . when a dataset is saturated, phylogenetic reconstruction may be misled by homoplasious signal [23] , which is evidenced by no further increase in transitions observed despite increasing genetic distance, indicating that multiple substitutions at nucleotide positions have occurred. the gradual increase of transitions/transversions observed with respect to genetic distance for b-marker suggests a lack of saturation for this selected region. correspondingly, xia's test supported the same outcome. xia's test is based on the ratio of observed entropy to the entropy of full substitution saturation defined as the index of substitution saturation (i ss ). if this value is not significantly smaller than the critical i ss (i ssc ) (value at which the sequences start to fail to recover the correct tree), the sequences have experienced severe substitution saturation and should not be used for phylogenetic reconstruction [23] . from the results obtained for b-marker we can infer that this region does not experience substitution saturation and can be used for phylogenetic studies. an important aspect for a successful phylogenetic experimental design is to predict the power of a dataset. phylogenetic noise from fast-evolving sites misleads phylogenetic inference [62] . therefore, identification of optimal levels of noise exclusion reduces the number of topologies that are not significantly worse than the optimal tree and allows a more robust inference of phylogeny and stronger conclusions about character evolution [63] . in our study, the phylogenetic noise associated to the b-marker proposed was just around 4% less, in comparison with whole segment b. this result strongly supports the use of the region proposed as phylogenetic marker since a value less than 30.0% for phylogenetic noise is accepted as reliable in phylogenetic inference [64, 65] . a high consistency between some particular regions and their whole genome in referring phylogenetic relationships can be considered as a good signature of a phylogenetic marker [66] . as a whole, we consider that the b-marker is a reliable phylogenetic marker for ibdv strains since it was able to reconstruct the same tree as the complete segment b of the viral genome. this outcome was also supported by s-h test, which has been proved as powerful indicator of the optimal level of phylogenetic noise reduction and topologies exclusion [62] . thus, the b-marker was able to discriminate between the defined lineages corresponding to the vvibdv and non-vvibdv strains. taking advantage of the reliability of this phylogenetic marker, the cuban ibdv strains were classified for this segment of the viral genome. previously classified vvibdv strains [16] were also classified as vvibdv for segment b. using the b-marker we also estimated the emergence of vvibdv-lineage in the global scenario around 1981. this date matches the range of years proposed by hon et al. [14] , who determined the emergence of tmrca for the vvvp1 sequences in the same year. in our previous work, the expansion of strains carrying hvr-vp2 sequences linked to high virulence of ibdv was fixed starting from iran in 1981 [16] . these results clearly suggest that around 1981, two important events took place. firstly, the emergence of the genetic background very virulent for both segments (a and b) of ibdv. subsequently, these two genetic backgrounds very virulent were put together into a reassorted strain that we know today as vvibdv. the demographic history of the segment b of the ibdv genome for non-vvibdv lineage, showed a trend toward a decrease in genetic diversity, possibly generated by the introduction of effective vaccination programs against classical and low virulent strains from early stages of the discovery of the disease [67, 68] . taking into account that most commercially available conventional live ibdv vaccines are based on classical virulent strains (reviewed in [69] ), the use of vaccine strains with similar genetic background to the field strains could have subjected ibdv's population to repeated bottleneck effects leading to a loss of fitness through a process of natural selection [70, 71] . on the contrary, the demographic history of the segment b of ibdv genome for vvibdv lineage showed a trend toward an initial growth of genetic diversity, possibly generated by the initial emergence of these strains. conventional live ibdv vaccines based on classical virulent strains exhibit only poor efficacy against vvibdvs [69] . thus, ibdv strains with this new genetic background had the possibility to express the potential advantages of their large quasispecies cloud making expedite their establishment and spreading around the world, especially during the first years of their emergence. the maintenance in genetic diversity of segment b for this lineage, suggesting stability in diversity of this population during 2001-2011, could be associated to the application of more strict control measures for these emergent strains during this period. in fact, novel vaccines were developed to be more effective against vvibdv strains, such as subunit vaccines, genetically engineered live ibdv vaccines, dna vaccines and others [69] . however, different factors including reversion to virulence of the vaccine strains [69] , non-sufficient induction of protective immune response [72] as well the intrinsic property of ibdv to evolve quickly, have made the task of controlling ibd by vaccination even more challenging. the expansion of vvibdv strains was linked to a reassortment event of the genome (segment b) of ibdv with a mutant vp2 background, which caused a sudden increase in virulence of these kind of strains [14] . thenceforth, vvibdv strains have been kept antigenically and genetically homogeneous, spreading to most of the countries (with the exception of australia) at least for two decades. however, the recent isolation of vvibdv strains with rare natural segment-b-reassorted [25, 26] have evidenced a possible change in the genetic structure and stability of vvibdv strains. thereby, ingrao et al. [73] suggested that it is probably only a matter of time until vvibdvs are replaced by an emerging strain with new antigenic or pathotypic properties. in fact, he et al. [25] have currently reported that reassortant ibdv strains were dominantly prevalent in southern china during 2000-2012 [25] . in the current work, the strain 117/96pir was defined as a natural reassortant between segments a from vvibdv and b from non-vvibdv. the date of the emergence of this natural reassortant in cuba was estimated to be 1994, after the introduction of vvibdv strains to cuba [16] . in fact, the ancestor for the cluster in which cuban strain 117/96pir96 was located was framed around the year 1976, coincident with the first introduction of the virus in the country [16] . therefore, the reassortment event that originated the strain 117/96pir96 seems to have occurred between the novel vvibdv strains introduced in cuba around 1991 and the atibdv strains, which had been circulating among the cuban poultry population since 1977. the pathogenicity and antigenicity of the strain 117/96pir reassortant is unknown. even though, several studies have confirmed that ibdv reassortants and vvibdv strains possess different biological characteristics [58, 74] . the pathogenicity and antigenicity of the strain 117/ 96pir reassortant need to be further investigated. additional pathogenicity and antigenicity studies would also be required for bf3hab97 and bf24hab11 strains. on the one hand, bf3hab97 strain showed a unique pattern of triplet amino acids 145-147, which has been associated with pathogenicity of ibdv [54] . on the other hand, bf24hab11 strain showed the signature 242d present in non-vvibdv strains suggesting a possible attenuation [29] . therefore, both strains could be useful to better understand the signatures and virulence factors for ibdv. regarding the evolution process of ibdv, our results for segment b showed that the substitution rate for non-vvibdv lineage was lower than vvibdv lineage (1.88x10 -4 per 4.80x10 -3 ). these different rates could be explained by several factors. on one hand, segment b from non-vvibdv lineage has been concomitant with a host for a longer period than vvibdv lineage. hence, non-vvibdv lineage may have a fitness advantage by keeping a narrow population rather than undergoing frequent changes, therefore facilitating a better host-virus equilibrium [75] . on the other hand, the application of different types of vaccines that did not fully protect chickens against infection by vvibdv strains could have induced a "selective noise" with greater chance for beneficial mutations to accumulate. this "selective noise" allows occasional slips from the first lightest mutational loads (originated by the immune response of the host induced by the vaccination) towards an increase of the weight of mutational loads (given by the quasispecies cloud) (reviewed in [76] ) with a consequent increase in the substitution rate. in this study, a powerful assessment of a phylogenetic marker included into segment b of the genome of ibdv was performed. this phylogenetic marker showed to be useful for classification and phylodynamic analyses for molecular epidemiology studies regarding segment b of ibdv strains. evolutionary rates and phylodynamic analyses from b-marker for ibdv showed difference in the mutation rates and the expansion pattern for non-vvibdv and vvibdv lineages. framed in cuba, the present work revealed the presence of a novel ibdv natural reassortant between segment a from vvibdv strains and segment b from non-vvibdv strains in cuban ibdv population. this study also contributed to a better understanding of the emergence of vvibdv strains, describing molecular epidemiology of ibdv using the state-of-the-art concerning to phylogenetic reconstruction approaches. the present work also proved the presence of a novel natural reassorted strain as possible manifest of change in the genetic structure and stability of the vvibdv strains. therefore, it also highlights the need to incorporate the phylogenetically useful information from segment b in the molecular epidemiology studies of ibdv. supporting information s1 fig. structural visualization of the residues translated from b-marker genome region on vp1 crystal. x-ray crystal structures of vp1, crystal structure 2r72 was downloaded from protein data bank; chimera software v1.6.2 was used for visualization. the residues translated from b-marker genome region were expanded from vp1. residues belonging to n-terminal domain are denoted in yellow. residues belonging to f domain are denoted in cyan. the remaining residues translated from b-marker genome region are denoted as heteroatoms. the remains of vp1 structure is maintained in gray. (tif) s2 fig. molecular analysis of the deduced amino acid sequences for the b-marker of the cuban field and reference strains. the pattern of the triplet amino acids 145-147 was framed in red rectangle, the position 242 associated with virulence was also framed in red rectangle. each main lineage (vvibdv and non-vvibdv) and cuban sequences were denoted. (tif) s1 table. ibdv sequences of the hypervariable region of segment a used for classification. table. comparison of topologies obtained for the complete segment b and the b-marker of ibdv using ml and bi methods. li: log-likelihoods, pkh: p value for kh normal test (kishino & hasegawa 1989) , prell: rell bootstrap proportions (kishino & hasegawa 1989) , psh: p value with multiple-comparison correction (mc in table 1 infectious bursal disease history of infectious bursal disease in the usa-the first two decades avian dis biophysical and biochemical characterization of five animal viruses with bisegmented double-stranded rna genomes biochemistry and immunology of infectious bursal disease virus vp1 of infectious bursal disease virus is an rna-dependent rna polymerase the last c-terminal residue of vp3, glutamic acid 257, controls capsid assembly of infectious bursal disease virus autoproteolytic activity derived from the infectious bursal disease virus capsid protein isolation and 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swine fever virus in endemic areas under c-strain vaccination dna vaccination with vp2 gene of very virulent infectious bursal disease virus (vvibdv) delivered by transgenic e. coli dh5alpha given orally confers protective immune responses in chickens infectious bursal disease: a complex host-pathogen interaction genomic sequencing and molecular characteristics of a very virulent strain of infectious bursal disease virus isolated in china reservoir host immune responses to emerging zoonotic viruses the relation of recombination to mutational advance key: cord-000588-3wok0n21 authors: sainz, juan; lupiáñez, carmen belén; segura-catena, juana; vazquez, lourdes; ríos, rafael; oyonarte, salvador; hemminki, kari; försti, asta; jurado, manuel title: dectin-1 and dc-sign polymorphisms associated with invasive pulmonary aspergillosis infection date: 2012-02-27 journal: plos one doi: 10.1371/journal.pone.0032273 sha: doc_id: 588 cord_uid: 3wok0n21 the recognition of pathogen-derived structures by c-type lectins and the chemotactic activity mediated by the ccl2/ccr2 axis are critical steps in determining the host immune response to fungi. the present study was designed to investigate whether the presence of single nucleotide polymorphisms (snps) within dc-sign, dectin-1, dectin-2, ccl2 and ccr2 genes influence the risk of developing invasive pulmonary aspergillosis (ipa). twenty-seven snps were selected using a hybrid functional/tagging approach and genotyped in 182 haematological patients, fifty-seven of them diagnosed with proven or probable ipa according to the 2008 eortc/msg criteria. association analysis revealed that carriers of the dectin-1 (rs3901533 t/t) and dectin-1 (rs7309123 g/g) genotypes and dc-sign (rs4804800 g), dc-sign (rs11465384 t), dc-sign (7248637 a) and dc-sign (7252229 c) alleles had a significantly increased risk of ipa infection (or = 5.59 95%ci 1.37–22.77; or = 4.91 95%ci 1.52–15.89; or = 2.75 95%ci 1.27–5.95; or = 2.70 95%ci 1.24–5.90; or = 2.39 95%ci 1.09–5.22 and or = 2.05 95%ci 1.00–4.22, respectively). there was also a significantly increased frequency of galactomannan positivity among patients carrying the dectin-1 (rs3901533_t) allele and dectin-1 (rs7309123_g/g) genotype. in addition, healthy individuals with this latter genotype showed a significantly decreased level of dectin-1 mrna expression compared to c-allele carriers, suggesting a role of the dectin-1 (rs7309123) polymorphism in determining the levels of dectin-1 and, consequently, the level of susceptibility to ipa infection. snp-snp interaction (epistasis) analysis revealed significant interactions models including snps in dectin-1, dectin-2, ccl2 and ccr2 genes, with synergistic genetic effects. although these results need to be further validated in larger cohorts, they suggest that dectin-1, dc-sign, dectin-2, ccl2 and ccr2 genetic variants influence the risk of ipa infection and might be useful in developing a risk-adapted prophylaxis. invasive pulmonary aspergillosis (ipa) is a life-threatening infection caused mainly by aspergillus fumigatus, an opportunistic fungal pathogen that frequently colonizes respiratory tracts and rapidly spreads to blood vessels and tissues [1] . the incidence of ipa infection has increased in the last years due to the use of immunosuppressive and immunomodulatory drugs and is still causing significant morbidity and mortality worldwide, especially in immunosuppressed and hematopoietic stem cell transplantation (hsct) patients [2] . early recognition of a. fumigatus by myeloid leukocytes is a crucial for down-stream immune response and conidia clearance and, therefore, in the development and progression of ipa infection [3] . myeloid leukocytes are activated by patternrecognition receptors (prrs), which directly recognize fungal cell wall structures and pathogen-associated molecular patterns (pamps) [4] . among the prrs expressed in neutrophils, pulmonary macrophages and dendritic cells, c-type lectin family members such as dc-sign (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin), dectin-1 (dendritic cell-associated c-type lectin-1) and dectin-2 (dendritic cellassociated c-type lectin-2) seem to be important in the effectiveness of innate immune response against a. fumigatus [5, 6] . recent studies have reported that the c-type lectins mediate a. fumigatus recognition, capture and internalization [7] and that their binding to fungal cell wall carbohydrates is highly specific and selective [8] . dc-sign and dectin-1 recognize b-(1-3)-glucans and galactomannans (gm) in the cell wall of a. fumigatus [7, 9] while dectin-2 interacts with a-mannans [10] . however, numerous studies have shown that c-type lectins are not only involved in the recognition of fungal pathogens but also in the induction of antifungal th1 and th17 immune responses [5, 11] . although the mechanisms underlying dc-sign-mediated immune response are still highly speculative, it has been suggested that dc-sign promotes dendritic cell (dc) migration and t-cell activation through the icam-3 binding [12, 13] and modulates tlr signalling by targeting raf-1, which regulates nfkb p65 activation and, consequently the production of pro-inflammatory cytokines [14] . likewise, dectin-1 and dectin-2 induce sykdependent canonical and noncanonical nfkb pathways [15, 16] promoting the production of some pro-inflammatory cytokines (il1a, il1b, il12 and il8), chemokines (ccl2/mcp-1, ccl3/ mip-1, cxcl2/mip-2 and cxcl10) [17] and the il17-mediated neutrophil recruitment [18] . it has also been described that dectin-1 and dectin-2 are able to work in collaboration with tlrs (mainly tlr2, tlr4 and tlr6) modulating immune responses through the production of il6, il12p70 and tnf [3, 16] . these findings support the hypothesis that c-type lectins, cytokines or chemokines are important mediators during infection with a. fumigatus. several polymorphisms in human prrs [19] [20] [21] [22] [23] [24] as well as in some cytokines [25] [26] [27] , chemokines [28] and their receptors [29, 30] have hitherto been associated with an increased risk of invasive aspergillosis in susceptible hosts. based on these observations, the objective of the present study was to investigate the role of tagging and potentially functional single-nucleotide polymorphisms (snps) located within the dc-sign, dectin-1, dectin-2, mcp-1/ccl2 and ccr2 genes on ipa susceptibility. all participants enrolled were caucasian and recruited in the university hospital virgen de las nieves (granada, spain) or in the university hospital of salamanca (salamanca, spain). all determinations and genetic analyses in hematological patients were performed with fully informed written consent, and anonymity of the data was guaranteed. the study protocol was approved by the ethical review committee of virgen de las nieves university hospital, granada, spain. the population included 182 hematological patients recruited between january 2004 and january 2011. all hematological patients in this study received a prolonged chemotherapy treatment or underwent hsct and were therefore considered susceptible to develop ipa infection. demographic information and clinical data were obtained by detailed review of hospital records. data were gathered on: site of infection; host factor criteria (severe neutropenia for .10 days, persistent fever for .96 h refractory to appropriate broad-spectrum antibacterial treatment, signs and symptoms indicating graft-versus-host disease [gvhd], corticoid therapy [.0.3 mg/kg per day], and invasive fungal infection during a previous episode of neutropenia), microbiological criteria (positive result for aspergillus antigen in $2 consecutive blood samples when considering an index of 0.5 or in only 1 sample when the index was higher than 0.8), and clinical criteria of lower respiratory tract infection [major criteria: any of the following new infiltrates on computed tomography (ct) imaging: halo sign, aircrescent sign, or cavity within area of consolidation; minor criteria: cough, thoracic pain, hemoptysis, pathologic pulmonary sound, and radiological evidence suggestive of invasive infection]. laboratory data were also recorded. proven and probable ipa was diagnosed based on the updated criteria (2008) reported by the european organization for research and treatment of cancer/invasive fungal infections cooperative group (eortc/ ificg) [31] . serum gm detection has been shown to be a useful test for the early diagnosis and follow-up of ipa and is now included in ipa diagnosis criteria [31] . in the present study, serum gm antigen was determined twice weekly during the hospital stay and at each outpatient visit until the end of their immunosuppressant or chemotherapeutic treatment. serum gm concentrations were determined by platelia aspergillus elisa kit (bio-rad, marnes-la-coquette, france) according to the manufacturer's instructions. this commercial kit has proven to offer good sensitivity to detect gm [32] , and gm concentration was found to correlate with the fungal tissue burden [33, 34] . a test sample was classified as positive when the optical density ratio was $0.5 in two consecutive positive samples or .0.8 in a unique serum sample. a careful review of concomitant treatments (piperacillin-tazobactam or amoxicillin-clavulonic acid) in each patient was necessary to detect possible false-positive gm determinations. likewise, tests were performed on the same day to avoid sample contamination and to ensure accuracy of results. twenty-seven tagging/functional snps within dc-sign, dectin-1, dectin-2, ccl2 and ccr2 were selected to genotype the entire panel of individuals ( table 1 ). the aim of the snp tagging was to identify a set of snps that efficiently tags all the known snps while the functional approach was used to determine the net impact of potentially functional variants within dc-sign, dectin-1, dectin-2, ccl2 and ccr2 genes on ipa risk. tagging snps were selected using haploview tagger program (http://www.broad.mit.edu/ mpg/haploview/; http://www.broad.mit.edu/mpg/tagger/) and a pairwise tagging with a minimum r2 of 0.8. in this selection we forced the inclusion of the dc-sign rs4804803 , ccl2 rs1024610 and ccl2 rs1024611 polymorphisms as their functionality has been suggested [35] [36] [37] . genomic dna was extracted from peripheral blood mononuclear cells (pbmcs) qiagen mini kit (qiagen, valencia, ca, usa). genotyping of dc-sign, dectin-1, dectin-2, ccl2 and ccr2 polymorphisms was carried out using kaspar assays (kbiosciences, hoddesdon, hertfordshire, uk) in a 384well plate format (applied biosystems, foster city, ca, usa) where hematological patient samples were randomly distributed. kaspar reactions were performed using kaspar assay mix (containing probes) and kaspar kit containing 26 reaction mix and mgcl2 (50 mm). pcr conditions were: denaturation at 94uc for 15 min, 10 cycles of denaturation at 94uc for 10 sec, annealing at 57uc for 5 sec and elongation at 72uc for 10 sec and 20 cycles of denaturation at 94uc for 10 sec, annealing at 57uc for 20 sec and elongation at 72uc for 40 sec. recycling conditions were 94uc for 10 sec, annealing and elongation at 60uc for 60 sec. pcr products were analyzed with the abi prism 7900ht detection system using the sds 2.4 software (applied biosystems). for internal quality control, about 5% of samples were randomly selected and included as duplicate. concordance between the original and the duplicate samples for the 27 snps analyzed was $99.5%. call rates for all snps were $97.8% with the exception of the dectin-1 rs11053599 snp with a call rate of 94.5%. the hardy-weinberg equilibrium (hwe) tests were performed in the control group (non-ipa patients) by a standard observedexpected chi-square (x 2 ) test at each polymorphic site (http://ihg2. helmholtz-muenchen.de/cgi-bin/hw/hwa1.pl). unconditional logistic regression was used to assess the main effects of the genetic polymorphisms on ipa risk using co-dominant, dominant and recessive inheritance models. for each snp, the more common allele in the control group was assigned as the reference category. all analyses were adjusted for age, gender, hematological malignancy and established risk factors for ipa infection (hsct, neutropenia, gvhd and corticoid therapy use) and were conducted using the statistical software ssps (version 14.0, spss inc., chicago, usa). all tests were considered to be statistically significant with a p value of ,0.05. adjustment for multiple testing was carried out following a conservative threshold for statistical significance, based on a revised version of the bonferroni method: we calculated for each gene the ''number of effective independent variables'' (m eff ) by use of the snp spectral decomposition approach (http://gump.qimr. edu.au/general/dalen/snpspdsuperlite/) [38] . we obtained a study-wise m eff value by adding up the gene m eff 's. snptool (http://www.dkfz.de/de/molgen_epidemiology/tools/ snptool.html) [39] and the haploview v4.2 software were used for ld blocks reconstruction and haplotype association statistics. block structures were determined according to the method of gabriel et al. [40] . we used the web-based tool fastsnp [41] available at http:// fastsnp.ibms.sinica.edu.tw for predicting the functional significance of the snps associated with ipa infection. fastsnp utilizes information from another online program polyphen (http://www. bork.embl-heidelberg.de/polyphen/) and from four different web resources (tfsearch, esefinder, rescue-ese and fas-ess) to determine whether snps are located at exonic splicing regulatory sites, cause a non-conservative amino acid change or whether they alter the transcription factor-binding site of a gene (for instance, acting as intronic enhancer). the score was given by this tool on the basis of levels of risk with a ranking of 0 (no effect), 1 (very low), 2 (low), 3 (medium), 4 (high), or 5 (very high). whole blood samples from 21 healthy donors were collected into paxgene rna tubes and stored at 280uc until use. total rnas were extracted using a paxgene blood rna isolation kit (preanalytix) and reverse transcribed to cdna using quantitect reverse transcription kit (catalog number: 205311; qiagen). real-time quantitative pcr was carried out using an abi prismh 7500 ht sequence detection system (applied biosystems) according to manufacturer's instructions. briefly, 2 ml of the cdna were loaded in a pcr reaction containing 12.5 ml of 26 quantitect sybr green pcr master mix with an appropriate concentration of mgcl 2 , 2.5 ml of primers (hs_cle-c7a_1_sg quantitect primer assay, catalog number: qt00024059; geneglobe, qiagen) and 8 ml of rnase-free water. pcr cycling conditions were as follows: 95uc for 15 minutes, followed by 40 cycles of denaturation at 95uc for 15 seconds combined with annealing at 60uc for 30 seconds and extension at 72uc for 30 seconds. all samples were run in duplicate. relative quantification of dectin-1 mrna expression was calculated with the 2 2ddct method. we obtained the fold changes in gene expression normalized to an internal control gene (gapdh, hs_gapdh_2_sg quantitect primer assay, catalog number: qt01192646; qiagen) and relative to one calibrator (first we also analyzed high-order interactions between snps using the multifactor dimensionality reduction (mdr) constructive induction algorithm. a detailed explanation on the mdr method has been described elsewhere [42, 43] . snps in ld (cut-off of r 2 ,0.8) were excluded from the mdr analysis. cross-validation and permutation testing were used to identify the best models. all possible two-, three-and four-way snp interactions were tested using 100-fold cross-validation and the exhaustive search. the model with the highest testing balanced accuracy (ta) and cross validation consistency (cvc) was selected as ''best model''. statistical significance was evaluated using a 1.000-fold permutation test to compare observed testing balanced accuracies with those expected under the null hypothesis of no association (using the mdr permutation testing module 0.4.9 alpha). mdr results were considered statistically significant at the 0.05 level. finally, interactions were visualized by constructing an interaction dendrogram according to the method described by moore et al [43] . mdr software and mdr permutation testing module are open-source and freely available from http://www.epistasis.org. population characteristics are described in table 2 . compared to non-ipa, ipa cases were more likely to have cough and pathologic pulmonary sound (p,0.001 and 0.011, respectively) and presented more often pathological chest radiographies and ct scans (p,0.001). established risk factors for ipa infection such as hsct, neutropenia, gvhd and corticoid therapy use were homogenously distributed between ipa and non-ipa patient groups. fifty-seven patients were diagnosed with proven or probable ipa infection according to the revised eortc/msg criteria (2008). the association of risk of ipa infection with the individual 27 snps in the c-type lectin and chemokine genes is shown in the table s1 . all analyzed polymorphisms fulfilled hardy-weinberg expectations for the control group (non-ipa patients). several polymorphisms were found to be associated with ipa infection ( table 3) table 3 and table s1 ). after correction for multiple testing using snpspd (number of independent marker loci, 21; p = 0.05/21 = 0.002), none of the snps retained significance although dectin-1 rs7309123 showed significance when the carrier status was analyzed (cc+cg vs. gg, p = 0.001; table 3 ). in order to assess the degree to which the selected snps and the positivity of gm correlated, we also performed lineal regression analysis. in the whole population, 3784 assays were performed in duplicate and 531 were considered as positive (14.03%). we found a significantly higher percentage of positive gm among patients carrying the dectin-1 rs3901533_t allele and among those patients bearing the dectin-1 rs7309123_g/g genotype suggesting a role of these polymorphisms in determining a defective recognition and clearance of aspergillus conidia (figure 1 ). no correlation was observed for the polymorphisms within dc-sign that were associated with the infection. we subsequently assessed whether snps associated with ipa infection showed capacity to change putative transcription factor binding sites using fastsnp. the predictive functional analysis suggested an intronic enhancer function for the dectin-1 rs7309123 snp due to its location in transcription factor cdxa (caudal type homeo-box transcription factor 1) binding site (gaaagac; score 1-2). these data suggest a central role of the rs7309123 in the susceptibility to ipa infection. none of the remaining snps associated with ipa infection was predicted to affect transcription factor binding sites or splicing or to introduce a damaging amino acid change. to explore the potential consequences of dectin-1 rs7309123 snp, dectin-1 mrna expression was measured in 21 healthy donors and was correlated with genotypes. our results showed that subjects harbouring gg genotype showed a significantly decreased level of dectin-1 mrna expression compared to c-allele carriers (p,0.001; figure 2 ). these results further supported our hypothesis suggesting that dectin-1 rs7309123_g allele may disrupt binding sites for potential transcription factors. the mdr analysis of the snps tested revealed statistically significant interactions. two snps (rs1024611 and rs4264222) were excluded from the mdr analysis as they were in ld with other snps using a cut-off value of r 2 ,0.80 in our sample set. the best interaction models selected by the turf filter algorithm along with its testing accuracy and cross-validation consistency are shown in table 4 . the overall best model with the highest crossvalidation consistency (cvc) consisted of a model that included the ccl2 rs4586 , dectin-1 rs3901533 , ccr2 rs3918358 and dectin-2 rs7134303 snps. this model had a significant testing accuracy of 0.7735 (permutation p,0.001) and a cross-validation consistency of 100/ 100. of note is that two snps showing genetic interaction in this model were not significantly associated with an increased risk of ipa infection in the univariate analysis (ccr2 rs3918358 and dectin-2 rs7134303 ). the best two-locus model consisted of the dectin-1 rs3901533 and dc-sign rs4804800 snps, two variants that showed also association in the single-locus analysis. this model had a testing accuracy of 0.6409 and a cross-validation consistency of 76/100. this model was not any more significant after 1.000-fold permutation testing. however, the entropy based information gain calculated for this pair of snps indicated strong synergy, which may be interpreted as the two snps acting together to increase the risk of ipa infection. the best three-locus model included the ccl2 rs4586 , dectin-1 rs3901533 and dectin-2 rs7134303 snps. in this model, testing accuracy raised to 0.7085 (permutation p = 0.025) whereas the cross-validation consistency was of 68/100. figure 3 illustrates an interaction dendogram that summarizes the estimates of interactions. the marked differences in susceptibility to ipa infection among hematological patients (with or without allo-hsct) suggest that the effective immune response against aspergillus is determined by both environmental and host genetic factors [44, 45] . studies on genetic polymorphisms in genes coding for components of the innate immunity have supported this hypothesis [19, [23] [24] [25] [26] [27] [28] [29] [30] 46] . in this report, we studied the influence of the tagging and potentially functional polymorphisms of dc-sign, dectin-1, dectin-2, ccl2/mcp-1 and ccr2 in susceptibility to ipa infection in a spanish population. polymorphisms in these genes have been reported to influence a number of infectious diseases including hiv-1 [47] [48] [49] , htlv-1 [50] , cmv [51] , tuberculosis [52] [53] [54] [55] , hcv [56, 57] , hbv [58] , dengue [35] and sars [59] among others, revealing their potential role in host defense against pathogens. we found that polymorphisms in dectin-1 (rs3901533 and rs7309123) and dc-sign (rs4804800, rs11465384, rs7248637 and rs7252229) were associated with an increased risk to develop ipa infection, which points towards their critical involvement in the pathogenesis of this invasive fungal infection. the highest risk of ipa infection was found for carriers of the dectin-1 rs3901533_t/t and dectin-1 rs7309123_g/g genotypes and the dc-sign rs4804800_g allele. patients carrying these genotypes/alleles had from 2 to 6 times increased risk of ipa infection. additionally, patients carrying the dc-sign rs11465384_t , dc-sign 7248637_a and dc-sign rs7252229_c alleles showed a 2-fold increased risk in comparison with patients harboring the wild-type allele. although it was not statistically significant, we also found that the dc-sign rs2287886 snp may be associated with a reduced risk of ipa infection. interestingly, this latter result was in agreement with our previously reported findings using the former eortc/msg classification criteria, 2005 [60] . the apparent effect of these snps on ipa susceptibility persisted even after adjustment for age, gender, hematological malignancy and several known risk factors (hsct, neutropenia, gvhd and corticoid therapy use), indicating that dectin-1 and dc-sign variants contribute independently to the risk of infection. another interesting finding of this study was the significantly greater positive gm percentage of patients carrying the dectin-1 rs3901533_t allele than those with the wild-type allele. additionally, patients harboring the g/g genotype for the dectin-1 rs7903123 snp showed an increased percentage of positive gm compared to those carrying the c allele (c/c+c/g). no differences were found when positive gm determinations were correlated with dc-sign polymorphisms. these data along with the remarkable degree of association of dectin-1 and dc-sign variants with risk of ipa infection provides a compelling evidence for a critical role for these prrs in immune response to ipa infection. in this regard, numerous studies have shown that dectin-1 and dc-sign are not only involved in the recognition of fungal pathogens but also in the induction of anti-fungal th1 and th17 immune responses [5, 11] . mezger et al. also demonstrated that dectin-1 is involved in the induction of several pro-inflamatory cytokines, chemokines and immune receptors [18] while werner et al. showed that dectin-1 is also regulating th17-mediated immune response in the lungs [61] . furthermore, dennehy and brown suggested a role of dectin-1 mediating its own signaling, as well as synergizing with tlrs to trigger nfkb-mediated immune response against fungal pathogens [62] . although it is now well recognized that snps in genes modulating immune response are likely to be determinants of host susceptibility to fungal infections, so far, little is known regarding the biological significance of these variants. in order to shed light into the potential functionality of dectin-1 (rs3901533 and rs7309123) and dc-sign (rs4804800, rs11465384, rs7248637, rs7252229 and rs2287886) variants, we investigated whether they were involved in disruption of a binding site for critical transcription factors that might influence transcription level of these genes. our predictive analysis showed that the carriage of the c allele for the dectin-1 rs7309123 snp creates a putative binding site for cdxa, a relatively unknown transcription factor, which might be involved in the control of dectin-1 gene expression. to assess whether the dectin-1 rs7309123 polymorphism might be associated with a decreased expression of dectin-1, we correlated dectin-1 mrna expression levels with dectin-1 rs7309123 genotypes. interestingly, we observed that individuals harbouring the gg genotype showed a relatively lower expression than those carrying the c allele (cc+gc). these results further supported our hypothesis suggesting that dectin-1 rs7309123 snp may have an effect on the dectin-1-mediated recognition of aspergillus conidia and subsequent immune responses. however, this predicted change in transcription activity is only suggestive at this stage and will need further validation using in vitro functional assays. several lines of evidence point to the relevance of epistatic effects in the etiology of complex diseases but, up to now, no studies have been carried out to analyze the presence of snp-snp interactions in ipa infection. for this reason, we decide to assess interactions among genetic polymorphisms within dc-sign, dectin-1, dectin-2, ccl2 and ccr2, genes and the risk of ipa infection. the mdr approach used in this study was able to determine two multilocus combinations associated with high risk to develop ipa infection. of the interactions identified, mdr indicated that the type of interaction in the two significant models was synergistic. these results support the hypothesis that multiple snp-snp interactions may play a role in determining the risk of ipa infection. this hypothesis is biologically plausible since the immune system would warrant prevention of fungal infection even when some genetic variants were present. recent population-based studies have even led to the identification of several snps involved in the early recognition of aspergillus and associated them with an increased risk for invasive fungal infection. it has previously been suggested that snps within c-type lectin genes are associated with fungal infections. platinga et al. (2009) and cunha et al. (2010) suggested that patients carrying the y238x (rs16910526) polymorphism in the dectin-1 gene were more likely to be colonized with aspergillus and candida species [19, 63] , compared with those harboring the wild-type allele. however, these results were not replicated in a very recent study [24] . in our study, such findings were neither evidenced even when hsct and non-hsct patients were analyzed separately (data not shown). this puzzling finding suggests that, although intronic polymorphisms within dectin-1 and dc-sign are indeed themselves a strong indication that these genes play an important role in the susceptibility to ipa infection, we cannot rule out the figure 3 . interaction dendrogram generated by the mdr software. the interaction dendrogram was used to confirm, visualize, and interpret the interaction model. the mdr analysis was performed by using the open-source mdr software package. the colors used depict the degree of synergy, ranging from red (highest information gain) to blue (highest information redundancy). note that the interaction between dectin-1 (rs3901533) and dc-sign (rs4804800) snps showed the highest degree of synergy (gain of information). doi:10.1371/journal.pone.0032273.g003 possibility that these snps are part of a bigger haplotype containing important other genetic variants in the neighboring genes. in any case, because all these population-based studies have been conducted using relatively small cohorts, additional studies in larger set of patients are needed to definitely establish the role of these variants in the susceptibility to invasive fungal infection. in summary, this study provides evidence of association between dectin-1 and dc-sign polymorphisms and the risk of ipa infection. by the inclusion of functional prediction analyses, the correlation of genotypes with positive gm determinations and dectin-1 mrna expression levels, the study strongly supported the role of dectin-1 gene variants in determining susceptibility to ipa infection. epistatic analyses also suggested the presence of a gene-gene interaction involving dectin-1 with ccl2 and ccr2 variants to determine ipa infection. despite all these evidences, additional studies using larger cohorts will be necessary to confirm the effect of these polymorphisms on the susceptibility to ipa infection. table s1 associations of polymorphisms involved in the phagocyte-immune related response against aspergil-lus. 1 models adjusted for age, gender, hematological malignancy, hsct, neutropenia (defined as absolute neutrophil count ,500 cells/mm3 for a period of more than 10 days), gvhd and corticoid therapy use (.0.3 mg/kg/day). { assuming a recessive model of inheritance. abbreviations: or, odds ratio; ci, confidence interval. differences in samples numbers are due to failures in genotyping. (doc) the clinical spectrum of pulmonary aspergillosis invasive aspergillosis in allogeneic stem cell transplant recipients: changes in epidemiology and risk factors phagocytosis of aspergillus fumigatus conidia by murine macrophages involves recognition by the dectin-1 beta-glucan receptor and toll-like receptor 2 mechanisms of phagocytosis in macrophages signalling through c-type lectin receptors: shaping immune responses the betaglucan receptor dectin-1 recognizes specific morphologies of aspergillus fumigatus dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin mediates binding and internalization of aspergillus fumigatus conidia by dendritic cells and macrophages differential highaffinity interaction of dectin-1 with natural or synthetic glucans is dependent upon primary structure and is influenced by polymer chain length and sidechain branching structural basis for selective recognition of oligosaccharides by dc-sign and dc-signr dectin-2 recognition of alpha-mannans and induction of th17 cell differentiation is essential for host defense against candida albicans dectin-1 directs t helper cell differentiation by controlling noncanonical nf-kappab activation through raf-1 and syk identification of dc-sign, a novel dendritic cell-specific icam-3 receptor that supports primary immune responses dendritic cells transport conidia and hyphae of aspergillus fumigatus from the airways to the draining lymph nodes and initiate disparate th responses to the fungus innate signaling by the ctype lectin dc-sign dictates immune responses syk-and card9-dependent coupling of innate immunity to the induction of t helper cells that produce interleukin 17 dectin-1 and dectin-2 in innate immunity against fungi pattern recognition: recent insights from dectin-1 proinflammatory response of immature human dendritic cells is mediated by dectin-1 after exposure to aspergillus fumigatus germ tubes dectin-1 y238x polymorphism associates with susceptibility to invasive aspergillosis in hematopoietic transplantation through impairment of both recipient-and donor-dependent mechanisms of antifungal immunity tolllike receptor 4 polymorphisms and aspergillosis in stem-cell transplantation polymorphisms in toll-like receptor genes and susceptibility to pulmonary aspergillosis tlr1 and tlr6 polymorphisms are associated with susceptibility to invasive aspergillosis after allogeneic stem cell transplantation influence of polymorphisms in innate immunity genes on susceptibility to invasive aspergillosis after stem cell transplantation the y238x stop codon polymorphism in the human beta-glucan receptor dectin-1 and susceptibility to invasive aspergillosis il1 gene cluster polymorphisms and its haplotypes may predict the risk to develop invasive pulmonary aspergillosis and modulate c-reactive protein level interleukin-10 promoter polymorphism as risk factor to develop invasive pulmonary aspergillosis protective role of interleukin-10 promoter gene polymorphism in the pathogenesis of invasive pulmonary aspergillosis after allogeneic stem cell transplantation polymorphisms in the chemokine (c-x-c motif) ligand 10 are associated with invasive aspergillosis after allogeneic stem-cell transplantation and influence cxcl10 expression in monocyte-derived dendritic cells tnfr1 mrna expression level and tnfr1 gene polymorphisms are predictive markers for susceptibility to develop invasive pulmonary aspergillosis variable number of tandem repeats of tnf receptor type 2 promoter as genetic biomarker of susceptibility to develop invasive pulmonary aspergillosis revised definitions of invasive fungal disease from the european organization for research and treatment of cancer/invasive fungal infections cooperative group and the national institute of allergy and infectious diseases mycoses study group (eortc/msg) consensus group detection of galactomannan antigenemia by enzyme immunoassay for the diagnosis of invasive aspergillosis: variables that affect performance prospective clinical evaluation of lower cut-offs for galactomannan detection in adult neutropenic cancer patients and haematological stem cell transplant recipients use of circulating galactomannan screening for early diagnosis of invasive aspergillosis in allogeneic stem cell transplant recipients a variant in the cd209 promoter is associated with severity of dengue disease a novel polymorphism in the mcp-1 gene regulatory region that influences mcp-1 expression ccl2 polymorphisms are associated with serum monocyte chemoattractant protein-1 levels and myocardial infarction in the framingham heart study accounting for decay of linkage disequilibrium in haplotype inference and missing-data imputation snp_tools: a compact tool package for analysis and conversion of genotype data for ms-excel the structure of haplotype blocks in the human genome fastsnp: an always up-to-date and extendable service for snp function analysis and prioritization power of multifactor dimensionality reduction for detecting gene-gene interactions in the presence of genotyping error, missing data, phenocopy, and genetic heterogeneity computational analysis of gene-gene interactions using multifactor dimensionality reduction genetic susceptibility to aspergillus fumigatus infections toll-like receptor 4 polymorphisms and aspergillosis plasminogen alleles influence susceptibility to invasive aspergillosis association of dc-sign promoter polymorphism with increased risk for parenteral, but not mucosal, acquisition of human immunodeficiency virus type 1 infection contrasting genetic influence of ccr2 and ccr5 variants on hiv-1 infection and disease progression san francisco city cohort (sfcc), alive study rantes -28g delays and dc-sign -139c enhances aids progression in hiv type 1-infected japanese hemophiliacs dc-sign (cd209) gene promoter polymorphisms in a brazilian population and their association with human t-cell lymphotropic virus type 1 infection investigation of promoter variations in dendritic cell-specific icam3-grabbing non-integrin (dc-sign) (cd209) and their relevance for human cytomegalovirus reactivation and disease after allogeneic stem-cell transplantation cd209 genetic polymorphism and tuberculosis disease joint effect of mcp-1 genotype gg and mmp-1 genotype 2g/2g increases the likelihood of developing pulmonary tuberculosis in bcg-vaccinated individuals a functional promoter polymorphism in monocyte chemoattractant protein-1 is associated with increased susceptibility to pulmonary tuberculosis promoter variation in the dc-sign-encoding gene cd209 is associated with tuberculosis variant in cd209 promoter is associated with severity of liver disease in chronic hepatitis c virus infection a novel mcp-1 gene polymorphism is associated with hepatic mcp-1 expression and severity of hcv-related liver disease association of common promoter polymorphisms of mcp1 with hepatitis b virus clearance cd209 (dc-sign) -336a.g promoter polymorphism and severe acute respiratory syndrome in hong kong chinese association between genetic polymorphism in the promotor region of cd209 and propensity to develop invasive pulmonary aspergillosis neutrophils produce il-17a in a dectin-1 and il-23 dependent manner during invasive fungal infection the role of the beta-glucan receptor dectin-1 in control of fungal infection early stop polymorphism in human dectin-1 is associated with increased candida colonization in hematopoietic stem cell transplant recipients we thank all participants in this study and rosa cano (laboratory technician) for sample collection and technical support. key: cord-000460-h3owwjao authors: xiong, jing; miller, virginia m.; hunter, larry w.; li, yunman; jayachandran, muthuvel title: leukocyteand platelet-derived microvesicle interactions following in vitro and in vivo activation of toll-like receptor 4 by lipopolysaccharide date: 2011-09-26 journal: plos one doi: 10.1371/journal.pone.0025504 sha: doc_id: 460 cord_uid: h3owwjao background: pro-coagulant membrane microvesicles (mv) derived from platelets and leukocytes are shed into the circulation following receptor-mediated activation, cell-cell interaction, and apoptosis. platelets are sentinel markers of toll-like receptor 4 (tlr4) activation. experiments were designed to evaluate the time course and mechanism of direct interactions between platelets and leukocytes following acute activation of tlr4 by bacterial lipopolysaccharide (lps). methodology/principal findings: blood from age-matched male and female wild type (wt) and tlr4 gene deleted (dtlr4) mice was incubated with ultra-pure e. coli lps (500 ng/ml) for up to one hour. at designated periods, leukocyte antigen positive platelets, platelet antigen positive leukocytes and cell-derived mv were quantified by flow cytometry. numbers of plateletor leukocyte-derived mv did not increase within one hour following in vitro exposure of blood to lps. however, with lps stimulation numbers of platelets staining positive for both plateletand leukocyte-specific antigens increased in blood derived from wt but not dtlr4 mice. this effect was blocked by inhibition of tlr4 signaling mediated by my88 and trif. seven days after a single intravenous injection of lps (500 ng/mouse or 20 ng/gm body wt) to wt mice, none of the platelets stained for leukocyte antigen. however, granulocytes, monocytes and apoptotic bodies stained positive for platelet antigens. conclusions/significance: within one hour of exposure to lps, leukocytes exchange surface antigens with platelets through tlr4 activation. in vivo, leukocyte expression of platelet antigen is retained after a single exposure to lps following turn over of the platelet pool. acute expression of leukocyte antigen on platelets within one hour of exposure to lps and the sustained expression of platelet antigen on leukocytes following a single acute exposure to lps in vivo explains, in part, associations of platelets and leukocytes in response to bacterial infection and changes in thrombotic propensity of the blood. acute and chronic infection, especially that induced by gramnegative bacteria is associated with increased risk of thrombosis and atherosclerotic disease [1, 2, 3, 4, 5] . little is known about the underlying cellular mechanisms responsible for these risks. lipopolysaccharide (lps), a component of the cell wall of gram-negative bacteria, is an antigen which initiates inflammation and innate immune responses by interacting with toll-like receptor 4 (tlr4). tlr4 is expressed on the surface of cells, including leukocytes and platelets [6, 7, 8] . under physiological conditions, platelets and leukocytes circulate in quiescent state and do not interact with each other. however, once activated under pathophysiological conditions such as those associated with infection, platelets change shape, secrete prothrombogenic inflammatory and cellular adhesion molecules from alpha-and densegranules which cause the platelets to adhere to each other or to leukocytes and/or vascular endothelium [9, 10, 11, 12] . the physiological consequences of stimuli associated with infection, like lps stimulation, are acute but can be sustained. for example, half-life of platelets was shortened and the activation state of newly formed platelets from bone marrow megakaryocytes increased within seven days following a single acute intravenous injection of lps in mice [13, 14] . however, cellular events, specifically those occurring among blood elements, contributing to the shortened half-life and increased activation state of platelets remains to be clarified. one mechanism offered to explain how infection contributes to the onset and progression of cardiovascular diseases is through increased production of proinflammatory cytokines [1, 3] . however, this explanation does not address how the production of inflammatory cytokines might proceed nor does it identity the cell types which are targets for the lps stimulation. platelets may represent one of the first blood borne elements to react to lps stimulation as changes in platelet reactivity via tlr4 seems to occur prior to sustained changes in circulating levels of cytokines [14] . alternatively, comparable activation of leukocyte as well as platelet result in formation of cell-derived microvesicles (mv) which may contribute to increased thrombogenic propensity of the blood, pro-inflammatory immune processes and thus cardiovascular risk [15, 16, 17, 18, 19, 20, 21, 22] . clarifying the interactions of these blood elements (platelets and leukocytes) in the setting of tlr4 activation might provide insight into how infection initiates or facilitates progression of cardiovascular disease. mv are cell membrane-derived vesicles ranging in size from 0.1 to 1 micron in diameter which are shed in response to cellular activation, cell-cell interaction and apoptosis [23, 24, 25, 26, 27] . these cell-derived vesicles are an interface of activation between cellular components of the blood with the vascular wall and between soluble components of the blood associated with immunity including response to infection [24, 28, 29] . for example, phosphatidylserine (ps) on the surface of mv provides catalytic sites for prothrombinase complex to generate thrombin needed for the conversion of fibrinogen to fibrin in formation of clots [25, 30, 31] . furthermore, exposure of diluted blood to lps in vitro increased production of platelet-derived as well as tissue factor positive mv within 3 to 6 hours [32, 33, 34] . while those experiments provide evidence that lps modulates platelet activation, they do not provide any insight about the interactions of platelet with other blood elements within the earliest stages of activation especially at time points prior to the period when measurable changes in circulating cytokines are observed in vivo [14] . therefore, the present study was designed to test the hypothesis that acute exposure to a sentinel dose of lps would induce mv production and exchange of specific proteins/ receptors between platelets and leukocytes via tlr4 activation. mv transport of biologically active cell contents including cell surface receptors among cells were identified using antibodies specific for cell antigens (i.e. cd41 or cd45, which is platelet-or leukocyte-specific antigens respectively) [24] ; mv derived from platelets and/or leukocytes were distinguished by cell specific fluorescein conjugated antigen staining using calibrated flow cytometry. four to eight month old, male and female c57bl10snj mice (wild type, wt) and c57bl10scn mice homozygous for deletion of tlr4 (dtlr4) were obtained from the jackson laboratory, bar harbor, maine. these mice do not express the il-12rb2 mutation that was originally described for this strain [35] . mice of each sex and age were used randomly in each of the various protocols. mice were housed in a temperature-controlled environment (2262uc; 5565% relative humidity), 12/12 light/dark cycle, and fed standard chow. experiments were approved by the institutional animal care and use committee, mayo clinic, rochester, mn. ultra-pure e.coli lipopolysaccharide (lps, 0111:b4 strain-tlr4 ligand, product number tlrl-pelps), pepinh-myd (product number tlrl-pimyd) or pepinh-trif (product number tlrl-pitrif) inhibitory peptide (invivogen, san diego, ca) were prepared as suggested by the supplier. mouse thrombin and bovine serum albumin were purchased from sigma chemical co., st. louis, mo, usa. cellular origin of antigens was determined using platelet (rat anti-mouse cd41 antibody) and total leukocyte (rat anti-mouse cd45 antibody) membrane specific fluorescein conjugated {phycoerythrin (pe)-or fluorescein isothiocyanate (fitc)-} antibodies by flow cytometry. pe-or fitc-conjugated annexin-v and matched isotype control antibodies were purchased from bd pharmingen international, san diego, ca. all other reagents and solvents used in this study were of analytical/ reagent grade. blood was collected from the retro-orbital sinus plexus (250-300 ml/mouse) of wild type and dtlr4 mice through siliconized capillary tubes coated with hirudin (thrombin inhibitor) and soybean trypsin inhibitor (sti, factor xa inhibitor) into 1.5 ml polypropylene tubes containing 20 ml of 100 mm hirudin and 1 mm sti [13] . for in vitro experiments, anticoagulated blood was aliquoted into pairs of tubes within 30 min after collection so that measurements from a vehicle-treated, control tube and lpstreated tube could be analyzed from each mouse at each time point. vehicle (saline) or lps (500 ng/ml) was added to one of each paired aliquots of blood. at designated time points (5, 30, and 60 minutes) after addition of vehicle or lps, 10 ml whole blood from each aliquot was diluted into 990 ml (1:100) hepes/ hanks' buffer (130 mm nacl, 5.4 mm kcl, 1.3 mm cacl 2 , 0.8 mm mgso 4 , 0.44 mm na 2 hpo 4 , 20 mm hepes, ph 7.4) with 1 mg/ml albumin and 1 mm sti for staining of platelets positive for leukocyte antigen and leukocyte positive for platelet antigen. the remaining blood was used to prepare platelet free plasma (pfp) for mv analysis. for in vivo experiments, male mice (8 month of age) received a single injection of lps (500 ng/mouse or 20 ng/gm body weight) into the tail vein. seven days after the injection, blood was collected as described above. for analysis of platelet antigen positive leukocyte sub-populations, flow cytometry was triggered with total leukocyte marker cd45-conjugated with allophycocyanin (cd45-apc). all cd45 positive events containing granulocytes, monocytes, lymphocytes and apoptotic bodies were separated by light forward and side scatter blot analysis and gated each sub-type of leukocytes based on their size. each gate was verified using cell specific antibodies (cd11b for granulocytes, cd14 for monocytes and cd3 for t-lymphocytes and cd19 for b-lymphocytes. differential leukocyte populations were then subjected to two-color analysis to differentiate leukocytes positive for phosphatidylserine and platelet antigen (annexin-v-fitc vs cd41-pe) from leukocytes negative for phosphatidylserine and platelet antigen. percentages of positive events were calculated above the set threshold from isotype control antibody. a blood sample from each mouse was divided into two 100 ml aliquots; one was diluted with 100 ml saline (control), the other with 100 ml saline containing lps (500 ng/ml final concentration). samples were incubated at 25uc. at time-points 0 (prior to lps or vehicle), 30 and 60 min, a 20 ml aliquot was removed from each sample, diluted in 20 ml 2% paraformaldehyde and incubated for an additional 30 min. each sample was then diluted in 250 ml pbs (0.1 mm pore membrane-filtered) and centrifuged at 456g for 12 min. the supernate, which contained platelets, was removed, placed into a new vial and used for imaging analysis. an aliquot of each platelet suspension was diluted 1:4 in pbs, and a 25 ml drop was placed on a glass slide, cover-slipped and sealed with glue. platelets were then imaged in dark-field mode using a light microscope (olympus bx41 with a 1006 oil-immersion lens) coupled to a cytoviva illumination system (cytoviva, inc., auburn, al). scanning from a corner of each cover-slip, platelets in each field were counted until 100 were totaled. platelets were categorized according to shape morphology (discoid, irregular, flattened, pseudopodia) or whether exhibiting membrane granules or in aggregates (each aggregate was counted as 1). the number of platelets in each category was expressed as percentage of total 100 platelets counted. diluted (1:100) whole blood (100 ml) was incubated with peconjugated platelet-and leukocyte-specific antibodies (cd41 and cd45, respectively), or separately with annexin v-fitc (binding to phosphatidylserine) for 30 min, after which 1% paraformaldehyde (400 ml) was added. matched fluorescein conjugated isotype control antibodies were used simultaneously staining to set the threshold and exclude nonspecific binding. interactions between cell (platelets or leukocytes) and cell-derived mv and ps expression on both platelets and leukocytes were analyzed by flow cytometry (facscalibur tm and facscanto tm , bd biosciences, san jose, ca). platelets were identified by forward and side scatter and with fluorescein conjugated cd41 antibody until 20,000 events gated for each sample, respectively ( figure 1a ). all buffers and antibodies were filtered twice through 0.2 mm membrane (millipore) filters for mv analysis. platelet free plasma (pfp) was prepared by double centrifugation at 30006g for 15 min. pfp was diluted (15 ml pfp+85 ml hepes/hanks' buffer) and incubated with 4 ml fitc-conjugated annexin-v and pe-conjugated cd41 or cd45 antibodies for 30 min in dark, at which time 350 ml hanks' balanced salts buffer (ph 7.4) with 2.5 mm cacl 2 was added. matched isotype control antibodies were used to set threshold and exclude nonspecific binding. mv were quantified based on counts of calibration beads (trucount tm beads) added immediately to the samples prior to analysis by flow cytometry (facscanto tm , bd biosciences, san jose, ca) as previously described [25, 26, 31] . in this study, mv were defined as events of ,1 mm in diameter using size calibration beads and positive for annexin-v and platelet-and leukocyte-specific markers. analysis of tlr4 intracellular signaling pathway lps stimulated tlr4 signaling was analyzed by introducing myd88 or trif inhibitory peptides as described in other studies [36, 37] . anticoagulated blood from wt mice was aliquoted into six tubes and treated with pepinh-control (vehicle), pepinh-myd (100 mm, myd88 inhibitory peptide which binds to myd88 to block tlr4 stimulated myd88 signaling) and/or pepinh-trif (100 mm which binds to trif to block tlr4 stimulated trif signaling) alone or in combination for 30 min after which either saline or lps (500 ng/ml) was added for one hour. at this time, an aliquot was diluted for analysis by flow cytometry to measure cellular origin of mv and platelet positive leukocyte antigen and leukocyte positive platelet antigen. data are presented as percentage or fold increase from the paired vehicle and lps treated blood samples at each time point. all data are presented as mean 6 sem; n = number of animals used in each experiment. statistical significance was evaluated by paired or unpaired two-tailed student's t-test. statistically significance was accepted at p,0.05. prior to addition of lps, platelets from wt mice were discoid, formed few aggregates and did not have extended psuedopodia (figure 2 ). surface expression of ps on platelets or leukocytes was similar in wt and dtlr4 mice. within each group of mice, expression of ps was significantly lower on leukocytes than on platelets (table 1) . during the first hour of incubation of the blood with lps, platelets from wt mice underwent a shape change, extended pseudopodia and formed aggregates (figure 2 ). within this first hour, neither surface expression of ps on either platelets or leukocytes or numbers of platelet-or leukocyte-derived mv changed significantly in wt mice or dtlr4 mice (table 1) . however, the number of platelets positive for leukocyte-specific antigen (defined by the platelet-gate on the flow cytometer, figure 1 ) increased significantly in a time-dependent manner in blood of wt mice, but not dtlr4 mice (figures 1) . using the total leukocyte specific antibody (cd45-apc) to define total leukocytes, numbers of leukocytes positive for platelet-specific antigen did not increase significantly in either wt or dtlr4 mice ( figure 1) . in blood from wt mice, the percentage of platelets positive for leukocyte antigen (cd45) was reduced significantly and to the same extent by myd88 and trif (figure 3 ). the combination of myd88 with trif did not reduce the percentage of aggregates to a greater extent than either alone (from 3.8360.48 to 1.3860.08). seven days following a single intravenous injection of a sentinel dose of lps (20 ng/gm body weight which is 500 ng/mouse), none of the platelets were positive for leukocyte antigen. on the contrary, compared to leukocytes obtained from animals treated with vehicle or a week before lps injection in the same mouse, granulocytes and monocytes were significantly positive for platelet antigen seven days after the single lps injection (figure 4) . apoptotic bodies also stained positive for platelet antigen (figure 4 ). understanding how infection alters cell-cell interactions and release of mv from specific blood borne elements may help to identify new targets for reducing cardiovascular/thrombotic risk with infection. this study demonstrates the acute, immediate interaction of platelets and leukocytes after incubation of whole blood with a sentinel dose of lps through tlr4 signaling. exchange of antigens and associations of specific cell-derived mv among cells is a mechanism for the transfers signaling molecules to specific cells. for example, mv derived from neutrophils induced platelet activation by binding to platelets gp1ba (glycoprotein 1b a) via activated a m b 2 on mv [38] , while platelet-derived mv mediate leukocyte-leukocyte aggregation, activate leukocyte phagocytic properties and amplify leukocyte-mediated tissue injury in thrombotic and inflammatory disorders [39] . results from the present study demonstrate that the number of platelets positive for leukocyte antigen increased within 60 min of exposure to lps. this increase was not accompanied by increased expression of ps on cell or mv surface. because expression of the leukocyte antigen on platelets was defined using the platelet size gate and platelet specific marker cd41 on the flow cytometry, larger leukocytes would be excluded. therefore, these leukocyte antigens may represent a membrane exchange during platelet-leukocyte adhesion or adhesion of leukocyte-derived mv to the platelets. the half-life of whole platelet-leukocyte aggregates may be shorter and therefore, we did not determine whole platelet-leukocyte aggregates in this study. with agonist binding, the tlr4 dimerizes and undergoes a conformational change required for the recruitment of signaling molecules [40] , such as the adaptor molecules myeloid differentiation protein 88 (myd88) and toll-interleukin-1(il-1) receptor figure 2 . representative dark-field micrographs of platelets from a wt mouse taken immediately before or after 30 and 60 min. incubation of the blood with 500 ng/ml lps. prior to lps application, platelet aggregates (of more than 5 platelets) were absent, and most platelets were discoid shaped (left panels). at 30 and 60 minutes incubation with saline, platelet morphology did not change (top panels); whereas, large aggregates of irregularly-shaped platelets were observed in those incubated with lps (bottom panels, inset shows platelets with pseudopodia). each bar represents 5 mm. similar results were observed with blood from two additional animals (n = 3). doi:10.1371/journal.pone.0025504.g002 domain containing adaptor inducing interferon-b (trif), which mediate myd88 dependent or independent pathway respectively [41, 42] . lps activates both myd88 and trif pathways, which are important in the tlr4 mediated intracellular signaling [42] . the acute effects of lps on platelet and leukocyte activation were most likely mediated through activation of tlr4 as platelet positive leukocyte antigen was not observed in blood from dtlr4 mice. furthermore, leukocyte antigen expression on platelet was reduced by inhibition of myd88-and trif-dependent pathways alone or in combination. these signaling pathways may be potential molecular targets to inhibit infection/inflammation induced interactions among formed elements in the blood. a single tlr4 sentinel dose injection of lps, such as used in this study, shortened platelet half-life and increased platelet production without increases in cytokine production within 3 hours of stimulation [14] . although in vivo interaction of plateletand leukocyte-aggregates with the vascular wall could stimulate or exacerbate proinflammatory immune responses [43, 44] , the half-life of these cell aggregates is not known. a significant finding of the present in vivo study is that leukocytes sustain or retain platelet antigen seven days after an in vivo injection of lps. this time point corresponds to turnover of the platelet pool and is consistent with observations that changes in platelet half-life and increases in platelet turnover are dependent on the concentration of agonist, i.e. lps, activation [13, 14] . quantification of dual-positive events is usually not considered in studies of cellular or mv quantification with lps stimulation. results from the present study, therefore, suggest that platelet-leukocyte antigen could be used as an additional biomarker of cellular activation for diagnostic or prognostic purposes in settings of sub-clinical or asymptomatic exposures to infective agents. monocytes showed the greatest expression of platelet antigen following lps injection. since monocytes are considered to be the primary leukocytic cell involved with development of atherosclerotic lesions [45, 46, 47] , the present results provide insight into a mechanism linking low to moderate levels of infection to progression of cardiovascular disease. to our knowledge, results of the present study represent the first to examine time-dependent changes in production of mv/ exchange of cell-specific antigens in cells derived from whole blood incubated with lps. in diluted blood, addition of lps (500 ng or 1 mg/ml) increased the number of mv from platelets and those positive for tissue factor but only after four hours of incubation [34] . because the blood was diluted, unlike the present study, cell-cell interactions were most likely attenuated and no evaluation was performed relative to interaction with leukocytes which could have indirectly affected platelet activation and production of cytokines. unexpectedly, ps expression on platelets (or leukocytes) did not increase significantly with acute exposure to lps. ps is expressed in the inner leaflet of plasma membrane but rapidly inverts to outer surface following activating stimuli [48, 49, 50] . exposure of ps to the outer membrane surface also occurs with release of mv [51] . results of the present study are consistent with reports that production of mv is not accompanied by ps expression on cell of origin but might be restricted to that portion of the membrane undergoing mv blebbing [52] . within one hour of exposure of whole blood to a concentration of lps that has threshold effect on cytokine production in vivo, platelets become positive for leukocyte antigen. platelet-leukocyte interactions require tlr4 signaling as the dual antigen positivity of platelets was observed in blood derived from wild type but not dtlr4 mice. furthermore, peptide inhibitors of tlr4 signaling molecules blocked the interaction. these events occur within 1 hour after the initial exposure to lps. in addition, effects of lps stimulation are sustained at least up to 7 days past the initial lps exposure, as leukocytes express platelet antigen at this time point. collectively, these results identify an acute and rapid signaling mechanism by which sentinel-grade acute infection through tlr4 alters blood hemostasis and sustained leukocyte activation which may contribute to progression of cardiovascular diseases. chlamydia pneumoniae binds to platelets and triggers p-selectin expression and aggregation: a causal role in cardiovascular disease? a major outbreak of severe acute respiratory syndrome in hong kong virulent strains of helicobacter pylori and vascular diseases: a meta-analysis association between chronic dental infection and acute myocardial infarction risk of myocardial infarction and stroke after acute infection or vaccination 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unresponsiveness of lpsdefective c57bl/10sccr mice peptide-mediated interference of tir domain dimerization in myd88 inhibits interleukin-1-dependent activation of nf-{kappa}b differential involvement of bb loops of toll-il-1 resistance (tir) domain-containing adapter proteins in tlr4-versus tlr2-mediated signal transduction expression, activation, and function of integrin alphambeta2 (mac-1) on neutrophil-derived microparticles platelet-derived microparticles and the potential of glycoprotein iib/iiia antagonists in treating acute coronary syndrome toll-like receptor signalling myd88-dependent and -independent activation of trem-1 via specific tlr ligands myd88-dependent and independent pathways of toll-like receptors are engaged in biological activity of triptolide in ligand-stimulated macrophages exovesicles from human activated dendritic cells fuse with resting dendritic cells, allowing them to present alloantigens in vitro study of pro-inflammatory and antitumour properties of microvesicles from bacterial cell wall of pantoea agglomerans evolving concepts in the triad of atherosclerosis, inflammation and thrombosis nanoparticle pet-ct imaging of macrophages in inflammatory atherosclerosis functional cd40 ligand is expressed on human vascular endothelial cells, smooth muscle cells, and macrophages: implications for cd40-cd40 ligand signaling in atherosclerosis back and forth: the regulation and function of transbilayer phospholipid movement in eukaryotic cells reconstitution of phospholipid scramblase activity from human blood platelets calcium involvement in aminophospholipid exposure and microparticle formation during platelet activation: a study using ca2+-atpase inhibitors phospholipid composition of cell-derived microparticles determined by one-dimensional high-performance thin-layer chromatography microparticles in cardiovascular diseases key: cord-002811-5hrydciz authors: hercik, christine; cosmas, leonard; mogeni, ondari d.; wamola, newton; kohi, wanze; omballa, victor; ochieng, melvin; lidechi, shirley; bonventure, juma; ochieng, caroline; onyango, clayton; fields, barry s.; mfinanga, sayoki; montgomery, joel m. title: a diagnostic and epidemiologic investigation of acute febrile illness (afi) in kilombero, tanzania date: 2017-12-29 journal: plos one doi: 10.1371/journal.pone.0189712 sha: doc_id: 2811 cord_uid: 5hrydciz introduction: in low-resource settings, empiric case management of febrile illness is routine as a result of limited access to laboratory diagnostics. the use of comprehensive fever syndromic surveillance, with enhanced clinical microbiology, advanced diagnostics and more robust epidemiologic investigation, could enable healthcare providers to offer a differential diagnosis of fever syndrome and more appropriate care and treatment. methods: we conducted a year-long exploratory study of fever syndrome among patients ≥ 1 year if age, presenting to clinical settings with an axillary temperature of ≥37.5°c and symptomatic onset of ≤5 days. blood and naso-pharyngeal/oral-pharyngeal (np/op) specimens were collected and analyzed, respectively, using afi and respiratory taqman array cards (tac) for multi-pathogen detection of 57 potential causative agents. furthermore, we examined numerous epidemiologic correlates of febrile illness, and conducted demographic, clinical, and behavioral domain-specific multivariate regression to statistically establish associations with agent detection. results: from 15 september 2014–13 september 2015, 1007 febrile patients were enrolled, and 997 contributed an epidemiologic survey, including: 14% (n = 139) 1<5yrs, 19% (n = 186) 5-14yrs, and 67% (n = 672) ≥15yrs. afi tac and respiratory tac were performed on 842 whole blood specimens and 385 np/op specimens, respectively. of the 57 agents surveyed, plasmodium was the most common agent detected. afi tac detected nucleic acid for one or more of seven microbial agents in 49% of afi blood samples, including: plasmodium (47%), leptospira (3%), bartonella (1%), salmonella enterica (1%), coxiella burnetii (1%), rickettsia (1%), and west nile virus (1%). respiratory tac detected nucleic acid for 24 different microbial agents, including 12 viruses and 12 bacteria. the most common agents detected among our surveyed population were: haemophilus influenzae (67%), streptococcus pneumoniae (55%), moraxella catarrhalis (39%), staphylococcus aureus (37%), pseudomonas aeruginosa (36%), human rhinovirus (25%), influenza a (24%), klebsiella pneumoniae (14%), enterovirus (15%) and group a streptococcus (12%). our epidemiologic investigation demonstrated both age and symptomatic presentation to be associated with a number of detected agents, including, but not limited to, influenza a and plasmodium. linear regression of fully-adjusted mean cycle threshold (c(t)) values for plasmodium also identified statistically significant lower mean c(t) values for older children (20.8), patients presenting with severe fever (21.1) and headache (21.5), as well as patients admitted for in-patient care and treatment (22.4). conclusions: this study is the first to employ two syndromic taqman array cards for the simultaneous survey of 57 different organisms to better characterize the type and prevalence of detected agents among febrile patients. additionally, we provide an analysis of the association between adjusted mean c(t) values for plasmodium and key clinical and demographic variables, which may further inform clinical decision-making based upon intensity of infection, as observed across endemic settings of sub-saharan africa. in low-resource settings, empiric case management of febrile illness is routine as a result of limited access to laboratory diagnostics. the use of comprehensive fever syndromic surveillance, with enhanced clinical microbiology, advanced diagnostics and more robust epidemiologic investigation, could enable healthcare providers to offer a differential diagnosis of fever syndrome and more appropriate care and treatment. we conducted a year-long exploratory study of fever syndrome among patients ! 1 year if age, presenting to clinical settings with an axillary temperature of !37.5˚c and symptomatic onset of 5 days. blood and naso-pharyngeal/oral-pharyngeal (np/op) specimens were collected and analyzed, respectively, using afi and respiratory taqman array cards (tac) for multi-pathogen detection of 57 potential causative agents. furthermore, we examined numerous epidemiologic correlates of febrile illness, and conducted demographic, clinical, and behavioral domain-specific multivariate regression to statistically establish associations with agent detection. from 15 september 2014-13 september 2015, 1007 febrile patients were enrolled, and 997 contributed an epidemiologic survey, including: 14% (n = 139) 1<5yrs, 19% (n = 186) 5-14yrs, and 67% (n = 672) !15yrs. afi tac and respiratory tac were performed on 842 whole blood specimens and 385 np/op specimens, respectively. of the 57 agents surveyed, plasmodium was the most common agent detected. afi tac detected nucleic acid plos with gains in vector control across sub-saharan africa and a significant decrease in overall malaria incidence rates, a better understanding of the prevalence of both malarial and nonmalarial agents potentiating fever syndrome is needed for several reasons including efforts to more rapidly detect and control diseases at their source to ensure global health security [1] [2] [3] [4] [5] . in low-resource settings, limited access to clinical laboratory diagnostics requires empiric case management of febrile illness. the use of comprehensive fever syndromic surveillance, with enhanced clinical microbiology, advanced diagnostics and more robust epidemiologic investigation, could enable healthcare providers to offer a differential diagnosis of fever syndrome and more appropriate care and treatment [6] . in order to inform clinical management and optimize treatment regimens for febrile patients, information on common causes and associations of febrile illness, particularly in areas endemic to malaria, is critically required. while previous studies have adopted a syndromic approach to estimate global burden of disease due to diarrhea and pneumonia, such an approach has not been taken to evaluate fever in the absence of other characterizing features (e.g., gastroenteritis, skin or soft tissue infections, etc.) [7] [8] . pathogen-specific approaches have been utilized to evaluate illness due to certain febrile diseases, predominately malaria, typhoid fever and arboviral infections, such as dengue virus; however, disease burden estimates due to other fever associated organisms, such as leptospira and coxiella burnetii, remain in question [9] [10] [11] [12] [13] . recent research evaluating the status of global fever surveillance has identified significant gaps in the field, including the lack of multi-pathogen diagnostic approaches, as well as limitations in geographic and temporal coverage to account for disease variations observed across time and space [7, [14] [15] . data regarding the cause of non-malarial febrile illness are currently lacking in tanzania's south-central region. to identify the type and prevalence of various bloodstream and respiratory agents among febrile patients in kilombero, tanzania, we conducted a yearlong exploratory surveillance project, surveying the presence of 57 different microbial agents in blood and naso/oro-pharyngeal specimens. additionally, we examined numerous correlates of febrile illness, and conducted demographic, clinical, and behavioral domain-specific multivariate regression to statistically establish associations with detection of identified agents. ethical clearance for this study was obtained from the national health research ethics sub-committee (nathrec) of medical research coordinating committee (mrcc) at the national institute for medical research (nimr/hq/r.8a/vol.ix/1735) in tanzania, and the us centers for disease control and prevention, center for global health (cgh hsr 2014-118). a written informed consent was obtained from each participant prior to the interview. prior to enrollment, all patients provided written assent for participation. pediatric patients (1 to 15 years-of-age) also needed documented parent consent in order to be enrolled. all pediatric patients were accompanied by an adult guardian at the time of interviewing. guardians participated in assisting with completion of the pediatric questionnaires when necessary. for all adult participants who are unable to read or write, a study member read through the full document twice to ensure the patient had complete understanding of the terms and conditions associated with their enrollment in this study. if illiterate, the participant printed an "x" rather than their name on the informed consent form. this consent, as marked by an "x," was then be verified with a signature of a witness. the participants were assured of anonymity in the analysis, presentation and publishing of the data. this surveillance project was conducted in tanzania's kilombero valley, a rural area where agro-industrial land use associated with sugarcane production has situated a growing human population in close proximity to areas of increased wildlife biodiversity (fig 1) . the illovo sugar limited estate in kilombero is the largest sugarcane production facility in tanzania. the estate is comprised of 13,000 hectares of land, of which 9,562 hectares are under cultivation. the estate consists of nine farms and two factories, and is further surrounded by small family-owned farms. the estate neighbors two national parks including: mikumi national park and udzungwa falls national park. this cross-sectional study was conducted at the illovo sugar limited estate hospital (k1) and clinic (k2). k1 hospital is a private 80-bed hospital, serving illovo employees and dependents as well community members from nine surrounding villages, including: nyandeo, msolwa ujamaa, mkula, mangula, kiberege, msolwa station, ruaha, kidogobasi and luhembe. k2 clinic is operational five days a week (monday-friday) and is only available to illovo employees and dependents. employees and dependents are offered medical care free-ofcharge; other community members who are not affiliated with the illovo company are offered care however must pay a fee for service, which is partially subsidized by the illovo company. kilombero (population >321,000) is situated at an elevation of 300m above mean sea level, in the morogoro region (population >2 million) of south-central tanzania. the climate is characterized by a long rainy period (march-may) and a short rainy period (october-december). malaria transmission intensity in this region is considered high, with 13% prevalence in community-based surveying of children (6-59 months) testing positive for malaria by rapid diagnostic testing [16] [17] . the objective of this hospital-based syndromic surveillance study was to examine exposure and epidemiologic associations with detection of bloodstream and respiratory agents among patients presenting to clinical settings with acute febrile illness (afi) in kilombero, tanzania. consenting patients, one year-of-age and above, with an axillary temperature of !37.5˚c and symptomatic onset of 5 days, were eligible for enrollment. blood and np/op specimens, along with demographic, clinical and behavioral data were collected in order to conduct epidemiologic analysis. patients at k1 hospital and k2 clinic were screened, and if found to be eligible for enrollment, they were directed to one of our local clinical staff members for processing. clinical, epidemiologic and laboratory data were entered electronically onto samsung tablets using an open data kit (odk) platform. following screening and informed consent, a clinical officer or assistant medical officer conducted a detailed clinical history followed by a physical examination. the attending officer entered all clinical data in a standardized electronic clinical case report form on the tablets using odk collect. data recorded on the ecrf included: patient vital signs, self-reported symptoms, self-reported previous and concurrent infections (i.e. malaria, hiv), provisional diagnoses, treatment recommendations, as well as final admission status. after the clinical examination and consultation was complete, clinical officers then administered a comprehensive epidemiologic survey to further capture demographic and behavioral data. patients under 15 years-of-age were administered a pediatric epidemiologic survey, whereas patients 15 years-of-age and above were administered an adult epidemiologic survey. these two survey questionnaires were similar in scope, however tailored to include or omit certain age-specific inquiries (e.g., vaccination history for children, occupational activities for adults). data recorded on the epidemiologic survey included: socio-economic data, household and residence status, travel history, wildlife and livestock animal interactions, recreational/ occupational activities, as well as preventative measures (i.e. vaccinations, vector control measures, etc.). wealth index was calculated using principal component analysis (pca) so to describe socio-economic status (ses) differentiation within our study group. in this regard, we pilot tested several examples of household assets that could be appropriate for creating ses indices for this target population. the following six household assets were chosen for calculation of wealth index by pca: radio, bicycle, mobile phone, light source, television, and refrigerator. prior to initiation of any antimicrobial therapy, blood was drawn from each enrolled participant. the venipuncture site was cleaned with an alcohol swab, followed by disinfection with povidone-iodine. for adults, a total of 15 ml of venous blood was extracted, including 10 ml of whole blood (in two 5ml edta tubes) for aliquotting and 5 ml of whole blood (in one 5ml non-edta plain tube) for further centrifugation of serum. for children, smaller volumes of 5-8 ml total whole blood were obtained. blood samples were stored in a -80˚c freezer on site, backed-up by a generator for emergency power supply. subsequently, at the time of enrollment, if any adult patient was suffering from respiratory conditions (cough, chest pain or sore throat) in addition to his/her febrile illness, staff collected an np/op swab. given that pulmonary infections in children may present with non-specific signs, especially early in the infectious course, staff collected an np/op swab from all enrolled pediatric febrile patients irrespective of pulmonary symptoms. np/op swabs were placed in viral transport media (vtm) and stored in a -80˚c freezer on site prior to specimen transport. an sd bioline (pan/pf) malaria rapid diagnostic test (standard diagnostics, inc.) was performed at point-of-care for all participants. if the patient yielded a positive rapid test result, a microscopic examination of giemsa-stained thick blood film smears was performed to determine intensity of infection. results were scored as 0 (no parasites), 1+ (10-19 parasites/μl), 2+ (20-29 parasites/μl), 3+ (30-39 parasites/μl), or 4+ (40 or more parasites/μl). whole blood and np/op specimens were transported by road, on dry-ice, to kenya medical research institute (kemri) laboratories in nairobi, kenya, on a quarterly basis, where they were stored in a -80˚c freezer prior to undergoing diagnostic testing using the syndromic taqman array card (tac) diagnostic platform [18] [19] [20] . for the purposes of this study, we utilized both acute febrile illness (afi) and respiratory illness tac assays to screen blood and np/op specimens respectively for a combined total of 57 viral, bacterial and parasitic organisms [18] [19] . for whole blood processing, total nucleic acid was extracted from one aliquot (2.5 ml) of blood using high pure viral nucleic acid large volume kit (roche diagnostics) and eluted in 100 μl of elution buffer. ms2 and phhv were added to each sample to serve as built-in controls to confirm success of the extraction process and amplification efficiency for rna and dna targets [19] . we mixed 46 μl of total nucleic acid extract with agpath one step rt-pcr reagents (life technologies corporation), in a 100 μl reaction, then pipetted into the inlet port of each channel. cards were centrifuged (1 min, 1200 rpm twice), sealed and the inlet ports removed as directed by the manufacturer's instructions. all afi tacs were run on the viia™ 7 real-time pcr system (life technologies corporation) using pcr cycling conditions comprising of 20 min at 45˚c, 10 min at 95˚c, followed by 45 two-step cycles of 15s at 95˚c and 1 min at 60˚c. confirmed afi tac detection of a bloodstream agent was defined by a 37 threshold cycle (c t ) value [19] . for 14 of the 27 agents detected by afi tac v2 including: chikungunya, crimean-congo hemorrhagic fever, dengue, hepatitis e, marburg, rift valley fever, yellow fever, brucella, c. burnetii, leptospira, rickettsia, salmonella enterica, salmonella serovar typhi, and yersinia pestis, duplicate ports were used to detect different conserved regions of the target organism. in the event of discordant results, individual real-time pcr (irtp) was performed to determine final detection status. for additional quality control, irtp was performed on 10% of all positives and 10% of all negatives. for np/op processing, total nucleic acid was extracted from specimens in the kingfisher ml extraction platform (thermo scientific, waltham, ma) using magmax nucleic isolation kit (life technologies, carlsbad, ca). briefly, 100μl of np/op specimen was mixed with 260μl of lysis-binding solution and then washed once with 600μl wash solution i, and twice with 450μl wash solution ii. nucleic acid material was eluted in 60μl elution buffer. forty-six microliters of total nucleic acid was mixed with agpath one step rt-pcr reagents (life technologies corporation), in a 100 μl reaction, and pipetted into the port of each channel in the cards. the assays were run on the viia™ 7 real-time pcr system (life technologies corporation) using pcr cycling conditions 45˚c for 10 min, 94˚c for 10 min, and 45 cycles of 94˚c for 30 s followed by 60˚c for 1 min [18] . confirmed positive respiratory tac detection of a np/op agent was defined by 37 cycle threshold (c t ) value. for all 30 agents detected by respiratory tac, duplicate ports were used to detect conserved regions of the target pathogen. in the event of discordant results, irtp was performed to determine final detection status. influenza a positives were further sub-typed by pcr to explore lineages based upon the hemagglutinin (ha) surface protein, that included infa h1n5, infa h3n2, infa h1n1, infa h5a and infa h5b. statistical analyses were performed using sas version 9.3 software (sas inc., cary, nc). descriptive statistics were presented as medians, ranges, and interquartile ranges (iqr) for continuous variables and as proportions and charts for categorical variables. given the high frequency of detection of plasmodium, we further examined detection status across three diagnostic platforms, and conducted a non-parametric tests kruskal-wallis test to compare mean c t values, as determined by qpcr, for patients grouped within each level of parasite intensity (1+, 2+, 3+, or 4+), as determined by blood smear. additionally, we conducted a linear multivariate regression analysis to determine mean plasmodium c t values associated with examined independent variables. clinical and epidemiologic correlates of agents detected among at least 10% of our patient population were evaluated using an agent-specific filtered multivariate logistic regression approach to determine statistically significant factors among test-positive participants against a test-negative control group. given the large number of potential risk factors examined in this study, we first assessed indicators by multivariate regression within respective domains, including socio-demographic, clinical and behavioral domains. statistically significant factors, as determined by domain-specific regression, were then evaluated in a combined multivariate logistic regression model. from 15 september 2014-13 september 2015, a total of 1104 afi patients were screened to determine eligibility for enrollment, of which 1007 were officially enrolled and 997 completed an epidemiologic survey. the majority of enrolled participants (66%) were male. the median age of enrollment was 23 years (range 1-79 years). of the 1007 patients enrolled, 139 were children (1<5 yrs; 14%), 186 were older children (5<14 yrs; 19%) and 672 were adults (!15 years-of-age; 67%). all enrolled patients were tanzanian, 851 (85%) lived on the grounds of the estate, and 823 (82%) were full-time residents of the kilombero area. among the enrolled adults, 249 (37%) were field laborers (e.g., sugarcane cutting and weeding) employed by the estate. educational levels varied among our adult population, with 479 (71%) patients having primary school-level education or below. socio-economic status, as denoted by the wealth index, also varied greatly among all enrolled participants, with the majority of patients (57%) falling within the two poorest quintiles ( table 1) . the most common presenting complaints, other than fever, were headache (80%), cough (32%) and abdominal pain (19%) ( table 2 ). the mean axillary temperature was 38.4˚c (range 37.5˚c-40.5˚c) which was consistent across all age groups. given similarity in fever distributions among younger children, older children and adults, we could define fever tertiles of all enrolled febrile participants, defined as mild fever (37.5˚c-38.0˚c), moderate fever (38.1˚c -38.6˚c), and severe fever (38.7˚c-40.5˚c). each tertile comprised approximately 33% of all enrolled participants. hiv positivity, albeit self-reported, was low (2.1%). of all enrolled participants, 463 (46%) were admitted for in-patient care and treatment ( table 2 ). the most common provisional diagnosis was malaria (39%), followed by "undefined fever" (19%), urinary tract infection (17%), and upper respiratory infection (16%). patient-level data, inclusive of particular clinical and epidemiologic data as well as diagnostic results, are included in a supplemental file (s1 table) . malaria rapid testing was performed at point-of-care on 968 (96%) patients. of these, 327 (34%) were positive for malaria, including 150 (16%) that were positive for p. falciparum, 6 (1%) that were positive for plasmodium non-falciparum species, and 171 (18%) that were positive for both p. falciparum and non-falciparum species. of the 327 malaria positives whereby a thick film blood smear was performed, 4 (1%) revealed no parasite found, 17 (5%) were denoted as 1+, 133 (41%) were denoted as 2+, 111 (34%) were denoted as 3+, and 62 (19%) were denoted as 4+. diagnostic evaluations using both afi and respiratory tac identified 31 different viral, bacterial and parasitic agents among 457 febrile patients contributing only blood specimens and 385 febrile patients contributing both blood and np/op specimens. there were 226 (25.6%) patients in which we did not detect nucleic acid for any of the 57 agents surveyed (table 3) . multiple codetections were also observed using both tac assays (tables 4 and 5 ). afi tac was performed on 842 febrile patients, of which we detected at least one bloodstream agent in 434 participants, including 7 (12%) of 58 younger children, 57 (37%) of 156 older children and 370 (59%) of 628 adults. among these 842 surveyed participants, we detected nucleic acid for 7 different bloodstream agents, including: plasmodium (399; 47%), leptospira (22; 3%), bartonella (4; 1%), s. enterica (4; 1%), c. burnetii (2; 1%), rickettsia (2; 1%), and west nile virus (1; 1%). malaria was found to be the most common agent detected, with the prevalence of malaria increasing with age, as observed among 12% of younger children, 34% of older children and 54% of adults. all 20 cases of leptospira were detected among adult patients. (13) 43 (23) 131 (19) 192 (19) chest pain 4 (3) 19 (10) 147 (22) 170 (17) vomiting 28 (20) 37 (20) 95 (14) 160 (16) diarrhea 18 (13) 10 (5) 73 (11) 101 (10) sore throat 3 (2) 25 (13) 69 (10) 97 (10) other 19 (14) 33 (17) 189 (28) cycle threshold (ct) values were detected by using real-time quantitative pcr for all surveyed agents. of the 399 plasmodium positive cases, the average c t was 17.9 with a range of 4.7-36.8. for the investigation of malaria, we used a tri-pronged diagnostic approach, including afi tac, an sd bioline (pan/pf) malaria rapid diagnostic test and microscopy. to more thoroughly examine detection across these three diagnostic platforms, we conducted a nonparametric kruskal-wallis test comparing mean c t values, as determined by qpcr, for patients grouped within each level of parasite intensity (1+, 2+, 3+, or 4+), as determined by blood smear. since low c t values correspond to high parasite loads, we would expect that lower mean c t values correlate with higher levels of parasite intensity. results were statistically significant (p-value = 0.0001) indicating differences among groups, and findings demonstrate an inverse relationship between mean c t values and level of parasite intensity. as depicted in fig 2, we observe lower mean c t values (i.e. higher detected numbers of plasmodium parasites), correlated with higher levels of parasite intensity. additionally, we conducted a linear multivariate regression analysis to determine if fully adjusted mean c t values were associated with specific explanatory variables (table 6 ). outputs from this analysis demonstrate statistically significant lower mean c t values for older children (20.8 ) as compared to younger children (27.8) . in addition, we observe significantly lower mean c t values for patients presenting with severe fever (21.1) and headache (21.5) , as well as those who were admitted for in-patient care and treatment (22.4) . fig 3 denotes the statistically significant adjusted mean c t values along a c t scale of 1-37. clinical and epidemiologic correlates of agents detected by tac among at least 10% of our patient population were evaluated using an agent-specific filtered multivariate logistic regression approach to determine statistically significant factors. table 7 demonstrates the statistically significant findings from the final combined multivariate regression model. this study provides an informative and valuable diagnostic and epidemiologic assessment of febrile illness in kilombero, tanzania among participants ! 1 year of age. of 842 patients who contributed a blood specimen for diagnostic investigation, we were able to detect at least one microbial agent in 434 (52%) patients. our study eligibility criteria and case definitions were not optimized to detect any specific agent. we maintained broad and inclusive enrollment criteria given the exploratory nature of our investigation. using our afi tac assay, we detected 7 different bloodstream agents among the 27 agents surveyed, including: plasmodium (399; 47%), leptospira (22; 3%), bartonella (4; 1%), s. enterica (4; 1%), c. burnetti (2; 1%), rickettsia (2; 1%), and west nile virus (1; 1%). other than one 12%) . while many of these agents are known to colonize in the human pharynx asymptomatically and thus not contribute to disease status, these organisms may have the capacity to launch an infectious course, especially in a host suffering from a weakened immune system and/or plagued by other concurrent infections. previous studies have demonstrated that when influenza is identified, detection usually signifies etiology of clinical illness; thus, we are more confident in suggesting disease causality for the 101 (26%) individuals who presented with influenza a/b [24] [25] [26] . among influenza cases, we detected influenza a in 91 (24%) patients, and detected influenza b in 11 (3%) patients. these results are similar to findings from a recent 30-month (2008-2010) nationwide sero-prevalence study of influenza in tanzania, which found influenza a to be around eight times more prevalent than influenza b [27] ; however, our results vary from analysis conducted the same year (2015-2016) in east africa by the world health organization's global influenza programme, which detected similar prevalence of influenza a (55%) and influenza b (45%) [28] . in viewing the distribution of viral and bacterial respiratory agents across age groups ! 1 year of age, we observe increased frequency of detection of influenza a/b, s. aureus and p. aeruginosa with age, and a decreased frequency of detection of adenovirus, enterovirus, respiratory syncytial virus (rsv) and m. catarrhalis. a recent cdc tac pilot study investigating the contribution of respiratory pathogens to hospitalizations of pneumonia in rural western kenya also utilized the respiratory tac assay, among other diagnostic measures [29] . this research group found similar detection frequency of adenovirus (8%) and s. pneumoniae (60%); however, detected lower frequency of influenza a (8%) as compared to our study in tanzania [29] . in another respiratory illness investigation by jain et al. where tac, pcr and serology were employed, researchers evaluated community-acquired pneumonia requiring hospitalization among us children [30] . while testing algorithms varied somewhat from the measures employed by this study, results from the us-based study demonstrated similar prevalence of influenza a (7%) and human rhinovirus (27%); lower prevalence of s. aureus (1%) and s. pneumoniae (4%); and higher prevalence of rsv (28%) and m. pneumoniae (8%), as compared to the younger pediatric cohort enrolled in our tanzania-based study [30] . the variation observed in detection frequency across these distinct settings as well as others, suggests perhaps different existing exposures and/or epidemiologic risk depending upon geography [29] [30] [31] [32] . further multi-country tac studies, using a standardized approach, with rigorous case definitions and uniform enrollment criteria, would help facilitate pooled analyses for more generalizable results. given that kilombero is located in a highly endemic area for malaria, results are not generalizable and different diagnostic outcomes would be expected from non-endemic settings. additionally, detection of plasmodium in older children and adults is not unexpected, and positive detection is not necessarily indicative of fever etiology or resultant of new, active infection. to further examine quantification of plasmodium detections across multiple diagnostic platforms, we conducted a non-parametric kruskal-wallis test comparing mean c t values, as determined by qpcr, for patients grouped within each level of parasite intensity (1+, 2+, 3+, or 4+), as determined by blood smear. findings from this analysis demonstrate an inverse relationship between mean c t values and level of parasite intensity. moreover, we conducted a linear multivariate regression analysis to determine fully adjusted mean c t values associated with specific demographic and clinical variables. outputs form this analysis show statistically significant lower mean c t values for older children (20.8 ) as compared to younger children (27.8) . additionally, we observe lower mean c t values for patients presenting with severe fever (21.1) and headache (21.5), as well as those patients who were admitted for in-patient care and treatment (22.4) . as the clinical research community begins to shift away from traditional microscopy approaches and towards molecular diagnostics for the diagnosis of malaria, this type of analysis could be valuable for directing patient management and clinical decision-making based upon intensity of infection, as determined by quantitative c t outputs [33] . while other investigational studies have relied upon laboratory outputs alone to characterize febrile participants, we supplemented our diagnostic findings with a robust epidemiologic analysis to further establish associations with detection status. to this end, clinical and epidemiologic correlates of agents detected among at least 10% of our patient population were evaluated using an agent-specific filtered multivariate logistic regression approach to determine statistically significant factors. results demonstrated several statistically significant factors to be considered, particularly age and symptomatic presentation. age was found to be statistically associated with detection of h. influenzae, s. pneumoniae, m. catarrhalis, s. aureus, human rhinovirus, influenza a, enterovirus, and plasmodium. symptomatic presentation of at least one clinical indicator examined was found to be statistically associated with detection of group a streptococcus, p. aeruginosa, influenza a and plasmodium. from this analysis, we also observe that detection of plasmodium was most prevalent among participants presenting with severe fever (38.7˚c-40.5˚c) across all age groups. these results are in agreement with findings from another recent fever study by d'acremont et al which found malaria, along with pneumonia and typhoid, to be more common among patients presenting with severe illness as compared to those with mild illness in tanzania [14] . our study had several limitations, particularly with regard to limitations in our diagnostic approach. afi-tac analyzes whole blood specimens derived from venous blood, whereas respiratory tac screens np/op swabs derived from a non-sterile pharyngeal source. while agents detected in whole blood indicate viremia, bacteremia, and/or parasitemia, agents detected from the pharynx may only be representative of microbial carriage. in this regard, for the purposes of this study, we did not state final determination of disease etiology. in order to keep consistent with our diagnostic definitions of positive detection, we only determined pathogen detection if quantitative pcr cycle thresholds (c t ) were at or below 37. on certain occasions, particularly in the case of s. typhi, patients presented with ct values within just one point of our a priori determined c t cutoff value (i.e., 37.04; 37.23; 37.99). it could be argued that c t cutoffs need to be re-adjusted for certain pathogens included on the afi tac assay, particularly bacterial pathogens that may present with low copy numbers in blood. while uniform approaches to c t cutoffs of syndromic tac cards may enable diagnostic efficiency and ease-of-use for laboratory technicians, it may not account for the natural variations in microbial load for the diverse suite of viral, bacterial and parasitic agents surveyed by this assay. a better understanding of the diagnostic value of quantitative diagnostics in determining febrile disease etiology, particularly in situations of co-detection, as well as the utility of re-calibrating diagnostic thresholds (c t cutoffs) for molecular tests, is critically needed [14] . finally, while enrollment spanned one full calendar year to account for seasonal variation, we also note a limitation with temporal coverage, given the possibility of yearly variation. we suggest that syndromic surveillance projects of this kind be extended for a minimum of 3-5 years, in order to ascertain true prevalence estimates. in light of these limitations, this study provides a novel platform for exploratory diagnostic and epidemiologic assessment of febrile illness in a previously un-surveyed region of sub-saharan africa. while previous studies have targeted specific epidemiologic variables and their relationship with diagnostic outcomes, this study surpassed normal epidemiologic investigative procedures, by exploring over 150 independent variables included in the clinical case report form (crf) and epidemiologic survey. given the large number of variables assessed in this study, the domain-specific and full multivariate approach to risk factor assessment provided a competent mechanism for data reduction. in this regard, our chosen statistical strategy allowed us to control for potential confounding factors, while adjusting for many factors that may demonstrate interaction. finally, a strong advantage of this study was our use of highly sensitive multi-pathogen molecular diagnostics. quantitative diagnostics of this kind can not only expand the number of agents surveyed in our investigation, but can also assist in revealing the complexity of infectious disease diagnoses when multiple detections are observed. previous studies suggest that co-infection of multiple microbial agents is likely to lead to pathogen interactions, which can have significant clinical ramifications [14] . the combination of multiple detections observed in this study underscores the difficulty in providing a simple diagnosis for febrile illness. however, as shown in the case of plasmodium, c t can add a useful quantitative metric for determining severity of infection and facilitate febrile patient processing, particularly in endemic settings when detection of parasitemia is not necessarily indicative of fever etiology. given that the afi taqman array card is optimized for afi surveillance in the african region, it can serve as a rapid screen for outbreak investigation and/or for preliminary surveillance of circulating pathogens and is an appropriate diagnostic choice to fulfill the needs of our initial exploratory investigation. however, once baseline prevalence has been established, more target, pathogen-specific diagnostic approaches (such as rdts) may be more costeffective for directing public health surveillance and proper patient management. this study provides an informative and valuable diagnostic and epidemiologic assessment of febrile illness in kilombero, tanzania ! 1 year of age. the novelty of this study is proven in our use of highly sensitive multi-pathogen molecular diagnostics to better characterize the type and prevalence of agents detected among febrile patients which can lead to more rapid detection and control of diseases and enhanced global health security. additionally, we provide an analysis of the association between adjusted mean c t values for plasmodium and key clinical and demographic variables, which may further inform clinical decision-making based upon intensity of infection, across endemic settings of sub-saharan africa. supporting information s1 table. clinical, epidemiologic and diagnostic findings from enrolled participants. (xls) jie liu for providing technical assistance in tac development and validation, as well as mr. daniel macharia for providing geo-spatial mapping services for this project. this body of work served as part of the dissertation for christine hercik in pursuit of a phd in the global infectious disease program at georgetown university. we would like to thank her committee members for their constant advisement and unwavering support of her work, including: joel montgomery, phd, christopher loffredo, phd, seble kassaye, md, and daniel lucey, md. the findings and conclusions of this study are those of the authors and do not necessarily represent the views of the us centers for disease control and prevention (us cdc), the kenya medical research institute (kemri), the national institute for medical research (nimr), or georgetown university. funding acquisition: christine hercik writing -review & editing typhoid fever and the challenge of nonmalaria febrile illness in sub-saharan africa 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and arusha regions of tanzania-serology and farmers' perceptions syndromic approach to arboviral diagnostics for global travelers as a basis for infectious disease surveillance association of respiratory viruses with outcomes of severe childhood pneumonia in botswana detection and characterization of respiratory viruses causing acute respiratory illness and asthma exacerbation in children during three different season results from the first 30 months of national sentinel surveillance for influenza in tanzania world health organization. weekly epidemiological record the contribution of respiratory pathogens to fatal and non-fatal respiratory hospitalizations: a pilot study of taqman array cards in kenya communityacquired pneumonia requiring hospitalization among u.s> children improved detection of respiratory pathogens by use of high-quality sputum with taqman array card technology identification of bacterial and viral co detections with mycoplasma pneumoniae using the taqman array card in patients hospitalized with communityacquired pneumonia the merits of malaria diagnostics during an ebola virus disease outbreak we would like to thank the illovo management team, in particular the chief medical officer of illovo sugar limited estate hospital, dr. sagumo chotta, for granting us permission to conduct our study at his facility. we sincerely acknowledge the local clinical and laboratory officers of illovo k1 hospital and k2 clinic, including: dr. hamis yusuf, mr. wilson gyunda, mr. rogers abisai, mr. bumija mruma, and mr. felix nyambuya, for providing exceptional contribution to this surveillance project. we would also like to thank dr. eric houpt and dr. key: cord-000905-1rhlu59c authors: cyktor, joshua c.; carruthers, bridget; beamer, gillian l.; turner, joanne title: clonal expansions of cd8(+) t cells with il-10 secreting capacity occur during chronic mycobacterium tuberculosis infection date: 2013-03-05 journal: plos one doi: 10.1371/journal.pone.0058612 sha: doc_id: 905 cord_uid: 1rhlu59c the exact role of cd8(+) t cells during mycobacterium tuberculosis (mtb) infection has been heavily debated, yet it is generally accepted that cd8(+) t cells contribute to protection against mtb. in this study, however, we show that the mtb-susceptible cba/j mouse strain accumulates large numbers of cd8(+) t cells in the lung as infection progresses, and that these cells display a dysfunctional and immunosuppressive phenotype (pd-1(+), tim-3(+), cd122(+)). cd8(+) t cell expansions from the lungs of mtb-infected cba/j mice were also capable of secreting the immunosuppressive cytokine interleukin-10 (il-10), although in vivo cd8(+) t cell depletion did not significantly alter mtb burden. further analysis revealed that pulmonary cd8(+) t cells from mtb-infected cba/j mice were clonally expanded, preferentially expressing t cell receptor (tcr) vβ chain 8 (8.2, 8.3) or vβ 14. although vβ8(+) cd8(+) t cells were responsible for the majority of il-10 production, in vivo depletion of vβ8(+) did not significantly change the outcome of mtb infection, which we hypothesize was a consequence of their dual il-10/ifn-γ secreting profiles. our data demonstrate that il-10-secreting cd8(+) t cells can arise during chronic mtb infection, although the significance of this t cell population in tuberculosis pathogenesis remains unclear. the factors that are responsible for the reactivation of latent mtb infection are not well understood, but likely involve contributions from both the host and the pathogen. to appreciate the role that the host immune system plays in mtb reactivation, we used relatively resistant (c57bl/6) or susceptible (cba/j) mice, whose susceptibility phenotype is most apparent during late stages of infection, to represent differences in the natural progression of tb between different human populations. cba/j mice have low numbers of antigen-specific cd4 t cells that produce relatively small amounts of ifn-c [1] [2] [3] [4] . cba/j mice also have elevated amounts of il-10 during mtb infection [5, 6] , contributing to their increased susceptibility to infection. however, the importance of cd8 + t cells during mtb infection in this mouse strain remains unclear. cd8 + t cells are an important component of the protective immune response to mtb, as defined by studies showing that mice deficient in cd8 + t cells had impaired control of mtb infection [7] [8] [9] [10] . although there is no consensus on the specific requirement for cd8 + t cells during mtb infection, cd8 + t cells can contribute to mtb control by secretion of ifn-c [11, 12] and cytotoxic lysis of host cells [13, 14] , yet their ability to maintain maximal effector function is dependent on cd4 + t cells [15] [16] [17] . studies have also reported that cd8 + t cells are most important during latent mtb infection in mice, and that cd8 + t cell depletion early after infection had little effect on disease outcome [18] . conversely, other studies suggest that cd8 + t cells are dispensable during mtb infection [19] [20] [21] . in chronic viral infection models, cd8 + t cells can become dysfunctional after chronic antigenic stimulation, characterized by a lack of functional or proliferative capability, secretion of il-10 [22] [23] [24] and surface expression of inhibitory molecules, such as programmed cell death-1 (pd-1) and t cell immunoglobulin and mucin protein-3 (tim-3) [25, 26] . pd-1 has classically been used as a marker of t cell exhaustion in viral infection and in cancer [27] [28] [29] [30] , while other studies have found that cells expressing tim-3 are dysfunctional and lack regulation [31, 32] , and that coexpression of pd-1 and tim-3 leads to extensive dysfunction of cd8 + t cells [33] . furthermore, cd8 + t cells expressing both pd-1 and cd122 (the b subunit of the il-2 receptor) have been shown to have suppressive qualities and secrete il-10 [34] . we, and others, have previously demonstrated that mtb susceptibility in cba/j mice is mediated by excessive pulmonary il-10 during infection [1, 2, 5, 35, 36] , yet the underlying mechanism remains unclear. although numerous cell types are capable of producing il-10, studies have previously shown that il-10-producing t cells can actively suppress the immune response in tb patients [37] , supporting an investigation into the il-10-producing properties of cd8 + t cells during mtb infection in cba/j mice. in this study we show that mtb-susceptible cba/j mice accumulated large numbers of cd8 + t cells in their lungs as mtb infection progressed that could not be fully accounted for by an expansion of ifn-c-producing cd8 + t cells. cd8 + t cell expansions expressed the inhibitory molecules pd-1, tim-3, and/ or cd122, and were capable of secreting il-10. cd8 + t cells from cba/j mice also preferentially expressed tcr vb8 and vb14, severely limiting the diversity of the cd8 + t cell repertoire. although vb8 cd8 + t cells could secrete il-10, in vivo depletion of this specific t cell clonal population during chronic infection did not overtly change the mtb burden in the lungs in the timeframe tested, although the amount of il-10 in the lung was reduced indicating some biological impact of depletion. comparing mouse strains that are relatively resistant and susceptible to mtb has enabled us to uncover a previously unappreciated role for cd8 + t cells in mtb susceptibility, and links the poor t cell function previously described by us [4, 6, 36] with increased production of il-10 in the cba/j mouse strain. this study was carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health. the protocol was approved by the institutional animal care and use committee of the ohio state university. specific pathogen-free, age/sex-matched cba/j wild-type (national cancer institute, nih, frederick, md), c57bl/6 wild-type (jackson laboratories, bar harbor, maine), or cba/j il-10 2/2 mice were maintained in ventilated cages inside a biosafety level 3 (bsl3) facility and provided with sterile food and water ad libitum. to generate cba/j il-10 2/2 mice, cba/j mice (jackson laboratories, bar harbor, maine) were crossed with c57bl/6 il-10 2/2 mice (jackson) for eight generations. at each cross progeny mice were ear-punched and dna was screened for the presence of a neomycin cassette at the il10 gene locus. il-10 +/ 2 mice were selected for further breeding. at the eighth generation, heterozygotes were crossed and il-10-deficient homozygote cba/j mice were selected. a homozygous breeder colony of cba/j il-10 2/2 mice was maintained thereafter. all protocols were approved by the ohio state university's institutional laboratory animal care and use committee. mtb erdman (atcc 35801) was obtained from the american type culture collection (manassas, va). stocks were grown in proskauer-beck liquid medium containing 0.05% tween 80 to mid-log phase and frozen in 1 ml aliquots at 280uc. mice were infected with mtb erdman using an inhalation exposure system (glas-col) calibrated to deliver 50-100 cfu to the lungs of each mouse, as previously described [38] . at specific time points post mtb infection mice were sacrificed and lungs were aseptically removed into sterile saline. organs were homogenized and serial dilutions plated onto 7h11 agar supplemented with oadc as previously described [4] . plates were incubated at 37uc for 21 days in order to enumerate bacterial colonies and calculate the bacterial burden. mice were euthanized by co 2 asphyxiation and lungs perfused with cold phosphate buffered saline containing 50 units/ml of heparin through the right ventricle of the heart. lungs from individual mice were mechanically disrupted using a gentlemacs dissociator (miltenyi biotec, boston, ma) followed by collagenase a (type xi) (0.7 mg/ml, sigma) and type iv bovine pancreatic dnase (30 mg/ml, sigma) digestion at 37uc for 30 minutes in gentlemacs c-tubes. lung cell suspensions were passed through a 70 mm nylon cell screen and residual erythrocytes were lysed with gey's solution. viable cells were determined by trypan blue exclusion. single lung cell suspensions were adhered to sterile tissue culture dishes for 1 hr at 37uc. non-adherent cells were washed and removed from the plates. cd4 + and cd8 + t cells were obtained from the non-adherent cell fraction by magnetic cell separation (bd imag anti-cd4 + particles gk1.5, anti-cd8 + particles 53-6.7) and either placed directly into trizol reagent (invitrogen, grand island, ny), homogenized, and frozen at 280uc or used for culture as described below. cd8 + vb8 + t cells were obtained from the cd4 neg fraction after treatment with anti-cd4 magnetic beads (bd), then stained with pe anti-vb8 (ebioscience) and purified using anti-pe magnetic particles (bd). purity of all cd4 + and cd8 + t cell populations was determined to be greater than 90% for all experiments by flow cytometry using an lsrii flow cytometer (bd biosciences, san jose, ca). for elispot, bone marrow-derived dendritic cells (bmdcs) were obtained from the tibiae and femora of age and sex matched non-infected wild-type or il-10 2/2 c57bl/6 or cba/j mice. cells were differentiated into dendritic cells using complete dmem supplemented with 10% conditioned media derived from gm-el4 cells, a gm-csf-producing clone kindly provided by arthur a. hurwitz (nci). 2610 6 bone marrow cells were plated at 37uc in 1 ml of gm-el4 conditioned media in sterile 24-well tissue culture plates. gm-el4 conditioned media was replaced on days 2, 4 and 6. 3610 4 bmdcs were infected overnight with mtb erdman at an moi of 1:1 then fixed in 2% paraformaldehyde (cba/j il-10 2/2 bmdcs were used unfixed). infected bmdcs were cultured with 2610 5 cd8 + t cells or cd8 neg t cells for 72 hr at 37uc in media containing either tissue culture media alone or 10 mg/ml anti-cd3 (145-2c11) and 1 mg/ml anti-cd28 (37.51). elispot reagents were obtained from ebioscience (e, f) 24 hr prior to necropsy mice were injected with brdu, and lung cells were analyzed for expression of brdu + cd4 + or cd8 + t cells. (g, h) absolute numbers of cd4 + or cd8 + t cells in wild-type or il-10 2/2 cba/j mice as determined by flow cytometry. results representative of at least three independent experiments with 5 mice per group, per timepoint. * p,0.05, ** p,0.01, *** p,0.001 as obtained by student's t test. (b, h) + p,0.05, ++ p,0.01, +++ p,0.001 as obtained by twoway analysis of variance comparing day 90 to day 150 post-infection. doi:10.1371/journal.pone.0058612.g001 ready-set-go! spot-forming units (sfu) were enumerated with an elispot plate counter (c.t.l.). for elisa, 1610 5 purified pulmonary cd8 + t cells were cultured with 10 mg/ml anti-cd3 (145-2c11) and 1 mg/ml anti-cd28 (37.51) for 72 hr at 37uc with 5% co 2 . after incubation, plates were frozen at 280uc until all timepoints were completed. elisa antibodies and standards were obtained from bd biosciences and processed as previously described [38] . colormetric reactions were read on a spectramax plate reader (molecular devices, sunnyvale, ca). isolated lung cells or mln were suspended in deficient rpmi (mediatech, manassas, va) supplemented with 0.1% sodium azide (sigma-aldrich). surface targets were detected as previously described. specific antibodies and isotype controls were purchased from bd biosciences: percp-cy5.5 anti-cd3e (145-2c11), allophycocyanin-cy7 anti-cd4 + (gk1.5), pe-cy7 anti-cd8 + (53-6.7), percp-cy5.5 anti-cd8 + (53-6.7), pe-cy7 anti-ifn-c (xmg1.2), pe anti-pd-1 (j43), fitc anti-cd122 (tm-beta 1), and fitc vb screening kit. pe anti-tim-3 (havcr2) antibody was purchased from ebiosciences. cytokine levels were determined according to the manufacturer's instructions for intracellular cytokine staining (cytofix/cytoperm fixation/permeabilization solution kit with bd golgistop, bd biosciences), following a 4 hr incubation with 1 mg/ml anti-cd3 (145-2c11) and 0.1 mg/ ml anti-cd28 (37.51). samples were read using an lsrii flow anti-cd8 + (53.6.72) depletion antibody (bioxcell) or whole rat igg2a (2a3) (bioxcell), were diluted to 2.5 mg/ml in pbs and stored at -80uc. thawed stocks were maintained at 4uc for up to 1 month. anti-vb8 (f23.1) antibody-secreting cell line [39] was kindly provided by dr. michael bevan. antibody administration was modified based on silva et al. [40] as follows: at day 90 postinfection, 0.5 mg of anti-cd8 + (53.6.72), anti-vb8 (f23.1), or control antibody was injected into the peritoneal cavity of each mouse, followed by 0.5 mg at weekly intervals thereafter until the designated experimental time points. at the initiation of antibody treatment cba/j mice consistently harbor 6.596 se 0.12 log 10 mtb cfu [4] . statistical analysis performed using graphpad prism software for the students t test per individual time point of each graph. any comparisons between timepoints of the same experiment utilize a two-way analysis of variance test with bonferonni post-tests for multiple comparisons. * p,0.05, ** p,0.01, *** p,0.001. following aerogenic infection of cba/j and c57bl/6 mice with mtb we observed a gradual accumulation of cd4 + t cells in the lungs of c57bl/6 mice and significantly fewer cd4 + t cells within the lungs of cba/j mice (fig. 1a) , as we have previously described [3, 4] . in contrast to the reduced number of cd4 + t cells, cba/j mice demonstrated a significant late accumulation of cd8 + t cells within the lungs as mtb infection progressed (fig. 1b) , eventually reaching or surpassing the number of pulmonary cd8 + t cells observed in c57bl/6 mice. this late accumulation was absent from c57bl/6 mice which reached a plateau at day 60. skewing of t cell subset proportions in cba/j mice could be better appreciated by determination of cd4:cd8 ratios throughout the course of infection (fig. 1c) , where a significant decline in the ratio of cd4 + to cd8 + t cells was observed by day 90 of mtb infection, preceding the increasing cfu within the lungs of cba/j mice (fig. 1d) [4, 6] . brdu staining of pulmonary cd4 + (fig. 1e) and cd8 + t (fig. 1f) cells from cba/j mice was not dramatically altered throughout infection, suggesting that the increased numbers of cd8 + t cells that were evident in cba/j mice may not be due to local proliferation, but a consequence of enhanced cellular recruitment. cba/j mice are known to produce abundant il-10 in their lungs as mtb infection progresses [5] and il-10 can stimulate the proliferation of cd8 + t cells [41] . therefore, we examined the numbers of cd4 + and cd8 + t cells present in the lungs of cba/j il-10 2/2 mice over the course of mtb infection (fig. 1g, h) and did not observe any differences between wild-type and il-10 2/2 cba/j mice in cd8 + t cell accumulations at late stages of infection, demonstrating that cd8 + t cell expansions occur independent of il-10. a significant increase in cd4 + and cd8 + t cells was observed in cba/j il-10 2/2 mice at day 30, reflecting enhanced t h 1 immunity in these mice (unpublished observations). these data demonstrate that the accumulation of cd8 + t cells in the lungs of cba/j mice during chronic infection is not mediated by il-10. cd8 + t cell expansions from mtb-infected cba/j mice do not align with ifn-c producing capacity we examined the number of t cells that were capable of secreting ifn-c after ex vivo tcr stimulation. as expected, c57bl/6 mice had increasing numbers of cd4 + [4] and cd8 + t cells that could produce ifn-c as infection progressed (fig. 2a) , which paralleled the increasing numbers of t cells within the lung (fig. 1) . cba/j showed an equivalent increase in ifn-cproducing cd4 + t cells (fig. 2b) , albeit at a significantly lower number compared to c57bl/6, as we have previously described [3, 4] . where the data differed, however, was in the finding that the number of ifn-c-producing cd8 + t cells reached a plateau as early as day 30 post-infection and did not increase any further, despite a significant accumulation of cd8 + t cells within the lungs of cba/j mice. as mtb infection progressed we also observed a significant increase in the number of cd8 + t cells from cba/j mice that could express cd69 (fig. 2c) , with over 40% of all cd8 + t cells in the lungs of cba/j mice being activated during chronic infection. cd8 + t cells also expressed the suppressor of t h 1 responses tim-3 (fig. 2d) , pd-1 (fig. 2e) , with a small population expressing both pd-1 and cd122 (fig. 2f) . therefore, although highly activated, a proportion of cd8 + t cell expansions from chronic mtb infected cba/j mice had a phenotype that was not associated with t h 1 cytokine secretion but was instead linked to immuno-suppressive properties including il-10 production [34] . because il-10 production has been closely linked to mtb susceptibility of cba/j mice [5] , we next examined the capacity of cd8 + t cells to produce il-10. cd8 + t cells from mtb-infected cba/j mice are capable of secreting il-10 cd8 + and cd8 neg cells were purified from the lungs of mtbinfected cba/j and c57bl/6 mice and cultured in il-10 elispot plates for 72 hours with autologous bone marrow-derived dendritic cells (bmdcs) that had been infected with mtb for 24 hours, in the presence or absence of anti-cd3 and anti-cd28. cd8 + cells from cba/j mice, and not c57bl/6 mice, were capable of secreting il-10 in response to tcr cross-linking (fig. 3a) . in contrast, cd8 neg cells from both mouse strains were capable of secreting il-10 under these same conditions (fig. 3b) . il-10-secreting cd8 neg cells were predominantly cd4 + as adherent cells were removed during cell purification and we have failed to detect il-10 within b cells and neutrophils in both mouse strains (not shown). we also observed that cd8 + t cells from cba/j and c57bl/6 mice were capable of secreting ifn-c under the same culture conditions (measured in the il-10 elispot supernatants) (fig. 3c) . culture of purified cd8 + and cd8 neg cells from mtb infected cba/j mice with mtb-infected bmdcs in the absence of tcr cross-linking, indicative of an mtb-specific response, resulted in the secretion of il-10 from both cd8 neg and cd8 + cells (fig. 3d) . interestingly, the capacity of cd8 + t cells to produce il-10 increased over time, in parallel to the increasing cfu and cd8 + t cell numbers in the lung. the low sfu, relative to tcr cross-linking likely reflects the challenges of delivering of mtb antigen into the appropriate processing pathway for presentation to cd8 + t cells. we determined the clonal repertoire of the cd8 + t cells that accumulated in the lungs of cba/j mice during mtb infection to determine whether the reactivity of cd8 + t cells was potentially driven by a dominant antigen, a concept supported by the finding of t cell clonal expansions in tb patients [42, 43] . at late stages of mtb infection, we observed that cd8 + t cells from cba/j mice primarily expressed two variable regions of the tcr beta chain (vb). vb8 (8.2,8.3) and vb14 were expressed by approximately 45% of all cd8 + t cells in cba/j mice at day 120 after mtb infection, compared to a less restrictive repertoire in c57bl/6 mice (fig. 4a) . vb expression was comparable on cd4 + t cells from both mouse strains (fig. 4b) , indicating a unique expansion within the cd8 + t cell pool in cba/j mice as mtb infection progressed. more detailed examination over the course of mtb infection showed that the percentage of cd8 + t cells expressing vb8 or vb14 was always significantly higher throughout mtb infection in cba/j mice, and this population significantly expanded further at late time points of mtb infection (fig. 4c, d) . similar to our findings with cd8 + t cell expansions, we found that both vb8 and vb14 cd8 + t cells were highly activated (fig. 4e) . we determined the il-10 producing capacity of cd8 + vb8 + t cells using magnetic bead separation, which purified the dominant vb8 expressing cd8 + population. cd8 + vb8 + and cd8 + vb8 neg t cells were isolated from mtb-infected cba/j mice or c57bl/6 mice (controls) at day 150 post mtb infection and cultured in il-10 elispot plates with autologous il-10 deficient bmdcs in the presence of anti-cd3/cd28. significantly more cd8 + vb8 + t cells from mtb-infected cba/j mice were capable of producing il-10 than cd8 + vb8 neg t cells from cba/j mice or from cd8 + vb8 + and cd8 + vb8 neg t cells from c57bl/6 mice (fig. 4f) . these data indicate a dominant role for cd8 + vb8 + expressing t cells in the il-10 production we previously observed in purified cd8 + t cell cultures. culture supernatants were also assayed for ifn-c by elisa (fig. 4g) and our data indicate that cd8 + vb8 + t cells can have dual secretion of il-10 and ifn-c, or represent a mixed population, as has been described by others [44] . in vivo depletion of il-10 producing cd8 + t cells during chronic mtb infection alters pro-inflammatory responses but fails to modify the bacterial load cd8 + t cells or vb8 + cells were depleted from wild-type cba/ j mice from day 90-120 after mtb infection, a time when il-10 and vb8 + cd8 + t cells were increasing in the lungs of mtbinfected cba/j mice [5] . following cd8 + depletion, mtb cfu (fig. 5a) , total cd4 + t cell numbers (fig. 5b) and ifn-cproducing cd4 + t cells (fig. 5c) were all moderately altered but data did not reach statistical significance. interestingly, cd8 + t cell depletion led to a significant decrease in the total amount of (fig. 5d) , reflecting the il-10secreting capacity of cd8 + t cells we observed in vitro. depletion of cd4 + t cells led to significantly increased mtb burden and mortality before day 120 (not shown) showing that depletion of a known protective t cell subset increased susceptibility. specific depletion of vb8 + cells also failed to significantly impact the mtb burden (fig. 5e) , although a modest reduction in cfu was similarly observed. this modest reduction, similar to our findings with cd8 + t cell depletion, suggests that although il-10 producing cd8 + t cells may not negatively impact the growth of mtb during the timeframe we investigated, they do not provide protection. we have demonstrated that clonal expansions of cd8 + t cells from cba/j mice accumulate in the lung over the course of mtb infection. this accumulation did not yield an equivalent increase in ifn-c + cd8 + t cells and, upon further phenotypic examination, we discovered that highly activated cd8 + t cells from cba/j mice expressed the t cell dysfunction markers pd-1 and tim-3, as well as co-expression of pd-1 and cd122 suggesting possible immunosuppressive activity. after ex vivo purification and culture, it was shown that cd8 + t cells from cba/j mice were capable of secreting il-10 after tcr stimulation and in response to mtb infected bmdcs. depletion of vb8 + cells or the entire cd8 + t cell population in mtb-infected cba/j mice led to a significant reduction in pulmonary il-10 levels, however, we observed no significant change in pulmonary cfu. using an mtb-susceptible mouse strain, we reveal that cd8 + t cells that are capable of producing il-10 can accumulate within the lung during mtb infection. cd8 + t cells within the lung expressed cd69, indicative of activation and functional capacity, yet failed to show a concomitant enhancement of ifn-c-producing capacity. these findings indicate that highly activated cd8 + t cells within the lung have alternate function, which we show here to be the capacity to secrete il-10, measured by elispot due to the known difficulties of measuring il-10 by intracellular flow cytometry. altered function was associated with the co-expression of a variety of receptors know for negative regulation of cell function (pd-1, cd122, tim-3) [25, 28, 33, 34] . in support of our findings, previous studies have shown that in chronic murine mtb infection pd-1 + t cells can proliferate but fail to secrete ifn-c unless this inhibition is overcome by direct tcr stimulation [45, 46] . it is unclear at this time why cd8 + t cells with inhibitory properties arise in cba/j mice as mtb infection progresses but we can hypothesize that this is a consequence of enhanced immune activation and subsequent exhaustion due to increasing bacterial loads in this mouse strain (fig. 1) [4, 6] . our failure to observe any significant change in cfu following cd8 + or vb8 + t cell depletion can be interpreted in several ways, the simplest being that il-10-producing cd8 + t cells have no biological influence on the control of mtb infection. while this is a possibility, we would reason that at the least the presence of il-10producing cd8 + t cells can be a putative biomarker of tb disease progression as they are only observed in a mouse strain of mtb susceptibility, and il-10 producing t cells have been previously described in the blood of tb patients [37] . in support of a negative role for cd8 + t cells in cba/j mice, in vivo depletion led to a moderate, albeit not significant, decrease in cfu. thus is in contrast to cd4 + t cell depletion in which all the mice were either dead or moribund before the necropsy time point (data not shown). these findings indicate that some cd8 + t cells from cba/j mice fail to contribute to protection in a similar manner than might be expected from studies of other mouse strains [47] [48] [49] . one possibility for our failure to detect significant changes in cfu following cd8 + t cell depletion is our observation that cd8 + t cells can secrete both il-10 and ifn-c. depletion of all cd8 + t cells would also remove a protective population, leading to a neutral effect. previous studies from our laboratory have shown that blocking the action of il-10 in cba/j mice during mtb infection provides enhanced protection [5] , and we consider that blocking the action of il-10 from il-10/ifn-c-secreting cd8 + t cells contributed to this phenotype. in addition to il-10 production, we also observed that cd8 + t cells from cba/j mice had severely restricted diversity of their tcr repertoire which would significantly limit the breadth of antigens that cba/j cd8 + t cells can recognize. cba/j mice have an endogenous mouse mammary tumor virus (mtv-6) that selectively deletes various tcr vb chains such as vb8.1, and vb 17a [50, 51] however this alone is not sufficient to explain the limited tcr diversity in cba/j mice. it is possible that mtb infection leads to specific deletion of certain subsets of protective cd8 + t cells or is driving clonal expansion of less protective cells (vb8, vb14). alternatively vb8/vb14 cd8 + t cells in cba/j mice may respond to a dominant mtb antigen(s) expressed in vivo, where persistent responsiveness leads to immune exhaustion and subsequent regulation through receptors such as pd-1 and tim-3. clonal cd8 + expansions have been described in tb patients [52] , including pediatric tb [42] , supporting the relevance of our findings to tb in man. clonal cd8 + expansions are also common in the elderly and in many viral models [42, 43, [53] [54] [55] [56] [57] [58] , yet their importance in mtb infection is still unclear. in summary, we show using an mtb-susceptible mouse strain (cba/j) that clonal expansions of cd8 + t cells with the capacity to produce il-10 can arise during chronic mtb infection. in combination with our previous finding that blocking the action of il-10 can improve outcome of mtb infection [5] , these data further support a negative role for il-10 in the generation and maintenance of protective immunity against mtb infection. our findings are of significance because vaccines that specifically stimulate cd8 + t cells are currently under development [59] [60] [61] [62] [63] . given that il-10-secreting cd8 + t cells with the potential to negatively impact control of mtb infection can naturally arise, generating such cells in response to vaccination should be considered. cba/j mice generate protective immunity to soluble ag85 but fail to respond efficiently to ag85 during natural mycobacterium tuberculosis infection h-2 alleles contribute to antigen 85-specific interferon-gamma responses during mycobacterium tuberculosis infection immunological basis for reactivation of tuberculosis in mice peripheral blood gamma interferon release assays predict lung responses and mycobacterium tuberculosis disease outcome 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vaccination new tb vaccines: is there a requirement for cd8 t cells? activation of cd8 t cells by mycobacterial vaccination protects against pulmonary tuberculosis in the absence of cd4 t cells cd8 cytotoxic t cells and the development of new tuberculosis vaccines key: cord-000352-qzkpik3z authors: carvalho, gabrielle; poalas, konstantinos; demian, catherine; hatchi, emeline; vazquez, aimé; bidère, nicolas title: participation of the cell polarity protein pals1 to t-cell receptor-mediated nf-κb activation date: 2011-03-30 journal: plos one doi: 10.1371/journal.pone.0018159 sha: doc_id: 352 cord_uid: qzkpik3z background: beside their established function in shaping cell architecture, some cell polarity proteins were proposed to participate to lymphocyte migration, homing, scanning, as well as activation following antigen receptor stimulation. although pals1 is a central component of the cell polarity network, its expression and function in lymphocytes remains unknown. here we investigated whether pals1 is present in t cells and whether it contributes to t cell-receptor (tcr)-mediated activation. methodology/principal findings: by combining rt-pcr and immunoblot assays, we found that pals1 is constitutively expressed in human t lymphocytes as well as in jurkat t cells. sirna-based knockdown of pals1 hampered tcr-induced activation and optimal proliferation of lymphocyte. we further provide evidence that pals1 depletion selectively hindered tcr-driven activation of the transcription factor nf-κb. conclusions: the cell polarity protein pals1 is expressed in t lymphocytes and participates to the optimal activation of nf-κb following tcr stimulation. establishment and maintenance of cell polarity is chiefly orchestrated by a tightly regulated interplay between three multi-protein complexes: i) scribble (scrib)/discs large (dlgh1)/lethal giant larvae (lgl) complex, ii) partitioning-defective (par) 3 and par6/ atypical protein kinase c (apkc) complex, and iii) crumbs (crb)/ protein associated with lin seven 1 (pals1)/ pals1-associated tight junction protein (patj) complex [1, 2] . however, each complex is not exclusive, as par6 links pals1 to par3/par6/apkc [3] . in t lymphocytes, cell polarity proteins were shown to partition the leading edge from the uropod at the cell rear, and therefore participate to cell migration, homing, and scanning [4, 5, 6] . in addition, scrib and dlgh1 are transiently recruited to the nascent immunological synapse formed with an antigen-presenting-cell (apc) [4] . their depletion in lymphocytes has been associated with an alteration of antigen receptor-mediated activation [7, 8, 9, 10] . the adaptor pals1 is crucial for cellular architecture as it maintains the apico-basal polarity in epithelial cells and authorizes indirect interactions between crb and patj [11, 12] . interestingly, dlgh1 and pals1 share a cooh-terminal part composed of a psd-95/dlg/zo-1 (pdz) domain followed by an sh3 domain adjacent to an inactive guanylate kinase (gk) homology region [2] . this unique sequence of pdz/sh3/gk defines the so-called membrane-associated guanylate kinase (maguk) proteins family, a group of molecules that serve as scaffolds to organize multi-protein signalosomes through their protein-protein interaction domains [13] . for example, the maguk-containing carma1 emerges as a central regulator of lymphocytes activation and proliferation downstream of antigen receptor stimulation [14] . indeed, carma1 operates as scaffold to recruit the heterodimer bcl10/ malt1 (cbm complex), a key step for conveying nf-kb signaling [14, 15, 16] . in addition to its established role in polarity, dlgh1 was shown to modulate lymphocyte proliferation upon t-cell receptor ligation, possibly through p38 recruitment or via the transcription factor nf-at [9, 10, 17, 18] . although the maguk pals1 plays a central role in the establishment of cell polarity, its contribution to lymphocyte activation remains elusive [8] . here we show that pals1 mrna and protein is expressed in human lymphocytes. furthermore, knocking down of pals1 with small interfering rnas (sirnas) led to a decreased proliferation of human t lymphocytes, resulting from a reduced activation of the transcription factor nf-kb. although several cell polarity proteins have been characterized in lymphocytes [4, 5] , pals1 expression in t cells remains to be determined [8] . to address this question, we first performed rt-pcr analysis on resting human cd3 + t cells and jurkat lymphocytes extracts, and detected mrna for pals1 ( figure 1a ). these mrna were efficiently translated into protein, as antibodies against pals1 detected a band, which was absent from pals1-sirna transfected primary t lymphocytes lysates ( figure 1b ). similar results were obtained with jurkat t cells ( figure 1b ). of note, pals1 levels remained unchanged in cells stimulated with antibodies to cd3 and cd28, or with pma and ionomycin ( figure 1c ). we next investigated pals1 subcellular location by confocal microscopy. in contrast to epithelial cells where it accumulate to tight junctions [12] , pals1 did not reach membrane domains and remains essentially cytosolic with punctuate structures. additional staining revealed that these structures coalesced with the golgi apparatus ( figure 1d and figure s1 ). accordingly, brefeldin a-triggered disassembly of the golgi apparatus also disrupted pals1 punctuate structures ( figure 1d ). this is reminiscent of pals1 relocation to the golgi apparatus in cells infected with sars coronovirus [19] . last, we observed that tcr-mediated stimulation only promoted a discrete redistribution of pals1 within the cytosol of jurkat cells ( figure s1 ). altogether, our results suggest that similarly to dlgh1, scrib, crb3, and pkcf [4, 5] , the cell polarity protein pals1 is expressed in lymphocytes at both mrna and protein level. because scrib and dlgh1 were proposed to modulate lymphocyte proliferation [7, 8, 9, 10] , we evaluated whether pals1 might also participate to t cell activation. to this end, peripheral blood lymphocytes (pbl) were purified on ficoll-isopaque gradients. primary human t cells were nucleofected for three days with sirna targeting pals1, prior stimulation with anti-cd3 and anti-cd28 antibodies. pals1 knockdown led to a significant decrease in tcr-mediated induction of the activation markers cd69 and cd25 on cell surface (figure 2a , b). this was accompanied by a reduction in carboxyfluorescein succinimydyl ester (cfse) dilution, which reflects cell proliferation ( figure 2c ). collectively, these data suggest that pals1 participates to the optimal lymphocyte activation and subsequent proliferation upon tcr stimulation. to further explore how pals1 impacts on lymphocyte proliferation, early signaling pathways emanating from the tcr were examined in jurkat cells transfected with pals1 sirna. we did not detect major alteration in the general pattern of tyrosine phosphorylation, or mitogen-activated protein kinase (mapk) extracellular signal-regulated kinases (erk) 1/2 phosphorylation upon tcr stimulation ( figure 3a ). only a slight but consistent increase in tcr-mediated phosphorylation of p38 was noted ( figure 3a ). moreover, cd3-induced calcium mobilization was largely normal in pals1-knockdown jurkat cells ( figure 3b ). we next analyzed tcr-mediated activation of nf-at and nf-kb transcription factors. sirna-treated jurkat t cells were cotransfected with firefly luciferase constructs driven by nf-at or nf-kb binding sequences and with a renilla luciferase control. pals1 knockdown had only a marginal effect on nf-at activity following stimulation with pma and ionomycin, or with antibodies to cd3 and cd28 ( figure 3c and figure s2 ). in sharp contrast, nf-kb activity was significantly reduced without pals1 ( figure 3d ). interestingly, tumor necrosis factor-a (tnfa)induced nf-kb activation remained essentially unaffected, underscoring the selective involvement of pals1 in the tcr-nf-kb pathway ( figure s3 ). altogether, our data unveiled an unexpected role for pals1 in tcr-mediated nf-kb activation. to gain insights on how pals1 modulate nf-kb, we first investigated the transcription factor binding ability by electrophoretic mobility shift assay (emsa). less nf-kb bound to its specific probe in nuclei extracts from pals1-sirna transfected cells following tcr stimulation ( figure 4a ). as expected, oct-1 binding remained unchanged without pals1. consistent with a diminished nf-kb activity, both the phosphorylation and subsequent proteasomal degradation of nf-kb inhibitor, ikba, were severely decreased in the absence of pals1 ( figure 4b ). because tcr-induced nf-kb activation relies on the assembly of the cbm complex [15] , bcl10 was immunoprecipitated from nonspecific (ns-) and pals1-sirna transfected jurkat cells. malt1, which forms an heterodimer with bcl10, coprecipitated with bcl10 regardless of stimulation. although pals1 was not found bound to bcl10, its absence diminished carma1 recruitment ( figure 4c , and data not shown). hence, our data suggest that pals1 participates to the optimal translocation and activation of nf-kb upon tcr stimulation, possibly by favoring the cbm assembly. since pals1 nucleates a ternary complex containing crb3 and patj, and further binds par6 to maintain cell polarity [3, 20, 21] , their contribution to tcr-mediated nf-kb was evaluated. similarly to pals1, mrna for patj, crb3, par6, were efficiently detected by rt-pcr ( figure 5a ). the same hold true for the unrelated cell polarity protein scrib ( figure 5a ). sirna-based knockdown of pals1 and crb3 significantly decreased nf-kb activation in cells treated by antibodies against cd3 and cd28, or with a mixture of pma and ionomycin. although less dramatic, similar results were observed with par6 or patj knockdown. by contrast, nf-kb was normally activated in the absence of scrib ( figure 5b ). in agreement, ikba phosphorylation was diminished in lysates from crb3-depleted cells, and to a lesser extent from patj-or par6-sirna transfected cells, and not from scrib-depleted cells. again, erk phosphorylation occurred normally ( figure 5c , d, e, and f). altogether, our data suggest that pals1 implication in the tcr-nf-kb pathway is inextricably linked to its cell polarity partners. in summary, our data show that the cell polarity protein pals1 is expressed in lymphocytes and contributes to their optimal activation. although dlgh1 and scrib were proposed to modulate nf-at or p38 [9, 10, 17, 18] and nf-at [7] respectively, a distinct scenario likely occurs for pals1. our results support a model in which pals1 participates to nf-kb activation, upstream of ikba phosphorylation and degradation. however, how precisely pals1 modulates nf-kb remains unclear. because maguk function as scaffold units to organize and integrate multi-molecular signaling complexes [13] , it is tempting to speculate that pals1 nucleates its own signalosome. for example, carma1 anchors a .900 kda complex including the heterodimer bcl10/malt1 [22] , and dlgh1 was reported to bind to lck, zap70, wasp [17] , and p38 [10] . in our hands, pals1 did not integrate the cbm, but its absence reduced carma1 binding to bcl10. it will therefore be interesting to identify pals1 partners in the context of lymphocyte activation. in line with this, crb3, patj and par6, which all bound pals1 to maintain cell polarity [2] , also participate to nf-kb signaling upon tcr ligation in lymphocytes, and might therefore complex with pals1 in lymphocytes. altogether, our results strengthen the unexpected function of cell polarity proteins in lymphocyte proliferation [7, 8, 9, 10] , and unveil an original role for pals1 during tcr-mediated nf-kb activation. jurkat t cells e6.1 were purchased from atcc. cd3 + human t lymphocytes from healthy donors (etablissement francais du sang) were isolated with the midimacs system (miltenyi biotec). cells were activated with a mixture of soluble anti-cd3e (hit3a, bd biosciences) and anti-cd28 (bd biosciences), or with 20-40 ng.ml 21 phorbol 12-myristate 13-acetate (pma, sigma) and 300 ng.ml 21 ionomycin (calbiochem). carboxyfluorescein succinimydyl ester (cfse) and brefeldin a were purchased from sigma, and the calcium-sensitive dye fluo-4 was from invitrogen. cells were washed twice with pbs 1x and lysed with 50 mm tris ph 7.4, 150 mm nacl, 1% triton x-100, 1% igepal, 2 mm edta, supplemented with complete protease inhibitors (roche). lysates were cleared by a centrifugation at 10,000g at 4 o c, and protein concentration determined (micro bca kit, pierce). samples were resolved on 5-20% sds-page gels and transferred to nitrocellulose membranes (amersham). for immunoprecipitations, samples were precleared with protein g-sepharose beads (roche) for 30 min prior to overnight incubation with antibodies and additional protein g-sepharose beads at 4uc, as previously described [23] . antibodies to bcl10 (a-6), ikba (c-21), malt1 (b-12), tubulin (tu-02), pals1 (h-250), scrib (c-6), par6 (g-9) and p65 (c-20) were purchased from santa cruz. phosphospecific antibodies against ikba, erk, p38, and antibodies to carma1 and to erk were from cell signaling technologies. anti-phosphorylated tyrosine (4g10, millipore), anti-gapdh (sigma), and immobilon (millipore) chemiluminescent substrates were also used. firefly luciferase constructs downstream of promoters for nf-kb or nf-at were co-transfected with renilla luciferase prl-tk (int -) plasmid (promega). luciferase activities were analyzed using the dual-luciferase kit (promega), with firefly fluorescence units normalized to renilla luciferase fluorescence units (bmg microplate reader). were purified from blood on ficoll-isopaque gradients. pbl were nucleofected with the nucleofactor system and t cell solution (amaxa, program u14), and left for three days in culture medium prior treatment. nuclear protein extraction and electrophoretic mobility shift assay (emsa) 4 mg of nuclear extracts from jurkat cells were examined for nf-kb-and oct1-binding activity by electromobility shift assay (panomics kit). samples were resolved on a 6% native polyacrylamide dna retardation gel in 0.5x tbe buffer and analyzed using a fuji la4000 system. cells were left for 10 min on poly-lysine coated slides (thermo scientific) prior fixation with pbs1x containing 4% paraformaldehyde. for tcr crosslinking experiments, cells were incubated with 5 mg.ml 21 anti-cd3 at 4uc for 15 min. after two washes, cells were incubated with 5 mg.ml 21 of goat antimouse (jackson) for 20 min either at 4uc or 37uc. to disassemble golgi apparatus, cells were treated with 10 mg.ml 21 brefeldin a for 60 min. samples were permeabilized with 0.05% triton-x100 in pbs1x for 5 min, and non-specific sites blocked with 10% fcs in pbs1x. antibodies used were: pals1 (millipore), 58k golgi (abcam), alexa-488 conjugated goat anti-rabbit igg or alexa-594 conjugated goat anti-mouse igg (invitrogen). samples were analyzed using a leica confocal microscope sp6. cells were incubated for 30 min at 4uc with fitc-and peconjugated antibodies against cd25 and cd69 (immunotools) and the respective isotype controls in pbs containing 0.5% bsa. after one wash with ice-cold pbs-bsa, cells were analyzed by flow cytometry with a facscalibur (bd biosciences). crosstalk between small gtpases and polarity proteins in cell polarization polarity complex proteins direct interaction of two polarity complexes implicated in epithelial tight junction assembly a network of pdz-containing proteins regulates t cell polarity and morphology during migration and immunological synapse formation cutting edge: atypical pkcs regulate t lymphocyte polarity and scanning behavior upsides and downsides to polarity and asymmetric cell division in leukemia the pdz domain-binding motif of the human t cell leukemia virus type 1 tax protein induces mislocalization of the tumor suppressor hscrib in t cells maintenance and modulation of t cell polarity dlgh1 is a negative regulator of t-lymphocyte proliferation scaffold protein dlgh1 coordinates alternative p38 kinase activation, directing t cell receptor signals toward nfat but not nf-kappab transcription factors molecular cloning and characterization of pals, proteins associated with mlin-7 the maguk protein, pals1, functions as an adapter, linking mammalian homologues of crumbs and discs lost membrane-associated guanylate kinases regulate adhesion and plasticity at cell junctions carma1-mediated nf-kappab and jnk activation in lymphocytes antigen receptor signaling to nf-kappab via carma1, bcl10, and malt1 interplay between bcl10, malt1 and ikappabalpha during t-cell-receptormediated nfkappab activation dlgh1 coordinates actin polymerization, synaptic t cell receptor and lipid raft aggregation, and effector function in t cells discs large (dlg1) complexes in lymphocyte activation the sars coronavirus e protein interacts with pals1 and alters tight junction formation and epithelial morphogenesis patj regulates tight junction formation and polarity in mammalian epithelial cells the crumbs3-pals1 complex participates in the establishment of polarity in mammalian epithelial cells malt1 ubiquitination triggers nf-kappab signaling upon t-cell activation casein kinase 1alpha governs antigen-receptor-induced nf-kappab activation and human lymphoma cell survival we thank d. arnoult and j. gavard for helpful discussions and critical reading of the manuscript. conceived and designed the experiments: gc nb. performed the experiments: gc cd kp eh nb. analyzed the data: gc cd kp eh av nb. wrote the paper: gc nb. key: cord-001162-z8cbbit3 authors: yun, heather c.; fugate, william h.; murray, clinton k.; cropper, thomas l.; lott, lisa; mcdonald, j. matthew title: pandemic influenza virus 2009 h1n1 and adenovirus in a high risk population of young adults: epidemiology, comparison of clinical presentations, and coinfection date: 2014-01-08 journal: plos one doi: 10.1371/journal.pone.0085094 sha: doc_id: 1162 cord_uid: z8cbbit3 background: in 2009, pandemic h1n1 influenza virus (2009 h1n1) emerged worldwide, causing morbidity and mortality that disproportionately affected young adults. upper respiratory infection (uri), largely due to adenovirus, is an endemic cause of morbidity in military training. whether clinical presentations differ or excess morbidity results from coinfection is unclear. methods: the center for advanced molecular detection evaluates epidemiology and rapid diagnostics of respiratory pathogens in trainees with uri. from may 1, 2009, to november 30, 2009, demographic, clinical, and pcr data from throat and nasal specimens for adenovirus and 2009 h1n1 were prospectively collected. results: 375 trainees with uri enrolled and were tested for both adenovirus and 2009 h1n1 by pcr (median age 20; 89% male). adenovirus pcr was positive in 72% (96% serotype e-4) and 2009 h1n1 in 20%. males were more likely to have adenovirus and females more likely to have 2009 h1n1 (p = 0.047). subjects with 2009 h1n1 presented an average of 1 week earlier in training, had shorter illness duration before enrollment, less sore throat, diarrhea, and fewer abnormal findings on throat exam. coryza and cough were more common with 2009 h1n1 compared to adenovirus. subjects with 2009 h1n1 were less likely to have adenovirus than those without, despite persistently high frequencies of adenovirus detections during peak 2009 h1n1 weeks (15% vs. 83%, p < 0.01). coinfection with adenovirus and 2009 h1n1 was rare (4%). rates of hospitalization and pneumonia did not differ between the adenovirus, 2009 h1n1, or coinfected groups. conclusion: military trainees with 2009 h1n1 vs. adenovirus have differing clinical presentations, and males are more likely to have adenovirus. despite high frequencies of adenovirus infection, coinfection with adenovirus and 2009 h1n1 is rare and apparently does not result in increased morbidity. non-influenza related upper respiratory infections (uri) are universally experienced illnesses that, despite their typically selflimited nature, lead to billions of dollars of lost income, and predispose to serious illnesses including pneumonia. [1] when influenza is responsible, pandemics can result and cause millions of deaths. in 2009, a novel h1n1 influenza virus (2009 h1n1) emerged and rapidly spread worldwide, causing excess mortality in children and young adults. although the global estimate of deaths has been lower than seen in several previous pandemics, the number of life years lost is estimated to be five times higher than those lost to seasonal h1n1 viruses and comparable to the number lost during the 1968 pandemic. [2, 3] military trainees, along with other groups of crowded, stressed individuals, are disproportion-ately affected by respiratory illnesses due to a variety of pathogens. with the exception of the prior adenovirus vaccine era from 1980-1996, adenoviruses have historically been the most common causes of febrile uri in this population, and have also led to serious illness and fatalities. [4] [5] [6] [7] in one large study of transmission dynamics of adenovirus in a military training setting, approximately one-third of incoming trainees were already immune, one-third developed a febrile uri due to adenovirus, and the remainder seroconverted with subclinical or asymptomatic infection. [8] large influenza outbreaks are less common, given the universal immunization of basic trainees and routine use of ring antiviral chemoprophylaxis in training units with known influenza cases, if cases occur within the first two weeks after immunization. [9, 10] however, in 2009, type-specific influenza vaccine was not widely available until well into the full wave of illness. [11] with large numbers of concurrently circulating respiratory pathogens occurring year round in this diverse group of individuals, coming from a variety of geographic locations and backgrounds, and living in close contact for months, coinfection with multiple organisms would be expected to be a regular occurrence. however, whether coinfection contributes to differing clinical presentations or outcomes in this young, healthy adult population is unknown. while coinfections with viral pathogens including 2009 h1n1 have been described in patients with respiratory infections, few prospective studies have related these to clinical presentation and outcomes in adults since molecular diagnostics became available, and none in the setting of high background rates of adenovirus. [12] [13] [14] [15] [16] [17] we sought to describe the epidemiology of 2009 h1n1 and adenovirus in a basic training population, and to correlate differences in clinical presentations and outcomes with each respective pathogen and in coinfections. joint base san antonio-lackland is the only air force location for basic military training with approximately 43,000 recruits per year, 6,000-7,000 recruits training at any given time, and a training period lasting 8.5 weeks. basic military trainees (bmts) are assigned to training units called ''flights'' of 50-60 individuals, with whom they train and reside in bay dormitories; tobacco product use is not allowed. ill trainees present for care at an outpatient clinic; if they are febrile with a respiratory illness they are then cohorted to a ''fever flight'' where they recover until they are afebrile and able to return to training. trainees who require hospitalization are admitted to the tertiary care hospital on base. trainees routinely receive chemoprophylaxis against streptococcus pyogenes during their first week of training; this consists of benzathine penicillin or azithromycin for penicillin allergic recruits. immunizations against meningococcus, hepatitis a and b, and measles, mumps and rubella are also administered during the first week of training. trivalent seasonal influenza vaccine was administered during the first week of training throughout the study period, but 2009 h1n1 vaccine was not available until december 1, 2009 . during the study period, oseltamivir was routinely used for treatment of ill trainees with confirmed 2009 h1n1 infection. oseltamivir was also routinely used for chemoprophylaxis of well trainees in close contact with a confirmed case. the center for advanced molecular detection (59 th medical wing/science and technology, air education and training command) was established in 2003 for prospective evaluation of epidemiology and novel technologies to rapidly detect respiratory pathogens in trainees with uri. subjects were approached for enrollment at the point of care for their uri, and met inclusion criteria if they were bmts 17 years of age or older and had any symptom of upper respiratory tract infection or pneumonia. demographic data, including age, race, gender, week of training, city/state of previous residence, and smoking history, were recorded. additionally, a symptom questionnaire (including respiratory and gastrointestinal symptoms), perceived stress level on a 10-point likert scale, and clinical signs, including vital signs, height and weight, physical exam findings, and physician diagnosis were recorded, as was the ward of hospital admission (intensive care unit vs. ward) where applicable. for this substudy, cases were included if they enrolled in the study and were tested for both adenovirus (using study methodology) and 2009 h1n1 (as part of clinical care). duplicate cases (for numerous presentations for uri nucleic acid extraction primers and probes used have been reported for adenovirus by heim et al. [19] all qpcr was conducted using applied biosystems 7900 and 7500 real-time pcr instruments (applied biosystems, ca). for adenovirus testing, cycling was conducted with 500 nm concentrations of both forward and reverse primers and 300 nm concentration of probe. reaction conditions included an initial 10 min denaturation at 95uc, followed by 45 cycles of 95uc for 15 sec and 60uc for 1 min. a specimen was considered positive if its cycle of threshold (ct value) was equal to or less than 40, as described previously. [19, 20] . all subjects provided written, voluntary informed consent in the presence of an ombudsman. the study was approved by wilford hall medical center/brooke army medical center institutional review board (irb). gender and ethnicity were self-reported. per department of defense (dod) directive 3216.02, for purposes of legal capacity to participate in dod-conducted or -supported research involving human subjects, all active duty service members in a federal duty status are considered to be adults. the participation of such members is not subject to requirements regarding research involving children or minors. when service members are under 18 years of age, students at service academies, or trainees, the irb shall carefully consider the recruitment process and the necessity to include such members as human subjects. the wilford hall medical center/brooke army medical center irb carefully considered the bmt recruitment process for the study and ruled that consenting bmts 17 years of age or older who presented to the health clinics could be enrolled. data were entered in duplicate for quality control. analysis was performed using existing software (spss, version 19.0; spss). continuous variables were analyzed by student's t-test or mann-whitney u test for parametric and nonparametric data, respectively. categorical variables were evaluated by chi-squared, fisher's exact test, or spearman correlation. multiple nonparametric groups of continuous variables were analyzed by kruskal-wallis testing. all p-values are two-tailed and statistical significance at p ,0.05. table 2 , and seasonal variation presented in figure 1 . during the peak of 2009 h1n1 activity among trainees, weeks 33-42, out of 130 subjects, adenovirus was found alone in 58, 2009 h1n1 alone in 51, and 9 were coinfected; neither virus was recovered from 12 subjects. during that time frame, there was a negative association between 2009 h1n1 and adenovirus (p,0.01). subjects with 2009 h1n1 were much less likely to be adenovirus positive than those without 2009 h1n1 (15% vs. 83%). subjects with adenovirus were much less likely to have 2009 h1n1 than those without adenovirus (13% vs. 81%). a comparison of demographic information in subjects infected with 2009 h1n1 vs. adenovirus is presented in table 3 . significant findings include gender disparities of 2009 h1n1 and adenovirus infection, with a larger proportion of males presenting with adenovirus (p = 0.047). including subjects with coinfections, adenovirus was detected in 60% of female subjects vs. 74% of male subjects, and 2009 h1n1 in 30% of female subjects vs. 19% of male subjects. 2009 h1n1 infected subjects also presented one week earlier in training. however, after exclusion of cases diagnosed in the first week of training, the distributions of the epidemic curves were similar and both peaked in the fifth week (data not shown). adenovirus detections peaked earlier in the year compared to 2009 h1n1 infections. the most commonly reported symptoms were fever (100%), cough (90%), headache (88%), and sore throat (87%), and the median duration of symptoms prior to enrollment was 3 days (table 4 ). six patients were diagnosed with pneumonia, and three were admitted to the hospital, all to regular internal medicine wards. clinical differences, presented in table 4 , included a shorter duration of illness prior to presentation for clinical care in the 2009 h1n1 infected group compared to the adenovirus group (2 days vs. 3 days, p , 0.01), increased proportion of subjects complaining of coryza and cough in the 2009 h1n1 group, and increased predominance of sore throat and diarrhea in the adenovirus group. physical exam revealed increased abnormal findings on throat exam in the adenovirus group. there were no differences in pneumonia or hospitalization rates between the two groups, but both of these events were rare. clinical data in subjects infected with adenovirus, with or without concomitant 2009 h1n1, are also presented in table 4 . sore throat was less common in the coinfection group compared to adenovirus alone (71% vs. 94%, p = 0.01), and abnormalities on throat exam were less common in the coinfection group compared to adenovirus alone. oral temperature on presentation was higher for the coinfected group than for those infected with adenovirus alone (101.9 0 f vs. 101.5 0 f, p = 0.03). there were no hospitalizations or diagnoses of pneumonia in the coinfection group; this was not significantly different than the rates seen in the adenovirus group. [16, 21] . a number of studies have also evaluated whether 2009 h1n1was associated with either negative or positive effects (predominantly in terms of acquisition rather than severity) on other respiratory viruses, and, taken together with reference to adenovirus, the results of these are inconclusive. one study performed in the united kingdom in 2009-2010 suggested negative associations between 2009 h1n1 and human metapneumovirus as well as rhinovirus, though not adenovirus. however, few adenovirus detections were found compared to our population (,5%), and most of these were in young children. [22] a south african study of respiratory viruses in hospitalized patients found only six patients with adenovirus and 2009 h1n1 coinfection out of over 8000 subjects enrolled. [23] to our knowledge, negative associations between adenovirus and influenza a virus (either seasonal or 2009 h1n1) detection have not been demonstrated. it is also unclear, if there is a negative association between the two viruses, whether adenovirus is protective against influenza infection or vice versa. in this data set, influenza patients presented earlier, although not after excluding those presenting during the first week of training, who likely would have arrived with their infection. the policy of cohorting together all bmts with fever and uri, regardless of the causative pathogen, would seem to increase the likelihood of coinfection, rather than skewing the data towards the appearance of a negative association. it will also be worthwhile to see whether influenza epidemiology will change since the late 2011 reintroduction of adenovirus serotypes 4 and 7 vaccines in military trainees, or whether issues arise with concurrent administration of both live attenuated influenza and adenovirus vaccines, which could affect current trainee vaccine policies. in the meantime, concerns about cohorting patients that may have either adenovirus or 2009 h1n1 on the basis of syndromic presentation can be alleviated on the relative scarcity of coinfection and on the absence of any evidence of increased illness severity among coinfected subjects. the differing demographics and clinical presentations of adenovirus vs. 2009 h1n1 infection in this study are also of interest. first, the gender differences seen for each virus are intriguing. males represented 89% of the study population, enrolled after presenting with respiratory illness. however, males generally represent only 80% of the air force basic training population, so the study population was already disproportionately male. adenovirus has long been suggested to be predominantly an illness of men in this population, and this study is no exception to this trend. [24, 25] 2009 h1n1, like all influenza a viruses, has similar mechanisms of transmission, yet in this study population disproportionately affected females. the reasons for this gender difference are unclear. both of these infections as captured in this study population would meet the cdc definition for influenzalike-illness (fever plus cough or sore throat), used for surveillance and cohorting purposes, but had differences in presentation of statistical and arguably clinical significance. [26] adenovirus was consistently more likely to produce signs and symptoms referable to the throat, while 2009 h1n1 produced a predominance of cough, as well as shorter illness duration prior to presentation for care, potentially reflecting more rapid development of uncomfortable symptoms. these findings may have importance for both infection control and empiric therapy. the strengths of this study include the molecular characterization of respiratory pathogens together with capture of detailed clinical data in a large cohort of otherwise healthy adult patients with few complicating comorbidities, as well as the closed nature of the population, which lends itself to capture of events such as hospital admissions and pneumonia diagnoses. limitations include the absence of serologic data, or the ability to serially characterize pathogens from the same cohort of individuals, which would provide more granular detail about the time course of developing each infection. subject enrollment was variable throughout the study period, depending on rates of clinical illness within the training population, as well as availability of study personnel to enroll trainees, and given that 2009 h1n1 influenza virus pcr was done as part of clinical care, there could have been some differences in those who enrolled vs. did not enroll, or those who received 2009 h1n1 testing (and thus were included in this study) and those who did not. due to this variability in total enrollment, and because 2009 h1n1 testing became infrequent clinically after november 2009, inferences about the impact of h1n1 towards the end of 2009 are limited. however, data from the naval health research center's febrile respiratory illness surveillance update show a rate of febrile respiratory infection that increased in december of 2009, 82% of which was associated with adenovirus, with no influenza virus detected. [27] additionally, the use of oseltamivir, both for prophylaxis and for treatment, would have impacted both epidemiology and severity of illness. finally, without inclusion of asymptomatic or minimally symptomatic individuals, conclusions can only be drawn about the scarcity of coinfections with individuals at the point of presentation to care. in summary, this epidemiologic survey of young adults in military training presenting with fever and uri demonstrated significant differences in 2009 h1n1 vs. adenovirus in terms of gender predilection and presenting symptoms. in addition, coinfections with 2009 h1n1 and adenovirus were rare despite high endemicity of adenovirus before and during the 2009 h1n1 epidemic, and, beyond a higher temperature on presentation, coinfections were not associated with increased clinical severity compared with adenovirus alone. the economic burden of non-influenza-related viral respiratory tract infection in the united states portrait of a year-old pandemic preliminary estimates of mortality and years of life lost associated with the 2009 a/h1n1 pandemic in the us and comparison with past influenza seasons adult adenovirus infections: loss of orphaned vaccines precipitates military respiratory disease epidemics. for the adenovirus surveillance group acute respiratory disease in military trainees: the adenovirus surveillance program genotype prevalence and risk factors for severe clinical adenovirus infection, united states adenovirus-associated deaths in us military during postvaccination period transmission dynamics and prospective environmental sampling of adenovirus in a military recruit setting respiratory diseases among u.s. military personnel: countering emerging threats influenza a in a basic training population: implications for directly observed therapy cdc: 38 million doses of h1n1 vaccine available do rhinoviruses reduce the probability of viral co-detection during acute respiratory tract infections? simultaneous detection of influenza a, b, and c viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-pcr assay mixed respiratory virus infections pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity rate and influence of respiratory virus co-infection on pandemic (h1n1) influenza disease high burden of non-influenza viruses in influenza-like illness in the early weeks of h1n1v epidemic in france cdc protocol of realtime rtpcr for influenza a (h1n1) (world health organization collaboration center for influenza at the rapid and quantitative detection of human adenovirus dna by real-time pcr evaluation and validation of a real-time pcr assay for detection and quantitation of human adenovirus 14 from clinical samples respiratory viral coinfection among hospitalized patients with h1n1 2009 during the first pandemic wave in brazil respiratory viral infections during the 2009-2010 winter season in central england, uk: incidence and patterns of multiple virus co-infections respiratory viral coinfections identified by a 10-plex real-time reversetranscription polymerase chain reaction assay in patients hospitalized with severe acute respiratory illness-south africa outbreak of severe respiratory disease associated with emergent human adenovirus serotype 14 at a us air force training facility in 2007 epidemic of adenovirus-induced respiratory illness among us military recruits: epidemiologic and immunologic risk factors in healthy, young adults self-reported influenza-like illness during the 2009 h1n1 influenza pandemic-united states febrile respiratory illness (fri) surveillance update, department of respiratory diseases research, naval health research center we appreciate the administrative assistance of francine stotler with the execution of this study.disclaimer: the opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the department of the army, department of the air force, department of defense or the us government. this work was prepared as part of their official duties and, as such, there is no copyright to be transferred. key: cord-001420-b4zcvd04 authors: crescenzo-chaigne, bernadette; barbezange, cyril; frigard, vianney; poulain, damien; van der werf, sylvie title: chimeric np non coding regions between type a and c influenza viruses reveal their role in translation regulation date: 2014-09-30 journal: plos one doi: 10.1371/journal.pone.0109046 sha: doc_id: 1420 cord_uid: b4zcvd04 exchange of the non coding regions of the np segment between type a and c influenza viruses was used to demonstrate the importance not only of the proximal panhandle, but also of the initial distal panhandle strength in type specificity. both elements were found to be compulsory to rescue infectious virus by reverse genetics systems. interestingly, in type a influenza virus infectious context, the length of the np segment 5′ nc region once transcribed into mrna was found to impact its translation, and the level of produced np protein consequently affected the level of viral genome replication. influenza viruses are members of the orthomyxoviridae family and are classified into three antigenic types, a, b and c. they have a negative-polarity rna genome segmented into eight (type a and b) or seven (type c) single-stranded molecules. type c virus has a unique envelope glycoprotein (hef) whereas type a and b viruses have two (ha and na) [1] . each genomic viral rna (vrna) is encapsidated by multiple nucleoproteins (np) and associated with the polymerase complex (p) formed by three subunits named pb1, pb2 and pa for influenza a viruses and pb1, pb2 and p3 for influenza c viruses. in the nucleus of infected cells, the messenger rnas (mrnas) are products of a transcription process involving a cap-snatching mechanism: the mrna synthesis is initiated with capped rna primers that are cleaved from host cell mrnas. transcription into mrna terminates 17 to 22 nucleotides (nt) upstream of the 59 end of the genomic vrna template at a stretch of five to seven uridine residues used as polyadenylation signal. the syntheses of the complementary rnas (crnas) and of the vrnas are primer-independent. anti-termination occurs at the poly u sequence during the crna synthesis which, itself, is used as a template for the synthesis of the genomic vrnas [1] . for each genomic vrna, the coding region is flanked by non coding (nc) sequences. the nc region of each genomic vrna can be divided into two parts: the conserved sequence common to all the viral segments and specific for each type, and the non conserved sequence [2] . for the 39 and 59 ends, respectively, the conserved sequences are 12 and 13 nt for type a and 11 and 12 nt for type c influenza viruses [3, 4, 5] . the total length of the nc sequences of each segment of influenza a and c viruses is very variable and depends on the non conserved sequence length [6] . this is particularly true for the np segment, for which the 39 and 59 nc sequences are 45 and 23 nt long for type a and 29 and 102 nt long for type c influenza viruses, respectively (all numbers excluding the atg and stop codons). within each genomic rna molecule, the nc ends form secondary structures. based on potential base-pairing, two elements were defined within the nc ends: the proximal element or region i (nt 1-9 of 39 end and 1-10 of 59 end) and the distal element or region ii involving sequences downstream of nt 10 and 11 from the 39 and 59 ends respectively [7] . two possible secondary structures formed by the 39 and 59 ends of each segment have been described: the 'panhandle structure' resulting from the base-pairing of the respective proximal and distal elements of each ends [8, 9, 10] ; and the 'corkscrew structure' that consists of hairpin loops formed by the respective proximal elements followed by base-pairing of the distal elements [11, 12, 13, 14] . these structures are known to be critical for the virus multiplication, in particular for the transcription and the replication of the vrnas [7] . the distal element can in fact be further divided into two sub-elements: the initial distal panhandle corresponding to the first nine nucleotides (for illustration, see a1 and c1 in fig. 1 and 2 , respectively) and the remaining distal element. we previously studied the role in type specificity of the nc regions of the ns segment, which are quite similar in length (26 nt for the 39 end of both types a and c, and 26 and 47 nt for the 59 end of type a and c, respectively). we tried to rescue, by reverse figure 1 . rescue of influenza a viruses harboring type a/c substitutions and/or mutations in the nc regions of the genomic np segment. nc region nucleotide sequences and predicted panhandle conformation for the different np genomic segments used in type a influenza virus genetic backbone. sequences of a/wsn/33 origin are in bold and those of c/jhb/1/66 origin are in plain. the mutations introduced by sitedirected mutagenesis are shown in boxes. the sequence of both ends of each segment of each rescued virus was verified as described [9] , and no influenza np non coding region role in translation plos one | www.plosone.org genetics, type a and type c viruses with one or both heterotypic ends. we showed for the ns segment that a homotypic proximal panhandle was necessary to obtain infectious influenza a or c viruses by reverse genetics [15] . here we report the results of a similar approach on the np segment, which is characterized by a large difference in the length of the nc sequences between type a and c influenza viruses, i.e. 45 and 29 nt long for the 39 end, and 23 and 102 nt long for the 59 end, respectively [6] . using heterotypic constructs and sitedirected mutagenesis, we studied the importance of the nc sequences to rescue infectious viruses by reverse genetics. our results supported the importance of the homotypic proximal panhandle, as seen for the ns segment, and that the length of the genomic 59 end influences the expression of the np protein. a series of recombinant viruses based on type a and c influenza virus reverse genetics systems were constructed to identify the parts in the nc regions of the np segment that are important for type specificity. both wild type (wt) viruses (a1 and c1, fig. 1 and 2 , mutation was detected. the energy barrier of the canonical pairs c:g and u:a, and of the wobble base pair g:u (represented by a black dot) described by vendeix et al [19] were used to calculate a score to evaluate the panhandle strengths. titers in log 10 (pfu/ml) are the mean 6 standard deviation (sd) of 2 to 4 independent reverse genetics experiments. doi:10.1371/journal.pone.0109046.g001 figure 2 . rescue of influenza c viruses harboring type c/a substitutions and/or mutations in the nc regions of genomic np segment. nc region nucleotide sequences and predicted panhandle conformation for the different np genomic segments used in type c influenza virus genetic backbone. sequences of c/jhb/1/66 origin are in plain and those of a/wsn/33 origin are in bold. the mutations introduced by sitedirected mutagenesis are shown in boxes. the sequence of both ends of each segment of each rescued virus was verified as described previoulsy [9] and no mutation was detected. the energy barrier of the canonical pairs c:g and u:a, and of the wobble base pair g:u (represented by a black dot) described by vendeix et al [19] were used to calculate a score to evaluate the panhandle strengths. titers in log 10 (pfu/ml) are the mean (6 sd) of 2 to 4 independent reverse genetics experiments. doi:10.1371/journal.pone.0109046.g002 respectively) were rescued with similar efficiencies to what was previously described [16, 17, 18] . in type a reverse genetics system, no virus was rescued when one or both np segment ends were of type c (constructs a2, a3 and a4, fig. 1 ). the strength of the proximal panhandle is highly affected in these constructs (energy barrier 14.09, 11.03, 11.72 for a2, a3, a4, vs 8.66 for wt a1, based on vendeix's formulas) [19] . we previously showed for the ns segment that a homotypic proximal panhandle is compulsory to rescue influenza virus by reverse genetics [15] . for the np segment, point mutations to restore a type a proximal panhandle were insufficient to rescue recombinant viruses (constructs a5 and a6, fig. 1 ). we nonetheless hypothesized that a homotypic proximal panhandle was required and we decided to maintain it in all further np constructs, as it was previously shown to be needed for viral polymerase binding and rna promoter activities [10, 20, 21, 22] . since the strength of the initial distal panhandle was also affected in a2, a3 and a4 (energy barrier 14.04, 10.34 and 8.03 respectively) and still remained affected in a5 and a6 (energy barrier 14.04 and 8.03, respectively, vs 11.38 for wt a1), point mutations were introduced to modify this strength in order to match that of wild type (constructs a7 and a8, energy barrier 11.38, fig. 1 ). only the virus with type a 39 end was rescued (a8), but with a dramatically lower efficiency. type c 59end is much longer than that of type a (102 nt vs 23 nt) [6] . to test whether this might affect the recovery efficiency, deletions were introduced in both a7 and a8 constructs to generate a9 and a10, respectively ( fig. 1) . surprisingly, a9 could not be rescued. the a10 virus was rescued with the same efficiency as wt. this was not surprising, since a1 and a10 differed by only two nucleotides. the role of the initial distal panhandle strength was further verified by modifying a10 to generate constructs a11 and a12 (energy barrier 11.38, 8.03 and 11.38, fig. 1 ). no virus was rescued when the strength of the initial distal panhandle was reverted to that of a6 while maintaining the short 59 end (construct a11). on the contrary, modification of the sequence of the initial distal panhandle while maintaining the strength (a12) led to efficient rescue. these data based on type a influenza virus np segment showed that it is possible to obtain virus by reverse genetics when a homotypic proximal panhandle and a homotypic strength of the initial distal panhandle are maintained, and that the length of the 59 end plays an important role in the efficiency of rescue. . the supernatants were collected at the indicated times after infection and virus titers were determined by plaque assay. results are expressed as the mean (6 sd) of two independent growth kinetics. b) and d) minireplicon assays. 293t cells were co-transfected with the expression plasmids coding for the nucleoprotein and the three subunits of the viral polymerase, and with the reverse genetics plasmid of interest, to reconstruct functional vrnp. twenty four hours post transfection, total rna were extracted, and viral vrna and mrna were quantified by rt-qpcr, along with the quantification of gapdh mrna as normalizer. results are expressed as fold changes calculated compared to the respective wt construct mean using the ddct method, and are the mean (6 sd) of several independent transfections (four for type a and two for type c). in order to quantify only np mrnas produced from the reverse genetics plasmid and not from the expression plasmid, h1n1pdm09 proteins had to be used in a type a context (b) since the primers used [48] also match pr8 np sequence. similarly, in the type c context (d), type a pr8 proteins were used as they were previously shown to efficiently transcribe and replicate type c pseudo-vrna [44] . doi:10.1371/journal.pone.0109046.g003 influenza np non coding region role in translation the fact that no virus was rescued for construct a9 pointed out the potential role of the 39end in type specificity. construct a13 differed from its parental construct a9 by an extension of the 16 nucleotides that precede the start codon in type a 39end in order to match the length of type a 39 end (fig. 1) . no virus was rescued and we supposed that there might be some important specific nucleotides in the type a 39 end. to test this hypothesis, we generated constructs a14 to a17 (fig. 1) , where point mutations were introduced in the wt a1 to replace specific nucleotides by those found in type c 39 end. the efficiency of rescue was only affected for a16, for which the strand interaction strength around the stop codon was modified (energy barrier 3.99 for a16 vs 20.7 for a1), resembling that of a9 and a13. however, all a14-a17 viruses were rescued and this could not, on its own, explain the impossibility to recover a9 and a13. type c np segment was demonstrated to be efficiently transcribed and replicated by type a polymerase in an artificial system based on cat expression [6] . although only the proximal and initial distal panhandles were described as required to initiate transcription and replication [23] , minireplicon assays using the same plasmids used for reverse genetics to generate vrna templates showed that transcription and replication levels were only slightly affected for a16 (fig. 3b ) and a13 constructs (data not shown), but were largely impaired for a9 construct (reduced by about a 100 fold compared to wt a1 construct). we speculate that impaired transcription/replication processes of the np segment might not be the main obstacle to reverse genetics recovery and that, somehow, packaging of the np segment could be prevented for a9 and a13. influenza virus non conserved non coding regions are known to carry packaging signals [24, 25] and to be involved in intermolecular vrna-vrna interactions [26] . the influence of the sequences surrounding the stop codon thus needs to be further investigated, as several positions differ between a13 and a16. the role of the np segment 59 and 39 ends in type c virus cycle was then analyzed. constructs with one or both type a ends were attempted (c2, c3 and c4, fig. 2 ). however, based on type a results, we decided to directly maintain a homotypic type c proximal panhandle and the homotypic initial distal panhandle strength in those constructs and all the subsequent ones. only c3 virus, with the remaining distal parts of type c 59end and of type a 39 end, was successfully and efficiently rescued. to confirm that the length of the 39 end was not essential to rescue type c viruses, a deletion of 16 nt in the 39 end of c3 was performed to generate the c5 construct, which was recovered as efficiently as c3 (fig. 2) . the importance of the initial distal panhandle strength was demonstrated by the introduction of point mutations in c5 (to generate c6). as expected, construct c6 with a weaker initial distal panhandle could not be rescued (fig. 2) . the role of the 59 end length on recovery efficiency was further explored by a series of deletions. on the contrary to type a virus, for which it was observed that increasing the length of the 59 end (from 23 nt to 102 nt) impaired the recovery efficiency, one could expect that, for type c virus, the efficiency would be affected by shortening the 59 end. a reduction of 22 or 41 nt (leading to 80 and 61 nt long 59 ends, respectively) in c1 and c3 constructs had no effect on the recovery efficiency (constructs c7-c8 and c10-c11 for c1 and c3, respectively, fig. 2 ). it was, however, affected when the 59 end length was reduced to 42 nt (constructs c9 and c12). these results confirmed the impact of the length of the np segment 59 end on reverse genetics efficiency, as seen for type a virus. they are also in agreement with the variability in length that was observed for the two other sequenced strains of influenza type c viruses (c/california/78 and c/ann arbor/1/50), whose np 59 ends are 80 nt long [6] . finally, they could explain why c4 and c2 viruses were not rescued, since the length of their 59 end was identical to that of type a (23 nt long), so probably too short in a type c context. extending the length in c4 59 end by 19 nucleotides to match that of c12 59 end allowed the recovery of recombinant virus by reverse genetics with a very low efficiency, but the virus could not be amplified from the reverse genetics supernatant (c13, fig. 2) . after one round amplification from plaque cloning purified viruses, sequencing did not reveal any changes in the nc regions of all segments of all viable viruses compared to the plasmids used for reverse genetics. the growth abilities of the particularly interesting viruses were then evaluated by growth kinetics at low multiplicity-of-infection (m.o.i). growth abilities of the type a virus with a long 59 end (a8) were reduced compared to that of viruses with a short 59 end (a1 and a10) (fig. 3a) . conversely, type c viruses with a long 59 end (c1 and c3) grew better than those with a short one (c9 and c12) (fig. 3c) . the sequences of the nc (2 pfu/cell), viral vrna and mrna levels for each segment were evaluated by specific two-step rt-qpcrs previously described [48] . results are expressed as the cp-mrna/cp-vrna ratios and are the mean (6 sd) of two independent infections, with six replicate points of qpcr being performed on each sample. doi:10.1371/journal.pone.0109046.g005 regions of all segments were checked for the viruses sampled at the last kinetic points, and no mutation was observed for these constructs (a1, a8, a10, c1, c3, c9, c12) compared to the stock viruses used for growth kinetics and the plasmids used for reverse genetics. minireplicon assays revealed that transcription and replication processes were only marginally affected ( fig. 3b and 3d) and could not account for the differences observed in growth kinetics. thus, growth kinetics confirmed the importance of the np segment 59 end length in influenza virus cycle. interestingly, it seems that a short 59 end is needed for efficient growth of type a viruses, whereas, on the contrary, type c viruses require a long 59 end. the growth of a16 virus was delayed compared to wt a1 at one day post infection (fig. 3a) . however, at two days post infection, the difference in titer was lower, suggesting an adaptation of a16. sequencing of a16 nc regions at that time point revealed a mutation at position 24 in the 39 end of the np segment (fig. 1) . intriguingly, this a24c modification reduced the strength of the putative interaction around the stop codon (energy barrier modified from 3.99 to 2.66). so far, it is however unclear whether or not, and, if so, how the changes introduced in the 39 end sequence of a16 or the strength of the interaction around the stop codon affect the growth abilities of type a influenza virus. overall, growth abilities of a8, a16, c9 and c12 highlighted the discrepancy that might be observed between the possibility to obtain a virus by reverse genetics and the fitness of the thenobtained virus. although a8 was not efficiently rescued by reverse genetics, its fitness was only slightly affected and it remained genetically stable. on the contrary, a16, with similar initial characteristics, seemed to have gained fitness through a single mutation. to try and understand the impact of 59 end length on type a influenza virus cycle, the level of expression of the np protein was assessed at early stages of infection with a1, a10 and a8. westernblot was performed on cellular extracts collected at six hours post infection at an m.o.i. of 2 and np/ns1 ratios were calculated (fig. 4) . the np protein expression was clearly reduced for the virus with a long 59 end compared to wild type (np/ns1 ratio of 0.8 vs 2.0 for a8 and a1, respectively). no difference was found for a10 virus compared to a1 (ratio of 2.0), indicating that the long 59 end is responsible for the lower expression of the np protein for a8. the mrna levels were relatively similar between the three viruses (mean cp values = 19.9760.87, 19.4760.48, 20.6560.47 for a1, a10 and a8, respectively), suggesting that translation, rather than transcription, could be mainly affected. the 59 end of np viral mrna is entirely complementary to the 39 end of the genomic vrna, but the viral mrna 39 end includes only the complementary to the non conserved nc sequence up to the polyu signal in the genomic vrna 59 end. consequently, whereas the np mrna sequences are almost identical for a1 and a10 (with only a two nucleotide difference in the sequence between the stop codon and the polya, and no difference in the np protein expression levels), the np mrna of a8 is characterized by a longer 39 untranslated region (utr). it was shown that the 39utr of cellular mrnas has a critical role in gene expression regulation [27] . the mrna 39utr could indeed regulate every steps of translation (initiation, elongation and termination) through different mechanisms involving rna-binding proteins, micro-rnas or the 'closed-loop' structure formed by the mrna 59 and 39 utrs via the eif4 translation initiation factors [28, 29] . several viral families were reported to regulate the translation of their own mrnas by such mechanisms at either the initiation (dengue virus [30, 31] ; coxackievirus [32] ), the elongation (tobacco mosaic virus [33] ) or the termination (hepatitis c virus [34] ) step. we thus postulate that, in a type a influenza virus context, a long 39utr in the np mrna, or specific sequence within such long 39utr, could affect the level or the speed of translation through interactions with yet unknown cellular factors. several functions of the np protein of influenza viruses have been described. among them, np would play a major role in the replication of the virus genome. more specifically, np would be involved in the switch from capped rna-primed transcription (protein expression) to primer-independent replication (copy of the genome). since negative-sense rna molecules cannot be synthesized directly from negative-sense rnas, full-length positive-sense replicative intermediates complementary to each segment, namely crnas, are required as templates in influenza virus replication. the synthesis of mrnas and crnas by the viral polymerase thus uses the same genomic vrna molecules as templates, but with different initiation and termination mechanisms [35] . different models for the role of np in this switch from primer-dependent transcription to primer-independent replication have been proposed. the switch was first attributed to the rna-binding properties of np, either by stabilizing the nascent crna and vrna transcripts through encapsidation [36] or by altering the rna promoter structure [37] . however recent studies showed that changes in the equilibrium between transcription and replication could be determined by the interaction of np with the components of the viral polymerase [38, 39] . as a consequence of this role of np, it can be anticipated that a lower level of np protein for a8 virus compared to wt a1 could delay the replication. such a delay was evaluated by comparing the levels of mrnas and vrnas for all the segments of these two viruses at the early stages of infection (6 hours post infection at an m.o.i of 2). the crossing-point (cp) values obtained for each kind of rna were combined to calculate a ratio cp-mrna/cp-vrna and these ratios were compared between the two viruses. as expected according to our hypothesis, a moderate but consistent reduction of the cp-mrna/cp-vrna ratio was observed for all segments of a8 (fig. 5) . this reduced cp-mrna/cp-vrna ratio could be attributed to a lower level of vrna production for a8 compared to wt a1 or to a slight delay in the shift from primerdependent transcription to primer-independent replication. these results confirmed that the consequence of a lower level of np observed by western blot for a8 was a reduction or delay in the genome replication; and this provided a convincing explanation for the slightly impaired growth abilities of a8 virus compared to wt a1 (fig. 3a) . using an original approach that aimed at exchanging the nc regions of influenza virus type a by those of type c and reciprocally, we showed the importance of the proximal panhandle and of the strength of the initial distal panhandle in type specificity for the np segment. without respecting the backbone type for these structures, no infectious virus could be rescued by reverse genetics in both type a and c systems. this confirmed the results we previously obtained with the ns segment [15] , at least for the proximal panhandle, but the role of the initial distal panhandle for the np segment highlighted the differences that exist between the different genomic segments. a similar approach on the other segments would undoubtedly reveal more intriguing features of influenza virus non coding regions. our results in an influenza virus infectious context also showed that the length of the 59 nc region of type a np segment seems to be involved in the translation regulation at the mrna level and that the amount of np produced subsequently affects the replication of the genome. we hypothesize that the lower growth abilities observed for type c viruses with a short 59end (c9 and c12, fig. 3c) were also a consequence of less np protein due to a slightly lower level of or to a slightly slower translation. it is unclear, however, how the length would have opposite effects in type a and c influenza viruses, but this could represent another example of the numerous differences that have been described between these two influenza virus types [40, 41, 42, 43] . plasmids phmg-pb1, -pb2, -pa/3 and -np, which express the pb1, pb2, pa/3 and np proteins, respectively, of influenza virus a/puerto rico/8/34 (pr8) or c/johannesburg/1/66 (c/jhb/ 1/66) under the control of the hydroxymethylglutaryl coenzyme a reductase (hmg) have been described previously [44, 45] . pb1, pb2, pa and np sequences of a/paris/2590/2009 (h1n1pdm09) were subcloned in phmg vector. the series of eight or seven plasmids containing the sequences of the genomic segments of a/wsn/33 or c/jhb/1/66 viruses under the control of human rna polymerase i promoter and upstream of hepatitis delta virus ribozyme sequence have been described previously [16, 17] . the modified heterotypic np plasmids were constructed by pcr using primers containing the total 39 or 59 nc sequence of the respective np segment, followed by cloning. point mutations in the np nc regions were introduced by site-directed mutagenesis, using quikchange ii site-directed mutagenesis kit (agilent) according to the manufacturer's instructions. the sequences of the primers will be provided upon request. all plasmids were verified by sequencing using a big dye terminator sequencing kit and an automated sequencer (perkin-elmer). madin-darby canine kidney cells (mdck) cells, human embryonic kidney 293t cells and human skin melanoma cells (sk93/2) [46] were cultured in dmem supplemented with 5%, 10% and 10% fetal calf serum (fcs), respectively. all cells were grown at 37uc with 5% co 2 . twelve or eleven plasmid based reverse genetics systems were used to produce the recombinant type a (a/wsn/33) and type c (c/jhb/1/66) influenza viruses, and were adapted from previously described procedures [16, 17, 18] . briefly, using 35 mm plates and dmem supplemented with 10% fcs, co-cultures of 293t (4610 5 /well) and mdck cells (3610 5 /well) for type a viruses and 293t cells (6610 5 /well) on poly l-lysin plates for type c viruses, were transfected with the four plasmids driving the expression of the homotypic np and polymerase proteins, together with the eight (for type a) or seven (for type c) plasmids that direct the synthesis of the vrna templates, using 0.5 mg of each plasmid and 18 or 24 ml of fugene hd (roche) for type a or type c reverse genetics, respectively. the dna and the transfection reagent were first mixed, then incubated at room temperature for 15 min, and finally added to the cells and incubated at 35uc or 33uc for type a and type c viruses, respectively. sixteen hours later, the dnatransfection reagent was removed, the cells were washed twice in dmem, and 2 ml (type a) or 6 ml (type c) of dmem containing 0.5 mg/ml or 0.25 mg/ml of l-1-tosylamido-2-phenyl chloromethyl ketone treated trypsin (tpck-trypsin, from worthington), respectively, were added. the cells were incubated at 35uc or 33uc for type a or type c, respectively. three or ten days post transfection for the type a or c, respectively, supernatants were collected for virus titration in a standard plaque assay as described below. viruses rescued by reverse genetics were titrated and plaquepurified by plaque assay using an agarose overlay. the plaque assay protocols slightly differed for type a and c viruses and have been described previously [16, 47] . briefly, mdck cells were used for type a viruses and mdck cells supplemented with bovine brain gangliosides (bbg) were used for type c viruses. the agarose overlay contained complete dmem with additional tpck-trypsin at 1 or 0.5 mg/ml for type a or c, respectively. finally, overlaid cells were incubated at 35uc for 3 days or at 33uc for 5 days for type a or c, respectively. type a or c plaque-purified viruses were then amplified on mdck or sk cells, respectively. growth kinetics were performed at an m.o.i. of 0.001 on mdck or sk cells for type a or c viruses, respectively. all these infections used dmem supplemented with 1 or 0.5 mg/ml of tpck-trypsin for type a or c viruses, respectively. viral titers in the supernatant were determined at the different indicated time points post infection by plaque assay, as described aboved. the sequence of the 39 and 59 nc regions of all genomic rna segments were verified for all rescued viruses. viral genomic rna was extracted using the qiaamp viral rna mini kit (qiagen) from 140 ml of culture supernatant following the manufacturer's recommendations. the rna was eluted in 60 ml of rnase-free water, and the 39 and 59 nc regions were amplified by rt-pcr, as previously described [9] . after purification, the pcr products were sequenced using a big dye terminator sequencing kit and an automated sequencer (perkin elmer). all sequences of the primers are available from the authors upon request. minireplicon assays were performed in 293t cells transfected using 10 ml fugene hd with 0.5 mg of pb1, pb2, pa and 1 mg of np type a h1n1pdm09 or pr8 expression protein plasmids for type a or type c, respectively, in presence of 0.1 ng of np constructs. twenty four hours post-transfection, total rnas were extracted, reverse transcribed using the 39 universal primer for the vrnas (59agggctcttcggccagcraaagcagg [48] or 59agcagaagcag for type a and type c influenza virus, respectively), or an oligo dt for the mrnas. the cdnas were used as templates for quantitative pcr (qpcr) using a light-cycler 480 (roche) pcr machine. primers designed by marsh et al. [48] and matsuzaki et al. [49] were used for the detection of the type a and type c np segment, respectively. as normalizer, onestep qrt-pcr targeting the gapdh sequences was performed [50] . mdck cells infected at an m.o.i. of 2 with the studied influenza a viruses were collected at six hours post-infection, solubilized in lds sample buffer (invitrogen) complemented with b-mercaptoethanol, and separated by 4-12% polyacrylamide gel electrophoresis (nupage invitrogen) followed by western blotting. an anti b-actin antibody (abcam, cambridge, uk) was used to control loadings. an in-house produced rabbit anti-influenza virus pr8 polyclonal antibody was used to detect np [51] . the anti-ns rabbit polyclonal antibody was a kind gift from d. marc (inra, tours, france). mdck cells were infected at an m.o.i. of 2 with the studied influenza a viruses. at six hours post-infection, total rnas were extracted, and reverse transcriptions and qpcr were performed as described above. separate qpcrs were performed with segmentspecific primers [48] using a lightcycler 480 (roche) pcr machine. the relative amounts of vrnas and mrnas were estimated by calculating the ratio mrna/vrna using the crossing point values (cp). orthomyxoviridae: the viruses and their replication mutations in the nonconserved noncoding sequences of the influenza a virus segments affect viral vrna formation the 39 and 59-terminal sequences of influenza a, b and c virus rna segments are highly conserved and show partial inverted complementarity 59 and 39 terminal nucleotide sequences of the rna genome segments of influenza virus segment-specific and common nucleotide sequences in the noncoding regions of influenza b virus genome rnas non coding extremities of the seven influenza virus type c vrna segments: effect on transcription and replication by the type c and type a polymerase complexes orthomyxovirus replication, transcription, and polyadenylation structure of influenza virus panhandle rna studied by nmr spectroscopy and molecular modeling differential effect of nucleotide substitutions in the 39 arm of the influenza a virus vrna promoter on transcription/replication by avian and human polymerase complexes is related to the nature of pb2 amino acid 627 the influenza virus panhandle is involved in the initiation of transcription nucleotides at the extremities of the viral rna of influenza c virus are involved in type-specific interactions with the polymerase complex mutational analysis of the influenza virus crna promoter and identification of nucleotides critical for replication promoter elements in the influenza vrna terminal structure a hairpin loop at the 59 end of influenza a virus virion rna is required for synthesis of poly(a)+ mrna in vitro the panhandle formed by influenza a and c virus ns non-coding regions determines ns segment expression rescue of influenza c virus from recombinant dna rescue of influenza a virus from recombinant dna generation of influenza a viruses entirely from cloned cdnas free energy calculation of modified base-pair formation in explicit solvent: a predictive model characterization of the rna-fork model of virion rna in the initiation of transcription in influenza a virus in vitro transcription and polymerase binding studies of the termini of influenza a virus crna: evidence for a crna panhandle sequence-specific binding of the influenza virus rna polymerase to sequences located at the 59 ends of the viral rnas amplification, expression, and packaging of a foreign gene by influenza virus the genome-packaging signal of the influenza a virus genome comprises a genome incorporation signal and a genome-bundling signal genome packaging in influenza a virus selective packaging of the influenza a genome and consequences for genetic reassortment hyper conserved elements in vertebrate mrna 39-utrs reveal a translational network of rna-binding proteins controlled by hur regulation and dysregulation of 39utr-mediated translational control translational control by the 39-utr: the ends specify the means enhancement of dengue virus translation: role of the 39 untranslated region and the terminal 39 stem-loop domain translational regulation by the 39 untranslated region of the dengue type 2 virus genome polypyrimidine tract-binding protein interacts with coxsackievirus b3 rna and influences its translation eukaryotic elongation factor 1a interacts with the upstream pseudoknot domain in the 39 untranslated region of tobacco mosaic virus rna hepatitis c virus 39utr regulates viral translation through direct interactions with the host translation machinery the influenza virus rna synthesis machine: advances in its structure and function model suggesting that replication of influenza virus is regulated by stabilization of replicative intermediates the influenza virus nucleoprotein: a multifunctional rna-binding protein pivotal to virus replication sequence in the influenza a virus nucleoprotein required for viral polymerase binding and rna synthesis interaction of the influenza a virus nucleocapsid protein with the viral rna polymerase potentiates unprimed viral rna replication isolation of a novel swine influenza virus from oklahoma in 2011 which is distantly related to human influenza c viruses clinical features of influenza c virus infection in children influenza c virus uses 9-o-acetyl-n-acetylneuraminic acid as a high affinity receptor determinant for attachment to cells comparison of the three large polymerase proteins of influenza a, b, and c viruses comparative analysis of the ability of the polymerase complexes of influenza viruses type a, b and c to assemble into functional rnps that allow expression and replication of heterotypic model rna templates in vivo a plasmid-based reverse genetics system for influenza a virus a molecular mechanism of complement resistance of human melanoma cells genetic analysis of animal viruses. new approaches and new vistas specific residues of the influenza a virus hemagglutinin viral rna are important for efficient packaging into budding virions detection and quantification of influenza c virus in pediatric respiratory specimens by realtime pcr and comparison with infectious viral counts development of a quantitative assay for sars coronavirus and correlation of gapdh mrna with sars coronavirus in clinical specimens naked rna immunization with replicons derived from poliovirus and semliki forest virus genomes for the generation of a cytotoxic t cell response against the influenza a virus nucleoprotein we are very grateful to g. brownlee for providing the ppoli expression plasmids of influenza a/wsn/33, j. pavlovic for the four phmg recombinant plasmids of influenza a/pr/8/34 and r. frade (saint antoine hospital, paris, france) for sk93/2 cells. we thank claire denis for technical assistance, d. marc (inra, tours, france) for anti-ns1 polyclonal antibody and n. naffakh, s. munier and g. abou-jaoud for helpful discussions and critical reading of the manuscript. conceived and designed the experiments: bcc sw. performed the experiments: bcc vf dp. analyzed the data: bcc cb. contributed reagents/materials/analysis tools: bcc. wrote the paper: bcc cb sw. key: cord-000638-ss1435el authors: beq, stephanie; rozlan, sandra; pelletier, sandy; willems, bernard; bruneau, julie; lelievre, jean-daniel; levy, yves; shoukry, naglaa h.; cheynier, rémi title: altered thymic function during interferon therapy in hcv-infected patients date: 2012-04-16 journal: plos one doi: 10.1371/journal.pone.0034326 sha: doc_id: 638 cord_uid: ss1435el interferon alpha (ifnα) therapy, despite good efficacy in curing hcv infection, leads to major side effects, in particular inducement of a strong peripheral t-cell lymphocytopenia. we here analyze the early consequences of ifnα therapy on both thymic function and peripheral t-cell homeostasis in patients in the acute or chronic phase of hcv-infection as well as in hiv/hcv co-infected patients. the evolution of t-cell subsets and t-cell homeostasis were estimated by flow cytometry while thymic function was measured through quantification of t-cell receptor excision circles (trec) and estimation of intrathymic precursor t-cell proliferation during the first four months following the initiation of ifnα therapy. beginning with the first month of therapy, a profound lymphocytopenia was observed for all t-cell subsets, including naïve t-cells and recent thymic emigrants (rte), associated with inhibition of intrathymic precursor t-cell proliferation. interleukin (il)-7 plasma concentration rapidly dropped while lymphocytopenia progressed. this was neither a consequence of higher consumption of the cytokine nor due to its neutralization by soluble cd127. decrease in il-7 plasma concentration under ifnα therapy correlated with the decline in hcv viral load, thymic activity and rte concentration in blood. these data demonstrate that ifnα-based therapy rapidly impacts on thymopoiesis and, consequently, perturbs t-cell homeostasis. such a side effect might be detrimental for the continuation of ifnα therapy and may lead to an increased level of infectious risk, in particular in hiv/hcv co-infected patients. altogether, this study suggests the therapeutic potential of il-7 in the maintenance of peripheral t-cell homeostasis in ifnα-treated patients. the hepatitis c virus (hcv) causes persistent infection in approximately two thirds of cases leading to chronic liver disease, liver failure, and, eventually, hepatocellular carcinoma in a substantial proportion of infected individuals. the most common therapy for chronic hepatitis c consists of pegylated interferon-a (ifna) and ribavirin administration which results in viral clearance in 43-46% (genotype 1) to 80%, (genotype 3) of treated patients [1] . interferon will continue to be a major component of new direct acting antivirals for treatment of hcv [2] . ifna is produced in large amounts during the acute phase of many viral infections [3, 4, 5, 6] . direct activation of interferonstimulated genes enhances naïve t-cell survival through increased bcl-2 and reduced bax activation [7] and contributes to clonal expansion of antigen-specific t-cells [8] . recent data suggest that early therapeutic intervention with pegylated ifna rescues polyfunctional memory t-cells expressing high levels of the il-7 receptor alpha chain (cd127) and bcl-2, allowing a higher rate of sustained viral response [9] . however, despite good efficacy, ifna-based therapies lead to sustained anemia, thrombocytopenia, neutropenia and lymphocytopenia [10, 11, 12, 13, 14] . moreover, pegylated ifna therapy enhances the risk of infection in older hcv-infected patients and hiv-infected individuals, independently from neutropenia [15, 16, 17] . the mechanisms of action of ifna include inhibition of different hematopoietic growth factors [18, 19] , possibly affecting lymphoid differentiation at an early stage [20] , and modifications in cell homing [12, 21, 22] . the mechanisms involved in ifna therapy-associated leukocyte depletion remain poorly understood. others and we have documented a strong reduction in the ability of hiv-infected patients to sustain thymic production as a direct consequence of a drop in intrathymic precursor t-cell proliferation [23, 24, 25] . similar thymic impact was also seen during early siv-infection in the rhesus macaque model [26] . the capacity of the thymus to produce recent thymic emigrants (rtes) is, in large part, dependent on thymocyte proliferation [27] . indeed, extensive thymocyte proliferation occurs between t-cell receptor beta (tcrb) and alpha (tcra) chain rearrangements. the extent of this proliferation directly correlates with thymic export [28] . the extent of cell proliferation in the thymus can be measured in patients through estimating, in peripheral blood cells, the ratio (sj/btrec ratio) between the frequency of signal joint t-cell receptor excision circles (sjtrec), produced during the excision of the tcrd locus prior to tcra chain rearrangement, and that of dbjbtrec t-cell receptor excision circles (trecs) produced during tcrbd to tcrbj rearrangement [29] . these by-products of tcr rearrangement processes are generated by the circularization of the chromosomal dna excised during tcr rearrangements respectively occurring at the dn3 (dbjbtrec) and dp (sjtrec) stages of differentiation. due to the fact that trecs do not replicate during mitosis, increased proliferation between dn3 and dp leads to the reduction of dbjbtrec frequency in rtes as compared to sjtrec frequency and consequently to an increase of the sj/btrec ratio [23] . the correlation between initial plasma ifna levels and the speed of thymic dysfunction observed during hiv primary infection suggested that ifna, produced as part of the innate immune response to infection, participates in the impairment of thymopoiesis. however, no direct evidence of the relationship between ifna production and thymic dysfunction was provided by these studies. in contrast, arizcorreta and colleagues showed that ifna and ribavirin therapy induces a substantial reduction of circulating sjtrecs, in hiv/hcv co-infected patients, accompanied by sustained naïve cd4 + t-cell defect, suggesting thymic dysfunction [10] . similarly, in the siv-infected rhesus macaque model, we showed that ifna therapy induced a strong decrease of circulating rte numbers as defined either by sjtrec frequency and numbers or by cd31 hi expression on naïve t-cells [30] . interestingly, in these animals, recombinant interleukin (il)-7 therapy more than abrogated the deleterious effects of ifna therapy [30] . il-7 is a key cytokine implicated at various levels of thymocytes differentiation. it allows cell survival during the rearrangement processes, and is implicated in the extensive thymocyte proliferation, in particular in intermediate single positive (isp) and early dp cells [31, 32, 33, 34] . this proliferation participates in the development of naïve t-cell diversity [35] . while up regulated by ifna [36, 37] , the cyclin-dependent kinase inhibitor p27/kip1 is negatively regulated by il-7 [38] , allowing isp and early dp thymocytes to proliferate. moreover, ifna also inhibit il-7 dependent proliferation through down modulation of the common c chain, that participates, together with cd127 to the il-7 receptor [39] . we here investigated the early impact of ifna therapy on thymic function and naïve t-cell homeostasis in both hcv-infected and hiv/hcv co-infected patients who started ifna therapy. we first evaluated the evolution of naïve t-cell subsets in three groups of hcv infected individuals: 1) acute hcv infection (n = 8), defined as ,6 months post estimated date of infection; 2) chronic hcv infection (n = 8), defined as .6 months post estimated date of infection; and 3) hiv/hcv co-infected individuals (n = 10). in all groups, patients were enrolled at the beginning of ifna therapy and were followed for a total of 4 months. while, for any group of patient's, naïve cd4+ and cd8+ t-cell counts were not significantly different from healthy individuals (figure 1a), as early as one month following treatment initiation, naïve cd4+ t-cell counts were significantly reduced in chronically hcv-infected patients (39%, 58%, 46% and 35% decrease at m1, m2, m3 and m4 respectively; p#0.025; figure 1b , top central panel). a similar trend was also observed in the cd8 compartment (40%, 39%, 33% and 33% decrease; figure 1b , bottom central panel). a comparable effect was also observed in most co-infected patients (mean cell count declines were 19%, 32%, 52% and 43% at m1, m2, m3 and m4 in the cd4+ t-cell compartment and 9%, 21%, 41% and 42% in cd8+ t-cell subset; p#0.05 by m2-m3; figure 1b , right panels). in contrast, naïve t-cell counts were only barely affected in acutely-hcv infected patients under ifna therapy ( figure 1b , left panels). similarly, central memory cd4+ t-cells (cd45 ra-ccr7+; tcm) demonstrated 38% and 28% decrease in hcv and hiv/hcv patients respectively (59% and 60% in cd8+ tcm) while effector memory (cd45ra2 ccr7-; tem) cd4+ t-cell counts declined by 45% and 10% in the same groups (61% and 65% in cd8+ tem) ( figure s1 ). within cd4+ naïve t-cells, rtes can be identified by their higher expression of the platelet endothelial cell adhesion molecule-1 (pcam-1 or cd31) [40] . while the number of rtes was similar in hcv-infected patients at study entry and healthy individuals ( these data demonstrate that, as early as one month following treatment initiation, ifna induces stronger alterations of naïve tcell subsets, and more specifically in the rte compartment than in any other t-cell subset, suggesting a specific effect on thymopoiesis. we thus analyzed the evolution of intrathymic precursor t-cell proliferation, peripheral t-cell cycling, il-7 plasma concentration and il-7 receptor alpha chain (cd127) expression, different factors affecting naive t-cell homeostasis. despite differences between the 3 groups at study entry, rte cycling rate, as estimated through measurement of ki-67 expression, did not change significantly during the follow-up period ( figure 3a ). these data demonstrate that the observed changes in sjtrec frequencies were not a consequence of variations of rte proliferation during ifna therapy but more probably due to reduced thymic production. we thus estimated thymic output through quantification of the sj/btrec ratio in all groups of patients ( figure 3b ). the sj/ btrec ratio estimates the extent of thymocyte proliferation between tcrb rearrangement and the excision of the t-cell receptor delta (tcrd) locus [23] . this parameter directly reflects the extent of thymic production and, contrarily to sjtrec values, is independent from peripheral rte proliferation or survival capacity [28] . the sj/btrec ratio was already low in hivinfected patients (p,0.005 as compared to healthy control donors; figure 3b bottom left panel) and did not evolve further under ifna therapy in co-infected patients ( figure 3b , bottom right panel). in contrast, acutely hcv-infected patients demonstrated higher than normal sj/btrec ratio at baseline (p,0.05 as compared to aged matched healthy controls), showed a significant reduction in sj/btrec ratio at m1 (p = 0.014) and m2 (p = 0.001; figure 3b , top panel). finally, a similar decline in the sj/btrec ratio was observed during ifna therapy in chronically hcv-infected patients (p,0.02 at m1, m2 and m3; figure 3b , central panel). precursor t-cell proliferation in the thymus is, at least in part, dependent upon il-7. we thus quantified plasma il-7 concentration in all groups of patients. at study entry, hcv-and hiv/ hcv-infected patients presented with elevated plasma il-7 (median = 10.3 pg/ml, range (6.7-12.9) in acutely hcv-infected patients; 8.3 pg/ml (6.3-10.5) in chronic hcv-infected patients and 7.15 pg/ml (4.3-13.5) in co-infected subjects), as compared to that observed in healthy control individuals (p,0.001 for any patients' group; figure 4a) . surprisingly, while lymphocytopenia established, il-7 plasma concentrations significantly decreased in both groups of hcv-infected patients (30, 54, 18 and 29% decrease at m1 to m4 in acute infection, p,0.05; 25, 46, 26 and 16% decrease at m1 to m4 in chronic infection, p,0.05; figure 4b left and central panels). in contrast, il-7 plasma levels did not significantly evolve in co-infected individuals during the first month of ifna therapy ( figure 4b right panel) . only patients with the highest il-7 plasma levels showed a reduction in the concentration of this cytokine. decreased plasma il-7 concentrations could be a consequence of reduced il-7 production, increased consumption by t-cells or sequestration by soluble il-7 receptor (scd127). in both hcvinfected and hiv/hcv co-infected patients, neither scd127 considering the variations in all the parameters we used to evaluate thymic function, we then sought to evaluate the impact of changes in il-7 plasma levels on de novo production from the thymus and on the number of both sjtrec and circulating cd4+ rtes. in a majority of patients, il-7 plasma level, sj/btrec ratio, sjtrec/ml and blood rte concentration fluctuated in parallel ( figure s2 ). variation of il-7 plasma concentration (dil-7) during the first month of therapy correlated with variations in naïve t-cell counts (cd4+ + cd8+; dnaïve t-cell counts) and rte cd4+ t-cell counts (drte t-cell counts) in both hcv (r = 0.521, p = 0.039 and r = 0.595, p = 0.025; figure 5a and 5b, left panels) and, to a lesser extent, hiv/hcv co-infected patients (r = 0.636, p = 0.048 and r = 0.539, p = 0.108; figure 5a and 5b, right panels). moreover, in hcv-infected patients, dil-7 also correlated with variations in intrathymic precursor t-cell proliferation (dsj/btrec ratio; r = 0.601, p = 0.020; figure 5c ). variations in plasma il-7 levels also correlated with changes in the proportions (d%ki-67+ in cd4+rtes; r = 0.806, p = 0.0002; figure 5d , left panel) and numbers (dki-67+rtes; r = 0.706, p = 0.002; figure 5e , left panel) of cycling rtes in acute and chronic hcv infected patients and with d%ki-67+rte counts in co-infected patients (r = 0.709, p = 0.022; figure 5e , right panel). overall, il-7 concentration was associated with reduced thymopoiesis and rte proliferation, lower consequently leading to limited circulating rte and naïve t-cell counts. these data strongly suggest that changes in il-7 plasma levels during ifna therapy directly impact the homeostasis of rtes. we herein demonstrated that ifna-based therapy leads to major lymphocytopenia in naïve t-cell compartments, in particular in the rte subset. several mechanisms could be implicated in the establishment of such a lymphocytopenia [41] . among these, enhanced apoptosis [42, 43] , cell sequestration in lymphoid or non-lymphoid organs [12, 21, 22] and regulation of peripheral t-cell homeostasis [20] . in our study, no major change in cell survival (bcl-2 expression) or t-cell activation (cd25 and cd69 expression) was observed during the follow-up period (data not shown). moreover, we did not observe any significant modification in ki-67 expression in any t-cell subset during the first month of therapy (data not shown and figure 3 ). finally, ifna-induced t-cell homing, although rapid and massive, is only a transient process [22] suggesting that this mechanism marginally contributes to the observed long lasting lymphocytopenia. interestingly, both sjtrec quantification (sjtrec/ml) and intrathymic precursor t-cell proliferation (sj/btrec ratio) were affected very early on after initiation of therapy ( figures 2b and 3b ). while sjtrec frequency and concentration in peripheral blood can be affected by modifications of parameters that impact on peripheral t-cell homeostasis (cycling, survival/apoptosis, homing), the sj/btrec ratio is a marker of the intrathymic proliferation history of rtes. indeed, this parameter is generated by cell proliferation that occurs between tcrb chain rearrangement and the excision of tcrd locus. further cell cycling after tcra chain rearrangement does not modify the sj/btrec ratio as both type of trecs are similarly diluted upon cell proliferation. accordingly, while exported to the periphery, the sj/btrec ratio of mature t-cells cannot be modified. therefore, while the observed decrease in sjtrec concentration (figure 2) can be a consequence of modifications of circulating t-cell homeostasis, the decline of the sj/btrec ratio observed during the first months of ifna therapy (figure 3) defines changes in thymocyte proliferation, thus in thymic output [28] . acutely infected patients demonstrated a higher sj/btrec ratio at baseline than patients in the chronic phase. however, this group was younger (median = 31.5 (26-47)) versus median = 53.5 (37-61)) than the chronic group (p,0.01; data not shown) and demonstrated normal sj/btrec ratio for their age. similar evolution of thymic function and circulating t-cell subsets were observed in both groups of hcv-infected patients, irrespective of the development stage of hcv pathology. the lack of effect of ifna therapy in hiv/hcv co-infected patients might be due to the fact that, as expected for chronically hiv-infected individuals, these patients already had a low thymic function at study entry. the impairment of thymopoiesis in hcv-infected patients under ifna therapy is reminiscent of that observed during the acute phase of hiv-1 infection [23] which suggested that long term production of ifna, as part of the anti-hiv innate immune response, may play a role in the observed thymic defect. the correlation between decline in il-7 plasma levels under ifna therapy and both thymic dysfunction and reduced t-cell counts, in particular in the naïve and rte compartments ( figures 5a and 5b) , confirms this hypothesis. finally, in a recent study, we showed that ifna treatment leads to decreased sjtrec frequency as well as reduced naïve t-cell and rte counts in siv-infected rhesus macaques [30] . such an effect was accompanied by a 30-40% decrease in il-7 plasma levels in these animals and could be counteracted by injection of recombinant simian il-7 [30] . one could expect that such an effect of type i ifns is not restricted to hiv-infection as many viral infections induce ifna responses and cause transient lymphocytopenia in the infected hosts [3, 4, 5, 6] . moreover, the ifna-induced reduction of thymic function and its probable consequences on naïve t-cell diversity may contribute to the higher infectious risk associated with ifna therapy, in particular observed in older patients [15, 16, 44] . there are multiple sources for circulating il-7 during viral infections including lymphoid organs, epithelial cells and recently the liver was identified as a major source of il-7. moreover, increased plasma il-7 levels can also be observed during viral infection in non-lymphopenic individuals ( [33] and unpublished data), suggesting a role in the development of immune responses. indeed, this cytokine participates to t-cell homing in various lymphoid and non-lymphoid tissues through stimulation of local chemokine productions [45] . increased il-7 plasma levels in lymphopenic individuals is likely due to reduced consumption [46] yet augmented production to counteract lymphopenia cannot be excluded [33] . the recent identification of the liver as an il-7 producing tissue upon tlr stimulation [47] makes it tempting to speculate that hcv-infection can also, through tlr activation, stimulate il-7 production by the liver. indeed, non-lymphopenic hcv-infected patients demonstrate similar il-7 plasma levels than lymphopenic hiv-infected individuals [33, 48] suggesting that most of the il-7 production in untreated hcv-infected patients was not linked to circulating t-cell counts. the reduction of il-7 plasma levels while lymphocytopenia establishes under ifna therapy, the absence of a correlation between il-7 plasma levels and cd127 expression and the concomitance of decreases in il-7 plasma levels and hcv viral load under therapy suggest that viremia might be driving il-7 production before initiation of therapy. our data suggest that, before initiation of ifna therapy, actively replicating hcv leads to the overproduction of il-7. subsequent reduction of il-7 production upon initiation of therapy probably reflects the elimination of il-7 producing hcv-infected hepatocytes. this sudden reduction of il-7 plasma levels may lead to diminished thymopoiesis. the fact that il-7 plasma levels did not reach normal levels when hcv became undetectable may suggest that, after the initial decline that follows the drop in viremia, il-7 plasma levels were regulated, as in hivinfected patients [33] and in ifna-treated siv-infected rhesus macaques [30] , as a consequence of lymphocytopenia through either reduced consumption or increased production in lymphoid organs [49] . future studies with a longer follow-up period, in particular after the end of ifna therapy and recovery from lymphocytopenia are required to further elucidate this point. we herein demonstrated that a substantial reduction in thymic export was observed in hcv-infected patients, during the first months of ifna therapy. this effect directly paralleled ifnainduced lymphocytopenia and decreased il-7 plasma levels, initially high in hcv-infected patients. these data suggest that il-7 production by the liver, a consequence of active hcv replication, was reduced while patients controlled hcv viremia. restricted il-7 plasma levels might, in association with the antiproliferative effect of ifna, limit t-cell production in the thymus. our study highlights the therapeutic potential of il-7 as a complement to the standard ifna based treatment to help hcvinfected patients to sustain normal circulating t-cell counts, and restore the diversity of the peripheral t-cell repertoire through its central thymopoietic effect. restoring the breadth and intensity of t-cell control over the hcv virus might be immediately beneficial for the hiv/hcv co-infected population and offer new promising avenues for chronic hcv in the context of massive drop of hcv viral load after short term treatment with new antiviral compounds that will continue to be administered in combination with ifna [50] . sixteen hcv-infected patients (c-1 to c-16) and ten hiv/ hcv co-infected patients (i-1 to i-10) naïve to ifna therapy were enrolled in this study. a summary of the virological and immunological status of patients at baseline is shown in table 1. all the hiv/hcv co-infected patients but one were under haart with undetectable viremia (,40 hiv copies/ml). chronically infected patients (c-9 to c-16 and i-1 to i-10) initiated pegylated ifna/ribavirin treatment (ifna-2a: pegasys, 180 mg weekly, ribavirin: copegus, 800 mg to 1000 mg daily) and were followed over a 4 months period. patients included in the acute phase of hcv infection (c-1 to c-8) were treated with pegylated ifna (ifna-2a: pegasys, roche, 180 mg weekly) [51, 52] . blood samples were taken monthly on edta. two milliliters of total blood were 2-fold diluted in fcs/20%dmso frozen at 280uc and conserved in liquid nitrogen. these total blood samples were subsequently used for flow cytometry analyses. plasma was separated from the remaining eight milliliters and mononuclear cells were purified on ficoll hypaque (eurobio, courtaboeuf, france) and frozen for further analyses. patients from the hcv mono-infection group were followed at the centre de recherche du chum, hôpital saint luc, montreal, qc, canada and its collaborators as previously described [9, 53] . patients from the hiv-hcv groups were followed at the hôpital henri mondor, creteil, france. clinical protocols conformed to figure 5 . variations in il-7 plasma levels correlate with evolution of rte production. correlations. between variations in il-7 plasma levels (dil-7) and either variations in (a) total (cd4 + + cd8 + ) naïve t-cell counts (dnaïve t-cell counts), (b) rte defined as cd31 hi naïve cd4 + t-cells (drte cd4 counts), (c) the sj/btrec ratio (dsj/btrec ratio), (d) the frequency of ki-67 + cells in the rte cd4 + t-cell subset (d%ki-67 + in cd4 + rtes) or (e) the number of circulating ki-67 + cd4 + rtes (dki-67 + rte counts) between study entry and month 1 of therapy were calculated for acutely (black symbols) and chronically (white symbols) hcv-infected patients (left panels) and hiv/hcv co-infected patients (right panels). correlation coefficients (spearman's r) and the associated probabilities (p) are shown. doi:10.1371/journal.pone.0034326.g005 ethical guidelines of the authors' institutions and the us department of health and human services' human experimentation guidelines. this study was approved by both the ethical committee of centre hospitalier de l'université de montreal (chum) and the ethical committee of hôpital henri mondor, créteil, france. samples were obtained with the written subjects' informed consent. immunophenotyping and flow cytometry analysis facs analyses were performed on cryopreserved samples. after thawing blood cells were incubated for 15 minutes at 4uc with conjugated monoclonal antibodies (mabs). for intracellular labeling, cells were permeabilized with the cytofix/cytoperm kit (becton dickinson) before incubation with specific mabs according to the manufacturer's instructions. samples were then washed, fixed in 2% paraformaldehyde phosphate-buffered saline (pbs/pfa 2%) and acquired using a cyan cytofluorometer (dako) and analyzed with flowjo 8.7 software. the monoclonal antibodies used in this study were: cd3-pacific blue (pb) (clone ucht-1; dako, trappes, france), cd4-peridin chlorophyll protein-cyanine 5.5 (percp-cy5.5) (clone l200; bd, le-pont-de-claix, france), cd45ra-phycoerythrin (pe) (clone hi100; bd), ccr7-allophycocyanin (apc) (clone 150503; r&d systems europe, lille, france); cd8-phycoerythrin-cyanine 7 (pe-cy7) (rpa-t8; bd), cd31-biotin (clone wm59; abdserotec, düsseldorf, germany); ki-67-fluorescein isothiocyanate (fitc) (clone mib-1; dako), bcl-2-fitc (clone 124; dako) and strepatavidin-pe-texas-red (bd). il-7 was quantified in the plasma using the il-7 quantikine hs kit according to the manufacturer's instructions (r&d systems europe). plasma soluble-cd127 quantification soluble plasma il-7 receptor (scd127) quantification was performed as previously described [54] . parallel quantification of the sjtrec and the 13 djbtrecs, together with cd3c gene (used as a housekeeping gene) was performed for each sample using lightcyclertm technology (roche diagnostics) with a technique adapted from [29] . intrathymic precursor t-cell proliferation was evaluated through calculation of the sj/btrec ratio as described [23] . hcv rna quantification was performed using an in-house quantitative real-time reverse transcription-pcr assay as previously described [9] , cobas amplicor hcv monitor test tm , version 2.0 (sensitivity 600 iu/ml)), qualitative cobas ampli-prep/cobas amplicor hcv test tm , version 2.0 (sensitivity 50 iu/ml) or abbott realtime hcv assay tm (sensitivity 12 iu/ ml). statistical analyses (spearmans rank correlations and wilcoxon matched -paired signed-rank tests) were performed using the stata/ic 10.0 (stata corporation, college station, tx u.s.a.). due to the exploratory nature of the study there was no correction for multiple comparisons, and calculated p values are reported herein. mechanism of action of interferon and ribavirin in treatment of hepatitis c a new standard of care for the treatment of chronic hcv infection selective lymphocyte depletion during the early stage of the immune response to foot-and-mouth disease virus infection in swine quantitation of t lymphocyte subsets helps to distinguish dengue hemorrhagic fever from classic dengue fever during the acute febrile stage effects of severe acute respiratory syndrome (sars) coronavirus infection on peripheral blood lymphocytes and their subsets extensive lymphopenia due to apoptosis of uninfected lymphocytes in acute measles patients a dual role of ifnalpha in the balance between proliferation and death of human cd4+ t lymphocytes during primary response type i interferons act directly on cd8 t cells to allow clonal expansion and memory formation in response to viral infection early interferon therapy for hepatitis c virus infection rescues polyfunctional, long-lived cd8+ memory t cells t cell receptor excision circles (trecs), cd4+, cd8+, and their cd45ro+, and cd45ra+, subpopulations in hepatitis c virus (hcv)-hivco-infected patients during treatment with interferon alpha plus ribavirin: analysis in a population on effective antiretroviral therapy efficacy and safety of combination therapy with interferon-alpha2b and ribavirin for chronic hepatitis c in hiv-infected patients efficacy and safety of alpha-interferon treatment for chronic hepatitis c in hivinfected patients. hiv-hepatitis spanish study group future trends in managing hepatitis c treatment of hepatitis c and anemia in human immunodeficiency virus-infected patients use of pegylated interferons is associated with an increased incidence of infections during combination treatment of chronic hepatitis c: a side effect of pegylation? incidence of neutropenia and infections during combination treatment of chronic hepatitis c with pegylated interferon alfa-2a or alfa-2b plus ribavirin opportunistic infections and cd4 lymphocytopenia with interferon treatment in hiv-1 infected patients regulation of cytokine expression by interferon-alpha in human bone marrow stromal cells: inhibition of hematopoietic growth factors and induction of interleukin-1 receptor antagonist effects of recombinant alpha and gamma interferons on the in vitro growth of circulating hematopoietic progenitor cells (cfu-gemm, cfu-mk, bfu-e, and cfu-gm) from patients with myelofibrosis with myeloid metaplasia impairment of t and b cell development by treatment with a type i interferon cd4+ t-lymphocytopenia in hiv-infected patients receiving interferon therapy for chronic hepatitis c. hiv-hepatitis spanish study group type i interferons directly regulate lymphocyte recirculation and cause transient blood lymphopenia hiv infection rapidly induces and maintains a substantial suppression of thymocyte proliferation changes in thymic function with age and during the treatment of hiv infection slow disease progression and robust therapy-mediated cd4+ t-cell recovery are associated with efficient thymopoiesis during hiv-1 infection il-7 induces immunological improvement in siv-infected rhesus macaques under antiviral therapy t cell homeostasis: thymus regeneration and peripheral t cell restoration in mice with a reduced fraction of competent precursors the magnitude of thymic output is genetically determined through controlled intrathymic precursor t cell proliferation estimating thymic function through quantification of t-cell receptor excision circles interleukin-7 treatment counteracts ifn-{alpha} therapy-induced lymphopenia and stimulates siv-specific cytotoxic t lymphocyte responses in siv-infected rhesus macaques interleukin-7 promotes survival and cell cycle progression of t-cell acute lymphoblastic leukemia cells by down-regulating the cyclin-dependent kinase inhibitor p27(kip1) interleukin-7 mediates the homeostasis of naive and memory cd8 t cells in vivo increased production of il-7 accompanies hiv-1-mediated t-cell depletion: implications for t-cell homeostasis the role of interleukin-7 in early t-cell development a direct estimate of the human alphabeta t cell receptor diversity how cells respond to interferons augmentation of antitumor activity of 5-fluorouracil by interferon alpha is associated with up-regulation of p27kip1 in human hepatocellular carcinoma cells down-regulation of p27kip1 expression is required for development and function of t cells endogenous interferon-alpha production by differentiating human monocytes regulates expression and function of the il-2/il-4 receptor gamma chain two subsets of naive t helper cells with distinct t cell receptor excision circle content in human adult peripheral blood tlymphocyte populations in hepatitis c and hiv co-infected patients treated with interferon-alfa-2a and ribavirin interferon alpha augments activation-induced t cell death by upregulation of fas (cd95/apo-1) and fas ligand expression ifn-alpha suppresses activation of nuclear transcription factors nf-kappa b and activator protein 1 and potentiates tnf-induced apoptosis rapid decline of cd4+ cells after ifn alpha treatment in hiv-1 infection injection of glycosylated recombinant simian il-7 provokes rapid and massive t-cell homing in rhesus macaques interleukin 7 signaling in dendritic cells regulates the homeostatic proliferation and niche size of cd4+ t cells hepatic interleukin-7 expression regulates t cell responses loss of il-7 receptor alpha-chain (cd127) expression in acute hcv infection associated with viral persistence interleukin-7 receptor expression: intelligent design hepatitis c virus (hcv) genotype 1 subtype identification in new hcv drug development and future clinical practice determinants of antiviral treatment initiation in a hepatitis c-infected population benefiting from universal health care coverage response rates to pegylated interferon and ribavirin in hcv/hiv coinfection: a research synthesis rare birds in north america: acute hepatitis c cohorts a validated assay to measure soluble il-7 receptor shows minimal impact of il-7 treatment the authors acknowledge the subjects who participated in this study. we also thank c. chesnel for clinical and logistical support. key: cord-001021-nag4at49 authors: shaheen, hussam h.; prinz, bianka; chen, ming-tang; pavoor, tej; lin, song; houston-cummings, nga rewa; moore, renee; stadheim, terrance a.; zha, dongxing title: a dual-mode surface display system for the maturation and production of monoclonal antibodies in glyco-engineered pichia pastoris date: 2013-07-10 journal: plos one doi: 10.1371/journal.pone.0070190 sha: doc_id: 1021 cord_uid: nag4at49 state-of-the-art monoclonal antibody (mab) discovery methods that utilize surface display techniques in prokaryotic and eukaryotic cells require multiple steps of reformatting and switching of hosts to transition from display to expression. this results in a separation between antibody affinity maturation and full-length mab production platforms. here, we report for the first time, a method in glyco-engineered pichia pastoris that enables simultaneous surface display and secretion of full-length mab molecules with human-like n-glycans using the same yeast cell. this paradigm takes advantage of homo-dimerization of the fc portion of an igg molecule to a surface-anchored "bait" fc, which results in targeting functional “half” iggs to the cell wall of pichia pastoris without interfering with the secretion of full length mab. we show the utility of this method in isolating high affinity, well-expressed anti-pcsk9 leads from a designed library that was created by mating yeasts containing either light chain or heavy chain igg libraries. coupled with glyco-engineered pichia pastoris , this method provides a powerful tool for the discovery and production of therapeutic human mabs in the same host thus improving drug developability and potentially shortening the discovery time cycle. the use of surface display for discovery of novel monoclonal antibodies (mabs) has evolved dramatically over the last decade. multiple antibody display formats have been proposed and implemented in both prokaryotic and eukaryotic systems [1] [2] [3] [4] [5] . despite these advances, the majority of these efforts have focused on the utilization of antibody fragments as surrogates for mab lead identification and maturation rather than full-length immunoglobulin g proteins (iggs). while the antigen binding domains of antibodies can be used to evolve binding affinity, they poorly predict the physicochemical characteristics and expressibility within the context of the fulllength cognate iggs. in addition, these approaches necessitate conversion and reformatting in order to express the identified leads in the preferred production hosts [6, 7] . these additional steps account for delayed timelines and result in lead attrition due to the unpredictable differences between formats, and switching between display and expression hosts. recent reports have described novel platforms dedicated for the discovery of full-length iggs in bacterial, mammalian, and yeast cell lines [1, 8, 9] . to date, even with extensive engineering, bacterial systems are unable to generate fully human glycosylated proteins, and thus require additional conversion steps for expression and selection of the discovered leads in mammalian cell lines. furthermore, while bacterial display has been successful in isolating mabs which could bind with nano-molar affinities to their desired target [10] [11] [12] , the prokaryotic expression may not predict how proteins would behave once produced in the eukaryotic hosts that possess post-translational modifications such as glycosylation. in the case of mammalian display, direct anchoring of the igg to the cell membrane was required [9] . this entailed the introduction of an additional ectopic sequence to the igg heavy chain thus leading to additional conversion steps and further selection to enable soluble expression of fulllength mabs. surface re-capture technologies for full-length mabs in yeast were developed to enable display and secretion in the same cell. these methods utilized post-secretory binding of igg molecules in the culture medium to an anchored moiety on the cell surface [13, 14] . binding was achieved directly through protein-protein or protein-ligand interactions. although re-capture technologies couple secretion to display for full length iggs, they suffer drawbacks of their own, including the need for modifying the protein sequence to allow surface binding in particular cases. moreover, re-capture following secretion introduces the risk of "crosstalk" between clones that could lead to the loss of the required genotype-to-phenotype relationship that is critical for efficient lead-cell isolation. without a strong genotype-to-phenotype relationship built into the display system the issue of "cross talk" among clones in the same culture must be addressed through tedious experimental modifications [15] . until very recently, no methodology has been reported that utilizes cell surface display to facilitate discovery of full-length mabs that unify discovery and production hosts while preserving the molecular integrity of the igg [16] . glycoengineered pichia pastoris has been successfully used for production of monoclonal antibodies and therapeutic development [17] [18] [19] [20] . here, we describe a dual-mode technique for engineering and production of full-length mabs in glyco-engineered pichia pastoris. this approach preserves cosecretion of full-length unmodified bivalent mabs into culture medium while utilizing the natural antibody biogenesis pathway to covalently display monovalent functional half igg molecules on the cell wall. this facilitates the simultaneous selection of an antibody sequence with desired binding properties and its subsequent production in the same host. in addition, we also describe the first attempt to utilize this approach alongside haploid yeast mating by combining full-length heavy chain and full-length light chain libraries and the application of this display system to mature the expressibility of a monoclonal antibody lead while maintaining its affinity. dna recombination and cloning were performed with restriction endonucleases and dna modification enzymes from new england biolabs (beverly, ma) and e. coli strain top10 from invitrogen (carlsbad, ca). oligonucleotides were synthesized by integrated dna technologies (coralville, ia) and pcr reactions were performed with pfuturbo from stratagene (la jolla, ca). gpi protein genes were codon optimized for p. pastoris and synthesized by geneart ag (regensburg, germany). pichia transformations were conducted using electroporation. table 1 lists the yeast strains used in this study. all glyco-engineered pichia pastoris expression strains used were constructed from wild-type pichia pastoris strain nrrl-y11430 (northern regional research laboratories, peoria, il) using methods described in [21] [22] [23] [24] . anti-pcsk9 yeast display mating library construction was described in chen et al. [25] . to create the plasmid containing the fc bait cassette, a codon optimized sequence of human igg1 fc fragment was synthesized using an ecori forward pcr primer containing the nucleic acid sequence of s. cerevisiae α-mating factor signal sequence fused upstream of the sequence encoding the igg1 fc n-terminus (dkthtcppc.), and a sali reverse primer encoding the c-terminus of igg1 fc that terminates in a sequence encoding a gggg linker. a plasmid containing the human igg1 heavy-chain gene sequence was used as a pcr template for amplification of an ecori-α-mating factor signal sequence-fc-gggg-sali fragment. both pcr product and pgly3033 [3] were digested using ecori and sali endonucleases. the ecori-sali fragment encoding the fc was ligated in frame to ecori-sali pgly3033 backbone to generate plasmid pgly9008. this plasmid enables delivery of the fc-scsed1 cassette under the control of the pichia pastoris aox1 promoter sequence. like the parent plasmid, it contains the pichia pastoris ura6 gene sequence, which serves as an integration locus in the genome, and the arsenite resistance gene, to allow selection on media containing sodium arsenite. one liter bioreactor and micro24 cultivations were performed as described previously [25] . the binding affinity of the anti-pcsk9 antibodies was measured on a biacore t100 instrument with a carboxymethylated dextran (cm5, cat# br-1006-68) chip and 1× hbs-ep+ (10 mm hepes, 150 mm nacl, 3 mm edta, and 0.05% surfactant p20) as the running buffer. the cm5 chip was immobilized on all flow cells with mouse anti-human igg (fc specific) according to the biacore human antibody capture kit (cat# br-1008-39) to ~ 7000 ru. anti-pcsk9 antibodies were captured on the chip to ~ 500 ru followed by analyte injections of wild-type human pcsk9 from 0.156 nm to 2.5 nm, except for the 96-well affinity measurements were crude supernatants were captured followed by a single injection of 2.5 nm of rhpcsk9. each flowcell was regenerated between each analyte injection with 3 m mgcl for 40 s at 10 μl/min. data was analyzed with biacore t100 evaluation software using the 1/1 binding model. after induction on methanol, 2 od 600 of cells(~ 10 7 cells) were collected into a 1.5-ml microfuge tube and washed twice with phosphate-buffered saline (pbs, sigma, st. louis, mo) and then suspended in 100 μl of pbs containing 1 μl (2 μg) of goat anti-human fc dyelight 488, or anti-human kappa light chain apc 635 (invitrogen, carlsbad, ca) at room temperature for 30 min. when labeling for both expression and affinity, pcsk9 conjugated with biotin (merck, whitehouse station, nj) was also added at a final concentration of 20 nm and detected with streptavidin conjugated with dyelight 488 or apc 635. after incubation with detection antibodies/reagents the cells were washed twice with pbs and suspended in 100 μl of pbs for flow cytometric analysis and sorting (when required). the labeled cells were kept on ice and protected from light throughout the experiment. flow cytometric analysis and cell sorting were performed on a facsaria cell sorter with blue and red lasers (bd biosciences, san jose, ca) equipped with facsdiva software. the procedure was performed according to lin et al. [3] . gating in a dot plot of fsc vs. ssc was routinely applied to exclude cell debris and to include a population of single cells with similar size for analysis and sorting. for each sort, the 1% of cells with the brightest signal were gated and 5000-10,000 total cells were collected. flow cytometry data were analyzed with software flowjo v 7.1.2 (tree star, inc., ashland, or). mabs were purified using a one-step, 96-well format streamline rprotein a methods described in [26] . proteins were resolved using labchip® gx ii (perkinelmer, waltham, ma). pngase f, 2-aminobenzamide (2-ab) labeling and high performance liquid chromatography (hplc) analysis of glycans has been described previously [22] . we designed a dual-mode antibody display system for presenting antibody molecules on the surface of pichia pastoris. in this system, the hinge region along with ch2-ch3 domains comprised in the crystalizable fragment (fc) of the igg1 molecule was directly fused to the n-terminus of the previously described glycophosphoinositol (gpi) protein scsed1p [3] . the introduction of this fusion in yeast results in the targeting of the fc to the cell wall. as described in figure 1 , once this anchored fusion is co-expressed with a full-length igg molecule, the fc portion of the fc-sed1p fusion is expected to hetero-dimerize and form inter-disulfide linkages, through their respective hinge regions, in the endoplasmic reticulum (er) with the fc portion of the heavy chain (hc) of the half igg molecule. the complex interaction of fc-scsed1p-fc(hc+lc) coupled to the association between the heavy chain and the light chain (lc) within the antigen binding fragment (fab) results in the display of the monovalent hc+lc moiety on the cell surface. the concomitant and independent association of the soluble heavy and light chain units of the full length igg molecule would result in the secretion of fully assembled antibody molecules. to reduce the above concept into practice, we introduced the fc-scsed1p expression cassette into three glyco-engineered pichia pastoris strains. as described in table 1 these strains included yeast ygly18483 which expresses full-length antihuman pcsk9, and ygly13979 which expresses full-length anti-human her2 iggs. we assayed antibody display and expression in each strain following cultivation in 1l bioreactors by two independent methods. first, we examined the display of monovalent iggs on the cell surface by monitoring the expression of the light chain on the yeast cell surface. cells were labeled with anti-human igg kappa chain and their fluorescence intensity was analyzed using flow cytometry ( figure 2a ). we reasoned that the presence of the light chain on the cell surface would be indicative of the successful heterodimerization and assembly of cell wall anchored fc-scsed1p and monovalent hc+lc igg molecule. second, the supernatants of cultures in (figure 2a ) were passed through a protein a column to capture secreted full-length igg. the eluents were subsequently resolved by electrophoresis to establish the presence of full-length fully assembled iggs ( figure 2b ). as demonstrated in figure 2a (i; ii), only strains containing both fc-scsed1p bait and full-length igg expression cassettes reacted with anti-human kappa light chain antibodies indicating the ability of the system to display the (hc+lc) half antibody molecules. moreover, electrophoresis of protein acaptured pools confirmed that the supernatants of the display strains contain levels of secreted full-length iggs that are similar to those of the non-display parents without fc-scsed1p bait ( figure 2b ). we also employed 2-aminobenzamide (2-ab) labeling and high performance liquid chromatography (hplc) to analyze the glycan content on iggs at fc n297 site produced in strains with or without fc-scsed1p. we observed that the glycosylation signatures in the display-secretion strains (with "fc bait") were similar to those of non-display parent strains without "fc bait" (figure 2c ). taken together these experiments prove that this system is capable of displaying monovalent antibodies while maintaining simultaneous secretion of soluble full length assembled (hc2+lc2) molecules. furthermore, the secreted iggs are modified with human-type n-glycans that are comparable to those that occur on molecules produced by the "non-display" strains. to demonstrate that the robustness of dimerization between the "bait" fc-scsed1p and fc region of igg (hc+lc) occurs through hinge region disulfide linkages, we subjected cultures displaying anti-human pcsk9 to various denaturation conditions and then probed the cell surface for the presence of iggs. as described earlier, we labeled cells with fluorescentlyconjugated anti-human fc and anti-human igg kappa chain antibodies followed by flow cytometry. only when cultures were treated with 0.5m dtt in the presence of 6m urea, did we observe a complete loss in the ability to detect the light chain on the surface of these cells (50% of fc was still detectable due to the presence of cell wall-anchored fc-sed1p bait) ( figure 2d ). we concluded that in this method, the capture of the half antibody (hc+lc) on the cell surface occurs via both hydrophobic and covalent interaction in a manner similar to the assembly of full length iggs. we next sought to establish whether the displayed monovalent antibodies are properly assembled and are able to bind to their cognate antigens. to this end, we probed the ability of displayed anti-pcsk9 (hc+lc) antibody to bind specifically to biotinylated human pcsk9. an anti-her2 display strain was used as a negative control. we incubated both display strains with fluorescently labeled anti-human fc and biotinylated human pcsk9. as shown in figure 3a , yeasts displaying anti-her2 on cell surface were able to bind to the anti-fc reagent only, while cells displaying anti-pcsk9 reacted with both biotinylated pcsk9 antigen and anti-human fc antibody ( figure 3b ). the goal of developing this novel antibody surface display is its application in antibody discovery and maturation while preserving full-length igg secretion. for that purpose, we designed a proof-of-concept study to demonstrate the utility of this display method in maturing the expressibility of an antibody lead in glyco-engineered pichia pastoris while maintaining the affinity to its known antigen. an anti-pcsk9 mab (mab1) was originally identified from a phage display library with low nanomolar affinity. in order to isolate novel anti-pcsk9 leads with optimal expression in glyco-engineered pichia pastoris, two synthetic dna libraries covering heavy and light chain cdr3 regions were designed and synthesized by site directed mutagenesis. the final diversity of the heavy chain library was 4,320 while the final diversity of the light chain library was 1,296. both heavy chain and light chain libraries were introduced separately into haploid pichia pastoris strains containing the fc-scsed1p expression cassette. to create a full-length antibody library, haploid libraries of the heavy and light chains were combined by mating to generate a diploid fulllength igg library with a diversity of ~5 x 10 6 . we sequenced the heavy and the light chain genes from 40 diploid strains. this confirmed both the presence of heavy and light chains, and the amino acid sequence diversity in the library, as all of the clones tested contained unique antibody genes. we also analyzed affinities of secreted iggs produced in these diploids by surface plasmon resonance (spr). although the majority of these diploid clones did not produce high levels of full-length antibodies as assessed by elisa and sds-page (data not shown), no pcsk9 binders were observed in this randomly selected pool where full length igg was produced, thus indicating majority of mutations that were introduced to this antibody resulted in loss of optimal expression and antigen binding (see figure 4b, panel l1) . we subsequently employed facs to isolate highly-expressed mabs that retained binding to pcsk9. to this end, three rounds of sorting were carried out. twenty nm biotinylated pcsk9 antigen was used in each round, and the top 1% binders were isolated based on fluorescence intensity. after each round, ~1000 clones were plated on solid medium. forty clones were selected, and their heavy chain and light chain genes were pcr amplified and sequenced to determine their amino acid sequence. since this method enables simultaneous display and production of mabs, leads selected by facs were directly cultured in 96-well plates to produce full-length secreted igg for in vitro pcsk9 binding studies. as depicted in figure 4b , when compared to the anti-pcsk9 positive control, we were able to enrich for a pool of positive pcsk9 binders following three rounds of facs ( figure 4b : l1 > s1 > s2 > s3). interestingly, the final round of sorting enriched a single mab candidate ( figure 4b : s3), as suggested by gene sequencing data, this clone was highly represented after two sorting rounds which indicates the high efficiency of facs enrichment (table 2 ). using single-point (2.5 nm rhpcsk9) spr, the affinities of a select number of co-secreted iggs from round two clones were determined. as listed in table 2 we were able to isolate a novel mab against psck9 with affinity similar to the original anti-pcsk9 lead. the anti-kappa elisa protein titers for the novel leads reached up to 30 µg/ml compared with 10 µg/ml for the mab1 control. we sought to generate novel anti-pcsk9 binders with optimized expressibility in glyco-engineered pichia, while maintaining affinity of the original lead. to confirm this in a bioreactor setting, the selected clones: high affinity s2-10, and medium affinity s2-15 were cultivated and fermented in smallscale bioreactors to generate full-length mabs. originator anti-pcsk9 (ax) expression strain was included as a control. following fermentation, mabs were purified using protein a chromatography. the concentration of antibody was 4 . the construction of a proof of principle mating library and the isolation of high affinity, and high expression antigen binders using fc-sed1p. a) haploid pichia pastoris strains containing fc-sed1p expression cassette were transformed with a library of anti-pcsk9 hc or a library of anti-pcsk9 lc. following mating and selection, diploids were cultured to express full length igg library. cultures were labeled using 20nm biotinylated human pcsk9 and apc 635 labeled antihuman kappa. dyelight 488 labeled streptavidin was used to detect biotinylated pcsk9. b) analysis and enrichment of high affinity anti-pcsk9 binders using three rounds of sequential sorting (s1 > s2 > s3). known anti-pcsk9 strain co-expressing fc-sed1p was labeled as above as a control. c) sensograms showing binding and dissociation kinetics of rhpcsk9 to immobilized anti-pcsk9 antibodies. each anti-pcsk9 antibody lead was captured on the chip to ~ 500 ru followed by analyte injections of wild-type human pcsk9 at concentrations ranging from 0.156-2.5nm. kinetics for the highest pcsk9 concentration (2.5 nm) is depicted for these antibodies. doi: 10.1371/journal.pone.0070190.g004 determined using protein a hplc analysis and affinities were measured by spr by injecting rhpcsk9 at concentrations ranging from 0.156 to 2.5 nm. figure 4c and table 3 show that the ranking of affinities of these novel iggs were in agreement with data obtained in 96-well experiment, however the affinity values for lead s2-15 appeared lower in the 96-well experiment ( table 2 ). this is primarily due to the nature of the biacore data that is obtained by assaying crude supernatants from 96-well plates using a single concentration of the antigen. for that reason this assay can only be used for highthroughput clone selection, and data from the purified samples (table 3) are reported for the actual affinities. the newly identified s2-10 exhibited a slightly slower "off" rate but maintained almost identical "on" rate compared to the original molecule (ax), which might account for the difference in affinity (2.14 nm compared to 4.32 nm, respectively) ( table 3) . moreover, the expression levels of newly identified leads reached up to 0.5 g/l compared with ~100mg/l observed for the original anti-pcsk9 lead ax. this demonstrates that this display method is capable of discovering novel high affinity antibody leads with improved production titers. we have presented the development of a novel dual-mode display and secretion technology in glyco-engineered pichia pastoris, and demonstrated the utility of this method in selecting mab leads with variable affinities and high productivity. the combination of display and secretion in the same clone enables the continuity and fidelity of the antibody discovery process and could lead to shortened maturation cycle and desirable lead developability. here, we describe, a novel system for the display of monovalent functional antibodies on the surface of glycoengineered pichia pastoris. this technology takes advantage of the antibody dimerization in the er to achieve capture of the fc region of a half mab to an anchored fc molecule through covalent disulfide bond formation in the hinge region, while preserving the assembly and secretion of free soluble fulllength iggs. previous methods have utilized fab anchoring through pairing of protein partners in the er, where one of the monomers is anchored to a surface molecule. examples of such display systems include the aga1-aga2 [27] and the gr1-gr2 [3] systems. the utilization of the native fc region as an anchoring surrogate, nevertheless, simulates the process of antibody assembly, and thus eliminates expression or stability biases that could be introduced through ectopic anchoring sequences. this surface display module in combination with facs could be employed to screen large igg libraries for high affinity and well-expressed leads that also possess desirable stability profilesyeast libraries could be subjected to various robustness tests to select clones that maintain antigen binding following denaturing conditions. n-glycans can impart desired biophysicochemical properties on proteins including solubility and formulability [28] [29] [30] [31] . antibody libraries in glycoengineered pichia can be designed to include canonical glycosylation sites in the fab region. facs can be used to obtain sequences containing n-glycosylation that do not interfere with antigen binding affinity. in our studies, we used glyco-engineered pichia strains to establish proof of principle of the anchored fc bait as a dual-mode antibody surface display and production platform. in a similar manner, it is conceivable that any eukaryotic antibody production cell line could be used to display monovalent (hc+lc) while simultaneously secreting full-length iggs. other membrane or cell wall proteins can be used to anchor fc regions of any igg subclass. however, glyco-engineered pichia offers advantages over other established antibody discovery platforms. it is able to generate igg molecules with human glycoforms while maintaining the high transformation efficiency of yeast. furthermore, the tractability of a fungal system enables coevolving the cell line and molecule of interest in the same experiment to generate "genetically-customized" production hosts for each discovered lead. it is of importance to note that this system as well as other display formats may be biased towards selecting antibodies with lower affinity and higher expression over ones with higher affinity but lower expression. for that reason extra care should be employed in designing and performing facs experiments to include clones with such criteria. surface display boasts the ability to link genotype to phenotype. in the classical antibody display paradigms, sorted clones are isolated and their genotypes are subcloned into new production hosts. this cloning step involves screening multiple clones to isolate ones with defined properties. the anchored fc system side-steps the need for these additional steps by combining selection of antibody affinity and production host in a single experiment. libraries can be generated and displayed in the cell line of choice, such as glyco-engineered pichia pastoris. selected clones can be fermented directly in small or large scale vessels to generate material for in vitro assays such as affinity measurements, cell culture assays, or physicochemical properties. successful candidates can be fed directly into in vivo preclinical models via simple scale-up production in stirred tank reactors ( figure 5 ). if desired, once a clone is selected, genetic approaches can be utilized to evict the anchored fc cassette to generate an antibody production strain. the development of a dual antibody surface display concept that preserves the ability to simultaneously secrete full-length igg simplifies the process of therapeutic antibody discovery and development. it also has the potential to shorten development timelines and increase the probability of success. when combined with glycofi mabs expression platform in glyco-engineered pichia pastoris, it provides a bona fide nextgeneration, end-to-end antibody discovery and production platform. secretion-and-capture cell-surface display for selection of targetbinding proteins phage antibodies: filamentous phage displaying antibody variable domains a novel fragment of antigen binding (fab) surface display platform using glycoengineered pichia pastoris bivalent antibody phage display mimics natural immunoglobulin multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (fab) heavy and light chains recombinant human antibodies: linkage of an fab fragment from a combinatorial library to an fc fragment for expression in mammalian cell culture baculovirus expression cassette vectors for rapid production of complete human igg from phage display selected antibody fragments isolation of engineered, full-length antibodies from libraries expressed in escherichia coli development of a novel mammalian cell surface antibody display platform anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from escherichia coli-expressed libraries engineering of recombinant antibody fragments to methamphetamine by anchored periplasmic expression increased antibody affinity confers broad in vitro protection against escape mutants of severe acute respiratory syndrome coronavirus isolation of antigen-binding camelid heavy chain antibody fragments (nanobodies) from an immune library displayed on the surface of pichia pastoris construction of a novel synergistic system for production and recovery of secreted recombinant proteins by the cell surface engineering a flow cytometric assay for screening improved heterologous protein secretion in yeast simultaneous surface display and secretion of proteins from mammalian cells facilitates efficient in vitro selection and maturation of antibodies optimization of humanized iggs in glycoengineered pichia pastoris glycoengineered pichia produced anti-her2 is comparable to trastuzumab in preclinical study optimization of a glycoengineered pichia pastoris cultivation process for commercial antibody production improved production of monoclonal antibodies through oxygenlimited cultivation of glycoengineered yeast use of combinatorial genetic libraries to humanize n-linked glycosylation in the yeast pichia pastoris humanization of yeast to produce complex terminally sialylated glycoproteins cloning and disruption of the pichia pastoris arg1, arg2, arg3, his1, his2, his5, his6 genes and their use as auxotrophic markers a combinatorial genetic library approach to target heterologous glycosylation enzymes to the endoplasmic reticulum or the golgi apparatus of pichia pastoris generation of diploid pichia pastoris strains by mating and their application for recombinant protein production purification process development of a recombinant monoclonal antibody expressed in glycoengineered pichia pastoris isolating and engineering human antibodies using yeast surface display glycosylation influences on the aggregation propensity of therapeutic monoclonal antibodies post-translational modifications differentially affect igg1 conformation and receptor binding the antibody paradigm: present and future development as a scaffold for biopharmaceutical drugs effect of posttranslational modifications on the thermal stability of a recombinant monoclonal antibody we thank previous and current glycofi members for supporting the work in this study, and for their suggestions and comments in preparing this manuscript. the authors would also like to thank ann marie norris for assistance with figures and art. key: cord-001078-5m29nugu authors: chen, xiaorong; yang, zongguo; lu, yunfei; xu, qingnian; wang, qiang; chen, liang title: clinical features and factors associated with outcomes of patients infected with a novel influenza a (h7n9) virus: a preliminary study date: 2013-09-17 journal: plos one doi: 10.1371/journal.pone.0073362 sha: doc_id: 1078 cord_uid: 5m29nugu objective: the present study aimed to analyze clinical features and factors associated with treatment outcomes of h7n9 influenza a virus infection. methods: the clinical progress in 18 h7n9-infected patients was monitored and recorded. the clinical features of h7n9 infection were noted and factors associated with treatment outcomes were analyzed by univariate analyses. results: the average ages of patients in recovered and critical conditions were 67.0±10.83 years and 72.75±12.0 years, respectively. renal insufficiency developed more frequently in critically ill patients (p = 0.023). the duration of traditional chinese medicine (tcm) therapy was longer in recovered patients than in critically ill patients (p = 0.01). laboratory tests showed that levels of c-reactive protein, serum creatinine, and myoglobin were significantly higher in critically ill patients than in recovered patients (p = 0.011, 0.04, and 0.016, respectively). meanwhile, levels of all t cell subsets examined including total cd3(+), cd4(+), cd8(+), and cd45(+) t cells were lower in critically ill patients than in recovered patients (p = 0.033, 0.059, 0.015, and 0.039, respectively). logistic regression analysis demonstrated that c-reactive protein level, myoglobin level and tcm therapy duration were likely associated with treatment outcomes of h7n9 infection (p = 0.032, 0.041 and 0.017, respectively). conclusion: elderly people may have increased risk for h7n9 virus infection. t cell-mediated responses play an important role in defense against the h7n9 virus. c-reactive protein level, myoglobin level and tcm duration may be associated with treatment outcomes of h7n9 infection. avian influenza caused by influenza a viruses (iav) has become a serious threat to human health in recent years. infections with avian influenza viruses, which usually occur after exposure to poultry and/or wild birds, are commonly characterized by conjunctivitis, upper and/or lower respiratory tract disease, pneumonia, and multiple organ failure [1] [2] [3] . wild birds are believed to constitute the natural reservoir for iavs and play a central role in the spread of the viruses. iavs are classified into subtypes based on different combinations of 16 hemagglutinin (ha: h1 -h16) and 9 neuraminidase (na: n1 -n9) surface antigens, and two pathotypes (low and high pathogenicity) based on lethality for chicken. all highly pathogenic avian iavs known to date belong to the h5 or h7 subtype [4] . the h7n9 viruses, a subgroup of h7 viruses that normally spread among birds have currently been found to cause human infections in china, prompting intensive research to address the challenge of a potential epidemic/pandemic. although h7n9 family clusters have been reported in shanghai and shandong province [5] , no evidence of human-to-human h7n9 virus transmission has been found to date [6] . presently, there is a general lack of information about h7n9 infections, e.g., the transmission mechanism and the number of mild infections in china are unknown. unlike h5n1, which could wipe out a flock of poultry within days, h7n9 infections in poultry are usually asymptomatic or mild. nonetheless, based on what has been learned from the h5n1 avian flu virus which has killed 371 people in 15 countries since 2003 and proved extremely difficult to control, the risk of an h7n9 epidemic/pandemic should not be underestimated, especially in china where poultries are reared with diverse methods without strict biosecurity measures [7] . studies on h7n9 transmission mechanisms and clinical features/outcomes are critical to prevent and control potential h7n9 epidemic/pandemic. overall, few cases of h7 virus transmission to mammals have ever been reported in asia and n9 virus infections in human have never been documented anywhere in the world [8] except h7n9 [7] . the h7n9 variants currently in circulation most likely evolved through a combination of genes from viruses in beijing bramblings, zhejiang ducks, and korean wild birds according to report by chinese scientists. human infections with highly pathogenic h7 viruses generally resulted in conjunctivitis or uncomplicated influenza [3, 8] study protocol for this preliminary investigation and informed consent documents were reviewed and approved by the ethics committee of shanghai public health clinical center affiliated with fudan university. all participants provided written informed consent after receiving information of the aims, methods, anticipated benefits, and potential hazards of the study. all work reported herein was conducted in china. diagnosis of h7n9 infection in patients was made by positive test for h7n9 viral rna. throat-swab specimens were collected from patients and tested for h7n9 viral rna by real-time reverse transcription polymerase chain reaction (rt-pcr), which was carried out using standard rt-pcr protocol at shanghai centers for disease control and prevention. all 18 patients who participated in the study were admitted to shanghai public health clinical center and diagnosed with h7n9 infection from april 6 to april 20, 2013. their medical records were obtained and analyzed. laboratory tests of blood, liver function, renal function, myocardial enzyme, t cells, and immunoglobulins were carried out at medical inspection department of shanghai public health clinical center. other information about patients was either retrieved from patients' medical records or acquired directly from patients via a questionnaire. factors analyzed for possible correlation with clinical features and treatment outcomes in patients included 1) baseline characteristics of patients, such as age, sex, occupation, underlying conditions, exposure to poultry and/or wild birds in the past seven days, date of symptom onset and hospital admission, date of specimen collection, and date of positive diagnosis; 2) results from laboratory tests and imaging examinations; 3) treatment regimen including basic supporting therapy, antibiotic therapy, antiviral therapy, traditional chinese medicine (tcm) therapy, and other therapies if applicable; and 4) current condition of patients including the length of stay in the hospital. following guidelines from the influenza a h7n9 clinic program published by the national health and family planning commission of p. r. china (the 2nd edition, 2013) [9] , we considered patients who met the following criteria recovered: 1) throat-swab specimens tested negative for h7n9 viral rna, and 2) disappearance of fever and other clinical symptoms and signs. following the same set of guidelines, we considered patients who met the following criteria critically ill: 1) pneumonia with two or more complications, such as acute respiratory distress syndrome, heart failure, renal failure, septic shock, encephalopathy, and secondary infections; and 2) no improvement in at least one of the above mentioned complications after 3 d of active treatment. all participating patients were treated by twice daily oral administration of 75 mg oseltamivir. patients were also given antibiotics based on blood and/or throat-swab specimens/sputum tests for bacterial infections. if no specific bacterial pathogens were detected from the specimens, advanced treatment was considered. antibiotics given to h7n9-infected patients if applicable included moxifloxacin, sulbactam and cefoperazone, levofloxacin, meropenem, piperacillin, imipenem, and cilastatin. some patients also received glucocorticoid therapy, intravenous immunoglobulin therapy, and tcm therapy. only chinese herbs prescribed according to specific syndromes were considered tcm in this study. proprietary chinese medicines and injections of chinese medicines were excluded because they could contain certain western drug ingredients. these herbal tcms were prescribed following group discussions of tcm experts from longhua hospital, shanghai university of tcm, department of tcm of zhongshan hospital, fudan university, and xr chen in our hospital. based on syndrome differentiation criteria from wei-qi-ying-xue and clinic programs of influenza a h7n9 implemented by the national health and family planning commission of p. r. china, yinqiao powder and hoisting powder were prescribed for patients with mild syndromes and qingwen-baidu-decoction was prescribed for critically ill patients. tcms were taken orally twice daily at 150 ml decoction per dose. the purchase, decoction, and administration of chinese herbs were supervised by pharmacy department of tcm in shanghai public health clinical center. baseline data were expressed as mean 6 standard deviation (sd) or median values. the kolmogorov smirnov test was used for the test of normality on quantitative data. to analyze differences between baseline data, student's t-test analysis was performed on mean numeric data, mann-whitney u-test analysis was performed on non-numeric data, and fisher's exact test was used for categorical variables. logistic regression models were used to evaluate possible factors contributing to different outcomes in h7n9-infected patients. results were reported as odds ratios (or) with their 95% confidence intervals (ci). all baseline characteristics and laboratory tests were analyzed using univariate analysis. multivariate analysis was not considered suitable for such a small sample study. statistical analyses were performed using pasw statistics software version 18.0 from spss inc. (chicago, il, usa). all p values were two-tailed, and differences with p,0.05 were considered statistically significant. patients were grouped according to their conditions. patients in group a were in recovered condition or had recovered, and patients in group b were in critical condition or had died. there were 14 males and four females in the 18 h7n9-infected patients. as shown in table 1 , 10 patients were in group a and eight were in group b. average ages of patients in group a and group b were 67.0610.83 and 72.75612.0 years, respectively. four patients, including three from group a and one from group b, had a history of smoking. in group a, seven patients had underlying conditions: five had hypertension, one had coronary heart disease, and one average time from onset of symptoms to treatment with antiviral agents was 5 or 6 days in both groups. five patients, including one from group a and four from group b, developed acute respiratory distress syndrome during the course of the disease. seven patients, two from group a and five from group b had heart failure. four patients experienced renal failure and three experienced septic shock, all from group b. three patients in group b were diagnosed with encephalitis based on their clinical symptoms, dysphoria and one in lethargy condition. all three patients developed poor cognitive ability as observed in medical examinations although no csf test was conducted on these patients. two patients died while the third in critical condition with no improvement observed in their neurological condition throughout treatment. moreover, three patients from group b who tested positive in the sputum culture fungal spore test also had secondary infections. renal failure rates were significantly different between the two groups (p = 0.023). as shown in figure 1 , more than half of the 18 h7n9-infected patients suffered from fever (88.9%), cough (77.8%), expectoration (55.6%), fatigue (50.0%), poor appetite (83.3%), dry month (72.2%), thirst (72.2%), dyspnea (66.7%), chest distress (66.7%), and bitter taste in month (61.1%). in addition, five patients suffered from hemoptysis and two suffered from dysphoria. other symptoms such as muscle soreness (3), aversion to cold (2), perspiration (5), pharyngodynia (1), short breath (4), deep yellow urine (3), and cold-limbs (1) also occurred in patients with h7n9 infection. although no statistically significant differences were found in routine blood indices between the two groups, the c-reactive protein level was significantly higher in group b patients than in group a (table 2 ). also, serum creatinine level in group b was higher than that in group a (126.03644.63 mmol/l in group b vs. 76.91610.69 mmol/l in group a, p = 0.04), indicating more severe renal impairment in critically ill patients. while critically ill patients had significantly higher myoglobin levels than recovered patients (p = 0.016), no differences in immunoglobulin concentrations were observed between the two groups. however, all t cell subsets examined including total cd3 + , cd4 + , cd8 + , and cd45 + t cells, were lower in group b patients than in group a. baseline characteristics were included in the univariate analysis to identify possible factors associated with critical outcomes (table 3 ). our univariate analysis indicated that 1 mg/l increase in c-reactive protein (p = 0.032), 1 mmol/l increase in total bilirubin (p = 0.096), 1 mmol/l increase in serum creatinine (p = 0.1), 1 mg/l increase in myoglobin (p = 0.041), and 1 cell/ml increase in cd3 + /cd4 + /cd45 + t cells were likely associated with a critical outcome (p = 0.089, 0.093, and 0.08, respectively). one-day increase in tcm therapy was found to be significantly associated with a critical outcome (p = 0.017). these results might be limited by the small sample size in our study. further studies with larger sample sizes should be conducted to verify our preliminary results. the recent discovery of h7n9 virus infections by a new influenza virus strain in three critically ill patients, as reported by gao et al. [8, 10] , is of major public health significance. laboratory testing conducted in china showed that h7n9 viruses were sensitive to anti-influenza drugs known as neuraminidase inhibitors. these drugs were effective against seasonal influenza and h5n1 virus infections when given early in the course of the illness. however, these drugs had not yet been used to treat h7n9 infections. to date, the national health and family planning commission of p. r. china has released two editions of the influenza a h7n9 clinic programs in which supporting therapies including oxygen therapy, antipyretic therapy, and cough and phlegm relief and medicinal therapy with antiviral drugs such as oseltamivir and zanamivir were recommended. particularly, respiratory support including extracorporeal membrane oxygenation was recommended for critically ill patients. tcm therapy was also recommended as a second-line treatment for h7n9 infection. previous studies have demonstrated that tcms may have beneficial effects on patients with avian influenza [11, 12] . our univariate logistic regression analysis indicated that the duration of tcm therapy could be partially associated with the prognosis in h7n9-infected patients. however, tcm therapy was not considered for four critically ill patients with ards, which might have accounted for the short duration of tcm therapy in the critically ill group. consequently, the therapeutic value of tcm therapy in critically ill patients might have been underestimated. further studies including randomized controlled trials are needed to fully evaluate tcm therapy in this patient population. nine patients were also treated with intravenous immunoglobulins (ivig). six of the nine patients recovered, one was in severe condition, and two died. examination of baseline characteristics of h7n9-infected patients revealed that most patients were elderly males, suggesting that elderly people were at higher risk for h7n9 infection. in contrast, a high proportion of severe cases and deaths in h5n1 infection occurred in children [13] . the high proportion of elderly patients in h7n9 infection might be attributed to higher exposure of the elderly to h7n9 virus or to age-related body functions, such as decreased immune function [14] . although h7n9 infections preferentially occurred in males, our logistic regression analysis did not show any statistically significant differences in clinical outcomes between genders. our results suggested that history of smoking and/or underlying clinical conditions may increase risk for infection, especially for patients with hypertension, diabetes, and heart-pulmonary insufficiency. four patients, including three from group a and one from group b, had a smoking history of 30 to 40 years with 20 to 40 cigarette smoking per day, which could have contributed to their poor respiratory function. nearly half of the patients (8/18) had hypertension, two had diabetes, three had coronary heart diseases, and three had copd, indicating that chronic heart and lung diseases would increase risk for h7n9 infection. poultry and wild birds have been recognized as natural reservoir of iavs [15] . in our study, only three patients had confirmed exposure to poultry or wild birds 7 d before onset of illness. however, according to previous report, 60% of h7n9-infected patients in china had epidemiological links to poultry and/or wild birds [16] . li et al. also reported that 77% of h7n9-infected patients in their study had a history of exposure to live animals, including chicken (76%) [17] . taken together, avoiding contact with poultry and wild birds may effectively reduce the risk of infection. meanwhile, placing infected poultry and wild birds in quarantine is important for the control and prevention of h7n9 infection. our results also suggested that early diagnosis and treatment of h7n9 infection were critical in achieving favorable clinical outcomes. in our study, nearly all h7n9-infected patients were treated as common pneumonia in the early stage of the illness. the average time from onset of symptoms to h7n9 virus confirmation was 7.162.03 days in group a and 6.3862.07 days in group b. antiviral treatment was not initiated until 5-6 days after the onset of illness. the delayed diagnosis and treatment for this highly pathogenic avian influenza virus infection might have partially contributed to the poor prognosis in h7n9-infected patients. we believe that early surveillance and early diagnosis are of great importance for favorable clinical outcome. full characterization of clinical features of h7n9 infection should speed up diagnosis and treatment. most h7n9-infected patients in our study had developed severe conditions with one or more complications, such as acute respiratory distress syndrome, heart failure, renal failure, septic shock, encephalopathy, and secondary infections. our results suggested that such complications significantly worsened the clinical outcomes of h7n9-infected patients. effective management of complications in the course of the disease would be critical to achieve favorable clinical outcomes and prevention of death. our results showed that h7n9-infected patients in critical conditions had higher c-reactive protein level and more severe renal insufficiency and myocardium injury. c-reactive protein level might be used as an early marker for clinical prognosis in h7n9 patients based on results from our logistic regression analyses, which was in agreement with previous studies showing that c-reactive protein level might serve as a marker for severity of illness in influenza infections [18, 19] . since multivariate analysis was not performed in our small sample study, further research is needed to verify this. interestingly, levels of t cell subsets including total cd3 + , cd4 + , cd8 + , and cd45 + t cells, were all lower in patients in critical conditions than those in recovered conditions. the protective role of cd8 + t cells in aiv infections has been discussed in previous studies [20, 21] . it has been shown that cd8 + t cell-mediated responses in patients were mainly directed against conserved viral proteins, enabling cd8 + t cells to provide protection against heterologous influenza strains [22] . cd4 + t cells have also been reported to contribute to immunity against influenza. previous studies showed that cd4 + t cells were induced after influenza virus infection and played a central role in acquired immunity through induction and maintenance of cd8 + memory t cells and assisting b cells in antibody production [23, 24] . cd45 + t cells were found to be not only essential for efficient t-and bcell antigen receptor signal transduction but also involved in cytokine signaling [25, 26] . cd45ko mice and humans who lacked cd45 expression were severely immunodeficient and had very few peripheral t lymphocytes with impaired t and b cell responses [27] [28] [29] . results of our analysis suggested that t cellmediated responses might play an important role in immune defense against h7n9 virus infection. further studies on t cellmediated responses during h7n9 infection might provide useful information to help develop effective therapies such as h7n9 vaccines. we used glucocorticoid therapy for most h7n9-infected patients in our observational study. some controversies exist in the use of glucocorticoid therapy for iav infection. han et al. reported that early use of parenteral glucocorticoid therapy for fever reduction and pneumonia prevention in patients with pandemic influenza a (h1n1) infection increased the risk of critical disease and death [30] . likewise, he et al. advised that glucocorticoids should not be used in critically ill patients with h1n1 influenza infection [31] . however, carter argued that while steroids should not be used as monotherapy in the treatment of avian influenza, they might provide therapeutic benefits as an adjunct therapy to antiviral agents if prescribed at appropriate dosages [32] . we found no clear association between glucocorticoid therapy and clinical outcomes in h7n9-infected patients in our study. future studies with larger patient populations are required to evaluate the benefits and risks asscocaited with glucocorticoid therapy in treatment of h7n9 infections. in conclusion, successful treatment of h7n9 influenza depends on early diagnosis of h7n9 infection at the onset of clinical symptoms even if they are mild. knowledge of risk factors, clinical features, and potential complications discussed in our paper would help clinicians who triage patients with suspected or confirmed h7n9 infection determine appropriate treatment strategies and fight potential epidemic/pandemic. increased research and surveillance are required to further understand the pathogenesis of h7n9 infection and epidemiological factors that contribute to severe conditions. further studies are also needed to identify h7n9 natural reservoirs and delineate mechanisms of transmission. the preliminary results reported in the present article were from a small sample study. further studies with larger sample sizes are needed to verify and extend our findings. avian influenza: recent developments the 2009 h1n1 influenza outbreak in its historical context influenza a: from highly pathogenic h5n1 to pandemic 2009 h1n1. epidemiology and clinical features highly pathogenic avian influenza expert review: the clinical symptoms between the father and the son with h7n9 virus infection in shandong province were obviously different, difficult to support the probability of human-to-human transmission of h7n9 virus h7n9 avian flu kills seven and infects 23 in china h7n9 avian flu infects humans for the first time human infection with a novel avian-origin influenza a (h7n9) virus national health and family planning commission of the people's republic of china. diagnosis and treatment program of humankind h7n9 avian influenza infection global concerns regarding novel influenza a (h7n9) virus infections chinese herbal medicine for severe acute respiratory syndrome: a systematic review and meta-analysis a preliminary study on the medical expenditure of chinese medicine and integrative medicine treatment for influenza a (h1n1) in the fever clinics the world health organization influenza at human-animal interface, cumulative number of confirmed human cases of avian influenza a (h5n1) reported to who h7n9 incident, immune status, the elderly and a warning of an influenza pandemic a review of avian influenza in different bird species the beijing news. 40 percent of avian influenza had no contact with poultry preliminary report: epidemiology of the avian influenza a (h7n9) outbreak in china creactive protein serum levels as an early predictor of outcome in patients with pandemic h1n1 influenza a virus infection correlation of c-reactive protein to severity of symptoms in acute influenza a infection cd8 t cells utilize trail to control influenza virus infection protection against diverse highly pathogenic h5 avian influenza viruses in chickens immunized with a recombinant fowlpox vaccine containing an h5 avian influenza hemagglutinin gene insert identification of novel avian influenza virus derived cd8+ t-cell epitopes cd4 t cell responses to influenza infection cd4 + t-cell memory: generation and multi-faceted roles for cd4 + t cells in protective immunity to influenza cd45: a critical regulator of signaling thresholds in immune cells cd45: new jobs for an old acquaintance cd45-null transgenic mice reveal a positive regulatory role for cd45 in early thymocyte development, in the selection of cd4+cd8+ thymocytes, and b cell maturation mutations in the tyrosine phosphatase cd45 gene in a child with severe combined immunodeficiency disease a deletion in the gene encoding the cd45 antigen in a patient with scid early use of glucocorticoids was a risk factor for critical disease and death from ph1n1 infection clinical analysis of critically ill patients with h1n1 influenza a rationale for using steroids in the treatment of severe cases of h5n1 avian influenza key: cord-000689-8lvzab4i authors: qi, yilin; operario, darwin j.; georas, steve n.; mosmann, tim r. title: the acute environment, rather than t cell subset pre-commitment, regulates expression of the human t cell cytokine amphiregulin date: 2012-06-14 journal: plos one doi: 10.1371/journal.pone.0039072 sha: doc_id: 689 cord_uid: 8lvzab4i cytokine expression patterns of t cells can be regulated by pre-commitment to stable effector phenotypes, further modification of moderately stable phenotypes, and quantitative changes in cytokine production in response to acute signals. we showed previously that the epidermal growth factor family member amphiregulin is expressed by t cell receptor-activated mouse cd4 t cells, particularly th2 cells, and helps eliminate helminth infection. here we report a detailed analysis of the regulation of amphiregulin expression by human t cell subsets. signaling through the t cell receptor induced amphiregulin expression by most or all t cell subsets in human peripheral blood, including naive and memory cd4 and cd8 t cells, th1 and th2 in vitro t cell lines, and subsets of memory cd4 t cells expressing several different chemokine receptors and cytokines. in these different t cell types, amphiregulin synthesis was inhibited by an antagonist of protein kinase a, a downstream component of the camp signaling pathway, and enhanced by ligands that increased camp or directly activated protein kinase a. prostaglandin e2 and adenosine, natural ligands that stimulate adenylyl cyclase activity, also enhanced amphiregulin synthesis while reducing synthesis of most other cytokines. thus, in contrast to mouse t cells, amphiregulin synthesis by human t cells is regulated more by acute signals than pre-commitment of t cells to a particular cytokine pattern. this may be appropriate for a cytokine more involved in repair than attack functions during most inflammatory responses. different functional subsets of cd4 t cells are crucially involved in immune defense against diverse pathogens. at least four effector subsets are derived by differentiation from naïve cd4 t cells, and each expresses a characteristic combination of transcription factors, soluble mediators and surface molecules [1, 2] . th1 cells predominantly produce interferon-c (ifnc) and protect against intracellular pathogens; th2 cells produce interleukin (il)-4, il-5 and il-13 and help to eliminate extracellular parasites; th17 cells produce il-17a and il-17f and are crucial in fighting against extracellular bacteria and fungi; whereas induced t regulatory cells (itreg) produce il-10 and transforming growth factor b (tgfb), and suppress t and b cell effector responses. although the initial th1 and th2 subsets are relatively stable, recent studies have demonstrated some flexibility and plasticity, particularly in other cd4 t cell subsets. cytokines and other soluble mediators in the lymph node and inflamed tissue can further affect the cytokine expression profile of effector cd4 t cells [3] , in some cases by changing the differentiation status of the effector cd4 t cells. primed precursor cd4 t (thpp) cells, that produce mainly il-2 and chemokines when stimulated, remain uncommitted with respect to their effector cytokine pattern and can later differentiate into either th1 or th2 cells [4] [5] [6] . suppressive treg cells, expressing the forkhead transcription factor foxp3, can lose the expression of foxp3 and acquire the ability to produce pro-inflammatory cytokines during autoimmunity [7] . th17 cells can acquire the ability to produce ifnc in th1 polarizing conditions [8, 9] . adoptively transferred il-4-producing th2 effector cells can produce ifnc during viral challenge infections [10] . th9 cells develop from th2 populations in the presence of tgfb [11, 12] and t follicular helper (tfh) cells may represent a further differentiation step from several of the other subsets [13] . acute modifications of cytokine patterns can also occur. il-12+ il-18 enhance the secretion of ifnc by th1 cells [14, 15] , and il-2 enhances cytokine production [16, 17] . in contrast, il-10, tgfb, prostaglandin e2 (pge2) and adenosine inhibit inflammatory cytokine production [18] [19] [20] . mouse th2 cells, but not naive or th1 cells, express amphiregulin (ar), a member of epidermal growth factor (egf) family. like other egf members, ar is expressed as a transmembrane precursor protein and released by proteolytic cleavage [21, 22] . soluble ar binds to egf receptors and promotes proliferation and differentiation of epithelial cells, fibroblasts and keratinocytes [23] [24] [25] . ar-deficient mice [26] showed slower kinetics of clearance [27] of the helminth parasite, trichuris muris, that is cleared most effectively by th2-biased responses. ar production is also induced in human mast cells by ige cross-linking [28, 29] , in human eosinophils by il-5 [30] , and in human basophils by il-3 [31] . thus ar is induced by activation of at least four cell types contributing to type 2 inflammation, suggesting a role for ar during an allergic immune response. in addition, production of ar by immune cells is potentially important for tissue remodeling and repair [21, 26] during and after damaging immune responses. very little is known about the regulation of ar gene expression in human t cells. we examined the regulation of ar synthesis by human t cells, and found that in contrast to mice, many subsets of human t cells, including cd4 and cd8, naive and memory, th1 and th2, all express ar in response to tcr stimulation. factors that elevate camp levels synergized with tcr stimulation to enhance ar expression, while inhibiting expression of most inflammatory cytokines. thus in human t cells, ar production is regulated strongly by the environmental context during stimulation, but not restricted to particular precommitted effector subsets of t cells. biotinylated goat anti-human ar and biotinylated goat normal igg (isotype control), and apc conjugated anti-human ccr4 (205410) were obtained from r&d systems (minneapolis, mn). leaf tm purified anti-human cd3e (okt3), apc-cy7 conjugated anti-human cd4 (rpa-t4), pacific blue or apc-cy7 conjugated anti-human cd69 (fn50), pe-cy5 conjugated antihuman cd154 (24-31), percp-cy5.5 conjugated anti-human cd27 (o323), alexa fluor 700 conjugated anti-human cd62l (dreg-56), pacific blue conjugated anti-human cxcr3 (tg1/ cxcr3), pe-cy7 or pe-cy5 conjugated anti-human cd123 (6h6), alexa fluor 700 conjugated anti-human il-2 (mq1-17h12), fitc conjugated anti-human il-4 (mp4-25d2), and percp-cy5.5 conjugated anti-human il-17a (bl168) were purchased from biolegend (san diego, ca). functional grade purified anti-human cd28 (cd28.2), pe conjugated anti-human cd45ra (hi100), fitc conjugated anti-human cd45ro (uchl1), pe-cy5 conjugated anti-human cd19 (hib19), pe-cy7 conjugated anti-human ifnc (4s.b3), and apc-conjugated streptavidin were obtained from ebioscience (san diego, ca). alexa fluor 488 conjugated anti-human cxcr5 (rf8b2) and pe-cy7 conjugated anti-human ccr7 (3d12) were purchased from bd bioscience (san jose, ca). qdoth 605 conjugated antihuman cd3 (ucht1), pe-texas red and qdoth 705 conjugated anti-human cd8a (3b5), pe-texas red conjugated anti-human cd4 (s3.5), tri-color and qdoth 800 conjugated antihuman cd14 (tük4), qdoth 655 conjugated anti-human cd45ra (mem-56), pacific blue conjugated anti-human tnfa (mp9-20a4), and live?dead fixable yellow dead cell stain kit were obtained from invitrogen (carlsbad, ca). 7-aminoactinomycin d (7-aad) and tapi-1 was obtained from calbiochem (gibbstown, nj). camp agonist (8-cpt-camp) and camp antagonist (rp-8-br-camp) were purchased from biolog (bremen, germany). phorbol 12-myristate 13-acetate (pma), ionomycin, monensin, pge2, forskolin and 3-isobutyl-1methylxanthine (ibmx), adenosine were obtained from sigma (st.louis, mo). heparinized blood was obtained from healthy donors under a protocol approved by the university of rochester medical center research subjects review board. written, informed consent was obtained from all subjects. pbmc were isolated by ficoll-hypaque (cellgro, herndon, va) density gradient centrifugation. cells were suspended in complete rpmi-8 (rpmi-1640 medium containing 100u penicillin/streptomycin (invitrogen) supplemented with 8% heat-inactivated fetal calf serum (fcs, hyclone, logan, ut)). in the experiment treating cells with adenosine, serum-free medium x-vivo tm 20 (lonza, walkersville, md) was used. to purify human naïve and memory cd4 t cells from pbmc, fresh pbmc were stained with antibodies specific for cell surface markers and cd4+cd8-cd14-cd123-cd45ra+cd45ro-(naïve cd4 t cells) and cd4+cd8-cd14-cd123-cd45ra-cd45ro+ (memory cd4 t cells) were sorted on a facsaria (bd bioscience, san jose, ca). purified human naïve cd4 t cells were stimulated with irradiated (100gy) allogeneic epstein-barr virus (ebv) -transformed b cells (1:1 ratio) in complete rpmi-8 medium at 10 5 cells/ml in round-bottom 96-well plate. th1-biased cultures contained recombinant human il-2 (5 ng/ml, peprotech), recombinant human il-12 (20 ng/ml, peprotech) and anti-il-4 (5 mg/ml, r&d systems). th2-biased cultures contained recombinant human il-2 (5 ng/ml), recombinant human il-4 (20 ng/ml, r&d systems), anti-il-12 (5 mg/ml, ebioscience) and anti-ifnc (5 mg/ml, r&d systems). fresh medium containing 5 ng/ml il-2 was added if necessary to cultures showing strong proliferation. the cultures were restimulated and expanded every seven days. to enrich for cells with the th1 or th2 phenotypes, after 14 days priming, th1 and th2 cells were stimulated with platebound anti-cd3+ anti-cd28 for 8 hours. ifnc+ th1 cells and il-5+ th2 cells were stained and sorted by the macs cytokine secretion assay (miltenyi biotec, auburn, ca) according to the manufacturer's instructions. the enriched ifnc+ th1 cells and il-5+ th2 cells were expanded as previously for 14 days. for intracellular staining (ics) of ar, pbmc (10 6 per well) were stimulated with medium alone, anti-cd3 (5 mg/ml) + anti-cd28 (1 mg/ml), staphylococcal enterotoxin b (seb, 1 mg/ml), pma (10 ng/ml) + ionomycin (500 ng/ml), influenza h1n1 peptides (h1n1 [new caledonia/new york], 20 ng/ml/peptide), fel d1 (50 mg/ml, indoor, charlottesville, va), der p1 (50 mg/ml, indoor) or tetanus peptides (3 mg/ml/peptide) in round-bottom 96-well plate (costar, corning inc., corning, ny). th1 or th2 cultures were treated with medium alone or pma + ionomycin. after 10 hours stimulation (with 2 mm monensin present for the last 8 hours), the cells were first stained with live?dead fixable yellow dead cell stain kit, and then stained for cell surface markers cd4, cd8, cd14, cd123, and cd45ra. after cells were fixed and permeabilized using fix-perm (bd bioscience), the cells were stained with anti-ar, anti-ifnc, anti-il-2, anti-il-4, anti-il-17a, anti-tnfa, anti-cd3 and anti-cd69 (or anti-cd154) intracellularly. for cell surface staining of ar, pbmc were stimulated with medium alone or 1 mg/ml seb in the presence or absence of 50 mm tapi-1, an adam17 protease inhibitor [32] , for 6, 12, and 24 hours. after stimulation, the cells were stained with antibodies against ar, cd3, cd4, cd8, cd14, cd123, cd69 and 7-aad. data were acquired using an lsr ii flow cytometer (bd bioscience), and analyzed with flowjo software (tree star inc., ashland, or). total rna was extracted using trizol (invitrogen) according to the manufacturer's instructions. cdna was prepared by reverse transcription from total rna using multiscribe tm reverse transcriptase (applied biosystems, foster city, ca) with random hexamer primers (applied biosystems). quantitative real-time pcr (rt-pcr) was performed using the applied biosystems 7900ht sequence detection system. primers and probes specific for ar, heparin-binding egf-like growth factor (hb-egf), il-2, ifnc, il-3, il-4, il-5, il-10, il-13, cd3d, egf, neuregulin (nrg) 1-4, epiregulin (ereg), betacellulin (btc) and tgfa were all obtained from taqman gene expression assays (applied biosystems). cd3d gene expression was used as an endogenous control for normalizing mrna amounts. all samples were run in duplicate and data were analyzed using sds software (applied biosystems). purified cd4 t cells were treated with medium alone or cd3/ cd28 beads (cells:beads 2:1) in the presence or absence of 50 mm tapi-1. after 24 hours, the supernatants were collected and ar was measured using the human amphiregulin duoset elisa development kit (r&d systems). the detection limit of the assay was 7.8 pg/ml. because the antiserum for the elisa was produced by immunization with bacterial recombinant human ar, and the standard is also non-glycosylated ar, this elisa probably underestimates the concentration of normal human glycosylated ar. mouse th2 cells produce ar in response to tcr-mediated activation [27] , and the expression of ar by hemopoietic cells contributes to the clearance of a helminth parasite. however, our recent studies showed that basophils were the major human pbmc type that produced ar in response to anti-cd3/cd28 stimulation [31] , whereas production of ar by t cells was much lower. therefore we examined human t cells in more detail, to determine whether human t cells could produce ar, and if so, whether this was produced preferentially by human th2 cells. human pbmcs were stimulated with soluble anti-cd3+ anti-cd28, seb, or pma + ionomycin for 10 hours (protein secretion inhibitors were added during the last 8 hours). cd69 staining increased on almost all anti-cd3/cd28-and p+i-stimulated cells, and a subset of seb-stimulated cells ( figure 1a ). ar staining was increased, only in the cd69+ population, and this increase was most obvious in the p+i-stimulated cells. for all three stimulation conditions, the staining intensity for ar increased for the whole cd69+ population, i.e. separate positive and negative populations were not resolved, and so the percentage of cells in the ar+ gate may be an underestimate of the total number of cells expressing ar. the specificity of ar staining was demonstrated by using a control goat antiserum (right column). similar results were obtained with cd8 t cells ( figure 1a) . to independently confirm ar expression by human t cells, and to test whether t cells produced ar as a direct result of tcr stimulation, human cd4 and cd8 t cells were purified by sorting, and stimulated with beads coated with anti-cd3+ anti-cd28 antibodies. at different times, rna was extracted from the cells, and levels of ar and il-2 mrna measured by rt-pcr. ar mrna levels increased rapidly after stimulation, and returned to low levels after ten hours, whereas il-2 showed slower kinetics ( figure 1b) . the kinetics of ar production were similar in cd4 and cd8 t cells. thus human t cells directly express ar in response to polyclonal tcr stimulation. as demonstrated by other studies [33] , hb-egf mrna was also upregulated in activated human cd4 t cells (figure 2a) , although the levels were lower than ar and peaked at a later time ( figure 2b ). tgfa and ereg mrna were also detected in resting cd4 t cells, but not increased during tcr activation. other egf members were undetectable. in our previous mouse experiments [27] , ar and hb-egf were also the only egf family members induced by tcr stimulation (data not shown). expression of hb-egf protein was confirmed by cell surface and intracellular staining (data not shown). egf family members (including ar) are initially expressed as transmembrane proteins and released into the extracellular region after cleavage by metalloproteases, particularly adam17 [22] . to determine whether t cells also initially expressed surface ar and then released the soluble cleavage product, surface ar was stained during tcr activation in the presence or absence of the adam17/tace inhibitor tapi-1 [32] . tapi-1 increased ar expression on the surface of both cd4 and cd8 t cells measured by frequency ( figure 3a ) or fluorescence intensity (data not shown). conversely, tapi-1 decreased soluble ar in the supernatant ( figure 3b ). in the absence of tapi-1, ar expression on t cells gradually decreased and was barely detectable after 24 hours. as adam17 mrna was detected by rt-pcr in resting human t cells and upregulated on activation (data not shown), these results suggested that ar was first synthesized as a membrane protein on human t cells and then released by adam17 cleavage, as in other cell types [34, 35] . in mice, ar was expressed selectively in tcr-activated th2 cells [27] but not th1 ( figure s1 ) or naive cd4 t cells. this was a pre-committed, intrinsic property of the th2 cells, as th2 but not th1 cells expressed ar even when in vitro-derived mouse th1 and th2 cell lines were activated together in the same culture (data not shown). however, most human cd4 (and cd8) t cells expressed ar in response to pma plus ionomycin stimulation ( figure 1a ). we therefore examined in more detail which human t cell subsets were responsible for ar production. naive and memory cd4 and cd8 t cells produce ar. to examine the ability of naive and memory t cell subpopulations to express ar, we stimulated pbmc with an allogeneic ebv-transformed b cell line, which would be expected to activate a small fraction of both memory and naive cd4 and cd8 t cells. alloantigens stimulated a fraction of both cd4 and cd8 t cells to produce ar ( figure 4a ), relative to the unstimulated control. the specificity of staining was confirmed by isotype control antibodies. the cells producing ar (and other cytokines) were included in the cd69+ population. ar was induced by allogeneic stimulation in both cd45raand cd45ra+ subsets of cd4 and cd8 t cells at frequencies ranging from 0.028% to 0.35%. these levels were comparable to the frequencies of cd4+ cd45ra+ or cd45ra-t cells producing il-2, or cd4+ cd45ra-memory t cells producing ifnc. as expected, il-2 was produced by both memory (cd45ra-) and naive (cd45ra+) cd4 t cells, whereas ifnc (and il-4 at low levels) were produced mainly by memory cells ( figure 4b ). the expression of ar by both cd45ra-and cd45ra+ subsets of cd4 t cells was tested at the mrna level in sebstimulated cells sorted according to ar and cd45ra expression ( figure 4c ). confirming the specificity of the anti-ar antibody ar is produced by memory cd4 t cell subsets expressing different cytokine phenotypes. although naive cd4 t cells are relatively homogeneous, the memory population includes a wide range of differentiated effector subsets. as ar is expressed selectively by mouse th2 cells, we examined whether ar production by human cd4 memory t cells was preferentially associated with expression of a particular cytokine or surface marker pattern. th1-and th2-biased human cd4 t cell populations were induced by stimulation of sorted naive human cd4 t cells with an allogeneic b cell line in th1-or th2-biasing cytokine conditions. the populations were further enriched by using the cytokine secretion assay to sort ifnc-or il-5producing cells, respectively. the resulting populations were strongly polarized, but unlike mouse t cells, both th1 and th2 human cell lines expressed ar ( figure 5a ). these results were confirmed using ex vivo human cd4 t cell populations. human pbmc were stimulated with seb, and ar and other cytokines measured by intracellular staining. naive cells (cd45ra+) expressed high levels of il-2 and ar, but very low levels of either ifnc or il-4 (data not shown). memory cells produced all cytokines tested, at varying frequencies. to determine whether ar expression was associated positively or negatively with subset-specific cytokines, the frequencies of cells expressing ar plus each of the other cytokines were measured from the ics results. these values were then compared with the double-producing frequencies predicted for random association of each cytokine pair, by multiplying the individual frequencies for each cytokine. figure 5b shows that ar was expressed in association with tnfa, il-2, ifnc, il-4 and il-17 at slightly higher frequencies than predicted by random association. similarly, tnfa and il-2 showed positive associations with all other cytokines. in contrast, the subset-specific cytokines ifnc, il-4 and il-17 showed mostly negative associations between each other, as expected. these results were confirmed at the rna level by sorting seb-stimulated human pbmc according to surface ar expression. both ar+ and ar-memory cd4 t cell populations expressed similar levels of il-4 and ifnc as measured by rt-pcr ( figure 5c ). il-2 mrna levels were higher in ar+ t cells, in both cd45ra-and cd45ra+ cells. ar is produced in response to antigen stimulation. we next tested whether human cd4 t cells expressed ar during antigen/apc stimulation in response to influenza peptides, allergens or tetanus antigens to stimulate type 1, type 2 and thpp-biased recall responses, respectively [6] . pbmcs were stimulated with antigens for 10 hours, and ar and other cytokines measured by ics. although these three antigens induced in vivo recall responses with characteristically different levels of il-2, ifnc and il-4, all three antigens induced substantial production of ar in the activated (cd154+) cells ( figure 5d ). similar results were obtained with cells from multiple subjects, although the magnitudes of the antigen responses were variable for all cytokines. thus ar can be expressed by all the conventional defined subsets of t cells that we have tested, including cd4 and cd8, naïve and memory, thpp, th1 and th2. to confirm the protein results, influenza-specific cd69+ ifnc+ cells were sorted from two subjects (results from one subject are shown in figure 5e) , and rt-pcr demonstrated that ar mrna levels were strongly elevated in the ifnc+ influenza-specific cells ar is produced by t cell subsets expressing different chemokine receptors and surface markers. chemokine receptors expressed selectively by t cell subsets lead to different homing and chemotactic properties. expression patterns of chemokine receptors are partly but not entirely related to cytokine commitment patterns [36] [37] [38] [39] . additional surface markers, including cd27 and the homing receptor cd62l are also expressed heterogeneously on human cd4 t cells. we therefore examined ar expression within subsets of memory cd4 t cells defined by the expression of these proteins. ar was produced at approximately similar frequencies by cd4 t cells positive or negative for the chemokine receptors ccr4, ccr7, cxcr3 and cxcr5, as well as cd62l and cd27 ( figure 6 ). however, expression of the activation-induced protein cd69 was strongly correlated with ar expression, as seen in previous figures. taken together with the data described above, ar expression appears to be a general ability of most or all subtypes of human t cells after tcr activation. as our results had demonstrated that ar production was not limited to a pre-committed subset of t cells, we then tested whether ar production was regulated by acute signals in the immediate milieu during tcr stimulation. in many cell types ar is strongly regulated by the camp-pka-creb signaling pathway. ar expression was significantly up-regulated by camp-elevating agents in both resting and anti-cd3 stimulated human pbmc populations enriched for human t cells [40] . however, in that study the negatively-selected t cell population would also have contained basophils, and we have shown that basophils express ar rapidly in response to il-3 and camp agonist ( [31] and unpublished data). thus anti-cd3 stimulation of the cd4 t cell + basophil population could have induced il-3 production by t cells, indirectly resulting in ar production by basophils. we have now re-examined the effect of camp elevation on the expression of ar by different t cell subsets. tcr and camp signals synergize to induce ar expression. tcr activation alone (which transiently elevates camp [41] ) induced transient ar mrna expression (figure 7a) , and a strong camp agonist (pka activator 8-cpt-camp) also induced low levels of ar in the absence of other signals. however, tcr and pka signaling synergized to induce higher and more sustained levels of ar and hb-egf mrna ( figure 7a ), as well as high levels of ar protein (supernatant plus cell-associated, figure 7b ). this strong synergy contrasts with a previous study [40] , possibly due to the presence of basophils in the responding population in that study. as both naive and memory cd4 t cells produce ar (figure 4 ), we tested whether pka activation would enhance ar expression in both populations. figure 7c shows the response of purified figure 5 . several human cd4 t cell subsets can produce ar. (a) allogeneic th1 and th2 cell lines from three subjects were stimulated with pma + ionomycin for 6 hours. the percentage of cells expressing ifnc, il-4, and ar was analyzed by ics. (b) the expression of ar and other cytokines was measured in seb-stimulated pbmc from four subjects by ics, calculating the frequencies of single cytokine producers, and all possible combinations of double-producers, among the cd154+ cd4+ t cells. the figure shows the ratio between the observed frequencies of doubleproducing t cells for each cytokine pair, and the expected frequencies (calculated as the product of the individual frequencies for each cytokine). values represent the ratios for the double-producer combination defined by the row and column labels. ratios above or below 1 are indicated by solid or open symbols, respectively. (c) il-4, ifnc and il-2 mrna levels were measured by rt-pcr in the sorted populations described in figure 4c . (d) pbmc were treated with influenza h1n1 peptides or tetanus (five subjects each), or the allergens fel d1 (solid symbols) or der p1 (open symbols)(three subjects each). the numbers of memory cd4 t cells expressing ar and other cytokines were measured by ics. the backgrounds (no antigen) have been subtracted. each symbol represents one individual and the filled bar is the mean of all tested subjects. (e) cd69+ cd4+ t cells (control_cd69+) were sorted from pbmc incubated in medium alone. cd69+ifnc+ and cd69+ifnc-cd4 t cells were sorted from influenza peptidetreated pbmc using the cytokine secretion assay. the mrna levels of ifnc and ar were measured by rt-pcr. results in (a-c) are representative of at least three experiments, (d) represents two experiments using a total of 5 independent subjects, and (e) represents two experiments. doi:10.1371/journal.pone.0039072.g005 ar expression is modified by natural and synthetic modulators of camp signaling. in the experiments described above, camp signaling was altered by an agonist (8-cpt-camp) that directly targeted pka to mimic the increase of intracellular camp levels. to further confirm that the camp-pka-creb signaling pathway regulates ar expression, we tested natural and pharmacological agents that increase the intracellular levels of camp by acting at two additional steps: pge2 and adenosine are natural ligands for g-protein coupled receptors that activate adenylyl cyclase [20, [42] [43] [44] ; forskolin activates adenylyl cyclase directly [20] ; and ibmx is a broad inhibitor of camp-degrading phosphodiesterases [45] . consistently, all four camp elevating agents upregulated ar mrna and protein expression in anti-cd3-stimulated t cells. in each case, the elevated signal was blocked by the camp antagonist ( figure 7d ). the enhancement of ar by the pde inhibitor suggested that pde reduced the moderate levels of camp induced by tcr activation in cd4 t cells [41] . ar and other cytokines are regulated reciprocally by camp signals. in contrast to the enhancement of ar expression, the camp agonist inhibited expression of many other cytokines ( figure 7e ), and all four pka-activating agents described in figure 7 inhibited expression of il-2 and ifnc (data not shown). these results are consistent with previous studies with camp agonists and natural camp elevating agents, such as pge2 and adenosine [19, 20] . thus ar expression in t cells is enhanced under conditions that suppress the production of many other cytokines. in contrast to the preferential expression of ar by mouse th2 cells, we have now shown that synthesis of human ar is not restricted to a particular human t cell subset. ar can be produced by activated naive and memory cd4 and cd8 t cells, including th1 and th2 phenotypes. our results suggest that ar is not a specific product of certain pre-committed effector subsets of human cd4 t cells, but instead is regulated mainly by additional signals present during t cell activation, particularly signals influencing the camp signaling pathway. the lack of precommitment suggests that, in contrast to the memory of effector functions carried by t cells committed to th1, th2, th17 etc phenotypes, the amount of ar produced in a particular immune response is regulated by the local environment during that response, but is less influenced by previous immune priming. the discrepancy we have identified between mouse and human t cell regulation highlights the importance of performing cross-species comparisons of effector t cell phenotypes. ar production was not restricted to a defined t cell effector subset, but ar and il-2 levels were moderately correlated in both naïve and memory cd4 t cells. although this could indicate the existence of a previously-unrecognized subset, it is possible that the correlation could be the result of shared transcriptional or mrna stability regulatory factors, or to similar activation thresholds for il-2 and ar. expression of ar also showed moderate correlation with the expression of tnfa. high levels of ar mrna and protein were induced by synergy between tcr signals and signals that elevated camp or activated pka. this contrasts with a previous report suggesting that both resting and anti-cd3 stimulated t cells significantly up-regulated ar in response to a camp agonist [40] . however, the enriched t cell population used in that study was purified by negative selection and very likely included basophils, which we have shown are potent producers of ar in response to il-3 [31] . ar expression is also strongly enhanced by camp agonists in basophils (y. qi and t.r. mosmann, unpublished data) and so it is possible that basophils may have produced the ar in response to the camp agonist without tcr stimulation. in contrast to the induction of ar by camp elevating agents, these mediators suppress inflammatory responses by inhibiting cytokine expression and t cell proliferation. synthesis of several pro-inflammatory or type 1 cytokines is inhibited by camp ( figure 7e and [19, 20, 46, 47] , whereas camp can either inhibit or enhance production of type 2 cytokines such as il-4, il-5 and il-13 ( figure 7e and [47] ) depending on the stimulation conditions [48, 49] . natural mediators that elevate the camp pathway and lead to pka activation include pge2 (mainly via the g protein-coupled receptors e2 and e4 on t cells) and adenosine (mainly via the a 2a receptor on t cells). both mediators are produced at sites of immune inflammation, adenosine by degradation of atp from dying cells, and pge2 by activated macrophages. pka activation signals also synergized with tcr signals to induce hb-egf mrna and protein expression in human cd4 t cells (data not shown). thus during the progression of an inflammatory response, there may be a switch from pro-inflammatory cytokine production to ar (and hb-egf) production. our findings allow us to construct a model of the role of t cell derived ar in adaptive immunity. during an immune response, initial immune attack mechanisms that destroy the pathogen are superseded at later times by suppression that reduces immunopathology, and tissue repair that restores normal structure and function. t lymphocytes are major cellular contributors to all three phases, and are thought to play a role in repair by producing hb-egf and bfgf [33] . ar and hb-egf, as members of the egf family, promote the proliferation of fibroblasts, epithelial cells, and smooth muscle cells, which are major cell types repaired or remodeled at local tissue sites during an inflammatory response. tissues with chronic inflammation show extensive cell proliferation, tissue thickening and reduced elasticity. regulation of the balance between attack and repair cytokines produced by t cells is thus crucial to the successful outcome of the response. . tcr and camp synergize to induce ar production in human cd4 t cells. purified cd4 t cells were incubated with or without tcr stimulation (anti-cd3/cd28 beads) and the camp agonist. (a) ar and hb-egf mrna expression was measured by rt-pcr. (b) the concentrations of ar in the supernatant and cell lysates were measured by elisa. (c) enriched cd45ra+cd45ro-(naïve) and cd45ra-cd45ro+ (memory) cd4 t cells were treated with medium alone, or anti-cd3/cd28 beads in the presence or absence of camp agonist (1 , 1000 mm) . the concentration of ar in the supernatant at 24 hours was measured by elisa. (d) purified cd4 t cells were treated with medium alone, or anti-cd3/cd28 beads in the presence or absence of the camp-modifying agents shown. rna was extracted at 4 hours, and ar mrna was measured by rt-pcr. the concentration of ar in the 24-hour supernatant was measured by elisa. (e) pbmc were treated with anti-cd3+ anti-cd28 antibodies in the presence or absence of camp agonist or antagonist for 8 hours. cd4 t cells were purified by cell sorting and rna was extracted. the mrna levels of ar and other cytokines were measured by rt-pcr. all results are representative of at least three experiments. doi:10.1371/journal.pone.0039072.g007 in this model, ar derived from human t cells would be expressed mainly in response to tissue injury, consistent with the importance of the local environmental signals for ar regulation. this contrasts with the requirement for specific effector mechanisms to combat different pathogens, in which pre-commitment to cytokine effector phenotypes (thus linking antigen and effector specificities) may be more effective for regulating clearance functions. collectively, the coordinate inhibition of pro-inflammatory cytokines and induction of tissue-remodeling cytokines of the egf family may represent a switch from pathogen clearance to tissue repair mechanisms by effector human t cells. figure s1 mouse th2 but not th1 cells express ar in response to tcr activation. in vitro induced allogeneic th1 and th2 cell lines [50] from b6pl or ar 2/2 mice were stimulated with plate-coated anti-cd3 (2 mg/ml) + anti-cd28 (1 mg/ml) antibodies for 6 hours. expression of ar, ifnc and il-4 in cd4 t cells was analyzed by ics. biotinylated goat antimouse ar antibodies were obtained from r&d systems. leaf tm purified anti-mouse cd3e (145-2c11) and leaf tm purified antimouse cd28 (37.51) were purchased from biolegend. apc-cy7 conjugated anti-mouse cd3 (17a2), alexa fluor 700 conjugated anti-mouse cd4 (gk1.5), pacific blue conjugated anti-mouse cd44 (im7), percp-cy5.5 conjugated anti-mouse cd69 (h1.2f3), apc conjugated anti-mouse il-2 (jes6-5h4), pe-cy7 conjugated anti-mouse il-4 (bvd6-24g2), pe conjugated antimouse il-5 (trfk5), and fitc-conjugated streptavidin were obtained from ebioscience. pe-alexa fluor 610 conjugated antimouse ifnc (xmg1.2) was obtained from invitrogen. similar results were obtained in at least three experiments. (tif) two types of murine helper t cell clone. i. definition according to profiles of lymphokine activities and secreted proteins peripheral cd4+ t-cell differentiation regulated by networks of cytokines and transcription factors mechanisms underlying lineage commitment and plasticity of helper cd4+ t cells single il-2-secreting precursor cd4 t cell can develop into either th1 or th2 cytokine secretion phenotype in vivo priming of cd4 t cells that produce interleukin (il)-2 but not il-4 or interferon (ifn)-gamma, and can subsequently differentiate into il-4-or ifn-gamma-secreting cells protein vaccines induce uncommitted il-2-secreting human and mouse cd4 t cells, whereas infections induce more ifn-gamma-secreting cells instability of the transcription factor foxp3 leads to the generation of pathogenic memory t cells in vivo late developmental plasticity in the t helper 17 lineage highly purified th17 cells from bdc2.5nod mice convert into th1-like cells in nod/scid recipient mice longlived virus-reactive memory t cells generated from purified cytokine-secreting t helper type 1 and type 2 effectors transforming growth factor-beta 'reprograms' the differentiation of t helper 2 cells and promotes an interleukin 9-producing subset il-4 inhibits tgf-beta-induced foxp3+ t cells and, together with tgf-beta, generates il-9+ il-10+ foxp3(-) effector t cells functional and epigenetic studies reveal multistep differentiation and plasticity of in vitrogenerated and in vivo-derived follicular t helper cells il-1 family members and stat activators induce cytokine production by th2, th17, and th1 cells il-18-stimulated gadd45 beta required in cytokine-induced, but not tcr-induced, ifngamma production two types of mouse t helper cell. iv. th2 clones secrete a factor that inhibits cytokine production by th1 clones il-2 absorption affects ifn-gamma and il-5, but not il-4 producing memory t cells in double color cytokine elispot assays contextual regulation of inflammation: a duet by transforming growth factor-beta and interleukin-10 interleukin-4 gene expression in activated human t lymphocytes is regulated by the cyclic adenosine monophosphate-dependent signaling pathway prostaglandin e2 and other cyclic amp elevating agents inhibit interleukin 2 gene transcription by counteracting calcineurin-dependent pathways membrane-anchored growth factors distinct roles for adam10 and adam17 in ectodomain shedding of six egfr ligands structure and function of human amphiregulin: a member of the epidermal growth factor family the epidermal growth factor receptor couples transforming growth factor-alpha, heparin-binding epidermal growth factor-like factor, and amphiregulin to neu, erbb-3, and erbb-4 a functional screen for genes inducing epidermal growth factor autonomy of human mammary epithelial cells confirms the role of amphiregulin targeted inactivation of the egf and amphiregulin genes reveals distinct roles for egf receptor ligands in mouse mammary gland development amphiregulin, a th2 cytokine enhancing resistance to nematodes fcepsilonrimediated amphiregulin production by human mast cells increases mucin gene expression in epithelial cells amphiregulin expression in human mast cells and its effect on the primary human lung fibroblasts amphiregulin production by human eosinophils human basophils express amphiregulin in response to t cell-derived il-3 tumor necrosis factor-alpha convertase (adam17) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (sars-cov) receptor, angiotensin-converting enzyme-2 (ace2) t lymphocytes synthesize and export heparin-binding epidermal growth factor-like growth factor and basic fibroblast growth factor, mitogens for vascular cells and fibroblasts: differential production and release by cd4+ and cd8+ t cells targeting tace-dependent egfr ligand shedding in breast cancer mammary ductal morphogenesis requires paracrine activation of stromal egfr via adam17-dependent shedding of epithelial amphiregulin differential expression of chemokine receptors and chemotactic responsiveness of type 1 t helper cells (th1s) and th2s expression of the chemokine receptors ccr4, ccr5, and cxcr3 by human tissue-infiltrating lymphocytes rules of chemokine receptor association with t cell polarization in vivo flexible programs of chemokine receptor expression on human polarized t helper 1 and 2 lymphocytes cytokine networks are pre-activated in t cells from hiv-infected patients on haart and are under the control of camp antibody binding to cd5 (tp67) and tp44 t cell surface molecules: effects on cyclic nucleotides, cytoplasmic free calcium, and camp-mediated suppression delineation of the mechanism of inhibition of human t cell activation by pge2 e prostanoid 2 (ep2)/ep4-mediated suppression of antigen-specific human t-cell responses by prostaglandin e2 a2a adenosine receptor (ar) activation inhibits pro-inflammatory cytokine production by human cd4+ helper t cells and regulates helicobacter-induced gastritis and bacterial persistence tcr-and cd28-mediated recruitment of phosphodiesterase 4 to lipid rafts potentiates tcr signaling lymphokine regulation of activated (g1) lymphocytes. i. prostaglandin e2-induced inhibition of interleukin 2 production prostaglandin e2 inhibits production of th1 lymphokines but not of th2 lymphokines prostaglandin e2 differentially modulates il-5 gene expression in activated human t lymphocytes depending on the costimulatory signal differential modulation of t helper type 1 (th1) and t helper type 2 (th2) cytokine secretion by prostaglandin e2 critically depends on interleukin-2 synthesis of several chemokines but few cytokines by primed uncommitted precursor cd4 t cells suggests that these cells recruit other immune cells without exerting direct effector functions we thank jason weaver for providing influenza h1n1 and tetanus peptides, john looney for helpful advice and assistance with subject recruitment, and jennifer scantlin and deanna maffett for obtaining human blood samples. key: cord-000719-o7ttiu97 authors: jonsson, colleen b.; camp, jeremy v.; wu, albert; zheng, huaiyu; kraenzle, jennifer l.; biller, ashley e.; vanover, carol d.; chu, yong-kyu; ng, chin k.; proctor, mary; sherwood, leslie; steffen, marlene c.; mollura, daniel j. title: molecular imaging reveals a progressive pulmonary inflammation in lower airways in ferrets infected with 2009 h1n1 pandemic influenza virus date: 2012-07-20 journal: plos one doi: 10.1371/journal.pone.0040094 sha: doc_id: 719 cord_uid: o7ttiu97 molecular imaging has gained attention as a possible approach for the study of the progression of inflammation and disease dynamics. herein we used [(18)f]-2-deoxy-2-fluoro-d-glucose ([(18)f]-fdg) as a radiotracer for pet imaging coupled with ct (fdg-pet/ct) to gain insight into the spatiotemporal progression of the inflammatory response of ferrets infected with a clinical isolate of a pandemic influenza virus, h1n1 (h1n1pdm). the thoracic regions of mockand h1n1pdm-infected ferrets were imaged prior to infection and at 1, 2, 3 and 6 days post-infection (dpi). on 1 dpi, fdg-pet/ct imaging revealed areas of consolidation in the right caudal lobe which corresponded with elevated [(18)f]-fdg uptake (maximum standardized uptake values (suvmax), 4.7–7.0). by days 2 and 3, consolidation (ct) and inflammation ([(18)f]-fdg) appeared in the left caudal lobe. by 6 dpi, ct images showed extensive areas of patchy ground-glass opacities (ggo) and consolidations with the largest lesions having high suvmax (6.0–7.6). viral shedding and replication were detected in most nasal, throat and rectal swabs and nasal turbinates and lungs on 1, 2 and 3 dpi, but not on day 7, respectively. in conclusion, molecular imaging of infected ferrets revealed a progressive consolidation on ct with corresponding [(18)f]-fdg uptake. strong positive correlations were measured between suvmax and bronchiolitis-related pathologic scoring (spearman’s ρ = 0.75). importantly, the extensive areas of patchy ggo and consolidation seen on ct in the ferret model at 6 dpi are similar to that reported for human h1n1pdm infections. in summary, these first molecular imaging studies of lower respiratory infection with h1n1pdm show that fdg-pet can give insight into the spatiotemporal progression of the inflammation in real-time. in march of 2009, an outbreak of a novel variant of h1n1 influenza a virus was reported in cases of influenza illness in mexico [1] . by june 11, the world health organization raised the pandemic alert level to its highest level, declaring the first influenza pandemic in over 40 years [1] . unlike seasonal influenza viruses, this novel h1n1 pandemic strain (h1n1pdm) tended to affect younger healthier populations and had an increased risk of morbidity and mortality [2] [3] [4] with 12-30% of the population developing clinical influenza, 4% of those requiring hospital admission, and 1 in 5 requiring critical care [5] . in general, however, infection of the h1n1pdm was relatively mild in most persons, although a fatal viral pneumonia with acute respiratory distress syndrome occurred in approximately 18,000 cases. in contrast to seasonal influenza in human cases, h1n1pdm infections showed a tropism for the lung similar to h5n1 [6] . the ability of h1n1pdm viruses to infect the lower respiratory track has been attributed to a broader specificity in the binding of the viral hemagglutinin (ha) to a2-3in addition to a2-6-linked sialic acid (sa) receptors [7, 8] . it is reasonable that the lung tropism of the h1n1pdm contributed to the severity of disease in those individuals with preexisting complications such as asthma and chronic obstructive pulmonary disease (copd) [6, [9] [10] [11] [12] . data from limited human autopsies and animal studies of various pandemic strains also suggest contribution of the host innate immune response and the virus in the progression of disease [13] [14] [15] [16] . molecular imaging can potentially play a strong role in basic infectious disease research and clinical response by providing a noninvasive, spatiotemporal measurement of viral infection and host inflammation [17, 18] . to explore the potential utility of molecular imaging in influenza infection, we chose the ferret (mustela putorius furo) model. ferrets have been used as an animal model of influenza infection and pathogenesis since 1934, when they were reported to develop an acute respiratory tract illness when exposed to influenza viruses from humans and swine [19] . in contrast to mice, the ferret can be infected by human isolates without adaptation and display signs and symptoms of infection such as sneezing and nasal secretions that are similar to what is seen in humans [20] [21] [22] [23] . the ferret is an attractive model for imaging influenza pulmonary infections given the ferret's long trachea, large lung capacity, and bronchiolar branching. these anatomical features can potentially bridge imaging with histopathologic evaluation. finally, the ferret more closely mimics humans in distribution of sialic acid (sa) receptors in the respiratory tract with higher a-2-6 sa in the upper respiratory tract and sa with a-2-3in the lower [24] . historically, plain film (x-ray) radiography and computed tomography (ct) have been useful for clinical assessments of influenza disease severity in clinical cases [25, 26] . these imaging modalities are limited by characterizing only anatomic changes in the lung parenchyma, such as ground-glass opacity (ggo) and consolidations, which represent different degrees of interstitial and alveolar filling by cells, edema, and inflammatory exudate [27] . in contrast, positron emission tomography (pet) imaging with the radiotracer, [ 18 f]-2-fluoro-2-deoxy-d-glucose ([ 18 f]-fdg), can provide data on metabolic activity of cells by measuring sites of increased glycolysis from leukocyte chemotaxis and accumulation, and provide increased sensitivity in detection of cells during inflammation. [ 18 f]-fdg, an analog of glucose that is moved into cells via facilitated transport, is commonly used in pet imaging as a radiotracer in clinical and basic science research. recent studies have demonstrated the utility of the [ 18 f]-fdg in assessment of infectious disease burden in animal models of schistosomiasis and tuberculosis [28] [29] [30] . pet/ct has been used in the assessment of one hospitalized h1n1pdm patient and revealed an intense inflammatory response [31] . the uptake of [ 18 f]-fdg in humans and animals suggests the predominant presence of activated neutrophils [32] [33] [34] [35] . in studies of mice infected with influenza a virus, neutrophils play a critical role in protection and recovery from infection, and participate in the process of adaptive immunity to the virus [36] . coupled with molecular virology and pathology, molecular imaging with important probes of disease has enormous potential to reveal early critical factors that contribute to the clinical progression of illness as well as accelerate screening, the efficacy and mechanistic studies of vaccines and antiviral therapies [17, 18] . to test the hypothesis that fdg-pet/ct imaging could reveal the spatiotemporal nature of h1n1pdm inflammation and disease progression, we chose the ferret model of h1n1pdm influenza infection. further, for these studies we chose a low passage clinical isolate, a/kentucky/180/2010 or ky/180, herein, which has a change in the ha1 gene, d222g, which correlates with increased severity of disease in patient cases from several countries [37] [38] [39] [40] . in mice, infection with h1n1pdm engineered to include this change show increased viral titers and pathology, however, in ferrets there do not seem to be any major differences in clinical signs, transmissibility or pathogenicity [41, 42] . our results show for the first time, the spatiotemporal progression of inflammation with ct and pet using [ 18 f]-fdg in ferrets infected with h1n1pdm in conjunction with histopathology and viral titers over a seven day period. importantly, the extensive areas of patchy ggo and consolidation seen in the ferret model at 6 days post-infection (dpi) are similar to that reported for human h1n1pdm infections [31] . in vivo imaging with these modalities for anatomic (ct) and molecular (pet) data suggests increased pulmonary inflammation as the amount of circulating virus becomes undetectable. these results suggest that molecular imaging will be a great asset in gaining insight into the temporal and spatial progression of the inflammatory process caused by influenza virus infection. due to the size of the siemens trimodal gantry for pet/ct imaging we chose four month old females rather than male ferrets. the pandemic h1n1 isolate ky/180 employed in these studies was isolated from nasal swab sample provided by the severe influenza pneumonia surveillance project, a clinical study of hospitalized patients with influenza pneumonia in kentucky. the patient had a severe course of influenza disease and died after 19 days. sequencing of the ha1 gene from this isolate revealed the d222g mutation, which has been associated with severe disease in human cases [37] [38] [39] [40] . the second passage of the ky/180 seed was employed in the characterization of infection in the female ferrets. the 50% infectious dose in female ferrets was determined to be 10 0.07 tcid 50 (data not shown). in group 1, six ferrets were mock infected with pbs. in group 2, six ferrets were infected intranasally (i.n.) with ky/180 with a 0.5 ml dose of 0.5610 5.7 tcid 50 per naris. ferrets were monitored for temperature for 10 dpi, and for body weight and clinical symptoms for 28 dpi. two animals in each group were euthanized on days 2, 14 and 18 to determine virus and hi titers in blood, lung and several additional organs at 2 dpi. in figure 1a , the body weight changes are shown for mock-and ky/180-infected ferrets. body weight showed a drop on day 2 where the weight remained for the remainder of the study. figure 1b shows the average temperatures of the mock-and ky/ 180-infected ferrets for the first 10 dpi. temperature peaked on days 1 and 5 for ky/180 infected animals with a mean temperature of 103.4uf (sd = 1.71uf) and 103.2uf (sd = 0.52uf), respectively. the average hemagglutinin inhibition (hi) serum antibody titer in the blood on day 14 was 540 (reciprocal dilution) and the average total serum influenza-specific igg by elisa was 7610 (reciprocal dilution) (fig. 1c) . in studies to determine the infectious dose, additional tissues were taken from animals infected at with ky/180 with a 0.5 ml dose of 0.5610 4.7 tcid 50 per naris. on day 2, viral titers were the highest in the nasal turbinates (10 6.25 tcid 50 /g) followed by the caudal lung (10 6.0 tcid 50 /g) and trachea (10 3.75 tcid 50 /g). the lowest levels of virus were observed in the cranial lobe of the lung (10 3.2 tcid 50 /g). viral titers were measured by tcid 50 in nasal turbinates, trachea, right cranial lobe of the lung, right caudal lobe, brain, liver, spleen, kidney, duodenum, jejunoilieum, colon and rectum. ky/180 was detected in jejunum in one animal (10 2.0 tcid 50 /g), but was not detected in any other tissues (data not shown). female fitch ferrets were divided into five groups, with two per group of animals that were mock-infected with pbs (group 1) or intranasally infected with ky/180 (groups 2-5), in a 0.5 ml dose of 0.5610 6.0 tcid 50 per naris, on day 0 (table 1) . group 5 was the only group that was imaged each day; while groups 2-4 were imaged and sacrificed on days 1, 2 and 3 post-infection. this study design permitted evaluation of the progression of imaging with infection and pathology in two animals each day as well as continuous imaging of the lungs in one cohort over the seven-day time-period. fdg-pet and ct images of the h1n1pdm-infected and mock-infected ferrets were successfully obtained and fused for two ferrets on days 1, 2, 3 and 6 ( fig. 2 and 3 ). volumes of interest (voi) and corresponding maximum standardized uptake values (suvmax) were generated for any metabolically active lesions in the lung as well as background activity in the lungs, liver, heart, thymus, and thoracic lymph nodes. baseline imaging prior to infection showed no focal areas of lung consolidation on ct and background standard uptake values (suvmax) of the [ 18 f]-fdg levels ranged from 0.7-1.0 for pet ( fig. 2a and fig. 3a ). each figure shows the one two-dimensional coronal plane that were standardized across days to provide a similar orientation and do not necessarily represent the suvmax as that plane may be out of view. by 1 dpi, an area of consolidation was identified on ct in the right caudal lobe with corresponding radiotracer uptake on pet (fig. 2b , suvmax of 4.7). consolidative areas in the right caudal lobe increased by day 2, with a persistently elevated suvmax of 3.1(data not shown). by day 3, the consolidation increased in the right caudal lobe ( fig. 2c and fig. 3c , suvmax of 3.7 and 4.4, respectively) and also appeared in the left caudal lobe (suvmax of 3.2) of ferret 2214 (fig. 3c ). by 6 dpi, there were widespread areas of patchy consolidation on ct with multiple areas of increased radiotracer uptake in both ferrets in caudal and cranial lobes ( fig. 2d and fig. 3d , suvmax of 6.0 and 7.6 on the right, 4.2 and 4.6 on the left, respectively). these results suggest that inflammation progresses into the lower respiratory airways after infection into the upper part of the lower respiratory system. a ferret from the uninfected cohort was also imaged on day 6, with no focal appearance of consolidation on ct and no evidence of increased [ 18 f]-fdg uptake on pet (image not shown, background suv of 0.6). to measure viral shedding, each day each ferret was swabbed in the nasal, throat and fecal passages and the viral titer was measured by tcid 50 ( table 2 ). the highest levels of viral shedding were measured in the throat swabs. nasal swabs also showed viral shedding for most animals, while the presence of virus in rectal swabs was low although detectable in a few animals. replication of h1n1pdm in nasal turbinates and lungs were determined post-mortem from the right caudal lobe of the lung taken on euthanasia (table 3) . four sections were taken per lobe to provide greater insight into the spread of the virus in the tissue (fig. 4) . high levels of virus were detected in all nasal turbinate samples at 1, 2, and 3 dpi (95% c.i. = 5.43+/21.00 tcid 50 / ml). virus was also detected in a majority of lung sections from 1, 2, and 3 dpi. it was absent in the lung sections from one animal on 2 dpi, although it was present in the ferret's nasal turbinates, suggesting that the timing of infection in this animal was slower than the others. of note, this same animal had a focus of consolidation on ct and radiotracer uptake on pet. this observation also suggests that, while the virus may have been undetectable by tcid 50 , low levels of virus had entered the lower respiratory system. no animals on day 7 post-infection had detectable virus in the nasal turbinates or the sampled lung tissue. virus was not detected in nasal, throat and fecal passages or the sampled lung tissue from ferrets in any of the controls. upon necropsy, all but the right caudal lobe of the ferret lung was fixed with paraformaldehyde. following fixation, sections were taken for histopathology from the right and left cranial lobes, left caudal lobe and the middle accessory lobe. representative photographs from slides of the left caudal lobe are shown in figure 5 . the ferrets in the control group had intact bronchiolar walls with very minimal infiltration by neutrophils with the exception of the left caudal lobe from control animal 2206 sacrificed on day 1. possible causes of this pattern of change may be an underlying systemic vasculopathy which is typically confirmed by evaluation of other organs that were not collected (e.g., kidney, spleen, liver). in general pulmonary lesions associated with influenza infection were roughly comparable at days 1 and 2 and consisted of variable suppurative or necrosuppurative bronchiolitis and mixed cell alveolitis at minimal to moderate severity levels. by 1 dpi, there were some small foci of inflammation without much infiltration of the bronchi or bronchioles. there was an increased severity of inflammatory findings in lung lobes from infected ferrets on day 3. specifically, more extensive infiltration of neutrophils can be seen within the bronchiolar lumen, along with necrosupprative bronchiolitis and mixed cell alveolitis. at day 7, lesions observed in the lung lobes continued to exhibit an increased severity compared to the majority of lung lesions seen at 1 and 2 dpi. bronchiolar epithelial hyperplasia and cytokaryomegaly were noted in addition to bronchiolitis. to evaluate potential correlations between pet/ct with histopathology, the suvmax of lesions in the right and left lung of each ferret were compared with the cumulative histopathology scores assigned by the veterinary pathologist (fig. 6 ). on average, the suvmax was higher in the right lung than the left lung but the slopes and spearman's correlation coefficients (r) were similar between the two sides. the highest correlation was seen between the cumulative bronchiolitis score and suvmax (r of 0.71 and 0.75 on the right and left, respectively). the next highest was between the cumulative bronchitis score and suvmax (r of 0.69 on the right and 0.67 on the left). a weaker positive correlation was seen between the cumulative alveolitis score and suvmax (r of 0.47 on the right and 0.57 on the left). herein, we show for the first time the feasibility of utilizing [ 18 f]-fdg pet coupled with ct imaging of h1n1pdm in ferret to track the progression of pulmonary disease in real-time. we chose a low passage clinical isolate, ky/180, which has a change in the ha1 gene, d222g. the d222g change in h1n1pdm correlates with increased severity of disease in patient cases from several countries [37] [38] [39] [40] . the patient from which we obtained the ky/180 isolate also had a severe course of influenza illness over a period of 19 days that resulted in death. recently, studies in mice and ferrets infected with pandemic influenza viruses a/california/04/2009 and a/netherlands/602/2009 engineered with the d222g mutation have shown that the d222g mutation are lethal in mice, but not ferret [41, 42] . the lethality in mice, but not ferrets, has been attributed to the greater abundance of a2-3-sa in the mouse model [42, 43] . all of these viruses have an affinity for a2,6-sas associated with attachment to and replication in cells of the upper respiratory tract as shown by the high levels of viral replication in the nasal turbinates. thus, infection of ferrets with these h1n1pdm isolates engineered with d222g and our clinical isolate have not correlated with clinical findings in patients. these results in ferrets are not surprising given that 80% of the fatal cases of h1n1pdm had underlying medical conditions and bacterial infections [6] . discovery of the molecular components of the host response that may promote pathogenesis will be critical for defining new treatments. noninvasive imaging can provide real-time in vivo monitoring of the progression of infection, inflammation and disease that may give insight into the mechanisms that modulate disease progression. recently, veldhuis kroeze et al, presented data on the monitoring of pulmonary lesions of h1n1pdm influenza virusinfected ferrets with ct scanning which correlated with disease progression and severity [44] . as those studies demonstrate, ct is a powerful tool, but it will not give the molecular details that can be provided by pet or spect imaging of probes that target critical host responses such as neutrophil invasion. in our study we coupled ct scanning with the [ 18 f]-fdg radiotracer and show infection and inflammation of influenza infection in the lower respiratory system with foci of increased [ 18 f]-fdg uptake corresponding to areas of lung opacity on ct, with underlying inflammation on necropsy. in comparison to human ct imaging studies of influenza, the molecular images in the ferret show strong similarity. ct findings in patients with confirmed influenza infection show patchy ground-glass opacities in segmental multifocal distributions, mixed with areas of consolidation in the lung [12, 25, 45] . moreover, the few case reports of human influenza in which lungs were imaged by [ 18 f]-fdg pet demonstrate areas of high uptake in these ground-glass opacities and consolidation [31] . our study similarly demonstrates this pattern in the ferret model, also showing patchy opacities on ct with high uptake of radiotracer on pet, with necroscopy-based confirmation of inflammation in the left caudal lobe. specifically, we show the ferret lung demonstrated progressive consolidation on ct and fdg uptake on pet predominantly in the right caudal lobe, which progressed to the left caudal lobe by day 3 p.i. by day 6, the diffuse metabolically active lesions seen on pet/ct were similar to what has been reported in the human literature during the 2009 h1n1 pandemic [31] . histopathologic evaluation of the lungs confirmed the progressive nature of the pulmonary lesions and corroborated the radiologic data. suppurative and necrosuppurative bronchiolitis seen on days 1 and 2 became progressively worse by days 3 and 7 post-infection. the inflammation tended to be patchy or multifocal and an entire lung lobe was never uniformly affected, corresponding to the multiple patchy lesions seen on pet/ct by the end of the study. this also agreed with the analyses of the viral titers in various sections of the right caudal lobe and the pet/ct imaging. our analyses of viral titers in the four representative sections suggest different levels of infiltration of the lobe. the histopathologic scoring for bronchiolitis correlated the best with the suvmax of the lesions seen in the right and left lungs on pet. in general, the severity of infection and inflammation on imaging can be represented by (1) the volume of affected lung (i.e. the percentage of diseased lung relative to total lung capacity), and (2) the extent of parenchymal destruction (disruption of pulmonary architecture) and inflammatory cell migration. our study first aims to correlate fdg uptake measurements with histology, thereby analyzing the extent of parenchymal destruction and cellular infiltrates. it should be considered that there can be variation in matching fdg uptake with histologic severity because more severe architectural distortion can lead to necrosis with more dead cells, therefore showing less uptake of radiotracer among metabolically inactive dead cells and nonviable tissue. our study, however, shows that progressing inflammatory infiltrates on histology in the studied time period after acute influenza infection corresponds to radiologic trends. additionally, our study demonstrates spatial progression with increased size and number of abnormal foci in the lung parenchyma during acute infection. ultimately, utilizing these new imaging tools, we envision a number of future experiments to delineate potential differences in the course of h1n1pdm and h5n1 infection in ferrets. we also plan to explore additional radiotracers that might reveal potential differences in host responses in the immune system and the process of acute injury in the lung. future studies will assess differences in presentation of those who recover from infection versus those who eventually succumb to infection such as with more lethal isolates such as h5n1. this model should be valuable in rapid assessment of the effect of various treatments on pulmonary inflammation and damage. finally, these first pet/ct imaging approaches could be extended to a number of other important pulmonary infections caused by pathogens such as hantaviruses, respiratory syncytial virus, and sars cov, to gain further insight into the spatiotemporal in vivo dynamics of disease progression [18] . the influenza h1n1pdm virus, a/kentucky/180/2010, (ky/ 180; genbank cy99332 and cy99333) was isolated from the nasal swab of a severe hospitalized case (hospitalized in march 2010) provided by the severe influenza pneumonia surveillance project, an ongoing clinical study of hospitalized patients with influenza pneumonia in kentucky (courtesy of dr. julio ramirez). the virus was isolated and passaged in the allantoic cavity of tenday-old embryonated hens' eggs at 37uc. the allantoic fluid was harvested 72 h after inoculation, pooled and stored in stored in aliquots at 280c until use. the infectious virus titer of the resulting seed stock was determined by tcid 50 (50% tissue culture infectious dose) and the titer calculated by reed and muench [46] and confirmed by plaque assay on mdck cells. passage e2 was used for the studies reported herein. ferret studies were approved by the university of louisville institutional animal care and use committee. university of louisville has veterinary medicine tasked to monitor and support all animal experiments. research was conducted in compliance with the animal welfare act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the guide for the care and use of laboratory animals, national research council, 1996. the facility where this research will be conducted is fully accredited by the association for assessment and accreditation of laboratory animal care international. all female fitch ferrets were obtained from triple f farms (sayre, pa). ferrets were selected after screening blood samples for the presence of influenza antibodies using a hemagglutination inhibition assay (hi). ferrets that were seronegative for seasonal and pandemic viruses were shipped directly to the university of louisville regional biocontainment laboratory and acclimated for seven days prior to initiation of the studies. animals were fed teklad laboratory diet #2072 (harlan/teklad, madison, wi) and water ad libitum. for the characterization of the progression of infection of the ky/180 clinical isolate we utilized four month old, female ferrets. prior to infection with virus, ferrets were anesthetized with 0.05 mg/kg atropine, 5.0 mg/kg ketamine, and 0.08 mg/kg dexmedetomidine intramuscularly. subsequently, six animals were inoculated intransally (i.n.) with 0.5 ml of infectious virus per naris as a bolus, which was diluted to 10 5.7 tcid 50 /ml in phosphate buffered saline (pbs). six additional animals were inoculated by i.n. with 0.5 ml of infectious virus per naris as a bolus with pbs (mock). anesthesia was reversed with 0.4 mg/kg atipamezole. ferrets were monitored daily for temperature and clinical symptoms. on day 2 post-infection, two ferrets were taken to measure viral titers in blood, lung, brain, trachea, nasal turbinates, spleen, kidney, thymus, liver, duodenum, jejuno-ileum, large intestine, and rectum. gross pathology was defined during necropsy for the lung. at 14 and 28 days post-infection two additional ferrets from each group were analyzed for viral titers and pathology in the lung. for molecular imaging studies, twelve, four-month-old female fitch ferrets were utilized. animals were fed food and water ad libitum except 4 h prior to and during ct/pet imaging. on day 0, ferrets were anesthetized prior to inoculation with virus or pbs with ketamine, dexmedetomidine and atropine. eight animals were inoculated i.n. with 0.5 ml of infectious virus per naris as a bolus, which was diluted to 10 6.0 tcid 50 /ml in phosphate buffered saline (pbs). four ferrets were inoculated i.n. with 0.5 ml pbs per naris as a mock-infected control. for imaging, on days 0, 1, 2, 3 and 6, anesthetic induction and maintenance were achieved with 1-3% isoflurane. blood glucose levels were checked prior to administration of the radiolabeled tracer to ensure that they were within normal limits, which typically range from 62-134 mg/dl in ferrets [47] . glucose was provided to animals to compensate for body fluids lost during imaging. typically 30 mls of lactated ringer's solution (hospira) was administered subcutaneously (s.q.) following completion of the imaging. animals were monitored for body temperature and vital signs during imaging. imaging was performed on 0, 1, 2, 3, and 6 days post-infection (dpi). each day, four ferrets were imaged with ct and pet on hardware designed for preclinical animal studies, including microct and micropet, respectively. two ferrets were euthanized the each day and necropsied to obtain tissue samples for virologic and histopathologic analyses (please see study design table 1 ). image acquisition was conducted with a siemens inveon trimodal scanner (siemens preclinical, knoxville, tn), which is a small animal imaging platform that combines micropet, microct, and microspect modalities within one unit. this combination facilitated co-registration of pet and ct images as the study subject was kept in a uniform position on the scanner bed, minimizing potentially large motion artifacts as a result of repositioning the animal between each scan. the inveon microct scanner features a variable-focus tungsten x-ray source with an achievable resolution of 20 mm and a detector with a maximum field of view (fov) of 8.4 cm65.5 cm. the source-to-object distance was 263.24 mm and the source-to-detector distance was 335.67 mm. the inveon pet detector provided an axial field of view (fov) of 12.7 cm with a spatial resolution of 1.44 mm. pet images were reconstructed using a 2d-filtered backprojection algorithm with attenuation correction provided by microct imaging. for the microct scan, the following imaging settings were used: two bed positions, 80 kvp, 500 ma, 500 ms exposure time, and 464 binning. after each ferret underwent microct imaging, the bed position was reset and micropet imaging with 18f-fdg (petnet, louisville, ky) began immediately. for each ferret, 2 mci of 18f-fdg was administered (i.p.) with a 60-90 min uptake period. radioactive dose was confirmed with an atomlab 500 dose calibrator (biodex medical systems inc., ny). all imaging data were processed with pmod software (v3.1; pmod technologies ltd., zurich, switzerland). microct data were received from the inveon platform as dicom files and pet data as micropet files. scans were imported into the program's local sql database with the units for the pet radiotracer in kbq/ cc. pet images were co-registered with the ct images with reslicing done as necessary to facilitate later calculations. for analysis of 18f-fdg levels, the standardized uptake value (suv) was used. suv is a widely used semi-quantitative measure that normalizes radiotracer uptake in a given region of interest based on body weight, and calculated for this study as follows: for all calculations, animal weights were expressed in kilograms and fdg activity in megabecquerels. for each image series, suvs for each voxel were calculated using an external filter in pmod, with the radionuclide half-life set at 6586.2 sec for 18f-fdg. for each pulmonary lesion, an ellipsoid volume of interest (voi) was generated that encompassed the structure. then, automatic isocontour detection was used to refit the voi by setting a threshold of 50-60% of the difference between the maximum and minimum intensity suvs in the ellipsoid voi such that 0.5*(suv max 2suv min ). in cases where the automated threshold included contiguous structures in the voi, manual refitting in conjunction with the co-registered ct scan was used to exclude those surrounding structures. for all vois, maximum suv (suv max ) and average and standard deviation of all pixels in the volume (suv mean 6 sd) were calculated. for ct analysis, image interpretation was performed by a radiologist (in consultation with the scientific team) having more than ten years of diagnostic experience along with formal certifications by the american board of radiology (abr) and the american board of nuclear medicine (abnm). lesions on ct were identified using conventional criteria and terminology; ground-glass opacity (ggo) is defined in this study as hazy increased lung opacity, with discernible underlying lung architecture such as visible bronchial and vascular structures, representing partial displacement of air in interstitial and alveolar airspaces; consolidation is defined in this study as high density lung lesions (more dense than ggo) in which vascular and bronchial margins are obscured, representing complete displacement of alveolar air [27] . on days 1, 2, 3 and 6 and prior to euthanasia, swabs were taken from each ferret from nasal, throat and rectal regions. following scheduled euthanasia, the nasal turbinates and the right caudal lobe of the lung from each ferret, which was divided laterally into four segments, were isolated. all swab and tissue samples were snap-frozen in liquid nitrogen and stored at 280uc until analyzed for virus titer by tcid 50 . frozen tissues were weighed and diluted 10% weight per volume into cold dmem with 1% penicillin/ streptomycin and 0.2% bsa before being homogenized and centrifuged to remove debris. tissue homogenate and swabs were serially diluted 10-fold in dmem with 2 mg/ml tpck-trypsin, 0.2% bsa, 4.5 g/l glucose, 1% penicillin/streptomycin, 2 mm l-glutamine, and 25 mm hepes. each sample was analyzed in quadruplicate following incubation in 96-well plates with madin-darby canine kidney (mdck) cell monolayers at 37uc in 5% co 2 for three days. supernates were collected from each well were assayed for hemagglutination activity using 0.5% turkey red blood cells as an indicator of infection. viral titers were expressed as log 10 tcid 50 /ml and were calculated using the reed-muench method [48] . the hi test quantitates serum antibody to influenza virus which can prevent agglutination of turkey rbcs (fitzgerald industries international inc., ma). heat-inactivated serum samples were treated with receptor-destroying enzyme (sigma-aldrich) for removing nonspecific inhibitors (followed by rbc adsorption) and were diluted 2-fold serially from initial dilution of 1:10. ha antigen (8 ha units in 25 ml) were added onto each well and incubated for 1 h at rt. following antigen-antibody reaction, 50 ml of 0.5% turkey rbc were added to each well and incubated for 1 h at rt. hi negative wells were scored based upon a diffuse sheet of agglutinated rbcs covering the bottom. hi positive wells were scored if they showed a well circumscribed button of nonagglutinated rbcs. lungs were inflated and stored in 10% neutral-buffered formalin. three lung sections were placed into cassettes per lung section (right cranial, left cranial, left caudal, and right middle lobe) until they were trimmed, paraffin-embedded, and sectioned. sections were mounted on glass slides and stained with hematoxylin and eosin for microscopic evaluation at experimental pathology laboratories, inc. by a veterinary pathologist. sections were examined for the presence of abnormal findings including supprative and necrosupprative inflammation; epithelial hyperplasia and cytokaryomegaly; and fibrinous and exudative changes. changes were graded with a standardized scale of 0-5, with 0 classified as ''not present'', 1 as ''minimal'', 2 as ''slight/mild'', 3 as ''moderate'', 4 as ''moderately severe'', and 5 as ''severe/high.'' for each ferret, a composite score for pathological changes was generated based on the locations in the respiratory tract (alveoli, bronchioli, bronchi) for statistical evaluation. all statistics were performed using r version 2.13.0 and graphpad prism 5. for each image, mean suvmax and standard deviations were obtained. for each ferret, suvmax values were correlated with 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receptor binding effect of d222g mutation in the hemagglutinin protein on receptor binding, pathogenesis and transmissibility of the 2009 pandemic h1n1 influenza virus influenza virus receptor specificity and cell tropism in mouse and human airway epithelial cells pulmonary pathology of pandemic influenza a/h1n1 virus (2009)-infected ferrets upon longitudinal evaluation by computed tomography novel influenza a (h1n1) infection: chest ct findings from 21 cases in seoul a simple method of estimating fifty percent endpoints haematological and serum chemistry profiles of ferrets (mustela putorius furo) a simple method of estimating fifty percent endpoints we thank dr. michael bray for helpful discussions during the design of the experiments and dr. julio ramirez for helpful discussions regarding our findings. key: cord-000322-8ctsa9sd authors: ninove, laetitia; nougairede, antoine; gazin, celine; thirion, laurence; delogu, ilenia; zandotti, christine; charrel, remi n.; de lamballerie, xavier title: rna and dna bacteriophages as molecular diagnosis controls in clinical virology: a comprehensive study of more than 45,000 routine pcr tests date: 2011-02-09 journal: plos one doi: 10.1371/journal.pone.0016142 sha: doc_id: 322 cord_uid: 8ctsa9sd real-time pcr techniques are now commonly used for the detection of viral genomes in various human specimens and require for validation both external and internal controls (ecs and ics). in particular, ics added to clinical samples are necessary to monitor the extraction, reverse transcription, and amplification steps in order to detect false-negative results resulting from pcr-inhibition or errors in the technical procedure. here, we performed a large scale evaluation of the use of bacteriophages as ics in routine molecular diagnosis. this allowed to propose simple standardized procedures (i) to design specific ecs for both dna and rna viruses and (ii) to use t4 (dna) or ms2 (rna) phages as ics in routine diagnosis. various technical formats for using phages as ics were optimised and validated. subsequently, t4 and ms2 ics were evaluated in routine real-time pcr or rt-pcr virological diagnostic tests, using a series of 8,950 clinical samples (representing 36 distinct specimen types) sent to our laboratory for the detection of a variety of dna and rna viruses. the frequency of inefficient detection of ics was analyzed according to the nature of the sample. inhibitors of enzymatic reactions were detected at high frequency in specific sample types such as heparinized blood and bone marrow (>70%), broncho-alveolar liquid (41%) and stools (36%). the use of t4 and ms2 phages as ics proved to be cost-effective, flexible and adaptable to various technical procedures of real-time pcr detection in virology. it represents a valuable strategy for enhancing the quality of routine molecular diagnosis in laboratories that use in-house designed diagnostic systems, which can conveniently be associated to the use of specific synthetic ecs. the high rate of inhibitors observed in a variety of specimen types should stimulate the elaboration of improved technical protocols for the extraction and amplification of nucleic acids. real-time (rt) pcr and reverse transcription (rt) pcr techniques are rapid and versatile diagnostic procedures broadly used in clinical virology where there are mostly considered as diagnostic ''gold standards'' [1] . monitoring rt-pcr and rt-rt-pcr assays and validation of the results rely on the use of relevant external or internal controls (ecs or ics) [1, 2] and commercial kits including such control systems are being increasingly improved for the molecular diagnosis of a number of pathogens such as hiv, hepatitis viruses, influenza viruses etc.. however, one of the main strengths of rt-pcr is versatility, which provides the opportunity to set-up ''in-house'' protocols for specific pathogens. the scientific literature now includes an impressive number of 'home made'' assays for various viral agents. whilst most commercial kits include both ics and ecs allowing accurate validation of the results [3] , ''home made tests'' are frequently performed in the absence of ics and therefore without any possible individual monitoring of each diagnostic reaction. for example, the detection of technical errors or pcr amplification inhibitors is intrinsically impossible if only ecs are used. in addition, ecs are usually undistinguishable from the native genome. here, our objective was to develop and test on a large number of clinical samples a bacteriophage-based ic system suitable for a standard laboratory of medical virology. we present results obtained by using t4 and ms2 bacteriophages as ics in a routine-based evaluation including important criteria for the design of ecs include (i) the possibility to use quantified or semi quantified controls (in order to manage the detection level of the diagnostic test used), (ii) the possibility to distinguish amplicons obtained from ecs from those obtained from the detected pathogen (in order to detect false positives due to accidental amplification of ec) and (iii) the use of relevant nucleic acids (ie, rna and dna molecules for rna and dna viruses, respectively). simple protocols for preparing plasmid dna or synthetic rna quantified controls are described in supporting information s1 and schematised in figure 1. such controls include specific sequences for the hybridisation of detection primers and probe, but also an exogenic ''not i'' sequence (detectable by a specific probe or cleavable by the not i restriction enzyme). 2a. rt-pcr assays for the detection of t4 and ms2 bacteriophages. the criteria to be addresses regarding ics were: (i) to monitor all steps of the diagnostic procedure (extraction, rt, pcr); (ii) to be amenable for dna and rna viruses ; (iii) to rely on a detection system specific of the phage(s) to avoid complex molecular constructs; (iv) to be usable in either simplex or multiplex format, and in one-step or two-step rt-pcr reactions. we used freeze-dried e. coli enterobacteria phage t4 (t4) and enterobacteria phage ms2 (ms2) obtained from the american type culture collection (atcc ref. 11303-b4 & 15597-b1, respectively). protocols for real time pcr detection of phages ta and ms2 were elaborated in various formats and are described in supporting information s2. briefly, primers and probes targeting t4 phage (t4f ccatccatagagaaaatatcagaacga, t4r taaataattcctcttttcccagcg, t4probe vic-aaccagtaatttcatctgcttctgatgtgaggc-tam-ra) and ms2 phage (ms2f ctctgagagcggctctattggt, ms2r gttccctacaacgagcctaaattc, ms2probe vic-tcagacacgcggtccgctataacga-tamra) were designed from genomic sequences. optimised oligonucleotide concentrations were 10pmol for primers and 4pmol for probes for both t4 and ms2 detection assays. 2b. spiking and validation procedures. a suspension of t4 and ms2 phages was used for spiking clinical samples. both phages were diluted to provide a similar level of detection by rt-pcr. the following format was used: similarly, the influence of spiking on the sensitivity of rt-pcr detection was evaluated by comparing detection of enterovirus (ev) and cmv in serial dilutions of cell culture supernatant media and a series of positive clinical samples spiked with t4-ms2 mix in either two-step or one-step rt-pcr format (see supporting information s4). table 1 were included in the study. the approval of the relevant ethics committee (ifr48, marseilles, france) was obtained for investigating the benefit of using phage spiking, but individual consent from patients was not required since french national regulations under the term of biomedical research (loi huriet-sérusclat (loi 881138)) indicate that the signature at the hospital entrance office warrants that all samples taken during hospitalization for diagnostic purposes are accessible for research (excluding human genetic research) without the specific consent of the patient. 2b. spiking and rt-pcr assays. a 200ml volume of each 8,950 clinical specimens was spiked (according to the procedure described above) before extraction which was performed onto the magna pure lc instrument (roche) using the high pure viral nucleic acid kit (dna extraction) or the magna pure lc rna isolation high performance kit (rna extraction) according to manufacturer's recommendations. csf, amniotic fluids, biopsy tissues, effusions and aqueous humor were processed for dna+rna extraction using the biorobot ez1 with the virus mini kit v2.0 (both from qiagen). reverse transcription was performed for rna viruses using the taqman reverse transcription reagents kit (roche) and random hexanucleotides as per manufacturer's instructions. for each specimen: (i) rt-pcr reactions were carried out according to medical prescription (table 1) , and (ii) distinct rt-pcr reactions for detection of t4 or ms2 were performed under a 15 ml reaction format (7,5 ml of mastermix, 3 pmol of each primer and 1,2 pmol of probe) and a standard cycling protocol (50uc for 2 min, 95uc for 10 min and 45 cycles 95uc for 15 sec, 60uc for 1 min). 2c. interpretation of results. for each series of t4 and ms2 rt-pcr, the mean ct value and the standard deviation within the series were calculated. each individual reaction was subsequently analysed as follows: (i) if the ct value was equal to or lower than the mean ct value of the series +1sd, it was recorded as ''correct detection of the phage'' (cdp), and associated with the absence of detectable inhibitor or technical problem while processing the corresponding sample. (ii) if the ct value was higher than the mean ct value of the series +1sd (or undetectable), it was recorded as ''inefficient detection of the phage'' (idp), and associated with the presence of amplification inhibitor(s) or technical problem while processing the corresponding sample. n when idp was associated with a positive pcr (detection of a pathogen), this result was validated despite the presence of inhibitors (this would not apply to the case in which quantification of viral load is necessary). n when idp was associated with negative pcr detection results, a new assay was performed using a tenfold dilution of the nucleic acid extract. all negative results were considered unresolved (unr). positive results were validated. the detection of ev and cmv in serial dilutions of supernatant cell culture media or in series of positive clinical samples spiked with t4-ms2 mix was performed and the comparison of the ct values showed that neither the addition of the phage mix itself (wilcoxon test, p = 0.18 for cmv and 0.45 for ev), nor the presence of phage-specific primers and probe in the reaction mix (wilcoxon test, p = 0.77 for cmv and 0.18 for ev) did interfere with the ct value associated with viral detection in two-step (table 2 and 3) and one-step rt-pcr. during the period of study, 8,950 clinical samples were spiked with phages and tested: 7,397 for the presence of dna viruses only, 337 for the presence of rna viruses only and 1,216 for both, corresponding to a total of 45,530 results transmitted to the prescribers (see details in figure 2 ). inhibitors could be detected for all types of samples, but with highly variable rates (analysis performed for series including a minimum of 10 samples): less than 10% in the case of pleural effusions and sputum samples, genital and pharynx swabs, lymph nodes and placentas; 10% to 30% for edta whole blood, white blood cells, sera, biopsies, pericardial and peritoneal effusions, bronchial aspiration, csf, amniotic fluids, others swabs, urine, aqueous humor and culture cells; 30% to 50% for plasma, bal and stools; more than 50% for heparinized plasma and bone marrow. idps were significantly more frequently detected for ms2 (rna) than t4 (dna) in plasma, bal and csf samples (p,0.05, khi2 test, yates corrected). conversely, idps were significantly more frequently detected for t4 than ms2 in stools (p,0.05, khi2 test, yates corrected). finally, it was noted that clinical samples stored at 280uc prior extraction exhibited an idp rate lower than 30%. rt-pcr and rt-rt-pcr techniques are now widely used for the molecular diagnosis of viral infections. our study was focused on the importance of quality controls for such diagnostic assays. we proposed a simple protocol for synthesizing specific external controls which combines standard techniques for obtaining quantified dna or rna positive controls. importantly, these controls were designed to include an extrinsic sequence that contains a (very rare) cutting site for the noti restriction enzyme. this provides a simple tool for using adapted and reproducible amounts of positive controls, and also for identifying pcr contamination due to carry over of the positive control. this detection can be performed by real time amplification using the specific not i probe (figure 1 [4, 5, 6, 7] . our study confirms that the performance of 'home made' tests can be significantly improved by the used of phage-based internal controls, but, most importantly, shows that such controls can be used for routine virological diagnosis and usable for a variety of clinical samples. here, clinical samples were spiked with both t4 and ms2 phages, allowing the detection of inhibitors for both dna and rna viruses. thirty-six different types of clinical samples were tested (including various blood samples, cerebrospinal fluids, stools, respiratory samples, swabs, biopsies or effusion fluids) and a large number of samples were tested in the context of an hospital routine molecular virology laboratory. the use of phages as internal controls proved to be extremely versatile and could be adapted to a broad range of methods and pathogens. it was validated for both pcr and rt-pcr real time techniques, in simplex or multiplex format and, in the case of rt-pcr assays, one-step or two-step amplification formats. in addition, whilst the current report relies on probe-based real time amplification techniques, the method could also be conveniently adapted to a real time sybr green detection assay [8] . this is important and suggests that a strategy including phage-based internal controls can be implemented in diagnostic laboratories irrespective of the technical characteristics of the amplification methods used for routine tests. in our experience, the simplex format strategy (i.e. based on testing phages and viral pathogens in distinct amplification reactions) proved to be the most simple and costeffective for routine molecular diagnosis since it does not require the specific development of multiplex reactions and relies on a unique control reaction for dna viruses and another for rna viruses. our study identified different frequencies of inhibitors according to the nature of the clinical samples. in samples such as csf, sera or pharynx swabs, inhibitors were identified in ,10% and ,15-20% for dna and rna virus detection, respectively. these values are unexpectedly high, and imply that a significant number of samples with results believed to be ''negative'' in the absence of an internal control should be considered ''unresolved''. specific samples such as heparinized blood (72,9% of inhibitors for dna virus detection), bone marrow (73.8% of inhibitors for dna virus detection) and stools (36.6% and 20.8% of inhibitors for dna and rna viruses detection, respectively) may also benefit from the detection of inhibitors and the identification of ''unresolved'' tests. it should be noted that the frequency of amplification inhibition not only relates to the nature of the sample tested, but is also intrinsically linked with the technical protocols used for the extraction and amplification of nucleic acids. in our experience, no major differences were observed when different silica column-or magnetic beads-based extraction techniques, or different commercialized pcr or rt-pcr kits, were tested (data not shown). however, a detailed analysis may reveal that specific techniques give better results when used for testing specific samples. we suggest that, in the future, phage-based internal controls may constitute cost-effective tools with which to measure the frequency of amplification inhibition in specific samples. estimation of this parameter may become a major criterion for the evaluation of extraction (and to a lesser extent amplification) techniques. finally, in the context of diagnostic virology laboratories, our study shows that standard extraction and amplification techniques used for the molecular diagnosis of human pathogens led to a significant proportion of 'unresolved' results, which cannot be identified if an internal control is not used. in the absence of an internal control, such samples are commonly identified as 'negative', which is with hindsight incorrect: in our hands, detecting amplification inhibition using phages as internal controls, and testing tenfold dilutions of the nucleic acid extracts demonstrated that some of these samples were actually positives. this cost-effective and convenient strategy can therefore be used for enhancing the quality of routine molecular diagnosis, but it may also be adapted in other contexts such as testing of large numbers of animal or environmental samples. supporting information s1 preparation of dna and rna synthetic ecs. real-time pcr in virology practical considerations in design of internal amplification controls for diagnostic pcr assays preparation of armored rna as a control for multiplex real-time reverse transcription-pcr detection of influenza virus and severe acute respiratory syndrome coronavirus use of bacteriophage ms2 as an internal control in viral reverse transcription-pcr assays an internally controlled, one-step, real-time rt-pcr assay for norovirus detection and genogrouping implementation of a t4 extraction control for molecular assays of cerebrospinal fluid and stool specimens enhanced reverse transcription-pcr assay for detection of norovirus genogroup i a simple method for molecular detection of swine-origin and human-origin influenza a virus detection of herpes simplex virus dna by real-time pcr quantification of human cytomegalovirus dna in bone marrow transplant recipients by real-time pcr real-time quantitative pcr for human herpesvirus 6 dna quantitative analysis of human herpesvirus 8 viral load using a real-time pcr assay detection and differentiation of human polyomaviruses jc and bk by lightcycler pcr a quantitative, internally controlled real-time pcr assay for the detection of parvovirus b19 dna rapid diagnosis of enterovirus infections by real-time pcr on the lightcycler using the taqman format development of a taqman rt-pcr assay without rna extraction step for the detection and quantification of african chikungunya viruses simultaneous detection of influenza viruses a and b using real-time quantitative pcr applicability of a real-time quantitative pcr assay for diagnosis of respiratory syncytial virus infection in immunocompromised adults lower respiratory viral illnesses: improved diagnosis by molecular methods and clinical impact reverse transcription, real-time pcr assay for detection of toscana virus rapid detection of west nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a taqman reverse transcriptase-pcr assay development and validation of real-time one-step reverse transcription-pcr for the detection and typing of dengue viruses key: cord-001126-uqr00nzd authors: zhang, zhicheng; dai, wei; dai, dingzhen title: synonymous codon usage in ttsuv2: analysis and comparison with ttsuv1 date: 2013-11-26 journal: plos one doi: 10.1371/journal.pone.0081469 sha: doc_id: 1126 cord_uid: uqr00nzd two species of the dna virus torque teno sus virus (ttsuv), ttsuv1 and ttsuv2, have become widely distributed in pig-farming countries in recent years. in this study, we performed a comprehensive analysis of synonymous codon usage bias in 41 available ttsuv2 coding sequences (cds), and compared the codon usage patterns of ttsuv2 and ttsuv1. ttsuv codon usage patterns were found to be phylogenetically conserved. values for the effective number of codons (enc) indicated that the overall extent of codon usage bias in both ttsuv2 and ttsuv1 was not significant, the most frequently occurring codons had an a or c at the third codon position. correspondence analysis (coa) was performed and ttsuv2 and ttsuv1 sequences were located in different quadrants of the first two major axes. a plot of the enc revealed that compositional constraint was the major factor determining the codon usage bias for ttsuv2. in addition, hierarchical cluster analysis of 41 ttsuv2 isolates based on relative synonymous codon usage (rscu) values suggested that there was no association between geographic distribution and codon bias of ttsuv2 sequences. finally, the comparison of rscu for ttsuv2, ttsuv1 and the corresponding host sequence indicated that the codon usage pattern of ttsuv2 was similar to that of ttsuv1. however the similarity was low for each virus and its host. these conclusions provide important insight into the synonymous codon usage pattern of ttsuv2, as well as better understangding of the molecular evolution of ttsuv2 genomes. it is well known that the 64 codons of the genetic code encode the 20 standard amino acids as well as three translation termination signals (uaa, uag, uga). each amino acid is encoded with at least one codon (e.g., met and try); however, due to the degeneracy of the genetic code, some amino acids are encoded with up to six codons (e.g, leu, ser and arg). codons encoding the same amino acid are referred to as synonymous codons. studies have indicated that synonymous codon usage is non-random and species-specific [1] . some synonymous codons are more frequent than others both within and between genes, and this phenomenon is termed synonymous codon usage bias [2] . in general, genome dynamics, primarily mutation pressure, facilitate the evolution of novel viruses and strains and contribute to adaption to environment and host [3] . hence, codon usage variation is considered to be an indicator of the type of force that influences genome evolution. investigation of codon bias and the forces that influence it provides insights into the fundamental mechanisms of viral evolution. thus, understanding codon bias is essential to understand the interplay between a virus and its host. it was well established that mutational pressure and natural selection [4, 5] were presented as the two major factors accounting for codon usage variation in mammalian, protozoan and endosymbiotic bacterial genes [6] . in their investigate of codon usage variation, shackelton et al (2006) found that codon usage bias was strongly correlated with overall genomic gc content, indicating that compositional constraint under mutation pressure rather than natural selection was the main factor for specific codons [7] . naya et al (2001) examined the chlamydomonas reinhardtii genome, which has a high gc content, and found no evidence that base constraint under mutation pressure was responsible for determining the codon usage pattern [8] . recently, it was also reported that codon usage variation is related to gene function and length [9, 10] , dna replication and selective transcription [11] , protein secondary structure [12, 13] and environmental factors [14] . torque teno virus (ttv) is a small, single-stranded, negative-sense non-enveloped, circular dna virus [15] , which has been classified as a member of the recently discovered anelloviridae family [16] . it was first identified in a japanese patient with post-transfusion hepatitis of unknown aetiology in 1997 [17] . subsequently, ttv has been detected in humans, chimpanzees, poultry, swine, cattle, sheep, cats and dogs [18, 19] . ttv was first detected in swine in 1999 and two genetically distinct species, torque teno sus virus 1 (ttsuv1) and 2 (ttsuv2), have been identified based on the low sequence identity between the two variants [20] . recently, torque teno sus virus (ttsuv) infection of pigs has become widespread in many countries, including the usa, canada, spain, germany, china, japan, korea and brazil [21] . despite the fact that ttv infection in humans is not yet directly associated with any disease [22] , ttsuvs have been shown to be involved in co-infection with other diseases, including the experimental induction of porcine dermatitis and nephropathy syndrome in combination with porcine reproductive and respiratory syndrome virus infection [23] and post-weaning multisystemic wasting syndrome (pmws) in combination with porcine circovirus type 2 (pcv2) infection in a gnotobiotic pig model [24] . moreover, kekarainen et al. (2006) found that ttsuv2 was detected at a significantly higher rate in pmws pigs than in healthy pigs [25] . other research comfirmed that the replication of ttsuv2, but not of ttsuv1, was upregulated in the pigs with pmws [26, 27] . this result was supported by taira et al (2009) , who examined animals suspected of infection with pmws and porcine respiratory disease complex [28] . however, due to the limited number of animal species examined and the lack of information about viral cell and tissue tropism, the characteristics and evolution of ttsuv are not fully understood. we previously investigated synonymous codon usage in ttsuv1 [29] and began to suspect that this method might be important for elucidating the molecular mechanism and evolutionary process of ttsuv. in this study, synonymous codon usage bias was analyzed in the coding sequences (cds) from the 41 available ttsuv2 genomes, and the codon usage patterns of ttsuv2 and ttsuv1 were compared. complete genome sequences from 41 ttsuv2 isolates were downloaded from the national center for biotechnology information (http://www.ncbi.nlm.nih.gov/genbank/). each ttsuv2 cds was analyzed using dnastar version 7.1 (dnastar, madison, wi). table 1 summarizes relevant details about these viral sequences. the recombination analysis tool (rat, http://cbr.jic.ac.uk/ dicks/software/rat/) was used to detect recombination events in ttsuv2 and ttsuv1 sequences. recombination is a prevailing drive that shapes genome evolution, and it is believed to influence the efficacy of natural selection on codon usage [30] . rat uses a distance-method-based algorithm to perform pair-wise comparisons with multiple sequence alignments (dna or protein). the rat graph represents the genetic distance of each sequence in the alignment to a reference sequence (y-axis) for each position in the sequence (x-axis). a putative recombination event is detected when the lines representing two sequences intersect in the graph [31] . general nucleotide composition (a%, c%, t% and g%) and nucleotide composition at the third position of each codon (a 3s %, c 3s %, t 3s % and g 3s %) were analyzed for ttsuv2 cdss using molecular evolutionary genetics analysis (mega) software version 5.0 [32] . the gc and gc 3s index was used to calculate the overall g + c content in the gene sequence and at the third position of synonymous codon (excluding met, trp and termination codons). relative synonymous codon usage (rscu) values and effective number of codons (enc) values were calculated using codonw software version 1.4 (http:// codonw.sourceforge.net). the rscu is defined as the ratio between the usage frequency of one codon in the gene and its expected frequency in the synonymous codon family (i.e., the observed frequency of a codon adjusted for amino acid composition). rscu value is calculated according to the following published equation [33] : the enc is the most useful estimator of absolute synonymous codon usage bias [34] and can indicate the degree of synonymous codon bias in a codon family. enc values range from 20 (only one synonymous codon occurs in the cds) to 61 (all synonymous codons occur with equal frequency). a gene with an enc value lower than 35 is generally considered to have significant codon usage bias. correspondence analysis (coa), also known as principal component analysis, was performed with codonw software version 1.4. coa is the most commonly used multivariate statistical analysis method [35] . in this analysis, coa was used to study the major trends in sequence variation and distribute genes along continuous axes according to these trends. each gene was represented as a 59-dimensional vector, each dimension corresponding to the rscu value for each sense codon (excluding met, trp and termination codons). major variation trends within this dataset can be determined with the relative inertia: genes were positioned according to the major inertia to determine the major factors affecting codon usage bias in the gene. correlation analysis was performed to compare the relationship between nucleotide composition and synonymous codon usage pattern using spearman's rank correlation analysis method. a phylogenetic tree was constructed by the neighbor-joining method with a bootstrap of 1000 replicates, based on the clustal w alignment produced with mega software version 5. cluster analysis was performed using the hierarchical cluster method, and the distances between selected sequences were calculated by the euclidean distance method. all statistical results were analyzed using student's ttest, spss software version 11.6 for windows (p > 0.05, no difference; 0.01 < p < 0.05, non-significant difference; p < 0.01, significant difference). recombination is believed to influence the efficacy of natural selection on codon usage [30] . a single recombinant sequence present in an alignment can seriously influence the branch order and branch length of the trees generated using standard phylogenetic methods [36] . therefore, it was necessary to exclude any ttsuv2 and ttsuv1 sequences found to be recombinant from further analysis. recombination analysis of a nucleotide sequence alignment including all 41 ttsuv2 sequences and 29 ttsuv1 sequences was performed using rat software ( figure 1 ). the resulting graph provided no evidence for recombination within or between ttsuv2 and ttsuv1 sequences. however, the graph indicated that the sequences diverged at nucleotide position 2282 into branches corresponding to ttsuv2 and ttsuv1. the 41 ttsuv2 sequences were further analyzed for codon usage bias and the synonymous codon usage pattern between ttsuv2 and ttsuv1 (previously analyzed) [29] was compared, as described in the following sections. the nucleotide content of the ttsuv2 genomes is provided in table 2 . in the cdss from the 41 genomes, a and g occurred more frequently than c and t. a occurred most frequently at the third codon position (average a 3s % = 41.77%) and t occurred the least frequently (average t 3s % = 27.67%). the overall nucleotide composition and the composition at the third codon position in ttsuv2 genomes suggest that compositional constraint might be influencing the codon usage pattern of this genome. the gc% of ttsuv2 genomes (42.9% to 46.7%, average 45.1%) is lower than for other vertebrate dna viruses. the gc 3s % ranged from 43.2% to 48.2% with a mean value of 46.2%. due to this compositional constraint, it was expected that a would occur most frequently at the third codon position in ttsuv2 genomes. the enc values of these ttsuv2 genomes were much higher than genomes of other dna viruses, varying from 55.20 to 58.18 with a mean value of 56.21. this result indicates that codon usage bias is not remarkable in ttsuv2 genomes and is apparently maintained at a stable level. the overall rscu values for the 59 codons in all 41 ttsuv2 genomes indicated that a and c occurred most frequently at the third codon position (i.e., gua for val, gca for ala, caa for gln and aac for asn) as shown in table 3 . in addition, the ccu, acu and uau codons, encoding pro, thr and tyr, respectively, occurred more frequently than the other synonymous codons for these amino acids. two codons encoding arg, cga and cgc, also occurred more frequently than their synonymous codons. these results support the hypothesis that compositional constraint is a major contributing factor in codon usage pattern in ttsuv2 genomes. for ttsuv2 sequences, enc was plotted against both the gc content at the third synonymous codon position (gc 3s %) and the expected enc values, as determined by codonw analysis (figure 2 ). all actual codon usage indices were lower than expected, although differences were small. in addition, a positive correlation (r = 0.316, 0.01 < p < 0.05) between gc 3s and enc values was found. these results taken together support the conclusion that factors other than compositional constraint under mutation pressure (the major factor accounting for codon usage bias) have influenced ttsuv2 evolution. to investigate rscu variation, coa was performed using the 41 ttsuv2 genomes as a single dataset. as described in the "materials and methods" section, the distribution of genes on the coa axis was used to identify the source of the variation among a set of multivariate data points. a major trend in the first axis (f 1 ') accounted for 16.91% of total synonymous codon usage variation, and the second major trend in the second axis (f 2 ') accounted for 13.72% of the total variation (data not shown). coa was performed for ttsuv1 and ttsuv2 genomes separately and the first two axes of the plots are shown in figure 3 . although ttsuv1 and ttsuv2 genes occupied all four quadrants of the rectangular coordinate system, the points were generally separated from each other. this result reveals that variation in codon usage might be one of the factors driving the observed aspect of ttsuv evolution. to explore whether the evolution of codon usage bias in ttsuv2 cds had been driven by mutation pressure alone or whether translation selection from its host has also contributed, we first compared the correlation between general nucleotide composition (a%, t%, g%, c%, gc%) and nucleotide composition at the third codon position (a 3s %, t 3s %, g 3s %, c 3s %, gc 3s %) using the spearman's rank correlation analysis method (table 4) . a significant positive correlation was observed between a% and a 3s % (r = 0.761, p < 0.01), c% and c 3s % (r = 0.392, 0.01 < p < 0.05), gc% and gc 3s % (r = 0.645, p < 0.01) and significant negative correlation was observed for most of heterogeneous nucleotide comparisons. taken alone, these results suggest that compositional constraints under mutation pressure determine the codon usage pattern for ttsuv2. however, a significant positive correlation between g furthermore, g + c content at the first and second codon positions (gc 1 % and gc 2 %) was compared with the g + c content at the third codon position (gc 3 %). a highly significant correlation was observed between gc 1 % with gc 2 % (r = 0.551, p < 0.01), gc 3 % (r = 0.699, p < 0.01), and gc 2 % with gc 3 % (r = 0.490, p < 0.01). since the effects were present at all codon positions, the results further support the hypothesis that nucleotide constraint under mutation pressure was a main determinant for synonymous codon usage pattern in ttsuv2. coa was also performed for the first two principle axes (f 1 ' and f 2 ') and a%, t%, g%, c%, gc%, a 3s %, t 3s %, g 3s %, c 3s %, gc 3s % ( table 5 ). the first principle axis (f 1 ') exhibited a significant positive correlation with g%, c%, gc%, c 3s %, gc 3s % and a negative correlation with a%, a 3s %. it was interesting to note that, except g 3s % (r = -0.357, 0.01 < p <0.05), the second principle axis (f 2 ') had no correlation with any nucleotide content. these results further support the conclusion that composition constraints under mutational bias is an important factor determining synonymous codon usage pattern in ttsuv2, and but that other factors, such as natural selection, contributed. in the enc plot (figure 2 ), most points were near to and under the expected curve, which suggested that other factors contributed to codon usage bias in addition to mutation pressure. to examine this further, a comparative analysis of rscu values was performed for ttsuv2, ttsuv1 and swine, the natural host for this virus. we found that the codon usage pattern of ttsuv2 was mostly coincident with that of ttsuv1 and that the similarity between the viruses and the host was low. in particular, except for ccu encoding pro and uau encoding tyr, all the preferentially used codons in ttsuv2 and ttsuv1 had an a or c in the third codon position: uua for leu, aua for ile, uca for ser, cac for his, gac for asp and ugc for gly (table 3 ). in contrast, most frequent codons in swine had a t or a at the third codon position. although some codons frequent in swine, such as cac for his, aaa for lys, gac for asp and aaa for glu, were also frequent in ttsuv2 and ttsuv1, the high frequency codons in swine (cug for leu, ucu for ser, ugu for cys) were generally low frequency codons in ttsuv2 and ttsuv1. it was worth noting that the similarity to swine was higher for ttsuv1 than it was for ttsuv2. the rscu values of synonymous codons in ttsuv1 and swine, including gug for val, gcu for ala, cag for gln, aau for asn, were clearly different than ttsuv2 values. this suggests that ttsuv1 might have adapted to its host under natural selection to some degree for improved translation efficiency and that selection pressure from the host had less effect on codon usage pattern of ttsuv2. a cluster tree was generated with the rscu values from all 41 ttsuv2 genomes using a hierarchical cluster method. as shown in figure 4 , the ttsuv2 cds were divided into three main lineages (i-iii). lineage i comprised two strains isolated from the usa, one from germany and five from china. twentytwo strains isolated from brazil, spain and china were grouped into lineage ii. lineage iii was comprised of strains isolated from china only. some genes from different isolates were classified into the same lineage, while others genes from the same isolate were classified into different lineages; thus lineage did not correspond well with geographical distribution. the phylogenetic analysis of all 41 ttsuv2 (black dots) and 29 ttsuv1 sequences (white dots) was performed to determine the conservation and variation of codon usage pattern within ttsuv lineages ( figure 5 ). the two major branches of the resulting phylogenetic tree corresponded to ttsuv2 and ttsuv1, and each branch had several minor branches. thus, phylogenetic analysis of the two viruses did not reveal correlations between sequence differences and geographical distribution. ttsuv is an emerging small dna virus, widely distributed in pig-farming countries. although reports implicate ttsuv in coinfection with other diseases, in depth studies on molecular characteristics and pathogenic mechanism are lacking [37, 38] . synonymous codon usage is a well established technique for analyzing genetic information from viral genomes. most codon usage studies have focused on higher organisms or microorganisms with large genomes and viruses that pose a great threat to human health, such as human immunodeficiency virus, human bocavirus [39] , hepatitis virus [40] and influenza a virus [41] . results from analyzing codon usage bias in ttsuv genomes are expected to contribute to the knowledge of the characteristics and molecular evolution of this virus. this report furthers our investigation of synonymous codon usage variation in ttsuv1 and provides the first analysis of ttsuv2. recombination is an important event in viral evolution and epidemiology [42] . it is interesting to note that recombinant viruses appear to be highly pathogenic, suggesting that recombination events either preserve or increase the pathogenicity of the original strains. various studies have demonstrated that natural inter-and intra-genotypic recombination occurs frequently in viruses, as shown for highly pathogenic porcine reproductive and respiratory syndrome viruses [43] , pcv2 [44] , humane enterovirus 71 [45] , and rabbit haemorrhagic disease virus [46] . thus, before analyzing codon usage bias for ttsuv2, we first conducted recombination analysis of 41 ttsuv2 sequences and 29 ttsuv1 sequences, and found no evidence for recombination between the two viruses ( figure 1) . in this study, we analyzed synonymous codon usage bias in ttsuv2 cds, as well as the relationship between codon usage patterns of ttsuv2 and ttsuv1. most frequent codons in both ttsuv2 and ttsuv1 had a or c at the third codon position. mean enc values for h5n1 influenza a virus [47] , severe acute respiratory syndrome [48] and human bocavirus [39] , reported as 50.91, 48.99 and 44.45, respectively, are lower than the enc values for ttsuv2 and ttsuv1 (56.21 and 56.46, respectively), indicating a relatively low codon usage bias for these two viruses. codon usage patterns for ttsuv2 and ttsuv1 were remarkably similar. in addition, no significant relationship was found between the codon usage pattern of ttsuv2 and its host; although ttsuv1 codon usage was comparatively more similar to swine than that of ttsuv2 (table 3) . this observation might be the result of genome composition evolution and dynamic processes of mutation and selection that enabled the ttsuv1 virus to escape the antiviral cell responses and adapt its codon usage to its host environment [49] . in this study, nucleotide frequency at the third codon position of synonymous codons correlated to general composition for some codons but not for others ( table 4 ). the gc content was similar at all codon positions in ttsuv2 genomes, presumably as a result of mutational pressure. in addition, the general correlation between codon usage bias and composition constraint suggest that mutational pressure was an important factor determining codon usage in ttsuv2, as seen in the highly significant correlation between gc 1 %, gc 2 % and gc 3 % (p < 0.01), and remarkable correlation between f 1 ' values with respect to a%, g%, c%, gc%, a 3s %, g 3s %, gc 3s % (p<0.01) ( table 5) . furthermore, in all enc plots, values for ttsuv2 genomes were below the expected curve ( figure 1 ). taken together, the above evidence indicates that compositional constraint under mutational pressure significantly contributed to the variation of synonymous codon usage in ttsuv2 genomes. natural selection has been shown to influence the synonymous codon usage pattern in viruses [50] and this conclusions is supported by this study. first, although the gc 3s % for the ttsuv2 genome is lower than average (46.20%), the most frequent codons had a or c at the third codon position (table 3) . second, a significant positive correlation existed between g% and c 3s %, and gc% and t 3s % (p < 0.01), whereas no correlation was detected between t% and t 3s % or g% and g 3s % (p > 0.05) ( table 4 ). except g 3s %, no correlation was found between f 2 ' values and a%, t%, g%, c%, gc%, a 3s %, t 3s %, c 3s % or gc 3s % (p > 0.05) in this study (table 5) . third, most points in the enc plot were close to the expected curve, although all were below it ( figure 2 ). the above evidences suggests that, in addition to mutation pressure, natural selection played an important role in determining codon usage bias for ttsuv2 genomes as well. thus, codon bias in the ttsuv2 genome is multi-factorial. we believe that these characteristics of ttsuv2 genomes might have conferred adaptive advantage resulting in a highly efficient dissemination of this virus through different modes of transmission. the analysis of ttsuv genome sequences identified two genetically distinct species, ttsuv1 and ttsuv2. coa was performed to detect possible codon usage variation between these two viruses. unexpectedly, the distribution of the two viruses showed that genetically distinct species were distantly located in the plane defined by the first two axes of the analysis (figure 3) . a cluster tree analysis based on the rscu values of ttsuv2 genomes revealed that geographic factors failed to correspond to the codon usage pattern of this virus (figure 4) . further, the phylogenetic tree had two major branches corresponding to the two different species, and no specific geographical correlation was detected in this analysis ( figure 5 ). it seems likely that, given extensive international communication and various modes of transmission for this virus, geographical distance is a weak factor in the distribution of ttsuv2 in different countries. in summary, our investigation of synonymous codon usage pattern in ttsuv2 cds revealed that codon usage bias is not remarkable, possibly representing the interactions between compositional constraint under mutation pressure and natural selection. however, both ttsuv1 and ttsuv2 genomes exhibited significant synonymous codon usage bias favoring a or c at the third codon position, presumably determined by compositional constraint under mutation pressure. although the analysis of synonymous codon usage does not perfectly reflect the genetic variation of ttsuv2 nor does it distinguish between ttsuv1 and ttsuv2, our results provide an insight into the codon usage variation in ttsuv2 genes that may also facilitate understanding of ttsuv evolution. â�¢ represents ttsuv2 and â�� represents ttsuv1. doi: 10.1371/journal.pone.0081469.g005 synonymous codon usage in lactococcus lactis: mutational bias versus translational selection evolution of codon usage patterns: the extent and nature of divergence between candida albicans and saccharomyces cerevisiae avian influenza virus exhibits rapid evolutionary dynamics mutation pressure shapes codon usage in the gc-rich genome of foot-and-mouth disease virus factors influencing the synonymous codon and amino acid usage bias in at-rich pseudomonas aeruginosa phage phikz codon usage in regulatory genes in escherichia coli does not reflect selection for 'rare' codons evolutionary basis of codon usage and nucleotide composition bias in vertebrate dna viruses translational selection shapes codon usage in the gc-rich genome of chlamydomonas reinhardtii codon usage and gene function are related in sequences of arabidopsis thaliana low affinity and unstable hemoglobin variant caused by aac--atc (asn--ile) mutation at codon 108 of the beta-globin gene replicational and transcriptional selection on codon usage in borrelia burgdorferi second codon positions of genes and the secondary structures of proteins. relationships and implications for the origin of the genetic code studies on the relationships between the synonymous codon usage and protein secondary structural units codon usage in nucleopolyhedroviruses molecular and biophysical characterization of tt virus: evidence for a new virus family infecting humans classification of ttv and related viruses (anelloviruses) a novel dna virus (ttv) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology genomic and evolutionary characterization of tt virus (ttv) in tupaias and comparison with species-specific ttvs in humans and non-human primates genomic characterization of tt viruses (ttvs) in pigs, cats and dogs and their relatedness with species-specific ttvs in primates and tupaias rolling-circle amplification of torque teno virus (ttv) complete genomes from human and swine sera and identification of a novel swine ttv genogroup molecular characterization of porcine tt virus, an orphan virus, in pigs from six different countries isolation of multiple tt virus genotypes from spleen biopsy tissue from a hodgkin's disease patient: genome reorganization and diversity in the hypervariable region expression of all six human torque teno virus (ttv) proteins in bacteria and in insect cells, and analysis of their igg responses effect of coinfection with genogroup 1 porcine torque teno virus on porcine circovirus type 2-associated postweaning multisystemic wasting syndrome in gnotobiotic pigs prevalence of swine torque teno virus in post-weaning multisystemic wasting syndrome (pmws)-affected and non-pmws-affected pigs in spain torque teno sus virus 1 and 2 viral loads in postweaning multisystemic wasting syndrome (pmws) and porcine dermatitis and nephropathy syndrome (pdns) affected pigs dynamics of torque teno sus virus 1 (ttsuv1) and 2 (ttsuv2) dna loads in serum of healthy and postweaning multisystemic wasting syndrome (pmws) affected pigs prevalence of swine torque teno virus genogroups 1 and 2 in japanese swine with suspected post-weaning multisystemic wasting syndrome and porcine respiratory disease complex analysis of synonymous codon usage patterns in torque teno sus virus 1 (ttsuv1) does recombination improve selection on codon usage? lessons from nematode and fly complete genomes recombination analysis tool (rat): a program for the high-throughput detection of recombination mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods an evolutionary perspective on synonymous codon usage in unicellular organisms an evaluation of measures of synonymous codon usage bias analysis of synonymous codon usage in foot-and-mouth disease virus the effect of recombination on the accuracy of phylogeny estimation porcine genogroup 1 torque teno virus prrsv infections in gnotobiotic swine evaluation of the effects of porcine genogroup 1 torque teno virus in gnotobiotic swine analysis of synonymous codon usage in 11 human bocavirus isolates analysis of codon usage in type 1 and the new genotypes of duck hepatitis virus codon usage bias and the evolution of influenza a viruses. codon usage biases of influenza virus recombination in evolutionary genomics recombination is associated with an outbreak of novel highly pathogenic porcine reproductive and respiratory syndrome viruses in china molecular analysis of porcine circovirus type 2 strains from uruguay: evidence for natural occurring recombination evidences for intertypic and intratypic recombinant events in ev71 of hand, foot and mouth disease during an epidemic in hubei province, china evidence for recombination in the major capsid gene vp60 of the rabbit haemorrhagic disease virus (rhdv) analysis of synonymous codon usage in h5n1 virus and other influenza a viruses analysis of synonymous codon usage in sars coronavirus and other viruses in the nidovirales natural infection with torque teno sus virus 1 (ttsuv1) suppresses the immune response to porcine reproductive and respiratory syndrome virus (prrsv) vaccination after the bottleneck: genome-wide diversification of the mycobacterium tuberculosis complex by mutation, recombination, and natural selection conceived and designed the experiments: zz wd. performed the experiments: zz wd. analyzed the data: zz wd.contributed reagents/materials/analysis tools: zz wd dd. wrote the manuscript: zz wd. key: cord-002044-9xgt3tf4 authors: hendiger, jacek; chludzińska, marta; ziętek, piotr title: influence of the pressure difference and door swing on heavy contaminants migration between rooms date: 2016-05-12 journal: plos one doi: 10.1371/journal.pone.0155159 sha: doc_id: 2044 cord_uid: 9xgt3tf4 this paper presents the results of investigations whose aim was to describe the influence of the pressure difference level on the ability of contaminants migration between neighbouring rooms in dynamic conditions associated with door swing. the analysis was based on airflow visualization made with cold smoke, which simulated the heavy contaminants. the test room was pressurized to a specific level and then the door was opened to observe the trail of the smoke plume in the plane of the door. the door was opened in both directions: to the positively and negatively pressurized room. this study focuses on the visualization of smoke plume discharge and an uncertainty analysis is not applicable. unlike other studies which focus on the analysis of pressure difference, the present study looks at the contaminants which are heavier than air and on “pumping out” the contaminants by means of door swing. setting the proper level of pressure difference between the contaminated room and the neighbouring rooms can prove instrumental in ensuring protection against toxic contaminants migration. this study helped to establish the threshold of pressure difference necessary to reduce migration of heavy contaminants to neighbouring rooms. an effective and well-designed ventilation system is essential when protection of indoor environment against external conditions is required, and on the premises of special use where hazardous airborne contaminants could be released. control of contaminants migration between rooms or areas is crucial in both cases. the common strategy to direct the airflow and thus the contaminants transfer is room pressurization. it is widely utilized in chemical or biological laboratories and clean rooms. room pressurization is also the main method used for the protection of isolation rooms in hospitals. in health care facilities, this way of ventilation system operation is utilized in both the airborne infection isolation and protective environment rooms, however, the key difference is the required direction of airflow between the room and the adjacent space, such as a corridor, which determines the application of negative or positive pressure in the protected room, respectively. positive pressure ventilation is also designed in operating rooms. there are many different suggestions with regard to the level of pressure difference which should be created to avoid contaminants migration between rooms. useful information can be found in the guidelines for health services buildings, biological and chemical laboratories, as well as for smoke control systems, but the specific value depends on the local standards, law or recommendations. according to the us center for disease control and prevention [1] , a minimum pressure difference needed to direct airflow between rooms should be equal to 2.5 pa. a similar value is recommended by the american institute of architects [2] . the uk department of health in the best practice guidance for health buildings recommends 5 pa as a minimum for negative pressure isolation and, instead of a negative pressure in the isolation room, allows a positive pressure of outside corridor of 8-12 pa (10 pa nominally) [3] . guidelines in taiwan suggest a minimum negative pressure of 8 pa in relation to the adjacent room or corridor [4] . ashrae [5] recommends 12.45 pa as a value of pressure difference for the rooms of enhanced cleanliness requirements (class), which is confirmed by the study conducted by ahmed et al. 1993 [6] . similar guidelines can be found in the guidance for industry sterile drug products [7] , where, in the case of clean areas, the value of 10-15 pa is recommended as a minimum value of pressure difference protecting the room. in the case of protection of aseptic isolations, where it is recommended to achieve complete physical separation from the external environment, the values range from 17.5 to 50 pa. some publications refer to the airflow control as a primary means of protection of rooms and thus having an indirect influence on diversification of pressure between adjacent rooms nih (2003) [8] ; aia (2001) [2] ; hitchings (1994) [9] ; gill (1994) [10] and coogan [11] . such publications recommend a range of 126-510 m 3 /h as the difference between the exhausted and supplied air in a contaminated room. however, since the value of the difference of airflow and the generated pressure difference value are closely connected, streifel (2000) [12] recommends 212 m 3 /h as a difference in the airflow and simultaneously points out 2.5 pa as a minimum pressure difference necessary to protect rooms and 7.5 pa as an optimum value. additionally, gill [10] presents a minimum velocity on the room leakages at the level of 0.508 m/s. however, all the above guidelines and considerations refer to the steady-state conditions with the doors closed. another ratio recommended in the guidelines, especially for health care rooms, is the required air change per hour (ach), which, depending on the source, ranges from 6 to 12 ach [1, 3] . research with tracer gas sf 6 conducted by tung et al. [4] indicates that the ventilation system of negative pressure differential 15.0 pa in the isolation room demonstrates the best ventilation efficiency to extract contaminants, for both 12 and 24 ach, which were tested. at lower values of pressure differential the more important factor was ach, and higher ach gives better results. can be the case in operating rooms [19, 20] . door opening immediately causes pressure equilibration between the rooms [21, 15, 22] . moreover, it was demonstrated that door swinging makes the air mass from both sides blend, especially at the top edge of the door wing, while the difference in the airflow, which protects the rooms when the door is open, is too low to direct the airflow appropriately with the door open [15, 22] . thus, it was noted that the door should not be opened rapidly. similar conclusions can be found in [14] where it is pointed out that the negative pressure gradient may have been transiently reversed if the door-opening motion was too rapid, and sliding doors were recommended instead of hinged one. under the study [15, 22] permanent conditions with the door open were also verified. the limit value, which prevents the air from coming out of the room was the pressure difference at the level of 2 pa. to compare, the american industrial hygiene association [23] recommends minimum airflow velocity of 0.25 m/s through any opening, including open doorways, and preferred velocity of 0.51 m/s in a desired direction. airflow through the doorway is also influenced by the air density difference which results from temperature difference between neighbouring rooms [16, 17, 24, 25] . when the temperature difference is high enough, the airflow is directed by the gravity rather than by the pumping effect of door swing [16, 17] . the research described in the available literature, concerning migration of air between rooms in the conditions of pressure difference was conducted using tracer gases, cfd simulations and smoke visualization. it also took into consideration such factors as door operation, temperature difference or ach level. however, most investigations focused on the general contaminant without taking into account its weight and location in the room. whenever it is required to protect rooms against migrating contamination, contamination may also contain elements which are heavier than air. in laboratories, these may be chemical compounds connected with the production taking place in the laboratory, while in hospitals these may be anesthetic gases or even aerosols containing hazardous bacteria or viruses. research on heavy contaminants behaviour is also important due to the fact that among the heavier than air contaminants there are a lot of toxic substances belonging to the group of chemical weapons. [26] . literature does not define specific requirements for the contaminants heavier than air, apart from the layout of air exhausts (if any). therefore, in room protection focused design the same pressure difference values are usually used, regardless of the kind, density or weight of the contaminants. in the present paper we focused on heavier-than-air contaminants, accumulated in the lower part of a room. the tests were made with dense smoke visualization in order to investigate the relation between the value of pressure difference, door swing and migration level of heavy contaminants between the rooms. in addition, disturbances in contaminant transfer resulting from movement of a person from a contaminated room to a protected one were also considered. the main method of conducting tests and observation was visualization of contaminated air flow, carried out by means of smoke. the tests were conducted on a suitably prepared testing stand. the measuring stand was configured in a testing room equipped with air-supply/air-exhaust systems (fig 1) . both adjacent rooms, where the tests were conducted, had an area of 6x8 m and were 3 m high. they were connected by a single-wing swing door, 2x0.9 m in size. the measuring set-up let us precisely set and monitor the pressure difference between test rooms, measure velocity of the air in the apertures, airflow rate of the air supplied or exhausted from the rooms and visualize the contaminant transfer. a set of measurement series was performed with different values of maintained pressure difference, starting with small values, i.e. 2.5 pa till 50 pa used in standard smoke control systems. the main components of the test installation were (fig 2) : • a device for adjustment and measurement of supply/exhaust airflow rate, adjustment dampers, measuring area reducers and orifice plate, pressure transducers, • simulated source of smoke-the low fog machine, • camera for visualization of smoke transfer with adjustment elements, • lighting for measurement area. the area near the door in both test rooms was lined with black fabric in order to enhance contrast between the smoke and the background. the smoke, generated by the low fog machine, was let in, each time, just above the floor level, at the distance of approx. 1.5 m from the doors. the airflow volume was measured by a system containing a measuring tube (venturi flow meter) and a pressure transducer. the measuring tube was a standard made element with external diameter of 160mm and measurement accuracy not lower than 5%. the pressure transducer connected with the measuring tube had a measuring range from 0 to 500 pa, resolution of 1 pa and measurement accuracy of 0.5%. the airflow volume was calculated as follows: where: k-constant value for the measuring tube, δpm-measurement pressure, pa. the maximum measurement error of the airflow volume v, calculated as an error of a compound value was 5.3%. positive pressure in the room was measured by means of a pressure transducer with measuring range of 0-100 pa, resolution of 0.1 pa and measurement accuracy of 0.5%. during all measurement sessions a constant temperature of 23°c was maintained both in the rooms and in the ventilation system which generated pressure difference. pressure difference between the rooms provoked air leakage through the air leakage points in the room envelope, and in the closed elements of the ventilation system. functional relationship between the airflow volume and the pressure difference it generated as well as geometry of the openings, the so called power law equation, confirmed in numerous sources in the literature, was proposed by ashrae in [27] : where: q-airflow through opening, m 3 /s, c-flow coefficient, m 3 /(s ápa n ), n-pressure exponent, [-], δp-pressure difference across opening, pa. exponent (n) and coefficient (c) depend on the kind of openings through which the air passes. the research conducted [28] also proved that the results obtained depend significantly on the geometrical parameters of the openings and on the inflow and outflow of air from the opening. the value of exponent (n) on the basis of tests performed by walker [29] for the process of air infiltration through the openings, is constant and equals approx.0.6-0.7. most frequently, exponent (n) has a typical value of 0.65. the value of flow coefficient (c) and that of pressure exponent (n) can be determined empirically by means of appropriate pressure tests. during the tests parameters of the test room were determined, with the door and other openings closed. for this purpose, volume of the airflow supplied to the test room with gradually increasing pressure values was measured. the results of airflow measurements are shown in table 1 . the above results were approximated by means of the function described in eq 1. the following relationship was obtained: with correlation coefficient r = 0.998 adopting a typical coefficient n = 0.65 resulted in a very good representation of room characteristics in the form of the following relationship: with correlation coefficient r = 0.998 room characteristics are shown in fig 3. as it can be seen, the test room used in the test has typical characteristics describing leakage. the tests were performed in measurement series with different values of maintained initial level of pressure difference (from low values of approx. 2.5 pa to the values typically used in desmoking systems, i.e. 50 pa) and the direction and width of the door swing shown in tab.2. the testing variants assumed certain behavioural patterns of the users. for this reason, the case of opening and closing the door adjacent to the contaminated area was considered, i.e. a scenario in which a person is looking out of the door in order to assess the situation. for this testing variant, two widths of door swing were taken into account i.e. 10 cm (s = 10) and 50 cm (s = 50). variant s = 10 was realized at two values of door swing velocity, the so called "slow" and "fast". moreover, during the measurement series the influence exerted by movement of a person on the contaminants migration was also taken into consideration. such a scenario corresponds to the case of "escape" from the endangered zone. however, with variants s = 10 and s = 50 the door was opened and then closed while in the escape mode the door was opened to 50 cm and left open after a person transition. the process of opening and closing the door was manual so there could have occurred small differences in velocity and time of its opening. testing variants used in measurement series are shown in table 2 . simulation of contamination was carried out using a low fog machine. during measurements the machine was always placed on the negatively pressurized side, approx. 1.5 m away from the door. smoke was introduced at the floor level (5 cm above the floor) in a low turbulent manner, so the mixing of smoke with indoor air was limited. the height of smoke layer was about 10-15 cm at the beginning of each test. temperature of air above the smoke layer in the negative pressure room was equal to the air temperature in the positive pressure room, and no significant temperature gradient was observed in both rooms. the course of a single measurement series was: • closing of the door, • generating appropriate pressure difference, • start-up of a simulated source of contamination (low fog machine), • generating appropriate conditions: opening and closing of the door, "escape" mode. selected results obtained in different measurement series and notes about each series are presented below. case "i" refers to the door opening towards the negatively pressurized zone, while case"ii" refers to the door opening towards the positively pressurized zone. descriptions of the series and conclusions were prepared on the basis of visualization and observation of the smoke movement. temperature during the measurements remained constant, therefore, the influence of the buoyant force on contamination movement through the door can be excluded. the door was opened manually and therefore small differences in velocity and time of door opening may have occurred. however, control measurements showed that these differences would not have been greater than 5%. figs 4-9 shows the results for cases i and ii and pressure difference: 2.5, 12.5 and 25 pa. in all the cases, with 2.5 pa pressure difference, a considerable amount of smoke migrated to the adjacent room with the door closed. in the "escape" mode, a plume of smoke trailed after with 12.5 pa, when the door was opened and closed at the width of 10 cm, the smoke practically did not migrate out of the room. at 50 cm wide opening and in the "escape" mode, a relatively small amount of smoke remained outside the room. above the pressure difference of 25 pa, the smoke practically did not migrate out of the room when the door was opened and closed. only at 25 pa, a passage of a person still caused moderate migration of the smoke outside. in all the cases, with pressure difference up to 5 pa, the smoke remained outside the room after the door was closed. in the "escape" mode the smoke trailed after the person leaving the room. a 25 pa the degree of smoke migration when the door was closed and opened, fell to a moderate level. when a person passed through the door, the smoke practically did not trailed after the person and soon withdrew. figs 10 and 11 show the results obtained in different series. during the measurement series with the door opened towards a negatively pressurized zone (fig 10) , in the case of the lowest values of pressure difference (2.5 pa), a considerable degree of contaminants migration to the influence of the pressure difference and door swing on heavy contaminants migration between rooms room was observed. after closing the door, it was noted that a certain portion of the smoke left migrated further towards an adjacent room or remained at the doorsill. increased pressure difference (5, 7, 12.5 pa) between rooms made it possible to arrest smoke in a room when the door was open to the width of 10 and 50 cm. however, a passage of a person through the door resulted in intensive turbulences causing discharge of certain portion of smoke plume outside. when the door was left ajar, the airflow provoked by generating initial pressure difference of 25 pa, prohibited smoke from getting outside. the same case was noted in previous measurement scenarios. additionally, like in other cases, also in those conditions, the opening of the door and passage of a person through that door resulted in a small discharge of smoke plume. initial pressure difference generated at the level of 50 pa caused a complete blockage of smoke transfer even when a person was passing through the door, not only when the door was partly open. during the measurements conducted with the door directed towards a positive pressure zone (fig 11) a slightly bigger smoke plume discharge was noted in comparison to the corresponding measurements for the door swing towards negative pressure. pressurization at the level of 2.5 pa, 5.0 pa, irrespective of the width of the door swing, does not prevent migration of the smoke plume outside, which remains in a protected room after the door has been closed. it is only with the door left ajar that a slow withdrawal of smoke plum to a contaminated room was observed. a similar phenomenon was noted during the measurement series with pressure difference of 7.0 and 12.5 pa; the amount of smoke plume migrating outside was visibly smaller. in comparison to the measurements with lower pressurization, initial generation of pressure difference at 25 pa noticeably minimizes migration of smoke plume outside, irrespective of the width of the door swing and of a passage of a person through the door. only minute quantities of smoke migrate. opening of the door between the rooms of different pressure levelled pressure value on both sides, which was proved by the tests performed, and confirmed in some publications [21, 15, 22] . the studies showed that door swing outside the room towards a positively pressurized zone was more disadvantageous in comparison to the cases when the door was opened into the contaminated room (with negative pressure). with the door opened towards negative pressure, reduction of the amount of contamination to the level specified as moderate or small occurred when the pressure difference was at approx. 7 pa. that was the case for all the examined modes of door swing (s = 10 fast, s = 10 slow, s = 50, "escape" mode). similar reduction in the amount of contamination in the case of opening of the door towards positive pressure was observed only with the door swing at 12.5 pa, and even at 25 pa. this finding is confirmed by the studies of sansone and keimig [15] published earlier. the reason for such a phenomenon is the effect of drawing in contamination through the opposite to the leading side of the door opening towards the room with positive pressure and further pushing of the contamination into the room when the door was closed. in the case of door swing towards the negative pressure, the smoke was drawn in through the eddies that formed around the edge of travelling door, but the air passing through the door left ajar made the smoke withdraw. different observations were recorded by wiesman in [22] , where the author decided that it is more advantageous to open the door towards the positive pressure. however, his studies assumed only opening of the door in the required direction without closing the door afterwards, which did not take into account the door pumping effect. this effect has a considerable impact on contamination spreading. the studies performed showed that the amount of contamination depends on the velocity of contamination pumping. faster pumping made more contamination migrate. this was demonstrated by both measurement sessions for cases ii 12.5 and 50 pa carried out in variants s = 10 slow and s = 10 fast, and also by the comparison of the "escape" mode and s = 50 mode. in both cases longer time of door swing was connected with a smaller amount of contamination getting out. these observations confirm the conclusions put forward by sansone and keimig [15] showing that it is necessary to swing the door more slowly. door pumping presents a considerable difficulty for the effective protection against contamination migration. the tests show that it causes greater contaminants migration as compared to the door left ajar in stable conditions. the measurements performed showed that in order to protect the rooms against contaminants migration during door pumping, it is necessary to have an airflow ensuring velocity of approx. 0.6-1.0 m/s on the door left ajar. it is a three times greater value in comparison with the values recommended by the aiha [2] in the case of the door left ajar in stable conditions (0.254m/s). moreover, measurement results presented in the article indicate that contaminants migration depends on two factors: door pumping and transferring contaminants on the feet of the person passing through the door in the "escape" mode. however, with a lower pressure difference and with lower velocity values with the door left ajar, door pumping plays a much bigger role. with the pressure and velocity values growing with the door left ajar, the influence of door pumping decreases, while the contamination is transferred mainly on the person's feet. the presented studies, unlike the other ones which also examine the influence of pressure difference, included the "pumping" effect, i.e. transfer of contaminants by means of the door swing. the observations provided the grounds to conclude that the door effect can be a contributing factor to intensification of contaminants transfer, even in comparison to the cases when the door was left open. although, in terms of contamination, it is more advantageous to swing the door towards negative pressure of 2.5 pa, as shown in some publications, it is definitely not sufficient in order to protect the rooms if the door swings. door swinging towards negative pressure causes reduction in the contaminants transfer to the level specified as moderate or small already at the pressure of 7 pa. the same effect with the door opened in the opposite direction is obtained with pressure difference of 12.5 pa and even 25 pa. increase of pressure difference from 25 to 50 pa did not help to prevent migration with the door swing towards negative pressure. moreover, it was shown that quick door swing causes a greater transfer of contaminants regardless of the width of the door opening, which shows that it is necessary to open the door more slowly. in order to protect the rooms against contaminants transfer during door pumping it is necessary to have the airflow which guarantees velocity of approx. 0.6-1.0 m/s on the door left ajar. contaminants migration is also influenced by their 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formulations for air infiltration calculations key: cord-000959-nk2thkme authors: downer, eric j.; jones, raasay s.; mcdonald, claire l.; greco, eleonora; brennan, sabina; connor, thomas j.; robertson, ian h.; lynch, marina a. title: identifying early inflammatory changes in monocyte-derived macrophages from a population with iq-discrepant episodic memory date: 2013-05-06 journal: plos one doi: 10.1371/journal.pone.0063194 sha: doc_id: 959 cord_uid: nk2thkme background: cells of the innate immune system including monocytes and macrophages are the first line of defence against infections and are critical regulators of the inflammatory response. these cells express toll-like receptors (tlrs), innate immune receptors which govern tailored inflammatory gene expression patterns. monocytes, which produce pro-inflammatory mediators, are readily recruited to the central nervous system (cns) in neurodegenerative diseases. methods: this study explored the expression of receptors (cd11b, tlr2 and tlr4) on circulating monocyte-derived macrophages (mdms) and peripheral blood mononuclear cells (pbmcs) isolated from healthy elderly adults who we classified as either iq memory-consistent (high-performing, hp) or iq memory-discrepant (low-performing, lp). results: the expression of cd11b, tlr4 and tlr2 was increased in mdms from the lp group when compared to hp cohort. mdms from both groups responded robustly to treatment with the tlr4 activator, lipopolysaccharide (lps), in terms of cytokine production. significantly, mdms from the lp group displayed hypersensitivity to lps exposure. interpretation: overall these findings define differential receptor expression and cytokine profiles that occur in mdms derived from a cohort of iq memory-discrepant individuals. these changes are indicative of inflammation and may be involved in the prodromal processes leading to the development of neurodegenerative disease. neuroinflammatory changes develop with age and are a feature of most neurodegenerative diseases including alzheimer's disease (ad). epidemiological studies indicate that long-term nonsteroidal anti-inflammatory treatment reduces the risk of developing ad [1] suggesting that inflammatory processes may contribute to very early changes in the pathogenesis of the disease. the inflammatory changes in the central nervous system (cns) in normal ageing and ad are characterised by microglial activation with resultant changes in microglial phagocytic activity and neurotoxic consequences [2] [3] [4] ; the evidence indicates that some of these cns changes have systemic parallels. the number of blood-derived monocytes is increased in ad patients [5] , and it has been reported that circulating monocytes express specific cell adhesion molecules (cams) that are altered with age, and in patients with ad and mild cognitive impairment (mci) [6, 7] . interestingly, circulating monocytes readily infiltrate the brain in murine models of ad [8, 9] and evidence indicates that bloodborne monocytes can act as amyloid phagocytes and may be involved in amyloid-b (ab) clearance [10] . these findings, alongside others which show that monocytes, in addition to in vitro differentiated macrophages, express several ad-related genes [11] , suggest that circulating monocytes may be a valuable model system in assessing the role of microglial cells in the pathogenesis of neurodegenerative conditions. human monocytes express an array of cams and receptors that are crucial for cell-cell and cell-matrix interactions, transmigration and innate immune responses. of these, cd11b is a receptor for intercellular adhesion and is a phenotypic monocytic cell marker, the expression of which increases with age, indicating a more activated phenotype [6] . monocytes/macrophages also express a diverse array of toll-like receptors (tlrs), a family of signalling pattern recognition receptors that mediate innate immunity by recognising conserved motifs of microbial origin known as pathogen-associated molecular patterns (pamps) and endogenous damage-associated molecular patterns (damps) that are released by injured tissue [12] . human blood monocytes express tlr1, tlr2, tlr4, tlr5, tlr6, tlr8, and tlr9 mrna, with tlr2 and tlr4 being the most highly expressed [13] . tlrs continue to emerge as key players in cns diseases and much data link tlrs with ad pathology, with polymorphism in the tlr2 and tlr4 gene linked with the disease [14] . the classical monocyte marker, cd14, a glycosylphosphatidyl inositol (gpi)anchored myeloid glycoprotein, acts as a co-receptor for tlrs and is responsible for the uptake of gram-negative bacterial lipopolysaccharide (lps) via macropinocytosis [15] . indeed, tlr4 acts as the primary signalling receptor for lps [16] , with tlr2 further required for lps-induced tlr4 signalling [17] . in an attempt to quantify relative decline and explore discrepancies between targeted cognitive domains that are central to ageing (e.g. episodic memory) and a proxy measure of lifetime cognitive capacity, we have identified a group of otherwise healthy adults who we classify as low performers (lp) because their memory performance is discrepant with their estimated iq. the advantage of adopting such an approach is threefold. first, it allows quantification of true age-related changes in the strength or weakness of memory relative to an estimate of lifetime cognitive capacity. second, it allows one to dissociate domain-specific changes in memory function. third, it allows classification of individuals as high or low memory performers based on standardised measures of memory relative to iq that can be utilised, not only in research, but also in a clinical setting. the purpose of the study was to compare the expression of receptors (cd11b, tlr2 and tlr4) on circulating monocyte-derived macrophages (mdms) and the response of these cells to lps in samples prepared from the lp cohort and a cohort which we classified as iq memory-consistent (high-performing, hp) individuals. we show that mdms isolated from the lp group display enhanced expression of cd11b, tlr2 and tlr4, compared to the hp group. we further show that treatment of mdms with lps promoted pro-inflammatory cytokine production in hp and lp groups, but that the effect was exacerbated in mdms prepared from the lp group. adults (35 female, 12 male) with a mean age of 71.5 years (range of 65 to 82) were recruited from the older adult participant panel of the trinity college institute of neuroscience, dublin, ireland. participants were assigned to low and high performing sub-groups (lp and hp respectively) based on their memory performance relative to an estimate of their intelligence. z-scores were used to relate their performance on the wechsler memory scale story recall test [wms iii uk; 18] to their scores on the national adult reading test [nart ; 19] . participants were defined as lp if they scored more than 0.75 standard deviations below their nart-estimated iq on the wms. all other participants were classified as hp. this approach yielded 35 hps (26 female, 9 male) with a mean age of 71.9 (sd = 4.7) and 12 lps (9 female, 3 male) with a mean age of 71.1 (sd = 4.8). no significant difference was observed in mini mental state exam (mmse) scores between the two groups (table 1) . participants received reimbursement of travel expenses to the maximum value of j20. this study was approved by the ethics committee of the school of psychology at trinity college dublin, and all participants provided informed consent. all participants completed a detailed questionnaire about their own health and current medications, as well as any relevant health issues in their family. participants who had a history of head injury, stroke, epilepsy, neurological conditions, major psychiatric disorder, heart attack or diabetes were excluded from the study. a battery of tests was administered in one session, and focused on memory and executive function. the mmse was used as a screening measure. the nart was used as a proxy measure of general intellectual status. memory was assessed using 3 subtests of wechsler memory scale: logical memory i and ii verbal paired associates i and ii visual reproduction i and ii. human pbmcs were prepared from heparinised venous whole blood samples (50 ml per donor) from hp and lp individuals by density separation over lymphoprep tm (axis-shield, norway). plasma samples were separated following centrifugation, aliquoted and stored at -80uc. cd14 + monocytes were isolated from pbmcs using positive selection with macs microbeads (miltenyi biotec, germany) and an automacs cell sorting instrument using a method which results in up to 95% purity as estimated by flow cytometry [20] . the mean percentage monocytes in pbmcs from hp and lp groups was 20.561.0 and 18.4861.7, respectively (p = 0.289; 2-tailed unpaired t-test). in this study, cd14 + cells, obtained from pbmcs following macs sorting, were seeded (6610 5 cells/ml) on 24-well plates and cultured in rpmi supplemented with fbs (10%), antibiotics and human granulocyte macrophage colony-stimulating factor (10 ng/ml; r&d systems, uk) for 7 days. at the end of this period, cells were assessed again by flow cytometry; 90.2% of the cells were cd14 + ( figure s1 ), which is consistent with previous studies that have been shown to yield .85-92% cd14 + cells [20] [21] [22] . we refer to these cells as mdms in the present study. freshly-isolated pbmcs and mdms were washed 3 times in facs buffer (2% fbs, 0.1% nan 3 in pbs) and blocked for 20 min at 4uc with purified human igg (1 mg/ml in facs buffer; sigma, uk). cells were incubated with anti-human cd11b-apc (clone icrf44), anti-human tlr2-fitc (clone t2.5) and anti-human tlr4-pe-cy7 (clone hta125) (all at 1:100 in facs buffer; biosciences, uk). a minimum of 300,000 events were collected and the percentage positive staining for each cellular target was calculated. total viable pbmcs were initially gated via forward and side scatter (demonstrated in figure s2 ) and mdms were stimulated with lps (100 ng/ml) and after 24 h rna was extracted using a nucleospinh rnaii isolation kit (macherey-nagel inc., germany). the concentration of lps used is in line with those used in various inflammatory paradigms in human monocytes and macrophages [23] [24] [25] . the concentration of rna was determined using a uv/vis spectrophotometer (beckman coulter inc., ireland). cdna synthesis was performed on 1 mg rna using a high capacity cdna rt kit (applied biosystems, usa) according to the manufacturer's instructions. equal amounts of cdna were used for rt-pcr amplification. real-time pcr primers were delivered as ''taqmanh gene expression assays'' containing forward and reverse primers, and a fam-labeled mgb taqman probe for each gene (applied biosystems, usa). primers used were as follows: cd11b, tlr2 and tlr4 (taqmanh gene expression assay no. hs00355885_m1, hs00610101_m1 and hs00152937_m1, respectively). a 1 in 4 dilution of cdna was prepared and real-time pcr performed using an applied biosystems 7300 real-time pcr system. cdna was mixed with qpcr tm mastermix plus (applied biosystems, usa) and the respective gene assay in a 25 ml volume (10 ml of diluted cdna, 12.5 ml taqmanh universal pcr mastermix, 1.25 ml target primer and 1.25 ml gapdh). human gapdh was used as an endogenous control and expression was conducted using a gene expression assay containing forward and reverse primers and a vic-labeled mgb taqman probe (#4326317e; applied biosystems, usa). samples were run in duplicate and 40 cycles were run as follows: 10 min at 95uc and for each cycle, 15 s at 95uc and 1 min at 60uc. gene expression was calculated relative to the endogenous control and analysis was performed using the 2 2ddct method. in all experiments no change in relative gapdh mrna expression between treatment groups was observed. mdms were stimulated with lps (100 ng/ml; 24 h) and supernatants were assessed for tumor necrosis factor (tnf)a, il-6, il-10 and il-12p70 production using a human pro-inflammatory-7-plex assay (mesoscale discovery, usa) according to manufacturer's instructions. briefly, plates were blocked in diluent 2 for 30 min at room temperature and duplicate samples and standards (0-10,000 pg/ml) added for 2 h at room temperature with vigorous shaking. detection antibody solution was added for 2 h at room temperature with vigorous shaking. plates were washed again and read buffer was added to the plate. the plate was read immediately using a mesoscale sector imager plate reader and pro-inflammatory cytokine concentrations in test samples were evaluated with reference to the standard curve prepared using the 7-plex calibrator blend. plasma samples from hp and lp groups were analysed for concentrations of crp by elisa (duoset, r&d systems, uk) according to manufacturer's instructions. data were analysed using a student's t-test for independent means or two-way analysis of variance (anova) as appropriate. when analysis by anova indicated significance (p,0.05), the post hoc student newman-keuls test was used. data are expressed as means 6 standard errors of the mean (sem). human mdms isolated from the lp group display enhanced expression of cd11b, tlr2 and tlr4, compared to hps since neurological symptoms are often exacerbated by infection [26] , we initially assessed plasma levels of the inflammatory reactant crp in plasma from hp and lp groups. no significant difference in plasma crp concentration was found between groups ( figure 1a ). pbmcs predominantly constitute lymphocytes and monocytes, with lower numbers of natural killer (nk) and dendritic cells. cd11b is an adhesion molecule primarily expressed on the surface of monocytes, but also on activated lymphocytes and a subset of nk cells, where it mediates leukocyte adhesion and migration to orchestrate an inflammatory response [27] . indeed, cd11b expression is upregulated by many inflammatory mediators including cytokines [28] . cd11b expression was unchanged on pbmcs isolated from the hp and lp groups ( figure 1b , c, p = 0.45) and no difference in mdm cd14 mrna was observed between groups ( figure 1d ) suggesting that differentiation of monocytes to macrophages was similar in lp and hp individuals. however, the percentage of cd11b + cells in the mdm preparation was significantly greater in the lp group, compared with the hp group ( figure 1e , f, p,0.05) and the cd11b staining intensity was also higher on mdms from the lp group, compared with the hp group ( figure 1g , p,0.05). although cd11b mrna was also enhanced in mdms from the lp, compared with the hp, group the difference did not reach statistical significance ( figure 1h , p = 0.19). since macrophages and other cells of the innate immune system recognise distinct microbial products via tlrs, we next assessed the expression of tlr2 and tlr4 since they are specifically implicated in inflammatory changes associated with neurodegeneration [14, 29] . tlr2 expression on cd11b + cells was similar on pbmcs isolated from the hp and lp groups ( figure 1i , j) but expression in mdms from the lp group was higher than the hp group, although it did not reach statistical significance ( figure 1k , l, p = 0.056); mfi values for the hp and lp groups were 88.1 and 77.2 respectively. tlr2 mrna was significantly greater in mdms prepared from the lp group compared with the hp group (p,0.05; figure 1m ). tlr4 expression on cd11b + cells was similar on pbmcs isolated from the hp and lp groups ( figure 1n , o) but expression on mdms from the lp group was significantly greater than from the hp group (p,0.05; figure 1p , q); mfi values for the hp and lp groups were 37.1 and 28.3 respectively. tlr4 mrna was significantly greater in mdms prepared from the lp group compared with the hp group (p,0.05; figure 1r ). overall, this indicates that differentiated mdms, but not freshly prepared pbmcs, isolated from the lp cohort display elevated expression of cd11b and tlr2/4. since tlr4 acts as the primary signalling receptor for gramnegative bacterial lps [16] and because tlr4 expression was increased in mdms prepared from the lp cohort, we examined the effect of lps on cytokine production in mdms from hp and lp groups. production of the cytokines tnfa, il-6, il-10 and il-12p70 was similar in unstimulated mdms from hp and lp groups ( figure 2 ). however, lps significantly increased cytokine production in mdms from both groups, and importantly the production of tnfa (figure 2a ), il-6 ( figure 2b ), il-10 ( figure 2c ) and il-12p70 ( figure 2d ) was exacerbated in mdms prepared from the lp group, compared with the hp group (p,0.05). this indicates that mdms prepared from the lp group are hyper-responsive to lps, at least in terms of cytokine production, and the increase in tlr4 expression provides a plausible mechanism for this effect. we investigated the expression of tlr2 and 4, and the impact of in vitro treatment with lps on the production of cytokines in mdms prepared from an iq memory-discrepant lp cohort, compared with an iq memory-consistent hp cohort. the significant finding is that mdms from the lp group expressed higher levels of tlr2 and tlr4, as well as cd11b, and exhibited a more robust response to lps, than mdms from the hp group. ageing is associated with defects in both the innate and adaptive arms of the immune system. indeed, elderly individuals display reduced b cell production [30] and t cell memory [31] , ensuring greater susceptibility than young individuals to bacterial and viral infection. much data also indicate that ageing affects cells of the innate immune system, particularly associated with alterations in neutrophil [32] and monocyte/macrophage phagocytic capacity [33] , and ability to produce cytokines and chemokines [33] . overall, cumulative data indicates that defective innate immunity is associated with advancing age. intermediate stages between normal ageing and dementia are recognised by several classification systems; the best recognised are the several clinical subtypes of mci, a preclinical stage of ad [34] . the lack of diagnostic tools which enable early detection of conditions like mci and/or ad is a major stumbling block for treatment of disease and a method of detecting early changes in cognitive function relative to previous levels would be a significant advance. structural changes which are indicative of conversion from normal ageing to mci and ad have been identified using image analysis [35] . in addition, a number of peripheral blood-based biomarkers have been suggested, however the most compelling data have been obtained from analysis of cerebrospinal fluid (csf) in which the ratio of ab/ phosphorylated tau combined with total tau levels yielded a positive predictive value in terms of conversion from mci to ad [36] . myeloid cells, constituting blood-borne monocytes, tissue resident macrophages and parenchymal microglia, have defined functions in neurodegenerative diseases. monocytes and macrophages not only share the same origin with microglia in the brain, but also have the same antigens, functions, and regulatory mechanisms [37] . much research has focused on characterising the neurodegenerative role of microglial cells which accumulate at senile plaques in ad [9] , secreting proteolytic enzymes that degrade ab [38] and expressing receptors that promote the phagocytosis of ab [39] . monocytes and macrophages are professional phagocytic cells and central players in orchestrating the innate immune response. monocytes/macrophages have proinflammatory and cytotoxic properties, with the proclivity to produce inflammatory molecules such as tnfa and inducible nitric oxide synthase [37] . blood-borne monocytes are highly mobile and rapidly recruit to inflamed cns tissue during bacterial infection [40] , autoimmune disorders [41] and neurodegenerative disease [8] . indeed, circulating monocytes can infiltrate the cns parenchyma and differentiate into microglia [42] , where they are important for clearing amyloid and limiting its deposition in murine models of ad [43] . furthermore, macrophages display an activated phenotype in neurodegenerative disease, typified by increased expression of cell surface markers such as cd11b [44] , cns infiltration [45] and enhanced production of inflammatory cytokines [46] . here we show that expression of tlr4 was greater in mdms obtained from the lp cohort compared with the hp cohort and, consistently, that incubation of cells from the lp cohort responded more robustly to lps with significantly greater production of tnf, il-6 and il-12 than that produced by mdms from the hp group. mdms are a type i or classically activated (m1) macrophage that display a pro-inflammatory phenotype [47] . mdms are considered a peripheral counterpart of microglia, as they share the same progenitor and antigen markers, and they have similar biological behaviours that mirror microglial function in the brain [48, 49] . previous evidence has shown that they produce inflammatory cytokines in response to lps treatment [50] and that they express tlr4 [51] , which is confirmed here. interestingly monocytes from older, compared with younger, individuals have higher intracellular levels of tnf both at baseline figure 1 . enhanced cd11b, tlr2 and tlr4 expression in mdms from lps. a) plasma isolated from hp and lp groups displayed comparable levels of crp. data are presented as mean 6 sem and represent triplicate determinations from 35 and 12 samples per hp and lp groups, respectively. b) cd11b expression on pbmcs was unchanged between hp and lp subjects. c) representative plots of cd11b + cells in pbmc populations. numbers beside gated areas indicate the percentage positive cells in that area. d) cd14 expression in mdms was similar in lp and hp individuals following 7 days in culture. e) percentage cd11b expression was increased in mdms derived from the lp group, compared with the hp group (p,0.05). f) representative dot plots of cd11b + cells in mdms (with values beside gated areas indicating the percentage positive cells in that area) show marked differences between cohorts. g) cd11b mean fluorescence intensity on mdms was increased in the lp group compared with the hp group (p,0.05). h) cd11b mrna in mdms derived from the lp group was slightly, though not significantly, greater than values from the hp group (p = 0.19). i) tlr2 expression on cd11b + pbmcs was similar in hp and lp groups and this is also shown in the representative dot plots of tlr2 + cells (j) which indicate the percentage positive cells. k) tlr2 expression on cd11b + mdms was increased in the lp group, compared to the hp group (p = 0.056) and this is also shown in the representative dot plots of tlr2 + cells (l) which indicate the percentage positive cells. m) tlr2 mrna expression was increased on mdms derived from the lp group compared with the hp group (p,0.05). n) tlr4 expression on cd11b + pbmcs was similar in hp and lp groups and this is also shown in the representative dot plots of tlr4 + cells (o) which indicate the percentage positive cells. p) tlr4 expression on cd11b + mdms was increased in the lp group compared with the hp group (p,0.05) and this is also shown in the representative dot plots of tlr4 + cells ( and following lps stimulation [6] , while pro-inflammatory cytokine [52] and chemokine [53, 54] levels are elevated in peripheral blood monocytes isolated from the elderly after lps stimulation. in addition, pbmcs from ad patients have been reported to produce enhanced levels of il-6 following lps stimulation compared with controls [55] . these findings suggest that monocyte function is dysregulated both with age and in ad, but the present study significantly extends these observations to show that similar changes can be picked up in mdms prepared from a group of otherwise healthy adults whose memory performance is discrepant with their estimated iq. cd11b is a receptor for intercellular adhesion molecule family members cd54, cd102 and cd50, enabling leukocyte adhesion and migration to mediate the inflammatory response [27] . cd11b is also a typical monocyte/macrophage marker, the expression of which is increased following activation [44] . indeed, cd11b expression is increased in monocytes from aged individuals [6] and on microglial cells in aged animals [3] . furthermore the number of cd11b + activated microglia is increased in mouse models of ad [56] and ms [57] . the present data indicate that cd11b expression was enhanced in mdms, but not pbmcs, from the lp cohort, indicating that these cells displayed an enhanced activated phenotype. this cell type-specific effect indicates that altering the phenotype of human monocytes in vitro may provide a mechanism which identifies alterations of myeloid cell function in cohorts with the most subtle cognitive changes. it is of interest to note that modifications of macrophage phenotype have been suggested in conditions where clear pathology is identified, i.e. in chronic inflammatory conditions [58] , autoimmune disorders [59] and ad [60] . human monocytes express an array of tlrs [13] and our data indicate that both cd11b + pbmcs and mdms express tlr2 and tlr4. in the present study we focused on examining expression of tlr2 and tlr4 since much evidence indicates the role of these receptors in neurodegenerative conditions. monocytes derived from elderly individuals display defective tlr signalling, particularly tlr1/2, with further decreases in relative tlr1 and tlr4 expression determined on peripheral blood monocytes from older subjects [6, 61] . tlr2/4 2/2 mice are protected from cognitive impairment following ab immunisation [29] with polymorphism in the tlr2 and tlr4 gene linked with ad [14] . knockout studies suggest differential effects of tlrs on ad progression; deletion of cd14, a co-receptor for tlr2/4, reduces plaque burden in a murine ad model [62] , but deletion of myd88 enhances memory deficits [63] . importantly, evidence indicates that ab binds microglial tlr2 and 4 [64] suggesting that tlrs function as members of the ab receptor complex. our findings indicate that the expression of tlr2 and tlr4 is increased in mdms from the lp group, compared with the hp group. since increased tlr expression does not occur on monocytes in healthy elderly individuals [6] , while increased expression of tlr2 and tlr4 has been reported in pbmcs from patients with late-onset ad [65] , the data may suggest a role for increased tlr expression in progression from the mildest manifestation of episodic memory loss to more profound cognitive decline. our results identify changes in the expression of receptors cd11b, tlr2 and tlr4 on mdms isolated from a unique cohort of iq memory-discrepant older adults. consistent with the increased expression of tlr4, stimulation with lps promoted enhanced cytokine production in mdms from this lp group, compared with the hp iq memory-consistent group. it is important to note that bacterial and(or) viral infections are associated with reduced cognitive performance [26] , but no differences in plasma concentrations of crp were identified between the cohorts in this study. these peripheral cell changes, which are indicative of inflammation, may provide a biological substrate indicative of the earliest discernible changes in episodic memory decline which may be a precursor to more profound changes that are associated with the development of neurodegenerative disease. figure s1 flow cytometric analysis of mdms. flow cytometric analysis of mdms and selection of cd14 + cells. cd14 + monocytes were separated from pbmcs using a macs sorter. the cd14 + monocytes were cultured for 7 days with gm-csf (10 ng/ml), and stained with a cd14 antibody to determine purity by flow cytometry. gates were made using the isotype controls. after one week in culture 90.2% of cells were cd14 + . (tif) figure s2 flow cytometric analysis of pbmcs. a) flow cytometric analysis of pbmcs and selection of live cells. pbmc gate location determined via forward/side scatter analysis of pbmcs. b) using a logical gating strategy, live pbmcs gated for cd11b. 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n-cyano-n'-(2-{[8-(2,6-difluorophenyl)-4-(4-fluoro-2-methylphenyl)-7-oxo-7,8-dihy dropyrido[2,3-d] pyrimidin-2-yl]ami-no}ethyl)guanidine (sb706504) apolipoprotein a-i mimetic 4f alters the function of human monocyte-derived macrophages signal transduction in lps-activated aged and young monocytes cytokine production and aging: overproduction of il-8 in elderly males in response to lipopolysaccharide rantes and mip-1alpha production by t lymphocytes, monocytes and nk cells from nonagenarian subjects il-6 release by lps-stimulated peripheral blood mononuclear cells as a potential biomarker in alzheimer's disease modest amyloid deposition is associated with iron dysregulation, microglial activation, and oxidative stress )c]dac-pet for noninvasively monitoring neuroinflammation and immunosuppressive therapy efficacy in rat experimental autoimmune encephalomyelitis model macrophages in allergic asthma: fine-tuning their pro-and anti-inflammatory actions for disease resolution macrophage subpopulations in systemic lupus erythematosus a changing perspective on the role of neuroinflammation in alzheimer's disease age-associated defect in human tlr-1/2 function deletion of cd14 attenuates alzheimer's disease pathology by influencing the brain's inflammatory milieu myd88-adaptor protein acts as a preventive mechanism for memory deficits in a mouse model of alzheimer's disease cd14 and tolllike receptors 2 and 4 are required for fibrillar a{beta}-stimulated microglial activation increased expressions of tlr2 and tlr4 on peripheral blood mononuclear cells from patients with alzheimer's disease our co-author and colleague, tom connor, passed away on 26th february 2013; we would like to dedicate this publication to his memory.the authors gratefully acknowledge the support of science foundation ireland (07/in.1/b949) and atlantic philanthropies (neuroenhancement for independence in elder lives (niel). the authors would also like to thank l. penny for her assistance with phlebotomy, and b. moran for technical help with facs. conceived and designed the experiments: ejd rsj tjc ihr mal. performed the experiments: ejd rsj eg sb. analyzed the data: ejd rsj cm eg sb. wrote the paper: ejd. key: cord-000050-tfcerilc authors: rao, srinivas; kong, wing-pui; wei, chih-jen; yang, zhi-yong; nason, martha; styles, darrel; detolla, louis j.; sorrell, erin m.; song, haichen; wan, hongquan; ramirez-nieto, gloria c.; perez, daniel; nabel, gary j. title: multivalent ha dna vaccination protects against highly pathogenic h5n1 avian influenza infection in chickens and mice date: 2008-06-18 journal: plos one doi: 10.1371/journal.pone.0002432 sha: doc_id: 50 cord_uid: tfcerilc background: sustained outbreaks of highly pathogenic avian influenza (hpai) h5n1 in avian species increase the risk of reassortment and adaptation to humans. the ability to contain its spread in chickens would reduce this threat and help maintain the capacity for egg-based vaccine production. while vaccines offer the potential to control avian disease, a major concern of current vaccines is their potency and inability to protect against evolving avian influenza viruses. methodology / principal findings: the ability of dna vaccines encoding hemagglutinin (ha) proteins from different hpai h5n1 serotypes was evaluated for its ability to elicit neutralizing antibodies and to protect against homologous and heterologous hpai h5n1 strain challenge in mice and chickens after dna immunization by needle and syringe or with a pressure injection device. these vaccines elicited antibodies that neutralized multiple strains of hpai h5n1 when given in combinations containing up to 10 has. the response was dose-dependent, and breadth was determined by the choice of the influenza virus ha in the vaccine. monovalent and trivalent ha vaccines were tested first in mice and conferred protection against lethal h5n1 a/vietnam/1203/2004 challenge 68 weeks after vaccination. in chickens, protection was observed against heterologous strains of hpai h5n1 after vaccination with a trivalent h5 serotype dna vaccine with doses as low as 5 µg dna given twice either by intramuscular needle injection or with a needle-free device. conclusions/significance: dna vaccines offer a generic approach to influenza virus immunization applicable to multiple animal species. in addition, the ability to substitute plasmids encoding different strains enables rapid adaptation of the vaccine to newly evolving field isolates. the highly pathogenic h5n1 influenza virus causes lethal multi-organ disease in poultry, resulting in significant economic losses and a public health concern in many parts of the world. the greatest threats posed by this virus are its ability to cause mortality in humans, its potential to compromise food supplies, and its possible economic impacts. viral maintenance in poultry potentiates the risk of human-to-human transmission and the emergence of a pandemic strain through reassortment. an effective, safe poultry vaccine that elicits broadly protective immune responses to evolving flu strains would provide a countermeasure to reduce the likelihood of transmission of this virus from domestic birds to humans and simultaneously would protect commercial poultry operations and subsistence farmers. dna vaccines have been shown to elicit robust immune responses in various animal species, from mice to nonhuman primates [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] . in human trials, these vaccines elicit cellular and humoral immune responses against various infectious agents, including influenza, sars, siv and hiv. in addition to their ability to elicit antibody responses, they also stimulate antigenspecific and sustained t cell responses [1] [2] [3] 6, 12, 13] . dna vaccination has been used experimentally against various infectious agents in a variety of mammals, including cattle (against infectious bovine rhinotracheitis/bovine diarrhea virus, leptospirosis and mycobacteriosis) [14, 15] , pigs (against classical swine fever virus and mycoplasmosis) [16] , and horses (against west nile virus and rabies) [17] . in addition, dna vaccines have been tested against avian plasmodium infection in penguins [18] and against influenza and infectious bursal disease in chickens [7, 8, 19] , duck hepatitis b virus in ducks [6] , and avian metapneumovirus and chlamydia psittaci in turkeys [20, 21] (reviewed in ref. [22] ). while they have been used in chickens to generate antisera to specific influenza viruses and confer protection against the low pathogenicity h5n2 strain [23] , there is only one previous report of a monovalent dna vaccine effective against h5n1 (and that only against a matched h5n1 isolate) [24] ; no protection with multivalent dna vaccines against heterologous strains has been reported. development and characterization of a dna vaccine modality for use in poultry offers a potential countermeasure against hpai h5n1 avian influenza outbreaks. the virus can infect humans, typically from animal sources, including commercial and wild avian species, livestock, and possibly other non-domesticated animal species [25] [26] [27] . while there is marked diversity in the host range of type a influenza viruses, many experts have speculated that a pandemic strain of type a influenza could evolve in avian species or avian influenza viruses could contribute virulent genes to a pandemic strain through reassortment [28, 29] . thus, there is reason to consider vaccination of poultry that would stimulate potent and broad protective immune responses [7, 30, 31] . in undertaking such efforts, it is important that there be a differentiation of infected from vaccinated animals [32] so that animals can be protected and permit monitoring of new infections using proven and sensitive methodologies. in this study, we used an automated high capacity needle-free injection device, agro-jeth (medical international technology, inc., denver, co) to explore the feasibility of dna vaccination of poultry. after optimization of injection conditions, alternative multivalent dna vaccine regimens were analyzed and compared for magnitude and breadth of neutralizing antibodies, as well as protective efficacy after challenge in mouse and chicken models of hpai h5n1 infection. the findings suggest that it is possible to develop a multivalent dna vaccine for poultry that can protect against multiple hpai h5n1 strains and that could keep pace with the continued evolution of avian influenza viruses. immunogenicity and neutralizing antibody specificity of alternative ha dna vaccines in mice to evaluate the efficacy of multivalent dna vaccines, initial studies were performed in mice. expression vectors encoding has from ten phylogenetically diverse strains of influenza viruses [33] were generated by synthesis of cdnas (see materials and methods) in plasmid expression vectors, pcmv/r or pcmv/r 8kb, which mediates high level expression and immunogenicity in vivo [34, 35, 36] . animals were immunized with each expression vector intramuscularly (im) at three week intervals, and the antisera were evaluated on day 14 after the third immunization for their ability to neutralize hpai h5n1 pseudotyped lentiviral vectors as previously described [35, 36] . we have previously shown that lentiviral assay inhibition (lai) yields similar results to microneutralization and hai analyses with higher sensitivity in mice [35, 36] to determine whether immunization with multiple has simultaneously could expand the breadth of the neutralizing antibody response without significant loss of magnitude, a combination of 10 ha dna vaccine immunogens was administered im at proportionally lower concentration (1.5 mg per immunogen) into groups of 10 mice (see materials and methods). remarkably, despite a log lower dna concentration of each component, significant neutralizing antibody titers were generated to each of the 10 immunogens, with .80% neutralization against 6 out of 12 h5 ha pseudoviruses at dilutions of up to 1:400 ( fig. 2a) . to evaluate whether similar breadth of immunity could be generated with fewer immunogens, two different combinations of 5 immunogens were selected, based on the phylogenetic diversity of ha among the avian influenza viruses [33] and the crossreactivity of the neutralizing antibody responses of select individual immunogens (fig. 1 ). as expected, there were substantial differences in the breadth of neutralization between these two sets of 5 immunogen multivalent vaccines (fig. 2 , b vs. c). in one set, while neutralization of homologous strains was comparable to the monovalent and the 10 immunogen multivalent immune response, fewer cross-reactive antibodies were detected, directed most prominently against a/iraq protection of dna-vaccinated mice against challenge with heterologous h5n1 a/vietnam/1203/2004 influenza virus mice immunized as described above were challenged with a heterologous h5n1 virus 68 weeks after the final dna vaccination. animals were then challenged with 10 ld 50 of the highly pathogenic a/vietnam/1203/2004 virus intranasally, and morbidity and mortality were monitored for 21 days after the viral challenge. the control animals, injected with the plasmid expression vector with no insert, died within 10 days of infection. complete survival was observed in the groups immunized with the 10 component and set 2 of the 5 component multivalent dna vaccines (fig. 3) . immunization with ha derived from the a/ indonesia/05/2005 strain or set 1 of the 5 component multivalent dna vaccine showed a survival rate approaching 90%. in contrast, animals injected with ha plasmid dna derived from a/ anhui/1/2005, which has diverged more from a/vietnam/ 1203/2004, showed a lower percent survival (70%) after lethal viral challenge. survival differences between groups were assessed using a log-rank test and the gehan-wilcoxon test on the survival curves for pairs of groups. a test was deemed significant if the pvalue was ,0.01. mice injected im with different has, a/ indonesia/5/05, a/anhui/1/05, 10ha, 5 ha (set 1), or 5 ha (set 2) showed a significant difference compared to control (all p values,0.001). among the ha-immunized groups, there was no significant difference between any two groups (p.0.08 for all comparisons). for example, no significant difference was observed between the a/anhui/1/05 group, which had the least survival among the ha immunized groups (7 out of 10), and other ha groups: a/indonesia/5/05 (p = 0.377), 10 ha (p = 0.082), 5 ha (set 1) (p = 0.101), or 5 ha (set 2) (p = .411). therefore, we cannot exclude the possibility that the 3 deaths in the a/anhui/1/05 group may have been due to random chance. since it is desirable to confer protective immunity in poultry and ha dna vaccination was effective in mice, we next examined the breadth and potency of single or multiple ha plasmid immunization in chickens. the ability of chickens to generate specific antibodies was assessed with three strains that showed broad cross protection in mouse studies (a/vietnam/1203/2004, a/anhui/ 1/2005 and a/indonesia/05/2005), administered individually or in combination, by different injection methods. in addition to needle injection, a needle-free repetitive injection device, agro-jeth (medical international technology, inc., denver, co), was analyzed. this device disperses the 0.1 to 5 ml injection doses into the dermal, subcutaneous, or intramuscular tissue depending upon the pressure adjustments, powered by a co 2 gas pressure plunger [39] . the injection conditions were determined by histologic analysis of tissues that received injections of india ink; a pressure of 48 psi was chosen since it enabled consistent delivery into intradermal and subcutaneous tissues (fig. s1 ). immunization of chickens with the control plasmid (cmv/r) without an ha gene insert elicited minimal neutralizing antibody titers compared to ha-immunized animals 1 week after 3 dna immunizations. nearly all chickens immunized with either monovalent or multivalent ha dna vaccines generated significant neutralization titers ( fig. 4 and table s1 ). in general, there was a progressive increase in the amount of neutralization after each successive dna vaccination (data not shown) with maximal response at 1 week after the 3 rd dna immunization, with highest and most consistent levels in the trivalent vaccine group delivered with the agro-jeth device. neutralization of the indonesia ha strain was the most robust, with neutralization nearing 100% at titers greater than 1:3200. both the monovalent and multivalent vaccines elicited robust homologous ( vaccine (fig. 4 ). even though one chicken (238) in the multivalent vaccine group produced almost the same degree of neutralization at each time point and was protected, it did not produce a high neutralizing antibody titer for reasons that were uncertain but possibly related to a non-specific inhibitor in the sera. to determine whether chickens immunized with single or multiple dna vaccines were protected from a lethal challenge of a heterologous hpai h5n1 virus, vaccinated chickens were in panels b and c, mice (n = 10) were immunized with 15 mg of plasmid (3 mg each) three times at 3 week intervals. serum pools from the immunized animals were collected 14 days after the third immunization. the antisera were tested against the 12 indicated pseudotyped lentiviral vectors at varying dilutions. error bars at each point indicate the standard deviation; each sample was evaluated in triplicate. in general, the immunized serum neutralized all tested pseudotyped lentiviruses at low dilutions while differences were often observed at high dilution. doi:10.1371/journal.pone.0002432.g002 inoculated with 20 ld 50 of highly pathogenic a/vietnam/1203/ 2004 heterologous virus intranasally using standard methods [25, 40] and monitored for morbidity, mortality, viral shedding and serum antibodies. while all the control animals died within 2 days of infection, 100% survival was noted in the rest of the chickens (fig. 5a ). the animals that were healthy, showing no signs of clinical disease or malaise, were euthanized on day 14. there was no evidence for viral shedding monitored via tracheal and cloacal swabs of infected chickens 2-14 days after challenge as determined by embryonal inoculation (data not shown: egg infectious dose 50 (eid 50 ) limit of detection ,100 virus particles). to compare the relative efficacy of dna vaccines delivered im by needle and syringe versus the needle-free agro-jeth device injection, a dose-response study was performed with amounts of dna vaccine ranging from 500 to 0.5 mg with two inoculations. in these experiments, the ha derived from a/chicken/nigeria/641/ 2006 was substituted for a/vietnam/1203/2004 since it represented a more contemporary isolate. the observed rate of protection was higher among the animals receiving 5 mg by agro-jet (8/8) than by im injection (6/8) (fig. 5, b vs. c). both modes provided complete protection for all animals at doses higher than this, and 25% protection for the animals receiving 0.5 mg doses (fig. 5b, c) . survival differences between consecutive doses were assessed using a log-rank test on the survival curves for pairs of groups. a test was deemed significant if the p-value was ,.01, and marginally significant if the p-value was ,.05 but ..01. chickens injected im showed a marginally significant difference between 0.5 and 5 mg (p = .047). in the same group there was a significant difference between control and 5, 50 and 500 mg (p,.001 for all comparisons) and the difference between control and 0.5 mg was marginally significant (p = .016). chickens that were injected using agro-jeth showed a significant difference between 0.5 and 5 mg (p = .004) and between control and 5, 50, and 500 mg (p,.001 for all comparisons). there were no differences between control and 0.5 mg or between 5, 50, and 500 mg. lastly, the survival differences between agro-jeth and im for each dose group were not significant. the neutralizing antibody response to homologous and heterologous has corresponded with protection and correlated with dose, with higher titers elicited by injection with agro-jeth compared to needle (table s2) . we assessed viable viral shedding after inoculation by chick embryo inoculation three days after virus challenge (week 8). while we noted some embryonic lethality at the 0.5 mg dose, there was no embryonic lethality at 5, 50 or 500 mg groups (data not shown). since the hpai h5n1 virus first appeared ten years ago, this highly pathogenic avian influenza virus has shown increasing diversification and dissemination in asia, africa, and europe [28, [41] [42] [43] [44] . in addition to its effects on human health by crossspecies transmission [28, 45, 46] and ability to compromise food sources, it poses a continuing threat to public health as it evolves and adapts in different species. the pandemic potential of this virus, especially as it relates to the poultry industry and for reservoir avian hosts, underscores the need for a vaccine that offers broad spectrum immunity and protection against lethal viral challenge. while the virus remains restricted in its ability to infect humans and undergo efficient human-to-human transmission [28, 47] , its persistence and spread in poultry increases the risk of the emergence of a pandemic strain. one approach to pandemic risk reduction is to limit the propagation of the virus in poultry and other relevant avian species. we have previously reported that dna vaccines encoding ha can confer protection against a highly lethal human pandemic influenza virus, the 1918 h1n1 virus, in mice [36] . dna vaccines offer several advantages, including the ability to express diverse antigens, tolerability in various hosts, ease of delivery, and stability for storage and distribution without the necessity of maintaining a cold chain; they have been shown to be safe and efficacious in a variety of animal models [2, 4, 12, 22, 48] . because they do not contain other viral proteins used to screen for infection, they also address the need to differentiate vaccinated from infected animals. there is evidence that dna vaccination elicits cell-mediated immunity against influenza ha in addition to inducing an antibody response [36] , an effect that could significantly contribute to protective immunity as viruses show genetic drift and reduced susceptibility to neutralization. ideally, a highly effective influenza vaccine should not only be able to let the host develop a protective immune response against a matching live virus challenge but also elicit robust protective immune responses against a broad range of homologous and heterologous h5 influenza strains. a multivalent h5 vaccine containing diverse serotypes could expand the antigenic breadth sufficiently to provide protection against heterologous challenge and may preclude the emergence of vaccine-resistant strains that may arise due to evolutionary vaccine pressure on the virus. due to the antigenic drift and shift of the influenza virus genome, it has been very difficult to predict the next dominant strain of an avian endemic outbreak. dna vaccines can be synthesized in a relatively short period of time, and the targeted mutations can be tailored to specific viral serotypes. the mutations promote a focused and enhanced immune response [3, 49, 50] that may be particularly important in the event of an outbreak where specificity is the key to epidemic control. the use of modified codons ensures maximal expression in the host and eliminates the possibility of recombination with influenza viruses that might potentially generate new strains. a more broadly protective murine vaccine was developed here by including more has from varying strains in the multivalent vaccine (figs. 2 and 3) . however, it is less practical to include large numbers of different has in one vaccine due to the cost and complexity of manufacturing such a vaccine. therefore, we simplified the vaccine regimen based on cross-neutralization studies and phylogenetic relationships. a trivalent vaccine was subsequently identified for further studies. due [51] . while three dna immunizations were used initially to demonstrate protective immunity and have been used previously to elicit protection in mice [36] , we found that effective protective immunity could be induced with two dna vaccinations and as little as 5 mg trivalent dna immunization using the id/sc route with the agro-jeth device. in addition, based on the chick embryo inoculation data, we believe that there is effective neutralization of the virus and lack of infectious viral shedding in chicken vaccinated with as little as 5 mg of dna. the device's capacity for rapid repetitive injection and the lower quantity and stability of dna enhance the practicality and utility of this approach for vaccination of endangered species in captivity or administration to poultry or other animals. a/vietnam/1203/2004 (h5n1) (a/vn/1203/04) was obtained from the repository at the centers for disease control and prevention (cdc), atlanta, georgia. the virus was propagated in 10-day old embryonated chicken eggs at 35uc and stored at 270uc until use. the virus was titrated by the reed and muench method to determine eid 50 [52] . genbank abd28180) were synthesized using human-preferred codons (geneart, regensburg, germany) [36] . ha cdnas from diverse strains of influenza viruses were then inserted into plasmid expression vectors, pcmv/r or pcmv/r 8kb, which mediates high level expression and immunogenicity in vivo [34, 35, 36] . for initial trivalent immunizations in chickens, the a/vietnam/1203/ 2004, a/anhui/1/2005 and a/indonesia/05/2005 strains were used and in the dose response study, the vietnam strain was replaced with a/chicken/nigeria/641/2006. the immunogens used in dna vaccination contained a cleavage site mutation (pqrerrrkkrg to pqretrg) as previously described [35, 36] . this mutation was generated by site-directed mutagenesis using a quickchange kit (stratagene, la jolla, ca). dna immunization of mice [6] [7] [8] week old female balb/c mice were purchased from the jackson laboratory and maintained in the aaalac-accredited vaccine research center animal care facility (bethesda, md) under specific pathogen-free conditions. all experiments were approved by the vaccine research center animal care and use committee. the mice were immunized as previously described [5] . briefly, mice (10 animals for all test groups, 20 animals for the the study was carried out in the aaalac-accredited animal facility at the university of maryland school of medicine. six groups of 8 one-day-old male and female spafas white leghorn chickens, gallus domesticus, were obtained from charles river laboratories (connecticut). the animals were housed in brooder and grower cages (mcmurray hatcheries, iowa). feed (teklad japanese quail diet -3050, harlan-teklad, wi) and water were provided to the animals ad libitum. the study was performed in strict accordance with the ''guide'' after approvals from the animal care and use committees of the vaccine research center, nih and the university of maryland. dna immunizations were performed as described at 0, 3 and 6 weeks. a total dose of 500 mg of one or a combination of the following dna plasmids in a volume of 250 ml was administered to each animal: pcmv/ r, pcmv/r-ha agro-jeth is a needle-free device used for mass delivery of vaccines and drugs in livestock and poultry. the device is semiautomatic and requires a small co 2 tank or compressed air for low pressure delivery. upon trigger activation, co 2 disperses the injectate at a precise dose into the muscle, dermis or subcutaneous tissue depending on the setting that was standardized for our use. we used an effective volume of 0.1 ml in our injectate [39] . in this study we were able to effectively deliver 0.1 ml of injectate into the animal's dermis/subcutaneous tissue at a pressure of 48-55 psi. sixty-eight weeks after the last immunization, female balb/c mice were lightly anesthetized with ketamine/xylazine and inoculated intranasally with 10 ld 50 of a/vietnam/1203/2004 virus diluted in phosphate-buffered saline in a 50 ul volume. mice were monitored daily for morbidity and measured for weight loss and mortality for 21 days post infection. any mouse that had lost more than 25% of its body weight was euthanized. all experiments involving the hpai virus were conducted in an aaalac accredited facility (bioqual inc., gaithersburg, md) under bsl 3 conditions that included enhancements required by the usda and the select agent program. white leghorn chickens were challenged one week after the last immunization with 20 lethal dose 50 (ld 50 ) of a/vietnam/1203/04 (h5n1) influenza a virus, equivalent to 2610 4 eid 50 based on previous challenges [53] . chickens were infected with 200 ml virus intranasally. tracheal and cloacal swabs were collected days 3 and 5 post-challenge and stored in glass vials containing bhi medium (bbl tm brain heart infusion, becton dickinson) at 280uc. blood was collected 14 days post-challenge and serum was titered by microneutralization assay. chickens were observed and scored daily for clinical signs of infection, morbidity and mortality. chickens that survived the study were bled and humanely euthanized at day 14 post-challenge. lungs, heart, intestine and kidney were collected and samples were stored in formalin for histopathology. experiments were carried out under bsl3+ conditions with investigators wearing appropriate protective equipment and compliant with all institutional animal care and use committee-approved protocols and under animal welfare act regulations at the university of maryland, college park, maryland. representative tracheal and cloacal swabs were chosen to run an eid 50 assay for comparison and virus titers were determine by the method of reed and meunch [52] . briefly, swabs were used to infect 10 day-old embryonated chicken eggs in 10-fold dilutions. three eggs were inoculated per dilution and incubated for 48 hours before titration. neutralizing antibodies were titrated from serum samples collected week 5 and 7 post-vaccination and day 14 post-challenge. the microneutralization assay was performed using a 96-well plate format. serum was treated with receptor-destroying enzyme (denka seiken co.) and treated at 37uc per the manufacturer's instructions. after an overnight incubation and subsequent inactivation samples were brought to a final dilution of 1:10 using pbs and each sample was serially diluted and virus, diluted to 100 tcid 50 , was added to each well. the plates were then incubated at 37uc, 5% co 2 for 1-2 hours. following incubation, supernatants were used to infect a second 96-well plate of mdck cells. microplates were incubated at 4uc for 15 minutes and then 37uc, 5% co 2 for 45 minutes. supernatants of serum and virus were then discarded and 200 ml of optimem (containing 1x antibiotics/antimycotics, 1 mg/ml tpck-trypsin) was added and incubated at 37uc, 5% co 2 for 3 days. after 3 days, 50 ml of the supernatant from each well was transferred into a new 96-well microplate, and an ha assay was performed to calculate the antibody titers. virus and cell controls were included in the assay. two-fold dilutions of heat-inactivated sera were tested in a microneutralization assay as previously described [54] for the presence of antibodies that neutralized the infectivity of 100 tcid 50 (50% tissue culture infectious dose) of the a/vietnam/ 1203/2004 h5n1 virus on mdck cell monolayers by using two wells per dilution on a 96-well plate. the recombinant lentiviral vectors expressing a luciferase reporter gene were produced as previously described [35, 36] . for the neutralization assay, antisera from immunized animals were heat-inactivated at 55uc for 30 minutes and mixed with 50 ml of pseudovirus at various dilutions. the sera/virus mixture was then added to 293a cells in 96-well b&w tc isoplates (wallac, turku, finland; 12,000 cells/well). two hours later, the plates were washed and fresh medium was added. cells were lysed in mammalian cell lysis buffer (promega, madison, wi) 24 hrs after infection and luciferase activity was measured using the luciferase assay system (promega, madison, wi). the following strains were used for the production of pseudotyped viruses: for ha we used a/thailand/1(kan the ha/hi titers were determined as previously described [54] . briefly, ha titers were calculated using 50 ml of 0.5% chicken red blood cell suspension in pbs added to 50 ml of twofold dilutions of virus in pbs. this mix was incubated at room temperature for 30 minutes. the ha titers were calculated as the reciprocal value of the highest dilution that caused complete hemagglutination. hi titers were calculated by titrating 50 ml of antiserum treated with receptor-destroying enzyme and an equivalent amount of a/vietnam/1203/2004 virus (four hemagglutinating doses) was added to each well. wells were incubated at room temperature for 30 minutes and 50 ml of a 0.5% suspension of chicken red blood cells was added. hi titers were calculated after 30 minutes as the reciprocal of the serum dilution that inhibited hemagglutination. table s1 hemagglutination inhibition (hi), microneutralization titer (nt), and lai of sera from individual chickens immunized with different vaccines. sera from immunized animals were obtained at week 5 or 7, a week before or after the final boost, and neutralization was assessed by hi, microneutralization (nt) and lai (shown as ic 50 ). individual animal serum of each group is shown and was analyzed as described in the materials and methods section. figure s1 characterization of needle-free (agro-jeth) dna immunization in chickens. to evaluate the distribution of fluid into superficial or deep layers of subcutaneous tissues after delivery by agrojeth, 4 or 7 week old chickens were injected with a solution containing india ink with this needle-free device at various pressures, ranging from 45 to 55 mm hg. three sites (thigh, wing and breast) were used, and biopsies were taken for routine hematoxylin and eosin staining. representative sections of thigh injections are shown from 7 week old chickens and were similar at 4 weeks (data not shown). while the 48 mm hg pressure deposited the injectate into the dermis/subcutaneous region (left), the higher pressure injections, 52 and 58 mm hg, deposited the injectate into the subcutaneous and muscle layers (middle, right). 48 mm hg consistently provided an optimal pressure to deposit the injectate into the dermis and subcutaneous tissue and was chosen for all agrojeth immunizations. found at: doi:10.1371/journal.pone.0002432.s003 (10.74 mb doc) the 10 plasmid combination group (10 ha) received 1.5 mg dna for each of the 10 ha plasmids (total 15 mg) as used in the single plasmid groups mentioned above. for the two 5 plasmid combination groups pcmv/r 8kb-ha biological features of genetic immunization dna vaccines dna vaccines: immunology, application, and optimization strategies for inducing protection against avian influenza a virus subtypes with dna vaccines a dna vaccine induces sars coronavirus neutralization and protective immunity in mice immunotargeting with 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influenza a/duck/singapore/3/97(h5) vaccines against infection with a/vietnam/1203/04(h5n1) virus in ferrets vesicular stomatitis virus vectors expressing avian influenza h5 ha induce cross-neutralizing antibodies and long-term protection efficacy of the agro-jet mit-ii needle-less jet injector for iron dextran administration in piglets molecular determinants within the surface proteins involved in the pathogenicity of h5n1 influenza viruses in chickens are we ready for pandemic influenza? breakthrough of the year. avian influenza: catastrophe waiting in the wings the threat of an avian influenza pandemic avian influenza and pandemics-research needs and opportunities public health. public health risk from the avian h5n1 influenza epidemic host range restriction and pathogenicity in the context of influenza pandemic probable person-to-person transmission of avian influenza a (h5n1) comparative evaluation of three different intramuscular delivery methods for dna immunization in a nonhuman primate animal model hiv-1 dna vaccines amino acid 226 in the hemagglutinin of h9n2 influenza viruses determines cell tropism and replication in human airway epithelial cells ab and t cell epitopes of influenza a virus, knowledge and opportunities a simple method of estimating fifty percent endpoints a new generation of modified liveattenuated avian influenza viruses using a two-strategy combination as potential vaccine candidates eight-plasmid system for rapid generation of influenza virus vaccines replication and transmission of influenza viruses in japanese quail we thank ati tislerics, tina suhana, and catherine murray for help with manuscript preparation, michael cichanowski and brenda hartman for figure preparation, alida ault, john paul todd and vi dang for technical assistance, and members of the nabel lab for helpful advice and discussions. we are grateful to louis detolla, aruna panda, elisa luna and their staff for housing, care, and management of the chickens in their aaalac-accredited facility at university of maryland medical school, baltimore. additionally we would like to thank the influenza genome project of the nih and the depositors of the sequences in genbank. key: cord-000851-uylgyhs8 authors: wang, zhenya; chai, weidong; burwinkel, michael; twardziok, sven; wrede, paul; palissa, christiane; esch, bettina; schmidt, michael f. g. title: inhibitory influence of enterococcus faecium on the propagation of swine influenza a virus in vitro date: 2013-01-07 journal: plos one doi: 10.1371/journal.pone.0053043 sha: doc_id: 851 cord_uid: uylgyhs8 the control of infectious diseases such as swine influenza viruses (swiv) plays an important role in food production both from the animal health and from the public health point of view. probiotic microorganisms and other health improving food supplements have been given increasing attention in recent years, but, no information on the effects of probiotics on swine influenza virus is available. here we address this question by assessing the inhibitory potential of the probiotic enterococcus faecium ncimb 10415 (e. faecium) on the replication of two porcine strains of influenza virus (h1n1 and h3n2 strain) in a continuous porcine macrophage cell line (3d4/21) and in mdbk cells. cell cultures were treated with e. faecium at the non-toxic concentration of 1×10(6) cfu/ml in growth medium for 60 to 90 min before, during and after swiv infection. after further incubation of cultures in probiotic-free growth medium, cell viability and virus propagation were determined at 48 h or 96 h post infection. the results obtained reveal an almost complete recovery of viability of swiv infected cells and an inhibition of virus multiplication by up to four log units in the e. faecium treated cells. in both 3d4/21and mdbk-cells a 60 min treatment with e. faecium stimulated nitric oxide (no) release which is in line with published evidence for an antiviral function of no. furthermore, e. faecium caused a modified cellular expression of selected mediators of defence in 3d4-cells: while the expression of tnf-α, tlr-3 and il-6 were decreased in the swiv-infected and probiotic treated cells, il-10 was found to be increased. since we obtained experimental evidence for the direct adsorptive trapping of swiv through e. faecium, this probiotic microorganism inhibits influenza viruses by at least two mechanisms, direct physical interaction and strengthening of innate defence at the cellular level. swine influenza is an infectious disease caused by rna viruses which are highly contagious. multiple strains of this virus are common throughout pig populations worldwide and cause significant economic losses in industry. at least three swiv subtypes h1n1, h1n2 and h3n2 are currently circulating in the swine population despite regular vaccinations, and exchange of influenza viruses between human and swine is common and not a one-way street [1, 2] . the traditional vaccine is less effective because it cannot possibly include all the strains actively infecting people in the world. therefore, therapeutic alternatives for preventing infections and maintaining the health of livestock are highly warranted. probiotics are defined as live microorganisms which when administered in adequate amounts confer a health benefit on the ''host'' (fao/who, 2001) . although targeting the intestinal tract probiotics can also affect mucosal defence in general, including immune responses in the respiratory tissues [3, 4] . probiotic bacteria, as a part of gut microflora, are reported to promote the host defense and to modulate the immune system [5] . probiotics have been recently shown to mediate antiviral effects against certain viruses in vitro and in vivo [6, 7, 8, 9] and the effect of various strains of probiotics on the course of virus infections in pigs is being studied intensively. however, while some descriptive information on the effect of probiotics on model viruses such as vesicular stomatitis virus (vsv), transmissible gastroenteritis virus (tgev) and rotaviruses [6, 7, 8, 9] are available, no such data are yet available for swine influenza viruses which are most important in view of their exquisite zoonotic capacity. it is commonly believed that the mammalian influenza viruses are restricted to the respiratory tissue and thus may hardly be affected by probiotics acting in the intestine. however, a recent report on the pathogenesis of seasonal influenza virus h1n1 in ferrets shows that this virus is also present in the intestine [10] . furthermore it is world acknowledged that avian influenza viruses frequently infect in the intestine of the avian host [2] . therefore it appears justified to include influenza viruses in studies on the probiotic inhibition of virus multiplication both in vitro and in vivo. enterococcus faecium ncimb 10415 is authorized in the eu for safe use as a probiotic feed additive and therefore represents a suitable probiotic to study its possible anti-viral properties. we had previously carried out experiments with this probiotic in the context of bacterial infection which showed that e. faecium modulates intestinal immunity in piglets [11, 12] . in the present study we explored if e. faecium affects the replication of swine influenza virus h1n1 and h3n2 in a macrophage (3d4/21) and epithelial cell line (mdbk). assessing the effect of e. faecium on the viability of 3d4/ 21 and mdbk cells cytotoxicity of e. faecium on both 3d4/21 and mdbk cells is shown in fig. 1 . compared to control cells (100% cell survival rate), the application of e. faecium on the examined cell lines did not lead to any detrimental effects on cell integrity or metabolism unless the concentration exceeded the concentration of 1610 7 cfu/ml. as seen from the results compiled in fig. 1 , e. faecium at a concentration of 1610 8 cfu/ml had a severe cytotoxic effect especially for the macrophage cell line 3d4/21. under the same conditions a proportion of about 60% of the mdbk-cells still survived in the presence of this probiotic. based on these results, 1610 6 cfu/ml of e. faecium was applied for the interference studies described below. as expected from the above cytotoxicity study 1610 6 cfu/ml of e. faecium did not affect the viability of uninfected 3d4/21 and mdbk cells (fig. 2 , first two bars for each of the cell types). while swiv at 48 or 96 hpi had destroyed the cell monolayers completely (defined as 0% survival, not shown) each of the treatment modalities with the above concentration of e. faecium resulted in a protection of the cells from swiv infection. among these the setup ''competition'', where the probiotic bacteria and swiv-inoculum are added to the monolayers together for 60 min, resulted in an 80% protective effect for 3d4/21 and in a 70% protective effect on mdbk cells. but even a pretreatment of the cells with e. faecium and the addition of the probiotic after completion of swiv-infection both resulted in a significant rescue of the cells from ''death'' through the swiv-infection (grey and horizontally marked bars in fig. 2 ). these results were confirmed by another viability assay in which pi staining of dead cells is measured by flow cytometry (data not shown). the effect of e. faecium treatment on viral titers was validated by the tcid 50 assay. as shown in fig. 3 , the virus titer was decreased significantly after treatment of both types of host cells with e. faecium, but the degree of inhibition differed depending on whether the probiotic was present before, during or after infection with swiv. an up to 4 log 10 tcid 50 reduction was obtained when e. faecium and swiv were present on the monolayers simultaneously indicating that direct competition between swiv and the probiotic for presently unknown entities results in the most effective inhibition of virus production (see below) during the 48 h period before swiv was quantified in the growth medium. these results are in line with those from the cell viability assay of swiv-infected cells treated or non-treated with the probiotic shown in fig. 2 . qualitatively the probiotic induced inhibition of swiv appears to be the same with both types of host cells, but it appears to be somewhat more effective in the macrophage line 3d4/21. however, this could also be due to the lower swiv-titers reached in the non-treated mdbk-cells which was about one log-unit less than in the non-treated 3d4/21-cells. it is known from the literature that, beside multiple other functions, nitric oxide (no) is an important physiological messenger and effector molecule for antiviral effects. assessment of the secretion of no under the influence of e. faecium revealed a most significant stimulating effect for 3d4/21 cells. as shown in fig. 4 , e. faecium increased the production of no in both noninfected (bar on right side) and swiv-infected cells (first three bars). as with the results shown above, the strongest stimulation was reached by e. faecium added to the host cells simultaneously with the virus (''competition'', white bar). in mdbk-cells stimulation on no was much less pronounced. however the results shown in fig. 4 indicate the same tendency as for 3d4/21cells and are significant for the probiotic induced stimulation of no in non-infected cells [13] . it is possible that influenza virus particles could be engaged in direct physical interaction with the probiotic bacteria which may lead to a loss of infectivity. to address this question we included an experiment where virus particles were mixed with probiotic bacteria in a test tube and incubated for 1.5 h at room temperature (''preincubation''). after low speed centrifugation of the mixture to sediment e. faecium, the bacterium-free supernatants were titrated for infectious swiv in a tcid 50 assay. as seen from the data in table 1 , virus titers were reduced by about two logunits in both types of host cells. if virus was bound to bacterial cells during the preincubation period, it should be present in the bacterial sediment after the centrifugation step. to probe this possibility, the bacterial sediments were subjected to real timequantitative pcr in which primers for swiv m-protein were applied. this resulted in the finding that up to 50% of the input virus was detectable in the bacterial sediment (data not shown). as shown in table 1 , swiv in the supernatants from low speed centrifugation had lost infectivity significantly both on 3d4/21and mdbk-cells. however, when no was measured in the same cultures, an increase was recorded for both cell types. the observed inhibition of virus multiplication and stimulation of no in the experiments where the probiotic was added to the cells before swiv was in play or after the virus had entered the cells (fig. 1, fig. 3 ) indicate, that e. faecium may influence cellular factors which affect virus growth. we therefore analysed the 3d4/ 21 cell expression of cytokines (il-6, il-10, tnf-a, ifn-a) and tlr3 which are known for their potential to modulate virus production. the results from quantitative rt-pcr shown in fig. 5 reveal a decreased expression of these mediators when compared to the non-treated samples (swiv-infected 3d4/21-cells without e. faecium). on the other hand, the immunosuppressive cytokine il-10 showed a low expression at 2 h, but increased strongly at 6 h and 24 h in the probiotic-treated cultures (fig. 5 ). furthermore, e. faecium promoted an increased expression of ifn-a, at 2 h, 6 h and 24 h post swiv infection although without significance of the values for the virus group and e. faecium treated group, so this effect can only be regarded as a tendency. probiotic microorganisms have been mainly studied in the context of bacterial infections of the gastrointestinal tract which is the natural target tissue of probiotics. however, there are a few reports which indicate that upon oral intake, probiotics can also affect infections of the respiratory tract [14] . there are also reports in the literature where probiotics induce antiviral activity in vitro and are even applied as a medical treatment against persistent virus infections in humans and animals. we chose the zoonotic swine influenza viruses as a novel study object to test for the antiviral potential of the probiotic e. faecium and to elucidate its mechanisms of action and we present the results from in vitro experiments using porcine h1n1-and h3n2influenza virus in mdbk-and 3d4/21 cells, respectively. our results demonstrate that the probiotic e. faecium effectively protects host cells from swine influenza virus infection and are in support of the above author's hypothesis, that probiotics are not only useful to inhibit enteric viruses, but may also have potential for the control of respiratory viruses. two different swiv strains were chosen which are currently circulating in the pig population, h1n1 (a/swine/greven/ idt2889/2004) and h3n2 (a/swine/bondelum/idt5959/ 2007). as an established model for the present in vitro study an epithelial-(mdbk-cells) and a porcine alveolar macrophage cell line (3d4/21-cells) were utilized. it can be argued that the concentration of the probiotic utilized may not reflect the situation in the target tissue in vivo. however, the concentration chosen for treatment of the cell cultures (10 6 cfu/ml) reflects the same concentration which was determined in the gut of piglets fed e. faecium as a supplement during previous feeding trials in our research consortium [15] . in order to find out during which period of the swiv replication cycle the probiotic has the most stringent effect, e. faecium was added for a brief period of time (60 or 90 min) to the host cells either before, during or after virus infection (fig. 6) . the results indicate that the simultaneous addition of virus and e. faecium to the host cell monolayer was the most effective timing for the inhibition of virus multiplication. as shown in fig. 3 and fig. 4 , this experimental setup (termed ''competition'') resulted in a 4 logunit reduction of virus titer and in a concomitant rescue of cell viability. since a 1 h exposure of the monolayers to e. faecium before swiv-infection and a 1 h treatment after completion of virus infection also led to a 2-3 log-unit loss of virus titer, the probiotic must alter host cell factors which apparently results in an inhibition of influenza virus multiplication. obvious candidates for such factors are mediators of cellular defence processes. the expression of no and its subsequent increased activity has previously been reported to play a role in the host response to multiple viral families, and in various host species [16, 17] . in addition to its antiviral properties, no has been described to modulate intestinal barrier function, gut motility, iron transport, and has been implicated in numerous infections and noninfectious diseases of the intestine [18] . we found that e. faecium increased the expression of no in both 3d4/21 and mdbk cells (fig. 5) . all the samples collected after treatment with e. faecium showed significantly increased no-values when compared to the non-treated counterparts on 3d4/21 cell line. this is consistent with the hypothesis that high no levels are associated with decreased virus production. in addition to stimulating no-release, probiotics could also affect the expression of cytokines and other immune mediators relevant for the innate immune response to viral infections. we therefore determined the expression of selected mediators in swiv-infected host cells (3d4/21-cells only). as seen from fig. 5 , e. faecium promoted an increased expression of ifn-a. since the difference between the values of non-treated and e. faecium treated cells were found to be non-significant, ifn-a can be ruled out as the main immunoregulatory cytokine that could lead to e. faecium induced inhibition of swiv-infection. another cytokine stimulated by the probiotic treatment was il-10, which is a typical th2 cytokine that is initially repressed in virus infected cells but then expressed at higher levels later in infection to control the initial inflammatory response to infection. interestingly, this cytokine is clearly enhanced in the macrophage cell line upon e. faecium treatment and thus could support cellular control of swiv infection. two pro-inflammatory cytokines were found to be clearly reduced in the swiv-infected 3d4/21-cells treated with the probiotic, il-6 and tnf-a. secretion of il-6 by macrophages is known to play an indirect immunoregulatory role in the immune response to viral infection [19] , and tnf-a acts as an inflammatory cytokine by triggering a cascade of cytokine production [20] since both il-6 and tnf-a are downregulated by e. faecium in swiv-infected 3d4/21 cells, the reduced inflammatory response caused by some cytokines at the cellular level may contribute to the antiviral effect of the probiotic. tolllike receptor 3 (tlr-3) was the first identified antiviral tlr to have a central role in the host response to viruses [21] . our experimental data show that the treatment of swiv-infected 3d4/ 21-cells with e. faecium led to an decreased expression of tlr-3 at 2 h and 6 h post infections compare to virus alone which suggest that the probiotic induced modulation of this receptor may have a role in the antiviral function of e. faecium. since e. faecium acts most inhibitory when it is added together with the virus particles, we assessed whether swiv might be physically trapped or inactivated by the probiotic bacteria in a mixed incubation as detailed as ''preincubation assay'' in the experimental design shown in fig. 6 (lower panel). the results summarized in table 1 show that a substantial portion of the input virus particles are indeed trapped by the bacteria since virus infectivity is lost from the supernatants and viral genome equivalents are detected in the bacterial sediments after low speed centrifugation of the incubation mixture (data not shown). thus under such experimental conditions two antiviral functions of the probiotic may operate synergistically and add up to produce a more severe inhibition of swiv. the results presented altogether show that the probiotic e. faecium quite effectively inhibits the multiplication of swine influenza viruses in relevant cell culture systems. the antiviral mechanism of this probiotic is probably manifold since it was found to act on both the virus particles and the host cells. however, at least a few inhibitory parameters could be identified: e. faecium bacteria are able to adsorb swiv-particles and to alert the cells by mediating a rapid antiviral response through modulating the expression of defence relevant mediators. amongst these il-6, tnf-a, il-10, ifn-a and tlr-3 were identified as entities modulated by the probiotic treatment. it is realized that e. faecium can induce much more complex reactions in a treated tissue since only a few mediators could be assessed in this study. one common denominator of probiotic action could be no which is a mediator affected by many cellular signaling cascades. in line with publications for other virus-host cell systems, our results point to a central role of no which is stimulated upon the treatment with the probiotic and which may mount an improved cellular defence response against swiv-infection in tissues which were stimulated with a probiotic. based on the in vitro data shown here for a porcine influenza virus, we hypothesize that the use of e. faecium as a probiotic feed (or food) additive has the potential of reducing influenza virus infections in mammalian tissues. swiv challenge experiments with piglets which are fed e. faecium as a supplement are presently in progress in order to test this hypothesis. the swiv strains h1n1 (a/swine/greven/idt2889/2004), h3n2 (a/swine/bondelum/idt5959/2007), madin-darby bovine kidney (mdbk) cells [22] used in this study were a generous gift from dr. r. dürrwald (impfstoffwerk dessau-thornau, germany). stock virus of h1n1 and of h3n2 was propagated in mdbk and in mdck cells, respectively. the h1n1 strain was used on mdbk cells and the h3n2 strain was used on 3d4/21 macrophages. mdbk and mdck cells were maintained in dulbecco's modified eagle's medium (dmem; pan biotech) supplemented with 5% fetal calf serum (hyclone), and 1% penicillin/streptomycin (biochrom). the porcine continuous monomyeloid cell line 3d4/21 established from primary porcine alveolar macrophages [23] were kindly provided by prof. a. cencič (university of maribor, slovenia) and dr. hana weingartl. 3d4/21 cells were maintained in advanced dulbecco's modified eagle's medium (gibco) supplemented with 10% fetal calf serum (hyclone), and 1% penicillin/streptomycin (biochrom). e. faecium ncimb 10415 (cylactinh, cerbios-pharma sa) was maintained in todd-hewitt broth (roth). all experiments were done in triplicate. to determine possible cytotoxic effects of e. faecium, different concentrations (1.00e+05, 1.00e+06, 1.00e+07 or 1.00e+08 colony forming units (cfu)/ml) were added to 3d4/21 and mdbk cell monolayers in 96-well plates (greiner bio-one) for 72 h and cell viability was monitored by a methylthiazolyldiphenyl-tetrazolium bromide (mtt) viability assay. briefly, cell monolayers were washed after the 72 h treatment period, 20 ml mtt in pbs was added to each well and the plates were further incubated at 37uc in a co 2 incubator for 1.5 h. solubilisation of the formazan crystals formed during this period was achieved by the addition of dmso solution. the absorbance (od) at 570 nm was measured using a microplate reader (tecan, germany). cell survival rate was determined as bacteria average od value/ control average od value. for interference studies, infection of cells with both strains of swiv was done at a multiplicity of infection (moi) of 0.01. e. faecium was applied at a concentration of 10 6 cfu/ml. in avoid of carry over effects, after e. faecium was washed off two times, 1% penicillin/streptomycin was used to stop the propagation of e. faecium. the schematic in fig. 6 depicts our experimental setup for studying the interference between e. faecium with swiv-infection in the two cell culture systems. it allows us to define at what time during virus growth the addition of the probiotic is most effective. the preincubation setup should reveal whether the probiotic bacteria have a direct effect on the virus particles without any involvement of host cells. the mtt assay was used to measure the mitochondrial function, which serves as a viability index of metabolically active cells. after the experimental incubation period, the mtt assay was applied as described above. the percentage of metabolically active cells treated with probiotic bacteria and the percentage of protection from cytopathic effect achieved was then calculated. all data represent the average values for a minimum of six wells of three independent experiments. virus titers were calculated as 50% tissue culture infectious doses (tcid 50 ) by titrating supernatants containing h1n1 or h3n2 swiv in tenfold steps on 3d4/21 cells or mdbk indicator cells, respectively. three days after infection, indicator cells were stained by giemsa (sigma) and the cytopathic effect (cpe) was observed macroscopically and under the microscope. the results of all tcid 50 assays were calculated according to the reed and muench method [24] . assessment of nitric oxide (no) release no release was determined by measuring the amount of no 2 2 released into the culture medium by use of the griess-assay (promega) according to the manufacturer's instructions. briefly, 50 ml of each experimental sample was transferred into a 96 well plate in triplicate. defined standard samples were assessed in parallel to produce a standard curve. 50 ml of a sulfanilamide solution (1% sulfanilamide in 5% phosphoric acid) were added to each well for 10 minutes at room temperature. then 50 ml of the ned solution were dispensed at room temperature for 10 minutes and absorbance (od) at 570 nm measured using a microplate reader (tecan). no release in each sample was calculated by use of the nitrite standard curve generated in parallel. to investigate if virus could be trapped by probiotics, e. faecium (1.00e+06 cfu/ml) were mixed with 0.01 moi swiv in a total of 1 ml dmem for 90 min co-incubation at 37uc in a co 2 incubator for 1.5 h. the mixture was then centrifuged at 3,500 rpm for 10 min (compare fig. 6 -preincubation assay). sediments were prepared for quantitative pcr and supernatants were used to infect cells. total rna was isolated from sediments and m protein of swiv was amplified and compared to virus control. the virus titer and no release assays mentioned above were carried out at 48 or 96 hpi. after the various treatment periods, 3d4/21 cells were collected from the wells at 2 h, 6 h and 24 h. total rna was isolated from cell samples by use of the gene matrix rna purification kit (eurx). reverse transcription (rt) was performed using the revertaidtm first strand cdna synthesis kit (fermentas) according to the manufacturer's instructions. pcr reactions were performed in a total volume of 25 ml in an icycler iq detection system (bio-rad laboratories). the expression of each gene was analyzed using the relative quantification method [25] . the designations of genes, the primer sequences, the annealing temperatures, and the sizes of the amplification are listed in table 2 . all calculations were performed with ibm spss 19. data analysis for virus titers and no release were performed by two factorial anova followed by a post hoc test (scheffe). data analysis for cytokine expression was performed by paired, two tailed t-test. p values of ,0.05 were considered statistically significant. p values of ,0.01 were considered statistically very significant. all data are given as the mean 6 standard deviation. preparing for avian influenza: lessons from the ''swine flu affair effect of long term consumption of probiotic milk on infections in children attending day care centres: double blind, randomised trial immunomodulatory function of lactic acid bacteria immunobiotics and the probiotic evolution porcine small intestinal epithelial cell line (ipec-j2) of rotavirus infection as a new model for the study of innate immune responses to rotaviruses and probiotics putative probiotic lactobacillus spp. from porcine gastrointestinal tract inhibit transmissible gastroenteritis coronavirus and enteric bacterial pathogens influence of probiotic lactobacilli colonization on neonatal b cell responses in a gnotobiotic pig model of human rotavirus infection and disease effect of orally administered lactobacillus casei on porcine reproductive and respiratory syndrome (prrs) virus vaccination in pigs transmission and pathogenesis of swine-origin 2009 a(h1n1) influenza viruses in ferrets and mice effects of bacillus cereus var. toyoi on immune parameters of pregnant sows influence of a probiotic enterococcus faecium strain on development of the immune system of sows and piglets interactions of macrophages with probiotic bacteria lead to increased antiviral response against vesicular stomatitis virus probiotics and non-intestinal infectious conditions an interdisciplinary study on the mode of action of probiotics in pigs protection of insulin secreting cells from nitric oxide induced cellular damage by crosslinked hemoglobin inhibition of influenza virus replication by nitric oxide characterization of turkey inducible nitric oxide synthase and identification of its expression in the intestinal epithelium following astrovirus infection production of human tumor necrosis factor alpha, interleukin-6, and interleukin-10 is induced by lactic acid bacteria pro-and anti-inflammatory cytokines in human immunodeficiency virus infection and acquired immunodeficiency syndrome tlr3 in antiviral immunity: key player or bystander? receptor ganglioside content of three hosts for sendai virus. mdbk, hela, and mdck cells continuous porcine cell lines developed from alveolar macrophages: partial characterization and virus susceptibility a simplified multiwell plate assay for the measurement of hepatitis a virus infectivity a mechanistic model of pcr for accurate quantification of quantitative pcr data we are grateful to ralf dürrwald for generously supplying the swiv strains through idt-company (dessau, germany). we thank avrelija cencič (maribor, slovenia) and hana weingartl (winnipeg, canada) for supplying 3d4/21 cells. we are most grateful to stephan ludwig for critical reading of the manuscript. key: cord-002621-sq5iod1w authors: attia, mohamed i.; eldehna, wagdy m.; afifi, samar a.; keeton, adam b.; piazza, gary a.; abdel-aziz, hatem a. title: new hydrazonoindolin-2-ones: synthesis, exploration of the possible anti-proliferative mechanism of action and encapsulation into plga microspheres date: 2017-07-25 journal: plos one doi: 10.1371/journal.pone.0181241 sha: doc_id: 2621 cord_uid: sq5iod1w the synthesis and molecular characterization of new isatin-based hydrazonoindolin-2-ones 4a-o and 7a-e are reported. the in vitro anti-proliferative potential of the synthesized compounds 4a-o and 7a-e was examined against ht-29 (colon), zr-75 (breast) and a549 (lung) human cancer cell lines. compounds 7b, 7d and 7e were the most active congeners against the tested human cancer cell lines with average ic(50) values of 4.77, 3.39 and 2.37 μm, respectively, as compared with the reference isatin-based drug, sunitinib, which exhibited an average ic(50) value of 8.11 μm. compound 7e was selected for further pharmacological evaluation in order to gain insight into its possible mechanism of action. it increased caspase 3/7 activity by 2.4and 1.85-fold between 4 and 8 h of treatment, respectively, at 10 μm and it caused a decrease in the percentage of cells in the g1 phase of the cell cycle with a corresponding increase in the s-phase. in addition, compound 7e increased phosphorylated tyrosine (p-tyr) levels nearly two-fold with an apparent ic(50) value of 3.8 μm. the 7e-loaded plga microspheres were prepared using a modified emulsion-solvent diffusion method. the average encapsulation efficiency of the 7e-loaded plga microspheres was 85% ± 1.3. while, the in vitro release profile of the 7e-loaded microspheres was characterized by slow and continuous release of compound 7e during 21 days and the release curve was fitted to zero order kinetics. incorporation of 7e into plga microspheres improved its in vitro anti-proliferative activity toward the human cancer cell line a549 after 120 h incubation period with an ic(50) value less than 0.8 μm. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 cancer is one of the most formidable health burdens with increasing annual frequency worldwide [1] . in spite of precluding many of its causative risk factors, cancer still gives rise to about 550,000 deaths in the world every year and is the second leading cause of death next to heart disease [2] . the currently available cancer therapies usually suffer from poor selectivity between normal and invaded cells, with serious side effects and the development of drug resistance [3] . therefore, the continued search to identify novel antitumor compounds bestowed with low toxicity and minimum side effects remains critically important. the 1h-indole-2,3-dione (isatin) moiety constitutes the backbone of a number of agrochemicals, dyes and bioactive molecules, owing to its appropriate size and unique electronic properties [4] [5] [6] [7] . various 3-substituted isatins have been utilized as anticancer drugs or drug surrogates. example of this class of isatin derivatives is sunitinib (1, su11248, sutent tm , pfizer, inc., fig 1) which was clinically approved in 2006 and is used now as the standard first-line treatment for the management of gastrointestinal stromal cancers and renal cell carcinoma due to its multi-targeting tyrosine kinase inhibiting activity [8] . nintedanib (2, fig 1) is another potential anticancer agent of this class that possesses triple angiokinase inhibiting activity toward vascular endothelial growth factor receptors (vegfrs),platelet-derived growth factor receptors(pdgfrs) and fibroblast growth factor receptors (fgfrs) [9] . semaxanib (3, fig 1) was developed as a new anticancer candidate with multiple tyrosinekinase receptor inhibitor activity with gi 50 values of less than 10 nm against 46 out of 53 nci cell lines, but it has been discontinued in clinical trials owing to its dangerous side effects [10, 11] . schiff bases of isatin are acknowledged to have a broad spectrum of biological activities including anticonvulsant [12] , antibacterial [13] , antifungal [14] , anti-hiv [15] , antiviral [16] and anticancer activity [17] [18] [19] . indirubin (4, fig 2) is a bis-isatin isomer of the widely used colorant indigo (5, fig 2) , and it is the active ingredient of a traditional chinese remedy (danggui longhui wan) that is used for the treatment of chronic myelogenous leukemia (cml) [20] . moreover, indirubin (4) inhibits both glycogen synthase kinase 3 beta (gsk-3β) and cyclin-dependent kinases (cdks) via binding to the atp pocket with ic 50 values of 0.190 and 0.075 mm, respectively [21, 22] . azaindirubin (6, fig 2) was developed to overcome the poor water solubility and hence low bioavailability of indirubin (4) and it showed anti-proliferative activity toward ovarian adenoma cell lines [23] . in the same vein, the symmetric bis-isatin derivative (7, fig 2) displayed in vitro anti-proliferative activity against the hepg2 cell line with ic 50 value about 4.23 mm [21] . microsphere carrier systems made from the naturally occurring biodegradable polymers have attracted considerable attention for several years in the field of sustained drug delivery. these carrier systems can precisely control the release rates and target drugs to a specific body site with an enormous impact in formulation and development of novel drug delivery systems [24] . biodegradable polyesters such as copolymers of lactic and glycolic acid (plga) are attractive biomaterials for the encapsulation of pharmaceuticals and bioactive compounds. in this case, encapsulated drugs are not as readily available to biological systems as when they are in solution because the release of the drug will occur only after the onset of polymeric degradation [25] . in this context, certain potential functionalized schiff bases of isatin 4a-o were designed and synthesized to evaluate their in vitro anti-proliferative activity against a panel of human cancer cell lines. in addition, the non-symmetric bis-isatins 7a-e were also prepared and evaluated as new anti-proliferative agents. the compound exhibiting the best pharmacological activity was subjected to deeper pharmacological investigations in order to gain insight into its 4f [27] , 4i [28] and 6a, b [29] have been previously reported. the microspheres were prepared with poly (d, l-lactide co-glycolide) plga (50:50, mol. wt 30,000-60,000), which was purchased from sigma-aldrich (st. louis, usa). the emulsifier, low molecular weight polyvinyl alcohol (pva) was obtained from alfa aesar (karlsruhe, germany). dichloromethane (dcm) was purchased from avonchem (united kingdom). dimethyl sulfoxide (dmso) was obtained from loba chemie (mumbai, india). all ingredients used were of analytical grade. all cell lines have been purchased from the american type culture collection (atcc). 11 .01 (s, 1h, nh indolic ); 13 general procedure for the synthesis of target indolin-2-ones7a-e. a mixture of intermediates 6a, b (1 mmol) and the appropriate 3-hydrazonoindolin-2-one2a-d (1 mmol) in ethyl alcohol and catalytic amount of glacial acetic acid was heated under reflux for six hours, filtered while hot and the precipitate was washed with ethanol. the solid product was collected and re-crystallized from an ethanol/dmf mixture (3:1) to furnish compounds 7a-e with 67-79% yield. the detailed experimental procedures for pharmacological evaluation of the synthesized compounds were provided as supplementary materials. preparation of 7e -loaded plga microspheres. plga microspheres were prepared by a modified emulsion-solvent diffusion method as described previously [32] . briefly, the dcm (3 ml) phase containing plga was poured into the dmso (4 ml) phase containing compound 7e to obtain a 7e-polymer ratio of 1:10. the resulting organic phase was added dropwise with the help of a syringe into an aqueous phase (25 ml) containing 1% (w/v) pva using magnetic stirrer at 300 rpm and then homogenized at 13,500 rpm at room temperature for 3 min. the resulting emulsion was then transferred into water (25 ml) at room temperature and stirred at 200 rpm with a magnetic stirrer overnight until the solvent was evaporated. the resulting plga microspheres were recovered by centrifugation at 15000 rpm using a 3-30 k centrifuge (sigma, germany) for 15 min at 4˚c, washed three times with distilled water (50 ml) to remove the residual pva, re-suspended in distilled water (15 ml) and then lyophilized in a telstar freeze-dryer (terrassa, spain) at -50˚c with a pressure below 1 mbar for 24 h. plain microspheres were prepared in a similar manner except for the absence of compound 7e in the dmso phase. microsphere characterization. microsphere yield: the percentage yield of microspheres was calculated using the weight in mg of the final product after drying by lyophilization with respect to the initial total weight in mg of the drug and the polymer used for the preparation in the emulsifying process according to eq (1) [32, 33] . the equation below shows the percentage yield calculation. encapsulation efficiency and compound 7eloading: 10 mg of quantitatively weighed microspheres were dissolved in dmso (2 ml) and then sonicated for 15 min, then 0.1n hcl (5 ml) was added to precipitate the polymer. the solution was centrifuged at 10,000 rpm for 10 min. then the supernatant was diluted appropriately and analyzed for the content of 7e spectrophotometrically (biochrom libra s22, uk) at λ max of 318 nm to determine the 7e content. the polymer did not interfere with the absorbance of7e at the specified wavelength. theoretical 7eloading was determined by the entire 7e present in the polymer solution in the microspheres. the 7e-loading and entrapment efficiency were determined by formulas (2) and (3), respectively. all of the measurements were conducted in triplicate. determination of the particle size of microspheres: particle size of the prepared microspheres was measured using a dynamic light scattering particle size analyzer, model zetasizer nano-zs, zen 3500, malvern instruments (worcestershire, uk) at 25.0 ± 0.1˚c [34, 35] . lyophilized microspheres (1 mg) were re-suspended in distilled water (100 ml) and the particle size was recorded in triplicate. to investigate the morphological characterization of the prepared microspheres, they were mounted onto a double-sided adhesive tape attaching to an aluminum stub, then coated with gold and examined by scanning electron microscope (sem, jeol jsm-7001f, japan) at 20 kv acceleration voltage with a secondary detector. the magnification was adjusted until a clear image of the surface of the microspheres appeared and the picture was then recorded [36] . in vitro7e-release: studies on the in vitro release of 7e from microspheres were carried out using the vial method as reported by dubey et al. [34] . compound 7e-plga-microspheres containing 5 mg equivalent of compound 7e were suspended in a vial containing phosphate buffer (10 ml, ph 7.4) with 0.3% tween 80 to improve the solubility of 7e. the vial was incubated in a water bath at 37 ± 0.5˚c and vibrated horizontally at speed of 100 rpm. in vitro 7e release was assessed by intermittently sampling the vial (2 ml) at predetermined time intervals (1, 2, 3, 4, 5, 6, 10, 12, 15, 18, 20 and 21 days), then replacing the volume with 2 ml of fresh phosphate buffer. cell viability assay: the cell viability assay was performed as described in the section of antiproliferative activity in the supplementary materials. the synthetic strategy adopted for the preparation of the target derivatives is depicted in s3 and s4 files. indoline-2,3-diones 1a-c were refluxed with 99% hydrazine hydrate in methanol to obtain the corresponding hydrazone derivatives 2a-c.the reaction of hydrazones 2a-c with different substituted benzaldehydes 3a-e in ethanol in the presence of a catalytic amount of glacial acetic acid furnished the target derivatives 4a-o with 67-83% yields (s3 file). the ir spectra of compounds 4a-o displayed absorption bands around 3400 cm -1 for the indolic nh group in addition to the absorption bands of carbonyl groups near 1720 cm -1 which is consistent with the previously reported ones for compounds 4a [26] and 4f [27] . also, their 1 h nmr spectra revealed a d 2 o-exchangeable signals in the region δ 10.84-11.01 ppm attributable to the nh protons of the indolin-2-one moieties, in addition to the signal of the methine protons (-ch = n-) in the region δ 8.55-8.70 ppm which is in accordance with those previously reported for compounds 4a [26] , 4f [27] and 4i [28] . moreover, the 13 c nmr spectra of compounds 4a-o showed signals resonating around δ 163 ppm, characteristic of c = o carbons. direct methylation and benzylation of indoline-2,3-dione 1a with dimethyl sulfate and benzyl bromide were carried out in the presence of sodium hydroxide or potassium carbonate to furnish intermediates 6a and 6b, respectively. the second group of the target indolin-2-ones 7a-e was obtained in good yields (67-79%) through the reaction of the two intermediates 6a and 6b with the appropriate 3-hydrazonoindolin-2-one 2a-d in refluxing ethyl alcohol using catalytic amount of glacial acetic acid (s4 file). the ir spectra of the target compounds 7a-e revealed the presence of the characteristic absorption bands due to the nh and carbonyl groups of indolin-2-one moieties. moreover, the 1 h nmr spectra of 7a-e revealed the presence of the singlet indolic nh protons at a δ 11.02-11.15 ppm. also, the signals of the aliphatic protons (n-ch 3 ) of compounds 7a, b were observed as singlet signals around δ 3.22 ppm, while the benzylic protons (-ch 2 ) of 7c-e appeared as singlet signals close to δ 5.01 ppm. anti-proliferative activity. twenty compounds, i.e. 4a-o and 7a-e, were analyzed for cancer cell growth inhibitory activity. these studies were carried out using cells derived from human lung, colon and breast tumors (a549, ht-29 and zr-75 cells, respectively). this initial assessment of activity tested each compound in quadruplicate at a single concentration of 30 μm, if solubility permitted. as indicated in table 1 , the test compounds 4a-j, 4l-n and 7a-e exhibited anti-proliferative activity against a549, ht-29 and zr-75 cells with an average growth inhibition in the range from 3.5 to 97.7%, whereas compounds 4k and 4o stimulated the growth of the tested cell lines. compounds 7b, 7d and 7e were the most active compounds, showing an average growth inhibition from 95.4 to 97.7%. therefore, they were subjected to quantitative inhibitory concentration 50% (ic 50 ) determination for their cell growth inhibitory activity towards the a549, ht-29 and zr-75 cancer cell lines. the results are presented in table 2 and fig 3. extra sum-ofcompound 7e exhibited the best average ic 50 value (2.37 μm), being nearly three-fold more potent than the positive control, sunitinib (average ic 50 value = 8.11 μm). therefore, compound 7e was subjected to deeper pharmacological investigations in order to obtain insight into its pharmacological profile. apoptosis and caspase 3/7 activity. compound 7e was analyzed for apoptosis inducing activity in cancer cells. these studies were carried out using cells derived from human lung (a549). this follow-up assessment of activity tested compounds in quadruplicate at concentrations equivalent to the ic 50 value to inhibit growth and a concentration three-fold above the ic 50 concentrations over a time course ranging from 2 to 48 h. as indicated in fig 4, compound 7e at 10 μm increased caspase activity by 2.4-and 1.85-fold between 4 and 8 h of treatment, respectively, while the lower tested concentration had no effect on caspase 3/7 activity. cell cycle effects. compound 7e was analyzed for effects on various aspects of cell cycle progression in human cancer cells. these studies were carried out using cells derived from lung adenocarcinoma (a549). this follow-up assessment of activity tested compounds used immunofluorescent imaging of phosphorylated rb protein and the total dna content of each cell to determine the cell cycle phase. the ability of the test compounds to affect the cell cycle and rb phosphorylation was tested over a range of concentrations from less than 100 nm to 50 μm. as indicated in fig 5, compound 7e had dose-dependent effects on the tested parameters. it caused a significant reduction in the total cell number after 24 h of treatment with an ic 50 value = 2.67 μm and with an ic 50 value = 2.36 μm after 48 h (table 3) , reflective of the dosedependent growth inhibition observed in the homogenous assay. in addition, compound 7e caused a decrease in the percentage of cells in the g1 phase of the cell cycle with a corresponding increase in the s-phase. this suggests that part of the compound effects on growth may be attributable to a decreased rate of progression through the cell synthesis and anticancer activity of certain hydrazonoindolin-2-ones cycle and corresponding decreases in proliferation, similar to the positive control, sunitinib. arrest in g2 may represent a checkpoint blockade, whereas mitotic arrest may, in some cases, lead to mitotic catastrophe and subsequent programmed death of cells with multiple or aberrant nuclei. the increase in s-phase cells without a concomitant g2/m phase fraction may indicate a very rapid onset of apoptosis in the case of compound 7e. as with other cell cycle parameters, levels of phosphorylated rb protein were substantially reduced in a dose-dependent manner by the control and the test compound 7e. after 24 h of treatment, the ic 50 value was lower than the ic 50 value for reductions in the cell number caused by the test compound 7e (table 3 ). this may support the hypothesis that the inhibition of cyclin dependent kinases by isatin compounds plays a role in their growth inhibitory activity. however, the correlation was less apparent at the 48 h time point. furthermore, compound 7e was analyzed for its effect on total cellular levels of phosphorylated tyrosine (p-tyr) residues in human cancer cells. these studies were carried out using cells derived from lung adenocarcinoma (a549) and immunofluorescent imaging. the ability of the test compound 7e to affect acute serum stimulation of tyrosine phosphorylation was tested over a range of concentrations from less than 100 nm to 50 μm. compound 7e increased p-tyr levels nearly two-fold with an apparent ic 50 value of 3.8 μm, which is consistent with the growth inhibitory ic 50 value of this compound. in contrast, the positive control compound sunitinib caused a modest dose-dependent decrease in the level of cellular tyrosine phosphorylation activity with an ic 50 value of 0.9 μm, well below its growth inhibitory ic 50 value (fig 6) . synthesis and anticancer activity of certain hydrazonoindolin-2-ones selectivity. as an indicator of selectivity for tumor cells, compound 7e was analyzed for cell growth inhibitory activity in three non-tumorigenic cell lines.iec-6 cells derived from rat intestine exhibit morphologic and karyotypic features of normal intestinal epithelial cells [37] . cultures derived from human fibrocystic mammary tissue (mcf-10a) are non-tumorigenic and exhibit features of primary cultures of breast tissue, including dome formation [38] .fibroblasts derived from embryonic tissue from mice (swiss 3t3 fibroblasts) are both non-tumorigenic and contact inhibited [39] .for comparison, the a549 human non-small cell lung cancer (nsclc) cell line was included. this assessment of the growth inhibitory activity of the compounds was tested in quadruplicate at a maximum concentration of 25 μm, followed by 10 serially diluted concentrations. as indicated in fig 7 and table 4 , compound 7e inhibited growth in both normal and tumor cell lines >50%. compound 7e displayed a selectivity value of 1.7 while the positive control sunitinib showed a selectivity value of 1.4 (the difference between the mean ic 50 in non-tumor cell lines versus the nsclc cells). multidrug resistant lung cancer cell line. compound 7e was analyzed for cancer cell growth inhibitory activity in a sensitive nsclc cell line (a549) and a multidrug resistant lung cancer cell line (nci-h69ar) which expresses the abcc1 efflux pump protein. this assessment of activity tested compounds in quadruplicate at a maximum concentration of 25 μm, followed by 10 serially diluted concentrations. as indicated in fig 8 and table 5 , compound 7e inhibited growth in sensitive a549 and resistant nci-h69ar cell lines with ic 50 values of1.6 and 12.7μm, respectively. the resistant nci-h69ar cell line was nearly eight-fold less sensitive, indicating that this compound may be subjected to efflux by abcc1. the positive control sunitinib showed a lesser degree of resistance, i.e. the resistant nci-h69ar cell line was 1.9-fold less sensitive. encapsulation efficiency and 7e loading capacity. the prepared microspheres had high yield of 82% w/w recovery with respect to the initial amounts of polymer and 7e used in the microsphere formulation. the average encapsulated efficiency of 7e in plga microspheres was 85% ± 1.3 using a 1:10 7e:polymer ratio. the high encapsulation efficiency may be due to the presence of a high amount of the polymer, which can reduce 7e loss during the fabrication process. the percentage 7e-loading capacity was 9.84 ± 0.15. fourier transform infrared spectroscopic (ft-ir) analysis. the ft-ir spectra of pure 7e, plga, their physical mixture, plain microspheres and 7e-loaded microspheres were obtained to verify the chemical interaction between 7e and the polymer. the functional group bands of 7e remained the same in the spectra of both pure 7e as well as in the physical mixture and the formulation. this indicated that no interaction took place between 7e and the polymer. in the ft-ir spectrum of 7e-loaded microspheres, it was found that there was no significant spectral shift or disappearance of the bands of 7ein any spectrum of 7e with the polymer, as shown in fig 9, indicating compatibility between 7e and the plga polymer. particle size determination. the particle size of drug-loaded microspheres is considered an important characteristic as it could affect drug release properties. the particle size determination revealed that the produced microspheres were uniform with a size range of 1-3μm. the small polydispersity index (pdi) suggested that the size distribution of the particles was fairly monomodal (fig 10) . the loading of 7edidnot alter the particle size, as the average particle size of the plain microspheres and the loaded microspheres was 2.26±0.2 and 2.287±0.17 μm, respectively (mean ± sd, n = 3). fig 10 shows the particle size distribution of the 7e-loaded microspheres around 3 μm. a narrow particle size distribution is an important aspect in passive targeting of microspheres as well as for stability issues. the results demonstrate that the particle size of the prepared microspheres was uniform. morphological analysis of 7e-loaded microspheres. scanning electron microscope (sem) images revealed that the microspheres were intact spheres showing the formation of spherical and smooth surface microspheres with almost no pores or cavities, which may prolong the release of the encapsulated 7e over an extended period of time. no crystals of 7e were detected, and there was no clogging or deformation and few fragments of polymer adhering, as shown in fig 11. no difference was observed in the morphological properties of microspheres due to the presence of compound 7e. the particle size observed by sem was consistent with that obtained from the zetasizer particle size analyzer. these results indicate the efficiency of the preparation method and optimization of the preparation parameters such as the concentration and viscosity of the polymer solution. the obtained results are consistent with the previously reported studies for different drugs [40] [41] [42] . drug release evaluation. drug release from biodegradable polymeric particles occurs through a combination of several mechanisms. generally, it occurs through desorption of surface-bound drug, diffusion of the drug through the polymeric matrix and erosion of the polymer particles. in vitro 7e release was assessed in phosphate buffer (ph 7.4) containing a surfactant to simulate the physiological milieu and maintain sink conditions. samples were incubated at 37˚c, and the amount of 7e was determined using a uv spectrophotometer. fig 12 shows the release profile of raw 7e powder and the prepared 7e-plga microspheres. linear prolonged 7e release rates from the microspheres were observed for 21 days without an initial burst release (fig 12) . on the other hand, the release of compound 7e from the raw powder was faster than its release from the microspheres, as 65% of 7e was released after 5 days. this uncontrolled burst release of compound 7e from raw powder could result in too high local concentration which could lead to severe local tissue injure and hamper appropriate delivery to the target. it can be seen that 7e-loaded microspheres exhibited biphasic drug release kinetics [34, 43] with an initial release up to 6% after 48 h and 27% after six days followed by cumulative release of 90% after 20 days. no initial burst release was observed, suggesting low 7e density at the surface of the microspheres and hence the homogenous distribution of 7e within the microspheres. the high polymer concentration led to the formation of a dense polymer matrix structure in the microspheres and no pores as observed in the sem images, which resulted in a significant decrease in the initial burst release from the microspheres. the kinetics of 7e release from microspheres was close to the zero order model. the correlation coefficient (r 2 ) was 0.9884 and the release curve was fitted to zero order kinetics. this release pattern was due to diffusion and degradation of the microspheres. fig 12 shows the initial slow release of 7e from microspheres, which could be attributed to the hydrophobicity of 7e. moreover, the interaction of the hydrophobic 7e molecule with the polymer matrix via hydrophobic binding forces might cause a trapping of the drug inside the particles and, therefore, slower release. the rate of 7e release from the loaded microspheres gradually increased, as 10% release was observed after three days (fig 12) .this was probably due to the diffusion of the drug, which was present at the surface of the microspheres. thereafter, constant release was observed, which may have been due to 7e diffusion and matrix synthesis and anticancer activity of certain hydrazonoindolin-2-ones erosion mechanisms of the biodegradable plga polymer. slow drug release from microspheres maybe due high encapsulation of the drug with low swelling of polymer in the release medium, leading to slow diffusion of the drug particles from polymeric matrices. furthermore, the degradation of plga 50:50 is slow. therefore, the release of 7e from microspheres may depend on drug diffusion, the plga surface and bulk erosion or swelling [34] .conclusively, compound 7e was slowly and continuously released from the plga microspheres with no obvious burst release. this behavior is consistent with a previous study using plga microspheres for sustained drug release [44] and with several anticancer drugs incorporated into microspheres [33, 40, [45] [46] [47] . this formula was used for further studies on the human lung cancer cell line a549. cell viability assay. the human lung cancer a549 cell line was incubated with various concentrations (0.8, 1.6, 3.13, 6.25, 12.5, 25 and 50 μm) of both free 7e and 7e-loaded microspheres to evaluate the anti-proliferative activity by assessing their effect on cell viability. unloaded plga microspheres (lacking compound 7e) were coincubated with the same cell line to show that tumor growth was not inhibited due to the plga microspheres alone. in fig 13a, the cytotoxicity of free 7e was evaluated at0.8, 1.6, 3.13 and 6.25 μm. after 24 h of incubation time, no cytotoxicity was observed at low concentrations (0.8 and 1.6 μm), while after 72 h, only a 30% reduction in cell viability could be seen at 1.6 μm. moreover, at 3.1 μm, 20 and 70% reductions in the cell viability were noted after 24 h and 72 h of incubation, synthesis and anticancer activity of certain hydrazonoindolin-2-ones respectively, and almost total inhibition was observed after 120 h of incubation. a remarkable reduction in cell viability was observed at 6.25 μm = after 24 h of incubation and was maintained for 120 h of incubation. therefore, the cytotoxicity effect of free 7e needs higher concentrations to obtain minimal to no growth of the human lung cancer cell linea549. synthesis and anticancer activity of certain hydrazonoindolin-2-ones fig 13b illustrates the obtained results when 7e-loaded microspheres were incubated with the human lung cancer cell linea549for incubation periods and concentrations similar to that of the free 7e. there was no cytotoxicity observed at 0.8 μm after 24 h of incubation, but at 1.6, 3.13 and 6.25 μm, 30, 40 and 50% reductions in cell viability were observed, respectively. after 72 h of incubation, cell viability was decreased to 10, 7 and 5%, respectively, for the same concentrations. this assay demonstrated that entrapped 7e was more effective in arresting cell growth as compared with free 7e. the enhanced cytotoxicity of 7e-loaded microspheres was observed with increasing incubation time in a concentration dependent manner. therefore, the decrease in percentage cell viability was not immediate but rather gradual and continuous due to the polymer that allowed for slow release of 7e from microspheres and hence its gradual cytotoxic effect. the extended activity of 7e from 7e-loaded microspheres might be explained by the fact that they can be adsorbed onto the cell membrane, generating a drug concentration synthesis and anticancer activity of certain hydrazonoindolin-2-ones gradient near the cell surface that could favor 7e to penetrate into the cell. furthermore, the cell can phagocytize 7e-loaded microspheres, allowing 7e to be released inside the cytoplasm, thus contributing to a sustained 7e concentration [48] . quantitative in vitro cytotoxicity of 7e-loaded microspheres was investigated via the determination of theic 50 value toward a549cells as compared with that of free 7e. the ic 50 value was almost the same for both the free 7e (ic 50 value = 6.51 μm) and the 7e-loaded microspheres (ic 50 value = 6.50 μm) after 24 h of incubation (fig 14) . increasing the incubation time of 7e-loaded microspheres to 72 and 120 h resulted in a significant reduction in its ic 50 value by approximately 85 and 90%, respectively. on the other hand, the ic 50 value of 7eloaded microspheres was reduced to approximately 60 and 70% after 72 and 120 h, respectively, as compared with the ic 50 values of free 7e. statistical analysis revealed that these values were significantly different (p< 0.05) from the ic 50 values of free 7e. the enhanced7e activity mediated by its incorporation into microspheres can be attributed to its sustained release, as shown in fig 12. microspheres can act as a reservoir for 7e, protecting the drug from hydrolysis and provide not only sustained release of the drug, but also contribute to the maintenance of drug activity. several reported studies have emphasized that incorporation of anticancer drugs in such delivery systems improves their cytotoxic effects [49, 50] . the synthesis and molecular characterization using different spectroscopic tools of certain isatinbased hybrids 4a-o and 7a-e are described. compounds 4a-o and 7a-e were subjected to in vitro anti-proliferative assessment against ht-29 (colon), zr-75 (breast) and a549 (lung) human cancer cell lines. the most active congeners in the preliminary in vitro anti-proliferative screening, compounds 7b, 7d and 7e, showed average ic 50 values of 4.77, 3.39 and 2.37 μm, respectively, as compared with the reference drug sunitinib which exhibited an average ic 50 value of 8.11 μm towards the tested human cancer cell lines. compound 7e was selected for deeper pharmacological testing to gain insight into its possible in vitro anti-proliferative mechanism of action. it increased caspase activity by 2.4-and 1.85-fold between 4 and 8 h of treatment, respectively, at 10 μm. moreover, compound 7e decreased the percentage of cells in the g1 phase of the cell cycle with a corresponding increase in the s-phase. the increase in s-phase cells without a concomitant g2/m phase fraction may indicate a very rapid onset of apoptosis. in addition, compound 7eincreased p-tyr levels by nearly two-fold with an apparent ic 50 value of 3.8 μm, which is consistent with its growth inhibitory ic 50 value. compound 7e was incorporated into biodegradable plga microspheres to improve its in vitro anti-proliferative activity. the results of the in vitro anti-proliferative assay indicated that 7e-loaded plga microspheres exhibited better cytotoxicity as compared with free 7e toward the human lung cancer cell line a549 after 3 and 5 days of incubation. design, synthesis and apoptosis inducing effect of novel (z)-3-(3'-methoxy-4'-(2-amino-2-oxoethoxy)-benzylidene)indolin-2-ones as potential antitumour agents benzothiazoles: search for anticancer agents circumventing tumor resistance to chemotherapy by nanotechnology anticonvulsant activity of hydrazones, schiff and mannich bases of isatin derivatives isatin-derived antibacterial and antifungal compounds and their transition metal complexes isatin compounds as noncovalent sars coronavirus 3c-like protease inhibitors novel one-pot, three-component synthesis of spiro sunitinib versus interferon alfa in metastatic renal-cell carcinoma effect of bibf 1120 on reversal of abcb1-mediated multidrug resistance small molecule inhibitors of protein kinases in cancerhow to overcome resistance structure-activity-relationship studies of conformationally restricted analogs of combretastatin a-4 derived from su5416 anticonvulsant activity of schiff bases of isatin derivatives synthesis and antimicrobial activity of 1-[(n, n-disubstituted amino) methyl]-3-[(2-phenyl-3, 4-dihydro-4-oxoquinazoline-3-yl]indole-2-one synthesis, antibacterial, antifungal and anti-hiv evaluation of schiff and mannich bases of isatin derivatives with 3-amino-2-methylmercapto quinazolin-4(3h)-one synthesis, antibacterial, antifungal and anti-hiv evaluation of schiff and mannich bases of isatin and its derivatives with triazole synthesis of some 3-(4'-nitrobenzoylhydrazono)-2-indolinones as potential antiviral agents synthesis, anticancer, and cytotoxic activities of some mononuclear ru(ii) compounds 1-arylmethyl-2,3-dioxo-2,3-dihydroindole thiosemicarbazones as leads for developing cytotoxins and anticonvulsants synthesis and biological evaluation of 2-(5-substituted-1-((diethylamino)methyl)-2-oxoindolin-3-ylidene)-n-substituted-hydrazinecarbothioamides indirubin and meisoindigo in the treatment of chronic myelogenous leukemia in china synthesis, in vitro and in vivo antitumor activity of symmetrical bis-schiff base derivatives of isatin indirubin, the active constituent of a chinese antileukaemia medicine, inhibits cyclin-dependent kinases synthesis and antitumor activity of 7-azaindirubin preparation and characterization of mucoadhesive microcapsules of gliclazide with natural gums preparation and characterization of paclitaxel-loaded pldla microspheres novel coordination complexes of the trivalent ruthenium, rhodium and iridium with hydrazones derived from isatin hydrazide and various aldehydes with spectral and biological characterization synthesis and antimicrobial activity of 5-chloroindoline-2, 3-dione derivatives synthesis of bis-schiff bases of isatins and their antiglycation activity simple and efficient microwave assisted n-alkylation of isatin design, synthesis and qsar study of certain isatin-pyridine hybrids as potential anti-proliferative agents synthesis and biophysical insights into the bindingof a potent anti-proliferative non-symmetricbis-isatin derivative with bovine serum albumin:spectroscopic and molecular docking approaches preparation and characterization of simvastatin loaded plga microparticles for tissue engineering applications formulation and evaluation of sustained release implantable microspheres of temozolomide for brain targeting prepared by a novel technique development of docetaxel-plga-nanoparticles and in vitro antitumor activity in pc3 cells targeted to prostate tumor chlorambucil encapsulation into plga nanoparticles and cytotoxic effects in breast cancer cell in vitro and in vivo evaluations of plga microspheres containing nalmefene epithelioid cell cultures from rat small intestine. characterization by morphologic and immunologic criteria isolation and characterization of a spontaneously immortalized human breast epithelial cell line, mcf-10 quantitative studies of the growth of mouse embryo cells in culture and their development into established lines prevention of local tumor growth with paclitaxel-loaded microspheres preparation and characterization of δ9-tetrahydrocannabinol-loaded biodegradable polymeric microparticles and their antitumoral efficacy on cancer cell lines preparation and in-vitro evaluation of controlled release plga microparticles containing triptoreline preparation, characterization and in vitro cytotoxicity of indomethacin-loaded plla/plga microparticles using supercritical co 2 technique sustained release donepezil loaded plga microspheres for injection: preparation, in vitro and in vivo study cytotoxicity of pharmaceutically optimized nanometric systems of a chemotherapeutic drug on breast and liver tumor cells controlled release of dutasteride from biodegradable microspheres: in vitro and in vivo studies preparation, characterization and evaluation of drug release properties of simvastatin-loaded plga microspheres formulation, in vitro drug release study and anticancer activity of 5-fluorouracil loaded gellan gum microbeads the effect of temozolomide/poly (lactide-co-glycolide) (plga)/nano-hydroxyapatite microspheres on glioma u87 cells behavior formulation, characterization and evaluation of curcumin-loaded plga nanospheres for cancer therapy key: cord-001120-fxd533b4 authors: everitt, aaron r.; clare, simon; mcdonald, jacqueline u.; kane, leanne; harcourt, katherine; ahras, malika; lall, amar; hale, christine; rodgers, angela; young, douglas b.; haque, ashraful; billker, oliver; tregoning, john s.; dougan, gordon; kellam, paul title: defining the range of pathogens susceptible to ifitm3 restriction using a knockout mouse model date: 2013-11-21 journal: plos one doi: 10.1371/journal.pone.0080723 sha: doc_id: 1120 cord_uid: fxd533b4 the interferon-inducible transmembrane (ifitm) family of proteins has been shown to restrict a broad range of viruses in vitro and in vivo by halting progress through the late endosomal pathway. further, single nucleotide polymorphisms (snps) in its sequence have been linked with risk of developing severe influenza virus infections in humans. the number of viruses restricted by this host protein has continued to grow since it was first demonstrated as playing an antiviral role; all of which enter cells via the endosomal pathway. we therefore sought to test the limits of antimicrobial restriction by ifitm3 using a knockout mouse model. we showed that ifitm3 does not impact on the restriction or pathogenesis of bacterial (salmonella typhimurium, citrobacter rodentium, mycobacterium tuberculosis) or protozoan (plasmodium berghei) pathogens, despite in vitro evidence. however, ifitm3 is capable of restricting respiratory syncytial virus (rsv) in vivo either through directly restricting rsv cell infection, or by exerting a previously uncharacterised function controlling disease pathogenesis. this represents the first demonstration of a virus that enters directly through the plasma membrane, without the need for the endosomal pathway, being restricted by the ifitm family; therefore further defining the role of these antiviral proteins. intrinsic cellular defense molecules are able to detect and restrict invading pathogens at the level of the infected cell and constitute an initial repertoire of proteins that prevent infection. such intrinsic defenses have the ability to detect the pathogen, and either directly block a component of the pathogen's life cycles and / or signal to the innate and adaptive immune system to further control the infection. in certain cases, these intrinsic restriction factors recognise non-self pathogenassocated molecular patterns, such as lipids, proteins and nucleic acids, from a broad range of pathogens through pathogen recognition receptors. this ability allows the host to detect bacterial, viral, fungal and protozoan pathogens [1] . for example, toll-like receptor 4 alone is able to detect gram-negative bacteria, fungi, trypanosomes and surface proteins on several viruses [2] . alternatively, other restriction factors, particularly those that target viruses, appear to have a more reduced range of pathogens that they can block, as is the case for many interferon-stimulated genes (isgs) [3] . however, restriction factors that work in defined cellular locations against a common pathogen feature of infection may also have broad anti-influenza properties. we and others have found that the isg interferon-inducible transmembrane 3 (ifitm3), initially defined as playing a developmental role in germ cell homing [4] , has a profound role in the restriction of viruses entering the cell through the acid endosomal pathway [5, 6] , including influenza and dengue viruses [7] . since the discovery of ifitm3's antiviral role, the number of viruses restricted by the ifitm family has expanded considerably [5, [7] [8] [9] [10] [11] [12] [13] [14] . this has led to the generation of hypotheses about how the ifitm family achieves restriction; namely through preventing the fusion of viral and cellular membranes [15, 16] . recently, the role of ifitm3 has been expanded by the discovery of nonenveloped reoviruses' restriction [9] . this has important implications, as nonenveloped viruses do not rely on membrane fusion to gain release from late endosomes. instead, it is hypothesised that these viruses may physically disrupt the endosomal membrane through their surface proteins [17, 18] . this therefore potentially broadens the actions of ifitm3 beyond enveloped viruses and may also include other non-viral pathogens. the role of ifitm3 in vivo shows it is crucial restriction factor for preventing the onset of severe influenza viral infections in a knockout mouse model [6] . further, the overrepresentation of a single nucleotide polymorphism (snp), rs12252 c allele in the human ifitm3 gene in two cohorts of patients hospitalised with influenza virus during the 2009 h1n1 pandemic shows that the rs12252 cc genotype confers an 4-5 fold increased risk developing a severe influenza virus infection [6, 19] . here we sought therefore to expand and define the role of ifitm3 in pathogen restriction by assessing the susceptibility of ifitm3-deficient (ifitm3 -/-) mice to bacteria (salmonella typhimurium, citrobacter rodentium, mycobacterium tuberculosis), a parasite (plasmodium berghei) and a virus (respiratory syncytial virus, rsv) to determine the specificity of this crucial antimicrobial protein. we show that ifitm3 is specifically an antiviral protein; yielding no significant phenotype in mice when challenged with bacteria and protozoa, despite studies implicating the ifitm family in restriction of these pathogens [20, 21] . we also show a novel role for ifitm3 in vivo in restriction of rsv: a virus that does not enter cells through the endosomal pathway, adding further to the role of ifitm3 as a central antiviral restriction factor that targets cellular entry. background-matched 8-10 week old wild type, ifitm3 -/-(wellcome trust sanger institute [22] ) and ifngr -/-mice (jackson laboratories), all of which were >95% c57bl/6, were maintained in accordance with uk home office regulations, uk animals scientific procedures act 1986 under the project license ppl 80/2596. animals were supplied with food and water ad libitum and were monitored daily for signs of illness. ifitm3 -/-mice were phenotyped through pipelines at the wellcome trust sanger institute as described previously [23, 24] . immunohistochemistry 5-µm sections of paraffin-embedded tissue were incubated with anti-ifitm3 antibody (abcam), which was subsequently bound to a secondary horse radish peroxidase-conjugated antirabbit antibody (dako). sections were counterstained with hematoxylin (sigma-aldrich) and were assessed for expression by microscopy. groups of 8 ifitm3 -/-and 8 c57bl/6j mice were challenged intravenously with 5 x 10 5 colony forming units (cfu) salmonella typhimurium m525 containing tetc, (fragment c of tetanus toxin, to act as an antigen for subsequent antibody quantification), and followed for 28 days. on day 14 postinfection (pi), four from each group of mice are culled and organs (spleen, liver and caecum) removed. a small piece of the spleen and liver was fixed in 4% formalin and then later processed to paraffin blocks as a biobank of infected tissue for histological study if interesting. the rest of the organs were weighed then homogenized, serially diluted and plated to determine viable bacterial load. at day 28pi, the remaining four mice are culled following a terminal bleed and the organs removed and processed as above. the blood was allowed to clot then centrifuged when the serum was removed and used to detect tetc antigen specific antibodies by enzyme-linked immuosorbent assay (elisa). mice were weighed and monitored daily for signs of clinical illness. groups of 8 ifitm3 -/-and 8 c57bl6j mice were orally infected by gavage with 1 x 10 9 cfu of and followed for 28 days. every 2-3 days faeces from infected mice are collected. these were then weighed and homogenized in 1 ml per 100 mg of faeces. this was then serially diluted and plated to determine viable bacterial load. on day 14pi four mice per group were culled and organs (spleen, liver, caecal contents, caecum, 6cm of colon) removed. a small piece of the distal colon was fixed in 4% formalin and processed to paraffin blocks as a biobank of infected tissue for histological analysis. the rest of the organs were weighed then homogenized, serially diluted and plated to determine viable bacterial load. on day 28pi the remaining four mice were culled and the above is repeated. mice were weighed and monitored daily for signs of clinical illness. mice were infected by low-dose aerosol exposure with h37rv m. tuberculosis using a glas-col (terre haute, in) aerosol generator calibrated to deliver approximately 100 bacteria into the lungs. bacterial counts in the lungs at each time point of the study were determined by plating serial dilutions of individual lung homogenates on duplicate plates of middlebrook 7h11 agar containing oadc enrichment. colony-forming units were counted after 3-4 weeks incubation at 37 °c. a transgenic plasmodium berghei anka reporter line, pbgfp-luc con (rmgm-28), that constitutively expresses a fusion protein of gfp and firefly luciferase [25] , was maintained by passage in balb/c mice. to induce experimental cerebral malaria (ecm), infected blood containing 5 x 10 5 infected red blood cells was injected intraperitoneally. from day six pi mice were monitored twice daily and scored for clinical signs of neurologic disease using a ten parameter murine coma and behaviour scale adapted from that of carroll et al. [26] . mice classified as having ecm were killed by cervical dislocation. on days 2 and 3 pi parasite growth was monitored by luciferase measurements in 3 μl of blood, which were collected from a tail vein into citrate-dextrose acd freezing buffer (sigma) and stored at -80°c until analysis using the promega bright-glo luciferase assay system with a berthold orion ii microplate luminometer. parasitemia was also monitored using giemsa-stained thin blood smears. plasma samples from p. berghei infected mice were analysed for cytokines using a cytometric bead array inflammation kit (bd biosciences) according to manufacturer's instructions. samples were acquired on a bd facs aria ii, and data analysed using bd fcap array software. rsv strain a2 (from prof p. openshaw, imperial college london) was grown in hep-2 cells and viral titer determined by plaque assay. mice were infected intranasally (i.n.) with 5 x 10 5 plaque forming units (pfu) under isoflurane anesthesia. weight was measured daily to monitor disease severity. to collect bronchoalveolar lavage (bal) fluid, the lungs of each mouse were inflated five times with 1 ml of pbs and bal fluid kept on ice; 100 μl was centrifuged onto glass slides and stained with hematoxylin and eosin for cell differentiation. the remainder was centrifuged, the supernatant retained at −80°c, and the pellet resuspended in rpmi medium with 10% fetal calf serum. lungs were removed, the smaller lobe was snap frozen in liquid nitrogen for rna extraction and the remainder was homogenized by passage through 100-μm cell strainers (falcon). red blood cells in the lung sample were lysed in ammonium chloride buffer, and the remaining cells resuspended in rpmi medium with 10% fetal calf serum. viable cell numbers were determined by trypan blue exclusion and lung cells types were differentiated by flow cytometry on a bd facs aria ii using antibodies from bd and ebioscience. rsv viral load was measured by quantitative rt-pcr for the rsv l gene using primers and probes previously described [27] , with l gene copy number determined using a rsv l gene standard and presented relative to μg lung rna. lungs were homogenised with a rotor-stator homogeniser, centrifuged and the supernatant collected for cytokine analyses. cytokines in lung homogenate and bal fluid were quantified using duosets from r&d systems. all results are expressed as mean +/-s.d.; statistical significance was calculated by analysis of variance (anova) followed by bonferroni's multiple comparison test when there were more than two groups and student's t-tests for the comparison of two groups. non-normally distributed data were assessed by mann-whitney u test. results regarding mouse survival were analysed by a log-rank (mantel-cox) test. all data regarding mouse phenotyping, including how individual traits were statistically analysed can be found at the wellcome trust sanger institute's mouse resources portal (http:// www.sanger.ac.uk/mouseportal/) all calculations were performed using graphpad prism 5.0 software, and results were considered significant at p < 0.05. many genes are embryonically lethal or lead to no overt phenotype when knocked out in mice. indeed, ifitm3 -/-mice have no discernable phenotype [22] . to examine in greater detail, uninfected mice were assessed against a panel of phenotypic assays [24] , incorporating a robust set of adult traits that are capable of detecting phenotypic variations. we observed no statistically significant differences across all of our key phenotyping categories, including those assessing immune functions compared to wild type control mice ( table 1) . each of the categories listed in table 1 can be further subdivided into a number of additional categories, again all being wild type in phenotype. the complete list of phenotyping can be accessed through the wellcome trust sanger institute's mouse resources portal (http://www.sanger.ac.uk/mouseportal/). we assessed the tissue distribution of ifitm3 by immunohistochemistry of wild type compared to ifitm3 -/-mice, including lymph node, lung, spleen, liver and intestine. the expression of ifitm3 was absent in all ifitm3 -/-mouse organs, as expected, but was highly expressed in all wild type organs ( figure 1 ). in wild type mice, the expression pattern of ifitm3 was noteworthy. the spleen and lymph nodes indicated that ifitm3 was predominantly expressed in the red pulp, but was absent from the white pulp. similarly, intestinal staining revealed ifitm3 expression to be high in the lamina propria, but not on the villus epithelium. conversely, lung and liver showed ubiquitous expression of ifitm3 throughout the tissues, with protein present in respiratory epithelial cells and hepatocytes, respectively. as the first indication of the crucial role of ifitm3 only appeared upon infection with influenza [6] and the tissue distribution suggests ifitm3 is important in multiple organ systems, we challenged the ifitm3 -/-mice with a number of different pathogens. wild type and ifitm3 -/-mice were intravenously dosed with 1 × 10 6 cfu of s. typhimurium m525 bacteria and observed for 28 days pi for signs of morbidity and weight loss (figure 2a ). all mice survived the challenge and gained weight over the time course of the study. ifitm3 -/-mice appeared to gain proportional weight at a slower rate, however, this was due to these mice being on average 5g heavier at the start of challenge and based on lack of bacterial counts and pathology, is unlikely to be due to infection. on day 28 pi, anti-s. typhimurium antibody titres were determined from the sera of wild type and ifitm3 -/-mice. this indicated that both genotypes of mice produced statistically similar antibody profiles ( figure 2b) . further, the bacterial load was determined in the spleen, liver and faecal contents ( figure 2c-e) . similarly, bacterial counts revealed no significant differences between wild type and ifitm3 -/-mice; together showing that ifitm3 does not play a role in resistance or susceptibility to salmonella infection. wild type and ifitm3 -/-mice were orally gavaged with 1 × 10 9 cfu of c. rodentium bacteria and monitored for 28 days pi for signs of morbidity. weight loss profiles showed that neither wild type nor ifitm3 -/-mice had any overt signs of illness over the course of infection ( figure 3a ). bacteria shed in the faeces of these mice also revealed no significant differences between the genotypes, with clearance of infection achieved by day 25 pi in ifitm3 -/-mice ( figure 3b ). mice were sacrificed on days 14 and 28 pi to determine any differences in the bacterial burden between wild type and ifitm3 -/-mice. counts in the caecum (total, caeceal patch and contents) and colon showed no significant differences in bacterial colonisation and clearance ( figure 3c-f) . similarly, analysis of the liver and spleen revealed no instances of bacteraemia in either wild type or ifitm3 -/-mice ( figure 3g,h) . taken together, these data suggest ifitm3 does not impact on c. rodentium infection or pathogenesis. wild type and ifitm3 -/-mice were aerogenically infected with an aerosolised dose of approximately 100 cfu of h37rv m. tuberculosis bacteria and monitored for signs of morbidity for the following 28 days. to determine whether ifitm3 was involved in the control of the bacterial infection, mice were sacrificed on days 0, 7, 14 and 28 pi to calculate the bacterial burden in the lungs. there were however, no significant differences between wild type and ifitm3 -/-mice (figure 4) , with bacterial growth kinetics indicating that ifitm3 does not effect on m. tuberculosis infection and pathogenesis. mice were intraperitoneally injected with 5 × 10 5 red blood cells infected with a p. berghei anka reporter line, pbgfp-luc con (rmgm-28), that constitutively expresses a fusion protein of gfp and firefly luciferase [25] . interferon gamma (ifnγ) receptor knockout mice (ifngr -/-) mice were included to act as control, as these mice do not succumb to lethal episodes of ecm. the experimental challenge revealed there to be no significant difference in phenotype seen in ifitm3 -/-mice compared with wild type littermate controls, with both showing susceptibility to ecm ( figure 5a ). the ~50% survival of wild type mice falls within acceptable boundaries owing to inherent inefficiencies in the delivery of parasites into the mice [28] . in contrast, ifngr -/-mice infected in parallel were fully protected from infection. analysis of parasite burden revealed that all mice were infected with p. berghei ( figure 5b ), but with no significant differences at day three pi. additionally, levels of the inflammatory cytokines ifnγ, tumor necrosis factor alpha (tnfα) and monocyte chemotactic protein-1 (mcp-1) were analysed by cytometric bead array, again no significant differences between wild type and ifitm3 -/-mice were observed ( figure 5c ). wild type and ifitm3 -/-mice were intranasally infected with 5 × 10 5 pfu of rsv-a (a2 strain) and were monitored daily for weight loss for seven days pi. cohorts of mice were sacrificed on days four and seven pi to quantify viral burden and immunological changes over the course of the challenge. ifitm3 -/-mice showed highly significant weight loss on days six and seven pi compared to wild type littermates (p < 0.01) ( figure 6a ). furthermore, ifitm3 -/-mice showed a significantly higher peak in viral load on day four pi (p < 0.05), which remained higher than wild type mice at seven days pi ( figure 6b ). bronchoalveolar lavage (bal) was performed at days four and seven pi and lungs harvested at day seven for cell and cytokine measurements. cellular infiltrate was quantified over the course of infection, which showed a significant increase in total cells resident in the lungs on day seven pi in ifitm3 -/-mice (p < 0.05, figure 6c ) and a similarly significant increase in total cellular infiltrate in the bal fluid on day four pi (p < 0.01, figure 6d ). flow cytometry revealed an increase in all cellular subpopulations in ifitm3 -/-mice relative to wild type littermates on day seven pi. in particular, numbers of total cd3+ t-cells (p < 0.05) in the lungs ( figure 6e) (p < 0.01, figure 6f ). analysis of inflammatory cytokines, including ifnγ, il-6 and il-1β revealed differences in their levels between genotypes of mice in the lungs and bal fluid on day seven pi ( figure 6g ,h), with significantly higher levels of ifnγ (lung: p < 0.05, bal: p < 0.05) and il-1β (p < 0.05) in ifitm3 -/-mice relative to wild type controls. the role of ifitm3 in restricting virus infections, where the virus enters the cell through the acidic endosomal pathway, is well established [5, 15, 29] . however, ifitm3's role in other infections or the effect in removing ifitm3 in vivo in the absence of infection is not well understood. here we show ifitm3 is expressed in many different murine tissues and cell types and does affect the response to rsv infection in mice. ifitm3 does not contribute to the infection phenotype of citrobacter, salmonella or mycobacterium bacterial infections. these bacterial species encompass a range of physiological and pathogenic niches. salmonella enterica serovar typhimurium (s. typhimurium) is an intracellular bacteria that enters cells through phagocytosis or by a bacterial-triggered entry mechanism and replicates within endosomal-like structures known as salmonella-containing vacuoles [30] . in contrast, citrobacter rodentium (c. rodentium) is a noninvasive, gram-negative bacterium used to model enteropathogenic and enterohaemorrhagic e. coli infections of the gut [31, 32] , and mycobacterium tuberculosis (m. tuberculosis) is an intracellular respiratory bacterium that replicates primarily within macrophages and dendritic cells, before forming latent granulomas in the infected organs [33] , and is the causative agent of tuberculosis (tb). we found no evidence for control of m. tuberculosis bacterial growth in murine lungs, despite the fact that the pathogen triggers a type i ifn response [34] , which subsequently up-regulates ifitm3 expression. further, a recent study implicated a snp (rs3888188) in the promoter of ifitm3 with susceptibility to tb [21] , wherein the minority rs3888188-g allele was significantly overrepresented in patients with tb compared to healthy controls in a han chinese population. we found no evidence in our murine model that the ablation of ifitm3 expression impacted on m. tuberculosis infection. however, it should be noted that we only assayed for initial infection and colonisation of the lungs and that the long term dormancy characteristic of human tb is not observed in the mouse model. furthermore, these differences may have arisen due to our challenge strain differing from that used in the human study; therefore inducing different gene expression signatures. further, we found that ifitm3 does not impact on the development of ecm in plasmodium berghei anka infection, although it is well established that malarial parasites elicit strong type i and type ii ifn responses in their hosts, which have been shown to impact on the severity of infection [35, 36] , with ifnα and ifnγ contributing to lethality in murine models. furthermore, eight snps in the ifn receptor, ifnar1, have been associated with the development of cerebral malaria in children; a finding that is corroborated in ifnar -/-mice, which also do not develop cerebral malaria [37] . ifitm3, along with several other isgs, are significantly up-regulated in patients that have become infected with p. falciparum [20] . it was shown that deletion of several of these isgs, including tbk1 and the double knockout of irf3 and irf7 prevented mice from developing lethal ecm [20, 36] . our work shows ifitm3 is not an isg involved in the pathogenesis of experimental cerebral malaria. however, we saw that ifitm3 was important in the control of rsv infection, leading to more severe disease in ifitm3 -/-mice, as assessed by weight loss, viral load and a dysregulated immune response. although these trends were seen with influenza virus infection of ifitm3 -/-mice, the phenotype seen in the rsv challenge is not as striking [6, 38] . this may be due to the response to the different viruses and the genetic background of the mice (c57bl/6 taconic), which we [39] and others [40] have shown is influential in rsv disease severity. rsv is one of the commonest respiratory pathogens in children that necessitates hospitalisation [41] ; accounting for three times more admissions to hospital than influenza viruses [42] . rsv, like influenza virus, is an enveloped virus that initially causes a mild upper respiratory tract infection that can develop into bronchiolitis and cause acute respiratory distress. our discovery that the lack of ifitm3 can alter the pathogenesis of rsv infection suggests ifitm3 either directly restricts rsv cell infection in vivo, or exerts a hitherto uncharacterised function controlling virus infection in vivo is novel and supports associations seen in the mouse model [43, 44] , in airway epithelial cultures [45] and in blood from hospitalised infants [46] . strikingly, rsv infects cells through the plasma membrane and does not require the endosomal pathway. rsv enters airway epithelial cells via f protein binding of nucleolin, which is situated in cholesterol rich microdomains / lipid rafts [47, 48] . rsv is proposed to bind to nucleolin via its f protein, which initiates hemifusion of the rsv envelope with the cell membrane [48] ; thus delivering the viral content directly into the cytoplasm without the need for endosomes. recently, li and colleagues [49] suggested that the ifitm family of proteins are capable of restricting viral hemifusion and the formation of syncytia by reducing membrane fluidity [15, 16] . ifitm3, however, is primarily distributed intracellularly on endosomal membranes [50] . of the ifitm family members, ifitm1 is primarily localised to the cell surface [15] : the site of rsv-cell fusion. therefore ifitm1, which is functional in the ifitm3 -/-mice [6] , may provide the strongest block to rsv infection. previous studies have shown a degree of overlap of function between ifitm1, -2 and -3, with certain ifitms having specificity for restricting particular viruses [7, 8] . importantly, ifitm1, -2 and -3 interact and may function co-operatively [15] , possibly with ifitm3 potentiating an ifitm1 restriction of rsv. indeed, ifitm1 has been shown to be up-regulated during rsv infection [51] . with the recent recognition of ifitm3-mediated restriction of nonenveloped reoviruses, the pleotropic effect of ifitm3 on diverse virus infections, or the co-operative role of all antiviral ifitm proteins as a layered defense to different virus infections, remains to be determined. mice were intraperitoneally injected with red blood cells containing p. berghei anka and were monitored for survival for 12 days pi. n = 9 for wild type, n = 2 for each of ifitm3 -/and ifngr -/-mutants (a). parasite biomass was monitored by measuring the activity of a luciferase reporter gene constitutively expressed by the parasite and expressed as relative light units (rlu) (b), and cytokine dysregulation was measured from the sera on day three pi by cytometric bead assay (c). ■: wild type, □: ifitm3 -/-, ○: ifngr -/-. results show means ± s.d. (n > 2). pattern recognition receptors and inflammation pathogen recognition and innate immunity the broad-spectrum antiviral functions of ifit and ifitm proteins ifitm/mil/fragilis family proteins ifitm1 and ifitm3 play distinct roles in mouse primordial germ cell homing and repulsion ifitm3 inhibits influenza a virus infection by preventing cytosolic entry ifitm3 restricts the morbidity and mortality associated with influenza the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus interferon inducible transmembrane protein 3 (ifitm3) restricts reovirus cell entry interferon-induced cell membrane proteins, ifitm3 and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms identification of five interferon-induced cellular proteins that inhibit west nile virus and dengue virus infections a diverse range of gene products are effectors of the type i interferon antiviral response ifitm-2 and ifitm-3 but not ifitm-1 restrict rift valley fever ifitm1 is a tight junction protein that inhibits hepatitis c virus entry the cd225 domain of ifitm3 is required for both ifitm protein association and inhibition of influenza a virus and dengue virus replication the antiviral effector ifitm3 disrupts intracellular cholesterol homeostasis to block viral entry strategy for nonenveloped virus entry: a hydrophobic conformer of the reovirus membrane penetration protein î¼1 mediates membrane disruption adenovirus protein vi mediates membrane disruption following capsid disassembly interferon-induced transmembrane protein-3 genetic variant rs12252-c is associated with severe influenza in chinese individuals innate immune recognition of an at-rich stem-loop dna motif in the plasmodium falciparum a functional promoter polymorphism of <italic>ifitm3</italic> is associated with susceptibility to pediatric tuberculosis in han chinese normal germ line establishment in mice carrying a deletion of the ifitm/ fragilis gene family cluster mouse large-scale phenotyping initiatives: overview of the european mouse disease clinic (eumodic) and of the wellcome trust sanger institute mouse genetics project genome-wide generation and systematic phenotyping of knockout mice reveals new roles for many murine malaria parasite sequestration: cd36 is the major receptor, but cerebral pathology is unlinked to sequestration a rapid murine coma and behavior scale for quantitative assessment of murine cerebral malaria preexposure to cpg protects against the delayed effects of neonatal respiratory syncytial virus infection plasmodium berghei anka (pba) infection of c57bl/6j mice: a model of severe malaria interferon-inducible transmembrane proteins of the innate immune response act as membrane organizers by influencing clathrin and v-atpase localization and function salmonella enterica: a surprisingly well adapted intracellular lifestyle citrobacter rodentium of mice and man enhanced susceptibility to citrobacter rodentium infection in microrna-155-deficient mice immunology of tuberculosis mycobacterium tuberculosis triggers host type i ifn signaling to regulate il-1beta production in human macrophages cytokines: accelerators and brakes in the pathogenesis of cerebral malaria type i interferons suppress cd4(+) t-cell-dependent parasite control during blood-stage plasmodium infection ifnar1 controls progression to cerebral malaria in children and cd8+ t cell brain pathology in plasmodium berghei-infected mice ifitm3 limits the severity of acute influenza in mice genetic susceptibility to the delayed sequelae of neonatal respiratory syncytial virus infection is mhc dependent genetic susceptibility to respiratory syncytial virus infection in inbred mice respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology the burden of respiratory syncytial virus infection in young host transcription profiles upon primary respiratory syncytial virus systemic signature of the lung response to respiratory syncytial virus plasticity and virus specificity of the airway epithelial cell immune response during respiratory virus global gene expression profiling in infants with acute respiratory syncytial virus broncholitis demonstrates systemic activation of interferon signaling networks cholesterol-rich microdomains as docking platforms for respiratory syncytial virus in normal human bronchial epithelial cells advances in understanding respiratory syncytial virus infection in airway epithelial cells and consequential effects on the immune response ifitm proteins restrict viral membrane hemifusion the n-terminal region of ifitm3 modulates its antiviral activity by regulating ifitm3 cellular localization a systemsbased approach to analyse the host response in murine lung macrophages challenged with respiratory syncytial virus we would like to thank c. brandt for maintaining mouse colony health and wellbeing. key: cord-002100-dt5zvebj authors: he, yonghua; schmidt, monica a.; erwin, christopher; guo, jun; sun, raphael; pendarvis, ken; warner, brad w.; herman, eliot m. title: transgenic soybean production of bioactive human epidermal growth factor (egf) date: 2016-06-17 journal: plos one doi: 10.1371/journal.pone.0157034 sha: doc_id: 2100 cord_uid: dt5zvebj necrotizing enterocolitis (nec) is a devastating condition of premature infants that results from the gut microbiome invading immature intestinal tissues. this results in a life-threatening disease that is frequently treated with the surgical removal of diseased and dead tissues. epidermal growth factor (egf), typically found in bodily fluids, such as amniotic fluid, salvia and mother’s breast milk, is an intestinotrophic growth factor and may reduce the onset of nec in premature infants. we have produced human egf in soybean seeds to levels biologically relevant and demonstrated its comparable activity to commercially available egf. transgenic soybean seeds expressing a seed-specific codon optimized gene encoding of the human egf protein with an added er signal tag at the n’ terminal were produced. seven independent lines were grown to homozygous and found to accumulate a range of 6.7 +/3.1 to 129.0 +/36.7 μg egf/g of dry soybean seed. proteomic and immunoblot analysis indicates that the inserted egf is the same as the human egf protein. phosphorylation and immunohistochemical assays on the egf receptor in hela cells indicate the egf protein produced in soybean seed is bioactive and comparable to commercially available human egf. this work demonstrates the feasibility of using soybean seeds as a biofactory to produce therapeutic agents in a soymilk delivery platform. each year in the united states, more than 530,000 babies, approximately 12% of total births, are born before 37 full weeks of gestation [1] . as a growing health issue the rate of premature birth has increased by 36 percent since the early 1980s. one of the major problems associated with prematurity is the development of a condition known as neonatal necrotizing enterocolitis (nec) [2] . this is observed clinically as the abrupt development of bloody diarrhea, abdominal swelling, and tenderness in a premature infant who is otherwise doing well [3] . current treatment often requires surgical removal of the damaged and dead intestine, often resulting in mortality (about 40%) or, if the infant survives, to manifest significant resulting lifetime problems [3] [4] [5] . although the direct cause of nec is not known, the most significant contributing factor is premature birth. post-partum establishment of an abnormal gut microbiome creates the opportunity for bacterial invasion into gut due to immature intracellular junctions of the intestinal mucosa [6, 7] . experimental and clinical evidence suggest that prematurity and nec is associated with deficient endogenous production of epidermal growth factor (egf), which is necessary for normal intestinal development and repair [8, 9] . egf is a critical growth factor found in multiple fluids that bathe the developing intestine including amniotic fluid, fetal urine, breast milk, bile, and saliva [2, 10, 11] . in the amniotic fluid, there is an increasing concentration of egf as gestation progresses [12] . egf amounts in mother's milk is highest first days after parturition with mothers of extreme pre-term neonates having 50-80% higher than mother's milk of full term infants [13] . human studies have demonstrated that egf is resistant to proteolytic degradation across a range of gastric ph [14] . while egf is produced to some extent in duodenal brunner's glands and kidney, the vast majority of egf is produced in the salivary glands [15] . exogenous infusion of egf in utero has been shown to accelerate the maturation of intestinal enzyme activity as well as stimulate intestinal growth [16, 17] . the importance of egf to gut development is highlighted by the fact that knockout of the egf receptor in some mice strains results in death due to a bloody diarrhea that is remarkably similar to human nec [18] . transgenic mice directed to intestinally overexpress egf displayed a number of beneficial effects, including increased body weight and villus height, after a small bowel resection compared to nontransgenic mice [19] . conversely, inhibition of egf receptors impairs intestinal adaption following a small bowel resection [20] . a prospective, multi-center trial demonstrated that infants fed regular formula (not containing growth factors) were 6 to 10 times more likely to develop nec than infants fed breast milk [21] . while a large number of biologically active peptides and growth factors have been identified in breast milk, egf is one of the major peptides present in significant concentrations [22] . the concentration of egf in milk is found to be inversely proportional to the gestational age of the infant, therefore, the more premature the infant, the more egf is present in the breast milk [13] . this may be a compensatory response to the premature removal of the fetus from the egf-rich amniotic fluid. it has been demonstrated in several animal models of nec that administration of exogenous egf has been shown to significantly reduce the severity of intestinal injury [23, 24] . the proactive treatment of infants at nec risk with egf supplementation could therefore accelerate intestinal maturation, thus preventing the development of nec. if the proactive egf feeding strategy is effective to induce the maturation of the neonate intestinal tract then this simple approach may mitigate the development of nec with its resulting high costs in medical resources, pain and possible life-long debilitation and for the 40% infants with nec that proves fatal [25] [26] [27] . to accomplish such a proactive therapeutic approach adapting infant formula for egf delivery would be simple and economic and mimic the delivery of egf in mother's milk. the need and potential delivery makes infant formula containing egf a good model for food-sourced plant biotechnology. soybean-derived formula encompasses a significant fraction of the total infant formula market. soybean milk and derived products are a potential food-therapy delivery platform that could include a variety of medically-necessary products including drugs such as egf but might also include oral vaccines [28, 29] . the economy of production and simple conversion into therapeutic materials that has long-shelf life makes soybean biotech products a potentially desirable commodity for use in cost-sensitive scaled applications. addressing the devastating disease of nec through a simple proactive treatment protocol is an excellent platform to explore the potential of soybean-produced therapeutics. here we report the accumulation of human egf (hegf) in geneticallyengineered soybean seeds and show that the recombinant egf is indistinguishable from authentic human egf and is bioactive at stimulating egf receptor (egfr) activity. epidermal growth factor protein from humans was produced in soybean seeds by constructing a plant gene expression cassette that involved a synthetic codon optimized egf nucleotide sequence (protein sequence from genbank accession ccq43157). this 162 bp open reading frame was placed in-frame behind a 20-amino acid endoplasmic reticulum (er) signal sequence from the arabidopsis chitinase gene [30, 31] . the er-directed egf encoding open reading frame was developmentally regulated by the strong seed-specific storage protein glycinin regulatory elements [31] . the entire seed specific cassette to direct egf production was placed in a vector containing the hygromycin resistance gene under the strong constitutive expression of the potato ubiquitin 3 regulatory elements as previously described [31] [32] [33] . the result plasmid pgly::shegf was sequenced using a glycinin promoter primer (5' tcattcac cttcctctcttc 3') to ensure the egf open reading frame was placed correctly between the regulatory elements. somatic soybean (glycine max l. merrill cv jack (wild type)) embryos were transformed via biolistics using 30 mg/l hygromycin b selection and regenerated as previously described [34] . embryos from resistant lines were analyzed by genomic pcr to confirm the presence of inserted hygromycin cassette using primers specific to the hygromycin gene (hygf 5'ctcactattcctttgccctc3' and hygr 5'ctgacctattgcatctcccg3'), cetyl trimethyl ammonium bromide (ctab) extraction genomic dna isolation and the following amplification conditions: 150 ng genomic dna in 25 μl total reaction containing 200 nm primers and 3 u taq polymerase (neb) and the following cycling parameters (initial 95°c 4 min then 45 cycles of 95°c 30 s, 55°c 45 s, 72°c 90s; followed by a final extension of 72°c 7 min). dry seeds from two successive generations of pcr positive plants were analyzed by elisa for the expression of egf protein until all 7 lines were confirmed to be homozygous. egf transgenic soybean plants along with nontransgenic control wild type cultivar plants were grown side by side in a greenhouse at 25°c under 16 h daylight with 1000 μm -2 /s. total soluble protein was extracted from dry seeds of two homozygous egf lines and a nontransgenic control by repeated acetone washes followed by acetone precipitation with the protein pellet dissolved in water. proteins with molecular weight 10 kda and under were isolated by separately passing each extract through an amicon ultra centrifugal filter (merck, kenilworth nj). the samples were each suspended in sample buffer (50mm tris hcl, ph6.8 2% sds (w/v), 0.7 m β-mercaptoethanol, 0.1% (w/v) bromphenol blue and 10% (v/v) glycerol) and then denaturated 5 min 95°c. protein content was determined by bradford assay [35] . a 15% sds-page gel was used to separate 30 μg protein for each of the three samples: negative control wild type, lines 4 and 5 of egf transgenic soybean dry seeds. commercially available human egf (gibco, life technologies,united kingdom) was used at 0.5 μg as positive control. gel was electroblotted onto immobilon p transfer membrane (millipore, bedford ma) and blocked with 3% milk solution in tbs for at least 1 hr. primary antibody was a commercially available anti-egf (calbiochem, san diego ca) and was used in a 1:100 ratio in 3% bsa-tbs buffer overnight at room temperature. after 3 washes of 15 mins each with tbs buffer, the blot was incubated with a 1:10,000 ratio in tbs of secondary antibody anti-rabbit igg fabspecific alkaline phosphatase conjugate (sigma, st. louis mo). after 3 washes, the presence of the egf protein was detected by using a color substrate (bcip/nbt: final concentrations 0.02% (w/v) 5-bromo-4-chloro-3-indoyl phosphate and 0.03% (w/v) nitro blue tetrazolium in 70% (v/v) dementhylformadmide) (kpl, gaithersburg ma). total soluble protein was extracted from dry soybean seeds as described previously [31, 32] from all 7 lines of pgly::shegf transgenic plants along with nontransgenic seeds as a negative control. egf was quantitated by commercially available human egf elisa assay (quantikine elisa kit from r&d systems, minneapolis mn) according to the manufacturer's instructions. the provided positive control was used to create a standard curve in order to determine the amount of egf in each soybean protein extract. each homozygote egf transgenic line was assayed with three biological replicates and results displayed as mean +/-standard error. total soluble proteins were extracted, quantitated and suspended in sample loading buffer as previously described [31, 32] . approximately 30 μg of protein extract from dry seeds of 4 homozygous egf lines were separated on a 4-20% gradient sds-page gel (biorad, hercules ca) along with extract from a nontransgenic seed. the gel was subsequently stained with 0.1% total soluble protein was extracted from 3 biological egf transgenic soybean dry seed samples, lines 4, 5 and 6. as described above, proteins with molecular weights lowers than 10 kda were concentrated using an amicon ultra centrifugal filter (merck, kenilworth nj). non-transgenic seeds were used as a negative control and 5 μg commercially available egf (as above in immunoblot section) was the positive control. protein was precipitated by adjusting the solution to 20% (v/v) trichloroacetic acid and allowed to sit at 4°c overnight. precipitated proteins were pelleted using centrifugation, washed twice with acetone and then dried using vacuum centrifugation. the commercial egf was not filtered or precipitated, only dried. dried pellets were rehydrated with the addition of 10 μl 100 mm dithiothreitol in 100 mm ammonium bicarbonate and placed at 85°c for 5 minutes to reduce disulphide bonds. samples were then alkylated with addition of 10 μl iodacetamide in 100 mm ammonium bromide and placed at room temperature in the dark for 30 minutes. two μg trypsin in 200 μl 100 mm ammonium bromide was added to each samples and placed in 37°c overnight for enzymatic digestion. post trypsin digest samples were desalted using a peptide reverse phase microtrap (michrom bioresources, auburn ca), dried and ultimately resuspended in 2 μl of 2% (v/v) acetonitrile, 0.1% (v/v) formic acid. separation of peptides was performed using a dionex u3000 splitless nanoflow hplc system operated at 333 nl minute using a gradient from 2-50% acetonitrile over 60 minutes, followed by a 15 minute wash with 95% acetonitrile and a 15 minute equilibration with 2% acetonitrile. the c18 column, an in-house prepared 75 μm by 15 cm reverse phase column packed with halo 2.7 μm, 90å c18 material (mac-mod analytical, chadds ford pa) was located in the ion source just before a silica emitter. a potential of 2100 volts was applied using a liquid junction between the column and emitter. a thermo ltq velos pro mass spectrometer using a nanospray flex ion source was used to analyze the eluate from the u3000. scan parameters for the ltq velos pro were one ms scan followed by 10 ms/ms scans of the 5 most intense peaks. ms/ms scans were performed in pairs, a cid fragmentation scan followed a hcd fragmentation scan of the same precursor m/z. dynamic exclusion was enabled with a mass exclusion time of 3 min and a repeat count of 1 within 30 sec of initial m/z measurement. spectra were collected over the entirety of each 90 minute chromatography run. raw mass spectra were converted to mgf format using msconvert, part of the proteowizard software library [36] x!tandem 2013.09.01.1 [37] and omssa [38] algorithms were employed via the university of arizona high performance computing center to perform spectrum matching. precursor and fragment mass tolerance were set to 0.2 daltons for both omssa and x!tandem. trypsin cleavage rules were used for both algorithms with up to 2 missed cleavages. amino acid modifications search consisted of single and double oxidation of methionine, oxidation of proline, n-terminal acetylation, carbamidomethylation of cysteine, deamidation of asparagine and glutamine and phosphorylation of serine, threonine, and tyrosine. x!tandem xml and omssa xml results were filtered using perl to remove any peptide matches with an evalue > 0.05 as well as proteins identified by a single peptide sequence. the protein fasta database for glycine max was downloaded on august 5, 2015 from ncbi refseq with the addition of the egf amino acid sequence. a randomized version of the glycine max fasta was concatenated to the original as a way to assess dataset quality. the mass spectrometry proteomics data have been deposited to the proteomexchange constortium (http://proteomecentral. proteomexchange.org) via the pride partner repository [39] with the dataset identifier pxd003326 and 10.6019/pxd003326. hela cells (obtained from american tissue culture collection) were cultured in minimum essential media (mem) complemented with 10% fetal bovine serum (fbs), 100 units/ml penicillin, and 100 μg/ml streptomycin. for western blotting assay, cells grown in 6-well plate were kept in serum free mem media for 24 hours. cells were then either kept in serum free medium (control) or stimulated with soy milk alone, soy egf or commercial recombined human egf for different time period as indicated. cells were lysed by directly adding 1× sds sample buffer (50 mm tris-hcl, ph 6.8, 10% glycerol, 2% sds and 5% β-me) to the cells after washing 3 times with 1x pbs. egf bio-activity was determined via egfr phosphorylation and downstream akt phosphorylation. total egfr was also measured since egfr is known to undergo internalization when stimulated with egf. antibodies used in western blot are anti-p-egfr (tyr1068) (#2234, cell signaling technology), anti-total egfr (#06-847, millipore), anti-p-akt (#4060, cell signaling technology) and anti-lamin b1 (# 13435, cell signaling technology) [40] . for immunocytochemistry assay, cells were grown on coverslip in 6-well plate and kept in serum free media for 24 hours before stimulation, cells were then either kept in serum free media (control) or stimulated with human or soy egf for 6 hours. cells were washed with pbs and fixed with 4% formalin. egfr was labeled using anti-egfr antibody (#4267, cell signaling technology) and detected with alexa fluor 594 goat anti-rabbit igg (#a11012, life technology). the cell nucleus were shown using mounting medium with dapi (#h-1200, vectorshield). to produce hegf in soybean a strong soybean seed-specific promoter and terminator was used to regulate gene expression of a synthetic soybean codon optimized hegf (shegf) gene that included an n-terminal 60 nucleotide er-signal sequence (fig 1a) . in the engineering strategy for the hegf expression in soybean, the components of the prepro portions of hegf were eliminated in preference to produce only the final recombinant hegf product. to facilitate the co-translational transfer of the egf into the er lumen for disulfide bond formation a plant signal sequence was added so that the hegf synthesized would be as a pre-hegf. the gly::shegf construct was used for biolistic transformation of soybean somatic embryo cells as outlined in [31] [32] [33] [34] . embryos were selected in liquid culture by hygromycin b and individual regenerated lines were separated, propagated, and induced to form cotyledonary embryos. the cotyledonary embryos were evaluated for hegf production using egf-specific elisa that indicated a variation of heterologous protein production (data not shown). the most promising egf expressing lines were moved forward for regeneration by desiccating and subsequent germination. the initial t 0 generation egf transgenic plants were grown in the greenhouse and further selected by genomic pcr for an additional 2-3 generations. additionally, each generation of seeds produced by the selected lines were assayed for hegf content by elisa. the hegf content of each line in seeds representative of the homozygous population is shown in fig 1b. the lines varied in hegf content but seeds within each line had a narrow range of hegf accumulation. the egf transgenic line 5 produced in excess of 100 μg hegf per gm dry seed weight, a level calculated to be much in excess of potential therapeutic requirements. by comparison, yeast stains have been used as an expression system for both human egf [41] and mouse egf [42] with the highest levels produced being from a multicopy insert pichia pastoris clone secreting 49 μg egf/ml. in both the mouse and human egf yeast production systems, truncated versions of the egf were detected. the hegf soybeans and nontransgenic soybeans were evaluated to determine the biochemical authenticity of the soybean-produced egf protein. using 1d sds/page and parallel immunoblots probed with anti-egf, the soluble low molecular weight (<10 kda) seed proteins and the mr of the soybean-produced hegf was evaluated. the total protein polypeptide of the hegf expressing lines appeared to be identical to the standard parental control (fig 2) . immunoblots of the 1d sds/page probed with anti-egf showed a lack of an immunoreactive band in the nontransgenic soybean seed control and recognized a 6 kda mr band in the hegf expressing lines 5 and 4. the soybean-produced hegf has the same apparent mr as authentic recombinant hegf fractioned in an adjacent lane (fig 3) . to further assess the soybean-synthesized hegf the seed lysates were enriched in low mr total proteins and concentrated. the crude low mr proteins were reduced, alkylated, and cleaved with trypsin prior to analysis by mass spectrometry. the resulting data was queried with the hegf sequence and exact matches for peptides encompassing the majority of the sequence of the complete mature hegf protein were obtained (fig 4) . together the data shows that transgenic soybeans successfully produced and accumulated hegf that is the correct mr, is immunoreactive with antibodies directed at authentic egf in both elisa and immunoblot assay, and that a majority mass spectrometry of fragments of the soybean-produced hegf match the human egf sequence. the delivery of any biopharma product in the context of compositionally complex food presents the potential that the components of the food may act to modulate bioactivity. plantsource foods in particular pose problems because plant tissues often possess a wide range of intrinsic biologically active components including proteins and natural products. the natural products of food could mask or enhance the effects of an expressed biopharma product. to evaluate the potential of egf activity in soymilk delivery commercial recombinant human egf (rhegf) was added as a supplement to soymilk and the intrinsic activity of the egf was tested with a hela cell assay. fig 5 shows the effects of soymilk on the display of the egf receptor (egfr) on hela cells and the effect of commercial rhegf supplement to soymilk. soymilk does not modify the display of egfr on hela cells showing that soymilk alone is biologically inactive. the binding of egf to egfr results in the decrease of displayed egfr as it is internalized into the hela cells. hela cells treated with commercially available recombinant rhegfsupplemented soymilk display the same decrease in egfr as cells treated with rhegf in media without soymilk. parallel time-course experiments show that the effect of rhegf binding to efgr is rapid with a reduction of displayed efgr occuring within 5 min of treatment and continuing out to at least 30 min (data not shown). together these assays show that soymilk has no apparent negative bioactivity with respect to both the binding of commercial rhegf to the hela cell egfr or the viability of the hela cells over the course of the assay. to assess the bioactivity of soybean-produced hegf, samples were prepared from both shegf transgenic soybean lines and nontransgenic controls that were used to stimulate hela cells to induce egfr internalization, degradation and phosphorylation. in results shown in fig 5, soybean-produced hegf induces the internalization, degradation and phosphorylation of egfr that is indistinguishable from the bioactivity of commercial rhegf delivered in control samples. in contrast, samples prepared from control nontransgenic soybeans exhibited no apparent bioactivity showing the degradation and phosphorylation of egfr is the result of egf binding of either commercial rhegf added to the media or from the hegf produced by the transgenic soybeans. together these results show that nontransgenic soybean seeds have no intrinsic egf-mimic activity able to induce egfr degradation or phosphorylation, while soybeans producing hegf have identical activity in comparison to commercial rhegf. soybean produced egf displayed comparable bioactivity to commercially available egf. panel a. soybean produced hegf induces a rapid phosphorylation of hela cell egfr. serum free media (sf) and sf media with soymilk alone does not induce egfr phosphorylation and degradation. soymilk from seeds producing shegf added at different concentrations (0.1, 0.05, 0.025 μg/ml) induced concentrationdependent egfr degradation comparable to the effect of rhegf. serum free media and serum free media with nontransgenic soybean soymilk (negative controls) showed no effect on inducing pegfr. in contrast soymilk from shegf soybeans given at different concentrations (0.1, 0.05, 0.025 μg/ml) induced pegfr comparable to control rhegf. pakt indicates the functional activation of egfr. lamin b1 was used as a loading control. panel b. exogenous commercial rhegf and shegf induces an internalization and degradation of egfr in hela cells shown as a decrease in abundance assayed by immunoblot. the results shown demonstrate that soymilk alone has no intrinsic bioactivity with respect to egfr abundance. the rhegf is not degraded in soymilk over 24 hours having the same bioactivity as control recombinant rhegf.-ctrl-sf media alone. soy egf and rhegf are at 0.1 μg/ml. lamin b1 was used as a loading control. panel c. shown is an immunohistochemical assay of hela cells showing that shegf induces internalization of the egfr comparable to that from control rhegf. in c, the cells were first treated with soy/egf or human egf for 6 hours, fixed and then immunostained with egfr antibody overnight. egfr shows red staining while nucleus was stained by dapi and shows blue staining. in developing a food-based delivery platform for biopharma it is important to address the question of whether there are significant collateral consequences in seed composition resulting from the genetic modification. ideally a consumption plant biotechnology platform, such as soymilk, should be fully equivalent to the standard type other than the intended modification. seeds in general, including soybeans, possess an inventory of bioactive proteins and small molecules that will affect the metabolism of consumers in both advantageous and disadvantageous manner. for soybeans some of the relevant molecules are allergens, anti-metabolite proteins, and small molecules especially isoflavones. to test for potential collateral composition in the hegf-producing soybeans, the shegf transgenic and nontransgenic control soybeans were analyzed by non-targeted proteomics and metabolomics. among the significant proteins identified include various well-documented allergens and anti-metabolite proteins. a comparison of standard soybeans with hegf-producing soybean lines showed that there was no significant difference (p = .01) between nontransgenic control and shegf transgenic soybeans aside from the targeted production of hegf for any other proteins of concern. this data is available in pride partner repository with the dataset identifier pxd003326 and 10.6019/pxd003326. non-targeted small molecule metabolomics was used to conduct a parallel analysis of the nontransgenic and hegf soybeans. again there were insignificant differences between nontransgenic soybean seeds and the shegf transgenic seeds (fig 6) with one notable exception. soybean highly regulates sulfur availability and its allocation into protein. from a nutritional perspective soybean is considered a somewhat sulfur deficient crop. there have been a number of biotechnology experiments to increase sulfur content be either modifying assimilation and biosynthesis pathways leading to methionine or over-expressing high-methionine proteins such as maize zeins. modifying sulfur by pathway or competition has an effect on sulfurresponsive proteins including the bowman-birk trypsin inhibitor (bbi) and beta chain of the storage protein conglycinin. egf is a high sulfur content protein that broadly mimics bbi as a small globular protein synthesized by the er and presumptively competing for sulfur amino acid charge trna. expressing hegf in soybean has an effect on metabolites involved in sulfur amino acid metabolism that is consistent with producing a protein of egf's composition. a complete dataset of all metabolite abundance of the standard and hegf-expressing lines is available as an on-line spreadsheet (s1 table) . among the assayed molecules of particular note is the soybean molecule genistein, an isoflavone that has been shown to affect the activity of tyrosine phosphatase in the signal cascade associated with egf signaling [43] [44] [45] [46] . genistein levels were determined to be the same in both the nontransgenic and hegf-expressing soybean lines. this too demonstrates that the expression of hegf in soybeans does not produce any incidental collateral consequences of concern for its potential therapeutic use. since the inception of plant biotechnology its potential use for biopharma applications has been assessed [47] [48] [49] [50] . several different plant organs proposed as production for food/feed delivery systems [51] [52] [53] . for many vaccine applications fruit are a highly advantageous delivery system providing a broadly accepted platform for even the most recalcitrant consumer (for example, [54, 55] ). although fruit are perhaps one of the best delivery systems from the perspective of point of delivery, fruit also has logistical issues with relatively short time that ripened fruit are palatable requiring a tightly coordinated effort to produce, distribute, and use biopharma product that could be challenging for deployment in mass quantities. an alternative fig 6. relative proportion of non-targeted metabolites detected in soybean seeds shown as amount in egf transgenic compared to nontransgenic (wt). complete list of non targeted metabolites quantitated in samples in s1 table. doi:10.1371/journal.pone.0157034.g006 transgenic soybean production of bioactive human epidermal growth factor (egf) is to develop a biopharma platform that is broadly acceptable for food and feed delivery but can be lightly processed to preserve bioactivity and can be massively scaled to maximize the distribution potential of the product. soybean is a potentially useful biopharma platform that could have broad application in both food and feed end uses [29, 30, 56, 57] . soybean has been demonstrated as a platform to produce heterologous proteins at a standard that far exceeds the levels typically needed for biopharma [31] . soybeans can be used to produce both soymilk and formula for potential delivery to human infants or children as well as for production animals such as swine and calves. soybean can also be used to produce protein concentrates for inclusion in industrial food and feed or more simply as protein aggregates as tofu. soybean production is efficient and economic that can be massively scaled if needed. recently developed technology makes it feasible to increase the amount of recombinant protein product by silencing and exchange with a storage protein(s) [31, 58, 59] . as a platform, soybean is an industrial crop with vast majority of its total production being directed toward products including processed food, protein used as animal feed, and its oil for food, feed, fuel, and chemical feedstock uses. many of the goals of further enhancing and modifying soybeans are largely directed at improving its utilization for industry products rather than direct food use. as a biopharma platform to produce soymilk derived products soybean seeds can be stored for years anticipating future needs while retaining the potential to be rapidly processed into formula/milk or tofu using adaptations of traditional technology in use for over a millennium. soybeans like many other seeds produce an array of intrinsic small globular proteins with secondary disulfide bonds accumulated at relatively high levels (>1% of total proteins). soybean in particular accumulates the bowman-birk trypsin inhibitor that is 8.5 kda with 3 intra-chain disulfide bonds [60] . this suggests that soybean seeds are optimized as a potential bioreactor to produce and store proteins like egf, a 6.9 kda protein with 3 intra chain disulfide bonds paralleling intrinsic seed proteins. in a predecessor experiment a mutant inactive bbi was expressed in transgenic soybeans showing that alternate small proteins can be expressed in soybean [60] . expression of a construct encoding shegf regulated by the soybean seed storage protein promoter results in the accumulation of hegf at > 100 μg /gm of dry soybean seed, a level to be many fold over the estimated therapeutic requirements of 50 μg/kg weight of treated individual [61] . soybean-produced hegf appears to be completely comparable to authentic hegf in its mr, immunoreactivity with specific antibodies, correspondence of fragment sequence in mass spectrometry assay, and in bioactivity inducing the internalization, degradation and phosphorylation of efgr. together the results shown demonstrate that soybean seeds will produce hegf at proto-therapeutic levels and the derived hegf from these seeds are bioactive for egf activity in a model hela cell assay. the expression of hegf in soybean has little collateral impact on seed composition soybeans have been used as an expression platform for a wide variety of heterologous proteins with investigative as well potential food/feed and biopharma goals [33, [62] [63] [64] [65] [66] . prior biopharma expressions have included prototype expression of vaccine models [67] as well as proinsulin and fibroblast and human growth factor [68, 69] . in this study potential collateral changes in prototype product mature soybean seeds resulting from hegf expression was evaluated by non-targeted proteomics and metabolomics to assess both large and small molecules. these assessments showed that there was no significant difference in the seed proteome of the egf transgenics compared to nontransgenics. this is a pertinent result as soybeans are regulated in the us under falpa (the 2004 food and allergen labeling protection act) and unintended alterations of any of the known seed allergens or anti-nutritional proteins can be of concern. similarly the non-targeted metabolomics of the soybean seeds showed a significant lack of alteration of the small molecule profile in response to hegf accumulation. among the molecules assessed the lack of change in genistein is among the most significant as this isoflavonoid has been shown to have activity with tyrosine phosphatase that is in the signal cascade of animal and human cells that includes egf/egfr signaling [43, 44, 70] . in the hela cell assessments there was no synergistic effect of standard soybean milk and authentic egf on egfr activity indicating that the identical genistein concentration in the standard and hegf expressing soybeans is below the threshold of effect in the assays conducted. the one significant alteration in the small molecule profile was in methionine-related metabolism. egf is a sulfur rich protein containing three disulfide bonds that has some general resemblance to the soybean bowman-birk inhibitor. soybean is a relatively sulfur deficient crop and much effort has been made to increase its sulfur amino acid content by either the co-expression of sulfurrich proteins such as zeins [71] or by increasing the sulfur flux by altering the sulfur amino acid pathways [72] [73] [74] . these studies have shown that within limits the increase of a sulfur sink such as expressing a high-sulfur content protein will collaterally induce modest increases in sulfur amino acid source. the results of increases in sulfur amino acid metabolites accompanying hegf expression in soybean is in accord with these prior experiments. together the results of the non-targeted proteome and metabolome assessments show that converting soybean into a prototype biopharma delivery platform of hegf does not result in any adverse alterations of the soybean seed's composition. soybeans could be used to produce biopharma products that might be delivered as milk or formula. as a test of this concept human epidermal growth factor (hegf) has been produced in soybeans to potentially address the devastating disease of neonatal necrotizing enterocolitis. this is a disease of premature infants of low birth weight. these infants have underdeveloped organs including the intestinal tract. the resulting gangrenous infection is treated by emergency surgery to remove dead portions of the intestinal tract that even under most optimistic situations has a high mortality rate and high cost of treatment. an alternative approach is to proactively treat infants at risk immediately post-partum to attempt to improve the integrity and maturity of the lining epithelial cells. the bioactivity results with model hela cells shows that hegf can be produced and accumulated in soybean seeds and as crude soy-milk lysate is capable of stimulating a response from the egf receptor (egfr) that occurs on epidermal surfaces such as the intestinal tract. soybean-produced hegf has potential other applications in cosmetics, burn and injury treatment, stimulating improved adaptation of the bowel to massive intestinal loss. supporting information s1 table. non-targeted metabolome set. births: final data for 2010 role of human milk in extremely low birth weight infants' risk of necrotizing enterocolitis or death necrotizing enterocolitis: the search for a unifying pathogenic theory leading to prevention neonatal necrotizing enterocolitis: a nine year experience: ii outcome assessment long-term survival and parenteral nutrition dependence in adult patients with the short bowel syndrome 16s rrna gene-based analysis of fecal microbiota from preterm infants with and without necrotizing enterocolitis fecal microbiota in premature infants prior to necrotizing entercolitis establishment of the gut microbiota in western infants bacterial colonization and gut development in preterm neonates epidermal growth factor: biology and mechanism of action early human milk feeding is associated with a lower risk of necrotizing enterocolitis in very low birth weight infants epidermal growth factor (egf) concentrations in amniotic fluid and maternal urine during pregnancy increased epidermal growth factor levels in human milk of mothers with extremely premature infants human epidermal growth factor: isolation and characterization and biological properties immunocytochemical localization of human epidermal growth factor/urogastrone in several human tissues transforming growth factor alpha and epidermal growth factor in protection and healing of gastric mucosal injury epidermal growth factor enhances intestinal adaptation after massive small bowel resection epithelial immaturity and multiorgan failure in mice lacking epidermal growth factor receptor intestinal overexpression of egf in transgenic mice enhances adaptation after small bowel resection selective inhibition of the epidermal growth factor receptor impairs intestinal adaptation after small bowel resection breast milk and neonatal necrotizing enterocolitis human milk for the premature infant epidermal growth factor reduces the development of necrotizing enterocolitis in a neonatal rat model intestinal barrier failure during experimental necrotizing enterocolitis: protective effect of egf treatment short bowel syndrome management of the short bowel syndrome in the pediatric population pediatric shortbowel syndrome: the cost of comprehensive care plant made pharmaceuticals: from edible vaccines to ebola therapeutics towards using biotechnology to modify soybean seeds as protein bioreactors. in, recent advancements in plant expression in crop plants production of escherichia coli heat labile toxin (lt) b subunit in soybean seed and analysis of its immunogenicity as an oral vaccine the collateral protein compensation mechanism can be exploited to enhance foreign protein accumulation in soybean seeds a rnai knockdown of soybean 24 kda oleosin results in the formation of micro-oil bodies that aggregate to form large complexes of oil bodies and er containing caleosin transgenic soybean seeds accumulating β-carotene exhibit the collateral enhancements of high oleate and high protein content traits towards normalization of soybean somatic embryo maturation a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding proteowizard: open source software for rapid proteomics tools development tandem: matching proteins with tandem mass spectra open mass spectrometry search algorithm retinoblastoma protein (prb), but not p107 or p130, is required for maintenance of enterocyte quiescence and differentiation in small intestine the proteomics identifications (pride) database and associated tools: status in 2013 characterization of recombinant human epidermal growth factor produced in yeast production of mouse epidermal growth factor in yeast: high level secretion using pichia pastoris strains containing multiple gene copies genistein, a tyrosine kinase inhibitor, reduces egf-induced egf receptor internalization and degradation in human hepatoma hepg2 cells genistein alters growth factor signaling in transgenic prostate model (tramp) genistein enhances relaxation of the spontaneously hypertensive rat aorta by transactivation of epidermal growth factor receptor following binding to membrane estrogen receptors-α and activation of g protein-coupled, endothelial nitric acid synthase-dependent pathway genistein increases epidermal growth factor receptor signaling and promotes tumor progression in advanced human prostate cancer the production of recombinant pharmaceutical proteins in plants sowing the seeds of success: pharmaceutical proteins from plants recombinant pharmaceuticals from plants: the plant endomembrane system as bioreactor commercialization of biopharmaceutical and bioindustrial proteins from plants efficacy of a food plant-based oral cholera toxin β subunit vaccine oral immunization with hepatitis β surface antigen expressed in transgenic plants immunogenicity of recombinant lt-b delivered orally to humans in transgenic corn expression of hepatitis b surface antigen (hbsag) gene in transgenic banana (musa sp) severe acute respiratory syndrome (sars) s protein production in plants: development of recombinant vaccine expression and immunogenicity of an escherichia coli k99 fimbriae subunit antigen in soybean protein expression systems: why soybean seeds?" in soybean-molecular aspects of breeding cosuppression of the α-subunits of β-conglycinin in transgenic soybean seeds induces the formation of endoplasmic reticulum-derived protein bodies silencing of soybean seed storage proteins results in a rebalanced protein composition preserving seed protein content without major collateral changes in the metabolome and transcriptome reduction of protease inhibitor activity by expression of a mutant bowman-birk gene in soybean seed epidermal growth factor augments adaptation following small bowel resection: optimal dosage, route, and timing of administration expression of functional recombinant human growth hormone in transgenic soybean seeds processing and localization of bovine β-casein expressed in transgenic soybean seeds under control of a soybean lectin expression cassette embryo-specific silencing of a transporter reduces phytic acid content of maize and soybean seeds metabolically engineered oilseed crops with enhanced seed tocopherol an alternative to fish oils: metabolic engineering of oilseed crops to produce omega 3 long chain polyunsaturated fatty acids correct targeting of proinsulin in protein storage vacuoles of transgenic soybean seeds high-level expression of basic fibroblast growth factor in transgenic soybean seeds and characterization of its biological activity expression of correctly processed human growth hormone in seeds of transgenic tobacco plants genistein analogues: effects on epidermal growth factor receptor tyrosine kinase and on stress-activated pathways increased sulfur amino acids in soybean plants overexpressing the maize 15 kd zein protein enhanced levels of methionine and cysteine in transgenic alfalfa (medicago sativa l.) plants over-expressing the arabidopsis cystathionine γ-synthase gene transgenic soybean plants overexpressing o-acetylserine sulfhydrylase accumulate enhanced levels of cysteine and bowman-birk protease inhibitor in seeds effects of proteome rebalancing and sulfur nutrition on the accumulation of methionine rich δ-zein in transgenic soybeans front key: cord-000547-adfigzc1 authors: beniac, daniel r.; melito, pasquale l.; devarennes, shauna l.; hiebert, shannon l.; rabb, melissa j.; lamboo, lindsey l.; jones, steven m.; booth, timothy f. title: the organisation of ebola virus reveals a capacity for extensive, modular polyploidy date: 2012-01-11 journal: plos one doi: 10.1371/journal.pone.0029608 sha: doc_id: 547 cord_uid: adfigzc1 background: filoviruses, including ebola virus, are unusual in being filamentous animal viruses. structural data on the arrangement, stoichiometry and organisation of the component molecules of filoviruses has until now been lacking, partially due to the need to work under level 4 biological containment. the present study provides unique insights into the structure of this deadly pathogen. methodology and principal findings: we have investigated the structure of ebola virus using a combination of cryo-electron microscopy, cryo-electron tomography, sub-tomogram averaging, and single particle image processing. here we report the three-dimensional structure and architecture of ebola virus and establish that multiple copies of the rna genome can be packaged to produce polyploid virus particles, through an extreme degree of length polymorphism. we show that the helical ebola virus inner nucleocapsid containing rna and nucleoprotein is stabilized by an outer layer of vp24-vp35 bridges. elucidation of the structure of the membrane-associated glycoprotein in its native state indicates that the putative receptor-binding site is occluded within the molecule, while a major neutralizing epitope is exposed on its surface proximal to the viral envelope. the matrix protein vp40 forms a regular lattice within the envelope, although its contacts with the nucleocapsid are irregular. conclusions: the results of this study demonstrate a modular organization in ebola virus that accommodates a well-ordered, symmetrical nucleocapsid within a flexible, tubular membrane envelope. viruses have evolved as genome packaging machines to efficiently transfer nucleic acids between susceptible host cells, ensuring replication. the majority of viruses have hollow, quasispherical shells rather than tubular structures, perhaps because this gives the most efficient packaging of nucleic acid with a fixed copy number of coat protein subunits. in non-enveloped viruses, the volume enclosed by the (usually) icosahedral structure is a constraint on the size of the genome, giving a limited capacity to encode capsid proteins, and usually restricts the genome copy number, or ploidy of the virion, to one [1] . most membraneenveloped viruses are also quasi-spherical, but their symmetry is frequently less well-ordered, which is usually described as pleomorphic. this feature allows some flexibility in volume, which could accommodate variation in the size of the genome or its copy number. nevertheless, most viruses, irrespective of their architecture, appear to have evolved to encapsidate only a single copy of their genome within the protein or protein/lipid shell, or a dimeric copy in retroviruses. notable exceptions are the paramyxoviridae and the birnaviridae where particles may contain up to four copies of the rna genomes [2, 3] . although some strains of influenza can produce elongated virions, there is a mechanism that selectively encapsidates only one set of genome segments in each virion [4] . the filoviridae family, including the ebolavirus and marburgvirus genera, cause haemorrhagic fevers with high mortality in humans, and no effective treatments are currently approved [5] , although candidate vaccines are promising [6] . the 18.9 kb single-stranded negative-sense non-segmented rna genome of ebola virus (ebov) codes for at least eight proteins. the ribonucleoprotein complex is composed of the nucleoprotein (np), polymerase protein (l), vp24, vp30, and vp35. the trimeric transmembrane glycoprotein (gp) forms surface spikes on the virion envelope and also has a soluble form, while the matrix protein, vp40, is associated with the inner surface. [5, [7] [8] [9] . the gp spike, a class i fusion protein, mediates cellular attachment and entry and is extensively glycosylated, especially in the glycan-rich mucin-like domain [10] [11] [12] [13] . three proteins, vp24, vp35 and np are essential for nucleocapsid formation [14] . although some of the major protein interactions that occur during ebov morphogenesis have been characterised [14, 15] , the three-dimensional (3d) structure and molecular arrangements have not been previously determined. structural details are essential to understand how protection of the genome, cell binding, entry, and immune evasion are achieved in a filamentous animal virus, and to determine how this unique morphology plays a role in pathogenesis. research on filoviruses has been hampered by their status as biosafety level 4 pathogens. previous investigations of filovirus structures within embedded, sectioned and metal-stained cells by electron tomography revealed few details of the high resolution oligomeric structure [16, 17] . it has been demonstrated that aldehyde-fixation alone, and subsequent cryo-electron microscopic imaging in the frozen-hydrated state preserves structures, at least up to 12 angstroms resolution [18] , and in some cases, fixation improves the resolution achievable [19] . in addition, it has also been shown that high-resolution x-ray structures can also be obtained in the presence of aldehyde fixatives [20] . therefore, we analyzed purified and isolated ebov and ebola virus-like structures using cryo-electron microscopy (cryo-em), and cryoelectron tomography (cryo-et). in the current study, the zaire strain of ebov was purified and inactivated by paraformaldehyde fixation: excess fixative was then removed by dialysis to reduce beam damage for imaging in the frozen-hydrated state. the flashfreezing at liquid ethane temperatures used in cryo-electron microscopy preserves the structural and molecular detail, avoiding artifacts associated with conventional em methods, such as dehydration and/or sectioning or staining, that usually prevent detailed structural analysis. digital image processing reveals the 3d organization of ebov, including the structural arrangement of component molecules at resolutions of 14-19 å . we identified filamentous ebov particles 20 microns or longer, with a well ordered internal structure, and a helical nucleocapsid giving an internal ''herring-bone'' appearance using cryo-em and cryo-et (figures 1, 2 , s1, s5). the nucleocapsid, as observed within intact viral particles, has a uniform helical structure (figures 1, 2, 3, 4) and is enveloped by a membrane coated by an external layer of gp spikes. from the same image data set, we combined extracted volumes from tomograms with 2-d single particle processing to determine the structure of the gp spikes ( figure 5 ) to a resolution of 14 å as measured by the fourier shell correlation (fsc) 0.5 criterion. virions are rarely straight. variation in the overall length of virions is non-random, and they fit into ordered size classes ( figure 1 ). analysis of 2090 distinct intact virions with a nucleocapsid from cryo-electron micrographs shows that the most common class length (53%) of virus particles is 982679 nm ( figure 1a , table s1 ). the other size classes are multiples of this length. enveloped filovirus particles have several empty and linked ebov structures were excluded from the histogram data. a single g1-single/comma shaped ebov is shown (inset on the right, g1 = 1 copy of genome). (b) low magnification cryo-images showing: g1-single/comma shape, g1-single/ linear, g5-continuous (g5 = 5 copies of genome). (c) high magnification of a g1-(single genome) virion with a region filtered to emphasize the nucleocapsid. (d) low magnification image of a g4-linked ebov, each genome copy is indicated and numbered, the red arrows show the transition points between nucleocapsids. the circular holes (filled with vitreous ice) appear as lighter regions and the support film (''quantifoil'') appears dark grey. a ''linker'' region is shown at higher magnification (inset). doi:10.1371/journal.pone.0029608.g001 different morphologies (shown in figures 6, s1 , s5). these configurations are ''single'' particles, containing a nucleocapsid of uniform length, (which we postulate to contain one copy of the genome), ''continuous'' particles, with nucleocapsids of a length of the single virion multiplied by an integer of 2 or greater; and ''linked'' virions composed of a series single-genome nucleocapsids connected by short sections of empty envelope. negative staining can cause drying and staining artefacts which hamper accurate measurements and preclude 3d analysis. nevertheless, in previous studies using this technique, the average length of marburg virus figure 2 . image processing of ebola virus. linear 2d averaging of ebov: the envelope and nucleocapsid are prominent features (a). the line trace is colour-coded as follows: red, spike; beige, lipid envelope; green, membrane-associated proteins; white, membrane-nucleocapsid gap; blue and purple, outer and inner nucleocapsid. (b) 2d class averages of envelope plus inner face. (c) vp40 vlps, showing 2d averages from the from side regions (first two) and end-on/central regions (last three). in (a-c) representative individual repeats have been highlighted in color using the same scheme as in (a). (d) schematic model of the nucleocapsid and envelope, highlighting the relative distribution of np to vp40. (e,f) 3d reconstruction of the nucleocapsid with the same colour scheme as in (a). the location of the inner nucleocapsid, and the bridge are indicated. the reconstruction is presented at a volume threshold that would encompass a single copy of each of these proteins, and the viral rna. in (e) the vertical (protein-protein) and horizontal (protein-rna) contacts are indicated by yellow and white arrows, respectively. (g) various recombinant nucleocapsid-like structures, and authentic ebov, which have been studied by electron microscopy [14, 15, 21, 28] . 3d schematics of these structures highlighting the rna and protein composition and the diameter of these structures, at the same scale for comparison to (e). doi:10.1371/journal.pone.0029608.g002 was measured as 790 nm, and ebov as 970 nm [21, 22] : the latter is very similar to the mean length of 982 nm measured in the current study by cryo-tem of frozen hydrated specimens. in addition, discrete sub-populations of virions of double or triple the average length were also previously reported in centrifuged virus preparations [21] . we also observed ''empty'' filaments that lack nucleocapsids, with a random length and a smaller diameter ( figure s1c ). these empty nucleocapsids are also visible in previously published thinsection micrographs of human ebov infected pathology specimens, e.g. [23] , and are thus probably not an artifact of cell culture. it is also unlikely that the polyploidy we have observed in ebov is a peculiarity of the particular viral isolate or of the vero cell line that we used for this investigation. previous studies using negative stain em have shown that both marburg virus and several different ebov species can produce filamentous virions up to 14 mm long when grown in several different cell lines [21, 22] . blood specimens from guinea-pigs and monkeys that were inoculated with primary isolates of the marburg agent, when centrifuged and observed by negative stain, also showed filamentous virions of variable length, with the average reported as about 1 mm in length but with a smaller proportion being over 2 mm in length [24] . in addition, viral filaments of lengths from 1.4 to 1.6 mm long, and occasionally as long as 2.6 mm, can also be seen in published ultrathin sections of ebov infected human and monkey tissues [23, 25] . thin-section em always underestimates the lengths of virions, since filament profiles are frequently truncated by the section plane: in addition, tissue shrinkage of 10 to 20% during dehydration and resin embedding is common. thus, it is probable that polyploid ebov with multiple nucleocapsids are also produced in naturally infected humans and animals, though it is not possible to make a direct comparison with cell cultured virus: it would be very difficult to obtain large enough quantities of concentrated virus from animals to carry out detailed cryo-tem analysis and measurements. the ebola nucleocapsid structure was solved to 19 å resolution (fsc 0.5 criteria) using linear regions of 34,605 images (taken at 3-4 mm defocus, figure 2 ). since virus particles were not straight enough for conventional helical image processing, a combination of tomography, sub-tomogram averaging, single particle averaging, and the iterative helical real-space construction method were used [26, 27] . the ebov nucleocapsid is a right-handed doublelayered helix with an outer diameter of 41 nm and a hollow inner channel 16 nm in diameter as determined by image analysis and tomography (figures 2, 3 , 4, s2, s4, movie s1). the pitch is 6.96 nm, with 10.81 repeats per helical turn ( figure 2 ). the inner nucleocapsid, composed of large subunits, is linked by vertical and horizontal contacts between the large subunits ( figure 2e ,f). the horizontal contacts occur between the large subunits at a diameter of 22.3 nm. the vertical contacts linking the coils have a higher density than the horizontal ones, so we interpret the horizontal contacts as involving viral rna (white arrow, figures 2e, 4e ; movie s1) and the vertical contacts as protein-protein interactions (yellow arrow, figure 2e ). the np subunits are also linked by an outer horizontal layer at a diameter of 37 nm. this layer consists of a ring of bridges between adjacent large subunits ( figure 2 ). the bridges joining the np subunits are composed of two lobes, one of which of which is slightly bigger than the other. previous studies that produced recombinant nucleocapsid-like structures showed that expressed vp24 and vp35 both independently associate with np, but that all three proteins together are necessary to produce ,50 nm diameter helical nucleocapsid-like structures. when vp35, vp30, vp24, and np were transfected together, approximately 50 nm diameter helical nucleocapsid-like structure was also generated, whereas np alone generated helical np-rna complexes ,20-25 nm in diameter, which were nuclease sensitive [14, 28, 29] . taken together these results suggest that vp24 and vp35 are the structural components of the bridge located on the periphery of the nucleocapsid ( figure 2g ). it is not possible to accurately delineate vp24 and vp35 within the bridge at this resolution, however the density of the bridge is consistent with a predicted total mass of 60 kd, thus each bridge is composed of one molecule of vp24 and one molecule of vp35. it is likely that the larger lobe is vp35 and that vp24 therefore resides within the smaller lobe ( figure 2f ). thus, each bridge is composed of a vp24-vp35 heterodimer that holds adjacent np molecules together horizontally. this structure explains how vp24 and vp35 are able to independently interact with np, and why all three are required for the formation of a double-layered nucleocapsid. in addition, it implies that vp24 and vp35 can interact with each other as well as each interacting with a different site on the np molecule in order to make an oligomeric structure. the recombinant nucleocapsid data indicates that both vp35-vp30-vp24-np and vp35-vp24-np produce approximately 50 nm diameter helical structures that are indistinguishable from nucleocapsids produced by ebov. this indicates that vp30 does not increase the diameter of the nucleocapsid. we propose that vp30 lies in the interior of the nucleocapsid and is not part of the bridge on the periphery of the nucleocapsid. this localization is consistent with previous work showing that vp30 is a component of the nucleocapsid, and associates with np, but is non-essential for nucleocapsid formation [14, 30, 31] . our model, in which the inner layer at 22.3 nm diameter is rna-np, and the outer bridge centered at 37 nm is composed of vp24-vp35 heterodimers, with vp30 bound to np, is thus consistent with these previous observations [14, 28, 29] , and suggests that the outer vp24-vp35 heterodimer bridge functions in the stabilization and/or protection of the nucleocapsid. we have modeled the arrangement of the rna within the ebov nucleocapsid (table s2 ). since no atomic resolution data on the ribonucleoprotein structure of filoviruses have yet been reported, the ribonucleoprotein ring-structures of other members of the mononegovirales are useful for comparison. although the mass of the nucleoproteins, and the number of nucleotides per subunit differs amongst these virus families, a model of the 18.9 kb ebov genome with the rna following a circular fixed radius as in respiratory syncytial virus (rsv) [32] would make a nucleocapsid containing one copy of the ebov genome about 914 nm long (table s2 ). this fits closely with the 982 nm length class (53% of the particles observed in this study) containing a single copy of the ebov genome threaded through the np at a fixed radius. allowing 33 nm of space for the membrane envelope to curve around at each end of the virus particle gives an estimated length for a single-genome virus particle of 980 nm, which is very close to that observed (982679 nm). the majority of the virus particles fall into size categories that are a multiple of the putative single genome length (g1), giving size classes of 1.960.15 mm (g2: 18.7% of the particles having 2 genomes); 2.960.2 mm (g3: 12% of particles having 3 genomes) and so on ( figure 1a and table s1 ) . the length of the longest particle measured was consistent with having 22 genome copies. our model predicts a nucleotide to np ratio of 13.0 (table s2) , which is within the range of 12 to 15 nucleotides per np molecule as measured by biochemical studies of marburg virus [33] . in partially full virions, the membrane envelope is constricted at the transition point where the nucleocapsid ends and the empty membrane tube begins (figures 1, s1c ). empty virions ( figure s1c ) have a similar structure to vp40-gp virus-like particles ( figure 3a ). both full and empty virions have a continuous layer of spikes projecting from the surface (figures s1, s2, s3, movie s2), giving an overall diameter of 120 nm for full particles. the majority of virions are linear (figures 1, s1a, s5 ), others have a ''comma-shaped'' appearance, with a globular head containing portions of the nucleocapsid that are curled-up or bent at one end ( figures 1a,b and s1b). it is clear that ''toroidal'' virions previously identified by negative staining [22, 34] are a variation of comma-shaped virions. internal vesicles of 20-40 nm in size are also sometimes observed at the ends of virions ( figure s1d ). these vesicles appear to be formed during the process of envelopment of the nucleocapsid, since they were not observed in preparations of vp40 or vp40-gp vlps. vp40 vlps had wavy envelopes with an irregular diameter ranging between 48 nm and 142 nm (n = 49) compared to vp40-gp vlps which were more ordered with a diameter between 50 nm and 91 nm (n = 26), (figure 3 ). thus the presence of gp, and certain contacts between the gp and vp40, play a part in stabilizing the tubular membrane envelope structure, and our observed structure agrees with previous reports that gp enhances vp40 vlp budding [35, 36] . the previously reported ''branched'', filamentous forms [34] were rarely observed: these consist of empty tubes (data not shown). it is possible that centrifugal virus purification disrupted most branched structures that were seen previously with negative staining of cell culture supernatant. the vp40 matrix protein shows a regular 5 nm lattice spacing ( figure 2b , c). both the nucleocapsid and the vp40 layers are ordered, however the contacts between them appear to be nonsymmetrical ( figure 2d ), implying some flexibility in their intermolecular contacts. it has been shown that vp35 interacts with the vp40 and can be packaged into vp40 vlps [37] . the localization of the vp24-vp35 bridges on the periphery of the nucleocapsid, may allow interactions of one or more of the nucleocapsid proteins with vp40, possibly through projecting low-density protein loops. there is a 6-7 nm gap of low density between the nucleocapsid and the vp40 layer, but tomography also shows discrete areas of connectivity between the nucleocapsid and matrix protein layers, which may be connections between the envelope and the nucleocapsid ( figure 3i ). these results are substantiated by the analysis of sub-tomograms where helical symmetry is clearly evident but was not imposed (figures 4, s4) . the use of sub-tomogram analysis improves the resolution of the data by averaging sub-tomograms together which are at different angular orientations. the helical nucleocapsid is clearly identified in the structure, including the gap between the envelope-vp40, and the nucleocapsid-vp40. the right-handed pitch of the helix is clearly discernable (figure 4e ), as well as the putative location of several vp40 proteins, although the resolution of the tomographic data by itself is slightly lower than with single particle analysis. while showing the overall organization of ebov, tomography also allows estimation of the stoichiometry of the major structural proteins (figure 3 , movie s2). both ebov and vp40-gp vlps have an irregular distribution of the gp spikes on the surface (figures 3, s3) demonstrating the lack of any ordered lattice-like arrangement with the matrix protein vp40. the spikes are clustered, and the average centre-to-centre spacing is 15.2 nm ( figure s3, and movie s3) . we calculate that a virion of 982 nm in length would have about 1888 copies of the gp spike, and 8391 copies of vp40. the wide spacing of the ebov gp in the viral envelope allows plenty of space for free access and binding of any neutralizing antibodies directed at both the club-shaped head and stem region without stearic hindrance (figures 3j, s3) . the nucleocapsid structure implies equimolar ratios of np, vp24, vp30, and vp35. a genome of 18.9 kbp with 13 bases per np with a single genome copy gives 1454 molecules of np, vp24, vp30, and vp35 per virion. previous analyses of coomassie blue stained gels of purified ebov predicted 625, 1208, 833, and 2686 protein molecules of np, vp24, vp30, and vp35, per virion respectively [38] , which is in the same stoichiometric range as predicted by our model for the nucleocapsid, taking into account the variability of individual protein band staining by coomassie blue, which is affected by factors such as distance of migration [39] , basic amino acid content [40] , and the extent of glycosylation [41] . since np is glycosylated, we anticipate underestimation of the np content of virions [14, 41] . the gp spike is necessary for cellular attachment and fusion of ebov. a definitive cell surface ligand for the receptor binding domain has not been identified, and a number of different cell surface proteins are able to enhance infection [42, 43] . cleavage of gp results in two domains: gp1 containing the receptor binding domain and gp2 that contains the fusion and transmembrane domains. the structure of a smaller engineered fragment of gp1-gp2 has recently been determined by x-ray crystallography (3csy pdb [42] ). we determined the structure of the entire ebov gp trimeric spike at a resolution of 14 å (fsc 0.5 criteria), by combining sub-tomograms from the spikes of vp40-gp vlps (234 images) with ebov images for the side perspective data (8084 images) using projection matching as previously described [44] ( figure 5 and movie s4). in our reconstruction, the spike is in situ in the viral membrane, thus the transmembrane region and base of the spike, adjacent to the membrane, are less well defined than the distal region of the spike, due to the smaller differences in contrast between lipid and protein versus water and protein, as well as fresnel fringes at the edge of the viral membrane. the spike extends 10 nm from the surface of the envelope, with a clubshaped head 6.5 nm in height, and a 3.5 nm long stalk. docking of the previously determined x-ray structure into our cryo-em map shows a good fit, with a correlation coefficient of 0.75 using the docking and correlation program situs [45] . the difference map calculated between our cryo-em map and the gp1-gp2 structure identified volumes corresponding to the domains deleted to generate the 3csy gp1-gp2 structure ( figure 5 ). we show that the mucin-like domains (connected at v310 and e502 -shown in green in figure 5 ) completely fill the previously described bowllike chalice which contains the putative receptor binding sites [42] . the proximity of glycosylation sites in the gp1-gp2 structure suggests that the distal density of the spike contains the glycans that were deleted in order to construct the gp1-gp2 structure. in addition, each mucin-like domain has an ''arm-like'' projection, which extends radially at the distal end of the spike, to a maximum diameter of 13 nm. the localization of the mucin-like domain is consistent with previous studies showing that endosomal proteolysis plays a role in enhancing infectivity as well as binding of the ebola gp to the plasma membrane [46, 47] . the other two major deletions in the 3csy structure (n278-r299 and a189-y214) are situated at the midpoint of the structure just above the stalk (shown in pink in figure 5 ). inclusion of the kz52 fab in the docked structure demonstrates that this neutralizing epitope (from a human survivor) is localized on the side of the stem region of ebov gp trimer at the base of the clubshaped head, and that the fab domain lies close to the lipid envelope when bound, and approximately tangential to the viral envelope. the densities putatively corresponding to n278-r299 and a189-y214 are close to the kz52 neutralizing site, but do not obstruct antibody binding. the mucin-like domains are out of the way and cannot interfere stearically with kz52 fab binding. this is consistent with previous results indicating that kz52 binding does not require cathepsin cleavage [48] . we have thus delineated the low resolution structure of the glycocalyx or ''glycan cap'' that covers the distal end of the uncleaved ebov gp spike, which is consistent with a proposed role in immune evasion [42] . our data will enable docking of future structures to investigate receptor binding, antigenicity, and fusion mechanisms. we have shown that ebov particles are capable of a high degree of polyploidy, made possible by the extreme length polymorphism of budding virus particles. polyploidy in filoviruses may be more extensive than in any other virus family, with 46% of virions having more than one genome copy, and some having up to 22 copies. polyploidy has been shown to increase infectivity rates in paramyxovirus and birnavirus [2, 3] . attempts to investigate infectivity rates of ebov by centrifugal fractionation of the different sized particles are stymied by the extreme filamentous morphology, as well as that fact that ebov of different lengths have the same buoyant density (personal observations). a complex double layered helical nucleocapsid appears to be unique to filoviruses. in the case of rhabdoviruses, the bullet-shaped nucleocapsid precludes them from being linked sequentially. although some influenza strains can produce filamentous virions, this morphology appears to be driven by the matrix protein only [49] . a filamentous morphology may have evolutionary implications by allowing genome length flexibility. it could also enhance the ability for viral dissemination in infected tissues, for example by diapedesis of budding filamentous virions through epithelial layers. zaire ebolavirus was propagated in vero e6 cells and purified as previously described [50] . ebola enriched samples were checked by sds-page and western blotting, and rendered non-infectious by fixation with 4% paraformaldehyde. excess fixative was removed by placing the fixed samples in a slide-a-lyzer g2 cassette with a 0.5 ml capacity, and a 10,000 mwco (thermo scientific pierce protein research products, rockford, illinois, usa), followed by dialysis against pbs. virus-like particles were produced as previously described [51] . all work with infectious ebola virus (virus culture and purification) was performed in the biosafety level 4 laboratories at the national microbiology laboratory of the public health agency of canada, winnipeg, manitoba. samples for cryo-electron microscopy (cryo-em), and cryoelectron tomography (cryo-et) were mixed with bsa coated accessories, wageningen, the netherlands) at a ratio of 2:1 (virus:gold) for cryo-et, and (9:1) for cryo-em. specimens (4 ml) were then applied to glow-discharged quantifoil grids with 2 mm holes spaced at 1 mm intervals (quantifoil microtools gmbh, jena, germany). grids were subsequently plunge cooled in liquid ethane using a vitrobot mark iv (fei company, hillsboro, oregon, usa). specimens were transferred to a tecnai 20 g2 transmission electron microscope (fei) operated at 200 kv, equipped with a gatan ct3500tr single tilt rotation lowtemperature specimen holder. for cryo-em imaging was conducted at temperatures of ,2185uc. images were recorded using an eagle 4k ccd camera (fei company, hillsboro, oregon, usa). for single particle image analysis, images were taken at 50,0006 or 80,0006 magnification at 2-4 mm defocus, with a dose of 10 electrons/å 2 . this corresponded to a pixel size at the ccd detector of 2.147 å /pixel and 1.353 å /pixel, respectively. for virus length measurements low magnification cryo-em images were taken at 5,0006, 3,5006 and 2,5006. for cryo-et single axis tomograms were taken at 25,0006, 29,0006 or 50,0006 magnification, at 28 m or 26 m defocus, with angle steps of 2u24u. data were collected within tilt ranges of 660u, or 652u, with a total dose/tomographic data set of 47-60 electrons/å 2 . for cryo-em, data collection was done using the low-dose unit and software coupled with the tem imaging & analysis (tia) software (fei company, hillsboro, oregon, usa). automated eucentricity determination, and focusing were performed using the xplore3d data acquisition software (fei company, hillsboro, oregon, usa). for cryo-et, data collection was done using the xplore3d data acquisition software, the low-dose unit, and the tia software (fei company, hillsboro, oregon, usa). the exact magnification in the microscope at the ccd detector was determined using a calibration grid (pelco international, redding, ca). ebola virus length measurements (n = 2090) were made using the image j software package [52] using the free hand line tool, and the analyse/measure function. for this analysis only viruses containing a continuous nucleocapsid were measured. viruses with linked nucleocapsids and empty viruses were omitted. the measurements that were made in image j were then collated, analysed, and plotted, using microsoft excel. tomographic image analysis of cryo-et data was carried out with the inspect3d xpress software package (fei company, hillsboro, oregon, usa). the tomographic images were aligned to each other by a two-step process. the first step involved alignment of adjacent images by cross correlation. this process was repeated several times until the shift between adjacent images was below one pixel in either the x or y plane. the second step involved the alignment of the entire image stack using 10 nm colloidal gold particles as fiducial markers. in this instance the term ''image stack'' refers to all the images collected in a single tomographic tilt. this procedure involves the selection of ten to twenty of the 10 nm gold particles and the subsequent identification and tracking of these particles in all of the images in the image stack. the locations of these particles are then used in conjecture with the tilt angles of each image to globally align all of the images to each other. the last step in this process was to calculate the three dimensional reconstruction of the tomogram from the aligned images. in this study we used the simultaneous iterative reconstruction technique (sirt) algorithm with 10 iterations to calculate the final three-dimensional reconstruction (tomogram). sub-tomogram image analysis of cryo-et data was carried out with the automated recognition of geometries, objects, and segmentations (argos) software package (fei company, hillsboro, oregon, usa). for this analysis an 80 3 pixel subtomogram was extracted from a tomogram using the chimera [53] software package. in this analysis the tomogram used contained a linear region of the ebola virus ( figure s4 ), and the 80 3 pixel sub-tomogram contained a single linear segment. this template was then used by the argos software to conduct an exhaustive search of the original tomogram for similar structures. this analysis involved a six dimensional search matrix (three positional variants, and three rotational variants). the entire search process was sped up by the argos software by utilizing parallel processing on the computer's graphics processing unit (gpu). once individual sub-tomograms were selected based on correlation, they were inspected and compared to the initial template sub-tomogram. the extracted and aligned sub-tomograms were subsequently averaged with a filter that minimized the missing wedge artifact. this average structure then was used as the reference and the entire procedure was repeated several times. single particle image analysis: software and hardware single particle cryo-em image processing was carried out using the eman/eman2 and spider/web image processing program packages [54, 55] . particle selection (eman) and contrast transfer function correction (eman2) were conducted on an apple inc. mac pro computer (12-core, intel xeon nehalem processors 2.93 ghz, 32 gb ram, mac os x 10.6.7). all subsequent calculations were performed on a dell poweredge r900 4-way 64-bit xeon x7460 processors, six core 2.67 ghz cpus with 256 gb ram running linux (centos 5.2). images were corrected for contrast transfer function (ctf) using the ''e2ctf.py'' function in the eman2 software package 5 , which estimates defocus and corrects for ctf by phase-flipping. images of the spike (n = 8084 side perspective; n = 234 end-on perspective) and nucleocapsid (n = 34,605) were selected for image analysis. the resolution of the cryo-em reconstruction was estimated by fourier shell correlation using the fsc 0.5 criteria. in all subsequent sections image analysis procedures were conducted using the spider software package unless otherwise stated. analysis of initial images of the ''straight'' linear segments of the ebola virus using fourier transformation indicated that there was sufficient bending of the helical nucleocapsid to make standard helical analysis problematic. therefore, an initial reference free single particle 2d analysis was conducted in eman using the ''startnrclasses'' program to identify any potentially recurring motifs within linear regions of the ebola virus. in order to further investigate the nucleocapsid repeat identified in the 2d analysis the iterative helical real space reconstruction method (ihrsr) was implemented [26, 27] . this procedure requires an initial 3d helical reference structure which is used for image alignment. in this investigation a linear region of the ebola virus which was extracted from a tomogram was used to generate this helical reference structure. this initial 3d structure was first pre-treated with a gaussian mask to select only the nucleocapsid-containing region of the virus tomogram. an auto correlation function was then performed in which the volume was rotated around the helical axis, and translated along the axis of the nucleocapsid. at each rotational and translational position and autocorrelation value was calculated (between the shifted and unshifted volume). the net result of this process was the determination of the helical symmetry present in the tomogram. in order to analyse the data generated by this procedure the microsoft excel spread sheet program was used. the correlation plots generated by this process solved the handedness, pitch, and number of repeats per turn for the nucleocapsid. these symmetry parameters were then applied to the tomogram to generate the initial 3d model. the ihrsr method was then applied to the 34,605 single particle images of the nucleocapsid as previously described [26, 27] . for the spike dataset two image populations were combined composed of side, and end-on perspectives. for the side view perspective images were subfield directly off of the ebola virus cryo-em images. for the end-on perspectives sub-tomographic volumes were extracted from the tomographic reconstructions of the ebola vlp. the 3d volumes were then added in the ''z'' plane to generate 2d projection averages which were then used for the subsequent single particle image analysis. the data were the processed using eman to generate an initial 3d reconstruction, which was then refined in spider using the projection matching technique as previously described [44, 56] . the docking of the 3csy.pdb [42] structure to the cryo-em structure of the spike was accomplished using the situs [57] software package with the exception that only the gp1 and gp2 components of the 3csy structure were used for the docking process. the ''floodfill'' program in situs was used to segment the spike component of the 3d cryo-em reconstruction from the envelope component of the reconstruction. the segmented volume was then used for the docking procedure using the 'colores' function in situs. once docked the entire 3csy.pdb structure which included the fab of the kz52 neutralizing antibody was superposed over the docked gp1/gp2 component of the structure. the 3d cryo-em reconstructions, cryo-et reconstructions, 3d models of the ebola virus, and the atomic resolution structure 3csy.pdb were visualized using ucsf chimera software package (computer graphics laboratory, university of california, san francisco, supported by nih p41 rr-01081) [53] . the 3d images and movies presented in this manuscript were generated directly by ucsf chimera software package. , region three is shown, with the locations of correlation maxima shown with an x. the angular distance between each maximum was calculated from several plots. a total of 71 measurements gave an average angular distance of 33.6u+/ 28.5u between helical repeats, resulting in 10.7 repeats per turn. using the 6.96 nm pitch (fig. s8 ) the step in z per helical repeat was calculated as 0.65 nm. these helical symmetry values were then imposed on the nucleocapsid tomogram and this structure was used as the initial reference volume for refinement using the iterative helical real space reconstruction method. (tif) figure s3 surface spike distribution in the ebola vlp. longitudinal z-slices through the top and middle of the particle are shown, as well as the end-on view (a). the tomogram is shown as a shaded surface at a density threshold that indicates the spikes (b). the volume from one side of the tomogram has been extracted, and a red-blue color scheme shows the depth at which the spikes are located. this region of the envelope has a surface area of 15,651 nm 2 . selected spikes have been identified by red circles, the single particle reconstruction of the spike is shown at the same scale to the right in a red square for comparison. the same region in (b) is shown in (c) with a solid orange cylinder to provide a visual cue for the viral envelope. eighty-six individual spikes were counted (white spheres) and have a patchy distribution (d), each spike would occupy an average area of 182 nm 2 , giving an average spacing between spikes of 15.2 nm. the reconstruction of the spike (blue) with the docked kz52 fab (purple) has been included to show that there is ample room for antibody attachment. (tif) figure s4 extraction of ebola nucleocapsid structure for sub-tomographic analysis. the tomogram of a linear region of the ebola virus was used as the first reference for subtomogram analysis (a). when viewed along the helical axis (y) or from the end perspectives (x,z) the basic components are visible. the tomographic volume was also cylindrically masked along the x-axis, selecting only the density containing the nucleocapsid, to highlight the components of the nucleocapsid in the tomogram (b). two-dimensional single particle image analysis was carried out with cryo-images (c) (not tomographic data sets), for comparison to the 3d tomographic data. the average shown in this panel was generated by reference free classification, using the ''startnrclasses'' program in eman [54] . the 6.96 nm helical pitch can be easily seen in the 2d average, but is also visible in the projections of the tomographic volume in (a, b) . (tif) figure s5 representative low-magnification images of ebola virus. frozen hydrated virus is clearly visible with sections of the filamentous virus over both the support film and across the holes in the quantifoil film. individual g1 (single genome copy) virus is circled in red, several sections containing a nucleocapsid are indicated by a blue arrowhead, and regions without a nucleocapsid are indicated by a magenta arrowhead. globular heads are identified by yellow arrowheads. in this image the circles (light grey, 2 m diameter) are filled with frozen hydrated virus in a thin aqueous layer, and the quantifoil support film appears as darker grey. table s1 length analysis of ''continuous'' ebola virus particles. the length of 2090 ebov particles were measured using imagej [52] . the values in the ''model length'' column are based on multiples of the g1 mean length. the values in the in the ''mean length'' column were calculated directly from the data. only full particles containing a continuously packaged nucleocapsid were measured, all others (linked-nucleocapsid and empty particles) were omitted from this analysis. the terms g1-g22 indicate the number of genomes/viral particle (i.e. g22 = 22 genomes). all measurements are in mm. modeling of rna in the ebola nucleocapsid. two previously determined atomic resolution structures of negative stranded rna viruses (vsv (2gic.pdb) [58] , and rsv (2wj8.pdb) [32] ) were used to estimate the ebov nucleocapsid length and number of nucleotides per nucleoprotein. images of vsv (a) and rsv (b) are shown as a molecular surface with the protein in orange and the rna as a green ribbon. from left to right, they show a surface view from the side, a side-on cross section, an end-on view, the rna density alone in projection, and a rotational average of the projection. the vsv-based estimate, with a saw-tooth pattern of rna in the helix, gave a nucleocapsid which 614.37 nm long, too short for the measured length of the g1 ebov (982 nm). the rsv-based model, with a relatively straight/circular pattern of rna in the helix predicted a nucleocapsid 914.55 nm long, which closely fits the measured length of g1 virions, after allowing ,34 nm space at each end to accommodate the curve of the envelope containing gp spikes and matrix proteins. the rsv-like model gives 13 nucleotides per nucleocapsid protein which is similar to previous biochemical estimates of 12-15 for marburg virus [33] , suggesting that the rna in the ebov nucleocapsid is arranged in a smooth helical pattern at a diameter of ,22 nm. (tif) movie s1 3-d reconstruction of ebola virus nucleocapsid. this movie shows the three-dimensional structure of the of the ebola virus nucleocapsid as shaded surface representation. the surface is set at a density threshold which would include one copy of np, vp24,vp30, vp35, and the rna. the nucleocapsid rotates, and then is sliced through the z-axis to show the internal components of the structure. movie s3 ebola virus spike distribution. this movie shows one surface of a cryo-electron tomogram of an ebola viruslike particle, generated by expressing the vp40 and gp proteins. the structure rotates showing the distribution of spikes. the locations of individual spikes are identified by white spheres, and the reconstruction is replaced by a cylinder to show the patchy distribution of spikes on the surface of the virus-like particle. movie s4 3-d reconstruction of ebola virus spike. this movie shows the three-dimensional structure of the of the ebola virus gp spike. the structure rotates showing views from different angles, and indicates the location of the docked 3csy.pdb [42] structure with the gp1 and gp2 domains and kz52 antibody (purple). (mov) physical principles in the construction of regular viruses studies on the pleomorphism of hvj virons infectious bursal disease virus is an icosahedral polyploid dsrna virus architecture of ribonucleoprotein complexes in influenza a virus particles ebola virus: new insights into disease aetiopathology and possible therapeutic interventions live attenuated recombinant vaccine protects nonhuman primates against ebola and marburg viruses ebola virus: from discovery to vaccine the virion glycoproteins of ebola viruses are encoded in two reading frames and are expressed through transcriptional editing gp mrna of ebola virus is edited by the ebola virus polymerase and by t7 and vaccinia virus polymerases ebola virus glycoprotein 1: identification of residues important for binding and postbinding events conserved receptor-binding domains of lake victoria marburgvirus and zaire ebolavirus bind a common receptor comprehensive analysis of ebola virus gp1 in viral entry identification of two amino acid residues on ebola virus glycoprotein 1 critical for cell entry the assembly of ebola virus nucleocapsid requires virion-associated proteins 35 and 24 and posttranslational modification of nucleoprotein functional mapping of the nucleoprotein of ebola virus nucleocapsid-like structures of ebola virus reconstructed using electron tomography electron tomography reveals the steps in filovirus budding grafix: stabilization of fragile macromolecular complexes for single particle cryo-em grafix: sample preparation for single-particle electron cryomicroscopy post-crystallization treatments for improving diffraction quality of protein crystals differentiation of filoviruses by electron microscopy ebola and marburg viruses: i. some ultrastructural differences between strains when grown in vero cells ultrastructure of ebola virus particles in human liver on the etiology of an unknown human infection originating from monkeys apoptosis induced in vitro and in vivo during infection by ebola and marburg viruses a robust algorithm for the reconstruction of helical filaments using single-particle methods the iterative helical real space reconstruction method: surmounting the problems posed by real polymers assembly and budding of ebolavirus characterization of the ebola virus nucleoprotein-rna complex filoviridae: marburg and ebola viruses the ebola virus ribonucleoprotein complex: a novel vp30-l interaction identified crystal structure of a nucleocapsid-like nucleoprotein-rna complex of respiratory syncytial virus morphology of marburg virus np-rna ebola and marburg viruses: ii. their development within vero cells and the extra-cellular formation of branched and torus forms analysis of ebola virus and vlp release using an immunocapture assay contribution of ebola virus glycoprotein, nucleoprotein, and vp24 to budding of vp40 virus-like particles ebola virus vp35-vp40 interaction is sufficient for packaging 3e-5e minigenome rna into viruslike particles descriptive analysis of ebola virus proteins quantitative densitometry of 1-50 g protein in acrylamide gel slabs with coomassie blue why does coomassie brilliant blue r interact differently with different proteins? a partial answer interference of the carbohydrate moiety in coomassie brilliant blue r-250 protein staining structure of the ebola virus glycoprotein bound to an antibody from a human survivor biochemical and structural characterization of cathepsin l-processed ebola virus glycoprotein: implications for viral entry and immunogenicity architecture of the sars coronavirus prefusion spike using situs for flexible and rigid-body fitting of multiresolution single-molecule data endosomal proteolysis of the ebola virus glycoprotein is necessary for infection proteolysis of the ebola virus glycoproteins enhances virus binding and infectivity antibody-mediated neutralization of ebola virus can occur by two distinct mechanisms structural organization of a filamentous influenza a virus replication-deficient ebolavirus as a vaccine candidate the creation of stable cell lines expressing ebola virus glycoproteins and the matrix protein vp40 and generating ebola virus-like particles utilizing an ecdysone inducible mammalian expression system ucsf chimera-a visualization system for exploratory research and analysis eman: semiautomated software for high-resolution single-particle reconstructions spider and web: processing and visualization of images in 3d electron microscopy and related fields conformational reorganization of the sars coronavirus spike following receptor binding: implications for membrane fusion situs: a package for docking crystal structures into low-resolution maps from electron microscopy structure of the vesicular stomatitis virus nucleoprotein-rna complex we thank b. szklarczuk and s. silcox for compiling the movies. key: cord-001460-eo2bxxbq authors: padhi, siladitya; burri, raghunadha reddy; jameel, shahid; priyakumar, u. deva title: atomistic detailed mechanism and weak cation-conducting activity of hiv-1 vpu revealed by free energy calculations date: 2014-11-13 journal: plos one doi: 10.1371/journal.pone.0112983 sha: doc_id: 1460 cord_uid: eo2bxxbq the viral protein u (vpu) encoded by hiv-1 has been shown to assist in the detachment of virion particles from infected cells. vpu forms cation-specific ion channels in host cells, and has been proposed as a potential drug target. an understanding of the mechanism of ion transport through vpu is desirable, but remains limited because of the unavailability of an experimental structure of the channel. using a structure of the pentameric form of vpu – modeled and validated based on available experimental data – umbrella sampling molecular dynamics simulations (cumulative simulation time of more than 0.4 µs) were employed to elucidate the energetics and the molecular mechanism of ion transport in vpu. free energy profiles corresponding to the permeation of na(+) and k(+) were found to be similar to each other indicating lack of ion selection, consistent with previous experimental studies. the ser23 residue is shown to enhance ion transport via two mechanisms: creating a weak binding site, and increasing the effective hydrophilic length of the channel, both of which have previously been hypothesized in experiments. a two-dimensional free energy landscape has been computed to model multiple ion permeation, based on which a mechanism for ion conduction is proposed. it is shown that only one ion can pass through the channel at a time. this, along with a stretch of hydrophobic residues in the transmembrane domain of vpu, explains the slow kinetics of ion conduction. the results are consistent with previous conductance studies that showed vpu to be a weakly conducting ion channel. the viral protein u (vpu) is one of the four accessory proteins encoded by hiv-1 that has an n-terminal transmembrane (tm) domain and a c-terminal cytoplasmic domain [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] . a schematic representation of the protein is shown in figure 1 (a). the cytoplasmic domain has two a-helices [11, 12] , and is involved in the degradation of cd4 molecules in the host cell [13, 14] . the tm domain, which is known to have a helical topology [15] [16] [17] [18] [19] [20] , has been suggested to enhance virus release via two mechanisms: formation of ion-conducting channels [21, 22] and degradation of the antiviral protein tetherin [23] . the ion channel is formed via oligomerization of monomeric vpu to form a pentamer [24] [25] [26] 16] . because of its importance in virus release, an understanding of the mechanism of ion permeation is of great importance. vpu has been reported to show ion channel activity in lipid bilayers, xenopus oocytes, and in the plasma membrane of escherichia coli [21, 22] . vpu channel has been shown to be selective towards monocations (na + and k + ), and cannot differentiate between the two [21, 22] . channel recordings on full length vpu (vpu ) and the vpu tm region (vpu 1-32 ) excluding the cytoplasmic domain showed that the conductance states and ion-conduction recordings are similar for the two [27] . the channel has been suggested to exist in a number of conductance states, which are believed to correspond to different open state conformations. furthermore, the duration for which the channel remains open is independent of the voltage, and the channel shows biphasic voltage activation. the variation of conductivity with respect to salt concentration has been measured by mehnert et al., which showed that ion conduction follows michaelis-menten behavior [28] . the ion transport activity is rather weak, though the channel shows pore-like characteristics. a mutant vpu with ser23rleu mutation in the tm domain does not conduct ions, suggesting a critical role for serine in ion transport [28] . two possible roles have been ascribed to the serine residue. firstly, it might act as a weak ion binding site, and secondly, it creates a hydrophilic environment in the vicinity, thereby reducing the length of the hydrophobic stretch that occurs inside the channel, thus making it easier for an ion to pass through. the passage of ions through the pore has been previously modeled using steered molecular dynamics (smd) simulations [29] . the possibility of the existence of a number of conformations of oligomeric vpu has recently been suggested by computational studies that have modeled the assembly of monomeric vpu into oligomers [30] . this supports experimental channel recordings that suggested the existence of a number of conductance states of vpu [27] . molecular dynamics (md) simulations have been successfully used to study transmembrane proteins in general [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] . a powerful approach is the use of umbrella sampling md simulations, which are in general very helpful in understanding free energy changes corresponding to rare events [42] [43] [44] [45] . this method has been used by roux and coworkers for elucidating the energetics of ion permeation through channels [46] [47] [48] [49] . this method also has been used to obtain a multi-ion free energy profile for the kcsa k + channel [46] . results from that study showed that an ion trapped in the selectivity filter is able to proceed further into the channel because of electrostatic repulsion from ions that subsequently approach the selectivity filter. based on this, it was proposed that rapid conduction in the channel is driven by interionic repulsion. the method has subsequently been used for calculating the free energy profile of ion transport through the gramicidin channel as a function of both axial and radial positions [47] , and for elucidating how the kcsa channel preferentially allows k + rather than na + ions to pass through [49] . the present study employs umbrella sampling md simulations to elucidate the mechanism of ion conduction through the vpu channel. we have recently reported a molecular model for the pentameric state of the tm domain of vpu protein, which has been used here [24] . available experimental information on vpu was used to validate the proposed structure. since only the tm domain is involved in channel activity [22] and the conductance properties of channels formed by the vpu tm region are almost the same as those formed by full length vpu [27] , the cytoplasmic domain of vpu has not been included in the model. this study explains the molecular and energetic basis of ion selectivity in the channel, and suggests that a hydrophobic stretch in the channel might control the kinetics of the ion permeation process. finally, a mechanism for the transport of ions is proposed based on a multiion free energy landscape. the vpu channel structure used in the study is a pentameric form of the tm region modeled in a recent study [24] . in addition to the protein, the system includes 95 lipid (popc) molecules, 5496 water molecules, 13 k + ions and 18 cl 2 ions, with a total of 31984 atoms. prior to the current study, the system was equilibrated for 30 ns without any restraints and was found to be adequately equilibrated, as evidenced from the convergence of rmsd, the occurrence of several stabilizing intra-protein and protein-lipid interactions, and the retaining of the structural integrity of the channel, in general. the transport of two different ions through the channel was studied, namely na + and k + . for modeling the permeation of a k + ion, the coordinates of a k + ion from bulk water were first swapped with a water molecule at the c-terminus of the channel. conformations with the k + ion occurring at different positions along the axis of the pore were generated by pulling the ion along the pore axis using steered molecular dynamics (smd) simulations. the ion was pulled at a constant velocity of 0.01 å ps 21 by applying an external force on a dummy atom connected to the permeating ion via a spring with a harmonic constant of 10 kcal mol 21 å 22 . the structural model for the channel was aligned along the z-axis [24] , and hence the pulling force was applied along the z-axis, thereby making the ion move from the cterminus to the n-terminus. positional restraints with force constant 1 kcal mol 21 å 22 were applied on the heavy atoms of the protein to prevent the drifting of the helices during the pulling of the ion. constant temperature and pressure were employed by using langevin dynamics and a langevin pressure piston, respectively. it must be noted that the smd simulations were used to obtain the initial structures for the umbrella sampling simulations and not for investigating the permeation mechanism. the namd program [50, 51] was used for the smd simulations with the charmm22 all-atom protein force field with cmap corrections [52, 53] , the charmm36 all-atom lipid force field [54] , optimized parameters for ions [55] , and the tip3p water model [56] . for the studies on the permeation of na + , the conformations were generated by replacing the k + ion in the pore with an na + ion. umbrella sampling was performed by applying a biasing potential on the permeating ion, with the potential having a parabolic form. independent simulations were performed for different positions of the ion along the pore axis, so that each simulation sampled the conformations of the system with the ion at the respective region. for a window i, the biasing potential has the form where k is the harmonic constant, z i,0 is the center of the window i, and z is the position of the ion along the reaction coordinate at any given instant. the potential energy function then has the form where r n denotes the positions of all the atoms in the system, and e u (r n ) is the unbiased potential energy function. the unbiased probability distribution of the ion along the pore axis p i u (z) is computed from the biased probability distribution p i b (z), and is given by the free energy along the reaction coordinate is then given by where f i is a constant for window i. there are several approaches for determining f i , of which the most popular is the weighted histogram analysis method (wham) [57] [58] [59] . it calculates the unbiased global distribution p u (z) using the relation where p i (z) is the weight for window i which ensures that the statistical error for p u (z) is minimized. it is given by where n i is the number of steps sampled for window i. f i is calculated using the relation and f i are calculated in a self-consistent manner using the above equations. conformations with the ion placed at different places along the pore axis were extracted from the smd simulation, and were used as initial structures for the different windows in the umbrella sampling calculations. the ion position ranged from z = 230 å to z = 36 å , giving a total of 133 windows along the z-axis spaced 0.5 å apart (the model structure used in the study had previously been aligned along the z-axis). a distribution of ion positions around the center of a given window was obtained by applying a biasing potential with a force constant of 20 kcal mol 21 å 22 on the ion using the mmfp module in the charmm program [60] . positional restraints with force constant 1 kcal 21 mol 21 å 22 were applied on the backbone c a atoms of the protein to prevent the drifting of the channel. ions other than the permeating ion were excluded from the pore region by using a repulsive sphere of radius 15 å , with the repulsive force acting on ions only when they entered this sphere. covalent bonds involving hydrogen were constrained using shake [61] . simulations were carried out in the npt ensemble using the charmm program [60] with the same force field parameters as those used in the pulling simulations mentioned above. simulations for each window involved 100 ps of equilibration followed by 1 ns of production, with a simulation time of (133 * 1.1) ns for each free energy profile. for calculating the potential of mean force (pmf) for the transport of the ion across the channel, the umbrella sampling trajectories were unbiased using wham [57] [58] [59] . the permeation of two k + ions through the channel was modeled by considering different positions of one ion relative to the second ion, with the spacing between the two ions varying from 4 å to 10 å (additional windows with 11 å and 12 å spacing between the ions were sampled for the middle region of the channel). a total of 395 windows spaced 1 å apart were sampled with the two ions at different positions along the pore axis. a biasing harmonic potential of force constant 10 kcal mol 21 å 22 was applied on the two ions. parameters for the other restraints used were the same as those used for the calculations with a single ion in the pore. equilibration and production runs for each window were carried out for 100 ps and 200 ps, respectively. the total simulation time combining all the single ion and two-ion simulations was more than 0.4 ms, with a total of 661 independent simulations. all molecular images were rendered using the visual molecular dynamics (vmd) program [62] . an electrostatic surface representation of the channel was generated using the pdb2pqr server [63] . atomistic structure of tm domain of vpu, and features of the lumen of the pore a representation of the channel modeled in a hydrated lipid bilayer is shown in figure 1 (b). a few pore water molecules can be seen, especially in the bottom half of the pore, towards the cterminal opening. the channel consists of five helical tm domains held together predominantly by van der waals forces, with the interface between the helices being formed by nonpolar residues [24] . the channel is wider at the c-terminal end than at the nterminal end, leading to a relatively greater degree of hydration at this end. the residues arg30 and tyr29 at the c-terminal end of the tm domain face the exterior side of the channel, and they stabilize the protein in the lipid bilayer by forming hydrogen bonds with phospholipid headgroups [24] . the sidechain of arg30 of each of the monomers is also able to form a salt bridge with a glu28 residue on the adjacent monomer. although lys31 is directed towards the interior of the channel, it lies in a region that is sufficiently solvated, so that the residue is shielded from exerting any influence on any ion passing through the channel. the orientation of these residues in the channel is shown in figure s1 (a). a serine at position 23 faces the lumen of the pore, and it marks the end of the small hydrophilic space at the c-terminal entrance of the channel. figure 2 (a) shows the serine residue facing the interior of the pentameric channel. the pore is constricted in the middle, which is a largely dehydrated region, owing to the occurrence of hydrophobic residues. the kinetics of ion conduction through the pore is likely to be controlled by this long hydrophobic stretch. initially, the nature of the pore was investigated by examining the electrostatic representation of the pore-lining residues in the channel shown in figure 2 (b). in the surface representation, one monomer of vpu has been omitted in the figure for clarity. the figure shows electronegative regions in red, and electropositive regions in blue. it can be seen that the top and middle region of the channel is constricted and lined entirely by nonpolar residues. further down the channel, towards the cterminal end, the pore widens, and the occurrence of a serine residue gives rise to a slightly hydrophilic region. the widening of the pore was shown to be due to the kink around the ile17 residue (see figure s1 (b)) [24] , which is in agreement with experimental data [19] . the serine residue can be seen as a red region at the bottom of the channel. the occurrence of such a hydrophilic region is expected to stabilize the permeating ion, thereby assisting ion transport. it follows that the length of the hydrophobic stretch would have been greater in the absence of this serine residue. the serine therefore reduces the effective length of the hydrophobic stretch, and notably, it has been reported previously that the ser23rleu mutant of vpu is inactive towards ion permeation [28] . it is not practical to model events that occur at time scales longer than the ones that are practical with md simulations. additionally, insufficient sampling issues preclude calculation of free energies associated with such rare events. use of umbrella sampling, which applies a harmonic potential along the predetermined reaction coordinate, allows sampling of intermediate states in the process of ion permeation with relatively less difficulty and with significantly less computing time, while at the same time allowing the calculation of the free energy change along the reaction coordinate using methods like wham [57] [58] [59] . an important step in employing umbrella sampling is the selection of the reaction coordinate. the axis of the pore has proved to be a valuable reaction coordinate in the study of ion conduction through channels [46] [47] [48] [49] 64, 65] . using the pore axis as the reaction coordinate, the present study illustrates the energetics and atomistic mechanism of the ion channel activity of vpu. the pmfs for the transport of na + and k + ions are shown in figure 3 . the pmf along the pore axis is shown, with the cterminus (or the intracellular side) on the left and the n-terminus (extracellular side) on the right. the residues lining the pore are also marked appropriately in the plots. the free energy profiles presented correspond to the ion permeating from the bulk water region outside the c-terminal end of the channel to the bulk water region on the other end of the channel. for determining the errors, the sampling data of last 1 ns/window was divided into five parts with 200 ps/window, and the pmf was computed separately for each part. the mean, standard deviation, and standard error were then calculated from the pmf obtained for the different intervals. the energy barriers for this process are about 43.9 kcal/mol for na + and 43.3 kcal/mol for k + . such a high energy barrier is to be expected, given the highly hydrophobic environment that prevails inside the channel. the energy barrier observed is considerably higher than that for other known ion channels [46] [47] [48] [49] 64, 65] , and it suggests that the ion channel activity of vpu is weaker than that of typical ion channels. this is in good agreement with conductance experiments suggesting that vpu is expected to behave as a weakly conducting ion channel [28] . the high energy barrier encountered by the permeating ion is likely to make the process kinetically slow. the features of the two free energy profiles (na + and k + ) were found to be very similar to each other. the movement of the ion into the channel from the c-terminal side is accompanied by the release of 2 kcal/mol of energy, with a shallow minimum occurring around z = 210 å (the region is marked with an arrow in figure 3 ). this corresponds to the position of the ring of ser23 residues, and suggests the role of serine as a weak binding site. this is consistent with previous conductance studies on mutant forms of vpu, which have proposed that the serine can act as a weak binding site for ions, since replacing the serine with a hydrophobic residue results in loss of ion channel activity. the fact that the minimum seen around the serine is shallow rather than deep indicates that the binding site is at best only a weak one. as the ion moves further into the channel, there is a surge in the free energies brought about by the unfavorable interactions that the positive ions have with the hydrophobic environment of the channel. the maximum is reached around two valine residues, namely val9 and val12, which occur in the middle of the hydrophobic stretch in the channel. the ions experience a soft minimum around 15 å , which arises from the stabilization of the ion at this position by coordinating water molecules (see later). figure 4 shows snapshots of the na + ion at two places inside the channel. the first snapshot shows the ion near the ring of ser23 residues, while the second snapshot shows the ion near the ring of val12 residues. as can be seen in the figure, the latter corresponds to the narrow stretch of the channel, where the energy barrier reaches a maximum. the remarkably similar free energy profiles corresponding to the permeation of na + and k + explain why the channel does not discriminate na + over k + , as reported previously based on experimental studies [22] . given that the windows are close to each other, 1.1 ns long md simulations for each of the 133 windows per free energy profile is deemed adequate. the total simulation time for each of the two ions was therefore (1.1 ns * 133)<146 ns. the pmfs obtained at different durations of the simulations and for the whole duration are given in figure 5 . the pmfs calculated agree well with each other with only minor variations in the free energy values. based on this, modeling of the multi-ion permeation was also modeled using a similar protocol (see below). solvation of the ion during the permeation process and its relationship with the free energy profiles were examined based on the hydration number, the number of water molecules in the first solvation shell of the ion and the interaction energies (see below). figure 6 shows the average hydration number of the ions with respect to the position along the channel. the hydration numbers were calculated based on cutoff values of the distance between the ion and the oxygen atom of water molecules (2.8 å for na + and 3.2 å for k + ). these cutoff values were chosen accounting for the size of the respective ions [49] . as the ion enters the c-terminal side of the channel, it retains its hydration shell, and proceeds spontaneously into the channel. the hydrophilic environment in this region is created by the polar ser23 sidechain (this corresponds roughly to the position 210 å in the figure). as the ion proceeds towards the n-terminal side, it gradually loses waters of hydration, with the hydration number reaching a minimum in the middle region of the channel. the loss in hydration number leads to a destabilization of the ion in the middle region relative to the c-terminal region, which is reflected in the rise in pmf. this is consistent with the earlier discussion on the nature of the pore in this region that this region is rich in hydrophobic residues, which results in stripping of water molecules from the ion and drying of the whole region in general. it must be noted that there are relatively more number of water molecules around the ion when the ion is in the region between ile5 and val9 (corresponding to z = 15 å ). this is explained by a slight widening of the pore around this region [24] , making it possible to accommodate more water molecules. the stabilization of the ion by this solvation explains the occurrence of the soft minimum seen in the pmf at this position ( figure 3 ). as the ion proceeds towards bulk water beyond the n-terminal end of the pore, the hydration number of the ion increases, and is associated with a sharp fall in the pmf. figure s2 shows the radial distribution function of water molecules around the permeating ion. the first hydration shell is seen as a thick band in the figure. it can be seen that although there are some water molecules beyond the first hydration shell, there is no distinct second hydration shell. the permeating ion encounters two principal interacting partners inside the channel -the pore water molecules and the channel itself, and such interactions are expected to affect the free energy profiles significantly. accordingly, interaction energies, which include the contributions of the electrostatic and lennard-jones (lj) terms of the potential energy function, were calculated to examine the solvation energy. mean values of the interaction energies between ion and water, and ion and protein are shown in figure 7 . as can be seen clearly, the major contribution comes from the solvating water molecules, whose contribution towards ion stability is several orders of magnitude higher than that offered by the protein. it is to be noted that such interaction energy calculations have limited applicability, since these are calculated based on the potential energy equation only, and do not include entropy effects, for instance. the loss of solvation in the middle region, which is seen in the figure as an increase in the solvation energy, also explains the rise in pmf in this region. the energy barrier that is seen in the free energy profile therefore arises from the desolvation of the ion. this energy barrier, in turn, is likely to make the process of ion conduction kinetically slow, making vpu a weakly conducting ion channel. previous biophysical studies have suggested that ser23 might act as a weak binding site for ions [28] . the interaction of the ion with the protein shown in figure 7 reveals a shallow minimum for both na + and k + near the serine residue, suggesting weak binding ability. on the other hand, there is a remarkable enhancement of stability due to solvation as the ion moves towards the serine; this is seen as a fall in the solvation energy of the permeating ion. the serine residue therefore enhances ion movement into the channel by giving rise to a sufficiently solvated region on the c-terminal side. there are thus two ways in which the ser23 makes conditions conducive for ion transport: firstly, it acts as a weak binding site, and, secondly, it gives rise to a hydrophilic region around the entrance of the pore. to investigate if more than one ion can pass through the channel at a time, the free energy profile was calculated as a function of the positions of two permeating k + ions. the free energy landscape with respect to the position along the z-axis of the first ion and the distance of the second ion from the first is shown in figure 8 . the probability distributions obtained from the biased simulations were checked for adequate sampling along both the reaction coordinates prior to construction of the free energy surface. the lowest energy pathway with respect to the position of the first ion is also marked in the plot. the structures presented below the plot show the conformations of the channel corresponding to this path at three different positions. the free energy surface as a whole shows that permeation of two ions at the same time is not thermodynamically favorable compared to the movement of one ion. if one follows the minimum energy path given in the figure, one can see that when the two ions are near the c-terminal side of the pore (bottom left corner of the figure) , the ions are close in space. this is because this region is reasonably well solvated, and interactions between the two ions are shielded. as the two ions cross the hydrophilic region around ser23 and enter the dehydrated middle region of the channel, interionic repulsion becomes significant. the reduced number of water molecules hydrating the ions, and the dry nature of this part of the pore leads to longer distances between the two ions. this means that two ions cannot enter the middle region of the channel simultaneously, and only one ion can pass at a time. once the first ion reaches the n-terminal region, the second ion starts crossing the hydrophobic stretch in the pore. thus a mechanism is hypothesized in which only one ion crosses the hydrophobic region of the channel at a given time while the next ion waits near the c-terminal region, until the first one reaches the n-terminal end of the pore. the kinetics of the process of ion transport is therefore seen to be controlled primarily by the hydrophobic stretch. such a hypothesis is also consistent with previous experimental studies. fischer and coworkers have proposed that the conductivity of vpu follows michaelis-menten behavior upon increasing the salt concentration [28] . such behavior is explained by the fact that a single ion can pass through the channel at a time, as suggested by the mechanism discussed here. it may be noted that, although the windows for the two ion permeation were chosen such that the spacing between the two ions varied from 4 å to 12 å , the conformations sampled during the simulations cover a range of 3 å to 14 å . while conformations with the spacing close to 3 å are sampled in the terminal regions of the channel, conformations with the spacing increasing to 14 å are seen in the middle region of the channel. this suggests a propensity of the two ions to stay close together while they are near the hydrated terminal regions of the channel. on the other hand, in the middle region of the pore, the ions tend to stay as far apart as possible, thereby minimizing interionic repulsion. perspectives the high energy barrier reported here for the transport of k + and na + through the vpu channel can be attributed to the long stretch of hydrophobic residues which constrict the pore, and cause the dehydration of the pore lumen ( figure 1b) . it might be argued that the use of restraints on the backbone c a atoms of the protein (to prevent drifting) during the umbrella sampling simulations might hinder relaxation of the protein, and thereby cause the pmf to rise. however, it must be mentioned that the protein structure used here was previously subjected to unrestrained equilibration simulations in explicit solvent and bilayer for 30 ns, which is expected to be long enough for the protein backbone and sidechain atoms to relax. furthermore, unlike transporters, ion channels do not usually undergo large scale domain motions during the process of solute transport, since they undergo gating prior to solute transport [66] . thus the use of weak restraints on c a atoms during the umbrella sampling is not likely to affect the dynamics in a significant way. an estimate of the rate of ion conduction can be made using the arrhenius equation. an energy barrier of around 43 kcal/mol that is reported here suggests that the rate for the ion conduction to be several orders of magnitude lower than that estimated using the conductivity measurements previously reported by mehnert et al. [28] . thus, although the present study is able to qualitatively account for the weak channel activity of vpu, it is not able to quantitatively reproduce the conductance reported in experiments. the difficulty in accurately reproducing experimentally determined conductances from the height of energy barriers derived from pmfs has been reported by sansom and coworkers [65] . one factor that could lead to inaccuracies in the pmf is the nonpolarizable nature of the force field used [47, 48] . apart from the protein, the polarizability of the water model is also greatly important, since the pore water molecules play a crucial role in stabilizing the ion inside the pore [47] . another factor that could affect the quantification of properties of the channel is an inadequate representation (in simulations) of the conditions prevailing in electrophysiological experiments. experimental channel recordings investigating selectivity in vpu have been carried out under the application of a voltage [17, 21, 22, 27, 28] . it is likely that the presence of an electric potential brings about conformational changes in the channel, leading to different conductance states of the channel. a possible approach for obtaining insights into these conductance states is to model the channel in the presence of a voltage. however, vpu has been shown to exhibit a variety of conductance states even if a very small potential is applied [27] . furthermore, the open time for the channel has been shown to be voltage-independent [27] . the factors leading to the occurrence of the different conductance states, therefore, are still not properly understood. it has been suggested that gating in the channel might be regulated by the lateral membrane pressure and the lipid composition [27] . thus, an understanding of the different conductance states might be possible by modeling ion permeation through the channel in a number of phospholipid environments with varying lateral pressure. further studies are necessary in understanding the role of applied external voltage on the structure and dynamics of vpu, and the resultant changes in ion permeation thermodynamics/ mechanism if any. free energy profiles for conduction of monovalent cations across the vpu channel have been characterized using umbrella sampling free energy calculations. the calculations reveal reasonably high energy barriers for ion movement through the channel, owing to the highly hydrophobic environment of the pore. the high energy barriers suggest that ion transport across the pore is kinetically slow, making vpu a weakly conducting channel. the weak ion conducting activity is consistent with experimental biophysical studies, and has been explained based on the free energy calculations and hydration properties. the energy barriers arise from the desolvation of the ion as it moves from bulk water to the middle hydrophobic region of the channel. the permeation of na + and k + involves comparable energetic costs, explaining why the channel does not discriminate na + over k + . the results show that the ser23 residue plays a role in ion channel activity by giving rise to a hydrophilic region in the pore, and by serving as a weak binding site. the possibility of multi-ion permeation has been investigated by computing the free energy as a function of the positions of two permeating ions. results show that only one ion can pass at a time through the middle hydrophobic region of the channel. the mechanism proposed is able to account for the slow kinetics of ion transport across the channel, and it suggests a role for the hydrophobic stretch in controlling the kinetics of the process. figure s1 orientation of important residues in the channel. (a) glu28, tyr29, arg30, and lys31 residues. arg30 and tyr29 are seen interacting with nearby lipid headgroups. arg30 also forms a salt bridge with glu28 on an adjacent helix. lys31 faces the pore, but is very unlikely to exert any influence on the permeating ion, since it is shielded by a large number of water molecules. (b) kink in the helix around the ile17 residue. (tif) identification of a protein encoded by the vpu gene of hiv-1 a novel gene of hiv-1, vpu, and its 16-kilodalton product human immunodeficiency virus type 1 vpu protein is an oligomeric type i integral membrane protein hiv-1 accessory proteins-ensuring viral survival in a hostile environment misdirection of membrane trafficking by hiv-1 vpu and nef viral proteins function as ion channels viral channel forming proteins-modeling the target hiv-1 vpu-an ion channel in search of a job viral channel proteins in intracellular protein-protein communication: vpu of hiv-1, e5 of hpv16 and p7 of hcv structural modeling of vpu from hiv-1 based on solid-state nmr observables solution structure of the hydrophilic region of hiv-1 encoded virus protein u (vpu) by cd and 1h nmr spectroscopy solution structure of the cytoplasmic domain of the human immunodeficiency virus type 1 encoded virus protein u (vpu) human immunodeficiency virus type 1 vpu protein induces rapid degradation of cd4 the two biological activities of human immunodeficiency virus type 1 vpu protein involve two separable structural domains solution structure and orientation of the transmembrane anchor domain of the hiv-1-encoded virus protein u by high-resolution and solid-state nmr spectroscopy vpu transmembrane peptide structure obtained by site-specific fourier transform infrared dichroism and global molecular dynamics searching correlation of the structural and functional domains in the membrane protein vpu from hiv-1 expression, purification, and activities of full-length and truncated versions of the integral membrane protein vpu from hiv-1 three-dimensional structure of the channel-forming trans-membrane domain of virus protein ''u'' (vpu) from hiv-1 threedimensional structure of the transmembrane domain of vpu from hiv-1 in aligned phospholipid bicelles the vpu protein of human immunodeficiency virus type 1 forms cation-selective ion channels identification of an ion channel activity of the vpu transmembrane domain and its involvement in the regulation of virus release from hiv-1-infected cells tetherin inhibits retrovirus release and is antagonized by hiv-1 vpu molecular dynamics simulations reveal the hiv-1 vpu transmembrane protein to form stable pentamers oligomerization of the human immunodeficiency virus type 1 (hiv-1) vpu protein-a genetic, biochemical and biophysical analysis molecular dynamics investigation of membrane-bound bundles of the channel-forming transmembrane domain of viral protein u from the human immunodeficiency virus hiv-1 towards a mechanism of function of the viral ion channel vpu from hiv-1 biophysical characterization of vpu from hiv-1 suggests a channel-pore dualism reconstructing potentials of mean force from short steered molecular dynamics simulations of vpu from hiv-1 assembling viral channel forming proteins: vpu from hiv-1 setting up and running molecular dynamics simulations of membrane proteins molecular dynamics simulations of membrane channels and transporters molecular simulation approaches to membrane proteins ion selectivity in channels and transporters showcasing modern molecular dynamics simulations of membrane proteins through g protein-coupled receptors visualizing functional motions of membrane transporters with molecular dynamics simulations membrane assembly of simple helix homooligomers studied via molecular dynamics simulations pore opening and closing of a pentameric ligandgated ion channel in silico investigations of possible routes of assembly of orf 3a from sars-cov the determinants of hydrophobic mismatch response for transmembrane helices molecular mechanism of membrane binding of the grp1 ph domain umbrella sampling frontiers in free-energy calculations of biological systems computational approaches for investigating base flipping in oligonucleotides nmr imino proton exchange experiments on duplex dna primarily monitor the opening of purine bases energetics of ion conduction through the k + channel energetics of ion conduction through the gramicidin channel ion permeation through a narrow channel: using gramicidin to ascertain all-atom molecular dynamics potential of mean force methodology and biomolecular force fields ion selectivity of the kcsa channel: a perspective from multi-ion free energy landscapes namd2: greater scalability for parallel molecular dynamics scalable molecular dynamics with namd all-atom empirical potential for molecular modeling and dynamics studies of proteins extending the treatment of backbone energetics in protein force fields: limitations of gas-phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations update of the charmm all-atom additive force field for lipids: validation on six lipid types finite representation of an infinite bulk system: solvent boundary potential for computer simulations comparison of simple potential functions for simulating liquid water the weighted histogram analysis method for free-energy calculations on biomolecules. i. the method the calculation of the potential of mean force using computer simulations wham: the weighted histogram analysis method charmm: the biomolecular simulation program numerical integration of the cartesian equation of motions of a system with constraints: molecular dynamics of n-alkanes vmd: visual molecular dynamics pdb2pqr: an automated pipeline for the setup, execution, and analysis of poisson-boltzmann electrostatics calculations ion selectivity mechanism in a bacterial pentameric ligand-gated ion channel energetics of multi-ion conduction pathways in potassium ion channels capturing functional motions of membrane channels and transporters with molecular dynamics simulation we thank dr. semparithi aravindan and dr. prabhakar bhimalapuram for fruitful discussions. conceived and designed the experiments: udp sj. performed the experiments: sp rrb. analyzed the data: sp rrb udp. wrote the paper: sp udp. key: cord-000868-vnwpzsu8 authors: eissmann, kristin; mueller, sebastian; sticht, heinrich; jung, susan; zou, peng; jiang, shibo; gross, andrea; eichler, jutta; fleckenstein, bernhard; reil, heide title: hiv-1 fusion is blocked through binding of gb virus c e2d peptides to the hiv-1 gp41 disulfide loop date: 2013-01-22 journal: plos one doi: 10.1371/journal.pone.0054452 sha: doc_id: 868 cord_uid: vnwpzsu8 a strategy for antiviral drug discovery is the elucidation and imitation of viral interference mechanisms. hiv-1 patients benefit from a coinfection with gb virus c (gbv-c), since hiv-positive individuals with long-term gbv-c viraemia show better survival rates than hiv-1 patients without persisting gbv-c. a direct influence of gbv-c on hiv-1 replication has been shown in coinfection experiments. gbv-c is a human non-pathogenic member of the flaviviridae family that can replicate in t and b cells. therefore, gbv-c shares partly the same ecological niche with hiv-1. in earlier work we have demonstrated that recombinant glycoprotein e2 of gbv-c and peptides derived from the e2 n-terminus interfere with hiv entry. in this study we investigated the underlying mechanism. performing a virus-cell fusion assay and temperature-arrested hiv-infection kinetics, we provide evidence that the hiv-inhibitory e2 peptides interfere with late hiv-1 entry steps after the engagement of gp120 with cd4 receptor and coreceptor. binding and competition experiments revealed that the n-terminal e2 peptides bind to the disulfide loop region of hiv-1 transmembrane protein gp41. in conjunction with computational analyses, we identified sequence similarities between the n-termini of gbv-c e2 and the hiv-1 glycoprotein gp120. this similarity appears to enable the gbv-c e2 n-terminus to interact with the hiv-1 gp41 disulfide loop, a crucial domain involved in the gp120-gp41 interface. furthermore, the results of the present study provide initial proof of concept that peptides targeted to the gp41 disulfide loop are able to inhibit hiv fusion and should inspire the development of this new class of hiv-1 entry inhibitors. introduction gb virus c (gbv-c) is a common human virus that can be transmitted sexually, parenterally and vertically from mother to child [1, 2] . infection of immunocompetent individuals usually leads to clearance of gbv-c viraemia within the first years; however, gbv-c can cause persistent infection in approximately 25% of cases [3] . as a member of the flaviviridae family, for which a fourth genus, termed pegivirus, was recently proposed, it is related to the hepatitis c virus (hcv) [4] . however, acute and persistent infection with gbv-c does not appear to be associated with hepatitis or any other diseases in humans (reviewed in [5, 6] ). the gbv-c genome is a positive-sense, single-stranded rna (9.4 kb) that contains one long open reading frame (orf) [7] [8] [9] . the polyprotein is post-translationally processed by cellular and viral proteases into the structural proteins such as e1 and e2, as well as the nonstructural proteins ns2, ns3, ns4a/b and ns5a/b [10] . in most industrialized countries 1% to 5% of healthy individuals are viraemic for gbv-c and 10% to 15% have developed anti-e2 antibodies as evidence of past gbv-c infection [11] . in contrast to hcv, in the majority of gbv-c infected individuals, the occurrence of anti-e2 antibodies is associated with clearance of the virus [12] . however, gbv-c elimination has been documented without the appearance of anti-e2 antibodies, indicating that a diagnosis based merely on the detection of anti-e2 antibodies in serum might lead to an underestimation of prevalence of prior infection. based on the common transmission routes, the prevalence of gbv-c in hcv and hiv-1-positive individuals is much higher than in the general population. gbv-c rna prevalence rates range from 20% to 30% in hcv coinfected individuals and from 15% to 40% in hiv-1 patients (reviewed in [3] ). several, but not all, epidemiological studies and a metaanalysis have reported that gbv-c viraemia has a beneficial effect on the course of hiv-1 disease progression and survival [13] [14] [15] [16] [17] [18] [19] [20] . furthermore, mother-to-child transmission of gbv-c reduces the vertical transmission of hiv-1 from gbv-c/hiv-1 coinfected mothers [21] , and recently it was reported that accidental gbv-c acquisition via transfusion is associated with a significant reduction in mortality in hiv-infected individuals [22] . gbv-c is a lymphotropic virus that replicates primarily in b and t cells [23] . therefore, it occupies, at least in part, the same ecological niche as hiv-1. several effects have been proposed to explain the interference with hiv-1, including induction of chemokines, cd4 receptor and coreceptor modulation, prevention of th2 cytokine profile, reduction of t cell activation, as well as downmodulation of fas-mediated apoptosis in gbv-c coinfected individuals [24] [25] [26] [27] [28] [29] [30] [31] . cell culture experiments revealed a direct influence on hiv-1 replication by at least two gbv-c proteins, i.e., the nonstructural protein ns5a and the e2 envelope protein [31, 32] . whereas ns5a leads to decreased hiv-1 receptor expression and stabilization of the th1 profile, the e2 protein prevents hiv-1 entry by a mechanism as yet unknown [31, [33] [34] [35] . using synthetic peptides presenting different regions of e2, we recently demonstrated that this interference with hiv-1 entry can be ascribed to the n-terminal part of the gbv-c e2 protein ranging from residue 29 to 72 (according to genbank accession no. af121950) [33] . two overlapping 20 mer peptides (p4-7 and p6-2) presenting amino acids 37 to 56 (wdrgnvtllcdcpngpwvwv) and 45 to 64 (lcdcpngpwvwvpafcqavg), respectively, of the gbv-c e2 protein, were shown to be most potent with ic 50 s between 2 and 0.2 mm in a tzm-bl-based hiv-1 replication assay. cell entry of hiv-1 is mediated by noncovalently associated trimers of gp120 and gp41 subunits assembled into functional spikes on the surface of virions. the noncovalent association between the two subunits appears to be maintained by interaction of the n-and c-termini of gp120 with the disulfide loop region of gp41 and parts of the flanking n-and c-terminal heptad repeat regions (nhr and chr) (reviewed in [36] ). the primary receptor for gp120 is cd4, which is expressed on the surface of monocytes, macrophages, and on subsets of dendritic and t cells [37] . upon engagement of cd4, gp120 undergoes a conformational shift, enabling binding to the chemokine coreceptors cxcr4 or ccr5, respectively [38, 39] . this interaction is thought to induce additional conformational changes in the envelope trimer that result in exposure of the hydrophobic fusion peptide of gp41 and its insertion into the host cell membrane. subsequently, the nhr and chr regions of gp41 fold back and interact with each other, in order to form a six-helix bundle (6-hb), that promotes the convergence of the viral and cellular membrane and the formation of the membrane pore (reviewed in [40] ). in this study, we aimed to elucidate the mechanism of action by which gbv-c e2 20 mer peptides p4-7 and 6-2, representing the e2 residues from 37 to 64, inhibit hiv-1 entry. we demonstrate that e2 peptides act late after cd4 and coreceptor engagement via binding the gp41 disulfide loop region, a new promising target for hiv-1 entry inhibition. peptides n-acetylated or fluorescein-conjugated peptides derived from the gbv-c glycoprotein e2 (p4-7, p4-7s, p6-2, p6-2s, p9, and p28) were obtained from emc microcollections (tübingen, germany). all other peptides (listed in table s1 ) were synthesized by a standard solid-phase fmoc method using a peptide synthesizer. the n-and c-termini of n36 and c34, as well as the gp120 n-terminus peptides, were acetylated and amidated, respectively. the gp41 disulfide peptides (loop36ox and loop36s) were n-terminally biotinylated for site-selective attachment to streptavidin-coated assay plates. loop36o6 was cyclized by formation of a disulfide bridge between the two cysteine residues through air oxidation at ph 8. the peptides were purified to homogeneity (.95% purity) by high-performance liquid chromatography and verified by laser desorption mass spectrometry (perseptive biosystems, framingham, ma) and by esi mass spectrometry, respectively. peptide stocks were dissolved in 75% dmso/h 2 o and diluted for experiments in respective buffers or medium. hiv-1 virions containing the blam-vpr chimera were produced as previously described [33] . briefly, 293t cells were cotransfected with pnl4-3 proviral dna, pcmv-blam-vpr, and padvantage vectors. after 2 days of cultivation, the viruscontaining supernatant was centrifuged for 10 min at 3006g to remove cellular debris. the hiv-1 virion-containing supernatant was overlaid onto a 20% sucrose cushion and ultracentrifuged at 350006g at 4uc for 90 min. the resulting pellet was resuspended in medium and aliquots were frozen at 280uc until usage. pha/il-2-stimulated pbmc or tzm-bl cells were incubated with e2 peptides (25 mm), phorbol 12-myristate 13-acetate (pma; 40 ng/ml), sdf-1a (1 mg/ml) or rantes (50 nm) for 6 hr. cd4, cxcr4 and ccr5 surface expression was analyzed by using standard facs staining protocols. cells were washed with pbs and incubated for 30 min at 4uc with pe-labeled antibodies (cd4 clone sk3, cxcr4 clone 12g5, and ccr5 clone 2d7, bd biosciences, germany). subsequently, washed cells were resuspended in pbs and analyzed with a facscalibur tm flow cytometer (bd biosciences, germany). the virion-based fusion assay was performed essentially as described elsewhere [41] . briefly, 5610 4 tzm-bl cells were infected with sucrose-purified hiv-1 nl4-3 virions containing blam-vpr (5 ng p24-gag per approach). under temperaturearrested state (tas) conditions, binding of the hi virions to target cells was allowed at low temperature via spinning inoculation, for which virions were added to cells and centrifuged at 20956g, 4uc for 30 min. to remove unbound virus particles, cells were washed with cold medium and incubated with 20 mm of each e2 peptide or respective controls (amd-3100 [10 mm], 2f5 [0.5 mg/ml], b12 [0.3 mg/ml], and b4 [0.3 mg/ml]) at 23uc for 1 h. virus-cell fusion was initiated by a temperature shift to 37uc for a total of 2 hr. under standard conditions, e2 peptides, or defined control agents, were added to cell culture shortly before hiv-1 inoculation, and cells were incubated at 37uc for 2 hr. subsequently, cells were washed with hbss, loaded with the ccf2-am substrate, as described by the manufacturer (invitrogen, germany), and incubated overnight at 20uc. blam activity was quantified using a fluorescence plate reader. the extent of viruscell fusion was determined from the ratio of blue (460 nm) and green (510 nm) emission upon exciting the cells at 405 nm. hiv-1 gp120 binding to cd4, ccr5, and cxcr4 hiv-1 gp120 binding studies using flow cytometry were performed as previously described [42] . briefly, supernatants containing soluble fc-gp120 jrcsf fusion protein or the fc control protein were obtained from transiently transfected 293t cells. for binding studies, 293t cells were transiently transfected with pcd4 expression plasmid or an empty plasmid as control, and preincubated with 40 mm of the e2 peptides, 1 mm scd4, 3 mg/ml vrc01, or 10 mm of the ccr5 inhibitor tak-779. similar amounts of soluble fc-gp120 or fc control, were added for 45 min on ice, and cells were washed and stained with anti-human igg, fitc-conjugated secondary antibody (dako, germany) for 30 min at 4uc. subsequently, washed cells were resuspended in pbs and analyzed with a facscalibur tm flow cytometer. data were collected with bd cellquest tm and analyzed with fcs express v3. in parallel, cd4 and ccr5 expression was determined by staining with the antibody clones sk3 and 2d7, respectively (bd biosciences, germany). hiv-1 gp120 binding studies by immunoblotting were performed as previously described [43] . briefly, 293t cells were transiently transfected with either pcd4 or pccr5 expression plasmids. verification of expression was performed 2 days after transfection via flow cytometry. after 2 days of cultivation, the cells were resuspended in binding buffer (50 mm hepes, 5 mm mgcl 2 , 1 mm cacl 2 , 5% bsa, 0.1 mm nan 3 , ph 7.5). in a 50 ml binding assay, either 40 mm tak-779 or maraviroc, 0.8 mm scd4, 40 mg/ml vrc01, 160 mg/ml 447-52d, 200 mg/ ml f425b4e8 (all: nih aids research and reference reagent program), 40 mg/ml 5f3 (polymun scientific), 0.6 mm e2 340 -fc or fc control, 0.2 mm p4-7, p6-2 or p28 and additionally 10 mg/ml hiv-1 bal or hiv-1 iiib gp120 protein (nih aids research and reference reagent program) were added. after incubation for 1 h at 37uc, cells were washed with cold pbs and lysed in triton x-100 and tween-20 containing buffer. lysates were separated by a 5% sds-page and verified with monoclonal gp120 antibody f425 b4a1 (nih aids research and reference reagent program) or with sheep a-hiv-1-gp120 (aalto bio reagents ltd.) in western blot analyses. hiv-1 gp120 (5 ng) was used as a standard. detection was achieved with hrp-conjugated antihuman antibody and chemiluminescence reagents. 293t cells were transiently transfected with an hiv-1 env expression vector, coding for the cxcr4-tropic nl4-3 gp160 precursor (x4env) for surface expression of gp120-gp41 env trimers or empty plasmid as control. after 2 days of cultivation, cells were incubated with 1 mg scd4 and 1 mm fitc-conjugated e2 peptides for 1 h at 37uc respectively. subsequently, cells were washed, resuspended in pbs, and analyzed by flow cytometry using a bd tm lsr ii flow cytometer (becton dickinson biosciences, germany). flow cytometer data were collected with bd facsdiva tm and analyzed with fcs express v3. in parallel, gp160 expression was determined by staining with hiv-1 gp120 mabs (2g12 and 17b) and an anti-human igg-fitc pab (dako, germany). flat-bottom, 96-well microtest elisa plates (sarstedt, germany) were coated overnight at 4uc with recombinant hiv-1 gp120 (0.5 mg/ml), gp41 (0.22 mg/ml) proteins, or with streptavidin (4 mg/ml) in coating buffer (0.1 m nahco 3 , 0.1 m na 2 co 3 , ph 9.87). nonspecific binding was blocked (blocking buffer: 1% bsa, 0.1% tween-20 in phosphate buffer, ph 7.2) for 1 h at room temperature, followed by washing steps (wash buffer: 0.1% tween in phosphate buffer, ph 7.2). streptavidin-coated plates were then incubated with gp41 disulfide loop peptides (loop36ox or loop36s; 2.5 mm). all plates were then incubated with recombinant e2 340 -fc or fc protein in serial dilutions in sample buffer (phosphate buffer, ph 7.2) for 3 hr at room temperature. to perform the competitive elisas, the recombinant e2 340 -fc protein was adjusted at 5 mg/ml and the monoclonal gp41 antibodies (246-d, f240, t32, d50, 2f5, 4e10, chessie 8 [all: nih aids research and reference reagent program], and 5f3 [polymun, austria]) at 100 mm. after washing procedure, the plates were incubated for 1 h with polyclonal rabbit anti-human igg peroxidase antibody (dako, germany) 1:6000 in sample buffer and washed. plates were developed with tmb peroxidase substrate solution (kpl, usa), or with opd/h 2 o 2 solution in the dark. absorbances (ods) were read at 450 or 492 nm, respectively, using a multi-channel photometer (elisa-reader axsym, abbott laboratory, usa). inhibitory activity of the peptides (p4-7, p6-2, and p28) on 6-hb formation was measured by a modified elisa-based method as previously described [44] . briefly, 96-well polystyrene plates were coated with the 6-hb-specific monoclonal antibody, nc-1 igg, (described by [45] ) (2 mg/ml in 0.1 m tris, ph 8.8), and then blocked with 2% non-fat milk. n36 (0.25 mm), the tested peptides and c34 as control were added at graded concentrations into the wells. after incubation for 30 min at 37uc, c34-biotin (0.25 mm) was added, followed by incubation for 45 min at 37uc and washing with wash buffer (pbs containing 0.05% tween-20) six times. streptavidin-labeled horseradish peroxidase (invitrogen, grand island, ny) and the substrate 3,39,5,59-tetramethylbenzidine (sigma, st. louis, mo) were added sequentially. absorbance at 450 nm (od 450 ) was measured. the percent inhibition of 6-hb formation by the peptides was calculated as described before [46] . globular and disordered regions in e2 were identified using globplot2.3 [47] with standard settings. secondary structure prediction was performed using the consensus prediction method provided by the nps@ server [48] . structural homologs were identified with the bioinfo.pl meta prediction server [49] , and modelling was performed with modeller6.2 [50] , using the crystal structure of a single-variable-domain antibody (pdb code: 2z8w) as template [51] . sequence comparison between different e2 proteins and between e2 and gp160 was performed with lalign (http://www.ch.embnet.org/software/lalign_form.html) using the local alignment option. the gp160 signal peptide comprising residues 1 to 30 was removed prior to the similarity searches because it is not present in the mature protein. all statistical analyses were performed using wilcoxon rank-sum test (two-tailed). it has been shown that the hiv-1 receptor/coreceptor density plays a critical role in the efficiency of hiv-1 fusion and infection [52] . thus, we addressed the question of whether or not the two overlapping gbv-c e2 20 mer peptides p4-7 and p6-2 have an impact on the cell surface presentation of the hiv-1 receptors cd4 and cxcr4 or ccr5, respectively. the cell surface presentation of these receptors was quantified via flow cytometry after incubation of primary peripheral blood mononuclear cells (pbmcs) and of hela-derived hiv indicator cells expressing cd4, ccr5 and cxcr4, designated as tzm-bl cells, with the e2 peptides p4-7 and p6-2. the non-hiv-1-inhibitory peptide p28 derived from the c-terminal part of e2 (residue 271 to 290), served as a negative control [33] . as shown in figure 1 , both cd4 and coreceptor expression on pbmc or tzm-bl cells remained largely unaffected by e2 peptides, thereby eliminating a change in receptor density as a mode of action of the e2 peptides. the effect of gbv-c e2 peptides arises after gp120/cd4 and gp120/coreceptor engagement to find out whether early or late steps of hiv-1 entry are affected by the e2 peptides, the e2 peptide activity was determined before and after gp120/cd4 engagement using the vpr-b-lactamase (vpr-blam) enzyme-based virus-cell fusion assay under standard (pre-cd4 binding) and temperature-arrested state (tas; post-cd4 binding) conditions, respectively. based on the temperature sensitivity of envelope-mediated membrane fusion, low temperature (,28uc) during and after cd4/gp120 engagefigure 3 . e2-derived peptides do not influence hiv-1 gp120 binding to cd4 and ccr5. (a) 293t cells expressing cd4 were preincubated with e2-derived peptides and specific controls that bind to gp120 (scd4; vrc01, directed against cd4 binding site [bs]) or ccr5 (tak-779). subsequently, cells were incubated with soluble fc-gp120 (hiv-1 jrcsf ) and binding to cd4 was analyzed by flow cytometry using a fitc-conjugated anti-human igg pab. binding to cells in the absence of inhibitor was set as 100%. columns show average values 6sd of three independent experiments each performed in duplicate. *: p,0.05 in (b) and (c) 293t cells expressing cd4, ccr5, or cxcr4 were preincubated with e2-derived peptides, e2 340 -fc and fc protein as well as specific controls that bind to gp120 (scd4, vrc01, 447-52d, f425 b4e8), gp41 (5f3), or ccr5 (tak-779, maraviroc). soluble hiv-1 gp120 was added to the cd4-, ccr5-, or cxcr4-expressing cells and binding was investigated by separating the cell lysates on sds-page and visualized by performing western blot analyses. no influence of e2 peptides or intact e2 protein on the binding efficiency between gp120 and cd4, ccr5, or cxcr4 respectively, was observed after normalization of band intensity with the gel loading control hsp90. doi:10.1371/journal.pone.0054452.g003 ment leads to an arrest of the fusion process at an intermediate state and the blockade of coreceptor binding, 6-hb formation, and final membrane fusion [53] [54] [55] . as shown in figure 2 , the effect of antibodies that interfere with the initial cd4/gp120 interaction (b12 and b4) was largely abrogated under tas conditions. in contrast, p4-7 and p6-2 showed full activity under both conditions, comparable to known fusion inhibitors like amd-3100 and the neutralizing mper antibody 2f5 that act after cd4 engagement. therefore, these data provide evidence that p4-7 and p6-2 implement their hiv-1 inhibitory activity post-cd4 binding of gp120. to substantiate this conclusion, next we assessed the impact of e2 peptides on gp120 binding to cd4. immunoblotting and flow cytometry data revealed that e2 peptides and the whole e2 ectodomain fused to the fc domain of human igg (e2 340 -fc) that showed hiv-1 inhibitory activity in earlier studies [32] do not interfere with the interaction of recombinant gp120 with cellular expressed cd4 ( figure 3a, b) . in a related approach, the effect of e2 peptides or intact e2 protein on the interaction between gp120 and the coreceptors ccr5 and cxcr4 expressed on cells was tested by immunoblotting. to ensure efficient binding of gp120 to the coreceptors, soluble cd4 (scd4) was added simultaneously. use of ccr5-antagonists maraviroc and tak-779 or anti-gp120 coreceptor binding site (v3-loop) antibodies 447-52d or f425b4e8, respectively, as positive controls revealed a clear reduction in the gp120/ccr5 and a less pronounced decline (densitometric analysis ,50%) in the gp120/cxcr4 interaction. however, after normalization of the band intensity (hsp90; data not shown) there was no indication for any e2-effect on the binding efficiency between gp120 and the hiv-1 coreceptors ccr5 or cxcr4, respectively ( figure 3c , d). recombinant gbv-c e2 protein interacts with gp41, while n-terminal gbv-c e2 peptides abrogate this interaction previously, we have shown that the target of the hiv-1inhibitory e2 peptides is most likely located on the hiv-1 particle, rather than on the cell [33] . the surface of the hiv-1 particle consists of a lipid bilayer with incorporated host cell proteins and a limited number of functional envelope (env) trimers (on average 1467) [56] . therefore, we tested whether the hiv-1-inhibitory e2 peptides interact with trimeric hiv-1 env proteins expressed on the surface of transfected cells. for this purpose, 293t cells were transiently transfected with an hiv-1 env expression vector, coding for the gp160 precursor of the cxcr4-tropic nl4-3 envelope (x4env). binding of fitc-labeled e2 peptides was monitored by flow cytometry. although we could observe a background attachment of p4-7 and p6-2 to mock-or vectortransfected 293t cells, there was a significant increase in the binding of p4-7 and p6-2 when cells expressed hiv-1 env and were pretreated with scd4 ( figure 4a ). in contrast, the increase of binding was not observed with the negative control peptide p28. these data suggest that the hiv-inhibitory e2 peptides bind to hiv-1 env and that this interaction seems to be cd4-dependent, since the induction of conformational changes in gp120 and/or the increase in availability of gp41 after cd4 engagement facilitates e2-binding. in order to prove the assumption that gbv-c e2 interacts with hiv env, subsequent binding assays were performed with plate immobilized subunits of hiv-1 envelope proteins (gp120 and gp41). to improve the significance of this experiment, here we used the whole e2-fc fusion protein (e2 340 -fc) as a ligand. the fc domain alone served as a negative control. ninety-six-well plates were either coated with recombinant full-length gp120 (gp120 iiib [immuno diagnostics, 1001-10], and gp120 mn [immune technology, it-001-002mnp]), expressed in eukaryotic cells, or with gp41, expressed in e. coli, consisting of the complete (gp41 iiib [abcam, ab68129]) or truncated gp41 ectodomain (gp41 mn [nih, 12027] ). in gp41 mn , the hydrophobic fusion peptide (fp), the transmembrane region (tm), and most of the c-terminal part of the cytoplasmic tail (cp) were omitted (illustrated in figure 5a ). whereas no binding of the recombinant e2 340 -fc fusion protein to gp120 was observed, e2 340 -fc protein was shown to interact with both gp41 variants in a dose-dependent manner ( figure 4b ). to test whether gbv-c e2 binds hiv gp41 with the nterminal domain, competition assays were performed with the nterminal e2 peptides p4-7 and p6-2. the results of this experiment revealed that both hiv-1-inhibitory e2 peptides abrogated the protein-protein interaction between hiv-1 gp41 and gbv-c e2 in a dose-dependent manner, whereas increasing amounts of the negative control peptides p9 and p28 had no effect ( figure 4c , left panel). the e2 peptide p9 (residue 81 to 100 of gbv-c e2, see table s1 ) was chosen as an additional control [33] , due to its more comparable content of cysteine and hydrophobic residues. noteworthy, p4-7 and p6-2 contain either two or three cysteines, respectively. to elucidate the characteristics of the e2-gp41 interaction, next we tested the competition ability of peptide variants p4-7s and p6-2s in which the cysteine residues were replaced with serine. the results revealed that the serine variants p4-7s and p6-2s had lost their ability to abrogate the e2-gp41 binding, suggesting that the cysteine residues within the hivinhibitory e2 peptides play a crucial role for gp41 recognition ( figure 4c, right panel) . in combination with these findings, we are able to show that the n-terminal region of gbv-c e2 (,residue 37 to 64), that is represented by the hiv-1-inhibitory peptides p4-7 and p6-2, interacts with hiv-1 gp41 and that the cysteine residues within this region appear to contribute substantially to the gp41 binding. we next sought to localize the e2 peptide binding site within gp41. the transmembrane protein gp41 consists of several functional regions ( figure 5a ). the fp, tm and cp regions of gp41 do not appear to be involved in the interaction with gbv-c e2 or e2 peptides, since the absence of these regions (gp41 mn ) did not abrogate or decrease the e2 binding to gp41 ( figure 4b ). to test whether the e2 peptides interact with either the nhr or chr regions of gp41, which would lead to a blockade of 6-hb formation, a competitive elisa was performed. in this assay, peptides n36 and biotinylated c34, which present the nhr and the chr region of gp41, respectively, interact with each other, forming stable 6-hb structures in vitro [57, 58] . the 6-hb-specific mab nc-1 was used as a capture antibody [45] and the gbv-c e2 peptides were tested for competition with n36 and c34, respectively. neither p4-7 nor p6-2 was able to interfere with the formation of 6-hb, suggesting that the binding site of the e2 peptides is neither located in the nhr (n36) nor in the chr (c34) regions of gp41 ( figure 5b ). to further dissect the e2 binding site within gp41, we tested whether the e2 peptides compete for gp41 binding with different monoclonal anti-gp41 antibodies with known specificities, including mabs 246-d, f240, t32, d50, 5f3, 2f5, 4e10, and chessie 8. of the eight antibodies, only the gp41 interaction of f240 and t32 was inhibited in a dose-dependent manner by p4-7 and p6-2, but not by the negative control p28 ( figure 5c ). no unspecific binding between the e2 peptides (p4-7 and p6-2) and the antibodies f240 or t32 was observed that could have account for the same competition results (data not shown). therefore, we conclude that p4-7 and p6-2 compete with f240 and t32 considerably for the same binding region. f240 and t32 represent two of three tested antibodies that recognize the immunodominant disulfide loop region of gp41. their epitopes overlap partially with each other, implying that the e2 peptides bind approximately between residues 596 and 612 of gp160. the epitopes of all tested anti-gp41-mabs are shown in figure 5a and table s2 . again, the serine variants of p4-7 and p6-2 lost their ability to compete for the binding target with the disulfide loop antibody f240 or were at least less efficient (t32). finally, we performed a direct binding assay with biotin-tagged 36mer gp41 disulfide loop peptides (residues from 588 to 623 according to hiv gp160 hxb2 ) and gbv-c e2 340 -fc. we immobilized via streptavidin an oxidized (closed loop) or a linear variant of the disulfide loop, in which the cysteine residues were replaced with serine (table s1 ) onto plates and the binding of graded concentrations of gbv-c e2 340 -fc was monitored with hrp-anti-human antibodies. indeed, a distinct binding of recombinant e2 340 -fc protein to the gp41 disulfide loop peptides was evident in a dose dependent manner, whereas for the fc control only a low background binding was observed. the binding of e2 340 -fc was clearly diminished when the linear serine variant of the gp41 disulfide loop was tested ( figure 5d ). specific intra-and intermolecular interactions within hiv-1 env are essential for the formation of a native and functional hiv-1 envelope trimer, as well as for concerted conformational changes during the entry process. therefore, peptides or small molecules that mimic env regions involved in these interactions are potential inhibitors of the formation of properly folded tertiary and quaternary env structures. examples of this strategy are potent peptide fusion inhibitors, which resemble the gp41 nhr or chr sequences, including enfuvirtide (t20) [59] , sj-2176 [60] , or n36 [61] . hence, we performed a similarity search between the gbv-c e2 ectodomain and hiv-1 gp160 and identified a local similarity of two sequence stretches of the gbv-c e2 n-terminus to the hiv-1 gp120 n-terminus ( figure 6a ). the sequence stretches 33-46 and 54-70 of e2 exhibit sequence similarities of 85.7% and 76.5% to the n-terminus of gp120. this region of similarity also agrees very well with the sequence region spanned by e2-derived peptides (residues 29-72) that proved to be potent in hiv-entry inhibition [33] . we have also put this sequence similarity in the structural context of gp120 and gp41. high-resolution structural information on gp120-gp41 interaction remains elusive (reviewed in [36] ). however, several mutagenesis studies and a recent crystal structure of gp120 suggest that the n-and c-termini of gp120 interact predominantly with the disulfide loop region of gp41 [62, 63] . interestingly, the n-terminal sequence stretch of gp120 that has been proposed to be involved in the gp120-gp41 interaction is located within the gp120 region that resembles the hiv-1-inhibitory n-terminal gbv-c e2 peptides ( figure 6b ). furthermore, residues within the gp41 trimer, which have been proposed to be involved in the gp120 interface, are located near or within the epitopes of the two antibodies t32 and f240 whose interaction with gp41 was disrupted by the e2 peptides p4-7 and p6-2 ( figure 6c ). to prove the assumption that a similarity between the gbv-c e2 and hiv gp120 n-termini enables e2 or e2 peptides to bind to gp41 in a gp120 manner, a corresponding n-terminal gp120 peptide should be able to disturb the e2-gp41 interaction. therefore, we assessed in e2-gp41 competition assays the effect of a gp120 peptide (n35, gp120 residue 31 to 65) that includes the respective e2-similarity region (figure 7) . we observed that n35 efficiently competed with e2 for gp41 binding, implying that e2 and gp120 share the same gp41 interaction site. not surprisingly, the gp41 affinity was more pronounced for n35 (ic 50 :0.7 mm) than for the e2 peptides (ic 50 s: 7.0 mm for p6-2 and 11.7 mm for p4-7). the c54s variant of this peptide (n35s) still abrogated the e2-gp41 interaction in a dose dependent manner, but less efficiently than the wild-type n35 peptide (ic 50 :4.6 mm). together, these results strongly support the hypothesis that a mimicry phenomenon between the n-termini of the non-related viral glycoproteins e2 gbv-c and gp120 hiv, enables the e2 n-terminus to approach the gp120 binding site on gp41 that is believed to be the disulfide loop region. since no structural information on the gbv-c e2 protein is currently available, we performed structure predictions and globularity analyses of the gbv-c e2 protein in order to get an idea whether the n-terminus of e2 is accessible to the interaction with hiv-1 env involved in membrane fusion. our analysis consistently suggests the presence of a folded region spanning mn) and peptides (n36, c34) used in elisa as well as the binding epitopes of different monoclonal gp41 antibodies. gp41 consists of the n-terminal fusion peptide (fp), the n-and c-heptad repeats (nhr, chr) that are connected by the disulfide loop region (c-c loop), the membrane proximal external region (mper), the a-helical transmembrane-spanning domain (tm), and the cytoplasmic tail (cp). (b) inhibitory activity on 6-helix-bundle (6-hb) formation of peptides p4-7, p6-2, and p28, as well as c34 as control was measured by elisa using the nc-1 monoclonal antibody that detects the 6-hb formation between c34-biotin and n36 peptides. each sample was tested in triplicate. this experiment was repeated twice and similar results were obtained. the statistical significance (p,0.01) was achieved for all measure points (c34 vs. p28). (c) competitive binding of e2 peptides and different gp41 targeting mabs to immobilized recombinant gp41 mn . simultaneously to mab incubation e2 peptides were added with increasing amounts. the graphs show average values of three independent experiments each performed in duplicate. *: p,0.05 (p4-7 or p6-2 vs. p28) (d) binding of recombinant e2 340 -fc protein and fc protein as control to cyclic (loop36ox) and linear (loop36s) peptides presenting the hiv-1 gp41 disulfide loop (residues 588-623). the graphs show average values of three independent experiments each performed in duplicate. the statistical significance (p,0.05) was achieved with more than 0.003 mm concentrations of each protein (loop36ox+e2 340 -fc vs. loop36ox+fc). doi:10.1371/journal.pone.0054452.g005 figure 6 . local similarities between the n-termini of gp120 and e2. (a) similarities of the two local sequences in the n-termini of hiv-1 gp120 and gbv-c e2. the detected e2 stretches (residues 33-46 and 54-70) are shown in the context of the entire e2 n-terminus and are color coded in green and orange, respectively. illustrated are the sequences of the peptides that proved to be potent in hiv-1 entry inhibition [33] . these peptides almost exclusively cover the e2 sequence stretch that exhibits similarities to the gp120 n-terminus. molecules are shown in space-filled presentation and the functionally important regions are colored. (b) structure of monomeric gp120. the n-terminal region of gp120 that exhibits local similarity with the active e2-derived peptides is shown in blue. residues that were deduced from mutational analyses to be relevant for the gp120-gp41 interaction (reviewed in [36] ) are additionally shown in green. (c) structure of trimeric gp41. the disulfide-bonded loops that are recognized by the t32 antibody (residues 596-612) are shown in red. residues 592-596 additionally present in the f240 epitope are shown in orange. residues that were deduced from mutational analyses to be relevant for the gp120-gp41 interaction (reviewed in [36] ) are additionally shown in green. the coordinates for structure presentations were taken from pdb entries 3jwd and 2ezo for gp120 and gp41, respectively. doi:10.1371/journal.pone.0054452.g006 residues 76 to 195 of gbv-c e2. the remaining extracellular part, in particular the n-terminal region including amino acids 1 to 75 that comprise the hiv-1-inhibitory domain, does not contain significant amounts of regular secondary structure and is therefore predicted to form no globular conformation ( figure 8) . consequently, the e2 n-terminus is likely to be rather flexible which could plausibly account for its interaction with gp41 during hiv-1 entry. in this study, we have explored the molecular mechanism underlying the previously reported interference with hiv-1 entry by two overlapping peptides derived from the n-terminal part of the gbv-c envelope protein e2. we are able to demonstrate that the e2 peptides interfere with late stages of hiv-1 entry. this notion is evidenced by the observations that neither the cell surface expression of hiv-1 receptors, nor the binding of gp120 to cd4 and coreceptors, respectively, appears to be affected. instead, hiv-inhibitory e2 peptides showed full activity on hiv entry at a post-binding stage. in agreement with these findings, binding experiments revealed an interaction between e2 or e2 peptides and the hiv-1 transmembrane protein gp41 responsible for late membrane fusion activity. in an earlier study, we demonstrated that the hiv-1 inhibitory region ranges from residue 29 to 72 of the gbv-c e2 n-terminus [33] . in this study we show that the e2 peptides p4-7 and p6-2 (representing e2 residues 37 to 64) compete with e2 protein for gp41 binding. therefore, we assume that the hiv-inhibitory region or at least the inner domain ranging from residue 37 to 64 reflects a gp41-binding site of gbv-c e2. interestingly, the sequence stretch covered by the hiv-inhibitory peptides includes the strongest conserved part of the nonglobular n-terminus of related flaviviridae e2 proteins ( figure s1 ). in particular, several cysteines (c46, c48, c60) and tryptophans (w55, w65) are highly conserved among gbv-c and gbv-a isolates, suggesting that the respective e2 proteins might also exhibit a similar anti-lentiviral activity. the alignment shown in figure s1 also reveals that the sequence conservation is less pronounced for the more distantly related e2 proteins of gbv-b and gbv-d. the n-termini of the e2 proteins from gbv-c and hcv show no detectable sequence homology at all rendering functional similarity rather unlikely. the results of several experiments aimed at dissecting the e2binding domain within gp41 suggest that e2 interacts with the gp41 disulfide loop region. the gp41 disulfide loop region structurally connects the nhr and chr domains and contains two conserved cysteines [64] . noteworthy, the n-terminal e2 region contains three cysteine residues (cys46, cys48 and cys60) as well. previously we could show that variants of the e2 peptides (p4-7s and p6-2s), in which cysteines were replaced with serine residues, lost their hiv-inhibitory capacity [33] . in agreement with these observations, in this study, p4-7s and p6-2s lost their ability to compete with e2 for gp41binding. in addition, the cysteine residues within the disulfide loop peptide appeared to be crucial for the interaction with recombinant e2 protein. this implies that the cysteine residues within the hiv-inhibitory e2 peptides may interfere with the oxidation state of the respective cysteines in the gp41 disulfide loop region. a variety of evidence suggests that a number of viral envelope glycoproteins depend on a dynamic thiol/disulfide balance to mediate virus-cell fusion (reviewed in [65] ). for hiv-1 it has been shown that after cd4 binding a cell surface-associated reductase activity leads to cleavage of disulfide bonds at least within gp120 and that this event is obligatory for triggering membrane fusion [66] . however, the insights into the mechanistic role of the disulfide loop cysteines for the fusion reaction are still limited and need further evaluation. future studies will show, whether reducing agents would change the interference effects of gbv-c e2-derived peptides. based on cryo-em structural information, the gp41 transmembrane protein is expected to be at least partially buried in the trimeric gp41-gp120 structure [67] . thus, the transient states of gp41 appears to be valid hiv-1 inhibitor targets, as evidenced by a number of known hiv-1 fusion inhibitors, including gp41targeted peptides and low-molecular-weight inhibitors. these inhibitors typically bind to the nhr or chr regions during the prehairpin stage in order to prevent the formation of the 6-hb. however, münch et al. [42] isolated the natural hiv-1 entry inhibitor virip from human hemofiltrates, targeting the gp41 fusion peptide, and broad neutralizing antibodies, like 2f5, 4e10, and z13e1, bind the mper, an epitope that is also transiently accessible at a late stage of hiv-1 entry. our results show that the interaction of a peptide with the gp41 disulfide loop region can block hiv-1 fusion, thus introducing the gp41 disulfide loop as a new and promising target for hiv-1 entry inhibition. the relevance of disulfide loop-targeting hiv-1 inhibitors is further supported by two reports that describe low-molecular-weight hiv-1 inhibitors causing resistance-conferring mutations within that disulfide loop region [68, 69] . the gp41 disulfide loop is an immunodominant region termed cluster i [70] , suggesting that this region is permanently or at least transiently accessible [71] . however, unlike the e2 peptides studied here, antibodies directed against cluster i appear to have no relevant hiv-1 neutralizing capacity. the difference of action between the cluster i antibodies and e2 peptides is an interesting question that needs further investigation. one possible explanation may be the fact that steric forces prevent globular antibodies to gain access to the gp41 disulfide loops at the right time, whereas peptides may have sufficient flexibility for this purpose. however, it is tempting to speculate whether in vivo the intact e2 ectodomain is able to reach the gp41 disulfide loop in a peptide-like manner? while no structural information on e2 is available, our computerbased structural analysis revealed that the n-terminal region of e2 (residues 1 to 75) appears to be largely unstructured, suggesting that this protein domain may be sufficiently flexible to interact with the disulfide loop of gp41 during hiv-1 entry. in this context, experiments addressing the amount of circulating e2 (virus-versus possibly exosome-associated or processed e2) in plasma of gbv-c coinfected hiv-positive individuals appear appropriate. the surface glycoprotein gp120 and the transmembrane gp41 subunits are noncovalently associated on the viral surface. however, little is known at the atomic level about the critical gp120-gp41 interface. several mutagenesis studies suggest that the n-and c-termini of gp120 interact with the disulfide loop of gp41 and flanking parts of the nhr and chr regions [62, 63, [72] [73] [74] . , for which a structural model could be generated, is shown in backbone presentation and colored according to the secondary structure type. the n-terminus (residues 1-75), which is not predicted to adopt a globular three-dimensional structure, is schematically depicted as circles indicating the identity of the respective amino acids. cysteines, which may form disulfide bonds, are shown as diamonds and their sequence position is indicated. residues belonging to the p4-7 and p6-2 peptides investigated in the present study are highlighted in red and blue, respectively. overlapping residues present in both peptides are shown in magenta. doi:10.1371/journal.pone.0054452.g008 however, our knowledge about the dynamic gp120-gp41 interplay during the different phases of hiv-1 entry is sparse. we do not know which parameters allow gp120 to stabilize gp41 in a metastable conformation in the unliganded trimer and after cd4 engagement but support gp41 rearrangements after coreceptor binding. a recent crystal structure of an hiv-1 gp120 core with intact gp120 n-and c-termini revealed three topologically separate and structurally plastic layers [75] . the conformational mobility of these layers together appears to be relevant for buffering movements of gp120 from gp41. it should be noted that the extended gp120 n-terminus appeared particularly flexible, such that interactions with gp41 in the viral spike could easily induce termini movements [75] . this plasticity of the termini might also offer an explanation for the fact that the interactions of the gp120 n-terminus with gp41 can be mimicked by flexible peptides. computational and experimental data from this study indicate that n-terminal gbv-c e2 peptides mimic the hiv-1 gp120 n-terminus and are therefore able to compete with gp120 for binding to the disulfide loop region of gp41. competition experiments confirmed that e2 peptides (p4-7 and p6-2) and a corresponding n-terminal gp120 peptide ranging from residue 31 to 65 of gp120 share the same binding region in gp41. therefore, we conclude that the respective e2 peptides may displace gp120 and disturb the interface between gp120 and gp41 after cd4 or coreceptor binding. this phenomenon may result in impeding the concerted interplay between stabilization (native, unbound state) and destabilization (receptor-bound and following states) of the gp120-gp41 complex, which is critical for hiv-1 entry to take its proper course. in particular, binding of e2 or e2-derived peptides to the gp41 disulfide loop may influence the proper disulfide formation of gp41, the flexibility of gp41 that is needed to promote the 6-hb formation, or the membrane accessibility of gp41 that is essential for lipid mixing. however, the elucidation of the precise mode of action of the disulfide loop targeting peptides will be an interesting subject for future evaluations. in this context, it would be interesting to consider chimeric peptides that incorporate sequences of gbv-c e2 and the hiv-1 n-terminus of gp120 as a new approach to enhance the antiretroviral activity of disulfide loop-targeted peptide inhibitors. previously, herrera et al. [76] reported that peptides derived from the n-terminus of gbv-c e2 (ranging from residues 31 to 78), as well as several more downstream regions of e2, interact with the hiv-1 fusion peptide, resulting in suppression of hiv-1 replication. our data do not confirm the association of the gbv-c e2 n-terminus with the hiv-1 fusion peptide during hiv-1 entry. however, it is very plausible that other e2 regions interact with the n-terminal gp41 fusion peptide, in addition to the interaction of the gbv-c e2 n-terminus with the gp41 disulfide loop proposed in this study. most recently, xiang et al. [35] reported that the gbv-c e2 protein or a peptide representing the e2 residues 276 to 292 interfered with hiv-1 entry, when expressed in jurkat cells. however, when added to cells the respective peptide did not inhibit hiv-1 entry unless it was fused to tat for cellular uptake. taken together, these observations support the relevance of gbv-c e2 for hiv-1 entry interference but suggest that additionally to the n-terminus at least the c-terminal part of the e2 ectodomain is involved in hiv-1 entry inhibition. this may also explain the modest effect on cxcr4 cell surface presentation upon expression of full length e2 that we do not see after cell incubation with n-terminal e2 peptides. however, it needs further evaluation to what extent e2-presenting gbv-c particles or gbv-c infected cells via a bystander effect, proposed by xiang et al. [35] , contribute to the e2-mediated hiv-1 entry impairment. in conclusion, we propose a new strategy of hiv-1 entry inhibition based on sequence resemblance between the n-termini of gbv-c e2 and hiv-1 gp120. our results demonstrate that peptides targeting the gp41 disulfide loop are able to inhibit hiv-1 fusion, introducing a novel design concept for hiv-1 fusion inhibitors. figure s1 multiple sequence alignment of related e2 proteins. multiple sequence alignment of e2 proteins from gbv-c (black), gbv-a (green), gbv-b (red), and gbv-d (blue). the gbv-c isolate used in the present study is shown in the first line and the sequence stretch covered by the active peptides is underlined. the second line shows a distantly related e2 protein from chimpanzee gbv-c to highlight the sequence divergence within the gbv-c isolates. conserved cysteines and aromatic residues are shown as bold letters. (tif) table s1 sequences of synthetic gbv-c e2 and hiv-1 peptides. *numbering follows gbv-c e2 genbank accession no. af121950 or hiv-1 hxb2 gp160 (hiv databases: http://www. hiv.lanl.gov), respectively x: e-aminohexanoic acid. (doc) gb virus c transmission by blood products prevalence of gb virus type c/hepatitis g virus rna and of anti-e2 in individuals at high or low risk for blood-borne or sexually transmitted viruses: evidence of sexual and parenteral transmission gb virus type c interactions with hiv: the role of envelope glycoproteins the gb viruses: a review and proposed classification of gbv-a, gbv-c (hgv), and gbv-d in genus pegivirus within the family flaviviridae g-pers creepers, where'd you get those papers? a reassessment of the literature on the hepatitis g virus gb virus type c/hepatitis g virus sequence and genomic organization of gbv-c: a novel member of the flaviviridae associated with human non-a-e hepatitis molecular cloning and disease association of hepatitis g virus: a transfusiontransmissible agent isolation of novel virus-like sequences associated with human hepatitis hepatitis g virus encodes protease activities which can effect processing of the virus putative nonstructural proteins humoral immune response to the e2 protein of hepatitis g virus is associated with longterm recovery from infection and reveals a high frequency of hepatitis g virus exposure among healthy blood donors age-dependent acquisition of hepatitis g virus/gb virus c in a nonrisk population: detection of the virus by antibodies gb virus c/hepatitis g virus infection: a favorable prognostic factor in human immunodeficiency virus-infected patients? carriage of gb virus c/hepatitis g virus rna is associated with a slower immunologic, virologic, and clinical progression of human immunodeficiency virus disease in coinfected persons infection with gb virus c and reduced mortality among hiv-infected patients effect of gb virus c/hepatitis g virus coinfection on the course of hiv infection in hemophilia patients in japan persistent gb virus c infection and survival in hiv-infected men effect of coinfection with gb virus c on survival among patients with hiv infection effect of hepatitis g virus infection on progression of hiv infection in patients with hemophilia. multicenter hemophilia cohort study effect of early and late gb virus c viraemia on survival of hiv-infected individuals: a meta-analysis reduced mother-to-child transmission of hiv associated with infant but not maternal gb virus c infection acquisition of gb virus type c and lower mortality in patients with advanced hiv disease gb virus c replicates in primary t and b lymphocytes inhibition of hiv strains by gb virus c in cell culture can be mediated by cd4 and cd8 t-lymphocyte derived soluble factors gb virus type c infection modulates t-cell activation independently of hiv-1 viral load gbv-c coinfection is negatively correlated to fas expression and fas-mediated apoptosis in hiv-1 infected patients regulation of cc chemokine receptor 5 in hepatitis g virus infection slower progression of hiv-1 infection in persons with gb virus c co-infection correlates with an intact t-helper 1 cytokine profile gb virus c coinfection in advanced hiv type-1 disease is associated with low ccr5 and cxcr4 surface expression on cd4(+) t-cells inhibition of hiv-1 replication by gb virus c infection through increases in rantes, mip-1alpha, mip-1beta, and sdf-1 an 85-aa segment of the gb virus type c ns5a phosphoprotein inhibits hiv-1 replication in cd4+ jurkat t cells hiv entry inhibition by the envelope 2 glycoprotein of gb virus c peptides derived from a distinct region of gb virus c glycoprotein e2 mediate strain-specific hiv-1 entry inhibition gb virus type c infection polarizes t-cell cytokine gene expression toward a th1 cytokine profile via ns5a protein expression characterization of a peptide domain within the gb virus c envelope glycoprotein (e2) that inhibits hiv replication hiv envelope: challenges and opportunities for development of entry inhibitors the t4 gene encodes the aids virus receptor and is expressed in the immune system and the brain cc ckr5: a rantes, mip-1alpha, mip-1beta receptor as a fusion cofactor for macrophage-tropic hiv-1 hiv-1 entry cofactor: functional cdna cloning of a seven-transmembrane, g protein-coupled receptor entry inhibitors in the treatment of hiv-1 infection a sensitive and specific enzyme-based assay detecting hiv-1 virion fusion in primary t lymphocytes discovery and optimization of a natural hiv-1 entry inhibitor targeting the gp41 fusion peptide the thetadefensin, retrocyclin, inhibits hiv-1 entry identification of a critical motif for the human immunodeficiency virus type 1 (hiv-1) gp41 core structure: implications for designing novel anti-hiv fusion inhibitors a conformation-specific monoclonal antibody reacting with fusion-active gp41 from the human immunodeficiency virus type 1 envelope glycoprotein n-substituted pyrrole derivatives as novel human immunodeficiency virus type 1 entry inhibitors that interfere with the gp41 six-helix bundle formation and block virus fusion globplot: exploring protein sequences for globularity and disorder protein structure prediction. implications for the biologist 3d-jury: a simple approach to improve protein structure predictions comparative protein structure modeling. introduction and practical examples with modeller structure of an ignar-ama1 complex: targeting a conserved hydrophobic cleft broadens malarial strain recognition sensitivity of hiv-1 to entry inhibitors correlates with envelope/coreceptor affinity, receptor density, and fusion kinetics the temperature arrested intermediate of viruscell fusion is a functional step in hiv infection a covalent inhibitor targeting an intermediate conformation of the fusogenic subunit of the hiv-1 envelope complex evidence that the transition of hiv-1 gp41 into a six-helix bundle, not the bundle configuration, induces membrane fusion distribution and three-dimensional structure of aids virus envelope spikes core structure of gp41 from the hiv envelope glycoprotein atomic structure of the ectodomain from hiv-1 gp41 peptides corresponding to a predictive alpha-helical domain of human immunodeficiency virus type 1 gp41 are potent inhibitors of virus infection hiv-1 inhibition by a peptide a trimeric structural subdomain of the hiv-1 transmembrane glycoprotein alanine scanning mutants of the hiv gp41 loop peptide mimic of the hiv envelope gp120-gp41 interface model for the structure of the hiv gp41 ectodomain: insight into the intermolecular interactions of the gp41 loop cell entry by enveloped viruses: redox considerations for hiv and sars-coronavirus glycosaminoglycans and protein disulfide isomerase-mediated reduction of hiv env subunit organization of the membrane-bound hiv-1 envelope glycoprotein trimer sensitivity to a nonpeptidic compound (rpr103611) blocking human immunodeficiency virus type 1 env-mediated fusion depends on sequence and accessibility of the gp41 loop region a lowmolecular-weight entry inhibitor of both epitope map of human immunodeficiency virus type 1 gp41 derived from 47 monoclonal antibodies produced by immunization with oligomeric envelope protein epitope mapping of two immunodominant domains of gp41, the transmembrane protein of human immunodeficiency virus type 1, using ten human monoclonal antibodies a recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits is an antigenic mimic of the trimeric virion-associated structure human immunodeficiency virus type 1 gp120 envelope glycoprotein regions important for association with the gp41 transmembrane glycoprotein role of the hiv gp120 conserved domain 1 in processing and viral entry structure of hiv-1 gp120 with gp41-interactive region reveals layered envelope architecture and basis of conformational mobility effect of synthetic peptides belonging to e2 envelope protein of gb virus c on human immunodeficiency virus type 1 infection a small-molecule, nonpeptide ccr5 antagonist with highly potent and selective anti-hiv-1 activity we thank lu lu key: cord-000143-2xvd5ogf authors: napthine, sawsan; lever, robert a.; powell, michael l.; jackson, richard j.; brown, t. david k.; brierley, ian title: expression of the vp2 protein of murine norovirus by a translation termination-reinitiation strategy date: 2009-12-22 journal: plos one doi: 10.1371/journal.pone.0008390 sha: doc_id: 143 cord_uid: 2xvd5ogf background: expression of the minor virion structural protein vp2 of the calicivirus murine norovirus (mnv) is believed to occur by the unusual mechanism of termination codon-dependent reinitiation of translation. in this process, following translation of an upstream open reading frame (orf) and termination at the stop codon, a proportion of 40s subunits remain associated with the mrna and reinitiate at the aug of a downstream orf, which is typically in close proximity. consistent with this, the vp2 start codon (aug) of mnv overlaps the stop codon of the upstream vp1 orf (uaa) in the pentanucleotide uaa ug. principal findings: here, we confirm that mnv vp2 expression is regulated by termination-reinitiation and define the mrna sequence requirements. efficient reintiation is dependent upon 43 nt of rna immediately upstream of the uaa ug site. chemical and enzymatic probing revealed that the rna in this region is not highly structured and includes an essential stretch of bases complementary to 18s rrna helix 26 (motif 1). the relative position of motif 1 with respect to the uaa ug site impacts upon the efficiency of the process. termination-reinitiation in mnv was also found to be relatively insensitive to the initiation inhibitor edeine. conclusions: the termination-reinitiation signal of mnv most closely resembles that of influenza bm2. similar to other viruses that use this strategy, base-pairing between mrna and rrna is likely to play a role in tethering the 40s subunit to the mrna following termination at the vp1 stop codon. our data also indicate that accurate recognition of the vp2 orf aug is not a pre-requisite for efficient reinitiation of translation in this system. for most eukaryotic mrnas, translation initiation is a 59-enddependent process beginning with recognition of the cap structure by the cap-binding complex eif4f [1] and (usually) recognition of the aug codon of the first open reading frame (orf) on the mrna by the scanning ribosome complex [2] . this 59-end dependence is a problem faced by many rna viruses with polycistronic genomes and elaborate strategies have been developed to facilitate access of ribosomes to downstream open reading frames (orfs). amongst these, a number of unconventional translation strategies have been described [3] . these include leaky scanning of 40s subunits past the start codon of the first orf [4] , the possession of intercistronic internal ribosome entry signal [5] , programmed ribosomal frameshifting during elongation [6] and stop codon suppression at the termination step [7] [8] . another strategy that has evolved to allow expression of a downstream orf is termination-reinitiation (also referred to here as stop-start). in this process, ribosomes translate the upstream orf but following termination, a proportion of 40s subunits remain tethered to the mrna and go on to reinitiate at the start codon of the downstream orf. this termination-dependent reinitiation strategy allows the coupled expression of products from adjacent orfs and thus the production of a defined ratio of gene products. termination-reinitiation in virus systems [9] was first described in the synthesis of the bm2 protein of the orthomyxovirus influenza b virus [10] and subsequently in expression of vp2 of feline calicivirus (fcv) of the genus vesivirus [11] [12] [13] and vp10 of the calicivirus rabbit haemorrhagic disease virus (rhdv) of the genus lagovirus [14] . a related phenomenon is also seen in expression of the m2-2 protein [15] [16] of the paramyxovirus respiratory syncytial virus (rsv) and the m2-2 protein [17] of pneumovirus of mice (pvm). in fcv, the stop codon (uga) of the major capsid stop-start protein vp1 overlaps the start codon of the minor capsid protein vp2 (auga) (the stop-start ''window''). efficient termination-reinitiation depends upon several factors, including the close proximity of the stop and start codons, the transit of ribosomes along the vp1 mrna up to the stop codon and a stretch of some 70-80 nucleotides (nt) of mrna upstream of the stop-start window whose primary sequence, rather than the encoded protein, is key. this region of the mrna, termed the termination upstream ribosomal binding site (turbs), is needed for the retention of post-termination 40s subunits [11] . a short sequence of the turbs (termed motif 1) that is complementary to part of helix 26 of 18s rrna likely acts to tether the 40s ribosomal subunit to the mrna post-termination, allowing time for the ribosome to acquire the factors necessary to initiate on the downstream orf [12, 14, 18] . the turbs may also act by recruitment of eukaryotic initiation factor 3 (eif3) or eif3/40s complexes [13] . recent studies of termination-reinitiation in the expression of the orthomyxovirus influenza bm2 protein have revealed a requirement for a shorter stretch of mrna (45 nt) upstream of the stop-start window, but nevertheless, the rna contains a similar turbs motif 1 [19] . from rna secondary structure probing, it has been proposed that this stretch may be displayed on the apical loop of a stem-loop structure that may form following transit of the ribosome through the region and termination at the upstream orf stop codon [9, 19] . in this paper, we describe an analysis of termination-reinitiation in the expression of the vp2 protein of murine norovirus (mnv), a calicivirus of the genus norovirus. the vp2 start codon (aug) of mnv overlaps the stop codon of the upstream vp1 orf (uaa) in the pentanucleotide uaaug, consistent with a terminationreinitiation strategy, and a stretch of bases (59 uaugggaa 39) complementary to 18s rrna helix 26 is present upstream. using a luciferase-based reporter plasmid, we show that vp2 is expressed by termination-reinitiation and provide evidence consistent with a functional interaction between the coding region of the vp1 mrna and 18s rrna. the formation of mrna secondary structure within the turbs is also investigated. overall, our data suggest that the mechanism of vp2 expression is broadly similar to that of the other caliciviruses and influenza b. however, in contrast to what was observed with the fcv signal [13] and seen here with influenza bm2, termination-reinitiation at the mnv signal shows resistance to the initation inhibitor edeine. thus the mechanism by which the aug of the downstream orf is recognised may differ. to investigate termination-reinitiation in the synthesis of the mnv vp2 protein, a 255 bp fragment of viral cdna was cloned between the sali and bamhi sites of the dual-luciferase reporter vector p2luc [20] . the cloned fragment, which contained 203 bp of sequence information upstream of the uaaug stop-start window, and 52 bp downstream was suspected, on the basis of work with other viruses (see introduction), to contain all of the required sequences for termination-reinitiation. the cdna fragment was cloned in such a way that the renilla and firefly luciferase orfs were in frame with the stop and start codons respectively of the termination-reinitiation motif to give an orf configuration 59 rlucvp1-vp2fluc 39 ( figure 1 ). this vector, named p2luc-mnvwt, contains a t7 rna polymerase promoter allowing synthetic mrnas to be generated to investigate the stopstart process in in vitro translation reactions. the translation of in vitro synthesised wild-type (wt) mrna from p2luc-mnvwt was carried out in flexih rabbit reticulocyte lysate (flexihrrl) supplemented with 140 mm kcl (see materials and methods) and gave products of the expected sizes (upstream rlucvp1 orf, ,42 kda, downstream vp2fluc orf, ,64 kda, figure 1b ). the molar ratio of vp2fluc to rlucvp1 (taking into account the methionine content of the two proteins) was typically in the region of 1:10. thus, initiation on the downstream orf occurred at a frequency of about 10% of that of the upstream orf. that this was indeed the product of the second orf was further confirmed by comparing the migration of rrl translation products from mrnas derived from p2luc-mnvwt that had been linearised at different points within the second orf (data not shown). termination-reinitiation is distinct from ires-mediated expression of downstream orfs as translation through the upstream orf is an absolute requirement [11, 14, 16] . in order to establish whether this is also the case for mnv expression, a premature in-frame stop-codon was inserted close to the end of the rluc orf but upstream of vp1 sequence information (219 bp upstream of the authentic rlucvp1 termination codon). if the expression of vp2fluc is a result of termination-reinitiation, translating ribosomes would be unable to reach the aug start figure 1 . minimal sequence requirements for mnv termination-reinitiation. a) schematic of the p2luc-mnv reporter mrna. the termination-reinitiation region (203 nt upstream and 52 nt downstream of the uaaug motif) was cloned into the sali and bamhi sites of the p2luc reporter plasmid. hpai run-off transcripts for in vitro translation were generated using t7 rna polymerase. the location of the t3 promoter present in the structure mapping construct p2luc-mnv-t3 is indicated. b) deletion analysis of mnv termination-reinitiation. a series of p2luc-mnv variants were prepared with stepwise, in-frame deletions from the 59 end of the inserted viral sequence. the wild-type (wt), premature stop (ps) and deletion mutant plasmids were linearised with hpai and run-off transcripts translated in flexih rrl at a final rna concentration of 50 mg/ml in the presence of [ 35 s]-methionine and 140 mm added kcl. the products were resolved by 12% sds-page and visualised by autoradiography. the number of nucleotides of viral sequence remaining up to the aug start codon of the mnv orf is shown below the gel. the product of the full-length or truncated versions of the rlucvp1 orf (predicted size of mnvwt is 42 kda) is marked rluc, and the vp2fluc product (predicted size, 62 kda) is marked fluc. the mnv ps rluc product is the shortest (predicted size, 33 kda). rrf denotes the relative reinitiation frequency in comparison to mnvwt (set at 100). the figure in brackets represents the ratio of the intensity of the fluc and rluc products (adjusted for methionine content and expressed as a percentage) for the mnvwt mrna. doi:10.1371/journal.pone.0008390.g001 codon of vp2fluc in the mutant mrna and the orf could not be translated. as is clear in figure 1b , the introduction of a premature stop codon into the rluc/m1 orf abolished expression of the vp2/fluc product, but had no effect on synthesis of the upstream orf (rlucvp1ps, ,33 kda). these data are thus consistent with a termination-reinitiation strategy for the expression of the vp2 protein and confirm a requirement for translation through the upstream orf. expression of mnv vp2 is dependent on ,40-43 nt upstream of the uaaug motif previous work has suggested that viral termination-reinitiation events show little dependence on sequence information downstream of the ''stop-start'' window but require 45-250 nt of upstream primary sequence [11,14,16.19] . in order to determine the minimal sequence requirements for termination-reinitiation in vp2 expresssion, deletions of increasing size were made from the 59 end of the inserted viral information (figure 1b) . the stop-start product was synthesised efficiently with up to 43 nt of vp1 information present upstream of the uaaug motif, and to a lesser extent with 40 nt. however, deletion to 37 nt or less abolished expression of the termination-reinitiation product ( figure 1b) . these data indicate that only 40 nucleotides of vp1 primary sequence immediately upstream of the stop-start window are required for termination-reinitiation in vitro, although 43 nt are required for full activity. termination-reinitiation of mnv vp2 synthesis is dependent upon an mrna sequence with complementarity to 18s rrna in fcv, rhdv and influenza b, it has been shown that termination-reinitiation requires a closely conserved primary sequence element (referred to as motif 1) that is complementary to a region of helix 26 of 18s rrna [11] [12] 14] ). the position of motif 1 varies somewhat, with the 59 base 73 nt (rhdv), 63 nt (fcv) or 34 nt (influenza b) upstream of the stop codon of the first orf. mutational analysis has revealed that this sequence is essential for the stop-start process [12] [13] [14] [18] [19] . within the ,43 nt minimal region of the mnv vp1 rna required for vp2 expression, a stretch of bases with a similar level of complementarity to 18s rrna is also found (figure 2a , complementary bases are shown in italics). to investigate whether this region plays a role in termination-reinitiation in vp2 expression, two point mutations were made to disrupt potential mrna:rrna pairs ( figure 2a ). in the first, the a at -31 was mutated to a g (p2luc-mnv gu), creating a presumably slightly weaker putative u-g base pair between the rrna and mrna. in the second, the g at -32 was changed to a c (p2luc-mnv cc), which would act to disrupt the interaction between 18s rrna and mrna. as can be seen in figure 2b , the latter mutation greatly reduced expression of the vp2fluc product, supporting the idea that an interaction between the 18s rrna and the mrna just upstream of the termination-reinitiation site is required. in the mutant where pairing was predicted to be maintained (p2luc-mnv gu) termination-reinitiation was clearly detectable, although the efficiency was reduced somewhat compared to that of wild-type mrna. the experiments described above confirm the existence of motif 1 and its role in reintiation in mnv. it was therefore of interest to determine the context of this 18s rrna complementary region within the global rna secondary structure of the minimal functional sequence, and to compare the structure with that determined for the influenza bm2 signal [19] . to achieve this, a bacteriophage t3 promoter was inserted upstream of the viral sequence of the p2luc-mnv.61 plasmid ( figure 1b ). the plasmid was linearised with bamhi, t3 run-off transcripts synthesised and the rna end-labelled with [ 33 p]-catp. the labelled transcripts were subjected to limited chemical and enzymatic probing prior to analysis on denaturing polyacrylamide gels. the chemical probes used were imidazole and lead acetate, specific for cleavage of single stranded regions. enzymatic probes were rnases t1, u2 and cl3, which preferentially cleave single-stranded g, a and c residues respectively, and rnase cv1, which cuts in helical regions in double-stranded or stacked conformations. a representative stucture mapping gel is shown in figure 3 and in figure 4 , the data are mapped onto mfold predictions of the secondary structure of the ''stop-start'' region. structure probing analysis of the mnv signal revealed that, like the bm2 signal, the mrna in the region essential for terminationreinitiation is not highly structured. this was especially evident from the chemical probes, with most residues sensitive to imidazole and lead cleavage. the enzymatic probes were also active against the majority of bases in the region and consistent with this, cv1 probing identified very few double-stranded or stacked bases. we also noticed a few cl3 cuts at residues other than c, although the reason for this is uncertain. minimal free energy mfold predictions, performed using the online server of zuker (http://mfold.bioinfo.rpi.edu/cgi-bin/rna-form1.cgi) indicated that the most stable rna fold was the bulged stem-loop shown in figure 4 . however, the correspondence between this mfold and the mapping data was not absolute. whilst in general, the single-stranded probes displayed more activity against regions of the model predicted to be single-stranded than they did against predicted helices, there were anomalies. for example, residues g51-52 were sensitive to rnase t1, yet were predicted to be in a double-stranded region (stem 2). generally, the predicted duplexes showed more reactivity to single-stranded probes than one would expect for stable double-stranded stretches. therefore, it seems likely that the rna in this region is metastable, potentially adopting a number of co-existing structures. in our model, the sequence complementary to 18s rrna is sequestered between two putative stems (stems 2 and 3; figure 4 ) at a location similar to that found with bm2 [19] . given that the termination-reinitiation process requires the ribosome to translate through the vp1 orf, secondary structure in the rna upstream of the ''stop-start'' window would be unwound and perhaps remodelled as the ribosome transits to the termination codon. toeprinting of ribosomes paused at initiation codons has shown that the 59 edge sites of cleavage were identified by comparison with a ladder of bands created by limited alkaline hydrolysis of the rna (oh-) and the position of known rnase u2 and t1 cuts, determined empirically. products were analysed on a 10% acrylamide/7m urea gel containing formamide. data was also collected from 6% and 15% gels (gels not shown). enzymatic structure probing was with rnases t1, u2, cl3 and cv1. uniquely cleaved nucleotides were identified by their absence in untreated control lanes (0). the number of units of enzyme added to each reaction is indicated. chemical structure probing was with imidazole (i, hours) or lead acetate (pb; mm concentration in reaction). the water lane (w) represents rna which was dissolved in water, incubated for four hours and processed in parallel to the imidazole-treated sample. the sequence of the probed rna and the inferred secondary structure is shown in figure 4 . of the ribosome is some 12 to 13 nt from the first base of the aug [21] . this would place the 59 edge of the terminating ribosome (with the uaa codon in the a-site) close to residue c78 on our mrna. thus a terminating ribosome would prevent formation of the secondary structure, conceivably releasing the 18s rrna complementary region for interaction with the ribosome (see discussion). an alternative structure can be predicted under such circumstances, shown in the inset box in figure 4 . in this structure, the 59 arm of the original stem 2 is predicted to pair with alternative bases to generate a new stem (stem 29) with motif 1 forming part of the apical loop. this alternative fold is attractive for a number of reasons. by displaying motif 1 on an apical loop, this could promote 18s rrna binding and ribosome tethering [19] . until ribosomes transit through this region, motif 1 would remain within a larger structure with potentially reduced access to the ribosome which could, at least in part, account for the observation that the signal does not appear to function as an ires. the deletion analysis of figure 1 is also consistent with a role for this alternative structure as the functional ''end-point'' maps to the start of the 59 arm of stem 29. furthermore, most, if not all, viral turbs have the potential for base-pairing between regions flanking motif 1 [18] . nevertheless, it should be noted that the structure mapping data are not fully consistent with this alternative structure, for example, there is considerable sensitivity to rnase t1 cleavage within the 59 arm of stem 29. this is considered further in the discussion section. efficient termination-reinitiation seems to require the close proximity of the stop and start codons [12, 14, 19] . to investigate whether this is also the case for mnv, the authentic stop codon of rlucvp1 (in the context of the fully functional mnv49; see figure 1b ) was mutated from uaa to caa such that the first orf was extended by 13 amino acids (mnv49.1 figure 5 ). the separation of stop and start codons by such a distance in bm2 is known to reduce reinitiation about 10-fold [19] , but with the mnv signal, only a three-fold reduction in flucvp2 synthesis was observed ( figure 6 ). a possible explanation for this lies in the fact that the ''new'' stop codon is itself embedded within a second potential stop-start sequence (ugaug) which could facilitate some reinitiation, but perhaps at a lower frequency, as it would not necessarily be spaced appropriately with respect to motif 1. another possibility is that 40s subunits terminating at the downstream stop-start sequence can reinitiate, at a reduced frequency, at the correct (upstream) aug, despite the increased spacing, with the 40s subunit remaining tethered to the mrna and ''snapping-back'' to the normal position of reinitiation. in an attempt to distinguish between these possibilities, additional constructs were prepared in which point mutations were introduced into pmnv.49 such that the termination and start codons in the two stop-start regions were changed separately and in combination (mnv49.2 to mnv49.8; see figures 5 and 6) . from this analysis, it is evident that modification of the authentic termination-reinitiation motif reduces reinitiation, irrespective of whether the stop or start codon is eliminated. alteration of the aug codon had the most effect, with reinitiation reduced to 8-38% of the wild-type level. when the natural termination site was changed such that termination now took place 13 or 15 amino acids downstream, the frequency of termination-reinitiation was also reduced, to 25-49% of the wild-type level. in mnv49.8, where termination of the upstream orf occurred 30 amino acids downstream of the natural site, very little reinitiation was seen (8% of the wild-type level), indicating that the ribosome is unable to locate the authentic aug from such a distal termination site. in the translation of this mrna, an additional product was seen (asterisked in figure 6 ) whose size is consistent with a fusion of the encoded orfs (this is considered in the discussion section). reinitiation events that take place at either the authentic or the downstream stop-start motifs would produce polypeptides that differ in size by only 13 amino acids, thus we would not expect to be able to distinguish them by sds-page, and this is clear in figure 6 , where the reinitiation products show very similar electrophoretic mobilities. thus we cannot say with confidence whether a particular aug (or both) is used. however, the substantial reintiation activity displayed by mnv49.7, an mrna in which both augs were changed, indicates that non-aug codons can act as reinitiation codons, although probably at reduced efficiency. this is consistent with other work demonstrating that reinitiation can occur at non-aug codons within the context of a termination-reinitiation signal [12, 14, 19] . whilst in principle, reinitiation of translation of the mnv 49.7 vp2fluc orf, following termination, could occur at the next available aug, this is located 54 amino acids from the natural stop-start signal and initiation here would produce a substantially shorter product that would have been detectable by sds-page. thus in this mrna, a significant proportion of ribosomes (25% of the wild-type level) that terminate 13 amino acids downstream of the authentic stop-start site can reinitiate in an aug-independent manner within the stop-start window. the precise mechanism of termination-reinitiation is not known, but the sensitivity of fcv vp2 protein expression to the translation initiation inhibitor edeine suggests that the reinitiation process bears at least some similarity to standard initiation at aug codons [13] . to ascertain whether edeine sensitivity is a general feature of termination-reinitiation, we analysed the effect of the peptide on the activity of the mnv and bm2 signals (with fcv as a control) using translation time-courses (figure 7 ). reactions were programmed with the relevant mrna and at various times an aliquot was removed, edeine added (to 5 mm) and the aliquot reincubated such that the total time of translation was 60 minutes. to determine the time of first appearance of the termination and reinitiation products, identical reactions were also performed in which the elongation inhibitor cycloheximide replaced edeine. in the edeine experiments, it was evident that for fcv and bm2, only a trace of ''stop-start'' product was synthesised at the early time points. in these experiments, the vast majority of ribosomes did not reach the stop-start window until at least 7.5 minutes had passed (as shown in the cycloheximide time course experiments [data not shown; see legend to figure 7 ]), thus the trace of vp2fluc seen likely corresponds to the product of infrequent internal initiation at the vp2fluc aug or is derived from those few ribosomes that had reached the stop-start window prior to edeine addition. at later time points, however, the termination-reinitiation product steadily accumulated, with the ratio of the upstream and downstream orfs stabilising after 30 minutes (at a reinitiation frequency of ,4%). thus for the fcv and bm2 signals, when edeine is present prior to arrival of ribosomes at the stop-start signal, it greatly inhibits termination-reinitiation, but has little effect on translation post-reinitiation. unexpectedly, the mnv signal responded differently, with the termination-reinitiation product being more evident at early times post-edeine addition (in comparison to fcv and bm2). at these early time points, few ribosomes would have reached the stop-start window prior to edeine addition, thus the mnv signal shows increased resistance to the effects of edeine. examination of the kinetics of synthesis of the two orfs (figure 7d ) reveals that in all cases, the frequency of termination-reinitiation at early time points was higher than that seen at the steady state. this is indicative of a titration effect; early in the time course, when fewer ribosomes have loaded onto the mrna (due to the earlier addition of edeine), the greater frequency of reinitiation may reflect the increased relative abundance of a necessary factor. the molecular basis of the resistance to edeine seen with the mnv signal is difficult to explain. it may be that recognition of the stop-start motif is indeed blocked by edeine but somehow, a proportion of initiation complexes still recognise the aug present in the second pentanucleotide motif (ugaug; see above) on the mrna. in this paper we show that expression in vitro of the murine norovirus vp2 protein occurs by coupled translation terminationreinitiation. the process requires the close proximity of stop and start codons, a defined region of mrna upstream of the stop-start window that includes a functional turbs motif 1 and translation by the ribosome through this region up to the site of terminationreinitiation. secondary structure mapping indicates that the rna in this region is weakly structured, with motif 1 loosely embedded in the 59 arm of a putative stem-loop structure. the mnv signal thus exhibits many of the features and functional characteristics of the stop-start signals of fcv, rhdv and influenza b. the figure 1 ] which acts as the ''wild-type'' reference construct [wt] in these experiments), in which the stop and start codons of the termination-reinitiation signal were altered. the figure shows the primary sequence and three-frame translation of the relevant region of the mrna encoded by each construct. the natural stop-start motif is shown in pink and emboldened text, the downstream fortuitous stop-start motif in pink. mutations within the mrna sequence are highlighted by uppercase, red emboldened characters. the upstream rlucvp1 orf is highlighted in grey, as is the downstream vp2fluc orf where this is known. likely key methionines (start codons) or their replacement amino acid are highlighted in green. doi:10.1371/journal.pone.0008390.g005 molecular mechanism of termination-reinitiation remains to be fully elucidated, however. central to the discussion is the turbs and in this context the purpose of the identified motifs, the role (if any) of rna secondary structure, and the functional requirement for translation through the turbs. regarding motif 1, it is clear that in all studies so far, mrna mutations that would destabilise an interaction with 18s rrna reduce or abolish reinitiation and changes not predicted to affect pairing having a lesser effect or none at all. recently, the reciprocal experiment was performed, where mutations were introduced into the relevant region of (yeast) 18s rrna. their effect on termination-reinitiation was found to be highly consistent with a role for mrna-18s rrna pairing [18] . these experiments confirm a role in tethering through rrna, although do not rule out the contribution of other factors, for example, binding of eif3 [13] . a comparative alignment of the mnv signal with other known or suspected termination-reinitiation signals (figure 8 ) reveals that motif 1 is always present and that the stop and start codons of the termination-reinitiation site are in close proximity to each other. what does vary is the spacing between the two elements, from only 26 nt in the case of bm2 to 29 nt in mnv, 53 nt in fcv, 61 nt in rhdv and 62 nt (the longest) in the lagovirus european brown hare syndrome virus. it is not clear whether the ''additional'' sequences present in viruses with longer turbs have a role in termination-reinitiation. deletion analysis of the fcv and rhdv turbs has revealed some dispensible sequences -there may be some flexibility in the spacing of motif 1 that allows other biological information to be accommodated into the turbs without affecting function in stop-start. however, there is little sequence conservation between the signals of viruses of different genera, arguing against the presence of other primary sequence motifs. another stretch of bases of functional consequence has been identified in fcv and rhdv, namely turbs motif 2, which is located closer to the stop-start window than motif 1 and is speculated to help position ribosomes correctly at the reinitiation codon [12, 14] . recent work has shown that the functional requirement for motif 2 is in its participation in a basepaired region that forms between this motif and a stretch of bases immediately upstream of motif 1 [18] ; see figure 8 . this basepairing has previously been noted from structure predictions of the signal of fcv [13] and direct rna secondary structure probing of bm2 stem 2 [19] and the mnv stem 2 (see figure 4) . based on the observations of luttermann and meyers [18] , the formation of this stem is likely to be important to termination-reinitiation in the bm2 and mnv systems. indeed, it is noticeable that in the deletion analysis of the mnv signal, and that of bm2 [19] , those deletions that would affect formation of stem 2 showed reduced activity in termination-reinitiation (figure 1b, figure 4) . despite this progress, the occurence and role of rna secondary structure within viral turbs is poorly understood. direct structure probing and mfold analysis indicates that the rna upstream of the stop-start window is metastable and whilst the secondary structures proposed for fcv [13] , bm2 [19] and mnv (this study) are superficially similar, the largely single-stranded nature of the turbs weakens these models and their comparison. the insertion of a premature termination codon upstream of the turbs blocks reinitiation, ruling out the possibility that vp2 expression occurs by ribosome recruitment to a conventional, structured, viral ires or by shunting from the untranslated region of the upstream orf. the requirement for translation through the turbs may simply reflect the need to deliver ribosomes to the stop-start window, but it could also indicate a requirement to remodel the turbs, conceivably by alteration of rna secondary structure or displacement of a bound factor. based on chemical and enzymatic rna structure probing of the bm2 signal and folding predictions (mfold), it has been suggested that transit of the ribosome to the stop-start window leads to melting of one stemloop structure and the formation of an alternative structure that has motif 1 displayed on its apical loop [19] . the position of mnv motif 1 relative to the stop-start window is very similar to that of bm2 suggesting that the same remodelling could operate (figures 4 and 8) . however, whilst transit and termination of the ribosome would destabilise the identified secondary structure (figure 4) , liberating turbs motif 1 in close proximity to helix 26, it is not clear whether this motif would subsequently be displayed as part of an alternative secondary structure. whilst mfold analysis of the mnv region present locally upstream of the terminating ribosome does suggest an alternative secondary structure, further work will be needed to confirm this possibility. studies on the fcv signal have revealed that the reinitiation process occurs in the standard fashion by the criterion of sensitivity to edeine, but it is distinct in being completely independent of eif4g or the eif4f complex [13] . analysis of the mnv signal here provides further evidence that the process deviates from the standard mechanism. first, like bm2 [19] , there appears to be efficient use of non-aug codons to reinitiate translation, indicating a relaxed requirement for the full complement of initiation factors, which would include eif1 and eif1a, thought to play important roles in locating and correct recognition of the aug start codon [22] [23] . secondly, in contrast to what has been observed with fcv and bm2, the mnv signal is more resistant to treatment with edeine. edeine does not inhibit binding of the eif2/gtp/met-trnai ternary complex to the 40s ribosomal subunit, nor met-trnai/40s complex scanning, but there is a complete failure of aug codon recognition, so that scanning continues past all aug codons, and, probably as a secondary consequence, there is no ribosomal subunit joining [24] [25] . the relative insensitivity of the mnv signal to edeine suggests that recognition of the aug start codon during reinitiation may not require a scanning ternary complex. it is not clear why the fcv and bm2 signals respond differently to edeine, especially as the organisation of the bm2 signal (with regard to the position of motif 1 and the primary sequence of the stop-start window) is so similar to that of mnv. another observation that hints at nonstandard reinitiation mechanisms relates to the the translation pattern seen with the mnv49.8 transcript. in this mrna, the two termination-reinitiation windows (the natural uaaug and the fortuitous downstream ugaug) were mutated to eliminate the stop codon in each case. in translations of this mrna, where termination occurs 30 amino acids downstream of the authentic site, very little termination-reinitiation product was seen, but an additional product was synthesised whose size is consistent with that of a fusion of the two reporter orfs (asterisked in figure 6 ). the origin of this protein is uncertain. it could have arisen through a ribosomal frameshift event, although no obvious conventional frameshift signals are present in the region of overlap between the two orfs [26] [27] . it could also represent the outcome of a failed attempt to terminate and subsequent resumption of translation by ribosomes on the downstream orf. further work will be required to elucidate the nature and origin of this product and how it relates to the mechanism of termination-reinitiation. plasmids used to assay termination-reinitiation were based on the p2luc reporter vector [20] . sequences encompassing the stopstart signal of mnv (203 bp of sequence information upstream of the vp1 stop codon and 52 bp downstream) and fcv (97 bp upstream of the vp1 stop codon and 14 bp downstream) were generated by pcr (using pfu polymerase [roche]) from, respectively, plasmids pt7:mnv (kind gift of dr ian goodfellow, imperial college, london) and psg-2/3* [13] , a kind gift of dr tuiya pã¶yry, university of cambridge. the pcr products and p2luc were digested with sali and bamhi and ligated together. sequences were confirmed by dideoxy sequencing (using the facility at the department of biochemistry, university of cambridge). the influenza b termination-reinitiation assay plasmid (p2luc-bm2wt; 250 bp upstream of m1 stop-codon, 18 bp downstream) was described previously [19] . site-directed mutagenesis was performed using the quikchange ii site-directed mutagenesis kit (stratagene) according to manufacturer's instructions. for large deletions (greater than 48 bp) a modification of the manufacturer's protocol was used with the primers containing ,30 bp of complementary sequence either side of the site of deletion, as described previously [28] . mutagenesis to introduce insertions longer than 6 bp was performed in two steps [29] , by first subjecting mutagenesis reactions (containing either the sense or antisense primer) to three cycles of pcr, then mixing the reactions and performing a further 18 cycles according to manufacturer's instructions. reporter plasmids were linearised with hpai and capped run-off transcripts generated using t7 rna polymerase as described [30] . messenger rnas were recovered by a single extraction with phenol/chloroform (1:1 v/v) followed by ethanol precipitation. remaining unincorporated nucleotides were removed by gel filtration through a nucaway spin column (ambion). the eluate was concentrated by ethanol precipitation, the mrna resuspended in water, checked for integrity by agarose gel electrophoresis and quantified by spectrophotometry. unless otherwise stated, mrnas were translated in flexih rabbit reticulocyte lysate (flexihrrl, promega) programmed with 50 mg/ml template mrna. typical reactions were of 10 ml and composed of 60% (v/v) flexihrrl, 20 mm amino acids (lacking methionine), 500 mm mgoac, 2 mm dtt, 5u rnase inhibitor (rnaguard, ge healthcare life sciences), 130 mm-160 mm kcl (optimised for each batch of flexihrrl) and 0.2 mbq [ 35 s]methionine. reactions were incubated for 1 h at 30uc and stopped by the addition of an equal volume of 10 mm edta, 100 mg/ml rnase a followed by incubation at room temperature for 20 minutes. samples were prepared for sds-page by the addition of 10 volumes of 2x laemmli's sample buffer [31] , boiled for 3 minutes and resolved on 12% sds-page gels. the relative abundance of products on the gels was determined by direct measurement of [ 35 s]methionine incorporation using a packard instant imager 2024. a plasmid encoding the putative termination-reinitiation signal of mnv (p2luc-mnvwt) was modified by site-directed mutagenesis to include a t3 rna polymerase promoter 30 bp upstream of the minimal required viral sequence generating plasmid p2luc-mnv-t3. rna for structure mapping was prepared by in vitro transcription of bamhi-digested p2luc-mnv-t3 using t3 rna polymerase. transcription reactions were performed on a 200 ml scale essentially as described [30] . structure mapping was performed using a 59 endlabelling procedure as described previously [30, 32] . all probing reactions were performed in a final volume of 50 ml and contained ,40,000 c.p.m. 59 33 p-end-labelled transcript, 10 mg escherichia coli rrna and the relevant enzymatic or chemical probe. further details are provided in the legend to figure 3 . molecular mechanisms of translation initiation in eukaryotes translation of insulin-related polypeptides from messenger rnas with tandemly reiterated copies of the ribosome binding site translational control of viral gene expression in eukaryotes regulation of expression of human immunodeficiency virus naturally occurring dicistronic cricket paralysis virus rna is regulated by two internal ribosome entry sites frameshifting rna pseudoknots: structure and mechanism endless possibilities: translation termination and stop codon recognition translational control in positive strand rna plant viruses translational terminationreinitiation in viral systems eukaryotic coupled translation of tandem cistrons: identification of the influenza b virus bm2 polypeptide translation of the minor capsid protein of a calicivirus is initiated by a novel termination-dependent reinitiation mechanism characterization of the sequence element directing translation reinitiation in rna of the calicivirus rabbit hemorrhagic disease virus the mechanism of an exceptional case of reinitiation after translation of a long orf reveals why such events do not generally occur in mammalian mrna translation a bipartite sequence motif induces translation reinitiation in feline calicivirus rna expression of the orf-2 protein of the human respiratory syncytial virus m2 gene is initiated by a ribosomal termination-dependent reinitiation mechanism coupled translation of the respiratory syncytial virus m2 open reading frames requires upstream sequences coupled translation of the second open reading frame of m2 mrna is sequence dependent and differs significantly within the subfamily pneumovirinae the importance of inter-and intramolecular base pairing for translation reinitiation on a eukaryotic bicistronic mrna characterization of the termination-reinitiation strategy employed in the expression of influenza b virus bm2 protein a dualluciferase reporter system for studying recoding signals ribosome pausing and stacking during translation of a eukaryotic mrna dissociation of eif1 from the 40s ribosomal subunit is a key step in start codon selection in vivo the eukaryotic translation initiation factors eif1 and eif1a induce an open conformation of the 40s ribosome migration of 40s ribosomal subunits on messenger rna in the presence of edeine migration of 40s ribosomal subunits on messenger rna when initiation is perturbed by lowering magnesium or adding drugs mutational analysis of the rna pseudoknot component of a coronavirus ribosomal frameshifting signal mutational analysis of the ''slipperysequence'' component of a coronavirus ribosomal frameshifting signal generation of deletion and point mutations with one primer in a single cloning step two-stage pcr protocol allowing introduction of multiple mutations, deletions and insertions using quikchange site-directed mutagenesis structure-function analysis of the ribosomal frameshifting signal of two human immunodeficiency virus type 1 isolates with increased resistance to viral protease inhibitors cleavage of structural proteins during the assembly of the head of bacteriophage t4 characterization of the frameshift signal of edr, a mammalian example of programmed -1 ribosomal frameshifting distinct protein forms are produced from alternatively spliced bicistronic glutamic acid decarboxylase mrnas during development we wish to thank dr ian goodfellow for providing the mnv plasmid and dr tuija pã¶yry for plasmid psg-2/3* and for helpful advice and discussion. conceived and designed the experiments: db ib. performed the experiments: sn ral mlp. analyzed the data: sn ral mlp rjj db. contributed reagents/materials/analysis tools: rjj. wrote the paper: rjj db ib. key: cord-001343-3euy4u9k authors: wang, yadong; li, xiangrui; yuan, yiwen; patel, mahomed s. title: a multi-method approach to curriculum development for in-service training in china’s newly established health emergency response offices date: 2014-06-27 journal: plos one doi: 10.1371/journal.pone.0100892 sha: doc_id: 1343 cord_uid: 3euy4u9k objective: to describe an innovative approach for developing and implementing an in-service curriculum in china for staff of the newly established health emergency response offices (heros), and that is generalisable to other settings. methods: the multi-method training needs assessment included reviews of the competency domains needed to implement the international health regulations (2005) as well as china’s policies and emergency regulations. the review, iterative interviews and workshops with experts in government, academia, the military, and with hero staff were reviewed critically by an expert technical advisory panel. findings: over 1600 participants contributed to curriculum development. of the 18 competency domains identified as essential for hero staff, nine were developed into priority in-service training modules to be conducted over 2.5 weeks. experts from academia and experienced practitioners prepared and delivered each module through lectures followed by interactive problem-solving exercises and desktop simulations to help trainees apply, experiment with, and consolidate newly acquired knowledge and skills. conclusion: this study adds to the emerging literature on china’s enduring efforts to strengthen its emergency response capabilities since the outbreak of sars in 2003. the multi-method approach to curriculum development in partnership with senior policy-makers, researchers, and experienced practitioners can be applied in other settings to ensure training is responsive and customized to local needs, resources and priorities. ongoing curriculum development should reflect international standards and be coupled with the development of appropriate performance support systems at the workplace for motivating staff to apply their newly acquired knowledge and skills effectively and creatively. the outbreak of severe acute respiratory syndrome (sars) in 2003 was a watershed moment for china [1] . it triggered major health reforms [1±4] and led to an increase in public health funding of about 100% by 2007, accounting for a rise in spending from 0.75% to 0.89% of the gross domestic product [1] . subsequent emergencies that echoed the imperative for reforms included the outbreaks of influenza h5n1 in birds and humans [1±4] , melamine contamination of milk formula that affected over 294,000 chinese children [5] , and the earthquake in sichuan that resulted in over 69 000 deaths, displaced about 15 million people and led to the mobilization of over 10 000 medical workers [6] . the reforms to strengthen china's emergency preparedness and response capabilities included new laws and regulations [7±10], increased support for training and research, and the adoption of international best practice [2±4,11] . to help implement the new laws, health emergency response offices (hero) were established within china's health administration units at the national, provincial and municipal levels, in centers for disease control (cdc) and in tertiary hospitals [12] . the heros within any one province now collectively employ around 1000 staff members, and their role is to help develop and coordinate preparedness planning and emergency response within their jurisdictions, as well as implement the international health regulations (2005) (ihr) [13] . however, because staff members were recruited opportunistically from diverse professional backgrounds into the newly established heros, they had not been trained systematically in emergency preparedness and response previously. while they received in-service training, this was offered in an ad hoc manner, was not preceded by a needs assessment [14] , and was aimed primarily at improving knowledge of the new laws that did not translate directly into strengthening performance of the heros [15, 16] . at the global level, perennial threats of the pandemic spread of infectious diseases like sars and influenza, as well as the sequelae of earthquakes, tsunamis, bioterrorism and complex humanitarian emergencies, heightened awareness of the need to strengthen national, regional and global capacity in prevention, preparedness and response to public health emergencies. in 2004, the world association for disaster and emergency medicine (wadem) proposed a framework for disaster health to facilitate the development of educational programs in the field [17] . efforts were directed at identifying and defining criteria for assessing disaster health-related competencies, standards for guiding curricular development, and exploring the methods, duration and desired outcomes of training [17±19]. australia for example, developed a national framework for disaster health education to provide guidance for educators to achieve a standardized and integrated approach across the country [20] . australia adapted the wadem recommendations to target seven educational levels, and outlined the curricular contents and outcomes for each level; the seven levels were defined as 1) informing the community, 2) raising awareness of health workers, 3) providing basic knowledge and skills for health workers, 4) advancing the knowledge of health workers who play lead roles in disaster management, 5) enhancing expert knowledge among a small group of health workers, 6) targeting specialist level amongst a small group of individuals, and 7) encouraging research and innovation to further develop the knowledge base of disaster health [20] . in recognition of the national need for an in-service program to target the``level four'' health staff as defined by wadem, i.e.`h ealth workers who played lead roles in disaster health management'' [20] , china's ministry of health (moh) commissioned the capital medical university in 2010 to develop and implement a competency-based curriculum to help strengthen the performance of the new cadre of hero staff. an immediate priority was to address the who requirements for countries to meet the core capacity requirements for implementing the ihr by june 2012 [21] . this paper outlines the consultative process used to conduct the training needs assessment and develop the curriculum for implementation across china, with support from who. the multi-method approach to curriculum development can be applied in other settings to ensure training is responsive and customized to local needs, resources and priorities. we did not submit the study proposal for ethics approval because we conducted meetings and interviews with study informants on a voluntary basis for the sole purpose of identifying training needs at the workplace; we did not gather any personal information or attributes about individual informants beyond their age and past work experience. figure 1 outlines the approach for developing the in-service curriculum using addie as the basic framework for instructional design [22] . each of the five phases of this model ± assessment, design, development, implementation, and evaluation ± were reviewed critically by a technical advisory panel (tap). the panel offered suggestions for strengthening the curriculum development process, as well as the content, relevance and quality of the curriculum. the multi-method training needs assessment and subsequent steps of the addie model are detailed below. we defined a competency as``a cluster or related knowledge, skills, and attitudes that reflects a major portion of one's job (a role or responsibility), that correlates with performance on the job, that can be measured with well-accepted standards, and that can be improved with training and development'' [23] . a competency domain incorporated an inter-related group of competencies for performing specified tasks. in order to identify the list of competency domains against which training needs of hero staff could be assessed, we collated data from three sets of references. the first was from the ihr core capacities necessary to detect, assess, report and respond to emergencies [21] . the second set of references was from interrelated chinese laws and regulations: the emergency response law which is the legal framework for managing emergencies [7] , the national health emergency training outline which identifies the knowledge and skills to be targeted by training programs [11] , and the regulations on preparedness for and response to emerging public health hazards which outlines preparedness and response activities [8, 10] . the final set of references was selected from chinese publications on general guidelines for training health workers [24] and from curricula for training technical staff based in disease surveillance units, laboratories and the environmental health sector [14±16,24±27]. we assessed tasks, roles and responsibilities, and training needs of hero staff through face-to-face interviews of eleven experienced key informants; they included health emergency experts from the government, the military and the academic sector, and senior staff of heros. we also explored their awareness of existing training activities and the associated relative strengths and weaknesses, their preferences on modes of curriculum delivery and the optimal duration of in-service training. we collected data on the same issues through self-administered questionnaires to a group of 115 hero staff members from across china when they attended a training workshop in beijing. china's ministry of health (moh) appointed a technical advisory panel (tap) to critically review and offer comments and suggestions on all aspects of the needs assessment and curriculum development process, as well as on the relevance and quality of the curriculum. the eight panel members were nationally acknowledged experts in public health emergencies from the moh, cdcs, heros and the academy of military medical science. we used the results of the needs assessment to design the draft curriculum that included the topics listed in figure 1 . each of the identified competency domains was targeted through a selfcontained module that could be run independently of the other modules. each module encompassed theories, laws, concepts and tools and techniques essential for hero staff to perform effectively in their work. we developed questionnaires for hero staff to invite their comments and suggestions on the curriculum design, and the expected level of need and potential demand for the training. the questionnaires were disseminated with the draft curriculum to 700 hero staff members at the provincial and municipal levels and 1000 health staff responsible for responding to emergencies at the local level. we also invited comments on the curriculum from the technical advisory panel and staff of the who office in china. we used their feedback to revise the draft curriculum. the revised curriculum was endorsed by the technical advisory panel and the ministry of health before it was implemented, monitored and evaluated between june and november 2010. the key tasks of hero staff in their respective jurisdictions were identified as coordination of the development and implementation of preparedness plans and early warning surveillance systems, mobilization of emergency response teams, and implementation of the ihr [13] . the list of competency domains associated with these tasks is shown in table 1 . domains 1 to 8 were transcribed from both the ihr [13, 21] and the chinese references [7, 10, 11] . domains 9 to 18 were explicit only in the latter set of references; although implicit in the ihr capacities, study respondents suggested they be considered as discrete competency domains. of the 1700 health professionals to whom questionnaires were distributed, 1606 (94.5%) responded with comments and suggestions; selected characteristics of these respondents are summarized in table 2 . of the respondents, just under two-thirds were aged 40 years and over, 45% had worked previously as clinical practitioners, 35% as preventive health (public health) practitioners, and 8% as health administrators. overall, 58% had less than 5 years experience in emergency response work. the major weaknesses the respondents, key informants and the technical advisory panel identified in existing training programs were the didactic methods used for classroom teaching and the emphasis on hazard-specific approaches (such as for responding to sars or pandemic influenza or earthquakes), and on acquiring knowledge of the new laws and regulations rather than on addressing the challenges to their implementation. furthermore, while training programs in the past had targeted knowledge and skills required by technical staff such as epidemiologists, laboratory scientists and environmental staff, they had not incorporated skills in mobilizing, managing and coordinating teams across multiple sectors, disciplines and agencies. the respondents stressed the need for adopting interactive learning methods and problemsolving exercises in the classroom, as had been suggested in chinese publications [26±29]. they supported replacing thè hazard-specific' approach with the`all hazards' approach to managing emergencies, development of a competency-based curriculum with modules that addressed specific sets of competency domains, and limiting the duration of in-service training to about 2 to 3 weeks so these interfered minimally with trainees' regular work responsibilities. after discussions with the key informants and tap, 11 of the 18 competency domains shown in table 1 were to be developed into the individual modules shown in table 3 . they included six of the eight ihr-related competencies, while the remaining two, national legislation' and`national co-ordination' were omitted because they were directed mainly at national-level decisionmakers. the other five domains were specified in the chinese references, namely on-site organization during response, decisionmaking processes for alerting the community, resource management and stockpiling, risk assessment and management, and evaluation of response to emergencies. the remaining seven domains shown in table 1 were not developed into modules because they were considered to be a lower priority for health managers and could not be included in the first round of in-service training to be conducted over a limited 2±3 week timeframe. the proportion of the 1606 respondents who expressed a high level of need for these modules are shown in table 3 . case studies and desktop simulation exercise were both ranked as the preferred training method by 94% of respondents, followed by lectures (85%) and case studies (85%). the median preferred time for each module was 2.5 days. to accommodate this request and complete the course within 3 weeks, we included only nine 8. laboratory [10, 11, 21] domains not explicit in the ihr core capacities 9. health promotion and community education [7] 10. resource management and stockpiling [7, 10, 11] 11. risk assessment and management [7, 10, 11] 12. monitoring compliance with laws and regulations [7, 10] 13. decision-making processes for alerting the community [7, 10] 14. medical rescue [10] 15. on-site organization during response [7, 10] 16. managing recovery after an emergency [7, 10, 11] 17. evaluation of response to emergencies [7, 10] 18. research on emergencies [7] doi:10.1371/journal.pone.0100892.t001 modules, and excluded modules for two domains considered not to be priorities for heros: human resources and laboratory. the curriculum was revised to incorporate feedback from the stakeholders. two trainers prepared each module to be run over 2 to 2.5 days; one was typically a university academic with nationally acknowledged expertise in emergencies, and the other, a senior staff member of a hero. each module would be delivered through three sessions: a lecture on relevant theories, concepts and tools and techniques that were followed by a case study and a desktop simulation exercise; the latter two sessions were conducted with small groups of 6 to 8 participants as an opportunity to apply, experiment with, and consolidate the knowledge acquired from the lectures. before the course, trainees were to be provided with readings for each module, and invited to prepare appropriate material from their workplace for a case study relevant to at least one module; this could include a preparedness plan, report on a risk assessment, a risk communication plan, or other reports on an emergency event. the trainers then prepared case studies either from the participants' material, or designed them de novo based on their own experiences. an example of the former was a case study based on a jurisdictional preparedness plan and where participants had to identify strengths, weaknesses and contentious issues, and to explore alternative options in the light of the newly acquired knowledge from the lectures. on returning home, they were expected to review and revise their jurisdictional plan using a similar learning approach with their local team members. an example of a case study designed by the facilitator was the assessment and management of the risk of an infectious disease outbreak at a conference attended by about 1000 international participants. each small group would then be expected to present a 20 minute report for further discussions at a plenary, with a synthesis of key learning points. an example of a desktop simulation exercise developed by the facilitator was an earthquake scenario where participants had to convene a national response team, to mobilize them within a safe operational base near the affected zone, and then arrange the logistics for transporting and storing essential medical supplies and equipment. each small group would prepare and submit written responses to the evolving scenario to which they would receive feedback from the facilitators. the delivery of the modules was to be monitored to document attendance of trainers and trainees, the content of the training materials and the mode of delivery of each module. the evaluation plan was derived from the four kirkpatrick levels of evaluation [30] ; the first two levels,`reaction' to the learning content and environment and`learning' (or acquisition of new knowledge) were both assessed immediately after each module. the next two levels,`behavior' at the workplace and`results' (or the benefits of change in behavior at the workplace) were assessed three months later. the results of the needs assessment and curriculum were presented, debated and finalized at a workshop with key informants, tap members and senior hero staff. the first in-service course was delivered to a class of 37 hero staff members from 31 provinces across china; each participant had at least one undergraduate degree and over two-year's work experience in emergency response. the monitoring and evaluations activities were implemented as planned, and the results will be published separately. this study on developing an in-service curriculum for new professional cadre of hero staff from across the country adds to the literature on china's health reforms and enduring efforts to strengthen emergency response capabilities following on the sars outbreak and other emergencies [1±4] . it describes the multimethod approach used to identify training needs systematically, and to adopt international best practice in partnership with senior decision-makers and content experts from the government, academia and the military. the study revealed the overwhelming need to replace didactic teaching, the classical method used across china, with active learning and training methods. the consultative process for developing the curriculum was designed to address the scale of the challenge for coordinating planning and training activities across jurisdictions that cover a population of over 1.3 billion people, and where most provinces have more than 60 million residents. the process required active engagement with experts in government, academia and the military, as well as inputs from 1606 immediate potential beneficiaries. the expert technical advisory panel played a critical role through each step in the addie model, and endorsed the curriculum development process as well as the contents of the curriculum. the ihr (2005) core capacities are aimed at minimizing the international impact of communicable disease emergencies and chemical, radiation and nuclear accidents [13, 21] . however, they are also essential for timely and effective detection of, and responses to, the emerging threat at its source. china's reforms to strengthen preparedness and response capabilities [2±4,7±11] therefore incorporated the ihr core capacities as well as the competency domains identified as priorities based on china's recent experiences with emergencies [1±6]. the overwhelming rejection of didactic teaching methods by the study respondents and the need to replace these with active learning and teaching methods was encouraging news for the curriculum designers. this approach is consistent with the two chinese symbols that depict``learning'' (f`: xue â xõ â) -`acquisition of knowledge' and`repeated practice' as inseparable sides of the one coin. training aimed at strengthening performance has to create opportunities for active learning and experimentation with problem solving exercises to apply and consolidate newly acquired knowledge and skills. the curriculum marks the beginning of a new journey for strengthening the performance of the recently established heros in jurisdictions across china. this initial effort focused strongly on meeting the who requirements for developing the core capacities required to implement the ihr [21] , and in particular, to strengthen the competencies of the``level four'' health staff [20] who play lead roles in emergency planning and management. our approach for identifying and developing competencies and standards are consistent with those published by wadem in 2013 [18, 19] , and in many ways resemble those used in australia [20] . the initial list of 18 competency domains were derived from the eight core capacities required to implement the ihr [21] and the disaster management cycle of prevention, preparedness, response and recovery [17] , both of which are embedded in chinese laws and regulations [7±12] . the scope of the task and all aspects of the needs assessment and identification of the competencies were developed through iterative inputs from subject matter experts in the ministry of health, academia and the military. the mode of curriculum delivery was based on blended learning methods implicit in bloom's taxonomy as outlined in the australian framework [20] . the duration of training for the level four workers in australia is recommended for 40 hours [20] ; by contrast, our course was conducted over 100 hours because the target group had received little training in the past and included workers with diverse sets of past experience. however, consistent with the wadem guidelines [17±20], china needs to reconcile its curriculum with international standards and expand its efforts to develop and standardize training frameworks for the other six wadem levels [20] , as well as interconnect with the``crosscutting'' competencies of workers from the other disciplines that participate in emergency responses [19] . adopting the international standards and practice will also enhance communications, inter-agency cooperation and inter-operability across china's national borders. the major limitation of our training is that it helps develop only the knowledge, skills and attitudes essential for working in the field of emergency preparedness and response. we recognize that training constitutes but one component of a package of activities needed to strengthen the performance of hero staff. training must be linked with an appropriate performance support system [31] that motivates staff to apply the newly acquired knowledge and skills, and in this way, to start contributing effectively and creatively to the workplace. the support system includes a congenial work environment; explicit guidelines, supervision as well as feedback on the quality of their performance; appropriate resources such as materials, equipment and computer software; and incentives such as financial and non-financial rewards for good performance as well as opportunities for career development [31] . the multi-method approach to curriculum development by engaging actively with senior policy-makers, researchers, and experienced practitioners can be applied in other country settings to ensure training is responsive and customized to local training needs, resources and priorities. china's engagement with global health diplomacy: was sars a watershed communicable disease control in china: from mao to now progress in tuberculosis control and the evolving public-health system in china emergence and control of infectious diseases in china melamine and the global implications of food contamination emergency medical rescue efforts after a major earthquake: lessons from the 2008 wenchuan earthquake order of the president of the people's republic of china national emergency plan for responding to public health emergencies order of the state council of the people's republic of china regulations on preparedness for and response to emergent public health hazard ministry of health of the people's republic of china the major points of work to health emergency office in 2004 fifty-eighth world health assembly resolution wha58.3: revision of the international health regulations assessment of capacity for logistics investigation and analysis on current status of emergency training to staffs of centers for disease control and prevention in heilongjiang province chinese health economics 307: 66±69 the current situation and development of emergency management training system in china china emergency management 8: 12±17 international guidelines and standards for education and training to reduce the consequences of events that may threaten the health status of a community white paper on identifying and assessing competencies in disaster health world association for disaster and emergency medicine white paper on setting standards for selecting a curriculum in disaster health world association for disaster and emergency medicine a national framework for disaster health education in australia ihr monitoring framework: checklist and indicators for monitoring progress in the development of ihr core capacities in states parties. who designing effective instruction the quest for competence beijing: china financial & economic publishing house investigation on demand for public health emergency system of health workers in guangzhou evaluating the effectiveness of an emergency preparedness training programme for public health staff in china improving emergency preparedness capability of rural public health personnel in china the survey on china adult teaching mode study in recent twenty years adult teaching methods and patterns evaluating training programs: the four levels san francisso: berrett updating the behavior engineering model key: cord-000266-xwfptmmv authors: liao, qiuyan; cowling, benjamin; lam, wing tak; ng, man wai; fielding, richard title: situational awareness and health protective responses to pandemic influenza a (h1n1) in hong kong: a cross-sectional study date: 2010-10-12 journal: plos one doi: 10.1371/journal.pone.0013350 sha: doc_id: 266 cord_uid: xwfptmmv background: whether information sources influence health protective behaviours during influenza pandemics or other emerging infectious disease epidemics is uncertain. methodology: data from cross-sectional telephone interviews of 1,001 hong kong adults in june, 2009 were tested against theory and data-derived hypothesized associations between trust in (formal/informal) information, understanding, self-efficacy, perceived susceptibility and worry, and hand hygiene and social distancing using structural equation modelling with multigroup comparisons. principal findings: trust in formal (government/media) information about influenza was associated with greater reported understanding of a/h1n1 cause (β = 0.36) and a/h1n1 prevention self-efficacy (β = 0.25), which in turn were associated with more hand hygiene (β = 0.19 and β = 0.23, respectively). trust in informal (interpersonal) information was negatively associated with perceived personal a/h1n1 susceptibility (β = −0.21), which was negatively associated with perceived self-efficacy (β = −0.42) but positively associated with influenza worry (β = 0.44). trust in informal information was positively associated with influenza worry (β = 0.16) which was in turn associated with greater social distancing (β = 0.36). multigroup comparisons showed gender differences regarding paths from trust in formal information to understanding of a/h1n1 cause, trust in informal information to understanding of a/h1n1 cause, and understanding of a/h1n1 cause to perceived self-efficacy. conclusions/significance: trust in government/media information was more strongly associated with greater self-efficacy and handwashing, whereas trust in informal information was strongly associated with perceived health threat and avoidance behaviour. risk communication should consider the effect of gender differences. pandemic influenza a/h1n1 has a clinical profile similar to seasonal influenza, despite initially appearing more severe [1] . respiratory infectious diseases (rids) such as influenza are a major public health issue best dealt with by prevention, ideally vaccination. however, in the first six-months or so of a newlyemergent rid epidemic/pandemic vaccines are generally unavailable and non-pharmacological interventions can play a major role in minimizing rid spread [2] [3] [4] . government health education messages are a major source of information for promoting self-protective practices against rids. these preventive messages generally emphasize improved hygiene, face-mask use by infected persons, and social distancing measures, including avoiding crowds during epidemics [5] [6] [7] . predictors of population uptake of health protective behaviours in rid epidemics have begun to be studied [8] [9] [10] [11] [12] [13] , yet related theory remains nascent and this is problematic: to effectively predict behaviour during future epidemics robust theory is critical. effective models that enable comprehensive prediction of health protective behaviours remain limited mainly to two overlapping theoretical paradigms: the theories of reasoned action/planned behaviour (tpb) [14] [15] [16] and bandura's concept of self-efficacy [17] [18] [19] (the belief that one can successfully execute some behaviour), particularly regarding the core tpb concept of perceived behavioural control, which controversially is claimed by some to be largely synonymous with self-efficacy [19] [20] [21] and by others to be indistinguishable from intent [22] (the intention to execute a particular behaviour), the key predictive element of tpb [16] . when used to account for health-related behaviours tpbbased models typically account for ,35% of variance in outcomes [16] , while self-efficacy accounts for ,25% of variance in outcomes [23, 24] . however, neither tpb nor self-efficacy allow for the social and affective influences that might be expected logically to be important in rid [25, 26] . we report on a theoretical model that incorporated elements of influenza causal knowledge, perceived self-efficacy and also social and affective influences ( figure 1 ) because these latter variables have been less frequently studied in combination, but have theoretical and logical support for their potential importance in the context of rids. we tested this model against data collected in the early phase of the influenza a/h1n1 pandemic (table s1 ) to examine how levels of trust in formal and informal sources of risk/prevention information associated with hand washing and social distancing. ethics approval was obtained from the institutional review board of the university of hong kong/hospital authority hong kong west cluster. for this telephone interview, written informed consent was waived by the irb but verbal consent was required from all the respondents and agreement to participate in the interview was taken as further consent. before the interview began, a brief introduction about the study aims and interview contents was given and then respondents were asked whether the interview could start. if approval was received this was recorded and the interview performed. if not, respondents were thanked and the call was terminated. more than 98% of hong kong households have landline telephones and all local calls are free. random-digit dialled telephone numbers and within-household random-sampling grids (kish grids) are a cost-effective way to survey highly representative random population samples. kish grids are matrices containing random numbers for different sized households that facilitate random selection of individuals within households and help minimize sampling bias. the number of eligible household residents, ''n'', is determined by asking the person of first contact in the household. the kish grid provides a randomly generated number ''k'' between 1 and ''n'' which is used by the interviewer. ordering by age and starting from the oldest eligible member in the household, the k'th member is then invited to participate in the survey. different grid values are used for each household. as part of a series of surveys to monitor a/h1n1 epidemic activity, a commercial polling organization administered the questionnaire using this telephone-survey methodology, targeting 1,000-1,500 participants on each occasion, a sample size calculated to give an estimate of a/h1n1 health protective behaviours with a precision of 63%. the survey with the largest sample was selected for this analysis. sampling was performed during the evening to minimize exclusion of young working adults. data on attitudes, knowledge, situational awareness, risk perception and preventive behaviours (table s1) were collected by household telephone interviews, based on random digit dialling. one cantonese-speaking adult (age$18) who lives .4 nights per week in each household was selected using a kish grid. all interviews were conducted between 8:30pm-10:45pm from 23 rd -25 th june, 2009, two weeks after the first community transmission had been identified in hong kong. existing theoretical frameworks of behaviour change have been adapted to predict health-related behaviour-change for chronic, non-communicable diseases [15, 16] , but we lack a comprehensive evidence-based model of protective behaviour against rid threat [11] . a recent review of 26 papers on rid prevention behaviours concluded that 23 lacked a theoretical basis [13] . existing applications of health behaviour change models in communicable disease are almost exclusively limited to hiv/aids research [24, 27] and to a lesser extent hepatitis b and c, which share the same transmission pathways as hiv. there are good reasons why sexually-transmitted diseases embody a different set of influences than do rids. for example people are highly motivated to seek sexual contact (or injection drug use) and have a high degree of potential control (e.g. condom use) over the nature of these encounters, even though they may be situationally constrained from executing that control, and are infected only by direct exchange of bodily fluids. in contrast, one can acquire an rid transmitted by air droplets, hand contact or fomites for up to 72 hours after the person who is the source departs [28] , or immediately by being sneezed on. infection is much more casual. clearly, the controllability of rids requires different behavioural imperatives to those in stds and hence different psychological influences should be considered. attempts by the tpb to accommodate social influences had relied on incorporating social norms [14] , the behavioural expectations within a group. however, norms, and hence theoretical models reliant on norms to account for social processes, cannot accommodate the fact that communicable respiratory diseases make other humans ambiguous sources of threat: one can usually control sexual encounters but not who shares public transport. in this respect social factors in communicable respiratory disease differ significantly from those in non-communicable diseases and warrant greater consideration than existing hbc models allow. outbreaks of new infectious diseases constitute situations that are uncertain, dynamic, and embody highly personal threat, requiring rapid decisions on appropriate action [29] . under such circumstances timely and relevant information on the best preventive actions become critical to such decision-making. hence, health protective behaviour during the early stages of a novel epidemic would be more likely to resemble situational reactions using established or known default actions such as avoiding crowds (social distancing), rather than intention-based planning before any behavioural change, such as deciding to consult a doctor to administer a vaccination. later in the epidemic as threat familiarity increases, different factors such as planned behaviour may become important. reporting delays, uncertainty and other biases affect publicly available information on the characteristics of newly-emergent communicable diseases, such as a/h1n1 lay knowledge of infection-related risks can be limited. the resulting uncertainty about disease severity and transmissibility at the epidemic onset extends to the utility and timing of adopting preventive measures. information cues to individuals about initiating protective action must therefore be synthesized from various sources. perceived information reliability or trustworthiness influences decisions to utilize any given information source [30] to inform awareness of the situation. more trustworthy sources are therefore likely to be more influential. epidemic situational awareness is likely derived from formally-announced public information like news items, government press releases and health education messages, and also from informal, social sources [25, 29] ; observation of other peoples' behaviour and communications from family, peers and neighbours. noting how others behave informs action decisions in the observer [19] . if those around you are wearing masks, this indicates others might have knowledge you do not possess, and that the threat level might be locally high and imminent, suggesting prudent precautionary or rid preventive behaviour. observers are also subject to social conformity influences that can help adoption of group patterns of behaviour. maintaining situational awareness, involving elements of perception, comprehension and prediction [31] , during epidemics probably relies on these two types of information. however, when uncertainty is high and widespread, or when there is low confidence in social and other information sources then individuals' hpbs might be expected to be more independent of formal and informal information sources. perceived risk is influenced by several stimulus characteristics, including unfamiliarity, invisibility, dreadfulness and inequity [32] , and by recipient characteristics, including demographics and trust in information source and content [33] . perceived risk is an important determinant of protective behavioural responses [12, 34, 35, 36] , but is subject to optimistic bias, where for example people distort their risk of contracting influenza downwards relative to others [35, 37] . nonetheless, susceptibility to risk remains an important measure in understanding variation in behavioural responses to threat and reflects the key element of perceived risk in an epidemic/pandemic situation. worry is a cognitive process linked to anxiety [26, 38] and reflects negative affectivity, interacting with perception of susceptibility to risk [26, 39] and may also influence rid protective responses such as social distancing [13] . because data were collected using telephone interviews we had to adapt measures to suit a brief format in order to avoid people hanging up mid-way or providing invalid answers to hurry the interview, a problem encountered with this data collection method. we therefore used parsimonious measure to minimize assessment fatigue and low response rates which threaten representativeness. trust in government/media (formal) information: we asked about respondents' agreement with three statements (table s1 ). responses were made on categorical five-point scales ranging from ''strongly disagree'' to ''strongly agree''. scalability of these three items was assessed using cronbach's alpha, which at 0.61 indicated that the internal consistency between items was low, but acceptable. however, to minimize potential measurement error arising from the low internal consistency, this construct was treated as a latent variable in the subsequent analysis [40] . a latent variable is a concept opposed to an observed variable. a latent variable can not be measured directly but is inferred from one or more variables that are directly measured (observed variable) while an observed variable can be directly measured with a specific question or item or observed by the researchers. for example, an ''attitude'' is a concept that is difficult to measure directly with single items but can be inferred from various questions asking about different aspects of that attitude. then within the analysis ''attitude'' is treated as the latent variable while the questions used to infer it are the observed variables. trust in interpersonal (informal) information: respondents' agreement with two statements (table s1 ). responses were made on categorical five-point scales ranging from ''strongly disagree'' to ''strongly agree''. scalability of these two items was assessed using cronbach's alpha, which at 0.50 indicated that scalabilty was unsuitably low for two items. this suggests that these two items measure different aspects of social information. again to minimize potential measurement error this construct was treated as a latent variable in the subsequent analysis. understanding cause of a/h1n1 (''i understand how swine flu is caused'') and self-efficacy (confidence in one's ability to act in a way that achieves desired future outcomes) for a/h1n1 prevention (''i am confident that i can protect myself against swine flu''): each was assessed using responses on 5-point scales of agreement with these two single item statements (table s1) . perceived personal susceptibility: two items, one assessing absolute susceptibility (perceived absolute probability of developing a/h1n1) and another assessing relative susceptibility (perceived probability of developing a/h1n1 relative to peers) formed a latent variable for perceived personal susceptibility (table s1 ). the cronbach alpha of these two items was 0.66. worry about contracting h1n1. respondents were asked to indicate their level of worry over the past one week about contracting influenza a/h1n1. responses were 5-point scales of worry ranged from ''never thought about it'' to ''extremely worried'' (table s1 ). hand hygiene. respondents were asked to indicate frequencies of use of four hand hygiene practices over the three days prior to interview: hand washing after sneezing, coughing and touching nose; hand washing after returning home, use of liquid soap for hand washing, and hand washing after touching common objects. responses were on a 4-point scale of frequency: 1 ''never'', 2 ''sometimes'', 3 ''usually'' and 4 ''always''. cronbach's a was 0.62 (table s1 ). social distancing behaviours: a. social avoidance. respondents were asked to indicate if they had adopted any of four avoidance behaviours due to influenza a/h1n1 in the past 7 days: avoiding eating out, avoiding using public transport, avoiding going to crowded places, and rescheduling travel plans responses were coded as 1 ''yes'' and 0 ''no''. cronbach's a was 0.61 (table s1 ). we first compared the demographic structure of the sample against that of the general population derived from the hong kong government general household survey to identify any sample differences. our model proposes that trust in formal (government and media sources) and informal (from other people) information affects rid epidemic health protective behaviours, the former by informing about generic risk and response characteristics for dealing with a potential threat (causes and protective responses), the latter about threat imminence, severity and response effectiveness (seeing how others behave). we refer to the product of these combined processes as situational awareness, and propose that rather than driving behaviour directly information acts through altering the cognitive/affective domain of situational awareness. thus the model is predicated on several premises: that understanding of the disease and perceived personal susceptibility influence self-efficacy [17, 18, 31] ; that the effect of perceived susceptibility to influenza on hpbs acts through increasing worry about the disease [26, 33, 38, 41, 42] ; and that more worry from perceived susceptibility prompts hpbs [39, 41, 42] . these cognitive/affective processes are represented in the hypothesized model ( figure 1 ). structural equation modeling (sem) is a method for simulating and testing multiple and interrelated causal relationships simultaneously in statistical data, making it suitable for theory development and testing [40] . sem was applied to test the hypothesized model. sem is usually performed when a model contains latent variables assessed with specified measurement models. despite including estimations of a series of multiple regression equations, sem differs from regression analysis in several ways, which make it advantageous for this kind of analysis. first, sem is usually theoretically based because it is performed after researchers specify the hypothesized model. second, it can be used to refine the hypothesized model by estimating the measurement model and structural model simultaneously. finally sem analysis can accommodate measurement errors of the constructs in the model [40] . in our hypothesized model, trust in formal information, trust in informal information, perceived personal susceptibility, hand washing and social distancing behaviours were entered as latent (inferred) variables while other constructs were entered as observable (directly measured) variables because they were assessed with only one item. two different health protective behaviors, hand washing and social distancing, were entered as the hpb outcomes because we hypothesized that different influences may act on each of these. we assumed that the ''disturbances'' of the two health behavior outcomes were correlated. disturbance represents the unexplained variances of the latent variables predicted by the specified independent variables [40] . in making this assumption, we assumed that unexplained variance in the outcome variables could be correlated and the variables in question jointly influenced by other unknown factors, and so allowed for such constraints within the model by using more conservative criteria. previous studies have shown that hand hygiene and social distancing behaviours during a pandemic could be influenced by some common causes which were not fully explored in our study such as current health, past experience of disease and cues to action [14] . in particular, in our study, the two kinds of health protective behaviours occurred in the same situation of the 2009 influenza pandemic, and so it is sensible and reasonable to assume that they could be influenced by some common causes which were not fully explored in our studies. adequacy of the measurement models was tested before testing the full structural model. to test the full structural model, all constructs ( figure 1) were entered into the model and all factor loading, specified paths, covariance, measurement errors and disturbances were estimated simultaneously. since the model contained categorical variables, weighted least square with mean-and variance-adjusted estimation (wlsmv) was used to estimate the standardized parameter (b) for each path [43] . with this kind of estimation, chi-square difference testing is inappropriate. we therefore used the comparative fit index (cfi), tucker-lewis index (tli) and root mean squared error of association (rmsea) to evaluate the model fit to the data. a cfi.0.95, tli.0.95 and rmsea,0.05 indicate a good fit of data to the model [43] . the analysis was conducted in mplus 6.0 for windows [43] . the proportion of missing values ranged from 0.1% for ''in the past one week, have you ever worried about catching influenza a/h1n1'' to 10.1% ''did you wash hands after sneezing, coughing or touching nose in the past 3 days''. missing data were handled with multiple imputation to generate 10 datasets which were summarized into one for subsequent analysis. multiple imputation was performed in ameliaview [44] . responses are likely to differ by sociodemographic factors [13] . we therefore stratified the sample by gender and by age (,45 years old vs. .45 years old). education is also likely to have a significant effect but there are difficulties in education stratification in hong kong. the age cut-off of 45 years was adopted to account for the introduction in hong kong of 6-year compulsory education in 1971 and 9-year compulsory education in 1978 [45] . this means that people aged 45 or above are much less likely to have a tertiary (college/university) level education and less secondary (high school) education than people aged ,45 years old [45] . moreover, in traditional families in china, a son (who lived with his parents after marriage) was usually more educationally-favoured over daughters (who moved to their in-laws' home on marriage) to ensure support for the parents in their old age, so males usually obtained more education than females [45] . these distinctions were somewhat evidenced by our data which showed that 98% of the respondents aged ,45 compared to 71% of the respondents aged 45 or above (x 2 = 147.69, p,0.001), and 89% of male compared to 80% of female respondents, obtained at least secondary education (x 2 = 17.05, p,0.001). since the numbers of tertiary educated respondents and primary (elementary) educated respondents were too small to produce stable models, we limited stratification to gender and age only and acknowledge that this also incorporates indefinable education and income effects. consequently, we used a multi-group sem to assess the invariance of the model (figure 1 ) across gender and age group (respondents aged 18-44 and aged 45 or above). we tried to test the model by stratifying the sample into four subgroups (female aged 18-44, female aged 45 or above, male aged 18-44 and male aged 45 or above). however, the sample size for males aged 18-44 was relative small (table s2) . moreover, all the model variables were treated as categorical variables and we used the wlsmv method to estimate the model. this method requires that each subsample covers all the categories of each variable. in the case of one category, younger males, not all variable values were present. to meet the assumptions for analysis we would need to recode all variables, intrinsically altering the model. in order to avoid this, we relinquished a combined four-group comparison and instead compared the model across gender and the two age groups separately. to perform multigroup comparison we first ran a model with all parameters unconstrained. we then identified factor loadings that were not significantly different (p$0.05) and set these as equal, while loadings that were significantly different were allowed to vary, and finally paths that did not differ significantly were constrained to be equal while those that differed significantly were allowed to vary and estimated separately by groups. the ''difftest'' option in mplus 6.0 was used to obtain a correct chi-square difference test for the wlsmv estimators and was used to estimate the differences between the least constrained model (with all the paths freely estimated) and the most constrained model (with all the paths constrained to be equal) as well as the partially constrained model (with some of the paths freely estimated and others constrained to be equal) [43] . a p-value.0.05 for the ''difftest'' indicate a nonsignificant difference between the models. finally, to help interpret these multigroup sem comparisons, we performed a post-hoc examination of the model variable means for different gender and age groups and tested differences using the mann-whitney test, which tests differences between two groups on ordinal scales of measurements. a total of 1,001/1,449 (69.1% response rate) hong kong adults successfully completed the interview. the characteristics of the sample were compared against the hong kong 2006 by-census population data [46] , showing respondents to be better educated and more likely to have been born in hong kong compared to the general population (table 1 ) but otherwise representative. both formal and informal information trust were correlated with all situational awareness variables except worry about contracting a/h1n1 (''worry''), while formal information trust was also independent of perceived personal susceptibility (''susceptibility''). in turn, understanding of h1n1 cause (''understanding'') and perceived self-efficacy (''self-efficacy'') were significantly associated with hand washing while worry and susceptibility were significantly associated with social distancing (table s3 ). the sem model fitted well to the data with cfi = 0.977, tli = 0.969 and rmsea = 0.026. standardized coefficients indicated two primary features in the model; the first one linking formal information and hand hygiene and a second linking informal information and social distancing ( figure 2 ). paths were seen via formal information trust and self-efficacy (b = 0.25) and self-efficacy and hand hygiene (b = 0.23), and via formal information trust and understanding (b = 0.36), and understanding and hand hygiene (b = 0.19) while understanding and selfefficacy were independent. these associations formed the first feature. marginal associations between worry and hand hygiene and between self-efficacy and social distancing were seen, but the small standardized coefficients of b = 0.13 suggest that these paths are minor. susceptibility and worry were associated, but otherwise were functionally independent, both upstream from formal information trust, and downstream from hand hygiene. the second feature of the model is reflected in a different set of paths associating informal information trust with social distancing. trust in informal information sources was inversely associated with susceptibility (b = 20.21), which was associated positively with worry (b = 0.44), and inversely with self-efficacy (b = 20.42). however, more confidence in informal information sources was associated with more worry (b = 0.16) and finally, only worry was associated with social distancing (b = 0.36). trust in informal information was independent of understanding and self-efficacy. the only remaining notable feature of the model was a strong inverse association (b = 20.42) between susceptibility and selfefficacy. this suggests some interaction between these two variables that could strongly influence both sets of paths mentioned so far. overall, the model explained 11.3% of the variance in hand hygiene and 16.1% of the variance in social distancing behaviors. across gender, both the least constrained model and the most constrained models fit the data well with cfi.0.970, tli$0.970, rmsea = 0.025. the most constrained model did not differ significantly from the least constrained model (x 2 for ''difft-est'' = 29.30, d f = 19, p = 0.061). however, three sets of associations differed significantly between females and males: those between formal information trust and understanding, from informal information trust to understanding, and from understanding to self-efficacy. these paths were set free and estimated separately in female and male. the model with these paths freely estimated fit well to the data with cfi = 0.978, tli = 0.976 and rmsea = 0.023, and did not differ significantly from the least constrained model (x 2 for ''difftest'' = 15.07, d f = 16, p = 0.519). figure 3 presents the results of multigroup comparison of the model applied to males and females with the three path parameters unconstrained. for a given path, if the path coefficients did not differ significantly between males and females, only the path coefficient for males is presented; if the path coefficients differed significantly between males and females, the path coefficients for both genders are presented with the coefficients for males presented on the left of the slashes and for females presented on the right of the slashes. by comparison, the model shows that for both genders while the association between formal information trust and understanding was positive this association was stronger amongst females (b = 0.50) than males (b = 0.25); the association between informal information trust and understanding was weakly positive in males (b = 0.12) but weakly negative in females (b = 20.14), and; the association between understanding and self-efficacy was positive (b = 0.12) in males but non-significant in females (b = 20.01). across the two age groups, both the least constrained model and the most constrained model fit well to the data with cfi.0.960, tli$0.950, rmsea#0.030. the most constrained model did not differ significantly from the least constrained model (x 2 for ''difftest'' = 15.85, d f = 19, p = 0.667). no path was found to be significantly different between the two age groups. means and standard deviations for all model variables by gender and age group showed differences (table s4 ). all the constructs did not differ by gender except for hand hygiene and social distancing with female being more likely to wash their hands and adopt social distancing behaviours. trust in formal and informal information sources, self-efficacy, and hand hygiene significantly differed by age groups, with respondents of older age group being more likely to trust the information from both sources, perceive higher self-efficacy and wash their hands. we tested a hypothesized model of associations between trust in (formal/informal) information, situational awareness variables (causal understanding, self-efficacy, susceptibility and worry) and different types of health protective behaviours (hand hygiene and social distancing) for influenza protection. the model suggested that two different sets of influences relate trust in information to hand hygiene, and to social distancing respectively. the strongest associations observed were between susceptibility and self-efficacy neither age nor gender contributed significant variation to the association between trust in formal information and self-efficacy, and self-efficacy and hand hygiene. these findings are consistent with other studies showing self-efficacy is enhanced by procedural information [18, 19, 47, 48] and that attitudinally and actionoriented interventions are more successful in changing behaviour for communicable disease protection, such as in the case of hiv [24] . similarly, exposure to relevant media stories during the 2009 a/h1n1influenza pandemic was associated with higher efficacy beliefs regarding hygiene, which in turn was associated with greater frequency of reported tissue access and sanitising gel purchase among british people [49] . however, there is evidence that coping style interacts with the ability of procedural information to enhance self-efficacy and under circumstances of high threat, such as during sars-type epidemics where mortality is high, procedural information might be counter productive for some segments of the community who use an information avoidance (''blunting'') coping style [50] . self-efficacy was only weakly associated with social distancing. people are limited in their ability to avoid crowds in hong kong, one of the most densely populated cities on earth, despite the hong kong government recommending this in order to limit the pandemic [51] . however, the relatively mild impact of a/h1n1 meant that people saw no reason to jeopardize their economic well-being and curtail other social activities, given such a low perceived threat [49, 52] . hand washing was probably seen as sufficient protection. the association between trust in formal information and understanding of influenza cause differed by gender but not age, with females showing a stronger association. men tend to have poorer health knowledge than women [53] . we found that females were more likely to wash their hands than were males. older respondents reported significantly greater trust in formal information, marginally-significantly better understanding of influenza cause and were more likely to wash their hands. this is consistent with other studies reflecting that preventive practice is enabled by knowledge of causes [49, 54] . however, increasing knowledge is not itself sufficient to always ensure preventive behaviour [55] . in this context, understanding has an independent contribution to hand washing practice only. trust in informal information seems to be associated with less perceived susceptibility to health threat. this may reflect rational processes or cognitive bias. trusting social cues involves comparison and conformity influences, and can enhance optimistic bias (the tendency to view oneself less likely to experience negative events but more likely to experience positive events) in personal risk estimates [56] , thereby reducing perceived susceptibility. conversely, others' behavioural cues about health threat proximity can arouse motivating worry and anxiety producing protective action [17, 29] . we found trust in informal information was independent of both understanding of influenza cause and self-efficacy. however, when stratified by gender, the trust in informal information-understanding association was positive among males but negative among females. education is probably an important influence in understanding and may have a bearing on these patterns which await clarification. susceptibility was strongly associated with both self-efficacy (negatively) and worry (positively). neither worry nor susceptibility varied significantly by gender or age group. this is plausible and theoretically consistent [26, 34, 35, 39] . worry was strongly associated with social distancing, again consistent with british data [49] . although worry was also significantly associated with hand hygiene, the association was weak. elsewhere, using a generic measure of personal hygiene practices we have found a stronger association between disease worry and hygiene, suggesting a moderate effect of level of disease worry [57] . the model tested explained only a modest proportion of the variance in adoption of hbps, suggesting that there are significant theoretical gaps that remain to be filled. these await further research. social distancing is unassociated with formal hpb messages, suggesting potential susceptibility to a ''herd-like'' response in this chinese community, particularly if confidence in formal (government or doctors) information is low. voerten and colleagues describe such a pattern of response in the early stages of sars [25] . these models support the hypothesis that social distancing is more likely to occur when perceived health threat is high [25] . logically, when others seem to be behaving in a way that is informed and probably consistent then their actions provide clear information. if mixed social messages occur signalling uncertainty then the utility of social information will fall. this is likely to be associated with increase perceived susceptibility, and possibly greater worry and distancing behaviour. this pattern of responses would be most likely early in a novel rid epidemic where disease characteristics and behaviour are often uncertain. high threat uncertainty then drives social avoidance of potentially high-risk others. high levels of worry are associated with greater social distancing. around 50% of 997/14,297 (response rate 7%) british respondents agreed that social avoidance would minimize risk of a/h1n1 infection, and respondents reporting more anxiety were more likely to engage in preventive actions; severity and likelihood of infection were the most important determinants of preventive action [12] . further research on social influences on hpb during epidemic and pandemic rids is warranted. providing more knowledge about disease causes can improve hand hygiene but is unlikely to influence social avoidance, which appears less amenable to formal health messages. however, as formal messages achieve acceptance across the population, and uptake of hpbs increases, then under circumstances where a critical mass of the population are practicing precautions trust in informal information should increase, reducing susceptibility and worry and leading to declines in social avoidance. because others are likely adopting hpbs this makes them less of a contagion risk. conversely maintaining a high level of hand washing practices may require sustained public education activities. finally, different segments of the population probably communicate different types of information with their peers. self-efficacy in preventing a/h1n1 influences hand hygiene but has little influence on social distancing. formal health education messages that focus on enhancing the public's sense of their ability to protect themselves by adopting hygiene practices would seem to be the most effective to improve hand hygiene, but where the practice is already established, high levels of trust in these messages are not likely to significantly increase hand hygiene. this study is limited in being cross-sectional and relying on hypothesized modeling to infer causality. this is potentially errorprone and can only be confirmed by specific longitudinal tests of the hypotheses proposed above. there are potential limitations related to measurement imposed by the need to be parsimonious in questioning due to use of telephone interviews. where this is not done refusal rates would have been unacceptably high [12] raising serious questions about representativeness. as a consequence, construct validity for some latent variables was weaker than expected, for example, only two items were scaled to measured trust in informal information giving a low internal consistency. we re-ran the sem treating the two trust items as separate which gave almost identical associations with different situation awareness variables, so we entered their combined score as a latent variable in the final model. only one item measured self-efficacy. this is generally not considered adequate but does have precedent indicating it is valid for predicting behavioral change [9] . finally, this random sample, closely representative of the population of hong kong and collected early in the epidemic phase, nonetheless was slightly older and less-well educated than the general population. this was likely due to unavoidable sampling bias from surveying in the early evening to 10pm. many young adults do not return home from work until after this time and were thereby not sampled. the results may in part reflect this bias. otherwise the response rate was high at 69% and excellent compared to similar studies [12] . some of these above limitations may also have contributed to the low explained variance of the model. many factors influence rid protective behaviour. this study has examined a very limited number of these. confidence in formal information such as health education messages is associated with greater compliance to recommended preventive measures for influenza a/h1n1 [12] . however, the mechanisms for this were unclear. we have shown that this probably involves different mechanisms for hand washing and social distancing, and suggest how these might function. formal messages may not reduce social distancing behaviours until such time that preventive behaviours are widely adopted in the community. social distancing seems more likely to occur when there is high influenza-related worry and uncertainty, such as in the initial stages when epidemic circumstances are unknown, or if an epidemic is severe and appears poorly controlled, as during early sars. this would seem to be largely worry/affect-driven. if so, then social distancing is likely to occur irrespective of government messages as population anxiety about an epidemic increases. susceptibility may also increase and this may inhibit self-efficacy regarding hand washing. finally, high levels of community uncertainty or rumour are likely to increase distancing by exacerbating perceived susceptibility and worry. a simple version of our findings can be found it the supporting file (text s1). table s1 found at: doi:10.1371/journal.pone.0013350.s001 (0.05 mb doc) text s1 this is a simple version of our study findings for nonspecialists. found at: doi:10.1371/journal.pone.0013350.s005 (0.03 mb doc) pneumonia and respiratory failure from swineorigin influenza a (h1n1) in mexico efficacy of soap and water and alcohol-based hand-rub preparations against live h1n1 influenza virus on the hands of human volunteers facemasks and hand hygiene to prevent influenza transmission in households: a cluster randomized trial school closure and mitigation of pandemic (h1n1) 2009, hong kong cdc says ''take 3'' actions to fight the flu pandemic influenza prevention and mitigation in low resource communities prevention of avian influenza the impact of community psychological responses on 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gastrointestinal endoscopy: self-efficacy enhancement and coping style human swine influenza on sars type economic effects during infectious disease outbreaks determinants of health knowledge: an investigation of age, gender, abilities, personality, and interests knowledge, attitudes, and preventive practices about colorectal cancer among adults in an area of southern italy knowledge, attitude and practice about sexually transmitted diseases among university students in kampala do moderators of the optimistic bias affect personal or target risk estimates? a review of the literature social-cognitive factors and personal hygiene practices to protect against influenzas: a structural equation model comparing avian influenza a/h5n1 and 2009 pandemic a/h1n1 in hong kong we thank the hku public opinion programme for assistance in administering the telephone survey. we thank dennis ip, angela shen and joseph wu for helpful discussions. we also thank lincoln lau and vicky fang for technical assistance. analyzed the data: ql. wrote the paper: ql. planned the study: rf, bc. key: cord-001129-gi2kswai authors: lemos de matos, ana; mcfadden, grant; esteves, pedro j. title: positive evolutionary selection on the rig-i-like receptor genes in mammals date: 2013-11-27 journal: plos one doi: 10.1371/journal.pone.0081864 sha: doc_id: 1129 cord_uid: gi2kswai the mammalian rig-i-like receptors, rig-i, mda5 and lgp2, are a family of dexd/h box rna helicases responsible for the cytoplasmic detection of viral rna. these receptors detect a variety of rna viruses, or dna viruses that express unusual rna species, many of which are responsible for a great number of severe and lethal diseases. host innate sentinel proteins involved in pathogen recognition must rapidly evolve in a dynamic arms race with pathogens, and thus are subjected to long-term positive selection pressures to avoid potential infections. using six codon-based maximum likelihood methods, we were able to identify specific codons under positive selection in each of these three genes. the highest number of positively selected codons was detected in mda5, but a great percentage of these codons were located outside of the currently defined protein domains for mda5, which likely reflects the imposition of both functional and structural constraints. additionally, our results support lgp2 as being the least prone to evolutionary change, since the lowest number of codons under selection was observed for this gene. on the other hand, the preponderance of positively selected codons for rig-i were detected in known protein functional domains, suggesting that pressure has been imposed by the vast number of viruses that are recognized by this rna helicase. furthermore, the rig-i repressor domain, the region responsible for recognizing and binding to its rna substrates, exhibited the strongest evidence of selective pressures. branch-site analyses were performed and several species branches on the three receptor gene trees showed evidence of episodic positive selection. in conclusion, by looking for evidence of positive evolutionary selection on mammalian rig-i-like receptor genes, we propose that a multitude of viruses have crafted the receptors biological function in host defense, specifically for the rig-i gene, contributing to the innate species-specific resistance/susceptibility to diverse viral pathogens. the mammalian innate immune system operates as the first line of defense against microbial pathogen invasion [1] [2] [3] . this system recognizes infectious agents through a limited number of germline-encoded pattern-recognition receptors (prrs) predominantly expressed on sentinel cells [2, [4] [5] [6] . the host prrs recognize and react with specific microbial components, known as pathogen-associated molecular patterns (pamps), which includes bacterial lipopolysaccharides, peptidoglycans, lipoteichoic acids and cell-wall lipoproteins, fungal β-glucan and viral nucleic acids [2, 3, 5, 6] . the host prrs exhibit distinct expression patterns and following sensing of their cognate ligands, activate specific signaling pathways that lead to the expression of a variety of inducible self-defense genes involved in the collective inflammatory and immune responses [2] . to date, four different classes of prrs have been identified, including the cell membrane-associated c-type lectin receptors (clrs), the toll-like receptors (tlrs) at the cell surface and at the membrane of intracellular vesicles (endosomes and lysosomes), and the cytoplasmic detection systems for intracellular pamps, namely the rig-i-like receptors (rlrs) and the nod-like receptors (nlrs) [3, [6] [7] [8] . the rlrs are a family of dexd/h box rna helicases critically and exclusively involved in the recognition of "nonself" rna from actively replicating viruses in the cytoplasm of infected cells [9] . this receptor family consists of three members, the retinoic acid-inducible gene-i (rig-i/ddx58), the melanoma differentiation associated factor 5 (mda5/ifih1) and the laboratory of genetics and physiology 2 (lgp2/dhx58) [10] [11] [12] [13] [14] . rig-i and mda5 share high sequence similarity and several structural features, including an n-terminal region consisting of tandem caspase activation and recruitment domains (cards), a central dexd/h box rna helicase domain and a c-terminal domain (ctd). the two n-terminally located cards function as a signaling and interaction domain with other card-containing proteins [13, 15, 16] . the helicase domain retains catalytic activity to bind and unwind double stranded rna (dsrna) in an atp hydrolysis-dependent manner [10, 17] . the ctd plays a predominant role in highaffinity binding with dsrna, encoding a repressor domain (rd) in rig-i, but not in mda5, which harbors an rd-like domain that does not participate in autoregulation [18] . these two rlrs detect a variety of both dna and rna viruses, particularly at early phase of viral replication, and signal the production of type i interferons (ifns) and induction of an antiviral response [10, 17] . the third element of the rlr family, the lgp2 protein, lacks any cards but contains the helicase domain and the ctd also harbors a rd. the role of lgp2 in anti-viral immunity is less clear, but it has been suggested in different studies to serve both as a negative and a positive regulator of rig-i and mda5 signaling [10, [19] [20] [21] . despite the similarities between rig-i and mda5, they were shown to play different roles in anti-viral immunity by recognizing and protecting from specific classes of viruses [22] . rig-i detects preferentially and most effectively short rna sequences marked with 5'-triphosphate group (5'-ppp) and blunt-end of short double-stranded rnas (dsrnas) or singlestranded rna (ssrna) hairpins [23] [24] [25] [26] [27] . as a key sensor of ssrna viruses, rig-i is implicated in the response to arenaviridae [28] , bunyaviridae [28] , filoviridae [28] , flaviviridae [18, 29] , orthomyxoviridae [22, 30] , paramyxoviridae [22, 28, 30, 31] and rhabdoviridae [22, 23] . on the other hand, mda5 is activated by high-molecular-weight poly(i:c) fragments [22, 32] , and also by long-duplex rnas from the genomes of dsrna viruses [30] or dsrna replication intermediates of positive-strand viruses, such as caliciviridae [33] , coronaviridae [34] and picornaviridae [22, 32] . regardless the virus recognition specificity by rig-i and mda5, some viruses are redundantly sensed by both rlrs, such as the west nile virus and the dengue virus [30, 35] . in addition to the extensively described recognition of rna viruses by rig-i and mda5, a role in anti-viral signaling in response to several dsdna viruses has also been observed. as an rna sensor, rig-i does not detect dna directly; however, not only do many dna viruses create dsrna products by virtue of convergent transcriptional units derived from opposite strands, but also the host rna polymerase iii can mediate the transcription of cytoplasmic dna templates (such as transfected poly da:dt) into dsrna containing 5'-triphosphate, which will activate rig-i and trigger the production of type i ifn [36, 37] . both epstein-barr virus and myxoma virus are detected by rig-i, while vaccinia virus is sensed by mda5 [38] [39] [40] . it is also likely that the precise rlrs utilized for the sensing of specific viruses also operate within cell-specific contexts as well. interaction between host and pathogen results in a dynamic arms race. whenever pathogens develop strategies to overtake the host immune system, the host proteins involved in pathogen recognition have to respond by evolving to avoid or reduce potential infections. these dynamics result in hostpathogen adaptation and counter-adaptation, which in turns lead to the rapid co-evolution of both parties. particularly for the host, this accelerated molecular evolution is often reflected in host defense genes that exhibit strong signatures of ongoing diversifying selection [41, 42] . because viruses are responsible for a great number of severe and lethal diseases, together with the important role that rlrs play in mammalian innate immune system, we expect that rig-i, mda5 and lgp2 genes may have been under intense selective pressures in all mammals. we have previously demonstrated that one other class of mammalian prrs, the tlrs, exhibit striking evidence of positive genetic selection as a result of selective pressures exerted by pathogens [43] . using six different codon-based maximum likelihood (ml) methods, we searched for evidence of long-term selective pressures in the three rlr genes present in the available sequenced mammalian genomes and, where possible, pinpoint positively selected residues that might be involved in the host-virus interactions that have shaped their rapid diversification. specific lineages subject to episodic positive selection have also been identified in the three rlr genes by using two different branch-site models. publicly available mammalian rig-i, mda5 and lgp2 gene sequences were collected from ensembl and ncbi databases (table s1 ) for phylogenetic and selection analyses. the nucleotide coding sequences for each of the three rlr gene orthologous were aligned and are represented in figure s1 (rig-i alignment), figure s2 (mda5 alignment) and figure s3 (lgp2 alignment). the translation into deduced protein sequences is also represented in figure s4 (rig-i alignment), figure s5 (mda5 alignment) and figure s6 ( lgp2 alignment). the inherent limitations of using solely publicly available mammalian rlrs sequences should be highlighted, although several studies have used the same source of data for general conclusions about other genes in mammals [43] [44] [45] [46] [47] [48] [49] . the analyses performed ahead use only an individual representative of each included species and therefore, any drawn conclusions should be carefully considered. in mammalian rig-i phylogenetic reconstruction, the monophyly of six of a total of eight taxonomic orders was observed ( figure 1 ). however, an interesting fact was registered for the two remaining orders, rodentia and lagomorpha, when the european rabbit (order lagomorpha) grouped with the rodent cluster. when looking carefully at the european rabbit rig-i deduced protein sequence ( figure s4 ), a great number of conserved regions between this species and the other mammalian species was observed, with the exception of a region between codons 840 and 879. this 40 amino acid domain region was unique to the european rabbit rig-i. we originally speculated that this difference might have been the result of a gene conversion event with adjacent genes. however, when we examined the genes that are chromosomally adjacent to european rabbit rig-i (ndufb6, topors and frp), no clear evidence of gene conversion was detected by the software gard [50, 51] . for the mammalian mda5 gene sequences alignment, a significant recombination breakpoint was detected (nucleotide 903; p<0.01). therefore, two ml trees were reconstructed for the resulting segments, one for the first 903 nucleotides ( figure 2a ) and another ml tree for the remaining 2211 nucleotides ( figure 2b ). the gene phylogeny was also reconstructed for the whole alignment without testing recombination ( figure 2c ) to compare its topology with the other two resulting trees. the monophyly of the eight taxonomic orders included in the mda5 alignment was roughly recovered, with the clear exception of chiroptera in figure 2a and primates in figure 2b . regarding the lgp2 gene, no clear evidence of recombination was detected. the ml tree obtained (figure 3 ) supported the monophyly of the nine mammalian orders collected for this gene. all the molecular evolutionary analyses in this study were performed for both the complete nucleotide alignments ( figure s1 , figure s2 and figure s3 ) and for a trimmed version of the same genes to remove alignment gaps. figure s7 (rig-i alignment), figure s8 (mda5 alignment) and figure s9 (lgp2 alignment) correspond to the alignments where gaps present in all sequences, with the exception of one or two, have been removed, while gaps present in only one or two sequences were kept. we observed no significant differences in the results when using one or the other alignment for each gene (data not shown), but ultimately only the results from the trimmed version are presented here. evidence for positive selection on mammalian orthologous for rig-i ( figure s7 ), mda5 ( figure s8 ) and lgp2 ( figure s9 ) genes was detected using paml package [54, 55] site-specific models m1a versus m2a and m7 versus m8. these models test at the codon level whether a hypothesis that allows for positive selection (models m2a and m8) is a better fit to the data when compared to a null neutral hypothesis (models m1a and m7). results on the likelihood ratio test (lrt) performed between the likelihood scores of the null neutral and alternative selection models for each gene is indicated in table 1 . models which allow for positive selection (m2a and m8) gave a significantly better fit to the data for both rig-i and lgp2 alignments, suggesting that at least some of the codons within each set of orthologous gene sequences are subject to positive selection [56] . since a recombination breakpoint was detected on the mda5 alignment, each resulting segment (identified as 1 st and 2 nd segments) was tested individually for paml package [54, 55] site-specific models. although the comparison between the null neutral site model m1a and the selection site model m2a did not allow for rejection of the null hypothesis of neutral selection, the comparison between the more powerful pair of site-specific models m7 (neutral) and m8 (selection) yielded significant lrts ( table 1) . the parris method [57] implemented in the datamonkey web server [52, 53] was also applied to each rlr trimmed gene alignment ( figure s7 , figure s8 and figure s9 ) to look for evidence of positive selection, but no selective pressures were detected in any of the three genes (table s2 ). for each orthologous gene sequences alignment, the tree length parameter is indicated in table 1 . higher values of tree length, i.e. the expected number of nucleotide substitutions per codon, correspond to higher sequence divergence [58, 59] . the tree length values registered for the three genes fell into an intermediate and realistic level of sequence divergence which confers power to the codon models indicated by the lrt scores and to the bayes empirical bayes (beb) approach for site-specific inference of positive selection [58, 60] . model m8 implemented in the paml package [54, 55] and datamonkey web server [52, 53] slac, fel, rel, meme and fubar methods [61] [62] [63] were used to detect specific codons under selection in the three rlr genes. based on the methodology adopted by other authors and in previous studies [43, 47, 48, 64] , only codons identified by at least three of the six used methods are considered to be under positive selection (table s3 ). since the breadth of species included in each alignment is wide, by applying several methods to detect codons under positive selection and by overlapping the results, we should be decreasing the incidence of false positives. a total of sixteen codons for rig-i (figure 4 ), twenty for mda5 ( figure 5 ) and ten for lgp2 ( figure 6 ) were identified as candidate codons under selective pressure. regarding their location in each corresponding protein, the greatest number of these codons are located in protein functional domains, more specifically, eleven out of the sixteen rig-i codons (~ 69%), ten out of the twenty mda5 codons (50%) and seven out of the ten lgp2 codons (70%). to estimate the percentage of positively selected codons in the three proteins, we used human deduced protein sequences as a reference. human lgp2 exhibited 1.47% (10/678) of codons under positive selection. higher values were obtained for human mda5 and rig-i, 1.95% (20/1025) and 1.73% (16/925) of codons under selective pressure, respectively. to detect signatures of episodic positive selection in specific lineages of each rlr orthologous gene sequences alignment we performed two branch-site model analyses. these models allow the selective pressure indicated by the nonsynonymous to synonymous substitution rate ratio ω (d n /d s ) to vary both across sites in the gene and across lineages on the tree [65] . since no biological hypothesis existed to specify a priori branches to be examined for positive selection, the branch-site model a implemented in the paml package [54, 55, 66] was applied to all species branches on each rlr gene phylogenetic tree. the lrt performed for each branch was significant for 2δlnl > 3.84 [55, 66] . our analyses suggest that nine species branches in rig-i are under selective pressure (table 2 and figure 4b ). branch-site model a was applied to the two mda5 trees resultant from recombination testing and, for each tree, positive selection has operated only in two species branches (table 3 and figure 5b ). for lgp2, a total of six species branches had significant lrts corresponding to candidate lineages under selection (table 4 and figure 6b ). some of the species branches recognized by the branch-site model a were also identified by the branch-site rel method [67] (table 5 ) available in the datamonkey web server [52, 53] . for both rig-i and mda5, two species branches were simultaneously identified by the two methods, consisting in dog (calu) and european rabbit (orcu) branches for rig-i ( figure 4b ) and giant panda (aime) and guinea pig (capo) branches for mda5 ( figure 5b ). only one lgp2 species branch, corresponding to the giant panda (aime), was simultaneously identified by the branch-site model a and the branch-site rel method ( figure 6b ). in a human population genetics context, the first study on rlrs evolutionary history and selective footprints has been recently published [68] . nevertheless, to the best of our knowledge, our study is the first that searches for selective pressure acting on mammalian orthologous of the three rlrs and, in fact, we provide strong evidence of positive selection as well as identify a significant number of codons under probable selective pressures for rig-i, mda5 and lgp2. furthermore, our results on the rig-i rd in specific hosts suggest that certain viruses might be exerting long-term selective pressures on this gene. tlrs adaptive evolution has been the most extensively characterized of all the prrs in several animal groups, such as echinoderms [69] , birds [70] and different mammals [43, 64, [71] [72] [73] [74] . studies on known viral-recognition tlrs (tlr3, 7, 8 and 9) of closely related animal groups, like birds [70] , or within species, like humans and chimpanzees [64] , demonstrated that this class of prrs exhibits a background of strong purifying selection to keep their functional integrity, albeit in the birds study [70] significant instances of positive selection acting on a few amino acid sites were identified. nevertheless, when different ml codon-based methods were applied to detect evidence of acting positive selection in broader groups where a great number of species are included, like primates [64] and mammals [43] , most of the viral tlrs exhibited strong evidence of positive selection and specific codons with a high probability of being under selection were identified. similarly, in our study the codon-based analyses strongly support that the three rlr genes, rig-i, mda5 and lgp2, have all been subject to long-term selective pressures during mammalian evolution. also, we applied several methods that identified specific rlr codons with a high probability of being under selection, which may directly perturb downstream immune responses in a particular host infected by a viral pathogen. one of the major concerns when using large scale divergent species to infer positive selection acting on a set of orthologous genes and across lineages on the phylogenetic tree is the effect of saturation in synonymous substitutions, since they may saturate quickly as sequences diverge [75, 76] . as codon models consider both synonymous and nonsynonymous substitutions, the saturation of the first could cloud the information provided by nonsynonymous substitutions. nevertheless, the sequence divergence in our study, inferred through rlrs tree length values, fit into intermediate and realistic levels that should confer power to the lrt used to compare nested codon-models and robustness to the branchsite models, and to the beb approach for codon-specific detection of positive selection [58] [59] [60] . also, in this study the mammalian species collected for each of the three rlr genes were nearly the same, thus this host species spectrum should not influence the codon-based analyses and our observations when comparing the level of selective pressure between genes. in our study, mammalian mda5 showed the highest number and percentage of positively selected codons. nonetheless, the percentage of mda5 codons under selection located in the known protein functional domains was the lowest. this should reflect the imposition of functional and structural constraints in mda5 defined domains. on the other hand, we observed that lgp2 is apparently less prone to evolutionary change with the lowest number and percentage of codons under selective pressures. for rig-i, the greatest number of codons identified as candidates under selective pressures were located in known protein functional domains, which might reveal the pressure imposed by the great number of viruses recognized by this rlr [13, 14] . vasseur and colleagues [68] came to different conclusions in their study, once they were focused on intraspecies (human populations) polymorphisms and on the comparison of nonsynonymous to synonymous rates ratio ω (d n /d s ) between human and chimpanzee lineages for the three rlr genes. rig-i exhibited a stronger evolutionary constraint [68] , as attested by its low levels of nucleotide diversity, population differentiation and low tolerance of amino acid-altering variation. it also exhibited a dramatic decay in the ω ratio when compared to the other two rlrs [68] . this is the expected outcome in evolutionary studies when using closely related species, or genetic information for population of the same species, which result in a background of strong purifying selection to keep the protein functional integrity. in the same study [68] , the strongest signatures of positive selection were found in mda5 and lgp2 by exhibiting higher ω ratios than (table 2 ) are colored in green; branches simultaneously identified by paml branch-site model a and hyphy branch-site rel method (table 5) rig-i. besides, mda5 and lgp2 also appear to have evolved adaptively in specific human populations, presenting a great number of nonsynonymous mutations in both helicase and cterminal domains [68] . rig-i and mda5 contain two n-terminal cards [10, 17] . the interaction of these domains with an adaptor protein named ips-1 (also known as mavs, visa or cardif) is a crucial process to activate a wide range of downstream response factors, including type i ifns and other essential anti-viral proteins to induce intracellular immune responses [77] . interestingly, in our study, the cards of both rig-i and mda5 concentrated a large number of the deduced codons under selection. some of these are radical in terms of their physicochemical properties changes across mammalian species (figure 4 and figure 5 ), strengthening the case for positive selection. since the two cards are fundamental for (table 3) are colored in green; branches simultaneously identified by paml branch-site model a and hyphy branch-site rel method (table 5) (table 4 ) are colored in green; branch colored in blue has been simultaneously identified by paml branch-site model a and hyphy branch-site rel method (table 5 ). (c) positively-selected codons are exhibited in the table and numbered according to the mammalian lgp2 deduced protein sequences alignment ( figure s6 downstream rig-i and mda5 signaling, which implies functional constraints, the observed variability across species can be perceived as a great structural plasticity for mammalian cards. the helicase domain in the rlr family is generally described as exhibiting affinity for dsrna [78] . the existence of six highly conserved sequence motifs within this domain is a characteristic of the helicase superfamily 2 which integrates dexd/h box rna helicases. also, different aspects of helicase functions have been assigned to specific motifs [79] . bamming and horvath [11] compared the amino acid sequences of the three human rlr helicase domains with the established consensus sequences of helicase families elements and, despite slight differences, the sequences in individual motifs are highly conserved within rig-i, mda5 and lgp2. indeed, in our study the six helicase motifs of the three proteins were evolutionary conserved (data not shown) in the mammalian species collected. minor alterations occur in some species, but the extent of those differences concerning the involvement in substrate interaction, signal transduction and/or the whole antiviral response profile, is difficult to predict. rig-i rd is responsible for recognizing and binding to its rna substrates in a 5'-triphosphate (5'-ppp)-dependent manner. besides, binding studies clearly established that the ppprna binding site resides within the rd [14, 26, 80] . the function described for rig-i rd makes our current results worthy of note, since the rd is the rig-i domain that exhibits the strongest evidence of trans-acting selective pressures (figure 4 ). whether these differences play a role in rig-i activation after binding to the rnas from different viral pathogens that infect distinct mammalian hosts is a complex question. nevertheless, we can assume that the rd variability in mammals is related to the fact that rig-i senses a large variety of viruses [13, 14] . the performance of branch-site models in our study imposes a careful interpretation of data, since only one representative element of each species was included. still, some branches of the three rlr phylogenetic trees exhibited evidence of positive selection. the two species under episodic positive selection on [23, 39] . such results suggest that these lethal pathogens, and possibly other re-occurring viral infections in these specific hosts, might be exerting longterm selective pressures on the susceptible host rig-i gene. therefore, the changes on rig-i sequences across species, with special focus on the rd as suggested above, should be the result of a co-evolutionary process between speciesspecific infecting viruses and this host rna sensor protein. by detecting the extension of acting positive selection on mammalian rlrs, this study provides further insights into their biological functions in host defense against viral pathogens in general. differences in these genes across mammalian species may consequently impact downstream immune responses and, as a result, contribute to the species-specific resistance/ susceptibility profiles against many diverse viral pathogens. the coding region of the three rlr genes, rig-i, mda5 and lgp2, were collected for different mammalian species from ncbi (http://www.ncbi.nlm.nih.gov) and ensembl (http:// www.ensembl.org/index.html) databases (table s1 ). each set of mammalian orthologous gene sequences was aligned with clustalw [81] implemented in bioedit v7.0.9 [82] . the nucleotide sequences alignment corresponding to each gene coding region is represented in figure s1 (rig-i alignment), figure s2 (mda5 alignment) and figure s3 ( lgp2 alignment). translation into protein sequences was performed using also bioedit [82] . figure s4 , figure s5 and figure s6 represent the alignments of the deduced protein sequences for rig-i, mda5 and lgp2, respectively. for the evolutionary analyses, representative alignment gaps in figure s1 , figure s2 and figure s3 had to be removed: gaps present in all sequences, with the exception of one or two, have been removed, while gaps present in only one or two sequences were kept. figure s7 (rig-i alignment), figure s8 (mda5 alignment) and figure s9 (lgp2 alignment) correspond to trimmed versions of the nucleotide sequences alignment of each rlr gene. the nucleotide sequences alignment of each gene was firstly tested for recombination, as this biological process can mislead phylogenetic and positive selection analyses [83] . we used the software gard (genetic algorithm for recombination detection) [50, 51] , implemented in the datamonkey web server [52, 53] , to detect possible recombination breakpoints on each alignment. the nucleotide substitution model for each phylogenetic reconstruction was indicated by the akaike information criterion (aic) implemented in jmodeltest v0.1.1 [84] . regarding the rig-i gene one breakpoint was identified, but it was not supported by the kishino-hasegawa test. therefore, the complete alignment was used for the gene phylogeny reconstruction and gtr+g nucleotide substitution model was indicated as the best-fitting model. on the other hand, the software gard found evidence of two breakpoints in the mda5 gene alignment. however, only one of the breakpoints (nucleotide 903) reflected a significant topological incongruence (kishino-hasegawa test, p<0.01), suggesting that the multiple tree model can be preferred over the single tree model. we reconstructed mda5 phylogeny for the first 903 nucleotides of the mammalian alignment as also for the remaining 2211 nucleotides. to compare the different mda5 trees topology, we also used the complete alignment (no recombination testing) to reconstruct the gene phylogeny and gtr+g nucleotide substitution model was indicated by the aic as the best-fit. for the mda5 segments which resulted from recombination detection, the best-fitting nucleotide substitution models were tim3+g (first segment) and tim3+i+g (second segment). finally, we found no evidence of recombination for the lgp2 gene alignment. the best-fitting nucleotide substitution model determined for this alignment was the tpm2uf+i+g model. ml phylogenetic reconstruction was performed for the three genes using garli v2.0 (genetic algorithm for rapid likelihood inference) [85] . the analyses were performed with 1,000,000 generations and 1,000 bootstrap searches. ml trees were displayed using figtree v1.3.1 (http://tree.bio.ed.ac.uk/). codon substitution models implemented in the codeml program in paml v4.4 (phylogenetic analysis by maximum likelihood) package [54, 55] were applied to the trimmed alignments of rig-i ( figure s7 ), mda5 ( figure s8 ) and lgp2 ( figure s9 ). the codon frequency model f3x4 was fitted to all the alignments. two pairs of site-specific models were used, m1a (nearly neutral) versus m2a (selection) and m7 (neutral, beta) versus m8 (selection, beta & ω). in these comparisons, m1a and m7 neutral models (null hypothesis) do not admit positive selection, while m2a and m8 alternative models allow positive selection. a lrt with 2 degrees of freedom was performed, where a significant lrt demonstrates that the selection model fits better than the neutral model [56, 86, 87] . from the hyphy software available on the datamonkey web server [52, 53] , the parris method [57] was also applied to detect if a proportion of sites in each rlr alignment evolved with ω (d n /d s ) > 1. six different codon-based ml methods were applied to detect codons under positive selection on mammalian rig-i, mda5 and lgp2 trimmed alignments, and based on the methodology adopted by other authors and in previous studies [43, 47, 64] , only codons identified by at least three of the six used methods were considered to be under positive selection. model m8 from paml package [54, 55] was one of the codonbased methods used to detect codons under positive selection, and a bayes empirical bayes (beb) approach was employed to detect codons with a posterior probability >90% of being under selection [88] . five other methods, using hyphy software implemented in the datamonkey web server [52, 53] , were also applied to detect sites under selection for the three genes: the single likelihood ancestor counting (slac) method, the fixed effect likelihood (fel) method, the random effect likelihood (rel) method [61] and the recently described mixed effects model of evolution (meme) [62] and fast unbiased bayesien approximation (fubar) [63] methods. to avoid a high falsepositive rate [61] , sites with p-values <0.1 for slac, fel and meme models, bayes factor >50 for rel model and a posterior probability >0.90 for fubar were accepted as candidates for selection. two branch-site models allowing ω ratios to vary both among lineages and amino acid sites were performed: the paml branch-site model a [66] and the hyphy branch-site rel method [67] . when performing paml branch-site model a [66] , every species branch was analyzed as a foreground branch independently. for each analysis of a foreground branch, the remaining lineages were denominated as background branches. in branch-site model a, three ω ratios are assumed for foreground (0 < ω 0 < 1, ω 1 = 1, ω 2 > 1) and two ω ratios for background (0 < ω 0 < 1, ω 1 = 1). the null model is the same as model a, but ω 2 = 1 is fixed [66] . the beb approach was also used to calculate the posterior probability of a specific codon site and to identify those most likely to be under positive selection (posterior probability >90%) [88] . on the other hand, the branch-site rel method [67] was applied to identify branches where a proportion of sites evolved under episodic diversifying selection. figure s1 . mammalian rig-i nucleotide coding region sequences alignment. rig-i nucleotide coding region sequences for twenty-six mammalian species were collected from ensembl and ncbi databases, and aligned with clustalw implemented in bioedit. the symbol "." represents the same nucleotide as the reference sequence of human rig-i gene, "?" symbolizes an undetermined nucleotide and "-" represents a gap or deletion in the alignment. the figure s2 . mammalian mda5 nucleotide coding region sequences alignment. mda5 nucleotide coding region sequences for twenty-six mammalian species were collected from ensembl and ncbi databases, and aligned with clustalw implemented in bioedit. the symbol "." represents the same nucleotide as the reference sequence of human mda5 gene, "?" symbolizes an undetermined nucleotide and "-" represents a gap or deletion in the alignment. the lgp2 nucleotide coding region sequences for thirty mammalian species were collected from ensembl and ncbi databases, and aligned with clustalw implemented in bioedit. the symbol "." represents the same nucleotide as the reference sequence of human lgp2 gene and "-" symbolizes a gap or deletion in the alignment. the figure s4 . mammalian rig-i deduced protein sequences alignment. rig-i deduced protein sequences for twenty-six mammalian species were collected from ensembl and ncbi databases, and aligned with clustalw implemented in bioedit. the symbol "." represents the same codon as the reference sequence of human rig-i protein, "?" symbolizes an undetermined codon and "-" represents a gap or deletion in the alignment. the figure s5 . mammalian mda5 deduced protein sequences alignment. mda5 deduced protein sequences for twenty-six mammalian species were collected from ensembl and ncbi databases, and aligned with clustalw implemented in bioedit. the symbol "." represents the same codon as the reference sequence of human mda5 protein, "?" symbolizes an undetermined codon and "-" represents a gap or deletion in the alignment. the lgp2 deduced protein sequences for thirty mammalian species were collected from ensembl and ncbi databases, and aligned with clustalw implemented in bioedit. the symbol "." represents the same codon as the reference sequence of human lgp2 protein and "-" symbolizes a gap or deletion in the alignment. the used abbreviations correspond, by order of appearance, to the following species: hosa -human; patr -chimpanzee; papa -bonobo; gogo -gorilla; poab -orangutan; mamu -rhesus macaque; sabo -blackcapped squirrel monkey; caja -marmoset; mimu -mouse lemur; otga -bushbaby; bota -cow; ovar -sheep; susc -pig; tutr -dolphin; mylu -little brown myotis; ptva -large flying fox; ptal -black flying fox; loaf -elephant; mupu -ferret; aime -giant panda; calu -dog; feca -cat; eqca -horse; ocpr -american pika; orcu -european rabbit; ictr -squirrel; crgr -chinese hamster; mumu -mouse; rano -rat; capo -guinea pig. (tif) figure s7 . mammalian rig-i nucleotide trimmed sequences alignment. rig-i nucleotide trimmed sequences for twenty-six mammalian species were collected from ensembl and ncbi databases, and aligned with clustalw implemented in bioedit. the symbol "." represents the same nucleotide as the reference sequence of human rig-i gene, "?" symbolizes an undetermined nucleotide and "-" represents a gap or deletion in the alignment. the mammalian mda5 nucleotide trimmed sequences alignment. mda5 nucleotide trimmed sequences for twenty-six mammalian species were collected from ensembl and ncbi databases, and aligned with clustalw implemented in bioedit. the symbol "." represents the same nucleotide as the reference sequence of human mda5 gene, "?" symbolizes an undetermined nucleotide and "-" represents a gap or deletion in the alignment. the lgp2 nucleotide trimmed sequences for thirty mammalian species were collected from ensembl and ncbi databases, and aligned with clustalw implemented in 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gap penalties and weight matrix choice bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt the effect of recombination on the accuracy of phylogeny estimation jmodeltest: phylogenetic model averaging genetic algorithm approaches for the phylogenetic analysis of large biological sequence datasets under the maximum likelihood criterion likelihood models for detecting positively selected amino acid sites and applications to the hiv-1 envelope gene codonsubstitution models for heterogeneous selection pressure at amino acid sites bayes empirical bayes inference of amino acid sites under positive selection key: cord-002834-2htnywef authors: tsuchiaka, shinobu; naoi, yuki; imai, ryo; masuda, tsuneyuki; ito, mika; akagami, masataka; ouchi, yoshinao; ishii, kazuo; sakaguchi, shoichi; omatsu, tsutomu; katayama, yukie; oba, mami; shirai, junsuke; satani, yuki; takashima, yasuhiro; taniguchi, yuji; takasu, masaki; madarame, hiroo; sunaga, fujiko; aoki, hiroshi; makino, shinji; mizutani, tetsuya; nagai, makoto title: genetic diversity and recombination of enterovirus g strains in japanese pigs: high prevalence of strains carrying a papain-like cysteine protease sequence in the enterovirus g population date: 2018-01-11 journal: plos one doi: 10.1371/journal.pone.0190819 sha: doc_id: 2834 cord_uid: 2htnywef to study the genetic diversity of enterovirus g (ev-g) among japanese pigs, metagenomics sequencing was performed on fecal samples from pigs with or without diarrhea, collected between 2014 and 2016. fifty-nine ev-g sequences, which were >5,000 nucleotides long, were obtained. by complete vp1 sequence analysis, japanese ev-g isolates were classified into g1 (17 strains), g2 (four strains), g3 (22 strains), g4 (two strains), g6 (two strains), g9 (six strains), g10 (five strains), and a new genotype (one strain). remarkably, 16 g1 and one g2 strain identified in diarrheic (23.5%; four strains) or normal (76.5%; 13 strains) fecal samples possessed a papain-like cysteine protease (pl-cp) sequence, which was recently found in the usa and belgium in the ev-g genome, at the 2c–3a junction site. this paper presents the first report of the high prevalence of viruses carrying pl-cp in the ev-g population. furthermore, possible interand intragenotype recombination events were found among ev-g strains, including g1-pl-cp strains. our findings may advance the understanding of the molecular epidemiology and genetic evolution of ev-gs. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 next-generation sequencing was conducted on cdna libraries constructed from total rna of 222 fecal samples. via a blast search, 59 ev-g-like contigs that were longer than 5,000 nt, including the entire vp1-coding sequence with more than threefold coverage of sequence reads, were identified in 35 (15.8%) normal fecal samples, five (2.3%) fecal samples for mild diarrhea, and 10 (4.5%) diarrheic fecal samples (tables 1 and 2 ). eight of 50 samples revealed more than two ev-g-like contigs (table 1) . apart from three samples (ishi-ya3, ishi-ka3, and ishi-ka7), 47 samples were found to contain other viruses: rotaviruses a, c, or h; orthoreovirus; kobuvirus; picobirnavirus; astrovirus; porcine epidemic diarrhea virus (pedv); posavirus; sapelovirus; st-valerien virus; sapovirus; or teschovirus (table 1 and s3 table) . because ev-g discrimination is based on sequence identities of the complete vp1 gene (http://www.picornaviridae.com/), a phylogenetic tree using the complete vp1 nt sequence was constructed. japanese ev-g strains clustered with the reference strains of g1 (17 strains), g2 (four strains), g3 (22 strains), g4 (two strains), g6 (two strains), g9 (six strains), and g10 (five strains). one strain named ishi-ka2 branched independently and did not cluster with any reference strains (fig 1) . because >25% difference in complete vp1 nt sequences between isolates is a criterion for genotype classification [13] [14] , a pairwise comparison of complete vp1 nt sequences was conducted (table 3 and s1 table) . bu6-5, bu8-2, bu8-4, and ishi-ya4-2 formed one cluster but were found to be slightly related to the g3 group. although complete vp1 nt sequence identities of these strains to those of other g3 strains (except for ishi-ka3, ishi-ka3-1, ishi-ka4, ishi-ka5-1, ishi-ka6, and ishi-ka7) were <75.0% (69.8%-74.9%), these four strains shared !75.0% nt sequence identities with ishi-ka3, ishi-ka3-1, ishi-ka4, ishi-ka5-1, and ishi-ka6 (s1 table) . therefore, we tentatively classified these strains as g3-lineage 2 (fig 1) . ishi-ka2 revealed low nt sequence identities (57.5% to 73.1%) with other genotypes and thus ishi-ka2 may represent a new serotype of ev-g (table 3) . complete or nearly complete aa sequences of the coding-sequence region (cds) of 59 japanese ev-g strains were aligned and compared. we found that 17 of the 59 strains contain extra 633 to 651 nt (211 to 217 aa) within the 2c-3a coding region. according to blast analysis, these sequences have sequence homology to the pl-cp sequence variants that were recently identified in ev-g strains from the usa and belgium [24] [25] [26] . each inserted sequence is located between the coding regions 2c and 3a as in the usa and belgium strains. the insertion sequences were aligned and compared with those of the pl-cp of ev-g strains from the usa and belgium and with the pl-vp sequences in the genome of nidoviruses including porcine and bovine toroviruses by phylogenetic analysis and pairwise sequence comparison (fig 2 and s2 table) . the pl-cp sequence of japanese ev-g1 and that of japanese ev-g2 revealed !74.0% nt and !74.6% aa sequence identities to each other and to the usa and belgium ev-gs and clustered in one group but are distantly related to those of porcine and bovine toroviruses, showing lower sequence identities (57.0% to 64.6% in the nt sequence and 49.6% to 58.7% in the aa sequence). ev-g hgya2-1 and porcine torovirus hgya2-2 were identified on the same farm at the same time; however, the nt and aa sequence identity between the pl-cp sequences of those strains was 62.3% and 54.3%, respectively (s2 table) . japanese ev-g strains carrying pl-cp were subdivided into g1-pl-cp lineage 1, g1-pl-cp lineage 2, and g2 in the vp1 phylogenetic tree (fig 1) ; however, these groups were not clearly detectable in the pl-cp tree (fig 2) to further investigate the genomic relations among ev-g strains, phylogenetic trees based on nt sequences of three regions (vp4-vp3, vp1, p2, and p3) were constructed. the tree for vp4-vp3 was similar to that of vp1, but the p2 and p3 trees showed topologies different from each other and no clear-cut ev-g types could be defined (fig 3a) . ev-g1-pl-cp lineage 3 was found to be related to g1-pl-cp lineage 2 and 3 in the trees for vp4-vp3 and vp1, whereas g1-pl-cp lineage 3 was closely related to g3 and g9 strains in the p2 tree and to g3 and g1-pl-cp strains in the p3 tree. the g2-pl-cp strain hgya2-1 showed high homology genetic diversity and recombination of enterovirus g strains in japanese pigs to g2 strains in the tree for vp4-vp3 and vp1, whereas hgya2-1 showed high similarity with g1-pl-cp lineage 1 strains in regions p2 and p3 (fig 3a) . by simplot analysis, the crossover site was mapped to the 2a region. the g2-pl-cp strain hgya2-1 revealed that the downstream region of the crossover site has high similarity to the g1-pl-cp strain moi2-2-1 ( fig 3c) . to find a possible recombination breakpoint, a bootstrap scanning analysis was conducted. a possible recombination breakpoint was identified in the middle of the 2a region (fig 3d) . ishi-ka2 branched independently in the trees for vp4-vp3 and vp1, whereas ishi-ka2 clustered with g3-lineage 1 and formed a cluster with g3 strains identified on the same farm (s1a fig). simplot analysis suggested that ishi-ka2 has extremely high similarity to g3-lineage 1 strain ishi-ka7 in regions 2c and p3 (s1c fig). g3-lineage 2 strains showed a topology similar to that of the vp1 tree in vp4-vp3; however, the g3-lineage 2 strains branched separately from the g3-lineage-1 strains in the p2 and p3 trees (s1a fig) and were found to be closely related to each other throughout the genome (s1d fig) . although we did not initially aim to determine ev-g prevalence among pigs in japan in this study, contigs that were longer than 5,000 nt were found in 22.5% (50/222) of pigs and on 23.4% (18/77) of farms, suggesting that ev-gs are widespread among japanese pigs. fortyfour strains out of 59 (74.6%) were detected in healthy pigs, indicating that ev-gs seem not to be associated with diarrhea in pigs, in accordance with other reports [13] [14] 38] . because the detection limit of the method was not tested, a true prevalence study is needed in the future. ev-g genotyping is based on >25% divergence between vp1 nucleotide sequences [14, 39] . in the present study, according to the criteria, seven genotypes (g1, g2, g4, g6, g9, g10, and g?) were found in the feces samples from japanese pigs, and the predominant genotypes were g3 (37.3%; 22/59) and g1 (28.8%; 17/59; table 1 , fig 1) . there are few studies on the genotyping of ev-gs in pigs, and limited information is available from ddbj/embl/genbank databases. g1 and g6 types are predominant genotypes in vietnam, whereas g3, g2, and g4 types appear to be common genotypes in spain (however, that study did not analyze complete vp1 sequence) [40] . to date, g1-g16 genotypes and at least three ev-gs with an unassigned genotype, including ishi-ka2 in this study, have been reported [13-14, 25, 41] . owing to the limited number of reports on a specific geographic area, probably not all genotypes of ev-gs are known at present. further studies are needed for a comprehensive understanding of the genetic diversity of ev-gs in other geographic areas. picornaviruses show significant genetic variability driven by both mutations and recombination events [42] [43] . ishi-ka2 manifested >25% vp1 nucleotide sequence divergence from other strains; therefore, ishi-ka2 can be considered a new serotype of ev-gs. nonetheless, ishi-ka2 shares high sequence homology with the g3-lineage 1 strain ishi-ka7, which was identified in a pig kept on the same farm, except for the p1 region. it is likely that ishi-ka2 emerged by possible recombination events; however, the putative recombination points could not be identified, and the origins of these recombination events are unclear because the recombination counterparts of these strains could not be found in the ddbj/embl/genbank databases or our dataset. g3-lineage 2 strains have sequence homology to g3-lineage 1 in the p1 region, but they are distantly related to g3-lineage 1 on the basis of regions p2 and p3. because vp1 induces a host immune response, serological properties can be hypothesized based on sequence homology of the vp1 gene. on the other hand, these results suggest that full genome analysis may be needed in addition to the genotyping approaches based solely on the vp1 gene for precise ev-g classification. rna recombination events contribute to genetic diversity and may lead to changes in virulence, escape from host immunity, and adaptation to a new host [42] [43] [44] [45] [46] [47] [48] [49] [50] . ev-g strains carrying pl-cp in pigs with diarrhea have been reported in the usa and belgium [24] [25] [26] . in these cases, ev-g-pl-cps were detected solely or with low abundance of pedv. shang et al. constructed an infectious clone of the ev-g-pl-cp strain, 08/nc_usa/2015, and compared it with a pl-cp knockout recombinant virus. they found that the pl-cp knockout virus showed impaired growth and induced higher expression levels of innate-immunity genes, suggesting that ev-g-pl-cp strains acquire pathogenesis via a recombination event [25] . four out of 17 japanese ev-g-pl-cp strains were detected in diarrheic cases of pigs; however, 13 ev-g-pl-cp strains were isolated from healthy pigs. in all cases of detection of ev-g-pl-cp in japan, ev-g-pl-cp strains were identified together with other enteric viruses, such as astrovirus, sapelovirus, posavirus, rotavirus, picobirnavirus, sapovirus, teschovirus, torovirus, pedv, st-valerien virus, or kobuvirus (table 1) . mixed infection with ev-g-pl-cp and other enteric viruses may influence the pathogenicity of ev-g-pl-cp strains. the sequences of pl-cp of japanese ev-g pl-cp strains are distantly related to the sequences derived from orf1 of toroviruses, even though they were simultaneously identified on the same farm, and they have homology to those of usa and belgium strains (fig 2) , suggesting that a recombination event between an ev-g and torovirus occurred in the past. by recombination analyses, possible recombination events between ev-g-pl-cp strains were uncovered and a recombination breakpoint was identified in the middle of region 2a (fig 3) , in agreement with another report that describes a recombinant event between ev-g8 and ev-g9 [14] , suggesting that this point may be a hotspot of recombination events of ev-g. furthermore, vp1-2a junction is a known recombination hot-spot in human enteroviruses and this was discussed in many papers [51] [52] [53] [54] [55] . the present recombination profile in ev-g described here apparently mirrors that in human enteroviruses. ev-gs that received pl-cp have been evolving independently and gaining genetic diversity via recombination events. by a metagenomics approach, high genetic diversity of ev-gs, including new genotypes and high prevalence of ev-gs carrying pl-cp, was observed among ev-g isolates from the feces of japanese pigs. ev-gs comingle and pose a risk of coinfection in the current growing and high-density pig husbandry system of japan. coinfection of a single animal with multiple ev-gs, including ev-g-pl-cp strains, may lead to recombination events, which may in turn promote genetic diversity of ev-gs and ev-g-pl-cps. these findings may 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recombination and reemergence of ancient lineages recombination strategies and evolutionary dynamics of the human enterovirus a global gene pool mizutani. we received three funding including jsps kakenhi 15k07718 to makoto nagai, global innovation research of tokyo university of agriculture and technology to tetsuya mizutani, and the research project for improving food safety and animal health of the ministry of agriculture, forestry and fisheries of japan. we are very grateful to ms. kanako satoh and ms. megumi mori for technical assistance. conceptualization: tetsuya mizutani, makoto nagai. key: cord-000539-uh3q65we authors: zhang, yi; sun, honglei; fan, lihong; ma, yuan; sun, yipeng; pu, juan; yang, jun; qiao, jian; ma, guangpeng; liu, jinhua title: acute respiratory distress syndrome induced by a swine 2009 h1n1 variant in mice date: 2012-01-03 journal: plos one doi: 10.1371/journal.pone.0029347 sha: doc_id: 539 cord_uid: uh3q65we background: acute respiratory distress syndrome (ards) induced by pandemic 2009 h1n1 influenza virus has been widely reported and was considered the main cause of death in critically ill patients with 2009 h1n1 infection. however, no animal model has been developed for ards caused by infection with 2009 h1n1 virus. here, we present a mouse model of ards induced by 2009 h1n1 virus. methodology principal findings: mice were inoculated with a/swine/shandong/731/2009 (sd/09), which was a 2009 h1n1 influenza variant with a g222d mutation in the hemagglutinin. clinical symptoms were recorded every day. lung injury was assessed by lung water content and histopathological observation. arterial blood gas, leukocyte count in the bronchial alveolar lavage fluid and blood, virus titers, and cytokine levels in the lung were measured at various times post-inoculation. mice infected with sd/09 virus showed typical ards symptoms characterized by 60% lethality on days 8–10 post-inoculation, highly edematous lungs, inflammatory cellular infiltration, alveolar and interstitial edema, lung hemorrhage, progressive and severe hypoxemia, and elevated levels of proinflammatory cytokines and chemokines. conclusions/significance: these results suggested that we successfully established an ards mouse model induced by a virulent 2009 h1n1 variant without previous adaptation, which may be of benefit for evaluating the pathogenesis or therapy of human ards caused by 2009 h1n1 virus. a novel influenza a (h1n1) virus of swine origin emerged among humans in mexico during the spring of 2009 and rapidly spread worldwide [1] . the pandemic prompted the world health organization (who) to raise the alert level to the highest rating of six, the pandemic phase, within 2 months [2] . in august 2010, who officially declared that the disease was in the post-pandemic period [3] ; however, it is still circulating among humans, together with seasonal viruses. although most influenza cases caused by 2009 h1n1 virus infection typically display mild upper respiratory tract syndrome, some cases progress to severe pneumonia and acute respiratory distress syndrome (ards) [4, 5] . many studies have shown that ards caused by 2009 h1n1 virus results in 17.3-56% mortality [4, 6, 7, 8] , which was regarded as the major cause of death by 2009 h1n1 virus infection [9] . ards is the result of acute injury to lung tissue, commonly resulting from sepsis, trauma, and severe pulmonary infections [10] . infectious factors, most of which are viruses, have become one of the most important causes of ards in humans [11, 12, 13] . clinical cases and established animal models have revealed that the pathogenesis and pathological features of ards induced by different viral pathogens are distinct [14, 15] . however, knowledge of the pathogenesis of 2009 h1n1 virus, especially ards induced by 2009 h1n1 virus, is still limited and hinders therapeutic strategies. therefore, it is necessary to evaluate the pathogenesis of ards caused by 2009 h1n1 virus infection in an appropriate animal model to assess potential therapies. mice are a good model for evaluating the pathogenesis and antiviral therapy of influenza pneumonia, due to the general fidelity of the illness in mice to the human disease [16] . moreover, a mouse model of ards caused by highly pathogenic h5n1 avian influenza virus infection has been well established [13] . the typical 2009 h1n1 virus, such as a/california/04/2009 (ca/04), can efficiently replicate in mouse lungs without prior host adaptation. however, it only causes moderate lung lesions and no mortality, even when inoculated at a high dose of 10 6 pfu [17, 18] . thus, such typical 2009 h1n1 viruses may not be able to induce ards in a mouse model. in the present study, we used a virulent variant 2009 h1n1 virus, which was isolated from a pig and possessed a virulenceassociated ha-d222g mutation, to establish an ards mouse model. the model established here provides a useful tool to explore the mechanism of ards, as well as screening and therapeutic options. six-week-old female mice were infected intranasally (i.n.) with 10 2.5 pfu sd/09 virus. some of the infected mice showed signs of illness, such as altered gait, inactivity, ruffled fur, and anorexia on day 2 post-infection (p.i.). from day 2 p.i., the body weight of most mice significantly decreased ( figure s1 ). by day 6 p.i., most mice presented with more severe clinical signs of respiratory disease, including labored respiration and respiratory distress, and most mice lost almost 20% of their initial body weight. on day 8 p.i., most mice were nearly unable to respond to exterior stimuli, and acute respiratory rates and labored respiration were observed (video s1, and video s2 for control). approximately 60% of mice died between days 8 and 10 p.i. gross observation of infected mice showed that the lungs were highly edematous, with profuse areas of hemorrhage and consolidation. no obvious gross lesions were observed in the kidneys, liver, spleen or brain of infected mice. mice were infected i.n. with 10 2.5 pfu sd/09 virus, and three mice were euthanized on days 2, 4, 6, 8, 10 and 14 p.i., and the virus titers in viscera were determined. as shown in figure 1a , the virus titer in the lung gradually increased between days 2 and 6 p.i., and reached a peak on day 6 p.i. the virus titers in the lung gradually decreased from day 6 p.i., and only one of three mice possessed detectable virus in the lungs on day 10 p.i. no viruses were detected in other organs, including heart, spleen, liver, kidneys, blood and brain, at the indicated time. these results indicate that sd/09 virus could replicate efficiently in mouse lung but did not cause systemic infection. as shown in figure 1b , the effect of sd/09 viral infection on lung wet:dry weight ratio did not change significantly within 2 days p.i. however, a dramatic increase was observed from day 4 p.i., and reached a peak on day 8 p.i., which was nearly twice that observed in control group lungs (p,0.01). the change in lung wet weight:body weight ratio was similar to the change in lung wet:dry weight ratio ( figure 1c ). the results indicated that the sd/09 virus could induce acute lung edema in mice. kinetic observation of lung lesions of sd/09-virus-infected mice is shown in figure 2 . on day 4 p.i., lung lesions were characterized by dropout of mucous epithelium and inflammatory cells adhering to the bronchiolar surface ( figure 2c , d). on day 6 p.i., severe edema could be seen around blood vessels ( figure 2e ); interstitial pneumonia was also observed that showed interstitial edema and thickening of the alveolar walls; and the alveolar lumen was flooded with detached alveolar cells, erythrocytes, and inflammatory cells ( figure 2e , f). on day 8 p.i., the virus caused more severe interstitial pneumonia and peribronchiolitis, characterized by edema and extensive of lymphocytes, neutrophils and plasma cells around the area of bronchiolitis ( figure 2g , h). lesions in the lungs of infected mice were still severe on day 14 p.i., with extensive alveolar collapse, and remaining alveoli were filled with fibrin, desquamated alveolar cells, and inflammatory cells. lymphocytes and alveolar macrophages were the predominant inflammatory cells observed at high magnification ( figure 2j ). masson's stain revealed that alveolar walls and spaces were filled with collagen fibers ( figure 3a , b), indicating that proliferative fibroblastic lesions may develop. in comparison, lungs from mockinfected control mice had no apparent histological changes ( mice were inoculated i.n. with 10 2.5 pfu sd/09 viruses; tissues were collected at indicated times p.i. and viruses were titrated in mdck cells. body weight, lung wet and dry weight were determined and recorded. the lung wet weight:body weight ratio and lung wet:dry weight ratio were calculated and used as an indicator of lung edema. *p,0.05, **p,0.01, ***p,0.001, comparison between ratios obtained from the virusinfected and control groups. bars represent means 6 sd of data from three mice. doi:10.1371/journal.pone.0029347.g001 immunohistochemistry revealed viral antigens in the epithelial cells of the bronchioles ( figure 3d ), terminal bronchioles, and alveolar epithelial cells ( figure 3e ). these data indicated that sd/ 09 virus could infect the epithelia of the lower airway and cause viral pneumonia in mice. as shown in table 1 , virus-infected mice showed a slightly decreased partial pressure of arterial oxygen (pa o2 ), saturation of arterial oxygen (sa o2 ), and slightly increased partial pressure of arterial carbon dioxide (pa co2 ) from days 4 to 6 p.i. most infected mice presented with severe clinical signs of respiratory distress on day 8 p.i., and blood gas analysis also showed that pa o2 and sa o2 dramatically decreased compared with the controls (p,0.05). these results suggested acute respiratory dysfunction and severe hypoxemia in virus-infected mice. the number of leukocytes in balf from sd/09-infected mice showed an increase from day 4 p.i. ( table 2 ). the balf of virusinfected mice on day 6 p.i contained 1.1610 6 cells/ml and was significantly different from the 1.6610 5 cells/ml observed for pbsinoculated mice (p,0.001). these data indicate a dramatic increase in inflammatory cells in the lungs of sd/09-infected mice. to quantify the immune cell subpopulations responding to viral infection, we next determined cell differential counts in the infected lungs by wright staining. compared with pbs-inoculated animals, mice infected with sd/09 virus exhibited an increase of neutrophils from 4 days p.i., and the peak was 18-fold greater than that of the control group on day 8 p.i. leukopenia was detected on day 4 p.i., and was statistically significant on day 6 p.i. (p,0.05); the lowest value appeared on day 8 p.i. (p,0.001). furthermore, differential blood counts revealed that the number of lymphocytes sharply decreased in infected mice. the lowest number of lymphocytes observed occurred on day 8 p.i. (figure 4b ), which dropped to ,20% of the control group number (p,0.001). to determine the cytokine responses that occur after sd/09 virus infection, we measured the levels of five cytokines and chemokines in lungs of infected mice on days 2, 4, 6, 8 and 10 p.i. as shown in figure 5 , all five were significantly different between virus-infected and control mice. interleukin (il)-6 and il-10 in the virus-infected mice reached peak levels as early as day 2 p.i. and were significantly higher than those of the control group (p,0.001). interferon (ifn)-c, monocyte chemotactic protein (mcp)-1, and tumor necrosis factor (tnf) dramatically increased in mouse lungs on days 6-8 p.i. (p,0.001), consistent with the appearance of pulmonary lesions. these results showed that infection with sd/09 viruses resulted in elevated amounts of proinflammatory chemokines and cytokines in the lungs of mice. in the spring of 2009, a novel influenza a(h1n1) virus rapidly spread worldwide, resulting in the first influenza pandemic of the 21st century [1] . critically ill cases caused by 2009 h1n1 virus retrospectively showed that most had progressed or died due to ards [4, 6] . however, the pathogenesis and therapeutic intervention of ards caused by 2009 h1n1 infection have still not been elucidated. animal models of disease are important for characterizing pathogenesis and developing the preclinical evidence for revised approaches to ventilating patients with ards [19] . here, we present a mouse model for the study of ards induced by sd/09 virus, a virulent 2009 h1n1 variant. previous studies have indicated that typical 2009 h1n1 viruses such as ca/04 bind only to a-2,6-linked sialic acid (sa) receptor [17] , but only the a-2,3-linked sa receptor is found in the mouse respiratory tract [20] . therefore, such a typical 2009 h1n1 virus may not be able to induce ards in mice. in fact, we used ca/04 virus to induce ards in mice; however, animals inoculated with a high dose of ca/04 virus (10 6.5 pfu) only showed moderate respiratory symptoms, and no lethality was observed (unpublished data). it has been shown that 2009 h1n1 virus possessing a d222g mutation in hemagglutinin (ha) could increase the pathogenicity in mice [21, 22] and binding to the a-2,3 sa receptor [23] . moreover, clinical data indicate that such variants are only associated with severe h1n1 human infection [24] . therefore, we suggest that the variant possessing the d222g mutation in ha can induce ards in a mouse model. the virus used in the present study was isolated from swine in 2009, and sequence analysis revealed that all the eight genes of the isolate had a close relationship with the 2009 h1n1 influenza virus circulating in humans. notably, the swine isolate, sd/09, had a d222g mutation in ha. compared with ca/04 virus (ld 50 .10 6 pfu), sd/09 showed significantly increased virulence in mice, with an ld 50 of 10 2.25 pfu, which was nearly identical to that of the mouse-adapted strain a/hong kong/415742md/09 (ld 50 = 10 2.2 pfu) [17, 21] . mice infected i.n. with 10 2.5 pfu sd/ 09 virus showed obvious respiratory symptoms, including visually prominent signs of respiratory distress and abdominal respiration, with approximately 60% mortality between days 8 and 10 p.i. the lungs of virus-infected mice were highly edematous, which was also demonstrated by dramatically increased lung wet:dry weight ratio. pathological changes presented a progressive pattern, typically diffuse alveolar damage, interstitial and alveolar edema, neutrophil and macrophage-dominant inflammatory cellular infiltration, and areas of hemorrhage and necrotizing bronchiolitis. arterial blood gas saturation is a key parameter of ards in humans [25] . in the present mouse model, pao 2 and sao 2 of infected mice were significantly lower than in the control group from day 2 p.i., especially on day 8 p.i., where these parameters sharply decreased, and most virus-infected mice began to die. these changes in arterial blood gas demonstrated that most infected mice developed severe hypoxemia consistent with of the appearance of clinical signs and lung lesions of ards. previous studies showed that mice infected with typical 2009 h1n1 virus only exhibited mild interstitial inflammatory infiltration and limited alveolitis [18, 21] , whereas severe lung damage was found in the sd/09-infected mice, including severe edema around the blood vessels and bronchiolitis, and extensive inflammatory accumulation from 4 to 8 days p.i. at 10 days p.i., the surviving mice developed an irreversible fibrosis involving collagen deposition in alveolar walls and spaces, which was similar to that observed in human ards patients with 2009 h1n1 infection [26] . our histopathological results were consistent with ards induced by other influenza viruses. mice infected with mouse-adapted virus of the a/puerto rico/8/34 (h1n1), or high pathogenic h5n1 virus also showed a progressive series of pathological changes from interstitial pneumonia to diffuse alveolar damage [13, 27] . however, in contrast to highly pathogenic h5n1 virus, mice infected with sd/09 virus did not show viral spread to extrapulmonary organs. immunohistochemical examination revealed the presence of viral antigens in the bronchioles, terminal bronchiolar epithelium, and alveolar epithelial cells. perhaps sd/09 virus infection of the alveoli, particularly type ii pneumocytes, rather than bronchioles, is a key to the development of ards. type ii pneumocytes are responsible for the production and secretion of surfactant to lower the surface tension of water and allow membrane separation, and insufficient pulmonary surfactant in the alveoli may result in alveolar collapse [28, 29] . the 1918 pandemic h1n1 and high pathogenic h5n1 viruses preferentially infect type ii pneumocytes and alveolar macrophage in mice [15, 30] . alveolar macrophages may play a critical role in disease pathogenesis, not through production of infectious virus but rather through the upregulation of proinflammatory cytokines that may further damage alveolar pneumocytes [31] . these phenomena suggest that viral cell tropism may determine the processes of ards. pulmonary aberrant immune response is considered a significant feature of ards induced by 2009 h1n1 virus [19, 32] . in the present mouse model, the number of leukocytes observed in the balf of virus-infected mice significantly increased compared with the control mice on day 8 p.i. different counts in balf showed that the proportion of neutrophils dramatically increased. these innate immune cells were capable of reducing the virus load in the lung [33] ; however, they could cause lung injury through direct or indirect mechanisms. neutrophil oxidants and proteases can cause direct injury of cells in the alveolar-capillary membrane [34] . neutrophils and macrophages can secrete copious amounts of chemokines and cytokines that can recruit more immune cells into lung tissues, and produce a ''cytokine storm'', one of the most important factors in the production of ards [35] . a retrospective cohort study of 74 2009 h1n1 patients found that higher levels of proinflammatory cytokines and chemokines in plasma were observed in the ards-death group compared with the survived-without-ards or the mild-disease groups [5] . another study in critically ill patients with ards caused by 2009 h1n1 virus infection has shown that the hallmarks of disease severity were elevated levels of il-6, il-15, il-8 and tnf-a [36] . we examined the levels of five cytokines and chemokines in infected mouse lungs and found significant differences between the virus-infected and mock groups. it has proved that high levels of il-6 were able to mediate acute lung injury [37] , and had a negative correlation with the pa o2 :fi o2 ratio in severely affected patients with 2009 h1n1 virus infection [36] . our data showed sd/09 viral infection induced high levels of il-6 in mouse lung, which may also play an important role in the course of ards. hagau etc. found the levels of tnf-a increased significantly in the 2009 h1n1-related ards patients [36] . in present study, tnf levels also dramatically increased in the lungs of virus-infected mice, and were consistent with the clinical symptoms and reached peak levels when mice began to die. in addition, high levels of il-10, ifn-c and mcp-1 were also present in the virus-infected mouse lungs, similar to observations found in severely affected humans with 2009 h1n1 infection [38] . in summary, we successfully established an ards mouse model induced by a virulent 2009 h1n1 variant, which demonstrated key human ards clinical and pathological features, such as respiratory distress, low pa o2 , exudative, proliferative and fibrotic lung, and high levels of inflammatory cells and cytokines. the mouse model may contribute to the study of the pathogenesis and therapy of ards induced by 2009 h1n1 virus. to determine ld 50 of sd/09 virus, eight 6-week-old female balb/c mice per group were inoculated i.n. with 10 1 -10 6 pfu (50 ml) viruses and monitored for 14 days. the value of mld50 was calculated using the spearman-karber method and expressed by pfu per mld 50 [39] . we evaluated the pathogenicity of the virus in mice and found that it could efficiently replicate in the lungs of mice with high lethality (10 2.25 pfu per mld 50 ). to determine the optimal dose of inoculation, 10 mice in each group were infected i.n. with 10 1.5 , 10 2.5 or 10 3.5 pfu viruses, and the signs, body weight, and mortality were monitored daily for each group for 14 days. pilot experiments indicated that a dose of 10 2.5 pfu was optimal, because the course of the disease was prolonged and the mice presented with obvious signs of respiratory illness. balb/c mice were lightly anesthetized and inoculated i.n. with 50 ml 10 2.5 pfu sd/09 virus in pbs. mock-infected animals were inoculated i.n. with 50 ml pbs. at the indicated time, infected mice were sacrificed, and the parameters that present the course of the disease were determined. twenty mice (10 infected with sd/09 virus and 10 inoculated with pbs) were used to investigate clinical signs and mortality for 14 days. three mice were euthanized on days 2, 4, 6, 8, 10 and 14 p.i. and their organs were collected. the collected tissues were weighed, and 10% homogenates were prepared in cold pbs. the homogenates were centrifuged at 3000 rpm for 10 min to remove cell debris, and then the supernatants were 10-fold serially diluted for viral titer determination by plaque assay in mdck cells. virus titers were expressed as mean log pfu/g 6 standard deviation (sd). three mice were euthanized on days 2, 4, 6, 8, 10 and 14 p.i., and the lungs were removed and weighed and then desiccated in an oven at 60uc for 72 h. the lung wet weight:body weight ratio and lung wet:dry weight ratio were calculated and used as an indicator of lung edema, as previously described [40] . three mice were euthanized on days 4, 6, 8 and 14 p.i. the lungs were fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. lungs on day 14 p.i. were also stained with masson's trichrome. lung tissue sections taken on day 6 p.i. were stained for influenza a virus antigens. an anti-influenza nucleoprotein monoclonal antibody (aa5h; abcam, hong kong) was used to identify influenza a virus nucleoprotein in sections. secondary antibody (millipore, billerica, ma, usa) against the primary antibody was labeled with horseradish peroxidase, and the color reaction was developed with a horseradish peroxidase reaction kit (diaminobenzidine-tetrahydrochloride; sigma, st. louis, mo, usa). blood gas analysis was performed as previously described [13, 41] . three mice were anesthetized with zoletil (tiletamine-zolazepam; virbac; 20 mg/g) on days 4, 6, 8, 10 and 14 p.i. arterial blood samples were withdrawn into a heparinized syringe by percutaneous left ventricular sampling of lightly anesthetized mice that were spontaneously breathing room air. blood gas analysis was immediately performed using a vetstat electrolyte and blood gas analyzer (idexx laboratories, westbrook, ma, usa). leukocyte counts in balf were performed as previously described [42, 43] . briefly, three mice were euthanized on days 4, 6, 8 and 10 p.i., and the lungs were lavaged twice in situ with the chest cavity opened by midline incision with a total volume of 1.0 ml saline (4uc) inserted through an endotracheal tube. the rate of recovery of balf was not less than 90% for all animals tested. after the amount of fluid recovered was recorded, an aliquot of balf was diluted 1:1 with 0.01% crystal violet and 2.7% acetic acid for leukocyte staining and erythrocyte hemolysis. the number of leukocytes in the balf was counted with a hemacytometer under a microscope. for differential counts, the balf samples from each mouse were stained with wright stain, and the numbers of monocytes, neutrophil and lymphocytes were determined, on the basis of morphologic criteria, under a light microscope, with evaluation of at least 200 cells per slide. all slides were counted twice by different observers blinded to the status of the animal. heparinized blood samples were collected on days 4, 6, 8 and 10 p.i. the total numbers of leukocytes and differential blood counts for three individual mice were analyzed using an automated hematology analyzer. il-6, il-10, tnf, ifn-c and mcp-1 levels were determined in lung homogenates using a cytometric bead array technique (bd cytometric bead array mouse inflammation kit; bd bioscience, san diego, ca, usa) according to the manufacturer's instructions. briefly, 50 ml mouse inflammation capture bead suspension and 50 ml pe detection reagent were added to an equal amount of sample standard dilution and incubated for 2 h at room temperature in the dark. subsequently, samples were washed by adding 1 ml wash buffer and centrifugation at 2006 g at room temperature for 5 min. supernatants were discarded and 300 ml wash buffer was added. samples were analyzed on a bd facsarray bioanalyzer (bd bioscience) according to the manufacturer's instructions. standard curves were prepared similar to the method above. data were analyzed using bd cba software (bd bioscience). finally, the chemokine or cytokine levels were recorded as pg/ml homogenate. data were analyzed by two-way analysis of variance using graphpad prism version 5.00 (graphpad software, san diego, ca, usa). when a significant effect was observed, pairwise comparisons were performed using the bonferroni post-hoc test. all 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enos-null mice is hyperresponsive to mild hypoxia respiratory reovirus 1/l induction of intraluminal fibrosis, a model of bronchiolitis obliterans organizing pneumonia, is dependent on t lymphocytes role of p38 mitogen-activated protein kinase in a murine model of pulmonary inflammation the authors thank dr. yanxin hu and dr. deping han for technical assistance in histopathologic observation. key: cord-001781-afg1nmib authors: saksena, sumeet; fox, jefferson; epprecht, michael; tran, chinh c.; nong, duong h.; spencer, james h.; nguyen, lam; finucane, melissa l.; tran, vien d.; wilcox, bruce a. title: evidence for the convergence model: the emergence of highly pathogenic avian influenza (h5n1) in viet nam date: 2015-09-23 journal: plos one doi: 10.1371/journal.pone.0138138 sha: doc_id: 1781 cord_uid: afg1nmib building on a series of ground breaking reviews that first defined and drew attention to emerging infectious diseases (eid), the ‘convergence model’ was proposed to explain the multifactorial causality of disease emergence. the model broadly hypothesizes disease emergence is driven by the co-incidence of genetic, physical environmental, ecological, and social factors. we developed and tested a model of the emergence of highly pathogenic avian influenza (hpai) h5n1 based on suspected convergence factors that are mainly associated with land-use change. building on previous geospatial statistical studies that identified natural and human risk factors associated with urbanization, we added new factors to test whether causal mechanisms and pathogenic landscapes could be more specifically identified. our findings suggest that urbanization spatially combines risk factors to produce particular types of peri-urban landscapes with significantly higher hpai h5n1 emergence risk. the work highlights that peri-urban areas of viet nam have higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture than rural and urban areas. we also found that land-use diversity, a surrogate measure for potential mixing of host populations and other factors that likely influence viral transmission, significantly improves the model’s predictability. similarly, landscapes where intensive and extensive forms of poultry production overlap were found at greater risk. these results support the convergence hypothesis in general and demonstrate the potential to improve eid prevention and control by combing geospatial monitoring of these factors along with pathogen surveillance programs. two decades after the institute of medicine's seminal report [1] recognized novel and reemerging diseases as a new category of microbial threats, the perpetual and unexpected nature of the emergence of infectious diseases remains a challenge in spite of significant clinical and biomedical research advances [2] . highly pathogenic avian influenza (hpai) (subtype h5n1) is the most significant newly emerging pandemic disease since hiv/aids. its eruption in southeast asia in 2003-4 and subsequent spread globally to more than 60 countries fits the complex systems definition of "surprise" [3] . in this same year that iom had published its final report on microbial threats which highlighted h5n1's successful containment in hong kong in 1997 [4] , massive outbreaks occurred in southeast asia where it remains endemic, along with egypt's nile delta. since 2003, hpai h5n1 has killed millions of poultry in countries throughout asia, europe, and africa, and 402 humans have died from it in sixteen countries according to who data as of january 2015. the threat of a pandemic resulting in millions of human cases worldwide remains a possibility [5] . lederberg et al. [1] first pointed to the multiplicity of factors driving disease emergence, which later were elaborated and described in terms of 'the convergence model' [6] . the model proposes emergence events are precipitated by the intensifying of biological, environmental, ecological, and socioeconomic drivers. microbial "adaptation and change," along with "changing ecosystems" and "economic development and land use" form major themes. joshua lederberg, the major intellectual force behind the studies summed-up saying "ecological instabilities arise from the ways we alter the physical and biological environment, the microbial and animal tenants (humans included) of these environments, and our interactions (including hygienic and therapeutic interventions) with the parasites" [6] . combining such disparate factors and associated concepts from biomedicine, ecology, and social sciences in a single framework remains elusive. one approach suggested has been to employ social-ecological systems theory that attempts to capture the behavior of so-called 'coupled natural-human systems', including the inevitable unexpected appearance of new diseases, themselves one of the "emerging properties" of complex adaptive systems (cas) [7, 8] . the convergence model can be so adapted by incorporating the dynamics of urban, agricultural, and natural ecosystem transformations proposed with this framework. these associated multifaceted interactions including feedbacks that affect ecological communities, hosts and pathogen populations, are the proximate drivers of disease emergence. the initial hpai h5n1 outbreaks in vietnam represent an ideal opportunity to adapt and test a cas-convergence model. emergence risk should be highest in the most rapidly transforming urban areas, peri-urban zones where mixes of urban-rural, modern-traditional land uses and poultry husbandry coincide most intensely. specifically we hypothesized a positive association between the presence of hpai outbreaks in poultry at the commune level and: 1) peri-urban areas, as defined by saksena et al. [9] , 2) land-use diversity, and 3) co-location of intensive and extensive systems of poultry. we used the presence or absence at the commune level of hpai h5n1 outbreaks in poultry as the dependent variable. vietnam experienced its first hpai h5n1 outbreak in late 2003, since then, there have been five waves and sporadic outbreaks recorded over the years [10, 11] . we chose to study the first wave (wave 1) that ended in february 2004 and the second wave (wave 2) that occurred between december 2004 and april 2005. we used data from the viet nam 2006 agricultural census to develop an urbanicity classification that used data collected at a single point in time (2006) but across space (10,820 communes) to infer processes of change (urbanization, land-use diversification, and poultry intensification) [9] . the 58 provinces in vietnam (not counting the 5 urban provinces that are governed centrally) are divided into rural districts, provincial towns, and provincial cities. rural districts are further divided into communes (rural areas) and towns, and provincial towns and cities are divided into wards (urban subdistricts) and communes. a commune in viet nam is thus the third level administrative subdivision, consisting of villages/hamlets. for the purpose of simplicity we will henceforth use the term "commune" to refer to the smallest administrative unit whether it is a commune, town, or ward. we included risk factors documented in previous work. we also aimed to understand the differences, if any, in risk dynamics at different scales; comparing risks at the national scale to those at two sub-national agro-ecological zones. for this purpose we chose to study the red river and mekong river deltas, well known hot spots of the disease. hence we conducted two sets of analyses (waves 1 and 2) for three places (nation, red river delta, and mekong delta) producing a total of 6 wave-place analyses. data on outbreaks were obtained from the publicly available database of viet nam's department of animal health. given the highly complex dynamics of the epidemics and in keeping with recent methodological trends, we used multiple modeling approaches-parametric and non-parametric-with a focus on spatial analysis. we used both 'place' oriented models that can take into account variations in factors such as policies and administration as well as 'space' oriented models that recognize the importance of physical proximity in natural phenomenon [12] . very few empirical studies have attempted to determine whether urbanization is related to eid outbreaks or whether urbanization is associated primarily with other factors related to eid outbreaks. one immediate problem researchers face is defining what is rural, urban, and transitional (i.e., peri-urban). some studies have used official administrative definitions of urban and rural areas, but this approach is limited in its bluntness [13] . other studies prioritized human population density as a satisfactory surrogate [11, [14] [15] [16] [17] [18] [19] [20] , but this approach ignores the important fact that density is not a risk factor if it is accompanied by sufficient infrastructure to handle the population. spencer [21] examined urbanization as a non-linear characteristic, using household-level variables such as water and sanitation services. he found evidence that increased diversity in water supply sources and sanitation infrastructure were associated with higher incidences of hpai. these studies employed a limited definition of urbanization that lacked a well-defined characterization of peri-urbanization. still other studies have mapped the relative urban nature of a place, a broad concept that is often referred to as 'urbanicity' [22] [23] [24] [25] . while these studies show differences in the rural/ urban nature of communities across space and time, they have been limited to small-to medium-scale observational studies; and they have failed to distinguish between different levels of "ruralness". perhaps the best known model of peri-urbanization is mcgee's concept of desakota (indonesian for "village-town") [26] . mcgee identified six characteristics of desakota regions: 1) a large population of smallholder cultivators; 2) an increase in non-agricultural activities; 3) extreme fluidity and mobility of population; 4) a mixture of land uses, agriculture, cottage industries, suburban development; 5) increased participation of the female labor force; and 6) "grey-zones", where informal and illegal activities group [26] . saksena et al. [9] built on mcgee's desakota concepts and data from the 2006 viet nam agricultural census to establish an urbanicity classification. that study identified and mapped the 10,820 communes, the smallest administrative unit for which data are collected, as being rural, peri-urban, urban, or urban core. this project used the saksena classification to assess associations between urbanicity classes, other risks factors, and hpai outbreaks. researchers have estimated that almost 75% of zoonotic diseases are associated with landcover and land-use changes (lcluc) [27, 28] . lcluc such as peri-urbanization and agricultural diversification frequently result in more diverse and fragmented landscapes (number of land covers or land uses per unit of land). the importance of landscape pattern, including diversity and associated processes, which equate to host species' habitat size and distribution, and thus pathogen transmission dynamics is axiomatic though the specific mechanisms depend on the disease [29, 30] . landscape fragmentation produces ecotones, defined as abrupt edges or transitions zones between different ecological systems, thought to facilitate disease emergence by increasing the intensity and frequency of contact between host species [31] furthermore, fragmentation of natural habitat tends to interrupt and degrade natural processes, including interspecies interactions that regulate densities of otherwise opportunistic species that may serve as competent hosts [32] , although it is not clear if reduced species diversity necessarily increases pathogen transmission [33] . rarely has research connected land-use diversification to final health endpoints in humans or livestock; this study attempts to link land-use diversity with hpai h5n1 outbreaks. human populations in the rapidly urbanizing cities of the developing world require access to vegetables, fruits, meat, etc. typically produced elsewhere. as theorized by von thünen in 1826 [34] , much of this demand is met by farms near cities [35] , many in areas undergoing processes of peri-urbanization [26] . due to the globalization of poultry trade, large-scale chicken farms raising thousands of birds have expanded rapidly in southeast asia and compete with existing small backyard farmers [36] . large, enterprise-scale (15,000-100,000 birds) operations are still rare in viet nam (only 33 communes have such a facility). on the other hand, domestic and multinational companies frequently contract farmers to raise between 2,000 and 15,000 birds. recent studies have examined the relative role of extensive (backyard) systems and intensive systems [15, [17] [18] [19] 37] . in much of asia there is often a mix of commercial and backyard farming at any one location [36] . experts have suggested that from a biosecurity perspective the co-location of extensive and intensive systems is a potential risk factor [38] . intensive systems allow for virus evolution (e.g. low pathogenic avian influenza to hpai) and transformation, while extensive systems allow for environmental persistence and circulation [39] . previous studies of chicken populations as a risk factor have distinguished between production systems-native chickens, backyard chickens; flock density; commercial chickens, broilers and layers density, etc. [15, [17] [18] [19] 37] . in isolation, however, none of these number and/or density based poultry metrics adequately measures the extent of co-location of intensive and extensive systems in any given place. intensive and extensive systems in viet nam have their own fairly well defined flock sizes. a diversity index of the relative number of intensive and extensive systems of poultry-raising can better estimate the effect of such co-location; this study attempts to link a livestock diversity index with the presence or absence of hpai h5n1 outbreaks at the commune level. this study investigated for the 10,820 communes of viet nam a wide suite of socio-economic, agricultural, climatic and ecological variables relevant to poultry management and the transmission and persistence of the hpai virus. many of these variables were identified based on earlier studies of hpai (as reviewed in gilbert and pfeiffer [40] ). three novel variables were included based on hypotheses generated by this project. all variables were measured or aggregated to the commune level. the novel variables were: • degree of urbanization: we used the urbanicity classification developed by saksena et al. [9] to define the urban character of each commune. the classification framework is based on four characteristics: 1) percentage of households whose main income is from agriculture, aquaculture and forestry, 2) percentage of households with modern forms of toilets, 3) percentage of land under agriculture, aquaculture and forestry and 4) the normalized differentiated vegetation index (ndvi). the three-way classification enabled testing for non-linear and non-monotonous responses. • land-use diversity: we measured land-use diversity using the gini-simpson diversity index [41] . the gini-simpson diversity index is given by 1-λ, where λ equals the probability that two entities taken at random from the dataset of interest represent the same type. in situations with only one class (complete homogeneity) the gini-simpson index would have a value equal to zero. such diversity indices have been used to measure land-use diversity [42] . we used the following five land-use classes: annual crops, perennial crops, forests, aquaculture and built-up land (including miscellaneous uses) for which data were collected in the 2006 agricultural census. the area under the last class was calculated as the difference between the total area and the sum of the first four classes. the following variables are listed according to their role in disease introduction, transmission and persistence, though some of these factors may have multiple roles. • human population related transmission. human population density [11, 14-16, 18, 19, 44, 45] . • poultry trade and market. towns and cities were assumed to be active trading places [10, 18, 37, 44, 46] . so, the distance to the nearest town/city was used as indicator of poultry trade. trade is facilitated by access to transportation infrastructure [37, 47, 48] . so, the distance to the nearest a) national highway and b) provincial highway was used as indicator of transportation infrastructure. • disease introduction and amplification. the densities of chicken were calculated based on commune area [15, 19, 37, 49] . • intermediate hosts. duck and geese densities were calculated using total commune area [11, 19, 49] . as previous studies have shown a link between scavenging in rice fields by ducks and outbreaks, we also calculated duck density using only the area under rice. • agro-ecological and environmental risk factors. previous studies have shown that the extent of rice cultivation is a risk factor, mainly due its association with free ranging ducks acting as scavengers [10] . we used percentage of land under rice cultivation as a measure of extent. rice cropping intensity is also a known risk factor [11, 17, 37] . we used the mean number of rice crops per year as a measure of intensity. the extent of aquaculture is a known risk factor [10] , possibly because water bodies offer routes for transmission and persistence of the virus. the percentage of land under aquaculture was used as a metric. proximity to water bodies increases the risk of outbreaks [47, [50] [51] [52] , possibly by increasing the chance of contact between wild water birds and domestic poultry. we measured the distance between the commune and the nearest: a) lake and b) river. climatic variables-annual mean temperature and annual precipitation-have been associated with significant changes in risk [48, 53] . elevation, which is associated with types of land cover and agriculture, has been shown to be a significant risk factor in vietnam [10] . compound topographical index (cti, also known as topographical wetness index) is a measure of the tendency for water to pool. studies in thailand and elsewhere [54] have shown that the extent of surface water is a strong risk factor, possibly due to the role of water in long-range transmission and persistence of the virus. in the absence of reliable and inexpensive data on the extent of surface water we used cti as a proxy. cti has been used in ecological niche models (enm) of hpai h5n1 [55, 56] . however, given the nature of enm studies, the effect of cti as a risk factor has been unknown so far. cti has been used as a risk factor in the study of other infectious and non-infectious diseases [57] . some studies have shown that at local scales, the slope of the terrain (a component of cti) was significantly correlated with reservoir species dominance [58] . cti is a function of both the slope and the upstream contributing area per unit width orthogonal to the flow direction. cti is computed as follows: cti = ln (a s / (tan (β)) where; a s = area value calculated as ((flow accumulation + 1) ã (pixel area in m 2 )) and β is the slope expressed in radians [59] . though previous studies have indicated that normalized difference vegetation index (ndvi) is a risk factor [10, 20, 55, 60, 61], we did not include it explicitly in our models, as the urban classification index we used included ndvi [9] . we obtained commune level data on hpai h5n1 outbreaks from the publicly available database of the department of animal health [10] . viet nam experienced its first major epidemic waves between december 2003 and february 2006 [10] . we chose to study the first wave (wave 1) that ended in february 2004 and the second wave (wave 2) that occurred between december 2004 and april 2005. in wave 1, 21% of the communes and in wave 2, 6% of the communes experienced outbreaks. we used data from the 1999 population census of viet nam to estimate human population per commune. we relied on data from two agriculture censuses of viet nam. this survey is conducted every five years covering all rural households and those peri-urban households that own farms. thus about three-fourths of all of the country's households are included. the contents of the survey include number of households in major production activities, population, labor classified by sex, age, qualification, employment and major income source; agriculture, forestry and aquaculture land used by households classified by source, type, cultivation area for by crop type; and farming equipment by purpose. commune level surveys include information on rural infrastructure, namely electricity, transportation, medical stations, schools; fresh water source, communication, markets, etc. detailed economic data are collected for large farms. we used the 2006 agriculture census for most variables because the first three epidemic waves occurred between the agricultural censuses of 2001 and 2006 but were closer in time to the 2006 census [10] . however, for data on poultry numbers we used the 2001 agriculture census data set because between 1991 and 2003 the poultry population grew at an average rate of 7% annually. however, in 2004, after the first wave of the h5n1 epidemic, the poultry population fell 15%. only by mid-2008 did the poultry population return close to pre-epidemic levels. thus, we considered the poultry population data from the 2001 census to be more representative. we aggregated census household data to the commune level. a three-way classification of the rural-to-urban transition was based on a related study [9] . raster data on annual mean temperature and precipitation were obtained from the world-clim database and converted to commune level data. the bioclimatic variables were compiled from the monthly temperature and precipitation values and interpolated to surfaces at 90m spatial resolution [62] . this public database provides data on the average climatic conditions of the period 1950-2000. elevation was generated from srtm 90 meter digital elevation models (dem) acquired from the consortium for spatial information (cgiar-csi). compound topographical index (cti) data were generated using the geomorphometry and gradient metrics toolbox for arc-gis 10.1. prior to risk factor analysis we cleaned the data by identifying illogical values for all variables and then either assigning a missing value to them or adjusting the values. illogical values occurred mainly (less than 1% of the cases) for land-related variables such as percentage of commune land under a particular type of land use. next we tested each variable for normality using the bestfit software (palisade corporation). most of the variables were found to follow a log-normal distribution and a log-transform was used on them. we then examined the bi-variate correlations between all the risk factors (or their log-transform, as the case may be). correlations were analyzed separately for each place. certain risk factors were then eliminated from consideration when |r| ! 0.5 (r is the pearson correlation coefficient). when two risk factors were highly correlated, we chose to include the one which had not been adequately studied explicitly in previously published risk models. notably, we excluded a) elevation (correlated with human population density, chicken density, duck density, percentage land under paddy, annual temperature and compound topographical index), b) human population density (correlated with elevation and cti), c) chicken density (only at national level, correlated with cti), d) duck and goose density (correlated with elevation, chicken density, percentage land under paddy, land use diversity index and cti), e) annual temperature (correlated with elevation and cti) and f) cropping intensity (correlated with percentage land under paddy). considering the importance of spatial autocorrelation in such epidemics, we used two modeling approaches: 1) multi-level generalized linear mixed model (glmm) and 2) boosted regression trees (brt) [63, 64] with an autoregressive term [65] . glmm is a 'place' oriented approach that is well suited to analyzing the effect of administrative groupings, while brt is a 'space' oriented approach that accounts for the effects of physical proximity. we began by deriving an autoregressive term by averaging the presence/absence among a set of neighbors defined by the limit of autocorrelation, weighted by the inverse of the euclidean distance [65] . the limit of the autocorrelation of the response variable was obtained from the range of the spatial correlogram ρ (h) [66] . to determine which predictor variables to include in the two models, we conducted logistic regression modeling separately for each of them one by one but included the autoregressive term each time. we finally included only those variables whose coefficient had a significance value p 0.2 (in at least one wave-place combination) and we noted the sign of the coefficient. this choice of p value for screening risk factors is common in similar studies [15, 18, 45, 67] . we used a two-level glmm (communes nested under districts) to take account of random effects for an area influenced by its neighbors, and thus, we studied the effect of spatial autocorrelation. we used robust standard errors for tests of fixed effects. boosted regression trees, also known as stochastic gradient boosting, was performed to predict the probability of hpai h5n1 occurrence and determine the relative influence of each risk factor to the hpai h5n1 occurrence. this method was developed recently and applied widely for distribution prediction in various fields of ecology [63, 64] . it is widely used for species distribution modeling where only the sites of occurrence of the species are known [68] . the method has been applied in numerous studies for predicting the distribution of hpai h5n1 disease [16, 51, [69] [70] [71] . brt utilizes regression trees and boosting algorithms to fit several models and combines them for improving prediction by performing iterative loop throughout the model [63, 64] . the advantage of brt is that it applies stochastic processes that include probabilistic components to improve predictive performance. we used regression trees to select relevant predictor variables and boosting to improve accuracy in a single tree. the sequential process allows trees to be fitted iteratively through a forward stage-wise procedure in the boosting model. two important parameters specified in the brt model are learning rate (lr) and tree complexity (tc) to determine the number of trees for optimal prediction [63, 64] . in our model we used 10 sets of training and test points for cross-validation, a tree complexity of 5, a learning rate of 0.01, and a bag fraction of 0.5. other advantages of brt include its insensitivity to co-linearity and non-linear responses. however, for the sake of consistency with the glmm method, we chose to eliminate predictors that were highly correlated with other predictors and to make log-transforms where needed. in the glmm models we used p 0.05 to identify significant risk factors. the predictive performances of the models were assessed by the area under the curve (auc) of the receiver operation characteristic (roc) curve. auc is a measure of the overall fit of the model that varies from 0.5 (chance event) to 1.0 (perfect fit) [72] . a comparison of auc with other accuracy metrics concluded that it is the most robust measure of model performance because it remained constant over a wide range of prevalence rates [73] . we used the corrected akaike information criteria (aicc) to compare each glmm model with and without its respective suite of fixed predictors. we used spss version 21 (ibm corp., new york, 2012) for glmm and r version 3.1.0 (the r foundation for statistical computing, 2014) for the brt. for calculating the spatial correlogram we used the spdep package of r. the fourteen predictor variables we modeled (see tables) were all found to be significantly associated with hpai h5n1 outbreaks (p 0.2) in at least one wave-place combination based on univariate analysis (but including the autoregressive term) ( table 1) . land-use diversity, chicken density, poultry flock size diversity and distance to national highway were found to have significant associations across five of the six wave-place combinations. power of the glmm models, as measured by the auc, is very good with auc values ranging from 0.802 to 0.952 (tables 2-7 ). the predictive power of the national models was higher than that of the delta models. the predictive power of the brt models is good, with aucs ranging from 0.737 to 0.914. the brt models also had a better predictive power at the national level than at the delta level. these values are higher than those reported for wave 1 (auc = 0.69) and wave 2 (auc = 0.77) by gilbert et al. [11] . both gilbert et al. [11] and this study found that at the national level the predictive performance for wave 2 was higher than that for wave 1. wave 2 mainly affected the mekong river delta. previous studies indicated the duck density was an important predictor [11] ; our results, however, indicated that the diversity of duck flock size was a more important predictor than duck density. both the glmm and brt models found annual precipitation to be a significant factor. the glmm model indicated a negative association; similar to what was found by studies in china [51] and in the red river delta [53] . a global study of human cases also found occurrence to be higher under drier conditions [74] . generally, the role of precipitation was found to be far more significant in the deltas than for the country as a whole. the unadjusted relative risk (rr) of peri-urban areas in comparison with non-peri-urban areas was 1.41 and 1.60 for waves 1 and 2, respectively. in terms of urbanicity, we found that chicken density, percentage of land under rice, percentage of land under aquaculture, flock size diversity for duck and geese, and the compound topographical index (cti) to be highest in peri-urban areas (fig 1a-1e) . we also found that land-use diversity was higher in rural areas, but peri-urban areas had diversity levels only marginally lower (fig 1f) . the urbanicity variable alone, however, was not found to be significantly associated with hpai h5n1 in any place according to the glmm model except for the urban level in red river delta for wave 2 and in the mekong river delta for wave 1. the brt model ranked urbanicity as one of the least influential variables. land-use diversity was found to be significantly associated with hpai h5n1 in both waves for viet nam according to the glmm model, but at the delta level the association was significant only for wave 2 in the mekong river delta. the brt model indicated that land-use diversity highly influenced hpai h5n1 at the national level in wave 2. for the remaining waveplace combinations land-use diversity had middle to below-middle rank of influence. both the glmm and brt models indicated that the diversity of chicken flock-size had a strong association with hpai h5n1 for both waves at the national level. this was generally found to be true at the delta levels with some exceptions. the diversity of duck and goose flock size was also significantly associated with hpai h5n1 in all places, but the associations were much stronger in wave 2 than in wave 1. the glmm model indicated that the cti had a very strong association with hpai h5n1 at the national level in both waves although this was not true in the two deltas. the cti is a steady state wetness index commonly used to quantify topographic control on hydrological processes. accumulation numbers in flat areas, like deltas, are very large; hence the cti was not a relevant variable in the glmm model in these areas. the brt model however indicated that cti had middle to low influence in all waves and places. we found very high spatial clustering effects as indicated by the fact that in all waves and places the brt model found the spatial autocorrelation term to have the highest rank of influence. as expected, the relative influence of the autocorrelation term at the national level was higher (60-78%) than at the delta levels (14-35%). in the glmm models we found the akaike information criterion (aic) using the entire set of 14 variables to be much lower than the aics of a glmm model without fixed effects. this indicated that though clustering effects were significant, our theory driven predictor variables improved model performance. a limitation of using surveillance methods for the dependent variable (poultry outbreaks) is that the data may have reporting/detection biases [11] . under-reporting/detection in rural areas as compared to peri-urban areas is possible. we believe that the urbanicity and the shortest distance to nearest town risk factors serve as rough proxies for reporting/detection efficiency. previous studies have tended to use human population density as a proxy for this purpose. in our study we found a strong association between human population density and urbanicity. but we acknowledge that a categorical variable such as urbanicity may provide less sensitivity than a continuous variable such as human population density in this specific context. this study explored the validity of a general model for disease emergence that combined the iom 'convergence model' [6] and the social-ecological systems model [7, 8] , for investigating the specific case of hpai in vietnam. we sought to test the hypotheses that measures of urbanization, land-use diversification, and poultry intensification are correlated with outbreaks in poultry. our results generally support the hypothesis that social-ecological system transformations are associated with h5ni outbreaks in poultry. the results presented here highlight three main findings: 1) when relevant risk factors are taken into account, urbanization is generally not a significant independent risk factor; but in peri-urban landscapes emergence factors converge, including higher levels of chicken densities, duck and geese flock size diversities, and fraction of land under rice or aquaculture; 2) high land-use diversity landscapes, a variable not previously considered in spatial studies of hpai h5n1, are at significantly greater risk for hpai h5n1 outbreaks; as are 3) landscapes where intensive and extensive forms of poultry production are co-located. only one other study has explicitly examined urbanicity in the context of hpai h5n1. loth et al. [17] found peri-urban areas in indonesia were significantly associated with hpai h5n1 cases, even based on multivariate models. our study, however, attempted both to associate hpai h5n1 with degree of urbanicity and to determine the features of peri-urban areas that place them at risk. when those features (i.e., chicken densities, duck and geese flock size diversities, and the fraction of land under rice or aquaculture) are included in multivariate models, the role of the urbanization variable per se diminishes. we found in the main river deltas in viet nam (red river and mekong), urbanization had no significant association with hpai h5n1. this may be due to the fact that the deltas are more homogenous, in terms of urbanization, than the country as a whole. this is the first study to examine land-use diversity as a risk factor for hpai h5n1. measured by the gini-simpson diversity index of the five land-use classes on which data were collected in the 2006 viet nam agricultural census, and the presence or absence of hpai outbreaks at the commune level, our results indicate a strong association between land-use diversity and hpai h5n1 at the national level and in the mekong river delta. this metric captures both the variety of habitats and of the complexity of geospatial patterning likely associated with transmission intensity. our results are similar to what has been observed by studies of other eids using fragmentation metrics (e.g. [75] [76] [77] . this is one of the few studies, however, to link landscape fragmentation to an eid disease in poultry and not just to the vector and/or hosts of the eid. previous studies have focused on poultry production factors such as type of species, size of flocks, and extent of commercialization (e.g. [15, [17] [18] [19] . this study expands on those findings by providing evidence that when intensive and extensive systems of chicken and/or duck and geese production co-exist in the same commune, the commune experiences higher risk of disease outbreak. future studies need to examine the biological causal mechanisms in this context. we suggest that national census data (particularly agricultural censuses) compiled at local levels of administration provide valuable information that are not available from remotely sensed data (such as poultry densities) or require a large amount of labor to map at national to larger scales (land-use diversity). mapping land-use classes at the national scale for local administrative units (i.e., the 10,820 communes in viet nam) is not an insignificant task. future studies, however, could examine the correlation between a census-based metric with metrics derived from remote sensing used to measure proportional abundance of each landcover type within a landscape [78] . vietnam is relatively advanced in making digital national population and agricultural census data available in a format that can be linked to administrative boundaries. while other nations are beginning to develop similar capacities, in the short term the application of this method to other countries may be limited. ultimately, both census and remotely sensed data can be used independently to map the urban transition and diversity of land use; these tools, however, may provide their greatest insights when used together. another important contribution of this study was the discovery of the importance of cti. so far cti had been used only in ecological niche modeling studies of hpai h5n1; the specific role and direction of influence of cti had has so far been unknown. our study, the first to use cti as a risk factor, found it had a large positive influence on hpai h5n1 risk at the national level. previous studies have highlighted the role of surface water extent in the persistence and transmission of the hpai h5n1 virus. these studies measured surface water extent as area covered by water, magnitude of seasonal flooding, distance to the nearest body of water, or other variables that are often difficult to map using remotely sensed data, especially for large area studies. cti on the other hand has the potential to serve as an excellent surrogate which can easily be measured in a gis database. the national and regional (delta) models differed quite considerably, both in terms of performance and significant risk factors. in the deltas we commonly found only chicken density, duck flock size diversity and annual precipitation to be significant. this suggests dynamics of risk at the commune level are strongly dependent on the spatial range of analysis, consistent with another study in the mekong delta [61] . though that study's model initially included three dozen commonly known risk factors, the significant risk factors were limited to poultry flock density, proportion households with electricity, re-scaled ndvi median may-october, buffalo density and sweet potato yield. another study in the red river delta [79] found that in addition to the typical poultry density metrics, only the presence of poultry traders was significant. we speculate that for smaller regions, especially for known hot-spots, the relevant risk factors are those that reflect short-range, short-term driving forces such as poultry trading, presence of live bird markets and wet markets etc. improving model performance for smaller regions would require highly refined and nuanced metrics for poultry trading, road infrastructure, water bodies, etc.-data that are typically not available through census surveys. the differences between the national and regional models suggest that our results can inform planners making decisions at different hierarchical levels of jurisdiction: national, region and local. our study has the potential to inform the design of future research related to the epidemiology of other eids in viet nam and elsewhere. for example, we speculate that in southeast asia, japanese encephalitis, the transmission of which is associated with rice cultivation and flood irrigation [80] , may also show a strong association with peri-urbanization. in some areas of asia these ecological conditions occur near, or occasionally within, urban centers. likewise, hantaan virus, the cause of korean hemorrhagic fever, is associated with the field mouse apodemus agrarius and rice harvesting in fields where the rodents are present [80] . our work has demonstrated that the percentage of land under rice in peri-urban areas and rural areas is similar. hence diseases associated with rice production are likely to peak in peri-urban areas given other risk factors such as land-use diversity, cti, and distance to infrastructure. our poultry flock-size diversity findings may also be relevant to understanding the dynamics of other poultry related infections such as newcastle disease. finally, these results suggest the validity of a general model of zoonotic disease emergence that integrates iom's convergence model with the subsequently proposed social-ecological systems and eid framework. 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on the accuracy of distribution models: ecological phenomenon or statistical artefact? seasonal patterns in human a (h5n1) virus infection: analysis of global cases integrated mapping of establishment risk for emerging vector-borne infections: a case study of canine leishmaniasis in southwest france fragmentation analysis for prediction of suitable habitat for vectors: example of riverine tsetse flies in burkina faso the impact of habitat fragmentation on tsetse abundance on the plateau of eastern zambia spatial pattern analysis program for quantifying landscape structure risk factors of highly pathogenic avian influenza h5n1 occurrence at the village and farm levels in the red river delta region in vietnam factors in the emergence of infectious diseases we thank nargis sultana, university of hawaii, manoa for assistance with compiling a gis database. we thank the following for giving us advice and suggestions on the statistical model key: cord-001447-oi7bkm4z authors: dhanasekaran, sakthivel; biswas, moanaro; vignesh, ambothi r.; ramya, r.; raj, gopal dhinakar; tirumurugaan, krishnaswamy g.; raja, angamuthu; kataria, ranjit s.; parida, satya; subbiah, elankumaran title: toll-like receptor responses to peste des petits ruminants virus in goats and water buffalo date: 2014-11-04 journal: plos one doi: 10.1371/journal.pone.0111609 sha: doc_id: 1447 cord_uid: oi7bkm4z ovine rinderpest or goat plague is an economically important and contagious viral disease of sheep and goats, caused by the peste des petits ruminants virus (pprv). differences in susceptibility to goat plague among different breeds and water buffalo exist. the host innate immune system discriminates between pathogen associated molecular patterns and self antigens through surveillance receptors known as toll like receptors (tlr). we investigated the role of tlr and cytokines in differential susceptibility of goat breeds and water buffalo to pprv. we examined the replication of pprv in peripheral blood mononuclear cells (pbmc) of indian domestic goats and water buffalo and demonstrated that the levels of tlr3 and tlr7 and downstream signalling molecules correlation with susceptibility vs resistance. naturally susceptible goat breeds, barbari and tellichery, had dampened innate immune responses to pprv and increased viral loads with lower basal expression levels of tlr 3/7. upon stimulation of pbmc with synthetic tlr3 and tlr7 agonists or pprv, the levels of proinflammatory cytokines were found to be significantly higher while immunosuppressive interleukin (il) 10 levels were lower in pprv resistant kanni and salem black breeds and water buffalo at transcriptional level, correlating with reduced viralloads in infected pbmc. water buffalo produced higher levels of interferon (ifn) α in comparison with goats at transcriptional and translational levels. pre-treatment of vero cells with human ifnα resulted in reduction of pprv replication, confirming the role of ifnα in limiting pprv replication. treatment with irs66, a tlr7 antagonist, resulted in the reduction of ifnα levels, with increased pprv replication confirming the role of tlr7. single nucleotide polymorphism analysis of tlr7 of these goat breeds did not show any marked nucleotide differences that might account for susceptibility vs resistance to pprv. analyzing other host genetic factors might provide further insights on susceptibility to pprv and genetic polymorphisms in the host. peste des petits ruminants (ppr), also known as ovine rinderpest or goat plague, is an acute, highly contagious viral disease of goats and sheep, caused by the peste des petits ruminants virus (pprv), a morbillivirus in the family paramyxoviridae. the disease is characterized by high fever, nasal and ocular discharges, pneumonia, necrotic and ulcerative lesions of the mucus membranes and inflammation of the gastro-intestinal tract [1] . pprv infection results in great economic losses and affects productivity of sheep and goats subsequent to the global eradication of rinderpest [2] . for example, in 2004, the economic cost of pprv in india was estimated to be 1800 million indian rupees (us$ 39 million) per year [2, 3] . pprv replication and seroconversion has been demonstrated in large ruminants [4] . there is a solitary report on clinical pprv occurring in water buffalo [5] , although it has not been confirmed in later studies. in september 2004, outbreaks of ppr in sudan affected both sheep and camels [6] . ppr is generally considered a more serious disease in goats than sheep, however, increased susceptibility of sheep, goat and outbreaks involving both sheep and goats have been equally reported [2, 3, 7, 8, 9] . goats appear not to be affected in some outbreaks, while sheep suffer with high rates of mortality and morbidity [10] . strain specific virulence of pprv has been reported when the same breed of goats were experimentally infected [11] , and different breeds of goat have been shown to respond differently to infection with the same virus [12] . speciesspecific disease occurrence has been observed with foot and mouth disease, where cattle were highly affected while sheep had less severe infection with the virus [13] . epizootic haemorrhagic disease virus affects cattle but sheep do not suffer from this disease [14] . it is well recognised that ducks were generally resistant to avian influenza virus (aiv) whereas chickens suffer from severe disease with rapid death following infection with highly pathogenic aiv [15] . the reason for this species specificity is unclear at present. the natural susceptibility to pprv in goats could be attributed to several host-derived or virus-derived factors. one such hostderived factor could be the differential presence or distribution of specific viral receptors in these species, such as the signalling lymphocyte activation molecule (slam) that has previously been observed to be associated with pprv and other morbilli viruses such as measles virus and canine distemper virus [16, 17, 18] . host immune mechanisms could also account for this differential susceptibility, although this has not been explored in detail in ruminant species or breeds. toll like receptors (tlr) are type 1 transmembrane proteins expressed in almost all cell types and activate the innate immune system upon sensing pathogen associated molecular patterns (pamps). intracellular tlr that sense viral nucleic acids include tlr3 (double stranded rna), tlr7 and tlr8 (single stranded rna) and tlr9 (cpg motifs in dna) [19] . imiquimod and poly i:c are standard agonists used to induce tlr7 and tlr3 respectively leading to the production of inflammatory cytokines including type i interferons (ifn) and immune cell maturation [20, 21] . tlr are differentially expressed in various tissues and immune cells of water buffalo and goats, and have been shown to induce differential immune responses [22, 23] . the cell specific location and basal expression levels of tlr mrna could indicate the natural pamp load of that tissue as well the innate host resistance to pathogens [24] . in addition to the differential expression profiles of tlr, ligand induced downstream cytokine profiles and/or levels could also play a role in the innate disease resistance of a species or breed. for example, mastitis is an economically important inflammatory disease of the udder that has been shown to be more prevalent in the holstein breed of cows than in jersey cows [25] . this has been linked to temporal differences in the onset and duration of immune responses, including cytokines such as tumor necrosis factor alpha (tnfa) and ifnc, following intramammary inoculation of pathogenic e. coli. india has 23 genetically well characterized indigenous breeds of goats (http://www.icar.org.in/en/node/4688). barbari is a common breed of goat reared for meat and milk production in northern india. tellicherry, kanni and salem black are indigenous breeds of goats prevalent in southern india [26] . outbreaks of ppr have been reported in newly introduced barbari goats to southern india with mortality rates of 16.67% to 65.0% [27] . recently, a severe outbreak of ppr was reported in tellicherry breed of goats with 100% mortality in kids and 87.5% mortality in adults [28] . pprv infection appears to be subclinical in kanni and salem black breeds of goats. similarly, native water buffalo in india are resistant to pprv. although such anecdotal evidence and field observations on differential resistance within goat breeds and water buffalo are available, experimental evidence is lacking for the observed differences in susceptibility. we hypothesized that the differential susceptibility of various indian goat breeds and native water buffalo to pprv could be related to innate immune resistance mechanisms. infection of peripheral blood mononuclear cells (pbmc) from four breeds of goats and water buffalo resulted in differential viral replication kinetics and inflammatory cytokine profile including ifna, ifnc and tnfa with differential activation of tlr3 and tlr7. analysis of single nucleotide polymorphisms (snps) in the complete gene sequences of tlr7 between goat breeds did not show any differences that could account for this. we examined the relative basal expression levels of tlr3 and tlr7 mrna in naive pbmc by quantitative real-time rt-pcr (qrt-pcr) in nine individual animals per breed (n = 9). the kanni and salem black breeds revealed significantly higher basal tlr3 (p,0.001) and tlr7 (p,0.001) transcripts than barbari goats while there were no significant difference (p.0.05) between tellicherry and barbari breeds ( figure 1 ). to understand their contribution to virus replication, pbmc from each of these four goat breeds (n = 5) were infected with 1610 3.0 mean tissue culture infective dose (tcid 50 ) of pprv and the virus load analyzed at 24 h post infection (pi) by qrt-pcr, using primers specific to the pprv-h gene and tcid 50 . pbmc from barbari and tellicherry goats supported significantly (p,0.01) higher pprv replication than those from kanni and salem black (figure 2a ) with the yields being similar in these two breeds. there was a significant reduction in virus yield by one log 10 in kanni/salem black pbmc (p,0.05) compared to barbari/tellicherry goats ( figure 2a ). we then examined if pre-stimulation of pbmc with the tlr agonists poly i:c and imiquimod would result in reduction in pprv replication. a difference of about 6-8 ct values in pprv-h gene mrna levels was observed between unstimulated and poly i:c treated pbmc ( figure 2b ). in the case of pre-stimulation with imiquimod, this difference was about 2-3 ct values for barbari and tellicherry breeds and about 4-5 ct values for kanni and salem black breeds ( figure 2c ). comparison of viral load (pprv-h gene mrna levels and infective viral titres) in all the breeds following imiquimod treatment indicates a significant (p,0.05) reduction of viral load in kanni and salem black breeds than barbari and tellicherry breeds. tcid 50 was not determined for poly i:c treated and pprv infected pbmc since the 40-ct values of pprv h mrna expression levels across these breeds were not significantly different ( figure 2b ). in order to investigate the species specific disease outcome, we compared the permissiveness of goat or buffalo pbmc's to pprv replication in vitro. water buffalo supported significantly lower (about 4.89 fold) replication of pprv than goat pbmc (p,0.001). approximately 2 log 10 difference in the virus yield was evident between buffalo and goat pbmc upon pprv infection ( figure 3a & b). to understand the mechanistic basis for this differential permissiveness for virus replication in different goat breeds and goat vs water buffalo, we analyzed the downstream effector molecules of tlr3 and tlr7 engagement. pbmc stimulated with poly i:c, or imiquimod and further infected with pprv were analyzed for the expression of il1b, il6, il8, il10, il12p40, tnfa, ifnc and ifna mrna ( figure 4a-d) . though upregulation of both pro-and anti-inflammatory cytokines were observed in all goat breeds, levels of tnfa was seen to be consistently higher in kanni and salem black breeds in all treatment groups (poly i:c, imiquimod and pprv). this effect was prominent in the case of pprv infected pbmc, where levels of tnfa and more importantly, ifna and ifnc, were significantly higher in kanni and salem black breeds as compared to barbari ( figure 4c ). significant differences were not observed between these breeds and tellicherry in the levels of pro-inflammatory cytokine mrna. consistent with this, levels of the immunosuppressive cytokine il10 were significantly lower in these breeds than in barbari ( figure 3c ). lower mrna expression levels of il10 in kanni and salem black breeds were also observed on stimulating tlr3 and tlr7 with poly i:c and imiquimod ( figure 4a , b). we further validated these results by cytokine elisa for tnfa ( figure 5a ), ifna ( figure 5b ) and ifnc ( figure 5c ). cytokine levels were similar to the mrna expression profiles observed by qrt-pcr ( figure 5a -c). pprv infected pbmc from kanni and salem black breeds had higher production of tnfa, ifna and ifnc than barbari breed. poly i:c and imiquimod treated pbmc, similarly, showed higher production of tnfa and ifna in kanni and salem black breeds than in barbari suggesting tlr3 and tlr7 engagement by pprv. to determine whether there are differences in the induction of pro-inflammatory cytokines between water buffalo and goats, we examined the levels of these after infecting respective pbmc with pprv. increased mrna levels of il1b and ifna in buffalo pbmc and il10, il12p40 and ifnc in goat pbmcs were observed. ifna activation was greater in buffalo than in goats, 2360 vs 1498 fold, respectively ( figure 4d ). to further confirm this observation, ifna protein levels were assayed from culture to further define the association between differential pprv replication and ifna expression, the ability of ifna in limiting the viral replication was confirmed in cells treated with human recombinant ifna. the mean 40 -corrected ct value of pprv h gene expression in vero cells without ifna treatment was 12.1060.42. upon pre-treatment of vero cells with increasing concentrations of ifna, pprv h gene expression decreased. even the lowest dose of ifna tested (100 ng/ml) could reduce the replication by 451 fold (figure 6 ). to confirm the role of tlr7 signalling in ifna induction by pprv unequivocally, goat and buffalo pbmcs were treated with a predetermined optimum concentration (10 mg/ml) of a tlr7 antagonist irs 661 [] 24 h prior to imiquimod treatment or pprv infection. ifna mrna levels were significantly reduced by irs 661 treatment, when pbmc were stimulated either with imiquimod or pprv ( figure 7a, b) . to further confirm the role of tlr7 induced ifna in limiting virus replication, conditioned medium (cm) from pprv infected cells were used to pre-treat pbmc before virus infection ( figure 7c ). the percent inhibition in virus replication was significantly higher (p,0.01) in cm pretreated buffalo pbmc than goat pbmc ( figure 7c ). to determine whether the differential ifn and pro-inflammatory cytokine production between kanni/salem vs barbari/ tellicherry breeds of goats are dependent on single nucleotide polymorphisms (snps) in tlr genes, we examined the complete gene sequence of tlr7. the tlr7 gene was amplified and seven overlapping pcr amplicons were sequenced to obtain the full length tlr7 gene using the goat tlr7 sequence from genbank as a template (accession # gu289401). the sequences obtained for barbari, tellicherry and kanni goat breeds were submitted to genbank (accession # kc127658, kc127659, kc127660, kc127661, kc127662, kc127663, kc127657). nucleotide variations were detected across goat breeds. sequence analysis revealed five nucleotide changes in the tlr7 coding region and two nucleotide changes in the 39 untranslated region (39utr). changes were observed at nucleotide positions 777 (c/t in salem while c/c in other breeds), 2082 (a/a in salem while a/g in other breeds), 2730 (c/t in barbari while c/c in other breeds), 3006 (a/a in tellicherry while a/g in other breeds) and 3069 (g/a in barbari while g/g in other breeds) that corresponded to amino acid positions 259, 694, 910, 1002 and 1023. however, all of these changes were synonymous and no amino acid changes were observed between different goat breeds. in addition, two changes in the 39utr, at nucleotide positions 151 (g/a in barbari and salem while g/g in other two breeds) and 161 (g/a in barbari while g/g in other breeds) were also observed. following eradication of rinderpest, focus has shifted to pprv as a potential virus for eradication efforts. the single serotype of pprv, availability of an efficacious live attenuated vaccine, sensitive diagnostic tests and the economic impact of this disease, coupled with its ability to spread into new geographical regions, render it an attractive target [30] . ppr was first reported in india in sheep with a lineage iv virus in 1989 [31] , with no further reports until 1996, when a massive outbreak occurred in goats throughout northern india with a lineage iv virus [32] . three live attenuated vaccines, specific to lineage iv pprv strains have so far been tested in india [33] . goats have been reported to be more susceptible to pprv than sheep, while cattle and buffalo do not contract clinical disease [5, 32, 34] . increased mortality in lambs/kids, and increased susceptibility of west african goats, especially dwarf goats, compared to their european counterparts have been documented [11] . differential susceptibility of goat breeds within india have also been reported [35] . host genetic factors, in particular the major histocompatibility complex (mhc) genes, may influence susceptibility to disease. virus recognition can be influenced by genetic mutations in the interaction domains between virus and host receptors. in particular, non-mhc genetic variations in host tlr may cause reduced pathogen recognition and hamper innate immune activation [36] . studies on maedi-visna infection in sheep indicate that breed dependent susceptibility to the disease as well as individual susceptibility within the breed may be defined by specific polymorphisms in tlr7 and tlr8 genes [37] . in a related morbillivirus, snps in tlr 3, 4, 5, 6 and associated signalling molecules like myeloid differentiation primary response gene 88 (myd88) and md2 affected immune responses to the measles vaccine in human subjects [38] . single and double stranded rna are recognized by tlr7/8 and tlr3, respectively. tlr3 is a key sensor of viral infection, as most viruses will produce dsrna at some stage of its life cycle. tlr7 is highly expressed in immune cells like plasmacytoid dendritic cells (pdc), which produce substantial amounts of type i ifns in response to viral rna. in our study, the basal levels of tlr3 and tlr7 were significantly higher in the pbmcs of pprv resistant goat breeds, kanni and salem black. engagement of both tlr3 and tlr7 with the synthetic ligands poly i:c and imiquimod respectively, led to the suppression of pprv rna and infectious virus yield in pbmc of goats. this indicates that tlr3 and 7 play a role in the recognition of pprv rna by goat pbmc, though the role of cytosolic rna sensors like retinoic acidinducible gene 1 (rigi) and melanoma differentiation-associated protein 5 (mda5) have not been analyzed in this study and cannot be ruled out. almost complete abrogation of viral gene expression was observed after stimulation by poly i:c. this may be because poly i:c can also be recognized by other sensors, including rigi, mda5 and protein kinase r (pkr) [39] . if factors other than the receptor expression for pprv determine clinical disease, this should be at the level of virus replication and clearance by innate and adaptive responses. the cell surface receptor for pprv, the slam is expressed at lower levels in buffalo than goats [16] . however, we did see virus replication in infected pbmc although at considerably reduced levels suggesting that the cells of water buffalo are permissive to pprv infection. therefore, we questioned whether these differences are reflected at the level of multi-cycle virus replication. pprv replication in water buffalo pbmc was significantly lower than in goats, possibly because of enhanced type i ifn production in these species upon virus infection. pprv replication in human ifna pre-treated vero cells or in pbmc pre-treated with cm from virus infected cells was significantly lower even at very low doses, confirming the role of type i ifn in limiting virus replication. human ifna has been shown to effectively suppress the replication of bovine viral diarrhea virus and bovine parainfluenza virus [40, 41] . cytokines play a pivotal role in the induction and modulation of immunological responses. tlr signaling events lead to the activation of nuclear factor kapp-light-chain-enhancer of activated b cells (nfkb) and interferon regulatory factor (irf), which switch on expression of a specific panel of pro-inflammatory cytokines and chemokines such as tnfa, il6, il8 and regulated on activation, normal t cells (rantes) [19] . activation of tlr by viruses also results in the production and release of type i ifns [39] . tlr3 and tlr7 engagement by synthetic ligands lead to cytokine expression profiles similar to pprv infection except for a weak il1b, il6 and il8 production in goat pbmc. a predominantly inflammatory cytokine repertoire, with expression of tnfa, ifna and ifnc was observed at both mrna and protein levels. thus, it could be inferred that tlr engagement upon pprv infection results in inflammatory cytokine production via the canonical nfkb pathway and type i ifn production via the activation of irfs [42] . stimulation of tlr7 with synthetic rna oligonucleotides has earlier been shown to induce production of il-12, tnfa and ifnc in pbmc of cattle [43] . interestingly, in our study, ifnc levels were higher in pprv infected pbmc, compared to the engagement of tlr3/7 by their respective agonists. ifnc production by nk cells can be induced by il12 secreted by tlr stimulated dcs [44] . in buffalos, approximately 1.5 fold higher levels of ifna at mrna and protein levels were induced in pbmc compared to goats after infection with pprv suggesting that type i ifn may play a role in limiting virus replication in buffalo. further, we found that tlr7 mediated ifna production is critical because tlr7 antagonist inhibited ifna production both in imiquimod or pprv-treated goat and buffalo pbmc. this effect was more prominent in buffalo pbmc suggesting that tlr7 mediated ifna production determines pprv replication efficiency in this species. consistent with the inflammatory cytokine environment induced by pprv infection, expression of the immunomodulatory cytokine il10 was also observed, but its levels were high in the pprv susceptible goat breeds, barbari and tellicherry. il10 is a key regulatory cytokine with immunosuppressive properties that helps to regulate an uncontrolled inflammatory response [45] . in addition to preventing the maturation of antigen presenting cells, il10 can also regulate the proliferation and differentiation of th1 cells, which induces t cell-dependent suppression of antiviral responses [46, 47] . dexamethasone, a well-known immunosuppressive drug, induces immunosuppression by altering the expression levels of il10 and tnfa [48] . experimental immunosuppression of goat with dexamethasone and challenge with virulent pprv indicated that, immunosuppressed goats had a shorter viremia, more extensive and severe disease advancement, significant decrease in the number of leucocytes, high antigen load in various organs and higher mortality rate than the nonimmunosuppressed goats [49, 50] . taken together, it appears that a higher basal expression of tlr3 and tlr7 in kanni and salem breeds may correlate with increased inflammatory cytokine expression with lower levels of immunomodulatory cytokines leading to an enhanced antiviral state thus affording reduced susceptibility to pprv infection. similarly, in buffalo, the tlr-7 mediated type i ifn response upon infection may afford resistance to pprv. the goat tlr7 gene is 3.4 kb long, with a 3141 nucleotide open reading frame (orf), coding for 1046 amino acids. nucleotide sequence homology studies have shown a close relationship with other ruminant species, particularly sheep tlr7 [51] . earlier studies in 12 indian goat breeds have shown a total of 22 polymorphic sites, out of which 19 were present within the coding region and three in the 39utr [41] . the toll/ interleukin-1 receptor (tir) domain sequence is highly conserved between species, as it plays a crucial role in tlr downstream signaling [52] . in our study, sequence analysis revealed five nucleotide changes in the tlr7 coding region and two nucleotide changes in the 39utr. all the changes were synonymous and it is difficult to establish a correlation with specific snp and altered susceptibility to pprv in the goat breeds examined. there were no differences in the leucine repeat regions of tlr7 between different breeds of goats. though, we were unable to demonstrate a positive association between snp and differential susceptibility to pprv in the goat tlr7 gene, analyses of other immune genes including tlr3 and tlr4 may indicate the relationship between susceptibility to pprv infection and genetic polymorphisms in the host. earlier studies in buffalo tlr7 gene have also reported four different polymorphic positions (a/g-1400, a/g-1480 (d234n), c/t-2029 (l417f), a/g-2640), of which two were non synonymous snp's in leucine rich repeats (lrr) [53] . given the presence of snp's in lrr of buffalo tlr7 gene, which is responsible for sensing the pamp's, these snp's could be associated with differential sensing of pprv. further studies are required to correlate the reported snp's in buffalo tlr7 with observed differential response of buffalo pbmc to pprv. by qrt-pcr assays using sybr green chemistry. fold change in mrna expression induced by imiquimod or pprv stimulation was calculated using mock induced cytokine mrna expression levels as a calibrator. c) pprv replication in goat and buffalo pbmc (at 24 h and 48 h) in the presence of conditioned medium (cm) from pprv infected cells. the expression levels of viral hemagglutinin (h) mrna levels expressed as a percentage inhibition in viral replication in the presence of cm when compared with the control (pbmc+pprv we also found a role for basal and induced tlr3 expression levels in pprv infection in our study. in addition to tlr, variations in downstream intracellular signaling molecules such as myd88 and md2 may also play a role. in conclusion, our studies suggest that higher basal levels of tlr3/7 and augmented innate antiviral immune responses upon infection may afford resistance to pprv infection in kanni and salem breeds of goats compared to barbari and tellicherry breeds. compared to goats, elevated type i ifn levels after pprv infection in water buffaloes may afford reduced virus replication and possibly early virus clearance. lrr prediction revealed goat tlr7 has 21 lrrs and buffalo tlr7 has 15 lrrs. the difference in lrr numbers could be a critical factor in determining the signaling responses of goat and buffalo tlr7. future studies may provide insights into understanding the immunogenetic mechanisms underlying variations in the immune response to pprv. all animal studies have been conducted as approved by the ethics committee of the tamil nadu veterinary and animal sciences university, chennai-600 051, india. apparently healthy, 12-18 month old, barbari, tellicherry, kanni and salem black breeds of goats of either sex were maintained under similar conditions at the livestock research station, tamil nadu veterinary and animal sciences university, india. these animals were not vaccinated against pprv and had no record of any other disease during the course of the study. pbmc from nine animals of each breed were used for tlr mrna expression analysis, while pprv infection and tlr stimulationstudies were carried out on pbmc from five animals of each breed. live attenuated pprv (strain ar-87) was obtained from the department of veterinary microbiology, madras veterinary college, india and has been described elsewhere [31] . imiquimod r837 (2.5 mg/ml) and polyionosinic-polycytidylic acid, a synthetic analog of dsrna (poly i:c) (25 mg/ml) (invivogen, san diego, ca) were diluted in endotoxin-free water. aliquots were tested by the e-toxate kit (limulusam ebocyte lysate assay, sigma aldrich, st. louis, mo) and found to be free of endotoxins. blood was collected aseptically from the jugular vein into sterile ethylene diamine tetra acetic acid (edta) coated vacutainer tubes (becton dickenson, cambridge, uk) and processed for pbmc isolation. briefly, 5 ml of anti-coagulated blood was diluted in equal volumes of rpmi-1640 (invitrogen, paisley, uk) medium containing antibiotic and antimycotic solution, overlaid on 2.5 ml of histopaque, specific gravity 1.077 (sigma aldrich, st. louis, mo) and centrifuged at 15006g for 25 min. mononuclear cells were collected from the interface and washed three times in rpmi-1640 by centrifugation at 2006g for 10 min. viability was determined by trypan blue dye exclusion method. pbmc were stimulated with predetermined doses of tlr3 and tlr7 agonists, poly i:c and imiquimod (r837), respectively. cells were harvested at 24 h for cytokine transcript analysis. in a separate experiment, tlr ligand stimulated pbmc were infected with 10 3.0 tcid 50 of pprv. virus yields from tlr 3/7 stimulated and un-stimulated pbmc were assessed at 24 h pi by sybr green quantitative real time reverse transcription polymerase chain reaction (qrt-pcr), using primers specific for the pprv-h gene [35] . infective virus in the supernatants of pprv infected pbmc cultures were determined on vero cells and expressed as tcid 50 /ml [54] . total rna from pbmcs was extracted using tri reagent solution (sigma-aldrich, st. louis, mo) as permanufacturer's instructions. rna concentration and purity was determined using the biophotometer plus (eppendorf, hamburg, germany). two mg of total rna was reverse transcribed with oligo (dt) 18 primers using the high capacity cdna archive kit (applied biosystems, carlsbad, ca). basal expression levels of tlr3 and tlr7 mrna in pbmc were determined using gene-specific primers and taqman probes (fam-nfq) (applied biosystems, carlsbad, ca) ( table 1) . qrt-pcr was performed in triplicate under the following cycle conditions, 2 min at 50uc, 10 min at 95uc and 40 cycles of 95uc for 15 sec and 60uc for 1 min (applied biosystems 7500 real time pcr system, carlsbad, ca). cytokine gene expression levels were compared by sybr green qrt-pcr using gene specific primers (applied biosystems, carlsbad, ca) (primer sequences available upon request). unstimulated pbmc were used as control. corrected ct was calculated as: ct + (nt -ct')6s/s', where ct is the mean sample ct, nt is the mean of the house keeping genes gapdh/actin from the control group, ct9 is the mean of the gapdh/actin from treatment, s is the target gene slope, and s' is the gapdh/actin slope. the slope values were calculated using serial dilutions of cdna and the respective ct values for each dilution and pcr efficiency (e = 10 21 /slope) was determined. results were expressed as 40-ct values [54, 55] . changes in cytokine expression were expressed as fold change (2 2ddct ) over the respective basal levels of mockinduced pbmcs after normalizing for the endogenous control gene and using the corrected ct. antigen capture elisa kits for tnfa (abdserotec, kidlington, uk), ifna (mabtech, sweden) and ifnc (cusabio biotech, china) were used to determine cytokine concentrations in the culture supernatants of tlr ligand stimulated and/or pprv infected pbmc. elisa was performed according to the manufacturer's instructions and values were obtained spectrophotometrically on an elisa reader (epoch micro-volume spectrophotometer system, biotek, winooski, vt) at 492 nm. mock infected cell culture supernatant served as a control. tnfa and ifnc levels were expressed as the corrected optical density [od] of tlr-ligand stimulated or pprv infected culture supernatants from which the od of mock infected supernatants is subtracted. ifna concentrations in the experimental samples were extrapolated from the values generated from standards. blood samples were collected from barbari, tellicherry, kanni and salem black breeds of goat and genomic dna was isolated using the blood dna isolation kit (biobasic, usa). the concentration of extracted dna was determined using biophotometer plus (eppendorf, germany). seven pairs of overlapping primers were designed to amplify the full-length tlr7 gene (primers available upon request) and the pcr fragments were directly sequenced in both directions using the big dye terminator v3.1 cycle sequencing kit (applied biosystems, carlsbad, ca). sequences were assembled into a 3.4 kb contig, which contained a 3141 bp open reading frame. sequence contigs of tlr7 from each animal were further subjected to multiple alignments to identify nucleotide variations, using the lasergene software (dnastar, madison, wi). heterozygous nucleotides were scored manually across samples from different breeds by visualizing the individual chromatogram in chromas lite 2.01 (technelysium, queensland, australia). each polymorphic nucleotide was further analyzedfor its amino acid position and change. statistical analysis was performed with the graphpad prism software. the 40-corrected ct values of tlr3 and tlr7 mrna, fold changes in the ligand induced cytokine mrna expression, pprv h gene levels and virus yield estimated by tcid 50 determination was compared by two-way anova with bonferroni test for multiple comparisons. elisa values for each cytokine and pprv-h gene levels upon ifna treatment were compared by one-way anova with bonferroni test for multiple comparisons. means were considered significantly different when the p value was ,0.05. peste des petits ruminants prevalence and distribution of peste des petits ruminants virus infection in small ruminants in india global distribution of peste des petits ruminants virus and prospects for improved diagnosis and control the detection of antibody against peste des petits ruminants virus in sheep, goats, cattle and buffaloes isolation of pestes des petits ruminants virus from an outbreak in indian buffalo (bubalus bubalis) current situation of peste des petits ruminants (ppr) in the sudan peste des petits ruminants in ethiopian goats the isolation of peste des petits ruminants virus 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pathogenesis of peste des petits ruminants (ppr) virus infection in goats. microb detection of polymorphism and sequence characterization of toll-like receptor 7 gene of indian goat revealing close relationship between ruminant species structural basis for signal transduction by the toll/interleukin-1 receptor domains sequencing, characterization and phylogenetic analysis of tlr genes of bubalus bubalis a simplified objective method for quantification of peste des petits ruminants virus or neutralizing antibody rapid detection of newcastle disease virus replication in embryonated chicken eggs using quantitative real time polymerase chain reaction we gratefully acknowledge the funding received through virginiatech's open access subvention fund to defray the cost of open access publication of this manuscript. this work was partially supported through bbsrc-dbt fund # bb/l004801/1 ---awarded to sp and gdr.] key: cord-000131-ugbwvy6j authors: jones, james holland; salathé, marcel title: early assessment of anxiety and behavioral response to novel swine-origin influenza a(h1n1) date: 2009-12-03 journal: plos one doi: 10.1371/journal.pone.0008032 sha: doc_id: 131 cord_uid: ugbwvy6j background: since late april, 2009, a novel influenza virus a (h1n1), generally referred to as the “swine flu,” has spread around the globe and infected hundreds of thousands of people. during the first few days after the initial outbreak in mexico, extensive media coverage together with a high degree of uncertainty about the transmissibility and mortality rate associated with the virus caused widespread concern in the population. the spread of an infectious disease can be strongly influenced by behavioral changes (e.g., social distancing) during the early phase of an epidemic, but data on risk perception and behavioral response to a novel virus is usually collected with a substantial delay or after an epidemic has run its course. methodology/principal findings: here, we report the results from an online survey that gathered data (n = 6,249) about risk perception of the influenza a(h1n1) outbreak during the first few days of widespread media coverage (april 28 may 5, 2009). we find that after an initially high level of concern, levels of anxiety waned along with the perception of the virus as an immediate threat. overall, our data provide evidence that emotional status mediates behavioral response. intriguingly, principal component analysis revealed strong clustering of anxiety about swine flu, bird flu and terrorism. all three of these threats receive a great deal of media attention and their fundamental uncertainty is likely to generate an inordinate amount of fear vis-a-vis their actual threat. conclusions/significance: our results suggest that respondents' behavior varies in predictable ways. of particular interest, we find that affective variables, such as self-reported anxiety over the epidemic, mediate the likelihood that respondents will engage in protective behavior. understanding how protective behavior such as social distancing varies and the specific factors that mediate it may help with the design of epidemic control strategies. an ongoing outbreak of novel influenza a(h1n1), colloquially referred to as ''swine flu,'' has caused over 200,000 confirmed cases (as of 28 august 2009 [1] ). because of under-reporting, the actual number of people infected is substantially larger, particularly considering that many countries have ceased testing for a(h1n1) [1] . as human-to-human transmission became widespread in at least one region of the world, who rapidly declared the outbreak an imminent pandemic [2] and with widespread human infection, who declared a phase 6 pandemic on 11 june 2009, where it remains at the time of submission [3] . the virus appears to have a higher reproduction number and somewhat higher case fatality ratio than recent seasonal influenza viruses [4, 5] , and has certainly caused great concern in the population, fueled by both extensive media coverage and an initially high level of uncertainty about mortality rates and transmissibility of the virus. mathematical and computational models are useful for predicting the fate of an epidemic, and while such models have become increasingly complex and realistic in recent times, a key ingredient is often ignored: human behavioral responses to the threat and/or presence of a disease [6] . how people assess risk of infection and how such risk assessment drives behavioral change is of great interest as individual social distancing can greatly affect the spread of an epidemic [7, 8, 9] . while the effect of behavioral change in response to perceived health threats on the spread of infectious diseases has been investigated theoretically for some time, particularly in the context of sexually transmitted diseases [8] , recent work has started addressing the problem in a broader context that is also applicable to the spread of influenza [6, 7] . this work has a strong, though as yet under-explored relationship to work on risk perception and health threats [10, 11, 12] . data on risk perception and behavioral response in the general population have rarely been collected right from the outset of an epidemic. instead, they are usually gathered with a substantial delay in the case of influenza a(h1n1) [13] , after the epidemic has run its course, as in the case of sars [9] , or before sustained human to human transmission is established, as in the case of highly pathogenic avian influenza a(h5n1) [14] . however, the feasibility of halting or mitigating the spread of an infectious disease is highest during the very early phases of an outbreak, and thus data on behavioral response during this time would provide valuable information for public health policy and research. here, we report the results from an online survey that gathered data (n = 6,249) about risk perception of the outbreak during the first few days of widespread media coverage (april 29 -may 5, 2009) of the emergence of novel swine-origin influenza a(h1n1). the research presented here was approved by the stanford university non-medical human subjects institutional review board on 28 april 2009 (protocol #16782). we fielded in internet-based survey starting on 29 april 2009 using opinio survey software [15] . the url for the survey is (https://opinio.stanford.edu/opinio/s?s = 1403). the sampling universe for this study was adults 18 and older with access to a networked computer. the initial seed for the sample was generated using social networking software, and a request sent to a standing subject pool comprised of stanford alumni and social science students at a nearby community college maintained by the institute for research in the social sciences at stanford university. the survey was picked up by a variety of internet media sources including several science general media blogs. directly following publicity in these blogs, we received the most responses. table 1 summarizes the sample. the survey was designed to get a rapid assessment of respondents' affective state, sources of information on the emerging pandemic, and the behaviors undertaken for protection while minimizing respondent burden. as such, it included only 17 questions. questions probing subjective assessment of risk perception, level of anxiety, and ability to avoid flu infection were asked on a 9 point ordinal scale with anchors at the extrema (''very high'', ''very low'') and the center (''intermediate''). subjective emotional status (i.e., respondents' affective state regarding the epidemic) was anchored by the terms ''very calm'' through ''intermediate'' to ''very anxious.'' comparative questions of subjective risk percep-tion for eight health threats were asked using a five-point ordinal scale with anchors at all points: ''very low,'' ''low,'' ''intermediate,'' ''high,'' and ''very high.'' questions regarding media (both respondents' frequency of getting information from a particular source and their judgment of each source's accuracy) were asked on a five point ordinal scale with anchors at all points (''very often/ accurate'' to ''never/almost certainly inaccurate''). respondents' knowledge of swine flu was assessed with a series of six true/false questions. respondents gave free-text responses to questions about their current age, the number of people currently living in their household (including themselves) and their zip code if they currently live in the united states. respondents who reported not currently living in the united states were asked to report their current country of residence in a free-text field. a screen-shot of the full survey instrument is included in the supplementary material. for our analysis of participants' response to the threat of swine flu, we use a variable we call ''survey day.'' the survey went online in the evening of 28 april, pacific time, so we combined responses from 28 and 29 april into a single day. this combined day of 28-29 april represents survey day 1. subjects were asked to state the number of contacts in the past 24 hours. contacts were defined by close physical contact as operationalized by a face to face conversation of more than two words in the presence of another individual or physical exposure involving skin contact such as a handshake, hug, or contact during sporting activities. respondents were provided five ordered categories: less than 5, 5-10, 11-20, 21-50, 51-100, more than 100. handcock and jones [16] discuss the phenomenon of heaping and related problems for statistical inference in answering epidemiological questions regarding contact number. structuring responses within broad ordinal categories avoids many of the pitfalls of contact-heaping encountered in epidemiological investigations. to measure the response in epidemiologically relevant behavior to information on the potential pandemic, we asked a series of questions about protective actions taken by the respondents. in the survey, we asked: ''given the current status of the epidemic, which of the following precautionary actions will you take?'' avoid people who cough/sneeze avoid large gatherings of people wash hands more often avoid people who are in contact with infected people avoid public transportation avoid school/work avoid travel to infected areas use disinfectant wear a mask not all of these behaviors are necessarily effective or recommended protective measures (e.g., wearing a mask), but our aim was to gauge people's attempt at self protection so even non-efficacious behavioral change is interesting in that it indicates willingness to act on the part of the respondent. we constructed an index of protective behavior by summing the answers to the questions regarding actions taken to avoid influenza infection. the index ranged from 0-9, with larger values indicating more protective measures taken. using a binomial glm with canonical logit-link, we modeled the protection index as a function of covariates. our primary interest was the possible mediating effect of affective variables on action taken to protect against swine flu infection. to evaluate the hypothesis that respondents' affective state (subjective anxiety, fatalism about infection) predicts protective measures, we include in the model demographic (age, gender), epidemiological (household size, number of contacts, survey day), and media (source of information on the outbreak) conditioning variables. for the media variables, we constructed dummies with a value of 1 corresponding to answers of ''very often'' or ''often'' and a value of 0 for all other responses to the question of ''how often do you use the following sources to get information about swine flu?'' with such a large number of conditioning variables, in addition to the affective variables of greatest interest, there is a distinct danger of overfitting the glm. to address this problem, we used likelihoodbased model selection [17] to search the model space set up by our conditioning variables [18] . of the nine protective behaviors, increased hand-washing is both the simplest and probably most effective at curbing transmission. as such, it is strongly advocated in infection control educational material [19] . in addition to our tests for predictors of the protection index, we therefore also tested the effect of measured covariates on the odds of increased hand washing using a binomial glm again with canonical logit-link. a concern regarding the relationship of people's self-reported anxiety and their protective behavior is that some people might generally be more anxious regarding health. we probed general anxiety by asking about respondents' anxiety with regard to a number of infectious, chronic, and violent threats to health. we asked a series of questions probing respondents' perceived subjective risk on a 5-point scale for a variety of health threats, including other infectious diseases (a(h5n1) ''bird flu'', seasonal flu, hiv/aids), chronic diseases (heart disease, diabetes, cancer), and violence (unintentional accidents, terrorism). we calculated the correlation matrix for answers to these threat questions and used principal components analysis (pca) to explore potential structure in the responses to different categories of threat [20] . we begin by presenting descriptive results of the survey and follow with our primary analytical questions from the survey, namely, testing the hypothesis that respondents' affective state mediates their protective action. we gathered 6,249 responses from 28 april to 5 may 2009. table 1 presents descriptive statistics of the sample. figure 1 presents the distributions of respondents' contacts within the 24 hours prior to taking the survey. figure 2 presents the means of the subjective threats. swine flu had a mean second only to injury, and the highest among the infectious sources of threat. the mean of perceived threat from swine flu fell above the bonferroni-corrected 95% confidence interval for all other threats but unintentional injury. figure 3 presents the frequency distribution of perceived personal risk. there is a notable bimodality to this plot. this apparent bimodality is not simply attributable to sampling error since the difference between the responses = 4 vs. those = 5 vs. those = 6 is in excess of 300. further analysis using finite mixture models [21] provides strong statistical support for the reality of the bimodal pattern (results not shown). while the majority of respondents felt that their personal risk was low, there is a second mode rating their risk as intermediate ( = 5) . this same bimodal pattern can be seen in the frequency distribution of personal empowerment (i.e., ability to avoid infection) shown in figure 4 . while most respondents indicate that they are confident they can avoid infection, a substantial second mode appears at the intermediate value. figure 5 shows the frequency distribution of protective behaviors. we can see that nearly 80% of respondents report washing hands more frequently, while very few avoid work or school or wear protective masks. figure 6 shows the means for respondents' information sources. not surprisingly, the most common source of information reported was the internet. again, mean values are plotted with their 95% bonferroni-corrected confidence intervals. with the exception of social-networking tools (e.g., facebook, twitter), all other media sources are statistically indistinguishable from each other, with the social-networking tools being used significantly less. the results of the model for the protection index show a number of robust trends (table 2). in particular, we find that age increases and male gender decreases the protection index. receiving a large amount of information from the internet, television, and health officials all increase the protection index while receiving large amounts of information from print media, friends, or social networking media has no effect. the number of household members has no discernible effect, though the number of contacts outside the home does. for the ordered factor ''contacts,'' the first category (,5 contacts in the past 24 hours) is the reference category. interestingly, relative to respondents with the fewest number of contacts, all other contact categories have reduced protection indices, indicating that people with fewer contacts take more protective actions. not surprisingly, residence in mexico has a large positive effect, while residence in canada or europe decreased the index. the day that the survey was taken (29 april = 1) had a negative effect on the index, indicating that respondents took less protective action as the epidemic proceeded. respondents' reported subjective anxiety has a substantial impact on the index with high anxiety increasing protection, supporting our hypothesis that affective state mediates protective behavior. increased hand-washing showed similar trends to the model for the protection index (table 3) . male gender decreases while age and survey day increase the odds of increasing hand-washing. receiving a large amount of information from the internet, radio, television, and health officials increase, while living in europe or australia/new zealand decrease the odds. as with the overall protection index, perception of risk and subjective anxiety significantly increase the odds of increased hand-washing modestly. a key epidemiological question is how people's affective status and protective behaviors undertaken change as the epidemic proceeds. to develop a measure for this, we cross-tabulated individual values of the protection index and affective status by survey day. pearson's chi-square test for independence of both tables was strongly significant (affective: x 2 = 135.6, df = 48, p,0.001; protection: x 2 = 113.1, df = 54, p,0.001), indicating substantial departure of cells from the expected values. to visualize the pattern of departure from the expected values, we calculated an expected tables taken as the cross-product of the marginals of the observed table normalized by the grand sum. we combined rows of these tables to simplify the presentation, plotting the difference between observed and expected tables for a high, medium and low emotional status/protection index respectively. for example, a value of 251 on the calm affective status on day one means that there were 51 fewer responses in the calm categories than would be expected by the overall marginal distribution of responses across all days. in figures 7 and 8, we plot the change in respondent's protective behavior and emotional status over the first week of the survey. the lines represent the differences between observed and expected frequencies of responses for the 9-point scale simplified to three levels each. we see that by day three of the survey (may 1st), the relative number of people reporting a calm emotional state was very high, while the number of people reporting high values of the protective index declined dramatically. we interpret these results to indicate that people's response to a potential pandemic is quite sensitive to media reports. in general, individuals' survey responses to perceived risk for the eight health threats were only moderately correlated, with pairwise correlations typically well under 0.5. pca did not reveal that a substantially reduced number of dimensions explained these correlated data -six principal components were required to explain 85% of the variance. nonetheless, some intriguing pc loadings revealed themselves. in particular, the second pc, which explained 15.6% of the variance in the data, showed strong positive correlations with swine flu (r = 0.516), bird flu (r = 0.530), and terrorism (r = 0.467). all three of these threats receive a great deal of media attention and their fundamental uncertainty are likely to generate an inordinate amount of fear vis-a-vis their actual threat [22] . our results indicate that respondents' behavior varies systematically with covariates from demographic, epidemiological, media, and affective domains. people's anxiety about swine flu and the preventative actions they took to avoid infection declined as the perceived gravity of the novel outbreak waned. overall, subjective risk perception was low and people's belief in their ability to avoid infection was high. both of these distributions nonetheless showed a marked bimodality, with a large proportion of respondents indicating a higher subjective risk and more protective actions taken than the majority (figures 3-4) . the results of our statistical modeling suggest that respondents' deployment of protective behavior is mediated by their subjective anxiety level, controlling for demographic, epidemiological, and geographic variables. there is good and bad news in this result. the literature on risk perception and public health shows that there is generally a very weak correlation between people's anxiety over a particular risk and the probability of death or disability arising from that risk [11, 12] . overall, it is unclear whether anxiety over perceived risk will lead to efficacious protective behaviors [10] . this said, by far the most common protective behavior reported in our survey was increased hand-washing, which has been shown to be effective at removing influenza a(h1n1) virus from subjects' hands [23] and is promoted by cdc and other health organizations as an effective infection control intervention [19] . one curious result from the model for the protection index is that people with the fewest contacts have marginally higher protection indices. there are two potential explanations for this finding: (1) individuals with small social support networks (and consequently, few contacts outside the home) are more anxious, making it more likely that they will take greater protective actions or (2) people concerned about infection and taking relatively many protective actions also reduce the number of contacts they have and therefore had a small number of contacts in the past 24 hours. the first explanation is consistent with work in social epidemiology on the role of social networks in mediating infection risk [24, 25] because of the nature of the sample, we are unable to evaluate the direction of causality that leads to this result. nonetheless, it remains an intriguing hypothesis. many questions about this novel swine-origin influenza a(h1n1) virus remain. of particular concern is the possibility that the virus could mutate during the flu season in the southern hemisphere and selection could drive it to become more virulent as it returns to the northern hemisphere in autumn. worryingly, such a pattern of multiple waves with an increased proportion of the total influenza-associated mortality burden has been reported for all three past influenza pandemics [26, 27] . finding a means to simultaneously communicate to the public the structural uncertainty inherent in projecting pandemics and the seriousness of a pandemic after the media frenzy about its emergence has died down remains a major challenge to public health. pharmaceutical interventions such as vaccines and antiviral drugs may form a strong line of defense, but the efficacy of such measures remains unclear and depends on the particular biology of a given pathogen. this is exacerbated by people's reluctance to be vaccinated [28] . with more than 300 infectious diseases emerging within less than a century [29] , the threat of pandemic influenza is only the latest out of many public health threats posed by infectious diseases in a globalized world. unlike pharmaceutical interventions, non-pharmaceutical interventions like social distancing may be effective in halting or at least mitigating an epidemic independent of the specific biology of a pathogen, and thus provide a reliable set of strategies to combat infectious diseases that warrant further attention [30] . our results that people do not rely on social networking tools to the extent that they rely on other media may have implications for cdc strategies for spreading public health information via social media [19] . in particular, public health messages spread via social media will need to backed up by information spread via more traditional channels, which respondents list as being common sources of trusted information on the outbreak. our study is subject to a number of weaknesses. the advantage of our internet-based sampling strategy is the ability to quickly deploy a survey and thereby track responses in near real-time. the clear disadvantage of this strategy is a sacrifice of population representativeness. despite its general availability on the internet, our sample shows a pronounced bias for highlyeducated respondents living in the western united states. these biases clearly limit the generality of our results, but the large number of respondents filing out the survey as information on the potential pandemic changed nonetheless provides a uniquely valuable data source. within one week (the cutoff point for the current analysis), we had a sample of 6,249 responses. in contrast, the telephone-based study of rubin et al. [13] employed a random-digit-dial sampling design, allowing a more representative sample of the general uk population, but their sample was only 997 respondents and the survey was undertaken after media attention had abated, beginning 8 may 2009. nonetheless, the results reported in this paper are largely congruent with our own results and we see the studies as strongly complementary. our respondents began filling out the survey on the day that who upgraded the pandemic threat category of the h1n1 outbreak from 4 to 5 and spans the week in which the number of who-confirmed cases increased tenfold. while our sampling design is subject to many of the usual criticisms of internet-based surveys and is not necessarily representative of the general population, the unparalleled immediacy, longitudinal nature, and the large number of respondents it contains make our data set unique and scientifically important for the study of the spread of information and distribution of risk perception and behavioral change during the most uncertain time (i.e. the initial phase) of an epidemic of a virus novel to the human population. pandemic (h1n1) 2009 -update 63 influenza a (h1n1): pandemic alert phase 6 declared, of moderate severity pandemic potential of a strain of influenza a (h1n1): early findings. science a preliminary estimation of the reproduction ratio for new influenza a(h1n1) from the outbreak in mexico the spread of awareness and its impact on epidemic outbreaks modeling targeted layered containment of an influenza pandemic in the united states a tale of two futures: hiv and antiretroviral therapy in san francisco the impact of community psychological responses on outbreak control for severe acute respiratory syndrome in hong kong the social amplification of risk: a conceptual framework relative risk in the news media: a quantification of misrepresentation medicine in the popular press: the influence of the media on perceptions of disease public perceptions, anxiety, and behaviour change in relation to the swine flu outbreak: cross sectional telephone survey avian influenza risk perception likelihood-based inference for stochastic models of sexual network evolution model selection and inference: a practical information-theoretic approach modern applied statistics with s social media at cdc-novel h1n1 flu research methods in anthropology: qualitative and quantitative approaches finite mixture models dealing the with the dangers of fear: the role of risk communication efficacy of soap and water and alcohol-based hand-rub preparations against live h1n1 influenza virus on the hands of human volunteers assessing the physical health-effects of social networks and social support social networks, host-resistance, and mortality -9-year follow-up-study of alameda county residents influenza: the mother of all pandemics the signature features of influenza pandemics -implications for policy willingness of hong kong healthcare workers to accept pre-pandemic influenza vaccination at different who alert levels: two questionnaire surveys global trends in emerging infectious diseases interim pre-pandemic planning guidance: community strategy for pandemic influenza mitigation in the united states-early, targeted, layered use of nonpharmaceutical interventions. atlanta: department of health and human services, cdc we thank chris thomsen, karen cook, and especially vijoy abraham from the stanford institute for research in the social sciences for help getting the survey online so quickly. dan salkeld provided helpful comments. we are also very grateful to two anonymous reviewers for substantially improving the original manuscript. conceived and designed the experiments: jhj ms. analyzed the data: jhj ms. wrote the paper: jhj ms. key: cord-001359-c1uom5f7 authors: oslund, karen l.; zhou, xu; lee, boram; zhu, lingxiang; duong, trang; shih, robert; baumgarth, nicole; hung, li-yin; wu, reen; chen, yin title: synergistic up-regulation of cxcl10 by virus and ifn γ in human airway epithelial cells date: 2014-07-17 journal: plos one doi: 10.1371/journal.pone.0100978 sha: doc_id: 1359 cord_uid: c1uom5f7 airway epithelial cells are the first line of defense against viral infections and are instrumental in coordinating the inflammatory response. in this study, we demonstrate the synergistic stimulation of cxcl10 mrna and protein, a key chemokine responsible for the early immune response to viral infection, following treatment of airway epithelial cells with ifn γ and influenza virus. the synergism also occurred when the cells were treated with ifn γ and a viral replication mimicker (dsrna) both in vitro and in vivo. despite the requirement of type i interferon (ifnar) signaling in dsrna-induced cxcl10, the synergism was independent of the ifnar pathway since it wasn’t affected by the addition of a neutralizing ifnar antibody or the complete lack of ifnar expression. furthermore, the same synergistic effect was also observed when a cxcl10 promoter reporter was examined. although the responsive promoter region contains both isre and nfκb sites, western blot analysis indicated that the combined treatment of ifn γ and dsrna significantly augmented nfκb but not stat1 activation as compared to the single treatment. therefore, we conclude that ifn γ and dsrna act in concert to potentiate cxcl10 expression in airway epithelial cells via an nfκb-dependent but ifnar-stat independent pathway and it is at least partly regulated at the transcriptional level. influenza pneumonia remains a major cause of morbidity and mortality worldwide. airway epithelial cells are the first line of defense against viral infections in the lung and are instrumental in coordinating the early inflammation leading to an adaptive immune response. cxcl10 (ifn gamma inducible 10 kda protein, ip10) is a non-elr cxc chemokine with potent biological effects including monocyte stimulation, natural killer and activated t cell migration, modulation of adhesion molecule expression, inhibition of angiogenesis [1] as well as antimicrobial effects at high concentrations [2] . the role of cxcl10 in viral pneumonia has not been thoroughly characterized but evidence suggests it is important for the migration of nk cells, macrophages, t cells, neutrophils and plasmacytoid dendritic cells into the lung [3, 4] . in a mouse model of rsv infection, antibody-mediated neutralization of cxcl10 resulted in a significant increase in disease symptoms including impaired viral clearance, reduced pulmonary dendritic cell numbers and maturation and a reduction in viral specific cd8(+) t cells [5] . synergistic up-regulation of cxcl10 has been described in vitro in several cell types and in response to different pro-inflammatory molecules but always in conjunction with ifn c. tnfa and ifn c induce synergistic levels of cxcl10 in a human endothelial cell line hmec-1 via an erk dependent pathway [6] , il-1b and ifn c in human intestinal epithelial cell lines [7] , pdgf and ifn c in blood derived macrophages [8] , tnfa and ifn c in primary human airway smooth muscle cells and gastric epithelial cells [9, 10] , prolactin and ifn c [11] as well as substance p and ifn c in human keratinocytes [12] and hyaluronan fragments with ifn c in mouse macrophages [13] . interestingly, synergistic induction of cxcl10 has been described with ifn c in conjunction with hiv-1 in human astrocytes and macrophages and thought to play a role in hiv induced encephalopathy [14, 15] . this marked variety of pro-inflammatory molecules that elicit a synergistic response of cxcl10 in a wide variety of cell types is indicative of a highly conserved and likely biologically important cellular response. however, synergistic induction of cxcl10 in response to influenza and ifn c in airway epithelial cells has not been previously reported. in this study, we demonstrate synergistic induction of cxcl10 in well differentiated primary human bronchial epithelial (hbe) cells following influenza virus infection and the treatment with ifn c. we further demonstrate that this synergy was mediated by the interaction between dsrna (an intermediate of viral replication) and ifn c in vitro and in vivo. in the follow-up mechanistic study, this synergism was found to be transcriptionally regulated as demonstrated by a chimeric promoter reporter gene assay. in addition, it was not dependent of the ifnar pathway as neither the neutralizing antibody to ifnar nor the ifnar deficiency affected the synergism. finally, nfkb, but not stat1, appeared to mediate this synergism. culture and treatments of primary human bronchial epithelial (hbe) cells human bronchial tissues were purchased from the commercial source (national disease research interchange, philadelphia, pa) as described before [16, 17] . no tissues from patients diagnosed with lung-related diseases were used. all those lungs were either autopsy leftovers or were rejected for transplant. they were sent to us with arbitrary numerical code. no identity link to the actual patient can be identified. protease-dissociated hbe cells were plated on transwellh chambers (corning) and were maintained in immersed culture conditions until they reached confluence when they were transferred to an air-liquid interface (ali) culture condition [16, 17] . at air-liquid interface, the cells were maintained in a ham's f12/dmem (1:1) with the addition of transferrin (5 mg/ml), insulin (5 mg/ml), cholera toxin (10 ng/ml), epidermal growth factor (10 ng/ml), dexamethasone (0.1 mm), bovine hypothalamus extract (15 mg/ml), bsa (0.5 mg/ml) and all-transretinoic acid (30 nm). cells were maintained at ali for 7 days and were placed in basal media devoid of the additives, with the exception of retinoic acid, overnight prior to the experiments. recombinant ifn c was purchased from r&d systems and synthetic double stranded (ds) rna (i.e. poly i:c) from emd biosciences. ifn c was used at 50 ng/ml and poly i:c at 25 mg/ ml. a neutralizing antibody to ifnar was obtained from us biologics and used at 2.5 mg/ml [18] . both ikb kinase and jak inhibitors were purchased from emd biosciences and used at 5 mm. for influenza infection, cells were infected with the influenza virus strain mem at 1200 hau. for in vivo study, balb/c mice were purchased from charles rivers laboratories and used at 6-8 weeks of age for intratracheal instillations. for primary airway epithelial cell cultures, ifnar null mice were a kind gift from dr. nicole baumgarth (ucd) and the wild-type controls mice were c57bl/6j. mice were used at 8-10 weeks of age. all protocols described were approved by the university of california, davis and university of arizona, which were responsible for the proper care and use of experimental animals. the protocol was performed as described previously [19] . briefly, at the time of necropsy, the chest and cervical region were exposed. a small puncture was placed in the proximal trachea to allow cannulation with sterile 0.86 mm polyethylene tubing (intramedic clay adams) which was secured in place with a 3.0 silk suture. a loose suture was placed at the distal end of the trachea just proximal to the carina. the trachea was dissected free and immediately placed in dmem at 4uc. and each trachea gently inflated with 0.2% protease through the tracheal cannula after tightening the distal suture. dissociated epithelial cells were gently harvested by injecting 5 milliliters of cell culture media through the trachea. mte cells from all tracheas were pooled and re-suspended in cell culture media prior to plating on transwellß chambers (corning) coated with purcolß (advanced biomatrix). mte cells were maintained in submerged conditions until confluent at which time they were kept in air-liquid interface conditions for one week in the presence of 100 nm retinoic acid. for the experiments, mte cells were treated with 50 ng recombinant murine ifn c (r and d systems) and/or 25 mg dsrna for 24 hours. the protocol was performed as described previously [20, 21] . briefly, balb/c mice were anesthetized with isoflorane. in dorsal recumbancy, the tongue was gently pulled out, and a pipette tip was gently inserted into their laryngeal region through the oral cavity. 2 mg of dsrna and/or 5 mg of recombinant murine ifn c in 50 ml of lps free pbs was deposited and breathed in. lps free pbs was used as the control. mice were kept in an upright position until recovery from anesthesia. twenty four hours later, mice were euthanized, and lungs were lavaged with 1 ml pbs. all lungs were flash frozen in liquid nitrogen for subsequent rna isolation. total rna was extracted with trizolß reagent (invitrogen) and cdna was generated from an equal amount of rna (1 mg per reaction) by moloney's murine leukemia virus-reverse transcriptase (applied biosystems) using random hexamers (invitrogen). sybr green master mix (roche applied science) and the abi7900ht detection system (applied biosystems) were used following the manufacturer's protocol for real-time pcr analysis. the relative mrna amount of each sample was calculated based on its threshold cycle, ct, in comparison to the ct of the housekeeping gene, beta actin or glyceraldehyde 3-phosphate dehydrogenase (gapdh). the results are presented as 2 2(ct cxcl10-ct of housekeeping gene) as fold induction over the control condition. the purity of the amplified product was determined as a single peak of the dissociation curve. throughout the study, there was no observable fluctuation in the ct values of the housekeeping genes from different treatment conditions. primers to human and murine cxcl10 were purchased from sa biosciences. the primer sequences for human beta actin are as follows: forward: tgtgtccgtcgtggatctga and reverse: cctgcttcaccaccttcttgat. the primer sequences for murine gapdh are as follows: forward: tcctccaccttt-gacgctg and reverse: accaccctgttgctgtagcc. in preparation for collecting conditioned media from primary human airway epithelial cells, the cells were washed twice with sterile pbs. fresh media containing ifn c and/or poly i:c was placed basally with 100 ml of media placed apically. following a 24 hour incubation, the media was harvested, centrifuged to remove any cellular debris and stored at 280uc. bronchoalveolar lavage fluid was used for the murine cxcl10 elisa. commercially available elisa kits were used according to the manufacturer's directions (r&d systems). results were normalized to the amount of conditioned media or lavage fluid and expressed as ng/ ml or pg/ml. the construct contains 735 bp upstream of the transcription start site of cxcl10 was cloned into pgl3 luciferase reporter vector (promega). the construct was confirmed by dna sequencing. early differentiated hbe cells (7 days) were transfected with cxcl10 promoter-luciferase construct and prl-tk using lipofectamine 2000 (invitrogen) according to the manufacturer's specifications. prl-tk was used as the internal control for normalizing transfection efficiency. the empty vector, pgl3 basic was used as a negative control. in brief, cells were plated onto 12 well plates at 80-90% confluency. the next day, cells were washed twice with opti-mem (invitrogen) before transfection. the cells were incubated at 37uc with the mixture of dna constructs and lipofectamine 2000 in opti-mem for 5-6 hours at which time cell culture media was also added to the cells. transfected cultures were treated with 50 ng/ml ifn c and/or 25 mg poly i:c for 24 hours. a dual luciferase reporter assay kit (promega) was used following the manufacturer's protocol. for each transfection, the relative firefly luciferase activity was normalized to the renilla luciferase activity. neutralization of ifnar 2.5 mg/ml of a mouse monoclonal antibody to chain 2 of the human alpha, beta, omega interferon receptor (ifnar) was added to culture media of cells for 1 hour prior to the start of the experiment. this antibody has previously been documented to neutralize the extracellular domain of the human type i interferon receptor with high affinity at the dose used in this study [18] . ifn c and/or poly i:c were added to the culture media following the pre-incubation period and with the ifnra antibody still present. mouse igg was used as the control. following a 24 hour incubation, rna isolation and qpcr were performed as previously described. data are expressed as mean 6 se. experiments were conducted in triplicate with at least three independent cultures. group differences were calculated using an analysis of variance (anova) followed by a bonferroni multiple comparison test. differences were considered significant for p values less than 0.05. as shown in fig. 1a , well differentiated hbe cells demonstrated significant synergistic induction of cxcl10 mrna following infection with the mem influenza virus and treatment with ifn c. cxcl10 induction was also significantly elevated following the combined treatment of mem and ifn c treatment as compared to the treatment with ifn c alone. consistently, potentiation of cxcl10 protein production by the combined treatments was detected in the cell culture supernatant from hbe cells (fig. 1b) . apical release of cxcl10 was enhanced in all treatment conditions with more pronounced levels detected in the combined mem infection and ifn c treatment (fig. 1b) . interestingly, significant levels of cxcl10 were also released basally except that they were much lower than the apical secretion. these results demonstrate that influenza virus in combination with ifn c synergistically induce cxcl10 mrna and protein production from primary human airway epithelial cells. because viral replication and its intermediate-double stranded (ds) rna have been shown to mediate many influenza induced phenotypes. we then examined the possibility if dsrna could synergize with ifn c in the induction of cxcl10. as shown in fig. 2a , hbe cells demonstrated synergistic induction of cxcl10 mrna in a time dependent manner starting as early as 3 hours after treatment. treatment with ifn c and dsrna resulted in significant synergistic induction of cxcl10 mrna levels at all the time points. combined treatment for 24 hours resulted in an over 8000 fold induction of cxcl10 mrna. consistently, fig. 2b demonstrates potentiation of cxcl10 protein in cell culture supernatant from hbe cells treated for 24 hours. apical release of cxcl10 was enhanced in all treatment conditions with more pronounced levels detected in the combined ifn c and dsrna treatment. significant levels of cxcl10 were also released basally, although the levels were much lower than the apical secretions. these results suggest that the synergy between influenza infection and ifn c treatment may be caused by the interaction between dsrna-and ifn c-mediated signaling pathways. to determine whether ifn c and dsrna synergistically induce cxcl10 in vivo, we performed intra-tracheal delivery of ifn c and dsrna in balb/c mice and examined cxcl10 mrna and protein levels. as shown in fig. 3a , dsrna alone induced a significant increase in cxcl10 mrna, and ifn c and dsrna treatment together synergistically induced cxcl10 mrna 54 fold in mouse lung. likewise, fig. 3b demonstrates a significant induction of cxcl10 protein in bronchoalveolar lavage fluid after treatment with dsrna and synergistic induction following ifn c and dsrna treatment. these results demonstrate synergistic induction of cxcl10 mrna and protein in vivo following ifn c and dsrna treatment. because dsrna is known to induce the type i interferon pathway, we investigated the involvement of this pathway by using a neutralizing antibody to human ifnar. fig. 4a demonstrates that the ifnar neutralizing antibody did not affect synergistic induction of cxcl10 mrna in hbe cells, although it did repress dsrna-induced cxcl10. to confirm this result, we isolated mte cells from ifnar null mice, which were completely lack of ifnar expression. fig. 4b demonstrates that the lack of ifnar did not affect the synergistic induction of cxcl10. interestingly, induction of cxcl10 by dsrna alone was significantly impaired in the mte cells from ifnar null mice as compared to the cells from wild-type mice. taken together, these results demonstrate that synergistic induction of cxcl10 mrna was independent of the type i interferon receptor in both human and mouse airway epithelial cells. in contrast, dsrna-induced cxcl10 appeared to depend on the type i interferon receptor, suggesting that the synergy may be mediated via a different pathway. because of the rapid elevation of cxcl10 by ifn c and/or dsrna ( fig. 2a) , we tested the possibility if this synergy occurred at transcriptional level by transient transfection of the hbe cells with a cxcl10 promoter/luciferase chimeric construct containing 735 bp of the upstream regulatory region of cxcl10. fig. 5 demonstrates the combined treatment of ifn c and dsrna significantly increased the luciferase reporter gene activity as compared with the treatment of ifn c or dsrna alone. these results support that the synergism occurred at least partially through a direct transcriptional mechanism and the proximal 735 bp region of the cxcl10 promoter was critical for the induction. because both nfkb and isre sites were present in this cloned cxcl10 promoter region, we further tested nfkb and stat pathways in this synergism as both pathways have been shown to be responsible for cxcl10 gene expression. indeed, the specific inhibitor targeting the kinase either upstream of nfkb (ikb kinase) or stat1 (jak) significantly blocked cxcl10 induction by ifn c, by dsrna, or by the combined treatment (fig. 6a) , which confirms the indispensable role of both pathways in . the synergism between ifn c and dsrna(dsr) was independent of ifnar. a) hbe cells were treated with 2.5 mg/ml of either an isotype control antibody (black bar) or a monoclonal neutralizing antibody ifnar (grey bar) for one hour prior to the addition of 50 ng/ml ifn c and/ or 25 mg/ml dsrna for 24 hours. rna was isolated followed by qpcr. ns: not significant. $: p,0.05. isotype control antibody vs. ifnar. b) primary mte cells isolated from either wild-type (black bar) or ifnar deficient (grey bar) mice were grown in vitro and treated with 50 ng/ml ifn c and/or 25 mg/ml dsrna for 24 hours. rna was isolated followed by qpcr. ns: not significant. $: p,0.05. wild-type vs. ifnar deficient. triplicate wells were used for each experiment and the experiment was repeated at least three times. doi:10.1371/journal.pone.0100978.g004 regulating cxcl1 expression. when the signaling pathway was further examined, the treatment of ifn c or dsrna alone was found to induce robust degradation of ikb, a surrogate of the activation of nfkb pathway which is initiated by the reaction catalyzed by ikb kinase. and the combined treatment resulted in much greater ikb degradation as compared to the single treatment (fig. 6b) . in contrast, despite the activation of stat1a and stat1b by these treatments, no synergism was observed (fig. 6b) . therefore, the synergism of ifn c and dsrna on cxcl10 expression was mediated by nfkb pathway. airway epithelial lining is the first defense against respiratory viral infection. in this study, we seek to understand the modulation of an important chemokine-cxcl10 by the combined effects of ifn c and viral infection. this is the first report documenting the observation and mechanism underlying the synergism between ifn c and viral infection (or dsrna treatment) in airway epithelial cells. ifn c is a type ii interferon and oftentimes elevated in the context of viral infection. it is generally accepted that direct innate antiviral defense is mediated by type i ifn. indeed, we and others have shown that dsrna (or virus)-induced epithelial derived type i ifn plays critical role in the airway antiviral defense [16, 21, 22] . in the present study, we demonstrate the synergy between dsrna-and ifn c-induced signaling in the regulation of cxcl10. interestingly, although dsrna-induced cxcl10 depended on type i ifn pathway, the synergy between dsrna and ifn c was completely independent of it in both human and mouse epithelial cells. this lack of dependence was not due to the remaining residue of the ifnar functionality in the antibody neutralization assay, because the epithelial cells from ifnar deficient mice used in the fig. 4a still preserved the synergistic response despite the loss of entire ifnar expression. thus, in the presence of ifn c, dsrna (or virus)-induced signaling is very likely to be altered, which emphasizes the importance of studying the pathway crosstalk as demonstrated in the present study. previous studies have demonstrated that ifn c in conjunction with different pro-inflammatory molecules leads to a synergistic and dramatic induction of cxcl10 in a variety of cell types including keratinocytes, macrophages, endothelial cells and smooth muscle cells [6, 8, 9, 12] . several of these studies have demonstrated that the transcription factor, nf-kb, is involved in cxcl10 induction in a variety of systems [7] [8] [9] . in contrast, very few studies have demonstrated that cxcl10 induction is dependent on an isre site in the promoter region. kanda and coworkers found substance p and ifn c induced synergism of cxcl10 in human keratinocytes was dependent on an isre site and two nf-kb sites in the cxcl10 promoter located 2210 to 2 221 of the transcription start site [12] . in addition, majumder and co-workers found evidence that ifn c and tnf a act in synergy via p48 complexes with stat-1a binding to this same isre site in the cxcl10 promoter of human fibrosarcoma lines [23] . evidence that similar synergism occurs at the transcriptional level in mouse cells has been demonstrated in a small number of in vitro studies [13, 24] . using the murine fibroblast nih 3t3 cell line, ohmoir and co-workers established that an isre site and one of two nf-kb sites in a 243 bp fragment flanking the transcription start site of the murine cxcl10 gene were critical for ifn c and tnf a induced synergy [24] . our study has further extended these studies to the airway epithelial system in the context of viral infection, and the results apparently support the importance of nfkb. figure 6 . nfkb-dependent signaling is responsible for the synergism between ifn c and dsrna (dsr). a) well differentiated primary hbe cells were treated with 50 ng/ml ifn c and/or 25 mg/ml dsrna for 24 hours in the presence or absence of ikb kinase or jak inhibitor. rna was isolated followed by qpcr. solvent control: black bar; ikb kinase inhibitor: grey bar; jak inhibitor: light grey bar. $: p,0.05. inhibitor treatment vs. solvent control. b) cells were treated with 50 ng/ml ifn c and/or 25 mg/ml dsrna for 3 hours. cellular protein was collected and analyzed by western blot analysis. actin was used as a loading control. pstat1: phosphorylated stat1. this is the representative image from three replicates. doi:10.1371/journal.pone.0100978.g006 the present study demonstrates the significant contribution of cxcl10 from epithelial cells, which is consistent with the emerging role of active defense by these cells in the respiratory viral infection. cxcl10 secretion by hbe cells appeared to be polarized. the treatment with ifn c, dsrna and combined treatments resulted in a progressively enhanced secretion of cxcl10 mainly from apical surface. in contrast, the basal secretions of cxcl10 protein in these cultures were much less. the gradient between the apical and basal compartments may have important implications in vivo leading to enhanced emigration of cxcr3 positive effector cells (primarily cd8+ t cells) to the airway epithelial microenvironment. although dsrna has been used extensively as a synthetic dsrna to model viral infections, its stabilized derivatives are also being investigated for clinical use as an immunomodulatory agent for use as vaccine adjuvants in anti-viral treatment and cancer therapies [25] [26] [27] [28] . while dsrna was used in this study to simulate a viral infection, it is important to note that it, in and of itself, can also be involved in synergistic induction of cxcl10 in airway epithelial cells. this fact could be an important consideration in the treatment of patients with pre-existing diseases in which ifn c is part of the pathogenesis such as viral infections. in summary, we demonstrate ifn c and the influenza virus synergistically induce cxcl10 in human airway epithelial cells. this synergism is likely to be mediated by dsrna-induced signaling both in vitro and in vivo, which is independent of the type i interferon receptor pathway. furthermore, we demonstrate the 735 bp proximal region of the cxcl10 promoter plays a critical role in regulating this synergistic induction. this region of the cxcl10 promoter contains punitive isre and nfkb transcription factor binding sites. therefore, further study has been carried out to demonstrate the involvement of nfkb, but not stat1, in this synergism. this capacity for airway epithelial cells to markedly up-regulate cxcl10 likely has important consequences for cd8+ t cell and nk cell migration to the airway epithelial microenvironment following influenza virus infection. the immunobiology of interferongamma inducible protein 10 kd (ip-10): a novel, pleiotropic member of the c-x-c chemokine superfamily cutting edge: ifn-inducible elr-cxc chemokines display defensin-like antimicrobial activity cellular immune responses to severe acute respiratory syndrome coronavirus (sars-cov) infection in senescent balb/c mice: cd4+ t cells are important in control of sars-cov infection ip-10 mediates selective mononuclear cell accumulation and activation in response to intrapulmonary transgenic expression and during adenovirus-induced pulmonary inflammation cxcl10/cxcr3-mediated responses promote immunity to respiratory syncytial virus infection by augmenting dendritic cell and cd8(+) t cell efficacy molecular mechanisms underlying the pro-inflammatory synergistic effect of tumor necrosis factor alpha and interferon gamma in human microvascular endothelium nf-kappab-dependent synergistic regulation of cxcl10 gene expression by il-1beta and ifn-gamma in human intestinal epithelial cell lines pdgf synergistically enhances ifn-gamma-induced expression of cxcl10 in blood-derived macrophages: implications for hiv dementia regulation of tnf-alpha-and ifn-gamma-induced cxcl10 expression: participation of the airway smooth muscle in the pulmonary inflammatory response in chronic obstructive pulmonary disease ifngamma synergizes with tnf-alpha but not with viable h. pylori in up-regulating cxc chemokine secretion in gastric epithelial cells prolactin enhances interferon-gamma-induced production of cxc ligand 9 (cxcl9), cxcl10, and cxcl11 in human keratinocytes substance p enhances the production of interferon-induced protein of 10 kda by human keratinocytes in synergy with interferon-gamma hyaluronan fragments synergize with interferon-gamma to induce the c-x-c chemokines mig and interferon-inducible protein-10 in mouse macrophages molecular mechanism(s) involved in the synergistic induction of cxcl10 by human immunodeficiency virus type 1 tat and interferon-gamma in macrophages proinflammatory cytokines and hiv-1 synergistically enhance cxcl10 expression in human astrocytes rhinovirus induces airway epithelial gene expression through double-stranded rna and ifndependent pathways characterization of human mucin 5b gene expression in airway epithelium and the genomic clone of the aminoterminal and 59-flanking region identification of a novel subunit of the type i interferon receptor localized to human chromosome 21 stimulation of airway mucin gene expression by interleukin (il)-17 through il-6 paracrine/ autocrine loop vanadium pentoxide (v(2)o(5)) induced mucin production by airway epithelium rhinovirus-induced major airway mucin production involves a novel tlr3-egfr-dependent pathway negative control of tlr3 signaling by ticam1 down-regulation ) p48/ stat-1alpha-containing complexes play a predominant role in induction of ifn-gamma-inducible protein, 10 kda (ip-10) by ifn-gamma alone or in synergy with tnf-alpha the interferon-stimulated response element and a kappa b site mediate synergistic induction of murine ip-10 gene transcription by ifn-gamma and tnf-alpha treatment of viral and neoplastic diseases with double-stranded rna derivatives and other new agents a north american brain tumor consortium phase ii study of poly-iclc for adult patients with recurrent anaplastic gliomas synthetic double-stranded rna poly(i:c) combined with mucosal vaccine protects against influenza virus infection antiviral role of toll-like receptor-3 agonists against seasonal and avian influenza viruses key: cord-002913-k5b6abyk authors: kim, ha kyun; kim, hyun jung; kim, jae hyung; kim, tae hoon; lee, sang hag title: asymmetric expression level of clock genes in left vs. right nasal mucosa in humans with and without allergies and in rats: circadian characteristics and possible contribution to nasal cycle date: 2018-03-13 journal: plos one doi: 10.1371/journal.pone.0194018 sha: doc_id: 2913 cord_uid: k5b6abyk numerous peripheral tissues possess self-sustaining daily biologic rhythms that are regulated at the molecular level by clock genes such as per1, per2, clock, and bmal1. physiological function of nasal mucosa exhibits rhythmic variability to a day-night environmental cycle. nevertheless, little is known of the expression and distribution pattern of clock genes in nasal mucosa. the present study investigates the expression level and distribution pattern of per1, per2, clock, and bmal1 genes in nasal mucosa of healthy controls, allergic rhinitis patients, and normal rats. in human and rat nasal mucosa, the levels of these genes are asymmetrically expressed in nasal mucosa derived from right and left cavities in normal controls, allergic patients, and rat. in human nasal mucosa, the expression levels of these genes were higher in the decongested side than the congested mucosa. in rat nasal mucosa, these clock genes are expressed in a rhythmic circadian manner under the regular light/dark cycles. the expression levels of muc5ac, a key mucin genes produced in superficial epithelium, are higher in decongested side than that congested side in human nasal mucosa. in rat nasal mucosa, muc5ac levels showed a circadian rhythm which was associated with different expression levels in nasal mucosa derived from the right and left nasal cavities. taken together with these results, the present study shows that the clock genes such as per1, per2, clock, and bmal1 are present in human and rat nasal mucosa, and suggest that these clock genes may control the pathophysiological function of nasal mucosa as circadian oscillators and affect the maintenance of the nasal cycle. introduction spontaneous periodic changes in the congestion state of the nasal mucosa associated with reciprocal fluctuations in nasal resistance to airflow are referred to as the nasal cycle. cyclical shrinkage and dilation of the venous sinusoids distributed in the nasal mucosa contributes to the spontaneous alteration in nasal airflow [1] [2] [3] [4] [5] . this physiological phenomenon is present in 80% of normal human subjects and is also found in several animal species [5] [6] [7] [8] [9] . although the function of the nasal cycle has not been clearly established, the cyclical changes in the congestion state of the nasal mucosa associated with nasal cycle may result in plasma extravasation and nasal fluid formation [3] . during the nasal cycles, one nasal cavity becomes more open, and its mucosal glands increase their secretion, while nasal mucosa in the opposite cavity becomes engorged, which diminishes mucosal gland secretion [10, 11] . eccles proposed that the nasal cycle may have a role in respiratory defense by alternating air conditioning between the two nasal passage and by generating the nasal fluid [3] . furthermore, nasal cycle are accompanied by change in dilation of the venous sinuses distributed in the nasal turbinate and nasal septum. the dilation state is controlled by the autonomic nervous system that regulates vasomotor activity of the nasal mucous membranes [9, 12] . it is believed that the action of the sympathetic nerve controlling reciprocal changes of nasal airflow is regulated by the vasomotor control areas of the medulla [13, 14] . the hypothalamus participates in regulation of the nasal cycle via the sympathetic nervous system distributed in the nasal mucosa [15] . data reported in numerous studies have shown that many mucosal functions in nasal cavities are subject to a circadian rhythm. in normal subjects, a prominent circadian fluctuations in concentration of igg, iga, and albumin was revealed in nasal secretions [16, 17] . circadian changes of the mucociliary transport times have also been shown [18] . concentrations of albumin, elastase, and il-8 in nasal lavage samples from healthy volunteers were significantly different between morning and afternoon [19] . the amount of collected nasal secretion was significantly higher at 6 a.m. than at 3 p.m. after a metacholine nasal challenge. furthermore, numerous studies showed the diurnal manifestation and 24-h variation in the severity of allergic rhinitis [20, 21] . symptoms of allergic rhinitis are relatively intense nocturnally, during sleep, and in the early morning [22] . a statistically higher concentration of inflammatory mediators was detected in the nasal secretions of allergic rhinitis patients after a challenge in the morning than in the afternoon. such a circadian variation of nasal reactivity suggests that the ability to induce inflammatory activity in nasal mucosa depends on a circadian rhythm [23] . however, the regulator of these circadian rhythms at the molecular level has yet to be determined. the molecular basis of the biological circadian rhythm is believed to be determined by the clock genes, which are expressed within the suprachiasmatic nucleus as well as in the peripheral tissues [24] [25] [26] [27] [28] [29] . the molecular analysis of the biological clock genes has revealed that a basic mechanism of the clock machinery composes the autoregulatory positive and negative feed-back loops of a set of clock genes [30, 31] . these feedback loops are composed of the basic helix-loop-helix transcription factors clock and bmal1. these molecules dimerize, and activate transcription of the period genes per1 and per2 [31] [32] [33] [34] [35] . recent studies have revealed circadian variations in expression of clock genes in peripheral tissues, indicating that the analysis of clock gene expression profiles in peripheral tissues may provide useful clues for the elucidation of their individual circadian functions [24, 36, 37] . in this respect, we hypothesized that clock genes may be rhythmically expressed in nasal mucosa. therefore, the present study had the following aims: 1. to investigate the expression levels and distribution pattern of per1, per2, clock, and bmal1 genes in normal and allergic human nasal mucosa and to establish whether their levels correlate temporally with alternating congestion and decongestion of nasal mucosa; 2. to elucidate whether these genes exhibit asymmetrical expression levels in nasal mucosa of opposite nasal cavities in rat and whether their levels are subject to a circadian rhythm; 3. to determine if the expression levels of these genes are associated with the production of muc5ac and aquaporin 5. this study was approved by the ethics committee of korea university and the committee of care and use of laboratory animals of korea university. normal control subjects and patients with moderate/severe persistent allergic rhinitis were selected for this study. the clinical characteristics of normal controls and allergic patients are presented in table 1 . patients who were admitted to hospital for augmentation rhinoplasty were considered as normal control subjects. normal controls did not have any significant clinical symptoms as revealed by a physical examination, nasal examination, and medical history. moderate/severe persistent allergic rhinitis was defined according to the allergic rhinitis and its impact on asthma criteria [38] . all subjects were nonsmokers without any pulmonary diseases and were not taking any medication during the preceding 3 months. acoustic rhinometry was performed in all subjects enrolled in this study in the morning between 7:00 am and 8:00 am. immediately after measurement of nasal cavity volume, nasal mucosa was respectively sampled from both inferior turbinate under local anesthesia. this study was approved by the institutional ethics committee, and all participants gave written informed consent. all tissue samples were divided into three parts. the first and second parts were stored at -70˚c for subsequent rna and protein isolation. the third part of samples was used for immunohistochemistry. the committee of care and use of laboratory animals of our institution approved all animal care and experimental procedure. experimental procedures for animals are reported in accordance with the animal research reporting of in vivo experiments (arrive) guidelines [39, 40] . male 5-6 week old sprague-dawley rats (dbl, chungcheong buk-do, korea) were maintained at a temperature of 23˚c ± 2˚c under a 12 light-dark cycle per day. animals were nasal mucosa samples were analyzed using rt-pcr and real time pcr for per1, per2, clock, bmal1, muc5ac, and aquaporin 5 (for primers see table 2 ). total rna was extracted from each sample using trizol reagent (invitrogen, burlington, on, ca) and used for cdna synthesis in a reaction mixture containing 2.5 u of mml-v (gibco brl, grand island, n.y., usa). thereafter, resulting cdna was amplified by rt-pcr or real time pcr. real-time pcr was carried out in a bio-rad icycler using sybr green. amplification was conducted after a 30-s denaturation at 95˚c followed by 45 cycles of incubation at 95˚c for 15 s and at 60˚c for 20 s. all reactions were performed in triplicate, and expression of each target gene was normalized to gapdh or eef1a1expression. the relative amount of target gene in each sample was determined as 2 δδct . to investigate the localization of clock genes protein in human and rat nasal mucosa, tissue sections were incubated overnight at 4˚c with a mouse monoclonal anti-per1 antibody (1: 200 dilution, sc-81574), a mouse monoclonal anti-per2 antibody (1: 200 dilution, sc-377290), a mouse monoclonal anti-clock antibody (1:100 dilution, sc-271603), or a mouse monoclonal anti-bmal1 antibody (1:100 dilution, sc-373955), (all antibodies were from santa cruz biotechnology, inc., santa cruz, ca, usa). as a negative control, the primary antibodies were replaced with nonimmune normal serum (santa cruz biotechnology). diaminobenzidine (dab; sigma-aldrich, st. louis, mo, usa) was used for color development. for western blot analysis, the extracted protein from frozen tissue samples was resolved on 4-12% tris glycin sds-page gels and transferred to immobilon (millipore, bedford, mass., usa). the blots were blocked with pbs-t containing 1% skim milk and then incubated with anti-per1, anti-per2, anti-bmal1 (all diluted to 1:500), anti-clock antibody (diluted to 1: 200), or a mouse monoclonal anti-aquaporin 5 (1;100 dilution, sc-514022) (santa cruz biotechnology, inc., santa cruz, calif., usa) in pbs-t overnight at room temperature. as an internal control, β-actin expression level was measured in parallel blots using a β-actin antibody (santa cruz biotechnology, inc., santa cruz, calif., usa). the intensity of detected bands was quantified using scion image beta 4.0.2 statistical analyses were carried out using spss for windows (version 16.0.0; spss, chicago, ill., usa). the expression levels of clock genes in normal and allergic patients analyzed by two-way analysis (anova). associations between the expression levels of genes and rhinometric data were analyzed using the mann-whitney u test. the level of significance was set at p < .05. if significant group differences were detected by the anova, then a post hoc analysis was applied. cosinor analysis was then performed using fold increase over zt0 to test for the presence of a circadian rhythm. changes in clock gene expression were referenced to the levels of expression at the 9 am. time point (zt0). data are presented as means ± sd (n = 6 mice per time point). rt-pcr performed with primers specific for per1, per2, clock, and bmal1 mrna showed that these genes are expressed in normal nasal mucosa of rat as well as in normal and allergic human nasal mucosa (figs 1a, 1b and 2). real-time pcr was conducted to evaluate the expression levels of per1, per2, clock, and bmal1 genes in total rna isolated from nasal mucosa of both inferior turbinate of the human nasal cavity. interestingly, these results revealed that these genes had asymmetric expression levels in nasal mucosa from both turbinates in normal control and allergic patients and showed that the expression levels of these genes were higher in decongested mucosa than congested mucosa, irrespective of gapdh or eef1a1 usage as housekeeping gene (fig 1d and 1e ). asymmetric expression levels of these genes were found in all subjects enrolled in the present study. per1, per2, clock, and bmal1 protein expression in the human nasal mucosa was confirmed by western blot (fig 1c) . expression of these proteins in all samples was also confirmed by immunohistochemistry. immunoreactivity of these genes was detected in normal and allergic human nasal mucosa, where per1, per2, clock, and bmal1 localized to the epithelial cells, submucosal glands, and vascular endothelium (fig 2d) . we then evaluated the possibility that asymmetric expression levels of these genes may be related to the nasal cycles, particularly to congested or decongested phase evaluated by acoustic rhinometry. the expression levels of per1(f = 25.7, p <0.05), per2 (f = 6.6, p <0.05), clock (f = 8.7, p<0.05), and bmal1 (f = 10.8, p<0.05) genes were higher in decongested mucosa than in congested mucosa in normal controls and allergic patients (fig 1d and 1e) . however, the levels of these clock genes expressed in decongested mucosa were not different between normal and allergic patients (fig 1d and 1e) . rat nasal mucosae obtained from both nasal cavity of rat showed also asymmetric expression in mrna and protein levels of per1, per2, clock, and bmal1 (fig 2a, 2b and 2c ). rhythmic expression patterns for per1, per2, clock, and bmal1 genes were observed for two consecutive days in the nasal mucosa of rat (fig 3) . multiple peaks in per1 mrna levels in mucosa from the right nasal cavity appears at zt3 and zt12 (95% ci, cosinor, p<0.001) (fig 3a) . then per1 mrna levels decreased at zt24 before increasing again at zt30 (95% ci, cosinor, p<0.001). in contrast, per1 mrna level in mucosa from the left nasal cavity was low until zt18. thereafter, its levels rose, showing a double peak at zt27 and zt36 (95% ci, cosinor, p < 0.001) (fig 4a) . per2 mrna levels in mucosa from the right nasal cavity peaked at zt3 and zt12 (95% ci, cosinor, p< 0.001) (fig 3b) , demonstrating a double peak, and then declined between zt21 and zt27. double peaks also were found on the 2 nd day at zt33 and zt39 (95% ci, cosinor, p<0.045) in mucosa from the left nasal cavity, per2 mrna level dynamics were opposite to those detected in right nasal cavity. clock and bmal1 mrna levels in mucosa from the right and left nasal cavities showed peaks at zt21 and zt45 (95% ci, cosinor, p< 0.036), and then, their mucosal levels were reversed between both nasal mucosa, showing different expression levels between both nasal mucosa. the expression levels of per1, per2, and clock genes were significantly different in mucosa from the right and left nasal cavities suggesting their asymmetrical expression levels in the two nasal cavities. however, although the mucosal expression levels of bmal1 genes in the right and left nasal cavities were different during peak times, no statistical differences in bmal1 expression were noted during the decreased circadian phase (fig 3a, 3b, 3c and 3d ). these antiphase expression patterns between per1/per2 and clock/bmal1 suggest that there is a functional peripheral circadian core oscillator in the nasal mucosa of rat. furthermore, immunohistochemical experiments also showed that the expression of protein products of these genes was found in the respiratory mucosa of rat nasal cavity. in contrast to their expression patterns in the human nasal mucosa, rat per1 and per2 proteins were predominantly distributed in the superficial epithelial layer and submucosal glands, whereas rat clock and bmal1 proteins were mainly localized to the vascular endothelium (fig 4) . to evaluate whether the expression levels of muc5ac and aquaporin 5 genes may be associated with circadian expression levels of clock genes in human and rat nasal mucosa, real time pcr and western blots were performed. the results of these experiments showed that muc5ac expression levels were higher in the decongested side in human nasal mucosa ( fig 1a, 1b, 1d and 1e) and were subject to circadian rhythm in the rat nasal mucosa (fig 3e) . in contrast, although aquaporin 5 gene levels showed circadian rhythms in rat nasal mucosa, but its levels were not different between the right and left nasal cavities during several circadian time points (fig 3f) . furthermore, aquaporin 5 levels were not different between congested and decongested sides in the human nasal mucosa. (fig 1d and 1e ). in this study, we present molecular and immunologic evidence of circadian clock-genes in the human and rat nasal mucosa. notably, per1, per2, clock, and bmal1 were asymmetrically expressed in nasal mucosa derived from the right and left nasal cavities of normal control and allergic patients. the expression levels of these clock genes were higher in decongested mucosa than in congested mucosa in normal control and allergic rhinitis. asymmetric expression of these four clock genes was also found in nasal mucosa derived from the left and right nasal cavities of rat, each clock gene showing a circadian rhythm. in rat nasal mucosa, per1 and per2 expression levels showed a robust rhythm and were accompanied by antiphase oscillations in clock and bmal1 levels. these findings provide evidence that a functional circadian clock is present in human and rat nasal mucosa, acting as distinct, circadian oscillators to set the circadian rhythm. furthermore, the present results suggest the possibility that asymmetric expression of clock genes may be associated with the nasal cycle. numerous studies have reported that clock genes are expressed in peripheral human tissues or cells. clock genes in those cell function as peripheral oscillators of the circadian rhythm, [41] [42] [43] [44] [45] [46] . in this respect, the existence of a circadian clock within nasal mucosa was anticipated because numerous studies reported circadian variations in the pathophysiological functions of nasal mucosa. although circadian variation in human biopsy samples could not be determined, expression levels of all clock genes tested in the present study were subject to robust circadian rhythms in rat nasal mucosa under light-dark cycle conditions. based on these results, we conclude that clock genes are expressed in human and rat nasal mucosa. moreover, a circadian rhythm in nasal mucosa is likely to function independently of the central clock distributed in the brain. our results are consistent with previous results that showed significant circadian variation in the levels of per1 and per2 mrna in mouse nasal mucosa, where per2 was localized to the epithelial cells, vascular endothelial cells, and nerve termini [47] . in the present study, the cellular localization of the four clock proteins was different in human and rat nasal mucosa. in human nasal mucosa, positive immunoreactivity of four clock genes was mainly detected in the superficial epithelium, submucosal glands, and vascular endothelial cells. however, in rat nasal mucosa, strong immunoreactivity of per1 and per2 was mainly observed in the superficial epithelium and in a small number of submucosal glands, whereas the vascular endothelium was only lightly stained. in contrast, clock and bmal1 were mainly localized to the vascular endothelium. based on their characteristic localization pattern, we hypothesized that these clock genes may play their divergent roles in the physiological function of human and rat nasal mucosa. because clock proteins in human nasal mucosa were identified in the superficial epithelium, submucosal gland, and vascular endothelium, it is conceivable that their expression might be involved in circadian rhythm of mucosal function such as nasal secretion and blood supply of microvasculature. in rat nasal mucosa, per1 and per2 might be involved in secretory function, whereas clock and bmal1 genes might regulate blood supply. further studies are warranted to determine whether there is clock gene-mediated interplay between blood flow and secretion. it has been generally accepted that cyclical changes in nasal airway resistance are caused by autonomic tone that regulates blood supply to the mucosal vessels and is under the control of a hypothalamic center [15] . nevertheless, the control process underlying the nasal cycle is not fully understood. interestingly, we found that there are significant differences in the expression levels of clock genes in nasal mucosa in both human nasal cavities. the expression levels of these clock genes in decongested mucosa were higher than in congested mucosa. these results show that clock genes may possibly be involved in the nasal cycle of congestion and decongestion of the nasal mucosa. similar findings were also noted in nasal mucosa of rat. it has been demonstrated previously that vessels in nasal mucosa are divided into resistance vessels and capacitance vessels [48] . the resistance vessels are composed of small arteries, veins and arterio-venous anastomosis. especially, arterio-venous anastomosis, controlled by the autonomic nervous system, regulates nasal mucosa blood flow by alternating smooth muscle tone. in contrast, the major capacitance vessels in nasal mucosa are composed of vascular sinusoid, and its swelling is mainly affected by nasal mucosa blood flow and filling pressure. the volume of blood flow in nasal mucosa affects nasal resistance, and the dilation of vascular sinusoid can result in nasal congestion, contributing to nasal obstruction [49] [50] [51] [52] [53] . the existence of a circadian rhythm in the function of blood vessels has long been recognized. the clock genes are ubiquitously expressed and exhibit rhythmic expression in blood vessels. cumulative evidence has demonstrated that these 24-h timers exerts a significant influence on the vasculature, whereby disruption of circadian clock components results in vascular stiffness and altered endothelial progenitor cell function [54] . moreover, mutations of clock genes have been reported to have detrimental effects on the vascular function in mice [55] . mice deficient in bmal1 and clock genes showed a reduced vasodilatory response to acetylcholine and had impaired endothelial function [56] . selective deletion of bmal1 from smooth muscle attenuated the circadian rhythm of blood pressure [57] . in this respect, we hypothesize that clock genes involved in the control of blood flow may contribute to the regulation of the nasal cycle. more studies are required to evaluate the molecular mechanisms of clock gene functions in cyclical congestion and decongestion of nasal mucosa. significant changes in the circadian amplitude of clock gene expression has been shown to cause cellular dysfunction and chronic diseases [58, 59] . the expression levels of clock proteins such as bmal1 and per2 are reduced in inflamed lung tissue and peripheral blood mononuclear cells in patients with chronic obstructive pulmonary disease [60] . viral respiratory infection in the lung induced molecular clock dysfunction and increase mortality in bmal1 knockout mice, suggesting that altered clock function impairs the immune response [61] . circadian alterations in expression levels of clock, cry1, and per2 mrna were found in schizophrenia [62] . the expression levels of bmal1 and per1 are normally in antiphase with each other, as demonstrated, for example, in the synovial membrane samples obtained from patients with osteoarthritis, where high bmal1 expression coexisted with low per1 expression. this expression pattern is commonly found in all clock works in the body [26] . however, this antiphase phenomenon was not found in rheumatoid arthritis patients. these results indicate that the clock is dysfunctional in rheumatoid arthritis, suggesting that inflammation may disturb circadian time-keeping [63] . in this respect, although the relationships between allergic rhinitis and clock genes functions remains unclear, we hypothesized that clock genes expression levels might be altered in allergic nasal mucosa in comparison with their levels in normal nasal mucosa. however, no significant differences in the expression levels of per1, per2, clock, and bmal1 in human control and allergic nasal mucosa samples were noted in the present study. in a recent study, the expression pattern of clock genes showed a clear circadian rhythm with a peak at zt13 in both control and asthmatic mice. no significant differences in the daily rhythmic pattern of per1 and per2 expression were observed in control and ova-treated mice [64] , which suggests that allergic responses in the lung may not interact with clock functions. recently, an association between the molecular circadian clock and the immune system and inflammation has been recognized. for example, passive cutaneous and systemic anaphylactic reactions were observed in wild-type, but not in per2-mutant mice [65] . these reactions were also absent in mice with mechanical disruption of the central scn clock [66] . ear swelling, serum ige levels, and the number of mast cells were significantly increased in clock mutant mice, providing evidence that clock mutation induces the t-helper type 2 immune response and aggravates contact hypersensitivity in skin [67] . bone marrow-derived basophils in wild-type mice exhibited circadian variation in ige-mediated il-4 and histamine production, which was not observed in bone marrow-derived basophils of mice bearing a mutation in the clock gene [68] . therefore, functional analyses of resident cells including nasal epithelial cells or inflammatory cells infiltrated into nasal mucosa in clock genes mutant mice are further required. since robust rhythms of per1 and per2 were observed in superficial epithelial layer of rat nasal mucosa, we investigated whether the expression of genes encoding muc5ac and aqua-porin5, secretory substances produced in superficial epithelium, is subject to circadian rhythm in nasal mucosa. in human nasal mucosa, the expression of muc5ac is higher in the decongested side than in congested side. in rat nasal mucosa, muc5ac levels showed a circadian rhythm associated with different expression levels in the right and left nasal cavities. expression levels of per1 and per2 was the highest at zt3 and zt12 on the right side and at zt27 and zt36 on the left side. muc5ac levels was the highest at zt3, zt9, and zt21 on the right side and at zt27 and zt33 on the left side. however, the expression levels of aquporin5 were not different between the two nasal cavities in human and rat nasal mucosa and exhibit a lower amplitude oscillation. these findings suggests that both muc5ac and aquaporin5 production are modified by circadian change, but aquaporin5 oscillates with a lower amplitude. it is known that muscarinic m1, m2, m3 receptors are expressed in nasal mucosa [69, 70] . because muscarinic receptors in the mouse lung show a circadian rhythm in their expression levels, and because in nasal mucosa, muscarinic type 3 receptors participate in mucin secretion [70, 71] , it is speculated that the circadian regulation of muc5ac secretion is controlled by the muscarinic receptors. nevertheless, further experiments are needed to assess the circadian regulation of mucin secretion. in conclusion, the present study revealed that the clock genes per1, per2, clock, and bmal1 are present in human and rat nasal mucosa, displaying asymmetric expression levels between nasal 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supported by the basic science research program through the national research foundation of korea (2016r1d1a1a02936927). this research was also supported by a korea university grant, korea (q1524681). key: cord-002222-rgqwm3vb authors: olarte-castillo, ximena a.; hofer, heribert; goller, katja v.; martella, vito; moehlman, patricia d.; east, marion l. title: divergent sapovirus strains and infection prevalence in wild carnivores in the serengeti ecosystem: a long-term study date: 2016-09-23 journal: plos one doi: 10.1371/journal.pone.0163548 sha: doc_id: 2222 cord_uid: rgqwm3vb the genus sapovirus, in the family caliciviridae, includes enteric viruses of humans and domestic animals. information on sapovirus infection of wildlife is limited and is currently lacking for any free-ranging wildlife species in africa. by screening a large number of predominantly fecal samples (n = 631) obtained from five carnivore species in the serengeti ecosystem, east africa, sapovirus rna was detected in the spotted hyena (crocuta crocuta, family hyaenidae), african lion (panthera leo, family felidae), and bat-eared fox (otocyon megalotis, family canidae), but not in golden or silver-backed jackals (canis aureus and c. mesomelas, respectively, family canidae). a phylogenetic analysis based on partial rna-dependent rna polymerase (rdrp) gene sequences placed the sapovirus strains from african carnivores in a monophyletic group. within this monophyletic group, sapovirus strains from spotted hyenas formed one independent sub-group, and those from bat-eared fox and african lion a second sub-group. the percentage nucleotide similarity between sapoviruses from african carnivores and those from other species was low (< 70.4%). long-term monitoring of sapovirus in a population of individually known spotted hyenas from 2001 to 2012 revealed: i) a relatively high overall infection prevalence (34.8%); ii) the circulation of several genetically diverse variants; iii) large fluctuations in infection prevalence across years, indicative of outbreaks; iv) no significant difference in the likelihood of infection between animals in different age categories. the likelihood of sapovirus infection decreased with increasing hyena group size, suggesting an encounter reduction effect, but was independent of socially mediated ano-genital contact, or the extent of the area over which an individual roamed. environmental contamination might be an important route for fecal-oral transmission of sapovirus, for example when spotted hyenas sniff virus infected feces or ingest water contaminated with virus infected feces. if so, individuals with a limited range may be less likely to encounter virus infected feces than those with an extensive range. in the serengeti np, adult and subadult hyenas (i.e. those !12 months of age) not only range throughout the approximately 56 km 2 of their clan's territory, but also undertake long distance foraging trips (of approximately 140 km distance round-trip) from their clan territory [46, 47] . the ranges of cubs (<12 months of age) are by comparison extremely limited, being restricted to the communal den area within the clan territory [47] . if the extent of an animal's range determines its chance of encountering virus infected feces, then all else being equal, cubs should be less often infected with sapovirus than older animals. finally, sapovirus transmission might depend on basic population parameters such as population density, principally represented by clan (group) size. if the chance of transmission increases with animal density, then individuals living in larger clans should be more likely to be infected than those in smaller clans. however, if an encounter reduction effect operates [48, 49] , then we expect the chance of susceptible individuals encountering an infected animal to decline with clan size. in humans, sapovirus infection is currently thought to provide immunological protection, at least to antigenically homologous sapoviruses, although specific immunological responses are still unknown [16] . currently nothing is known about the immunological responses of spotted hyenas to sapovirus infection, or the length of immunological protection following sapovirus infection. even so, if sapovirus infection induces long-term immunity against reinfection regardless of strain-type, we would expect cubs (i.e. naïve animals) to be more prone to infection than adults, as is the case for coronavirus infection in this species [35] . however, if sapovirus infection provides only short-term immunity, we would expect re-infections among animals of all ages. if immune responses are strain-specific, re-infection would also be expected in animals of all ages, following the appearance of a divergent strain. this study aims to advance knowledge of sapovirus infection in wild carnivore communities in africa. we report the identification of sapoviruses in wild carnivores in africa and investigate the genetic diversity of strains infecting sympatric carnivore species in the serengeti ecosystem. we assess temporal changes in sapovirus infection in a large population of individually known spotted hyenas during a period spanning more than a decade and investigate whether sapovirus infection provides long-term immunity against future infection. furthermore, we test three mechanisms likely to affect the fecal-oral spread of sapovirus infection in spotted hyenas. this study was conducted in the serengeti np, from february 2001 to march 2012. fresh fecal samples (n = 514) were collected shortly after deposition from individually known spotted hyenas including 146 samples from adults (females n = 93, males n = 53), 41 samples from subadults (females n = 20, males n = 21) and 327 samples from cubs (females n = 152, males n = 175) from three large clans (denoted in fig 2 as i, p, and m). fecal samples were also collected from other carnivores in the serengeti np (african lion, panthera leo, n = 9; bat-eared fox, otocyon megalotis, n = 9; silver-backed jackal, canis mesomelas, n = 74 samples; golden jackal, canis aureus, n = 25). following collection, feces were thoroughly mixed and divided in aliquots. tissue samples (5 intestine, 2 liver, 9 lung, 10 lymph node, 12 spleen, 10 blood, 1 muscle, 1 saliva) were also collected opportunistically from dead spotted hyenas which were mostly killed by lions or when hit by motor vehicles [40] and hence were not necessarily members of study clans, and from two other carnivore species (bat-eared fox: 2 intestines; silver-backed jackal: 1 intestine, 2 liver, 3 lung). both fecal and tissue samples were stored and transported frozen at -80°c, or were preserved in rnalater (sigma-aldrich inc., st. louis, mo, usa), stored initially at -10°c, and finally stored at -80°c until analyses [35, 36] . currently, porcine enteric calicivirus (pec) cowden strain [50, 51] is the only known sapovirus that can be cultured. hence, viral detection and initial characterization involves mostly molecular methods based on sequence data of the well-conserved rna-dependent rna polymerase (rdrp) and the variable structural vp1 genes [16, 21] . in this study, sapovirus rna was detected by targeting the highly conserved rdrp gene. total rna was extracted from 200μl of 10% (wt/vol) fecal suspension in depc-treated water using the qiaamp minelute virus spin kit (qiagen, hilden, germany), according to manufacturer's instructions. sapovirus rna was detected with the broadly reactive primer pair p289 and p290 [52] targeting highly conserved motifs in the rdrp protein of caliciviruses. based on the sequences initially generated, nested primers were designed, cali2f (5'-cag tga cag cca cat cct tg-3') and cali2r (5'-agc act gca gca gca aag ta-3'), targeting the rdrp gene. rt-pcr was performed using superscript tm iii one-step rt-pcr system with platinum 1 taq dna polymerase (invitrogen, karlsruhe, germany) following the user manual's instructions in a total reaction volume of 25μl. amplicons of the expected size were purified using the qiagen pcr purification kit (qiagen, hilden, germany). in order to avoid rnases, all surfaces were cleansed with rnase away (molecular bioproducts, san diego, ca, usa). the purified products were sequenced bidirectional using the big dye terminator cycle sequencing kit 1.1 (applied biosystems [abi], darmstadt, germany) following the manufacturer's instructions. a 3130 genetic analyzer (abi) was used for the sequencing. subsequently, sequences were assembled in geneious v 9.0.2 (biomatters ltd, auckland, new zealand) or bioedit 7.0.9.0 [53] . samples that could not be sequenced were considered positive when bands of the expected size were present with both primer pairs. for these samples the rt-pcrs were run in duplicate to ensure that the results were reliable. to obtain a longer segment of the rdrp gene, the primer 90r (5'-rcc ctc cat ytc aaa cac ta-3') was used together with the primer calir2.. genbank accession numbers for 20 sequences identified by this study are designated kt777545-kt777564. these accession number are included in our phylogenetic tree (fig 2) , together with the host species, the year in which the variant was collected and for spotted hyenas also clan membership, denoted as i,m,p if know or z if not known. all partial rdrp genes sequences (210 nucleotides, 70 amino-acids) presented the characteristic caliciviral glpsg motif. one sample from a spotted hyena in 2011 was sequenced for a longer fragment of the rdrp gene (700 nucleotides, 233 amino-acids, accession number kt777560) which presented both the glpsg motif and the ygdd motif. sapovirus sequences obtained in this study for the partial rdrp gene together with others retrieved from genbank were aligned using the muscle algorithm [54] in geneious v 9.0.6 (biomatters ltd, auckland, new zealand). at least one reference sequence of each of the five genogroups of sapovirus (gi-gv) was included in the analysis (gi, n = 5, accession numbers ay237422, ay694184, dq366345, u95644, u73124, gii, n = 3, ay646855, ay237420, ay603425, giii, n = 3, fj715800, af182760, fj387164, giv, n = 1, dq058829, gv, n = 1, ay646856). additional sapovirus sequences from domestic dog (jn387135, jn387134), california sea lion (jn420370), mink (ay144337) and bats (jn899072, jn899074, jn899075) were included. viruses from other genera in the caliciviridae family known to infect carnivores were also included, such as feline calicivirus (af098931-32) and canine calicivirus (af053720, ab070225) from the vesivirus genus, and a norovirus reported from a captive african lion (genus norovirus, ef450827). average nucleotide and amino-acid similarities were calculated using discovery studio visualizer 4.0 (accelrys software inc, san diego, usa). phylogenetic relationships were reconstructed using maximum likelihood (ml) and bayesian markov chain monte carlo (mcmc) phylogenetic inferences. the ml analysis was performed in paup ã 4.0b10 [55] using 1,000 bootstrap replicates to estimate the statistical support of the branches. the bayesian analysis was carried out using mrbayes version 3.1 [56, 57] . the mcmc search was set to 10,000,000 iterations, with trees sampled every 1,000 th iteration. the nucleotide substitution model used in the ml analysis was obtained using modeltest 3.7 [58] and for the bayesian analysis using mrmodeltest 2.3 [59] . for both cases the akaike information criterion (aic) was used to select the best-fitting model. to determine factors influencing the likelihood of sapovirus infection and changes in long-term infection prevalence we screened feces from individually recognized spotted hyenas in three study clans (i,m,p). age was estimated when individuals were first sighted as cubs, to an accuracy of ± 7 days [60] using pelage characteristics, whether their ears were flattened or upright, and their coordination during locomotion [61, 62] . we classified animals as cubs when less than 12 months of age, as subadults when between 12 and less than 24 months of age, and as adults when ! 24 months of age [63] . sex was determined by the dimorphic glans morphology of the erect phallus [64] . total clan size comprised all adults, subadults and cubs of both sexes. access to food resources in clan territories is determined by social status: all immigrant males are socially subordinate to female clan members and their offspring at food resources in the clan territory [65] . we determined the rank of adults in separate female and breeding male linear dominance hierarchies using the outcome of submissive responses in dyadic interactions within each sex, as detailed in [43, 60, 63] . to compare individual ranks across clans of different sizes, we used standardized ranks. we calculated the standardized rank of each individual within its clan on the date it was sampled using the method described by [66] . this method assigns standardized ranks between -1 (held by the animal with the lowest rank) and +1 (held by the animal with the highest rank) [60, 63] . adult females with standardized ranks higher or equal to the median standardized rank of 0 were classified as holding high social status, those with standardized ranks below 0 as low social status [43] . cubs and subadults were assigned the social status of their mother [60] . all immigrant males held a social status below adult clan females [63] . if sapovirus infection depends on intra-specific contact rates, we would expect the dynamics of social interactions within each clan to determine exposure to pathogens. for this purpose we constructed an index of social (ano-genital during greeting ceremonies) contact rates in spotted hyenas as follows. we combined social status and sex in that high ranking females and their offspring were given a high score (for contact rate), low ranking females and their offspring were given a medium score, and immigrant and reproductively active natal males were given a low score. in order to assess whether the range of an animal, the size of the area over which an individual typically roams, determines the chance of exposure to pathogens, we classified adults and subadults of both sexes with an extensive geographical foraging range as 'roaming' , because they range both within their clan territory (~55-75 km 2 ) and undertake long distance foraging trips outside the clan territory [46, 47] . cubs were classified as 'den-bound' , i.e., with a small range restricted to the vicinity of the communal den inside the clan territory. for the purpose of considering the effect of basic population parameters such as population density on incidence of infection we used total clan size on the date each animal was sampled. to investigate whether sapovirus infection provided immunity against re-infections we genetically screened feces from 91 individually known spotted hyenas from which fecal samples were obtained on at least two different dates. of these, 76 individuals were screened on two different dates, 10 individuals on three different dates and 5 individuals on four different dates. using these screening results we calculated the average interval duration between two successive sampling dates. we used nonparametric models, including the mann-whitney u-test and the kruskal-wallis test, to compare medians [67] and the kaplan-meier survivorship and the logrank test in survival analyses to compare the survivorship curves of intervals between different combinations of incidences of infection [68] . to investigate differences in the prevalence of sapovirus in the spotted hyena population studied between 2001 and 2012 we first tested for differences in the prevalence of infection across years, using a log-likelihood ratio-test. for this test we only considered years with a sample size of at least 20 individuals, thus years 2001, 2002 and 2012 were excluded where sample sizes were 17, 11 and 5, respectively. we also checked for possible differences between age categories, using the same statistical test. these analyses were run in systat version 13 (systat software inc., richmond, va, usa). we then ran models to assess which of three possible mechanisms influenced the likelihood of sapovirus infection in our study population. for this purpose we used binary logistic regression models [69] , with predictor variables contact rate, lifetime range and clan size, and ran these as mixed models with animal identity as a random variable to account for the fact that some individuals contributed more than one tissue or fecal sample to the data set. if a genetic screening result was available for more than one organ or fecal sample for an individual on the same sampling date, only one result was included in the dataset for the prevalence models; if we obtained both a positive and negative result from an animal on the same day, the positive result was selected. this applied to 7 individuals where we had two fecal samples from the same day, and to 7 individuals from which altogether 16 tissue samples were examined. we included data from all individuals sampled during the years which could either be classified as outbreak or non-outbreak years (see results). models were run with the glmer function of package lme 4 version 1.1-8 in in r (r development core team, v. 3.1.1). we used log-likelihood ratio tests and information criteria (aic and schwartz's [bic s ] and raftery's bayesian information criterion [bic r ]) to check whether the final model was superior to an intercept-only or a reduced model. models were considered similar if differences in aic were less than 2.5 and preferable if the difference exceeded 6.0 [70] ; similar if differences in bic r were less than 2.0, a positive degree of preference if values of bic r varied between 2.01 and 6.0 and a strong degree of preference if values of bic r differed by more than 6 (a. raftery in [71] , p73). as the evaluation of our models with all information criteria produced similar conclusions, we report only aic values. the significance of each predictor variable was assessed in the following way. we calculated the marginal contribution of each parameter to the full model by subtracting from the full model the log-likelihood ratio of a second model with each variable removed and testing the difference against a chi-square distribution with the appropriate degrees of freedoms (see discussions in [69, 71] ). in order to illustrate the effect of clan size on the chance of infection, we proceeded as follows. we calculated "covariate adjusted estimates" of the logits for each record over the observed range of values by adjusting them to the median of the remaining covariates (contact rate, lifetime range) of their log-odds (logit) for being infected ( [69] , p80), and then converted the resulting estimates into probabilities using the logistic equation. this permitted us to show the effect of clan size on the likelihood of infection whilst controlling for the covariates contact rate and lifetime range at their middle values. the significance threshold for all tests was fixed at 5% and all tests were two-tailed. the data used for the statistical analyses is contained in s1 table. ethics statement the study was approved by the tanzanian commission of science and technology (cost-ech) and the tanzania wildlife research institute (tawiri). permission to work in the serengeti national park was granted by the tanzanian national parks authority (tanapa). the work was also approved by the internal ethics committee of the leibniz institute for zoo and wildlife research (izw), approval no. 2011-04-03. screening targeting the conserved rdrp gene revealed sapovirus rna in feces from spotted hyena (33.3%, 171/514 samples), african lion (33.3%, 3/9 samples) and bat-eared fox (22.2%, 2/9 samples). no sapovirus rna was found in fecal samples from golden (0/25) or silverbacked jackals (0/74). sapovirus rna was found in tissue samples collected opportunistically from dead spotted hyenas (spleen, 6/12 samples, liver 1/2 samples, lymph node 2/10 samples), but not in intestine (0/5 samples) or lung samples (0/9 samples). animals with positive spleen samples were negative for sapovirus rna in their other available tissues (2 lymph nodes; 1 liver; 1 lung). a total of 20 partial rdrp gene sequences (16 from spotted hyenas, 3 from african lions and 1 from bat-eared foxes) were obtained and used for the phylogenetic analysis, together with publically available sequence data from 25 representatives of all sapovirus genogroups, divergent unclassified sapoviruses, and other genera in the caliciviridae family, including norovirus and vesivirus. the sapovirus strains from wild carnivore species in the serengeti ecosystem were placed together in one independent monophyletic cluster (fig 2) , and separately from all recognized sapovirus genogroups (gi to gv) and other unclassified sapoviruses. nucleotide sequence comparison between strains within the african wild carnivore group and other sapoviruses revealed low nucleotide similarity, ranging from 70.4% ± 1.4 with two domestic dog strains to 56.2% ± 1.5 with sequences from genogroup gii strains. at the amino acid level the highest similarity was with strains within genogroup giv (84.9% ± 1.5) and the lowest with strains from one bat species in asia (74.8% ± 0.6). nucleotide sequence comparison with members of other genera in the calicivirus family known to infect carnivores also showed low similarity values (feline and canine calicivirus; 60.3% ± 1.7 and 59.3% ± 2.1, respectively, norovirus from a captive african lion; 56.1% ± 0.9). within the group of african wild carnivore strains, the sapovirus strains from spotted hyenas grouped together and separately from those obtained from african lions and bat-eared foxes (fig 2) . one strain from a member of the p clan (all from members of the p study clan), all the clusters contained variants obtained from members of at least two of the three study clans. notably, one variant obtained from a bat-eared fox in 2011 was placed separately from those obtained from spotted hyenas in that same year, but close to the three variants obtained in 2010 from african lions. comparison of nucleotide sequences revealed that the average similarity between all strains from spotted hyenas (96.1% ± 3.8) was lower than between strains from african lions and bateared foxes (99.1% ± 0.3). the percentage similarity from the variant in 2007 placed separately from the other spotted hyena variants was lower (87.4% ± 0.6) than that of all the other spotted hyena variants that grouped more closely (97.4% ± 1.6). however, comparison at the amino acid level revealed that all strains from spotted hyenas were identical. the average similarity of the sequences from african lions and bat-eared foxes was 99.2% ± 0.9 (with differences at two amino acid positions). the average nucleotide and amino acid similarity between the spotted hyena strains and those from african lions and bat-eared foxes was 85.1% ± 1.0 and 99.6% ± 0.7, respectively. overall, sapovirus infection prevalence (combining results from fecal and tissue samples) in spotted hyenas was 34.8% (180/517 samples, table 1 ). infection prevalence between 2001 and 2012 (fig 3) fluctuated substantially between years (log likelihood ratio = 69.157, df = 11, p < 0.00001). we considered infection prevalence in any given year equal to or above 40% as indicative of an outbreak of sapovirus infection in that year. by this definition, 2003, 2004, 2006, 2007, 2010 were considered 'outbreak years' , and 2005, 2008, 2009, 2011 were considered 'non-outbreak years' in which infection prevalence was below 40%. of the 16 partial rdrp gene sequences obtained from spotted hyenas, 11 of these were from outbreak years, three were from non-outbreak years and two were from years that could not be classified (2002). in the phylogenetic tree strains from non-outbreak years clustered with those from outbreak years (fig 2) . to determine whether the prevalence of sapovirus infection was affected by age, we screened feces from animals in different age categories (i.e., cubs, subadults and adults). we found no significant differences in infection prevalence between different age categories across all years (chi square test, likelihood ratio = 2.045, df = 2, p = 0.36), in non-outbreak years (likelihood ratio = 3.860, df = 2, p = 0.15), or outbreak years (likelihood ratio = 3.331, df = 2, p = 0.19). we screened for sapovirus rna in feces obtained from 91 individuals on two separate occasions (s2 table) . of these, 15 individuals were sampled on at least three separate occasions, and from five of these animals on a fourth occasion ( table 2) . results revealed 32 transitions of an individual from sapovirus rna negative to positive and 15 transitions from positive to negative. in many cases the infection status did not change between sampling dates, for both initially negative (negative to negative, 53 cases) and initially positive individuals (positive to positive, 11 cases). we found no cases of transitions from positive to negative to positive ( table 2) . transition intervals were similar between first and second, second and third, and third and fourth sampling date (kruskal-wallis test, h = 2.567, df = 2, p = 0.28). we therefore analyzed the relationship between the duration of time intervals between successive samples and changes in infection status without regard to the number sampling repeats per individual. the duration significantly varied between different categories of changes of infection status (survival analysis, log-rank test, log-likelihood ratio = 10.114, df = 3, p = 0.018). similar intervals were observed for changes in infection status from negative to positive (mean: 466.6 days, 95% c.i. 285.5-647.6 days, median 158 days, n = 32) and from positive to negative (mean: 602.8 days, 95% c.i. 185.8-1019.8 days, median 180 days, n = 15). the shortest and longest intervals were observed when there was no change in status: negative to negative (mean: 241.1 days, 95% c.i. 132.5-349.7 days, median 82 days, n = 53) and positive to positive (mean: 769.0 days, 95% c.i. 412.5-1125.5 days, median 715 days, n = 11), respectively. we used a mixed-effects binary logistic regression model to test factors influencing the likelihood of infection in spotted hyenas (log-likelihood ratio = 16.717, df = 4, p = 0.0022, n = 484 samples from 380 individuals with complete information). the results revealed that infection was not significantly altered by either contact rates or the extent of an individual's range ( table 3 ). the likelihood of infection significantly declined as clan size increased: with every additional individual in the clan, clan members were 1.02 times less likely to be infected with sapovirus ( table 3) . as the actual clan sizes ranged from 65 to 145 individuals, this implied a more than two-fold change in the likelihood of infection across the observed range of clan sizes (fig 4) . correspondingly, median clan sizes were significantly lower during outbreak (median = 77, mean = 83.6, with 95% c.i.: 81.4-85.9) than non-outbreak (median = 89, table 2 . rt-pcr fecal screening results for known spotted hyenas sampled at least three dates. this is the first report of sapovirus infection in wildlife species in africa. our results extend the host species range for this genus to include the spotted hyena, african lion and bat-eared fox. prior to our study, sapovirus infection in carnivores worldwide was not known from any species belonging to the felidae (including the domestic cat) [72] , or hyaenidae, but was reported only for species in the families otariidae (californian sea lion) [23] , mustelidae (mink) [25] and canidae (domestic dog) [26] . our phylogenetic analysis based on partial rdrp gene sequences revealed that sapovirus strains from wild carnivores in the serengeti ecosystem formed a monophyletic group that was distinct from other sapovirus strains worldwide, including strains from the three previously identified carnivore hosts (fig 2) . strains from spotted hyena formed a separate sub-group from those obtained from african lions and bat-eared foxes, even within the same sampling year (fig 2) , suggesting that strains circulating in the spotted hyena population are distinct from those in the african lion and bat-eared fox populations. evidence for a degree of speciesspecificity in host range is apparent in other viruses of carnivores in the serengeti ecosystem. genetically distinct alphacoronavirus variants infect spotted hyenas and sympatric silverbacked jackal during the same year [35] , genetically distinct strains of kobuvirus infect domestic dogs and wild carnivores [36] , and during the 1993/1994 canine distemper epidemic in the serengeti np, genetically distinct strains circulated in non-canids (african lion and spotted hyena) and canids (domestic dog and bat-eared fox) [73] . more extensive characterization of sapovirus strains infecting carnivore species in the serengeti ecosystem would clarify their host range and help identify which species in the large carnivore guild are infected with sapovirus. currently it is not known whether or not domestic dogs and domestic cats in africa are infected with sapovirus. our results support the conclusion of previous studies, which emphasize the importance of long-term monitoring when documenting the genetic diversity of sapovirus strains [20, 28, 31] . clearly we would have detected far less genetic diversity in our partial rdrp gene sequence data had our sampling of spotted hyenas been limited to a time frame of one or two years (fig 2) , and particularly if sampling was (by chance) only undertaken during non-outbreak years when infection prevalence was low (fig 3) . samples obtained during outbreak years revealed considerable genetic diversity; for example from 2006 to 2007 we obtained sequence data from five different variants, including the distinct 2007 variant (kt777556) which was the least similar to all others from this host species. as we were not able to sequence data from all rt-pcr positive samples, we cannot exclude the possibility that the genetic diversity among spotted hyena strains was higher than our results indicate. even so, in line with a previous study [74] , our result show that sequence data from the non-structural rdrp gene yields useful information on the genetic diversity of circulating sapovirus strains. some outbreaks of sapovirus infection in humans can be linked to the emergence of specific genotypes [18, 75, 76] , suggesting that herd immunity against prevailing genotypes may be evaded by the emergence of genetically novel strains. our long-term monitoring of sapovirus infection in spotted hyenas revealed significant changes in yearly prevalence during the study (fig 3) and the occurrence of three outbreaks of infection. the highest infection prevalence (above 72.4%) occurred during an outbreak from 2002 to 2004, whereas infection prevalence in two later outbreaks (from 2006 to 2007 and in 2010) was considerably lower. presumably the 2002/2004 sapovirus outbreak induced herd immunity to the genetic strains that circulated in spotted hyenas during this period, but our phylogenetic analysis (fig 2) did not reveal the emergence of genetically distinct strains in response to this. even so, our partial sapovirus rdrp gene sequence data are insufficient to draw strong conclusions. for this, a more extensive genetic investigation is needed, particularly using the vp1 gene used to place sapoviruses in genogroups [16, 21] , that may reflect the antigenic relationships between sapovirus [77] . overall sapovirus infection prevalence in spotted hyenas (34.8%) in the serengeti np was several magnitudes higher than the prevalence reported for the domestic dog (< 2%) [26, 72] and the bat h. pomona (1.6%) [28] . moreover, our long-term monitoring reveals that infection prevalence in spotted hyenas was typically high, being above 20% in most years (fig 3) . there has been much discussion about the effect of human age on sapovirus infection (reviewed by [16] ), mostly based on studies on individuals with gastrointestinal infections. however, there is growing evidence from research on humans with and without clinical symptoms which demonstrates sapovirus infection across a wide range of ages [76] , including elderly people [78] . our results on sapovirus infection across different age categories indicate that the likelihood of infections in spotted hyenas was not significantly influenced by age ( table 1) . the long-term perspective of our study allowed us to assess the sapovirus infection status of several individually known spotted hyenas on different sampling dates. several animals transitioned from positive to negative, and we interpret this to indicate that they successfully cleared the virus following infection. if, following an initial infection, spotted hyenas gained long-term immunity against further infection, infection prevalence should decline with age. as prevalence amongst adults reached almost 50% during outbreak years, our results do not provide strong evidence for long-term immunity in this species (table 1) . even so, we found no individual that changed from rt-pcr positive to negative to positive (table 2) which would have provided direct evidence of re-infection. during outbreaks of sapovirus in humans and pigs, cases of re-infection with sapovirus belonging to different genogroups have been reported in both species, suggesting genogroup-specific immunity for sapoviruses [74, 79, 80] . more extensive investigation of the genetic diversity across strains circulating in our spotted hyena population is needed to determine whether sapovirus strains induce (1) short-term immunity, which would permit re-infection with strains from the same sapovirus genogroup, (2) genogroup-specific immunity, in which re-infection would involve strains from different genogroups, or (3) possibly a complex interplay between the two, as hypothesized for the genetically and antigenically diverse norovirus which is closely related to the sapovirus genus [81, 82] . interestingly, infection status depended on the length of time between repeated samples. animals that were negative on two separate dates (table 2 ) had the shortest median period between sampling dates (82 days), those that changed from negative to positive (158 days) and from positive to negative (180 days) had an intermediate median number of days, whereas animals that were positive on both sampling days (715 days) had the longest median period. taken together, these results suggest that when a spotted hyena is infected, infection is cleared, and reinfection is unlikely within a period of several months, which is consistent with the idea that exposure to sapovirus does not provide long-term immunity against further infection. in humans, sapovirus shedding often subsided 14 days after the onset of illness [83] , but can persist for up to 38 days [74] . hence, we speculate that the spotted hyenas which were positive on two sampling dates several months apart were animals that were re-infected rather than individuals with persistent long-term infections. however, currently we cannot exclude the possibility that there may be spotted hyenas that shed sapoviruses for periods spanning several months. spotted hyena social and ranging behavior has been shown to structure transmission routes and the likelihood of infection by a broad range of pathogens [35, [41] [42] [43] [44] ]. yet when we tested whether these two factors influenced sapovirus infection in this species, neither the predicted effect of contact rates based on known patterns of greeting ceremonies, nor the extent of an individual's range significantly influenced the likelihood of infection in our study population (table 3) . clan size was a significant factor, but contrary to our expectation, the likelihood of infection declined with increasing clan size. this phenomenon is known in the behavioral literature as the attack-abatement effect [84] or as the encounter-reduction effect [48, 85, 86] . in multi-species host assemblages the same phenomenon in the ecological literature is termed a 'dilution' effect. at least five non-exclusive mechanisms have been identified [49] that can cause a reduction in infection incidence as the number of host species increases, but not all of them are relevant to intraspecific sapovirus infections in spotted hyenas. we have no evidence that sapovirus infection increases the death rate of infected individuals (mechanism 1), as no obvious clinical signs are associated with sapovirus infection in most spotted hyenas. although we lack a precise measure of the recovery rate (mechanism 2), the general absence of obvious clinical signs of sapovirus infection suggest that the recovery rate is already very high, so that a change in this factor is likely to be modest. a decrease in the density of susceptible individuals (mechanism 3) is unlikely unless this results from a substantial increase in the proportion of clan members immune to sapovirus infection, even if immunity is relatively transient. a substantial increase in the prevalence of immune clan members would result in far fewer sapovirus susceptible individuals. moreover, such an increase in herd immunity would probably also lead to a decrease in the prevalence of sapovirus excreting clan members, thereby reducing the probability of: (1) sapovirus transmission per encounter (mechanism 4) between clan members and (2) the encounter rate between susceptible and infectious individuals (mechanism 5) in a clan. further research and more detailed modeling of the interplay between clan size and the prevalence of clan members in different sapovirus infection states (susceptible, infected/excreting virus, and immune) is required to test this idea. in the context of our study, mechanism 5 (encounter rate between susceptible and infectious individuals) is similar to mechanism 3 (decrease in the density of susceptible individuals) because a reduced density of susceptible clan members caused by a rise in the prevalence of transient immunity would also curb the number of infected animals in a clan and may prevent their number growing in proportion to increasing clan size, and possibly holding them at or below a fixed number (threshold). an increase in the risk of internal pathogen infection with a decrease in a group size component has been reported by other studies [3, 42] . as sapoviruses cannot be cultured, with the exception of the strain pec cowden [50, 51] , knowledge of viral tropism and the receptor use for entry to host cells is limited. moreover, sapovirus typically infects intestinal tissue and to our knowledge is not known to infect other tissues, as illustrated by the absence of viral rna in any extra-intestinal tissues in gnotobiotic pigs inoculated with pec cowden [87] . however, in a few dead spotted hyenas (typically road casualties) from which we opportunistically obtained tissue samples, we detected sapovirus rna most often in the spleen and occasionally also in lymph nodes, intestines and the liver. to our knowledge this is the first report of sapovirus rna in extra-intestinal tissues following natural infections. the possibility that sapoviruses disseminate to extra-intestinal tissues may be of clinical importance [88] . notably, in asymptomatic mice shedding murine norovirus in feces, viral rna was also found in several extra-intestinal organs, including the liver, spleen and lymph nodes [89] . studies on caliciviruses infecting wild carnivores have focused on feline calicivirus (fcv, genus vesivirus, family caliciviridae). for example, serological surveys documented that african lions [90] and spotted hyenas in the serengeti ecosystem were exposed to fcv [91] with a high prevalence. our phylogenetic analysis shows that the variants we report from wild carnivores in africa are distinct from both fcv and canine calicivirus (fig 2) . a norovirus (genus norovirus, family caliciviridae), genetically related to human noroviruses was reported from a captive african lion [92] and subsequently identified in domestic dogs and domestic cats [93, 94] . combining these findings with our results suggests that the spotted hyena and african lion can be infected by viruses belonging to three different genera of the family caliciviridae. we provide the first report of sapovirus infection in wild carnivore species in africa, including the spotted hyena, african lion and bat-eared fox. long-term monitoring revealed considerable genetic diversity of variants from these species which were phylogenetically distinct from previously reported sapovirus strains from other geographical areas worldwide. sapovirus prevalence in the spotted hyenas varied between 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human and medical virology prevalence of antibodies to feline parvovirus, calicivirus, herpesvirus, coronavirus, and immunodeficiency virus and of feline leukemia virus antigen and the interrelationship of these viral infections in free-ranging lions in east africa antibodies to canine and feline viruses in spotted hyenas (crocuta crocuta) in the masai mara national reserve norovirus in captive lion cub (panthera leo). emerg infect dis detection and molecular characterization of a canine norovirus discovery and genomic characterization of noroviruses from a gastroenteritis outbreak in domestic cats in the us key: cord-000721-leedutqo authors: nawaz, sameena; allen, david j.; aladin, farah; gallimore, christopher; iturriza-gómara, miren title: human bocaviruses are not significantly associated with gastroenteritis: results of retesting archive dna from a case control study in the uk date: 2012-07-24 journal: plos one doi: 10.1371/journal.pone.0041346 sha: doc_id: 721 cord_uid: leedutqo gastroenteritis is a common illness causing considerable morbidity and mortality worldwide. despite improvements in detection methods, a significant diagnostic gap still remains. human bocavirus (hbov)s, which are associated with respiratory infections, have also frequently been detected in stool samples in cases of gastroenteritis, and a tentative association between hbovs, and in particular type-2 hbovs, and gastroenteritis has previously been made. the aim of this study was to determine the role of hbovs in gastroenteritis, using archived dna samples from the case-control infectious intestinal disease study (iid). dna extracted from stool samples from 2,256 cases and 2,124 controls were tested for the presence of hbov dna. all samples were screened in a real time pcr pan-hbov assay, and positive samples were then tested in genotype 1 to 3-specific assays. hbov was detected in 7.4% but no significantly different prevalence was observed between cases and controls. in the genotype-specific assays 106 of the 324 hbov-positive samples were genotyped, with hbov-1 predominantly found in controls whilst hbov-2 was more frequently associated with cases of gastroenteritis (p<0.01). a significant proportion of hbov positives could not be typed using the type specific assays, 67% of the total positives, and this was most likely due to low viral loads being present in the samples. however, the distribution of the untyped hbov strains was no different between cases and controls. in conclusion, hbovs, including hbov-2 do not appear to be a significant cause of gastroenteritis in the uk population. in 2005 a novel parvovirus was discovered in respiratory secretions of young children and was termed human bocavirus (hbov-1) [1] . other important members of the parvoviridae family include b19 which causes fith disease and human parvovirus 4 (parv 4) which has not yet been associated with a disease [2] . parvoviruses in animals are generally associated with systemic disease but also with respiratory and enteric symptoms [3, 4] . since the discovery of hbov-1 three other hbov genotypes have been described, hbov-2, hbov-3 and hbov-4. the association between hbov-1 and respiratory disease has previously been well established [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19] . although all hbovs have also been detected in stool samples with prevalences ranging from ,1% to 20%, only hbov-2 has been reported to be associated with symptoms of gastroenteritis [20] . nevertheless, the role of hbov2 as an aetiological agent of gastroenteritis has not been clearly confirmed, furthermore, to date, no clear association between the presence of hbov-3 and hbov-4 and disease has been established [21, 22] . recent seroepidemiological studies indicate that exposure to hbovs occurs early in life and 90% of the population are seropositive by the age of 5, although differences were reported in the seroprevalence of type-specific antibodies to the different hbovs, which suggested that hbov-1 infections are more prevalent [23] . the infectious intestinal disease study (iid study) was a large case control study of gastroenteritis carried out in the uk between 1993-1996 [24] with the aim to determine the burden and aetiology of sporadic cases iid in the uk population. initially, the use of classical microbiology diagnostic methods and electron microscopy (for virus detection) failed to detect a potential aetiological agent or toxin in 49% of the cases [25] . retesting of the archived samples from this study using molecular methods for the detection of enteric viruses, bacteria and protozoa revealed viruses to be the most common aetiological agents of gastroenteritis,the diagnostic gap for iid was reduced to 25% from 49% [26] . the aim of the present study was to evaluate the role of hbovs in iid in the uk population, using archived dna samples from the matched case-control iid-1 study [26] . in addition, the presence specifically of hbov-1, 2 or 3 was investigated in order to determine any possible associations between specific hbov genotypes and iid. a total of 4,380 archived dna from the iid study [26, 27] were tested for the presence of hbov dna. this archive comprised dna extracted from stool samples from 2,256 cases and 2,124 controls. the qpcr assay targeted the ns1 gene (ratcliff et al., unpublished method, personal communication) and was performed using an abi taqman7500. oligonucleotide primer and probe sequences and positions are described in table 1. the reaction consisted of 0.1 m ddt (invitrogen), 1x platinum quantitative pcr supermix-udg (invitrogen), pan-hbov-f and pan-hbov-r primers each at a concentration of 100 mm, pan-hbov-ns1 probe at 10 mm concentration, rox 25 mm (invitrogen) 2.5 ml of template dna and rnase free water to a final reaction volume of 25 ml. the amplification consisted of an initial denaturation at 95uc for 10 min, followed by 40 cycles with denaturation at 95uc for 15 sec, annealing at 55uc for 30 sec and extension at 60uc for 45 sec. hbov-1, 2 and 3-specific primer pair and probes were designed in house through alignment of sequence data available in genbank. the reaction conditions are as follows; 1x platinum quantitative pcr supermix-udg (invitrogen), hbov-ns1-1f, hbov-1r, hbov2-r and hbov3r primers each at a concentration of 20 mm, the hbov1,2 and 3 probe at 10 mm concentration, rox 25 mm (invitrogen), 2.5 ml of template and rnase free water was added to a final reaction volume of 25 ml. the amplification conditions for the typing assay are the same as those described in the hbov ns1 detection assay above. plasmids containing a 1773 bp and a 1737 bp region of the ns1 encoding gene of hbov-1 and hbov-3 respectively were used for assay optimisation and as controls. control material was kindly provided by r. ratcliff, adelaide, australia. the controls were also used in order to generate a standard curve for use with the pan-hbov assay in order to allow for normalisation of the data generated including the comparison of relative sensitivities of the different assays and for quantitation of dna present in each of the positive samples. the standard curve was generated using the plasmid containing a genome segment of the hbov-1 and consisted of a series of 10 fold dilution containing from 300,000 copies/ml down to 3 copies/ml. inter-and intraassay reproducibility was analysed by performing replicate testing of the standards in a single run (x11) and repeated runs (x2), and the standard curve was also included in each assay run for quality control and normalisation of results. a subset of 17 samples positive in the pan-hbov assay but which failed to amplify in the type-specific assays were confirmed using an alternative method published elsewhere [28, 29] , and 6 were further confirmed though direct sequencing of the amplicons obtained after purification either from solution or agarose gels using agencourt ampure (beckman coulter, usa) and geneclean spin kit (qbiogene), respectively, following manufactures protocols. the chi-squared test was used in order to evaluate the significance of differences observed between groups. for comparison of median values (analysis of ct values) the mann witney utest was used. prevalence odds ratio (por = pcases/(1-pcases)/ pcontrols/(1-pcontrols)) was calculated in the total cohorts and by age group. table 1 . hbov-specific oligonucleotide primers and probes (all located at the ns gene). sequence ( a total of 7.4% of the samples tested were positive for hbov. no statistically significant differences were seen in the prevalence of hbov between cases and asymptomatic controls, por = 0.79 (table 2) . peak hbov infection was observed in children under the age of 5, both in cases and controls, with significantly higher hbov incidence in children between 1 and 4 in asymptomatic controls than in the cases of gastroenteritis (por = 0.6; p,0.02). the number of hbov positives in older age groups was too small for meaningful statistical analysis. the average ct values were 34.5 and 34.8, and the median ct values were 36.3 and 37.4 in cases and controls respectively (see distribution in figure 1 ). the majority of hbov-positives in both cases and controls had copy numbers ranging between 30 and 299 copies/reaction (or between 4.5610 3 and 4.5610 4 copies/ml of feaces). the distribution of hbov viral loads between cases and controls was comparable and the median ct values between cases and controls were not significantly different (u-test; z = 0.458139, p.0.05). hbov dna was found in 149 (46%) samples in the absence of other co-pathogens (table 3) . no statistically significant differences were observed in the proportion of cases or controls in which hbov was found as a single organism or in the presence of one or more pathogens in the cohort as a whole, however in the 1-4 years of age group, hbov in the absence of any other enteric pathogens was found in 29% of the cases, but in 56% of the controls (p,0.05). hbov infections were detected year round although a peak was observed in the spring/early summer months, between april and june 1994 (figure 2 ). hbov dna was found in 48.8% and 45.7% of female cases and controls, respectively. the distribution of hbov among females and males was not significantly different from the distribution of females and males in the entire cohort which was 53% and 47%, respectively. a total of 106 (32.7%) hbov positives were genotyped, whilst 218 (67.3%) remained untyped after testing in the hbov types 1, 2 or 3 specific assays (table 4 ). hbov-1 detection was found predominantly in controls, (p,0.001) and hbov-2 was predominantly associated with cases (p,0.01). the prevalence of hbov-3 was not significantly different between cases and controls. hbov-1 and -3 were predominantly found in children (table 4) . hbov-2 in the absence of any other pathogen was detected in 17 (81.3%) of the cases, compared to 9 (47.4%) of the controls. in cases, hbov-2 was found across the age groups, although more frequently in children ,5, whereas in controls they were found predominantly in children ,5 with only 1 example in an adult ( table 4 ). the prevalence of hbov-2 in children ,5 years old was however not significantly different between cases and controls, 2.8% and 2.6%, respectively. a subset of hbov that were negative in the type 1,2 or 3specific assays were confirmed in an alternative pan-hbov pcr, and sequencing of a small number confirmed them as types 1, 2 or 3. the majority of the untyped samples (70%) had a ct value of .37 in the screening pan-hbov pcr, indicative of low viral loads being present in the samples. this represents the largest study to date investigating the role and distribution of hbovs infections in community acquired sporadic gastroenteritis and in asymptomatic controls. the prevalence of hbov infection in the uk population was found to be 7.4% across all ages, with a higher percentage of the infections occurring in children ,5 years of age (19%). however, the prevalence of hbov infections was comparable in cases of gastroenteritis and in age-matched asymptomatic controls. although the presence of enteric pathogens, eg norovirus or rotavirus, in asymptomatic individuals is well documented, a significantly higher prevalence of the pathogen is seen in cases than in the controls [26] . therefore, our data suggests that hbov are not causally associated with gastrointestinal disease in the uk population as a whole, nor in children. the prevalence of detection of hbov in stool samples in previous studies varies widely (see summary in table 5 ), but most coincide in reporting the highest prevalence in children. hbov infections were detected all year round in the uk although a tentative peak was observed in the spring/early summer months in 1994 (between april and june). different seasonal patterns in the peak prevalence of hbov have been reported in different countries (see table 5 ), of the 324 hbov positive samples, 106 (32.7%) were genotyped in the type-specific assays. hbov-1 was found predominantly in controls (p,0.001) and the prevalence of hbov-3 was similar in cases and controls. both hbov-1 and -3 were predominantly found in children. hbov-2 was predominantly associated with gastroenteritis cases (p,0.01). the overall prevalence in cases was 1.4% and 0.8% in controls, however, in children ,5 year of age, the prevalence in cases and controls was similar, 2.8% and 2.6%, respectively. the prevalence of hbov-2 in children in the uk was significantly lower than that reported in a study in australia, in which hbov-2 was detected in 17.2% and 8.1% of the cases and controls, respectively [22] . the findings of the study in australia lead to the proposal of hbov-2 as an important aetiological agent of infantile gastroenteritis. it is noteworthy however, that in the australian study, the association of hbov-2 with gastroenteritis was only significant when cases with a bacterial co-pathogen were included in the analysis. although in the present study hbov-2 in the absence of other enteric pathogens was found more frequently in cases than in controls, the small numbers found in such large study suggest that the role of these viruses in iid, if any, is likely to be small. a lack of correlation between hbovs or hbov-2 and paediatric gastroenteritis was also reported in several smaller studies published elsewhere [30, 31, 32] . a total of 67% of the hbov-positive samples could not be genotyped using the genotype-specific pcr assays. the majority of these untyped samples (70%) had ct values .37. this suggests that failure to type may be associated with low viral loads and differences in the relative sensitivities of the genotyping assays compared to the detection assay. although under experimental conditions and using plasmid controls the sensitivities of all assays were comparable, it is likely that when applied to true clinical samples the sensitivity of the type-specific assays was inferior, possibly due to as yet not identified strain variability within genotypes. also, a hbov type 4-specific assay was not included in this study, therefore, any possible hbov4 infections would not have been typed. of the panel of samples that were tested in an alternative pan-hbov pcr, the strains typed through sequencing were hbov-1 (2 samples), hbov-2 (1 sample) and hbov-3 (3 samples). furthermore, the distribution of untyped hbovs was not significantly different in cases and controls. hbovs in the absence of other enteric pathogens were seen in 46% of the hbov-positive samples, and more frequently in the controls, 50.3% vs 40.9% in cases. no significant difference in hbov load was observed between cases and controls, or between the samples positive for hbov alone or in the presence of other pathogens. previous studies have investigated the relationships between viral load and disease severity [33, 34, 35, 36, 37] . in respiratory infections significantly higher hbov loads were seen in samples collected from children positive for hbov alone than in those from children with co-infections. in respiratory infections also, viral loads .10 4 were associated with disease, whereas loads ,10 4 were associated with asymptomatic children [33, 38] . this lead to the suggestion that higher viral loads are indicative of a causative role of hbov in respiratory infections [33, 38] . however, brieu et al [38] found no significant correlation between viral load and clinical symptoms or disease severity. in conclusion, the results obtained from investigating for the presence of hbov dna in archived dna samples from a large and previously well described case-control study of iid suggest that hbov, including hbov-2,do not appear to be a significant cause of gastroenteritis in the uk population, and particularly in the paediatric population. although hbovs are relatively frequent across all ages, and in particular in preschool age children, they are found just as frequently among children and adults without symptoms of gastroenteritis. cloning of a human parvovirus by molecular screening of respiratory tract samples new dna viruses identified in patients with acute viral infection syndrome epidemiological studies of parvovirus infections in calves on endemically infected properties minute virus of canines (mvc, canine parvovirus type-1): pathogenicity for pups and seroprevalence estimate frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections human bocavirus: prevalence and clinical spectrum at a children's hospital detection of human bocavirus in canadian children in a 1-year study complete coding sequences and phylogenetic analysis of human bocavirus (hbov) the association of newly identified respiratory viruses with lower respiratory tract infections in korean children human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand human bocavirus quantitative dna detection in french children hospitalized for acute bronchiolitis human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus clinical features and complete genome characterization of a distinct human rhinovirus (hrv) genetic cluster, probably representing a previously undetected hrv species, hrv-c, associated with acute respiratory illness in children detection of human bocavirus in japanese children with lower respiratory tract infections epidemiological profile and clinical associations of human bocavirus and other human parvoviruses human bocavirus in iranian children with acute respiratory infections human bocavirus infection, people's republic of china human bocavirus in hospitalized children frequent detection of bocavirus dna in german children with respiratory tract infections a newly identified bocavirus species in human stool human bocaviruses are highly diverse, dispersed, recombination prone, and prevalent in enteric infections a novel bocavirus associated with acute gastroenteritis in australian children seroepidemiology of human bocaviruses 1-4 a report of the study of infectious intestinal disease in england.: food standard agency. the stationery office a study of infectious intestinal disease in england: microbiological findings in cases and controls detection by pcr of eight groups of enteric pathogens in 4,627 faecal samples: re-examination of the english case-control infectious intestinal disease study (1993-1996) detection of viral, bacterial, and parasitological rna or dna of nine intestinal pathogens in fecal samples archived as part of the english infectious intestinal disease study: assessment of the stability of target nucleic acid human bocavirus in children hospitalized for acute gastroenteritis: a case-control study evidence of human coronavirus hku1 and human bocavirus in australian children human bocavirus 2 in children detection of human bocavirus-2 in children with acute gastroenteritis in south korea high prevalence of human bocavirus 2 and its role in childhood acute gastroenteritis in china human bocavirus and acute wheezing in children human respiratory syncytial virus (hrsv) rna quantification in nasopharyngeal secretions identifies the hrsv etiologic role in acute respiratory tract infections of hospitalized infants quantitation of group a rotavirus by real-time reverse-transcription-polymerase chain reaction: correlation with clinical severity in children in south india diagnosing rotavirus a associated iid: using elisa to identify a cut-off for real time rt-pcr diagnosing norovirus-associated infectious intestinal disease using viral load human bocavirus infection in children with respiratory tract disease this study was conducted using existing material and data form the iid-1 study, which was funded by the food standards agency. we would like to thank the iid study executive committee for their support in approving the use of the archive dna material for this study. key: cord-002560-pue5q5wp authors: moreno, paloma s.; wagner, josef; mansfield, caroline s.; stevens, matthew; gilkerson, james r.; kirkwood, carl d. title: characterisation of the canine faecal virome in healthy dogs and dogs with acute diarrhoea using shotgun metagenomics date: 2017-06-01 journal: plos one doi: 10.1371/journal.pone.0178433 sha: doc_id: 2560 cord_uid: pue5q5wp the virome has been increasingly investigated in numerous animal species and in different sites of the body, facilitating the identification and discovery of a variety of viruses. in spite of this, the faecal virome of healthy dogs has not been investigated. in this study we describe the faecal virome of healthy dogs and dogs with acute diarrhoea in australia, using a shotgun metagenomic approach. viral sequences from a range of different virus families, including both rna and dna families, and known pathogens implicated in enteric disease were documented. twelve viral families were identified, of which four were bacteriophages. eight eukaryotic viral families were detected: astroviridae, coronaviridae, reoviridae, picornaviridae, caliciviridae, parvoviridae, adenoviridae and papillomaviridae. families astroviridae, picornaviridae and caliciviridae were found only in dogs with acute diarrhoea, with astroviridae being the most common family identified in this group. due to its prevalence, characterisation the complete genome of a canine astrovirus was performed. these studies indicate that metagenomic analyses are useful for the investigation of viral populations in the faeces of dogs. further studies to elucidate the epidemiological and biological relevance of these findings are warranted. interest in the virome, or the entire population of viruses present in a biological sample, has increased recently due to improved availability of high throughput sequencing or next generation sequencing (ngs) technologies, and improved metagenomic analytical methods [1, 2] . a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the virome comprises all types of viruses, including those that infect prokaryotic and eukaryotic organisms, dna or rna viruses, and viruses that cause acute or chronic infections. many of these viruses are difficult or impossible to propagate in cell culture, and molecular detection is difficult as no common gene such as the ribosomal 16s gene that is present in bacterial species exists in viruses. these limitations have hindered the identification and characterisation of uncultured viruses [3, 4] . recently, due to the advent of molecular enrichment protocols, high throughput sequencing and new metagenomic analytical methods we are now able to explore, identify and characterise viruses from different biological and environmental samples with a greater capacity [2, [5] [6] [7] [8] [9] [10] [11] in studies of human faeces, the virome has been shown to include viruses that infect eukaryotic organisms and viruses that infect prokaryotes (bacteriophages) [2, 5, [12] [13] [14] [15] [16] [17] [18] . bacteriophages have been reported in many studies to be the most frequently detected viral constituent in the gut of humans [1, 2, 5, 8, 16, 19, 20] . the faecal virome has been characterised for several animal species including pigs, bats, cats, pigeons, horses and ferrets [2, 6, 7, [9] [10] [11] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] . in dogs, the presence of enteric viral pathogens such as canine parvovirus, coronavirus, rotavirus and distemper virus (paramyxoviridae) have been identified only through targeted studies [32] [33] [34] [35] . to date, only one published study has used high throughput sequencing to investigate the faecal viral population in diarrhoeic dogs [6] . these investigators analysed faeces from dogs with acute diarrhoea and detected two new virus species, canine sapovirus and canine kobuvirus; known canine enteric viruses such as canine coronavirus, canine parvovirus, canine rotavirus as well as plant and insect viruses were also reported [6] . the aim of this study was to describe the faecal virome of samples collected from healthy dogs, and compare these findings to the faecal virome of dogs with acute diarrhoea in australia, using an illumina miseq shotgun metagenomic sequencing approach. a total of 16 faecal samples (8 from healthy and 8 from diarrhoeic dogs) were subjected to viral nucleic acid extraction, followed by nucleic acid enrichment, reverse transcription, random amplification and the creation of two libraries for each sample (dna and cdna), before being sequenced by illumina miseq platform (table 1) . after sequencing, a total of 93,744,624 raw sequences were generated. all raw sequences are available in ncbi, (bioproject id: prjna380672). after trimming by quality 80,414,313 high quality reads (hqrs) were available. all sequences corresponding to dog and cellular organisms (383,785 and 27,825,631 respectively) were removed and the resultant reads were de novo assembled (fig 1) generating in total 1,672,615 contigs and singletons (reads). from these contigs/singletons 1,285,171 (76.8%) had no hits in the database (s1 table) . further analysis of contigs/singletons with no hits confirmed most sequences had no hits, while a limited number matched bacterial, human or animal sequences with a very low coverage. in addition to the contigs/singletons with no hit in the database, some contigs/singletons matching to cellular organisms and some with low complexity were identified, however, were not analysed any further (s1 fig) . sequences similar to twelve viral families were identified in faecal samples from healthy and diarrhoeic dogs after analyses with two different bioinformatic pipelines and comparison against viral and ncbi databases (fig 1) . eight of these viral families infect eukaryotic organisms, and the remaining four infect prokaryotes. despite the known bias of sispa in the resultant sequences after de novo assembly [36], we report the number of contigs/singletons matching viral families and the subsequent analysis of alignments with the lowest common ancestor according to megan v5.2.1 [37]. faecal samples were collected from eight healthy dogs (table 1) . genetic analyses identified 659,696 contigs/singletons with no hits and 3968 contigs/singletons were classified as viral, matching to five viral families that infect eukaryotes and four that infect prokaryotes. 75.9% (3012 contigs/singletons) of the total number of viral contigs/singletons were classified as bacteriophages in the healthy canine faecal virome. bacteriophages were detected in the faeces of all dogs in this group and belonged to caudovirales order and microviridae family. viral contigs/singletons from five eukaryotic virus families were identified in faecal samples from 4 of the 8 healthy dogs (table 1) . three out of five viral families detected were dna viruses. adenoviridae and papillomaviridae were detected in a single sample containing only (table 1) , however, these were all detected in one sample. after analysis, only 76 contigs/singletons matched the reference sequence of alphacoronavirus 1 (feline infectious peritonitis virus, nc_002306.3) and covered only 0.5% of the complete genome (minimum match: 75% and minimum overlap: 50), which represented 3.2% of fipv_gp02 (receptor binding molecule) region. contigs/singletons belonging to the reoviridae family were found in only one sample. genetic analysis revealed they covered between 11%-35.5% of vp1, vp2, vp3 and vp4 genes of reference sequences of rotavirus a (nc_011506-nc_011510). another eukaryotic viral family found in one healthy dog sample was parvoviridae, genetic analysis of the 3 contigs/singletons showed a coverage of approximately 3.5% of the complete genome of canine parvovirus reference sequence (nc_001539), or 9.3% of the polyprotetin ns1-ns2. in eight faecal samples from dogs with acute diarrhoea, a total of 625,475 contigs/singletons had no hits and 17,242 were identified as viral contigs/singletons comprising 6 eukaryotic and 4 prokaryotic viral families (table 1) . bacteriophages comprised 98.19% of the total of viral contigs/singletons and they were present in all individuals and were identified as belonging to order caudovirales and microviridae family. eukaryotic families found in this group were coronaviridae, parvoviridae, reoviridae, caliciviridae, astroviridae, and picornaviridae ( table 1) . the most common eukaryotic viruses identified were rna viruses (5/6 viral families). interestingly, all 8 samples in this group contained at least one eukaryotic family each. co-infection was identified in 6 individual dog samples from this group. from the 8 samples from dogs with acute diarrhoea, 2 different eukaryotic virus families were detected in five samples and 3 eukaryotic families were detected in one sample ( table 1 ). the most prevalent family identified was astroviridae, present in 5 dogs followed by reoviridae present in 4 of 8 dogs with acute diarrhoea (table 1) . astroviridae contigs/singletons from 5 dogs were compared with reference sequence of canine astrovirus (nc_026814.1), the lowest common ancestor according to megan, and they covered between 2.4% and 5% of compete genome and between 6.3% and 12.9% of the orf2. the sample with the most contigs/singletons was later characterised. reoviridae contigs/singletons found in 4 dogs were compared with reference sequence of rotavirus a (nc_011503.2) and 3 of them covered between 10.5% and 23.4% of the vp4 gene and the other sample covered 23.6% of the vp7 gene. furthermore, contigs/singletons matching to canine parvovirus, were found in 2 dog samples with acute diarrhoea. one of the samples covered approximately 5.8% of the complete genome of canine parvovirus reference sequence (nc_001539), corresponding to 17.6% of vp2. the other sample contained 41 contigs/singletons that matched to this same reference sequence and in total they cover 100% of vp1, 66.2% of polyprotein ns1 and ns2 (cpvgp1) and 87.9% of vp2 genes. in this group were also found contigs/singletons matching to the coronaviridae family, covering between 0.6% and 1.7% of the complete genome of reference sequence alphacoronavirus 1 (fipv, nc_002306.3). one dog with acute diarrhoea contained contigs/singletons similar to a canine norovirus (jf930689.1), covering approximately 7.9% of the complete genome. other dog sample had contigs/singletons similar to a canine kobuvirus (jn387133.1), covering 2.2% of complete genome. to further explore the high abundance of contigs/singletons from astroviridae family in dogs with acute diarrhoea and their absence in healthy dogs, a near complete full genome of a representative canine astrovirus was generated through sanger sequencing (dd1, table 1 ). the genome encoded the complete three open reading frames (orfs): orf1a, orf1b and orf2. the total length was 6513 nucleotides, excluding the 3' poly (a) tail and the nucleotide composition was 28% a, 22% g, 26% t, 23% c. the g/c composition was 45%. the genbank accession number for the canine astrovirus sequence is kx756441. a phylogenetic tree was constructed using the protein alignment from the conserved region of the capsid (orf2) of the astrovirus characterised in this study (dd1) and other canine astrovirus orf2, together with mamastrovirus sequences from different mammalian species, including a chicken astrovirus as an outgroup. the phylogenetic analysis grouped our canine astrovirus within the canine astrovirus clade. the closest canine astroviruses to our australian sample were from uk and china with an identity between 98.82%-99.41% (fig 2) . using next generation sequencing and metagenomics analysis, the virome in faecal samples from 8 healthy dogs and 8 dogs with acute diarrhoea is described. only a single previous shotgun metagenomic study investigating the faecal virome of dogs with diarrhoea has been reported. in that study, mammalian viruses were found in 15 samples and two new virus species were described [6] . our study analysed 16 faecal samples from dogs (8 healthy and 8 diarrhoeic), and identified eukaryotic viruses in 12 samples, including all diarrhoeic samples and 50% of the healthy samples. thus, 70% of canine faeces contained eukaryotic viruses, suggesting that mammalian viruses are a common component of the enteric microbial population in dogs. our results must be interpreted with caution, due to bias created by sispa. areas of exaggerated depth appear when the sispa method is used, creating artefacts during de novo assembly. this results in regions of repetitive sequences [36] . in order to overcome this bias, all contigs/singletons were analysed at family level and only for viral eukaryotic families were the results further analysed to evaluate what percentage they covered to some specific viral species. the most common viral contigs/singletons identified in both groups were bacteriophages, similar to previous findings from human and other animal faecal virome studies [2, 5, [11] . bacteriophages modify diversity of bacterial populations due to their lytic life cycle and also promote different characteristics in the bacterial population due to their lysogenic life cycle transferring genes such as encoding toxins or resistance to antibiotic [38] . this life-cycle may lead to bacteriophages conferring advantage to some bacterial species in the environmental niche [39]. therefore, it is possible that the greater amount of contigs/singletons corresponding to bacteriophages identified in the group of dogs with acute diarrhoea, when compared to healthy dogs, means a higher amount of bacteriophages. if so, bacteriophages could have generated a change in the normal balance of bacterial population resulting in dysbiosis, and ultimately causing diarrhoea. conversely, it could be that an initial change in the bacterial population in these dogs resulted from the acute diarrhoea [40, 41] is the cause of the variation in the bacteriophage population. in our sample population, the latter explanation is most likely, because in a shelter environment a higher number of circulating pathogens, changes in diet and a stressful environment could contribute to the dysbiosis associated with acute diarrhoea [42] . the bacterial microbiome and the analysis of contigs/singletons matching specific bacteriophages were not assessed in this study, therefore further microbiome/virome cross analysis is necessary to elucidate the association between bacteria and bacteriophages in dogs. however, even this analysis would be unlikely to determine the cause or effect relationship between bacteriophages and dysbiosis at a single point in time. the analysis of the lowest common ancestor of eukaryotic viral families, according to megan, identified eight eukaryotic virus species (table 1) . however, each of these results require validation by targeted pcr, or whole genome characterisation of each species, as the ngs results after sispa amplification may be biased and not accurate depiction at a species level [36] . sequences matching those of human viruses (adenovirus and papillomavirus) were found in one sample from a healthy dog. only one contig from each virus covering a very small percentage of the genome in both cases. this finding could suggest contamination during collection or processing. known enteric pathogenic families parvoviridae and coronaviridae were identified in samples from both healthy dogs and dogs with acute diarrhoea. interestingly, almost all positive samples were from puppies (between 4-8 months) that had been vaccinated less than one month prior to sampling. the lowest common ancestry analysis in megan of the contigs/singletons matching parvoviridae family, suggested they were canine parvovirus (cpv), but as cpv positive dogs (as tested by faecal antigen tests) were not included in this study it is highly likely these results represent vaccine derived sequences not detected by the cpv antigen detection kit, or represent a virus load below the level of detection. previous studies have demonstrated that modified live vaccine virus can be detected in faecal samples for extended periods of time after vaccination [43] . further genome characterisation of these canine parvovirus is warranted to confirm this hypothesis. three individual samples contained coronaviridae contigs/singletons, two of which were from puppies [nd10 and dd8] ( table 1 ) and one from an adult dog [dd1] ( table 1 ). our results are consistent with li et al 2011, who also reported the highest number of coronaviridae reads in one sample collected from a puppy [6] . canine coronavirus can be shed in faeces in high numbers for up to 156 days [44, 45] . these findings validate the affinity of the coronaviridae viral family to infect young individuals [46] , and present as a common enteric pathogen in a shelter environment [42, 45, 47] . the uncommon viruses, canine kobuvirus and canine norovirus, were identified only in samples from dogs with acute diarrhoea. previous studies have suggested these viruses may have some association with enteric disease in dogs, however, both viral species have been detected in both healthy dogs and dogs with diarrhoea [6, 48] our shotgun metagenomic sequence data indicated that the most frequent rna viral family in dog samples with acute diarrhoea was astroviridae, being identified in more than half of the diarrheal samples. [49] . in dogs, astrovirus has been previously detected mainly in puppies with diarrhoea, but has also been occasionally reported in healthy dogs [50] [51] [52] [53] [54] . the only previous report of a possible canine astrovirus in australia was described in canine faeces in the 1984, where astrovirus-like particles were detected using electronic microscopy in healthy dogs [55] . to date, canine astrovirus has been reported in usa [56] , china [51] , italy [50, 57, 58] , uk [52] , france [53] , brazil [59] , korea [60] and japan [54] . the first description of the complete genome of two canine astroviruses was reported by a group of researchers from the uk in 2015 [52] . the current study contributes the first description of the complete genome of a canine astrovirus identified in australia. in our study, using sanger sequencing a near complete genome of a canine astrovirus was assembled from one dog with acute diarrhoea. a phylogenetic tree, analysing the capsid region (orf2) of this australian canine astrovirus and other astrovirus sequences present in genbank, determined that it belonged to the canine astrovirus clade, very closely related to the canine astrovirus strains from the uk and china (fig 2) . it is interesting to note that all canine astrovirus positive samples, were collected from the same shelter and obtained within a short period of time (sept-nov 2012). we could infer that this virus was endemic at that time in that shelter, and or maybe could represent an outbreak of diarrhoea in the shelter within that period of time. a more sensitive test (i.e.: quantitative pcr) in a larger number of samples from cases and controls may be useful to better understand the potential role of astroviruses as an aetiological agent in acute diarrhoea of dogs. in this study we analysed the faecal virome in healthy dogs and compared these findings with the faecal virome of dogs with acute diarrhoea. known dna and rna viruses were found, together with different proportions of bacteriophages in each group. in addition, we described and characterised the first complete genome of a canine astrovirus in australia. future longitudinal studies analysing viruses, bacteria and other potential pathogens should be performed to assess the aetiology of diarrhoea in dogs and further elucidate the pathological importance of viruses found in dog intestines. faecal samples from a total of 16 dogs were obtained between september 2012 and march 2013. all dogs were aged between 2.5 months and 7 years; and comprised 5 females and 11 males of various breeds ( table 1) . all faecal samples were collected from a single shelter in melbourne (lost dogs home), australia. all samples were maintained at 4ë�c for up to 24 hrs, then were transported on dry ice before storing up to five aliquots of 500 mg of faeces each at -80ë�c until further analyses. information about age, sex, breed, diet, vaccination and deworming status was recorded for each dog (university of melbourne animal ethics committee approval ids 1413272.2 and 1112035.1). animals were determined to be healthy based on physical examination by a veterinarian and absence of any clinical signs of disease. faecal consistency was considered normal as per published criteria (faecal scoring chart, purina), and all dogs had been treated with deworming drugs for prophylaxis (ilium pyraquantal, troy or milbemax, novartis). all samples were lifted from the floor, first thing in the morning before cleaning, during november 2012. faecal samples from 8 dogs with an acute onset of diarrhoea (less than 3 days of duration), were collected by a veterinarian from within the animal's enclosure. all dogs with acute diarrhoea were tested for the presence of canine parvovirus antigen in faeces using the anigen rapid cpv/ccv ag test kit, (bionote). positive samples were excluded from the study. none of the dogs had been treated with antimicrobial drugs within the previous 8 weeks of sample collection. the majority of healthy dogs were receiving commercial dry food and some of the dogs with diarrhoea were being fed a high-fibre prescription veterinary diet (hill's i/d diet). faecal samples were processed as described previously [6, 12] . briefly, aliquots of 500 mg of faecal sample were thawed and re-suspended in saline buffer (0.01m tris solution (ph7.5), 0.15m nacl, 0.01m cacl 2 ) at 3:1 ratio of solid mass. one mm zirconia/silica beads were added to the stool solution, filling around 150î¼l of an eppendorf tube, and vortexed vigorously for 3 minutes. the samples were then centrifuged at 17900 x g for 5 min, collecting the supernatant and repeating this step three more times. to reduce solid faecal matter and bacterial contamination, 500 î¼l of this solution was filtered through a 0.45 î¼m tube filter (corning costar spin x) by centrifugation at 3800 x g for 5 minutes, then the filtrate was transferred to 2 ml tubes. to enrich for viral dna and rna, a dnase/rnase step was incorporated using a modified protocol described previously [6, 12] . each sample was treated with a cocktail of dnases (turbo dnase, from ambion, baseline-zero from epicentre, benzonase from novagen and dnase i from roche) and rnase a (qiagen). this mixture was incubated in a water bath at 37ë�c for 3 hours. to stop the enzymatic activity, edta (amresco) was added in a final concentration of 15 mm to each sample and incubated at 75ë�c for 10 min. viral dna/rna protected from digestion within viral capsids were extracted using qiaamp viral rna mini kit (qiagen), according to manufacturer's recommendations. a second dnase/rnase step was performed on the extracted viral rna for elimination of genomic dna, using dnase i recombinant, rnase free (10u/î¼l) (roche) and protector rnase inhibitor (40 u/î¼l) (roche). after digestion of the dna, the viral rna was transcribed with sensiscript reverse transcriptase kit (qiagen; sensiscript rt kit) to generate cdna, according to manufacturer's instructions with minor modifications. briefly, for a more sensitive detection in the subsequent pcr, a mixture of oligo-dt primers (oligo (dt)15 primer, promega) and random primers (random hexamers, taqman reverse transcription reagents, roche, applied biosystems) were used and a rna denaturation step (95ë�for 3 minutes) was added. viral cdna and genomic dna were randomly amplified using a modified sispa protocol [61, 62] . briefly, a second strand synthesis was performed with large (klenow) fragment (new england biolabs) and random hexamers (roche, biosystems, 50î¼m) followed by digestion of the second strand product with the restriction enzyme cviqi (csp6.1), (new england biolabs). then a csp11/nbam24 adaptor was ligated to the digested dna using t4 dna ligase (invitrogen) followed by pcr amplification of the adaptor-ligated product with nbam24 pcr primers. an aliquot of the pcr product was validated on a 1% agarose tbe gel, where a positive smear with multiple bands confirmed the random sispa amplification of nucleic acid products. the amplified pcr products were cleaned up using wizard sv gel and pcr clean-up system (promega) following manufacturer's recommendations and two libraries with dual indexing for each sample were generated (dna and cdna) with illumina nextera xt dna sample preparation kit, according to manufacturer indications. after visualise it with agilent 2200 tape station system (agilent technologies), the libraries were submitted to the australian genome research facility (agrf) for a 250 bases paired-end sequencing on the miseq illumina platform. all raw sequences were deposited under bioproject id: prjna380672 at ncbi database. raw sequences were trimmed by quality score with prinseq software (v0.20.3) [63] , filtering for low quality reads from both ends using the dust score [64] with a threshold of 7. poly a/t tails in both ends (ten nucleotides of each end) and sispa primers sequences were also removed using this software. the mothur software v.1.31.2 [65] was applied and the sequences were trimmed again, eliminating homopolymers, ambiguous bases and sequences less than 100bp. after these trimming steps, high quality reads (hqr) were obtained and all bad quality reads were removed from the group file (fig 1) . subsequently, these dog free sequences were compared against a bacterial database (cam-era prokaryotic nucleotide database 10572.v7, nov 2012; http://camera.calit2.net/) [66] to eliminate bacterial sequences, using the blastn (blast 2.2.29+ standalone) algorithm with an 80% identity cut off. to extract cellular organism sequences from the group file, megan v5.2.1 and mothur software were used as described above (fig 1) . the host and bacteria free sequence reads, were de novo assembled with metavelvet (velvet 1.2.08, kmer51) [67] using kmer size 51 and contigs and singletons were created. these singletons were clustered with a 98% similarity using cd-hit-est (version.4.5.4 2011) [68] (fig 1) . all contigs and singletons clusters were analysed through two pipelines. (1) contigs and singletons clusters were compared against the camera viral nucleotide sequence database 10570.v9, using tblastx search with an e-value cut off 10 â��5 ; (2) contigs and singletons clusters were compared against the ncbi nucleotide database (2012) using blastn search with an e value cut off 1. all blast searches were performed in blast 2.2.29+ standalone. these files were then analysed by megan v5.2.1 [37] and the lowest common ancestor of known viral sequences were identified (fig 1) . finally, all viral contigs and singletons of eukaryotic organisms present in both analyses were aligned and compared with the ncbi reference sequence of the lowest common ancestor given by megan v5.2.1. all alignments were made using sequencher version 5.0.1 sequence analysis software (gene codes corporation, ann arbor, mi usa) with minimum match percentage of 70%-80% and minimum overlap 50 as assembly parameters, evaluating the percentage of coverage of the genome. all contigs and singletons with no hits were re-evaluated using online blastn with an e value cut off 10 and visualised with megan v5.2.1 to evaluate the alignment with its lowest common ancestor. in order to acquire the complete genome of canine astrovirus, multiple sets of primers were selected from the literature or designed based on sequences obtained from illumina reads (s2 table) . nucleic acids from a single faecal sample from a dog with acute diarrhoea (dd1), which had 18 contigs/singletons of canine astrovirus (after tblastx analysis) was used to determine the complete genome sequence. rna was extracted directly from the centrifuged sample after faecal extraction, previous to enrichment of viral nucleic acids as outlined before. rt-pcr was performed with superscript iii one-step rt-pcr system with platinum taq (invitrogenâ�¢). pcr conditions used were: 45ë�c for 60 min and 95ë�c for 5 min, 35 cycle of 94ë�c for 40 sec, 55ë�c for 1min and 72ë�c for 5 min, and a final elongation step of 72ë�c for 5 min, followed by final hold at 4ë�c. pcr products were run on a 1.2% agarose tbe gel stained with redsafeâ�¢ nucleic acid staining solution (intron biotechnology). all pcr products were excised and cleaned up with wizard sv gel and pcr clean-up system (promega) following manufacturer's protocol and sequenced using sanger sequencing at the agrf. the near complete genome of the canine astrovirus was assembled using sequencher version 5.0.1 sequence analysis software (gene codes corporation, ann arbor, mi usa) with minimum match percentage 80 and minimum overlap 50 as assembly parameters. phylogenetic analysis of this canine astrovirus was performed aligning protein sequences of the 172 conserved amino acids of the capsid region (orf2) from different species (s2 fig), using clustal w, from mega version 6.0 [69] with default settings. a phylogenetic tree with 1000 bootstrap was generated using the maximum likelihood method based on the jtt matrix-based model [70] , using mega version 6.0. the percentage of identity was calculated with clustalo 1.2.4 [71] supporting information s1 fig. megan viruses in the faecal microbiota of monozygotic twins and their mothers metagenomic analyses of an uncultured viral community from human feces viral metagenomics. reviews in medical virology viral metagenomics as an emerging and powerful tool in veterinary medicine. veterinary quarterly viral diversity and dynamics in an infant gut viruses in diarrhoeic dogs include novel kobuviruses and sapoviruses bat guano virome: predominance of dietary viruses from insects and plants plus novel mammalian viruses the human gut virome: inter-individual variation and dynamic response to diet the fecal virome of pigs on a high-density farm feline fecal virome reveals novel and prevalent enteric viruses viral metagenomic analysis of feces of wild small carnivores metagenomic analyses of viruses in stool samples from children with acute flaccid paralysis rna viral community in human feces: prevalence of plant pathogenic viruses metagenomic analysis of human diarrhea: viral detection and discovery study of the viral and microbial communities associated with crohn's disease: a metagenomic approach characterization of microbial dysbiosis and metabolomic changes in dogs with acute diarrhea common and emerging infectious diseases in the animal shelter. veterinary pathology long-term viremia and fecal shedding in pups after modified-live canine parvovirus vaccination m gene evolution of canine coronavirus in naturally infected dogs. the veterinary record an update on canine coronaviruses: viral evolution and pathobiology canine coronavirus-associated puppy mortality without evidence of concurrent canine parvovirus infection infectious diseases of the dog and cat novel norovirus in dogs with diarrhea fields virology,. 1. 6th ed. philadelphia detection and characterization of canine astroviruses isolation and characterization of canine astrovirus in china complete genome sequence of canine astrovirus with molecular and epidemiological characterisation of uk strains prevalence and risk factors of astrovirus infection in puppies from french breeding kennels detection of canine astrovirus in dogs with diarrhea in japan viruses and virus-like particles in the faeces of dogs with and without diarrhoea astrovirus-like, coronavirus-like, and parvovirus-like particles detected in the diarrheal stools of beagle pups genetic characterization of a new astrovirus detected in dogs suffering from diarrhoea enteric disease in dogs naturally infected by a novel canine astrovirus molecular characterisation of calicivirus and astrovirus in puppies with enteritis phylogenetic analysis of astrovirus and kobuvirus in korean dogs a virus discovery method incorporating dnase treatment and its application to the identification of two bovine parvovirus species sequence-independent, single-primer amplification (sispa) of complex dna populations quality control and preprocessing of metagenomic datasets a fast and symmetric dust implementation to mask low-complexity dna sequences introducing mothur: opensource, platform-independent, community-supported software for describing and comparing microbial communities community cyberinfrastructure for advanced microbial ecology research and analysis: the camera resource metavelvet: an extension of velvet assembler to de novo metagenome assembly from short sequence reads cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences mega6: molecular evolutionary genetics analysis version 6.0 the rapid generation of mutation data matrices from protein sequences. computer applications in the biosciences fast, scalable generation of high-quality protein multiple sequence alignments using clustal omega the authors gratefully acknowledge the helpful assistance of all staff at lost dogs home and university of melbourne u-vet werribee animal hospital with the faecal sample collection. also to dr celeste donato for her valuable scientific input and her assistance with the construction of the astrovirus phylogenetic tree. minot key: cord-001090-qg2r691d authors: twin, jimmy; bradshaw, catriona s.; garland, suzanne m.; fairley, christopher k.; fethers, katherine; tabrizi, sepehr n. title: the potential of metatranscriptomics for identifying screening targets for bacterial vaginosis date: 2013-09-27 journal: plos one doi: 10.1371/journal.pone.0076892 sha: doc_id: 1090 cord_uid: qg2r691d background: the ribosomal rna content of a sample collected from a woman with bacterial vaginosis (bv) was analysed to determine the active microbial community, and to identify potential targets for further screening. methodology/principal findings: the sample from the bv patient underwent total rna extraction, followed by physical subtraction of human rrna and whole transcriptome amplification. the metatranscriptome was sequenced using roche 454 titanium chemistry. the bioinformatics pipeline mg-rast and desktop dna analysis platforms were utilised to analyse results. bacteria of the genus prevotella (predominately p. amnii) constituted 36% of the 16s rrna reads, followed by megasphaera (19%), leptotrichia/sneathia (8%) and fusobacterium (8%). comparison of the abundances of several bacteria to quantitative pcr (qpcr) screening of extracted dna revealed comparable relative abundances. this suggests a correlation between what was present and transcriptionally active in this sample: however distinct differences were seen when compared to the microbiome determined by 16s rrna gene amplicon sequencing. to assess the presence of p. amnii in a larger pool of samples, 90 sexually active women were screened using qpcr. this bacterium was found to be strongly associated with bv (p<0.001, or 23.3 (95%ci:2.9–190.7)) among the 90 women. conclusions/significance: this study highlighted the potential of metatranscriptomics as a tool for characterising metabolically active microbiota and identifying targets for further screening. prevotella amnii was chosen as an example target, being the most metabolically active species present in the single patient with bv, and was found to be detected at a high concentration by qpcr in 31% of cohort with bv, with an association with both oral and penile-vaginal sex. the advent of molecular-based screening utilising massively parallel dna sequencing has increased our capability to characterise the microbial ecology of human clinical samples, both rapidly and economically [1] . in addition it has allowed a better understanding of the normal endogenous microbiota. the vaginal microbiota is complex, varying at different stages of reproductive life, as well as during the menstrual cycle. bacterial vaginosis (bv) is a condition affecting the vaginal microbiota where in the childbearing age woman the natural microbiota (typically lactobacillus spp.) is depleted, and replaced by an overgrowth of mixed, primarily anaerobic bacteria [2] . this condition has significant associations with miscarriage, premature birth and pelvic infections, and can increase a woman's risk of acquiring sexually transmitted infections and hiv [3] . no single aetiological agent has been identified yet for bv, and is now generally considered likely to be of polymicrobial aetiology. bv is typically diagnosed using either the nugent scoring method [4] that examines bacterial composition via a gram smear or the amsel criteria [5] that considers factors such as presence of discharge, amine production, presence of clue cells and a vaginal ph greater than 4.5. through microbiome studies, the microbiology of bv has been better characterised. these studies have generally been carried out using pcr-derived 16s rrna gene fragments, using ''universal'' bacterial 16s rrna gene primers [2] . while this approach is able to provide a comprehensive understanding of the bacterial community membership, it is not able to determine which members are transcriptionally active. metatranscriptomics is the analysis of the rna transcripts being expressed by a community at a given point of time. for bacteria, the predominant rna classes are ribosomal (primarily 16s rrna and 23s rrna), which are able to be used as taxonomic identifiers. the metatranscriptomic screening of clinical samples has been described for gut and dental microbiomes [6, 7] , with only one study to date applying this methodology to bv [8] . an important difficulty faced with the application of this technique to clinical samples is the low levels of rna present in many samples and the overabundance of host rna. this requires reduction of host rna and subsequent linear amplification of rna for transcriptomic analysis [9, 10] . this study describes a method to analyse microbial rrna from vaginal samples that is applicable to variety of clinical sample types. it demonstrates how the data gained can provide valuable information for larger qpcr based screening studies. a 26 year old woman presenting with abnormal vaginal discharge, odour, 4/4 amsel criteria and a nugent score of 10 was recruited with written consent from melbourne sexual health centre (mshc), victoria, australia. this patient reported no recent antibiotic use, had no recent male sexual partner, and reported only having one female partner in the prior 3 months and three in the past 12 months. vaginal discharge was collected using ten flocked swabs and immediately rotated and pooled in 4 ml rnalater (life technologies, grand island, usa). the rnalater solution was then stored at 280uc until processing. for the second component of this study, extracted dna (stored at 230uc) was utilised from a randomly selected subset of 90 samples obtained from a cross sectional study of sexually active women attending mshc in 2003/4 [11] . this set of samples comprised 34 women with normal microbiota (nugent 0-3), 20 with intermediate microbiota and 36 with bv . written consent for all de-identified participants and ethical approval for this study was obtained previously from the alfred hospital human research ethics committee. figure 1 provides an overview of the workflow for this study. initially, total rna was extracted from 2 ml rnalater solution [12] . total rna extraction was performed using trizol (life technologies) with use of 1-bromo-3-chloropropane in place of chloroform followed by rna purification using a rneasy column (qiagen, hilden, germany) [13] . any remaining dna was digested using the turbo dna-free enzyme (life technologies). human ribosomal content was subtracted using the mi-crobenrich kit (life technologies) as per manufacturer instructions followed by removal of small rnas with the megaclear kit (life technologies). rna concentration was determined by agilent 2100 bioanalyzer (agilent technologies inc., santa clara, usa). to increase the rna content needed for sequencing, whole transcriptome amplification (wta) and reverse-transcription using the transplexh complete whole transcriptome amplification kit (sigma-aldrich corporation, st. louis, usa) was utilized. this resulted in a cdna concentration of 50 ng/ml (from original rna content of 3.5 ng/ml) with 2 mg submitted to agrf (australian genome research facility ltd., brisbane, australia) for 454 sequencing (flx titanium chemistry; roche/454 life sciences, branford, usa). genomic dna was precipitated from the remaining trizol buffer using 0.3 ml of 100% ethanol per 1 ml of trizol buffer followed by two washes of the dna pellet with 0.1 m trisodium citrate in 10% ethanol [14] . an amplicon-based metagenomic library was generated from the extracted dna using the universal bacterial pcr primers 27f and 338r that target the v1-v2 hypervariable regions of the 16s rrna gene as previously described [15] . both cdna and 16s rrna gene amplicon libraries were subsequently bar-coded using mid tags and sequenced together utilising a quarter region of a 454 sequencing plate. subsequent to sequencing, all raw sequencing data was demultiplexed according to their mid tags and the data obtained from each sample was then imported into genomics workbench version 4.5.1 (clc bio, aarhus, denmark), for removal of wta primers and random primer sequences from each applicable read and data was filtered (low quality score limit of 0.05; maximum 2 ambiguous nucleotides allowed; minimum sequence length of 100 nt). each dataset was then screened for chimeric sequences using uchime [16] , with the trimmed and filtered data submitted to the mg-rast (http://metagenomics.anl.gov) bioinformatics pipeline for analysis (cdna library mg-rast id = 4461586.3; dna amplicon mg-rast id = 4461792.3) [17] . a 90% cut-off was used for database searches within mg-rast as an arbitrary cut-off for genus identities using the rdp database, and a 98% cut-off was used for species level identification [18] . mg-rast generates abundance counts based on the number of unique hits a particular sequence has against a particular database. it is therefore highly likely that a single read may have multiple abundance counts assigned to it if there is an equal relatedness. the identities for each read were sorted using excel 2007 (microsoft corporation, redmond, usa) to eliminate multiple identical hits for individual reads, with manual analysis being carried out using the blast algorithm for discrepant samples. graphical representation of bacterial abundances was achieved using krona charts [19] . functional genes from the cdna library were characterised by seed analysis within mg-rast. reads derived from the cdna library assigned to each major genus (comprising .10% of total population) using mg-rast were also imported into lasergene 8 (dnastar inc., madison, usa) for manual sequence alignment was carried out using a 98% sequence match cut-off. the consensus sequences of alignments were assigned an identity using the blast algorithm with a $98% identity required to assign a species name. all assays were performed on the lc480 real-time instrument (roche diagnostics, mannheim, germany) using 5 ml dna in a 20 ml reaction. the bacteria targeted were atopobium vaginae, leptotrichia/sneathia spp., megasphaera type i [20] , gardnerella vaginalis [21] , and the genera prevotella [22] and lactobacillus [23] . a primer set for prevotella amnii was developed using the forward and reverse primers 27f and 338r [24] with the incorporation of a taqman probe 59-[6fam] aaa gtt ggc cta atg ccc tat g[bhq1]-39, specific to p. amnii, based on 454 sequencing data from this study and all available relevant sequences on the genbank database. the total bacterial content of each sample was determined by the modification of an assay by nikkari et al. (2002) [25] with the incorporation of a taqman probe, 516f (59-[6fam] tgc cag cag ccg cgg taa [bhq1]-39) to calculate relative bacterial abundances and to determine sample adequacy for stored dna samples. relative bacterial abundances were compared using a paired t test or chi square test. the association of p. amnii with bv, defined as a nugent score of 7-10, was made using a chi square test, reported with odds ratios (or) with 95% confidence intervals (ci). all analyses were carried out using stata version 12 (statacorp lp, college station, usa) [26] . in total, 89969 raw sequencing reads were obtained with an ultimate post data filtration of 78285 reads (average length = 268691 bp; gc content = 4965%). overall, this library consisted of 72826 (93%) bacterial reads, 3865 (4.9%) human reads and 34 (0.04%) either plastid, fungal or viral reads (table 1) . of the bacterial reads, 31857 (43.7%) were of the 16s rrna gene and 4349 (6.0%) were non-ribosomal rna (eg. mrna) with the remaining matching other ribosomal regions such as the 23s rrna gene and intergenic regions. of the 16s rrna gene reads, the most dominant genus identified was prevotella (36% of the reads), followed by the genera megasphaera (19%), leptotrichia/ sneathia (8%), fusobacterium (8%), and 9% of the reads matching uncultured members of the fusobacteriales (figure 2 ). of the prevotella reads, 78% formed contigs of $1000 bp in length that matched p. amnii. analysis of assembly free reads of the prevotella spp. reads at the species level (11725 reads with $98% match to the rdp database) showed similar results, however distinction between p. amnii and p. bivia was not possible for the majority of these sequences (data not shown). the major fusobacterium was f. nucleatum, and the majority of megasphaera reads were $98% similar to the currently uncharacterised veillonellaceae bacterium s3pf24 (genbank accession jx104009). the bioinformatics pipeline of mg-rast was able to annotate 2053/4349 of the non-ribosomal rna reads, which seed analysis revealed to consist of genes involved primarily in protein metabolism (20.6%), carbohydrate/lipid utilisation (16.3%) and cluster based subsystems (13%). the most abundant functional gene read identities were primarily represented by genes expressed by the genus prevotella ( table 2) . comparison of cdna to dna from nugent 10 patient all of the predominant bacteria with .1% total abundance identified in the cdna library were also detected using the 16s rrna gene amplicon-based approach on extracted dna. there was a significant difference in the relative abundances given between both libraries for the most dominant taxa, such as the genus lactobacillus (primarily l. iners) that comprised 1.4% of the cdna reads and 23% of the dna amplicon library (p,0.0001). also of particular note were reads pertaining to the genus gardnerella that were present in 7% of the cdna library, but were nearly non-existent in the dna amplicon library with only seven reads detected (0.006%). when comparing the relative abundances of the seven taxa screened for using qpcr to the abundances given for the cdna library, there was no significant difference (p = 0.9093). we found that the proportion of lactobacillus spp. determined by qpcr to be 1.9% (cdna = 1.4%; dna amplicon = 23.1%), and the proportion of prevotella spp. was 48% (cdna = 35%; dna amplicon = 11%) ( table 3) . qpcr screening of sexually active women p. amnii was detected in 11/36 (30.6%) women possessing a nugent score of 7-10, whereby nine had a nugent score of 7 and two with a nugent score of 10. this bacterium was also detected in a single case possessing intermediate microbiota with a nugent table 4 ). the bacterial load of p. amnii in these 12 positive cases was high, averaging 1.41610 9 copies per swab (range = 7.74610 6 to 4.66610 9 ). this bacterium was found to be strongly associated with bv (p,0.001, or 23.3 (95%ci:2.9-190.7)) among the 90 women. when the 90 women were stratified by different sexual exposures the association between bv and p. amnii persisted for different sexual exposures in men. while the associations between bv and p. amnii were not significant among women reporting receptive oral sex with a woman or sex with a woman in the last 3 months, these analyses involved only small numbers (table 4 ). this study explored the active microbial diversity of a vaginal sample taken from a lesbian woman with symptomatic bv to demonstrate the potential of metatranscriptomics for identifying targets for screening studies. prevotella amnii was found to be the most active species present in this sample and was also detected at a high concentration by qpcr in 30.6% of bv cases in sexually active women. although we identified p. amnii as the predominant prevotella species in this sample (78% of prevotella reads; 28.1% of total bacterial population), there may be a possible overestimation in the cdna abundance given, as conserved regions shared by multiple prevotella spp. may be involved through chimeric assembly. this must be kept in mind when comparing to qpcr data (33.1% of prevotella reads; 15.8% of total bacterial population) that would otherwise suggest that this species is more active than others in this sample. this bacteria has only recently been associated with bv in the literature [27] . its closest relative, p. bivia, is highly cited as being associated with this condition, and in vitro studies have shown this bacterium to form a symbiotic relationship with g. vaginalis [28] . these two prevotella spp. share many phenotypic traits and it has been noted that culture-based methodologies may not differentiate between these two species, with full length 16s rrna gene sequencing recommended for species differentiation [29] . the other predominant bacteria in this case were identified through 16s rrna gene as the currently uncharacterised isolate veillonellaceae bacterium s3pf24 (phylogenetically belonging to the genus megasphaera) that was originally isolated from a vaginal environment, and the bacterium fusobacterium nucleatum. f. nucleatum has been described in cases of bv previously [30] . interestingly it has been implicated in amniotic infection and preterm birth in cases where the bacterium was also detected in the patients' oral cavity [31, 32] . f. nucleatum, as well as both prevotella and megasphaera spp. are regularly associated with periodontal disease [33, 34, 35] , and it is of interest to note that the single nugent score 10 participant analysed in this study had a history of recent female-female oral sex. these three bacteria were more abundant than the usual predominant g. vaginalis and a. vaginae in the cdna library, which collectively only comprised 10% [2] . further work is warranted to determine the role of these bacteria, in particular p. amnii, in the pathogenesis of bv. of particular interest is whether their association with oral sex represents a possible route of transmission. although not a focus of this study, mrna reads were also identified in this study that were primarily associated with protein metabolism and carbohydrate/lipid utilisation pathways of the genus prevotella. the low abundance of mrna reads (5.6%) was not surprising, given the low general relative abundance of mrna in relation to total rna in bacterial cells [36] . to focus more upon this class of rna transcripts, an additional enrichment step, such as the removal of bacterial rrna can be employed [37] , in the same manner as human rrna was reduced in this study. a comparison was made between the cdna and dna amplicon libraries to determine what proportion of the microbial community was active, and differences were observed between these libraries. this may suggest that there was a significant difference between what is present and what is transcriptionally active, although these findings are limited by the fact that this is a single sampling point. the broad range amplification of bacterial dna using the 16s rrna gene may have potential bias in the community structure, as has been found in numerous studies [38, 39, 40, 41] . our data suggests that g. vaginalis was underrepresented in the dna amplicon library when compared to the qpcr data from the same dna sample. despite being one of the most utilized primers for microbiome analysis, there has been suggestion in the literature that the forward pcr primer 27f may not reliably amplify this species [42] , and our analysis has demonstrated this similarly through qpcr for g. vaginalis. also, given that l. iners was found to be underrepresented in the cdna library when compared to the dna amplicon library, we applied a genus-specific lactobacillus qpcr assay to the dna sample, as well as one that targeted the genus prevotella that was present at a much higher abundance in the cdna library. we found that there was no statistical difference in the abundances given between qpcr and the cdna library for these genera. based on this limited data, we can suggest misrepresentation of several taxa in the dna amplicon library occurred, and that in this case, the vast majority of the bacteria physically present in this sample were also transcriptionally active. this further highlights the advantage of transcriptomics for such analysis as our data suggest that this analysis gives better representation of bacterial levels across the board. mg-rast for the analysis of metatranscriptomic work can be carried out using a standard desktop or laptop computer. however, this method may lead to misclassification of certain reads. for example, in this study it was found that many gardnerella reads were being classified as the genus bifidobacterium, megasphaera reads as the genera veillonella and dialister, and those of the genera leptrotrichia/sneathia as streptobacillus. therefore care must be taken in the interpretation of results, and manual checking of representative reads is required to ensure an accurate identification of sequencing reads to the genus level. in summary, this study has shown how metatranscriptomics can be utilised as a useful tool to carry out an in-depth examination of the active microbial ecology of an environment, and is capable of generating more taxonomic and potential functional data than an amplicon sequencing-based approach alone, and avoid potential misrepresentation issues caused by pcr primer selection. we have demonstrated that this kind of metatranscriptomic approach can be used to identify targets for screening in larger studies. as this study focussed on a single individual, generalisations based on the data are not possible and transcriptomic analyses on a large cohort is warranted. this being said, our limited findings reinforce that p. amnii may be an important bv-associated bacterium, and should be included in future studies investigating the pathogenesis of bv. its previously known association with periodontal disease, and the association with oral sex in this study, make it interesting to speculate about transmission of pathogenic bacteria between the oral and vaginal cavities, and their role in the development of bv. metagenomic analysis of bacterial infections by means of high-throughput dna sequencing the vaginal microbiome: new information about genital tract flora using molecular based techniques the aetiology of bacterial vaginosis reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation nonspecific vaginitis. diagnostic criteria and microbial and epidemiologic associations effect of periodontal pathogens on the metatranscriptome of a healthy multispecies biofilm model metatranscriptomic approach to analyze the functional human gut microbiota composition of the vaginal microbiota in women of reproductive age -sensitive and specific molecular diagnosis of bacterial vaginosis is possible? representation is faithfully preserved in global cdna amplified exponentially from subpicogram quantities of mrna broadspectrum respiratory tract pathogen identification using resequencing dna microarrays higher-risk behavioral practices associated with bacterial vaginosis compared with vaginal candidiasis simultaneous extraction of high-quality rna and dna from small tissue samples substitution of chloroform by bromochloropropane in the single-step method of rna isolation a reagent for the single-step simultaneous isolation of rna, dna and proteins from cell and tissue samples vaginal microbiome of reproductive-age women uchime improves sensitivity and speed of chimera detection the metagenomics rast server -a public resource for the automatic phylogenetic and functional analysis of metagenomes study of inter-and intra-individual variations in the salivary microbiota interactive metagenomic visualization in a web browser changes in vaginal bacterial concentrations with intravaginal metronidazole therapy for bacterial vaginosis as assessed by quantitative pcr detection of bacterial vaginosis-related organisms by real-time pcr for lactobacilli, gardnerella vaginalis and mycoplasma hominis comparative assessment of human and farm animal faecal microbiota using real-time quantitative pcr diversity of cervicovaginal microbiota associated with female lower genital tract infections inverse association of h 2 o 2 -producing lactobacilli and vaginal escherichia coli colonization in women with recurrent urinary tract infections broadrange bacterial detection and the analysis of unexplained death and critical illness stata statistical software: release 12 bacterial communities in women with bacterial vaginosis: high resolution phylogenetic analyses reveal relationships of microbiota to clinical criteria evidence for a commensal, symbiotic relationship between gardnerella vaginalis and prevotella bivia involving ammonia: potential significance for bacterial vaginosis prevotella amnii sp. nov., isolated from human amniotic fluid molecular analysis of the diversity of vaginal microbiota associated with bacterial vaginosis the origin of fusobacterium nucleatum involved in intra-amniotic infection and preterm birth abnormal bacterial colonisation of the genital tract and subsequent preterm delivery and late miscarriage pattern of distribution of prevotella species/phylotypes associated with healthy gingiva and periodontal disease identification of candidate periodontal pathogens and beneficial species by quantitative 16s clonal analysis the detection of eight putative periodontal pathogens in adult and rapidly progressive periodontitis patients: an institutional study chemical composition of escherichia coli validation of two ribosomal rna removal methods for microbial metatranscriptomics coverage evaluation of universal bacterial primers using the metagenomic datasets capturing greater 16s rrna gene sequence diversity within the domain bacteria improved detection of bifidobacteria with optimised 16s rrna-gene based pyrosequencing selection of primers for optimal taxonomic classification of environmental 16s rrna gene sequences the human vaginal bacterial biota and bacterial vaginosis we would like to thank the staff and students of melbourne sexual health centre and women's centre for infectious diseases for their assistance in this study, which was also supported by the victorian government's operational infrastructure support program. key: cord-000336-57es391o authors: liao, qiuyan; cowling, benjamin j.; lam, wendy wing tak; fielding, richard title: factors affecting intention to receive and self-reported receipt of 2009 pandemic (h1n1) vaccine in hong kong: a longitudinal study date: 2011-03-11 journal: plos one doi: 10.1371/journal.pone.0017713 sha: doc_id: 336 cord_uid: 57es391o background: vaccination was a core component for mitigating the 2009 influenza pandemic (ph1n1). however, a vaccination program's efficacy largely depends on population compliance. we examined general population decision-making for ph1n1 vaccination using a modified theory of planned behaviour (tbp). methodology: we conducted a longitudinal study, collecting data before and after the introduction of ph1n1 vaccine in hong kong. structural equation modeling (sem) tested if a modified tpb had explanatory utility for vaccine uptake among adults. principal findings: among 896 subjects who completed both the baseline and the follow-up surveys, 7% (67/896) reported being “likely/very likely/certain” to be vaccinated (intent) but two months later only 0.8% (7/896) reported having received ph1n1 vaccination. perception of low risk from ph1n1 (60%) and concerns regarding adverse effects of the vaccine (37%) were primary justifications for avoiding ph1n1 vaccination. greater perceived vaccine benefits (β = 0.15), less concerns regarding vaccine side-effects (β = −0.20), greater adherence to social norms of vaccination (β = 0.39), anticipated higher regret if not vaccinated (β = 0.47), perceived higher self-efficacy for vaccination (β = 0.12) and history of seasonal influenza vaccination (β = 0.12) were associated with higher intention to receive the ph1n1 vaccine, which in turn predicted self-reported vaccination uptake (β = 0.30). social norm (β = 0.70), anticipated regret (β = 0.19) and vaccination intention (β = 0.31) were positively associated with, and accounted for 70% of variance in vaccination planning, which, in turn subsequently predicted self-reported vaccination uptake (β = 0.36) accounting for 36% of variance in reported vaccination behaviour. conclusions/significance: perceived low risk from ph1n1 and perceived high risk from ph1n1 vaccine inhibited ph1n1 vaccine uptake. both the tpb and the additional components contributed to intended vaccination uptake but social norms and anticipated regret predominantly associated with vaccination intention and planning. vaccination planning is a more significant proximal determinant of uptake of ph1n1 vaccine than is intention. intention alone is an unreliable predictor of future vaccine uptake. influenza contributes significantly to worldwide morbidity and mortality [1] . periodically, influenza viruses mutate into antigenically-different strains leading to global pandemics [2] . the 2009 influenza pandemic (ph1n1) was caused by a triple reassortment of human, swine and avian influenza viruses [3] . vaccination is the most effective intervention for preventing influenza [4] and a core part of national pandemic plans for pandemic mitigation. lead times of at least 6 months in producing a vaccine against a novel strain means that while vaccines may be unavailable in time to prevent the first wave of a pandemic [5, 6] , effective public uptake of a vaccine may mitigate subsequent waves [7] . significant health promotion activities regarding influenza prevention have been prominent in hong kong since well before the onset of ph1n1, arising largely from the severe acute respiratory infection (sars) epidemic and a/h5n1 bird flu outbreaks. seasonal influenza vaccination is widely promoted each year. hong kong's ph1n1 epidemic started on 11 june 2009, peaking in september, and by early november had petered out ( figure 1 ). by the end of december 2009, the hong kong government had recorded 37,174 human ph1n1 cases [8] in a population of ,7 million. to minimize any potential second wave, significant televised and other publicity was given to the launch of a ph1n1 vaccination programme on 21 december 2009, initially for five priority groups: healthcare workers, persons with chronic illness and pregnant women, children aged 6 months to 6 years, adults aged 65 years or above, and pig farmers and slaughtering industry personnel [9] . on 26 january 2010 ph1n1 vaccination was extended to the general public. the vaccination was free for priority group members [10] , but cost hk$100-150 (us$13-20, 1-1.5% of hong kong's median monthly income of hk$10,000/ us$1,286/j991) per dose for the general population. a study in july 2009 of 301 respondents projected that vaccine uptake would be influenced by end-user cost, with 45%, of hong kong's general population being ''highly likely'' to take up ph1n1 vaccine if free, and 24% if costing hk$100-200 (us$ [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [11, 12] . from november 2009 onwards, television, radio, newspaper and official websites strongly encouraged priority groups to have ph1n1 vaccination [13] . however, the hong kong government did not make recommendations for the general population, who were asked to judge for themselves whether to be vaccinated or not. shortly after the vaccine launch for priority groups, local media prominently attributed several adverse events to ph1n1 vaccination, including, a case of guillain-barre syndrome (gbs) diagnosed a week after ph1n1 vaccination, reported on 6th january 2010, and an intrauterine death (iud) 3 weeks following the mother's vaccination, reported on 20th january 2010 ( figure 2 ). in both cases local health agencies presented convincing evidence challenging the link between vaccination and the two adverse events but were largely ignored. retrospectively, a drop in ph1n1 vaccination uptake among priority groups was observed [14] . it seems probable that the adverse media reports had impeded vaccination uptake among general population. we collected baseline data between 12-25 january, 2010, immediately before ph1n1 vaccine was made available to the general population and then two months later (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) march 2010) we recorded their reported vaccination status ( figure 2 ) with the intention of modelling how general population decision-making regarding ph1n1 vaccination might predict subsequent vaccine uptake. empirical studies have found that history of seasonal influenza vaccination [12, [15] [16] [17] [18] , perceived risk of pandemic influenza [17, [19] [20] [21] [22] [23] [24] [25] [26] , worry [17, 22, 26, 27] , and attitudes towards vaccine, such as vaccine efficacy and side-effects [12, 15, 20, [24] [25] [26] were significantly associated with intention to receive a vaccine against the influenza pandemic. this is consistent with the findings related to determinants of vaccination against seasonal influenza [28] [29] [30] [31] [32] . however, there are some common and significant limitations to these empirical studies. first, all except one [24] relied on vaccination intention to predict the actual vaccination uptake. in one study, since only a few respondents reported having received the ph1n1 vaccine, the authors combined those intending to get vaccinated with those who had already received the vaccine into one ''intending'' group and examined factors associated with this 'vaccination intention' [20] . this is problematic because factors associated with vaccination intention and actual vaccination receipt probably differ. moreover, the reliability of intention as a predictor of actual behavior remains controversial. harris et al. found that only about half of ''intending'' recipients of seasonal influenza vaccination actually take it and almost all those who do not intend to take it remained unvaccinated [33] . moreover, most studies conducted before the pandemic occurred or before the vaccine was available [11, 12, [16] [17] [18] [19] 21, 23] of dutch respondents reported intending to take ph1n1 vaccination prior to or at the onset of the (potential) pandemic phase, respectively [23] . similarly, in hong kong 45% of 301 respondents in july 2009 reported being ''highly likely'' to receive ph1n1 vaccine if offered for free [11, 12] . however, by the time vaccination became available intention appeared much lower with only 10-15% of study respondents in france and in turkey intending to take the ph1n1 vaccine [20, 24] . second, all the studies are cross-sectional rather than longitudinal; none assessed subsequent actual vaccination status. thus, although associations have been identified, there is no way to infer causality. third, most of the studies are atheoretical. although some of the studies developed their study questions based on theoretical framework such as hbm [17, 23, 24] , none have conducted model analysis and evaluated the model fit. therefore, due to these three reasons, there remains a significant concern about how valid such results are and a significant knowledge gap about how the observed pattern of influences could be explained. a major limitation of previous empirical studies [12, [15] [16] [17] [18] [19] [20] [21] [22] [23] 25, 26] is failure to accommodate the intention-behaviour gap. although several behavioral theories such as protection motivation theory (pmt) [34, 35] , theory of reasoned action (tra) [36, 37] and theory of planned behaviour (tpb) [38, 39] propose that intention is the proximal determinant of behaviour, intention does not necessarily translate into actual behaviour. empirical studies of the intention-behavior relationship showed that intention had a medium effect (a correlation of ,0.4-0.5) on behavior [40] [41] [42] , but a recent review including 47 experimental studies found that a medium-to-large change in intention induced by manipulated interventions caused only a small-to-medium change in behavior [43] , where an effect size of 0.5 is medium and one of 0.2 is small [44] . sheeran found that about 47% of those intending to take action fail to act [42] , consistent with harris et al's findings [33] . factors that are prime contenders to moderate/ mediate the relationship between intention and behaviour include behavioural control/efficacy, action planning and anticipation of consequences [41] [42] [43] . perceived behavioural control/self-efficacy. the tpb is an extension of the tra incorporating the concept of perceived behavioural control (pbc) as an intervening variable predicting both intention and also actual behavioural change directly [38, 39] . the direct effect of pbc on actual behavioural change partly explains why not all intention translates into behaviour. previous reviews suggested that intention-behaviour relationships could be moderated by perceived behavioral control, with higher levels of perceived behavioural control improving prediction of intention on behaviour [42, 43] . although some researchers suggested that pbc differs from self-efficacy because selfefficacy emphasized perceived internal control more while pbc also considers external control factors [45] , a systemic review on the efficacy of tbp found that pbc and self-efficacy had comparable effects on intention and behaviour [41] . despite being a dominant theory of behavioural change, because the tpb is limited in predicting behaviour we sought to enhance its predictive power by replacing pbc with self-efficacy and incorporating enhanced social effects to accommodate external control factors. implementation of intention/planning. implementation of intention, termed ''planning'', is a potentially important factor facilitating translation of intention into behaviour [42, 43, 46, 47] . planning is specific to situations (e.g., when, where, and how) within which one will perform the behaviour [46] . it activates the situational context for goal attainment and thereby makes the goal become more accessible [46, 47] . a meta-analytic review showed that implementation of intention as planning consistently caused a medium-to-large effect on behavioural change [47] . anticipated regret. anticipated regret is the expectation of feeling regret or upset if one does or does not conduct certain behaviours. anticipated regret has been found to be a strong predictor of vaccine uptake against seasonal influenza [31, 32] , playing the lottery [48] and exercise [49] . anticipated regret might also moderate the intention-behavior relationship: the higher anticipated regret for inaction, the better the prediction of intention on behaviour [48, 49] . a robust theoretical framework comprehensively explaining behavior change that elucidates population decision-making for health protective and promoting behaviour has long been sought. as the main contender, the tpb explains ,34% of variance in health behavioural change related to addictive behaviour, automobile-related behaviours, clinical and screening behaviour, eating behaviour, exercising behaivour, hiv/aids-related behaviour and oral hygiene behaivour [40] . the standard version of tpb proposes that attitudes towards the behaviour, subjective norm and pbc predict behavioral intention while intention and pbc predict the actual behavioural change [38, 39] . additional predictors that significantly improve the model's predictive power are needed [39] . two previous studies have examined modified versions of tpb to predict vaccination uptake against seasonal influenza [50, 51] . one study used tpb plus two additional factors: influenza vaccination history and anticipated regret, to predict intention to receive vaccine against seasonal influenza among elderly from social clubs [50] : vaccination history and anticipated regret respectively accounted for an additional 10.7% and 13.7% of total variance in influenza vaccination intention [50] . however, again the study was cross-sectional and actual vaccination uptake was not assessed. a second study of healthcare workers [51] adopted an extended version of tpb that included additional elements of anticipated regret, moral norm, descriptive norm and professional norm. the study found that controlling for the original tpb variables, moral norm and anticipated regret were significant determinants of actual receipt of seasonal influenza vaccine [51] . the study provides useful information for future application of the extended version of tpb. however, since the study was conducted among healthcare workers, some of the variables such as moral norm and professional norm which emphasize obligation and professional convictions may not be applicable among the general population. factors influencing ph1n1 vaccine uptake at the later stage of a pandemic might be more cognitively driven unlike behavioral responses during the early stage of a pandemic which might be more affect driven [52] . therefore, taking into account prior work on seasonal influenza vaccination uptake [50, 51] , extending the tpb could provide theoretical utility for understanding public decision on taking ph1n1 vaccination. starting with tpb and existing literature, we therefore built a conceptual model of public decision-making for ph1n1 vaccination ( figure 3 ). in addition to the original tpb components, seasonal influenza vaccination history, anticipated regret and vaccination planning were included in the model. the model proposed that attitudes towards vaccination (perceived benefits of ph1n1 vaccination and concerns regarding possible adverse effects of ph1n1 vaccination), perceived social pressures from significant others and other people around regarding ph1n1 vaccination (social norms regarding ph1n1 vaccination), perceived self-efficacy in taking vaccination (perceived self-efficacy), anticipated regret for not taking the ph1n1 vaccination (anticipated regret) and seasonal influenza vaccination history would predict vaccination intention, which in turn predicts vaccination planning and future vaccination uptake; anticipated regret and perceived self-efficacy could also predict vaccination status directly; finally, vaccination planning was proposed to bridge the intention-behavior gap and predict vaccination status directly ( figure 3 ). we conducted a longitudinal study of influences on ph1n1 vaccination behaviour in hong kong to test this model ( figure 3) , and subsequently followed up participants to record their selfreported receipt of ph1n1 vaccine. in this study, we aimed to answer the following research questions: how well does intention predict future uptake of ph1n1 vaccine? does vaccination planning mediate the relation between intention and future vaccination uptake? and do the original tpb components and the additional components (extended social norms, anticipated regret and seasonal influenza vaccination history) contribute to peoples' decisions on vaccination uptake? the study obtained ethics approval from the institutional review board of the university of hong kong/hospital authority hong kong west cluster. written informed consent was waived by the irb because all the data were analyzed anonymously, but verbal consent was obtained from all the subjects before the interview started. hong kong has 99% landline telephone penetration, local calls are free and telephone interviews are common and representative methods of survey data collection [53] . we conducted 13 main cross-sectional telephone surveys of psychological and behavioural responses to the first wave of the 2009 influenza a/h1n1 pandemic in hong kong from april through november 2009 (the parent study) [53] in order to monitor these variables. as an extension, the present study re-contacted subjects from some of these surveys and sought to understand public decision-making regarding ph1n1 vaccine uptake for mitigating the potential second wave of the pandemic. between 12-25 january, 2010 a baseline assessment for the present study was performed, immediately prior the local ph1n1 vaccination campaign extending to the general community (vaccination for high risk groups started from december 21, 2009), and we again contacted participants for follow-up two months later, between 15-30 march 2010. sample size determination. we estimated that a sample of at least 500 was required to achieve 80% power at an a = 0.05 to reject a model of the specified complexity ( figure 3 ) if the model fit index root mean square error of approximation (rmsea) exceeded 0.08 [54, 55] . to allow for a response rate ,60% in the follow-up and the baseline surveys, we need to target at least 1,389 subjects in the baseline survey. subject selection and inclusion criteria. a flow chart showing subject selection is provided in figure 4 . a total of 12,965 subjects participated in the parent study [53] . all these subjects were cantonese-speaking adults (aged$18) selected within households using a kish grid methodology, who were capable of and willing to answer a telephone interview. additional details about inclusion criteria are available elsewhere [53] . respondents in the 7 th , 9-12 th surveys of the 13 surveys comprising the parent study who, in the parent study agreed to be re-contacted and who had not received ph1n1 vaccine were invited to complete the baseline assessment for the present study. these five surveys (the 7 th , 9-12 th surveys) were selected because participants in these surveys had not had any follow-up contact either in the parent study or otherwise. this minimizes interview fatigue thereby improving response rates. these surveys were all of a comparable sample size, between 1,000-1,007 [53] . the five selected surveys were conducted between 21 july and october 23, 2009, and generated a representative [53] pool of 5,014 respondents of whom 61.4% (3,079/5,014) gave consent for further contact. from a list of the 3,079 subjects who agreed to be re-contacted, 1,648 calls were randomly selected and successfully made by a university telephone polling organization. unanswered calls were tried at least four times at different hours and weekdays before being replaced by new numbers. finally, a total of 1,511 (92%, 1,511/1,648) respondents agreed to participate in the baseline survey. of these 78 (5%, 78/1,511) reported already having received ph1n1 vaccination and were therefore excluded as ineligible, leaving 1,433 respondents who completed baseline interviews. the interview questionnaire for the baseline survey was derived from literature review, our previous cross-sectional surveys [53] and the theoretical framework constructed for this study (figure 3 ). specialists in health psychology, statistics, infectious disease and public health jointly determined the measures comprising the final questionnaire, guided by the need to maintain low assessment load and parsimony to ensure good response rates. the finalized questionnaire consisted of five sections: section 1 addressed respondents' self-rated health and their experience of influenzalike illness in the past six months; section 2 addressed risk perceptions regarding ph1n1; section 3 addressed perceived trust in information related to ph1n1 and ph1n1 vaccination from different information sources; section 4 addressed attitudes, beliefs and social norms regarding ph1n1 vaccine/vaccination, vaccination intention and planning; section 5 addressed key respondent demographics. overall, the baseline assessment consisted of 44 questions, which took less than 15 minutes to complete. other demographic data were obtained from the parent study [53] . prior to baseline assessment for the present study, subjects were reminded of their prior participation and that they had agreed to participate in a further study. the study was introduced as a survey of attitudes towards swine flu vaccination. we sought their willingness to participate. those agreeing were asked about their vaccination status. subjects who reported that they had already received ph1n1 vaccination were excluded. the remaining interview was performed. a follow-up survey was conducted 2 months later wherein respondents were reminded of the earlier survey and asked about their vaccination status and reasons for having had or not having vaccination. all the data were collected through telephone interview in both baseline and follow-up surveys. the measures comprising the study instruments were used to build the conceptual model ( figure 3 ) and are described below and in table 1 . perceived benefits of ph1n1 vaccination, and, concerns regarding adverse effects of ph1n1 vaccination. these two constructs assessed attitudes towards ph1n1 vaccination. perceived benefits of ph1n1 vaccination was assessed by measuring agreement on five-point ordinal scales (from 1 ''strongly disagree'' to 5 ''strongly agree'') with three statements (table 1) . a cronbach's alpha (a) of 0.71 indicated an acceptable internal consistency for this scale and these two items were treated as the indicators of a latent scale (perceived benefits of ph1n1 vaccination). concerns regarding adverse effects of ph1n1 vaccination were assessed by measuring agreement, using fivepoint scales, with two statements. the cronbach's a for these two items was 0.64, considered acceptable by some researchers [56] , though clearly less than desirable. we therefore treated the items as reflecting a latent variable (concerns regarding adverse effects of ph1n1 vaccination). social norms regarding ph1n1 vaccination. while tbp considers the influence of solely coercive social pressure from significant others to perform a behaviour, previous studies suggest that it is also important to consider the generalized tendency to adopt behaviours demonstrated by others encountered in daily life for imitative reasons [48, 57] . we use the term social norms rather than subjective norm to represent these broader coercive and imitative social influences. social norms were assessed by agreement on a 5-point scale with two statements. the internal consistency for these two items was weaker, with a = 0.53, which suggests each item appropriately measures different social influences. we initially incorporated these items separately in the structural equation model but except for the path weights dividing almost equally between the two items, no difference was otherwise seen. we therefore retained them as indicators of a combined latent construct in the model for purposes of model parsimony [58] . anticipated regret. anticipated regret was assessed with two statements asking about respondents' likelihood of feeling regret. responses of these two items were on a 7-point categorical scale (from 1 ''definitely not'' to 7 ''certain''). the internal consistency a for these two items was 0.68. the two items were used to indicate the latent variable ''anticipated regret'' in the modeling analysis. perceived self-efficacy. one item was used to measure selfefficacy, asking about respondents' agreement on a 5-point scale with the statement ''i am confident that i can go independently to get human swine flu vaccination''. a standard scale of self-efficacy was not adopted to minimize assessment load. however, a single item for self-efficacy has been shown elsewhere to have validity in predicting behavioural change [59, 60] . seasonal influenza vaccination history. respondents were asked whether they had received any seasonal influenza vaccination in the past three years (yes/no/don't know). vaccination intention. respondents were asked how likely it was that they would get vaccinated against ph1n1 during the winter flu season, using a 7-point likert scale (from 1 ''definitely not'' to 7 ''certain''). vaccination planning. we measured vaccination planning by assessing respondents' agreement on a 5-point scale with three statement items, such as ''i have planned when and where to get my human swine flu vaccination this winter''. the internal consistency a for these three items was 0.59, though less than the most common acceptable level of above 0.7, remaining at the minimal acceptable level (a ranged between 0.5-0.6) of reliability for preliminary research [56] . these items were also treated as indicators of a latent variable for modeling purposes. reported vaccination uptake. in the follow-up survey, respondents were asked to confirm if they had received ph1n1 vaccine within the past three months. respondents were also asked to indicate their major reasons for having or not having taken the ph1n1 vaccination using open-ended questions. multiple reasons could be given by each respondent. we first compared demographic differences between follow-up and lost-to-follow-up respondents with pearson chi-square test while demographic differences of the respondents who completed both the baseline and follow-up survey and the general population [61] were assessed using cohen's effect sizes [44] . proportions were calculated to describe patterns of vaccination intention, reported vaccination uptake, and major reasons for taking or not taking ph1n1 vaccination. structural equation modeling was then applied to examine the determinants of ph1n1 vaccination, vaccination intention and vaccination planning based on the extension of tbp. mplus 6.0 for windows (muthén & muthén, 1998 -2010 was employed because the model comprised dichotomous (vaccination status) and ordinal (vaccination intention) outcome variables. before testing the full structural model, zeroorder correlations between the measures of related constructs were calculated. confirmatory factor analysis was performed to assess the adequacy of the measurement model including perceived benefits of ph1n1 vaccination, concerns regarding adverse effects of ph1n1 vaccination, social norms regarding ph1n1 vaccination, anticipated regret and vaccination planning. to test the full structural model, all variables were entered into the model simultaneously. mean and variance adjusted weighted least squares estimation was applied to evaluate the standardized parameters (beta, b). since chi-square test is very sensitive to sample size and non-normally distributed data, several other model fit indices were evaluated including the comparative fit index (cfi), the tucker lewis index (tli), and rmsea. a cfi.0.90 and tli.0.90 indicates a good fit. rmsea less than 0.05 and one ranging between 0.05-0.08 respectively indicate a good and acceptable model fit [55] . misfitting models were respecified guided by theoretical soundness and modification indices [55] . missing proportions ranged from 0.1% for seasonal flu vaccination history to 5.5% for the item ''i have planned when and where to get my ph1n1 vaccination this winter''. there was no missing data for reported vaccination uptake. missing data were handled with multiple imputation [62] . of the 1433 respondents who completed the baseline assessment, 896/1433 (63%) respondents agreed to participate and completed the march follow-up survey (figure 4 ). demographic characteristics of respondents in the baseline and followup surveys are shown in table 2 . compared to respondents completing both baseline and follow-up surveys, respondents lost to follow-up were younger (x 2 = 14.24, p = 0.001) and more likely to be single (x 2 = 20.26, p,0.001). overall, the low cohen effect sizes (,0.3) showed that the demographics of respondents who completed both the baseline and follow-up surveys were comparable to those of the general population of hong kong [61] . of the 1,433 respondents who completed the baseline survey, 36% (510/1,433) reported that they would ''definitely not'' take ph1n1 vaccination during the winter flu season; 36% (521/1,433) reported being ''very unlikely/unlikely'' to take it; 19% (278/ 1,433) reported their ph1n1 vaccination likelihood as ''evens'' (50:50/equal likelihood); and only 8% (119/1,433) reported vaccination likelihood as ''likely/very likely/certain''. within the subset of 896/1,433 respondents who completed both baseline and follow-up surveys, 7% (67/896) had reported at baseline that they would be ''likely/very likely/certain'' to receive ph1n1 vaccination. however, in the follow-up survey, only 7/896 (0.8%) respondents reported having received ph1n1 vaccination in the intervening period, 4 of whom had reported being ''likely/very likely/certain'' to receive ph1n1 vaccination at baseline. reporting higher intention to receive ph1n1 vaccination in the baseline was associated with greater likelihood to vaccinate by follow-up (fisher's exact test, x 2 = 24.24, p,0.001). the 7 respondents who reported taking ph1n1 vaccination gave the major reasons for deciding on vaccination as follows: three choose vaccination because of the 'high risk of swine influenza' characterized by statements like ''swine flu is serious'', ''i am worried that swine flu will become more serious'', and ''i feel vulnerable to swine flu''; two reported that their decision was due to 'doctors' advice' and two reported 'belief of the vaccine efficacy'. other reasons provided by one respondent only were 'belief in the vaccine's safety', 'government recommendation', 'convenient availability', and 'protection of patients'. reasons for not having vaccination given by the 889 respondents who did not receive ph1n1 vaccination ( figure 5) were, most frequently 'low risk of or from swine influenza' (529/889, 60%) and 'concerns regarding adverse effects of the vaccine' (328/889, 37%). around 11% (100/889) of the respondents reported both 'low risk of/from swine influenza' and 'concerns regarding adverse effects of the vaccine'. table 3 for the final full structural model (figure 6 ), two additional paths were added and estimated based on the modification indices including a path from social norms to vaccination planning and path from anticipated regret to vaccination planning while the path from perceived self-efficacy to vaccination and the path from anticipated regret to vaccination were removed, coefficients for these two paths being nonsignificant and too small to be meaningful. the final model indicated a good fit with cfi = 0.96, tli = 0.93 and rmsea = 0.06 ( figure 6) . the model showed that respondents perceiving greater ph1n1vaccination benefits (b = 0.15), less concerns regarding vaccine adverse effects (b = 20.20), greater sensitivity to social norms b = 0.39), higher anticipated regret if not vaccinated (b = 0.47), higher perceived self-efficacy in taking ph1n1 vaccination (b = 0.12) and receiving seasonal influenza vaccination in the past three years (b = 0.12) reported greater intention to take ph1n1 vaccination, and accounted for 59% of variance in vaccination intention scores. greater adherence to social norms (b = 0.70), higher vaccination intention (b = 0.31) and higher anticipated regret (b = 0.19) were associated with more vaccination planning, together accounting for 67% of variance in vaccination planning. both vaccination intention (b = 0.30) and vaccination planning (b = 0.36) significantly predicted actual ph1n1 vaccination, accounting for 36% of variance in ph1n1 vaccination ( figure 6 ). the world health organization recommended a stepwise use of ph1n1 vaccines for protecting people against the ph1n1 influenza pandemic in july 2009 [63] . however, a vaccination program's efficacy largely depends on the public's compliance. our study found that only 5% of 1,511 subjects reported having received ph1n1vaccination and of 1,433 subjects remaining unvaccinated, only 8% reported intending (being likely/very likely/certain) to take the ph1n1 vaccine. two months later in the follow-up survey, an even smaller proportion, 0.8% of the respondents who completed both the baseline and follow-up survey reported having been vaccinated against ph1n1. perceived low risk of ph1n1 and concerns regarding vaccine-related adverse effects were the two most frequently cited reasons for refusing the vaccination. the extended tpb model suggests that both the original tpb components and the additional components contribute to people's decisions on vaccination uptake but that social norms and anticipated regret for not taking vaccination were the strongest determinants of vaccination intention and vaccination planning. finally vaccination planning partially-mediated the relation between intention and reported vaccination uptake. compared to previous studies, vaccination intention was much lower in our study than that found in surveys conducted prior to the influenza pandemic [23] or before the vaccine was available [11] , but was comparable to the findings of surveys conducted in france [20] and turkey [24] after ph1n1 vaccination programmes were launched there. an earlier hong kong study that relied on expressed intent to predict vaccination uptake [11] failed to accurately predict the subsequent meager population uptake of ph1n1 vaccination by, at best, an order of magnitude [64] , suggesting that intention alone is insufficient for predicting future vaccination uptake, consistent with empirical findings in other areas [33, 42] . despite predictions that intended ph1n1 vaccination uptake would decline if there was insufficient data on novel vaccine safety and efficacy [11] , safety issues were not the predominant barrier to vaccination in the present study. while 37% of our study respondents who remained unvaccinated cited vaccine safety concerns, despite good evidence that the vaccine is effective with a risk profile similar to that of seasonal influenza vaccine [65] , almost twice as many, 60%, cited 'low risk of/from swine influenza' as their reason for not getting vaccinated, suggesting that these respondents felt no advantage would be gained by vaccination. around 11% of respondents, cited both 'low risk of/ from swine influenza' and 'concerns regarding adverse effects of vaccine' as the reasons for not getting vaccinated, seemingly adopting a risk-benefit approach to vaccination decision-making. however, in the setting of low influenza risk, with the reports of vaccine related adverse events in the media after the vaccine was available for the priority groups (figure 2) , people may shift their perceived risks away from influenza and towards vaccination, suggestive of availability bias (risk distortion by easily recalled events) [66] . we believe that perceived vaccine risk would become progressively less of a barrier to vaccination as perceived influenza risk increases, and vice versa. moreover, despite recent reports that hong kong residents would be sensitive to vaccination pricing when considering whether to vaccinate [11, 12] , only 2.5% of our respondents cited high vaccine cost as the reason for rejecting vaccination. major reasons for taking ph1n1 vaccination corresponded to reasons for not taking it, with perception of ph1n1 risk most frequently cited. however, the few respondents receiving ph1n1 vaccination prohibited meaningful comparison. the extended version of tpb model fits well to the survey data. the model showed that an expanded social norms and anticipated regret accounted for most of the variance in vaccination intention, rather than the more core elements of tpb. in turn, social norms independently accounted for more than twice the variance in vaccination planning than did intention, and vaccination planning accounted for more variance in vaccination uptake than did intention. thus it seems that social norms comprise the major influences on vaccination uptake through modifying vaccination intention and planning. a meta-analytic review of tpb efficacy concluded that the tpb variable subjective norm (perceived coercive social pressure from significant others) weakly predicted intention compared to other tpb components, mainly due to poor measurement [41] . ''descriptive norm'' (perception of what other people do, imitation or conformity behaviour) is reportedly a more important predictor for intention [48, 57] . here we combined table 3 . correlations, means, standard deviations, and standardized factor loadings for the measurement model. measures of subjective and descriptive norms, treated as a latent variable (social norms), because they were found to have much the same predictive direction and weight. multiple item measures of norms should have better predictive power than single item measures [41] . this model importantly informs public health approaches to population behaviour during respiratory epidemics. first, information uncertainty or untrustworthiness, for example regarding vaccine safety, is likely to prompt people look to others for their cues to action: the social environment, namely what other people believe and do powerfully influences decisions to action [45, 67] . people often tend to imitate others, so establishing a ''vaccination trend'' may help uptake. for example, it could be effective to encourage those who remain unvaccinated with feedback from vaccinated peers and by providing an updated total of numbers vaccinated. what the general public think and do may prove to be as influential as information from scientists or health professional [68, 69] . second, encouraging uptake of a new vaccine will be problematic if the associated threat element is low, irrespective of vaccine pricing, particularly for novel and untested vaccines. vaccine safety and efficacy data should be provided wherever possible at all levels including through health-care providers, media and the general public. to effectively communicate the risk and benefit of a novel vaccine, it is important to establish an effective surveillance system to monitor vaccination progammes and rapidly respond to any reported adverse events [70] . the media have an important influence and both reactionary and opinionated news items should be recognized as potentially detrimental to vaccination uptake. in particular, the need to develop stories that generate revenue increasingly overrides balanced reporting in contemporary media. hence risk amplification remains a problem. public health agencies need to improve their liaison with influential media outlets to minimize this, where possible. third, omission bias, a phenomenon where people view vaccination as more risky than remaining unvaccinated, could be a barrier for vaccination [71] . omission bias arises when there is anticipation of greater regret about adverse effects of vaccination, if taken, than the regret about being infected with influenza if vaccination is rejected [72] . therefore, social marketing emphasizing the far greater likelihood of regret for consequences due to refusing vaccination than the regret over an improbably low adverse event due to taking vaccination may help to reduce this bias. for example, previous studies found that simply asking two questions about feeling regret for inaction could increase respondents' intention to play a lottery or do exercise [48, 49] . finally, vaccination planning is a key intervening variable between vaccination intention and actual vaccination. this is to be expected given that it is more proximal to actual behaviour than intention is. in those who may be undecided, interventions facilitating planning may prompt action. this could include suggesting where, when and how to get vaccination, improving and publicizing accessibility of vaccination centres and opening times. even so, intention and planning explained only 36% of the variance in the reported vaccination behaviour, suggesting that other factors, such as intention stability [42] , influencing vaccination behaviour await identification. study limitations include baseline attitudes/beliefs, vaccination intention and planning being measured at the same time point, prohibiting exploration of causality in observed associations. some study measures were constrained due to length of telephone interviewing, and while sub-optimal were necessary methodological compromises. although most researchers recommended cronbach's a of 0.7 as the minimal acceptable for internal consistency of multi-item scales, others accept 0.6 or 0.5-0.6 for preliminary research as the cut-off point [56] . other than dimensionality concerns, lower a can reflect too few items comprising the putative scale [73] . this is more likely for complex variables, such as social norms which have a broad spectrum of elements. though less than perfect, measurement errors can be reduced by incorporating the items as a latent variable in sem [55] , an approach we adopted. additionally, collinearity between exogenous indicators, such as social norms and perceived benefit can be potentially problematic, perhaps lowering the accuracy of sem estimation. however, since high associations between measures of the constructs were not observed (table 3) then collinearity-related error is probably small [74] . despite being randomly selected for the parent study, subjects of this study were not randomly selected from the general population, although demographics suggest the current sample is comparable to the hong kong general population [61] (table 2 ). moreover, subject recruitment was based on voluntariness and all data were selfreported. all could cause social desirability and selection bias, so caution is needed before extrapolation to the general population. also refusal at follow-up could have influenced patterns of responses. our study examined public decision-making regarding a novel influenza pandemic vaccine. our findings may not apply to vaccination against seasonal influenza due to numerous differences in beliefs towards the vaccination. for example, although perceived low risk remains the major reasons for refusing vaccination against seasonal influenza as in our study, vaccine safety is seldom cited as a barrier [28, 29] whereas we found that about one third of respondents had vaccine safety concerns. cultural differences in influenza and vaccination-related beliefs are possible [75] , but these differences may gradually diminish with the increasing identical news information available through the three dominant news agencies and common public health strategies being increasingly universal. related stories, such as use of preservatives and adjuvants in vaccine manufacture may enhance knowledge and reduce trust in product safety [76] . the role of media remains much under-researched in this regard. finally, data was insufficient to reliably report the reasons for ph1n1 vaccination uptake among the population. nonetheless, compared with other cross-sectional studies [12, [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] , the longitudinal design of this study strengthens understanding of influences on population decision-making for pandemic influenza vaccination uptake and represents a 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additives, or residuals? we thank the hku public opinion programme for assistance in administering the telephone survey. we thank diane ng for coordinating the data collection and data management. analyzed the data: ql. contributed reagents/materials/analysis tools: ql bjc wwtl rf. wrote the paper: ql bjc wwtl rf. key: cord-001421-6t5puo6p authors: marfà, santiago; crespo, gonzalo; reichenbach, vedrana; forns, xavier; casals, gregori; morales-ruiz, manuel; navasa, miquel; jiménez, wladimiro title: lack of a 5.9 kda peptide c-terminal fragment of fibrinogen α chain precedes fibrosis progression in patients with liver disease date: 2014-10-02 journal: plos one doi: 10.1371/journal.pone.0109254 sha: doc_id: 1421 cord_uid: 6t5puo6p early detection of fibrosis progression is of major relevance for the diagnosis and management of patients with liver disease. this study was designed to find non-invasive biomarkers for fibrosis in a clinical context where this process occurs rapidly, hcv-positive patients who underwent liver transplantation (lt). we analyzed 93 lt patients with hcv recurrence, 41 non-lt patients with liver disease showing a fibrosis stage f≥1 and 9 patients without hcv recurrence who received antiviral treatment before lt, as control group. blood obtained from 16 healthy subjects was also analyzed. serum samples were fractionated by ion exchange chromatography and their proteomic profile was analyzed by seldi-tof-ms. characterization of the peptide of interest was performed by ion chromatography and electrophoresis, followed by tandem mass spectrometry identification. marked differences were observed between the serum proteome profile of lt patients with early fibrosis recurrence and non-recurrent lt patients. a robust peak intensity located at 5905 m/z was the distinguishing feature of non-recurrent lt patients. however, the same peak was barely detected in recurrent lt patients. similar results were found when comparing samples of healthy subjects with those of non-lt fibrotic patients, indicating that our findings were not related to either lt or hcv infection. using tandem mass-spectrometry, we identified the protein peak as a c-terminal fragment of the fibrinogen α chain. cell culture experiments demonstrated that tgf-β reduces α-fibrinogen mrna expression and 5905 m/z peak intensity in hepg2 cells, suggesting that tgf-β activity regulates the circulating levels of this protein fragment. in conclusion, we identified a 5.9 kda c-terminal fragment of the fibrinogen α chain as an early serum biomarker of fibrogenic processes in patients with liver disease. early detection of fibrosis progression and the development of portal hypertension is of major relevance in the prognosis and treatment of patients with chronic liver disease [1] . indeed, early recognition of subjects prone to develop these alterations may allow prompt initiation of therapeutic interventions. therefore, identification of noninvasive biomarkers related to the activation of the fibrogenic process is of major relevance, particularly in those subjects with sustained liver injury [2] . however, despite the numerous attempts to uncover such molecules, this objective has resulted elusive. this is likely related to the natural history of liver disease. with the exception of fulminant hepatic failure, liver disease is an insidious process in which clinical detection and symptoms of hepatic decompensation may occur weeks, months or many years after the onset of injury, and healing may occur without clinical detection [3] . however, in particular clinical circumstances, i.e. patients infected with the hepatitis c virus (hcv), submitted to liver transplantation (lt), it is possible to expect recurrence of hepatic fibrosis and portal hypertension to occur within a short period of time [4] . thus, these patients constitute a population particularly suitable to identify noninvasive markers of early fibrogenesis. the current investigation took advantage of the faster development of hepatic fibrosis in hcv-positive lt patients. serum samples were collected shortly after lt and high-throughput proteomic techniques were used to ascertain whether the proteomic pattern of these samples differs from the proteomic pattern expression obtained from serum samples of non-infected lt patients. ultimately, the investigation was aimed to identify early circulating serum biomarkers of active fibrogenesis in patients with liver disease. one hundred and nineteen patients admitted to the liver unit to undergo a liver biopsy from june 2001 to january 2006 were prospectively considered for this study. exclusion criteria were presence of ascites, chronic kidney failure in hemodyalisis and moderate or severe acute graft rejection during the first three months, biliary complications or antiviral treatment during the first year after lt in the case of lt recipients. in addition 16 healthy volunteers were also included in the study. the design of the study was two folded: first we assessed whether the serum proteomic profile of recurrent hcv-lt patients differs from that of non-recurrent hcv-lt patients. the serum proteomic profile and routine liver and renal function tests were initially analyzed in a training set of 10 hcv-rna recurrent lt patients 6 months post lt that showed a fibrosis stage f$1 at 1 year after lt. paired hepatic venous pressure gradient (hvpg) determination was also available in 7 of these patients. the control group consisted in 9 patients without hcv-rna recurrence, who underwent antiviral treatment before lt and achieve sustained virological response. in addition, serum samples were also collected from 41 non-lt patients with advanced liver disease. the hcv or hepatitis b virus (hbv) was present in 8 and 3 of these patients, respectively, whereas the etiology of liver disease was other than viral in the remaining (9 nonalcoholic steatohepatitis, nash; 10 alcoholic liver disease, ald; 4 autoimmune hepatitis, ah; and 7 cryptogenic). thereafter, the results were validated in a test set of 83 hcv recurrent lt patients. serum samples were also collected 6 months post-transplantation and the proteomic profile was evaluated along with liver and renal function tests. hvpg measurement in 53 of these patients was also performed. percutaneous and transjugular liver biopsies and hvpg measurements were performed as we have previously described [5] . fibrosis stage was scored using the scheuer classification: no fibrosis (f0), minimal portal fibrosis (f1), periportal fibrosis (f2), fibrosis beyond the portal tract making septums (f3) and cirrhosis (f4) [6] . see (data s1). protein profiling was performed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (seldi-tof-ms) using the eight-spot format cm10 (weak cationic exchange) proteinchip arrays (bio-rad). in a preliminary study performed to set up the experimental conditions, 2 pooled serum samples from the 9 patients without hcv-rna recurrence and the 10 patients included in the training set were loaded onto three different types of protein chip arrays: h50 (that binds proteins through reverse phase or hydrophobic interactions), cm10 (negatively charged surface that acts as a weak cation-exchanger) and imac-30 (immobilized metal affinity capture surface preactivated with copper). the resulting spectra from each pool were compared and the cm10 array showed the highest number of peaks detected and the highest total signal intensity compared to h50 and imac-30; therefore only the cm10 array was used in the subsequent studies. prior to sample loading, spots were equilibrated two times with 200 ml of cm binding/washing buffer (0.1 m sodium acetate, ph 4). each sample was loaded in duplicate randomly in order to minimize any systematic error. forty microliters of fractionated serum sample was incubated in 60 ml of cm binding buffer for 30 minutes on a shaker at room temperature. afterwards, arrays were washed three times with 200 ml cm washing buffer for 5 minutes at room temperature. unbound serum proteins were removed by washing twice with deionized water. thereafter, arrays were air-dried and 1 ml of energy-absorbing matrix (saturated sinapinic acid in an aqueous solution containing 50% acetonitrile and 0.5% tfa) was added twice to each spot. the surface was allowed to air dry between each application. the array was read by using the proteinchip pbs ii reader (biorad). each spot was read at low (2500 nj), medium (3000 nj) and high (3500 nj) energy laser intensities. the mass-to-charge ratio (m/z) was set from 1.000 to 25.000 m/z for the low-energy laser intensity, between 2.500 and 200.000 m/z for the medium-energy laser intensity and from 5.000 to 200.000 for the high-energy laser intensity. all spectra were calibrated using two external calibration standards (all-in-one peptide standard and all-in-one protein standard, biorad). a peak resolution was optimized within 5.000 m/z, 12.000 m/z or 19.000 m/z according to low, medium or high energy laser intensity, respectively. all data were processed with the proteinchip data manager client 4.1 software (bio-rad). to minimize the possible random error and spectral outliers, all the raw data was normalized by the average total ion current across the group and all spectra differing by twice the standard deviation or more from the mean were deleted. furthermore, the baseline was also corrected by adjusting the parameter to 30 times the expected peak width. for the peak selection, several parameters were selected for the identification of peak clusters. thus, only peaks with a signal to noise equal or greater than 5; with a valley depth superior than three; found in a minimum of 20% of all spectra and with an m/z error below the 0.3% for the low-energy laser intensity spectra and below 2% for the medium-and high-energy laser intensity spectra, were considered. subsequently, all peak clusters detected were verified manually. relabeling, removal or addition of peaks was performed when necessary. to test the quality of the assay, pooled normal sera from two individuals was assessed. five protein peaks randomly selected over the course of the study were used to calculate the coefficient of variance (cv) as described [7] . we then determined the reproducibility of the seldi spectra, both within and between arrays (intra-assay and inter-assay, respectively). the intra-assay (spot to spot) cv was 11.95% for peak intensity and 0.02% for mass accuracy. the inter-assay (chip to chip) cv was 21.96% for peak intensity and 0.03% for mass accuracy. see (data s2). hepg2 cells were obtained from american type culture collection (atcc, manassas, va, usa). this immortalized, stable cell line can be repeatedly frozen, thawed and propagated. hepg2 cells were seeded (2610 6 cells/well) in vented t-75 flasks and grown to confluence in dulbecco's modified eagle medium (dmem), supplemented with 50 u/ml penicillin, 50 mg/ml streptomycin and 10% of fetal calf serum (fcs). thereafter, cells were switched to 1% fcs and incubated (37uc) under normoxic (21% o 2 , 5% co 2 ) or hypoxic conditions (5% o 2 , 5% co 2 ) in a controlled o 2 water-jacketed co 2 incubator (forma scientific series ii, 3131, marietta, oh) or treated with tnf-a (10 ng/ml, sigma, st louis, mo), lipopolysaccharide (lps, 10 ng/ml, the same day of the liver biopsy, 20 ml of blood were obtained in a fasting status. serum was stored at 280uc, and serum albumin, aspartate aminotransferase (ast), alanine transaminase (alt), bilirrubin and blood urea nitrogen (bun) were measured with the advia 2400 instrument (siemens healthcare diagnostics, tarrytown, ny, usa). amino-terminal propeptide of type iii procollagen (piiinp), hyaluronic acid (ha), and tissue inhibitor of matrix metalloproteinase type-1 (timp-1) were measured in all patients by a ce-marked random-access automated clinical immunochemistry analyzer that performs magnetic separation enzyme immunoassay tests (advia centaur, siemens healthcare diagnostics, tarrytown, ny, usa). the enhanced liver fibrosis (elf) score was calculated using the algorithm recommended in the ce-marked assay [elf = 2.278 + 0.851 ln(ha) + 0.751 ln(piiinp) + 0.394 ln(timp-1)] blood tests. statistical analysis of the results was performed by the non parametric mann-whitney u test and the kruskal-wallis test with the dunn post hoc test as appropriated. quantitative data were analyzed using graphpad prism 5 (graphpad software, inc. san diego, ca). we obtained written informed consent from all patients included in the study and the investigation was approved by the investigation and ethics committee of the hospital clinic of barcelona following the ethical guidelines of the 1975 declaration of helsinki. the principal demographic values of patients included in the definition group are shown in table 1 . as per the selection criteria, most recurrent hcv patients showed higher fibrosis and elf scores, elevated hvpg measures and greater ast and alt values than non recurrent hcv subjects. figure 1 depicts a portion of the spectra of all the samples investigated in this training group, ranging between 3000 and 11000 daltons; the mass to charge ratio analyzed. the expression pattern of the spectrograms obtained from non recurrent hcv patients clearly differed from those of recurrent hcv subjects. six statistically different peaks, identified in the figure as peptides a, b, c, d, and e, were detected. as shown in table 2 the signal intensity of four of these peaks (a, b, c, and f) was markedly higher in non recurrent than in recurrent patients whereas an inverse situation was observed in the remaining two peaks (d and e). the most remarkable difference was detected on analyzing peptide a (5905 m/z), since it was fully evident in all samples obtained from non recurrent hcv patients but almost suppressed in the serum of recurrent hcv individuals. an intriguing question arising from the above described results was to elucidate whether these findings result from the particular characteristics of hcv transplanted patients rather than to a differential feature characterizing early fibrogenic processes. thus, the serum proteomic spectrum was next analyzed in healthy individuals, in non-lt fibrotic hcv infected patients and in non hcv fibrotic patients. the clinical and demographic characteristics of these subjects are shown in table 3. fibrosis scores and liver function tests of the two groups of fibrotic patients were similar to those obtained in hcv lt patients. the mass spectrograms of all individuals included in this portion of the investigation are shown in figure 2 . the upper, middle and lower parts correspond to healthy subjects, fibrotic non hcv subjects and fibrotic hcv patients, respectively. since in the previous experiments the most striking differences were observed with peptide a, in this and the subsequent experiments we focused on the peak with a mass/charge ratio of 5905 da. all serum samples analyzed from healthy subjects showed a spectrogram compatible with the presence of this peptide whereas pathological serum samples, either from fibrotic non lt hcv patients or fibrogenic non hcv subjects showed the absence of this peptide or very low intensity peaks. these results indicate that neither hcv nor lt account for the suppressed expression of the a peptide in serum samples of fibrotic patients. to further confirm that peptide a behaves as an early serum biomarker of fibrosis, mass spectrometric analysis was performed in a test group of serum samples obtained from 83 hcv recurrent patients 6 months after lt. hvpg was assessed in 53 of these patients and the average value was of 5.560.8 mm hg. all the serum samples showed a quite similar expression pattern and coincidences included both the different peptide fragments detected and the signal intensity of these fragments (data s4). the most relevant finding was, however, that the mass spectrum corresponding to peptide a showed a very low peak intensity in all samples obtained from hcv recurrent patients. in fact, in 53 out of the 83 patients the peak intensity at m/z 5905 was under the background levels of 10 ma and in the remaining samples, intensities ranged between 11.5 and 81.4 ma ( figure 3) . thus, these results confirm the findings obtained in the training group in a larger group of subjects. to isolate the protein of interest and to determine candidate protein identity, serum samples from two healthy subjects containing high seldi intensity were pooled and separated by tricine-sds-page (figure 4b). the band at 5.9 kda was excised trypsinized and analyzed by lc-ms/ms. as shown in figure 4c , two peptide sequences were identified, which matched with the human fibrinogen a c-chain at 3.23% coverage, suggesting that suppression of the fibrinogen a c-chain 5.9 kda fragment is an early surrogate indicative of active fibrogenesis in patients with liver disease. to unveil potential mechanisms governing the release of the 5.9 kda peptide c-terminal fragment of the fribrinogen a chain in the serum of patients under an early fibrogenic process, hepg2 cells were treated with well known proinflammatory stimuli (tnfa, lps and il-1b) or profibrogenic substances (aii, et-1, apelin, fibronectin and tgf-b) for 6 hours. with the exception of tgfb, none of these substances induced significant changes in the expression of human fibrinogen chains messenger. however, evaluation of the extension and aggressiveness of the fibrogenic process in the injured liver is of major relevance for the diagnosis, prognosis and treatment of patients with hepatic disease [8] . the methods currently available to assess liver fibrosis include the serological determination of several parameters related to liver function and hepatic remodeling, imaging techniques, such as fibroscan or arfi and the use of invasive procedures such as hvpg measurement or liver biopsy, the latter still being the most widely accepted gold standard method for assessing liver fibrosis [9] . the specific limitations of each of these methods have been extensively discussed previously [10] . the risk of complications and low sensitivity for mild or moderate fibrosis are among the most remarkable limitation for invasive and non invasive methods, respectively [11] . recently, a liver fibrosis score, namely elf, which combines the serum concentrations of substances related to collagen metabolism (piiinp) and tissue remodeling (timp-1 and ha), has progressively been incorporated among the most common diagnostic tools to evaluate liver fibrosis. however, whereas this technique was found to be highly accurate in patients with advanced fibrosis (f3-f4 stage) [12] [13] [14] it appeared to be less efficacious in the diagnosis of mild or moderate fibrosis (f1-f2 stage) [15, 16] . early detection of active fibrogenic activity, therefore, still remains an open challenge in liver disease. fibrosis progression evolves over long periods of time, with this representing one of the most relevant difficulties to identify specific early biomarkers of fibrosis. in the current investigation this issue was overcome by assessing the proteomic profile of hcv-positive lt recipients in a training set of serum samples. blood samples were obtained at 6 months after lt and a liver biopsy was performed 1 year after surgery to define fibrosis stage. it is well known that fibrosis progression is accelerated in recurrent hepatitis c, with 15% to 47% of lt recipients developing fibrosis/cirrhosis within the first 3 years post transplantation [17] . therefore, rapid fibrosis progression is a major characteristic of this group of patients and for this reason they are particularly suitable to uncover serum tags of hepatic fibrosis. seldi-tof-ms technology or protein chip profiling combines mass spectrometry with a surface enhanced biochip which allows uniform and reproducible binding and desorption of biomarkers [18] . seldi-tof-ms also incorporates sample prefractionation. this markedly decreases the complexity of protein rich fluids, such as serum, allowing comparison of peak intensity between samples using large sample sets [19] . in the current study, serum proteins were fractionated by anion exchange chromatography based on their isoelectric points using a ph gradient. the resulting fraction was bound to a weak cation exchange surface to create an array of protein chip spots. this surface was selected according to its higher accuracy and reproducibility yields. using this technology we were able to simultaneously detect relative protein expression levels over a range of molecular masses of 2 to 180 kda, although the 2-20 kda range appeared to be the most sensitive. by means of this profiling system, we found at least 6 serum biomarkers that were differentially increased or decreased in recurrent hcv patients. among them, a protein of 5.9 kda (protein a) was fully suppressed in the serum of all the hcv patients included in the training set. in contrast, readily detectable levels of this protein were detected in all non-recurrent hcv patients. we assessed whether lt and/or hcv infection account for the different expression patterns of peak a in serum samples of nontransplanted hcv positive and hcv negative subjects with fibrosis. the demographic and biochemical characteristics of patients with fibrosis included in the training set of samples were quite similar to those displayed by the fibrotic patients of this protocol with the exception of hepatic enzymes which, as expected, were higher in lt recurrent hcv patients than in fibrotic non-transplant patients. both, the proteomic profile of the hcv positive samples and the proteomic profile of fibrotic patients non-infected sera, showed no or very low intensity peaks at the 5.9 kda spectra. this markedly differed from the proteomic analysis of the serum samples of healthy subjects included in this set of experiments because all displayed consistent amounts of protein a. interestingly, different etiologies (nash, ald, hbv, ah, cryptogenic) accounted for liver fibrosis in negative hcv patients, further emphasizing the close relationship between the lack of the 5.9 kda protein and the fibrogenic process. next, the spectral data obtained in the test set were applied for validation purposes. all serum samples included in the test set showed an intensity m/z 5905 peak well below the values found in both healthy subjects and non recurrent hcv patients. indeed, in most of these samples the a peak was not detected (figure 3). overall, our results showing markedly decreased expression of the m/z 5905 in the spectral profile of all samples from patients with fibrosis further strengthen the highly sensitive diagnostic performance of this peak. a major limitation of seldi-tof-ms technology is related to the unfeasibility of directly identifying the protein of interest. in fact, for the majority of protein identifications it is necessary to achieve the enrichment of the specific peak by chromatography procedures and purification by sds gel electrophoresis with subsequent triptic digest. in our investigation, amino acid sequencing of the trypsin digest of the 5.9 kda protein revealed it to be a fragment of the fibrinogen a c-chain. human fibrinogen is a circulating 340 kda glycoprotein which has been shown to be of hepatic origin in vivo. moreover, inflammatory stimuli may induce in vitro secretion of this glycoprotein in non hepatic cells including epithelial cells, granulosa cells, cervical carcinoma cells and trophoblasts [20] . however, current evidence strongly suggests that the largest site of human plasma fibrinogen is the hepatocytes [21] . it is comprised of two symmetric half molecules bound by a disulphide knot, each consisting in one set of three different polypeptide termed aa, bb and c. each of these polypeptides is encoded by a separate gene located on chromosome four. the predominant aa of circulating fibrinogen contains 610 aa (63.5 kda), the bb chain 461 aa (56 kda) and the c chain is heterogeneous, but the most abundant form consists of 411 aa (48 kda). the protein shows extensive post translational modification including phosphorylation, sulphation, glycosylation and hydroxylation. the fibrinogen a c-domain of the human fibrinogen is the c-terminal two-thirds of the aa chain that extends from the coiled oil portion of each half of the dimeric fibrinogen molecule [22, 23] . the a c-fragments are released into circulation as natural by-products of fibrinolytic systemic activation and are therefore, found in the systemic circulation in healthy individuals [24] . our results showing almost suppressed expression of the 5.9 kda fragment of the a c-chain of fibrinogen in patients undergoing a fibrogenic process are in agreement with those previously reported by nomura f et al in heavy drinkers [25] . furthermore, these authors showed that serum levels of this fragment were recovered when alcohol intake has ceased for more than 3 months and they also extended their findings to hcv infected patients [26] . later, this fragment was described as having diagnostic value in patients with acute respiratory syndrome [27] , breast cancer [28] and pancreatic adenocarcinoma [29] . the regulation of total human fibrinogen by a number of proinflammatory agents has been previously investigated using the hepg2 hepatocellular carcinoma cell line [30] . this in vitro model faithfully recapitulates fibrinogen expression including a, b and c fibrinogen [31] and has been used to study fibrinogen production and regulation in vitro [32] . accordingly we subsequently assessed the potential regulatory role of several candidate mediators on a-fibrinogen expression in hepg2 cells. a number of proinflammatory/profibrogenic agents that have previously been involved in the pathogenesis of the fibroproliferative processes [33] [34] [35] were tested. among them, only tgf-b showed significant regulatory activity on a-fibrinogen mrna expression and decreased 5.9 kda fibrinogen ac-fragment intensity. of note was, however, that the fold change in the fibrinogen ac-fragment induced by tgf-b in hepg2 cells was makedly lower than that observed in samples from fibrotic patients. the marked differences between the in vivo and in vitro experimental conditions likely account for this discordance. for instance, hepg2 is a human derived carcinoma cell line that shows altered abundance of tgfb receptors [36, 37] which in turn could result in some sort of resistance to this cytokine. on the other hand it is well known that regulation of acute-phase proteins is mediated by a combination of cytokines thus raising the possibility that additional factors involved in inflammatory processes also regulate the expression of the 5.9 kda fragment of fibrinogen [38] . our results are in line with past studies in which tgf-b inhibited the induction of fibrinogen produced by il-6 and decreased the synthesis of fibrinogen in hepg2 and hepb cells [38] , respectively. these latter experiments also showed a parallel diminution in afibrinogen mrna levels. this phenomenon seems to be mediated by post-transcriptional mechanisms since tgf-b did not modify fibrinogen gene transcription, suggesting that the effect of this cytokine in liver cells is regulated at the level of mrna stability [39] . overall, all these results indicate that tgf-b may regulate the synthesis of a-fibrinogen at the postranscriptional level. in summary, the current investigation took advantage of the faster development of hepatic fibrosis in hcv-positive lt patients to identify early circulating serum biomarkers of active fibrogenesis in patients with liver disease. using high throughput seldi-tof-ms technology we unveiled a differential protein pattern profile between early fibrosis recurrence and non recurrent lt patients. six protein peaks displaying statistically significant different intensities were observed within a range of 1000 to 25000 m/z. the peak located at 5905 m/z showed the most remarkable difference, since it was fully detected in non-recurrent lt patients but was almost suppressed in recurrent lt patients. similar results were found when comparing samples of healthy subjects with those of non lt fibrotic patients both hcv positive and negative, indicating that our findings were not related to either lt or hcv infection. identification of this protein peak showed more than a 99% coincidence with a c-terminal fragment of the fibrinogen a chain. moreover, cell culture experiments demonstrated that tgf-b downregulates a-fibrinogen mrna expression and decreases the peak intensity of the m/z 5.9 kda protein in hepg2 cells. in conclusion, we identified a 5.9 kda c-terminal fragment of the fibrinogen a chain as a serum biomarker of early fibrogenic processes in patients with liver disease. since tgf-b inhibited a-fibrinogen mrna expression in hepg2 cells it is temptative to speculate that the activation of this cytokine in the early phases of liver injury could be responsible for the impairment in the circulating levels of the fibrinogen a c-chain fragment in patients with active hepatic fibrogenesis. data s1 materials and methods corresponding to the serum fractionation procedure. fibrosis and cirrhosis reversibility: clinical features and implications biomarkers of hepatic fibrosis, fibrogenesis and genetic pre-disposition pending between fiction and reality pathogenesis of liver fibrosis liver fibrosis hepatic venous pressure gradient identifies patients at risk of severe hepatitis c recurrence after liver transplantation the nomenclature of chronic hepatitis: time for a change proteomic profiling of cholangiocarcinoma: diagnostic potential of seldi-tof ms in malignant bile duct stricture evaluation of liver fibrosis: a concise review biopsy and non-invasive methods for the diagnosis of liver fibrosis: does it take two to tango? non invasive markers of liver fibrosis noninvasive assessment of liver fibrosis serum markers detect the presence of liver fibrosis: a cohort study the enhanced liver fibrosis (elf) score: normal values, influence factors and proposed cut-off values assessment of liver fibrosis before and after antiviral therapy by different serum markers panels in patients with chronic hepatitis c noninvasive assessment of liver fibrosis arfi, fibroscanß, elf and their combinations in the assessment of liver fibrosis: a prospective study progression of liver fibrosis in post-transplant hepatitis c: mechanisms, assessment and treatment evaluation of serum protein profiling by surface-enhanced laser desorption/ionization timeof-flight mass spectrometry for the detection of prostate cancer: i. assessment of platform reproducibility clinical proteomics: searching for better tumour markers with seldi-tof mass spectrometry fibrinogen and fibrin human plasma fibrinogen is synthesized in the liver the structure and biological features of fibrinogen and fibrin comparative structural and functional features of the human fibrinogen alpha c-domain and the isolated alpha c fragment. characterization using monoclonal antibodies to defined coohterminal a alpha chain regions identification of novel and downregulated biomarkers for alcoholism by surface enhanced laser desorption/ionization-mass spectrometry serum fibrinogen a c-chain 5.9 kda fragment as a biomarker for early detection in hepatic fibrosis related to hepatitis c virus serum proteomic fingerprints of adult patients with severe acute respiratory syndrome serum proteomic analysis identifies a highly sensitive and specific discriminatory pattern in stage 1 breast cancer serum diagnosis of pancreatic adenocarcinoma using surface-enhanced laser desorption and ionization mass spectrometry human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis b surface antigen recombinant human fibrinogen and sulfation of the gamma' chain effects of cytokine combinations on acute phase protein production in two human hepatoma cell lines hypoxia and proinflammatory factors upregulate apelin receptor expression in human stellate cells and hepatocytes bacterial lipopolyshaccaride inhibits cb2 receptor expression in human monocytic cells apelin mediates the induction of profibrogenic genes in human hepatic stellate cells transforming growth factor-b and response to anticancer therapies in human liver and gastric tumors in vitro and in vivo differential inhibition of the tgf-b signaling pathway in hcc cells using the small molecule inhibitor ly2157299 and the d10 monoclonal antibody against tgf-b receptor type ii transforming growth factor beta 1 regulates production of acute-phase proteins transformin growth factor-b1 induced smad signaling, cell cycle arrest and apoptosis in hepatoma cells the authors are indebted to drs. f. elortza and i. iloro for their collaboration in the identification of the 5.9 kda protein peak. key: cord-000140-5kapn32k authors: wang, pei-gang; kudelko, mateusz; lo, joanne; siu, lewis yu lam; kwok, kevin tsz hin; sachse, martin; nicholls, john m.; bruzzone, roberto; altmeyer, ralf m.; nal, béatrice title: efficient assembly and secretion of recombinant subviral particles of the four dengue serotypes using native prm and e proteins date: 2009-12-15 journal: plos one doi: 10.1371/journal.pone.0008325 sha: doc_id: 140 cord_uid: 5kapn32k background: flavivirus infected cells produce infectious virions and subviral particles, both of which are formed by the assembly of prm and e envelope proteins and are believed to undergo the same maturation process. dengue recombinant subviral particles have been produced in cell cultures with either modified or chimeric proteins but not using the native forms of prm and e. methodology/principal findings: we have used a codon optimization strategy to obtain an efficient expression of native viral proteins and production of recombinant subviral particles (rsps) for all four dengue virus (dv) serotypes. a stable hela cell line expressing dv1 prme was established (hela-prme) and rsps were analyzed by immunofluorescence and transmission electron microscopy. we found that e protein is mainly present in the endoplasmic reticulum (er) where assembly of rsps could be observed. biochemical characterization of dv1 rsps secretion revealed both prm protein cleavage and homodimerization of e proteins before their release into the supernatant, indicating that rsps undergo a similar maturation process as dengue virus. pulse chase experiment showed that 8 hours are required for the secretion of dv1 rsps. we have used hela-prme to develop a semi-quantitative assay and screened a human sirna library targeting genes involved in membrane trafficking. knockdown of 23 genes resulted in a significant reduction in dv rsp secretion, whereas for 22 others we observed an increase of rsp levels in cell supernatant. conclusions/significance: our data describe the efficient production of rsps containing native prm and e envelope proteins for all dengue serotypes. dengue rsps and corresponding producing cell lines are safe and novel tools that can be used in the study of viral egress as well as in the development of vaccine and drugs against dengue virus. dengue is one of the most important vector-borne viral diseases in humans. however, the interaction between dengue virus (dv) and host cells is only partly understood. therefore, there is an urgent need to develop new tools to gain insight into the viral journey through host cells. as a member of the flavivirus genus in the flaviridae family, dv is a small, positive strand rna enveloped virus. there are four serotypes of dengue virus (dv1-4). their genome encodes a polyprotein precursor of at least seven non-structural proteins and three structural proteins which are the capsid protein (c), the membrane protein (m) and the envelope glycoprotein (e) [1] . the polyprotein is processed co-and post-translationally by cellular signalase in the lumen of the rough endoplasmic reticulum (er) and by a viral protease in the cytosol [1, 2, 3] . the nascent c protein contains a c-terminal hydrophobic domain that acts as a signal sequence for translocation of the immature form of m, the prm, into the lumen of the rough er. two adjacent prm c-terminal transmembrane domains are responsible for prm membrane anchoring and e translocation into the er [2] . prm and e associate into heterodimers at er membranes [4, 5] where they assemble with the viral rna/c complex to form progeny virions [1] . during the egress of virions through the secretory pathway, prm protein is cleaved by the trans-golgi resident furin protease to form the m envelope protein and the soluble pr segment, which is released into the extracellular medium upon particle secretion [6] . prm cleavage marks maturation of flavivirus virions [7, 8] . cleavage of prm is intimately correlated to change of conformation of envelope protein complexes. although it was thought that prm cleavage is a prerequisite for e dimerization, recent studies show that change of conformation most probably occurs at low ph in the tgn and allows cleavage of prm by furin [6, 9, 10] . prm and e proteins from flaviviruses, such as yellow fever virus [11] , japanese encephalitis virus (jev) [12, 13] , west nile virus (wnv) [14] and tick-bone encephalitis virus (tbev) [15, 16] , are able to assemble into subviral particles in the absence of any other viral component. subviral particles and infectious virions are coproduced in infected cells, assemble in an immature form, and subsequently undergo the same maturation process and display similar fusion activity as infectious viruses [17, 18] . therefore, subviral particles could be a precious tool for research on cell biology of dvs. although there have been attempts by several groups to obtain dv rsps, either their production was inefficient or the sequence of dvs structural proteins had to be substantially modified [19] before they could efficiently generate rsps. for example, the furin cleavage site on prm had to be mutated to establish the dv2 rsps producing cho cell line because it was found that the dv2 rsps cause cell-cell fusion [20, 21] . others replaced a portion of the carboxy-terminal region of dv1 and dv2 e genes with the corresponding sequence of jev to observe significant rsp secretion [22, 23] . in either case, these modifications may interfere with the interactions with cytosolic proteins and, possibly, with the maturation and folding of the structural proteins. here we describe the efficient production of native rsps of the four dv serotypes by using a codon optimization strategy. codons of the dv prme gene were replaced with those preferentially used in mammalian cells. optimization was first applied to the dv1 prme gene. we found that this experimental approach could efficiently increase the intracellular expression level of the e glycoprotein in human cells and therefore enhance the production of dv1 rsps. we have established a dv1 rsps producing stable cell line (hela-prme). our data by immunofluorescence and electron microscopy show that most of e protein co-localizes with markers of the er, where rsps accumulate. biochemical analysis of secreted rsps demonstrates that they contain e homodimers and m, indicating that rsps have trafficked through the secretory pathway and that maturation process has occurred. using the hela-prme cell line, we have developed a semi-quantitative assay to screen a 122 genes cellular membrane trafficking sirna library and identified genes that may be involved in the secretion of dv. from our screen, 23 genes show a significant reduction in dv1 rsps secretion whereas for 22 genes rsps levels were increased in cell supernatants. these data provide a proof of concept that rsps of dvs and the producing cell line are safe tools that can be used in the study of viral egress. finally, the optimization strategy was applied to dv2, dv3 and dv4 prme, and rsps for all four dv serotypes were efficiently produced. these native rsps are, therefore, a safe mimicry of virions that can be used to study viral and cellular requirements for virus assembly and egress. to generate dv rsps, we first transfected hela cells with native dv1 prme gene derived from the dv1 fga/na d1d [24] strain, which contained the signal sequence of vesicular stomatitis virus g (vsv-g) envelope glycoprotein inserted in frame upstream of the prme cdna. 48 hours post-transfection, expression of dengue e protein was monitored by flow cytometry on permeabilized transfected hela cells with the 4e11 monoclo-nal antibody against dengue e protein. e protein was detected in transfected cells at low expression level ( figure 1a) . analysis of the prme gene sequence revealed that 15% of nucleotide triplets were rarely used in mammalian cells so that its codon usage was not adapted for efficient expression in this cell system. thus we designed and synthesized an optimized dv1 prme (prmeopt) gene in which the rarely used codons were replaced with those preferred by mammalian cells, without changing the amino acid sequence ( figure s1 ). more than 70% codons were modified for prme optimization. in addition, we also removed the negative cisacting sites (such as splice sites, poly(a) signals, etc) which might have negatively influenced expression. the optimized gene resulted in high and stable expression levels in transfected mammalian cells ( figure 1b ). both the mean fluorescence intensity (mfi) and percentage of positive cells increased by almost 3 fold. e protein was hardly detected without cell permeabilization, indicating that its localization was intracellular and was not expressed at cell surface (data not shown). a hela cell line that stably expresses dv1 prm and e envelope proteins was then established by transducing hela cells with a retroviral vector pchmws-prmeopt-ires-hygromycin. hygromycin-selected cells were designated hela-prme. several single colonies of hela-prme were used in this study and they showed similar results. the hela-prme cell line has already been propagated for more than thirty passages in the presence of hygromycin and has kept a stable prme expression level. culture of hela-prme cells in the absence of hygromycin resulted in a significant reduction of prme expression (data not shown). the intracellular distribution of dv1 e glycoprotein was analyzed in fixed hela-prme cells by co-labeling with erp72, ergic-53 and golgin-97, which are proteins of the endoplasmic reticulum (er), the er-golgi intermediate compartment (ergic) and the golgi apparatus, respectively. as expected, the dv1 e glycoprotein mainly distributed in the er in hela-prme cells, as shown by immunofluorescence, where it colocalized with erp72 ( figure 2a -d), whereas it showed no colocalization with ergic-53 ( figure 2e -h) and golgin 97 ( figure 2i -l). interestingly, e and erp72 were also enriched in a perinuclear compartment, which is not usually labeled by anti-erp72 antibody and other er markers (data not shown), suggesting that it has been induced by prm and e viral protein expression. in addition, no staining was observed at the plasma membrane, confirming the previous flow cytometry result. altogether, our data demonstrate that the dv1 e glycoprotein is efficiently expressed and enriched in the endoplasmic reticulum in the hela-prme stable cell line. the endoplasmic reticulum is the assembly site for flaviviruses. in order to verify whether this er staining reflects the presence of e-containing assembled particles, we analyzed the hela-prme stable cell line by transmission electron microscopy (tem) on thin sections of fixed, epon-embedded cells ( figure 3a ). we found that hela-prme, but not control hela cells (data not shown), displayed dilated er membranes with aligned round particles and elongated parallel tubules. particles were ,20 nm in diameter and homogenous in size and shape. the aligned particles that we have observed in hela-prme cells are similar to the stacked viral particles which have been described in cisternae of the rough er in infected insect and mammalian cells [25, 26, 27, 28] . to confirm that these structures contain viral proteins, we labeled thawed cryosections with the 4g2 antibody that recognizes the e protein ( figure 3b -d). we found antibody binding for the e protein figure 2 . subcellular localization of the dv1 e protein in hela-prme cells. hela-prme cells were fixed, permeabilized, and stained for e protein (green) and for cellular marker antigens (red). dapi staining was used to label cell nuclei (blue). erp72, ergic-53 and golgin-97 are proteins of the endoplasmic reticulum (er), the er-golgi intermediate compartment (ergic) and golgi apparatus, respectively. the scale bar represents 10 mm. doi:10.1371/journal.pone.0008325.g002 present in the lumen of cisternae, where it is localized to electron lucent, round particles and tubular structures. in addition, e protein is also found on amorphous, electron dense material present in these cisternae ( figure 3b ). to confirm the nature of these cisternae by other than morphological criteria we performed a double labeling of the 4g2 antibody with the er resident protein calreticulin. we found antibody binding for calreticulin present on the 4g2 positive cisternae ( figure 3c ), unequivocally identifying them as er. in addition, we found a lower but significant amount of label for the e protein present in the golgi stack and in vesicles in the golgi area ( figure 3d ). thus, our observations indicate that expression of prm and e dv1 envelope proteins induces the formation of rsps in an erderived compartment. we then investigated if the rsps observed by em in hela-prme cells could undergo maturation and be secreted like dv. cell lysate (cl) and clarified supernatant (sn) concentrated by ultra-centrifugation were analyzed by western blotting followed by incubation with either the anti-e monoclonal antibody 4e11 ( figure 4a -b) or an anti-dv1 serum from a human patient ( figure 4c -d). in cl samples, a 50 kda monomeric form of the e glycoprotein was predominantly observed ( figure 4a ,c), whereas high levels of 100 kda e glycoprotein homodimers were readily detected in sn samples ( figure 4a -d). this suggests that whereas in hela-prme cells the majority of viral proteins are localized in the early pre-golgi secretory pathway in an immature state, secreted rsps have passed through maturation stages in the golgi apparatus thus resulting in homodimerization of e proteins. to further confirm the presence of e dimers in sn and exclude the possibility that dimerization resulted from treatment for electrophoresis, the sn were incubated in the presence or absence of a cross linker (3, 39-dithiobis [sulfosuccinimidylpropionate]; dtssp) and then subjected to western blotting using the anti-e 4e11 antibody. under these conditions, we observed that e dimer/ monomer ratio was lower in non-treated rsps compared to the figure 3 . electron microscopy analysis of the hela-prme cell line. hela-prme (dv1) cells were fixed and either prepared for epon embedding (a), or for immuno labeling on thawed cryosections (b-d). a), round particles are found aligned in the lumen of the er (arrows) together with tubular structures (arrowheads). b) labeling for the e protein is present in the lumen of cisternae. it is localized to electron lucent, round particles (arrows) and tubular structures (arrowheads). in addition e protein is also found on amorphous, electron dense material present in these cisternae (asterix). c) double labeling of e protein (10 nm gold, black arrows) and the er marker calreticulin (15 nm gold, white arrows). calreticulin is present on the limiting membrane of the cisternae, which contain round particles positive for e protein labeling. d) label for the e protein is also found within the golgi stack (g) and on vesicles in the golgi area. n = nucleus, all scale bars represent 200 nm. doi:10.1371/journal.pone.0008325.g003 dtssp treated (data not shown). these data indicate that, in sn, a significant proportion of e protein is present as a homodimer and detection of e monomers most likely results from partial dissociation under the denaturing conditions of electrophoresis. e homodimers were no longer detected when samples were heated in presence of dithiothreitol ( figure 4b ). in addition, the prm protein could be readily detected with the anti-dv1 serum in cl but not sn samples ( figure 4c ). by contrast, when the hela-prme cells were treated with nh 4 cl, which inhibits acidification of the trans-golgi compartment and, hence, the activity of furin protease and prm cleavage, prm was also found in sn ( figure 4d -e). to our surprise e dimers were also detected in these conditions. interestingly, the e monomers from sn exhibited slower electrophoretic mobility than those of cl samples, which suggests that e glycoproteins have acquired complex n-glycans in the golgi apparatus. this was confirmed by n-glycosidase f (pngase f) and endoglycosidase h (endoh) digestion ( figure 4 f-g), which cleaves nearly all types of n-glycan chains or only high mannose and some hybrid n-glycan chains from glycoproteins, respectively. whereas e protein in cl was sensitive to both treatments, only pngase f digestion increased the electrophoretic mobility of e protein in sn, demonstrating that rsps had acquired endoh-resistant, complex n-glycans in the golgi apparatus before secretion. moreover, we found that secretion of dv rsps was altered by incubation of cells at 15uc and 20uc, which block cargo transport from ergic to cis-golgi and exit from the trans-golgi, respectively, and by brefeldin a treatment, which inhibits exit from er and induces fusion of golgi membranes with er (data not shown). altogether, homodimerization of e, acquisition of complex sugars and efficient cleavage of prm indicate that prm and e viral proteins have correctly assembled in the er into rsps which have trafficked through the secretory pathway before secretion into the cell medium. to study the dynamic of rsp production, we performed a pulse chase experiment on overnight starved hela-prme cells that were metabolically labeled with s 35 -methionine for 1 hour and subsequently chased for 24 hours in normal medium. supernatant and cell lysate were collected at the specified time points after chase; rsps were immuno-precipitated using the 4e11 anti-e monoclonal antibody and then analyzed by autoradiography. detection of prm or m in this assay results from co-immunoprecipitation and, therefore, indicates efficient interaction with e protein. secretion of e and m proteins was detected from the 8 hours time point on, with a significant increase at 24 hours postchase ( figure 5a ). consistently, high levels of e and m proteins were found in cell lysates at all time points with a substantial figure 5b ). two major protein products were found in cell lysates, with an apparent molecular weight corresponding to e and prm proteins. bands of higher molecular weight were also present and could be oligomeric forms of e and prm or m. interestingly, the pattern of immunoprecipitated proteins from cell supernatant was slightly different. higher proportions of e dimers were found and a band corresponding to the molecular weight of m but not prm was detected at the 24 hours time point, indicating that most secreted rsps are mature. our data demonstrate that newly synthesized proteins need 8 hours to be translocated through the secretory pathway and released into the supernatant as mature rsps. to further characterize secreted dv1 rsps, we performed sucrose gradient fractionation on rsps concentrated from supernatant of hela-prme cells. supernatants were ultracentrifuged and rsps-containing pellets either resuspended in pbs or in 0.5% triton-x100 containing pbs to solubilize e from lipid membranes, as described [29] . as expected, triton-x100solubilized e protein and rsp-associated e protein appeared in distinct fractions of a discontinuous 20 to 60% sucrose gradient ( figure 6 ). the amount of e glycoprotein in each fraction was quantified. e glycoprotein in non-treated sample sedimented in fractions containing 20% to 30% sucrose ( figure 6 , black bars) whereas in triton-x100 treated samples, e protein was solubilized and detected in fractions at the top of the gradient (figure 6 , white bars). these data further confirm that the rsps formed in hela-prme are secreted into culture medium from which they can be easily purified by ultracentrifugation. moreover, we have obtained preliminary evidence that dv1 rsps and dv1 viral particles isolated from hela-prme and mammalian infected cells, respectively, sediment at similar densities on 10-60% continuous gradient of sucrose (dr philippe despres, institut pasteur, personal communication). our results show that the dv1 rsps produced by hela-prme cell line mimic maturation and secretion of dv1, thus providing a useful tool to study the interaction between dv and host cells during viral egress. to identify host factors that could either enhance or reduce production of dv rsps, we first developed a quantitative assay to relatively quantify levels of secreted particles in supernatant of hela-prme cells. the chemiluminescence dotblot (cldb) assay is based on the concentration of rsps from cell supernatant on pvdf membranes, followed by detection of e with a specific horseradish peroxidase (hrp)-conjugated antibody and quantification of substrate-induced luminescence using a luminometer. our data using purified e protein of known concentrations showed that, when ranging between 400 pg to 40 ng, e protein on pvdf membrane displayed a very good linear correlation with the luminescence density in cldb assay ( figure 7a ). the cldb was first used to estimate the rsps yield from hela-prme cell line, and we found that the concentration of e protein in supernatant of hela-prme cell line was around 500-1000 ng/ml under the culture condition used for sirna transfection (data not shown). we then screened a sirna library that consisted of 122 genes which target cellular membrane trafficking using the hela-prme cell line. non-targeting sirna (nt) and sirna targeting dv1 prme were added as controls. library and control sirnas were transfected in triplicates on 96-well plates. levels of rsps secreted by sirna transfected hela-prme were measured by cldb assay from 40 microliters of supernatant from each well. levels of e protein in cell supernatant were expressed in relative luminescence units and ratios to that of nt controls were shown in figure 7b . t test was used to assess the statistical significance of differences between each sample and nt. differences were considered statistically significant when p,0.05. we observed that targeting of 23 genes resulted in significant reduction of dv1 rsps amounts in supernatants whereas targeting of 22 other genes induced a significant increase. for instance, our results showed that downregulation of adp-ribosylation factor 1 (arf1), which regulates secretory membrane transport [30] , resulted in 3 fold decrease of dv1 rsps in cell supernatant, suggesting its involvement in the secretion of dv. some genes whose down-regulation enhanced levels of dv rsps in the supernatant are involved in endocytosis, such as the three dynamins which show a 2 to 4 fold increase in dengue rsps secretion. these proteins are involved in the budding process or in the transport of vesicles [31] . such an enhancement might have been due, at least in part, to a blockade in the re-internalization of secreted rsps by hela-prme cells. although further experiments are required to confirm the screening data, our study validates the use of dv rsps-producing hela-prme cell line in combination with the cldb-based quantification strategy as a promising system to facilitate the identification of cellular factors involved in dv secretion. the codon usage of dv2, dv3 and dv4 prme genes differed substantially from that of mammalian cells and, therefore, was optimized as described for dv1 by replacing more than 70% of the codons ( figure s1 ). rsps of the four serotypes were first produced in 293t cells by transient expression of prme genes. particles were purified and detected by western blotting using either the anti-e 4e11 monoclonal antibody or a mixture of sera from four patients who were infected by all dengue serotype ( figure 8a-b) . with the anti-e 4e11 antibody, monomeric and dimeric forms of the dv1 e protein could be readily detected whereas weaker signals were observed for the other three serotypes, because of limited affinity of the antibody ( figure 8a ) [32] . e proteins of all dvs were detected using the mixture of sera ( figure 8b ) with a signal of similar intensity, which suggested that rsps of four serotypes could be generated to comparable levels. monomeric e proteins of four serotypes displayed slightly different electrophoresis mobility, which could be due to a differential level of n-glycosylation. e glycoproteins of dv1 and dv3 have two n-glycosylation sites at asn 67 and asn 153, whereas those of dv2 and dv4 have only one at asn 67 [33] . dimeric e protein was observed in dv1, dv3 and dv4 but not in dv2. besides e, the prm protein was also detected in rsps produced by transient transfected 293t cells. recently, we have established hela-prme and 293t-prme cell lines of dv1, dv2 and dv3 using the same procedure described for dv1 hela-prme. we have compared the maturation of rsps produced by both cell types using sds-page and silver staining of the gel ( figure 8c ). supernatants from parental hela and 293t cells were used as controls in the experiment. we found that, whereas only a small fraction of prm was cleaved in the rsps produced by 293t-prme cell lines, cleavage of prm was much more effective in rsps from the hela-prme cell lines. this result indicates that efficacy of maturation is cell type dependent. rsps of the four serotypes were further analyzed by sucrose gradient. as for dv1, rsps of dv2, dv3 and dv4 were concentrated in fractions containing 20% to 30% sucrose ( figure 8d) . altogether, our results demonstrated that the strategy of codon optimization was successful in leading to the production of rsps of all serotypes with comparable efficacy and similar sedimentation properties. in this study, we have used a codon-optimization strategy to establish and characterize stable cell lines that produce rsps for the four dengue serotypes. previous studies had reported the production of dv rsps in which the glycoproteins were mutated either to avoid host cell fusion or to delete the er retention signal. for example, dv1 rsps were not secreted effectively and dv2 rsps could not form unless the transmembrane domain of the e glycoprotein, which contains an er retention motif was replaced with that of jev [22, 23, 34] . our ability to obtain the efficient production of rsps of all serotypes without any change in the amino acid sequence is likely the result of codon optimization for expression in mammalian cells, which has been proven to be an figure 6 . sucrose gradient analysis of dv1 rsps. the supernatant from hela-prme cells was concentrated and resuspended in pbs or 0.5% triton-x 100 containing pbs. rsps were then centrifuged in a 20 to 60% sucrose gradient at 28,000 rpm (beckman sw-41ti rotor) for 2.5 hours at 4uc. fractions of 0.5 ml were collected and e content was measured using cldb. the percentage of e protein in each fraction is displayed on the y axis. doi:10.1371/journal.pone.0008325.g006 figure 7 . application of the dv rsp-producing hela-prme cell line and cldb to screen a small library of sirna which targets 122 genes involved in membrane trafficking. a) the correlation between luminescence density and amount of e protein on pvdf membrane in cldb assay. b) the screen results of the sirna library which targets 122 genes involved in membrane trafficking. non-targeting sirna (nt) and sirna targeting dv1 prme were used as controls. level of e protein in cell supernatant of each sirna was expressed as its ratio to that of nt controls. two-sided student's t test was used to assess the statistical significance of differences between each sample and nt. differences were considered statistically significant when p,0.05. genes inducing either a significant decrease or increase in rsps production are shown in gray and black columns, respectively. doi:10.1371/journal.pone.0008325.g007 , respectively (b) . c) production of rsps by 293t-prme and hela-prme stable cell lines. dv1-dv3 rsps in supernatants were analyzed by sds-page and silver staining of polyacrylamide gels. bands corresponding to the approximate molecular weight of e monomers and dimers, as well as prm and m are indicated by arrows. supernatants from parental 293t and hela cells were used as controls. d) analysis of rsps of 4 serotypes by sucrose gradient. rsps were centrifuged in a 20 to 60% sucrose gradient at 28,000 rpm for 2.5 hours in 4uc. fractions of 0.5 ml were collected and measured using cldb. the percentage of e protein in each fraction is displayed on the y axis. doi:10.1371/journal.pone.0008325.g008 effective method to increase the expression level of glycoproteins from various viruses [35, 36] . thus, gene optimization significantly enhanced expression of prme glycoproteins in transfected cells, and this over-expression increased both rsp production and secretion. however, it is also possible that using other viral strains may result in different efficacy of rsps production, regardless of codon optimization. to our knowledge, this is the first report of rsps using the native prm and e envelope proteins for the four serotypes of dvs in mammalian cells. we have characterized the maturation of dv1 rsps produced by the hela-prme cell line. sucrose gradient and sedimentation analysis demonstrated that the dv1 rsps are concentrated in fractions containing 20-30% sucrose and are sensitive to detergent treatment. although prm/e heterodimers were not found in our experiments, possibly because prm/e interaction is weak, we observed processing of prm into m protein in hela-prme cells. this cleavage was sensitive to nh 4 cl treatment, which inhibits acidification of the trans-golgi compartment and, hence, the activity of furin protease. newly synthesized e proteins first form heterodimers with prm proteins in the er of host cells and then rearrange as homodimers during the process of secretion [37, 38] . the rearrangement from heterodimer to homodimer was first thought to require cleavage of prm by furin in the trans-golgi to form m and soluble pr proteins [39] . a recent study, however, has shown that such rearrangement is mainly caused by the progressive acidification of the milieu along the secretory pathway, which facilitates prm cleavage by furin [6, 10] . if rearrangement is a pre-requisite for prm cleavage to occur, it could explain, at least in part, why e homodimers were still observed in our study following treatment with the acidotropic reagent nh 4 cl. the precise mechanism underlying their presence in rsps under these experimental conditions, however, requires further investigation. finally, we found by pulse-chase experiments that 8 hours are necessary for production and secretion of rsps. altogether, these results show that mature dv1 rsps are efficiently produced by the stable hela-prme cell line. we have studied the subcellular localization of dv1 e and rsps in the hela-prme cell line. by immuno-staining of fixed cells and fluorescence microscopy, we have shown that most of the e protein is localized in the er compartment, where dengue glycoproteins are synthesized [1] . the fact that we did not detect any significant signal of e in ergic and golgi apparatus could be explained by a very low amount of protein in these organelles. in these conditions, saturating signals in the er would mask its detection in other compartments along the secretory pathway. the fluorescent microscopy results were in accordance with our electron microscopy data. analysis of cell sections by transmission electron microscopy has revealed that e proteins in the er are associated with both round particles and tubular structures. the tubules could either be intermediate forms of rsps assembly or result from accumulation of prm and e proteins in the er. budding of virions from cellular membranes depends on assembly of viral structural proteins that generate pushing and/or pulling forces simultaneously to induce curvature of membranes, which is necessary for particle formation and membrane fission. it is possible that assembly of overexpressed prm and e proteins in the er allows formation of long tubules but that the fission event is a limiting factor. the secretion pathway was also investigated by temperature block or pharmacological experiments. incubation at either 15uc, to stop traffic between the intermediate compartment and the cis-golgi, or at 20uc, to block exit from the trans-golgi network, or bfa treatment, to block the exit from er, significantly reduced secretion of rsps. these results demonstrate that rsps traffic through er, ergic and golgi compartments before being secreted out of the cell. the efficient production of dv1 rsps by hela-prme cell line and the fact that rsps could mimic the maturation and secretion processes allowed us setting up an assay to study the interaction between dv and host cells during egress, a step which has received little attention in comparison to dv viral entry and replication [40, 41, 42, 43] . we have validated our assay by using a sirna library preferentially targeting genes involved in cellular transport. as expected, a number of genes involved in the secretory pathway resulted in reduced release of rsps in the cell supernatant, whereas other factors were associated with an opposite effect. clearly, the identification of cellular factors involved in the egress process will be helpful to understand the maturation of dv and its pathogenicity. moreover, our system will allow comparing the effect of cellular factors using rsps assembled from the four dengue serotypes to test whether there are strain-specific interactions with host proteins. hela and 293t cells maintained in our lab [44] were cultured in dmem containing 10% fetal bovine serum. purified anti-e 4e11 and 4g2 monoclonal antibodies were provided by dr. a. amara and p. despres (institut pasteur, france), respectively. purified anti-dv1 mouse igg and sera from four patients infected by the four dengue serotypes (1) (2) (3) (4) , respectively, were kindly provided by dr. philippe buchy (institut pasteur, cambodia). the native (non-codon optimized) dv1 (strain fga/na d1d) prme gene containing construct was provided by dr a. amara. the prme sequences of dv1 (strain fga/na d1d), dv2 (strain fga/02), dv3 (strain pah881/88) and dv4 (strain 63632) were codon-optimized and synthesized by the geneart company (regensburg, germany) and subcloned into pcdna or retroviral vector pchmws-ires-hygromycin (kindly provided by dr. rik gijsbers, from molecular medicine at katholieke universiteit leuven) using bamhi and xhoi restriction sites. a nucleotide sequence encoding for the signal peptide of vsv-g (mkclly-laflfigvnc) was included upstream of each prme gene. sequences of codon-optimized prme genes are provided as supporting information ( figure s1 ). to produce the retroviral vector for delivery of the prme-opt gene into hela cells, the pchmws-prme-opt-ires-hygromycin, pcdna-vsv-g and p8.71 (modified hiv provirus coding for gag and polymerase) plasmids were co-transfected into 293t cells. the cell supernatant containing infectious vsv-g-pseudotyped retroviral particles was harvested 48 hours post-transfection and used to infect hela cells. two days after infection, cells were selected in culture medium containing 500 mg/ml of hygromycin for two weeks. selected cells (hela-prme) were tested by flow cytometry for e expression and were maintained in dmem +10% fbs +500 mg/ml of hygromycin. hela cells were transfected with pcdna-prme, pcdna-prme-opt or a pcdna empty vector using calcium phosphate precipitate method. two days after transfection, cells were detached by incubation in 10 mm edta at 37uc for 10 min, fixed in 2% paraformaldehyde, and then permeabilized in 0.1% triton x-100. after washing, the cells were incubated with a mouse anti-e antibody (4e11 for fluorescence microscopy on fixed cells, hela-prme cells were grown on glass coverslips. cells were fixed, permeabilized, and incubated with anti-dv human sera (1:200) and anti-erp72 rabbit polyclonal ab (1:200, stressgen bioreagents, ann arbor, mi, usa), anti-ergic-53 mab (1:1000, alexis biochemicals, farmingdale, ny, usa), or anti-golgin-97 mab (1:50, invitrogen, carlsbad, ca, usa), followed by the incubation with corresponding secondary antibody conjugated with fitc or tritc. nuclei were stained with dapi and coverslips were mounted on glass slides for analysis. fixed cells were visualized under axioobserver z1 inverted motorized fluorescent microscope using the apotome module and piloted through the axiovision 4.6 software and images were acquired through the mrm axiocam high resolution ccd camera (carl zeiss, germany). hela-prme cell line was fixed at 28 hours post-trypsination and processed for em and immuno-em. for conventional tem, cells were fixed in 2.5% glutaraldehyde in 0.1 m cacodylate buffer (ph 7.2) for 1.5 hr at room temperature and post-fixed with 1% osmium tetroxide in 0.1 m cacodylate buffer (ph 7.2) for 1 hour at room temperature. then cells were embedded in 2% agarose and cell blocks were post-fixed with 2% uranyl acetate in 30% ethanol, dehydrated in graded series of ethanol and embedded in epoxy resin. ultrathin sections were cut with a leica ultramicrotome uct (leica microsystems; vienna, austria) and collected on 400-mesh formvar coated copper grids. sections were stained 45 minutes with 4% aqueous uranyl acetate and 5 minutes with lead citrate. for immuno-em, cells were fixed with 4% formaldehyde in 0.1 m phosphate buffer (ph 7.4), and embedded in 12% gelatin. blocks were infiltrated with 2.3 m sucrose for cryoprotection, mounted on specimen holders and frozen in liquid nitrogen. cryosections were cut with a leica em uc6/fc6 microtome (leica microsystems, vienna, austria). thawed cryosections were labeled with anti-e 4g2 antibody, rabbit polyclonal antibody against er resident protein calreticulin (abcam, cambridge, ma, usa) and protein-a gold (10 nm and 15 nm) obtained from utrecht university (utrecht, the netherlands) and used as described before [45] . double labeling was performed sequentially, using for each antibody a different size of protein a gold. unspecific binding of the second protein a to the first antibody was blocked by incubation with 1% glutaraldehyde as described before [46] . the grids were viewed with philip cm10 electron microscope at 80 kv and images were taken with keenview camera (soft imaging system, lakewood, co, usa) using item 5.0 software (soft imaging system gmbh). supernatants of hela-prme or its parent hela cells were harvested and cleared by centrifugation at 3,000 rpm for 15 minutes and 10,000 rpm for 30 minutes. clarified supernatants were then concentrated by ultracentrifugation at 28,000 rpm for 2.5 hours. pellets were then resuspended in 100 ml of phosphate buffered saline (pbs). for the production of immature rsps, the hela-prme cells were cultured in medium containing 20 mm of ammonium chloride (nh 4 cl). to generate rsps of dengue 2, 3 and 4, pcdna constructs containing the optimized prme genes (10 mg each) were transfected into 293t cells separately. the dv1 pcdna-prme construct was transiently transfected as control. supernatants of transfected 293t cells were harvested, clarified and concentrated as mentioned above. after ultracentrifugation, rsps were resuspended in 100 ml pbs to which 33 ml of 4x nupage lds (lithium dodecyl sulfate) sample buffer (invitrogen) was added. rsps were then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) using 4,12% nupage novex bis-tris gels (invitrogen). for analysis of cell lysates by sds-page, hela-prme cells were detached by incubation in 10 mm edta at 37uc for 10 min and resuspended in 500 ml of pbs. cell suspensions were then mixed with 167 ml 4x of sample buffer and then sonicated before electrophoresis of samples as described above. for immunodetection, proteins were blotted from gels onto polyvinylidene difluoride (pvdf) membranes. the membrane was blocked overnight in 5% milk in pbst solution and then incubated with anti-e antibody (4e11, 1:1000) for 1 hour. after washes, the membrane was incubated for 1 hour at room temperature with a horseradish peroxidase-labeled goat anti mouse igg polyclonal antibody. the membrane was finally visualized using ecl western blot detection reagents (invitrogen) and amersham hyperfilm ecl (ge healthcare, waukesha, wi, usa). for silver staining, the gel was fixed for 30 minutes and incubated with sodium thiosulfate for 30 minutes at room temperature. after three washes, the gel was incubated with silver nitrate for 40 minutes and developed for 15 minutes in sodium carbonate solution (25 g/l). edta solution (40 mm) was used to stop the development. for the endoglycosidase treatment, rsps or hela-prme cell lysates were treated with 500 u of endoh or pngase f at 37uc for 3 hours according to the manufacturer's instructions (new england biolabs, beverly, ma, usa), and subsequently analyzed by western blot. a chemiluminescence dot-blot (cldb) method was developed to quantify rsps. briefly, 40 ml of supernatant of either hela-prme cell line or purified e protein solution at known concentration were blotted onto pvdf membrane through a dot blot 96 system (biometra, goettingen, germany). the membrane was blocked overnight in 5% milk in pbst solution, incubated with anti-e antibody (4e11, 1:10,000) for 1 hour and then for 1 additional hour with a peroxidase-labeled goat anti mouse igg polyclonal antibody (1:10,000; zymed). ecl western blot detection reagents (invitrogen), diluted five times, were mixed and added to the membrane and the luminescence intensity was measured using the microbeta luminometer (perkinelmer, waltham, ma, usa). for sucrose gradient analysis, concentrated rsps were ultracentrifuged in a 20 to 60% discontinuous sucrose gradient at 28,000 rpm (beckman sw-41ti rotor) for 2.5 hours in 4uc. all sucrose solutions were prepared with hepes buffer (20 mm). fractions of 0.5 ml were collected and levels of e were measured using the cldb assay. alternatively, rsps were treated with 0.5% triton x-100 for 1 hour before sucrose gradient fractionation, for rsps denaturation. the human membrane trafficking sirna library targeting 122 genes and the corresponding transfection reagents were purchased from dharmacon (#g-005500; dharmacon research inc, lafayette, co, usa). for the screen, the hela-prme cells were seeded in eight 96-well plates with 10,000 cells per well. 24 hours later, 10 pmol of each sirna was added to each well together with the transfection reagent. sirna targeting dv1 prme (target sequence: agatc-cagctgaccgatt) and non-targeting sirna (nt) (dharmacon research inc) were used as controls. each plate contained in triplicates 15-16 sirnas from the library, as well as positive (dv1 prme) and negative (nt) sirna controls. culture medium was changed two days post-transfection, the supernatant from each well was harvested after an additional 48 hour incubation and then cleared by centrifugation at 4000 rpm for 15 min. 40 ml of supernatant from each well were used to measure by cldb the levels of rsps secreted by sirna transfected hela-prme. two-sided student's t test was used to assess the statistical significance of differences between each sample and the non-targeting control. differences were considered statistically significant when p,0.05. levels of e protein in cell supernatant were expressed in relative luminescence units and ratios of experimental conditions to controls, set as unity, were calculated. figure s1 the four optimized dv prme sequences. each optimized prme gene has a bamh i restriction enzyme site, a kozak sequence gccacc, a signal sequences from vsv-g, and a xho i restriction enzyme site. found at: doi:10.1371/journal.pone.0008325.s001 (0.03 mb doc) a structural perspective of the flavivirus life cycle enzymatic characterization and homology model of a catalytically active recombinant west nile virus ns3 protease cell-associated west nile flavivirus is covered with e+pre-m protein heterodimers which are destroyed and reorganized by proteolytic cleavage during virus release alphaglucosidase inhibitors reduce dengue virus production by affecting the initial steps of virion morphogenesis in the endoplasmic reticulum association of the pr peptides with dengue virus at acidic ph blocks membrane fusion cleavage of protein prm is necessary for infection of bhk-21 cells by tick-borne encephalitis virus proteolytic activation of tickborne encephalitis virus by furin the flavivirus precursor membrane-envelope protein complex: structure and maturation structure of the immature dengue virus at low ph primes proteolytic maturation recombinant vaccinia virus producing the prm and e proteins of yellow fever virus protects mice from lethal yellow fever encephalitis a recombinant particulate antigen of japanese encephalitis virus produced in stably-transformed cells is an effective noninfectious antigen and subunit immunogen japanese encephalitis virus-vaccinia recombinants produce particulate forms of the structural membrane proteins and induce high levels of protection against lethal jev infection west nile virus recombinant dna vaccine protects mouse and horse from virus challenge and expresses in vitro a noninfectious recombinant antigen that can be used in enzyme-linked immunosorbent assays synthesis and secretion of recombinant tick-borne encephalitis virus protein e in soluble and particulate form molecular organization of a recombinant subviral particle from tick-borne encephalitis virus membrane fusion activity of tick-borne encephalitis virus and recombinant subviral particles in a liposomal model system recombinant subviral particles from tick-borne encephalitis virus are fusogenic and provide a model system for studying flavivirus envelope glycoprotein functions recombinant vaccinia viruses co-expressing dengue-1 glycoproteins prm and e induce neutralizing antibodies in mice dengue type 2 virus subviral extracellular particles produced by a stably transfected mammalian cell line and their evaluation for a subunit vaccine histidine 39 in the dengue virus type 2 m protein has an important role in virus assembly enhancing biosynthesis and secretion of premembrane and envelope proteins by the chimeric plasmid of dengue virus type 2 and japanese encephalitis virus secretion of noninfectious dengue virus-like particles and identification of amino acids in the stem region involved in intracellular retention of envelope protein determinants in the envelope e protein and viral rna helicase ns3 that influence the induction of apoptosis in response to infection with dengue type 1 virus histopathological and ultrastructural aspects of mice lungs experimentally infected with dengue virus serotype 2 ultrastructural aspects of the dengue virus (flavivirus) particle morphogenesis morphogenesis of yellow fever virus in aedes aegypti cultured cells. ii. an ultrastructural study composition and three-dimensional architecture of the dengue virus replication and assembly sites two distinct size classes of immature and mature subviral particles from tick-borne encephalitis virus arf proteins: roles in membrane traffic and beyond the dynamin superfamily: universal membrane tubulation and fission molecules? mapping of a dengue virus neutralizing epitope critical for the infectivity of all serotypes: insight into the neutralization mechanism the envelope glycoproteins of dengue 1 and dengue 2 viruses grown in mosquito cells differ in their utilization of potential glycosylation sites a strong endoplasmic reticulum retention signal in the stem-anchor region of envelope glycoprotein of dengue virus type 2 affects the production of virus-like particles codon usage limitation in the expression of hiv-1 envelope glycoprotein highly infectious sars-cov pseudotyped virus reveals the cell tropism and its correlation with receptor expression structure of dengue virus: implications for flavivirus organization, maturation, and fusion alterations of pr-m cleavage and virus export in pr-m junction chimeric dengue viruses c-src protein kinase inhibitors block assembly and maturation of dengue virus antiviral compounds discovered by virtual screening of small-molecule libraries against dengue virus e protein discovery of insect and human dengue virus host factors molecular targets for flavivirus drug discovery the m, e, and n structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles improving structural integrity of cryosections for immunogold labeling immunolocalization of the insulin regulatable glucose transporter in brown adipose tissue of the rat we thank a. amara, p. despres (institut pasteur), and p. buchy (institut pasteur-cambodia) for providing antibodies. special thanks to a. amara for sharing the native (non codon optimized) prme construct (strain fga/ na d1d). we also grateful to p. despres for helpful discussions throughout the project and suggestions in the choice of dengue strains to be used in this study. we express our gratitude to marie-christine prevost, (plate-forme de microscopie ultrastructurale, institut pasteur, paris) for her expert advice with electron microscopy experiments and for hosting jl in her lab. we acknowledge the support of the electron microscopy unit of the university of hong kong, li ka shing faculty of medicine. we thank dr. rik gijsbers (katholieke universiteit leuven) for the pchmws-ires-hygromycin plasmid. key: cord-000912-6l6c7jpq authors: vitelli, alessandra; quirion, mary r.; lo, chia-yun; misplon, julia a.; grabowska, agnieszka k.; pierantoni, angiolo; ammendola, virginia; price, graeme e.; soboleski, mark r.; cortese, riccardo; colloca, stefano; nicosia, alfredo; epstein, suzanne l. title: vaccination to conserved influenza antigens in mice using a novel simian adenovirus vector, panad3, derived from the bonobo pan paniscus date: 2013-03-11 journal: plos one doi: 10.1371/journal.pone.0055435 sha: doc_id: 912 cord_uid: 6l6c7jpq among approximately 1000 adenoviruses from chimpanzees and bonobos studied recently, the pan adenovirus type 3 (panad3, isolated from a bonobo, pan paniscus) has one of the best profiles for a vaccine vector, combining potent transgene immunogenicity with minimal pre-existing immunity in the human population. in this study, we inserted into a replication defective panad3 a transgene expressing a fusion protein of conserved influenza antigens nucleoprotein (np) and matrix 1 (m1). we then studied antibody and t cell responses as well as protection from challenge infection in a mouse model. a single intranasal administration of panad3-npm1 vaccine induced strong antibody and t cell responses, and protected against high dose lethal influenza virus challenge. thus panad3 is a promising candidate vector for vaccines, including universal influenza vaccines. influenza continues to pose a global health problem, as highlighted by the 2009 swine influenza pandemic and sporadic human infections with avian h5n1 influenza viruses. antigenic changes in influenza virus, primarily in the surface antigens hemagglutinin (ha) and neuraminidase (na), are referred to as antigenic shift (subtype changes by reassortment) and antigenic drift (mutation). this variability among influenza viruses hinders vaccination efforts. currently, annual surveillance is necessary to identify circulating viral strains for use in vaccine production. new vaccines are often required, and take about 6 months to become available [1] . thus new approaches are needed. in contrast, so-called ''universal'' vaccines targeting relatively conserved components of influenza virus can provide protection regardless of strain or subtype of virus, and may provide an alternative to the use of traditional vaccines. this immunity to conserved antigens would not necessarily prevent infection completely, but might decrease severity of disease, speed up viral clearance, and reduce morbidity and mortality during the initial stages of an outbreak until strain-matched vaccine became available [2] . furthermore, vaccines based on t cell immunity could be used in combination with a seasonal vaccine to improve efficacy, especially in the elderly who are at high risk of severe disease and show reduced responses to current flu vaccines. peptide scanning of t cell responses of healthy human individuals has shown that matrix 1 (m1) and nucleoprotein (np) are among the prominent targets of cd8 + and cd4 + t cell cross-recognition [3] , so they are of interest as vaccine candidates. by sequence homology, np is .90% conserved among influenza a isolates [4] . both murine [5] and human [6] cytotoxic t lymphocytes induced by np of one virus strain have been shown to cross-react with np from different influenza a strains. the strong immune responses to np in mice contribute to protection against challenge [7] via cd8 + t cells [5, 8] , as well as contributions from cd4 + cells [9, 10] and antibodies [11] [12] [13] . the influenza a matrix (m) gene encodes two highly conserved proteins: an ion channel protein, m2, and the capsid protein, m1. m1 is not a major protective antigen in the mouse and is not well recognized by mouse t cells [14] , but has long been known to be recognized by human t cells [15] . thus its potential contribution to vaccine protection may be underestimated by mouse studies. while epitopes providing targets widely shared among influenza viruses have been identified in multiple viral proteins, not all of them are highly immunogenic when presented by classical vaccines. more potent immunization can be achieved using recombinant vectors to express the influenza antigens and focus immunity on these targets. recombinant adenovirus vectors are especially effective at eliciting strong t cell responses to transgene products [16] [17] [18] . recombinant adenovirus vectors expressing np [19] or both np and m2 [20, 21] can protect mice against a range of influenza virus challenges, including highly pathogenic avian h5n1 strains. while potential interference by prior immunity to human adenoviruses has been suggested as a barrier, this issue can be circumvented by use of vectors based on animal adenoviruses [22] [23] [24] [25] . chimpanzee adenoviruses have been shown to be useful vaccine vectors in a variety of animal studies [26] [27] [28] [29] [30] , and the prevalence of neutralizing antibodies against chimpanzee adenoviruses is low in human populations [31] [32] [33] , but not all of them are equally immunogenic. in this study, we use a simian adenovirus, panad3, isolated from the bonobo pan paniscus. this novel adenovirus strain was identified in a study of more than 1000 adenoviruses isolated from chimpanzees and bonobos in order to increase the available repertoire of vectors [34] . in the large scale screening experiments, panad3 was among the most potently immunogenic in mice and was also among the least frequently recognized by neutralizing antibodies in human sera. we have generated a replication incompetent panad3 vector deleted of e1 and e3 regions and expressing a fusion protein of the np and m1 antigens of influenza a, chosen as targets of broad and cross-reactive t cell immunity in humans [3] . the panad3-based vaccine was tested for induction of antibody and t cell responses in the systemic and mucosal compartments in mice, as well as for protection against lethal influenza virus challenge. we demonstrate that panad3 expressing conserved influenza virus antigens provided highly effective protection after a single intranasal administration. thus it shows considerable promise as a vaccine candidate. all animal protocols and procedures were approved by the institutional animal care and use committee at the center for biologics evaluation and research (protocol #1991-06) and conducted in an spf animal facility accredited by the association for assessment and accreditation of laboratory animal care international. all experiments were performed according to institutional guidelines. during influenza challenge studies, animals that had lost 25% of their initial body weight were humanely euthanized to avoid further suffering. highly virulent, mouse-adapted virus a/fort monmouth/1/ 47-ma (h1n1) [a/fm] has been previously described [35] and was kindly provided by earl brown, university of ottawa, canada. it was prepared as a pooled homogenate of lungs from balb/c mice infected with the virus by the intranasal (i.n.) route 4 days earlier. pan adenovirus type 3 (panad3) was isolated from a stool specimen collected from a bonobo (pan paniscus). the panad3 isolate was amplified and the virus genome was then cloned in a plasmid vector and fully sequenced. as shown in a phylogenetic tree based on hexon sequences [34] , panad3 is a member of adenovirus species c, closely related to species c human and chimpanzee adenoviruses already used in preclinical and clinical trials (human ad5, chad3). panad3 vectors were constructed by homologous recombination in e. coli strain bj5183 by co-transformation with panad3 purified viral dna and a panad3-egfp shuttle vector. homologous recombination between pix genes, right itr dna sequences present at the ends of linearized panad3-egfp shuttle and viral genomic dna allowed its insertion in the plasmid vector, simultaneously replacing the e1 region with a human cytomegalovirus (hcmv) promoter-driven egfp expression cassette containing the bovine growth hormone polyadenylation signal (bgh polya), generating ppanad3de1-egfp. the e3 region (nucleotides 28684 to 32640) was then deleted through several cloning and homologous recombination steps to generate the ppanad3de1de3 backbone, which was propagated in hek 293 cells. expression cassettes containing consensus sequences of np and m1 plus the human cytomegalovirus promoter and bovine growth hormone polyadenylation signal were constructed. the influenza expression cassette contains consensus sequences of np and m1. influenza a np and m1 sequences were obtained from the ncbi influenza virus resource database (http://www.ncbi.nlm.nih. gov/genomes/flu/flu.html). protein sequences were chosen from among different subtype strains isolated between 1990 and 2009 that caused infection in humans worldwide. the np consensus sequence was derived by alignment of all non-identical sequences (h1n1: 88 of 629 sequences, h1n2: 5 of 26, h3n2: 244 of 1557, h5n1: 50 of 121) using muscle version 3.6, and applying the majority rule. further, the np sequence used in the panad3 vaccine lacks the nuclear localization signal residing in aa 6-8 (tkr mutated to aaa), which results in increased cytoplasmic accumulation. the m1 consensus sequence was similarly derived by the alignment of non-identical sequences (h1n1: 51 of 808 sequences, h1n2: 3 of 34, h3n2: 115 of 2150, h5n1: 23 of 145). np and m1 sequences were spaced by a flexible linker (gggsggg). the resulting npm1 sequence was codonoptimized for expression in eukaryotic cells. a diagram of the insert and its full sequence are given in figure 1 . the npm1 expression cassette was inserted into the panad3de1de3 backbone via homologous recombination in e.coli. sequences for hiv gag protein or a respiratory syncytial virus (rsv) fusion protein of f protein, nucleoprotein n and transcription factor m2-1 were inserted in constructs to be used as specificity controls. expression cassettes were inserted into a pneb shuttle vector and then transferred into the snabi linearized ppanad3de1de3-egfp plasmid by homologous recombination in e. coli, exploiting the homology between the hcmv promoter and bgh polya sequences. the panad3 vectors were produced in procell 92 cells, which were derived from the hek 293 cell line originally banked at the university of leiden in 1973 [36] and obtained from frank graham at macmaster university (hamilton, canada), and further adapted at okairòs to be suitable for manufacturing by incorporation of a plasmid carrying a tet repressor expression cassette and g418-resistance gene. the protocol for generating the procell 92 cell line followed essentially that published by matthews et al. [37] . briefly, hek 293 cells were transfected with an expression vector containing a cassette encoding the tet repressor under control of the human phosphoglycerate kinase-1 (pgk) promoter, and the g418-resistance gene. single clones were selected by growing the transfected cells in the presence of 1 mg/ ml g418 in culture medium. single clones were amplified and tested for tet repressor expression by western blot analysis. the stability of tet repressor expression in the selected clone was tested up to passage 63. panad3 vectors grown in these cells were purified by cesium chloride gradients and stored in buffer a195 [38] . viral particle (vp) measurements of adenovirus stocks were made by measurement of absorbance at 260 nm as described [39] . peptides np 147-155 (tyqrtralv) and sars m 209-221 (hagsndniallvq) were synthesized by the cber core facility. an mhc-i restricted peptide of adenovirus dna-binding protein (dbp 419-427 : falsnaedl), present in panad3 [40] and recombinant m1 (rm1) protein from strain a/pr/8/34 (h1n1) were purchased from genscript (piscataway, nj). recombinant nucleoprotein (rnp) from strain a/pr/8/34 (h1n1) was purchased from imgenex (san diego, ca). hela cells were infected with panad3-npm1 at indicated multiplicities of infection (moi). extracts were prepared 48 hours after infection using ten buffer (20 mm tris ph 7.5, 150 mm nacl, 1 mm edta ph 8, 1% triton x100 and protease inhibitors). nuclei and cell debris were spun out by centrifugation at 7,500 g, 30 minutes at 4uc. glycerol was added to supernatants to 10% and stored at 220uc. expression was assessed by western blotting with a mouse hyperimmune serum raised against the npm1 antigen. mice were euthanized and bronchoalveolar lavage (bal) fluid and lung cells obtained as in price et al., 2009 [20] . briefly, for bal fluid, lungs were flushed with 1 ml phosphate-buffered saline (pbs). lung cells were isolated by gradient centrifugation of minced and collagenase-digested lung tissue. splenocytes were depleted of erythrocytes by treatment with ack lysis buffer. sera from blood collected from the abdominal vena cava were isolated using bd microtainers (franklin lakes,nj), and decomplemented by heat-treating at 56uc for 30 minutes. t cell elispot assays were performed as described previously [41] . briefly, anti-interferon (ifn)-c mab an18 (bd pharmingen, san jose, ca) was used to coat elispot plates (millipore, billerica, ma). splenocytes or lung cells were added to wells at a concentration of 250,000 cells/well (and, when necessary, also 62,500 cells/well to bring results into the countable range) in ct medium [42] . peptides were added at a final concentration of 1 mg/ml. plates were incubated for 36-48 hr at 37uc in 5% co 2 . bound ifn-c was detected with biotinylated mab r4-6a2 (bd pharmingen) followed by incubation with alkaline phosphataselabeled streptavidin (kpl, gaithersburg, md). 100 ml 5-bromo, 4chloro, 3-indolylphosphate/nitroblue tetrazolium was used as the developing substrate (kpl). spots were counted with an eli-spot reader (zeiss; thornwood, ny). antibody levels in serum and bal were measured by enzymelinked immunosorbent assay (elisa) as in benton et al. [43] . specifically, nunc 96-well plates were coated at 4uc overnight with 1 mg/ml of rnp or 5 mg/ml of rm1 in pbs, then blocked. next individual mouse sera or bal were added to the plates. bound antibody was detected using human-adsorbed alkaline phosphatase-conjugated goat anti-mouse igg, or iga (southern biotechnology associates, birmingham, al) followed by the substrate p-nitrophenyl phosphate (sigma). od was measured at 405 nm. ad5 and panad3 neutralizing antibody titers were assayed as previously described [31] with some modifications. briefly, 3.5610 4 hek293 cells per well were seeded in a 96 well plate and cultured for 2 days. each adenoviral vector expressing secreted alkaline phosphatase (seap) was incubated for 1 hour at 37uc alone or with serial dilutions of serum, and then added to the 95-100% confluent hek293 cells and incubated for 1 hour at 37uc. supernatant was then removed and replaced with 10% fcs in dmem. seap expression was measured 24 hours later using the chemiluminescent substrate (cspd), from the phospha-lighttm kit (tropix cat no t1016, applied biosystems, bedford, ma) without heat inactivation. light emission (relative light units, rlu) was monitored 45 minutes after the addition of the cspd substrate, using the envision 2102 multi-label reader (perkin elmer, waltham, ma). survival data for vaccine groups vs. controls were compared by log-rank analysis and the bonferroni method using prism (graphpad software, inc., la jolla, ca). the panad3-npm1 construct was designed using two conserved influenza antigens important in human immunity, np and m1. to analyze the level of transgene expression, hela cells were infected with panad3-npm1 at various moi, and triton extracts prepared. western blot analysis of the extracts was performed using a mouse hyperimmune serum raised against the npm1 antigen. the 80 kd major band seen is consistent with the fusion npm1 protein (fig. 2) . the 80 kd band was also detected if the western blot was developed with a monoclonal antibody to np (data not shown). immune responses to panad3-npm1, comparing i.m. and i.n. routes intranasal (i.n.) immunization is especially efficient for induction of local immunity in the respiratory tract, including recruitment of memory t cells to the airways [44] . for a given vaccination, i.n. induces greater mucosal immune responses than intramuscular (i.m.) immunization, but weaker systemic responses [20, 21] . in pilot studies, we included both routes of immunization, in order to characterize the responses induced by the new vector. antibody responses. sera from individual mice 4 weeks after immunization were tested for antibodies to np. as shown in figure 3a , equivalent igg responses to np were detected in serum responses to panad3-npm1 given either i.m. or i.n. at a dose of 10 9 viral particles (vp). serum antibody responses were reduced when animals were immunized with a lower dose (10 7 vp) of panad3-npm1. in the bal, anti-np igg antibodies were induced by i.n. but not i.m. immunization (fig. 3b) . panad3-npm1 induced very little iga in the bal (fig. 3c) . a reagent control provided by bal from a/np+m2-rad5 immunized mice, a system known from previous studies to induce iga [21] , showed that the assay could detect iga antibodies if present. t cell responses. functional t cell responses to vaccination were measured by ifn-c elispot. figure 3d shows responses in the spleen and lungs to np 147-155 peptide, the immunodominant mhc i epitope of cd8 + t cells in balb/c mice [45] . immunization with panad3-npm1 i.m. produced much higher frequencies of np-specific t cells in the spleen than i.n. immunization, while the reverse was true in the lungs. these results show anatomical localization of the immune response, with i.n. more efficiently priming t cells in the respiratory tract, consistent with previous studies [20, 21, 44] . no response to np was seen in mice immunized with constructs containing an irrelevant transgene (hiv gag), and none of the mice responded to the sars 209-221 control peptide. a pilot experiment showed protection against challenge four weeks post-vaccination with 10 9 vp of panad3-npm1 given i.n. (data not shown). thus the panad3 vector was promising, and we pursued more detailed studies. given the superiority of i.n. administration for inducing t cell responses in the lungs, we further explored the immune responses to vaccination by this mucosal route, using panad3-npm1 or as a control panad3 with an irrelevant rsv insert. mice were immunized with doses of 10 9 ,10 7 , or 10 5 vp per mouse. antibody responses. serum and bal were analyzed for igg and iga antibodies to np and m1. figure 4a shows results for igg antibodies to np in serum and bal. at the highest vaccine dose, 10 9 vp per mouse, strong igg responses were seen for panad3-npm1. if the vaccine dose given to the mice was reduced to 10 7 vp per mouse, antibody responses were greatly reduced in serum and absent in bal. the iga antibody response to panad3-npm1 was undetectable in serum and marginal in bal (fig. 4b) . as in figure 3 , a reagent control provided by bal from a/np-rad5 immunized mice showed that the assay could detect iga antibodies if present. the antibody response to the m1 component of panad3-npm1 did not include iga (data not shown) and the igg response to m1 was only substantial in the serum (fig. 4c) . t cell responses. t cell responses were again measured by ifn-c elispot. at a dose of 10 9 vp, panad3-npm1 induced a strong t cell response in the lungs to the dominant np 147-155 epitope. both panad3-npm1 and the panad3-rsv control induced modest responses to the adenovirus peptide dbp 419-427 (fig. 5) . a hundred-fold lower dose of panad3-npm1 induced little response to either np 147-155 or dbp 419-427 . immune protection against challenge infection. one month after immunization mice were challenged with 100 ld 50 (10 4 tcid 50 ) of a/fm, a relatively high dose of a highly pathogenic virus. as shown in figure 6 , a dose of 10 7 vp or less did not protect, but at a dose of 10 9 vp panad3-npm1 provided considerable protection against this stringent challenge. most mice survived and displayed much less morbidity than controls, as shown by weight loss. cross-neutralization by human antibodies to ad5. ad5 and panad3 are closely related viruses, both belonging to adenovirus group c [34] . as one aspect of whether panad3 vectors are likely to be blocked by pre-existing immunity to ad5, we tested neutralization of a panad3 virus by human sera selected for particularly high neutralizing antibody to ad5 (titers .1000). as shown in table s1 , many of these high-titered sera had no neutralizing activity on panad3. some of the human sera with very high neutralizing titers ranging from 1628 to 4608 on ad5 had low neutralizing titers of 28-63 on panad3. in the efforts to develop a universal influenza vaccine, various platforms for immunization to conserved antigens have been studied. replication incompetent adenovirus vectors are promising, since their strong induction of innate immune responses provides a built-in adjuvant, and the antigen-specific b and t cell responses they induce are sustained for a long time [21] . animal adenoviruses have the potential advantage that humans have no prior exposure to them. for that reason chimpanzee adenoviruses have recently begun to be explored for use as vaccine vectors in humans, where they showed good safety and excellent immunogenicity [27, 29, 46] . furthermore, in tests of ad5 and four chimpanzee adenovirus vectors, prior immunization with a gfpexpressing construct blocked subsequent responses to the transgene product only for homologous vector; cross-blocking was minimal [34] . in this study, we tested a new vector based on bonobo virus panad3. both ad5 and bonobo virus panad3 belong to the adenovirus species c [34] . species b (for example ad35) has been shown in other studies to be less immunogenic than species c [47] . since they are so closely related, ad5 and panad3 may share certain structural features providing powerful internal adjuvant effects and thus higher immunogenicity. despite the structural similarity, human sera containing high anti-ad5 neutralization titers (.1000) show no or marginal neutralization capacity on panad3. moreover, in a screening study, panad3 was shown to be a potent vector in mice and in primates [34] . we show here that a single administration of panad3-npm1 influenza vaccine given i.n. provided highly effective protection against lethal challenge with mouse-adapted a/fm, with greatly reduced morbidity and mortality compared to controls. protection was comparable to that in previously published studies of ad5 expressing conserved influenza virus antigens for the same challenge virus and dose [20, 21] . the panad3 vaccine induced both antibody and t cell responses to np. as mentioned earlier, the t cell response to np is well-known to contribute to protection [5, [8] [9] [10] , and recent studies have reported that antibodies to np can also contribute to protection [12] . the fusion protein of np with m1 expressed by the panad3 vaccine has the advantage of including another major target of human immunity. using multiple target antigens may invoke different immune mechanisms, reduce the likelihood of generating escape mutants, and provide a larger range of epitopes that may be suitable for different mhc types. although m1 is not expected to play much of a role in protection in mice, it is a prominent target of t cell immunity in humans [3] , and might contribute to the performance of the panad3-npm1 vaccine in humans. the results presented here support the use of the panad3 vector as a vaccine candidate that is highly effective at inducing t cell and antibody immunity, while at the same time having the advantage that it is not neutralized by human sera [34] . thus panad3, when used to express conserved influenza virus antigens, has promise as a ''universal'' influenza vaccine candidate. table s1 sera from healthy human individuals from different geographical areas in europe and the united states had been screened previously for neutralizing activity to ad5 [34] . selected sera with high ad5 neutralizing activity (titers .1000) were tested for neutralization of panad3 as described in materials and methods, using vectors expressing the secreted alkaline phosphatase (seap) reporter gene. * arbitrary sample numbers. ** results of two tests. ethics statement: all volunteers gave written informed consent before participation, and the studies were conducted according to the principles of the declaration of helsinki and in accordance with good clinical practice. (doc) the annual production cycle for influenza vaccine cross-protective immunity to influenza a viruses memory t cells established by seasonal human influenza a infection cross-react with avian influenza a (h5n1) in healthy individuals biological and genetic evolution of the nucleoprotein gene of human influenza a viruses cytotoxic t cell recognition of the influenza nucleoprotein and hemagglutinin expressed in transfected mouse l cells recognition of homo-and heterosubtypic variants of influenza a viruses by human cd8 + t lymphocytes protective cd4 + and cd8 + t cells against influenza virus induced by vaccination with nucleoprotein dna influenza a virus nucleoprotein is a major target antigen for cross-reactive anti-influenza a virus cytotoxic t lymphocytes resistance to and recovery from lethal influenza virus infection in b lymphocyte-deficient mice vaccination with dna encoding internal proteins of influenza virus does not require cd8 + ctl: either cd4 + or cd8 + t cells can promote survival and recovery after challenge a novel role for non-neutralizing antibodies against nucleoprotein in facilitating resistance to influenza virus contributions of antinucleoprotein igg to heterosubtypic immunity against influenza virus regulation of antinucleoprotein igg by systemic vaccination and its effect on influenza virus clearance specificity and function of t lymphocytes induced by influenza a viruses identification of viral molecules recognized by influenza-specific human cytotoxic lymphocytes-t a replication-defective human adenovirus recombinant serves as a highly efficacious vaccine carrier replicationdefective adenovirus serotype 5 vectors elicit durable cellular and humoral immune responses in nonhuman primates neutralizing antibodies and cd8 + t lymphocytes both contribute to immunity to adenovirus serotype 5 vaccine vectors protection against multiple influenza a subtypes by vaccination with highly conserved nucleoprotein vaccination focusing immunity on conserved antigens protects mice and ferrets against virulent h1n1 and h5n1 influenza a viruses singledose mucosal immunization with a candidate universal influenza vaccine provides rapid protection from virulent h5n1, h3n2 and h1n1 viruses canine adenovirus vectors: an alternative for adenovirus-mediated gene transfer pathogenesis and immunogenicity of bovine adenovirus type 3 in cotton rats (sigmodon hispidus) characterization of a family of chimpanzee adenoviruses and development of molecular clones for gene transfer vectors construction of ovine adenovirus recombinants by gene insertion or deletion of related terminal region sequences chimpanzee adenovirus vaccine protects against zaire ebola virus a novel chimpanzee serotype-based adenoviral vector as delivery tool for cancer vaccines single-dose immunogenicity and protective efficacy of simian adenoviral vectors against plasmodium berghei chimpanzee-origin adenovirus vectors as vaccine carriers efficacy of severe acute respiratory syndrome vaccine based on a nonhuman primate adenovirus in the presence of immunity against human adenovirus quantitative adenovirus neutralization assays based on the secreted alkaline phosphatase reporter gene: application in epidemiologic studies and in the design of adenovector vaccines evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy immunogenicity of heterologous recombinant adenovirus prime-boost vaccine regimens is enhanced by circumventing vector cross-reactivity vaccine vectors derived from a large collection of simian adenoviruses induce potent cellular immunity across multiple species the influenza-virus variant a/fm/1/47-ma possesses single amino-acid replacements in the hemagglutinin, controlling virulence, and in the matrix protein, controlling virulence as well as growth characteristics of a human cell line transformed by dna from human adenovirus type 5 development and use of a 293 cell line expressing lac repressor for the rescue of recombinant adenoviruses expressing high levels of rabies virus glycoprotein development of stable liquid formulations for adenovirus-based vaccines unexpected pulmonary uptake of adenovirus vectors in animals with chronic liver disease t-cell response to adenovirus hexon and dna-binding protein in mice matrix protein 2 vaccination and protection against influenza viruses, including subtype h5n1 cd4 + t cell priming accelerates the clearance of sendai virus in mice, but was a negative effect on cd8 + t cell memory heterosubtypic immunity to influenza a virus in mice lacking either iga, all ig, nkt cells, or cd t cells the route of priming influences the ability of respiratory virus-specific memory cd8 + t cells to be activated by residual antigen mhc affinity, peptide liberation, t cell repertoire, and immunodominance all contribute to the paucity of mhc class i-restricted peptides recognized by antiviral ctl prevalence of serum neutralizing antibodies against chimpanzee adenovirus 63 and human adenovirus 5 in kenyan children, in the context of vaccine vector efficacy comparative seroprevalence and immunogenicity of six rare serotype recombinant adenovirus vaccine vectors from subgroups b and d we thank anthony ferrine, mary belcher and the cber animal facility staff for care of experimental animals, and marian major and andrew byrnes for review of the manuscript. key: cord-001601-tsuz3j40 authors: ngan, luong thi my; jang, myeong jin; kwon, min jung; ahn, young joon title: antiviral activity and possible mechanism of action of constituents identified in paeonia lactiflora root toward human rhinoviruses date: 2015-04-10 journal: plos one doi: 10.1371/journal.pone.0121629 sha: doc_id: 1601 cord_uid: tsuz3j40 human rhinoviruses (hrvs) are responsible for more than half of all cases of the common cold and cost billions of usd annually in medical visits and missed school and work. an assessment was made of the antiviral activities and mechanisms of action of paeonol (pa) and 1,2,3,4,6-penta-o-galloyl-β-d-glucopyranose (pgg) from paeonia lactiflora root toward hrv-2 and hrv-4 in mrc5 cells using a tetrazolium method and real-time quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. results were compared with those of a reference control ribavirin. based on 50% inhibitory concentration values, pgg was 13.4 and 18.0 times more active toward hrv-2 (17.89 μm) and hrv-4 (17.33 μm) in mrc5 cells, respectively, than ribavirin. the constituents had relatively high selective index values (3.3–>8.5). the 100 μg/ml pa and 20 μg/ml pgg did not interact with the hrv-4 particles. these constituents inhibited hrv-4 infection only when they were added during the virus inoculation (0 h), the adsorption period of hrvs, but not after 1 h or later. moreover, the rna replication levels of hrvs were remarkably reduced in the mrc5 cultures treated with these constituents. these findings suggest that pgg and pa may block or reduce the entry of the viruses into the cells to protect the cells from the virus destruction and abate virus replication, which may play an important role in interfering with expressions of rhinovirus receptors (intercellular adhesion molecule-1 and low-density lipoprotein receptor), inflammatory cytokines (interleukin (il)-6, il-8, tumor necrosis factor, interferon beta, and il-1β), and toll-like receptor, which resulted in diminishing symptoms induced by hrv. global efforts to reduce the level of synthetic drugs justify further studies on p. lactiflora root-derived materials as potential anti-hrv products or lead molecules for the prevention or treatment of hrv. human rhinoviruses (hrvs) (picornaviridae) are the most common cause of upper respiratory tract infection (or common cold) and are responsible for more than half of all cases of the common cold [1, 2] . they are also associated with more severe diseases such as acute otitis media in children [3] and sinusitis in adults [4] . hrvs can also cause severe lower respiratory tract symptoms such as pneumonia, wheezing, bronchiolitis, and exacerbations of asthma and chronic obstructive pulmonary disease in infants and children as well as fatal pneumonia in elderly and immunocompromised adults [1, 2] . although hrv-induced upper respiratory illness is often mild and self-limiting, the socioeconomic burden caused by medical visits and missed school and work by hrv infection is enormous [2, 5, 6] . the degree of drug misuse and abuse is significant and antihistamine and antibiotic usages have caused many side effects [7] . attempts to develop effective treatments or vaccination have been relatively limited and unsuccessful because of more than 100 serotypes of hrv [1, 2, 8] . there is a need for the development of selective antiviral agents with novel target sites to establish an effective hrv management strategy and tactics because currently no effective antiviral therapies have been approved for either the prevention or treatment of diseases caused by hrv infection [2] . plants may provide potential sources of antiviral products largely because they constitute a potential source of bioactive secondary metabolites that have been perceived by the general public as relatively safe, with minimal impacts to human health, and often act at multiple and novel target sites [9] [10] [11] [12] . certain plant preparations and their constituents are regarded as potential sources for commercial antiviral products for prevention or treatment of hrv infection. previous studies have shown that a methanol extract from the root of chinese peony, paeonia lactiflora pallas (paeoniaceae), possessed good antiviral activity toward hrv-2 and hrv-4. no work has been obtained concerning the potential use of p. lactiflora to manage hrv, although historically p. lactiflora root (2-4 g of dried root/3 times/day) is used as analgesic, hemostyptic, and bacteriostatic agents [13, 14] . the aim of the study was to assess the cytotoxic and antiviral effects on two cell lines (hela and mrc5) and two hrv serotypes (hrv-2 and hrv-4) of paeonol (pa), gallic acid (ga), and 1,2,3,4,6-penta-o-galloyl-β-d-glucopyranose (pgg) from p. lactiflora root using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (mtt) assay. the antiviral activities of these materials were compared with those of ribavirin, a broad-spectrum antiviral agent currently used clinically to treat various dna and rna virus infections [15] . the antiviral properties and mechanisms of action of the constituents also were elucidated using real-time quantitative reverse transcription polymerase chain reaction (qrt-pcr) with sybr green dye and specific enzyme-linked immunosorbent assay (elisa). instrumental analysis 1 merck silica gel 60 rp-18 f 254s plates (for rp-tlc) and an agilent 1200 series high-performance liquid chromatograph (hplc) (santa clara, ca) were used for isolation of active principles. ribavirin (>98% purity) and mtt were purchased from tokyo chemical industry (tokyo, japan) and sigma-aldrich (st. louis, mo), respectively. anitbiotic-antimycotic and minimum essential medium (mem) were purchased from invitrogen (grand island, ny). fetal bovine serum (fbs) was supplied by paa laboratories (etobicoke, ontario, canada). the protein molecular weight standards (precision plus protein all blue standards) were purchased from bio-rad life sciences (hercules, ca). ripa buffer and 1% mammalian cell protease inhibitor cocktail were purchased from sigma-aldrich. the primary antibodies used in this study were as follows: anti-icam-1 antibody [rabbit polyclonal to intercellular adhesion molecule-1 (icam-1)], anti-low-density lipoprotein receptor (ldlr) antibody (rabbit polyclonal to ldlr), and anti-actin antibody (rabbit polyclonal to actin) purchased from abcam (cambridge, uk). the secondary antibody (horseradish peroxidase conjugated goat polyclonal to rabbit) was supplied by abcam. all of the other chemicals and reagents used in this study were of analytical grade quality and available commercially. hela (atcc ccl-2), a human epithelial adenocarcinoma cervix cell line, and mrc5 (atcc ccl-171), a human lung fibroblast cell line, were purchased from the american type culture collection (atcc) (manassas, va). these cell lines were maintained in mem supplemented with 10% fbs and antibiotic-antimycotic solution (100000 units/l of penicillin, 100 mg/l of streptomycin, and 250 μg/l of amphotericin) in a humidified incubator at 37°c and 5% co 2 . hrv-2 (atcc vr-1112as/gp) and hrv-4 (atcc vr-1114as/gp) were purchased from atcc. virus titers were determined by cytopathic effects (cpe) in hela and mrc5 cells and were expressed as 50% tissue culture infective dose (tcid 50 ) per ml as described previously by morgan [16] . extraction procedures of air-dried root of p. lactiflora were performed as described previously by ngan et al. [17] . for isolation of active principles, viral cpe inhibition assay [18] toward hrv-4 in hela cell was used. the hexane-soluble fraction was most biologically active (table 1 ) and was chromatographed as described previously [17] . finally, an active principle 1 (94 mg) was isolated at a retention time of 10.9 min. the other active ethyl acetate-soluble fraction (8 g) was chromatographed on a 55 × 5 cm silica gel column (550 g) by elution with a gradient of chloroform and methanol [100:0 (1 l) (fig 1) by spectroscopic analyses, including ms and nmr. pa (1): compound 1 was isolated as an antibacterial principle from p. lactiflora root in our previous work [17] , and the spectral data of compound 1 was largely identical to the published data [17] . ga (2) was identified on the basis of the following evidence: white powder. [19] . pgg (3): compound 3 was isolated as an antibacterial principle from p. lactiflora root in our previous work [17] , and the spectral data of compound 3 was largely identical to the published data [17, 20] . the cytotoxicity of the test materials to two human cell lines was evaluated using an mtt assay described previously by morgan [16] . in brief, a 10× stock solution of mtt was prepared by adding 5 mg/ml mtt in phosphate-buffered saline (pbs) (ph 7.4). the stock solution was sterile-filtered and stored at −20°c. hela and mrc5 cells were seeded onto 96-well culture plates at a density of 3 × 10 4 cells per well for 1 day. the culture medium was removed and the plates with monolayer cells were replaced with media containing several different concentrations (1-1000 μg/ml) of the test materials in dimethylsulfoxide (dmso). after incubation at 37°c and 5% co 2 for 2 days, the culture plates were then washed once with 200 μl pbs. a volume of 100 μl medium containing 0.05% mtt were added to each well and then incubated for 4 h at the same condition. after then, mtt solution was removed and 150 μl dmso was added to each well. finally, the plate was shaken for 15 min to dissolve the purple formazan crystals that had formed. absorbance was read at 560 nm by using a versamax microplate reader (molecular devices, sunnyvale, ca) with a reference absorbance at 670 nm. monolayers of hela and mrc5 cells were seeded onto a 96-well culture plate as stated previously. subsequently, 90 μl media containing several different concentrations (1-1000 μg/ml) of each test material in dmso was put into the wells, and then 10 μl of 10×tcid 50 of the virus stock was added to produce an appropriate cpe within 2 days after infection. ribavirin served as a reference control and was similarly prepared. negative controls consisted of the dmso solution. viral inhibition rate (vir) (%) was calculated according to the formula [21] , vir = (a tv -a cv )/(a cd -a cv ) × 100, where a tv is the optical density measured with a given concentration of the test compound in hrv infected cells; a cv is the optical density measured for the control untreated hrv infected cells; a cd is the optical density measured for the control untreated hrv uninfected cells. virus titers in infected mrc-5 cultures treated with pa and pgg were measured as tcid 50 using mtt-based titration method [22] . in brief, mrc5 cells were seeded at 10 5 cells/ml in a 6-well plate. after 24 h, the cell monolayers were treated with 100 μg/ml pa or 20 μg/ml pgg and infected with hrv-2 or hrv-4 in a concentration of 10×tcid 50 /ml. uninfected untreated and infected untreated cultures were included in the assay. the 100 μg/ml of ribavirin was used as a reference control. after 48 h incubation at 37°c, the cultures were frozen and thawed at −80°c/25°c. cell debris was removed by centrifugation (2000 rpm) and the virus supernatants were collected. virus titration was then performed in hela cell cultures. initially, a 1:10 dilution of each supernatant was prepared followed by 10 serial 2-fold-dilutions, and added to hela cell monolayers. after 48 h, cell mortality was measured using an mtt assay stated previously. graphs were built by plotting dead cell percentages toward virus dilution factors of each virus supernatant. the 50% infectivity point was calculated through a linear regression analysis of the curve. the effects of the test compounds on the infectivity of hrv-4 particles were elucidated as described previously by choi et al. [23] . approximately 1.5-fold quantities of the ic 50 values of each test compound were applied. hrv-4 was preincubated with 100 μg/ml pa, 20 μg/ml pgg, or 100 μg/ml ribavirin for 1 h at 4°c. monolayers of mrc5 cells were infected with the pretreated or untreated hrv-4 for 1 h at 37°c. unbound virus was removed by washing the wells with pbs twice, and then cells were incubated in fresh medium supplemented with or without test compounds at 37°c. after 2 days, mtt test and antiviral activity were carried out as stated previously. the time-of-addition effects of all compounds on hrv-4 were examined according to the method of choi et al. [23] . in brief, monolayers of mrc5 cells were seeded onto a 96-well culture plate as stated previously. after washing with pbs, 100 μg/ml pa, 20 μg/ml pgg, and 100 μg/ml ribavirin were separately added onto the cells at either before (-1 h), during (0 h), or after (1, 2, 3, 4, 6, 8, 12, 16, 20, and 24 h) hrv-4 infection at 37°c. after 2 days, mtt test and antiviral activity were carried out as stated previously. to evaluate the level of gene expression, real-time qrt-pcr with sybr green dye was carried out. hrv-2 or hrv-4 infected and noninfected cultures of mrc5 monolayers grown in 25 cm 2 cell culture flasks (corning, ny) were treated with 100 μg/ml pa or 20 μg/ml pgg. after incubation at 37°c and 5% co 2 for 2 days, total rna was extracted from the culture cells using the rneasy plus mini kit (qiagen, hilden, germany) according to the manufacturer's instructions. contaminated genomic dna was removed using rq1 rnase-free dnase (promega, madison, wi). complementary dna (cdna) was synthesized using 1 μg total rna through a reverse transcription reaction using the superscript first-strand synthesis kit (invitrogen, carlsbad, ca). five log10-fold dilutions of cdna for each rna were performed to determine pcr efficiency (100 ng-10 pg per reaction). qrt-pcr was performed in 96-well plates using the steponeplus real-time pcr system (applied biosystems, foster, ca). each reaction mixture consisted of 10 μl of maxima sybr green/rox qpcr master mix (2×) (thermo scientific, foster, ca), 2 μl of forward and reverse primers (5 pmol each), 1 μl of cdna (8 ng), and 7 μl of double-distilled water in a final volume of 20 μl. oligonucleotide pcr primer pairs are listed in table 2 cell lysates from infected and noninfected mrc5 cultures 2 days after incubation with or without test compounds were obtained in ripa buffer and 1% mammalian cell protease inhibitor cocktail according to the manufacturer's instructions. working dilutions of primary antibodies were dilulted 500, 2000, and 1000 times for anti-icam-1, anti-ldlr, and anti-actin antibodies, respectively. working dilutions of secondary antibody was 1000, 1000, and 800 for anti-icam-1, anti-ldlr, and anti-actin, respectively. ten micrograms of cell lysates from different treatments were mixed with an equal volume of 5× laemmli sample buffer, boiled in 10 min, and resolved by electrophoresis in 11% sodium dodecyl sulfate-polyacrylamide gels (sds-page) [24] . after electrophoresis at 120 v in 2 h, proteins from the gels were transferred onto a polyvinyl difluoride membrane (pall corporation, pensacola, fl) using a electroblotting apparatus (bio-rad, hercules, ca). the membranes for anti-icam-1 and anti-ldlr were incubated in blocking solution containing 5% nonfat dry milk for 4 h to inhibit nonspecific binding. these membranes were then incubated overnight at 4°c with the primary antibodies. after washing with pbs (each 10 min) three times, the membranes were further incubated with the secondary antibody for 2 h and washed with pbs containing 0.5% tween-20 (v/v) (0.5% pbs-t) four times (each 15 min). the membranes for anti-actin were incubated in pbs blocking solution containing 5% bsa overnight to inhibit nonspecific binding, and then incubated with anti-actin antibody in the blocking solution for 3 h at 25°c and washed four times (each 15 min) in 0.1% pbs-t at room temperature before incubation with the second antibody. finally, all the membranes were developed with an ecl chemiluminescence reagent (amersham bioscience, buckinghamshire, uk) and exposed to a cp-pu x-ray film (agfa, mortsel, belgium). differences in protein expressions were quantified using a molecular imager gel doc xr system (bio-rad, hercules, ca) and normalized to actin expression on the same membrane. the expressions of icam-1 and ldlr were examined using real-time qrt-pcr and western blot. furthermore, concentrations of soluble icam-1 (sicam-1) in cell-free culture supernatants 2 days after treatment were measured using a sicam-1 elisa kit (pierce biotechnology, rockford, il) according to the manufacturer's instructions. the sensitivities of the assays were 0.3 ng/ml. the concentrations of icam-1 in the test samples were determined from od values using standard curve of each assay. the concentrations of il-6, il-8, and tnf in cell-free culture supernatants 2 days after treatment were measured using specific elisa. opteia il-6, il-8, and tnf elisa kits (bd biosciences, san diego, ca) were used for the assays. the sensitivities of these elisa assays were 2.2, 0.8, and 2.0 pg/ml, respectively. the concentrations of il-6, il-8, and tnf in the test samples were determined from od values using standard curve of each assay. cytotoxicity was expressed as 50% cytotoxic concentration (cc 50 ) of each compound that reduced the viability of cells to 50% of the control. fifty percent inhibitory concentration (ic 50 ) was defined as the compound concentration required to reducing the viral cpe to 50% of the control. the cc 50 and ic 50 values were determined using graphpad prism 5 software program (graphpad software, la jolla, ca). the ic 50 values for each serotype and their treatments were considered to be significantly different from one another when their 95% confidence limits (cls) did not overlap. the selectivity index (si) was determined as the ratio of cc 50 to ic 50 [23] . all data represent the mean ± sd of duplicate or triplicate samples of three independent experiments. statistical analyses were carried out using sas 9.13 program (sas institute, cary, nc). data from two groups were analyzed by a student's t-test, and multiple groups were analyzed by a one-way analysis of variance and bonferroni multiple comparison post-test. the antiviral activity of pgg, ga, and pa toward hrv-2 and hrv-4 in hela cells was compared with that of ribavirin using an mtt assay (table 3) . based on ic 50 effects of pa and pgg on hrv titers were compared with those of ribavirin (table 5) due to their antiviral activity with high selectivity, the effects of pa and pgg on the infectivity of hrv-4 particles were likewise compared with those of ribavirin (fig 2) . the inhibition rates of preincubation with 100 μg/ml pa, 20 μg/ml pgg, and 100 μg/ml ribavirin were 10.6, 11.2, and 10.0%, respectively. continuous presence of pa, pgg, and ribavirin during infection led to a significant increase in the inhibition rate (57.2, 54.5, and 58.2%). to investigate the mode of action of pa, pgg, and ribavirin, time-of-addition experiments were performed (fig 3) . treatment with 100 μg/ml pa or 20 μg/ml pgg considerably suppressed hrv-4 infection only when added just after the virus inoculation (0 h) (57.2 and 54.5% inhibition). the inhibition of these compounds declined to 40% or less when added at either prior (-1 h) or post (1-24 h) infection. similar results were observed with 100 μg/ ml ribavirin. further evidence of the inhibitory effects of pa and pgg on hrv replication in mrc-5 cells was provided by real-time qrt-pcr analysis (fig 4) . in the presence of 100 μg/ml pa or 20 μg/ml pgg in mrc5 cell cultures infected with hrv-2, the rna replication levels were reduced by 30.1 and 14.3 fold, respectively, compared to the levels in the cell cultures without the compounds (fig 4a) . similarly, the replication levels of the hrv-4 in the mrc5 cell culture treated with pa or pgg were also reduced by 16.3 and 15.1 fold, respectively, compared with the untreated cultures ( fig 4b) . unbound viruses were removed and washed by phosphate-buffered saline twice, and then cells were incubated in fresh medium with or without test compounds. after 2 days, inhibition was evaluated by tetrazolium method and expressed as the inhibition rate. each bar represents the mean ± sd of triplicate samples of three independent experiments. ***p<0.001, using a student's t-test. effect on icam-1 and ldlr expressions mrna expression of icam-1 in mrc5 2 days after hrv infection in the presence of 100 μg/ml pa or 20 μg/ml pgg were investigated using real-time qrt-pcr (fig 5a) . hrv-2 or hrv-4 infection increased icam-1 mrna expression, but these increases were reduced by the addition of pa and pgg. furthermore, the icam-1 mrna expression level in the group of pgg or pa treatment was lower than those in the group without the treatment with the test compounds. mrna expression of ldlr in mrc5 was likewise assessed (fig 5 b) . ldlr mrna expression in the hrv-2 or hrv-4 infected untreated cells was similar to that in the mock untreated cells and the expression decreased in the group of pa or pgg treatment. using elisa (fig 5c) , it was found that sicam-1 levels in supernatants of hrv-2 or hrv-4 infected cultures were higher than those in supernatants of noninfected cultures, and sicam-1 levels in supernatants of cultures treated with pa or pgg were significantly lower than those in cultures without the treatment with the test compounds. however, there was no difference in sicam-1 levels between mock cultures with or without constituents. western blot analysis of micam-1 and ldlr expressions in mrc5 cells 2 days after hrv infection in the presence of 100 μg/ml pa or 20 μg/ml pgg was performed. micam-1 protein levels in noninfected cultures were similar to those in cultures infected with hrv-2 or hrv-4, and the protein levels in cultures treated with pa or pgg at the test concentrations were also similar to those in cultures without treatment of the test compounds (fig 6a for hrv-2; fig 6b for hrv-4) . the presence of 20 μg/ml pgg in cultures resulted in the sharp decline in the ldlr protein expression, compared with the cultures without pgg treatment, regardless of hrv-2 infection. however, 100 μg/ml pa had no effect on expression of ldlr protein (fig 6c) . similar results were also observed with hrv-4 ( fig 6d) . effect on expression of cytokines mrna expressions of various cytokines in mrc5 2 days after infection with hrv-2 or hrv-4 in the presence of 100 μg/ml pa or 20 μg/ml pgg were assessed using real-time qrt-pcr. hrv-2 and hrv-4 evoked a significant increase in il-6 mrna expression levels in the nontreated cell cultures, but the expression levels were significantly reduced in the cell cultures treated with pa or pgg, although there was no difference in il-6 mrna levels between mock cultures with or without constituents (fig 7a) . similar results were also observed with il-8 mrna (fig 7b) . the expression of tnf mrna in the culture challenged with hrv-2 or hrv-4 was similar to that in the noninfected cell cultures. the treatment with pa and pgg did not remarkably reduce the tnf mrna expression, compared to the untreated cells ( fig 7c) . although the ifn-β mrna expression did not occur in the noninfected cell cultures, ifn-β mrna was expressed in the cell cultures infected with hrv-2 or hrv-4. the ifn-β mrna expressions were meaningfully inhibited by pa or pgg treatment (fig 7d) . il-1β mrna expression was also highly induced by hrv-2 or hrv-4, and the induction disappeared or was significantly reduced by pa or pgg treatment (fig 7e) . the concentrations of il-6 detected by elisa in the hrv-2 or hrv-4 infected cell supernatants were significantly higher than those in the noninfected cell supernatants. il-6 concentrations in the mock cultures treated with pa were higher than those in the mock cultures without the treatment. however, il-6 concentrations in infected cultures were reduced by treatment with 100 μg/ml pa or 20 μg/ml pgg. the decrease in hrv-induced il-6 secretion by 20 μg/ml pgg treatment was higher than that by 100 μg/ml pa treatment. pa and pgg affected il-6 protein expressions in a concentration-dependent manner (fig 8a) . similar results were observed with il-8 ( fig 8b) . however, tnf protein was not detectable in supernatants of all the cultures (data not shown). effect on tlr3 mrna expression tlr3 mrna expression in hrv-2 or hrv-4 infected cells was increased, compared with uninfected cells (fig 9) . tlr3 mrna expression in the cultures treated with 100 μg/ml pa or 20 μg/ml pgg was significantly lower than that in the cultures without the treatments with the test compounds. many plants and their constituents such as phenolics, terpenoids and alkaloids manifest antiviral activity toward different viruses [12, 25] and have been proposed as alternatives to conventional antiviral drugs. anti-hrv constituents derived from plants include alkaloids (e.g., arborinine and (s)-ribalinine, ic 50 3.19 and 82.95 μm [26] ; glaucine, ic 50 doi:10.1371/journal.pone.0121629.g008 [34] ; gallic acid, ic 50 294.55 μm [35] ). it has been reported that hrv capsid-binding compounds toward all hrv serotypes showed the existence of group a and b, based on a wide range of susceptibilities to antiviral compounds [36] . in the current study, the antiviral principles of p. lactiflora root was determined to be the aryl ketone pa (1), the simple benzoic acid ga (2) , and the gluco-hexose pgg (3). these constituents exhibited antiviral activity toward both group a (hrv-2) and group b (hrv-4). ic 50 of pgg, ga, and pa toward two hrvs was between 11.56 and 17.89 μm, between 426.99 and 448.10 μm, and between 492.17 and 608.38 μm, respectively, although ic 50 of the natural compounds stated previously is between 0.22 and 294.55 μm. pgg exhibited greater antiviral activity than ribavirin (ic 50 , 240.49-324.07 μm) toward two hrvs and high selectivity. the virus titration assay results also proved that the constituents had antiviral activity toward the hrvs. this original finding indicates that materials derived from p. lactiflora root can hold promise for the development of novel and effective naturally occurring antiviral agents for two different hrv groups a and b. pgg was reported to possess antiviral activity toward hepatitis b virus [37] and influenza a virus [38] . investigations on the modes of action of natural antivirals may contribute to the development of selective hrv therapeutic alternatives with novel target sites. the modes of antiviral action of plant secondary substances have been well documented by rollinger and schmidtke [1] and kitazato et al. [39] . targeting virus molecules is likely more specific and less toxic, but there is a narrow spectrum of viruses and a higher risk of creating resistant viruses [39] . on the contrary, chemicals which target cellular molecules may possess a broader antiviral activity spectrum and less risk of developing virus resistance, but may be more toxic to the host cell [39] . in the current study, pgg and pa do not interact with the hrv-4 particles, as preexposure of the virus to the constituents did not alter the infectivity of hrv-4 particles. based on time-course tests, pgg and pa can inhibit hrv-4 infection only when they were added during the virus inoculation (0 h), but not after 1 h or later. this finding suggests that pgg and pa may interact with the human cells in the early stage of hrv infections to protect the cells from the virus destruction. the mechanism of action of these constituents on cellular protection toward hrv-4 still remains unclear, although ribavirin was reported to enter host cells through nucleoside transporters in several studies [40, 41] . in addition, real-time rt-pcr analysis revealed that the rna replication levels of hrvs were remarkably reduced in the mrc5 cultures treated with these constituents. this finding suggests that pgg and pa may block or reduce the entry of the viruses into the cells in the early stage of hrv infections to protect the cells from the virus destruction and abate virus replication. detailed tests are needed to fully understand the modes of action of the constituents. it has been suggested that the mode of anti-hrv action of gallic acid might be derived from the inhibition of virus absorption [35] . interference with specific virus-host cell receptor interactions can be one of the potential interventions in antiviral chemotherapy [42] . hrvs are classified into the major and minor groups, based on their binding to icam-1 [43, 44] or to members of the ldlr family [45] , respectively. icam-1 is a critical target-docking molecule on epithelial cells for 90% of the hrv serotypes [43, 44] . the major group uses icam-1 as a mechanism to gain entry to the host cell [46] . it has been suggested that antagonism of the virus-receptor interaction would appear to be an effective way to inhibit a broad spectrum of hrvs [47] . macrolide antibiotics such as erythromycin inhibit infection by the major group of hrvs via a reduction in icam-1 expression [48] . micam-1 expression is equally induced by both major and minor group hrvs [49] . the hrv-induced icam-1 up-regulation is not receptor-restricted [49] . moreover, it has been reported that pretreatment of hrv-2 with sicam did not alter the ability of hrv-2 to induce icam-1 [46] . the current experiments demonstrated that both hrv-2 and hrv-4 induced the both forms of icam-1 expressed in mrc5. elisa results revealed that sicam-1 protein levels of infected cultures treated with pa or pgg were significantly lower than those in cultures without the compound treatment, although there was no difference in sicam-1 levels between mock cultures with or without the constituents. however, icam-1 mrna expression levels were decreased in the presence of pgg or pa in both infected and noninfected cell cultures. the findings indicated that these constituents interfered with host cell protein expression of this receptor through inhibition of the receptor rna expression or reduction of hrv replication. in addition, ldlr mrna expressions were also reduced in the presence of pgg and pa in the cultures. however, ldlr protein expression was inhibited in pgg treatment only. the findings suggest that pgg interfere with ldlr expression through inhibiting expression of ldlr rna or reduction of hrv replication, whereas pa interfered with ldlr expression through only reduction of hrv replication. pa was reported to suppress icam-1 expression in tnf-stimulated human umbilical vein endothelial cells [50] . hrv infection induces the production of numerous components of innate immunity including tlr3 and imflamatory mediators, such as kinins, leukotrienes, histamine, interleukins (il-1, il-6, and il-8), tnf, and regulated by activation normal t cell expressed and secreted [2, 51] . the surface-expressed tlr3s were reported to have an important function in response to a common human viral infection of natural host cells and play an important role in innate immune responses toward hrv infection [52, 53] . during the replication cycle of hrv, viral double-stranded rna (dsrna) is recognized by tlr3 [2] . the interaction between dsrna and tlr3 activates a signaling cascade that triggers increase of cytokine production in host cells and stimulates innate immune responses [2] . it was supposed that inhibition of tlr3 expression have potential modulatory effects on the pathophysiologic changes related to hrv infection [53] . cytokines such as tnf, il-1β, il-6, and il-8 induce upregulation expression of the adhesion molecules such as icam-1 in both epithelial and vascular endothelial cells, thereby increasing cell susceptibility to hrv infection [54] . the concentrations of il-6 and il-8 in nasal secretions correlate with the severity of the symptoms in patients with colds [55] . hrv infections were reported to be responsible for triggering exacerbations of asthma through inducing gene expression of these cytokines in asthmatic subjects [56] . elisa results revealed that pa induced il-6 and il-8 expression. therefore, the expression levels in the infected cell cultures treated with pa were lower than those in the infected cell cultures without the constituent as a consequence of the hrv replication reductions. pgg reduced il-6 and il-8 expression and the decreases of expression levels in the infected cell cultures treated with pgg were resulted from the interference of the constituent with both of host cell protein expression and hrv replication. the current study also indicated that the constituents reduced expressions of tlr3 and cytokines, such as tnf, ifn-β, and il-1β, which resulted in diminishing symptoms induced by hrv. it may be that effects of the constituents on blocking or reducing the entry of the viruses into the cells, which results in reducing hrv-rna replications, play an important role in interfering with hrv-induced gene expression. in conclusion, p. lactiflora root-derived preparations containing pgg and pa could be useful as an antiviral agent in the prevention or treatment of hrv infection. the antiviral action of pgg and pa may be an indication of at least one of the pharmacological actions of p. lactiflora. for the practical use of the preparations as novel anti-hrv products to proceed, further research is needed to establish their safety with respect to humans and whether this activity is exerted in vivo after consumption of p. lactiflora root-derived products by humans. lastly, detailed tests are needed to understand how to improve anti-hrv potency and stability for eventual 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human tracheal epithelial cells ifn-gamma regulation of icam-1 receptors in bronchial epithelial cells: soluble icam-1 release inhibits human rhinovirus infection paeonol suppresses intercellular adhesion molecule-1 expression in tumor necrosis factor-α-stimulated human umbilical vein endothelial cells by blocking p38, erk and nuclear factor-κb signaling pathways cooperative effects of rhinovirus and tnf-alpha on airway epithelial cell chemokine expression toll-like receptor 3 is induced by and mediates antiviral activity against rhinovirus infection of human bronchial epithelial cells levocetirizine inhibits rhinovirus-induced icam-1 and cytokine expression and viral replication in airway epithelial cells carbocisteine inhibits rhinovirus infection in human tracheal epithelial cells association between interleukin-8 concentration in nasal secretions and severity of symptoms of experimental rhinovirus colds rhinovirus-induced modulation of gene expression in bronchial epithelial cells from subjects with asthma key: cord-000077-d441jam3 authors: zhang, hao-jie; wang, yong-xiang; wu, hao; jin, dong-yan; wen, yu-mei; zheng, bo-jian title: the y271 and i274 amino acids in reverse transcriptase of human immunodeficiency virus-1 are critical to protein stability date: 2009-07-03 journal: plos one doi: 10.1371/journal.pone.0006108 sha: doc_id: 77 cord_uid: d441jam3 reverse transcriptase (rt) of human immunodeficiency virus (hiv)-1 plays a key role in initiating viral replication and is an important target for developing anti-hiv drugs. our previous study showed that two mutations (y271a and i274a) in the turn rt (gln(269)-arg(277)) abrogated viral replication, but the replication capacity and rt activity was discordant. in this study, we further investigated why alanine substitutions at these two sites would affect viral replication. we found that both rt activity and rt protein were almost undetectable in viral particles of these two mutants, although the pr160(gag-pol) mutants were properly expressed, transported and incorporated. using protease inhibition assay, we demonstrated a correlation between the degradation of the rt mutants and the activity of viral protease. our native gel analysis indicated that the mutations at 271 and 274 amino acids might cause conformational changes, leading to the formation of higher order oligomers instead of dimers, resulting in increased protein instability and susceptibility to viral protease. thus, residues 271 and 274 are critical to rt stability and resistance to viral protease. the conservation of the two amino acid residues among different strains of hiv-1 lent further support to this conclusion. the knowledge gained here may prove useful in drug design. reverse transcriptase (rt) of human immunodeficiency virus (hiv)-1, encoded by pol gene, is a multifunctional enzyme that possesses rna-and dna-dependent polymerase activities as well as rnase h activity [1] . rt is indispensable for hiv-1 and it converts the single-stranded viral rna into double-stranded dna upon viral entry into host cells. due to its important role in viral life cycle, rt is one attractive target for antiviral drug design [2] . the biologically active form of hiv-1 rt is a heterodimer consisting of two subunits, p66 (66 kda) and p51 (51 kda). the p51 subunit is derived from p66 by proteolytic cleavage of its cterminal domain [3] . the polymerase domain of p66 and p51, resembling a right hand configuration, consists of four subdomains, which are known as fingers, palm, thumb and connection. the fingers, palm and thumb subdomains of p66 form a nucleic acid binding cleft and the connection subdomains of the two subunits form the floor of the nucleic acid binding site [4] [5] [6] [7] . the thumb subdomain has four a helices. two antiparallel ahelices of them, a-h (asn 255 to ser 268 ) and a-i (gln 278 to thr 286 ), are important for holding the primer/template in position during the translocation in polymerization. the primary sequence (val 254 to ala 288 ) in the vicinity of these two a helices has been found to share homology with several other nucleic acid polymerases and has been termed the ''helix clamp'' [5, 8] . extensive studies have been carried out to shed light on the relationship between the ''helix clamp'' and function of rt. the effects of alanine-scanning mutations in a-h and a-i on polymerase activity, primer/template (p/t) binding, fidelity and enzyme kinetics have been determined. while mutations in a-i do not affect p/t binding or fidelity significantly, several a-h mutants exhibit lower binding affinity, processivity and frameshift fidelity [9] [10] [11] [12] . previous studies have demonstrated that mutations in these two helices can have significant effect on rnase h activity, minus-strand dna transfer activity and removal of polypurine track primer [13, 14] . although alanine substitutions at sites 269, 270, 271 and 277 have been investigated in two studies [9, 10] , detailed studies on the functional structure of the ''turn'' (gln 269 -arg 277 ) between a-h and a-i were limited. in our previous study on hepatitis b virus rt, conserved residues located at the ''turn'' of helix clamp motif were found important for pregenomic rna encapsidation during the assembly of nucleocapsids [15, 16] . since this homologous helix clamp motif is also present in hiv-1 rt, we hypothesized that residues in the turn may play important roles in viral life cycle. our recent study showed that alanine substitutions at 271 and 274 of hiv-1 rt drastically affected viral replication, but discordance between viral replication and rt activity was observed [17] . in this study, we confirmed our previous observations and further investigated why the two mutations abrogated viral replication. our study demonstrated that these two mutations lead to rapid degradation of rt in viral particles, indicating that the residues of 271 and 274 are critical for maintaining the stability of hiv rt. the parental hiv-1 proviral plasmids, plai.2, pnl4-3-de-egfp and phef-vsv-g, were obtained through the nih aids research and reference reagent program, division of aids, niaid, nih [18] [19] [20] . the pnl4-3-de-egfp based mutants (y271a, g273a, i274a, k275a, v276a, r277a) and plai2 based mutants (y271a and i274a) were constructed by sitedirected mutagenesis using quikchange ii xl site-directed mutagenesis kit (stratagene, usa) according to manufacturer's instruction. the open reading frame of rt p66 subunit was amplified from wild type or mutant pnl4-3-de-egfp using a pair of primers and cloned into pet-28b vector (novagen, shanghai) to obtain expression plasmid pet-p66 with the 66 his tag at 39 terminus. the paired primers used for site-directed mutagenesis and construction of plasmids are listed in table 1 . cell lines 293ft, hela and u373-magi-cxcr4 cem [21] were maintained in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal bovine serum and antibiotics (invitrogen, usa). mt2 cells [22, 23] were maintained in rpmi 1640 supplemented with 10% fetal bovine serum and antibiotics. to prepare pseudoviruses and live viruses, 293ft cells were cotransfected with 7.5 mg wild-type or mutant pnl4-3-de-egfp, and 2.5 mg phef-vsv-g, or 10 mg wild-type or mutant plai.2 using lipofectamine 2000 (invitrogen, usa) according to the manufacturer's instructions. supernatants were collected 48 h after transfection and stored at 280uc after spinning down and filtering to remove cell debris. the cells were rinsed with ice-cold phosphate-buffered saline (pbs), scraped from each plate and lyzed in cell lysis buffer (boehringer, germany) and 16 protease inhibitor cocktail (roche, usa) on ice for 30 min. cell lysates were stored at 220uc after centrifugation at 13,0006g at 4uc for 15 min to remove cell debris. one cycle infection assay was carried out using normalized pseudoviruses as described previously [20] . briefly, jurkat cells (0.5610 6 ) were infected with viral supernatants containing 250 ng p24, which was measured by vironostika hiv-1 antigen microelisa kit (biomerieux bv boxtel, netherlands). the virus and cells mixture was spun at 1,800 g at 30uc for 2 h. after the 2h spin infection, jurkat cells were washed with 2 ml culture medium twice, then cultured in 24-well plates at 37uc for 48 h. the cells were collected and washed twice with pbs. after the cells were fixed with 1% paraformaldehyde in pbs for 30 min on ice, the infected cells, as determined by the expression of gfp, were measured using a facscalibur instrument (becton dickinson, usa) and analyzed with cell quest software (becton dickinson, usa) as described previously [20] . infection with live viruses was conducted in u373-magi-cxcr4 cem and mt-2 cells. u373-magi-cxcr4 cem cells were infected with normalized wild type or mutant live viruses in 24-well plate as described previously [21] . briefly, triplicate wells (6610 4 cells/well) were infected with live viruses (60 pg p24/well) which were diluted in dmem containing 20 mg/ml of deaedextran (amersham biosciences). after cultured at 37uc in 5% co 2 incubator for 48 h, the cells were fixed in 1% formaldehyde-0.2% glutaraldehyde in pbs for 5 min. after washing twice with pbs, the cells were stained with 400 mg/ml of x-gal (5-bromo-4chloro-3-indolyl-b-d-galactopyranoside), 4 mm mgcl 2 , 4 mm potassium ferrocyanide, and 4 mm potassium ferricyanide in pbs for 2 h at 37uc. the plate was washed twice with pbs and blue foci were observed under microscope. infection with live viruses was also carried out in mt-2 cells as described previously [22, 23] . in brief, mt-2 cells (1610 4 cells/well) in 96-well plate were infected with live viruses (20 pg p24/well). after cultured for 6 days, the virus-specific cytopathic effect (cpe) was observed under microscope. the rt activity in pseudoviruses was measured by a rt assay using colorimetric kit (roche, usa). briefly, viral supernatants containing 2 mg p24 were centrifuged at 4uc for 2 h at 40,0006g and the viral pellets were resuspended in 50 ml lysis buffer. lyzed viral pellets were 10 fold serially diluted and the subsequent procedures were carried out according to the manufacturer's instructions. rt activities of mutants were calculated and compared to that of wild type pseudovirus. expression and subcellular localization of precursor gal-pol polyprotein were detected by immunofluorescence microscopy as described previously with some modifications [24] . briefly, hela cells cultured on coverslip in 24-well culture plate were transfected with 600 ng pnl4-3-de-egfp and 200 ng phef-vsv-g. two days post-transfection, the cells were fixed with 500 ml 4% paraformaldehyde (pfa) at room temperature for 15 min. after washing 3 times with pbs, 500 ml of 50 mm ammonium chloride was incubated with the cells for 10 min to neutralize residual pfa. the cells were washed 3 times with pbs and treated with 0.05% triton x-100 for 3 min. after washing 3 times with pbs, 500 ml 10% normal rabbit serum (nrs) in pbs was added to block the slip overnight at 4uc. primary antibody (mouse monoclonal antiintegrase, 1:100, santa cruz) and secondary antibody (texas red dye-conjugated rabbit anti-mouse igg, 1:100, jackson immu-noresearch) were incubated with samples in dark at room temperature. following each incubation, samples were subjected to 3 washes with 1% nrs in pbs. with 3 additional pbs washes, the coverslip was mounted using fluorescence mounting medium (dako) and observed under lsm510 meta confocal microscope (carl zeiss). gal-pol polyprotein and its products were tested by western blotting as described previously with some modifications [25] . briefly, viral proteins were separated by 10% sds-polyacrylamide gel electrophoresis (sds-page) and electro-blotted onto hybond-p pvdf membrane (ge healthcare, bio-sciences). after blocking with 5% skim milk for 1 h at room temperature, the membrane was incubated with primary antibodies (rabbit polyclonal anti-rt, 1:3000; mouse monoclonal anti-rt, antiintergrase, anti-protease or anti-capsid, 1:500) for 1 h. membrane was washed three times with pbs containing 0.1% tween 20 (pbs-t) and then incubated with horseradish peroxidase (hrp)-conjugated secondary antibody (goat anti-rabbit igg or goat anti-mouse igg, 1:4000) for 1 h. after the blots were rewashed three times in 0.1% pbst, signals were visualized using ecl western blotting substrate reagents (amersham biosciences, usa) and kodak biomax scientific imaging film (eastman kodak). wild type and mutant pet-p66 expression vectors were respectively transformed into e. coli bl21(de3) and the expression was induced with 0.3 mm isopropyl b-d-1-thiogalactopyranoside (iptg) when e. coli grew up to an od600 of 0.7,1.0. e. coli expressing p66 was spun down and re-suspended in the binding buffer (50 mm nah 2 po4, 300 mm nacl, 10 mm imidazole). after the bacteria were disrupted by ultrasonication and centrifuged at 15 000 g for 30 min at 4uc, the wild type and mutant p66 in supernatants were purified using ni-nta magnetic agarose beads (qiagen, germany). the purified p66 proteins were subjected to nativepage novex bis-tris gel analysis and western blotting according to the protocol recommended by invitrogen, usa. rt sequences of 1083 hiv-1 strains obtained from the hiv complete sequence database (http://www.hiv.lanl.gov/content/ sequence/newalign/align.html) were aligned and compared. based on the x-ray crystal structures of hiv-1 rt (1rth) from the research collaboratory for structural bioinformatics protein data bank (rcsb pdb), structural models of wild type and mutant rts were analyzed using swiss-pdbviewer software (http://spdbv.vital-it.ch/). statistical analysis of rt activities was performed by student's t test using stata statistical software. results were considered significant at p#0.05. the effect of six rt mutations at the helix clamp turn in hiv-1 rt on viral replication was tested with pseudoviruses. when jurkat cells were infected with the wild type and mutant pseudoviruses (250 ng p24 virus/5610 5 cells), viral replication was almost completely inhibited in the mutants y271a and i274a, while replication of other mutants did not show significant difference as compared to that of the wild type (fig. 1a) . a similar result was also obtained when the cells were infected with lower amount (150 ng p24) of pseudoviruses (data not shown). to confirm the above results, replication of wild type, y271a and i274a live viruses was further monitored in u373-magi-cxcr4 cem and mt2 cells. mutants y271a and i274a were undetectable in u373-magi-cxcr4 cem cells (fig. 1b) and no virus-specific cpe was found in mt-2 cells (fig. 1c) . since reverse transcription is the first step for hiv replication after viral entry into host cells, our results suggested that the two mutations might have affected the rt activity. rt activity and rt proteins were undetectable in pseudoviral particles of mutants y271a and i274a we thus interrogated the pseudovirus mutants for rt activity. the rt activity recovered from mutants y271a and i274a were almost unnoticeable, as compared with that of wild type ( fig. 2a) . this result was further confirmed using wild type, y271a and i274a live viruses (data not shown). next, we asked if the viral particles contain dysfunctional rt or the rt was not incorporated into the viral particles. by western blotting, rt protein was basically undetectable in viral particles of these two mutants (fig. 2b) . the results suggested that the mutations could affect the incorporation of rt into the viral particles, leading to a defect in reverse transcription which initiates viral replication. mutations y271a and i274a did not affect pr160 gag-pol expression and transportation we next investigated whether the loss of rt in viral particles of the mutants was attributed to altered expression and transportation of the precursor protein, polyprotein pr160 gag-pol , during virus assembly. pr160 gag-pol in lysates of cells transfected with the wild type and mutants was detected by western blotting. as shown in fig. 3a , similar levels of pr160 gag-pol , gag protein (pr55 gag ) and capsid protein p24 (ca p24) were found in cells transfected with the wild type or mutant constructs, indicating that the expression and stability of rt precursor protein were not affected by the mutations. immunofluorescence staining further demonstrated a normal subcellular localization of mutant gag-pol polyproteins as compared to that of the wild type, suggesting that the transportation of gag-pol was unlikely to be affected by alanine substitutions (fig. 3b) . the gag-pol polyprotein was incorporated into mutant virions of y271a and i274a, but the rt was degraded by viral protease to investigate whether the precursor gag-pol polyprotein was indeed incorporated into pseudoviral particles of mutants y271a and i274a, products of the gag-pol polyprotein, integrase (in), protease (pr) and p24, in wild type and mutant pseudoviral particles were examined by western blotting. the results showed that, except for rt (fig. 2b) , all products of the gag-pol polyprotein, in p32, pr p11 and ca p24, were detected in the mutants y271a and i274a at a level similar to that of the wild type pseudoviral particles (fig. 4a) . thus, pr160 gag-pol was indeed incorporated into the virions and processed properly. to study if the rts in the viral particles of y271a and i274a mutants were degraded by proteolysis that made them undetectable, pseudoviruses of wild type and mutants were generated in the presence or absence of indinavir, a highly specific inhibitor of hiv-1 protease. indinavir treatment was effective, because the treatment resulted in a dose-dependent inhibition of pr55 gag processing into p24 for wild type and mutant viruses (fig. 4b ). as shown in fig. 4c , in the absence of indinavir, both rt p66 and p51 were readily detected in the wild type pseudovirus, but not in the mutant viruses. in the presence of indinavir, however, both rt p66 and p55 were markedly reduced in the wild type virus but became detectable in mutant y271a viral particles, while rt p66 was also detectable in mutant i274a virus. we also detected the rt subunits in the viral supernatants collected at earlier time points (12, 24 and 36 hours post-transfection), but the mutant rts were still undetectable (data not shown). these results suggested that the y271a and i274a rts were degraded after incorporation of gag-pol polyprotein into the virions, which might be attributed to the activity of viral protease. since rt dimer is the stable form which remains resistant to the proteolysis, we tested whether treatment of dimerization enhancer could reduce the mutant rt proteolysis. wild type and mutant pseudoviruses were generated in the presence or absence of efavirenz (efv), the most potent dimerization enhancer [26] . the results showed that the rt mutants were still undetectable in the presence of efv (fig. 5a) . the conformation of the wild type and mutant rt p66 was further analyzed by native gel electrophoresis followed by western blotting (fig. 5b) . under native conditions, wild type p66 subunit formed homodimer. however, the formation of homodimer in mutant y271a was markedly reduced and a higher order oligomer appeared, which might be tetramer according to its molecular weight, while mutant i274a existed as at least two higher order oligomers, which were likely tetramer and octamer based on their molecular weights. however, the exact nature of the oligomers formed by mutant p66 subunits remained to be clarified. furthermore, the treatment with b-mercaptoethanol could partially disrupt the higher order oligomers and improve dimer formation. this result implicated that dimer formation might be necessary for the stability of rt and its resistance to proteolysis after incorporation of gag-pol polyprotein into the viral particles. amino acids at 271 and 274 are relatively conserved and big side chains may be important in maintaining rt stability and resistance to proteolysis by comparing 1083 complete sequences of hiv-1, it was found that the amino acids at 271 and 274 were relatively conserved. as shown in table 2 , tyrosine (y) is the predominant naturally existent amino acid at 271 residue (99.19%), while phenylalanine (f), histidine (h) and cysteine (c) occur rarely (,1%). similarly, isoleucine (i) is the predominant naturally existent amino acid at 274, which accounts for 98.43%, while in less than 2% of all cases, valine (v) and leucine (l) are found at this site. this finding suggested that these amino acids might probably play important role in maintaining the active conformation of rt. consistently, structural analysis suggested that amino acids at these two sites are buried in the thumb region of rt (fig. 6a) . it was further revealed that all wild type rts have relatively big side chains, but they are absent from the mutants 271a and 274a (fig. 6b & 6c) . thus, the loss of the side chains in the rt mutants plausibly leads to conformational change of rt, leading to aberrance in dimer formation and susceptibility to proteolysis by hiv-1 protease. hiv rt plays a key role in viral replication and is an important target for development of anti-hiv drugs. however, the turn between helices h and i of hiv-1 rt thumb region (amino acid residues 269 to 277) has not been well characterized yet. to understand the structure-function relationship of this turn in details, we investigated whether alanine substitutions in this region would affect rt activity. except for residues 269 and 270, which have been reported to have no significant influence on rt activity [9, 10] , and for residue 272, which is originally an alanine, six mutant pseudoviruses (residues 271 and 273 to 277) were constructed and viral replication was compared with that of the wild type virus. the replication of two mutants, y271a and i271a, were found to be almost completely abolished (fig. 1a) . this result was further confirmed in live virus system (fig. 1b & 1c) . since it has been reported that bacterially expressed y271a mutant has only 1% activity of wild type enzyme [9] , we asked whether the mutants would similarly affect rt activity in the viral particles. the results showed that the rt activity was basically undetectable in viral particles of these two mutants ( fig. 2a) . it was also found that the loss of rt activity in the viral particles might be attributed to the absence of rt in the viral particles rather than the incorporation of dysfunctional rt into the virions (fig. 2b) . after viral entry into the cells, the first step of hiv-1 replication is reverse transcription. our results thus suggested that replication of mutant viruses was abrogated, because the mutant rt did not exist in the virion. since hiv-1 rt is incorporated into the virion in the form of pr160 gag-pol , which is transported to cell membrane where the virus is packaged, through interaction with pr55 gag [27] [28] [29] [30] , we thus investigated whether the mutations would affect pr160 gag-pol expression and transportation. our results showed that pr160 gagpol was properly expressed and transported in cells transfected with mutant constructs, y271a and i271a (fig. 3) , indicating that these mutations did not affect either the production of the precursor or the interaction between pr160 gag-pol and pr55 gag . it was further found that the pr160 gag-pol was indeed incorporated into the virions of these two mutants, because except for rt, all other products of pr160 gag-pol including p24, protease and integrase [31, 32] , could be detected in the viral particles of mutants y271a and i271a (fig. 4a) . although it has been reported that the domain between residues 183 and 305 of rt is likely responsible for rt incorporation [33] , our study ruled out the involvement of rt was detected in wild type and mutant pseudoviruses generated in the presence and absence of efv, the most potent dimerization enhancer, by western blotting using mouse monoclonal anti-rt and anti-ca p24 antibodies, respectively. (b) purified wild type and mutant rt subunit p66 were analyzed by native gel electrophoresis followed by western blotting using mouse monoclonal anti-rt antibody in the presence or absence of b-mercaptoethanol (b-me). compared with wild type p66, which basically existed as homodimer, p66 subunit of the 271a and 274a mutants formed higher order oligomers, suggestive of conformational change. doi:10.1371/journal.pone.0006108.g005 the turn (gln 269 -arg 277 ). another report has suggested that two mutations in rt, l234d and w239a, led to premature cleavage of the gag-pol precursor and reduced levels of viral enzymes in the virions [34] . our results also excluded that the mutations of y271a and i271a affected the cleavage of the gag-pol precursor. by a viral protease inhibition assay using indinavir, we finally demonstrated that the rt mutants were degraded after incorporation of the precursor polyprotein into the virions and the degradation was associated with the activity of viral protease (fig. 4c) . our results indicated that the mutations in residues 271 and 274 would reduce the stability of rt inside the viral particles, rendering it susceptible to viral protease. post-incorporation degradation of rt has been reported previously. wapling et al. [35] have reported that mutations of w401l and w401a in rt can inhibit rt dimerization, resulting fig. 6a . a big side chain (blue) is found in naturally occurring residues but not in 271a. (c) structural models of naturally existing 274i, 274v and 274l residues as well as the lethal 274a mutation of p51 subunit. the same region was highlighted above in the right panel of fig. 6a . a big side chain (pink) is found in naturally occurring residues i, v and l, but not in 274a. doi:10.1371/journal.pone.0006108.g006 [36] . another mutation, l289k in p66 subunit, has also been reported to be able to abrogate dimerization [37] . another group, when they tried to generate infectious molecular clones of simian immunodeficiency virus, found that glutamic acid replacement at position 287 affected the stability of rt [38] . the biologically active and stable form of hiv-1 rt is a heterodimer consisting of p66 and p51, while the immediate precursor of p66/p51 heterodimer is the p66 homodimer. proteolytic removal of the rnase h domain in one of the p66 homodimer subunit by hiv-1 protease leads to the formation of stable heterodimer p66/p51 [39] . rt exists as an equilibrium mixture of monomers and dimers that include the p66/p51 heterodimer and the p66/p66 and p51/p51 homodimers, among which the heterodimer is the most stable and the p51/p51 homodimer is the most unstable [40] [41] [42] [43] . plausibly, the degradation of rt as reported in previous studies may be ascribed to the inhibition of rt p66 dimerization by the mutations. the mechanism of rt degradation in the virions observed in this study, however, may be different. our results showed that rt mutants were still undetectable in the viruses generated in the presence of a potent dimerization enhancer (fig. 5a ). our native gel analysis showed that the dimer form of rt mutant 271a was detected, although it was markedly reduced as compared to the wild type, while mutant 274a could not form dimer. instead, higher order oligomers of rt were detected in both mutants (fig. 5b ). it has been reported that higher order oligomerization may occur in hiv-1 rt [41, 44, 45] . our results have also indicated that y271a and i274a substitutions may change the conformation of rt, leading to oligomerization. the higher order oligomers formed in these 2 mutants, perhaps tetramer and octamer, may be associated with instability of rt mutants and their susceptibility to viral protease. furthermore, according to the locations of residues 271 and 274 (fig. 6a) , it is highly probable that alanine substitutions at 271 and/or 274 can affect the conformation of p51 thumb region and subsequently its interaction with rnase h domain of p66, resulting in the exposure of the 7-amino-acid p51-rnas h cleavage sequence in the p66 subunit [7, 46] and consequent proteolytic degradation by hiv-1 protease. the relative conservation of rt 271 and 274 residues and the loss of big side chains at these two sites in the mutants as shown in the structural models (fig. 6b & 6c ) also support their important roles. we tried to demonstrate this in vitro, by treating wild type and mutant rts with recombinant protease, but wild type rt, as well as mutant rts, could not be digested in vitro (data not shown). this result is consistent with that reported by abram and praniak [36] . this could be explained by different proteolytic stability of rt in vitro and in virions. taken together, our study showed that alanine substitutions at residue 271 or 274 of hiv-1 rt could cause conformational changes, rendering rt unstable and susceptible to viral protease. thus, it is demonstrated that the ''turn'' (gln 269 -arg 277 ) between two helices may be important in maintaining protein stability and formation of bioactive dimer. mutations in residues 271 and 274 of rt may inactivate the virus, which may enter cells but can not replicate. these findings may prove useful in anti-hiv drug design and vaccine development. considering the emergence of resistance to the current rt inhibitors, this turn, especially amino acid residues 271 and 274, may be an ideal new target for antiviral drug design. the agents targeting this region may be able to affect the stability or dimerization of hiv-1 rt and subsequently inhibit viral replication. moreover, the potential drug resistant mutations in this region, especially at residues 271 and 274, may probably inactivate the virus, which prevents the appearance of drugresistant virus. hiv reverse transcriptase structure-function relationships molecular targets for aids therapy expression of soluble, enzymatically active, human immunodeficiency virus reverse transcriptase in escherichia coli and analysis of mutants structure of unliganded hiv-1 reverse transcriptase at 2.7 a resolution: implications of conformational changes for polymerization and inhibition mechanisms crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded dna at 3.0 a resolution shows bent dna the structure of unliganded reverse transcriptase from the human immunodeficiency virus type 1 crystal structure at 3.5 a resolution of hiv-1 reverse transcriptase complexed with an inhibitor the 'helix clamp' in hiv-1 reverse transcriptase: a new nucleic acid binding motif common in nucleic acid polymerases structure/ function studies of human immunodeficiency virus type 1 reverse transcriptase. alanine scanning mutagenesis of an alpha-helix in the thumb subdomain role of the ''helix clamp'' in hiv-1 reverse transcriptase catalytic cycling as revealed by alanine-scanning mutagenesis reduced frameshift fidelity and processivity of hiv-1 reverse transcriptase mutants containing alanine substitutions in helix h of the thumb subdomain a minor groove binding track in reverse transcriptase effects of mutations in the polymerase domain on the polymerase, rnase h and strand transfer activities of human immunodeficiency virus type 1 reverse transcriptase residues in the alphah and alphai helices of the hiv-1 reverse transcriptase thumb subdomain required for the specificity of rnase h-catalyzed removal of the polypurine tract primer a putative new domain target for anti-hepatitis b virus: residues flanking hepatitis b virus reverse transcriptase residue 306 (rtp306) mutational analysis revealed that conservation of hepatitis b virus reverse transcriptase residue 306 (rtp306) is crucial for encapsidation of pregenomic rna mutational analysis of the ''turn'' of helix clamp motif of hiv-1 reverse transcriptase efficacy and safety analyses of a recombinant human immunodeficiency virus type 1 derived vector system changes in growth properties on passage in tissue culture of viruses derived from infectious molecular clones of hiv-1lai, hiv-1mal, and hiv-1eli novel single-celllevel phenotypic assay for residual drug susceptibility and reduced replication capacity of drug-resistant human immunodeficiency virus type 1 indicator cell lines for detection of primary strains of human and simian immunodeficiency viruses metabolism and anti-human immunodeficiency virus-1 activity of 2-halo-29,39-dideoxyadenosine derivatives infection of htlv-iii/lav in htlv-i-carrying cells mt-2 and mt-4 and application in a plaque assay severe acute respiratory syndrome-associated coronavirus 3a protein forms an ion channel and modulates virus release immunogenicity in mice of tandem repeats of an epitope from herpes simplex gd protein when expressed by recombinant adenovirus vectors nonnucleoside reverse transcriptase inhibitors are chemical enhancers of dimerization of the hiv type 1 reverse transcriptase rapid localization of gag/ gagpol complexes to detergent-resistant membrane during the assembly of human immunodeficiency virus type 1 the nonmyristylated pr160gag-pol polyprotein of human immunodeficiency virus type 1 interacts with pr55gag and is incorporated into viruslike particles requirements for incorporation of pr160gag-pol from human immunodeficiency virus type 1 into virus-like particles characterization of deletion mutations in the capsid region of human immunodeficiency virus type 1 that affect particle formation and gag-pol precursor incorporation characterization of ribosomal frameshifting in hiv-1 gag-pol expression hiv expression strategies: ribosomal frameshifting is directed by a short sequence in both mammalian and yeast systems incorporation of human immunodeficiency virus type 1 reverse transcriptase into virus-like particles mutations in the primer grip of human immunodeficiency virus type 1 reverse transcriptase impair proviral dna synthesis and virion maturation mutations that abrogate human immunodeficiency virus type 1 reverse transcriptase dimerization affect maturation of the reverse transcriptase heterodimer virion instability of human immunodeficiency virus type 1 reverse transcriptase (rt) mutated in the protease cleavage site between rt p51 and the rt rnase h domain structure/function studies of hiv-1(1) reverse transcriptase: dimerizationdefective mutant l289k generation of infectious molecular clones of simian immunodeficiency virus from fecal consensus sequences of wild chimpanzees the packaging and maturation of the hiv-1 pol proteins conformational stability of dimeric hiv-1 and hiv-2 reverse transcriptases human immunodeficiency virus-1 reverse transcriptase heterodimer stability dimerization of human immunodeficiency virus type 1 reverse transcriptase. a target for chemotherapeutic intervention effects of efavirenz binding on the subunit equilibria of hiv-1 reverse transcriptase a model for reverse transcription by a dimeric enzyme chemical crosslinking of the subunits of hiv-1 reverse transcriptase structural basis of asymmetry in the human immunodeficiency virus type 1 reverse transcriptase heterodimer the following reagents were obtained through the aids research and reference reagent program, division of aids, niaid, nih: plai. conceived and designed the experiments: hjz yxw ymw bjz. performed the experiments: hjz yxw hw. analyzed the data: hjz yxw hw dyj ymw bjz. wrote the paper: hjz dyj ymw bjz. key: cord-000408-pt3b4yc7 authors: lu, sydney x.; kappel, lucy w.; charbonneau-allard, anne-marie; atallah, renée; holland, amanda m.; turbide, claire; hubbard, vanessa m.; rotolo, jimmy a.; smith, marsinay; suh, david; king, christopher; rao, uttam k.; yim, nury; bautista, johanne l.; jenq, robert r.; penack, olaf; na, il-kang; liu, chen; murphy, george; alpdogan, onder; blumberg, richard s.; macian, fernando; holmes, kathryn v.; beauchemin, nicole; van den brink, marcel r. m. title: ceacam1 separates graft-versus-host-disease from graft-versus-tumor activity after experimental allogeneic bone marrow transplantation date: 2011-07-06 journal: plos one doi: 10.1371/journal.pone.0021611 sha: doc_id: 408 cord_uid: pt3b4yc7 background: allogeneic bone marrow transplantation (allo-bmt) is a potentially curative therapy for a variety of hematologic diseases, but benefits, including graft-versus-tumor (gvt) activity are limited by graft-versus-host-disease (gvhd). carcinoembryonic antigen related cell adhesion molecule 1 (ceacam1) is a transmembrane glycoprotein found on epithelium, t cells, and many tumors. it regulates a variety of physiologic and pathological processes such as tumor biology, leukocyte activation, and energy homeostasis. previous studies suggest that ceacam1 negatively regulates inflammation in inflammatory bowel disease models. methods: we studied ceacam1 as a regulator of gvhd and gvt after allogeneic bone marrow transplantation (allo-bmt) in mouse models. in vivo, ceacam1(−/−) t cells caused increased gvhd mortality and gvhd of the colon, and greater numbers of donor t cells were positive for activation markers (cd25(hi), cd62l(lo)). additionally, ceacam1(−/−) cd8 t cells had greater expression of the gut-trafficking integrin α(4)β(7), though both cd4 and cd8 t cells were found increased numbers in the gut post-transplant. ceacam1(−/−) recipients also experienced increased gvhd mortality and gvhd of the colon, and alloreactive t cells displayed increased activation. additionally, ceacam1(−/−) mice had increased mortality and decreased numbers of regenerating small intestinal crypts upon radiation exposure. conversely, ceacam1-overexpressing t cells caused attenuated target-organ and systemic gvhd, which correlated with decreased donor t cell numbers in target tissues, and mortality. finally, graft-versus-tumor survival in a ceacam1(+) lymphoma model was improved in animals receiving ceacam1(−/−) vs. control t cells. conclusions: we conclude that ceacam1 regulates t cell activation, gvhd target organ damage, and numbers of donor t cells in lymphoid organs and gvhd target tissues. in recipients of allo-bmt, ceacam1 may also regulate tissue radiosensitivity. because of its expression on both the donor graft and host tissues, this suggests that targeting ceacam1 may represent a potent strategy for the regulation of gvhd and gvt after allogeneic transplantation. ceacam1 is a member of a large family of carcinoembryonic antigen proteins [2] . it is primarily a type i transmembrane protein with multiple splice variants [9] , though soluble forms also exist. ceacam1 is widely expressed on a variety of tissues including endothelium [10] , epithelium [11] , hematopoietic cells [12] and both hematologic and solid tumors, and interacts in a homophilic and heterophilic fashion with physiological and pathogenassociated ligands, including carcinoembryonic antigen and the neisseria spp. proteins [13] . some ceacam1 isoforms contain intracellular itim motifs, and activation of ceacam1 results in the recruitment of the shp-1 and shp-2 phosphatases [8, 14] , which dephosphorylate substrates across a range of signaling pathways. ceacam1 thus inhibits t cell receptor (tcr) signaling and suppresses multiple aspects of t cell function. ceacam1 agonists attenuate cytokine secretion, t cell polarization and cytolytic function. in vivo, ligation of ceacam1 with soluble ligands or over-expression of itim-containing ceacam1 isoforms on t cells attenuates experimental colitis [7, 8] . additionally, ceacam1 is also expressed on intestinal t cells in patients with celiac disease [15] and ulcerative colitis [16] , and may represent an attempt by the immune system to negatively regulate these inflammatory processes. in addition to immune regulation, ceacam1 exerts a wide variety of other biological functions. it is a cell-cell adhesion molecule [17, 18] , and a receptor for a variety of commensal and pathogenic microbes in mouse and man [17, 19, 20, 21] . ceacam1 also regulates angiogenesis [6] , energy homeostasis [22] , and tumor biology [23, 24, 25] . ceacam1 regulates the tumorigenesis of colon cancers, and is a prognostic factor in lung adenocarcinoma. tumor expression of ceacam1 may regulate tumor angiogenesis and invasion, and the expression of both ceacam1 and cea by tumors may inhibit the functions of tumor infiltrating lymphocytes. allo-bmt is an established therapy with curative intent for a variety of hematologic malignancies and non-malignant conditions [26] . alloreactive t cells of donor origin play a criticial role in both gvhd, a major complication of allo-bmt, and graft-versustumor activity, a major contributor to the efficacy of allo-bmt as a cancer therapy. donor-recipient antigenic disparity, donor t cells, and tissue injury resulting in inflammation due to the conditioning regimen all contribute to gvhd, which primarily affects intestines, liver, skin and thymus [27] . ceacam1 is expressed both on leukocytes (especially t cells), as well as on epithelial and endothelial cells, which are prominent components of the parenchyma of the above-mentioned gvhd target organs. in addition, ceacam1 is upregulated on many tumors. in this report, we assess the impact of ceacam1 on alloreactive t cells in the donor allograft, as well as the effects of ceacam1 deficiency on recipients of allo-bmt with respect to gvhd and gvt activity. we assessed ceacam1 regulation of ghvd on donor t cells or recipients in two well-described major histocompatibility complex (mhc) class i/ii-disparate models c57bl/6 (b6, h-2 b )rbalb/ c (h-2 d ) and balb/crb10.br (h-2 k ). we used ceacam1 2/2 b6 mice [28] , ceacam1-transgenic (tg) b6 mice (described in figure s1 ), and ceacam1 2/2 balb/c mice [28] as the source of donor t cells or recipients. in all experiments, recipients received splitdose lethal irradiation (balb/c: 8.5 gy, b10.br: 11 gy) and a graft of 5610 6 allogeneic t cell depleted bone marrow (tcd-bm) of wildtype (wt) origin, with or without splenic t cells. we first transplanted irradiated balb/c mice with b6 tcd-bm with wt or ceacam1 2/2 t cells, and observed that recipients of ceacam1 2/2 t cells had significantly increased mortality compared to recipients of wt t cells ( figure 1a , left). we confirmed this in a second mhc-disparate allo-bmt model, balb/crb10.br ( figure 1a, right) . we next asked whether t cells overexpressing ceacam1 would cause less disease, and transplanted balb/c recipients with 0.5610 6 or 1610 6 donor wt or ceacam 1-tg t cells. at both doses, recipients of ceacam1-tg t cells showed attenuated mortality ( figure 1b) . finally, we assessed the role of ceacam1 on tissues of allo-bmt recipients, and transferred tcd-bm+t cells into wt vs. cea-cam1 2/2 balb/c recipients. this revealed that ceacam1 2/2 recipients had increased early (but not overall) mortality, with nearly 50% of mice succumbing within the first week ( figure 1c ). we next asked whether ceacam1 regulated gvhd target organ damage, and again assessed effects of ceacam1 deficiency or overexpression on donor t cells, and ceacam1 2/2 allo-bmt recipients. we observed that recipients of ceacam1 2/2 t cells had more severe large intestinal gvhd (figure 2a) . surprisingly however, these mice exhibited less thymic gvhd, as determined by thymic cellularity and numbers of cd4 + cd8 + double-positive (dp) thymocytes ( figure 2 ). thymic cellularity from age/sex-matched non-transplanted animals are shown in figure s2 . we also observed a modest trend towards less skin gvhd in recipients of ceacam1 2/2 t cells ( figure 2c ), suggesting that ceacam1 2/2 t cells caused preferential damage to the (large) intestines. in experiments comparing recipients of wt and ceacam1-tg t cells on day 21 post-transplant, we found that recipients of ceacam1-tg t cells demonstrated significantly less gvhd of the liver, intestines, and thymus compared to recipients of wt t cells, but similar skin gvhd ( figures 2d-f ). this appears to suggest that ceacam1-tg t cells caused less gvhd overall, with relatively little organ specificity. finally, we assessed ceacam1 2/2 allo-bmt recipients on day 14 post-transplant. in correspondence with increased early gvhd mortality, ceacam1 2/2 allo-bmt recipients showed increased large bowel damage and thymic gvhd ( figure 2g -h). we next assessed the numbers of donor cd4 and cd8 effector t cells after transfer of ceacam1 2/2 or ceacam1-tg t cellcontaining allografts, or in ceacam1 2/2 allo-bmt recipients. comparing recipients of wt t cells with those receiving ceacam1 2/2 t cells, we observed increased numbers of ceacam1 2/2 donor alloactivated effector t cells in the spleen, mln, and iel of allo-bmt recipients ( figure 3a) , which was associated with a concomitant decrease in the number of ceacam1 2/2 alloactivated cd4 and cd8 t cells in the pln and liver. when we analyzed organs of recipients of allografts containing wt vs. ceacam1-tg t cells, we noted decreased numbers of donor effector t cells in the mln, pln, and liver ( figure 3b ). via histopathological analysis, we also observed decreased numbers of total lymphocytic infiltrates into the liver, small and large bowels by histopathology ( figure 3b ), as well as decreased neutrophilic infiltrates in these organs (data not shown). finally, we assessed infiltrating t cells in wt and ceacam1 2/2 allo-bmt recipients, and observed increased numbers of donor alloactivated effector t cells in the mln and iel of ceacam1 2/2 allo-bmt recipients, but decreased numbers of these cells in the pln and liver ( figure 3c ). ceacam1 regulates the sensitivity of the small intestine to radiation injury the accelerated early mortality of ceacam1 2/2 allo-bmt recipients, together with increased accumulation of donor t cells in gi tract and mesenteric lymph nodes, but decreased numbers peripheral lymph nodes ( figure 3c ), led us to ask whether ceacam1 had differential effects in regulating gvhd target organ damage for various target organs and tissues. in the context of ceacam1 2/2 recipients, we therefore tested the radiation sensitivity of ceacam1 2/2 mice used as hosts, by irradiating wt and ceacam1 2/2 balb/c mice and assessing survival. ceacam1 2/2 animals showed increased kinetics of mortality, and in some cases, overall mortality after radiation injury ( figure 4a ). similar results were obtained on the b6 background ( figure 4c ). we then enumerated regenerating and surviving crypts in the small intestine (terminal ileum) at 84 hours after irradiation to assess intestinal radiation damage, and observed that ceacam1 2/2 mice had fewer regenerating and surviving crypts as compared with wt counterparts ( figure 4b and d), indicating greater damage to the small intestine across a wide range of radiation doses. it is thus quite likely that our radiation-containing conditioning regimen for transplant recipients also contributes in part to their survival kinetics, selective gvhd target organ damage ( figure 2g -h), and the selective accumulation of donor t cells in lymphoid tissues and target organs ( figure 3c ) in these recipients. donor ceacam1 2/2 cd8 t cells express higher levels of integrin a 4 b 7 post-transplant next, we studied a variety of possible mechanisms by which ceacam1 may regulate donor t cell function. we analyzed donor wt and ceacam1 2/2 alloactivated splenic t cells on day 14 after allo-bmt for trafficking molecules, and found that ceacam1 2/2 cd8 + cd44 + cd62l 2 effector t cells expressed higher levels of integrin b 7 subunit and the gut homing integrin a 4 b 7 ( figure 5a) , which is important for intestinal gvhd [31, 32, 33] . however, wt vs. ceacam12/2 cd4 effector t cells had similar integrin b 7 subunit expression, yet also accumulated in greater numbers in the gut ( figure 3a) , suggesting that regulation of target organ damage by ceacam1 is very likely to involve multiple additional mechanisms beyond trafficking molecule expression. additionally, levels of the a e subunit, which forms integrin a e b 7 , were similar, as were levels of ccr9, cd31, psgl1, ccr7, cxcr3, and lfa1 (data not shown). when we assessed the expression of trafficking molecules in recipients of wt vs. ceacam1-tg t cell allografts, we found no significant differences in levels of b 7 subunit, integrin a 4 b 7 ( figure 5b ), or any other molecules. this is consistent with the systemic reduction in gvhd in recipients of ceacam1-tg t cells. finally, we assessed trafficking molecules in irradiated wt vs. ceacam1 2/2 recipients of identical donor allografts, and observed again that donor cd8, but not cd4 splenic t cells in ceacam1 2/2 ceacam1 can be found on activated t cells [7, 8, 29 ] and, we thus performed a kinetic analysis of ceacam1 expression on t cells during alloactivation. we adoptively transferred cfse-labeled b6 t cells into irradiated balb/c recipients, and observed transient expression only on day 2 after alloactivation ( figure 6 and data not shown). furthermore, only cfse lo alloactivated t cells, which have divided $4 times in 48 hours, expressed low but consistently detectable levels of ceacam1 ( figure 6a ). these kinetics are consistent with a role for ceacam1 in regulating early events in t cell alloactivation. because the expression of ceacam1 on alloreactive t cells after adoptive transfer occurred in vivo with similar kinetics as t cell alloactivation [30] , we asked whether ceacam1 on either donor alloreactive t cells or radio-resistant cells in allo-bmt recipients could regulate this process. we transferred cfse-labeled purified b6 wt or ceacam1 2/2 splenic t cells into irradiated balb/c recipients and analyzed donor t cells in spleens on day 3. we observed that relative to isotype control staining, an increased percentage of alloactivated cfse lo cd4 ceacam1 2/2 t cells were positive for the alloactivation marker cd25, and that a greater percentage of these cells downregulated cd62l than wt t cells ( figure 6b-c) , suggesting that more of them became activated. additionally, an increased percentage of donor ceacam1 2/2 cd4 t cells had divided to a cfse lo alloactivated state ( figure 6d) , suggesting enhanced proliferation in the absence of ceacam1. we repeated these experiments with alloreactive ceacam1-tg t cells and as expected, observed a decrease in numbers of cfse lo t cells as assessed by cfse dilution ( figure 6e ). this is consistent with an inhibitory role for ceacam1 in the proliferation of alloreactive t cells. however, we did not observe significant differences in alloactivation between ceacam1-tg vs. wt donor t cells (data not shown). lastly, we assessed the role of ceacam1 expression on radioresistant cells in allo-bmt recipients for donor t cell alloactivation. we transferred cfse-labeled b6 t cells into irradiated wt vs. ceacam1 2/2 balb/c mice, and analyzed donor t cells in spleens on day 3. here, we did not observe differences in proliferation (data not shown), but donor cd4 t cells in ceacam1 2/2 allogeneic recipients did exhibit an increase in alloactivation as measured by cd25 ( figure 6f ). we measured serum cytokines in recipients of wt, ceacam1-tg and ceacam1 2/2 t cells on days 7 and 14 post-transplant, and observed that levels of ifnc, tnf, il-2, il-4, il-6, il-10, and il-12p70 were similar (data not shown). percentages of foxp3 + donor regulatory t cells and expression of t-bet were also similar between recipients of wt, ceacam1-tg and ceacam1 2/2 t cells (data not shown and table 1 ), and stimulation of splenocytes harvested on day 14 after bmt post-transplant from these three groups revealed essentially no il-17 + donor t cells (not shown), and similar percentages of donor ifnc + t cells (data not shown and table 1 ). as ceacam1 can regulate the cytolytic responses of lymphocytes [34, 35, 36, 37, 38] , we assessed the cytolytic function of wt vs. ceacam1 2/2 alloactivated cd8 t cells from the spleens of allo-bmt recipients on day 14. ceacam1 2/2 cd8 t cells and wt cd8 t cells demonstrated similar cytolysis against 51 cr-radiolabeled allogeneic a20 b cell lymphoma cells and el4 controls ( table 1) . lastly, we found no differences in dc numbers, activation state (cd80, cd86, mhc class ii) from the infusion of ceacam1 2/2 or ceacam1-tg t cells (table 1) , or in ceacam1 2/2 allo-bmt recipients. finally, we assessed the gvt activity of ceacam1 2/2 donor alloreactive t cells against a20 lymphoma and renca renal cell carcinoma. recipients of ceacam1 2/2 donor t cells had improved survival in the a20 lymphoma model ( figure 7a) , but both t cell replete groups showed comparable survival in the renca solid tumor model ( figure 7b ). when we analyzed these two tumor lines for ceacam1 expression, we noted that all a20 lymphoma cells uniformly expressed high levels, while only a subset of renca cells expressed some ceacam1 ( figure 7c ). in this report, we show that ceacam1, which is found on both donor alloreactive t cells as well as non-hematopoietic tissues such as gastrointestinal and hepatic epithelium, can regulate both donor t cell function and the sensitivity of allo-bmt recipients to radiation-containing preparative regimens. in addition, ceacam1 on donor t cells and tumors may modulate gvt activity. ceacam1 on both the donor allograft and recipient tissues thus appears to represent an important regulator of gvhd and gvt morbidity and mortality via both t cell dependent and independent mechanisms, suggesting that therapeutic approaches which modulate ceacam1 may need to assess and balance gvhd vs. gvt. ceacam1 on t cells has previously been shown to restrain cd4 t cell polarization, cytokine secretion and cytotoxicity. in our gvhd model systems however, we found similar t cell polarization and cytokine secretion when we analyzed donor alloreactive t cells ex vivo ( table 1) . we ascribe this to the strongly proinflammatory cytokine milieu found in recipients following myeloablative radiation treatment, as well as the ubiquitous presence of alloantigen, which together promote strong th1 differentiation regardless of ceacam1 expression. however, in our model systems ceacam1 regulated t cell activation, and numbers of donor alloactivated t cells in both lymphoid tissues and gvhd target tissues, in patterns that generally correlated with levels target organ damage (figures 2 and 3) . we also assessed the role of ceacam1 in allo-bmt recipients. in our model systems, wt t cells in a ceacam1-deficient environment showed a phenotype similar to that of ceacam1 2/2 alloactivated t cells: both showed increased activation, selective damage to the large intestines, and preferential accumulation in the mln and intestinal parenchyma of mice with gvhd, and correspondingly decreased infiltration of the liver and pln, ultimately leading to exacerbation of disease, with accelerated mortality in the first two weeks post-transplant. this suggests that ceacam1 on donor t cells interacts with recipient tissues, and that ceacam1 ''fraternal'' interactions between cells of the donor graft, were not sufficient to restrain gvhd in ceacam1 2/2 recipients. however, the increased early mortality of ceacam1 2/2 allo-bmt recipients with gvhd also led us to ask whether ceacam1 2/2 mice were sensitive to radiation injury. while ceacam1 2/2 mice were not significantly defective for hematopoiesis after sublethal irradiation at 3.5 and 4.5 gy (data not shown), they did exhibit significantly increased damage to the small intestines after lethal irradiation ( figure 4) . ceacam1 also directly regulates intestinal epithelia. due to enhanced wnt/b-catenin signaling, ceacam1 2/2 jejunal and ileal enterocytes exhibit higher levels of the positive cell cycle regulators c-myc and cyclin d1 [41] . dysregulated c-myc may sensitize cells to apoptosis [42, 43] , and higher levels of these proteins may render ceacam1 2/2 enterocytes more sensitive to radiation injury. finally, ceacam1 also regulates cell-cell adhesion [17, 18, 44, 45] under normal and pathological conditions; it may therefore also be possible that loss of ceacam1 regulates radiationinduced sloughing of intestinal epithelium. it is difficult to directly assess the relative importance of gastrointestinal radiation sensitivity versus increased gvhd in ceacam1 2/2 allo-bmt recipients, as radiation-induced gut damage may both be directly manifested in intestinal pathology, yet transmural migration of bacterial superantigens is an important first step for the initiation of gvhd [1, 39, 40] , and increased damage to the intestines of ceacam1 2/2 mice may thus amplify the development of gvhd in these mice, and also explain in part the specifically increased large intestinal gvhd we observed. in experiments with ceacam1 2/2 donor t cells, we also observed a trend for splenic donor cd8 alloactivated t cells to express higher levels of a 4 b 7 . although integrin a 4 b 7 is important for gvhd pathogenesis, and we have previously shown that b 7 2/2 t cells cause a sustained decrease in acute systemic and intestinal gvhd [31] , differential expression of integrin a 4 b 7 by ceacam1 2/2 t cells is almost certainly only one part of how ceacam1 regulates target organ gvhd. indeed, donor alloactivated cd4 t cells expressed comparable levels of integrin a 4 b 7 as wildtype cells, yet were also found in increased numbers in the gut ( figure 3a ). this suggests that other mechanisms, such as ceacam1 regulation of donor t cell activation ( figure 6 ) may also contribute to its regulation of gvhd target organ damage. moreover, recipients of ceacam1-tg t cells also had reduced intestinal infiltrates despite similar integrin a 4 b 7 expression, suggesting that ceacam1 regulates the accumulation of donor t cells in target tissues via multiple mechanisms. thus, our results on donor lymphocyte infiltrates into gvhd target tissues and secondary lymphoid tissues must be interpreted cautiously, as they must be influenced by t cell proliferation, retention and apoptosis, in addition to trafficking. although ceacam1 2/2 and ceacam1-tg t cells displayed overall symmetric and opposite phenotypes, we also noted differences. ceacam1-tg t cells primarily showed decreased proliferation, whereas ceacam1 2/2 t cells showed changes in proliferation, but also trafficking and activation. some of these differences may be due to our models: on wt t cells, ceacam1 is only briefly and transiently upregulated during activation. consequently, ceacam1 2/2 t cells are ''missing'' ceacam1 only transiently, while ceacam1-tg t cells constitutively over-express ceacam1. furthermore, while ceacam1 2/2 t cells are effectively insensitive to all ceacam1 ligands and interactions, ceacam1-tg t cells which over-express the protein may have increased fraternal ceacam1 interactions with other donor t cells, but may not necessarily experience increased ceacam1 interactions with donor bm or host hematopoietic and non-hematopoietic components. these differences may explain why their activation and trafficking phenotypes are not directly opposed. we were interested to note that in our gvt experiments, recipients of ceacam1 2/2 t cells had significantly improved survival when challenged with a20 lymphoma but not renal cell carcinoma. although both a20 lymphoma and renal cell carcinoma express ceacam1, a20 cells uniformly expressed ceacam1 at high levels, while only a subset of renca cells showed (somewhat lower) expression. indeed, a number of hematologic tumors, including el4 leukemia, p815 mastocytoma, and c1498 myeloid leukemia all express substantial levels of ceacam1 (data not shown), whereas some solid tumors, such as mouse 4t1 breast epithelial cancer and ct51 colon tumor normally express only lower or even minimal levels of ceacam1, similar to the lower level of expression we found with renca (not shown). therefore, one possibility is that the gvt activity of t cells can be negatively regulated by tumors expressing high levels of ceacam1, but is less important for tumors that express low levels or only on a subset of cells in the first place. however, renca in our gvt model systems is found primarily in the liver, and to a lesser extent, the lungs. since donor allografts with ceacam1 2/2 t cells showed decreased numbers of donor alloreactive t cells in the liver as compared with wildype in gvhd experiments ( figure 3a) , interpretation of gvt activity against renca with respect to ceacam1 on t cells must also consider this aspect of its biology. in conclusion, our results show that ceacam1 on both donor t cells and allo-bmt recipients controls the proliferation, activation, and trafficking of donor alloreactive t cells, and the sensitivity of gastrointestinal tissues to irradiation. consequently, ceacam1 may represent a viable target for reducing radiation-associated gastrointestinal toxicity, for the control of gvhd and gvt activity after allo-bmt. all animal protocols were approved by the memorial sloan-kettering cancer center (mskcc) institutional animal care and use committee (protocol #99-07-025). mice c57bl/6 (b6, h-2 b ), balb/c (h-2 d ), and b10.br (h-2 k ) mice were obtained from the jackson laboratory (bar harbor, me). b6 and balb/c ceacam1 2/2 mice, and b6 ceacam1-tg mice were generated at mcgill university (b6 and balb/c from harlan (montreal, quebec, canada)), and maintained at memorial sloan-kettering cancer center. mice used were between 8 and 12 weeks old. bm cells removed from femurs and tibias were t cell-depleted (tcd) with anti-thy-1.2 and low-tox-m rabbit complement (cedarlane laboratories, hornby, on, canada). enriched splenic t cells were obtained by nylon wool column passage. cells were resuspended in dmem and injected into lethally irradiated recipients on day 0 after total body irradiation ( 137 cs source) as a split dose 3 hours apart. purified splenic t cells were incubated with cfse (invitrogen, carlsbad, ca) at a concentration of 2.5-5 mm in pbs (5610 7 cells/ml) at 37uc for 20 minutes, washed twice with pbs, resuspended in dmem and infused intravenously into lethally irradiated allogeneic recipients. splenocytes from recipients were harvested at varying time points and analyzed by facs as described. survival was monitored daily, and mice were scored weekly for 5 clinical parameters (weight, posture, activity level, fur ruffling, and skin lesions) on a scale from 0 to 2. a clinical gvhd score was generated by summation of the 5 criteria scores; mice scoring 5 or greater were considered moribund and euthanized. small and large bowel, liver, and skin were assessed by experts in a blinded fashion. organs were preserved in formalin, transferred to 70% ethanol, and then embedded in paraffin, sectioned, stained with hematoxylin and eosin, and scored with a semi-quantitative scoring system. bowel and liver were scored for 19 to 22 different parameters associated with gvhd (detailed in table s1 ); skin was evaluated for number of apoptotic cells/mm 2 of epidermis via terminal deoxynucleotide transferase dutp nick end labeling (tunel). histopathologic scores, median fluorescence intensities and cell counts were compared between groups using the nonparametric unpaired mann-whitney u test; the mantel-cox log-rank test was used for survival data. additional methods are described in methods s1. figure s1 generation of ceacam-1 transgenic mice and expression of ceacam1 on transgenic t cells. a) the cc1-4l cdna, expressing 4 ig domains and the long cytoplasmic domain was inserted into the unique ecor1 site within the vahcd2 vector containing the hcd2 promoter and 2 polyadenylation sites (polya1,2) . b) the linearized construct was microinjected into c57bl/6 oocytes to produce transgenic mice that were identified by southern blot with a 1.3 kb 32p-labelled probe. this probe cross-reacts with the endogenous ceacam1, ceacam2 and ceacam10 genes and also identifies the 1.7 kb ecor1-digested transgene. c) tg mice were identified by pcr amplification of a 320 bp fragment from tail genomic dna with the cyt2 oligo within the cc1-l cytoplasmic domain and oligo cd2a2 within the hcd2 lcr region. d) western blots of lysates from thymi and spleens from 5 and 8-week-old wt and tg littermates with the rabbit polyclonal anti-ceacam1 ab 2457. e-f) cell surface ceacam1 on thymic (e) and splenic (f) cd3-gated t cells of wt and tg mice (n = 4) was revealed with anti-ceacam1 ab 2457. controls were normal rabbit serum. (tiff) figure s2 . ceacam1 2/2 mice exhibit increased thymic and splenic cellularity compared with wildtype animals, but do not exhibit skewing towards particular leukocyte lineages or subsets, while ceacam1-tg transgenic mice have similar numbers of splenocytes, thymocytes, and bone marrow cells compared with wt animals. a) 2 month old ceacam1 2/2 male balb/c mice were analyzed with age and sex-matched wildtype balb/c, n = 5. similar results were observed with age and sex-matched female wildtype and ceacam1 2/2 balb/c mice (n = 5, not shown), and in age and sex-matched wildtype and ceacam1 2/2 b6 mice (n = 5, not shown). b) thymocytes from mice in (a) were analyzed by flow cytometry. n = 5. c) 8-week old b6 and ceacam1-tg mice were analyzed for splenic, thymic and bone marrow (bm) cellularity. n = 3/group. (tiff) table s1 histopathological scoring scheme for gastrointestinal gvhd target organs. methods s1 (doc) pathophysiology of graft-versus-host disease ceacam1: contact-dependent control of immunity ceacam1 modulates epidermal growth factor receptor-mediated cell proliferation expression of the adhesion molecule ceacam1 (cd66a, bgp, c-cam) in breast cancer is associated with the expression of the tumor-suppressor genes rb, rb2, and p27 cd66 expression in acute leukaemia cea-related cell adhesion molecule 1: a potent angiogenic factor and a major effector of vascular endothelial growth factor specific regulation of t helper cell 1-mediated murine colitis by ceacam1 shp1 phosphatasedependent t cell inhibition by ceacam1 adhesion molecule isoforms redefined nomenclature for members of the carcinoembryonic antigen family cd66 nonspecific cross-reacting antigens are involved in neutrophil adherence to cytokine-activated endothelial cells cd66a (bgp), an adhesion molecule of the carcinoembryonic antigen family, is expressed in epithelium, endothelium, and myeloid cells in a wide range of normal human tissues carcinoembryonic antigen-related cell adhesion molecule 1 expression and signaling in human, mouse, and rat leukocytes: evidence for replacement of the short cytoplasmic domain isoform by glycosylphosphatidylinositol-linked proteins in human leukocytes the putative role of members of the cea-gene family (cea, nca an bgp) as ligands for the bacterial colonization of different human epithelial tissues the carboxylterminal region of biliary glycoprotein controls its tyrosine phosphorylation and association with protein-tyrosine phosphatases shp-1 and shp-2 in epithelial cells regulation of human intestinal intraepithelial lymphocyte cytolytic function by biliary glycoprotein (cd66a) locally inducible cd66a (ceacam1) as an amplifier of the human intestinal t cell response biliary glycoprotein, a member of the immunoglobulin supergene family, functions in vitro as a ca2(+)-dependent intercellular adhesion molecule intercellular adhesion of rat hepatocytes. identification of a cell surface glycoprotein involved in the initial adhesion process differential opa specificities for cd66 receptors influence tissue interactions and cellular response to neisseria gonorrhoeae interaction of mouse hepatitis virus (mhv) spike glycoprotein with receptor glycoprotein mhvr is required for infection with an mhv strain that expresses the hemagglutinin-esterase glycoprotein tumor suppressive role of an androgen-regulated epithelial cell adhesion molecule (c-cam) in prostate carcinoma cell revealed by sense and antisense approaches mechanism of glucose intolerance in mice with dominant-negative mutation of ceacam1 expression of ceacam1 in adenocarcinoma of the lung: a factor of independent prognostic significance the human tumor suppressor ceacam1 modulates apoptosis and is implicated in early colorectal tumorigenesis the tumour suppressor gene ceacam1 is completely but reversibly downregulated in renal cell carcinoma pathophysiologic mechanisms of acute graft-vs.-host disease the primacy of the gastrointestinal tract as a target organ of acute graft-versus-host disease: rationale for the use of cytokine shields in allogeneic bone marrow transplantation ceacam1a2/2 mice are completely resistant to infection by murine coronavirus mouse hepatitis virus a59 activation-induced expression of carcinoembryonic antigen-cell adhesion molecule 1 regulates mouse t lymphocyte function in vivo analyses of early events in acute graft-versus-host disease reveal sequential infiltration of t-cell subsets absence of beta7 integrin results in less graft-versus-host disease because of decreased homing of alloreactive t cells to intestine l-selectin and beta7 integrin on donor cd4 t cells are required for the early migration to host mesenteric lymph nodes and acute colitis of graft-versus-host disease lpam (alpha 4 beta 7 integrin) is an important homing integrin on alloreactive t cells in the development of intestinal graft-versus-host disease the mechanisms controlling nk cell autoreactivity in tap2-deficient patients the critical role of residues 43r and 44q of carcinoembryonic antigen cell adhesion molecules-1 in the protection from killing by human nk cells biological function of the soluble ceacam1 protein and implications in tap2-deficient patients pivotal role of ceacam1 protein in the inhibition of activated decidual lymphocyte functions cd66a interactions between human melanoma and nk cells: a novel class i mhcindependent inhibitory mechanism of cytotoxicity pathophysiology of acute graft-versus-host disease immunobiology of acute graft-versus-host disease intestinal tumor progression is promoted by decreased apoptosis and dysregulated wnt signaling in ceacam12/2 mice the influence of the oncogenes nras and myc on the radiation sensitivity of cells of a human melanoma cell line induction of apoptosis by tumor suppressor genes and oncogenes the cell adhesion molecule c-cam is a substrate for tissue transglutaminase cea adhesion molecules: multifunctional proteins with signalregulatory properties the authors thank the staff of the memorial sloan-kettering cancer center research animal resources center for excellent animal care. key: cord-000556-uu1oz2ei authors: kumar, ranjit; lawrence, mark l.; watt, james; cooksey, amanda m.; burgess, shane c.; nanduri, bindu title: rna-seq based transcriptional map of bovine respiratory disease pathogen “histophilus somni 2336” date: 2012-01-20 journal: plos one doi: 10.1371/journal.pone.0029435 sha: doc_id: 556 cord_uid: uu1oz2ei genome structural annotation, i.e., identification and demarcation of the boundaries for all the functional elements in a genome (e.g., genes, non-coding rnas, proteins and regulatory elements), is a prerequisite for systems level analysis. current genome annotation programs do not identify all of the functional elements of the genome, especially small non-coding rnas (srnas). whole genome transcriptome analysis is a complementary method to identify “novel” genes, small rnas, regulatory regions, and operon structures, thus improving the structural annotation in bacteria. in particular, the identification of non-coding rnas has revealed their widespread occurrence and functional importance in gene regulation, stress and virulence. however, very little is known about non-coding transcripts in histophilus somni, one of the causative agents of bovine respiratory disease (brd) as well as bovine infertility, abortion, septicemia, arthritis, myocarditis, and thrombotic meningoencephalitis. in this study, we report a single nucleotide resolution transcriptome map of h. somni strain 2336 using rna-seq method. the rna-seq based transcriptome map identified 94 srnas in the h. somni genome of which 82 srnas were never predicted or reported in earlier studies. we also identified 38 novel potential protein coding open reading frames that were absent in the current genome annotation. the transcriptome map allowed the identification of 278 operon (total 730 genes) structures in the genome. when compared with the genome sequence of a non-virulent strain 129pt, a disproportionate number of srnas (∼30%) were located in genomic region unique to strain 2336 (∼18% of the total genome). this observation suggests that a number of the newly identified srnas in strain 2336 may be involved in strain-specific adaptations. systems biology approaches are designed to facilitate the study of complex interactions among genes, proteins, and other genomic elements [1, 2, 3] . in the context of infectious disease, systems biology has the potential to complement reductionist approaches to resolve the complex interactions between host and pathogen that determine disease outcome. however, a prerequisite for systems biology is the description of the system's components. therefore, genome structural annotation or the identification and demarcation of boundaries of functional elements in a genome (e.g., genes, non-coding rnas, proteins, and regulatory elements) are critical elements in infectious disease systems biology. bovine respiratory disease (brd) costs the cattle industry in the united states as much as $3 billion annually [4, 5] . brd is the outcome of complex interactions among host, environment, bacterial, and viral pathogens [6] . histophilus somni, a gramnegative, pleomorphic species, is one of the important causative agents of brd [6] . h. somni causes bovine infertility, abortion, septicemia, arthritis, myocarditis, and thrombotic meningoencephalitis [7] . h. somni strain 2336, the serotype used in this study and isolated from pneumonic calf lung, has a 2.2 mbp genome and 2044 predicted open reading frames (orfs), of which 1569 (76%) have an assigned biological function. genome structural annotation is a multi-level process that includes prediction of coding genes, pseudogenes, promoter regions, repeat elements, regulatory elements in intergenic regions such as small non-coding rnas (srna), and other genomic features of biological significance. computational gene prediction methods such as glimmer [8] or genmark [9] use hidden markov models which are based on a training set of well annotated genes. although these methods are quite efficient, they often miss genes with anomalous nucleotide composition and have several well-described shortcomings: because bacterial genomes do not have introns, detecting gene boundaries is comparatively difficult; due to the usage of more than one start codon, computational genome annotation methods may predict overlapping orfs [10] ; prediction programs use arbitrary minimum cutoff lengths to filter short orfs, which may lead to under-representation of small genes. in case of srna (small non-coding rna) prediction, the lack of dna sequence conservation, lack of a protein coding frame, and the limited accuracy of transcriptional signal prediction programs (promoter/rho terminator prediction) confound computational prediction [11, 12] . computational prediction methods are a ''first pass'' genome structural annotation. whole genome transcriptome studies (such as whole genome tiling arrays [13, 14, 15] and high throughput sequencing [16, 17] ) are complementary experimental approaches for bacterial genome annotation and can identify ''novel'' genes, gene boundaries, regulatory regions, intergenic regions, and operon structures. for example, a transcriptomic analysis of mycoplasma pneumoniae identified 117 previously unknown transcripts, many of which were non-coding rnas, and two novel genes [18] . transcriptome analyses identified novel, non-coding regions in other species, including 27 srnas in caulobacter crescentus [15] , 64 srnas in salmonella typhimurium [17] , and a large number of putative srnas in vibrio cholerae [16] . srnas found in pathogen genomes are known to be involved in various housekeeping activities and virulence [19] . in this study we used rna-seq for the experimental annotation of the h. somni strain 2336 genome and to construct a single nucleotide resolution transcriptome map. novel expressed elements were identified, and where appropriate, computational predictions of previously described gene boundaries were corrected. in 2008 the complete genome sequence of the h. somni strain 2336 became available (genbank cp000947). the 2,263,857 bp circular genome has a gc content of 37.4%, and 87% of the sequence is annotated to coding regions. the genome has 2065 computationally predicted genes, of which 1980 are protein coding. we sequenced the transcriptome of h. somni using illumina rna-seq methodology, and obtained 9,015,318 reads, with an average read length of approximately 76 bp. we mapped approximately 9.4% reads onto the reference dna sequence of h. somni strain 2336 using the alignment program bowtie [20] . to determine expressed regions in the genome, we estimated the average coverage depth of reads mapped per nucleotide/base. we used pileup format, which represents the signal map file for the whole genome in which alignment results (coverage depth) are represented in per-base format. regions where coverage depth was greater than the lower tenth percentile of expressed genes were considered significantly expressed [21] ; in the current study, this corresponded to a coverage depth of 7 reads/bp in pileup format. as another measure for estimating background expression level, we analyzed the coverage in the intergenic regions of the genome. we assumed that at least half of the intergenic region is not expressed (considering the presence of known expressed regions, such as 39 and 59 utr of genes, intergenic region of the operons, and srnas) and calculated the coverage, which corresponded to #6 reads per base, lower than our first cutoff estimate. we retained the most conservative cutoff for expression, i.e., 7 reads per base for describing the expression map of h. somni. nucleotides in the genome sequence with coverage depth above our threshold value were considered to be expressed. this resulted in the generation of a whole genome transcriptome profile of h. somni 2336 at a single nucleotide resolution. figure 1 show the steps involved in the analysis of expressed intergenic regions. we compared the rna-seq based transcriptome map with the available genome annotation to identify expressed, novel, and intergenic regions in the genome. promoters and terminators were predicted across the genome to add confidence to the identified novel elements. for the first time, we report the identification of 94 srnas (table 1) in the h. somni genome. the start and end for srna in table 1 refer to the boundaries of transcriptionally active regions (tar, putative srnas). of these, twelve were similar to wellcharacterized srna families that are described in many bacterial species, such as tmrna, 6s, and fmn ( figure 2 ). the total of 82 novel srnas reported in this study has not been reported earlier. the majority of the identified srnas (.75%) were shorter than 200 nucleotides (length range 70-695 nucleotides). the average gc content of srna at 39.3% was slightly higher compared with the 37.4% gc content of the genome. promoters within 50 nt upstream/downstream of the tar boundaries were predicted for 68 srna. similarly, rho-independent transcription terminators were predicted within 50 bp upstream/downstream of 40 srna. figure 3 shows the depth of coverage for one of the identified novel srna ''hs46'' viewed in the artemis genome browser [22] . blast analysis of the srna sequences against the nonredundant, nucleotide database at ncbi revealed that 31 of the srna sequences were unique to the h. somni 2336 genome. another 41 were highly conserved (.95% identity with .95% coverage) only in h. somni strain 129pt, which is a commensal, preputial isolate. a set of 11 srnas were conserved in the related pasteurellaceae family, which includes genomes such as p. multocida, h. influenzae, h. parainfluenzae, and h. ovis. only 11 srnas were conserved in distant bacterial genomes from genera streptococcus, clostrodium, actinobacillus, vibrio, and others. this lack of srna sequence conservation beyond the species could indicate that srna sequences are under strong selection pressure, and that they could be responsible for the adaptation of many species to different environmental niches. we searched all h. somni srna sequences against the rfam database [23] to determine their putative functions. we found that 12 srnas were homologs to well characterized srnas in other genomes. the identified functional categories included fmn riboswitches, gcvb, glycine, intron_gpii, lysine, alpha_rbs, lr-pk1, isrk, mocorna, rnasep_bact_a, tmrna, and 6s. srnas for which no rfam function could be predicted represent a completely novel set of non-coding srnas. functions of these novel srna need to be determined by further experiments. we evaluated the coding potential of all expressed intergenic regions, by conducting blastx based sequence searches against the non-redundant protein database at ncbi followed by manual analysis and interpretation. we identified 38 novel protein coding regions ( table 2 ). the average length of the identified novel proteins was around 60 amino acids (ranged from 19 to 135 amino acids). the majority of the novel proteins (30) were conserved hypothetical proteins present in related species such as h. somni 129pt, m. haemolytica, and h. influenzae. some of the novel proteins had predicted functions, such as dnak suppressor protein, toxic membrane protein tnac, and predicted toxic peptide ibsb3 ( table 2 ). figure 4 shows an example of a novel protein ''hsp7'' that is similar (74% similarity and 100% coverage) to a putative, phage-related dna-binding protein of neisseria polysaccharea. the single nucleotide resolution map described in this study enabled us to correct the start site for five genes based on the current genome annotation (table 3) . these genes were annotated as phospholipid synthesis protein, ribosomal protein s2, aconitate hydratase 2, peptide chain release factor 2, and duf411, a protein of unknown function. based on evidence from rna-seq data, we performed a blast comparison with other phylogenetically similar proteins to confirm the new gene boundaries (table 3) . the comparison of the transcriptome map of the h. somni genome with predicted proteins revealed the presence of frameshift mutations. four genes have non-functional start codons, resulting in a predicted protein, truncated at the amino terminus (based on blast comparison with homologous proteins in other species), although full length mrna was present. an example is presented for the gene ''hsm_0748'', annotated as ''alpha-lfucosidase'' ( figure s1 ). the other three genes, hsm_0603, hsm_1666 and hsm_1668, encode a hypothetical protein, type iii restriction protein res subunit, and ctp synthase, respectively. two genes with frameshifts causing protein truncations (based on blast comparison with homologous proteins) are hsm_1385 (beta-hydroxyacyl dehydratase, faba) and hsm_1744 (alcohol dehydrogenase zinc-binding domain protein). the transcriptome map revealed a full length mrna for these two genes that code for truncated proteins. our transcriptome map of h. somni identified expression from 1636 (approximately 80%) of the predicted genes. the expressed genes were distributed evenly across all tigrfam functional categories (table s1 ). the transcriptome map allowed identification of operon structures at a genome scale, critical for identifying co-expressed genes and for understanding coordinated regulation of the bacterial transcriptome. we identified co-expression for 452 pairs (total 730 genes) of h. somni genes ( table s2 ) that were transcribed together and constituted a minimal operon. by joining consecutive overlapping pairs of co-expressed genes, we identified 278 distinct transcription units (table s3) . we compared our experimentally identified co-expressed genes with computationally predicted operons. the overlap between computational prediction of co-expressed genes using door [24] and this study was 86% (394 gene pairs) (table s4) . thus, our dataset validates expression of 394 computational gene-pair predictions. we identified 59 new gene pairs that are co-expressed and were not predicted by door, which could be part of unidentified, new operon structures. for example, further in-depth analysis indicated a new operon consisting of three genes: hsm1354, hsm1355 and hsm1356, annotated as ribosomal protein l20, ribosomal protein l35, and translation initiation factor if-3 respectively, which were not predicted computationally ( figure 5 ). the orthologs of these genes are well known to form a functional operon of ribosomal proteins (if3-l35-l20) in escherichia coli [25] . in this study using rna-seq we describe the whole genome transcriptome profile of h. somni 2336, a bovine respiratory disease pathogen. the single nucleotide resolution map helped uncover the structure and complexity of this pathogen's transcriptome and led to the identification of novel, small rnas and protein coding genes as well as gene co-expression. prokaryotic genome annotation is performed often using computational gene prediction programs [8, 9] . however, these prediction algorithms are not able to identify the non-coding srnas, antisense transcripts, and other small proteins. to overcome the shortcomings of computational genome structural annotation, various experimental methods are used for identification of novel expressed elements [13, 14, 15, 16, 17, 18, 26, 27, 28] . deep transcriptome sequencing (rna-seq) has emerged recently as a method that enables the study of rna-based structural and regulatory regions at the genome scale. rna-seq technology has many advantages compared with existing array based methods for transcriptome analysis. in particular, rna-seq does not require probes, so the process is free from probe design issues or bias from hybridization issues. also, the transcriptome coverage from rna-seq is very high [29, 30] . rna-seq was demonstrated to be effective for the discovery of bacterial non-coding rnas, accurate operon definition, and correction of gene annotation [27, 31, 32] . therefore, in the current study, we used rna-seq for profiling h. somni 2336 transcriptome. mapping of rna-seq reads onto the h. somni genome sequence resulted in more than 94% coverage with at least one read per base. this observation is consistent with the reported 94% genome expression in bacillus anthracis, 89.5% in sulfolobus solfataricus, and 95% in burkholderia cenocepacia, studied under one or more experimental growth conditions using rna-seq [32, 33, 34] . these results indicate that most of the bacterial genome sequence is expressed at some basal level. to identify significantly expressed regions above this baseline, we used two alternative methods (discussed in results section) to estimate the background expression. both methods yielded similar results (6-7 reads per base). we selected the higher stringency cutoff of 7 reads per base to minimize the number of false positives. we identified a total of 95 srnas in the h. somni genome. twelve of these were predicted by rfam [23] and are similar to conserved srna (e.g., 6s, tmrna, fmn) in other bacterial species, which helps validate our approach. the 83 novel h. somni srnas may have housekeeping function, regulatory activity, or participate in virulence as described in other pathogenic bacteria [19, 35, 36] . the identified srnas did not show any location specific bias across the genome. similarly, genes known to be associated with virulence are known to be scattered across bacterial genomes [37, 38] . however, the tendency to form clusters was observed with srnas, which could indicate that functionally related srnas tend to be located in close proximity. the rna-seq based transcriptome map of h. somni identified 38 novel protein coding genes that were missed by the initial annotation. the average length of the proteins coded by these genes exceeds 60 amino acids, suggesting that length based cutoff was not the main reason that these genes were missed by computational gene prediction programs. the novel protein coding genes identified in the current study could serve as a training set to improve gene prediction algorithms. the transcriptome map helped to identify incorrect annotation of start codons in the genome. transcriptional mapping does not provide direct evidence of translational start sites. however, location of identified transcriptional start sites suggest that the annotated start codons are incorrect, an observation that is confirmed by blast comparisons against homologous genes in other bacterial species. transcriptional mapping revealed genes where the 59 untranslated sequence extended well beyond the translational start. blast comparisons indicated that these genes have either nonsense or missense base changes relative to homologous genes in other bacterial species, causing apparent ''truncated'' proteins compared with those in other species. further work is needed to determine whether these 59 untranslated regions serve regulatory functions or they are vestigial. rna-seq data enabled us to determine operon structures at a genome scale, and it allowed identification of some operons not predicted by the computational operon prediction method. operon structures that include genes not expressed under the experimental growth condition used in the current study, could not be identified. our results support the notion that using a combination of experimental operon identification by rna-seq and computational prediction can improve operon identification in bacterial genomes [39] . for the first time, we report the rna-seq based transcriptome map of h. somni 2336 and describe novel expressed regions in the genome. whereas the results are interesting, we are aware of the limitations of the study. because the rna-seq protocol was not strand specific, we could not determine the strand specificity of expressed novel transcripts. therefore, table 1 lacks information about srna orientation in the genome. because strand specific information was missing, we could not describe antisense expression in the genome. for protein coding genes, we derived strand specificity based on alignment of the blast hit. despite this shortcoming, we identified novel expressed regions and transcriptional patterns across the whole genome at a high coverage, which is not possible by other transcriptome analysis methods. overall, this study describes rna-seq based transcriptome map of h. somni for identification of functional elements in a pathogen of importance to agriculture. our genome-wide survey predicts numerous, novel, expressed regions that need biological characterization for understanding disease pathogenesis. description of all functional elements in the h. somni system is a prerequisite for conducting holistic systems approaches to understand the complex pathogenesis of bovine respiratory disease. we propagated h. somni 2336 on three tsa-blood plates (with 5% sheep red blood cells) for 16 hr or until a fresh lawn of cells was visible. ibc approval was not required for acquiring the plates as they were purchased through a commercial vendor: fisher scientific (pittsburgh, pa), and manufactured by becton dickinson diagnostic systems, (franklin lakes, nj). we washed the plates with brain heart infusion (bhi) broth, adjusted the culture to an od620 nm = 0.8, and supplemented with rnaprotect reagent. the cells were harvested by centrifugation and stored at 280uc. we extracted total rna using the rneasy mini kit (qiagen, valencia, ca) following the manufacturer's protocol. total rna was treated with rnase-free dnase (invitrogen, carlsbad, ca). using bioanalyzer 2100 (agilent technologies, santa clara, ca), we determined the rna integrity number (rin) of total rna to be greater than 8. microbexpress tm kit (ambion, tx, usa), which specifically removes rrnas, was used for mrna enrichment. small rnas (i.e., trna and 5s rrna) are not removed with this enrichment step (confirmed by bioanalyzer). we used 100 ng enriched mrna with illumina mrna-seq sample preparation kit (illumina, san diego, ca) for library construction following the manufacturer's protocols. briefly, mrna was fragmented chemically by divalent zinc cations and randomly primed for cdna synthesis. after ligating paired-end sequence adaptors to cdna, we isolated fragments of approximately 200 bp by gel electrophoresis and amplified. we we checked all illumina reads for quality, and removed sequence reads containing ''ns''. custom perl script was written to convert illumina reads into fastq format. the script ''fq_all2std.pl'' from maq [40] converted fastq format to sanger fastq format. reads in sanger fastq format, were mapped onto the histophilus somni 2336 genome sequence (genbank accession number. cp000947) using the alignment tool bowtie [41] , allowing for a maximum of two mismatches. the reads that mapped to more than one location were discarded. we used samtools [42] to convert data into sam/bam format, and to generate alignment results in a pileup format. pileup format provides the signal map file and has per-base format coverage. custom perl scripts were written to calculate the background expression. processed data was deposited in geo with the accession number gse29578. we used in-house perl scripts to extract novel expressed intergenic regions to identify novel small rnas, riboswitches, and putative novel proteins. srna ,70 bp in length were discarded to minimize the number of false positives. for each novel expressed region, blast sequence searches were performed against the non-redundant protein database at ncbi to identify potential protein coding regions. intergenic regions within predicted operons [24] represent expressed regions and can be mis-classified as srnas. therefore, these regions were excluded. we analyzed blast results manually, to identify novel protein coding regions and start codon corrections. if no protein coding region was found in the intergenic expressed regions, the presence of a promoter or a rho-independent terminator allowed us to classify the regions as srna. bacterial promoter sequences were predicted by neural network promoter prediction program (http://www.fruitfly.org/seq_tools/promoter.html) [43] . rho-independent transcription terminators were identified using the program transtermhp [44] . for functional annotation, all identified identified srna sequences were searched against the rfam database [23] . srna sequence conservation among other genomes was determined by blastn searches against nonredundant nucleotide database at ncbi. we mapped srnas, along with additional features, onto genome browsers like igv [45] and artemis [46] for further visualization, manual analysis, and interpretation. gene expression: expressed reads with coverage above background were mapped onto the annotated genes of h. somni 2336. genes that had a significantly higher proportion of their length (.60%) covered by expressed reads were considered to be expressed. operons: rna-seq can identify and predict operon structures in bacteria. we considered two or more consecutive genes to be part of an operon, if they fulfilled the following criteria: (a) they are expressed; (b) they are transcribed in the same direction; and (c) the intergenic region between the genes is expressed. overlapping pairs of such genes were joined together to identify large operon structures. we used in-house perl scripts for the analyses. figure s1 mutated start codon. the figure shows that the predicted protein coding frame (mh_748) is shorter at the 59 end than the corresponding transcript level shown by the rna-seq coverage. although the transcript is longer near 59 end, no start codon is found in that region which might be a result of the mutation in that region of the start codon. this was further validated using homology searches of the full length transcript which shows high homology (95% identity and .95% coverage) to a alpha-l-fucosidase protein from m. haemolytica phl213. (tif) host-pathogen systems biology a systems biology approach to infectious disease research: innovating the pathogen-host research paradigm virus-host interactions: from systems biology to translational research infectious bovine rhinotracheitis, parainfluenza-3 and bovine respiratory coronavirus economic impact associated with respiratory disease in beef cattle the immunology of the bovine respiratory disease complex bovine platelets activated by haemophilus somnus and its los induce apoptosis in bovine endothelial cells microbial gene identification using interpolated markov models genemarks: a self-training method for prediction of gene starts in microbial genomes. implications for finding sequence motifs in regulatory regions large gene overlaps in prokaryotic genomes: result of functional constraints or mispredictions? computational approaches for the discovery of bacterial small rnas computational prediction of srnas and their targets in bacteria transcriptome analysis of escherichia coli using high-density oligonucleotide probe arrays wholegenome tiling array analysis of mycobacterium leprae rna reveals high expression of pseudogenes and noncoding regions small non-coding rnas in caulobacter crescentus experimental discovery of srnas in vibrio cholerae by direct cloning, 5s/trna depletion and parallel sequencing deep sequencing analysis of small noncoding rna and mrna targets of the global post-transcriptional regulator transcriptome complexity in a genome-reduced bacterium identification of small rnas in diverse bacterial species lack of development of new antimicrobial drugs: a potential serious threat to public health pathogen proteomes during infection: a basis for infection research and novel control strategies artemis: sequence visualization and annotation rfam: annotating non-coding rnas in complete genomes door: a database for prokaryotic operons resistance or decreased susceptibility to glycopeptides, daptomycin, and linezolid in methicillin-resistant staphylococcus aureus deep rna sequencing improved the structural annotation of the tuber melanosporum transcriptome bacillus anthracis genome organization in light of whole transcriptome sequencing identification of novel non-coding small rnas from streptococcus pneumoniae tigr4 using high-resolution genome tiling arrays studying bacterial transcriptomes using rna-seq next generation sequencing of microbial transcriptomes: challenges and opportunities a strand-specific rna-seq analysis of the transcriptome of the typhoid bacillus salmonella typhi mapping the burkholderia cenocepacia niche response via high-throughput sequencing structure and complexity of a bacterial transcriptome a single-base resolution map of an archaeal transcriptome small noncoding rnas controlling pathogenesis regulatory rna in bacterial pathogens vfdb: a reference database for bacterial virulence factors a genomic window into the virulence of histophilus somni the relative value of operon predictions extended-spectrum beta-lactamase-producing enterobacteriaceae: an emerging public-health concern ultrafast and memoryefficient alignment of short dna sequences to the human genome bad bugs, no drugs: no eskape! an update from the infectious diseases society of america application of a time-delay neural network to promoter annotation in the drosophila melanogaster genome rapid, accurate, computational discovery of rho-independent transcription terminators illuminates their relationship to dna uptake biologicalnetworks-tools enabling the integration of multi-scale data for the host-pathogen studies host-microbe interaction systems biology: lifecycle transcriptomics and comparative genomics we thank dr. john harkness and dr. stephen b. pruett for editing the final version of the manuscript. key: cord-003507-22ylifqo authors: kelly, j. daniel; worden, lee; wannier, s. rae; hoff, nicole a.; mukadi, patrick; sinai, cyrus; ackley, sarah; chen, xianyun; gao, daozhou; selo, bernice; mossoko, mathais; okitolonda-wemakoy, emile; richardson, eugene t.; rutherford, george w.; lietman, thomas m.; muyembe-tamfum, jean jacques; rimoin, anne w.; porco, travis c. title: projections of ebola outbreak size and duration with and without vaccine use in équateur, democratic republic of congo, as of may 27, 2018 date: 2019-03-07 journal: plos one doi: 10.1371/journal.pone.0213190 sha: doc_id: 3507 cord_uid: 22ylifqo as of may 27, 2018, 6 suspected, 13 probable and 35 confirmed cases of ebola virus disease (evd) had been reported in équateur province, democratic republic of congo. we used reported case counts and time series from prior outbreaks to estimate the total outbreak size and duration with and without vaccine use. we modeled ebola virus transmission using a stochastic branching process model that included reproduction numbers from past ebola outbreaks and a particle filtering method to generate a probabilistic projection of the outbreak size and duration conditioned on its reported trajectory to date; modeled using high (62%), low (44%), and zero (0%) estimates of vaccination coverage (after deployment). additionally, we used the time series for 18 prior ebola outbreaks from 1976 to 2016 to parameterize the thiel-sen regression model predicting the outbreak size from the number of observed cases from april 4 to may 27. we used these techniques on probable and confirmed case counts with and without inclusion of suspected cases. probabilistic projections were scored against the actual outbreak size of 54 evd cases, using a log-likelihood score. with the stochastic model, using high, low, and zero estimates of vaccination coverage, the median outbreak sizes for probable and confirmed cases were 82 cases (95% prediction interval [pi]: 55, 156), 104 cases (95% pi: 58, 271), and 213 cases (95% pi: 64, 1450), respectively. with the thiel-sen regression model, the median outbreak size was estimated to be 65.0 probable and confirmed cases (95% pi: 48.8, 119.7). among our three mathematical models, the stochastic model with suspected cases and high vaccine coverage predicted total outbreak sizes closest to the true outcome. relatively simple mathematical models updated in real time may inform outbreak response teams with projections of total outbreak size and duration. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 on may 8, 2018 , the world health organization (who) announced the occurrence of an outbreak of ebola virus disease (evd) in the democratic republic of congo (drc). [1] from april 4 through may 7, 21 suspected evd cases were reported in iboko and bikoro, équateur province. on may 7, blood samples from five hospitalized patients had been sent to kinshasa for ebola-pcr testing, and two were confirmed pcr-positive. [2] on may 21, vaccination of healthcare workers started. [3] by may 27, the ring vaccination campaign was being rolled out as 906 contacts and contacts of contacts were being actively monitored. six suspected, 13 probable and 35 confirmed evd cases had been reported, and 25 (52%) of 48 probable and confirmed evd cases had died. [1] this outbreak had several features that were worrisome for widespread transmission. cases were reported over a 168-kilometer distance, including four confirmed cases in thẽ 1,200,000-inhabitant provincial capital of equateur, mbandaka, which is situated on the congo river and bordering congo-brazzaville. [2] moreover, travel to kinshasa is frequent from mbandaka. given these risk factors, early epidemic growth profiles, [4] and evidence of unreported infection from previous outbreaks, [5, 6] the risk of a substantially larger outbreak could not be ignored. the factors causing epidemic growth to peak have been debated. delayed detection of evd outbreaks and resulting widespread distributions of evd have significantly contributed to epidemic growth. [7] in addition to traditional burial practices, ebola treatment units with low quality care and/or high mortality rates have discouraged ebola suspects from presenting to care and contribute to community-based transmission. [8] [9] [10] fragile, overwhelmed public health surveillance systems have also contributed to higher rates of unreported cases, who endanger urban communities, [11] which potentially have had higher transmission rates than rural communities. [12] change to subcritical transmission (reproduction number below 1) tends to occur when ebola response organizations deploy control, prevention and care measures, [13, 14] communities adopt more protective behaviors, [15, 16] and/or transmission decreases in a social network. [17, 18] scientific advances with rapid diagnostics and vaccines from the west africa outbreak were deployed in the april-july 2018 evd outbreak in drc and had the potential to limit ebola virus transmission. [19] [20] [21] we used reported case counts during evd outbreak in drc and/or time series from 18 prior outbreaks to estimate the total outbreak size and duration with and without the use of vaccines. these projections were intended to help organizations anticipate and allocate sufficient resources for the duration of the april-july 2018 evd outbreak. the following methods were used to generate projections: a stochastic branching process model, [22] statistical regression based on prior outbreaks, and gott's law. [23] on may 27, 54 evd cases (6 suspected, 13 probable and 35 confirmed) were reported in three locations (iboko, bikoro, and mbandaka) (fig 1) . based on evd situation reports from drc, we assumed the ring vaccination program started the week of may 21, so we used may 23 as the time point that vaccines were implemented in the model at high and low coverage levels. data on suspected, probable, and confirmed case counts were available from who situation reports in may 2018 and used as the basis for analysis. the published situation reports reflected data available up until may 11, 13, 16, 23 , and 27, respectively. during the outbreak, the ebola response team tested suspected cases for evd and depending on positive or negative results, cases were classified as confirmed or not a case. the final outbreak case count was 54 probable and confirmed cases (no suspected cases). [1] due to this reporting process, probable and confirmed cases were used to parameterize the stochastic branching process model, regression models, and gott's law. we added suspected cases to create an additional projection, using stochastic branching process model. we modeled ebola virus transmission using a stochastic branching process model, parameterized by transmission rates estimated from the dynamics of prior evd outbreaks, and conditioned on agreement with reported case counts from the 2018 evd outbreak to date. we incorporated high and low estimates of vaccination coverage into this model. then we generated a set of probabilistic projections of the size and duration of simulated outbreaks in the current setting. to estimate the reproduction number r as a function of the number of days from the beginning of the outbreak, we included reported cases by date from thirteen prior outbreaks and excluded the first historical outbreak reported in those countries (e.g., 1976 outbreak in yambuko, drc) (s1 table) . [25] [26] [27] [28] [29] [30] [31] [32] as there is a difference in the ebola response system as well as community sensitization to evd following a country's first outbreak, we employed this inclusion criterion to reflect the ebola response system in drc during what is now its ninth outbreak. the wallinga-teunis technique was used to estimate r for each case and therefore for each reporting date in the outbreak. [33] the serial interval was defined as the interval between disease onset in an index case and disease onset in a person infected by that index case. the serial interval distribution used for this estimation was a gamma distribution with a mean of 14.5 days and a standard deviation of 5 days, with intervals rounded to the nearest whole number of days, consistent with the understanding that the serial interval of evd cases ranges from 3 to 36 days with mean 14 to 15 days. [11, [34] [35] [36] given that serial interval distribution, which we can denote as a probability w (t) of a t-day interval between given primary and secondary cases, wallinga-teunis estimation works by defining a relative likelihood p ij for each possible source j of a given case i: and then deriving from that an estimated reproduction number rj for each case: after using this technique to derive estimated reproduction numbers for each case in an outbreak, we use these estimated r values and cases' onset dates d to estimate an initial reproduction number r 0 and quenching rate τ for each past outbreak by fitting an exponentially quenched curve: to each outbreak's r and d values (s1 fig) . transmission was modeled using a stochastic branching process model in which the number of secondary cases si caused by any given primary case i was drawn from a negative binomial distribution whose mean is the reproduction number r: [37, 38] si � nbðr; kþ; where r is reproduction number as a function of day, k is a dispersion parameter, and nb() denotes the negative binomial distribution. all transmission events were assumed to be independent. the serial interval between date of detection of each primary case and that of each of its secondary cases is assumed gamma distributed with mean 14.5 days and standard deviation 5 days, rounded to the nearest whole number of days, as above. the pair of parameters r 0 and τ estimated for the different past outbreaks used, and dispersion parameter k, were used in all possible combinations (with r 0 and τ taken as a unit) to simulate outbreaks. this model generated randomly varying simulated outbreaks with a range of case counts per day. after the ministry of health and who conducted epidemiological investigations about the beginning of the evd outbreak in équateur province, they concluded that the outbreak began on april 4, 2018, with a single case. [1] the simulation process occurs as follows: proposed epidemic trajectories are generated in an initial step based on the above branching process, and these are then subsequently filtered by discarding all but those whose cumulative case counts match the known counts of the april-july 2018 evd outbreak on known dates. the filtration accepts epidemics within a range of 3 cases more or less than each recorded value. this one-step particle filtering technique produced an ensemble of model outbreaks, filtered on agreement with the recorded trajectory of the outbreak to date. this filtered ensemble is then used to generate projections of the eventual outcome of the outbreak. [12] to model vaccination coverage with respect to total transmission (unreported and reported), we multiplied the estimate of vaccine effectiveness by low and high estimates of reported cases. in a ring vaccination study at the end of the west africa outbreak, the overall estimated rvsv-vectored vaccine efficacy was 100% and vaccine effectiveness was 64.6% in protecting all contacts and contacts of contacts from evd in the randomized clusters, including unvaccinated cluster members. [20] estimates of vaccine effectiveness were used in our stochastic model. the ring vaccination study found the vaccine to be effective against cases with onset dates 10 days or more from the date of vaccine administration, so we modeled the vaccination program as a proportionate reduction in the number of new cases with onsets 10 days or more after the program start date. then, past estimates of the proportion of unreported cases were used to estimate the proportion of exposed individuals not covered by the vaccination process. based on a sierra leonean study from the 2013-2016 outbreak, [11] we estimated that the percentage of reported cases in drc would rise over time from a low of 68% to a high of 96%. given these low and high estimates of reported cases and the estimate of vaccine effectiveness (64.6%), a low estimate of vaccination program coverage was 44% (68% multiplied by 64.6%) and a high estimate of vaccination program coverage was 62% (96% multiplied by 64.6%). the course of the outbreak with and without the vaccination program was modeled based on approximate dates available from situation reports. [1] for simulation based on probable and confirmed cases, from 122,683,392 simulated outbreaks, 21,036 were retained after filtering on approximate agreement with drc case counts. for simulation based on probable, confirmed, and suspected cases, from 122,683,392 simulated outbreaks, 32,431 were retained after filtering. the simulated outbreaks that were retained after filtering were continued until they generated no further cases. this ensemble was used to derive a distribution of outbreak sizes and durations. mean and median values and 95% prediction intervals were calculated using the 2.5 and 97.5 percentiles of simulated outbreak size and duration. these analyses were conducted using r 3.4.2 (r foundation for statistical computing, vienna, austria). for contrast with the stochastic model above, a simple regression forecast was conducted based solely on outbreaks of size 10 or greater. time series for all 18 such prior outbreaks were obtained, including seven prior ones from drc, dating back to 1976 (s1 table; the beginning of each outbreak was not reliably characterized; therefore, all time-series were aligned on the day they reached 10 cases. in the april-july 2018 outbreak, we observed cases over the period from april 4 to may 27 (day 0 to day 53). may 27 corresponded to day 34 since reaching 10 reported cases. for the prior 18 outbreaks, linear interpolation was used to obtain the number of cases on day 34 (after reaching 10 cases). to reduce the influence of outliers and high leverage points, and to improve linearity, we calculated the pseudologarithm transform f(x) = arcsinh(x/2), asymptotically logarithmic but well-behaved at 0. we used nonparametric theil-sen regression (r-package mblm) followed by calculation of the resulting prediction interval for a new observation. [46, 47] finally, we reported the median and 95% central coverage intervals for the prediction distribution conditional on the value being no smaller than the observed value on day 34. sensitivity analysis was conducted using ordinary least squares regression. these analyses were conducted using r 3.4.2 (r foundation for statistical computing, vienna, austria). gott' s law was used to estimate the outbreak size using data through may 27 and may 11. we included a projection using data through may 11 because we hypothesized that this method performs better when the first situation report is posted than at later in the outbreak period. [23] then we included a projection with the regression models using data through may 11 for comparison. with gott's law, we assume we have no special knowledge of our position on the epidemic curve. if we assume a non-informative uniform prior for the portion of the epidemic that still remains, the resulting probability distribution for the remaining number of cases y is: the median outbreak size was estimated, along with the two-sided 95% prediction interval. each of the above models assigned a probability to any possible value of the total outbreak size. the final outbreak size was 54 probable and confirmed cases, so we identified the probability of this equivalent number (53) from each model, as of may 27. probabilistic projections were scored using a log-likelihood (ignorance) score. [48] as of may 27, 2018, there were 6 suspected, 13 probable and 35 confirmed evd cases. bikoko had ten confirmed cases, 11 probable cases, and one suspected case. iboko had 21 confirmed cases, two probable cases, and one suspected case. mbandaka had four confirmed cases and one suspected case (fig 1) . with the stochastic model, we projected outbreak size and duration of probable and confirmed cases. in the absence of any vaccination program, the projected median outbreak size was 213.0 cases (mean 360.2; 95% prediction interval: 64.8, 1450.2). median duration of projected outbreaks was 175.0 days (mean 182.8; 95% prediction interval: 86.0, 282.0). using a lower estimate of 44% vaccination coverage, the median outbreak size was 104.0 cases (mean 118.8; 95% prediction interval: 58.0, 271.0) and median duration was 121.0 days (mean 124.6; 95% prediction interval: 74.0, 187.0). using a higher estimate of 62% vaccination coverage, the median size was 82.0 evd cases (mean 88.1; 95% prediction interval: 55.0, 156.0), and the median duration was 101.0 days (mean 103.4; 95% prediction interval: 68.0, 153.0). these projections with the stochastic model were repeated to estimate suspected, probable and confirmed cases (table 1 and table 2 with the regression based on past outbreaks, the median outbreak size was estimated to be 65.0 probable and confirmed cases (95% prediction interval: 48.8, 119.7), while use of ordinary least squares produced a median size of 97.9 probable and confirmed cases (95% prediction interval: 58.3, 169.9). outbreak projections were also reported using data through may 11 in table 3 . gott's law suggests that given 48 probable and confirmed cases, the median estimate of outbreak size was 96.0 cases (95% ci: 49.0, 1920.0). using the 34 probable and confirmed cases as of may 11, the median estimate of outbreak size was 68.0 cases (95% ci: 35.0, 1360.0). of the mathematical models employed, the stochastic model that included suspected cases and high vaccination coverage had the best probabilistic score (log likelihood of -1.31). likelihood scores of each model can be found in table 4 . when we were conducting our projections in late may, this outbreak still had the potential to become the largest outbreak in drc since 2007. vaccine use, regardless of coverage levels, was projected to prevent more than half of the total outbreak size. vaccines, however, were only part of concurrent prevention, control, and care strategies. [8, [49] [50] [51] we also found that the stochastic model with vaccine use projected that rare, large outbreaks (tail of the distribution of the model without vaccinations) were prevented, suggesting that repeat epidemics such as the 2013-2016 west african outbreak may have been highly unlikely once vaccines were rolled out. multiple models were used to estimate total outbreak size. this study exemplified how mathematical models, including simple regressions, can be useful for advising real-time decision making because they provided rapid projections and similar estimates of r as compared to complex models, [35] even though real-time modeling projections historically overestimated outbreak size and duration. [52, 53] our projections that included suspected cases did not suggest that vaccines had as much of an impact as our model using only probable and confirmed cases. the trends associated with suspected cases were subject to several factors, including operational choices of response teams and maturity of the outbreak. nevertheless, suspected case counts may at times provide a better glimpse into the near future of an outbreak than the confirmed and probable case counts. in our case, use of the time series of confirmed, probable, and suspect cases yielded a forecast closer to the final outbreak size. however, as model projections can be highly sensitive to inclusion of suspected cases and use of exact case counts, particularly the last few counts in the available data, conclusions must be taken with caution. thus far, there had been a strong local and international response, and deployment of vaccines and rapid diagnostic tests (rdt) occurred early in response efforts. [1] rdts were being used to screen ebola suspects while the vaccines are being administered to high-risk groups for evd, including healthcare workers, contacts, and contacts of contacts. to further limit epidemic growth from unreported cases, particularly those who have non-specific symptoms but table 1 . distribution of projected outbreak size from stochastic branching process model. mean, median and 95% prediction interval of outbreak size, by proportion of vaccine coverage, using probable and confirmed cases with and without suspected cases. probable table 2 . distribution of projected outbreak duration from stochastic branching process model. mean, median and 95% prediction interval of outbreak duration, by proportion of vaccine coverage, using probable and confirmed cases with and without suspected cases. there are limitations to our projections. projection distributions were right-skewed, with long tails (and we therefore reported the median instead of the mean). while there have been 22 observed evd outbreaks with a case count greater than ten cases, we were unable to include all prior outbreaks in our estimates due to data availability. [45, 54] note that the simple regression projection is based entirely on past outbreaks of evd (measured and reported in different ways), and cannot account for the improved control measures and vaccination in the way that a mechanistic model does. we included, however, as much real-time information into our estimates as possible, but situations such as the introduction of evd into a large urban population and implementation of rdts and vaccines are new to drc. we did not include vaccination of healthcare workers in the stochastic model. our estimates of vaccination effectiveness and reported cases were obtained from west africa because these estimates were not available for the evd outbreak in équateur. these modeling assumptions may not have been consistent with estimates in drc and should be carefully considered prior to use in other evd outbreaks. a strength of our approach was the use of multiple methods to estimate the outbreak size, although we note that gott's law has not been validated for outbreak projections in other evd outbreaks. additional limitations of the models were that they did not include parameters to address spatial spread, urban settings, conflict zone, or other factors that may have influenced the accuracy of the predictions, particularly in the 2018-2019 evd outbreak in northeastern drc (ongoing in january 2019). while it can also be useful and achievable to use models of these kinds to make short-term forecasts for evaluation of model performance and to inform outbreak response, [55] the present study was limited to projections of final outbreak size and duration. among our three mathematical models, the model that performed the best (stochastic model with suspected cases and high vaccine coverage) predicted total outbreak sizes close to the true outcome. when evd cases were introduced into mbandaka, there was concern that the total outbreak size could exceed most prior evd outbreaks in drc. indeed, our projections were consistent with this concern because models without vaccine coverage projected higher total outbreak sizes. in our stochastic model projections, vaccine use reduced mean total outbreak size by more than half, regardless of coverage levels (p<0.001, welch's t-test). as vaccine coverage was scaled up, an influx of support was warranted to support and bolster the evolving rapid response; however, continued efforts to strengthen the health system are equally as warranted so that we can respond to future outbreaks before they become epidemics. relatively simple mathematical models updated in real time may inform outbreak response teams with projections of total outbreak size and duration. supporting information s1 table. list of 21 prior ebola outbreaks from 1976 to 2016 by time period, country, confirmed/probable reported and time series case count, outbreak inclusion into the regression and stochastic models. world health organization regional office for africa. health topics: ebola virus disease world health organization. emergencies preparedness, response excitement over use of ebola vaccine in outbreak tempered by real-world challenges mathematical models to characterize early epidemic growth: a review minimally symptomatic infection in an ebola 'hotspot': a cross-sectional serosurvey anatomy of a hotspot: chain and seroepidemiology of ebola virus transmission after ebola in west africa-unpredictable risks, preventable epidemics the ebola suspect's dilemma biosocial approaches to the 2013-2016 ebola pandemic ebola virus disease in west africa-clinical manifestations and management unreported cases in the 2014-2016 ebola epidemic: spatiotemporal variation, and implications for estimating transmission heterogeneity in district-level transmission of ebola virus disease during the 2013-2015 epidemic in west africa the impact of control strategies and behavioural changes on the elimination of ebola from lofa county dynamics and control of ebola virus transmission in montserrado, liberia: a mathematical modelling analysis risk communication and ebola-specific knowledge and behavior during 2014-2015 outbreak a theory-based socioecological model of communication and behavior for the containment of the ebola epidemic in liberia modeling household and community transmission of ebola virus disease: epidemic growth, spatial dynamics and insights for epidemic control ebola control: effect of asymptomatic infection and acquired immunity rapid antigen test for ebola. for use under emergency use authorization (eua) only efficacy and effectiveness of an rvsv-vectored vaccine in preventing ebola virus disease: final results from the guinea ring vaccination, open-label, cluster-randomised trial (ebola ç a suffit!) ebola control: rapid diagnostic testing on the probability of the extinction of families implications of the copernican principle for our future prospects high-resolution global maps of 21st-century forest cover change report of a who/international study team ebola virus disease in southern sudan: hospital dissemination and intrafamilial spread ebola hemorrhagic fever outbreaks in gabon, 1994-1997: epidemiologic and health control issues the reemergence of ebola hemorrhagic fever, democratic republic of the congo prise en charge des malades et des défunts lors de l'épidémie de fièvre hé morragique due au virus ebola d'octobre à décembre a limited outbreak of ebola haemorrhagic fever in etoumbi, republic of congo ebola virus disease in the democratic republic of congo filovirus outbreak detection and surveillance: lessons from bundibugyo different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures ebola virus disease in west africa-the first 9 months of the epidemic and forward projections a systematic review of early modelling studies of ebola virus disease in west africa transmission dynamics and control of ebola virus disease (evd): a review comparing methods for estimating r0 from the size distribution of subcritical transmission chains superspreading and the effect of individual variation on disease emergence ebola virus disease in the democratic republic of the congo outbreak(s) of ebola haemorrhagic fever, congo and gabon potential for large outbreaks of ebola virus disease world health organization. weekly epidemiological record. abonnement annuel university of bergen dissertation for phd ebola virus disease outbreak in nigeria: transmission dynamics and rapid control ebola (ebola virus disease): history of ebola virus disease: 2014-2016 ebola outbreak in west africa: case counts estimates of the regression coefficient based on kendall's tau a rank-invariant method of linear and polynomial regression analysis. i, ii, iii. nederl akad wetensch, proc scoring probabilistic forecasts: the importance of being proper beyond vaccines: improving survival rates in the drc ebola outbreak food insecurity as a risk factor for outcomes related to ebola virus disease in kono district, sierra leone: a cross-sectional study the symbolic violence of 'outbreak': a mixed methods, quasiexperimental impact evaluation of social protection on ebola survivor wellbeing estimating the future number of cases in the ebola epidemic -liberia and sierra leone models overestimate ebola cases centers for disease control and prevention. outbreaks chronology: ebola hemorrhagic fever real-time projections of epidemic transmission and estimation of vaccination impact during an ebola virus disease outbreak in the eastern region of the democratic republic of congo we thank the ebola responders for their efforts in the 2018 evd outbreak in équateur, democratic republic of congo. key: cord-000208-th0wddvc authors: cornelissen, lisette a. h. m.; de vries, robert p.; de boer-luijtze, els a.; rigter, alan; rottier, peter j. m.; de haan, cornelis a. m. title: a single immunization with soluble recombinant trimeric hemagglutinin protects chickens against highly pathogenic avian influenza virus h5n1 date: 2010-05-14 journal: plos one doi: 10.1371/journal.pone.0010645 sha: doc_id: 208 cord_uid: th0wddvc background: the highly pathogenic avian influenza (hpai) virus h5n1 causes multi-organ disease and death in poultry, resulting in significant economic losses in the poultry industry. in addition, it poses a major public health threat as it can be transmitted directly from infected poultry to humans with very high (60%) mortality rate. effective vaccination against hpai h5n1 would protect commercial poultry and would thus provide an important control measure by reducing the likelihood of bird-to-bird and bird-to-human transmission. methodology/principal findings: in the present study we evaluated the vaccine potential of recombinant soluble trimeric subtype 5 hemagglutinin (sh5(3)) produced in mammalian cells. the secreted, purified sh5(3) was biologically active as demonstrated by its binding to ligands in a sialic acid-dependent manner. it was shown to protect chickens, in a dose-dependent manner, against a lethal challenge with h5n1 after a single vaccination. protected animals did not shed challenge virus as determined by a quantitative rt-pcr on rna isolated from trachea and cloaca swabs. also in mice, vaccination with sh5(3) provided complete protection against challenge with hpai h5n1. conclusions/significance: our results demonstrate that sh5(3) constitutes an attractive vaccine antigen for protection of chickens and mammals against hpai h5n1. as these recombinant soluble hemagglutinin preparations can be produced with high yields and with relatively short lead time, they enable a rapid response to circulating and potentially pandemic influenza viruses. influenza a viruses are enveloped, negative-strand rna viruses with a segmented genome. they infect a large variety of animal species, although birds are considered to constitute the reservoir from which all influenza a viruses in other species originate [1, 2] . on the basis of the antigenic properties of their two surface glycoproteins, hemagglutinin (ha) and neuraminidase (na), influenza a viruses are classified into 16 ha (h1-16) and 9 na (n1-9) subtypes. the highly pathogenic avian influenza (hpai) virus h5n1 causes multi-organ disease and death in poultry, resulting in significant economic losses in the poultry industry. hpai h5n1 also poses a major public health threat as it can be transmitted directly from infected poultry to humans with very high (60%) mortality rate. it is widely accepted that continued human exposure to influenza viruses circulating in wild and domestic avian species poses a permanent pandemic threat [3, 4] . effective vaccination against hpai h5n1 would protect com-mercial poultry and would thus provide an important control measure by reducing the likelihood of bird-to-bird as well as birdto-human transmission. therefore, the development of efficacious influenza vaccines is of high veterinary and public health importance. conventional vaccine preparations against hpai h5n1 are produced by propagating virus in embryonated chicken eggs or mammalian cells. the vaccine viruses used are either reassortants or low pathogenic wild-type avian viruses. either way, the viruses need to be selected for their ability to multiply in eggs or cultured cells, which may preclude the best genetic match to the circulating hpai h5n1 strains. in addition, these vaccines have several other limitations as reviewed elsewhere [5, 6] . hence, other vaccine strategies against hpai h5n1 have been explored including the use of, among others, live attenuated influenza vaccines [7, 8] , live vaccines based on heterologous viral vectors such as pox virus [9] , adenovirus [10] , baculovirus [11] and newcastle disease virus [12] , and dna vaccination [13] . while these different strategies often showed promising results, their applicability ultimately will depend on various important issues including safety, efficacy, production and costs [5] . protective immunity against influenza virus infection and disease is primarily conferred through ha via the induction of anti-ha antibodies. as protection from influenza virus infection correlates with anti-ha titers, nearly all vaccine approaches aim to induce high levels thereof. therefore, the ha protein is the antigen of choice for the development of recombinant subunit vaccines to protect against hpai h5n1. an influenza vaccine based on recombinant purified ha could offer the following advantages: i) the ha antigen can be produced using safe, quality-controlled and scalable conditions. ii) there will be no need for virus cultivation, thus avoiding the necessity a) to obtain viruses that replicate efficiently in eggs or cell culture, b) to use biocontainment facilities and c) to inactivate the virus using procedures that may affect antigenicity and raise safety concerns. iii) the recombinant ha protein can be highly purified thereby limiting adverse reactions caused e.g. by the presence of egg contaminants. iv) immunization with recombinant ha will allow the serological differentiation of naturally infected from vaccinated animals/flocks (the so-called diva principle; [14] ). iv) recombinant ha vaccines are manufactured with a relatively short lead time, allowing an accelerated response to emerging influenza strains. recombinant ha can be produced using different expression systems. when expressed in e. coli the resulting ha protein gave rise to the induction of hemagglutination inhibition (hi) titers upon immunization of mice [15] . however, as proper folding and trimerization of the ha protein requires multiple posttranslational modifications including glycosylation and disulfide bond formation, expression of the ha protein in higher eukaryotic systems is likely to result in superior antigenicity. thus, mammalian cellderived ha trimers were found to induce much higher levels of neutralizing antibodies than similarly produced monomeric ha protein [16] . the baculovirus expression system has been used to produce strain specific ha antigens in insect cells, which were shown to protect against a hpai h5n1 challenge [17] . such fullsize ha proteins may, however, be limited in their efficacy because the membrane proteins may not retain their native membrane-bound structure upon purification [18] . hence, the production of recombinant, soluble, stable ha trimers that are secreted from the cells seems like an attractive alternative approach. such ha trimers, expressed either in insect or mammalian cells, were indeed shown to elicit neutralizing antibodies [16] and to partially protect mice against hpai h5n1 challenge infection [19] . in view of their promising potential we have evaluated recombinant soluble ha trimers in chickens and mice for their ability to induce protective immunity against infection with hpai h5n1. to this end, recombinant soluble h5 proteins provided with a gcn4 trimerization motif and a strep-tag ii, the latter for purification purposes, were expressed in mammalian cells. the recombinant soluble h5 trimers (sh5 3 ) were purified from the culture supernatants using a simple one-step purification protocol and characterized with respect to their oligomeric state and bioactivity. subsequently, vaccination with the sh5 3 preparation was shown to provide complete protection against challenge with hpai h5n1 both in mice and in chickens, in the latter already after a single immunization. a cdna clone corresponding to residues 18 to 523 (h3 numbering) of the ha from a/viet nam/1203/2004 (h5n1) (genbank accession no. abw90137.1) was synthesized using human-preferred codons by genscript usa inc. in this clone the predicted ha ectodomain protein lacks a multibasic cleavage site. the cdna was cloned into the pcd5 expression vector for efficient expression in mammalian cells [20] . the pcd5 vector had been modified such that the ha-encoding cdna was cloned in frame with dna sequences coding for a signal sequence, an artificial gcn4 isoleucine zipper trimerization motif (krmkqiedkieeieskqkkieneiarikk) [21] and the strep-tag ii (wshpqfek; iba, germany). the resulting vector encodes the soluble trimeric h5 protein designated as sh5 3 . pcd5 expression vectors containing the ha ectodomainencoding sequences were transfected into hek293s gnti(2)cells [22] using polyethyleneimine i (pei) in a 1:5 ratio (mg dna: mg pei). at 6 h post transfection, the transfection mixture was replaced by 293 sfm ii expression medium (invitrogen), supplemented with sodium bicarbonate (3.7 g/liter), glucose (2.0 g/liter), primatone rl-uf (3.0 g/liter), penicillin (100 units/ml), streptomycin (100 mg/ml), glutamax (gibco), and 1,5% dimethyl sulfoxide. tissue culture supernatants were harvested 5-6 days post transfection. ha protein expression and secretion was confirmed by sodium dodecylsulfate (sds)-polyacrylamide gel electrophoresis (page) followed by western blotting using a mouse anti-strep-tag antibody (iba, germany). ha proteins were purified using strep-tactin sepharose beads according to the manufacturer's instructions (iba, germany). the concentration of purified protein was determined by using a nanodrop 1000 spectrophotometer (isogen life sciences) according to the manufacturer's instructions. the oligomerization status of the sh5 3 proteins was determined by analyzing the elution profile using a superdex200gl 10-300 column (ge healthcare). sialic acid-binding activity of sh5 3 was assessed using a fetuin solid phase assay. 1 mg/ml fetuin per well was used to coat 96-well nunc maxisorp plates. when indicated in the figure legend, fetuin was treated with vibrio cholera derived neuraminidase (vcna; roche) followed by three washing steps. sh5 3 was pre-complexed with horseradish peroxidase (hrp)linked anti-strep-tag antibody (2:1 molar ratio) for 30 min at 0uc prior to incubation of limiting dilutions on the fetuin-coated plates (60 min, room temperature [rt]). ha-binding was subsequently detected using tetramethylbenzidine substrate (biofx) in an elisa reader (el-808 [biotek]), reading the od at 450 nm. animal studies were conducted at the central veterinary institute (cvi), lelystad, under bsl3 conditions and after approval by the animal ethic committee. in the first chicken experiment, 6 weeks old spf white leghorn chickens (lohmann, cuxhaven, germany) were used (10 chickens per group). one group of animals was immunized twice (on day 0 and 21) by intramuscular (i.m.) injection (0.5 ml) of 10 mg for the next experiment, 70 one-day-old layer hens (white leghorn) were purchased from a local breeder. the chickens had been vaccinated against newcastle disease virus and infectious bronchitis virus at the age of one day according to the farm's routine and were raised in cvi's animal facility. at the age of 6 weeks, the birds were transported to the bsl-3 facility and allocated to 7 experimental groups of 10 birds each. the animals of six groups were immunized once (on day 21) or twice (on day 0 and 21) by i.m. injection of 10, 2 or 0.4 mg sh5 3 antigen adjuvanted with stimune. when immunized twice, the same doses were given on day 0 and 21. as a challenge control, one group was mock-vaccinated twice (on day 0 and 21) with pbs in stimune. four weeks after vaccination (day 49), blood samples were taken and the chickens were challenged as above and observed daily for clinical signs during 14 days. trachea and cloaca swabs were taken from each chicken on day 2, 4 and 7 p.i. female, specified pathogen-free (spf) 9-week-old balb/c mice (charles river laboratories; 10 animals per group) were immunized once (on day 21) or twice (on day 0 and 21) by i.m. injection (0.2 ml) of 2 mg sh5 3 adjuvanted with stimune. as a challenge control, one group of mice was mock vaccinated. three weeks after the vaccination, mice were anaesthetized with ketamin/xylazin by intraperitoneal injection and inoculated intranasally with 50 ml of h5n1a/viet nam/1194/04 containing ,10 median lethal dose (ld 50 ; 3.7 10 log tcid 50 ; provided by dr. alan hay from the who influenza centre at the national institute for medical research, london). the mice were weighed daily and examined for signs of illness during the next 14 days. clinical signs were recorded using a scoring system (0 = no clinical signs; 1 = rough coat; 2 = rough coat, less reactive, passive during handling; 3 = rough coat, rolled up, laboured breathing, passive during handling; 4 = rough coat, rolled up, laboured breathing, unresponsive). animals reaching a score of 4 were euthanized. surviving animals were bled and sacrificed on day 14 p.i. trachea and cloaca swabs were stored in cold tryptose phosphate broth supplemented with antibiotics. the medium was clarified by low-speed centrifugation and the supernatant was harvested, aliquoted and stored at 270uc. upon thawing, trachea and cloaca swabs sampled from the same bird on the same day were pooled and the viral rna was extracted from 200 ml using a magna pure lc total nucleic acid isolation kit (roche). subsequently, cdna was synthesized using reverse primer 59-cactgggcacggtgagc-39 and part of the m1 gene was amplified by running 45 cycles of light cycler pcr using primer 59-cttctaaccgaggtcgaaacgta-39 as the reverse primer in the presence of the taqman fluorescent probe 59-6fam-tcaggccccctcaaagccga-x-ph. negative and positive control samples were tested in parallel. the lower limit of detection was determined to be approximately 500 tcid 50 . some samples gave inconclusive results, meaning that they gave only a very weak signal (fluorescence,0.07) after .31 cycles. heat-inactivated immune sera from chicken blood samples were tested for hemagglutination inhibition (hi) activity with 1% chicken red blood cells and 4 hemagglutinating units (hau) of h5n1 (a/viet nam/1194/04 nibrg-14). in addition, the chicken sera were tested for hi activity using 8 hau (67 ng) of recombinant soluble trimeric ha protein. to this end the recombinant proteins were precomplexed with the anti-strep-tag antibody as described above, mixed with limiting dilutions of the chicken sera and incubated with 0.5% chicken red blood cells. red button formation was scored as evidence of hemagglutination. antibody titers were expressed as the reciprocal of the highest serum dilution showing hi. immune sera prepared from mouse blood samples were treated with vcna, heat-inactivated at 56uc for 30 min and tested by hi assay using 8 hau of sh5 3 as described above. total antibody titers against sh5 3 were determined by using a sh5 3 -specific elisa. to this end, 96-well nunc maxisorp plates coated with 0.5 mg sh5 3 per well were incubated with limiting dilutions of chicken or mouse sera. after extensive washing, the plates were incubated with goat-anti-chicken or goat-anti-mouse antibodies conjugated with hrp. peroxidase activity was visualized using tetramethylbenzidine substrate (biofx) and an elisa reader (el-808 [biotek]), reading the od at 450 nm. the od values of the 250-fold diluted samples, which were in the logarithmic phase of the curve, were plotted. in order to express soluble trimeric subtype 5 ha (sh5 3 ) in mammalian cells, the h5 ectodomain-coding sequence was first cloned into an appropriate expression vector. in the pcd5 vector used, the h5-sequence was preceded by a signal peptide-encoding sequence, to allow efficient secretion of the recombinant protein, and followed by sequences coding for the gcn4 isoleucine-zipper trimerizaton motif [21] and the strep-tag ii, the latter for purification purposes (fig. 1a) . expression of the h5 ectodomain was achieved by transient transfection of the expression plasmid into hek cells. expression and secretion of the h5 protein was verified by subjecting cell culture supernatant to gel electrophoresis followed by western blotting using an antibody directed against strep-tag ii (fig. 1b) . the results show that the recombinant h5 protein could be readily detected in the cell culture supernatant after transfection of the cells with the expression plasmid, but not after mock transfection. the secreted h5 protein was purified in a single step protocol by using streptactin sepharose beads. protein yields varied between 0.4-1 mg of recombinant protein per 100 ml cell culture medium. after purification of the h5 protein from the cell culture supernatant, the oligomeric state of the h5 protein was analyzed by gel filtration column chromatography (fig. 1c) . the bulk of the h5 protein eluted with the velocity of an oligomer, while only a minor fraction was found as aggregates in the void volume. the trimeric nature of the h5 oligomer was confirmed using blue-native gel electrophoresis followed by western blotting (fig. 1d) . when the h5 preparation was heat-denatured for increasing time periods prior to electrophoresis, the initially trimeric ha species dissociated into dimers and monomers. the biological activity of the purified sh5 3 was studied using a solid phase-binding assay with the sialylated blood glycoprotein fetuin. binding of sh5 3 was measured by means of the hrp conjugated to the anti-strep-tag ii antibody as detailed in the material and methods section. the h5 preparation exhibited a concentration dependent binding to the fetuin. this binding was sialic acid-dependent, as no binding was observed when the fetuin had been treated with neuraminidase (vcna; fig. 1e ). in conclusion, biologically active sh5 3 was efficiently produced using a mammalian expression system and readily purified. to examine the immunogenicity of sh5 3 and its potential as a vaccine a first experiment in chickens was performed, in which 10 chickens were vaccinated twice (on day 0 and 21) i.m. with 10 mg of stimune-adjuvanted sh5 3 and challenged 3 weeks later by i.n./i.t. inoculation of a lethal dose of a/viet nam/1194/04 virus. another 10 birds were mock-vaccinated to serve as challenge controls. as shown in fig. 2 , the boost vaccination with 10 mg sh5 3 conferred complete protection. none of the vaccinated chickens died or showed symptoms indicative of influenza-related disease, whereas all mockvaccinated chickens succumbed within 2 days. serological analysis showed that none of the mock-vaccinated animals contained antibodies against sh5 3 as determined by a sh5 3 -specific elisa (fig. 2b) . in contrast, all immunized animals demonstrated appreciable levels of ha antibodies already after a single immunization and these levels increased further after the boost. these total antibody levels against sh5 3 correlated nicely with the hi titers against h5n1 (fig. 2c) . all mock-vaccinated chickens had a hi titer below the detection limit. hi antibody titers observed after the sh5 3 boost reached with a maximum of 1024 and a minimum of 64, which was apparently still sufficient to protect the animal against the lethal challenge. interestingly, 50% of the birds developed hi antibody titers equal to 64 or higher already three weeks after the first vaccination. in addition, hi titers were also determined by using sh5 3 rather than h5n1 virus as the hemagglutinating agent, which gave essentially similar results (compare fig. 2d and 2c) , demonstrating the reliability of the assay. in summary, the results show that chickens vaccinated twice with sh5 3 are protected against a lethal challenge with h5n1. the hi titers observed suggested that one vaccination might already be sufficient to confer protection against hpai h5n1. these results prompted us to determine the minimal sh5 3 dose required to confer protection and to examine whether a single dose could already be sufficient. in the second vaccination-challenge experiment, chickens were thus vaccinated with 10, 2 or 0.4 mg of sh5 3 either once or twice and challenged four weeks later by infection with a lethal dose of a/viet nam/1194/04 as described above. the results are shown in fig. 3 . again, all mock-vaccinated birds succumbed to the infection within 2 days. vaccinating twice with a dose of 0.4 mg of sh5 3 was sufficient to protect 90% of the chickens against mortality, while all chickens survived when a dose of 2 or 10 mg was similarly administered (fig. 3a) . interestingly, also single vaccination with sh5 3 could induce sufficient immunity to protect chickens against lethal infection (fig. 3b) ; when a dose of 2 mg was given only one chicken died (90% protection), whereas a dose of 10 mg was protective to all birds. even after a single dose of 0.4 mg, 60% of the chickens were protected against death. serological analysis showed that protection against the lethal h5n1 challenge correlated well with the observed antibody levels against sh5 3 as determined by elisa (fig. 3c-d) and by hi assay (fig. 3e-f) . both assays revealed a dose-dependent antibody response, which was substantially enhanced after the booster immunization. relatively high ha antibody levels were observed after two immunizations, even with the lower dose, except in two animals, one of which did not survive the challenge (fig. 3c) . also after a single immunization, significant antibody levels were measured, except again in the low dose group. here, 5 animals had hardly measurable elisa titers. consistently, 4 of these animals succumbed to the challenge. also the one animal that died after a single immunization with 2 mg of sh5 3 did not have detectable sh5 3 antibodies. essentially the same results as with the elisa were obtained with the hi assay using sh5 3 as the hemagglutinating agent ( fig. 3e-f) . thus, the animals that succumbed to the lethal challenge after a single immunization also exhibited the lowest hi titers. we analyzed whether vaccination with sh5 3 decreased or prevented chickens from shedding challenge virus. for practical reasons, virus shedding was analyzed by a quantitative rt pcr rather than by measuring infectious virus titers. to this end, trachea and cloaca swabs were taken from the chickens of the vaccine dose titration experiment of fig. 3 at 2, 4 and 7 days after the challenge inoculation. the tracheal and the cloacal swab sampled from each chicken at each particular day were pooled and the presence of viral rna in the pooled swabs was analyzed using a quantitative rt pcr assay detecting the m1 gene. the results are shown in table 1 . of the chickens that received a booster vaccination, only 2 birds, both of which had received the lowest amount of antigen, tested positive. notably, these were the two animals that developed the lowest sh5 3 -specific antibody titers (fig. 3c) . one of these animals did not survive the challenge. virus shedding could not be detected in any of the other birds, although 3 swabs gave inconclusive results. of the chickens vaccinated only once, all animals that died tested positive. none of the birds vaccinated with 10 mg sh5 3 tested positive. of the chickens vaccinated with a lower dose and surviving, two tested positive, but only at day 2 p.i. in conclusion, the vaccinated birds that could control the lethal hpai h5n1 challenge infection exhibited minimum or no virus shedding. finally we examined whether sh5 3 would also confer protection in mice. therefore, 2 groups of 10 mice were vaccinated either once (on day 21) or twice (on day 0 and 21) with 2 mg of sh5 3 adjuvanted with stimune and challenged three weeks later by intranasal inoculation with ,10 ld 50 of h5n1 a/viet nam/ 1194/04. the percentage of mice surviving the infection, median clinical scores and body weights per group observed after the challenge inoculation are shown in fig. 4 . all mock-vaccinated mice succumbed to infection or had to be euthanized by day 9 p.i.. these mice showed severe clinical signs, including respiratory distress and significant weight loss, which continued until the animals died. a booster vaccination with sh5 3 provided 100% protection against the lethal dose of a/viet nam/1194/04; none of the mice showed significant signs of disease and their body weights remained constant. a single vaccination with sh5 3 did not protect mice against disease and concurrent weight loss; yet 40% of the mice survived. these mice started to recover from day 9 p.i. onwards as demonstrated by their regain of body weight. (fig. 4c ). fig. 4d shows the pre-challenge anti-sh5 3 antibody levels in individual serum samples,as determined by elisa. such antibodies could be detected in most vaccinated animals. however, after a single immunization, these levels remained very low compared to those in animals that received a booster vaccination. these results were essentially confirmed by determining the hi titers against sh5 3 in the same serum samples. with the exception of one animal, the serum of which demonstrated high levels of auto-agglutination, all mice displayed high hi titers when vaccinated twice and low hi titers when vaccinated once. thus, mice vaccinated twice with sh5 3 were protected against a lethal challenge with hpai h5n1, while a single vaccination provided partial protection. the differences in protection correlated with the observed serum hi titers. despite all the-understandable-attention drawn in the past year by media and scientific community to the new pandemic influenza a virus h1n1, a large influenza threat continues to be posed by hpai h5n1. in 2009, hpai h5n1 was found in wild birds in germany, china, mongolia and the russion federation, several outbreaks of the virus in poultry were reported in viet nam, hong kong, nepal, india, bangladesh, egypt, lao dpr, and cambodia, while h5n1 remained endemic in large areas of indonesia. that same year also dozens of confirmed human cases were reported (who timeline of major events; http://www.who.int/csr/disease/avian_influenza/ ai_timeline/en/index.html). even though the virus has so far remained restricted in its ability to infect humans and initiate efficient human-to-human transmission, its persistence and spread among wild birds and poultry holds a continual risk of the emergence of a pandemic strain. this threat can be reduced by vaccination of poultry against h5n1 as this would limit the propagation of the virus and minimize the risk of bird-to-human transmission. in addition, in case a human pandemic h5n1strain would emerge, there would be an immediate need for effective and reliable vaccines matching the pandemic strain. in the present study we evaluated the vaccine potential of a recombinant soluble h5 protein (sh5 3 ) to protect chickens and mice against a lethal infection with hpai h5n1. the recombinant ha vaccine, which has a short development cycle, proved to be protective after a single immunization in its natural host, while a booster vaccination was needed to confer complete protection in a mammalian model. in addition, the sh5 3 induced immunity prevented viral shedding from chickens. these promising results warrant further research into the development of recombinant soluble ha as a fast, safe and effective alternative vaccine not only against h5n1, but against other influenza a viruses as well. the recombinant ha expression cassette was constructed such that the ha protein was produced and secreted by cells in high yields as a bioactive trimer. the importance of the oligomeric state of the ha protein for the induction of neutralizing antibodies has recently been demonstrated [16] . high-molecular-weight oligomers and trimers, but not monomers, were found to efficiently induce neutralizing antibodies in mice. this difference was attributed to the preferential induction of antibodies against epitopes present in the monomeric, but not in the trimeric form [16] . while the soluble recombinant ha trimers were purified using metal affinity chromatography followed by ion-exchange chromatography in previous studies [16, 19] , we used a protocol based on the strep-tag system [23] . proteins with a strep-tag exhibit high affinity towards strep-tactin, an engineered form of streptavidin. by exploiting this highly specific interaction, streptagged proteins can be isolated in one step. furthermore, because the strep-tag elutes under gentle, physiological conditions it is especially suited for the generation of native proteins [24] , a characteristic that in the case of ha may contribute to the ability of the recombinant protein to induce neutralizing antibodies. the efficacy of the sh5 3 vaccine was first studied in chickens. adjuvanted with stimune, a water-in-oil adjuvant also known as specol, the sh5 3 protein formulation induced an immunity that was completely protective against a lethal h5n1 challenge after administration of two doses ($2 mg sh5 3 /dose). importantly, our vaccine preparation also protected chickens after a single immunization. while 100% of the chickens were protected after vaccination with 10 mg sh5 3 , 90% were protected when using 2 mg. similar ha doses were previously needed to protect chickens with a vaccine preparation consisting of full length ha proteins, which had been purified from insect cell cultures infected with recombinant baculovirus expressing the h5 gene, emulsified in a water-in-oil adjuvant [17] . these results are consistent with the observation that mammalian cell-produced ha trimers elicited similar levels of neutralizing antibodies as trimeric ha produced in insect cells [16] . the efficacy of our sh5 3 vaccine preparation was furthermore demonstrated by the absence of viral rna in the protected birds. this would imply that a vaccinated flock can pose a barrier against further spread of circulating virus. most conventional influenza vaccines require two vaccination rounds to produce antibody titers sufficiently high to confer full protection to chickens. in this regard, vaccination with sha 3 provides potential advantages over other vaccine approaches. however, the production costs per dose of sha 3 compared to egg-cultured inactivated whole-virus vaccines might be higher, even though recombinant protein expression in mammalian cell culture systems has been shown to be highly scalable and productive, with expression levels up to the order of grams of protein per liter [25, 26] . sha 3 vaccination could however be economically feasible in epidemic situations when millions of chickens have to be vaccinated individually, provided that a single preventive vaccination would suffice. moreover, vaccinated flocks housed in endangered regions rapidly achieve a state of protective immunity. this may be a particularly valuable feature in the event of a pandemic, when the virus transmission cycle needs to be interrupted as soon as possible and the risk of exposure of farmers, veterinarians and people in monitoring teams to potentially zoonotic hpai should be limited to the upmost extent. the efficacy of the sh5 3 vaccine was also studied in mice. the vaccine preparation was completely protective against a lethal h5n1 challenge after 2 doses. immunization with a single dose resulted in 40% survival. these differences in protection levels correlated with the observed anti-sh5 3 titers in the animals' sera. as the dose (2 mg) received by the mice is at least comparable, relative to their body weights, to the dose that conferred complete protection in chickens after a single immunization (10 mg), these results appear to suggest that the sh5 3 is more effective in conferring a protective immune response in chickens than in mice. the reason for this apparent discrepancy is unclear and warrants further investigations. in the only other study so far that used soluble ha trimers as a vaccine preparation in mice, much less protection against challenge with h5n1 was observed after two immunizations [19] . although the h5 trimer produced in this latter study differed from the one that we used, with respect to the trimerization motif (t4 foldon vs gcn4 trimerization motif) and the purification tag (his tag vs strep tag ii), the difference is more likely to be explained by the different adjuvant used (alum vs stimune). alum is known to induce low antibody titers when used with subunit vaccines [27] , while stimune has been reported to generate long-lasting, functional antibody responses [28, 29, 30] . stimune is however not licensed for human use. in conclusion, we have shown that the sh5 3 protein produced in mammalian cells elicited protective immune responses in mice and chickens when adjuvanted with stimune. chickens protected against the lethal h5n1 challenge also did not shed the virus at day 7 post infection. as these recombinant ha vaccines can be manufactured with high yields and a relatively short lead time, they offer an attractive alternative vaccination strategy, which will allow a rapid response to circulating and potentially pandemic influenza viruses. evolution and ecology of influenza a viruses avian influenza virus: of virus and bird ecology global epidemiology of human infections with highly pathogenic avian influenza a (h5n1) viruses pandemic influenza as a current threat influenza vaccines baculovirus-expressed influenza vaccine. a novel technology for safe and expeditious vaccine production for human use vaccines for pandemic influenza: summary of recent clinical trials immunization with live-attenuated influenza viruses 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2004 influenza virus comparative efficacy of neutralizing antibodies elicited by recombinant hemagglutinin proteins from avian h5n1 influenza virus baculovirus-derived hemagglutinin vaccines protect against lethal influenza infections by avian h5 and h7 subtypes nanodisc-incorporated hemagglutinin provides protective immunity against influenza virus infection glycans on influenza hemagglutinin affect receptor binding and immune response structure of coronavirus hemagglutinin-esterase offers insight into corona and influenza virus evolution a switch between two-, three-, and four-stranded coiled coils in gcn4 leucine zipper mutants structure and function in rhodopsin: kinetic studies of retinal binding to purified opsin mutants in defined phospholipid-detergent mixtures serve as probes of the retinal binding pocket overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems strep-tag ii for one-step affinity purification of active bhlhzip domain of human c-myc high level expression of functional human igms in human per expression systems for therapeutic glycoprotein production aluminum compounds as vaccine adjuvants investigation of several selected adjuvants regarding their efficacy and side effects for the production of a vaccine for parakeets to prevent a disease caused by a paramyxovirus type 3 immunization of syrian golden hamsters with f subunit vaccine of human metapneumovirus induces protection against challenge with homologous or heterologous strains evaluation of several adjuvants as alternatives to the use of freund's adjuvant in rabbits the authors would like to thank marius dwars for providing us with chicken erythrocytes and the groups of peter van rossum and simon riemersma for assistance with the animal experiments. conceived and designed the experiments: lc rdv pjmr camdh. performed the experiments: lc rdv eadbl ar. analyzed the data: lc rdv camdh. wrote the paper: lc pjmr camdh. key: cord-000877-usz7pnvu authors: abdel-moneim, ahmed s.; kamel, mahmoud m.; al-ghamdi, abdullhamid s.; al-malky, mater i. r. title: detection of bocavirus in children suffering from acute respiratory tract infections in saudi arabia date: 2013-01-30 journal: plos one doi: 10.1371/journal.pone.0055500 sha: doc_id: 877 cord_uid: usz7pnvu human bocavirus (hbov) was recently discovered in children with respiratory distress and/or diarrhea. to our knowledge, no previous study has reported the existence of bocavirus in saudi arabia. swabs samples from 80 children with respiratory tract infections were examined for the presence of hbov. real-time polymerase chain reaction was used as a sensitive method to detect the hbov. direct gene sequencing was used to determine the genotype of the detected virus isolates. hbov was detected in 22.5% of the examined patients. the np1 partial gene sequence from all patients showed that the circulated strains were related to hbov-1 genotype. most of hbov infected patients showed evidence of mixed coinfection with other viral pathogens. the current study clearly demonstrated that genetically conserved hbov1 circulates in saudi arabia. interestingly, most of the hbov1 infected cases were associated with high rates of co-infections with other viruses. bocavirus is a single-stranded dna virus belonging to the family parvoviridae, subfamily parvovirinae, genus bocavirus. bocaviruses are unique among parvoviruses because they contain a third orf between the non-structural and structural coding regions [1] [2] . the genus bocavirus includes viruses that infect bovine, canine, feline, porcine and some simian species as well as sea lions [3] [4] [5] [6] [7] [8] . human bocavirus (hbov) was first found in children with acute respiratory tract infections in 2005 [1] . it was then detected in children with respiratory tract infections in addition to gastroenteritis worldwide [9] [10] [11] [12] . the virus exists in four different serotypes hbov1-4 [1] [2] [13] [14] . although hbov 1 and 2 were reported in respiratory samples, all the 4 genotypes of hbov have been identified in children with acute gastroenteritis. hbov has been reported in various countries, indicating its worldwide endemic nature. the virus has been identified in europe [15] [16] [17] , america [18] [19] , asia [9, 20] , australia [21] [22] , africa [23] , and the middle east [24] . the prevalence of hbov ranges between 1.5 to 19.3% [18, 25] . primary infection with hbov seems to occur early in life and children between the ages of 6-24 months seem to be mostly affected [9] [10] , but older children can also be infected. newborn children may become protected by maternally derived antibodies [9] . hbov infections are rarely found in adults [26] [27] . lindner et al. detected anti-hbov antibodies in 94% of healthy blood donors .19 years of age [28] . hbov detection has been mostly performed on nasopharyngeal aspirates and swabs and relies mostly on classical [10, 18, 19, 21, 22, 26] and real-time pcr [10, 23, 29] . real-time pcr possesses many advantages over conventional pcr, as it offers greater sensitivity, specificity, and reduced expenditure of time. the current study aims to screen the epidemiological status and molecular phylogeny of hbov isolates prevailing in pediatric patients with respiratory infection in saudi arabia. the current study investigated the prevalence of hbov in patients suffering from respiratory tract infections in saudi arabia. the presence of the major viral causes of the respiratory distress in hbov positive cases was also screened. hbov was detected in 18/ 80 of the examined patients (22.5%) with ages ranging from 2 months to 10 years, (table 1-2). clinical findings for hbovpositive patients were indistinguishable from those for patients with other respiratory viruses. previously, hbov has been detected in samples from patients aged between 5 months and 2 years [1, 29] . ma et al, speculated that the antibody against hbov derived from the mother might protect children under 5 months of age from hbov infection [9] , however, we detected hbov in two cases below 5 months: in a 2-month-old and 4-month-old child ( table 1 -2) that may indicate the possibility of hbov infection in very young children. the rate of hbov in respiratory tract infections has been reported to be 1.5 to 19.3% [18, 25] . real-time pcr was used in the current study to screen hbov due to its high diagnostic sensitivity that could be responsible for the higher rate of hbov infection in saudi arabia than the widely accepted upper limit of infection rates worldwide. meanwhile, a recent study showed 21.5% prevalence among children [30] . the evidence of hbov as the main initiator of the disease in the infected cases is still uncertain because of its high co-infection rate with other pathogens, and it remains unclear whether hbov is the sole etiologic agent or just a concomitant virus bystander. in previous studies, none of the nasal swabs obtained from healthy children yielded a positive hbov test. this suggests that hbov is not a frequent commensal virus inhabiting the respiratory tract [16, 19] . hbov infections are frequently present in concomitant with other viruses and often occur in more than 50% of the tested samples [31] . in the current study, only one case was found to be infected only by hbov as a single virus entity while most of isolates (17/18) showed coinfection with other viral pathogens. the most frequently detected co-pathogens were rsv (13/18; 72.2%), iav (12/18 cases, 66.66%), respiratory adenovirus (6/18 cases, 33.33%) while only 1/18 (5.5%) case was coinfected with piv-3 and none was coninfected with piv-1 ( table 2 ). it is assumed that the rate and frequency of coinfections may be higher if more viruses were screened. consistent with other studies [1, 16] , the prevalence rate of bocavirus was higher in children under 2 years of age (table 1) . partial np-1 gene sequence of the eighteen detected hbov strains were obtained in our study. multisequence analysis showed complete identity (100%) between each other, and phylogenetic analysis demonstrated that they belonged to hbov1 (data not shown). blast analysis revealed complete homology to the published sequence of hbov1. furthermore, the phylogenetic analysis results of three selected sequences showed that the saudi hbov1 strains obtained from respiratory samples belonged to group i human bocaviruses (fig. 1) . to the best of our knowledge, this is the first report of hbov1 in saudi arabia. continuous surveillance and genome sequence analysis are needed to obtain more information on the genotypic variation and molecular evolution of hbov in the country. the study protocol was approved by the medical ethics review board of the college of medicine, taif university and by the pediatric hospital ethics committee in accordance with the guidelines for the protection of human subjects. informed written consents from the next of kin of the participants involved in the study were taken. nasopharyngeal swabs from 80 children suffered from moderate to severe lower respiratory tract infections were collected from january to may 2012 from the governmental pediatric hospital-al-taif, saudi arabia. the children's age ranged from two months to ten years of age. clinical manifestations and case histories were recorded. individual swabs were kept in 1 ml sterile saline containing gentamycin sulphate. swabs were routinely processed and kept at 280uc until further analysis. the real-time pcr assay was performed using commercial, taqman hydrolysis probe based, real time pcr bocavirus detection kit (liferiver, shanghai, china) in eppendorf master-cyclerh ep realplex 2 . the detection of the amplified amplicon was performed in fluorimeter channel fam with the fluorescent quencher bhq1. amplification reactions were performed in a volume of 25 ml containing 2.5 ml of dna template, 21.5 ml reaction mix, 0.4 ml enzyme mix, 1 ml internal control according to the manufacturer's instructions. the thermal cycling conditions were as follows: 2 min at 37uc, an initial denaturation of 2 min at 94uc and 40-cycles of 15 sec at 93uc and annealing/elongation step of 1 min at 60uc. hbov positive samples were screened for the presence of respiratory syncytial virus (rsv), influenza a virus (iav), parinfluenza virus 1(piv-1) and parainfluenza virus 3(piv-3), as well as respiratory enteric virus (adenv) using real-time pcr kits (shanghai zj bio-tech co., ltd). pcr amplification of a 354-base pair fragment of the np1 was performed as described previously [1] . the reaction mix contained 20 pmol of each primer and dna master mix (koma bioteck, inc., seoul, korea). the thermal cycling conditions were as follows: an initial denaturation of 5 min at 94uc, 35 cycles of 1 min at 94uc, 1 min at 54uc and 2 min at 72uc, final extension of 10 min at 72uc.positive pcr products were purified using a qiaquick pcr purification kit (qiagen) and were sequenced commercially (macrogen inc., seoul, korea). the nucleotide sequences of the ns1 gene were compared with those of hbov strains available at the genbank site. phylogenetic analyses were conducted with mega, version 4.1. the 3/18 partial sequences of the ns1 gene were submitted to genbank (accession numbers jx982976-jx982978). cloning of a human parvovirus by molecular screening of respiratory tract samples human bocaviruses are highly diverse, dispersed, recombination prone, and prevalent in enteric infections virus taxonomy: the eighth report of the international committee on taxonomy of viruses identification and nearly full-length genome characterization of novel porcine bocaviruses identification and characterization of a new bocavirus species in gorillas widespread infection with homologues of human parvoviruses b19, parv4, and human bocavirus of chimpanzees and gorillas in the wild the fecal viral flora of california sea lions identification and characterization of bocaviruses in cats and dogs reveals a novel feline bocavirus and a novel genetic group of canine bocavirus detection of human bocavirus in japanese children with lower respiratory tract infections epidemiological profile and clinical associations of human bocavirus and other human parvoviruses human bocavirus, a respiratory and enteric virus human bocavirus in children hospitalized for acute gastroenteritis: a case-control study a novel bocavirus associated with acute gastroenteritis in australian children a newly identified bocavirus species in human stool human bocavirus in french children human bocavirus in italian patients with respiratory diseases isolation of human bocavirus from swiss infants with respiratory infections human bocavirus infection human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus quantification of human bocavirus in lower respiratory tract infections in china frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections evidence of human coronavirus hku1 and human bocavirus in australian children human bocavirus in hospitalized children human bocavirus infection among children frequent detection of viral coinfection in children hospitalized with acute respiratory tract infection using a real-time polymerase chain reaction severe pneumonia and human bocavirus in adult surveillance and genome analysis of human bocavirus in patients with respiratory infection in guangzhou, china humoral immune response against human bocavirus vp2 virus-like particles human bocavirus and acute wheezing in children high prevalence of human bocavirus 1 in infants with lower acute respiratory tract disease in argentina human bocavirus commonly involved in multiple viral airway infections key: cord-001716-lbtdex4p authors: gilca, rodica; skowronski, danuta m.; douville-fradet, monique; amini, rachid; boulianne, nicole; rouleau, isabelle; martineau, christine; charest, hugues; de serres, gaston title: mid-season estimates of influenza vaccine effectiveness against influenza a(h3n2) hospitalization in the elderly in quebec, canada, january 2015 date: 2015-07-22 journal: plos one doi: 10.1371/journal.pone.0132195 sha: doc_id: 1716 cord_uid: lbtdex4p background: the 2014/15 influenza season in canada was characterized by an early epidemic due to vaccine-mismatched influenza a(h3n2) viruses, disproportionately affecting elderly individuals ≥65-years-old. we assessed vaccine effectiveness (ve) against a(h3n2) hospitalization among elderly individuals during the peak weeks of the 2014/15 epidemic in quebec, canada. methods: nasal specimens and clinical/epidemiological data were collected within 7 days of illness onset from elderly patients admitted with respiratory symptoms to one of four participating hospitals between november 30, 2014 and january 13, 2015. cases tested rt-pcr positive for influenza a(h3n2) and controls tested negative for any influenza. ve was assessed by test-negative case-control design. results: there were 314 participants including 186 cases (62% vaccinated) and 128 controls (59% vaccinated) included in primary ve analysis. median age was 81.5 years, two-thirds were admitted from the community and 91% had underlying comorbidity. crude ve against a(h3n2) hospitalization was -17% (95%ci: -86% to 26%), decreasing to -23% (95%ci: -99 to 23%) with adjustment for age and comorbidity, and to -39% (95%ci: -142 to 20%) with additional adjustment for specimen collection interval, calendar time, type of residence and hospital. in sensitivity analyses, ve estimates were improved toward the null with restriction to participants admitted from the community (-2%; 95%ci: -105 to 49%) or with specimen collection ≤4 days since illness onset (8%; 95%ci: -104 to 43%) but further from the null with restriction to participants with comorbidity (-51%; 95%ci: -169 to 15%). conclusion: the 2014/15 mismatched influenza vaccine provided elderly patients with no cross-protection against hospitalization with the a(h3n2) epidemic strain, reinforcing the need for adjunct protective measures among high-risk individuals and improved vaccine options. the 2014/15 influenza season in quebec, as elsewhere in canada, was characterized by an early and intense influenza epidemic due almost exclusively to antigenically-drifted and vaccinemismatched a(h3n2) viruses [1, 2] . as expected with influenza seasons dominated by a (h3n2) subtype activity, the elderly 65 years-old were disproportionately affected by excess hospitalizations and deaths [3, 4] . by mid-season in some jurisdictions, the number of longterm care facility (ltcf) outbreaks in 2014/15 exceeded even the full-season tallies of recent prior seasons, including those also distinguished by dominant, vaccine-mismatched a(h3n2) activity, such as 2012/13 [2, 5, 6] . in response to surveillance signals suggesting suboptimal vaccine performance, several midseason analyses assessed effectiveness of the 2014/15 influenza vaccine against the a(h3n2) epidemic strain. canada's sentinel physician surveillance network (spsn) measured vaccine effectiveness (ve) against medically-attended laboratory-confirmed outpatient a(h3n2) illness of -8% (95%ci:-50-23%) overall and 2% (95%ci:-49-36%) in non-elderly (<65-year-old) adults, indicating little or no vaccine protection even among individuals capable of mounting an effective immune response [2] . the canadian immunization research network (cirn) assessed ve against influenza a(h3n2)-related hospitalization, reporting estimates partially adjusted for age and comorbidity of 8% (95%ci:-102-58%) in non-elderly adults, substantially lower in elderly adults at -33% (95%ci:-104-13%) [7] . although canadian mid-season inpatient and outpatient ve findings for the 2014/15 season have been consistent with null vaccine effects (statistically non-significant and spanning zero) in both age groups, the cirn finding of a lower and negative point estimate of ve against a(h3n2) hospitalization in the elderly, more closely broaching statistical significance, warrants further clarification. here we assess ve against a(h3n2) hospitalization in the elderly during the peak epidemic weeks of the 2014/15 season in quebec, canada. this study was conducted under the surveillance mandate of the quebec ministry of health without requirement for institutional review board approval. as part of routine patient care, all patients admitted to hospitals participating in the project with respiratory symptoms are assessed for influenza by per-nasal specimen collection at local laboratory. all patients admitted 24 hours at one of these sentinel hospitals with cough, sore throat, or fever/feverishness of unknown etiology were invited by a research nurse to participate in the study. authors themselves did not have direct contact with patients or access to patient identifying information and no additional samples were collected for the purposes of research. verbal consent was elicited from the patient and/or guardian to test the specimen for influenza and other respiratory viruses at the provincial public health laboratory and to record demographic and clinical information such as influenza vaccination, and date of illness onset on standardized questionnaires. patient charts were also reviewed at discharge to collect information on clinical progress. verbal consent was documented on the questionnaire. capacity to consent was determined by the nurse; the number of patients unable or refusing to give consent was recorded weekly on recruitment files and qualified as exclusions. the annual recruitment period for the quebec sentinel hospital surveillance system, implemented since 2011, spans the peak of the influenza season defined as two consecutive weeks during which at least 15% of weekly samples from the quebec sentinel laboratory surveillance system test positive for influenza [8] . for the 2014/15 season, this threshold was surpassed beginning in week 48 (november 23-29, 2014) (fig 1) . systematic respiratory virus surveillance was then conducted among the four acute-care regional hospitals (2 community, 2 academic/tertiary care) serving as sentinel sites and providing care to about 10% of the quebec population overall. only elderly participants with specimen collection within 7 days of illness onset were eligible for inclusion in primary ve analysis. patients with respiratory symptom onset >72 hours after hospital admission were considered healthcare-associated and were excluded. elderly individuals in quebec are eligible for publicly-funded trivalent influenza vaccine (tiv). inactivated split or subunit tiv is primarily available for this age group but for the second consecutive season, elderly patients in ltcfs received an mf-59 adjuvanted subunit tiv. per recommendation of the world health organization, all 2014/15 tiv retained the same three influenza vaccine antigens as were also used in 2013/14, including the a/texas/50/2012 (h3n2)-like strain [9] . nasal specimens were tested at the provincial public health laboratory using the luminex rvp fast version-2 assay which detects influenza a and b and 14 other respiratory viruses. details are presented elsewhere [8] . influenza a subtypes were confirmed by reverse transcription polymerase chain reaction (rt-pcr) to detect a(h3) and a(h1)pdm2009 subtypes where otherwise non-subtypeable by luminex [10, 11] . comparison of proportions was by χ2 or fisher's exact test and for continuous variables was by wilcoxon and kruskal-wallis nonparametric tests. ve was estimated by test-negative case-control design [12] . patients diagnosed with laboratory-confirmed influenza a(h3n2) were considered cases and those testing negative for any influenza were controls. ve was defined as (1-odds ratio)x100% for hospitalization with laboratory-confirmed influenza a(h3n2) among vaccinated compared to non-vaccinated patients. participants who received the 2014/15 tiv 2weeks before illness onset were considered vaccinated. those for whom vaccination timing was unknown or <2weeks before onset were excluded but explored in sensitivity analyses as indicator variables. multivariable analyses by logistic regression adjusted for age, underlying comorbidity placing individuals at higher risk of influenza-related complications [13] , interval between symptom onset and specimen collection (4 days, 5-7 days), hospital site, epidemic week based on hospital admission date (49-51, 52, 53 and 1-2), and primary residence (community, ltcf or other institutional/group setting). sensitivity analyses explored ve by type of residence, comorbidity, specimen collection interval, and re-classification of patients with unknown vaccination status as vaccinated or as unvaccinated. during the recruitment period, nasal specimens were collected from 714 elderly patients among whom 537 were eligible for study participation. further inclusion and exclusion criteria as applied to the current data set for the primary ve analysis are shown in the median age of participants was 81.5 years and 30% were 85years-old (table 1) . twothirds were community-dwelling and underlying comorbidity was reported in 91%. the 2014/ 15 tiv was received by 62% of cases and 59% of controls, the latter comparable to coverage estimates for quebec elderly overall (62%) [14] . median age of vaccinated elderly was slightly greater than unvaccinated participants (81.8 vs. 79.0 years; p = 0.0003). a smaller proportion of community-dwelling elderly were vaccinated in 2014/15 compared to those in ltcf (55% vs. 84%; p = 0.002), more comparable to those living in other kinds of institutional/group settings (68%; p = 0.04). those with underlying comorbidity were more often vaccinated compared to those without (63% vs. 37%; p = 0.008). there was also variation in vaccination coverage by hospital site, lowest in hospital a, located in a region that was affected earliest in the epidemic. virtually all who were vaccinated in 2014/15 reported also receiving tiv at least once in the past (98% overall, 99% of those for whom this was known). fewer cases than controls had underlying comorbidity (89% vs. 95%;p = 0.04) and cases were hospitalized earlier in the epidemic. conversely, vaccinated participants were hospitalized later in the epidemic (table 1) . a greater proportion of cases than controls were vaccinated among those admitted to icu (80% vs. 60%) or dying during their hospital stay (83% vs. 60%), but sample size was small and differences were not statistically significant (p>0.05). specimen inclusion/exclusion criteria for primary vaccine effectiveness analysis. 1 patients whom nurses were not able to approach because of early discharge or other operational considerations (i.e. workload demands during peak weeks of respiratory admissions); 2 symptoms onset >72h after hospital admission; 3 exclusions are not mutually exclusive; 4 15 respiratory syncytial viruses, 13 entero/rhinoviruses, crude ve against elderly a(h3n2) hospitalization was -17% (95%ci: -86 to 26%). when adjusted for age and comorbidity, ve was -23% (95%ci: -99% to 23%) and with full adjustment for recognized confounders was -39% (95%ci: -142% to 20%)( table 2 ). in sensitivity analyses, fully-adjusted ve point estimates were improved toward the null with restriction to interval from illness onset to specimen collection participants admitted to hospital from the community (-2%; 95%ci: -105 to 49%) or with specimen collection 4 days since illness onset (-8%; 95%ci: -104 to 43%). conversely, the fullyadjusted ve point estimate varied further away from the null with restriction to participants with underlying comorbidity (-51%; 95%ci: -169 to 15%). however, in all analyses confidence intervals spanned the null and were wider as expected with inclusion of more covariates and with subset restriction. this study corroborates earlier outpatient and inpatient findings from canada showing that the 2014/15 influenza vaccine provided little or no protection against the dominant but vaccine-mismatched a(h3n2) strain. here, we report no vaccine protection against the serious outcome of hospitalization with the 2014/15 antigenically-distinct a(h3n2) epidemic strain among elderly citizens of quebec, canada. our ve point estimates against elderly a(h3n2) hospitalization for the 2014/15 season, whether partially-adjusted for age and comorbidity alone (-23%; 95%ci: -99% to 23%) or for a fuller range of potential confounders (-39%; 95%ci: -142 to 20%) are similar (within 10%) of the partially-adjusted ve estimate reported by the cirn hospital-based network (-33%; 95% ci: -104 to 13%) [7] . these ve estimates are substantially lower than cirn estimates for nonelderly adults for 2014/15 (8%; 95%ci: -102 to 58%). they are also lower than cirn estimates for the elderly for prior seasons including their mid-season 2013/14 ve against a(h1n1) pdm09-related hospitalization (63%; 95%ci: 35 to 79%) [15] or as reported by cirn in conference proceedings for the 2012/13 season also against vaccine-mismatched a(h3n2) hospitalization (crude ve = 29%; 95%ci: 18 to 39%) [16] . our 2014/15 ve estimates are also lower than mid-season ve estimates against outpatient medical visits reported among younger adults by canada's spsn (2%; 95%ci: -49% to 36%) [2] , by the united states (us) (12%; 95%ci: -26 to 39%) [17] or by the united kingdom overall (-2%; 95%ci: −56 to 33%) [18] . ve estimates specific to the elderly were not separately reported in any of these outpatient studies, but among participants 50 years-old in the us, ve against outpatient a(h3n2) illness was 14% (95%ci: -31 to 43%) [17] . none of these ve estimates are statistically significant and confidence intervals broadly overlap so that it is not possible to conclude whether ve in hospitalized elderly patients is lower than outpatient ve estimates for elderly or non-elderly adults. taken together, however, these results challenge assertions [19, 20] that vaccine provides better protection against severe complications than against infection per se, particularly during vaccinemismatched seasons. in fact, even in young adults in canada, point estimates of ve against influenza-confirmed hospitalizations reported by cirn have been consistently lower than estimates against ambulatory illness published by the spsn each season since 2011 [2, 15, 16, 21] . consistent findings of negative ve point estimates in relation to hospitalization outcomes in the elderly during the 2014/15 season require some explanation. confidence intervals around these negative point estimates are wide and cross the null, but broach statistical significance in some analyses. chance statistical variation, methodological bias, or a true biological phenomenon are among possible explanations. with vaccine coverage of 60%, our sample of just over 300 participants, approximately equally cases and controls, would have been sufficient to detect a statistically-significant ve of at least 50% (in either direction of the null), with 80% power. sample size requirements increase dramatically as ve more closely approaches zero, as illustrated also by cirn's failure to achieve significance despite more than triple the sample size [7] . in that regard negative ve estimates may reflect statistical variation around a true null effect although we cannot rule out that with additional sample size, ve estimates may have crossed toward statistically-significant negative ve. our study was predicated on the test-negative design, and, as for all observational studies, residual bias and confounding cannot be ruled out. with adjustment for recognized confounders ve estimates were generally reduced but showed greater variability, likely owing to greater sample size requirements to support multiple covariates. it is reassuring, however, that ve estimates were improved toward the null with restriction to more uniform and majority subgroups of participants including those with primary residence in the community, comprising two-thirds of our data set, or among participants with specimen collection 4 days since illness onset, comprising three-quarters of participants. ve was reduced among patients with comorbidity, comprising >90% of participants but including a wide variety of conditions, in whom bias related to propensity to vaccinate or to hospitalize likely varies a lot. factors confounding the association between vaccination and hospitalization risk are more complex than for outpatient medical visits, and this requires more in-depth evaluation generally in the interpretation of ve estimates for hospitalization outcomes, especially for seniors. although statistically most consistent with a null vaccine effect overall, it is also prudent to consider whether negative point estimates of ve in the elderly may reflect a true epidemiological finding. in subgroup analysis for the current 2014/15 season, canada's spsn also reported ve estimates against medically-attended a(h3n2) illness that were reduced and slightly negative in patients vaccinated in both 2014/15 and 2013/14 (-15%; 95%ci: -67% to 21%) but positive (i.e. protective) in participants who had received only the 2014/15 vaccine (43%; 95%ci: -29% to 75%) [2] . spsn findings in repeat vaccine recipients for the 2014/15 season were also associated with wide and overlapping confidence intervals, consistent also with null vaccine effects in both subgroups. a number of other recent studies in canada, the united states, and europe have also reported interference from prior receipt of seasonal influenza vaccine [22] [23] [24] [25] [26] . while these other studies showed negative interference that sometimes reduces protection (i.e. a lower but still positive ve point estimate), a negative ve estimate would suggest that vaccine interference may sometimes also be associated with increased disease risk. for several decades, the elderly have been a highly and recurrently immunized group, and virtually all of the vaccinated elderly in our study had received tiv in the past. we were not able to stratify by current and/or prior vaccination history and neither has cirn explored these possible influences, which require further evaluation. the most noteworthy precedent of negative ve arose during the spring-summer of 2009, and warrants mention here to highlight differences from the current context. prior receipt of 2008/09 seasonal vaccine was associated with negative ve against the markedly mismatched 2009 pandemic a(h1n1) virus, observed predominantly in non-elderly individuals. this observation was reported by canada's spsn and at least four other studies in canada [27] , subsequently also from hong kong [28] , the us [29] and japan [30] and thereafter also in a randomized ferret trial [31] . the canadian studies showed statistically significant two-fold increased risk (ve of -100%) for medically-attended outpatient a(h1n1)pdm09 illness but risk was not increased for hospitalization outcomes. this is different from the current season's finding of lesser magnitude, statistically non-significant and more variable vaccine effects (ve of -39%) against a(h3n2) hospitalization in the elderly, for which chance variation around the null and/or methodological considerations may be more likely explanations. additional studies are needed to definitively resolve the potential concern of vaccine-associated increased risk and/or to clarify the conditions of vaccine mismatch under which it may recur (e.g. antigenic distances [32] , immunological cohort effects based on original antigenic prime vs. boost exposures). in fact, the underlying mechanisms and virus-host immunological interactions to explain variability in disease burden and ve by age and influenza subtype require better understanding generally. the reason why elderly people suffer disproportionately from a(h3n2) subtype infections, as per the current season [3, 4] remains a longstanding but unanswered question. immuno-senescence alone is unlikely to provide the complete explanation since the same exceptional vulnerability is not observed in the elderly in relation to influenza a(h1n1) infection. our study has other limitations. a substantial proportion of elderly patients were excluded because they were unable to recall or report important information, such as vaccination status and date of symptom onset. however, their inclusion in indicator sensitivity analyses did not meaningfully alter ve estimates. vaccine status was self-reported and this may have resulted in exposure misclassification; however, this information was collected prior to influenza diagnosis, minimizing differential misclassification. studies in other settings, including hospitalized elderly, have reported consistency between self-reported and registry-based influenza vaccine status although that may not directly apply here [33, 34] . we think accuracy of self-reported vaccine status in our study may even be better because information was collected from patients within a shorter period of time since vaccination campaign as compared to the studies cited above. in addition, vaccination coverage in influenza-negative patients enrolled in our study was consistent with that reported from other sources during previous years of the study [8, 14] . our study was conducted during peak weeks of the influenza season; this may raise concerns about the particular impact of outcome misclassification (i.e. false negative cases) on our ve estimates. we have previously shown that other respiratory viruses remain an important cause of respiratory hospitalization even during peak weeks of the influenza epidemic [8] . the assay we used for influenza diagnosis has been reported elsewhere to have sensitivity >95% (98% for influenza a virus), with comparable proportions testing respiratory virus positive as per individual nucleic acid amplification testing across age groups [35] . even with sensitivity for influenza detection as low as 70%, in the context of near-perfect specificity, outcome misclassification has been shown to have negligible impact on ve estimates [36] . although test sensitivity may be lower in elderly adults, it is unlikely to drop below 70%. finally, in the test-negative study design, when patients with influenza are not censored and can also contribute as controls during another respiratory illness episode, the odds ratio directly estimates the relative risk and is not affected by the rare disease assumption [12] . in conclusion, we report negative point estimates that are statistically non-significant for ve against a(h3n2) hospitalization in the elderly for the 2014/15 season. our findings are consistent with other outpatient and inpatient studies from canada, indicating little or, as here, no vaccine protection against the dominant but vaccine-mismatched a(h3n2) epidemic strain. these findings reinforce the need for adjunct measures to protect high-risk individuals, including the elderly, from serious influenza outcomes during vaccine-mismatched seasons and for improved vaccine options over the long-term. public health agency of canada interim estimates of 2014/15 vaccine effectiveness against influenza a(h3n2) from canada's sentinel physician surveillance network influenza-associated hospitalizations in the united states mortality associated with influenza and respiratory syncytial virus in the united states low 2012-13 influenza vaccine effectiveness associated with mutation in the egg-adapted h3n2 vaccine strain not antigenic drift in circulating viruses interim estimates of 2014/15 influenza vaccine effectiveness in preventing laboratory-confirmed influenza-related hospitalization from the serious outcomes surveillance network of the canadian immunization research network other respiratory viruses are important contributors to adult respiratory hospitalizations and mortality even during peak weeks of the influenza season recommended composition of influenza virus vaccines for use in the 2014-2015 northern hemisphere influenza season. geneva: world health organization evidence of viremia in 2 cases of severe pandemic influenza a h1n1/09 real-time rt-pcr (rrt-pcr) protocol for influenza; revised the test-negative design: validity, accuracy and precision of vaccine efficacy estimates compared to the gold standard of randomised placebo-controlled clinical trials national advisory comittee on immunization. statement on seasonal influenza vaccine for enquête québécoise sur la vaccination contre la grippe saisonnière et le pneumocoque interim estimates of 2013/ 14 influenza clinical severity and vaccine effectiveness in the prevention of laboratory-confirmed influenza-related hospitalisation effectiveness of 2012/13 seasonal influenza vaccines in the prevention of influenza-related hospitalization in canadian adults: a public health agency of canada/canadian institutes of health research serious outcomes surveillance network study. option for the control of influenza early estimates of seasonal influenza vaccine effectiveness-united states low effectiveness of seasonal influenza vaccine in preventing laboratory-confirmed influenza in primary care in the united kingdom: 2014/15 mid-season results influenza-the need to stay ahead of the virus cochrane rearranged: support for policies to vaccinate elderly people against influenza interim estimates of 2013/14 vaccine effectiveness against influenza a(h1n1)pdm09 from canada sentinel surveillance network impact of repeated vaccination on vaccine effectiveness against influenza a(h3n2) and b during 8 seasons influenza vaccine effectiveness in the community and the household influenza vaccine effectiveness in the 2011-2012 season: protection against each circulating virus and the effect of prior vaccination on estimates /13 influenza vaccine effectiveness against hospitalised influenza a(h1n1)pdm09, a(h3n2) and b: estimates from a european network of hospitals association between the 2008-09 seasonal influenza vaccine and pandemic h1n1 illness during spring-summer 2009: four observational studies from canada seasonal influenza vaccine and increased risk of pandemic a/h1n1-related illness: first detection of the association in british columbia protective efficacy of seasonal influenza vaccination against seasonal and pandemic influenza virus during the 2009 in hong kong clinical and epidemiologic characteristics of an outbreak of novel h1n1 (swine origin) influenza a virus among united states military beneficiaries association between seasonal influenza vaccination in 2008-2009 and pandemic influenza a (h1n1) 2009 infection among school students from kobe randomized controlled ferret study to assess the direct impact of 2008-09 trivalent inactivated influenza vaccine on a (h1n1)pdm09 disease risk variable efficacy of repeated annual influenza vaccination validity of self-reported influenza and pneumococcal vaccination status among a cohort of hospitalized elderly inpatients evaluation of self-reported and registrybased influenza vaccination status in a wisconsin cohort comparison of the luminex xtag respiratory viral panel with in-house nucleic acid amplification tests for diagnosis of respiratory virus infections methodologic issues regarding the use of three observational study designs to assess influenza vaccine effectiveness we thank the quebec ministry of health (ministère de la santé et des services sociaux du québec) for providing financial support for this surveillance project. we are grateful to all participating hospitals and the molecular biology unit at quebec public health laboratory (laboratoire de santé publique du québec) for their dedication and strenuous effort during the peak of influenza season. key: cord-002624-59nznqsd authors: ti, jinfeng; li, zhijie; li, xiuli; lu, yunjian; diao, youxiang; li, fang title: identification of one b-cell epitope from ns1 protein of duck tembusu virus with monoclonal antibodies date: 2017-07-26 journal: plos one doi: 10.1371/journal.pone.0181177 sha: doc_id: 2624 cord_uid: 59nznqsd this study describes the identification of one linear b-cell epitope on tmuv ns1 protein with monoclonal antibody (mab) 3g2 by indirect enzyme-linked immunosorbent assay (elisa). in this study, ns1 protein was expressed in prokaryotic expression system and purified. one mab against ns1 protein was generated from balb/c mice immunized with recombinant protein ns1. a set of 35 partially-overlapping polypeptides covering the entire ns1 protein was expressed with pgex-6p-1 vector and screened with mab 3g2. one polypeptide against the mab was acquired and identified by indirect elisa and western-blot. to map the epitope accurately, one or two amino acid residues were removed from the carboxy and amino terminal of polypeptide sequentially. a series of truncated oligopeptides were expressed and purified. the minimal determinant of the linear b cell epitope was recognized and identified with mab 3g2. the accurate linear b-cell epitope was (269)dekeiv(274) located in ns1 protein. furthermore, sequence alignment showed that the epitope was highly conserved and specific among tmuv strains and other flavivirus respectively. the linear b-cell epitope of tmuv ns1 protein could benefit the development of new vaccines and diagnostic assays. tembusu virus infection in ducks is caused by tembusu virus (tmuv), which was first reported in april 2010 in china [1] . the virus can infect more varieties of ducks such as beijing duck, golden duck, shaoxing duck, cherry valley, campbell ducks, jinyun duck, etc. laying ducks infected mainly demonstrated drops in egg production, follicular rupture and bleeding, and yolk peritonitis. ducklings mainly displayed standing instability and paralysis, retarded growth, with 10 to 30% mortality rates [2, 3] . other birds such as chickens, geese, sparrows, etc were also infected and displayed obvious clinical signs [4] [5] [6] . so far, the disease has resulted in a great economic loss to the poultry industry and caused wide public concern. there is no specific treatment available for tmuv and the vaccination is an effective way to prevent tmuv infection in waterfowl. the inactivated vaccines and live attenuated vaccines against plos tmuv have been successfully developed and already used in clinical production [7, 8] . but the live attenuated vaccines could display disadvantages of reversion of virulence and spread, and the inactivated vaccines didn't display an effective cellular immunity. so the development of a new type of vaccine is very urgent. tmuv is a mosquito-borne flavivirus which belongs to the ntaya virus group within flavivirus genus, flaviviridae family [9] . tmuv genome encodes a single polyprotein which is cleaved into three structural proteins (c, prm and e) and seven non-structural proteins (ns1, ns2a, ns2b, ns3, ns4a, ns4b and ns5) [3] . among them, ns1 protein is a glycoprotein which is present in cell-surface or in the form of cell-associated protein [10] . ns1 protein possesses several unusual and interesting performances which is closely related to the membrane function and indispensable in the early viral replication, assembly and release of the virus [11] . ns1 protein contains multiple protective t-cell and b-cell epitopes which can induce both humoral and cell-mediated immunity without the risk of antibody-dependent enhancement [12, 13] . the b-cell epitopes mainly refer to multiple continuous amino acid residues on the protein surface or the spatial conformation of discontinuous amino acid residues. accurate analysis of the epitopes on ns1 protein is critical for further understanding the mechanism of ns1-mediated immune protection. in recent years, epitope-based marker vaccines have increasingly attracted wide attentions in public [14] . the identification of linear epitopes on ns1 protein would be conducive to the development of epitope-based marker and subunit vaccines, preparation of protective antibodies and understanding protein functions [15] . it has been demonstrated that ns1 protein of flavivirus has more virus-specific epitopes than cross-reactive ones in contrast to e protein which has more cross-reactive than specific epitopes [16] . therefore, it is very interesting and valuable to screen and identify epitopes on ns1 protein for specific serological diagnosis that can differentiate between infections caused by flaviviruses [16] [17] [18] . in this study, one linear b-cell epitope was identified and characterized with one monoclonal antibody against tmuv ns1 protein. this study can lay the foundation for comprehending the antigenic structure of tmuv ns1 protein, development of a specific serological diagnostic assay and a new type of vaccine for tmuv infection. this research was approved by the committee on the ethics of animal of shandong (permit number 20147620). myeloma cell line sp2/0 and baby hamster kidney bhk-21 cells (ccl-10, american type culture collection) were cultured in dmem/high glucose (hyclone, thermo scientific, usa) in a humidified 5% co 2 atmosphere at 37˚c. all culture media were added with 10% inactivated fetal bovine serum (transgen, beijing, china), 100 u/ml penicillin and 100 mg/ml streptomycin (solarbio, beijing, china). the sdsg strain (accession number: kj740747.1) was isolated from a duck farm in shandong province in 2013. the virus titer was 10 4.8 eld50/ 0.2ml (median embryo lethal dose), calculated according to the reed and muench method (reed and muench, 1938) . the duck and mouse sera against tmuv were also provided by dr. ke-xiang yu. the ns1 gene was amplified by conventional pcr using a duck tmuv cdna template. after agarose gel purification and restriction enzyme digestion, the recombinant plasmid pet-28 -ns1 was constructed and identified. the recombinant plasmid pet-28-ns1 and the control plasmid pet-28a(+) were transformed into bl21 (de3) cells and induced by isopropyl-thiogalac-topyranoside (iptg) with the final concentration of 1mmol/l at 37˚c for 1~6h. after induction, bacterial cells were centrifugated and washed with phosphate buffered saline (pbs, ph 7.4). bacterial cells after washing were lysed by sonication. the recombinant protein ns1 was identified with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and western-blot. and then it was purified by washing with urea solutions of different concentrations (0.5m, 1m and 3m) and the concentration was measured using a bca protein assay kit (kang wei, china). finally, the protein was stored in -80˚c. production of monoclonal antibodies (mabs) against tmuv ns1 protein 6-week-old balb/c mice were purchased from experimental animal center of shandong province and housed in spf isolators that were ventilated under negative pressure. feed and water were provided ad libitum. 6-week-old balb/c mice were immunized with the purified ns1 protein and freund's adjuvant (sigma, st. louis, mo, usa) for three times. three days after the final immunization, the balb/c mice were euthanized by carbon dioxide and spleen cells were harvested. subsequently, mouse splenocytes were fused with sp2/0 murine myeloma cells using 50% polyethylene glycol (peg 4000, sigma) according to the procedure described previously [19] . the fusion cells were separated into ninety six well plates and cultured selectively in dmem/high glucose containing 10% fetal bovine serum, 100 mm hypoxanthine, 16 mm thymidine and 400 mm aminopterin. on day five, 50μl of hat medium was added additionally in each cell of the 96 cell plates. on day twelve, hat medium was replaced with fresh ht medium completely. after hat/ht medium screening, culture supernatants were analyzed by the indirect enzyme-linked immunosorbent assay (elisa), western blotting, ifa and blocking elisa. the positive hybridoma cells were subcloned repeatedly by the limiting dilution method till monoclonal hybridoma cells were obtained. positive hybridoma cells were cultured in the abdominal cavity of paraffin-primed balb/c mice to obtain ascitic fluid. the characteristics of monoclonal antibodies were identified using pierce rapid isotyping kit (thermo, rockford, il, usa). the cell supernatants and ascitic fluids were respectively diluted from 1:10 to 1:20480 by double-dilution method. the titers of diluted antibodies were measured by indirect elisa. to screen the epitopes of monoclonal antibodies, 35 pairs of primers about 16-amino acid peptides partially-overlapping covering the entire ns1 gene were designed (table 1) . after the forward and reverse primers annealing, the genes encoding 35 pairs of 16-amino acid peptides were amplified. 35 polypeptide fragments were cloned into the pgex-6p-1 plasmid respectively, identified by sequencing and expressed as fusion proteins with gst as described previously [20] . the fusion proteins were identified by sds-page and purified by pierce gst protein interaction pull-down kit according to the manufacturer's instructions (thermo, usa). the concentrations of purified proteins were measured by a bsa protein assay kit (kang wei, china). in order to precisely locate the exact sequences of linear b-cell epitopes on ns1 protein, one or two amino acid residues were removed from the carboxy terminal of the peptide sequentially ( table 2 ). ten pairs of primers were designed according to the shortened peptide sequences. the primers were annealed and gene sequences of truncated peptides were amplified. ten oligopeptide fragments were cloned into pgex-6p-1 vectors respectively and expressed as fusion proteins with gst as previously [20] . the oligopeptide proteins were purified by pierce gst protein interaction pull-down kit according to the manufacturer's instructions (thermo, usa). the concentrations of purified proteins were measured by a bsa protein assay kit (kang wei, china). each well of 96-well plates was coated with purified fusion proteins subsequently in 0.1 m carbonate buffer (ph 9.6) and incubated at 4˚c overnight. after washing three times with pbst (0.5% tween-20 in pbs), the plates were blocked with 5% skimmed milk for 2 h at 37˚c. after washing, cell supernatants of different hybridomas were added to each well respectively and incubated at 37˚c for 1h. after the plates were washed three times with pbst, a goat antimouse hrp-conjugated polyclonal serum diluted 1:5000 with 5% skimmed milk (transgen, usa) . in the process of screening mabs against tmuv ns1 protein, 96-well plates were coated with recombinant protein ns1 and bl21 (de3) cells containing plasmid pet-28a(+) respectively. in the process of screening antigenic epitopes on ns1 of tmuv, 96-well plates were coated with glutathione purified gst-ns1 and s-transferase (gst). mouse sera against tmuv and murine myeloma cells supernatants were used as positive and negative controls. results depict the average of duplicate assays. the expressed fusion proteins were subjected to gel electrophoresis on 12% sds-page after denaturation with 2 × sds loading buffer at 100˚c for 3~5 min. and protein bands were transferred to a nitrocellulose membrane. the nitrocellulose membrane was blocked with 5% skimmed milk overnight at 4˚c. after washing three times with tbst(20 mm tris-hcl, 150 mm nacl, 0.01% tween-20, ph7.5), the membranes were incubated with duck serum against tmuv or mab against ns1 protein at 37˚c for 1 h. after washing three times with tbst, the membranes were immersed into a goat anti-duck or anti-mouse hrp-conjugated polyclonal serum diluted 1:5000 with 5% skimmed milk (kpl, 04-25-6, usa; transgen, fs101-02, beijing, china) for 1 h at 37˚c. after washing in the same way, membranes were colored in dab liquid at dark for 1~3 mins. running water was used to stop the reaction. bhk-21 cells were separated into a 24 well plate (costar corning, corning, ny) and inoculated with tmuv (10 3.5 eld 50 /0.2ml duck embryo allantoic fluid). when 80% cytopathic effect (cpe) of cells occurred, cells were fixed with methanol and acetone (1:1) for 20 min at -20˚c. after washing three times with pbs, the monoclonal antibody against tmuv ns1 protein diluted 1:100 with pbs was added and incubated for 1 h at 37˚c. after washing three times with pbs, cells were inoculated with a goat anti-mouse igg(h+l) with fitc conjugate (transgen, beijing, china) for 1 h at 37˚c. finally, the reaction result was observed under the fluorescence microscope (nikon, eclipse, te2000-s, japan). bhk-21 cells without inoculation with tmuv were used as the control. each well of 96 well plates was coated with purified ns1 protein in 0.1 m carbonate buffer (ph 9.6) and incubated at 4˚c overnight. after washing three times with pbst (0.5% tween-20 in pbs), plates were blocked with 5% skimmed milk for 2 h at 37˚c. after washing three times with pbst, mouse positive serum against tmuv ns1 protein was added to wells and incubated at 37˚c for 1h. after washing three times with pbst, mab against tmuv ns1 protein was added to wells and incubated at 37˚c for 1h. after plates were washed three times with pbst, a goat anti-mouse hrp-conjugated polyclonal serum diluted 1:5000 with 5% skimmed milk (transgen, fs101-02, beijing, china) was added and incubated for 1 h at 37˚c. after washing three with pbst, tmb was added into the wells as a substrate for hrp and incubated in dark for 15 min. 2m h 2 so 4 was used to stop the reaction and the absorbance was recorded at 450 nm by a microplate absorbance reader (biotec, usa). results depict the average of duplicate assays. the adajacent value of blocking elisa was determined using 25 mouse negative sera. the nitrocellulose (millipore, usa)a was punched a series of small circles by a hole puncher. about 2 μg of each purified fusion protein was spotted onto small circles of the nitrocellulose membrane. after the nitrocellulose membrane was dried, it was blocked with 5% skimmed milk at 37˚c for 2h. after washing three times with tbst, the membrane was incubated with different mabs respectively at 37˚c for 1 h. after washing three times with tbst, the membrane was incubated with a goat anti-mouse hrp-conjugated polyclonal serum (transgen, fs101-02, beijing, china) at 37˚c for 1 h. after washing three times with tbst, membranes were colored in dab liquid. running water was used to stop the reaction. recombinant protein ns1 was successfully expressed in bl21 (de3) and the highest expression level emerged at 5 hours after induction with 1mm iptg (fig 1a) . the fusion protein was about 46kda and present in inclusion bodies of bacterial lysates. it could react with the tmuv-positive serum from ducks ( fig 1b) . purified ns1 proteins were used to immunize balb/c mice. one hybridoma cell line was acquired by subcloning and screening, which produced mab that could strongly recognize native ns1 protein of tmuv and recombinant ns1 protein by indirect immunofluorescence assay (ifa) (fig 2) and western blot (fig 2) . the mab was named 3g2 which belonged to the subtype of igg2a and the light chain was kappa. the titers of culture supernatants and ascitic fluids were 1:5120 and 1:10240 measured by indirect elisa. the adajacent value of blocking elisa in this study was 18.67%. the inhibition rate of mouse positive serum against tmuv ns1 protein to mab 3g2 was 69.34% and much higher than the adajacent value. to screen the antigenic epitope of tmuv ns1 mab, 35 short peptide fusion proteins were successfully expressed in prokaryotic expression system and purified. according to the results of indirect elisa, one 16 aa peptide protein was recognized by mab 3g2 (fig 3) . the peptide was ns1-27 which exhibited a positive immunoreactive band of approximate 26 kda with mab 3g2 by western-blot (fig 4) . no immunoreactivity was detected in the control bands. to further map the accurate position of the epitope, one or two amino acid residues were cut down from both sides of ns1-27 polypeptide. ten truncated oligopeptides were expressed in the prokaryotic expression system and purified by different concentrations of urea solution. the truncated fusion peptides were detected using mab 3g2 by indirect elisa. the results showed that mab 3g2 can react with ns1-27-f-2, ns1-27-f-4, ns1-27-f-6, ns1-27-f-7, ns1-27-f-8 and ns1-27-r-2 oligopeptides, while not with ns1-27-f-9, ns1-27-r-3, ns1-27-r-4 and ns1-27-r-6 ( fig 5) . so the accurate epitope recognized by mab 3g2 was the 269 dekeiv 274 sequence which was mapped by truncating 8 aa and 3 aa from 5' end and 3' end of ns1-27 polypeptide respectively. in order to identify conservation of the ns1-27 epitope ( 269 dekeiv 274 ), the multiple sequence alignment was performed among 24 different tmuv isolates published in genbank. the results showed that the b-cell epitope of 269 dekeiv 274 was highly conserved and the amino acid homology was 100% among 24 tmuv strains (fig 7) . in order to identify the specificity of b-cell epitope, the sequence alignment was performed among other flaviviruses such as jev, denv, wnv, yfv, bagv, and mvev isolates published in genbank. the results showed that the b-cell epitope of 269 dekeiv 274 had 67% identity with jev ns1 sequences and 50% identity with bagv. in addition, the amino acid homology of the epitope was below 50% among other flaviviruses. especially there was no identity between the epitope and denv (1-4) ns1 sequences. so ns1-27 epitope had high specificity among other flaviviruses (table 3) and could be applied to the development of tmuv epitope-based vaccine and serological diagnosis method. tembusu virus infection has been an emerging flavivirus disease in waterfowls in recent years and resulted in severe economic losses to poultry industry. the study of detection, diagnosis, and prevention and control of the disease is a hot issue in the field of tembusu virus research. ns1 protein is the non-structural protein of flavivirus, which has important functional properties in virus rna replication, anti-viral immunity and host recognition of virus-associated molecular patterns [11, [21] [22] [23] . it is reported that ns1 protein or mabs against ns1 protein can confer active or passive immunity protection from the flavivirus challenge [24] [25] [26] , which is closely related to the epitopes on ns1 protein. therefore, it is important to identify the epitopes on ns1 protein for epitope-based vaccine designment and development of epitopebased serological tests. in our study, one b-cell epitope on tmuv ns1 protein was precisely mapped and analyzed which led to a better understanding of host immune responses and the development of new vaccines and diagnostic tools for tmuv. mabs against definite epitopes can provide an important experimental way for studying protein structure, and developing diagnostic and therapeutic reagents for pathogen control [27] [28] [29] . precise analysis of epitopes on flavivirus ns1 protein is very critical for comprehending the mechanism of ns1-mediated immune function. so far, a few reports have described the mapping b-cell epitopes on ns1 protein of jev [20] , wnv [14] , denv [30, 31] , and tbev [32] . and moreover there are also several methods of screening b-cell epitope such as peptide scanning technique, phage peptide library presenting technolog, eukaryotic expression presenting system and prokaryotic expression presenting system. the method of prokaryotic expression system [15, 33] is simple and easy to operate and need not special instruments and technologies. moreover it is at low cost and common laboratory can afford it in comparison to other methods s. but the method is generally applicable to the screening of linear epitopes and it has extensive workload. although this method has certain defects, it is very accurate and effective in epitope screening. and so far its application is very broad. in this study, one b-cell epitope was screened by prokaryotic expression system to express a series of small peptides of one b-cell epitope from tmuv ns1 protein truncated protein and analyzed by western blotting. tmuv ns1-specific mab was acquired with prokaryotic expression ns1 protein of tmuv. a pane of 16-aa polypeptides of ns1 protein was expressed and one 16-aa polypeptide ns1-27 was screened and identified by 3g2 mab. in order to accurately map the b-cell epitope, a set of truncated fusion oligopeptides of ns1-27 were expressed. one linear b-cell epitope of 269 dekeiv 274 was confirmed by probing the shortened oligopeptide proteins with 3g2 mab. in recent years, epitope-based vaccine and specific diagnostic tools have received extensive attentions. the b-cell epitope of tmuv ns1 protein could apply into the development of detection methods to investigate whether the detected antibody was a result of inactivated vaccine immunization or live virus infection. sequence alignment showed that ns1-27 epitope of 269 dekeiv 274 was highly conserved among tmuv strains. and furthermore, ns1-27 epitope had 100% identity among 24 tmuv strains. compared with other flavivirus, the epitope had less 50% identity than denv (1-4), yfv, wnv, and mve excluding jev and bagv. moreover, jev and bagv couldn't infect one b-cell epitope from tmuv ns1 protein poultry. so the highly conserved and specific nature of the identified epitope is beneficial for the application in vaccine design and epitope-based diagnosis. in summary, one highly conserved b-cell epitope on tmuv ns1 protein were precisely screened and identified which could provide an important basis and data for understanding the antigenic structure of ns1 protein and the clinical application of epitope-mediated detecting and diagnostic methods. tembusu virus in ducks, china analysis of the complete genome of tembusu virus, a flavivirus isolated from ducks in china complete genome sequence of a novel flavivirus, duck tembusu virus, isolated from ducks and geese in china genomic and antigenic characterization of the newly emerging chinese duck egg-drop syndrome flavivirus: genomic comparison with tembusu and sitiawan viruses duck tembusu virus exhibits neurovirulence in balb/c mice characterization of a tembusu virus isolated from naturally infected house sparrows (passer domesticus) in northern china development of a live attenuated vaccine candidate against duck tembusu viral disease efficacy evaluation of an inactivated duck tembusu virus vaccine duck egg-drop syndrome caused by byd virus, a new tembusu-related flavivirus vascular leakage in severe dengue virus infections: a potential role for the nonstructural viral protein ns1 and complement trans-complementation of yellow fever virus ns1 reveals a role in early rna replication flavivirus nonstructural protein ns1: complementary surprises japanese encephalitis virus structural and nonstructural proteins expressed in escherichia coli induce protective immunity in mice. microbes and infection identification of two linear b-cell epitopes from west nile virus ns1 by screening a phage-displayed random peptide library comprehensive mapping antigenic epitopes of ns1 protein of japanese encephalitis virus with monoclonal antibodies epitope-blocking enzyme-linked immunosorbent assay to differentiate west nile virus from japanese encephalitis virus infections in equine sera. clinical and vaccine immunology development and evaluation of an enzyme-linked immunosorbent assay for quantifying antibodies to japanese encephalitis virus nonstructural 1 protein to detect subclinical infections in vaccinated horses non-structural protein 1 (ns1) antibody-based assays to differentiate west nile (wn) virus from japanese encephalitis virus infections in horses: effects of wn virus ns1 antibodies induced by inactivated wn vaccine identification of two antigenic epitopes on sars-cov spike protein identification of a virus-specific and conserved b-cell epitope on ns1 protein of japanese encephalitis virus predominance of hla-restricted cytotoxic t-lymphocyte responses to serotype-cross-reactive epitopes on nonstructural proteins following natural secondary dengue virus infection immunization of mice with recombinant vaccinia virus expressing authentic dengue virus nonstructural protein ns1 protects against lethal dengue virus encephalitis reactivity of serum samples from patients with a flavivirus infection measured by immunofluorescence assay and elisa dna immunization with japanese encephalitis virus nonstructural protein ns1 elicits protective immunity in mice construction of recombinant pseudorabies virus expressing ns1 protein of japanese encephalitis (sa14-14-2) virus and its safety and immunogenicity the dengue virus nonstructural protein 1 (ns1) increases nf-kappab transcriptional activity in hepg2 cells identification of a transiently exposed ve-cadherin epitope that allows for specific targeting of an antibody to the tumor neovasculature fine and domain-level epitope mapping of botulinum neurotoxin type a neutralizing antibodies by yeast surface display structure and function analysis of therapeutic monoclonal antibodies against dengue virus type 2 identification of b-cell epitope of dengue virus type 1 and its application in diagnosis of patients comprehensive mapping of immunodominant and conserved serotype-and group-specific b-cell epitopes of nonstructural protein 1 from dengue virus type 1 a synthetic peptide based on the ns1 non-structural protein of tick-borne encephalitis virus induces a protective immune response against fatal encephalitis in an experimental animal model comprehensive mapping of common immunodominant epitopes in the west nile virus nonstructural protein 1 recognized by avian antibody responses the authors would like to thank dr. ke-xiang yu for providing pgex-6p-1 plasmid and sera against tmuv. key: cord-001455-n7quwr4s authors: rapin, noreen; johns, kirk; martin, lauren; warnecke, lisa; turner, james m.; bollinger, trent k.; willis, craig k. r.; voyles, jamie; misra, vikram title: activation of innate immune-response genes in little brown bats (myotis lucifugus) infected with the fungus pseudogymnoascus destructans date: 2014-11-12 journal: plos one doi: 10.1371/journal.pone.0112285 sha: doc_id: 1455 cord_uid: n7quwr4s recently bats have been associated with the emergence of diseases, both as reservoirs for several new viral diseases in humans and other animals and, in the northern americas, as hosts for a devastating fungal disease that threatens to drive several bat species to regional extinction. however, despite these catastrophic events little information is available on bat defences or how they interact with their pathogens. even less is known about the response of bats to infection during torpor or long-term hibernation. using tissue samples collected at the termination of an experiment to explore the pathogenesis of white nose syndrome in little brown bats, we determined if hibernating bats infected with the fungus pseudogymnoascus destructans could respond to infection by activating genes responsible for innate immune and stress responses. lesions due to fungal infection and, in some cases, secondary bacterial infections, were restricted to the skin. however, we were unable to obtain sufficient amounts of rna from these sites. we therefore examined lungs for response at an epithelial surface not linked to the primary site of infection. we found that bats responded to infection with a significant increase in lungs of transcripts for cathelicidin (an anti-microbial peptide) as well as the immune modulators tumor necrosis factor alpha and interleukins 10 and 23. in conclusion, hibernating bats can respond to experimental p. destructans infection by activating expression of innate immune response genes. bats, members of the mammalian order chiroptera, have evolved a range of characteristics that allow them to adapt to changing environmental conditions. they are the only mammals capable of powered flight, most bat species undergo torpor to conserve energy and species that inhabit high northern latitudes hibernate for up to eight months with body temperatures below 10uc [1] . bats are extremely diverse, making up a fifth of all known mammalian species. they occupy a variety of niches across most of the world where they contribute in many ways to ecological balance [2] . bats have been implicated as reservoirs for human bacterial [3] [4] [5] and viral pathogens [6] . in recent years bats have also been associated with the emergence of several devastating viral diseases in humans and other animals [7] [8] [9] [10] [11] [12] [13] [14] [15] . circumstantial evidence even suggests that most human and domestic animal pathogens of at least four viral families -coronaviridae, paramyxoviridae, lyssaviridae and filoviridae may have arisen in bats [15] [16] [17] and bats are more likely than rodents to host zoonotic viruses [18] . interestingly, with the exception of rabies and other lyssaviruses, viruses do not appear to cause overt pathology in bats, suggesting the evolution of benign relationships between bats and their pathogens [14, 19] . despite the growing realization of the importance of bats in environmental and human health and disease, we know relatively little about how bats interact with their pathogens and commensal microbes. data on their immune responses to infection during torpor or long-term hibernation are particularly scarce because individuals of some species spend as long as 8 months a year in hibernation (norquay and willis, in press) and are therefore difficult to sample. the few studies on bats and other mammals suggest that the energy conserving benefits of torpor and hibernation may be enhanced by a state of immunosuppression [20] . the fungus pseudogymnoascus destructans (formerly known as geomyces destructans) is a cold-adapted saprophytic fungus that targets hibernating bats. it appears to be a recent introduction to the northern americas [21] and in recent years has been responsible for the death of an estimated 6 million bats and the potential regional extinction of some species [22, 23] . this disease is called white nose syndrome (wns) for the white fungal mycelia on the faces and wings of affected bats. although damage caused by the fungus is restricted to the superficial skin, infected bats clearly show signs of systemic physiological perturbation such as dehydration, hypovolemia and metabolic acidosis [24] . infected bats arouse from torpor more frequently than uninfected bats [25, 26] possibly leading to emaciation. recently, we investigated the pathogenesis of p. destructans [24, 25] . we used samples collected during the experiment to address the question: can hibernating bats respond to infection by activating genes responsible for innate immune and stress responses? while the site of primary site of fungal infection and occasional secondary bacterial infection was the skin of the wings [25] , most of this tissue was needed for histopathology and fungal detection and we were unable to obtain sufficient amounts of rna from these sites. to test the hypothesis that bats mount a systemic response to infection we examined lungs as an example of an epithelial surface which could have come in contact with aerosolized fungal spores despite being remote from the site of infection or may have responded to infection of peripheral areas [27, 28] . fifty four male m. lucifugus bats were collected from a wnsfree cave in manitoba, canada. protocols for collecting and transporting bats, infection with p. destructans, maintenance of bats in hibernation and sample collection have been described previously [24, 25] . briefly, bats in groups of 18 were either sham inoculated or inoculated with n. american or european isolates of p. destructans. bats were housed at 7uc and greater than 97% humidity with ad libitum water. all bats were equipped with data loggers to monitor skin temperatures. bats were euthanized during the experiment when humanely required or at the termination of the experiment 120 days after inoculation. immediately following euthanasia samples from segments of wing as well as various tissues were preserved in rnalater (life technologies, am7021) or in formalin. samples in rnalater were kept at 220uc until they were processed. we did not find p. destructans isolate-specific differences and treated the bats as either infected or sham-infected (control). during necropsy we collected representative samples for histopathology from all major organ systems. in addition, representative samples were taken from all areas of the wing and rolled on dental wax before placing in 10% neutral buffered formalin. tissues were processed routinely for histology, 5 mm sections cut and stained with periodic acid-schiff stain to highlight fungal hyphae. liver and other tissues were processed routinely and stained with hematoxylin and eosin. wings were subjectively scored on a scale of 0 to 5 with 5 being very severe with .50% of wing covered in fungal hyphae. bacterial score was from 0 to 5 with 5 indicating wide-spread and abundant bacteria being present in many areas within the dermis and underlying connective tissues. interstitial lung neutrophil assessment was similarly evaluated on a scale of 0 to 5, with 5 being very severe. tissues were homogenized in 2 ml sealed vials with a 5 mm steel bead, 0.1 g of 0.1 mm zirconium silica beads, 350 ml of rlt buffer (with b-mercaptoethanol) (rneasy plus kit, qiagen, 74136) using a retsch mm400 tissue homogenizer at 30 hz twice for 2 minutes each. rna from tissues was extracted using the procedure provided with the rneasy plus kit. cdna synthesis cdna synthesis was performed with 1 mg of rna per reaction using the quantitect reverse transcription kit (qiagen, 205313). cdna samples were either stored at 4uc if proceeding to use in qrt-pcr, or stored at 280uc until they were needed. to identify myotis genes for various cytokines and proteins involved in immune responses we scanned the m. lucifugus genome for regions with sequences similar to transcripts of proteins from human, mouse and rat. for genome sequences that were not annotated, m. lucifugus exons detected were compared with known exon-intron junctions in corresponding genes in human, mouse and rat to ensure that the sequences did indeed represent homologous genes. for mrnas where qrt-pcr primers had been described for other species, m. lucifugus primers were designed from the sequence of corresponding m. lucifugus genes. for mrnas where such information was not available we designed primers to optimize for annealing temperature and specificity, and to minimize dimer formation. in all cases the veracity of the pcr products was determined by electrophoresis and confirmed by sequencing. details of primers are in table 1 . stratagene's mx3005p pcr cycler was used in conjunction with quantifast sybr green pcr kit (qiagen, 204056). in addition to the primer sets mentioned, gapdh and cytochrome b were both used for every sample as normalizers. for each sample tested, 2 ml (1 mm, final concentration) of diluted forward primer, 2 ml (1 mm) of diluted reverse primer, 12.5 ml of sybr green master mix, and 8.5 ml of cdna was used. samples were tested in duplicate in each run and values used in the analysis were averages of the duplicates. the thermal profile used was as follows: 95uc for 5 minutes for initiation, then 40 cycles of 95uc for denaturing for 10 seconds, and 60uc for annealing and extension for 30 seconds with readings at the end of every cycle, then 1 cycle of 95uc for 1 minute, 55uc for 30 seconds, and 95uc for 30 seconds. the denaturation characteristics of the products were determined by incremental increase from 55uc to 95uc. a no template control (ntc) was used in every sample tested. reactions that generated primer dimers or products with spurious denaturation profiles were not considered. in all cases the identity of the pcr products was confirmed by determining their nucleotide sequences (macrogen). only results from reactions that yielded unambiguous results (products of the expected size and sequence, homogenous denaturation profiles, no products for no-template controls) were used for analysis. data from qrt-pcr and histopathological scores were analysed with spss statistics version 21. for qrt-pcr the relative levels of a transcript for each bat were calculated as cycle threshold (ct) normalized separately (dct) for levels of transcripts for two ''house-keeping'' genes -glycerol 6 phosphate dehydro-genase (gapdh) and cytochrome b. a lower ct (or dct) of one (1) indicates approximately a two-fold higher concentration of rna. the significance of differences of mean values of dct between infected and mock-infected (control) bats were determined using a student t-test. pearson's coefficients were calculated for the dct levels for cytokine transcripts for bats in each treatment class and lung interstitial neutrophil scores and mean bacterial and hyphae scores for 5 wing sections for each bat. methods were approved by the university of saskatchewan committees on animal care (protocol #20100120) and biosafety (permit #vmb03). recently we demonstrated that the european and north american strains of p. destructans were equally pathogenic for hibernating m. lucifugus [24, 25] . during these experiments histological sections from wings and other tissues were examined and graded for the presence of hyphae, edema, necrosis, bacteria (epidermal or invasive), neutrophil infiltration and inflammation. we also preserved samples which were then used in this study. we determined levels of transcripts for several immune and stress response genes (table 1) in lungs from infected and control bats. only those qrt-pcr reactions that yielded unambiguous results were considered. there were significant differences for transcripts for the interleukins (il) 10 and 23, the pro-inflammatory cytokine tumour necrosis factor alpha (tnfa), and the anti microbial and immunoregulatory peptide cathelicidin (figure 1 ). in figure 1a lower dct of one indicates approximately a two-fold higher concentration of rna. infected bats as a group had lower dct values (on an average 8 to 14 fold higher concentrations of rna, histograms in figure 1 ) for the four genes. while the group of infected hibernating bats had higher levels of transcripts for il10, il23, cathelicidin and tnfa than hibernating uninfected bats, the levels varied over a wide range with some infected bats having levels comparable to (or lower than) those of uninfected animals. since the level of fungal infection and pathology varied considerably among infected bats, we attempted to correlate transcript levels with scores for fungal infection, fungal hyphae and bacterial infection in wings and neutrophil accumumyotis homologues for innate response genes were identified by scanning the myotis genome for sequences that matched those of well-characterized reference organisms. where qrt-pcr primers for reference organisms d had been validated in the literature b,c , we used the corresponding myotis sequences as primers. myotisspecific substitutions in these primers are in bold a . for other genes (hsp70, tnfa, nos2, il-10, grp78) myotis-specific primers were designed from potential myotis exon sequences using parameters for optimal use in pcr reactions. in all cases the identity of pcr products were confirmed by sequencing. primers were used in qrt-pcr at a final concentration of 1 mm. doi:10.1371/journal.pone.0112285.t001 lation in lungs (table 2 ). il23 and cathlicidin levels correlated with all four parameters of disease and inflammation. il10 correlated with level of infection, and hyphae and bacterial scores in the wing, and tnfa correlated with level of infection and wing hyphae score. there was an inverse relationship between transcript levels and dct. the correlation coefficients are therefore negative. our results suggest that hibernating m. lucifugus are capable of inducing the expression of genes for cytokines in response to infection. the bats we examined were kept under conditions that closely mimicked temperature and humidity encountered in their natural hibernacula (i.e., 7uc and .97% relative humidity) [24, 25] . during hibernation m. lucifugus body temperature is maintained within 1-2uc of ambient temperature for weeks at a time with return to normothermic temperatures during periodic arousal [24, 25] . it is difficult to imagine mammalian gene expression at temperatures this low and it is possible that the genes were transcribed during the arousal periods. our previous study indicated that p. destructans-infected bats aroused more frequently than uninfected bats [25] , a pattern which has also been observed in the wild [26] and this may also have contributed to higher basal levels of gene expression in their cells. however, our observation that the levels for il 23, cathelicidin, il 10 and tnfa and not the other transcripts examined (dectin 1, defensin b1, hepcidin, il 17, cytchrome b, heat shock protein 70, nitrogen oxide synthetase) were higher in the lungs of infected bats, suggest that the response was a specific to infection. we observed mild to moderate levels of interstitial neutrophils in the lung of infected bats. on average, the lungs contained 8 to 20 times more transcripts for the anti-microbial peptide cathelicidin as well as for the immune modulators tnfa and il 10 and 23. the increase in levels of il 23, cathelicidin and il10 correlated with the lung interstitial scores ( table 2) . little is known about the role in bats of these bioactive proteins, information from other species indicates that they are intimately involved in the response to infection. for instance: cathelicidins belong to a family of antimicrobial peptides with an intrinsic ability to kill bacteria and fungi but they also function as chemoattractants for inflammatory cell recruitment and cytokine release. they are found in the lysosomes of neutrophils, macrophages and epithelial cells after activation or infection with a wide variety of pathogens [29] . humans, mice and dogs have a single gene for cathelicidin while several related genes have been discovered in pigs, horses, cattle and chickens. m. lucifugus has at least two genes with similarity to the human gene. our primers were designed to amplify transcripts for the gene most similar to that for human cathelicidin. interleukin 23 is a multifunctional pro-inflammatory cytokine involved in both innate and adaptive responses [30] . it is an important mediator of dectin-dependent response of the protective neutrophils to lung infections by aspergillus [31] . it is also important in the maturation of t helper cells. in addition to being protective il23 may also play a negative role by inducing chronic inflammation and exacerbating the effects of aspergillus and candida infections [32] . interleukin 10 is also known as cytokine synthesis inhibitory factor (csif) because of its role in modulating the inflammatory response. its effect is to suppress th1 responses and stimulate th2 responses leading to b-cell survival and proliferation and antibody synthesis. il10 is produced by a number of cell types including monocytes, and activated subsets of regulatory and helper t-cells b-cells. its synthesis is tightly regulated and is mediated by the nfkb and ap1 pathways after stimulation by commensal and pathogenic microorganisms [33] . if the role of il10 in wns is indeed to direct the immune system to a th2 response, our results are in contrast with those of moore et. al. [34] who found increased levels of il4 in the serum of wns-affected bats. work in other species suggests that il4 directs the immune system to a th1 response. tumour necrosis factor alpha (tnfa) is a monocyte derived cytotoxin implicated in tumour regression, septic shock and cachexia. tnfa binding to its receptor stimulates the activation of the transcription factor, nfkb, which activates gene expression required for several defensive responses [35] . the presence of neutrophilia in the lung interstitia of infected bats, despite a lack of obvious pathology or the presence of microorganisms, is puzzling. however, these observations may be explained by: a) injury in the skin can lead to complement-induced tnfa expression in the lungs followed by inflammation and neutrophil infiltration [27] . b) immune responses at mucosal surfaces are coordinated, with stimulation at one surface leading to responses at other mucosae [36] . the pulmonary response may have been due to stimulation of the upper respiratory tract by fungal spores. c) there have been suggestions that hibernating mammals show marked leukopenia [20] raising the possibility that circulating leukocytes are sequestered in some organ, possibly the lungs. the infected animals may have responded to inflammation in the wings with an increase in neutrophils, which then migrated to the lungs. d) lung neutrophils in infected bats, and the accompanying increase in the transcripts of the immune modulators may represent the end of the infectious process in the lungs and the removal of fungal spores and hyphae. this suggests that bats are capable of suppressing a lung or systemic infection by p. destructans and may explain why wns, the disease, occurs only after bat-to-bat contact rather than by infection through inhalation. while relatively little is known about the response of bats to infections, work with other species indicates that the host's response to pathogens is complex involving the interconnected innate, relatively non-specific mechanisms as well as the more specific adaptive systems of humoral and cellular immunity. the increased transcripts we detected in infected bats represent but a few components of this complex network. while our study shows that hibernating bats can respond to an infection, it does have some limitations and our results should be interpreted with caution. for instance, we have used increase in the stable levels of transcripts for various cytokine genes as a surrogate for increases in their respective proteins. however mrna and protein levels do not always correlate, largely because of complex regulatory processes that separate transcription of mrnas from their translation into protein [37] [38] [39] . in addition, only one of the transcripts, for cathelicidin, is directly linked to an anti-fungal response. the remainder, il 23, il 10 and tnfa may be indirectly related or may represent a non-specific response to the stress of infection. the importance of temporal heterothermy in bats bat ecology bats and bacterial pathogens: a review hemotropic mycoplasmas in little brown bats (myotis lucifugus) bats as reservoir hosts of human bacterial pathogen, bartonella mayotimonensis bats and viruses: friend or foe? bats: important reservoir hosts of emerging viruses isolation of nipah virus from malaysian island flying-foxes virology. what links bats to emerging infectious diseases? isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus fruit bats as reservoirs of ebola virus middle east respiratory syndrome coronavirus in bats, saudi arabia bat flight and zoonotic viruses isolation of genetically diverse marburg viruses from egyptian fruit bats bats host major mammalian paramyxoviruses evolutionary insights into the ecology of coronaviruses a comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special? antiviral immune responses of bats: a review hibernation: the immune system at rest? bat white-nose syndrome: an emerging fungal pathogen an emerging disease causes regional population collapse of a common north american bat species experimental infection of bats with geomyces destructans causes white-nose syndrome pathophysiology of white-nose syndrome in bats: a mechanistic model linking wing damage to mortality inoculation of bats with european geomyces destructans supports the novel pathogen hypothesis for the origin of white-nose syndrome frequent arousal from hibernation linked to severity of infection and mortality in bats with white-nose syndrome chemotactic mediator requirements in lung injury following skin burns in rats lung injury secondary to chemotactic factor-induced leukocyte activation innate immune defense system of the skin the il23/th17 pathway as a therapeutic target in chronic inflammatory diseases neutrophils produce interleukin 17a (il-17a) in a dectin-1-and il-23-dependent manner during invasive fungal infection th17 cells in the setting of aspergillus infection and pathology the regulation of il-10 production by immune cells hibernating little brown myotis (myotis lucifugus) show variable immunological responses to white-nose syndrome molecular basis of nf-kappab signaling role of inducible bronchus associated lymphoid tissue (ibalt) in respiratory immunity aebersold r (1999) correlation between protein and mrna abundance in yeast correlation of mrna and protein in complex biological samples insights into the regulation of protein abundance from proteomic and transcriptomic analyses regulation of cathelicidin antimicrobial peptide expression by an endoplasmic reticulum (er) stress signaling, vitamin d receptor-independent pathway dectin-1 expression at early period of aspergillus fumigatus infection in rat's corneal epithelium expression of human beta-defensins 1 and 2 in kidneys with chronic bacterial infection the small molecule, genistein, increases hepcidin expression in human hepatocytes expression of interleukin-17 associated with disease progression and liver fibrosis with hepatitis b virus infection: il-17 in hbv infection the immunomodulatory effect of bone marrow stromal cells (bmscs) on interleukin (il)-23/il-17-mediated ischemic stroke in mice discovery of herpesviruses in bats key: cord-000866-dr2uow4m authors: picard-jean, frédéric; bougie, isabelle; shuto, satoshi; bisaillon, martin title: the immunosuppressive agent mizoribine monophosphate is an inhibitor of the human rna capping enzyme date: 2013-01-17 journal: plos one doi: 10.1371/journal.pone.0054621 sha: doc_id: 866 cord_uid: dr2uow4m mizoribine monophosphate (mzp) is a specific inhibitor of the cellular inosine-5′-monophosphate dehydrogenase (impdh), the enzyme catalyzing the rate-limiting step of de novo guanine nucleotide biosynthesis. mzp is a highly potent antagonistic inhibitor of impdh that blocks the proliferation of t and b lymphocytes that use the de novo pathway of guanine nucleotide synthesis almost exclusively. in the present study, we investigated the ability of mzp to directly inhibit the human rna capping enzyme (hce), a protein harboring both rna 5′-triphosphatase and rna guanylyltransferase activities. hce is involved in the synthesis of the cap structure found at the 5′ end of eukaryotic mrnas, which is critical for the splicing of the cap-proximal intron, the transport of mrnas from the nucleus to the cytoplasm, and for both the stability and translation of mrnas. our biochemical studies provide the first insight that mzp can inhibit the formation of the rna cap structure catalyzed by hce. in the presence of mzp, the rna 5′-triphosphatase activity appears to be relatively unaffected while the rna guanylyltransferase activity is inhibited, indicating that the rna guanylyltransferase activity is the main target of mzp inhibition. kinetic studies reveal that mzp is a non-competitive inhibitor that likely targets an allosteric site on hce. mizoribine also impairs mrna capping in living cells, which could account for the global mechanism of action of this therapeutic agent. together, our study clearly demonstrates that mizoribine monophosphate inhibits the human rna guanylyltransferase in vitro and impair mrna capping in cellulo. the synthesis and maturation of eukaryotic mrnas are crucial events for gene expression. following mrna synthesis, eukaryotic mrnas undergo a series of critical modifications before being exported to the cytoplasm where they are translated into proteins. these processing events include the addition of a cap structure at the 59 terminus, the splicing out of introns, the editing of specific nucleotides, and the acquisition of a poly(a) tail at the 39 terminus. the cap structure found at the 59 end of eukaryotic mrnas is critical for the splicing of the cap-proximal intron, the transport of mrnas from the nucleus to the cytoplasm, and for both the stability and translation efficiency of mrnas [1] . synthesis of the cap structure occurs co-transcriptionally on nascent mrnas and involves three enzymatic reactions. first, an rna 59-triphosphatase (rtase) hydrolyzes the c-phosphate at the 59-end of the nascent pre-mrna to generate a 59-diphosphate end. an rna guanylyltransferase (gtase) then catalyzes a two-step reaction in which it initially utilizes gtp as a substrate to form a covalent enzyme-gmp (epg) intermediate, with the concomitant release of pyrophosphate (ppi). the gmp moiety is then transferred to the 59-diphosphate end of the nascent rna transcript in the second step of the reaction to form the gppprna structure. finally, using s-adenosyl-methionine as a substrate, an rna (guanine-n7) methyltransferase catalyzes the transfer of a methyl group to the n-7 position of the guanine to produce the characteristic m7 gppprna cap structure [2] . in humans, a bifunctional rna capping enzyme catalyzes both the rtase and gtase reactions through distinct domains, while a separate polypeptide mediates the subsequent n-7 methylation [3] . the importance of the cap structure for rna metabolism is highlighted by genetic analyses in saccharomyces cerevisiae that showed that the triphosphatase, guanylyltransferase and methyltransferase components of the capping apparatus are essential for cell growth [4, 5, 6] . nascent mrna capping is a rapid, dynamic, and regulated cotranscriptional process that is subjected to quality control. transcription initiation is associated with the rna polymerase ii (rna pol ii) carboxy-terminal domain (ctd) ser 5 phosphorylation, which recruits the capping apparatus [7] . nascent mrnas are rapidly capped (as they are only 20-30 nt long), followed by rna pol ii ctd ser 2 phosphorylation, hce dissociation and mrna elongation [8] . messenger rna capping represents a quality control checkpoint as uncapped rna are degraded by the xrn2 59r39 exonuclease in order to avoid generation of uncapped mrna which are not likely to be translated [9, 10, 11] . uncapped mrnas are not recognized by the initiation factor eif4e and are degraded by the 59r39 xrn1 [12, 13] . given that the rna pol ii synthesizes 10-30 bases per second, the entire fate of an unsuccessfully capped mrna can be sealed within few seconds, stressing the importance of rapid and efficient mrna capping [14] . the rate-limiting activity of the capping apparatus is the twostep ping-pong gtase activity [15, 16] . a general mechanism for phosphoryltransfer involving conformational changes between an open and closed form of the enzyme has been previously solved based on various gtases crystal structures [17, 18] . the first step of the reaction is initiated by the binding of gtp to the open form of the enzyme followed by the closure of the c-terminal oligomerbinding (ob) fold domain and the n-terminal nucleotidyl transferase (nt) domain. this closure is stabilized by interactions between the bound nucleotide and residues from both nt and ob fold domain. once in the catalytically active close conformation, the gtp substrate is hydrolyzed to produce the enzyme-gmp covalent intermediate. interactions between the bound guanylate and the ob fold domain are disrupted upon gtp hydrolysis, which leads to the reopening of the enzyme concomitant with the release of pyrophosphate. the open conformation exposes the rna-binding site, thereby allowing the subsequent transfer of the gmp moiety onto the acceptor rna. figure 1 summarizes the mechanistic and structural pathway used by gtases. very few inhibitors of the gtase activity have been identified. recent in vitro studies have shown that foscarnet, an antiviral drug that targets the dna polymerase of human cytomegalovirus, is a potent inhibitor of the gtase reaction [19] . inhibition of the gtase reaction likely occurs through binding of foscarnet to the active site of the enzyme on account of its analogous nature to pyrophosphate, a product of both the polymerase and the rna guanylyltransferase reaction. the intracellular triphosphorylated form of ribavirin, a broad-spectrum antiviral nucleoside analogue, can also inhibit the activity of viral gtases. mechanistic studies have demonstrated that ribavirin triphosphate can actually be used as a substrate by viral gtases [20] . however, rnas capped with ribavirin are relatively inert to methylation by viral rna (guanine-n7) methyltransferases, thus resulting in mrnas that are stable but not efficiently translated into viral proteins [21] . ribavirin is a pleiotropic agent that can also inhibit the cellular inosine-59monophosphate dehydrogenase (impdh), which is critical in the metabolism of nucleic acid precursors [22] . the enzyme catalyzes the conversion of inosine monophosphate (imp) to xanthosine monophosphate (xmp) with the concomitant reduction of nad via a covalent intermediate (e-xmp). this conversion has been shown to be the rate-limiting step in de novo guanine nucleotide biosynthesis [23] . this reduction in intracellular concentrations of guanosine is a key factor which contributes to the decrease in viral replication [22] . mizoribine monophosphate (mzp) is another compound which has been shown to specifically inhibit the cellular impdh [24] . mzp ( fig. 2a) is the active metabolite of the immunosuppressive pro-drug mizoribine (bredinin), a nucleoside analog of the imidazole class that was originally isolated from eupenicillium brefeldianum [25, 26, 27] . crystallographic studies have demonstrated that the binding of mzp to impdh results in a transition state analogue complex reminiscent of the e-xmp intermediate formed during the conversion of imp to xmp [25] . mzp is therefore a highly potent antagonistic inhibitor of impdh that blocks the proliferation of t and b lymphocytes that use the de novo pathway of guanine nucleotide synthesis almost exclusively. guanosine nucleotide depletion impairs g protein coupled receptor transduction and cyclic gmp signaling, ultimately resulting in the interruption of the s phase of the cell cycle through specific inhibition of impdh [28] . the use of mizoribine has been approved in japan for induction and maintenance of immunosuppressive therapy after renal transplantation [29] . since ribavirin triphosphate and mzp share functional similarities, we investigated the attractive possibility that mzp could also inhibit the gtase activity of the human rna capping enzyme (hce). in the present study, we demonstrate that mzp can inhibit the formation of the rna cap structure catalyzed by hce. the biological implications of this inhibition are discussed. an expression plasmid containing the full-length hce protein (597 amino acids) was generated by inserting the corresponding cdna between the nhei and xhoi cloning sites of the pet28a plasmid (novagen). in this context, the hce protein is fused in frame with an n-terminal 6-his tag, and expression of the protein is driven by a t7 rna polymerase promoter. upon transformation of the pet-hce plasmid into escherichia coli bl21(de3), cultures were grown at 37uc in luria-bertani medium containing 30 mg/ml kanamycine until the a 600 reached 0.5. induction was initiated with 400 mm isopropyl b-d-thiogalactopyranoside (iptg) and 2% ethanol, protein expression was allowed for 20 h at 18uc. all subsequent procedures were performed at 4uc. the bacteria were harvested by centrifugation at 5000 rpm and resuspended in 50 ml of lysis buffer [50 mm tris-hcl (ph 7.5), 150 mm nacl, and 10% sucrose]. 50 mg/ml lysozyme and 0.1% triton were added prior to sonication. after removal of insoluble material by centrifugation at 13,000 rpm, the soluble extract was applied to a nickel2nitrilotriacetic acid2agarose (qiagen) column. the protein was eluted stepwise with elution buffer [50 mm tris-hcl (ph 8.0), 100 mm nacl, and 10% glycerol] containing 50, 100, 200, 500, and 1000 mm imidazole. the polypeptide composition of the fractions was monitored by sds-page. the recombinant hce protein was recovered in the 200 mm imidazole eluate. this fraction was dialyzed against buffer c [50 mm tris-hcl (ph 8.0), 50 mm nacl, 2 mm dithiothreitol (dtt), and 10% glycerol] that was supplemented with 5 mm potassium pyrophosphate to ensure a homogeneous non-guanylylated enzyme. the protein concentration was determined by the bio-rad dye binding method and stored at 280uc. the amino acids 1-219 corresponding to hce rtase domain (hce-t 1-219 ) and 229-597 corresponding to hce gtase domain (hce-g 229-597 ) were also expressed separately using the same procedure. an rna substrate of 32 nucleotides (59-gggcacaca-cagtcgaccacacaaaaccaccc-39) was synthesized with the maxiscript kit (ambion) using t7 rna polymerase. the 59triphosphate rna substrate was purified on a denaturing 8% polyacrylamide gel and visualized by ultraviolet shadowing. the corresponding band was excised and then eluted from the gel by an overnight incubation in 0.1% sds and 0.5 m ammonium acetate. the rna was then precipitated with ethanol and quantitated by spectrophotometry. alternatively, radiolabeled rna substrates were also synthesized by adding radiolabeled nucleotides to the transcription reaction. a 59-diphosphate rna substrate was also synthesized by subjecting the 59-triphosphate rna to hydrolysis by the saccharomyces cerevisiae rtase (cet1). the reaction was carried out in a buffer containing 50 mm tris-hcl, ph 7.5, 1 mm mgcl 2 , 5 mm dtt, 1 mm of cet1, and 5 nmoles of the 59-triphosphate rna for 30 min at 30uc. this 59-diphosphate rna was then purified on an 8% polyacrylamide gel, excised, and precipitated. the rna substrate was quantified by spectrophotometry and stored at 220uc. reaction mixtures containing 5 mm [a-32 p]gtp, 5 mm of 59triphosphate rna, 0.1 mm hce, 50 mm tris-hcl, ph 7.5, 5 mm mgcl 2 , 500 mm dtt, 0.5 ng/ml pyrophosphatase (roche), and 1 u of rnase out (invitrogen) were incubated for 15 min at 37uc. reactions were stopped by the addition of 20 ml of phenolchloroform. 10 ml of loading dye (97% formamide) was added to the aqueous phase and submitted to electrophoresis on a denaturing 10% polyacrylamide gel. formation of the 32 pradiolabeled gppprna was quantified with a phosphorimager. reaction mixtures containing 50 mm tris-hcl, ph 7.5, 5 mm mgcl 2 , 500 mm dtt, 3 mm of c-32 p-radiolabeled 59-triphosphate rna substrate (32 nt), 0.1 mm hce, and various concentration of mzp (as indicated) were incubated for 10 min at 37uc. gtase assays were conducted as described in the ''capping of a 59-triphosphate rna'' section except that a 59-diphosphate rna was use as a substrate. alternatively hce was substituted by either 0.08 mm d1r (vaccinia virus), 0.4 mm hce-g 229-597 , 1.2 mm a103r (chlorella virus) or 0,5 mm ceg1 (s. cerevisiae). the enzyme-gmp complex formation assays were carried out for 3 min at 37uc in a buffer containing 1 mm [a-32 p]gtp, 4 mm hce, 50 mm tris-hcl, ph 7.5, 5 mm mgcl 2 , 500 mm dtt, 0.5 ng/ml pyrophosphatase (roche), and various concentration of mzp (as indicated). the reactions were stopped by the addition of the lost of interactions between the bound guanylate and the ob fold domain, upon gtp hydrolysis, destabilizes the close conformation of the enzyme and leads to its reopening (structure 5) concomitant with the release of the pyrophosphate product. this exposes the rna-binding site of the enzyme (exact location unknown), thereby allowing 59-diphosphate rna binding (structure 6) and subsequent gmp moiety transfer onto the acceptor rna (structure 7). the capped rna is then released and the apo-enzyme (structure 1) is regenerated allowing reinitiation of the pathway. formation of the enzyme-gmp complex was initially performed for 15 min at 37uc in a buffer containing 1.5 mm [a-32 p]gtp, 1 mm hce, 70 mm tris-hcl, ph 7.5, 7.5 mm mgcl 2 , 70 mm dtt, 0.7 ng/ml pyrophosphatase (roche), and 1 u of rnase out (invitrogen). the transfer reaction was then initiated by adding a 59-diphosphate rna (32 nt) to a final concentration of 0.5 mm with various concentration of mzp (as indicated). after 30 sec at 37uc, the transfer reaction was stopped by the addition of 20 ml of phenol-chloroform. 10 ml of loading dye (97% formamide) was added to the aqueous phase and submitted to electrophoresis on a denaturing 10% polyacrylamide gel. the formation of the 32 p-radiolabeled gppprna was quantified with a phosphorimager. reaction mixtures containing 50 mm tris-hcl, ph 7.5, 5 mm mgcl 2 , 500 mm dtt, 0.7 ng/ml pyrophosphatase (roche), and 1 u of rnase out (invitrogen), 1 mm of 59-diphosphate rna substrate (32 nt) harboring a a-32 p-radiolabeled 59-phosphate, 0.2 mm hce were incubated in absence or in presence of 2 mm gtp or mzp for 2 h at 37uc. the reaction ph was adjusted to 5.2 with 50 mm naoac followed by the addition of 5 mg of nuclease p1 and incubation at 37uc for 60 min. the digestion were further adjusted with 50 mm tris-hcl to ph 8.0, and digested with 1 u of alkalie phosphatase (roche) for 30 min at 37uc. the reaction products were analyzed by thin-layer chromatography (tlc) on a polyethyleneimine-cellulose plate developed with 0.4 m ammonium sulfate. hek 293t cells were cultured in dulbecco's minimum essential medium (dmem) supplemented with 10% fetal calf serum. cell were transfected according to the manufacturer instruction using genecellin reagent (biocellchallenge, toulon, france). on day 1, 70% confluent 10 cm petri dishes where transfected with either pcdna3.1+ (invitrogen) (control vector), pcdna3.1+ construct expressing either the full-length hce wild-type protein (557 amino acids) fused to an ha tag (hce-wt-ha), the gtase defective hce k294a mutant fused to an ha tag (hce-k294a-ha), or the green fluorescent protein (gfp). eighteen hours later, 5610 4 transfected cells were seeded per well on 24-well tissues cultures plates. at 24 h mizoribine (sigma) was added to a final concentration of 0, 40, or 120 mm. at 66 h cells were submitted to a second transfection using the firefly luciferase encoding vector pgl3 (promega) and were harvested at 96 h to be submitted to luciferase assay (promega) according to manufacturer instructions. luciferase activities were normalized to untreated cells. western blot were performed using 10 mg of total protein extracted at 96 h. the protein were separated on a 12% sds-page and transferred onto a polyvinylidene fluoride membrane. the membrane was blocked and incubated overnight with either 1:1000 anti-ha (ha-probe (f-7), sc-7392, santa cruz biotechnology), 1:1000 anti-gfp (g1546, sigma-aldrich), or 1:10000 anti-actin (b-actin antibody #4967, cell signaling) primary monoclonal antibody followed by 1 h incubation with 1:5000 hrp-anti-mouse (ge healtcare) secondary antibody. revelation was done using ecl detection (perkinelmer) and exposure to x-ray film (denville scientific). metazoans initiate mrna capping with a bifunctional enzyme that harbors the rtase and gtase activity on the same peptide. in order to evaluate the inhibitory potency of mzp on the human capping apparatus, a hexa-histidine tagged full-length bifunctional hce protein (amino acid 1-597) was expressed in e. coli and purified by nickel-agarose chromatography. to ensure the purification of the apo-enzyme, hce was further dialyzed against potassium pyrophosphate and magnesium to remove any residual hce-gmp complex. the 69-kda hce protein was the predominant polypeptide in the purified fraction (fig. 2b ). in the presence of a 32-nt 59-triphosphate rna, [a-32 p]gtp and magnesium cofactor, the full-length hce was also shown to sequentially hydrolyze the c-phosphate from the rna substrate and transfer the gmp moiety from the gtp substrate onto the newly 59-diphosphorylated rna to form the gppprna cap structure (fig. 2c ). in order to investigate if mzp could inhibit the rna capping reaction, we initially addressed its impact on the complete rna capping reaction (rtase+gtase). a cold 59-triphosphate rna was incubated in the presence of hce, [a-32 p]gtp, magnesium and increasing concentrations of mzp. as shown in figure 2d , the addition of increasing concentrations of mzp to the reaction efficiently prevented the formation of the radiolabeled capped rna. mzp inhibited the reaction with an ic 50 of 150 mm (fig. 2e ). to ensure that the inhibition by mzp was specific, the reaction was performed in the presence of increasing concentrations of gmp which is chemically related to mzp. the rna capping reaction was not significantly altered by the presence of increasing concentrations of gmp; concentrations as high as 10 mm did not reduce the rna capping by more than 25% (fig. 2e ). we next investigated the susceptibility of the rtase domain of hce to mzp inhibition. to specifically monitor the effect of mzp on the rtase activity, a c-32 p-radiolabeled 59-triphosphate rna was incubated with hce and magnesium. rtase activity was monitored through the analysis of the 32 pi product released from the radiolabeled rna by tlc and autoradiography. our data indicate that increasing concentrations of mzp does not affect the rtase activity up to 10 mm (fig. 3a) . it should be noted that similar results were obtained when the assay was performed with only the rtase domain of hce (hce-t 1-219 ) (data not shown). the gtase reaction is a two-step ping-pong reaction, which involves the formation of a covalent epg intermediate upon gtp hydrolysis, followed by the transfer of this gmp moiety onto a 59diphosphate rna. we initially investigated the inhibitory potential of mzp on the complete two-step gtase reaction in steady-state condition (multiple turnover). a 59-diphosphate rna, [a-32 p]gtp, magnesium, pyrophosphatase and various concentration of mzp were preincubated together, and the reaction was initiated by the addition of hce. a concentration of 80 mm mzp resulted in a 50% decrease in the formation of the radiolabeled capped rna (fig. 3b) . interestingly, when the same experiment was conducted with the isolated gtase domain of hce (hce-g 229-597 ) the activity was still inhibited by mzp, albeit with a 10fold decrease in the potency ( fig. 3b and 4a) . in order to address if it was the presence (or the activity) of the rtase domain that could influence the susceptibility of the hce gtase domain to mzp inhibition, we generated a point mutation of the catalytic cysteine 126 of the rtase domain from the classical phosphatase core motif (i/v)hcxxgxxr(s/t)g [30] . our results show that this rtase-defective full-length hce mutant (hce-c126s) displayed a similar inhibition profile than the wild-type enzyme (fig. s1) . consequently, we conclude that the gtase reaction represents the catalytic step which displays the highest susceptibility to mzp inhibition during the synthesis of the rna cap structure. we next set out to identify which step (if any) of the gtase reaction is more susceptible to mzp inhibition. in order to investigate the inhibitory potency of mzp on hce-gmp complex formation (first step), hce was incubated with [a-32 p]gtp, magnesium and increasing concentrations mzp. since this gtase reaction is known to be highly inhibited by its pyrophosphate product, pyrophosphatase was also added to the reaction in order to drive the reaction forward. the formation of the epg covalent complex in single turnover condition was analyzed by sds-page and quantified by phosphorimaging. as seen in figure 3c , increasing concentrations of mzp only prevented formation of the epg complex at elevated concentrations of mzp; a concentration of 3 mm mzp resulted in a 50% inhibition of the hce-gmp complex formation. despite the low millimolar mzp concentration required to prevent the epg formation, this inhibition is not due to magnesium cofactor sequestration since the addition of an equimolar concentrations of mgcl 2 to mzp did not influence this effect (data not shown). nevertheless, mzp inhibition of the epg complex formation is weak and could not be solely responsible for the global inhibitory potency of mzp. we next investigated if the second step of the gtase reaction could be impaired by mzp. epg complex formation was allowed to initially form upon incubation of hce with excess [a-32 p]gtp, magnesium and pyrophosphatase. next, various concentrations of mzp and a cold 59-diphosphate rna were added to initiate the transfer of the [a-32 p]gmp moiety onto the rna in single turnover condition. as seen in figure 3d , our data indicate that mzp only has a minor impact on the second gtase step. we conclude that the inhibition of this step does not contribute significantly to the general inhibition effect caused by mzp. quantitative analysis of the mzp inhibition for the complete gtase reaction, or its first step alone, were conducted. the formation of gppprna structure or the epg covalent complex was monitored as a function of gtp concentration in the presence of increasing concentrations of mzp. the mzp concentration had no impact on the maximal velocity of the epg formation (gtase first step), which suggested a competitive mechanism of inhibition (fig. 3e) . interestingly, for the complete gtase reaction, the k m appears to be independent from the inhibitor concentration, which would suggest a non-competitive mechanism of inhibition (fig. 3f) . it should be noted that hce was not able to use the mzp as a substrate and transfer it onto a radiolabeled acceptor rna to form an atypical cap structure (fig. s2) . these results highlight 2 potentially different mechanisms of action of mzp. the weak inhibition of the epg formation appears to be competitive while the global inhibition seems to be non-competitive. the later raises the possibility that mzp could primarily bind to an allosteric binding site. in order to investigate if mzp specifically inhibits hce, and gain additional insight on the degree of conservation of the main mzp binding site, we conducted a wide array of gtase inhibition studies. the mammalian vaccinia virus gtase (d1r), the saccharomyces cerevisiae gtase (ceg1) and the gtase (a103r) from chlorella virus (pbcv-1) were submitted to mzp inhibition for both the complete gtase reaction and the epg formation (gtase first step). as compared to the hce (ic 50 = 80 mm), the other gtases showed reduced susceptibility to mzp inhibition for the complete gtase reaction. this difference ranges from 5-to 25-fold for the gtase from the mammalian vaccinia virus (ic 50 = 500 mm), the chlorella virus (ic 50 = 750 mm), and the yeast s. cerevisiae (ic 50 .2 mm) (fig. 4a) . mzp also inhibited the epg formation by all gtase assessed, albeit with smaller variations (fig. 4b ). both the human and vaccinia virus gtase (hce and d1r) had ic 50 values of 3 mm, the chlorella virus gtase (a103r) presented an ic 50 of 2 mm, while the yeast gtase (ceg1) seemed slightly less affected by mzp. unlike the complete gtase reaction, the low potency inhibition of the epg formation by mzp is fairly similar for all gtases assessed. these results support the hypothesis that the inhibition of the hce-gmp complex by mzp could only account for a minor contribution to the complete gtase activity inhibition. this raises the possibility that a conformational change hindrance could be the mechanism of action by which mzp inhibits hce. this hypothesis will be further investigated in the discussion. we next set out to investigate if the in vitro inhibitory potency of mzp on hce could be translated into the inhibition of the capping apparatus in living cells. monitoring the capping efficiency in mammalian cells is a great challenge. rna quality control systems ensure that unsuccessfully capped mrnas are rapidly degraded. as a net result, capping inhibition would not lead to uncapped mrna accumulation, but rather to a global decrease of mature mrnas. this potentially allows to indirectly evaluate the ability of mzp to inhibit rna capping by monitoring the transcription and translation of a reporter gene in a controlled environment. on average, an mrna is transcribed every 30 min, has a half-life of 9 hours, and is translated 40 times per hour to yield an average of 500-1000 proteins over its life span [31] . this gives nearly 3 orders of magnitude of amplification over every capping event and provides a very sensitive assay. as an attempt to rescue the capping inhibition induced by mzp, we chose four identical human embryonic kidney (hek 293t) cell lines, diverging only by the over-expression (or not) of hce-wt-ha, hce-k294a-ha (gtase defective mutant) or gfp protein (fig. 5c) . unlike hce-wt-ha, hce-k294a-ha might be slightly toxic (negative dominant effect), which would explain its lower over-expression, nevertheless, hce-k294a-ha can be over-expressed in mamalien cells and is easily detectable. both the activity and the stability of the reporter gene (firefly luciferase) are not influenced by high concentration of hce (fig. s3) . the mizoribine prodrug was added to a final concentration of 0, 40 or 120 mm to the four cell lines. the reporter gene expression was initiated upon transfection of the pgl3 vector (bearing the firefly luciferase reporter gene under the control of an rna pol ii promoter). luminescence quantification of the reporter level was performed after 30 h of reporter protein expression (fig. 5a) . our results indicate that cells treated with 40 mm or 120 mm mizoribine show a global reduction in protein expression, likely due to the partial gtp depletion induced by impdh inhibition. interestingly, the translation of the reporter gene was partially rescued only in cells over-expressing hce-wt-ha when compared the hce-k294a-ha, gfp, and control cells. in the presence of 40 mm and 120 mm of mizoribine, the cells overexpressing hce-wt-ha maintained a significantly higher reporter gene translation rate compared to all the other cell lines that did not harbor a functional capping apparatus (fig. 5b) . although this experiment does not allow for precise quantification of the capping inhibition, it demonstrates that the over-expression of the active capping apparatus in human cells was partially able to rescue the mizoribine-induced phenotype on a reporter gene that is dependent on the cap structure for its proper transcription and translation. our study provides the first biochemical evidences that mizoribine monophosphate can directly inhibit the human capping enzyme. the ability of mzp to inhibit a purified rna capping enzyme has not been previously documented and has implications on our understanding of the catalytic mechanism of rna capping enzyme. our results indicate that the overall gtase reaction is inhibited by mzp. hce is a bifunctional protein harboring both rtase and gtase activity. in the presence of mzp, the rtase activity appears to be relatively unaffected while the gtase activity is inhibited, indicating that the gtase activity is the main target of mzp inhibition. the gtase catalysis is a two-step reaction. interestingly, the inhibition of the complete gtase reaction by mzp could not be explained completely by the inhibition of its individual steps. while the gmp transfer onto an acceptor rna (second step) does not contribute significantly to the general inhibition effect caused by mzp, the epg formation (first step) is inhibited by mzp with an ic 50 25-times higher than the complete gtase reaction. this weak inhibition, led us to hypothesize that two distinct mzp binding sites could be present on hce. the main binding site responsible for the drug inhibitory potency is speculated to be allosteric and will be further discussed. the second would be the active site and is believed to bind mzp with a lower affinity, likely due to the drug chemical similarity with gmp. the weak binding of mzp to the active site would be coherent with epg complex formation being competitively inhibited by mzp. in silico docking of mzp on the highly conserved active site of an open conformation homology model of hce gtase domain provides additional information on the mechanism of mzp binding to this site ( fig. s4 and table s1 ). mzp appears to be coordinated by the most conserved amino acids, which would thereby explain the low inhibition specificity toward various gtase (fig. 4c and d) . nevertheless, the mechanism of inhibition of the enzyme-gmp complex, which is mediated by the weak binding of mzp to the active site, is fundamentally different and weaker than the mechanism of the complete gtase inhibition. a complete gtase catalytic round does not only imply the formation of the epg intermediate complex and the transfer of the gmp moiety to an acceptor rna, but also involves complex conformational changes where the ob fold domain leans toward or away from the nt domain. mzp could bind hce and block this conformational change, possibly through stabilization of the closed conformation, thereby preventing the reopening of the protein upon gtp hydrolysis. this hypothesis would likely imply an allosteric mzp binding site. interestingly, the kinetics studies, although performed in steady-state condition, point toward a non-competitive mechanism of inhibition, which would indicate that mzp binds elsewhere than the active site. this is also coherent with the inability of mzp to be used as a substrate and transferred onto an acceptor rna. despite the very high degree of conservation among this specific family of nucleotidyltransferases, mzp harbors a 5-to 25-fold gain in specificity for hce when compared to other gtases. this result additionally supports the presence of an allosteric mzp binding pocket. oddly enough, the lone gtase domain of hce (hce-g 229-597 ) is less susceptible by 10-fold to mzp inhibition than the full-length hce. this evidence, not only supports the presence of an allosteric site, but can also provide additional information about its localization on hce. it is tempting to speculate that mzp could bind near the n-terminal of the gtase domain (close to the rtase-gtase interdomain) or on a region of interaction between both the rtase and gtase domains since the abolition of the rtase domain reduces hce mzp susceptibility. in order to gain additional details on the mzp main binding site, molecular docking could be used. unfortunately, the structure of both the rtase and the gtase domain are separately available, but the structure of the full-length protein is still not available. nevertheless, we ran a molecular docking experiment of mzp on the gtase domain from hce. the lack of rtase domain, which is predicted to participate in mzp binding, and the moderate flexibility of the n-terminal domain, which introduces a structural incertitude, does not allow for definitive conclusions to be reached but it is interesting to note that, in preliminary experiments, mzp favorably docks on the n-terminal region of the hce gtase (near the rtase-gtase inter-domain). together, our results reveal that mzp inhibits the hce gtase activity with a 5-to 25-fold specificity in comparison to other gtases. although more work is yet required to confirm our hypothesis, these results raise the possibility that the gtase inhibition could be mediated by a conformational change hindrance upon binding of mzp to an allosteric binding site that is speculated to reside near the rtase-gtase inter-domain. nevertheless, mizoribine is one of the first compounds to demonstrate a certain degree of specificity toward a single gtase, despite the high degree of conservation of this crucial family of enzyme. mzp displays a higher in vitro inhibition potency for the gtase reaction (ic 50 = 80 mm) in comparison to the complete rna capping reaction (rtase+gtase) (ic 50 = 150 mm). this may simply be due to our experimental conditions (5 mm mg 2+ and low nucleotide triphosphate concentration) where the rtase activity of hce, which is partially inhibited by free mg 2+ , becomes the rate-limiting step [30] . however, in cellulo the rtase harbors a higher turnover rate than the gtase, which catalyzes the limiting step in rna capping [15] . in a cellular context we expect the efficiency of mzp to be dictated solely by its interaction with the gtase. historically, very few gtase inhibitors have been developed, neither as scientific tools nor as therapeutic agents. more recently however, novel gtase inhibitors have been discovered [32] . they include the allosteric inhibitor mycophenolic acid, the pyrophosphate analog foscarnet which acts as a product inhibitor, and ribavirin triphosphate, a gtp analog that is transferred to acceptor rnas by gtase, leading to stable but inefficiently translated pseudo-capped rna. the current study identifies mzp as a novel allosteric gtase inhibitor, which is speculated to block a crucial conformational change. the gtase activity being the ratelimiting step of the essential capping apparatus, all these gtase inhibitors are promising lead candidates for the development of novel selective capping inhibitors and lead the way to a new class of anti-cancer, antifungal, and antiviral drugs. what is the biological relevance of the present finding? numerous studies have demonstrated the potency of mzp to inhibit the cellular impdh and to lower the intracellular guanosine nucleotide pool thereby limiting cell growth [28] , but none have addressed its impact on the capping apparatus. monitoring the capping efficiency in living cells is a great challenge as the cellular quality control machinery degrades unsuccessfully capped mrnas [9, 10, 33] . since proper capping is crucial for mrna transcription, export, stability and translation, it is possible to monitor the capping efficiency based on the translation of a reporter protein. in order to evaluate if mzp could impair in cellulo capping, we monitored its indirect impact on the translation of the firefly luciferase reporter gene. as cellular protein levels are not only dictated by capping efficiency, we selected a cellular model where all variables unrelated to capping were constant. cells originating from the same population were trasfected in order to over-express either the active hce-wt-ha, the gtase-defective hce-k294a-ha mutant, the gfp control protein or no protein (control vector). they were submitted to concentrations of 0 mm, 40 mm or 120 mm of mizoribine. all cell lines treated with mizoribine showed a global reduction in reporter protein expression when compared with untreated cells. this expected effect is likely due to partial guanosine pool depletion induced by impdh inhibition [28, 34, 35] . interestingly, the reduction in transcription and translation of the reporter was significantly less severe only in cells over-expression hce-wt-ha for both mizoribine concentrations. the ability of hce-wt-ha over-expression to partially rescue the luciferase expression in the presence of mizoribine demonstrates that hce is one of the mizoribine pharmacological targets. furthermore, the inability of the gtase defective mutant hce-k294a-ha to rescue the reporter expression under mizoribine treatment further demonstrates, in agreement with our in vitro results, that in a cellular context it is the gtase activity of hce that is targeted by mzp. although indirect, this is strong evidence that mizoribine is able to impair capping in a cellular environment. as expected, capping could not be fully inhibited in cellulo at mizoribine concentrations of 40-120 mm, which is approximately the in vitro ic 50 of 80 mm. despite its oral bioavailability (underlying membrane permeability) and its low binding to serum proteins (2.3%), mizoribine was not expected to reach intracellular concentration higher then its ic 50 [36, 37, 38, 39] . of notice, the effect of mizoribine on cellular capping was however greater than anticipated. we hypothesize that the competition between hce and xrn2 for the nascent mrna 59 end could explain the potency of mzp in cellulo, as slowing (or inhibiting) the capping activity could be sufficient to shift the balance towards quality control take-over (degradation) and reduce downstream protein expression. what is the exact contribution of the capping apparatus inhibition to the global mizoribine mechanism of action? this question has yet to be addressed, but the immunosuppressive effect of mizoribine on t-cell is mainly mediated by gtp depletion (which not only blocks t-cells proliferation but also promotes tcell apoptosis) and might be exacerbated by the reduction of mrna capping and downstream cap-dependent translation [40, 41] . our study clearly demonstrates that the therapeutic agent mizoribine monophosphate inhibits the human rna guanylyltransferase in vitro and impairs mrna capping in cellulo. docking calculations were carried out using the docking server software and the dreiding force field was used for energy minimization of mzp using built-in chemaxon tools in docking server (23) . pm6 semi-empirical charges calculated by mo-pac2007 were added to the ligand atoms. nonpolar hydrogen atoms were merged and rotatable bonds were defined (24) . docking calculations were carried out using the coordinated of the structural model of hce. essential hydrogen atoms, kollman united atom type charges and solvation parameters were added with the aid of autodock tools (25) . affinity (grid) maps of 20620620 å grid points and 0.375 å spacing were generated using the autogrid program (25) . autodock parameter set-and distance-dependent dielectric functions were used in the calculation of the van der waals and the electrostatic terms, respectively. docking simulations were performed using the lamarckian genetic algorithm (lga) and the solis & wets local search method (26) . initial position, orientation and torsions of the ligand molecules were set randomly. each docking experiment was derived from 100 different runs that were set to terminate after a maximum of 2,500,000 energy evaluations. the population size was set to 150. during the search, a translational step of 0.2 å , and quaternion and torsion steps of 5 å were applied. the predicted distance are indicated in angströms. (tif) viral and cellular mrna capping: past and prospects structure, mechanism, and evolution of the mrna capping apparatus messenger rna capping enzymes from eukaryotic cells mutational analysis of the saccharomyces cerevisiae abd1 gene: cap methyltransferase activity is essential for cell growth isolation and characterization of the yeast mrna capping enzyme beta subunit gene encoding rna 59-triphosphatase, which is essential for cell viability mrna capping enzyme. isolation and characterization of the gene encoding mrna guanylytransferase subunit from saccharomyces cerevisiae different phosphorylated forms of rna polymerase ii and associated mrna processing factors during transcription in vivo transcriptional pausing and cap formation on three drosophila heat shock genes identification of a quality-control mechanism for mrna 59-end capping mrna capping enzyme activity is coupled to an early transcription elongation the yeast 59-39 exonuclease rat1p functions during transcription elongation by rna polymerase ii cocrystal structure of the messenger rna 59 cap-binding protein (eif4e) bound to 7-methyl-gdp the enzymes and control of eukaryotic mrna turnover distinction and relationship between elongation rate and processivity of rna polymerase ii in vivo genetic, physical, and functional interactions between the triphosphatase and guanylyltransferase components of the yeast mrna capping apparatus capping enzyme in eukaryotic mrna synthesis crystallization of the rna guanylyltransferase of chlorella virus pbcv-1 x-ray crystallography reveals a large conformational change during guanyl transfer by mrna capping enzymes kinetic and thermodynamic characterization of the rna guanylyltransferase reaction the broad spectrum antiviral nucleoside ribavirin as a substrate for a viral rna capping enzyme ribavirin is not a functional mimic of the 7-methyl guanosine mrna cap mechanisms of action of ribavirin against distinct viruses key enzymes of imp metabolism: transformation and proliferation-linked alterations in gene expression recombinant human inosine monophosphate dehydrogenase type i and type ii proteins. purification and characterization of inhibitor binding the immunosuppressive agent mizoribine monophosphate forms a transition state analogue complex with inosine monophosphate dehydrogenase'ä { studies on bredinin. i. isolation, characterization and biological properties genetic and biochemical studies on the activation and cytotoxic mechanism of bredinin, a potent inhibitor of purine biosynthesis in mammalian cells mizoribine: mode of action and effects in clinical use a multicenter trial of mizoribine compared with placebo in children with frequently relapsing nephrotic syndrome mammalian capping enzyme binds rna and uses protein tyrosine phosphatase mechanism global quantification of mammalian gene expression control the rna capping machinery as an anti-infective target structure and function of the 59-.39 exoribonuclease rat1 and its activating partner rai1 inhibitory effect of mizoribine and ribavirin on the replication of severe acute respiratory syndrome (sars)-associated coronavirus inhibitory effect of mizoribine on matrix metalloproteinase-1 production in synovial fibroblasts and thp-1 macrophages membrane transport mechanisms of mizoribine in the rat intestine and human epithelial ls180 cells potential value of high-dose mizoribine as rescue therapy for ongoing acute humoral rejection role of mizoribine in renal transplantation molecular properties that influence the oral bioavailability of drug candidates mizoribine-mediated apoptotic signaling pathway in human t-cell line prolonged depletion of guanosine triphosphate induces death of insulin-secreting cells by we thank dr. aaron shatkin for the generous gift of the cdna encoding the human capping enzyme. we also thank drs. guy lemay, james van etten, and simon labbé that have kindly provided genomic dnas for the cloning of the vaccinia virus d1r gene, chlorella virus a103r gene, and s. cerevisiae ceg1 gene, respectively. key: cord-002043-z1b7pj3s authors: wang, xue-yang; yu, hai-zhong; geng, lei; xu, jia-ping; yu, dong; zhang, shang-zhi; ma, yan; fei, dong-qiong title: comparative transcriptome analysis of bombyx mori (lepidoptera) larval midgut response to bmnpv in susceptible and near-isogenic resistant strains date: 2016-05-11 journal: plos one doi: 10.1371/journal.pone.0155341 sha: doc_id: 2043 cord_uid: z1b7pj3s bombyx mori nucleopolyhedrovirus (bmnpv) is one of the primary pathogens causing severe economic losses in sericulture. however, the molecular mechanism of silkworm resistance to bmnpv remains largely unknown. here, the recurrent parent p50 (susceptible strain) and the near-isogenic line bc9 (resistance strain) were used in a comparative transcriptome study examining the response to infection with bmnpv. a total of 14,300 unigenes were obtained from two different resistant strains; of these, 869 differentially expressed genes (degs) were identified after comparing the four transcriptomes. many degs associated with protein metabolism, cytoskeleton, and apoptosis may be involved in the host response to bmnpv infection. moreover, some immunity related genes were also altered following bmnpv infection. specifically, after removing genetic background and individual immune stress response genes, 22 genes were found to be potentially involved in repressing bmnpv infection. these genes were related to transport, virus replication, intracellular innate immune, and apoptosis. our study provided an overview of the molecular mechanism of silkworm resistance to bmnpv infection and laid a foundation for controlling bmnpv in the future. the silkworm, bombyx mori l. (lepidoptera: bombycidae) has been domesticated for production of cocoons for more than 5000 years. silkworm rearing and the silk industry still play an important role in china, india and many other developing countries. b. mori is also a good model for the study of insect genetics and immunology [1] [2] [3] [4] . bombyx mori nucleopolyhedrovirus (bmnpv) is the principal silkworm pathogen and causes serious economic losses in sericulture every year. among numerous silkworm strains, most are susceptible to bmnpv infection, although a few strains exhibit resistance [5] . the heredity of silkworm resistance against bmnpv infection is a relatively complicated process because resistance is controlled both by major dominant genes and multiple genes of micro-effect [6] . a series of studies have made significant progress in understanding silkworm resistance against bmnpv infection. xu et al. reported that bms3a was potentially involved in resistance to bmnpv infection [7, 8] . b. mori lipase-1, serine protease-2 and alkaline trypsin protein extracted from the digestive juice of larvae midguts showed strong antiviral activity in vitro [9] [10] [11] . using comparative proteomics, arginine kinase was found to be involved in the antiviral process of different resistant strains of silkworm [12] . in our laboratory, a total of 12 proteins that are potentially involved in viral infection were identified using one-and two-dimensional electrophoresis followed by virus overlay assays. these proteins could be categorized into the following groups: endocytosis, intracellular transportation, and host responses [13] . immune responses were found to be synergistically regulated by the toll, janus kinase/signal transducers and activators of the transcription (jak/stat) and immune deficiency (imd) pathways, which could act as an important defense against exogenous pathogenic infection in conjunction with subsistent pathogen recognition receptors and response proteins [14] [15] [16] [17] [18] . however, the molecular mechanisms of silkworm resistance to bmnpv infection are still not fully elucidated. in recent years, the high-throughput nature of next generation sequencing (ngs), using platforms such as illumina hiseq tm 2500 have provided fascinating opportunities in the life sciences and dramatically improved the efficiency and speed of gene discovery, especially in the research of host cell responses to exogenous pathogenic infection [19] . for example, hu et al. obtained numerous differentially expressed genes (degs) involved in metabolism, immunity, and inflammatory responses in microtus fortis following infection with schistosoma japonicum based on comparative transcriptome analysis [20] . diege et al. examined different fish tissues infected with salmon anemia virus (isav) using high-throughput transcriptomics and found a strong correlation between functional modules and viral-segment transcription [16] . ngs technology was also used to explore the molecular mechanism of silkworm resistance against exogenous pathogens. kolliopoulou et al. reported that several genes related to physical barriers, immune response, proteolytic/metabolic enzymes, heat-shock proteins, and hormonal signaling were possibly involved in silkworm resistance against bombyx mori cytoplasmic polyhedrosis virus (bmcpv) infection; although these genes might be induced by the virus in order to increase infectivity [21] . additionally, several candidate genes, such as bmets, bmtoll10-3 and hsp20-1, have been identified in the initial stage of bmnpv infection by analyzing the global transcriptional profile of silkworm cell lines and heads following bmnpv infection [22, 23] . in order to gain a global view of the molecular changes in silkworms during bmnpv infection, we selected near-isogenic line bc9 and recurrent parent p50 for transcriptome sequencing. through comparative analysis of the transcriptomes from these two strains, a total of 869 degs were obtained, which included many genes potentially related to bmnpv-resistance. our results may provide some reliable evidence to clarify the bmnpv-resistance molecular mechanism in silkworm. bmnpv (t3 strain) was maintained in the key laboratory of sericulture, anhui agricultural university, hefei, china. the virus was obtained from the hemolymph of infected larvae and purified by repeated and differential centrifugation according to the protocol developed by rahman et al. [24] . the concentration of the virus (ob/ml) was determined by hemocytometer. the recurrent parent p50 (susceptible strain), the donor parent a35 (resistant strain) and the near-isogenic line bc9 were maintained in our laboratory. the near-isogenic line was constructed according to the protocol used by yao et al. [6] . in brief, the recurrent parent p50 was crossed to the donor parent a35; progeny were repeatedly backcrossed with the recurrent parent for nine generations and each progeny was screened by bmnpv. the first three instar larvae were reared on a fresh artificial diet at 26±1°c, 75±5% relative humidity, and a 12 hours day/night cycle. the rearing temperature for the last two instars was reduced to 24±1°c; other conditions were unchanged. on the first day of fifth instar, all larvae were starved for 24 hours and then fed with 5 μl bmnpv suspended in sterile water (1.0×10 5 ob/ml) per larva orally; the control group was treated with sterile water. bmnpv occlusion bodies (ob) began fast proliferation at approximately 24 hours post inoculation (hpi) [25] ; thus, this time was considered optimal for sample collection. silkworm larvae were dissected and the midgut tissues were removed and then washed in pbs (137 mm nacl, 2.7 mm kcl, 4.3 mm na 2 hpo 4 , and 1.4 mm kh 2 po 4 , ph 7.4) prepared with diethy pyrocarbonate (depc) (sangon, china) treated h 2 o. thirty larvae midguts were mixed together to minimize individual genetic differences. samples were flash-frozen in liquid nitrogen and pulverized, and 100 mg of sample were added directly into a rnaase free microcentrifuge tube containing 1.0 ml trizol reagent (invitrogen, usa) and stored at -80°c for later use. the level of silkworm resistance to bmnpv was tested following the protocol developed by cheng et al. [26] . the fourth instar larvae were inoculated with bmnpv at different concentrations; inoculations were conducted in triplicate. the level of silkworm resistance was calculated using ibm spss statistics 20 (ibm, usa). the silkworm midguts dissolved in trizol reagent were homogenized. total rna of midguts were extracted according to the manufacturer's protocol. concentrations were quantified using a nanodrop 2000 spectrophotometer (thermo scientific, usa). the purity of all rna samples were assessed at an absorbance ratio of a 260/280 and a 260/230 , and the integrity of the rna was confirmed by 1% agarose gel electrophoresis. library construction, illumina sequencing and read assembly fragment interruption, cdna synthesis, addition of adapters, pcr amplification and rna--seq were performed by beijing biomarker technologies (beijing, china). the standard illumina methods and protocols were adopted to prepare and sequence the cdna libraries. nebnext poly(a) mrna magnetic isolation module (neb, usa) was used to enrich mrna, and then the cdna library was constructed using the nebnext mrna library prep master mix set for illumina (neb, usa) and nebnext multiplex oligos for illumina (neb, usa). the size of the library insert fragments was determined by 1.8% agarose gel electrophoresis, and the fragments were quantified using a library quantification kit/illumina ga universal (kapa, usa). suitable fragments were selected as templates and sequenced on an illumina hiseq tm 2500 using paired-end technology. three biological replicates were used to minimize sample differences. in order to obtain clean and high-quality reads for sequence assembly, the raw reads were filtered by removing adaptor sequences, low-quality sequences (reads with ambiguous bases 'n') and reads with 10% > q < 20% bases [27, 28] . the trinity assemble program was used to assemble the clean reads into contigs, which covered more full-length transcripts through a broad range of expression levels [29] . the resultant contigs were added to transcripts based on paired-end information. the longest transcript from alternative splicing transcripts was selected as the unigene. these unigenes were combined to produce the final assembly and used for annotation. to annotate unigenes, different sequences were searched by blastx against the ncbi nonredundant protein (nr) database and other databases, including swiss-prot protein database, the kyoto encyclopedia of genes and genomes (kegg) and the cluster of orthologous groups (cog) database. gene ontology (go) annotations, including molecular functions, biological processes, and cellular components, were obtained using the blast2go program (https://www.blast2go.com/) [30, 31] . all searches were performed with an e-value < 10 −5 . fragments per kilobase of transcript per million fragments mapped (fpkm) was calculated to represent the expression abundance of the unigenes [32] . fpkm may reflect the molar concentration of a transcript by normalizing for rna length and for the total read number. after normalizing genes expression levels, degs were obtained by pair-wise comparison of the four transcriptome libraries using ideg6 software [33] . an fpkm fold change of 1.2 or 0.83 between two libraries was defined as the reference standard, with the benjamini-hochberg false discovery rate (fdr < 0.01) used to adjust the p-values. in order to validate the results from our transcriptome sequencing analysis, the relative expression levels of 15 randomly selected genes were confirmed by reverse transcription quantitative pcr (rt-qpcr). additionally, 9 genes with well reported previously were selected to further validate the genes of interest that might be involved in bmnpv resistance. all the primers are listed in table 1 . rt-qpcr reactions were prepared with the sybr premix ex taq tm kit (takara), following the manufacturer's instruction. reactions were carried out in bio-rad cfx96tm real-time system (bio-rad, usa). the thermal cycling profile consisted of an initial denaturation at 95°c for 30 s, 40 cycles at 95°c for 5 s, and 60°c for 30 s. all samples were performed in triplicate. relative expression levels were calculated using the 2 -44ct method following the protocol of livak et al. [34] . in this study, b. mori ribosomal protein s3 (bmrps3) gene was used as a reference gene. statistical analysis was conducted using the spss software (ibm, www.ibm.com). the lc 50 value was used to evaluate the resistant level of silkworm to bmnpv infection. the lc 50 value of a35 was approximately 26-fold greater than that of bc9 and over 500-fold greater than that of p50. the value of bc9 was 23-fold greater than that of p50 ( table 2 ). transcriptome sequencing is an efficient technology for comparing gene expression levels in different samples, and in our study, it was used to search and analyze degs among p50, bc9 control and treatment groups. a total of four cdna libraries were sequenced: p50-(p50 treated with sterile water), p50+ (p50 infected with bmnpv), bc9-(bc9 treated with sterile water), bc9+ (bc9 infected with bmnpv), with each group created in triplicate. after removing the adaptors and low quality sequences, 144,439,382 sequence reads were obtained ( table 3 ). the gc content of each of the four libraries was approximately 50%, and cycleq30% was greater than 89.91% for each library. thus, the quality and accuracy of the sequencing data were sufficient for further analysis. most of the reads matched silkworm genomic locations. all the unigenes matched previously described sequences with approximately 70% coverage. the length distribution of unigenes had similar patterns among the four libraries, suggesting that there was little bias in the construction of the four cdna libraries (fig 1) . in order to annotate the unigenes, reference sequences were searched using blastx against the ncbi nr database (e-value < 10 −5 ). a total of 12,591 of 13,342 unigenes provided a blast result (s1 table) . s2 table shows the species with the closest match for each unigene. most of table 1 . primers used in rt-qpcr for validation of degs. forward primer reverse primer doi:10.1371/journal.pone.0155341.t001 in order to determine the reliability of the transcriptome sequencing, the relative expression levels of 15 randomly selected genes were analyzed by rt-qpcr (fig 2a) . the results were consistent with the transcriptome data. for example, the gene encoding peptidoglycan-recognition protein was down-regulated in both rna-seq and rt-qpcr analyses, with a similar fold change. the lipase 1 gene was significantly up-regulated in the resistant strain bc9 after infection with bmnpv, which was also consistent with previously reported results [10] . linear regression analysis of the correlation between rt-qpcr and ran-seq ( fig 2b) showed an r 2 (goodness of fit) value of 0.9169 and a corresponding slope of 1.5281, suggesting a strong positive correlation between rt-qpcr and transcriptome data. therefore, the transcriptome data were satisfied for further analysis. to further elucidate which degs had a potential role in antiviral response, a venn diagram was constructed. a total of 285 degs were found to be differentially regulated when comparing p50+ and p50-, of which 122 were up-regulated and 163 were down-regulated. similarly, 193 degs were found to be differentially regulated in the comparison of bc9+ vs. bc9-, with 56 genes up-regulated and 137 down-regulated. in addition, there were 154 degs differentially regulated in the comparison of bc9-and p50-, among which 78 genes were up-regulated and 76 genes were down-regulated (fig 3, table 4 , s3 table) . there were 197, 119, 82 unique degs in p50+ vs. p50-, bc9+ vs. bc9-and bc9-vs. p50-, respectively. go assignments were used to assign a functional classification to these degs. for cellular components, the number of unique degs fell into the macromolecular complex classification was distinct in bc9 and p50 following bmnpv infection. for molecular functions, the number of transporter activity related unique degs was distinct in bc9 and p50 following bmnpv infection. for biological progresses, the number of unique degs involved in metabolism processes and localization was distinct in bc9 and p50 following bmnpv infection (fig 4) . the comparisons of bc9+ vs. bc9-and p50+ vs. p50-identified many degs that might either be involved in silkworm defense against bmnpv or facilitate bmnpv infection. these genes could be divided into three categories: protein metabolism, cytoskeleton, and apoptosis. most of the degs (70%) associated with protein metabolism were down-regulated in bc9 following bmnpv infection. in contrast, 80% of the genes in p50 were up-regulated after bmnpv infection. most of the degs (87.5%) associated with the cytoskeleton were up-regulated in p50 after bmnpv infection, but the number of up-regulated genes (50%) decreased in bc9. a total of 19 degs associated with apoptosis were identified following bmnpv infection. ten of these degs were altered in bc9 after bmnpv infection. the other degs were all overexpressed in p50 following bmnpv infection (table 5) . pathogen infection stimulated both cellular and humoral responses of insects [33] [34] [35] [36] . genes participating in innate immunity pathways were identified and analyzed in regards to their potential role in bmnpv infection (table 6 ). thirty degs were identified, which could be classified into toll pathway, imd pathway, polyphenol oxidase (ppo) pathway, pattern recognition receptor, and antimicrobial peptide. as shown in table 6 , 14 (47%) of these genes were downregulated and 9 (30%) were up-regulated in bc9 after infection with bmnpv, while 17 (57%) were down-regulated and 6 (20%) were up-regulated in p50 after bmnpv infection. after removing the genetic background and strain specific immune stress response genes, a total of 22 degs were identified as possibly being involved in silkworm resistance to bmnpv (fig 3, table 7 ). for bc9-vs. p50-, 13 genes were up-regulated and the rest were down-regulated. however, the 22 genes were all down-regulated in bc9 following bmnpv infection ( fig 5) . some genes, including prostatic acid phosphatase, protease inhibitor 6, actin cytoskeletonregulatory complex protein pan1, and ef-hand domain-containing protein, were further analyzed by rt-qpcr (fig 6) . after bmnpv infection, the expression levels of 4 genes were down-regulation in bc9 and a35 (resistant strain) (fig 6) , which was consistent with the transcriptome data. thus, we deduced that the 22 genes were possibly involved in resistance to bmnpv infection. gene functions fell into the following categories: transport, virus replication, intracellular innate immunity, and apoptosis. despite the confirmation of an association between many genes and proteins and resistance to bmnpv, the molecular mechanism of antiviral activities was still unclear. here, transcriptome sequencing was carried out to identify genes related to bmnpv-resistance in silkworm across the genome. by using the near-isogenic line bc9 (resistant strain) and the recurrent parent p50 (susceptible strain) to study silkworm antiviral mechanisms, some degs responding to bmnpv infection were successfully identified after comparing infected groups and controls in the two strains. based on the go analysis, more degs were found to be involved in metabolic processes in bc9 (17.8%) than in p50 (9.2%) following bmnpv infection (fig 4) , which was consistent with previous reports for sogatella furcifera (hemiptera: delphacidae) and campoletis sonorensis (hymenoptera: ichneumonidae) [35, 36] . approximately twice as many degs related to transporter activity were identified in bc9 (12.2%) than in p50 (6.3%) (fig 4) . down-regulation of transporter related genes, such as lactase-phlorizin hydrolase (lph), b(0, +)-type amino acid transporter 1 (bat1), actin cytoskeleton-regulatory complex protein pan1 (pan1), mfs-type transporter (mfs), and otoferlin, could repress virus infection in host cells [37] [38] [39] [40] [41] [42] . therefore, the increased number of these genes with altered expression levels in bc9 might be related to bmnpv infection. moreover, the number of macromolecular complex genes in bc9 (14%) following bmnpv infection that showed an increase in expression compared with p50 (5.1%) were similar to previous reports [43, 44] . protein metabolism, cytoskeleton, and apoptosis may play an important role in host response to bmnpv infection cellular and metabolic processes will be dramatically changed after viral infection [35] . in our study, several degs that participate in protein metabolism were found to be of interest. for example, solute carrier family 12 is involved in transporting extraordinarily diverse solutes [45] , and cystathionine gamma-lyase participates in hydrolysis of cystathionine [46] . viruses may have to rely on cell proteins to accomplish replication in intercellular regions [47] , therefore, the downregulation of the genes involved in protein metabolism could inhibit the replication of bmnpv in the host cell. we speculated that the down-regulation of these genes affected virus replication. cytoskeleton-dependent intracellular transport is an important strategy for transport of viral particles to different destinations [48] . in this study, some cytoskeleton related genes were found to be of interest, including actin cytoskeleton-regulatory complex protein pan1 and actin-binding protein. these genes related to actin-coupled endocytosis could promote viral transport [49, 50] . we speculated that the down-regulation of the cytoskeleton genes might affect bmnpv transport. apoptosis plays a vital role in regulating cell response in lepidopteran insects during viral infections, where larvae use selective apoptosis and subsequent sloughing of the infected cells in the midgut epithelium to resist virus infection [51, 52] . in this study, some genes related to activation of apoptosis were found to be of interest, including cytochrome c, inhibitor of caspase-activated dnase, amyloid precursor protein and b-cell lymphoma protein 2. the activity of caspase-activated dnase was blocked by hepatitis c virus core at physiological levels, resulting in the inhibition of apoptotic cell death [53] . amyloid precursor protein is a member of several signaling pathways that are involved in abnormal cell cycles, subsequently leading to apoptosis [54] . b-cell lymphoma protein 2 could bind to bh3 domains of various pro-apoptotic regulators to activate apoptosis [55] . the overexpression of cytochrome c in rabies virus showed a decreased pathogenicity in vitro and in vivo [56] . based on their role in apoptosis activation, hosts need to increase the expression level of these genes to promote apoptosis when exposed to a virus; this supposition explains the up-regulation of genes involved in apoptosis in the transcriptome following bmnpv infection. additionally, a significantly higher relative transcriptional level of cytochrome c was detected by rt-qpcr in bc9 and a35 (fig 6) , which indicated the up-regulation of this gene could activate apoptosis to repress further bmnpv infection. however, some genes were down-regulated, including cysteine aspartic acid specific protease 9l, protein kinase a, apoptosis-inducing factor, apoptosis signal-regulating kinase 1, tnf-receptor-associated factor 6, tgf-beta-activated kinase 1, and p90 ribosomal s6 kinase. the inhibition of these genes may strongly impair viral infectivity and virus-induced apoptosis [57] [58] [59] [60] [61] [62] . although some differential expression of immune genes was observed, this might not be considered biologically important due to low expression levels; low expression levels were also found after bmcpv infection [21] . in this study, most immune related genes were down-regulated, with a few exceptions, such as the 2-fold up-regulation of toll-like protein and lysozyme. the expression of several other genes, including 18 wheeler, macrophage mannose receptor 1, and slit homolog 3 protein, were also up-regulated during the bmnpv infection. unfortunately, the relationship between immune genes and bmnpv remains unclear and requires further study. we presumed that the low expression levels of immunity related genes may be associated with the disruption of the immune system by bmnpv, similar to the pathogenicity of human immunodeficiency virus (hiv). based on the venn diagram, 22 genes of interest were identified, all of which were down-regulated in bc9 following bmnpv infection. these genes were grouped based on their functions, as reported in the literature. these groups, including transport, virus replication, intracellular innate immune, and apoptosis, may play an important role in the process of silkworm resistance to bmnpv. several genes related to virus transport, including lactase-phlorizin hydrolase (lph) [37] , b (0,+)-type amino acid transporter 1 (bat1) [38, 46] , actin cytoskeleton-regulatory complex protein pan1 (pan1) [63] , and otoferlin [41, 42] were identified in this study. the expression level of these genes were all down-regulated in bc9 following bmnpv infection (fig 5) . the resistant strain a35, a donor parent, was used to validate our results. the expression level of pan1 was chosen for further testing by rt-qpcr. expression levels of pan1 were down-regulated in both bc9 and a35 following bmnpv infection (fig 6) . the down-regulation of virus transport-related genes could inhibit the transmembrane and intracellular transport of bmnpv thereby preventing further infection. mfs-type transporter (mfs) [39, 40] , a transmembrane facilitator, was induced to increase intracellular monovalent ion concentrations, which led to lysis and cell death after hiv-1 infection. the down-regulation of this gene in silkworm could potentially block bmnpv infection. several genes related to virus replication were found to be of interest, including engrailed nuclear homeoprotein-regulated protein [64] , 1-acyl-sn-glycerol-3-phosphate acyltransferase alpha (asgpa) [65, 66] , alanine aminotransferase 2 (alt2) [67, 68] , u2af domain protein (u2af) [69, 70] , adenylate cyclase type 5 (act5) [71] , ef-hand domain-containing protein (efhp) [72, 73] , prostatic acid phosphatase (pap) [74] , and zinc ribbon domain protein (zrdp) [75] . in this study, the expression level of these genes were all down-regulated in the transcriptome of bc9 following bmnpv infection (fig 5) . in order to validate the results, rt-qpcr was conducted as described above. the expression levels of efhp and pap were lower following bmnpv infection in both bc9 and a35 (fig 6) . the down-regulation of these genes could directly or indirectly participate in repressing bmnpv replication in host cells. of the genes related to apoptosis, the expression level of pyruvate dehydrogenase kinase (pdk) was obviously inhibited after treatment with apoptosis-inducing agents [76] ; such down-regulation might activate apoptosis in response to bmnpv infection. protease inhibitor 6 is a member of the protein superfamily that contains til functional domain. zhao et al. used genome sequences to demonstrate that the expression level of the til superfamily were down-regulated following bmnpv infection [77] , which was consistent with our results (fig 6) . furthermore, peptidoglycan-recognition protein 2, hemolin, facilitated trehalose transporter tret1, integument esterase 2, defense protein precursor, and antimicrobial protein 6tox precursor also showed differential expression following bmnpv infection. the relationship of these genes to bmnpv is an important area for further study. four degs were down-regulated in bc9 following bmnpv infection, including rasresponsive element-binding protein 1, gtpase-activating protein, trypsin alkaline a and peroxidase (fig 5) . previous studies revealed that these proteins play a role in virus infections. mdv1-mir-m4 encoded by marek's disease virus efficiently targeted the 3' untranslated regions of ras-responsive element-binding protein 1 (rreb1) [78] . tbc domain proteins belonging to a gtpase-activating protein were knocked out by double stranded rna interference (rnai), which led to a decrease in the level of transcripts of white spot syndrome virus genes [79] . trypsin in the myocardium was able to trigger acute myocarditis following influenza a virus infection [80] . overexpression and rna silencing studies revealed that peroxidase was involved in the production of hepatitis c virus particles [81] . moreover, rt-qpcr results indicated that the expression levels of all genes were down-regulated in bc9 and a35 following bmnpv infection (fig 6) . therefore, we speculated that the down-regulation of these genes might be involved resistance to bmnpv infection. we hypothesized that the 22 degs discussed above played a role in the process of host response to bmnpv infection. the endocytosis process is triggered when the bmnpv nucleocapsid containing envelope binds to the cytomembrane. vacuolar atp synthase is activated by lph to promote the fusion of the envelope and endosome thereby releasing the nucleocapsid into the cytoplasm. this process can be promoted by pan1 and otoferlin. however, the transmembrane transport channel is an alternative pathway for virus to enter the cytoplasm, a process which can be facilitated by bat1. the released nucleocapsid is transported into the nucleus with the help of the cytoskeleton (efp). once viral dna is released into the nucleus, it will utilize host nucleotides to complete replication. in the final step of replication, viral dna has to rely on host cell amino acids for assembly on the cytoskeleton (table 5 ) [82] . in the cytoplasm, efhp, asgpa, alt2, u2af, act5 and zrdp play an important role in facilitating virus replication, although the exact mechanism is still unclear. moreover, transmembrane protein, mfs, is induced by bmnpv to increase cell volume, leading to lysis and cell death. we speculated that the down-regulation of these genes may affect the entry of virus into host cells and virus replication. the apoptosis process could also be triggered by pdk to inhibit bmnpv from further infecting other cells once bmnpv entered a host cell (fig 7) . taken together, our results provide useful information on silkworm resistance to bmnpv infection. supporting information s1 the genetics and genomics of the silkworm, bombyx mori silkworm genomics-progress and prospects genetic diversity among silkworm (bombyx mori l., lep., bombycidae) germplasms revealed by microsatellites lipopolysaccharide elicits expression of immune-related genes in the silkworm, bombyx mori gene expression profiling of resistant and susceptible bombyx mori strains reveals nucleopolyhedrovirus-associated variations in host gene transcript levels screening of molecular markers for npv resistance in bombyx mori l. 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pyruvate dehydrogenase kinase 4 (pdk4) could be involved in a regulatory role in apoptosis and a link between apoptosis and insulin resistance genome-wide identification and immune response analysis of serine protease inhibitor genes in the silkworm, bombyx mori marek's disease virus microrna designated mdv1-pre-mir-m4 targets both cellular and viral genes pmtbc1d20, a rab gtpase-activating protein from the black tiger shrimp, penaeus monodon, is involved in white spot syndrome virus infection ectopic trypsin in the myocardium promotes dilated cardiomyopathy after influenza a virus infection quantitative proteomics identifies the membrane-associated peroxidase gpx8 as a cellular substrate of the hepatitis c virus ns3-4a protease baculovirus infectivity and the actin cytoskeleton key: cord-001123-n2e4s7bu authors: lin, yue-zhi; yang, fei; zhang, shu-qin; sun, liu-ke; wang, xue-feng; du, cheng; zhou, jian-hua title: the soluble form of the eiav receptor encoded by an alternative splicing variant inhibits eiav infection of target cells date: 2013-11-22 journal: plos one doi: 10.1371/journal.pone.0079299 sha: doc_id: 1123 cord_uid: n2e4s7bu equine lentivirus receptor 1 (elr1) has been identified as the sole receptor for equine infectious anemia virus (eiav) and is a member of the tumor necrosis factor receptor (tnfr) superfamily. in addition to the previously described membrane-associated form of elr1, two other major alternative splicing variant mrnas were identified in equine monocyte-derived macrophages (emdms). one major spliced species (elr1-in) contained an insertion of 153 nt, which resulted in a premature stop codon situated 561 nt upstream of the predicted membrane spanning domain. the other major species (elr1-de) has a deletion of 109 nt that causes a shift of the open reading frame and generates a stop codon 312 nt downstream. because elr1-de presumably encodes a peptide of a mere 23 residues, only elr1-in was further analyzed. the expression of a soluble form of elr1 (selr1) by elr1-in was confirmed by western blot and immunofluorescence analyses. similar to elr1, the transcription level of elr1-in varied among individual horses and at different time points in the same individuals. the ratio of elr1-in mrna species to elr1 mrna was approximately 1∶2.5. pre-incubation of the recombinant selr1 with eiav significantly inhibited eiav infection in equine macrophages, the primary in vivo target cell of the virus. fetal equine dermal (fed) cells are susceptible to eiav in vitro, and the replication of eiav in fed cells transiently transfected with elr1-in was markedly reduced when compared with replication in cells transfected with the empty vector. finally, the expression levels of both forms of the eiav receptor were significantly regulated by infection with this virus. taken together, our data indicate that selr1 acts as a secreted cellular factor that inhibits eiav infection in host cells. for most retroviruses, the viral envelope binds to receptors in a ph-independent manner, suggesting that the virions can fuse directly to the cell membrane [1] . therefore, viral receptors on the cell membrane provide binding sites for the virus and are also involved in the structural modulation of viral envelopes, leading to the fusion of the cellular and viral membranes and virion entry, the first step in viral infection of target cells [2] . accordingly, studies of the role of viral receptors in the invasion of the virus are important to the development of antiviral reagents and vaccines. the equine infectious anemia virus (eiav) is a member of the genus lentivirus, family retroviridae, and its structure is the simplest out of all the known lentiviruses [3] . the receptor of eiav is equine lentivirus receptor 1 (elr1), which was identified by zhang et al. in 2005 using a functional cloning approach [4] . in contrast to most other lentiviruses, such as human immunodeficiency virus (hiv)-1, simian immunodeficiency virus (siv) and feline immunodeficiency virus (fiv), which require co-receptors for successful infection, eiav appears to depend only on a functional elr1 for the invasion of target cells. based on its sequence and structural characteristics, elr1 belongs to the tnf receptor (tnfr) superfamily [4, 5] , and many receptors of this superfamily, such as the growth factor receptor, leptin receptor and fas, also have soluble forms. soluble forms have also been identified for some immunoglobulins and chemokine receptors [6] [7] [8] . soluble receptors can be processed posttranscriptionally or posttranslationally. the release of membrane-associated forms from the cell surface contributes significantly to the formation of soluble receptors at the posttranslational level; this process is usually catalyzed by enzymes and highly is regulated. in addition, the alterative splicing of mrnas during the maturation of eukaryotic pro-mrna is another mechanism for the formation of soluble receptors. the translation of receptor mrna can be prematurely terminated due to alterative splicing, which produces receptors that lack the transmembrane and cytoplasmic domains [6, 7, 9] . much evidence has demonstrated that soluble viral receptors are functionally important for viral infections [10] [11] [12] [13] . the soluble receptors for hiv-1, ebv (epstein-barr virus) and rhinovirus are reportedly able to inhibit infection by the corresponding viruses [11, 14, 15] . another study found that the soluble form of the avian sarcoma leukosis virus subgroup a (aslv-a) receptor tva (stva) inhibited the infectivity of this virus by 90% at a low concentration (25 pm) but mediated aslv-a infection in cells lacking the receptor at a high concentration (5 nm) [16] . brindley et al. demonstrated that preincubation of eiav with the soluble ectodomain of elr1 dramatically reduced the viral infectivity on the target cells [17] . these data further implicate soluble viral receptors in the interaction between viruses and their host cells. as mentioned above, the investigation of the alternative splicing isoforms of a given receptor facilitates a better understanding of its functions. to the best of our knowledge, there are no reports on naturally expressed soluble eiav receptors. therefore, the present study had three objectives: 1) to identify alternative splicing variants for elr1, 2) to determine whether any of these variants encode soluble elr1 and 3) to characterize the roles of any soluble forms of the receptor in eiav infection. horses and related experimental protocols used in this study were approved by the institutional animal care and use committee (iacuc) of the harbin veterinary research institute (hvri), chinese academy of agricultural sciences. there were no animals scarified specifically for this study. fetal equine dermal (fed) cells and 293t cells were proliferated from the stock of the cell bank of epidemic disease examination and service center of hvri. equine monocyte-derived macrophages (mdms) were prepared from peripheral blood mononuclear cells (pbmc) that were isolated from 200-300 ml horse peripheral blood taken from the jugular vein by veterinarians. horses were infected with eiav in a previous study [19] , and the animals infected with pathogenic eiav strains were euthanized at the end of experiment or when demonstrated severe disease-associated symptoms resulting in distress by intravenous injection of pelltobarbitalum natricum (100 mg/kg of body weight, dissolved in saline) in the jugular vein by veterinarians according to protocols approved by iacuc of hvri. fetal equine dermal (fed) cells, equine monocyte-derived macrophages (mdms) and human 293t cells were used for the transfections and the expression of recombinant proteins. the cells were maintained in high-glucose dulbecco's modified eagle's medium (dmem, gibco, usa) supplemented with penicillin and streptomycin. the medium was supplemented with 10% fetal calf serum (fcs) for the fed and 293t cells. equine mdms (emdms) were enriched from whole blood as described previously [18] . eiav dlv34 (dlv34) is a cell culture-adapted eiav pathogenic strain, which was derived by 33 passages of the virulent eiav ln40 strain in donkey mdms. similar to eiav ln40 , inoculation with 10 4 tcid50 of dlv34 resulted in acute eia in all the experimentally infected horses [19] . the bdna is a sandwich nucleic acid hybridization assay, in which target mrna molecules are captured through cooperative hybridization of multiple probes. unlike pcr, in which a region of the intended target is exponentially amplified in order to generate detectable signal, in bdna assays only signals are amplified. bdna assays are consequently not susceptible to contamination risks associated with pcr-based assays [20, 21] . equine mdms were cultured and elr1-in mrna was extracted and quantified using bdna assay as described previously [18] . samples were read by a luminex 200 (molecular devices, usa) and all data were analyzed using the luminex is2.3 program. an acquisition gate between 5000 and 20,000 was set to exclude any doublet events and ensure that only single microspheres were measured. the construction of vectors for the expression of ha-and gfp-tagged elr1 and elr1-in the elr1 cdna was cloned by reverse transcription pcr (rt-pcr) using equine mdm with primers based on the published sequence of equine elr1 [4] . the green fluorescent protein (gfp) gene was subcloned from the pgfp-n3 vector (clontech, usa), and the gfp-elr1 fusion gene fragment was amplified by overlapping pcr. a human influenza hemagglutinin (ha) fragment was linked to the 39 of the elr1 cdna for the detection of the expressed recombinant elr1. the elr1-ha fusion fragment was amplified using the primers 59-gcgaattc-ttagcacagggacgcgtagtccgggacgtcgtatggg-taggcctggcagctct-39 for ha and 59-gcgaattctt-agcacagggacgcgt agtccgggacgtcgtatgggt-aggtctgaaggac-39 for elr1. both of these pcr products were digested with ecori and hindiii and inserted into the pcdna3.1 (+) vector (invitrogen, usa) at the same cloning sites. vectors for the expression of gfp-and ha-tagged elr1-in were constructed using the same procedures. plasmids containing only the gfp or ha gene fragment were used as the controls for gfp and ha expression, respectively. the translation of selr1, i.e., the protein predicted to be encoded by elr1-in, was confirmed using western blot and confocal imaging. briefly, 293t cells were transfected with plasmids expressing ha-tagged elr1 and elr1-in using polyjet transfection reagents (roche, usa). the expression of membraneassociated or soluble elr1 was examined by western blot using a monoclonal antibody against the ha tag (sigma, usa) fused to the receptor at the c-terminus. protein bands reacting with the ha antibody were visualized using a horseradish peroxidase (hrp)-conjugated goat anti-mouse antibody (sigma, usa). to perform confocal imaging, 293t cells seeded on coverslips were transfected with the ha-tagged selr1 or elr1 expression vectors. after 72 h of incubation, transfected cells were fixed with 4% paraformaldehyde in pbs for 30 min and permeabilized with 0.1% triton x-100 for 15 min. cells were blocked with pbs/ fish gelatin (5%) for 2 h, reacted with anti-ha monoclonal ab for 1 h and then incubated with an anti-mouse igg (whole molecule)-fluorescein isothiocyanate (fitc)-labeled antibody (f2012, sigma). cells were washed with pbs for three times and then stained with 4,6-diamidino-2-phenylindole (dapi, sigma) for 30 min and examined using a leica tcs sp5 confocal system (leica microsystems, germany). appropriate igg isotype controls were included to rule out non-specific binding. in addition, a 3d live cell imaging system (ultraview vox, perkinelmer, usa) was applied to observe the dynamically distribution of the selr1 and elr1 that fused to gfp at the n-terminus after being transiently expressed in 293t cells. an in vitro eiav infection inhibition assay was applied to examine the ability of selr1 to interact with eiav. specifically, selr1 was transiently expressed in 293t cells by transfection of the expression plasmid pcdna3.1-elr1-in. the culture medium that contained the secreted selr1 was collected at 48 h after transfection and incubated with 10 3 tcid50 of the dlv34 strain at 4uc for 30 min. the viruses that were pre-incubated with selr1 were then added to emdms in 6-well plates. cell-free culture medium was collected at 1, 3, 5 and 7 days post-infection (dpi) and measured for viral reverse transcriptase (rt) activity using a reverse transcriptase assay colorimetric kit (roche, switzerland). the dlv34 strain incubated with the culture medium of cells transfected with the empty pcdna3.1 vector was used as the control for non-specific bindings. the effect of selr1 overexpression on the eiav infection of target cells selr1 was transiently overexpressed in fed cells by transfection with pcdna3.1-elr1-in using polyjet transfection reagents. the fed cells were infected with 100 ml of appropriately diluted dlv34, 24 h after the transfection. the virion release into the medium was quantified at 2, 4, 6 and 8 dpi by measuring the viral rt activity. the mrna levels of elr1 and elr1-in in equine mdms infected with eiav were determined using quantitative real-time pcr. cultivated emdms were infected with 0.1 plaque-forming units (pfu) of dlv34 and collected at 4, 8 and 12 hours and 3 and 5 dpi. mrna samples were prepared from these cells and reverse transcribed into cdna using m-mlv reverse transcriptase (invitrogen, usa), and the expression levels of elr and elr1-in were then quantified using real-time pcr with a sybr green amplification and detection kit (qiagen, germany). the expression levels of the elr1 transcripts were normalized to that of the b-actin mrna. plasmids containing either the elr1 or elr1-in cdna were constructed to establish standard curves for copy number determination. the elr1 and elr1-in fragments were amplified by pcr, cloned into a pmd18-t vector and verified by dna sequencing. the standard plasmid was serially diluted from 1610 8 to 1610 2 copies/ml and then measured using real-time pcr. for each reaction, 5 ml of standard plasmid or cdna sample was added to a 20 ml reaction mixture containing 12.5 ml sybr green pcr master mix and 3 mm of each primer. the amplification protocol was as follows: 95uc for 10 min, followed by 35 cycles at 95uc for 30 s, 60uc for 30 s and 65uc for 30 s; the final extension was at 68uc for 10 min. all the samples were measured twice. the elr1 and elr1-in mrna levels were calculated by dividing the receptor copy number by the b-actin copy number. statistical analysis and data presentation were performed using student's t-test (two-tailed, confidence intervals of 95%). and graphpad prism version 4.0 (graphpad software, usa) programs, respectively. a probability value less than 0.05 was considered statistically significant. the identification of alternative splicing variants for elr1 the elr1 cdna was amplified from mrna templates extracted from emdms by reverse-transcription pcr using primers designed based on the sequence published in genbank. polyacrylamide gel electrophoresis revealed three bands migrating at approximately 1000 bp (fig. 1a) , and subcloning of the amplified fragments further confirmed the existence of three elr1 cdna fragments with different molecular masses (fig. 1b) . nucleotide sequencing revealed two other species of elr1: one containing an insertion of a 153 nucleotide (nt) fragment of intron 6 (elr1-in) and another with a deletion of 109 nt (elr1-de). to avoid the contamination of introns of chromosomal dna, the elr1-in mrna samples were pre-treated with dnase to remove any contaminating chromosomal dna and then pcr amplified with a pair of primers, one of which targeted the insert. the 125 bp elr1 cdna fragment was amplified from both mrna samples with or without dnase pre-treatment, indicating that elr1-in originated from the mrna. as a control, we confirmed that a 501 bp fragment from intron 3 of the equine mhc-i gene was amplified from the dna preparation without dnase treatment but not a preparation that was pre-incubated with the nuclease (data not shown). in addition, to exclude the possible artificial effect resulting in the elr1-in molecule during reverse transcription and pcr amplification, the presence of elr1-in mrna was further measured by a bdna assay, which detects specific mrna species directly from total rna preparations by hybridizing with signalamplified probes specific for the inserted sequence without being revers transcribed and amplified. as shown in fig. 1c , the average light unit of elr1-in mrna was 1,594 while that of the blank control was 53.5. to further investigate the status of the elr1 transcript variants in vivo, the variants were examined by cloning and sequencing the pcr-amplified specific cdna fragments from the pbmcs of three horses collected at different time points. all three major variants of elr1 mrna were found in all the horses and at all the time points examined. however, the ratios of each isoform were varied among the different horses and at the different sampling times. among the total 126 clones analyzed, the elr1 species was predominant (83/126), followed by elr1-in (31/126) and elr1-de (12/126) ( fig. 2a) . furthermore, additional isoforms with insertions or deletions were identified. among the 31 clones of elr1-in, 29 contained a fragment of intron 6, and the other two contained a fragment of intron 2 or intron 7. in addition, five types of deletions at different sites of the coding region were detected (fig. 2b) . all the insertions and deletions resulted in a shift of the open reading frame (orf). for the major form of elr1-in, the inclusion of a 153 nt fragment of intron 6 created an isoform with five different amino acid residues and then a premature stop codon 18 residues upstream of the predicted transmembrane domain (fig. 2c) . the cdna of elr1-de, which lacked exon 3, contained two orfs; the first encoded a peptide of only 27 residues, and the second encoded a receptor lacking the eiavbinding crd1 (fig. 2c) . because both of these predicted proteins were considered not to function as eiav receptors, they were not further evaluated in the present study. the receptor encoded by elr1-in was predicted to be a truncated form lacking the transmembrane domain and the cytoplasmic domain and, therefore, was presumed to be a soluble form of the receptor. the expression of soluble elr1 (selr1) was examined by western blot, flow cytometry and confocal microscopy. an selr1-ha fusion protein was overexpressed in 293t cells, and the 26 kda selr1-ha protein was detected in both the cell lysate and the culture medium by western blot using an anti-ha antibody. as a control, membrane-associated elr1-ha was detected only in the cell lysate (fig. 3a) . in addition, both selr1 and elr1 were transiently overexpressed in 293t cells with green fluorescence protein (gfp) fused at the n-terminus. flow cytometry analysis showed that, although the transfection efficiency was similar for both of the transfected cell populations, the fluorescence intensity of the gfp-selr1-expressing cells was markedly weaker than that of the gfp-elr1expressing cells, indicating a dramatic reduction in the intracellular accumulation of gfp-selr1 relative to gfp-elr1 (fig. s1 ). the aforementioned experiment suggested that the eiav receptor encoded by elr1-in was expressed as a soluble form of the receptor. to further examine the dynamic cellular distribution of selr1 and compare with that of elr1, a live cell imaging of the dynamic expression and distribution of the gfp-elr1 and gfp-selr1 was taken. as shown in fig. 3b , the membrane-associated gfp-elr1 was accumulated as a few large spots in the cell plasma at the early phase post transfection (fig. 3b) . the fluorescence extended and covered to the whole cell surface and plasma thereafter (intermediate phase). at the late phase of cell life, the intracellular fluorescence was seen as bands and islands, which was considered the remaining membrane-bound elr1 after the damage of plasma membrane. on the other hand, the transmembrane domain-free gfp-selr1 was observed as diffused fluorescence, distributed like donuts at the early phase, evenly covered to the whole cell at the intermediate phase and gradually attenuated to weak and faint images at the late phase. at certain time points, the leak of fluorescent content from some cells could even be observed (see the video of 3d live cell image in movie s1, e.g. at the recombinant selr1 was expressed as a fusion protein with an ha tag at the c-terminus in 293t cells, and its presence in the cell lysate or in the culture medium was examined by western blot using an anti-ha antibody. b-actin was applied as the internal control protein. (b) live cell imaging analysis of elr1 and selr1 expression in hek 293t cells. these two forms of receptor were expressed as proteins with a gfp fused at the nterminus of each. the three images of selr1 were enhanced at a higher level than elr1. (c) cellular location of elr1 and selr-1. ha-tagged elr1 and selr1 were expressed in 293t cells for 72 h. cells were then examined by confocal microscopy after being fixed, stained with dapi for the nucleus (blue) and dii for the cell membrane (red) and reacted with an anti-ha antibody and a fitc-labeled anti-mouse igg (green). appropriate igg isotype controls were included to rule out non-specific binding. doi:10.1371/journal.pone.0079299.g003 05:00:00). these images vividly demonstrated the behaviors of gfp-selr1 as a soluble protein. the cellular location of elr1 and selr1 was also detected by confocal microscopy. these two forms of eiav receptor were expressed as ha-tagged proteins in 293t cells and were detected by an ha monoclonal antibody and visualized by a fitc-labeled anti-mouse antibody. as displayed in fig. 3c , the elr1-ha was mostly detected on the cell membrane, so as the selr1-ha, but at a much lower level. combined with all the results submitted in fig. 3 and supplementary materials, it indicates that selr1 is expressed as a soluble protein and can be secreted out of the cell. however, some molecules of this soluble form of receptor are associated with the cell surface membrane. the ability of selr1 to inhibit eiav infection on target cells is the most important characteristic of the soluble receptor studied here. the recombinant selr1 was overexpressed in 293t cells, and eiav preparations pre-incubated with either the culture medium of selr1-expressing cells or the culture medium of cells transfected with the empty pcdna3.1 vector were used to infect emdms. the replication of the infected eiav was then examined by measuring the viral rt activity. as shown in fig. 4 , eiav incubated with the medium of pcdna3.1-transfected cells and the untreated virus replicated at similar rates. in contrast, the virus that was pre-incubated with medium from selr1-expressing cells showed a dramatically retarded replication that was characterized by delayed detectable rt activity and low rates of increase. this result strongly indicated that overexpressed recombinant selr1 was able to inhibit eiav infection in vitro, probably by competing with the membrane-associated receptor for the binding of virions on target cells. this result also suggests that the receptor encoded by elr1-in was a secreted protein. because selr1 interacted with eiav in culture medium, significantly blocking the subsequent infection of target cells by the selr1-preincubated virus, it was logical to examine whether the expression of the soluble receptor by the target cells interferes with viral infection. thus, the soluble form of elr1, tagged with ha, was overexpressed in fed cells, a major target of eiav in vitro, by transfection with the selr1 expression vector pcdna3.1-elr1-in. the cells were then infected with eiav 24 h after the transfection. the results revealed that eiav replicated at a significantly slower rate in the fed cells transfected with the selr1 expression vector than in the cells transfected with the empty vector at 6 and 8 dpi (p,0.05, see fig. 5a ), indicating the inhibitory effect of selr1 expressed in the target cells upon eiav infection. in addition, the presence of recombinant selr1 in the culture medium was confirmed by western blot using an anti-ha antibody. there was no b-actin detected in the medium, which ruled out the possibility that these soluble receptor molecules were leaked from broken cells (fig. 5b ). as shown in fig. 2 , the level of elr1 expression varied in different hosts and at different detection times. thus, we tested the effect of eiav infection on the expression of the membraneassociated and soluble forms of elr1. the elr1 and selr1 mrnas were quantified in emdms infected with eiav for 4 hours to 5 days using sybr green real-time pcr with b-actin as the internal control. the fold changes of elr1 and selr1 mrna (elr1-in) after eiav infection at each time point are shown in fig. 6 , with the fold changes of the mrna levels in mock-infected cells as the baseline (indicated as 1). both the elr1 and elr1-in levels showed no apparent change or a slight decrease during the 4 to 12 h post-infection. however, a notable increase (approximately 1.5-fold, p,0.05) was observed for the elr1 and selr1 mrnas in cells infected with eiav for 3 and 5 d when compared with the untreated controls. alternative splicing is an important posttranscriptional regulation mechanism in eukaryotic cells. most genes in eukaryotes are alternatively spliced into several forms, and the percentage of these genes can be as high as 93% in humans [22] . through such a mechanism, multiple proteins with different functions can be produced from a single gene, thus providing an opportunity for adaptation to different environments [23] . multiple alternative splicing variants of elr were identified in this study for the first time. the predominant isoform that produces a functional elr1 is a species of 942 bp containing exons 1-9. the other two major isoforms include elr1-in, which contains an insertion of a fragment of intron 6, and elr-de, in which exon 3 is deleted following splicing. these three major forms of spliced mrna exhibit distinct expression levels and ratios in different individuals and at different time points. the inserted fragment in elr1-in was determined to be a fragment of an intron, and intron retention is considered to occur in unprocessed or incompletely spliced pre-mrna [24, 25] . it is known that abnormal transcript variants, such as intron retentions, are occasionally generated in cells but normally do not exist long enough to have a functional impact. in addition, it has been reported that, although intron retention events are common in human genes, their frequency relative to the dominant transcript is the culture medium, containing secreted selr1, was incubated with an eiav strain dlv34 at 4uc for 30 min. virus that had been pre-incubated with selr1 was used to infect emdms in 6-well plates. dlv34 pre-incubated with medium from cells that had been transfected with the empty vector pcdna3.1 or untransfected cells were also used to infect emdms as controls. eiav replication was monitored by measuring viral rt activity at 1, 3, 5 and 7 days post infection (dpi). the data shown represent the means and standard errors of the means from three separate experiments. *p,0.05, compared with empty vector transfected cells. doi:10.1371/journal.pone.0079299.g004 generally low [22, 25] . our results demonstrated that elr1-in differed from a typical intron retention transcript: it contained a fragment of an intron and appeared with a high frequency compared with the predominant transcript of elr1 (approximately 25%). elr1-in was consistently detected in different individuals at different times, and it was translated into a functional soluble elr1 when the recombinant molecule was expressed. in addition, we did not find the typical ''gt/ag'' and ''gc/ag'' splicing sites in these spliced elr1 variants. however, due to the absence of a sufficient equine alternative splicing database, the splicing model of elr1-in remains unclear [24] . the protein encoded by elr1-in was confirmed to be a soluble receptor by several different approaches, including western blot, live cell imaging, confocal microscopy and flow cytometry, which demonstrated that most of the receptor molecules were secreted out of the cell after being expressed. however, 3d live cell imaging and confocal microscopy showed that a small portion of selr1 associated with the cell surface, although the fluorescent signal was much weaker than that of elr1, the membrane-associated form of eiav receptor. because the predicted hydrophobic transmembrane domain is absent in the selr1, the association of this soluble protein on cell membrane is mediated by the glycoside chains, like that observed for the eiav gp90 surface protein [4, 5] . because soluble viral receptors containing virus-binding motif(s) can be used as valuable tools for evaluating viral entrance into target cells, these molecules have played important roles in studies of virus-host interactions [26, 27] . it was reported that the coincubation of soluble cd4 with hiv-1 envelope protein resulted in the dissociation of the gp120 surface protein and gp45 transmembrane protein [6, 28] . cd4 is also the cellular receptor of hiv-1, hiv-2 and simian immunodeficiency virus sivmac and sivagm. the soluble form of cd4 (scd4) can block hiv-1 and hiv-2 infection in human lymphoma cell lines, but it enhances sivagm infection in these cell lines by 10 to 100-fold [29] . a similar augmenting effect of soluble viral receptors on the invasion of viruses was also observed in studies on herpes simplex virus (hsv) [30, 31] . in addition, a study on jaagsiekte retrovirus (jsrv) found that although the soluble form of its receptor, hyal2 (shyal2), did not mediate the entrance of a jsrv-pseudotype retroviral vector into cells lacking the integrated hyal2, this purified soluble protein significantly inhibited the infection of the pseudotyped vector in the target cells of jsrv [13] . moreover, some soluble receptors in the plasma can be considered markers of disease progression, such as soluble tnf receptor (stnfr) ii and soluble il-2 and il-6 receptors in hiv-1-infected patients [10, 14, 32] . therefore, soluble receptors play important roles in the interactions between viruses and hosts [33] [34] [35] [36] [37] . studies on the mechanisms regulating the formation of soluble viral receptors should lead to a better understanding of the cellular responses to invading viruses. in this study, the soluble form of the eiav receptor selr1 markedly inhibited eiav infection of its target cells when examined by in vitro competition studies, and this inhibitory effect appeared to increase with increasing infection time. additionally, the expression levels of elr1 and its alternative splicing variants were regulated by eiav infection. in a separate study on the restriction of eiav superinfection, we found that the up-regulation of selr1 induced by the initially infected viral strain was correlated with protection against subsequent viral infection (unpublished data). at present, it remains unclear whether the inhibitory effect of selr1 on eiav infection is based on the competition of the binding of the virus to membraneassociated elr1 or on the polymerization with eir1, which may trigger or may reduce signal transduction of membrane-associated receptors, therefore interfering with the expression of downstream cellular proteins [6] . however, our data indicate that selr1 is not merely a truncated product of a spliced elr1 transcript but is a functional protein with important biological roles. elr1 is a member of the tnfr superfamily, which is known to have important roles in regulating immune responses and inducing pathological outcomes [4, 38] . previous studies have shown that plasma tnfa concentration is positively correlated with the clinical symptoms of eiav-infected horses [18] . it was also reported that stnfr was significantly elevated in patients with hiv-1 infection [10] . because elr1 acts as both the receptor of eiav and a member of tnfr family, the finding that selr1 is regulatable and can compete its membrane-associated prototype form implicates the involvement of this soluble protein in both of the viral infectivity and the host response, especially that tnfa is closely related with eia symptoms. the possible role of selr1 in elr1 signaling is an interesting question to be explored. the results of this study indicate that the ability to inhibit eiav infection of target cells in vitro may be related to reduced viral entry into host cells in vivo. the regulated expression levels of both the soluble and membrane-associated forms of elr1 by eiav reveal the possible role of selr1 in the interaction between virus and host. therefore, selr1 may be considered a secreted cellular factor that regulates susceptibility to eiav and the pathological and immunological responses of the host. furthermore, the identification of elr1-in and other alternative splicing variants of elr1 suggests additional approaches for further studies on the pathogenesis and immunogenicity of eiav infection. figure s1 recombinant selr1 was expressed in 293t cells as a fusion protein with a gfp tag linked at the nterminus. the percentage of gfp-expressing cells and the fluorescence intensity of selr1-gfp in cells were examined by flow cytometry. the gfp-tagged elr1 was also expressed and analyzed in parallel in the experiments shown in this figure. all the experiments were performed for three times, and a representative result is shown. movie s1 a 3d live cell imaging of the dynamic expression and distribution of the gfp-elr1 and gfp-selr1 was taken to examine the dynamic cellular distribution of selr1 and compare with that of elr1. the images of selr1 were enhanced at a higher level than elr1. 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retrovirus receptor, hyal2 cytokines and soluble receptor changes in the transition from primary to early chronic hiv type 1 infection soluble recombinant cr2 (cd21) inhibits epstein-barr virus infection production and characterization of a soluble, active form of tva, the subgroup a avian sarcoma and leukosis virus receptor an equine infectious anemia virus variant superinfects cells through novel receptor interactions the pathogenic and vaccine strains of equine infectious anemia virus differentially induce cytokine and chemokine expression and apoptosis in macrophages an attenuated eiav vaccine strain induces significantly different immune responses from its pathogenic parental strain although with similar in vivo replication pattern a facile, branched dna assay to quantitatively measure glucocorticoid receptor auto-regulation in t-cell acute lymphoblastic leukemia a sensitive branched dna hiv-1 signal amplification viral load assay with single day turnaround pre-messenger rna 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infection soluble il-2 receptor serum levels-a marker for disease progression in patients with hiv-1 infection soluble interleukin-6 receptor induces motor stereotypies and co-localizes with gp130 in regions linked to cortico-striato-thalamo-cortical circuits soluble il-2ralpha (scd25) exacerbates autoimmunity and enhances the development of th17 responses in mice splicing fidelity, enhancers, and disease levels of soluble endothelial protein c receptor are associated with cd4+ changes in maraviroc-treated hiv-infected patients synergistic effects of soluble pd-1 and il-21 on antitumor immunity against h22 murine hepatocellular carcinoma tnf receptor (tnfr)-associated factor (traf) 3 serves as an inhibitor of traf2/5-mediated activation of the noncanonical nf-kappab pathway by traf-binding tnfrs key: cord-000375-fvfl0bn1 authors: shen, ching-i; wang, ching-ho; shen, shih-cheng; lee, hsiu-chin; liao, jiunn-wang; su, hong-lin title: the infection of chicken tracheal epithelial cells with a h6n1 avian influenza virus date: 2011-05-06 journal: plos one doi: 10.1371/journal.pone.0018894 sha: doc_id: 375 cord_uid: fvfl0bn1 sialic acids (sas) linked to galactose (gal) in α2,3and α2,6-configurations are the receptors for avian and human influenza viruses, respectively. we demonstrate that chicken tracheal ciliated cells express α2,3-linked sa, while goblet cells mainly express α2,6-linked sa. in addition, the plant lectin mal-ii, but not maa/mal-i, is bound to the surface of goblet cells, suggesting that sa2,3-linked oligosaccharides with galβ1–3galnac subterminal residues are specifically present on the goblet cells. moreover, both α2,3and α2,6-linked sas are detected on single tracheal basal cells. at a low multiplicity of infection (moi) avian influenza virus h6n1 is exclusively detected in the ciliated cells, suggesting that the ciliated cell is the major target cell of the h6n1 virus. at a moi of 1, ciliated, goblet and basal cells are all permissive to the aiv infection. this result clearly elucidates the receptor distribution for the avian influenza virus among chicken tracheal epithelial cells and illustrates a primary cell model for evaluating the cell tropisms of respiratory viruses in poultry. sialic acids (sas), consisting of a core of nine-carbon monosaccharide, are usually linked to the outermost capping position of glycans that are conjugated to cell-surface glycoproteins or glycolipids [1] . sialyltransferase adds sa to the terminal sugar residues, such as galactose (gal), n-acetylglucosamine (glcnac) or n-acetylgalactosamine (galnac) [1] . the conjugation between gal and sa can be either in the form of an a2,3 or an a2,6 glycosidic linkage. in mammals, n-acetylneuraminic acid (neu5ac) and nglycolylneuraminic acid (neu5gc) are the two most common types of sa, but neu5ac is the major type of sa in birds [2] . plant lectins extracted from maackia amurensis (m. amurensis leukoagglutinin, mal) and sambucus nigra (s. nigra agglutinin, sna) are usually applied for the detection of neu5aca2-3gal (saa2-3gal) and saa2-6gal glycans in tissues, respectively [3] . two types of m. amurensis lectins were discovered: one that can agglutinate erythrocytes (hemagglutinin) (mah, also known as mal-ii) and one that can agglutinate leukocytes (mal, also known as mam, maa, mal-i). although both mal-i and mal-ii recognize the saa2-3gal glycan, previous studies [4] and recent glycan microarray data [5] demonstrated that subterminal sugars affect their binding affinity to these two lectins. for example, mal-i, rather than mal-ii, showed the highest affinity to the saa2-3galb1-4galnac and did not bind to this oligosaccharide when the subterminal b1,4-linkage was replaced by a b1,3-linkage [6] . cell entry by influenza virus depends on the recognition of a terminal sa-capped glycosylated molecules by the viral hemag-glutinin (ha) protein [3] . generally, human influenza viruses preferentially bind to cell surface oligosaccharides that have the saa2-6gal linkage, while avian influenza viruses (aivs) prefer saa2-3gal [3] . especially, the glycans containing the saa2-3galb1-4galnac or saa2-3galb1-4(6oso3)galnac, show a high affinity to maa/mal-i [4] and aivs [7, 8] . tracheal/ bronchial epithelium, mainly consisting of ciliated cells, goblet cells and basal cells, is the initially attacked tissue by the invading influenza viruses. the infected chicken trachea shows necrosis and detachment of ciliated cells, suggesting that ciliated cell is one of the cells targeted by aiv [9] . previous analyses of the expression of sas in chicken have revealed that a2,3-linked sa was localized on tracheal ciliated cells and a2,6-linked sas was also present in the tracheal tissues [10, 11, 12] . however, the detailed distribution of sas among chicken tracheal epithelial (cte) cells remains unclear. clarification of the distribution and the expression intensity of influenza receptor on cte cells will help us to understand the viral tropism, viral spreading and the pathogenesis of avian influenza viruses. here, the sa distribution on cte cells were identified by the staining of biotin-labeled plant lectins. a taiwan-isolated aiv h6n1 was applied to characterize the cell tropism of aiv and the correlation of the cell tropism of aiv to the sa distribution on cte cells was also explored. the saa2-3gal expression of primary cte cells the distribution of saa2-3gal expression on the primary cte cells was determined by the staining with a plant lectin, maa (ey laboratories). the ciliated cells were revealed by the intense fluorescent microvilli, labeled by the b-tubulin specific antibody [13] . mucin 5ac glycoprotein and cytokeratin-14 (k14) were the specific markers for the goblet and basal cells, respectively [14, 15] . immunocytochemistry (icc) staining illustrated that most btubulin + cells expressed saa2-3gal terminal glycan (ratio of maa + /b-tubulin + cells, 0.8760.06, n = 317) (fig. 1a) . in a high magnification field, the intense spots of maa signal were surrounded by cilia bundles on the ciliated cells (fig. 1b) . in addition, maa lectin could also be detected on the primary k14 + basal cells (ratio of maa + /k14 + cells, 0.4660.12, n = 267) (fig. 1g) . interestingly, the maa signals did not colocalized with the mucin-expressing cells (ratio of maa + /mucin + cells, 0.00760.01, n = 223) (fig. 1d) , indicating that goblet cells may not express the saa2-3gal glycans which show high affinity to the maa lectin. however, for the immunohistochemistry in tissue sections, the distribution of saa2-3gal expression may show inconsistent distribution when different maa/mals were applied or the same lectins were from different providers [16, 17] . to further address the sa expression on goblet cells, mal-i, same as maa, was given by vector laboratories and applied to stain the cte cells. we found that abundant mal-i was detected on the b-tubulin + cells (ratio of mal-1 + /b-tubulin + cells, 0.8260.10, n = 301) (fig. s1), rather than the mucin + cells (ratio of mal-1 + /mucin + cells, 0.0260.02, n = 201) (fig. 1e ), similar to the result for maa. subtypes of saa2-3gal glycans with different subterminal sugars present varied affinity to maa/mal-i and mal-ii. whether the saa2-3gal glycan is not expressed on goblet cells was further evaluated by mal-ii (vector laboratories). surprisingly, 0.3760.12 mucin + cells (n = 594) were double-stained with mal-ii (fig. 1f ), indicating that a glycan subtype that shows a high affinity for mal-ii only, was expressed on the goblet cells. we also found that only 0.3360.09 b-tubulin + cells (n = 230) can be labeled with mal-ii (fig. 1c ). in addition, about half basal cells were mal-ii positive (0.5360.12, n = 265) (fig. 1h ). the saa2-6gal expression of primary cte cells the distribution of saa2-6gal on the cte cells was determined by staining with sna (vector laboratories). in contrast to the finding for maa/mal-i, abundant sna signals were mainly restricted to the mucin + cells (ratio of sna + /mucin + cells, 0.8560.09, n = 368) ( fig. 2a) , indicating that the goblet cells expressed saa2-6gal terminal glycan. in addition, sna lectin can also be detected in a one-third proportion of k14 + cells (ratio of sna + /k14 + cells, 0.3660.09, n = 284) (fig. 2b) . especially, the sna signal did not localize on the b-tubulin + cells (ratio of maa + /b-tubulin + cells, 0.0160.01, n = 237) (fig. 2c ). testing the sna-i, which was provided by ey laboratories, showed no difference to the result of sna, evidenced by the undetectable binding by sna-i on the ciliated cells (fig. 2d ). the saa2-3gal and saa2-6gal expression on basal cells although both maa and sna bound to the surfaces of a subpopulation of the k14 + cells ( fig. 1g and fig. 2b ), their detected signals were relatively weak (figs. s2a and s2b). to determine whether a single basal cells can express both saa2-3gal and saa2-6gal, cte cells were triple-stained with k14 primary antibody (cy5-2u ab, purple), fitc-conjugated maa (green) and biotin-labeled sna (detected by cy3-streptovidin, red) (figs. 3a, 3b and 3c). their cell nuclei were revealed by dapi staining (blue). when viewed in the same field under a confocal microscope, the icc result indicated that maa and sna can be co-expressed on single k14 + cells (indicated by the arrows). the distribution and relative expression levels of saa2-3gal and saa2-6gal are summarized in table 1 . it should be noted that because the relative affinities of the lectins to their targeted glycans may differ, comparing the intensity of staining between the maa and sna cannot be used as a quantitative comparison of the relative saa2-3gal and saa2-6gal expressions. in addition, the h6n1 viral antigens detected in the infected cells at a moi of 0.1 were mostly restricted to the b-tubulin + cells at 6 h.p.i. (fig. 5a ). few h6n1 antigens were detected in the goblet cells or basal cells, possibly due to the low expression of aiv receptors (figs. 5b and 5c). these results indicate that the ciliated cell, rather than the goblet or the basal cells, is the primary target cell for the aiv. in addition, these data also illustrate that the distribution of saa2-3gal expression among cells determines the cell tropism of the aiv h6n1 virus (as summarized in fig. 6 and table 1 ). the infectivity of h6n1 in cte cells was further characterized at a moi of 1. icc results showed that the ciliated cells (fig. 5d ), goblet and basal cells were all susceptible to infection by h6n1 as measured at 6 h.p.i. (figs. 5e and 5f ). although saa2-3gal expression was relatively low in goblet and basal cells, still 21.360.05% of goblet cells and 51.1610.9% of basal cells were infected. we speculated that the low infection of goblet cells by influenza virus at a high moi might mediate through the binding to mal-ii specific sa or through a sa-independent pathway [18] . however, this hypothesis still requires the support from further experiments. in the human tracheal/bronchial epithelium, ciliated cells display mainly a2,3linked sas and goblet cells express a2,6linked sas [19] . about one third of ciliated cells also display the a2,6-linked sas and goblet cells also express the a2,3-linked sas at a low degree [19, 20] . the distribution of the a2,3and a2,6-linked glycans was primarily on the apical surface of both ciliated and goblet cells, respectively [19, 20] . as revealed by specific lectins and icc staining in the primary cte cells, our results clearly illustrate that all three types of cte cells express saa2-3gal in a varied degree. saa2-3gal showed abundant expression on chicken ciliated cells but only a detectable level on basal cells ( fig. 6 and table 1 ). revealing the distribution and the expression intensity of influenza virus receptor on the tracheal basal cells will help us to understand the cell tropism of influenza virus and the pathogenesis of aiv infected tissues. our previous study indicated that the replication of infectious bronchitis virus (ibv) is restricted to ciliated cells and goblet cells, but not basal cells [21] . the tracheal/bronchial basal cells, assumed as multipotent stem cells, are responsible for epithelial recovery and reestablish normal respiratory function after desquamation of the ciliated and goblet cells [14, 15] . in an uncomplicated ibv-infected chick, clinical signs persist for only 1 week and the unaffected basal cells may be responsible for epithelial reconstruction of the injured respiratory tract [22] . in contrast, basal cells are susceptible to the infection of avian influenza virus at a moi of 1. the susceptibility of basal cells to aiv infection suggests that infection with aiv alone may cause the cell death of basal cells, and consequently affect basal membrane integrity and severe inflammation in the aiv infected trachea. interestingly, maa/mal-1 did not bind to the surface of goblet cells, suggesting that certain saa2-3gal glycans that show high affinity for maa/mal-i lectins, such as saa2-3galb1-4glcnacb and saa2-3galb1-4(6oso3)glcnacb [4, 5] , are not present to a significant extent on the cell membranes of goblet cells. however, one-third of the goblet cells showed strong mal-ii staining, suggesting that the mal-ii specific glycans, such as saa2-3galb1-3(saa2-6)galnaca and saa2-3galb1-3(6oso3)-galnaca are highly expressed on goblet cells [4, 5, 6, 23] . the expression profiles of sas on cte cells exhibits distributions that are distinct from those of human. in chickens, mal-i bind to the surface of ciliated cells and basal cells, but not goblet cells. the goblet cells express a mal-ii specific saa2-3gal glycan. in addition, both sna and sna-i failed to label the btubulin + cells, indicating that saa2-6gal is exclusively expressed on non-ciliated cells. in humans, by contrast, both ciliated and goblet cells can be labeled with maa/mal-i and sna [19, 20] , indicating that these two epithelial cells have both types of influenza viral receptors. moreover, both ciliated and goblet cells cells are permissive to infection by human influenza viruses [19, 20] . interestingly, using duck influenza a viruses to infect human airway epithelial cells, the viral antigen could only be detected in the ciliated cells, but not in the goblet cells, possibly due to the low saa2-3gal expression on the surface of goblet cells and a low moi infection [19, 20] . although the saa2-6gal glycans was detected in human tracheal/bronchial goblet cells [19, 20] and was capped on mucin protein [24] , it was also proposed that tracheal mucin in the airway contains saa2-3gal [25] (fig. 6a) . especially, this secreted mucin was shown to prevent infection by influenza viruses with a saa2-3gal preference in the airway [26] . this false-receptor effect may mask the ha of the aiv, disable the viral access to the goblet cells and also account, at least in part, for the hindrance of aiv infection to human goblet cell [26] . in chicken trachea, goblet cells exhibit high affinity for sna, suggesting that the mucin may contain saa2-6gal glycans. the viral neutralization effect of mucin in humans may be recapitulated in chickens to aid the clearance of the invading influenza viruses with a saa2-6gal preference. in addition to the sa2,6-linkage glycan, sa2,3-linked oligosaccharides with galb1-3galnac subterminal residues, which show a mal-ii preference, may be present in the goblet cells (table 1) [4, 5, 6, 23] . notably, although aivs can bind to the terminal saa2-3gal, duck and chicken influenza viruses exhibit different affinities for glycans with different subterminal residues [7, 8] . for example, duck influenza viruses prefer galb1-3glcnac or galb1-3galnac, but chicken influenza viruses prefer galb1-4glcnac or galb1-4(6oso3)glcnac [7, 8] . our study suggests that saa2,3-capped oligosaccharides following a galb1,3-linkage [6] are candidate sugars conjugated to the chicken tracheal mucin protein. determining the sa composites of chicken mucin protein and the neutralizing effect of chicken mucin on duck influenza viruses will be an interesting task that will provide important information about the innate defense mechanisms in the chicken trachea and about the interspecies transmission of influenza viruses between ducks and chickens. both the hindrance of the mucin barrier and the limited number of proper receptors on the epithelial cells of the host restrict cross-species infection by influenza viruses [6, 20] . nevertheless, genetic mutations or recombinations in the ha of an influenza virus can render interspecies transmission by altering the receptor tropism [27, 28, 29] . the 226 and 228 residues in the ha of the influenza h3 and h2 viruses are particular important for determining the receptor specificity and host range restrictions [30, 31] . the ser-to-gly mutation at ha position 228 and the leuto-gln mutation at ha position 226 shift the receptor preference from saa2-6gal to saa2-3gal and enhance the infectivity of human influenza virus in duck intestinal cells [31] . the ha of the chicken h6n1 virus we applied possesses the gqrsri sequence, which corresponds to the 225 to 230 positions in h3 ha [32] . we showed that most maa + cells, but few sna + cells, were permissive to infection by the h6n1 virus. in addition, at a low moi, most h6n1 proteins were detected in the saa2-3gal-rich ciliated cells, and few in the goblet and basal cells. these results indicate that the chicken h6n1 virus, even with a ser228 (using h3 ha numbering) in its ha, still possesses a saa2-3gal preference. similarly, the ha 226 residue, critical to cell tropism in human h3 and h9n2, has been shown to be an insufficient binding site for determining the receptor tropism of the human h1, h2, h5 and the avian h6, h9 and h5n1 viruses [4, 33, 34, 35] . these results emphasize that receptor tropism is determined by several critical residues in the ha proteins, but these sites in ha are not highly conserved among different influenza viruses. a taiwan aiv h 6 n 1 strain, 2838v (virulent), was obtained by serial inoculation of an original field isolate, h 6 n 1 2838, into specific pathogen free (spf) chickens [36] . the virus was further amplified by passage into the amnion of spf eggs. cell debris in the collected fluid was clarified by centrifuging at 1000 rpm for 10 min and passing through a 0.45 mm filter. the viral titer (50% of the egg infectious dose, eid 50 ) of 2838v was 1610 8 /ml. tracheas were obtained from one-day-old spf chicks (animal health research institute, tansui, taipei, taiwan) and rinsed in a dmem medium (invitrogen, carlsbad, ca, usa) under a sterile condition. the procedure for removing epithelial sheets from the tracheas and the detailed culture conditions for the cte cells have been described previously [21] . briefly, tracheas were digested with dispase i solution (2.5 u/ml dispase i, roche) for 2 h at 37uc. the detached cell sheets of tracheal epithelium from the tracheal lumen were harvested and further digested with collagenase i (1 mg/ml, roche) for 5 min at 37uc. the disrupted tracheal epithelial sheets were gently pippeted and homogenized into small cell clumps. the cell pellets were resuspended in a cte medium [21] and were seeded on 2% matrigel-coated 24-well plates (corning). the cells were cultured at 37uc with 5% co 2 for 3 days. the animal use protocol in this study had been reviewed and approved by institutional animal care and use committee in national chung hsing university. the approval number is 99-09. the tested lectins for the saa2-3gal capped glycan were maa (ey laboratories, san mateo, ca, usa), mal-i (vector laboratories, burlingame, ca, usa) and mal-ii (vector laboratories). the lectins for the saa2-6gal were sna (vector laboratories) and sna-i (ey laboratories). the maa lectins were conjugated with biotin or a green fluorescent dye, fluorescein isothiocyanate (fitc). the signal of the biotin-labeled lectins was enhanced and visualized by staining with a red-fluorescent cy3conjugated streptavidin (rockland, gilbertsville, pa, usa). the cte cells were fixed in 4% cold paraformaldehyde and blocked using the carbo-free tm blocking solution (vector laboratories). immunocytochemistry (icc) was performed using the biotin-labeled lectins (maa, mal i, mal ii, sna and sna i, 1:500) or the fitc-conjugated maa (1:50, ey laboratories). the used primary antibodies were b-tubulin (1:500, sigma-aldrich, st. louis, mo, usa), mucin 5ac (1:100, sigma-aldrich) and cytokeratin 14 (k14, 1:100, convance, princeton, nj, usa). the specificity of the anti-aiv 2838 polyclonal antibodies (1:500, from infected chickens) has been evaluated in our previous report [37] . appropriate fluorescence-tagged secondary antibodies (2u ab, jackson immunoresearch, west grove, pa, usa) and cy3conjugated streptavidin (rockland) were used for visualization. blue 49,6-diamidino-2-phenylindole (dapi) was used for nuclear staining. the number of icc-staining positive cells in a 24-well plate was manually counted from five individual fields. images of the immunostaining were captured using a confocal microscope (lsm510 meta, zeiss). for comparing the expression intensity of sa among cte cells, the images were collected with the same setting and same objective (406) under the confocal microscope. figure s1 the saa2-3gal expression of ciliated cells (btubulin + ) was characterized by the double-staining of biotin-labeled mal-1 (vector laboratories, 1:500). scale bar, 50 mm. (eps) figure s2 expression levels of saa2-3gal (labeled by maa)(a) and saa2-6gal (labeled by sna)(b) in tracheal basal cell (k14 + ) were shown by immunocytostaining. the arrows in a and b indicate cells with high-intensity maa and sna staining, respectively. the cell nuclei were stained with dapi (blue). scale bar, 50 mm. 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low-pathogenicity h6n1 avian influenza virus field isolate detection of h6 influenza antibody by blocking enzyme-linked immunosorbent assay we thank dr. yi-ling lin at the institute of biomedical sciences in academia sinica for critical reading of this manuscript and valuable comments. key: cord-000269-v4jochbe authors: wittekindt, nicola e.; padhi, abinash; schuster, stephan c.; qi, ji; zhao, fangqing; tomsho, lynn p.; kasson, lindsay r.; packard, michael; cross, paul; poss, mary title: nodeomics: pathogen detection in vertebrate lymph nodes using meta-transcriptomics date: 2010-10-18 journal: plos one doi: 10.1371/journal.pone.0013432 sha: doc_id: 269 cord_uid: v4jochbe the ongoing emergence of human infections originating from wildlife highlights the need for better knowledge of the microbial community in wildlife species where traditional diagnostic approaches are limited. here we evaluate the microbial biota in healthy mule deer (odocoileus hemionus) by analyses of lymph node meta-transcriptomes. cdna libraries from five individuals and two pools of samples were prepared from retropharyngeal lymph node rna enriched for polyadenylated rna and sequenced using roche-454 life sciences technology. protein-coding and 16s ribosomal rna (rrna) sequences were taxonomically profiled using protein and rrna specific databases. representatives of all bacterial phyla were detected in the seven libraries based on protein-coding transcripts indicating that viable microbiota were present in lymph nodes. residents of skin and rumen, and those ubiquitous in mule deer habitat dominated classifiable bacterial species. based on detection of both rrna and protein-coding transcripts, we identified two new proteobacterial species; a helicobacter closely related to helicobacter cetorum in the helicobacter pylori/helicobacter acinonychis complex and an acinetobacter related to acinetobacter schindleri. among viruses, a novel gamma retrovirus and other members of the poxviridae and retroviridae were identified. we additionally evaluated bacterial diversity by amplicon sequencing the hypervariable v6 region of 16s rrna and demonstrate that overall taxonomic diversity is higher with the meta-transcriptomic approach. these data provide the most complete picture to date of the microbial diversity within a wildlife host. our research advances the use of meta-transcriptomics to study microbiota in wildlife tissues, which will facilitate detection of novel organisms with pathogenic potential to human and animals. information about the commensal and pathogenic microbial communities associated with host species, including humans, is limited. the endemic microbial community of a healthy host is important to characterize because its perturbation can be a cause of disease [1, 2] . pathogenic microbes often escape detection if the clinical consequences of infection are similar to known pathogens or if they infect non-domestic species [3] . the maintenance of unknown pathogens in wildlife species is particularly problematic because many emerging human and livestock infections arise from contact with wild animals [4] [5] [6] [7] . with the advent of meta-genomics methods, the entire community of microorganisms that exist in a given environment can potentially be identified. pyrosequencing and other high throughput sequencing approaches have been applied to determine the microbial population in environmental samples such as soil and seawater [8] [9] [10] [11] and more recently to investigate the community of microbes on human mucosal surfaces [12] [13] [14] [15] , both of which are rich in microorganisms. next generation sequencing methods have also been successfully applied to identify the microbial agents of several new diseases [16] [17] [18] [19] [20] . recently, rna based meta-transcriptomic studies [21] [22] [23] , which profile both protein-coding transcripts and ribosomal rna (rrna), have been used to study both functional and structural features of environmental microbial communities. the key question behind this study was whether viable microorganisms could be detected within healthy mammalian lymphoid organs by employing massively parallel sequencing coupled with computational techniques able to detect transcripts of microorganisms among the abundant transcripts of the mule deer host. lymph nodes are the specific replication sites for certain pathogenic viruses and bacteria [24] [25] [26] [27] [28] [29] . moreover, although the blood and the lymph systems are considered to be essentially free of viable microorganisms in healthy individuals, the transient and often asymptomatic presence of both bacteria and viruses have been detected in the circulation [30, 31] . phagocytic cells engulf these microbes and migrate to lymph nodes. thus, lymph nodes should concentrate the commensal, endemic, and potential pathogenic microbial communities of a host species. we evaluated the microbial community in retropharyngeal lymph nodes of mule deer to assess microbial exposure via the oral or respiratory route. because ungulates browse and receive small punctures from sharp forage, we reasoned that healthy animals would potentially be exposed to microorganisms from their environment or to resident oral and rumen microorganisms that would be cleared in draining nodes. we used mule deer to highlight the utility of this approach in a wildlife host, but the method is broadly applicable to any host species. our studies document for the first time that there is a community of viable microorganisms in retropharyngeal lymph nodes of healthy wild ungulates. furthermore, our findings demonstrate the applicability of meta-transcriptomic techniques for the detection of novel bacteria and viruses in internal organs. the microbial community of mule deer lymph nodes detection of protein-coding and ribosomal rna transcripts provides strong support for the presence of viable and replicating microorganisms. therefore, we enriched the total rna obtained from lymph nodes for poly(a) + rna to prepare cdna libraries and subjected them to pyrosequencing on a roche gs flx sequencer (roche-454 life sciences). properties of sequencing runs are given in table s1 . all reads were compared against the nonredundant ncbi protein database. the composite metatranscriptomic species profile for five individual and two pools of 4 or 8 mule deer samples, determined using the software megan [32] , is depicted in fig. 1a . on average, 51% of total transcript-tags could be assigned to known taxa with a bit score cutoff of 50 (see table s2 ). of the assigned tags, 99.3% were of eukaryotic origin, predominantly matching to bos taurus and other close relatives of mule deer that are represented in the protein database. approximately 0.3% of the assigned tags were to bacteria. proteobacteria represented 60% of all bacterial hits; enterobacteriaceae in the gammaproteobacteria were the most commonly identified within this group. firmicutes and actinobacteria represented 22% and 5% of the identified bacterial taxa. table s3 lists all bacterial genera detected in the seven data sets. transcripts assigned to archaea, family halobacteriaceae, were identified in both pooled samples but none of the individual libraries. only 37 transcripts were assigned to viruses. twenty-nine of these matched to the retroviridae and poxviridae while the remaining were to phages, insect viruses, and a single assignment to herpesvirus. these results suggest that representatives of many bacterial phyla, archaea, and two major virus families are transcriptionally active in mule deer retropharyngeal lymph nodes. meta-genomics studies evaluating microbial rich communities were pioneered based on genomic dna sequences [8] [9] [10] 13] . thus, we compared genomic libraries prepared from retropharyngeal lymph node tissue of md 72360, md 80228, md 84709, and md 84730 with our data from transcript libraries derived from those animals (fig. 1 ). many sequences from the genomic dna libraries were to non-coding regions and could not be used for taxonomic profiling (fig 1b, table s2 ). based on proteincoding sequences, only four bacterial genera were identified in the comprehensive megan analysis of the four genomic data sets. xylella and burkholderia were identified in md 72360, acidovorax was found in md 84709, and bartonella was found in both md 84709 and md 84730. bartonella and xylella, as well as a member of the beta retroviruses (found in md 80228 and md 84709), were identified only in the genomic dna data, suggesting that they might not represent actively replicating organisms. these findings indicate that meta-transcriptomics may be the preferred method for detecting the viable endemic microbial community in the tissues of healthy animals. the most commonly detected microorganisms in the transcriptome libraries comprised intestinal and skin-dwelling bacteria and soil and freshwater bacteria. ruminococcus, which is part of the commensal intestinal microbial community of ungulates, was detected in all seven libraries ( fig. 1a and table s3 ). other bacteria found in at least three of the seven data sets were propionibacterium, a commensal bacterium of skin and the gastrointestinal tract, and the environmental soil or water inhabitants magnetospirillum, streptomyces and pseudomonas. members of the latter genus are able to colonize a wide range of niches and are also potential pathogens. other animal and human pathogenic genera detected in at least three different libraries were burkholderia, streptococcus, flavobacteria, and members of the enterobacteriaceae (escherichia, providencia). the overall bacterial diversity and the number of unique transcripts assigned to each bacterial taxon varied among the samples. notably, helicobacter was only detected in the library constructed from md 257 but there were 12 unique transcript-tags assigned to this genus. more commonly, bacterial taxon identification was based on a single tag. many of the single transcript-tags came from md 80228, which had the highest bacterial diversity profile of all libraries analyzed, and from md 84730. bacterial genera detected solely in either one or both of these two samples include acinetobacter, legionella, enterobacter, salmonella, yersinia, vibrio, listeria, mannheimia, and members of the corynebacterineae, all of which contain known pathogens. in addition, both specimens depicted by far the highest numbers of reads taxonomically assigned to the family enterobacteriaceae. the lowest diversity of bacterial genera was found in the md oct-pool, which was derived from eight different mule deer. pooling rna from several animals potentially increases the representation of transcripts common to all animals but might decrease the ability to detect transcripts that are unique to one animal. consistent with this, the md bonner-pool, which was derived from four animals, provided a broader spectrum of bacterial genera than the md oct-pool. thus, pooling samples did not improve our ability to detect microbial diversity in lymph node samples. in contrast, viruses were detected in both pooled samples, although the total number of transcript-tags was low. of the individual libraries, only md 257 had evidence of viral transcripts (fig. 1a) . the majority of viral transcripts were from a cervid poxvirus [33] , and a novel gamma retrovirus. the computational analysis described above identified putative microorganisms in mule deer tissue based on detection of proteinencoding transcriptional activity. although the cdna used in our analyses was derived from total rna enriched for polyadenylated rna, it retained a considerable amount of the abundant ribosomal rna (rrna). these sequences contribute to the 'no hits' category in figure 1 and table s2 . bacterial rrna derived from the same dataset can, therefore, be used to provide additional support for species identification. by classifying the rrna-tags from each library using the rdp rrna classifier tool [34, 35] (http://rdp.cme.msu.edu/) we increased the number of bacterial genera identified (see fig. 2 for md 257, fig. s1 for md 80228 and md oct-pool, and table s4 ). abiotrophia, which is a component of the normal oral and intestinal microbial commu-nity, was detected in six of the seven libraries; environmental bacteria such as thermoanaerobacter, which is frequently found in hot springs, were detected in four of the seven samples. other genera that were identified based on rrna in at least two of the libraries were actinomyces, campylobacter and mycoplasma. of particular importance, rrna-tags supported the presence of helicobacter in the md 257 library (fig. 2) , of acinetobacter, escherichia, pseudomonas, salmonella, shigella and variovorax in the md 80228 library, and of shigella in the md bonner-pool library ( fig. 1 and fig. s1 , tables s3 and s4). the support for helicobacter in the md 257 library was particularly compelling because there were 12 unique transcripttags and one rrna-tag to this genus. we evaluated the phylogenetic relatedness of the mule deer helicobacter with other helicobacter based on four of the protein-coding transcripts and on the single 16s rrna sequence. all analyses demonstrated that the helicobacter detected in the mule deer lymph node is a unique organism that affiliates with the h. pylori cluster ( fig. 3a and 3b, and fig. s2 ). because 16s rrna sequence data is available for more species, we were able to further demonstrate that the closest figure 1 . megan comparison of the taxonomic profiles of (a) cdna transcript-tags from 454 sequencing five individual lymph node samples and two lymph node sample pools and (b) genomic dna-tags from four individual lymph node samples. depicted are assignments with bit score cutoffs $50. circle sizes are scaled logarithmically. not assigned: sequencing-tags matching to sequences in the ncbi database that are not assigned to taxa; no hits: sequencing-tags not matching to any sequences in the ncbi database. doi:10.1371/journal.pone.0013432.g001 figure 2 . megan comparison of taxonomic profiles of md 257 cdna transcript-tags analyzed against the protein database (red) and the ribosomal database (blue), and of v6 amplicon 16s rrna-tags analyzed against the ribosomal database (green). bit score cutoff for the protein database comparison was set at 50, and confidence cutoffs for the ribosomal database comparisons were set at 80%. doi:10.1371/journal.pone.0013432.g002 relative to mule deer helicobacter is a newly described h. cetorum isolated from different dolphin species (lagenorhynchus acutus, lagenorhynchus obliquidens, and tursiops truncatus) and a beluga whale (delphinapterus leucas) [36] (fig. 3a) . we also evaluated the phylogenetic placement of acinetobacter detected in md 80228 based on both 16s rrna and rpo-b sequences. the number of rpo-b sequences for acinetobacter in the database is limited. however, we demonstrated that the md 80228 transcript-tag clustered with those of acinetobacter (fig. 3d ) [37] . moreover, based on 16s rrna, we determined that the acinetobacter species identified in the md 80228 cdna library was distinct from all known acinetobacter and was most closely affiliated with acinetobacter schindleri (fig. 3c) . the low representation of viral sequences was not unexpected because viruses causing acute infections should be difficult to detect in healthy animals. retroviruses integrate into the host genome as part of their replication cycle, thus transcription of viral genes can be persistent in infected animals. overall four transcript-tags were assigned to gamma retroviruses of the family retroviridae. based on the transcript-tag from the md bonner-pool and an upstream region that is conserved in gamma retroviruses, pcr fragments were amplified and sequenced from md 191 cdna, which was used in the md bonner-pool, and from genomic dna of md 80228. these sequences were compared to other gamma retrovirus sequences using maximum likelihood methods (fig. 4) . the mule deer gamma retrovirus forms a distinct clade within the gamma retroviruses, which has many well-described members of primate, murine, and feline origin. a newly described gamma retrovirus from killer whale (orcinus orca) [38] is the closest relative of this mule deer retrovirus. the killer whale virus was described as an endogenous retrovirus based on its finding in various tissues and individuals. however, our detection of transcripts to this virus in only three of the libraries and the sequence variation in the pcr fragment between genomic (md 80228) and transcript-derived (md 191) mule deer samples suggest that both endogenous and exogenous gamma retroviruses might be present. as an alternative approach to identifying bacterial microorganisms present in lymph node tissue, we utilized amplicon dna library sequencing technology. the hypervariable region v6 of the 16s rrna gene was used because it has been reported to differentiate between many bacterial species [39] . amplicon libraries of v6 were generated from the 454 cdna libraries of md 257, md 80228, and md oct-pool and subjected to multiplex pyrosequencing on a roche gs flx sequencer (for properties of amplicon sequencing runs, see table s1 ). the v6 amplicon rrna tags were evaluated using the rdp classifier tool (table s5) . the assigned bacterial genera cluster in the gamma-and betaproteobacteria, the actinobacteria and in the order bacilli. a comparison of the three methods used to detect bacteria in mule deer lymph node samples is shown for md 257 in figure 2 and for md 80228 and md oct-pool in figure s1 . acinetobacter, burkholderia, corynebacterium, escherichia, providencia, salmonella, pseudomonas, ralstonia, staphylococcus, streptococcus and variovorax were identified by both amplicon and cdna sequencing in md 257, md 80228, and/or md oct-pool (tables s3 and s5) . although the overall taxonomic diversity in the v6 rrna amplicon libraries was lower than that detected in the cdna transcript libraries, the diversity within bacterial classes was higher. newly identified genera comprised predominantly environmental soil, sediment and water inhabitants (e.g. aeromicrobium and bdellovibrio), and the potential pathogens stenotrophomonas, rhodococcus, rothia, and gardnerella [40] [41] [42] [43] . these findings indicate that the v6 rrna amplicon sequencing technology is a valuable tool in complementing information about the bacterial community in host tissues. microbiome profiles of environmental samples and animals have mostly been based on the analysis of genomic dna [8] [9] [10] 13, 44, 45] . further, studies on the microbiomes of humans or animals have been restricted to habitats known to harbor large collections of microorganisms, in particular skin, oral cavity or gut [12] [13] [14] [15] [46] [47] [48] . in this study, we sought evidence for viable microorganisms in lymph nodes, an organ hitherto believed to be largely amicrobic in the absence of overt disease [26] [27] [28] [29] . our data demonstrate that transcriptional activity of a variety of bacteria and a limited number of archaea and viruses, including novel organisms, can be confirmed in healthy animals using a meta-transcriptomic approach. in our study, we faced the computational challenge of detecting a rare microbial community in a dominant pool of host genetic material. we utilized transcript-based libraries because there is an amplification of protein-coding sequences during transcription, which increased our detection ability and provided support that the identified microorganisms were viable. further, the database for protein-coding regions is more extensive than that for noncoding regions for non-reference organisms. thus, focusing on transcripts should facilitate classification of novel organisms and those without complete genome coverage. indeed, our study demonstrates that at the moderate sequencing depth employed, there were more assignable sequencing tags to protein-coding regions utilizing cdna compared to genomic dna, which consequently increased our ability to detect microbial taxa. in addition, transcriptome sequencing yields bacterial ribosomal rna, which is highly expressed in metabolically active microorganisms and is well documented as a taxonomic tool for bacteria. because single protein-coding or rrna transcript-tags from a putative microorganism were frequently encountered, our confidence in taxonomic assignment increased by employing bioinformatics methods to classify organisms based on both types of transcripts. amplicon 16s rrna sequencing increased the sensitivity to detect members of some bacterial classes. however, primer specific methods do not provide as comprehensive a perspective on the microbiota due to a possible amplification bias towards more abundant taxa or those exhibiting higher primer specificity. therefore, neither our metatranscriptomic nor amplicon sequencing approaches should be considered quantitative. we note that in samples that are highly enriched for actively replicating microbial organisms, such as environmental samples or gastrointestinal tract specimens, cdna-based approaches can yield an abundance of small rna produced by complex microbial communities, which can facilitate studies on microbial ecology but be less useful for identification of individual microbes [49] . in addition established metagenomics or metatranscriptomic [11, 50, 51] approaches that utilize sample fractionation methods for microbial enrichment will likely provide a more comprehensive profile of the community structure. these methods were not applicable to our samples, which included phagocytized microorganisms and viable microbes that were not robustly proliferating. nevertheless, as deeper sequencing of cdna libraries using newer high-throughput sequencing methods becomes more accessible, it could complement the roche-454 pyrosequencing data, potentially covering the entire viable microbial community. our study confirms that there are viable microorganisms in intact lymph nodes of apparently healthy mule deer. in the analyzed samples, we identified members of all bacterial phyla, as well as archaea, a dna virus and a retrovirus. the bacteria were representative of organisms that are commensal to mule deer and to their external environment. for example, we detected the common rumen and intestine dwellers, ruminococcus and abiotrophia, based on transcript-and rrna-tags, respectively, in most libraries, indicating that commensal gut and mucosal microorganisms may routinely be sampled in secondary lymphoid tissue, presumably from transient bacteremia. streptomyces was the most common soil dwelling bacteria identified. of interest, legionella, which is found near hot springs, was identified only in an individual mule deer from the yellowstone region. the finding of a considerable number of archaeal transcripts in md oct-pool and md bonner-pool libraries implies that members of this domain of life are likely present in mule deer habitats or resident in mule deer gastrointestinal tracts, as has been recently documented in humans [52] . correspondingly, environmental bacteria identified in healthy deer lymph nodes may reflect the animal's habitat. few viruses were identified with our analysis methods. this could represent the paucity of viruses in healthy animals. however, viral detection may be more difficult than bacterial identification using this technology in part due to extensive sequence diversity among viruses in the same family. for example, we were only able to detect the gamma retrovirus because a transcript was present which was homologous to a conserved region of the viral env gene, and the cervid poxvirus was detected because sequence data for this virus was present in the database. other persistent viruses, such as herpes viruses (for which we detected a single transcript), would be expected to be present in some animals. however, detection of latent herpes virus infection may be difficult because protein-coding transcript levels are low and latent viruses express non-coding rna [53] . in addition, viral detection can be compromised if viral sequence tags were misassigned to the host organism because of homology of viral and host genes. thus, many virus tags might be found among the host transcripts or in the not-assigned or no-hits groups of the megan analysis, which together comprise nearly half of the total sequenced transcript-tags of our data. in addition to our finding of a novel gamma retrovirus, we also identified new species of helicobacter and acinetobacter. phylogenetic evaluation of helicobacter transcripts and 16s rrna from the md 257 cdna library placed this new organism in the helicobacter pylori/helicobacter acinonychis/helicobacter cetorum complex. all members of this complex have been associated with gastritis and peptic ulcer disease in humans and animals [36, [54] [55] [56] . our detection of this bacterium in only one animal suggests that this helicobacter is not a mule deer commensal. of interest in this respect is the high incidence of h. pylori infections and gastric ulcers in american indian populations from the same geographical area in central montana [57] . acinetobacter and pseudomonas were identified in md 80228 libraries based on all detection methods used (cdna transcripts for protein-coding and rrna, and amplicon rrna). phylogenetic evaluation of acinetobacter transcripts and 16s rrna from the md 80228 cdna library placed the respective reads in close relationship to acinetobacter schindleri. acinetobacter species are important environmental organisms, however they also are notable pathogens. in particular, acinetobacter schindleri infections appear to be increasing in prevalence in hospitalized patients [37, 58] . therefore, both of the newly identified bacteria are potential mule deer pathogens. in conclusion, our study demonstrates that endemic microbiota can be detected in lymph nodes of healthy animals using metatranscriptomic approaches. these results suggest that metatranscriptomic analyses of secondary lymphoid organs could be valuable in monitoring endemic infections in wildlife or livestock as well as in detecting novel infectious organisms with the potential for causing emerging zoonotic or epizootic infectious diseases. further, these studies have the potential to cast new light on the diversity of life within and among individuals. retropharyngeal lymph nodes were obtained from a total of seventeen individual montana mule deer that were presented by hunters to check stations approximately 5 hr (range 2-11 hr) of being shot. because our samples were obtained from legally killed animals, the study is exempt from montana state university guidelines governing animal experimentation. lymph nodes were dissected from animals with sterile scalpel and forceps, and rinsed in 70% ethanol. after dissection from the animal, the lymph nodes were either frozen directly or stored in rnalater (applied biosystems, ambion, ca) until further processing. lymph node tissue was taken from mule deer in several geographical regions. the bonner pool consisted of tissue from four mule deer (167, 191, 196, 200) preparation of genomic dna, total rna, poly(a) + rna and cdna lymph node tissue cores were dissected into small pieces and further disrupted, lysed and homogenized using a tissuelyser with steel beads (qiagen, germany). genomic dna was isolated from lymph nodes of four individual mule deer (md 72360, md 80228, md 84709, and md 84730) using either the genomic dna buffer set with 20/g genomic-tips (qiagen, germany) or the allprep dna/rna mini kit (qiagen, germany). total rna was isolated using the rnaqueous-midi kit (applied biosystems, ambion, ca). for the bonner-and oct-pools, equal quantities of total rna from lymph nodes of four or eight individual mule deer, respectively, were combined. poly(a) + rna was enriched from total rna using the micropoly(a) purist kit (applied biosystems, ambion, ca). poly(a) + rna (0.9-5.0 mg each) was used for cdna synthesis (just cdna double-stranded cdna synthesis kit, stratagene, ca) after elimination of residual contaminating genomic dna using the turbo dna-free kit (applied biosystems, ambion, ca). in one case we explored an alternative empirical approach to enrich for rare microbial transcripts, using total rna of the md oct-pool. reverse transcription and amplification of cdna was done as described by cheung and coworkers [59] and included a normalization step, which effectively decreased overexpressed reads. the data resulting from this approach are included in the md oct-pool data. up to 5.0 mg of cdna or genomic dna was subjected directly to preparation of 454-dna libraries and subsequently to pyrosequencing without any prior pcr or cloning steps. library preparation and pyrosequencing were performed as described previously [60] on a roche gs20 sequencer flx (roche applied sciences/454 life sciences, branford, ct), producing sets of rna-tags or dna-tags, respectively. the runs were performed on either quarter or half plates, resulting in read numbers between 10,673 and 176,878 and base numbers in the range of 1,411,420 to 41,066,808. the md oct-pool cdna library was run twice due to low read and base numbers of the first run, and the transcript-tags of these two runs and of the run following the normalization approach (see above) were combined for all subsequent data analysis. sequences are deposited to the sequence read archive (in progress). the data of individual 454 runs (and the compilation of normal and normalized md oct-pool data) was compared against the ncbi non-redundant protein database (blastx-nr) with an evalue of 1e -4 to identify transcript rna-derived tags. to filter repetitive elements, repeatmasker (http://www.repeatmasker. org) was used to scan the mule deer sequences, with the latest version of repbase 13.04 [61] . the output files were analyzed with the program megan [32] version 3.7.2. the 16s ribosomal rna content of the cdna pyrosequencing reads was analyzed by comparison to the ribosomal database of the ribosomal database project (rdp) version 10 (http://rdp.cme. msu.edu/) [34] . the selected output reads were classified by the rdp classifier tool (naã¯ve bayesian rrna classifier version 2.0) using the taxonomic outline of the bacteria and archaea, release 7.8, for the setup of the taxonomical hierarchy [35] . the output files were analyzed with megan version 3.7.2 [32] . for the md octpool, the combined data of three individual 454 runs was used. the cdna from md 191, which was used in the md bonner pool, and genomic dna from md 80228 were subjected to pcr using forward primer 5-atgtgggggagttgattctttt-ta and reverse primer 5-ctgcgcctgagtggtctacata. pcr conditions were 40 cycles of 95uc for 30 sec, 56uc for 30 sec and 72uc for 90 sec. fragments were gel isolated, cloned using the stratagene pcr cloning kit (stratagene, la jolla, ca) and sanger sequenced. partial nucleotide sequences of 16s rrna and rpo-b for helicobacter and acinetobacter and of flgk, gdp-d-mannose dehydratase and udp-3-o[3-hydroxymyristoyl] glucosamine nacyltransferase for helicobacter from cdna sequencing, and of env gene for the retrovirus from a pcr product were aligned with the respective homologous sequences available in genbank using the mega version 4 [56] software. the appropriate nucleotide substitution model for each data set was selected by the akaike information criterion implemented in the modeltest version 3.7 [62] , and maximum likelihood (ml) trees were reconstructed using phyml version 2.4.4 [63] . using the same program (phyml) nodal supports were estimated with 100 bootstrap replicates. the trees were visualized in figtree version 1.2.2 (http://tree.bio.ed.ac.uk/software/figtree/). fusion-primers were designed including the sequences of the 454-amplicon dna library specific primers a and b, respectively, (gs flx amplicon dna library preparation method manual, www.roche-applied-science.com), 4-base barcode sequences for identifying amplicon products derived from mule deer specimen md 257, md oct-pool, and md 80228 (tgca, acgt, and cgat, respectively), and the ''universal'' v6-specific pcr primer sequences v6f: 59 tcgatgcaacgcgaagaa 39 and v6r: 59 acatttcacaacacgagctgacga 39 (designed to conserved regions flanking v6 based on comparison of 110 bacterial dna sequences [39] ). the md 257 template for amplicon generation was based on the total rna fraction depleted of poly(a) + rna (see ''preparation of genomic dna, total rna, poly(a)+rna and cdna''). the supernatant was cleared of small rna molecules using the megaclear kit (applied biosystems, ambion, ca) and depleted of host ribosomal rna performing two cycles of the microbenrich (applied biosystems, ambion, ca) protocol. subsequent depletion of bacterial ribosomal rna yielded an rna sample enriched for bacterial transcripts (microbexpress, applied biosystems, ambion, ca), which was subjected to cdna synthesis (just cdna double-stranded cdna synthesis kit, stratagene, ca) after elimination of residual contaminating genomic dna using the turbo dna-free kit (applied biosystems, ambion, ca). either cdna derived from rna enriched for non-polyadenylated bacterial mrna (md 257) or cdna sequencing library samples derived from reverse transcribed poly(a) + rna (for md oct-pool and md 80228) were used as templates for the generation of 16s rrna v6 hypervariable region-specific amplicons using the faststart high fidelity pcr system (roche, switzerland). pcr conditions were 50 cycles of 94uc for 30 sec, 55uc for 45 sec and 72uc for 45 sec. the yielded amplicon products were purified using ampure, and the resulting individual amplicon dna libraries were clonally amplified by multiplex emulsion pcr followed by sequencing using the gs flx pyrosequencing platform. the sequencing output data were computationally divided into subsets according to the barcodes (and the corresponding mule deer sample) and the primers a or b. figure s1 comparative megan analysis of (a) md 80228 and (b) md oct-pool transcript-tags analyzed by comparison to the protein database (red) and the ribosomal database (blue), and of amplicon 16s rrna-tags compared to the ribosomal database (green). bit score cutoff for the protein database comparison was set at 50, and confidence cutoffs for the ribosomal database comparisons were set at 80% and 80%, respectively. environmentally-acquired bacteria influence microbial diversity and natural innate immune responses at gut surfaces analysis of adherence, biofilm formation and cytotoxicity suggests a greater virulence potential of gardnerella vaginalis relative to other bacterial-vaginosis-associated anaerobes a newly discovered human pneumovirus isolated from young children with respiratory tract disease 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repbase update, a database of eukaryotic repetitive elements modeltest: testing the model of dna substitution a simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood the authors acknowledge the support of field staff in collecting lymph node samples. excellent technical support was provided by josh marceau. we also thank several anonymous reviewers for helpful comments. key: cord-001099-jirkkkvy authors: yousuf, mohammad a.; zhou, xiaohong; mukherjee, santanu; chintakuntlawar, ashish v.; lee, jeong yoon; ramke, mirja; chodosh, james; rajaiya, jaya title: caveolin-1 associated adenovirus entry into human corneal cells date: 2013-10-11 journal: plos one doi: 10.1371/journal.pone.0077462 sha: doc_id: 1099 cord_uid: jirkkkvy the cellular entry of viruses represents a critical area of study, not only for viral tropism, but also because viral entry dictates the nature of the immune response elicited upon infection. epidemic keratoconjunctivitis (ekc), caused by viruses within human adenovirus species d (hadv-d), is a severe, ocular surface infection associated with corneal inflammation. clathrin-mediated endocytosis has previously been shown to play a critical role in entry of other hadv species into many host cell types. however, hadv-d endocytosis into corneal cells has not been extensively studied. herein, we show an essential role for cholesterol rich, lipid raft microdomains and caveolin-1, in the entry of hadv-d37 into primary human corneal fibroblasts. cholesterol depletion using methyl-β-cyclodextrin (mβcd) profoundly reduced viral infection. when replenished with soluble cholesterol, the effect of mβcd was reversed, allowing productive viral infection. hadv-d37 dna was identified in caveolin-1 rich endosomal fractions after infection. src kinase activity was also increased in caveolin-1 rich endosomal fractions after infection, and src phosphorylation and cxcl1 induction were both decreased in caveolin-1-/mice corneas compared to wild type mice. sirna knock down of caveolin-1 in corneal cells reduced chemokine induction upon viral infection, and caveolin-1-/mouse corneas showed reduced cellular entry of hadv-d37. as a control, hadv-c2, a non-corneal pathogen, appeared to utilize the caveolar pathway for entry into a549 cells, but failed to infect corneal cells entirely, indicating virus and cell specific tropism. immuno-electron microscopy confirmed the presence of caveolin-1 in hadv-d37-containing vesicles during the earliest stages of viral entry. collectively, these experiments indicate for the first time that hadv-d37 uses a lipid raft mediated caveolin-1 associated pathway for entry into corneal cells, and connects the processes of viral entry with downstream proinflammatory cell signaling. human adenoviruses (hadvs) cause a broad range of infections at mucosal sites [1] . additionally, hadv vectors are widely used for the study of cellular processes, and are chosen as gene and vaccine delivery vehicles for clinical use. a shortcoming of hadvs in therapy for human diseases is their intrinsic tendency, as with infection by naturally occurring hadvs, to induce innate immune responses and inflammation [2, 3] . one potential mechanism for hadv induced inflammation can be found in the process of viral entry, in which cellular components involved in viral internalization directly induce subsequent inflammatory gene expression by infected cells [4] [5] [6] [7] . therefore, to understand basic mechanisms of hadv induced inflammation, and to mitigate complications of adenoviral gene therapy, it is important to understand modes of hadv entry into various cell types. endocytosis is a multi-step process that involves internalization of macromolecules and particles into specific transport vesicles derived from a cell's plasma membrane. clathrin mediated endocytosis is a well studied endocytic pathway used by both enveloped and non-enveloped viruses [8] .the cell surface is abundantly endowed with other components that allow detection and response to external stimuli. lipid rafts represent one of the major constituents of the cell membrane, contain a heterogeneous population of proteins, and act as centers for downstream cell signaling. caveolae are a special type of lipid raft that by electron microscopy appear as uncoated, flask-shaped invaginations of the plasma membrane, and are responsible for uptake and efflux of cholesterol esters. caveolar, lipid raft mediated endocytosis plays an important role in cell adhesion and growth [9, 10] . caveolins are the major integral membrane protein of caveolae [11] and include three types with a similar molecular weight of 22 kda [12, 13] . caveolin-1 and -2 are found in most cell types, while caveolin-3 is present only in muscle cells. knockout of the gene for caveolin-1 in mice also reduces caveolin-2 expression, and leads to loss of morphologically defined caveolae [14] . caveolae-like structures were identified as possible pathways of viral entry for sv40 in 1989 [15] , however, sv40 entry via caveolae was recently disputed [16] . in addition to sv40, caveolae and lipid rafts have been implicated in the entry of filovirus [17] , human enterovirus [18] , echovirus [19] and human immunodeficiency virus [20, 21] , among others [22] [23] [24] [25] [26] . importantly, the specific means of host cell entry by viruses appears to be dependent on the type of cell and its state of activation, and may be redundant [27, 28] . signaling molecules are an integral part of the host immune response to viral infection, and recent studies have shown a role for cell signaling in viral entry [29] [30] [31] [32] and viral replication [33] . for adenoviruses, viral attachment to specific integrins activates pi3k which in turn activates the small gtpases, rac and cdc42 [34] . inhibition of cell signaling can drastically reduce virus infection [35, 36] . sv40 entry is regulated by at least five different kinases [37] . virus attachment to cells can induce lipid raft formation by clustering of specific surface proteins and lipids. interestingly, many viruses bind glycophosphatidylinositol (gpi)-anchored proteins and gangliosides, associated with the outer leaflet of the plasma membrane, and this may lead directly to activation of cellular kinases [30] , adding to increasing evidence that virus binding to lipid rafts specifically initiates intracellular signaling. for the entry of adenoviruses, existing evidence suggests entry begins with a requisite binding of the adenovirus capsid fiber knob to a cellular receptor such as the coxsackie adenovirus receptor (car) [38] , cd46 [39, 40] , or in particular for viruses causing epidemic keratoconjunctivitis, gd1a glycan [41] . initial binding is followed by a secondary interaction between the arginine-glycine-aspartic acid (rgd) motif in the viral capsid penton base and cellular integrins α v β 3 , α v β 5, and α v β 1 [42] [43] [44] [45] this secondary interaction is thought to induce a host cell signaling cascade resulting in clathrin mediated endocytosis [46] [47] [48] , and possibly, activation of the rab5 dependent classical endosomal pathway [49] . herein, we show that hadv-d37, an etiologic agent of epidemic keratoconjunctivitis, enters primary human corneal fibroblasts predominantly via lipid rafts and caveolae, suggesting both redundancy and cell specificity in mechanisms of adenoviral entry. to test whether viral entry is cell and/or virus specific as previously shown for hadv-c [50] , we directly compared pathways of infection in human corneal fibroblasts and a549 cells using hadv-d37 and hadv-c2. individual confocal image slices were loaded to measure co-localization pixels (amira 5.2.2) in cells (green: caveolin-1 or lamp1, a late endosomal marker associated with conventional endosomes [51] , red: cy3-labeled hadv-d37). in human corneal cells, cy3-labeled hadv-d37 co-localized to a greater degree with caveolin-1 than with lamp1 (p=.0068), while in a549 cells hadv-37 predominantly co-localized with lamp1 (p=.0025), indicating an entry pathway other than caveolae ( figures 1a and b) . hadv-c2, not known to infect the cornea, failed to infect corneal cells in culture, and in a549 cells co-localized predominantly with caveolin-1 (p=.02). (figures 1a and b) . cholera toxin b subunit (ctxb) has been used to track and identify lipid rafts. to further examine the role of lipid raft microdomains in hadv-d37 entry, we treated corneal cells with both cy3-labeled virus and 488-ctxb, and tracked their cellular localization over time ( figure 1c ). co-localization of virus and ctxb was observed in the cell membrane at 30 min, cytoplasm at 60 min, and perinuclear region at 90 min, supporting a role for caveolin-1 and lipid rafts in hadv-d37 entry into corneal cells. in order to directly determine a role for cholesterol-laden lipid rafts in hadv-d37 entry, primary corneal cells were pretreated with mβcd (5 mm) to deplete cholesterol prior to infection with cy3-labeled hadv-d37. earlier studies showed an essential role for both integrins and src kinase in hadv entry [31, 35, 45, 46, 52] . therefore, control pretreatments also included an rgd-containing 15-mer (50 μm) designed to match the amino acid sequence in the hadv-d37 penton base (davp(rgd)nyasaaea) [53, 54] , in order to block integrin aggregation, and its kge-containing 15-mer control (davp(kge)nyasaaea), otherwise identical to the rgd 15mer. the chemical pp2 (10 μm) was used to inhibit src kinase activity. at both 30 min and 1 hr post infection, viral entry appeared nearly completely blocked by pretreatment with either mβcd or rgd, and partially by pp2, but not by kge ( figure 2a ). replenishment of cholesterol (0.4mg/ml in 0.2mm mβcd) for 1 hr appeared to restore both normal cell morphology and viral entry, as assessed by confocal microscopy ( figure 2b ). western blot analysis of detergent free lipid raft preparations at 1 hr post infection revealed increased caveolin-1 in lipid rafts from virus infected cells as judged collectively in fractions 4, 5 and 6 ( figure 2c ). caveolin-1 was seen at much lower levels and only in the 5 th fraction of mock infected cells. in cells that were mβcd treated prior to virus infection, caveolin-1 was reduced. we also observed increased phospho-src (psrc) in virus infected cell fractions 5 and 6. reductions in both caveolin-1 and psrc were observed in rgd pretreated cells, whereas pp2 pretreatment reduced only psrc and not caveolin-1 ( figure 2c ). these results suggest a relationship between caveolin-1 and src in hadv-d37 entry. our results indicate that cholesterol depletion in corneal cells presents an impediment to entry of hadv-d37 past the cell membrane. to investigate the type of endosome used in figure 1 . hadv-37 uses caveolin-1 to enter human corneal cells. a. human corneal and a549 cells grown on slide chambers were infected with cy3-labeled hadv-d37 (red). cells were then stained with caveolin-1 or lamp1 antibodies as indicated followed by alexa-fluor488 (green) secondary antibodies. original magnification: 63x. insets represent similarly magnified squares from each photomicrograph. b. three random frames from different experiments were chosen in masked fashion from each experiment (n=3) to quantify co-localization using amira 5.2.2. in a549 cells, hadv-c2 co-localized predominantly with caveolin-1, while hadv-d37 co-localized with lamp1 (*p=.02 and *p=.0025, respectively). in corneal cells (cc), cy3-labeled hadv-d37 co-localized predominantly with caveolin-1 (*p=.0068), and hadv-c2 was not seen. students t test was used for all comparisons. c. human corneal cells were grown in chamber slides, cy3-labelled hadv-d37 and 488-cholera toxin b (ctxb) were added to the cells on ice and then warmed to 37°c, and incubated for 30, 60, and 90 min, prior confocal microscopy. hadv-d37 (red) co-localized with ctxb (green) at all time points. at 30 min after warming, hadv-d37 and ctxb co-localization was observed at the cell membrane, at 60 min in the cytoplasm, and at 90 min at the perinuclear region. , and then infected with cy3-labeled hadv-d37 (red). mβcd pretreatment prevented virus from entering the cells both at 30 min and 1 hr post infection as compared to no pretreatment. rgd and pp2 reduced entry as compared to kge treated or untreated cells. co-staining with alexa-fluor488 phalloidin (green). original magnification: 63x. insets represent similarly magnified squares from each photomicrograph. b. cholesterol was added to mβcd pretreated cells for one hr and then infected with cy3-labeled hadv-d37. confocal microscopy revealed entry of viruses into the corneal cells. c. detergent free lipid raft preparations in hadv-d37 infected corneal cells, pretreated with mβcd, rgd 15-mer, kge 15-mer, or pp2, and lysed at 1 hr post infection, followed by immunoblotting for caveolin-1 or phosphorylated src (psrc). blots show increased caveolin-1 and psrc upon viral infection, but reduced expression with cholesterol depletion or blocking of integrin aggregation (mβcd and rgd treatment, respectively). pp2 treatment did not effect caveolin-1 levels in infected cells, but did reduce psrc. hadv-d37 entry, flotation gradient fractionation of post nuclear supernatants was performed to separate different endosomal compartments [55] . upon viral infection, fractions 4-6 maximally expressed lamp1 indicating the presence of late endosomes [56] , while fractions 9-11 were enriched with caveolin-1, indicating the presence of caveolae ( figure 3a ). when the blots were stripped and reprobed, the caveolin-1 containing fractions were found to also contain psrc. rab5, a marker for early endosomes [57] was observed in fractions 14-16 upon viral infection, but was not evident in the fractions expressing psrc. we confirmed src activation in fractions 10 and 11 using a src kinase assay in which substrate fluorescence and kinase activity are inversely proportional ( figure 3b ). these results suggest that a pathway mediated by caveolin-1, and involving src kinase, is an important route of hadv entry into human corneal cells. mβcd pretreatment altered the fractionation of caveolin-1, and resulted in reduced identification of endosomal markers ( figure 3a ). however, we cannot exclude alternative possible effects of mβcd on clathrin mediated mechanisms, as previously shown [58, 59] . real-time pcr was then performed to amplify hadv-d37 genomic e1a nucleotide sequence [54] in order to determine the presence of viral dna in different endosomal fractions. upon infection of corneal cells, significantly more viral dna was present in the fraction 10, containing caveolin-1and psrc, than in fraction 6, which was abundant in lamp1 ( figure 3c ) fractions were immunoblotted for caveolin-1 (cav 1), psrc, rab5, and lamp1. b. src kinase assay was performed on endosomal fraction #'s 5-11. in this assay, fluorescence is inversely proportional to kinase activity. as shown, fractions 10 and 11, which previously showed increased caveolin-1, also demonstrated greater src kinase activity. c. fractions that had maximum expression of lamp1 (#6) and caveolin-1 (#10) were then subjected to real time pcr. for both fractions, the quantity of viral dna was reduced by mβcd pretreatment (*p<. 05). cholesterol replenishment resulted in restoration of e1a expression (*p<.05). there was no difference in e1a expression between virus infected cells and those pretreated with mβcd but replenished with cholesterol and then virus infected. the data presented is representative of four experiments each run in duplicate. doi: 10.1371/journal.pone.0077462.g003 (fraction 6 vs. fraction 10 in virus infected cells: p=.0027). mβcd pretreatment reduced viral dna content in both endosomal fractions (p<.05, both comparisons). viruses can often circumvent artificial barriers to cell entry by use of alternate entry pathways [50, 60, 61] . however, in our study, mβcd pretreatment reduced the presence of viral dna in both the caveolin-1 containing fraction and in the lamp1 containing fraction. we also examined for the presence of viral dna in lipid raft fractions from mβcd treated cells when replenished with cholesterol, as compared to those treated with mβcd alone prior to viral infection. in lipid raft fractions from mβcd treated, cholesterol replenished cells, e1a content was equivalent to that from cells not treated with mβcd prior to infection ( figure 3c ). caveolin-1 is a critical protein to caveolae [62] , and upon reduction in caveolin-1 expression, cells lose the ability to form caveolae. we prepared a sirna oligo against caveolin-1 that by real-time pcr reduced its expression in corneal cells tõ 5% of normal (p=.0002, figure 4a ). western blot analysis after sirna transfection also showed reduction in caveolin-1 protein as compared to untransfected or control sirna ( figure 4b ). specificity of the caveolin-1 sirna was validated by showing expression of unrelated protein kinase c was not reduced compared to control (data not shown). however, hadv-d37 infection of corneal cells after caveolin-1 knock down by sirna reduced il-8 expression by ~ 50% (p=.0001, figure 4c ). using the mouse adenovirus keratitis model [5, 63, 64] , we then analyzed the role of caveolin-1 in viral infection. cy3labeled hadv-d37 was injected into the corneal stroma of caveolin-1 -/-mice on a c57bl/6j background or wild type c57bl/6j controls. by confocal microscopy performed at 30 min, 1 hr, and 24 hr post infection, viral entry, but not viral attachment, appeared markedly reduced in caveolin-1 -/-as compared to wild type mice at these time points ( figure 5a ). western blot analysis of wild type and caveolin-1-/-mice corneas after infection revealed increased psrc in wild type corneas at 1 and 24 hr post infection, compared to caveolin-1 -/-mice, suggesting that caveolin-1 is necessary for src activation after infection (fig, 5b) . similarly, upon viral infection, cxcl1 protein expression was reduced in caveolin-1 -/-mice as compared to wild type mice (p=.0001, figure 5c ). these results suggest that caveolin-1 is important to viral entry, kinase activation, and subsequent chemokine expression. src kinase has been previously shown critical to both hadv-d37 entry [35] and inflammatory consequences of infection of human corneal cells [35, 52, [65] [66] [67] . data presented above further suggest that src may play an important role in lipid raft mediated hadv entry into corneal cells. therefore, we applied the src kinase inhibitor pp2 (10 μm) to the mouse adenovirus keratitis model. when injected 30 min prior to hadv-d37 infection, pp2 reduced corneal inflammation as assessed by histology 4 days post infection ( figure 5d ) and also reduced cxcl1 expression by elisa at 16 hr post infection (p<.05, figure 5e ), while the pp2 solvent dmso alone did not reduce inflammation or cxcl1 expression. taken together, these data suggest hadv-d37 enters corneal cells at least in part via caveolae, in an integrin dependent and src kinase mediated pathway. caveolae are submicroscopic, membrane associated vesicles found abundantly in some but not all mammalian cells. a caveola has a characteristic flask or bulb-shaped appearance [68] [69] [70] , without the obvious coat of clathrin pits [ 71] . initially, sv40 was reported to use the caveolar pathway to enter cells [15, 72] , but was later observed to use a caveolae independent pathway [16, 60, 73] . each caveolae contains approximately 140-150 caveolin-1 molecules [74] . to confirm caveolin associated viral entry into corneal fibroblasts, we performed ultrastructural studies with and without viral infection ( figure 6 ). electron micrographs of uninfected fibroblasts demonstrate abundant membrane associated vesicles, visible as flask shaped invaginations of the cell membrane ( figure 6a ). within 1 hr after hadv-d37 infection, viruses were seen in some of these caveola-like structures ( figure 6b-d) . in some tissue sections, it appeared that caveolae were fusing to form caveolar vesicles (caveosomes) as reported earlier [75] . viruses also accumulated in these presumed caveosomes ( figure 6d ). in order to confirm that the membrane associated vesicles seen ultrastructurally are indeed caveolae, we applied immuno-electron microscopy with primary antibodies against caveolin-1 or clathrin, and secondary antibody bound to protein-a nanogold. membraneassociated vesicles in human corneal fibroblasts labeled well with antibody against caveolin-1 ( figure 6e ). within 30 min after viral adsorption, many caveolin-1 expressing vesicles were seen to contain virus ( figure 6f ). the size of the caveolin-1 containing vesicles in corneal cells ranged from 80-140nm in diameter (data not shown). therefore some vesicles appeared able to accommodate the ~ 90 nm diameter adenovirus. notably, clathrin staining vesicles in the same cells were much less abundant and no clathrin staining vesicles were seen to contain virus (data not shown). these results confirm that hadv-d37 uptake into human corneal fibroblasts occurs in vesicles containing caveolin-1. endocytosis is used by pathogens as a gateway to cellular entry. the specific pathway of entry seems to be host and pathogen specific [76] . the most well studied mechanism of receptor-mediated entry is clathrin-dependent endocytosis. most previous reports suggest that hadvs use clathrin mediated endocytosis for cellular entry [77, 78] , with macropinocytosis as an alternate pathway [77, [79] [80] [81] . caveolae are ubiquitous on the surface of cells, particularly in fibroblasts [82] , and appear large enough to accommodate human adenoviruses [83] [84] [85] , which typically measure ~ 90 nm in diameter. recently, hadv-c was shown to utilize lipid raft mediated, caveolar endocytosis in non fibroblast cell lines [50, 61] . because of our longstanding interest in pathogenesis of hadvs in the human cornea, we sought to test viral entry in primary human corneal fibroblasts. we used two cell types, primary fibroblasts cultured directly from human donor corneas, and as control, a549 cells, a human alveolar epithelial cell line that was previously shown to support hadv-1 virion production [86] and commonly used for the culture of hadvs. our results clearly demonstrate caveolin-1 associated entry into corneal cells by the highly cornea-tropic hadv-d37. in contrast, hadv-d37 entry into a549 cells was less robust and appeared to predominantly involve the lamp1 pathway. as an additional control, we also tested the entry of hadv-c2, a non corneal pathogen, but the degree of entry into human corneal cells was too low to permit quantitative analysis. caveolae mediated entry of another non-enveloped dna virus, sv40, is controversial [16, 60, 73] , and in the absence of caveolin-1, sv40 still successfully enters cells [60] , suggesting redundant and alternative mechanisms. however, in corneal stromal cells, hadv-d37 appears to rely largely on lipid raft mediated, caveolin-1 associated endocytosis. cholera toxin b subunit (ctxb) has been shown to bind glycosphingolipid ligand, gm1, and has been used to identify distinct, lipid raft endocytic pathways [87] . in our study, ctxb co-localized with hadv-d37 in corneal cells during entry. we show the presence of viral dna predominantly in caveolin-1 associated membrane fractions. it is important to acknowledge that clathrin-mediated endocytosis is also sensitive to acute depletion of cholesterol [58, 88] and that raft recruitment has been shown to precede clathrin-dependent endocytosis for anthrax toxin, egfr, and bcr, respectively [89] [90] [91] . in our study, cholesterol depletion also reduced viral dna in lamp1 rich fractions in corneal cells, indicating a similar redundancy in cholesterol function. when cholesterol was replenished in cells treated with mβcd, there was a gain in entry as determined by confocal microscopy and viral dna expression. we also previously showed the critical role of src in hadv infection [35, 52, 66, 67, 92] . the observation of increased src kinase activity specifically in caveolin-1 enriched fractions in infected cells strongly suggests a role for caveosomal signaling upon viral infection. at 1 hr post infection, pp2, a chemical inhibitor of src kinases, partially reduced viral entry, whereas both mβcd and rgd more completely blocked viral entry. using a mouse keratitis model [63] , caveolin-1-/-mice showed reduced entry of virus as compared to wild type mice and showed reduced levels of both psrc and cxcl1 expression complimenting our in vitro data. taken together, these findings suggest a role for src kinase in the caveosome during viral entry, with downstream effects on chemokine expression by infected cells. viruses even within the same virus family can utilize different endocytic pathways, and there may be extensive cross talk between pathways. for example, within the polyomaviruses, jc virus enters cells via clathrin mediated endocytosis [93] and is later sorted into caveosomes [94] , while bk virus uses ph dependent caveolar endocytosis [95] . other polyomaviruses enter cells using uncoated vesicles independently of both caveolin and clathrin [96] . these studies do not account for potential differences in cell types used to determine viral entry. our ultrastructural studies confirm the association between hadv-d37 with caveolin-1 in corneal fibroblasts during viral entry, but not clathrin in the same cells. our data also suggests that hadv-d37 may utilize different entry pathways in different cell types. time course studies with a panel of cell types relevant to corneal infection will be necessary to determine the range of entry mechanisms utilized by hadv-d37, and to better understand the infectious and inflammatory outcome of different entry pathways. hadv-d37 replicates readily in human corneal fibroblasts [53] , and in our hands, knockdown of caveolin-1 reduced chemokine expression, suggesting that caveolin contributes to both productive infection and corneal inflammation. however, our data may be relevant only to the earliest stages of infection, and does not address possible sorting of virus later in infection to different endosomal compartments. in summary, we have shown that hadv-d37 enters corneal stromal cells via a caveolin-1 associated, lipid raft specific pathway, inducing caveosomal signaling and expression of proinflammatory mediators. lipid raft mediated endocytosis may prove a useful target for antiviral therapy in corneal infection. the animals involved in this study were procured, maintained, and used in accordance with the laboratory animal welfare act of 1966, as amended and nih 80-23, guide for the care and use of laboratory animals, national research council, and upon approval from the animal care committee at the mass. eye and ear infirmary. the derivation of corneal cells from deceased human donors was approved by the massachusetts eye and ear infirmary human studies committee, and conformed to the tenets of the declaration of helsinki; written informed consent for the use of the corneas from the deceased donors or their families was deemed unnecessary by the human studies committee, because the corneal tissues arrived without identifying data, and were tissues that were found by the eye banks to be unsuitable for corneal transplantation and would have been discarded if not used in research. primary human corneal fibroblasts were derived from human donor corneas as previously described [97] . cells from multiple deceased donors from multiple eye banks were pooled and the cell monolayers used at passage two or three. the a549 cells and hadvs used in this study were purchased from american type culture collection (atcc, manassas, va). virus purification was performed by cesium chloride gradient. cy3labeled virus was prepared as previously described [98] . eight to 12-week-old wild type c57bl/6j and caveolin-1 -/mice were obtained from jackson laboratories (bar harbor, me). for experimental infection, female mice were anesthetized by intramuscular injection of ketamine (85 mg/kg) and xylazine (14 mg/kg). anesthetic drops (0.5% proparacaine hydrochloride, alcon, fort worth, tx) were applied topically to the right cornea, followed by injection with 1 μl pp2 (10 μm), dmso carrier, and then hadv-37 after 1 hour. one μl of virus (10 5 tissue culture infective dose (tcid)), or virus-free dialysis buffer was injected into the central corneal stroma with a glass micropipette needle fitted with a gas-powered microinjection system (mdi, south plainfield, nj) under an ophthalmic surgical microscope (carl zeiss meditec, inc., thornwood, ny). at 4 days post infection, mice were euthanized using co 2 inhalation prior to removal of corneas for histology. cells grown on slide chambers (nunc, rochester, ny) were treated with mβcd (5mm; sigma, st. louis, mo), rgd 15-mer (50mm, genscript, piscataway, nj ), kge 15-mer (50mm, genscript, piscataway, nj) or pp2 (10 μm, calbiochem, la jolla, ca) for 1 hr, and then infected with cy3-labeled hadv for 30 min and 1 hr. cells were partially fixed in 0.05% paraformaldehyde for 10 min, washed in pbs containing 2% fbs, and permeabilized in solution containing 0.1% triton x-100 for 5 min. after 30 min blocking in 3% fbs-pbs, the cells were incubated in 5 μg/ml of alexa fluor 488 phalloidin (invitrogen, eugenia, or) for 30 min at room temperature, washed three times in 1x pbs containing 2% fbs. cells were then fixed in 2% paraformaldehyde, and mounted using vectashield (vector labs, burlingame, ca) mounting medium containing dapi. images were taken in a leica sp5 confocal microscope using a 63x glycerol immersion objective. mouse corneas were harvested at various time points after hadv-d37 injection and stained with alexa fluor 488 phalloidin, (invitrogen), fixed, and mounted using vectashield. for colocalization studies after infection, cells were fixed and stained with antibodies to caveolin-1, (santa cruz biotechnology, ca) lamp1, (santa cruz biotechnology), or 488-ctxb, (invitrogen, or). individual confocal image stacks were loaded to measure co-localization pixels by amira 5.2.2 (amira, burlington, ma) from cells stained green (caveolin-1 or lamp1) and red (cy3labeled hadv-d37). three frames each from 3 separate experiments were chosen in masked fashion and, used to generate mean and standard deviation. cells were treated with mβcd, washed thoroughly, and infected with purified hadv at a multiplicity of infection (moi) of 10 for 30 min or mock treated with dialysis buffer. after infection, lr fractions were isolated using detergent free methods. briefly, cells were washed once in ice cold pbs, suspended in 500mm na 2 co 3 , and lysed by 20 strokes in a prechilled dounce homogenizer. further disruption of cell membranes were achieved by passing the lysate through a 23 gauge needle 5 times, followed by 3 cycles of sonication for 15 s. the subsequent lysate was mixed with an equal volume of 90% sucrose-mbs (25mm mes{2-(nmorpholinoethanesulfonic acid) plus 0.15m nacl [ph 6.5]), overlaid on an equivalent volume of 35% sucrose-mbs-na 2 co 3, and centrifuged for 16-18 hr at 4°c. 500μl fractions were collected and the protein concentrations measured. the lysates were immunoblotted with anti-psrc (cell signaling, danvers, ma) to identify phosphorylated src in lr along with the lr marker caveolin-1. flotation gradient fractionation of post nuclear supernatants was performed with 62%,(2.351m) 35%, (1.177m) and 25%, (0.806m) sucrose gradients [55] and 100 μl fractions collected. one hr post hadv infection after pretreatment with mβcd (5mm), untreated controls and mock infected cells were collected and post nuclear supernatants were prepared by using nucbuster (novagen, germany). post nuclear supernatants were then mixed with an equal amount of 62% sucrose, layered over with 1.5 volume of 35% sucrose and 1 volume of 25% sucrose, and centrifuged for 1 hr at 149,000 x g at 4°c. a series of 100 μl fractions were collected from the surface and subjected to western blot analysis for caveolin-1, phosphorylated src, rab5, and lamp1 (santa cruz biotechnology), and real-time pcr for the hadv-d37 e1a gene in order to determine the presence of viral dna in the endosomes. the sirna for caveolin-1 and its control scrambled sirna (table 1) were constructed using scitools from integrated dna technology (idt, coralville, ia). one nm was transfected using lipofectamine rnaimax (invitrogen, carlsbad, ca) following the manufacturer's instructions. at 48 hr post transfection, cells were infected with hadv for 2 hr, and then analyzed for caveolin-1 and il-8 message using real-time rt-pcr (table 1) . for western blot analysis, 20 μg of protein was separated in a 4-20% gradient gel (invitrogen) and immunoblotted with antibodies against caveolin-1 and actin (thermo scientific, pittsburgh, pa), the latter as an internal control. to quantify the presence of viral dna in endosomal compartments, the e1a gene was amplified from endosomal fractions after sucrose gradient with primers (idt, table 1 ) designed based on the hadv-d37 genomic e1a nucleotide sequence [54] . real-time pcr was performed with fast sybr green master mix (applied biosystems, foster city, ca) with the following conditions, 40 cycles at 95°c (10 s), 55°c (20 s), and a final extension at 72°c for 10 min. to determine mrna expression for caveolin-1 and il-8, total rna was extracted from caveolin-1 or scrambled sirna transfected, hadv-d37 infected cells, at 2 hr post-infection using trizol (invitrogen) according to the manufacturer's instructions. total rna was then treated with dnase i (1 unit) (new england biolabs, ipswich, ma) at 37°c for 1hr. two μg of the dnase treated rna was subjected to reverse transcription with m-mlv reverse transcriptase (promega, madison, wi) and oligo dt 15 primers (idt). primers for caveolin-1 and il-8 were designed using primer3 plus software (http://www.bioinformatics.nl/cgibin/primer3plus/primer3plus.cgi), and synthesized by idt ( table 1 ). the cdnas were diluted by 1:10 and 1 µl was used for qrt-pcr using fast sybr green master mix under the conditions: 40 cycles oat 95°c (30 s), 60°c (1 min), 72°c (30 s), and a final extension at 72°c for 10 min. a non-template control and endogenous control (human gapdh) were measured for the relative quantification. the related expression levels were calculated by the 2 -δδct method against the untransfected/uninfected controls [99] . src activity assays were performed by using profluor® src-family kinase assay (promega, madison, wi). one microgram of each endosomal fractions (peptide substrate) and 25 μl of 100 μm atp was added to the reaction buffer and incubated for 60 min at room temperature, followed by the addition of protease reagent for another 60 min at room temperature. the reaction was then stopped by addition of stabilizer reagent. when there is no kinase activity, the peptide substrate remains nonphosphorylated, and the protease will remove all amino acids from the peptide substrate and liberate the highly mouse corneas were removed at 16 hr post infection (n = 3/ group). corneas were then homogenized in 400 µl of pbs with 1 mm phenylmethylsulfonyl fluoride (pmsf), 1 µg/ml aprotinin, and 10 µg/ml leupeptin (sigma-aldrich, st. louis, mo). the lysates were centrifuged at 10,000 x g for 10 minutes at 4°c, and the supernatants were used for elisa. mouse cxcl1 (kc) (r&d systems, minneapolis, mn) protein detection was performed with commercially available sandwich elisa kits with capture and detection antibodies, according to the manufacturer's instructions. each sample and standard was analyzed in duplicate. the plates were read on a microplate reader (molecular devices, sunnyvale, ca) and analyzed (softmax; molecular devices). upon euthanasia at 4 d post infection, mouse corneas were removed surgically, rinsed in pbs, and fixed with 10% neutral buffered formalin for 24 hr at room temperature. after paraffin embedding, whole eyes were cut into 5-μm-thick sections, mounted on positively charged slides and air dried overnight. after deparaffinization and rehydration, slides were stained with hematoxylin and eosin for histology, coverslipped, and photographed (axiovert 135; carl zeiss meditec, inc.), using a 40× objective. human corneal fibroblasts plated on 10 cm dishes and grown to 95% confluence were infected with cesium chloride purified hadv-d37 virus at a moi of 10, and incubated at 30 min at 4° c, followed by incubation at 37° c for 30 min. the cells were then washed with pbs, and fixed in 2 % paraformaldehyde containing 2.5 % gluteraldehyde, 0.1 m cacodylate and 2.5 mm cacl 2 for 1 hr, and harvested in 2 % agarose. the cell pellet was fixed for 1.5 hr in 2 % aqueous oso 4 , and dehydrated. following dehydration, the cell pellet was embedded in epon and sectioned at 70-90 nm. the specimens were stained with saturated aqueous uranyl acetate, and sato's lead stain. photomicrographs were taken on a philips cm-10 electron microscope (koninklijke philips electronics n.v., amsterdam, netherlands) operating at 80 kv and fitted with a ccd camera. for immunostaining, cells were adsorbed with purified hadv-d37 or mock infected with buffer for 30 min at 4° c, followed by incubation at 37° c for 30 min, washed in pbs, fixed in 4% paraformaldehyde for 1 hr, and harvested. cells were treated with 2.3 m sucrose in pbs for 15 min, frozen and sectioned at -120° c at 50-80 nm thickness, and transferred to formvarcarbon coated copper grids. grids were blocked with 1% bsa for 10 min and incubated for 30 min with 5 µl of rabbit polyclonal caveolin-1 antibody, (bd biosciences, san jose, ca) at 25 µg/ml in 1% bsa, followed by wash in pbs, and incubation with protein-a nanogold (cell microscopy center, department of cell biology, university medical center, utrecht, the netherlands) [100] with a particle size of 10 nm, for 20 min. alternate sections were stained with mouse igg1 raised against rat clathrin heavy chain (bd biosciences) followed by rabbit antiserum to mouse igg (icn biomedicals, irvine, ca) at 1:100 dilution, followed by protein-a nanogold with a particle 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insights into corneal tropism subcellular fractionation of tissue c cellsg taylor immunocytochemical characterization of the endocytic and phagolysosomal compartments in peritoneal macrophages rab5 controls early endosome fusion in vitro extraction of cholesterol with methyl-beta-cyclodextrin perturbs formation of clathrin-coated endocytic vesicles productive entry of type c foot-and-mouth disease virus into susceptible cultured cells requires clathrin and is dependent on the presence of plasma membrane cholesterol clathrin-and caveolin-1-independent endocytosis: entry of simian virus 40 into cells devoid of caveolae efficient species c hadv infectivity in plasmocytic cell lines using a clathrin-independent lipid raft/caveola endocytic route multiple functions of caveolin-1 adenovirus type 37 keratitis in the c57bl/6j mouse chemokine cxcl1/kc and its receptor cxcr2 are responsible for neutrophil chemotaxis in adenoviral keratitis ceacam6 attenuates adenovirus infection by antagonizing viral trafficking in cancer cells specific nfkappab subunit activation and kinetics of cytokine induction in adenoviral keratitis human adenovirus type 19 infection of corneal cells induces p38 mapk-dependent interleukin-8 expression high-resolution 3d quantitative analysis of caveolar ultrastructure and caveola-cytoskeleton interactions the shape of caveolae is omega-like after glutaraldehyde fixation and cup-like after cryofixation caveolae as plasma membrane sensors, protectors and organizers structure of caveolae caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the er gm1 structure determines sv40-induced membrane invagination and infection kinase-regulated quantal assemblies and kiss-and-run recycling of caveolae ultrastructural identification of uncoated caveolin-independent early endocytic vehicles mechanisms of viral entry: sneaking in the front door adenovirus triggers macropinocytosis and endosomal leakage together with its 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of its receptor via a lipid raftmediated clathrin-dependent process relationships between egfr signaling-competent and endocytosiscompetent membrane microdomains lipid rafts unite signaling cascades with clathrin to regulate bcr internalization jnk regulates mcp-1 expression in adenovirus type 19-infected human corneal fibroblasts jc virus enters human glial cells by clathrin-dependent receptor-mediated endocytosis invasion of host cells by jc virus identifies a novel role for caveolae in endosomal sorting of noncaveolar ligands infection of vero cells by bk virus is dependent on caveolae early steps of polyomavirus entry into cells adenovirus keratitis: a role for interleukin-8 fluorescent virions: dynamic tracking of the pathway of adenoviral gene transfer vectors in living cells analysis of relative gene expression data using real-time quantitative pcr and the 2 cryosectioning and immunolabeling key: cord-000947-psguw47w authors: feng, jianyu; guo, hong; li, sen; lu, tun title: a study of the mechanism of the chaperone-like function of an scfv of human creatine kinase by computer simulation date: 2013-04-24 journal: plos one doi: 10.1371/journal.pone.0062147 sha: doc_id: 947 cord_uid: psguw47w a new application of antibodies is to use them as macromolecular chaperones. protein antigens usually have multiple epitopes, thus, there may be a plurality of antibodies binding to one antigen. however, not all antibodies that bind to one antigen could act as a chaperone. experiments show that some screened anti-human creatine kinase single chain antibodies (scfv) could assist in the folding and stabilizing of the enzyme, while others could not. we built the model of the single chain antibody (scfv-a4) that increased the stability of human creatine kinase (hck) by the homology modeling method. epitopes of human creatine kinase were predicted by computer and then the binding of scfv-a4 and hck was modeled with computer. the calculation results were further combined with the peptide array membrane experiment results to obtain reliable models for the scfv-a4-hck complex. based on the above study we gave an explanation about how scfv-a4 could act as a macromolecular chaperone assisting the folding of hck. this study provides an approach for predicting antigen-antibody binding mode and also a useful theoretical guidance for the study of antibodies' chaperone-like function. in recent years, a number of human diseases, such as alzheimer's, huntington's, parkinson's, and creutzfeldt-jakob's diseases, were reported to be related to the misfolding and aggregation of proteins [1, 2] . molecular chaperones are a kind of protein that are capable of assisting nascent peptides in correctly folding to functional proteins by binding to the folding intermediate to avoid kinetic traps, suppressing aggregation of the substrate [3, 4] . traditional molecular chaperones could be classified according to their molecular weights and sequences to families such as hsp90, hsp70, hsp60 and nucleoplasmin. they have low specificity and react with many kinds of proteins. the low specificity of conventional molecular chaperones enables them to help many house-keeping proteins simultaneously. but a protein-misfolding disease might be caused by only one specie of protein which carries point mutants while most other housekeeping proteins are normal [5] . thus the use of traditional molecular chaperones as therapeutic molecules for misfolding diseases may have problems such as low efficiency and undesired side-effects. a new field in development is to design or screen specific macromolecules which could be used as chaperones for target proteins, inhibiting their misfolding or coagulation to cure the related protein-misfolding diseases [6] [7] [8] . antibodies are the most common macromolecules that can bind specifically to target proteins. previous researches had shown that some antibodies could excert a chaperone-like function on their antigens [9] . therefore antibodies with a chaperone-like function were considered as the therapeutic drug candidates for protein misfolding diseases because they only affect mutant proteins, leaving normal proteins intact. in addition, antibodies with a chaperone-like function were helpful research tools for protein folding researches. human creatine kinase (hck) is a protein of important physiological function, which is closely related to intracellular energy operation, muscle contraction and atp regeneration [10] . according to existing researches in the folding of hck, the dysfunction of hck could be a highly possible pathogenic factor of many serious diseases [11, 12] . our previous studies [13] indicate that hck expressed in e. coli existed as inclusion bodies. antibodies produced by using hck expressed by e. coli as antigen could be used to study the renaturation of inclusion bodies, such as capturing the intermediates during the folding process of hck to study the structural characteristics of the intermediates. an scfv is a fragment of a conventional antibody which is constructed by connecting the variable domains of the antibody heavy chain and the light chain by a segment of linker peptide. scfvs with high affinity and specificity to their antigens had been isolated from phage display libraries by many groups [14] [15] [16] [17] . schlattner [18] have successfully isolated several scfv clones from a human antibody phage display library that recognize cytosolic bb-ck. in our previous work [13] several scfvs had been screened out from a phage library using recombinational hck as antigen. only one of the scfvs named scfv-a4 has a significant chaperonelike function, preventing the aggregational precipitation of hck during its folding and accelerating its recovery to nature conformation. in order to comprehend the unique chaperone property of scfv-a4, the binding between scfv-a4 and hck must be analyzed. the top priority would be identifying the portion of hck bound by scfv-a4. molecular docking by computer has been widely used in the study of the binding mechanism of protein-protein or proteinligand systems, including themes such as protein-folding mechanism [19, 20] , protein-protein surface modification [21, 22] , improving the binding between proteins and polypeptides or small molecules [23] [24] [25] . the number of degrees of freedom of protein-protein interactions was so large that no docking program nowadays can search the conformational space thoroughly even at the classical mechanics level of theory. therefore, no current software is able to correctly dock two proteins ab initio. one approach to reduce the search space for protein-protein interactions is to predict protein-protein interaction sites first and then use the orientational docking method to test the possibility of the binding. this is the approach used in this work. as to antigen-antibody binding, the parts of the antibodies bound to antigens were defined as cdr regions (complementarity determining regions) which could be identified by sequence analysis, while the interaction sites on the antigens were called the epitopes. many bioinformatics software tools had been developed to predict epitopes. b-cell epitopes typically belong to one of the two classes: linear (continuous) epitopes or conformational (discontinuous) epitopes. at present the prediction methods for linear epitopes had been extensively studied and the prediction accuracy had been improved to be as high as 73.37% [26] . thus linear epitopes prediction programs could be used to predict the potential binding sites of hck. one technique recently developed to screen potential linear epitopes was the peptide array membrane technique [27] . peptide fragments covering the whole sequence of a protein antigen were synthesized and spotted on the membrane, the membrane was then treated with antibodies. after washing away the unbound antibodies and labeling the bound antibodies with secondary chromogenic antibodies, the peptide segments of potential epitopes could be identified. such identified peptides were only potential epitopes because when they were fixed on the membrane their conformation might not be the same as when they were parts of a protein. but we expected more reliable results could be deduced by combining the two methods. after the potential interaction sites of both interacting proteins had been identified, molecule docking software packages were used to generate 3d models for the scfv-antigen complexes. the quality of the 3d complex models can be used to verify the validity of the previous prediction based on 1d sequence. the chaperone-like function of the antibody was explained by investigating their interactions. the steps of this research were summarized as below. first, the three-dimensional structure of scfv-a4 was constructed by the homology modeling method. then potential epitopes of hck were predicted by software tools and the peptide array membrane experiment. the 3d models of scfv-a4-hck complex were built by orientational docking. finally, we analyzed the interaction between hck and scfv-a4 and speculated the mechanism underlying the chaperone-like function of the scfv-a4. this research also provided a new approach for studying antibodyantigen interactions based on the peptide array membrane experiment and bioinformatics software tools. the cdrs and the linker of scfv-a4 were inferred from the alignment as shown in figure 1 . the model of the antibody was constructed by the modeller 9v7 program as shown in figure 2a . the distributions of w-y dihedral angles of the backbone were checked by the procheck program [28] . a ramachandran map was used to describe the dihedral angles of the low-energy conformation ( figure 2b ). it showed that 87.4% of the residues fell into the best area, 12.1% of the residues into the other license area, 0.5% of the residues into the permitted area, 0.0% of the residues into the not allowed area. therefore, dihedral angles of the structure of the model were reasonable. for 87.65% residues the 3d-1d compatibility score is greater than 0.2 by verify-3d [29] (a high score indicated the compatibility of the sequence and the tertiary structure is good). the amino acids with scores less than 0.2 were gly112-lys142 which belonged to the linker segment ( figure 2c ). since no template was used for modeling the linker the poorer model result of this area was expected. the above evaluations verified that the three-dimensional model of scfv-a4 was reasonable. the peptide array membrane was shown in figure 3 . each square contained a 12 mer peptide. first set of peptides covered hck sequence was spotted in squares a1 to m9. peptides at the spots n9 to t5 were the peptides picked from first set in which first amino acid sequence numbers were odd number; this just served as a repeat experiment. after secondary antibodies and chromogenic substrates were added, six sets of continuous spots were developed. each set of the continuous spots corresponds to a peptide segment of hck. spots a2-a6 correspond to asn5-tyr14 of hck; spots of e3-e13 correspond to ser129-lys138 of hck; spots of f17-f21 correspond to lys170-met179 of hck; spots of i3-i12 correspond to glu248-leu257 of hck; spots of i22-i29 correspond to lys265-asn274 of hck; spots of k5-k14 correspond to thr313-thr322 of hck. these six peptides were potential scfv-a4 binding sites on hck. there were 11 linear epitopes predicted by ellipro [30] and 15 linear epitopes predicted by fbcpred [26] . these predicted epitopes were potential binding sites for all antibodies. for example, some predicted peptides might be the epitopes for anti-hck antibodies with no chaperone-like functions. there were a total of eight overlapping epitopes in the two groups of epitopes predicted by both methods. these eight epitopes were shown in table 1 . for these eight peptides, four peptides fell in the nterminal domain, one peptide fell in the linker area and three fell in the c-terminal domain. comparing to the peptide array membrane experiment results, there were two overlapping sequences, lys170-met179 and lys265-asn274, which were positive in both methods. an orientational docking of scfv-a4 and hck epitopes was carried out by zdock [31] docking server. if we combined the results from peptide array membrane experiment and bioinformatics predictions, we could reduce the workload by only docking the two overlapping peptides. for experimental completeness, we did the orientational docking for all of the six peptides screened out by the peptide array experiment. zdock did not generate any reasonable models for four of the six peptides (ser129-lys138, glu248-leu257, thr313-thr322, asn5-tyr14). it was interesting because it agreed with the bioinformatics prediction that they were not epitopes. to look into the details of the six peptides, we investigated their structure feature. as shown in figure 4 , the first three peptides were embedded inside the hck monomers, thus they were not accessible by scfv-a4. for peptide asn5-tyr14, it was buried between the dimerization interfaces of the two hck monomers therefore it was also inaccessible for scfv-a4. only the rest two peptides (lys170-met179 and lys265-asn274 could be docked to scfv-a4 by zdock server and two complex models were obtained. because zdock used rigid body approximation, to obtain a more accurate protein complex structure, complex models generated by zdock were redocked by rosettadock [32] . which implemented the flexible docking method and came with a more accurate scoring function. rosettadock generated 1000 new complex models for each of the two epitopes respectively. the output results were ranked by docking scores. we discarded models in which the binding sites of the antibody were outside the cdr regions and picked the best complex models by the best rank scores. two final complex models for the two epitopes were named as complex-i and complex-ii respectively and shown in figure 5 . antibodies and their functional derivatives such as scfv and fab are known for their specific binding and have already been used in protein folding researches [9] . antibodies with a chaperone-like function had the potential to be used as therapeutic agents for misfolding diseases. more researches need to be done to elucidate the mechanism of antibodies' chaperone-like function. a protein antigen usually carries more than one epitope, therefore an antigen can interact with several different types of antibodies. obviously the different types of antibodies have different impacts on the antigen's properties. we had previously screened out several scfvs for hck and one of them named scfv-a4 showed strong chaperone-like activity. to elucidate why scfv-a4 had the chaperone-like function, we combined computer modeling and peptide array membrane technique to study the interactions between scfv-a4 and hck. firstly, bioinformatics software tools were used to predict the interacting sites between scfv-a4 and hck and then the results were combined with the peptide array membrane experiment results to build the 3d models of the binding complex. the prediction of protein-protein interactions by computer is still a challenging subject, and it is inappropriate to rely entirely on the energy scoring function to filter the results directly. thus, we used the orientational docking method followed by redocking, the cumulated results were then sorted by the scoring function to get reliable protein-protein binding models. first the binding sites of the antigen and the antibody were predicted respectively. after that the two proteins were orientationally docked according to the predicted binding sites. sequence based epitope prediction was still difficult especially for the conformational epitopes. we combined the prediction results of two different software tools, and only those epitopes reported by both methods were considered as potential epitopes. the accuracy of each method was 73% (fbcpred) and 84% (ellipro) respectively, so the accuracy of the prediction of the two methods combined could reach 95%. the number of predicted epitopes was further reduced by comparing them with the peptide array membrane experiment results. the epitopes we studied were linear epitopes, and for linear peptide epitopes, the binding solely depends on amino acid sequences. if an antibody could bind to a linear peptide epitope, it will bind to the peptide irrespective of whether the peptide was on the protein, on the folding intermediate or on the membrane. consequently only the epitopes which were positive in peptide array membrane experiment were selected for further modeling. two antigenantibody model complexes were screened out as the final result. there are two major entry points for scfv-a4 to exert its chaperone-like function on hck, on the hck folding intermediate or on the folded hck. yan et al found that the c-terminal domain of hck was more likely to be the binding site during rabbit muscle ck (rmck) aggregation [33] and the linker played a crucial role in rmck folding [34, 35] . after further investigate, they found that the linker act as a lid to protect the hydrophobic side chains of phe250 and val347 against exposure to solvent [36] . in complex model-i, scfv-a4 bound to the opposite site of hck thus has not impact on these residues (figure 5a ). in complex model-ii, the binding position of scfv-a4 was close to the two hydrophobic residues and actually formed a bulky protrusion (figure 5b) , therefore for complex model-ii, scfv-a4 bound to hck would hinder the aggregation of hck by steric effects.webb [37] found that the c-terminal portion of the chick ck (gly215 to lys380) was much more resistant to digestion than the n-terminal portion (pro1 to gly133), and the middle of the hck sequence (arg134 to arg214) was most sensitive to proteolysis. they proposed that the folding intermediate consisted of a folded c-terminal domain and a partially folded n-terminal domain separated by an unfolded central linker. gross et al.'s studies suggested that the c-terminal fragment (168-380) of mitochondrial ck represented an autonomous folding unit, and that the folding of the c-terminal domain might precede the conformational stabilization of the n-terminal moiety in vivo [38] . the binding site of complex model-i was located between these two terminal domains, which indicated that in complex model-i, scfv-a4 would not significantly interfere with the major folding path ways of the two domains. it may stabilize the folded c-terminal domain and assist in the folding of the nterminal domain. there was a v8 endoproteinase sensitive fragment in the chick ck intermediate. when the chick ck had been resurrected, this fragment resisted to the proteinase, since it became less exposed in the folded kinase [39] . therefore, this fragment can be regarded as a characteristic part of the creatine kinase intermediate. this fragment identified by v8 endoproteinase was ifkkag [37] , the corresponding sequence of hck was ile263-gly268. it is interesting that in complex model-ii, scfv-a4 also bound to this segment which indicated that scfv-a4 could bind with the intermediate and participate in hck folding or resurrecting process. to summarize, in complex model-ii scfv-a4 can exhibit a chaperone-like function either by hindering the aggregation by the steric effects or by assisting the folding of hck and stabilizing the folded conformation. in complex model-i, scfv-a4 exhibits chaperone-like function by assisting the folding of hck and stabilizing the whole structure. in this study we combined computer modeling and the peptide array membrane method to investigate the interaction between scfv-a4 and hck. the mechanisms underlying scfv-a4's chaperone-like function were discussed. the traditional method of studying antigen-antibody interactions is by constructing many antigen mutants and observing the changes of the binding activity. the whole process is tedious and time consuming. epitopescanning by peptide array membrane method has been made cheap and fast nowadays, our work demonstrated that the combination of the peptide array membrane technique with bioinfomatics methods could be a more efficient approach for epitope mapping. for the two complex models we proposed, further study can be done to verify the models by engineering point mutants on the predicted epitopes of hck and investigating the change of the system's behaviors. homology modeling in our study was done in the following three steps: (1) speculating the linker, the light and the heavy chain segments of the antibody. the sequence of scfv-a4 was used to do a blast search in the non-redundant protein sequences database of ncbi. a scfv sequence (id: acz28696.1) [40] which had the highest score and the lowest e value was picked out to be used as reference. the sequences of acz28696.1 and scfv-a4 were aligned by clustalx1.81 [41] and each area of the scfv-a4 sequence was appointed by comparing the sequence with acz28696.1. (2) homology modeling templates preparation. since linker peptides of the single chain antibody were often absent in the pdb database [42] , the sequence of the linker peptide would be excluded when aligned with the template antibody. the sequence of scfv-a4 without linker was used to search the pdb database. the light chain and the heavy chain were processed separately. we obtained a template (2dd8 [43] ) with a higher identity which was a fab antibody fragment. the sequence identity of the light chain of the fab was 100%, while that of the heavy chain was 93%. both chains of the fab were used as templates for building the 3d model of scfv-a4 by modeller 9v7 [44] . (3) optimization of the model. since the linker was modeled without an appropriate template, optimization work was focused on the linker. the dope-based loop modeling protocol [44] was used to optimize the linker structure. the cdrs are the major region of the antibodies that bind to antigens. since the cdrs of the scfv acz28696.1 was known, the cdrs of scfv-a4 was inferred from the alignment result of the two sequences ( figure 1 ). the resulted cdr assignment was checked to be complied with the kabat numbering rules [45] . the crystal structure of hck was available in pdb file 1i0e [46] . hck is a dimmer in nature but the 1i0e only contains a each peptide epitope was noted by its sequence region on hck. there were total eight epitopes predicted by both software and their overlapping region was listed at the right column. doi:10.1371/journal.pone.0062147.t001 figure 4 . positions of the peptides screened out by the peptide array membrane method on the hck 3d structure. the six experimental positive peptides were colored in salmon and represented by using the stick model. as shown in the picture, asn5-tyr14 is at the position of dimerization interface of the two monomers; ser129-lys138 is completely embedded inside the structure; glu248-leu257 is in a dent of the surface; lys265-asn274 which contains v8 endoproteinase fragment was also in a dent of the structure; lys170-met179 is located at the bottom of the structure; thr313-thr322 is located in the largest groove of the structure. doi:10.1371/journal.pone.0062147.g004 monomer and the structure of two peptide fragments, met1-his7 and gly323-gly331, was missing. thus we use another ck structure, the 1u6r for rabbit ck in pdb as the homology modeling template. the sequence identity between rabbit ck and hck is 96% and the 1u6r file contains the structure of the two missing fragments peptides of the 1i0e file. we used fbcpred [26] based on the machine learning method and ellipro [30] based on the 3d structure for epitope prediction. an epitope predicted by both methods would be regarded as a potential epitope. as for fbcpred, default parameters were adopted for linear epitope prediction, the length of the linear epitopes was set to 14, and the specificity was 75% as default. no parameter was needed for ellipro. 12 mer peptides from the hck sequence were synthesized on nitrocellulose membrane as individual spots. peptide sequences were started from hck n-terminal and each subsequent peptides shift one position to the right. a set of such peptides completely sampled the whole hck sequence and formed an array on the membrane. the peptide array membrane was incubated with purified scfv-a4 antibodies in the mp buffer (30 mm tris-hcl, ph 7.6, 170 mm nacl, 6.4 mm kcl, 0.05% tween 20, 5% sucrose) overnight at 4uc with gentle shaking. unbound antibodies were removed with tbs (4uc) andthe antibodies bound were electro-transferred onto a pvdf membrane using a semi-dry blotter (0.8 ma/cm2, three hours). the membrane was blocked with 5% non-fat dry milk for 2 h and incubated with the primary antibody (anti-v5 antibody) overnight at 4uc. after being washed with tbs buffer for three times, the membrane was incubated with the alkaline phosphatase-conjugated secondary antibodies for 1 hour and developed using the enhanced chemiluminescence method. the zodck server was used to dock scfv-a4 to hck. the zdock server allowed user to pick contact and blocking residues of the proteins for docking. the hck residues of the predicted epitopes were chosen as contact residues for docking. there were six linear peptides exhibiting positive reactivity with scfv-a4 in the peptide array membrane experiment and the corresponding portions of hck were designated as the orientational docking sites of hck. no blocking residues of hck were selected during docking, but for scfv-a4, residues of the linker segment were selected as the blocking residues. rosettadock was used to optimize the models build by zdock because it implemented the flexible docking method and had a scoring function of better performance. the perturbation docking method of the rosetta-dock program was applied and the three default perturbation parameters were used. one thousand models were produced for each selected zdock model complex. conceived and designed the experiments: jyf tl. performed the experiments: hg sl. analyzed the data: jyf tl. contributed reagents/ materials/analysis tools: sl. wrote the paper: jyf tl. figure 5 . the scfv-a4-hck complexes. model i is shown at left. model ii is shown at right.scfv-a4 is colored in marine. hck is shown as dimmer. one hck monomer is colored in lime. the n-terminal of the other hck monomer is colored in slate and c-terminal in cyan, the linker is colored in orange. the v8 endoproteinase sensitive fragment is colored in magentas. the binding residues are represented by using the stick model. the phe250, val347 are represented using the sphere model. 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structure modeling using modeller sequences of proteins of immunological interest: us dept. of health and human services structure of human muscle creatine kinase key: cord-003270-vu9b5a14 authors: panahi, heidar ali; bolhassani, azam; javadi, gholamreza; noormohammadi, zahra title: a comprehensive in silico analysis for identification of therapeutic epitopes in hpv16, 18, 31 and 45 oncoproteins date: 2018-10-24 journal: plos one doi: 10.1371/journal.pone.0205933 sha: doc_id: 3270 cord_uid: vu9b5a14 human papillomaviruses (hpvs) are a group of circular double-stranded dna viruses, showing severe tropism to mucosal tissues. a subset of hpvs, especially hpv16 and 18, are the primary etiological cause for several epithelial cell malignancies, causing about 5.2% of all cancers worldwide. due to the high prevalence and mortality, hpv-associated cancers have remained as a significant health problem in human society, making an urgent need to develop an effective therapeutic vaccine against them. achieving this goal is primarily dependent on the identification of efficient tumor-associated epitopes, inducing a robust cell-mediated immune response. previous information has shown that e5, e6, and e7 early proteins are responsible for the induction and maintenance of hpv-associated cancers. therefore, the prediction of major histocompatibility complex (mhc) class i t cell epitopes of hpv16, 18, 31 and 45 oncoproteins was targeted in this study. for this purpose, a two-step plan was designed to identify the most probable cd8+ t cell epitopes. in the first step, mhc-i and ii binding, mhc-i processing, mhc-i population coverage and mhc-i immunogenicity prediction analyses, and in the second step, mhc-i and ii protein-peptide docking, epitope conservation, and cross-reactivity with host antigens’ analyses were carried out successively by different tools. finally, we introduced five probable cd8+ t cell epitopes for each oncoprotein of the hpv genotypes (60 epitopes in total), which obtained better scores by an integrated approach. these predicted epitopes are valuable candidates for in vitro or in vivo therapeutic vaccine studies against the hpv-associated cancers. additionally, this two-step plan that each step includes several analyses to find appropriate epitopes provides a rational basis for dnaor peptide-based vaccine development. hpvs are a large branch of the papillomaviridae family, grouped in different genera (alpha-, nu-/mu-, beta-and gamma-papillomaviruses), with more than 200 genotypes [1] [2] [3] [4] . the classification of papillomaviruses (pvs) has been based on l1 gene sequence. they are clinically divided into two groups: low-risk hpvs, like hpv 6 and 11, which cause benign lesions (warts and benign papillomas), and high-risk hpvs (hrhpvs), like hpv16 and 18, which are a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 response. however, the addition of cd4+ t cell epitopes can significantly augment its strength and duration [49, 52, 53] . cd8+ ctls commonly recognize intracellular-originated peptides presented by mhc-i molecules. they accommodate peptides with 8-11 residues; the ideal length is 9 residues. while cd4+ helper t lymphocytes (htls) commonly recognize extracellular-originated peptides presented by mhc-ii molecules. they accommodate peptides with 10-30 residues or even more; the ideal length is [12] [13] [14] [15] [16] . the strength of the interaction between a t cell receptor and a peptide-mhc complex (pmhc), depends on the presented peptide and the mhc structure [49, 54] . the binding of a peptide to mhc-i molecule is the most selective stage in the way of peptide presentation [55] . bioinformatics tools can predict the potential immunogenic epitopes from thousands of epitopes in a short time [56] . generally, the algorithms of these tools range from ones programmed to determine peptide-mhc molecule binding data to those based on structural similarity, molecular modeling, and molecular docking [57] . peptides that bind to a specific mhc molecule have sequence similarity. therefore, peptide sequence patterns have been used to predict their binding to mhc molecules [58] . in recent years, the accuracy of these methods has increased strikingly, and more than 90% of natural epitopes have been recognized at a high specificity of 98% [59] . this improvement in performance was achieved by the expanding experimental binding data, available in the immune epitope database (iedb) and analysis resource (http://www.iedb.org/), and by the improvement of machine-learning algorithms [60] . regarding the fundamental importance of epitope prediction in vaccine development, we investigated the best potential cd8+ t cell epitopes from the e5, e6, and e7 oncoproteins of four prevalent hrhpv genotypes (16, 18, 31 and 45) in the world and iran [61] , as shown in a two-step plan was designed to identify the most probable cd8+ t cell epitopes (fig 2) . for the first step, mhc-i and ii binding, mhc-i processing, mhc-i population coverage and mhc-i immunogenicity prediction analyses, and for the second step, mhc-i and ii protein-peptide docking, epitope conservation, and cross-reactivity with host antigens analyses were considered. the second step analyses were performed only for the selected peptides in the first step. in jan 2018, in order of priority, the refseq, reviewed or unreviewed sequences of hrhpv oncoproteins (e5, e6, and e7) were retrieved from the national center for biotechnology information database (ncbi) (http://www.ncbi.nlm.nih.gov/) and uniprotkb/swiss-prot database (http://www.uniprot.org/). the isoform sequences of hpv16, 18, 31, and 45 oncoproteins were retrieved from hpv t cell antigen database (http://cvc.dfci.harvard.edu/hpv/ html/search.php). all the sequences are accessible in supporting information (s1 file). binding of epitopes to mhc-i molecules is an essential step for antigen presentation to ctls. herein, it was predicted by four online servers, as illustrated in table 1 . the hla supertypes and frequently occurring hla-i alleles provided by the servers were included in the analysis. however, when an allele (e.g., hla-b � 14:02) was not provided, but its allele group (i.e., hla-b � 14) was available, we used the allele group instead of the allele. the used human and mouse alleles, or allele groups are provided in supporting information (s1 table) . currently, eight prediction methods are available in the iedb mhc-i binding prediction tool, i.e., iedb recommended [62] , consensus [69] , netmhcpan3 [59, 70] , artificial neural network (ann) [71, 72] , smm with a peptide-mhc binding energy covariance matrix (smmpmbec) [73] , stabilized matrix method (smm) [74] , comblib_sidney2008 [75] , pickpocket [76] , netmhccons [77] and netmhcstabpan [78] . the iedb-recommended and consensus are not independent methods; they use ann, smm and comblib_sidney2008 methods to generate a representative index for each predicted pmhc; the median of percentile ranks (prs) or binding scores obtained from the used methods is reported as a representative pr or consensus score in the iedb-recommended or consensus method respectively. the pr is calculated by comparing the half maximal inhibitory concentration (ic 50 ) of subjected peptide against a group of random peptides from swiss-prot database. the ic 50 value, expressed as nanomolar, shows binding affinity. the lower ic 50 or pr means higher binding affinity. as a rough guideline, peptides with ic 50 values <50 nm are considered as high affinity, 50-500 nm intermediate affinity and more than 500-5000 nm low affinity. no known t cell epitope has got an ic 50 value >5000 nm to date [60] . in this study, iedb recommended method was used. the outputs for each pmhc in this method consisted of a median pr, a method-specific ic 50 , and a method-specific pr. predictions were made against 76 frequently occurring human mhc-i alleles (including 12 hla supertypes) and 6 mhc-i mouse alleles. epitope length was set on 8, 9, 10, and 11mer. peptides with median pr <2.0 are applied for the analysis. netmhcpan4 mhc-i binding prediction. netmhcpan4 server predicts binding of peptides to the known mhc molecules using anns method. it is trained on a combination of naturally eluted ligands (55 human and mouse mhc-i alleles) and binding affinity data (172 mhc molecules from human, mouse, cattle, primates, and swine). besides, the user can perform a prediction against any custom mhc-i molecule by uploading its full-length sequence [66] . in this study, predictions were performed for 8, 9, 10, and 11mer peptides against 76 frequently occurring human mhc-i alleles and 8 mhc-i mouse alleles. pr thresholds for strong and weak binders were set on 0.5 and 2.0, respectively. peptides with pr <2.0 were applied for the analysis. rankpep mhc-i binding prediction. rankpep predicts binder peptides of a given protein sequence or sequence alignments to mhc-i and ii molecules. the algorithm of rankpep based on the comparison of sequence similarities, using position-specific scoring matrices (pssms) method. it employs profiles of a group of aligned peptides recognized to bind to a specific mhc molecule and creates a consensus sequence by determining the most common residue for each position. then, it allocates an optimal score to the consensus sequence, compares the score of the subjected peptide with the optimal score, and gives the peptide a percentile optimal value for comparison. finally, it highlights strong binders in red [67, 68] . herein, the prediction was made against 31 frequently occurring hla-i and 7 h2-i alleles. the server did not provide all common lengths of epitopes for all the mhc alleles. thus, the used alleles and their provided epitope lengths are shown together, as given in supporting information (s1 table) . syfpeithi mhc-i binding prediction. syfpeithi (http://www.syfpeithi.de/0-home. htm/) is a database of over 7000 published and verified peptide sequences of human, mouse, and other organisms, known as natural binders of mhc-i and ii molecules. when syf-peithi analyzes a peptide for binding prediction against a specific mhc-i allele, its scoring system evaluates every residue of the query and gives it an arbitrary value between 1 and 15, according to whether it is an anchor, auxiliary anchor, or preferred residue. it allocates the value 1 to those residues which slightly preferred in that particular position, 15 to the ideal anchor residues, and -1 to -3 to those residues which exhibit an adverse effect on the binding ability. the sum of these values is the score of the peptide. the maximal score could vary between different mhc alleles [54, 79] . herein, the prediction was made against 26 frequently occurring hla-i alleles and 5 h2-i alleles. epitope length was set on 8, 9, 10, and 11mer. every predicted pmhc which got a score less than 70% of the reference sequence score was excluded from the analysis. the allele-specific reference sequence was selected from rankpep's consensus sequence [68] , or our syfpeithi predicted epitopes, whichever got the highest score in syfpeihi server. the reference sequences, their sources, and their scores are given in supporting information (s2 table) . recognition of high immunogenic cd8+ t cell epitopes was the primary aim of this study. therefore, all predictions were primarily made against epitopes with 8-11 residue length. however, it was valuable to determine that which 9mer mhc-i epitope is the core peptide of the mhc-ii epitope(s) too. the core peptide lies on the mhc-ii molecule grooves, and play the central role in constructing pmhc. with this strategy, the short minimal predicted epitopes could be used in designing of synthetic long peptides (slps), resulting in peptide loading to both mhc-i and ii molecules. iedb mhc-ii binding prediction. in this study, the mhc-ii binding prediction was made by iedb mhc-ii binding predictor (http://tools.iedb.org/mhcii/) [60, 63, 64] . iedb possess seven prediction methods for mhc-ii binding prediction: iedb-recommended, consensus [63] , netmhciipan [80] , nn-align [81] , smm-align [82] , combinatorial libraries [75] and sturniolo's method [83] . herein, the iedb-recommended method was used, and all peptides with pr<2.0 were selected for the analysis. the prediction was made against 35 human alleles (iedb reference set) and three mouse alleles, given in supporting information (s3 table) . the server has fundamentally set the epitope length on 15mer. each iedb-recommended method participated in the prediction process offered a core peptide (9mer) for each predicted epitope (15mer). we associated the 9mer mhc-ii core peptides with the 9mer mhc-i predicted epitopes to determine that which mhc-i epitope is the core peptide of the mhc-ii epitope(s) too. mhc-i t cell epitope processing predictions of e5, e6, and e7 oncoproteins are made by the iedb combined predictor (http://tools.iedb.org/processing/). this tool combines predictors of three main steps of mhc-i antigen presentation pathway (proteasomal processing, transporter associated with antigen processing (tap) transport, and mhc-i binding) and calculates a total processing score for each predicted epitope. it allows the user to choose a method from ann, smm, smmpmbec, comblib_sidney2008, netmhcpan, netmhccons and pickpocket methods for the binding prediction. in the current update (2018), the iedb team has changed the choice of the recommended prediction method for the processing tool to be netmhcpan 3.0 rather than a consensus, since the processing tools requiring an ic 50 value, which the consensus method does not provide. furthermore, netmhcpan 3.0 has provided all mhc alleles and has performed the predictions very well in recent comparisons [65] . there are two types of proteasomes, the housekeeping types which are expressed instinctively, and immuno types which are provoked by ifn-γ secretion. the immunoproteasomes are believed to improve the efficiency of antigen presentation [62, 65] . in this study, the immunoproteasome option was selected. the program outputs for every predicted epitope consisted of proteasome score, tap score, mhc score, processing score (proteasome + tap score), total score (proteasome + tap + mhc score), and mhc-i ic 50 . the tap scoring system calculates a-log (ic 50 ) value for the binding of a peptide (or n-terminal of its precursors) to the tap molecules. the higher tap score, the higher transport rate. [62, 65, 84] . herein, the analysis was made against the human and mouse mhc-i alleles used later in the iedb binding prediction, with the iedb-recommended method and other default settings of the program. epitopes with ic 50 <1000 nm for hla-i alleles and <5000 nm for h2-i alleles were included in the analysis. several factors could clarify the difference between epitope and non-epitope peptides; an essential factor is epitope immunogenicity, i.e., it could be recognized by t cells. some amino acids, particularly those with large and aromatic side chains (especially tryptophan, phenylalanine, and isoleucine), are associated with immunogenicity. moreover, the positions p4-6 of a peptide are more critical for immunogenicity [85] . in this study, the mhc-i immunogenicity of all predicted epitopes was determined by the iedb web server (http://tools.iedb.org/immunogenicity/) [85] . this tool uses the properties of amino acids and their locations to predict the immunogenicity of a pmhc. the default option was selected to specify which positions of the query peptide to be masked from the analysis, because it masked positions which are also suggested for the most frequent human mhc-i allele, hla-a � 02:01. iedb population coverage prediction tool (http://tools.iedb.org/population/) [86] is used to predict the hla-i population coverage of all 8-11mer predicted epitopes in the first step. this tool can accept a target population by two query levels: 1) area-country-ethnicity and 2) ethnicity alone. it can integrate allele frequency information retrieved from the allele frequency net database (afnd) (http://www.allelefrequencies.net/default.asp) [87] . iedb also accepts custom populations with allele frequencies defined by users. since, hla-i and hla-ii t cell epitopes elicit immune responses from two different t cell populations (ctl and htl, respectively), the server provided three different population coverage modes: 1) hla-i lonely, 2) hla-ii lonely, and 3) hla-i and hla-ii together. herein, the mhc-i promiscuous predicted epitopes and their binding hla-i alleles (ic 50 <500 nm or pr<2.0) were entered as inputs for the analysis against the world population. the primary aim of molecular docking is the prediction of the binding site of a ligand at a protein receptor surface, and then docking and modeling the ligand into the recognized site. in this study, the binding ability of the first step selected peptides to human and mouse mhc molecules, was analyzed by cabs-dock (http://biocomp.chem.uw.edu.pl/cabsdock/) server. the server uses a multistage procedure that involves multiple programs, with the cα-cβ-side chain (cabs) model at its heart. the detailed information about these stages is given in supporting information (s2 file) [88, 89] . also, fig 3 shows the pipeline of cabs-dock protocol [88] . cabs-dock gets the 3d structure of the receptor and the sequence of the peptide as obligatory inputs. furthermore, there are some non-obligatory inputs as recommendations which could improve outputs. in this study, duplicate dockings for each peptide (6240 dockings in total) were done against the most significant human/mouse mhc-i and ii molecules which had at least one well-structured protein data bank (pdb) file in the rcsb protein data bank (https://www.rcsb.org/), as shown in table 2 . these pdb files are in the complex with their peptidic ligand and some x-ray crystallography solution molecules (heteroatoms). thus, these excess molecules, as well as redundant mhc molecules were removed before executing docking process. since, the binding site of epitopes on the mhc molecules was well-known previously, the unlikely regions to bind masked before the analysis. cabs-dock returns ten representative models (medoids) as the best-simulated models and ranks them by cluster density (cd). cluster density is equal to the number of elements in a cluster divided by their average ligand root mean square deviation (rmsd). the higher cd value implies greater accuracy. ligand rmsd value shows the differentiation measure between cluster elements. as a guideline; rmsd < 3.0 å means high accuracy; rmsd � 3.0 and � 5.5 å means medium accuracy and rmsd > 5.5 å means low accuracy [88] . herein, the rmsd and cd of the best-simulated models were selected for the analysis. the best model, which has the highest cd value, is not necessarily the top-ranked model, because, in some cases, peptides were not attached to their binding site properly. thus, these malformed models were excluded from the analysis. it is important to note that, due to the different frequency of mhc alleles in human populations, the equal cd value of different mhc alleles, don't have equal value regarding population coverage. thus, to involve the effect of population coverage, the cd value of every model was multiplied by its allele population coverage (divided by hundreds for more facility) to obtain a weighted index. then, the sum of all hla-i or ii weighted indexes of each peptide was calculated to get a total docking score (tds), used as a score to compare the candidate peptides. it is the first time that the tds has been formulated and used for this purpose. this formula is also applicable to the similar docking scores obtained from other servers. the use of highly conserved epitopes in a vaccine formulation reduces the risk of tumor immune escape and provides broader protection against different virus strains or genotypes. thus, the conserved areas are preferred to use in therapeutic vaccines, if they are appropriate epitopes. herein, the epitope conservancy analyses for the first step selected peptides were done in three levels: 1. inter-isoform conservancy: the percent of conservancy between all isoforms of each e5, e6, or e7 oncoprotein. 2. inter-type conservancy: the percent of conservancy between hpv16 and 31 (alpha-papillomavirus 9), as well as between hpv18 and 45 (alpha-papillomavirus 7). 3. inter-hrhpv conservancy: complete (100%) conservancy between all hrhpvs (hpv16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59). the selected peptides in the first step were analyzed to find inter-strains and inter-types conservancy percentages by iedb tool, conservation across antigens, (http://tools.iedb.org/ conservancy/). the inter-hrhpvs conservancy analysis was done by the iedb and expasy clustalw servers (https://embnet.vital-it.ch/software/clustalw.html). cross-reactivity with host antigens can cause adverse immune responses. therefore, the selected peptides in the first step were checked for similarities with the mouse and human proteomes by the ncbi blastp tool (https://blast.ncbi.nlm.nih.gov/blast.cgi). regarding the studies, different peptides usually get different scores/ranks in different analyses. this inconsistency indicates that these results needed to be analyzed with an integrated approach. indeed, integrated approach is more practical and efficient in such conditions in comparison with analysis by analysis filtering approach, in which those epitopes are chosen for the next analysis that have gotten an acceptable score in the previous analysis. herein, the integrated approach was applied in both steps of epitope selection. since the ultimate goal of the discovery of therapeutic epitopes is to use them in human vaccines, only the scores/ranks of human alleles were used to rank epitopes in some studies. however, investigators usually test therapeutic vaccines on mouse species in preclinical trials, thus in the current study, the binding status of the predicted epitopes to mouse mhc-i alleles was also studied by several binding predictors and molecular docking, as well. as stated above, ctl-mediated responses play a crucial role in killing the malignant cells. besides, the binding of epitopes to mhc-i molecules is the most selective step for antigen presentation to ctls. therefore, in the first step, the selection was made primarily by the comparison of obtained mhc-i binding, processing and immunogenicity scores/ranks, and population coverage percentages. however, the mhc-ii binding ranks were actually of secondary importance to the selection process as an added advantage. additionally, the population coverage has a dual application. first, it determines the coverage of a given peptide in the target population. second, it is the best index for summarizing and evaluating of the hla-i binding predictions too, since it is calculated from the results of hla-i binding prediction analyses. in the first step, ten peptides (tables 3-5 ) from each hpv genotype oncoprotein (120 peptides in total), which got better results in the first step analyses were selected for the second step analyses, including protein-peptide molecular docking, epitope conservation, and crossreactivity with host antigens. the individual detailed results of the mhc-i and ii binding (s3 file), mhc-i immunogenicity (s4 file) and mhc-i population coverage (s5 file) predictions, as well as, mhc-i and ii molecular docking (s6 file) and epitope conservation (s7 file) analyses are given in supporting information, as 15 excel files. indeed, cabs-dock returns ten representative medoids as the best-simulated models and ranks them by cluster density (cd). cluster density is derived from two factors (the number of elements in a cluster and their average ligand rmsd) that is an advantage for this server. in the second step, five peptides out of ten selected peptides in the first step (tables 6-8) , which got better results in all analyses of both steps, were selected as the final-predicted epitopes. none of the final predicted epitopes showed more than 90% sequence similarity with mouse and human proteomes. high prevalence and mortality of oncogenic infectious pathogens such as hpv and helicobacter pylori have caused serious problems for humans. currently, people who are infected with hrhpvs but show normal cytology or precancerous lesions do not have any treatment option, causing the disease progress toward invasive carcinoma in some cases. unfortunately, no fda-approved immunotherapy exists for pre-existing hpv infections or their related cancers to date. immunotherapy of hpv-associated cancers by dna or peptide-based vaccines, depends on the recognition of highly immunogenic epitopes, inducing robust and specific immune responses, particularly cell-mediated responses against the malignant cells. the primary aim of this study was the prediction of cd8+ t cell epitope from the e5, e6 and e7 oncoproteins, using a comprehensive two-step selection plan. these proteins chose because they play a pivotal role in the cell transformation, immune evasion, and maintenance of malignancy, as well as, their permanent expression (e6 and e7) by the malignant cells [24] [25] [26] . expression of e5 oncoprotein occurs in the early phase of hpv infection. evidence indicates that e5 play a prominent role in the genesis of hpv-associated cancers, but is not essential for cancer progression [90] , since when hpv genome integrates into the host genome, it usually results in the disruption of e1, e2, and e5 genes. therefore, targeting e5 protein provides an opportunity for treatment of hpv infections and preventing the precancerous lesions from the progression to established carcinomas [20, 91] . some genotypes of hrhpvs are more involved in the genesis of epithelial tissue malignancies [61] . thus, in this study, hrhpv16, 18, 31 and 45 were targeted due to their high prevalence in the hpv-associated cancers, especially cervical carcinoma. there are several limitations for epitope prediction: 1) the major drawback of peptidebased vaccines is low immunogenicity [92, 93] . many studies have focused on enhancing immunogenicity using immune stimulating agents or adjuvants to avoid this problem. another solution is the use of agonist epitopes [94] . epitope immunogenicity is a crucial factor in vaccine development. however, many of known natural epitopes when are analyzed in silico by iedb mhc-i immunogenicity predictor, do not obtain a high score. therefore, in this study, epitope selection was based on the integrated approach, in which one analysis does not play an important role alone. 2) there are certain drawbacks associated with the function of each method invented for the mhc-peptide binding prediction [95] . for this reason, several predictors and a molecular docking program were used to augment the prediction accuracy. 3) some web tools have been developed for mhc-ii epitope prediction. since mhc-ii groove can bind to peptides with variable lengths, and different peptides have the different number of residues between their n-terminus and first anchor [54], the exact assignment of mhc-ii core peptide would be a difficult problem which reduces the success rate of these prediction tools. therefore, most mhc-ii prediction tools did not usually make epitope predictions as accurately noted for mhc-i molecules [64, 96] . in cancer immunotherapy, the ctl-mediated responses play the central role in eradication of malignant cells, and the binding of epitopes to mhc-i molecules is an essential step for antigen presentation to ctls. thus, in this study, predicted epitopes were primarily selected by their mhc-i binding and processing scores. however, the mhc-ii binding scores were actually of secondary importance to the epitope selection process as an extra advantage. additionally, there are several other essential determinants which significantly affect the outcomes, such as antigen processing, immunogenicity, population coverage, conservancy and cross-reactivity with host antigens. vaccine development requires a comprehensive approach to cover all these effectual elements, covered in this study. the primary aim of molecular docking is the recognition of binding site of a ligand at a protein receptor surface, and docking and modeling the ligand into this recognized site. in this study, cabs-dock server was used for molecular docking analyses. cabs-dock has several main advantages: 1) the method does not require any data about the peptide structure and its binding site. 2) during docking process, peptide conformation is entirely flexible. 3) it is possible to apply dynamic conformational changes in the receptor structure and 4) to exclude some receptor regions from the docking search, leading to the more efficient search in the vicinity of the binding site at a sensible time. [88, 89] . in comparison with protein-ligand (small molecules) docking, protein-peptide docking analysis is more problematic, since significant conformational changes occur during the process. as a general rule, how much the length of the query peptide to be longer, there are more torsions and conformational flexibilities. additionally, in comparison to protein-protein interactions, protein-peptide dockings are more transient, and their binding affinities are notably weaker [88] . these factors make structural predictions of long peptides very challenging. therefore, in this study, 9mer peptides were preferred for selection compared to other possible lengths. they are also preferred by all mhc-i molecules as epitope and by mhc-ii molecules as the core peptide of epitopes. moreover, expansion of 9 or 10mer ctl epitopes to longer peptides may create a practical alternative, containing both cd4+ htl and cd8+ ctl epitopes; especially, when cd4+ htl epitopes, covering ctl epitopes, are not recognized [97] . [94, 96, [98] [99] [100] [101] [102] [103] . however, the prediction of t cell epitopes inducing strong responses has remained a big challenge. for therapeutic hpv vaccines, many candidates have been designed to trigger the activation of ctls or htls, mostly by targeting two major hpv oncoproteins, e6 or e7 [104] , and in a few studies, e5 oncoprotein [98, 99] . as well as, several clinical trials have been launched for immunotherapy of hpv-associated cancers [46], although, they have not been so immunogenic, to induce a sufficient cellular immunity and eradicate malignant cells completely. some studies have suggested that the use of e6 and e7 slps, containing both cd4+ htl and cd8+ ctl epitopes, led to more potency and durability of cd8+ t cell reactivity in vivo, in comparison with the minimal ctl epitopes [97, 105] . in 1993, as pioneers in hpv epitope studies, feltkamp et al. recognized the hpv16-e7 sequence rahynivtf as an mhc-i epitope that can provoke ctl-mediated responses and eradicates established hpv l6-induced tumor cells in mice [106, 107] . this sequence is the first hpv16-e7 predicted epitope in our study as well. in 2015, kumar et al. studied hpv16-e5 oncoprotein to predict the candidate t-cell and bcell epitopes [98] . they have screened 11 potent epitopes for mhc-i molecules according to pr and the immunogenicity score, using iedb mhc-i binding and immunogenicity predictors. they found a 14mer potent epitope, safrcfivyiifvy, having the lowest pr and the highest immunogenicity score, i.e., 0.5 and 0.70, respectively. notably, our second hpv16-e5 predicted epitope, safrcfivy, is the n-terminal part of safrcfivyiifvy, and our first predicted epitope, flihtharf, is the c-terminal part of the third epitope of their study, vyiplflihtharf. in 2017, tsang et al. scanned the hpv16-e6 and e7 oncoproteins for the match peptides with the consensus motif of hla-a2 binding peptides [94] . the bimas algorithm [108] was employed to rank probable binding peptides according to the predicted one-half-time dissociation of pmhcs. three potential ctl predicted epitopes of the e6 protein (klpqlctel, kiseyr-hyc, and qqynkplcdl) and three of the e7 protein (ymldlqpet, tlheymldl, and rtledllmgt) were selected. they showed the immunogenicity of these peptides was enhanced when their agonist epitopes were used. the klpqlctel and tlheymldl sequences are the seventh and the fifth predicted epitopes of hpv16-e6 and hpv16-e7 in our study, respectively. experimental evidences about hrhpv-derived epitopes in literatures are mostly limited to e6 and e7 oncoproteins of hpv16 and 18. among our first-step predicted epitopes: fllcfcvll and yiifvyipl from the e5-derived epitopes [109] , fafrdlcivy [110] , cyslygttl [111] , vydfafrdl [111, 112] , kfyskisey [113] , klpqlctel [114] [115] [116] , iseyrhycy [117] , eyrhycysl [111] , klpdlctel [116, [118] [119] [120] , fafkdlfvv [119, 120] and klpdlctel [116, [118] [119] [120] from the e6-derived epitopes, rahynivtf [121] , ledllmgtl [122] , tlheymldl [115, [122] [123] [124] , llmgtlgiv [115, 116, 125, 126] , qaepdrahy [117] , gtlgivcpi [115, 126] , fqqlflntl [127] and tlqdivlhl [119] from the e7-drived epitopes were reported as t-cell epitopes experimentally. besides, ivyrdgnpy, cyslygttl, klpqlctel and iseyrhycy from the e6-derived epitopes, and rahynivtf and gtlgivcpi from the e7-derived epitopes were also reported as hla ligands [128] . others are novel epitopes that they also require experimental studies for validation. as far as we know, this is the first time that in a laborious in silico study for epitope prediction, e5, e6 and e7 oncoproteins of hrhpv16, 18, 31 and 45 have been investigated altogether. moreover, in previous studies, usually only one predictor tool was used for making epitope prediction, or if several tools were used, no integrated approach was employed to make the conclusion. we believed that our predicted epitopes are valuable candidates for further in vitro and in vivo therapeutic vaccine studies. additionally, the introduction of the ten epitopes for each hpv genotype oncoprotein in the first step of the study shows which region of each oncoprotein is rich of the epitope, and thus, is more suitable for use in the design of slps. notably, the previous in vivo studies have been conducted using slps of hrhpv-e6 and/or-e7 oncoproteins, in particular hpv16 oncoproteins [92, [129] [130] [131] [132] [133] . furthermore, the two-step plan of this in silico study, which each step includes several analyses to find proper epitopes by an integrated approach, would provide a basis for rational epitope prediction. however, it could be more efficient by adding other useful analyses. further studies are recommended on the peptide binding assays, the design of polyepitope constructions including e5, e6 and e7 epitopes, the expansion of the minimal ctl epitopes to longer peptides (slps), the use of various adjuvants, involvement of delivery routes, mouse immunization with the designed constructs, evaluation of immune responses such as cytokines, antibodies, ctls and tumor growth for finding the best construct for clinical trials. it is important that improper vaccine design and immunosuppressive microenvironment were known as the main reasons of the failure in cancer immunotherapy by therapeutic cancer vaccines [134] . cross-roads in the classification of papillomaviruses hpv vaccine: current status and future directions the natural history of human papillomavirus infection. best practice & research clinical obstetrics & gynaecology classification of papillomaviruses (pvs) based on 189 pv types and proposal of taxonomic amendments global burden of human papillomavirus and related diseases carcinogenic human papillomavirus infection human papillomavirus molecular biology and disease association tumour virus vaccines: hepatitis b virus and human papillomavirus global burden of cancers attributable to infections in 2008: a review and synthetic analysis. the lancet oncology worldwide burden of cancer attributable to hpv by site, country and hpv type comprehensive control of human papillomavirus infections and related diseases human papillomavirus genome variants human papillomavirus types in 115,789 hpv-positive women: a meta-analysis from cervical infection to cancer human papillomaviruses. iarc monographs on the evaluation of carcinogenic risks to humans e5 protein of human papillomavirus 16 downregulates hla class i and interacts with the heavy chain via its first hydrophobic domain the bovine papillomavirus oncoprotein e5 retains mhc class i molecules in the golgi apparatus and prevents their transport to the cell surface the e6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53 degradation of the retinoblastoma tumor suppressor by the human papillomavirus type 16 e7 oncoprotein is important for functional inactivation and is separable from proteasomal degradation of e7 human papillomavirus and related diseases in the world modeling the mhc class i pathway by combining predictions of proteasomal cleavage, tap transport and mhc class i binding peptide binding predictions for hla dr, dp and dq molecules a systematic assessment of mhc class ii peptide binding predictions and evaluation of a consensus approach immune epitope database and analysis resource (iedb) 3.0. mhc-i processing predictions-tutorial netmhcpan-4.0: improved peptide-mhc class i interaction predictions integrating eluted ligand and peptide binding affinity data prediction of peptide-mhc binding using profiles. immunoinformatics: predicting immunogenicity in silico prediction of mhc class i binding peptides using profile motifs a consensus epitope prediction approach identifies the breadth of murine t(cd8+)-cell responses to vaccinia virus netmhcpan, a method for mhc class i binding prediction beyond humans gapped sequence alignment using artificial neural networks: application to the mhc class i system netmhc-3.0: accurate web accessible predictions of human, mouse and monkey mhc class i affinities for peptides of length 8-11 derivation of an amino acid similarity matrix for peptide: mhc binding and its application as a bayesian prior generating quantitative models describing the sequence specificity of biological processes with the stabilized matrix method quantitative peptide binding motifs for 19 human and mouse mhc class i molecules derived using positional scanning combinatorial peptide libraries the pickpocket method for predicting binding specificities for receptors based on receptor pocket similarities: application to mhc-peptide binding netmhccons: a consensus method for the major histocompatibility complex class i predictions pan-specific prediction of peptide-mhc class i complex stability, a correlate of t cell immunogenicity information on syfpeithi. institute for cell biology-department of immunology-heidelberg accurate pan-specific prediction of peptide-mhc class ii binding affinity with improved binding core identification nn-align. an artificial neural network-based alignment algorithm for mhc class ii peptide binding prediction prediction of mhc class ii binding affinity using smm-align, a novel stabilization matrix alignment method generation of tissue-specific and promiscuous hla ligand databases using dna microarrays and virtual hla class ii matrices identifying mhc class i epitopes by predicting the tap transport efficiency of epitope precursors properties of mhc class i presented peptides that enhance immunogenicity predicting population coverage of tcell epitope-based diagnostics and vaccines allele frequency net 2015 update: new features for hla epitopes, kir and disease and hla adverse drug reaction associations modeling of proteinpeptide interactions using the cabs-dock web server for binding site search and flexible docking cabs-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site human papillomavirus type 16 e5 protein as a therapeutic target recombinant adeno-associated virus expressing human papillomavirus type 16 e7 peptide dna fused with heat shock protein dna as a potential vaccine for cervical cancer immunotherapy of established (pre) malignant disease by synthetic long peptide vaccines more than one reason to rethink the use of peptides in vaccine design identification and characterization of enhancer agonist human cytotoxic t-cell epitopes of the human papillomavirus type prediction of mhc-peptide binding: a systematic and comprehensive overview prediction of epitope-based peptides for vaccine development from coat proteins gp2 and vp24 of ebola virus using immunoinformatics cd8+ ctl priming by exact peptide epitopes in incomplete freund's adjuvant induces a vanishing ctl response, whereas long peptides induce sustained ctl reactivity identification of immunotherapeutic epitope of e5 protein of human papillomavirus-16: an in silico approach computational prediction of linear b-cell epitopes in the e5 oncoprotein of the human papillomavirus type 16 using several bioinformatics tools multi epitope peptide vaccine prediction against sudan ebola virus using immuno-informatics approaches a systematic bioinformatics approach for selection of epitope-based vaccine targets a novel multi-epitope peptide vaccine against cancer: an in silico approach epitope-based vaccine target screening against highly pathogenic mers-cov: an in silico approach applied to emerging infectious diseases advances in peptide-based human papillomavirus therapeutic vaccines therapeutic vaccination with papillomavirus e6 and e7 long peptides results in the control of both established virus-induced lesions and latently infected sites in a pre-clinical cottontail rabbit papillomavirus model vaccination with cytotoxic t lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16-transformed cells cytotoxic t lymphocytes raised against a subdominant epitope offered as a synthetic peptide eradicate human papillomavirus type 16-induced tumors scheme for ranking potential hla-a2 binding peptides based on independent binding of individual peptide side-chains cytotoxic t-lymphocyte responses to human papillomavirus type 16 e5 and e7 proteins and hla-a*0201-restricted t-cell peptides in cervical cancer patients hla class i binding promiscuity of the cd8 t-cell epitopes of human papillomavirus type 16 e6 protein novel oligomannose liposome-dna complex dna vaccination efficiently evokes anti-hpv e6 and e7 ctl responses identification of an hla-a24-restricted cytotoxic t lymphocyte epitope from human papillomavirus type-16 e6: the combined effects of bortezomib and interferon-gamma on the presentation of a cryptic epitope identification of human papillomavirus 16-e6 protein-derived peptides with the potential to generate cytotoxic t-lymphocytes toward human leukocyte antigen-a24+ cervical cancer human papillomavirus 16 e6-specific cd45ra+ ccr7+ high avidity cd8+ t cells fail to control tumor growth despite interferon-gamma production in patients with cervical cancer a conserved e7-derived cytotoxic t lymphocyte epitope expressed on human papillomavirus 16-transformed hla-a2+ epithelial cancers immunization with a poly (lactide co-glycolide) encapsulated plasmid dna expressing antigenic regions of hpv 16 and 18 results in an increase in the precursor frequency of t cells that respond to epitopes from hpv 16, 18, 6 and 11 identification in humans of hpv-16 e6 and e7 protein epitopes recognized by cytolytic t lymphocytes in association with hla-b18 and determination of the hla-b18-specific binding motif up-regulation of hla class-i antigen expression and antigen-specific ctl response in cervical cancer cells by the demethylating agent hydralazine and the histone deacetylase inhibitor valproic acid human t-cell responses to hla-a-restricted high binding affinity peptides of human papillomavirus type 18 proteins e6 and e7 synthetic peptides of human papillomavirus type 18 e6 harboring hla-a2.1 motif can induce peptide-specific cytotoxic t-cells from peripheral blood mononuclear cells of healthy donors establishment of an hla-a*0201 human papillomavirus type 16 tumor model to determine the efficacy of vaccination strategies in hla-a*0201 transgenic mice different methods of identifying new antigenic epitopes of human papillomavirus type 16 e6 and e7 proteins naturally processed and hla-b8-presented hpv16 e7 epitope recognized by t cells from patients with cervical cancer human ctl epitopes encoded by human papillomavirus type 16 e6 and e7 identified through in vivo and in vitro immunogenicity studies of hla-a* 0201-binding peptides dendritic cell-based tumor vaccine for cervical cancer ii: results of a clinical pilot study in 15 individual patients human ctl epitopes encoded by human papillomavirus type 16 e6 and e7 identified through in vivo and in vitro immunogenicity studies of hla-a*0201-binding peptides identification of a naturally processed hla-a*0201 hpv18 e7 t cell epitope by tumor cell mediated in vitro vaccination role of hla-a motifs in identification of potential ctl epitopes in human papillomavirus type 16 e6 and e7 proteins prospects of combinatorial synthetic peptide vaccine-based immunotherapy against cancer experience with synthetic vaccines for cancer and persistent virus infections in nonhuman primates and patients mechanisms of peptide vaccination in mouse models: tolerance, immunity, and hyperreactivity therapeutic vaccination against human papilloma virus induced malignancies immunologic treatments for precancerous lesions and uterine cervical cancer therapeutic cancer vaccines the authors sincerely thank dr. ali namvar and miss elnaz agi for their valuable guidance and comments during preparation of the paper. key: cord-001213-gxqufddb authors: butt, azeem mehmood; nasrullah, izza; tong, yigang title: genome-wide analysis of codon usage and influencing factors in chikungunya viruses date: 2014-03-04 journal: plos one doi: 10.1371/journal.pone.0090905 sha: doc_id: 1213 cord_uid: gxqufddb chikungunya virus (chikv) is an arthropod-borne virus of the family togaviridae that is transmitted to humans by aedes spp. mosquitoes. its genome comprises a 12 kb single-strand positive-sense rna. in the present study, we report the patterns of synonymous codon usage in 141 chikv genomes by calculating several codon usage indices and applying multivariate statistical methods. relative synonymous codon usage (rscu) analysis showed that the preferred synonymous codons were g/c and a-ended. a comparative analysis of rscu between chikv and its hosts showed that codon usage patterns of chikv are a mixture of coincidence and antagonism. similarity index analysis showed that the overall codon usage patterns of chikv have been strongly influenced by pan troglodytes and aedes albopictus during evolution. the overall codon usage bias was low in chikv genomes, as inferred from the analysis of effective number of codons (enc) and codon adaptation index (cai). our data suggested that although mutation pressure dominates codon usage in chikv, patterns of codon usage in chikv are also under the influence of natural selection from its hosts and geography. to the best of our knowledge, this is first report describing codon usage analysis in chikv genomes. the findings from this study are expected to increase our understanding of factors involved in viral evolution, and fitness towards hosts and the environment. chikungunya virus (chikv), a member of the genus alphavirus of the family togaviridae, is a small (60-70 nm), enveloped, singlestrand positive-sense rna virus. the genome is approximately 12 kb in size and comprises two open reading frames (orfs) encoding non-structural and structural proteins, respectively [1] . the chikv genome is arranged in the order of 5-9cap-nsp1-nsp2-nsp3-nsp4-(junction region)-c-e3-e2-6k-e1-poly(a)-39 [1] . since the first isolation of chikv from a febrile individual in tanzania in 1953 [2] , chikv has caused several outbreaks in asia, africa, and indian ocean islands, emerging as a serious public health concern [3] [4] [5] [6] . chikv infection is characterized by abrupt onset of high fever, headache, rashes, arthralgia and myalgia. the typical clinical sign of the disease is poly-arthralgia, which is a very painful condition affecting joints and may persist for several months to years in some cases [7] . being an arthropodborne virus, the mode of transmission is the mosquitoes of the aedes spp. it is generally accepted that chikv originated from africa, where it is primarily maintained in a yellow fever-like zoonotic sylvatic cycle and depends upon non-human primates and arboreal, peridomestic mosquitoes as reservoir hosts. however, the spread of chikv in asia and urban endemics are associated with a dengue-like ''human-mosquito-human'' direct transmission cycle, where a. aegypti and a. albopuctus serve as primary transmission vectors and humans serve as hosts [7] [8] [9] . the genetic code comprises 64 codons that can be divided into 20 groups, where each group consists of one to six codons, and each group corresponds to each of the standard amino acids. alternative codons within the same group coding for the same amino acid are often termed 'synonymous' codons, although their corresponding trnas might differ in their relative abundance in cells and in the speed by which they are recognized by the ribosome. this redundancy of the genetic code, in which most of the amino acids can be translated by more than one codon, represents a key step in modulating the efficiency and accuracy of protein production, while maintaining the same amino acid sequence of the protein. on the other hand, the synonymous codons are not chosen randomly both within and between genomes, which is referred to as codon usage bias [10, 11] . this phenomenon of synonymous codon usage bias has been studied in a wide range of organisms, from prokaryotes to eukaryotes and viruses [12] [13] [14] [15] [16] [17] . studies on codon usage have determined several factors that could influence codon usage patterns, including mutational pressure, natural or translational selection, secondary protein structure, replication and selective transcription, hydrophobicity and hydrophilicity of the protein and the external environment. among these, the major factors responsible for codon usage variation among different organisms are considered to be compositional constraints under mutational pressure and natural selection [12, [18] [19] [20] . previous studies on codon usage in different viruses have highlighted mutational pressure as the major factor in shaping codon usage patterns compared with natural selection [12, [21] [22] [23] ; however, as our understanding of codon usage increases, it appears that although mutational pressure is still a major driving force, it is certainly not the only one when considering different types of rna and dna viruses [24] [25] [26] [27] . considering their comparatively small genome size and other viral features, such as dependence on host's machinery for key process including replication, protein synthesis and transmission in comparison with prokaryotic and eukaryotic genomes, the interplay of codon usage among viruses and their hosts is expected to affect overall viral survival, fitness, evasion from host's immune system and evolution [15, 28] . therefore, knowledge of the codon usage in viruses can not only reveal information about molecular evolution, but also improve our understanding of the regulation of viral genes expression and aid vaccine design, where the efficient expression of viral proteins may be required to generate immunity. in the present study, we report the detailed codon usage data and analysis of various factors shaping the codon usage patterns in chikv genomes. codon usage bias, or preference for one type of codon over another, can be influenced greatly by the overall nucleotide composition of genomes [21] . therefore, we first analyzed the nucleotide composition of coding sequences from chikv genomes. as shown in table 1 , the mean a% (28.91) was the highest, followed by similar composition of g% (25.75) and c% (25.19) , with the u% being the lowest (20.16) . the mean gc and au compositions were 50.91% and 49.06% respectively. this appears to suggests there might be equal or almost equal distribution of a, u, g, and c nucleotides among codons of chikvs, with potentially more preference towards a-ended codons followed by g/c-ended codons. however, a clearer picture of overall nucleotide composition that could influence the codon usage preference in chikv genomes emerged from the analysis of the nucleotide composition of the third position of codons (a 3 , u 3 , g 3 , c 3 ) and of gc 1 , gc 1,2 , gc 3 and au 3 ( table 1) . the mean c 3 and g 3 were the highest, followed by a 3 and u 3 . the gc 3 values ranged from 54.9% to 57.2%, with a mean of 55.86% and a standard deviation (sd) 0.40 compared with that of au 3 , whose values ranged from 42.8% to 45.1%, with a mean of 44.14% and an sd of 0.41. the gc 1 ranged from 50.6% to 53.8%, with a mean of 53.56% and an sd 0.27. the gc 1,2 values ranged from 48.2% to 48.7%, with an average of 48.45% and an sd of 0.07. therefore, from the initial nucleotide composition analysis, it is expected that g/c-ended codons might be preferred over a/u-ended codons in chikv genomes. to determine the patterns of synonymous codon usage and to what extent g/c-ended codons might be preferred, we performed rscu analysis and calculated the rscu values. among the 18 most abundantly used codons in chikv genomes, eleven (uuc, cug, auc, gug, ccg, uac, ugc, cac, cag, aac and gac) were g/c-ended (c-ended: 7; g-ended: 4) and the remaining seven (aca, gca, uca, aga, aaa, gaa, gga) were a-ended codons; none of the preferred codons were u-ended ( figure 1a and table 2) . from rscu analysis, we observed that chikv exhibits comparatively higher codon usage bias towards g/c-and less towards a-ended codons. however, it is also interesting to note that the mean gc% and au% values are very similar (table 1 ), yet the g/c-ending codons were used in a comparatively biased manner, indicating that the g/c content at the third position of the codons influenced the shaping of the overall synonymous codons usage patterns. the overall general trend of the 59 synonymous codon usages was also relatively consistent among different genotypes of chikv, indicating that the evolutionary processes of the three genotypes of chikv are restricted by the synonymous codon usage pattern to some extent ( figure 1b and table 2 ). furthermore, analysis of over-and underrepresented codons showed that codons with an rscu.1.6 are infrequently observed in chikv genomes. the rscu values of the majority of preferred and non-preferred codons fell between 0.6 and 1.6. we further divided the rscu data into three groups; (a) codons with rscu,0.6 (under-represented), (b) codons with rscu values between 0.6 and 1.6 (unbiased/randomly represented), and (c) codons with rscu values .1.6 (over-represented). among 59 codons, only cug (leu) and aga (arg) had an rscu.1.6. however, the under-represented codons (rscu,0.6), were identified as follows: cuu, cuc for leu, guu for val, and cgu, cgg for arg. the remaining 52 codons had rscu values between 0.6-1.6 ( figure 1 and table 2 ). these findings suggested that despite being an rna virus with a high mutation rate in its lifecycle, chikv has evolved to form a relatively stable genetic composition at some specific levels of synonymous codon usage. this was further confirmed by enc and cai analysis as discussed in coming sections. combining nucleotide composition and rscu analysis, we deduced that the selection for preferred codons has been mostly influenced by compositional constraints, which also accounts for the presence of mutational pressure. however, we suspect that the compositional constraints may not be the sole factor associated with codon usage patterns in chikv, because although the overall rscu values could reveal the codon usage pattern for the genomes, it may hide the codon usage variation among different genes in a genome [29] . to quantify the extent of variation in codon usage among different genomes of chikv arising from different geographical regions and genotypes, the enc values for each genome were calculated. the enc values among chikv genomes ranged from 54.55 to 56.41, with a mean of 55.56 and an sd of 0.34 (table 1 ). an average value of 55.56 (enc.40) represents stable enc values and indicates a relatively conserved genomic composition among different chikv genomes. in general, there is an inverse relationship between enc and gene expression; i.e., a lower enc value indicates a higher codon usage preference and higher gene expression and vice versa [30] . our results show that the overall codon usage bias and gene expression among different chikv genomes is lower, slightly biased and would be mainly affected by the base composition. previous studies on codon usage analysis among other rna viruses, such as bovine viral diarrhea virus (enc: 50.91) [22] , classical swine fever virus (enc = 51.7) [17] and hcv (enc = 52.62) [31] , have also reported lower codon usage bias. the same is also true in the case of arthropodborne rna viruses, including west nile virus (enc: 53.81) [15] and dengue virus (denv) (enc: 49.70: denv-1; 48.78: denv-2; 49.52: denv-3; and 50.81: denv-4) [14] . a possible explanation for the weak codon bias of rna viruses is that it might be advantageous for efficient replication in host cells, with potentially distinct codon preferences [21] . the codon adaptation index (cai) is often used as measure of level of gene expression and to assess the adaptation of viral genes to their hosts. highly expressed genes exhibit a strong bias for particular codons in many bacteria and small eukaryotes. in comparison to the enc, which is another way of calculating codon usage bias and measures deviation from a uniform bias (null hypothesis), cai measures the deviation of a given protein coding gene sequence with respect to a reference set of genes [32] . here, being parasitic organisms, it can be expected that the codon usage patterns of viruses would be affected by its hosts to some extent [33] . for instance, the codon usage pattern of poliovirus is reported to be mostly coincident with that of its host [34] , while the codon usage pattern of hepatitis a was reported to be antagonistic to that of its host [35] . we therefore computed and compared the codon usage of chikv with its two hosts (homo sapiens and pan troglodytes), and transmission vectors (a. aegypti and a. albopictus). the results showed that the codon usage patterns of chikv were a mixture of coincidence and antagonism to its hosts and vectors (table 2 ). in detail, the preferred codons for 12 out of 18 amino acids were common between chikv and h. sapiens. this included uuc (phe), cug (leu), auc (ile), gug (val), uac (tyr), aga (arg), ugc (cys), cac (his), cag (gln), aac (asn), aag (lys) and gac (asp). furthermore, all common preferred codons between chikv and h. sapiens were g/cended (c-ended: 7; g-ended: 4), with exception of an a-ended preferred codon for amino acid arg. similarly, preferred codons for 10 out of 18 amino acids were common between chikv and p. troglodytes. in case of the two transmission vectors, 10 out of 18 preferred codons were common among both mosquito species and chikv. it is also interesting to note that, except for amino acid arg, the remaining 10 highly preferred codons were same among chikv, h. sapiens, a. aegypti and a. albopictus. moreover, the preferred codon usage profiles of a. aegypti and a. albopictus were also very similar: 16 out of 18 preferred codons were common between, with exceptions for the preferred codons for asp and gly ( table 2 ). these results indicated that selection pressures from hosts and vectors have influenced the codon usage pattern of chikv and the possible fitness of the virus to adjust among its dynamic range of hosts and vectors. a mixture of coincidence and antagonism has also been reported previously in the case of hcv [31] and enterovirus 71 [13] . it was suggested that the coincident portions of codon usage among viruses and their hosts could enable the corresponding amino acids to be translated efficiently, while the antagonistic portions of codon usage may enable viral proteins to be folded properly, although the translation efficiency of the corresponding amino acids might decrease [31] . although the comparative analysis of individual rscu values as given above is frequently employed as a method of estimating the effect of synonymous codons usage of the hosts on that of specific viruses, it has its limitations in revealing the effect of the overall codon usage of the hosts on the formation of codon usage patterns of the viruses. therefore, we took advantage of a method proposed recently that estimates the similarity degree of the overall codon usage patterns comprehensively between viruses and their hosts by treating the 59 synonymous codons as 59 different spatial vectors. the advantage of this formula, as reported by the authors in the case of dengue viruses, is that the comparative overall codon usage takes the place of the direct estimation of each synonymous codon usage; thus, the new method avoids the situation that the variations of 59 synonymous codon usage confuse the correct estimation of the effect of the host on the virus for codon usage [36] . the similarity index d(a,b) was therefore calculated for each genotype of chikv in relation to its hosts and vectors. the similarity index was found to be highest for a. albopictus vs. chikv group followed by p. troglodytes vs. chikv, a. aegypti vs. chikv and lowest in the case of h. sapiens vs. chikv (figure 2) , indicating that the effect of a. albopictus and p. troglodytes on the formation of the overall codon usage patterns of chikv is relatively higher than that of the a. aegypti and h. sapiens. secondly, we computed the effect of transmission vectors on the formation of the overall codon usage patterns of three genotypes of chikv. a. aegypti had the strongest effect on the east central south african (ecsa) genotype, followed by west african (wa) and asian genotypes. in the case of a. albopictus, the strongest effect was noted on the ecsa genotype, followed by asian and wa genotypes. as for the effects of the two primates on the formation of the overall codon usage of chikv, the strongest effect of h. sapiens was on the asian genotype, closely followed by the ecsa and wa genotypes. by contrast, p. troglodytes had its strongest and equal effect on ecsa and asian genotypes, followed by wa genotype (figure 2 ). therefore, from the similarity index analysis, we observed that selection pressure from hosts and vectors have contributed to shaping the molecular evolution of chikv at the respectively, on the formation of the overall codon usage patterns of chikv (figure 2 ). the stronger effect of p. troglodytes than h. sapiens could also be attributed to the maintenance of chikv in a yellow fever-like zoonotic sylvatic cycle and its dependence upon non-human primates as reservoir hosts [7, 9] . moreover, the similarity index of codon usage was also the highest between chikv and a. albopictus, as compared with a. aegypti, p. troglodytes and h. sapiens. the successful human-to-human transmission of chikv depends on aedes mosquitoes [7, 9] ; therefore, the stronger effect of a. albopictus on all three genotypes of chikv suggests that this vector might be a more efficient reservoir for viral replication and transmission compared with a. aegypti. these results are in agreement with recent studies showing more efficient dissemination and transmission of chikv by a. albopictus, which contribute to its ongoing re-emergence in a series of large-scale epidemics [37, 38] . correspondence analysis (coa). codon usage is multivariate by its very nature; therefore, it is necessary to analyze the data using multivariate statistical techniques, such as coa [39] . therefore, to determine the trends in codon usage variation among different chikv genomes, we performed coa on the rscu values, which were examined as a single dataset based on the rscu value of each coding region (figure 3 ). the first principal axis (f9 1 ) accounted for 53.57% of the total variation, and the next three axes (f9 2 2f9 4 ) accounted for 25.16%, 7.62%, and 2.06% of the total variation in synonymous codon usage, respectively. for further analysis, plots were reconstructed based on different geographical locations ( figure 4 ) and genotypes of chikv isolates ( figure 5 ). as expected the chikv isolates belonging to ecsa genotype were distributed across all planes of axes. when these plots were accessed on regional basis, it was found that different genotypes are circulating in single country. this analysis showed that the three different genotypes of chikv might have common ancestor. this further implies that the geographical diversity and associated factors, such as presence of favorable transmission vectors, climate features, host range and susceptibility, have also contributed to shaping the molecular evolution and codon usage in chikv, even though it appears to be less influential than mutational pressure (based on the current analysis). effect of mutational pressure in shaping the codon usage patterns in chikv. mutational pressure and natural selection are considered the two major factors that shape codon usage patterns [40] . a general mutational pressure, which affects the whole genome, would certainly account for the majority of the codon usage among certain rna viruses [21] . to determine the extent of the influence of these two factors on chikv codon usage, we performed correlation analysis between different nucleotide constraints. a complex correlation was observed among different nucleotide constraints (table 3 ). u 3 % had a significant positive correlation with u% (r = 0.621, p,0.01) and g% (r = 0.185, p,0.05), whereas it had significant negative correlations with c% (r = 20.606, p,0.01) a% (r = 20.278, p,0.01) and gc% (r = 20.806, p,0.01). c 3 % had significant positive correlation with c% (r = 0.621, p,0.01), a (r = 0.261, p,0.01) and gc% (r = 0.798, p,0.01), and negative correlations with u% (r = 20.5877, p,0.01) and g% (r = 20.217, p,0.01). a 3 % had positive correlations with a (r = 0.625, p,0.01), c% (r = 0.327, p,0.01) and negative correlations with u% (r = 20.373, p,0.01) and g% (r = 20.576, p,0.01), whereas no correlation was observed between a 3 % and the gc%. g 3 % was positively correlated with g% (r = 0.658, p,0.01) u% (r = 0.354, p,0.01), and negatively correlated with c% (r = 20.377, p,0.01) and a% (r = 20.610, p,0.01); the correlation with the gc% was nonsignificant. in the case of gc 3 %, positive correlation was noted with c% (r = 0.498, p,0.01) and gc% (r = 0.852, p,0.01), and negative correlation with u% (r = 20.480, p,0.01); the correlation with g% was non-significant. finally the gc and gc 12 were also compared with gc 3 and a highly significant positive correlations (r = 0.28, p,0.01; gc 12 versus gc 3 ) (r = 0.85, p,0.01; gc versus gc 3 ) was observed as shown in figure 6a and 6b respectively. furthermore, a significant negative correlation between gc 3 and enc values was also observed (r = 20.756, p,0.01). this analysis collectively indicates that mutational pressure is most likely responsible for the patterns of nucleotide composition and, therefore, codon usage patterns, because the effects were present at all codon positions. in addition to correlation analysis, linear regression analysis was also performed to determine correlations between the first two principle axes (f9 1 and f9 2 ) and nucleotide constraints of chikv genomes. again, several significant correlations were observed between the two principle axes and nucleotide contents ( table 4) . figure 7a ) and gc% (r = 20.21, p,0.01). in the case of f 2 , a 3 %, g 3 % and c% had non-significant correlations. f9 2 axis showed significantly positive correlations with c 3 (r = 0.69, p,0.01), gc 3 % (r = 0.74, p,0.01; figure 7b ), gc% (r = 0.64, p,0.01), a% (r = 0.17, p,0.05) and g% (r = 0.39, p,0.01) whereas, negative correlations with u 3 % (r = 20.66, p,0.01), and u% (r = 20.34, p,0.01) ( table 4 ). our analysis shows that mutational pressure has played a major role in shaping the dynamics of codon usage patterns within chikv genomes. correlation analysis between enc and gc 3 values. a plot of enc versus gc 3 (nc plot) is widely used to study codon usage variation among genes in different organisms. it has been postulated that an enc-plot of genes, whose codon choice is constrained only by a g 3 + c 3 mutational bias, will lie on or just below the continuous curve of the predicted enc values [30] . although, the nucleotide composition correlation analysis showed that codon usage in chikv genomes is mainly caused by compositional constraints or mutational pressure, we were interested to determine the possible influence of other factors, such as natural selection. therefore, we constructed a corresponding relation distribution plot between the enc and gc 3 values. as table 3 . summary of correlation analysis between nucleotide constraints in chikv genomes. shown in figure 8 , all points aggregated closely towards the right side under the expected enc curve, indicating that, apart from mutation pressure, the codon usage patterns have also been influenced by other factors to some extent. relationship between dinucleotide and codon usage patterns in chikv. it has been suggested that dinucleotide bias can affect overall codon usage bias in several organisms, including dna and rna viruses [41] [42] [43] . to study the possible effect of dinucleotides on codon usage in chikv genomes, we calculated the relative abundances of the 16 dinucleotides from the coding sequences of chikv. the occurrences of dinucleotides were not randomly distributed, and no dinucleotides were present at the expected frequencies (table 5) . under-representation of cpg dinucleotides in different rna and dna viruses has been reported [41] . in the case of chikv, the relative abundance of cpg showed deviation from the ''normal range'' (mean 6 (table 2) . on the other hand, despite slight overrepresentation of the gpc dinucleotide, all gpc-containing codons were also under-represented (rscu,1.6) and were not preferred codons for their respective amino acids, with two exceptions; gca (ala, rscu = 1.43) and ugc (cys, rscu = 1.33) ( table 2 ). it has been proposed that cpg deficiency in pathogens is associated with the immunostimulatory properties of unmethylated cpgs, which are recognized by the host's innate immune system as a pathogen signature [28] . recognition of umethylated cpgs by toll like receptor 9 (tlr9), a type of intracellular pattern recognition receptor (prr), leads to activation of several immune response pathways [44] . the vertebrate immune system relies on unmethylated cpg recognition in dna molecules as a signature of infection, and cpg under-representation in rna viruses is exclusively observed in vertebrate viruses; therefore, it is reasonable to suggest that a tlr9-like mechanism exists in the vertebrate immune system that recognizes cpgs when in an rna context (such as in the genomes of rna viruses) and triggers immune responses [45] . compared with differential (over-and under-) representation of cpgs in different organisms, upa under-representation also exists in several organisms, including vertebrates, invertebrates, plants and prokaryotes [41] . the presence of tpa in two out of three canonical stop codons and in transcriptional regulatory motifs (e.g., the tata box sequence) is believed to be responsible for its under-representation. therefore, upa under-representation is expected to reduce the risk of nonsense mutations and minimizes improper transcription [43, 46] . in the case of chikv, the relative abundance of upa also deviated from the ''normal range'' (mean 6 sd = 0.85960.022) and was under-represented, similarly to cpg. the six codons containing upa (uua, cua, gua, uau, uac and aua) were also under-represented (rscu,1.6) and were not preferred codons for their respective amino acids. the cpa (mean 6 sd = 1.12560.017) and upg (mean 6 sd = 1.27560.022) dinucleotides were over-represented compared with the rest of the 14 dinucleotide pairs (table 5) . similarly, the eight codons containing cpa (uca, cca, aca, gca, caa, cag, cau and cac) and five codons containing upg (uug, cug, gug, ugu and ugc) were also overrepresented compared with the rest of the codons for their respective amino acids and a majority of them were also preferential codons for their respective amino acids, based on rscu analysis ( table 2 ). over-representation of cpa and upg in different organisms has been observed and is regarded as a consequence of the under-representation of cpg dinucleotides. one possible explanation is that methylated cytosines are prone to mutate into thymines through spontaneous deamination, resulting in the dinucleotide tpg and the subsequent presence of a cpa on the opposite strand after dna replication [47] . however, this theory cannot explain under-representation of cpgs in rna viruses. moreover, under-representation of cpgs has also been observed in several vertebrate viruses, where it is independent of their genomic composition and replication cycles. recently, two studies performed large-scale dinucleotide analyses in different viruses and suggested that the cpg usage of +ssrna viruses is affected greatly by their hosts. as a result, most +ssrna viruses mimic their hosts' cpg usage and the existence of an rna dinucleotide recognition system, probably linked to the innate immune system of the host, has also been proposed [41, 48] . finally, the relative abundance of dinucleotides was also correlated with the first two principal axes. among the 16 dinucleotides, 11 significantly (positive and negative) correlated with the first axis and 16 significantly (positive and negative) correlated with the second axis (table 5 ). these observations indicated that the composition of dinucleotides determines the variation in synonymous codon usage. therefore, from the present dinucleotide composition analysis, it is evident that selection pressure associated with (i) maintenance of efficient replication and transmission cycles among multiple hosts, and (ii) evolution of escape mechanisms to evade from the host antiviral responses, have contributed to shaping the overall synonymous codon usage in chikv. effect of natural selection in shaping the codon usage patterns in chikv. it has been suggested that if synonymous codon usage bias is affected by mutational pressure alone, then the frequency of nucleotides a and u/t should be equal to that of c and g at the synonymous codon third position [26] . however, in case of chikv genomes, variations in nucleotide base compositions were noted (table 1 ), indicating that other factors, such as natural selection, could also influence overall synonymous codon usage bias. as the role of natural selection is also evident from previous codon usage analysis studies in several viruses [25, 26, 49] , we were interested to determine to what extent natural selection might be involved in the codon usage patterns of chikv. for this purpose, we computed the gravy and aromaticity (aro) values for each chikv isolate (table s1 ) and a linear regression analysis was performed between gravy, aro and the f9 1 , f9 2 , enc, gc and gc 3 values. the analysis results showed that the gravy values were not significant for f9 1 and were highly significant for f9 2 , enc, gc 3 and gc. in the case of aro, an opposite trend was observed: aro values were significantly negatively correlated with f9 1 and correlations with f9 2 , enc, gc 3 and gc were not significant ( table 6 ). these results indicated that, although natural selection has influenced codon usage of chikv genomes to some extent, it is much weaker compared with mutational pressure. taken together, our analysis showed that overall codon usage bias in chikv is slightly biased, and the major factor that has contributed to shaping codon usage pressure is mutational pressure. in addition, contributions of other factors, including hosts, geography, dinucleotides composition and natural selection, are also evident from our analysis. our data suggested that codon usage in chikv is undergoing an evolutionary process, probably reflecting a dynamic process of mutation and natural selection to re-adapt its codon usage to different environments and hosts. to the best our knowledge, this is first report of codon usage analysis in chikv and is expected to deepen our understanding of the mechanisms contributing towards codon usage and evolution of chikv. the complete genome sequences of 141 chikv isolates (in fasta format) were obtained from the national center for biotechnology (ncbi) genbank database (http://www.ncbi.nlm. nih.gov). the accession numbers and other detailed information of the selected chikvs' genomes, such as isolation date, isolation place, host and genome size were also retrieved ( table 7) . the following compositional properties were calculated for the chikv genomes; (i) the overall frequency of occurrence of the nucleotides (a %, c %, u/t %, and g %); (ii) the frequency of each nucleotide at the third site of the synonymous codons (a 3% , c 3% , u 3% and g 3% ); (iii) the frequencies of occurrence of nucleotides g+c at the first (gc 1 ), second (gc 2 ), and third synonymous codon positions (gc 3 ); (iv) the mean frequencies of nucleotide g+c at the first and the second position (gc 1,2 ); and (v) the overall gc and au content. the codons aug and ugg are table 5 . summary of correlation analysis between the first two principal axes and relative abundance of dinucleotides in chikv genomes. the rscu values for all the coding sequences of chikv genomes were calculated to determine the characteristics of synonymous codon usage without the confounding influence of amino acid composition and the size of coding sequence of different gene samples, following a previously described method [18] . the rscu index was calculated as follows: where g ij is the observed number of the ith codon for the jth amino acid which has n i kinds of synonymous codons. rscu values represent the ratio between the observed usage frequency of one codon in a gene sample and the expected usage frequency in the synonymous codon family given that all codons for the particular amino acid are used equally. the synonymous codons with rscu values .1.0 have positive codon usage bias and were defined as abundant codons, while those with rscu values ,1.0 have negative codon usage bias and were defined as less-abundant codons. when the rscu values is 1.0, it means there is no codon usage bias for that amino acid and the codons are chosen equally or randomly [50] . moreover, the synonymous codons with rscu values .1.6 and ,0.6 were treated as over-represented and under-represented codons, respectively [23] . for the comparative analysis of codon usage between chikvs and its vectors and hosts; codon usage data for two transmission vectors (a. aegypti, a. albopictus), and hosts (h. sapiens, p. troglodytes) were obtained from the codon usage database (http://www. kazusa.or.jp/codon/) [51] . zhou et al. proposed a method recently to determine the potential impact of the overall codon usage patterns of the hosts in the formation of the overall codon usage of viruses [36] . here, we applied the same approach in case of chikv and the similarity index d(a,b) was calculated as follows: where r(a,b) is defined as a cosine value of an included angle between a and b spatial vectors representing the degree of similarity between chikv and a specific host at the aspect of the overall codon usage pattern, a i is defined as the rscu value for a specific codon among 59 synonymous codons of chikv coding sequence, b i is termed as the rscu value for the same codon of the host. d (a,b) represents the potential effect of the overall codon usage of the host on that of chikv, and its value ranges from zero to 1.0 [36] . the relative abundance of dinucleotides in the coding regions of chikv genomes was calculated using a previously described method [43] . a comparison of actual and expected dinucleotide frequencies of the 16 dinucleotides in coding regions of the chikv was also undertaken. the odds ratio was calculated using the following formula: p xy~f xy f y f x where f x denotes the frequency of the nucleotide x, f y denotes the frequency of the nucleotide y, f y f x the expected frequency of the dinucleotide xy and f xy the frequency of the dinucleotide xy, etc,. for each dinucleotide were calculated. as a conservative criterion, for pxy.1.23 (or ,0.78), the xy pair is considered to be over-represented (or under-represented) in terms of relative abundance compared with a random association of mononucleotides. the cai is used as a quantitative method of predicting the expression level of a gene based on its codon sequence. the cai value ranges from 0 to 1. the most frequent codons simply have the highest relative adaptiveness values, and sequences with higher cais are preferred over those with lower cais [32] . the enc is used to quantify the absolute codon usage bias of the gene (s) of interest, irrespective of gene length and the number of amino acids [30] . in this study, this measure was calculated to evaluate the degree of codon usage bias exhibited by the coding sequences of chikvs. the enc values ranged from 20 for a gene showing extreme codon usage bias using only one of the possible synonymous codons for the corresponding amino acid, to 61 for a gene showing no bias using all possible synonymous codons equally for the corresponding amino acid. the larger the extent of codon preference in a gene, the smaller the enc value is. it is also generally accepted that genes have a significant codon bias when the enc value is less than or equal to 35 [30, 52] . the enc was calculated using the following formula: where f k (k = 2,3,4,6) is the mean of f k values for the k-fold degenerate amino acids, which is estimated using the formula as follows: where n is the total number of occurrences of the codons for that amino acid and s~x k i~1 n i n 2 , where n i is the total number of occurrences of the i th codon for that amino acid. genes, whose codon choice is constrained only by a mutation bias, will lie on or just below the curve of the expected enc values. therefore, for elucidating the relationship between gc 3 and enc values, the expected enc values for different gc 3 were calculated as follows: enc expected~2 zsz 29 s 2 z(1{s 2 ) where s represents the given gc 3 % value [30] . coa is a multivariate statistical method that is used to explore the relationships between variables and samples. in the present study, coa was used to analyze the major trends in codon usage patterns among chikvs coding sequences. coa involves a mathematical procedure that transforms some correlated variable (rscu values) into a smaller number of uncorrelated variables called principal components. to minimize the effect of amino acid composition on codon usage, each coding sequence was represented as a 59 dimensional vector, and each dimension corresponded to the rscu value of each sense codon, which only included several synonymous codons for a particular amino acid, excluding the codons aug, ugg and the three stop codons. correlation analysis was carried out to identify the relationship between nucleotide composition and synonymous codon usage patterns of chikv. this analysis was implemented based on the spearman's rank correlation analysis. all statistical processes were carried out using the statistical software spss 16.0 for windows. table s1 hydrophobicity (gravy) and aromaticity (aro) indices in chikv genomes. (docx) ind-ka51 fj000068 11812 2006 human india ecsa 50 ind-mh51 fj000067 11812 2006 human india ecsa 52 ind-gj53 fj000065 11813 2006 human india ecsa 53 ind-kr51 fj000066 11812 2006 human india ecsa 54 ind-gj51 fj000064 11807 2006 human india ecsa 55 ind-06-guj jf274082 11829 2006 human india ecsa 56 ind-ka52 fj000063 11812 2006 human india ecsa 57 rgcb05/kl06 gq428211 11764 2006 human india ecsa 58 rgcb03/kl06 gq428210 11764 2006 human india ecsa 59 chik31 eu564335 11810 2006 human india ecsa 60 sl10571 ab455494 11829 2006 human -ecsa 61 sl11131 ab455493 11829 2006 human -ecsa 62 ind-06-ka15 ef027135 11729 lk(pb)ch5808 fj513637 11710 2008 human sri lanka ecsa 89 lk(pb)ch3008 fj513632 11693 2008 human sri lanka ecsa 90 lk(pb)ch1608 fj513629 11716 2008 human sri lanka ecsa 91 lk(pb)ch5308 fj513635 11726 2008 human sri lanka ecsa 92 lk(pb)chik6008 gu013529 11718 2008 human sri lanka ecsa 93 lk(pb)ch1008 fj513628 11722 2008 human sri lanka ecsa 94 lk(pb)chik3408 gu013528 11715 2008 human sri lanka ecsa 95 lk(eh)ch6708 fj513654 11717 2008 human sri lanka ecsa 96 lk(eh)ch7708 fj513657 11696 2008 human sri lanka ecsa 97 lk(eh)ch4408 fj513645 11714 2008 human sri lanka ecsa 98 lk(eh)ch20108 fj513679 11717 2008 human sri lanka ecsa 99 lk(eh)ch18608 fj513675 11716 2008 human sri lanka ecsa 100 lk(eh)chik19708 gu013530 11714 2008 human sri lanka ecsa lkehch13908 fj445426 11717 nc/2011-568 he806461 11621 2011 human new caledonia ecsa 131 v0603310_kh11_btb jq861260 11743 2011 human cambodia ecsa 132 v1024311_kh11_pvh jq861256 11754 2011 human cambodia ecsa 133 v1024308_kh11_pvh jq861254 11750 2011 human cambodia ecsa 134 v1024314_kh11_pvh jq861258 11733 2011 human cambodia ecsa 135 v1024306_kh11_pvh jq861253 11745 2011 human cambodia ecsa 136 v1024310_kh11_pvh jq861255 11736 2011 human cambodia ecsa 137 v1024313_kh11_pvh jq861257 11755 2011 human cambodia east central south african, ecsa; democratic republic of congo, drc; central african republic, car; west african the 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frequency of cpg in animal dna patterns of evolution and host gene mimicry in influenza and other rna viruses optimization of codon usage of poxvirus genes allows for improved transient expression in mammalian cells an evolutionary perspective on synonymous codon usage in unicellular organisms codon usage tabulated from international dna sequence databases: status for the year 2000 an evaluation of measures of synonymous codon usage bias key: cord-000366-u4649rtx authors: shan, tongling; lan, daoliang; li, linlin; wang, chunmei; cui, li; zhang, wen; hua, xiuguo; zhu, caixia; zhao, wei; delwart, eric title: genomic characterization and high prevalence of bocaviruses in swine date: 2011-04-15 journal: plos one doi: 10.1371/journal.pone.0017292 sha: doc_id: 366 cord_uid: u4649rtx using random pcr amplification followed by plasmid subcloning and dna sequencing, we detected bocavirus related sequences in 9 out of 17 porcine stool samples. using primer walking, we sequenced the nearly complete genomes of two highly divergent bocaviruses we provisionally named porcine bocavirus 1 isolate h18 (pbov1-h18) and porcine bocavirus 2 isolate a6 (pbov2-a6) which differed by 51.8% in their ns1 protein. phylogenetic analysis indicated that pbov1-h18 was very closely related to a ∼2 kb central region of a porcine bocavirus-like virus (pbo-likev) from sweden described in 2009. pbov2-a6 was very closely related to the porcine bocavirus genomes pbov-1 and pbov2 from china described in 2010. among 340 fecal samples collected from different age, asymptomatic swine in five chinese provinces, the prevalence of pbov1-h18 and pbov2-a6 related viruses were 45–75% and 55–70% respectively, with 30–47% of pigs co-infected. pbov1-a6 related strains were highly conserved, while pbov2-h18 related strains were more diverse, grouping into two genotypes corresponding to the previously described pbov1 and pbov2. together with the recently described partial bocavirus genomes labeled v6 and v7, a total of three major porcine bocavirus clades have therefore been described to date. further studies will be required to elucidate the possible pathogenic impact of these diverse bocaviruses either alone or in combination with other porcine viruses. members of the parvoviridae family are small non-lipid enveloped viruses with a diameter of 18-26 nm, icosahedral symmetry (t = 1), encoded by a single-stranded linear dna genome of approximately 4,000 to 6,000 nucleotides (nt) [1] . the family includes two subfamilies: densovirinae, parvovirinae. the subfamily of densovirinae contains four genera: densovirus, iteravirus, brevidensovirus and pefudensovirus, which infect only invertebrates [1] . the parvovirinae subfamily is currently subdivided into five genera: parvovirus, erythrovirus, dependovirus (adeno-associated virus), amdovirus, and bocavirus, infecting vertebrates [1] . the bocavirus genus was recently assigned by the international committee on taxonomy of viruses (ictv) [1] to parvovirus genomes containing a third orf (labeled np1) between the ns1 and vp1/vp2 genes [2] . bocaviruses were first identified in bovine and canine [3, 4] , samples from which it derives its genus name [1, 5] . presently, the bocavirus genus contains eight members: bovine parvovirus, canine minute virus (cnmv), human bocavirus 1-4 (hbov1-4), a gorilla bocavirus and a partially sequenced chimpanzee bocavirus [1, 6, 7] . the first human bocavirus (hbov) was found in the nasopharyngeal secretion of a child with respiratory problems using a methodology closely related to that used here [8] . hbov has been associated with lower respiratory tract symptoms and possibly diarrhea [5, [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] , and shows a very low degree of genetic variability worldwide [6, 23, 24] . hbov2 was first reported in the stool of pakistani children with non-polio acute flaccid paralysis (afp) [10] , and then in australian children and chinese children with diarrhea [9, 25] . hbov3 was first reported in the stool of australian children with diarrhea [9] and then in stool from nigerian, tunisian, nepalese and us children [5] . hbov4 was reported in the stool of children with afp from nigeria and tunisia [5] . hbov 1/2/3 were also detected in untreated sewage water from throughout the us [26] . recently, a novel bocavirus was identified in the feces of captive gorillas with diarrhea [6] and from wild gorillas and chimpanzees [7] . in 2009, a porcine bocalike virus (pbo-likev) was reported in swine feces with postweaning multisystemic wasting syndrome in sweden and 1854 bp of its partial genome sequenced [27] . in 2010, the nearly complete genomes of distinct porcine bocaviruses provisionally named pbov1 and pbov2 were characterized from feces of swine in china [28] . finally, partial genome sequences of 2.4 kb from another clade of porcine bocaviruses labeled 6v and 7v where also identified yielding three major bocavirus groups in swine (pbo-likev, pbov1/pbov2, and 6v/7v). random amplification and sequencing has been used to discover novel virus in human and animal [8, 10, . in this study, we found in swine feces highly distinct bocavirus whose genome we tentatively named porcine bocavirus 1 (pbov1-h18), and porcine bocavirus 2 (pbov 2-a6). the nearly complete genomes of both viruses were acquired and are described here. pbov1-h18 and pbov2-a6 were also screened for in 340 stool samples of asymptomatic swine from five provinces of china. a total of 340 porcine stool samples from different aged swine were collected from 17 middle or large-scale porcine farms (200-2,000 sows each) in five provinces of china from april 2008 to october 2009, of which 80 were collected from four farms located in shanghai, 60 from three farms located in jiangsu province, 120 from six farms located in anhui province, 20 from a farm located in shandong province, and 60 from three farms located in guizhou province and stored at 280uc (table 1 ). one stool sample was randomly selected from each of the 17 farms. the samples were suspended in pbs (0.01 m phosphate, ph7.2-7.4, 0.15nacl), vortexed, centrifuged at 15000 g, and filtered through a 0.22-mm filter to remove eukaryotic-and bacterial-cell-sized particles [45, 46, 54, 55] . the filtrates were then treated with benzonase, dnase and rnase to digest non-particleprotected nucleic acid as reported [54, 55] . viral nucleic acids were then extracted using the tianamp virus dna/rna kit (tiangen biotech, beijing, co., ltd.). viral cdna synthesis was performed by incubation of the extracted viral rna/dna with 100 pmol of primer k-8n [56] with a degenerate 39 end and the use of superscript reverse transcriptase, and the opposite strand of the cdna was generated after melting and reannealing and primer extension using klenow dna polymerase [45, 46, [54] [55] [56] . pcr of extension products was performed as reported previously using the k-8 primer (k-8n without the degenerate 39 end) [54] . this protocol amplifies both viral rna and dna genomes [54, 55] . random rt-pcr dna products ran as smears on agarose gel and were gel purified (axygen, ca, usa), then subcloned into pmd-18t plasmid vector (takara, japan) for sequencing. the sequences were then screened for sequence similarities using tblastx and blastn against the nr database in genbank. sequences of each pcr product were assembled using seqman ii program (dnastar, inc). the identification of open reading the near-full genomes of pbov1-h18, pbov2-a6 and the partial ns1 and vp1 sequences from the diagnostic npcr have been deposited in genbank under accession numbers hq291308-hq291309 and hq291310-hq291343. seventeen porcine samples stool supernatants from 17 farms were analyzed using a generic viral particle-protected nucleic acid enrichment procedure followed by random amplification of extracted rna and dna (see materials and methods) [46, [54] [55] [56] . amplified dna was then subcloned and 1190 plasmid inserts were sequenced (70 for each of 17 samples). nine samples (totaling 82 subclones) showed the presence of fragments whose virtual translation products were related to canine and human bocaviruses using blastx. twenty-four clones from pig sample h18 and 26 clones from pig sample a6 showed significant similarity with bocaviruses. h18 derived sequences showed high (.99%) identity with the recently described porcine bocavirus-like virus (pbo-likev), the only porcine bocavirus reported at the time of these experiments (genbank gu902971) [27] . sequences from porcine sample a6 showed low identity with those of h18 and pbo-likev. the h18 and a6 samples were selected for targeted viral genome amplification and sequencing. the 24 sequences from h18 were assembled to form a continuous sequence of approximately 2700 nucleotides that appeared to lack .1300 and 1000 nucleotides from the 59and 39 ends of its genome. pcr primers based on the available h18 sequences and regions highly conserved between hbov2 (fj170278) and canine bocavirus (fj214110) were used to amplify the nearly complete bocavirus genome we provisionally called pbov1-h18 (5267 nt). to confirm this genome sequence, this sequence was re-amplified using 6 sets of pcr primers generating overlapping fragments of the genome which were directly sequenced. using the same method, the nearly complete bocavirus genome (5117 nt) from sample a6 was also sequenced and was provisionally labeled pbov2-a6. using an open reading frame (orf) finder (http://www.ncbi. nlm.nih.gov/gorf/gorf.html), three orf were found in both genomes (figure 1 ). the orfs of pbov1-h18 were 636 aa for ns1, 219 aa for np1 and 621 aa for vp1/vp2. the orfs of pbov2-a6 were 703 aa for ns1, 221 aa for np1 and 704 aa for vp1/vp2. the possible splicing of bocavirus ns1 transcripts recently shown to extend the length of ns1 proteins was not investigated here [6, 57] . nucleic acids were extracted from 340 porcine stool samples. pbov1-h18 related sequences were screened for using nested pcr with primers amplifying a 530-bp fragment of the np1/vp1 region. the electrophoretic bands of the expected size were subcloned and sequenced. the results showed that the prevalence of pbov1-h18 related viruses was high in china with 215 out of 340 (63.2%) porcine samples positive ( to determine the genetic relationship of pbov1-h18 and pbov2-a6 with recently described porcine bocaciruses and bocaviruses from other host species, both nucleotide and amino acid alignments were generated and used for phylogenetic analyses. when the whole genomes were considered porcine bocaviruses as a group (except for the v6/v7 variants with only np1 sequences available), were most closely related to the canine bocavirus cnmv (figure 2a ). phylogenetic analyses of the 3 orfs -ns1, np1 and vp1/vp2 -were also performed ( figure 2b-g) . in all three regions, pbov1-h18 was most closely related to the chinese pbov1 and pbov2 recently reported by cheng et al [28] . pbov2-a6 was closely related in np1 to the first reported partial porcine bocavirus sequence pbo-likev from sweden, the only region available for comparison (fig. 2c, f ) [27] . table 2 numerically shows the protein similarities between pbov1-h18 and pbov2-a6 and other porcine and non-porcine bocaviruses. the partial vp1 sequence of pbov2-a6 related variants from different farms was also phylogentically analyzed and fell into two major clades we named pbov2 genotype 1 and 2 (pbov2-g1 and pbov2-g2). the two previously described chinese pbov1 and pbov2 ''species'' grouped within these two genotypes. pbo-likev was originally found in swine with postweaning multisystemic wasting syndrome (pmws) in 2009 when approximately 35% of its genome sequence was reported [27] . the nearly full genomes of two distantly related porcine bocaviruses labeled pbov1 and pbov2 as well as two partial genomes labeled v6 and v7 were then reported in 2010 [28] . these bocaviruses grouped into 3 phylogenetic clades containing pbo-likev, pbov1/pbov2, and v6/v7. in the present study, we characterize two nearly complete bocavirus genomes, one of which (pbov1-h18) grouped with the pbo-likev clade while the second (pbov2-a6) fell with the pbov1/pbov2 clade. we provisionally named the genome from the h18 sample pbov1-h18 since its closest homologue, pbo-likev, was the first reported porcine bocavirus [27] . the virus from sample a6 was provisionally named pbov2-a6 since it phylogenetically groups with the second reported set of porcine bocaviruses containing both pbov1 and pbov2 [28] . no close homologues of the v6/v7 clade were identified in this study. under this proposed classification scheme, the viruses labeled pbov1 and pbov2 by cheng et al, therefore both belong to the pbov2 clade, the second reported clade of porcine bocaviruses [28] . under this proposed taxonomic classification, the v6 and v7 bocaviruses [28] belong to the still only partially characterized pbov3 clade. sequence analysis of the pbov2 clade showed that their vp1/ vp2 genes were highly diverse and could be classified into two genotypes ( figure 3 ). the partial np1 and vp1 genes of pbov1-h18 related viruses detected in this study were more highly conserved (99-100% identity), consistent with a recent report by zhai et al reporting pbov1 in chinese pigs using partial vp1/ vp2 nested pcr and sequencing [58] . zhai et al also found a high prevalence of pbov1 (69% in weanling piglets) with a higher frequency of pbov1 in animals also infected with pcv2, prrsv, pttv or csfv and in pigs with respiratory symptoms versus healthy pigs [58] . members of the bocavirus genus contain an 3 rd orf of unknown function labeled np1 gene. recently another parvovirus (ppv4) was identified in porcine feces that also contained a central 3 rd orf, although unrelated in sequence to the bocaviruses np1 [59] . none of the orfs of ppv4 clustered phylogenetically with the bocaviruses (data not shown) but instead clustered with members of the parvovirus genus [59] . ppv4 is therefore unrelated to the bocaviruses reported here. hbov1 has been associated with respiratory symptoms while other hbov may be associated with diarrhea and acute flaccid paralysis [5, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] 25] . a gorilla bocavirus was detected in captive animals in the us experiencing severe diarrhea [6] . pbov1 was found in pigs with pmws in sweden [27] and in pigs with respiratory tract symptoms in china [58] . in the present study, both pbov1 and pbov2 were highly prevalent in both asymptomatic swine from five provinces of china. further studies are needed to examine possible associations between infections with these different porcine bocaviruses, the viral loads excreted, the presence of co-infections and various porcine diseases. virus taxonomy: the eighth report of the international committee on taxonomy of viruses animal bocaviruses: a brief review complete 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the family picornaviridae, is globally widespread novel borna virus in psittacine birds with proventricular dilatation disease new adenovirus species found in a patient presenting with gastroenteritis new dna viruses identified in patients with acute viral infection syndrome discovery of a novel human picornavirus in a stool sample from a pediatric patient presenting with fever of unknown origin a highly divergent picornavirus in a marine mammal a highly prevalent and genetically diversified picornaviridae genus in south asian children recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: identification of a candidate etiologic agent genomic characterization of novel human parechovirus type a new arenavirus in a cluster of fatal transplant-associated diseases characterization of a novel coronavirus associated with severe acute respiratory syndrome identification of a novel gammaretrovirus in prostate tumors of patients homozygous for r462q rnasel variant a newly discovered human pneumovirus isolated from young children with respiratory tract disease rapid identification of known and new rna viruses from animal tissues metagenomic analyses of viruses in stool samples from children with acute flaccid paralysis characterization of virus isolates by particle-associated nucleic acid pcr characterization of the gene expression profile of human bocavirus high prevalence of a novel porcine bocavirus in weanling piglets with respiratory tract symptoms in china identification and molecular cloning of a novel porcine parvovirus key: cord-000434-ff2zadol authors: zhao, rongmao; cui, shujuan; guo, li; wu, chao; gonzalez, richard; paranhos-baccalà, gláucia; vernet, guy; wang, jianwei; hung, tao title: identification of a highly conserved h1 subtype-specific epitope with diagnostic potential in the hemagglutinin protein of influenza a virus date: 2011-08-19 journal: plos one doi: 10.1371/journal.pone.0023374 sha: doc_id: 434 cord_uid: ff2zadol subtype specificity of influenza a virus (iav) is determined by its two surface glycoproteins, hemagglutinin (ha) and neuraminidase (na). for ha, 16 distinct subtypes (h1–h16) exist, while nine exist for na. the epidemic strains of h1n1 iav change frequently and cause annual seasonal epidemics as well as occasional pandemics, such as the notorious 1918 influenza pandemic. the recent introduction of pandemic a/h1n1 iav (h1n1pdm virus) into humans re-emphasizes the public health concern about h1n1 iav. several studies have identified conserved epitopes within specific ha subtypes that can be used for diagnostics. however, immune specific epitopes in h1n1 iav have not been completely assessed. in this study, linear epitopes on the h1n1pdm viral ha protein were identified by peptide scanning using libraries of overlapping peptides against convalescent sera from h1n1pdm patients. one epitope, p5 (aa 58–72) was found to be immunodominant in patients and to evoke high titer antibodies in mice. multiple sequence alignments and in silico coverage analysis showed that this epitope is highly conserved in influenza h1 ha [with a coverage of 91.6% (9,860/10,767)] and almost completely absent in other subtypes [with a coverage of 3.3% (792/23,895)]. this previously unidentified linear epitope is located outside the five well-recognized antigenic sites in ha. a peptide elisa method based on this epitope was developed and showed high correlation (χ(2) = 51.81, p<0.01, pearson correlation coefficient r = 0.741) with a hemagglutination inhibition test. the highly conserved h1 subtype-specific immunodominant epitope may form the basis for developing novel assays for sero-diagnosis and active surveillance against h1n1 iavs. influenza a viruses (iavs), members of the orthomyxoviridae family, are highly contagious to a variety of avian and mammalian species. iavs cause seasonal influenza epidemics annually and recurring pandemics with severe consequences for public health and global economy [1, 2] . at least three iav-pandemics emerged in the last century (1918 a/h1n1, 1957 a/h2n2, and 1968 a/h3n2). the 1918 spanish flu was the most serious influenza pandemic that killed over 50 million people worldwide [3] . the latter two pandemics, although mild compared to the 1918 incidence, resulted in significant mortality, with close to 2 million and 1 million deaths, respectively [4] . the latest pandemic influenza, and newest global health challenge, occurred in 2009 due to the emergence of an a/ h1n1 pandemic iav (h1n1pdm virus). the h1n1pdm virus has been detected in more than 214 countries and territories and has caused 18,389 deaths as of july 30, 2010 [5] . the viral genome of iav consists of eight single-stranded negative sense rna segments that encode at least 11 viral proteins, including two surface glycoproteins, hemagglutinin (ha) and neuraminidase (na) [6] . based on the antigenic properties of ha and na, iavs have been classified into 16 ha subtypes and 9 na subtypes [7] . all 16 ha subtypes have been identified in avian species, while only 6 ha subtypes (h1, h2, h3, h5, h7 and h9) are known to infect human beings [8, 9, 10] . h1, h2 and h3 subtypes have caused pandemics, while h1 and h3 also dominate seasonal epidemics together with influenza b virus. ha, encoded by segment 4 of the iav genome, is a glycoprotein of approximate 560 amino acid. the biologically active ha is a homologous trimeric molecule that is attached to the virion membrane through its carboxy terminus [11] . ha plays a critical role in the pathogenesis of iavs. ha mediates iavs' binding to the cellular receptor n-acetylneuraminic (sialic) acid as well as the subsequent membrane fusion process [12] . ha also stimulates host protective immunities, specifically the production of neutralizing antibodies. the generation of anti-ha neutralizing antibodies has been the major target for influenza vaccine development [11, 13] . due to its specificity in immune response, ha is also an important target for iav subtyping using immunoassays [7, 14] . active serological surveillance for viral antibodies is of great importance for influenza control and prevention. several iav subtype-specific serological tests have been developed. at present, subtyping of iav mainly relies on a hemagglutination inhibition (hi) test using ha and na subtype-specific reference sera [15] . however, there are a number of drawbacks to hi testing. this assay is 1) relatively laborious; 2) low in sensitivity; 3) requires preparation of antigen from viable viruses which are potentially hazardous and 4) contains low signal to noise ratio, e.g. the assay exhibits inter-variability and subtype cross-reactivity [16, 17] . moreover, the hi test can be confounded by steric hindrance from na antibodies, leading to nonspecific inhibition and misidentification [18] . microneutralizing test is an alternative method to type and subtype influenza viruses. however, due to the needs of cell culture process, this method is labor-intensive and requires biological safety containments (particularly for high pathogenic strains). as such, it is not suitable for large scale investigations [19, 20] . recently, subtyping of iav antibodies using different categories of elisa assays have also been reported [16, 17, 21] . however, present elisa assays mainly rely on an ha antigen, which can lead to nonspecific detection to some extent due to the possible cross-reaction of different subtypes [22, 23] . virus-derived epitopes are useful tools to accurately evaluate immune response and to differentiate which responses are specific or due to cross-reactivity [24, 25, 26] . several studies have reported the existence of ha subtype-specific as well as inter subtypeconserved epitopes [27, 28, 29] . elisa assays based on epitopes that are highly conserved and specific for one certain ha subtype will be useful for rapid and simple subtyping of iavs. such epitopes in iavs have not been fully addressed although many studies have been performed. in the present study, we report the successful identification of a new epitope, which is highly conserved among the majority of iav strains of h1 subtype. moreover, we developed an elisa assay for h1 antibody subtyping based on this epitope. results derived from this new assay correlate with results obtained through the use of hi test. to identify the immunodominant epitopes in the ha protein, a peptide scanning assay was performed. a set of 50 peptides with five residues overlapping with the adjacent peptides spanning the ectodomain sequences of the ha protein of the h1n1pdm virus strain a/california/04/2009 were synthesized. the binding between these peptides and the convalescent serum samples from 11 h1n1pdm patients were examined by elisa using these peptides as coating antigens. five of these peptides (p3, p5, p15, p16 and p31) were found to react well with the sera tested. these peptides corresponded to the sequences of amino acid (aa) residues 38-52, 58-72, 158-172, 168-182, and 318-332 in the ha molecule, respectively ( fig. 1a and table 1 ). among them, the p3 peptide reacted with 54.5% (6/11) of the sera, the p15 and p16 peptides reacted with 81.8% (9/11) of the sera, while the p5 and p31 peptides reacted with 100% (11/11) of the sera. these data indicated that these peptides may contain h1n1pdm virus b cell epitopes. to visualize the location of the peptides on the ha protein, we mapped the peptides on the crystal model of this protein (fig. 1b) . the various colors in figure 1b represent the different peptides. although p3 (residues 38-52, indicated by blue) and p31 (residues 318-332, indicated by red) are parts of ha1 in primary sequence, they are located in the middle of helix a and b in the trimeric structure and are partially surface exposed. p5 (residues 58-72, indicated by magenta) seems to be a dispatch that links the stem region and the globular region and is fully surface exposed (fig. 1b) . p15 and p16 (residues 158-172 and 168-182, indicated by orange) are located in the receptor binding domain [11] . to confirm the immunogenicity of these peptides in vivo, we analyzed sera derived from peptide-immunized mice. the five positive peptides and two control peptides (p6 and p30) were coupled with keyhole limpet hemocyanin (klh) and were used to immunize balb/c mice ( table 1 ). the antisera were collected five days after the third immunization and titrated by elisa using corresponding peptide as a coating antigen. our results showed that all of the peptide conjugates except p15 induced potent antibody titers. the endpoint titers of antisera in elisa from mice immunized with p3, p5, p6, p16, p30, and p31 peptides were 1:6,400, 1:51,200, 1:51,200, 1:12,800, 1:51,200, and 1:25,600, respectively ( fig. 2a) . these data indicate that most of the positive peptides elicite humoral immunity and are highly immunogenic in mice. to confirm that these antibodies can recognize the ha antigen, the reactivity of the anti-peptide sera were evaluated by western blot and elisa against the purified ha0 protein of h1n1pdm virus. our data demonstrate that sera against p3, p5, and p31 but not those against p6 and p30 (controls) react to the ha0 protein ( fig. 2b and 2c ). the anti-p16 sera did not react to the ha0 protein, although it exhibited a high elisa reactivity to the ha0 protein ( fig. 2b and 2c ). taken together, our results demonstrate that p3, p5, and p31 peptides contain dominant epitopes of h1n1pdm virus. we then characterized these three peptides in the following studies. to determine if the epitopes identified in this study can stimulate neutralizing antibodies, a ha-pseudotype neutralization test was performed against the anti-peptide sera using the h1n1pdm pseudotyped lentivirus. none of the sera against p3, p5, p16, and p31 could efficiently inhibit (90% inhibition [30] ) the entry of h1n1pdm ha pseudotypes ( figure 2d ), indicating that these epitopes do not contain neutralizing activity. western blot analysis was used to determine the specificity of the epitopes present in the peptides p3, p5, and p31. the h1-h16 recombinant ha proteins were obtained by transient expressions of corresponding genes by the pcaggs vector in 293t cells. the lysates of these cells were used to examine the specificity of antibodies elicited by peptide-conjugates. as shown in fig. 3a , the anti-p3 serum reacted with h1 (including 07h1 and 09h1 viruses), h2, h5, and h6 ha proteins, while anti-p5 and anti-p31 sera only reacted with the h1 ha proteins. these findings indicated that p5 and p31 may contain h1-subtype specific epitopes. to evaluate the subtype-specificity of epitopes in p5 and p31 further, additional ha proteins of three epidemic human strains from different years (1918, 1934 and 1977) as well as a swine strain were expressed by pcaggs vector in 293t cells. the reactivity of anti-p5 and p31 sera with the cell lysates was determined by western blot analysis. our results showed that the anti-p5 serum strongly reacted with all of the six h1 ha proteins in a manner similar to an antibody against the ha1 of h1n1pdm virus (fig. 3b ). we found that the anti-p31 sera reactivity was weak against ha proteins from the 1918 and 1977 virus strains (fig. 3b ). these data indicated that the epitopes in p5 and p31 peptides are relatively conserved among h1-subtype iavs though these viruses have circulated in the world for almost a century. to test if anti-p5 sera cross-react with influenza type b virus, the reactivity of anti-p5 sera with two representative influenza type b virus strains (b/hubeiwujiagang/158/2009,yamagata lineage and b/heilongjianghulan/116/2010, victoria lineage) and an influenza type a virus strain (a/h1n1/pr8/34) was examined by western blot analysis. the results showed that anti-p5 serum reacted well with a/h1n1/pr8/34 virus but not with influenza type b virus strains (fig. 3c) , further confirming the specificity of the epitope in p5. to determine the conservation of the identified epitopes among iavs, the aa sequences of p3, p5, and p31 were aligned with the corresponding aa sequences of all the 16 subtype has available in the genbank. fig. 4 is a representative of the alignment analysis, showing that p5 is identical to ha of h1 subtype strains. p3 is identical to ha of the h1 subtype, as well as highly identical to ha of the h2, h5, and h6 subtypes. these data are consistent with the specificity analysis by western blot (fig. 3a) . although anti-p31 antibody only recognizes the h1-subtype ha, it is similar to multiple subtypes. to assess the identity levels of p5 and p31 sequences among the known iav strains, in silico coverage analysis was performed. this analysis showed that the p5 peptide sequence could be identified in 91.6% (9860/10767) of the h1-subtype ha sequences available in the influenza research database (http://www.fludb.org) ( table 2) . notably, this sequence scarcely presented (3.3% 792/23895) among the has of h2-h16 subtype iavs. however, despite a high identity in the h1 ha proteins (93.1%), the peptide sequence of p31 also presented among the has of h2-h16 viruses (78.8%). taken together, these findings indicate that the p5 peptide is h1-subtype specific and is conserved among h1 virus strains. to define the epitope contained in p5 precisely, a peptideinhibition elisa was performed. this experiment is reliable and is a standard methodology to determine the fine specificity of antigen-antibody reactions [31, 32] . a panel of short peptides derived from p5 (n6-n14, with c-terminus truncation; and c6-c14, with n-terminus truncation) were used to block the binding of anti-p5 antibody to coated p5. as shown in fig. 5 , antibody induced by the p5-klh conjugate was inhibited by peptide n10-n14 and the parental peptide p5 to similar extents, whereas peptides n6-n9 only showed inefficient inhibition even at high molar concentrations. a similar pattern of inhibition was observed with the c-terminal conservative derivatives. peptides c12, c13, c14, and the parent peptide p5 demonstrated comparable and efficient inhibition, whereas only slight inhibition was observed in peptides c6-c11 (fig. 5b) . since the amino acid sequence lrgvapl overlapped both peptides n10 and c12 (fig. 5) , we speculate that this sequence met the minimum requirements of binding to the anti-p5 antibody. however, the synthetic peptide lrgvapl did not block the binding between p5 peptide and its antibody, nor did it directly bind to the p5 antibody (fig. 6 ). as p5 (1918, 1934, 1977, 2007, 2009 ) and a swine h1n1 strain. 293t cells transfected with an empty vector was used as a control (ctrl). b-actin was used as a loading control. for the backgrounds of various subtype iav strains, see inspired by the high specificity of p5 among the h1-subtype viruses, we developed an indirect elisa assay using the p5 peptide to evaluate its performance as a diagnostic tool for h1 antibodies. the hi test was used as a reference method. as shown in table 3 , the overall agreement of these two methods was 87%, showing that the two methods have good correlation (pearson correlation coefficient r = 0.741). the sensitivity and specificity of peptide-elisa versus hi test was 96.5% and 74.4%, respectively, indicating the potential of the peptide-elisa method in detecting antibody against h1-subtype iavs. in the present study, we identified immunodominant linear b cell epitopes on the h1n1pdm virus ha protein by a peptide scanning approach using h1n1pdm patients sera. we confirmed that an unidentified epitope was highly conserved among h1 subtypes viruses and showed a good correlation with results obtained using the hi test. these findings demonstrate the potential of epitope-based antibody detection in iav diagnosis and surveillance. iav escapes the human immune system by continuous antigenic drifts and occasional antigenic shifts [33] . attempts to develop universal vaccines and reliable diagnostic tools based on conserved epitopes of iav are big challenges. several epitopes that can elicit broad spectrum neutralizing antibodies have been identified recently. for example, sui et al. identified a universal neutralizing epitope for group 1 ha [34] . yoshida et al. reported a universal epitope in antigenic site b shared by h1, h2, h3, h5, h9, and h13 subtypes [35] . all these epitopes are conformationdependent. in this study, we identified two epitopes (p5 and p31) which have not been identified previously (fig. s1 ). the p5 (aa58-72) seems to be a dispatch that links the stem region and the globular region and is fully exposed on the surface, while p31 (aa 318-332d) is located in the middle of helix a and b on ha2. in contrast to previous studies, we found p5 to be a linear b cell epitope. our data demonstrate that this epitope is highly conserved among h1 viruses (9860/10767, 91.6%). because viral mutants that are resistant to conformational epitopes are more easily generated, the conserved linear epitope is more suitable for differentiating subtypes than conformational epitopes [33] . hence, the epitope in p5 provides a new target for reliable diagnostics of h1-subtype iavs. antigenic sites in iav ha proteins of h1, h2, and h3 subtypes had previously been characterized by sequence analysis on antigenic variants and amino acid substitutions. these previously identified antigenic sites were mainly located in the globular head in the three-dimensional structure of the ha1 subunit of the ha molecule [36, 37, 38, 39] . for instance, five antigenic sites have been identified in ha of influenza virus a/pr/8/34, a well-known reference strain of h1n1 iav [39] . recently, several epitopes were identified in the ha2 unit [34, 40, 41] . together with these reports, our results indicate that there are more epitopes than what we have imaged and the epitopes of iav need to be further characterized. the difference between our findings and previously identified epitopes can be explained by the difference of screening method used between our study and those of others. in previous studies, monoclonal antibodies from murine hybridoma cells were used to identify antigenic sites, while in this study we used a peptide scanning approach, which involves overlapping peptide library and human convalescent antisera-a strategy that is widely used for viral epitope identification [27, 42] . given the fact that viral antigen can be recycled and presented as short peptides with different conformation during humoral immune response and these short peptides can be selected by b cell clones [43] , and that convalescent sera from patients were much more complex than monoclonal antibodies from mice and can reflect the real immune responses during viral infection [44] , our approach adds to the available techniques currently being used to identify linear epitopes in serologic tests. because ha pseudotyped lentivirus has been widely applied in the study on neutralizing antibodies against iavs [30] , we used this method to evaluate if the epitopes identified in this study could stimulate neutralizing antibodies. our data showed that these epitopes could not elicit neutralizing antibodies in pseudovirion neutralizing assays due to their linear nature. previous studies have shown that most neutralizing epitopes are conformation dependent [34, 35] . the length of b cell epitopes can vary from 5 to 20 amino acids [45, 46] . to map the epitope contained in p5, we performed a peptide-inhibition elisa using a series of n-terminal and cterminal truncated peptides. however, we found that the fulllength p5 (15 aa in length) rather than truncated peptides showed strongest binding to the corresponding antibody ( fig. 5 and fig. 6 ). as peptides n10 and c12 are the shortest truncated p5 that can bind to anti-p5 antibody and share a core sequence of lrgvapl, we tested whether this sequence could be the epitope. however, the synthetic peptide lrgvapl did not block p5-antibody interactions nor bind p5 antibody (fig. 6) . thus, we speculate that adjacent amino acids to this sequence are also involved in the binding of antibody elicited by p5-klh conjugates. the concept of using linear epitopes in influenza virus diagnostics and control has not been extensively investigated. in a recent study, an epitope-blocking elisa, which can universally detect antibodies to human h5n1 virus, has been developed [17] . our results show that a peptide-elisa based on the highly conserved h1 subtype-specific epitope can also be used for the detection of h1 antibodies, displaying good correlativity with the hi test. our results indicate the potential of the p5 epitope in h1subtype iav diagnosis. however, the performance of this assay needs to be further evaluated in studies with large scale samples. in conclusion, our data provide evidence that the h1 subtype ha harbors more epitopes than what has been found previously. the conservation of an epitope (p5, aa 58-72) in the h1-subtype ha of iav and its near complete absence in other subtypes indicate that this epitope meets the critical requirements for diagnosis of h1 subtype influenza virus infections. the peptide-elisa developed in our study may be applicable for serodiagnosis and may serve as a universal diagnostic tool for h1subtype iav surveillance. to screen the h1-subtype specific epitopes, a set of 50 peptides spanning the amino acid sequences of the ha protein ectodomain of pandemic a/h1n1 2009 (h1n1pdm) influenza virus strain a/ california/04/2009 were synthesized. each peptide is 15 amino acids in length with five residues overlapping with the adjacent peptides [46] (fig. 1) . the peptides selected for immunization experiments are shown in table 1 . these peptides were conjugated with a carrier protein, the keyhole limpet hemocyanin (klh; sigma, st. louis, mo), to improve their immunogenicity [47] . as the water solubility of peptides p5, p15, p16 and p31 were too low to conjugate with klh directly, these peptides were first linked to 6-aminocaproic acid and then to the tripeptide kkc prior to being conjugated with klh [48] . in addition, a family of short peptide homologs to the p5 peptide was also synthesized to fine map the epitope contained in the p5 peptide (fig. 5) . all of the peptides and their conjugates used in this study were synthesized by sangon (shanghai) biotechnol co., ltd. (shanghai, china). each peptide was purified to homogeneity (.95% purity) by highperformance liquid chromatography and verified by mass spectrometry. the reference influenza virus strains a/pr8/34 (h1n1) (abbreviated pr8), b/hubeiwujiagang/158/2009 (yamagata lineage, abbreviated by) and b/heilongjianghulan/116/2010 (vic-toria lineage, abbreviated bv) were kindly provided by the beijing center for disease control and prevention. the viruses were grown in mdck cells as described elsewhere [5] . the titers of virus strains were determined by hemagglutination tests and expressed as hemagglutinating units (hau). for western blot analysis, the inactivated viruses were lyzed in a lysis buffer (50 mm tris-hcl, 150 mm nacl, 5 mm edta, 1% triton-x, ph 7.4) supplemented with a protease inhibitor cocktail (roche, indianapolis, in). serum samples were collected from 11 convalescent patients during the early 2009 h1n1 pandemic in beijing. the diagnostic criteria for h1n1 influenza virus infection of these patients fully followed the who's descriptions. sera from 10 healthy blood donors were used as negative controls. in addition, serum samples collected from 100 blood donors were recruited to evaluate the peptide-elisa assay developed in this study. all these samples were kindly provided by the beijing municipal center for disease control and prevention (beijing, china) and written informed consent was obtained from all participants. all samples were coded prior to analysis to ensure anonymity and the procedures were approved by the institutional medical ethic review board of the institute of pathogen biology, chinese academy of medical sciences (beijing, china). the full-length cdna fragments corresponding to h2-h16 ha subtypes of iav were inserted into the pcaggs vector (purchased from addgene) to express entire ha proteins (unpublished data). for h1 proteins, ha gene representing human iav strains from different years (1918, 1934, 1977, 2007 and 2009 ) and a swine influenza virus strain were expressed by inserting the corresponding cdna fragments into the pcaggs vector in a similar manner. for the details of these influenza virus strains, please refer to fig. 4 . recombinant plasmids were transfected into 293t cells (atcc number crl-11268) using lipofectamine 2000 (invitrogen, carlsbad, ca) according to the manufacturer's instructions. the cells were harvested and lyzed 72 hours after transfection. the expression of ha proteins was verified by western blot analysis using murine antibodies against corresponding ha1 proteins (unpublished data). the reactivities of the synthetic ha peptides or purified ha0 protein (eenzyme llc, montgomery village, md) with the convalescent-phase sera from h1n1pdm patients and the serum samples from mice immunized with peptide conjugates were determined by elisa. briefly, each peptide (1 mg/well) or protein (0.1 mg/well) was used to coat 96-well microtiter plates (corning costar, acton, ma) in 0.1 m carbonate buffer (ph 9.6) at 4uc overnight. after blocking with 1% bovine serum albumen (bsa), the plates were incubated with indicated diluted serum samples (human or mouse) at 37uc for 2 hr, then washed four times with pbs containing 0.1% tween 20 (pbs-t). bound igg antibodies were detected with horseradish peroxidase (hrp)-conjugated goat anti-human igg or anti-mouse igg (sigma) at 37uc for 1 hr. after four washes with pbs-t, the reaction was visualized by addition of the substrate 3,39,5,59-tetramethylbenzidine (tmb, sigma). color development was stopped by the addition of 50 ml/well of 2 m sulphuric acid after 15 min. the optical density at 450 nm (od 450 nm ) was measured by an elisa plate reader (molecular devices, sunnyvale, california). to evaluate the reactivity of the p5-derived short peptides with the anti-p5 antibody, peptide-inhibition elisa assays were performed by adding dilutions of the peptides to a constant amount of the antibody elicited by the p5-klh conjugates (1:5000 dilution). maximum binding to antigen-coated wells was observed in the absence of a peptide inhibitor. the antibody bound was expressed as a percentage of the maximum level of binding. female balb/c mice of 6-8 weeks old were immunized subcutaneously with various peptide-klh conjugates mixed with freund's complete adjuvant (sigma) at 100 mg per injection. boost injections were given at 2-week intervals with 50 mg antigen in freund's incomplete adjuvant (sigma) [49] . the antibodies were collected five days after the third boost. all the animal experiments were carried out in the facilities of the institute of laboratory animal sciences (ilas), chinese academy of medical sciences (cams). all the experimental procedures were approved (permit number slxkj2009-0007) and supervised by the animal protection and usage committee of ilas, cams. at 72 hr post-transfection, the cells transfected with haexpressing plasmids were harvested, pelleted, and lyzed in a lysis buffer (50 mm tris-hcl, 150 mm nacl, 5 mm edta, 1% triton-x, ph 7.4) supplemented with a protease inhibitor cocktail (roche, indianapolis, in). aliquots of cell lysates (50 mg) or virus lysates were blotted after 10% sds-page onto nitrocellulose membranes (pall, port washington, ny). the membranes were blocked with 5% non-fat milk and then incubated with the primary antibodies indicated in the figures at 4uc overnight. this was followed by incubation with the goat anti-mouse irdyeh fluor 800-labeled igg secondary antibody (1:10, 000) (li-cor, lincoln, ne). after washing, the membranes were scanned by the odysseyh infrared imaging system (li-cor) and analyzed with odyssey software. the molecular sizes of the developed proteins were determined by comparison with the pre-stained protein markers (fermentas, maryland, ca). hi test was carried out as described elsewhere [5] . rde treated serum samples were inactivated at 56uc for 30 min and two-fold serially diluted at an initial dilution of 1:10. twenty five ml of the diluted serum were incubated with 25 ml of the four hemagglutination units from reference influenza strains for 30 min at room temperature. the reference h1n1 iav strains for hi test were a/ tianjinjinnan/15/2009(h1n1) and a/california/04/2009 (h1n1), respectively. 50 ml of 1% chicken erythrocyte suspension was added to each well and incubated for 30 min at 4uc. positive reactions were recorded when the hi antibody titer was equal to or greater than 40. h1n1pdm virus pseudotyped lentiviruses were produced in 293t cells co-transfected with pnl4.3-r 2 e 2 , ha and na constructs using a polyethylenimine (pei)-based transfection protocol [50] . cell culture supernatants were collected 48 hr post-transfection, filtered through a 0.45 mm-pore size filter (millipore, billerica, ma ) and used in pseudotype neutralization test. serum samples, heat inactivated at 56uc for 30 min, were diluted 40-fold in culture medium and mixed with an equal volume of diluted h1n1pdm influenza pseudovirions. after incubation at 37uc for 1 hr, 100 ml of pseudovirions (containing 50 ng/ml of hiv p24 gag protein) and serum mixtures were added into 96-well plates that contained 293t cells grown for 24 hr at initial 1610 4 cells. infectivity was evaluated at 72 hr post-infection by luciferase assay. the percentage of infectivity of pseudovirions treated by tested serum to that of negative serum (as control) was calculated. 90% reduction in infectivity by serum addition is considered to be neutralizing activity [30] . tests were run at least as duplicates. to assess the identity of the ha epitopes in iavs, in silico analysis was performed by utilizing bioinformatics tools at the influenza research database (http://www.fludb.org) [51] . the two programs used in this study were search for protein sequences and identify short peptides in flu proteins. the former program was used to define the number of total sequences in ha proteins according to the subtype parameter (h1 or h2-h16). the latter program defined the number of hits (p5 or p31) in the h1 or h2-h16 total sequences. because there are no standards for evaluating short peptide sequence homology, we chose the fuzzy match analysis to represent the identical level of a peptide sequence to ha proteins. the analysis type was chosen as fuzzy match, which meant .50% of characters were identical to the searched aa string. for example, entering gilgfvftl may also find ailgfvfti but not aligfvfsi. the pearson correlation coefficient was calculated by pearson chi square test for crosstab tables using spss software. the sensitivity and specificity of the peptide-elisa versus hi test was determined by roc curve analysis using spss software. figure s1 localization comparison between the identified peptides and the classical five antigenic sites in stereo view. the ha monomer surface view of influenza virus a/pr/8/34 (pdb id:1ru7) is shown and colored to illustrate the five antigenic sites (sa, sb, ca1, ca2, and cb) and the identified peptides. from most membrane distal to proximal: p3 (blue), p31 (red), p5 (black), cb (green), ca1 (magenta), ca2 (rainbow), sa (yellow), and sb (cyan). (tif) influenza: lessons from past pandemics, warnings from current incidents influenza: old and new threats the origins of pandemic influenza-lessons from the 1918 virus the 2009 influenza a (h1n1) pandemic: what have we learned in the past 6 months fields virology: orthomyxoviridae. 5th edn characterization of a novel influenza a virus hemagglutinin subtype (h16) obtained from black-headed gulls continuing evolution of h9n2 influenza viruses in southeastern china avian influenza a virus (h7n7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome avian influenza a (h5n1) receptor binding and membrane fusion in virus entry: the influenza hemagglutinin the structure and function of the hemagglutinin membrane glycoprotein of influenza virus influenza vaccines for the future characterization of a novel influenza hemagglutinin, h15: criteria for determination of influenza a subtypes manual of diagnostic tests and vaccines for terrestrial animals development of blocking elisa for detection of antibodies against avian influenza virus of the h7 subtype development of epitope-blocking elisa for universal detection of antibodies to human h5n1 influenza viruses a laboratory manual for the isolation and identification of avian pathogens comparison of the hemagglutination-inhibiting and neutralizing antibody responses of volunteers given 400 chick cellagglutinating units of influenza a/new jersey/76 split-virus vaccine role of the laboratory in diagnosis of influenza during seasonal epidemics and potential pandemics development of rha1-elisa for specific and sensitive detection of h9 subtype influenza virus prokaryotic expression and purification of ha1 and ha2 polypeptides for serological analysis of the 2009 pandemic h1n1 influenza virus utility of pandemic h1n1 2009 influenza virus recombinant hemagglutinin protein-based enzymelinked immunosorbent assay for serosurveillance ab and t cell epitopes of influenza a virus, knowledge and opportunities epitope analysis for influenza vaccine design quantifying influenza vaccine efficacy and antigenic distance antigenic characterization of recombinant hemagglutinin proteins derived from different avian influenza virus subtypes epitope mapping of the hemagglutinin molecule of a highly pathogenic h5n1 influenza virus by using monoclonal antibodies evaluation of the subtype specificity of monoclonal antibodies raised against h1 and h3 subtypes of human influenza a virus hemagglutinins establishment of retroviral pseudotypes with influenza hemagglutinins from h1, h3, and h5 subtypes for sensitive and specific detection of neutralizing antibodies minimum requirements for immunogenic and antigenic activities of homologs of a synthetic peptide of influenza virus hemagglutinin genetic control and fine specificity of the immune response to a synthetic peptide of influenza virus hemagglutinin one step closer to universal influenza epitopes structural and functional bases for broad-spectrum neutralization of avian and human influenza a viruses crossprotective potential of a novel monoclonal antibody directed against antigenic site b of the hemagglutinin of influenza a viruses antigenic structure of the haemagglutinin of human influenza a/h2n2 virus structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 a resolution structural identification of the antibodybinding sites of hong kong influenza haemagglutinin and their involvement in antigenic variation the antigenic structure of the influenza virus a/pr/8/34 hemagglutinin (h1 subtype) a simple slot blot for the detection of virtually all subtypes of the influenza a viral hemagglutinins using universal antibodies targeting the fusion peptide monoclonal antibodies against the fusion peptide of hemagglutinin protect mice from lethal influenza a virus h5n1 infection identification of immunodominant epitopes on the membrane protein of the severe acute respiratory syndrome-associated coronavirus the who, how and where of antigen presentation to b cells screening of random peptide library of hemagglutinin from pandemic 2009 a(h1n1) influenza virus reveals unexpected antigenically important regions predicting flexible length linear b-cell epitopes b cell epitope mapping using synthetic peptides approaches to augmenting the immunogenicity of melanoma gangliosides: from whole melanoma cells to ganglioside-klh conjugate vaccines universal antibodies and their applications to the quantitative determination of virtually all subtypes of the influenza a viral hemagglutinins production of antipeptide antisera analysis of hemagglutinin-mediated entry tropism of h5n1 avian influenza biohealthbase: informatics support in the elucidation of influenza virus host pathogen interactions and virulence understanding sensitivity and specificity with the right side of the brain we thank dr. fang huang for her assistance in serum sample collection. key: cord-000346-9b6yz3f4 authors: holder, benjamin p.; simon, philippe; liao, laura e.; abed, yacine; bouhy, xavier; beauchemin, catherine a. a.; boivin, guy title: assessing the in vitro fitness of an oseltamivir-resistant seasonal a/h1n1 influenza strain using a mathematical model date: 2011-03-24 journal: plos one doi: 10.1371/journal.pone.0014767 sha: doc_id: 346 cord_uid: 9b6yz3f4 in 2007, the a/brisbane/59/2007 (h1n1) seasonal influenza virus strain acquired the oseltamivir-resistance mutation h275y in its neuraminidase (na) gene. although previous studies had demonstrated that this mutation impaired the replication capacity of the influenza virus in vitro and in vivo, the a/brisbane/59/2007 h275y oseltamivir-resistant mutant completely out-competed the wild-type (wt) strain and was, in the 2008–2009 influenza season, the primary a/h1n1 circulating strain. using a combination of plaque and viral yield assays, and a simple mathematical model, approximate values were extracted for two basic viral kinetics parameters of the in vitro infection. in the st6gali-mdck cell line, the latent infection period (i.e., the time for a newly infected cell to start releasing virions) was found to be 1–3 h for the wt strain and more than 7 h for the h275y mutant. the infecting time (i.e., the time for a single infectious cell to cause the infection of another one) was between 30 and 80 min for the wt, and less than 5 min for the h275y mutant. single-cycle viral yield experiments have provided qualitative confirmation of these findings. these results, though preliminary, suggest that the increased fitness success of the a/brisbane/59/2007 h275y mutant may be due to increased infectivity compensating for an impaired or delayed viral release, and are consistent with recent evidence for the mechanistic origins of fitness reduction and recovery in na expression. the method applied here can reconcile seemingly contradictory results from the plaque and yield assays as two complementary views of replication kinetics, with both required to fully capture a strain's fitness. influenza is the most important respiratory disease in terms of mortality and morbidity. each year, between 3 and 5 million severe cases and 250,000 to 500,000 deaths due to seasonal influenza are reported worldwide [1, 2] . cyclic pandemics due to antigenic shifts constitute an important threat [3] as was demonstrated by the swine-origin pandemic of 2009 [4] . since vaccines for novel influenza virus strains require approximately 6 months to develop and produce [5] , antivirals remain the first line of defense. there are only two classes of antivirals approved for treatment of influenza [6] . the adamantanes, such as amantadine and rimantadine, are ineffective against b-type viruses [7] and have recently become ineffective against most a/h3n2 and some a/h1n1 viruses due to a mutation in the m2 gene [8] . the neuraminidase inhibitors (nai), which include zanamivir and oseltamivir, were approved a decade ago and have shown excellent activity against all influenza a subtypes and b viruses [9] . a recent rapid increase in resistance to oseltamivir, however, has become a cause for concern. the h275y mutation in the neuraminidase (na) gene (h274y in n2 numbering), first described in 2000 [10] , is the most frequent mutation associated with oseltamivir-resistance in the n1 subtype, but it had long been thought to critically reduce viral fitness [11] . with a location on the framework residue of the enzyme catalytic site [12] , the mutation has been shown to cause a reduced affinity for the substrate in enzyme activity assays [12, 13] , an impaired viral fitness in vitro [14] [15] [16] , and up to a 100-fold reduction in transmission efficiency in ferrets [14, 17] . for these reasons, strains carrying the h275y mutation were not thought to be a great concern for public health [10, 18] . during the 2007-2008 influenza season, however, the a/ brisbane/59/2007-like (h1n1) h275y mutant emerged and rapidly disseminated worldwide in the apparent absence of antiviral pressure [8, [19] [20] [21] . recently, our group performed a study on the replicative capacities of the a/brisbane/59/2007 h275y mutant strain where we showed that its fitness, based on in vitro and animal studies, was similar to that of its wild-type (wt), oseltamivir-susceptible, counterpart [22] . these observations, and those of others [23] , correlate with the clinical situation encountered in the 2008-2009 season where almost 100% of the a/h1n1 viruses isolated in north america and europe were resistant to oseltamivir due to the h275y mutation [1, 19, 23] . recent work suggests that the origin of both the fitness reduction conferred by the h275y mutation and the unique fitness of the a/brisbane/59/2007 mutant strain is found in the virus na activity and surface expression. specifically, the reduction of na activity conferred by the h275y mutation has been associated with a reduced expression of surface neuraminidase, possibly due to defects in the folding of the molecule or its transport through the cellular membrane [24] . it has been shown, however, that two other mutations in the na gene (v234m and r222q) can provide a compensatory effect by increasing na surface expression, and that these two substitutions indeed occurred in the evolution of the h1n1 seasonal strain between 1999 and 2007 [24] . the neuraminidase of contemporary (a/brisbane/59/2007-like) strains susceptible to oseltamivir have shown a higher affinity for the substrate in na activity assays than older h1n1 seasonal strains (e.g., a/new caledonia/20/99 and a/solomon islands/3/06); contemporary oseltamivir-resistant strains (with the h275y substitution) have shown a decrease in that affinity, but remain above the level of older strains [25] . thus, it seems that these pre-existing mutations led to an over-expression of na and provided a favorable environment for the appearance of the h275y mutation. the eventual dominance of the h275y mutant may be due to a better balance between the hemagglutinin (ha) and na activity [25, 26] . it remains an open question, however, precisely how these mechanistic changes lead to viral fitness changes. the answer to this should be found in the details of the infection kinetics in the interaction of virus and cell. in this paper, we present a method to extract the values of viral kinetics parameters, specific to a particular strain, from parallel experiments of plaque and viral yield assays. our previous study [22] assessed the in vitro replicative capacity and fitness of pairs of wt influenza virus strains and their h275y mutant counterparts by use of viral yield assays and by qualitatively comparing plaque sizes. here, we show that it is possible to use these experimental measures to quantitatively characterize the kinetics parameters responsible for the replicative efficiency of influenza virus strains. as a proof of concept, we apply our method for extracting the viral kinetics parameters to the oseltamivir-susceptible/-resistant pair of a/brisbane/59/2007 influenza virus strains in order to determine how the known genotypic differences in these two strains map to quantitative changes in the viral kinetics parameters characterizing their replicative efficiency. the kinetics parameters extracted through our method suggest that the h275y mutant has weaker na activity compared to its wt counterpart -confirmed by na activity assays -which manifests itself as a longer phase of latent infection before viral release -confirmed by single-cycle viral yield experiments. however, the results also indicate that this longer latent infection period for cells infected by the h275y mutant is compensated for by a shorter infecting time required for that cell, once releasing virions, to successfully infect other cells. in order to obtain two complementary views of the infection kinetics for the a/brisbane/59/2007 wt and h275y mutant strains, virus growth over time was observed in two different in vitro systems: the viral plaque assay and the multiple-cycle viral yield assay. viral plaque assays. figure 1 shows representative plaques for the wt and h275y mutant strains of a/brisbane/59/2007 (h1n1) viruses at each time point. the average plaque radius of each strain over time, calculated by averaging three independent experiments of three such wells at each time point, is shown in figure 2 . the plaque growth is characterized by an initial delay where no growth is observed, followed by a period of linear increase of the plaque radius over time. after 60 h, the rate of plaque growth declines and the linear approximation is no longer valid. the growth attenuation could be due to a number of factors including a hardening of the overlay, a depletion of nutrients required for viral production and cell maintenance, and the widespread destruction of the cell monolayer leading to holes and irregularities disrupting and limiting further growth. the plaque assay is a long-standing and standard technique in virology [27, 28] and plaque sizes have been used in many in vitro studies to qualitatively evaluate the phenotypes of various viruses [22, 29, 30] . plaque assays are often used for strain comparison and, in that context, plaque diameters at a single time point are reported. these plaque diameters are then typically used to conclude that, for example, if the plaques observed at 48 h for strain a are larger than those for strain b, then strain a must have a higher replicative fitness than strain b. however, one should question whether such conclusions are valid and robust to experimental variability. looking at figure 2 , one can see that at 36 h, the plaque radius of both a/brisbane/59/2007 wt and its h275y mutant counterpart are comparable in size. yet at 60 h, the wt strain has significantly larger plaques than the h275y mutant, and the situation is reversed at 96 h. thus, relying on single time point measurements for comparing strains can be misleading. here, instead, we exploit the fact that the average plaque growth is approximately linear in time between 36 and 60 h. this allows us to extract a novel measure, the plaque velocity, which is the slope in the linear regression of the plaque radius to the 36, 48 and 60 h time points. the plaque velocity, unlike the plaque radius at a given time point, is a robust measure in that it takes into account plaque radius at several time points and is not affected by differences in the length of the delay period which precedes the period of linear growth. using this method, the measured plaque growth was more rapid for the wt than the h275y mutant, with a plaque velocity of 1:79+0:1d cell =h compared to 1:48+ 0:1d cell =h, where d cell is the diameter of one cell. thus, using plaque velocity alone, it would appear that the wt strain has a replicative fitness advantage over the h275y mutant. multiple-cycle viral yield assays. in order to complement the information provided by the plaque assay, namely the plaque velocity, we also conducted multiple-cycle viral yield experiments. the results of these experiments for the wt and h275y mutant strains are shown in figure 3 . the kinetics of the viral yield experiments can be broken into two different phases: an exponential growth of virus concentration, characterized by the viral titer growth rate, followed by an exponential decay of virus concentration, characterized by the viral titer decay rate, after the viral titer peak. the viral titer decay rate was the same for both strains at 0:19+0:02h {1 . the viral titer growth rate of the h275y mutant was 0:83+ 0:02h {1 , slightly greater than that of the wt which was 0:75+0:04h {1 . thus, it would appear from the viral titer growth rate alone, that the h275y mutant has a replicative advantage over the wt strain, in contrast with the findings using the plaque velocity extracted from the plaque assay alone. this discrepancy between the conclusions drawn from each experimental measure points to a complementarity between the two assays: they appear to emphasize different aspects of viral replication. thus, combining the information provided by these two assays is key to obtaining a complete and consistent picture of what shapes a particular strain's replicative fitness. the plaque growth experiment yields a single experimental measure for each strain: the plaque velocity. the multiple-cycle viral yield assay provides two quantities: the viral titer growth rate, and the viral titer decay rate. it is the goal of this paper to associate these broad experimental measures to the values of fundamental infection parameters, specific to each strain, which quantitatively characterize replicative efficiency. to this aim, we have constructed mathematical models which allow us to simulate each in vitro assay in a computer experiment. the basic mathematical model used here is similar to other within-host models of viral infection [31] [32] [33] [34] [35] [36] . a cell can be in one of four states -target (uninfected), latently infected (infected but not yet releasing virus), infectious (releasing virus) and dead (no longer releasing virus) -and its passage through these states ( figure 4 ) is determined by five infection kinetics parameters. target cells interacting with virus become latently infected at a constant infection rate per virus, b. the average time a cell remains latently infected is called the latent infection period, t l , and the average time a cell releases virus is called the infectious lifespan, t i . virus is produced by infectious cells at a constant viral production rate, p, and this free virus loses infectivity exponentially at a constant rate of viral infectivity loss, c (as is observed in experiments [35] ). in applying this model, it is assumed that the growth of a particular influenza virus strain in a particular cell line is determined by a single, unique set of values for these five parameters. thus, although the mathematical structure of the models used for each experimental assay is different (see materials and methods for a detailed description of each), the parameter values for a particular virus strain are assumed to be constant from assay to assay. with only three experimentally measured quantities, it would be impossible to uniquely identify all five parameters for a particular virus strain. fortunately, it is possible to reduce the number of parameters considered and obtain unique identification of a few key parameter values. one parameter can be determined immediately from the multiple-cycle viral yield results. the viral titer decay rate, characterizing the decline of the virus concentration after the peak (figure 3 ), corresponds to the slowest of the rate of loss of virus-producing cells and the rate of viral infectivity loss [37] . since prior in vitro experiments have shown that infectious cell death is nearly complete shortly after the viral titer peak [38, 39] , we set the rate of viral infectivity loss, c, equal to the viral titer decay rate. because this decay rate was determined to be 0:19h {1 for both a/brisbane/59/2007 strains, we have fixed the rate of viral infectivity loss to this value for all simulations. this corresponds to a virion half life of approximately 3.6 h, which is consistent with prior measurements for influenza virus in the experimental literature (see, e.g., [35, 40] ). having fixed the rate of viral infectivity loss, we are left with four undetermined parameters and two experimental measures. for the experiments considered here, the infection rate per virus, b, and the viral production rate, p, can be combined into a single parameter, leaving only three parameters to be determined. the rationale for this simplification is the fact that, during an infection, the two parameters play equivalent roles: doubling the rate at which virus is produced by cells will have the same effect on new infections as doubling the rate at which virus infects cells. therefore, the only identifiable quantity is the product of the two rates, pb. since their product has units of inverse time squared, we have chosen to express this quantity as a new characteristic time, the infecting time, t inf~ffi ffiffiffiffiffiffiffiffiffiffiffiffiffi 2= pb ð þ p , which corresponds to the average time it takes a single virus-producing cell to cause the latent infection of one more (see materials and methods). we are left then with two experimental measures -the viral titer growth rate and the plaque velocity -whose values may depend on three unknown infection kinetics parameters: the infecting time, t inf ; the latent infection period, t l ; and the infectious lifespan of a cell, t i . to determine how each of these parameters affect the infection dynamics, we varied each individually about a base value and measured the effect on the simulated experimental quantities ( figures 5a and 5b ). one parameter, the infectious lifespan of a cell, t i , had very little effect on either the plaque velocity or the viral titer growth rate. in the latter case, this parameter was explicitly neglected in the derivation of the growth rate, because earlier viral yield experiments have shown little cell death prior to the peak of the virus concentration (see, e.g., [38, 39] ). the fact that, over a wide range of infectious lifespan values, the resulting plaque velocity remained unchanged, is perhaps more surprising. indeed, a shorter infectious lifespan will lead to the earlier appearance of plaques, resulting in larger plaque sizes at any given time. we have shown, however, in earlier work where influenza virus plaques were observed by immunostaining [41] , that the same plaque velocity can be measured from both the progress of dead cells, as we consider here, and the progress of newly infected cells. this indicates that plaque velocity is established at the advancing edge of an infection wave, and is likely unaffected by cell death in the wake of that wave. in those experiments, the infectious lifespan of a cell appears only as a time-delay between the infected cell plaque growth and the dead cell plaque growth. this has also been observed for the plaques of other viruses [42] . since the infectious lifespan has little effect on the experiments we consider, and is therefore not identifiable here, we have fixed its value for both strains and for all simulations to value of t i~1 2h, obtained from the literature ( table 1) . this leaves only two parameters, the infecting time, t inf , and the latent infection period, t l , to be determined from our two experimental measures, the plaque velocity and viral titer growth rate. the full dependence of each experimental measure on the two remaining parameters are presented as contour plots in figures 5c and 5d . because the plaque velocity and the viral titer growth rate depend on both the infecting time, t inf , and the latent infection period, t l , the experimental measurement of either quantity alone is not sufficient to specify the values of these infection parameters for a given strain. the measurement of both, however, can provide enough information for this specification, provided that the dependence on the parameters is sufficiently different for the two quantities. to demonstrate this concept using the a/ brisbane/59/2007 (h1n1) wt and h275y mutant strains, we have plotted the experimentally-measured values of plaque velocity and viral titer growth rate as functions of the infecting time and latent infection period, using the model dependence determined above ( figure 6 ). figures 6a and 6d show the values of the kinetics parameters most consistent with the measured plaque velocities of the a/ brisbane/59/2007 wt and h275y mutant strains, respectively. rather than plot a single line at the average measured value, we have accounted for the error in the measurement of the plaque velocities by plotting regions of contour denoting the one-and two-standard deviations (for a detailed description see materials and methods). we can see that while the plaque velocity does constrain the two parameters to a specific region, that region is too large to allow any useful comparison of the two strains. similarly, figures 6b and 6e show the values of the kinetics parameters most consistent with the measured viral titer growth rate. the consistency of a particular pair of parameter values with each of the two experimental measures can be combined by finding the intersection of the two parameter regions. this region of intersection corresponds to those parameter values most consistent with the parallel plaque and viral yield experimental measurements for a particular strain. the extent of these regions, shown in figure 6c for the a/brisbane/59/2007 wt strain and figure 6f for the h275y mutant strain, is summarized in table 2 . the region of intersection suggests that the latent infection period for the h275y mutant (w7 h) is longer than that of the wt strain (1-3 h), while the infecting time of the mutant (v 5 min) is much shorter than that of the wt (30-80 min). in order to test the predictions made in the previous section by applying the mathematical model to parallel plaque and viral yield assays, we performed two additional experimental tests which could provide some qualitative and quantitative confirmation. to independently estimate the latent infection period for the two a/brisbane/59/2007 influenza virus strains, we performed a single-cycle viral yield experiment. single-cycle experiments were performed at an moi of 1 such that most cells would be infected simultaneously and pass through the phases of latency and viral release at the same time. therefore, the observed virus production of the cell culture can be considered roughly proportional to that of an individual cell. the results of two independent experiments for each strain are shown in figure 7a ; one experiment shows the viral titer over one full day post-infection and the other over only 14 h but with more frequent sampling. for each replicate, the viral titer of each strain was observed to grow rapidly after 4 h postinfection, with the wt viral titer reaching a plateau at approximately 8 h post-infection and that of the h275y mutant reaching a plateau between 10 h and 14 h post-infection. although the viral titer data in each replicate followed a relatively smooth curve, the inter-replicate variation was quite large, with peak virus titer varying from 2|10 3 pfu ml to almost 10 5 pfu ml. it is also notable that all of these peak values were well below the values seen in the multiple-cycle viral yield assay (figure 3) , by a factor of * > 1000. both of these features could be explained by the action of a relatively large defective interfering particle population [43, 44] within the viral stock, which is not uncommon for the influenza virus [45] . the delay in the peak of viral titer between the two strains is qualitatively consistent with the model predictions of the previous section: the h275y mutant strain appears to have a longer latent infection period than the wt strain. to make this comparison more quantitative, we scaled each experimental data set such that the peak virus was equal to one and then performed a least-squares fit to the full set of normalized data for each virus strain ( figure 7b ). we utilized a model similar to that used for the analysis of the multiple-cycle viral yield assay, but allowed for a normal distribution of the latent infection period among cells rather than a fixed value for all cells, as assumed previously (see materials and methods). the fitted value of the average latent infection period, t l , was found to be 5.6 h for the wt strain and 7.5 h for the h275y mutant, with fitted values of the standard deviation in the normal distribution, s l , of 0.5 h and 1.2 h, respectively. these results are summarized in table 2 , along with 95% confidence intervals determined by fitting 1000 bootstrap replicates [46] . the longer latent infection period predicted for the h275y mutant strain, could be the result of poorer na activity. this would also explain the shorter infecting time for the mutant strain in that its virions would more easily bind to new cells with less interference from its na activity. to investigate whether the h275y mutant had poorer na activity compared to the wt, we directly measured the enzymatic activity of the na of each virus strain using the through modeling and simulation of two common in vitro experiments, the plaque and viral yield assays, we have extracted we have shown that seemingly contradictory results from the two experiments -plaques of the susceptible strain grow more quickly than the resistant strain, while the reverse is true of their titer growth in viral yield assays -can be considered complementary views of the infection kinetics which allow for the determination of parameter values controlling the replication of each strain. specifically, we have found that the latent infection period of the h275y mutant strain -equal to the time elapsed between the successful infection of a cell by a virion and the significant release of virus progeny by the newly infected cell -is much longer than that of the wt strain (by 4-10 h). the infectivity of the mutant strain, however, was found to be much higher than the wt, as quantified by the infecting time -equal to the time for a single infectious cell to cause the latent infection of one other, within a completely susceptible cell population. independent single-cycle viral yield assay results lend support to the hypothesis of a longer latent infection period for the mutant strain than the wt, but suggest a more moderate (*2h) difference between the two. these results are consistent with the larger na activity of the susceptible (wt) strain compared to the h275y mutant, reported here and by others [25] , and its increased na surface expression [24] . since neuraminidase is the viral surface enzyme responsible for cleaving the virus from its sialic-acid receptors at the cell surface [47] , it can be expected that an increase of its expression would lead to more rapid viral release (a shorter latent infection period for the wt strain) but may also hinder the subsequent attachment of virions to other cells, leading to decreased infectivity (longer infecting times). a complete understanding of the viral kinetics requires investigation of the ha/na balance. it has been shown that the a/brisbane/59/2007-like strains of the 2007-2009 influenza seasons differ from earlier h1n1 seasonal strains by a few amino acid substitutions in the ha gene [25] , but none of these involve interaction with the receptor and are therefore not likely to have influenced the changes in fitness. we have recently sequenced the entire genomes of our a/brisbane/59/2007 strains, and found three amino acid substitutions in the ha gene for the h275y mutant compared to the wt strain. two substitutions, g189v and l264f, do not involve interaction with the receptor, but the third, a193t, lies within the receptor-binding site. this latter substitution has been noted in earlier work in relation to oseltamivir-resistant strains of the influenza virus [48] and an investigation of its influence on viral kinetics is a necessary direction for future work. mathematical models have been successfully applied previously to characterize the in vivo virus replication kinetics of hiv [31, 32] , hepatitis b and c [33, 49] , and influenza [34, 36, 50] , as well as in vitro viral yield experiments studying the effects of antiviral drugs [35] and the optimization of vaccine production [51, 52] . models of viral plaques have also been considered [53] [54] [55] , although these were primarily directed at phage growth in an agar suspension of bacteria, a slightly different system than the cell monolayers considered here. the method presented here for the determination of the infection parameters differs from previous mathematical modeling approaches to viral dynamics in that we have considered the explicit dependence of two experimental quantities on the parameters, rather than fitting a full dynamical model to the time-course of an experiment. there are a number of benefits to this approach. first, we have been careful to determine that the two experimental quantities under consideration, plaque velocity and viral titer growth rate, depend on only two unknown infection parameters, the infecting time, t inf , and the latent infection period, t l . this ensures that the problem of parameter extraction is theoretically solvable, which is often not the case when fitting a multi-parameter model to experimental data (see, e.g., [56] ). second, the experimental quantities themselves are robust and easily measurable in repeated experiments. the viral plaque is formed by the progression of an infection wave across the monolayer of cells [42] whose constant velocity is determined by the infection kinetics averaged over many thousands of cells. therefore the measured plaque velocity depends on the average interaction of virus and cell, and is insensitive to stochastic effects on a small scale. similarly, the viral titer growth rate is due to the collective infection of thousands of cells and is independent of the details of initial infection (i.e., the precise value of the multiplicity of infection) or the total number of cells in the system. other quantities of in vitro experiments, such as the time and value of the viral peak in a yield experiment, are much more sensitive to experimental details. finally, the method we have applied here is robust to changes in the construction of the mathematical model itself. we have, for example, performed the same analysis of the plaque and yield assays using a stochastic model with more general assumptions about the cell transitions from latently infected to infectious and found nearly identical results (not shown here). it is important to note that the results we present here are preliminary, a proof of concept for the method which requires further verification and refinement. in particular, it would be useful to develop an experimental assay which could measure the infecting time for a given strain, in the same way that single-cycle viral yield experiments give an approximate measure for the latent infection period. it is also of interest to design a set of experiments which may be less expensive and laborious than those presented here, perhaps using fluorescent or photographic observations of cell cultures rather than virus titrations, and which can identify a fuller set of viral kinetics parameters. we are currently designing competition experiments for the a/brisbane/59/2007 wt and h275y mutant strains in which the predictions which follow from the parameters extracted here can be tested directly. when verified, the basic method of analyzing parallel plaque and viral yield experiments introduced here should be useful in other contexts. for example, the investigation of other drug-resistant viruses (e.g., that of the pandemic a/h1n1), the rapid characterization of fitness for emerging strains, and assays measuring the activity of new antivirals would all be enhanced through the application of our method. the a/brisbane/59/2007-like (h1n1) strains used were the oseltamivir-susceptible a/québec/15230/08 (wt) and the oseltamivir-resistant a/québec/15349/08 (na-h275y mutant). these clinical isolates were obtained from two distinct, untreated, immunocompetent patients during the 2007-2008 influenza season [22] . all experiments were performed on st6gali-expressing mdck cells [29] which over-express the a -(2,6) sialic acid receptor predominantly found in the human upper respiratory tract. prior to infection, cells were grown to confluence, achieving an average diameter of *20mm (used herein as a unit of length, d cell ). plaque assays were prepared using a semi-solid overlay of 1.2% avicel rc-581 (fmc biopolymers, newark, delaware, usa) as described by matrosovich et. al. [57] and stained with crystal violet. six-well plates (corning life sciences, lowell, ma, usa) were infected with 25+10pfu=well, representing a multiplicity of infection (moi) of approximately 10 {5 , and stained every 12 h for 96 h. the plates were then photographed using a dslr camera (fujifilm s2 with a 60 mm nikkor macro objective) and the areas of viral plaques were measured using the threshold and analyze particle features of imagej, an nih opensource image analysis software [58, 59] . all plaque radii at one timepoint (three independent experiments of three wells each) were averaged and the standard error of the mean was calculated. the radial growth rate was determined by linear regression to the average radii at time points prior to 72 h. multiple-cycle viral yield assays for the a/brisbane/59/2007 wt and h275y mutant were performed with moi&10 {5 . supernatants were harvested every 6 h for the first 36 h of infection and every 12 h subsequently, then titrated by plaque assay as previously described [22] . the geometric average and standard deviation was determined from three replicates at each time point. high moi single-cycle yield assays were performed as described by hurt et. al. [60] . monolayers of st6gali-mdck cells were grown to confluence in 12-well plates and infected with 10 6 pfu well (moi = 1) in 1 ml of infection medium. virus was adsorbed for 1 h at 37 0 c in a co 2 incubator. the supernatant was then removed and cells were quickly washed once with acidic saline (0.9% nacl in water, ph 2.2) and twice with pbs (ph 7.4). fresh maintenance medium was added and plates were returned to the incubator. supernatants were harvested every two hours for 24 h ( figure 7a , experiment 1) or every hour for 14 h (experiment 2), in duplicate. samples were frozen at {80 0 c until titrated in duplicate by plaque assay [57] . the enzyme kinetics of the neuraminidase was measured in duplicate for each strain as described in [61] , using the munana reagent (4-methyl-umbelliferyl-n-acetyl neuraminic acid (sigma-aldrich, st-louis, co, #m8639)). briefly, 10ml of live viruses diluted to 1:2|10 6 pfu ml were incubated at 37 0 c in opaque black microfluor b cs50 96-well plates (vwr, montreal, qc, #62402-983) with 30ml of munana reagent ranging from 0 to 3000mm final concentration and 10ml of enzyme buffer [1:1 mix of 325mm mes (2-[n-morpholino]ethanesulfonic acid) ph 6.5 (sigma-aldrich, st-louis, co, #m8250) and 10mm cacl 2 (sigma-aldrich, st-louis, co)]. fluorescence was measured in a viktor 3 multilabel counter (perkinelmer, waltham, ma) every 90 seconds for 45 minutes. the excitation wavelength was 365nm and the emission wavelength 450nm with a 2:5nm excitation slit and a 20nm emission slit. k m and v max were calculated using a homemade excel macro, created following [62] , and confirmed using the built-in ''enzyme kinetics'' features of the graphpad prism 5.01 software (graphpad software, la jolla, ca). plaque growth was simulated using a one-dimensional, timedelayed, partial differential equation (pde) model: where t(r,t) and i(r,t) are the densities of target and infectious cells, respectively, and v (r,t) is the virus concentration. s (20-fold smaller than the stokes-einstein value for a 100 nm particle in 37 0 c water); the rate of viral infectivity loss was fixed at c~0:19h {1 based on the observed viral titer decay rate for both a/brisbane/59/2007 strains in the multiple-cycle viral yield assays (see results); and the infectious lifespan was held fixed at 12 h (table 1) . simulations were initialized with a ''top-hat'' central region of infectious cell density with radius d cell =2, and with all other cells in the target state. all fields rapidly take the form of traveling waves t(z), i(z) and v (z), where z~r{vt, with the same velocity, v. the multiple-cycle viral yield assay was modeled using a meanfield, delay-differential system of equations: where t and i are now the number of target and infectious cells, n is the total number of cells, v is the homogeneous virus concentration and all parameters have the same meaning as in the pde model above. these equations can be derived from equation (1) by assuming spatial homogeneity and integrating over space. an expression for the exponential growth rate, l g , of viral titer in the multiple-cycle yield assay can be derived from this system by assuming that in the early phases of an infection (well before the viral titer peak): the number of target cells is approximately constant t(t)~t 0 &n; there is an exponential growth of infectious cells i(t)~i 0 e lgt and virus v (t)~e lgt with common rate l g ; and infectious cell death can be neglected (t i ??). substituting these expressions into (2) yields a transcendental equation for the viral titer growth rate: for any values of p, b, t l and c, this equation can be solved numerically for the viral titer growth rate, l g . the assumptions made in deriving the expression require that the viral titer growth rate be measured early in the course of infection, well before the time of peak, when the number of infected cells is small compared to the total number of cells. both the plaque velocity and viral titer growth rate depend on the infection and production rates only through pb. since this quantity has units of inverse time squared (units of virus cancel), it is useful to rewrite the dependence on these rates as a characteristic time, ffiffiffiffiffi ffi 2 pb s . a physical meaning can be ascribed to this quantity by considering equation (2) in the case of a single infectious cell (i 0~1 ), within a completely susceptible cell population (t 0~n ). if loss of viral infectivity, c, is neglected, the equations can be then integrated to show that t inf~ffi ffiffiffiffi ffi 2 pb s is the time for that single infectious cell to cause the (latent) infection of one more cell. therefore, we call this characteristic time the infecting time. the contour plots in figure 6 were created using the functional dependence of the plaque velocity and viral titer growth rate on the infecting time, t inf , and the latent infection period, t l , as determined by model simulation, along with the experimentally measured values of these quantities and their associated measurement error, under the assumption that these errors are normallydistributed. for example, the function f v , plotted in figure 6a and figure 6d , takes values between zero and one, according to where v exp is the experimentally-measured plaque velocity with measurement error s v and v mod (t inf ,t l ) is the theoretical dependence of the plaque velocity determined by model simulation. contours for the one and two-s values are drawn at f v~0 :6065 and 0:1353. a function on the parameter space, f lg , for the viral titer growth rate is constructed analogously. the product of these two functions is plotted in figures 6c and 6f to show the likely regions of viral kinetics parameters controlling growth for each virus strain. in fitting the single-cycle viral yield data, a more biologicallyrealistic model was used which assumes that the set of latent infection periods for a collection of cells is normally-distributed about t l , rather than fixed [63] . in this model, target cell and virus dynamics are identical to that of equation (2) where l 0 and i 0 are the number of cells latently infected and infectious, respectively, at the start of the experiment, f l (t) is the probability density function for the latent infection period and p i (t) is the probability that a cell remains infectious for at least a time t after the latent-infectious transition. if a dirac delta function is used for f l (t) and a heaviside step function for 1{p i (t), then the infectious cell dynamics of equation (2) are recovered. in the fits to the single-cycle data (figure 7 ), f l (t) was taken to be normal (truncated at t~0 and renormalized) with parameters t l and s l ; the function p i (t) was also derived from a normal distribution f i (t) with p i (t)~1{ d dt f i (t) , with fixed parameters t i~1 2h and s i~1 h. oseltamivir for treatment and prophylaxis of influenza infection inhibition of neuraminidase inhibitor-resistant 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parameter identifiability and estimation of hiv/aids dynamic models new low-viscosity overlay medium for viral plaque assays a simple and fast method for determining colony forming units assessing the viral fitness of oseltamivir-resistant influenza viruses in ferrets, using a competitive-mixtures model fluorometric assay of neuraminidase with a sodium (4-methylumbelliferyl-a-d-n-acetylneruaminate) substrate advanced biochemistry: protein properties and kinetics (btc 560) -course website exploring the effect of biological delays in kinetic models of influenza within a host or cell culture induction of programmed cell death (apoptosis) by influenza virus infection in tissue culture cells apoptosis: a mechanism of cell killing by influenza a and b viruses ns1 protein of influenza a virus down-regulates apoptosis impairment of multicycle influenza virus growth in vero (who) cells by loss of trypsin activity mode of action of the anti-influenza virus activity of plant flavonoid, 5,7,49-trihydroxy-8-methoxyflavone, from the roots of scutellaria baicalensis the primary function of rna binding by the influenza a virus ns1 protein in infected cells: inhibiting the 29-59 oligo(a) synthetase/ rnase l pathway the computational aspects of this work were made possible by the facilities of the shared hierarchical academic research computing network (sharcnet: www.sharcnet.ca) and compute/calcul canada. we wish to thank marc auger for photographing the plaques. key: cord-001446-mpuovmeb authors: bratcher, preston e.; gaggar, amit title: factors influencing the measurement of plasma/serum surfactant protein d levels by elisa date: 2014-11-03 journal: plos one doi: 10.1371/journal.pone.0111466 sha: doc_id: 1446 cord_uid: mpuovmeb background: extensive variations in human surfactant protein d (sp-d) levels in circulation as measured by elisa exist in the published literature. in order to determine the source of these variations, factors influencing the measurement by elisa were explored. materials and methods: peripheral blood from healthy individuals was collected into various vacutainers during the same blood draw. recombinant sp-d was diluted into different matrices and used for a standard curve. samples were analyzed by capture elisa using one of two distinct detection antibodies. results: the type of matrix had some effects on detection of recombinant sp-d. the type of anticoagulant used and dilution factor had very little effect, except for in plasma collected in edta vacutainers. the extent of variation in published values seemed to be due to the elisa configuration employed, and, in agreement with this, we found that by switching the detection antibody, there was a 50% decrease in the extrapolated sp-d value of serum and plasma samples. storage of samples resulted in slight changes in measured sp-d levels. conclusions: the elisa configuration employed to measure circulating levels of sp-d has a significant effect on the extrapolated values. in both configurations tested, the use of edta as a coagulant resulted in inconsistent values, and we, therefore, suggest the avoidance of this anticoagulant when assaying for sp-d by elisa. while the demonstrated effects of several factors on measurement of sp-d may not account for all the disparities amongst the previous studies, they stress that variations in methodologies for measuring the same protein can result in very inconsistent results. surfactant protein d (sp-d) is a pulmonary collectin involved in regulation of inflammation, innate immune defense, and surfactant homeostasis. it is expressed by clara cells and alveolar type ii cells in the lung. sp-d has a multimeric structure which gives it the ability to agglutinate pathogens, as well as aid in the clearance of apoptotic cells, cellular debris, and foreign particles in the lung [reviewed in [1] ]. circulating levels of sp-d have been examined for their potential use as a biomarker in various diseases including dermatitis [2, 3] , acute lung injury (ali)/acute respiratory distress syndrome (ards) [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] , periodontitis [14] , interstitial pulmonary fibrosis (ipf) [10, 12, [15] [16] [17] [18] [19] [20] [21] [22] [23] , chronic obstructive pulmonary disease (copd) [15, [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] , emphysema [37] , cystic fibrosis (cf) [15, 38, 39] , coronary disease [40, 41] , sclerosis [42] [43] [44] [45] [46] , cancer [47, 48] , sarcoidosis [21, 49] , allergies [28, [50] [51] [52] , rheumatoid arthritis [53, 54] , and respiratory infections [18, [55] [56] [57] [58] [59] [60] . sp-d levels have also been proposed to correlate with genetic elements [61] [62] [63] , body mass index (bmi) [64] [65] [66] [67] [68] , age [65] , circadian rhythm [69] , and with particle exposure [70, 71] and cigarette smoking habits [25, 27, 28, 31, 37, [72] [73] [74] [75] [76] [77] . in addition, there have been studies examining the levels of sp-d in subjects with turner syndrome [78] , paraquat intoxication [79] , swimming in variably treated waters [80] , lung transplant patients [81] , patients undergoing neurosurgical operations [82] , drowning victims [83] , polymyositis/dermatomyositis [84] , dementia [85] , lupus [86] , and sleep apnea [87] . similarly to cc-16 and kl-6, sp-d is thought to be a marker of pulmonary leak into the vasculature [88] , and therefore alveolar destruction would result in an increase in levels of these pulmonary proteins in the blood. however, protein levels in lung do not always correlate with protein levels in blood [35] , suggesting the possibility of alternative mechanisms affecting sp-d levels in circulation. various commercially available kits and non-commercially available elisa configurations have been used to compare sp-d levels in plasma and serum from normal, healthy controls and the various disease states described above. interestingly, there is a very substantial discrepancy between the reported values of the healthy control populations between studies, as well as in the magnitude of the range of values in this population. while the elisa configuration used to measure sp-d seemed to have a large impact on the values reported, there are significant variations in the healthy control sp-d levels and range amongst reports using the same configuration. the purpose of this study is to determine factors affecting the measurement of sp-d by elisa that may, therefore, explain the variations of serum and plasma sp-d levels reported in the published literature. all human studies were approved by the university of alabama at birmingham institutional review board for human use with all subjects providing written consent. peripheral blood was collected from healthy volunteers by venipuncture into serum, heparin sulfate, k 2 edta, and sodium citrate vacutainers (bd biosciences) during a single draw. samples were kept at room temperature until blood in the serum tube was coagulated. afterwards, samples were centrifuged at 400xg for 10 minutes to separate blood cells from serum or plasma. samples were either directly aliquoted and stored at 280uc or given an additional round of centrifugation at 3000xg for 10 minutes to separate platelets from serum or plasma. all serum and plasma sample values depicted in figures were free of platelets. all experiments had 6 samples per group except for the experiments depicted in mouse antihuman sp-d capture antibody, biotinylated mouse antihuman sp-d detection antibody, streptavidin conjugated to horse radish peroxidase, and recombinant human sp-d standard were purchased as a kit from r&d systems (catalog # dy1920) and used according to the manufacturer's protocol. for experiments testing the effects of different matrices on the detection of recombinant human sp-d, a concentrated stock was made in pbs with 1% bsa from separately purchased recombinant human sp-d expressed in nso cells (r&d systems, catalog # 1920-sp-050). for studies comparing detection antibodies, polyclonal goat antihuman sp-d antibody (r&d systems, catalog # af1920) was used instead with a monoclonal mouse antigoat conjugated to horse radish peroxidase (sigma) used as a secondary. all sample values were extrapolated from a second order polynomial curve fit of 9 concentrations of the standard (two-fold dilutions with a high concentration of 40 ng/ml) diluted in 1% bsa pbs. statistical analyses were performed as described in the figure legends using prism 5 (graphpad software), and all reported p values are twotailed. all error bars represent standard deviation. in order to determine the mean and range of sp-d levels in the serum/plasma of normal, healthy individuals, we performed a non-exhaustive search of the literature which revealed more than 60 publications in which these values were described. interestingly, an unexpected amount of variation on these reported values was observed. when grouping according to the elisa configuration used, one configuration consistently resulted in significantly higher values than the two other configurations for which multiple references were obtained (figure 1a ). for each configuration, a substantial range of means/medians was observed in the healthy control population. in addition to the variations seen in the averages, the spectrum of values in all populations ranged from 55.9 pg/ml [22] to 3.9653 mg/ml [89] , representing a more than 70,000 fold difference. when examining the range of values seen in healthy subjects from each individual study and grouping according to the elisa configuration used, a large variation in range was observed ( figure 1b) . overall, the study with the smallest range for any population (as a percentage of the average) reported a 95% confidence interval of 95.7-109.0 [54] , while the study containing the population with the largest range reported a standard deviation of 327% of the mean value [42] . based on the above observations, we can infer that the largest difference in the measured levels of sp-d is due to the elisa configuration employed, but that there are still significant differences between the averages of healthy individuals from studies using the same configurations. in order to compare the results of various studies in another manner, we examined the fold change from healthy populations for the four diseases/conditions for which a substantial number of publications were available (ipf, cf, cigarette smoking, and systemic sclerosis). this would allow us to control for elisa configurations employed, technical protocol followed for the elisa as well as for sample collection and processing, and any differences in the populations (as controls have been appropriately matched). the expectation was that the fold change from healthy would be very similar for each disease/ condition. however, large differences in the extent of this change were observed (figure 1c ). in order to determine if the matrix used to generate the serial dilutions of recombinant human standard (rhsp-d) had any effects upon measurement, we compared values produced by dilutions in pbs, solutions of bsa (in pbs), and solutions of fbs (diluted in pbs) (figure 2 ). at higher concentrations of protein (i.e. 5% bsa, 50% fbs, and 100% fbs), a decrease in the amount of rhsp-d detected was observed, with the amount measured in samples containing 10 ng/ml rhsp-d being the most variable of any samples measured in this assay. using the manufacturer's recommended matrix (1% bsa) produced results very similar to a 10% fbs matrix, with both giving very consistent measurements and the greatest values. the values for pbs matrix gave similarly consistent measurements, but at a slightly lower value. this effect may be due to adsorption of rhsp-d without a carrier protein to the tubes used for serial dilutions [90] . serum matrix (millipore), which had background levels of sp-d, also inhibited detection of recombinant sp-d at higher concentrations. all further standard curves were established by serial dilutions of recombinant sp-d in 1% bsa in pbs. one factor that was found to differ between studies and could, therefore, be a source of variation of reported healthy population values, was the type of anticoagulant (or lack thereof) in the collection container. when blood was simultaneously collected in various vacutainers, serum and heparin plasma gave similar measurements of sp-d, while citrate plasma gave values significantly lower than serum values (figure 3 ). edta plasma gave the most inconsistent results, and values were also significantly lower than serum values. in order to determine if calcium concentration in the sample had a significant effect upon the measurement of sp-d, rhsp-d was assayed in serially diluted cacl 2 or edta. the detection of rhsp-d was only slightly effected by the changes in calcium concentration in the sample (figure 4a ). additionally, we examined the effects of calcium concentration in the human samples by adding calcium to edta plasma samples and by adding edta to serum samples. while reconstituting the free calcium in the edta samples had little effect, the addition of edta to the serum samples had a dramatic effect that was inconsistent amongst the patient samples. while many elisas employ the use of pbs lacking divalent cations as a buffer during the detection of antibody-captured antigens, the elisa configuration employed by holmskov et al. uses a tris-buffered saline solution containing 5 mm calcium chloride, as the monoclonal detection antibody binds to sp-d in the presence of calcium [58] . in addition, it was previously demonstrated that sp-d has the ability to bind to various immunoglobulins in a calcium-dependent manner [91] . in order to determine the effects of calcium in the context of sp-d detection by elisa, elisas were performed with antibodies diluted in buffer with or without calcium. we examined these effects on both recombinant sp-d and sp-d in plasma with either the elisa kit's detection antibody or a polyclonal goat anti-sp-d antibody produced by the same manufacturer. in all cases but one, the addition of 5 mm calcium to the dilution and wash buffer resulted in a small (,17%) but significant increase in sp-d concentration relative to detection in the absence of calcium (figure 4b ). it is important to note that with the kit reagents, since the relative increase in detection of recombinant sp-d and sp-d in plasma in the presence of calcium is very similar, the extrapolated sp-d concentration in plasma when using a standard curve of recombinant sp-d should not significantly change. interestingly, while the inclusion of calcium increased the recognition of native sp-d by the polyclonal antibody, it had no effect on the detection of recombinant sp-d by this antibody. although it is beyond the scope of this study, future work will explore whether this effect is due to an increase in antibody recognition of antigen, increase in non-specific binding of antibodies by captured sp-d, or an increase through another mechanism. given that some of the variation seen in the published literature might be explained by the use of different antibodies, we detected sp-d in serum and plasma samples using either the elisa kit's detection antibody or the polyclonal goat anti-sp-d antibody. while there is no significant difference between detection by the [12, [16] [17] [18] [19] 21] , cf [15, 38, 39] , cigarette smoking [28, 31, [72] [73] [74] [75] , or sclerosis [42] [43] [44] 46] was different between publications. an asterisk (*) denotes p#0.001 by one-way anova with tukey's multiple comparison test. doi:10.1371/journal.pone.0111466.g001 kit antibody versus the polyclonal goat antibody with regard to the type of collection tube used, overall, there is a ,50% reduction in the extrapolated value produced by detection with the polyclonal goat antibody compared to the kit antibody ( figure 5 ). in both cases, the same capture antibody was employed, and it is therefore possible that using a different capture antibody could have a similar effect on varying the extrapolated sp-d concentration in the sample. another factor that fluctuated from study to study was whether the sample was assayed neat or the sample was prediluted (and the amount the sample was diluted). given this fact and that diluting recombinant sp-d into 100% fbs had some influence in the detection of said sp-d, we compared sp-d values detected in undiluted and diluted samples. there was a very minimal difference between values produced by undiluted and 10 fold diluted serum, citrate plasma, and heparin plasma ( figure 6 ). ten fold diluted edta plasma, however, produced a significantly higher extrapolated sp-d value when compared to the same sample undiluted. this same effect was seen when the polyclonal goat antibody tested above was used for detection (data not shown). while the most common processing technique involved separating the blood cells (but not platelets) from serum and plasma and storing this sample at 280uc until assayed, some variations in processing and storage were present. to address this, we assayed serum and heparin plasma without platelets immediately after processing or after storage at 4uc, 220uc, or 280uc ( figure 7) ; there were no significant differences between conditions. additionally, there were only subtle differences between a sample stored at 220uc and 2 80uc, and depending on whether the sample went through a freeze/ thaw (ft) cycle, contained platelets or not, or was spun after thawing to separate any precipitates/debris (data not shown). sp-d concentrations of samples stored at 220uc and 280uc for two weeks were not different from the same samples at one week (data not shown). this study provides experimental evidence that variations in the anticoagulant used and elisa configuration can have dramatic effects upon the measured sp-d level. storage and processing of samples as well as diluent used for the standard have minor effects on the level of sp-d extrapolated by elisa. although these results may partially explain the variations seen in reported sp-d values in the blood, it is important to note the caveats associated with this review of the literature. for the comparison of healthy control groups amongst various studies, it is possible that differences in ethnic background and/or geographical location from which subjects were recruited may contribute to the variations seen when the same elisa configuration was employed, as could the type of anticoagulant. however, we would expect the range of samples between healthy populations of different studies to be similar. in addition, assuming that an appropriately matched control population was used for comparison to the disease population, the fold change between healthy and disease should be more similar than what was observed in the literature. measuring sp-d by elisa presents a problem due to its multimeric structure. for each molecule (a complete dodecamer) of multimerized protein, there are at least 12 of the same epitopes present. due to this property, it is not necessary to use discrete antibodies in a sandwich elisa format to capture and then detect sp-d; even the same monoclonal antibody could be used for both. however, this may cause a difference in read out compared to using antibodies that detect different epitopes, due to the fact that the degree of multimerization has been shown to vary in the lungs during different disease states [92] and using the same monoclonal antibody would fail to detect a monomer. even using two monoclonals that recognize distinct epitopes to measure sp-d in its native state would be confounded by differences in the degree of multimerization. for example, if an antibody against the carbohydrate recognition domain of sp-d was used to capture and an antibody against the neck region of sp-d was used to detect, a single, captured monomer of sp-d would be able to bind a single anti-neck antibody. however, a single, captured dodecamer would be able to bind 12 anti-neck antibodies. in order to accurately establish absolute concentrations by elisa, it would be important that the multimeric structure of the standard was equivalent to that of the samples, as there could be differences in antigen binding in higher order multimers due to steric hinderance. unfortunately, we did not have the necessary resources to examine the influence of the degree of multimerization on elisa measurements. in addition to the possible effects caused by its multimeric nature, there is evidence in the literature to variable posttranslational modifications of sp-d in native samples. these modifications may, in fact, mask the epitope recognized by an elisa antibody. this may account for one of the largest differences seen in the reported elisa measurements of sp-d in serum/plasma, as the highest concentrations of sp-d came from the non-commercially available elisa which used purified, native sp-d as a standard; this is opposed to almost all others, which used recombinantly expressed sp-d as a standard. regardless of whether the antibodies used were raised against recombinant sp-d or purified native, because the post-translational modifications can vary from one patient to the next, this presents a significant obstacle to accurate measurements of natural sp-d using antibodies. the results of this study demonstrate the influence that the antibody may have on the sp-d concentration as measured by elisa. in our experiments, it is important to note that the same recombinant standard and human serum/plasma samples were analyzed. given the confounders mentioned above, it is possible that the antibody from the kit recognizes an epitope that is not post-translationally modified or altered in human serum/plasma sp-d, while several of the epitopes recognized by the polyclonal antibody are modified in human serum/plasma sp-d, but were unmodified in the recombinantly expressed sp-d (which was used as the immunogen). this would result in the observed relative decrease in binding of the native sp-d from the blood compared to the recombinant sp-d. accuracy of sp-d measurements is critically important to the validation of this protein as a biomarker in pulmonary disease. standardization of sample processing and storage, including the avoidance of edta as an anticoagulant, is necessary to ensure consistent results. although absolute values may vary greatly due to the elisa configuration employed, relative differences in sp-d concentrations amongst various disease groups should be consistent throughout the published literature. we are currently exploring alternative methods to quantify sp-d levels in the blood, as we believe this is necessary in order to establish the absolute concentration. surfactant proteins sp-a and sp-d: structure, function and receptors surfactant protein d (sp-d) and systemic scleroderma (ssc) surfactant protein d in atopic dermatitis and 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victims clinical significance of serum surfactant protein d (sp-d) in patients with polymyositis/ dermatomyositis: correlation with interstitial lung disease serum surfactant protein d is correlated to development of dementia and augmented mortality circulating surfactant protein d is decreased in systemic lupus erythematosus comparison of biomarkers of subclinical lung injury in obstructive sleep apnea lung epithelium-specific proteins: characteristics and potential applications as markers the association between bmi and plasma cytokine levels in patients with acute lung injury characterization and prevention of the adsorption of surfactant protein d to polypropylene collectin surfactant protein d binds antibodies and interlinks innate and adaptive immune systems increased prevalence of low oligomeric state surfactant protein d with restricted lectin activity in bronchoalveolar lavage fluid from preterm infants the authors would like to thank tarek abdalla for his assistance with sample collection and processing. conceived and designed the experiments: peb. performed the experiments: peb. analyzed the data: peb. contributed reagents/materials/ analysis tools: peb ag. wrote the paper: peb ag. key: cord-001707-piyo00yg authors: murray, jillian; cohen, adam; walaza, sibongile; groome, michelle; madhi, shabir; variava, ebrahim; kahn, kathleen; dawood, halima; tempia, stefano; tshangela, akhona; venter, marietje; feikin, daniel; cohen, cheryl title: determining the provincial and national burden of influenza-associated severe acute respiratory illness in south africa using a rapid assessment methodology date: 2015-07-08 journal: plos one doi: 10.1371/journal.pone.0132078 sha: doc_id: 1707 cord_uid: piyo00yg local disease burden data are necessary to set national influenza vaccination policy. in 2010 the population of south africa was 50 million and the hiv prevalence was 11%. we used a previously developed methodology to determine severe influenza burden in south africa. hospitalized severe acute respiratory illness (sari) incidence was calculated, stratified by hiv status, for four age groups using data from population-based surveillance in one site situated in gauteng province for 2009–2011. these rates were adjusted for each of the remaining 8 provinces based on their prevalence of risk factors for pneumonia and healthcare-seeking behavior. we estimated non-hospitalized influenza-associated sari from healthcare utilization surveys at two sites and used the percent of sari cases positive for influenza from sentinel surveillance to derive the influenza-associated sari rate. we applied rates of hospitalized and non-hospitalized influenza-associated sari to census data to calculate the national number of cases. the percent of sari cases that tested positive for influenza ranged from 7–17% depending on age group, year, province and hiv status. in 2010, there were an estimated 21,555 total severe influenza cases in hiv-uninfected individuals and 13,876 in hiv-infected individuals. in 2011, there were an estimated 29,892 total severe influenza cases in hiv-uninfected individuals and 17,289 in hiv-infected individuals. the incidence of influenza-associated sari was highest in children <5 years and was higher in hiv-infected than hiv-uninfected persons in all age groups. influenza virus was associated with a substantial amount of severe disease, especially in young children and hiv-infected populations in south africa. influenza virus causes substantial disease in low-and middle-income countries each year, but data on the disease burden are limited [1] . data on influenza from country-level surveillance can show the distribution of disease among the population and identify groups at high risk of infection. this information can subsequently help policy makers make evidence-based decisions on how to target influenza treatment and prevention programs, such as vaccination, and can lead to more effective allocation of resources within a country [2] . south africa is a middle-income country, but there is great variation in socioeconomic status with some provinces that are more similar to low-income countries [3, 4] . this results in some populations within the country having a disproportionately higher level of exposure to risk factors for communicable disease. these include environmental risk factors, such as crowded living conditions and exposure to indoor air pollution, as well as biological risk factors, such as malnutrition and underlying infections [5] [6] [7] . these risk factors may drive the burden of influenza in south africa to be greater than other countries with similar income level [3, 8, 9] . in particular, the high hiv prevalence in south africa likely leads to higher numbers of severe influenza-associated illness and thus more hospitalized cases [9] [10] [11] [12] [13] [14] [15] . the influenza season in south africa occurs in the austral winter months of may to august [14, 16, 17] . influenza vaccine has been available in the public sector in south africa for many years but coverage is only approximately 5%. the recommended target groups for annual vaccination include pregnant women, persons with underlying medical conditions (including hiv), children less than 5 years of age and persons over 65 years of age [18] . the estimation of national influenza burden is challenging particularly in low resource settings where data on the national number of consultations, hospitalizations and deaths are lacking in most instances. we estimated influenza-associated severe acute respiratory illness (sari) by hiv status in south africa using a rapid assessment methodology [2] . the protocol for the sari surveillance system was approved by the research ethics committees of the universities of the witwatersrand and kwazulu-natal. the surveillance data used for the model was deemed non-research by the united states centers for disease control and prevention and did not need human subjects review by that institution. all data that was analyzed was de-identified and consent was obtained from all patients before they were enrolled into surveillance. information from the surveys used was publically available. we utilized a multiplier model to estimate numbers of individuals with hospitalized and nonhospitalized influenza-associated sari for each province in south africa in four age groups (<5, 5-24, 25-44 and 45 years) for 2009-2011, stratified by hiv serostatus (fig 1) . non-hospitalized sari includes cases from the population that do not reach the hospital due to health care access barriers. this method was previously used in kenya and guatemala but was modified to include stratification by hiv serostatus to reflect the impact of hiv on influenza burden in south africa [2] . [19, 20] . sentinel surveillance is conducted in four out of the nine south african provinces (gauteng, kwazulu-natal, mpumalanga and north west). hospitalized children aged <5 years are enrolled in surveillance with physician-diagnosed lower respiratory tract infection, and individuals 5 years are enrolled if they meet a modified who case definition for sari (sudden onset of fever with cough or sore throat and shortness of breath or difficulty breathing with 7 days from presentation to the hospital) [21] . population-based surveillance is implemented at the chris hani baragwanath academic hospital (chbah) in gauteng province. chbah is a large public academic hospital in the soweto township in gauteng, south africa. it serves approximately 1.4 million lower income south african individuals [12] . estimation of the rate of hospitalized sari cases in soweto (gauteng province) (fig 1, step 1). we estimated the total number of sari hospitalizations using the number of enrolled sari cases at chbah, adjusting for non-enrollment (refusal to participate and nonenrolment during weekends) by age groups and hiv status as previously described [12] . the rates of hospitalized sari cases in soweto were obtained by dividing the estimated number of sari cases by the mid-year study population estimates by age group and hiv status. the hiv prevalence in the study population was obtained from the projections of the actuarial society of south africa (assa) aids and demographic model. the population of soweto represents approximately 11% of the population of gauteng province (referred thereafter as the base province). we assumed that the incidence rates for soweto reflected the incidence rate for the province. at the time of this study, chbah was the only public hospital serving soweto, where approximately 10% have private medical insurance ( [22] . more than 80% of uninsured and 10% of insured persons seek care at public hospitals such as chbah. therefore, the majority of individuals in soweto that seek care do so at chbah [12] . estimation of the rate of non-hospitalized sari in soweto (gauteng province) (fig 1, step 2). we estimated the rate of non-hospitalized sari in the base province using the data from two healthcare utilization surveys for sari that were conducted in soweto and klerskdorp, north west province in 2013. non-hospitalized cases of sari refer to cases of sari identified in the population that had symptoms severe enough to be hospitalized but did not seek care at a hospital, for reasons such as limited access to healthcare. briefly, between 4,000 and 6,000 community members were surveyed in each of the two sites as part of a cross-sectional door-to-door household survey and asked if they had a severe pneumonia in the past year (as defined as fever and cough and difficult breathing for 2-30 days or a physician diagnosis of pneumonia) and whether they were admitted to a hospital for the event. the ratio of those that sought care to those that did not was applied to the rate of hospitalized sari to get the rate of non-hospitalized sari in the base province [2] . estimation of the rates of hospitalized and non-hospitalized sari in the other provinces (fig 1, step 3). estimates of hospitalized sari rates for the other eight provinces in south africa were derived by adjusting the soweto (gauteng province) sari incidence rates for the provincial-level prevalence of known risk factors for pneumonia (from the south africa demographic and health survey and the assa model). risk factors included exposure to indoor air pollution, crowding, malnutrition, low birth-weight and non-exclusive breastfeeding; the last three were only included as risk factors for children under five years of age [2, 8] . the relative risks of sari associated with each risk factor were determined from the published literature [8] . in addition, we adjusted the provincial rates by the proportion of ari seeking care in the given province to the proportion of ari cases seeking care in the base province (gauteng province) using health and demographic surveys (dhs) [4] . an upward adjustment factor (greater than one) resulted in a greater incidence of sari in the province relative to soweto (gauteng province). similarly, a downward adjustment factor (less than one) resulted in a decrease in sari incidence in the province relative to soweto (gauteng province). the equations used for the provincial adjustment are provided in the technical s1 appendix. the rates of non-hospitalized sari in the remaining provinces were estimated using the provincial prevalence of known risk factors for pneumonia as described for the hospitalized cases. in addition, we adjusted the provincial rates by the proportion of ari not seeking care in the given province to the proportion of ari cases not seeking care in the base province (gauteng province) using health and demographic surveys (dhs) [4] . estimation of the rates of influenza-associated hospitalized and non-hospitalized sari (fig 1, step 4) . the estimates of hospitalized and non-hospitalized sari rates were subsequently multiplied by the percent of sari associated with influenza virus from surveillance (i.e., the influenza detection rate) to give the incidence for influenza-associated sari. data on the percent of sari due to influenza by age group in separate hiv-infected and-uninfected populations were pooled from all sentinel surveillance sites to give an average percentage that was used in the calculations for each province. estimating the provincial and national numbers of hospitalized and non-hospitalized sari cases and influenza-associated sari cases (fig 1, step 5). the number of hospitalized and non-hospitalized sari cases and influenza-associated sari cases was obtained by multiplying the estimated rates by the mid-year population estimates. provincial population estimates stratified by age group and hiv status were obtained from the assa 2008 population model. in an alternate method, we utilized a non-hiv-stratified model. to account for hiv prevalence in this model, we included hiv as a risk factor for sari in calculating the provincial adjustment factors rather than estimating separate incidences for hiv-infected and-uninfected individuals. the relative risk for hiv was determined from the literature to be 7.3 for children under five years of age and 5.6 for children and adults over five years of age, and was used in previous applications of this methodology [2, 8, 23, 24] . the base sari incidence rate in this method was also obtained from the population-based surveillance at chbah and the same healthcare utilization adjustments were made. data on the proportion of sari cases that were influenza-associated was pooled across surveillance sites to give an average percentage that was applied to all provinces. confidence intervals were estimated by bootstrapping each parameter included in the calculations 1000 times, including the sari rates in the base province, the influenza detection rate among sari cases, the prevalence of the provincial risk factors, the proportion of sari cases seeking care from the hus in the base province and the proportion of ari cases seeking care in each province form the dhs. the upper and lower limits of the 95% confidence intervals were the 2.5 th and 97.5 th percentile of these estimates, respectively. the highest rates of sari in both hiv-infected and hiv-uninfected populations were among children less than five years of age, ranging from 2,253-5,507 per 100,000 in the hiv-infected population and 1,609-2,027 per 100,000 in the hiv-uninfected population over the three year study period [12] . the incidence rate of sari in the hiv-infected population was consistently greater than the incidence in the hiv-uninfected population for all age groups. the rate ratio between hiv-infected and hiv-uninfected persons was highest in the 5-24 year old age group (range [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] and lowest in the under-5 year olds (range 1-3). sari rates varied by year, with 2009 having the highest incidence rate for most age groups among both hiv-infected and hiv-uninfected populations. base hospitalized influenza-associated sari rates, soweto, gauteng similar to the base incidence of sari, the base influenza-associated sari incidence rate was greater among hiv-infected individuals for all three years and highest in children <5 year of age, ranging from 140-844 and 93-366 per 100,000 person-years in the hiv-infected anduninfected populations, respectively, over the three year time period (table 1) . among adults, influenza-associated sari rates were highest among the oldest age group (>45 years) for hivinfected (range 143-266 per 100,000 person-years) and hiv-uninfected populations (20-54 per 100,000 person-years) ( table 1) . as the base province, gauteng had a risk factor adjustment factor of 1. the eastern cape consistently had the largest downward adjustment factor (range 0.9-1.1), while mpumalanga consistently had the largest upward adjustment factor (range 1.5-1.9; s1 table) . across the three surveillance years studied, mpumalanga and the northern cape consistently had the highest sari incidence rate across both hiv-infected and uninfected groups and the four age groups studied (s2 table) . rates for hospitalized influenza-associated sari stratified by age and province are given in table 1 . the estimated incidence of influenza-associated sari was consistently highest among the hiv-infected population across all age groups in all of the nine provinces for the surveillance years 2009-2011. within both the hiv-infected and hiv-uninfected populations, the highest incidence of influenza-associated sari was found in the <5 year olds for all provinces and surveillance years. in 2009, the national number of hospitalized influenza-associated sari cases was estimated to be 22,525 in hiv-uninfected individuals and 8,715 in hiv-infected individuals ( table 2) . at two sites where healthcare utilization surveys were conducted, 61% of influenza-associated sari cases were not hospitalized. in 2009, the total number of hospitalized and non-hospitalized influenza-associated sari cases was estimated to be 47,711 in hiv-uninfected individuals and 17,993 in hiv-infected individuals ( table 2 ). in 2010 and 2011, there were fewer overall estimated influenza-associated sari cases compared to 2009. in 2010, there were an estimated 21,555 total severe influenza cases in hiv-uninfected individuals and 13,876 in hiv-infected individuals ( table 2 ). in 2011, there were an estimated 29,892 total severe influenza cases in hiv-uninfected individuals and 17,289 in hiv-infected individuals ( table 2) . in our alternate method in which hiv was adjusted for as a risk factor rather than a stratifying variable, the greatest impact on the adjustment factors was for the 25-44 year old age group where the hiv prevalence is highest. the eastern cape province still consistently had the largest downward adjustment factor, except in the 25-44 year old age group where limpopo had the largest downward adjustment. however, kwazulu-natal province consistently had the largest upward adjustment factor across all age groups, due to the high hiv prevalence in that province. the results from method 2 yielded similar incidence of influenza-associated sari and number of hospitalized cases across the age groups and years included in the analysis (s3 table) . the non-stratified alternate method estimated a total of 74,558 cases of hospitalized influenza-associated sari for 2009-2011, only 6% more than the 70,607 cases estimated by the stratified model for the same years. influenza-associated sari represents a substantial burden of disease in south africa. using a rapid assessment methodology, we found that the highest rate was seen in children <5 years of age irrespective of hiv status. among adults, the highest number was seen in hiv-infected adults aged 25-44 years. previous studies of influenza in south africa have not calculated a national burden [25] [26] [27] [28] and have not estimated the non-hospitalized sari burden. our methodology allowed estimation of the number of cases in each province by age group and hiv serostatus, which can support decisions on targeted interventions within the confines of limited resources. our study highlights the important role that hiv infection plays when estimating severe influenza burden in settings with high hiv prevalence. among the age group with the highest hiv prevalence (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) (35) (36) (37) (38) (39) (40) (41) (42) (43) (44) year olds), hiv-infected persons accounted for 80% of the national burden of influenza-associated sari, despite representing only 30% of the national population [29] . hiv-specific incidences for influenza-associated disease in south africa have been reported previously to be high in hiv-infected individuals [11, 12] . a study in children 2-60 months of age in 1997 reported influenza-associated severe lower respiratory tract infection incidence rates of 1,268 and 148 per 100,000 for hiv-infected and hiv-uninfected children, respectively [11] . more recently, an analysis of population-based data from chbah found influenza-associated lower respiratory tract infections (lrti) to be highest in the hiv-infected population [12] . some countries may not be able to calculate hiv-specific influenza incidence rates due to lack of reliable hiv-stratified influenza data in the population. in the non-hiv-stratified alternate method, we derived similar estimates of burden using a model incorporating adjustment for hiv prevalence by province. this suggests that hiv-specific influenza incidence rates are not necessary to derive valid overall burden estimates because hiv prevalence by province is available in most african countries through demographic and health surveys or multicluster indicator surveys [4] . there were several limitations to our methodology. our study relies on extrapolating incidence rates from one surveillance site in one province to the rest of the provinces of south africa. although a high percentage of the base province is under surveillance from this one large surveillance site and we adjusted this incidence based on province-level risk factor for sari, there are likely additional unmeasured factors that can lead to variation in rates regionally. we assumed that the relative risk of sari for the different risk factors determined for the populations in the literature are the same relative risks for the population of south africa, which might not be the case [8, 23, 24] . second, the denominator estimates also might have been inaccurate. we used the assa 2008 population model to estimate the population and hiv prevalence by province and age group. these population estimates are extrapolations from the 2001 census. third, the healthcare utilization surveys used to calculate non-hospitalized cases was undertaken in only two sites in two provinces. moreover, the surveys did not stratify health-seeking by hiv status or age, which is likely to influence health-seeking behavior. fourth, these estimates reflect the dsease burden in the public sector; however, a similar study using a different modelling approach in the private health sector of south africa found similar incidence rates of influenza-associated respiratory hospitalizations [30] . lastly, we assume that the percent of influenza-associated sari is the same across all age groups, provinces and hiv status. this rapid assessment methodology has been used in kenya, guatemala and now south africa [2] . other countries that have influenza surveillance in place can also implement this methodology to estimate their national burden of severe influenza disease. we found that provinces in south africa vary in the prevalence of important risk factors for sari and influenza and that these impact the burden of severe disease. current recommendations in south africa include the vaccination of hiv-infected individuals with the seasonal influenza vaccine; however, the vaccine tends to be under-utilized in this population and in south africa in general [31] . the results of this rapid assessment can be used to focus efforts targeting influenza vaccination by demonstrating the distribution of disease across south africa. supporting information s1 global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis estimation of the national disease burden of influenza-associated severe acute respiratory illness in kenya and guatemala: a novel methodology surveillance and management of influenza in africa: an urgent need still to be met undernutrition can affect the invading microorganism social mixing patterns within a south african township community: implications for respiratory disease 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virus strains isolated from a localised influenza outbreak in south africa in 2003 south african national hiv prevalence, incidence, behaviour and communication survey 2008: a turning tide among teenagers? cape town hospitalizations associated with influenza and respiratory syncytial virus among patients attending a network of private hospitals in south africa coverage of high risk groups for influenza vaccination in south africa, 2011-2013 influenza seasons. options for the control of influenza cape town key: cord-002889-fie121ns authors: white, michael; freistaedter, andrew; jones, gwendolyn j. b.; zervos, emmanuel; roper, rachel l. title: development of improved therapeutic mesothelin-based vaccines for pancreatic cancer date: 2018-02-23 journal: plos one doi: 10.1371/journal.pone.0193131 sha: doc_id: 2889 cord_uid: fie121ns pancreatic cancer is the 5(th) leading cause of cancer deaths, and there are no effective treatments. we developed a poxvirus platform vaccine with improved immunogenicity and inserted the mesothelin gene to create an anti-mesothelin cancer vaccine. mesothelin expression is mostly restricted to tumors in adult mammals and thus may be a good target for cancer treatment. we show here that the modified vaccinia virus ankara (mva) virus expressing mesothelin and the enhanced mva virus missing the immunosuppressive a35 gene and expressing mesothelin were both safe in mice and were able to induce ifn-gamma secreting t cells in response to mesothelin expressing tumor cells. in addition, the mva virus has oncolytic properties in vitro as it can replicate in and kill panc02 pancreatic adenocarcinoma cell line tumor cells, even though it is unable to replicate in most mammalian cells. deletion of the a35 gene in mva improved t cell responses as expected. however, we were unable to demonstrate inhibition of panc02 tumor growth in immunocompetent mice with pre-vaccination of mice, boosts, or even intratumoral injections of the recombinant viruses. vaccine efficacy may be limited by shedding of mesothelin from tumor cells thus creating a protective screen from the immune system. pancreatic cancer is the 5 th leading cause of cancer deaths in the united states [1] and has the lowest 5-year survival rate (7%) for solid tumors [2] largely owing to the fact that radiation, surgery, and current chemotherapy options are ineffective. new treatment strategies are essential. human mesothelin is normally expressed only in mesothelial cells of the pleura, peritoneum, and pericardium. however, mesothelin is overexpressed in a high percentage of ovarian cancers, pancreatic cancers, non-small lung cancers, and mesotheliomas, making it a potential target for anti-cancer treatments [3] [4] [5] [6] [7] . human and mouse mesothelin share sequence similarity, expression patterns, and biochemical characteristics, [7] , and the homeostatic function of mesothelin in mammals is unknown: the gene can be deleted without apparent effect in mice plos c57bl6 mice and thus can be grown in syngeneic mice to allow for study of an anti-tumor immune response in an immunocompetent mouse model. this has a great advantage over tumor studies performed with xeno-or allografts in immunocompromised mice. to determine whether we can improve vaccine immunogenicity and efficacy, we test the parental normal mvameso virus vaccine against the mva mesothelin expressing virus that has had the immunosuppressive a35 gene removed (mvamesoa35del). all animal experiments were conducted with the approval of the east carolina university animal care and use committee (k157) in an aaalac accredited animal facility. isoflurane anesthesia was used for tumor injections and for euthanasia. the c57bl/6 chemically-induced pancreatic adenocarcinoma cell line, panc02, was a kind gift from dr. keping xie (md anderson cancer center, houston, tx). panc02 cells were grown in rpmi, with 10% fetal bovine serum, 2 mm glutamine, 100 u/ml of penicillin and streptomycin. mesothelin over expressing panc02 cell lines are described in zervos, et al [7] . cells were incubated at 37˚c with 5% co 2. vaccinia virus, mva, and the mva a35 deletion mutant viruses were previously described [29, 31] . baby hamster kidney (bhk) cells were employed for growth of mva and were cultured using dulbecco's modified eagle medium (dmem), supplemented with 10% fetal bovine serum, 2 mm glutamine, 100 u/ml of penicillin and streptomycin. in order to create a therapeutic anti-mesothelin vaccine, we inserted mouse mesothelin into the mva viral genome. we used the normal wild type mva or the mva in which the a35 virulence gene had been deleted [31] to create the mvameso virus and the mvamesoa35del recombinant viruses respectively. in both cases, mesothelin was cloned into a vector (plw44, a gift from l. wyatt, national institutes of health) designed for insertion into an area of the mva genome where genes had been naturally deleted during passage of the virus [44] . recombinant viruses expressing the gfp marker protein were selected, purified, and analyzed to confirm the genotype by raid prep pcr analysis [45] . the presence of the mesothelin gene was confirmed using mesothelin-specific primers (ggctggctatggctgtaagac and agcagtgt tggatgagtccatcgt) with an annealing temperature of 51˚c. in order to assess the presence of the a35 gene, recombinant viruses were analyzed using a35 specific primers (tgacta taacagatacatt and cagattatctgaaggattgat) at an annealing temperature of 40˚c. to confirm the insertion sites of the mesothelin, we used primers in the mva flanking regions, (cataagtataaagtccgactattgttc and gataaagttgcatcatcacctat). the pcr amplicons were run on an 0.8% agarose gel. peptides representing murine mesothelin coding sequence, gvygfqvseadvralgglac and cppgkepykvdedlifyqn, were synthesized and conjugated to klh carrier proteins via the terminal cysteine residues (underlined, genemed synthesis), and used for immunization of two rabbits [7] . sera were confirmed for reactivity with the immunizing peptides by elisa. bhk cells (2x10 6 cells/well) were uninfected or infected with the 3 mva viruses at a multiplicity of infection (moi) of 5 plaque forming units (pfu, infectious virus particles) of virus per cell for 13 h in 5% co2 atmosphere. cells were lysed with disruption buffer and heat inactivated at 95˚c for five minutes. dna was sheered using a 22-gauge needle and samples were loaded onto a pre-cast 4-20% gradient gel (thermo scientific) for sds-page and transferred overnight. membrane was blocked for 30 minutes at room temperature with 1% fat free milk in tbs, and incubated with rabbit anti-mesothelin antibody (1:10,000) for 2 h at room temperature, washed, and then incubated in anti-rabbit igg (fc) ap conjugate (promega), 1:7500 at room temperature for one h, washed and developed with western blue stabilized substrate (promega). hek transected cells, previously described [7] , were used as control. for analysis of released mesothelin, supernatants were collected from wells containing the normal or mesothelin over-expressing panc02 cells [7] , uninfected bhk cells or cells infected with mva viruses. the cell supernatants were run on an sds-page gel, transferred to a pvdf membrane and probed with a rabbit anti-mesothelin antibody and an alkaline phosphatase conjugated 2˚antibody as described above. female c57bl/6 mice (charles river, 10-14 weeks of age) were used for experiments. all mice were housed in the east carolina university department of comparative medicine aaalac accredited animal facility and kept in conventional conditions with full access to food and water throughout the study. all procedures were approved by the institutional animal care and use committee and in accordance with recommendations for proper care and use of laboratory animals. in order to assess the safety of injecting mva virus expressing mesothelin, we injected mice i.m. (n = 5) with pbs, normal wild type mva, mvameso, or mvamesoa35del (3.8 x 10 7 pfu/mouse i.m. in 25 ul, and boosted 1 month later with 1.5 x 10 7 pfu/mouse) and monitored mouse health daily by body score condition and weight up to 125 days, similar to previous work [29] . numbers of gamma interferon (ifn-gamma)-secreting spleen cells were enumerated similarly to methods described previously [29, 31] . ninety-six-well plates (immulon h2b; thermo electron) were coated overnight with purified rat anti-mouse ifn-gamma (1 mg/ml; pharmingen) at 4˚c. plates were washed with blocking buffer before adding a titration of murine splenocytes in rpmi 1640 medium. splenocytes were harvested from pbs, mva, mvameso, or mvame-soa35del infected mice 1 month after vaccination. the in vitro stimulation of splenocytes was achieved either by the addition of panc02 cells, wild type vaccinia virus (moi = 3), or lewis lung cells, which do not express mouse mesothelin [7] , followed by incubation for 40 h at 37˚c. plates were then washed (pbs with 0.05% tween 20, 0.1% sodium azide) and incubated with biotinylated rat anti-mouse ifn-gamma (0.5 mg/ml; pharmingen) for 2 h at 37˚c. plates were washed and incubated with streptavidin-ap for 1 h at 37˚c. plates were developed with an agarose-bcip (5-bromo-4-chloro-3-indolylphosphate)-amp mixture, and spots were counted by using a dissection microscope. to assess viral replication over time [41, 42] , 350,000 cells were plated in a 24-well plate. bhk cells were used as a positive control since mva is known to replicate in them. bhk or panc02 cells were infected with mva at an moi of 5 pfu/cell for 2 h, then inocula were removed and the cells were washed once. supernatants and cells were harvested separately at six time points: 0, 12, 16, 24, 36, and 48 h post-infection. these samples were frozen and thawed three times before being titered in bhk cells in 6-well plates [31] . panc02 cells (10,000 cells/well) were plated in triplicate in a 96-well plate. mva was added to the wells at a multiplicity of infection (moi) of 0.5 and 5.0 pfu/cell. after 24 h, 10 ul 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulphophenyl)-2h-tetrazolium, inner salt/phenazine methosulphate (mts/pms) (2.0 mg/ml mts, promega and 0.1 mg/ml pms, sigma) was added to each well, and the absorbance was read at 492 nm [42] . media only was used to define the background control level. we used the syngeneic heterotopic tumor model employing the murine pancreatic adenocarcinoma panc02 cell line [7] . female c57bl/6 mice (charles river, 10-14 weeks of age, n = 4-8 per group) were injected s.c. in the right flank with panc02 cells (6.4x10 5 ), one injection per mouse, under isoflurane (3%) inhalation anesthesia [7] . tumor size was measured three times per week by a digital caliper, and volume was calculated as [(smallest diameter 2 ) x largest diameter]/2. mice were humanely euthanized if they reach endpoints of body condition score, tumor size, ulceration, or impairment. for analysis of safety and immunogenicity, mice were injected with 3.8 x 10 7 pfu/mouse i.m. in 25 ul, and boosted 1 month later with 1.5 x 10 7 pfu/ mouse. for treatment of tumor bearing mice, mice were injected s.c. in the right flank with panc02 cells (6.4x10 5 ), and then injected i.m. contralaterally with 7.6 x 10 7 pfu/mouse 25 ul 3 times, on days 11, 18, 25 after tumor cell injections. for pre-vaccination of mice, mice were injected i.m. with 3.8 x 10 7 pfu/mouse, and then boosted with 1.5 x 10 7 pfu/mouse, and challenged with panc02 tumor cells as above. for viral injections into tumors, tumors were allowed to grow, and viral injections (3.5 x 10 6 pfu/mouse in 25 ul) into tumors began when tumors were 100 mm 3 . experiments were repeated at least three times, and representative data are shown. a twotailed student's t test was used to compare groups. p values < 0.05 were considered significant. while tumor associated antigens, such as mesothelin, often do not induce a robust immune response, evidence suggests it is possible to generate an anti-tumor response by presenting the tumor antigen in an immunogenic context, such as by expressing the protein in a viral infection [19, 46] . in order to create a putative therapeutic anti-mesothelin vaccine, we inserted the mouse mesothelin gene into the poxvirus mva genome under a viral promoter so that mesothelin would be expressed in any cells infected with the recombinant virus. we constructed both a normal wild type mva virus expressing mesothelin (mvameso) and a mesothelin-expressing mva virus in which the viral a35 immunosuppressive virulence gene had been deleted (mva-mesoa35del) [31] . the purified viruses were analyzed by pcr to confirm the presence of the mesothelin gene in the expected loci in the genomes and the presence or absence of the a35 gene. next, we wanted to confirm the expression of mesothelin protein from the recombinant viral genomes in infected cells. as shown in fig 1, mesothelin protein was detected as a broad band~50 kda. mesothelin is glycosylated, so differences in glycosylation can be seen as different mw bands, and glycosylated forms may vary in different cell types [7] . mesothelin protein was strongly expressed in both mva mesothelin virus-infected bhk cells, with monomers and apparent dimers (~100 kda), but not in cells infected with mva parental wild type virus or uninfected bhk cells (fig 1) . this indicated that the recombinant mesothelin expressing viruses were able to express significant quantities of mesothelin protein in infected cells. cells were lysed and samples were loaded onto a pre-cast 4-20% gradient gel. rabbit anti-mesothelin antibody and anti-rabbit igg (fc) ap conjugate (promega) detected mesothelin protein (arrow). hek transected cells, previously described [7] , were used as control. https://doi.org/10.1371/journal.pone.0193131.g001 as a control, mesothelin was detected by western blot in hek cells transfected with a mesothelin expressing plasmid with a strong promoter [7] , but not in hek cells transfected with the control plasmid vector. mesothelin could also be detected in the panc02 murine pancreatic adenocarcinoma cell line as we have shown previously by western blot and flow cytometry [7] . while mesothelin is a tumor associated antigen that can be detected in panc02 cells grown in vitro or as a tumor grown in the mouse, it is not a highly abundant protein [7] . multiple forms and sizes of cleaved mesothelin proteins have been reported to be released from cells expressing it [6, 7] . we have previously shown that 2 forms of the mesothelin protein are found in supernatants of panc02 cells, a carboxy terminal region released by furin cleavage and one derived from the amino terminus [7] . we assessed what forms of mesothelin proteins are present in the supernatants of bhk cells infected with the 3 mva viruses (wild type mva, mvameso, and mvamesoa35del) and for comparison cultured panc02 cells overexpressing mesothelin [7] . as seen in fig 2, several protein bands that react with rabbit anti mesothelin antisera were detected by western blot, with large quantities of released mesothelin apparent in the cells infected with the recombinant viruses encoding mesothelin. in order to assess the safety of injecting mva viruses expressing mesothelin, we injected mice i.m. (n = 5) with pbs, wild type mva, mvameso, and mvamesoa35del. mice were boosted 1 month later. we monitored mouse health by body score condition daily and weight for 125 days as shown in fig 3. there were no significant differences between the 4 groups. we observed no adverse effects after the vaccination. this suggests that no significant anti-mesothelin autoimmune disease was induced in the mice. to determine whether the viruses expressing mesothelin protein were able to induce an immune response in mice, we first attempted to measure anti-mesothelin antibody in vaccinated mouse sera. we attempted to measure reactivity to panc02 cells, which express mesothelin on the surface by flow cytometry as we have done previously [7] , however we got very low responses, possibly due to released mesothelin (fig 2) binding the anti mesothelin antibodies in vivo. we then measured the frequency of anti-mesothelin ifn-gamma secreting t lymphocytes by elispot assay. mice vaccinated with pbs or mva had very few (<5) splenocytes that secreted ifn-gamma in response to panc02 cells (fig 4) . mvameso vaccinated mice had an average of 53 ifn-gamma secreting t cells per 10 6 splenocytes, and mvamesoa35del had an average of 80, significantly more (p<0.05) than mva and pbs treated mice. in comparison, there were very few spots (1-3) in response to stimulation with lewis lung cells that do not express mouse mesothelin, and mice vaccinated with mva, mvameso and mvamesoa35del viruses all had good responses to restimulation with vaccinia virus (124, 147, and 148 spots respectively). these results indicate that while all mice responded to the virus infection, and the mesothelin expressing viruses induced an immune response in mice that reactivated in response to mesothelin expressing panc02 cells. additionally, vaccination with the mvame-soa35del virus induced 52% more responding t lymphocytes compared to the mvameso virus, suggesting that removal of the a35 induced a stronger immune response, but the difference did not reach statistical significance. some oncolytic poxviruses have been shown to specifically target and replicate in tumor tissues in vivo [19] because the tumor or microenvironment supports viral replication. we therefore wanted to assess if mva virus was able to infect and replicate in panc02 cells by a one-step viral growth curve [47] . mva is unable to replicate in almost all mammalian cells: baby hamster kidney (bhk) cells are one known exception in which mva can replicate. bhk and panc02 cells were infected at an moi of 5 pfu/cell so that each cell would be infected, and the supernatants and cells were harvested separately and titered for virus quantity over time. as shown in fig 5, over 48 h the number of infectious mva virions (plaque forming units, pfu) increased in bhk cells more than 2 logs. mva replication in panc02 cells also increased by more than 2 logs, suggesting that the mva viruses might be effective oncolytic viruses in vivo since they can infect and replicate selectively in the panc02 tumor cells, while unable to replicate in normal mammalian tissues. viruses released into the supernatants also increased during mva infection of both cell lines. these results were promising, because they indicated that the virus could potentially amplify in situ and spread to cells that were not initially infected at the time of virus inoculation. since mva was able to replicate in panc02 cells, we wanted to assess whether mva was able to kill panc02 cells in vitro. panc02 cells were uninfected or infected with an moi of 0.5 or 5 pfu/cell and compared to media with no cells. the infection was allowed to proceed for 24 h, and mts reagent was added to measure cell metabolism as a measure of viability. to test the hypothesis that administration of mesothelin expressing viruses could induce an immune response and reduce mesothelin expressing tumor growth, we established tumors in mice [7] and then treated with the mva mesothelin viruses. to test if the removal of the immunosuppressive a35 gene improved this therapeutic vaccine efficacy, we assessed the mvameso virus compared to the mvamesoa35del virus. tumors were established by s.c. injection of tumors in one flank of the mice. at 11, 18, and 25 days post tumor injection, mice , and survival (b) was not improved. we have previously described the construction of stable mesothelin over expressing panc02 cells and shown that tumors formed by these cells were significantly smaller than the tumors expressing wild type levels of mesothelin [7] . we also tested whether the mesothelin expressing viruses might have greater efficacy against panc02 cells that expressed higher levels of the panc02 target. however, there was also no efficacy against the panc02 tumor cells expressing increased levels of panc02 protein, and the tumors continued to grow rapidly. since the panc02 tumors grow rapidly, we tested whether inducing an immune response to mesothelin prior to tumor induction would be protective against tumor growth. mice were injected i.m. with pbs, wild type mva, mvameso, or mvamesoa35del, and boosted 2 weeks later. two weeks after the boost, tumor cells were injected s.c. contralaterally in the flank. as shown in fig 8a, the prior vaccination with mvameso or mvamesoa35del did not significantly decrease tumor growth or increase survival times. next, we attempted direct intratumoral injections since mva is able to replicate in and kill panc02 cells in vitro. we injected tumor cells s.c. and when tumors reached 100 mm 3 , we injected pbs, wild type mva, mvameso, or mvamesoa35del directly into tumors 3 times, 5 days apart, however, we were unable to detect a benefit (fig 8b) . we also attempted to treat stable mesothelin overexpressing panc02 [7] tumors by intratumoral injection, however there was no apparent effect, as tumors continued to grow and mice had to be euthanized. we have shown that murine mesothelin can be expressed from the mva virus genome and detected as a~50 kda monomer protein on sds-page immunoblot, and a~100 kda presumed dimer. mesothelin is released from cells and can be detected in the supernatants of cultured cells [7] and in much higher quantities in cells infected with mva viruses that expresses mesothelin under a viral promoter. injection of the mesothelin expressing viruses into animals caused no apparent ill effect to at least 4 months after vaccination, suggesting that mvameso vaccine is safe and does not initiate a deleterious autoimmune response. the mvameso vaccine induced a strong response to the mesothelin expressing panc02 tumor cell line, only slightly smaller than the amount of anti-vaccinia virus t lymphocytes induced by injection of vaccinia virus [29] . these results indicate that the mesothelin protein was expressed in an immunogenic form by the virus in the infected mice. furthermore, mva virus (with and without mesothelin expression) was able to replicate in and kill panc02 tumor cells grown in vitro, suggesting that it would be an effective oncolytic virus in vivo. our previous results suggested that mesothelin might be a good target for control by an immune response [7] , as mesothelin overexpression reliably caused a decrease in a heterotopic tumor growth in an immunocompetent syngeneic mouse model while in vitro proliferation was not inhibited [7] . together, these results suggested that mvameso might be an effective treatment for pancc02 tumors, and that the mvamesoa35del might be more effective given its improved immunogenic properties [31] . however, we were unable to demonstrate any efficacy of the mesothelin mva vaccines prophylactically or therapeutically, even with multiple boosts or when injected directly into the tumors. one possibility is that the panc02 tumor cell line is simply too aggressive for control in an animal model where the tumor microenvironment may act to protect and promote tumor cell growth [7] . while we had hoped that intratumoral injection would allow the virus to replicate in, spread, and kill tumor cells, we did not see a potent oncolytic effect in animals. perhaps while the mva virus can kill panc02 tumor cells in vitro, the anti-viral immune response in vivo limits the replication and spread of the virus. an ineffective anti-tumor immune response may also be caused by the shedding of mesothelin by the tumor in vivo (decoy mesothelin) blocking the immune effector antibodies and t cells. mesothelin is known to be detectable in supernatants of panc02 cells [7] and in sera from patients with mesothelin expressing tumors [6, 48, 49] . this shed soluble mesothelin may be taken up by other cells and bind anti-mesothelin antibodies rendering them ineffective. these problems may be worsened by administration of these mesothelin-expressing viruses because they also release soluble mesothelin at high levels. another possibility is that the shed mesothelin induces a state of anergy/tolerance in the animal, but our data show that mesothelin specific t lymphocytes are generated in these animals even though there is soluble circulating mesothelin shed from the mesothelin virus infected cells. perhaps new developments with cart cells [10] may be able to more effectively facilitate t cell targeting and killing of cancerous cells. our data showed that both mvameso and mvamesoa35del vaccines were able to generate a t lymphocyte response that was activated by the panc02 cells, and the mvamesoa35del induced a stronger response, however it may be possible that the panc02 cells have developed mutations that protect them from effective cytotoxic t lymphocyte killing, even if t lymphocytes are appropriately expanded and targeted to recognize them. tumors that develop in humans and animals have already been highly selected in vivo to avoid the immune responses that control cancers. further improvements in these oncolytic therapeutic vaccines may be made by including cytokines such as il-10 and gmcsf, which have been reported to improve oncolytic potential of vaccinia virus and ultimately host survival [50, 51] . in addition, depletion of t regulatory cells may counteract homeostatic or cancer cell driven immunosuppression and enhance the development of mesothelin-specific cd8 t cells capable of killing tumor cells [52, 53] . although some results suggest that immune responses can be counter-productive: the anti mesothelin immune response may induce autoantibodies to the target antigen as well as other proteins by epitope spreading, and these antigen-spreading autoantibodies may cause increased damage to the animal. it was previously shown that antigen spreading was associated with a trend toward decreased survival in patients with prostate cancer treated with a recombinant viral vaccine [46] . finally, given the difficulties with targeting an antigen like mesothelin that is shed, it may be more effective to target an antigen that is not shed. recent results targeting a different pancreatic tumor associated antigen, the oncofetal fucose-rich glycovariants of the pathological bile salt-dependent lipase, suggest it may be a superior tumor target antigen [54] . united states cancer statistics: 2008-20012 incidence and mortality web-based report cancer statistics mesothelin is overexpressed in the vast majority of ductal adenocarcinomas of the pancreas: identification of a new pancreatic cancer marker by serial analysis of gene expression (sage) localization of mesothelin in epithelial ovarian cancer mesothelin is a malignant factor and therapeutic vaccine target for pancreatic cancer mesothelin expression in human lung cancer murine mesothelin: characterization, expression, and analysis of growth and tumorigenic effects in a murine model of pancreatic cancer mesothelin is not required for normal mouse development or reproduction sirna-mediated erc gene silencing suppresses tumor growth in tsc2 mutant renal carcinoma model mesothelin immunotherapy for cancer: ready for prime time? control of human mesothelin-expressing tumors by dna vaccines analysis of cloned fvs from a phage display library indicates that dna immunization can mimic antibody response generated by cell immunizations control of large, established tumor xenografts with genetically retargeted human t cells containing cd28 and cd137 domains identification of novel human ctl epitopes and their agonist epitopes of mesothelin mesothelin-specific cd8(+) t cell responses provide evidence of in vivo cross-priming by antigen-presenting cells in vaccinated pancreatic cancer patients mesothelin-targeted immunotherapies for malignant pleural mesothelioma phase i study of ss1p, a recombinant anti-mesothelin immunotoxin given as a bolus i.v. infusion to patients with mesothelin-expressing mesothelioma, ovarian, and pancreatic cancers new high affinity monoclonal antibodies recognize non-overlapping epitopes on mesothelin for monitoring and treating mesothelioma replicating poxviruses for human cancer therapy efficacy of vaccination with recombinant vaccinia and fowlpox vectors expressing ny-eso-1 antigen in ovarian cancer and melanoma patients raccoonpoxvirus safety in immunocompromised and pregnant mouse models the evolution of poxvirus vaccines serum antibodies to blood group a predict survival on prostvac-vf the poxvirus vectors mva and nyvac as gene delivery systems for vaccination against infectious diseases and cancer phase i trial of recombinant modified vaccinia ankara encoding epstein-barr viral tumor antigens in nasopharyngeal carcinoma patients genome sequence and comparative virulence of raccoonpox virus: the first north american poxvirus sequence vaccination with alvac and aidsvax to prevent hiv-1 infection in thailand identification of poxvirus cd8+ t cell determinants to enable rational design and characterization of smallpox vaccines the poxvirus a35 protein is an immunoregulator poxvirus safety analysis in the pregnant mouse model, vaccinia and raccoonpox viruses deletion of the a35 gene from modified vaccinia virus ankara increases immunogenicity and isotype switching clinical development of modified vaccinia virus ankara vaccines modified vaccinia virus ankara-based vaccine vectors induce apoptosis in dendritic cells draining from the skin via both the extrinsic and intrinsic caspase pathways, preventing efficient antigen presentation assessment of the protective effect of imvamune and acam2000 vaccines against aerosolized monkeypox virus in cynomolgus macaques clonal vaccinia virus grown in cell culture as a new smallpox vaccine epithelial immunization induces polyfunctional cd8+ t cells and optimal mousepox protection immunogenicity of a highly attenuated mva smallpox vaccine and protection against monkeypox shared modes of protection against poxvirus infection by attenuated and conventional smallpox vaccine viruses the evolving role of immunotherapy in prostate cancer prostvac-vf: a vector-based vaccine targeting psa in prostate cancer vaccinia virus a35r inhibits mhc class ii antigen presentation vaccinia virus decreases mhc class ii antigen presentation, t cell priming, and peptide association with mhc class ii antigen presentation assays to investigate uncharacterized immunoregulatory genes severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice rapid preparation of vaccinia virus dna template for analysis and cloning by pcr clin cancer res. a viral vaccine encoding prostate-specific antigen induces antigen spreading to a common set of self-proteins in prostate cancer patients characterization of the vaccinia virus a35r protein and its role in virulence establishment of the enzymelinked immunosorbent assay system to detect the amino terminal secretory form of rat erc/mesothelin establishment of a novel specific elisa system for rat n-and c-erc/mesothelin. rat erc/mesothelin in the body fluids of mice bearing mesothelioma gmcsf-armed vaccinia virus induces an antitumor immune response effective depletion of regulatory t cells allows the recruitment of mesothelin-specific cd8 t cells to the antitumor immune response against a mesothelinexpressing mouse pancreatic adenocarcinoma an iscom vaccine combined with a tlr9 agonist breaks immune evasion mediated by regulatory t cells in an orthotopic model of pancreatic carcinoma a pancreatic tumorspecific biomarker characterized in humans and mice as an immunogenic onco-glycoprotein is efficient in dendritic cell vaccination the authors wish to acknowledge the expert technical help of melinda carver. key: cord-048446-gaemgm0t authors: white, laura forsberg; pagano, marcello title: transmissibility of the influenza virus in the 1918 pandemic date: 2008-01-30 journal: plos one doi: 10.1371/journal.pone.0001498 sha: doc_id: 48446 cord_uid: gaemgm0t background: with a heightened increase in concern for an influenza pandemic we sought to better understand the 1918 influenza pandemic, the most devastating epidemic of the previous century. methodology/principal findings: we use data from several communities in maryland, usa as well as two ships that experienced well-documented outbreaks of influenza in 1918. using a likelihood-based method and a nonparametric method, we estimate the serial interval and reproductive number throughout the course of each outbreak. this analysis shows the basic reproductive number to be slightly lower in the maryland communities (between 1.34 and 3.21) than for the enclosed populations on the ships (r(0) = 4.97, se = 3.31). additionally the effective reproductive number declined to sub epidemic levels more quickly on the ships (within around 10 days) than in the communities (within 30–40 days). the mean serial interval for the ships was consistent (3.33, se = 5.96 and 3.81, se = 3.69), while the serial intervals in the communities varied substantially (between 2.83, se = 0.53 and 8.28, se = 951.95). conclusions/significance: these results illustrate the importance of considering the population dynamics when making statements about the epidemiological parameters of influenza. the methods that we employ for estimation of the reproductive numbers and the serial interval can be easily replicated in other populations and with other diseases. the emergence of the highly pathogenic avian influenza strain h5n1 has raised concerns of an imminent influenza pandemic. public health workers, government officials and disaster planners have an increasing interest in better understanding the potential impact of an influenza pandemic and possible strategies for containment. crucial in this planning is an understanding of the basic epidemiology of the disease in various settings. this has led to a growing interest in the analysis and understanding of past epidemics, particularly that of 1918, the most virulent and deadly influenza epidemic of the 20th century. mortality has been estimated at 50-100 million people worldwide as a result of influenza in the 1918 pandemic [1] . it is reasonable to suppose that by better understanding the transmission dynamics of the highly pathogenic virus in 1918, we can gain greater insight into the dynamics, and thus potential methods of control, for a future pandemic [2] . important parameters for understanding disease transmission are the reproductive number and the serial interval [3] . the basic reproductive number is defined as the average number of secondary infections created from a primary infection in an entirely susceptible population [4, see also 5] . a more complex, but perhaps meaningful parameter is the effective reproductive number which defines the average number of secondary infections an infected will create at a given point in the epidemic. this parameter takes into account that not all contacts of an infected individual are with susceptible persons, as well as the impact of public health control measures. control strategies are typically targeted to drive this number below one and maintain it there, as this will lead to eventual extinction of the epidemic. an example of this is herd immunity, or immunity to a disease that is incurred from a sufficiently large proportion of the population being immune to a disease. modeling techniques are often used to determine the proportion of the population that should be vaccinated in order to have the reproductive number low enough to avoid outbreaks of disease [6] . the serial interval can be defined as the time interval between a primary case presenting with symptoms and its infectee developing symptoms [7, 8] . thus this quantity is completely observable. this is a mixture of the incubation period and the infectious period, both of which are useful to understand, but difficult to measure. the sars outbreak of 2003 had a relatively long serial interval, estimated to be between 8 and 10 days on average and following a weibull distribution [9] making case isolation extremely effective in containing the epidemic. methods for the estimation of basic epidemiological parameters are still in development phase. [10] provides a thoughtful summary of methods for estimating the reproductive number. one particularly interesting and useful method has been previously described by [7] for estimating the daily reproductive number, r t , or the average number of cases an infected individual on day t would cause. one interesting feature of this method is that for days where no cases are observed, the estimated effective reproductive number is zero. another observation is that this method essentially estimates a curve for the effective reproductive number that traces the epidemic curve, lagged by the average serial interval length. this nonparametric method presupposes information on the serial interval distribution. this is typical as most methods for estimating the reproductive number rely on knowledge of the serial interval. few have described analytical methods for estimating the serial interval, making most methodologies dependent on contact tracing data, which is often difficult and expensive to attain. [11] describe a method to estimate the reproductive number that relies on limited contact tracing information but not a full estimate of the serial interval. [12] have recently described a method to estimate the serial interval and then used this estimate with the estimator proposed in [7] of the daily reproductive number and have applied their method to data from outbreaks of avian influenza in poultry farms in europe. several researchers have studied the 1918 pandemic and estimated some of these key epidemiological parameters. estimates have ranged from 2-3 for the basic reproductive number, r 0 , when using an seir model with a mean latent period of 1.9 days and infectious period of 4.1 days [13, 14] . using an exponential model and assuming the serial interval to be four days (somewhat based on the assumptions of [13] ), [15] estimated r 0 to be 2.6-10.6 for confined settings (such as prison and ships) and 2.4-4.3 for community settings. the estimates for the mean latent and infectious periods come from [16] and were used again by [17] and [18] . it appears that the original estimates were derived from epidemic data, although their source is not well documented. in what follows, we introduce new methodology for the estimation of both the daily reproductive number and the serial interval. we apply this method to data from two outbreaks on military ships in the 1918 influenza outbreak, as well as welldocumented outbreaks in five maryland communities. the results from this method are compared to that of [12] . the results illustrate the differences in infectious disease dynamics between outbreaks in a closed population and a dynamic community. we analyze data from several well-documented influenza outbreaks in 1918. first we consider data from two troop ships that embarked in the late fall of 1918 [19] . the medic reported two initial cases on november 11. out of 989 passengers (156 crew members, 829 soldiers, 4 civilians) 313 became sick with influenza over a 40 day period (attack rate, ar, = 0.32), though most of the cases occurred within the first fourteen days. the boonah left durban and in five days, on november 29, reported the first three definitive cases of influenza. those who collected the data note that there were likely some initial cases that were not identified. out of 1095 on board (164 crew members and 931 troops), 470 cases were reported (ar = 0.43) in the 40 days of the epidemic. the united states public health service created special surveys of 18 localities during the pandemic [20] . reported results from six communities in maryland are derived from house-to-house surveys requesting the date of onset of influenza for all infected, and the sex and age of each case of pneumonia and influenza. a summary of these populations is provided in table 1 . we describe a likelihood based methodology for estimating the reproductive number at each day in the epidemic as well as the serial interval. the method builds on that described by [21] . we assume that the population is closed, that all cases are observed, and use daily case counts only (i.e. number of new cases each day). let n = {n 0 , n 1 , n 2 ,…, n t } represent the daily cases counts of influenza for the t days of the epidemic and x ij represent the number of cases that appear on day j that are infected by individuals that appeared sick on day i. following is a representation of the disease transmission model in the population. . . we assume that the total number of cases produced by those on day i, x i? , are poisson distributed with parameter n i r i , where r i is the reproductive number for cases on day i. we further assume that x i = {x i,i+1 , x i,i+2 ,…,x i,i+k } follows a multinomial distribution with parameters x i? , p, k, where p = {p 1, p 2,…, p k } represent the distribution of the serial interval. using these assumptions we can construct a likelihood function (see details in the supplemental information), which, when simplified, yields the following convenient form where m i~ri ( x k j~1 p j n i{j ) [21] . maximization of this likelihood with respect to r i and p yields estimates of these parameters. to further simplify this process and create a more parsimonious model, we parameterize p by allowing it to follow a traditional parametric form for a serial interval (for instance a weibull, gamma, log normal, or exponential distribution). then the p j are functions of the parameters of the density (for instance in the case of the gamma distribution, the p j only depend on the shape and rate parameters of the gamma). similarly r i can be modeled parametrically as a function of time. one example of a reasonable model for this is the four parameter logistic curve [22] [23] [24] given by the parameters of this curve describe the initial height of the curve (approximately a+b), the point of inflection (d), the curvature over the inflection (c) and the final height of the curve (a). these parameters have biological meaning in this setting where the initial height corresponds to the values of r i prior to intervention and significant depletion of the susceptible population. the inflection point and its steepness would describe the timing of intervention and the rapidity with which it impacts transmission. the final height would describe the ultimate value of r i, which typically is less than one, indicating that disease transmission is in a sub epidemic state. in our analysis, we also implement the method described by [12] (hereafter referred to as the garske et al. method) and compare the results of the two methodologies. this method first estimates the generation time distribution using a likelihood based method. then the effective reproductive number is estimated using the method described by [7] (hereafter referred to as the wt method). we fit the likelihood for both methods using a nelder-mead maximization procedure and use 576 starting values in order to ensure that we reach the global maximum. all analyses were done using r 2.4.1. both methods assume homogenous mixing in the population, no missing data (clearly violated with the data from the maryland communities), that a primary case experiences symptom onset prior to any cases that it infects and a completely closed system where all cases are infected by a case that has been observed. in the case of the maryland data, where only a sample of the total number of cases was surveyed, we can observe the efficacy and robustness of these methods with sample data. certainly results should be interpreted with caution, however, as we will show, the results that are obtained are consistent with previous estimates for influenza. standard errors were calculated for the mle method using a parametric bootstrap. one thousand epidemics were simulated using the parameter estimates and estimates were obtained from each of these simulated epidemics. the standard deviation of the 1000 estimates was used as the standard error estimates. we used the method described in [12] to estimate the standard error for their estimates, however our simulations based on their assumption of asymptotic normality yielded a large number of negative estimates for the parameters. it is possible that this is due to the non-independence in the data and lack of theoretical underpinnings for the method that they propose. these results make their standard error estimates infeasible to estimate in this case. therefore we do not present standard error estimates for the results obtained using their methodology. in order to determine the accuracy and relative merit of the estimates obtained from each methodology, we compute one-stepahead residuals and implement a cross validation approach to analyze the generalizeability of the estimates obtained. the onestep-ahead residuals were calculated by first using the estimates from a particular location along with the data to predict the next days' number of cases,ñ i as follows: eachñ i is calculated using n 0 , n 1 , …, n i21 . then the one-stepahead residuals are calculated as we present these residuals averaged over the t days observed. generalizeability of the results was studied using an ad hoc cross validation (cv) technique. this is done by using the estimates obtained from one location to calculate the one step ahead residuals for another location. specifically we use the boonah ship estimates to calculate residuals with the medic data and then use the medic estimates to calculate the residuals for the boonah data. for the maryland communities, we report the average of the residuals obtained using the estimates from one community to predict the epidemics in each of the other four communities, creating five cv estimates (one for each community). table 2 gives the results for the serial interval distribution estimates. notable in these results is the striking consistency in the estimates of the first moment, with the exception of cumberland. the second moments vary much more, however. in general they tend to be much larger for the ships when using the garske et al. method compared to the mle method. for the communities, we observe that they are consistently around 10 for the garske et al. method and vary much more for the mle method. also of interest in these results are the large error estimates, particularly for cumberland, but also to a smaller extent for frederick. this is perhaps indicative of the model not fitting the data as well, for instance the logistic model may not be the best fit in this scenario, or that the lack of census data on cases might be more problematic here. in table 3 and figure 1 , we present the results for estimation of the effective reproductive number. evident in these results, is the large initial reproductive number for the boonah ship. this is likely due to some of the missing data at the beginning of the epidemic and thus the model attributing the large number of cases that rapidly develop to the few individuals who were initially reported. the logistic model fits this as accurately as possible, but perhaps the important message is the qualitative result, indicating that initial transmission in this susceptible, non-quarantined population was very high and rapidly decreased as many became infected. the result is similar for medic though the initial value is not high. we also note that the reproductive number dropped to sub epidemic levels rapidly (around 10 days for both ships). in the maryland communities the initial reproductive number tended to be slightly lower (ranging from 1.34 in salisbury to 3.21 in table 4 , we present the results of the residual analysis. we notice here that the garske et al. method often does better than the mle method. it is important to point out that the wt method of fitting the effective reproductive model over fits the model and suffers from generalizeability. this method essentially traces the epidemic curve, lagged by the mean of the generation time distribution. thus, according to the residuals, it appears that the wt method outperforms the mle. however, considering the importance of external validation and reproducibility, the model suffers somewhat as evidenced by the cv measures. the exceptions to this are in the case of the boonah where the cv measure is impacted by the large initial mle estimate of the reproductive number and in cumberland where it appears that either the parametric model chosen may not represent the best fit to the data or there were sensitivities to the survey data. we have presented results that are informative with regard to the dynamics of the 1918 influenza pandemic in different populations and provide insight into two methodologies for estimating basic epidemiological parameters. both methods assume that the population is closed, there are no missing cases and no migration to or from the population. the second of these assumptions is clearly violated with the data from maryland; however the results appear to be reasonably robust to this discrepancy, except in the case of cumberland. the purpose of this exercise determines to some extent which methodological approach we might favor. if the intent is to simply estimate the parameters for a specific epidemic and better understand what exactly was occurring in that setting, then the method presented by [12] (garske et al.) appears to provide good fit. the caveat that we see in this method is that by estimating the effective reproductive number with the methodology of [7] (wt) there is an over fit of this parameter and it essentially traces the epidemic curve, lagged by the mean of the serial interval. it is not clear if this is a desirable or informative property. the mle method has greater promise for generalizeability. while it can be argued that adhering to a parametric definition of the shape of the effective reproductive number leads to a greater chance of lack of fit, it can also lead to a result that can be interpretable for other settings that are similar to that being studied. one can choose any reasonable parametric form for modeling the effective reproductive number. here we have only shown the four parameter logistic model, and feel that it is suitable in most cases where the epidemic curve has a single peak. it is feasible that this model may not apply well in all situations. another approach might be to analyze the data using the garske et al. method and then smooth the plot of the effective reproductive number and from this determine a parametric form that closely approximates the smoothed curve. multiple models could be implemented, then the residual analysis that we have shown provides a valuable tool for model assessment and comparison. the results of these models can be sensitive to underreporting initially in the epidemic. we see this clearly in boonah, where it was acknowledged that there was underreporting early on and this led to us getting very high estimates for the initial reproductive number. similarly, in cumberland, if we remove the first five days of data (three cases on the first day, six cases on the second and then no cases the following three days) we get much more reasonable estimates (m~6:00,ŝ 2~1 0:32) with smaller residuals (6.00). therefore, it is important to note that unusual observations in the first few days can impact the estimates and one should pay careful attention to this possibility. overall both methodologies presented are valuable tools that can be used in tandem for understanding the dynamics of infectious disease epidemics. these methods are easy to implement and interpret. the results that we have presented suggest that the average serial interval for pandemic influenza in 1918 was consistently between three and four, regardless of the setting. the standard deviation for the serial interval distribution varied much more for the mle method depending on the location. garske et al. estimates indicate that the value was consistently smaller in the communities than in the ships. it is not clear exactly how to interpret this result. further, we consistently see a large initial value for the reproductive number. in the ships, this value is higher and rapidly drops off, perhaps due to the close quarters and extremely rapid transmission that could take place in these very vulnerable populations. in the communities, the reproductive number tended to drop off later, typically around day thirty. this could be due to a larger initial susceptible population and more complicated dynamics for the disease to spread, leaving large pockets of susceptible individuals unexposed for a longer period of time than in the ships. the 1918 influenza pandemic: insights for the 21 st century pandemic influenza: past, present and future the interval between successive cases of an infectious disease infectious diseases of humans definition and estimation of an actual reproduction number describing past infectious disease transmission: application to hiv epidemics among homosexual men in denmark herd immunity and herd effect: new insights and definitions different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures a note on generation times in epidemic models transmission dynamics and control of severe acute respiratory syndrome estimating individual and household reproduction numbers in an emerging epidemic estimation in real time the efficacy of measures to control emerging communicable diseases the transmissibility of highly pathogenic avian influenza in commercial poultry in industrialized countries transmissibility of 1918 pandemic influenza transmission dynamics of the great influenza pandemic of estimates of the reproduction numbers of spanish influenza using morbidity data an influenza simulation model for immunization studies community trials of vaccination and the epidemic prevention potential containing pandemic influenza with antiviral agents influenza in maryland. preliminary statistics of certain locations a likelihood based method for real time estimation of the serial interval and reproductive number of an epidemic a flexible growth function for empirical use radioligand assay statistical analysis of radioimmunoassay data these results confirm the high pathogenicity of influenza and its ability to rapidly spread through populations. it also appears that the greatest difference between the spread of influenza in a closed population without the ability to implement control measures is a large initial reproductive number that declines rapidly. in more diffuse communities with complicated dynamics, it is likely that the reproductive number will not decline as rapidly. conceived and designed the experiments: lw mp. performed the experiments: lw. analyzed the data: lw. wrote the paper: lw. key: cord-001254-y2knt8g0 authors: parkhomenko, taisiya a.; doronin, vasilii b.; castellazzi, massimiliano; padroni, marina; pastore, michela; buneva, valentina n.; granieri, enrico; nevinsky, georgy a. title: comparison of dna-hydrolyzing antibodies from the cerebrospinal fluid and serum of patients with multiple sclerosis date: 2014-04-15 journal: plos one doi: 10.1371/journal.pone.0093001 sha: doc_id: 1254 cord_uid: y2knt8g0 it was found that high-affinity anti-dna antibodies were one of the major components of the intrathecal igg response in multiple sclerosis (ms) patients [williamson et al., pnas, 2001]. recently we have shown that iggs from the sera of ms patients are active in the hydrolysis of dna. here we have shown, for the first time, that average concentration of total proteins (132-fold), total iggs (194-fold) and anti-dna antibodies (200-fold) in the sera is significantly higher than that in the cerebrospinal fluid (csf) of fifteen ms patients. the relative activities of total protein from sera and csfs varied remarkably from patient to patient. it was surprising that the specific dnase activity of the total protein of csf reparations were 198-fold higher than the serum ones. electrophoretically and immunologically homogeneous iggs were obtained by sequential affinity chromatography of the csf proteins on protein g-sepharose and fplc gel filtration. we present first evidence showing that iggs from csf not only bind but efficiently hydrolyze dna and that average specific dnase activity of homogeneous antibodies from csf is unpredictably ∼49-fold higher than that from the sera of the same ms patients. some possible reasons of these findings are discussed. we suggest that dnase iggs of csf may promote important neuropathologic mechanisms in this chronic inflammatory disorder and ms pathogenesis development. multiple sclerosis (ms) is a chronic demyelinating pathology of the central nervous system presenting a serious medical and social problem. its etiology remains unclear, and the most valid theory of its pathogenesis assigns the main role in the destruction of myelinproteolipid shell of axons to inflammation related with autoimmune reactions ( [1] and refs therein). although the t-cell immune system plays a leading role in ms pathogenesis, normal functioning of the b-cell system is also important for the disease development. an enhanced synthesis of immunoglobulins (usually iggs), their free light chains, and of polyspecific dna binding antibodies (abs) interacting with phospholipids are observed in ms patients [1] . new keys for understanding ms pathogenesis have appeared after cloning igg repertoire directly from active plaques and periplaque regions in ms brains and from b-cells recovered from the cerebrospinal fluid of a patient with ms with a subacute disease [2] . it was found that high affinity anti-dna abs were a major component of the intrathecal igg response in ms patients. furthermore, dna-specific monoclonal abs derived from two ms individuals as well as a dna-specific ab derived from a systemic lupus erythematosus (sle) patient bound efficiently to the surface of neuronal cells and oligodendrocytes. for these abs, cell-surface recognition was dna-dependent. the findings indicate that anti-dna abs may promote important neuropathologic mechanisms in chronic inflammatory disorders, such as ms and sle [2] . artificial abzymes (catalytic antibodies against transition state analogues of chemical reactions) and natural abzymes are novel biological catalysts that have attracted much interest in the last years (reviewed in [3] [4] [5] [6] [7] ). artificial abzymes are abzymes against analogs of transition states of catalytic reactions [3] [4] [5] [6] [7] or antiidiotypic abs induced by a primary antigen, which may show some of its features including the catalytic activity (for review also see [8] [9] [10] [11] [12] ). during past two decades it has become clear that auto-antibodies (auto-abs) from the sera of patients with different autoimmune diseases can possess enzymatic activities and that their occurrence is a distinctive feature of autoimmune diseases (reviewed in [13] [14] [15] [16] [17] ). it is thought that abzymes may play a significant role in forming specific pathogenic patterns and clinical settings in different autoimmune conditions through their broadened auto-ab properties [13] [14] [15] [16] [17] . patients with autoimmune diseases produce abs to nucleoprotein complexes, to dna and to enzymes that participate in nucleic acid metabolism [13] [14] [15] [16] [17] . in autoimmune diseases, there can be a spontaneous induction of anti-idiotypic antibodies, which are abs elicited by a primary antigen, including some with catalytic activity, or transition from polyreactive catalytic activity to autoantigen-directed activity. natural abzymes hydrolyzing dna, rna, polysaccharides, oligopeptides, and proteins are present in the serum of patients with several autoimmune and viral diseases (reviewed in [13] [14] [15] [16] [17] ). healthy humans do not develop abzymes with detectable dnase and rnase activities, their levels being usually on the borderline of sensitivity of the detection methods [13] [14] [15] [16] [17] . it has recently been shown that myelin basic protein (mbp)hydrolyzing activity is an intrinsic property of iggs, igms, and igas from the sera of ms patients [17] [18] [19] [20] [21] [22] . recognition and degradation of mbp peptides by serum auto-abs were confirmed as a novel biomarker for ms [21] [22] . the established ms drug copaxone appears to be a specific inhibitor of mbp-hydrolyzing abzyme activity [22] . at the same time, as mentioned above, anti-dna abs were found to be a major component of the intrathecal igg response in ms patients [2] . first dnase abzymes were found in the sera of sle patients [23] . then, it was shown that iggs and igms from the sera of ms patients effectively hydrolyzed dna, rna, and oligosaccharides [24] [25] [26] [27] [28] . whereas only 18 and 53% of ms patients contained increased concentrations of abs to native and denatured dna, respectively, as compared with healthy donors, dnase abzymes were found in ,80-90% of ms patients [17, 24, 25] . since dnase abzymes of ms patients [14] similarly to sle patients [28] are cytotoxic, cause nuclear dna fragmentation and induce cell death by apoptosis, they can play an important role in sle and ms pathogenesis. taking these observations into account, analysis of relative concentrations of proteins, canonical enzymes and dnase abzymes in the cerebrospinal fluid (csf) of ms patients is of special interest. in the present study we have used different approaches to provide, for the first time, a very strong direct evidence that dnase activity is intrinsic to iggs from csf of ms patients and compared some other parameters characterizing the ms csf and sera. fifteen patients (11 women and 4 men) satisfying the criteria for clinically or laboratory-supported definite ms according to [29, 30] were retrospectively selected for the study. of these, 13 were relapsing-remitting (rr), and 2 were primary progressive (pp) in agreement with the criteria of lublin and reingold [31] . clinical course (rr and pp), clinical activity (relapse at time of sampling), and mri activity (the presence of gadolinium enhancing lesions at mri examination) were analyzed as described previously [32] . the characteristics of the ms patients are summarized in table 1 . it was interesting to compare some different indexes for csf and sera of ms patients. therefore, first we measured a relative concentration of total protein in csf and sera of ms patients ( table 2 ). the relative concentrations of total protein of csfs (range 0.26-0.66 mg/ml) and sera (47-74 mg/ml) of fifteen ms patients varied in different ranges. the average concentration of the total protein in the serum (62.566.7 mg/ml) was ,130-fold higher compared with csf (0.4860.09 mg/ml) and these values did not demonstrate good correlation (coefficient correlation (cc) = 20.12), table 2 ). the relative concentrations of total iggs in the serum and csf were first measured using an immunoblotting test system. the relative concentration of total iggs in the serum (range 7.9-16.6 mg/ml; average value 11.761.8 mg/ml) was 167-fold higher than that for the csf (range 0.02-0.19 mg/ ml; average value 0.0760.04 mg/ml) and there was not good correlation between these values, cc = +0.07. iggs from the serum and csf were purified by affinity chromatography on protein-g sepharose and igg-protein concentration was estimated in the peaks eluted from the sorbent by an acidic buffer (see below). the relative concentration of total iggs determined by two different methods was the same within the experimental error. interestingly, the concentration of total protein in csf was 6.9-fold higher than total iggs, while this difference in the case of the serum (5.4-fold) was by a factor of 1.3 lower. we estimated the relative concentration of anti-dna antibodies in csf and serum. the relative concentration of anti-dna antibodies in the csf of all patients was measured after dilution of csfs 2-fold; the values for all csfs were comparable (range 0.21-0.24 a 450 units counting on a cerebrospinal fluid without dilution; average value 0.2260.008 a 450 units), while in the case of the serum (dilution 1000-fold) it was significantly varied (range 110-2000 a 450 units counting on serum without dilution; average value 4376311 a 450 units). since the relative concentration of anti-dna antibodies in csf was relatively low, we have removed all abs from csfs by passage through two columns: protein g-sepharose and protein a-sepharose. csfs passed through the columns and controls containing only a standard buffer demonstrated the same a 450 values, which were #5% in comparison with those for untreated csfs. thus, we have shown that the relatively low a 450 values corresponding to untreated csfs are caused by anti-dna antibodies in these csfs. in addition, we have shown that dna-hydrolyzing activity is an intrinsic property of iggs from csf (see below). there was no good correlation between anti-dna antibodies in the sera and csfs, cc = +0.57 (table 2 ). in addition, the correlation was very low between the total concentration of iggs and anti-dna antibodies both in csf (cc = +0.40) and the serum (cc = 20.32). at the same time, the average differences in the concentration of total iggs for the serum and the csf (167-fold) were 11.9-fold lower than the difference for anti-dna antibodies of the serum and the csf (1986-fold) ( table 2) . it is known that human sera contain several major and especially minor proteins. the average concentration of the total protein in the serum was ,130-fold higher than in csf (table 2) . it was interesting to compare major proteins of the sera and csfs using sds polyacrylamide gel electrophoresis (sds-page). fig. 1 demonstrates major proteins in some samples of the sera and the csfs. one can see that the sera contain more major proteins, but in both cases iggs (150 kda) and human serum albumin (,67 kda) are most major proteins. at the same time, in contrast to csfs, the sera contain several major proteins with mol. masses higher than 150 kda as well as lower than 150 kda, but higher than 67 kda (fig. 1) . it is interesting to see that in csfs there are several additional good visible proteins with electrophoretic mobility lower than 67 kda in comparison with serum preparations. it was interesting to compare total dnase relative activity (ra) of all dna-hydrolyzing enzymes and abs in the sera and csf of ms patients. preparations of total protein were capable to hydrolyze supercoiled plasmid dna (scdna), forming single breaks in one strand of supercoiled dna (relaxed dna) and then multiple breaks yielding linear dna. finally, they hydrolyzed dna into short and medium-length oligonucleotides. however, after such a deep hydrolysis of dna, it was very difficult to estimate the relative activity of these preparations. the ras of total protein from sera and csf varied remarkably from patient to patient. fig. 1 illustrates typical examples of cleavage of scdna by total protein from several ms patients after 2 h of incubation. therefore, in order to estimate the dnase activity quantitatively, we found the concentration for each preparation and the time of incubation sufficient to convert scdna into the relaxed form (10-40%; for example, lanes 2, 6, 7, 9, and 10 of fig. 1c ) without further noticeable fragmentation after 1-2 h of incubation. since all measurements (initial rates) were taken within the linear regions of the time courses and protein concentration, the measured ras were normalized to standard conditions (pmole dna/1 mg of protein/1 h; standard units (su)) ( table 3 ). the relative specific dnase activity of total protein of the serum preparations was varied in the range 0.26-0.88 pmole dna/1 mg of protein/1 h (average value 0.5160.16 su). it was surprising that the specific activity of the total protein of csf reparations were 198-fold higher (range 25-263 su, average value 101650 su) than the serum ones (table 3) . at the same time, concentration of total protein in the case of csf was 130-fold lower than that of sera (table 2) . therefore, in calculation on 1 ml of liquid the average ra of total protein corresponding to the sera was only 1.5-fold lower than that for csfs. recently, several strict criteria have been applied to show that the dnase activity is an intrinsic property of iggs from sera of ms patients but not from healthy donors [24] [25] [26] . when searching abzymes in csf of ms patients, the igg fraction was purified by chromatography on protein-a sepharose in conditions to remove non-specifically bound proteins, followed by gel-filtration as in [24] [25] [26] [27] . the relative amount of csf preparations from only thirteen ms patients was enough for the purification of igg preparations. to analyze the ''average'' situation for ms iggs, we prepared a mixture of equal amounts of iggs from sera of thirteen patients (igg mix ). the homogeneity of the 150 kda igg mix was confirmed by sds-page, which showed a single band before, and two bands corresponding to the h and l chains after ab reduction with dtt (silver staining) ( fig. 2a) . to prove that dnase activity of csf igg mix is its intrinsic property and is not due to copurifying enzymes, we applied some of well known rigid criteria [13] [14] [15] [16] [17] 33] ; a) electrophoretic homogeneity of igg mix ( fig. 2a) ; b) gel-filtration of igg mix in conditions of ''acidic shock'' (ph 2.6) did not lead to the disappearance of ab activity and the peak of activity tracked exactly with 150 kda abs (fig. 2c) ; c) complete adsorption of the activity by sepharose bearing monoclonal mouse abs against human igg light chains and its elution from the adsorbent with buffer of low ph (fig. 2d ). in order to exclude possible artifacts due to any hypothetical traces of contaminating enzymes we analyzed additionally the dnase activity of igg mix using an in situ assay in sds-page gels containing dna. after incubation, in order to allow dnase renaturation and staining with ethidium bromide, a sharp dark band at the position of dna hydrolyzing proteins was revealed on a fluorescent background of dna-bound ethidium bromide (fig. 2b) . after the dissociation of the igg mix using dtt, dnase table 3 . relative dnase activity of total proteins and iggs from csf and sera of patients with ms.*. serum (8) csf (9) serum (10) csf (11) serum ( activity was revealed only in the band of the separated light chains. similar data was obtained earlier for abzymes from serum of ms patients [24] [25] [26] . since sds dissociates any protein complexes, and the electrophoretic mobility of hypothetical contaminating dnases cannot coincide at the same time with that of intact igg and its lchains, the detection of dnase activity in the gel region corresponding only to igg and its light chains, together with the absence of any other bands of the activity or protein, provides direct evidence that dnase activity is an intrinsic property of ms igg mix and is not due to copurifying enzymes. dnase activity was detected earlier in the case of iggs from sera of 71 of 75 ms patients (,95%) but in none of 50 healthy donors [24] [25] [26] . there was not any possibility to get csf preparations from healthy donors. however, one can assume that csf, similarly to serum preparations from healthy donors, does not contain iggs with dnase activity. we compared the ras in the hydrolysis of dna of igg preparations from sera and csfs of thirteen ms patients. the ras of iggs from sera and csf varied markedly from patient to patient. fig. 3 illustrates typical examples of cleavage of scdna by iggs (0.2 mg/ml) from the sera and csfs (0.003 mg/ml) of several patients after 2 h of incubation at their fixed concentrations. to estimate the dnase activity quantitatively, we found the concentration for each igg preparation corresponding to the linear part of the rate dependences on ab concentration (the conditions of the reaction of the pseudo-first order), and the time of incubation sufficient to convert scdna into the relaxed form fig. 3a ). since all measurements (initial rates) were taken within the linear regions of the time courses and ab concentration curves, the measured ras for individual iggs were normalized to standard conditions similarly to those for the preparation of total protein (table 3) . it was surprising, but csf iggs possess significantly higher specific dnase activity than serum ones. average specific dnase activity of serum iggs (average value 11.264.3 pmole dna/1 mg of ab/1 h; range 3.6-20.8 units) is 48.5-fold lower than that for csf abs (average value 543.76239.7 units; range 117.3-1190.5 units) (table 3 ). at the same time, there is no good correlation between catalytic activities of iggs from the sera and csf, cc = + 0.26 (table 3) . interestingly, average specific ras of serum iggs are ,22-fold higher than those of serum total proteins, while this difference in the case of csf is significantly lower, only ,5.4-fold (table 3 ). there are no published data concerning csf abzymes with any catalytic activities. data reported in this paper provide strong evidence that dnase activity is an intrinsic property of iggs present in csf of ms patients: it is not due to copurifying enzymes. the presence of anti-dna abs and dnase iggs in csf of ms patients is in agreement with the detection of b-cells producing anti-dna abs directly in active plaques and periplaque regions in ms brain and cerebrospinal fluid of a patient with ms [2] . since sera of ms patients contain greater number of different proteins than csfs (fig. 1a ) and the average concentration of total protein in the sera is ,130-fold higher than in csfs, it is not surprising that average specific ra of csf total protein is 198-fold higher than that of the serum one. the question is why specific ras of csf total iggs are significantly (48.5-fold) more active than those from sera. in addition, relative concentration of abs (of a total pool) interacting with dna in sera is ,1986-fold higher than in csf, while a difference in the concentration of total iggs is only 198-fold, a difference of 10.3-fold (table 2) . thus, a small specific fraction of anti-dna abs from the csf may be 10-fold more active (totally ,485-fold) than a similar small fraction of abs interacting with dna from the sera. in this context, some data from literature should be mentioned. overall, abzymes of ms patients may be significantly more active in the hydrolysis of dna than what we have found ( table 3) . as it was previously shown, the fractions of abzymes with different catalytic activities, including nuclease ones, in the serum of autoimmune patients usually do not exceed 1-7% of total immunoglobulins [13] [14] [15] [16] [17] . since the specific activity was calculated using the total concentration of iggs, the specific dnase activities of the individual monoclonal subfractions in a polyclonal igg pool may be significantly higher than those of the nonfractionated iggs. in addition, the repertoire of polyclonal abs against different antigens in the case of sera from ms patients may be significantly wider than that of csfs. it may be one of the possible reasons of a lower specific activity of sera iggs. at the same time, an ever-growing number of observations suggest that autoimmune diseases originate from defects in hematopoietic stem cells [34] . it was recently shown that the specific reorganization of immune system during the spontaneous development of a profound sle-like pathology in mrl-lpr/lpr mice is associated with changes in the differentiation profile and the level of proliferation of bone marrow hematopoietic stem cells and with the production of dnase, atpase, and amylase abzymes [35, 36] . immunization of healthy mice with dna also leads to a production of abs with dnase activity; however, it is only associated with increased lymphocyte proliferation and suppression of apoptosis of lymphocytes in different organs (especially spleen), but not with a change in the differentiation of bone marrow cells [35, 36] . thus, it is reasonable to suggest that b-cells of csf of ms patients can produce not only abs interacting with dna [2] , but also specific anti-dna abzymes with higher dnase activity. abzymes produced by lymphocytes against dna in different organs of ms patients (and circulating in the blood system) may have a lower dnase activity in comparison with anti-dna abs of csf or may be different ratio of abzymes and anti-dna abs without catalytic activity in the csfs and sera of ms patients. we have estimated ccs between different characteristics of csf and sera. it was shown that there was no good correlation between several identical indexes characterizing csfs and sera of ms patients, ccs varying in the range of 20.12 to +0.57 (tables 2 and 3 ). the correlation between the total protein concentration and: a) total concentration of iggs in csf (cc = +0.17; columns 1 and 3) and sera (cc = +0.47; columns 2 and 4); b) relative concentration of anti-dna abs in csf (cc = +0.11; columns 1 and 5) or sera (cc = 20.04; columns 2 and 6) was low ( table 2) . at the same time, low but still the best positive correlation was observed between the total protein concentration and relative dnase activity of total csf protein (cc = +0.61; columns 1 and 7), whereas in the sera these values showed negative correlation (cc = 20.49; columns 2 and 8) (tables 2 and 3) . similar low ccs were observed for other estimated parameters. cc between the relative concentrations of iggs and anti-dna abs was relatively low in csf (cc = +0.4; columns 3 and 5), and negative in the sera (cc = 20.32; columns 4 and 6) ( table 2) . interestingly, cc between the concentration of iggs and the relative specific dnase activity of csf abs (cc = +0.16; columns 3 and 11) was lower than that of the sera (cc = 20.58; columns 4 and 12) (tables 1 and 2 ). finally, the relative concentration of total anti-dna abs correlated with the relative specific igg dnase activity better in the sera (cc = +0.51; columns 6 and 12) than in csf (cc = + 0.11; columns 5 and 11) (tables 2 and 3 ). an additional question is why there is no good correlation between various indexes, characterizing different ms patients. an analysis of correlation between titers of abs to dna as well as to mbp and 13 different standard clinical parameters including poser criteria (indexes for evaluation of damage to functional systems: pyramidal functions; cerebellar functions; functions of brain stem; sensitive functions; functions of intestines and urinary bladder; visual functions; cerebral (psychical) functions and sum of these characteristics) in the case of 49 patients with ms was carried out [17] . for the whole group of ms patients, the absolute values of positive ccs between titers of anti-dna or anti-mbp abs and clinical poser indexes were very low (between 0.01 and 0.19), absent (,0), or even negative (20.02 to 20.07) and statistically non-significant. several ccs became higher and reached values up to 0.1 to 0.55 and 20.04 to 20.47 after the division of cohort into subgroups of patients with primary progressing, secondary progressing and remitting course of the disease [17] . the groups of primary progressing remitting course and secondary progressing course of ms patients were not ''homogenous'' with respect to the patients' characteristics, and their further subdivision using cluster and factorial analysis revealed high statistically significant correlation coefficients [17] . for example, for one sub-subgroup of the remitting course subgroup, a direct dependence between titers of anti-mbp and symptoms of lesions of the pyramidal tract was observed (cc = 0.92). in some cases, correlations of the opposite sign were observed for the same pairs of analyzed parameters for the three subgroups with different ms courses and their sub-subgroups obtained by cluster analysis from the subgroups. the absence of a definite dependence between titers of anti-dna and anti-mbp abs and these parameters with standard clinical indices may be due to several reasons. ms is an extremely multifactorial disease, in which similar pathomorphological and clinical indices manifested as ms may result from very different underlying processes and conditions [37, 38] . for example, in each ms patient, the ''relative stability'' of different organs and their functions to the destructive effect of transient immune system errors can be significantly different depending on the genetic background and environmental stress factors, including geographic ones [37] [38] [39] . some proteins of influenza, herpes, polyoma, epstein-barr and other viruses and of some bacteria have been reported to mimic human myelin proteins, and these infections can therefore lead to immunization with their proteins and stimulate the subsequent formation of abs to myelin and finally to the development of autoimmune reactions [38, [40] [41] [42] [43] . in individual ms patients, the development of autoimmune reactions can be stimulated by different viral or bacterial infections as well as various toxic chemicals. furthermore, it should also be taken into account that ms is pathology of at least two-phases [44] . the cascade of reactions corresponding to the first inflammatory phase is very complicated and involves many proteins, enzymes, cytokines, and chemokines inducing macrophages and other cells producing no n radicals and osteopathin [38, 44] . the complex and coordinated action of t-and b-cells, complement system, inflammation mediators, and auto-abs result in the formation of demyelinization nodi and interruption of axon conductivity. the neurodegenerative phase of ms that ensues later is directly connected with the neural tissue destruction in these patients [38, 44] . therefore, any analysis of biochemical, immunological and clinical indices must take into account of the current stage of the disease. obviously, quite different characteristics of pathologic processes can be obtained in individual patients as the disease progresses against the background of the continually changing immunoregulation, including exhaustion of different compensatory and adaptive mechanisms and systemic metabolic changes. this makes the clinical course of ms hardly predictable in individual patients [38, 44] . therefore, it is not surprising that we could not find a high statistically significant correlation of titers of abs to dna and ras of abzymes with all parameters measured, since each patient can be characterized by an individual combination of genetic, environmental, chronic, inflammatory, autoimmune, demyelinating, neurodegenerative and other factors. overall, all data obtained demonstrate that the dnase activity is an intrinsic property of iggs deriving from csf and sera of ms patients. these iggs are polyclonal and may consist of extremely different repertoires of dnase subfractions in the case of csf and sera. we have shown previously that the appearance of abzymes specifically hydrolyzing dna is among the earliest and clear signs of autoimmune reactions in a number of autoimmune diseases when titres of abs to dna or other auto-antigens have not yet increased significantly and correspond to their ranges for healthy donors [16, 17, [24] [25] [26] . therefore, detection of dnase abs in the sera and csf of peoples can be considered as an additional index for early diagnostic of this pathology. most chemicals, proteins, protein g-sepharose, and the superdex 200 hr 10/30 column were from sigma or ge healthcare. fifteen consecutive ms patients (11 women and 4 men; mean age = 39612.5 years) satisfying the criteria for definite ms according to the classification of mcdonald [29] and admitted to the multiple sclerosis center of the university of ferrara during the period from january 2012 to october 2012 were retrospectively selected for the study. disease severity was scored in all ms patients at the time of sample collection using kurtzke's expanded disability status scale (edss) [30] (mean at entry = 1.861.4; range from 0 to 4.0). clinical course (rr and pp), clinical activity (relapse at time of sampling), and mri activity (the presence of gadolinium enhancing lesions at mri examination) were analyzed as described previously [32] . at entry none of the patients had fever or other symptoms or signs of acute infections. moreover, at the time of sample collection none of the patients had received any potential disease-modifying therapies during the 6 months before the study. the blood and csf sampling protocols confirmed the local committee for medical ethics in research (comitato etico della provincia di ferrara) that approved our study in accordance with helsinki ethics committee guidelines including written consent of patients confined to present of their blood and csf for diagnostics of a possible disease and scientific purposes. the protocol was approved at 31 may 2007 and it was focused on the creation of a biological bank of csf and serum samples, and related clinical data of patients with ms and other neurological diseases including: a) a study of potential markers (especially proteins) for diagnostic and prognostic significance in diseases of the nervous system; b) specific antibodies directed against antigens potential exogenous and/or endogenous; c) presence of pathogens (mostly viruses or bacteria) for association studies and pathogenesis; d) neurotransmitters and their metabolites; e) a study of different properties of different markers. csf and serum samples were collected under sterile conditions and stored in aliquots at 80uc until assay. ''cell-free'' csf samples were obtained after centrifugation at room temperature of specimens taken by atraumatic lumbar puncture performed for purposes of diagnosis in the absence of contraindications. serum samples derived from centrifugation of blood specimens with-drawn by puncture of an anterocubital vein at the same time of csf extraction. paired csf and serum samples from ms patients were stored and measured under exactly the same conditions. informed consent was given by all patients before inclusion and the study design was approved by the regional committee for medical ethics in research. csf and serum igg levels were measured by immunochemical nephelometry with the beckman immage 800 immunochemistry system (beckman instruments, inc. fullerton, ca. usa) according to the procedure of salden et al. [45] . in all cases, protein concentration in the intact csf, sera of ms patients and final solutions of abs was measured using bradford assay with a bovine serum albumin standard. the concentration of iggs after their purification by affinity chromatography on protein g-sepharose (see below) was measured in the same way. relative concentrations of iggs in the intact csf and the sera of ms patients were analyzed using special quantitative isoelectrofocusing and immunoblotting test system in italy according to the standard manufacturer's protocol and equipment (igg ief, helena laboratories, gateshead, tyne and wear, uk). in addition, the relative concentrations of iggs in the intact csfs and the sera of ms patients were measured after abs purification by affinity chromatography on protein g-sepharose (see below). the titers of anti-dna abs were determined using standard assay plates with immobilized double-stranded dna, horseradish peroxidase-conjugated mouse abs against human igg, and tetraethyl benzidine as substrate according to the standard manufacturer's protocol (vector, russia). the preparations of human blood serum and csf were diluted respectively 1000 and 2 times and 100 ml of final solution was added to the strips. the reaction was stopped with sulphuric acid and optical density (a 450 ) of the solutions was determined using a uniskan ii plate reader (mtx lab systems, usa). the relative concentration of anti-dna abs in the samples was expressed as the difference in the relative absorbance at 450 nm (average of three measurements) between the experimental samples and the control samples containing no abs. as additional controls, we have used preparations complete devoid of abs after passage of csfs through protein g-sepharose and protein a-sepharose. there was no difference in the relative absorbance at 450 nm of csf preparation containing no abs and controls containing a buffer only. finally, a 450 values were recalculated on respective biological fluids without dilution. electrophoretically and immunologically homogeneous iggs were obtained by sequential affinity chromatography of the csf and serum proteins on protein g-sepharose and fplc gel filtration similarly to [24] [25] [26] [27] . in each case the protein corresponding to the central part of igg peaks was concentrated and used in further purification or analysis. iggs from csf were incubated in 50 mm glycine-hcl (ph 2.6) containing 0.2 m nacl for 20 min at 25uc. separation of the iggs under ''acid shock'' conditions was done by fplc gel filtration on a superdex 200 hr 10/30 column equilibrated with 50 mm glycine-hcl (ph 2.6) containing 0.1 m nacl as previously described [24] [25] [26] [27] . after 1-2 weeks of storage at 4uc in order to refold after the acid shock, the abs were used in the activity assays described below. in some cases, electrophoretically homogeneous iggs were chromatographed on sepharose bearing immobilized monoclonal mouse abs against light chains of human iggs as in [24] [25] [26] [27] . the protein was applied to the column (1 ml) equilibrated with 20 mm tris-hcl (ph 7.5) containing 0.1 m nacl and the column was washed with the same buffer containing 0.3 m nacl. abs were eluted in 0.05 m glycine-hcl (ph 2.6), neutralized, dialyzed and sterilized as described above. dna-hydrolyzing activity of total protein and igg preparations was analyzed using supercoiled (sc)dna as earlier described for the analysis of dnase i and human serum catalytic antibodies [24] [25] [26] . the reaction mixture (20 ml) contained 20 mg/ml scdna pbluescript, 5 mm mgcl 2 , 1 mm edta, 20 mm tris-hcl (ph 7.5), and 0.003-0.2 mg/ml abs or initial preparations of the sera or csf (total protein) finally diluted respectively 1000-and 15fold. reaction mixtures were incubated for 0.5-3 h (standard time, 2 h) at 37uc. the cleavage products were analyzed by electrophoresis in 1% agarose gel. the images of ethidium bromidestained gels were captured on a sony dsc-f717 camera and a relative amount of dna in different bands was analyzed using imagequant v5.2 (molecular dynamics). the activities of igg preparations were determined as a decrease in the percentage of dna converted from the initial supercoiled form to the relaxed form, corrected for the distribution of dna between these bands in the control (incubation of pbluescript in the absence of abs). all measurements (initial rates) were taken within the linear regions of the time courses (15-40% of dna hydrolysis) and then recalculated to the standard conditions (see tables) . sds-page analysis of abs for homogeneity and for the polypeptide spectrum of the sera and csf was performed in a 5-16% gradient gel containing 0.1% sds (laemmli system) as described in [24] [25] [26] . iggs were used before and after their treatment with 10 mm dithiothreitol. the polypeptides were visualized by silver and coomassie blue staining [24] [25] [26] . in situ, experiments dnase activity of iggs after sds-page was analyzed in gel containing calf thymus dna (5 mg/ml) under non-reducing conditions as in [24, 25] . before the electrophoresis, igg samples were incubated at 22uc for 10-20 min in 20 mm tris-hcl (ph 7.5) containing 0.1% sds. to restore the enzymatic activity after sds-page, sds was removed by incubating the gel for 1 h at 22uc in 20 mm tris-hcl (ph 7.5) and washing the gel five times with the same buffer. to refold the protein after sds treatment and to assay it for dnase activity, longitudinal slices of the gel were incubated at 25uc for 15-48 h in the reaction buffer containing 20 mm tris-hcl (ph 7.5), 4 mm mgcl 2 , and 0.2 mm cacl 2 . to visualize the products of dna hydrolysis, the gel was stained with ethidium bromide. the same ethidium bromidestained or parallel longitudinal slices were used to detect the position of igg in the gel by coomassie blue staining. the results are reported as mean 6 s.e. of at least three independent experiments for each sample analyzed. errors in the 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polyclonal dna-hydrolyzing antibodies from the sera of patients with multiple sclerosis immunoglobulins from blood of patients with multiple sclerosis like catalytic heterogeneous nucleases iggs containing light chains of the l and k type and of all subclasses (igg1-igg4) from sera of patients with multiple sclerosis hydrolyze dna amylolytic activity of igm and igg antibodies from patients with multiple sclerosis novel functional activities of anti-dna autoantibodies from sera of patients with lymphoproliferative and autoimmune diseases recommended diagnostic criteria for multiple sclerosis: guidelines from the international panel on the diagnosis of multiple sclerosis rating neurological impairment in multiple sclerosis: an expanded disability scale (edss) defining the clinical course of multiple sclerosis: results of an international survey intrathecal production of chlamydia pneumoniae-specific high-affinity antibodies is significantly associated to a subset of multiple sclerosis patients with progressive forms catalytic hydrolysis of vasoactive intestinal peptide by human autoantibody organ-specific and systemic autoimmune diseases originate from defects in hematopoietic stem cells formation of different abzymes in autoimmune-prone mrl-lpr/lpr mice is associated with changes in colony formation of haematopoetic progenitors antibodies with amylase activity from the sera of autoimmune-prone mrl/mpj-lpr mice multiple sclerosis: molecular and cellular mechanisms oil and gas linkage disequilibrium between the mbp tetranucleotide repeat and multiple sclerosis is restricted to a geographically defined subpopulation in finland viral infections trigger multiple sclerosis relapses: a prospective seroepidemiological study concepts of viral pathogenesis of multiple sclerosis myelin basic protein and human coronavirus 229e cross-reactive t cells in multiple sclerosis affinity and catalytic heterogeneity of polyclonal myelin basic protein-hydrolyzing iggs from sera of patients with multiple sclerosis multiple sclerosis: a two-stage disease analytical performance of the three commercially available nephelometers compared quantifying protein in serum and cerebrospinal fluid this study was supported in part by grants from the presidium of the russian academy of sciences (molecular and cellular biology program, dnase antibodies cerebrospinal fluid plos one | www.plosone.org april 2014 | volume 9 | issue 4 | e93001 6.2; russian foundation for basic research (13-04-00208, 13-04-00205, and 14-04-31281), funds from the siberian division of the russian academy of sciences and funds from the regione emilia romagna, italy (ricerca sanitaria finalizzata). key: cord-000786-ofpcgxce authors: chua, brendon y.; johnson, douglas; tan, amabel; earnest-silveira, linda; sekiya, toshiki; chin, ruth; torresi, joseph; jackson, david c. title: hepatitis c vlps delivered to dendritic cells by a tlr2 targeting lipopeptide results in enhanced antibody and cell-mediated responses date: 2012-10-16 journal: plos one doi: 10.1371/journal.pone.0047492 sha: doc_id: 786 cord_uid: ofpcgxce although many studies provide strong evidence supporting the development of hcv virus-like particle (vlp)-based vaccines, the fact that heterologous viral vectors and/or multiple dosing regimes are required to induce protective immunity indicates that it is necessary to improve their immunogenicity. in this study, we have evaluated the use of an anionic self-adjuvanting lipopeptide containing the tlr2 agonist pam(2)cys (e(8)pam(2)cys) to enhance the immunogenicity of vlps containing the hcv structural proteins (core, e1 and e2) of genotype 1a. while co-formulation of this lipopeptide with vlps only resulted in marginal improvements in dendritic cell (dc) uptake, its ability to concomitantly induce dc maturation at very small doses is a feature not observed using vlps alone or in the presence of an aluminium hydroxide-based adjuvant (alum). dramatically improved vlp and e2-specific antibody responses were observed in vlp+e(8)pam(2)cys vaccinated mice where up to 3 doses of non-adjuvanted or traditionally alum-adjuvanted vlps was required to match the antibody titres obtained with a single dose of vlps formulated with this lipopeptide. this result also correlated with significantly higher numbers of specific antibody secreting cells that was detected in the spleens of vlp+e(8)pam(2)cys vaccinated mice and greater ability of sera from these mice to neutralise the binding and uptake of vlps by huh7 cells. moreover, vaccination of hla-a2 transgenic mice with this formulation also induced better vlp-specific ifn-γ-mediated responses compared to non-adjuvanted vlps but comparable levels to that achieved when coadministered with complete freund’s adjuvant. these results suggest overall that the immunogenicity of hcv vlps can be significantly improved by the addition of this novel adjuvant by targeting their delivery to dcs and could therefore constitute a viable vaccine strategy for the treatment of hcv. hepatitis c virus (hcv) infection affects an estimated 200 million individuals worldwide and contributes to significant morbidity and mortality rates associated with liver cirrhosis and hepatocellular carcinoma. approximately 80% of infected individuals do not clear the virus following acute infection and will develop chronic infection that can lead to end-stage liver disease and complications. although treatment options using a combination of pegylated interferon-a and ribavirin are available, sustained clearance of the virus is only achieved in approximately 40% of individuals infected with hcv genotype 1 and 60-70% of those who are infected with genotypes 2 or 3 [1] . recent advances in the treatment of hcv using directly acting antiviral agents (daas) such as boceprevir and telaprevir have improved svr rates in both treatment naïve and experienced patients (reviewed in [2] ). however, treatment can be prolonged, expensive and also associated with substantial side effects. the development of an effective vaccine that can significantly reduce the number of new infections and improve sustained virological response rates could therefore be a useful adjunct to current therapeutic approaches and reduce the impact of infection on global health care systems. whilst the immune correlates mediating the clearance of virus are still not entirely clear or defined, there is substantial evidence demonstrating that the development of a broad multifunctional t cell response against an array of key viral proteins such as core, e1, ns3, ns4 and ns5 during acute hcv infection is associated with disease resolution [3, 4] and may also provide a level of protection against reinfection [5] . it is also becoming increasingly apparent that such responses alone are not enough [6] and that neutralising antibodies also play an integral role in conferring protection [7, 8] and facilitating viral clearance by mechanisms including antibodydependent cellular cytotoxic mechanisms [9] . an effective hcv vaccine will need to induce antibody and cell-mediated responses and also provide cross protection against different viral genotypes and quasispecies. neutralising antibodies induced against conserved, conformational epitopes in the viral envelope e1 and e2 glycoproteins [10] [11] [12] , notably antigenic region 3 (ar3) [13] of e2, including the critical neutralisation contact residues contained within domain i of e2 [14] and amino acids 313-327 of e1 [15] , can be broadly cross-neutralising. the fact that these antibodies neutralise different hcv genotypes highlights the importance of including epitopes from both envelope proteins for a vaccine strategy to be effective. virus-like particles (vlps) possess features which make them ideal vehicles for the delivery of viral antigens to the immune system; (i) antibody epitopes are presented in the native conformation for induction of potentially neutralising antibodies (ii) multiple t cell, cd4 + and cd8 + , epitopes are packaged in vlps (iii) vlps lack regulatory proteins as well as genetic material that could pose a risk of reversion or mutation (iv) encouraging results have been obtained using insect cell-derived recombinant vlps expressing hcv antigens which induce virus-specific humoral and cellular responses [16] [17] [18] (v) hcv vlps appear to possess properties favourable for dendritic cell uptake [19] and (vi) they exhibit superior immunogenicity and antigenicity over recombinant protein and dna-based vaccine approaches [16, 17] . an important consideration in the manufacture of hcv-based vlps is the cell-type used for their manufacture. for example, it has been shown that vaccination with recombinant hcv envelope proteins expressed in mammalian cells, but not in yeast or insect cells, protect chimpanzees from primary infection by an homologous hcv isolate [20] . similarly, rosa et al have demonstrated that mammalian cell-derived recombinant envelope proteins bind to human cells with higher affinity than those produced in yeast or insect cells and appear to be antigenically and functionally similar to the viral proteins produced in an infected host cell [21] . more recently, vaccination of macaques using vlps in a prime-boost regime has been reported to induce broadly neutralising antibody responses against different hcv genotypes [22] . although all of these studies provide encouraging results supporting the development of hcv vlp-based vaccines, the fact that heterologous viral vectors and/or unrealistic dosing regimes are required to induce protective immunity indicates that it is necessary to improve their immunogenicity. in this study we evaluate the immunogenicity of mammalian cell-derived vlps containing structural proteins (core, e1 and e2) of hcv genotype 1a when delivered directly to dendritic cells (dcs) using a toll-like receptor 2 (tlr2) targeting lipopeptide. this lipopeptide contains the tlr2 agonist dipalmitoyl-sglyceryl-cysteine (pam 2 cys) and associates electrostatically with protein antigens significantly improving their ability to induce both humoral and cell-mediated responses [23] . the ability of lipopeptide-vlp complexes to facilitate dc uptake, induce antibody capable of inhibiting vlp entry into target cells and to elicit cell-mediated antigen-specific responses were each determined. hcv virus-like particles were constructed using a recombinant adenovirus containing encoding the hcv structural proteins (core, e1 and e2) of hcv 77h, genotype 1a. briefly, the core/e1 genes were amplified from pbrtm_hcv 1-3011 plasmid containing the genome of hcv h77 genotype 1a (a gift from prof c rice). the core/e1 genes were amplified from pbrtm_hcv 1-3011 plasmid containing the genome of hcv h77 genotype 1a (a gift from prof c rice). the forward core/e1 primer (59gcctctagagccaccatgcatcaccatcaccat-cacacaagcacgaatcctaaactcaaagaaaaacc 39) was designed to introduce an xbai enzyme restriction site followed by a kozak sequence, a start codon and a his(6) tag at amino terminal end of the core protein. the reverse primer of core/e1 (59 ggcttaagcccggtgacgtgggtttcc gcgtcgac 39) was designed to amplify from the sequence downstream of the region corresponding to e1/e2 cleavage site (amino acids 383/384) and to introduce an afl ii restriction site at the 39 end. next, the e2 genome was amplified using a forward primer (59cgactagt-gaaacccacgtcaccgggggaagtgccggccgc 39) that also introduced a spei site at the 59 end. the reverse e2 primer (59cggatatctcatcac gcctccgcttgggatatgagtaa-catcatcc 39) was designed to introduce a double stop codon and an ecorv restriction site at the 39 end of the amplicon. the core/e1 and e2 amplicons were cloned into pgemeasy (promega) and subsequently subcloned and ligated to produce a construct which was verified by dna sequencing. this construct was subsequently subcloned into padtrack-cmv (provided by b vogelstein, howard hughes medical centre, baltimore), digested with pmei and transformed into adeasier-1 cells by electroporation (bio-rad gene pulser) as previously described [24] . high titres of recombinant adenovirions encoding the hcv proteins (radhcv-ce1e2) were produced in 293t cells by serial passaging and the equivalent multiplicity of infection (moi) was determined as described previously [24] . to produce hcv vlps, huh7 cells were infected with radhcv-ce1e2 at a moi of 1. at 72 hours post-infection, cells were collected and disrupted using a dounce homogeniser and centrifuged at 17,000 g for 5 min. the supernatant was further centrifuged through a 30% sucrose cushion (containing 20 mm tris ph 7.4 and 150 mm nacl) at 178,000 g for 4 hours at 4uc. the resulting pellet was resuspended in 50 mm tris ph 7.and 100 mm nacl and purified through a 33% caesium chloride gradient by ultracentrifugation at 14uc at 143,000 g) for 72 hours. twelve 1 ml gradients were recovered and dialysed against sterile pbs at 4uc overnight. fluorescent labelling of vlps was achieved by adding vlps (6 mg/ml) to 2 mg/ml of fluorescein isothiocyanate (fitc; sigma aldrich) in 50 ml of dmso. the suspension was vortexed vigorously and incubated overnight at 4uc before dialysis against pbs the next day. all vlp preparations were stored in aliquots at 270uc until use. huh7 and 293t cells were grown in dulbecco's modified eagle's medium (dmem; invitrogen usa) supplemented with 10% fetal calf serum (fcs) and streptomycin 50 mg/ml at 37 c in 5% co 2 . the syntheses of the branched anionic peptide construct containing eight n-terminal glutamic acid residues (e 8 ) using traditional fmoc chemistry has been described previously [23] . briefly, synthesis was carried out manually using peg-s ram solid support (rapp polymere, tübingen, germany; substitution factor 0.27 mmol/g). fmoc-lysine(mtt)-oh (novabiochem, läufelfingen, switzerland) was first coupled to the support and the fmoc protecting group present on the a-amino group then removed and fmoc-lysine(fmoc)-oh was then coupled to the exposed n-terminal amino group. subsequent de-protection and acylation of another two rounds of fmoc-lysine(fmoc)-oh yielded eight branch points to which glutamic acid residues were coupled. the primary amino groups of the glutamic acid residues were then acetylated using a 20-fold excess of acetic anhydride and a 40-fold excess of diisopropylethylamine (dipea; sigma, australia) to generate e 8 which has an overall charge of 28. lipidation of e 8 was then carried out by removing the mtt protective group present on the e-amino group of the c-terminal lysine followed by acylation of the exposed e-amino group with two serially added serine residues. the pam 2 cys lipid moiety was then coupled according to zeng et al [25] to generate e 8 (pam 2 cys) ( figure 1a ). following assembly, lipopeptides were cleaved from the solid phase support and all side-chain protecting groups removed with 88% tfa, 5% phenol, 2% tips, 5% water for 3 hours at room temperature. lipopeptides were analysed by reversed phase highpressure liquid chromatography (rp-hplc) using a vydac c4 column (4.66300 mm) installed in a waters hplc system. the chromatogram was developed at a flow rate of 1 ml/min using 0.1% tfa in h 2 o and 0.1% tfa in acetonitrile as the limit solvent. lipopeptides were purified if necessary. all products presented as a single major peak on analytical rp-hplc and had the expected mass when analysed using an agilent series 1100 ion trap mass spectrometer. a line of murine balb/c-derived dcs (d1 cells) was prepared and propagated according to the method described by chua et al [26] . after a minimum of 21 days in culture, cells were stained for class ii mhc using fitc conjugated anti-ia/ie antibody (clone m5/114.15.2; becton dickinson, usa) and pe-conjugated cd11c (clone 2g9; becton dickinson, usa) prior to use. cells were verified to be cd11c + mhc class ii + by flow cytometry using a facscaliber (becton dickinson, usa). d1 cells (2610 5 ) were seeded onto a petri dish in 1 ml of fresh d1 media [26] and incubated at 37uc and 5% co 2 in the presence or absence of 5 mg fitc-labelled vlps alone or with vlps mixed with e 8 pam 2 cys (0.2 pmole/ml). cells were harvested 24 hours later and washed with facs wash (1% fcs/5 mm edta in pbs) before fixation in 1% paraformaldehyde in pbs. to examine cellular association of vlps, cell fluorescence was analysed by flow cytometry (facscaliber, becton dickinson, usa). for examination of intracellular uptake of vlps, extracellular fluorescence was quenched by addition of an equal volume of 0.1 m citrate buffer (ph 4.0) containing 250 mg/ml trypan blue (merck, damstadt, germany) prior to analysis [27] . data were analysed using flowjo software (tree star, san carlos, ca). to assess the degree of dc maturation resulting from exposure to different adjuvants, cells were exposed to varying concentrations of aluminium hydroxide gel, alhydrogel (sigma aldrich, missouri, usa), e 8 pam 2 cys or lipopolysaccharide (5 mg/ml) as a positive control (lps; sigma aldrich, milwaukee, usa). in some experiments, cells were incubated with vlps alone (5 mg/ml) or in the presence of e 8 pam 2 cys (0.032 mg/ml). after 16 hours, cells were harvested, washed and analysed for expression of surface class ii mhc antigen. all experimental procedures involving animals were approved by the university of melbourne's animal ethics committee under the aec numbers 0707207 and 061061. hla-a2k b transgenic mice (hhd mice) were obtained from the queensland institute for medical research and bred in the animal house facility of the department of microbiology and immunology at the university of melbourne under specific pathogen free conditions. these mice do not express h-2d b but instead express the chimeric monochain of the a1 & a2 domains of hla-a2.1 and the a3 cytoplasmic and transmembrane domains of h-2d b linked at its n-terminus to the c terminus of human b2 microglobulin [28] . female hhd mice (3 per group) were inoculated subcutaneously on each side of the base of tail (50 ml per dose) with vlps (30 mg) either alone, emulsified in an equal volume of complete freund's adjuvant (cfa) or with an equal amount of e 8 pam 2 cys on days 0 and 14. spleens were removed 28 days after the second dose and splenocytes restimulated in vitro at a concentration of 3610 6 cells/ml in rf-10 medium consisting of rpmi 1640 medium (gibco, usa) supplemented with 10% fetal calf serum (csl, parkville, australia) 7.5 mm hepes, 2 mm l-glutamine, 76 mm 2-mercaptoethanol, 150 u/ml penicillin, 150 mg/ml streptomycin, 150 mm non-essential amino acids and 10 u/ml of recombinant il-2 (roche, indianapolis, usa) at 37uc in an atmosphere of 5% co 2 . restimulation was carried out in the presence of 10 mg of vlps or 10 mm of an irrelevant hcvderived hla-a2-restricted epitope (ns5b2594-2602) that is not contained in the vlp construct. cells were harvested 5 days later, washed and serial dilutions commencing at 5610 5 cells/ml performed in polyvinylidene fluoride (pvdf) membrane-lined 96-well plates (millipore, ireland) previously coated with 5 mg/ml anti-ifn-c capture antibody (clone r4-6a2-bd pharmingen, san diego, usa). cells were then cultured in the presence of 3.75610 5 irradiated autologous vlp-pulsed (10 mg) splenocytes for 40 hours at 37uc and 5% co 2 . after washing with pbst (pbs containing 0.05% tween 20), biotinylated anti-ifn-c detection antibody (clone xmg1.2; becton dickinson, usa) was added and incubated for 2 hours at room temperature in a humidified atmosphere. plates were then washed and streptavidin-alkaline phosphatase (becton dickinson, usa) added and incubated for a further 2 hours. spots representative of ifn-c-producing cells were developed by the addition of 100 ml of 1 mg/ml 5-bromo-4-chloro-3-indolyl phosphate in 2-amino-2-methyl-1-propanol buffer (sigma-aldrich, usa) for 30 minutes. individual spots were counted using an aid ispot elispot reader (gmbh, strassberg, germany). analysis of variance in all experiments and all p values in this study were conducted and obtained using one-way anova nonparametric statistical analysis and tukey's post-hoc range tests performed with prism 5 (graphpad software, la jolla, california usa). flat bottom 96-well polyvinyl plates were coated with either purified hcv vlps (10 mg/ml) or recombinant e2 protein (5 mg/ ml) in pbsn 3 overnight at 4uc. prior to coating plates with hcv vlps, wells were pre-incubated with galanthus nivalis lectin (2 mg/ml; sigma aldrich australia) in carbonate buffer (15 mm naco 3 , 35 mm nahco 3 , 0.21 mm nacl) for 30 minutes at room temperature following removal of antigen, 100 ml of bsa (10 mg/ml) in pbs was added and plates incubated for 1 hour at room temperature before washing four times with pbst (pbs containing v/v 0.05% tween-20 [sigma aldrich, milwaukee, usa]). serial dilutions of sera obtained from immunised mice were added to wells and incubated in a humidified atmosphere overnight. after washing, bound antibody was detected using horseradish peroxidase-conjugated rabbit antimouse igg antibodies (dako, glostrup, denmark) in conjunction with enzyme substrate (0.2 mm 2,2_-azino-bis 3-ethylbenzthiazoline-sulfonic acid in 50 mm citric acid containing 0.004% hydrogen peroxide). the reaction was stopped by addition of 50 ml of 0.05 m naf. titers of antibody are expressed as the reciprocal of the highest dilution of serum required to achieve an optical density of 0.2. for the detection of specific antibody secreting cells by elispot, pvdf membrane-lined 96-well plates (mabtech, nacka strand, sweden) were coated with 100 ml of pbs containing vlps (10 mg/ml), recombinant e2 (10 mg/ml) or anti igg antibody (10 mg/ml) overnight at 4uc. plates were washed 5 times with pbs and blocked for 2 hours using rpmi 1640 medium (gibco, usa) supplemented with 20% bsa (sigma, australia). wells were emptied before 5610 5 splenocytes in 200 ml of rf-10 medium was added and incubated for 36 hours at 37uc in an atmosphere of 5% co 2 . spot forming units representative of specific antibody-producing cells were developed as previously described for the detection of ifn-c-secreting cells except that biotinylated anti-igg antibody and streptavidin-conjugated horseradish peroxidase (both from mabtech, nacka strand, sweden) were used as detecting reagents. in order to determine any inhibition of cell entry by vlps using antibodies present in sera of vaccinated animals, huh7 cells were enhancement of hcv-vlp immunogenicity plos one | www.plosone.org first incubated with pbs (10% fcs) for 15 min at 4uc to reduce non-specific binding of antibodies subsequently added. cells were washed twice, resuspended in pbs (0.1% fcs) and incubated with fitc-labelled vlps at 4uc for 1 hr. serial dilutions of sera from vaccinated or non-vaccinated mice were then added and incubated for a further 1 hour at 37uc. for each reaction, 5610 5 huh7 cells and 200 ng of fitc-labelled vlps were used in a total volume of 500 ml. at the end of this incubation period, cells were washed with pbs (0.1% fcs) and then fixed in bd cytofix (becton dickinson, usa). inhibition of vlp entry was determined by flow cytometry and analysed using weasel 2.0 software (walter and eliza hall institute, melbourne, australia). sera from vaccinated mice that demonstrated a decrease in specific cellular binding of 50% or more compared to sera from naïve mice were considered to contain neutralising antibodies [18] . hcv genotype 1a vlps were produced by transducing a human hepatocyte-derived cell line with recombinant adenovirus containing encoding the hcv structural proteins (core, e1 and e2) of genotype 1a to produce particles that harbour antigenic resemblance to virions produced in an infected host cell. because of the essential role that dcs play in the induction of both humoral and cell-mediated responses, we first examined the ability of a spleen-derived dc line (d1 cells) to take up fluorescein isothiocyanate-labelled hcv vlps (fitc-vlps). flow cytometric analysis revealed that dcs incubated with fitc-vlps exhibited higher whole cell fluorescence intensities compared to untreated dcs indicating the presence of cell-associated vlps ( figure 1b) . exposure of dcs to vlps pre-mixed with the lipopeptide e 8 pam 2 cys also resulted in higher levels of cell fluorescence compared to untreated dcs. the percentage of fluorescenated cells in these cultures was similar to cultures that contained fitc-vlps alone ( figure 1c) . to determine the magnitude of vlp cell uptake, intracellular fluorescence was measured by quenching extracellular fluorescence after exposure of cells to trypan blue prior to flow cytometric analysis [27] . although the resulting fluorescence intensities of dcs incubated with fitc-vlps were now lower following this treatment, the levels were still notably higher than untreated cells confirming the presence of intracellular fitc-vlps ( figure 1d ). equivalent fluorescence cell intensities were also observed in dcs that were incubated with fitc-vlps pre-mixed with e 8 pam 2 cys. however, a higher percentage of fluorescenated cells were detected in those cultures compared to those exposed to fitc-vlps alone ( figure 1e ) indicating that an increase in uptake of these constructs is facilitated using the lipopeptide. to investigate the ability of hcv vlps and e 8 pam 2 cys to cause activation of dcs, we measured the expression of surface mhc class ii molecules following incubation with the various antigens. the results (figure 2a) indicate that untreated dcs contained two populations of cells which were mhc class ii low and mhc class ii high , the latter comprising ,24% of the population analysed. while the distribution of these populations was not affected by exposure to hcv vlps alone, incubation with hcv vlps mixed with e 8 pam 2 cys caused a dramatic shift in the distribution of mhc class ii expressing cells such that 83% of cells were mhc class ii high . the upregulation of mhc class ii expression on these cells were comparable to those cultured in the presence of lps which is a potent dc maturation stimulus. further dosing experiments showed that increasing the concentrations of hcv vlps to 20 mg/ml, did not induce dc activation because the percentage of mhc class ii high dcs in cultures containing escalating doses of vlps remained similar to those containing untreated dcs ( figure 2b ). in contrast, exposure to as little as 0.1 nmoles of e 8 pam 2 cys was sufficient at inducing a greater than two-fold increase in activation of dc compared to untreated cells ( figure 2c) and was similar to the levels of activation observed with lps. no dc activation was observed in the presence of alhydrogel ( figure 2d ). the presence of e 8 pam 2 cys in hcv vlp-containing formulations however, not only promotes uptake of hcv vlps by dcs but also considerably increases the level of dc activation. to determine if the dc activating properties of vlp formulations containing e 8 pam 2 cys translate to an improvement in hcv vlp immunogenicity, balb/c mice were inoculated with vlps alone or with vlps mixed with e 8 pam 2 cys. hcv vlp-specific antibody titres in sera obtained after one, two or three doses of each formulation were then determined by elisa. administration of vlps alone in saline was able to elicit detectable titres of specific antibody that were marginally increased after each dose of antigen ( figure 3a ). in animals that received hcv vlps mixed with e 8 pam 2 cys, however, antibody levels were significantly higher, in some cases by up to ten-fold more than those from animals that received the same dose of hcv vlps alone. in fact the titre of specific antibody induced following administration of 3 doses of hcv vlps alone was achieved using a single dose only of hcv vlp mixed with e 8 pam 2 cys. when compared to animals that were inoculated with hcv vlps formulated with alhydrogel, an adjuvant widely used to induce antibody responses to both human and veterinary vaccines [29] , lower antibody titres were observed in these animals than in those that received the vlp-lipopeptide formulation. in examining levels of e2 specific antibodies elicited by vaccination, higher titres were once again demonstrated in animals that received 3 doses of vlps mixed with e 8 pam 2 cys compared to those that were inoculated with vlps alone or with alhydrogel ( figure 3b ). the hierarchical pattern of antibody responses induced by e 8 pam 2 cys and alhydrogel was also confirmed by the numbers of specific antibody secreting cells that were detected in the spleens of vaccinated mice. once again significantly higher numbers of cells secreting both hcv vlp ( figure 4a ) or e2-specific antibodies ( figure 4b ) were detected in animals that received hcv vlps mixed with e 8 pam 2 cys than those that were inoculated with hcv vlps alone or vlps formulated with alhydrogel. to assess the neutralising activity of antibodies induced by vaccination, we first set out to investigate if vlp entry into human hepatocyte cell line huh7 could be inhibited. pre-incubation of fitc-labelled vlps (vlp-fitc) with pbs or naïve serum resulted in minimal inhibition of vlp entry ( figure 5a ). however, the presence of an antibody against cd81, a cell surface molecule implicated in hcv entry into hepatocytes [30] , was able to prevent vlp entry into these cells by .90% confirming that these vlps also utilise this molecule to facilitate cell entry. we next analysed the ability of sera obtained from mice inoculated with vlps to inhibit the binding and entry of vlps into huh 7 cells ( figure 5b ). neutralisation of binding of vlps to huh7 cells was significantly greater in sera obtained from mice inoculated with vlps in e 8 pam 2 cys (,50%) compared to sera obtained from mice inoculated with vlps administered in alhydrogel (,30%) or in saline (,20%). the ability of vlp formulations containing e 8 pam 2 cys to induce a cell-meditated immune response was examined by inoculating transgenic mice expressing the mhc class i (hla-a2) allele but not endogenous h-2d b molecules [28] . control transgenic animals were inoculated with vlps alone or vlps emulsified with an equal amount of complete freund's adjuvant (cfa). splenocytes from vaccinated animals were obtained 28 days post-inoculation and re-stimulated with antigen in vitro. the results ( figure 6 ) of an elispot assay carried out revealed significantly higher numbers of hcv vlp-specific ifn-c producing cells in the spleens of mice inoculated with vlps in the presence of e 8 pam 2 cys or vlps emulsified in cfa than in those that received vlps alone. the development of novel, effective anti-viral vaccine strategies in recent times has seen a notable shift away from the use of traditional formulations which utilize whole inactivated or live attenuated viruses towards approaches based on recombinant subunit protein antigens which are more easily characterised and defined. vlps offer features that make them a useful platform for delivering viral antigens in a single vaccine construct which not only minimises the risks that may be associated with preparations containing or requiring the use of a replicating pathogen but will also closely resemble native viral antigens from which they are derived. the most convincing demonstration of vlps efficacy is the quadrivalent vlp-based vaccine gardasil which prevents persistent infection and associated disease caused by human papillomavirus [31] . other studies of vlp-based vaccination strategies have also shown promising results and led to phase i testing against a number of disease indications including seasonal and pandemic influenza, hepatitis b, malaria and hiv (reviewed in [32] ). depending on the type of virus used to manufacture a vlp construct, studies have shown that protective responses induced by vlps can be elicited without co-administration of adjuvant [33] [34] [35] . in many cases, however, the induction of useful immune responses may require multiple doses [36, 37] , a regime that may be impractical to implement in the field or involve a viral vector to provide an initial priming dose followed by a boost using vlps [22, 38] . of relevance to the present study, the use of adjuvants to enhance vlp immunogenicity has been shown to induce strong antibody responses using dose-sparing amounts of hiv [39] or norwalk virus-derived vlps [40] and also elicits cell-mediated were incubated with vlps (5 mg) alone or formulated with e 8 pam 2 cys (0.01 nmoles/ml) or alhydrogel (5 mg) in a total volume of 1 ml. for comparative purposes within all experiments, cells were also either left untreated, exposed to lps (5 mg/ml) or to similar amounts of each adjuvant alone. cell surface mhc class ii expression was determined after 16 hours using a peconjugated anti-ia/ie antibody. cells expressing low levels of mhc class ii molecules were deemed to be immature whilst those expressing high levels were considered to be mature. shown are representative histograms depicting cell surface mhc class ii expression from one of three experiments conducted separately. mhc class ii high expressing cells are shaded in grey. for dosing experiments, cells were also incubated with increasing amounts of (b) vlps, (c) e 8 pam 2 cys, (d) alhydrogel or vlps (5 mg) formulated with increasing amounts of (e) e 8 pam 2 cys, or (f) alhydrogel. doi:10.1371/journal.pone.0047492.g002 responses that culminate in improved protection against lethal influenza viral [41, 42] and tumorigenic challenge [43] . our previous studies have shown peptide epitope and proteinbased antigens can be made far more immunogenic when covalently attached to pam 2 cys in order to target their delivery via tlr2 to dendritic cells (dcs) [44] . this results in the induction of robust antibody and cd8 + t cell-mediated immune responses and has been shown for multiple indications [44] [45] [46] [47] . each of these vaccine candidates demonstrated the ability of this simple lipid structure to dramatically enhance the immunogenicity of antigens that are otherwise immunologically inert. nevertheless, the approach introduces complexities into the vaccine manufacturing process due to the requirement for covalent attachment of pam 2 cys to an antigen. the use of the anionic lipopeptide e 8 pam 2 cys overcomes many of the technical complexities related to this process, especially the use of covalent chemistries, by making use of electrostatic association with antigen [23] . the ability of pam2cys to dramatically enhance the immunogenicity of hcv vlps was demonstrated in the improved overall antibody responses that we observed. not only are greater antibody titres induced following vaccination with vlps formulated with e 8 pam 2 cys compared to the use of vlps alone or when co-administered with alhydrogel but up to 3 doses of nonadjuvanted or traditionally adjuvanted antigen were required to match the titres obtained with a single dose using lipopeptide. most importantly in the context of hcv the trend translates to improved e2-specific antibody responses and the use of lower doses of vlps to achieve this while maintaining efficacy has major advantages by providing cost benefits to vaccine manufacturers. our studies examining the interaction of vlps with dcs indicate that while improvements in vlp uptake mediated by this lipopeptide is minimal, its ability to concomitantly induce the maturation of dcs at very small doses is a feature not observed using vlps alone or vlps administered in the presence of alum. our previous work also demonstrated that association of antigen with charged lipopeptide facilitates trafficking of antigen to lymph cys. supernatants were clarified by centrifugation, incubated with huh7 cells (5610 5 ) in a total volume of (500 ml) for 1 hour. cells were then harvested and cellular fluorescence levels analysed by flow cytometry. all bar graphs represent the percentage reduction in vlp entry relative to baseline levels obtained using serum from naïve mice. doi:10.1371/journal.pone.0047492.g005 figure 6 . cell-mediated responses elicited by vaccination. hla-a2k b transgenic mice (n = 3 per group) were inoculated (100 ml) subcutaneously at the base of the tail on days 0 and 14 with 30 mg of vlps alone, emulsified with an equal amount of complete freund's adjuvant (cfa) or pre-mixed with 30 mg of e 8 pam 2 cys in saline. splenocytes were obtained 28 days later and restimulated for 7 days in the presence of 10 mg vlps or an irrelevant hcv-derived hla-a2restricted epitope not part of the vlp construct. the frequency of peptide-specific t cells producing ifn-c was determined in an elispot assay. each bar represents the average number of ifn-c producing t cells and standard deviation in each group after subtracting nonspecific responses from corresponding samples stimulated with the irrelevant peptide. doi:10.1371/journal.pone.0047492.g006 enhancement of hcv-vlp immunogenicity plos one | www.plosone.org nodes draining from the vaccination site [23] and together with the results presented in this study provide an explanation for the dose-sparing neutralising antibody responses that we observe. coadministration of vlps using charged lipopeptide has the added benefit of eliciting vlp-specific cell-mediated responses in hla-a2 transgenic mice, a fact that may also be attributed to dc targeting and activation. given the results of the work described in this study, we conclude that the use of this branched anionic lipopeptide together with vlps containing hcv antigens in order to provide a broad spectrum of conformational epitopes can provide benefits in terms of inducing improved levels of neutralising antibody titres and eliciting cell-mediated responses. this strategy could therefore constitute a valuable addition to the armamentarium of current vlp-based vaccine developments against hcv. peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis c: a randomised trial antiviral strategies in hepatitis c virus infection full-breadth analysis of cd8+ t-cell responses in acute hepatitis c virus infection and early therapy differential antigenic hierarchy associated with spontaneous recovery from hepatitis c virus infection: implications for vaccine design hepatitis c virus reinfection in injection drug users cd4+ immune escape and subsequent t-cell failure following chimpanzee immunization against hepatitis c virus rapid induction of virus-neutralizing antibodies and viral clearance in a single-source outbreak of hepatitis c spontaneous clearance of chronic hepatitis c virus infection is associated with appearance of neutralizing antibodies and reversal of t-cell exhaustion serum antibodies against the hepatitis c virus e2 protein mediate antibodydependent cellular cytotoxicity (adcc) exploiting information inherent in binding sites of virus-specific antibodies: design of an hcv vaccine candidate cross-reactive with multiple genotypes characterization of the hepatitis c virus e2 epitope defined by the broadly neutralizing monoclonal antibody ap33 induction of neutralizing antibody responses to hepatitis c virus with synthetic peptide constructs incorporating both antibody and t-helper epitopes broadly neutralizing antibodies protect against hepatitis c virus quasispecies challenge the disulfide bonds in glycoprotein e2 of hepatitis c virus reveal the tertiary organization of the molecule isolation and characterization of broadly neutralizing human monoclonal antibodies to the e1 glycoprotein of hepatitis c virus hepatitis c virus-like particles induce virus-specific humoral and cellular immune responses in mice immunization with hepatitis c virus-like particles protects mice from recombinant hepatitis c virus-vaccinia infection inhibition of hepatitis c virus-like particle binding to target cells by antiviral antibodies in acute and chronic hepatitis c uptake and presentation of hepatitis c virus-like particles by human dendritic cells vaccination of chimpanzees against infection by the hepatitis c virus a quantitative test to estimate neutralizing antibodies to the hepatitis c virus: cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells a prime-boost strategy using virus-like particles pseudotyped for hcv proteins triggers broadly neutralizing antibodies in macaques soluble proteins induce strong cd8+ t cell and antibody responses through electrostatic association with simple cationic or anionic lipopeptides that target tlr2 modulation of mapk pathways and cell cycle by replicating hepatitis b virus: factors contributing to hepatocarcinogenesis synthesis of a new template with a built-in adjuvant and its use in constructing peptide vaccine candidates through polyoxime chemistry comparison of lipopeptidebased immunocontraceptive vaccines containing different lipid groups dendritic cell acquisition of epitope cargo mediated by simple cationic peptide structures h-2 class i knockout, hla-a2.1-transgenic mice: a versatile animal model for preclinical evaluation of antitumor immunotherapeutic strategies aluminium compounds for use in vaccines binding of hepatitis c virus to cd81 safety, immunogenicity, and efficacy of quadrivalent human papillomavirus (types 6, 11, 16, 18) recombinant vaccine in women aged 24-45 years: a randomised, double-blind trial developments in virus-like particle-based vaccines for infectious diseases and cancer chimeric severe acute respiratory syndrome coronavirus (sars-cov) s glycoprotein and influenza matrix 1 efficiently form virus-like particles (vlps) that protect mice against challenge with sars-cov a microbial platform for rapid and low-cost virus-like particle and capsomere vaccines vaccine potential of nipah virus-like particles vaccination with multimeric l2 fusion protein and l1 vlp or capsomeres to broaden protection against hpv infection effect of mucosal and systemic immunization with virus-like particles of severe acute respiratory syndrome coronavirus in mice dna-vlp prime-boost intra-nasal immunization induces cellular and humoral anti-hiv-1 systemic and mucosal immunity with cross-clade neutralizing activity effects of adjuvants on igg subclasses elicited by virus-like particles an intranasally delivered toll-like receptor 7 agonist elicits robust systemic and mucosal responses to norwalk virus-like particles adjuvants that stimulate tlr3 or nlpr3 pathways enhance the efficiency of influenza virus-like particle vaccines in aged mice intranasal immunization with influenza vlps incorporating membrane-anchored flagellin induces strong heterosubtypic protection murine polyomavirus virus-like particles carrying full-length human psa protect balb/c mice from outgrowth of a psa expressing tumor a totally synthetic vaccine of generic structure that targets toll-like receptor 2 on dendritic cells and promotes antibody or cytotoxic t cell responses protection against heterologous human papillomavirus challenge by a synthetic lipopeptide vaccine containing a broadly cross-neutralizing epitope of l2 intranasal lipopeptide primes lung-resident memory cd8+ t cells for long-term pulmonary protection against influenza a selfadjuvanting multiepitope immunogen that induces a broadly cross-reactive antibody to hepatitis c virus key: cord-001865-ji83zmy7 authors: kuroda, kengo; kiyono, tohru; isogai, emiko; masuda, mizuki; narita, moe; okuno, katsuya; koyanagi, yukako; fukuda, tomokazu title: immortalization of fetal bovine colon epithelial cells by expression of human cyclin d1, mutant cyclin dependent kinase 4, and telomerase reverse transcriptase: an in vitro model for bacterial infection date: 2015-12-01 journal: plos one doi: 10.1371/journal.pone.0143473 sha: doc_id: 1865 cord_uid: ji83zmy7 cattle are the economically important animals in human society. they are essential for the production of livestock products such as milk and meats. the production efficiency of livestock products is negatively impacted by infection with zoonotic pathogens. to prevent and control infectious diseases, it is important to understand the interaction between cattle tissue and pathogenic bacteria. in this study, we established an in vitro infection model of an immortalized bovine colon-derived epithelial cell line by transducing the cells with lentiviral vectors containing genes encoding cell cycle regulators cyclin d1, mutant cyclin dependent kinase 4 (cdk4), and human telomerase reverse transcriptase (tert). the established cell line showed continuous cell proliferation, expression of epithelial markers, and an intact karyotype, indicating that the cells maintained their original nature as colon-derived epithelium. furthermore, we exposed the established cell line to two strains of salmonella enterica and ehec. interestingly, s. typhimurium showed higher affinity for the established cell line and invaded the cytoplasm than s. enteritidis. quantitative rt-pcr revealed that gene expression of toll-like receptor 1 (tlr1), tlr 2 and tlr 3, whereas tlr 4, 5 and 6 were not detectable in established cells. our established immortalized colon-derived epithelial cell should be a useful tool for studies evaluating the molecular mechanisms underlying bacterial infection. cattle are the economically important domestic animal source of livestock-related products such as meat and milk. the efficiency of their production is largely affected by various zoonotic pathogens such as escherichia coli o157:h7 [1] and salmonella enterica [2] . infection can be established by a variety of routes, including fecal contamination of feed, or transmission from humans or wild animals. many pathogens in cattle are living as commensal bacteria at the mucosal surface without invading the reservoir host, however, exponential growth of the bacteria and invasion into the intestinal epithelial cells are critical steps to establish infection in infected animals. an in vitro cell culture system is essential for molecular studies of bacterial affinity for epithelial cells. however, as far as we know, intestinal cell lines from cattle are not available from worldwide cell banks such as the american type culture collection (atcc). bacterial adhesion and invasion are detectable using relatively simple methods such as fluorescent immunohistochemical staining [3] . thus, an established bovine colon epithelial cell line would be a powerful tool for studies that assess the effects of infectious bacteria on host colon epithelial cells. over the past several decades, primary cells have typically been immortalized by the introduction of simian vacuolating virus 40 large t antigen, or human telomerase reverse transcriptase (tert) with human papilloma virus-derived e6/e7 protein [4] [5] [6] . although expression of these oncogenic proteins is effective for immortalization, these oncogenic proteins promote genomic instabilities such as chromosome structure abnormalities [7, 8] . furthermore, the expression of these oncogenic proteins can change the original nature of the primary cells. recently, sasaki et al. and shiomi et al. have demonstrated that co-expression of the human cyclin d1, mutant cdk4 (cdk4r24c), and tert immortalizes human ovarian epithelial cells and myogenic cells [9, 10] . we also previously demonstrated that co-expression of human cyclin d1, mutant cdk4, and tert efficiently immortalizes fibroblast cells derived from several kinds of animals such as pigs, cattle [11] , and monkeys [12] . this immortalization was effective, regardless of the cell or tissue type or the species of origin, and retained the original karyotype pattern in a high percentage of the cells. thus, this method is an excellent system for establishing cell lines that keep their original phenotypes. this study is one of the national projects associated with the great east japan earthquake and has been entirely endorsed and supported by the japanese government through the ministry of education, culture, sports, science and technology, japan, and the detailed description of the animal care and protocols is described in our previous study [13] . in briefly, we collected organs and tissues from the euthanized cattle by the combined unit of veterinary doctors belonging to the livestock hygiene service center (lhsc) of fukushima prefecture and those belonging to the ministry of agriculture, forestry and fisheries, japan. cattle were sacrificed by these veterinarians by the following method according to the regulation for animal experiments and related activities at tohoku university (regulation no: 2014kado-037). cattle were sacrificed by exanguination from the jugular vein in their unconscious state by a pentobarbital (2 mg/kg) and suxamethonium (10 ml/kg) after intramuscular injection of hypnotics (xylazine hydrochloride, 0.2 mg/kg). colon epithelial tissues were obtained from a fetus of japanese black cattle (male, about 5 age in month), which was resected from euthanized parent cattle that were raised in the evacuation zone surrounding the fukushima daiichi nuclear power plant accident. all procedures were authorized by the animal experiments and related activities office at tohoku university (regulation no: 2014kado-037). the colon tissue was cut in parallel to intestinal tract that is 3 cm long in inside 1cm from anus. the tissue was gently washed with phosphate buffered saline (pbs) (nissui phamaceutical co., ltd., tokyo, japan). the epithelial layer including mucosa was scraped with a sterilized knife into a 100 mm dish coated with atelocollagen (koken co., ltd, tokyo, japan) and containing dulbecco's modified eagle's medium (nacalai tesque, kyoto, japan) supplemented with 10% fetal bovine serum (invitrogen, carlsbad, us) and 1% antibiotic-antimycotic mixed solution (nacalai tesque). the dish maintained at 37°c in an atmosphere containing 5% co 2 and medium change was conducted every three days. to immortalize fetal bovine colon cells, csii-cmv-tert, csii-cmv-cyclin d1, and csii-cmv-hcdk4r24c were simultaneously introduced into the primary cells. to monitor the transfer efficiency, csii-cmv-egfp was introduced into the primary cells in the independent well. the preparation and recombination of lentiviral constructs have previously been described [9] . the production of recombinant lentiviruses with vesicular stomatitis virus g glycoprotein was also described in a previous study [9] . primary cells were seeded at a density of 1.0×10 5 cells/well in a 6-well plate and inoculated with csii-cmv-tert, csii-cmv-cyclin d1, and csii-cmv-cdk4r24c lentiviruses at a multiplicity of infection (moi) of five for each virus in the presence of 6 μg/ml polybrene. primary bovine colon cells and newly established bovine fetal colon epithelial cells in this study (bfce-k4dt cells) were seeded at a density of 1×10 5 cells/well on a 6-well plate (bd biosciences, franklin lakes, us). when the cells reached confluency, both primary cells and recombinant cells were harvested, and the total number of cells in each well was determined using a coulter automated cell counter (invitrogen). population doubling (pd) was used as the measure of cell growth rate. pd was calculated from the formula pd = log2 (a/b), where a is the number of harvested cells and b is the number of plated cells [6, 14] . experiments were carried out in triplicate, and the averages and standard deviations (sd) were calculated. after population doubling assay, bfce-k4dt cells were seeded at a density of 100 cells to 100 mm dish, and cultured for 1 week. cloning of bfce k4dt cells from single cell were performed by cloning cylinders methods. cells were washed with pbs. the cloning cylinder (sigma, st. louis, us) was set gently over a colony and 100 μl 0.25% trypsin (nacalai tesque) was added to the cloning cylinder. after incubation of the dish at 37°c for 5 minutes, 100 μl culture medium was added. cell suspension was aspirated and transferred a medium-filled 6-well plate. cloning cells were maintained as same methods as the primary culture. following experiments were done using this cloned immortalized cells (passage number > 15) and primary cells (passage number = 2). primary cells and bfce-k4dt cells were homogenized in lysis buffer (1 m tris-hcl at ph 7.4, 3 m nacl, 1% triton x-100, 6 mm sodium deoxycholate, and 0.5% protease inhibitor cocktail; nacalai tesque). the detailed method for western blotting was described in our previous report [15] . a rabbit polyclonal antibody against human cyclin d1 (1:5000, medical & biological laboratories co., ltd., nagoya, japan), a mouse monoclonal antibody against human cdk4 (1:2000, medical & biological laboratories co., ltd.), and an antibody againstá-tubulin (1:1000, santa cruz biotechnology, inc., dallas, us) were used as the primary antibodies. the corresponding secondary antibodies (sheep anti-rabbit and anti-mouse igg conjugated to horseradish peroxidase, 1:2000; ge healthcare, little chalfont, buckinghamshire, uk) were used for detection. signals on the membranes were detected using an ecl kit (ge healthcare), and the image was obtained with an imagequant las-4000 mini system (ge healthcare) under the condition (exposure time; 2 min, sensitivity; high). telomerase activity was detected with the telomere repeat amplification protocol (trap) method [16] , using a trapeze telomerase detection kit (millipore, billerica, us). cell extracts were obtained from the established cells cultured on 6-well plates. cell extract from 293 t cells was used as a positive control. the telomerase extension reaction was performed at 37°c for 90 min. pcr and polyacrylamide gel electrophoresis (page) were conducted according to the manufacturer's recommendations. to visualize dna products, the gel was stained with gelred™ (biotium, hayward, us), and the images were captured by using a uv transilluminator at 254 nm. cells were treated with colcemid at a final concentration of 0.02 mg/ml on the day before harvesting in 15 passage numbers. after trypsinization, the cells were suspended in hypotonic solution and fixed in carnoy's fluid. fixed cells were dropped onto a glass slide and stained using the g-band staining method. fifty metaphase cells were analyzed for their karyotype. the results obtained from this analysis were displayed according to the international system for human cytogenetic nomenclature (iscn). this analysis was conducted in nihon gene research labolatories inc. (miyagi, japan, test number: 15000611). an immunohistochemical study was performed on cells fixed with 4% paraformaldehyde in pbs for 20 min at room temperature on a 12-well plate. the fixed cells were washed with pbs, and incubated with 0.5% triton x-100 in pbs for 30 min at room temperature for permeabilization. as a negative control for this staining, bovine fetal fibroblast cells (bff ncc) were used [11] . after a wash with pbs, the cells were treated with 1% bovine serum albumin (sigma) in pbs as blocking buffer for 1 h at room temperature. purified mouse anti-e-cadherin (bd bioscience) and monoclonal antibody to cytokeratin 8 (exbio, vestec, czech republic) were used as primary antibodies. the samples were exposed to primary antibody in blocking buffer at 4°c overnight. cells were washed three times with pbs for 5 min and treated with alexa fluor 488-conjugated secondary antibody (invitrogen) for 1 h at room temperature, while protecting from exposure to light. the nuclei were visualized by staining with 1 μg/ml of 4',6-diamidino-2-phenylindole (dapi) (dojindo laboratories, kumamoto, japan). the stained images were detected by a fluorescence microscope system (fsx100, olympus). salmonella enterica serovar enteritidis strain zse1 isolated from zambia [17] and typhimurium wild type strain st1wt [18] bacteria were grown routinely in trypcase soy broth (tsb) at 37°c for 18 h. enterohemorrhagic escherichia coli (ehec) obtained by recloning of edl931 (strain atcc 35150) was grown routinely in brain heart infusion (bhi) broth at 37°c for 20 h. bfce primary cells and bfce k4dt cells were seeded in a 12-well plate to reach a density of 5.0 × 10 4 cells/well and incubated for 48 hours at 37°c in an atmosphere containing 5% co 2 . the medium was changed to dmem with 10% fbs (without antibiotics) 2 hours before bacterial infection to eliminate any potential effects of the antibiotics, and cells were infected with bacteria at 3.0 × 10 6 cells/well. to count the adhesive bacteria at 30, 60, and 120 minutes after the infection, wells were washed with pbs 3 times and disrupted with lysis buffer (pbs containing 0.1% (wt/vol) sodium dodecyl sulfate (sds) (wako pure chemical industries, ltd., osaka, japan) and 1% (vol/vol) triton x-100). lysate containing ehec was 10-fold diluted with pbs and plated on nutrient agar for determination of adhesion bacterial counts. conditions of cell seeding and infection of salmonella were same as the methods of ehec adhesion assay. to count the total (adhesive and invasive) bacteria at 30, 60, and 120 minutes after the infection, wells were washed with pbs 3 times and disrupted with lysis buffer. to the count for the invasive bacteria, cells were washed with pbs 3 times and incubated with 100 μg/ ml gentamicin-containing fresh culture medium for 2 hours post-infection, which results in adhesive bacteria were killed. gentamicin treated cells were lysed with the lysis buffer after pbs wash 3 times and harvested. each lysis buffer containing bacteria were 10-fold diluted with pbs and plated on nutrient agar for determination of total and invasive bacterial counts. for the assay, bfce-k4dt cells were seeded on 24-well tissue culture plates at a density of 1 × 10 5 cells/well and incubated overnight. before infection, cells were incubated for 2 h in a medium without antibiotics. they were then infected for 120 min at 37°c with a bacterial suspension at 5.0 × 10 5 cells/well. cells were washed in pbs to remove non-adherent bacteria. after 1 h blocking with 1% bsa in pbs at room temperature, extracellular bacteria were stained with an anti-salmonella serotype-specific antibody (s. typhimurium, o9 serum and s. enteritidis, o4 serum; diluted 1:300, denka seiken co., ltd., tokyo, japan), which was followed by incubation with alexa 594-labelled goat anti-rabbit antibody (life technologies, diluted 1:200) for 1 h at room temperature. cells were permeabilized for 5 min with 0.2% triton x-100, and total bacterial counts were determined by staining with antiserum against s. typhimurium, o9 serum, or antiserum against s. enteritidis, o4 serum (diluted 1:300, denka seiken co., ltd.), followed by incubation with alexa 488-labelled goat anti-rabbit antibody (life technologies, diluted 1:200) for 1 h at room temperature. in this way, internalized bacteria (green) were distinguished from extracellular bacteria (yellow, due to an overlay of red and green fluorescence). the nuclei were visualized by staining with 1 μg/ml of dapi, and the staining patterns were detected by a fluorescence microscope (fsx100, olympus, tokyo, japan). bfce cells were seeded on the 8 well millicell 1 ez slide (millipore) at 5.0 ×10 4 cells. cells were incubation overnight to detect bacterial adhesion. for the detection of bacterial adhesion, cells were changed media without antibiotics 2 h before the bacterial infection. they were then infected for 120 min at 37°c with a bacterial suspension at 3.0 ×10 6 . to prepare the sem observation, cells were washed pbs and fixed with 2% glutaraldehyde in 0.1 m cacodylate buffer for 2 h at 4°c and dehydrated by 70%, 90% and 100% ethanol for 15 min respectively. fixed and dehydrated cells were coated with platinum-palladium and observed by sem (s4200, hitachi, tokyo, japan). bfce-k4dt cells were seeded at 3.0 ×10 5 cells to a 6 well plate and incubated for 48 hr. total rna was isolated by using nuclespin rna plus and reverse transcription was performed by using primescript™ rt master mix (takara bio inc., shiga, japan) according to manufacturer's instructions. realtime pcr was done on a thermal cycler dice single (takara) using sybr premix ex taq (takara). quantitative pcr was performed with appropriate primers (s1 table) to test for statistical differences in this research, we used student's t test. a p-value less than 0.05 was considered statistically significant. data were expressed as mean ± standard deviation (sd). to monitor the efficiency of recombinant virus infection, we exposed primary bovine fetal colon cells to csll-cmv-egfp as well as simultaneously introduction of csll-cmv-tert, -cyclin d1, and -hcdk4r24c. primary bovine colon cells were efficiently infected with the egfp-expressing virus, resulting in a high percentage of green fluorescence-positive cells ( fig 1a) . we named the bfce cells transduced with mutant cdk4, cyclin d1, and tert as "bfce-k4dt" (bovine fetal colon epithelial cell established with cdk4, cyclin d1 and tert). to determine if the bfce-k4dt cells were fully immortalized, we evaluated the cell proliferation rate by measuring the population doubling (pd) value. fig 1b shows the pd value of bfce primary cells (diamond) and bfce-k4dt cells expressing cdk4r24c, cyclin d1, tert (square). bfce-k4dt cells proliferated more rapidly than did bfce primary cells, and bfce primary cells arrested cell proliferation at the 5th passage. bfce-k4dt cells showed epithelial cell-like morphologies, in both low and high cell density conditions (fig 1c) . when we compared the cell morphology of bfce-k4dt cells with that of the original bfce, there was no significant difference between them (data shot shown). the expression of mutant cdk4 and cyclin d1 proteins was detected using western blotting, and the results are shown in fig 2a. as expected, primary bfce cells did not show any signals for these proteins (fig 2a, lane 1) , whereas bfce-k4dt cells showed high protein expression for both cdk4 and cyclin d1 proteins (fig 2a, lane 2) . we next investigated telomerase activity with the trap assay [18] . we observed 6-bp ladders in 293t cells (used as positive control) and minimal activity levels in the negative control, verifying the reliability of the detection method. bfce primary cells were negative for the activity (fig 2b, lane 3) , but both bfce-k4dt 1 and 2 showed high levels of telomerase activity ( fig 2b, lanes 5) , indicating that the introduced tert was properly expressed in bfce-k4dt cells. chromosomal analysis demonstrated that the established bfce cells exhibited a normal 60xy diploid karyotype (fig 3) . this indicated that introduction of the human cyclin d1, detected in bfce-primary cells and bfce-k4dt cells (fig 4) . interestingly, these epithelial markers expression was detected throughout the cytoplasm in bfce-k4dt cells. on the other hand, minimal signals were detected in bff ncc cells (fig 4) . these findings indicate that bfce-k4dt cells are derived from epithelial cells. with this staining assay, s. typhimurium showed positive signals with alexa 488 (fig 5a and 5e , black arrowheads) and alexa 594 (fig 5b and 5f , white arrow heads). in merged images, adherent s. typhimurium yielded a yellow fluorescence pattern (fig 5d and 5h , stars) and intracellular s. typhimurium was revealed by green fluorescence (fig 5, 5d and 5h, black arrows). these results indicate that s. typhimurium has affinity for the surface cellular membrane and can invade the established bfce-k4dt cells. however, bfce-k4dt cells exposed to s. enteritidis, as well as non-infected bfce-k4dt cells, did not show any intense dots of staining in the cytoplasm (fig 5, 5i-5l and 5m-5p ). fig 6a is showing the results of gentamycin protection assay using s. enteritidis and s. typhimurium. in bfce-primary cells, s. enteritidis was detectable as total bacteria in time-dependency but not as invasion bacteria, which indicates that s. enteritidis hardly invade bfce-primary cells while can adhere the cell surface. although this tendency was observed in bfce-k4dt cells, invasive s. enteritidis was able to detect even in 30 min after infection. s. typhimurium could adhere both bfce-primary and-k4dt cells as same as primary cells, and also invade in time-dependency more than s. enteritidis. results of fluoroscopy assay described above and these quantifications suggest that established bfce-k4dt cells have host specificity originated from primary cells, and immortalized bfce-k4dt cells can be more susceptible for invasion of salmonella than primary cells. we sought to identify the adhesion of other bacterium, ehec, and show the result in fig 6b. the number of adhesion ehec in both primary and k4dt cells increased time-dependent manner. at 120 min after infection, adhesion of ehec on bfce-primary was more than -k4dt cells. this bias could also be confirmed in salmonella infection (fig 6a) , however observed more prominently in ehec infection. scanning electron microscopy produced detailed images that show bacterial adhesion on the cell surface. s. enteritidis (fig 6ca) and s. typhimurium (fig 6cb) tended to attach the cell edge while ehec was adhering cell surface (fig 6cc) . quantitative rtpcr revealed that established bfce-k4dt cells transcribed tlr 1, tlr 2 and tlr 3, but tlr4, 5 and 6 were could not detectable. this result was different from previous study that primary colonocyte cells expressed tlr 1, tlr 3, tlr 4 and tlr 6 [19] (table 1) . . total bacteria were stained green (a, e, i, and m; black arrows), adherent bacteria were stained red (b, f, j, n; white arrows), nuclei of cells were stained blue, and merge images of extracellular bacteria (stars) and intracellular bacteria (black arrows) appear yellow and green respectively (d, h, l, p). each staining was carried for 3 times and representative pictures were shown here. intestinal epithelial cells are exposed to serious risk of the pathogenic microbes although have an important role for the uptake of nutrients and fluids. previous studies have attempted to establish intestinal epithelial cell lines from various animals, such as cat [20] , mouse [21] and cattle [22, 23] by the introduction of simian vacuolating virus 40 large t antigen. transfection of this gene is the most frequently used to immortalization not only intestinal cells but also various cells. these established cell lines, however, include the risk for the change of the original nature of the primary cells [7, 8] . for the establishment of a cell line, the first difficulty to overcome is the hayflick limit [24] . primary cells cannot proliferate indefinitely due to cellular senescence. as a solution to this limitation, we introduced human cyclin d1, mutant cdk4, and tert. the version of cdk4 we transfected has a mutation (r24c) at the binding site for the senescence protein p16. when a normal cell is exposed to cellular stress, or reaches the senescence point, p16 protein accumulates in the cells [25, 26] . the p16 protein binds to cdk4 and negatively regulates the activity of cdk4-cyclin d1 complex. however, p16 cannot suppress the cdk4r24c-cyclin d1 complex. due to the constitutive activity of the cdk4r24c-cyclin d1 complex, phosphorylation and inactivation of retinoblastoma protein (prb) is maintained, resulting in an accelerated rate of cell growth. this method of cell immortalization was initially reported in humans [9] , and we previously reported that this immortalization method could be applied to a wide variety of animals, since cdk4 and cyclin d1 are evolutionally conserved [11, 12] . in the present study, we established a bfce-k4dt cell line, derived from bovine fetal colon epithelial cells via transfection of three genes, human cyclin d1, mutant cdk4, and telomerase. this cell line showed an accelerated speed of cell proliferation and kept on proliferating after stopping its growth in bfce-primary cells (passage number = 5) as a result of the expression of these three genes. furthermore, karyotype analysis revealed that established bfce-k4dt cells maintain an intact karyotype after passage number 15. from these results, we succeeded in establish immortalized cell line from primary cells having comparatively weak proliferative capacities. the bfce-k4dt cells showed positive expression of cytokeratin 8 and e-cadherin, which are markers of epithelium-derived cells although these expressions were not strong in the cellto-cell contact region. thus, tight junction of bfce-k4dt cells is not strong even when cells are confluence. furthermore, bfce-primary cells were also not strong expression levels of these epithelial markers in the cell-to-cell contact region, which suggests that this feature was not caused by the method in this study but the native character. furthermore, sem analysis in this study revealed surface of bfce-k4dt cells (fig 6c) , and we could not recognized rough and slender microvilli-like structure observed in other research reported [22] even when we maintained bfce-k4dt cells for 1 week after confluency in the culture (data not shown), but microvilli-like projections were detectable (fig 6cc) . it was known one of the reasons why a loss of the epithelial characteristics including cell-to-cell junction is caused by epithelial mesenchymal transition (emt) [27] resulting from cell disaggregation during the period of epithelial [29] . we sought to establish the new cell line because the expression of these oncogenic proteins could lead to change the original nature of the primary cells. these observations suggested that further challenges are necessary to understand the flexible and dynamic cell environments by using various types of cell lines, and established bfce-k4dt cells could be a valuable tool. salmonellosis is an important disease of cattle, most often caused by s. dublin and s. typhimurium. s. typhimurium is frequently detected in calves around 2 months of age, in which it induces diarrhea [30] [31] [32] and also infect a wide range of domesticated or wild animals, as well as humans [33] . s. enteritidis is most often isolated from chickens and eggs [34] , and is also one of the major causes of human food-borne gastroenteritis worldwide [35] . interestingly, many s. typhimurium were detected as intense dots of positive staining when added to bfce-k4dt cells, which was explained as the presence of the bacterium in the cytoplasm of the cells. however, s. enteritidis did not show any intense positive signals in the cytoplasm, indicating a difference in the ability of s. typhimurium and s. enteritidis to invade bovine colon epithelium. furthermore, gentamycin protection assay revealed that the numbers of invasive s. enteritidis were smaller than that of s. typhimurium in support of immunofluorescent staining despite higher dose of salmonella than fluorescent immunohistochemistry. previous study revealed that there are differential immune responses to s. typhimulium, s. dublin, and s. enteritidis in bovine peripheral blood leukocytes [36] . these suggests that this detected difference of invasion number between s. typhimurium and s. enteritidis might account for their distinct host infection associations and can be a possible factor of immune response differentiation. toll-like receptors (tlrs) contribute to host resistance to microbial pathogens, and each tlr bind targets as microbial ligands, such as tri-acylated lipopeptide (tlr 1), lipoprotein (tlr 2), double strand rna (tlr 3), lipopolysaccharide (lps) (tlr 4), flagellin (tlr 5) and di-acyl lipopeptide (tlr 6) [37, 38] . in this research, we could detect the tlr 1, tlr 2 and tlr 3, but not tlr 4, tlr 5 and tlr 6. on the other hand, bridger et al. have reported that bovine primary colonic cells established by them harbored mrna specific for tlr 1, tlr 3, tlr 4 and tlr 6 [19] . these results show there are differences of expressing tlr genes among bovine colonic cells, and new bovine colon cell line establishment will lead to an opportunity for deeper understanding of the interactions between pathogenic bacteria and bovine host. tlr 2 mediates cellular responses to various infectious pathogens and their products including yeast cell walls, whole mycobacteria, peptidoglycan [39] [40] [41] [42] [43] [44] . tlr 2 activities for recognition of these ligands, however, are known to complex other tlrs, such as tlr 1 and tlr 6 [45] . q-rtpcr showed tlr 1 and tlr 2 were transcribed in bfce-k4dt cells, so that this cell line is a useful model for investigation of the interaction between infectious pathogens and tlr 1/2 heterodimer complex. although established bfce-k4dt cell did not show gene expression tlr 6, they could be useful in vitro model for discussing about interaction with tlr 2 and the downstream signaling of tlr 2/6 heterodimer complex. tabeta et al. have reported that tlr3 deficient mice are hyper-susceptible to mouse cytomegalovirus infection, which indicates tlr3 has a protective role against viral infection [45] . tlr3 is expressed in cell endosomal compartments can recognize viral nucleic acids [46] . established bfce-k4dt cells are expressing tlr3, thus this cells are suitable for the study accompanied with not only bacterial infection but also viral infection. in this study, tlr 4 playing a key role in the recognition of lps and tlr 5 that is triggering cardiac innate immune responses and causing acute contractile dysfunction when combined with its ligand flagellin [47] were not detectable. thus, established cells can be considered pathways via these receptors' ligand were inactivated, such as myd88-and tirap-dependent pathways [48] . tlrs-deficient mice were developed to examine the role of tlrs in vivo [49] [50] [51] [52] [53] [54] . bfce-k4dt cells can be suited for in vitro monitoring the effects of these ligands without these tlrs and them downstream pathways as well as tlr 1, 2, 3-mediated reaction confirmed in bfce-k4dt. these results need to be further evaluated with various types of bacterial strains, and our established immortalized colon epithelium-derived cell line having many distinctive features will continue to be a useful tool to investigate the molecular mechanisms underlying intestinal bacterial and viral infection. supporting information s1 table. sequences of primers for detection of tlrs gene expression. primers were designed according to previous report [19] . 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compromising differentiation potential bovine and porcine fibroblasts can be immortalized with intact karyotype by the expression of mutant cyclin dependent kinase 4, cyclin d, and telomerase establishment of cell lines derived from the genus macaca through controlled expression of cell cycle regulators distribution of artificial radionuclides in abandoned cattle in the evacuation zone of the fukushima daiichi nuclear power plant effects of bisphenol a exposure on the proliferation and senescence of normal human mammary epithelial cells the anti-proliferative effects of the chfr depend on the forkhead associated domain, but not e3 ligase activity mediated by ring finger domain detection of telomerase activity in human cells and tumors by a telomeric repeat amplification protocol (trap) detection of salmonella inva by isothermal and chimeric primer-initiated amplification of nucleic acids (ican) in zambia derepression of salmonella pathogenicity island 1 genes within macrophages leads 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the preexisting cytokeratin network during epithelial-mesenchymal transition of cultured neonatal rat hepatocytes development of an in vitro model for bovine placentation: a comparison of the in vivo and in vitro expression of integrins and components of extracellular matrix in bovine placental cells. cells, tissues, organs establishment and characterization of immortalized bovine endometrial epithelial cells molecular screening and risk factors of enterotoxigenic escherichia coli and salmonella spp. in diarrheic neonatal calves in egypt the estimation of doses of salmonella typhimurium suitable for the experimental production of disease in calves aromatic-dependent salmonella typhimurium as modified live vaccines for calves current perspectives in salmonellosis. the british veterinary journal results of salmonella isolation from poultry products, poultry, poultry environment, and other characteristics mechanisms of egg contamination by salmonella enteritidis differential innate immune responses of bovine peripheral blood leukocytes to salmonella enterica serovars dublin, typhimurium, and enteritidis. veterinary immunology and immunopathology pathogen recognition with toll-like receptors. current opinion in immunology toll-like receptors: critical proteins linking innate and acquired immunity cellular responses to bacterial cell wall components are mediated through myd88-dependent signaling cascades. international immunology human toll-like receptor 2 mediates monocyte activation by listeria monocytogenes, but not by group b streptococci or lipopolysaccharide human toll-like receptors mediate cellular activation by mycobacterium tuberculosis peptidoglycan-and lipoteichoic acidinduced cell activation is mediated by toll-like receptor 2. the journal of biological chemistry cutting edge: recognition of gram-positive bacterial cell wall components by the innate immune system occurs via toll-like receptor 2 toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products the repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors subcellular localization of toll-like receptor 3 in human dendritic cells bacterial flagellin triggers cardiac innate immune responses and acute contractile dysfunction pathogen recognition and innate immunity cutting edge: role of toll-like receptor 1 in mediating immune response to microbial lipoproteins differential roles of tlr2 and tlr4 in recognition of gram-negative and gram-positive bacterial cell wall components cutting edge: toll-like receptor 4 (tlr4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for tlr4 as the lps gene product detection of pathogenic intestinal bacteria by toll-like receptor 5 on intestinal cd11c+ lamina propria cells discrimination of bacterial lipoproteins by toll-like receptor 6 recognition of double-stranded rna and activation of nf-kappab by toll-like receptor 3 we thank dr. hiroyuki miyoshi (riken, bioresource center) for providing the lentivirus. we express our appreciation to dr. shin-ichi ohno and ms. takako ishiyama for their technical assistance. key: cord-003712-mafz21no authors: perez vidakovics, maria laura a.; ure, agustín e.; arrías, paula n.; romanowski, víctor; gómez, ricardo m. title: junín virus induces autophagy in human a549 cells date: 2019-06-19 journal: plos one doi: 10.1371/journal.pone.0218730 sha: doc_id: 3712 cord_uid: mafz21no autophagy, a highly regulated degradative process that promotes cellular homeostasis, is increasingly recognised as a fundamental component of the cellular response against viral infection. in this study, we investigated the role of autophagy during junín virus (junv) multiplication using human a549 cells. we found that junv infection induces an increment of the lc3-ii/lc3-i ratio, an accumulation of punctate pattern in rfp-lc3-transfected cells and the colocalisation of viral nucleoprotein and lc3 protein, suggesting autophagosome formation. junv infection also induced the degradation of the autophagy receptor p62, suggesting that complete autophagic flux was triggered. in addition, we showed that inhibition of autophagy with bafilomycin a1 or 3-methyladenine significantly reduces viral multiplication. moreover, viral yield was increased when autophagy was induced using rapamycin. furthermore, junv infection induced the colocalisation of p62, atg16, rab5, rab7a and lamp1 with the autophagosomal lc3 protein. that suggests that phagosomes undergo the maturation process during viral infection. finally, we demonstrated that sirna experiments targeting essential autophagy genes (atg5, atg7 and beclin 1) reduce viral protein synthesis and viral yield. overall, our results indicate that junv activates host autophagy machinery enhancing its multiplication. autophagy is a highly conserved and tightly regulated process in which cellular components are engulfed into double-membrane vesicles (autophagosomes) and degraded to maintain cellular homeostasis. this process begins with a negative membrane curvature which expands and encloses the cytosolic cargo, forming the autophagosome, that later fuses with late endosomes or lysosomes for degradation [1, 2] . the initiation of autophagosome formation is negatively regulated by the mammalian target of rapamycin complex 1 (mtorc1) kinase. when the mtorc1 complex is inhibited, the ulk1 kinase complex summons autophagy-related (atg) proteins to the site of nucleation of the autophagosome precursor (phagophore) [3] . downstream and small interfering rnas targeting critical components of the autophagic machinery and analysed the effect on junv multiplication. our results suggest that junv exploits the host autophagy to enhance its multiplication. for the detection of junín virus, rabbit polyclonal antibody against junv n protein [30] or viral stocks of the virulent p3441 strain [31] of junv and tcrv were amplified as previously described [31] . briefly, monolayers of bhk21 cells were infected with each strain and clarified supernatants (5,000 × g) were collected 4 to 5 days post-infection (d p.i.). uv inactivation of viral stocks was performed by irradiation at 254 nm using a uv lamp (camag) for 1 h as described [32] . samples of virus stocks uv-inactivated were analysed by plaque assay as described below, to ensure complete inactivation. viral manipulations were conducted in a biosafety cabinet class ii type a2 under biosafety level 3 (bsl3) conditions. all laboratory personnel is vaccinated with the candid#1 vaccine. a549 (atcc no. crm-ccl-185), vero e6 (atcc no. crl-1586) and bhk-21 (atcc no. ccl-10) cells were grown in minimum essential medium (mem, life technologies) containing 10% fetal bovine serum (fbs, life technologies). cells were incubated at 37˚c in a 5% co 2 incubator. all cell cultures were tested for the presence of mycoplasmas using a pcr detection kit (8208) from sciencell research laboratories. for virus infection experiments, a549 cells cultured in 24 or 6-well plates were infected with junv or tcrv at a moi of 3 and the virus inoculum was removed after adsorption for 1 h. the cell monolayers were incubated in complete fresh medium at 37˚c for the indicated times, afterwards supernatants and cells were harvested. optimal concentrations of different drugs (3-methyladenine, bafilomycin a1 and rapamycin) were used to explore the effects of autophagy on the multiplication of junv. briefly, a549 cells were pretreated for 3 h with the indicated drugs prior to junv or mock infection. subsequently, the cells were infected with junv as above and further incubated in fresh media in the absence or presence of these drugs at the same concentrations as for the pretreatments. corresponding dmso or dd h 2 o were used as vehicle controls. at the indicated times, supernatant and cells were collected and analysed for viral titre and protein expression as described below. a549 cells were seeded on glass coverslips in 24-well tissue culture plates (corning glass works, corning). the following day, the cells were transfected at 60% confluence with the plasmid at 3,0 μg/well using the transfection reagent (roche) according to the manufacturer's instructions. the medium was replaced after 6 h with mem containing fbs, and the cells were cultured for another 24 h before infection. western blot analysis was performed as previously described [33] . briefly, the cells were washed with pbs for three times and then scraped off, incubated on ice with cell lysis buffer (150 mm nacl, 50 mm tris-hcl ph 7.4, 1 mm edta, 1% n-40) containing a protease inhibitor cocktail (roche molecular biochemicals) and 0.1 mm pmsf for 2 h. the cell lysates were centrifuged at 14,000 × g for 20 min at 4˚c. the protein concentration was determined using the bradford assay. equal amount of protein samples were diluted in 6 × laemmli sample buffer and separated on sds-page gels. the proteins in the gel were transferred to polyvinylidene fluoride (pvdf) membranes (millipore, iseq00010) which were then blocked with 5% non-fat dry milk in tbst for 2 h and incubated overnight at 4˚c with the primary antibodies. the membrane was then incubated for 2 h with the appropriate secondary antibodies. immunoreactive bands were visualised using the enhanced chemiluminescence system (ecl, perki-nelmer life sciences). the intensities of the western blot bands were analysed using imagej software. ten-fold dilutions (from 10 −1 to 10 −3 ) of the viral culture supernatants were added to 24-well plates with a 40-50% confluence monolayer of vero e6 cells. the plate was then incubated at 37˚c for 1 h with gentle rocking. following adsorption, the inoculum was removed and overlaid with 2 ml of mem containing 0.8% methylcellulose and 2% fbs and further incubated at 37˚c in a humid atmosphere with 5% co 2 . plaques were allowed to develop for either 4-6 days before being fixed (4% w/v paraformaldehyde) and stained with a 1% crystal violet in 20% ethanol and d h2o. for confocal imaging, the cells were grown on glass coverslips and then infected with junv as indicated at a moi of 3. at the indicated h p.i, the cells were washed once in pbs and fixed with 4% pfa for 15 min at room temperature (rt). to stain endogenous lc3, fixed cells were permeabilised in 100% cold methanol for 10 min at −20˚c, washed three times in pbs and incubated in blocking buffer (2% fbs, 1% normal goat serum in pbs) for 1 h at rt as previously described [34] . alternatively, fixed cells were permeabilised with 0.1% saponin-5% normal goat serum in pbs for 15 min. immunostaining of lc3 was performed overnight at 4˚c by incubating cells in primary antibody solution, then washed with pbs and followed by 2 h incubation with the corresponding secondary antibody. to detect colocalisation of lc3 and rab5, rab7a or lamp1 primary antibodies and their respective secondary antibodies were used as indicated. the nuclei were stained with 4 0 -6-diamidino-2-phenylindole (dapi, d9542, sigma aldrich). coverslips were mounted with prolong gold and stored at 4˚c in the dark until observation. the confocal images were collected on a leica tcs sp5 ii microscope. this microscope was equipped with an hcx pl apo cs 63.0x 1.40 oil uv objective, a 543 nm line of an helium/neon laser, a 488 nm line of an argon/ion laser and las af version 2.2.1 4842 software. images were processed using imagej software (nih). for colocalisation analysis, pearson's correlation was calculated using "coloc 2" tool [35] . pearson's score above 0.5 was considered a strong co-localization [36] . a549 cells were grown to 60% confluence in 6-well cell culture plates and transfected with sitran transfection reagent (tt300001, origene) following the manufacturer's recommendations. a final concentration of 5 nm sirna against becn1 (sr305711), atg5 (sr306286), atg7 (sr307159) or rab7a (sr305302) was used (origene). the efficient knockdown of the target protein was evaluated by wb. the results were expressed as the mean ± s.d. of three independent experiments. student's unpaired t-test was used to evaluate the difference between the test samples and controls. a pvalue <0.05 was considered statistically significant. several approaches need to be combined in order to reliably demonstrate the autophagy status of human cell lines. established assays used to monitor autophagy include biochemical detection of the conversion of the cytosolic lc3-i to membrane-bound lipidated lc3-ii by immunoblotting, observation of changes in subcellular distribution of autophagosomal protein markers by immunofluorescence (e.g. lc3 puncta formation), and detection of autophagosomes using fluorescent-tagged probes to monitor autophagy flux [5, 37] . in order to assess autophagosome formation, we examined lc3 status after infection of a549 cells with junv. protein samples from harvested cells at increasing times p.i. were subjected to western blot (wb) analysis using an anti-lc3 antibody that recognises both forms of lc3 (fig 1a and 1b) . simultaneously, rabbit antibodies against n were used to track the progression of infection (fig 1a and 1c ). the expression level of lc3-ii was significantly increased from 12 to 24 h p.i. in junv infected cells compared to the mock, indicating the activation of autophagy during junv infection of a549 cells. in parallel, junv n increased sharply 24 h p.i., which was consistent with the increase of lc3-ii and the progression of infection (fig 1a, 1c and 1e ). in contrast, no significant change in the lc3-ii level was observed in mock-treated cells at 48 h. to establish if junv infection altered the autophagic flux involving the turnover of autophagosomal proteins, we analysed the expression level of p62, a polyubiquitin-binding protein (also known as sequestosome-1 (sqstm1)), which interacts with lc3 [38] . the p62 and p62-bound polyubiquitinated proteins become incorporated into the completed autophagosome and are degraded in autolysosomes [37] , thus serving as an indicator of autophagic degradation. inhibition of autophagy correlates with increased levels of p62 in mammals, suggesting that steady-state levels of this protein reflect the autophagic status [37] . immunoblotting analysis revealed significant progressive degradation of p62 in junv infected cells compared to mock-infected cells (fig 1a and 1d ), suggesting that autophagosomes were able to fuse with lysosomes to degrade the cargos. taken together, these data suggest that the autophagic flux in junv infected cells is activated and remains complete. during autophagy, lc3 can be redistributed from a diffuse cytoplasmic localization to a distinctive punctate cytoplasmic pattern, which reveals the recruitment of lc3 to autophagic vesicles [5, 39] . in order to assess the localization of endogenous lc3, a549 cells were infected with junv and after 24 h p.i. the cells were fixed and subjected to immunoconfocal microscopy analyses. the localization of viral proteins in infected cells was analysed using a specific given that autophagy is increased during junv infection, we then determined whether cellular autophagy regulates junv multiplication. to accomplish this, we pretreated a549 cells with 3-methyladenine (3-ma), an autophagy inhibitor acting on the pi3k pathway [40] . as indicated by wb analysis, treatment of cells with 3-ma ( fig 3a) reduced the expression of n in infected cells when compared to untreated cells. moreover, a decrease in viral progeny in the supernatant medium of the cells treated with 3-ma was confirmed by titration of infectious particles ( fig 3a) . to ensure that 3-ma was inhibiting autophagy, lc3-ii was assayed in parallel samples ( fig 3d) . next, bafilomycin a1 (baf) was used to analyse the turnover of autophagosomes induced by junv infection. baf is an inhibitor of the vacuolar (v)-type atpase, which avoids the fusion between autophagosomes and lysosomes and thereby prevents maturation of autophagosomes [41] . junv or mock infected a549 cells were pre-treated with baf for 3 h. the expression of junv n was determined at 24 h p.i. by wb (fig 3b) . a decrease of n expression in infected cells was observed in response to baf treatment. furthermore, we observed a significant decrease of the viral titre in the supernatant of junv infected cells treated with baf ( fig 3b) . as expected, increased lc3ii levels were detected, confirming the inhibition of autophagic degradation by baf ( fig 3d) . the blockage of endosomal acidification negatively affects the infectivity of viruses that require a low ph for membrane fusion and release of the viral genome. in order to dissect cells were fixed and processed for if 24 h p.i. lc3 was detected using a mab rabbit anti-lc3b from cell signaling technology and a pab cy3-conjugated donkey anti-rabbit igg as secondary ab. junv n was detected using a mab mouse anti-junv n from bei resources followed by incubation with alexa fluor 488-conjugated donkey anti-mouse igg as secondary antibody. (b) images were analysed using image j software. a highermagnification view was included. pearson's coefficient was used for assessing colocalisation. https://doi.org/10.1371/journal.pone.0218730.g002 the stage of viral cycle targeted by each one of the autophagy inhibitors, cells were either treated with the different drugs three hours before the infection (viral adsorption) or three hours after the infection ( fig 4a) . later, they were cultured in mem containing each drug at the same concentration as for the treatment. after 24 h p.i., the viral titre from cell supernatant and the expression levels for p62, n and the lc3-ii/lc3-i ratios were assessed. either with the early autophagy inhibitor (3-ma) or the late inhibitor (baf), the treatment pre or post-infection resulted in reduced expression of n and lower viral titre as compared to junv-infected cells that received no treatment (nt) (fig 4b-4d) . additionally, a combination of ammonium chloride and leupeptin (nl) treatment to inhibit total lysosomal proteolysis also resulted in a decrease of n expression and viral production in infected cells. together, these results show that inhibition of lysosomal degradation reduces infectious viral progeny. importantly, the effect of baf and nl treatments was clearly observed upon addition of drugs 3 h post-infection, when attachment, internalisation and uncoating have been completed (viral yield reduction: baf, 51% (pre-infection) and 34% (post-infection); nl, 64% (pre-infection) and 54% (post-infection)). this result would indicate that inhibition by these compounds is at least partially targeted to events that take place after virus uncoating. to further determine the involvement of autophagy during junv infection, we investigated the effect of autophagosome induction using rapamycin (rap), which induces autophagy by blocking the mtor pathway [42] . under these conditions, viral yields of junv infected cells were higher than in untreated cells (fig 3c) . in parallel, experiments performed adding rap post-infection resulted in a similar increase in viral yields of junv, which indicates that the activation of autophagy benefits both viral entry/uncoating and later steps as assembling and viral budding (fig 4) . the effect of rap in junv infection suggests that autophagy promotes junv yield and increases viral progeny, which is consistent with the results of our autophagy inhibition assays. in order to provide further evidence of junv-induced autophagosome formation, cells were transfected with a plasmid expressing a red fluorescent protein-tagged lc3 (rfp-lc3), and redistribution of lc3 was established by fluorescence microscopy. after junv infection, a redistribution of rfp-lc3 from a diffuse (mock, no treatment) to a punctate pattern was apparent (fig 5a and 5b we also compared the effects of rap, baf and 3-ma on the generation of rfp-lc3 punctate pattern in infected a549 cells. the cells infected with junv presented similar numbers of puncta compared to mock-infected cells treated with rap-commonly used as positive control-but the cells treated with rap and junv-infected showed even higher values of puncta per cell than the corresponding control ( fig 5b) . as expected, the number of puncta in mock-infected cells treated with baf was higher than in the nt control, in agreement with previous reports indicating that the treatment with baf promotes the accumulation of autophagosomes by blocking their fusion with lysosomes [43] . no significant difference was detected in the number of rfp-lc3 puncta per cell in mock versus infected cells pre-treated with baf, although a change in the size and morphology of the induced puncta was observed. however, 3-ma reduces the formation of rfp-lc3 puncta in cells infected with junv when compared to non-treated infected cells (fig 5b) . to analyse the status of the autophagy flux during junv infection, we used a tandem mcherry-gfp-tagged lc3 expression vector. as gfp is sensitive to the acidic conditions in the lysosome, only the fluorescence from mcherry persists in this environment. as a consequence, the colocalisation of mcherry and gfp signals indicates a vesicular compartment that has not fused with an acidic compartment as in the case of the phagophore or autophagosome. on the other hand, the red fluorescence of mcherry without gfp signal corresponds to an autophagosome fused with an endosome or lysosome, called amphisome or autolysosome, respectively [44] . a549 cells were transfected with mcherry-gfp-lc3 plasmid, and 24 h posttransfection were treated with rapamycin, baf or infected with junv. when compared to control cells, an increase in the number of mcherry puncta following rapamycin treatment was observed, indicating promoted autophagy and fusion between autophagosomes and acidic compartments (fig 5c) . in contrast, the blockage of organelle acidification triggered by baf resulted in the accumulation of lc3 puncta containing both, gfp and mcherry signal (fig 5c and 5d ). cells infected with junv displayed an accumulation of mcherry puncta (fig 5c and 5d) , where only a few of them colocalised with gfp puncta. this result indicates that there is an accumulation of acidified autophagosomal structures in junv-infected cells and suggests that the virus induced both the initiation and maturation of autophagosomes but did not block the process at the autophagosome stage. t n e m t a e r t t n e m t a e r t -3 -3 a a a a a a a a a a a a a a a a a a a a a e e e e e e e e e e e e e e e e e e e e e r r r r r r r r r r r r r r r r r r r r r t a a a a a a a a a a a a a a a a a a a a a e e e e e e e e e e e e e e e e e e e e e r r r r r r r r r r r r r r r r r r r r r t the activation of autophagy by junv infection could be caused either by incoming virions or by viral replication products. to determine whether junv replication is required for the induction of autophagy, we challenged rfp-lc3-transfected cells with either live junv or uv-inactivated junv and measured the effect on autophagy by monitoring aggregation of rfp-lc3 using fluorescence microscopy and the conversion of lc3-i to lc3-ii by wb. previously, we used the plaque formation assay to verify that the uv-inactivated virus was replication defective (s5 fig). at 24 h post exposure to junv, there was no difference between the number of rfp-lc3 puncta per cell induced by uv-inactivated junv and replication competent junv (fig 6a and 6b) . moreover, wb analysis revealed similar levels of lc3-ii/lc3-i ratio in a549 cells treated either with uv-inactivated or replication competent junv ( fig 6c) . overall, these results suggest that junv particles play a role, but junv replication is not required for the induction of autophagosomes. we also evaluated the capacity of another nw arenavirus to activate autophagy by performing similar experiments with tacaribe virus (tcrv) as shown in fig 6. the rfp-lc3 punctate pattern and the lc3-ii level induced by tcrv infection closely resembled the results observed with junv infected cells (fig 6a-6c ). upon maturation, autophagosomes may fuse with late endosomes and lysosomes, forming autolysosomes. we examined virus-infected cells for the association between autophagosomes (lc3) and early endosomes (rab5), late endosomes (rab7a) or the lysosomal-associated membrane protein 1 (lamp1). as demonstrated in fig 7a and 7b , the colocalisation of lc3 and early and late endosomal markers was detected in junv-infected cells, while uninfected cells exhibited almost no overlap. moreover, junv-induced vesicles are positive for both lc3 and lamp1, indicating that these vesicles have likely fused with lysosomes ( fig 7c) . no colocalisation of viral n and lamp1 was observed in infected cells (fig 7f) . lc3 also colocalised with atg16l1 and p62 in junv infected cells, which represent isolation membrane (fig 7d and 7e ). these results suggest that junv infection induced the formation of novel autophagosomes without alteration of their maturation. in addition to the studies with pharmacological agents, we used sirna targeting key autophagy genes, beclin 1, atg5, atg7 and rab7a, in order to evaluate the role of these cellular factors on viral yield. as mentioned earlier, beclin 1 is part of class iii pi3k complex involved in the initiation of autophagy [45] . atg5 contributes to lc3-pe conjugation, while atg7 acts as an e1-like enzyme for the ubiquitin-like proteins atg12 and lc3, making both essential regulators of autophagosome assembly [46] . finally, rab7a has a crucial role in the final maturation of autophagosomes into autolysosomes [47, 48] . to deplete these proteins, we treated a549 cells with sirna for 24 h and then challenged the cells with junv. wb analysis of cell lysates revealed that levels of endogenous atg5, atg7, beclin 1 or rab7a protein were significantly reduced in cells treated with the specific sirnas compared to scrambled sirna ( fig 8a and 8c ). in parallel, we observed a decrease in the expression of n in junv infected cells treated with autophagy-specific sirna compared to the control sirna (fig 8a and 8c) . also, depletion of autophagic proteins led to reduced viral titres (fig 8b) . viral load in the supernatant medium of infected cells declined by 49%, 44%, 76% and 90% after silencing beclin 1, atg5, atg7 and rab7a, respectively. collectively, the data demonstrate that inhibition of autophagy affects junv yield. in the present work, we studied the interaction between the cellular autophagy pathway and the nw arenavirus junv. lc3 puncta were detected by immunostaining for endogenous lc3 or following the redistribution of mcherry-gfp-lc3 or rfp-lc3 signal after infection. this, coupled with the observation that lc3-i was converted to lc3-ii, suggests that the lc3 puncta induced by junv were autophagosomes. in addition, our results show that junv infection promoted p62 degradation, indicating that complete autophagic flux was triggered upon junv infection. moreover, increasing the number of autophagosomes prior to viral infection by rapamycin treatment promoted viral yield. finally, we observed colocalisation of junv n, the major nucleocapsid structural protein, and lc3, indicating that viral proteins were associated with autophagic structures. we further showed that the pharmacological modulation of autophagy altered the outcome of junv multiplication. class iii pi3k plays a pleiotropic role in autophagy and protein sorting pathways [49] . the compound 3-ma blocks the formation of autophagosomes by inhibiting the activity of class iii pi3k. our data demonstrate that 3-ma affects junv yield, suggesting that an early step in the formation of the autophagosome such as recruitment of single-membrane secretory pathway-derived vesicles or newly formed double-membrane vesicles could provide a site for rna replication. moreover, 3-ma reduces virus capacity to induce rfp-lc3 puncta suggesting that autophagosomes induced by junv require class iii pi3k activity, and consequently, their formation may be dependent on the beclin 1-vps34 complex. in agreement with our results, linero and scolaro found that junv infection activates pi3k/akt signalling pathway at an early stage of infection [24] . in fact, uv-inactivated junv infected cells redeemed the pattern of akt phosphorylation observed for the replication-competent virus, indicating that pi3k/akt signalling is triggered at an early stage of the viral cycle [24] . similarly, we observed that uv-inactivated virus induces a rfp-lc3 punctate pattern comparable to infectious virus, suggesting that the autophagosome formation induced by junv would be triggered by early events of the virus-cell interaction. other viruses also activate autophagy independently of viral replication as is the case of foot-and-mouth disease virus [50] , vesicular stomatitis virus [51] and human cytomegalovirus [52] . in addition, the non-canonical raf/mek/erk signalling pathway, which modulates autophagy by regulating beclin 1 [53] [54] [55] , could participate in early events during junv-induced autophagy. supporting this idea, the activation of the raf/mek/erk signalling pathway induced by junv infection is also required to ensure efficient junv replication [56, 57] . even though inhibition of erk1/2 signalling pathway does not affect junv adsorption, internalisation and uncoating [57] , we cannot exclude that early events that take place after uncoating but before viral replication, such as sensing of viral cargo molecules, may activate this pathway, and in consequence regulate autophagy. we demonstrated that silencing critical genes of the different stages of autophagy, namely atg5, atg7, beclin 1 or rab7a, reduces viral protein expression and viral progeny. impaired virus multiplication after atg7 knockdown, an essential regulator of autophagosome assembly, has been observed for various rna viruses, including coxsackievirus b3, hcv and rotavirus [58] [59] [60] . in the following steps of autophagosome biogenesis, atg5, atg12, and atg16 form the autophagic elongation complex determining the site of lc3 lipidation [61] . in particular, the contribution of atg5 to viral replication has been demonstrated for several rna viruses [62] [63] [64] . more recently, fahmy and labonté [65] found that the autophagy elongation complex is recruited at the membranous web-an intracellular membrane rearrangement composed of double-membrane vesicles-and promotes hcv replication and assembly. likewise, we found that junv induces the colocalisation of lc3 with p62 and atg16, suggesting the formation of the autophagy elongation complex. moreover, atg5 interacts transiently with the hcv rna polymerase as a proviral factor during the onset of viral infection [64] . another autophagy component evaluated in this study-beclin 1-sits at the core of autophagy regulation [66, 67] . the knockdown of beclin 1 significantly reduced the yield of junv, similarly to other viruses such as hcv [59] and influenza a virus [68] . finally, the small gtpase rab7a is considered a multifunctional regulator of autophagy and endocytosis [48, 69] . rab7a controls the maturation of endosomes and autophagosomes, regulates the trafficking of endosomal multivesicular bodies to lysosomes and participates in the fusion step with lysosomes. it has been shown that individual knockdown of lamp2 and rab7a, which impairs complete autolysosome maturation, inhibits both hcv rna replication and viral protein expression [70] . our results showing that junv infection induces the colocalisation of rab5, rab7a or lamp1 and lc3, indicate that phagophore or autophagosome fusion with early and late endosomes is not being blocked by junv infection. on the other hand, silencing of rab7a had a strong inhibitory effect on viral multiplication, reflecting the role of endosomal trafficking on viral infection outcome. other studies performed with the nw arenavirus pichindé and junv, have shown that viral entry is trafficked through the clathrin-and dynamin-2-mediated endocytosis, by which the virus travels through rab5-mediated early endosomes and rab7a-mediated late endosome [22, 71] . although the silencing of rab7a is expected to affect the uncoating of junv, we can not rule out that late fusion events essential for the production of viral infectious particles are also compromised. further experiments are needed to dissect the effect of rab7a on junv multiplication. based on several studies describing the life cycle of rna viruses, nunberg et al. [72] proposed that arenavirus replication was likely to be compartmentalised to specialized virusinduced organelles referred to as replication-transcription complexes (rtcs). they found that candid#1, the vaccine attenuated strain of junv, and tcrv rna synthesis takes place in discrete cytosolic puncta, the formation of which is induced in part by n and suggests that rtcs also include a membrane component. although we did not detect rtcs or rna directly, the fact that we observed colocalisation of n and lc3 in a similar cytoplasmic punctate pattern suggests that autophagosome membranes could be involved in rtc assembly. however, these authors did not find colocalisation of rtcs induced by tcrv and autophagosomes [72] . this alleged discrepancy could probably be related to immunostaining methods or even the use of different cell line and virus. further studies will be needed in order to establish the relationship between the lc3-n puncta and rtcs induced by junv infection. it is also likely that the autophagosome provides a physical scaffold where junv may be assembled or viral rna synthesised. recruitment of components of the autophagic machinery to assemble viral rtc could be involved in both mechanisms. for example, rotavirus initiates autophagy and hijacks this membrane trafficking pathway to transport viral proteins to viroplasms [73] . a recent study shows that lcmv rna aggregates colocalise with the early endosomal marker rab5c and the viral glycoprotein in infected cells, and propose that lcmv uses the surface of a cellular membrane-bound organelle as a site for the pre-assembly of viral components [74] . for some viruses, the events related to vesicular acidification are used to assist with their multiplication. in particular, junv requires a low ph in the entry vesicle for the success of the glycoproteins-mediated membrane fusion [23, 75] . that is consistent with entry by clathrinmediated endocytosis, as clathrin-coated vesicles deliver their cargo into endosomes with an acidic content [21] . in agreement with previous studies [76] , the pre-treatment with lysosomotropic agents such as baf and nl severely affects viral yield in a549 cells. nevertheless, when these agents were added after the early steps of the viral cycle (binding, receptor-mediated endocytosis, uncoating and genome release), the viral yield was still affected. these results suggest that vesicle acidification also plays a role in the late events of the viral cycle. in the experiment performed by castilla et al. [76] , vero and bhk cells were used as a model to assess the effect of baf and concanamycin a (ca), another specific inhibitor of vacuolar type-atpase, in the production of junv infectious particles. even when ca was added 5 h p.i., both, extracellular virus titre and infectious cell-associated virus quantified 24 h p.i., were significantly reduced. castilla et al., suggest that ca could also interfere with the egress of cell-associated infectious virus to the extracellular medium besides its early effect on viral endocytosis. these results are in agreement with our experimental findings using baf (pre vs post-infection experiments, fig 4) . for some viruses like poliovirus, vesicle acidification promotes maturation of the assembled, encapsidated virus particles into infectious virions [77] . in the case of junv, we can speculate that late membrane fusion events related to the viral assembly and budding, as could be the fusion of autophagosomes and mvbs or the plasma membrane, are also affected by post-infection treatment with baf. in summary, our findings suggest that junv activates autophagy in a549 human cells favouring viral multiplication. to our knowledge, this is the first report showing that the infection of an arenavirus induces autophagy. our studies provide novel insights into arenavirushost interactions and open a new window to examine the pathogenesis of this viral family. this knowledge could contribute to the development of antiviral strategies against arenavirus infection. autophagosome formation: core machinery and adaptations the machinery of macroautophagy the ulk1 complex molecular definitions of autophagy and related processes lc3, a mammalian homologue of yeast apg8p, is localized in autophagosome membranes after processing lysosomal turnover, but not a cellular level, of endogenous lc3 is a marker for viral evasion of autophagy autophagy fights disease through cellular self-digestion autophagy and selective deployment of atg proteins in 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participation of the phosphatidylinositol 3-kinase/akt pathway in junín virus replication in vitro involvement of cytoskeleton in junín virus entry correlation between endogenous interferon and the clinical evolution of patients with argentine hemorrhagic fever regulation mechanisms and signaling pathways of autophagy production of interferon α by human immunodeficiency virus type 1 in human plasmacytoid dendritic cells is dependent on induction of autophagy autophagy-dependent viral recognition by plasmacytoid dendritic cells. science (80-) arenavirus nucleocapsid protein displays a transcriptional antitermination activity in vivo endothelial cell function alteration after junin virus infection tacaribe virus but not junin virus infection induces cytokine release from primary human monocytes and porcine reproductive and respiratory syndrome virus induces autophagy to promote virus replication measuring autophagy in stressed cells fiji: an open-source platform for biological-image analysis bridging the gap between qualitative and quantitative colocalization results in fluorescence microscopy studies guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes p62/sqstm1 binds directly to atg8/lc3 to facilitate degradation of ubiquitinated protein aggregates by autophagy guidelines for the use and interpretation of assays for monitoring autophagy the phosphatidylinositol 3-kinase inhibitors wortmannin and ly294002 inhibit autophagy in isolated rat hepatocytes a comprehensive glossary of autophagy-related molecules and processes mtor interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery infection, and the immune response guidelines for the use and interpretation of assays for monitoring autophagy beclin1: a role in membrane dynamics and beyond two ubiquitin-like conjugation systems that mediate membrane formation during autophagy multiple roles of the small gtpase rab7 role for rab7 in maturation of late autophagic vacuoles the autophagosome: origins unknown, biogenesis complex foot-and-mouth disease virus induces autophagosomes during cell entry via a class iii phosphatidylinositol 3-kinase-independent pathway autophagy is an essential component of drosophila immunity against vesicular stomatitis virus early induction of autophagy in human fibroblasts after infection with human cytomegalovirus or herpes simplex virus 1 regulation of autophagy by kinases raf/mek/erk can regulate cellular levels of lc3b and sqstm1/p62 at expression levels a non-canonical mek/erk signaling pathway regulates autophagy via regulating beclin 1 raf/mek/erk pathway activation is required for junin virus replication role of the erk1/2 signaling pathway in the replication of junín and tacaribe viruses autophagosome supports coxsackievirus b3 replication in host cells knockdown of autophagy-related gene decreases the production of infectious hepatitis c virus particles rotavirus increases levels of lipidated lc3 supporting accumulation of infectious progeny virus without inducing autophagosome formation the atg16l complex specifies the site of lc3 lipidation for membrane biogenesis in autophagy autophagic machinery activated by dengue virus enhances virus replication coronavirus replication complex formation utilizes components of cellular autophagy autophagy protein atg5 interacts transiently with the hepatitis c virus rna polymerase (ns5b) early during infection the autophagy elongation complex (atg5-12/16l1) positively regulates hcv replication and is required for wild-type membranous web formation physiological functions of atg6/beclin 1: a unique autophagy-related protein beclin-1 targeting for viral immune escape autophagy is involved in influenza a virus replication maturation of autophagosomes and endosomes: a key role for rab7 activation of the unfolded protein response and autophagy after hepatitis c virus infection suppresses innate antiviral immunity in vitro pichindé virus is trafficked through a dynamin 2 endocytic pathway that is dependent on cellular rab5-and rab7-mediated endosomes arenavirus infection induces discrete cytosolic structures for rna replication autophagy hijacked through viroporin-activated calcium/ calmodulin-dependent kinase kinase-β signaling is required for rotavirus replication visualization of the lymphocytic choriomeningitis mammarenavirus (lcmv) genome reveals the early endosome as a possible site for genome replication and viral particle pre-assembly intersubunit interactions modulate ph-induced activation of membrane fusion by the junin virus envelope glycoprotein gpc involvement of vacuolar proton atpase in junin virus multiplication intracellular vesicle acidification promotes maturation of infectious poliovirus particles we thank dr maria isabel colombo for providing us with the plasmid rfp-lc3. the following reagents were obtained through bei resources, niaid, nih: monoclonal anti-junin virus, clones sa02-bg12 (nr-49274) and na05-ag12 (nr-48834), both produced in vitro. key: cord-000695-g5sum116 authors: hou, yanxia; guo, yingying; wu, chunyan; shen, nan; jiang, yongping; wang, jingfei title: prediction and identification of t cell epitopes in the h5n1 influenza virus nucleoprotein in chicken date: 2012-06-20 journal: plos one doi: 10.1371/journal.pone.0039344 sha: doc_id: 695 cord_uid: g5sum116 t cell epitopes can be used for the accurate monitoring of avian influenza virus (aiv) immune responses and the rational design of vaccines. no t cell epitopes have been previously identified in the h5n1 aiv virus nucleoprotein (np) in chickens. for the first time, this study used homology modelling techniques to construct three-dimensional structures of the peptide-binding domains of chicken mhc class ι molecules for four commonly encountered unique haplotypes, i.e., b4, b12, b15, and b19. h5n1 aiv np was computationally parsed into octapeptides or nonapeptides according to the peptide-binding motifs of mhc class i molecules of the b4, b12, b15 and b19 haplotypes. seventy-five peptide sequences were modelled and their mhc class i molecule-binding abilities were analysed by molecular docking. twenty-five peptides (ten for b4, six for b12, two for b15, and seven for b19) were predicted to be potential t cell epitopes in chicken. nine of these peptides and one unrelated peptide were manually synthesized and their t cell responses were tested in vitro. spleen lymphocytes were collected from spf chickens that had been immunised with a np-expression plasmid, pcaggs-np, and they were stimulated using the synthesized peptides. the secretion of chicken ifn-γ and the proliferation of cd8(+) t cells were tested using an elisa kit and flow cytometry, respectively. the significant secretion of chicken ifn-γ and proliferation of cd8(+) t lymphocytes increased by 13.7% and 11.9% were monitored in cells stimulated with peptides np(89–97) and np(198–206), respectively. the results indicate that peptides np(89–97) (pkktggpiy) and np(198–206) (krgindrnf) are np t cell epitopes in chicken of certain haplotypes. the method used in this investigation is applicable to predicting t cell epitopes for other antigens in chicken, while this study also extends our understanding of the mechanisms of the immune response to aiv in chicken. the introduction into the human population of animal-derived influenza a viruses with a novel haemagglutinin (ha), or a novel ha and neuraminidase (na), and their subsequent spread could result in global influenza pandemics [1] . since 2003, the highly pathogenic h5n1 avian influenza virus (aiv) has caused numerous cases of severe disease and death in humans [2] . an influenza pandemic could ensue if this virus developed the capacity to spread easily among humans [3] [4] [5] . migratory birds constitute the natural reservoir for aivs, but chickens may play a key role in the transmission to humans [6] . epitopes can be used for accurately monitoring immune responses to aiv and for the rational design of protective vaccines. however, only two epitopes from the h5n1 avian influenza a/vietnam/1194/2004 virus are included in the immune epitope database and analysis resources (iedb). the majority of t cell and b cell epitopes have been identified in mouse, human, or rabbit hosts. few epitopes have been described in chicken [7] . the structural basis of peptide binding to mammalian major histocompatibility complex (mhc) class i molecules is well understood [8] . the peptide-binding groove is formed by the a1 and a2 domains. each domain contributes four strands to an eight -stranded anti-parallel b-sheet. two long interrupted helices, one from each domain, pack against the side of this sheet in an orientation directed away from the cell membrane. there is a series of pockets (a-f) along the peptide-binding groove where highly polymorphic amino acids mediate recognition via haplotype-specific associations with antigens and t cell receptors. however, highly conserved residues are found at both ends of the peptide-binding groove that form a network of hydrogen bonds, which directly interact with hydrogen bonds at the peptide's nterminus and c-terminus [9] [10] [11] . peptides that bind to mhc class i molecules are usually octamers or nonamers, where only one or a few residues can interact with polymorphic residues in the groove. these residues are known as anchor residues when they are found in an anchoring position [12] [13] [14] [15] . several public databases and prediction services are available for mhc molecular ligands and peptide motifs, including syfpeithi and rankpep [16, 17] . unlike mammalian studies, the majority of investigations of mhc class i molecules in chicken remain limited to primary sequences and the structure of mhc class i molecule from the b21 haplotype was solved only recently [18, 19] . the chicken mhc b system is located on chicken chromosome 16 (chr16) and is composed of tightly linked polymorphic regions: bf (class i) and bl (class iib) and a large family of polymorphic ig-superfamily (igsf) genes called bg-the latter sharing sequence similarities with mammalian mhc butyrophilin, myelin oligodendrocyte glycoprotein, and trim genes [20, 21] . the mhc b-f molecules are structurally and functionally similar to mammalian mhc class i molecules. the two class i genes are known as bf1 and bf2, although the bf2 gene is mainly expressed. the mhc class i (b-f) molecules present antigen peptides to the cd8 + t lymphocytes, which have a central role in the immune system. the mhc class i molecules of different haplotypes have specific peptide-binding tropisms, and b-f-associated peptide-binding motifs have been determined for several haplotypes, including b4, b12, b15, and b19 [22] . thirteen peptides derived from the v-src gene of the rous sarcoma virus (rsv) prague strain were predicted to fit the peptide-binding motif of the mhc class i molecules of b12 haplotype and all 13 synthetic peptides actually bound to the bf12 class i molecule in subsequent binding tests [23] . this demonstrates that peptide-binding motifs can be used to predict the antigen peptides presented by chicken mhc class i molecules. however, no t cell epitope of h5n1 aiv np has yet been identified in the chicken [7] , while no information is available on the structure of chicken mhc class i molecules belonging to the b4, b12, b15, and b19 haplotypes. for the first time, the current study reports the homology modelling structures of the peptidebinding domains of chicken mhc class i molecules for the b4, b12, b15, and b19 haplotypes. potential t cell epitopes were predicted by molecular docking of 25 peptides in the h5n1 aiv np in chicken. np 89-97 and np 198-206 were shown to be t cell epitopes of h5 aiv np by analysing the cd8 + t cell proliferation and the interferon (ifn-c) expression. to our knowledge, this is the first report to describe the structure of chicken mhc class i molecules from the b4, b12, b15 and b19 haplotypes and the t cell epitopes of h5n1 aiv np in chicken. all computations were conducted using the discovery studio 2.5 (ds2.5) program developed by accelrys software inc and the sybyl 8.1 program developed by tripos inc on an sgi fuel workstation running red hat enterprise 5.3 and a dell server running the red hat enterprise 5.2 linux operating system. spf chickens were housed in hepa-filtered isolators. chicken lymphocytes collected from immunized chickens, which were approved by harbin veterinary research institute, chinese academy of agricultural sciences and performed in accordance with animal ethics guidelines and approved protocols. the animal ethics committee approval number is heilongjiang-syxk-2006-032. dna vaccine pcaggs-np was constructed and assessed according to ma and jiang [24, 25] . the synthetic gene for np of the isolate a/goose/gongdong/1/96 (h5n1) with codons optimized for chicken usage was synthesized by pcr assembly of long single-strand dna templates (100 bases in length) (the oligonucleotide sequences are available upon request). the synthetic optinp was cloned into the plasmid vector pcaggs under the control of the chicken b-actin promoter. the plasmid was named pcagg-np. the expression of the np protein from the plasmid was confirmed by indirect immunofluorescence assay and western blotting of plasmid transfected cef cells. nine of the predicted peptides (table 1) were selected to test their t cell responses in vitro. they were synthesized to .90% purity by the genscript corporation (nanjing, china): np [7] [8] [9] [10] [11] [12] [13] [14] (krsyeqme), amino sequence of h5n1 aiv(gd/1/96) np was parsed into octamers or nonamers according to the peptide-binding motifs of mhc class i molecules belonging to the b4, b12, b15, and b19 haplotypes. the 3d structures of those peptides and the mhc class i molecules were predicted using homology modelling method and then the binding affinity between each peptide and mhc class i molecule was analysed using molecular docking. those peptides, which have correct binding conformation and high binding affinity, were predicted to be potential t cell epitopes in chicken. predicted peptides 9 of 25 were synthesized and used to stimulate splenic lymphocytes collected from np dna vaccineimmunized spf chickens. mhc class i molecule-restricted potential t-cell epitopes were confirmed by detecting of cd8 + t cell proliferation and interferon (ifn-c) expression. the amino acid sequences of chicken mhc class i molecules belonging to the b4, b12, b15, and b19 haplotypes were obtained from genbank (b4, genbankid: cak54654.1; b12, genbankid: bag69386.1; b15, genbankid: bag69413.1; and b19, gen-bankid: bag69441.1). blastp was performed to search for homologous proteins in the protein data bank (pdb) using the blosum62 scoring matrix optimized with a gap penalty of 11 and a gap extension penalty of 1. the modeler [27] program was then executed in ds2.5 to construct the 3d structures of chicken mhc class i molecules for the four haplotypes. the quality of the 3d models was evaluated with a ramachandran plot using the procheck [28] program and the verify protein (profiles-3d) program [29] in ds2.5. to improve the quality of the models, unsatisfied loop regions in each model were refined using the loop-refinement protocol based on the modeler energy in ds 2.5. all structures were then minimized using the charmm force field [30] and an explicit solvent model (tip3p water) with a steepest descent method for 8000 steps and a conjugated gradient minimization for a further 8000 steps. the cavity depth, lipophilic potential (lp), flexibility (fx), and electrostatic potential (ep) of the four structures was analysed using the molcad program in sybyl8.1. during this process, the gasteiger-hückel charges were assigned to all atoms, while surface maps were generated and visualized using sybyl8.1. the np protein sequence of h5n1 isolate a/goose/gongdong/1/96 (h5n1) was downloaded from the uniprot database (uniprotid: ncap_i96a0) and automatically parsed as octapep-tides or nonapeptides using a computer program, which was developed in our laboratory, based on the peptide-binding motifs of chicken mhc class i molecules belonging to the b4, b12, b15, and b19 haplotypes [22] . the motifs were as follows: the structures of the octapeptides and nonapeptides were modelled using ds2.5. in total, seventy-five peptide structures were prepared for docking analysis. surflex-dock [31] is used to dock ligands into a protein-binding site and it is particularly successful at eliminating false positive results [32] while offering unparalleled enrichment in virtual highthroughput screening combined with state-of-the-art speed, accuracy, and usability [33, 34] . to test the feasibility of using surflex-dock for docking mhc molecules and peptides, 50 peptide-mhc complexes retrieved from the pdb database were separated and re-docked using the surflex-dock program. the rmsd was calculated for each modelled pair and crystal structure pair to evaluate the docking program. surflex-dock was used to dock the octapeptides and nonapeptides to their corresponding mhc class i molecule receptors, where the parameters of the threshold and bloat values of the program were optimized to 0.51 and 1. the interaction modes and binding energies of the docked complexes were analysed using ds2.5. peptides with a higher docking score and rational conformation were predicted as candidate t cell epitopes for each haplotype. for the dna vaccine immunizations, 13 three-week-old spf chickens were separated into two groups, eight were immunized twice with 100 mg of pcaggs-np in their leg muscle at threeweek intervals, while five chickens were injected with the same volume of pbs as controls. sera were collected weekly for detecting of np antibody. serum antibodies to h5n1 aiv np were detected using an indirect elisa method as described by [24] , which used prokaryotically-expressed np as the antigen. the testing steps as follows, after washing of the plates, 50 ml of the test serum mixed in the test wells with 50 ml of antigen diluted 1:10 in elisa buffer. after incubation at 37uc for 1 h, 100 ml horseradish peroxidase conjugate, diluted 1:1000 in elisa buffer, was added to each well and plates were further incubated at 37uc for 1 h. after two washing steps, 100 ml of tmb substrate was added and incubated at room temperature for 10 min. the reaction was stopped by adding 100 ml h 2 so 4 2 m. the extinctions were measured at 490 nm with a micro elisa reader (bio-rad). to prepare splenic lymphocytes, all eight pcaggs-np immunized chickens were killed by cardiac puncture blood collection. sterile spleens were collected and meshed through a sieve screen using a syringe plunger to obtain a single-cell suspension in tissue culture medium (rpmi 1640, gibco brl ny, usa). cell suspensions were overlaid onto histopaque 1077 density gradient medium and centrifuged at 1800 rpm for 20 min at 18uc. lymphocytes were collected from the interface and washed three times in rpmi, before cells were counted using a trypan blue dye exclusion assay. splenic lymphocytes collected from the 8 immunized chickens were stained with 5 mm cfse (invitrogen) in pre-warmed pbs for 10 min, washed three times, and suspended in rpmi 1640 containing 2 mm l-glutamine, 100 iu ml -1 penicillin, 100 iu ml -1 streptomycin, and 10% foetal bovine serum (r10 medium). cells were plated at 10 6 well -1 in 24-well plates with rpmi 1640 medium, before stimulation for 5d with 100 mg ml -1 of the nine peptides generated from np and the unrelated peptide n 71-78 . cells were then washed with pbs and stained using anti-cd8-pe before flow cytometric analysis, which was performed on cytomics fc500 mcl(beckman) and analysed with its embedded software cxp. two repeats were performed simultaneously for each peptide during the flow cytometric analysis. splenic lymphocytes collected form the 8 vaccinated chickens were plated at 10 6 well -1 in 24-well plates with rpmi 1640 medium, before stimulation for 48 h using 100 mg ml -1 of the nine peptides generated from np and the unrelated peptide n 71-78 . cells were then collected and centrifuged at 1000 rpm for 5 min. the cell culture supernatants were then analysed to determine the chicken ifn-c using chicken ifn-c cytosets tm (invitrogen), according to the manufacturer's instructions [35] . the extinctions were measured at 450 nm with a micro elisa reader (bio-rad). for each peptide, two copies of splenic lymphocytes from one chicken were treated and analysed simultaneously. results were expressed as mean 6 s.e.m. for two replicates. statistical analyses were performed using spss 16.0 for windows. significant differences (p,0.05) between means were tested by one-way anova, followed by tukey's honestly significant difference test. the four haplotypes selected in this study belonged to 11 commonly encountered unique haplotypes (b2, b4, b5, b6, b7, b12, b13, b14, b15, b19, and b21) and the motifs of their binding peptides were previously reported [22, 23] . the peptide-binding groove of the chicken mhc class i molecule contains a1 and a2 domains [18] , so only those two domains were used for modelling and analysis in this study. blastp search results showed that the chicken mhc class i molecule 3bev (pdb accession number) belonging to the b21 haplotype had the highest sequences identity and similarity with the four target sequences. the sequence identity and similarity between 3bev and b4, b12, b15, and b19 were 84.3%, 84.3%, 87.1%, and 89.9%, and 89.3%, 89.9%, 91%, and 93.3%, respectively. the sequence alignments of the four target sequences and 3bev are shown in fig. 1. 3bev has been crystallized to a high resolution of 2.1 å , so it was selected as the template for modelling the four protein structures. the initial crude homology models were built using mod-eller and refined using modules for loop refinement and charmm minimization in ds2.5. thus, the final structures were of high quality. the profiles-3d scores of all amino acid residues in the four structures were greater than zero (fig. 2) , while an evaluation of the stereochemical quality of the models using procheck showed that no residues were in the disallowed regions of the ramachandran plots (fig. 3) . this indicated that the backbone dihedral angles, phi and psi, of the four structure models were reasonably accurate. the four models are similar to the previously reported b21 haplotype. the peptide-binding domain is formed by two helices at the top and an eight-stranded sheet at the bottom (fig. 4a) . the pairwise root mean-square differences (rmsd) between the ca positions of 3bev and the structures of the b4, b12, b15, and b19 haplotypes are very small (0.23, 0.20, 0.24, and 0.30 respectively). however, there are some variations around the peptide-binding grooves and these differences may determine differences in their peptide-binding properties. specific residues in the binding groove have an essential role in peptide binding. figure 4b shows the key residues comprising the binding site, when all the models and the template were superimposed. peptide binding to a given class i mhc molecule requires the presence of anchor residues that complement the physicochemical characteristics of the specificity pockets [36] . anchor residues are usually found at peptide position 2 (p2), where they interact with pocket b, but sometimes at position 5 or 6 (p5 or p6), where they interact with pocket c or e. a c-terminal residue (pc) also typically binds in pocket f [8] . the anchor residues glu or asp (p2) in b4 have an electrostatically favourable interaction with the side-chain of the pocket b residue aspa9, which points up from the b-sheet. similarly, anchor residues p2 arg in b15 and b19 provide an electrostatically favourable interaction with the negatively-charged residues aspa34 and glua62, the side-chains of which point towards the binding groove from the a-helix. the presence of glu8 or asp8 as major residues in the c-terminal position of the b4 motif is unprecedented, and there are no previous mammalian examples of negatively-charged residues in the c-terminal anchor position [16] . it is possible that the positively-charged arga80 residue of the f pocket is the key to the binding of anchor residues p8glu or p8asp. there is an anchor residue in the central cavity region corresponding to pocket c in b12 and b4. this is very similar to the mammalian mhc class i molecules h-2 kb and h-2 db, and it may be related to the glya68(69) residue without a side-chain located in the a-helix (the residue number in parentheses is derived from mammals, whereas that without parentheses is from the chicken). the volumes of the binding grooves in b4 and b12 are 662.45 å 3 and 637.677 å 3 , respectively, which are greater than those of b15 (582.595 å 3 ) and b19 (581.676 å 3 ) (fig. 5) . this may explain why the former have anchor residues in the middle region of their binding peptides. in general, anchor residues point downwards into the binding groove, which allows them to fill the pocket and maintain the stability of the peptide-mhc complex. the electrostatic potential of the peptide-binding groove of the b4 haplotype is highly positively-charged, whereas those of the b15 and b19 haplotypes are negatively-charged and that of b12 is neutral (fig. 6) . the peptide-binding grooves are highly lipophilic in all models (fig. 7) . the b pockets of the peptide-binding grooves . verification score plots of the models (plots generated using ds2.5). the verification scores of all residues in b15 were greater than zero, whereas one residue in b12 and b19 and three residues in b4 were less than zero. doi:10.1371/journal.pone.0039344.g002 are more flexible than the f pockets in all structures (fig. 8) , which suggests that the variation of the anchor residues in the b pocket is greater than in the f pocket. as described in the materials and methods, 50 peptide-mhc complexes were selected from the pdb database to validate the docking process. all the peptides were extracted from the complexes, before they were re-docked to the corresponding mhc molecules using surflex-dock. when compared with the corresponding initial complexes, the average rmsd was 1.8 and the docking scores were all greater than 8.0 (table 2) . docking was also successfully performed with v-src c-tail peptide 517-524 (lpacvlev) and an identified t cell epitope [23] to the modelled b12 mhc class i molecule, where the docking score was 10.91 and the docking complex conformation was rational. thus, it was appropriate to use surflex-dock for screening t cell epitopes based on their homology-modelled structures. based on the assessment results, the criteria for t cell epitope prediction using surflex-dock were defined as follows: a) the peptide made close contact with the groove and it docked in the correct direction; b) the anchor residues bound to the anchor site in a rational conformation; and c) the docking score of the complex was greater than 8.0. a total of 25 potential t cell epitope peptides were predicted (eight for b4, six for b12, two for b15, and seven for b19). nine peptides, which marked in bold in table 1, were selected to test their t cell responses in vitro. the binding energies of those complexes were also calculated. the results are shown in table 1 . peptide stimulation experiments were conducted using splenic lymphocytes derived from np dna vaccine-immunized chickens to verify some of the predicted potential t cell epitopes. to assess the immune effects of the vaccine, serum np antibody was detected using the elisa method. compared to the control group, a significant increase (p,0.01) in blood np antibody was observed in the immunized group two weeks after the first vaccination. after the boost, the blood np antibody level of the immunized group increased further and it remained at a high level throughout the duration of the experiment (fig. 9 ). this indicated that the immune systems of the chickens were activated by the vaccine immunization and that the splenic lymphocytes of chickens were sensitized. activation of lymphocytes using peptides np to verify the predicted t cell epitopes in np, ten synthetic peptides (top nine of the 25 predicted peptides from np and one unrelated peptide) were incubated with sensitized splenic lymphocytes for 5 d. flow cytometry analysis showed that the proliferation of cd8 + t lymphocytes increased by 13.7% and 11.9% in cells stimulated with the peptides np 89-97 and np 198-206, respectively (fig. 10) . chicken ifn-c concentration in cells stimulated using peptides np 89-97 and np 198-206 were significantly higher than the control and unrelated peptide-stimulated cells (fig. 11) . these results demonstrate that the peptides np 89-97 and np 198-206 are np t cell epitopes in chickens of certain haplotypes. the objective of this study was to predict and verify t cell epitopes in the h5n1 aiv np in chicken. using a motif combined with a structure-based method, 25 potential t cell epitope peptides were predicted in the h5n1 aiv np in chickens of b4, b12, b15, and b19 haplotypes. np 89-97 and np 198-206 were found to induce a significant proliferation of cd8 + t lymphocytes and they increased the secretion of chicken ifn-c in sensitized splenic lymphocytes. these data suggest that peptides np 89-97 and np 198-206 are np t cell epitopes in chickens of certain haplotypes. this study is important for the following two reasons. first, this is the first study to determine the structural characteristics of the peptide-binding domains of chicken mhc class i molecules belonging to the b4, b12, b15, and b19 haplotypes using a combined motif-structure method to predict t cell epitopes in chickens. second, np 89-97 and np 198-206 are the first two t cell epitopes to be identified in aiv np in chickens of certain haplotypes. homology modelling is widely used in many areas of structure-based analysis and study [37, 38] . however, there are few studies of chicken mhc class i molecules compared with those of human or mouse. the only chicken mhc class i molecule structure that has been solved is the b21 haplotype [18] . thus, there is little information available on the structure and function of chicken mhc class i molecules. therefore, the current study used homology modelling to investigate the peptide-binding domains of chicken mhc class i molecules belonging to the b4, b12, b15 and b19 haplotypes. to the best of our knowledge, this is the first attempt to understand the peptide-binding properties of these molecules based on their structures. the assessment indicated that the models were of high quality in terms of their folding and they were suitable for structure-based t cell epitope prediction. the structural characteristics of the peptide-binding properties of these mhc class i molecules were described in the results section. only four haplotypes with known motifs were selected in this study. however, it is possible to use this solution to predict t cell epitopes of mhc class i molecules belonging to different haplotypes for other antigens in chickens, because the only difference would be the increased number of peptides required for molecular docking. this study found that 25 out of 75 peptides were potential t cell epitopes in the h5 aiv np in chickens of the four haplotypes. an analysis of previous results showed that some of those peptides have been identified as t cell epitopes in humans. there is evidence that np 91-99 (ktggpiykr), np 361-375 (rgvqias-nenmetme), and np 174-184 (rrsgaagaavk), which are derived from the h3n2, h3n2, and h1n1, respectively, are t cell epitopes, with mhc restriction alleles of hla-a68, h-2db, and hla-b27, respectively [39] . of these peptides, np 89-97 (pkktggpiy) and np 362-369 (gvqiasne) are completely conserved in all influenza virus strains, while np 179-186 (agaavkgv) is relatively conserved. some potential epitopes were also shared by several haplotypes. chickens used for meat and egg production are always heterozygotes, so the identification of these shared t cell epitopes will facilitate the development of broad-spectrum protective vaccines for chickens. furthermore, epitopes shared by birds and humans are of great importance in the design of rational vaccines for protecting humans and birds from aiv infection. this study used a dna vaccine plasmid expressing the np from a/goose/gong dong/1/96 (h5n1). a dna vaccine expressing ha from a/goose/gong dong/1/96 (h5n1) has been found to elicit antibody responses and protect chickens against challenge with hpai virus [25] . methods used for producing the ha-expression dna vaccine were adopted when preparing the dna expression plasmid pcagg-np. np antibody responses were detected in immunized chickens, suggesting that this vaccine elicits successful immune responses to the np antigen in chickens. the t cell responses were not detected directly in this study, but it was inferred that a successful antibody response would have been accompanied by a successful t cell response based on our knowledge of the dna vaccine. nine of the peptides were synthesized and used to stimulate sensitized splenic lymphocytes to verify that they were epitopes. increases in the proliferation of cd8 + t lymphocytes and the secretion of chicken ifn-c demonstrated the antigenicity of these peptides. np 89-97 (pkktggpiy) and np 198-206 (krgindrnf) induced significant t cell responses in splenic lymphocytes. an analysis of the prediction results showed that np 89-97 was predicted to be a t cell epitope for both b15 and b19, while np 198-206 also belonged to the b19 haplotype. thus, it is suggested that the major haplotype of the experimental spf chickens might be b19. np 89-97 also overlapped with a previously identified hla-a68 restriction t cell epitope np 91-99 (ktggpiykr) of the h3n2 aiv [7] . this further verifies the significance of this epitope, which could be used in human and chicken vaccines to provide protection against different influenza virus subtypes. using in silico and in vitro approaches, this study identified two novel t cell epitopes np 89-97 (pkktggpiy) and np [198] [199] [200] [201] [202] [203] [204] [205] [206] (krgindrnf) in the h5n1 aiv np in chickens of certain haplotypes. the method used in this investigation is applicable to predicting t cell epitopes for other antigens in chicken, while this study also extends our understanding of the mechanisms of the immune response to aiv in chickens. characterization of an avian influenza a (h5n1) virus isolated from a child with a fatal respiratory illness avian influenza. h5n1 moves into africa, european union, deepening global crisis emerging and re-emerging infectious diseases: influenza as a prototype of the host-pathogen balancing act pandemic influenza threat and preparedness highly pathogenic avian influenza (h5n1): pathways of exposure at the animalhuman interface, a systematic review ab and t cell epitopes of influenza a virus, knowledge and opportunities the three-dimensional structure of peptide-mhc complexes complex assembly, crystallization and preliminary x-ray crystallographic studies of the swine major histocompatibility complex molecule sla-1*1502 complex assembly, crystallization and preliminary x-ray crystallographic studies of duck mhc class i molecule structural diversity of class i mhc-like molecules and its implications in binding specificities comment on ''characterizing the n-terminal processing motif of mhc class i ligands consensus motifs and peptide ligands of mhc class i molecules chemistry of peptides associated with mhc class i and class ii molecules characterizing the n-terminal processing motif of mhc class i ligands syfpeithi: database for mhc ligands and peptide motifs enhancement to the rankpep resource for the prediction of peptide binding to mhc molecules using profiles structures of an mhc class i molecule from b21 chickens illustrate promiscuous peptide binding complex of a b21 chicken mhc class i molecule and a 11 mer chichen peptide the chicken b locus is a minimal essential major histocompatibility complex nomenclature for the chicken major histocompatibility (b and y) complex peptide motifs of the single dominantly expressed class i molecule explain the striking mhcdetermined response to rous sarcoma virus in chickens v-src oncogenespecific carboxy-terminal peptide is immunoprotective against rous sarcoma growth in chickens with mhc class i allele b-f12 construction of a recombinant vector expressing avian influenza virus nucleoprotein with an avian-derived promoter and detection of its immunogenicity enhanced protective efficacy of h5 subtype avian influenza dna vaccine with codon optimized ha gene in a pcaggs plasmid vector localization of a t-cell epitope within the nucleocapsid protein of avian coronavirus modeller: generation and refinement of homology-based protein structure models procheck: a program to check the stereochemical quality of protein structures assessment of protein models with three-dimensional profiles charmm: a program for macromolecular energy, minimization, and dynamics calculations surflex-dock 2.1: robust performance from ligand energetic modeling, ring flexibility, and knowledge-based search surflex: fully automatic flexible molecular docking using a molecular similarity-based search engine comparative evaluation of eight docking tools for docking and virtual screening accuracy fast structure-based virtual ligand screening combining fred, dock, and surflex effects of reticuloendotheliosis virus and marek's disease virus infection and co-infection on ifn-gamma production in spf chickens refined structure of the human histocompatibility antigen hla-a2 at 2.6 ?resolution homology modeling in drug discovery: current trends and applications a homology modeling of ferredoxin-nitrite reductase from arabidopsis thaliana epitope-specific tcrbeta repertoire diversity imparts no functional advantage on the cd8+ t cell response to cognate viral peptides key: cord-002305-qq73gr9y authors: anson, marie; amado, inês; mailhé, marie-pierre; donnadieu, emmanuel; garcia, sylvie; huetz, françois; freitas, antonio a. title: regulation and maintenance of an adoptive t-cell dependent memory b cell pool date: 2016-11-23 journal: plos one doi: 10.1371/journal.pone.0167003 sha: doc_id: 2305 cord_uid: qq73gr9y we investigated the ability of monoclonal b cells to restore primary and secondary t-cell dependent antibody responses in adoptive immune-deficient hosts. priming induced b cell activation and expansion, aid expression, antibody production and the generation of igm(+)igg(-) and igm(-)igg(+) antigen-experienced b-cell subsets that persisted in the lymphopenic environment by cell division. upon secondary transfer and recall the igm(-)igg(+) cells responded by the production of antigen-specific igg while the igm(+) memory cells secreted mainly igm and little igg, but generated new b cells expressing germinal center markers. the recall responses were more efficient if the antigenic boost was delayed suggesting that a period of adaptation is necessary before the transferred cells are able to respond. overall these findings indicate that reconstitution of a functional and complete memory pool requires transfer of all different antigen-experienced b cell subsets. we also found that the size of the memory b cell pool did not rely on the number of the responding naïve b cells, suggesting autonomous homeostatic controls for naïve and memory b cells. by reconstituting a stable memory b cell pool in immune-deficient hosts using a monoclonal high-affinity b cell population we demonstrate the potential value of b cell adoptive immunotherapy. immune responses to infectious agents have different out-comes that can either protect or fail to control disease. protection from re-infection relies on the establishment of efficient secondary immune responses that require the generation of antigen-specific "memory" b and t lymphocytes. the generation and selection of t-cell dependent "memory" b cells involves distinct molecular mechanisms: immunoglobulin isotype recombination and somatic hyper mutation, both dependent on the expression of aid [1] . therefore, a long-standing paradigm defined memory b cells as igm -igg + isotype switched cells [2] . different lines of evidence indicate that this is not always the case. in humans, it has been shown that some igm + b cells bear the phenotype of other memory cells, being cd27 + , and carry frequent point mutations in the v region of the ig genes, suggesting that they must represent highly selected b cell populations [3] . in mice, populations of cd19 + igm + able to mount secondary responses have been identified [4] [5] [6] [7] . overall these findings suggest that the t-cell dependent memory b cell pool comprises distinct subsets of memory b cells with different properties and effector functions [4] [5] [6] . the biological properties that ensure the long-term persistence of memory and efficient secondary antibody responses have not been yet completely established. while initial studies proposed that after transfer memory b cells faded rapidly [8, 9] suggesting that long-lasting memory required the continuous recruitment of new cells [8] and/or antigen persistence [9, 10] , others suggested that memory b cells were able of extended survival without cell division [11] in the absence of antigen [2] . long-term persistence of antibody responses has also been attributed to populations of long-lived plasma cells mainly resident in the bone marrow following immunization [12, 13] . the demonstration of the compartmentalization of "antibody memory" into different cellular layers suggested that the separate subsets of memory b cells behave differently. accordingly, it has been reported that igg + cells that could rapidly respond upon challenge did not persist long, while igm + cells could generate a second wave of germinal center responses allowing persistence of memory [4] [5] [6] 14] . currently, immunotherapy approaches using passive antibody transfer [15, 16] ) is limited by the short half-life of immunoglobulin. therefore new therapy strategies may require the adoptive transfer of high-affinity memory b cells, ready to respond and able to persist. the development of these new strategies requires a profound understanding of the mechanisms that regulate memory b cell numbers and ensure long persistence upon adoptive transfer. moreover, knowledge of the mechanisms that determine the size of the memory b cell pool may be also critical to device new reconstitution strategies. so far, studies comparing populations of naïve and memory b cells have been hindered both by the vast clonal heterogeneity of the cells involved and by our inability to generate significant numbers of antigen specific memory b cells. indeed in a normal laboratory mouse the population of b cells bearing a "memory igg + phenotype' represent a small fraction of the total b cell pool (<0.5%) and upon immunization the number of the clonal diverse antigen-specific memory b cells generated is generally very limited (<10 3 ) [1, 6] . to circumvent these limits, we decided to compare the properties of homogeneous populations of naïve and memory b cells of known antigen specificity, belonging to the same clone. we used sw hel transgenic mice where b cells bear a high-affinity bcr specific for hel and are capable of class switch recombination and somatic hypermutation (shm) [17, 18] . to identify "memory b cells" the sw hel mice were crossed with mice where aid transcription provokes the permanent expression of an yfp reporter in post-germinal center lymphocytes [19] . these mice were in a rag2-deficient background and therefore contain a pure population of monoclonal hel-specific b cells. to generate memory cells, purified naïve b cells from the sw hel .aid/yfp.rag2 -/mice were transferred into adoptive hosts together with monoclonal ova-specific cd4 + t cells from otii.rag2 -/-tcr transgenic mice. upon immunization with ova-hel complexes, we obtained a significant number of persisting hel-specific igm + ig-g -yfp + and igm -igg + yfp + memory b cells, number that did not correlate to the number of precursor naïve cells initially injected suggesting that the memory b cell pool is regulated independently. we characterized the functional capacity of these two memory cell types in immune deficient hosts. mice b6 and b6.rag2-/[20] mice were kept at the centre des techniques avancées (cdta), centre national de la recherche scientifique (cnrs), orleans, france; swhel.aid/yfp.rag-/-mice, obtained by crossing swhel (18)(a gift of dr. robert brink) and aid/yfp [19] (a gift of dr. rafael casellas) with b6.rag2-/-mice. otii.rag-/-mice were kept in our animal facilities at the pasteur institute. experiments were preformed according to pasteur institute safety committee in accordance with french and european guidelines and the ethics committee of paris 1 (permits 2010-0002, -0003 and -0004). euthanasia of the mice was performed by cervical dislocation. this specific study was approved by the european research council (erc) committee related to the grant adg09 249740-qsis. the general status of the mice was controlled daily by monitoring the appearence of obvious pain, distress or suffering (prostration, respiratory issues, loss of weight). the end-point of the experiment was determined by a loss of more than 20% of the weight or as soon as the distress signs appeared. in this case, experiment was stopped and the animals were euthanized. single-cell suspensions of b cells from spleens and lymph nodes of sw hel .aid/yfp.rag -/mice together with cd4 + t cells from spleens and lymph nodes of otii.rag -/mice were transferred intravenously into the retro-orbital sinus of b6.ly5 a igh a or b6.rag2 -/recipient mice. mice received 10 6 hel + b cells and 10 6 cd4 + t cells unless stated otherwise. mice were immunized 24h later with 1 mg of ovalbumin coupled to hen egg lysozyme (ova-hel) in 50μg of alu-s-gel (serva) we determined as the optimal dose of ag (data not shown). naive cells from sw hel .aid/yfp.rag -/mice and memory b cells subsets from immunized b6.rag -/hosts mice were purified from spleens and lymph nodes by flow cytometry sorting. single-cell suspensions containing 5×10 4 b cells and 10 6 t cells were transferred intravenously into b6.rag2 -/recipient hosts. the purity of sorted cells was above 98%. 24 h after transfer, mice were immunized with 1 mg of ova-hel. spleen, bone marrow, inguinal and mesenteric lymph nodes single-cell suspensions were stained for cell surface or intracellular proteins with appropriate combinations of the following monoclonal antibodies conjugated to pacific blue, qdot-655, brillant violet 605, allophycocyanin, peridinin chlorophyll protein-cyanine 5.5, phycoerythrin, phycoerythrin-cyanine7: anti-cd19 (6d5), anti-igm (r6-60.2), anti-igg1 (x56), anti-cd138 (281-2), anti-gl7 (gl7), anti-cd95 (jo2), anti-cd62l (mel-14), anti-cd69 (h1-2f3), anti-baffr (7h22-e16), anti-cxcr5 (l138d7), anti-ia b (af6-120.1), anti-cd80 (16-10a1), anti-cd73 (ty-11-8) and anti-pdl2 (ty25) and anti-ki-67 (mm1) purchased from becton dickinson pharmingen, biolegend, invitrogen and ebioscience. cells were also stained with hel (sigma) coupled with af594 using alexa fluor1 594 protein labeling kit from life technologies. before staining, cells were treated with fc-block (cd16/cd32, becton dickinson pharmingen). dead cells were excluded during analysis according to their light-scattering characteristics. for intracellular stainings, cells were first stained with antibodies specific for cell surface antigens. then, cells were fixed and permeabilized according the manufacturer's recommendations (bd bisciences). for proliferation assay, mice were injected i.p. with 50 mg/kg of brdu (sigma-aldrich) and were killed 24 or 72 hours later. incorporated brdu was detected intracellularly using anti-brdu apc-conjugated antibodies according to the manufacturer's recommendations (bd biosciences). all data acquisitions and analyses were performed with lsrfortessa (becton dickinson) interfaced with bd facsdiva (becton dickinson) and flowjo (tree star) software. subsets of memory b cells were sorted as cd19 + hel + yfp + igm + or igg + and naive cells as cd19 + hel + yfp -igm + using a facsariaiii flow cytometer. the purity of the sorted populations varied from 90-95%. sera hel-specific ig concentrations were quantified by elisa. plates were coated with hel and saturated with pbs-5% milk. dilutions of sera were added. after incubation (2 hours, 37˚c) and washing, hrp-labeled anti-mouse igm or igg antibodies were added. after incubation and washing, bound antibodies were revealed with the substrate o-phenylenediamine and h2o2. the reaction was stopped after 10 min. by addition of 10% sds and the absorbance read at 492nm in a multiscan spectrometer. ig concentrations were determined by comparing the displacement of the dilution curves in the linear interval between standards at a concentration of 1 mg/ml and the serum samples. the quantification of igg or igm secreting cells was assayed by elispot technique. briefly, plates were coated with hel. after saturating, the cells were distributed into the micro wells in rpmi1640-2%fcs. the plates were incubated for 12 h at 37˚c, 5% co2 atmosphere. after extensive wash, plates were incubated with goat anti-mouse igm or anti-igg labeled with alkaline phosphatase. after washing, the revealing substrate was added (2,3 mm 5-bromo-4-chloro-3-indolyl phosphate diluted in 2-amino-2-methyl-1-proprenolol buffer). spleens from 14 day-immunized mice were initially fixed with paraformaldehyde and embedded in 4% low-gelling-temperature agarose (type vii-a; sigma-aldrich) prepared in pbs. 150μm slices were cut with a vibratome (vt 1000s; leica) in a bath of ice-cold pbs. for immunolabeling, samples were saturated with pbs supplemented with 10% of fetal calf serum, then were labeled with primary antibodies anti-b220-apc (clone ra3-6b2) and anti-igd-pe (clone 11-26c.2a) and analyzed with a spinning disk confocal microscope equipped with a coolsnap hq2 camera (photometrics) and a 20x objective. images were acquired and analyzed with metamorph 7 imaging software molecular devices). sample means were compared using the student's t test. sample means were considered significantly different at p < 0.05. during an immune response the complexity of determinants expressed by immunizing antigen and the degeneracy of antigen-specific recognition results in a vast heterogeneity of responding cells rendering impossible the direct comparison of the properties of naïve and memory b cells belonging to the same clone. we have devised an experimental system that permits the comparison between naïve and memory b cells expressing the same antigen receptor and allows marking permanently memory b cells. for that purpose we used sw hel transgenic mice in a rag2-deficient background holding a single population of monoclonal b cells, all bearing a high-affinity bcr specific for hel and capable of class switch recombination and somatic hypermutation (shm) [17, 18] . to identify antigen-experienced b cells the sw hel . rag2 -/mice were crossed with mice where aid transcription induces the permanent expression of an yfp reporter in post-germinal center lymphocytes [19] . since in intact tg mice immune responses were not traceable, probably because of the presence of low level pre-existing anti-hel antibodies that neutralize the immunizing protein, we used an adoptive cell transfer strategy to study the ability of the high affinity monoclonal b cell to reconstitute response in immune-deficient hosts and generate antibody memory. purified naïve b cells from the sw hel .aid/yfp.rag2 -/mice were transferred into rag2-deficient mice together with monoclonal ova-specific cd4 + t helper cells from otii.rag2 -/-tcr transgenic mice. the day after, host mice were immunized with ova-hel complexes (fig 1a) . in these conditions, antigenic challenge resulted in b cell activation and the development of significant numbers of cd19 + hel + aid/yfp + b cells, which were not detected in non-immunized mice or in mice immunized in absence of helper t cells (fig 1b) . we followed the early kinetics of this response. the number of hel-specific b cells increased from the initial 2x10 6 transferred to about 15x10 6 at day 14 (fig 1c left) the b cells expressing aid/yfp being the dominant population (fig 1c right) . a fraction of the hel-specific b cells underwent class switch recombination and at day 14 we recovered both igm + igg -aid/yfp + and igm -igg + aid/yfp + cell populations (fig 1b) . b cell expansion and phenotypic changes were accompanied by the production of igm and igg hel-specific antibodies ( fig 1d) . two weeks after antigenic challenge we observed the formation of germinal centers in the spleen of the host mice ( fig 1e) . coherently we found that while upon adoptive transfer all b cells expressed cd95, only after antigenic challenge most yfp + b cells expressed the germinal center specific marker gl7 (fig 1f) . in conclusion, the adoptive cell transfer strategy allowed the development of a primary immune response with b cell activation and expansion, induction of aid expression, class switch recombination, antigen-specific igm and igg antibody production and germinal center formation. we studied the evolution of the b cell response. from two weeks onwards the total number of b cells contracted and at four weeks we recovered about 2-4x10 6 cells, number that remained stable up to week 20 (fig 2a) . high titers of hel-specific igg were kept from week 3 to 8, declined thereafter, but were still significantly elevated 20 weeks later (fig 2b) . a population of cells secreting hel-specific igs was present in the spleen (fig 2c) , but not in the bm (not shown) even at the late time points. about 60% of the recovered cells exhibited the phenotype of antigen-experienced ("memory") cd19 + hel + aid/yfp + expressing either igm or igg ( fig 2d and 2e) . we compared the phenotype of the two aid/yfp + igm + and aid/yfp + igm -igg + memory cell populations recovered with that of the naïve b cells (fig 2f) . we found that antigen-experience and naïve b cells expressed similar levels of cd62l, cd69 and baffr (not shown). antigen-experienced cells presented sustained expression of cd95 and increased levels of pna, but the vast majority lost expression of the germinal center marker gl7 present at earlier times post-immunization ( fig 2f compare to fig 1f) . compared to naïve b cells, aid/ yfp + cells expressed higher levels of cd80 and mhc class ii and down-regulated expression of cxcr5 (fig 2f) . these findings indicate that the post-germinal center aid/yfp + b cells express an activated phenotype [5, 21] , have increased antigen-presenting capacity [22] , but may loose the ability to re-enter primary follicles being cxcr5 low [23] . we have also compared the patterns of gene expression (rnaseq) by naïve, activated (yfpcells of immunized mice) and both populations of yfp + memory cells. the data shows a clear discrimination of naïve and activated/memory cells while indicating only minor differences between both subsets of yfp + memory cells (fig 3) . mrna was isolated from sort-purified naïve (cd19 + hel + yfp -igm + ) igm + iggor igm -igg + hel + cd19 + yfp + memory b cells from spleen of different recipient mice. total recommended by the manufacturer. the validated libraries were then subjected to dna sequencing. the analysis is performed using the r software, bioconductor packages including deseq2 and the pf2tools package (version 1.2.9) developed at pf2 (institut pasteur). normalization and differential analysis are carried out according to the deseq2 model and package (version 1.8.1). fig 3a shows a representative heat map of the different cells populations. fig 3b shows late in the immune response persistent b cell numbers were kept by active cell division as a significant fraction of the cells were ki67 + (fig 2g left) and incorporated brdu (fig 2g middle) . the frequency of brdu + cells was higher among the aid/yfpcells (15%) than in the major aid/yfp + memory population (3%) and similar between the igm + and igm -aid/ yfp + populations ( fig 2g middle and not shown) . three days after brdu pulse populations were clear of brdu + cells (fig 2g right) attesting their high division rate. in spite of their increased proliferation rate, memory cells numbers were stable indicating that proliferation may be compensated by cell death as suggested by the frequency of caspase3 + cells (fig 2h) . the frequency of caspase3 + cells was higher among the aid/yfp + cells suggesting that a fraction of these cells may represent cells undergoing terminal differentiation. importantly, these findings demonstrate that the transfer strategy allowed the generation of significant numbers of persisting antigen-experienced yfp + cells. it is not yet known whether the number of antigen-experienced memory b cells correlated to the number of naïve b cells or if it is controlled independently of the initial number of antigenspecific b cells present. to approach this question we transferred different numbers of mature naïve b cells from sw hel .aid/yfp.rag2 -/donors (ranging from 10 5 to 5.10 6 ) into rag2-deficient mice together with an excess of cd4 + t helper cells (10 6 ) and immunize the hosts the day after cell transfer with ova-hel in optimal non-limiting quantities. to directly compare the results obtained after the transfer of different all numbers we allowed the responses to reach steady-state eight weeks after antigenic challenge. we studied the amplitude of the immune response by measuring the serum titers of hel-specific igg antibodies and enumerating the number of hel-specific b cells recovered. we found that in the presence of excess t cell help, the levels of the hel-specific iggs (fig 4c) , and both the total number of hel-specific ( fig 4a) and of memory yfp + b cells recovered (fig 4b) , did not correlate to the number of antigen specific naïve b cells initially transferred. memory b cells are defined functionally by their ability to induce secondary igg antibody responses upon secondary antigenic challenge. we investigated whether the subsets of aid/ yfp + igm + and aid/yfp + igm -igg + antigen-experienced (memory) b cells persisting at late time points could mount secondary igg responses and persist after secondary transfer. for this purpose we followed two different experimental strategies. in the first, 5x10 4 cells of either igm + or igm -igg + memory b cells, were transferred with an excess helper otii cd4 + t cells into secondary rag-deficient hosts that were boosted with ova-hel the day after cell transfer. in the absence of immunization antibody levels were undetectable (not shown) and three weeks after transfer recovery of both memory b cell subsets was about 10-20% of the initial cell input, exceeding naïve b cell recovery (fig 5a) , supporting the notion that memory b cells may not require specific ligand recognition to survive (2). one cannot exclude, however, that cross-reactivity of the bcr transgene with environmental antigens may allow signaling sufficient to maintain naïve and memory cell survival in the absence of hel [24] . following immunization, the secondarily transferred aid/yfp + igm -igg + cells responded promptly with the exclusive production of significant levels hel-specific igg thus confirming their memory statute (11) . the aid/yfp + igm + b cells in response to antigenic boost produced only limited amounts of igm antibodies (fig 4b) , little igg antibodies, but did generate gl7 + b cells more efficiently than the igg + memory b cell population (fig 5d) . thus the igm + subset may contain precursors able to generate a secondary germinal center reaction and a new progeny of igg + effectors (4). with time antibody levels decayed rapidly suggesting that the number of transferred memory b cells declined in the secondary hosts after antigenic boost. indeed, igm + and igg + memory b cells failed to expand and 3 weeks after immunization cell recovery was similar to the retrieval observed in the non-immunized hosts (compare fig 5e and 5a) . in similar experimental conditions, naïve b cells following immunization expanded, acquired aid/ yfp expression and their numbers more than doubled the number initially injected (figs 5f and 2a). these data suggest that a significant fraction of the memory b cells generated have a reduced expansion capacity being programmed for rapid differentiation for effector functions. besides long-term survival memory b cells must maintain functional activity in the absence of nominal antigen to be fully effective. to test this we used an alternative approach where memory cells were parked in secondary rag-deficient hosts for 30 days before re-immunization. we found that under these conditions antigenic challenge resulted in the production of hel-specific igg antibodies and in a 100 fold increase in the number of cells recovered, expansion that largely exceed that observed after immediate challenge (fig 5g) . the aim of this study was to characterize the fate of activated b cells and the generation of memory b cells. to do this, we adoptively transferred monoclonal b cells into immune deficient hosts followed by immunization in presence of t cell help. this strategy resulted in the development of different b cell memory subsets, namely igm + and igg + , as described for in situ generated memory cells [4, 6, 14] . these findings indicate that distinct memory b cell subsets are not the result of the heterogeneity of initially responding naive cells, but originate from the differentiation of a single b cell clone. while studying the respective rate of proliferation of both types of memory b cells, we found the same high rate of proliferation for igm + and igg + memory b cells. these results contrast with previous published data. first it was been reported that "in situ" memory b cells persist as resting non-dividing cells [11, 25] . however, we have shown that upon adoptive transfer and in absence of competing cells, b cells increase their division rate to occupy the available empty niche [26] , which may explain the higher division rate observed here using this adoptive cell transfer strategy. secondly, comparing life spans among heterogeneous memory b cell populations it was previously reported a lower division rate among the igm + subset compared to the igg + polyclonal subset [6] . differences in bcr affinity between igm + and igg + memory clones may explain the higher division rate previously observed among the igg + cells [6] . in contrast we compared memory b cell subsets belonging to the same clone bearing the same high affinity bcr. overall these observations support the notion that lymphocyte division rates and life spans are not an intrinsic cell property, but rather determined by the environment and the presence of competing populations [27] . they demonstrate that upon the correct conditions memory b cells can persist by cell division. an important question was whether the number of memory b cells depends on the number of initial naïve b cells. we found that, in the presence of an excess of t cell help, that was not the case. however, it was previously reported during polyclonal responses that serum titers of anti-hsa was proportional to the number of cells transferred into irradiated mice [28] . it is possible that limited antigen-specific t-b cell encounters may constraint the number of responding b cells and thus determine linear precursor-progeny between naïve and memory b cells. our findings indicate that within a single clone the number of precursor naive b cells present in the peripheral b cell pool does determine neither the intensity nor the final number of memory b cells in response to an optimal dose of antigen. they suggest that the size of memory b cell pool may be controlled independently of the number of naïve b cell precursors and that in the absence of clonal competition the memory niche can be filled with a single monoclonal population. considering diverse polyclonal populations, the limited niche for memory cells will imply strong competition among clones resulting in the selection of best fit (high affinity) cells: rare mutated clones being able to out compete more frequent but less avid clones. in our settings, the transgenic memory b cells are likely to counter select any new mutant clones since they express a very high affinity bcr selected in the course of a secondary immune response [29] . thus, notwithstanding the expression of aid and proliferation we did not detect any bcr vh and vl ig-chain nucleotide mutations among the recovered memory b cells (not shown). these findings may have implication for vaccination protocols as they indicate that each new antigenic exposure or unrelated immunization would add extra competing clones supporting the need for repeated antigenic boosts to prevent memory b cell attrition. they also demonstrate that the memory b cell pool can be reconstituted from a relatively small number of antigen-specific cells. it is likely that the relatively poor memory b cell expansion observed after immediate boost after adoptive transfer could be due to the lack in rag-deficient hosts of the appropriate environment required for memory b cell survival and function. it should be pointed out that b cell transfer into transgenic ml5 rag-deficient hosts expressing low levels of hel [29] resulted in rapid cell loss and recovery suggesting that in these hosts, b cells are trapped by antigen in locations were they are unable to survive (not shown). nevertheless, it has been shown that b cells can drive the maturation of follicular dendritic cells and the organization of lymphoid follicles [30] . similarly, transferred helper cells may also modify their immediate environment. thus, by allowing lymphocytes to adapt and modify their immediate environment we improved their response and more important, we recovered the memory b cell pool size present in the original donor mice. in this study we show that it is possible to fully reconstitute a primary response and the establishment of antibody memory in immune deficient mice after adoptive transfer of antigen-specific monoclonal b cells together with a population of monoclonal helper t cells. indeed, it is generally believed that in immune deficiencies, b cell therapy has restricted application due to intrinsic defects of host's lymphoid organs structure that may prevent development of immune responses, germinal center formation, establishment of antibody memory and limit cell survival. in contrast we showed that after adoptive transfer in immune deficient hosts antigen immunization induced b cell activation and expansion, induction of aid expression, class switch recombination, antigen-specific igm and igg antibody production, germinal center formation and the generation of two subsets of aid/yfp + igm + iggand aid/yfp + ig-m -igg + antigen-experienced b cell subsets able to persist in a lymphopenic environment by cell division mimicking responses obtained in intact non-tg mice [4] . upon challenge the aid/yfp + igm -igg + cells responded promptly with the production of hel-specific igg while the aid/yfp + igm + b cells secreted only limited amounts of igm antibodies and fail to produce igg. in contrast the aid/yfp + igm + b cells could give rise to new gl7 + b cells, suggesting that full reconstitution of the memory b cell pool may require transfer of the different antigen-experienced b cell subsets. importantly, we found that the recall responses were more efficient if the transferred memory cells were given the required time to adapt to their new environment, suggesting that a period of accommodation is necessary before the transferred cells are fully capable to respond. our findings also show that different processes can modify the survival conditions of memory b cells. finally, we found that the generation of the memory b cell pool in response to an optimal dose of ag did not rely on the number of the initially responding b cells, suggesting autonomous homeostatic controls for naïve and memory b cells a property that may allow reconstitution of the memory pool in immune-deficient hosts using a limited number of precursor naïve b cells. an autonomous control of the memory b cell pool where each antigenic exposure adds new competing clones supports the notion of vaccination strategies using antigenic boosting to prevent memory b cell attrition. overall the findings reported demonstrate that it is possible to reconstitute the memory b cell pool of an immune deficient host with an artificially induced population of monoclonal high affinity memory b cells. class switch recombination and hypermutation require activation-induced cytidine deaminase (aid), a potential rna editing enzyme memory b-cell persistence is independent of persisting immunizing antigen cd40-cd40l independent ig gene hypermutation suggests a second b cell diversification pathway in humans multiple layers of b cell memory with different effector functions cutting edge: hierarchy of maturity of murine memory b cell subsets different b cell populations mediate early and late memory during an endogenous immune response a germinal center-independent pathway generates unswitched memory b cells early in the primary response secondary antibody responses to thymus-independent antigens. decline and life-span of memory b-cell memory is short-lived in the absence of antigen induction of long-lived germinal centers associated with persisting antigen after viral infection maintenance of b-cell memory by long-lived cells generated from proliferating precursors maintenance of serum antibody levels b cell memory and the long-lived plasma cell plasticity and heterogeneity in the generation of memory b cells and long-lived plasma cells: the influence of germinal center interactions and dynamics maintenance of serological memory by polyclonal activation of human memory b cells an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus altered migration, recruitment, and somatic hypermutation in the early response of marginal zone b cells to t cell-dependent antigen b cell receptor-independent stimuli trigger immunoglobulin (ig) class switch recombination and production of igg autoantibodies by anergic self-reactive b cells regulation of aid expression in the immune response induction of self-tolerance in mature peripheral b lymphocytes immunological memory: contribution of memory b cells expressing costimulatory molecules in the resting state identification of murine germinal center b cell subsets defined by the expression of surface isotypes and differentiation antigens a coordinated change in chemokine responsiveness guides plasma cell movements the clone size of peripheral cd8 t cells is regulated by tcr promiscuity cellular dynamics of memory b cell populations: igm+ and igg+ memory b cells persist indefinitely as quiescent cells transfer of small resting b cells into immunodeficient hosts results in the selection of a self-renewing activated b cell population lymphocyte lifespans: homeostasis, selection and competition quantitative studies of the adoptive immunological memory in mice. ii. linear transmission of cellular memory altered immunoglobulin expression and functional silencing of self-reactive b lymphocytes in transgenic mice the sequential role of lymphotoxin and b cells in the development of splenic follicles the authors thank malika serra for technical help. key: cord-004017-gcmpatlb authors: errecaborde, kaylee myhre; macy, katelyn wuebbolt; pekol, amy; perez, sol; o’brien, mary katherine; allen, ian; contadini, francesca; lee, julia yeri; mumford, elizabeth; bender, jeff b.; pelican, katharine title: factors that enable effective one health collaborations a scoping review of the literature date: 2019-12-04 journal: plos one doi: 10.1371/journal.pone.0224660 sha: doc_id: 4017 cord_uid: gcmpatlb advocates for a one health approach recognize that global health challenges require multidisciplinary collaborative efforts. while past publications have looked at interdisciplinary competency training for collaboration, few have identified the factors and conditions that enable operational one health. through a scoping review of the literature, a multidisciplinary team of researchers analyzed peer-reviewed publications describing multisectoral collaborations around infectious disease-related health events. the review identified 12 factors that support successful one health collaborations and a coordinated response to health events across three levels: two individual factors (education & training and prior experience & existing relationships), four organizational factors (organizational structures, culture, human resources and, communication), and six network factors (network structures, relationships, leadership, management, available & accessible resources, political environment). the researchers also identified the stage of collaboration during which these factors were most critical, further organizing into starting condition or process-based factors. the research found that publications on multisectoral collaboration for health events do not uniformly report on successes or challenges of collaboration and rarely identify outputs or outcomes of the collaborative process. this paper proposes a common language and framework to enable more uniform reporting, implementation, and evaluation of future one health collaborations. ongoing and emerging health challenges such as infectious disease epidemics, bioterrorism, antimicrobial resistance, and natural disasters require a coordinated response from a highly diverse, collaborative, and trained health workforce. "one health" is a concept and approach intended to meet such demands. though loosely defined, a broadly accepted definition of one health describes it as "the integrative effort of multiple disciplines working to attain optimal health for people, animals, and the environment [1] [2] [3] [4] world organisation of animal health (oie), n.d.; world health organization, n.d). a one health approach recognizes that complex health challenges are beyond the purview of any one sector or discipline working in isolation [5] and that a resilient health workforce must be capable of effective and collaborative prevention and detection of, as well as response to emerging health challenges. a one health approach, therefore, calls for collaboration across disciplines, sectors, organizations, and national borders in support of increasingly complex health challenges [1] [2] [4] [5] [6] [7] [8] . while one health advocates increasingly support collaborative and multi-sectoral approaches to health challenges, no common language or metrics exist to uniformly describe and evaluate such efforts. few studies explicitly analyze the factors and conditions that support effective one health practices and collaborations. this hinders the ability of health professionals to learn from past experiences and improve upon current and future one health policies, partnerships, and practices. this paper seeks to address this gap by analyzing and identifying factors that enable effective multisectoral collaboration and response to health events. in this study, a multidisciplinary team of researchers reviewed a broad scope of literature describing collaborative and multi-sectoral approaches to past health events to understand how such collaborations are commonly described and evaluated and to identify and synthesize enabling factors for one health collaborations. this paper identifies twelve factors related to effective one health implementation and collaboration and concludes with a proposed framework for evaluating future multisectoral one health collaborations. the ultimate aim of this work is to support and improve multisectoral preparedness and response efforts. although its conceptual foundations date back hundreds of years, the formal global health construct known today as one health wasn't officially recognized by international and scholarly bodies until 1984 [8] . the hiv/aids pandemic in the 1980s and the hanta virus outbreak in 1993, made clear that emerging disease threats can cross national borders, cultures and species. with that came a broader recognition that animal and zoonotic diseases pose a serious threat not only to human health but to global health security. policy makers and health practitioners looked to collaborative health efforts as a response to these emerging challenges [3] . the subsequent decades which were marked by unprecedented global interconnectedness and human mobility [9] were associated with threats to global health security, including manmade threats, such as the use of anthrax as a bioweapon, and emerging diseases like sars and avian influenza. these challenges necessitated the need for a more formal coordinated action from countries, regions, and the global health community at large to address such health threats. in order to address the afore-mentioned challenges, there have been emergence of major health initiatives and frameworks such as the global health security agenda, the international health regulations-joint external evaluations (ihr-jee), the world health organization (who)-world organisation for animal health (oie) operational framework for good governance at the human-animal interface [10] , and the world bank's one health operational framework [11] . a common thread among these initiatives is the emphasis on multisectoral and transdisciplinary collaboration and a call for strengthening human, animal and environmental health systems through a one health approach. the global health community, including those already engaged in one health, continue to grapple with the fundamental questions of what characterizes a successful one health approach, including how to set goals, establish frameworks, facilitate collaborative work, and how to process and measure outcomes [12] . efforts to measure one health programmatic outcomes and operations are necessary for the improvement of collaborative efforts. this article supports such efforts by 1) identifying key factors that support effective collaboration around health events and 2) proposing a framework for documenting and evaluating one health collaborations in a more uniform and systematic manner. collaboration is an inherent and explicit part of the one health approach which calls for the active engagement of institutions, managers, and health practitioners across disciplines and sectors [1] [2] [3] [4] . despite widespread recognition of the importance of a one health approach, there exists a gap in the literature regarding what constitutes a successful one health collaboration. this study draws upon the existing public affairs literature on collaborative, or 'crossboundary' collaborations to understand which factors enable successful collaboration around health events. review of the literature on collaboration. scholars of public policy, organizational partnerships, team science, and multisectoral collaboration have produced a series of theoretical frameworks to describe cross-boundary collaborations and identify which practices make them successful [13] [14] [15] . the focus on collaboration and partnership is not unique to any one discipline, yet there is very little cross-fertilization of research across disciplines. this research builds upon the existing literature on cross-boundary collaborations and applies it to one health collaborations. the conceptual framework for this study focuses on three critical phases of a successful cross-boundary collaboration: adequate starting conditions, an effective process of collaboration, and attention to the outcomes of collaboration [16] [17] [18] [19] [20] . starting conditions. there is a general consensus in the literature on cross-boundary collaborations that starting conditions-the conditions in place before any collaborative process begins-impact the process, structures and outcomes of collaborative engagement. these include prior history (e.g. successes, failures, existing partnerships), the environment (e.g. resource imbalances, stakeholder incentives), and relational dynamics (e.g. balances of power, who convenes or facilitates the collaboration, and how and by whom problems are defined) [16, 17] . the presence or absence of such conditions influences successes and challenges encountered during the collaborative process. process. beyond starting conditions, many scholars point to the process of collaboration itself and the structures in place to support effective collaboration [13, 14, 20, 21] . although the terms used for collaboration vary, scholars focusing on the process of collaboration point to the importance of leadership, shared goals, trust and mutual understanding, institutional structures and resources, communication, and data management. measuring outcomes. a review of the literature on collaboration suggests a lack of validated metrics for measuring collaborative effectiveness and performance. several scholars of cross-boundary collaborations, citing works published between 2005 and 2019, highlight the importance of measuring the outcomes of collaboration and lament the challenges of describing and evaluating collaborations in a uniform way [12, 14, 16, 17, 20, [22] [23] [24] . this underscores the importance of understanding which factors support collaborative efforts, and how teams can evaluate their performance and outcomes in association with these factors. the literature on cross-boundary collaborations and its attention to the starting conditions, processes, and outcomes of collaborative approaches have informed this study on the factors that enable effective one health collaborations. the following questions guided this study: what factors (systems, structures, processes, skills, competencies, decisions, and actions) enabled two or more disciplines or sectors to collaborate effectively in a health event? a scoping literature review was conducted to identify key factors that facilitate multisectoral collaborations around major health events such as disease outbreaks using published accounts of actual health events. a scoping review, in contrast to a systematic review, is well-suited for a field such as one health that is still relatively new and evolving, as the method allows for assessment of emerging evidence, as well as a first step in research development [25] [p. 12]. due to the lack of a common language and framework for describing one health collaborations, this scoping review builds that foundation by providing a broad overview of one health collaborations and supporting the synthesis of key concepts, evidence, and research gaps [26, 27] . the scoping review was initiated by a multidisciplinary team in january 2017. the team members were composed of individuals with expertise in veterinary medicine, public health, public policy, organization and management leadership studies, international development, monitoring and evaluation, and education. because the researcher is central to the methods and analysis of qualitative research, it was important to select a transdisciplinary research team that could work effectively to address the research questions and to illustrate the disciplines that were represented in the transdisciplinary approach employed for this scoped review. selection of relevant articles. the search included peer-reviewed articles available todate in the u.s. national library of medicine's pubmed database that were searched using specified mesh (medical subject headings) terms. although the multidisciplinary research team has extensive experience in one health, they were not trained in sensitive search strategies [26] . the research team thus elected to work with a university of minnesota research librarian to develop mesh terms for this study. table 1 provides a list of the key terms used to identify articles discussing multisectoral health events and collaborations. to avoid tautology, it was a deliberate decision to not use "one health" as a search term. instead, drawing upon the researchers' extensive experience in one health, various terms were used to describe one health and similar multidisciplinary and cross-sectoral health collaborations. the underlying assumption was that any articles explicitly addressing one health would be captured using these key terms. this initial mesh search identified 2,630 non-duplicated articles. this scoping review was an inductive study of the literature and was conducted in order to support more hypothesis driven research for one health. by design, the authors elected to limit this literature review to the pubmed database at the outset of the study. pubmed is peer-reviewed and peer-led database. articles are selected and included based on scholarly and quality criteria by literature review committees and are tagged by keyword and by article structure, contributing to more accurate retrieval than other databases (e.g. google scholar); accurate retrieval supports the search results are reproducible and reportable, which is critically important for a scoping review of the literature in which it is important for other researchers, no matter their location, to repeat the study. the decision to use one database reflects the exploratory nature of this study and the author's intent to propose further hypothesis-driven research that may include additional databases. this methodological choice is in line with arksey and o'malley (2005) who attest that decisions must be made at the outset of a study to clarify reasons for the coverage of the review in terms of time span and language [26] [p. [23] [24] . initially, citations and abstracts of these articles were screened in two phases. the articles were reviewed for inclusion based on the criteria outlined in table 2 . in the first screening, 179 abstracts met initial inclusion criteria and full articles were procured and reviewed. in the second phase of screening, two further criteria were included to better achieve scoping review objectives. the research team divided into transdisciplinary pairs which included a reviewer from the health sciences and one from the social sciences. each of the articles that met the initial inclusion criteria were divided among the team members and then independently reviewed according to the modified screening criteria. articles were included if both reviewers agreed that they met all initial requirements. in instances where the transdisciplinary reviewers did not agree, the articles were brought to a full research team meeting and reviewed jointly until consensus among all researchers was achieved. this same method of collaborative review was used for the second round of screening and resulted in 50 articles for the final analysis. the prisma diagram below (fig 1) illustrates the article search, screening, and review process. 3. the health event discussed involved an infectious disease challenge; and 4. the case or event involved at least two sectors or disciplines. inclusion criteria: 1. articles met initial screening criteria and were included if they met the following targeted requirements: 2. the article provides a retrospective analysis of an actual health event; 3. the case or health event involved a noteworthy (describing successes or challenges encountered during health event) interaction among at least two sectors or disciplines. exclusion criteria: articles were excluded if they failed to discuss any specific aspects of collaboration, even if they generally acknowledged the importance of multisectoral collaboration. https://doi.org/10.1371/journal.pone.0224660.t002 analysis. the interdisciplinary team conducted an analysis of the 50 articles that explicitly addressed multisectoral collaboration in response to an actual health event. each reviewer coded approximately 5-10 selected articles using the qualitative data analysis software, maxqda [28] . descriptive codes were identified in advance to ensure that baseline data reflected the one health aspects of the articles reviewed. all other codes emerged from the data using a grounded theory approach [29, 30] . preliminary and axial coding procedures are outlined in the following section and ensured that inductive and deductive thinking could be related. preliminary coding. a set of predetermined, descriptive codes were used to denote the location and nature of the health event in the articles, including specific infectious agents, factors that enable one health collaboration: a scoping review relevant disease vectors or hosts, and the various entities involved in the collaboration. each paper was coded for the predetermined codes outlined in table 3 . predetermined codes were also used to identify the entities involved in each health event response. the team used the code "roles" to identify individuals or groups who participated in the coordinated response in a formal role based on individual expertise and formal training. while many of these roles represent professions in the health sciences, this category also included representation from the social sciences, media and community relations, government, and engineering. other articles focused on types of training, identified by the research team as "disciplines," (e.g., clinical epidemiology [31] or food safety [32] ), or specific professions (e.g., toxicologist [33] or information technology specialist [34] versus specific professions). a third type of classification in the literature was more general categorization of sectors involved, such as the traditional designations of public, non-profit, and private/for-profit. axial coding. axial coding was used to construct linkages between "data sets" or, in this scoping review, articles regarding intersectoral collaboration. axial coding is a qualitative research technique to relate data together in order to reveal codes, categories, and subcategories, as well as patterns in the data [35] . this grounded theory is an iterative process that combines inductive and deductive thinking. each article was first coded to identify any area of text where authors analyzed collaboration around a specified health event. in this process, it became quickly apparent that the review team would need to differentiate between actual and hypothetical forms of collaboration reported. all articles included in the analysis at this stage were retrospective analyses of actual health events, yet many were actually prospective in their analysis and discussion. as an example, several of these articles included suggestions based on what should happen in an ideal scenario, rather than what occurred in practice, thus leaving out key details of the actual event. therefore, a first round of organizing codes differentiated between collaborations that actually happened versus ideal scenarios and hypothetical lessons, allowing the research team to focus the analysis on what actually happened ( table 4 ). the text was further coded to reflect whether the authors were reporting a success factor of collaboration, or a challenge of collaboration. both the successes and challenges reported in the literature were related during the grounded theory thematic analysis and informed the final thematic results reported. after the first round of axial coding was conducted to organize the data, the authors employed a deductive framework developed from the review of literature on multisectoral collaboration [13] . aligned with this framework, the research team distinguished between starting conditions for collaboration, the process of collaboration itself, and the outcomes of collaboration (table 5) . finally, the review team re-examined the passages coded as "actually happened" and "successes". these codes were then related to the deductive codes of starting conditions and process-based factors. an excel table was used to organize axial codes into a table of final results. limitations. the primary limitations of this scoping review are three-fold. first, the literature analysis relies on peer reviewed publications alone, which may have underrepresented collaborative efforts that are more commonly encountered in grey literature. future work may be expanded to include these types of sources. second, there was no consistent framework or language for reporting the successes or challenges of collaboration, and thus, important content may have been missed during the search and review [36] . the scoping review team tried to overcome this with two strategies, which included building an expanded list of search terms and conducting an iterative review process using two independent transdisciplinary reviewers. both methods offset this limitation and might have minimized the likelihood of missing specific content. third, the researchers could not identify specific metrics for evaluating performance and collaboration in the literature. this meant that an evaluation baseline was not present. however, the research team believes that the final subset of articles represents a diverse crosssection of transdisciplinary efforts around emerging health events. the scoping review yielded 50 peer-reviewed publications explicitly addressing multisectoral collaboration in response to an actual health event. this section describes the nature of the one health collaborations analyzed as well as the various factors that enable one health collaboration. types of one health events analyzed include natural disasters, infectious disease outbreaks, endemic disease, bioterrorism, and biosecurity preparedness. in each of these cases, the underlying multisectoral collaboration was either a preparedness (planned or ongoing collaboration) or response (emergency or ad hoc collaboration) effort. the sample included one health events from around the world. most articles addressed health events in europe/eurasia (25%), the americas (25%), and asia (23%). less represented in this sample were health events taking place in africa (11%), oceania (10%), and the middle east (6%). most health events involved a specific infectious agent (97%), while the remaining 3% focused on infectious disease challenges such as hospital infections, pest management, or tsunami response. a total of 67 different infectious agents were coded. among the infectious agents identified, 58% were bacterial, 40% were viral, and 2% were protozoal (e.g. malaria). 39% of these agents primarily affect humans and 33% are predominantly animal-related. 16% of the agents were food and water-related, 10% were insect related, and an additional 2% were related to environment. overall, 60% of the infectious agents were considered zoonotic, meaning they spread between humans and animals. relevant disease vectors or hosts represented in more than one publication included bats, cattle, poultry, horses, swine, humans, mosquitoes and midges. involved parties or entities played varied roles and represented diverse disciplines and sectors, as illustrated in table 6 . thematic findings are presented according to the one health collaboration framework, which distinguishes between individual, organizational, and network factors that enable multisectoral and transdisciplinary collaboration at the onset and in the process of addressing a one health event. the team ultimately created organizing categories that reflected the individual, organizational and network levels of collaboration (table 7) . these categories were informed by a review of the literature; for the purposes of this discussion, the definition of network is provided by emerson and nabatchi [18] , and is defined as "the processes and structure of public policy decision making and management that engage people constructively across the boundaries of public agencies [organizations], levels of government, and or the private and civic spheres in order to carry out a public purpose that could not otherwise be accomplished," [p.2]. within each level, the review team created groups of subcategories to further organize codes. the research team identified 12 key factors that support successful multi-sectoral collaborations around major health events. at the individual level, these factors include 1) education & training and 2) prior experience & existing relationship. organizational factors include 3) organizational structures, 4) organizational culture, 5) human resources, and 6) communication. finally, network-level factors include 7) network structures, 8) relationships, 9) leadership, 10) management, 11) available & accessible resources, and 12) the political environment. these final individual, organizational and network factors were then further characterized factors that enable one health collaboration: a scoping review according to their relevance at the start of a collaboration "starting condition" or during the process "process-based" factors of collaboration. the researchers identified that the organizational thematic factors were relevant to both starting conditions and process-based factors so were not separated. the final results of this literature review are thus presented in table 8 . the researchers also coded each paper for outcomes. of all the articles coded, only 4 articles reported on outcomes of collaborations. the outcomes reported included: (1) cost reduction; (2) decreased mortality; (3) decreased morbidity, (4) multisectoral development opportunities resulted from the collaboration; (5) improved safety; (6) effective use of available resources. table 8 . final axial coding process included both inductive and deductive codes and reflects emerging themes for successful collaboration. starting condition factors initial deductive code (table 5) process factors initial deductive code (table 5) individual (emergent axial code table 7 ) preemptive technical training/ continuing education [37] [38] [39] [40] [41] [42] [43] [44] [45] disease specific technical training [34, 45, 46] preemptive collaborative training [47, 48] strong public-sector led training [39] training and capacity building provided a platform for better collaboration for outbreak [49] ngos support gov. through staff training [50] participatory epidemiology training [51] prior experience & existing relationships (informal/formal) pre-existing multisectoral relationships [45, [52] [53] [54] [55] previous experience collaborating for health events [34, 56, 57] ad hoc "just-in-time" training shared training & organizational alignment; aggressive, rigorous, just-in-time, and critical trainings for key positions and critical events with monthly follow-up meetings to support compliance [31, 58] training and capacity building provided a platform for better collaboration for outbreak response [49] instituting multisectoral disease specific training; ongoing training-for new and existing systems [39, 45] organizational (emergent axial code table 7 ) shared response guidelines [42, 50] structures frequently included policies/protocols [59, 60] reporting -management protocol -task management -response plan -communications/ communication strategy [34, 61] infection planning, control and traceback procedures [62] systems reporting, laboratory systems [59] surveillance systems [41, 58, 59] planning; iterative improvement of systems [46, 48, 60] information management system/ database [41, 48, 63, 64] information sharing (data available and useful) [45, 48] ) tool sharing during response [65] lab systems in place [59] online system for hr recruitment [45] intentional multidisciplinary engagement, collaborative capacity [43, 48, 66, 67] standard operating procedures [55] interoperability [42] needs assessment and prioritization [38, 48] culture leadership, accountability, ownership, trust, transparency of processes, systems based thinking, cultural awareness and engagement leadership to support the iterative and developmental review of collaborative processes [58] strong, engaged leadership [32, 35, 52, 68] accountability; ownership [67, 68] cultural engagement; engagement; diversity/ involvement of community [67, 69] trust [38, 41, 49, 70] transparency [31, 34, 61] need to understand each other's' processes [53, 70] systems based thinking/ approach [34, 48] cultural awareness; engagement of diverse stakeholders to reflect community needs [53] credibility [38] existing relationships [49] institutional knowledge (experience and relationships) [31, 45] revise and revisit mandates based on lessons learned [37, 71] clearly defined roles and responsibilities [35, 42, 65] resources available and accessible (including human resource allocation) [35, 45] informed staff/ staff are aware of systems in place, increased engagement of staff [31, 45] reflexive workforce reflexive human resource protocol to ensure positions are adequately filled & workers are incentivized [31, 57] reflexive approach [31, 45] adaptability to rapidly changing context [42] rapid start-up response; shared response guidelines [42] (continued ) factors that enable one health collaboration: a scoping review multi sectoral coordinating mechanisms/ platforms for engagement [34, 41, 45, 52, 60] memoranda of understanding, terms of reference or bilateral agreements to support the development of existing relationships that promote ongoing engagement [41, 45, 48, 72] in this discussion, the research team suggests 12 thematic factors that may be used by practitioners involved in one health activities to more systematically assess the successes and challenges of multisectoral collaboration, including those contributing to successful outcomes. further research is needed to refine and validate these factors and ultimately support more uniform and rigorous assessments of one health collaborations. the axial coding process allowed for factors reported to facilitate or discourage successful collaboration to be categorized as either a relevant starting condition of collaboration, or as relevant to the process of collaboration. during the data analysis, certain themes within each category of individual, organizational and network factors emerged as relevant to "setting the stage" for effective collaborative processes, while other factors were essential to maintaining the process of collaboration itself. the researchers found that this distinction was critical in our understanding of how successful collaborative processes are initiated. the starting conditions presented in this paper represent the collaborative preparedness and planning necessary to support effective one health processes. in addition, the process of collaboration allows for the emergence of new ways of collaborating. this symbiotic relationship between starting conditions and process, allows us to view the entire system of collaboration. in this system, starting conditions influence the process of collaboration, and the process itself can lead to improvement of structures and processes that will now inform improved starting conditions. this cyclical and emergent process is inherent in collaboration and must be accounted for when considering evaluation and systems-based improvements. individual factors. relevant success factors at the onset of a one health event include an individual's education and training, as well as prior experience and existing relationships. many authors identified existing or previous education and training as enabling factors for collaborative success [37, [40] [41] [42] 44, 47, 49, 51] . formal technical education and training of individual workers prior to a health event was critical to prepare the necessary human resources for response efforts. authors noted that foundational technical training during an event was often not possible [41, 42] , but that preemptive and collaborative planning did support the development of key relationships, and in some cases, the development of shared protocols used in the response. the absence of formalized training opportunities before an event, both individual technical training and collaborative, were frequently reported as a gap and a challenge to effective one health response [40] [41] [42] 49] . shared competencies were suggested as a strategy for standardizing protocols and performance across multiple individuals and organizations [49] . multiple sources also reported the importance of prior experience in collaborative response efforts and how this established existing relationships to support the work, both formal (i.e. required communication through standard operating procedures) and informal (i.e. loosely structured and based on personal relationships and ongoing professional engagement) [34, 45, 52, 53, 56, 57] . when instituted before a health event occurs, these starting conditions were reported to support a more effective collaboration processes. individual factors that supported the process of collaboration were most frequently reported as workers having access to necessary education and training that was available ad hoc or as "just in time" training to support operations during the health event. examples reported include the use of shared training across organizations to additionally support institutional alignment and partnership with community-level organizations to provide training [39, 42, 49] . many of these trainings were reported to be rigorous and responsive with continuous follow-up to support compliance [31, 45, 58] . williams et al [45] discussed how ongoing multisectoral disease specific training supported workers to operate within new and existing systems while simultaneously sharpening their technical competence. these training and capacity-building opportunities were reported to provide a platform for better collaboration for outbreak response [49] . however, ad hoc trainings do not replace or diminish the need for foundational technical training, as formalized education and training were reported as a critical starting condition to facilitate quick mobilization in the case of a health event. our literature review uncovered the role for both strong university-based education and training, and the role that ad hoc or "just-in-time" training can play to meet immediate operational needs during process-based response. organizational factors. factors reported to enable organizational-level collaboration were broadly applicable to both the starting conditions and the processes of collaboration. organizational structures, culture and resources were cited as important elements for creating an enabling environment for effective one health collaboration. the organizations serve to connect the individual worker with a network of one health actors. the organizational structures that support collaboration were often discussed as a success factor. these structures include, but are not limited to, the policies and protocols or systems established within organizations to support technical implementation and collaborative efforts. policies and protocols reported to be supportive included technical guidelines and standard operating procedures, as well as management, response and communication strategies and protocols [34, 42, 50, 59, 60, 62] . in addition, organizations reported the need for functional systems for information and resource management and sharing and reporting both surveillance and laboratory results [41, 43, 45, 48, 55, 59, 63, 64, 66, 67] . these systems were reported to benefit from being adaptive, flexible/reflexive and improved through iterative feedback and monitoring and evaluation [38, 46, 48, 60] . organizational culture was reported in multiple key areas [31, 35, 38, 41, 48, 49, 60, 61, [68] [69] [70] 81] . the role of organizational leadership was discussed at length in many of the reviewed publications. authors recognized and identified the importance of having strong and engaged leadership [31, 34, 52, 68] and the need for leadership to support the iterative and developmental review of collaborative processes [60] . in addition, organizations benefited from having a culture that supported accountability, ownership, cultural engagement and diversity [53, 68] . trust and credibility were consistently reported as a key element of organizational success [38, 41, 49, 70] , as was the need for both an understanding of each other's processes and systems based thinking [34, 48, 53, 70] . authors reflected on the importance of cultural awareness, transparency of communication processes [31, 34, 53, 61] and the engagement of diverse stakeholders who were able to reflect community needs [53] . human resource-related factors appeared in all three levels of analysis. research suggests that workers need to be trained at an individual level, have defined roles and responsibilities at an organizational level, and need to be able to mobilize their efforts at a network level. at an organizational level, human resources are made up of individual contributors and also function as collective entities that reflect employees' prior experiences, existing relationships, and the collective institutional knowledge of its members [31, 34, 45, 49] serve to benefit the organizations in which they work. clear roles and responsibilities were consistently reported [34, 42, 45] , as well as awareness of systems in place to support ongoing engagement, operations and information sharing [31, 45] . additionally, several authors highlighted the importance of a reflexive workforce, i.e. human resources that were readily available and could be mobilized quickly and efficiently to respond to health event in a rapidly changing context [31, 42, 45, 57] . network factors. starting condition factors reported to enable collaboration at the network level included network structures, existing relationships, available resources in the face of a health event, and the political environment in place to support these efforts. pre-existing network structures were reported to provide a foundation for effective collaborative efforts to occur across participating organizations. established multisectoral coordinating mechanisms (mcms), also referred to as one health platforms or joint task forces, were often reported as key to assisting with collaboration across a network [34, 41, 42, 45, 48, 52, 60, 72] . organizational and network structures provided operational standards that crossed relational and organizational boundaries at all levels of the system-individual, organizational and network-which supported the development of formal relationships at each level. analysis suggested that these systems and relationships need to be in place before the health event. mcms provide a formalized operating foundation in which organizations and individuals could contribute, and formalized roles and responsibilities supported effective human resource mobilization in both organizations and networks [34, 42, 45, 72] . these structures were often supported by formal policies or agreements such as bilateral agreements or memoranda of understanding (mous) [41, 45, 72] . in addition, operating procedures such as the incident command system (ics) also supported effective mobilization of multiple organizations within the mcm [60] . finally, the importance of formal structures were repeatedly emphasized as a response to "lessons learned" during challenging responses. on the contrary, lack of existing structures was reported to prevent efficient multisectoral engagement in the preparedness and response to health events [37, 42, 71] . several sources indicated that reporting structures and policies at local, regional, national and international levels support continuity of response and effective implementation in response to health events [45, 48, 49, 58, 60, 62, 65, 72, 73] . these reporting structures and policies allowed for information flow between stakeholders, and the coordination of response efforts across a diversity of individuals and organizations participating in preparedness or response efforts [31, 49, 58, 65, 74] . established structures created a foundation for network relationships that support effective outbreak response to a health event [31, 40, 42, 45, 47, 49, 54] . development of formal and informal relationships prior to a health event allowed individuals, organizations and networks to more effectively respond once an emergency arose. the existence of structural agreements in any form such as mcms, mous, shared standard operating procedures (sops) or bilateral agreements were reported to support the further development of existing relationships to promote ongoing engagement prior to and throughout a health event [55, 74] . preemptive planning for potential disease threats was reported to strengthen connections and relationships and support multisectoral disease training, sometimes leading to shared protocols [45] . additionally, the creation of common goals [34, 47] and clearly defined, previously established, roles and responsibilities for individual actors and network partners were reported as necessary in network operations [34, 40, 42, 45, 49, 54] . cultural awareness and the inclusion of diverse stakeholders from government, nonprofit, and private sectors from the national to community level, was consistently reported as a success factor for collaborative efforts if included from the start [33, 34, 37, 42, 51, 52, 56, 59, 61, 75] . availability of resources, including human resources, that can be easily and efficiently mobilized in a health event was considered an important factor for response [31, 34, 37, 41, 44, 45, 49, 52, 54, 74, 82] . authors also noted the importance of a supportive political environment to aid in the development and institutionalization of effective collaborative structures [41, 48, 65, 72] . a supportive political environment was reported to enable the flow of available financial, human and material resources and empowered decision making [72] . readily available resources supported rapid mobilization of collaborative efforts when a health emergency occurred. this is particularly impactful given that the absence of these resources and actions was noted across the literature as challenges to effective health response. network leadership and management processes were critical to effective multisectoral response efforts. leadership engagement during a health event allowed for the mobilization and needed support for management processes. by utilizing existing structures and decision-making power, leaders and lead agencies can support managers and the process of management across organizations and networks. emergency response protocols, such as the ics, were frequently reported as mechanisms to this end, by concretely providing a leadership and management structure to support ongoing multi-organizational response. it was particularly useful for identifying a lead agency and establishing structures for regular meetings and communications. in the process of collaboration, relevant network factors included network leadership, management, and the effective and efficient mobilization of resources for response. for example, strong and engaged network leadership was noted as an important success factor for collaboration. when established prior to a health event, factors reported to support network collaborations included identifying a lead agency [41, 52] , promoting information sharing and joint decision-making across a network [49, 60, 65] , and convening regular multisectoral meetings [53, 58, 60] . in addition, strong leadership was integral for strategic risk communication across the network [45] . effective network management during an outbreak was reported in the areas of management practices, monitoring and evaluation (m&e), communication, awareness and ongoing stakeholder engagement. management practices included case and task management through the mcm [41] , regularly scheduled meetings [53, 58, 60] and development of shared response guidelines and management protocols across the network [58, 81] . these management practices, when paired with existing structures, can support rapid start up response in the face of health events [51] . monitoring and evaluation allowed for the iterative review of the collaborative processes during response efforts, as well as the outcomes of the collaborative process [31, 34, 38, 45, 48, 55, 60] . monitoring and evaluation was reported as integral in being able to show the outcome of interventions as beneficial or not [31, 38, 45] . the importance of communications cannot be overemphasized and was repeatedly reported as an integral factor for building relationships, trust and supportive organizational culture, and for contributing to effective response processes. both the characteristics and the methods of communications were highlighted as important. characteristics of successful communication included the need to be frequent and honest [44, 45] ; timely and consistent [45] ; reflexive and flexible [59] and prioritized (risk-based) [45] , and streamlined [54, 70] . additionally, characteristics included the need for communications that build trust [49] and have a clear purpose [31, 44, 70] . these elements were widely reported to support effective communication within and across organizations [31, 41, 44, 48, 49, 51, 53, 54, 58, 60, 70, 77, 78, 80] . communication was deemed most effective when it was regular, frequent, and designed to foster awareness and support the engagement of a range of stakeholders, from local through national, regional and international levels. the mcms, or the use of ics, were often cited as important organizing structures for ongoing communication during a health event, supporting meetings, data collection and information sharing [43, 48, 58, 60] , underscoring the importance of starting conditions to support communications. multiple methods of communication were reported including electronic communications, list-servs, contact lists and regular meetings; in many cases these were supported through existing mcms [48, 58] monthly meetings [53, 58, 60] and establishing clear lines of communication [31, 44, 51, 77, 78] were reported as critical. these methods were supported by the use of a variety of methods and platforms such as press briefings, websites, television, newspaper, teleconferencing, listserv, available contact lists, local/ regional/ cross-border meetings and periodic reporting [44, 45, 49, 53, 58, 62, 77] . additionally, leadership and management processes played a key role in supporting or challenging communication; high-level support, resource allocation, and use of good practices across an organization are foundational for good internal and external communication. closely linked with communication was the reported importance of building shared awareness and diverse stakeholder engagement. awareness included information sharing, education campaigns, jointly coordinated communications and public release of reports with all members of the network and with the public [31, 38, 44, 45, 52, 54, 55, 61, 70] . engagement of diverse stakeholders before, during, and after the response was reported as essential; these stakeholders included community and local actors, national governments, intergovernmental organizations, and operating partners [45, 49, [51] [52] [53] 75] . to facilitate communication across stakeholder groups, adams et al. [60] and butler et al. [70] underscored the importance of transparent joint communications specifically between responders and community leaders for efficient and effective response. butler et al. [70] further reported the success of joint interviews held with stakeholders to support shared understanding of response needs. diverse partners, including foreign militaries, were reported to support foundational infrastructure that allowed other international partners to stay involved when supporting a response effort when they would not have been able to serve effectively on their own [39, 71, 79] . common goals, common interests, and perspective sharing amongst stakeholders were reported to support an effective response to a health event [38, 49, 53] . resource mobilization and allocation during an event, relies heavily on the starting conditions, as well as the communication, leadership and management during the process of collaboration. a number of authors pointed to the importance of being able to mobilize both the material and human resources. once again, the involvement of diverse stakeholders, the use of mcms and management systems such as ics were attributed with the ability to draw upon existing resources. processes characterized as successful included establishing a supply chain with standardized access, delivery, allocation and flow [34, 41, 68, 80] . human resource mobilization benefited from online recruitment [45] as well as reflexive human resource protocols to ensure that positions were filled and workers are incentivized and rewarded for participation [31, 57] . outcomes reported. although the researchers created a code to capture reported outcomes of collaborative efforts, only a small number of authors reported outcomes of their collaborative processes. outcomes were consistently missing or under-reported in the literature reviewed, and this is likely a result of one health outcomes being difficult to characterize. the few reported included the outcomes of cost reduction and improved safety [34] , decreased mortality [41] , reduction in mrsa (methicillin-resistant staphylococcus aureus) cases [31] , increased stakeholder buy-in [45] , and a report that multisectoral professional development opportunities resulted from the response [47] . however, implementation of m&e activities was one of the major gaps in the reports of one health collaboration. the majority of articles reviewed never discussed the evaluation of either the process of collaboration or the resulting outputs or outcomes. this creates a pivotal challenge in understanding how to improve one health operations. the authors noted that outcomes of collaborative efforts were consistently missing or underreported in the literature reviewed. language in collaboration. language used to describe one health work continues to be a challenge when working across disciplines. each discipline contributing, and specifically those authors reporting on these interactions, bring their own nomenclature and vernacular [36] . as also discussed in the limitations of this work, we encountered challenges in how authors reported on which entities were involved in the response to a health event. organizations, sectors and disciplines were characterized in different ways, making is difficult to find a standard classifying system for the coding. considerations for the evaluation of one health. despite an emphasis on the importance of iterative improvements to collaboration, the implementation of monitoring and evaluation activities was one of the major gaps in the reports of one health collaboration. the majority of articles reviewed never discussed the evaluation of either the process of collaboration or the resulting outputs or outcomes. this creates a pivotal challenge in understanding how to improve one health operations. it became clear in the literature review that there was no standard framework for how to evaluate one health processes [12, 36] . although networks and collaborators such as the network on the evaluation of one health are making important advances, practical evaluation tools are still needed [83] . some authors from public affairs, such as emerson and nabatchi (2015) et al. [19] have proposed a framework for evaluating outputs, outcomes, and what they refer to as "adaptations" of collaborative processes [18, 19] . their work is one of the first to propose an integrated framework that captures collaborative results at all levels of the system, from the target population to the participating organizations, and the network as a whole. the results of this scoping review are intended to support the next steps in supporting one health evaluation. using the 12 factors uncovered in this review, the authors have outlined a reporting framework ( table 9 ) that may help practitioners consider their activities in light of important collaborative starting conditions and process-based factors. the researchers propose this to the one health factors that enable one health collaboration: a scoping review community as a tangible next step that may lead to more effective reporting and potentially evaluation of one health efforts in the future. the proposed framework in table 9 recognizes that each factor will be operationalized within the context of the health event and that flexibility in reporting is imperative. this framework may be useful in providing a common language on how practitioners discuss and report on their one health efforts. in the process of conducting this research, the research team encountered many of that same collaborative challenges as described in the articles reviewed. the research team had to negotiate and re-negotiate ways of working, integrate differing points of view and assign roles in ways to leverage expertise but not reinforce bias. additionally, the researchers had to establish and meet internal standards while also achieving the outward facing objective of finishing the analysis and writing of this article. finally, as with any transdisciplinary work, language was consistently a problem. the inherent challenge of interdisciplinary work is in how we talk about collaboration and the terminology we use to describe both theory and practice. for the research team, creating clear definitions supported the development of a common language. differing approaches can be a significant barrier when active collaboration is not structurally supported, valued, and continuously monitored for health and effectiveness. our efforts reinforce the need for training for those skill sets that fall beyond technical sector-specific training. when grappling with the question of which skills were most important in our collaborative process, we determined that the shared objective of collaboration was the foundation for our ability to integrate the differing expertise that each team member brought to the process. simply, we took continual action to achieve our combined goal including reading new literature, considering new frameworks, learning new things, and asking many questions. the subsequent challenge is, of course, that there are very few formal opportunities to gain access to training around these competencies and mindsets in one health teams. most often, as in our case, it is an ad hoc process that rests on the motivations, shared values, and time available within a team to develop in this way. our review suggests that, while this approach worked for us, it would not be a time or resource-effective approach within the context of a health emergency. thus, one health approaches need to be evaluated to help practitioners decide when and how to most effectively collaborate for their intended purposes. of the 2,630 article abstracts screened, only 179 met initial inclusion criteria and the full research articles were obtained. of that subset of articles, only 50 discussed the successes, challenges and lessons learned from operational one health response to a health event. a majority of the articles focused broadly on the need for collaboration between multiple sectors or disciplines with little attention to what factors enable an effective one health response effort. the low number of included articles reflects a broader challenge for the one health community, suggesting the necessity that one health researchers move beyond discussing the inherent need for one health, to actually reporting on the processes, outputs and outcomes of their collaborative efforts. as such, no consistent framework or language was found to report on the process, outputs or the outcomes of one health work in the articles reviewed. in the analysis, the research uncovered 12 factors that supported successful health event response. the researchers were able to make important advances by characterizing these factors as important at the start of collaboration or relevant to the process of collaboration. using these 12 factors, the researchers propose a one health reporting framework which when used to report on one health collaborations, can support the further refinement and identification of success factors for one health. these factors may serve as the basis for developing evaluation metrics and the 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central role of prompt information sharing and public communication public health communication with frontline clinicians during the first wave of the 2009 influenza pandemic department of defense west nile virus surveillance in 2002 collaboration, cholera, and cyclones: a project to improve point-of-use water quality in madagascar a collaborative strategy to tackle tuberculosis in england north india: delivering dots via collaboration with private providers and non-governmental organizations a blueprint to evaluate one health. front public heal key: cord-001958-2gt3fwpy authors: meseda, clement a.; atukorale, vajini; kuhn, jordan; schmeisser, falko; weir, jerry p. title: percutaneous vaccination as an effective method of delivery of mva and mva-vectored vaccines date: 2016-02-19 journal: plos one doi: 10.1371/journal.pone.0149364 sha: doc_id: 1958 cord_uid: 2gt3fwpy the robustness of immune responses to an antigen could be dictated by the route of vaccine inoculation. traditional smallpox vaccines, essentially vaccinia virus strains, that were used in the eradication of smallpox were administered by percutaneous inoculation (skin scarification). the modified vaccinia virus ankara is licensed as a smallpox vaccine in europe and canada and currently undergoing clinical development in the united states. mva is also being investigated as a vector for the delivery of heterologous genes for prophylactic or therapeutic immunization. since mva is replication-deficient, mva and mva-vectored vaccines are often inoculated through the intramuscular, intradermal or subcutaneous routes. vaccine inoculation via the intramuscular, intradermal or subcutaneous routes requires the use of injection needles, and an estimated 10 to 20% of the population of the united states has needle phobia. following an observation in our laboratory that a replication-deficient recombinant vaccinia virus derived from the new york city board of health strain elicited protective immune responses in a mouse model upon inoculation by tail scarification, we investigated whether mva and mva recombinants can elicit protective responses following percutaneous administration in mouse models. our data suggest that mva administered by percutaneous inoculation, elicited vaccinia-specific antibody responses, and protected mice from lethal vaccinia virus challenge, at levels comparable to or better than subcutaneous or intramuscular inoculation. high titers of specific neutralizing antibodies were elicited in mice inoculated with a recombinant mva expressing the herpes simplex type 2 glycoprotein d after scarification. similarly, a recombinant mva expressing the hemagglutinin of attenuated influenza virus rga/viet nam/1203/2004 (h5n1) elicited protective immune responses when administered at low doses by scarification. taken together, our data suggest that mva and mva-vectored vaccines inoculated by scarification can elicit protective immune responses that are comparable to subcutaneous vaccination, and may allow for antigen sparing when vaccine supply is limited. the eradication of smallpox, a disease that caused the death of hundreds of millions of people over many centuries, was accomplished primarily by the use of replication-competent vaccinia virus strains as vaccines. traditional (first-generation) smallpox vaccines, as well as more recently developed cell culture-derived second-generation smallpox vaccines such as acam2000 [1, 2] , the currently licensed smallpox vaccine in the united states, are inoculated into vaccine recipients by scarification of the skin surface, also known as percutaneous, skin or cutaneous vaccination [3] . rare but severe adverse reactions caused by these vaccines, including generalized vaccinia, eczema vaccinatum, and the more recently recognized cases of myopericarditis [4, 5, 6, 7] , limit the use of these vaccines for routine preventative vaccination of the general populace in the absence of any immediate risk of exposure to variola virus (the etiologic agent for smallpox) or other pathogenic orthopoxviruses such as monkeypox virus. thus, as early as the 1930s, efforts were made to develop safer smallpox vaccines by attenuating existing strains of vaccinia virus [8, 9] . as part of this effort, the modified vaccinia virus ankara (mva) was developed in the early 1970s. mva was derived from the chorioallantois vaccinia virus ankara (cva) strain of vaccinia virus, by more than 570 passages in chick embryo fibroblast (cef) cells [10] . during the course of passage of cva in cef cells, several genes (mainly host-range and immunomodulatory genes) were lost, resulting in the severely attenuated mva. about 15% of the viral genome was lost during passage in cef cells, and mva does not replicate productively in most mammalian cells [11, 12, 13] . mva has been extensively evaluated in different animal models [14, 15, 16, 17] and in clinical trials, and found to be less reactogenic when compared with replication-competent first and second generation smallpox vaccines [18, 19] . mva is licensed as a smallpox vaccine in europe and canada, and currently undergoing clinical development in the united states. the severe attenuation of mva and its consequent loss of the capacity to replicate efficiently in mammalian cells is evident in its inability to produce a "vaccine take", a pustular lesion that develops at the inoculation site, when vaccinia virus is inoculated on the skin surface. apart from its potential use as a smallpox vaccine in immunocompromised individuals, mva has the capacity to accommodate heterologous dna, and express encoded proteins, thus serving as a useful viral vector in vaccine development against different types of pathogens. several recombinant mva vectors expressing heterologous proteins of different human pathogens are at various phases of clinical development [20, 21] some of the mva-vectored vaccines in clinical trials include those expressing human immunodeficiency virus antigens [22, 23, 24] , mycobacterium tuberculosis 85a antigen [25, 26, 27] , malaria antigens [28, 29, 30] , human papilloma virus antigen [31] , hepatitis c antigens [32, 33] , respiratory syncytial virus antigens [34] , influenza virus antigens [35, 36, 37] , epstein-barr virus antigen [38, 39] and more recently, ebola virus antigens [40] . several other mva-vectored vaccines have also been evaluated in preclinical studies [41, 42, 43] . in most preclinical and clinical studies, mva or recombinant mva vectors, unlike replication-competent vaccinia virus strains, are inoculated into subjects via the intramuscular, intradermal or subcutaneous routes. although mva has been demonstrated to have a better safety profile than replication-competent vaccinia virus, a relatively large inoculum volume of 0.05 to 0.10ml and 0.5 to 1ml of mva or recombinant mva is typically used in small animal models and in non-human primates/humans, respectively. this often results in local reactions at the site of inoculation, including muscle ache, pain, and tenderness [44, 36] . in addition, the inoculation of prophylactic or therapeutic regimens with needles and syringes can be problematic for some people, a global problem commonly called needle phobia or fear of the needle in common phraseology. although the use of needle-free injection devices such as the jet injector [45] has become increasingly popular, hypodermic syringes and needles remain in wide use for the delivery of prophylactic and therapeutic remedies. previously, we [46] described a recombinant vaccinia virus, a33r/b5rko, that was severely attenuated for plaque formation in permissive cell lines due to deletions of the a33r and b5r genes, encoding the a33 and b5 proteins, respectively, of the extracellular virion form of vaccinia virus. the severe attenuation and growth characteristics of the a33/b5rko virus is reminiscent of the properties of mva. a33r/b5rko elicited vaccinia-specific igg and neutralizing antibodies when balb/c mice were inoculated by scarification at the base of the tail. in an intranasal challenge model, using the western reserve strain of vaccinia virus, the a33r/ b5rko virus conferred comparable levels of protection on mice as those vaccinated with a clonal isolate of dryvax, dv-3. during the course of the work above [46] , antibody [47] and robust cell-mediated immune responses after the inoculation of recombinant mva vectors through the skin, were also reported [48, 49, 50] , suggesting that percutaneous inoculation of mva may elicit equivalent or higher immune responses than subcutaneous injection. in the work described here, we demonstrate in mouse models that percutaneous inoculation of mva elicited protective immune responses against lethal intranasal challenge with the western reserve (wr) strain of vaccinia virus, and at low doses of mva, lower morbidity was recorded in mice that were vaccinated via the percutaneous route than in those immunized via the intramuscular or subcutaneous routes. in addition, we show in two models that percutaneous inoculation of recombinant mva expressing heterologous antigens elicited specific immune responses, including neutralizing antibodies, at levels that are comparable to subcutaneous inoculation. a recombinant mva expressing herpes simplex virus 2 (hsv-2) glycoprotein d elicited gd2-specific igg and hsv-2 neutralizing antibodies, and a recombinant mva expressing the hemagglutinin of influenza rga/vietnam/1203/04, h5n1, elicited protective immune responses when inoculated by the percutaneous route. our data suggest that vaccination via the percutaneous route is efficient in stimulating protective immune responses, and may find clinical relevance in immunizations with mva and mva-vectored vaccines. the modified vaccinia virus ankara (mva; atcc # vr-1508) was provided by drs. linda wyatt and bernard moss (niaid/nih) and vaccinia virus strain western reserve (vv-wr; atcc # vr-1354) was provided by dr. bernard moss. we have previously described the construction of a recombinant mva expressing the herpes simplex virus type 2 glycoprotein d (mva-gd2), in which the us6 gene of hsv-2 (strain ms) was inserted into the deletion ii region in mva by homologous recombination [51] . expression of glycoprotein d is driven by the synthetic vaccinia early/late promoter [52] . recombinant mva expressing the hemagglutinin (h5) of influenza virus rga/viet nam/1203/2004 (h5n1) (mva-ha) was constructed by homologous recombination of the h5 gene into the deletion iii region of mva [53] . expression of the influenza h5 is driven by the vaccinia h5r promoter. mva and recombinant mva vectors were prepared from primary chick embryo fibroblast cells (cef) or df-1 cells (atcc # crl-12203) that had been infected with the appropriate virus as previously described [54] , and partially purified viruses were obtained by passing infected cell lysates through a 36% sucrose cushion. mva and mva recombinants were titered on df-1 cell monolayers. vv-wr was prepared from infected bsc-1 cells (atcc # ccl26) , and was also partially purified through a 36% sucrose cushion, and titered on bsc-40 cell monolayers (atcc # crl-2761). the attenuated influenza virus rga/viet nam/1203/2004 (h5n1) was grown in the allantoic cavity of embryonated eggs and titrated on mdck cells. cells were cultivated and regularly maintained in dmem containing 10% fetal bovine serum (5% for mdck cells) and 5μg/ml gentamicin. balb/c mice (4-5 weeks old) were obtained from the jackson laboratories, bar harbor, maine. mice received feed and water freely. the animal study protocol for the work described in this manuscript was approved by the institutional animal care and use committee (iacuc) of the center for biologics evaluation and research, usfda (cber/fda). intramuscular inoculation into the muscles of the hind legs and subcutaneous inoculation at the base of the tail using 1ml tuberculin syringes affixed with 25-gauge needles, were performed as previously described [17] . tail scarification of anesthetized animals was also performed as previously described [17] . briefly, the inoculum was diluted in sterile endotoxin-free phosphate-buffered saline (pbs) such that the desired unit dose is contained per 2μl. mice were anesthetized by intraperitoneal injection of 1x avertin (2,2,2,-tribromoethanol in tert-amyl alcohol (sigma-aldrich, st. louis, missouri)) diluted in pbs at 20 μl per gram body weight. using a 25-gauge needle, 15 to 20 needle scratches/puncture were made at the base of the tail and 2μl of the inoculum was applied to the scarified surface. vaccinia virus (strain wr) and attenuated influenza a virus (rga/viet nam/1203/2004 (h5n1)), a 6+2 reassortant vaccine strain bases on influenza a/puerto rico/8/34, were used in challenge experiments. for intranasal mouse challenge, a dose of ten 50% lethal dose (10 ld 50 ) (equivalent to 1.5 x 10 5 pfu) of vv-wr was used. for challenge with rga/viet nam/1203/2004 (h5n1)), a dose of 10 6 pfu was used. we previously determined this dose to be lethal in our influenza virus challenge model. mice were anesthetized by intraperitoneal injection of 1x avertin (20 μl per gram body weight), followed by inoculation of the challenge virus in a total of 20 μl suspension into the nares (10μl per naris) as previously described [17] . mice were monitored and weighed daily for two weeks post challenge. mice that lost 25% of their initial body weight were considered to have reached the study endpoint, and were humanely euthanized per the iacuc-approved animal study protocol. testing of antisera for antigen-specific antibodies (igg) was performed by standard enzymelinked immunosorbent assay (elisa). elisa for vaccinia-specific antibodies was performed using psoralen/uv-inactivated vaccinia virus (dryvax) as the antigen, as previously described [17] . hsv-2 gd-specific igg elisa using partially-purified hsv-2 glycoproteins (50 ng/well) as the antigen, and hsv-2 neutralization assay were performed in 96-well immulon 2 hb plates (fisher scientific) and 96-well tissue culture plates (corning), respectively, as previously described [51] . influenza virus h5-specific igg was quantified using sanofi pasteur's inactivated rga/viet nam/1203/2004 vaccine (cber reference antigen #50) as the coating antigen. immulon 2 hb plates were coated with cber reference antigen at 1 μg/well, and plates were stored at 4°c overnight. plates were washed with phosphate-buffered saline containing 0.5% tween-20 (pbst), and then blocked with pbst containing 10% fetal bovine serum for 2 hours. subsequent incubation of the antigen with the test serum samples and completion of the assay followed standard elisa protocol as previously described [51] . testing of antisera for the neutralization of influenza a virus rga/viet nam/1203/2004 (h5n1), an attenuated vaccine strain generated by reverse genetics, was performed using a microneutralization assay as previously described [55] . the presence of virus was detected using biotin-conjugated antibody to influenza np (clone a; milipore, billerica, ma, usa) followed by hrp-labeled streptavidin (kpl, gaithersburg, md, usa). mice were vaccinated with 10 7 pfu of mva-gd2 via the subcutaneous or percutaneous route. a control group was vaccinated with 10 7 pfu of mva. seven (7) days after vaccination, mice were euthanized, spleens were collected from dissected mice, and lymphocytes were isolated from spleen homogenates as previously described [51] . spleen cells were cultured in 24-well tissue culture plates and re-stimulated with live hsv-2 (strain ms) at a multiplicity of infection of 1.0. culture supernatants were harvested after 48 hours, clarified of cells by centrifugation, and tested for il-2 and ifn-γ by capture elisa as described [51] , and reagents obtained from bd pharmingen. unpaired, two-tailed student's t-test for statistical comparison of antibody titers was performed using graphpad prism 6.02 software (graphpad software, inc.). the kaplan-meier survival logrank test was performed using the sigmaplot 13.0 software (systat software, inc.), and was used to compare differences in the number of surviving animals in the various treatment groups after virus challenge. in all cases, a p value <0.05 indicates statistically significant differences between treatment groups. in a preliminary experiment to investigate the utility of the percutaneous route for the delivery of mva, we observed that mva delivered by tail scarification, while statistically insignificant (p = 0.298), elicited a higher vaccinia-specific igg response and protection in mice than the same dose (10 6 pfu) delivered by the intramuscular route (s1 fig) . the experiment using 10 6 pfu of mva did not allow us to observe any differences in antibody response, disease progression and protection between the two immunization routes. thus in subsequent experiments, lower doses of mva were evaluated. groups of mice were vaccinated with 10 5 pfu of mva via the intramuscular, subcutaneous, or percutaneous routes. for comparison, a set of three groups of mice were similarly vaccinated with 10 5 pfu of the licensed acam2000 smallpox vaccine, via the same three routes. an untreated group was included as a control. three weeks after vaccination, serum samples were obtained from all mice and tested for vaccinia-specific antibodies by elisa, using inactivated dryvax as the antigen. among mice in the mva treatment cohort, an igg response was detectable in 2/5 mice in the subcutaneous group, and 1/5 in the percutaneous group. the untreated mice and all mice in the mva intramuscular group had no detectable igg at this time point. by contrast, all but three mice (2/5 and 1/5 in the intramuscular and subcutaneous groups, respectively) in the acam2000 cohort had detectable levels of igg (fig 1a) . at 4 weeks postimmunization, all mice were challenged intranasally, with a lethal dose (10 ld 50 ) of vv-wr. all mice in the untreated group showed severe symptoms of infection, reaching the study endpoint of 25% weight loss by day 7 post-challenge, and had to be euthanized (fig 1b & 1c) . except for a mouse in the mva-intramuscular group, all other mice vaccinated with mva or acam2000, survived. among the mva vaccinated animals, mice in the intramuscular group lost the most weight (fig 1b) , with a mean peak loss of about 16% on day 9 post-challenge. weight loss among the mva subcutaneous and percutaneous groups were similar, with peak losses of 9.6% (day 6) and 8.9% (day 7), respectively. however, mice in the mva percutaneous group recovered more quickly (98.4% of original mean body weight on day 14) than the subcutaneous group (94.4% mean weight on day-14). among the acam2000 treatment groups ( fig 1c) , the average peak weight loss was 8.8% (day-5), 7.7% (day-6), and 6.2% (day-6), for the intramuscular, subcutaneous, and percutaneous groups, respectively. in another experiment, mice in groups of five were vaccinated with 10 3 pfu or 10 5 pfu of mva by scarification. two other groups were similarly vaccinated with 10 3 pfu or 10 5 pfu of acam2000, and a control group was scarified with pbs. antisera were collected after three weeks and mice were challenged with 10 ld 50 of vv-wr. none of the mice in the pbs group had detectable igg and all succumbed to vv-wr infection ( table 1 ). all mice in the 10 3 pfu mva group had no detectable igg and 2/5 in the 10 5 mva group had detectable igg. however, 1/5 and 5/5 of mice survived in the 10 3 pfu and 10 5 pfu mva, respectively. in the acam2000 cohort, 2/5 and 5/5 of mice in the 10 3 pfu and 10 5 pfu groups, respectively, were seropositive. three of five (3/5) and . morbidity, as measured by mean weight changes post-challenge, is shown for the mva treatment groups (b), and for the acam2000 treatment groups (c). the "untreated" control group was the same for both the mva and acam2000 groups. a "+" sign represents a mouse that succumbed to infection. 5/5 of mice in the acam2000 cohort survived vv-wr challenge (table 1) . these sets of data suggest that in this mouse model, mva inoculation by the percutaneous route elicits equivalent or greater protective immune responses than inoculation via the intramuscular or subcutaneous routes. percutaneous vaccination with recombinant mva-gd2 elicits hsv-2-specific immune responses mva is an attractive vector being used for the expression of transgenes and has been used in the expression of antigens of a variety of pathogens. similar to studies investigating mva as a smallpox vaccine, preclinical and clinical evaluation of mva-vectored vaccines in development has relied predominantly on the use of intramuscular, intradermal or subcutaneous routes of mva delivery. in order to determine whether the percutaneous route will be useful for the evaluation of mva-vectored recombinant vaccines, a recombinant mva (mva-gd2) expressing the glycoprotein d of herpes simplex virus type-2 (hsv-2, strain ms) was evaluated for immunogenicity in the balb/c mouse model. in this recombinant mva, the hsv-2 us6 gene encoding glycoprotein d was inserted into the deletion ii site in mva by homologous recombination [51] . groups of mice were vaccinated with mva-gd2 at three dose levels: 10 5 pfu, 10 6 pfu, and 10 7 pfu; with each dose level administered subcutaneously or percutaneously. the control group received 10 7 pfu of mva subcutaneously. all treatment groups were vaccinated using a prime/boost immunization strategy as previously described [51] . antisera were collected 3 weeks after the priming vaccination, as well as at 3 weeks after the boost, and were tested for hsv-2-specific igg at both time points, using partially-purified hsv-2 glycoproteins as the antigen (fig 2a) . antisera collected at the 6-week time point were also tested for hsv-2 neutralizing antibodies (fig 2b) . antisera obtained from mice vaccinated with the mva vector had no detectable hsv-2 specific antibodies at both time points. hsv-2 specific antibodies were detected in antisera obtained from mice vaccinated with mva-gd2 at both time points (fig 2a) . the mean igg titers (log 10 the serum samples obtained at the 6-week time point were tested for the ability to neutralize hsv-2 infectivity in vero cells. similar to the igg elisa result, antisera from the mva control group did not neutralize hsv-2 (mean serum neutralization (sn) titer was below the level of quantitation of 0.30 log 10 ). low to modest neutralizing antibody titers were detected in mva-gd2 vaccinated mice (table 2 ). in the subcutaneous vaccination sub-groups, mean sn titers (log 10 ) of 0.96 (±0.13), 1.08 (±0.46), and 2.41 (±0.43), were obtained for the 10 5 pfu, 10 6 pfu, and 10 7 pfu groups, in another experiment, cellular immune response to mva-gd2 was evaluated. mice were vaccinated with 10 7 pfu of mva subcutaneously, or with 10 7 pfu of mva-gd2 either subcutaneously or percutaneously. spleen cells were harvested from mice 7 days after vaccination and tested for cytokine (il-2 and ifn-γ) secretion as previously described (meseda et al., 2002) . spleen cells were re-stimulated in vitro by live infection with hsv-2 (strain ms) at a multiplicity of infection of 1.0. supernatants were collected from the cultured spleen cells and tested for levels of il-2 and ifn-γ. whereas the levels of il-2 and ifn-γ in the supernatants from the spleen of mva-infected mice were below detection, all mice in both mva-gd2 groups had detectable levels of il-2 and ifn-γ, ( table 2 ). mean il-2 levels of 40.3 ± 21 pg/ml and 52.3 ± 40.1 pg/ml were obtained for the mva-gd2 subcutaneous and percutaneous groups, respectively. similarly, ifn-γ levels were 28.5 ± 17.4 pg/ml, and 82.3 ± 114.3 pg/ml, for the subcutaneous and percutaneous mva-gd2 groups, respectively. taken together, this set of data suggests that the inoculation of recombinant mva-gd2 by scarification is capable of eliciting antigen-specific immune responses that are comparable or higher than delivery by subcutaneous inoculation. mva is used as a vector for the expression of heterologous antigens. the deletion sites in mva, including the deletion ii and deletion iii sites, are commonly used for the insertion of transgenes. in order to broaden our understanding of the utility of the percutaneous route for the delivery of mva-vectored vaccines, we further evaluated a recombinant mva, mva-ha, in which the hemagglutinin gene of influenza a virus rga/viet nam/1203/2004 (h5n1), was inserted in the deletion iii site of mva. in a series of experiments, the antibody response following vaccination via the subcutaneous or percutaneous routes was characterized, and the protective effectiveness of vaccination via these routes was evaluated in a mouse intranasal challenge model. in the first experiment, groups of mice (five per group) were vaccinated with 10 5 , 10 6 , or 10 7 pfu of mva-ha on a prime-boost schedule at an interval of 3 weeks between vaccinations. each dose of mva-ha was administered via the subcutaneous or percutaneous routes. a control group was vaccinated with 10 7 pfu of mva via the subcutaneous route. three weeks after vaccination, serum samples were obtained from mice and tested for h5-specific igg, and all mice were challenged with 10 6 pfu of attenuated influenza rga/viet nam/1203/2004 (h5n1) via the intranasal route. except for a mouse in the mva control group that had a low level of non-specific igg, all other mice in the control group had no h5-specific igg, and 4/5 (including the one with detectable igg) succumbed to influenza virus challenge. all mice that were vaccinated with mva-ha, irrespective of the route, had high levels of h5-specific igg titers in the second experiment, mice in groups of mice (five per group) were vaccinated with 10 4 , 10 5 , or 10 6 pfu of mva-ha via the subcutaneous or percutaneous route on a prime-boost schedule at an interval of 3 weeks between vaccinations. a control group was vaccinated with 10 6 pfu of mva, subcutaneously. antisera were obtained from mice three weeks after the booster vaccination, and all mice were challenged with 10 6 pfu of influenza rga/viet nam/ 1203/2004. all mice in the mva group had no detectable h5-specific igg. by contrast, all mice inoculated with mva-ha, irrespective of the dose, had high titers of h5-specific igg (fig 3a) . the differences in igg levels between the subcutaneous and percutaneous cohorts at each dose level of mva-ha were not statistically significant, although mean igg titers were slightly higher in the subcutaneous cohort. following intranasal challenge with influenza rga/viet nam/1203/2004, all mice in the mva group succumbed to infection, and all mice vaccinated with mva-ha, irrespective of the dose, survived with varying degrees of morbidity. among mva-ha vaccinated mice, the 10 4 pfu/subcutaneous group recorded the severest weight loss (fig 3b) , and the difference in weight changes between the subcutaneous and percutaneous groups vaccinated with 10 4 pfu was statistically significant (two-tailed p-value = <0.001). a summary of the neutralizing antibody titers of pooled antisera for each treatment group from the two mva-ha experiments described above is presented in table 3 . the mva control group had no detectable neutralizing antibody in the serum samples obtained at any time point. among the groups vaccinated with mva-ha, neutralizing antibody was below detection in post-prime antisera, except in the 10 7 pfu subcutaneous group where a titer of 10 was obtained. however, following the administration of booster inoculations, all mva-ha treatment groups had detectable levels of neutralizing antibody that increased with increase in vaccine dose. neutralizing antibody titers in mice in the subcutaneous cohort were 15, 40, and 80, for the 10 4 , 10 5 , and 10 6 pfu, respectively. similarly, neutralizing antibody titers among the percutaneous cohort were 15, 60, and 80, for the 10 4 , 10 5 , and 10 6 pfu, respectively. interestingly, the levels of neutralizing antibody for the percutaneous treatment groups were similar to the subcutaneous group at the same mva-ha dosage, in spite of slightly higher total h5-specific igg levels in the subcutaneous cohort (difference is not statistically significant). this set of data suggests that the antibody response, both total igg and neutralizing antibodies, elicited by mva-ha was comparable between mice that were inoculated via the subcutaneous route and those inoculated via the percutaneous route. further, we observed that all mice inoculated with 10 4 pfu of mva-ha by prime-boost, were protected from influenza virus challenge. in the experiments with mva-ha described above, all mice that were vaccinated on a primeboost schedule with 10 4 pfu of mva-ha elicited antibody responses and protection that were indistinguishable between subcutaneous and percutaneous treatment cohorts. in order to further scrutinize the differences between the two inoculation routes, lower doses of mva-ha were used in experiments. in one experiment, groups of mice (five per group) were vaccinated with mva-ha at doses of 10 2 , 10 3 or 10 4 pfu, each dose administered via the subcutaneous or percutaneous route. a control group was inoculated subcutaneously with 10 4 pfu of mva. booster inoculations in the same amount of mva-ha or mva were administered three weeks after mice were primed. at three weeks after the booster doses, mice were challenged with attenuated rga/viet nam/1203/2004, at 10 6 pfu per mouse, and were evaluated daily for two weeks as described above. none of the animals vaccinated with mva survived. all mice vaccinated with 10 3 pfu mva-ha in this prime/boost schedule survived. in the 10 2 pfu mva-ha group, 2/5 and 1/5 survived in the subcutaneous and percutaneous groups, respectively. influenza virus pathogenesis in these mice, as measured by weight loss (fig 4) shows a doseresponse with respect to mva-ha, with weight loss being inversely proportional to the dose of mva-ha. there were no major differences in survival rates between mice in the subcutaneous and percutaneous groups inoculated with the same dose of mva-ha. at 10 2 pfu of mva-ha, the mean weight loss was higher in the percutaneous group than the subcutaneous group, but the difference was not statistically significant (two-tailed p-value = 0.084). finally, in two independent experiments, mice in groups of five in each experiment were vaccinated with low doses of mva-ha that were administered once, and were challenged three weeks after vaccination. groups of mice were inoculated at doses of 10 2 , 10 3 , or 10 4 pfu of mva-ha subcutaneously or percutaneously. control groups received 10 4 pfu of mva subcutaneously. a summary of the number of surviving mice is presented in table 4 . none of the mice vaccinated with mva survived. among mice in the mva-ha subcutaneous vaccination cohort, 3/10, 5/10, and 8/10 survived in the 10 2 pfu, 10 3 pfu, and 10 4 pfu vaccination groups, respectively. similarly, among mice in the percutaneous vaccination cohort, 5/10, 6/10, and 8/ 10 survived viral challenge. the mean weight loss among mice in these experiments are shown in fig 5. statistical comparisons of the number of surviving mice show that differences in survival between mice that were vaccinated with 10 4 pfu of mva-ha (irrespective of vaccination route) and the mva control group, were statistically significant (table 4 ). however, a comparison of the observed differences in survival between the percutaneous and subcutaneous groups at the 10 2 pfu dose level indicates the difference is not statistically significant. this set of data suggests that even at low vaccination doses, differences between mouse groups vaccinated via the subcutaneous and percutaneous routes are not apparent in this challenge model. the modified vaccinia virus ankara (mva) is licensed in europe and canada as a third generation smallpox vaccine, and currently in clinical development for licensure in the united states. the relatively better safety record of mva compared to first and second generation smallpox vaccines is well documented. this, in addition to its large capacity to accommodate heterologous genes, express encoded proteins, and elicit both humoral and cell-mediated immune responses also makes mva an attractive vector for the delivery of several candidate vaccines for a variety of infectious and non-infectious human and veterinary diseases [56, 57, 58, 59, 60] . evidence for the delivery of antigens through the skin in asia dates back to the 1500s with the practice of variolation and continued with the advent of the smallpox vaccine in the late 18 th century [3] . thus replication-competent smallpox vaccines, including those that were used in the successful eradication of smallpox, such as dryvax, lister, livp, temple of heaven, and em-63, were mostly administered by skin scarification [61] . the current us-licensed secondgeneration smallpox vaccine, acam2000, is also administered by skin scarification, a procedure that is believed to be partly responsible for the success of the global eradication of smallpox by provoking robust innate and adaptive immune responses [62] . due to the severe attenuation of mva, as epitomized by its inability to replicate productively in many mammalian cells [11, 12, 13] , mva and mva-vectored vaccines are usually administered via routes other than percutaneous in preclinical studies. clinical investigations of mva-vectored vaccines have mostly used intramuscular [23, 34, 35, 36] and intradermal routes [38, 39] , and to a lesser extent, the subcutaneous injection route [63] . local reactogenicity following vaccination with mva or mva-vectored vaccines is believed to be more severe with subcutaneous and intradermal inoculations than via intramuscular route [57, 63, 64] . in a comparison of the safety and immunogenicity of an mva-vectored hiv vaccine, individuals vaccinated with mva.hiva by the subcutaneous and intradermal routes were found to develop more severe local reactions than those vaccinated via the intramuscular route [63] . however, intramuscular and subcutaneous tissues have relatively fewer antigen presenting cells than the skin tissue and may not be adequate for optimal immune responses [65] . administering vaccines against infectious diseases through the skin has generated significant interest in recent years, including its use in the delivery of the bcg tuberculosis vaccine [66] , and has been further boosted by the development of the microneedle patch technology, which delivers vaccines intradermally. microneedle inoculation of vaccines has been used in preclinical evaluation of several vaccines, including inactivated polio vaccine [67] , influenza vaccine [68, 69, 70] , and measles vaccine [71] . clinical application of vaccines to the skin has also been documented for a number of vaccines, including influenza vaccine [72, 73] , and rabies vaccine [74] . recent studies have suggested that vaccine delivery through the skin takes advantage of the abundant presence of skin-resident antigen-presenting cells, including different subsets of dendritic cells and langerhans cells, as well as infiltrating antigen presenting cells, to provoke robust immune responses that include both humoral and cell-mediated immune responses [65] , and induce long-lived cd8 + t cell memory [75, 76] . moreover, data on the delivery of different types of vaccines through the skin suggest that both live vaccines and subunit vaccines can be administered through the skin, with successful immunization outcomes [67-71, 73,74] . earlier data from our laboratory [17] as well as from clinical trials [57, 77] suggest that delivery of the modified vaccinia virus ankara into the intradermal layer of the skin elicited robust immune responses that were higher than intramuscular or subcutaneous inoculations, and protected mice from intranasal challenge with vaccinia virus [17] . we further showed that a severely attenuated recombinant vaccinia virus that fails to form visible plaques in several mammalian cell lines, reminiscent of mva, elicited protective immune responses when used to vaccinate mice by scarification [46] . in the work described here, we expanded our investigation on the delivery of mva as well as mva-vectored antigens through the skin. in a preliminary experiment, we observed that igg titers were higher in mice that received 10 6 pfu of mva by skin scarification than in mice that received the same dose of mva by intramuscular inoculation. in subsequent experiments, antibody responses and protection of mice that were vaccinated with mva subcutaneously or by tail scarification were higher than in those vaccinated via the intramuscular route. melamed et al. [47] compared the antibody response elicited in response to mva and two recombinant mvas that had been genetically modified to replicate in vero and bsc-1 cells, after inoculating mice by intramuscular injection or tail scarification. their data indicate that tail scarification was efficient at inducing an antibody response, although the intramuscular route elicited higher geometric mean titers of antibody and conferred higher survival rates. the difference between their observation and the one reported here may be due to differences in experimental procedures and/or assay methods. for instance, in our work, mva for tail scarification is typically in 2 μl volume per dose, making it easier to handle than the 10μl used by melamed et al [47] . in subsequent experiments, the route comparison was limited to subcutaneous versus percutaneous routes, since the subcutaneous route is more commonly used in vaccination studies of mva vectors. as the utility of mva as a viral vector for the expression of heterologous antigens is expanding [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] 78] , we also compared the antibody responses and protection conferred by vaccination with two mva recombinants, one expressing the hsv-2 glycoprotein d, and the other expressing the h5 hemagglutinin of influenza virus rg/a viet nam/1203/2004 (h5n1). mice that were vaccinated with mva-gd2 by tail scarification, elicited higher than or similar titers of hsv-2 gd2-specific igg and neutralizing antibodies to those vaccinated by subcutaneous inoculation. the observed differences in igg titers were not statistically significant between the two routes at 10 6 or 10 7 pfu, but were statistically significant at 10 5 pfu, suggesting that the percutaneous delivery of mva-gd2 may be more effective than subcutaneous inoculation in eliciting hsv-2 neutralizing antibodies at lower vaccine doses. consistent with previous reports [48, 49] , percutaneous inoculation of mva-gd2 also elicited cell-mediated immune responses, as evident in the secretion of ifn-γ and il-2 by re-stimulated immune splenocytes. mva vectors expressing influenza antigens have been shown to elicit protective immune responses in animal models [79] , including mice [80] [81] [82] [83] , ferrets [84] and macaques [85, 86] . in the second recombinant mva vaccine model, mva-ha, expressing influenza virus h5, was used to vaccinate mice by subcutaneous injection or by tail scarification, and the h5-specific antibody response and protective effectiveness against intranasal challenge with the homologous attenuated influenza virus rga/viet nam/1203/2004, were assessed. the data indicate that comparable levels of antibody titers and protection were conferred by the two immunization routes. interestingly, higher survival rates among mice vaccinated by tail scarification were recorded at low vaccine doses. among the advantages attributed to skin delivery of vaccines is the possibility of antigen dose sparing [70, 72] . the data described in this manuscript support these earlier reports as lower doses of mva or mva-gd2 or mva-ha were found to elicits full or partial protection of mice. in summary, we showed that mva, and recombinant mva vectors expressing hsv-2 gd2 or influenza virus h5 elicited protective immune responses in the mouse model. taken together, the data presented in this work suggest that mva and mva-vectored vaccines can be effective when delivered through the skin. with the advantage that antigen delivery to the skin requires a volume that is at least 25 times (in murine models) to 200 times (in humans) less than the volume used in subcutaneous vaccination, a more comprehensive investigation of the clinical benefit of delivering mva and mva-vectored vaccines through the skin is necessary. apart from its efficiency in provoking robust immune responses, it may also help to ameliorate or obliterate some of the commonly reported volume-related local reactions associated with subcutaneous or intramuscular vaccine delivery, such as pain at the site of injection, and may be more acceptable in people with needle phobia, thus enhancing compliance with scheduled immunization programs. although the preclinical evaluation of vaccines by skin scarification, as described in our study, involves the use of improvised needles for the scarification process prior to the application of vaccines, advances in the development of the microneedle patch [87, 88, 89] should facilitate painless application of vaccines to the skin without the use of hypodermic injection needles or the bifurcated needle used in administering smallpox vaccines. in preclinical studies, microneedle delivery of mva-vectored vaccines has been shown to be effective in eliciting robust immune responses against malaria [90] that are comparable to the levels attained by intradermal vaccination. thus with further refinement, the use of microneedle delivery appears to hold a promising future for the application of viral-vectored vaccines through the skin. were vaccinated subcutaneously or percutaneously with 10 5 , 10 6 , or 10 7 pfu of mva-ha by prime-boost at an interval of 3 weeks between vaccinations. a control group received 10 7 pfu of mva prime-boost, subcutaneously. serum samples obtained 3 weeks after priming (week-3) and 3 weeks after boosting (week-6) were tested for h5-specific igg (a). error bars represent standard deviation. mice were subsequently challenged with 10 6 pfu of influenza rga/viet nam/1203/2004, and weighed daily for two weeks (b). a "+" sign represents a mouse that succumbed to infection. (tif) acam2000 clonal vero cell culture vaccinia virus (new york city board of health strain)-a second-generation smallpox vaccine for biological defense acam2000: a newly licensed cell culture-based live vaccinia smallpox vaccine cutaneous vaccination: antigen delivery into or onto the skin cardiac adverse events following smallpox vaccination-united states myopericarditis following smallpox vaccination among vaccinia-naive us military personnel incidence and followup of inflammatory cardiac complications after smallpox vaccination smallpox vaccination-associated myopericarditis is more common with the newest smallpox vaccine jennerian prophylaxis by means of intradermal injections of culture vaccine virus smallpox vaccination and its consequences: first experiences with the highly attenuated smallpox vaccine mva the smallpox vaccination strain mva: marker, genetic structure, experience gained with the parenteral vaccination and behavior in organisms with a debili modified vaccinia virus ankara undergoes limited replication in human cells and lacks several immunomodulatory proteins: implications for use as a human vaccine host range and cytopathogenicity of the highly attenuated mva strain of vaccinia virus: propagation and generation of recombinant viruses in a nonhuman mammalian cell line highly attenuated modified vaccinia virus ankara replicates in baby hamster kidney cells, a potential host for virus propagation, but not in various human transformed and primary cells highly attenuated smallpox vaccine protects mice with and without immune deficiencies against pathogenic vaccinia virus challenge immunogenicity of a highly attenuated mva smallpox vaccine and protection against monkeypox modified vaccinia virus ankara protects macaques against respiratory challenge with monkeypox virus enhanced immunogenicity and protective effect conferred by vaccination with combinations of modified vaccinia virus ankara and licensed smallpox vaccine dryvax in a mouse model 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clinical trial of mva.hiva vaccine administered to infants born to human immunodeficiency virus type 1-positive mothers in nairobi safety and efficacy of mva85a, a new tuberculosis vaccine, in infants previously vaccinated with bcg: a randomised, placebo-controlled phase 2b trial safety and immunogenicity of a candidate tuberculosis vaccine mva85a delivered by aerosol in bcg-vaccinated healthy adults: a phase 1, double-blind, randomised controlled trial safety, immunogenicity, and efficacy of the candidate tuberculosis vaccine mva85a in healthy adults infected with hiv-1: a randomised, placebo-controlled, phase 2 trial a phase ia study to assess the safety and immunogenicity of new malaria vaccine candidates chad63 cs administered alone and with mva cs a human phase i/ iia malaria challenge trial of a polyprotein malaria vaccine evaluation of the efficacy of chad63-mva vectored vaccines expressing circumsporozoite protein and me-trap against controlled human malaria infection in malaria-naive individuals immune therapy for human papillomaviruses-related cancers a human vaccine strategy based on chimpanzee adenoviral and mva vectors that primes, boosts, and sustains functional hcv-specific t cell memory efficacy of immunotherapy with tg4040, peg-interferon, and ribavirin in a phase 2 study of patients with chronic hcv infection safety and immunogenicity of novel respiratory syncytial virus (rsv) vaccines based on the rsv viral proteins f, n and m2-1 encoded by simian adenovirus (panad3-rsv) and mva (mva-rsv); protocol for an open-label, doseescalation, single-centre, phase 1 clinical trial in healthy adults a t cell-inducing influenza vaccine for the elderly: safety and immunogenicity of mva-np+m1 in adults aged over 50 years safety and immunogenicity of a modified-vaccinia-virus-ankara-based influenza a h5n1 vaccine: a randomised, double-blind phase 1/2a clinical trial induction of influenza (h5n8) antibodies by modified vaccinia virus ankara h5n1 vaccine a recombinant modified vaccinia ankara vaccine encoding epstein-barr virus (ebv) target antigens: a phase i trial in uk patients with ebv-positive cancer phase i trial of recombinant modified vaccinia ankara encoding epstein-barr viral tumor antigens in nasopharyngeal carcinoma patients use of chad3-ebo-z ebola virus vaccine in malian and us adults, and boosting of malian adults with mva-bn-filo: a phase 1, single-blind, randomised trial, a phase 1b, open-label and double-blind, dose-escalation trial, and a nested, randomised, double-blind, placebo-controlled trial high, broad, polyfunctional, and durable t cell immune responses induced in mice by a novel hepatitis c virus (hcv) vaccine candidate (mva-hcv) based on modified vaccinia virus ankara expressing the nearly full-length hcv genome a novel poxvirus-based vaccine, mva-chikv, is highly immunogenic and protects mice against chikungunya infection comparative assessment of vaccine vectors encoding ten malaria antigens identifies two protective liver-stage candidates safety and immunogenicity of imvamune, a promising candidate as a third generation smallpox vaccine needle-free vaccine delivery effect of the deletion of genes encoding proteins of the extracellular virion form of vaccinia virus on vaccine immunogenicity and protective effectiveness in the mouse model attenuation and immunogenicity of host-range extended modified vaccinia virus ankara recombinants epidermal injury and infection during poxvirus immunization is crucial for the generation of highly protective t cell-mediated immunity microneedle array design determines the induction of protective memory cd8+ t cell responses induced by a recombinant live malaria vaccine in mice dry-coated live viral vector vaccines delivered by nanopatch microprojections retain long-term thermostability and induce transgene-specific t cell responses in mice prime-boost immunization with dna and modified vaccinia virus ankara vectors expressing herpes simplex virus-2 glycoprotein d elicits greater specific antibody and cytokine responses than dna vaccine alone compact, synthetic, vaccinia virus early/late promoter for protein expression production and characterization of mammalian virus-like particles from modified vaccinia virus ankara vectors expressing influenza h5n1 hemagglutinin and neuraminidase preparation of cell cultures and vaccinia virus stocks detection of antibody to avian influenza a (h5n1) virus in human serum by using a combination of serologic assays protective efficacy of recombinant modified vaccinia virus ankara delivering middle east respiratory syndrome coronavirus spike glycoprotein safety and immunogenicity of modified vaccinia ankara (acam3000): effect of dose and route of administration cardiac safety of modified vaccinia ankara for vaccination against smallpox in a young, healthy study population efficacy assessment of an mva vectored rift valley fever vaccine in lambs evaluation of mva-5t4 as a novel immunotherapeutic vaccine in colorectal, renal and prostate cancer smallpox vaccines for biodefense protective properties of vaccinia virus-based vaccines: skin scarification promotes a nonspecific immune response that protects against orthopoxvirus disease studies of a prophylactic hiv-1 vaccine candidate based on modified vaccinia virus ankara (mva) with and without dna priming: effects of dosage and route on safety and immunogenicity comparing the safety and immunogenicity of a candidate tb vaccine mva85a administered by intramuscular and intradermal delivery microneedle-mediated vaccine delivery: harnessing cutaneous immunobiology to improve efficacy efficacy of percutaneous versus intradermal bcg in the prevention of tuberculosis in south african infants: randomised trial inactivated polio vaccination using a microneedle patch is immunogenic in the rhesus macaque enhanced immune responses by skin vaccination with influenza subunit vaccine in young hosts delivery of subunit influenza vaccine to skin with microneedles improves immunogenicity and long-lived protection dose sparing enabled by skin immunization with influenza virus-like particle vaccine using microneedles measles vaccination using a microneedle patch dose sparing with intradermal injection of influenza vaccine comparative immunogenicity of trivalent influenza vaccine administered by intradermal or intramuscular route in healthy adults long-term anti-rabies antibody persistence following intramuscular or lowdose intradermal vaccination of young vietnamese children skin infection generates non-migratory memory cd8+ t(rm) cells providing global skin immunity skin vaccination with live virus vectored microneedle arrays induce long lived cd8(+) t cell memory comparison of lyophilized versus liquid modified vaccinia ankara (mva) formulations and subcutaneous versus intradermal routes of administration in healthy vaccinia-naive subjects modified vaccinia virus ankara as antigen delivery system: how can we best use its potential? candidate influenza vaccines based on recombinant modified vaccinia virus ankara recombinant modified vaccinia virus ankara-based vaccine induces protective immunity in mice against infection with influenza virus h5n1 vaccinia virus-based multivalent h5n1 avian influenza vaccines adjuvanted with il-15 confer sterile cross-clade protection in mice vectors based on modified vaccinia ankara expressing influenza h5n1 hemagglutinin induce substantial cross-clade protective immunity broad protection against avian influenza virus by using a modified vaccinia ankara virus expressing a mosaic hemagglutinin gene evaluation of a modified vaccinia virus ankara (mva)-based candidate pandemic influenza a/h1n1 vaccine in the ferret model preclinical evaluation of a modified vaccinia virus ankara (mva)-based vaccine against influenza a/h5n1 viruses modified vaccinia virus ankara encoding influenza virus hemagglutinin induces heterosubtypic immunity in macaques coated microneedles for transdermal delivery microneedle-based vaccines coated microneedle arrays for transcutaneous delivery of live virus vaccines microneedle array design determines the induction of protective memory cd8+ t cell responses induced by a recombinant live malaria vaccine in mice the authors wish to thank drs. bernard moss and linda wyatt, niaid/nih, for the mva and vv-wr viruses. we also thank dr. maryna eichelberger and dr. alonzo garcia, cber/ fda, for reviewing this manuscript. this work was supported by intramural research funds key: cord-001546-ndz3oarf authors: ayithan, natarajan; bradfute, steven b.; anthony, scott m.; stuthman, kelly s.; bavari, sina; bray, mike; ozato, keiko title: virus-like particles activate type i interferon pathways to facilitate post-exposure protection against ebola virus infection date: 2015-02-26 journal: plos one doi: 10.1371/journal.pone.0118345 sha: doc_id: 1546 cord_uid: ndz3oarf ebola virus (ebov) causes a severe hemorrhagic disease with high fatality. virus-like particles (vlps) are a promising vaccine candidate against ebov. we recently showed that vlps protect mice from lethal ebov infection when given before or after viral infection. to elucidate pathways through which vlps confer post-exposure protection, we investigated the role of type i interferon (ifn) signaling. we found that vlps lead to accelerated induction of ifn stimulated genes (isgs) in liver and spleen of wild type mice, but not in ifnar(-/-) mice. accordingly, ebov infected ifnar(-/-) mice, unlike wild type mice succumbed to death even after vlp treatment. the isgs induced in wild type mice included anti-viral proteins and negative feedback factors known to restrict viral replication and excessive inflammatory responses. importantly, proinflammatory cytokine/chemokine expression was much higher in wt mice without vlps than mice treated with vlps. in ebov infected ifnar(-/-) mice, however, uninhibited viral replication and elevated proinflammatory factor expression ensued, irrespective of vlp treatment, supporting the view that type i ifn signaling helps to limit viral replication and attenuate inflammatory responses. further analyses showed that vlp protection requires the transcription factor, irf8 known to amplify type i ifn signaling in dendritic cells and macrophages, the probable sites of initial ebov infection. together, this study indicates that vlps afford post-exposure protection by promoting expeditious initiation of type i ifn signaling in the host. ebola viruses (ebovs) are enveloped, negative-sense rna filoviruses that can cause a severe hemorrhagic fever in humans and non-human primates (nhps) [1, 2] . mouse-adapted ebov causes similar acute disease in mice, offering a useful animal model to study ebov infection [3, 4] . ebov infection is characterized by rapid viral replication and dysregulated innate and adaptive immune responses. the disease follows profound suppression of type i ifn signaling and a contrasting excess inflammation that leads to mucosal hemorrhages and multi-organ failure resembling septic shock syndrome [5, 6] . virally encoded anti-ifn proteins, vp24 and vp35 play major roles in ebov virulence [7, 8] . vp35 blocks type i ifn induction in dendritic cells (dcs) and macrophages, and acts as a virulence factor necessary for a recombinant virus to attain infectivity in the host [9] [10] [11] [12] . vp24, on the other hand, blocks ifn signaling by interfering with ifn activated jak/stat pathways [7] . lines of evidence support the critical importance of type i ifn signaling in providing resistance against ebov infection; mice deficient in stat1, a transcription factor required for ifn induction, or ifnar1, encoding the membrane receptor for type i ifns, are susceptible to wild type zaire ebov, against which wild type mice are resistant [13] [14] [15] . a study of sudan ebov infection in humans showed that ifnα levels are significantly higher in surviving patients than those with fatal ebov infection, who had higher levels of proinflammatory cytokines/chemokines such as il-6, and mip-1β [16, 17] . high ifnα production is reported to correlate with increased resistance against ebov in mice as well [18] . administration of recombinant ifnα or ifnβ confers delayed time-to-death in nhps [19, 20] . furthermore, ifnα, used as an adjunct therapy for monoclonal antibody treatment, is shown to enhance protection in nhps [21] . ebov infection remains a potential threat to public health, which is compounded with the lack of effective prevention or treatment. to overcome this problem, various vaccine candidates have been developed, including various dna constructs, recombinant viruses, vlps, as well as treatment with anti-sense sirna [22] [23] [24] . vlps are subunit-based vaccines, extensively studied for a variety of infectious pathogens [25, 26] . vlps prepared from ebov and other filoviruses are composed of the matrix protein (vp40), glycoprotein (gp), and at times nucleoprotein (np) and represent a potentially promising candidate for ebov vaccine. ebov vlps have been shown to confer protection upon rodents and nhps when given prior to infection [27] [28] [29] . in the accompanying paper, we show that post-exposure administration of trivalent vlps protects mice from lethal ebov infection, further crediting the potential of vlps as a possible vaccine [30] . in that study, we show that vlp protection requires macrophages, dendritic cells (dcs) as well as b and either cd4 or cd8 t lymphocytes, indicating that both innate and adaptive immunity are involved in conferring protection. the aim of this study was to further investigate molecular bases of postexposure protection by vlps. based on our previous report that vlps stimulate type i ifn expression in dcs and macrophages, in vitro, we focused on the role of type i ifn signaling, and found that post-exposure vlp treatment leads to accelerated activation of ifn signaling, resulting in early induction of isgs. significantly, vlp stimulated isg induction coincided with the attenuation of proinflammatory cytokine surge in ebov infected mice. the reduced inflammatory responses was attributed to activation of type i ifn signaling, since vlp treated ifnar -/mice were unable to inhibit not only viral replication but proinflammatory responses, and succumbed to death. our results indicate that early type i ifn response is a major mechanism that contributes to vlp mediated protection against ebov infection. ifnar -/-, ifnar +/+ mice of balb/c background and irf8 -/and irf8 +/+ mice of c57bl/6 background were bred in the nichd animal facility and transferred to the facility of the united states army medical research institute of infectious diseases (usamriid) for ebov infection studies. research was conducted in compliance with the animal welfare act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the guide for the care and use of laboratory animals, national research council, 1996. the facility where this research was conducted is fully accredited by the association for assessment and accreditation of laboratory animal care international. the iacuc committee approving this protocol is the united states army medical research institute of infectious diseases (usamriid) iacuc. animals were monitored at least once daily and their status was evaluated according to an intervention score sheet approved by usamriid iacuc. monitoring increased to three times daily if the animals were given a score of three or four. euthanization was by co 2 inhalation followed by confirmatory cervical dislocation. analgesics and anesthetics were not used in this study and animals were euthanized for humane purposes if they reached a score of five or more, which would be indicated if the animals exhibited ruffled fur, weakness, unresponsiveness, and/or difficulty walking. otherwise, animals were euthanized at the end of the study. vlps were composed of ebov gp, np and vp40 and were generated in mammalian 293t cells as reported previously [31] . vlp preparations used in this study contained <0.03 endotoxin u/mg. mice were infected with~1000 pfu (~3,000 ld 50 ) of mouse-adapted ebov via intraperitoneal (i.p.) route [32] . mice were injected with vlps (50 μg) diluted in pbs through i.p. 24 h after ebov infection. morbidity and mortality of ebov infected mice were monitored twice daily for up to 14 days. total rna from liver and spleen of ebov infected mice were extracted by trizol method (invitrogen) and cdna was synthesized from 1 μg total rna by superscript ii reverse transcriptase (invitrogen). qpcr amplification was done with 3 ng cdna in 5 μl sybr green pcr master mix (applied biosystems) with 3 μm of both reverse and forward primers used in the abi prism 7500 sequence detection system (applied biosystems). mrna of expression of indicated genes were analyzed as described in detail elsewhere [33] . the primer pairs were used for ebov gp, 5'-tgggctgaaaactgctacaatc-3' and 5'-ctttgtgcacataccggcac-3'; nlrp3, 5'-tgctcttcactgctatcaagccct-3' and 5'-acaagcctttgctccagaccctat-3'. all other gene primer sequences were followed from the previous publications [10, 33] . transcript levels were normalized with hprt, and expressed as relative expression. statistical analysis was carried out by excel software using two-tail paired student's t test. data represent the mean of at least three independent assay ± sem. a p value < 0.05 was considered significant. to assess the role of type i ifn signaling in vlp-mediated protection against ebov infection, we tested ifnar1 -/mice for protection by vlps. in fig. 1a , wild type (wt) and ifnar1 -/mice (both balb/c background, n = 10) were injected with 50 μg of vlps 24h after infection by the mouse adapted (ma) ebov, and the morbidity and mortality were checked daily for the subsequent 14 days. wt mice without vlp injection all died between day 5 and day 7, whereas 80% of mice that received vlps survived after ebov infection, confirming that vlps protect mice even when they were given post-infection. in contrast, ifnar -/mice that received vlps all died before or at day 5 as those without vlp injection (fig. 1a) . these results are in agreement with previous report on early death of ma-ebov infected ifnar -/mice [15] . ebovs are thought to initially infect dcs and macrophages in liver and spleen, making these tissues the major sites of ebov replication in the mouse, although the virus infects many other organs later [3, 34] . to ascertain whether vlps inhibit viral replication, we measured ebov glycoprotein (gp) mrna expression in liver and spleen from wt and ifnar -/mice with or without vlp administration. qrt-pcr analysis in fig. 1b and 1c, found that levels of gp mrna rose sharply in ifnar -/mice on day 2 of infection when gp mrna was still at background in wt mice. ifnar -/mice that received vlps also expressed considerable amounts of gp mrna, although levels varied between liver and spleen in the vlp treated group. thus, ebov appeared to replicate faster and to a greater extent in ifnar -/mice than wt mice. it should be noted here that in wt mice, gp mrna levels began to increase rapidly after day 2, peaking on day 3 to day 5, and that vlp injection inhibited gp mrna expression by more than half (s1 fig.) . 1000 pfu ma ebov, followed 24 h later by injection i.p. with 50 μg of ebov vlps. one group of ifnar +/+ (wt) mice infected with ebov without vlp served as control. mortality is expressed as percent survival of each group on indicated days. results are a representative of three independent experiments, which gave very similar outcomes. qrt-pcr detection of ebov gp mrna level on day 2 post-ebov infection with or without vlps from liver (b) and spleen (c) of wt or ifnar -/mice. gp transcripts were normalized by hprt and values represent the mean ± sem of duplicate samples from three independent experiments. asterisk denotes significant differences compared to wt controls (*p 0.05, **p 0.01). these results are in line with the results that ifnar -/and stat1 -/mice are more susceptible to ebov infection, suggesting the possibility that vlp mediated protection is linked to the activation of type i ifn signaling [13] [14] [15] . however, vlp injection may not have prevented ebov pathogenesis in ifnar -/mice, possibly because the disease manifests more severely in these mice than in wt mice. on the other hand, it has been recently shown that adenovirus based vaccine can protect ifnar -/mice from lethal evob infection presumably through antibody responses, which indicates that ifnar -/mice are not universally vulnerable, and anti-ebov resistance can be attained in some cases [35] . we recently reported that ebov vlps activate type i ifn transcription in dcs and macrophages in vitro, leading to induction of many isgs in these cells [33] . here we asked whether vlps stimulate isg induction in vivo. wt mice were infected with ma ebov and received vlps 24 h later, and induction of isg mrnas was tested on days 1.5 and 2. isgs encoding anti-viral proteins were first examined, as they may provide early protection against ebov infection. upper panels in fig. 2a and 2b compare induction of anti-viral isgs, ifit1, mx1, oas1a and stat1 with or without vlp injection in liver and spleen. in this early stage, levels of these isgs were consistently higher in the vlp-injected groups than those without vlps. at later stages of infection, however, the situation reversed, in that mice without vlps had higher levels of isgs, as seen on day 3 (s1 fig. for complete kinetics) . these results indicate that vlp administration accelerated type i ifn and isg induction, which presumably provide early anti-viral activity, not afforded without vlps. we next tested whether vlps induce other isgs, particularly those with negative regulatory activities. this question was of interest to us, since mice that did not receive vlps expressed higher levels of proinflammatory cytokines and chemokines, which raised the possibility that ifn signaling exerts negative regulatory activity towards proinflammatory responses, perhaps by controlling nf-κb activation [36] . shown in the lower panels in fig. 2a and 2b is induction of irgm1, usp18, trim21 and trim30. irgm1 is an ifn inducible gtpase that inhibits lps induced endotoxin shock in mice [37] . usp18 is an isg15 deconjugating factor that negatively regulates tlr signaling and resultant cytokine induction [38] . trim21 and trim30 are members of the tripartite motif family that downregulate tlr induced inflammatory responses [39] [40] [41] [42] . expression of these isgs was also higher in the vlp injected group than that without vlps both in liver and spleen. similar to anti-viral isgs, expression of these negative regulatory factors changed at the later stage (s1 fig). these data indicate that vlps accelerate induction of anti-viral and negative regulatory isgs, which may help suppress ebov's anti-ifn antagonism (see discussion). to confirm that vlp induction of isg is dependent on type i ifn signaling, we next tested isg induction in ifnar -/mice. as expected, none of the isgs tested in fig. 2 were induced in ifnar -/mice after vlp treatment or ebov infection (s2 fig). vlps lower expression of proinflammatory cytokines in ebov infected mice ebov pathophysiology such as severe hemorrhagic symptoms and tissue damage is thought to be associated with dysregulated inflammatory cytokine production [2, 43] . given that vlps accelerated induction of negative regulatory isgs, we next evaluated whether vlps modulate expression of proinflammatory genes. in fig. 3 , expression of tnfα, il-6 and il-1β, chemokines such as mcp-1 (ccl2), mip-1α (ccl3), mip-1β (ccl4), kc (cxcl1) and inflammasome gene nlrp3 was measured in ebov infected mice with or without vlps. these genes were all strongly induced upon ebov infection and peaked on day 3 with a gradual decline on days 5 in all cases, their expression was significantly attenuated in the vlp-treated group as compared to the group without vlps. the difference was most dramatic in the early stage on day 3, where the expression was reduced at least by 50%. in agreement with these results, we noted that serum levels of some of these proinflammatory cytokines were higher in ebov infected mice that were treated with vlps as compared those without vlps [30] . these results support the view that limiting superfluous inflammatory responses contribute to vlp mediated protection. ifnar -/mice increasing evidence indicates that type i ifns antagonize inflammatory responses in a variety of settings [44] [45] [46] . in light of the results that vlps stimulate those isgs known to suppress proinflammatory responses, it was of importance to determine whether type i ifn signaling by and of itself affects ebov induction of proinflammatory cytokines and chemokines. results in fig. 4 and s3 fig compare expression of the above proinflammatory factors in ifnar +/+ and ifnar -/mice infected with ebov. all cytokines and chemokines tested were induced after ebov infection in both strains. importantly, their levels were much higher in ifnar -/mice than ifnar +/+ mice. these results indicate that type i ifn signaling downregulates ebov stimulated induction of proinflammatory factors, possibly through isgs with negative regulatory activities. the above results indicated that type i ifns attenuate proinflammatory responses during ebov infection. to explore whether type i ifns have a similar activity in settings other than ebov infection, we next tested lps and ifnβ induced inflammatory responses in macrophages in vitro. lps activates nf-κb mediated proinflammatory cytokine induction, which can result in endotoxin shock [36] . as shown in fig. 5 , combined treatment with lps and ifnβ led to hyper induction of tnfα, il-6, il-1β and a chemokine kc in ifnar -/macrophages as compared to wt cells. lps and ifnβ also induced negative feedback factors, trim21 and trim30, with much lower expression in ifnar -/cells than ifnar +/+ cells. these results support a model in which type i ifns negatively regulate proinflammatory cytokine/chemokine responses at least in some situations. we found that mip-1α, mip-1β and mcp-1 were not hyperinduced in ifnar -/cells, suggesting that some proinflammatory genes are regulated not only by type i ifns but other factors (data not shown). alternatively, these differences may reflect variances between in vivo and in vitro conditions. to further define pathways downstream of ifnar activity, important for vlp protection, we directed our attention on irf8, a transcription factor expressed in macrophages and dcs [47] [48] [49] . irf8 is induced by ifns and tlr ligands in a stat1 dependent manner, and plays a pivotal role in facilitating innate immune responses. although irf8 is not involved in initial triggering of type i ifn induction, it amplifies ifn transcription in dcs and macrophages [50] . irf8 promotes induction of multiple anti-microbial factors and is required for innate resistance against a variety of pathogens [50] [51] [52] . irf8 stimulates expression of mhc and costimulatory molecules to boost antigen presentation [48, 50] . we thus tested whether irf8 disruption affects vlp-mediated protection against ebov. survival data in fig. 6a show that approximately 80% of irf8 -/mice that received vlps died between day 6 and 8, which is nearly identical to the mortality curve of wt mice without vlps. as expected, the majority of wt mice that received vlps survived against ebov infection. it is of note that ifnar -/mice died 1 to 2 days earlier than irf8 -/mice, which may be attributed to the difference in the mouse background. correlating with the lack of protection, ebola gp mrna levels were much higher role of type i ifn in vlp-mediated protection against ebov infection in ebov infected irf8 -/mice than irf8 +/+ mice with or without vlps (fig. 6b) . we next examined whether induction of anti-viral and negative feedback isgs is dependent on irf8. data in fig. 6c illustrate that induction of these isgs was very meager in irf8 -/mice, in contrast to robust induction in irf8 +/+ mice. importantly, vlps did not rescue isg induction in irf8 -/mice. results were similar in liver and spleens ( fig. 6c and s4 fig). these results indicate that vlps, upon initial activation of type i ifn cascade, rely subsequently on the activation of downstream pathways represented by irf8 to confer protection against ebov. to gain insight into the pathways through which vlps confer resistance against ebov infection, we investigated the role of type i ifn signaling in vivo and found that it significantly contributes to vlp-mediated protection. this conclusion is supported by the observation that post-exposure vlp treatment accelerated isg induction in ebov infected mice, leading to reduced viral replication and inflammatory gene expression. further supporting the critical role of type i ifn signaling in the protection, vlps did not induce isgs in ifnar -/mice, and did not protect the mice from lethal ebov infection. these results are consistent with the report that post-exposure ifnβ or ifnα treatment increases protection against ebov infection in nhps [1, 6, 15, 19] . it is likely that vlps initially stimulated type i ifn genes, which in turn led to early induction of isgs. in line with this notion, we recently showed that exogenous vlps stimulate transcription of ifnα and ifnβ in dcs and macrophages in vitro, an event coupled with immediate and robust isg induction [33] . it may be reasonable to assume that ifnar -/mice were not protected by vlps primarily because isg induction was absent. however, ifnar -/mice may be susceptible to infection due to additional defects in innate immunity that are a secondary consequence of defective ifn signaling, which obliterates vlps protection. contouring this notion however, it is of note that ifnar -/mice can be protected against ebov by an adenovirus-based vaccine, indicating that ifnar -/mice are not totally without defense [35] . rather, it is possible that ifnar -/mice are not protected by vlps that rely on isg induction for protection, whereas they are protected by the adenovirus vaccine that depends on antibody response. vlp-induced isgs included anti-viral proteins known to inhibit replication of rna viruses such as ifit1, mx1 and oas1a, as well as negative feedback factors that curb excess inflammatory responses, such as irgm1, usp18, trim21 and trim30. although the question of which antiviral isgs are effective in inhibiting ebov replication awaits further research, it is anticipated that some of anti-viral isgs induced by vlps may interfere with ebov life cycle [53] . what is the significance of accelerated ifn response in vlp mediated protection? available evidence suggests that vlps may overcome ebov's anti-ifn antagonism. the virally encoded vp24 and vp35 disable the entire ifn system in the host; while vp24 blocks the jak/stat pathway of ifn signaling, vp35, an ebov virulence factor, inhibits type i ifn induction in many cell types [6, 7, 9, 11] . we previously showed that vp35 inhibits type i ifn induction in murine dcs by premature sumoylation and inactivation of irf7 [10] . it is thought that vp24 and vp35 have a decisive effect on the subsequent host resistance, since abated ifn signaling would impair proper innate immune responses, leading to deficiency in dc maturation, defective antigen presentation and aberrant inflammation. compromised innate immunity would consequently undermine development of adaptive immunity [6] (see a model in fig. 7) . it is remarkable that in the vlp treated mice, isg induction began early within 1.5 to 2 days after ebov infection (which was only 0.5 to 1 days after vlp treatment), when little to no isg induction was seen in mice without vlps. the delayed isg induction in ebov infected mice is reminiscent of the reports showing that influenza virus delays isg induction in lung epithelial cells through ns1, an influenza anti-ifn protein that is linked to disease pathology [54, 55] . an influenza virus strain deficient in ns1 is shown to induce isgs earlier than wild type virus, although the wild type strain does stimulate isgs later on [54, 55] . supporting the view that viral anti-ifn factors stall isg induction, rather than completely abrogate the induction, we also observed isg induction on day 3 and later in mice without vlps. it may be envisaged that vlps trigger ifn activation early on, thereby eluding the activity of the ebov anti-ifn proteins (a model in fig. 7) . the most striking observation made in this study is the vlp-dependent suppression of proinflammatory responses. this suppression was a result of type i ifn signaling, as ifnar -/mice expressed higher levels of proinflammatory cytokines and chemokines, observed not only after ebov infection but also by ifnβ and lps stimulation. these results are in accordance with the growing recognition that type i ifns are linked to attenuation of inflammatory responses [6, 44] . for example, pinto et al., reported, in the west nile virus infection model that ifnar -/mice express excess proinflammatory cytokines, including those found in this study, as compared to wt mice, which correlated with increased disease pathology. in this system, the overt inflammatory responses were attributed to ifn signaling in macrophages and dcs [46] . induction of proinflammatory cytokines and chemokines may be negatively regulated by ifn signaling through a series of negative feedback factors [37, [39] [40] [41] [42] [56] [57] [58] . irgm1, induced by ifn signaling restricts lps induced endotoxin shock without limiting ifnβ expression [37] . trim21 and trim30 inhibit proinflammatory cytokine induction, at least in part by interfering with the nf-κb dependent arm of transcription [39] [40] [41] . in addition, these factors may act by post-transcriptional mechanisms, affecting inflammasome activation [42] . in this regard, guarda et al. [45] reported that type i ifns inhibit production of il-1 by inhibiting activity of the nlrp1 and nlrp3 inflammasomes and by il-10 induction. thus, isgs with negative regulatory activity may preferentially attenuate proinflammatory pathways, while sparing ifn induction pathways. given our earlier observations that vp35 does not grossly affect nf-κb activation, while strongly inhibiting type i ifn activation, ebov may promote proinflammatory pathways at least in part through vp35 [10] . lastly, we show that the transcription factor irf8 is required for vlp mediated post-exposure protection. our results offer an added mechanistic insight into the pathways through which vlps provide protection. irf8 is expressed predominantly in macrophages and dcs, and helps to amplify type i ifn gene induction and boosts ifns biological activities [51] . given that macrophages and dcs are the putative early sites of ebov infection, vlps may exert a major impact on these cells to facilitate early innate immunity, in an irf8 dependent manner. in conclusion, vlps confer post-exposure protection upon ebov infected mice by rapidly inducing isgs, thereby permitting timely establishment of anti-viral and anti-inflammatory states in the host. vlps may act primarily by relieving ebov's antagonism against type i ifns, resulting in reduced systemic inflammation and subsequent enhancement in acquired immune responses (a model in fig. 7 ). pathogenesis of ebola hemorrhagic fever in primate models: evidence that hemorrhage is not a direct effect of virus-induced cytolysis of endothelial cells a compendium of 40 years of epidemiological, clinical, and laboratory studies mouse models for filovirus infections a mouse model for evaluation of prophylaxis and therapy of ebola hemorrhagic fever monocyte-derived human macrophages and peripheral blood mononuclear cells infected with ebola virus secrete mip-1alpha and tnf-alpha and inhibit poly-ic-induced ifn-alpha in vitro how ebola and marburg viruses 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regulation of toll-like receptor-mediated immune responses socs proteins, cytokine signalling and immune regulation the content of this publication does not necessarily reflect the views or policies of the us department of defense or the united states army medical institute of infectious diseases. we thank members of pgd for in depth discussions and critical reading of the manuscript. we also thank ms. monica gupta for breeding ifnar -/mice for this study. key: cord-252838-av7ducrk authors: lucchi, naomi w.; demas, allison; narayanan, jothikumar; sumari, deborah; kabanywanyi, abdunoor; kachur, s. patrick; barnwell, john w.; udhayakumar, venkatachalam title: real-time fluorescence loop mediated isothermal amplification for the diagnosis of malaria date: 2010-10-29 journal: plos one doi: 10.1371/journal.pone.0013733 sha: doc_id: 252838 cord_uid: av7ducrk background: molecular diagnostic methods can complement existing tools to improve the diagnosis of malaria. however, they require good laboratory infrastructure thereby restricting their use to reference laboratories and research studies. therefore, adopting molecular tools for routine use in malaria endemic countries will require simpler molecular platforms. the recently developed loop-mediated isothermal amplification (lamp) method is relatively simple and can be improved for better use in endemic countries. in this study, we attempted to improve this method for malaria diagnosis by using a simple and portable device capable of performing both the amplification and detection (by fluorescence) of lamp in one platform. we refer to this as the realamp method. methodology and significant findings: published genus-specific primers were used to test the utility of this method. dna derived from different species of malaria parasites was used for the initial characterization. clinical samples of p. falciparum were used to determine the sensitivity and specificity of this system compared to microscopy and a nested pcr method. additionally, directly boiled parasite preparations were compared with a conventional dna isolation method. the realamp method was found to be simple and allowed real-time detection of dna amplification. the time to amplification varied but was generally less than 60 minutes. all human-infecting plasmodium species were detected. the sensitivity and specificity of realamp in detecting p. falciparum was 96.7% and 91.7% respectively, compared to microscopy and 98.9% and 100% respectively, compared to a standard nested pcr method. in addition, this method consistently detected p. falciparum from directly boiled blood samples. conclusion: this realamp method has great potential as a field usable molecular tool for diagnosis of malaria. this tool can provide an alternative to conventional pcr based diagnostic methods for field use in clinical and operational programs. approximately 2 billion people are exposed to malaria with morbidity surpassing 250 million cases and close to 1 million deaths per year [1] . accurate diagnosis is critical for the proper treatment of malaria [2] . the existing tools for the diagnosis of malaria include microscopy, parasite antigen/enzyme detection kits (commonly referred to as rapid diagnostic tests (rdts)) and molecular tools (reviewed in [3] ). each of these diagnostic tools has its own advantages and limitations. at present, microscopy and rdts remain the only feasible options for malaria detection in many endemic countries. microscopic diagnosis is the oldest method, can provide quantitative data and can identify species when used appropriately. lack of infrastructure and training in most endemic countries has made microscopic diagnosis challeng-ing which has contributed to recent interest in deploying rdts more broadly. the current rdts detect parasite antigens such as histidine rich protein (hrp) -2, lactate dehydrogenase (ldh) and aldolase using immunochromatographic methods. the majority of the commercial rdts detect hrp-2 which is expressed only by p. falciparum but not other species and therefore this test offers specific diagnosis of falciparum malaria. a limitation of this test is that hrp-2 can persist in the blood for several days after the parasites are cleared therefore the assay cannot accurately tell whether someone has a current or recently treated infection. another concern about this test is the recent discovery that up to 40% of p. falciparum parasites in parts of south america have deleted the hrp-2 gene (which leads to false negative results) [4] . most non-hrp-2 based tests (ldh and aldolase) are commonly pan-species test that allow for the speciation of p. falciparum and/or non-falciparum species when used in conjunction with hrp-2 based tests. therefore, nucleic acid-based molecular methods are a potentially good alternative for malaria diagnosis as they can accurately differentiate all human-infecting plasmodium species and detect low levels of parasitemia. pcr-based diagnosis recently helped to identify p. knowlesi in humans which had been misdiagnosed using microscopy [5] . unfortunately, the current pcr-based methods are beyond the capacity of most malaria-endemic countries because they require sophisticated laboratory infrastructure and training which makes these techniques expensive and technically challenging to implement in simple clinical laboratories or field settings. however, as progress is made towards better malaria control and eventual goal of elimination, more sensitive diagnostic tools will be required in order to detect asymptomatic low level parasitemia. therefore, further efforts are needed to develop next generation molecular tools for field use with a goal that such tools can complement, or in some situations, replace the existing molecular methods for malaria diagnosis and operational programs such as monitoring and evaluation of control and elimination programs. the recently developed loop-mediated isothermal amplification (lamp) method is a relatively simple and field-adaptable technique [6] . parasite dna is amplified under isothermal conditions using a polymerase with strand displacement properties (usually the bacillus stearothermophilus (bst) polymerase); therefore, sophisticated and expensive thermal cyclers are not required. the amplification of dna results in the formation of magnesium pyrophosphate which appears as a precipitate as the reaction progresses. the appearance of this precipitate is used as a sign of a positive reaction. in addition, lamp was shown to amplify dna with high efficiency, amplifying a few copies of dna to 10 9 in less than 1 hour [6] . four lamp primers are used specific to six sites of the target sequence which makes them highly specific to the target [6] . the addition of two extra primers, known as loopprimers, was shown to accelerate the time to product formation [7] , thereby shortening the required reaction time (30 minutes to 1 hr). given that this method does not require a thermocycler or sophisticated training, it has the potential to be used as a molecular diagnostic tool for point-of-care (poc) diagnosis in both developing and developed countries provided further modifications are made. indeed, lamp has been used for the detection of several infectious diseases such as legionella bacteria [8] , west nile virus [9] , severe acute respiratory syndrome [10] , avian influenza virus [11] , and norovirus [12] . recently, the lamp method was used for the detection of malaria parasites using the 18s rrna gene as the target gene [13] [14] [15] [16] [17] . poon et al. 2006 [16] reported successful application of lamp for malaria diagnosis for the first time. they reported detecting p. falciparum directly from heat-treated clinical samples in which they boiled packed red blood cells at 99uc for 10 minutes, pelleted the cells by centrifugation and used the supernatant in the lamp assay. in this study the sensitivity and specificity of lamp was reported to be 95% and 99% respectively compared to an inhouse nested pcr. in 2007, han et al. reported a species specific lamp diagnostic method; using clinical samples and a conventional dna extraction method, they demonstrated sensitivity and specificity of 98.5% and 94.3% respectively compared to microscopy and a nested pcr [14] . detection limits of 10 copies of the target 18s rrna genes for p. malariae and p. ovale and 100 copies for p. falciparum and p. vivax were demonstrated [14] . the plasmodium-specific and species-specific primers were shown to require less than 40 minutes for amplification. in another study, paris et al. compared the lamp method with both microscopy and p. falciparum hrp-2 rdt [13] [14] [15] [16] [17] . they found that lamp had 100% specificity and 77.6% sensitivity when compared to hrp-2 rdt and 100% specificity and 73.1% sensitivity when compared to microscopy. however, in contrast to what was reported by poon et al. the sensitivity and specificity of lamp compared to a nested pcr based on primers designed by singh et al. [18] were shown to be 79.1% and 58.3% respectively when heat treatment for dna extraction was used [15] . chen et al. used p. vivax primers to detect microscopically positive p. vivax clinical samples using the lamp assay [13] . the limit of detection for p. vivax was shown to be 30 parasites per microliter (p/ml) [13] with 100% specificity and 98.3% sensitivity compared to microscopy. the utility of the lamp assay may be limited by the difficulty of visualization of precipitate especially at lower target dna concentrations. therefore, attempts have been made to use the intercalating dye sybr-green to measure the end reaction using a uv light [13, 15] or conventional real-time pcr fluorescence readers [19] [20] [21] [22] . however, paris et al. showed that the uv fluorescence method produced a high rate of false positives and suggested that this method be abandoned as a lamp read-out [15] . yamamura et al. combined the lamp method with a melting curve analysis using the genopattern analyzer gp1000 (yamato scientific, tokyo, japan) [17] . using clinical samples, they demonstrated lamp sensitivity and specificity of 97.8% and 85.7% respectively, as compared to microscopy. however, the use of sophisticated equipment for diagnostic applications is not feasible in many field settings due to the lack of appropriate infrastructure. therefore, there is a need for a simple field-usable method that can afford a quicker and objective readout for the diagnosis of malaria using the lamp method. here, we explored the utility of a simple portable device (tube scanner) in which both the amplification platform (heating block) and fluorescent detection unit for end point use (with the ability to acquire real time data) are combined into a single unit for lamp assay. we refer to this method as realamp. we demonstrate the utility of this method for the diagnosis of malaria by using published plasmodium genus specific primers and comparing it to microscopy and a nested pcr method as described by singh et al [18] . samples used in this study were obtained from a human clinical trial conducted in tanzania to assess the efficacy of anti-malarial drugs. this study was approved by both the ifakara health institute and the cdc institutional review board and informed written consent forms were obtained from each subject. the portable fluorescence reader (ese-quant tube scanner) used for this study was developed by a commercial manufacturer (ese gmbh, stockach, germany, figure 1a ). this device has an eight tube holder heating block with adjustable temperature settings and spectral devices to detect amplified product using fluorescence spectra. this equipment weighs about 2.2 lbs with the dimensions 74 mm6178 mm6188 mm (h 6 w 6 d). the unit is completely portable and can be operated with a li-ion rechargeable power pack without external power supply. a small lcd (monitor) is available to display the results (as positive or negative) without the need of a computer. however, the device can also be used together with a computer to generate real time amplification plots as the reaction progresses (as done in this study). p. falciparum (3d7) was cultured in our laboratory. the cultures were synchronized by the sorbitol method to select for the ring stage parasites which have single nuclei and therefore can be reproducibly used for quantitation of dna. a thin smear was made, stained with giemsa and the percentage parasitemia determined. the number of parasites/ml (p/ml) was determined by counting the total number of rbcs/ml using a coulter counter and using the percentage parasitemia data: (p/ml = rbcs/ml x percentage parasitemia). the other three human-infecting plasmodium species, p. vivax (sv4), p. malariae (uganda i), and p. ovale (nigeria i) were acquired from infected monkeys or chimpanzees at the cdc. the parasites/ml data for these species were obtained by microscopy. to test the limits of detection of realamp, the dna from these four species was diluted from 40,000 p/ml or 10,000 p/ml to 1 p/ml. in addition, p. knowlesi and seven other primate malaria parasites, p. inui, p. cynomolgi, p. coatneyi p. fieldi, p. semiovale, p. fragile, p. gonderi were tested. ninety four samples confirmed to be p. falciparum positive by microscopy obtained from a human clinical trial to assess the efficacy of antimalarial drugs and 12 samples known to be negative for p. falciparum (based on microscopy diagnosis) from a previous study [23] were used to assess the sensitivity and specificity of the realamp assay. non-malaria infected human dna was used as a control. dna was isolated from all the samples using a qiaamp dna mini kit (qiagen, valencia, ca-(qiagen method)). the dna was aliquoted and stored at 220uc. to determine the utility of the heat-treated method of dna extraction in realamp cultured p. falciparum parasites were subjected to the heat-treatment method as described by poon et al. [16] . briefly, freshly cultured 3d7 parasites were adjusted to 50% hematocrit using whole blood. a starting parasitemia of 40,000 p/ml was prepared from which six 10-fold serial dilutions were prepared to a final parasite concentration of 0.4 p/ml. fifty microliters each of these dilutions were heated on a heat-block at 99uc for 10 minutes. the tubes were then centrifuged at 13,000rpm for 3 minutes and the supernatant collected and used in the realamp and nested pcr assays. in parallel, an aliquot of each of these dilutions was also subjected to the qiagen method of dna isolation and also tested by both realamp and nested pcr methods. nested pcr was performed with primers and cycling conditions as described by singh et al. [18] with some modifications. reactions were performed in 20 ml total volume containing 1x buffer, 2.5 mm mgcl 2 , 200 mm dntps, 200 nm primers, and 1.25 units of taq polymerase (new england biolabs, ipswich, ma). the pcr amplified material was analyzed using gel electrophoresis (2% gel) to visualize the bands of appropriate size. the realamp method was performed using the commercially available loopamp dna amplification kit (eiken chemical co., ltd., tokyo, japan) following the manufacturer's instructions with the exception of the addition of 0.25 ml per 12.5 ml reaction volume of a 1:100 diluted sybr green (invitrogen) or by the use of an in-house reaction buffer. to test the utility of an in-house reaction buffer, pilot experiments were performed in a 12.5 ml total volume containing a 2x in-house buffer (40 mm tris-hcl ph 8.8, 20 mm kcl, 16 mm mgso 4 , 20 mm (nh 4 )so 4 , 0.2% tween-20, 0.8m betaine, 2.8 mm of dntps each), 0.25 ml of a 1:100 dilution sybr green and 8 units of bst polymerase (new england biolabs, ipswich, ma). genus specific primers, as described by han et al. [14] were used to amplify the gene coding for the 18s ribosomal rna. dna amplification was carried out at 63uc for 90 minutes using the ese-quant tube scanner (ese gmbh., stockach, germany) which was set to collect fluorescence signals at 1 minute intervals. a typical realtime amplification plot obtained using the realamp method is shown in figure 1b . in the plot, the y-axis denotes the fluorescence units in milli-volts (mv) and the x-axis shows the time in minutes. amplification of p. falciparum dna yielded sigmoid shaped amplification curve while the control tube (no dna) had no measurable fluorescence indicated by a flat line in the plot. the sensitivity and specificity of realamp method was calculated using both microscopy and a nested pcr assay [18] as reference tests. the percentage specificity and sensitivity were calculated using the formulae shown below: in addition, 95% confidence intervals (95%ci) for both sensitivity and specificity were calculated. we were able to amplify any of the four species of human malaria parasites (p. falciparum, p. vivax, p. malariae and p. ovale) within 20 minutes (figure 2 ). the fluorescence peak typically persisted for about 5 minutes and then declined over time. in addition, this assay was able to detect p. knowlesi and seven other primate malaria parasites (p. inui, p. cynomolgi, p. coatneyi p. fieldi, p. semiovale, p. fragile, p. gonderi (data not shown)). no amplification as observed with the non-malaria infected human dna control ( figure 2 ). the limits of detection of realamp were determined using dna obtained from p. falciparum, p. vivax, p. ovale and p. malariae. the dna was diluted from 40,000 p/ml (p. falciparum) or 10,000 p/ml to 1 p/ml. the limits of detection of realamp varied between 1-100 p/ml for the different species (table 1 ). this assay required at least 1-10 p/ml for the detection of p. ovale and p. malariae. p. vivax was detected consistently at10 p/ml. for the detection of p. falciparum a minimum of 10-100 p/ml was required ( table 1 ). the nested pcr detected up to 1 p/ml for all the four species (data not shown). the time to amplification varied between 15-60 minutes. more time to amplification was required for samples with lower parasite densities although no clear correlation was observed between time to amplification and parasite densities. clinical samples, with median parasitemia density of 3,200 p/ ml (range 61-248,950 p/ml), were used to test the utility of this platform for the diagnosis of field samples. the sensitivity and specificity of the realamp method compared to microscopy and nested pcr is shown in table 2 . of the 94 microscopically positive samples tested, 90 samples were confirmed to be positive by the nested pcr and 89 positive by realamp. eleven out of the 12 microscopically negative samples were shown to be negative by the two methods. overall, the sensitivity and specificity of realamp and nested pcr was similar when compared to microscopic data ( table 2 ). the realamp method showed 98.9% (95% ci: 93.1-99.9%) sensitivity and 100% (95% ci: 100%) specificity when compared to nested pcr. we compared dna obtained by the standard qiagen method of dna isolation and that obtained by direct heating for their performance in realamp method. as shown in table 3 , the realamp method was able to amplify up to 40p/ml of p. falciparum from heat-treated samples and occasionally up to 4 p/ml whereas, up to 0.4 p/ml were detected when dna obtained from the qiagen method was used. there was no consistent amplification below 40 p/ml especially with heat treated dna. no amplification was detected with heat-treated uninfected whole blood (data not shown). cost analysis of the realamp method compared to the nested pcr table 4 summarizes the cost of running the realamp method compared to that for nested pcr assay. the cost of performing realamp was lower when an in-house buffer was used. however, the cost of realamp increased when a commercially available buffer was used. the capital investment cost for both pcr and tube scanner was comparable (table 4 ). in addition, there are important practical advantages of using realamp as further discussed. the lamp molecular assay is a potentially useful alternative to the current molecular tools that require sophisticated equipment and techniques [6] . a few studies have clearly demonstrated that lamp can be used for malaria diagnosis [13] [14] [15] [16] [17] . in this study we integrated the amplification and detection stages of lamp into one portable and simple to use platform in an attempt to make this method easily usable even in field settings. as summarized in table 5 , results from the realamp method were comparable to the previously reported malaria lamp assays [13] [14] [15] [16] [17] demonstrating reasonable sensitivity and specificity profiles when compared to microscopy and nested pcr. the reported sensitivities and specificities ranged from 73.1% to 98.3% and 85.7% to 100%, respectively, using microscopy as a reference standard ( table 5) . two of these studies [15, 16] also compared malaria lamp assays with pcr-based assays (table 5 ) and one study used hrp-2 rdt as a reference [15] . the use of different reference tests, such as different pcr-based assays, clearly influences the sensitivity and specificity profile obtained. in addition, differences in the parasite densities of the samples used in the various studies may influence the sensitivities and specificities which could explain the variations observed across these studies. out of the 94 microscopically positive samples used in this study, 4 were negative by nested pcr and 5 by realamp assay. the parasite density determined by microscopy for these samples ranged from 240 p/ml to 191,320 p/ml. failure to amplify these samples by these two molecular methods was not due to low parasite density but most likely due to poor quality of the dna preparation since we could not amplify some of the same samples using other nucleic acid tests. further prospective studies in different transmission settings will be required to further evaluate the performance of the realamp method in comparison to other routine diagnostic tests. one of the limitations of the current study is the fact that the realamp method was evaluated using only plasmodium genus specific primers. although the lamp method can be used for the diagnosis of malaria parasites at the species level [14] , attempts to use these published species specific primers did not yield consistent results in our hands both by conventional lamp and realamp methods. we are currently evaluating the use of new dna targets to develop primer sets that can be used for malaria species diagnosis. despite this limitation, this genus-specific realamp method can be used for monitoring and evaluation of malaria control programs in the field. it can also be used as a confirmatory test for malaria infection in place of a standard pcr-based assay. the observation that the lowest level of parasitemia required to detect p. falciparum was one to two orders of magnitude higher than that needed for the other three species tested can be explained by the fact that the p. falciparum samples used were selected for the ring stages whereas all parasite stages (rings and schizonts, the latter containing more dna than the ring stage) were present in the other plasmodium species. these limits of detection with this genus-specific realamp method are similar or better than those reported for microscopy and rdts. improving on the limits of detection by realamp (or any other malaria diagnostic test) has real potential now more than ever as there is a concerted effort to increase malaria prevention and control programs. it is hoped that these programs will lead to a reduction in malaria transmission and therefore to lower infection levels. therefore, more sensitive tools that can be used in field settings will be needed for the evaluation of these control programs. the realamp method, as reported here, was not designed for the quantitation of parasitemia. we observed that the time to amplification was shorter for samples with high parasite densities than for samples with low parasite densities. however, this relationship was noticed only when the reactions were run simultaneously: a strict correlation was not observed when samples were compared between runs indicating that one cannot draw conclusions about parasitemia levels based on the amplification time. further efforts are needed to determine if this method can be improved for quantitative purpose. the use of the heat-treatment method for template preparation provides a good alternative to the expensive and labor intensive dna isolation methods that might not always be possible in field settings. in this study we were able to successfully use heat-treated samples for realamp amplification similar to results reported by poon et al [16] . we did not observe any inhibition of pcr amplification as, previously reported [16] , when using the heattreated sample for dna amplification in the nested pcr assay. the heat-treatment method yielded dna extract that could be used to reliably detect as low as 40 p/ml. at this level of detection limit, we hope it will yield results that can be comparable to microscopic diagnosis (100-200 p/ml) in the field. this method showed slightly lower efficiency compared to qiagen method (below 40 p/ml) and it is not clear if this difference was due to poor efficiency in dna extraction at low parasitemia level or due to other factors. nevertheless, these results clearly illustrate that the heat-treatment method can be further improved to make it an alternative to conventional dna isolation methods in the field. in our hands, the cost of running the realamp method was cheaper than that of the nested pcr when an in-house buffer was used in place of the commercially available buffer. the performance of our in-house buffer was as good as that of the commercial buffer and it consistently yielded similar results. our price estimates are arbitrary and may vary for other users depending on where and how their reagents and equipment are purchased. these cost estimates do not include labor and other infrastructure costs which will vary too, depending on the region. regardless of these cost factors, there are several important practical aspects of the realamp that makes it an attractive method for field use. this includes a) the fact that the tube scanner is light and small and is easily portable to even remote places while standard thermocyclers require an established laboratory setting, b) an alternate power source such as battery can be used to operate the tube scanner, c) no post-run manipulation such as gel electrophoresis is required to visualize the results contributing to shorter turnaround time, d) the realamp method is technically easier to perform than the nested pcr, e) this method has automation features to report results directly to remote locations, and f) this method can be modified to handle large sample numbers (e.g. using a 96-well plate holder). the tube scanner is comparable to the real-time turbidimeter used in some studies [16] in the sense that both are capable of detecting a positive sample in real-time resulting in similar amplification plots. the tubidimeter measures the turbidity of reaction mixture while the realamp measures the fluorescence units generated as the product is formed. however, the tube scanner has an added feature in which the results (as positive (+) or negative (2)) can be reported on the provided lcd without the need of a computer. it would be of interest to compare the utility of these two readout machines simultaneously to determine if there is any advantage of using one over the other. we would like to point out that the realamp method can be performed with any alternative equipment that is similar to the tube scanner used in the study. the utility of any diagnostic assay for point-of-care and field use will lie, among other things, on the fact that it is less expensive and simple to perform without compromising its sensitivity and specificity. the realamp method will be more attractive for field use if the lamp reagents can be stored at room temperature without requiring a cold chain. our preliminary data suggests that the realamp reagents can be kept at least for two weeks at room temperature without loss of activity but further studies are required to investigate this fully. in summary, this study has shown that the realamp method is a potential field usable tool for diagnostic purpose and for use in malaria control programs. further field studies in different endemic countries will help to optimize its use for various malaria control applications and as a point-of-care tool. who (2010) guidelines for the treatment of malaria diagnosis of malaria: challenges for clinicians in endemic and non-endemic regions a large proportion of p. falciparum isolates in the amazon region of peru lack pfhrp2 and pfhrp3: implications for malaria rapid diagnostic tests plasmodium knowlesi malaria in humans is widely distributed and potentially life threatening loop-mediated isothermal amplification of dna accelerated reaction by loop-mediated isothermal amplification using loop primers rapid and simple detection of legionella species by lamp, a mew dna amplification method real-time reverse transcription loop-mediated isothermal amplification for rapid detection of west nile virus development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus development of h5-rt-lamp (loop-mediated isothermal amplification) system for rapid diagnosis of h5 avian influenza virus infection rapid detection of norovirus from fecal specimens by real-time reverse transcriptionloop-mediated isothermal amplification assay recombinant mycobacterium bovis bcg producing the circumsporozoite protein of plasmodium falciparum fcc-1/hn strain induces strong immune responses in balb/c mice detection of four plasmodium species by genus-and species-specific loopmediated isothermal amplification for clinical diagnosis loopmediated isothermal pcr (lamp) for the diagnosis of falciparum malaria sensitive and inexpensive molecular test for falciparum malaria: detecting plasmodium falciparum dna directly from heat-treated blood by loop-mediated isothermal amplification evaluation of a new rapid molecular diagnostic system for plasmodium falciparum combined with dna filter paper, loop-mediated isothermal amplification, and melting curve analysis a genus-and species-specific nested polymerase chain reaction malaria detection assay for epidemiologic studies application of a loop-mediated isothermal amplification method for the detection of pathogenic leptospira loop-mediated isothermal amplification (lamp) method for rapid detection of trypanosoma brucei rhodesiense loop-mediated isothermal amplification (lamp) test for detection of trypanosoma evansi strain b use of reverse transcription loop-mediated isothermal amplification for the detection of plum pox virus plasma ip-10, apoptotic and angiogenic factors associated with fatal cerebral malaria in india we would like to thank the director of ifakara health institute for providing the field samples used in this study and vincent hill for the critical review of the manuscript. table 5 . summary of sensitivity and specificity of malaria lamp assays reported in the literature. key: cord-000720-5b936n3g authors: nannyonga, betty; sumpter, david j. t.; mugisha, joseph y. t.; luboobi, livingstone s. title: the dynamics, causes and possible prevention of hepatitis e outbreaks date: 2012-07-24 journal: plos one doi: 10.1371/journal.pone.0041135 sha: doc_id: 720 cord_uid: 5b936n3g rapidly spreading infectious diseases are a serious risk to public health. the dynamics and the factors causing outbreaks of these diseases can be better understood using mathematical models, which are fit to data. here we investigate the dynamics of a hepatitis e outbreak in the kitgum region of northern uganda during 2007 to 2009. first, we use the data to determine that [image: see text] is approximately 2.25 for the outbreak. secondly, we use a model to estimate that the critical level of latrine and bore hole coverages needed to eradicate the epidemic is at least [image: see text] and [image: see text] respectively. lastly, we further investigate the relationship between the co-infection factor for malaria and hepatitis e on the value of [image: see text] for hepatitis e. taken together, these results provide us with a better understanding of the dynamics and possible causes of hepatitis e outbreaks. outbreaks of diseases such as avian influenza, sars and west nile virus have alerted us to the potentially grave public health threat from emerging and re-emerging pathogens [1] [2] [3] . many important infectious diseases persist on a knife-edge: rapid rates of transmission coupled with brief infectious periods. such violent epidemic behavior has been observed in plague [4] , cholera [5] , pertussis [6] and more recently hepatitis e. the recent outbreak of hepatitis e in northern uganda, has left many dead and a number of infectives that continue to spread the infection [7] . hepatitis e is caused by infection with the hepatitis e virus (hev) which has a fecal-oral transmission route. it is a self-limiting disease but occasionally develops into an acute severe liver disease. as emerging and re-emerging infectious diseases increase in outbreak frequency, there is a compelling interest in understanding their dynamics [8] [9] [10] . the kitgum outbreak, which we study here, has been linked to contaminated water or food supplies [11] . an assessment conducted by the uganda red cross and district representatives in agoro revealed that for a population of about 28,045 with 6,039 households mainly living in camps for internally displaced people in potika as well as agoro and oboko satellite camps, the latrine coverage was as low as 3.7%. this means that there is one latrine for every 27 people. further, only 23 boreholes were functional implying that the bore hole coverage is 0:38%, or one bore hole per 263 households. another possible factor that could be implicated in the outbreak of hepatitis e is its possible relationship with malaria. malaria has been shown to disarm the immune system and increase susceptibility to viral infections such as hiv [11] . recently, in a 3-month follow-up study the pattern of co-infection of plasmodium falciparum malaria and acute hepatitis a (hav), in 222 kenyan children under the age of 5 years was observed [12] . the incidence of hav infections during p. falciparum malaria was found to be 6.3 times higher than the cumulative incidence of hav, suggesting that co-infection of the two pathogens may result from changes in host susceptibility. there is also evidence both for [13, 14] and against [15, 16] an association between hepatitis b viruses and malaria. hev transmission route is similar to the hepatitis a virus and thus for hev it is important to consider possible links to co-infection with malaria. this can be done using mathematical models of multiple pathogens [17] [18] [19] [20] [21] [22] . in this paper, mathematical models are used to study the effects of both environmental conditions and malaria on hepatitis e infections. the models designed are fit to data from the kitgum outbreak, to estimate the basic reproduction number and to relate them to the level of contamination of the environment. we assume that the small number of latrines [23] , leads to contamination of environment. this in turn leads to contaminated water. owing to the few number of bore holes in the region, lack of access to clean water gives rise to the viral infection of hepatitis e. we formulate two mathematical models: one for hepatitis eonly and another for the co-infection with malaria, based on prior work as in [4, 24, 25] . in their framework, an individual is categorized according to their infection status and passes sequentially through the series of non infectious, infectious and recovered classes. a system of ordinary differential equations are then designed, analyzed and later fit to data from the hepatitis e outbreak in kitgum district to estimate desired parameters. first we model the epidemiology of hepatitis e, an environmentally transmitted viral infection. the dynamics of the disease are an seir framework, i.e. susceptible, exposed, infectious and recovered. hepatitis e virus is mainly spread by the fecal-oral route. this results either from directly touching the contaminated environment and eat without washing hands, or drinking contaminated water. in kitgum district uganda, most people live in internally displaced camps. the number of latrines in the area are not enough for the entire population, [11] , and people use the local environment for this purpose. when rain falls, it washes the faeces into water bodies. in the kitgum region, few people have access to clean bore hole water [11] , and therefore collect water from the contaminated water sources. to model this phenomenon we use l, to denote the proportion of households in kitgum with access to latrines. therefore, the rate of change of contamination c of the environment is given by where r is the transmission rate of hev from the infected human, i, to the environment. in the human population, susceptibles, s, are recruited at a rate m that equals to the per capita natural mortality rate for each group. this assumption is made to keep the population constant, while keeping a turnover of individuals in the population. we assume that a fraction b of the population has access to clean bore hole water and cannot become infected. susceptible individuals without bore hole access become infected with the hepatitis e virus at a rate bc, where b is the transmission rate of hev from the contaminated environment c, to the human. this gives after successful infection, the individual is now exposed to hev and moves to the exposed class e. the incubation period takes a mean period of 1 s days. the equation for this group is given by at the end of the incubation period 1 s , the individual becomes infectious and moves to group i. at this point, they display signs and symptoms that include fever, fatigue, loss of appetite, nausea, vomiting, abdominal pain, jaundice, dark urine, clay-colored stool and joint pain [26] . the infected individual may recover at a rate c. these dynamics are given by di dt~s e{(mzc)i: of the total infected individuals, a fraction p of them die due to the infection, and (1{p) recover to join the immune group r. this implies that equations (1) to (5) provide a system of equations defining the transmission of hev between a contaminated environment and humans. assuming that the dynamics of the environment are fast. this means that the environment reaches a steady-state before the humans. the quasi-stationary-state (qss) for c can be obtained from equation (1) to give this steady state is then substituted in the human equations to give a reduced system of equations as follows: dr dt~( 1{p)ci{mr: as in [27, 28] , in this system, 1~szezizr. thus, the last equation in (7) is redundant. this system of equations will be analyzed and fit to data. the endemic stationary state is given by where is the basic reproduction number for hev. the term s mzs is the proportion of the exposed humans that survive the incubation period. the other fraction, br(1{l)(1{b) mzc is transmission rate of hev during the infectious period of the human. the disease-free equilibrium point is stable if r 0 v1 (see supporting information s1) when r 0 w1 the endemic equilibrium point in equation (8) exists and is stable. this equilibrium is attained via oscillatory dynamics, with period t*2p is the mean age at infection, and g~1 mzs z 1 mzc is the ecological generation length of the infection. figure 1a is a plot of the data for the outbreak from 2007 through 2009. to estimate model parameters and determine the critical level of control needed to eradicate the epidemic, the model described by the equations in (7) is fit to the data collected during the kitgum outbreak ( figure 1 ) during the invasion phase of hev, the prevalence is approximately taking the log of both sides of equation (10) and performing linear regression (details in supporting information s1) on this equation, (figure 1b gives an estimated value r 0~2 :15. to determine r 0 when natural mortality is not equal to zero, (i.e. mw0), we use a non-linear differential equation fitting tool, called the potterswheel toolbox [29] . in this fitting technique, the c 2 value of the sum of the squares of the differences between the observed and fitted values is minimized by searching through different parameter values. we set m~0:0004, p~0:0169, b~0:0038, l~0:0037, and fit the free parameters, s,c, and the force of transmission br. we repeat this process 50 times to produce a range of best fits. the basic reproduction number for each run is then calculated using the expression in equation (9) the fitted parameters estimate the basic reproduction number r 0 between 2.08-2.39 with average 2.25. this value is similar to that found from the linear regression fitting. figure 1c is a plot of the model outcome using the parameters generated from the fitting to the kitgum outbreak. in addition to hepatitis e, individuals in the kitgum region were at a risk of acquiring malaria which is endemic to uganda. to model possible co-infection we adopt the model to include a susceptible group which comprises both those with and without malaria. that is, the total susceptible population s9 = s+m where m is the proportion of individuals infected with malaria. the malaria dynamics will not be modelled in detail here but an assumption is made that malaria continuously invades the population, and individuals move back and forth between infection and recovery from the disease. this implies that the equilibrium state for this model is given by clearly, this assumption provides a much simplified model when compared to a full model of vector-borne malaria [3, 12, 27] . our concern here, however, is how background levels of malaria effect transmission dynamics of hev. in kitgum, at its lowest point during march 2009, 2,316 cases of malaria were reported out of a total population of 28,045 [7] . thus 8.3% of the population are infected with malaria at any time, m ã~0 :083. recovery rate for malaria is r~0:1429 per week, and thus we set f~0:0129. under the above assumption, equations (7) are rewritten to incorporate the malaria dynamics in equation (11) where j is a parameter that models change the increase (or decrease) in susceptibility to hepatitis e of malaria infected individuals [12] . the other parameters are as defined in equations (7) and remain as defined there. we assume here that after exposure to hev, both the susceptible and malaria infected groups join the exposed, e and subsequently the i group. in other words, individuals that harbor both infections are assumed to develop hev symptoms at the same speed as those with only hev. the dynamics of this model for standard parameter values are shown in figure 2 . using the next generation method as in van den driessche and watmough (2002), [30] , the basic reproduction number for hepatitis e in presence of malaria is given by where r 0 is as defined in equation (9) when r c v1 infected individuals will have more chances of recovery than of transmitting the disease further hence the epidemic will die out. when r c w1, there exists an endemic equilibrium point as shown in supporting information s2 given by p . this implies that the endemic stationary point is attained via damped oscillations. the stability of this point would depend on the sign of the real part, a. if a.0, then the steady state is an unstable spiral, otherwise, it is a stable spiral. if ½j(mzf)z(mzr) 2 4jm(mzfzr) w1, then we have real roots, and stability of this equilibrium state will depend on the signs of these roots. if both are positive, the steady state is an unstable node; if both are negative, it is a stable node. if one of them is positive and the other negative, the steady state is a saddle point. figure 2a shows the evolution of the malaria infected, m, the exposed, e, and the infected, i with time, while figure 2b is a phase space portrait in the si plane. from equation (14) it can be seen that the value for r c is determined by the proportions of susceptibles and malaria infectives in the population. rearranging and assuming that szm~1 this equation gives a criteria for an epidemic of this criteria is plotted in figure 3a . as expected, if jw1 then presence of malaria increases the probability of an outbreak of hepatitis e, while if jv1 the presence of malaria inhibits hepatitis e. assuming that malaria is at equilibrium in the population. (for example week of march 2009, with 2,316 malaria cases), gives m ã~0 :083, then equation (16) gives a direct relation between j and r 0 . in fitting the model we note that the transmission rates br and j are not independent. indeed, using potterswheel to fit the co-infection model shows that the range of values for br is between 1.28-4.69, c between 1.01-1.75 and j values are between 0.02-13.27 (figure 3b ) all of these values fall on line corresponding to r c between 2.19-2.48. this relationship follows the same curve as the analytical results in figure 3a . to test potential interaction between hepatitis e and malaria empirically, we now assume that in the absence of malaria, hepatitis e has r 0~1 and does not spread. thus malaria is required for the spread of hepatitis and jw1. since r c~2 :3 from the data and m ã~0 :083, then substituting these values in to equation 14 gives j~16:9. this implies that, under the assumption of co-infection as the factor which promotes hepatitis e, malaria infected individuals were infected with hepatitis e up to 16.9 times more than those not infected with malaria. as we gain more information about the role of co-infection, this relationship can be used to improve estimation of br. as in [25, 27] , the criterion under which hepatitis e will invade the population when malaria is endemic is derived in the supporting information s3. thus, hepatitis e virus invades if and the co-infection persists if j fzr(1{r 0 ) fr 0 : ð18þ as in the hepatitis e-only model, potterswheel toolbox is used to investigate the basic reproduction number r c , when natural mortality is not equal to zero, (i.e. mw0) a sequence of parameter estimates are generated, this time setting the fits in sequence to 20. the process is repeated until a set of 50 readings is obtained. the parameters are chosen in such a way that 0:1077vsv0:4777 (i.e. 15v1=sv64 days, the hepatitis e incubation period, [26, 31, 32] ), and x 2 value is less than 65. the basic reproduction number for each run is calculated using the expression in equation (14) . the global burden of disease (gbd) concept, first published in 1996, constituted the most comprehensive and consistent set of estimates of mortality and morbidity yet produced [33] . a gbd study aims to quantify the burden of premature mortality and disability for major diseases or disease groups, and uses a summary measure of population health, the daly (disability-adjusted life years), to combine estimates of the years of life lost and years lived with disabilities. a daly is defined as an indicator to quantify the burden of the disease and the functional limitation and premature mortality [34] . it can be used across cultures to measure health gaps as opposed to health expectancies, and the difference between a current and an ideal situation where everyone lives up to the age of the standard life expectancy, and in perfect health. in developing the daly indicator, murray and lopez (1996) , [33] since thedaly combines in one measure the time lived with disability, yld, and the time lost due to premature mortality, yll, then the yll metric essentially corresponds to the number of deaths, p, multiplied by the standard life expectancy, l, at the age at which death occurs. therefore, to estimate yld on a population basis, the number of disability cases is multiplied by the average duration of the disease and a weight factor that reflects the severity of the disease on a scale from 0 (perfect health) to 1 (dead) the basic formula (without applying social preferences) for one disabling event is given by where i is the number of incidence cases, dw is the disability weight, and d is the average duration of disability. since the reported cases are not specified according to age, the estimate will be done on a population basis. typical symptoms of hepatitis e include jaundice (yellow discoloration of the skin and sclera of the eyes, dark urine and pale stools), anorexia (loss of appetite), an enlarged, tender liver (hepatomegaly), abdominal pain and tenderness, nausea and vomiting, and fever and the disease may range in severity from sub-clinical to fulminant [35] . to calculate the yld, we will set the disability weight to that for a diarrhea disease episode, (equal to 0.11, [33] ), in untreated or treated form. in developing the daly indicator, additional social choices are taken into account. for example, is a year of healthy life gained now worth more to society than a year of healthy life gained sometime in the future? the daly is an incidence-based measure, rather than a prevalence-based measure. therefore, to estimate the net present value of years of life lost, a time discount rate to years of life lost in the future is applied, to adjust both costs and health outcomes [36] . discounting health with time reflects the social preference of a healthy year now, rather than in the future. to do this, the value of a year of life is generally decreased annually by a fixed percentage, d. therefore, equations (20) and (21) are respectively transformed to yld~i according the who [35] , the life expectancy for a ugandan male is 51 and 48 for a female. an average of 50 years will be used. the number of latrines and boreholes that would have prevented the hepatitis e outbreak in kitgum are calculated using our results in preceding sections. first, it is assumed that if the people had the necessary and sufficient number of latrines in addition to safe drinking, then the outbreak would not have occurred. then, the costs of constructing the required latrines and boreholes are computed. from the results, the cost of saving one life from hepatitis e, for one year is determined. the current number of latrines in kitgum is 1,038 [11] . this implies one latrine per 27 people. according to the rules and regulations of kampala city council authority, building inspection department, 1 latrine should be shared by a maximum of 5 people. we can use our estimated values of the basic reproduction number r 0 to determine the level of l and b that make r 0 v1. first, we use equation (9), the parameters in table 2 , and the value of r 0~2 :11, (linear regression) assume that contamination is due to insufficient latrines. then, for r 0~1 , this method estimates that the latrine coverage should be increased to at least 17.1%. this translates into increasing the number of latrines from 1,038 to 4,796 (i.e. 3,756 extra latrines): 1 latrine per 7 people. similarly, the boreholes should be increased from 23 to 230, that is, 17.7%, or 1 bore hole per 26 households. similar results are obtained using r 0~2 :25 found from the non-linear fitting tool. in this case of hepatitis e-only, the latrines should be increased to 16.1% and boreholes must cover 16.6% of the population. from the coinfection model, latrines should be increased to 17.5%: 4,908, (3,870 extra), 1 for 6 people. boreholes should be increased to 18.1%, a total of 234, or 1 bore hole per 26 households. our model suggests that to eradicate the epidemic, the minimum number of additional latrines required is 3,47. the average cost of digging and constructing a basic pit latrine is approximately usd 250.00 (quotation from city council official) therefore, 3,477 would cost a total of usd 869,250.00. thus, the cost per disability adjusted life year averted in kitgum, in the case of hepatitis e is 869,250/7,066 = usd 123.00. in addition to improving hygiene we should consider education. let us now consider the case of education to the camp dwellers. taking the simplest and cheapest scenario of hiring twenty (20) guidance and counseling officials to educate the dwellers about hepatitis e for about a month (that is 30) days, moving around the camp. let us assign each counselor, 10 households per day. we then calculate the total amount in usd that would facilitate such an exercise as shown in table 3 . from the calculations, it is seen that 104,000 usd would be required. assuming the success of such an operation this translates into 104,000/7,066 = usd 14.71 cost per disability adjusted life year. the epidemic of hev in kitgum lasted a period of over two years [7] . within this period, 160 individuals have lost their lives. as a result, the disease burden, the functional limitation and premature mortality have equaled a disability adjusted life years equal to 7,066, even allowing for the relatively low life expectancy in this part of uganda. this paper provides a case study of how a simple epidemic model can be fit to such an outbreak disease. two fitting methods have been used; the first, an analytical method and the other based on a freely available fitting tool. using these methods, a reliable estimate of r 0 &2:2 has been provided. we then use the model to find the measures to keep r 0 v1. the necessary levels of latrine and bore hole coverages needed to eradicate the epidemic are both around 16 to 18%. although the cost of construction of the required number of latrines is a one off cost, the benefits are large. here we show what the benefits would have been in terms of protection against hepatitis e. however, other diseases due to poor sanitation that have been reported in uganda, such as cholera and dysentery, could be prevented in the same way [7] . [35] c per capita rate of recovery from hev 0.0238-0.1429/day [11] p the proportion that died during the outbreak 160 9449 [23] b the latent period of hev 15v 1 s v64/day [26, 32] l the proportion of humans with latrines 3.7% [11] b the proportion of humans with bore hole water 0.38% [11] t outbreak of chicken flu rattles hong kong transmission dynamics and control of severe acute respiratory syndrome the epidemiology and control of malaria a contribution to the mathematical theory of epidemics inapparent infections and cholera dynamics impact of immunisation on pertussis transmission in england and wales ministry of health uganda weekly report mathematical approaches for emerging and re emerging infectious diseases: an introduction the global emergence/resurgence of arboviral diseases as public health problems emerging infectious diseases: a global fire brigade responds to disease outbreaks temporal association of acute hepatitis a and plasmodium falciparum malaria in children association of hepatitis b surface antigen carriage with severe malaria in gambian children short report: hepatitis b infection and severe plasmodium falciparum in vietnamese adults hepatitis b infection is associated with asymptomatic malaria in the brazilian amazon hostvirus interactions during malaria infection in hepatitis b virus transgenic mice the effect of antibody-dependent enhancement on the transmission dynamics and persistence of multiple-strain pathogens chaos persistence and evolution of strain structure in antigenically diverse infectious agents theoretical-studies of the effects of heterogeneity in the parasite population on the transmission dynamics of malaria the ecology of genetically diverse infections ecology and evolution of the flu refractory periods and climate forcing in cholera dynamics infectious diseases of humans: dynamics and control tracking the dynamics of pathogen interactions: modeling ecological and immune-mediated process in a twopathogen single-host system centers for disease control and prevention. hepatitis e information for health professionals modelling infectious diseases in humans and anilmals dynamical modeling and multi-experiment fitting with potterswheel reproduction numbers and subthreshold endemic equilibria for compartmental models of disease transmission the global burden of disease: a comprehensive assessment of mortality and disability from diseases injuries and risk factors in 1990 and projected to 2020 cost-effectiveness in health and medicine dual infection with hiv and malaria fuels the spread of both diseases in sub-saharan africa thank you to the eaump makerere university uganda, dr. sankaran, and the mathematical biology team of the department of mathematical sciences, bath university, uk. the authors thank prof. tom britton for many useful suggestions on an earlier version of this manuscript. analyzed the data: bn djts. wrote the paper: bn djts. model analysis: jytm lsl. we have also considered co-infection with malaria. if we assume that presence of malaria during a hepatitis e outbreak increases persistence infection, then we estimate that a malaria infective can be infected with hepatitis e up to 16 times more than one without malaria. the critical value of j determined in this study agrees with prior studies showing increased susceptibility to other infections for malaria infected individuals [37] . however, this last result is speculative and the more important point is the relationship given in figure 3 between co-infection and other model parameters. supporting information s1 linear regression on system 7. supporting information s2 the endemic stationary points for the co-infection model. supporting information s3 the invasability criterion for the co-infection model. (tex) key: cord-252739-1manzf3l authors: zheng, yueming; zhu, xuejing; zhou, pingzheng; lan, xi; xu, haiyan; li, min; gao, zhaobing title: hexachlorophene is a potent kcnq1/kcne1 potassium channel activator which rescues lqts mutants date: 2012-12-12 journal: plos one doi: 10.1371/journal.pone.0051820 sha: doc_id: 252739 cord_uid: 1manzf3l the voltage-gated kcnq1 potassium channel is expressed in cardiac tissues, and coassembly of kcnq1 with an auxiliary kcne1 subunit mediates a slowly activating current that accelerates the repolarization of action potential in cardiomyocytes. mutations of kcnq1 genes that result in reduction or loss of channel activity cause prolongation of repolarization during action potential, thereby causing long qt syndrome (lqts). small molecule activators of kcnq1/kcne1 are useful both for understanding the mechanism of the complex activity and for developing therapeutics for lqts. in this study we report that hexachlorophene (hcp), the active component of the topical anti-infective prescription drug phisohex, is a kcnq1/kcne1 activator. hcp potently increases the current amplitude of kcnq1/kcne1 expressed by stabilizing the channel in an open state with an ec(50) of 4.61±1.29 μm. further studies in cardiomyocytes showed that hcp significantly shortens the action potential duration at 1 μm. in addition, hcp is capable of rescuing the loss of function of the lqts mutants caused by either impaired activation gating or phosphatidylinositol-4,5-bisphosphate (pip2) binding affinity. our results indicate hcp is a novel kcnq1/kcne1 activator and may be a useful tool compound for the development of lqts therapeutics. kcnq (or kv7) channels are voltage-gated potassium channels. they mediate sub-threshold, noninactivating voltage-gated potassium currents that have important roles in controlling membrane excitability [1] . of the five known isoforms, kcnq1-5, kcnq1 is the only one predominantly expressed in heart. kcnq1 is the pore forming subunit, tetrameric kcnq1 complexes give rise to functional channels. in native cells such as cardiomyocytes, kcnq1 coassembles with a non-conductive accessory kcne1 subunit, a small single transmembrane protein encoded by kcne1 gene. the heteromultimeric kcnq1/ kcne1 was proposed to mediate a slowly activating current that accelerates the repolarization of action potential in cardiac tissues, also known as iks [2, 3] . loss-of-function mutations in kcnq1 lead to long qt syndrome (lqts), a severe arrhythmia characterized by an abnormality in cardiac repolarization leading to prolonged qt interval [4] [5] [6] . the severity of lqts varies from syncope to sudden death. lqts can be either congenital or acquired. more than 50% congenital lqts cases and 90% lqts occurring during exercise are linked to mutations in the kcnq1 gene. genetic studies of lqt patients have identified at least 113 kcnq1 mutations, including missense (86/113), nonsense (6/ 113), deletion (13/113), frame shift (1/113) and splice (7/113) mutations [7] . potentiation of the kcnq1 channel by small molecule activators is thought to be a potential and attractive strategy to treat lqts. recently, a number of activators of kcnq channels have been reported [8] [9] [10] [11] [12] . however, activators for kcnq1 are still rare and few are effective on the physiologically relevant kcnq1/ kcne1 complex [13] . two known examples include r-l3 and zinc pyrithione (znpy) [13, 14] . both potentiate homomeric kcnq1 channel but lack sensitivity to the kcnq1/kcne1 complex. to date, only three small molecule activators for the kcnq1/kcne1 complex have been identified. they are mefenamic acid (mfa), dids and phenylboronic acid (pba). initially, mfa and dids were identified as chloride channel blockers [15] . these compounds strongly potentiate the kcnq1/ kcne1 but exhibit little effect on homomeric kcnq1. in contrast, pba, an aromatic derivative of boronic acids, potentiates both the homomeric kcnq1 and the kcnq1/kcne1 complex with millimolar effective concentration [16] . we screened a collection of 1,280 drugs or drug candidates against homomeric kcnq channels and identified hcp as one of active compounds. hcp, also known as nabac, is the active component of phisohex, a topical anti-infective prescription drug [17] . we found that both the homomeric kcnq1 and the kcnq1/kcne1 complex were sensitive to hcp at micromolar concentrations and the effect on the kcnq1/kcne1 complex is much more potent than that on homomeric kcnq1. further studies showed that hcp was effective in cardiomyocytes and was capable of rescuing the lqts kcnq1 mutants. taken together, our study indicates that hcp as an effective kcnq1/kcne1 activator. cell culture and transfection cho cells were grown in 50/50 dmem/f-12 (gibco) with 10% fetal bovine serum (fbs), and 2 mm l-glutamine (invitrogen). to express the channels and mutants, cells were split at 24 h before transfection, plated in 60-mm dishes, and transfected with lipofectamine 2000 tm reagent (invitrogen), according to the manufacturer's instructions. a gfp cdna (amaxa, gaithersburg, md) was cotransfected to identify the transfected cells by fluorescence microscopy. fluxor thallium assay cho cells stably expressing the rat kcnq2 were routinely cultured in dmem/f12 medium, supplemented with 10% fbs and 500 mg/ml g418. the fluxor thallium assay protocol was the manufacturer's protocol. cho-kcnq2 cells were seeded in wells of 96-well plates at ,10,000 cells/well and grown until 80-90% confluence at 37uc in a 5% co 2 incubator. the medium was removed the following day and 80 ml of fluxor loading buffer was added to each well for 90 min at room temperature (rt) in darkness. after removing the loading buffer, 100 ml/well of assay buffer and 20 ml/well of 7x control/test compound were added to cells at rt in darkness. compounds to be tested were prepared using assay buffer; controls were assay buffer (ec 0 ), ec 50 of retigabine and ec 100 of ztz240. after 30 min, cell plates were loaded on fdss. after 10 seconds of recording, 20 ml/well of stimulus buffer was added. the plates were read every second for 110 s. the stimulus buffer contained 1.30 mm k2so4 and 9.80 mm tl 2 so 4 . the gpotentiation% ((r test -r control )/(r control -r buffer )*100%) was calculated for each well using the 35 second fluorescence ratio. to identify compounds with potentiation activity on kcnq channels, a thallium flux assay was developed and used to screen a microsource tm library of 1,280 compounds at 10 mm final concentration. in the pilot screening, hcp exhibited strong potentiation on the fluorescence signal of kcnq2. to record current of the expressed kcnq channels in cho cells, standard whole-cell recording was used. pipettes were pulled from borosilicate glass capillaries (tw150-4, world precision instruments). when filled with the intracellular solution, the pipettes have resistances of 3-5 megaohms. during the recording, constant perfusion of extracellular solution was maintained using a bps perfusion system (ala scientific instruments). pipette solution contained (in mm): 145 kcl, 1 mgcl 2 , 5 egta, 10 hepes and 5 mgatp (ph 7.3); extracellular solution contained (in mm): 140 nacl, 3 kcl, 2 cacl 2 , 1.5 mgcl 2 , 10 hepes and 10 glucose (ph 7.4). current and voltage were recorded using an axopatch-200b amplifier, filtered at 2 khz, and digitized using a digidata 1440a with pclamp 10.2 software (axon instruments). series resistance compensation was also used and set to 60-80%. all animal procedures were performed in accordance with the national institute of heath guide for the care and use of laboratory animals, under protocols approved and strictly followed by the institutional animal care and use committees (iacuc). the iacuc checked all protocols and approved this study. single myocytes were isolated from the left ventricle of adult guinea pig in a langerdorff perfusion system as previously described [18] . the hearts were removed quickly via midline thoracotomy and perfused with a ca 2+ -free tyrode's solution containing collagenase (6 mg/ml) and protease (0.1 mg/ml) for patch clamp data were processed using clampfit 10.2 (molecular devices, sunnyvale, ca) and then analyzed in graphpad prism 5 (graphpad software, san diego, ca). voltage-dependent activation curves were fitted with the boltzmann equation, g = g min +(g max -g min )/(1+exp(v-v 1/2 )/s), where g max is the maximum conductance, g min is the minimum conductance, v 1/2 is the voltage for reaching 50% of maximum conductance, and s is the slope factor. dose-response curves were fitted with the hill equation, e = e max /(1-(ec 50 /c)p), where ec 50 is the drug concentration producing half of the maximum response, and p is the hill coefficient. data are presented as means 6 sd. significance was estimated using unpaired two-tailed student's t tests. an effect was considered significant if p,0.05. structurally, hcp is distinct from all previously reported kcnq1 or kcnq1/kcne1 activators (fig. 1) . to obtain a better understanding for hcp activity on kcnq channels, we examined its effects on kcnq1, kcnq1/kcne1, kcnq2, kcnq3, kcnq2/3 and kcnq4 isoforms by electrophysiology. to elicit the currents, a fixed 210 mv depolarization was used for all tested channels. table 1 summarizes 10 mm hcp potentiation on the indicated outward currents. hcp potentiates all tested channels except kcnq3 (fig. 2) . noticeably, hcp significantly potentiates both kcnq1 and the kcnq1/kcne1 complex. the i/i 0 values were 1.4860.13 and 4.4760.70, respectively. the effects of hcp on g-v curves of these subtypes were examined and summarized in table 1 . the unique subtype selectivity, i.e., strong the homomeric kcnq1 mediates a characteristic outward current with detectable inactivation (fig. 3a) . in the presence of 10 mm hcp, steady-state outward currents at different depolarizing voltages were greatly potentiated. the potentiation on the kcnq1 channel mediated by previous reported kcnq1 activators, such as znpy and r-l3, involves a hyperpolarizing shift of v 1/2 . to determine the hcp effects on the homomeric kcnq1, we examined the g-v curves of the kcnq1 in the absence and presence of 10 mm hcp. before application of hcp, the v 1/2 value was 219.4361.39 mv (fig. 3e) . after application of hcp, the v 1/2 value was shifted to 249.3561.93 mv with a left-shifting around 30 mv. inactivation is a distinctive feature for kcnq1. the inhibition of inactivation by activators, such as znpy, is thought to be a contributing factor for an overall increase novel kcnq1/kcne1 activator hexachlorophene plos one | www.plosone.org of current amplitude [13] . we analyzed the inactivation kinetics at different depolarization steps and found no detectable difference before and after application of hcp (fig. 3b) . hence, hcp increases overall conductance of the kcnq1 by left-shifting the g-v curve but not affecting inactivation. in addition, hcp also induced delaying of deactivation in hyperpolarizing voltages (fig. 3c) . in the presence of 10 mm hcp, time constant of deactivation was slowed from 40.0567.52 ms to 265.51623.23 ms (n = 3, p,0.01). the reduction of deactivation rate is consistent with the overall increase of current amplitude or g max . to examine hcp modulation on kcnq1/kcne1 complex, we co-expressed the cdnas of kcnq1 and kcne1 (1:1) in cho cells. consistent with previous reports, the effects of kcne1 on kcnq1 include increase in overall current amplitude, slowing of the activation and deactivation kinetics, and removal of inactivation (fig. 4a) . we tested hcp effects at 210 mv, which is the same potential to elicit the kcnq1 channel. we found that in the presence of 10 mm hcp, after around 120 seconds, a large instantaneous current became evident. finally, the outward current was potentiated around 4.4760.70 fold, which is much higher than that of the kcnq1 (fig. 4c ). similar to effects on kcnq1, hcp also slowed the deactivation of the kcnq1/ kcne1 complex (fig. 4a ) and left-shifted the g-v curve (fig. 4b) . analysis of the dose-response curve of hcp on the kcnq1/ kcne1 complex revealed an ec 50 value of 4.6161.29 mm. because hcp potentiates the kcnq1/kcne1 complex, we tested its effects on iks in acutely isolated guinea pig cardiomyocytes. we found after application of 1 mm hcp, the iks was increased 1.3460.15 fold (n = 4) at +50 mv measured potential. the g-v curve was left shifted 27.1362.44 mv (n = 4) (fig. 5a , b & c). the effects of hcp on action potential were further examined. the action potential was elicited on isolated guinea pig ventricular myocytes by current injection. upon application of 1 mm hcp, the action potential duration (apd) was shortened by 10.2461.71%. consistently, the apd 90 (action potential duration at 90% repolarization) and apd 50 (action potential duration at 50% repolarization) were shortened by 10.5861.88% and 18.8263.65%, respectively (n = 7) (fig. 5e & f) . the inhibitory effects can not restore in five minutes after removal of the compound but can be reversed by choromonal 293b, a blocker of iks. choromonal 293b at 10 mm alone prolonged the apd around 30.0463.15% (n = 4). after co-application of 10 mm choromonal 293b, the action potential duration was restored (n = 4) (fig. 5e) . accordingly, the effects of 1 mm hcp on apd was reduced to 5.4361.54% (n = 4). hcp at 3 mm effects caused further reduction of action potential duration. the change of apd, apd 90 and apd 50 were 31.7167.3, 34.1168.09 and 37.6567.36, respectively (n = 9). r-l3, a reported iks activator which shortens apd effectively, was tested as a positive control [14] . in our system, 1 mm and 3 mm r-l3 significantly deceased the apd 50 about 9.5961.29% (n = 3) and 25.3062.29% (n = 3) (fig. 5d) , which were consistent with the previous study. thus, these data are consistent with the notion that hcp acts on the native kcnq channels. mutations in kcnq1 have been found to cause lqts [1, 19] . the common phenotype of these mutants is reduction of iks current, which are commonly thought to be mediated by the kcnq1/kcne1 complex [2, 3] , as a result of decrease in either channel activity or trafficking efficiency. hcp has exhibited potent effects on both the homomeric kcnq1 and the kcnq1/ kcne1 complex; we thus hypothesize its ability to rescue the function reduction of these mutants. we selected and expressed four mutants, r190q, t587m, r243c and r539w, which are located in different regions of kcnq1. among these mutants, r243c and r539w exhibited low but consistent currents, while the others did not exhibit detectable currents in cho cells. r243 is located near the c-terminal end of s4 transmembrane domain, while r539w is located at the c-terminal tail [20, 21] . mutations r243c and r539w were thought to impair activation gating or to reduce pip2 binding affinity, respectively. in the current study, both homomeric mutant channels were significantly potentiated by 10 mm hcp. the potentiation on r243c and r539w were comparable with that on kcnq1 wild type ( fig. 6 and table 2 ). we next tested whether hcp was effective on the mutant/ kcne1 and found that both complexes were also sensitive to 10 mm hcp. the outward currents of r243c/kcne1 and r539w/kcne1 were potentiated by 1.4460.33 (n = 5) and 2.9160.89 (n = 5) fold, respectively. the effects of hcp on voltage-dependent activation of the mutant channels were also examined and the results are shown in table 2 . besides r243c/ kcne1, the g-v curves of all tested mutant channels were leftshifted by 10 mm hcp. hcp, also known as nabac, is the active component of phisohex, a prescription drug widely used as an effective antibacterial skin cleanser in the treatment of acne [17] . in previous studies, hcp has been identified as an inhibitor of enoylacyl carrier protein reductase and 3cl protease of sars-cov [22, 23] . it was also found to attenuate wnt/b-catenin signaling pathway, which plays important roles in cell proliferation, differentiation and oncogenesis [24] . in the current study, we identified hcp as an activator of kcnq channels. among all reported activators for kcnq channels, hcp exhibits unique subtype selectivity. the potentiation on outward current for these homomeric kcnq channels at 210 mv is: q1,q2,q4. hcp lacks sensitivity to kcnq3. the wellcharacterized kcnq activator, retigabine, lacks sensitivity to kcnq1. ztz240, another kcnq activator that is distinct from retigabine and znpy, potentiates kcnq2 and kcnq4 but does not affect kcnq1 and kcnq3. although znpy shows similar selectivity to hcp, i.e. potentiates all kcnq isoforms except kcnq3, znpy lacks sensitivity to the kcnq1/kcne1 complex expressed in heterologous system [13] . here we show hcp exhibited more potent effects on the complex than that on the kcnq1. the unique subtype selectivity supports hcp may potentiate kcnq channels through a different mechanism from known activators. hcp represents a novel class of kcnq1/kcne1 activator r-l3 potentiates the homomeric kcnq1 channel at very low concentration, but does not affect the kcnq1/kcne1 complex. interestingly, potentiation effects of r-l3 on the complex seems dependent on the kcne1 to kcnq1 ratio. an increased ratio causes noticeable reduction of r-l3 activity [14] . alanine scanning revealed that r-l3 may interact with specific residues located in the s5 or s6 transmembrane domains of kcnq1 subunits [25] . consistently, our previous study showed that residues critical for the potentiation effects by kcne1 are clustered together in the s6 region in proximity with the critical residues for znpy [13] . different from r-l3 and znpy, hcp potentiates both the homomeric kcnq1 and the kcnq1/ kcne1 complex. mefenamic acid, dids and pba were reported kcnq1/kcne1 activators [15, 16] . although all three compounds exhibit potent effects on the kcnq1/kcne1 complex, similar to hcp, the phenotypes of the three activators easily differentiate hcp from them. mefenamic acid or dids also cause instantaneous opening of the kcnq1/kcne1 complex [26] . however, mefenamic acid and dids effects on the homomeric kcnq1 are much weaker than hcp. they left-shift the g-v curve and slow deactivation but do not increase overall conductance of homomeric kcnq1 [15, 27] . differently, hcp significantly increases the overall conductance of the homomeric kcnq1 (fig. 2) . pba potentiates both the kncq1 and the kcnq1/kcne1. of interest, pba exhibits inhibitory effect initially and then gradually potentiation effect on both homomeric kcnq1 and the kcnq1/kcne1 complex. these again argue that pba and hcp act via different mechanisms [16] . it is of interest that both pba and hcp have slow off rate; the effects once reaching steady state were difficult to wash off. taken together, these results suggest that hcp may represent a novel class of kcnq1/kcne1 activator. current therapy for lqts is inadequate. because the critical role of the current mediated by kcnq1/kcne1 in repolarization of cardiac action potential, augmenting kcnq1/kcne1 by small molecule may represent an attractive strategy to treat lqts. our study found that 1 mm hcp effectively potentiated native iks and left-shifted the g-v curve. iks was activated instantaneously in higher concentrations of hcp, including 3 mm or 10 mm. the effects of hcp on shortening the action potential duration in cardiomyocytes further supports its potential use for development therapeutics for lqts either as a tool compound or as a lead compound. the slight reduction of the action potential amplitude suggests that hcp may affect other channel(s). most of lqtscausing mutations were from kcnq1 among the identified lqtassociated genes including kcnq1, herg,scn5a, kcne1 and kcne2 [4, 5, 19] . these mutations cause reduction in the potassium current, thereby leading to prolongation of cardiac repolarization [7, 28, 29] . many mutations of lqts are trafficked to the plasma membrane, suggesting no major folding defects, but reduction of activity [7, 29] . among the tested mutants, r190q is a novel identified mutation located at the linker of s2-s3. it is thought to affect the activation gating of kcnq1/kcne1. r587m is a trafficking deficient mutation located at the cterminal end. loss of function of r190q and r587m is consistent to previous reports [30, 31] . r243c is a missense mutation located in s4, a transmembrane segment implicated in activation gating of potassium channels [20] . the suppressed current of r243c/ kcne1 is consistent with the notion that the mutation prevents normal channel gating. the additional mutant r539w was identified in patients with a dominantly inherited classical long qt (romano-ward) syndrome. r539 is located at the c-terminal tail and shows a reduced pip2 binding affinity [21] . it has been demonstrated that pip2 is necessary for the function of various ion channels including kcnq channels [32, 33] . a recent study showed that the pip2 affinity of r539w/kcne1 is reduced [34] . potentiation of both r243c and r539w suggests hcp is capable of restoring loss-of-function caused by various mutations, either with impaired gating (r243c) or reduced pip2 affinity (r539w). however, the effects of hcp on the kcnq1/kcne1 mutant channels are weaker than that on wild type kcnq1/kcne1 channel. for the wild type kcnq1/kcne1 channel, 10 mm hcp dramatically left shifts the g-v curve around 266.0566.15 mv. in contrast, the left shifting for kcnq1(r539w)/kcne1 is only 229.3562.74 mv, and furthermore, in the case of kcnq1(r243c)/kcne1, the shifting was negligible. considering physiological efficiency of an activator of kcnq channel more relies on its effect on the g-v relationship, the weaker or loss of effects of hcp on the g-v curves of these kcnq1/kcne1 mutant channels should be considered as major factors in developing therapeutic for lqts. in conclusion, we identified hcp, the active component of a prescription drug, as a potent kcnq channel activator with unique subtype selectivity. it exhibits strong potentiation effects on both the homomeric kcnq1 and the kcnq1/kcne1 complex. the distinct pharmacological phenotype and chemical structure of hcp from other reported activators suggest that hcp represents a novel class of kcnq1/kcne1 activators. the activity of shortening the action potential duration and the ability to rescuing the loss-of-function of lqts mutants make hcp a useful tool in development of therapeutics for lqts. kcnq potassium channels: physiology, pathophysiology, and pharmacology k(v)lqt1 and lsk (mink) proteins associate to form the i(ks) cardiac potassium current coassembly of k(v)lqt1 and mink (isk) proteins to form cardiac i(ks) potassium channel mechanisms of i(ks) suppression in lqt1 mutants evidence for a cardiac ion channel mutation underlying drug-induced qt prolongation and life-threatening arrhythmias spectrum of mutations in long-qt syndrome genes kcnq1 gene mutations and the respective genotype-phenotype correlations in the long qt syndrome neural kcnq (kv7) channels activation of kv7 (kcnq) voltagegated potassium channels by synthetic compounds activation of kcnq2/3 potassium channels by novel pyrazolo [1,5-a] pyrimidin-7(4h)-one derivatives the therapeutic potential of neuronal kv7 (kcnq) channel modulators: an update kv7 channels as targets for the treatment of pain desensitization of chemical activation by auxiliary subunits: convergence of molecular determinants critical for augmenting kcnq1 potassium channels a novel benzodiazepine that activates cardiac slow delayed rectifier k+ currents meclofenamic acid and diclofenac, novel templates of kcnq2/q3 potassium channel openers, depress cortical neuron activity and exhibit anticonvulsant properties discovery of a novel activator of kcnq1-kcne1 k+ channel complexes soap bacteriostats mitochondrial atpsensitive potassium channels inhibit apoptosis induced by oxidative stress in cardiac cells the genetic basis of long qt and short qt syndromes: a mutation update long qt syndrome-associated mutations in the s4-s5 linker of kvlqt1 potassium channels modify gating and interaction with mink subunits impaired kcnq1-kcne1 and phosphatidylinositol-4,5-bisphosphate interaction underlies the long qt syndrome evaluation of metal-conjugated compounds as inhibitors of 3cl protease of sars-cov inhibition of the staphylococcus aureus nadph-dependent enoyl-acyl carrier protein reductase by triclosan and hexachlorophene hexachlorophene inhibits wnt/beta-catenin pathway by promoting siah-mediated beta-catenin degradation pharmacological activation of normal and arrhythmia-associated mutant kcnq1 potassium channels the role of the isk protein in the specific pharmacological properties of the iks channel complex stilbenes and fenamates rescue the loss of i(ks) channel function induced by an lqt5 mutation and other isk mutants molecular biology of arrhythmic syndromes pathophysiological mechanisms of dominant and recessive kvlqt1 k+ channel mutations found in inherited cardiac arrhythmias novel mutations in kvlqt1 that affect iks activation through interactions with isk characterization and subcellular localization of kcnq1 with a heterozygous mutation in the c terminus regulation of ion channels by phosphatidylinositol 4,5-bisphosphate pip2 is a necessary cofactor for ion channel function: how and why? kcne1 enhances phosphatidylinositol 4,5-bisphosphate (pip2) sensitivity of iks to modulate channel activity key: cord-012461-v8d91fdo authors: marnissi, boutheina; kamali-moghaddam, masood; ghram, abdeljelil; hmila, issam title: generation of ssdna aptamers as diagnostic tool for newcastle avian virus date: 2020-08-13 journal: plos one doi: 10.1371/journal.pone.0237253 sha: doc_id: 12461 cord_uid: v8d91fdo aptamers are short single-stranded dna (ssdna), rna or synthetic xna molecules, which are used as a class of affinity binders recognizing target molecules with a very high affinity and specificity. the aim of this study was to generate and characterize ssdna aptamers for the detection of newcastle disease virus (ndv). these aptamers were selected using systematic evolution of ligands by exponential enrichment (selex) in combination with quantitative high-throughput dna sequencing. after three rounds of selections, a highly enriched ssdna pool was sequenced, and the results were analyzed using fastaptamer toolkit. sequencing reads were sorted by copy numbers and clustered into groups, according to their sequence homology. top aptameric sequences were used to develop a sandwich enzymatic linked aptamer assay (elaa) for rapid and sensitive detection of ndv in farm samples. the selected aptamers have an affinity within the nanomolar range, and a high specificity with no cross-reactivity towards other avian viruses. following optimization of the sandwich elaa method, the results demonstrated that both selected aptamers apt_ndv01 and apt_ndv03 with dissociation constant values of 31 nm and 78.1 nm, respectively, showed the highest specificity and affinity for ndv detection. the elaa results were verified by quantitative real-time pcr, demonstrating strong concordance, and showing outstanding accuracy for detection of ndv in field sample. in summary, combination of selex with high-throughput dna sequencing allowed rapid screening and selection of aptamers. the selected aptamers allowed recognition of ndv with high affinities. this is the first report that uses a validated sandwich elaa for rapid and specific detection of ndv in poultry samples. newcastle disease (nd) is an acute and highly contagious avian disease, which causes heavy economical losses to poultry industry, worldwide [1] . the causative agents are virulent strains of avian paramyxovirus type 1 (apmv1). in regard to their pathogenicity for chickens, nd virus (ndv) strains are divided into three pathotypes; velogenic (highly fatal), mesogenic (intermediate virulence) and lentogenic strains (low virulence). some members of the latter, such as lasota and v4 subtypes are used as live vaccines [2] . the virus belongs to the genus avulavirus of the family paramyxoviridae, in the order of paramyxovirinae. the viral genome is a non-segmented, negative-sense and single-stranded rna of approximately 15.2 kilobases (kb), encoding six main proteins; nucleoprotein (n), phosphoprotein (p), matrix protein (m), large polymerase protein (l), hemagglutinin-neuraminidase (hn) glycoproteins, and fusion protein (f) [1, 3] . the glycoproteins hn and the f protein are located on the surface of the viral membrane, and induce neutralization antibody production. they enable the virus to attach to sialic acid-containing receptors on the host cells, and are responsible for the neuraminidase activity [4] . the f protein is responsible for the fusion of the viral envelope with the cellular plasma membrane [5] . these two proteins are also considered as biomarkers for ndv. the ndv infects wild birds and poultry species. it has been reported as a major problem for the poultry sector in latin america (mexico, colombia, venezuela, and peru) [6] , in iran [7] and several other countries, worldwide. however, data on the prevalence of the disease are insufficient in tunisia, and the virus continues to affect the profitability of poultry production. biosecurity measures and vaccination programs should be enforced to ensure better control and mitigate virus incidence, avoiding the emergence of more pathogenic strains. to monitor this major viral disease and to avoid health crisis in poultry, the development of sensitive assays for early detection, enabling characterization of circulating viruses is needed and will have great economical and epidemiological significances. appropriate diagnosis is the key factor for better handling viral diseases. nevertheless, many avian viral diseases are difficult to distinguish, especially at the disease onset with a set of similar and nonspecific clinical symptoms. to develop efficient tools for the diagnostic of these major viral diseases, the potential of aptamers was explored as an affinity binder for ndv. aptamers are short single-stranded dna (ssdna), rna, synthetic xna oligonucleotides or peptide molecules, which can recognize their targets with high affinity and specificity [8] . their production, using an experimental procedure termed selex (systematic evolution of ligands by exponential enrichment), is easy and inexpensive [9] . since their discovery, the aptamers have rapidly emerged as a key factor in several biological processes such as diagnostic, therapy, drug discovery, food science, and drug delivery [10] . several studies have been conducted on the aptamers' potential use for the diagnostic of different diseases including cancer and infectious diseases [11, 12] . recently, aptamers against influenza virus have been demonstrated as an potential alternative tool to antibodies with equal or greater affinity to ha antigen [13, 14] . new aptamers against h9n2 influenza subtype have been characterized in our laboratory and have been used to develop a highly sensitive real-time immuno-pcr-based test [15] . similarly, characterization of aptamers against other viruses including respiratory syncytial virus (rsv), severe acute respiratory syndrome virus (sars) and measles virus have already been reported [16] . however, to date there is no reports on aptamers developed for avian virus detection. the present study reports on a rapid and an efficient approach for the selection and characterization of ssdna aptamers against ndv. the generated aptamers were then tested for their affinity and specificity to efficiently detect the viral antigens in farm samples. no vertebrate animals, embryos or tissues have been involved in the present work. for validation of the generated aptamers, the leftover chicken samples received for routine diagnostics, were used. no further ethical approval was obtained as no laboratory animals were used in this study. lasota vaccine strain of ndv, live h120 vaccine strain of infectious bronchitis virus (ibv), live ibdv vaccine strain of gumboro virus, attenuated live 1133 strain of avian reovirus and live avian influenza tunisian isolate a/ck/tun/145/12 subtype h9n2 were used for the specificity test. the study was performed on a total of 27 poultry samples including tracheal (et) and cloacal swabs (ec) and internal organs consisting of allantois (a), kidneys (k), lung (l), liver (li) and trachea (t) collected from chickens with suspected ndv infection. the field samples were collected from eight farms located in the following provinces: bizerte, nabeul, ben arous, sidi bouzid, beja, ariana, sfax and jendouba, and were sent by veterinarians to the diagnostic laboratory at institute pasteur of tunis for analysis, in the frame of their routine follow-up/monitoring of poultry farms, or in case of reporting a disease suspicion, especially for the diagnostic of ndv and avian influenza, which are under continuous surveillance in tunisia. samples information including the region, the veterinary or/and farm or/and company name as well as type of production and age of poultry are summarized in s1 table in s1 file. the ndv-bound ssdna aptamers were selected from a ssdna library, using a slightly modified selex protocol reported by hmila et al. and arnold et al. [15, 17] . the random dna library (wap40m) consisted of 80 nucleotides (nt) containing a 40 nt randomized central region flanked by two adaptor sequence regions each of 20 (integrated dna technologies, inc., coralville, ia). the library sequence-5'-agtgcaagcagtattcggtc-(n) 40 -taaa gctgatgcgtgatgcc-3'-was used as reported by lamont et al. [18] . before selection, the ssdna library was denatured by heating at 95˚c for 5 min then cooled on ice for 10 min to prevent inter-strand base pairing. selex was initiated with the immobilization of 100 μl of diluted lasota vaccine strain in 96-well microtiter plate overnight at 4˚c. the plate was then dry blotted, and 100 μl of the single-stranded aptamer dna library (10 μm) was mixed with the virus and incubated for 1 h with gentle shaking. the wells were dry blotted, and then 100 μl of phosphate-buffered saline (pbs; ph 7.2; 150 mm nacl + 5mm mgcl2) was added and incubated for 5 min at room temperature (rt). a nacl salt gradient was used for the elution of the enriched aptamers; the wells were sequentially washed using five different concentrations of 0.5, 1.0, 1.2, 1.4, and 1.5 m nacl, allowing gradual releasing of aptamers, starting from those with the weakest to strongest binding affinities. the molecular basis for this phenomenon is mainly related to the nacl's ability to sever electrostatic interactions, the key interactions between the aptamers and the protein [19] . then, 2 μl of the isolated dna oligonucleotides from the 1.5 m elution was amplified by pcr, and was used to recognize the virus pre-immobilized on a membrane nitrocellulose. products from the aptamer-spots were extracted and a symmetric pcr amplification was performed with an initial heating step for 5 min at 95˚c followed by 25 cycles of 95˚c for 30 s, 55˚c for 30 s, 72˚c for 30 s and a final extension step at 72˚c for 7 min. the reaction mixture contained 5 μl of 10x pcr buffer, 3 μl of selex pool, 0.5 μl (10 μm) of each forward wp20f1 (agt-gca-agc-agt-att-cgg-tc) and reverse wp20r1 (taa-agc-tga-tgc-gtg-atg-cc) primers (integrated dna technologies, inc., coralville, ia), 1 μl mgcl 2 (50 mm; thermofisher scientific), 1 μl dntp (10 mm; thermofisher scientific), 0.5 μl taq polymerase (thermofisher scientific), and the volume was adjusted to 50 μl with deionised water. an asymmetric pcr was performed using 2 μl symmetric pcr products, and an optimal ratio of forward and reverse primers of 25:1, respectively. the thermocycles were initiated with a heating step for 5 min at 95˚c followed by 9 cycles of 95˚c for 30 s, 63˚c for 15 s, 72˚c for 15 s, and a subsequent 10 cycles of 95˚c for 30 s, 55˚c for 15 s, 72˚c for 15 s, and a final step of 72˚c for 3 min. the reaction mixture was composed of 5 μl of 10x pcr buffer, 2 μl of symmetric pcr product, 1.2 μl of wp20r (1 μm), 3 μl of wp20f (10 μm), 1 μl of mgcl 2 (50 mm), 1 μl of dntps (10 mm), 0.5 μl taq polymerase in a total volume of 50 μl, adjusted with deionised water. the pcr product of the selex amplified products using wp20f1 primer was purified with the minelute1 pcr purification kit (qiagen). the sequencing library was conducted using the ab library builder™ system (thermofisher scientific) and amplified according to the ion xpress™ plus and ion plus library preparation for the ab library builder™ system protocol. it was then purified using the agencourt1 ampure1 xp reagent (beckman coulter). the library size and its concentration were assessed by a bioanalyzer high sensitivity chip (agilent technologies). the samples were pooled, followed by a template preparation on the ion chef™ system, using the ion pi hi-q chef kit (thermofisher scientific). the samples were then loaded on an ion pi™ v3chips and sequenced on the ion proton™ system, using the ion pi™ hi-q sequencing 200kit chemistry (thermofisher scientific). the dataset analysis allowed identification and removing of the specific 5 0 -and 3 0 -primer binding regions in each sequence, using a command line. subsequently, the sequences were filtered by discarding those longer than 50 nt and keeping the intern randomized regions of the original aptamer sequences. the majority of these sequences had an expected length of 40 ± 3 nt after pre-processing and filtration of the aptamer dataset. fastaptamer toolkit was used following the same steps previously described [19] . first, the fastaptamer-count package was used to rank and sort the sequences by their abundance by counting the number of times each sequence is sampled from a population, according to their copy numbers. second, the fastaptamer-cluster was used to align and classify the reads by sequence similarity. finally, sorted sequences were evaluated for their affinity and specificity. selected ssdna molecules were subjected to secondary structure prediction, using mfold software (http://mfold.rna.albany.edu/?q=mfold/dna-folding-form). the elaa was employed to test the binding affinity of the selected aptamers to ndv, using the following procedure. lasota vaccine virus was diluted in pbs and 100 μl were used to coat the wells of a 96-well microtiter plate at 4˚c overnight. the wells were then rinsed three times with pbs-t (10 mm pbs, ph 7.2; 0.05% tween-20) and blocked with 200 μl/well of 5% skim milk at rt, for 1 h. next, a volume of 100 μl/well of a 2-fold dilution serial of 5'-biotin-labeled dna aptamers from 10,000 to 0.15 nm was added to ndv coated wells and incubated at rt for 1 h. a naïve library at 10 nm in 100 μl was used as a non-specific binding control. the wells were then washed three times with pbs-t to remove unbound aptamers. streptavidinhorseradish peroxidase conjugate (1:5000) was added, and incubated at rt for 30 min. after five washes with pbs-t, opd (o-phenylene diamine dihydrochloride) was added for visualization. finally, the reactions were stopped by adding 50 μl/well of h 2 so 4 (2n), and the optical densities (od) were recorded at 492 nm, using a microplate reader. to determine the equilibrium dissociation constants (k d ) for the candidate aptamers, the od value mean of a naïve library was subtracted from the od value mean of each aptamer reading. to evaluate their specificity for ndv, the binding of the five selected aptamers apt_ndv01 to apt_ndv05 were tested against the lasota live vaccine strain of ndv, the h120 live vaccine strain of ibv, the isolate a/ck/tun/145/12 (h9n2) isolate of ia, ibdv vaccine strain of gomboro disease, attenuated 1133 strain of reovirus and a naïve library as negative control as described above. all the experiments were performed in triplicate. a sandwich elaa was carried out in streptavidin coated plates (thermofisher scientific), using one of the biotin-labeled apt_ndv02, apt_ndv03, apt_ndv04 or apt_ndv05 as a capture, and digoxigenin-labeled apt_ndv01 as a reporter molecule. each 96-well microtiter plate was coated with 100 μl of 10 nm biotin-aptamers and incubated at rt for 1 h, washed three times with 200 μl of washing buffer pbs-t (10 mm pbs, ph 7.2; 0.05% tween-20). next, lasota vaccine virus was added in 100 μl pbs (ph 7.2) and incubated for 1 h at rt. wells were then washed five times with pbs-t and 1 μm of the reporter digoxigenin-modified apt_ndv01 diluted in 100 μl pbs was added, and the incubation was continued for an additional 1 h. anti-digoxigenin antibody (1:2000) was added to the corresponding wells to react for 30 min, followed by three washes. finally, opd was added before stopping the reaction by adding h 2 so 4 (2n). the absorbance was measured at 492 nm and the results of each combination were calculated as the mean ± sd from three experiments. a total of 27 samples from poultry suspected to be infected with ndv, including tracheal (et) and cloacal swabs (ec) and internal organs consisting of allantois (a), kidneys (k), lung (l), liver (li) and trachea (t) were analyzed. the samples were assessed for ndv using the sandwich elaa, in streptavidin coated microtiter plates. first, 100 μl of 10 nm biotin-apt_ ndv03 were incubated at rt. after 1 h incubation, the wells were washed three times with 200 μl of pbs-t, and 1 μl of each farm sample diluted in 100 μl pbs was added into each well, and incubated for 1 h at rt. after three washes with pbs-t, 100 μl of 10 nm digoxigenin-labeled apt_ndv01 was added, incubated for 1 h at rt and the plate was washed five times. the anti-digoxigenin antibody (1:2000) was then added to each well and incubated for 30 min. after three washes, a solution of opd was added before the reaction was stopped by h 2 so 4 (2n), and the absorbance were recorded at 492 nm. one step qrt-pcr was conducted in a total volume of 12 μl, using agpath-id™ one-step rt-pcr kit (applied biosystems tm) by mixing 7.5 μl 2x rt-pcr buffer, 0.6 μl of 25x rt-pcr enzyme mix, 2 μl rna template, 0.3 μm of each forward (fr-5 0 -tygagggactt gaaygttgac-3') and reverse primer (rv-5 0 -cctgaggagaggcatttgcta-3') specific to polymerase (m) gene [20] and 0.2 μm taqman™ probe (p: 5 0 fam-ttctctagcagtgg gacagcctgc-tamra-3'). the reactions were conducted by heating the mixture at 45˚c for 10 min followed by 95˚c for 10 min and 45 cycles of 95˚c for 15 s and 60˚c for 45 s. a negative control lacking the rna template was included in each pcr run. lasota vaccine strain titrating 10 6 eid 50 /ml, was used to determine the limit of detection (lod) of qrt-pcr. a 100 μl volume of the vaccine was treated to extract the viral rna, which was used to prepare a two-fold serial dilution, and then the qrt-pcr was performed as described above. the data was calculated to determine the lod of the assay. also, to determine the lod of the sandwich elaa, an aliquot of 10 6 eid 50 /ml of lasota vaccine strain was used to prepare two-fold serial dilutions from which 100 μl was added to each well of a microtiter plate pre-coated with 100 μl of 10 nm biotinylated apt_ndv03. the plate was incubated at rt for 1 h, the wells were washed three times with 200 μl of washing buffer pbs-t, and then, 1 μm digoxigenin-apt_ndv01, diluted in 100 μl pbs, was added to each well. after 1 h incubation at rt, anti-digoxigenin antibody (1:2000) was added and the incubation continued for an additional 30 min. the wells were then washed once with pbs-t, the opd was added and the optical densities were recorded at 492 nm. statistical significance of the sandwich elaa and qrt-pcr results was determined by oneway anova test, using simple inter-active statistical analysis (sisa) online tool (http:// www.quantitativeskills.com/sisa/index.htm), and defined as significantly different if < 0.01. for more relevant statistical significance analysis, a 95% confidence interval (ci) of the mean, a range with an upper and lower number calculated from a sample was determined by oneway anova test. the mean and the standard deviation (sd) and the coefficient of variation percentage (cv %) of optical densities (od 492 nm) for sandwich elaa and the threshold cycle (ct) of qrt-pcr were further analyzed, using excel software. to measure the relative variability between triplicates of one sample, cv% was calculated as cv% = (stdev of the sample triplicates /mean of the sample triplicates) � 100 and defined as significantly different if cv% < 20%. statplus pro version 5.9.8 was used to calculate the lod, the lloq, the uloq, the mdd and the sensitivities of both methods. the lod is the lowest analyte concentration that can be accurately differentiated from the background and at which its detection is feasible. a conventional and standard lod estimation method consists on the measurement of replicates of background or blank sample, the determination of the mean values and sd and the calculation of the lod as the mean + 2 sd [21] . the lod was calculated as lod = background signal + (3 x sd mean ), where the background signal corresponds to the mean value of three negative control samples; the sd mean being the standard deviation of those values. additionally, the lower limit of quantification (lloq) is the lowest concentration at which the analyte can be accurately identified and at which certain predefined bias and imprecision targets are achieved. furthermore, the lloq value may be comparable to the lod or at a higher concentration. lloq were determined as lloq = lod + (10 x sd background ). the upper limits of quantification (uloq) was determined as uloq = f (x-(3 x sd x ), whereas the minimal detectable dose (mdd) was determined as mdd = 2 x sd background mean . dna-aptamers against ndv were generated using the selex protocol described by hmila et al. [15] . briefly, the lasota virus vaccine was incubated with a randomized dna pool and washed. the output of the selex was amplified by symmetric (s1 fig in s1 file) and asymmetric pcr, and the final pcr products were incubated with the virus immobilized on a nitrocellulose membrane, using an immune-blot test to monitor the enrichment of target-binding aptamers. the products from the aptamer-immobilized spots were subsequently used for the next rounds of selection. after three rounds of selex, an increased fluorescence signal was observed ( s2 fig in s1 file) . then the dna pool was sequenced using high-throughput sequencing. the dna sequence pools (s2 table in s1 file) were analyzed using fastaptamer software. the pre-processed sequences were filtered and then analyzed by fastaptamer-count to sort the reads based on their abundance, which is normalized for reads per million (rpm) and ranked according to the decreasing abundance (s3 table in s1 file). the results showed that the most abundant sequence was sampled 7,620 times, corresponding to 1,362.29 rpm. fas-taptamer-count output was used as fastaptamer-cluster input. using the command"fas-taptamer_cluster," closely-related sequences were grouped into clusters (s4 table in s1 file). as shown in table 1 , each generated random sequence was sorted by its rank, reads, rpm, cluster number, rank within that cluster and levenshtein edit distance that reflects the number of insertions, substitutions, or deletions between sequences. from the analyzed pool, the first five sequences with high rpm were selected for further characterization. to evaluate the binding affinity of selected aptamers to ndv, the virus was incubated with increasing concentrations of biotin-labelled aptamers, and subsequently analyzed by elaa, based on a nonlinear regression equation. the results showed that the five selected random sequences of aptamers have affinities at the lowest nanomolar range, and the aptamers apt_ndv01 and apt_ndv04 demonstrated the highest affinities (fig 1 and table 2 ). the specificity of the five selected aptamers, apt_ndv01-05, against ndv was evaluated by testing their affinities against various avian viruses besides ndv-lasota vaccine strain, including h120-ibv, ibdv-gomboro, 1133 reovirus, avian influenza-h9n2, and a naïve library which was used as a negative control. all five aptamers showed high specificity for ndv, while no affinity for the other virus strains was observed (p-values < 0.01; fig 2) . the performance of the apt_ndv01, in combination with the other four selected aptamers, was tested using the elaa. the results demonstrated that the apt_ndv01 in combination with apt_ndv03 was the most effective, closely followed by the apt_ndv01/apt_ndv02 combination as compared to apt_ndv01/apt_ndv05, which shows lower interaction with the antigen; no detectable interaction with the negative control was observed (fig 3) . a combination of dig-apt_ndv01 and biotin-apt_ndv03 was selected for their higher specificity and capture affinity for the detection of ndv in farm samples (s3 fig in s1 file) . to compare the sensitivity as well as the performance of both sandwich elaa and qrt-pcr methods, the optical densities (od at 492 nm) for sandwich elaa and the threshold cycle (ct) of qrt-pcr were used to determine the lod, the lloq, the uloq, the mdd and the sensitivities of the both methods ( s5 fig in s1 file) . as expected, the dynamic range for qrt-pcr was greater than that for sandwich elaa, which also demonstrates better lod, lloq, ulod and mdd. the analytical characteristics of both assays are summarized in (table 3) . to evaluate the ability of the selected aptamers to detect ndv in farm samples, 27 samples collected from suspected chickens were analyzed by the developed sandwich elaa, using the apt_ndv03 as a capture and the apt_ndv01 as a reporter affinity binder. the results were compared to those obtained by qrt-pcr as the golden standard method. out of the 27 analyzed farm samples, 15 were revealed positive by the elaa-based method, which were confirmed by the standard qrt-pcr method, indicating the good accuracy of the sandwich elaa test (table 4 ). in recent years, aptamers have been investigated as an alternative means for their bio-affinity and target recognition, which has become one of the promising nano-molecules with great interest for medical needs. the use of aptamers is widely extended, as therapeutic molecules, for the development of biosensor or specific delivery means of active molecules [22] . the aptamers are functional short chains of nucleic acids of 20 to 90 bases. their folding into a variety of 3d structures offers a large area of antigen recognition, allowing them to be powerful agents for targeting and binding to any type of molecules, ions, or whole cells. their specific recognition of targets and their high affinity in the range of nano-or sub-nanomolar range [23] make them very powerful tools for bio-recognition. they are often compared to antibodies with desirable and higher properties. they are chemically derivable and controllable, with low highly specific aptamers against ndv. the specificity of five aptamers with the highest affinity for ndv were tested using elaa to detect either vaccine strain of ndv-lasota, h120-ibv, ibdv-gomboro, 1133 reovirus and avian influenza-h9n2. a naïve library was used as a negative control. the test was performed in triplicate. p-values for apt_ndv01-05 were < 0.01, calculated using one-way anova test. https://doi.org/10.1371/journal.pone.0237253.g002 immunogenicity and quite high physical stability [24, 25] . their production is quite inexpensive and reproducible. another major advantage of aptamers is that they can be generated and selected in vitro by selex from a dna library of approximately 10 15 randomized sequences. selex is a robust combinatorial chemical screening in vitro method and selection process table 4 . cross-tabulation between sandwich elaa and qrt-pcr. results of the diagnosis of ndv in tracheal (et) and cloacal swabs (ec) and internal organs, consisting of allantois (a), kidneys (k), lung (l), liver (li) and trachea (t) collected from chickens with suspected ndv infection. results of sandwich elaa and qrt-pcr are reported as positive or negative for each sample. may extend between two to four weeks, which is significantly short in comparison with generation of specific antibodies (months). here, we describe the first study that identifies and characterizes dna aptamers specific to ndv, a major pathogen for poultry industry. dna aptamers were chosen rather than rna ones because of their stability and easier usage in the field. different selex protocols for the generation of dna aptamers against various targets have previously been reported. one of the described protocols, based on high salt concentration for elution of the binders, and limited number of amplifications of the aptamers that bind to the target on the dot blot was used in this study. this protocol that was first reported by arnold et al. [17] and later was successfully used in our previous work with some modification [15] , allowed generation of aptamers using very few selection rounds in comparison to other protocols, which often require up to 10 rounds of selection [26, 27] . here, the selex was performed using three rounds of selection. the first step was the elution of high affinity aptamers using high concentration of nacl. the sequential increased concentration of nacl allows gradual release of the aptamer species starting from those with the weakest to the strongest affinity by disturbing the electrostatic interactions. the selex was followed by an immuno-blot test, where the eluted dna oligonucleotides from the 1.5 m of nacl was amplified by pcr and used to recognize the virus immobilized on a nitrocellulose membrane. the specific aptamers were extracted from the aptamer-spots and then amplified. this step of amplification, isolation on blot and extraction of specific aptamers was repeated three times to enrich the highly specific aptamers with no requirement for counter selex. during the selex procedure, the eluted dna were amplified using asymmetric pcr. this represented one of the most essential steps in the selection process. if it is not controlled correctly, the amplification of the selected aptamers might give rise to a complete loss of the desired sequences [28] , and a failure of the selection process [29] . different protocols for the optimization of the pcr process have been used [30, 31] . in our approach, the enriched dna library from the third round of selex was sequenced using high-throughput sequencing, and analyzed by fastaptamer tool. high throughput sequencing was used instead of conventional cloning and sequencing, since high throughput sequencing is a powerful approach allowing identification of higher affinity and specificity of ssdna aptamers that might otherwise not be discovered through the conventional method [32] . after sequencing, the data were first processed using ion proton suite software, which performs base calling and quality filtering. after the control quality test, data were secondly processed using simple command line to scan sequences for forward and reverse primers and the insert length. in fact, proper reads with 80 nt, including 20 nt of 5'primer + 40 nt random sequence + 20 nt 3' primer, were sorted. concatemeric sequences containing only primer sequence were filtered out leaving 40 nt for downstream analysis, using different packages of fastaptamer toolkit. this software is open source licensing and designed for non-bio-informatics analyst. fastaptamer includes a collection of scripts that perfectly perform basic analysis steps for all combinatorial selections, regardless of the technology used for the selection process. thus, we identified five aptamers, which were analyzed and evaluated for their potential use in the sandwich elaa as a diagnostic assay. affinity and specificity tests were performed using dc-elaa, a variant of enzymelinked immuno-sorbent assay (elisa), which has been used to study protein-protein interactions [33] . dc-elaa results demonstrated that the five selected aptamers bound selectively to ndv with apparent k d values of 24.33 nm to 85.12 nm. elaa technique has previously been used to detect various targets such as thrombin, m. tuberculosis and cocaine detection [34] [35] [36] , but to our knowledge, this technique has not been used to detect avian viruses, in particular ndv. the elaa approach may be conducted under different configurations such as direct, indirect and sandwich detection test. compared to direct and indirect elaa, the sandwich elaa seems to be more accurate and stable, depending on the capture aptamers' efficiency for target recognition, but not on the immobilization, the purity or the concentration of the target [37] . the performance of elaa has previously been shown to be comparable to that of elisa technique, and it has been reported as a powerful tool for the diagnostic of viral diseases such as zika, bovine parainfluenza and influenza subtype a [38] [39] [40] . in our work, the sandwich elaa test was developed to allow detection of ndv in complex matrices without the need for any purification steps nor sample preparation. such approach has also been used in other studies to successfully detect, for instance, thrombin, m. tuberculosis and cocaine with greater performances as compared to assays utilizing antibodies [41, 42] . the sandwich elaa was initially evaluated using lasota vaccine strain and total ndv antigen from farm samples. optimal conditions, including time and temperature of incubation, as well as buffer composition and aptamer concentration were established, allowing the best detection efficiency. additionally, we found that the use of a combination of apt_ndv01 and apt_ndv03 in a sandwich elaa resulted in the best performing assay to detect and measure different levels of ndv with significant signals over a very low background. furthermore, the elaa based on apt_ndv01 and apt_ndv03 aptamers showed high specificity for ndv, and we did not observed any false positive signals when the assay was tested on other live avian viruses such as h120-ibv, ibdv, 1133 reovirus vaccine strains as well as avian influenza isolate -h9n2. however, further validation on larger sample cohorts and other viral agents may be needed. the obtained results using the sandwich elaa, when a total of 27 field samples were analyzed, perfectly correlated with the results from current golden standard qrt-pcr method. the advantages of the sandwich elaa are the rapid and reproducible production of synthetic aptamers as probes instead of antibodies, and the lack of requirement for sample preparation and viral genome extraction. in summary, we report the great performance of the combination of selex and high throughput sequencing for rapid screening and characterization of aptamers. dna aptamers targeting ndv were successfully selected after three rounds of evolved enrichment, using lasota vaccine as a target along with a selex process. fastaptamer toolkit was used to analyse the results obtained with the five selected aptamers. binding analysis revealed that selected aptamers recognize ndv with high specificity and with nano-molar affinities. the present study is the first report that uses validated novel aptamer-based sandwich detection method to detect ndv in field samples. supporting information s1 file. (pdf) newcastle disease and other avian paramyxoviruses pathogenesis of newcastle disease in chickens experimentally infected with viruses of different virulence sequence analysis of fusion protein gene of newcastle disease virus isolated from outbreaks in egypt during antigenic mapping and functional analysis of the f protein of newcastle disease virus using monoclonal antibodies rescue of newcastle disease virus from cloned cdna: evidence that cleavability of the fusion protein is a major determinant for virulence epidemiology, control, and prevention of newcastle disease in endemic regions: latin america. trop anim health prod prevalence of avian influenza, newcastle disease, and infectious bronchitis viruses in broiler flocks infected with multifactorial respiratory diseases in iran selection of aptamers against pathogenic bacteria and their diagnostics application systematic evolution of ligands by exponential enrichment: rna ligands to bacteriophage t4 dna polymerase aptasensors as the future of antibiotics test kits-a case study of the aptamer application in the chloramphenicol detection new prostate cancer targets for diagnosis, imaging, and therapy: focus on prostate-specific membrane antigen screening of nucleic acid aptamer of lung cancer cells based on cell exponential enrichment ligand system evolution and its application in tumor diagnosis and treatment rapid and highly sensitive method for influenza a (h1n1) virus detection exploiting enzyme catalysis in ultra-low ion strength media for impedance biosensing of avian influenza virus using a bare interdigitated electrode a novel method for detection of h9n2 influenza viruses by an aptamer-real time-pcr advancements in nucleic acid based therapeutics against respiratory viral infections one round of selex for the generation of dna aptamers directed against klk6 a combined enrichment and aptamer pulldown assay for francisella tularensis detection in food and environmental matrices fastaptamer: a bioinformatic toolkit for high-throughput sequence analysis of combinatorial selections development of a real-time reverse-transcription pcr for detection of newcastle disease virus rna in clinical samples limit of blank, limit of detection and limit of quantitation investigations on the interface of nucleic acid aptamers and binding targets increased inhibitory ability of conjugated rna aptamers against the hcv ires evolution of a t7 rna polymerase variant that transcribes 2'-omethyl rna new ntp analogs: the synthesis of 4'-thioutp and 4'-thioctp and their utility for selex magnetic separation-based multiple selex for effectively selecting aptamers against saxitoxin, domoic acid, and tetrodotoxin selection of aptamers by systematic evolution of ligands by exponential enrichment: addressing the polymerase chain reaction issue emulsion pcr: a high efficient way of pcr amplification of random dna libraries in aptamer selection development and validation of a new pcr optimization method by combining experimental design and artificial neural network optimization and troubleshooting in pcr quantitative selection of dna aptamers through microfluidic selection and high-throughput sequencing selection, characterization and interaction studies of a dna aptamer for the detection of bifidobacterium bifidum identification and application of ssdna aptamers against h(3)(7)rv in the detection of mycobacterium tuberculosis a self-assemble aptamer fragment/target complex based high-throughput colorimetric aptasensor using enzyme linked aptamer assay a colorimetric sandwich-type assay for sensitive thrombin detection based on enzymelinked aptamer assay aptamers as a replacement for antibodies in enzyme-linked immunosorbent assay. biosens bioelectron screening and identification of ssdna aptamers against hn protein for detection of bovine parainfluenza virus type 3 antibodies in serum an aptamer-based electrochemical sensor that can distinguish influenza virus subtype h1 from h5 selection of dna aptamers that bind to influenza a viruses with high affinity and broad subtype specificity displacement enzyme linked aptamer assay an enzyme-linked oligonucleotide assay our thanks are addressed to the institute pasteur of tunis, the ministry of health and the ministry of higher education and research of tunisia for their invaluable support towards the study. we are also grateful to the swedish scilifelab genome center, uppsala, for their valuable assistance with sequencing. key: cord-011798-uss38ped authors: li, guowei; luo, zhe; anwar, muhammad; lu, yuqiu; wang, xiantao; liu, xuening title: intellectual capital and the efficiency of smes in the transition economy china; do financial resources strengthen the routes? date: 2020-07-02 journal: plos one doi: 10.1371/journal.pone.0235462 sha: doc_id: 11798 cord_uid: uss38ped intellectual capital has been grabbed the attention of researchers due to its momentous role in sustainable competitive advantage and organizational success. there is a growing catalog of related assessments, publications and reviews that display the direct and indirect role of intellectual capital in business success and profitability. despite the bourgeoning literature, studies have not yet unleashed the influence of each dimension of intellectual capital; human capital, structural capital and customer capital on smes' efficiency with financial resources as a moderator. the present study fills the gap and assesses if financial resources strengthen the paths between the dimensions of intellectual capital and smes' efficiency. a survey method was used and collected evidence from 264 chinese smes. the findings exhibit that human capital directly enhances smes' efficiency but the presence of financial resources as a moderator weakens the influence. however, social capital and customer capital do not directly improve smes' efficiency but financial resources reinforce the paths social and customer capital and smes efficiency. this research recommends that owners and managers of smes need to use their financial resources complementary with structural and customer capital while human capital should be used exclusively. steered by globalization and advanced technology, many firms (especially in emerging economies) persistently search to discern drivers that can configure their performance and survival in the dynamic markets. doubtlessly, a variety of tangible (land, material, capital and technology etc.) and intangible (knowledge, r&d and reputation, etc.) have been recognized that spur business profitability (e.g, [1] [2] [3] ). considering the nature and characteristics of small and medium enterprises (smes), as they have a deficiency of resources, lack of support and smallness (e.g. [4] , typically make them unable to invest a huge amount of money in risky and physical assets. this sensation encourages them to recognize alternative opportunities (less risky, convenient and easy to adopt) in order to enhance their survival in the markets [5] . since the last decade, studies have shown their interest in discovering factors that can significantly boost and the path to smes performance such government support [6] , knowledge sharing [7] , creativity [8] , innovation [9] and it capabilities [10] and entrepreneurial orientation [11] etc. studies have also discussed the role of intellectual capital (ic) (human capital, customer capital and structural capital) in firm competitive advantage and performance in emerging and developed economies [12, 13] also, studies have assessed the direct and indirect influence of ic on performance [14] [15] [16] . nevertheless, it is still unclear "how each dimension of ic; human capital, customer capital and structural capital contributes to smes efficiency"? this research is an attempt to examine the influence of each dimension of ic on smes' efficiency. though, ic may not significantly contribute to efficiency as some studies pointed out the indirect influence of ic on performance [17] [18] [19] . in this regard, smes may need some other factors that can support and transpire ic into high efficiency. for instance, cheng and krumwiede [20] argue that all the intangible factors do not significantly enhance performance in environmental uncertainty but firm resources required to configure the situation. we argue that financial resources can strengthen the link between ic and smes' efficiency. this research, on the other hand, examines the moderating role of financial resources between each dimension of ic and smes' efficiency. there are ample evidence that confirm the significant role of ic in the financial performance of enterprises. for instance, [21] argued that efficient use of ic facilitates smes in exploiting growth opportunities and gaining high financial performance. [22] demonstrated that the dimensions of ic; human capital, structural capital and relationship capital are very vital for efficiency in the banking industry in the long run. alhassan and asare [23] also revealed that human capital and capital employed to boost the productivity of banks in emerging economies. similarly, oppong and pattanayak [24] revealed that productivity and competitive advantage of an enterprise are no longer dependent on tangible resources but heavily rely on the intangible resource, particularly ic. this notion has been supported by several authors. for instance, kamath [25] scrutinized that human capital is the most significant predictor of productivity in indian business organizations. additionally, mondal and ghosh [26] state that the banking industry enhances its efficiency through human capital. however, some studies shed light on the importance of structural capital in the business industry. bontis, janošević [27] scrutinized that many business firms rely on structural capital because it articulately works for them in improving productivity. soriya and narwal [28] indicated that many organizations configure their employee productivity through structural capital. there are several reasons why financial resources can significantly moderate the relationship between ic and efficiency. for instance, considering the high failure ratio of smes across the globe, studies have reported the major reason of lack of finance (e.g [29, 30] ). having enough finance enables smes to boost their operational activities effectively. for instance, fonseka et al, [31] demonstrate that out of many resources, financial capital is the most prominent for smes in emerging economies. moreover, cooper, gimeno-gascon and woo [32] also describe that human and financial capital help newly established ventures as a shield to respond to the external threats and changes. thus, financial resources enable firms to get benefits from the ic and transform their skills into the best use. additionally, some firms are unable to enter into profitable markets due to a lack of finance. thus sufficient finance encourages them to seize the benefits of new markets [31] . adequate financial capital facilitates firms in opportunity recognition that is helpful for efficiency e.g. [33] some firms are unable to effectively use their skills and resources [34] . in this case, financial capital, as a driver can assist managers to reorganize the resources and skills in a way to gain high profit [35] . this research aims to explore the role of financial resources in firm efficiency as well as how financial resources strengthen the relationship between ic and smes' efficiency. alternatively, as described earlier, this research aims to recognize less risky sources and resources that enable smes to gain efficiency in the turbulent markets. this research assesses the resource base view (rbv) theory which tells the role of tangible and intangible resources in firm competitive advantage and superior performance [36] . more interestingly, rbv theory gives more emphasis to intangible resources over tangible in terms of high performance [2, 36] . in this perspective, this research argues that intangible resources e.g. ic can give a greater advantage over tangible resources especially in emerging economies. this research is based on smes operating in emerging market china where more than 95% of firms are smes. the country heavily relies on smes but still, they face big pressure and a high failure ratio (e.g, [37] ). the findings of this research facilitate owners, managers and policy-makers to give due attention to the survival and performance of smes, so they will enable them to make prominent contributions to economic growth, gdp and propensity. there is a great need for financial institutions and smes banks in the country in order to provide interest-free loans or where enterprises can borrow a loan at a lower interest rate. efficiency is defined as "using minimum input to gain maximum output" there . smes across the globe face a shortage of resources [2] . therefore, they need to strengthen their ic strategy in order to compete in the market and gain technical efficiency. b. economies of scale: it is sometimes called cost advantage where a firm increases its production that minimizes the cost. when an enterprise produces more-it becomes more efficient and thereby lowering the costs. this type of efficiency is very popular in both small and large firms as both firms try to gain maximum benefits at a lower cost. however, in particular, small firms are more cost concentrated because of the deficiency of resources [2] . they intend to reduce different types of costs, resulting in high profitability [42] . c. technical change: this type of productivity explains production differential. it measures the change in efficiency between the current and next period (t-t+1). it is not necessarily technological change but can be occurred by the change in regulation, quantities of inputs and price of inputs etc. this type of efficiency is also very crucial in smes because of the internal structure that is not restricted they often modify their strategic posture when they see any benefits or loss [43] . they are more inclined towards change and innovation, thereby emphasizing on high profitability and outputs [9] . rbv theory [36] sheds light on the prominence of tangible and intangible resources in the realism of sustainable performance in a turbulent market. indeed both the resources are very crucial for high performance, productivity and competitive advantage [44] . however, recent studies have claimed that tangible resources are no longer work for productivity and sustainable competitive advantage in smes but intangible factors are more crucial (e.g., [24] ; [37]. this notion is supported by several studies [45] ; [2] . ic is deemed an intangible factor that has been remained a prominence piston in smes for profitability, sustainable positon and exploiting opportunities [21, 46] ). the reasons behind the significance of intangible resources (hereby ic) in smes are deficiency of resources in smes, smallness and lack of capacity to invest in tangible resources [45] . for instance, kraja [47] conducted a study check the comparative importance of tangible and intangible resources and revealed that intangible resources are more powerful for smes' performance. khan, yang [2] also argued that investment in intangible resources enables emerging smes to gain sustainable competitive advantage and superior performance in the dynamic markets. indeed, intangible resources are less expensive but give more advantages [37] . therefore, smes emphasize intangible resources, especially ic to enhance their position in the market [48] . human capital-a dimension of ic facilitates enterprises in efficient utilization of resources, thereby resulting in maximum benefits [49] . investment in human capital is one of the most critical decisions of enterprises because it significantly leads to productivity [50] . kong and kong [51] also dissected that human capital significantly and positively increases the productivity of private and public enterprises in china. haris, yao [52] displayed that structural capital significantly underpins the profitability of the banking sector in the emerging market pakistan. according to [53] (p. 139-140) "structural capital is, what romer would call "ideas", because it can be replicated at a large scale and low costs. this implies that structural capital is the true force behind productivity. furthermore, leal-millán, roldán [54] describe that customer capital is the key to sustainability and performance in a turbulent environment because customer knowledge gives superior advantages. to summarize, all the considerable ample evidence indicates that ic-being an intangible asset plays a crucial role in the productivity of smes that is the main theme of the rbv theory. even though ic has been growing since the last several decades but still universal definition is not yet confirmed, rather it involves more rigorous conceptualization in theory and practice. most managers and scholars have vague concepts about how to manage invisible resources; human capital, customer capital and structural capital [55, 56] . according to stewart [57] , ic as the intellectual material of information, knowledge, experience and intellectual property that can be utilized to create wealth. studies have discussed various dimensions that can encompass ic such as human capital, relational capital, customer capital, structural capital, organizational capital and social capital etc. (e.g [58, 59] . however, many studies agreed on the three dimensions; human capital, customer capital and relational capital that are used in this research. ic is an essential part of firm activities related to financial and non-financial. it affects the internal processes and reporting system of firms that are aligned to profitability and effectiveness [60] . according to a 'resource-based view' of competition, ic is considered as an important source of competitive advantage. in prior studies, efficiency is measured in two dimensions; technical efficiency and cost-efficiency. ic as knowledge, intellectual property, information, analytical skills, competencies and expertise that are used by a company to enhance its competitive advantage that ultimately influences shareholders' wealth [61] . in the current turbulent markets, companies face a dramatic change and high pressure from the external environment. in these circumstances, ic such as human capital, structural capital and relationship capital is the best strategy sustain for the long run and gain high efficiency [62] . ic is a good type of activity that helps the organization to use the knowledge effectively to gain high financial performance and efficiency [63] . though some studies have scrutinized that all the dimensions of ic are not significantly related to firm competitiveness and efficiency. for instance, yaseen, dajani and hasan [64] claimed that only relational and structural capital significantly enhance a firm competitive position. besides, knowledge in the economy or an organization can be circulated through human, structural or relationship capital-components of ic. well managed ic can help firms to gain success and competitive advantage in the markets that cannot be achieved by competitors and industry rivals [65] . firms often use hrm practices for smooth operation. however, ic can be a prominent factor for highly innovative and competitiveness [66] . ic is very necessary for manufacturing firms as they are engaged in manufacturing activities that required high analytical and intellectual abilities. ic help manufacturing firms to redesign their internal structure and strategic process which in turn facilitate firms to gain efficiency [20] . a firm efficiency can be improved by several factors but intangible resources (especially ic) can significantly enhance efficiency as compared to other factors [67] . firms, especially in emerging markets, face several problems including lack of resources and capabilities. hence, they adjust the shortcomings through ic to gain a highly competitive position and effectiveness over a longer period [68] . in a competitive market, where a firm is trying to be highly innovative and successful, it needs efficiency that can be gained through social capital [69] . it is argued that firms should emphasize on intangible resources (e.g., ic) to achieve effectiveness and innovative performance in a dynamic market [20] . all the dimensions of ic (human, structural and relational capital) are significantly positively related to firm efficiency [70] . recently, ic has deemed an essential intangible asset for business organizations, especially in those business industries that are characterized by advanced technology and high intensive knowledge capital. in addition, ic is used to configure the business's success and to encourage innovativeness, creativity, value creation and competitive edge among firms [71] . to summarize, ic provides capabilities and resources to build a competitive advantage for organizations. a firm without using ic may not be able to gain sustainable competitive advantage and superior performance in a specific market or industry and without a competitive advantage, a firm can quickly exit the industry [48] . therefore; organizations possess various resources e.g. tangible and intangible that are either directly or indirectly influenced their performance, competitive advantage and survival [72] . ic is regarded as an intangible asset within organizations that can spur their performance [73, 74] . it is argued that human capital significantly contributes to firm value and success but the relationship can be affected by a firm internal and external factor [75] . considering the indirect influence of ic toward performance and profitability, we argue that financial resources can significantly tight the link between ic and firm efficiency. financial capability is considered the ability or capacity of a business to use financial resources as a medium of exchange for other productive resources [76] . the rbv theory suggests that a venture needs tangible and intangible resources to acquire sustainable status in the market [36] . hence, we perceive that ic and financial resources should work combine for enterprises to seize a competitive position and satisfactory efficiency. for instance, tseng, lin and yen [77] claimed that venture processing activities are influenced by both ic and financial capital. similarly, yan and ning [78] also scrutinized that the relative importance of ic and financial capital in business values and argued that a firm should not exclude any of these because both are very crucial for high corporate value. it is doubtless that intangible resources are essential for smes' growth and survival in transition and emerging economies. however, mere intangible resources do not boost operational activities of smes, they must have satisfactory finance to recognize new opportunities to enhance their innovative and financial performance [79] . the efficient use of financial resources saves ventures from wastage of capital in different investment activities and enables firms to utilize the resources properly [80] . other resources and capabilities facilitate business activities effectively. however, financial resources and quality financial management boost operational activities in the business sector [81] . when a venture tries to recognize new opportunities in a competitive market, it needs financial and non-financial resources. though the resources such as market and entrepreneurial orientation facilitate the discovery of opportunities for earning profitably, but financial resources amplify the paths towards high earning [82] . for instance, consider the theory of arbitrage finance [83] , that criticizes the role of external capital due to a low level of performance and gives worth to internal financial resources due to speed progress during high environmental uncertainty. hence, we align our model to the theory because our model also sheds light on internal capital that can configure other resources (e.g. ic) for business operations. this argument is supported by kaleka [84] who scrutinized that a firm with greater financial resources enjoys superior performance due to the unique role of the capital in effectively constituting other resources. for instance, penrose, [85] securitizes that a firm's resources such as financial capital, talented managers and knowledge etc. are the key inputs into the production process, value creation and high efficiency. indeed enough resources are very crucial for smes' success. however, financial capital is deemed very important in spurring a firm's efficiency and growth [86] . a firm with greater access to financial resources enables them to use their internal resources smoothly and articulately that can give high competitiveness and profit [87] . financial resources not only enable firms to recognize and exploit the internal opportunities but also help them to reconfigure their internal process to react external opportunities appear in international markets to improve their profit [88] . adequate finance enables firms in access to unique and scarce resources that are essential for growth and smooth running of operational activities [89] . having strong financial resources can encourage firms to get into a more risky situation which in turn provide higher financial benefits [90] . ic is created through the exchange and combination of intangible resources that may be characterized as explicit or implicit knowledge within organizations. consequently, organizations need to differentiate themselves and perform tasks and actions differently in order to succeed in the markets [91] . hence, a sustainable competitive advantage does not come automatically by offering final products and services to the customers but it comes from the resources that produce them. resources here also deemed financial and other that can reconfigure ic towards high performance and competitiveness. as pointed out by hunt and [92] , competitive advantage will not continue unless a firm uses its resources efficiently and effectively to deliver values to a specific segment in the markets. it posits that a firm needs to develop value-creating strategies from its sources for improving sustainable efficiency [36, 93] . one significant factor, especially in smes, is financial capital that does not only help to a successful venture in the long run but also shields them from unexpected loss [32] . therefore, we argue that an enterprise needs adequate finance that should be used combined with ic to gain desirable efficiency in the competitive market. consequently, in the way that the association will be stronger when enterprises have sufficient financial resources. h4. financial resources strengthen the association between structural capital and smes' efficiency in the way that the association will be stronger when enterprises have sufficient financial resources. h4. financial resources strengthen the association between customer capital and smes' efficiency in the way that the association will be stronger when enterprises have sufficient financial resources. the conceptualized model is illustrated in fig 1. this research is based on smes operating in the transition economy china. we targeted smes from three major cities; beijing, shanghai and shenzhen because majority ventures have their head offices in these regions. unlike the email survey which gives a lower response rate, we used a hard copy approach for data collection. it is difficult to estimate the number of enterprises in these regions. hence we used the nonprobability method and followed a convenience sampling method for the collection of the data. a total of 600 questionnaires were distributed among the firms (200 in each city). we requested owners and top managers as they are the people who know the financial position and strategic activities of their firms. additionally, where managers were not aware of exact financial figures, they recommended us to ask financial managers regarding efficiency and financial outcomes. we asked them that the survey is volunteer to be filled. to reduce social desirability bias, we have clearly mentioned in the covered letter of the survey that the data of this survey is exclusively used for research analysis, and the information will not be shared elsewhere. since chinese managers face difficulty in understanding an english version questionnaire, therefore we translated the questionnaire in the chinese language that was approved by the committee for research. once the questionnaire was approved by the committee of sichuan university china, we started data collection from the enterprises. in the questionnaire, it was ensured that the data (collected through this questionnaire) will be used only for research purpose and the firm information will not appear intellectual capital and the efficiency of smes; do financial resources strengthen the routes? anywhere else. we followed a hard copy approach as it gives a desirable response rate as compared to an online survey. managers and owners were asked to fill the survey voluntarily and during the busy schedule, we requested to fill the survey later. we received back 285 questionnaires but some of these questionnaires were incorrectly filled. intellectual capital. ic in this study is used as an independent variable. to derive useful insights, we used each dimension of ic; human capital, customer capital and structural capital. prior studies have used various dimensions to measure ic. the items for human capital, customer capital and structural capital were adopted from liu [14] and jain, vyas and roy [48] respectively. financial resources. financial resources indicate a firm capacity to have finance and capital to be used for the smooth running of operational activities. to measure fa, we used five items adopted from the prior study memon, an and memon [79] . firm efficiency. in large firms, researchers typically use a formula output/input to measure efficiency. smes do not publish their financial information with the general public which creates the problem in measuring their performance and efficiency. in this regard, researchers have recommended self-reported measures because they have several advantages over objectives measures; • it is difficult to obtain a financial statement and financial records of smes because they do not publicly share their financial information [94] . • several studies have confirmed a significant match in the results of self-reported and archived data used for performance [95, 96, 97] . • semrau, ambos [98] claimed that self-reported measures give better results as compared to archived data in emerging economies. therefore, we focused on a self-reported measure for the efficiency of the smes. to measure efficiency, owners/managers were asked how they use the least resources for maximum outputs in terms of return on investment, return on assets and return on sales, technology on production etc. the items were measured using five points likert scales illustrating strongly disagree 1 to strongly agree 5. however, for measuring efficiency, we used strongly declined 1 to strongly improved. control variables are used to attenuate the chances of spurious results in a research study. in smes research, the nature of the industry, age and size of the enterprises and educational background of top managers and owners are suggested by several studies to be controlled [95] ; khan et al., 2019; [45] . following the suggestions of these studies, we also control these particular variables in our structural model when testing the hypotheses. the nature of the industry is a categorical variable, hence we created group difference analysis to check it as a controlling factor. we created three groups; manufacturing1, trading2 and services3 and then compared the results of each group with others. we did not realize a significant difference between the groups, hereby dropped the nature of industry from the control list because of a minor role. however, the age and size of the enterprises and educational background of the owners/managers are the continuous variables that are used as a control in the structural model. our results displayed that the size of the enterprises does not play a significant role while the age of the firms and qualification of the top managers and owners indicate a significant role in the hypothesized model. we used spss to check the mean (m), standard deviation (s.d.), multicollinearity and normality of the data. our study shows (see table 2 respectively. considering the skewness and kurtosis, our data are normally distributed because all the constructs have acceptable skewness and kurtosis values ±2 as suggested by george [99] . moreover, there is no threat of multicollinearity in our study because there is no exceed value (above 3) of variance inflation factor (vif) and lower value (below 0.10) of tolerance in our sample [100] . hence, the descriptive statistics of the data are satisfactory and acceptable. the relationship between the factors is given in table 3 . scholars do not accept or reject hypotheses on the results of correlation values but it just initially supports the findings. it illustrates that hc, customer capital and financial resources are significantly positively related to efficiency (r = 0.175, r = 0.187 and r = 0.175) respectively. however, the association between structural capital and efficiency is not significant (r = 0.091). the correlation results confirm that the sample has no problem with multicollinearity because all the values are below 0.80 [101] . data gathered through closed-ended questions, from the same respondents and at the same time face common method variance problem [102] . hence, it is essential to test the common method variance problem in the data set. we used harman's single factor test in spss to check the threat of common method variance. our findings revealed only five factors of which the first factor displayed only 24.15%. this variance is in the acceptable range (below 50%) as recommended by [102] . however, harman's single factor test is criticized for lack of validity. therefore, we tested the impact of a common latent factor on the measurement model and compared the results of the models (one with a common latent factor and one without the common latent factor). we discovered that the fitness of the old model (without the common latent factor) are significantly better than the new model (with the common latent factor) which clarifies that our sample is free of common method variance. intellectual capital and the efficiency of smes; do financial resources strengthen the routes? early response and late response in a data set can cause non-response bias which can affect results [103] . there were 157 early responses and 107 late responses (after they had given a reminder) in our sample. after calculating mean values of the variables; human capital, structural capital, customer capital, financial resources and the efficiency of smes, we applied the t-test to assess if there is any non-response bias in the data. we compared the results of the two groups (late and early responses), we realized that the p-value of t-tests (greater than 0.05) did not indicate a significant difference between the groups, hereby confirmed the absence of non-response biases. the validity (convergent and discriminant) and reliability (see fig 2) of the constructs are checked through confirmatory factor analysis in amos. however, first, we guaranteed the fitness of the model and revealed that cmin/df = 2.804 provided satisfactory value (e.g. below 3) as per the suggestion of hinkin [104] . gfi = 0.81, cfi = 0.87, tli = 0.85 and nfi = 0.81 displayed acceptable values as followed by previous studies [104, 105] . additionally, rmr = 0.023 and rmsea = 0.079 are desirable (below 0.09) as argued by steiger [105] . we found (see table 4 ) that all the standardized factor loadings of the items (after divided by the average number of items toward each factor) provided satisfactory values for convergent validity (above 0.50) and discriminant validity (above 0.70, after taking the square root of ave). finally, composite reliability (c.r.) was assessed which exhibited pleasing value (above 0.70) as advocated by bagozzi [106] . a structural model was applied in amos for testing the hypothesized model that is given in fig 3. the main reason to apply a structural model is that it allows researchers to test all the hypotheses in a single [107] . hence, we tested all the paths (direct as well as interacted) via a single structural model. we found (see table 5 ) that human capital significantly contributes to smes efficiency (β = 0.163, c.r = 2.705, p = 0.007) that supported h1. structural capital does not significantly affect smes efficiency (β = -0.089, c.r = -1.560, p = 0.119) which did not support h2. customer capital also did not display a significant influence on smes efficiency (β = 0.049, c.r = 0.766, p = 0.444) that did not favor h3. considering the moderating role of fr, our findings exhibited that financial resources as a moderator significantly reduce the influence of human capital on smes efficiency (β = -0.030, c.r = -2.879, p = 0.004) which opposed the proposed hypothesis 4. however, our study confirmed that financial resources significantly strengthen the path between structural capital and smes efficiency (β = 0.037, c.r = 3.242, p = 0.001) as well as between customer capital and smes efficiency (β = 0.033, c.r = 3.131, p = 0.002) that supported h5 and h6 respectively. all the control factors; educational background, size and age of the enterprises exposed a significant role in the model. r square revealed that only 19% variance in the smes' efficiency is explained by ic in the presence of the moderating role of financial resources as well as the control factors. since we have a moderator (fr) between ic and smes efficiency in the model. we used unstandardized beta, m and s.d. of the factors to draw the interaction term. fig 4 shows that low and high financial resources do not strengthen the path between human capital and ef. however, high financial resources reduce the influence of human capital on efficiency even if there is high human capital with a venture. the p-value for the interaction term of hcxfr is significant 0.004 (see table 5 , opposite to hcxfr) which confirms that financial resources significantly moderate the link between human capital and efficiency of smes. fig 5 shows that low financial resources slightly strengthen the path while high financial resources significantly strengthen the link between structural capital and smes' efficiency. the p-value of the interaction term 0.001 displays the significant role of financial resources between structural capital and the efficiency of smes. intellectual capital and the efficiency of smes; do financial resources strengthen the routes? fig 6 displays that both low and high financial resources significantly improves the influence of customer capital on smes' efficiency. however, high financial resources show more strength in the influence of customer capital on smes' efficiency. the p-value of the interaction term ccxfr is 0.002, hereby confirms that financial resources significantly strengthens the association between customer capital and smes' efficiency. we also performed a robust check-in spss by applying regression analysis. our findings displayed some differences in terms of the interaction results as compared with the structural intellectual capital and the efficiency of smes; do financial resources strengthen the routes? model because, in the regression model, two insignificant interactions; hcxfr and ccxfr were reported as shown in table 6 . we conducted an in-depth interview with five managers/owners of the smes to enhance validity of the results and especially to know if there is social desirability bias in the data. we asked the following questions in the interview to get open ended answers. 2. ans: three managers shed light on the importance of their ic for high performance and efficiency while two firms replied that their ic averagely contribute to their performance. 3. how your existing financial resources moderate the path between ic and efficiency? 4. ans: four managers said that they get high benefits of ic when financial resources work as a moderator while one manager said that my firm faces financial constraint, and we are unable to get desirable efficiency. intellectual capital and the efficiency of smes; do financial resources strengthen the routes? 5. generally, do you agree that your performance and efficiency depend on ic and financial resources? 6. ans: three managers were agreed and said that ic and financial resources are the key to performance and efficiency in the current competitive markets. however, two managers said that other types of resources; it, online business activities and reputation. 7. to summarize the interview results, we hereby confirm that overall, the results match with the survey data where the dimensions of ic and financial resources are considered crucial factors of high efficiency and performance. based on the importance of intangible resources over tangible, this research tested the influence of each dimension of ic on firm efficiency with a moderating role of financial resources. our findings have new implications for practicing managers and policymakers. these implications are different from previous studies. for instance, in the past, one zone of the research relied on tangible resources [108] [109] [110] , while others, especially more recently give more weightage to intangible resources [2, 3, 45] in term of high performance and growth. this research collected empirical evidence from an emerging economy to discover how the intangible resources; human capital, customer capital and relational capital influence firm efficiency and how financial resources affect the relationship. consequently, this research favors rbv theory where more weightage is given to intangible resources over tangible resources toward competitive advantage and superior performance. though, the prior studies have mostly focused on the relationship between intangible resources and competitive advantage and performance. however, this research assessed the relationship between each dimension of ic and firm efficiency. in this research, we argue that ic-being an intangible resource significantly intellectual capital and the efficiency of smes; do financial resources strengthen the routes? contributes to efficiency when ventures have sufficient financial resources. as pointed out by the rbv theory, both tangible and intangible resources are crucial for venture success and profitability. our study scrutinized that only intangible resources are not worthy, but there should be sufficient tangible resources (e.g. finance) to enjoy high efficiency in the competitive market. we found that human capital significantly enhances firm efficiency that supported h1 of the research. in line with mehralian et al., [111] who scrutinized that human capital is the most significant predictor of competitive advantage in the business industry. similarly, veltri and silvestri [112] argued that human capital is a noteworthy factor in high growth and value in business firms. however, our study shows that financial resources significantly reduces the influence of human capital on smes' efficiency that is different from other studies. our study opposes chen, hsu and chang [113] who claimed that human capital helps managers to manage resources that in turn spurs their degree of internationalization and firm growth. similarly, our findings are different from delery and roumpi [114] who claimed that human capital provides a sustainable competitive advantage in the presence of sufficient resources and valuable opportunities. the reason for the negative moderation is that managers in smes are unable to use financial resources efficiently due to a lack of financial skills. for instance, klapper, lusardi and van oudheusden [115] claimed that the lowest rate of financial literacy is reported in emerging economies such as pakistan and china. another reason that the majority of smes are controlled by agents rather managed by the owners directly. perhaps the agents do not effectively manage the financial resources for operational activities. our study displayed that structural capital directly does not improve smes' efficiency unless the ventures have sufficient fr. in contrast to long kweh, lu and wang [70] who scrutinized that structural capital significantly contributes to smes' efficiency. our study also opposes andreeva and garanina [116] who exhibited a significant positive association between structural capital and values in russian firms. however, our findings are related to bontis, ciambotti, palazzi and sgro [117] who exposed that structural capital does not significantly influence corporate social performance. however, we found that financial resources significantly reinforce the impact of structural capital on smes' efficiency in the emerging market china. our findings support buckley et al., [87] who established that financial resources enable firms to manage and structure internal resources articulately that result in high efficiency and growth. our analysis revealed that customer capital does not directly mend smes' efficiency in emerging markets. unlike yang and kang [118] who reported a significant positive association between customer capital and business profitability, our study displays an insignificant path in emerging smes. however, our findings favor bianchi martini, corvino, doni and rigolini, [119] who did not find any significant association between relational capital and enterprises value. similarly, andreeva and garanina [116] also did not scrutinize any significant connection between relational capital and business profitability. our findings partially favor pedro, leitão and alves [120] who scrutinized that customer capital positively influences profitability but this relationship is fortified in the existence of other factors, resources and surroundings environment. in a similar vein, luo, griffith, liu and shi [121] exposed that hc, structural capital and customer capital influence financial performance but these links are moderated by ownership of the business. we argue that the direct relationship between ic and smes' efficiency is questionable, it can be moderated by venture internal resources. this research provides imperative operational guidelines for managers and owners of smes to improve their venture efficiency through ic and fr. first, smes should improve their human capital as it directly configures business efficiency in the dynamic markets. however, this study revealed that financial resources weaken the influence of human capital on smes' efficiency, hence it the management of financial resources should be investigated. perhaps managers cannot effectively use the financial resources for the operational activities due to a lack of financial skills. therefore, it is owners to assign financial investment to financial literature managers, because they can manage the capital in a better way [122] . another suggestion derived from this research is that managers with interpersonal human skills and with a lack of financial skills should not take responsibility for capital investment. they should use nonfinancial resources and information to enhance their venture efficiency. however, our study displays that structural capital and customer capital do not directly improve smes' efficiency unless there are adequate financial resources existed. hence, ventures need to bring sufficient finance to boost their operational activities because mere structural capital and customer capital do not help in high efficiency. smes should not rely directly on their structural capital and customer capital but they should first manage sufficient finance to get maximum benefits and efficiency. smes should not exclusively use structural capital and customer capital for efficiency goal but managers/owners should balance their finance that can strengthen the path between structural capital, customer capital and efficiency. it is also suggested for smes that financial capital should not be directly invested in projects because it does not significantly enhance efficiency. ventures need to use the financial resources as well as structural capital and customer capital complementary to configure their efficiency. the implications are not limited to smes but listed firms can also apply it for their objectives and goals. similarly, venture trade in developed markets can also get advantages of this study by modifying their strategic posture in terms of ic and capital. some of the key implications are highlighted below. ➢ smes need to use their human capital exclusively for enjoying high efficiency in the turbulent market. they should not use financial resources and human capital combine to getting efficiency and profitability in the emerging market china. ➢ however, structural capital and customer capital should not be used exclusively, but ventures should manage sufficient finance to get satisfactory efficiency via structural capital and customer capital. ➢ financial resources do not enhance efficiency directly, but it must be used with structural capital and structural capital to acquire a desirable advantage. despite having significant contributions of this research to rbv theory and the literature of ic, financial resources and firm efficiency, this research has some limitations that are needed to be addressed in future studies. the model is tested in the transition market china that may not be deemed the best representative of other emerging markets-hereby suggested to extend this model in other emerging markets. moreover, developed markets can be confirmed to enhance the validity of the model if intangible resources are very prominent for smes' efficiency. this research is based on empirical evidence that are collected from smes through a structured questionnaire, future researchers can use open-ended questions and interviews (with several managers instead of only a few, as done in this study) that can give useful information in this nature of studies. moreover, to reduce social desirability bias, the survey can be filled from different source or other person and other control factors can be applied to reduce the chances of spurious results. large firms can be surveyed or data from financial reports and statements can be collected for testing the model. in this study, the only moderating role of financial resources is checked. though, the other resources and capabilities such as technology and market knowledge can give fruitful insights. moreover, to avoid common method bias, a longitudinal study can be conducted in future studies. we tested the moderating role of financial resources between the dimensions of ic and smes efficiency and revealed unexpected results. we suggest considering the financial knowledge and financial literacy of owners/managers to discern if it is worthy of high efficiency in the presence of ic. we controlled the age and size of the enterprises in this research. however, it will better to unleash how small size, middle size and large ventures as well as new and matured ventures get benefits of ic in the presence of fr. we further suggest assessing the role in organization competitiveness, innovative and environmental performance if financial resources facilitate these outcomes. the present study tests the influence of the intangible resources (human capital, structural capital and customer capital) on smes' efficiency with a moderating role of financial resources. we surveyed 264 chinese smes via a self-administrated questionnaire to test the model. we used amos to test the hypotheses and generate results. our study exposed that only human capital directly improves smes' efficiency while structural capital and customer capital do not directly spur efficiency. however, we found that financial resources as a moderator reduce the influence of human capital on efficiency while significantly improve the influence of structural and customer capital on smes' efficiency. our study suggests that smes should use human capital exclusively while structural and customer capital should be merged with financial resources to get higher efficiency. our study also advises the chinese government to initiate financial programs for industrial growth, so the enterprises will able to borrow loan for their operational activities. supporting information s1 appendix. 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reporting and company performance: evidence from europe intellectual capital and performance: taxonomy of components and multi-dimensional analysis axes the effects of customer relationships and social capital on firm performance: a chinese business illustration is knowledge that powerful? financial literacy and access to finance: an analysis of enterprises in the uk key: cord-012891-heqsfzkm authors: blanco vázquez, cristina; alonso-hearn, marta; juste, ramón a.; canive, maría; iglesias, tania; iglesias, natalia; amado, javier; vicente, fernando; balseiro, ana; casais, rosa title: detection of latent forms of mycobacterium avium subsp. paratuberculosis infection using host biomarker-based elisas greatly improves paratuberculosis diagnostic sensitivity date: 2020-09-03 journal: plos one doi: 10.1371/journal.pone.0236336 sha: doc_id: 12891 cord_uid: heqsfzkm bovine paratuberculosis (ptb) is a chronic granulomatous enteritis, caused by mycobacterium avium subsp. paratuberculosis (map), responsible for important economic losses in the dairy industry. current diagnostic methods have low sensitivities for detection of latent forms of map infection, defined by focal granulomatous lesions and scarce humoral response or map presence. in contrast, patent infections correspond to multifocal and diffuse types of enteritis where there is increased antibody production, and substantial mycobacterial load. our previous rna-seq analysis allowed the selection of five candidate biomarkers overexpressed in peripheral blood of map infected holstein cows with focal (abca13 and mmp8) and diffuse (fam84a, sparc and des) lesions vs. control animals with no detectable ptb-associated lesions in intestine and regional lymph nodes. the aim of the current study was to assess the ptb diagnostic potential of commercial elisas designed for the specific detection of these biomarkers. the ability of these elisas to identify animals with latent and/or patent forms of map infection was investigated using serum from naturally infected cattle (n = 88) and non-infected control animals (n = 67). roc analysis revealed that the abca13-based elisa showed the highest diagnostic accuracy for the detection of infected animals with focal lesions (auc 0.837, sensitivity 79.25% and specificity 88.06%) and with any type of histological lesion (auc 0.793, sensitivity 69.41% and specificity 86.57%) improving on the diagnostic performance of the popular idexx elisa and other conventional diagnostic methods. sparc and mmp8 showed the highest diagnostic accuracy for the detection of animals with multifocal (auc 0.852) and diffuse lesions (auc 0.831), respectively. in conclusion, our results suggest that quantification of abca13, sparc and mmp8 by elisa has the potential for implementation as a diagnostic tool to reliably identify map infection, greatly improving early detection of map latent infections when antibody responses and fecal shedding are undetectable using conventional diagnostic methods. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 bovine paratuberculosis (ptb) is a chronic granulomatous enteritis caused by mycobacterium avium subsp. paratuberculosis (map), that is responsible for important economic losses due to reduced milk production, premature culling, reduced slaughter value and continued spread of infection [1, 2] . furthermore, map has a clear zoonotic potential since it has been postulated as a possible trigger factor in several autoimmune diseases in humans such as crohn's disease (cd) [3, 4] , type i diabetes (t1d) [5] , multiple sclerosis (ms) [6, 7] or rheumatoid arthritis (ra) [8, 9] . one mechanism that has been suggested to cause the onset and/or exacerbation of autoimmune disease is molecular mimicry, whereby map antigens share sequences or structural similarities with self-antigens [10] , so the immune response against map antigens could also induced undesirable immune responses against host proteins in genetically predisposed individuals [11] . two forms of infection, latent or patent, can be distinguished in map infected cattle [12] . the disease typically progresses from a latent form with low or moderate frequency of microbiological or humoral immunological evidence of infection, characterized by the presence of focal histological lesions in their intestinal tissues to more severe forms of the disease with a high frequency of microbiological or humoral immunological evidence of infection, in which the granulomatous lesions are patent (multifocal and diffuse lesions readily detected upon microscopic examination of the intestine and associated lymph nodes). a latent form would represent a form of silent ptb that causes no direct losses, but that maintains a hidden map reservoir in a herd, while a patent form often corresponds with a visibly clinical disease. transmission of map primarily occurs by the fecal-oral route through the ingestion of map contaminated feces, colostrum, or milk. infection usually occurs within the first months of life of the animal but remains subclinical for an average of 2-5 years before becoming clinical in a few cases. spread of ptb is mainly due to its extremely long latent period during which map can be shed intermittently into the environment through feces. thus, early detection and removal of animals in that stage from the herd is critical for ptb control. most ptb control programs are based on testing and culling test-positive cows combined with good management practices [13] . several diagnostic techniques are used to detect map infected cattle; however, their performance vary widely depending on the stage of map infection [14] [15] [16] . currently available diagnosis methods have low sensitivities and specificities for the detection of latent infection, as the bacteria is excreted in low numbers and animals have low titers of specific antibodies. fecal culture has been considered the gold standard for diagnosis of map but its sensitivity varies from 70% in cattle with ptb-associated clinical signs to 23-29% in infected cattle with no detectable clinical signs [17] . pcr offers a rapid method of assessing map status in cultures, feces, tissue and milk [18] . the sensitivity and specificity of fecal pcr were estimated to be 29% and 99.3%, respectively [19] . recently, taniguchi et al. [20] have established an association between the amount of map dna in feces, determined by real-time quantitative pcr and the histopathological classification of ileocecal valve lesions. elisas, used to detect anti-map antibodies, provide rapid results and offer a cheaper alternative to fecal culture and pcr. however, the appearance of an antibody response detectable by elisa is late as it is associated with disease progression, high shedding, clinical signs and the appearance of severe lesions in gut tissues [16, [21] [22] [23] . thus, the sensitivity of serum specific antibody elisa varies depending on the stage of infection, being 50-87% in cattle with clinical signs, 24-94% in cattle with no clinical signs but shedding map and 7-22% in infected cattle with no clinical signs and no shedding [17] . in general, elisa specificity varies between 40 and 100% [17] and depends on the particular test, exposure to environmental mycobacteria, concurrent infection with mycobacterium bovis, previous intradermal tuberculosis (tb) test and map vaccination. early stage diagnostics primarily target pro-inflammatory cellular immune responses, the interferon-gamma (ifn-γ) release assay (igra) uses a sandwich elisa to detect whether a t-cell mediated immune response has been elicited in response to map by measuring the difference in ifn-γ signals for whole blood samples activated by a map-specific antigenic extract and a non-specific antigen (m. phlei extract) (id vet, grabels, france) [24] . however, the igra only reflects map exposure, and thus cannot discriminate between individuals with controlled infection from those with sub-clinical disease, indicating that this method might lead to culling healthy individuals. this means that, even though detection of patent forms can be readily done through different methods, there is not an affordable, sensitive and specific method for detection of latent forms. therefore, novel diagnostic tools with high sensitivity and specificity are needed to detect latent map infections. the potential of emerging -omic approaches to complement and enhance the diagnosis of map infection in cattle has been previously reviewed [23] . host biomarkers, identified using transcriptomics and proteomics, have been proposed as tools to develop novel diagnostic methods for ptb [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] . however, the application of those biomarkers for ptb diagnosis has yet to be developed and properly validated for naturally infected cattle in various stages of infection [37] . park et al. [38] developed a real-time pcr method based on measuring the gene expression levels of 8 bovine biomarkers (timp1, hp, serpine1, tfrc, mmp9, defb1, defb10, and s100a8) in whole blood of cows that might be useful for the diagnosis of ptb disease, including subclinical stage cases. we previously identified several potential biomarkers for the diagnosis of map-infected animals [39] . whole rna-sequencing (rna-seq) was used to identify host genes differentially expressed in peripheral blood samples collected from animals with focal, or diffuse lesions in gut tissues vs. control animals without detected lesions nor bacterial load in gut tissues. genes encoding for bovine proteins mmp8 (matrix metallopeptidase 8) and abca13 (atp binding cassette subfamily a member 13) showed significantly higher expression in animals with focal lesions, while genes encoding for fam84a (family with sequence similarity 84 member a), sparc (secreted protein acidic and cysteine rich) and desmin (des) were up-regulated in animals showing diffuse lesions when compared with the control animals (table 1 ). in the present study, these five proteins were selected for their validation as biomarkers for the different ptb forms of infection by elisa according to their high expression levels in peripheral blood, their cellular location (extracellular proteins, present in sera) and commercial availability of elisa kits for its specific detection. the aim of this study was to evaluate the diagnostic potential of commercial elisas based on detection of these five bovine biomarkers to detect latent and patent forms of map infection in naturally infected cattle, using reference serum samples from well characterized animals with focal, multifocal and diffuse histological lesions in their intestinal or associated lymphoid tissues [40]. two groups of animals (n = 155) were included in this field study: slaughtered group) ninety-four holstein friesian cows (ranging from 0.81 to 12.66 years of age) came from 26 farms located in the principality of asturias (northwest of spain). in asturias, 32.6% of the herds and 1.9% of the animals were positive by serum elisa in 2019 (unpublished data from the regional government). specifically, fifty-six animals came from a dairy farm with a mean herd size of 105 cows (2016-2019) and a mean prevalence of ptb of 6.30% in the sampling period, based on serum elisa test (idexx laboratories, hoofddorp, the netherlands). the infection status of these 56 animals was also determined once a year by fecal culture and real time pcr (2016-2019). another thirty-four animals were randomly selected from cows slaughtered in a local abattoir (coming from 24 different farms) and four more cows were culled from the serida's friesian cow farm (3% mean prevalence of ptb in the period 2018-2019). the ptb infection status of these 94 animals at the time of slaughter was determined by histopathology, specific antibody serum elisa test, and bacteriological culture and specific real-time pcr of tissues and feces; and ptb-free group) sixty-one animals (ranging from 0.50 to 10.08 years of age) came from a farm in asturias with 0% prevalence of ptb. the ptbfree status of this farm was verified yearly by repeatedly negative idexx serum elisa results and lack of a history of clinical ptb in the period 2016-2019, as well as by bacteriological culture and specific fecal real-time pcr in 2019. samples of serum and feces included in this field study were collected from all animals while tissue samples were only taken from the slaughtered animals in situ at the local abattoir after evisceration and split for microbiological and histopathological processing. experimental procedures were approved by the serida animal ethics committee and authorised by the regional ministry of agro-livestocks and indigenous resources of the principality of asturias (authorization codes proae 29/2015 and proae 66/2019). all the procedures were carried out in accordance with directive 2012/63/eu of the european parliament (guidelines for the care and use of animals for research purposes). peripheral blood and fecal samples were collected by trained personnel and in accordance with good veterinary practice. tissue sections of distal jejunum, ileocecal valve (icv) and, jejunal and ileal lymph nodes were collected from the 94 slaughtered cows, fixed in 10% neutral buffered formalin, sliced and embedded in paraffin wax using standard procedures. afterwards, 4 μm sections were assessed by haematoxylin-eosin (he) and ziehl-neelsen (zn) staining for specific acid-fast bacteria (afb) detection. slices were observed using an olympus bh-2 light microscope (olympus, tokyo, japan) and photographed using an olympus dp-12 digital camera (olympus, tokyo, japan). the stained sections were examined by light microscopy for afb and evidence of map disease-specific pathological lesions, and were classified in four groups: focal, multifocal, diffuse and with no detectable ptb-associated lesions [40] . briefly, the focal lesions consist of granulomas, mainly located in the jejunal and ileal lymph nodes, and not affecting the intestinal lamina propia. the multifocal lesions consist of well-demarcated granulomas in the intestinal lymphoid tissue and also in the intestinal lamina propia. the diffuse lesions were characterized by severe and diffuse granulomatous enteritis and lymphadenitis, which markedly altered the normal histological structure. according to the inflammatory cell type present in the infiltrate and the amount of acid-fast bacilli (afb), diffuse lesions were subdivided into diffuse lymphoplasmocytic or paucibacillary, diffuse intermediate and diffuse histiocytic or multibacillary lesions [41] . the focal, multifocal, diffuse and with no detectable ptb-associated lesions groups correspond to the previously described latent (focal), patent (multifocal and diffuse) and apparently free (no lesions) forms of infection [42]. blood ( for bacteriological culture, a pool (2 gr) of ileocecal lymph nodes, distal jejunal lymph node, icv, and distal jejunum were decontaminated with 38 ml of hexa-decyl pyridinium chloride at a final concentration of 0.75% (sigma, st. louis, mo, usa) and homogenized in a stomacher blender. after 30 min of incubation at room temperature, a 15 ml aliquot of the suspension was transferred to a new tube and left overnight for decontamination and sedimentation. approximately, 200 μl of the suspension was taken from the layer right over the sediment and inoculated into two slants of herrolds egg yolk medium (heym; becton dickinson, sparks, md, usa) and into two slants of lowenstein-jensen medium (lj; difco, detroit, mi, usa), both supplemented with 2mg/l of mycobactin j (id.vet innovative diagnostics, grabels, france). feces were taken from the rectum of each animal, maintained at 4˚c and processed within 48 h after arrival at the laboratory. the fecal samples (2 g each) were decontaminated, blended in a stomacher, and cultured in heym and lj, as previously described for tissue culture. isolation of genomic dna from tissues and feces was performed using the magmax total nucleic acid isolation kit according to the manufacturer's instructions ( the concentration of the selected biomarkers in the serum of each animal were measured using commercially available elisas according to the manufacturers´instructions (mybio-source, san diego, ca. usa). quantitative sandwich elisa kits bovine matrix metalloproteinase 8 (detection range 3.12-100 ng/ml); bovine protein fam84a elisa kit (detection range 62.5-2000 pg/ml), bovine sparc elisa kit (detection range 0.78-50 ng/ml), and competitive bovine atp-binding cassette sub-family a member 13 elisa kit (detection range 1-5000 pg/ml) and bovine desmin elisa kit (detection range 0-25 ng/ml) were used for specific detection of mmp8, fam84a, abca13, sparc and des, respectively. a standard curve was used to determine the concentration of each biomarker in the serum samples (average od of each standard was plotted on the vertical axis against the concentration on the horizontal axis and the best fit drawn to generate a regression curve). standards and samples were tested in duplicate. the mean value of the blank control was subtracted from mean raw od values before result interpretation. the concentration of the biomarkers in each sample was interpolated from the standard curve. for optimization various dilutions of the serum were tested (for instance: undiluted, 1:2, 1:4 and 1:8) and the dilution which showed a larger number of samples with measurement values included within the range of the standard curve was considered optimal. regarding the repeatability and reproducibility of these elisas the supplier indicates that both the intra-plate and inter-plate variability expressed as coefficient of variation (cv% = standard deviation/mean of replicates x 100) for fam84a and mmp8 are less than 15%, the intra-plate cv is �6.6% and the inter-plate cv�11.3% for sparc and both the intra-lot and inter-lot cv are less than 10% for des and abca13. data obtained from biomarkers quantification was analyzed using the proc, optimalcutpoints and caret packages of r program statistical environment version 3.6.0 (http://www.rproject.org/), with confidence intervals stated at 95% for the final results. the auc (area under the curve) and optimal cut off value for each biomarker-based elisa was determined individually by receiver operator characteristic (roc) curve analysis. the optimal cut off values for sensitivity and specificity were based on maximum youden index (j = se+sp-1). the discriminatory power of each biomarker to discern between the different histopathological groups and the control group was determined as follows: biomarker-based elisas with auc values �0.9 were considered to have excellent discriminatory power; 0.8�auc<0.9 good discriminatory power; 0.7�auc <0.8 fair discriminatory power; and auc <0.7, poor discriminatory [38, 43] . multivariate binary logistic regression models (caret package of r) were used to assess the diagnostic capacity of the simultaneous use of several biomarkers providing auc, sensitivity and specificity values for the different biomarker combinations. comparison of roc curves to test the statistical significance of the difference between the areas under roc curves (derived from the same cases) was performed for the biomarkers with fair, good and excellent auc values (auc�0.7) within each histopathological group using the delong method [44] . the proc freq of the sas statistical package (sas inc., cary, nc, usa) was used for agreement analysis between pairs of diagnostic assays. the coefficient of agreement (kappa (κ)) was interpreted as follows: κ = 0.00-0.20, poor; κ = 0.21-0.40, fair; κ = 0.41-0.60, moderate; κ = 0.61-0.80, good; and κ = 0.81-1.00, excellent agreement. the histopathological, immunological and microbiological characteristics of the animals included in this study, slaughtered animals (n = 94) and control animals from a ptb-free farm (n = 61), are summarized in table 2 . the complete dataset is available in s1 table. pathological examination of intestinal tissue sections allowed the classification of the animals in four groups according to the type and extension of the histopathological lesion: focal (n = 55, 58.51%), multifocal (n = 18, 19.14%), diffuse (n = 15, 15.95%) and with no detectable histological lesions (n = 6, 6.38%). in the group of animals with focal lesions, 76.36% were positive by one or more diagnostic methods (zn, fecal and tissue real-time pcr, fecal or tissue bacteriological culture or serum elisa). specifically, 49.09% were positive by zn, 5.45% by both fecal bacteriological culture (low bacterial load, <10 cfu/gr), and serum elisa, 9.09% by fecal real-time pcr, and 28.30% by both tissue real-time pcr and by tissue bacteriological culture. none of the animals in the focal group (0 out of 28 animals with known clinical status) showed clinical signs associated with ptb. in the group of animals with multifocal lesions, 100% of the animals were positive for at least one of the following 6 techniques: zn, fecal and tissue real-time pcr, fecal and tissue bacteriological culture and serum elisa. specifically, 94.44% of the animals were positive by zn, 27.78% by serum elisa, 33.33% by fecal real-time pcr, 44.44% by both tissue real-time pcr and tissue culture and 16.67% by fecal culture (one animal with low and two with heavy bacterial load). in this group 21.43% of the animals (3 out of 14 with known clinical status) showed clinical signs. in the diffuse group, 100% of the animals were positive by zn. serum elisa and both fecal and tissue real-time pcr analysis showed, in each case 73.33% positives. fecal and tissue culture analysis revealed 26.66% and 66.67% positives, respectively, both with heavy bacterial loads (>50 cfu/gr). in this group, 64.28% (9 out of 14 with known clinical status) of the animals had ptb-associated clinical signs. only 6 out of the 94 animals analyzed by histopathology did not show any detectable histological lesion in their intestinal tissues and associated lymph nodes. these 6 animals were the diagnostic accuracies of each biomarker-based elisa to discriminate between the different histopathological groups and the control group was investigated by roc analysis ( table 3 ). the aucs, sensitivities, and specificities values were estimated for each biomarker individually and in combination based on optimal cut off values. the diagnostic performance of the biomarker-based elisas was also compared to that of the specific anti-map antibody elisa (idexx elisa). roc analysis of the elisas for the detection of 4 biomarkers (fam84a, des, mmp8 and spacr) and the idexx elisa was performed using 88 serum samples from 55 animals with focal, 18 with multifocal and 15 with diffuse lesions while roc analysis of the abca13-based elisa was performed using 85 serum samples from 53 animals with focal, 17 with multifocal and 15 with diffuse lesions. the expression levels of each biomarker in the serum of every single holstein friesian cattle within the different histopathological groups (focal, multifocal, and diffuse) and in the noninfected control group are shown in fig 1. roc curves of each selected biomarker for the different histopathological groups when compared to the non-infected control group are shown in fig 2. 3.2.1 detection of animals with focal histological lesions. three biomarkers had fair (mmp8, 0.7�auc<0.8) or good (abca13 and sparc, 0.8�auc<0.9) discriminatory power between the focal and control groups, while the rest of the biomarkers had poor discriminatory power (auc<0.7). the abca13-based elisa showed the most accurate diagnostic performance with an auc value of 0.837 (95% confidence interval [ci]: 0.757-0.917, p<0.001), a sensitivity of 79.25% and a specificity of 88.06%. comparison of the roc curves of biomarker-based elisas with aucs�0.7 (abca13, mmp8 and sparc) showed that there were no significant differences between the three roc curves (p>0.05 in all cases). we also tested whether using a combination of biomarker-based elisas increases sensitivity of the method. logistic regression analysis indicated that biomarkers fam84a and mmp8 were excluded and des, abca13 and sparc were included in the diagnostic model for discrimination of animals with focal lesions and control animals. the auc value of this diagnostic model reached 0.911 (95% ci: 0.859-0.964), and the model exhibited 84.62% sensitivity and 86.57% specificity. diagnostic performance of abca13, mmp8 and sparc for the detection of animals with focal lesions was better than that of the idexx elisa which had an auc value of 0.541 (95% ci: 0.437-0.646, p>0.05), a sensitivity of 14.55% and a specificity of 100.00%. two biomarkers had fair (mmp8, 0.7�auc<0.8) or good (sparc, 0.8�auc<0.9) discriminatory power between the multifocal and control groups, while the rest of the biomarkers had poor discriminatory power. the sparc-based elisa showed the most accurate diagnostic performance with an auc value of 0.852 (95% ci: 0.749-0.954, p<0.001), a sensitivity of 66.67% and a specificity of 92.54%. comparison of the roc curves of mmp8 and sparc-based elisas (aucs�0.7) showed that there were no significant differences between their roc curves (mmp8 vs. sparc, p = 0.154). logistic regression analysis indicated that abca13 and mmp8 were excluded and biomarkers fam84a, des and sparc were included in the diagnostic model for discrimination of animals with multifocal lesions and control animals. the auc of the diagnostic model was 0.851 (95% ci: 0.737-0.964) with a 70.54% sensitivity and 89.55% specificity. diagnostic performance of mmp8 and sparc-based elisas for detection of animals with multifocal lesions was better than that of the idexx elisa which had an auc value of 0.600 (95% ci: 0.446-0.754, p>0.05), a sensitivity of 27.78% and a specificity of 100.00%. abca13 and mmp8-based elisas had good discriminatory power between the diffuse and control groups (0.8�auc<0.9), while the rest of the biomarkers had poor discriminatory power (auc<0.7). the mmp8-based elisa showed the most accurate diagnostic performance with an auc value of 0.831 ((0.744-0.917, 95% ci), p<0.001) a sensitivity of 100.00% and a specificity of 71.64%. the diagnostic performance of the idexx elisa for the detection of animals with diffuse lesions was better than that of the biomarker-based elisas with an auc value of 0.918 (95% ci: 0.810-1.000, p<0.001), a sensitivity of 86.67% and a specificity of 97.01%. comparison of the roc curves of elisas with aucs�0.7 (abca13, mmp8 and idexx elisa) showed that there were no significant differences between the three roc curves (p>0.05 in all cases). logistic regression analysis did not find any significant diagnostic model for the discrimination of animals with diffuse lesions and control animals. abca13, mmp8 and sparc-based elisas had fair (0.7�auc<0.8) discriminatory power between animals with multifocal and diffuse lesions and the non-infected control group. the mmp8-based elisa showed the most accurate diagnostic performance with an auc value of 0.781 (95% ci: 0.692-0.869, p<0.001) a sensitivity of 96.97% and a specificity of 58.21%. diagnostic performance of the mmp8-based elisa for detection of animals with multifocal and diffuse lesions was better than that of the idexx elisa which had an auc value of 0.745 (95% ci: 0.632-0.858, p<0.001), a sensitivity of 54.55% and a specificity of 97.01%. comparison of the roc curves of elisas with aucs�0.7 (abca13, mmp8, sparc and idexx) showed that there were no significant differences between the four roc curves (p>0.05 in all cases). logistic regression analysis indicated that biomarkers des, abca13, mmp8 and sparc-based elisas can be included in a diagnostic model for discrimination of animals with multifocal and diffuse lesions and the control animals. the auc value of the diagnostic model reached 0.820 (95% ci: 0.727-0.910), and the model had 75% sensitivity (75%) and 85.07% specificity. for the detection of animals with any type of histological lesions (focal, multifocal or diffuse) we compared these animals with the control group. it must be taken into account that in this analysis the three different histopathological groups are not equally represented (focal n = 55, multifocal n = 18 and diffuse n = 15), however, this is a reflection of the real situation on farms (n = 67) . the number of animals with focal, multifocal, diffuse or with any type of lesions represented in the plot for the abca13-based elisa was 53, 16, 14 and 83, respectively. biomarkers were detected and quantified in bovine serum by specific elisas supplied by mybiosource, san diego, ca, usa. fam84a, bovine family with sequence similarity 84 member a; des, bovine desmin; abca13, bovine atp binding cassette subfamily a member 13; mmp8, bovine matrix metallopeptidase 8; sparc, bovine secreted protein acidic and cysteine rich; idexx elisa. the data are represented as scatter plots with each dot representing a single animal. the mean of each histopathological group is represented by a gross black point and the standard deviation by a vertical line. the asterisks indicate if the differences between each histopathological group and the control are or not significant ( � , p<0.05; �� , p<0.01; ��� , p<0.001). https://doi.org/10.1371/journal.pone.0236336.g001 [42]. abca13, mmp8 and sparc-based elisas had fair (0.7�auc<0.8) discriminatory power between animals with any type of lesion and the control group. the abca13-based elisa showed the most accurate diagnostic performance with an auc value of 0.793 (95% ci: 0.719-0.867, p<0.001), a sensitivity of 69.41% and a specificity of 86.57%. however, comparison of the roc curves of biomarker-based elisas with aucs�0.7 (abca13, mmp8 and sparc) showed that there were no significant differences between the three roc curves (p>0.05 in all cases). logistic regression analysis indicated that biomarker fam84a was excluded and biomarkers des, abca13, mmp8 and sparc-based elisas included in the diagnostic model for discrimination of infected animals and non-infected control animals. the auc value of the model reached 0.878 (95% ci: 0.824-0.932) with an 80.95% sensitivity and 85.07% specificity. the diagnostic performance of the abca13, mmp8 and sparc-based elisas was better than that of the idexx elisa which had an auc value of 0.618 (95%ci: 0.53-0.705, p<0.012), a sensitivity of 28.41% and a specificity of 100.00%. the coefficients of agreement (κ values) between the histopathological classification of lesions and the results of the elisas are also included in table 3 . the best coefficients of agreement between the focal, multifocal and diffuse histopathological groups and the biomarker-based elisa results were obtained for biomarker abca13 (good agreement, κ = 0.6771), sparc (good agreement, κ = 0.6043) and mmp8-based elisa (moderate agreement, κ = 0.4803), respectively. the agreement between the histopathological findings and the biomarker-based elisa results was worse when the multifocal and diffuse lesion groups were grouped. in this case, the best result was obtained for the mmp8-based elisa which showed a moderate agreement (κ = 0.4569). the best coefficient of agreement between the group of animals with any type of lesion and the biomarker-based elisa results was obtained for abca13-based elisa with a moderate κ value of 0.5437. an excellent agreement (κ = 0.8368) was obtained between the histopathological classification of animals with diffuse lesions and the idexx elisa results. in the rest of the histopathological groups, the coefficient of agreement of the biomarker-based elisas was better than that of the idexx elisa. the agreement coefficients were consistent with the auc values obtained by roc analysis. the diagnostic performance of the abca13, mmp8 and sparc-based elisas for the focal and any type of lesion groups was compared with the idexx elisa and additionally with other conventional ptb diagnosis methods such as specific fecal and tissue real-time pcr and bacteriological culture ( table 4 ). the biomarker-based elisas showed a better diagnostic value than the other diagnostic methods tested for the detection and discrimination of animals with focal lesions. for instance, the abca13-based elisa detected as positive 42 out of 53 animals with focal lesions (79.25% sensitivity) and as negative 59 out of the 67 controls (88.06% the number of animals with focal, multifocal, diffuse or with any type of lesions represented for abca13 was 53, 17, 14 and 84, respectively. fam84a, bovine family with sequence similarity 84 member a; des, bovine desmin; abca13, bovine atp binding cassette subfamily a member 13; mmp8, bovine matrix metallopeptidase 8; sparc, bovine secreted protein acidic and cysteine rich; idexx, idexx serum elisa. https://doi.org/10.1371/journal.pone.0236336.g002 specificity). specifically, the idexx elisa was 100% specific but only detected 3 out of 55 animals with focal lesions (5.45% sensitivity) when the cut off used was the one established by the supplier (55%) or 8 out of 55 (14.54%) when the cut-off point established by roc analysis was used (28.39%). ante-mortem assays like fecal culture and pcr were 100% specific but only detected 3 out of 55 (5.45%) and 5 out of 55 (9.09%) animals with focal lesions, respectively. post-mortem assays like tissue culture and pcr were 100% specific and detected a higher percentage of animals with focal lesions, 15 out of 53 (28.30%) and 15 out of 53 (28.30%), respectively. bovine biomarker-based elisas also showed a higher sensitivity and diagnostic value than the other diagnostic methods tested for overall detection of animals with any type of histological lesions ( table 4 ). the abca13-based elisa was able to detect 59 out of 85 infected animals (69.41%) and as negative 58 out of 67 (86.57%) while the idexx elisa was 100% specific but only detected as positive 19 out of 88 animals with lesions (21.59%) when the cut off was 55% or 25 out of 88 (28%) when the cut off used was 28.41%. ante-mortem assays like fecal culture and pcr were 100% specific but only detected 10 out of 88 (11.36%) and 22 out of 88 (25%) animals with any type of lesions, respectively. post-mortem assays like tissue culture and pcr were 100% specific and detected a higher percentage of animals with lesions, 33 auc, area under the curve; p-value, it is the p-value of the auc area, indicates whether the discrimination between animals with focal, multifocal, diffuse or any type of lesions and controls is significant; the cut-off point is expressed as ng/ml for the biomarkers and as a % of the positive reference sample for the idexx elisa; se, sensitivity; sp, specificity; dv, diagnostic value (semi-sum of the sensitivity and specificity); the control group consists of 67 animals, 6 animals with no lesions detected and 61 from a ptb-free farm; abca13, bovine atp binding cassette subfamily a member 13; a, indicate that the number of animals with focal, multifocal, diffuse or with any type of lesions analysed for abca13 was 53, 17, 15 and 85, respectively; b, the cut off value used to estimate the sensitivity and specificity of the assay was calculated by roc analysis; c, the cut off value used to estimate the sensitivity and specificity of the assay was the one established by the supplier � , the estimation of the specificity for this methods is based on the analysis of 6 animals with no lesions, there is not availability of tissues for the 61 live animals from the ptb-free farm. the diagnostic methods with the best diagnostic value are shown in bold face. https://doi.org/10.1371/journal.pone.0236336.t004 out of 86 (38.37%) and 34 out of 86 (39.53%), respectively. all the tested methods had lower sensitivity than the biomarker-based elisas for the detection of animals with focal lesions and for the overall detection of animals with any type of histological lesion. the goals of ptb control programmes may vary from eradication in areas of low prevalence, control in areas with high prevalence or increased surveillance in an area with no prior history of disease [37] . currently, the control of ptb at the herd level is based mainly on the identification and withdrawal of infected animals, especially map-shedding animals, to suppress sources of infection and maximize the productive life of the animals [45] . therefore, the effectiveness of these control programs is strongly conditioned by the diagnostic methods used for the detection of infected animals. host biomarkers may provide improved diagnostics for ptb increasing the effectiveness of control programmes. to the best of our knowledge this is the first study where the detection of biomarkers in serum samples by specific elisas has been used and validated as a diagnostic tool for detection of map-infected animals. our results indicate that the abca13, sparc and mmp8-based elisas have higher auc values and sensitivities than the idexx elisa and other current diagnostic methods for detection of animals with focal, multifocal and any type of histopathological lesions, respectively. specifically, the abca13-based elisa had the best diagnostic performance for detection of animals with any type of lesions, it was able to detect 69.41% of these animals while the idexx elisa, fecal culture and fecal pcr detected 28.41%, 11.36% and 25%, respectively. therefore, the abca13-based elisa greatly improves overall detection of animals with ptb-specific histological lesions. this is due to the fact that the biomarker-based elisas have better diagnostic accuracies for the focal and multifocal groups than the other diagnostic methods. in fact, the abca13-based elisa showed the most accurate diagnostic performance for detection of animals with focal lesions. it had a 79.25% sensitivity vs. the 14.55%, 5.45% and 9.09% sensitivities for the idexx elisa, fecal culture and fecal pcr, respectively. likewise, the sparc-based elisa showed the best discriminatory power between the multifocal and control animals. it identified 66.67% of animals with multifocal lesions while the idexx elisa, fecal culture and fecal pcr identified 27.78%, 16.67% and 33.33%, respectively. the mmp8-based elisa showed the highest diagnostic accuracy for detection of animals with focal and multifocal lesions when they were grouped, it has a sensitivity of 96.97% vs. the 54.55%, 51.51% and 21.21% of the idexx elisa, fecal culture and fecal pcr, respectively. these results indicate that the biomarker-based elisas consistently show higher sensitivity values than the current diagnostic methods. the specificity of the idexx elisa and the other conventional diagnostic methods (fecal and tissue culture and pcr) was higher (>97%) than that observed for the biomarker-based elisas in each histopathological group. the specificity values obtained for the best biomarker-based elisas in each histopathological group ranged from 58.21% of the mmp8-based elisa for detection of the grouped multifocal and diffuse animals to 92.54% of the sparc-based elisa for detection of the multifocal group. lower specificity values could be explained by the presence of infected animals that have not been recognized as such in the control group. this could be due to the low sensitivity of the diagnostic methods (idexx elisa, and fecal culture and pcr) used to determine the ptb-status of the ptb-free farm [14] . there was a lack of control animals with no ptb-associated histopathological lesions detected. but even using histopathology to determine the ptb-status it is possible that some animals with focal lesions were treated as negative. the sections of intestine examined by histopathology correspond to the areas where lesions are most consistently found but the actual fraction of intestine analysed is very small which is not representative of what can be in the entire intestine. another possible hypothesis to explain the lower specificity values obtained for the biomarker-based elisas is the presence in the ptb-free farm of animals infected by non-tuberculous mycobacteria that cause an increase in the levels of the selected biomarkers. however, this possibility needs to be explored before drawing any definitive conclusions. these considerations highlight the difficulty in establishing ideal control groups for the diagnosis of this disease. logistic regression analysis indicated that combination of biomarker-based elisas could improve the diagnostic performance (auc values) of the elisas used individually for detection of some histopathological groups. on the whole, the diagnostic models had higher auc values, increased sensitivities and decreased specificities. it will be necessary to assess whether the changes in sensitivity and specificity produced by the use of combined biomarkers are worthwhile as the use of such combined biomarkers would raise testing costs and complicate the diagnostic procedure. the sample size used in this study for validation of the biomarkerbased elisas and the diagnostic models was moderate (n = 155) so further studies with a larger collection of samples need to be performed to validate that these biomarkers can be used to accurately diagnose all manifestations of map infection. up-regulation of these genes is a host response to infection of map. the abca13 gene is a member of the abc gene subfamily a. abc proteins facilitate translocation of heterogeneous substrates (lipids, peptides, proteins, ions, etc) across the cell membrane using energy acquired by the hydrolysis of atp. gene ontology annotations related to this gene include atpase and cholesterol transporter activity. in humans, the expression of abca13 is elevated in subjects with several pathologies as leukemia, prostate tumor, colorectal cancer, and tumor cell lines in central nervous system [46, 47] . several studies have associated overexpression of abca13 with poor prognosis of cancer [48, 49] . abca13 is also considered a useful marker for predicting lymph node metastasis in resected gastric cancer patients in early stage [50] . disruption to abc transporter activity results in lipid accumulation and elevated levels of inflammatory cytokines in lung tissue [51] . therefore, increased values of abca13 protein might be related to the chronic inflammation observed in the small intestine of map-infected animals. there is evidence suggesting that map can induce the expression of matrix metallopeptidases (mmps), which are the main proteases in the pathogenesis of mucosal ulcerations such inflammatory bowel disease [52] . tissue inhibitors of mmp (timsps) have been suggested as potential biomarkers for tb. timps-1, -2 and -3 facilitate remodeling and repair of tissue following destruction by mmps. for instance, the concentration of mmp-8 (or collagenase-2) has been demonstrated to decrease rapidly during tb treatment [53]. mmp9 and timp1 are known to be up-regulated in tuberculosis infection and have been proposed as biomarkers for diagnosis of tuberculosis [54] . mmp8 is an mmp mainly produced by neutrophils and associated with many inflammatory conditions [55, 56] . up-regulation of mmp-8 expression in peripheral blood of map infected animals may indicate that mmp-8 plays an important role in the inflammation and destruction of tissue observed in the development of ptb. sparc, also known as osteonectin, is a matrix protein that binds collagen, and is required for the development of granuloma-like structures during chronic infections [57] . in our previous rna-seq study [39] , the protein-protein interaction analysis revealed a col1a2 centered network containing a coli1a2-sparc functional interaction. sparc and col1a2 were upregulated in the peripheral blood gene expression profiles from the cows with diffuse lesions which suggests that the expression of both proteins could lead to a bad prognosis in mapinfected cows. a sparc-centered network was also expressed more strongly in mycobacterium bovis-challenged monocyte-derived macrophages from bovine tb infected cows than in healthy cows [58] . currently, it is impossible to identify all infected animals which makes it difficult to improve control and eradicate ptb. even though, as we have confirmed here, the map specific antibody elisa performs well for patent forms of infection, latent forms are mostly overlooked. for this reason, sensitive biomarker-based diagnostic assays that widen the detection range could be used: 1) in eradication campains by identifying cows before they commence fecal shedding of the pathogen; 2) to prevent the purchase of infected cattle that escape current detection methods avoiding rapid spread of ptb between herds; and 3) to reduce the potential of a herd to transmit infection to other domestic and wild ruminants. that is especially relevant since ptb is a disease that affects not only cattle but also other domestic (sheep, goats) [59] and wild ruminants (deer, fallow-deer) [60, 61] , and other species like camelids [62] , that have similar digestive characteristics although they are not true ruminants [63] . improvement of the identification of map-infected animals through sensitive biomarker-based elisas could also help to study and confirm the potential role of map-infection as a common pathogenetic contributor to various autoimmune diseases [10] . in conclusion, biomarker-based elisas with good diagnostic performance could be used to improve ptb management. this is especially relevant for early detection of animals during latent stages of infection which are currently escaping detection. individual or combined biomarker-based elisas could provide significant improvements in current ptb control programs when used together with traditional diagnostic methods. supporting information s1 economic losses due to paratuberculosis in dairy cattle relationship between antibodies against mycobacterium avium subsp. paratuberculosis in milk and shape of lactation curves mycobacterium avium suespecies paratuberculosis and crohn's disease: a systematic review and meta-analysis the consensus from the mycobacterium avium ssp type 1 diabetes at-risk children highly recognize mycobacterium avium subspecies paratuberculosis epitopes homologous to human znt8 and proinsulin anti-mycobacterial antibodies in paired cerebrospinal fluid and serum samples from japanese patients with multiple sclerosis or neuromyelitis optica spectrum disorder epstein-barr virus and mycobacterium avium subsp. paratuberculosis peptides are recognized in sera and cerebrospinal fluid of ms patients interferon regulatory factor 5 is a potential target of autoimmune response triggered by epstein-barr virus and mycobacterium avium subsp. paratuberculosis in rheumatoid arthritis: investigating a mechanism of molecular mimicry rheumatoid arthritis patient antibodies highly recognize il-2 in the immune response pathway involving irf5 and ebv antigens antibody response to homologous epitopes of epstein-barr virus, mycobacterium avium subsp. paratuberculosis and irf5 in patients with different connective tissue diseases and in mouse model of antigen-induced arthritis association of mycobacterium avium subsp. paratuberculosis with multiple sclerosis in sardinian patients paratuberculosis control: a review with a focus on vaccination strategies for time of culling in control of paratuberculosis in dairy herds elisa and fecal culture for paratuberculosis (johné s disease): sensitivity and specificity of each method evaluation of three elisas for mycobacterium avium subsp. paratuberculosis using tissue and fecal culture as comparison standards age-specific characteristics of elisa and fecal culture for purpose-specific testing for paratuberculosis ante mortem diagnosis of paratuberculosis: a review of accuracies of elisa, interferon-γ assay and faecal culture techniques development and evaluation of a novel multicopy-element-targeting triplex pcr for detection of mycobacterium avium subsp. paratuberculosis in feces evaluation of a rapid fecal pcr test for detection of mycobacterium avium subsp. paratuberculosis in dairy cattle the association between detection of mycobacterium avium subsp. paratuberculosis dna in feces and histopathological classification evaluation of a commercial elisa for diagnosis of paratuberculosis in cattle detection of mycobacterium avium subsp. paratuberculosis: comparing fecal culture versus serum enzyme-linked immunosorbent assay and direct fecal polymerase chain reaction potential application of emerging diagnostic techniques to the diagnosis of bovine johne´s disease (paratuberculosis) composition and potency characterization of mycobacterium avium subsp. paratuberculosis purified protein derivatives biomarker discovery in subclinical mycobacterial infections of cattle proteomic analysis of plasma from holstein cows testing positive for mycobacterium avium subsp. paratuberculosis (map) analysis of transcriptional profiles to discover biomarker candidates in mycobacterium avium subsp. paratuberculosis-infected macrophages, raw 264.7 gene-expression profiling of calves 6 and 9 months after inoculation with mycobacterium avium subsp. paratuberculosis whole-blood gene-expression profiles of cows infected with mycobacterium avium subsp. paratuberculosis reveal changes in immune response and lipid metabolism host transcriptional profiles and immunopathologic response following mycobacterium avium subsp. paratuberculosis infection in mice transcriptional profiling of ileocecal valve of holstein dairy cows infected with mycobacterium avium subsp. paratuberculosis responses of bovine innate immunity to mycobacterium avium subsp. paratuberculosis infection revealed by changes in gene expression and levels of microrna application of transcriptomics to enhance early diagnostics of mycobacterial infections, with an emphasis on mycobacterium avium ssp. paratuberculosis mycobacterium tuberculosis extracelular vesicle-associated lipoprotein lpqh as a potential biomarker to distinguish paratuberculosis infection or vaccination from tuberculosis infection gene expression profiles during subclinical mycobacterium avium subspecies paratuberculosis infection in sheep can predict disease outcome identification of micrornas in bovine faeces and their potential as biomarkers of matrix metalloproteinase 8 (mmp8) gene polymorphisms in chronic periodontitis contrasting roles of sparcrelated granuloma in bacterial containment and in the induction of anti-salmonella typhimurium immunity transcriptome changes upon in vitro challenge with mycobacterium bovis in monocyte-derived macrophages from bovine tuberculosis-infected and healthy cows paratuberculosis in sheep and goats paratuberculosis in free-ranging wildlife in north america histopathological classification of lesions observed in natural cases of paratuberculosis in free-ranging fallow deer (dama dama) paratuberculosis in small ruminants, deer, and south american camelids evolutionary history and differences between camelids and ruminants we would like to acknowledge the expert technical assistance from the statistical advice unit of the scientific-technical services of the university of oviedo, our collaborating farms and the astega veterinary services for their collaboration in the sampling work. finally, we would like to acknowledge the daily work of serida´s farm operators in the care and maintenance of animals. we gratefully acknowledge kevin p. dalton for proofreading the manuscript. conceptualization: marta alonso-hearn, rosa casais. key: cord-254000-pbzcupjg authors: suvannachart, pukkapol; asawaphureekorn, somkiat; chansangpetch, sunee; inobhas, abhibol; pongpirul, krit title: repeatability, reproducibility, agreement, and safety of tono-pen tip cover for intraocular measurement using latex and polyethylene wrap date: 2020-09-28 journal: plos one doi: 10.1371/journal.pone.0239875 sha: doc_id: 254000 cord_uid: pbzcupjg purpose: to evaluate repeatability, reproducibility, and agreement of intraocular pressure measurement with tono-pen using ocufilm and polyethylene wrap tip cover in human eyes. methods: this is a cross-sectional, experimental study. a gas-sterilized, polyethylene wrap was used as an alternative for tono-pen tip cover. for the right eye, 4 measurements using polyethylene wrap tip cover were done by two examiners (a and b) in random order to assess intra-observer repeatability and inter-observer reproducibility. for the left eye, 4 measurements were done by examiner a using both polyethylene wrap tip cover and ocufilm in random order to assess intra-observer repeatability and agreement. bland-altman plot and intraclass correlation coefficient (icc) were used in all analyses. cost minimization analysis was evaluated. results: for examiner a, the repeatability of polyethylene wrap tip cover was -0.34, 95% limits of agreement (loa) were -3.04 to 2.36, and icc was 0.93 in the right eyes. as for the left eyes, the repeatability of polyethylene wrap tip cover was -0.33, 95% loa were -3.01 to 2.36, and icc was 0.93. for examiner b, the repeatability of polyethylene wrap tip cover was -0.02, 95% loa were -2.88 to 2.83, and icc was 0.92. the inter-observer reproducibility of polyethylene wrap tip cover was 0.36, 95% loa were -3.34 to 4.07, and icc was 0.90. the repeatability of ocufilm was -0.42, 95% loa were -2.75 to 1.91, and icc was 0.95. the agreement of polyethylene wrap tip cover and ocufilm was -0.71, 95% loa were -5.18 to 3.76, and icc was 0.83. there were no allergic reactions or serious complications. from the cost minimization analysis, the local cost for polyethylene tip cover was approximately 8 times lower compared to ocufilm. conclusions: tono-pen with ocufilm and polyethylene wrap tip cover were used to measure the intraocular pressure. the polyethylene wrap tip cover demonstrated acceptable repeatability, reproducibility, and agreement with ocufilm in normotensive eyes, and had a good safety profile. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 one of the most important procedure in routine ocular examination is the measurement of the intraocular pressure (iop). indirect iop measurement using different techniques, such as applanation and indentation, are generally used in clinics. tono-pen ⓡ (tp; reichert, new york, usa) is a handheld, digital tonometer that involves both indentation and applanation mechanisms. it demonstrates comparable measurement of iop compared to the goldmann applanation tonometry (gat) which is the gold standard method [1, 2] . it can measure the iop in a small area on the cornea and can be used on patient in any position. this easy-to-use device can be used by novice medical personnel without compromising the result [3] . there is a good intra-session repeatability in both glaucoma and healthy patients [4] . the use of tp requires a tip cover to prevent damage to the transducer tip and cross contamination. ocufilm ⓡ (of, reichert, new york, usa) is a commercially available disposable tip cover made from latex that can cause allergy. the prevalence of latex allergy in the general population worldwide is approximately 4.3% [5] . patients with severe latex allergy were reported to develop conjunctival injection, eyelid erythema, and eyelid edema after iop measurement with latex tip cover [6] . of tip cover is sanitized but not sterilized. this prevents its use in post-operative eyes that need sterile instrument for iop measurement. the cost of this single use tip cover may cause financial burden, especially in developing countries. in addition, sometimes there are shortages of of tip cover. in order to overcome these barriers, a previous study demonstrated that a fingertip of the surgical glove could be used as a tip cover and showed satisfactory repeatability and agreement with of [7] . however, the cost of latex surgical glove is significant and latex allergy is still possible. alternative material should be considered such as plastic wrap for packaging the food which has a smooth surface and barrier properties against moisture, gas, and organisms. this economical and readily available material can be attached to any surface without adhesive. the majority of food grade plastic wrap is made from either polyethylene or polyvinyl chloride. both materials can withstand heat up to 120-130 degrees celsius so they can be gas sterilized. both types of the plastics are widely used in medical applications, such as catheters and synthetic materials. in ophthalmology, polyethylene has long been used in ocular surgery [8] . its use can cause some postoperative reaction after being inserted into rabbit eyes [9] . plastic wrap has been reported to be used as a barrier in gat and contact a-scan ultrasonography [10] [11] [12] . for iop measurement with tp, our previous eye model and study conducted in canine eyes showed good repeatability and agreement between the custom-made polyethylene wrap (pw) tip cover and of without causing any ocular surface complications [13, 14] . the purpose of this study was to evaluate the repeatability, reproducibility, and agreement of iop measurement with tp using of and pw in human eyes. the safety and cost comparison between both tip covers were also evaluated. this is a cross-sectional, experimental study. it was approved by the institutional review board of the faculty of medicine, chulalongkorn university, bangkok, thailand, and adhered to the tenets of the declaration of helsinki. this trial was registered in the thai clinical trial registry (tctr) and its identification number is tctr20190108001. written informed consent was obtained from all participants. ophthalmic patients and healthy volunteers, at least 18 years old, were invited to enroll in this study. all participants underwent a thorough ocular examination, including visual acuity testing, and slit lamp examination of the anterior and posterior segments. those with a history of plastic or latex allergy, history of intraocular surgery rather than an uncomplicated small incision cataract surgery with phacoemulsification technique, or have corneal surface pathologies in either eye such as abrasion, infiltration, and scar, were excluded from the study. iop measurement was done by two examiners; examiner a was an ophthalmologist and examiner b was a general practitioner. tono-pen avia ⓡ was used in this study. although regular calibration is not necessary, it was performed per standard protocol recommended by the manufacturer once at the beginning of the day. a drop of 0.5% tetracaine was instilled to both eyes to achieve adequate anaesthesia. ten gentle applanations at the central cornea were performed for each measurement to obtain an average iop. only iop reading with a statistical confidence indicator of 95 was considered reliable and was used in the analysis. pw used in this study (cleanwrap ⓡ , seoul, korea) which was identical to that used in our previous studies [13, 14] . the thickness of the film was 10 micrometres. it was sent for cytotoxic testing at the national metal and materials technology center (mtec) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) assay which tested the viability of mouse fibroblast after 24-hour exposure to the material. the test demonstrated no cytotoxic potential with 100% viability of the cells (report no. mtec1200/62). pw tip cover was prepared by cutting the commercial pw to a size of 5 by 5 centimetres. a cover holder was created by cutting the paper ring from the of tip cover package in half widthwise ( fig 1a) . both materials were put in a sterilization pouch. this package was sent for ethylene oxide sterilization ( fig 1b) . when in use, the sterilized pouch was peeled off and the pw was carefully lifted out of the pouch by touching only its edge without touching the central part of the pw. then, the pw was placed over the tp tip and the cover holder was put on top of the pw and advanced to the neck of the tp head until the pw was secured in place and attached smoothly over the tp tip ( fig 2b) . the overall study flow diagram is shown in fig 3. the right eye of each participant was used to assess the intra-observer repeatability and inter-observer reproducibility of pw. four iop measurements, performed twice by examiners a and b, were done on the right eye. the left eye of each participant was used to study the agreement between of and pw tip covers and intra-observer repeatability of both types. four iop measurements, twice for pw and of, were done by examiner a. to balance the effect of decreasing iop after repeated measurements, the order of measurement by the tip covers and the examiners were done in random sequence. for safety evaluation, ocular surface examination with fluorescein staining under cobalt blue light was performed after each measurement to detect any damages that might have occurred during the procedure. any new pathologies compared to the baseline examination, including punctate epithelial erosion, corneal epithelial defect, conjunctival injection, conjunctival papillary reaction, and chemosis, were noted. all statistical analyses and graphs were generated using medcalc for windows, version 19.2 (medcalc software, ostend, belgium). the unit of analysis was the eye. baseline characteristics and complications were reported using descriptive statistics as appropriate. for intra-observer repeatability of of and pw, bland-altman plot (ba plot) was used to demonstrate mean differences (md), limits of agreement (loa) and their 95% confidence intervals (95% ci) [15] . for inter-observer reproducibility and agreement between of and pw, ba plot with multiple measurements per participant was used [16] . intraclass correlation coefficient (icc) estimates and their 95% ci were calculated based on the following parameters: mean rating (k = 2), absolute agreement, two-way model, single measures and same raters for all participants. icc estimates were interpreted using koo and li classification [17] . cost minimization analysis was performed by comparing the cost of materials and production of each tip cover. a total of 128 participants (256 eyes) were recruited into the study. majority of the participants were female (78.1%). the mean age ± standard deviation was 46.0 ± 16.6 years (range 18-83). nine of the right eyes and 4 of the left eyes had pseudophakia. for intra-observer repeatability of pw by examiner a (right eyes), the md (95% ci) was -0.34 (-0.58 to -0.10). the 95% loa (95% ci) were -3.04 (-3.46 to -2.63) to 2.36 (1.94 to 2.77) (fig 4) . the icc (95% ci) was 0.93 (0.90 to 0.95). for intra-observer repeatability of pw by examiner b (right eyes), the md (95% ci) was -0.02 (-0.28 to 0.23). the 95% loa (95% ci) were -2.88 (-3.31 to -2.44) to 2.83 (2.39 to 3.26) (fig 5) . icc (95% ci) was 0.92 (0.89 to 0.94). for inter-observer reproducibility, the md (95% ci) between examiner a and examiner b was 0.36 (0.09 to 0.64). the 95% loa (95% ci) were -3.34 (-3.84 to -2.93) to 4.07 (3.66 to 4.56) (fig 6) . the icc (95% ci) was 0.90 (0.85 to 0.93). for intra-observer repeatability of of by examiner a (left eyes), the md (95% ci) was -0.42 (-0.63 to -0.21). the 95% loa (95% ci) were -2.75 (-3.11 to -2.39) to 1.91 (1.55 to 2.26) (fig 7) . icc (95% ci) was 0.95 (0.92 to 0.97). for intra-observer repeatability of pw by examiner a polyethylene wrap tip cover for tono-pen (left eyes), the md (95% ci) was -0.33 (-0.57 to -0.09). the 95% loa (95% ci) were -3.01 (-3.42 to -2.60) to 2.36 (1.95 to 2.77) (fig 8) . icc (95% ci) was 0.93 (0.90 to 0.95). for agreement between of and pw by examiner a (left eyes), the md (95% ci) was -0.71 (-1.07, -0.35). the 95% loa (95% ci) were -5.18 (-5.83 to -4.63) to 3.76 (3.21 to 4.40) (fig 9) . icc (95% ci) was 0.83 (0.75 to 0.89). all analyses are summarized in table 1 . all of the eyes had no serious complications post-measurement by both of and pw. the only complication found in the study was punctate epithelial erosion (pee). for pw, 7 (5.5%) of the right eyes and 1 (0.8%) of the left eye had pee after all measurements were done. for of, 3 (2.3%) of the left eyes were found to have pee. none of the participants reported any post-measurement complications within the first 24 hours. there were no vision-threatening complications such as corneal epithelial defect and keratitis, or any allergic reactions. table 2 shows the detail of the cost to produce pw. from the cost minimization analysis, the average cost of of was around 0.8 usd. the average cost of one pw was 0.1 usd. the cost difference between of and pw tip cover was 0.7 usd. all costs were calculated from local purchase with local currency (thai baht). the exchange rate at the time of this study was around 32 baht per 1 usd. in the present study, we have demonstrated that pw could be used as an alternative to ocufilm for tp tip cover. both pw and of had comparable intra-observer repeatability and interobserver reproducibility with good agreement. according to published literature, test-retest variability of of was 0.1 mmhg, 95% loa were -3.3 to 3.5 mmhg, and the icc range was 0.82 to 0.85 [2, 18, 19] . in our study, the intra-observer repeatability of of was -0.42 mmhg but the loa were narrower (-2.75 to 1.91 mmhg) with excellent icc (0.95) ( table 1 ). the intra-observer repeatability of pw by both examiner a (right eye = -0.34 mmhg, left eye = -0.33 mmhg) and examiner b (right eye = -0.02 mmhg) were acceptable. the loa of examiner a (right eye = -3.04 to 2.36 mmhg, left eye = -3.01 to 2.36 mmhg) and examiner b (right eye = -2.88 to 2.83 mmhg) were acceptable. both examiners produced very similar loa which indicated that the iop measurement with pw was independent of the examiner's experience. all icc were classified as good to excellent. these results were similar to our previous studies that used pw in an eye model (intra-observer repeatability = -0.25 mmhg, 95% loa = -4.55 to 4.05 mmhg) [13] and in canine eyes (intra-observer repeatability = 0.27 mmhg, 95% loa = -2.74 to 3.27 mmhg) [14] . the inter-observer reproducibility of pw (0.36 mmhg) and loa (-3.34 to 4.07 mmhg) were acceptable (table 1 ) and comparable to our previous study done in canine eyes (interobserver reproducibility = -0.39 mmhg, 95% loa = -4.79 to 4.01 mmhg) [14] . pw and of are different in many aspects, including materials and thickness. the thickness of pw is 10 microns. of is grossly thicker, but its actual thickness is not available from the manufacturer. however, our study found an acceptable agreement between both tip covers, although pw produced slightly higher readings. in addition, the icc showed moderate to good agreement. similar results were found in our previous eye model (md = 0.29, 95% loa = -5.68 to 6.26) [13] and study conducted in canine eyes (md = 0.13, 95% loa = -3.92 to 4.18) [14] . thus, pw and of could be used as tp tip cover interchangeably. it is interesting that tp itself has a relatively wide range of 95% loa as previously mentioned, and the range is even larger when it is compared to other tonometers. compared to gat, the current gold standard, tp demonstrated varying results in different populations, with md varying from -0.27 to 1.63 mmhg and the range of 95% loa were 4.88 to 16.34 mmhg in normal individuals [1, 18, [20] [21] [22] [23] . for glaucoma patients, especially those who have uncontrolled and elevated iop, md could be as high as 6.8 (range of 95% loa = 25.9 mmhg) [24] . a systematic review denoted that only 48% of the results from tp were within 2 mmhg from gat value [25] . this high variation could partly be explained by the dynamic change of iop under several factors such as time of measurement, repeated measurements, area of cornea contact, patient's stress and unintentional valsalva maneuver, and the examiner's experience. another reason is that tp measures iop instantaneously and has a very short contact time resulting in a greater variation of iop especially in patients with a wider ocular pulse pressure [26] . in terms of safety, in this study, there were no sight-threatening complications and allergic reaction for both of and pw. there were only a few participants from pw and of groups that developed pee post-measurements. this might be explained by the mechanical damage from multiple measurements. furthermore, the contact area of tp was small (2.36 mm 2 ) compared to gat (7.35 mm 2 ), and the contact time was very short. none of the participants with pee required revisiting or further treatment. thus, pw was safe for iop measurement. pw has many advantages. the material is generally available. pw tip cover can be easily prepared in any hospital with a significant cost reduction compared to the commercial product. the cost minimization analysis found that the cost of pw was approximately 8-times lower than the cost for of. another advantage of using pw over of is that it can be gas sterilized. consequently, pw can be used in post-operative patients where sterility is of concern. the sterilization process with ethylene oxide is commonly used and has high efficiency in eradicating microorganisms without deleterious effects on the plastic material [27] . in addition, compared to of, pw can be used safely in patients with latex allergy. this method may also be suitable for a situation of high demand of use due to a concern of cross contamination like the recent outbreak of coronavirus disease 2019 (covid-19). there were some limitations in this study. firstly, most of the participants had iop within normal range. tp has lower accuracy when the patients have an extreme iop [28] . this should be considered prior to using pw in clinical practice. in addition, our previous study found greater md and wider loa in the eye model with higher iop range compared to the lower ones [13] . future studies are required to validate the agreement between pf and of tip cover in eyes with higher iop range. secondly, the efficacy of tp has high variation, thus, it would be better to compare the performance of both tip covers against another reliable instrument with less variability such as gat and manometer. furthermore, the central corneal thickness (cct) was not measured in our study because we made a comparison within the same eye of the individual. however, the difference in cct might affect the accuracy of the measurement [29] . we also did not measure the pw thickness after gas sterilization. the thickness may have been altered and become uneven after sterilization which could affect the iop measurement. pw tip cover was safe and demonstrated acceptable intra-observer repeatability, inter-observer reproducibility, with good agreement compared to of tip cover for iop measurement with tono-pen in normotensive eyes. pw could be used as an alternative tip cover for tono-pen. prospective comparative analysis of 4 different intraocular pressure measurement techniques and their effects on pressure readings repeatability of intraocular pressure measurements with icare pro rebound, tono-pen avia, and goldmann tonometers in sitting and reclining positions utility of the tono-pen in measuring intraocular pressure in trinidad: a cross-sectional study test retest variability of tonopen avia current prevalence rate of latex allergy: why it remains a problem? latex allergy associated with the latex cover on the tonopen agreement and reproducibility of tono-pen xl tip covered with ocufilm and fingertip of surgical glove in intra-ocular pressure measurement the present state of the use of plastics in eye surgery ocular reactions to plastic materials (polyethylene and teflon) cling film as a barrier against cjd in goldmann-type applanation tonometry disposable film cover for the tip of goldmann's tonometer cling film as a barrier against cjd in corneal contact a-scan ultrasonography agreement and repeatability of intraocular pressure measurement between ocufilm® and plastic wrap tonopen® tip cover. paper presented at: the 2nd asean ophthalmology society congress repeatability and agreement of a custom-made plastic wrap tono-pen® tip cover for intraocular pressure measurement. paper presented at: the 7th world glaucoma congress statistical methods for assessing agreement between two methods of clinical measurement agreement between methods of measurement with multiple observations per individual a guideline of selecting and reporting intraclass correlation coefficients for reliability research intradevice and interdevice agreement between a rebound tonometer, icare pro, and the tonopen xl and kowa hand-held applanation tonometer when used in the sitting and supine position intraocular pressure of supine patients using four portable tonometers. optometry and vision science: official publication of the american academy of optometry repeatability of intraocular pressure measurements with icare pro rebound, tono-pen avia, and goldmann tonometers in sitting and reclining positions comparison of three methods of tonometry in normal subjects: goldmann applanation tonometer, non-contact airpuff tonometer, and tono-pen xl can corneal biomechanical properties explain difference in tonometric measurement in normal eyes? optometry and vision science: official publication of the american academy of optometry agreement among goldmann applanation tonometer, icare, and icare pro rebound tonometers; non-contact tonometer; and tonopen xl in healthy elderly subjects comparison of three different tonometers in eyes with angle closure. optometry and vision science: official publication of the american academy of optometry systematic review of the agreement of tonometers with goldmann applanation tonometry the tono-pen. a manometric and clinical study ethylene oxide gas sterilization of medical devices clinical evaluation of the oculab tono-pen comparison of tono-pen avia intraocular pressure measurements performed at limbus with central corneal tono-pen avia intraocular pressure key: cord-013334-cptu0k7s authors: holst-hansen, joachim a.; bergenholtz, carsten title: does the size of rewards influence performance in cognitively demanding tasks? date: 2020-10-21 journal: plos one doi: 10.1371/journal.pone.0240291 sha: doc_id: 13334 cord_uid: cptu0k7s classic micro-economic and psychology theories propose different implications of monetary incentives on performance. empirical studies in sports settings show that athletes generally perform worse when the stakes are higher, while a range of lab studies involving cognitively demanding tasks have led to diverging results, supporting positive, negative and null-effects of higher (vs. lower) stakes. in order to further investigate this issue, we present a pre-registered, randomized, controlled trial of 149 participants solving both anagrams and math addition tasks. we do not find a statistically significant effect of the size of the reward on neither performance, self-reported effort nor intrinsic motivation. we propose that future studies should contrast the potential impact of rewards on different kinds of task, e.g. compare tasks that solely require cognitive effort vs. tasks that require motor skills, as in sports. how and if one should provide monetary incentives to individuals to improve their performance is a key question for the scientific disciplines economics and psychology, as well as managers in organizations. a general answer to this question is arguably unattainable, since different types of tasks, timeframes and organizational cultures seem to call for different reward systems [1] [2] [3] . when focusing on the nature of the tasks, meta-analyses reveal that in simple tasks, monetary incentives generally improve performance [3, 4] . this claim is in line with the micro-economic perspective, which argues that monetary incentives leads to increased motivation and effort, which in a task that depends on effort, will lead to higher performance [5, 6] . in contrast, a psychologist might argue that if the task is enjoyable, complex or embedded in an organization rather than in an artificial lab-experiment, the micro-economic explanation becomes inadequate, since intrinsic motivations will constitute a stronger influence on behavior, and these monetary incentives could crowd out intrinsic motivations [7] . because most studies have merely compared offering some monetary reward vs. not offering a monetary reward at all, it is less clear if a higher monetary incentive has a different effect than a lower monetary incentive [5] . a manager can, in principle, modify the size of the incentive, which means the question is both of theoretical and practical interest. the proponents of the micro-economic perspective will argue that higher stakes lead to an even higher incentive to increase the effort, and if the task allows for further increase in effort, higher stakes should lead to a better performance than lower stakes [6] . yet, too high stakes could also lead to a number of detrimental effects. for one, the opportunity to gain an exceptionally high reward could interfere with the participant's focus while performing the task [5] , which could lead to a decrease of the participant's performance. focusing on the magnitude of the potentially high reward might even make participants nervous, thus making them choke under pressure, further reducing the quality of their performance [5, 8] . second, the relatively high stake might enhance the crowding out effect. if the task invites an intrinsic motivation, a higher reward can reduce the positive effect on effort from this intrinsic motivation. if this reduction is higher than the potential positive effect from a higher reward, the overall effect will be negative [7] . third, if the given task can't be solved more efficiently than the level of performance a low monetary incentive allows, a higher stake can't have an additional positive effect due to a ceiling effect [3] . data from sports events involving high stakes allows an examination of the impact of higher stakes on performance in real life scenarios. studies ranging from basketball [9, 10] , tennis [11], biathlon [12] to golf [13] find that higher stakes generally reduces performance of the participant. more specifically, in golf the effect leads to golfers putting worse when more money is at stake [13] , while higher stakes implied diminished quality of basketball free throws in games compared to training [10] as well as worse free throws in crucial parts of the game [9] . whereas [14] did not find a general effect in an analysis of penalty kicks in soccer, players did perform worse at home, an effect similar in kind to influence on the performance of biathletes [12] . yet, two caveats should be added to these kinds of sports data. first, all these studies involve situations of fairly extreme pressure, exceeding the pressure an employee would usually meet in their daily work. second, these studies do not rely on randomized trials, and hidden confounding factors might be shaping the results; e.g. the size of the audience, self-selection into these sports events or the higher status that beating other competitors implies. lab-studies allow for randomized controlled trials, where only the size of monetary rewards is varied. in the following we review studies relying on tasks that require cognitive skills and effort while solving some form of problem, thus excluding pure memory tasks [15] and tasks that do not require a cognitive but only a physical effort, such as typing [5] . paradigmatic tasks relied upon are the monty hall problem, probabilistic challenges, anagram puzzles and additive math problems, i.e. cognitively demanding challenges most of which do not require particular experiences or educations. studies that have asked participants to solve various forms of cognitively demanding tasks have led to ambiguous findings; some studies find a positive effect on performance of higher stakes compared to lower [6, 16, 17] , some find a negative effect [5, 18] while others find no clear effect when comparing high vs. low stakes [17, [19] [20] [21] [22] . while the findings seem inconclusive, the reason might be systematic discrepancies in the used samples, type of tasks or size of rewards. yet, these factors do not seem to create clear demarcation lines. a range of studies have relied on a task that required bayesian reasoning, where some studies have found a positive effect of higher stakes [16, 17] , whereas [20] found no effect of increasing the size of the rewards. note, that while all three studies required bayesian reasoning, one could argue that the monty hall problem used by friedman [20] should be categorized as an insight problem, rather than requiring continuous probabilistic updating. furthermore, the exact same task (resembling an iq test) has been used in germany [18] and israel [6] . the latter study offered 0, 0.1, 1, and 3 nis (shekel) in addition to a flatrate of 60 nis, while the former study offered a similar reward structure (0, 1, 5 and 50 eurocents) in addition to a flatrate of 5 euros. gneezy and rustichini [6] found that the two highest reward levels performed statistically significantly better than the group not paid a piece-rate which in turn was better than the group paid 0.1 nis as a piece-rate. yet, in the replication study the very low piece-rate outperformed no piece-rate and high piece-rate, while other differences were not statistically significant [18] . finally, the size of the reward is also not clearly associated to higher performance. a high piece-rate reward of 50 cents vs. a low reward of 1 cent (factor of 50) did not improve performance in miller & estes' [22] study. in ariely et al. [5] a factor of 10 generally led to a negative effect, whereas the same factor led to no significant impact on quality in mason & watts [19] , while a factor of 3 and 1.5 has been shown not to lead to changes in performance [21] . note that mason & watts [19] also examined if a higher reward improved productivity, i.e. how many tasks participants on the platform amazon mturk were willing to carry out. participants thus had the opportunity to continue to solve mason & watts' [19] tasks or go to other, similar tasks, that were paid less/more. we only investigate performance in terms of relative number of correct solutions. given these inconclusive findings and the fact that relatively few studies showing no effect are available we worry that publication bias might be shaping the nature of the published results [23] [24] [25] . in order to contribute to the understanding of the implications of modifying monetary reward sizes, we present a pre-registered randomized controlled trial. we rely on a between-subject design, where primarily danish participants where to solve some of the same cognitively demanding tasks (additive math and anagram) as in the most cited of all the above mentioned studies on higher vs. lower incentives [5] . compared to former studies, we employed a medium level difference between higher vs. lower stakes. we expand on the experimental design in the next section. the overall aim is to test if a higher piece-rate reward leads to a different performance level. more specifically, we test if substantially but not radically higher monetary rewards (factor of five, i.e. 2 dkk (0.26 euros) vs 10 dkk (1.3 euros) for each correctly solved task) lead to a better performance when solving a cognitively demanding, yet relatively simple, task. since the two overarching theoretical frameworks, micro-economics and self-determination theory as well as the empirical studies outlined above [5, 6, [16] [17] [18] [19] [20] [21] [22] offer ambiguous results, we will test the following competing hypotheses. the hypotheses as well as all statistical analysis are preregistered, unless explicitly labelled exploratory (see https://osf.io/sb6ty/, an english version can be found in s1 appendix). a larger piece-rate reward will lead to better performance. a larger piece-rate reward will lead to worse performance. we carry out a pre-registered, randomized controlled trial in order to identify if a higher or lower monetary reward influences the performance level. participants solved two tasks in sequence, a math addition problem and an anagram. both tasks are cognitively demanding and requiring substantial and continuous effort, without requiring an advanced education beyond primary school. our study can be considered a conceptual replication of the aforementioned empirical studies, while being the most similar in kind to [5] ; albeit differing in terms of the incentive size, being a between-subject rather than a within-subject study, relying on an increase in the number of tasks each participant engages with as well as the length of the task duration. the study was exempted from ethical approval by a regional committee on health research ethics and was approved to be carried out at the university lab by the relevant human subjects committee. data is available at https://osf.io/fdgms/?view_only=3cc4847 e870e417586af6d186881b8da. our sample consists of 149 participants, collected over two rounds with respectively 81 and 68 participants in each round. the latter round was added during the review process to solidify the results and s4 appendix contains descriptive statistics for each round. this sample size allows the identification of an effect size of r = 0.161 at a two-tailed p-value threshold of 0.05 if relying on a simple linear regression. we note that funder and ozer [26] categorize 0.1 as a small effect size and 0.2 as a medium effect size, while gignac and szodorai's meta-analysis [27] shows that 0.2 is a typical effect. smaller effect sizes can be of interest if they accumulate [26] , but in contrast to, e.g., a study on growth mindset [28] one needs to continuously reinvest the higher reward to generate the effect. the effect of rewards will thus not accumulate. we note that ariely et al. [5] established an effect size of 0.351 for their equivalent math task, when comparing high vs. low rewards. this calculation is based on our re-analysis of their data (the authors kindly responded immediately to our request for their data), using a simple linear regression to assess how many more tasks were solved in the low reward condition. the participants are primarily from denmark (46.31% listed danish as their mother tongue), 54.36% were female, and the average age was 25.05 (6.35 sd). these characteristics fit the general distribution of participants in the university's pool of lab participants. to our knowledge this is the first study of its kind to be carried out in denmark. pokorny [18] and achtziger et al. [17] carried out their studies in germany, which culturally speaking probably resembles danish students the most, even though cultural differences between germany and denmark exist [29] . exercises and survey. we exposed participants to relatively simple, cognitively demanding tasks that mainly require effort, rather than a creative insight, without being pure memory tasks; math addition and anagram. we have chosen to employ two different tasks, which forces the participants to utilize different kind of skills, thus improving construct validity. the participants were given 10 minutes to solve two kinds of exercises (20 min in total), which each consisted of 50 tasks. all exercises and surveys were completed on paper. the first math addition exercise (cf. fig 1) was to find the two numbers that constitute a sum of ten in a box with 12 numbers like below, an exercise identical to study two in [5] . every box has exactly one solution. in the next anagram exercise (cf. fig 2) the participants were given 7-10 letters where the order had been randomized by an internet website [30] , which uses the fisher-yates-knuth shuffling algorithm to randomize the order of the letters. the participants had to construct a word in the english language using these letters. all the tasks in this exercise had at least one solution. some tasks proved to have more than one solution, e.g. the letters "telsetr" can spell both letters and settler. no participants gave more than one answer to any tasks in this exercise. achtziger et al. [17] discovered that immediate feedback could play a moderating role, influencing the behavior after receiving the feedback. in our experiment, the participants received no external feedback during the experiment. however, most of the submitted answers does the size of rewards influence performance? from the participants were correct and hence participants will probably have had some idea of their performance during the experiment. the participants were presented the exercises one at a time, and in both kinds of exercises, the participants were required to solve the tasks in the order presented (the tasks were numbered 1-50). the participants were given ten minutes for each of the exercises and were not allowed to transfer time from one exercise to another. the tasks were solved on paper. the average number of tasks solved was 22.51 (9.42 sd), i.e. 22.51% of all tasks. no participant solved all tasks in any exercise, while 2 out of 149 participants solved more than 40 out of 50 math tasks. no one solved more than 40 (out of 50) anagram tasks. therefore, performance in neither the low or high reward structure seems to face a ceiling effect cf. [3] . the allocated time span differs from the four minutes used by ariely et al. [5] . we have chosen to engage the participants for a longer duration, since the longer time period can help identify smaller differences. for example, if participant a solves 0.1 task more than participant b per minute, a four-minute trial might end up with them having solved the same number of tasks while a ten-minute trial will reveal the difference. on the other hand, we wanted to avoid inducing cognitive fatigue by having the participants work for hours. the participants were told that a session (including additional surveys) would last forty minutes in total. no sessions exceeded this length, and no sessions were substantially shorter. furthermore, since [5] relied on a within-subject design, participants ended up spending 16 minutes on the exercises within different incentives system, which is not very different from the 20 minutes our participants spend on solving the exercises. the participants completed the addition exercise first. hereafter they completed the tipi personality survey [31] followed by the anagram exercise. finally, they completed a survey aiming to measure three key variables identified in our literature review, i.e. effort (cf. microeconomics), intrinsic motivation (cf. self-determination theory) and focus [5] . the intention is to capture if these factors are correlated with the size of the reward. the questions are listed in s2 appendix. treatment. the treatment is the size of the reward, where the high stakes scenario involves a reward five times as big as the lower stakes scenario. in previous studies some have relied on much starker differences. ariely et al. [5] employed a factor of 10 and 100 in some of their experiments, while both miller & estes [22] as well as pokorno [18] relied on a factor of 50. in contrast, achtziger et al. [17] doubled the reward in the high stakes scenario. overall, compared to former studies a factor of five is at a medium level. we have tried to balance the dual aims of external validity and avoid the risk that an affect disappears in statistical 'noise'. a factor of five is somewhat closer to a realistic managerial scenario than if the high reward is 50 or 100 times higher than the low reward. even if a factor of 50 influenced behavior, it would be unlikely that such an intervention would be economically beneficial to implement. the participants in the condition with a low reward got 2 dkk (0.26 euros) per correctly solved task, while participants in the high reward scenario received 10 dkk. 30 dkk were does the size of rewards influence performance? added to the money earned from solving the tasks in order to ensure that participants reached the minimum baseline pay of 40 dkk that participants in the lab have to receive to participate in a study. only 3 participants solved too few tasks and had to be boosted to 40 dkk. the reward was taxable income and reported to tax authorities by the university. the best paid participant received 470 dkk (63 euros) for forty minutes of effort, which equals an hourly wage of 705 dkk, before taxes. this is a high wage, but it is not unlikely that some of the participants will earn a similar wage at some point in their life, especially considering most of them are university students. the following paragraph is the information regarding reward size given to the participants with the low [high] reward: your expected earnings for participating in the study are between 40 [40] and 230 [1.030] danish crowns. you will be rewarded with a fixed amount of 30 [30] dkk. for each correctly solved task, 2 [10] dkk will be added to your payment. you cannot earn less than 40 [40] dkk. the reward for solving one task is the same in both types of exercises. the participants were not informed that the experiment was about pay and performance, and thus were not aware that other participants were exposed to a different reward structure. participants were given the following piece of information regarding the aim of the experiment when signing up: "the aim of the study is to gain more knowledge as to how performance can be influenced by contextual factors outside the participant's control." deciding not to inform participants about the variation in reward structure also implies that we relied on a between-subject design instead of the within-subject design in ariely et al. [5] . earlier research showed that changing reward structure could influence behavior in the second round of exercises [16] . furthermore, in a within-subject design one risks that participants learn more efficient problem-solving strategies and become better over time. a betweensubject design thus reduces the risk of 'noise' from learning and switching effects. two sessions were run on any given day, one starting at 09:00 and one starting at 10:00. the same time of day was chosen since cognitive fatigue can influence students' performance on standardized tests [32] , and therefore running experiments at many different times of the day would induce unnecessary variation. a random number generated by excel was used to decide if the first session would be run with the low or the high reward. the other session was then run with the other reward structure. no sessions were run saturday or sunday, and two days involved only one session, due to lack of participants. the slots were posted on the laboratory's website for participants and the participants could then choose to participate in the experiment. the analysis has been preregistered (https://osf.io/sb6ty/) following the template from aspredicted [33] . an english version can be found in s1 appendix. non self-explanatory variables are described in the following. performance (dependent variable). performance is the sum of the number of solved tasks in the adding task and the anagram task. the sum is chosen as the dependent variable instead of the two exercises being analyzed separately. this pre-registered approach is selected in order to reduce the degrees of freedom and because the exercises are chosen to test the same thing; influence from size of reward on performance when solving cognitively demanding tasks. conscientiousness (control variable). conscientiousness has consistently been shown to be correlated with job performance [34] , which is why it is included as a control variable, and captured via the tipi [31] . we selected this short personality inventory due a worry about the potential cognitive fatigue that the long tests might imply and the fact that it is a control variable, not a main predictor. see s3 appendix for an overview of the questions. in order to further investigate how the reward size might influence behavior, we also assess if self-reported measures of effort [6] , intrinsic motivation [7] and focus [5] vary depending on the reward structure the participants have been exposed to. these three variables are described in the following. intrinsic motivation and effort. in order to measure intrinsic motivation (cf. self-determination theory) and effort (cf. microeconomics) we have used questions based on imi [35] , but worded according to our setting. for reference, [36] report a cronbach's alpha of 0.78 for the interest-enjoyment scale (our measure of intrinsic motivation) and 0.84 for effort. four questions capture the self-perceived effort that the participant put into solving the exercises, while four questions capture the self-perceived interest and enjoyment (i.e. intrinsic motivation). all questions are listed in s2 appendix. in order to calculate an intrinsic motivation score, answers to question 2, 4 (reversed), 7 (reversed), and 8 are averaged (cf. the appendix). in order to calculate an effort score, answers to question 1, 3 (reversed), 5 (reversed), and 6 are averaged (cf. the appendix). since three hypotheses are tested in the secondary analysis, the alpha level is adjusted using bonferroni's method. this means that the alpha level is 0.0167. we have tested for difference in mean using welch's t-test instead of student's t-test. this test's risk of type 1 error is closer to the significance level when the variances are unequal compared to both student's t-test and a choice between student's t-test and welch's t-test based on a preliminary test for equality of variances [37] . focus. in order to measure how focused the participants were, three questions have been developed. these are the three last questions from the questionnaire shown in s2 appendix. these questions aim to capture if the reward distracted the participants in their task solving. the average from these three answers (with the last question reversed) is the focus score for a participant. fig 3 illustrates how there were no substantial differences between the individuals in the two conditions. out of the 149 participants two persons did not inform their age, and two persons did not fill out the survey designed to measure effort, intrinsic motivation, and focus. the data has been analyzed using a multiple linear regression, using the size of the reward (low or high) as an independent variable, while gender and conscientiousness have been added as covariates. the only pre-registered dependent variable is the total number of solved tasks, which allows us to investigate if a high or low reward influences performance. homoskedasticity has not been assumed, and thus robust standard errors have been used. the regression was estimated using ols with the following regression equation: fig 4 reveals a p-value of 0. 12, and thus no strong support for either a positive or negative effect from a larger reward. however, we note that the confidence interval is rather wide, and a positive effect is still contained in the confidence interval. we provide an additional, exploratory analysis, since some participants on the first day of sessions (two sessions, 16 participants) did not solve the tasks in the order presented, despite this being noted as an explicit requirement in the written instructions. in the following analysis does the size of rewards influence performance? answers from these two sessions were corrected as if the instruction had not been given. in the remaining sessions this instruction was emphasized, and these answers were corrected with this instruction in mind. we include a dummy for the first day of sessions, in order to control for the difference, which leads to the following equation: the results in fig 5 are almost identical to the former model, showing a slightly higher pvalue (0.16) and very similar confidence interval. thus, including the dummy does not change the conclusion, and we again cannot reject the null hypothesis that size of reward does not influence performance. all three theories identified in the introduction rely on a mediating, explanatory variable. we therefore also analyse if the size of reward influenced effort (mediator in microeconomics), intrinsic motivation (mediator in self-determination theory), or focus (mentioned as a potential meditator by [5] ). this allows us to test if we might be unable to measure the effect of reward size on performance, but can identify a change in the level of effort, motivation or focus. does the size of rewards influence performance? the descriptive data only contain 147 observations since two participants chose to not fill out the questionnaire aimed at measuring these things. the data do not show a significant effect from size of reward on either focus, intrinsic motivation, or effort at the 0.0167 threshold. effort has the lowest p-value (p = 0.09), however the participants who received the low reward exerted more effort according to data-the opposite of the prediction offered by classical microeconomics. the data can be exploratory re-analysed excluding the results from the first two sessions. however, this does not result in a statistically significant difference in any of the tests (see s4 appendix). we also note that the low cronbach's î± for the questions measuring focus (0.55, cf. fig 6) imply the associated t-test should be interpreted with caution. we tested whether the self-reported effort, intrinsic motivation, and focus influenced performance, irrespective of the reward size. fig 7 shows that both effort (p < 0.01 for both models) and intrinsic motivation (p = 0.018 and 0.020 for model 3 and 4 respectively) are positively and significantly related to performance, as predicted by classical microeconomics and self-determination theory. participants that performed well thus self-reported higher levels of effort and intrinsic motivation than those that did not do well, corroborating that these factors are indeed important for the tasks. does the size of rewards influence performance? a statistically significant effect of focus on performance could not be detected (p = 0.318 and p = 0.294). however, this might be due to the fact that questions aimed at measuring focus did not fully capture this construct, cf. the relatively low cronbach's î±. these results are robust to model specifications (see fig 8 which includes covariates) and the exclusion of data from the first two sessions (see s4 appendix). results from nine former studies on the comparative impact of high vs. low rewards are remarkably mixed, since one can find support for all kinds of effects; positive, negative and no effect. we worry that publication bias might skew the available results, since documenting a null effect is difficult and have a higher risk of not being published [23, 38] , while documenting small effects require substantial sample sizes. in our pre-registered randomized, controlled trial we do not find strong evidence that supports higher stakes reduce or improve performance. the absence of evidence is not evidence of an absence of an effect from a higher reward, of course, but given our sample size we could have identified an effect size of r = 0.161 if relying on a simple linear regression (cf. [26] ). the correlation between high reward and performance was 0.125 in our study, and with this correlation we would have required a sample size of 247 to obtain a p-value below 0.05, if relying on a simple linear regression. while we cannot rule out the existence of a small positive effect of high rewards, our study reduces the prior belief one should have in the replicability of former, relatively large effect sizes, in either direction. furthermore, a small non-cumulative effect would generally not be economically efficient to implement for a manager. to illustrate, even if we assume that the difference our data implies are not just random fluctuations, the high rewards merely lead to approximately two extra solved tasks, out of an average of about 22 tasks solved. yet, a manager would have to pay a factor of 5 to gain such a relatively small improvement. we should add that higher stakes would be economically beneficial for the one receiving the higher reward. additional analysis supports the interpretation that we do not find strong support for reward structure influencing the participants in our sample. micro-economics argues that a higher reward should lead to a higher performance since the reward structure should influence the effort, while self-determination theory argues that a higher reward could harm intrinsic motivation. however, the size of the reward did not have a statistically significant effect on neither the self-reported level of effort nor intrinsic motivation, while both effort and intrinsic motivation was significantly positively related to performance. thus, we do not find strong support for the size of the reward shaping the dependent variable, or the mediating variables they are supposed to influence. overall, while former studies have shown that a piece-rate payment system generally leads to higher performance compared to flat-rate payments when individuals are solving relatively simple tasks [3] , we find no clear behavioral effect from manipulating the reward size. since our data did not imply a risk of a ceiling effect, one could speculate that getting some kind of monetary reward is a sufficient motivator for participants in a lab-study, while having the opportunity to gain relatively big rewards do not lead participants to choke due to pressure [5] . yet, a range of studies from the world of sports repeatedly indicate that large reward differences, tend to lead to high stakes reducing performance [9] [10] [11] [12] [13] . this discrepancy between sports and cognitive tasks in the lab might simply be the difference between a randomized, controlled trial and observational studies, which cannot exclude potentially confounding factors, such as the public scrutiny and the status competition that sports events also imply. ideally one would have data on sports events with only a single competitor or without an audience. however, in addition to these confounding factors, we also find good reasons to believe that different theoretical mechanisms are in play in the various settings. all sports disciplines studied require not only a basic cognitive effort (as in the typical lab-task) but also a concentrated, physical motor effort; e.g. when completing a golf putt or a basketball free throw [9, 10, 13] . these various skills arguably have a different evolutionary history: "we are all prodigious olympians in perceptual and motor areas, so good that we make the difficult look easy. abstract thought, though, is a new trick, perhaps less than 100 thousand years old. we have not yet mastered it." [39 p. 2] . one can therefore argue that we should expect performance differences depending on the skills required in the given setting. for example, being able to drive a golf ball relies on motor skills, while a golf putt requires not only motor skills but also a cognitive effort and ability to assess the length of the putt and slope of the green. we consider it a promising avenue for future research to disentangle these variables. we propose that future studies should investigate the contrast between a) simple, cognitively mundane tasks [5] , b) insight tasks [40, 41] , c) tasks only requiring motor skills (e.g. a golf drive) and d) tasks that require motor skills as well as cognition (e.g. a golf putt). if one relies on a typical pool of lab-participants, one also avoids the potential selection effect of the very experienced sports players that participate in high performance sports. we expect that tasks solely requiring high motor skills should see less of a negative effect of higher stakes compared to tasks that require motor skills as well as cognition. high stakes in insight tasks in particular and cognitively very demanding tasks in general could lead to worse performance levels compared to lower stakes scenarios. this negative effect is argued to be due to reduction in intrinsic motivation, ability to focus and that the reward structure can lead to a more conservative search strategy [1] inhibiting the opportunity to find good solutions to the task at hand. finally, all these investigations could involve a competitive element, as in sports, or not, in order to further identify the extent of this effect. to sum up, we did not identify an effect of the size of the stake involved, when solving a mundane, cognitive task. we worry that the lack of articles not showing an effect might reflect a file-drawer bias [23, 25] . only by providing public access to studies that cannot document an effect, can we update the relevant prior one should have concerning the 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growth mindset improves achievement hofstede insights. compare countries. hofstede insights letter shuffler world's simplest randomization tool a very brief measure of the big-five personality domains cognitive fatigue influences students' performance on standardized tests aspredicted: create. aspredicted article has been frequently cited: the big five personality dimensions and job performance: a meta-analysis intrinsic motivation inventory (imi) psychometric properties of the intrinsic motivation inventory in a competitive sport setting: a confirmatory factor analysis. research quarterly for exercise and sport a note on preliminary tests of equality of variances full publication of results initially presented in abstracts: a meta-analysis a critique of pure learning and what artificial neural networks can learn from animal brains differentiating insight from non-insight problems how to investigate insight: a proposal article collections published by the public library of science key: cord-252795-x66zqmgv authors: islam, md. akhtarul; barna, sutapa dey; raihan, hasin; khan, md. nafiul alam; hossain, md. tanvir title: depression and anxiety among university students during the covid-19 pandemic in bangladesh: a web-based cross-sectional survey date: 2020-08-26 journal: plos one doi: 10.1371/journal.pone.0238162 sha: doc_id: 252795 cord_uid: x66zqmgv the purpose of this study was to investigate the prevalence of depression and anxiety among bangladeshi university students during the covid-19 pandemic. it also aimed at identifying the determinants of depression and anxiety. a total of 476 university students living in bangladesh participated in this cross-sectional web-based survey. a standardized e-questionnaire was generated using the google form, and the link was shared through social media—facebook. the information was analyzed in three consecutive levels, such as univariate, bivariate, and multivariate analysis. students were experiencing heightened depression and anxiety. around 15% of the students reportedly had moderately severe depression, whereas 18.1% were severely suffering from anxiety. the binary logistic regression suggests that older students have greater depression (or = 2.886, 95% ci = 0.961–8.669). it is also evident that students who provided private tuition in the pre-pandemic period had depression (or = 1.199, 95% ci = 0.736–1.952). it is expected that both the government and universities could work together to fix the academic delays and financial problems to reduce depression and anxiety among university students. the outbreak of coronavirus diseases (covid -19) has been substantially influencing the life and living of people across the world, especially after the declaration of a global pandemic by the world health organization in the second week of march 2020 [1] . as of june 7, 2020, around 6.91 million people were infected with the covid-19, with a confirmed fatality of another 0.4 million worldwide [2] . hence, many countries implemented a range of anti-epidemic measures, such as restricting travel for foreign nationals [3] , closing down public spaces, and shutting down the entire transit system [4, 5] , to contain the transmission of the highly contagious infections from human-to-human. following the detection of first covid-19 case on march 8, 2020 [6] , bangladesh like many other countries put the lockdown strategy into effect on march 26, 2020 , to ensure 'social distance' through 'home quarantine' to curb the 'spread' among its population [7] [8] [9] , since a precise treatment or vaccine for the infected and people at risk are yet to achieved by the global health community [10, 11] . however, all education institutions were closed initially from march 18 to march 31, 2020 across the country and later extended to the mid of june 2020 in phases [12, 13] . this unprecedented experience of 'home quarantine' under lockdown with the uncertainty of academic and professional career has multifaceted impacts on the mental health of students. for example, a canadian study focusing on the effects of quarantine after the severe acute respiratory syndrome (sars) epidemic found an association between longer duration of quarantine with a high prevalence of anxiety and depression among people [14] . the ongoing covid-19 pandemic is creating a psycho-emotional chaotic situation as countries have been reporting a sharp rise of mental health problems, including anxiety, depression, stress, sleep disorder as well as fear, among its citizens [15] [16] [17] [18] [19] , that eventually increased the substance use [15] and sometimes suicidal behavior [20] [21] [22] . researchers in china observed that the greater exposure to 'misinformation' through social media are more likely contributing to the development of anxiety, depression, and other mental health problems among its population of different socioeconomic background [23] [24] [25] [26] . studies before the covid-19 pandemic also suggested an inverse relationship between media exposure and mental health [27, 28] . on the contrary, a study in south korea during the middle east respiratory syndrome (mers) reported a positive relationship between risk perception and media exposure [29] . given the unexpected circumstances, it is crucial to explore the psycho-social experience of university students in bangladesh, especially during the covid-19 pandemic. such a study is expected to measure the psychological impacts of an unforeseen emergency on students, as well as to formulate and execute effective interventions and strategies to mitigate the mental health of people at large. this study was designed to address the psychological problems experienced by university students in bangladesh. the survey was conducted in the second week of may, from may 6 to may 12, 2020. students enrolled in different universities across bangladesh were the target population. an easy to understand questionnaire was used to collect 'basic information,' 'depression,' and 'anxiety' related information. an online-based platform was used to distribute the e-questionnaire, developed by using the google form, to the students. university students from all the divisions in bangladesh were contacted through different social networks and interviewed (see fig 1) . the snowball sampling technique was used for collecting information from students. an informed consent form was attached to the e-questionnaire, and each participant consented to participate in the survey after reading the consent form. the participants were asked to share the e-questionnaire with their friends using their personal and institutional facebook and messenger. this study was formally approved by the ethical clearance committee of khulna university, bangladesh. the participants responded anonymously to the online survey by filling up an informed consent letter in the first section of the e-questionnaire. in the consent form, all the participants were provided with information concerning the research purpose, confidentiality of information, and right to revoke the participation without prior justification. basic information. 'basic information' contained the personal information of the respondents. current 'age' of students ('17-20', '21-24', '>24'), whether the student is 'lagging behind study' ('yes' and 'no'), doing any sorts of 'exercise during lockdown' ('yes' and 'no'), students who did 'tuition' before lockdown ('yes' and 'no'), the gender of the student ('male' and 'female'), 'place of residence' of students ('rural' and 'urban'), is he/she 'living with family' during lockdown ('yes' and 'no'). depression. depression was determined by using the patient health questionnaire (phq-9). phq-9 is an easy way to use in a questionnaire for screening depression of the responses that are used to predict depression of an individual and what state he/she is in during the survey. the scores in phq-9 range from '0 = not at all' to '3 = nearly every day' [30] . the reason for choosing phq-9 was that it proved to be a useful tool for detecting depression [31] . the levels of depression for the study were categorized as 'mild = 5-9', 'moderate = 10-14,' 'moderately severe = 15-19,' 'severe = � 20.' anxiety. anxiety was evaluated by using the generalized anxiety disorder (gad-7). in the questionnaire, the questions were used for screening anxiety state of an individual on a scale ranging from '0 = not at all sure' to '3 = nearly every day' [32] . gad-7 has been found successful in identifying anxiety among different populations and thus used for its reliability [33] . the levels of anxiety for the study were categorized as 'none-minimal = <5,' 'mild = 5-9,' 'moderate = 10-14 and 'severe = � 15.'. frequency tabulation was used to summarize basic information of respondents, as well as their response to depression and anxiety. binary logistic regression [34] was used to identify variables influencing depression and anxiety among students by categorizing the outcome variable into two categories, i.e., depressed = 'yes' and 'no' and anxious = 'yes' and 'no,' which would provide a clearer idea about how intensely different factors are influencing the outcomes. logistic regression generates the coefficients (and its standard errors and significance levels) of a formula to predict a logit transformation of the probability of the presence of the characteristic of interest: where p is the probability of the presence of the characteristic of interest. the logit transformation was defined as the logged odds: and, rather than choosing parameters that minimize the sum of squared errors (like in ordinary regression), estimation in logistic regression accepts parameters that maximize the likelihood of observing the sample values. table 1 shows the descriptive information of different selected variables of the university student in bangladesh. results show that 392 (82.4%) students were found to have mild to severe depressive symptoms, and 389 (87.7%) students were found to have mild to severe anxiety symptoms. more than 60% of the students were male (67.2%), and the rest were female. one in three students lived in rural areas (35.1%). less than a quarter percent of students (24.8%) believed that they were not academically lagging, and just over 30% reportedly have exercise regularly during the lockdown at home. table 2 shows the prevalence of depression and anxiety among bangladeshi university students. out of the total 476 valid participants, 392 (82.4%) were found to have mild to severe depressive symptoms. male (67.35%) had higher depressive symptoms than the female (32.65%) counterparts, whereas students in the early twenties (66.07%) showed higher depressive symptoms than other age groups. depression was also prevalent among students with no physical exercise (62.24%) and those who consider themselves lagging behind others in terms of academic activities (76.78%). besides, students living with families (96.93%) and in urban areas (65.05%) showed higher depressive symptoms. in the case of anxiety, 389 (87.7%) students exhibited mild to severe anxiety symptoms. out of the total students suffering from an anxiety disorder, females (33.67%) had lower anxiety symptoms than males (66.33%), whereas students in the early twenties (66.58%) showed higher anxiety. like depression, anxiety was also prevalent mostly among students with no physical exercise (61.95%), troubled with the thought of lagging behind others academically (76.60%). moreover, students living in urban areas (62.21%) with families (96.40%) also showed symptoms of anxiety. table 3 reveals that students who thought that s/he was lagging behind others in academic activities were 1.8 times (95% ci: 1.098, 2.935) more likely to be depressed than the student with no such worries. students living with families were 2.6 times (95% ci: 1.418, 4.751), more likely to be depressed than the students living apart from families. on the other hand, students providing supplementary classes before lockdown were 1.4 times (95% ci: 0.856, 2.227), more likely to show mild to severe anxiety symptoms than their counterparts with no such involvement. students who were worried about their academic activities were 1.8 times (95% ci: 1.099, 2.883) more likely to exhibit mild to severe anxiety symptoms than students with no such worries. students living with families were 1.8 times (95% ci: 1.021, 3.308), more likely to have mild to severe anxiety symptoms than students staying away from families during the lockdown. covid-19 pandemic came out as the most devastating and challenging crisis for public health in the contemporary world. apart from the soaring mortality rate, nations across the globe have also been suffering from a spike of the excruciating psychological outcomes, i.e., anxiety and depression among people of all ages. university students are no exception, as all the educational institutions are unprecedentedly closed for more than usual, and for bangladesh, it is more than two months in a row. such closure, in general, triggers a sense of uncertainty about academic and professional career among the educands and intensifies persistent mental health challenges among university students [33, 35, 36] . given such circumstances, the main goal of this study was to investigate the prevalence of depression and anxiety among the bangladeshi university students during the covid-19 pandemic and to explore the factors influencing the presence of depression and anxiety disorder. the findings of the web-based cross-sectional survey indicate that more than two-thirds of the students were experiencing mild to severe depression (82.4%) and anxiety (87.7%). earlier studies in bangladesh observed the presence of both depression and anxiety among students in higher academia. for example, a survey of medical students in 2015 suggested that more than 50% of students in medical colleges are suffering from depression (54.3%) and anxiety (64.8%) [37] . another study, on university students excluding the freshmen, complemented the previous work and found that the prevalence rate of depression and anxiety was 52.2% and 58.1%, respectively [38] . compared to the earlier studies, our study suggests that university students in bangladesh are experiencing an unparalleled growth of depression and anxiety under the current global pandemic situation. the results also suggest that the university students' involvement in private tuition is a critical factor in understanding the increased prevalence of depression and anxiety among them. in bangladesh, a significant number of students are involved in part-time jobs, such as private tuition, to finance their educational expenses, and sometimes to support their families, and their reliance on private tutoring as a part-time job is increasing gradually [39] . however, being unable to provide tuition under the lockdown situation means disruption of regular income and joblessness. the prolonged unemployment, together with financial insecurity, is the most significant stressors contributing to the increased rates of depression and anxiety among university students in bangladesh. a study suggests that unemployment is significantly associated with mental and somatic disorders, which could limit the individuals' chances for feelings of achievement, accomplishment, and satisfaction, and eventually lead to the impairment of psychological functioning [40] . self-esteem could also be affected by the loss of work as studies found that lack of family support during unemployment adversely affects the mental well-being of individuals [41, 42] . apparently, the sudden joblessness and financial insecurity are putting the university students in an unpleasant situation, affecting their socioeconomic and mental well-being [43] . it has been well accepted that living with families strongly generate reassurance among the individuals, therefore, reduce depression and anxiety. because positive family environments often benefit the mental health of the vulnerable youth experiencing depression or anxiety [44] . however, this pandemic has brought extreme financial pressure on families. most of the families have been suffering from unmanageable debts and a decline in income, thus, leaving the family members in a traumatized situation [45, 46] . university students, who used to earn and contribute to their families before lockdown, can hardly assist their parents in this crisis moment. the results of this study suggest that despite living with family, anxiety and depressive symptoms have been increasing among university students in bangladesh mainly due to financial insecurity. universities in developed countries put strict health protocols into action, such as washing hand, using face-mask, advising 'stay-home' strategy when sick, to facilitate continuation of education in higher academia and later switched to campus-wide online learning [47, 48] . in bangladesh, the protective interventions, such as wearing mask or using the personal protective equipment, are yet to be enforced largely due to limited supplies [49, 50] , hence, the government opted to implement the country-wide lockdown. approximately two-thirds of the students are getting depressed thinking they might be falling academically behind their contemporaries in other parts of the world during the prolonged closure of universities. they, however, reiterated that the online classes could not fulfill their requirements [51] and a significant percentage of the students are still out of the reach of the online class. in addition, their research projects and internships had to be ceased since they were instructed to leave the halls (dormitories for students) of their respective universities [3] . not only that, the covid-19 crisis also created a severe challenge of the global reversion for the graduates to accomplish their future academic and working goals [52] . although university closures were intended to keep students safe, for many, these notions came out with different sets of mental health issues. meanwhile, a study reported that graduate students generally experience significant amounts of stress and anxiety, which also affects their usual behavior [53] . the results in this study stressed on the fact that the nation-wide lockdown in bangladesh is going to cause a significant disruption in the academic programs and create a gap in both teaching and learning. the academic delays could have long-term impacts on the psychology of students as they are more likely to be graduated later than they have expected. in this regard, faculties, as well as university authorities, should stay connected with the students using social media platforms and motivate them to move forward together during this difficult time. apart from the issues mentioned above, this study found no significant differences between male and female students with relation to depression or anxiety, thus complement previous studies [36, 37, 54] . however, egyptian research remarked that female university students are more likely to suffer anxiety and less prone to depression than male students [55] . the current study did not find any statistically significant association between the socio-demographic variables (including place of residence and exercise) with depression and anxiety. a few studies, on the contrary, reported a significant association between socio-demographic variables [37] and exercise [56] with depression and anxiety. a malaysian study reported substantial differences concerning age and permanent residence with depression or anxiety, however, observed no significant association between some socio-demographic variables (including gender, ethnicity, study major, monthly family income) and the psychological problems [36] . the strengths and limitations of the current study are determined by several issues. the equestionnaire allows to assess the prevalence of anxiety and depression among university students while maintaining the who recommended "social distance" during the covid-19 pandemic, which otherwise would be impossible. moreover, the data for the e-survey were collected by globally validated standardized tools for quantitative analysis. on the contrary, given the limited resources available and the time-sensitivity of the covid-19 outbreak, the snowball sampling strategy was chosen instead of random samples. in this cross-sectional study, the identified factors are regarded as associated factors, which could be either be the causes or the results of depression or anxiety. furthermore, due to ethical requirements on anonymity and confidentiality, the contact details of the respondents was not collected. however, the use of validated screening e-questionnaire was considered as a cost-effective approach to explore the situation in general, therefore, used in this study. since the research methodology could not reach people with medically examined depression and anxiety symptoms, the provision of the results may not fully reflect the severity of depressive and anxiety symptoms among students. another limitation of this study is not using the tools designed specifically for the covid-19 pandemic, such as the coronavirus anxiety scale (cas). meanwhile, it would be ideal for conducting a prospective study on the same group of participants with tools developed especially for the covid-19 pandemic after a period to provide a concrete finding and to facilitate the demand for a focused public health initiative. despite some limitations, this study gives the first empirical evidence that a large percentage of bangladeshi university students have been suffering from depression and anxiety symptoms during the ongoing pandemic. in addition to academic and professional uncertainty, financial insecurity is contributing to the rise of depression and anxiety among university students. to minimize the growing mental health problems, the government, along with the universities, should work together to deliver promptly and accurately economy-oriented psychological support to the university students. to ensure the continuous involvement of students in educational processes, the universities should initiate all-inclusive online-based educational programs to reach out the students living in remote areas with or without devices in association with internet-service providers by providing scholarship or student loan. furthermore, parents should be encouraged, by providing pandemic response and recovery support from the government, to create a friendly and positive family environment for university students without imposing pressure on the future academic and working career. world health organization. who director-general's opening remarks at the media briefing on covid-19-11 coronavirus disease (covid-19): situation report-138 world health organization mental health care for international chinese students affected by the covid-19 outbreak epidemic of covid-19 in china and associated psychological problems china's ongoing battle against the 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economic challenges for a developing country from offline to online: challenges and opportunities for entrepreneurship education following the covid-19 pandemic. entrepreneurship education and pedagogy the impact of covid-19 on the sports medicine fellowship class of 2020 easing covid-19 lockdown: will wearing face masks be safe enough? dhaka tribune covid-19: use of face mask. the financial express online learning: a panacea in the time of covid-19 crisis closure of universities due to coronavirus disease 2019 (covid-19): impact on education and mental health of students and academic staff mental health and suicidal behavior among graduate students prevalence and socio-demographic correlates of mental health problems among iranian health sciences students. academic psychiatry inadequate sleep and exercise associated with burnout and depression among medical students depression, anxiety and stress among first year medical students in an egyptian public university we are grateful to the participants, as well as thankful to the editors and anonymous reviewers. key: cord-013356-y6vceq2x authors: peace, angela; pattemore, david; broussard, melissa; fonseka, dilini; tomer, nathan; bosque-pérez, nilsa a.; crowder, david; shaw, allison k.; jesson, linley; howlett, brad g.; jochym, mateusz; li, jing title: orchard layout and plant traits influence fruit yield more strongly than pollinator behaviour and density in a dioecious crop date: 2020-10-23 journal: plos one doi: 10.1371/journal.pone.0231120 sha: doc_id: 13356 cord_uid: y6vceq2x mutualistic plant-pollinator interactions are critical for the functioning of both non-managed and agricultural systems. mathematical models of plant-pollinator interactions can help understand key determinants in pollination success. however, most previous models have not addressed pollinator behavior and plant biology combined. information generated from such a model can inform optimal design of crop orchards and effective utilization of managed pollinators like western honey bees (apis mellifera), and help generate hypotheses about the effects of management practices and cultivar selection. we expect that the number of honey bees per flower and male to female flower ratio will influence fruit yield. to test the relative importance of these effects, both singly and simultaneously, we utilized a delay differential equation model combined with latin hypercube sampling for sensitivity analysis. empirical data obtained from historical records and collected in kiwifruit (actinidia chinensis) orchards in new zealand were used to parameterize the model. we found that, at realistic bee densities, the optimal orchard had 65-75% female flowers, and the most benefit was gained from the first 6-8 bees/1000 flowers, with diminishing returns thereafter. while bee density significantly impacted fruit production, plant-based parameters-flower density and male:female flower ratio-were the most influential. the predictive model provides strategies for improving crop management, such as choosing cultivars which have their peak bloom on the same day, increasing the number of flowers with approximately 70% female flowers in the orchard, and placing enough hives to maintain more than 6 bees per 1000 flowers to optimize yield. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 mutualistic plant-pollinator interactions play a vital role in plant reproduction in both natural systems and managed (i.e. agricultural) systems. animal-mediated pollination is important for 87.5% of angiosperms globally [1] , and 75% of the most important crop species benefit significantly from this service [2] , providing greater than us$170 billion in economic value annually [3] . functionally dioecious plants are especially reliant on pollination, as pollinators must cross from one plant to another. even in well-studied systems, such as kiwifruit (actinidia chinensis), the complexity of interacting variables limits the ability of researchers to provide clear recommendations to growers, with proposed stocking rates varying from 3-8 colonies per ha [4] . mathematical modeling of plant-pollinator interactions can help understand key determinants in pollination success [5] . such approaches could be valuable tools for designing optimal crop orchard layouts and for the effective use of managed pollinators in agricultural systems. this may be especially important in dioecious crops that have separate male and female plants which adds further complexity in conducting empirical field trials when these plants respond differently to environmental variables. in spite of this, pollination models have tended to focus on plant biology [6] [7] [8] [9] or insect behavior [10, 11] but few have examined both simultaneously [12, 13] . including variables such as flower phenology, the ratio of male to female flowers, pollinator abundance, and flower handling behavior could assist in the generation of robust models. combining information from both pollinators and plants in the same framework more realistically represents field conditions and enables us to directly compare their importance. a significant challenge in developing good models is sufficient data for parameterization. we chose kiwifruit as our model dioecious crop system as there are four decades of empirical data, examining many aspects of both insect behavior and plant biology [14] . kiwifruit is a deciduous vine, with male and female flowers borne on separate plants [15] . neither sex produces nectar, and the female flowers produce inviable pollen [15] , which is high in lipids [16] , to attract pollinators instead. plants are typically trained onto a pergola system, with male vines interplanted amongst a larger number of female vines at a 1:3 to 1:8 ratio [4, 17] . although male cultivars typically have 2-3x more flowers than female cultivars [18] , grower planting and pruning regimes ultimately determine the floral sex ratio in orchards. while kiwifruit have a number of pollinating species in their native range [19] [20] [21] , most growing regions rely on honey bees for pollination, representing the vast majority of all flower visitors in the united states [22] , france [23] , australia [24] , and new zealand [25, 26] . we expected that male-female kiwifruit flower ratio and pollinator density will influence fruit yield, along with various parameters of pollinator behavior. to test the relative importance of these effects, both singly and simultaneously, we used a system of delay differential equations (ddes) combined with latin hypercube sampling for parameter sensitivity analysis [27] . the model explicitly tracks pollinators (parameterized here based on data from honey bees), with varying pollen loads as they preferentially visit male and female flowers, as well as we develop and analyze a mathematical model of pollination dynamics that incorporates key aspects of both plant biology and insect behaviors. first, we present a sub-model of the flowering dynamics in an orchard, then in the following section, we add the pollinator dynamics. we assume homogeneous conditions across the field for both flower and pollinator densities. table 1 describes the model state variables and parameters. flowering dynamics. we consider a kiwifruit orchard made up of male and female trees and model the opening and closing of flowers throughout the bloom. to capture pollination dynamics, it is important to know how many male and female flowers are open at any given day. here, we assume that the total number of flower buds in the field is fixed and the rate they open follows a normal distribution. let b m and b f denote the total number of male and female flower buds. initially all flower buds are closed. let m and f denote the number of male and female flowers that have opened. the rates that these flowers open is modeled as mðtþ elsewhere pollinator dynamics. pollinator dynamics are modeled with differential equations that divide the population into subcompartments based on their pollen load. pollinators can have a high, medium, or low pollen load (denoted as p m1 , p m2 , and p m3 respectively) or be carrying no pollen (denoted at p f ). these states represent a division of empirical data on single-visit deposition, which often follows an exponential [28] or steeper than exponential decay [29] . we assume that pollinators completely load up on pollen with a visit to a male flower and deposit some pollen with a visit to a female flower. we assume that male pollen availability is not limiting in this scenario; to partially compensate for this short-coming of the model we limit active foraging to four hours per day as captured by the visitation rate, corresponding to field observations [30] . this four-hour window of pollen-foraging activity limits the total amount of pollination in a day (built into the visitation rate parameter). within this window, pollen availability is typically not a limiting factor in kiwifruit due to male flowers having up to twice as many pollen grains than female flowers, and the anthers continued to dehisce over this four-hour period. a diagram depicting the movement of pollinators between the compartments is shown in fig 2. a pollinator with a high pollen load can move between compartments p m1 , p m2 , p m3 , and p f with subsequence visits to female flowers. regardless of current pollen loads, whenever a pollinator visits a male flower it completely loads up on pollen and enters the p m1 compartment. the rate that pollinators visit male and female flowers is a crucial part of the model dynamics. we consider a pollinator visitation rate that depends on the search rate (α), the handling time (β) and the densities of open male (m) and female (f) flowers, as described above in eq (2) . for pollinator visitation rates, previous work suggests that saturating functions of flower densities such as holling type ii functional responses are typical of oligolectic consumers that use only a few plant species and holling type iii responses are typical of generalist consumers that switch between hosts [6] . while honeybees are generalist, here we use a holling type ii response because of the mono-culture orchard environment in the model. following previous studies [6, 31, 32] we defined the total pollinator visitation rate as: which has the units of per time. this visitation rate includes visits to both male and female flowers. the movement of pollinators between male and female flowers depends on the proportion of male vs female flowers, as well as pollinator preferences. previous studies suggest that honey bees have a preference to visits flowers of the same sex as the one they are currently on [22, 24, 33, 34] . we define the preference parameter δ such that a pollinator on a male flower can preferentially choose to next visit another male flower (0 < δ < 1). similarly, we define the preference parameter � such that a pollinator on a female flower preferentially next visits another female flower (0 < � < 1). pollinators have no preference if δ = � = 1. we used these preference parameters to define functional forms representing the probability of a pollinator to visit each type of flower, following the method used in [35] . these probabilities depend on the ratio of male to female flowers raised to the power of the preference, such that the movement of pollinators between flowers can be written as the following expressions: 1 à m f þ m � � d fraction on male flowers that move to a female flower ð4bþ � ε fraction on female flowers that move to a female flower ð4cþ � ε fraction on female flowers that move to a male flower ð4dþ values for strong preferences were used for the analyses in this paper, details are described in the parameterization section. note that a strong preference for remaining on the same type of flower corresponds with a low probability of switching between flower types. https://doi.org/10.1371/journal.pone.0231120.g003 full pollinator-flower model. the complete pollinator-flower model are described with the following system of differential equations: |ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl {zffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl } total visitation rate þ p f þ |ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl {zffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl } moves from female to male |ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl {zffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl } |ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl {zffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl } moves from male to female � p m2 |ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl {zffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl } moves from female to male :ð5dþ the incorporation of flowering dynamics given in eq (2) into the system of differential equations for the pollinators model (5) results in a system of ordinary differential equations when t � min{τ m , τ f }, before any open flowers begin to close, followed by a system of delay differential equations with a single delay τ = min{τ m , τ f } when min{τ m , τ f }�t � max{τ m , τ f }, and then by a system of delay differential equations with two fixed delays, τ m and τ f . this model tracks the number of open male and female flowers (m, f) and the number of pollinators of each type (p m1 , p m2 , p m3 , p f ) as they visit male and female flowers. pollination measurement. the total number of visits to female flowers is an important factor for pollination. visits to female flowers from the different classes of bees represent different pollen depositions needed to determine success of fruitset. we define the visits of pollinators to female flowers that result in pollen deposition as either type one, two, or three, as depicted in fig 2. we then define fruit set for a day t denoted by p(t) as where v n (t) for n = 1, 2, 3 represents the total number of type n visits that each flower has received at the time of closing (day t), and p n represents the percent chance that a single visit will fully pollinate a flower to set fruit, for each visit type n. the total predicted yield is the fruit set for each day multiplied by the number of female flowers closing on that day, summed over all the days, the total predicted yield ¼ where dfc(t) denotes the daily number of female flowers closing at day t. the total predicted yield proportion over all days is the number of flowers closing on each day multiplied by the fruit set for that day divided by the total number of female flowers, in our calculation we use the number of total female flower buds, the total predicted yield proportion ¼ all model parameters are listed in table 2 . in order to parameterize the visitation rate eq (3) we assume the pollinators are active in the field for only 4 hours per day. for the search rate α we assume a pollinator encounters 2 flowers per min, or 480 visits per day, assuming they forage only 4 hours a day. for the handling time β it has been observed that the average time a pollinator spends on a flower is 16 sec, or 0.0011 days [36] . we use an odds ratio to parameterize the preference parameters, δ and �. experimental observations on pollinator behaviors in environments with equal density of male and female flowers (1:1 ratio, which is typical across planting regimes even when the ratio of male to female plants differs, due to flower pruning practices) reveal that pollinators on male flowers have a 0.957 probability of remaining on male flowers and those on female flowers have a 0.951 probability remaining on female flowers [14, 24, 34] . this information was used to help parameterize the preference parameters. following the experimental conditions, we assume an equal density of male and female flowers and take δ = ln(0.957)/ln(0.5) and � = ln(0.951)/ln(0.5). it is important to note, that while these preference parameters are constant, the probability of switching from flower types depends on these preferences, as well as the open number of male and female flowers, see eq 4 and [30] , and we assume a baseline value of ρ = 6 pollinators. we assume the p 2 percent chance to set fruit from single type two visit 0.55 0.1-0.65 [14] p 3 percent chance to set fruit from single type three visit 0.22 0-0.5 [14] https://doi.org/10.1371/journal.pone.0231120.t002 modeling plant-pollinator interactions to predict fruit yield percent chance that a single type one visit (transitions a pollinator from group p m1 to group p m2 ) will fully pollinate a flower to set fruit is p 1 = 66%. a single type two visit (transitions from p m2 to p m3 ) will fully pollinate a flower with assumed p 2 = 55% and a single type three visit (transitions from p m3 to p f ) will fully pollinate a flower with assumed p 2 = 22% [14] . for the total number of flowers we assume the number of flower buds follows b m = b f = 600, 000 per ha. all simulations were conducted using matlab's differential equations solvers ode45 and dde23 with initial conditions such that 0% of pollinators were p m1 , p m2 , and p m3 , and 100% of pollinators were p f at time t = 0 for an orchard of sample size of 1 ha. parameter values for the total number of flower buds b m (male) and b f (female) along with the number of pollinators per 1000 female flowers ρ are used to determine the total number of pollinators for each simulation. base simulations testing model behaviors. model simulations for the set of baseline values given in table 2 are shown in fig 4. pollinators of type p m1 and p f fluctuate during the blooming period while the number of pollinators of types p m2 and p m3 remain low (fig 4a) . the accumulated number of visits to female flowers at the time of closing is almost identical across visit types (fig 4e) , and is driven by the number of open female and male flowers, since the number of pollinators is fixed. our model output measure (total predicted yield) is shown in fig 4f. as expected, type one visits have the highest fruit set rate while type three visits have the lowest fruit set rate, even though the total number of these visits are similar (fig 4f) . the results in fig 4(f) multiplied by the daily number of female flowers closing yields the daily predicted yield. then the summation of this yield returns the main output for our model; the total predicted yield (see eq (7)). under these baselines values total predicted yield is 545,120 fruit / ha with a predicted fruitset of 90%. this is on the high end of reported fruit set in "hayward" orchards (c.f. 80% in gonzales et al. 1998 [43] ), but matches the experience of the authors in field trials where fruit set is measured before harvest and thus is a higher figure than fruit set calculated for yield (pattemore d pers. obs., broussard m pers. obs.). accordingly, the fruit number per hectare is higher than the 200,000-300,000 often reported in the literature [44] [45] [46] , but again is within the range of possible outcomes. simulations exploring model outputs. we varied key model input parameters and investigated model predictions with numerical simulations and sensitivity analysis. model parameters are presented in table 2 . a major model output measure is the predicted yield, which is defined as the number of female flowers per ha that became fully pollinated fruit. a second important model output is the percentage of female flowers that became fully pollinated fruit, defined here as the fruit set. we used numerical simulations to explore variations in flowering dynamics including the percentage of buds that are female and shifts in the duration of time when male and female flowers are both opened (by varying the peak date in male flower opening rate). we also explored variations in pollinators dynamics including bee densities, preference parameters and pollinator handling time. parameter sensitivity analysis. in order to better assess the predictions of our model we investigate the uncertainty of the estimated parameter values using latin hypercube sampling (lhs) with the statistical partial rank correlation coefficient (prcc) technique, which provides a global parameter sensitivity analysis. lhs is a stratified monte carlo sampling method without replacement giving a global and unbiased selection of parameter values [27] . the prcc technique is used to assess the importance of each parameter for a given output measure. it is appropriate when the parameters have a nonlinear and monotonic relationship with table 2 with initial conditions that pollinators haven't collected any pollen yet (i.e., p m1 = p m2 = p m3 = 0 and p f = ρ � b f /1000). the output measures. using a model orchard of 1 ha we used lhs to sample the parameters listed in table 2 and used prcc to investigate the output measure of the total predicted fully pollinated fruit per hectare (yield). following marino et al. 2008 [27] we performed a z-test on the resulting prcc values and verified that, in general, higher magnitude prcc values correspond with a stronger influence on the output measure. most of the parameters had nonlinear and monotonic relationships to the total predicted yield. additional investigation on parameter values that were nonmonotonic was done by truncating the parameter space to monotonic regions, details are presented in the appendix. to investigate the role of key plant parameters we varied the ratio of male to female flowers in the orchard by fixing the total number of flowers and varying the percentage of flowers that are female, all other parameters were set to their base values shown in table 2 . increasing the fraction of flowers that are female (versus male) per hectare first increases the total predicted yield (fruit per hectare), peaking near 0.66, and then decreasing rapidly as female flowers make up the majority of the orchard (fig 5) . when the fraction of female flowers per hectare is low, nearly all female flowers produce fruit: predicted fruitset reaches above 97%. however, the total yield (fruit produced) is low due to the low quantity of female flower buds. on the other hand, when most flowers are female, predicted fruitset decreases to 20% along with an associated decline in yield. this is due to the fact that while the quantity of female flowers is high, the quantity of male flowers is low and the chances of successful pollination decreases other key plant parameters influence the timing of when male and female flowers are open and receptive. pruning and the use of chemical bioregulators are typically used to control the onset and duration of flowering by growers [47] . over a longer time frame, cultivar selection can be used to ensure adequate overlap of male and female flowering across a range of environmental conditions. the model assumes the rate that flowers open follow normal distributions with key parameters specifying the peak day of flower openings for both the male (t m ) and female (t f ) distributions. varying the peak day that male and female flowers open influences the duration of time with both types of flowers open simultaneously as well as the number of flowers open during these times (fig 6a) . in particular, differences between t m and t f shifts these distributions and affects the overlapping time when both flower types are open. in fig 6 we hold t f = 6 days constant and vary the peak day of male flowers opening from t m = 3-9 days. predicted yield is maximized (with associated fruit set rates above 91%) when both flower types open concurrently with the same peak opening day (fig 6b) . while shifting the peaks a day apart does not have a huge influence on the predicted yield, a shift of two or three days has significant consequences. to investigate the role of key pollinator parameters, we varied pollinator density based on data on observed honey bee densities. the total predicted yield increases rapidly as the number of bees increases from one to six bees per 1000 female flowers (fig 7) . here fruit set also increases from 39% with only one bee per 1000 female flowers to over 90% with six bees per 1000 female flowers. while continuing to increase the number of bees does increase fruit set rate and the total predicted yield, the increase slows down substantially above six bees per 1000 female flowers. pollinator behavior parameters also play important roles in the model. the model includes preference parameters for pollinators to remain on the type of flower they are visiting, based again on data from honey bee observations. for the baseline values, a pollinator on a male flower preferentially chooses to visit a male flower next (δ), likewise a pollinator on a female flower preferentially chooses to visit a female flower next (�). total predicted yield increases as the pollinators increasingly prefer to switch between male and female flowers in sequential visits (fig 8a) . the yield increases substantially when preference for switching is very small and saturates quickly after. the drastic increase in yield begins to plateau close to the baseline parameter values for the preferences, � and δ (dashed lines in fig 8a) . it is important to note that another relevant pollinator behavior is the speed of foraging. our model includes two parameters for this: the handling time and search rate. our analyses indicate that of these two, the handling time is the most influential; the total predicted yield decreases quickly as the pollinators' handling time increases (fig 8b) . when the handling time increases from 10 sec to 60 sec, fruit set rates decrease from 100% to 50%. the modeling framework enables us to vary key plant and pollinator parameters simultaneously. for a given percentage of female flower buds that make up the orchard, predicted yield increases as the number of bees per 1000 female flowers increases (fig 9) . when the female flower buds percentage is high (between 50% and 90%), maintenance of bee densities over 6 bees per 1000 female flowers will lead to better pollination and therefore ensure a high predicted yield. parameter sensitivity analysis shows that the percentage of female flowers, the total number of buds, and the bee density have the most significant effect on the total predicted yield ( fig 10) with a positive correlation. bee density, the pollinators' preference to switch from female to male flowers (�), the male flowering period (σ m ), and the pollinator's preference to switch from male to female flowers (δ) are the next most important parameters that are positively correlated with the predicted yield, while pollinator handling time is the only parameter with a strongly negative effect on the total predicted yield. flower density and the percentage of female flowers were highly influential parameters in predicting final fruit yield. also important was the width of the male blooming window. managed honey bees are the primary mode of kiwifruit pollination globally [2] , and several pollinatorrelated factors were found to influence yield, with bee density, flower handling time, and preference for moving between flowers of different sexes all highlighted by our sensitivity analysis. kiwifruit flowers may take up to 40 honey bee visits to be fully pollinated [48] , but this is partially due to the large numbers of bees which deposit little or no pollen. we found that increasing bee density will increase fruit production, but that there is a point of diminishing returns after the first 6-8 bees per 1000 female flowers and buds. this finding broadly agrees with the literature, which reports that densities of around 3-6 bees per 1000 flowers are sufficient for full pollination [25, 49, 50] , with sustained higher bee numbers being unusual, though sustained densities of 14 bees per 1000 flowers have been reported in cages [49] and densities of 30-60 bees per 1000 flowers may occur for a very brief period of time in rare circumstances [4, 34] . we found that a longer flower handling time was negatively correlated with fruit production in this model. although empirical data show that honey bee flower handling time is not correlated with pollen deposition [14] , the rate of flower visitation is a well-known factor in limiting the effectiveness of pollinators independently of pollen deposition. [51] . table 2 . pollinators prefer flowers of the same sex in sequential visits; in (a) low preference values near 0 correspond with strong tendencies to remain on either male or female flowers, higher preference values correspond with strong tendicies to switch flower type. blue dashed line in (a) depicts baseline values of δ (preference to remain on male flowers) and red dashed line depicts values of � (preference to remain on female flowers). https://doi.org/10.1371/journal.pone.0231120.g008 preference factors are less well-known, but highlighted here. honey bees are able to differentiate between male and female kiwifruit flowers without landing on them [34] , and they must travel from a male flower to a female flower to deposit viable pollen. this chance of switching can potentially be affected by other pollinators in the field [52] , as well as the attractiveness of the male and female cultivars. increasing the chance of switching between plant sexes may be a critical factor for kiwifruit pollination, as the baseline values in our model are right on the edge of a steep decline-if less switching happens than currently reported in the literature (as indicated by the base parameter values), there could be very significant, negative impacts on pollination. when examining the interaction of bee density and the proportion of female flowers, we found that, at typical bee densities (< 12 bees per 1000 flowers), the optimum proportion of table 2 . https://doi.org/10.1371/journal.pone.0231120.g009 female flowers was 65-75% of total flowers, representing a 'sweet spot' between having more possibilities for fruit development and risk from insufficient movement of bees between the two flower sexes. current orchard plantings have an approximately 50:50 ratio between male and female flowers [4] , highlighting an opportunity to increase yield by changing pruning practices to increase the proportion of female flowers-an easily achievable intervention compared with changing pollinator behavior. our model takes advantage of over 30 years of field-based data in new zealand and other parts of the world and provides a way to quantitatively assess how different plant-and insectrelated factors interact and their importance for final fruit set. our results suggest that choosing cultivars which have their peak bloom on the same day, planting and pruning to achieve approximately 70% female flowers in the orchard, having as many flowers as the vine can support to full fruit size, and placing enough hives to maintain more than 6 bees per 1000 flowers will optimize yield. there is the potential for future work to improve the predictive power of this model by accounting for multiple pollinators and spatial scale and pattern. supporting information s1 appendix. many of the parameters have monotonic relationships with the output measure (s1a fig) and the prcc statistics for those are reliable. however, we note that parameters σ m , σ f , t m , t f and the proportion of female flower buds in the field exhibit nonmonotic behaviors. therefore, we conducted additional lhe sampling by truncating the ranges of these parameters to monotonic regions. s1b and s1c fig depict the monotonicity of the truncated parameter space. the resulting prcc results for the entire parameter space as well as the truncated parameter spaces are compared in s2 fig. parameters for the total number of buds, percentage of female buds, bee density, and handling time are consistently identified as important parameters in all cases. we note that in the truncated case we split the percentage of female flower buds into the cases of 5-76% and 76-96%. in the first half this parameter shows a highly influential positive relationship with predicted yield (large positive prcc value) and in the second half the parameter is inversely related to the predicted yield. this is as expected as saturating the field with only female buds will eventually cause a decrease in yield. these dynamics are observed in the monotonicity plots as well. (pdf) s1 available code. matlab code for our model was provided as an online supplemental file and is available to download. 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in kiwifruit influence of pollination systems on fruit set and fruit quality in kiwifruit (actinidia deliciosa) kiwifruit yield efficiency, plant density, and bud number per surface unit effects of different bud loading levels on the yield, leaf and fruit characteristics of hayward kiwifruit spatial variation in 'hayward' kiwifruit fruit size and orchard yield within a growing region across seasons use of plant bioregulators in kiwifruit production how many bee visits to fully pollinate kiwifruit effect of honey bee saturation on the pollination of chinese gooseberries variety 'hayward' biology of honeybee (apis mellifera l.) pollination of kiwifruit (actinidia deliciosa why flower visitation is a poor proxy for pollination: measuring single-visit pollen deposition, with implications for pollination networks and conservation wild bees enhance honey bees' pollination of hybrid sunflower we would like to thank mark goodwin for his assistance in obtaining data for model parameterization, and ruth williams and warrick nelson for their feedback on the manuscript. key: cord-000341-d3a06n3f authors: xu, wanghui; han, lu; lin, zhanglin title: screening of random peptide library of hemagglutinin from pandemic 2009 a(h1n1) influenza virus reveals unexpected antigenically important regions date: 2011-03-18 journal: plos one doi: 10.1371/journal.pone.0018016 sha: doc_id: 341 cord_uid: d3a06n3f the antigenic structure of the membrane protein hemagglutinin (ha) from the 2009 a(h1n1) influenza virus was dissected with a high-throughput screening method using complex antisera. the approach involves generating yeast cell libraries displaying a pool of random peptides of controllable lengths on the cell surface, followed by one round of fluorescence-activated cell sorting (facs) against antisera from mouse, goat and human, respectively. the amino acid residue frequency appearing in the antigenic peptides at both the primary sequence and structural level was determined and used to identify “hot spots” or antigenically important regions. unexpectedly, different antigenic structures were seen for different antisera. moreover, five antigenic regions were identified, of which all but one are located in the conserved ha stem region that is responsible for membrane fusion. our findings are corroborated by several recent studies on cross-neutralizing h1 subtype antibodies that recognize the ha stem region. the antigenic peptides identified may provide clues for creating peptide vaccines with better accessibility to memory b cells and better induction of cross-neutralizing antibodies than the whole ha protein. the scheme used in this study enables a direct mapping of the antigenic regions of viral proteins recognized by antisera, and may be useful for dissecting the antigenic structures of other viral proteins. the 2009 a(h1n1) influenza virus, which is also referred to as the swine-origin influenza virus (s-oiv), caused the first global influenza pandemic in recent decades [1] . given continuous antigenic drift and reassortment of heterotypic influenza viruses circulating in human and animal reservoirs, global concerns have been raised regarding an increasing threat of an influenza pandemic [2] . current treatment strategies for influenza-a viruses, such as vaccines and drugs, have not provided broad and lasting protection, partly due to the constantly evolving nature of the viral surface glycoprotein, hemagglutinin (ha) that allows it to avoid host immune attack. ha is the key viral antigen in determining host specificity and inducing neutralizing antibody since it plays a major role in binding to host cell receptors and fusing with host cell membranes [3] . for many challenging diseases caused by viruses, the recognition of certain neutralizing epitopes by the immune system can indeed provide broad and potent protection [4, 5] . the antigenic structure of ha and the corresponding antibody response are not fully understood, complicating rational design of vaccines aimed at modulating antibody responses for targeting key epitopes. our previous knowledge of viral antigenic structure was based mainly on the structure of antibody-antigen complexes or mutational analysis of related antigenic drifts [6] [7] [8] . recently, many new approaches have emerged for the rapid extraction of monoclonal antibodies toward target antigens from antibody phage display libraries [5, 9, 10] , using direct sorting of memory b cells [11] , or by immortalization of igg expressing b cells that reflect the antibody repertoire [12, 13] . these methods provide much more information on viral epitopes, although they are often laborious. the major alternative approaches for epitope mapping are derived from scanning with antigenic peptides displayed on the surface of bacteriophage, bacteria and yeast [14] [15] [16] [17] [18] [19] [20] [21] , which in recent years have increasingly incorporated fluorescence-activated cell sorting (facs). in these efforts, short defined or random peptides with a narrow length range (,50 amino acid residues (aa)) are used to screen against monoclonal or polyclonal antibodies. inspired by advances in cell surface display technology [22, 23] and peptide fragment library construction [24] , we devised a highthroughput scheme that utilizes yeast display and facs that allows for direct screening of random viral peptide libraries against complex antisera instead of isolated antibodies (zuo t, shi xl, xu wh, lin z, and zhang lq, unpublished results). the peptide library is generated by random digestion of the gene encoding the target viral protein, followed by a pcr-based reassembly step that results in fragments with controllable lengths (normally in the range of 100-500 bp) [24] . these peptides are then expressed on the yeast cell surface by fusion to the yeast adhesion receptor aga 2 protein, which has previously been used to display defined short antigen fragments of various viral and non-viral proteins [19] [20] [21] . from the sequences of the antigenic peptides after screening, it is possible to determine the relative frequency of each amino acid residue involved in recognition by antisera, and how these residues are distributed in the three-dimensional structure of ha. using this approach, we analyzed the 2009 a(h1n1) influenza virus ha protein using mouse, goat and human antisera. unexpectedly, different antigenic profiles were seen for different antisera. moreover, five antigenic regions were identified, four of which are located in the conserved ha stem region responsible for membrane fusion. elisa binding assays and absorption experiments using peptides that encompass these regions have confirmed their antigenic activities. the procedure for construction, expression and screening of random viral peptide libraries using yeast surface display is shown in fig. s1 (see also materials and methods for details). in step 1, the full length ha gene of the pandemic 2009 a(h1n1) influenza virus (including the core ha protein of 519 aa, the signal peptide of 16 aa, and the inter membrane domain of 36 aa) was digested and re-assembled by pcr to form a pool of fragments enriched in 100-500 bp [24] . in step 2, the fragment library was ligated into the yeast display vector pctcon-t (derived from the yeast surface display vector pctcon-2 [23] shown in fig. 1 ), transformed into yeast eby100 cells and induced for expression, generating a typical library of 1610 5 -10 6 clones to cover most of the possible fragments [25] . because there are 3 stop codons on pctcon-t downstream of the peptide inserts to terminate every orf, the theoretical possibility for in-frame fusions is 1/3 for the n-terminal end of the peptide inserts. thus, the theoretical yield for cells expressing in-frame peptides is 1/6 (the fragments can be inserted in forward and reverse directions). subsequently in step 3, the cell library was incubated with antisera, labeled with secondary fluorescent antibody and subjected to facs in step 4, after which the cells harboring antibody-binding peptides were distinguished from those with non-binding peptides. in step 5, we isolated plasmids directly from the pool of yeast cells containing positive peptide sequences, which were retransformed into e. coli for sequencing. this reduced laborious plasmid extraction from individual yeast clones. plasmids containing in-frame sequences were transformed back into yeast for individual flow cytometry (fcm) verification. retransformation and fcm were also performed for plasmids containing out of frame sequences (false positive). antigenic peptide sequences were aligned with the ha sequence in step 6 (see also figs. 2 and 3). in step 7, statistical analyses both on the sequence and structural levels were then performed to map out the antigenically important regions of the ha protein, by summarizing the frequency of each amino acid residue appearing in the antigenic peptides (see also fig. 4) . the ha polypeptide is cleaved into two subunits linked by a disulfide bond, ha1 and ha2, to form the mature ha trimer. the majority of the ha1 subunit forms the viral membrane-distal globular head responsible for receptor binding, whereas the ha2 subunit together with the n-and c-termini of ha1 forms the membrane-proximal stem region that plays a major role in membrane fusion. thus two plasmids, pctcon-ha1, pctcon-ha2, respectively, were constructed to display the ha1 subunit of 328 aa (residues 17-344 of the ha protein) and the ha2 ecto-domain of 192 aa (residues 345-536 of the ha protein) as positive controls for fcm (fig. 1) . the ha1 and ha2 displayed on the yeast surface were recognized by the mouse, goat and human antisera, and confirmed by fcm (fig. s2 , panels b, e, and, h; and c, f, and i, respectively). screening of antigenic peptides recognized by mouse antisera immunized with ha protein we first screened the yeast library displaying the h1n1 ha peptides to study the murine immune response to recombinant ha protein of the 2009 a(h1n1) influenza virus. freshly induced library cells were incubated with mixed sera from immunized mice and subsequently labeled with fluorescent secondary antibodies. to avoid potential bias introduced by growth advantage of the yeast cells, only one round of sorting was performed. plasmids isolated from positive yeast cells were transformed into e. coli cells and plated. single clones (100) were picked randomly and sequenced, of which 82 (82%) were shown to contain in-frame inserts. these results indicate that the sorting process is able to distinguish in-frame sequences from the random library that contained only 1/6 (16.7%) in-frame inserts efficiently. after verification of individual peptides by fcm detection, the ratio of recombinant antigen expressed on the yeast surface (rays) was determined by dividing the mean fluorescence intensity of peptide expressing yeast cells by the mean fluorescent intensity of the nonexpressing (pctcon-2) yeast cells [26] . 56 positive clones with a rays ratio $2 were considered as antigenic and analyzed at both the sequence and structural level [26] . alignment of positive peptides with the original ha sequence shows that 87% are in the ha2 region (95627 aa), with just 13% in the ha1 region (259626 aa) which cover nearly the entire ha1 protein (fig. 2, panel a). the fcm histograms of several representative yeast clones are shown in fig. 3 , panel a. clones m-5 (corresponding to residues 55-313 of ha, or 70 aa shorter than ha1) and m-7 (residues 69-275, or 120 aa shorter than ha1) were strongly positive (rays ratio .5), and contained the entire receptor binding domain (rbd) and the vestigial esterase domain in the globular ha head. clones m-33 (residues 387-489) and m-52 (residues 423-492) displayed much shorter peptides but also showed strong fluorescent fcm signals (fig. 3 , panel a), albeit with relatively lower rays ratios of 3-4. judged from the mean fluorescence intensities, the binding affinities of these displayed peptides were generally weaker than the whole ha1 (fig. s2, panel b) . the sequencing results of the antigenic peptides alone are less informative (fig. 2) . nevertheless, when we perform a statistical analysis by summarizing the normalized frequency of each amino acid residue in all positive antigenic peptides (defined as the residue frequency map), a major peak with the full width at half maximum (fwhm) from residues 387-480 is revealed (fig. 4 , panel a). we also modify the residue frequency map by taking into account the respective rays ratios of positive peptides, which however results in an overall rather similar frequency map (fig. s3, panel b) . furthermore, even if we combine all the in-frame sequences from sorted yeast cells, a similar map (fig. s3 , panel c) is again obtained, implying that the noises from the in-frame but the residue frequency map is then overlaid onto the crystal model of the trimeric ha of the 2009 a(h1n1) influenza virus (pdb id: 3lzg) [27] , and annotated by colors from red (high frequency) to blue (low frequency) (fig. 4, panel b ). this structural frequency map reveals that the major peak (corresponding to residues 387-480 within the fwhm) on the residue frequency map can be divided into two dominant antigenic regions since they have independent structural features. one corresponds to the central coiled-coil helix cd in the ha2 subunit responsible for membrane insertion, designated as r1 (residues 424-480, indicated by red, see also fig. 5 ), while the other contains helix a in the ha2 subunit that is important for conformational change upon low ph exposure, designated as r2 (residues 387-423, indicated by faint red and white, see also fig. 5) [28] . r1 is present in a relatively recessed surface area while r2 is located near the exterior surface area of the stem region. for the ha1 subunit, no distinct antigenic region is identified, because of the limited number of peptides (7) with relatively longer lengths (all .200 aa), which are insufficient to form peak areas. we also screened the same yeast library against serum samples from goats immunized by the 2009 a(h1n1) influenza virus to examine whether different animal models would yield a similar profile of antigenic peptides. following sorting and sequencing, 78/100 clones were confirmed as in-frame, among which 55 tested positive by the goat anitsera in fcm (rays ratio $2). surprisingly, alignment of the positive antigenic peptides with the full ha protein shows a distribution strikingly different from the alignment obtained using the mouse antisera in several aspects. first, 78% of the antigenic peptides are located in the ha1 region and have shorter lengths (54632 aa), while only 9% are in the ha2 region (96619 aa). second, 13% of the peptides are in the region across ha1 and ha2 (66631 aa) (fig. 2, representative antigenic clones were subjected to fcm. as shown in fig. 3 , panel b, clones g-15 (residues 38-136 of ha) and g-29 (residues 70-162) in the ha1 region, g-46 (residues 308-339) near the ha1-ha2 cross region, and g-55 (residues 413-479) in the ha2 region, all shorter than 100 aa, demonstrated strong antigenicity (rays ratio .5). judged from the mean fluorescence intensities, the binding affinities of these displayed peptides were generally stronger than the whole ha1 (fig. s2 , panel e). by analyzing the residue frequency map, more peaks are revealed than those obtained from the mouse anitsera, with the largest one with its fwhm spanning residues 22-125 with a small split around residue 60, and a medium one with its fwhm at residues 303-350 (fig. 4 , panel c, and fig. s4 ). in the structural frequency map (fig. 4 , panel d), the largest peak is composed of two independent regions: residues 22-60 at the n-terminus of ha1 adjacent to helix a on ha2, designated as r3; and residues 61-125 located in the vestigial esterase domain of the globular head, designated as r5 (indicated by two red regions in the globular head and stem region, respectively, see also in order from most membrane proximal to distal: r1 (orange, residues 424-480 of ha) and r2 (yellow, residues 387-423) in ha2 were determined by screening against mouse and human antisera; r3 (purple, residues 22-60), r4 (green, residues 303-350), and r5 (cyan, residues 61-125) in ha1 were determined by screening against goat antisera. the ha monomer cartoon view is shown on the right and follows the same coloring scheme, with the third monomer shown in the back and colored in grey. doi:10.1371/journal.pone.0018016.g005 should be noticed that r1 and r2 identified in the mouse serum screening also appear in this residue frequency map, although in a much less dominant manner as illustrated by the small plateau between the end of r4 and residue 479 (fig. 4, panel c) . taken together, the antigenic peptides recognized by the goat antisera are different from those recognized by the mouse antisera, and in particular present a more diverse picture of antigenic sites in the ha1 region. the screening against human plasma immunized by the 2009 a(h1n1) influenza vaccine was carried out essentially as described above. among the 100 sequences obtained after facs analyses, 74 peptides were found to be in-frame, and 51 were confirmed to be antigenic (rays ratio $2). it is interesting to see that the distribution of theses peptides is similar to that obtained using the mouse antisera, with only 4% of peptides in the ha1 region (87 and 37 aa in length), 4% across ha1 and ha2 (96 and 39 aa in length), and 92% in the ha2 region (100630 aa) (fig. 2 , panel c). using fcm, three representative clones, h-11 (residues 359-479 of ha), h-26 (residues 390-480) and h-45 (residues 423-480), all in the ha2 region, showed strong signals (rays ratio .5). although only 2 antigenic peptides are in the ha1 region, a clone expressing h-1 (residues 108-194), which harbored the sa antigenic site in rbd of the ha protein, yielded a rays ratio greater than 3 (fig. 3, panel c) . again, judged from the mean fluorescence intensities, the binding affinities of these displayed peptides were generally weaker than the whole ha1 (fig. s2, panel h) . the residue frequency map also reveals a major peak (fwhm at residues 392 to 480). this peak position is similar to that obtained using the mouse antisera (fwhm at residues 387-480), resulting in an almost identical structural frequency map (fig. 4 , panels e and f, and fig. s5 ). thus, these antigenic regions are considered identical to r1 and r2 obtained from the mouse serum screening (fig. 5 ). the accessibility of short antigenic peptides displayed on the yeast cell surface [19] [20] [21] was confirmed in this study with fluorescence confocal microscopy, as exemplified by the antigenic clones g-29 (93 aa) and g-46 (32 aa), from the screening against the goat antisera (fig. s6) . as shown in the figure, the antigenic peptides were localized on the outer surface of the membrane, and well accessible to the antibodies in the complex antisera. three representative yeast clones displaying antigenic peptides g-15, g-55, and h-11, obtained from the screening against the goat antisera and human plasma samples, were characterized for neutralization titers (ic 50 ) by testing protection efficacy to mdck cells from the a/reassortant/nymc x-179a/california/07/ 2009(h1n1) exposure. cells containing pctcon2 were used as absorbent controls for evaluation of background ic 50 values. ha1 and ha2 were also incorporated to determine the ability of the globular head and stem regions to absorb antibodies in the antisera. as summarized in table s1 , all single clones reduced the ic 50 of the corresponding antisera, demonstrating qualitatively the neutralization efficacy of these antigenic peptides. binding affinities of purified peptides to mouse, goat and human antisera to further confirm the antigenic activities of the identified r1-r5 regions, five newly designed short peptides encompassing regions r1-r5 (fig. 6, panel a) were expressed as c-terminal fusions to thioredoxin (trx) and purified from e. coli [29, 30] . the binding affinities of these peptides to the mouse, goat and human antisera were characterized by the method of elisa, with thioredoxin as the control (fig. 6, panels b and d) . as can been seen from the figure, the elisa results generally correspond well to the screening results shown in figs. 4 and 5 . specifically, the peptide p1 (corresponding to the r1 region identified in all three screenings, figs. 4 and 5 ) was reactive to all three antisera. the peptides p3, p4 and p5, corresponding to the r3-r5 regions identified only in the screening against the goat antisera (fig. 4 , panels c, d, and fig. 5) were only reactive to the goat antisera (fig. 6, panel c) . there is one exception in that the peptide p2 (corresponding to the r2 region identified in the screenings using the mouse and human antisera, figs. 4 and 5) showed no measurable affinity to either mouse or human antisera, suggesting that the r2 region might be less conformation-independent than the other four regions. it should be noted that, after subtraction of the background binding of the negative control protein (trx), the binding affinity of the peptide p1 to the human antisera after vaccination increased only about one fold compared with the antisera before vaccination (fig. 6, panel d) . this might be contributed to cross-reactive antibodies to other influenza subtypes pre-existing in the human serum subjects, given the conservative nature of the p1 sequence or the r1 region [13] . we report here the identification of antigenic peptides of the 2009 a(h1n1) influenza ha protein from a combinational library of viral protein fragments displayed on the surface of yeast cells and sorted by facs. the peptide fragments were constructed in a way to have a broad but limited range of lengths [24] , and screened using antisera from mouse, goat and human. we then used a novel statistical approach to identify antigenically important regions of the viral protein based on the frequency of each residue appearing in the antigenic peptides at both the primary sequence and structural level. this approach reveals interesting antigenic features of the ha protein. first, the antibody responses to the 2009 a(h1n1) viral protein ha appear to vary significantly depending on the species, with the goat response being strikingly different from those of mouse and human (fig. 4) . these results imply possibly different immune responses among animal species, and in the case of human, the specific immunization background may also play a role [13] . moreover, the antigenic peptides obtained from the screening against the mouse or human antisera are predominantly located in the ha2 region, whereas those from the screening against the goat antisera are predominantly in the ha1 region. second, five antigenically important regions, r1 to r5 in the order from most membrane proximal to distal (fig. 5) , are identified. moreover, except for r5 which is located in the vestigial esterase domain of the ha globular head, all of the other regions are in the ha stem region. among them, r1 and r2 come together to form a major peak with a fwhm range from residues 387 to 480 (fig. 4, panels a and e) . although the peptide p2 corresponding to the r2 region showed no measurable affinity to either mouse or human antisera (fig. 6) , which suggests that this region might be more conformation-dependent than the rest four regions, it is plos one | www.plosone.org assigned as a separate region for the following reasons: (1) structurally it is distinguishable from the r1 region (fig. 5) , and (2) several recent studies have reported the epitopes of monoclonal antibodies targeting h1 subtype ha proteins, which fall into the r2 region of this study [10, 31, 32] (see below). previously most influenza ha antibodies have been found to recognize epitopes in the globular head and interfere with binding to target cells [6] [7] [8] [33] [34] [35] [36] , for example, the classical five antigenic sites (sa, sb, ca 1 , ca 2 , and cb) [6, 7] . however, technically the monoclonal antibodies used in these previous studies were obtained from murine hybridoma cells by radioimmunoassay (ria) or hemagglutination inhibition (hi) assay. the nature of these assays favored the isolation of antibodies that bound predominantly to the globular head of the virus and inhibited binding of the virus to the host cells. in this current study, however, complex antisera were used, which should contain antibodies recognizing other regions of the ha protein. on the other hand, only one antigenic region (r5) is identified in the globular head in this study, possibly because the epitopes in this head region are more conformation-dependent and may require longer peptides or even a complete domain of ha1 to be displayed for full antigenicity (at least for the mouse serum and human plasma samples). it is then noteworthy that the head region, especially the area around the receptor binding site, contains mostly beta structures, which more likely require stabilization by long-range interactions than the helical structures in other regions of ha (fig. 5) . however, the peptide library constructed in this study was enriched at 100-500 bp (or less than 170 aa), which was likely insufficient to map the conformational epitopes. nonetheless, the current screening method is a valuable complementation to the more classical approaches that are biased toward full ha1 or ha2 domain. several recent studies support our findings regarding antigenic regions r1-r4 in the stem region of ha. in efforts aimed at characterizing antibodies with cross-neutralizing activity for various influenza virus subtypes [10, 13, 31] , it was found that most of these antibodies bind to the ha stem region and interfere with conformational changes of the protein critical for membrane fusion. for example, in the case of antibody cr6261 (binding to ha of the sc1919 virus, h1 type), two antigenic regions on ha stem were revealed: helix a in the ha2 region, and the adjacent ha1 region [32] . the critical sites in helix a (corresponding to residues 390-391, 393-395, 398, 401-402, 405 in h1n1 ha) fall within the r2 region identified in this study, whereas the hydrophobic interaction sites in the adjacent ha1 region (corresponding to residues 38-42, 306-307 in h1n1 ha) fall into regions r3 and r4 of this study, respectively. more recently, it was reported that for a different cross-neutralizing antibody that targets a broad range of h3 subtype virus strains, 12d1, the dominant contacts on the antigen (corresponding to residues 425-455 in h1n1 ha) [37] correlate well with the r1 region found in this study. furthermore, vaccination in mice using a ha2-based synthetic peptide mimicking the 12d1 epitope was shown to provide protection against influenza viruses of h3n2, h5n1, and h1n1 subtypes [38] . taken together, our approach should be useful for dissecting antigenic regions on the ha stem, and providing clues for designing more potent peptide vaccines in inducing cross-neutralizing antibodies than the entire ha protein [32] . compared with other labor-intensive epitope identification methods that rely on identifying critical residues in escape mutants and crystal structures, the method used here gives a direct and panoramic mapping of the antigenic regions of viral proteins recognized by antisera in a facile and high-throughput way. our approach likely contains an inherent bias for shorter peptides, but nonetheless complements the more classical approaches that favor longer antigenic peptides or full antigenic domains. the scanning of antigenic peptides based on phage, bacterial or yeast surface display methods has been increasingly used in recent years. our approach presents two advantages. first, technically we adopt an easy-to-implement scheme for generating a controllable size of peptides and our screening typically involves only one round of facs-based sorting, in contrast to multiple rounds of panning or sorting used in the literature [15, 16, 18] . although the latter results in many repeat sequences, we argue that much information is lost in the enrichment process. second and more importantly, since simple alignment of antigenic peptides with the corresponding viral protein yields only limited information [15, 17, 39] , we employ a novel statistical means of summarizing the frequency for each residue appearing in the antigenic peptides at both the primary sequence and structural level, which enables a direct mapping of the antigenic structure of a viral protein. along this line, it is interesting to note that, using our statistical approach, we reevaluate the highly repeated antigenic peptides identified for the h5n1 ha protein in a recent report [15] . several major peaks are clearly revealed (fig. s7) , which should be useful for further characterization of the antigenic peptides for h5n1 ha. (h1n1) influenza vaccine, and a mixture of 9 samples with the highest activity among 110 samples as judged by elisa. these samples were provided by dr. boping zhou of shenzhen east lake hospital (shenzhen, china), and written informed consent was obtained from all study participants. all samples were de-identified prior to analysis, and the protocols were approved by the institutional review board (irb) of shenzhen east lake hospital. all viral strains are closely related. restriction enzymes and dna polymerases were purchased from new england biolabs (beverly, ma) or takara (dalian, china). dnase i was obtained from worthington (lakewood, nj). fluorescence-labeled secondary antibodies were purchased from invitrogen (shanghai, china) or santa cruz biotechnology (santa p5 (blue arrows), in relation to the five antigenic regions r1-r5. the coordinate of the ha protein is indicated by the grey bar, with the five antigenic regions represented in the same color as in fig. 5 . the peptides p1-p5 were expressed as c-terminal fusions to the thioredoxin (trx) tag. binding activities of the peptides against the mouse (b), goat (c) and human (d) antisera before and after immunization were characterized by the method of elisa, with the trx protein as the negative control. the x-axis shows the dilution ratios of corresponding antiserum samples. the y-axis shows the absorbance at 450 nm after development with the substrate 3,39,5,59-tetramethylbenzidine (tmb). doi:10.1371/journal.pone.0018016.g006 cruz, usa). oligonucleotides were synthesized by invitrogen or takara. the kits for dna purification, gel recovery, plasmid miniprep and a-tailing modification were obtained from tiangen (beijing, china) or takara (dalian, china). the kit for yeast plasmid dna isolation was from omega biotech (victoria bc, canada). sequencing was performed by invitrogen or by sinogenomax (beijing, china). escherichia. coli dh5a was obtained from takara. yeast strain saccharomyces cerevisiae eby100 was obtained from invitrogen. the pctcon-2 yeast display vector was kindly provided by prof. dane wittrup [23] . the dna fragments coding the ha1 and ha2 proteins were amplified from plasmid cmv-r-cali two xcm i restriction sites (ccannnnn/nnnntgg) were introduced between nhe i and bamh i sites of pctcon-2 to create pctcon-t. digestion of pctcon-t by xcm i restriction nuclease and gel extraction yielded the t-vector with two 39 t overhangs. random fragments with 39 a tails were inserted between the two xcm i sites by t-a ligation. the gene coding for the full ha protein of a/california/04/ 2009(h1n1) virus was amplified from plasmid cmv-r-cali-04-09 with the forward primer 59-cgccaccatgaaggc-tatcc-39 and reverse primer 59-ttagatgcagattctg-cactg-39. detailed procedures for fragmentation and reassembly were described previously, except that phusionh high-fidelity polymerase (neb) was used in reassembly [24] . the reassembled dna samples were purified and modified to add 59 a overhangs by an a-tailing kit (takara). the backbone vector pctcon-t was digested with xcm i and purified with a tiangen gel purification kit to reduce self-ligation. the gene fragments and backbone vector were ligated at 16uc for 16 h and then electroporated into e. coli dh5a competent cells for propagation. the plasmid library was then isolated and used to transform yeast eby100 cells by the li-ac method [40] . expression of peptide libraries on yeast eby100 cells was performed as reported by wittrup's group [23] . transformed yeast cells were grown for 24 h at 30uc with shaking in sdcaa medium (yeast nitrogen-based casamino acid medium containing 20 g?l 21 glucose) and passed one time into fresh medium to eliminate dead cells. the library cells were then centrifuged, resuspended in sgcaa medium (yeast nitrogen-based casamino acid medium containing 20 g?l 21 galactose) to an od 600 value of 0.5-1 and induced at 20uc for 36-48 h with shaking. prior to yeast library screening, nonspecific interactions of serum samples (i.e., mouse, goat or human antisera) with yeast proteins were removed by incubation with induced yeast cells carrying the control vector pctcon-2. to label cells for facs, 1610 7 (for yeast eby1001 cells, od 600 <1610 7 cells/ml) freshly induced library cells were pelleted at 8,000 g for 1 min in a 1.5 ml microcentrifuge tube and washed two times with 1 ml of 16phosphate buffered saline (pbs). cells were then incubated with 100 ml of pre-absorbed serum (1:50 diluted in pbs) at 4uc for 1 h. unbound antibodies were removed by washing two times with 1 ml of pbs. the library cells were incubated with 1-2 mg of fluorescein isothiocyanate (fitc) or phycoerythrin (pe) labeled secondary antibodies in 100 ml at 4uc for 45 min. after washing two times with pbs, cells were resuspended in 1 ml of pbs and loaded onto a bd-facs ariaii tm machine (shanghai, china) for sorting. yeast cells harboring the pctcon-2 vector were processed as described above and used as a negative control. the positive gate was set to exclude all negative control cells (,0.1% leakage). the total amount of cells used for sorting was adjusted to ensure that more than 1610 5 cells could be harvested, which were then collected in 5 ml of sdcaa medium and grown overnight at 30uc with shaking. after one passage of library cells into fresh medium, heterogeneous plasmid dna was isolated from the library and transformed into e. coli dh5a cells. for each library, a total of 100 colonies were randomly picked from the plates and sequenced using a pair of primers (pctcon2-seq-for: 59-gttccagac-tacgctctgcagg-39 and pctcon2-seq-rev: 59-gttc-cagactacgctctgcagg-39). the plasmids carrying gene fragments inserted in-frame with the original ha gene were retransformed into yeast eby100 cells, induced, and labeled as described for fcm detection on a bd-facs calibur cytometer (shanghai, china). the ratio of recombinant antigen expressed on the yeast surface (rays) was determined by dividing the mean fluorescence intensity of peptide expressing yeast cells by the mean fluorescence intensity of the non-expressing (pctcon-2) yeast cells. clones with a rays ratio $ 2 were considered positive and the corresponding peptide sequences were selected for further analysis [26] . yeast cells containing plasmids for displaying antigenic peptides were grown at 30uc for 24 h in sdcaa medium and induced at 20uc for 36 h in sgcaa medium. 1610 7 cells were harvested by centrifugation, incubated with goat antisera (1:100 diluted in 100 ml of pbs) and labeled with 1 mg of fitc conjugated antigoat igg secondary antibody as described above. labeled cells were resuspended to 5610 7 cells/ml, fixed with 4% paraformaldehyde and photographed on a zeiss 710 laser scanning confocal microscopy (carl zeiss, germany) at the center of biomedical analysis, tsinghua university. yeast cells containing nonexpressing vector pctcon-2 and ha1-expressing vector pctcon-ha1 were treated in the same way as negative and positive controls, respectively. elisa assays for purified representative peptides against three antisera five peptides newly designed to encompass the regions r1-r5 were expressed as fusions to the c-terminus of thioredoxin (trx) and purified from e. coli, using a modification of the self-cleaving elastin-like polypeptide tag method (elp) (with purity over 80% as judged by sds-page) [29, 30] . for comparison, the trx tag was used as the negative control. when mouse and goat antisera were employed as the first antibody, the wells were coated with 100 ng of peptides or trx. but for human antisera, 10 ng of peptides or trx control was used to decrease the background signal caused by unspecific interactions between the human antisera and the trace impurities from e. coli cells. after blocking with 10% fetal bovine serum (fbs) diluted in 16pbs with 0.25% tween-20 (pbst), serial dilutions were added to each well and incubated for 1 h at 37uc, followed by addition of 1:5,000 diluted hrp-conjugated secondary antibody. assays were developed by adding 100 ml of 3,39,5,59-tetramethylbenzidine (tmb) substrate solution and the reactions were stopped with 50 ml of h 2 so 4 (1 m). the assays were carried out in duplicates and each well was washed 4 times with pbst between steps. the absorbance at 450 nm was recorded on a spectromax 190 microtiter reader (molecular devices, ca). prior to the neutralization assay, 500 ml antisera (diluted 1:50, and pre-absorbed with induced yeast cells carrying the control vector pctcon-2) were mixed with freshly induced yeast cells (1610 8 ) displaying specific antigenic peptides on surface. after incubation at 4uc for 12 h, the mixtures were centrifuged at 8,000 g for 1 min and supernatants mixed with another batch of freshly induced yeast cells. this process was repeated four times, and then the supernatants were used for determining the neutralization titers, using a microneutralization assay with a/reassortants/nymc x-179a/california/07/ 2009(h1n1) virus and mdck cells according to standard procedures [41] . briefly, triplicate serial dilutions (1:50 to 1:1600) of antiserum samples were incubated with 100 50% tissue culture infective dose (tcid 50 ) of virus for 2 h at 35uc prior to adding mdck cells. cells were incubated at 35uc for 72 h, and the half maximal inhibitory concentration (ic 50 ) was determined. all the neutralization assays were performed at the aids research center, school of medicine, tsinghua university following standard procedures. figure s1 schematic outline of the screening approach. in step 1, the gene encoding the viral protein h1n1 ha was amplified, digested and re-assembled to generate the fragment library (the red and grey segments indicate the antigenic and nonantigenic peptides, respectively). in step 2, the fragment library was ligated into the display vector pctcon-t, transformed into yeast cells and induced for expression. in step 3, the yeast cells expressing random peptides were incubated with antisera and fluorescence-labeled second antibodies and subjected to facs in step 4. afterward in step 5, the gene fragments were isolated from the sorted cells and sequenced. the in-frame sequences were retransformed into yeast cells and verified by fcm detection individually. in step 6, an antigenic peptide profile was extracted for the sequences corresponding to the antigenic peptides, based on which the residue frequency and structural frequency maps were derived in step 7. (tif) [15] . the x-axis represents all h5n1 ha amino acid residues. the y-axis shows the normalized frequency of individual residue appearing in the 784 antigenic peptides (39 unique sequences) obtained from panning against h5n1 avian influenza convalescent sera. the six clusters (i-vi) defined by khurana et al. are graphically represented below the x-axis. several representative antigenic peptides are also shown as green arrows (numbered according to khurana et al.) . even though the antigenic peptides were enriched multiple times during the screening process and thus these peptides are less diverse and might be biased in sequences, several peaks are clearly identifiable, and predominantly in the ha2 region. (tif) antigenic and genetic characteristics of swine-origin 2009 a(h1n1) influenza viruses circulating in humans hemagglutinin receptor binding avidity drives influenza a virus antigenic drift receptor binding and membrane fusion in virus entry: the influenza hemagglutinin broad and potent neutralizing antibodies from an african donor reveal a new hiv-1 vaccine target combinatorial antibody libraries from survivors of the turkish h5n1 avian influenza outbreak reveal virus neutralization strategies the antigenic structure of the influenza virus a/pr/8/34 hemagglutinin (h1 subtype) antigenic structure of influenza virus haemagglutinin defined by hybridoma antibodies structural identification of the antibody-binding sites of hong-kong influenza hemagglutinin and their involvement in antigenic variation human monoclonal-antibodies against a plethora of viral pathogens from single combinatorial libraries structural and functional bases for broad-spectrum neutralization of avian and human influenza a viruses broad diversity of neutralizing antibodies isolated from memory b cells in hivinfected individuals an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus heterosubtypic neutralizing antibodies are produced by individuals immunized with a seasonal influenza vaccine phage display for engineering and analyzing protein interaction interfaces antigenic fingerprinting of h5n1 avian influenza using convalescent sera and monoclonal antibodies reveals potential vaccine and diagnostic targets epitope mapping and affinity purification of monospecific antibodies by escherichia coli cell surface display of gene-derived random peptide libraries epitope mapping of antibodies using bacterial surface display libraries against libraries for combinatorial selection of replicating antigen-antibody pairs domain-level antibody epitope mapping through yeast surface display of epidermal growth factor receptor fragments comprehensive antibody epitope mapping of the nucleocapsid protein of severe acute respiratory syndrome (sars) coronavirus: insight into the humoral immunity of sars fine antigenic variation within h5n1 influenza virus hemagglutinin's antigenic sites defined by yeast cell surface display display technologies: application for the discovery of drug and gene delivery agents isolating and engineering human antibodies using yeast surface display identification of viral peptide fragments for vaccine development dna fragmentation-based combinatorial approaches to soluble protein expression part i. generating dna fragment libraries recombinant antigen expression on yeast surface (rays) for the detection of serological immune responses in cancer patients structural basis of preexisting immunity to the 2009 h1n1 pandemic influenza virus structure of influenza hemagglutinin at the ph of membrane-fusion a thioredoxin gene fusion expression system that circumvents inclusion body formation in the e. coli cytoplasm simple bioseparations using self-cleaving elastin-like polypeptide tags heterosubtypic neutralizing monoclonal antibodies cross-protective against h5n1 and h1n1 recovered from human igm(+) memory b cells antibody recognition of a highly conserved influenza virus epitope a complex of influenza hemagglutinin with a neutralizing antibody that binds outside the virus receptor binding site identification of the binding-sites to monoclonal-antibodies on a/ussr/90/77 (h1n1) hemagglutinin and their involvement in antigenic drift in h1n1 influenza-viruses structural assignment of novel and immunodominant antigenic sites in the neutralizing antibody response of cba/ca mice to influenza hemagglutinin structure of influenza virus haemagglutinin complexed with a neutralizing antibody broadly protective monoclonal antibodies against h3 influenza viruses following sequential immunization with different hemagglutinins vaccination with a synthetic peptide from the influenza virus hemagglutinin provides protection against distinct viral subtypes fine and domain-level epitope mapping of botulinum neurotoxin type a neutralizing antibodies by yeast surface display high-efficiency yeast transformation using the liac/ss carrier dna/peg method who (2002) manual on animal influenza diagnosis and surveillance the authors wish to thank prof. dane witrrup for kindly providing the yeast expression vector, prof. paul zhou for the ha gene, dr. guoyang liao for the goat antisera, and dr. boping zhou for the human plasma samples. we would also like to thank prof. linqi zhang and dr. zhonghua liu for providing reagents for the facs, elisa and neutralization assays, key: cord-001383-hww0watl authors: li, wenchao; jin, hongyan; sui, xiukun; zhao, zhanzhong; yang, chenghuai; wang, wenquan; li, junping; li, gang title: self-assembly and release of peste des petits ruminants virus-like particles in an insect cell-baculovirus system and their immunogenicity in mice and goats date: 2014-08-12 journal: plos one doi: 10.1371/journal.pone.0104791 sha: doc_id: 1383 cord_uid: hww0watl peste des petits ruminants (ppr) is an acute, febrile, viral disease of small ruminants that has a significant economic impact. for many viral diseases, vaccination with virus-like particles (vlps) has shown considerable promise as a prophylactic approach; however, the processes of assembly and release of peste des petits ruminants virus (pprv) vlps are not well characterized, and their immunogenicity in the host is unknown. in this study, vlps of pprv were generated in a baculovirus system through simultaneous expression of pprv matrix (m) protein and hemaglutin in (h) or fusion (f) protein. the released vlps showed morphology similar to that of the native virus particles. subcutaneous injection of these vlps (pprv-h, pprv-f) into mice and goats elicited pprv-specific igg production, increased the levels of virus neutralizing antibodies, and promoted lymphocyte proliferation. without adjuvants, the immune response induced by the pprv-h vlps was comparable to that obtained using equivalent amounts of pprv vaccine. thus, our results demonstrated that vlps containing pprv m protein and h or f protein are potential “differentiating infected from vaccinated animals” (diva) vaccine candidates for the surveillance and eradication of ppr. peste des petits ruminants (ppr) is a highly contagious and economically important viral disease of domestic and some wild small ruminants, and in particular, of goats and sheep. it is notifiable to the office international des epizooties (oie). clinically, the disease is characterized by severe pyrexia, oculonasal discharges, necrotizing and erosive stomatitis, enteritis, and pneumonia. it was first described in the ivory coast, west africa, but has now become wide-spread in sub-saharan africa, arabia, the middle east, southwest asia, india, and other countries [1] . in china, ppr was first reported in tibetin 2007 [2] , and in december 2013, a ppr outbreak was reported in xinjiang yili; in this outbreak,1236 goats were infected, of which 203 died, and6671 goats in the susceptible population were killed [3] . thus, ppr outbreaks can cause severe economic losses, because they often result in high morbidity and mortality; therefore, development of an effective vaccine for the prevention and control of ppr is particularly important. the causative agent, ppr virus (pprv), is a member of the family paramyxoviridae and the genus morbillivirus [4] .the nonsegmented, single stranded, negative-sense rna genome of pprv encodes six structural proteins (nucleocapsid [n] , phosphoprotein [p] , matrix [m] , fusion [f], hemagglutinin [h] , and polymerase [l] proteins), and two non-structural proteins(c and v). proteins n, p, and l are required for reconstituting viral rna polymerase activity; m protein is required for particle formation and budding, and the two surface glycoproteins, h and f, are required for attachment and entry into the host cell. structurally, the virions are morphologically pleomorphic particles that are surrounded by a host-derived membrane, modified by the h and f transmembrane glycoproteins, as well as the m protein, which is associated with the inner surface of the viral membrane [5, 6] .as in other paramyxoviruses, the c protein of pprv is expressed from an alternative open reading frame (orf) within the p gene, whereas the v protein is expressed by rna editing [7] . these two proteins are thought to be involved in viral evasion of the host's immune responses, specifically by blocking interferon production during infection [8] . for the control of ppr, four attenuated vaccines are available commercially. in endemic areas, the virus is currently controlled using the nigeria 75/1 strain (nig75/1), which has been attenuated by serial passage in vero cells. in india, three other live attenuated vaccines are currently licensed for use: sungri 96, arasur 87, and coimbatore 97 [9] . however, a major disadvantage of these attenuated vaccines is that they cannot be used for differentiating infected from vaccinated animals(diva).recently, subunit vaccines have been developed based on the incorporation of pprv immunogens into various vector systems, such as sheep or goat pox, vaccinia virus, adenovirus, or baculovirus vectors [10, 11, 12, 13] . virus-like particles (vlps) recently emerged as alternatives to subunit vaccines; these have the advantage that they mimic the organization and conformation of the native virus capsid and envelope, but lack the viral genome [14, 15] .given their intrinsic immunogenic properties and high safety profile, vlps have been generated in different heterologous expression systems and have been tested as vaccine candidates for a variety of viral diseases [16] . in the human vaccines market, six vlp-based vaccines, including three for hepatitis b (recombivax hb, engerix b, and genhevac b), two for human papillomavirus (gardasil and cervarix), and one for influenza a (inflexalv), have been developed [16, 17] .in the veterinary field, a vlp-based vaccine against porcine circovirus type 2 (pcv2), viz., porcilis pcv, intervet, has recently been licensed [18] . to date, the assembly and release of pprv vlps have not been well characterized, and their immunogenicity remains unknown. it is known that the pprv glycoproteins h and f contain neutralizing antibody epitopes and several t cell epitopes [19, 20] , and that pprv-specific lympho proliferative responses can be elicited in goats immunized with a recombinant adenovirus expressing the h or f protein [10] . therefore, we hypothesized that immunization with vlps containing h or f proteins could induce a strong pprv-specific immune response. in this study, two types of vlps for pprv (pprv-h and pprv-f)were developed, using viral m protein co-expressed with h or f protein. mice and goats immunized subcutaneously with these pprv vlps developed both humoral and cellular immune responses. cell culture and viruses pprv vaccine strain (nig75/1) was obtained from the china institute of veterinary drug control, beijing, china. vero cells (the american type culture collection, atcc: ccl-81, manassas, va, usa) were cultured in dulbecco's modified eagle medium (dmem; invitrogen, grand island, ny, usa) containing 10% fetal bovine serum (fbs; invitrogen, grand island, ny, usa) at 37uc with 5% co 2 , and were used for pprv propagation and titration. spodoptera frugiperda sf21 insect cells were maintained in sf-900ii insect serum-free medium (invitrogen, grand island, ny, usa) as monolayer cultures or in suspension cultures maintained on temperate orbital shakers (120rpm) at 28uc. cloning of m, h, and f genes and construction of bacmid transfer plasmids pprv rna was extracted using trizol ls (invitrogen, carlsbad, ca, usa). reverse transcription(rt) and polymerase chain reaction (pcr) were performed on extracted viral rna with gene-specific oligonucleotide primers( table 1 ) that had been designed according to the sequence of the pprv nig75/1 strain (genbank accession no. x74443.2).following rt-pcr, the cdna fragments containing the pprv m, h, and f genes were cloned into the pmd18-t vector (takara, dalian, china). the integrity of the nucleotide sequences of the m, h, and f genes was verified by dna sequencing. the transfer plasmids were generated using the pfastbac dual vector (invitrogen, grand island, ny, usa), which contains two promoters. the m gene was cloned, as a1-kbxhoi-kpnidna fragment, downstream of the p10 promoter within the pfastbac-dual bacmid transfer vector that had been digested with xhoi and kpni. the resulting baculovirus transfer plasmid containing the pprv m gene was designated p-m. a bacmid transfer vector expressing both the m and h genes was prepared by cloning the h gene, as a 1.8-kb bamhi-hindiii dna fragment, into the bamhi/hindiii sites downstream of the autographacalifornica multiple nuclear polyhedrosis virus (acmnpv) polyhedron(p h ) promoter in p-m. the resulting plasmid, p-mh, encoded the m and h genes downstream from the p10 and p h promoters, respectively. similarly, a bacmid transfer vector,p-mf, which permitted expression of both the m and f genes, was prepared by cloning the f gene, as a 1.6-kb bamhi-xbai dna fragment, downstream of the p h promoter, into p-m (fig.1 ). recombinant bacmids were produced by site-specific transpositioning after transformation of bacmid transfer plasmids containing the pprv genes into e.coli dh10bac competent cells(invitrogen, carlsbad, ca, usa), which contained the acmnpv baculovirus genome. the recombinant bacmids were identified by pcr using m13 primers together with gene-specific primers ( table 1 ). the recombinant bacmid dna was transfected into sf21 insect cells using cell fectin ii (invitrogen, grand island, ny, usa), according to the bac-to-bac expression systems manual (invitrogen, grand island, ny, usa). briefly, a transfection mixture that contained 1 mg of recombinant bacmid dna, 8ml of cell fectinii, and 2 ml of unsupplemented grace's medium (invitrogen, grand island, ny, usa), was prepared. following incubation at room temperature for 30 min, the mixture was added to sf21 insect cells, which had been seeded at 1610 6 cells per well, in 6-well plates. the transfection mixture and cells were incubated for 5 h at 28uc; the transfection mixture was then removed and 2 ml serum-free sf-900ii medium was added. the cultures were incubated at 28uc for 5 days and the supernatants were harvested as the first passage (p1) viral stock. cells were continuously passaged in order to amplify baculovirus stock with the highest viral titer. the recombinant baculovirus stock was titrated by agarose plaque assay on sf21 insect cell cultures in 6-well plates. to produce pprv-h vlps, sf21 insect cells were infected in a 200-ml volume, at a cell density of 2610 6 cells per ml, with rbvs expressing pprv h and m proteins, at a multiplicity of infection (moi)of 4. similarly, pprv-f vlps were produced by infecting sf21 insect cells with rbvs expressing the pprvf and m proteins. cell culture supernatants were harvested 3 days post-infection, cleared by low-speed centrifugation (20006g,30 min at 4uc)to remove cells, and vlps in the supernatants were then pelleted by ultracentrifugation using a sorvallt1250 rotor (100,0006g, for 1 h, at 4uc). the resulting pellets were dissolved in 2 ml of phosphate-buffered saline solution (pbs, ph7.2), and were then further purified through a discontinuous sucrose gradient (20-30-60% w/v, in pbs) and sedimented by ultracentrifugation in a sorvallth641 swinging bucket rotor (100,0006g, for 3 h, at 4uc). as a control, we used the pprv nig75/1 virus. the visible opalescent bands in the sucrose gradient were collected, diluted in pbs, and pelleted by centrifugation in a sorvall t1250 rotor (100,000 6g, for 2 h, at 4uc) to remove the sucrose. the sedimented particles were resuspended in pbs and stored at 4uc. the concentration of the vlps was determined using a bicinchoninic acid protein assay kit (bca protein assay kit; beyotime, shanghai, china). to detect the protein reactivity of vlps, purified vlps were boiled for 5 min in reducing laemmli sample buffer and resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred to a polyvinylidene difluoride membrane by electroblotting (bio-rad, hercules, ca, usa). the membrane was blocked with 5% dried non-fat milk in pbst (0.05% tween-20 in pbs), overnight at 4uc, and then incubated with goat serum against pprv (diluted 1:200 in blocking buffer), for 1 h at 37uc. the membrane was washed three times with pbst, for 5 min each time, and then incubated with horseradish peroxidase (hrp)conjugated rabbit anti-goat igg antibody (diluted 1:2000 in blocking buffer; zhongshanjinqiao, beijing, china). following several washes with pbst, the specific bands were detected using the substrate 3,39-diaminobenzidine tetrahydrochloride (tiangen, beijing, china) for 5 min at room temperature. the reaction was stopped with water and membranes were photographed. to examine budding of vlps, sf21 insect cells that had been infected with rbvs expressing the m and h/f proteins were fixed with 0.25% glutaraldehyde and 1% osmium tetraoxide, dehydrated with ethanol, and then embedded in spi-pon 812 resin (structure probe, west chester, pa, usa). thin sections were stained with lead citrate and uranyl acetate and then observed by electron microscopy [21] . the presence and morphology of the purified vlps and pprv were evaluated by tem of negatively stained preparations. a carbon-coated grid was set on a 20-ml sample drop for 10 min, and washed with deionized water. washed samples were negatively stained with 1% uranyl acetate at ph 4.5. excess stain was removed using filter paper, and the grids were observed at magnifications ranging from 60,0006 to 200,0006 on a transmission electron microscope (h-7500, hitachi, tokyo, japan) operating at 80 kv. the designs of the animal experiments are shown in table 2 . the animal experiments were conducted with prior approval from the animal care and use committee of chinese academy of agricultural sciences, china. all animal experiments were carried out in accordance with the requirements of the regulations of experimental animal administration of the pr china. all efforts were made to minimize suffering. the proteins that were used to vaccinate animals were purified by sucrose gradient ultracentrigugation and the concentration was determined by using a bca protein assay kit (bca protein assay kit; beyotime, shanghai, china). for mouse experiments, female inbred balb/c mice (vital river, beijing, china), aged 6 to 8 weeks, were randomly divided into seven groups (n = 10 for each group). mice were immunized subcutaneously (s.c.) with different amounts (10 or 40 mg total protein per mouse) of pprv vlps or pprv nig75/1diluted in 100ml of pbs. all groups received a booster injection of the original dosage at 4 weeks after the primary immunization. no adjuvant was used for any of the mice. blood samples were collected by retro-orbital plexus puncture, at various time-points. after the blood samples were allowed to clot and then centrifuged, serum samples were collected and stored at 220ucuntil required for antibody titration. for goat experiments, 20 outbred goats (6-24 months of age) were randomly divided into four groups of five each, and housed separately. none of the goats had detectable levels (titers less than1:5) of pprv neutralizing antibodies. groups 1 and 2 were vaccinated subcutaneously with 300 mg pprv-h or pprv-f vlps diluted in 1 ml of pbs, respectively. group 3 goats were inoculated with 1 ml pprv nig75/1(10 5.5 tcid 50 ), as positive control. group 4 goats were inoculated subcutaneously with 1 ml pbs as negative control. booster vaccination was performed using the same dose, at 3 weeks after primary immunization. serum samples were collected at various time-points post-immunization, and stored at 220uc until analyzed. sera from immunized and control mice were analyzed for pprv-specific antibodies (igg, igg1, and igg2a) by indirect elisa. briefly, 96-well flat-bottomed plates were coated with 100mlof purified pprv whole-virus antigen at a concentration of 2 mg/ml per well in 0.1 m carbonate/bicarbonate coating buffer (ph 9.6),overnight at 4uc, and then the plates were blocked with 5% skim milk in pbst, for 2 h at 37uc. serum samples, serially diluted in pbst, were added to the wells, in duplicate, and incubated at 37uc for 1 h. after washing with pbst three times, each well received 100ml of a 1/4000-diluted hrp-labeled goat anti-mouse igg, igg1, and igg2a (southern biotech, birmingham, al, usa) and incubated at 37uc for 1 h. finally, after washing with pbst three times, 100 ml of freshly prepared tetramethylbenzidine (tiangen, beijing, china) solution was added to each well and color was allowed to develop for 10 min, before stopping the reaction with 100ml of 2 m h 2 so 4. optical density (od) values were measured at a wavelength of 450 nm using an elisa microplate reader (multiskan ascent; thermo, vantaa, finland). the mean titers were calculated from log 10 of the highest serum dilution that yielded a two-fold higher od value compared to that of sera from pbsvaccinated mice. all serum samples from immunized mice and goats were analyzed for the detection of pprv-specific virus neutralization antibody (vna) titers using a micro-neutralization assay, according to the 2013 oie terrestrial manual [22] . briefly, serum samples were complement-inactivated at 56uc for 30 min and were then serially diluted two-fold (mice sera, from 1:4; goat sera, from 1:5) table 2 . experimental designs used for the animal studies. in cell culture medium. the diluted sera (100ml) were mixed with an equal volume of pprv (1000tcid 50 /ml) in a 96-well cell culture plate and incubated at 37uc for 1 h; then, 50ml of vero cell suspension was added to each well and the cells were cultured at 37uc in the presence of 5% co 2 .a series of control wells for virus and uninfected cells were also included. cells were observed for a cytopathic effect (cpe) by microscope at day 8 after incubation, and the vna titer was calculated. vna titer was defined as the highest dilution of the test serum at which the cpe was inhibited by at least 50%. three mice from different groups were sacrificed at 7 days after booster immunization and splenocyte suspensions were prepared as previously described [23] . splenocytes were seeded in 96-well flat-bottom plates at a density of 2610 6 cells per well in rpmi1640 medium (invitrogen, grand island, ny, usa) containing 10% fbs. splenocytes were stimulated in vitro with 50ml of uvinactivated pprv nig75/1(10 4 tcid 50 ). concanavalin a (sigma-aldrich, st. louis, mo, usa) was used as positive control, and medium was used as negative control. after 3 days' incubation at 37uc in a 5% co 2 atmosphere, the proliferative response was determined using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h-tetrazolium, inner salt (mts) assay, using the cell titer 96 aqueous one solution cell proliferation kit (promega, madison, wi, usa), following the manufacturer's instructions. the lymphocyte proliferation rate was quantified using the stimulation index (si), which was calculated as the ratio of the od 490 of the stimulated cells to the od 490 of the negative controls. goats were bled via jugular vein puncture at week 2 after the booster injection. peripheral blood mononuclear cells were prepared as previously described [10] . cells(2610 5 cells per well) were plated in 96-well plates and stimulated with inactivated pprv nig75/1 for 72 h at 37uc under 5% co 2 . unstimulated cells, cultured in medium alone, were used as negative control. the proliferative response was determined by mts assay, as described above. interferon (ifn)-celispot assays splenocytes from inoculated mice were obtained and plated at a density of 2610 5 cells per well in a 96-well elispot plate, which had been coated with ifn-c. one hundred microliters of inactivated pprv nig75/1was added as stimulus. the cultures were then incubated in a 37uc humidified incubator, with 5% co 2 , for 16 h, and then spots were detected as per the manufacturer's instructions (mabtech, nacka, sweden). the data were analyzed with one-way anova in graphpad prism version 5.00 (graphpad software, san diego, ca, usa); pvalues less than 0.05 were considered statistically significant. the open reading frames of pprv m, h, and f genes from pprv strain nig75/1 were amplified by rt-pcr and sub-cloned into pfastbacdual vectors (fig.1 ).the insertion of the genes into the expression vector was confirmed by enzyme digestion analysis and dna sequencing. the rbvs were generated after transfection of sf21 insect cells with recombinant bacmids. the titers of rbvs-pprv mh and rbvs-pprv mf were titrated in a plaque-forming assay, and exhibited titers of 3.5610 7 and 4.7610 7 pfu/ml, respectively, at the third passage. pprv-h vlps and pprv-f vlps were produced and purified as described in the materials and methods section. the vlps bands were mainly distributed between 30% and 60%sucrose in the gradient after ultracentrifugation ( fig.2a) . electron microscopic examination of negatively stained samples revealed that both pprv-h and pprv-f vlps showed spherical shapes, with spikes on their surfaces, and with a diameter of approximately 80-100 nm, which is similar to the morphology and size of native pprv particles propagated in vero cells (fig.2e-g ).an ultrathin section of sf21 insect cells infected with rbvs at 48-h post-infection was investigated, revealing vlps with a diameter of approximately 100 nm budding from the plasma membrane, and some spikeless vlps distributed throughout the cytoplasm (fig. 2b-c) . incorporation of the h or f proteins, as well as m protein, into vlps was confirmed by western blotting using anti-pprv antibodies, by the presence of 37 kda, 70 kda, and 70 kda bands, corresponding to the m, h, and f proteins, respectively, on the blots (fig.3) . the sizes of the m and h proteins corresponded to those predicted from the deduced protein sequences, but the f protein band derived from vlps differed from the form of pprv particles propagated in mammalian cells. in the mammalian cell expression system, the f protein is synthesized as a precursor (f0) of about 60 kda; after translation, f0 is glycosylated and cleaved to yield a 46-kda (f1) and 25-kda (f2) disulfide-link-competent f protein. in the baculovirus-insect cell expression systems, the pprv f protein was generated as a 70-kda band in the western blot membrane, indicating that the f protein was not cleaved and was generated as the f0 precursor, which was also glycosylated. in total, the amount of the expressed vlps was about 3-4.5 mg/l in the cell culture medium. the specific antibody responses of mice immunized with pprv-h vlps or pprv-f vlps were determined by indirect elisa. the levels of total pprv-specific igg in the serum after primary and booster vaccinations were determined; the results are shown in fig.4 . total igg titers in all groups of mice immunized with vlps or pprv were higher after the booster than after the primary vaccination, indicating that booster immunization could stimulate the immune memory cells and induce an anamnestic response, promoting antibody production and exaltation. when mice were immunized with a lower antigen dose of 10 mg, total igg titers of mice immunized with pprv-h vlps were significantly higher than those of mice immunized with pprv-f vlps or pprv after the primary vaccination, indicating that the titers of antibodies against pprv-h vlp proteins increased faster than the titers of antibodies against pprv-f vlps or pprv. the maximal titers detected in both vlp-and virus-immunized mice were similar after the booster vaccination. similar patterns of total igg responses were found in mice immunized with 40 mg vlps or pprv. pprv-specific serum antibodies of igg1 and igg2a were also evaluated by elisa. these results demonstrated that mice immunized with pprv-h vlps and pprv using a lower dose of antigen (10 mg) induced high igg1 serum antibody titers(1:1000 to 1:2400), and lower specific igg2a serum antibody titers(1:200 to 1:250) after the first immunization. in contrast, the levels of igg1 antibody responses were lower (1:200) ,and the igg2a responses were higher (1:700),in mice immunized with pprv-f vlps relative to those observed in mice immunized with pprv-h vlps or pprv. the levels of igg1 and igg2a from all three immunized groups of mice increased after the booster immunization (fig.5a) . similar patterns of igg1 and igg2a responses, as well as igg1:igg2a ratios, were found in mice immunized with 40 mg vlps or pprv (fig.5b) . taken together, the data indicated that immunization with pprv-h vlps induced predominantly specific igg1 serum antibodies, favoring the stimulation of a t-helper class 2(th-2) immune response against pprv, while immunization with pprv-f vlps induced a stronger th-1 immune response, despite the use of a lower immunization dose. since the neutralizing activity against pprv is an indicator of functional antibodies that confer protective immunity, we investigated whether mice and goats immunized with pprv vlps could produce neutralizing antibodies, using a micro-neutralization assay. serially diluted sera from various time-points postimmunization were complement-inactivated, and incubated with live pprv nig75/1; the cpe assays were conducted in vero cells. as shown in table3, pprv-h or pprv-f vlps-vaccinated mice developed vna, and the titers ranged from 1:32 to 1:64 and from 1:16 to 1:32, after booster immunization. as shown in table 4 , at 2 weeks after primary immunization, the titers of vna in the pprv-h vlps-and pprv-vaccinated goat groups exceeded 10, a number that represents a margin considered to be protective in goats vaccinated with live attenuated pprv vaccines [22] . moreover, this value was significantly enhanced after the booster immunization and reached values of 80-160; these high vna titers were maintained for at least 6 weeks, and decreased gradually from 15 weeks after primary immunization. the titer of the pprv-f vlps group was lower compared to that of the pprv-h vlps-and pprv-vaccinated goat groups. however, 2weeks after the booster immunization, all the goats that had been immunized with pprv-f vlps showed vna titers above 10. taken together, the data of the mouse and goat vaccination experiments showed that serum vna levels were significantly higher in the pprv-h vlps-immunized group than in the pprv-f vlps-immunized group, and were comparable to that of the whole virus-immunized group. no neutralization activity against pprv was detected when using pre-immune serum or in the sera of experimental animals injected with pbs. to investigate the cell-mediated immune responses induced by vlps, the lymphocyte proliferation response of mice was analyzed after the booster immunization. as shown in table3, compared with the pbs control group, mice immunized with vlps or pprv exhibited significantly stronger lymphocyte proliferation responses (p,0.05). as shown in table4, the data from the lymphocyte proliferation assay of goats from the three groups immunized with pprv-h vlps, pprv-f vlps, or attenuated pprv indicated similarly strong cellular immune responses. there was no significant difference among mice immunized with different concentrations of vlps or pprv (p.0.05). we also evaluated the mounting of pprv-specific t-cell responses in vaccinated mice. splenocytes, obtained at 7days after the booster immunization, were cultured in the presence of inactivated pprv, and ifn-c production was measured by elispot assays (fig.6 ). both pprv-h vlps-and pprv-f vlps-vaccination induced specific ifn-c production in response to pprv exposure; the frequency of cells secreting ifn-c positively correlated with the immunization dose. no specific production of ifn-c in response to pprv exposure was detected in control mice inoculated with pbs. altogether, these data demonstrated that inoculation with pprv-h vlps and pprv-f vlps can generate specific t-cells that recognize the pprv h and f proteins in vivo. the minimum molecular requirement for efficient assembly and release of pprv virions is still unknown. in this study, we found that the major structural genes of pprv can be efficiently expressed in recombinant baculovirus-infected sf21 insect cells and that these genes can then support the production of pprv vlps. the expression of m protein was necessary and sufficient for the formation of vlps (data not shown); the additional expression of the h or f glycoproteins allowed formation of budding particles with the typical morphology and size of pprv and related paramyxoviruses. a recent study demonstrated that pprv spikeless vlps could be formed by co-expression of the m and n proteins in insect cells; however, expression of m protein alone in insect cells did not result in vlp formation, as assessed by tem [24] , which was contrary to our results. the finding that m protein alone could be released from cells and generate vlps has also been observed for some other paramyxoviruses, including human parainfluenza virus type1 [25] , sendai virus [26] , newcastle disease virus [27] , and nipha virus [28] , as well as for viruses in other related virus families, including ebola [29] and vesicular stomatitis virus [30] .in addition, some paramyxovirus m proteins, such as parainfluenza virus type5 [31] and mumps virus [32] , lack the ability to direct efficient vlp production when expressed alone in cells, but vlp production becomes highly efficient when m proteins are expressed together with other viral proteins. hence, although the requirements for efficient vlp production differ among paramyxoviruses, it appears as if, in general, m protein is the major requirement for paramyxovirus vlp production, as vlps cannot be formed in its absence [6, 33] . the baculovirus expression vector-insect cell system has been utilized extensively for vlp production, and particularly for production of enveloped vlp vaccines [34, 35, 36] . baculovirus expression of heterologous genes permits multiple post-translational modifications, like folding, oligomerization, phosphorylation, glycosylation, acylation, disulfide bond formation, proteolytic cleavage, and so on, which are similar or identical to those that occur in mammalian cells. however, in this study, the molecular size of the expressed f protein was 70 kda, but the f protein was cleaved into f1 and f2 protein upon expression in mammalian cells; the results indicated that there are differences in the posttranslational modification in the two expression systems. furthermore, insect cells are able to grow in serum-free medium cultures; this is highly desirable, because serum may be a cause of adventitious viruses. therefore, its use requires more intensive purification processes to remove serum proteins. additionally, these cells are perfectly adapted to suspension conditions, allowing for large-scale culture [37, 38] . these advantages make the baculovirus expression vector-insect cell system a promising platform for production of vlps [39] . in this study, we chose the pfastbacdual (invitrogen) baculovirus transfer vector, which contains two promoters, so that insect cells infected with a single recombinant bicistronic baculovirus could express two proteins concurrently. the coexpression strategy is particularly important for the efficient assembly of multi-protein complexes, where simultaneous expression of multiple viral proteins is essential [40] .co-expression and co-infection strategies have previously been compared in the production of triple-layered rotavirus vlps(vp2,vp6, and vp7), and sars vlps. for production of rotavirus vlps, co-expression was more efficient than co-infection [41] . sars vlps could not be generated in sf9 cells co-infected with three recombinant viruses expressing the m, e, and s proteins, but spontaneous assembly of highly stable vlps occurred when using a single recombinant baculovirus expressing all three sars proteins simultaneously [35] . the main limitation of the baculovirus expression vector-insect cell system is the co-production of recombinant baculoviruses (enveloped viruses) during the infection process; thus, when developing pprv vlp vaccines, the challenging task is the table 4 . peste des petitsruminants virus (pprv) neutralizing antibody titers and lymphocyte proliferation responses in goats immunized with pprv virus-like particles (vlps) or native pprv particles. purification of vlps from the recombinant baculoviruses, which share a similar density and enveloped structure [39, 42] . in our study, although purification was possible by centrifugation in sucrose density gradients, residual baculoviruses were consistently detected in vlp preparations (fig. 2d) .given the safety concerns, vlps should be purified via chemical (binary ethylenimine) or physical(uv irradiation) inactivation treatments, to eliminate any possible baculovirus infectivity when considering clinical trials. for many viruses, recombinant vlps have been shown to yield promising vaccines, because of their robust immunogenicity, elicitation of protective neutralizing antibodies (due to the presence of conformational epitopes on vlps), and their safety profiles (as they do not contain any viral genetic material). in this study, the effectiveness of pprv vlps as an immunogen was demonstrated in mice and goats. the results indicated that high specific antibody titers against pprv could be induced by both pprv-h vlps and pprv-f vlps, which were as high as those resulting from immunization with pprvnig75/1. the vna titers elicited by pprv-h vlps were much higher than those induced by pprv-f vlps, and the vna titers of pprv-h vlps were as high as the responses to the attenuated virus vaccine. previous reports have shown that the h protein of pprv is a more potent inducer of high vna titers than the f protein [11] , while the f protein seems to induce mainly a cellular immune response [43] .our results demonstrated that mice immunized with pprv-h and pprv-f vlps exhibited a similarly high level of cell-mediated immune responses. we further showed that subcutaneous immunization of mice with pprv-h vlps at a dose of 10 mg, in the absence of adjuvant, induced strong humoral immune responses. this immunogenicity is related to the structural features of the vlps, which are particulate structures that contain a dense and repetitive pattern of epitopes on their surfaces. in contrast, monovalent, non-particulate antigens do not always reflect the native structure of the protein, and the related antibody responses are not always specific for physiologically relevant epitopes. consequently, administration of these vaccines often requires use of strong adjuvants, as well as large and frequent doses of the antigen. in contrast, vlp-based vaccines are strongly immunogenic, often reducing or eliminating the need for exogenous adjuvants [44, 45] . an additional benefit of this vlp-vaccination approach is that it may allow improved surveillance through diva, as seroconversion to n protein will occur only in animals that have been infected with pprv, but not in vlp-vaccinated animals that have not been infected. a commercially available n protein-based competitive elisa kit might strengthen the usefulness of vlp vaccines [46] . we also detected pprv-specific antibodies in the vaccinated goats, using an n protein-based competitive elisa established in our laboratory. no n protein-specific antibodies were found in goats vaccinated with pprv h or pprv f vlps; in contrast, seroconversion to n protein was detected in goats vaccinated with the pprv nig 75/1 attenuated virus (data not shown). to our knowledge, this is the first report that demonstrated the construction of pprv vlps consisting of immunogenic proteins and that assessed their immunogenicity in two animal models, viz., mice and goats. taken together, the results of our study suggested that pprv vlps can be generated in insect cells simultaneously expressing the pprv m protein with h or f protein. the vlps had a morphology and size similar to those of native virus particles. humoral and cellular immunity against pprv were efficiently induced in mice and goats vaccinated with these pprv vlps, without the need for adjuvants. thus, pprv-h vlps or pprv-f vlps may be promising vaccine candidates for ppr, and diva tests may prove of great benefit in future control programs for this disease. . specific interferon (ifn)-cproduction in response to peste des petits ruminants virus (pprv) proteins in splenocytes from mice, as detected by elispot assay. splenocytes from mice inoculated with pprv virus-like particles (vlps), pprv per se, or phosphate-buffered saline (pbs) were isolated and cultured for 16h in the presence of inactivated pprv. the production of ifn-cwas measured using an elispot assay. samples were tested in triplicate and mean ifn-cvalues for each mouse are shown. doi:10.1371/journal.pone.0104791.g006 global distribution of peste des petits ruminants virus and prospects for improved diagnosis and control peste des petits ruminants virus in tibet /12/2013: peste des petits ruminants, (people's rep. of china) classification of peste des petits ruminants virus as the fourth member of the genus morbillivirus the molecular biology of the morbillivirus (measles) group molecular mechanism of paramyxovirus budding paramyxoviridae: the viruses and their replication morbillivirus v proteins exhibit multiple mechanisms to block type 1 and type 2 interferon signalling pathways comparative efficacy of peste des petits ruminants (ppr) vaccines recombinant adenovirus expressing f and h fusion proteins of peste des petits ruminants virus induces both humoral and cell-mediated immune responses in goats a goat poxvirus-vectored peste-des-petits-ruminants vaccine induces long-lasting neutralization antibody to high levels in goats and sheep mva recombinants expressing the fusion and hemagglutinin genes of pprv protects goats against virulent challenge baculovirus display of fusion protein of peste des petits ruminants virus and hemagglutination protein of rinderpest virus and immunogenicity of the displayed proteins in mouse model virus-like particles: promising platforms with characteristics of diva for veterinary vaccine design current strategies for subunit and genetic viral veterinary vaccine development virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development eleven years of inflexal v-a virosomal adjuvanted influenza vaccine one dose of a porcine circovirus 2 subunit vaccine induces humoral and cellmediated immunity and protects against porcine circovirus-associated disease under field conditions immune responses in goats to recombinant hemagglutinin-neuraminidase glycoprotein of peste des petits ruminants virus: identification of a t cell determinant mapping of b-cell epitopic sites and delineation of functional domains on the hemagglutinin-neuraminidase protein of peste des petits ruminants virus human annexin a6 interacts with influenza a virus protein m2 and negatively modulates infection manual of diagnostic tests and vaccines for terrestrial animals immune responses in mice vaccinated with a suicidal dna vaccine expressing the hemagglutinin glycoprotein from the peste des petits ruminants virus formation of peste des petits ruminants spikeless virus-like particles by co-expression of m and n proteins in insect cells human parainfluenza virus type 1 matrix and nucleoprotein genes transiently expressed in mammalian cells induce the release of virus-like particles containing nucleocapsid-like structures role of matrix and fusion proteins in budding of sendai virus requirements for the assembly and release of newcastle disease virus-like particles quantitative analysis of nipah virus proteins released as virus-like particles reveals central role for the matrix protein ebola virus vp40 drives the formation of virus-like filamentous particles along with gp viral liposomes released from insect cells infected with recombinant baculovirus expressing the matrix protein of vesicular stomatitis virus requirements for budding of paramyxovirus simian virus 5 virus-like particles mumps virus matrix, fusion, and nucleocapsid proteins cooperate for efficient production of virus-like particles paramyxovirus assembly and budding: building particles that transmit infections assembly and release of hiv-1 precursor pr55gag virus-like particles from recombinant baculovirus-infected insect cells efficient assembly and release of sars coronaviruslike particles by a heterologous expression system protective efficacy of baculovirus-derived influenza virus-like particles bearing h5 ha alone or in combination with m1 in chickens a kinetic and statistical-thermodynamic model for baculovirus infection and virus-like particle assembly in suspended insect cells recombinant protein vaccines produced in insect cells use of baculovirus expression system for generation of virus-like particles: successes and challenges coexpression vs. co-infection using baculovirus expression vectors in insect cell culture: benefits and drawbacks triple layered rotavirus vlp production: kinetics of vector replication, mrna stability and recombinant protein production influenza viruslike particles comprised of the ha, na, and m1 proteins of h9n2 influenza virus induce protective immune responses in balb/c mice development of a dual recombinant vaccine to protect small ruminants against peste-des-petits-ruminants virus and capripoxvirus infections assembly and biological and immunological properties of newcastle disease virus-like particles pandemic h1n1 influenza virus-like particles are immunogenic and provide protective immunity to pigs development of a competitive elisa for detecting antibodies to the peste des petits ruminants virus using a recombinant nucleoprotein we thank the electron microscopy core facility of the institute of agroproducts processing, science, and technology, caas, for tem analysis. we also thank dr. huailiang ma for kindly editing this manuscript. conceived and designed the experiments: wl gl. performed the experiments: wl xs. analyzed the data: wl zz hj. contributed reagents/materials/analysis tools: jl cy ww. wrote the paper: wl zz. requested financial support: gl. key: cord-012387-1ogcxd7b authors: kaufman, aaron r.; hersh, eitan d. title: the political consequences of opioid overdoses date: 2020-08-04 journal: plos one doi: 10.1371/journal.pone.0236815 sha: doc_id: 12387 cord_uid: 1ogcxd7b the united states suffered a dramatic and well-documented increase in drug-related deaths from 2000 to 2018, primarily driven by prescription and non-prescription opioids, and concentrated in white and working-class areas. a growing body of research focuses on the causes, both medical and social, of this opioid crisis, but little work as yet on its larger ramifications. using novel public records of accidental opioid deaths linked to behavioral political outcomes, we present causal analyses showing that opioid overdoses have significant political ramifications. those close to opioid victims vote at lower rates than those less affected by the crisis, even compared to demographically-similar friends and family of other unexpected deaths. moreover, among those friends and family affected by opioids, republicans are 25% more likely to defect from the party than the statewide average republican, while democrats are no more likely to defect; independents are moderately more likely to register as democrats. these results illustrate an important research design for inferring the effects of tragic events and speak to the broad social and political consequences of what is becoming the largest public health crisis in modern united states history. for over-marketing and over-prescribing highly addictive opioids [7] , while others point to social and economic determinants like poverty, unemployment, and limited economic mobility as important moderating factors of drug use and abuse. [8] [9] [10] . while the public health effects of the opioid epidemic are clear, the social and political effects are largely unstudied, though a study from the beginning of the crisis estimates economic costs of opioid users as $13,000 canadian dollars per year [11] . a broad literature in political science examines the consequences of traumatic events like terrorism [12] , race riots [13] , and war [14] , largely finding politically mobilizing effects; there is good reason to suspect that the opioid crisis has produced complex political aftershocks as well. and while the effects of such shocks to individuals may be pronounced, they may also serve as drivers of local or national policy, as victims of such tragic events often become spokespeople in advocating for large policy changes. this paper applies a validated causal research design to study the political consequences of the opioid epidemic on the individuals closest to it-the friends and family of overdose victims. leveraging recent computational advantages and newly available large-scale government databases, we show that the opioid epidemic has significantly altered patterns of party identification and turnout, but with important heterogeneity across partisanship. in contrast to other studies examining the effects of tragedy on political participation, we find that friends and family of opioid overdose victims are less likely to turn out to vote than they were before tragedy struck, even compared to victims of premature cancer or a demographically-matched sample of registrants without familial opioid overdoses. moreover, opioid overdoses seem to change the beliefs and preferences of those affected: independents are far more likely to re-register as democrats, and republicans are nearly 50% more likely to defect to other parties, but democrats' party affiliations are unchanged. more broadly, we expand the literature on the persistent effects of shocks to political behavior by showing a context in which participation declines rather than increases, and where shifts in party identification are causally identified, offering new insights into possible long-term consequences of large public health crises. how might friends and family of opioid overdose victims respond politically? we examine two sets of outcomes: political engagement and partisan preference. prior research finds positive engagement effects for crime victimization [15] , immigration [16] , race riots [13] and terrorist attacks [12] , so we have reason to suspect that those affected by the opioid epidemic may mobilize to affect policy change. another line of research, however, suggests that sadness and depression might decrease voter mobilization [17] , offering a contrasting hypothesis. on partisan preferences, too, prior research is split: whereas victims of terrorism become on average more conservative as national identity becomes more important [12] , those proximate to race riots become more liberal, perhaps as they become more aware of the struggles of racial minorities [13] . and in considering the psychological repercussions of opioid overdose, while anxiety may lead to more liberal preferences [18] , fear may lead to more conservative preferences instead [19] . our measure of partisan preference is the change in registered party affiliation, a strong behavioral indicator of preferences relative to survey reporting. finally, a large literature observes that apolitical events like sports victories, hurricanes, and shark attacks tend to disadvantage the incumbent party, whom voters blame for misfortunes [20] , suggesting that overdose victims' families may defect from the democratic party during the democratic administrations, or defect to it during republican administrations; an equally large literature points out that events like overdoses may increase the salience of healthcare-related issues in the minds of voters, pushing them toward the party with the most ownership over the healthcare debate [21, 22] . how do these conflicting hypotheses manifest in the friends and family of opioid overdose victims? our analysis strategy leverages panel data with voter behavior linked to accidental drug death records. we identify in public voter registration records the friends and family of opioid overdose victims, and compare their election turnout rates and party identification in the elections immediately before and after their loved one's death. opioid overdose victims are not a representative sample of the population, however, so any political differences we observe may be due to demographic differences instead. to avoid this inferential pitfall we identify a control group of individuals who prematurely died of cancer (which we define as 50 years old or younger, corresponding to approximately the youngest 10% of cancer deaths [23] ), and perform coarsened exact matching with mahalanobis distance [24] to obtain a sample of individuals who died of cancer that look demographically identical to our sample of individuals who died of opioid overdoses. in this way, we causally identify the effect of having a loved one die of an opioid overdose as compared to another type of premature death. to threaten our causal identification, an unobserved variable would have to confound the difference between the change in party identification among opioid overdose victims' families and the change in turnout or party identification among cancer victims' families. (figs 1 and 2 ). this comparison separates out the effect of premature deaths from the effects of an opioid overdose death in particular, isolating the bundle of treatments distinctive to opioid deaths such as politicization, media attention, and stigmatization. we begin with a panel of two connecticut voter files: a 2013 voter file with 2,421,897 individuals and a 2017 voter file with 2,428,165 individuals; connecticut uses a unique voter id which we use to match registrants from 2013 to 2017, producing 2,001,250 matched individuals. the connecticut voter file contains party identification information as well as election turnout for as more than a decade of previous federal, statewide, and local elections. we merge these data with geography-based census measures from the national historical geographic information system (nhgis, see table 1 ). the office of the chief medical examiner in connecticut makes free available a de-identified list of accidental opioid-related deaths; our version of this data contains 3,583 records. these records contain the location of the deceased individual, their age at death, their race, sex, town of residence, and the opioid(s) identified in their toxicology reports. we limit our analyses to those overdoses that occurred after the 2012 general election but before the 2016 primary election. to match these individuals to the voter file, we first re-identify them by searching for their dates, locations, and ages at death in publicly searchable obituary records. we take all uniquely identified opioid overdose obituaries and manually record their full names and precise birth dates. this results in 1,369 opioid deaths in our data set. we also identify the friends and family of these overdose victims by locating those individuals living at the same address as the overdose victim at the time of death, and find 1,972 such individuals. we exclude cases with more than 10 registered voters living at the same address as they may not share a friend or family relationship with the victim. for our comparison sample, we search obituary records for premature cancer deaths that occurred after the 2012 general election but before the 2016 primary election, then manually the political consequences of opioid overdoses record their full names and precise birth dates. we find 110 such deaths and, using the same procedure as for the overdose victims, we find 705 household members. using coarsened exact matching (cem) with mahalanobis distance, we identify two samples of matched voters: one that looks demographically similar to the family members of cancer victims, and another that looks demographically similar to the family members of opioid overdose victims. by comparing political behaviors of these matched groups to the original samples of family members, we control for all observed (but not unobserved) factors that might confound the relationship between cancer or opioid overdose deaths and voter turnout. the variables in our matching procedure include race, age, sex, county, household occupancy, census block median household income, and prior turnout history. we summarize our data in table 1 below. overdose deaths disproportionately fall on hispanics and african-americans, on men rather than women, and on the young more than the old, but otherwise resemble the population relatively well. unsurprisingly, cancer victims also have substantially more household members than opioid victims. however, we do observe that victims of overdose deaths and their families vote at lower rates than the statewide average. we first examine voter turnout (fig 1) . the statewide turnout rate in the 2016 general election in connecticut was approximately 65% (top panel rightmost grey dot and dashed grey line); republicans voted at a higher rate (dashed red line). the overall turnout rate among the families of cancer victims (dark grey dot) is about 8 percentage points lower from the overall turnout rate, while the rate among families of opioid victims is about 10 percentage points lower. democrats among the families of both sets of victims vote at lower rates; both democrats and republicans among the families of both sets of victims vote approximately 10 percentage points less than their copartisans who were unaffected, and to a lesser degree, less than the demographically-matched samples of copartisans. a different pattern emerges for primary election turnout: the friends and family of opioid overdose victims vote at a rate about 4 percentage points lower than the overall rate, and both democrat and republican friends and family of opioid overdose victims vote at lower rates than the average, but cancer victim families of neither party vote at lower rates. turning to party affiliation (table 2) , we examine the rates at which the friends and family of opioid overdose victims change parties relative to cancer victims' friends and family as well as the statewide average. statewide, both democrats and republicans left their party from 2012 to 2016 at a rate of 5.6%; among democrats, both cancer and opioid overdose victims' friends and family leave the party at approximately the same rate-5.2% and 5.3% compared to 5.6% statewide. among republicans, however, both cancer and opioid overdose victims' friends and family leave the party at much higher rates-both 7.4%-than the statewide average of 5.6%. in comparison, demographically-matched control republicans left the party at rates of 6.2% and 6.3%, indicating a 1 percentage point estimated treatment effect of premature death on republican party defection. this change is almost entirely due to republicans becoming independent (5.6% and 5.7%) rather than becoming democrats (1.9% and 1.8%). among independents, the friends and family of cancer victims join a major party at about the statewide rate, but independent friends and family of opioid overdose victims join the democratic party at a rate 1/3 higher than the statewide rate. to test the significance of these differences, we conduct chi-squared tests between pairs of columns represented as transition matrices. chi-squared tests indicate a statistically significant difference (p < 0.01) between the statewide and od families transition matrices and between the cancer families and od families transition matrices, but not between the statewide and cancer families transition matrices. as well, the difference between the od families and od matched control transition matrices is significant, but the difference between the cancer families and cancer matched control transition matrices is not. the opioid epidemic, much like covid-19, represents a political conflict rooted in a public health crisis, and it is therefore important to examine its political ramifications. in this study we have shown that the opioid epidemic substantially affects the political behavior among the friends and family of its victims. while tragic deaths in general reduce turnout in the general election, only opioid epidemic deaths reduce turnout in the primary election compared to other premature deaths. moreover, turning to partisanship, republican friends and family were increasingly likely to defect from the gop, independents were increasingly likely to join the democratic party, both suggesting a liberalizing trend. the shifting partisan allegiances suggest that, in the face of opioid-driven tragedy, voters do not appear to hold the democratic party accountable for opioid overdoses; note that during this period of study, both the us presidency and the connecticut governorship were held by democrats. to the contrary, voters act as though they prefer more liberal policies toward managing the opioid crisis in the face of self-interest or socialization [25, 26] , even if they may not know the differences between the parties' policies [27] . we do note third key limitations of our results. first, our analysis is restricted to a single, unrepresentative state. secondly, we only find obituary records for 38.2% of opioid overdose victims in the state, and that sample may be unrepresentative of opioid overdose victims as a whole. finally, by identifying cancer deaths through obituary records, we may have an unrepresentative sample of statewide cancer deaths. importantly, for this unrepresentativeness to bias our internal validity, cancer victims with and without obituaries that specifically mention cancer would need to be systematically different in a manner correlated with changing party registration. taken together, these three limitations restrict our ability to generalize our findings to other states and populations. however, if we could safely assume that the trends we observe in connecticut extrapolate to the entire us, based on results from [28] we would estimate that the opioid overdose resulted in 200,000 fewer cast votes in the 2016 presidential election, nearly 40,000 additional democratic registrants, and an equal number of fewer republican registrants merely among the friends and family of its victims (see s1 text). extending this line of inquiry may involve interrogating whether this change in party affiliation is a policy-driven response to self-interest or a broader shift in outlook and emotional state in response to tragedy. finally, our results add important nuance extant literature on the politically mobilizing effects of dramatic events like terrorism and race riots, and future research in this field may also examine and characterize which types of shocks are mobilizing and which depress turnout, or which shocks tend to liberalize or conservatize. supporting information s1 text. (pdf) the opioid crisis: a comprehensive overview. current pain and headache reports contribution of opioid-involved poisoning to the change in life expectancy in the united states a time-release history of the opioid epidemic the role of science in addressing the opioid crisis the road to h.: narcotics, delinquency, and social policy opioid discussion in the twittersphere. substance use & misuse opioid-prescribing patterns of emergency physicians and risk of long-term use public health and international drug policy. the lancet opioid crisis: no easy fix to its social and economic determinants viewing addiction as a brain disease promotes social injustice social costs of untreated opioid dependence long-term effect of september 11 on the political behavior of victims' families and neighbors can violent protest change local policy support? evidence from the aftermath of the 1992 los angeles riot. american political science review communication and political mobilization: digital media and the organization of anti-iraq war demonstrations in the us crime victimization and political participation revisiting lepanto: the political mobilization against islam in contemporary western europe. patterns of prejudice depression and political participation threat, anxiety, and support of antiterrorism policies. american journal of political science are all conservatives alike? a study of the psychological correlates of cultural and economic conservatism. the journal of psychology blind retrospection: why shark attacks are bad for democracy issue salience, issue ownership, and issue-based vote choice. electoral studies disputed ownership: parties, issues, and traits in the minds of voters. political behavior peer reviewed: heart disease and cancer deaths-trends and projections in the united states causal inference without balance checking: coarsened exact matching concentrated burdens: how self-interest and partisanship shape opinion on opioid treatment policy how should we wage the war on drugs? determinants of public preferences for drug control alternatives heuristics behaving badly: party cues and voter knowledge drug and opioid-involved overdose deaths-united states we thank sheng ha and two anonymous reviewers for invaluable feedback. we are indebted to neal beck, leo celi, brian libgober, luke miratrix, robert ward, and madeleine wolf for helpful comments during the preparation of this manuscript, and to obituarydata.com of generously providing access to national obituary data. all remaining errors are our own. this work was approved by the harvard university committee on the use of human subjects, irb protocol 19-0015; it was designated "not human subjects research". the authors have declared that no competing interests exist. key: cord-257603-ov0b8yub authors: azlan, arina anis; hamzah, mohammad rezal; sern, tham jen; ayub, suffian hadi; mohamad, emma title: public knowledge, attitudes and practices towards covid-19: a cross-sectional study in malaysia date: 2020-05-21 journal: plos one doi: 10.1371/journal.pone.0233668 sha: doc_id: 257603 cord_uid: ov0b8yub in an effort to mitigate the outbreak of covid-19, many countries have imposed drastic lockdown, movement control or shelter in place orders on their residents. the effectiveness of these mitigation measures is highly dependent on cooperation and compliance of all members of society. the knowledge, attitudes and practices people hold toward the disease play an integral role in determining a society’s readiness to accept behavioural change measures from health authorities. the aim of this study was to determine the knowledge levels, attitudes and practices toward covid-19 among the malaysian public. a cross-sectional online survey of 4,850 malaysian residents was conducted between 27(th) march and 3(rd) april 2020. the survey instrument consisted of demographic characteristics, 13 items on knowledge, 3 items on attitudes and 3 items on practices, modified from a previously published questionnaire on covid-19. descriptive statistics, chi-square tests, t-tests and one-way analysis of variance (anova) were conducted. the overall correct rate of the knowledge questionnaire was 80.5%. most participants held positive attitudes toward the successful control of covid-19 (83.1%), the ability of malaysia to conquer the disease (95.9%) and the way the malaysian government was handling the crisis (89.9%). most participants were also taking precautions such as avoiding crowds (83.4%) and practising proper hand hygiene (87.8%) in the week before the movement control order started. however, the wearing of face masks was less common (51.2%). this survey is among the first to assess knowledge, attitudes and practice in response to the covid-19 pandemic in malaysia. the results highlight the importance of consistent messaging from health authorities and the government as well as the need for tailored health education programs to improve levels of knowledge, attitudes and practices. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the coronavirus disease 2019 (covid-19) emerged in wuhan, china at the end of 2019. since then, it has spread to 200 countries and has been declared a global pandemic by the world health organisation (who). to date, there are more than 2.3 million positive covid-19 cases recorded with at least 150,000 deaths globally [1] . the first case of covid-19 in malaysia was detected on 25 th january 2020 involving three tourists from china [2] . the number of cases steadily increased before the nation's first two deaths were recorded on 17 th march [3] . as of 20 th april 2020, malaysia has recorded more than 5300 positive cases involving 89 deaths. a majority of these cases were traced back to a religious gathering which has now reached its fifth-generation infections [4] . the malaysian prime minister enforced a movement control order (mco) on 18 th march 2020 as a mitigation effort to reduce community spread and the overburdening of the country's health system. similar to lockdowns in china and italy, the mco restricted most non-essential activity outside the home. malaysians were only permitted to leave the house for basic activities such as buying groceries and seeking medical treatment. the mco also restricted malaysians from leaving the country and all foreigners from entry. non-essential sectors were ordered to close operations or allow employees to work from home. lockdown measures were perceived as necessary to curb the spread of the virus as rapid human-to-human transmission occurred and much about the virus remained unknown [5] . due to the obscurity of this novel virus, there has been a lot of confusion and misunderstanding about the virus itself, how it can spread and the necessary precautions that should be taken to prevent infection. this becomes increasingly challenging with the vast amount of misinformation and disinformation shared on social media that is clouding people's understanding of covid-19 [6] . when the initial mco announcement was made, malaysians reacted in panic and confusion. aside from panic buying, people crowded public transportation hubs to travel back to their hometowns, potentially increasing the risk of infection to other parts of the country. while this reaction to the mco was not unexpected, it raises questions regarding the level of understanding and attitudes toward covid-19 among malaysians. the knowledge, attitudes and practices (kap) toward covid-19 play an integral role in determining a society's readiness to accept behavioural change measures from health authorities. kap studies provide baseline information to determine the type of intervention that may be required to change misconceptions about the virus. assessing the kap related to covid-19 among the general public would be helpful to provide better insight to address poor knowledge about the disease and the development of preventive strategies and health promotion programs. among the lessons learned from the sars outbreak is that knowledge and attitudes are associated with levels of panic and emotion which could further complicate measures to contain the spread of the disease [7, 8] . the survey also gives a general picture of malaysians covid-19 prevention practices before the mco and this can better prepare the government to address future health crises involving infectious diseases. the results of this study are important to inform future efforts focusing on societal readiness to comply with pandemic control measures. a quantitative approach was utilised to achieve the objectives of this study. a survey is most appropriate as it allows large populations to be assessed with relative ease [9] . in this study, a cross-sectional survey was deemed most appropriate to gather information on covid-19 for the malaysian context. data collection was performed online using the survey monkey platform. the call for participation was made on social media. the ethics committee of universiti kebangsaan malaysia approved our study protocol, procedures, information sheet and consent statement (jep-2020-276). participants who gave consent to willingly participate in the survey would click the 'continue' button and would then be directed to complete the self-administered questionnaire. this cross-sectional survey was conducted in the second week of the mco, between 27 th march 2020 to 3 rd april 2020. the target sample size was 3,640, determined by identifying the smallest acceptable size of a demographic subgroup with a ±5% margin of error and a confidence level of 95% [10, 11] . as it was not feasible to conduct a systematic nationwide sampling procedure during this period, the researchers opted to use an online survey using survey monkey advantage annual. members of the malaysian public above the age of 18 and currently residing in the country were eligible to participate in the survey. we utilised several strategies to reach as many respondents as possible all over the country within the one-week data collection period. this includes relying on professional and personal networks of the researchers, reaching out to community leaders and social media influencers to broadcast and share the survey. two main platforms used in disseminating this survey were social media (facebook, twitter and instagram) and whatsapp. facebook and whatsapp were selected as two of the most popular communication and social platforms in malaysia [12] . while facebook is generally preferred by older malaysians, twitter and instagram are more popular among the younger generation. a standardised general description about the survey was given in the whatsapp message/social media postings before the link was provided to both english and malay language versions of the questionnaire. a total of 4,850 participants took part in the survey. the survey instrument is an adaptation of the measures developed in a study on chinese residents' knowledge, attitudes and practices (kap) towards covid-19 in china [13] . the questionnaire consisted of four main themes: 1) demographics, which surveyed participants' sociodemographic information, including gender, age, state of residence, occupation, and household income; 2) knowledge about covid-19; 3) attitudes toward covid-19; and 4) practices relevant to covid-19. the survey was offered in the english and malay languages. a backward-translation approach was used in translating the items between english and bahasa malaysia, so as to ensure linguistic and conceptual equivalence [14] . discrepancies between the two versions were rectified, and equivalence of measuring on all items was ensured through consultation with bilingual researchers. to measure knowledge about covid-19, 13 items were adapted from previous research [13] . these items include the participant knowledge about clinical presentations (items 1-4), transmission routes (items 5-8) and prevention and control (items 9-13) of covid-19. participants were given "true," "false," or "not sure" response options to these items. a correct response to an item was assigned 1 point, while an incorrect/not sure response was assigned 0 points. the maximum total score ranged from 0-13, with a higher score indicating better knowledge about covid-19. to measure attitudes towards covid-19, surveyed participants were asked whether they agreed, disagreed or were not sure that the pandemic would be successfully controlled. they were also asked about their confidence towards the government in winning the battle against covid-19 (yes or no) and about the ability of the government in handling the covid-19 crisis (agree, disagree, or not sure). to measure practices, participants were asked yes/no questions on whether they had avoided going to crowded places such as weddings; wore a face mask when leaving home; and whether they practiced proper hand hygiene in the week before the movement control order (mco). for this study, the collected data were analysed using the statistical package for the social sciences (spss), version 26. descriptive analysis focused on frequencies, and percentages while chi-square tests, independent samples t-tests and one-way analysis of variance (anova) were utilised to determine the differences between groups for selected demographic variables. the statistical significance level was set at p < 0.05. internal consistency of the knowledge measures was tested using a reliability test where the cronbach alpha coefficient aided in determining the reliability of the variables. the results showed that the cronbach alpha for knowledge (13 items) was 0.655. the result added credence where according to griethuijsen, the range of cronbach alpha within 0.6 to 07 is considered adequate and reliable [15] . it is attested that the items used to measure knowledge on covid-19 are therefore acceptable. a total of 4850 participants participated in the study. out of the total, the average age was 34 years (sd = 11.2, range = 18-73), 2808 (57.9%) were women, 1993 (41.1%) resided in central malaysia and 2173 (44.8%) were employed in the public sector. other demographic characteristics are detailed in table 1 . a total of thirteen questions were used to measure knowledge on the covid-19 virus. the average knowledge score for participants was 10.5 (sd = 1.4, range 0-13). the overall correct answer rate of the knowledge questionnaire was 80.5% (10.5/13 � 100) while the range of correct answer rates for all participants were between 46.2 to 100%. about 77.2% of participants were able to obtain scores above 10, representing an acceptable level of knowledge on covid-19. most participants knew that people who had contact with an infected person should be immediately isolated for a period of 14 days (99.1%) and that this is an effective way to reduce the spread of the virus (98.9%). even so, there was noticeable confusion among participants regarding transmission of the virus. only 43.3% of participants answered correctly when asked if the virus was airborne and just 35.7% answered correctly when asked if eating and touching wild animals could result in infection [ table 2 ]. differences in knowledge scores among different demographic characteristics were assessed using t-tests and anova. the results show that knowledge scores were significantly different across genders, age groups, regions, occupation groups and income categories. higher knowledge scores were obtained among female participants, those above the age of 50, people residing in central malaysia and in the higher income category. the results of the anova analyses show that the knowledge scores of people living in the central region were significantly higher than other regions. additionally, the average knowledge score of students were significantly lower than those of other occupation categories and those earning below rm3,000 per month showed significantly lower knowledge scores [ table 3 ]. participants were asked three questions in assessment of attitudes. the first question asked whether or not they agreed that the covid-19 situation would be successfully controlled; second, whether they thought malaysia would be able to win its battle against the virus; and third, whether they thought the malaysian government was handling the health crisis well [fig 1] . for the first question, a majority of participants agreed that covid-19 would successfully be controlled (83.1%). even so, 14% of participants were unsure whether the virus would be controlled and a smaller number of participants disagreed that it would be successfully controlled (2.1%). the attitude of successfully controlling covid-19 was significantly associated with age group, region and occupation. knowledge scores of those who were unsure were also significantly lower than those who agreed that the virus would be successfully controlled [ table 4 ]. for the second attitude question, the majority of participants had confidence that malaysia would be able to win the battle against covid-19 (95.9%), while a small percentage did not have that confidence (3.3%). the confidence that malaysia would be able to win the battle against covid-19 was associated with age group and occupation. no significant difference was found between the two confidence groups in terms of knowledge score. the third attitude question asked whether the participant agreed that the malaysian government was handling the covid-19 health crisis well. a large percentage of participants agreed with this statement (89.9%). rates of disagreement and uncertainty were at 3.8% and 5.4% respectively. agreement that the malaysian government was performing well in handling the covid-19 crisis was significantly associated with gender, age group, region and occupation. knowledge scores were also significantly different between those who agreed that the government was doing a good job at handling the crisis and those who were unsure. practices toward covid-19 were measured using three questions enquiring on: 1) avoidance of crowded places, 2) wearing of face masks; and 3) practising proper hand hygiene in the week before the movement control order (mco) was implemented in malaysia [fig 2] . for the first question, 83.4% of participants reported that they were avoiding crowded places in the week before the mco was implemented. the other 16.6% did not avoid crowded places. in examining the differences between demographic groups, it was found that there were significant associations between age group, income category and avoidance of crowded places. younger people and those earning below rm3,000 monthly were more avoidant of crowded places in the week before the mco. there were also significant differences in knowledge scores between those who did and did not avoid crowded places. those with higher knowledge scores did not avoid crowded places in the week before the mco [ table 5 ]. the second question asked participants if they were wearing face masks when outside the home during the week before the mco began. more than half of the participants reported wearing a face mask when going out in public (51.2%). the remaining participants did not wear a mask (48.8%). the wearing of face masks was found to be significantly associated with gender, age group, region, occupation and income group. males, people between the ages of 18 and 49, students and those earning less than rm3,000 per month showed higher percentages in wearing face masks when leaving the house. people living in the central region, those above the age of 50 and people with an income over rm12,000 per month were less likely to wear a face mask. the results also show that there was a significant difference between knowledge scores in terms of mask-wearing. those with higher knowledge scores did not wear masks when leaving the house in the week before the mco was enforced. lastly, when enquired about hand hygiene, a majority of participants reported that they practised proper hand hygiene by frequently washing their hands and using hand sanitiser (87.8%). even so, there was still a percentage of participants who were not practising proper hand hygiene in the week before the implementation of the mco (12.2%). there were significant associations found between proper hand hygiene and gender, age group, region and occupation. females, those living in the central region, people between the ages 18 to 29 and students were more likely to practise good hand hygiene. those above 50, public knowledge, attitudes and practices towards covid-19 in malaysia residents in the eastern region and retirees were among the highest percentage of participants who had not practised good hand hygiene in the week before the mco. covid-19 is a relatively new virus that has had devastating effects within the short time since it was first detected in december 2019. to date, there has been limited published data on population knowledge, attitudes and practices toward covid-19, specifically in malaysia. the novelty of this disease, along with its uncertainties, make it critical for health authorities to plan appropriate strategies to prepare and manage the public. it is therefore of utmost importance that the knowledge, attitudes and practices of the population be studied to guide these efforts. the average knowledge score of malaysians in regards to covid-19 was moderate at 10.5 ±1.4 with an overall correct rate of 80.5%. even so, correct rates of covid-19 knowledge ranged widely indicating that while some participants had high levels of knowledge on the disease, others did not. malaysians above the age of 50 held higher knowledge scores, possibly due to a higher risk perception of contraction and complications from the disease [16] . on the other hand, those with low monthly income scored among the lowest knowledge scores. this may indicate limited access to credible and timely information about the virus. this variation in levels of knowledge may be reflective of the current covid-19 information landscape in the country. although health authorities have been consistently disseminating covid-19 information since the disease was first detected in malaysia, there has also been a surge in false and inaccurate information [17, 18] . the overload of information may have caused confusion and difficulty ascertaining correct information. several studies conducted in other asian countries have indicated high levels of covid-19 knowledge among the general population [13] and healthcare workers [19] . differences in measurement and scoring systems do not make it possible for accurate comparisons of knowledge levels across these studies. the present study found that a large majority of participants held positive attitudes toward overcoming covid-19. roughly eight out of ten participants agreed that covid-19 would be successfully controlled. similarly, approximately nine out of ten participants were confident that malaysia would be able to win its battle against the virus and that the malaysian government was handling the health crisis very well. high levels of positive attitudes were also detected in the kap study conducted in china [13] . the authors attributed the positive attitudes to the drastic measures taken by the chinese government in mitigating the spread of the virus. in malaysia, the swift action taken by the malaysian government in enforcing the mco may have also contributed to these positive attitudes. although the percentage of participants reporting uncertainty toward success in fighting against covid-19 was low (14%), this was significantly associated with lower knowledge scores. these results reinforce conclusions from previous studies associating higher levels of knowledge with higher confidence and positive attitudes in health crises [20] . positive attitudes were higher among those working in the public sector. this group showed the highest confidence that covid-19 would be successfully controlled, that malaysia would win the battle against the disease and that the malaysian government were handling the health crisis well. this may be due to work duties or affiliations directly related to government efforts toward the containment of the virus. in the current study, most participants reported taking precautions such as avoiding crowded places and practising proper hand hygiene in the week before the mco was implemented. this indicates a general willingness for participants to make behavioural changes in the face of the covid-19 pandemic. even so, people above the age of 50 and people who earned more than rm12,000 per month were among those who did not avoid crowded places in the week before the mco. the week before the implementation of the mco coincided with the school holidays in malaysia, a common season for family holidays and gatherings such as weddings. those above the age of 50 were also more likely to attend daily religious congregations like praying at the mosque. cultural norms may have been influential in the decision to attend these gatherings despite the health risks, especially among the older generation. previous research has also shown that those with higher income were less willing to comply with health recommendations [21] perceived less fear and more control in pandemic situations [22] . interestingly, enquiry into the wearing of face masks garnered a mixed response. almost half of the participants indicated that they did not wear a face mask when leaving the home in the week before the mco. there are two possible explanations for this behaviour in the malaysian context. firstly, the use of face masks is not a norm in malaysian society. it is uncommon for the typical malaysian to wear a face mask when ill. the emergence of covid-19 caused an increase in demand for medical face masks (and hand sanitiser) in the country and supplies were short [23] . the scarcity of face masks meant that many regular members of the public were unable to obtain them. the shortage of personal protective equipment was not limited to malaysia. it had become a global problem due to increased demand in response to covid-19 [24] . secondly, the ministry of health malaysia has been adamant that medical face masks should only be worn by those who are showing symptoms of covid-19 or similar illnesses. this was to ensure ample supplies of personal protective equipment for medical workers on the frontline. even so, mixed messages had been communicated to the public by different authoritative bodies on the use of face masks. it is possible that the lack of supply and the confusion caused by the mixed messages led to the divided response on the wearing of face masks when out in public. admittedly, covid-19 has been a teething public health problem around the world. scientists are working diligently to explore different vaccines and treatment options. social table 5 . demographic characteristics of participants and practices toward covid-19 (n = 4850). in the week before the mco, did you avoid going to crowded places such as weddings? in the week before the mco, did you wear a face mask when leaving the home? in the week before the mco, did you practice proper hand hygiene by frequently washing your hands and using hand sanitizer? scientists, especially those in public health and health communication, are working to identify the levels of knowledge, attitudes and practices on covid-19 among the public as to design cost-effective public health campaigns and education programmes. the current survey, in fact, exposes the need for more comprehensive education programmes with focus on consistency of information from the government and related authorities. covid-19 education efforts should take a proactive approach and focus on dispelling misinformation in the form of conflicting opinions, old wives' tales and incorrect information. due to the levels of media and telecommunication usage in malaysian society [25] [26] [27] and evidence from prior research [28] , authorities would benefit from utilising both mainstream and social media in disseminating these messages. sampling for the study was conducted via a convenience sample through the networks of the researchers and disseminated through different social media platforms (whatsapp, facebook, twitter etc.). as a result, there is a possibility of bias as underprivileged populations may not have been able to participate in the study. additionally, when compared to current population statistics in malaysia [29] , the sample of the study were over-representative of women, people below the age of 50, and those employed in the public sector. therefore, there are limitations to the representativeness of the findings. a more systematic, inclusive sampling method is warranted to improve representativeness and generalisability of the findings. the second limitation is related to the kap instrument used in this study. the instrument was adapted from a survey that had been previously tested and utilised in china [13] . even so, a more thorough assessment of instrument validity and reliability would have produced a more robust instrument. due to the limited time and urgency of the survey, attitudes and practices were measured with only one item each. in addition to this, possible factors contributing to knowledge, attitude and practice such as risk perceptions and health literacy [30, 31] were not measured in this study. these would have been a useful addition in understanding the knowledge, attitudes and practices of covid-19 in malaysia. a further limitation of the present study is the possibility of participants giving socially desirable responses. as this study used self-reported data, it is possible that participants may have answered attitude and practice questions positively based on what they perceive to be expected of them [32]. in summary, the present study was able to provide a comprehensive examination of the knowledge, attitudes and practices of malaysians toward covid-19. the findings suggest that malaysians have an acceptable level of knowledge on covid-19 and are generally positive in their outlook on overcoming the pandemic. even so, consistent messaging from the government and/ or health authorities are key to aid public knowledge and understanding of covid-19. additionally, some categories of the population may benefit from specific health education programs to raise covid-19 knowledge and improve practices. supporting information s1 data. (sav) other" includes occupations such as manual labour and contract/ part-time work references 1. world health organization. coronavirus disease 2019 (covid-19): situation report-91 malaysia confirms first cases of coronavirus infection malaysia records first two covid-19 deaths; cases soar to 673 health ministry detects five 'generations' of covid-19 cases linked to tabligh cluster. the star clinical features and short-term outcomes of 102 patients with corona virus disease 2019 in wuhan, china covid-19 and communication planning for health emergencies fear and stigma: the epidemic within the sars outbreak an analysis on reasons of sars-induced psychological panic among students a quick guide to survey research sample size: a rough guide gainesville: university of florida internet users survey 2018: statistical brief number twenty-three knowledge, attitudes and practices towards covid-19 among chinese residents during the rapid rise period of the covid-19 outbreak: a quick online cross-sectional survey back-translation for cross-cultural research mansour net al. global patterns in students' views of science and interest in science letter to the editor: clinical features and short-term outcomes of 18 patients with corona virus disease 2019 in intensive care unit it's a national effort to fight fake news during covid-19, mco. malay mail five more probed for spreading fake news on covid-19. new straits times knowledge and attitude toward covid-19 among healthcare workers at district 2 hospital knowledge and attitudes of medical staff in chinese psychiatric hospitals regarding covid-19 knowledge, attitudes, and practices about influenza illness and vaccination: a cross-sectional survey in two south african communities. influenza other respir viruses lay perceptions of the pandemic influenza threat demand for face masks, hand sanitisers soars shortage of personal protective equipment endangering health workers worldwide the use of social media among adolescents in klang valley the relevance of radio broadcasts towards z generation teenagers in malaysia a qualitative exploration of the misconceptions, knowledge gaps and constructs of leptospirosis among rural and urban communities in malaysia social statistics bulletin: malaysia 2019. putrajaya: dosm influence of health literacy on health information seeking behavior among students in public university establishing the hls-m-q18 short version of the european health literacy survey questionnaire for the malaysian context we would like to express our appreciation to the faculty of social sciences and humanities, universiti kebangsaan malaysia, particularly to associate professor dr. kadaruddin aiyub and noraznita anor basri for their assistance and a sincere thank you to all members of the malaysian public who participated in the survey. resources: emma mohamad. key: cord-012889-dtil5xeo authors: holzer, joshua title: the effect of copartisan justice ministers on human rights in presidential democracies date: 2020-09-02 journal: plos one doi: 10.1371/journal.pone.0234938 sha: doc_id: 12889 cord_uid: dtil5xeo a body of literature suggests that states with independent courts are more likely to protect human rights. a recent article challenges this notion by arguing that when both the president and his or her justice minister share the same party—i.e., they are copartisans—that state is less likely to protect human rights, as justice ministers may value their loyalty to the president over their duty to enforce court decisions. in this article, i estimate government respect for human rights accounting for both copartisan justice ministers and an independent judiciary. in the end, i find copartisan justice ministers to be negatively associated with high government respect for human rights, even after controlling for judicial independence. many constitutions already seek to ensure an independent judiciary, but if copartisan justice ministers increase the likelihood that governments repress, then perhaps constitutional engineers should also consider options that would reduce the likelihood that both the president and his or her justice minister share the same party. in a recent article, i warn "of the human rights repercussions when one party controls both the presidency and the ministry of justice" ([1]: 8), as i find that when both the president and his or her justice minister share the same party-i.e., they are copartisans-that state is less likely to protect human rights. according to the literature, "independent courts constrain human rights abuses" ([2]: 1); however, this literature does not account for any negative effect resulting from copartisan justice ministers, despite the role justice ministers play in administering court decisions. similarly, while examining the influence of copartisan justice ministers on government respect for human rights, my recent article fails to account for the potentially positive effect of an independent judiciary. the purpose of this article is to address these omissions, and towards that end, i have re-estimated my previous model-this time controlling for independent courts. in the end, i still find copartisan justice ministers to have negative repercussions on the likelihood that a government respects human rights. de jure, de facto. . .does it matter? scholars have long emphasized the important role that an independent judiciary plays not only in the functioning of a legal system [3] , but also in the implementation of human rights a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 legislation [4] . an inverse relationship between judicial independence and human rights violations is now widely known to exist [5, 6] . regimes that protect human rights are generally found to be associated with independent courts [7] , and human rights are expected to be least protected in the countries where the courts are most dependent [8, 9] . raz ([10] : 185) argues that "[t]he rules concerning the independence of the judiciary. . .are designed to guarantee that they will be free from extraneous pressures and independent of all authority save that of the law," and as such, the rules "are, therefore, essential for the preservation of the rule of law." it is important to note, however, that there are conceptual and operational difficulties when defining 'the rule of law' [11] . defining and measuring the 'independence' of the judiciary can be just as murky [12] . typically, the concept of judicial independence is broken down into two subcategories: de jure independence and de facto independence. de jure independence refers to the formal protections for insulating the judiciary, such as the method of appointing judges and the security of their tenure. some even argue that judicial independence also requires budgetary control [13] , for "[a]s long as the congress controls the purse strings, members of the supreme court will not be totally autonomous" ( [14] : 146). these safeguards are often outlined within a country's constitution, in an attempt to prevent other branches from tilting the balance of power in their favor. while cross [5] finds significant linkages between de jure independence and improvements in human rights practices, keith [15] argues that such linkages are not nearly as encouraging as one would hope. in her examination of "the impact of constitutional provisions. . .on state abuse of the right to personal integrity," she finds that "none of the constitutional provisions for individual freedoms is statistically significant" ( [15] : 111). such results add to a growing chorus that argues that de jure "judicial independence alone does not ensure that the courts will play an active role in protecting rights" ( [16] : 29). many scholars now argue that the disconnect between de jure judicial independence and the expectation of better human rights practices can be explained by the difference between de jure and de facto independence. while de jure independence refers only to formally written protections, de facto independence "is often defined as the extent to which a court may adjudicate free from institutional controls, incentives, and impediments imposed or intimidated by force, money, or extralegal, corrupt methods" ( [17] : 286). as melton and ginsburg ([18] : 209) point out, "clever politicians can exploit the absence of any single de jure protection for judicial independence." "this," they argue, "helps us understand why, over the last 25 years, increases in de jure protection of judicial independence have not been followed by similar increases in de facto judicial independence" ( [18] : 210). so whereas de jure independence has not been reliably found to equate to high government respect for human rights, howard and carey ( [17] : 290) suggest that de facto independence "leads to greater political rights." the challenge, however, is that de facto judicial independence "is not directly observable" ( [19] : 107). in an attempt to address this, linzer and staton [20] have constructed a new latent de facto measure of judicial independence that draws upon previous direct and indirect indicators by keith [21] , howard and carey [17] , feld and voigt [22] , gwartney et al. [23] , cingranelli and richards [24] , marshall and jaggers [25] , johnson, souva, and smith [26] , and the political risks services group [27] . using linzer and staton's [20] measure, both crabtree and fariss [2] and crabtree and nelson [28] have found de facto judicial independence to be positively associated with high government respect for human rights. unfortunatley, independent courts only matters to the extent that the executive branch is willing to enforce that court's decision; neither crabtree and fariss [2] nor crabtree and nelson [28] take this into account. as such, i argue that in addition to focusing on the de facto independence of a court, scholars should also be looking at the 'independence' of the executive authority charged with implementing court decisions. as many have noted, government policies implemented as a result of court decisions are not always a perfect translation of those decisions [29] [30] [31] [32] . this is because "[j]udicial decisions are not self-executing" ( [33] : 27). in the federalist papers, hamilton ([34] : 239) argues that "the independence of the judges may be an essential safeguard against the effects of occasional ill humors in the society," yet hamilton ([34] : 236) also readily acknowledges that courts have "neither force nor will, but merely judgment; and must ultimately depend upon the aid of the executive arm even for the efficacy of its judgments." building upon this idea, spriggs ([35] : 1122-1123) suggests that the "imperfect translation of court opinions into agency policies" is deliberate, as sitting governments "are strategic and implement supreme court opinions based upon their expectations of the costs and benefits." typically, the ministry of justice is charged with administering justice, and the justice minister is legally bound to follow court decisions and implement them accordingly. however, while courts are ideally independent, justice ministers are usually selected by-and answerable to-a partisan executive. as such, justice ministers are often appointed as a result of party loyalty. can justice really be blind when wearing partisan blinders? in my recent article [1] , i find that when both the president and his or her justice minister share the same party, that state is less likely to protect human rights. why is this the case? first, a copartisan justice minister could aid a president that wishes to repress rights. second, a repressive justice minister may find themselves less constrained by a copartisan president. take, for example, the case of united states (us) president andrew jackson and attorney general roger b. taney. in 1831, worcester and several other non-native american missionaries were indicted in the us state of georgia under an 1830 act of the georgia legislature meant "to prevent white persons from residing within that part of the chartered limits of georgia occupied by the cherokee indians. . .and to enforce the laws of the state within the aforesaid territory" ( [36] : 539). worcester was ultimately convicted and "condemned to hard labour for four years in the penitentiary of georgia" ( [36] : 536). in an opinion delivered by chief justice john marshall, the us supreme court held that the georgia act, under which worcester had been prosecuted, violated the us constitution, treaties between the us and the cherokee nation, and "an act [of the us congress] to regulate trade and intercourse with the indian tribes" ( [36] : 540). in essence, the georgia act interfered with the federal government's authority and was deemed unconstitutional. upon hearing of marshall's decision, president jackson-who sided with georgia in this confrontation and opposed native american sovereignty-is alleged to have said: "john marshall has made his decision: now let him enforce it!" ( [37] : 384). today, jackson is derided for his role in tramping the rights of native americans and supporting slavery [38] , yet jackson was aided in these endeavors by a loyal attorney general, "political hack, roger b. taney" ( [39] : 554). at this time it is important to note that in some countries, government 'ministries' are called departments (e.g. the us department of justice). furthermore, note that in some countries, the 'justice minister' is called the minister of justice, the minister for justice, the secretary of justice, or in some cases (like in the us) the attorney general. for the sake of simplicity, i simply use the generic term 'justice minister' throughout this article, except for this us anecdote. continuing on, in his pulitzer-prize winning study on jackson, schlesinger ([40] : 65) refers to taney as jackson's "spearhead of radicalism." in addition to fighting native american sovereignty and supporting slavery, finkelman ([41] : 92) notes that "as attorney general, taney had believed that free blacks had no rights that any government had to protect." calhoun ([42] : 612) adds that "taney-as he sometimes did-found in the law what he wanted to find." ultimately, taney's "obsessive fidelity to jacksonian tenets" was later rewarded, when jackson appointed him to the supreme court as chief justice, upon chief justice marshall's death ( [39] : 584). taney was promoted to replace the man that he had been complicit in helping jackson ignore! as illustrated in this particular case, a copartisan 'justice minister' worked as an enabler to a president determined to trample rights (even in opposition to an independent supreme court). what about when the justice minister is not a copartisan? first, a repressive justice minister that is not a copartisan may find themselves more constrained by the president, and therefore less likely (and/or able) to repress rights. second, a copartisan justice minister could 'sound the alarm' in the face of a president that is repressing (or wishes to repress) rights. for instance, if a president were to propose a new policy (or the renewal of a current policy) that the justice minister disagreed with, the justice minister could attempt to effectively 'veto' said policy by privately-or publicly-threatening to withdraw their support of the president. the justice minister could also attempt to exercise autonomy over how (or whether) the president's policy is implemented. the president, if even aware of the insubordination, would then be in the awkward position of having to sack the justice minister or ask for their resignation, which often has a political cost. take, for example, the case of indonesian president abdurrahman wahid and justice minister yusril mahendra. in 1999, wahid became indonesia's first elected president after the end of suharto's 30-year rule. wahid's presidency started with high hopes, but soon after coming to power "[h]is erratic and amateur approach to administration" ( [43] : 273) led to a series of missteps. to make matters worse, "[h]e also interfered with legal processes" ( [44] : 95), and as a result, "[h]ostile parliamentarians accused him of a broad array of misdemeanours and failings" ( [43] : 273). in response, wahid "threatened to jail his political opponents," thereby revealing "an authoritarian tendency" that would become increasingly apparently throughout his presidency ( [44] : 95). by 2001, indonesian's parliament began considering the impeachment of wahid, and to preempt this, "wahid threatened to 'freeze' the parliament" ( [44] : 95). in response, "[t]he supreme court ruled that wahid lacked the constitutional power to dissolve the legislature" ([45]: 31), which is effectively what a 'freeze' would have been. during this time, "justice minister yusril ihza mahendra was sacked for making repeated public calls for the president to resign" ( [46] : 354). mahendra-who was not a copartisan-had previously suggested that wahid resign during a private cabinet meeting earlier in the year ( [46] : 350). when the suggestion was rebuffed, mahendra began speaking out in public. mahendra's sacking by wahid eventually led to a ministerial revolt, at which point most of those that had once supported wahid's "presidential bid turned against him" ([47]: 124). ultimately wahid was successfully impeached. if mahendra had been a copartisan, he might have been less willing to support wahid's removal; instead, mahendra put his job as justice minister in jeopardy by leading the charge against wahid. as illustrated in this particular case, a justice minister that was not a copartisan chose loyalty to the law over loyalty to an authoritarian president. to review, some argue that an independent judiciary promotes high government respect for human rights; however, these studies fail to account for any negative effect resulting from copartisan justice ministers. in a recent article, i argue that country-years where both the president and the justice minister are of the same party are more likely to be associated with poor human rights practices; however, my study fails to account for the potentially positive impact of an independent judiciary. this leads me to the primary question this article seeks to address: are copartisan justice ministers still negatively associated with high government respect for human rights when one controls for the independence of the judiciary? as my just stated research question suggestions, the purpose of this article is to ascertain whether copartisan justice ministers are still negatively associated with high government respect for human rights when one controls for the independence of the judiciary. this idea seeks to extend the findings of my previous article [1] , which first examined the relationship between copartisan justice ministers and government respect for human rights in presidential democracies. in order to build upon my previous empirical findings, my sample is modeled of that which was used in my previous study-i.e. presidential democracies. while many early human rights studies have utilized either the polity or freedom house measures to identify which regimes are democratic, this is problematic as both polity and freedom house classify regimes-in part-based on their respect for human rights, and therefore, using either measure would partially control for my outcome variable [2] . as such, following the trend of more recent human rights studies (e.g. [1, 2, 28, 48] ), i utilize cheibub, gandhi, and vreeland's democracy versus dictatorship dataset [49] to determine what constitutes a 'democracy'. per their classification, a regime is considered to be a 'democracy' when the president is elected, the legislature is elected, there is more than one party competing in elections, and an alternation under identical electoral rules has taken place ( [49] : 69). consistent with my previous study [1] , i utilize bormann and golder's democratic electoral systems around the world dataset [50] to further narrow my pool of democracies to specifically 'presidential' democracies. why only presidential systems? practically, i am limited to those particular country-years until such as time as more data becomes available. theoretically, this makes sense anyway, as there are important differences between presidential and parliamentary systems, particularly with regard to judicial matters. for instance, samuels and shugart ( [51] : 255) note that in presidential systems, "the separation of powers creates presidentialized parties." presidents "often engineer a de facto reversal of the party-executive principal-agent relationship" found in parliamentary systems, and as such, presidents "come to control parties for their own purposes" ( [51] : 251). in other words, presidents may have more influence over copartisans than prime ministers. furthermore, moreno, crisp, and shugart ( [52] : 89) argue that de facto judicial independence can only exist in democratic "presidential systems, where there is no notion of parliamentary sovereignty," as only in these countries do "courts typically have authority that overlaps with the elected bodies and may even overturn acts of the elected bodies on constitutional grounds." in essence, this line of argument asserts that parliamentary sovereignty is often incompatible with judicial review, which itself is a requirement for de facto judicial independence. "courts are independent to the extent that they are not accountable to the political bodies" ([52]: 90). when courts are not empowered to overturn laws-as is the case in many parliamentary systems-such courts are "essentially another part of the bureaucracy," subservient to those doing the legislating ([52]: 89). this echoes hamilton's ( [34] : 237) fear that if "the legislative body are themselves the constitutional judges of their own powers," this could "enable the representatives of the people to substitute their will to that of their constituents." however, "[i]n presidential systems. . . [c] ourts that have the authority to veto legislative acts are thus another actor in the process" ([52]: 90). in such systems, an independent judiciary serves as "an intermediate body between the people and the legislature, in order, among other things, to keep the latter within the limits assigned to their authority" ( [34] : 237). given data limitations, given the impact that 'presidentialized' parties may have on the relationship between presidents and copartisans, and given the importance of judicial review (which is not present in all parliamentary systems) to de facto judicial independence, both practically and theoretically it seems appropriate to limit my sample to presidential systems. as a result, however, note that my findings cannot likely be generalized beyond presidential democracies. with that said, although my sample size is limited to only about a decade, i see no reason why my findings cannot also be applicable to presidential democracies in earlier or more recent decades. for my primary dependent variable, i utilize the cingranelli-richards physical integrity rights index (ciri index), which is an additive nine-point index of the following four ordinal indicators of government respect for physical integrity rights: the right to be protected from political imprisonment, torture, disappearance, and extrajudicial killing [53] . ciri index scores ranges from '0' (no respect for any of the four physical integrity rights) to '8' (full respect for all of them). i also utilize an alternative dependent variable: the political terror scale (pts), which -like the ciri index-is coded using data from the us department of state's country reports on human rights practices and amnesty international's annual report [54] . while ciri index scores are determined by individually evaluating instances of political imprisonment, torture, disappearance, and extrajudicial killing (then adding together all four constituent scores), pts scores are determined by collectively evaluating the range of the population effected by instances of political imprisonment, torture, disappearance, and extrajudicial killing. countries are designated a level ranging from '1' to '5': '1' indicates that the state is under a secure rule of law, people are not imprisoned for their views, torture is rare or exceptional, and political murders are extremely rare; '2' indicates that there is a limited amount of imprisonment for nonviolent political activity, torture is exceptional, and political murder is rare; '3' indicates that there is extensive political imprisonment, and political murders are common; '4' indicates that disappearances, torture, and political murders are all common, though state terror only affects those who interest themselves in politics; finally, '5', which indicates that statesanctioned repression has been extended to the whole population, and state leaders place no limits on the means or thoroughness with which they pursue personal or ideological goals [55] . while ciri index scores aim to reflect "actual government practices" ( [24] : 406), pts scores aims to reflect "the 'range' of violence committed" ( [55] : 368). despite these differences, however, "pts and ciri essentially measure the same thing" ([56]: 88). as such, scholars that employ one of these indices as their dependent variable often report analogous estimations using the alternate index as a robustness check (e.g. [48, [57] [58] [59] ). in order to aid in comparability to the ciri index, i have followed the literature's trend by inverting pts scores, such that higher scores now indicate greater government respect for physical integrity rights. despite their widespread use, in recent years ciri index scores and pts scores have had to contend with some amount of criticism. for instance, clark and sikkink ( [60] : 567) argue that "[b]ecause of increased quality and quantity of information," both ciri index and pts scores "skew toward worse scores in later years." building upon this idea, fariss ([61] : 300) argues that over time, "the u.s. state department and amnesty international look harder for abuse, look in more places for abuse, and classify more acts as abuse." he calls "this process. . .a changing standard of accountability" ([61]: 297). to address this, he has created new latent scores for human rights which he claims are "unbiased estimates of repression" ( [61] : 297). he then uses his scores to "show that respect for human rights has improved over time," which seemingly supports the argument that ciri index scores and pts scores are skewed downward as a result of more critical us state department and amnesty international reporting in recent years. as a result, some scholars have abandoned the use ciri index scores and/or pts scores in favor fariss' latent human rights scores. in response, haschke and gibney ([56]: 89-90) note that clark and sikkink [60] and fariss [61] "repeatedly assert that the [us state department and amnesty international] annual reports are now longer and more detailed-and presumably more accurate-than they had been in the past," yet "the evidence that they marshal in this regard is selective and does not constitute compelling proof." in their analysis, haschke and gibney ([56] : 99) show that "any measurable bias is actually in the opposite direction of" what clark and sikkink and fariss claim. in another response, cingraneli and filippov ( [62] : 1088) note that they "have identified serious problems" with the alleged 'unbiased estimates' created by fariss. using fariss' own computer code they find that the upward trend identified by fariss "depended almost entirely on the inclusion of the mass killing indicators" ( [62] : 1086). they caution that "[t]hose who use fariss's scores should be aware that there is a strong built-in correlation between mass killings and those scores," and as such, "evaluators should remember that the trends in fariss's scores for capable and democratic countries are affected by frequencies of mass killing events in failed and authoritarian states" ( [62] : 1088). for this reason alone, it would be inappropriate for me to use fariss' scores as my sample is limited to democracies. however, cingraneli and filippov ( [62] : 1089) also caution "that latent scores should not be used as dependent variables in conventional regression analysis because doing so could produce inconsistent or severely biased estimates." to further drive the point home, they argue that in creating his scores, fariss "comes close to data manufacture, and scores fabricated in this way should not be used in place of carefully collected and consistently coded real human rights data" such as the ciri index and pts ([63]: 274). an even more recent article [64] further calls into question the validity of fariss' scores. in short, although some scholars have followed fariss in utilizing his new scores, these estimates are not without controversy and-given recent critiques-they are not the most appropriate for my particular research question. on final alternative i would like to briefly touch on is the latest version of the new varieties of democracy (v-dem) dataset [65] . given that the dataset does include a few human rights measures, some [66] has suggested that it could be used as an alternative to ciri index scores and pts scores. while this new version shows promise, v-dem's physical violence index (which would be the measure most comparable to ciri index scores and pts scores) is only an aggregation of their extrajudicial killings and torture indicators. note that unlike ciri index scores and pts scores, v-dem's physical violence index does not account for either disappearances or political imprisonment. in other words, ciri index scores and pts scores are much more similar to one another than either are to v-dem's physical violence index, and as such, i argue that if my primary dependent variable is ciri index scores, pts scores would be a more appropriate check for robustness than v-dem scores. to conclude, i would like to point out that neither ciri index scores nor pts scores are relics of some bygone era of human rights research; in 2020 alone, there has already been several human rights-related papers published that have opted to use these scores in place of (and despite the creation of) the new fariss and v-dem measures (e.g. [1, [67] [68] [69] ). my primary independent variable-copartisan justice minister-is pulled from my previous article [1] . this variable is coded as '1' for every country-year where the president and his or her justice minister are in the same party (and '0' when they are not in the same party). further details of this variable's construction can be found in my previous article. for my de facto judicial independence variable, i follow others [2, 28] in using linzer and staton's [20] measure. however, i use their most recently updated version [70] , and for the sake of brevity, i simply refer to this variable as judicial independence. beyond the copartisan justice minister and judicial independence variables, i use the exact same control variables as my previous article; these variables include a measure of constitutional checks on the executive, the level of civil conflict, population size, gross domestic product (gdp) per capita, and finally a control for the previous year's level of government repression. note that since poe and tate [71] , the inclusion of such variables has become common practice in models that estimate government repression, and as a result of the significance of their article (and later extensions [72, 73] ) many studies within the human rights literature now generically refer to similar model specifications as "poe and tate model[s]" ( [74] : 663). at this point, i will elaborate on some specifics of my particular poe and tate model. note that summary statistics are presented in table 1 . to begin, many poe and tate models include polity iv's xconst variable as a control (e.g. [74] [75] [76] ); this variable takes into account "the extent of institutional constraints on the decision-making powers of the chief executive" ([77]: 62). however, doyle and elgie ( [78] : 732) point out that while "some studies have estimated the effect of variation in the level of constraints on the executive in the system of checks and balances by operationalizing polity's xconst variable," this measure does not fully account for all the variation between various presidential systems. in other words, since polity iv's xconst measure was designed to estimate executive constraints across all countries, it does not fully capture the variation within specifically presidential democracies, which is the focus of this article. as such, i follow recent articles [79, 80] in using doyle and elgie's ( [78] : 734) quantification of "the constitutional power of presidents," which i have inverted and refer to as presidential constraints. the inclusion of this variable as a control is particularly important to this article's research question as "judges are less likely to invalidate legislation or governmental actions in countries possessing strong presidents" ( [81] : 435). continuing on, poe and tate ( [71] : 859) note that "regimes are more coercive when they are involved in civil conflict." the measure of civil conflict i utilize is drawn from the uppsala conflict data program [82] ; the use of this particular dataset is common within the human rights literature (e.g. [75, 79, 80, 83] ). my civil conflict variable is coded as '0' for each countryyear with less than 25 battle-related deaths, '1' for each country-year where there were between 25 and 999 battle-related deaths, and finally '2' for each country-year where there were more than 999 battle-related deaths. to conclude, poe and tate models typically include measures for population size (which seems to be negatively associated with high government respect for human rights), gdp per capita (which seems to be positively associate with high government respect for human rights), as well as a lag of the dependent variable (as a country's previous year's level of government repression seems to influence the following year's level of government repression). first, both my population size and gdp per capita measures are from the world bank [84] , and-following the literature-both of these measures have been logged to correct their distributional nature [85] . second, although poe and tate [71] include a lagged dependent variable, "[b] ecause the ciri [and pts] variables are non-linear, a simple lagged dependent variable is less appropriate because it does not efficiently model the autoregressive trend in the data" ([86]: 12). as such, i follow hafner-burton ( [87] : 615) in including a series of "binary indicators measuring a state's previous level of repression. . .to account for dependence across the categories of the dependent variable over time"; this particular technique has since been used in many other human rights-related studies (e.g. [48, 73, 86, 88] ). because these binary "variables are included for diagnostic rather than substantive reasons and given the large number of them," like others, i do not report their estimates when discussing my findings ( [73] : 298). however, note that they are all statistically significant, as can be seen using my replication files. at this point, i would like to remind the reader that my dependent variables (i.e. ciri index scores and pts scores) are both ordinal, and therefore, an ordinary least squares (ols) regression (which is linear) would lead to biased inferences. long and freese ( [10] : 309) warn that while "it is tempting to analyze ordinal outcomes with the linear regression model (lrm). . .an ordinal dependent variable violates the assumptions of lrm, which can lead to incorrect conclusions." instead, they argue that "[w]ith ordinal outcomes, it is much better to use models that avoid the assumption that the distances between categories are equal" ( [10] : 309). following long and freese's advice, the most appropriate way to test my research question would be to utilize ordered probit regression. indeed within the broader human rights literature, models that similarly estimate ciri index scores and/or pts scores commonly rely upon the use of ordered probit regression (e.g. [1, 48, 57, 58, 68, 75, 88] ). table 2 shows that my copartisan justice minister variable is found to be negatively associated with both high ciri index scores and high pts scores; these relationships are statistically significant at least at the 95% level. notably, both models control for judicial independence, which (as expected) is found to be positively associated with both high ciri index scores and high pts scores. consistent with previous human rights scholarship, across both models all remaining statistically significant control variables have signs pointing in the expected direction. for instance, presidential constraints and (logged) gdp per capita are both found to be positively associated with both high ciri index scores and high pts scores, while civil conflict and (logged) population size are both found to to be negatively associated with both high ciri index scores and high pts scores. in fig 1, i illustrate predicted probabilities (and 95% confidence intervals) for ciri index scores and pts scores when the justice minister goes from copartisan to not a copartisan. note that these probabilities are based upon each control variables' mean (or mode in the case of civil conflict, as it is categorical); this data can be seen in table 1 . these probabilities were estimated using the clarify software package [89] and help to provide substantive meaning to the models reported in table 2 . starting with the top-half of fig 1, you can see that for the 'average' state in my dataset (i.e. a state whose parameters match the mean/mode of my control variables), if that state were to have a justice minister that is not a copartisan (instead of a copartisan), the predicted probability of having a ciri index score of 7 or 8 goes up, while the predicted probability of having a ciri index score of 4, 5, or 6 goes down. recall that higher ciri index scores indicate greater government respect for human rights, and the scale tops out at 8. while ciri index scores can go down to 0, those scores are not listed in fig 1 as the predicted probability of such scores (as well as any change in such scores) is effectively zero. this means that for the 'average' state, switching from a copartisan justice minister to a justice minister that is not a copartisan increases the probability of high government respect for human rights, and decreases the probability of low government respect for human rights. the bottom-half of fig 1 follows this same trend; for the 'average' state, if that state were to have a justice minister that is not a copartisan (instead of a copartisan), the predicted probability of having a pts score of 4 or 5 goes up, while the predicted probability of having a pts score of 3 goes down. any change in the as previously mentioned, the ciri index is an additive index. ciri index scores are calculated by adding together ciri political imprisonment scores, ciri torture scores, ciri disappearance scores, and ciri extrajudicial killing scores; as can be seen in table 1 , each of these four ordinal indicators of government respect for specific physical integrity rights range from '0' (which indicates poor respect) to '2' (which indicates full respect). unlike pts, one of the benefits of the ciri index is that these these four ordinal indicators can be disaggregated. in fig 2, i illustrate predicted probabilities (and 95% confidence intervals) for disaggregated ciri index scores when the justice minister goes from copartisan to not a copartisan. as with fig 1, these probabilities correspond to the 'average' state in my dataset (i.e. a state whose parameters match the mean/mode of my control variables). starting with the left-side of fig 2, note that all the gray ciri political imprisonment and ciri disappearance confidence intervals overlap with zero, which indicates that the probability changes are not statistically significant (at the 95% level). this suggests that country-years with copartisan justice ministers are no more-and no less-likely to have different ciri political imprisonment scores or ciri disappearance scores versus country-years where the justice ministers is not a copartisan. moving to the right-side of fig 2, note that the predicted probability of having a ciri torture score or ciri extrajudicial killing score of 2 goes up when the justice minister is not a copartisan. furthermore note that these gray confidence intervals do not overlap with zero. this suggests that country-years where the justice ministers is not a copartisan are more likely than country-years where the justice minister is a copartisan to have the highest ciri torture score and ciri extrajudicial killing score. in other words, in comparison to when the justice minister and president share the same party, it appears that when the justice minister is not in the same party as the president, such governments are less likely to condone the use of torture and extrajudical killings. according to hamilton ([34] : 236), "the judiciary is beyond comparison the weakest" branch of government, as it "has no influence over either the sword or the purse. . .and can take no active resolution whatever." in light of hamilton, i argue that in addition to focusing on whether courts are independent of undue influence, scholars should also be looking at whether justice ministers are independent of undue influence, given the role justice ministers play in administering court decisions. a body of literature suggests "that independent courts constrain human rights abuses" ([2]: 1), yet these studies do not take into account copartisan justice ministers. similarly, while examining the influence of copartisan justice ministers on government respect for human rights, my recent article [1] fails to account for the impact of an independent judiciary. with this article, i have sought to remedy these omissions by re-estimating my previous model while controlling for the independence of the judiciary, and in the end, i still find copartisan justice ministers to be negatively associated with high government respect for human rights. at this point, many constitutions already include provisions to ensure de jure judicial independence, yet-to my knowledge-no constitution prohibits the justice minister from being a copartisan of the president. volcansek and lockhart ( [90] : 33) argue that "constitutional drafters should focus less on institutional elements related to judicial independence. . .[r]ather, those crafting constitutions intended to protect human rights should look to dispersing government power." 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dataset user's manual. center for systemic peace maximizing the reliability of cross-national measures of presidential power measuring presidential centrism and its effect on repression: does ideology influence whether democratic governments respect human rights? reevaluating the presidential runoff rule: does a provision promote the protection of human rights? the relationship between independence and judicial review in post-communist courts armed conflicts nationalism and human rights: a replication and extension world development indicators data analysis for politics and policy women and economic statecraft: the negative impact international economic sanctions visit on women trading human rights: how preferential trade agreements influence government repression the perils of plurality rule and the major(itarian) effect of cabinet composition on human rights in presidential democracies clarify: software for interpreting and presenting statistical results explaining support for human rights protections: a judicial role i would like to thank stephen gilpin at state technical college of missouri for his assistance with figs 1 and 2. key: cord-001207-yjaiybwf authors: sachsenröder, jana; twardziok, sven o.; scheuch, matthias; johne, reimar title: the general composition of the faecal virome of pigs depends on age, but not on feeding with a probiotic bacterium date: 2014-02-19 journal: plos one doi: 10.1371/journal.pone.0088888 sha: doc_id: 1207 cord_uid: yjaiybwf background: the pig faecal virome, which comprises the community of viruses present in pig faeces, is complex and consists of pig viruses, bacteriophages, transiently passaged plant viruses and other minor virus species. only little is known about factors influencing its general composition. here, the effect of the probiotic bacterium enterococcus faecium (e. faecium) ncimb 10415 on the pig faecal virome composition was analysed in a pig feeding trial with sows and their piglets, which received either the probiotic bacterium or not. results: from 8 pooled faecal samples derived from the feeding trial, dna and rna virus particles were prepared and subjected to process-controlled next generation sequencing resulting in 390,650 sequence reads. in average, 14% of the reads showed significant sequence identities to known viruses. the percentage of detected mammalian virus sequences was highest (55–77%) in the samples of the youngest piglets and lowest (8–10%) in the samples of the sows. in contrast, the percentage of bacteriophage sequences increased from 22–44% in the youngest piglets to approximately 90% in the sows. the dominating mammalian viruses differed remarkably among 12 day-old piglets (kobuvirus), 54 day-old piglets (boca-, dependoand pig stool-associated small circular dna virus [pigscv]) and the sows (pigscv, circovirus and “circovirus-like” viruses cb-a and rw-a). in addition, the shannon index, which reflects the diversity of sequences present in a sample, was generally higher for the sows as compared to the piglets. no consistent differences in the virome composition could be identified between the viromes of the probiotic bacterium-treated group and the control group. conclusion: the analysis indicates that the pig faecal virome shows a high variability and that its general composition is mainly dependent on the age of the pigs. changes caused by feeding with the probiotic bacterium e. faecium could not be demonstrated using the applied metagenomics method. the viral community present in faeces is composed of a variety of viruses originating from the gut tissue, from intestinal microorganisms or from ingested food. the totality of viruses present in faeces has also been frequently designated as the faecal virome [1, 2] . the functions of the faecal virome are supposed to be manifold, which include roles for the viruses as pathogens, regulators of bacterial growth, gene-transfer vehicles and modulators of the immune system [3] [4] [5] [6] . early insights into the composition of the human faecal virome were provided by random cloning strategies [7, 8] . later on, the availability of deep sequencing methods lead to more comprehensive analyses of faecal viromes [1, 9] , including the development of processcontrolled techniques enabling comparison of different analyses [10] . the composition of the faecal virome of pigs has been studied recently [2, 10, 11] . although samples derived from different continents had been analysed in these studies, the general composition was found to be similar. the majority of the detected virus sequences belonged to bacteriophages and pig viruses. only a few sequences belonged to plant viruses as well as other viruses. most of the bacteriophage sequences originated from viruses belonging to the families siphoviridae, microviridae and myoviridae [10] . the most abundant porcine viruses were kobuvirus, rotavirus, pig stool-associated small circular dna virus (pigscv), astrovirus, sapovirus and enterovirus b. most of them represent widely distributed enteric viruses of pigs [2, 10, 11] . whereas rotaviruses are well-known pathogens of piglets, which may lead to diarrhoea [12, 13, 14] , the clinical importance of the other viruses is a subject of controversy [10, [15] [16] [17] [18] [19] [20] [21] [22] . only little is known about the stability and dynamics of the faecal virome under different conditions. for the human faecal virome, reyes et al. [9] investigated the intra-and interpersonal variation by analysing faeces of monozygotic twins and their mothers at different time-points. by this, it was found that the viromes were unique to the individuals regardless of their degree of genetic relatedness. minot et al. [1] analysed the inter-individual variation of the human faecal virome and its dynamic response to diet. it was shown that the largest source of variance among the viromes was caused by interpersonal variations and not by the diet. a high interpersonal diversity of gut bacteriophages was also described in two humans which were monitored over a 2.5 year period [23] . in another study, a much lower diversity of the virus community was found in infants as compared to adults [24] . although this study has been conducted by cloning followed by classical sequencing, mathematical modelling of the derived sequence data indicated that the virome of adults was composed of approximately 2000 genotypes as compared to only 8 genotypes in one week-old infants. the observed beneficial effects of probiotic bacteria on enteric virus infections have been recently reviewed by colbere-garapin et al. [6] . this includes clinical studies showing beneficial effects of probiotic bacteria in children with rotavirus-caused diarrhoea [25] [26] [27] . feeding with probiotic microorganisms such as lactobacillus rhamnosus gg, saccharomyces boulardii or bifidobacterium lactis resulted in milder clinical symptoms, reduced virus shedding and shortened the duration of diarrhoea in children [27] [28] [29] . in pigs, enterococcus (e.) faecium ncimb 10415 is a commonly used probiotic bacterium [35, 37] . it has been shown recently, that feeding of pigs with this probiotic bacterium affected shedding of enteric viruses dependent on the virus species [30] . especially, rotavirus was shed later and in lower amounts in the group of piglets that received e. faecium ncimb 10415 as compared to the control group. the specific mechanisms responsible for this effect are not known so far. however, changes in the mucosal and systemic immunity due to feeding with e. faecium ncimb 10415 have been described [31] [32] [33] [34] . in addition, direct interactions of this bacterium with enteric virus particles have been observed in in vitro studies [35] . however, it is not known so far, whether probiotic bacteria can also influence the general composition of the faecal virome, e.g. by changing the composition of the bacterial community, which represents the host population for bacteriophages, or by direct interactions with specific viruses. the primary aim of the presented study was to analyze the effect of the probiotic bacterium e. faecium ncimb 10415 on the general composition of the faecal virome in pigs. faecal samples from sows and their piglets experimentally fed with or without the probiotic bacterium were analyzed using a process-controlled deep sequencing method. the populations of the detected virus sequences were compared between the feeding groups as well as the age groups and general insights into the stability and dynamics of the pig faecal virome under different age-related and feeding conditions were generated. the animal experiment (pig feeding trial) was approved by the local state office of occupational health and technical safety ''landesamt für gesundheit und soziales berlin'' (lageso reg. nr. 0347/09). animal experiment and sampling scheme the design of the pig feeding trial has been described in detail by martin et al. [36] and is schematically shown in figure 1 . briefly, sows and their piglets received either no probiotic bacterium or approximately 5610 6 cfu/g e. faecium ncimb 10415, which was fed with their diet starting at 28 days ante partum. the sows received a commercial diet (una-hakra, hamburg, germany). additional feeding of the piglets started at 12 days of age with a non-medicated non-commercial pre-starter diet [36] . after weaning at 28 days of age, they were fed with a non-commercial mash starter diet [37] . the homogenous distribution of the probiotic in feed has been previously demonstrated by a colony hybridization assay [37] . the faeces of 6 sows of each group were sampled at day 28 ante partum (before e. faecium diet) and at day 14 post partum. faeces of their piglets (6 from each group) were collected at day 12 and at day 54 of age (end of the experiment). piglets were euthanized at the end of the experiment by intracardial injection of a lethal dose of tetracaine hydrochloride, mebezonium iodide and embutramide (t61, intervet, unterschleißheim, germany). although the whole experiment included a larger number of animals [30, 37] , only faeces of piglets were analyzed, for which the faeces of their mother sows had also been analysed. the faeces of each group and time-point were pooled. the samples were stored at 220uc until analysis. dna was extracted from faecal samples and subsequently analyzed by real-time pcr specific for e. faecium ncimb 10415 as previously described [38] . the standards used for quantification were prepared from negative pig faecal samples spiked with known amounts of cultured e. faecium ncimb 10415 cells as described by starke et al. [37] results are expressed as log of cell numbers per g faeces. three different bacteriophages (m13, ms2, t4) were grown, titrated and used as process controls for monitoring the efficiency of the virome analysis procedure as described previously [10] . a total of 10 ml of the bacteriophage mixture containing approximately 10 5 plaque-forming units of each bacteriophage was added per 1 g faeces. virus particles were purified from the faecal samples by a combination of tangential flow filtration (tff) and caesium chloride (cscl) density gradient ultracentrifugation, and concentrated by centrifugal filtration and tff as described [10, 39] . a total of 17 g of pooled faecal samples from the sows were used. due to limited availability of faeces in the youngest age group, 1.7 g of the pooled faecal samples from the piglets was used. the samples were spiked with test-phages and resuspended 1:10 in smbuffer (100 mm nacl, 8 mm mgso 4 , 50 mm tris-hcl ph 7.5) by magnetic stirring. the sample was centrifuged at 10,000 g for 30 min in order to remove the large particulate debris and the supernatant was collected. the procedure was repeated by centrifugation for 3 hours at 10,000 g to remove smaller particular structures. afterwards, a first tff was performed using a 0.22 mm filter (pall corporation, middleton; ma, usa) to remove bacterial and eukaryotic cells and debris. the remaining filtrate was subjected to a second tff with a 50 kda filter (pall corporation, middleton; ma, usa) in order to concentrate the virus particles. the viral preparations were further concentrated by centrifugation through vivaspin 50,000 mwco concentrators (sartorius stedim biotech gmbh, götting, germany) at 3,500 g resulting in a final volume of 36 ml. the preparation was divided into two fractions of 18 ml, which were added separately onto preformed stepwise caesium chloride (cscl) density gradients with density layers of 1.7, 1.5, 1.35 and 1.2 g/ml (5 ml each) and ultracentrifuged at 20,000 g for 14 hours at 10uc. the 1.35-1.5 g/ml layers were collected from the gradients using a syringe. to eliminate free dna present in the virus concentrate, an aliquot of 1 ml cscl purified virus solution was treated with 50 units dnase i (2,000 u/mg, bovine pancreas grad ii; roche diagnostics gmbh, mannheim, germany) for 45 min at 37uc, followed by heat inactivation for 10 min at 65uc. thereafter, dna and rna were extracted simultaneously using nuclisens magnetic extraction (biomerieux, nürtingen, germany). the extracted nucleic acids (75 ng per reaction) were randomly primed for cdna synthesis using the transplexh complete whole transcriptom amplification kit (wta2, sigma-aldrich, st. louis, mo, usa) according to the protocol recommended by the supplier; however, the annealing temperature was decreased to 40uc (2 cycles) and 45uc (2 cycles) in order to enable the simultaneous amplification of dna and rna [10] . aliquots of 75 ml each were removed from the wta2 reaction at different cycle numbers, purified and size-selected using mobispin s-400 columns (mobitec, göttingen, germany). the dna concentration was measured from the preparations using a nanodrop spectrometer (analytic jena, jena, germany) and the preparation derived from a minimum of amplification cycles with a dna concentration above 50 ng/ml was chosen for deep sequencing. a total of 1 mg dna was used for deep sequencing on a 1/8 plate of the gs-flx sequencer 454 titanium (gs titanium sv empcr kit (lib-l) v2; gs titanium picotiterplate kit 70675; gs titanium sequencing kit xlr70t; life sciences, roche, branford, usa) according to the manufacturer's protocol. the raw sequence data have been submitted to the sequence read archive (sra) at genbank as bioproject prjna232620 with sra accession numbers srp034937 (srx396427-srx396434). primary sequence analysis was performed in two steps: identification of all virus species included in the samples and analysis of species abundances regarding selected sets of species. raw sequence reads were subjected to primer/adaptor trimming using seqman (dnastar, lasergene, usa) and selection for a minimum length of 50 nt. in parallel, all primary reads were subjected to de novo contig assemble using the 454 newbler assembler [40] software, with criteria of 90% minimum overlap identity and a minimum overlap length of 40 nt. in order to create a local database containing all virus sequences with significant homologies to the sequence reads, homology searches for all primary reads were performed with blastx [41] against the non-redundant nucleotide database of ncbi [42] . in parallel, homology searches for the contigs were performed with clc main workbench 6.2 [43] against the viral genome nonredundant reference sequence nucleotide database [44] and additional sequences from recently discovered viruses using the tblastx algorithms [39] . from both approaches, all blast results with an e-value , = 10 24 were selected and used for creation of the local sequence database. using this database, abundances of species were calculated. for bray curtis dissimilarity (see below), specific subsets, which consisted of mammalian viruses, bacteriophages or enterococcus phages, were used. in all cases, trimmed reads were mapped against the sequences of the local database to calculate species abundances with the readmapper bowtie 2 2.0.5 [45] . thereafter, numbers of mapped reads were corrected for multiple read assignments. the reads of the bacteriophages used as process control were subtracted from the number of the virus reads in subsequent analyses. shannon index [46] was calculated to compare the diversity of the species identified by primary reads. the shannon index is maximal for a sample with a balanced species distribution and it has a low value for a sample with an uneven species distribution; e.g. if some single species are a highly abundant. the maximal value depends on the number of species in a sample. bray curtis dissimilarity [47] was calculated for pairwise comparisons of samples and dendrograms were constructed by hierarchical clustering with the average linkage method. this analysis included counting of detected species and determination of their taxonomy, which was also used to determine the virus hosts (bacteria, vertebrates, plants etc.). a total of 8 pooled faecal samples were derived from sows and their piglets from an experimental feeding trial with the probiotic bacterium e. faecium ncimb 10415. four of the samples were derived from animals receiving the probiotic bacterium (group p) and four samples originated from the control group that did not receive probiotics (group c). a detailed scheme of the feeding trial is presented in figure 1 . the presence of e. faecium ncimb 10415 in the faeces of sows and their piglets was analyzed by quantitative real-time pcr. as shown in table 1 , e. faecium ncimb 10415 was not detected in the samples of the control group. also, no e. faecium ncimb was detectable in the sample taken from the sows of the probiotic group immediately at the beginning of the experiment (28 day ante partum) as well as in the samples from the 12 day-old piglets of this group, which were still suckled at this time-point. considerable amounts of e. faecium ncimb 10415 were demonstrated in the samples taken from the sows at 14 day post partum and from the 54 day-old piglets, both belonging to the probiotic group. the 8 pooled faecal samples were analyzed by processcontrolled deep sequencing. in total, 390,650 reads were generated, with an average of 48,831 reads per sample. the efficiency of the whole method was monitored by a process control consisting of three bacteriophages, which were added in constant amounts to the samples. in all samples the three test-phages could be detected representing 0.9% to 3.4% of all generated reads. the numbers of totally generated reads, test-phage reads and other virus reads is summarized for the individual samples in table 2 . the number of the test-phage reads in relation to the total virus reads ranged from 3.8% to 24.4% and is shown in figure 2 . using a cut-off e-value of , = 10 24 for the blastx homology search of the sequences, the viral reads could be assigned to 36 known virus families. only 10 of these families dominated the faecal viromes representing more than 1% in at least one of the samples. as shown in figure 3 and table s1 , the composition of the faecal viromes according to virus families varied remarkably among the samples. a grouping of the virus families according to the taxonomic kingdom of hosts of the contained viruses revealed that the main detected groups were mammalian viruses (colored red in fig. 3 ) and bacteriophages (colored blue in fig. 3 ). in contrast, viruses from other hosts (insects, plants, amphibians and fungi) ranked together between 0.2% and 3.4% only. a closer inspection of the proportion of the read numbers from mammalian viruses compared to that from bacteriophages revealed marked differences between the samples derived from different age groups. in the youngest piglet group (12 days of age), the main fraction consisted of mammalian viruses with 55% (control group) and 77% (probiotic group). in the group of 54 dayold piglets, the proportion of mammalian viruses was reduced to 24% (control group) and 30% (probiotic group). within the four groups of the sows (one year old) the amount of mammalian viruses ranged from 8% to 12%. in contrast to those findings, the proportion of bacteriophages increased with the age of the pigs. in the 12 day-old piglets, 44% (control group) and 22% (probiotic group) of the reads relate to bacteriophages. the percentage of bacteriophages increases in the 54 day-old piglets to 68% (control group) and 72% (probiotic group), whereas approximately 90% of the virus reads belong to bacteriophages in the four sow groups. no differences in the general composition of virus families or the respective hosts were evident, when the probiotic group was compared to the control group. in overall, sequences with significant identities to 524 known bacteriophage species were detected. the bacteriophages most abundant in the eight samples are shown in figure 4 and table s2 . in all cases, the bacteriophage population is dominated by 9 to 16 species, which represent 76-90% of all bacteriophage reads of the respective sample. the most abundant phages as identified by the highest number of reads with sequence identities to known bacteriophage genomes are lactococcus phage 1706, dragonflyassociated microphage 1, chlamydia phages 4, chp1 and chp2, bdellovibrio phage phimh2k, spiroplasma phage 4, microvirus ca82 as well as enterococcous phages efap-1 and efrm31. a comparison between the bacteriophage populations of the specific samples indicated that many of the most abundant bacteriophage species are present in all samples, however, with different relative frequency. apart from that, the composition of the faecal virome with regard to bacteriophage species was relatively variable between the samples and every sample contained its unique collection of bacteriophages. no consistent differences between age groups and feeding groups were obvious when the abundance of bacteriophage species was analyzed. interestingly, the sample taken at day 14 post partum from the enterococcus faecium-fed sows contained relatively high amounts of the enterococcous phages efap-1 (10.8%) and efrm31 (7.0%), which were only sporadically detected in the other groups (0.02% to 0.6%). by analysis of all virus reads excluding the bacteriophages, sequences with significant identities to 205 known virus species were detected. among that, 92.9% of the sequences belonged to viruses infecting mammalian animals. this percentage of mammalian viruses decreased with age, with an average of 99.1% in the 12 day-old piglet group, 91.8% in the 54 day-old piglet group and 79.0% in the sows. the relative percentage of the most abundant mammalian virus genera in the eight samples is shown in figure 5 and dant mammalian viruses. among the ''circovirus-like'' viruses, sequences with highest identities to the viruses cb-a and rw-a were most often detected. no consistent differences were obvious between the group fed with the probiotic bacterium and the control group. however, a relatively high proportion of mamastrovirus sequences (16.3%) was detected in the sample derived from the 54 day-old piglets; while this virus was not detected in the other groups (less than 2 reads per sample). the calculation of the shannon index was used to assess the diversity of the sequences detected in the specific samples (table 3) . generally, comparison of shannon indexes between piglets and sows indicated that the diversity increased with age. when only the bacteriophage sequences were analysed, the average shannon index of the piglet groups was 1.3 and that of the sows 1.7. for the mammalian virus sequences, the average shannon index for the piglets was 1.9 and that for the sows 3.0. calculation of bray-curtis distances determined similarities of the faecal viromes detected in the specific samples. figure 6 illustrates clustering of samples on the basis of bray curtis distance calculated by abundances of species-specific subsets. as shown in the dendrogram based on abundances of all virus species (including bacteriophages), a grouping according to age is evident (fig. 6a) . the two samples taken from the 12 day-old piglets cluster closely together and are separated from the other samples. among these other samples, the two samples taken from the 54 day-old piglets form one separate branch, whereas the four samples of the sows are all contained in the other branch. a branching according to the feeding group is not evident from this dendrogram. the same grouping is evident, when only the mammalian virus sequences are analysed (fig. 6b) . the analysis of the bacteriophages shows no evident grouping according to age or feeding group (fig. 6c) . also, no grouping according to age or feeding group was evident, when only the sequences of the enterococcus phages were used for the analysis (fig. 6d ). comparisons of the composition of intestinal viromes from different samples have been only scarcely described so far. a few studies investigated individual differences of faecal viromes and the influence of diet and age in humans [1, 9, 24] , whereas similar studies on faecal viromes of pigs are almost missing. technical problems with the use of deep sequencing methods for comparative virome analyses may represent one major problem in this context [10, 48] . here, we tried to overcome some of these problems by using a process-controlled deep sequencing approach [10] . by this, the efficiency of the analysis can be estimated for each sample, thereby enabling identification of major differences due to different performances of the method. we could show here, that all types of the bacteriophages used as process control could be detected in the final data sets of all samples. this indicates that the method has a reproducible performance and the generated data can generally be used for comparative analyses. however, the detection rates of the process control bacteriophages varied between the samples from 0.9% to 3.4%. as the detection rate of the bacteriophages is -besides technical factors -also dependent on the amount of viruses initially present in the analyzed sample, improved deep sequencing methods enabling quantitative analyses should be developed in future for comparative virome investigations. in the eight investigated pooled samples, the overall composition of the virus community was similar to that described for other pig faecal viromes [2, 10, 11] . the two major virus groups were bacteriophages and porcine viruses, whereas plant viruses and viruses with other hosts were only rarely detected. however, large differences were detected in the ratio between bacteriophages and mammalian viruses in the distinct samples; in addition, the diversity of detected virus species varied between the analysed viromes. these data indicate that the faecal virome of pigs is not uniform and static, but shows a remarkable variability. for human faecal viromes, a high variability even between the analyzed individuals has been described [1, 9] . as only pooled faecal samples have been analyzed in the study presented here, future investigations are necessary in order to assess the inter-individual variability of faecal viromes of pigs. the most obvious factor influencing the composition of the pig faecal virome was the age. the percentage of porcine viruses, which comprised the most abundant group in the youngest piglet samples, decreased dramatically in the samples from the older pigs. in parallel, the percentage of bacteriophages as well as the diversity of detected virus species increased by age. interestingly, porcine kobuvirus and pig scv, which both had been discovered only recently [10, 49] , were among the most frequently detected viruses in the faecal viromes of the youngest and oldest age groups, respectively. this underlines the importance of unfocused detection systems in order to get an undistorted picture of the abundance of viruses in a sample. as all samples analyzed here originated from an experimental feeding trial, the detected virus composition may vary in comparison to field-origin samples. however, the age-specific effect was strong and very similar in both analyzed groups, which were held completely separate during the whole period of the experiment. the differences may be explained by an age-related susceptibility to specific virus infections as well as by an increasing immunity to porcine viruses due to completed virus infections with higher age. in addition, the progressive diversification of the bacterial enteric flora, which serves as the host pool for bacteriophages, would also explain the increasing diversification of the virus flora by age. an increasing diversity of the virus species in faeces of humans has already been described [24] . in contrast to the age-related effect, no clear differences could be detected in the composition of the faecal viromes according to feeding with the probiotic bacterium e. faecium ncimb 10415. a relatively high percentage of enterococcus phages in the sample derived from an e. faecium-feeded group may indicate multiplication of the phage due to the application of its host. this explanation may indicate that a larger amount of the probiotic bacteria may be lysed by the bacteriophages and are therefore not available for the probiotic therapy; however, this interpretation is questionable as the bacteriophages were only found in one of the samples. also, a relative high proportion of astrovirus was found in one of the samples of the control group. interestingly, real-time rt-pcr analyses of samples derived from the same feeding trial confirmed the presence of astrovirus exclusively in the control group [30] . however, the same study indicated later shedding of rotaviruses with lower amounts in the probiotic group as compared to the control group, which was not detected by our virome analysis. a closer inspection of the data shows that up to 10 7 astroviruses per gram faeces were present in the samples, whereas only up to 10 5 rotaviruses per gram were detected [30] . therefore, a lower sensitivity of the virome analysis method may explain the discrepancies and still deeper sequencings may be necessary in future to detect more subtle changes in the faecal virome composition due to probiotic feeding. the composition of the identified bacteriophage species in the different samples revealed no consistent pattern. however, most of the detected sequence reads showed only moderate identities to the known bacteriophage sequences present in the database. therefore, it has to be considered that the majority of the detected sequences belong to so far unknown bacteriophages and that the identified bacteriophage species represent only their next relatives. a definitive assignment of a host to these sequences is therefore currently not possible. the quality of the database with regard to genomic sequences of bacteriophages is crucial for virome analyses. for example, the high proportion of the detected lactococcus phage 1706 may reflect the disproportionately high abundance of those phage sequences in the database as a consequence of intensive research on these bacteriophages, which are problematic agents for the diary cheese product industry [50] . in contrast, for another highly abundant bacteriophage, the dragonfly-associated microphage, the specific bacterial host is still unknown [51] . an increase of annotated bacteriophage sequences in the databases is therefore a prerequisite for studies on the interactions between bacteriophages and their host bacteria in the gut in future. alternatively, a deeper sequencing may enable the assembly of complete bacteriophage genome sequences from the metagenomic data set. by this, an assignment to their hosts was possible by identification of inserted host-related sequences as recently described [3] . in summary the data show a high variability of the pig faecal virome. most obvious are age-related differences in the proportion between pig viruses and bacteriophages as well as an increasing diversification of virus species by age. consistent differences due to probiotic feeding could not be identified by our metagenomic analysis. the results of comparative pig virome analyses may help to understand the complex interactions between viruses, bacteria and the pig within the intestinal tract. future research should focus on the optimization of the method in order to increase its sensitivity and on the improvement of the sequence databases, especially regarding annotated bacteriophage sequences. it will be interesting to apply the optimized techniques to analyse the diversity of faecal viromes in individuals and to identify further factors like geographical origin or disease-related changes influencing its composition. table s1 relative abundance of virus families in the analyzed faecal viromes. the table shows the number of reads with sequence identities to a certain virus family in relation to all virus reads (in %). families showing an abundance of less than 1% in a distinct faecal virome are subsumed to other families. families which were not classified by the international committee on taxonomy of viruses (ictv) are subsumed to not assigned. the group p received the probiotic bacterium e. faecium ncimb 10415 (p) and the group c (c) received no probiotic. table s2 relative abundance of bacteriophage species among all bacteriophages detected in the analyzed faecal viromes. the table shows the number of reads with sequence identities to a certain bacteriophage species in relation to all bacteriophage reads (in %). bacteriophage species showing an abundance of less than 1% in a distinct faecal virome are subsumed (,1%). the group p received the probiotic bacterium e. faecium ncimb 10415 (p) and the group c (c) received no probiotic. (pdf ) table s3 relative abundance of mammalian virus genera among all animal viruses detected in the analyzed faecal viromes. the table shows the number of reads with sequence identities to a certain mammalian virus genus in relation to all animal virus reads (in %). mammalian viruses, which are so far not assigned to a certain genus, are indicated in apostrophes. mammalian virus genera showing an abundance of less than 1% in a distinct faecal virome are subsumed (genera , 1%). viruses from non-mammalian hosts are subsumed to non mammalian viruses. the group p received the probiotic bacterium e. faecium ncimb 10415 (p) and the group c (c) received no probiotic. (pdf) the human gut virome: inter-individual variation and dynamic response to diet the fecal virome of pigs on a high-density farm antibiotic treatment expands the resistance reservoir and ecological network of the phage metagenome genome signature-based dissection of human gut metagenomes to extract subliminal viral sequences analysis of a viral metagenomic library from 200 m depth in monterey bay, california constructed by direct shotgun cloning prevention and treatment of enteric viral infections: possible benefits of probiotic bacteria. microbes infect rna viral community in human feces: prevalence of plant pathogenic viruses metagenomic analyses of an uncultured viral community from human feces viruses in the faecal microbiota of monozygotic twins and their mothers simultaneous identification of dna and rna viruses present in pig faeces using process-controlled deep sequencing diversity of viruses detected by deep sequencing in pigs from a common background different virulence of porcine and porcine-like bovine rotavirus strains with genetically nearly identical genomes in piglets and calves diversity and zoonotic potential of rotaviruses in swine and cattle across europe zoonotic aspects of rotaviruses occurrence and investigation of enteric viral infections in pigs with diarrhea in china molecular detection of porcine kobuviruses in italian swine identification and molecular characterization of porcine kobuvirus in u. s. swine. virus genes a divergent clade of circular single-stranded dna viruses from pig feces emerging and re-emerging swine viruses complete genome analysis of porcine enterovirus b isolated in korea characterization of a novel porcine enterovirus in domestic pig in hungary astrovirus infections in humans and animals -molecular biology, genetic diversity, and interspecies transmissions rapid evolution of the human gut virome viral diversity and dynamics in an infant gut current level of consensus on probiotic science-report of an expert meeting-london. gut microbes meta-analysis: the effects of lactobacillus rhamnosus gg supplementation for the prevention of healthcare-associated diarrhoea in children probiotics for prevention and treatment of diarrhea the comparition of the efficacy of two different probiotics in rotavirus gastroenteritis in children dose-dependent effect of lactobacillus rhamnosus on quantitative reduction of faecal rotavirus shedding in children feeding of the probiotic bacterium enterococcus faecium ncimb 10415 differentially affects shedding of enteric viruses in pigs enterococcus faecium ncimb 10415 supplementation affects intestinal immuneassociated gene expression in post-weaning piglets. manuscript number: accepted but unpublished so far impact of the probiotic bacteria enterococcus faecium ncimb 10415 (sf68) and bacillus cereus var. toyoi ncimb 40112 on the development of serum igg and faecal iga of sows and their piglets influence of a probiotic enterococcus faecium strain on development of the immune system of sows and piglets modulatory effects of lactobacillus salivarius on intestinal mucosal immunity of piglets antiviral effects of a probiotic enterococcus faecium strain against transmissible gastroenteritis coronavirus influence of age and enterococcus faecium ncimb 10415 on development of small intestinal digestive physiology in piglets individual response of mother sows to a probiotic e. faecium strain lead to different microbiota composition in their offspring accepted but unpublished so far effect of the probiotic enterococcus faecium ncimb10415 on cell numbers of total enterococcus spp., e. faecium and e. faecalis in the intestine of piglets laboratory procedures to generate viral metagenomes assembly algorithms for next-generation sequencing data basic local alignment search tool langmead b, salzberg sl (2012) fast gapped-read alignment with bowtie 2 a mathematical theory of communication an ordination of the upland forest communities of southern wisconsin metagenomic study of the viruses of african straw-coloured fruit bats: detection of a chiropteran poxvirus and isolation of a novel adenovirus candidate new species of kobuvirus in porcine hosts characterization of 1706, a virulent phage from lactococcus lactis with similarities to prophages from other firmicutes diverse circular ssdna viruses discovered in dragonflies (odonata: epiprocta) we thank robert pieper and his colleagues at the institute for animal nutrition (free university berlin, germany) for the coordination of the animal experiment and paul wrede (charité, berlin, germany) for helpful discussion. we also thank wilfried vahjen and ingo starke (institute for animal nutrition, free university berlin, germany) for their support in detection of e. faecium ncimb 10415 in the samples. key: cord-001898-ntqyjqqk authors: huang, chih-wei; lin, hui-chen; chou, chi-yuan; kao, wei-chuo; chou, wei-yuan; lee, hwei-jen title: lys-315 at the interfaces of diagonal subunits of δ-crystallin plays a critical role in the reversibility of folding and subunit assembly date: 2016-01-05 journal: plos one doi: 10.1371/journal.pone.0145957 sha: doc_id: 1898 cord_uid: ntqyjqqk δ-crystallin is the major structural protein in avian eye lenses and is homologous to the urea cycle enzyme argininosuccinate lyase. this protein is structurally assembled as double dimers. lys-315 is the only residue which is arranged symmetrically at the diagonal subunit interfaces to interact with each other. this study found that wild-type protein had both dimers and monomers present in 2–4 m urea whilst only monomers of the k315a mutant were observed under the same conditions, as judged by sedimentation velocity analysis. the assembly of monomeric k315a mutant was reversible in contrast to wild-type protein. molecular dynamics simulations showed that the dissociation of primary dimers is prior to the diagonal dimers in wild-type protein. these results suggest the critical role of lys-315 in stabilization of the diagonal dimer structure. guanidinium hydrochloride (gdmcl) denatured wild-type or k315a mutant protein did not fold into functional protein. however, the urea dissociated monomers of k315a mutant protein in gdmcl were reversible folding through a multiple steps mechanism as measured by tryptophan and ans fluorescence. two partly unfolded intermediates were detected in the pathway. refolding of the intermediates resulted in a conformation with greater amounts of hydrophobic regions exposed which was prone to the formation of protein aggregates. the formation of aggregates was not prevented by the addition of α-crystallin. these results highlight that the conformational status of the monomers is critical for determining whether reversible oligomerization or aggregate formation occurs. introduction δ-crystallin is a taxon-specific eye lens protein. it is the major soluble protein in the eye lens of reptiles and birds and functions as a structural protein to maintain the refraction properties of the lens [1, 2] . δ-crystallin and argininosuccinate lyase (asl) are homologous proteins. asl is in this study, the effects of this interaction on the folding pathway of wild-type and mutant proteins were investigated using urea as a denaturant. the different distributions of dissociated component from wild-type and mutant proteins, as measured by sedimentation velocity experiment, suggests the quaternary structure dissociates in different ways for wild-type and mutant proteins. structural simulation supports different dissociation processes for the two proteins. these results highlight the critical role of k315 in stabilizing the quaternary structure of δ-crystallin. the residue appears to control both the dissociation of dimers into monomers and the stability of the produced monomers. the monomers dissociated from the k315a mutant protein with a stable and compact conformation provided a good model for studying the folding mechanism of the δ-crystallin. this study reveals the conformational status of the monomers, which determines whether functional protein or aggregates are formed. the recombinant wild-type and the k315a mutant δ-crystallin or αa-crystallin plasmid were transformed and over-expressed in e. coli bl21 (de3) with induction by isopropyl-β-d-thiogalactopyranoside (iptg). proteins were purified as previously described [8, 17] . the supernatants of crude cell extracts were loaded onto a q-sepharose anion exchange column (hiprep 16/10 q xl, ge healthcare) pre-equilibrated in buffer a (50 mm tris-hcl buffer, ph 7.5) and eluted with a linear gradient of 0 to 0.4 m nacl in buffer a. recombinant protein was eluted at approximately 0.15 m nacl. the eluted protein was pooled and treated with ammonium sulfate to 1.2 m. after filtration, the sample was loaded onto a hydrophobic interaction column (source™ 15phe) pre-equilibrated in buffer a containing 10% (v/v) glycerol and 1.2 m ammonium sulfate and eluted with a linear gradient to the same buffer lacking ammonium sulfate. the retained proteins were eluted at~0.3 m ammonium sulfate. fractions were pooled and loaded onto s-300 sephacryl column (26 mm x 85 cm) pre-equilibrated in 50 mm tris-acetic acid buffer, ph 7.5. fractions were analyzed by sds-page and protein concentrations determined by the method of bradford [18] . proteins possessing a c-terminal his 6 tag were purified on ni affinity column (chelating sepharose ff, ge healthcare) then desalted using a sephadex g-25 column (26 mm x 12 cm) as previously reported [11] . equilibrium unfolding experiments were carried out by overnight incubation of δ-crystallin with various concentrations of urea or gdmcl in 50 mm tris-acetic acid buffer, ph 7.5 at 25°c. the refolding experiments were undertaken by dilution of equilibrium-denatured δ-crystallin to a series of urea or gdmcl concentrations in the same buffer. the experiments for equilibrium unfolding of monomeric δ-crystallin were undertaken by overnight incubation of δ-crystallin in 1.5 m urea at 25°c followed by addition of various concentrations of gdmcl to the solution followed by incubation for 2 hrs. the refolding experiments were carried out by dilution of the denatured δ-crystallin into a solution containing 1.5 m urea and 0.8, 3 or 5 m gdmcl in 50 mm tris-acetic acid buffer, ph 7.5. to analyze the conformation and quaternary structure of the refolded δ-crystallin, the refolding experiments were undertaken by 20-fold dilution of the denatured monomeric δ-crystallin with buffer. the transition region in fig 2b for the reversible dissociation of k315a mutant δ-crystallin in urea solution was analyzed using the following method. the unfolding process was described as a two-state transition for the conversion of the tetramer (t) into monomers (m). the thermodynamic parameters were obtained by global fitting of the tryptophan fluorescence signal to eq 1 [19] : where y is the observed signal from tryptophan fluorescence, y n and y u are the signals in the folded and unfolded states, and m f and m u are the slopes of the baselines preceding and following the transition region. δg u 0 is the free energy difference in the absence of urea and m is the variation in the free energy of unfolding with the urea concentration. reversible unfolding of monomeric k315a mutant δ-crystallin were described as a four state process (m$i 1 $i 2 $u). the unfolding curve from tryptophan fluorescence was analyzed using a four-state unfolding model described as the transition from n to u with two intermediates, i 1 and i 2 , in the process. the thermodynamic parameters were calculated by fitting the data to eq 2 [8, 20] : where y i1 and y i2 are the signals in the i 1 and i 2 states. δg 1 0 , δg 2 0 and δg 3 0 are the free energy differences in the absence of gdmcl for n to i 1 , i 1 to i 2 and i 2 to u transition, respectively, and m 1 , m 2 and m 3 are the variation in the free energy of unfolding with the gdmcl concentration. enzymatic activity assay δ-crystallin was assayed for asl activity by monitoring the absorption of fumarate at 240 nm in a perkin-elmer lambda 40 spectrophotometer. assays were performed at least in triplicate in 50 mm tris-hcl buffer (ph 7.5) with 1 mm sodium argininosuccinate as substrate. a molar absorption coefficient of 2.44 x 10 3 m -1 cm -1 was used for all calculations [21] . the fluorescence spectra were measured on a perkin-elmer ls-50 luminescence spectrophotometer at 25°c. intrinsic tryptophan fluorescence spectra of the protein were recorded with excitation wavelength set at 295 nm and using 5 nm band-width for both excitation and emission wavelength. all spectra were corrected for buffer or denaturant absorption. the average emission wavelength was utilized for data analysis [22] . the ans (1-anilinonaphthalene-8-sulfonic acid) (molecular probes; eugene, oregon) was used as probe to bind with proteins and the fluorescence was recorded from 450 to 550 nm the circular dichroism (cd) spectra were measured on a jasco j-810 spectropolarimeter at 25°c. experiments were performed in 20 mm tris-acetic acid buffer (ph 7.5) with a 1 mm path-length over a wavelength range from 200 to 250 nm. all spectra were averaged from three accumulations and were buffer corrected. the observed ellipticity (θ) (degrees) was converted into the mean residue ellipticity [θ] by the equation: [θ] = θ×m mrw /10×d×c [23] , where m mrw is the mean residue weight, d is the light path (cm), and c is the concentration of protein in g/ml. the protein sedimentation were performed on a beckman-coulter (palo alto, ca) xl-a analytical ultracentrifuge (auc) with an an50 ti rotor at 20°c with 130 000 g in standard double sectors aluminum centerpieces. the radial scans were recorded with a time interval of 7-min and a step size of 0.003 cm. all data were fitted to the continuous c(s) distribution model and a continuous size-distribution with respect to frictional ratio (f/f o ) model using the sedfit program [24, 25] . the partial specific volume of the protein, solvent density and viscosity were calculated using the sednterp program [26] . the solvent density and viscosity of varied urea concentrations were calculated and included in the fitting. electrophoresis was performed using a phastsystem (ge healthcare). the samples were subjected a phastgel 4-15% gradient gel which contains the native buffer strip (0.88 m l-alanine, 0.25 m tris/hcl ph 8.8) attached to the surface of the gel and both electrodes. the electrophoresis was carried out at 10 ma and 15°c for 400 vh. after electrophoresis, the gel was fixed in 20% (w/v) trichloroacetic acid and stained with coomassie brilliant blue r250. the turbidity of protein solution was measured using a perkinelmer lambda 40 spectrophotometer equipped with a peltier temperature control accessory to monitor the light scattering at 360 nm. all measurements were carried out at 25°c. spectra were corrected using measurements recorded for native protein in the absence of denaturants. the aggregation rate was calculated by fitting the data to the single or double exponential equation: where y t is the signal at time t, y 0 is the signal of the final state, y i is the change in amplitude, and k i is the rate constant for aggregation. data for the linear increase in signals was fitted to a linear equation using sigmaplot 10. the assay was performed by setting the excitation wavelength at 440 nm and measuring the emission spectrum from 460 to 600 nm. proteins unfolded in denaturant were diluted into 50 mm tris-acetic acid buffer (ph 7.5) to give 0.05 mg/ml of protein in the presence of thioflavin t (tht) (50 μm). the spectrum of tht alone was used as a correction for each assay. the crystal structure of goose δ-crystallin (pdb code: 1xwo) with all water molecule removed was subjected to the charmm force field and energy minimized with the smart minimization algorithm to satisfy (rms gradient~0.1 kcal/mol/å). the implicit solvent model of generalized born was included in the calculation. the structural model was used as a template to build the k315a mutant model using the build mutant protocol (accelrys discovery studio 3.5, accelrys inc.). the best scoring model conformation was selected for energy minimization. molecular dynamics (md) simulations were performed using the standard dynamics cascade protocol. the structures of wild-type and mutant δ-crystallin in the charmm force field were subjected to initial energy minimization for 500 steps by steepest descent followed by a conjugate gradient for 500 steps. the minimized models were then heated from 50 to 300 k in 2 ps md simulations followed by equilibration for 2 ps at 300 k in the absence of any structural restraint. finally, the equilibrated models were submitted to md simulations for 100 ps at nvt (constant number of particles, volume, and temperature) using the berendsen coupling algorithm [27] . wild-type and k315a mutant δ-crystallin purified to near homogeneity were used for all analysis. equilibrium unfolding experiments were conducted by incubation of proteins in buffer supplemented with different gdmcl or urea concentrations overnight. tryptophan fluorescence was used to monitor the conformational changes during the unfolding process in the microenvironment around the tryptophan residues (fig 2a and 2b ). there are two tryptophan residues, w74 and w169, in the structure of δ-crystallin. they are located in the solvent accessible domain 1 and the helix bundle of domain 2, respectively ( fig 1a and 1b) . unfolding of the wild-type and k315a mutant protein follows a multistep process in gdmcl solution and was not reversible after 20-fold dilution (fig 2a) . as shown in fig 2a, the dramatic changes in the signal for the first transition were due to subunit dissociation as reported previously, with the gdmcl concentrations for half transition ([d] 1/2 ) at 1 ± 0.05 and 0.5 ± 0.01 m for wildtype and k315a mutant protein, respectively [28] . unfolding of the two proteins in urea solution followed a three-state process, with the [d] 1/2 values in the first transition at 3.6 ± 0.1 and 1.7 ± 0.1 m urea, respectively ( fig 2b) [8, 12] . the differences in the denaturant concentration required for the half transition clearly show the potency of gdmcl when disrupting of the conformation of δ-crystallin [11] . in the presence of urea, the k315a mutant protein was dissociated and stayed in a stable conformation between 2 and 5 m urea. the conformation was more stable than for wild-type protein at urea concentrations exceeding 4 m. when the urea was removed by 20-fold dilution into buffer, the denatured mutant protein was able to refold into a conformation similar to the original state (fig 2b) . in contrast, dilution of 3.5 or 4 m urea denatured wild-type δ-crystallin or 6 m urea denatured k315a mutant protein did not result in the restoration of the properly folded conformation. these results suggest that the reversible assembly of the quaternary structure of δ-crystallin is correlated with the conformation of the dissociated monomers. the exposure of hydrophobic surfaces in the presence of urea was investigated using ans titration. a dramatic increase in fluorescence was observed at around 3 m and 1 m urea, for wild-type and the k315a mutant, respectively, indicating that subunit dissociation had occurred due to the exposure of hydrophobic areas (figs 2c and 3) . the result is consistent with the observed changes in the tryptophan micro-environment as probed by tryptophan fluorescence ( fig 2b) . the highest signals occurred around 2 and 5 m urea for the mutant and wildtype protein, respectively. dilution of the 2.5 m urea denatured k315a mutant protein resulted in restoration of a native-like conformation. however, dilution of 4 or 6 m urea denatured wildtype or mutant protein, respectively, resulted in higher levels exposure of hydrophobic areas. since α-helices are the major secondary structure in δ-crystallin, the ellipticity at 222 nm was used to analyze the structural changes induced by the presence of urea (fig 2b) . the results showed both proteins retaining relatively stable α-helical structure at concentrations of urea below 2 m. there was about 13% and 30% loss of the structure at 3 m and 4 m urea, respectively. effect of urea on the size-and-shape changes of δ-crystallin variants the size and size-and-shape changes of wild-type and k315a mutant protein in different urea concentrations were determined by sedimentation velocity measurements and using continuous c(s) distribution and c(s, f/f 0 ) distribution analysis, respectively (figs 3 and 4) (s1 and s2 figs) [29] . in the absence of urea, the two proteins appeared as one major component with sedimentation coefficients about 8.5 and 8.4 s, respectively (fig 3) . this peak corresponds to tetrameric δ-crystallin [11] . they possessed the native conformation as judged from the friction ratio (f/f 0 ) distribution profile (with the centre region below 1.5 as shown by red in the contour) (fig 4a and 4b ). at 2.5 m urea two components were observed for wild-type protein with sedimentation coefficients about 6.6 and 3.2 s, and these are assumed to be the dissociated dimeric and monomeric forms. the s values of the two peaks decreased with increasing urea concentration. the proportion of the second (monomeric) peak increased from 6% to 27% to 60% in the presence of 2.5, 3.0 to 3.5 m urea, respectively. dilution of the denatured wild-type protein at 3.5 m urea resulted in refolding into one major component with an s value of 8.1 (fig 3a) . measurement of asl activity showed that around 25% activity was recovered following refolding (table 1) . subunit dissociation was observed at about 1.2 m urea for the k315a mutant protein, with s values for the major peak of 6.8 and a shoulder at about 4.5 s (fig 3b) . a single peak with an s value of 4.5 was observed for the mutant protein at~1.5 m urea. this species is thought to be dissociated monomers possessing the native conformation as judged from the friction ratio (f/ f 0 ) distribution profile (fig 4c) . the monomers were reassembled into a similar quaternary structure of wild-type protein after removing the urea (fig 4d) . when the urea concentration was increased to 4.5 m, the s values for the single component were gradually decreased to about 2.4 ( fig 3b) . dilution of the protein denatured with 2.5 m urea resulted in refolding into one major peak with a s value of 8.1 s. the refolded protein showed around 80% asl activity was recovered (table 1) . since k315a mutant protein was reversible dissociated into stable monomers at 1.5 m urea, the conformational reversibility of monomers was investigated. monomeric k315a mutant protein that had been produced by equilibrium incubation of the native protein in 1.5 m urea was treated with various gdmcl concentrations. unfolding of monomeric k315a mutant proteins followed a multistep pathway as measured by both tryptophan and ans fluorescence ( fig 5a and 5b ). stable intermediates were identified from the unfolding curve of ans fluorescence which included the highest ans fluorescence region at around 1 m gdmcl and a steady state at~3 m gdmcl (fig 5b) . the monomeric protein had lost about 30 and 55% of its secondary structures at 1 and 3 m gdmcl, respectively. the changes in tryptophan fluorescence were dilution of monomeric k315a mutant protein denatured in 5 m gdmcl resulted in refolding to a similar conformation as the original monomeric state (fig 5a and 5b) . however, dilution of 1 and 3 m gdmcl denatured monomeric protein resulted in the increasing of the ans fluorescence, indicating higher exposure of hydrophobic area (fig 5b) . the results suggest the conformation of the partly unfolded intermediate could affect the folding reversibility of the monomeric k315a δ-crystallin mutant. k315a mutant δ-crystallin that denatured in 3 m urea was reversible folded back to the original conformation after dilution (fig 2b) . signal changes in the tryptophan fluorescence with different urea concentration were used to calculate the thermodynamic parameters by directly fitted to the two-state mechanism (eq 1) [19] . the free energy difference in the absence urea (δg 0 ) for the transition was determined to be 6.5 ± 0.3 kcal/mol ( table 2) . the changes of tryptophan fluorescence as a function of gdmcl concentration were used to calculate the thermodynamic parameters for the reversible unfolding of the monomeric k315a δ-crystallin mutant (fig 5a) . the unfolding curve was best fitted into a four-state model [8, 20] . the [gdmcl] 1/2 for the transitions from the m to i 1 , i 1 to i 2 and i 2 to u (denaturation) were about 0.6 ± 0.04 m, 2.1 ± 0.2 m and 3.9 ± 0.1 m, respectively. the thermodynamic parameters determined are summarized in table 2 . the total free energy difference (δg 0 ) for folding of monomeric k315a δ-crystallin mutant was determined to be 12.8 ± 0.7 kcal/mol. to determine the ability about refolding of denatured monomeric k315a mutant followed by reassembly to a tetrameric protein, the monomeric proteins that denatured by gdmcl were diluted with buffer to remove both of the urea and gdmcl. the results showed that the quaternary structure of k315a mutant protein was recovered from the denatured monomers instantly after dilution. the amount of the assembled protein was increased with the incubation time possessing about 60% of the activity recovered ( fig 5c and table 1 ). in contrast, without denaturant treatment, respectively. samples from refolding of 5 m gdmcl denatured wild-type and mutant protein for time period of zero or overnight are shown in lanes 3-4 and 5-6, respectively. the protein concentrations used in the assays were 0.03, 0.1 and 0.1 mg/ml for (a), (b) and (c), respectively. doi:10.1371/journal.pone.0145957.g005 is the concentration of denaturant at which the transition is half completed. the data was calculated by global fitting to eq 1. b the data were fitted to a 4-state unfolding model (eq 2) described as the transition from m to u. two intermediates, i 1 and i 2 , were assumed in the process before denatured species (u). these data are the mean ± sd of at least three independent experiments. doi:10.1371/journal.pone.0145957.t002 dilution of 5 m gdmcl denatured wild-type δ-crystallin, the quaternary structure was recovered after overnight incubation with no detectable activity ( table 1 ). the similar result was also shown for 5 m gdmcl denatured k315a mutant protein. these results suggested the different pathway for protein folding that seems due to the distinct conformation of the denatured protein caused by different means of denaturation. since refolding of partly unfolded monomeric mutant δ-crystallin resulted in a conformation with high exposure of hydrophobic regions, the occurrence of protein aggregation in the process was determined using light scattering measurement. no protein aggregation was detected upon dilution of 0.84, 3 and 5 m gdmcl denatured monomeric mutant protein into buffer containing 1.5 m urea. however, when 0.84 and 3 m gdmcl denatured monomeric mutant protein was diluted into buffer, protein aggregation was occurred. the rates for aggregate formation were calculated to be ca. 0.14 and 0.0004 min -1 , respectively (fig 6a) . when αa-crystallin, the chaperone protein, was added in a 5:1 ratio to 0.84 m gdmcl denatured monomeric mutant protein in folding buffer, no change in the rate of protein aggregation was observed. formation of aggregates by αa-crystallin alone did not occur under the same conditions. it is notable that upon dilution of 5 m gdmcl denatured monomeric mutant protein into buffer, no aggregation occurred. the structural features of the protein aggregate were investigated using the thioflavin t assay [30] . an increase in fluorescence intensity resulting from binding of tht with the aggregates over time was observed following dilution of 0.84 and 3 m gdmcl denatured monomeric mutant δ-crystallin into buffer (fig 6b) . the results suggest the possible formation of ordered aggregates. no changes in the signal were observed during the incubation period upon refolding of 5 m gdmcl denatured monomeric mutant protein. to determine the effect of the interactions provided by k315 at the diagonal subunits in disassembly of the quaternary structure, a md simulation were run for 100 ps for wild-type and mutant δ-crystallin in the absence of any structural restraints. from the simulation trajectory, the dynamic motion for disassembly of the quaternary structure and conformational changes in the tertiary structure were elucidated. the distances between the c α of d237 and r182, r302 and e330 and the two k315 or a315 residues were measured to evaluate the extent for subunit dissociation between the a-c, a-d and a-b dimeric pairs, respectively (fig 7a) . these residues interact with each other by hydrogen bonding or salt bridges at the dimeric pair interface in the native structure. these interactions are lost on replacement of k315 with a315. the results showed that the distances between d237 and r182, r302 and e330 and the two a315 residues increased linearly at a similar rate, except that the rate of change for the r302-e330 interaction in wild-type protein was about half of that for the mutant protein. in contrast, no changes in the distance between the k315 residues were observed before 80 ps. inspection of the timecourse at 20 ps in wild-type protein showed that the primary dimers of subunit a and c or b and d showed were separated, while the diagonal dimers of subunit a and b or c and d were connected by the interactions of residue k315 (fig 7b) . however, the subunits for both of the primary and diagonal dimers were separated from each other in the mutant protein. the results suggest a different disassembly process for tetrameric wild-type and mutant proteins. the quaternary structure of δ-crystallin is assembled as two pairs of closely associated dimers. previous mutagenesis studies have used to investigate the interactions at the interfaces of double dimers to elucidate their role in the stabilization of the quaternary structure [11] . the unique stable conformation from unfolding of k315a mutant protein in the presence of urea suggests that the interactions provided by this residue at the interfaces may play a critical role in stabilization of the quaternary structure of δ-crystallin. lys-315 is the only residue which is arranged symmetrically at the diagonal subunit interfaces (fig 1b) . the ε-amino group in the side-chain of this residue forms hydrogen bonds with the carbonyl groups of m312, v313 and k315 within the symmetric subunit (from pisa analysis: http://www.ebi.ac.uk/msd-srv/prpt_int/cgi-bin/piserver) (fig 1d) . substitution of this residue by alanine reduces the structural stability of the protein. the results from the previous study showed about a 9°c reduction in the thermal stability of the secondary structure and the changes in the micro-environment surrounding the tryptophan residues [11] . this mutant protein was also more susceptible to chemical denaturation, since about half of the concentration of denaturant was required to disrupt its quaternary structure compared to wild-type protein. both of the wild-type and mutant protein showed similar and not reversible denaturation in the presence of gdmcl. however, differences in the denaturation pathway were observed when urea was used as the denaturant. the results suggest that the non-covalent interactions between the intra and inter-subunits might be disrupted by the ionic character of gdmcl, with subunit dissociation and denaturation occurring simultaneously for both proteins [31] . the previous studies showing the presence of only a monomeric molten globule intermediate in the dissociation pathway of wild-type δ-crystallin in gdmcl supports this assumption [12, 13] . thus, the role of k315 in the folding process of δ-crystallin cannot be distinguished under the strong denaturant. around 2~5 m urea, the k315a mutant δ-crystallin is in a stable conformation, as judged by tryptophan fluorescence measurements (fig 2b) . subunit dissociation occurs under these conditions, resulting in the exposure of hydrophobic regions (figs 2c and 3b ). only monomers were identified in this state, as measured by sedimentation velocity analysis. the monomers that dissociated from the tetramers at~1.5 m urea possessed a nearly identical content of secondary structure as native protein and native-like conformation (figs 2b and 4c ). monomers in this conformation were able to refold and re-associate into tetramers with a similar conformation as the native protein, and possessed significant asl activity. when the urea concentration was increased to 2.5 m, the conformational changes led to further exposure of hydrophobic regions. however, following this conformational change at higher urea concentrations, protein folding was not reversible. the contrasting result found for wild-type protein was that the dissociation and denaturation were concurrent under the effect of urea (fig 3a) . in this condition, the conformation of the dissociated monomers was partly unfolded, as judged from the reduced level of α-helix (fig 2b) . the dissociated monomers seem to refold into alternative conformations then re-associate into tetramers with only part of the catalytic activity recovered (table 1 ). these results indicated that the conformation of the monomers seems related to the assembly pattern for functional protein. it is interesting that the interactions provided by k315 at the interfaces seem to affect the disassembly pathway of the quaternary structure of wild-type protein. it was found that both of the dimers and monomers were dissociated from the wild-type protein at around 2 to 4 m urea, as measured by sedimentation velocity while only monomers were dissociated from the mutant protein (fig 3) . these results suggest that the interactions provided by k315 at the interfaces seem to increase the energy barrier for dimeric dissociation. when these interactions were disrupted by mutation, the monomers could be isolated with intact conformation from the tetramers earlier than for the wild-type protein in urea. thus, the steps for dissociation and denaturation could be distinguished in the unfolding process of the k315a mutant δ-crystallin. in this study, the dynamic motion of protein structure in the process was simulated for elucidation the dissociation mechanism of δ-crystallin. from the trajectory, the tetramers were found to disassemble at the early stage. due to the interactions by k315, the interfaces between the diagonal dimers remain connected while distances increase between the subunits of the primary dimers in wild-type protein. in contrast, the subunits between both of the diagonal and primary dimers were dissociated in the k315a mutant protein (fig 7) . as δ-crystallin was assembled by two close contact dimers as transthyretin, dissociation at the two primary dimer interfaces would be expected to occur at the initial stage [4, 32] . the simulation result provides a novel pattern for dissociation of the double dimeric protein consistent with the results of sedimentation velocity experiments. a possible explanation for this earlier dissociation of the subunit from the primary dimers compared to diagonal dimers is differences in solvent accessibility. unlike the location of the interfaces between the subunits of the primary dimer, the position of k315 is buried at the interior interfaces away from solvent. thus, the interactions of k315 at the interfaces of the protein seem to elevate the stability of the quaternary structure. for wild-type protein, two diagonal dimers were presumed to disassemble initially from the tetramers followed by subunit dissociation of the diagonal dimers. however, dissociation at the interfaces of two primary dimers would assume to be the first step in the unfolding process of the k315a mutant protein (fig 8) . the detail mechanism for folding of monomeric protein remains elusive due to the monomers dissociated from wild-type δ-crystallin were in a molten globule conformation. thus, the monomers that reversible dissociated from k315a mutant δ-crystallin with a stable conformation and possessing similar level of secondary structure as the original state, and this would be a good model for studying the folding process. the monomeric protein was reversible denatured in a four-state mechanism in the presence of gdmcl and two intermediates were detected in the process (fig 5) . refolding of the partly unfolded intermediate was not reversible which in turn resulted in a conformation with more exposure of hydrophobic regions. only denatured δ-crystallin was reversible folded into the monomers with a similar conformation to the original state. it is interesting that the refolded monomers were able to reassemble into tetramer instantly upon dilutions, with substantial recovery of activity (fig 5 and table 1 ). this contrasts with the slow refolding of gdmcl denatured wild-type protein into its tetrameric form with no detectable activity. the slow recovery of the quaternary structure for the latter protein is due to an energy barrier for the appropriate assembly of double dimers, as reported previously [14] . the results suggest that the conformation of the denatured monomers which the tetrameric wild-type (t) and k315a mutant (t*) δ-crystallin was dissociated through the diagonal dimer (d) and primary dimer (d*) to monomers with partial unfolded (m) and stable (m*) conformation, respectively. monomers of the wild-type or the mutant protein were then denatured through intermediate (i or i*) into respective unfolded form (u or u*). the monomers (m) of wild-type protein in partial unfolded conformation was associated in alternative pathway to form dimers (d 1 ), and then assembled into tetramers (t 1 ) or aggregates (a). the aggregation was prevented by αa-crystallin. refolding followed by assembly of the intermediates (i 1 * and i 2 *) of the mutant protein resulted in the aggregates (a 1 and a 2 ) formation and the chaperone function of αa-crystallin was invalid in this process. doi:10.1371/journal.pone.0145957.g008 was unfolded by stepwise dissociation or directly unfolded with 5 m gdmcl could be different. the consequence of this might be that protein folding occurs via different pathways leading to the refolded monomers with different conformations to associate into native structure or alternative structures without function. protein aggregates are prone to form during the reassembly process from refolding of partly unfolded monomeric intermediates of δ-crystallin. the intermediate with the highest exposure of hydrophobic conformation is particularly prone to aggregate formation (fig 5) . aggregate formation by monomeric intermediates with defined conformations was also reported for transthyretin under mildly acidic conditions [33] . the result implies that the conformational status of the monomers influences subunit association. it is interesting that the presence of αcrystallin seems to increase the formation of aggregates from the monomeric intermediates of δ-crystallin with partly unfolded conformation, while α-crystallin alone was not affected under these conditions (fig 6) . the studies for αa peptide which induces the aggregation of soluble α-crystallin suggested that the mechanism for aggregate formation might due to the changes in the hydrophobicity of α-crystallin induced by the peptide [34, 35] . our previous study reported that aggregate formation during refolding of gdmcl denatured wild-type δ-crystallin was due to the improper assembling of double dimers and was prevented by the presence of α-crystallin [14] . in this study, the aggregate formation was caused by assembly of the refolded monomeric intermediate which facilitated the aggregate formation of α-crystallin. it thus postulated that the electrostatic interaction with the substrate seems to be key factors to determine the chaperon-like or anti-chaperone activity of the α-crystallin [34, 36] . the underlying mechanism requires further investigation. nonetheless, the result highlights the conformational status of the monomers which play a critical role in the folding pathway for reversible oligomerization or aggregate formation. in conclusion, the folding pathways of wild-type and mutant δ-crystallin are summarized as the working models shown in fig 8. this model depicts the key interactions from k315 at the interfaces of diagonal subunits not only to stabilize the quaternary structure of δ-crystallin but also to act as the energy barrier for dissociation of stable monomers. the stability might be one of the reasons for recruitment of the metabolic enzyme asl into the lens as a crystallin protein [37] . the single polypeptide chain of δ-crystallin after translation would be assumed to fold into functional tetramers as the proposed refolding pathway for k315a mutant. however, due to the interactions by k315, the tetrameric protein would be assumed to dissemble in an alternative manner to form the diagonal dimers, followed by simultaneous subunit dissociation and denaturation. monomers in this status might associate into dimers via a different pathway which then assemble slowly into a non-native tetrameric form or self-associate into aggregates which can be prevented by the presence of α-crystallin. the reversible folding of the monomers that dissociated from the k315a mutant protein with near native conformation provided the folding mechanism of the δ-crystallin. in this process, the ordered aggregate formation from re-association of the partly unfolded intermediate reveals a specific status of the protein to avoid the chaperone function of α-crystallin. this model proposes a possible mechanism about the aggregate formation for lens protein under stress effect and their interaction with α-crystallin. this study reveals the key role of monomers that dissociated from the oligomeric crystallin; their conformational status determines the levels of aggregate formation. , the panels show the raw sedimentation and theoretical fitted data (solid lines), and the fitting residual, respectively. (c) grayscale of residual bitmap. the raw sedimentation data were fitted to the continuous size distribution model using the sedfit program [25] . (tif) lens crystallins: gene recruitment and evolutionary dynamism the recruitment of crystallins: new functions precede gene duplication lens crystallins. innovation associated with changes in gene regulation the structure of avian eye lens δ-crystallin reveals a new fold for a superfamily of oligomeric enzymes human argininosuccinate lyase: a structural basis for intragenic complementation evidence for neutral and selective processes in the recruitment of enzyme-crystallins in avian lenses structural comparison of the enzymatically active and inactive forms of δ crystallin and the role of histidine 91 the effect of n-terminal truncation on double-dimer assembly of goose δ-crystallin structural studies of duck δ1 and δ2 crystallin suggest conformational changes occur during catalysis crystal structure of an inactive duck δii crystallin mutant with bound argininosuccinate substitution of residues at the double dimer interface affects the stability and oligomerization of goose δ-crystallin guanidine hydrochloride induced reversible dissociation and denaturation of duck δ2-crystallin monomeric molten globule intermediate involved in the equilibrium unfolding of tetrameric duck δ2-crystallin kinetic refolding barrier of guanidinium chloride denatured goose δ-crystallin leads to regular aggregate formation disruption of a salt bridge dramatically accelerates subunit exchange in duck δ2 crystallin guanidine hydrochloride-induced denaturation and refolding of transthyretin exhibits a marked hysteresis: equilibria with high kinetic barriers distinct interactions of αa-crystallin with homologous substrate proteins, δ-crystallin and argininosuccinate lyase, under thermal stress. biochimie a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding determination and analysis of urea and guanidine hydrochloride denaturation curves equilibrium unfolding of bombyx mori glycyl-trna synthetase metabolism of amino acids and amines resolution of the fluorescence equilibrium unfolding profile of trp aporepressor using single tryptophan mutants the application of circular dichroism to studies of protein folding and unfolding macromolecular size-and-shape distributions by sedimentation velocity analytical ultracentrifugation determination of the sedimentation coefficient distribution by least-squares boundary modeling analytical ultracentrifugation in biochemistry and polymer science molecular dynamics with coupling to an external bath d18g transthyretin is monomeric, aggregation prone, and not detectable in plasma and cerebrospinal fluid: a prescription for central nervous system amyloidosis? reversible unfolding of the severe acute respiratory syndrome coronavirus main protease in guanidinium chloride mechanism of thioflavin t binding to amyloid fibrils protein denaturation with guanidine hydrochloride or urea provides a different estimate of stability depending on the contributions of electrostatic interactions kinetic stabilization of the native state by protein engineering: implications for inhibition of transthyretin amyloidogenesis the acid-mediated denaturation pathway of transthyretin yields a conformational intermediate that can self-assemble into amyloid mechanism of the chaperone-like and antichaperone activities of amyloid fibrils of peptides from αa-crystallin the αa66-80 peptide interacts with soluble αcrystallin and induces its aggregation and precipitation: a contribution to age-related cataract formation amyloid-induced aggregation and precipitation of soluble proteins: an electrostatic contribution of the alzheimer's beta(25-35) amyloid fibril gene sharing by δcrystallin and argininosuccinate lyase we thank dr m. d. lloyd (university of bath, u. k.) for reading of this manuscript before publication. conceived and designed the experiments: hjl wyc. performed the experiments: cwh hcl wck cyc. analyzed the data: cwh cyc. wrote the paper: cwh hjl. key: cord-002848-w6q1x1zs authors: zhang, ailian; yang, xiumei; li, quanxiao; yang, yu; zhao, gan; wang, bin; wu, daocheng title: immunostimulatory activity of water-extractable polysaccharides from cistanche deserticola as a plant adjuvant in vitro and in vivo date: 2018-01-23 journal: plos one doi: 10.1371/journal.pone.0191356 sha: doc_id: 2848 cord_uid: w6q1x1zs a safe and effective vaccine adjuvant is important in modern vaccines. various chinese herbal polysaccharides can activate the immune system. cistanche deserticola (cd) is a traditional chinese herb and an adjuvant candidate. here, we confirmed that water-extractable polysaccharides of cd (wpcd) could modulate immune responses in vitro and in vivo. in a dose-dependent manner, wpcd significantly promoted the maturation and function of murine marrow-derived dendritic cells (bm-dcs) through up-regulating the expression levels of mhc-ii, cd86, cd80, and cd40, allogenic t cell proliferation, and the yields of il-12 and tnf-α via toll-like receptor4 (tlr4), as indicated by in vitro experiments. in addition, its immunomodulatory activity was also observed in mice. wpcd effectively improved the titers of igg, igg(1) and igg(2a) and markedly enhanced the proliferation of t and b cells, the production of ifn-γ and il-4 in cd4(+) t cells and the expression level of ifn-γ in cd8(+) t cells better than alum. furthermore, wpcd could markedly up-regulate the expression levels of cd40 and cd80 on dcs in spleen and down-regulate the treg frequency. the study suggests that polysaccharides of cistanche deserticola are a safe and effective vaccine adjuvant for eliciting both humoral immunity and cellular immunity by activating dcs via tlr4 signaling pathway. vaccines are important for controlling or preventing diseases. newly generated and current developing vaccines are highly purified recombinant antigens with a higher safety, but purified antigens cannot stimulate a satisfactory immune response compared with attenuated or inactivated pathogen preparations. with the aid of adjuvants, vaccines can induce persistent immune responses [1, 2] . new powerful adjuvants are widely concerned in both vaccine a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 development and human health. to date, various adjuvants have been identified and extensively studied. these adjuvants endow vaccines with several advantages, including decreasing the required amount of antigens, minimizing the number of immunizations required for normal immune responses, and inducing more rapid, broader, and stronger immune responses [3] [4] [5] . although many potent adjuvants have been developed, such as lipolysaccharide (lps) and freund's complete adjuvant (fca), they are not widely applied due to their toxicity. hence, only a few adjuvants, such as alum, are licensed for clinical use [6] . overall, more effective and safer adjuvants should be developed to promote the better prophylactic and therapeutic vaccines against infectious and noninfectious diseases. safety is an important consideration factor in the development of adjuvants. chinese herbaceous constituents include nutrients and important active composites, such as phenolic compounds and polysaccharides, which can act as powerful immunostimulants [7] [8] [9] . among them, polysaccharides from traditional chinese herbs have been widely explored due to their immunostimulatory activities and low toxicity. for example, inulin is an immune adjuvant without the toxicity of other adjuvants such as fca. advax tm delta inulin adjuvant also has successfully enhanced the immunogenicity of vaccine [10, 11] . astragalus, also known as huangqi in chinese and radix astragali in latin, is widely distributed throughout the world. polysaccharide is one of its major active ingredients responsible for the immunomodulatory activity. experimental studies have demonstrated that astragalus polysaccharide possessed strong immunomodulatory effects both in vitro and in vivo [12, 13] . lycium barbarum polysaccharides as vaccine adjuvant also showed good improvements and stimulating effects [14, 15] . these studies suggested that chinese herbral polysaccharides were ideal candidates for adjuvant development. cistanche deserticola y. c. ma (cd, "rou cong rong" in chinese) is a valuable traditional chinese herb and commonly considered as "ginseng of the deserts" because of its superior tonic effects. it is distributed in arid or semi-arid regions in xinjiang. in china, its dried fleshy stem has been used as a tonic food for hundreds of years [16, 17] . for many years, boiled cd has been used as a tonic food to treat overstrain-induced impairment, suggesting that it is safe for oral administration. this plant is widely concerned due to its broad medicinal functions. recent phytochemical and pharmacological studies demonstrated that polysaccharides were the main biologically active components and had various biological effects, including the immunomodulatory activity, antioxidant effect, and anti-inflammatory effects [18] [19] [20] [21] . however, the immune enhancement activity of water-extractable polysaccharides from c. deserticola in xinjiang was seldom reported. it is well known that dcs are important antigen presenting cells (apcs) providing signals required for initiating immune responses and modulating innate and adaptive cells. some adjuvants improving antigen uptake by dcs can increase co-stimulatory or mhc molecules and enhance the immunity. when dcs maturation starts, more co-stimulatory molecules and cytokines are produced and dcs show distinct phenotypes [22, 23] . in this study, we firstly exploited whether wpcd could promote activation of dcs via tlr4 signaling pathway in vivo. since dcs were the link between innate and adaptive immune responses, we hypothesized that dcs activation stimulated by wpcd would influence the outcome of adaptive immunity. we examined the specific immune response to ovalbumin (ova) in mice treated with wpcd, including titers of igg, igg 1 and igg 2a subclass, t-and b-proliferation, and the production of cytokine. we investigated whether wpcd treatment would increase the activity of dcs and treg cells in the spleen of these mice. our findings proved the potential immunomodulatory activity of natural polysaccharides from c. deserticola and expanded its application scope. eight-to ten-week old c57bl/6, balb/c or icr female mice were purchased from the first hospital of xinjiang medical university (urimuqi, xinjiang, china). the mice were housed under pathogen-free conditions according to the guidelines of the animal care and use committee (acuc) of xinjiang university for animal health and wellbeing. the animal experiment procedures had been approved by the aucc of xinjiang university. c. deserticola is a plant in xinjiang province in china. crude polysaccharide was obtained through water extraction and ethanol precipitation. briefly, 100 g of dried c. deserticola was ground into powder and then filtered. powder was refluxed with petroleum ether at room temperature repeatedly and then refluxed with anhydrous ethanol for 1 h to remove colored ingredients and lipids. residues were extracted with boiling water for three times and the extracts were pooled together, centrifuged at 4000 rpm for 10 min, and refluxed at 60˚c for 4 h. four times of volumes of 95% ethanol was added into the solution in order to precipitate crude polysaccharides. the crude polysaccharides were re-dissolved in distilled water and treated with sevage reagent to remove proteins. extracts were dissolved in pbs and sterilized through 0.22-μm filter. finally, the total sugar content of wpcd was 59.58%, as indicated by the phenol-sulfuric acid analysis [24] . bm-dcs were generated according to the previously described method with slightly modifications [25] . bone marrow dcs from c57bl/6 mice were flushed out with rpmi-1640 complete medium (gibco) supplemented with 10% fetal calf serum (hyclone). the collected cells were re-suspended in rpmi-1640 medium with 50 μm β-mercaptoethanol (sigma, st louis, mo) and 20 ng/ml murine gm-csf (peprotech, rocky hill, ny) and incubated at 37˚c in 5% co 2 atmosphere. half of the culture medium was replaced by fresh medium containing gm-csf every 1-2 days. cells were collected on day 6, treated with different doses of wpcd, lps (100 ng/ml) (sigma, st louis, mo) for 24 h and then assessed by flow cytometry. in vitro maturation effect of wpcd on bm-dcs was evaluated based on phenotypic analysis results by facs. on day 7, bm-dcs from c57bl/6 were induced in the presence of gm-csf and then treated with different concentrations of wpcd (0.01, 0.02, 0.05, 0.1 and 0.2 mg/ml) for 12 h in triplicate. lps (100 ng/ml) was used as a positive control. after bm-dcs were washed in pbs and fc block (bd biosciences, ca) was used to prevent nonspecific antibody binding, cells were stained in pbs by using the appropriate fitc-, pe-, or apc-conjugated cd40, cd11c, cd86, cd80, and mhc-ii antibodies (bd) for 20 min at 37˚c. the stained cells were detected by facs calibur (bd). the data analysis was carried out with flowjo software (tree star). in dcs maturation experiment in vitro, cell culture supernatants were also collected to analyze il-12 and tnf-α by using cytokine assay kits (boster, wuhan, china) according to the manufacturer's instructions. the absorbance at 450 nm was measured with an elisa plate reader (bio-rad, usa). cytokine quantities in the samples were calculated with standard curves of recombinant cytokines according to the regression linear method. to test their allogenic stimulatory activity, the experiment of mixed lymphocyte reaction (mlr) was performed according to the previous method [26] by mtt (sigma, st louis, mo) assay. briefly, bm-dcs from c57bl/6 mice used as stimulator cells were recovered on day 7 in rpmi-1640 medium plus gm-csf and treated with various concentrations of wpcd for 12 h at 37˚c. cells were washed and re-suspended in rpmi-1640 medium before plating into the 24-well plate. singleplenocyte suspensionas responder cells were isolated from balb/c mice. bm-dcs were mixed with splenocytes according to the ratios of 1:5 and 1:10. cells were cultured at 37˚c in 5% co 2 atmosphere for three days in triplicate. lps treatment was used as a positive control. cultures were subsequently added with 20 μl of mtt for the final 4-h cultivation. the absorbances at the measuring wavelength (490 nm) and the reference wavelength (650 nm) were measured for t cell proliferation analysis. all samples were measured against a background control. bm-dcs were pre-treated with 5 μm tak-242 (inhibitor of tlr4, medchemexpress inc. usa) for 1 h and then co-cultured with various concentrations of wpcd (100 and 200 μg/ ml) for 12 h in the presence or absence of golgi stop in triplicate. in the positive control, lps was added. cells and supernatants were detected by facs for analyzing surface markers cd86 and cd40 and by elisa for analyzing cytokines tnf-a and il-12, respectively. acute toxicity test. in oral acute toxicity test in this study, 40 healthy adult icr mice were used. icr mice were fasted (food but not water overnight) before dosing. wpcd was orally administered respectively in the doses of 0, 50, 500 or 5000 mg/kg body weight to each animal for 7 days. saline-treated and alum-treated mice were included in the control group. the animals were observed daily within 14 consecutive days. the mice were individually observed to record mortality and clinical signs. in addition, body weights and their food and water consumption were measured throughout the experiment. on day 14, the spleen or thymus index was calculated as: (spleen or thymus weight/bodyweight) × 10. detection of wpcd immunomodulatory activity in vivo. to investigate whether wpcd had the immunomodulatory activity, ovalbumin (ova) (sigma) was used as the model antigen and female icr mice were randomly separated into 7 groups (negative control group (0.9% nacl and wpcd 400 μg) and the positive control (alum 200 μg with ova)). the mice were administered subcutaneously twice with 10 μg ova alone or wpcd with ova according to the dose of 20, 100 or 400 μg with a 2-week interval. blood samples and spleen were obtained after the booster vaccination in order to detect igg titers, the igg subclasses, splenocyte proliferation and cytokine, dcs surface markers, and treg frequency. ova-specific igg titers and subclasses were examined by elisa according to the previous method [27] . elisa plates (nunc, thermo fisher scientific) were coated overnight and then blocked. serum samples were diluted serially for igg titer analysis or the detection of igg 1 and igg 2a (southern biotech, inc.). the plates were then incubated with pbst containing hrpconjugated anti-mouse igg, igg 1 , and igg 2a for 1 h at 37˚c. the colorimetric reaction was developed with tetramethylbenzidine (tmb) and then the absorbances at 450 nm/655 nm was measured and expressed as optical density (od) units. splenocyte proliferation was determined by the mtt assay. on day 21 after the first vaccination, single splenocyte suspension was obtained. cells were cultured in rpmi-1640 medium in 96-well plates according to the concentration of 1×10 6 cells/well in triplicate. the cultures were stimulated with ova (final concentration 10 μg/ml), cona (final concentration 5 μg/ ml) (sigma, st louis, mo), and lps (final concentration 100 ng/ml) for 48 h at 37˚c. cells were cultured for 48 h and 20 μl (5 mg/ml) of mtt (sigma, st louis, mo) was added into each well and incubated for another 4 h. after adding 50 μl of dmso into each well to stop the color development (sigma), plates were read at 570 nm by a microtiter plate reader (bio-rad, ca, usa). splenocyte proliferation was expressed as stimulation index (si), which was the od 570 nm ratio of a stimulated well to an unstimulated well. the yields of il-4 and ifn-γ in t cells were measured by intracellular cytokine staining according to a published protocol with minor modifications [27, 28] . single splenocyte suspension was prepared on day 21 after the first vaccination. after red blood cell lysis, the splenocyte (2×10 6 cells/ml) were incubated with ova (10 μg/ml) for 4-h stimulation and golgi stop (bd) was added for 12 h incubation, pma as positive control. cells were collected, washed with pbs, stained with cd4-fitc/cd8-fitc, and fixed and permeabilized with cytofix/cytoperm kit (bd) according to the manufacturer instructions. intracellular cytokine staining was performed with the appropriate concentration of il-4-pe or ifn-γ-apc antibodies at 4˚c for 20 min. stained cells were detected by facs. data analysis was carried out in the flowjo. for the analysis of dcs maturation from splenocyte in mice, cell surface staining was performed with cd11c-pe, cd40-fitc, and cd80-apc antibodies. on day 3 after the first vaccination, single splenocyte suspension (1×10 6 cells/ml) was obtained and cells were subjected to double staining. the fluorescent intensities were measured by the facs calibur and the measured data were analyzed by flowjo. in order to observe whether wpcd could down-regulate the frequency of cd4 + cd25 + foxp3 + treg cells. single splenocyte suspension was prepared on day 7 after the second vaccination. the splenocyte (2×10 6 cells/ml) was subjected to cell surface staining with cd4-apc antibody, followed by nuclear cytokine staining with the appropriate cd25-fitc and foxp3-pe antibodies with the mouse regulatory t-cell staining kit (ebiosciences) according to the manufacturer's instructions. the frequency of cd4 + cd25 + foxp3 + treg cells was tested on facs. data analysis was carried out in the flowjo. one-way analysis of variance (anova) tests (tukey's multiple-comparison test) were performed to analyze the differences among multiple experimental groups. all values are expressed as mean ±sd. p< 0.05 is believed to be statistically significant. the statistical analyses were performed using prism 5.0 software. adaptive immunity is determined by the activation of dcs, including the types of co-stimulatory molecules and cytokines [29, 30] . initially, we investigated whether wpcd could promote the expression of co-stimulatory molecules. according to different doses of wpcd, bm-dcs from c57bl/6 were administered. the expression levels of cd11c, cd86, cd80, cd40, and mhc-ii in cells were analyzed (fig 1) . the cells gated with ssc and fsc showed that the treatment with different doses of wpcd did not change the morphology of bm-dcs (data not shown). the expression levels of cd86, cd80, and cd40 were significantly up-regulated in a dose-dependent manner compared to the untreated group and reached the plateau under the dose of 20 μg/ml of wpcd, but the difference was not significant compared with lps group (fig 1a-1c) . the expression level of mhc-ii was significantly increased to its maximum value under the dose of 50 μg/ml (fig 1d) . the analysis results of cellular surface markers demonstrated that dcs treated with lps or wpcd showed the significantly increased expression levels of cd86, cd80, cd40, and mhc-ii and the promoted phenotypic maturation. some adjuvants could increase co-stimulatory molecules on dcs or directly induce the secretion of cytokines. il-12 and tnf-α is the major cytokines for activating th1 immune response. we analyzed the yields of cytokine in bm-dcs upon wpcd treatment. after bm-dcs were treated with different contents of wpcd for 12 h, the supernatant was collected to detect the contents of il-12 and tnf-α with elisa kit. wpcd could dose-dependently increase the productions of il-12 (fig 2a) and tnf-α (fig 2b) . the results demonstrated that wpcd could induce the functional maturation of dcs. in an immune response, it is necessary to functionally activate dcs. then, the higher level of allogenic t cell proliferation was induced by fully mature dcs. therefore, the functional response of dcs to wpcd was investigated by mlr. the allostimulatory capability of wpcd-induced dcs, similar to lps, was enhanced dramatically in a dose-dependent manner at the indicated ratio (fig 2c and 2d) . the results indicated that wpcd improved antigen presentation and optimized t-cell response by mlr in vitro. it could be inferred from the above results that bm-dcs activated by wpcd indicated similar dcs surface molecule expressions to lps (a tlr4 ligand). we thus hypothesized that bm-dcs are activated by wpcd via tlr4 pathway. after bm-dcs were pretreated with tak-242 (tlr4 inhibitor) and co-cultured with wpcd and lps for 12 h, the expression of cd40, cd86, tnf-a or il-12 were detected by facs or elisa. as shown in fig 3a and 3b , the tak-242 treatment resulted in the significantly down-regulated expressions of cd40 and cd86 induced by lps or wpcd, and cytokine production of il-12 or tnf-a were also significantly decreased (fig 3c and 3d) . the results indicated that wpcd could activate dc maturation through the tlr4 pathway. for the purpose of comparison, we evaluated the effects of wpcd on the elicitation of humoral immune response in ova-immunized mice. before vaccination and on days 14, 21, 35, and 49 after the first vaccination, blood was collected to detect igg titer and igg subclasses serum by elisa. the igg antibody response to ova increased with the increase in the dose of wpcd administration (100 and 400 μg) in a dose-dependent manner (fig 4) . the optimal concentration was 100 μg/ml of wpcd and increased igg responses by two times compared to the ova alone on days 21, 35 and 49 (fig 4a) . considerable increases in ova-specific igg 1 and igg 2a antibody levels under 20 and 100 μg of wpcd administration were obtained compared with ova group in serum on day 35 ( fig 4b) . meanwhile, alum promoted only the expression levels of ova-specific igg and igg 1 antibodies in the immunized mice. wpcd alone did not trigger ova-specific antibody. the above results obviously indicated that wpcd generated the higher antibody titers and the more balanced th1/th2 responses than alum. splenocyte proliferation is another indicator of cellular immunity. cona stimulates t-cells and lps stimulates b-cell proliferation. on day 21 after the first vaccination, single splenocyte suspension was obtained and respectively stimulated with ova (10 μg/ml), ova 323-339 (10 μg/ml) peptide, cona (5 μg/ml) and lps (5 μg/ml) for 48 h. then the proliferation of t cell was measured by mtt method. wpcd could significantly promote ova-antigen, con a-mitogen and lps mitogen-stimulated splenocyte proliferation in the mice immunized with ova and wpcd (fig 5) and the optimal concentration of wpcd was 100 μg/ml. these data indicated that wpcd as a vaccine adjuvant in ova-immunized mice could more effectively induce the activation of t-cells and b cells than alum. all tested wcpd adjuvants formulated with ova vaccines generated equally strong humoral and cellular immunity (figs 4 and 5) . on the basis of these data, to further evaluate wpcd influences on ova-specific t helper (th) cell response, we further analyzed cytokine expressions in cd8 + and cd4 + t cells by flow cytometry (fig 6) . on day 21 after the first vaccination, single splenocyte of mice was isolated and then co-cultured with the ova (10 μg/ ml). il-4 yield in cd4 + t cells in the group administered with 100 μg ova/wpcd was significantly higher than that in ova/alum group and ova group and similar to that in pma (fig 6a) . moreover, ifn-γ yield in cd8 + and cd4 + t cells was also significantly enhanced in the mice administered with ova/wpcd (20, 100, and 400 μg) (fig 6b and 6c) . compared with alum adjuvant, wpcd showed the better capability of inducing il-4 and ifn-γ secretions from t cells. dcs are recognized as the most potent apcs involved in initiating primary immune responses. thus, on day 3 after the first vaccination, we also investigated the effects of wpcd on cd40 and cd80 on dcs in the spleen in mice (fig 7a and 7b) . the mice in 100-μg ova/wpcd group produced the significantly higher levels of cd40 and cd80 on dcs than those in the ova/alum group. these data showed that wpcd could activate dcs and induce dcs maturation in mice. treg cells can balance the tolerance and immune responses. to further investigate how wpcd modulated the immune response, treg cells in the spleen in mice were stained with assessment of the immunostimulatory activity of wpcd in vitro and in vivo mouse regulatory t cell staining kit. on day 21 after the first vaccination, a decreased frequency of cd25 + foxp3 + treg cells was observed in the total cd4 + t cells (fig 7c) . compared with the ova and ova/alum groups, wpcd group showed the significantly reduced treg frequency. in order to estimate the oral acute toxicity, we carried out the acute toxicity test. the mice orally administered with 5000 mg/kg body weight showed no abnormal behavior or side effects and no mortality was found in the toxicity evaluation test. no significant difference in body assessment of the immunostimulatory activity of wpcd in vitro and in vivo weight gain, thymus index and spleen index was recorded among various groups of the mice administered with different doses of wpcd and had no significant difference (table 1) . no mortality was observed after 14 days. therefore, the ld 50 value of wpcd was more than 5000 mg per kg body weight. to test whether wpcd had negative effects on the growth of the mice, before and after subcutaneous vaccination, the body weight was respectively determined for each mouse (table 2 ). in the subsequent observation of the mice, side effects or abnormal behaviors were not observed. besides, the body weight of the mice administered with wpcd and the mice adjuvants are key components in vaccines [31] . due to the advances in genomics and proteomics, more and more recombinants and synthetic vaccine molecules are identified. therefore, more adjuvants and formulations are required. some potent adjuvants are generally related to the increased toxicity, for example fca. therefore, it is necessary to find a safe formulation containing different synergistic components eventually driving the desired immune response. polysaccharides from chinese herbs are nontoxic and show no significant side effect [32, 28] . a safe adjuvant is required for a certain vaccine. cistanche deserticola is an important tonic herb and widely exists in arid lands and warm deserts in the northwest of china. cistanche deserticola is widely concerned due to its bioactivities including immunomodulatory, antioxidative, antibacterial and antitumor effects [20, 21] . some progresses have been made in structural characterization and immunological activity of cistanche deserticola, which is a good candidate for developing adjuvants. we experimentally evaluated the adjuvant effects and mechanism of wpcd in vitro and in vivo. subcutaneous administration of wpcd significantly promoted humoral and cellular immune responses by increasing serum antibodies and lymphocyte proliferation, enhancing the expression of cytokines, up-regulating the dc maturation, and down-regulating the frequency of cd4 + cd25 + foxp3 + treg cells. dcs are key apcs for priming naive t cells. the activation of dcs is important for adjuvants. dcs, especially murine marrow-derived dcs, are often used to evaluate adjuvants and vaccines. flow cytometry can distinguish the cells which have been activated after capturing an antigen from surrounding cells. in fcm analysis, the degree of aggregation of stimulated assessment of the immunostimulatory activity of wpcd in vitro and in vivo cells is an indirect indicator of evaluating safety of immunomodulators [3] . thus, wpcd adjuvant activity data in dcs in vitro may provide valuable information for animal models. based on bm-dc model, the expression levels of mhc-ii, cd86, cd80, and cd40, cytokine production, and allogenic t cell proliferation were detected in the optimum wpcd concentration range. the expression levels of mhc-ii, cd86, cd80, and cd40 were up-regulated in bm-dcs and the yields of tnf-a and il-12 were increased in a dose-dependent manner. the allogenic t cell proliferation was observed. the morphology of bm-dcs was not changed. by inducing bm-dcs activation and the secretion of inflammatory cytokines in vitro, wpcd adjuvant might significantly increase the amount of antigen and the amount of apcs and stimulate t cells to secrete ifn-γ, which contributes to the enhancement of immunomodulatory activity. various chinese herbaceous polysaccharides are capable of activating the immune system and possess excellent adjuvant abilities through stimulating dcs maturation by tlr4 pathway [33, 28] . thus, we assume that tlr4 participates in the signaling pathway of wpcd-induced dcs maturation. as expected, tlr4 inhibitor treatments resulted in a significant decrease of tnf-a and il-12. furthermore, the inhibition of tlr4 pathway also hindered the expression of cd 40 and cd80 on dcs, indicating that the maturation of dcs was dependent on tlr4. therefore, it is obvious that tlr4 participates in the wpcd-induced dcs maturation. the optimum dose of adjuvants and vaccine formulation is empirically determined in many experiments. in order to explore the dose-response relationship of wpcd adjuvant and ova antigens in mice, we selected different wpcd doses based on previously reported data from bm-dcs in vitro to subcutaneously administer icr mice twice. we found that wpcd significantly increased the yield of ova-specific antibody and induced a balanced th1/th2 immune response with an enhancement of igg 1 and igg 2a levels. especially, the igg 2a level was higher than that treated with alum in the optimum dose. therefore, wpcd resulted in the higher levels of specific antibody and higher efficacy with the lower injections. new vaccines require the induction of strong cellular responses, which involve antibodies, t helper (th) cells, and cytotoxic t lymphocytes (ctls). therapeutic vaccine aims to induce stronger t cell responses. the ideal adjuvant enhances the potency of the vaccine and promotes cell-mediated immunity without causing toxic effects [34] . among the observations in immunized mice models, wpcd led to an increase in ova-specific and non-specific splenocyte proliferation compared with alum. some adjuvants up-regulate cytokines and the immune system. for example, saponins may stimulate cell-mediated immune responses to an antigen which normally induces only antibodies. we selected il-4 and ifn-γ as the indicators to indirectly evaluate the levels of immunities in mice. wpcd could induce more il-4 and ifn-γ secretions than alum. thus, strong helper t-cell responses and cytokine secretions were observed as a result of wpcd vaccination, suggesting that wpcd could more efficiently stimulate lymphocytes to secret the th1-type cytokine and the th2-type cytokine than alum. adjuvants influencing antigen presentation can affect complex immune processes. treg cells can modulate th1 and th2 responses [35] . an appropriate dose of wpcd may promote the maturation of dcs in mice via increasing the expression levels of cd80 and cd40 and amplify the capability of ova antigen presentation in the early immune response. the experimental results of the dcs activation and treg frequency demonstrated that wpcd induced maturation of dcs and decreased treg frequency in the spleen in mice. these results proved that wpcd enhanced the efficacy of ova vaccines by increasing targeting antigen-presenting cells and promoting t-cell specific activation. in the development of adjuvants, it is difficult to selectively induce the appropriate immune response against corresponding infection. suitable adjuvant should have low side effects and toxicity to human beings or animals. thus, wpcd safety should be considered, including immediate and long-term side effects. in this study, we explored acute toxicity of wpcd and the negative effect of wpcd on the growth performance of mice for 110 days. the results of acute toxicity of wpcd showed that body weight, thymus index, or spleen index had no significant difference. the ld50 value of wpcd was more than 5000 mg per kg body weight. throughout the experimental subsequent observation of the mice, no side effect or abnormal behavior in mice was found. these results indicated that wpcd was safe. in conclusion, in this study, wpcd, a crude polysaccharide extracted from the c. deserticola from xinjiang, exhibited some properties of adjuvants. for example, wpcd enhanced both th1 and th2 responses by activating dcs, increasing antibody responses, improving cytokine production. moreover, we explored the mechanism of wpcd efficacy by analyzing dcs maturation and function via tlr4 pathway in vitro. in the mice only treated with wpcd, no obvious side effect was observed. hence, wpcd possessed the higher immunostimulatory activity in cellular and humoral immune responses. adjuvants are a mixture of several compounds. a mixture of several crude extracts may have greater beneficial effects than a single plant extract. it is necessary to systematically explore wpcd extracts, test their efficacy and safety, and elucidate the mechanisms of their effects. supporting information s1 file. arrive checklist. (docx) overview of vaccine adjuvants: present and future history of vaccination the perfect mix: recent progress in adjuvant research new horizons in adjuvants for vaccine development key roles of adjuvants in modern vaccines alum adjuvanticity: unraveling a century old mystery complementary medicine: a review of immunomodulatory effects of chinese herbal medicines extraction, characterization, and biological activity of polysaccharides from sophora flavescens, ait the adjuvant effects of high-molecule-weight polysaccharides purified from antrodia cinnamomea on dendritic cell function and dna vaccines a novel hepatitis b vaccine containing advax™, a polysaccharide adjuvant derived from delta inulin, induces robust humoral and cellular immunity with minimal reactogenicity in preclinical testing severe acute respiratory syndrome-associated coronavirus vaccines formulated with delta inulin adjuvants provide enhanced protection while ameliorating lung eosinophilic immunopathology tlr-4 may mediate signaling pathways of astragalus polysaccharide rap induced cytokine expression of raw264.7 cells structural features and biological activities of the polysaccharides from astragalus membranaceus lycium barbarum polysaccharides as an adjuvant for recombinant vaccine through enhancement of humoral immunity by activating tfh cells the immunological activity of lycium barbarum polysaccharides liposome in vitro and adjuvanticity against pcv2 in vivo desert ginseng": a review chemical constituents and in vitro anti-inflammatory activity of cistanche violacea, desf. (orobanchaceae) extract anti-nociceptive and anti-inflammatory activity caused by cistanche deserticola in rodents analysis of chemical constituents in cistanche species phenylethanoid glycosides with anti-inflammatory activities from the stems of cistanche deserticola cultured in tarim desert extraction, purification, characterization and antioxidant activities of polysaccharides from cistanche tubulosa dendritic cells: unique leukocyte populations which control the primary immune response maturation of murine bone marrow dendritic cells induced by acidic ginseng polysaccharides colorimetric method for the determination of sugars generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor overcoming hbv immune tolerance to eliminate hbsag-positive hepatocytes via pre-administration of gm-csf as a novel adjuvant for a hepatitis b vaccine in hbv transgenic mice intranasal co-administration with the mouse zona pellucida 3 expressing construct and its coding protein induces contraception in mice dendritic cells in vivo: a key target for a new vaccine science immunomodulating activities of polysaccharides isolated from panax ginseng vaccines: science, health, longevity, and wealth specific medicinal plant polysaccharides effectively enhance the potency of a dc-based vaccine against mouse mammary tumor metastasis adjuvant-active aqueous extracts from artemisia rupestris l. improve immune responses through tlr4 signaling pathway adjuvant effect of a natural tlr4 ligand on dendritic cell-based cancer immunotherapy. cancer let adjuvants for human vaccines control of regulatory t cell development by the transcription factor foxp3 with special thanks to bing wang and daocheng wu for providing good ideas in the experiments. key: cord-000265-llilwq1u authors: gao, rongbao; dong, libo; dong, jie; wen, leying; zhang, ye; yu, hongjie; feng, zijian; chen, minmei; tan, yi; mo, zhaojun; liu, haiyan; fan, yunyan; li, kunxiong; li, chris ka-fai; li, dexin; yang, weizhong; shu, yuelong title: a systematic molecular pathology study of a laboratory confirmed h5n1 human case date: 2010-10-12 journal: plos one doi: 10.1371/journal.pone.0013315 sha: doc_id: 265 cord_uid: llilwq1u autopsy studies have shown that human highly pathogenic avian influenza virus (h5n1) can infect multiple human organs other than just the lungs, and that possible causes of organ damage are either viral replication and/or dysregulation of cytokines and chemokines. uncertainty still exists, partly because of the limited number of cases analysed. in this study, a full autopsy including 5 organ systems was conducted on a confirmed h5n1 human fatal case (male, 42 years old) within 18 hours of death. in addition to the respiratory system (lungs, bronchus and trachea), virus was isolated from cerebral cortex, cerebral medullary substance, cerebellum, brain stem, hippocampus ileum, colon, rectum, ureter, aortopulmonary vessel and lymph-node. real time rt-pcr evidence showed that matrix and hemagglutinin genes were positive in liver and spleen in addition to positive tissues with virus isolation. immunohistochemistry and in-situ hybridization stains showed accordant evidence of viral infection with real time rt-pcr except bronchus. quantitative rt-pcr suggested that a high viral load was associated with increased host responses, though the viral load was significantly different in various organs. cells of the immunologic system could also be a target for virus infection. overall, the pathogenesis of hpai h5n1 virus was associated both with virus replication and with immunopathologic lesions. in addition, immune cells cannot be excluded from playing a role in dissemination of the virus in vivo. influenza pandemic are characterized by the worldwide spread of novel influenza strains for which most of the population lacks substantial immunity [1, 2] . pandemic viruses typically cause heightened morbidity and mortality [1] . the continued circulation of highly pathogenic avian influenza (hpai) viruses h5n1 has resulted in occasional coincident infections among humans. since late 2003, when widespread h5n1 virus poultry outbreaks were reported in multiple countries in asia, there have been 467 laboratory confirmed human cases in ten countries reported to the world health organization as of december 2009 with a mortality rate of about 60% [3, 4] . global public health concerns surrounding h5n1 viruses include not only individual transmission events between infected poultry and individual humans, but also their pandemic potential, should these viruses acquire genetic changes that result in sustained human-to-human transmission. to date, several case clusters of h5n1 infections have been reported [5] and limited epidemiologic information has suggested person-to-person transmission of h5n1 in a few instances, usually involving family members. of additional concern to both human and animal health, is the extensive geographic spread of hpai h5n1 viruses in recent years and their isolation from multiple species of wild birds and mammals [6] [7] [8] [9] . despite the recent emergence of the 2009 h1n1 pandemic [10] , the pandemic threat from hpai h5n1 viruses has not diminished [11] . human h5n1 disease is clinically and pathologically distinct from that caused by seasonal human influenza a h3n2 or h1n1 viruses [12] . the majority of confirmed human hpai h5n1 virus infections have been characterized by a severe clinical syndrome including a rapid progression of lower respiratory tract disease, often requiring mechanical ventilation within days of admission to a hospital [13] [14] [15] [16] [17] [18] . in addition to pulmonary complications, other clinical manifestations of h5n1 virus infections may include severe lymphopenia, gastrointestinal symptoms, and liver and renal dysfunction [14, 16, 17, 19, 20] . reactive hemophagocytosis in multiple organs, and occasional detection of viral antigen or viral rna in extrapulmonary organs suggest a broader tissue distribution of h5n1 viruses compared with seasonal viruses in fatal human cases [21, 22] . patients with severe h5n1 disease have unusually higher serum concentrations of proinflammatory cytokines and chemokines. levels of plasma macrophage attractant chemokines cxcl10 (ip-10), cxcl9 (mig), and ccl-2 (monocyte chemoattractant protein 1, mcp-1) and of neutrophil attractant interleukin-8 (il8) were substantially higher in patients with h5n1 disease compared with those experiencing seasonal influenza virus and were significantly higher in h5n1 patients who died compared with those who recovered [23, 24] . the elevation of plasma cytokine levels was positively correlated with pharyngeal viral load [23] and may simply reflect more extensive viral replication, and consequently, direct viral pathology rather than being causative of the pathology observed in h5n1-infected patients. compared with human h1n1 and h3n2 influenza viruses, infection of human primary macrophage cultures in vitro with h5n1 viruses also lead to the hyper-induction of proinflammatory cytokines [25] . most studies on h5n1 pathology describe pulmonary features of human disease. although h5n1 virus infection of humans is primarily one of the lower respiratory tract, more recent reports suggested that influenza a h5n1 may in rare, severe cases, disseminate beyond the lungs and infect brain [26, 27] , intestines [20, 27] and lymphoid tissues [27] , and result in extra-pulmonary clinical manifestations including encephalopathy or encephalitis [15, 28] . this extrapulmonary dissemination of hpai h5n1 virus contrasts with seasonal influenza virus infection of humans which, even in fatal cases, is restricted to the respiratory tract. however, there have been relatively few reports describing histopathology and virus distribution in h5n1 cases [5, 16, 21, 24, 26, 27] . to better understand the pathogenesis of human h5n1 virus infection, and investigate the route of virus dissemination in vivo, we report on the use of different techniques to detect virus distribution and infection of 5 organ systems in a laboratory confirmed fatal human h5n1 virus infection, and analyze the relationship between viral load in tissues and host response. our results suggested that the virus can infect multi-organs besides pulmonary. high viral load is associated with increased host response though the viral load is significantly difference in various organs. cells of immunologic system could not be excluded to play a role in dissemination of the virus. virus culture, real-time rt-pcr, ihc and ish were used to identify virus distribution in different tissues. as shown in table 1 , live virus was recovered from respiratory tissues including lung, trachea, bronchus and aortopulmonary vessel. in the digestive system, virus was isolated from tissues collected from the ileum, colon and rectum, but not the stomach, duodenum or liver. of note, virus culture was also positive on tissues collected from brains, ureter and axillary lymph-node. sequencing results showed that the sequences of isolates are identical. real time rt-pcr results were consistent with the detection of virus by culture, except for the liver and spleen tissues which were positive by real time rt-pcr but negative for virus isolation. the tissue distribution of viral rna or antigen detected by ish and ihc stains respectively, was also generally consistent with virus isolation by culture or real time rt-pcr result. however, several exceptions were noted. there was a lack of detectable staining by either method in bronchus tissue, despite virus detection. on the other hand, both staining methods detected viral product in the kidney, although virus isolation and real time pcr were both negative. the viral load is associated with host response figured out by proinflammatory factors. the relative h5n1 viral load in different tissues by determining the ratio of viral ha copy number relative to the copy number of the beta actin gene for a given tissue sample. tissues of the respiratory system yielded higher copy number ratios than tissues from all to other organ systems with the lower left lung lobe yielding the highest viral load, overall. interestingly, ureter tissue had the highest viral load of non respiratory system tissues. all other tissues had lower viral loads that were not. among the digestive system tissues, the viral load in the liver was the highest (see fig. s1 ). additionally, we detected mrna copies of macrophage attractant chemokine cxcl10 (ip-10), macrophage inflammatory protein 3b (mip-3b), rantes, tumor necrosis factor (tnf)-related apoptosis-inducing ligand (trail) and tnfa in tissues of brain, respiratory organ, spleen and lymph-node. rantes and trail can be detected in all selected tissues. mip-3b is positive in selected tissues except right upper lobe lung with positive b-actin. ip-10 can be determined copies in cerebral context, left upper lobe lung, left lower lobe lung, right middle lobe lung, right lower lobe lung, and aortopulmonary vessel. tnf-a can be showed copies in left upper lobe lung, left lower lobe lung, right middle lobe lung, right lower lobe lung, aortopulmonary vessel and spleen. the correlation among levels of viral load and were showed by fig. 1 . the pearson's cross correlation analysis (see table s1 ) showed that high viral load is associated with high host response figured out by proinflammatory factors (p,0.05) except tnf-a (p.0.05), and the correlation is significant between proinflammatory factors except between trail and tnf-a (p.0.05), and between trail and ip-10(p.0.05) as well. the histopathologic features in different organs are shown in fig. 2 . lung showed diffused alveolar damages including intraalveolar edema, focal intra-alveolar hemorrhage, necrosis of alveolar line cells, focal desquamation of pneumocytes in alveolar spaces, interstitial mononuclear inflammatory cell infiltrates, and extensive hyaline membranes. trachea showed focal denudation of the epithelium with edema, and mononuclear inflammatory cell infiltrates. spleen showed depletion of lymphocytes with congestion and organized infarcts. axillary lymph-node was congested with depletion of lymphocytes. the central nervous system showed extensive edema with focal neuronal necrosis in hippocampus. diastem between purkinje cells layer and particle cells layer showed focal augmentation in cerebellum. liver was congested with edema and focal fatty degeneration. kidney was congested with edema. other selected tissues showed no significant histological changes. (fig. 3i ). as influenza virus is a negative-strand rna virus, ish stain with sense and antisense probes detected both virus rna (with sense probe), mrna and crna(with anti-sense probe). ish stain showed that the two sets of probes (for hemagglutinin and nucleoprotein) generated similar staining results. in the selected 24 autopsy tissue from 5 organ systems, positive staining of ish was detected in all samples except bronchus, stomach and duodenum. in the lung tissue samples, sense probes extensively hybridized in the nuclei of pneumocytes (fig. 4a) , whereas antisense probes hybridized in both the cytoplasm and nuclei. in all other organs with positive staining, both sense and antisense were present mainly in the cytoplasm of infected cells. in respiratory system, the positive staining was found in epithelial cell of lung ( we systematically studied the tissue tropism of h5n1 virus in a fatal human case, based on 24 autopsy tissue samples from 5 organ systems, and analyzed the relationship between viral load and host response level in selected tissues. we presented evidence suggesting that the h5n1 virus can infect selected tissue of selected 5 organ systems including respiratory, digestive, nervous, urinary and lymphoid system. high viral load can induce high proinflamatory factors level in tissues, and immune cells could be the target of the virus. there is a substantial amount of evidence that hpai h5n1 virus can infect extrapulmonary organ tissues [16] [17] [18] 27] and precede other clinical manifestation [20, 29] . our results presented that the viral antigens or viral rna can be found in trachea, lung, brain, intestines, liver, spleen, lymph-node and kidney which were reported as same before [20, 26, 27, 30] , as well as in aortopulmonary vessel and ureter which were not reported before. notably, the virus can be found in tissues of lower gastrointestinal tract including small intestine and large intestine but in stomach and duodenum. the origin of infection in the extrapulmonary organs could be blood-borne, which is supported by previous studies showing live h5n1 virus can be isolated from the serum [20] and plasma [31] . unfortunately, we haven't obtained blood samples to [25, 30, [33] [34] [35] [36] and t lymphocytes [37] . the viral load shows various levels in different organs although the virus can be found in multiple organs. quantitative detection of viral gene showed that viral load is the highest in tissues of respiratory system, especially, in left lower lobe of lung. the liver had the highest viral load in tissues of digestive system. interestingly, renal duct showed a high viral load although pcr detection of kidney was negative. and viral load was high in spleen and lymphonode. despite of the viral cells tropism, possible reasons of different viral load could include activation of low ph [38] , viral receptor distribution [39] , cell n-linked glycoprotein distribution [40] or other unknown mechanisms. on other hand, non-permissive or abortive infection is extra possible in some of the tissues where the virus cannot replicate effectively. additionally, it should be possible that phagocytosis of viral antigen from pathologically significant lytic/replicative infection caused positively viral detection in these tissues. high viral load is associated with increased host responses. to be different with seasonal influenza virus, the h5n1 virus caused intense transcription induction and secretion of inflammatory cytokines/chemokines, as documented in human cases [15, 22] , and increased induction of cytokines and chemokines has been demonstrated in h5n1-infected mice [41] . moreover, high virulence of h5n1 virus is associated with increase with increased host responses [42] . however, the relationship between viral load and host immuno-response has never been reported. our study suggested that the high viral load is correlated with proinflamatory factors including ip-10, rantes, mip-3b and trail. this finding is particularly relevant to the mechanism of h5n1 pathogenesis which is associated not only with the virus but also with the host responses. inflammatory protein can be produced by almost any infected cells and by immune cells, including alveolar macrophages [43] . however, type ii pneumocytes have a marked ability to secrete large amounts of cytokines, such as tnf-a, gm-csf, mcp, and il-8, in response to various insults [44, 45] and can be induced to secrete il-1a, il-6, rantes, and mcp-1, in response to tnf-a [45, 46] , the last being produced by alveolar macrophages during h5n1 infection. therefore, it is possible that the much greater cytokine induction by the h5n1 virus was as much a consequence of the response of individual, infected cells as it was a consequence of the numbers of infected cells. immune cells could be a target of the virus infection. previous findings showed that viral sequences and antigens have been detected in lymphocytes in lymph node tissue, as well as in hofbauer cells (macrophages of the placenta), kupffer cells (macrophages of the liver), and mononuclear cells in the intestinal mucosa [27] . this is consistent with our find. our presented evidence shows that virus can be detected in macrophage. moreover, our finding showed that viral rna can be detected in cd3+ t lymphocytes, progenitor cells and follicular dendritic cells of spleen. the spleen is a complex organ with several functions, including the removal of senescent or aberrant red blood cells from circulation, as well as the removal of circulating pathogenic organisms [47] . so circulationg immune cell with h5n1 virus could play a role in dissemination of virus when those cells with virus can not remove the virus in cells completely. we unprecedentedly investigated 24 autopsy tissues of 5 organ systems from a dead patient of h5n1 infection. our study results indicate that the h5n1 virus could be found in multiple organs. viral load in infected tissues is correlated with host response. and circulating immune cell can not be excluded to play a role in dissemination of virus in vivo. the patient was laboratory confirmed as an h5n1 infection by china cdc on february 20, 2008. he is a 42 years-old chinese male living in guangxi, china with a history of 6 days of fever, cough and dyspnea. two weeks before hospital admission, he bought 3 hens at an open-air market, one of which died later at same day. the remaining birds exhibited symptom of chicken attack next day. the patient killed and cooked the birds, and ate them together with other family members who later did not experience any clinical symptom. on admission, the patient was febrile with a temperature of 40.3uc, had infiltration of lower left lung lobe based on chest radiography, bilateral lower lung moist rales, and substantially reduced oxygen saturation. he was placed on a ventilator and treated with antibiotics, spasmolysis, corticosteroids, and the dissipatation of phlegm and fluids. despite treatment, the patient presented with function damage of multiple organ (lung, heart, liver and kidney) on the second day after admission, and died 59 h after admission, and 8 days after the onset of symptoms. throat swabs were collected and performed rt-pcr and rrt-pcr detection at the day patient died. no antiviral drug treatment was given since the patient died before finally laboratory diagnosis. after informed consent was obtained, the cadaver was stored at 4uc and underwent autopsy about 18 h after death. the autopsies were done following conventional protocols with strict adherence to biosafety procedures [48] . twenty-four tissues were collected from respiratory, digestive, nervous, urinary and lymphatic organ systems. duplicate tissue samples were collected; one sample was fixed in diethylpyrocarbonate (depc) treated 10% formalin for pathologic analyses, while a second sample was frozen at 280uc for virus isolation and molecular analyses. frozen tissues were thawed and homogenized before inoculation into embryonated eggs for viral culture. briefly, 1.0 ml of phosphate-buffered saline (pbs) was added to 100-150 mg of tissue, which was homogenized, and, then centrifuged; 0.1 ml of the recovered supernatant was injected into the allantoic cavity of 11 days-old specific pathogen-free embryonated chicken eggs. the allantoic fluids were tested for haemagglutation activity with turkey red blood cells after 72 h incubation at 35uc. samples containing virus were subjected to rt-pcr and sequencing. three blind passages were performed on hemagglutinationnegative samples to confirm the absence of infectious virus. all steps were performed in bsl-3 containment laboratory. total rna from the tissues samples was extracted using an axygen total rna extraction kit (axygen, usa) as described in the manufacturer's instructions. briefly, 400 ml of lysis buffer was added to 30-40 mg of ground tissue. the nucleic acids were eluted in 50 ml nuclease-free water and stored at 220uc. for every five samples, one negative control (water) was included to detect any possible contamination. control rna products derive from in vitro transcription of the matrix (m) gene rna of a/anhui/1/2005(h5n1), human house keeping gene beta-actin and human cytokines/chemokines (including ip-10, rantes, trail, mip-3b and tnf-a) genes were used as positive controls and to establish the detection limit of the assay. recombinant plasmids with entire m gene, beta-actin gene segment and cytokines/chemokines gene segment were linearised by restriction enzyme, and then purified using a dna clean-up kit. dna concentration was measured as od units at 260 nm. one mg of linearized plasmid dna was transcribed using riboprobe in vitro transcription system kit (promega, usa) according to the manufacturer's instructions. the transcribed rna was purified using phenyl/chloroform solution and was quantified by spectrophotometer. rna copy number was then determined following the method of fronhoffs [49] . to analyze the h5n1 viral load and quantify proinflammatory factors in different tissues, real-time rt-pcr was performed with a strategene detection system using a fluorescently labeled taqman probe to enable continuous monitoring of amplicon formation. the primer and probe of h5 ha gene is from the who-released primer sets [50] . the primers and probes of proinflammatory factors and beta-actin gene were obtained from the literature [51, 52] . the concentration of primer and probe used was 40 mm and 10 mm, respectively. the reaction was completed in a total volume of 25 ml performed by quantitect probe pcr kit (qiagen, germany). the reaction mixture was incubated with 5 ml dnase-treated total rna (the template which was used to amplify b-actin gene was performed by 100-fold dilution) at the following temperature cycles. first, the reverse transcription reaction was completed by 1 cycle at 50ucfor 30 min. next, h5n1 ha gene, cytokine/chemokine gene and house keeping (bactin) genes were amplified by 1 cycle at 94uc for 15 min and 45 cycles at 94uc for 15 s, 55uc for 30 s, and 72uc for 30 s each. as described in previous report [53] , the standard curve was generated using serial dilution of in vitro transcribed standard rna (from ,10 to 10 7 copies). the viral load and cytokines/ chemokine levels are presented as the log 10 value of the ratio between copies of the target gene and b-actin gene. immunohistochemical stain was performed on 5 mm thick deparaffinized sections using monoclonal antibodies against the nucleoprotein of influenza a (serotec, uk) by a two-step peroxidase method. for controls, we used unrelated antibodies in place of the primary antibody. briefly, sections were deparafinized by 2 washes in xylene and were rehydrated through decreasing concentrations of ethanol. after washing in pbs at ph 7.6 for 5 min at room temperature (rt), sections were heattreated with antigen-retrieval solution (tris/edta buffer, ph 9, dako, denmark) for 10 min using microwave antigen retrieval method. after blocking with 10% normal horse serum for 10 min at rt, the sections were incubated with the specific antibody (1:100) for 30 min at rt. unbound antibody was removed by 3 washes in pbs before adding hrp-labelled polymer for 30 min at rt (dako csa detection system, denmark). after washing unbound labeled polymer in pbs 3 times, peroxidase staining in tissue sections was revealed by dab solution (csa detection systems, dako, denmark). after stopping the reaction in running water, sections were counter-stained with a quick rinse in mayer's hematoxylin solution. after dehydrating with increasing concentrations of ethanol and xylene, the sections were mounted with dpx and examined by light microscopy (olympus bx51, japan). the development of probes was based in analysis of the full haemagglutinin and nucleoprotein gene sequences of all chinese human isolates of h5n1. oligonucleotide dna probes representing conserved gene regions were used. probes were labeled by digoxigenin-utp (roche diagnostics, penzberg, germany) and tested for specificty using human biopsy of gut tissue with in vitro infection of h5n1 virus. since h5n1 is a negative-stranded rna virus, sense probes were defined as the probes that detect the viral rna (negative-stranded), whereas antisense probes detected mrna and complementary rna (crna), which are both positive-stranded. briefly, before hybridization, all solutions were prepared with depc-treated water. after deparaffinization and rehydration, tissue sections of 5 mm thickness were treated with proteinase k digestion for 30 min. tissue sections were then incubated with a hybridization cocktail containing 25 mg/ml of probes at 37uc for 16,18 h. all sense and antisense probes were applied separately on consecutive tissue sections. after blocking with normal horse serum (1:100), sections were incubated with alkaline phosphatase-labelled digoxigenin antibody (1:1000, roche diagnostics, penzberg, germany) for 1 h, and the reaction products were colorised with nitroblue tetrazolium/5-bromo-4choloro-3-indolyl phosphate (roch diagnostics, penzberg, germany) for 1-1.5 h, and counterstained in 2% nucleic fast red liquid for 1 min. as a positive control, we used human lung and gut biopsy tissues with in-vitro infection of h5n1 virus. negative controls also included an unrelated antisense probe against the fragment of the heamaglutinin gene of the seasonal influenza virus h3n2 as well as h5n1 in-situ hybridization probes to tissues. after completeing the colorization reaction of the ish as outlined above, sections were incubated with 3% hydrogen peroxide to quench endogenous peroxidase activity. the sections were then blocked with 10% normal horse serum for 10 min at rt, before incubating with monoclonal antibodies (against cd3 + , cd68 + , cd34 + , d35 + ) for 1 h at rt. unbound antibody was removed by 3 washes in pbs before the addition of hrp-labeled polymer for 30 min at rt (dako csa detection system, denmark). after washing 3 times with pbs to remove unbound labeled polymer, peroxidase staining in tissue sections was revealed by dab solution (csa detection systems, dako, denmark). after stopping the reaction in running water, sections were counter-stained by 2% nuclear quick red solution for 30 s. for each tissue, double-labeled stain with ihc and ish was repeated three times for data analysis. to strengthen further the results of colocalization studies, we performed ish and ihc on consecutive sections. tissue sections showing ish-positive cells were carefully compared with consecutive tissue sections on which ihc with antibodies against specific cell markers was applied. co-localization of a specific cellular marker and viral genome was clearly identified. the correlation between viral load and quantitative proinflammatory factors profile was analyzed by pearson's correlation test using instat software (vision 5.0, graphpad prism). differences were considered significant at p,0.05. figure s1 the distribution of viral load in selected tissue samples. the viral ha gene and b-actin gene copies in tissues were determined by quantified real-time rt-pcr. the ratios between ha and b-actin gene copies which was showed by logarithm presented the viral-load level in different tissue. found at: doi:10.1371/journal.pone.0013315.s001 (0.11 mb tif) global epidemiology of influenza: past and present evolution and ecology of influenza a viruses characterization of an avian influenza a (h5n1) virus isolated from a child with a fatal respiratory illness cumulative number of confirmed human cases of avian influenza a/(h5n1) reported to who probable person-to-person transmission of avian influenza a (h5n1) avian flu: h5n1 virus outbreak in migratory waterfowl identification and subtyping of avian influenza viruses by reverse transcription-pcr highly pathogenic h5n1 influenza virus in smuggled thai eagles avian influenza virus (h5n1) mortality surveillance influenza a (h1n1): pandemic alert phase 6 declared, of moderate severity estimation of potential global pandemic influenza mortality on the basis of vital registry data from the 1918-20 pandemic: a quantitative analysis avian influenza virus (h5n1): a threat to human health the first confirmed human case of avian influenza a (h5n1) in mainland china avian influenza a (h5n1) in 10 patients in vietnam avian influenza a (h5n1) infection in humans human disease from influenza a (h5n1) clinical characteristics of 26 human cases of highly pathogenic avian influenza a (h5n1) virus infection in china clinical features and rapid viral diagnosis of human disease associated with avian influenza a h5n1 virus influenza a/ h5n1 virus infection in humans in cambodia fatal avian influenza a (h5n1) in a child presenting with diarrhea followed by coma pathology of fatal human infection associated with avian influenza a h5n1 virus influenza virus entry and infection require host cell n-linked 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immunopathology in influenza virus infection higher in vitro susceptibility of human t cells to h5n1 than h1n1 influenza viruses duck and human pandemic influenza a viruses retain sialidase activity under low ph conditions sialic acid species as a determinant of the host range of influenza a viruses pathology, molecular biology, and pathogenesis of avian influenza a (h5n1) infection in humans pathogenesis of hong kong h5n1 influenza virus ns gene reassortants in mice: the role of cytokines and b-and t-cell responses early and sustained innate immune response defines pathology and death in nonhuman primates infected by highly pathogenic influenza virus lethal h5n1 influenza viruses escape host anti-viral cytokine responses alveolar epithelial type ii cell: defender of the alveolus revisited proinflammatory response of alveolar epithelial cells is enhanced by alveolar macrophage-produced tnf-alpha during pulmonary ischemia-reperfusion injury type ii alveolar cells play roles in macrophage-mediated host innate resistance to pulmonary mycobacterial infections by producing proinflammatory cytokines massive destruction of malaria-parasitized red blood cells despite spleen closure biosafety level 3 laboratory for autopsies of patients with severe acute respiratory syndrome: principles, practices, and prospects a method for the rapid construction of crna standard curves in quantitative real-time reverse transcription polymerase chain reaction recommended laboratory tests to identify avian influenza a virus in specimens from humans functional tumor necrosis factor-related apoptosis-inducing ligand production by avian influenza virus-infected macrophages differential expression of chemokines and their receptors in adult and neonatal macrophages infected with human or avian influenza viruses a sensitive one-step real-time pcr for detection of avian influenza viruses using a mgb probe and an internal positive control the authors would like to thank the human avian influenza surveillance network of guangxi zhuang autonomous region and guangxi medical university for assisting sample collection, katz jackie m and wunju shieh from cdc for their assistance in experience. key: cord-000182-ni6iyzdn authors: he, zhisong; zhang, jian; shi, xiao-he; hu, le-le; kong, xiangyin; cai, yu-dong; chou, kuo-chen title: predicting drug-target interaction networks based on functional groups and biological features date: 2010-03-11 journal: plos one doi: 10.1371/journal.pone.0009603 sha: doc_id: 182 cord_uid: ni6iyzdn background: study of drug-target interaction networks is an important topic for drug development. it is both time-consuming and costly to determine compound-protein interactions or potential drug-target interactions by experiments alone. as a complement, the in silico prediction methods can provide us with very useful information in a timely manner. methods/principal findings: to realize this, drug compounds are encoded with functional groups and proteins encoded by biological features including biochemical and physicochemical properties. the optimal feature selection procedures are adopted by means of the mrmr (maximum relevance minimum redundancy) method. instead of classifying the proteins as a whole family, target proteins are divided into four groups: enzymes, ion channels, g-proteincoupled receptors and nuclear receptors. thus, four independent predictors are established using the nearest neighbor algorithm as their operation engine, with each to predict the interactions between drugs and one of the four protein groups. as a result, the overall success rates by the jackknife cross-validation tests achieved with the four predictors are 85.48%, 80.78%, 78.49%, and 85.66%, respectively. conclusion/significance: our results indicate that the network prediction system thus established is quite promising and encouraging. identification of drug-target interaction networks is an essential step in the drug discovery pipeline [1] . the emergence of molecular medicine and the completion of the human genome project provide more opportunity to discover unknown target proteins of drugs. many efforts have been made to discover new drugs in the past few years. however, the number of new drug approvals remains quite low (around only 30 per year). this is partially because many compounds or drug candidates have to be withdrawn owing to unacceptable toxicity. such failures have wasted a lot of money. it would be beneficial to develop computational methods for predicting the sensitivity and toxicity before a drug candidate was synthesized [2, 3, 4] . however, a number of problems need to be overcome in order to find out the exact effects of a drug. firstly, drugs could have numerous effects including positive and negative effects, and it is hard to find out and elucidate the possible effects; secondly, different people would have completely different responses to a drug even though the same gene products are only slightly different [5, 6, 7, 8] ; thirdly, it is very hard to trace the drug effects since the biological interaction pathways are extremely complicated in human beings. therefore, it would be very helpful for drug development if the interactions between drugs and target proteins could be predicted more accurately and the underlying mechanisms could be better understood. several computational approaches have been developed for analyzing and predicting drug-protein interactions. the most commonly used are docking simulations [9, 10, 11, 12] , literature text mining [13] , and combining chemical structure, genomic sequence, and 3d structure information [14] , among others (see, e.g., [15, 16, 17] ). machine learning and data mining methods have been widely used in the computational biology and bioinformatics area. many researchers have made lots of efforts to develop useful algorithms and softwares to investigate various drug-related biological problems, such as hiv protease cleavage site prediction [18, 19] , identification of gpcr (g protein-coupled receptors) type [20, 21] , protein signal peptide prediction [22] , protein subcellular location prediction [23, 24, 25] , analysis of specificity of galnac-transferase protein [26] , identification of protease type [27, 28] , membrane protein type prediction [29, 30, 31, 32] , and a series of relevant webserver predictors as summarized in a recent review [33] . here we propose a predictor for drug-target interactions based on the nearest neighbor algorithm [34] . since biochemical and physicochemical features [35] are important for characterizing proteins, in this study they are used to represent proteins as done by many previous investigators (see, e.g., [36, 37, 38] . to improve the predictor's performance, minimum redundancy maximum relevance (mrmr) algorithm [39] is used to rank the features. meanwhile, the incremental feature selection and forward feature selection are applied for feature selection. the protein targets for drugs are divided into enzymes, ion channels [40, 41, 42, 43] , gpcrs [44, 45] , and nuclear receptors [14] in this study. finally, four predictors for predicting the interactions of drugs with each of the four protein families are developed in hopes that they can help provide useful information for drug design. in addition to the dataset used by yamanishi et al. [14] , information about drug compounds and genes can be obtained from kegg [46, 47] by the ftp operations: ftp://ftp.genome.jp/ pub/kegg/ligand/drug/drug for the drugs, and ftp://ftp.genome. jp/pub/kegg/genes/fasta/gene.pep for the genes. after excluding the drug-target pairs that lack experimental information, we finally obtained a total of 4,797 drug-target pairs, of which 2,719 for enzymes, 1,372 for ion channels, 630 for gpcrs, and 82 for nuclear receptors. all these datasets were used as the positive datasets in the current study. the corresponding negative datasets were derived from the above positive datasets via the following steps: (1) separate the pairs in the above positive dataset into single drugs and proteins; (2) re-couple these singles into pairs in a way that none of them occurs in the corresponding positive dataset; (3) randomly picked the negative pairs thus formed until they reached the number two times as many as the positive pairs. the drug-target benchmark datasets thus obtained for enzymes, ion-channels, gpcrs, and nuclear receptors are given in online supporting information s1, s2, s3, and s4, respectively. representing drugs with chemical functional groups composition. the number of drugs is extremely large. however, most of them are small organic molecules and are composed of some fixed small structures, called functional groups. since functional groups usually represent the characteristics of a compound as well as its reaction mechanism with other molecules, features derived from its functional groups could be very effective in characterizing a drug. moreover, the number of common functional groups is quite small, and hence it is possible to use the functional group composition to uniquely represent a drug [48] . a number of functional groups are available in nature, and we selected the following 28 common groups for the current study: (1) alcohol, (2) aldehyde, (3) amide, (4) amine, (5) hydroxamic acid, (6) phosphorus, (7) carboxylate, (8) methyl, (9) ester, (10) ether, (11) imine, (12) ketone, (13) nitro, (14) halogen, (15) thiol, (16) sulfonic acid, (17) sulfone, (18) sulfonamide, (19) sulfoxide, (20) sulfide, (21) a_5c_ring, (22) ar_6c_ring, (23) non_ar_5c_ring, (24) non_ar_6c_ring, (25) hetero ar_6_ring, (26) hetero non_ar_5_ring, (27) hetero non_ar_6_ring, and (28) hetero ar_5_ring. thus, following the same treatment as in [23] , a drug compound can now be formulated as a 28-d (dimensional) vector given below: where g i (i~1, 2, á á á , 28) is the occurrence frequency of the i-th functional group in the drug d, and t the matrix transpose operator. representing target proteins with pseudo amino acid composition by incorporating biochemical and physicochemical features. now the problem is how to effectively represent a target protein. two kinds of representations are generally used in this regard: the sequential representation and the non-sequential representation. the most typical sequential representation for a protein sample is its entire amino acid sequence, which can contain the most complete information of a protein. to deal with this model, the sequence-similarity-searchbased tools, such as blast [49] , are usually used to find the desired results. unfortunately, this kind of approach failed to work when the query protein did not have significant homology to the proteins in the training dataset. thus, various non-sequential representations or discrete models were proposed. the simplest discrete model was based on the amino acid composition (aac) (see, e.g., [50] ). however, if using the aac model to represent a protein, all its sequence-order information will be lost. to avoid completely losing the sequence-order information, the pseudo amino acid composition (pse-aac) was proposed [36] to represent the sample of a protein. the pseaac can be used to represent a protein sequence with a discrete model yet without completely losing its sequence-order information. for further information about pseaac, see the web-page by clicking the link http://en. wikipedia.org/wiki/pseudo_amino_acid_composition. ever since the concept of pseaac was introduced, it has been widely used to study various problems in proteins and protein-related systems (see, e.g., [37, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66] ). meanwhile, many different forms of discrete models were also proposed (see, e.g., [20, 30, 32, 51, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82] ). however, regardless of how much different these models are, they just belong to different forms of pseaac, as elucidated in a recent comprehensive review [83] . here, we are to propose a different pseaac to represent drug-targeted proteins in terms of their biochemical and physicochemical features [84] . six different types of features were considered: (1) hydrophobicity, (2) polarizability, (3) polarity, (4) secondary structure, (5) normalized van der waals volume, and (6) solvent accessibility. each amino acid residue in a protein sequence can be represented by a set of different states according to its features. for instance, its hydrophobicity feature can be marked by one of the following three states: ''polar'', ''neutral'', or ''hydrophobic'' [85] ; its solvent accessibility feature by one of the two: ''buried'' or ''exposed to solvent'', as predicted by predacc [35] ; its secondary structure feature by one of the three: ''helix'', ''sheet'', or ''coil'', as predicted by the method in [86] ; and so forth. thus, a protein sequence can be translated to a series of codes according to the biochemical and physicochemical properties of its constituent amino acid residues. for example, if using ''p'', ''n'' and ''h'' to represent the three states of hydrophobicity: ''polar'', ''neutral'', and ''hydrophobic'', the protein sequence ''dmaeimsdkp-qagml'' can be translated to ''phnphhnppnpnnhh'' according to the codes of the hydrophobic property feature. the encoded sequences thus obtained would have different length for proteins of different sizes, which will make the prediction engine difficult to handle. to make the feature-encoded sequence to be a vector with a fixed number of dimensions, three properties of a sequence was used: composition (c), transition (t), and distribution (d). c represents the global composition of each letter in the sequence; t, the frequency of a code letter changing from one to another; d, the distribution pattern of the code letters along the sequence, measuring the percentage of the sequence length within which the first, 25%, 50%, 75%, and 100% of the amino acids of each code letter is located. take the above hydrophobic property sequence as an example: its c feature is 5/15 = 33.3% for all of p, h, and n, while the t feature is 2/10 = 20%, 3/10 = 30% and 5/10 = 50% for the changes between h and p, n and h, n and p, respectively. the measurement of feature d is a little more complicated. for the letter h, the first, 25%, 50%, 75% and 100% of hs in the sequence is located at the position of 2, 5, 6, 14, and 15. thus its d feature is ( , with a total of 21 components. likewise, for the sequences encoded by the other four biochemical properties, each is also corresponding to 21 components. but for the sequence encoded by the solvent accessibility with only two states (''buried'' or ''exposed to solvent''), the encoded sequence is corresponding to only 14 components. finally, by adding the 20 components of aac [87] into the vector concerned, the total number of components thus obtained for a given protein is 5|21z20z14~139; i.e., the protein can be formulated as a 139-d vector given by where p i (i~1, 2, á á á , 139) is the i-th component of the protein p. of the 139 components, 119 are derived according to the codes of the above six biochemical and physicochemical features, and 20 are the aac components of p. with all samples represented by a feature vector, now it is possible for us to construct our predictor using the machine learning approach. the nn (nearest neighbor) algorithm is quite popular in pattern recognition community owing to its good performance and simple-to-use feature. according to the nn rule [88] , the query sample should be assigned to the subset represented by its nearest neighbor. in this study, if the drug-target pair with the shortest distance is a positive sample, meaning that they can interact with each other, the sample for test is seen as a positive drug-target pair. otherwise, the test sample is seen as a negative one. there are many different definitions to measure the ''nearness'' for the nn algorithm, such as euclidean distance, hamming distance [89] , and mahalanobis distance [50, 90, 91] . in the current study, the following equation was adopted to measure the nearness between samples v x and v y where v x : v y is the dot product of the two vectors, and v x k k and v y their modulus, respectively. when v x :v y we have d(v x ,v y )~0, indicating the ''distance'' between these two sample vectors is zero and hence they have perfect or 100% similarity. after constructing the drug-target interaction predictor, we have to evaluate its performance. in statistical prediction, the following three cross-validation methods are often used to examine a predictor for its effectiveness in practical application: independent dataset test, subsampling (k-fold cross-validation) test, and jackknife test [92] . however, as elucidated by [24] and demonstrated by eq.50 in [93] , among the three cross-validation methods, the jackknife test is deemed the most objective that can always yield a unique result for a given benchmark dataset, and hence has been increasingly used and widely recognized by investigators to examine the accuracy of various predictors (see, e.g. [51, 53, 54, 55, 56, 57, 59, 62, 63, 64, 94, 95, 96] ).'' accordingly, in this study the jackknife cross-validation was adopted to calculate the success prediction rates as well. although we've constructed the drug-target predictor based on the original feature set described above, it is possible to improve its performance with a better feature set. apparently, not every feature in the feature set is equally relevant to the drug-target interaction. what's more, features may not be independent with each other. the ''bad'' will have negative impact on the accuracy and efficiency of the predictor, so it is possible to do the feature selection process to construct a more compact and effective feature set. the first step is using maximum relevance minimum redundancy (mrmr) [36] to do feature evaluation. maximum relevance minimum redundancy (mrmr) [39] was firstly developed for analysis of microarray data. it ranks each feature according to its relevance to the target and redundancy to other features. the better a feature is deemed to be, the higher the rank it will be assigned to. mutual information (mi), denoted by i to indicate the dependence of two features used to quantify the relevance and redundancy. mi is defined as following: based on mi, we can quantify relevance (d) and redundancy (r) as: where f candidate is the feature to be calculated, and c is the target variable. by combining the above two equations to maximize relevance and minimize redundancy, the following mrmr function is constructed: where v s and v t are the already-selected feature set and to-beselected feature set, respectively, and m and n are the sizes of these two feature sets, respectively. the earlier a feature is selected, the better it would be though of. finally, we can get an ordered feature list with a rank for every feature to indicate its importance in the feature set. in our study, the mrmr program is obtained from: http://research. janelia.org/peng/proj/mrmr/index.htm. to calculate mi, the joint probabilistic density and the marginal probabilistic densities of the two vectors were used. a parameter t is introduced here to deal with these variables. suppose mean to be the average value of one feature in all samples, and std to be the standard deviation, the feature of each sample would be classified into one of the three groups according to the boundaries: mean+(t : std). in our study, t was assigned to be 1. as mentioned above, the importance of each feature is rated according to its rank in the mrmr analysis. the next step is to determine which features should be selected as the optimal feature set for our drug-target predictor. here the ifs (incremental feature selection) procedure is used to solve the problem. each feature in the mrmr feature list was added one by one, and n different feature sets are obtained if the total feature number is n, while the i-th feature set is: based on each of the n feature sets, an nn algorithm predictor was constructed and tested with the jackknife cross-validation test. with all the n overall accurate rates calculated, we could draw an ifs curve with the index i to be the x-axis and the corresponding overall accurate rate to be the y-axis. thus, s opt~f f 1 , f 2 , :::, f n g is regarded as the optimal feature set if the curve reach its peak where the value of its x-axis is nƒn. because four independent predictors are needed for the four different classes of drug-target pairs, the ifs analysis procedure will be processed four times with each for a specific predictor. to refine feature selection, the ffs (forward feature selection) procedure based on the result of ifs was used. ffs is a feature selection method based on ifs results which tries every feature in the candidate feature set and adds the feature that achieves the highest prediction accuracy into the already-selected feature set in each goes. suppose the ifs curve reaches its peak with apex as its x-axis, the initial ffs-selected feature set was constructed as: more features in ffs-to-be-selected feature set would be added into the ffs-selected feature set one by one. the ffs-to-beselected feature set with m features covers the features with mrmr ranks between k+1 and k+1+m, where m is a user-defined positive integer smaller than n{k with n to be the size of the original feature set. in each round of ffs, each feature in ffs-tobe-selected feature set would be taken out and added to the ffsselected feature set. each predictor based on each new ffsselected feature set would be tested, and the feature set obtained the highest overall accurate rate would be used as the new ffsselected feature set. this process would be run for m times, until the ffs-to-be-selected feature set becomes a null set. an ffs curve similar to the ifs curve could be drawn with x-axis as the index and y-axis as the overall accurate rate. in this study, ffs was run for each of the four benchmark datasets based on the corresponding ifs result. m for all these processes was set to 50, while k for each ffs was set to be the index of the point with the first maximum value (i.e. the maximum point with the smallest index) in the corresponding ifs curve. to improve performance of the predictor of drug-target interaction, feature selection process was carried out. the first step of feature selection is feature evaluation. in this study, mrmr was used to evaluate every feature in original feature set. listed in online supporting information s5 are two kinds of outputs: the first one is the maxrel list which shows ranks of features for their relevance to the target; the second is mrmr list showing the mrmr ranks according to the feature order satisfying eq. 3. in this study, only the mrmr list was used as the results of feature evaluation. since there are four groups of samples, mrmr was run four times with each for one of them. with the four mrmr lists, ifs was processed for each of the four sample groups, generating four ifs curves. based on these results, we set k in ffs to be 16, 15, 14 and 19 for the data of enzymes, ion channels, gpcrs and nuclear receptors, respectively. each of these figures is the index of the point of the first maximum value in the corresponding ifs curve. shown in fig. 1 are the four ifs curves with their corresponding ffs curves. the peaks of the four ffs curves finally reach the overall success rates of 85.48% with 32 features, 80.78% with 37 features, 78.49% with 30 features, and 85.66% with 32 features for enzyme group, ion channel group, gpcr group and nuclear receptor groups, respectively. features selected by mrmr+ffs for the four different groups are quite different from each other, showing the intrinsic differences between them. although there are more features for target than those for drug in the original feature set, more drug features were selected, showing the important role of drugs. many of the selected target features are for protein secondary structure, especially for enzyme group (half of selected target features are for this). all types of features are selected in at least one group, showing that all biochemical and physicochemical features have their irreplaceable positions in drug-target interaction process. for the details of the optimal feature-set outputs by ffs for the four benchmark datasets, see the online supporting information s6. for the specificity and promiscuity, we divided the drug-protein interactions into four groups according to the targets of drugs: enzymes, ion channels, gpcrs, and nuclear receptors. we used all the known drugs and target proteins in the gold standard data as training data to predict the potential interactions between all human proteins annotated as members of the four classes in kegg genes and all compounds in kegg ligands. enzyme recognition is the primary event involved in the interaction of proteins with other proteins and with small molecules such as metabolites and therapeutics. predicting drugenzyme interactions has direct application for completing genome annotations, finding enzymes for synthetic chemistry, and predicting drug specificity, promiscuity and pharmacology. it is suggested that the secondary structure information plays the major role in determining the drug-enzyme interactions activity. for example, cytochrome p450 (cyp) induction-mediated interaction is one of the major concerns in clinical practice and for the pharmaceutical industry [97] . induction of cyp1a enzymes with a specific structure-stable state may activate some xenobiotics to their reactive metabolites, leading to toxicity [98, 99] . amino acid composition and hydrophobicity also contribute considerably to these interactions. an insertion/deletion (i/d) polymorphism of the angiotensin i-converting enzyme (ace) have an influence on the antihypertensive response, particularly when using ace inhibitors (acei) [100] , mirroring that the amino acid composition did contribute to the interactions. hydrophobicity plays a role in determining the coefficients of drug-enzyme interaction energy with the application to drug screening as well as in silico target protein screening [101, 102] . the g-protein coupled receptor (gpcr) superfamily, which is comprised of estimated 600-1,000 members, is another largest known class of molecular targets with varieties of physiological activities and proven therapeutic value [103] . they are integral membrane proteins sharing a common global topology that consists of seven transmembrane alpha helices, intracellular cterminal, an extracellular n-terminal, three intracellular loops and three extracellular loops [33, 44] . it is suggested that secondary structure and polarity would play a major role in determining the drug-gpcrs interactions activity. small secondary structures such as helices and loops are identified as entities potentially involved in stabilizing interactions with ligands [33] . these motifs were situated mainly in the apical region of transmembrane segments and included a few extracellular residues [104] . crystal structures of engineered human beta 2-adrenergic receptors (ars) in complex with an inverse agonist ligand, carazolol, provide threedimensional snapshots of an important g protein-coupled receptor (gpcr) with a beta-sheet structure and forms part of the chromophore-binding site [105] . glida provides interaction data between gpcrs and their ligands, along with chemical information on the ligands, as well as biological information regarding gpcrs [106] . some of the features reflect physical interactions that are responsible for the structural stability of the transmembrane, the formation of extensive networks of interhelical h-bonds and sulfur-aromatic clusters that are spatially organized as ''polarity'', the close packing of side-chains throughout the transmembrane domain. when more experimental 3d structures become available for gpcrs in the future, this will help building reliable models for a wider range of gpcrs that would be suitable for docking studies. joint use of ligand-based chemogenomic and docking would certainly improve the prediction. ion channels are a large superfamily of membrane proteins that pass ions across membranes and are critical to diverse physiological functions in both excitable and nonexcitable cells and underlie many diseases. as a result, they are an important target class which is proven to be highly ''druggable''. according to our analysis, secondary structure and polarity play the major role in determining the drug-ion channels interactions activity. secondary structure controls the membrane potential and interrogates ion channels in different conformational states. the drug-ion channels interaction needs gated state where they can switch conformation between a closed and an open state [42, 43] . simulations on model nanopores reveal that a narrow hydrophobic region can form a functionally closed gate in the channel and can be opened by either a small increase in pore radius or an increase in polarity [107, 108] . nowadays, intense research is being conducted to develop new drugs acting selectively on ion channel subtypes and aimed at the understanding of the intimate drug-channel interaction [109] . nuclear receptors (nr) are ligand-activated transcription factors that regulate the activation of a variety of important target genes, which are the most important drug targets in terms of potential therapeutic application. according to our results, secondary structure and polarizability play the major role in determining the drug-nrs interactions. the conservative motif of the nr is typically described as three stacked alpha-helical sheets. the helices that make up the ''front'' and ''back'' sheets are aligned parallel to one another. the helices in the middle sheet run across the two outer sheets and only occupy the space in the upper portion of the domain. the space in the lower part of the domain is relatively void of protein, and for most nrs, this creates an internal cavity for small-molecule ligands [110] . hydrogen bonds with polarizability activity play a crucial role in protein-drug interactions (see, e.g., [11] ). our approaches and the results thus obtained could be used to demonstrate how nuclear hormone receptors form a network of direct interactions. and this network increases in complexity to describe the interactions with target genes as well as small molecules known to bind a receptor, enzyme, or transporter. a comprehensive drug-target interaction network system has been established that contains four classifiers for predicting the drugable interaction of compounds with enzymes, ion-channels, gpcrs, and nuclear receptors, respectively. it is anticipated that the network predictor system may become a very useful tool for drug development. particularly it may help us find new or potential drug-target interactions. online supporting information s1 the benchmark dataset for the drug-target enzyme interaction system. it contains 8,157 genedrug pair samples, of which 2,719 are positive and 5,438 negative. the 1st column of the table indicates the nature of samples with 1 for positive and 2 for negative; the 2nd column shows the code of target gene; and the 3rd column shows the code of drug. all the detailed information for the genes and drugs listed here can be found in a guide to drug discovery: target selection in drug discovery predicting human safety: screening and computational approaches assessment of chemical libraries for their druggability review: progress in computational approach to drug development against sars 3d structure modeling of cytochrome p450 2c19 and its implication for personalized drug design molecular modeling of two cyp2c19 snps and its implications for personalized drug design review: pharmacogenomics and personalized use of drugs review: structure of cytochrome p450s and personalized drug structure-based maximal affinity model predicts small-molecule druggability a fast flexible docking method using an incremental construction algorithm review: structural bioinformatics and its impact to biomedical science binding mechanism of coronavirus main proteinase with ligands and its implication to drug design against sars a probabilistic model for mining implicit 'chemical compound-gene' relations from literature prediction of drug-target interaction networks from the integration of chemical and genomic spaces statistical prediction of protein chemical interactions based on chemical structure and mass spectrometry data integrating statistical predictions and experimental verifications for enhancing protein-chemical interaction predictions in virtual screening alignment-free prediction of a drug-target complex network based on parameters of drug connectivity and protein sequence of receptors a vectorized sequence-coupling model for predicting hiv protease cleavage sites in proteins review: prediction of hiv protease cleavage sites in proteins gpcr-ca: a cellular automaton image approach for predicting g-protein-coupled receptor functional classes gpcr-gia: a web-server for identifying gprotein coupled receptors and their families with grey incidence analysis signal-cf: a subsite-coupled and window-fusing approach for predicting signal peptides using functional domain composition and support vector machines for prediction of protein subcellular location cell-ploc: a package of web-servers for predicting subcellular localization of proteins in various organisms euk-mploc: a fusion classifier for large-scale eukaryotic protein subcellular location prediction by incorporating multiple sites a sequence-coupled vector-projection model for predicting the specificity of galnac-transferase protident: a web server for identifying proteases and their types by fusing functional domain and sequential evolution information prediction of protease types in a hybridization space prediction of membrane protein types and subcellular locations low-frequency fourier spectrum for predicting membrane protein types memtype-2l: a web server for predicting membrane proteins and their types by incorporating evolution information through pse-pssm support vector machines for predicting membrane protein types by using functional domain composition review: recent advances in developing web-servers for predicting protein attributes a k-nearest neighbor classification rule based on dempster-shafer theory predacc: prediction of solvent accessibility prediction of protein cellular attributes using pseudo amino acid composition using amphiphilic pseudo amino acid composition to predict enzyme subfamily classes digital coding of amino acids based on hydrophobic index feature selection based on mutual information: criteria of max-dependency, max-relevance, and min-redundancy insights from modelling three-dimensional structures of the human potassium and sodium channels the structure of phospholamban pentamer reveals a channel-like architecture in membranes mechanism of drug inhibition and drug resistance of influenza a m2 channel structure and mechanism of the m2 proton channel of influenza a virus prediction of g-protein-coupled receptor classes coupling interaction between thromboxane a2 receptor and alpha-13 subunit of guanine nucleotide-binding protein ligand: chemical database for enzyme reactions from genomics to chemical genomics: new developments in kegg predicting networking couples for metabolic pathways of evaluating the statistical significance of multiple distinct local alignments a novel approach to predicting protein structural classes in a (20-1)-d amino acid composition space prediction of protein secondary structure content by using the concept of chou's pseudo amino acid composition and support vector machine use of fuzzy clustering technique and matrices to classify amino acids and its impact to chou's pseudo amino acid composition using the concept of chou's pseudo amino acid composition to predict apoptosis proteins subcellular location: an approach by approximate entropy predicting protein subcellular location using chou's pseudo amino acid composition and improved hybrid approach the modified mahalanobis discriminant for predicting outer membrane proteins by using chou's pseudo amino acid composition predicting subcellular localization of mycobacterial proteins by using chou's pseudo amino acid composition prediction of subcellular localization of apoptosis protein using chou's pseudo amino acid composition prediction of g-protein-coupled receptor classes based on the concept of chou's pseudo amino acid composition: an approach from discrete wavelet transform using the augmented chou's pseudo amino acid composition for predicting protein submitochondria locations based on auto covariance approach predicting the cofactors of oxidoreductases based on amino acid composition distribution and chou's amphiphilic pseudo amino acid composition predicting lipase types by improved chou's pseudo-amino acid composition using chou's amphiphilic pseudoamino acid composition and support vector machine for prediction of enzyme subfamily classes using chou's pseudo amino acid composition to predict subcellular localization of apoptosis proteins: an approach with immune genetic algorithm-based ensemble classifier prediction of cell wall lytic enzymes using chou's amphiphilic pseudo amino acid composition medicinal chemistry and bioinformatics -current trends in drugs discovery with networks topological indices proteomics, networks, and connectivity indices application of pseudo amino acid composition for predicting protein subcellular location: stochastic signal processing approach weighted-support vector machines for predicting membrane protein types based on pseudo amino acid composition slle for predicting membrane protein types using pseudo amino acid composition to predict protein structural classes: approached with complexity measure factor using pseudo amino acid composition to predict protein subcellular location: approached with lyapunov index, bessel function, and chebyshev filter using cellular automata to generate image representation for biological sequences using cellular automata images and pseudo amino acid composition to predict protein subcellular location using pseudo amino acid composition to predict transmembrane regions in protein: cellular automata and lempel-ziv complexity using pseudo amino acid composition to predict protein structural class: approached by incorporating 400 dipeptide components predicting protein structural classes with pseudo amino acid composition: an approach using geometric moments of cellular automaton image using grey dynamic modeling and pseudo amino acid composition to predict protein structural classes predicting membrane protein type by functional domain composition and pseudo amino acid composition hum-ploc: a novel ensemble classifier for predicting human protein subcellular localization large-scale plant protein subcellular location prediction predicting membrane protein types by the llda algorithm predicting eukaryotic protein subcellular location by fusing optimized evidence-theoretic k-nearest neighbor classifiers pseudo amino acid composition and its applications in bioinformatics, proteomics and system biology recognition of a protein fold in the context of the structural classification of proteins (scop) classification the classification and origins of protein folding patterns seventy-five percent accuracy in protein secondary structure prediction predicting protein folding types by distance functions that make allowances for amino acid interactions a fuzzy k-nearest neighbours algorithm discriminant analysis; chapter 12 multivariate analysis of variance on the generalized distance in statistics this reference also presents a brief biography of mahalanobis who was a man of great originality and who made considerable contributions to statistics review: prediction of protein structural classes review: recent progresses in protein subcellular location prediction an intriguing controversy over protein structural class prediction some insights into protein structural class prediction subcellular location prediction of apoptosis proteins cyp induction-mediated drug interactions: in vitro assessment and clinical implications cyp1a1: friend or foe? inhibition and induction of human cytochrome p450 enzymes: current status angiotensin i-converting enzyme gene polymorphism and drug response genome scale enzymemetabolite and drug-target interaction predictions using the signature molecular descriptor svm-prot: web-based support vector machine software for functional classification of a protein from its primary sequence molecular tinkering of g protein-coupled receptors: an evolutionary success critical role for the second extracellular loop in the binding of both orthosteric and allosteric g protein-coupled receptor ligands structural basis for ligand binding and specificity in adrenergic receptors: implications for gpcr-targeted drug discovery glida: gpcr-ligand database for chemical genomics drug discovery-database and tools update investigation into adamantane-based m2 inhibitors with fb-qsar an in-depth analysis of the biological functional studies based on the nmr m2 channel structure of influenza a virus ion channel pharmacology the nuclear receptor superfamily and drug discovery key: cord-002935-jq1xumrh authors: postnikova, elena; cong, yu; dewald, lisa evans; dyall, julie; yu, shuiqing; hart, brit j.; zhou, huanying; gross, robin; logue, james; cai, yingyun; deiuliis, nicole; michelotti, julia; honko, anna n.; bennett, richard s.; holbrook, michael r.; olinger, gene g.; hensley, lisa e.; jahrling, peter b. title: testing therapeutics in cell-based assays: factors that influence the apparent potency of drugs date: 2018-03-22 journal: plos one doi: 10.1371/journal.pone.0194880 sha: doc_id: 2935 cord_uid: jq1xumrh identifying effective antivirals for treating ebola virus disease (evd) and minimizing transmission of such disease is critical. a variety of cell-based assays have been developed for evaluating compounds for activity against ebola virus. however, very few reports discuss the variable assay conditions that can affect the results obtained from these drug screens. here, we describe variable conditions tested during the development of our cell-based drug screen assays designed to identify compounds with anti-ebola virus activity using established cell lines and human primary cells. the effect of multiple assay readouts and variable assay conditions, including virus input, time of infection, and the cell passage number, were compared, and the impact on the effective concentration for 50% and/ or 90% inhibition (ec(50), ec(90)) was evaluated using the fda-approved compound, toremifene citrate. in these studies, we show that altering cell-based assay conditions can have an impact on apparent drug potency as measured by the ec(50). these results further support the importance of developing standard operating procedures for generating reliable and reproducible in vitro data sets for potential antivirals. ebola virus (ebov) infection in humans and nonhuman primates is often associated with high morbidity and mortality rates, as well as severe hemorrhagic fever [1] [2] [3] [4] . ebov is a biosafety level-4 pathogen transmitted by contact with bodily fluids, fomites, or droplets from plos infected patients. ebov is considered a significant threat to public health and global security due to its potential to be used as a bioweapon [5] [6] [7] [8] . currently, no fda-approved vaccine or therapeutic agents are available, and supportive care remains the standard for ebola virus disease (evd) treatment. therefore, accelerated efforts in the development of therapeutics is a key objective in the ebov research community, especially since the 2013-2016 evd epidemic in western africa. drug discovery and development requires considerable time and resources to identify an effective drug that will progress to clinical trials [9, 10] . as a result, research investigating the repurposing of drugs for additional indications have become increasingly more prevalent to accelerate the identification of therapeutic drugs for evd. the off-label use of fda-approved drugs is particularly advantageous as safety concerns and ethical problems have already been addressed [11] [12] [13] [14] . to effectively identify potential compounds of interest from large libraries of chemical compounds, share more reliable and reproducible data between laboratories, and provide data to the international community, appropriate methods or models need to be established. furthermore, these models should be evaluated to determine how predictive they are for identifying compounds most likely to be efficacious in humans. for evd, indications of efficacy could include successful treatment and survival of patients, alleviation of disease severity, or mitigation of clinical symptoms associated with ebov infection. a variety of methods are available to measure antiviral activity in vitro. however, the development of a screening assay to detect compounds with anti-ebov activity was previously limited due to the difficulty of developing a suitable high-throughput screening system for a biosafety level-4 viral pathogen. classical methods evaluate drug efficacy include the reduction of virus yield [15] or a decrease in viral rna transcription as determined by real-time polymerase chain reaction (pcr) [16] [17] [18] . recent therapeutic screening methods have transitioned from the classical methods of measuring viral inhibition to assays with the ability to be automated, resulting in higher-throughput. assay chemistries have been developed to enable the homogeneous measurement of a variety of different endpoints such as cytopathic effect, viral protein or reporter gene expression, which can serve as markers of viral replication [19] . the growing interest in identifying drugs with activity against ebov has resulted in a variety of assays and readouts for activity as well as cytotoxicity. the use of cell-based assays for highthroughput screening of compound libraries has increased steadily over recent years [20] [21] [22] [23] . as cell-based assays monitor specific viral proteins and provide the means to screen for potent viral inhibitors intracellularly, these assays identify drug candidates with desired pharmacological properties in the primary drug-discovery pipeline. in this study, the susceptibility to ebov using both immortalized cell lines and primary monocyte-derived macrophages (mdms) was investigated under a variety of conditions such as different multiplicities of infection (mois), times of exposure, and the cell passage numbers. a cell-based assay with ebov vp40-specific antibody was used to detect infected cells. fluorescence or chemiluminescence readout was used for determining signal-to-noise (s/n) ratio, and a high-content imaging system was applied to determine the percentage of ebov-positive cells. toremifene citrate, which the world health organization considered evaluating in a clinical trial for treatment of evd, was chosen as a positive control to measure ebov inhibition under each condition [24] [25] [26] [27] . the conditions under which drugs are tested can influence their apparent potency. while testing drugs for the world health organization (who) community, we received many requests to repeat experiments under specific conditions to confirm activity identified by another laboratory. the resulting data sets indicated just how variable the ec 50 value can be under varying assay conditions. the data presented here provides insight on how different assay parameters can impact the in vitro efficacy of potential anti-ebov antivirals using toremifene citrate as a model compound. vero e6 (african green monkey kidney; atcc 1586) cells were obtained from the american type culture collection (manassas, va). vero c1008 (e6) cells (african green monkey kidney, working cell bank nr-596) were obtained through bei resources (national institute of allergy and infectious diseases [niaid] , national institutes of health [nih], manassas, va). huh 7 cells (human hepatocellular carcinoma) were obtained from dr. hideki ebihara (niaid, rocky mountain laboratories, hamilton, mt). all cell lines were maintained at the integrated research facility (irf) following cell source instructions. a primary vero e6 and huh 7 cells culture were grown to 90% confluency in a t-175 (fisher scientific) or triple layer tissue culture flask (nunc) containing dulbecco's modification of eagle medium (dmem) (gibco) supplemented with 10% heat-inactivated fetal bovine serum (fbs) (sigma). cells were dispersed by trypsin (gibco) treatment and then reseeded into secondary cultures. the process of removing cells from the primary culture, diluting, and then transferring them to secondary cultures constitutes a passage. both cell lines were provided at passages 4-22, at which point a new culture was introduced and the previous passage series was ended. additionally, cell cultures were required to be a least 85% viable in order to achieve acceptance criteria and to be plated for use in a screening assay. the generation of mdms has been described in previous studies [28, 29] . briefly, pbmcs were isolated from human whole blood by density-gradient centrifugation over histopaque (1.077 g/ml, sigma-aldrich, st. louis. mo). monocytes were purified using human cd14-specific microbeads (miltenyi biotec, san diego, ca, 130-050-201) following manufacturer's instructions. cd14 + monocytes were differentiated into mdms by culturing for 6-7 days with recombinant human macrophage colony-stimulating factor (bio-techne, minneapolis, mn, 216-mc-005) and conditioned medium from kpb-m15 cells (kind gift from dr. atsunobu hiraoka, scgf research laboratory, kyoto, jp). media were replaced every 2-3 days during the incubation for a total of 6-7 days. the cells were harvested and plated on desired 96-well plates 1 day prior to the drug screen assay. the differentiated mdms were characterized by flow cytometry before assay initiation. toremifene citrate (oral solution) tested in this study was purchased from sigma-aldrich (cas 89778-27-8; t7204-5mg). the makona 05 isolate of ebov (h. sapiens-tc/gin/14/wpg-c05) (ebov/mak, genbank accession no. kp096420), a kind gift from dr. gary p. kobinger (public health agency of canada, winnipeg, ca), was used in these studies. to generate virus stocks, ebov/mak was inoculated at an moi of 0.01 in vero c1008 cells (bei resources, manassas, va, catalog nr-596). when the cytopathic effect was visible at day 5-7 after infection (51-75% of monolayer showing cpe), cell culture supernatants were harvested and clarified by centrifugation. the ebov/mak titer was determined by plaque assay in vero e6 cells. virus titers were measured using 10-fold serial dilutions of culture supernatant in triplicate infections of vero e6 cell monolayers in 6-well plates. after incubation at 37˚c for 1 h (plates were rocked every 15 minutes), 2 ml of medium containing 2x mem (gibco), 1.25% avicel (fmc biopolymer), and 1x antibiotic-antimycotic (gibco) were added to each well (2ml/well). after 7 days post-incubation, virus plaques were stained with 0.2% crystal violet (ricca chemical) in 10% neutral buffered formalin (thermo scientific) and infectivity titers were measured in plaque forming units per ml (pfu/ml). all procedures using live ebov were performed under biosafety level-4 (bsl-4) conditions. vero e6 and huh 7 cells were seeded overnight at 3 to 4 × 10 4 cells per well and mdm cells were plated at 1 × 10 5 cells per well in 100 μl of dulbecco's modified eagles's medium with 10% fetal bovine serum in black opaque (thermo fisher scientific, waltham, ma, corning 3916, 07-200-627) or clear bottom 96-well greiner microplates (greiner bio-one, monroe, nc, 655948). ebov/mak isolate was diluted in culture media to the specified mois (the titers used to determine moi hereby were generated on vero e6 cells) in 96-well plates. the cells were then infected by transferring 50 μl of ebov/mak isolate from the virus dilution plates to cell plates using the 96-well liquidator (rainin instrument, oakland, ca). the cells were incubated at 37˚c and 5% co 2 for the indicated periods of time. the plates were fixed by adding 200 μl of 20% neutral-buffered formalin (final concentration 10%) at 24, 48, 72 or 96 h postinoculation (hpi). after fixing for 24 h, the plates were transferred to a bsl-2 lab for antibody staining as described previously [28] . briefly, ebov was detected by exposure of the infected, fixed, and permeabilized cells to a monoclonal mouse antibody specific to the ebov vp40 matrix protein (b-md04-bd07-ae11, made by us army medical research institute of infectious diseases, frederick md under centers for disease control and prevention contract) [30] , followed by staining with an alexafluor 1 594 goat anti-mouse igg (heavy + light chains) antibody (life technologies, carlsbad, ca) at 37˚c for 1 h. fluorescence was quantified using a tecan plate reader (infinite 1 m1000, tecan us, morrisville, nc) or an high-content imaging (hci) system (operetta, perkinelmer, waltham, ma). hci images were collected at 20x magnification using 1 to 9 fields of view in each well to quantify the percent of ebov-positive cells. the viability of the cell layer was monitored by staining cell nuclei with the hoechst 33342 dye (molecular probes) at 37˚c for 2 h. columbus 2.4.2 software (perkinelmer) was used to analyze the hci data. the chemiluminescent enzyme-linked immunosorbent assay (celia) was performed by detecting ebov with the anti-ebov vp40 antibody followed by staining with the horseradish peroxidase (hrp)-conjugated goat anti-mouse secondary antibody (seracare, milford, ma, cat. #074-1802). chemiluminescence was quantified using pico chemiluminescent substrate (thermo fisher scientific inc., rockford, il) and a plate reader (infinite 1 m1000 tecan). vero e6, huh 7 and mdm cells were seeded as described above in 100 μl media overnight at 37˚c with 5% co 2 . compounds in dimethyl sulfoxide were prediluted to reduce dimethyl sulfoxide concentration to 0.05% or lower. compounds were prediluted in dilution blocks before performing a final 1:4 dilution by transferring 50 μl of each compound to cell plates containing 150 μl of cell culture media. this dilution achieved a desired compound concentration in 200 μl of cell culture media. the process of performing the drug screen assay is shown in fig 1. three plates were set up per experiment, two plates (clear bottom 96-well greiner microplates) for detecting inhibition of ebov, and one mock plate (black opaque plate) for determining drug cytotoxicity. after 1 h of predilution and transport to the bsl4 laboratory, 50 μl of virus (or mock control) at the desired moi was added to cells. at 48, 72 or 96 hpi, assay plates were fixed at final concentration of 10% nbf for 24 h before transferring to a bsl-2 lab for staining. infected cells were detected as described above. to further confirm the accuracy of assays with high background, chemiluminescence assay was performed afterwards. cytotoxicity in mock infected cell plates was measured 48 or 72 h after treatment with compounds using the celltiter glo luminescent cell viability assay kit according to the manufacturer's instructions (promega, madison, wi). luminescence was read on the infinite 1 m1000 tecan plate reader (fig 1) . non-linear regression analysis and curve fitting parameter were performed to calculate ec 50 s, ec 90 s and 50% cytotoxic concentration (cc 50 s) (graphpad software, la jolla ca) [12] using dose-response curves for the compounds (toremifene citrate). error bars of dose-response curves represent the standard deviation of three replicates. equations for the ratio of s/n, percentage of ebov-positive infected cell, and z' factor were defined previously [31, 32] , and ec 50 s were used as parameters for assay validation. the quality control of cell-based assay is at specific assay endpoints, cells are fixed and transferred to the bsl-2. immunostaining was performed with a ebov-specific antibody against vp40 and a fluorescent or chemiluminescent secondary antibody using a plate washer/dispenser. fluorescence is quantified on a plate reader. the hci system (operetta) is used to detect ebov-positive cells and count cells with a nuclei stain (hoechst 33342). in parallel, cytotoxicity assays (celltiter glo) with mock infected cells are performed at bsl-2. luminescence is read on the infinite 1 m1000 tecan plate reader. data are analyzed using graphpad prism and/or columbus software (operetta). https://doi.org/10.1371/journal.pone.0194880.g001 factors that influence ebola antiviral activities in cell-based assays plos one | https://doi.org/10.1371/journal.pone.0194880 march 22, 2018 represented by z' factor which is defined as equation z' = [1-((3 ã sd pos )+(3 ã sd neg ))/(imean pos -imean neg )]. in our assay, the z' criteria is as follows: z' = 0.5-1.0 corresponds to an excellentassay; z' = 0-0.5 corresponds to a suboptimal assay; z' <0 corresponds to a unsuccessful assay [32] . the susceptibility to ebov infection was evaluated in multiple cell types in cell-based assays measuring anti-ebov activity. vero e6, huh 7 and mdm cells were infected with ebov/ mak isolate at 8 different mois, and the assay was terminated after 24, 48, 72 or 96 hpi. cells were stained with a fluorescent antibody, and hoechst dye was used to visualize the cell nuclei. infectivity was measured using the high-content imaging (hci) system as percentage of vp40 positive cells. the growth of ebov/mak isolate in three cell types was compared over time. in vero e6 cells, ebov spread slightly slower, and the number of positive cells were overall lower compared to huh 7 cells (fig 2a-2d ). mdms were the most susceptible to ebov among the cell types. at 24 hpi, mdms already exhibited a typical dose response relative to virus input, while only minimal ebov replication was observed in vero e6 and huh 7 cells at 24 hpi at all mois tested. virus spread effectively at 48 hpi with higher (1 to 3.3) moi of vero e6, huh 7 cells and most of mois in mdms (fig 2a-2f ). the infection in mdms was saturated at 72 hpi at almost all mois (fig 2e and 2f ). the nuclear stain for all cell types showed that the cell layer deteriorated with increased virus inoculum and duration of infection as was clearly evident at the 96 hpi time point and at higher virus input (mois of 3.3) (fig 2b, 2d , 2f and 2g). the cell layer frequently became fragile, and at later time points, the cell layer would lift off the well surface possibly because of increased exposure to virus, higher moi, or manipulation of plates during the fixing/staining procedure (fig 2g) . the hci system proved to be invaluable for determining optimal assay conditions (end point and virus input) and in identifying potential issues such as cell layer integrity. different virus input and endpoints were assessed to identify an acceptable range for testing drugs in vero e6 and huh 7 cells (tables 1 and 2 ). the same experimental plates from fig 2a-2d were used for this analysis. the fluorescence s/n ratio was calculated using a tecan plate reader, and the percentage of ebov-positive cells was determined using hci. for reproducible data, we determined that the percentage of ebov-positive cells should be kept within a range of 10 to 80% and the s/n ratio at 4 to 10. at lower s/n ratios (<4) or lower percentage of infected cells (<10%) distinguishing activity of a drug from non-activity becomes increasingly difficult. on the other hand, at higher s/n ratios (>10), when cell infection rate trends towards 80-90% and cells overgrow at later time points, the risk of a disrupted cell layer is greater. cell layer disruption increases the variability of the assay. therefore, the optimization of assay conditions (i.e., duration of infection, virus input) was carefully calibrated resulting in a compromise between signal strength and cell viability. based on the data obtained from fig 2 and tables 1 and 2 , two optimized conditions for the fluorescence assays were selected. an moi of 1 with 48 hpi as endpoint and an moi of 0.1 with an endpoint of 72 hpi produced a robust signal for all 3 cell types, vero e6, huh 7 and mdms (fig 2a-2f , tables 1 and 2). vero e6 cells were infected with ebov at around 17.1%, and mdms and huh 7 cells usually had a stronger signal with around 80% or higher of ebov-positive cells (fig 2c-2f ). the two conditions had a similar signal or ebov infection during the assay development phase, vero e6 cells appeared less reliable in producing a consistent viral spread, and this lack of reliability appeared to be due to cell culture passage history. to address this matter, vero e6 and huh7 cells were analyzed at different cell passage numbers by determining percentage of ebov-positive cells (fig 3a and 3b ). both cell types were infected with a moi of 1 for 48 h. the viral spread of huh 7 cells were consistent over cell culture passages 6-30, indicating that the passage number does not impact ebov spread c the percentage of ebov-positive cells was determined with a vp40/alexa-594 antibody stain to detect ebov and a nuclear stain to quantify the number of cells in a high-content imaging system. d s/n is the ratio of the fluorescent signal (mean from 3 infected wells) to the noise (mean from 3 mock-infected wells) quantified using a plate reader. abbreviations: ebov + , ebola virus-positive; hpi, hours post-inoculation; moi, multiplicity of infection; s/n, signal-to-noise ratio. in huh 7 cells in the range tested. in contrast, ebov spread on vero e6 cells varied considerably between cell passages 6-28 with peak infection (replication efficacy) at cell culture passages 12-14 ( fig 3a) and equally lower for early or late passages. significant differences were observed at passages 8, 10, 12, 14, and 16 when compared to the passage 6 or 28 using an ordinary one-way anova following dunnett's multiple comparison in graphpad prism 7.0. occasionally, exceptions to this trend were observed (fig 4) . the infectivity of ebov in vero e6 and huh 7 cells were compared at an early (p6) and a late passage number (p28) at a range of mois (0.03-2.0) for 48 h. in this case, vero e6 cells tested at the later passage showed a lower infection rate (<40%) as expected, while the early passage demonstrated an unusually high infection rate (up to 80%). infectivity in huh 7 cells remained consistent regardless of passage number, vero e6 cells were inconsistent overall despite the trend originally observed. both the virus input and duration of the experiment can have an impact on drug activity. to address this, we evaluated the in vitro efficacy of toremifene citrate, an fda-approved drug with proven anti-ebov activity [33] , under different parameters using fluorescence as the read out. huh 7 cells were infected with a constant moi of 1 and treated with toremifene citrate for 48, 72, or 96 h (fig 5a) . later time points resulted in a decrease in activity with the ec 50 at 96 hpi 3.3-fold higher than at 48 hpi. when the time point remained constant at 72 hpi the activity of toremifene citrate increased as the virus input decreased (mois of 0.1, 0.3, 1, or 3) (fig 5b) . the ec 50 at a moi of 0.1 was 6.9-fold lower than at a moi of 3. in addition to time and virus input, the cell type used in the assay can result in differences in the calculated ec 50 . anti-ebov activity of toremifene citrate was measured using variable assay endpoints (48, 72 and 96 hpi), mois (0.001, 0.01, 0.1, and 1), and cell lines (vero e6 and huh 7 cells, fig 6) . ec 50 values for toremifene citrate increased with exposure time and virus input in both cell types. overall, activity in vero e6 cells was higher with maximum activity (ec 50 = 0.15 μm) at 96 hpi and an moi of 0.01. in huh 7 cells, maximum activity (ec 50 = 0.28 μm) was detected at 96 hpi and an moi of 0.001. ec 50 values for toremifene citrate increased with exposure time and virus input in both cell types indicating that more drug is required to produce the same anti-ebov effect. factors that influence ebola antiviral activities in cell-based assays to increase sensitivity of the drug screen assays, a chemiluminescent enzyme-linked immunosorbent assay (celia) was developed for evaluating compounds for anti-ebov activity. the celia combines the advantage of specificity of an immunoassay with the high sensitivity of a chemiluminescent enzyme detection assay and is a simple and low cost screening assay [34, 35] . the celia was compared to the fluorescent assay (detected by regular plate reader or the hci system) by testing the efficacy of toremifene citrate against ebov infection in huh 7 cells with a range of mois (0.1, 0.3, 1, and 3 ) at three different time points (24, 48 and 72 hpi) . assay parameters, signal-to-noise (s/n), and z' factor, were compared between the assays (table 3) . at the earliest time point (24 hpi), the celia had higher sensitivity than the fluorescent detection assay as the z' were in the acceptable range (>0.2) even at the lowest moi of 0.1. in contrast, the fluorescent assay required higher mois for reliable data sets (z'>0.2). at the 48 hpi time point, both detection methods generated high quality data sets with the celia providing improved z' factors (>0.8). at 72 hpi, the celia and the fluorescent assay also produced excellent data using hci for detection, while the fluorescent data detection by regular plate reader showed higher variability, maybe due to cytopathic effects. the ec 50 s and ec 90 s factors that influence ebola antiviral activities in cell-based assays were determined on those data sets with acceptable z' (>0.2) and overall there was good correlation between the different detection methods (table 4 ). all detection assays demonstrated that higher moi and longer times of exposure led to an increase of the ec 50 values for toremifene citrate. in contrast, the ec 90 values showed considerably lower fluctuations under the different conditions tested. the established celia was used to compare anti-ebov activity of toremifene citrate in 3 different cell types, vero e6, huh 7 and mdms using an moi of 0.5 and a time point of 48 h (fig 7) . huh 7 cells and mdms had s/n ratios in the range of 1000s with z' factors generally above 0.5 (fig 7a) . the s/n for vero e6 cells ranged from >100 to over 1000, leading to more variability as reflected in the wider range of z' factors (0.3-0.9) (fig 7a) . the activity (ec 50 ) of toremifene was compared, and considerable differences were detected for the 3 cell types ( fig 7b) . the efficacy of toremifene citrate was 3-fold higher in huh 7 cells than in mdms, and 2-fold higher than in vero e6 cells (fig 7b) . in summary, the chemiluminescent drug screen assay for evaluating anti-ebov activity of compounds is a very robust, reliable and reproducible method. the data presented in this study exemplify the importance of having guidelines for testing drugs in vitro for antiviral activity and generating reproducible data sets that can be shared and confirmed by outside laboratories. several parameters should be considered when testing factors that influence ebola antiviral activities in cell-based assays drugs in vitro for antiviral efficacy. cell type, assay endpoint, and the virus input are among the most important factors [36] . the vero e6 cell line, derived by immortalization of african green monkey kidney cells, is the most commonly used cell line for testing antivirals against filoviruses. ebov propagates very well in this cell line, and many laboratories, including ours, generate their virus stocks and determine virus titers in vero e6 cells. however, the argument to use a cell line with more relevance to human disease has come up repeatedly. hence, the human liver cancer cell line, huh 7 and human mdms were chosen for these studies. macrophages are relevant to human disease, and they are considered to play an important role in virus dissemination in pathogenesis of evd [37] . therefore, a drug that is highly effective at inhibiting ebov infection in mdms may better translate to in vivo potency than vero e6 cells. the susceptibility of three cell types was compared over a range of virus input and over time. all cell types were permissive to ebov infection, but to different degrees. mdms demonstrated optimal replication starting as early as 24 hpi at an of moi 0.1. in contrast, ebov grew slower in vero e6 and huh 7 cells. the optimal conditions for evaluation of drug efficacy using the fluorescent assay were at an moi of 0.1 for the 72 h time point or at an moi of 1 for the 48 h time point for all 3 cell types to ensure a strong virus signal and avoid destruction of cell layer (fig 2g) . however, an moi of 1 is not suitable for detecting inhibitors of later steps in the virus life-cycle. the ability to detect inhibitors of virus assembly or egress will be reduced with increasing moi, as higher proportions of the cells will be infected even in the factors that influence ebola antiviral activities in cell-based assays though vero e6 cells are one of the most broadly used cell lines for testing compounds in in vitro assays, our data show that the performance of these cells is not ideal especially when evaluating drugs for anti-ebov activity. although it is never a good idea to passage cells for too long [38, 39] , we found that at lower or higher passages the vero e6 cells can show a decreased percentage of ebov-positive cells. huh 7 cells showed more efficient and more reliable virus replication irrespective of passage number. immortalized cancer cell lines are in general an easy choice for in vitro drug testing because they are easy to handle, propagate, expand, and plate on a week-by-week basis with relative consistency. in contrast, primary cell types such as human primary macrophages do not proliferate in cell culture and are ideally generated fresh from human blood upon use. for large scale testing, procuring the blood volumes needed for generating mdms from a cumbersome isolation technique could be a challenge. mdms demonstrated very efficient spread of virus through cell culture over time. donor-todonor variability precludes consistency, and in general, macrophages from different donors are not pooled. despite of the constraints, testing a selection of drugs that are closer to human clinical studies in human macrophages makes sense to get a better idea on efficacy in a more relevant target cell. fluorescence was detected by two different read outs and each technique has its advantages. the plate reader will measure the average fluorescence across the whole well. readings are quick and conducive to handling large numbers of plates during screening. however, the quality of the cell layer cannot be monitored. hci provides images of 1 or more fields of each well, and a nuclear stain can be added in parallel to indicate the viability of the cell layer. in the assay development phase, this tool is especially useful in identifying conditions that will ensure a healthy cell layer or identify issues such as plate corner or edge effects for assay quality control. one drawback is that not the whole well is imaged, but only a certain number of fields inside the well. it is important to pick the fields wisely to avoid bias or skewing of data. scanning at least 8 fields per well is recommended for statistical purposes, but that will increase reading time per plate. the time to read and analyze the hci data can take considerably longer than the data acquired with the regular plate reader. hci measures different parameters such as percentage of ebov-positive cells and mean signal intensity per cell. both the regular plate reader and hci have unique applications in a drug testing program. while a plate reader is good for a quick readout of large number of plates, the hci system is used to cross check on quality of cells and to clarify issues of noise and signal variability. in addition to the fluorescent assay, we also implemented a celia using an hrp-labeled antibody, which amplified the signal and increased the sensitivity of virus detection. as expected, the celia showed an improvement in the quality of data sets compared to the fluorescent assay. s/n ratio and z' factor were in an acceptable range as early as 24 hpi with the lowest virus input detected at an moi of 0.1. an ec 50 could also be determined at this time point. at 48 or 72 hpi, the two fluorescent read outs were similar to the chemiluminescent read out. toremifene citrate is a selective estrogen receptor modulator reported to be active in vivo against ebov in mice [12, 33] . this is an fda-approved drug for treating breast cancer [12] . mechanism of action studies showed that toremifene citrate affects ebov virus entry at the stage of virus-fusion with the endosomal membrane [40] . toremifene citrate was chosen by the who as a potential candidate for clinical evaluation in evd patients. we evaluated the performance of toremifene citrate on anti-ebov activity using different conditions. a trend towards higher ec 50 s with increasing input virus and longer assay time was observed. the data indicate that if the amount of virus present in cells or tissues is high enough, the drug will be less active. also with longer time for the virus to replicate, drugs may be unable to stem high viral replication. in contrast, the ec 90 values were less affected by high moi or longer time points, which may, therefore, be a more reliable parameter for comparing data sets between laboratories. serum in the media and pretreatment of cells with the compound can also have a considerable effect on antiviral activity. toremifene was compared directly to brincidofovir under a panel of different conditions [41] showing that activity of brincidofovir was dependent on media conditions with low serum (< 5%) and 48 h pretreatment before adding virus. in contrast, the serum concentration had no effect on the activity of toremifene citrate, and pretreating cells for 48 h (instead of 1 h) decreased the activity of this drug. many reports on the repurposing of fda-approved drugs discuss and compare a drug's in vitro ec 50 (determined in cell cultures) with its maximum concentration in human plasma (determined in clinical trials) when evaluating the potential of the drug to have in vivo activity. however, our data show that the ec 50 of a drug can range over more than a log based on testing conditions (e.g, moi, time of endpoint, serum). johanson et. al. [12] reported ec 50 s of 1.73 and 0.97 μm for toremifene with two different ebov strains ebov/kik and ebov/may, respectively, at 48 hpi with 0.4 moi by celia using the 13c6 antibody. these data are comparable to the ec 50 of 1.10 ± 0.71 μm for ebov/mak determined in our assay under similar conditions (table 4 ; moi 0.3/ 48 hpi). understanding how assays are performed and the potential variables is important, and caution should be used when using in vitro data for making decisions to advance a drug to in vivo studies. addition, we acknowledge laura bollinger and jiro wada at the irf for technical writing services and figure preparation, respectively, for this manuscript. multiple ebola virus transmission events and rapid decline of central african wildlife ebola virus: from discovery to vaccine exotic emerging viral diseases: progress and challenges phase 1 clinical trial of apical membrane antigen 1: an asexual blood-stage vaccine for plasmodium falciparum malaria the soviet union's anti-agricultural biological weapons hemorrhagic fever viruses as biological weapons: medical and public health management transmission of ebola virus (zaire strain) to uninfected control monkeys in a biocontainment laboratory lethal experimental infections of rhesus monkeys by aerosolized ebola virus drug repositioning: identifying and developing new uses for existing drugs treatment of ebola virus disease productive replication of ebola virus is regulated by the c-abl1 tyrosine kinase fda-approved selective estrogen receptor modulators inhibit ebola virus infection a systematic screen of fda-approved drugs for inhibitors of biological threat agents screening of fda-approved drugs for treatment of emerging pathogens plaque assay for ebola virus rapid detection and quantification of rna of ebola and marburg viruses, lassa virus, crimean-congo hemorrhagic fever virus, rift valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-pcr identification of bisindolylmaleimides and indolocarbazoles as inhibitors of hcv replication by tube-capture-rt-pcr high-throughput molecular detection of hemorrhagic fever virus threats with applications for outbreak settings generation of egfp expressing recombinant zaire ebolavirus for analysis of early pathogenesis events and high-throughput antiviral drug screening novel small molecule entry inhibitors of ebola virus identification of a broad-spectrum antiviral small molecule against severe acute respiratory syndrome coronavirus and ebola, hendra, and nipah viruses by using a novel high-throughput screening assay identification of an antioxidant small-molecule with broad-spectrum antiviral activity identification of a small-molecule entry inhibitor for filoviruses toremifene for breast cancer: a review of 20 years of data phase ii clinical trial of high-dose toremifene as primary hormone therapy in aromatase inhibitor-resistant breast cancer toremifene citrate (fareston) evaluation of the activity of lamivudine and zidovudine against ebola virus characterization of yellow fever virus infection of human and non-human primate antigen presenting cells and their interaction with cd4+ t cells pathology of experimental ebola-zaire (mayinga) virus infection transmitted to guinea pigs by oral, conjunctival and tonsillar routes high-throughput screening assays for the identification of chemical probes a simple statistical parameter for use in evaluation and validation of high throughput screening assays a screen of approved drugs and molecular probes identifies therapeutics with anti-ebola virus activity determination of residual enrofloxacin in food samples by a sensitive method of chemiluminescence enzyme immunoassay chemiluminescence enzyme immunoassay for the determination of sulfamethoxydiazine development of high-content imaging assays for lethal viral pathogens drug targets in infections with ebola and marburg viruses use of multiple assay endpoints to investigate the effects of incubation time, dose of toxin, and plating density in cell-based cytotoxicity assays cytotoxicity testing: measuring viable cells, dead cells, and detecting mechanism of cell death inhibition of ebola virus entry by a c-peptide targeted to endosomes the lipid moiety of brincidofovir is required for in vitro antiviral activity against ebola virus we thank irf cell culture staff in preparing the cells used in this study. we thank dr. atsunobu hiraoka (scgf research laboratory, kyoto, jp) for the kind gift of conditioned medium from kpb-m15 cells. we thank dr. gary kobinger (public health agency of canada, winnipeg, ca) for the makona isolate of ebola virus (genbank accession no. kp096420). we thank dr. hideki ebihara (niaid, rocky mountain laboratories, hamilton, mt) for huh 7 (human hepatocellular carcinoma) cells. we thank dr. sheli radoshitzky (usamriid, frederick md) for her gift of vp40 ebov vp40 matrix protein (b-md04-bd07-ae11. in key: cord-001910-6zfz2ns5 authors: zhang, xianming; wu, weiliang; zhu, yongcheng; jiang, ying; du, juan; chen, rongchang title: abdominal muscle activity during mechanical ventilation increases lung injury in severe acute respiratory distress syndrome date: 2016-01-08 journal: plos one doi: 10.1371/journal.pone.0145694 sha: doc_id: 1910 cord_uid: 6zfz2ns5 objective: it has proved that muscle paralysis was more protective for injured lung in severe acute respiratory distress syndrome (ards), but the precise mechanism is not clear. the purpose of this study was to test the hypothesis that abdominal muscle activity during mechanically ventilation increases lung injury in severe ards. methods: eighteen male beagles were studied under mechanical ventilation with anesthesia. severe ards was induced by repetitive oleic acid infusion. after lung injury, beagles were randomly assigned into spontaneous breathing group (bipap(sb)) and abdominal muscle paralysis group (bipap(ap)). all groups were ventilated with bipap model for 8h, and the high pressure titrated to reached a tidal volume of 6ml/kg, the low pressure was set at 10 cmh(2)o, with i:e ratio 1:1, and respiratory rate adjusted to a paco(2) of 35–60 mmhg. six beagles without ventilator support comprised the control group. respiratory variables, end-expiratory volume (eelv) and gas exchange were assessed during mechanical ventilation. the levels of interleukin (il)-6, il-8 in lung tissue and plasma were measured by qrt-pcr and elisa respectively. lung injury scores were determined at end of the experiment. results: for the comparable ventilator setting, as compared with bipap(sb) group, the bipap(ap) group presented higher eelv (427±47 vs. 366±38 ml) and oxygenation index (293±36 vs. 226±31 mmhg), lower levels of il-6(216.6±48.0 vs. 297.5±71.2 pg/ml) and il-8(246.8±78.2 vs. 357.5±69.3 pg/ml) in plasma, and lower express levels of il-6 mrna (15.0±3.8 vs. 21.2±3.7) and il-8 mrna (18.9±6.8 vs. 29.5±7.9) in lung tissues. in addition, less lung histopathology injury were revealed in the bipap(ap) group (22.5±2.0 vs. 25.2±2.1). conclusion: abdominal muscle activity during mechanically ventilation is one of the injurious factors in severe ards, so abdominal muscle paralysis might be an effective strategy to minimize ventilator-induce lung injury. eighteen male beagles were studied under mechanical ventilation with anesthesia. severe ards was induced by repetitive oleic acid infusion. after lung injury, beagles were randomly assigned into spontaneous breathing group (bipap sb ) and abdominal muscle paralysis group (bipap ap ). all groups were ventilated with bipap model for 8h, and the high pressure titrated to reached a tidal volume of 6ml/kg, the low pressure was set at 10 cmh 2 o, with i:e ratio 1:1, and respiratory rate adjusted to a paco 2 of 35-60 mmhg. six beagles without ventilator support comprised the control group. respiratory variables, end-expiratory volume (eelv) and gas exchange were assessed during mechanical ventilation. the levels of interleukin (il)-6, il-8 in lung tissue and plasma were measured by qrt-pcr and elisa respectively. lung injury scores were determined at end of the experiment. for the comparable ventilator setting, as compared with bipap sb group, the bipap ap group presented higher eelv (427±47 vs. 366±38 ml) and oxygenation index (293±36 vs. mechanical ventilation is the main therapy for patients suffering from ards, which will improve oxygenation, reduce the work of breathing and prevent muscle fatigue. however, mechanical ventilation itself might aggravate lung injury [1, 2] . although there is a widely use of the mechanical ventilation strategies such as low tidal volume, lower airway plateau pressure, optimal positive end-expiratory pressure (peep), permissive hypercapnia, patients with ards still has a higher morbidity and mortality in the past two decades [3] . invasive mechanical ventilation for patients with ards include preserving spontaneous breathing (sb)and controlled mechanical ventilation (cmv). whether sb should be preserved has been debated for many years. animal experiments [4, 5] and clinical studies [6] have reported that cmv may increase alveolar collapse and inhomogeneity of pulmonary parenchyma, and thus induce further lung injury. sb during mechanical ventilation results in better aeration and less atelectasis in lung dependent zones, and less hyperinflation in nondependent lung zones in ards [7] . some researchers have claimed that, even in the most severe ards, preserving sb activity was associated with beneficial effects in pulmonary function, speeding of weaning, and discharge from the icu [8] . however, in a recent multicenter trial, papazian et al [9] found that, in patients with severe ards, muscle paralysis was associated with a lower adjusted 90 days morbidity than patients that received placebo.yoshida et al [10] also found that in animal with severe ards, muscle paralysis might be more protective for injured lung, and sb could worsen lung injury. however, the precise mechanism is unclear. until now, it is unknown whether the activity of abdominal muscles has any impact on vali. caironi et al [11] has recently proved that the amount of lung cyclically recruitment and decruitment, but not lung stress and strain, leads to the increase of mortality in ards patients. expiration in mechanical ventilation, as well as in normal breathing, is a passive phenomenon produced due to the lung elastic recoil forces. however, in the presence of increased respiratory driving, expiratory muscles, especially the abdominal muscles may actively participate in breathing and increase intra-abdominal pressure. it has been proved that the increase of intraabdominal pressure even 10 cmh 2 o may have injurious impacts on organ functions [12] and aggravate lung damage [13] . therefore, we hypothesized that abdominal muscle activity during mechanically ventilation increases lung injury in severe acute respiratory distress syndrome. in this study we explored the hypothesis. this study was approved by the ethics committee of guangzhou medical university. experimental animals obtained from the kangda laboratory animals science & technology company of gaoyao city and the care and treatment of the animals were in compliance with the university standard. position and anesthetize with propofol by continuous infusion (0.16-0.5 mg/ kg/ h). paralysis was achieved with pancuronium(bolus = 0.16 mg /kg, followed by 0.08 mg /kg/ h). after orotracheal intubation with an 8.0 mm id cuff tube, lungs were ventilated with the ventilator evita 4 (dräger medical ag, lübeck, germany). volume-controlled model with a tidal volume (vt) of 10 ml/kg, peep 5 cm h 2 o, i:e ratio 1:1, fio 2 1.0 and the respiratory rate(rr)was adjusted to maintain paco 2 between 35 and 45 mmhg. intravenous fluid (lactated ringer ' s; 6 ml /kg/ h) were administrated to maintain the mean arterial blood pressure more than 70 mmhg. the right jugular vein and femoral artery were catheterized and connected to picco system for measure of core temperature, mean arterial blood pressure(mpa) and obtain artery blood sample for gas analysis. a multipair esophageal electrode-balloon combined catheter was put into the esophagus, and airway occlusion technique was used to check the proper position [14] . airway pressure (paw), esophageal pressure (peso) and intragastric pressure (pgas) from a side tap was connected to the end tracheal. a mlt300l respiratory flow head was used to measure airflow, and integrated airflow to obtain tidal volume. signal of paw, peso, pgas, airflow, diaphragmatic esophageal surface electromyography (emgdi) and abdominal muscles surface electromyography (emgab) were recorded by powerlab 16/30 sp and chart 7.2 software on a mac book computer. body temperature was constant throughout the whole experiment at 37°c with a heating pad. after obtaining baseline measurements and 30 minute stabilization, lung injury was induced by the total dose of 0.30 ml/kg purified oleic acid. if necessary, additional injection oleic acid (0.2 ml each time) was given until pao 2 /fio 2 were below 100 mmhg. a stable severe ards model was established when the pao 2 /fio 2 value remain less than 100 mmhg within 30 min [15, 16, 17] . after obtaining the measurements at injury, ventilator mode switched to bipap mode and the animals were randomly divided into sb group (bipap sb group) and abdominal muscle paralysis group(bipap ap group). after muscles paralysis, the p high titrated to achieve a vt of 6 ml/kg, p low was set at 10 cm h 2 o, with fio 2 1.0, i:e was fixed at 1:1 to minimize changes in mean paw. the mandatory rr adjusted a paco 2 of 35 to 60 mmhg. in bipap sb group, in order to recover sb, stopped the injection of pancuronium and gradually reduced the dose of propofol. according to previous studies, in order to ensure a strong effort of unsupported sb during bipap, the mandatory rr was adjusted to maintain the percentage of minute ventilation (mv) of unsupported sb to total mv >50% (fig 1) .other ventilator settings remained the same as the above setting. sb was monitored by online registration of the peso signal. the amount of sb was quantified by measuring minute volumes before and after neuromuscular blockade. in the bipap ap group, the abdominal muscles paralysis method was described as warner do [18] , a flexible epidural catheter was placed percutaneous through the second coccygeal vertebra and advanced until the tip closed to the l4 or l5 vertebra in the epidural space as confirmed by visual observation and autopsy. through the epidural catheter, 2% lidocaine was injected in 0.5 ml increment until the emgab was abolished and followed by continuous infusion of ropivacaine hydrochloride 1-2ml/h. all the other ventilator setting were totally same with bipap sb group. all variables were recorded continuously. mean paw for the bipap mode can be calculated as follows [19, 20] : (p high × t high + p low × t low ) / (t high + t low ), where the t high is the length of time for p high ; t low : length of time during p low . if the ratio of t high : t low is fixed at 1:1, then the mean paw could be kept constant when we changed the rr.using above method adjust ventilator, we could maintain the level of mean paw comparable in our study. the transpulmonary pressure (p l ) was calculated as paw-peso. peak paw were recorded. peso swings as the frequency per minute of each type of breathing cycle was used to calculate the total rr (s1 fig) . the product of inspiratory peso vs. time (ptp) were determined. eelv eelv at peep or p low was measured by simplified helium dilution method. a flexible tube inserted between endotracheal tube and the circuit y and clamped during an end-expiratory pause at peep. anesthesia balloon filled with1.5 l known gas mixture of helium (13.4%) in oxyge was then connected to the tube. after releasing the clamp, 10 tidal volumes compressing were performed rhythmically to dilute the helium gas mixture with the gas contained in beagles lungs. concentration of the helium in the bag was then measured with the helium analyzer (c-square company, usa), the following formula was used to computed the eelv (ml) = viâci cf -vi, where vi: initial gas volume in the bag; ci: initial helium concentration; and cf: the final helium concentration [21] . a pressure differential pneumotachometer was used to measure end-tidal co 2 (etco 2 ). the alveolar dead space fraction (vd/vt) was calculated by [22] : vd/vt = paco2àetco2 plasma samples was collected at baseline, lung injury and at the end of 8h mv. after centrifuged at 3,000 rpm for 15 min,supernatant aliquots were frozen at -80°c for analysis. plasma levels of il-6 and il-8 were measured using an elisa kit for dogs (genequick, guangzhou, china) according to manufacturer protocol. expression levels of il-6 and il-8 mrna in lung tissues were measured by qrt-pcr as previously described [20] . gapdh primers were used as an internal control. the sense and antisense of the primers (5'-3') used for il-6 and il-8 were: il-6 f: tgaccactcctgacccaacc, r: tccagactccgcaggatgag; il8f: acttccaagctggctgttgc, r: ctggcatcgaagttctgaactg. after 8 hours of ventilation, all beagles were euthanized by intravenous injection of potassium chloride, and lung tissues were harvested (s2 fig). samples were obtained separately from the upper lobe, middle lobe and ventral, lateral and dorsal sections of the right lower lobe. samples were fixed in 10% formalin and were stained by he for histological analysis. all sections were examined by the same pathologist and were evaluated by the following lung injury scores system [4, 23] : 0, minimal changes; 1, mild; 2, moderate; 3, severe; 4, maximal changes. for each slide including the following criteria: congestion, alveolar and interstitial edema, granulocytes, lymphocytes and erythrocytes infiltration, fibrinous exudates and micro thrombi. the sum of pathological score was calculated by adding the cumulative lung injury sub-scores (maximal value is 44). all date were represented as means ± sds. normal distribution of the data were assessed by the kolmogorov-smirnov test. comparison of data between two experimental groups with each other was performed using the unpaired t test or mann-whitney tests as appropriate. comparison of the continuous date within the same group before and after the experiments were evaluated by paired t tests. anova or kruskal-wallis test were applied for multiple-group comparisons as appropriate. effects of time and group differences on respiratory variables were evaluated by repeated measures two-way anova. the lsd post-hoc test was used as appropriate. ibm spss statistics 21 was used for statistical analyses. differences were considered to be statistically significant if p was less than 0.05. shows tracing records of paw, pes, pgas, p l , airflow, emgab and emgdi for the two groups in representative animals. there were no difference in the value of mean paw, and the only difference was the absence of abdominal muscle activity in bipap ap group. sb occurred rarely at p high in the experimental groups. the average percentage of minute ventilation of unassisted sb relative to total minute ventilation in the bipap sb group was above 50%.after abdominal muscle paralysis, the percentage decreased from 50%-100% to 10% -50%. as shown in table 1 , at baseline, there are no differences in hr、map between the groups during the entire experiment. there were also comparable mean paw between the experimental groups. the paco 2 level was less than 60 mmhg in all of the animals. due to activity of the diaphragm and abdominal muscles, the bipap sb group presented higher swing of pes, pgas and peak p l than bipap ap group. after abdominal muscle paralysis, bipap ap group presented lower swing of peso, pgas, peak p l , more even p l and longer time on p high (s1 table) . moreover, the bipap ap group resulted in a higher eelv (427±47 ml) compared with the bipap sb group (366±38 ml) (fig 2) . meanwhile, bipap ap group showed a lower vd/vt than bipap sb group (fig 3) . bipap ap group showed a trend toward improving pao 2 /fio 2 , but not in bipap sb group. the difference in pao 2 /fio 2 between two groups was statistically significant after 2h mv (p = 0.025). ptp decreased gradually from bipap sb group to bipap ap group. as shown in , and all experimental groups were higher than the control group. as shown in table 2 , the sum of lung injury scores was lower in bipap ap group (22.5±2.0) than that in bipap sb group (25.2±2.1), but the sum of scores in the experimental groups was higher than that in the control group. the bipap ap group showed less congestion, alveolar edema, alveolar infiltration of neutrophils and interstitial, and less infiltration of lymphocyte. the bipap sb group showed increased alveolar collapse, alveolar congestion, infiltration of inflammatory cells, and interstitial edema with hyaline membrane formation (fig 5) . in an oleic acid-induced model of experimental ards in beagles, our findings suggested that abdominal muscle activity during mechanically ventilation increases lung injury in severe acute respiratory distress syndrome. to our knowledge, no previous experimental study has investigated the effect of abdominal muscle on lung damage in ards. we used an oleic acid-induced experimental ards because this model has reproduced many basic characteristics of ards [17] . for different animals, the same dose of oleic acid administered by the same route can induce a reasonably reproducible lung injury [24] . different ventilated strategies have been proposed in ards. bipap mode was chosen because it can be easily modulated to maintain comparable levels of ventilator support in the three groups. by the methods of adjusting ventilator setting, the levels of mean paw were comparable, the only difference was with or without abdominal muscles activity which were visible from electromyogram. in our study, we used a super syringe method to make a static pressure-volume curve and our results showed that the lower inflection points were 8-9cm h 2 o for lung injury. so we set p low (peep) at 10 cm h 2 o for the three groups. with comparable ventilator setting, the oxygenation index were significantly higher in bipa-p ap group than bipap sb group after 2h mv. this outcome proved that abdominal muscle values are means ± sd. ards = acute respiratory distress syndrome; bipap sb = biphasic positive airway pressure with sb; bipap ap = biphasic positive airway pressure with abdominal muscles paralysis; sb = spontaneous breathing; mv = minute ventilation; paco 2 = partial pressure of carbon dioxide; pao 2 /fio 2 = ratio of partial pressure of arterial oxygen to faction of inspired oxygen concentration; rr = respiratory rate; vtave = average tidal volume; p plat = plateau pressure; ptp = pressure time product; mean paw, = mean airway pressure; peak p l , = peak transpulmonary pressure; mean p l , = mean transpulmonary pressure; peso = esophageal pressure; pgas = intragastric pressure; δpes = change of esophageal pressure, ptp = pressure time product *p < 0.05,bipap ap vs. bipap sb group at the same time. activity may worsen gas exchange. there were several reasons to explain this phenomenon: first, activity of the abdominal muscle was associated with decreased eelv. douglas and colleagues [25] observed in their study that eelv was parallel to oxygenation. similarly, a better gas exchange was also observed after abdominal muscle paralysis. second, activity of the abdominal muscle was associated with increased ptp, which represented a decrease of total work of breathing and oxygen consumption.third, activity of the abdominal muscle resulted in an decreased p l on p high , which could recruit the collapse alveolar units and result in greater lung units available for oxygenation [26] . fourth, abdominal muscle activity increase of intraabdominal pressure which is associated with the decrease of p l and respiratory compliance on t high , which can lead to a loss in lung volume and hypoxemia episodes [27] . fifth, activity of the abdominal muscle resulted in worsen blood reperfusion to nondependent lung area, and led to a higher vd/vt and greater inhomogeneity of lung ventilation to perfusion. all the above factors can worsen gas exchange. ventilator-associated lung injury (vali) includes volutrauma, atelectrauma and biotrauma., and these injuries may eventually lead to severe systemic inflammatory response and multiple organ failure. no previous studies have proved the relationships between abdominal muscle and vali in ards. in an oleic acid-induced ards model, our study showed that bipap ap had lower mrna expression of il-6 and il-8 in lung tissues and less total cumulative histopathological lung injury scores compared with bipap sb group. these findings suggested that activity of the abdominal muscle during mechanically ventilation was one of the injurious factors in severe ards. various mechanisms may explain the findings:①activity of the abdominal muscle can increase the value of 4pes which has been shown to promote the formation of pulmonary edema and aggravate lung injury [26] . ②activity of the abdominal muscle can increase end-expiratory alveolar pressure at the start of expiratory. ③activity of the abdominal muscle can increase intra-abdominal hypertension. it has been proved that activity of the abdominal muscles can increase iap by up to 20 cmh 2 o [28] . when intra-abdominal hypertension existed, unopposed increase of intra-abdominal pressure by spontaneous expiratory can cause greater lung injury by reducing p l in dependent zones [13] . ④activity of the abdominal muscle can decrease eelv, and make part of lung units breathing in the lower inflection point of the p-v curve and cause so-called "low volume injury" which is associated with the cyclic opening-closing of lung units and aggregates lung injury by interfacial forces. ⑤activity of the abdominal muscle allows peep to be bad controlled and resulting in increased "atelectrauma". ⑥activity of the abdominal muscle resulted in decreased p l which was presumed to aggravate lung injury. ⑦activity of the abdominal muscle resulted in decreased eelv, so lung strain, a major determinant of vali, might be further increased. there are several limitations in this study. first, the work was done in an oleic acid-induced ards model. therefore, we are not sure whether these results can be reproduced in the other ards models. second, due to protective strategy with a ltv used in this experiment, we cannot preclude the protective effects of abdominal muscle on a high tidal volume injurious ventilation. third, the rr and nervous distribution of canine may not be the same as human being, so we cannot guarantee our data can be used for patients and further studies are needed. finally, in bipap ap group, lidocaine and ropivacaine hydrochloride were used for epidural anesthesia to paralyze abdominal muscles, so we cannot rule out the possibility that these drugs affected pulmonary inflammatory response. in conclusion, in a canine model of oleic acid-induced severe ards, abdominal muscle activity during mechanically ventilation increases lung injury in severe acute respiratory distress syndrome, so abdominal muscles paralysis minimize ventilator-induced lung injury in early, severe patients with ards. supporting information table. representative data in different groups. (xlsx) spontaneous breathing activity in acute lung injury and acute respiratory distress syndrome ventilator-induced lung injury early airway pressure release ventilation prevents ards-a novel preventive approach to lung injury spontaneous breathing with biphasic positive airway pressure attenuates lung injury in hydrochloric acid-induced acute respiratory distress syndrome pressure support ventilation and biphasic positive airway pressure improve oxygenation by redistribution of pulmonary blood flow spontaneous breathing during ventilatory support improves ventilation-perfusion distributions in patients with acute respiratory distress syndrome spontaneous breathing improves lung aeration in oleic acid-induced lung injury the impact of spontaneous breathing during mechanical ventilation neuromuscular blockers in early acute respiratory distress syndrome the comparison of spontaneous breathing and muscle paralysis in two different severities of experimental lung injury lung opening and closing during ventilation of acute respiratory distress syndrome intra-abdominal hypertension in the critically ill: it is time to pay attention effects of preserved spontaneous breathing activity during mechanical ventilation in experimental intra-abdominal hypertension a simple method for assessing the validity of the esophageal balloon technique partial liquid ventilation with perfluorocarbon improves gas exchange and decreases inflammatory response in oleic acid-induced lung injury in beagles chest wall disruption with and without acute lung injury: effects of continuous positive airway pressure therapy on ventilation and perfusion relationships overview of the pathology of three widely used animal models of acute lung injury chest wall motion during epidural anesthesia in dogs airway pressure release ventilation: theory and practice effect of spontaneous breathing on ventilator-induced lung injury in mechanically ventilated healthy rabbits: a randomized, controlled, experimental study lung volume in mechanically ventilated patients: measurement by simplified helium dilution compared to quantitative ct scan estimating alveolar dead space from the arterial to end-tidal co(2) gradient: a modeling analysis pumpless extracorporeal lung assist for protective mechanical ventilation in experimental lung injury animal models of acute lung injury improved oxygenation in patients with acute respiratory failure: the prone position airway pressure release ventilation: what do we know mechanisms of hypoxemia episodes in spontaneously breathing preterm infants after mechanical ventilation abdominal muscle activity and intraabdominal pressure after upper abdominal surgery the authors are thankful to ph.d. yuanda xu, dongming hua, for assistance in the experiments, to dr.ruibing wu, for pathological analysis, we also thank dr.zhimin lin,for excellent technical assistance, and dr. yuanzi hua for his careful review of the manuscript. key: cord-000063-tex6bgab authors: sui, hong-yan; zhao, guang-yu; huang, jian-dong; jin, dong-yan; yuen, kwok-yung; zheng, bo-jian title: small interfering rna targeting m2 gene induces effective and long term inhibition of influenza a virus replication date: 2009-05-22 journal: plos one doi: 10.1371/journal.pone.0005671 sha: doc_id: 63 cord_uid: tex6bgab rna interference (rnai) provides a powerful new means to inhibit viral infection specifically. however, the selection of sirna-resistant viruses is a major concern in the use of rnai as antiviral therapeutics. in this study, we conducted a lentiviral vector with a h1-short hairpin rna (shrna) expression cassette to deliver small interfering rnas (sirnas) into mammalian cells. using this vector that also expresses enhanced green fluorescence protein (egfp) as surrogate marker, stable shrna-expressing cell lines were successfully established and the inhibition efficiencies of rationally designed sirnas targeting to conserved regions of influenza a virus genome were assessed. the results showed that a sirna targeting influenza m2 gene (sim2) potently inhibited viral replication. the sim2 was not only effective for h1n1 virus but also for highly pathogenic avian influenza virus h5n1. in addition to its m2 inhibition, the sim2 also inhibited np mrna accumulation and protein expression. a long term inhibition effect of the sim2 was demonstrated and the emergence of sirna-resistant mutants in influenza quasispecies was not observed. taken together, our study suggested that m2 gene might be an optimal rnai target for antiviral therapy. these findings provide useful information for the development of rnai-based prophylaxis and therapy for human influenza virus infection. influenza a virus (iav) remains a scourge on human health [1, 2, 3] . its antigen drifts and shifts are an ever-changing challenge for available vaccines [4, 5] . the appearance of drug resistance is the main hurdle for the development of antiviral drugs [6, 7, 8, 9] . given the limitations of current anti-influenza a virus strategies, the need for novel strategies for prevention and treatment of iav is evident [10] . in this regard, rna interfering (rnai) technology holds great promise to inhibit the replication of iav, including h5n1 virus. rnai is a form of posttranscriptional gene silencing mediated by short double-stranded rna, known as small interfering rna (sirna) [11, 12] . in this process, the cellular complex dicer cleaves a double-stranded rna (dsrna) molecule to yield doublestranded duplexes 21-25 nucleotides in length. these sirnas then guide the rnai induced silencing complex (risc) to cleave target mrnas that share sequence identity with the sirna [13, 14, 15] . since it was first demonstrated that adding exogenous, synthetic sirna molecules to mammalian cells can induce rnai, there have been rapidly expanding efforts to develop rnai therapies that induce the degradation of target messenger rna (mrna) involved in genetically inherited diseases or acquired disorders [16, 17, 18, 19, 20, 21, 22] . iav is an enveloped, negative-stranded rna virus. the unique property of single-stranded rna virus itself makes rnai an attractive approach for development of anti-avian influenza therapeutics. the single-stranded viral genome, consisting of 8 segments contained at least 10 open reading frames (orfs), serves as template for both viral genome replication and subgenomic mrna synthesis. it has been reported that sirnas respectively targeting to the viral genes of polymerase 1 (pb1), polymerase 2 (pb2), polymerase a (pa), nucleocapsid protein (np), nonstructure proteins (ns1 and ns2), matrix proteins (m1 and m2), especially those specific for np, pa and pb1, can potently inhibit replication of influenza a viruses [16, 23, 24, 25, 26] . however, it has been reported that hiv and hcv may develop sirnaresistant mutations quickly [17, 27, 28] , and therefore abrogated the further rnai treatment. thus, the evaluation of long term inhibition efficiency of designed sirnas and screening of the emergence of sirna resistance mutants are also an important research target. in the present study, we identified an effective sirna targeting m2 gene (sim2), a highly conserved gene in iav, as compared to a reported effective sirna targeting np gene (sinp). we further established cell lines which stably expressing the shrnas by transducing lentiviral-shrna vectors to madin-darby cannie kidney (mdck) cells. using these two cell lines, we evaluated long term antiviral effects of these sirnas against iav subtypes h1n1 and h5n1 and further screened the potential sirna-resistant viral mutations. our results showed that rationally designed sim2 conferred long term effective inhibition for iav replication. it was further demonstrated that no sirna-resistant viral mutation appeared in sim2 targeting sequence even after the virus was cultured in the shrna expressing stable cell line for 40 passages. two sirnas targeting the m2 gene were rationally designed by sirna target designer (the sequences of sirnas are shown in the supporting information table s1 ) and their effect in inhibiting the virus replication was assessed in mdck cells. two sirnas targeting the np gene were included in the experiments as controls. the results showed the sirna m-950 exhibited a good inhibition effect with dose dependent manner, while another sirna m-126 just slightly inhibited virus replication even at a concentration of 100 nm (fig. 1a) . fig. 1b showed that the sirna np-1496 could inhibit influenza virus replication, while sirna np-336 had no inhibition effect, which is consistent with the previous report [25] . the sim2 exhibited higher inhibitory effect of h1n1 virus than sinp in stable cell lines based on the above results, the lentiviruses expressing the shrnas m2-950 or np-1496 were constructed and transduced into mdck cells to establish two stable cell lines, shm2-mdck and shnp-mdck. mdck cells and the mdck cells transduced by blank lentivirus (mock mdck) were used as controls. the cell lines were infected with h1n1 virus at a moi of 0.005 and culture supernatants were harvested at indicated time-points to determine the virus titer by plaque assay. as shown in fig. 2 , virus replication kinetics of mock mdck is similar with that of mdck, indicating that lentivirus integration didn't influence virus replication. virus titers in shnp-and shm2-mdck cell cultures were 2 to 10 folds lower than the controls mdck and mock mdck cultures, suggesting that virus replication had been suppressed by the expressed shrnas in both shm2-mdck and shnp-mdck cells. notably, sim2 exhibited a better inhibition effect, showing about 2-fold lower viral titer than sinp, although the expression levels of sim2 and sinp were similar (dct sim2 = 6.68, sinp = 6.95). the sim2 abolished not only m2 mrna but also sinp mrna accumulation in the stable cell lines we also measured the accumulation of mrna for np and m2 gene in infected mdck, mock mdck, shm2-mdck and shnp-mdck cells. the mrnas were extracted from the cells harvested at 1, 2, 4 and 24 hrs post-infection and tested by realtime rt-pcr. the mrna expression level is normalized by copy to further confirm whether the suppression of np mrna in shm2-mdck cells indeed affect np protein expression, the np protein level was tested by an indirect immunofluorescence assay. as shown in fig. 4 , egfp fluorescence, an indicator of shrna expression, was detected in mock mdck, shnp-mdck and shm2-mdck but not in mdck cells, while np protein was detected in mdck and mock mdck cells but not in shnp-mdck and shm2-mdck cells. the results were consistent with above viral mrna results, indicating that sim2 indeed suppressed the np protein expression. sim2 provided more potent anti-h5n1 viral effect than sinp in stable cell lines we further tested whether sim2 could also inhibit the replication of a highly pathogenic h5n1 avian influenza virus. as shown in fig. 5a , although numbers of plaques were similar in different mdck cell lines, smaller size of plaques were only found in shm2-mdck cells, suggesting that sim2 inhibited replication of h5n1 virus. the cell lines were also infected with different amounts of h5n1 virus and culture supernatants were collected at different time points to determine the virus titers by ha assay. the virus replication was significantly inhibited in shm2-mdck cells at all time-points, but shnp-mdck just offered a minor inhibition effect at early stage of the virus infection (fig. 5b ). these results further confirmed that sim2 could provide a more potent protection than sinp against h5n1 infection. to test if sim2 sirna-resistant virus mutant would quickly appeared when cultured in shm2-mdck cells, h5n1 virus was continually cultured in shm2-mdck cells for 40 passages. every 10 passages, the culture supernatant was collected and tested by plaque reduction assay. no obvious larger size of plaque was found. ten plaques with relative larger size were picked to further identify potential mutation in the sirna targeting region by sequencing. the results showed that no mutation appeared in the sim2 targeting region even after 40 passages of the cultures (fig. 6 ). the principal finding of this study is that rationally designed sirna targeting influenza m2 gene (m-950) conferred effective long term inhibition against influenza a virus replication. such high suppressive effect is not only against h1n1 influenza a virus but also against a highly pathogenic h5n1 subtype. in the previous related studies, ge and his co-workers [25] screened sirnas targeting to 6 conserved genes of influenza a virus and showed that np-1496 was the best since it can confer a more than 200-folds inhibition of h1n1 virus. li et al [29] and tomkines et al [23] further confirmed that np-1496 provided high anti-h5n1 effect. we therefore included np-1496 as a positive control in this study. our results showed that sirna m-950 exhibited similar (fig. 1 ) or even slight higher (fig. 2) inhibitory effect against iav replication as compared to that of np-1496. a recent report by zhou et al [30] also showed that several sirnas targeting np and m genes exhibited effective inhibition against influenza a virus replication in cultured mdck cells and in animal models. however, sequences of their reported sirnas targeting m2 gene are completely different from the sirna m2-950. furthermore, chemically synthesized sirnas or plasmid based shrnas were always delivered by transfection in previous related studies, whereas we used a lentivirus system to deliver selected shrnas. although the integration property of lentivirus has abrogated it to be used in human, it is helpful for our study purpose to successfully establish stable cell lines persistently expressing sirnas. in this study we found that sim2 not only decreased the level of m2 mrna but also the level of np mrna, suggesting that sim2 has a broad inhibition manner in the process of influenza virus replication. ge et al have reported a similar broad inhibition of sirnas [25] . in their study, np-1496 and pa-2087 provided a broad inhibition to h1n1 influenza virus, which not only abolished the accumulations of specific np or pa mrnas but also inhibited the accumulations of mrnas for m, ns1, pb1, pb2 and pa or np genes. a possible explanation is that some double stranded sirnas may result in ifn responses or activate a rna degradation pathway, e.g. phosphorylated protein kinase r (pkr) [9, 31, 32] . however, the mechanisms of this broad inhibition of some sirnas are still not very clear yet. from the standpoint of viral target choice in rnai based antiviral therapy, np protein is required for elongation and antitermination of nascent crna and vrna transcripts [33, 34] . without newly synthesized np, further viral transcription and replication are blocked. while, m2 plays a critical role in the assembly of infectious virus particles. thus, the potent antiviral effect of sim2 may be attributed to its broad inhibitory effect. depending on the stringency of sirna-target base pairing, sirna treatment may cause selection of sirna-resistant viruses, and this has been shown with hiv and hcv [17, 27, 28] , and therefore abrogated the further medication or treatments. using lentiviral delivery system, we established stable cell lines persistently expressing shrna, which provided a more convenient experimental approach to study long term inhibition effect of sirnas and screen for sirna resistant virus mutants in quasispecies in vitro. our results showed that h5n1 virus cultured in shm2-mdck were equally susceptible to sim2 as the original virus even after 40 passages. moreover, sequencing of sim2 targeted region in 10 such independent plaque purified virus isolates revealed sequence identical to the parental one. the current data have shown no insertion, deletion and nucleotide substitution in the sirna target sequence, therefore demonstrated sim2 possessed good long term inhibition effect for influenza virus replication without the problem of sirna resistant mutants. taken together, all the findings about effective rnai target, lentiviral vector delivery and the establishment of stable shrna expressing cell lines in our study provide rational information for the development of sirnas as prophylaxis and therapy for influenza virus infection in humans. mdck and human embryonic kidney 293t cells were respectively maintained in mem and dmem (invitrogene, usa) supplemented with 10% heat-inactivated fetal bovine serum (fbs) and antibiotics (100 u penicillin g/ml and 100 ug streptomycin/ml). influenza virus strains a/new caledonia/ 20/1999 (h1n1) and a/hong kong/486/97 (h5n1) used in these experiments were prepared in mdck cells and virus titers were determined by tcid 50 . all experiments with h5n1 virus were performed in bsl-3 laboratory. the sirnas targeting m or np gene of influenza a virus were designed by sirna target designer version 1.51 from promega (http://www.promega.com/sirnadesigner/program/). the duplexes of designed and previously reported sirnas were synthesized by invitrogene (usa) (the sequences were shown in the supporting information table s1 ). the sirnas were reverse transfected to mdck cells using lipofectamine tm rnaimax (invitrogene, usa) as described in company's instruction. after incubated the cells for 16,18 hrs, the cells were infected with the viruses and followed by detection of viral replication. 24 hours after infection, rna were extracted from the cells and followed by real time rt-pcr to detect the relative quantities of replicated viral rna. the h1-promoter-driven shrna cassettes were constructed by annealing two primers containing the 19-nt sense and reverse complementary targeting sequences with a 9-nucleotide loop -ttcaagaga-and flanking mlu1 and cla1 cloning sites (the sequences of shrna were shown in the supporting information table s1 ), and then cloned into the 39-end of the h1 promoter in the lvthm plasmid [35, 36] . the sequences of the insertions were confirmed by dna sequencing. lentiviral vectors with shrna expression cassette were produced by calcium phosphate-mediated, three-plasmid transfection of 293t cells [37] . briefly, 293t cells (2.5610 6 mdck and the stable shrna expressing cell lines in 24-well plates were infected with viruses at moi of 0.005,0.5 (2 mg/ml trypsin was used in the infection process of h1n1). after incubation for 1 hr, the infected medium was removed and mem without fbs was added. cell supernatants were collected at different time points. the viral load was detected by hemagglutination (ha) and/or plaque assays as described previously [39] . briefly, the ha assay was carried out in u-bottom 96 well plates. serial 2-fold dilutions of virus samples were mixed with an equal volume of a 0.5% suspension of turkey erythrocytes (lampire biologic laboratories, pipersville, usa) and incubated at room temperature (rt) for 45 mins. wells containing an adherent, homogeneous layer of erythrocytes were scored as positive. for plaque assay, serial 10-fold dilutions of virus sample were added into a monolayer of mdck cells. after 1 hr incubation, the virus was removed and the cultures were overlaid with 1% semi solid agar-mem. three days after infection, plaques were visualized by staining of crystal violent. real-time rt-pcr was carried out as described previously [39] . briefly, h1n1 or h5n1 virus infected mdck, mock mdck, shnp-mdck and shm2-mdck were harvest at 1, 2, 4 and 24 hr after infection. total rna was extracted from the infected cell samples using rneasy rna isolation kit (qiagen, germany) and reverse transcribed using superscript ii reverse transcriptase and oligo dt primer (invitrogene, usa), according to the manufacturer's protocol. viral mrna copies were measured by sybr green m63000 real-time pcr system indirect immunofluorescence assay was performed as described previously [40, 41] with some modification. mdck, mock mdck, shnp-mdck and shm2-mdck cells grew on micro cover glasses (thomas, usa) were infected with 1 moi of h1n1 virus for 6 hrs, after washed with pbs, the cells were fixed in 4% paraformaldehyde for 15 mins at rt and then permeabilized in 0.1% triton x-100 for 3 mins at rt. after washed with pbs again, the cells were incubated with 1:50 diluted mouse anti-np antibody (abcam, uk) for 30 mins in dark at rt. the cells were washed three times in pbs with 1% fcs and incubated with 1:500 diluted texas red-conjugated anti-mouse lgg (abcam, uk) for 30 mins in the dark at rt. the cells were washed and mounted. slides were viewed under an olympus fluorescence microscope (olympus, germany). the screening of potential sirna resistant mutants were performed in our established stable shrna-expressing cell lines according to previously described protocols [42] with some modification. briefly, the shm2-mdck cells in a t25 cm 2 flask were infected with h5n1 virus. after cultured for 2 days, the supernatants were harvested. part of the supernatants was inoculated to shm2-mdck for next passage of the virus culture, another part was subjected for plaque assay to determine if potential sirna-resistant virus appeared. every 10 passages, ten bigger size of plaques in the plaque assay were picked for sequencing to detect any mutation in the sirna targeting region using a pair of primers: forward, 59-aag gca gat ggt gca ggc aat-39 and reverse, 59-tac tcc agc tct atg ctg aca-39. table s1 found at: doi:10.1371/journal.pone.0005671.s001 (0.03 mb doc) three indonesian clusters of h5n1 virus infection in 2005 probable person-to-person transmission of avian influenza a (h5n1) probable limited personto-person transmission of highly pathogenic avian influenza a (h5n1) virus in china avian influenza a (h5n1) in 10 patients in vietnam virulence may determine the necessary duration and dosage of oseltamivir treatment for highly pathogenic a/vietnam/1203/04 influenza virus in mice amantadineresistant influenza a in a nursing facility catalytic and framework mutations in the neuraminidase active site of influenza viruses that are resistant to 4-guanidino-neu5ac2en amantadine-resistant influenza a in nursing homes. identification of a resistant virus prior to drug use antisense rna: function and fate of duplex rna in cells of higher eukaryotes avian influenza a (h5n1) infection in humans role for a bidentate ribonuclease in the initiation step of rna interference an rna-directed nuclease mediates post-transcriptional gene silencing in drosophila cells rna interference rna interference technology : from basic science to drug development shortinterfering-rna-mediated gene silencing in mammalian cells requires dicer and eif2c translation initiation factors using sirna in prophylactic and therapeutic regimens against sars coronavirus in rhesus macaque sirnaresistance in treated hcv replicon cells is correlated with the development of specific hcv mutations stable inhibition of hepatitis b virus proteins by small interfering rna expressed from viral vectors specific inhibition of bcr-abl gene expression by small interfering rna inhibition of bcr-abl gene expression by small interfering rna sensitizes for imatinib mesylate (sti571) rna interference targeting fas protects mice from fulminant hepatitis caspase 8 small interfering rna prevents acute liver failure in mice protection against lethal influenza virus challenge by rna interference in vivo inhibition of influenza virus matrix (m1) protein expression and virus replication by u6 promoterdriven and lentivirus-mediated delivery of sirna rna interference of influenza virus production by directly targeting mrna for degradation and indirectly inhibiting all viral rna transcription inhibition of influenza virus production in virus-infected mice by rna interference human immunodeficiency virus type 1 escapes from rna interferencemediated inhibition hiv-1 can escape from rna interference by evolving an alternative structure in its rna genome construction of influenza virus sirna expression vectors and their inhibitory effects on multiplication of influenza virus effective small interfering rnas targeting matrix and nucleocapsid protein gene inhibit influenza a virus replication in cells and mice mechanisms of inhibition of the host interferon alpha/ beta-mediated antiviral responses by viruses viruses and interferon: a fight for supremacy transcription antitermination during influenza viral template rna synthesis requires the nucleocapsid protein and the absence of a 59 capped end influenza virus rna replication in vitro: synthesis of viral template rnas and virion rnas in the absence of an added primer lentivirus-mediated rna interference of dc-sign expression inhibits human immunodeficiency virus transmission from dendritic cells to t cells a versatile tool for conditional gene expression and knockdown efficient lentiviral vectors for short hairpin rna delivery into human cells short-term cytotoxic effects and long-term instability of rnai delivered using lentiviral vectors delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza a/h5n1 virus immunogenicity in mice of tandem repeats of an epitope from herpes simplex gd protein when expressed by recombinant adenovirus vectors anti-tumor effects of human peripheral gammadelta t cells in a mouse tumor model antiviral methods and protocols the author would like to acknowledge tronolab group for kindly providing lentiviral vector system. key: cord-002426-5e1xn7kj authors: falcón-lezama, jorge abelardo; santos-luna, rené; román-pérez, susana; martínez-vega, ruth aralí; herrera-valdez, marco arieli; kuri-morales, ángel fernando; adams, ben; kuri-morales, pablo antonio; lópez-cervantes, malaquías; ramos-castañeda, josé title: analysis of spatial mobility in subjects from a dengue endemic urban locality in morelos state, mexico date: 2017-02-22 journal: plos one doi: 10.1371/journal.pone.0172313 sha: doc_id: 2426 cord_uid: 5e1xn7kj introduction: mathematical models and field data suggest that human mobility is an important driver for dengue virus transmission. nonetheless little is known on this matter due the lack of instruments for precise mobility quantification and study design difficulties. materials and methods: we carried out a cohort-nested, case-control study with 126 individuals (42 cases, 42 intradomestic controls and 42 population controls) with the goal of describing human mobility patterns of recently dengue virus-infected subjects, and comparing them with those of non-infected subjects living in an urban endemic locality. mobility was quantified using a gps-data logger registering waypoints at 60-second intervals for a minimum of 15 natural days. results: although absolute displacement was highly biased towards the intradomestic and peridomestic areas, occasional displacements exceeding a 100-km radius from the center of the studied locality were recorded for all three study groups and individual displacements were recorded traveling across six states from central mexico. additionally, cases had a larger number of visits out of the municipality´s administrative limits when compared to intradomestic controls (cases: 10.4 versus intradomestic controls: 2.9, p = 0.0282). we were able to identify extradomestic places within and out of the locality that were independently visited by apparently non-related infected subjects, consistent with houses, working and leisure places. conclusions: results of this study show that human mobility in a small urban setting exceeded that considered by local health authority’s administrative limits, and was different between recently infected and non-infected subjects living in the same household. these observations provide important insights about the role that human mobility may have in dengue virus transmission and persistence across endemic geographic areas that need to be taken into account when planning preventive and control measures. finally, these results are a valuable reference when setting the parameters for future mathematical modeling studies. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 dengue fever (df) is the most important arthropod-borne viral disease in the world. it is caused by infection with any of the four dengue virus (denv) serotypes. nearly half of the human population inhabits areas with denv transmission. in mexico dengue incidence and severe cases have been increasing in the last decade. to date, there is no specific treatment or vaccine for df and vector control stands as the cornerstone for df prevention [1, 2] . df is an important public health problem, especially in urban areas [3] , where it usually presents in large outbreak. the costs of treatment and management during a df outbreak are a serious burden for health systems, especially when there is a risk for saturation of health facilities [4] . the actors that are necessary for denv transmission are fairly well understood. nonetheless, some of the dynamical features about these actors still need to be elucidated in order to understand how they impact on transmission. human mobility has been studied in relation to other infectious diseases, where its role as an important driver for disease transmission has been proven [5, 6] . mathematical models have suggested that local scale human mobility may play a role in denv transmission, outbreak persistence, and control efficiency [7, 8] . however, little information is available on detailed human mobility patterns in geographic areas where df is endemic or on confirmed cases during an outbreak. recently, gps-based technologies have been tested and shown to be a reliable and acceptable tool for quantifying human mobility [9, 10] . human mobility has been described in relatively recent reports by using indirect measures [11, 12] . for these reasons, we studied the micro and macromobility of dengue virus-infected subjects in an endemic locality. here we present the results of a cohort-nested case-control study on a dengue endemic urban locality in mexico. the protocol of the project was reviewed and approved by the comité de etica y de prevención de conflictos de interes (institutional review board) ci 1046 no 1160 and departamento de investigación (external review) servicios de salud de morelos, mexico dei/cei/0281/2012. cohort-nested, case-control study. sample: 126 individuals (42 cases, 42 intradomestic controls and 42 population controls) with age older than 12, and residents in axochiapan, morelos state, méxico, were selected from the cohort "peridomestic infection as determinant for dengue virus transmission" [13] . they were assigned into three study groups: a. cases were individuals with laboratory evidence of recent, symptomatic or asymptomatic, denv infection, and identified as the only persons infected within their households during the study. b. intradomestic controls, were individuals with a negative serological result for recent denv infection, living in the same household with a case; c. population controls were individuals with a negative serological result for recent denv infection, randomly selected from the same locality. all controls tested negative for denv during the same period and using the same validated techniques than cases (igm or igg capture elisa) as reported in the cohort study [13] . partici-pant´s selection was performed as follows: cases were approached first, if accepted participation an intradomestic control was randomly assigned from the pool of subjects living in the same house that had both baseline and final negative elisa results. for each pair of in-house participants, a randomly selected population control was then assigned (fig 2) . given the limited number of available gps loggers and the three-month time frame for the follow-up, the recruitment was limited to a maximum of 50 cases with their respective intradomestic and population controls. between may and september, 2012, with prior signed informed consent, all participants were provided with a portable gps (gps data-logger, transystems mod. 747 a+), programmed for recording its position at 60-second intervals (variables date, time, latitude, longitude, altitude and speed), during 24 hours a day, for a minimum period of 15 days. participants were instructed to carry their gps at all times whenever they left their homes and to recharge the equipment's battery daily during their in-home resting times. this follow up was performed in cases identified during the immediate previous season, on average one year after diagnosis confirmation, in order to match the activities performed during the high transmission season, and under the assumption that their mobility patterns remained unchanged after disease, and constant through time. a web-based interface was developed to import the text files from gps equipment to a main database. variables were homogenized, waypoints in the initial and final days of each individual gps track (comprising incomplete days) were eliminated in order to standardize the period of time to be analyzed starting at 00:00:00 hours on the second day of follow up and ending at 23:59:59 on the last to final day of follow up. data were converted into a feature dataset and projected from the geographic coordinate system to a lambert coordinate system (from hexadecimal to metric units) with the purpose of performing arithmetic operations for distance calculations. origin or routinely residence sites were identified by means of an iterative algorithm (mean center) employing waypoints from 00:00:00 to 04:59:59 hours, monday to friday. routine residence coordinates were added to the database. distance to home variable (dhome) was calculated for each extradomestic waypoint using sql applying the following formula: where; x 1 = xhome (x coordinate from home), x 2 = xccl (x coordinate from each waypoint), y 1 = yhome (y coordinate from home) y, y 2 = yccl (y coordinate from each waypoint). waypoints within the peridomestic area (dhome < 50m) were identified. distance, speed, altitude and time differentials were created: where: xccl 1 = (x coordinate from previous waypoint), xccl 2 = (x coordinate from current waypoint), yccl 1 = (y coordinate from previous waypoint) and, yccl 2 = (y coordinate from current point) displacement and spatial permanency variables for each subject with complete data were generated. visit sites were defined as those areas out of the individual's home with a 50 m radius in which each participant remained static for a period enough to allow a potential effective interaction with local vectors. these sites were identified by generating an algorithm through which visit clusters were formed using the following criteria: stops lasting 5 minutes or longer, a distance from home of 50 m or farther, distance of the current waypoint from previous waypoint < 50 m, and speed for the current waypoint of 2 km / h or less. for each cluster (visit site) a centroid was calculated. common visit sites for cases were identified as hexagonal cells with 50 m radius [14] which were visited by at least two different cases at a given time, and where the proportion of different visiting cases was at least two thirds of the total visiting population for that cell. common visit sites for controls were identified as hexagonal cells with 50 m radius which were visited by at least two members from each control population and not visited by any of the cases. the geographic universe in the study was divided in five areas (1.-inside the house, 2.-out of the house but in the locality, 3.-out of the locality but in the municipality, 4.-out of the municipality but in the state, 5.-out of the state), limited by four buffers. a circular buffer with 50 m radius around each participant's home limited the first area and three additional polygonal buffers were drawn according to the administrative limits for the locality, municipality and state. arcgis arcinfo 10 was used for processing and analyzing spatial data, sql server was used to create a geodatabase, arc sde 10 was used as interpreter between entre sql and arcgis. statistical analyses were performed using stata 12. fifty randomly selected cases were asked to participate in the study from which 42 (84%) accepted participation. all approached controls agreed to participate. in total 126 individuals (42 cases, 42 intradomestic controls and 42 population controls) were recruited. our drop-out rate was lower than 1% (1/126) since one participant (intradomestic control) did not finish the follow-up due to the loss of the assigned gps logger. table 1 describes the main characteristics of the subjects in each group. no statistically significant differences were observed in most of variables except in age, since cases were significantly younger than the intradomestic or population controls (cases mean: 29.2, sd: 17.7; intradomestic controls mean: 35.9 sd: 13.5; population controls mean: 37.4 sd: 16.6. p = 0.0315). of 126 participants, 125 (99.2%) participants completed their follow up since one gps used by an intradomestic control went missing. the final database contains 3,064,887 waypoints from these 125 participants, and all participants were followed by a mean of 15.9 continuous days. as for the number of days of follow-up for each group no differences were recorded. as expected, most of the waypoints in the population fell within the intradomestic area (< 50 m radius from home centroid). as distance from home (absolute displacement) increased, we observed a marked decrease in the proportion of waypoints. all three groups presented a small peak when the distance reached the 100 m radius. from this point the proportion of waypoints quickly decayed (fig 3) . no differences were noticed for absolute displacement among the groups. the hourly distribution of recorded waypoints out of the participant's homes is shown in fig 4. as expected, participants usually left their homes early in the morning and returned by the end of the day. although we recorded waypoints out of the participants' homes in every hour of the day, the period comprised between 08:00 pm and 12:00 pm registered the peak in the number of waypoints recorded out of the homes, and this number decreased steadily as the day progresses. the pattern during weekdays ( fig 4a) suggests that cases leave their homes and return to them slightly earlier than control groups. as for the weekends (fig 4b) , both control groups show a similar pattern to that observed for weekdays, nonetheless, cases seem to remain in their homes more often and return earlier. table 2 shows values for different mobility variables. no significant differences were recorded among groups for the following variables: mean distance from home at all times, maximum recorded distance at any given time, and mean time spent in each geographic area at any speed or at static speed. nonetheless, when comparing the number of visits per geographic area, the cases had fewer recorded visits in the area out of the locality but in the municipality (3.1 vs 18.7, p = 0.0428), and more visits in the area out of the municipality (10.4 vs 2.9, p = 0.0282), both compared to intradomestic controls. these differences were statistically significant. consistent with this behavior, although non-statistically significant, cases visited more states, municipalities, and regions with high dengue incidence through their follow up, in comparison to both control groups. we next examined the proportion of waypoints recorded by the comparison groups in each area stratified by age (fig 5) . there is a notorious difference in the proportion of waypoints among the cases, observing an increase of nearly 8 percentile points in the intradomestic waypoints, recording the highest frequency in cases under age 25 (fig 5a. however the difference not statistically significant (cases < 25: median 90.2%, interquartile-range 75.5-95.5; cases ! 25: 80% iqr 66-86.4; p = 0.079). we also observed a difference in the area out of the municipality but in the state, where the group of cases aged 25 and older spent the highest proportion of time (age < 25: 0% iqr 0-1%; age ! 25: 1.1% iqr 0-4.6; p = 0.0233). there was no significant difference between cases and population controls, regardless of age. when comparing cases versus intradomestic controls, we observed a statistically significant difference in time spent in area out of the municipality but in the state (cases: 1.1% iqr 0-4.6; ic: 0% iqr: 0-0.6; p = 0.009). we found differences in mobility patterns when analyzing data by gender ( fig 5b) . women had a higher proportion of waypoints within the intradomestic area than men. these differences were statistically significant for intradomestic area (male: 77.4% iqr: 65.8-86.9; female: 89.4% iqr 80.5-93.5; p = 0.002), out of their homes but in the locality (male: 15.5% iqr 5.3-25; female: 7.2% iqr 4.6-16.5; p = 0.0063) and the area out of the locality but in the municipality (male: 1.1% iqr: 0.1-4.9; female: 0.1% iqr: 0-0.4; p<0.0001). nonetheless, linear mean (p = 0.061) and maximum (p = 0.1468) distances were not. a) cases vs. intradomestic controls. a conditional logistic regression analysis was performed, including variables identified in the bivariate analysis as having a p value < 0.20, and by data mining techniques using all variables as reported previously [15] . for bivariate analysis variables were age (continuous and dichotomic [under 25 or 25 and older]) gender, occupation (intra or extradomestic), education (dichotomic), and the proportion of time spent in each geographic area. for the data mining we considered the whole data base. the final model included age (or: 0.015 ic95% 0.0005-0.488; p = 0.018) and the area out of the municipality but in the state (or 2.61 ic 95% 1. 16-5.88 ; p = 0.021). we observed a protective effect in the 25 and older group, and a risk effect when the proportion of time spent in the area out of the municipality but in the state is increased. b) cases vs. population controls. a multiple logistic regression analysis was performed including variables identified with p value < 0.20 in the bivariate analysis and data mining techniques using all variables as reported previously [15] . for bivariate analysis, only the variables age (continuous and dichotomic [under 25 or 25 and older]), gender, occupation (intra and extradomestic), education (dichotomic), proportion of time spent in each area and linear distance were taken into consideration. for the data mining we considered the whole database. the final model included: occupation (or 3.02 ic 95% 1.02-8.88; p = 0.045), proportion of time in the area out of the municipality but in the state (1.42 ic 95% 1.02-1.98; p = 0.035), proportion of time in the area out of the locality but in the municipality (or 0.74 ic 95% 0.57-0.96; p = 0.023), and age (or 0.26 ic 95% 0.09-0.78, p = 0.017). next we analyzed the geographic distribution of the recorded waypoints for each group, both locally and regionally (fig 6) . all three groups recorded waypoints exceeding a 100 km radius from their homes (fig 6a, 6b and 6c ). as expected, most of recorded waypoints were located within the locality. nonetheless, all three groups recorded waypoints exceeding the locality, municipality and state limits. these trajectories were headed mainly to the east, north and west of the locality, and were consistent with the location of the main cities in the area, including cuernavaca (population 338,650) and cuautla (population 154,358), the capital city and the second most important city in the state of morelos, respectively. the geographic distribution of the visits performed by cases and df cumulative incidence for the central mexico region, during year 2012, is shown in fig 7. as seen, this group performed visits to locations with and without df transmission. the number of states, municipalities and regions with high dengue incidence is shown in table 2 . within the locality of axochiapan the most visited areas were identified by dividing the locality in 50 m-radius hexagonal cells (fig 8a) . the most visited cells were those located in the locality's central area, which correspond to the location of the main market, road junctions and main administrative and / or service offices. the location of the cells considered as common visit sites for cases are shown in fig 8b. unlike in fig 8a, the geographical distribution of these 15 cells tends to be peripheral with respect to the locality. using google earth™ we identified the geographic features of each of the 15 cells that was classified as a common visit site for cases. fourteen out of fifteen cells were geographically located within the locality of axochiapan, morelos, and the last one was in the central area of a neighboring small locality (town of tzicatlán) in the state of puebla. as for their typology, xix out of 15 cells clearly corresponded to residential areas (including that in the neighboring state), one cell was a residential area adjacent to a local large business, four cells included small processing plants, a warehouse and a local business, three peripheral cells were crop fields and one cell was clearly a soccer field. previous works have used gps tools for measuring exposure to infectious diseases [16] [17] [18] . in df, recent works in the endemic area of iquitos, peru, have elegantly described human population mobility [19, 9] . however, few data are available for infected cases so far. our work builds upon our knowledge of the role played by mobility in denv transmission documenting spatial mobility of subjects from an endemic region that had, or had not been recently infected by denv. our data show that the people from axochiapan stay within their houses or surrounding areas most of the time. this is consistent with previous observations in iquitos, where population rarely moves more than 1 km away from their homes [10] , however, some individuals recorded movements to very distant locations through the relatively short follow-up period. these movements were present in all three study groups and exceeded a 100-km radius from the center of the study, covering the neighboring states of morelos, state of mexico, puebla, mexico city, tlaxcala and hidalgo. all of these states are located in the mexican central plateau, which is also the best connected region in the country and therefore it is not difficult to reach those destinations by commute travel [20] . surprisingly, spatial mobility in humans was not geographically symmetrical in our study, since no movements were recorded to state of guerrero, which is a coastal, highly endemic area for dengue and also a popular destination for leisure activities. as for the reason why the mobility of the individuals is biased towards central plains in mexico and practically absent towards the southern regions, it was a surprising finding also for us, but we think that it has to do with two factors: first the economic activities in axochiapan are mainly related to agriculture, trade and services which are strongly influenced by the needs of mexico city and its metropolitan area, comprised also by the states of morelos, puebla, méxico and hidalgo. the main cities of these states were those that were visited by the cases and in a lesser extent by the controls. secondly, we did not perform any follow-up during summer and christmas holidays, which in mexico are specific periods for leisure. these activities are usually performed in places that might be different that those observed in our study, including the beaches in the southern coast. it is possible that had we performed our follow-up in vacation periods the observed mobility might have been different, and also leave us with a very interesting research question for the future. the large size of the area covered by these few individuals from a small locality (axochiapan has roughly 17,000 inhabitants) is of capital importance, given the fact that in mexico, and probably in many other places, epidemiological surveillance, prevention and control activities for df are mainly planned, supported and executed by local health authorities, who rely on the information generated by a number of systems, most of them automated [14] , but that are usually restricted to their local administrative limits, namely municipality, sanitary jurisdiction or state at best. thus when df outbreaks overcome those limits and a wider coordination is needed, it is probably that the outbreaks are already established and the window of time for effectively applying control measures has been lost. our data show that the cases group had the largest difference on the time spent in the home area with strong age dependence. older cases spent less time in their homes compared to younger cases. as far as the number of visits is concerned, subjects in the cases group, especially those aged over 25, performed many and more distant visits, than subjects in the intradomestic control group. this difference with the population control group was less marked. this scenario suggests that the population aged over 25 might play an important role in denv persistence and dispersion perhaps working as geographic spreaders. our group previously determined dengue incidence for this age group and proposed a dynamic model which seems to be corroborated by the results presented here [13] . infected individuals, both symptomatic but also asymptomatic [21] , may facilitate the infection of extradomestic mosquito populations in a local scale as models have suggested [8] , or at a regional scale introducing or exporting the virus. given the fact that we performed an uninterrupted 24-hour follow up, we were capable to register the time when individuals left their homes for whatever activity they performed. to our surprise, we recorded waypoints out of the participant's homes virtually at any hour of the day. although the majority of records show that people in axochiapan have a day-light pattern of activities, we recorded waypoints from cases, between 00:00 and 05:00 am, which were consistent with participants' declared jobs, which were related to nocturnal activities such as bakers and workers from the local stone processing plant. the dispersion patterns described above, both in space and time, might be of importance in the results obtained in the control of denv transmission in this and similar small localities. the usual schedule considered by local health authorities for applying preventive measures, which favors early hours for insecticide spraying and the visits by entomological control brigades, in a geographically focalized strategy might hinder the efficacy of the actions by the mere fact that people moves from their homes and remain away during the time these actions are normally applied. in our data, the hourly pattern for activity suggests that cases might leave earlier their homes during weekdays, and thus their homes might have a higher probability to remain closed by the time health authorities apply preventive or control measures. it is important to notice the high mean age of the cases in axochiapan, in both the participants in the study and those recorded historically in the state of morelos, in comparison to other endemic areas from mexico and the americas. this is however, consistent with a previous work in the area [13] , and is probably due to the fact that most (35 out of 42 members of the cases group) of the cases that we studied were asymptomatic, also, although not statistically significant, the mean age of the asymptomatic individuals was higher than that from symptomatic individuals (mean age asymptomatic: 31.54 vs mean age symptomatic: 17.43, t test p = 0.0535). thus, suggesting a possible stronger role of asymptomatic population with age above 25 as spreaders for denv transmission. previous studies have described that visiting other cases' households is a risk factor for denv dispersion [22] ; working sites have also been suggested as possible transmission sources outside of cases' households [23] . models have shown that sites outside homes can play a role in denv transmission and infection [7] , and a recently published work showed positive correlations in thailand between the aedes spp. house index and specific landscape features [24] . our findings are consistent with these data since cases coincided in houses different to their own in at least five different geographical locations. additionally we found cases that coincided in four potential working places, and a soccer field. as far as we know, this is the first time that leisure sites have been documented as possible areas for denv transmission. the lack of study of such sites has previously been pointed out as a weakness in the study of human mobility [25] . as for the common visit sites for controls, we identified 38 cells that were visited only by individuals from both control groups but not by cases. these cells included only one potential working site; whereas the cells commonly visited by cases included several likely workplaces. the identification and study of the extradomestic sites where people coincide is relevant: a recent simulation model has concluded that the selection of areas for df control out of the cases' homes is important not only in terms of the time the subjects spend in them, but also because of the local vector:host ratio, and the other habitual destinations of people that visit the same area [8] . it is possible that much of the movement in a given society is driven by specific population needs and the possibility to fulfill them within or outside from their own locality. axochiapan is a fairly small and well connected locality to other small cities, all of them endemic for df. it is not unrealistic to think that some of the population needs can be readily fulfilled within the same locality whereas other cannot, compelling the population to move away in a permanent or transitory fashion. if a specific population such as that with age older than 25 becomes infected and effectively play a role as spreaders, then perhaps small and peripheral localities to larger cities might have a key importance in sustaining denv transmission across large geographic areas. the assessment of such situations requires a critical review of existing data and the generation of specific studies that may help us to recast current models and more importantly, to completely understand urban denv transmission [26] . we have identified some weaknesses in our study. the most relevant is our small sample size which might have hindered our capability to identify clear patterns in the mobility from this mexican community. there is a possibility that any individual belonging to either control group might have get infected during the follow up; thus making her/him eligible to become a case, therefore disqualifying him/her for being a suitable control and consequently introducing an information bias; nonetheless, we believe that, although a possibility, this was negligible due to two reasons: first, the follow-up of 15 days was very short for this event to occur, and secondly because while recovering each gps, we asked all individuals whether they had experienced fever or any other symptom suggesting dengue infection during the follow-up. none of the participants reported any change in their health status. although we understand that a more robust argument to ensure the infected/uninfected status might be performing an elisa to each control after finishing their follow-up in order to be certain about their exposure, financial constrains made impossible this procedure. possible future improvements in our study are: increase the limited sample size, the inclusion of adequate representation for the population under the age of 12, which probably is a relevant group for transmission during the initial and focalized phases of a df outbreak. although no schools could not be identified in our study as a common site visited by cases, this observation needs to be taken cautiously since our study did not consider the follow-up of children usually studying elementary education. thus, we cannot rule out any role of these sites in dengue transmission at younger ages. additionally, extending the duration of the follow up might improve the chances of successful identification of patterns whose frequencies are longer than a week, such as wage collection, bill payments, and communitarian meetings, among others. this last topic is essential; nonetheless it is limited by technical issues that might be addressed as technology for massive and continuous long-lasting follow up becomes available. finally, the main reasons for which the participants move were not deeply explored in our study, thus we cannot be certain whether the recorded movements indeed depend on non-satisfied needs or on leisure activities. we can only assume that at least those movements performed during the mornings and afternoons between monday and friday correspond to real needs such as employment, education, and supply acquisition, and those performed during weekends are related to leisure. some causes for loss of gps information in field studies have been recently described [27] . although some of these causes might be present in our study, we believe they did not represent significant sources of bias or information loss, since we took some specific measures. for example, people were prevented of accidentally turning the gps off by strapping a tape in the controls. in order to diminish the probability that the participants could forget their gps units at home we performed a weekly phone call reminding them the importance of the usage attachment according the protocol during each individual follow-up. barriers to signal were not important in the studied area since it is located in a plateau with few elevations, and buildings taller than 3-stories are practically absent. finally, the gps equipment used in the study had battery autonomy of up to 32 straight hours and enough memory for recording up to five times the mean number of waypoints programmed to collect in each subject. based on our own data and that from recently published works we conclude that gpsbased technology is a solid tool for the study of detailed human mobility in denv transmission or other infectious diseases, which can and must be adopted in public health and epidemiology as a basic instrument. the important geographic dispersion in our results demonstrates the necessity for studying the potential role that human mobility has in denv transmission and outbreak duration and also a strong argument to study and clarify the role that asymptomatic cases might have in dengue virus dispersion. furthermore our data strongly suggest that the size of the areas considered for prevention and control of df outbreaks needs to be revised and that it is necessary to integrate this knowledge into the planning of preventive and control measures, which usually are prone to using basic shapes such as circles or squares as geographic references in order to define limits, ranges, trajectories and points of origin. it is clear that human populations move normally across geographical areas and not only during holidays or vacations. according to our data, the magnitude of these displacements is larger than that considered as an administrative responsibility for local health services providers. this is relevant for denv transmission if a large fraction of that mobile commuting population is also asymptomatic but viremic, facilitating with their movements the exposure of local uninfected mosquito populations with the virus, which might result in an increased geographical dispersion and persistence of the outbreaks due a continuous process of spreading and reintroduction of the virus to susceptible populations. finally, we believe that the data here reported should be valuable for parameterization of mathematical models exploring specific issues in dengue epidemiology such as geographical dispersion of human activities, contact rate among humans in intermediate spots, optimal range for vector control coverage, optimal target places for health promotion activities, impact of coordinated regional collaboration, and transmission dynamics among satellite and large cities. all essential topics that are still to be understood and weighed as drivers in the transmission of this and other mosquito-transmitted diseases. dengue and dengue heamorrhagic fever world health organization. dengue guidelines for diagnosis treatment, prevention and control urbanisation and infectious diseases in a globalised world the global economic burden of dengue: a systematic analysis travel implications of emerging coronaviruses: sars and mers-cov effect of travel on influenza epidemiology man bites mosquito: understanding the contribution of human movement to vector-borne disease dynamics day-to-day population movement and the management of dengue epidemics assessing and maximizing the acceptability of global positioning system device use for studying the role of human movement in dengue virus transmission in iquitos, peru using gps technology to quantify human mobility, dynamic contacts and infectious disease dynamics in a resource-poor urban environment understanding individual human mobility patterns the scaling laws of human travel peridomestic infection as a determining factor of dengue transmission nation-wide, web-based, geographic information system for the integrated surveillance and control of dengue fever in mexico a search space reduction methodology for data mining in large databases the use of a vest equipped with a global positioning system to assess water-contact patterns associated with schistosomiasis distribution and interspecies contact of feral swine and cattle on rangeland in south texas: implications for disease transmission health &demographic surveillance system profile: the kombewa health and demographic surveillance system (kombewa hdss) multiple outbreaks for the same pandemic: local transportation and social distancing explain the different "waves" of a-h1n1pdm cases observed in méxico during asymptomatic humans transmit dengue virus to mosquitoes house-to-house human movement drives denguevirus transmission epidemiology of dengue and dengue haemorrhagic fever in a cohort of adults living in bandung analyzing the spatio-temporal relationship between dengue vector larval density and land-use using factor analysis and spatial ring mapping population movement and vector-borne disease transmission: differentiating spatial-temporal diffusion patterns of commuting and noncommuting dengue cases recasting the theory of mosquito-borne pathogen transmission dynamics and control strengths and weaknesses of global positioning system (gps) data-loggers and semi-structured interviews for capturing fine-scale human mobility: findings from iquitos authors wish to thank to servicios de salud de morelos for its support to this project. the authors have declare that no competing interests exist. key: cord-003841-7uaj9hmx authors: desmonts de lamache, d.; moges, r.; siddiq, a.; allain, t.; feener, t. d.; muench, g. p.; mckenna, n.; yates, r. m.; buret, a. g. title: immuno-modulating properties of tulathromycin in porcine monocyte-derived macrophages infected with porcine reproductive and respiratory syndrome virus date: 2019-08-23 journal: plos one doi: 10.1371/journal.pone.0221560 sha: doc_id: 3841 cord_uid: 7uaj9hmx porcine reproductive and respiratory syndrome virus (prrsv) is a positive-stranded rna virus that grows in macrophages and causes acute pneumonia in pigs. prrsv causes devastating losses to the porcine industry. however, due to its high antigenic variability and poorly understood immunopathogenesis, there is currently no effective vaccine or treatment to control prrsv infection. the common occurrence of prrsv infection with bacterial infections as well as its inflammatory-driven pathobiology raises the question of the value of antibiotics with immunomodulating properties for the treatment of the disease it causes. the macrolide antibiotic tulathromycin (tul) has been found to exhibit potent anti-inflammatory and immunomodulating properties in cattle and pigs. the aim of this study was to characterize the anti-viral and immunomodulating properties of tul in prrsv-infected porcine macrophages. our findings indicate that blood monocyte-derived macrophages are readily infected by prrsv and can be used as an effective cellular model to study prrsv pathogenesis. tul did not change intracellular or extracellular viral titers, not did it alter viral receptors (cd163 and cd169) expression on porcine macrophages. in contrast, tul exhibited potent immunomodulating properties, which therefore occurred in the absence of any direct antiviral effects against prrsv. tul had an additive effect with prrsv on the induction of macrophage apoptosis, and inhibited virus-induced necrosis. tul significantly attenuated prrsv-induced macrophage pro-inflammatory signaling (cxcl-8 and mitochondrial ros production) and prevented prrsv inhibition of non-opsonized and opsonized phagocytic function. together, these data demonstrate that tul inhibits prrsv-induced inflammatory responses in porcine macrophages and protects against the phagocytic impairment caused by the virus. research in live pigs is warranted to assess the potential clinical benefits of this antibiotic in the context of virally induced inflammation and tissue injury. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 responsible for estimated losses exceeding us$600 million/year in the usa alone, porcine reproductive and respiratory syndrome (prrs) is a devastating disease in the swine industry [1] . first identified in europe and north america in the late 1980s [2] , this syndrome is currently prevalent in most swine-producing countries [3] . its causative agent, the porcine reproductive and respiratory syndrome virus (prrsv), is a small enveloped positive-sense singlestranded rna virus, member of the arterivirus genus [3] . sequence comparison between viral isolates demonstrated that prrsv exists in at least two distinct genotypes, the european genotype (eu type or type i) commonly referred to as prrsv-1, and the north american genotype (na type or type ii) known as prrsv-2 [4] . prrsv has a very narrow cell tropism, and may induce persistent asymptomatic infections [5, 6] . in its natural host, the virus targets alveolar macrophages (am) [7, 8] , and is able to infect most cells of the monocyte-macrophage lineage such as intravascular and lymph node macrophages [7, 9, 10] . these cells play a crucial role in immune surveillance, pathogen killing and adaptive immune response stimulation [11] . prrsv impairs macrophage phagocytic and bactericidal functions, induces host cell death often resulting in an inflammatory response, and perhaps most importantly predisposes the pig to secondary infections [12] [13] [14] [15] [16] . indeed, opportunistic pathogens, whether viral-swine influenza virus, pseudorabies virus-or bacterial-streptococcus suis, bordetella brochiseptica-potentiate prrsv-induced pneumonia [14] . these synergistic effects promote a self-sustaining inflammatory response increasing the severity and the duration of the disease [17] [18] [19] [20] [21] . the common occurrence of prrsv infection with bacterial infections combined with the lack of efficient vaccines begs the question of the value of antibiotics for the treatment of prrs. traditionally, antibiotic efficacy is evaluated solely based on their antimicrobial properties. however, some macrolides have been found to modulate ros and pro-inflammatory cytokines such as cxcl-8 and il-6, and to alter the production of lipid mediators that regulate inflammation [22] [23] [24] [25] [26] [27] . these antibiotics accumulate within leukocytes at concentrations that may reach 500 times the systemic levels, which in turn allows them to be transported directly to the site of infection and confers them superior pharmacodynamics [25] . there is little evidence supporting a direct anti-viral property for macrolides, but their effects on leukocytes support the hypothesis that such macrolides may be beneficial in the context of viral infections such as prrsv [24, 25] . in an attempt to uncover new mechanisms whereby macrolides may protect against the detrimental effects of prrsv, the present study investigated the effects of tulathromycin in porcine monocyte-derived macrophages. tulathromycin is a triamilide in which its 15 lactone-ring is comprised of 3 polar amine groups. it is used for the treatment and prevention of swine respiratory diseases associated with actinobacillus pleuropneumoniae a gram-negative bacteria often found in prrsv-infected pigs [14] . a. pleuropneumoniae exerts cytotoxic effects in macrophage and neutrophils and increases the production of pro-inflammatory il-6, cxcl-8 -also known as interleukin-8-and leukotriene b 4 , which ultimately leads to severe pulmonary tissue damage and death [28] [29] [30] [31] [32] . recent studies have demonstrated that in addition to its antimicrobial effects, tulathromycin inhibits cxcl-8 and ltb 4 production in stimulated neutrophils and macrophages [26, 30, 33] . in addition, tulathromycin promotes the apoptotic death of neutrophils and their phagocytic clearance by macrophages -a phenomenon known as efferocytosis-both crucial processes in the resolution of inflammation [26, 30, [33] [34] [35] . we hypothesized that tulathromycin may generate immunomodulatory benefits in prrsv-infected monocyte-derived macrophages. the findings indicate that tulathromycin, in the absence of a direct anti-viral effect, is able to restore the phagocytic function and to attenuate the pro-inflammatory phenotype of prrsv-infected monocyte-derived porcine macrophages. the african green monkey kidney cell line marc-145 (crl-12231) which is highly permissive to prrsv, was used for viral passage and plaque titration assay, as validated previously [36, 37, 36] marc-145 cells were cultivated in dulbecco's modified eagle's medium (dmem; thermo fisher scientific, waltham, ma, usa) supplemented with 10% fbs (invitrogen, carlsbad, ca, usa) and 100 iu/ml penicillin-streptomycin (thermo fisher scientific, waltham, ma, usa). the cells were maintained at 37˚c, 5% co2 and passaged twice weekly. prrsv-2 isolate nvsl 98-7895 (genbank accession no. ay545985.1) was used in all experiments as previously described [37] . viral titration was performed via plaque assay. briefly, marc-145 cells were seeded in 12 well plates (costar; sigma aldrich, saint-louis, mo, usa) and grown until confluency. once at confluency, cells were infected with prrsv, for 1 hour in serum-free dmem to allow attachment of viral particles. following attachment, marc-145 were overlaid with a solution of 2x mem diluted 1:1 with 1.5% agarose. infection was carried for 96h and plaques were revealed with neutral red (sigma-aldrich, saint-louis, mo, usa). dr. r. m. yates from university of calgary generously provided both marc-145 cell line and prrsv-2 isolate nvsl 98-7895. all animal experimental practices and care were conducted according to the standards of the canadian council of animal care guidelines and approved by the university of calgary life and environmental science animal care committee. blood was collected from healthy large white and landrace cross 10-to 22 weeks old (15-to 60 kg) female and castrated male piglets. the animals were housed at the veterinary science research station (university of calgary) at 22˚c ± 2˚c with 40% humidity, light cycles consisted of 12 hours continuous light exposure followed by 12 hours of darkness. piglets were fed twice with the antibiotic-free feed 16% hog grower (hi-pro feeds, okotoks, ab, canada), water was provided ad libitum. after 22 weeks, animals were euthanized and tissues made available for secondary teaching and research use. in accordance with the standards of the canadian council on animal care, pigs were euthanized by intracardiac injection with sodium pentobarbital. monocytes were obtained and differentiated into macrophages as described previously [30] . briefly, blood was pooled and centrifuged for 20 minutes at 1200 x g, 4˚c in a heraeus megafuge 16r (thermo fisher scientific, waltham, ma, usa). the plasma was removed, and the buffy coat layer was collected into and diluted 1:1 in filter-sterilized 0.9% nacl. sterile polysucrose and sodium diatrizoate gradient solution (histopaque; sigma-aldrich, saint-louis, mo, usa) was added into each tube before centrifugation for 40 minutes at 1200 x g, 4˚c. pbmcs located at the opaque interphase were then collected, washed with sterile-filtered 2x hank's balanced salt solution (hbss; thermo fisher scientific, waltham, ma, usa) and centrifuged for 10 minutes at 500 x g, 4˚c. contaminating erythrocytes were removed by three hypotonic lysis cycles with sterile ice-cold double-distilled water for 30 seconds followed by the addition of 2x hbss to restore tonicity. pbmcs were then resuspended in serum-free iscove's modified dubelcco's medium (imdm; thermo ficher scientific, waltham, ma, usa) supplemented with 100 iu/ml penicillin-streptomycin. cells were counted using a hemocytometer and viability was assessed by 0.1% trypan blue exclusion (flow laboratories). pbmcs purity was determined by diff-quick staining on cytospin slides (cytospin4 cytocentrifuge, thermo fisher scientific, waltham, ma, usa). the cells were then plated in tissue-culture treated 6, 12, 24 and 96 well plates (costar; sigma aldrich, saint-louis, mo, usa) or in labtek chamber slides (thermo fisher scientific, waltham, ma, usa) at a concentration of 1.0 x10 6 cells/ ml for two hours to allow attachment. following adhesion, non-adherent mononuclear cells were washed with warm hbss (37˚c). subsequent adherent monocytes were incubated for 7 days at 37˚c, 5% co 2 in imdm supplemented with 10% heat inactivated(hi)-pig serum (ge healthcare, chicago, il, usa), 100 iu/ml penicillin-streptomycin and 15% l929 supernatant to allow for differentiation into monocyte-derived macrophages (mdms). l929-conditionned medium is commonly used to potentiate monocytes to differentiate into homogenous populations of mature macrophages [37, 38] . culture media was changed every 3 days. flow cytometry was used to quantify the number of cells expressing cd163 (a known cluster of differentiation of monocytes and macrophages). more than 95% of the isolated cells expressed cd163 (s1 fig) . on day 7, as described previously [26] , macrophage differentiation was monitored by microscopic morphological changes using diff-quick, and esterase staining, a well known feature allowing to distinguish between monocytes and mature macrophages [39] . at day 7, more than 95% of the cell preparations were differentiated macrophages (data not shown). seven days-old differentiated macrophages were incubated with tulathromycin (draxxin; zoetis, parsippany-troy hills, nj, usa) diluted in imdm + 10% hi-pig serum at a concentration of 0.5 mg/ml or 1 mg/ml or with vehicle control (imdm + 10% pig serum), as established recently [26, 33] . at these concentrations and time points, the drug exhibits immunomodulating properties in bovine macrophages without inducing apoptosis [26] . antibiotics like tulathromycin accumulate within leukocytes at concentrations that may reach >500 times the systemic levels, which in turn allows them to be transported directly to the site of infection, and hence confers them with superior pharmacodynamics [25, 40] . this phenomen is critical to the mode of action of tulathromycin. the drug concentrations used in these present experiments are consistent with this knowledge, and with previous studies that showed that tulathromycin has immunomodulating effects in bovine and porcine neutrophils and macrophages [26, 30, 33] . these recent studies have reproduced the same immunomodulating effects seen in vitro at these drug concentrations than when using live infected cattle and pigs given tulathromycin at the recommended therapeutic dosage [26, 30, 33] . hence the concentrations used here reflect the physiological conditions in which the drug accumulates at high concentrations within these leukocytes. indeed, comparison of intracellular drug concentrations in treated versus untreated animals have been published previously [40] . using lc/ ms ms, in animals given the recommended dose of 2.5 mg/kg body weight, studies have measured the rapid and prolonged distribution of the drug into lung homogenates, pulmonary epithelial ling fluid (pelf), as well as in pelf cells. macrophages are the major constituents of pelf cells in such preparations. drug levels measured in pelf cells reached concentrations 565 times greater than those in plasma [40] . it is believed that this great affinity for cellular uptake may be related, in part, to the tri-basic chemical structure of the drug and the trapping of ionized drug within acidic phagolysosomes. macrophages were infected with prrsv 30 minutes after tul or vehicle treatment, or not infected (uninfected controls) and incubated for 1 h at 37˚c, 5% co 2 to allow virus attachment and entry (time 0; t = 0). prrsv was diluted in serum-free dmem to reach a multiplicity of infection (m.o.i) ranging from 0.1 to 1 depending on the experiment. culture media was replaced by pre-warmed imdm supplemented with 10% pig serum for all experimental groups. prrsv infection was performed for another 2 to 48 hours depending on the experiment. all functional assays contained the following experimental groups: untreated and uninfected control (control); tulathromycin-treated (tul); untreated and prrsv-infected (virus); tulathromycin-treated and prrsv-infected (tul+virus); lps-activated; pro-apoptotic positive control (staurosporine; 3μm) (sts); or pro-necrotic positive control (0.1% triton-x) (trit-x) where appropriate. to avoid l929 cytokine-induced polarization of macrophages, all macrophages activation experiments were performed on monocytes that were grown in l929 supernatant free. the effects of tulathromycin and prrsv on macrophage differentiation and activation was determined via microscopic observations and cytokine quantification. mdms were treated with tulathromycin (0.5 or 1 mg/ml) for 1 hour and infected with prrsv (m.o.i. of 0.1) for 2, 4, 12 or 24h at 37˚c, 5% co 2 . supernantants were collected and frozen at -80˚c until processed and macrophages were stained with diffquick (electron microscopy sciences, hatfield, pa, usa) and observed with a nikon eclipse t300 microscope to assess morphological changes. images were taken with a retiga 2000x camera (q imaging, surrey, bc, canada) on a leica dmr fluorescent microscope (leica, wetzlar, germany) and analyzed using imagej software. individual macrophage morphology was assessed and classified as "resting" or "fibroblast-like" morphology. at least 150 macrophages per group in 3 independent experiments were assessed. supernatants were processed to measure interleukin-8 (cxcl-8) and interleukin-10 (il-10) concentrations using the porcine cxcl-8 quantikine enzyme-linked immunosorbent assay (elisa; p8000, r&d systems, minneapolis, mn, usa) and the il-10 quantikine elisa (p1000, r&d systems, minneapolis, mn, usa) respectively. samples were processed as per manufacturer's instructions. ros production by mdms following tulathromycin treatment and/or prrsv infection was monitored with the oxiselect intracellular ros assay kit (cell biolabs, san diego, ca, usa). experiments assessed ros production in resting cells, as well as in a group of cells induced by lipopolysaccharide (lps) to determine the effects of the various stimuli under basal conditions in these cells, as well as when they were activated. mdms were infected for 2, 4, 12, or 24h (m. o.i of 0.5) or uninfected (uninfected control). the same treatments were performed on macrophage stimulated with lipopolysaccharide (1μg/ml lps from e. coli o26:b6 (sigma-aldrich, saint-louis, mo, usa). mdms were exposed to 2',7'-dichlorodihydrofluorescin diacetate (dcfh-da) a cell-permeable fluorogenic probe oxidized to highly fluorescent 2',7'-dichlorodihydrofluorescein (dcf) by ros. fluorescence intensity, proportional to ros levels within the cytosol was measured using a spectramax m2e microplate reader (molecular devices, san jose, ca, usa) reading at 480 nm (excitation) and 530 nm (emission). phagocytic capacity of mdms was assessed using non-opsonized zymosan particles and opsonized latex beads. non-opsonized phagocytosis was monitored using fluorescently labelled saccharomyces cerevisiae zymosan a particles (texas red; sigma-aldrich, saint-louis, mo, usa). mdms seeded on labtek chamber slides or on coverslips at 1x10 6 cells/ml were infected for 2 or 12 hours (m.o.i of 0.5) or not infected (uninfected control). following infection, experimental groups were incubated with zymosan a particles diluted in control media to a final ratio of 10:1 (zymosan:cells) for 1 hour. after exposure, extracellular zymosan a particles were washed away with warm pbs and the cells were fixed in ice-cold 80% acetone solution. actin was stained with the alexa fluor 488 phalloidin antibody (thermo fisher scientific, waltham, ma, usa) and the nucleus was revealed with dapi (thermo fisher scientific, waltham, ma, usa). enumeration of intracellular zymosan was performed using a leica dmr fluorescent microscope. fc-mediated phagocytic index was measured using carboxylate-modified 3μm diameter latex or silica beads (kisker biotech, steinfurt, germany) covalently coated with bsa and human igg (sigma-aldrich, saint-louis, mo, usa). the beads were subsequently incubated with macrophages for 45 minutes at a 10:1 (beads:cells) ratio. following phagocytosis, extracellular beads were washed away with warm pbs and the cells were stained with diffquick (electron microscopy sciences, hatfield, pa, usa) before microscopic observations. macrophages containing one or more zymosan particles or latex beads were considered as 'positive cells', the phagocytic index was calculated as the ratio of positive macrophages versus total macrophages. a minimum of 150 cells per experimental group were counted from randomly selected fields. all pictures were taken using leica dmr fluorescent microscope with a retiga 2000x (q imaging, surrey, bc, canada) and analyzed using imagej software. in order to prevent any counting bias slides labellings were covered with tape prior to microscopic observations. the pro-apoptotic effects of tulathromycin and prrsv were assessed using a cell death detection elisa kit (roche) according to the manufacturer's instructions as previously described [22, 26] . absorbance was measured using a spectramax m2e microplate reader (molecular devices, san jose, ca, usa) set at 405nm. mdms were incubated with tulathromycin (0.5 or 1 mg/ml) for 30 minutes and infected with prrsv (m.o.i. of 0.1) for 2, 12 or 24h at 37˚c, 5% co 2 . similar experimental treatments were conducted with cells stimulated with lps (1μg/ml from e. coli o26:b6; sigma-aldrich, saint-louis, mo, usa) to assess apoptosis in activated macrophages. for all experiments, cells incubated with imdm containing 10% hi-pig serum or staurosporine (1μm) were used as negative and positive controls respectively. annexin v staining (roche) was performed on the same experimental groups to further assess apoptotic cell death. staining was performed as per manufacturer's instructions and fluorescence was observed using a leica dmr fluorescent microscope equipped with a hcx pl fluotar x40 objective (aperture = 0.75). images were taken at 2, 12 and 24 hours post infection (p. i) with a retiga 2000x (q imaging, surrey, bc, canada). importantly, experiments measuring cytokines and ros followed those in which we measured apoptosis. this allowed to adjust concentrations of tulathromycin and virus to levels at which apoptosis was not detected in the cells, and hence cell death would not be a factor in the cellular cytokine and ros responses. necrosis was assessed through the determination of lactate dehydrogenase (ldh) levels using a cytotoxicity detection kit (roche). mdms were treated with vehicle medium alone (control) or with tulathromycin (1mg/ml) for 30 minutes. cells were then infected with prrsv for 2, 6, 12 or 24h (m.o.i. 0.1) or supplemented with control medium, and 1% triton x 100 in media was used as positive control. supernatants were collected and processed following manufacturer's instructions. a spectramax m2e microplate reader (molecular devices, san jose, ca, usa) was used to measure ldh concentrations in each sample at 492 nm. necrosis was expressed as the absorbance ratios of the experimental cell lysates versus absorbances from controls arbitrarily set at 1.0 (100%). all experimental groups were assessed in duplicates. to assess the potential anti-viral effects of tulathromycin, extracellular and intracellular viral particles counts were monitored with plaque titration assays. for extracellular counts, supernatants were harvested at 2, 12, 24 and 48 hours p.i and incubated with confluent marc-145 cells for 96 h as described above. for intracellular counts, macrophages were washed twice with warm (37˚c) phosphate buffer saline (pbs; sigma-aldrich, saint-louis, mo, usa) and lysed with double distilled water exposure and thorough mixing. cellular debris were spun down at 10,000 x g for 30 minutes and supernatants were harvested and incubated with confluent marc-145 cells as described previously. prrsv staining was performed to further characterize potential antiviral effects. marc-145 cells were seeded in labtek chamber slides, grown to 90% confluence and infected with prrsv (m.o.i. 0.1) for 24h at 37˚c, 5% co 2 . prrsv foci numbers and size were revealed using the sr-30f antibody (rti, llc, brooking, sd, usa). fluorescence ratio was calculated using imagej. five fields of view per well were counted per sample. expression levels of prrsv receptors in mdms in the presence and absence of tulathromycin were assessed by immunofluorescence. seven days old mdms cultivated with or without l929 conditioned medium were treated with hbss (control) or tulathromycin (1 mg/ml for 12 hours). the murine l929 fibroblast cell line, known to secrete macrophage-colony stimulating factor (m-csf) is widely used to induce macrophage differentiation from monocytes and prevent differentiation into monocyte-derived dendritic cells [38] . prior to staining, l929-grown cells were washed 3 times in ice-cold pbs to remove all l929 media and then fixed in 4% paraformaldehyde in pbs for 15 minutes. fixed cells were then washed 3 times in cold pbs and stained for 1 hour with a r-phycoerythrin (rpe) conjugated anti-cd163 antibody (bio-rad, hercules, ca, usa) and a fluorescein isothiocyanate (fitc) conjugated anti-cd169 antibody (bio-rad, hercules, ca, usa) at a dilution of 1 to 500 and 1 to 250 respectively. following staining cells were washed 3 times in cold pbs and observed under leica dmr fluorescent microscopy. fluorescence ratios from randomly selected fields were calculated using the software imagej. images were taken with a retiga 2000x camera. to prevent any bias, slide labelling was covered with tape prior to microscopic observations. all statistical analyses were made using prism 5 software and data were expressed as means + standard error from mean (sem). all data sets were tested for normality. data with parametric distribution were compared using student's t-test, or one-way anova with tukey's multiple comparision anlaysis where appropriate. non-parametric data were compared with a kruskal-wallis test. for every assay, a minimum of 3 separate, independent experiments were conducted with all experimental groups assayed in duplicates or triplicates. statistical significance was established at p < 0.05. in order to determine whether blood monocytes and mdms are susceptible to prrsv we isolated blood monocytes from healthy pigs and cultured them for a period of 7 days in medium supplemented with pig serum to mimic biological conditions. after plating, adherent monocytes exhibited a round shape morphology and were approximatively 10μm in diameter ( fig 1a) . by day 7, the cells displayed a larger, macrophage-like, morphology with characterisitic cytoplasmic vacuoles (fig 1c) . monocyte differentiation was also measured using non-specific esterase (nse) staining. by day 7 more than 95% cells were esterase-positive cells. oneday-old monocytes were significantly less susceptible to prrsv compared to 7 days old differentiated mdms (fig 2) . prrsv viral particle numbers increased by a 1.69 log (50-fold increase) in mdms, and by a 1.1 log (13-fold increase) in blood monocytes, between 2 and 48 hours p.i. in both cell types, prrsv infection reached a plateau at 24h p.i. (fig 2) . to optimize the macrophage differentiation protocol, mdms were also cultured in a l929-conditioned medium. monocytes cultivated in l929-conditioned medium showed the same morphology as those cultivated in medium devoid of l929-factors. consistent with previous data, microscopic observation and nse staining showed that by day 7, more than 95% cells were macrophages. viral titers in monocytes incubated with l929 were significantly higher at 48 hours p.i. compared to viral titers in monocytes cultivated without l929 supernatant (fig 2) . numbers of prrsv infectious particles were significantly elevated in mdms cultivated with l929-supernatants at all time points of the infection (except from the 8 hours p.i. time point) versus mdms cultivated in medium supplemented with hi-pig serum alone (fig 2) . in l929-cultivated mdms, prrsv infection peaked at 24 h p.i. and declined afterwards, whereas it continued to increase at 48 hours in l929-cultivated monocytes (fig 2) . based on these observations, we chose to use l929-cultivated mdms for functional experiments, unless stated otherwise. considering that l929 supernatants may contain cytokines other than m-csf (such as il-10 and il-4) that could influence macrophage polarization and confound our studies, we decided to grow our cells in medium containing only pig serum when assessing macrophage pro-inflammatory signaling. moreover, to limit the impact of tulathromycin and prrsv-induced apoptosis on macrophage numbers and functions, we treated our cells with tulathromycin at a concentration of 0.5 mg/ml and decreased prrsv m.o.i from 0.5 to 0.1. at these concentrations, neither the virus nor the drugs significantly induced mdm apoptosis at the experimental time points (data not shown). if the cells were treated at a concentration of 1mg/ml, functional analysis were performed before 12 hours of incubation. prrsv infection induced a sharp fibroblast-like morphological alteration and pseudopod projections in mdms (fig 3a) . in uninfected cells (control and tul), less than 20% of cells exhibited this change in morphology, while nearly 60% of the cells exhibited this phenotype upon prrsv infection (fig 3b) . tulathromycin pre-treatment significantly inhibited this morphological change in mdms (fig 3b) . since macrophage shape and function are correlated [41] , we hypothesized that prrsv-induced morphological changes were associated with a change in macrophage activation. infection of mdms with prrsv caused a 6-fold increase of cxcl-8 secretion after 24 hours (fig 4) . prrsv-induced cxcl-8 secretion was significantly inhibited when mdms were pretreated with tulathromycin (fig 4) . the positive control lps, also significantly increased the production of cxcl-8 (fig 4) . we then measured the production of mitochondrial ros, a hallmark of pathogenic oxidative damage in inflamed tissues [42, 43] . prrsv infection significantly increased intracellular ros (fig 5) . tulathromycin treatment abolished prrsv and lps-induced intracellular ros production, however, it did notrestore ros levels to control values in cells exposed to both lps and prrsv (fig 5) . interestingly, intracellular ros levels were significantly lower in mdms incubated with lps and prrsv compared to mdms stimulated only with lps (fig 5) . as our results indicated that tul inhibited macrophage pro-inflammatory signaling, another set of experiment assessed the effects of the drug on il-10, a cytokine with potent anti-inflammatory properties [44] . resting mdms produced approximately 300 pg/ml il-10 throughout the course of the experiments (fig 6) . prrsv infected cells secreted significantly less il-10 compared to control cells at 2 and 12 hours p.i. (fig 6) . il-10 levels did not significantly change versus controls when uninfected cells were treated with tulathromycin alone. however, immuno-modulating properties of tulathromycin in prrsv-infected porcine monocyte-derived macrophages prrsv-induced il-10 inhibition was abolished when the cells were pre-treated with tulathromycin at 2 and 12 hours post infection (fig 6) . tulathromycin (1 mg/ml), as well as prrsv alone (m.o.i = 0.5), or the positive control staurosporine induced mdms apoptosis 24 h post-infection (fig 7) . combined pre-treatment with tulathromycin (1mg/ml; 1h) and prrsv (m.o.i = 0.5) for 24 hours showed an additive effect to induce further mdms apoptosis versus single treatments (fig 8a) . to confirm these data, cells were stained with annexin v, a phospholipid-binding protein with high affinity for the early apotptic marker phosphatidylserine (ps) [45] . at 24 hours, both tulathromycin alone or prrsv alone induced significant levels of apoptosis compared to controls (4-fold increase vs. control) (fig 8b and 8c) . when cells were exposed to the combination of tulathromycin immuno-modulating properties of tulathromycin in prrsv-infected porcine monocyte-derived macrophages and prrsv, levels of apotosis were almost double those measured in cells exposed to single treatments (fig 8b and 8c ). prrsv infection (m.o.i = 0.5) significantly increased the levels of ldh produced during necrosis 12 and 24 hours p.i. (fig 9) . treatment with tulathromycin (1mg/ml) significantly reduced prrsv cell necrosis at 12 hours (fig 9) . this effect of tulathromycin could no longer be detected at 24 hours. tulathromycin alone did not alter levels of necrosis (fig 9) . triton-x (trit-x), used as a pro-necrotic positive control, induced necrosis in mdms (fig 9) . another set of experiments assessed the effects of prrsv, and of tulathromycin, on the nonopsonized and opsonized phagocytic functions of mdms. prrsv infection significantly immuno-modulating properties of tulathromycin in prrsv-infected porcine monocyte-derived macrophages inhibited both phagocytic functions of the cells (figs 10 and 11 ). prrsv-induced phagocytic inhibition was inhibited by tulathromycin (figs 10b and 11b ). tulathromycin treatment alone did not alter mdms phagocytosis versus controls (figs 10 and 11 ). during phagocytosis, macrophages can engulf multiple antigens at the same time [46, 47] . additional experiments assessed mdms that engulfed less than 5 particles (ie with basal phagocytic indices) versus cells that ingested more than 5 particles (i.e. with elevated phagocytic indices). prrsv infection significantly reduced the number of cells with high phagocytic indeces, an effect that was abolished by tulathromycin (figs 10c and 11c) . tulathromycin inhibited the prrsv-induced reduction of basal and high phagocytic indices (figs 10 and 11 ). tulathromycin alone did not change either of the mdms phagocytic indices versus controls. the same results were obtained when the cells were infected for 2 hours (fig 12) . another set of experiments assessed whether the effects of tulathromycin described above were associated with direct antiviral properties of the antibiotic, in porcine mdms (fig 13a) or marc-145 cells (fig 13b) . upon incubation with prrsv, extracellular and intracellular viral particles were enumerated via plaque assay. tulathromycin did not change intracellular or extracellular viral titers in either of the cell models (fig 13a and 13b ). to verify these results, marc-145 cells were stained with fitc-conjugated anti-prrsv nucleocapsid antibody sr30f antibody. size and numbers of viral foci were calculated in presence or absence of tulathromycin ( fig 13c) . again, tulathromycin pre-treatment did not alter viral titers compared to exposure to prrsv alone (fig 13c and 13d ). to further examine the effects of tulathromycin on prrsv infectivity, experiments measured viral receptor expression in mdms. to date, two major prrsv receptors have been extensively studied (cd163 and cd169) and it is not entirely clear which one of these two receptors is essential for prrsv infection [48] [49] [50] . since l929-conditioned medium increases viral titers, we hypothesized that it might be due to an increase in cell permissivity resulting from an increase in prrsv receptor expression. to test this hypothesis, we cultivated monocytes in medium containing pig serum alone or in l929-conditionned medium for 7 days and then treated them with tulathromycin. mdms differentiated in medium devoid of l929-supernatant expressed both receptors. approximatively 28% of cells expressed cd163 and 89% of cells expressed cd169. tulathromycin treatment did not significantly change the percentage of cd163 and cd169 positive cells (respectively 29% and 83% of positive cells) (fig 14a; upper panels; fig 14b) . mdms incubation in l929-supernatant supplemented medium was sufficient to significantly increase the number of cd163 positive cells (more than 90% of mdms were cd163 positive versus less than 30% in pig serum supplemented medium alone). in addition, following l929-supernantant exposure we were not able to detect any cd169 positive cells (fig 14a; lower panels; fig 14b) . tulathromcyin treatment following l929-incubation did not have any significant effect on viral receptor expression in these experiments (fig 14a; lower panels; fig 14b) . prrs is one of the most devastating diseases of the porcine industry [1, 3] . treatment options to control prrs outbreaks are limited and the efficacy of vaccines is thwarted by the antigenic variability of prrsv [51] . disease severity is closely related to the ability of the virus to dysregulate macrophages functions and induce inflammation. therefore, we hypothesize that targeting either of these components may represent a critical element of novel therapeutic approaches. anti-inflammatory and immunomodulatory properties of macrolides have been well established [23-25-24] . whether these effects may be beneficial in the context of viral diseases such as prrs remains obscure. the present study assessed the anti-viral and immunomodulating properties of tulathromycin (tul) in prrsv-infected porcine macrophages. the findings indicate that tul inhibits prrsv-induced inflammatory responses in porcine monocyte-derived macrophages and protects against the phagocytic impairment caused by the virus, in the absence of any direct anti-viral effects. the two most common cellular models are pams and marc-145 cells [12, 52] . both have significant limitations. the isolation of pams requires bronchoalveolar lavages, and the function of these cells depends on the age and environment of the animal [53] . moreover, shortly after the initiation of a respiratory infection, alveolar macrophages are replaced by monocytederived macrophages, which therefore represent a key cell population in host-prrsv immuno-modulating properties of tulathromycin in prrsv-infected porcine monocyte-derived macrophages interactions. monkey marc-145 epithelial cells do not originate from pigs. therefore, the present experiments developed and used a simple porcine monocyte-derived macrophage model system to characterize the impact of tulathromycin on prrsv infection. previous in vitro studies have shown that the virus could infect blood monocyte-derived macrophages (mdms) [54] . consistent with previous findings, monocytes were less susceptible to prrsv than differentiated monocyte-derived macrophages [54] . the addition of l929 supernatant during macrophage differentiation significantly increased their susceptibility to the virus compared to macrophages cultivated in medium supplemented with pig serum alone. it has been well established that l929 supernatant is a source of m-csf and is used to induce macrophage differentiation [38] . immunostaining of prrsv receptors showed that the addition of l929 immuno-modulating properties of tulathromycin in prrsv-infected porcine monocyte-derived macrophages strongly upregulated cd163 (29% positive cells to 89% positive cells) but abolished cd169 expression. these results indicate that l929 supernatant modulate the expression of prrsv receptors, and that cd163 alone is sufficient for prrsv infection. this is consistent with recent observations showing that cd163, but not cd169, enabled non-permissive cells to become susceptible, and that increased cd163 correlates with increased susceptibility to prrsv [49, 54, 55] . l929 supernatant is known to contain m-csf, but very little is known about other cytokines and chemokines present in this supernatant [38] . considering that cd163 and cd169 expression can be induced by il-10 and ifn-γ respectively, and that il-10 treatment increases prrsv infectivity while ifn-γ decreases it [54, 56, 57] , the role of these cytokine in the modulation of macrophage susceptibility to infection requires further investigation. the present findings demonstrate that mdms can readily be infected by prrsv, and hence represent a useful cellular model to study prrsv pathogenesis, as suggested recently [37] . a hallmark of prrsv pathogenesis resides in its ability to alter macrophages survival and function, hence predisposing the host to secondary infections [13, 58] . there is correlation between macrophage morphology and function, hence providing an easy way to monitor immuno-modulating properties of tulathromycin in prrsv-infected porcine monocyte-derived macrophages changes in macrophage polarization [41] . in this study we found that prrsv infection dramatically altered monocyte-derived macrophage morphology, inducing an elongated phenotype with numerous cytoplasmic pseudopods. recent findings indicate that these pseudopods promote intercellular junctions allowing prrsv to evade host immunity through direct intercellular spread [59] . tulathromycin pre-treatment was sufficient to prevent the prrsvinduced pseudopod formation and morphological alterations in macrophages. whether tul may prevent intercellular junctions and thus hinder prrsv immune evasion requires more research. to test the hypothesis that change in macrophage morphology was associated with altered function, we measured the production of pro-and anti-inflammatory cytokines (cxcl-8 and il-10 respectively) as well as the production of mitochondrial ros. cxcl-8 is a potent neutrophil chemoattractant secreted by macrophages and other cell types, and is a critical mediator of neutrophil infiltration in inflamed tissues [26] . the present findings demonstrate that prrsv is a potent inducer of cxcl-8 in monocyte-derived macrophages. virally induced cxcl-8 secretion was inhibited by tul. studies in live animals are warranted to assess whether these observations suggest that tul might attenuate prrsv-induced inflammation through cxcl-8 inhibition. mitochondrial ros production is a hallmark of cell stress and inflammation, and contributes to prrsv-induced tissue damage [42, 43] . other reports showed that mitochondrial ros production was implicated in prrsv-induced apoptotic death of marc-145 cells [43] . here we demonstrate that prrsv indeed induces ros production in porcine monocyte-derived macrophages, and that this production is inhibited when the cells are pre-treated with the antibiotic. tulathromycin was also able to restore ros levels to control in lps-stimulated cells but not in lps and prrsv exposed to both lps and prrsv. interestingly, in these conditions mdms showed a decrease in ros production compared to cells exposed only to lps. this suggest that prrsv may inhibit intracellular ros production of mdms during bacterial infections.another set of studies sought to determine whether tul inhibition of the viral-induced pro-inflammatory cxcl-8 coincided with an increase in antiinflammatory signaling. we found that the virus alone was able to inhibit il-10 secretion, and that tul blocked this effect. these data are in contrast with others from the scientific literature. indeed, it is generally accepted that prrsv induce il-10 production to increase its infectivity [54] . in fact, il-10 activated cells are more permissive to prrsv than unstimulated m1-polarized macrophages [54] . more research is necessary to explain the mechanisms whereby prrsv regulates the production of il-10. tulathromycin alone did not induce il-10 secretion suggesting that cxcl-8 and mitochondrial ros inhibition by tul was not dependent on il-10 production. taken together the present findings strongly support the hypothesis that tulathromycin may attenuate prrsv-induced inflammation by inhibiting production of pro-inflammatory cxcl-8, and by preventing the suppression of anti-inflammatory il-10. consistent with previous studies, we found that prrsv and tul induced macrophage apoptosis [33, 43, 60, 61] . the present findings also illustrate that tul and prrsv haver additive pro-apoptotic effects. morevoer, the data indicate that prrsv leads to cell necrosis, an effect that was inhibited by tul. necrosis is known to exacerbate local inflammation, to induce the release of cytotoxic molecules, and to lead to extensive tissue damage, while cell apoptosis contributes to the resolution of inflammation [62, 63] . more research in live prrsv-infected animals will help determine whether tul is able to promote the resolution of prrsv-induced pulmonary inflammation at least in part via such a mechanism, as well by shifting local cytokine release from pro-inflammatory to anti-inflammatory mediators. it is well established that prrsv infected pigs are often infected by secondary pathogens [13, 58] . at present, the mechanisms resulting in the increase of secondary infections during prrsv infections remain incompletely understood. studies have shown that prrsv is directly able to impair macrophage phagocytosis, which in turn may represent a key element of the development of secondary infection [12, 64, 65] . macrophage phagocytosis is triggered when phagocytic receptors including opsonic receptors (fcr) or pattern recognition receptors such as the mannose receptor, are activated [45, 66] . using non-opsonized zymosan particles or igg-coated latex beads, the present findings demonstrate that prrsv significantly inhibits both phagocytic pathways. these results are consistent with previous reports showing decreased phagocytosis of latex beads, or live bacteria (streptococcus suis) upon prrsv infection [67, 68] . recent findings suggest that prrsv-1 inhibits phagocytosis through its interaction with sialoadhesin (also referred to as cd169). however, in our model system, l929 cultivated mdms were negative for cd169 suggesting either that mechanisms for inhibition of phagocytosis are strain and/or genotype dependent, or that prrsv may inhibit phagocytosis through multiple pathways [16] . another report recently demonstrated that the same nsvl-98-7895 strain as used here may impair phagosomal maturation and nadph oxidasemediated respiratory burst, both implicated in the antimicrobial properties of macrophages [37] . tul blocked the prrsv-induced inhibition of non-opsonized and igg-mediated macrophage phagocytosis. these observations pave the way towards studies in vivo to assess whether this antibiotic might help control secondary infections during prrsv infections through this mechanisms in addition to its direct anti-microbial properties. the mechanisms whereby tul protects against prrsv-induced inhibition of phagocytosis require further elucidation. some macrolides such as tilmicosin and tylvalosin have been recently demonstrated to possess direct anti-viral effects against prrsv [69, 70] , while others like erythromycin do not [71] . in the experiments described herein, tul did not exhibit any direct anti-viral properties, nor did it significantly alter the expression of the two receptors used by the virus for entry, cd169 and cd163. together, the data indicate that in porcine mdms, tul is able to block prrsvinduced pseudopod formation, necrosis, pro-inflammatory cxcl-8 and mitochondrial ros production, and inhibition of macrophage phagocytosis. in addition tul also synergized with prrsv to induce pro-resolution cell apoptosis and the production of anti-inflammaotry il-10. the results also show that the protective modulation of macrophage structure, function, and behavior by tul occurs in the absence of a direct anti-viral effect. the present observations pave the way towards further studies with a prrsv-1 strain to determine whether the effects we observed in this study are conserved with the other prrsv genotype. studies in vivo will help determine whether and how these effects may translate into clinical benefits. assessment of the economic impact of porcine reproductive and respiratory syndrome virus on united states pork producers reproductive failure of unknown etiology porcine reproductive and respiratory syndrome virus changes to taxonomy and the international code of virus classification and nomenclature ratified by the international committee on taxonomy of viruses the molecular biology of arteriviruses arterivirus molecular biology and pathogenesis effects of origin and state of differentiation and activation of monocytes/macrophages on their susceptibility to porcine reproductive and respiratory syndrome virus (prrsv) virus quantification and identification of cellular targets in the lungs and lymphoid tissues of pigs at 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host-pathogen interplay at primary infection sites in pigs challenged with actinobacillus pleuropneumoniae anti-inflammatory benefits of antibiotic-induced neutrophil apoptosis: tulathromycin induces caspase-3-dependent neutrophil programmed cell death and inhibits nf-κb signaling and cxcl8 transcription. antimicrob. agents chemother macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving tgf-β, pge2, and paf resolution of inflammation: a new therapeutic frontier evaluation of porcine reproductive and respiratory syndrome virus replication in laboratory rodents infection of porcine bone marrow-derived macrophages by porcine respiratory and reproductive syndrome virus impairs phagosomal maturation granulocyte/macrophage colony-stimulating factor is expressed and secreted in cultures of murine l929 cells identification of markers that distinguish monocytederived fibrocytes from monocytes, 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for attachment/internalization of the porcine reproductive and respiratory syndrome virus improved vaccine against prrsv: current progress and future perspective enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma-104 cell line variability of neutrophil and pulmonary alveolar macrophage function in swine establishing porcine monocyte-derived macrophage and dendritic cell systems for studying the interaction with prrsv-1 cd163 expression confers susceptibility to porcine reproductive and respiratory syndrome viruses human monocytes express cd163, which is upregulated by il-10 and identical to p155 interferon-inducible cd169/ siglec1 attenuates anti-hiv-1 effects of ifn-α immune responses in pigs infected with porcine reproductive and respiratory syndrome virus (prrsv) porcine reproductive and respiratory syndrome virus utilizes nanotubes for intercellular spread adenoviral-expressed gp5 of porcine respiratory and reproductive syndrome virus differs in its cellular maturation from the authentic viral protein but maintains known biological functions apoptosis and porcine reproductive and respiratory syndrome virus corpse clearance defines the meaning of cell death differential effects of apoptotic versus lysed cells on macrophage production of cytokines: role of proteases effects of porcine reproductive and respiratory syndrome virus (isolate tw91) on porcine alveolar macrophages in vitro in utero infection with prrs virus modulates cellular functions of blood monocytes and alveolar lung macrophages in piglets mechanisms of phagocytosis in macrophages porcine reproductive and respiratory syndrome virus productively infects monocyte-derived dendritic cells and compromises their antigen-presenting ability transcriptional analysis of prrsv-infected porcine dendritic cell response to streptococcus suis infection reveals up-regulation of inflammatory-related genes expression antiviral activity of tilmicosin for type 1 and type 2 porcine reproductive and respiratory syndrome virus in cultured porcine alveolar macrophages tylvalosin exhibits anti-inflammatory property and attenuates acute lung injury in different models possibly through suppression of nf-κb activation antibiotic-mediated inhibition of porcine reproductive and respiratory syndrome virus (prrsv) infection: a novel quinolone function which potentiates the antiviral cytokine response in marc-145 cells and pig macrophages the authors thank troy feener, barbara smith, and the staff at the veterinary sciences research station at the university of calgary for their help with animal handling. we also thank dr. constance finney and dr. edina szabo for their help with flow cytometry. buret. key: cord-001142-puj74k7y authors: crescenzo-chaigne, bernadette; barbezange, cyril; van der werf, sylvie title: the panhandle formed by influenza a and c virus ns non-coding regions determines ns segment expression date: 2013-11-21 journal: plos one doi: 10.1371/journal.pone.0081550 sha: doc_id: 1142 cord_uid: puj74k7y exchange of the extremities of the ns segment of type a and c influenza viruses in reverse genetics systems was used to assess their putative role in type specificity. restoration of each specific proximal panhandle was mandatory to allow the rescue of viruses with heterotypic extremities. moreover, the transcription level of the modified segment seemed to be directly affected by the distal panhandle strength. influenza a, b and c viruses are members of the orthomyxoviridae family, a group of enveloped, segmented, single-stranded negative-sense rna viruses. reassortment between type a, b and c influenza viruses has never been reported to date. the general structural features and genome organization of influenza viruses suggest that they share a common ancestor [1] . the genome of influenza a and b viruses consists of eight segments, whereas that of influenza c virus has only seven segments since it has a single envelope glycoprotein (hef) instead of two for type a and b viruses (ha and na) [2] . the genomic viral rnas (vrnas) are associated with the nucleoprotein (np) and the polymerase complex (p). the latter is formed by three subunits named pb1, pb2 and pa for influenza a and b viruses and pb1, pb2 and p3 for influenza c virus, respectively. in the nucleus of infected cells, viral messenger rna (mrna) synthesis is initiated with capped rna primers that are cleaved from host cell mrnas (capsnatching mechanism), and terminates 17 to 22 nucleotides (nt) upstream of the genomic vrna template 5' end at a stretch of five to seven uridine residues used as a polyadenylation signal. genomic vrna replication requires a full-length positive-sense rna template (complementary rna or crna), and both crna and vrna syntheses are primer-independent [2] . the coding region of each genomic vrna is flanked by noncoding (nc) sequences that are divided into conserved and non conserved parts [3] . the length of the nc sequences differs for each segment and also varies between virus types [4, 5] . for the 3' and 5' ends, respectively, the conserved parts are 12 and 13 nt for type a, 12 and 11nt for type b and 11 and 12 nt for type c influenza viruses [6] [7] [8] . the 5' and 3' nc sequences base-pair to form two elements: the proximal element or region i (nt 1-9 of the 3' and 5' ends) and the distal element or region ii involving sequences downstream of nt 10 and 11 from the 3' and 5' ends, respectively [9] . two main secondary structures have been described: the "panhandle structure" resulting from complete base-pairing between the two ends [10] [11] [12] [13] [14] and the "corkscrew structure", where the proximal element form hairpin loops [15] [16] [17] [18] . these conformations are known to be critical for transcription and replication of the vrnas [9] . to investigate whether, or not, and how the complete nc regions of a given segment are involved in type specificity, we attempted to rescue, by reverse genetics, type a and c influenza viruses with chimeric non-coding sequences. all experiments were based on the ns segment, the smallest segment for both influenza virus types and for which the nc regions of both viruses are quite similar in length. we showed that type specificity of the proximal element is critical to rescue infectious viruses, and that the distal element might modulate viral transcription. the 12-or 11-plasmids based reverse genetic systems were used to produce recombinant type a (a/wsn/33) and type c (c/jhb/1/66) influenza viruses, and were adapted from previously described procedures [19] [20] [21] . the modified ns plasmids (chimeric) were constructed by pcr. point mutations in the ns nc regions were introduced by directed mutagenesis using quikchange ii site-directed mutagenesis kit (agilent technologies) according to the manufacturer's instructions. the primer sequences will be provided upon request. all plasmids were sequenced using a big dye terminator sequencing kit and an automated sequencer (perkin-elmer). human skin melanoma cells (sk93/2) [22] , 293t human embryonic kidney cells and madin-darby canine kidney cells (mdck) cells were cultured in dmem supplemented with 10% and 5% fetal calf serum (fcs), respectively. all cells were grown at 37 °c with 5 % co 2 . plaque assay using an agarose overlay was performed for titration of influenza a viruses in dmem with 1 µg/ml of l-1-tosylamido-2-phenyl chloromethyl ketone tpck-trypsin (worthington) for 3 days at 35 °c. titration by plaque assay of influenza c viruses on mdck cells at 33°c was described previously [21] . for influenza a viruses, the rescued viruses were plaque purified twice on mdck cells before final amplification on mdck cells at an m.o.i. of 0.01. growth kinetics were performed with the stock viruses. mdck cells were infected at an m.o.i. of 0.001 in dmem supplemented with 1µg/ml tpcktrypsin. the rescued influenza c viruses were plaque purified on mdck cells supplemented with bovine brain gangliosides (bbg) before amplification on sk cells. for growth kinetics, sk cells were infected at an m.o.i. of 0.001 in dmem supplemented with 0.25 µg/ml tpck-trypsin. supernatants were collected at indicated time points and virus titres were determined by plaque assay. viral genomic rna was extracted using qiaamp viral rna mini kit (qiagen) according to the manufacturer's recommendations and cdna, pcr and sequencing were performed as previously described [10] . all primer sequences are available from the authors upon request. mdck cells were infected at an m.o.i. of 2 with the wild-type or mutated influenza a viruses. at six hours post-infection, total rnas were extracted, reverse transcribed using the 3' universal primer for vrna (5'agggctcttcggccagcraaagcagg) or oligo dt for mrna, and used as a template for quantitative pcr (qpcr). separate pcrs were performed with segment-specific primers designed by marsh et al [23] using of a lightcycler 480 (roche). the relative concentrations of vrnas and mrnas were determined by calculating mrna/vrna ratios using the crossing point values (cp). as control, a one step qrt-pcr targeting the gapdh sequences was performed [24] . cells were solubilised in lds sample buffer (invitrogen) complemented with β mercaptoethanol, and proteins were separated by polyacrylamide gel electrophoresis (nupage invitrogen) followed by western blotting. rabbit polyclonal anti-ns antibody was a kind gift from d. marc (inra, tours, france). the rabbit anti-pr8, used to detect np, was produced in house [25] . anti beta-actin (abcam, cambridge, uk) antibody was used for loading controls. the role of nc sequences in type specificity was investigated for the ns segment of the most divergent type a and c influenza viruses using reverse genetics systems [19] [20] [21] . a set of type a and type c chimeric viruses was generated, in which one or both type-specific extremities of the ns segment were totally or partially replaced by their counterpart from type c or type a virus, respectively (figure 1) . in a type c backbone, no virus was rescued when one or both type a extremities were introduced, and only the wild-type 5'c/3'c virus was recovered (figure 1 ). in a type a backbone (figure 1 ), no virus harbouring the type c 5'end (5'c/3'c and 5'c/3'a) was rescued. only viruses with a type a 5' end (5'a/3'a and 5'a/3'c) could be rescued, but a c-to-u mutation in the 3'c proximal sequence was systematically observed at nucleotide 5 (5'a/3'c(c5u)). introduction of this reverse substitution into the wild-type 5'a/3'a construct (to generate 5'a/3'a(u5c)) systematically led to a wild-type uracil reversion, confirming the importance of this position. minigenome experiments previously showed that this position did not modify the transcription/replication processes [10] . interestingly, this mutation in 5'a/3'c, leading to 5'a/3'c(c5u), restored a proximal panhandle sequence identical to that of the wild-type 5'a/3'a. to demonstrate the necessity of the type specific proximal panhandles to recover viruses, chimeric heterotypic ends were created to include proximal panhandles homotypic of the reverse genetics backbone. both type a and c viruses with 5' and 3' modified ns ends (5'c/3'c-proxa and 5'a/3'a-proxc, respectively) were rescued as efficiently as wild-type viruses (figure 1 ). the fact that the proximal panhandle of the unsuccessful constructs is not homotypic strongly suggested that a homotypic proximal panhandle is required. all rescued type c and a viruses were plaque-purified and subsequently amplified in sk and mdck cells, respectively. kinetics assays at low multiplicity-of-infection (m.o.i.) were then performed in the respective cell types. no difference was observed for type c viruses; wild-type and 5'a/3'a-proxc grew with similar efficiencies (figure 2a) . the three rescued type a [20, 21] . the introduced mutation in 5'a/3'a(u5c) is underlined. the sequence of both ends of each segment of each rescued virus was verified as described [10] , and no mutation was detected except at position 5 in the 3' end for 5'a/3'c and 5'a/3'a(u5c) yielding viruses 5'a/ 3'c(c5u) and 5'a/3'a, respectively (indicated by arrows). in constructs 5'a/3'a-proxc in the type c system and 5'c/3'c-proxa in the type a system, only the distal panhandle was modified, but the homotypic proximal panhandle was conserved. the energy barriers of the canonical pairs c:g and u:a, and of the wobble base pair g:u (represented by a black dot) were described by vendeix et al [34] and were used to calculate a score to evaluate the panhandle strength. the proximal panhandle consists of 9 potential basepairs, when the distal panhandle was defined to include the potential base-pairs up to the type a poly-u signal. type c reverse genetics was performed in 293t cells and supernatants were collected and titrated at day 10 post-transfection (p.t.). type a reverse genetics was performed in a co-culture of 293t and mdck cells, and supernatants were collected and titrated at 72h p.t. titers in pfu/ml are the mean of 2 to 4 independent experiments. doi: 10.1371/journal.pone.0081550.g001 viruses replicated to similar final titers, although both 5'a/ 3'c(c5u) and 5'c/3'c-proxa were slightly delayed ( figure 2b) . overall, these results indicated that the major packaging signals were not located within the distal panhandle sequence, since it was possible to rescue the 5'a/3'c(c5u) virus. thus, the distal sequence of the type c 3' end was not restrictive for ns segment packaging. this is in agreement with published data showing that ns segment packaging does not require wild-type 3' end sequences [26] . the putative effect of modifying the extremities on the transcription and replication steps was then studied at an early stage of the viral cycle for the three rescued type a viruses. levels of mrna and vrna were analyzed by rt-qpcr [23] at 6 hours post infection (h.p.i.) at high m.o.i., using rt primers specific for mrna or vrna ( figure 3a ). crossing point values were used to evaluate the relative level of both processes, by calculating an mrna/vrna ratio. compared to wild-type 5'a/ 3'a, significant differences (p<0.05) were found only for 5'a/ 3'c(c5u), but not for 5'c/3'c-proxa. interestingly, the cpmrna/cpvrna ratio for 5'a/3'c(c5u) was higher than for wild-type 5'a/3'a for all segments, meaning that either more vrna or less mrna was synthesized for 5'a/3'c(c5u). the sole difference between 5'a/3'c(c5u) and wild-type 5'a/3'a viruses being the 3' distal extremity of the ns segment suggested that the level of ns encoded proteins (i.e. ns1 or/and ns2/nep) was affected at early stages of infection for this virus (5'a/3'c(c5u)). western-blot was used to evaluate the amount of ns encoded proteins two hours before the observed effect. the amount of np protein was used to normalize the results. at 4 h.p.i. at high m.o.i., only 5'a/ 3'c(c5u) showed a clear increase in the production of ns1 protein, as indicated by a higher ns1/np ratio ( figure 3b ). the putative roles of the proteins encoded by the ns segment in viral transcription and replication have been documented: (i) inactivation of ns1 protein reduces the amount of all vrnas without affecting the amount of mrnas [27] ; (ii) ns1 protein counteracts the effect of a polymerase regulating factor, possibly hnrnp-f [28] , that inhibits influenza virus full-length rna synthesis [29] ; and (iii) ns2/nep protein reduces transcription and increases replication [30] . the latter role of ns2/nep could be indirect by stimulating the synthesis of virus-derived small viral rnas, whose depletion was shown to reduce vrna synthesis [31] . thus, the decreased mrna/ vrna ratio observed at 6 h.p.i. at high m.o.i. could reflect an increase in replication, which would be consistent with the higher viral titer for 5'a/3'c(c5u) we observed at that time point (5x10 5 vs 1.6x10 7 pfu/ml for wild-type and 5'a/3'c(c5u), respectively). the most likely hypothesis for the increased amount of 5'a/ 3'c(c5u) ns proteins is that the level of transcription was affected and that more mrna was synthesized for 5'a/ 3'c(c5u) at earlier stages of infection (2-4 h.p.i.). such a trend, but no significant difference, in ns mrna quantification after normalization relative to gapdh was indeed observed between two and four h.p.i. (data not shown). the difference in the base-pairing strength of the distal panhandle formed by the heterotypic extremities in 5'a/3'c(c5u) compared to wild-type, evaluated by scores calculated based on the number and nature of the base-pairs (figure 1) , could favour the attachment of the polymerase. the stability of the duplex formed by the 5' and 3' ends was indeed shown to be an important parameter for polymerase binding [32] . a weaker distal panhandle for 5'a/ 3'c(c5u) (score 7.39 vs 10.74 for wild-type) could facilitate polymerase binding and/or transcription initiation, resulting in slightly more ns mrna synthesized. this is further supported by the results for the 5'c/3'c-proxa virus, whose distal panhandle base-pairing score is identical to that of wild-type 5'a/3'a (figure 1) , and for which ns1 expression levels ( figure 3b ) and mrna/vrna ratios ( figure 3a ) were similar to those obtained for wild-type. the major role of the proximal panhandle in type specificity that we identified and the hypothesized involvement of the distal panhandle in transcription need to be tested on the other (2 pfu/cell), viral vrna and mrna levels for each segment were evaluated by specific two-step rt-qpcrs previously described [23] . results were expressed as the mean of mrna/vrna cp (crossing-point) ratios calculated on data obtained from 12 independent qpcrs (corresponding to two infections, two independent cdna syntheses per infection and three independent qpcrs for each cdna). statistics were performed using the anova test. black bars: wild-type 5'a/3'a; hatched bars: 5'a/3'c(c5u); grey bars: 5'c/ 3'c-proxa. (b) levels of np and ns1 proteins after infection with the rescued influenza a viruses. four hours after mdck infection at a high m.o.i. (2 pfu/cell), cell lysates were analyzed by western-blot for viral ns1 and np proteins and for β-actin as cellular control. following chemiluminescence acquisition with a g. box (syngene, cambridge, uk), band densities for viral proteins were determined using genetools software (syngene, cambridge, uk), and were used to calculate ns1/np ratios for each virus. due to the high levels of viral protein expression during infection, protein extracts were diluted in uninfected cellular extracts prior to electrophoresis. results are from one representative assay out of three independent infections. doi: 10.1371/journal.pone.0081550.g003 segments. it is interesting to note that comparison of the distal panhandles formed by the extremities of each segment for a given virus shows differences in base-pairing scores (data not shown). in particular, regardless of the influenza a virus subtype, the distal panhandle of the ha segment was systematically the weakest one. it would be interesting to determine to what extent the distal panhandle base-pairing strength may have an impact on ha expression levels and consequently influence the ha/na molecule ratio found at the virion surface [33] . our approach to understand what in the ns segment noncoding regions was important for type specificity by attempting to rescue type a and c influenza viruses with chimeric nc sequences in the ns segment unraveled the requirement for a type specific proximal panhandle and the role of the distal panhandle in regulation of transcription. our results thus provide useful information to improve our understanding of influenza a virus biology. evolution and ecology of influenza a viruses orthomyxoviridae: the viruses and their replication mutations in the nonconserved noncoding sequences of the influenza a virus segments affect viral vrna formation non coding extremities of the seven influenza virus type c vrna segments: effect on transcription and replication by the type c and type a polymerase 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formation in explicit solvent: a predictive model we are grateful to g. brownlee for providing the original plasmids for the influenza type a virus reverse genetics system and r. frade (saint antoine hospital, paris, france) for the sk 93/2 cells. we thank d. marc (inra, tours, france) for anti-ns1 polyclonal antibodies, v. frigard for technical assistance, d. marc, g. abou-jaoudé, d. moisy and s. munier for helpful discussions and critical reading of the manuscript. conceived and designed the experiments: bcc sw. performed the experiments: bcc. analyzed the data: bcc cb sw. contributed reagents/materials/analysis tools: bcc cb. wrote the manuscript: bcc cb sw. key: cord-258243-2utl2mfl authors: chen, jeng-wen; liao, po-wu; hsieh, chi-jeng; chen, chu-chieh; chiou, shang-jyh title: factors associated with changing indications for adenotonsillectomy: a population-based longitudinal study date: 2018-05-29 journal: plos one doi: 10.1371/journal.pone.0193317 sha: doc_id: 258243 cord_uid: 2utl2mfl objective: adenotonsillectomy (at) is one of the most common surgical procedures performed in children and adults. we aim to assess the factors associated with changes in the incidence of and indications for at using population-level data. study design: this retrospective cohort study investigated patients who underwent at between 1997 and 2010 by using data from the taiwan national health insurance research database. we examined surgical rates and indications by the calendar year as well as age, sex, hospital level, and insured residence areas for the correlating factors. results: the average annual incidence rate of at was 14.7 per 100,000 individuals during 1997–2010. pediatric (<18 years) patients represented 48.2% of the total at population. more than 99% of the patients underwent the at procedures as an inpatient intervention. longitudinal data demonstrated an increasing trend in the pediatric at rates from 1997 (4.3/100,000) to 2010 (5.7/100,000) (p = 0.029). in the adult subgroup, a decreasing prevalence of infectious indications (p = 0.014) coincided with an increasing neoplastic indications (p = 0.001). in the pediatric subgroup, the prevalence of obstructive indications increased (p = 0.002). the logistic regression analyses indicated that the significant factors associated with the changing surgical indications for at were the age in the adult subgroup and hospital level in the pediatric subgroup. conclusions: this study revealed a low at rate in taiwan than that in other countries. pediatric at incidence increased during 1997–2010. although a rising prevalence of obstructive and neoplastic indications was noted, infection remained the most common indications for at. age in the adult subgroup and hospital level in the pediatric subgroup were factors associated with the changing indications for at. introduction tonsil surgery has been performed for more than 3,000 years [1, 2] . historically, tonsillectomy was mainly performed to treat the infectious complications of tonsillitis [2, 3] . with the introduction of oral antibiotics in the 1960s, the number of tonsillectomies declined; however, it is still one of the most common surgical procedures performed in western countries [3] [4] [5] [6] [7] . adenoidectomy is a regular surgical procedure performed by otolaryngologists. occasionally, an adenoidectomy is performed concurrently with tonsillectomy. an adenoidectomy performed alone or with other procedures such as tympanostomy tube placement, or nasal surgery may exhibit different characteristics. the variation in adenotonsillectomy (at) rates across different countries may be explained in part by the different cultural attitudes toward the protective role of these lymphoepithelial structures [8, 9] , use of antibiotics for throat infections, and absence of generally accepted indication criteria for surgery [10, 11] . a 22-year nationwide cohort showed a significant decrease in the incidence of tonsillectomy over time [12] ; however, regional variations were observed in the number of tonsillectomies. following the introduction of a national clinical guideline for the management of sore throat and indications for tonsillectomy, mcleod et al. reported a probable association between an increase in admissions with tonsillitis or peritonsillar abscess and a decrease in the rate of tonsillectomy [13] . in contrast to the abundance of information on the incidence trends of at in western countries [3-5, 14, 15] , limited data exist for eastern countries [16] . the role of recurrent throat infections as a primary indication for at is debatable. when the most stringent criteria for at were applied, a randomized clinical trial [17] showed that surgery was modestly beneficial. however, the same study also provided support for nonsurgical management because during the 3-year follow-up period, many participants in the nonsurgical groups had fewer than three episodes of throat infection, most of which were mild. by contrast, when less frequent or milder illness led to at, little to no benefit was found [18] . another study revealed a low level of consensus among pediatricians and otolaryngologists on the appropriateness of at [11] . evidence-based guidelines regarding repeated throat infections as an indication for at are still lacking, which leaves this procedure in the category of "preference-sensitive care" [19] . differences in clinical practice remain an obvious but inadequately documented problem [20] . the surgical rate of pediatric at varies notably among different countries [21] [22] [23] because of the high accessibility of medical resources when throat infections occur and the cultural acceptability of antibiotic treatment for upper respiratory tract infections. additionally, discrepancies exist between physicians and parents regarding the use of surgical intervention for recurrent throat infections. most parents are concerned about the effects of tonsil removal on the immune system and may seek a second opinion regarding surgical options. furthermore, physicians with different subspecialties may have dissimilar stances on the indications for surgical intervention [11] . in this study, we investigated the factors associated with the incidence and changing indications of tonsillectomy and at from 1997 to 2010 in taiwan. these epidemiological data will provide valuable information for the daily practice of physicians, and contribute to the development of an accepted clinical guideline for these procedures. we conducted a retrospective, population-based cohort study using data from the national health insurance research database (nhird); the nhi commenced in 1995 and covers over 99% of the population in taiwan [24] . the longitudinal health insurance database (lhid) contains the complete claims data from 2005 of 1 million beneficiaries. the authors used the registry files from 1997 to 2010 and all applications for reimbursements regarding the inpatient and outpatient healthcare services provided to each patient for analysis. the institutional review board of cardinal tien hospital approved this study (cth-103-3-5-035) and waived informed consent because the datasets consisted of anonymized, de-identified nationwide data. all patients who had undergone a tonsillectomy without adenoidectomy (international classification of diseases, ninth revision, clinical modification [icd-9-cm] procedure code 28.2) or tonsillectomy with adenoidectomy (icd-9-cm procedure code 28.3) between 1997 and 2010 were identified from the representative samples from the nhi program. patients who underwent an adenoidectomy without tonsillectomy (icd-9-cm procedure code 28.6) were excluded. all claims data, including demographic administrative and clinical information from both the inpatient and outpatient databases, were used in the analysis. the index date was defined as the time that the tonsillectomy or at was performed. a list of diagnoses (according to the icd-9-cm) was obtained from the database for each patient on the index date of surgery. these top three diagnoses were categorized into either infectious or inflammatory indications (recurrent infection or chronic inflammation [rici]), obstructive indications (tonsillar hypertrophy or upper airway obstruction [uao]), or neoplastic indications (suspicious benign or malignant neoplasms [tumor] ). if more than one diagnosis fit into a particular category, only the first appearing diagnosis was used to define the indication for at. the leading icd-9-cm codes for the indications of rici, uao, and tumor were 474, 780, and 146, respectively. table 1 shows the complete list of icd-9-cm codes (top three diagnoses) in these categories of surgical indications. we investigated the distribution of the three major categories of surgical indication according to sex, age group (<5 years, 5-11 years, 11-17 years, 18-40 years, and >40 years), hospital level (medical centers, regional hospitals, and local hospitals), and insured residence areas according to the nhi divisions (taipei, northern, central, southern, kaoping, and eastern). these variables were considered as the possible factors associated with the incidence of and indications for at. we presented the data in three different groups, i.e., a total study population, an adult subgroup (!18 years), and a pediatric subgroup (<18 years) for comparison. we evaluated the incidence of at by the calendar year from 1997 to 2010 according to the index date of surgery. we presented the distribution of the three major categories of surgical indication (rici, uao, and tumor) as the number and percentage of patients and analyzed the trends of changing surgical indications. sas version 9.2 (sas institute, inc., cary, nc, usa) was used for all the analyses. descriptive statistics were analyzed using pearson's chisquare test. we performed a simple linear regression model to examine the trends in surgical rates and indications by the calendar year. multinomial logistic regression was established after adjustment for potential confounding effects of age, sex, hospital level, and insured residence areas to identify the possible factors associated with the different surgical indications. the significance was set at a two-sided p < 0.05. several studies have confirmed the validity and reliability of the nhi lhid from 1 million representative beneficiaries in clinical research [25] [26] [27] . a total of 1,892 ats were performed between 1997 and 2010. the average annual incidence of at was 14.7 per 100,000 individuals during the study period. the sample comprised 41.3% female patients, with a mean age of 24.0 ± 18.2 years. patients aged <18 years formed 48.2% of the total at population. table 2 shows the number and percentage of patients who received tonsillectomy alone or tonsillectomy with adenoidectomy by age groups in this cohort. tonsillectomy alone is more commonly performed in adults (920/1195, 76.9%). in contrast, tonsillectomy with an adenoidectomy is most often performed in patients of 5-11 y/o (415/697, 59.5%). only two patients (2/1892, 0.1%) underwent at on an outpatient basis. mean hospital stay of at patients was 3.7 ± 2.9 days (median, three days). were icd-9-cm 146 (malignant neoplasm of oropharynx), 196 (secondary and unspecified malignant neoplasm of lymph nodes of head, face, and neck), and 210 (benign neoplasm of lip, oral cavity, and pharynx) at 32%, 18.0%, and 11.7%, respectively. the overall incidence of the tumor category demonstrated a noticeable increasing trend. among the total study population, rici showed a decreasing trend (y = -0.6571x + 89.571, p = 0.462), whereas uao and tumor showed an increasing trend (y = 2.2044x + 23.824, p = 0.091 and y = 1.4769x -0.9341, p = 0.001, respectively). in the adult subgroup (figs 2b and 3b) , the incidence and proportion of at performed for rici decreased from 46 (78%) in 1997 to 32 table 3 indicates the number and percentage of the three categories of surgical indications according to sex, age groups, hospital level, and insured residence areas in the total study population. the male patients had a higher overall at rate and were more likely to undergo at with the indications of uao and tumor, whereas the female patients were more liable to undergo surgery with the indication of rici. however, rici remained the most prevalent indication for at, followed by uao and tumor, in both the male and female patients. the distribution of the surgical indications across each age group differed significantly (p <0.0001). adolescence aged 12-17 years were the most likely to undergo at because of infectious indications, with 73.7% of these patients undergoing surgery for this reason. another 24.3% of the patients in this age category underwent surgery due to uaos. only 2% of these patients received surgery because of a suspected or confirmed neoplasm. the pattern of the distribution of surgical indications remained similar in the <5 years and 5-11 years age groups. however, the proportion of infectious indications decreased in the adult subgroups. the percentage of uao was relatively stable across all age groups. notably, the proportion of tumor indications increased dramatically in the >40 years age group (28.1%). compared with the pediatric age groups and 18-40 years group, that of >40 years had more tumor indications, which coincided with a decrease in the infectious indications. most of the ats were performed in the medical centers and regional hospitals (table 3) . infection was the most frequent cause of surgery at all hospital levels (61.1% in medical centers, 63.2% in regional hospitals, and 70.6% in local hospitals). the distribution of surgical indications across the hospital level was significantly different (p <0.0001). the distribution pattern of surgical indications was similar in medical centers and regional hospitals, those performed in local hospitals were more likely to have been caused by infection and less likely to have been caused by uao or tumor. the trend in the at rate and the proportion of surgical indications by the calendar year according to sex, age groups, hospital level, and insured residence areas in the total study population are shown in the supplemental data (s1 file). we examined the incidence rate and distribution of the surgical indications for at among the six distinct insured residence areas in taiwan ( table 3) . most of the ats were performed in the taipei district (42.9%, 800/1863), followed by the central (20.5%, 381/1863), south (12.7%, 237/1863), northern (10.9%, 204/1863), kaoping (10.4%, 193/1863), and eastern districts (2.6%, 48/1863). the distribution of the surgical indications among the insured residence areas was significantly different (p <0.0001). we performed multinomial logistic regression to identify the possible factors associated with the different surgical indications after adjustment for age, sex, hospital level, and insured residence areas, in which uao was the reference group. we compared rici with uao and tumor with uao in the same model (table a in s2 file) . after adjusted with other variables, rici occurred more often in the younger and female patients compared with uao. tumor occurred more often in the older patients compared with uao. in addition, we examined the related factors (sex, age groups, hospital level, and resident areas) with a time series (s1 file). the patients in the 5-11 years group had a higher proportion of at due to uao after 2006, whereas the proportion in the 18-40 and >40 years group declined after 2006. the patients in the 5-11 years of age had a higher incidence of at because of rici after 2000, whereas the incidence in the 18-40 years group decreased after 2000. moreover, the incidence of patients with rici for at shifted from medical centers to regional hospitals during the study period, whereas the incidence of surgical indications for uao and tumor did not change significantly. most patients with uao and tumor indications received at surgery at medical centers. tables 4 and 5 indicate the number and percentage of the three categories of surgical indications according to sex, age groups, hospital level, and insured residence areas in the adult and pediatric subgroups, respectively. in the adult subgroup, the distribution of the surgical indications in gender (p < 0.001), age (p < 0.0001), hospital level (p < 0.0001) and insured residence areas (p = 0.005) differed significantly. multinomial logistic regression (table b in s2 file) showed that age was the only significant factor associated with the different surgical indications in the adult subgroup. compared with rici, uao occurred more frequently in the higher hospital level. the effect of sex, an insured residence area, and hospital level was not statistically significant in the tumor group (vs. uao) after all the variables were controlled. by contrast, the distribution of the surgical indications in the pediatric subgroup differed significantly in hospital level (p < 0.0001). logistic regression (table c in s2 file) showed that hospital level was the only significant factor associated with the different surgical indications in the pediatric subgroup. this report is the first study on the trends and indications of at across all ages in taiwan during 1997-2010. the overall incidence rate of at was 14.7 per 100,000 population, and increased during 1997-2010. more than 99% of the study population received an at on an inpatient basis. we noticed a low incidence of at and a relatively small percentage of pediatric at procedures in taiwan. however, longitudinal data confirmed an increasing trend in the pediatric at rates from 1997 to 2010. regarding surgical indications for at, although we reported a declining trend of infectious indications, treatment of rici remained the most common cause of at regardless of the calendar year, sex, age groups, hospital level, or insured residence areas. in the adult subgroup, we identified a decreasing prevalence of infectious indications, coincided with an increasing neoplastic indications. in the pediatric subgroup, the proportion of obstructive indications increased significantly. the logistic regression analyses indicated that after adjustment for confounders, the significant factors associated with the changing surgical indications for at were the age in the adult subgroup and hospital level in the pediatric subgroup. since 2000, an increasing amount of evidence has shown that solitary tonsillectomy as a treatment for adult sdb patients might have a limited effect, even if they present with grade iv (kissing) tonsils. otolaryngologists usually recommend adult sdb patients to receive uvulopalatopharyngoplasty (uppp), instead of solitary tonsillectomy, in those who do not tolerate to or are unwilling to use continuous positive airway pressure devices or other conservative treatment such as oral appliances [28] . in contrast, uao has become a plausible indication for at, especially in pediatric patients [3] . because adenotonsillar hypertrophy is a major cause of obstructive sleep apnea (osa) in children, at is among the first-line therapies recommended for uncomplicated pediatric osa [29, 30] . two of the most common reasons for pediatric and young-adult at are infection and uao secondary to adenotonsillar hypertrophy [7, 31] . although at for a suspected or confirmed neoplasm is rarely performed in children, it is the third most prevalent indication for at in adults [6] . the nhi has encouraged almost all patients with at to undergo surgery on an inpatient basis. by contrast, ambulatory at procedures in children have nearly doubled over the past decade in the united states, with only <3% of tonsillectomies performed in an inpatient setting [22] . in taiwan, the inconvenience of admission may partly reduce the surgical rate in the pediatric population. one study found that even when physicians recommended a surgical procedure based on the current guidelines, parents often chose nonsurgical treatment options because of anecdotal reports or based on their own education or social experiences [26] . the percentage of surgical procedures in the <18 years subgroup (48.2%) is relatively small compared with the figures that have been reported by other studies [5, 16, 21, 31] , reflecting parental concerns regarding at in taiwan. the surgical indications for at have fundamentally changed over time. parker and walner [3] demonstrated a shift in surgical indications for pediatric at from infection toward obstruction. in the group aged <18 years, more than 85% underwent surgery due to a uao. koshy et al. [4] reported that incidence of osa diagnosis among children aged < 4 years who underwent at doubled between 2001 and 2011. clement [32] presented an increase in the rate of ambulatory at in children with conditions on the sdb spectrum from 26% in 2001 to 55% in 2011, whereas uao became the most common indication for at among preschool children. our results confirmed an increasing trend toward surgical management for upper airway obstructive symptoms rather than for mainly infectious reasons. however, treatment of rici remained the most common indication for at across all age groups. those results reveal a lack of awareness among pediatricians and otolaryngologists of the symptoms of sdb and the difficulty in diagnosing osa in the pediatric population. tonsillectomy for tonsil asymmetry and suspicion of malignant diseases is rarely performed in children, but it is an indication for tonsillectomy in the adult population [33] . compared with deep tonsil biopsies, tonsillectomies have a significantly higher probability of detecting occult tonsillar tumors [34] . in this study, the surgical indications for tonsillar neoplasm increased substantially from 1997 to 2010, especially after 2006. a possible reason for this trend was the taiwanese government's implementation of free oral and pharyngeal mucosal screening among high-risk groups (those who aged 18 or above and have a history of smoking or betel nut chewing) from 2007. by the end of this national cancer prevention and control project, 1440,000 high-risk candidates (aged 18 or above, with a history of smoking or betel nut chewing) had been screened. the screening rate was 28%. one thousand two hundred and forty-eight patients were diagnosed to have oral or oropharyngeal cancer during 2008-2009. the timing of this national policy correlated well with the increase in tumor tonsillectomy. furthermore, increasing incidence rates of oropharyngeal cancers were discovered on a global scale [35] . in the distribution of surgical indications according to hospital level, we discovered an increasing trend in the proportion of rici and a decreasing trend in the proportion of uao and tumor among local hospitals. this trend is also plausible because the diagnosis of sdb may require overnight polysomnography and sleep laboratory testing may not be available in local hospitals. additionally, patients diagnosed with neoplasms preferred regional hospitals or medical centers to local hospitals. in the analysis of the surgical indications by insured residence area, we found higher-than-expected (based on the population in each division) numbers of ats performed in the taipei and central districts. the surgical rate of at in taiwan seemed to be higher in the metropolitan areas than in rural areas. the abundance of medical resources in these metropolitan areas may be a contributing factor. however, insured residence area was not an independent factor associated with the distribution of the surgical indications for at after adjustment for confounders. this study had several limitations. first, the dataset did not include a chart review of the participants; therefore, detailed clinical data were not available. second, because the inherent preference of coding could not be assessed from our results; the accuracy of the type of procedures performed and the surgical indications may have been affected. third, this study included a homogeneous population of chinese patients in taiwan; the generalizability of our results to other ethnic populations may be limited. finally, selection bias may still exist when using lhid2005 for trend analysis. those patients who received surgery after 1997 but died before 2005 were not included in the lhid2005. the study population who became older after 2005 might have a higher risk of developing neoplasms. moreover, the number of surgery in the pediatric population less than five years of age was underestimated between 2005 and 2010. all these biases were potential confounders in this study. therefore, we also examined the lhid2000 and lhid2010, which concurred with our current finding, to substantiate our observed trends in lhid2001. however, a complete dataset from the entire population with an extended study period would allow for more accurate trend analysis and prediction. epidemiological trends in at have changed substantially. the annual incidence rate of at shifted significantly due to major public health events. ricis remained the most common surgical indication for at across all ages during 1997-2010. incidence of pediatric at increased with a shift in surgical indications from infections to obstruction. in the adult, the proportion of at performed for infections decreased, whereas those performed due to neoplasm increased significantly. the age in the adult subgroup and hospital level in the pediatric group were significant factors associated with the changing surgical indications for at. these epidemiological data will add information for the daily practice of physicians and otolaryngologists, and contribute to the development of an evidence-based clinical guideline for these procedures. a history of tonsillectomy: two millenia of trauma, haemorrhage and controversy factors influencing the indication for tonsillectomy: a historical overview and current concepts trends in the indications for pediatric tonsillectomy or adenotonsillectomy changing indications and socio-demographic determinants of (adeno)tonsillectomy among children in england-are they linked? a retrospective analysis of hospital data incidence of tonsillectomy in denmark adult tonsillectomy: current indications and outcomes changes in incidence and indications of tonsillectomy and adenotonsillectomy van cauwenberge pb. the immune response in adenoids and tonsils high rates of detection of respiratory viruses in tonsillar tissues from children with chronic adenotonsillar disease is there agreement among general practitioners, paediatricians and 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adenoidectomy/tonsillectomy rates among children of the veneto region changes and consistencies in the epidemiology of pediatric adenotonsillar surgery does adenotonsillectomy really reduced clinic visits for pediatric upper respiratory tract infections? a national database study in taiwan a 10-year experience with universal health insurance in taiwan: measuring changes in health and health disparity herpes zoster and subsequent risk of cancer: a population-based study validation of the national health insurance research database with ischemic stroke cases in taiwan risk of sepsis in patients with amyotrophic lateral sclerosis: a population-based retrospective cohort study in taiwan practice parameters for the surgical modifications of the upper airway for obstructive sleep apnea in adults diagnosis and management of childhood obstructive sleep apnea syndrome treatment options for pediatric obstructive sleep apnea indications for tonsillectomy and adenoidectomy day-case tonsillectomy for children in glasgow: the impact of changing indications and deprivation implementation by scottish otolaryngologists of the scottish intercollegiate guidelines network document management of sore throats and the indications for tonsillectomy: four years on tonsillectomy vs. deep tonsil biopsies in detecting occult tonsil tumors worldwide trends in incidence rates for oral cavity and oropharyngeal cancers this work was supported by research grants (cth-104-1-2d03, cth-106a-2a22 and cth-107-mf-02) from catholic cardinal tien hospital in cooperation with national taipei university of nursing and health sciences. the funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.the manuscript was edited for proper english language, grammar, punctuation, spelling, and overall style by the highly qualified native english-speaking editors at wallace academic editing (o-2017-006526). writing -review & editing: jeng-wen chen, shang-jyh chiou. key: cord-003503-t6cnjwpd authors: sung, ming-hua; lin, chao-nan; chiou, ming-tang; cheng, i-ju; thanh, quang-hien; chao, day-yu; lan, yu-ching title: phylogeographic investigation of 2014 porcine epidemic diarrhea virus (pedv) transmission in taiwan date: 2019-03-06 journal: plos one doi: 10.1371/journal.pone.0213153 sha: doc_id: 3503 cord_uid: t6cnjwpd the porcine epidemic diarrhea virus (pedv) that emerged and spread throughout taiwan in 2014 triggered significant concern in the country’s swine industry. acknowledging the absence of a thorough investigation at the geographic level, we used 2014 outbreak sequence information from the taiwan government’s open access databases plus genbank records to analyze pedv dissemination among taiwanese pig farms. genetic sequences, locations, and dates of identified pedv-positive cases were used to assess spatial, temporal, clustering, gis, and phylogeographic factors affecting pedv dissemination. our conclusion is that s gene sequences from 2014 pedv-positive clinical samples collected in taiwan were part of the same genogroup 2 identified in the us in 2013. according to phylogenetic and phylogeographic data, viral strains collected in different areas were generally independent of each other, with certain clusters identified across different communities. data from gis and multiple potential infection factors were used to pinpoint cluster dissemination in areas with large numbers of swine farms in southern taiwan. the data indicate that the 2014 taiwan pedv epidemic resulted from the spread of multiple strains, with strong correlations identified with pig farm numbers and sizes (measured as animal concentrations), feed mill numbers, and the number of slaughterhouses in a specifically defined geographic area. porcine epidemic diarrhea virus (pedv) causes acute diarrhea, vomiting, and dehydration, resulting in high mortality rates for suckling piglets [1] . since 2010, a new pedv variant belonging to genogroup 2 has spread throughout the united states and across multiple asian countries, including china and taiwan [2, 3] . an initial identification in the us was made in april of 2013 [4] . a 40.5% premises-level incidence of pedv caused the deaths of more than 8 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 million newborn piglets in a single year-an event that significantly affected the american swine industry [5] [6] [7] [8] [9] . subsequent genogroup 2 epidemics have been reported in major swineproducing countries such as canada, mexico, taiwan, korea and japan [8, [10] [11] [12] [13] . one of the most notable pedv outbreaks occurred in south korea in late 2013 [14] , more than a half million pigs died from pedv infections in japan between 2013 and 2016 [15] , and a significant increase in pedv outbreaks occurred in taiwan around the same time [16] . pedv is now considered the world's most catastrophic swine disease, with major financial impacts noted throughout the global pork industry. the pedv genome is comprised of at least seven open reading frames (orf1a, orf1b and orf2-6) encoding four structural (s), envelope (e), membrane (m), and nucleocapsid (n) proteins [17] . a high degree of genetic diversity has been observed in the s glycoprotein gene [18] [19] [20] . partial spike (s) polyprotein genes located in the virus envelope are central to pedv biological properties such as interactions with cellular receptors during virus entry, the neutralizing of antibody induction in natural hosts, growth adaptation in vitro, and virulence attenuation in vivo [19] . the pedv spike (s) protein is a type 1 transmembrane envelope glycoprotein with a 4,158 nucleotide sequence divided into s1 and s2 domains. the s1 region (aa 26-734) is responsible for viral binding, while the s2 domain (aa 735-1383) serves as an anchor for viral membrane and fusion activity [21, 22] . thus, the s glycoprotein is considered a primary target for pedv vaccine development. as the major envelope glycoprotein found in virion, s serves as an important viral component for studying genetic relationships among pedv isolates, and for determining pedv epidemiological status [23, 24] . pedv is believed to infect pigs by both direct and indirect fecal-oral routes. due to the scales and complexities of modern swine production systems, pedv is likely transmitted between farms via diarrheic feces or vomitus; contaminated environmental sources involving clinically or sub-clinically infected pigs; trailers used to transport livestock, manure, or food sources; farmers or visitors wearing contaminated clothes; or wild animals and birds [5, 25] . other potential sources include contaminated fomites (e.g., raw food, feed, sow milk), food ingredients or additives, and environmental features such as wind direction, farm altitude, terrain slope, and tree coverage [26] . after an initial outbreak, pedv may spread at an increasingly rapid rate due to inadequate farm hygiene management procedures such as improper disinfection and poor biosecurity. the virus can remain dormant in weaning pigs or growth finishing units, eventually triggering mild symptoms and resulting in low mortality rates [6] . although researchers believe that pedv infections primarily result via fecal-oral routes, the rapid regional spread of the disease raises the possibility of airborne transmission [4] . support for this hypothesis includes an identified correlation between disease-spread direction and prevailing wind direction [7] , with environmental features such as land coverage, altitude, and slope possibly influencing airborne disease dissemination [26] . to determine specific temporal and geographic relationships associated with pedv strain transmission, we used phylogenetic, phylodynamic and phylogeographic methods to systematically evaluate potential temporal and spatial transmission routes among taiwanese swine farms during the 2014 outbreak. epidemiological and geographic data were collected from 92 animal lab reports of pedv viral infections involving 8,557 pig farms, 37 pig feed mills, 57 slaughterhouses, and 5,806,237 animals. these reports are available from an open database maintained by the taiwan government. additional epidemiological and genetic information was gathered for purposes of determining details of the disease spread. a total of 48 global pedv whole genome sequences (s1 table) and 49 taiwan partial s1 gene sequences (648 nucleotide of pedv s1 gene position 1468-2115) (s2 table) were downloaded from genbank, and information for the tw4 whole genome sequence was collected from a previous study [27] . information datasets focused on pig feeding and disease were collected from the taiwan open data website (http:// data.gov.tw/), the animal health research institute of the taiwan executive yuan's agricultural council (http://eng.nvri.gov.tw/fmodule/default.aspx), and genbank (https://www.ncbi. nlm.nih.gov/genbank/). in the first stage of this study, pedv phylogenetic and phylogeographic data analyses were performed for purposes of organizing viral transmission evidence and tracking possible transmission routes. in the second, data for variables of interest associated with the pig feed industry were collected and combined with geographic information system (gis) data for investigation using open source quantum gis (qgis v3.2.3) software [28] . an sas mixed procedure was used to create linear regression models, with livestock breeding variables employed to predict pedv infections. gene sequences for the 49 global pedv whole genomes and 49 taiwan pedv partial s1 gene sequences were downloaded from genbank and aligned with a single taiwan pedv complete genome from a previous study [27] using the clustalw multiple alignment feature in bioedit [29] . the best-fit model, as determined using jmodeltest 2.1.7 [30] , had a gamma distribution (gtr+g+i). to ensure topological consistency, phylogenetic trees were constructed using maximum likelihood (ml) and bayesian methods (mega version 6.0 and beast v1.8.2, respectively). branch support was evaluated using bootstrap analyses based on 1,000 ml tree replications. bootstrap values >75% were considered as belonging to the same monophyletic group. to identify the specific locations of migration events, we grouped the 49 pedv isolates into different counties and used various pedv viral strains as references. spatial location reconstruction and viral migration activity were estimated using discrete coalescent tree and bayesian phylogeographic methods. bayesian markov chain monte carlo (mcmc) sampling using beast v1.8.2 [31] was employed to infer the time-scaled phylogenies of partial pedv s genes. hky+g and relaxed clock exponential models were used prior to setting coalescent population constants in the mcmc simulations. estimated convergence and effective sampling sizes were visually assessed using tracer v1.6. multiple chains were combined based on a 10% burnin using the version of logcombiner (v1.8.2) included in the beast package. maximum clade credibility trees with temporal and spatial annotations were summarized with the 10% burn-in removed using treeannotator (v1.8.2, also in the beast package). figtree (v1.4.2) was used to generate presentation figures. bayes factor (bf) tests were conducted to build statistical support for transmission routes among geographic locations using spread3 (v0.9.6; bf cutoff = 3) [32] . bf values were used to indicate differences between posterior and prior probabilities so that rates between any two locations were non-zero. routes with high bf values were considered as having greater potential for viral strain migration. to create animations of viral dispersion over time, annotated mcc trees were converted into a keyhole markup language (kml) file using spread3 (v0.9.6). the kml file can be visualized with an open-access earth map downloaded from natural earth (http://www. naturalearthdata.com) as a qgis software base layer. the maximum-likelihood phylogenetic tree (fig 1) was constructed from 49 global pedv whole genome sequences. sequences found in taiwan clustered with 100% bootstraps containing viruses from other asian countries and the us between 2011 and 2015. a correlation was determined between this cluster and the genogroup 2 originally identified in the us. among the taiwan sequences, some of the viruses collected and identified in 2014 were strongly correlated with one another (bootstrap values >75%), while others were mixed with viral strains collected in other countries at different times, suggesting multiple transmission events (fig 2 & table 1 ). to further evaluate transmission periods and to verify viral strain origins, all 49 partial s gene sequences determined from the taiwan pedv samples were used for phylodynamic data analyses (fig 3) . four clusters (a, b, c and d) exhibited statistically significant posterior probabilities (>0.8), including 9 viral strains on 4 monophyletic branches. the most recent common ancestor (mrca) for the 9 viral strains was traced to july 2013. all other coalescent tree sequences were determined as independent. viral branches had mrcas in 2013 or the spring of 2014, with none identified as statistically significant. thus, the phylodynamic coalescent tree established for this study indicates the involvement of multiple virus strains in the 2014 pedv outbreak. results from our phylogeographic analysis were matched to a map of the country to specify the geographic boundaries of the taiwan outbreak (fig 4) . next, molecular sequence data were combined with isolation time data and geographic coordinates to determine the spatiotemporal distribution of taiwan pedv strains. lineages were identified in several agricultural communities in the far south, with additional virus strains found in central taiwan at approximately the same time. the southern infections were spatially closer to each other. the data indicate an absence of natural and artificial barriers restricting the spread of the virus. the pig feed industry risk map shown was created to assist in the identification of significant risk factors associated with infections ( fig 5) . results from our gis system analysis of positive cases indicate correlations between pedv infection transmission and both pig farm size (number of pigs, not physical size) (0.000232, 95% ci [0.000102, 0.000362]) and slaughterhouse distribution (0.8043, 95% ci [0.4351, 1.1735]) ( table 2 ). our data also indicate higher infection rates in counties with fewer pig feed mills (-0.7857, 95% ci [-1.2298, -0.3415]). phylogeographic inferences are a potential tool for identifying the transmission and dissemination routes of pedv and other potentially much deadlier infectious diseases. however, to date very few research efforts in asia have utilized full genome sequencing for determining geographic structures due to the high costs and enormous amounts of computational time phylogeographic investigation of 2014 porcine epidemic diarrhea virus transmission in taiwan required for analyses [33, 34] . several researchers have suggested using partial s genes for phylogenetic tree construction and for phylodynamic analyses specifically aimed at studying the genetic relatedness of pedv strains [3, 10, 19] . in this study, we investigated temporal and geographic relationships among pedv strains identified as having been transmitted among farms in taiwan in 2014, using partial sequences from s genes extracted from porcine fetus samples and genbank sequences as reference panels. according to the phylogenetic tree we constructed based on these partial s gene sequences, the primary pedv strain in taiwan is related to the genogroup 2 strain identified in samples collected in the us in 2013 [35] . as previously suggested, the most recently identified taiwan pedv strains have greater similarity with us strains than with chinese or earlier taiwanese strains [3, 36] . transmission may have occurred as early as december 2013 [36] , but the crossborder route to taiwan remains unknown. results from our phylogenetic analysis of pedv viruses associated with the taiwan outbreak confirm independence in multiple counties. although these viruses share a common ancestry with the us genogroup 2 pedv, our coalescent tree data indicate that only 9 of the taiwanese viruses were significantly clustered (fig 3) . further, no recombination involving taiwanese strains was observed in the present study. five independent pedv strains were identified in taiwan on or before september 16, 2013 in wanda (km246707 and km246708) and jhutian (km246673, km246674 and km246675) townships. these are considered starting points for pedv complex dissemination throughout sections of taiwan up to may 15, 2014. according to a mix of phylogenetic and coalescent data ( table 2) , clustering was limited to 3 counties in southern taiwan (wanluan, jiouru and daliao) (fig 3) , with most of the identified viruses existing independently. combined, data for pedv divergence during the taiwan outbreak suggest a common ancestry shared by multiple virus lineages. since that time period, no evidence has been found indicating outbreaks involving multiple virus strains in taiwan. according to our phylogenetic analysis, most of the identified pedv strains from the 2014 outbreak were independent, despite sharing a common ancestor. multiple pedv invasions from abroad were also identified in japan during the 2014 outbreak [15, 37] . the widespread dissemination that followed presumably resulted from the movement of pigs, agricultural vehicles, farmers, farm visitors, commercial feed products, and other materials [15, 37] . reproducing novel approaches used in the molecular and spatial surveillance of the porcine reproductive and respiratory syndrome virus (prrsv) in the us and in pedv studies in japan [26, 38] , we utilized a combination of phylogeographic and gis approaches in our effort to profile the taiwan pedv outbreak. gis has been used to investigate correlations between diseases and factors that include pig farm size, number of feed mills, and number of pig slaughterhouses in a specified geographic zone (fig 5) . we used a mixed linear regression to identify factors associated with the number of pedv cases in taiwan. results indicate positive correlations between the number of cases and both slaughterhouse number and pig farm size, and a negative correlation with number of feed mills ( table 1 ). the highest concentration of pedv cases was identified in a multi-county section of southern taiwan characterized by a large number of pig farms with high animal densities. our results are in agreement with findings from previous studies suggesting that excessive farm capacity (measured as the total number of pigs on a farm) is a risk factor for the spread of pedv [5] . further, aerosol transmission is considered a viable dissemination route in environments marked by high pig densities and close animal proximities [4] . regarding the negative correlation between pedv cases and number of feed mills, our data indicate that pig farms at the end of feed routes likely have higher probabilities of infections. some reports suggest that vehicles used for the dual purposes of transporting swine to slaughterhouses and delivering feed to farms may increase the potential for pedv due to feedbag contamination [5, 25, 39, 40] . our finding of a high correlation between the number of pedv cases and the number of pig slaughterhouses suggests that transport trucks may have been a factor in the 2014 taiwan outbreak. other risk factors requiring further research include spray-dried porcine plasma (sdpp, an important blood-based component of nursery pig diets) and improper disposal procedures when pig corpses are collected and sent to rendering plants. there is a clear need to collect more data on feed truck routes, sdpp supplement distribution, rendering plant procedures (especially delivery), and slaughterhouse processes when trying to identify the sources of various strains in taiwan. due to the potential for significant financial losses, there is a strong need to act on these and other possible factors before detailed studies can be designed, funded, and completed. farms, slaughterhouses, and feed suppliers in counties with high pig densities need to immediately enhance their biosecurity measures to prevent future pedv outbreaks, and greater effort is required to monitor potential transmission routes. table. https://doi.org/10.1371/journal.pone.0213153.g004 supporting information s1 conceptualization: yu-ching lan. porcine epidemic diarrhea, diagnosis, and elimination introduction of pedv outbreak and prevention method evolutionary characterization of the emerging porcine epidemic diarrhea virus worldwide and 2014 epidemic in taiwan. infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases evidence of infectivity of airborne porcine epidemic diarrhea virus and detection of airborne viral rna at long distances from infected herds epidemiological factors associated to spread of porcine epidemic diarrhea in japan. preventive veterinary medicine porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus a porcine epidemic diarrhea virus outbreak in one geographic region of the united states: descriptive epidemiology and investigation of the possibility of airborne virus spread deadly pig virus slips through us borders risk factors for porcine reproductive and respiratory syndrome virus infection and resulting challenges for effective disease surveillance isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the united states fighting a deadly pig disease. industry, veterinarians trying to contain ped virus, new to the us comparison of porcine epidemic diarrhea viruses from germany and the united states novel porcine epidemic diarrhea virus variant with large genomic deletion, south korea. emerging infectious diseases isolation of porcine epidemic diarrhea virus during outbreaks in south korea phylogeographic investigation of 2014 porcine epidemic diarrhea virus transmission in taiwan plos one isolation and molecular characterization of porcine epidemic diarrhea viruses collected in japan in 2014 us-like strain of porcine epidemic diarrhea virus outbreaks in taiwan porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines novel porcine epidemic diarrhea virus (pedv) variants with large deletions in the spike (s) gene coexist with pedv strains possessing an intact s gene in domestic pigs in japan: a new disease situation. plos one heterogeneity in spike protein genes of porcine epidemic diarrhea viruses isolated in korea detection and molecular diversity of spike gene of porcine epidemic diarrhea virus in china the s2 glycoprotein subunit of porcine epidemic diarrhea virus contains immunodominant neutralizing epitopes cellular entry of the porcine epidemic diarrhea virus sequence analysis of the partial spike glycoprotein gene of porcine epidemic diarrhea viruses isolated in korea coronaviruses: structure and genome expression role of transportation in spread of porcine epidemic diarrhea virus infection, united states. emerging infectious diseases land altitude, slope, and coverage as risk factors for porcine reproductive and respiratory syndrome (prrs) outbreaks in the united states molecular characterization of the porcine epidemic diarrhea virus tw4/2014 in taiwan. austin virology and retro virology qgis geographic information system. open source geospatial foundation bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt jmodeltest 2: more models, new heuristics and parallel computing bayesian phylogeography finds its roots spread: spatial phylogenetic reconstruction of evolutionary dynamics origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis distinct characteristics and complex evolution of pedv strains us-like strain of porcine epidemic diarrhea virus outbreaks in taiwan molecular characterization of pig epidemic diarrhoea viruses isolated in japan from 2013 to 2014. infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases spatial dynamics of porcine epidemic diarrhea (ped) spread in the southern kyushu, japan. preventive veterinary medicine an evaluation of contaminated complete feed as a vehicle for porcine epidemic diarrhea virus infection of naive pigs following consumption via natural feeding behavior: proof of concept an evaluation of a liquid antimicrobial (sal curb(r)) for reducing the risk of porcine epidemic diarrhea virus infection of naive pigs during consumption of contaminated feed key: cord-000726-tonaaft2 authors: chang, binggong; petersen, robert; wisniewski, thomas; rubenstein, richard title: influence of mabs on prp(sc) formation using in vitro and cell-free systems date: 2012-07-27 journal: plos one doi: 10.1371/journal.pone.0041626 sha: doc_id: 726 cord_uid: tonaaft2 prp(sc) is believed to serve as a template for the conversion of prp(c) to the abnormal isoform. this process requires contact between the two proteins and implies that there may be critical contact sites that are important for conversion. we hypothesized that antibodies binding to either prp(c)or prp(sc) would hinder or prevent the formation of the prp(c)–prp(sc) complex and thus slow down or prevent the conversion process. two systems were used to analyze the effect of different antibodies on prp(sc) formation: (i) neuroblastoma cells persistently infected with the 22l mouse-adapted scrapie stain, and (ii) protein misfolding cyclic amplification (pmca), which uses prp(sc) as a template or seed, and a series of incubations and sonications, to convert prp(c) to prp(sc). the two systems yielded similar results, in most cases, and demonstrate that prp-specific monoclonal antibodies (mabs) vary in their ability to inhibit the prp(c)–prp(sc) conversion process. based on the numerous and varied mabs analyzed, the inhibitory effect does not appear to be epitope specific, related to prp(c) conformation, or to cell membrane localization, but is influenced by the targeted prp region (amino vs carboxy). prion diseases are a group of fatal neurodegenerative disorders that are associated with conformational conversion of the cellular prion protein, prp c , which is mainly a-helical with very few beta sheets, into a b-sheet-rich form, prp sc [1] [2] [3] [4] [5] . the mechanism by which prp c is converted to the abnormal isoform is still not clear, but it is presumed to involve a prp c -prp sc complex, with the latter serving as a conformational template [6] . in this model, prp sc serves as a template that binds to prp c and produces a conformational conversion into the abnormal isoform. this raises the issue of whether there are critical contact sites that mediate conversion. if this is the case, interfering with or blocking complex formation should prevent the prp c to prp sc conversion process. previous reports have described anti-prp antibodies that can stop or hinder the conversion process add reference 44 and renumber [7] [8] [9] [10] [11] [12] [13] [14] . protein misfolding cyclic amplification (pmca) is an assay that mimics the prp sc propagation process under cell-free conditions. in this method prp sc is amplified by converting prp c to a prp sc seed during incubation with periodic sonication [15] . prp sc generated by pmca is infectious in wild-type animals [16] and can be indefinitely propagated while preserving the properties of the original prp sc strain [16] [17] [18] . furthermore, pmca has been quite useful in studying the cofactors that influence prp conversion [19] [20] [21] [22] [23] [24] [25] [26] , and in detecting prp sc from biological samples of humans and animals [17, [27] [28] [29] [30] [31] [32] [33] [34] . we hypothesized that antibodies binding to prp c and/or prp sc might hinder or prevent the formation of the prp c -prp sc complex and thus prevent the conversion process. we compared the effect of individual prp-specific monoclonal antibodies (mabs) on the prp c -prp sc conversion process using both an n2a/22l cell culture model and the test-tube pmca system. our results demonstrate that the mabs have a range of inhibitory effects on the prp c -prp sc conversion process. the degree of inhibition is mab specific and more dependent on the antibody targeting region than on the specific epitope being recognized. furthermore, since the pmca-based method is dose-dependent and rapid, it may serve as an ideal screening assay for potential inhibitors of both prp sc accumulation and the progression of prion diseases. all procedures involving animals and their care were conducted in accordance with the united states department of agriculture animal welfare act and the national institute of health policy on humane care and use of laboratory animals. tissue samples from uninfected and prion agent-infected mice and hamsters were obtained using protocols approved by the institutional animal care and use committee of the suny downstate medical center (protocol #'s 07-250-09 and 07-251-09). a 10% normal hamster brain homogenate (nbh) was prepared in phosphate buffered saline (pbs) containing 1% triton x-100, 4mm edta and 1% protease inhibitor cocktail (abcam). prpspecific mabs were generated against recombinant (murine or hamster) prp or brain-derived proteinase k (pk)-resistant purified prp sc [35] from brains of clinical mice infected with the me7 mouse-adapted scrapie strain or clinical hamsters infected with the 263k hamster-adapted scrapie strain. the mabs used in this study were purified (montage antibody purification kit; millipore, billerica, ca), isotyped (elisa mouse antibody isotyping kit; thermo fisher, rockford, il), and epitope mapped ( table 1) . the immunoreactivity of all the mabs were analyzed on western blots against denatured, pk-digested and undigested prp derived from uninfected and infected brain homogenates as well as by elisa against recombinant prp. with the exception of mab 3f4, each of the individual mabs had equivalent immunoreactivity against murine and hamster prp sc on an immunoglobulin concentration basis. all of the mabs were highly reactive against both hamster prp c and prp sc isoforms and, for the pmca studies, were individually added to the 10% nbh at a final concentrations of 50 mg/ml. a 10% 263k brain homogenate was prepared in pbs only and diluted to a final concentration of 10 24 . a 100 ml aliquot of this homogenate was initially combined with 10 ml of 10% nbh (with or without added mab). each sample was sonicated (qsonic at 480w power, 60 amplitude, 40,000 j energy, 90 sec process time, 3 sec pulse on21 sec pulse off), then incubated at 37uc for 1 hr. this was defined as one cycle of serial pmca (spmca). at the completion of each cycle, an additional 10 ml of 10% nbh (with or without mab) was added. at the end of every five cycles, 100 ml of the total volume was transferred to a new tube containing an equal volume of 10% nbh (with or without mab) and the cycling reactions continued. at the completion of 40 cycles (spmca 40 ), 500 ml from each sample was pk-treated (100 mg/ml final concentration, 50uc, 30 min), followed by the addition of protease inhibitor cocktail. the sample was heated (100uc, 10 min) and then centrifuged at 16,0006g for 2 min at room temperature. the supernatant was combined with 6x laemmli sample buffer, and 50 ml was electrophoresed in a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) followed by transfer to nitrocellulose membrane. the membrane was blocked for 1 hr in pbs containing 0.1% tween 20 (pbst) with 5% non-fat dry milk and incubated with 2 mg/ml biotinylated mab 08-6/2f11. the membrane was washed 3 times (10 min each) with pbst, incubated for 60 min in hrp-conjugated streptavidin (invitrogen) (1:5000 in pbst containing 5% non-fat dry milk) followed by 3 additional pbst washes and detection of proteins with ecl supersignal west dura kit (thermo fisher). quantification of prp sc was performed by densitometric analysis using nih image j software. cellulose membranes spotted with 99 overlapping 13-mer prp peptides were produced as previously described [36] . the membranes were blocked with 5% non-fat dry milk/tris-buffered saline containing 0.1% tween 20 (tbst) probed with antibody diluted 1:5000 in 1% normal goat serum/tbs at 4uc overnight, followed by horseradish peroxidase (hrp)-conjugated goat antimouse secondary (cappel 55570) for 2 hours at room temperature, and detected using millipore immobilon western chemiluminescent hrp substrate (cat wbkls0500). membranes were regenerated for re-use by shaking with dimethylformamide for 30 minutes, then 8m urea/50mm tris-hcl ph 8.0/1% b-mercaptoethanol (b-mc)/1%sds overnight at 37uc, followed by a 30 min wash in the same buffer, and then twice for 30 minutes each in 50% methanol/glacial acetic acid, and finally three times for 5 minutes each in methanol. after air drying membranes were stored in a sealed container at room temperature. murine neuroblastoma n2a cells (atcc line ccl 131) were grown in the minimal essential medium supplemented with 10% fbs, penicillin and streptomycin and infected with 2% 22l brain homogenate as described previously [12] . following infection, the amount of prp sc in 200 mg cell lysate aliquots of the n2a/22l cells was determined by pk digestion (1 mg/ml pk for 30 min at 37uc), sds-page on 12.5% tris-tricine gels [37] and western blot analysis as previously described [12] . for treatment of cells with mabs, n2a/22l cells (from the fifth passage after infection and higher) were plated in six-well plates and once the cells were 70-80% confluent, mabs were added at a final concentration of 10 mg/ml and incubation was continued for 96 hr. each mab was tested in three independent experiments using independently infected cell lines. each experiment included both a positive control (untreated n2a/22l cells) and a negative control (n2a cells), which were subjected to pk digestion. the level of pk-resistant prp sc was measured in western blots using hrp-conjugated sheep anti-mouse igg as the secondary reagent and ecl supersignal west dura kit. membranes were exposed to x-ray film (x-omat blue xb-1; kodak, new haven, ct,) with a constant exposure time of 30 sec. the films were converted into eight-bit grayscale digital files. quantification of prp sc was performed by densitometric analysis using nih image j software v. 1.34. areas under the curves for three prp bands representing non-, mono-and diglycosylated isoforms of the protein were summarized from each sample to calculate the total amount of prp and expressed as percentages of the average value from a positive control (untreated n2a/22l), whereas the optic density of the background was taken from negative control lanes (n2a cells). the prp-specific mabs that were evaluated for their ability to prevent prp c to prp sc conversion have linear epitopes that span the entire prion protein from the amino to the carboxy terminus ( table 1 , fig. 1 ). we used n2a cells persistently infected with the 22l mouseadapted scrapie strain (n2a/22l) to evaluate the affect of each mab on prp sc formation ( fig. 2a and 2b) . treatment with the mabs did not result in any cytotoxicity to the n2a/22l cells throughout the incubation period. further, incubation of the. n2a/22l cells with 10 mg/ml purified, irrelevant mouse igg had no effect on prp sc formation compared to untreated n2a/22l cultures (figs. 2a and 2b). mab 3f4 did not reduce prp sc formation compared to control n2a/22l cultures lacking mab. mab 3f4 does not react with mouse prion protein so this was not surprising [38] . the ability of a singly added mab to inhibit prp sc formation was not related to a specific epitope since all of the remaining singly added mabs inhibited prp sc formation to varying degrees. of the individually added mabs, 5d6 was the most effective at inhibiting prp sc formation (95% inhibition) while 3a2 was the least effective (38% inhibition). targeting the amino terminus with mab 7e4 was effective at inhibiting 73% prp sc formation as was targeting the octapeptide repeat region using mab 10e4 which resulted in almost 90% inhibition. strangely, although their epitopes overlap, mab 11f12 was less effective than mab 5d6 at inhibiting prp sc formation (53% vs 95% inhibition). this is in contrast to mabs 8e9 and 1b11, which have overlapping epitopes with 8e9 being more expansive, and resulted in 52% and 42% inhibition, respectively. the combination of 5d6 and 11f12 did not result in an additive inhibitory effect and, in fact, resulted in less inhibition than either one alone. this was confirmed in studies where the addition of 8e9 to 5d6 and 11f12 caused a 45% prp sc inhibition, which was slightly better than 8e9 alone, although the predicted additive inhibitory effect of 63% for the three mab combination (48% for 8e9 plus 15% for the 5d6 and 11f12 combination) was not observed. studies were performed with pmca to determine whether a cell-free system can recapitulate the effect of mabs on prp c conversion observed in infected cells. this system also allowed us to evaluate whether accessibility of mab to membrane associated prp c in the living cells influences the prp c to prp sc conversion process. mabs (12-50 mg/ml final concentration) were added throughout the spmca 40 protocol along with the 10% nbh spiked with a 10 24 dilution of 263k infected brain homogenate as described in the methods section. this dilution of infected brain homogenate does not result in detectable pk resistant prp sc immunostaining (fig. 3a) and, therefore, did not interfere with the detection of newly formed prp sc . at the completion of spmca 40 , the samples were digested with pk (100 mg/ml) and analyzed on immunoblots using biotinylated mab 2f11 which reacts equally with both hamster prp c and prp sc . it is interesting to note that although the 263k-infected brain homogenate displayed the 3 band pattern typical for the multiple glycosylated forms of prp c and prp sc (fig. 3a) , the spmca 40 products in the positive controls and mab-treated reactions consisted of only a single diglycosylated 30 kda prp sc band observed after pk digestion at the higher levels of inhibition, .50%, but had two bands or a smear when there was less inhibition (fig. 3b) . pmca in the presence of mabs was also used to study the importance of binding site specificity in the prp c to prp sc conversion process (fig. 3) . we performed spmca with different mab concentrations to determine the minimum amount of mab necessary to inhibit the conversion process. using a 10 24 dilution of 263k-infected hamster brain homogenate as the prp sc seed and a 10% normal brain homogenate (nbh) as the source of prp c , we tested the ability of mabs to inhibit the conversion of prp c to prp sc . for each mab, final concentrations of 12 mg/ml (lanes 3, 6, 9, 12, 15, 18, 21 and 24), 25 mg/ml (lanes 4, 7, 10, 13, 16, 19, 22 and 25) and 50 mg/ml (lanes 5, 8, 11, 14, 17, 20, 23, and 26) were prepared in hamster nbh and used in the spmca reactions. compared to spmca 40 , which contained no mab (lane 1) and with the exception of 02-3/3a2, the majority of the prp-specific mabs inhibited the conversion process in a dose-related manner although some were more effective than others. mabs 7e4, 10e4, 11f12, 8e11, and 8e9 completely inhibited the conversion process at 50 mg/ml while mabs 1b11 and 2f7 inhibited the conversion process to a lesser degree. the inhibition caused by the mabs was a specific response since spmca 40 studies replacing mabs with purified normal mouse igg (at 12-50 mg/ml) in the 10% nbh did not cause any inhibition of prp sc formation (data not shown). it is interesting to note that, with the exception of only 8e9, the epitopes for all the mabs that caused complete inhibition are located in the amino half of the prp while those that caused incomplete inhibition are located in the carboxy half of prp. there was good correlation between the extent of prp sc inhibition when 10 mg/ml mab in cell culture was compared to 12 mg/ml mab with spmca 40 . a separate study using spmca 40 demonstrated that mabs 3f4 and 5d6 caused complete inhibition of prp sc formation at 12-50 mg/ml (fig. 4) . therefore we extended those studies and evaluated the effects of mabs 3f4 and 5d6 using a wider range of mab concentrations (1.5-50 mg/ml). compared to the other antibodies in this study, mabs 3f4 and 5d6 had the most pronounced effects on prp sc formation as demonstrated by the low concentrations of 3 and 6 mg/ml, respectively, causing complete inhibition (fig. 4) . the potent inhibitory effect of 5d6 on prp sc observed using spmca 40 coincides with its dramatic effect in the n2a/22l culture model. furthermore, the poor prp sc inhibition by 3a2 with spmca 40 (fig. 3b) corresponded well with the poor inhibition (only 32% reduction compared to negative control) observed in the cell culture system ( fig. 2a and 2b ). currently, there is no effective treatment for prion diseases. to date, hundreds of chemical compounds have been identified that antagonize prion propagation in vitro in cell culture-based assays and/or in vivo in animal studies [39] [40] [41] [42] [43] . unfortunately, many compounds efficient in in vitro studies were only effective in animal models if treatment was begun before or close to the time of inoculation with the infectious agent [44] . furthermore, many of the candidate compounds have limited usefulness clinically due to toxicity or their inability to cross the bloodbrain barrier [e.g. congo red [45] , iododoxorubicin, b-sheet breakers]. additional therapeutic and/or prophylactic strategies have been and continue to be pursued. vaccination with recombinant mouse prp delays the onset of prion disease in mice [46] . passive immunization with anti-prp antibodies was shown not only to inhibit formation of prp sc in a cell-free system [47] , but was also shown to prevent infection of susceptible n2a cells [7] and to inhibit prion replication in infected cells [8, 47, 48] . the effectiveness of these treatments were also dependent on when they were administered relative to the time of infection. in an initial passive immunization study using wild-type cd1 mice, mabs 8b4 (to mouse prp residues 34-52) and 8h4 (to mouse prp residues 175-185) given immediately after challenge with 139a scrapie by intraperitoneal (ip) injection (50 mg/week), resulted in a significant prolongation of the incubation period with 10% of the 8b4 treated animals remaining disease free in the group challenged with a lower dose of prp sc [10] . in another study using higher antibody doses (4000 mg/week ip) of either icsm 18 (to mouse prp residues 146-158) or icsm 35 (to mouse residues 95 to 105), prion infection from a peripheral source was completely prevented if treatment was continued for 7 or 30 days immediately following prp sc challenge [9] . furthermore, a transgenic mouse model that expresses mab 6h4 is resistant to prion infection via ip injection by a mechanism that involves either perturbation of cellular prp trafficking/prp c degradation or disruption of the prp c -prp sc interaction [49] . previous studies have reported that the 132-140 portion of prp c [8] or the 132-156 region of prp [50] [51] [52] [53] are important for the generation of prp sc . rigter et al. [54] found two high affinity binding regions for protein-protein interactions using ovine peptide-arrays: (i) sheep-prp peptides 43-102, including the amino-terminal octarepeats, and (ii) sheep-prp peptides 134-177 figure 2b for representative western blots). prp sc western blots were quantitated and the amount of inhibition was determined relative to n2a/22l control cultures. the controls consisted of cells both in the absence of mab and in the presence of normal mouse igg. the % prp sc inhibition plotted represents the mean 6 sd from three independent experiments as described in methods. doi:10.1371/journal.pone.0041626.g002 which encompasses most of the scrapie susceptibility-associated polymorphisms in sheep. moroncini et al. [55] found that residues within the 89-112 and 136-158 segments of prp c are key components of the prp c -prp sc complex. beringue et al. [11] reported that antibodies exclusively binding prp c were relatively inefficient inhibitors of prp sc accumulation compared with antibodies that additionally recognize disease-associated prp isoforms. féraudet et al. [56] screened 145 anti-prp mabs for their capacity to inhibit prp sc replication in infected n2a or rov9 cells. they identified four different linear epitopes that hindered the prp c to prp sc conversion: the amino terminal region 26-35, the octarepeat region 59-89, the intermediate region 97-102, and the central region 130-160. the observation that antibodies that bind to the amino terminus of the prion protein are capable of inhibiting conversion suggests that the figure 3 . a. western blot of 263k brain homogenate that was used as the seed for pmca. dilutions of the brain homogenate was prepared and either untreated (lanes 1 and 2) or pk-treated (lanes 3 and 4) prior to sds-page and western blotting. a 1023 dilution prior to and after pk demonstrates the three protein banding pattern typical for 263k brain homogenate whereas no bands are visible at a 1024 dilution of the same homogenate. b. western blotting of the pmca products following spmca40 in the absence and presence of prp-specific mabs. fourty cycles of serial pmca was carried out in the absence or presence of mabs as described in the text. each mab was added at a final concentration of 12, 25, and 50 mg/ ml. following pk treatment, the pmca products were subjected to sds-page, western blotted and immunostained for prpsc. the protein bands were quantitated and the level of prpsc inhibition, relative to the no mab and normal mouse igg controls, were determined. doi:10.1371/journal.pone.0041626.g003 figure 4 . influence of mabs 3f4 and 5d6 on prp sc formation following spmca 40 . mabs 3f4 and 5d6 were added to spmca 40 at final concentrations of 0-50 mg/ml. the pmca products were pk treated and western blotted. the prp sc was quantitated and the level of prp sc inhibition was determined relative to control reactions. doi:10.1371/journal.pone.0041626.g004 endogenous proteolytic cleavage occurs after the site of conversion. to more completely explore the possible therapeutic effect of anti-prp antibodies, and to establish another system to analyze the influence of abs on the conversion process, we screened mabs produced in our laboratory for their capacity to inhibit prp sc formation. this screening was performed using n2a/22l cells and cell-free spmca. in n2a/22l cultures, all mabs that react with mouse prp reduce prp sc formation although with varying efficiency. thus, similar to previous results [55] , we found that the ability to inhibit prp c to prp sc conversion was not restricted to a single epitope or limited to a specific region of the protein. however, the greatest inhibition was observed with mabs that targeted epitopes in the amino terminal, unstructured region of the prp. the greatest inhibition in the n2a/22l cells was with mab 5d6. this is consistent with a prior study using mab 6d11 (anti-prp residues 95-105) which in a screen of multiple mabs, only one produced the greatest inhibition (,100%) [12] . mab 6d11 has also been shown to have some efficacy in vivo prolonging the presymptomatic incubation period [57] . the mab inhibition results obtained using pmca were similar to that found in the cell culture system. pmca has the advantages over the cell culture model of being cost-effective, simple, rapid, sensitive, and more amenable to studies of dose dependence. for identification of potential candidate mabs that might have in vivo activity it is likely that such mabs would have to produce 90 to 100% inhibition in the much simpler in vitro systems. the interaction of prp c to prp sc is critically dependent on the structural compatibility of the molecules as supported by the existence of a species barrier for prion infection, related to minor differences in the primary sequence of prp c in different species. therefore, it is not surprising that antibodies that may alter or mask the critical epitopes on prp c and/or prp sc , involved during the mutual conformational complementarity required in prion propagation, will be inhibitory for prion replication. although many anti-prp antibodies targeting different regions of prp may have some therapeutic effect in vitro, it is not clear how this relates to their efficacy in vivo. on the one hand, it is tempting to speculate that only the antibodies exhibiting near complete inhibition in vitro would be effective in vivo given the obstacle of the blood brain barrier and access to prp in cells. however, it is also possible that only partial inhibition of conversion is required in vivo allowing the cells to ''recover''. in either case, it would be advantageous for these therapeutic antibodies to have high affinities of binding to prp c and/or prp sc , as well as targeting specific critical prp domains. one can hypothesize that the simultaneous targeting of more than one critical epitope will lead to greater benefits. however, co-treatment experiments performed with a mixture of two antibodies compatibly binding cell-surface prp c did not show any benefit with compared to treatment involving a single mab in our current experiments. in a previous study [58] , we demonstrated synergistic binding with one of our antibody pairs. synergistic binding of inhibitory mabs, i.e. reaction with an antibody that increases the binding of the second antibody, would be predicted to enhance the inhibitory effect. further studies with antibody pairs fitting this description will be required to test this hypothesis. in addition, determining the significance of the mab's ability to bind both prp c and prp sc may provide further insight into the conversion process. conceived and designed the experiments: rr tw. performed the experiments: bc rp tw rr. analyzed the data: bc rp tw rr. contributed reagents/materials/analysis tools: rp tw rr. wrote the paper: rr. secondary structure analysis of the scrapie-associated protein prp 27-30 in water by infrared spectroscopy perturbation of the secondary structure of the scrapie prion protein under conditions that alter infectivity conversion of a-helices into b-sheets features in the formation of the scrapie prion proteins jr (1993) conformational transitions, dissociation, and unfolding of scrapie amyloid (prion) protein prions. cold spring harb.perspect. biol3, a006833) novel proteinaceous infectious particles cause scrapie scrapie prion protein accumulation by scrapie-infected neuroblastoma cells abrogated by exposure to a prion protein antibody antibodies inhibit prion propagation and clear cell cultures of prion infectivity monoclonal antibodies inhibit prion replication and delay the development of prion disease anti-prp antibodies for prophylaxis following prion exposure in mice prpsc binding antibodies are potent inhibitors of prion replication in cell lines clearance and prevention of prion infection in cell culture by anti-prp antibodies could immunomodulation be used to prevent prion diseases? specific binding of normal prion protein to the scrapie form via a localized domain initiates its conversion to the proteaseresistant state sensitive detection of pathological prion protein by cyclic amplification of protein misfolding in vitro generation of infectious scrapie prions protein misfolding cyclic amplification for diagnosis and prion propagation studies coinfecting prion strains compete for a limiting cellular resource effect of transition metals (mn, cu, fe) and deoxycholic acid (da) on the conversion of prpc to prpres the stoichiometry of host prpc glycoforms modulates the efficiency of prpsc formation in vitro efficient in vitro amplification of a mouse-adapted scrapie prion protein formation of native prions from minimal components in vitro enhancement of protein misfolding cyclic amplification by using concentrated cellular prion protein source the role of glycophosphatidylinositol anchor in the amplification of the scrapie isoform of prion protein in vitro cellular factors implicated in prion replication plasminogen stimulates propagation of protease resistant prion protein in vitro presymptomatic detection of prions by cyclic amplification of protein misfolding detection of infectious prions in urine in vitro amplification and detection of variant creutzfeldt-jakob disease prpsc ultrasensitive detection of scrapie prion protein using seeded conversion of recombinant prion protein in vitro amplification of prpsc derived from the brain and blood of sheep infected with scrapie detection of sub-clinical cwd infection in conventional test-negative deer long after oral exposure to urine and feces from cwd+ deer a novel method for preclinical detection of prpsc in blood sulfated dextrans enhance in vitro amplification of bovine spongiform encephalopathy prpsc and enable ultrasensitive detection of bovine prpsc a rapid and efficient method to enrich safprotein from scrapie brains of hamsters sars corona virus peptides recognized by antibodies in the sera of convalescent cases endogenous proteolytic cleavage of normal and disease-associated isoforms of the human prion protein in neural and non-neural tissues immune surveillance and antigen conformation determines humoral immune response to the prion protein immunogen drug therapy in human and experimental transmissible spongiform encephalopathy pharmacological approaches to prion research prion diseases: from molecular biology to intervention strategies prion diseases-close to effective therapy? recent advances in prion chemotherapeutics porphyrin and phthalocyanineantiscrapie compounds screening congo red and its analogues for their ability to prevent the formation of prp-res in scrapie-infected cells immunization delays the onset of prion disease in mice cell-surface retention of prpc by anti-prp antibody prevents protease-resistant prp formation anti-prp antibodies block prpsc replication in prion-infected cell cultures by accelerating prpc degradation prevention of scrapie pathogenesis by transgenic expression of anti-prion protein antibodies propagation of prions with artificial properties in transgenic mice expressing chimeric prp genes a single hamster prp amino acid blocks conversion to protease-resistant prp in scrapie-infected mouse neuroblastoma cells species specificity in the cell-free conversion of prion protein to proteaseresistant forms: a model for the scrapie species barrier efficient conversion of normal prion protein (prp) by abnormal hamster prp is determined by homology at amino acid residue 155 mapping of possible prion protein self-interaction domains using peptide arrays motifgrafted antibodies containing the replicative interface of cellular prp are specific for prpsc screening of 145 anti-prp monoclonal antibodies for their capacity to inhibit prpsc replication in infected cells anti-prp mab 6d11 suppresses prp sc replication in prion infected myeloid precursor line fdc-p1/22l and in the lymphoreticular system in vivo prp antibody binding-induced epitope modulation evokes immunocooperativity key: cord-048492-4z38v9rg authors: tang, julian w.; ngai, karry l. k.; lam, wai y.; chan, paul k. s. title: seasonality of influenza a(h3n2) virus: a hong kong perspective (1997–2006) date: 2008-07-23 journal: plos one doi: 10.1371/journal.pone.0002768 sha: doc_id: 48492 cord_uid: 4z38v9rg background: the underlying basis for the seasonality of influenza a viruses is still uncertain. phylogenetic studies investigated this phenomenon but have lacked sequences from more subtropical and tropical regions, particularly from southeast asia. methodology/principal findings: 281 complete hemagglutinin (ha) and neuraminidase (na) sequences were obtained from influenza a(h3n2) viruses, collected over 10 years (1997–2006) from hong kong. these dated sequences were analyzed with influenza a(h3n2) vaccine strain sequences (syd/5/97, mos/10/99, fuj/411/02, cal/7/04) and 315 other publicly available dated sequences from elsewhere, worldwide. in addition, the na sequence alignment was inspected for the presence of any naturally occurring, known, neuraminidase inhibitor (nai) resistance-associated amino acid mutations (r292k and e119v). before 2001, the hong kong ha and na sequences clustered more closely with the older vaccine sequences (syd/5/97, mos/10/99) than did sequences from elsewhere. after 2001, this trend reversed with significant clusters containing ha and na sequences from different locations, isolated at different times, suggesting that viral migration may account for much of the influenza a(h3n2) seasonality during this 10-year period. however, at least one example from hong kong was found suggesting that in some years, influenza a(h3n2) viruses may persist in the same location, perhaps continuing to circulate, sub-clinically, at low levels between seasons, to re-emerge in the influenza season the following year, relatively unchanged. none of these hong kong influenza a(h3n2) na sequences contained any of the known nai-resistance associated mutations. conclusions/significance: the seasonality of influenza a(h3n2) may be largely due to global migration, with similar viruses appearing in different countries at different times. however, occasionally, some viruses may remain within a single location and continue to circulate within that population, to re-emerge during the next influenza season, with relatively little genetic change. naturally occurring nai resistance mutations were absent or, at least, very rare in this population. despite many hypotheses and studies, the underlying basis for the annual recurrence of seasonal influenza still remains a mystery [1] . hammond et al. [2] postulated a rapid, global dispersion of 'airborne aerosolized influenza virus' via the atmosphere, to account for the persistence and spread of the disease. recent reviews have discussed the various approaches to resolving this question, and identified various factors that may be involved, including: properties of the virus itself (mutation rates and immune escape), properties of the host (seasonal variation in host health and behavior, e.g. crowding and air travel, production and dissemination of bioaerosols through sneezing and coughing), and properties of the environment (temperature, humidity and weather variations, e.g. el nino) [3] [4] [5] [6] [7] . some of these factors have been incorporated into mathematical models to attempt to understand the driving forces behind influenza seasonality [6, [8] [9] [10] [11] [12] [13] . sequence-based analyses have become very popular recently and have shed some interesting insights into possible underlying mechanisms of influenza seasonality. many of these also urge for (or at least hint at) the need for more sequences from tropical regions to be made publicly available to increase the accuracy of such analyses [14] [15] [16] [17] [18] [19] [20] [21] . other studies have analyzed genetic data together with the even more scarcely available antigenic data, in attempts to understand and even predict the most likely emerging strains [22] [23] [24] [25] . even the application of mass spectrometry has been applied to influenza surveillance [26] . hong kong is a subtropical region of almost 7 million people, 95% of whom are ethnic chinese, with a mean temperature of 24uc and mean relative humidity of 79% [27] . it lies geographically in the northern hemisphere, and its influenza season occurs during february-april, sometimes with a second peak during june-august, each year. in contrast, other northern hemisphere countries usually have a more extended influenza season from november to march/april, whereas the influenza season of southern hemisphere countries usually occur from may to september [7, 28] . hence, hong kong may be unique in that its biphasic influenza seasonality seems to straddle those of the northern and southern hemisphere countries, making the molecular epidemiology of its circulating influenza viruses of great interest. in addition, hong kong and southern china have been referred to as the 'epicenter' for new influenza a viruses with pandemic potential for over 25 years now [29] . for all of these reasons, any investigation of the underlying basis for influenza seasonality may benefit greatly from a study of influenza viruses isolated from hong kong. in this study, an analysis is presented of 281 hong kong influenza a(h3n2) hemagglutinin (ha) and neuraminidase (na) full-length, dated sequences collected over 10 years (1997-2006) to assist the ongoing efforts to elucidate the underlying basis for the seasonality of influenza a(h3n2). the ha and na ml phylogenetic trees (with and without the additional, down-loaded contemporary sequences from publicly available archives) produced in this study are too large to include as separate figures in this paper and have been published as online supporting information in a scrollable pdf format for further inspection on the plos one journal website (http://www. plosone.org/home.action). for each of these trees, certain clusters of interest have been highlighted using annotated red boxes or ellipses, and will be specifically referred to, in the following text for further description and discussion. all of these 281 hong kong influenza a(h3n2) ha and na sequences have been deposited on genbank (accession nos.: eu856814-eu857094 for ha, and eu857095-eu857375 for na sequences). figures s1 and s2 show the relationship between the 281 ha and na sequences for the hong kong influenza a(h3n2) samples and 4 world health organization (who) influenza a(h3n2) vaccine strain ha sequences (syd/5/97, mos/10/99, fuj/411/02 and cal/7/04). these hong kong ha and na sequences were inspected to determine if there were any sequences from consecutive influenza seasons occurring on the same branch, indicating that viruses with the same or very similar ha and na gene sequences were occurring in adjacent influenza seasons. this would suggest that that particular virus carrying this gene may have remained 'latent' in that population, to re-emerge in the same population the following season. one example of such possible viral persistence between influenza seasons was found, with ha and na sequences from the same viruses (5251jan02 and 5267jan03, as indicated in figures s1 and s2 for the ha and na phylogenetic trees, respectively), showing a similar clustering pattern for both these genes, separated by at least one year. interestingly, the ha sequence from sample 5250jan02 clusters closely with those from samples 5251jan02 and 5267jan03 (figures s1 and s3), but its na sequence lies some distance away on a separate branch (figures s2 and s4 ). this may suggest a possible reassortment event, either with its ha or na gene segment. further full genome sequencing and analysis may resolve this issue. the the relationship between the who vaccine ha and na sequences and those from hong kong and elsewhere can be seen even more clearly in figures s3 and s4 when the 315 jcvi sequences are added to each tree. although these contemporary jcvi sequences are mainly drawn from just three additional locations, they still represent the northern hemisphere (new york, usa) and the southern hemisphere (western australia and various locations in new zealand). again, for reference, the january hong kong ha and na sequences from each year are again highlighted in red boxes. in addition, in figures s3 and s4 , red ellipses have been added to show where similar ha and na sequences from other, non-hong kong locations have clustered with hong kong sequences on the same branch. the dates of such sequences may be the same (within the limit of the one month temporal resolution used in this study), relatively similar, or very different. these highlighted clusters serve to demonstrate the mobility and ubiquity of this influenza a(h3n2) virus, worldwide, during this 10-year period, i.e. genetically similar viruses can appear in different parts of the world at similar and also different times. these examples are not meant to be exhaustive and other such examples may be found in these trees. these number and position of the clusters indicated by the red ellipses differ between figure s3 (ha sequences) and figure s4 (na sequences) probably because there are different selection pressures acting on these two genes as they have quite different functions (i.e. the ha protein is used by the virus to bind to the host cell for entry, whereas the na protein is an enzyme that enables new progeny viruses to leave the host cell). also, in figures 3 and 4 , for both the ha and na gene sequences, respectively, there is a large region of transition between mos/10/99-like and fuj/411/02-like viruses, containing sequences collected during 1999-2005. inspection of the protein-coding alignment for na sequences showed no evidence of any of the known n2 neuraminidase inhibitor (nai) resistance-associated mutations, r292k and e119v, in any of these hong kong influenza a(h3n2) isolates collected between 1997-2006. this comparative analysis of dated ha and na sequences from influenza a(h3n2) viruses from 4 geographical regions of the world (new york, western australia, new zealand and hong kong) attempts to elucidate more clearly the behavior of influenza a(h3n2). this study contributes an additional 10 years of influenza a(h3n2) ha and na sequences, from hong kong, to the publicly available sequence database (genbank), which should aid other researchers investigating an underlying basis for influenza a(h3n2) seasonality. the approach of the analysis in this study has been to compare accurately dated ha and na sequences using established phylogenetic techniques to examine which sets of sequences cluster together, and by examining the dates and locations from which they were collected, to infer the movements of the virus within those dated periods. a similar analysis was recently performed using dated whole genome influenza a(h3n2) sequences from new york, new zealand and australia, downloaded from publicly available databases, in an attempt to test two competing hypotheses: whether seasonal influenza a(h3n2) viruses continuously 'migrate' around the world, particularly between northern and southern hemispheres; or whether the virus remains 'latent' in one location and reactivates each year to produce the familiar pattern of influenza seasonality [19] . as these authors stated, ideally, whole genomes should be used for more accurate phylogenetic analyses of influenza virus as has been reported previously [14, 15] , with at least one good reason for this being the potentially misleading conclusions caused by influenza viral ha and na gene reassortments [19] . however, many laboratories worldwide do not have the resources to perform whole genome sequencing, which is expensive -particularly those in subtropical and tropical regions from where such influenza sequence data is significantly lacking. in addition, with any phylogenetic study such as this, there is always a limitation on the number of sequences that are available (i.e. the number of respiratory samples containing influenza viruses that have been collected and sequenced in any one influenza season), and the number that can be comfortably analyzed within a given time-frame (i.e. the limitations on computing power, which again may be more of a problem in tropical and subtropical countries that are more resource-limited). in this particular study, there is also a problem of sample bias as these sequences were obtained from only hospitalized children (rather than from those who remained in the community), and may therefore reflect the influenza virus population isolated from the more clinically severely ill patients (or those with more concerned, anxious parents). however, there was a rationale for deliberately selecting children's samples for this study. the reason for this is that, especially in hong kong, unlike adults, children of school age (1-10 years old) are more likely to stay close to home and not travel far from home, which would minimize any importation of influenza viruses from overseas. this would therefore reduce this confounding factor when assessing the migration vs latency hypotheses as an explanation for the underlying mechanism of influenza seasonality in hong kong. accepting all of these shortcomings, some of which are inevitable for such phylogenetic studies (since not all samples can be collected and sequenced from all infected individuals from all over the world for any particular virus), there are still some useful conclusions that can be gained from this study: i) from figures s1 and s2, there is at least one example of a virus that reappears, relatively unchanged between consecutive influenza seasons, and which can be seen as some evidence to support the 'latency' hypothesis [19] . since the same viruses show this same pattern of clustering in both their ha and na genes, this reduces the likelihood that this was due to a reassortment event in one of these genes, i.e. it is less likely for the same viruses to have reassorted both the ha and na genes during the same years -though of course this possibility cannot be ruled out. whole genome sequencing would be useful to confirm this if resources are available in the future. ii) from figures s3 and s4 , it is difficult to say whether viruses from hong kong preceded (or gave rise to) those from elsewhere, since viruses from outside hong kong can be found to both pre-date and post-date those isolated from hong kong. however, there are several examples of similar viruses being isolated in different parts of the world at about the same time (as shown in some of the red ellipses in figures s3 and s4 figures s3 and s4) , though this may not necessarily mean that the fuj/411/02-like viruses originated from there. hence, these results suggest that the seasonality of influenza a(h3n2) may, in fact, result from a combination of these 2 models postulated by nelson et al [19] , i.e. mainly migration, but with occasional examples of latency. again, due to the unavoidable, incomplete sampling and sequencing of influenza a(h3n2) viruses worldwide, this support for these hypotheses ('migration' and 'latency'), as presented here, is admittedly, only patchy at best. however, the fact that we can find at least one example supporting the latency hypothesis, in these 281 ha and na sequences spanning 10 years (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) , suggests that they may both play a part in the underlying mechanism governing influenza a(h3n2) seasonality. the large variations in both the ha and na sequences during the transitional period between mos/10/99 and fuj/411/02 ( figure s3 and s4) may have reduced the protective efficacy of any pre-existing influenza antibodies (from prior infection or immunization by mos/ 10/99-like viruses) before the inclusion of the fuj/411/02 strain in the world health organization (who) recommendations for the seasonal influenza vaccine in 2004. such a reduction in protective efficacy from the contemporary who influenza vaccine has indeed been suggested in several reports [14, 30, 31] . similarly, although, the na antigen is not usually specified in seasonal influenza vaccine, and its protective effect is unknown, its sequence variation in na during this transitional period, may have also contributed to the reduced protection provided by the seasonal influenza a(h3n2) vaccine component during this period, when the fuj/411/02-like viruses were emerging. in summary, this study has provided additional data from hong kong to support a mainly migratory mechanism to explain the underlying seasonality of influenza a(h3n2) viruses. however, there may be small localities of so-called 'latency' where viruses remain circulating at low levels within that local population, to reemerge during the influenza season of the following year, with relatively little genetic change. this may affect only a minority of these populations, with the majority being infected by newly imported influenza viruses from elsewhere. these concepts are summarized in figure 1 . two recent papers [37, 38] have suggested that the existing evidence tends to support a migration rather than a latency mechanism to explain the annual seasonality of influenza a. however, they did not have access to large numbers of influenza sequences from southeast asia. thus, in view of the new data presented in this study, it is hoped that such hypotheses may be revised, to include a contribution from the latency mechanism. in some populations (perhaps those more localized in southeast asia), this latency mechanism may contribute more significantly to the underlying basis for the seasonality of influenza a -a possibility not entirely ruled out by one of these studies [38] . more data is required to explore this hypothesis in those populations. admittedly, the fact that in this 10-year (1997-2006 ) data set of almost 300 influenza a(h3n2) ha and na sequences from hong kong, we have only found one particular example supporting the latency mechanism, does suggest that this is a relative rarity, and that the migration mechanism is probably responsible for the majority of influenza seasonality patterns seen worldwide. finally, it is interesting, and to a certain extent, reassuring, to note that during this 10-year period (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) , no influenza a(h3n2) viruses isolated from this cohort of nai-naïve children from hong kong, showed any evidence of naturally occurring, known nai resistance-associated amino acid mutations (r292k and e119v). there are many parts of the puzzle remaining, such as exactly how do some of these influenza viruses migrate so widely, yet still remain relatively similar over several years. how is the low level circulation of 'latent' influenza viruses accomplished between seasons? is this a property of the population's host immune responses, the virus, the environment or some combination of each of these factors? in a collaborative effort to answer these questions and perhaps to improve the accuracy of influenza strain forecasting and vaccine composition [38] , it is also hoped that this study will encourage more researchers in southeast asia to make their influenza sequences publicly available for analysis -especially whole genome sequences where resources permit, and particularly sequences from more tropical countries where influenza is prevalent all year round, with less well-defined seasonal peaks. approximately 30 influenza a(h3n2) isolates for each year of 1997-2006 obtained from the nasopharyngeal aspirates (npas) of children aged 1-10 years, admitted to the prince of wales hospital (pwh) in hong kong, were retrieved from long-term archives (stored either at 270uc or in liquid nitrogen) for hemagglutinin (ha) and neuraminidase (na) gene sequencing and analysis. these children presented with acute respiratory illness and did not receive anti-influenza therapy before or during their illness. as influenza a(h1n1) was the predominant circulating virus during 2006, very few h3n2 isolates were obtained for that year. table 1 shows the age and sex distribution of these children. verbal consent for the initial diagnostic testing of these samples for respiratory viruses, including influenza, was obtained from the parents of these children. such verbal (rather than written) consent is routine for such standard diagnostic tests in hong kong. the local joint chinese university of hong kong-new territories east cluster (joint cuhk-ntec) research ethics committee institutional review board awarded ethics approval for this retrospective sequencing and molecular epidemiological study (study reference number: cre-2005.390) without the need to obtain further, explicit, written consent. this is also in agreement with the american college of epidemiology guidelines [33] for such retrospective epidemiological/surveillance studies. the samples retrieved from the deep-freeze archive were first generation isolates, i.e. the original clinical samples (npas) had been inoculated, for routine diagnostic testing, into madin-darby canine kidney (mdck) cells. these mdck-cultured viral isolates were originally confirmed to be influenza a(h3n2) before being frozen and archived. for this study, these frozen isolates were retrieved from deep-freeze, thawed and then used directly for sequencing. if any of these newly-thawed, archived samples failed to amplify at this stage, as determined by ethidium bromide staining and gel electrophoresis, it was inoculated into mdck cells and re-cultured. after this additional step, most isolates were successfully sequenced. if this step still failed, then an alternative isolate was retrieved from deep-freeze for sequencing. total rna was extracted using the purelink tm viral rna/ dna kit (invitrogen, carlsbad, usa) according to the manufacturer's instructions, and resuspended in 50 ml of rnase-free water. reverse transcription-polymerase chain reaction (rt-pcr) was carried out with superscript iii one-step rt-pcr system with platinum taq dna polymerase kit (invitrogen, carlsbad, usa) according to the manufacturer's protocols. in brief, a 25-ml reaction mix containing 0.5 mm of each forward and reverse primers and 10 ml of extracted rna template were used for the rt-pcr. sets of primers was designed to amplify the complete influenza a(h3n2) ha and na genes ( table 2) . the rt reaction (55uc for 30 min) was followed by 94uc for 2 min and 40 cycles of pcr (94uc for 30 sec, 50uc for 30 sec, and 68uc for 1 min 45 sec, for each cycle) and a final extension at 68uc for 10 min. the pcr products were purified by microspin sephacryl s-400 hr columns (amersham biosciences, uk). sequencing reactions were performed using bigdyeh terminator v3.1 cycle sequencing kits (applied biosystems, foster city, usa) with 2.5 ml of template cdna. for sequencing the ha and na genes, the primers used are shown in table 3 . sequencing reactions were performed on an applied biosystems 3130 abi sequencer (abi, foster city, usa) and in both directions to cross-check the results. alignments of nucleotides sequences were carried out using seqscape v2.5 (applied biosystems, foster city, usa). these hong kong influenza a(h3n2) ha and na gene sequences were aligned and edited in bioedit v.7.0.9.0 [32] . after alignment and manual editing, to enable all sequences (i.e. both the hong kong and jcvi reference sequences) to have the same final length for the construction for each of the phylogenetic trees shown in figures s1 to s4 , the sequence lengths were: this was due to different ha and na sequence lengths being available in the respective jcvi and who sequence data bases. phylogenetic tree construction was performed with paup* version 4.0b10 [34] by using a maximum likelihood (ml) approach, under an optimum model of evolution as determined by modeltest v3.7 [35] . due to the large dataset and to reduce the time required for computation, optimal trees were searched for by using a nearest neighbor interchange (nni) heuristic search strategy. bootstrapping was performed within paup* and displayed using exported pdf files created using figtree v1.0 (previously available from: http://tree.bio.ed.ac.uk/ software/figtree/), which were subsequently annotated using adobe acrobat professional 6.0. the phylogenetic trees for these influenza a(h3n2) ha and na sequences for the period 1997-2006 were rooted against the reference ha and na sequences obtained from the influenza vaccine strain a/syd/5/97(h3n2) downloaded from the los alamos national laboratory (lanl) database (http://www.flu. lanl.gov/vaccine/) [36] . this strain was used as it was representative of the influenza a(h3n2) viruses circulating in 1997, i.e. at the start of this hong kong influenza a(h3n2) archive. other available ha and na sequences for other vaccine strains (mos/ 10/99, fuj/411/02, cal/7/04) were also downloaded and included in all the phylogenetic trees. for comparison with other contemporary influenza a(h3n2) sequences worldwide between 1997-2006, all publicly available (at the time of this analysis) dated ha and na sequences from children of similar ages (0-16 years old -the upper range was extended to include more sequences), spanning this period were downloaded from the then tigr (the institute for genomics presence/absence of established n2 neuraminidase inhibitor (nai) resistance-associated mutations, r292k and e119v once the na nucleic acid sequences were obtained, they were aligned, in-frame for protein coding, and converted to amino acids using the built-in function in bioedit. the presence or absence of the established nai resistance-associated mutations, r292k and e119v, was then determined by inspection of the resulting amino acid alignment, with reference to the influenza a/syd/5/ 97 (h3n2) na sequence, which was isolated before the licensing and widespread use of neuraminidase inhibitors that began after 1999/2000. table s1 the 315 downloaded tigr(jcvi) influenza a(h3n2) ha and na sequences used to construct the multi-country ha and na phylogenetic trees shown in figures s3 and s4 . ms excel file containing the genbank accession numbers of the 315 downloaded tigr(jcvi) influenza a(h3n2) ha and na sequences which were used to construct the multi-country ha and na phylogenetic trees shown in figures s3 and s4 author contributions global epidemiology of influenza: past and present impact of atmospheric dispersion and transport of viral aerosols on the epidemiology of influenza epidemiology and seasonality of respiratory tract virus infections in the tropics association of influenza epidemics with global climate variability dynamic patterns of avian and human influenza in east and southeast asia influenza seasonality: underlying causes and modeling theories influenza virus transmission is dependent on relative humidity and temperature dynamics of annual influenza a epidemics with immunoselection ecological and immunological determinants of influenza evolution the sirc model and influenza a influenza drift and epidemic size: the race between generating and escaping immunity epidemic dynamics and antigenic evolution in a single season of influenza a seasonal dynamics of recurrent epidemics whole-genome analysis of human influenza a virus reveals multiple persistent lineages and reassortment among recent h3n2 viruses large-scale sequencing of human influenza reveals the dynamic nature of viral genome evolution influenza in tropical regions long intervals of stasis punctuated by bursts of positive selection in the seasonal evolution of influenza a virus stochastic processes are key determinants of short-term evolution in influenza a virus phylogenetic analysis reveals the global migration of seasonal influenza a viruses the evolution of epidemic influenza global patterns in seasonal activity of influenza a/h3n2, a/h1n1, and b from 1997 to 2005: viral coexistence and latitudinal gradients mapping the antigenic and genetic evolution of influenza virus epochal evolution shapes the phylodynamics of interpandemic influenza a (h3n2) in humans predictability and preparedness in influenza control epidemiology. influenza escapes immunity along neutral networks global surveillance of emerging influenza virus genotypes by mass spectrometry influenzaassociated hospitalization in a subtropical city seasonality of infectious diseases and severe acute respiratory syndrome-what we don't know can hurt us an influenza epicentre? effectiveness of the 2003-2004 influenza vaccine among u.s. military basic trainees: a year of suboptimal match between vaccine and circulating strain influenza-associated deaths among children in the united states analysis for free: comparing programs for sequence analysis phylogenetic analysis using parsimony (*and other methods). version 4 modeltest: testing the model of dna substitution the value of a database in surveillance and vaccine selection the genomic and epidemiological dynamics of human influenza a virus the global circulation of seasonal influenza a (h3n2) viruses key: cord-048353-hqc7u9w3 authors: chis ster, irina; ferguson, neil m. title: transmission parameters of the 2001 foot and mouth epidemic in great britain date: 2007-06-06 journal: plos one doi: 10.1371/journal.pone.0000502 sha: doc_id: 48353 cord_uid: hqc7u9w3 despite intensive ongoing research, key aspects of the spatial-temporal evolution of the 2001 foot and mouth disease (fmd) epidemic in great britain (gb) remain unexplained. here we develop a markov chain monte carlo (mcmc) method for estimating epidemiological parameters of the 2001 outbreak for a range of simple transmission models. we make the simplifying assumption that infectious farms were completely observed in 2001, equivalent to assuming that farms that were proactively culled but not diagnosed with fmd were not infectious, even if some were infected. we estimate how transmission parameters varied through time, highlighting the impact of the control measures on the progression of the epidemic. we demonstrate statistically significant evidence for assortative contact patterns between animals of the same species. predictive risk maps of the transmission potential in different geographic areas of gb are presented for the fitted models. the 2001 fmd epidemic in the uk had a substantial cost in human, animal health and economic terms (alexandersen et al. [1] , kao [2] ). understanding the risk factors underlying the transmission dynamics of that epidemic and evaluating the effectiveness of the control measures are essential to minimise the scale and cost of any future outbreak. epidemic modelling [3, 4, [5] [6] [7] proved critical to decision making about control policies which were (in some cases controversially) adopted to control the 2001 epidemic [8] [9] [10] . modelling now has a 'peace-time' contingency planning role. one weakness of the modelling studies undertaken in 2001 was the relatively ad-hoc nature of the parameter estimation methods employed. in their first paper, ferguson et al. [4] used maximum likelihood methods to fit to the observed incidence time series, but did not attempt to fit to the spatio-temporal pattern of spread. in their later work, the same authors developed a more robust method for estimating species-specific susceptibility and infectiousness parameters and spatial kernel parameters (see supplementary information to [3] ), but at the time the statistical basis for the methods developed was lacking. in retrospect, the methods developed turned out to be closely related to those developed during the sars epidemic by wallinga and teunis [11] , although the earlier work incorporated population denominator data to allow for spatial-and species-based heterogeneity in disease transmission. nevertheless, the methods employed had the limitation of not being fully parametric, meaning they could not be extended to fit arbitrary transmission models to the observed data. keeling et al. [5] used maximum likelihood methods to estimate transmission parameters, but it was also supplemented by more ad hoc least-squares matching to regional incidence time series. therefore there remains a need to develop rigorous modern statistical approaches for parameter estimation of non-linear models for the 2001 fmd outbreak. bayesian markov chain monte carlo (mcmc) techniques are the best established such methods and have been successfully employed in the analysis of a range of spatiotemporal outbreak data in the past [12] [13] [14] , as well as to purely temporal incidence data [15, 16] . here we develop mcmc-based inference models for the 2001 fmd epidemic in gb. the models examine: the extent to which transmission was spatially localised and the temporal variation in transmission, species-specific variation in susceptibility, infectious-ness and heterogeneity in contact rates between and within species. we take the farm as the unit of our study and ignore the possible impact of within-farm epidemic dynamics. thus we implicitly assume disease spread within a farm is so rapid as to be practically instantaneous, with all animals on a farm becoming infectious at the same time. our data consists of information on all the farms in the uk listed in the 2000 agricultural census [see http://www.defra.gov. uk/footandmouth/cases/index.htm ]. there were a total of 134,986 farms listed in that dataset and uniquely identified by their county/parish/holding (cph) number. their spatial coordinates are provided together with the number of animals by species within each farm. a partition of all gb farms according to the animal types represented is shown in figure 1a . their geographical distribution is represented in figure 1b as the number of farms per 565 km. notice the high density areas in the north west (cumbria), south west (devon), wales and scotland where the main epidemic foci developed. there is also an area of high density in the shetland islands corresponding to very small crofter smallholdings. figure 1c and d show the numbers of sheep and cattle kept per 565 km square. during the 2001 fmd outbreak, a total of 2026 infected premises (ips) were recorded -farms where fmd was diagnosed, and which were subsequently culled. the ip dataset contains, for each farm, the estimated date of infection (determined by a clinical evaluation of the age of lesions on affected animals), and the dates of disease reporting, confirmation and culling. a total of 7457 other (non-ip) farms were also culled -mostly as contiguous premises (cps, about 3103) or dangerous contacts (dcs, about 1287), but some under other local culling policies used in cumbria and scotland. for instance about 1846 (79%) out of a total of 2342 sheep farms in cumbria had all sheep culled under the ''local 3 km radial sheep cull'' policy adopted there. some of the farms (about 30) were recorded both as dcs and cps. multiple records per farm were often found in the disease control management system dataset, and it was often unclear whether this was due to data entry errors or as a result of sequential species-specific culls on the same farm. in our analysis we therefore considered the whole farm to be culled at the last recorded date of culling. the most frequent species are cattle and sheep (see figure 1a ). there are less than 3% farms with pigs only and only 10 farms with just pigs were diagnosed as ips in 2001 (less than 1% of all the ips). this indicates a-priori that pigs contributed far less to the 2001 outbreak than many other fmd outbreaks (despite their high levels of shedding [1, 17] ), and we therefore decided to discard pigs-only farms from the current study to simplify the analysis. the sensitivity analysis section shows that this simplification does not significantly affect estimates of other epidemiological parameters. we discarded another three ips due to missing information or possible mistakes regarding their location or number of animals, leaving a total of 2013 ips in our analysed dataset. we model the epidemic as a space-time survival process [18] . the total observation time t is the 240 days between 7 th february and 5 th october 2001. each farm i at the location (x i , y i ) is associated with an infection time t i (if infected), a removal time r i (if slaughtered) and two integers n c i and n s i representing, respectively, the number of cattle and sheep on the farm. s c and s s represent per-capita cattle and sheep susceptibility, respectively, while i c and i s represent per capita cattle and sheep infectivity. the susceptibility is a relative measure of animal sensitivity to the disease whereas infectivity represents the infectious risk posed by an animal to others. we use a continuous kernel to describe how the probability of contact between farms scaled with distance. transmission is naturally assumed to decrease with the distance between farms according to the power law where d ij represents the euclidian distance between the infected farm i and the susceptible farm j. the parameters a (kernel offset) and c (kernel power) are to be estimated. the kernel captures all forms of movement and contact between farms and as such, the use of a simple 2 parameter function is inevitably a highly simplified representation of the true complexity of inter-farm contacts. we examined other functional forms for the kernel (such as those used in some other analyses [19] ) but the resulting model fits were much poorer than found using the power-law kernel above. given the susceptibility and infectiousness parameters and the kernel, the infection hazard from an infected farm j to a susceptible farm i is then quantified by this model is over-specified as stated, so we arbitrarily assume s s = 1 throughout, meaning s c represents the ratio of cattle-tosheep susceptibility. for a constant (distance-independent) kernel this is just a mass-action closed epidemic model with heterogeneous susceptibility and infectiousness. this model assumes susceptibility and infectiousness parameters scale linearly with the number of animals of different species on the farm, a relatively strong assumption imposed for model parsimony reasons. the mixing matrix embedded in (2) quantifies the 4 species-specific mixing rates between animals on different farms: cattle-to-cattle (s c i c ) sheep-to-cattle (s c i s ), cattle-to-sheep (s s i c ) and sheep-to-sheep (s s i s ). this model formulation is identical to that used by keeling et al. [5] , except for the functional form of kernel used. the force of infection on a susceptible farm i at time t depends on the whole history of events and is just where , if the farm i is susceptible and the farm j is infectious at the time t 0, otherwise by default, we assume a latent period of 1 day (latency is represented within the function l); i.e. farms are infectious the day after they are infected. however, we test the sensitivity of our estimates to the assumption by also examining latent periods of 2 and 3 days. the probability density function that farm i is infected at time t is then given by hence, the contribution that a farm i, observed to be infected at time t, makes to the log likelihood is just: a farm which is not infected contributes to the overall likelihood the probability that it escapes infection during the observation period, i.e. until the time it is culled (r i ) or for the duration of the epidemic t, whichever is shorter. its contribution to the log likelihood is therefore the total log likelihood of the model can be written as we then extend the simple model above by introducing an additional parameter to understand to what extent the transmission within species is altered by between species transmission. the parameter r quantifies the degree to which mixing between species is assortative -with r,1 representing assortative mixing and r.1 disassortative mixing. the interaction model still assumes constant parameters with respect to time along the whole observation period t. the mixing matrix defined in equation (2) becomes s c i c rs c i s rs s i c s s i s ð8þ where we again fix s s to be 1 to avoid model over specification. the force of infection (3) and model log likelihood equation (7) change accordingly. assuming transmission parameters were constant in time throughout the epidemic is obviously a crude simplification. however, allowing infectivity to vary continuously in time results in an over-specified model and problems of parameter identification and confounding. we therefore examined two sets of models in which changes in transmission parameter were restricted to 2 significant points in time denoted by t cut , namely 23 rd february (when the national ban on animal movements was introduced) and 31 st march (when control measures were intensified and the so called 24/48 hour ip/cp culling policy was introduced). models were respectively fitted to the individual case data from the start of the epidemic (conditioning on the first infection) or from after 23rd february (conditioning on the 54 farms that were already infected by that date). a detailed history of the epidemic is given by kao [9] . we separately fitted model variants which assumed a discrete change in parameters on 23 rd february and on 31 st march. confounding meant that only a very limited number of parameters could be varied in time, so we examined the effect of varying infectiousness and kernel parameters separately. we fitted four separate time-varying model variants: (i) varying the cattle infectivity by a factor and keeping sheep infectivity constant through time (cattle infectivity model); (ii) varying sheep infectivity by a factor but not cattle infectivity (sheep infectivity model); (iii) varying both cattle and sheep infectivity by the same ratio (cattle & sheep infectivity model); (iv) varying the kernel parameters (time varying kernel model). for the last model variant we also fitted a version which includes non-assortative mixing between species (see equation (8)). hence the most general mathematical expression of the transmission model is: where the scripts pre and post are self-explanatory for time varying parameters. when fitting models with time varying infectivity parameters we actually fit i post and the ratio m = i pre /i post we called infectivity factor. this is a within species ratio, a parameter directly fitted by the models, unlike the between species infectivity ratio additionally calculated as explained later in the text (see parameter estimates section). note that all models above treat the epidemic as fully observed, i.e. infection times are assumed to be known (when in fact only estimated infection times are known -see sensitivity analysis section), and only ips are assumed to be infectious. we adopt a bayesian framework for statistical inference and use mcmc methods for fitting the model to individual case data. this is not strictly necessary, given our simplifying assumption that the epidemic was completely observed, but it provides a more consistent and robust framework within which to relax that assumption in future work. we obtained parameter estimates and equal-tailed 95% credible intervals from the marginal posterior distributions of the fitted parameters. for the basic model for instance we estimated the relative cattle susceptibility, s c , two infectivity parameters (i c (t);i c and i s (t);i s for all t) and two kernel parameters (c(t);c post ;c pre ;c and a(t);a post ;a pre ;a for all t). we used the posterior mean deviance as a bayesian measure of fit or model adequacy as defined by spiegelhalter et al. [20] . the posterior density deviance is defined as: where log{p(y|h)} is the log-likelihood function for the observed data vector y given the parameter vector h and c is a constant which does not need to be known for model-comparison purposes (being a function of the data alone). the smaller the mean posterior deviance, the better the corresponding model fits the data. if the posterior deviance distributions for two different models overlap significantly, it is necessary to use additional criteria to compare model fit -namely a comparison of the relative complexity of the models. the deviance information criterion (dic) is perhaps the most general of such methods, being a generalisation of the akaike information criterion for bayesian hierarchical models [20] . we define the complexity of a model by its effective number of parameters, p d , defined as where e[ ] represents taking expectations (the posterior average). the dic is then defined as a lower value of dic corresponds to a better model. this criterion offers flexibility for comparing non-nested models [20] and it is straightforwardly computed within an mcmc algorithm. we applied the classic random walk metropolis hastings algorithm [21, 22] and a block-sampling of parameters due to the computationally expensive form of the likelihood [23, 24] . a log scale has been used for sampling as the parameters were all positive definite and were expected to potentially vary by orders of magnitude. however, linear scale sampling yielded similar results. the convergence of the chains was also very much improved (see robert [25] for more on perfect sampling and reparameterization issues) compared with sampling on a linear scale. the model was coded in c and parallelized using openmp 2.0. the mcmc sampler was allowed to equilibrate with convergence being evaluated visually from the likelihood and parameter traces. for the simpler models, 5,000 iterations were sufficient for equilibration, while this increased to 20,000 for the most complex models. also, using log scale sampling, we verified that the chains were able to converge even if started with initial parameter values far from the final posterior mean values. posterior distributions were estimated from 100,000 iterations. the rate of the acceptance varies from model to model. for the baseline model we achieved a 25% rate of acceptance and for the most complex model (8 parameters), a rate of approx 10%. these values compare well with the ''golden'' acceptance rate for random walk metropolis hastings of 23% (roberts [26] ). we did not encounter common problems in mcmc estimation like slow convergence and slow mixing (o'neill [27] ). there were some correlations between parameters, mostly having biological explanations (cattle and sheep infectivity for instance), but a careful parameterization lowers them. we verified parameter estimates were not dependent on parameterization choices -e.g. no difference was seen whether we fitted species infectivity individually, or just fitted sheep infectivity and then the ratio of cattle-to-sheep infectivity. table 1 lists the parameter estimates we obtained for a set of fitted models conditioned only on the first infection whereas table 2 presents the estimates for models conditioned on infections occurring up to 23 rd february. the posterior deviances for each set of models are plotted in figure 2a and figure 2b , respectively. figure 2a illustrates some clear conclusions. of the two models without time variation in parameters, the interaction model fits significantly better than the baseline model without heterogeneous mixing between species. however, fitting the interaction model broadened the credible intervals of the infectivity parameter estimates (table 1) , indicating (unsurprisingly) slight confounding between the 4 infectivity and susceptibility parameters. of the models which allowed infectivity to vary on 23 rd february, allowing only cattle infectivity variation gave a slightly better fit than varying sheep infectivity or both. however, of the models with parameters which vary on 23 rd february, the model variants which allow the 2 kernel parameters to vary at that time point fit substantially better (by both deviance and dic criteria, see table 1 ) than those which just allow a species-specific variation in infectivity. this is encouraging for the inference procedure, as the main control measure initiated on that date was the banning of (figure 3a and figure 3b ). the parameter estimates are less precise before 23 rd february (table 1) due to the relatively small number of ips (about 57) before that date. looking at the most complex model (namely the interaction model with time varying kernel), cattle were estimated to be 5.7fold (4.6, 6.8) more susceptible than sheep (see figure 3c and table 1 ). rather than mentioning animals' specific infectivity (see figure 3d and table 1 ), it is more informative to comment on the cattle:sheep infectivity ratio parameter for the most complex fit (this ratio does dot appear in the tables as it is not a model parameter). we calculated it within the mcmc algorithm as the ratio of the two species infectiousness for each sampled parameter point. the most complex model suggests that cattle are 5.95-fold (4.54, 7.63) more infectious than sheep (figure 3e) . the parameter quantifying assortativity in mixing was estimated at r = 0.45 (0.31, 0.61) -well below 1, the level at which mixing between species is random (figure 3f ). by comparison with the model with a time varying kernel but random mixing between species, the effect of heterogeneous mixing between species modified the between-species transmission as given by matrix (1.9) as indicated below. cattle-to-cattle and sheep-to-sheep transmission is higher (by 19% and 54% respectively) for the model with non-random mixing, whereas the sheep-to-cattle and cattle-to-sheep transmissions dropped by 41% and 37 % respectively. conditioned on 23 rd february, 7 model variants have been considered (table 2 and figure 2b) . we examined the baseline and interaction models (no change in parameters over time), allowing cattle infectivity to vary on 31 st march and both cattle and sheep infectivity to vary by the same factor after 31 st march (with and without heterogeneity in mixing) and allowing both kernel parameters to vary on 31 st march. unsurprisingly, the kernel parameters were not significantly different if allowed to be different before and after 31 st march, neither did this model prove to be the best fit. overall, while the variations in mean deviance (figure 2b ) seen between model variants were much smaller than for the models conditioned on the first infection (figure 2a ), the interaction model allowing for time varying cattle infectivity gave the most adequate fit (measured by both mean deviance and dic, see table 2 ). we cannot statistically compare the two sets of models in table 1 and table 2 , as the data used are different for the two cases. however, the parameter estimates from the best-fitting models of each table are largely consistent. each post-23 rd february estimated value from the best-fit model in table 1 is included in the corresponding pre-31 st march 95% credible interval of the best fit model in table 2 (and vice-versa). the most important message from the second set of models is that all models with cattle time varying infectivity (best fit) indicated higher values of infectivity after 31 st march than before (m = 0.73 (0.63, 0.83)) ( table 2 ). this may seem paradoxical but reflects the fact that while culling (the effect of which is explicitly included in the input data) dramatically reduced case incidence in april, from may to september 2001, case incidence maintained itself at a low level -but almost entirely within cattle farms. this increase in cattle infectivity may therefore really reflect the impact of reduced biosecurity and/or increased non-compliance with movement controls. it is informative to examine what our parameter estimates imply in terms of geographic variation in transmission potential. given the parameter estimates for each model, we can define the relative risk of transmission an infectious farm j would pose to all susceptible farms in the country r j : r j~ic n c j zi s n s j p i=j sc ss n c i zn s this quantity multiplied by the average duration of infectiousness of a farm (time from end of latency to culling) gives the reproduction number r 0j of the farm j. we divided the uk into 5 km squares and then calculated the average transmission risk of all farms in each square (local r 0 ). figure 4 shows how geographic risk changed before and after 23 rd february for our best fit model conditioned on the first infection. the kernel shape has a major influence on the average risk distribution throughout the country. figure 5 shows the corresponding risk maps for the estimates inferred from our best fit model conditioned on 23 rd february. a slightly higher risk is predicted after 31 st march by the model conditioned on 23 rd february due to the increase in the cattle infectivity after this date. the risk estimates after 23 rd february from the first set of models appear consistent with those obtained from the models conditioned on 23 rd february, though a rigorous statistical comparison is not appropriate. we have made the strong assumption for this study that the only infected farms during the 2001 epidemic were the reported ips, and hence that any farms which were infected but culled before clinical diagnosis were not responsible for causing any infections. it is therefore interesting to calculate how many of the proactively culled farms our model predicts might have been infected (but, by definition, not diagnosed). to calculate the probability p i that a particular proactively culled farm i was infected, we need to adjust the infection hazard by the probability that the farm would have not been reported as a clinical case before its culling date t i c . from the outbreak data, we calculate the probability density of the time from infection to report for reported ips and hence the cumulative probability distribution of the time from infection to report, denoted by f. then, with l i (t) being the force of infection on a proactively culled farm i at time t (from the best fit model conditioned on 23 rd february), the probability that that farm gets infected and escapes reporting between its potential infection time and culling time t i c is we calculate the expected number of infections in different classes (e.g. dcs, cps) of proactively culled farms culled within a particular time interval (t i c [ t 0 ,t 1 ½ ). for instance, the expected number of cps culled at the time t i c [ t 0 ,t 1 ½ which are predicted to have been infected can be formally written as this is a simplification, as in reality the delay from infection to report almost certainly depends on the size and species mix on a farm, but the result is nevertheless indicative of the expected level of infection in proactive culling. also, at this stage, the calculations are made as if culling was a non-informative censoring process. this is a reasonable assumption for all proactively culled farms except for dcs (which by definition had been identified by veterinarian as having had a high risk of exposure) but our method may underestimate the infection rate. in calculating the infection to report delay distributions, we divided the epidemic after 23 rd february into 3 time periods: 23 rd february-31 st march, 31 st march-1 st may and 1 st may-5 th october. in these intervals a total of 1332, 4498 and 1627 farms were slaughtered, respectively. our best fit model conditioned on 23 rd february predicts different infectivity regimes before and after 31 st march (see parameter estimates and table 2 ) but we split further the second period of time due to different delays in reporting to culling. the infection to report delay is 8.6 and 8.8 days for the last two periods of time respectively but the infection to cull delay drops from 9.4 and 8.8 days respectively. applying this approach to the interaction model with time varying cattle infectivity which conditioned on the 23 rd of february, we calculated the expected proportion of proactively culled farms which were infected. we estimate that approximately 1.3% (1%, 1.6%) of 7457 culled non-ip farms may have been infected -97 in total (figure 6a ). of the 1332 farms culled between 23 rd february and 31 st march, 1.7% (1%, 2.4%) may have been infected (23 farms). of the 4498 farms culled between 31 st march and 1 st may, we estimate 0.7% (0.5%, 1%) were infected (34 farms). in the period 1 st may to 5 th october, we estimate that 1.6% (1%, 2.3%) of 1627 farms culled were infected (27 farms). the proportion of cps estimated to have been infected is 2% (1.5%, 2.5%), equating to 62 farms (figure 6b ). over the whole epidemic, we estimated 1.5% (0.8%, 2.1%) of farms designated as dcs were infected (19 farms). this estimate (figure 6c ) does not allow for higher risk of infection implied by the veterinary judgement that led to those dcs being identified, which may mean that a higher proportion were in fact infected. if we assume that dcs were 3 times more likely to be infected due to their status than the model would predict, then the incidence of infection in dcs goes up accordingly, i.e. to 4.6% or 59 farms. farms culled neither as dcs or cps (typically those culled under the 3 km and local sheep cull policies in the cumbria, dumfries and galloway areas) had the lowest estimated rate of infectiona mere 0.5 % (0.2%, 0.8%) or 16 out of 3067 farms. in this section we examine the sensitivity of our results to a number of factors: leaving pigs out of the analysis, possible errors in the estimated ip infection dates, and the assumed latent period. to justify the simplification of the analysis by discarding the number of pigs in a farm, we present some more detailed statistics regarding this variable. we also fit the simplest model conditioned the last two farms are exclusively pig farms. we denote by n p i ,s p ,i p the number of pigs in farm i, pigs susceptibility and pigs infectivity respectively. the simplest model similar to (1.2) conditioned on the first infection has been fitted, reducing the number of parameters in the same manner. in addition we estimated pig:sheep susceptibility ratio and pig infectivity, assuming all parameters constant through time. we found that cattle:sheep susceptibility ratio is 6. table 1 shows parameter estimates for cattle and sheep are largely unaffected by ignoring the pig population, with none of the estimates from the two analyses being significantly different. we conclude that including pigs would not change the conclusions presented in table 1 regarding cattle and sheep (given the very small number of ips which had pigs) but it would decrease the power of the analysis and increase model complexity. to understand to what extent our estimates are affected by the assumption that the infection dates have been accurately observed, we randomized the estimated infection dates by adding a gaussian noise with zero mean and a standard deviation of 2 days. this is motivated by the substantial proportion in the observed standard deviation (73.5% less or equal than 2 days) of the distribution time from the estimated infection date to the report date of ips. we then fitted the simplest model (conditioned on both first infection and 23 rd february) to 10 such randomised datasets. the average estimates across them are given in table 3 . they lie well within the confidence intervals we predicted in table 1 . the average cattle:sheep infectivity ratio is also very close to the values estimated using the original data. the average estimates across 10 randomized datasets using the most appropriate model conditioned on 23 rd february (i.e. cattle infectivity and interaction model) are also in table 3 . the values are within the 95%ci presented in table 2 . we assessed a sensitivity analysis for the estimated proportion of infections in proactively culled farms (see the previous section) with respect to infection times. using the predicted parameters for each dataset, we calculated the average proportions across all of them, for each category of proactively culled farms. the average proportion of infections between dc farms is 1.37% (2%, 0.78% and 0.72% for each period of time, respectively). for cp farms, the same quantities evaluate to 1.9% with 1.8%, 1.3% and 1.98%, respectively. overall proactively culled farms, we obtained an average percentage of 1.25% with 1.64%, 0.81% and 1.6% for each considered period of time. all the values are well within the 95%cis predicted by the original data (see the previous section and figure 6 ). all the results presented above assume a fixed latent period of 1 day. we tested the sensitivity of parameter estimates to this assumption by examining latent periods of 2 and 3 days. overall, we would expect infectiousness parameters to increase to compensate for the shorter infectious period, and thus slightly increased generation time (namely the mean time from infection of one case and the time of infection of the cases that case generates). interestingly, however, it is the kernel parameter estimates which are altered as the latent period is varied with the kernel becoming slightly less local with increasing latent period. for two and three days latent period, pre 23 rd february, the values of c dropped from 1.69 (table 1) this paper has presented a statistical analysis of the spatiotemporal evolution of the 2001 foot and mouth outbreak in gb. qualitatively, the results agree with those obtained by keeling et al. [5] in identifying cattle as being the key species in the 2001 epidemic. using the interaction model conditioned on 23 rd february with time varying cattle infectivity, we estimated that 88% of ips between 23 rd feb-31 st march were infected by cattle and only 12% by sheep. sheep-to-sheep transmission only accounts for 3.1% of ips in that period. after 31 st march (when we estimated that cattle infectivity increased slightly, see table 2 ) allowing for non-random mixing between species indicates contacts between farms are assortative on the basis of species composition of the farm; i.e. like species mix with like. this agrees with intuition about the nature of farming practices (e.g. sharing of personnel and equipment is likely to be more common if 2 farms have the same livestock species). the implications of the moderate degree of assortativity we found for control measures remains to be explored. we did not use data collected during the epidemic on traced contacts between farms to fix the spatial kernel function in our analysis, since in the final version of the fmd epidemic data warehouse [http://www.defra.gov.uk/footandmouth/cases/index.htm] very few of the contacts apparently identified early in the epidemic remain confirmed. also we shared the concern of earlier work that the distribution of contact distances in traced contacts may well be biased [3] . we therefore estimated the kernel function, using an offset power-law functional form. the higher value of the kernel power parameter we estimated after 23 rd february (2.67 vs. 1.70 before figure 3a) is consistent with the expected dramatic shortening in the typical contact distance following the national movement ban. this localized spread together with the higher estimated level of infectivity in cattle after 31 st march explains the long tail of the epidemic seen in 2001. in estimating the transmission risk between farms, we assumed a dependence on the euclidian distance between them. in reality, other metrics (e.g. the time required to travel between two farms) might be more reasonable, and should be examined in future work. we also did not include information on landscape (e.g. height above sea-level, location of rivers, trees etc). the estimated risk maps (figure 4 and figure 5 ) match the areas of the country where highest case incidence rates were seen -with the notable exception of wales. the discrepancy between the high predicted risk in wales and the small number of cases observed may reflect inaccuracies in the input data set -keeling et al. [5] reduced farm-level sheep population numbers by 30% in wales and obtained a better geographic match to the data (matt keeling, personal communication). however, the discrepancy may also reflect model inadequacy. we have not here allowed for other farm-level risk factors, such as the farm fragmentation index considered by ferguson et al. [3] . we have not explored more complex non-linear models of the dependence of susceptibility and infectiousness on the number of animals on a farm or relaxed our implicit assumption that contact rates between farms scale linearly with the local density of farms. all these assumptions are being relaxed in ongoing work. the most important issue to be revised in future work is to allow for proactively culled farms which were not diagnosed as ips to be potentially infected and infectious to other farms. this requires modification of the inference model used to allow for an arbitrary number of unobserved infections. the very low numbers of proactively culled farms we estimated as infected suggested that the effect of this model refinement may be limited. it should be noted though that these infection prevalence estimates are in part a result of the relatively non-local kernel estimated simultaneously. if kernel estimates change in a refined analysis -and if dcs were attributed a much higher risk of infection than estimated here due to their status -then it is possible that estimated infection rates in dcs and other proactively culled farms may increase somewhat. however, even if these factors increased our estimated infection prevalence among proactively culled farms 5 fold (which seems unlikely from ongoing work), it would still mean that only a small proportion (,10%) of dcs and cps culled were infected. this does not imply that proactive culling had no effect on the epidemic -as the largest expected effect of such culling is via the targeted depletion of susceptible animals. in this regard, proactive culling has the same epidemiological impact as vaccination. future work will revisit past estimates of exactly how important such culling was for the control of the 2001 fmd epidemic. the pathogenesis and diagnosis of foot-and-mouth disease the impact of local heterogeneity on alternative control strategies for foot-and-mouth disease transmission intensity and impact of control policies on the foot and mouth epidemic in great britain the foot-and-mouth epidemic in great britain: pattern of spread and impact of interventions dynamics of the 2001 uk foot and mouth epidemic: stochastic dispersal in a heterogeneous landscape modelling vaccination strategies against foot-and-mouth disease predictive spatial modelling of alternative control strategies for the foot-andmouth disease epidemic in great britain mathematical modelling of the foot and mouth disease epidemic of 2001: strengths and weaknesses the role of mathematical modelling in the control of the 2001 fmd epidemic in the uk models of foot-and-mouth disease different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures markov chain monte carlo methods for fitting spatiotemporal stochastic models in plant epidemiology likelihood estimation for stochastic compartmental models using markov chain methods estimating parameters in stochastic compartmental models using markov chain methods modelling antigenic drift in weekly flu incidence bayesian inference for partially observed stochastic epidemics the importance of immediate destruction in epidemics of foot and mouth disease statistics for spatial data: wiley series in probability and mathematical statistics spatio-temporal point processes, partial likelihood, foot and mouth disease bayesian measures of model complexity and fit monte carlo sampling using markov chains and their applications teller e (1953) equations of state calculations by fast computing machines bayesian data analysis markov chain monte carlo in practice advances in mcmc: a discussion weak convergence and optimal scaling of random walk metropolis hastings algorithms mcmc methods for stochastic epidemic models bayesian analysis of experimental epidemics of foot-and-mouth disease the authors wish to thank christl donnelly for useful discussions. we are grateful to defra and bbsrc for research funding. conceived and designed the experiments: nf ic. performed the experiments: ic. analyzed the data: ic. contributed reagents/materials/analysis tools: nf ic. wrote the paper: nf ic. key: cord-000581-ewx5xhrb authors: rudge, james w.; hanvoravongchai, piya; krumkamp, ralf; chavez, irwin; adisasmito, wiku; ngoc chau, pham; phommasak, bounlay; putthasri, weerasak; shih, chin-shui; stein, mart; timen, aura; touch, sok; reintjes, ralf; coker, richard title: health system resource gaps and associated mortality from pandemic influenza across six asian territories date: 2012-02-21 journal: plos one doi: 10.1371/journal.pone.0031800 sha: doc_id: 581 cord_uid: ewx5xhrb background: southeast asia has been the focus of considerable investment in pandemic influenza preparedness. given the wide variation in socio-economic conditions, health system capacity across the region is likely to impact to varying degrees on pandemic mitigation operations. we aimed to estimate and compare the resource gaps, and potential mortalities associated with those gaps, for responding to pandemic influenza within and between six territories in asia. methods and findings: we collected health system resource data from cambodia, indonesia (jakarta and bali), lao pdr, taiwan, thailand and vietnam. we applied a mathematical transmission model to simulate a “mild-to-moderate” pandemic influenza scenario to estimate resource needs, gaps, and attributable mortalities at province level within each territory. the results show that wide variations exist in resource capacities between and within the six territories, with substantial mortalities predicted as a result of resource gaps (referred to here as “avoidable” mortalities), particularly in poorer areas. severe nationwide shortages of mechanical ventilators were estimated to be a major cause of avoidable mortalities in all territories except taiwan. other resources (oseltamivir, hospital beds and human resources) are inequitably distributed within countries. estimates of resource gaps and avoidable mortalities were highly sensitive to model parameters defining the transmissibility and clinical severity of the pandemic scenario. however, geographic patterns observed within and across territories remained similar for the range of parameter values explored. conclusions: the findings have important implications for where (both geographically and in terms of which resource types) investment is most needed, and the potential impact of resource mobilization for mitigating the disease burden of an influenza pandemic. effective mobilization of resources across administrative boundaries could go some way towards minimizing avoidable deaths. recent experience from the 2009-h1n1 pandemic highlights how health system capacities, even in developed countries, can be stretched by relatively mild pandemic scenarios [1] [2] [3] . indeed, the vast majority of previous health system analyses in relation to pandemic influenza have focused on developed countries [1, [3] [4] [5] [6] [7] [8] [9] [10] [11] , while the capacity of health systems in low and middle income countries remains largely unstudied. paradoxically, understanding outbreak response capacity in low and middle-income countries is arguably of greater importance than that in developed countries, not only because health systems are weaker [12] , but also because many of these countries are in regions where the risk of emerging infectious diseases is highest [13] . moreover, these countries may suffer disproportionately because of associations between morbidity, pandemic influenza and poverty [14] . proposed strategies for pandemic preparedness in many countries frequently focus on development and acquisition of pandemic vaccines and stockpiling and distribution of antiviral drugs [15] [16] . in the southeast asia region, while surveillance and outbreak response capacities have been strengthened in hope of early detection and control of outbreaks, there has been much less investment into preparing health systems for pandemic mitigation [17] . modeling studies have been used to inform optimum intervention strategies for responding to pandemic influenza, but often neglect to take into account feasibility of health systems to implement such a response and the potential impact of resource shortages on the pandemic burden. investigation of health system capacity in east and southeast asia is of particular interest, not only given the fertile conditions for the emergence and spread of new diseases [13, 18] , but also the wide socio-economic inequalities within the region, and focus of investment by the international community into pandemic influenza preparedness [19] . resource gaps for a pandemic response are likely to be wide and vary greatly between and within countries in asia [20] . but exactly how wide are these gaps, what are the consequences of the gaps in terms of the pandemic disease burden, and to what extent could these consequences be mitigated by improving resource allocation and mobilization? to address these questions, we conducted a health systems analysis across six asian countries and territories with widely varying socioeconomic conditions: cambodia, indonesia, lao pdr, taiwan, thailand and vietnam. in this analysis, mathematical modeling and health system resource data collected across the six territories were used to estimate and compare, within and across countries, the resource gaps, and potential consequences of those gaps in terms of expected mortalities, for a hypothetical pandemic influenza scenario. this study was conducted as part of the asiaflucap project (www.asiaflucap.org), the overall aim of which is to conduct health systems analyses to support capacity development for responding to pandemic influenza across six countries and territories in asia, specifically: cambodia, indonesia, lao pdr, taiwan, thailand and vietnam. for this comparative analysis we focus on four key health system resources: antiviral drugs (specifically oseltamivir), hospital beds, mechanical ventilators and healthcare workers (doctors and nurses), chosen due to their critical importance for responding to pandemic influenza. these resources, along with over 50 other resource items relating to health system infrastructure, equipment, materials, and human resources, were selected through a systematic literature review and a delphi consensus process by a panel of 24 experts, as described in [21] . quantities of these resource items were enumerated during march to september 2009 through questionnaires administered to hospitals and health offices in all districts of each of the six study countries (except indonesia, where data were collected only from districts in jakarta and bali due to the vast geographic scale of the country). additional questionnaires were sent to ministries of health to capture central stockpiles. we received 100% response rates from hospitals and district health offices in cambodia. overall response rates from district health offices were more than 95% in viet nam and indonesia (jakarta and bali), 86% in lao pdr, 72% in taiwan, and 59% in thailand. the response rates for hospital questionnaires were slightly lower at 93% for indonesia and vietnam, 70% for lao pdr, and approximately 46% for thailand and taiwan. data from the questionnaires were double-entered into an excel database. missing values, due to non-responses or incomplete questionnaires, were extrapolated using linear prediction models, specific to each country and resource item, based on a number of district characteristics such as total number of hospital beds or public hospital beds, population size, and geographic location (region/province). for oseltamivir and ventilators, two-step models were used to first estimate the likelihood of having any oseltamivir or ventilators, and then to predict the number of these items. extrapolation of missing data was carried out in stata version 11. in order to estimate health system resource needs and gaps for a pandemic influenza scenario, we used a mathematical model previously developed as part of the asiaflucap project to simulate the transmission dynamics of a pandemic influenza outbreak [22] . full details and equations of the model can be found in [22] , and are summarized in text s1. briefly, the model is based on a deterministic seir (susceptible-exposed-infectious-recovered/removed) model described by differential equations tracking number of people in each compartment over time. given that the primary aim of this analysis was to provide relative estimates of resource gaps within and across countries, rather than to accurately simulate the spread of pandemic influenza throughout each country, the model structure was kept relatively simple, with homogeneous mixing patterns and no age-structure assumed within the modeled population. however, novel complexity is incorporated through making parameters describing the clinical course of infected individuals conditional upon the availability of certain key health system resources (antivirals, beds, and ventilators). thus we could obtain relative indications of the consequences of resource shortages on the pandemic disease burden, specifically in terms of ''avoidable deaths'', which we define as deaths that would not have occurred in the presence of sufficient resources. the infectious compartment of the model was subdivided into three groups based on clinical severity: asymptomatic, mild and severe infections. all asymptomatically and mildly infected patients were assumed to recover, while severe cases were at risk of death and were assumed to need antiviral treatment and hospitalized care, although whether they received either of these depended upon the availability of oseltamivir and hospital beds, respectively. treatment with antivirals and hospitalization were assumed to reduce the infectious period and the probability of death for severe cases. furthermore, a proportion of severe cases are assumed to require mechanical ventilation, which they will receive as long as ventilators are available, otherwise these cases would die. the model parameters describing transmissibility and clinical severity were chosen based on data from the 2009-h1n1 pandemic, with a basic reproduction number of 1.32 [23] [24] . under the parameter values chosen (see table s1 and [22] ), in a population with sufficient resources (i.e. when there are no shortages of oseltamivir, hospital beds, or ventilators), the scenario predicts an overall attack rate of 35.6%, a clinical attack rate of 24.9%, a peak prevalence (of symptomatic cases) of 0.94%, and a case fatality rate of 0.018%. in the absence of any resources, the case fatality rate is substantially higher at 0.029%, while attack rates and peak prevalence remain very similar. estimating resource needs, gaps, and associated mortality in our baseline scenario, resource gaps were estimated assuming that 12% of ''general'' hospital resources (beds, ventilators and human resources) are available for care of pandemic influenza cases, with the remaining 88% required for maintaining essential healthcare services, as in a previous pilot study for thailand [20] , and based on previous reports [25] [26] . we also assumed that, in the event of a pandemic all available oseltamivir doses would be dedicated to severe influenza cases. these assumptions regarding resource spare capacity and oseltamivir usage were relaxed in a multivariate uncertainty analysis (described below). the resource data were aggregated at provincial level for all countries except taiwan, where they were aggregated at county level, and indonesia, where the data were kept at district level. (counties in taiwan and districts in indonesia are of comparable population size to the provinces of the other four countries.) we then ran the model separately for each province (lao pdr, cambodia, thailand, vietnam), county (taiwan) and district (indonesia), with the appropriate resources for each of these administrative areas, assuming a closed population and that resources could not be shared between these areas in a timely manner. for the purposes of narrative flow, we henceforth use the term ''province'' for counties in taiwan. all simulations started with one mild case entering a completely susceptible population. in addition to running the model with the available resources, based on the survey data, we also ran the model with unlimited resources, in order to calculate resource needs, and thus also the resource gaps (or indeed surpluses) by comparing with the resource availability data. the needed number of hospital beds, ventilators and humans resources were estimated from the peak number of cases requiring hospitalization and ventilation, while the needed number of oseltamivir doses was calculated from total number of severe cases occurring over the duration of the outbreak (full details on assumptions of resource depletion rates are detailed in [22] , and summarized in text s1). by comparing the number of deaths predicted by simulations with sufficient resources with those from simulations using actual resource data, we also estimated the number of deaths due to resource gaps, which we term as ''avoidable deaths''. a multivariate uncertainty analysis was conducted to approximate uncertainty surrounding avoidable deaths in light of uncertainty in resource spare capacity and effectiveness, which may vary between settings. specifically, the proportion of ''general'' healthcare resources within each province/district that would be available to care for pandemic influenza patients was allowed to vary between 5-20%, and parameters describing the effectiveness of each resource for reducing the risk of death in cases requiring those resources were allowed to vary independently between a wide range of 20-80%. we also explored the impact of relaxing the assumption that oseltamivir administration is restricted to severe influenza cases, by allowing between 0-5% of mild cases to be treated. one hundred combinations of values were chosen randomly from these ranges using latin hypercube sampling, and simulations were run using each combination. the medians, interquartile ranges (iqr) and 95 th percentile ranges of model outcomes were then calculated across the simulations. since the aim of this study was to compare resource capacities across geographic areas and resource types, rather than to evaluate how transmission dynamics may vary across geographic areas, epidemiological parameters of the model were kept fixed in the multivariate uncertainty analysis to ensure comparability of resource capacity outcomes. however, due to the unpredictability of pandemic scenarios, we also explored model outcomes for a range of values for r 0 and for the severe clinical attack rate in a separate univariate sensitivity analysis. the model was coded and run in r version 2.10.1, using the ''simecol'' package [27] with the runge-kutta 4 th order algorithm for numerical integration of the differential equations. arcgis version 10 was used to map the calculated resource gaps and avoidable mortalities at provincial level. figure 1 presents the geographical distribution of estimated resource gaps across provinces (or districts in the case of indonesia) in each study country for the modeled pandemic influenza scenario, under our baseline assumptions and point estimates for parameter values. the corresponding statistical distributions of resource capacities across areas within each country can be found in figure s1 . a summary of overall resource gaps for each country is presented in table 1 . there was substantial variation in resource gaps both between and within countries, and across resources types (figures 1 and s1) . overall, the biggest gaps were generally seen in cambodia and lao pdr, particularly when standardized by population size, with almost all provinces in these countries displaying gaps in all resources, with the exception of nurses which were estimated to be sufficient in approximately half of the provinces in these countries. in contrast, relatively few provinces in taiwan were estimated to have gaps, at least in general health system resources (beds, ventilators, and human resources), with quantities of these resources often considerably above those predicted to be needed for this scenario. nevertheless, almost half of provinces in taiwan were predicted to have insufficient oseltamivir supplies to treat all severe cases (although it should be noted that the results in figures 1 and s1 do not account for central stockpiles which might be mobilized in the event of a pandemic, as discussed later). thailand, indonesia and vietnam generally displayed a more mixed picture. results were comparable between vietnam and indonesia, with relatively few provinces of vietnam (7?9%) and only one district of jakarta (and none in bali), estimated to have insufficient oseltamivir to treat all severe cases, with supplies of this antiviral drug comparably high across most other areas in these countries. healthcare workers were also predicted to be mostly sufficient in vietnam, with only 3?1% and 1?6% of provinces predicted to have a shortage of doctors and nurses, respectively, for this scenario. however, gaps in hospital beds were observed in over half of provinces in vietnam and districts of jakarta and bali, and all of these provinces displayed a shortage of mechanical ventilators. indeed, of all the resources, the largest gaps were observed in ventilators across all countries except taiwan (table 1) . thailand had the second highest number of ventilators (absolute and per capita) after taiwan, but a shortage of this resource was nevertheless predicted in over 80% of thai provinces. a very heterogeneous pattern was observed in thailand, with around 50% of provinces showing a shortage of hospital beds and oseltamivir, while many other provinces showed a clear ''surplus'' of the latter. meanwhile, a shortage of medical doctors was predicted in over 80% of thai provinces, with gaps in doctors comparable to those lao pdr and cambodia ( table 1 ). the number of nurses in thai provinces was estimated to be somewhat more sufficient, however. a fairly distinct geographical pattern of resource gaps was evident in thailand, particularly for oseltamivir and ventilators, with north-eastern (and some southern) provinces showing shortages more comparable with those in neighboring lao pdr and cambodia than with other thai provinces ( figure 1 ). it should be noted that estimates of gaps in beds, ventilators and human resources were highly dependent on the spare capacity of resources assumed to be available to care for pandemic influenza patients. for example, if spare capacity was assumed to be 5%, rather than 12%, then even in taiwan most areas would suffer gaps in these resources for the modeled scenario. furthermore, use of oseltamivir on even a fairly small proportion (5%) of mild cases, resulted in much faster depletion of this resource, such that all countries are predicted to experience a shortage of oseltamivir for treating all severe cases. the geographic distribution of avoidable deaths, estimated by calculating the number of deaths that would be prevented by filling all resource gaps in each province, and standardized by population size, is presented in figure 2 . (a corresponding map showing absolute numbers of estimated avoidable death is given in figure s2 .) figure 3 shows estimated avoidable death rates attributable to gaps in each resource type (antivirals, beds and ventilators) aggregated across all provinces in each country, and accounting for uncertainty in resource effectiveness and spare capacity. avoidable deaths for a given resource gap were estimated by calculating the number of deaths that would be prevented by filling that resource gap only. figures 2 and 3 highlight how resource gaps could have a substantial impact on mortality rates during an influenza pandemic. a combination of the large population size and shortage of ventilators results in the estimation that, out of the five countries for which nationwide data were collected, vietnam would have the highest total number of avoidable deaths. however, the results for jakarta and bali suggest that indonesia would have the highest avoidable death toll if the data is extrapolated across the entire population of this country. when standardized by population size, the highest rates of avoidable deaths were estimated in cambodia and lao pdr, accounting for over half of all pandemic-associated mortalities in these countries. the median avoidable death rates for these countries were over 15 times higher than that for taiwan, where a relatively low proportion (median: 7.6%, iqr: 5.5-10.7%) of total deaths was estimated to be due to resource gaps. almost all avoidable deaths in taiwan were predicted to be due to local shortages of oseltamivir. in all other countries shortages of ventilators were estimated to be the biggest cause of avoidable deaths (figure 3 ). in indonesia, lao pdr, and vietnam, this result was largely robust to uncertainty surrounding resource effectiveness and spare capacity. for cambodia and thailand, however, the uncertainty analysis suggested that gaps in oseltamivir might also be a main cause of avoidable deaths (figure 3 ). when adding an additional layer of uncertainty to the model assumptions, by allowing for up to 5% of mild cases to be treated with oseltamivir, the increased shortages of the latter further increased uncertainty surrounding the relative importance of gaps in oseltamivir ( figure s3 ). given such uncertainties and the sensitivity of results to model assumptions, the proportion of mortalities that can be attributed to gaps in specific resources should be interpreted with some caution. a clear negative correlation was observed between estimated avoidable mortality rates and gdp per capita at country level ( figure 4a ). total funds per capita committed by donors towards avian and human influenza for each country, up to december 2009 [28] were positively correlated with avoidable mortality rates ( figure 4b ). within many countries it was evident that, while at least some provinces displayed resources gaps, other provinces were estimated to have more than sufficient resources for responding to the modeled scenario, with an overall ''surplus'' of some resources in several countries (table 1) . furthermore, central stockpiles of oseltamivir were present in all countries from which data on this could be obtained. thus we also investigated the proportion of avoidable deaths that might be averted in each country if the total available resources were equitably distributed across provinces according to provincial population size ( figure 5 ). when accounting for central stockpiles of oseltamivir, the overall supply of this drug was estimated to be sufficient to treat all severe cases in all countries (table 1) . thus it was estimated that, in each country (except for vietnam, and jakarta and bali, where provincial supplies of oseltamivir are already relatively high), effective mobilization of oseltamivir across administrative areas could potentially avert a significant proportion of the avoidable deaths estimated under current resource distributions (up to 100% of avoidable mortalities in taiwan; figure 5 ). the (less feasible) scenario of redistributing available beds and ventilators according to provincial need within each country was generally estimated to have less of an impact on the number of avoidable deaths, compared to mobilization of oseltamivir ( figure 5 ). in the case of ventilators, this highlights how the large numbers of deaths attributed to gaps in this resource (figure 3 ) are mostly due to overall nationwide shortages of ventilators, rather than an inequitable distribution of ventilators within most countries. in thailand, however, if all ventilators were distributed in proportion to provincial population sizes, the model predicts around 30% (iqr: 21-41%) fewer avoidable deaths than the number predicted under the observed ventilator distribution. estimates of resource gaps, and thus also avoidable mortalities, were very sensitive to the severity of the modeled pandemic scenario in relation to transmissibility and proportion of cases requiring hospitalization ( figure s4) . a sensitivity analysis showed that under more severe (yet still plausible) pandemic scenarios, even taiwan could experience substantial deaths due to shortages of hospital resources ( figure s4a and s4c) . furthermore, as the severity of the scenario increased, so too did the proportion of avoidable deaths that were attributable to gaps in hospital bed capacity (shown for cambodia in figure s4b and s4d) . it is important to note, however, that for the ranges of values explored for the basic reproduction number and the proportion of cases that become severely ill, consistent patterns were observed when comparing relative magnitudes of avoidable mortality rates across countries (and also across provinces within countries). our results indicate that health system resource gaps for responding to a mild to moderate pandemic influenza scenario are wide and vary greatly, both within and between countries in southeast asia, and that these gaps could have a profound impact on pandemic-associated mortalities. our estimates of resource gaps and avoidable mortality rates at country level show a clear association with national gdp. this result is consistent with a previous analysis of data from the 1918 influenza pandemic, which found that per capita income explained a large proportion of the variation in mortality across countries during the pandemic period [14] . moreover, extrapolation of these mortality rates to the 2004 world population suggested that around 96% of deaths from pandemic influenza would occur in developing countries [14] . our results suggest that, due to inequitable distribution of resources, the variation in pandemic burden is likely to be profound within, as well as between, countries. countries which have experienced the highest burden of highly pathogenic avian influenza (h5n1), namely vietnam and indonesia, appear to be most prepared in terms of the availability and geographical distribution of oseltamivir. in the other study countries, we estimated that central stockpiles of oseltamivir would be sufficient to cover any provincial gaps for treating all severe cases from the modeled scenario, and thus mobilization of this resource could potentially avert a large number of avoidable mortalities in these countries. indeed, in all countries except vietnam, we estimated that optimum mobilization of resources across administrative boundaries could save more than 10% of avoidable deaths. while timely mobilization of resources may be possible in taiwan, with its small geographical size and relatively developed infrastructure, the feasibility of this scenario is questionable in the poorer, and larger, countries of the mekong region, where it might be prudent to disburse central stockpiles of antiviral drugs to provincial and district health facilities prior to an outbreak. gaps in mechanical ventilators were predicted to be a major cause of avoidable deaths, with almost all provinces across all countries estimated to have severe shortages of this of this resource, with the exception of taiwan and some thai provinces. this pattern likely reflects the relatively high cost and human resource skills associated with acquisition and operation of ventilators, and highlights the importance of developing robust triage criteria as part of pandemic preparedness plans to ensure that this resource is allocated to the patients who are most likely benefit [29] [30] . a previous analysis similarly suggested that a dire shortage of mechanical ventilators would be a major limiting factor in responding to a pandemic influenza outbreak in the united states [31] . we found particularly wide variation in the availability of ventilators, and indeed other hospital resources, in thailand, where our results suggest that inequitable distribution of health system resources [32] , rather than simply an overall nationwide shortage, could lead to a high number of avoidable deaths from pandemic influenza. of course, hospital equipment such as beds and ventilators are useless unless sufficient and qualified human resources are available to treat influenza patients, and our results suggest that gaps in healthcare workers would also be an important limiting factor for responding to pandemic influenza in many countries, particularly for cambodia, lao pdr and thailand. it is encouraging that total donor funds committed to avian and human influenza broadly correspond to avoidable mortality rates estimated at country level. a recent paper on financial and technical assistance from the 2010 international ministerial conference on animal and pandemic influenza (imcapi) reports that over 50% of total donor funding committed towards avian and human influenza worldwide between 2005 and 2009 was allocated towards ''human health and pandemic preparedness'' (with other funds committed towards sectors such as animal health; monitoring, information, and internal coordination; and information, education and communication) [28] . however, the extent to which these funds have been, or will be, allocated towards mitigating the resource gaps identified in our study is unknown to us and beyond the scope of this analysis. this study is subject to several limitations, many of which relate to assumptions that were necessary for the modeled scenario. for example, in our baseline scenario we assumed that 12% of hospital capacity, across all provinces and countries, would be available to care for the surge of patients with influenza infections. in reality, surge capacity is likely to vary substantially between and within countries (and over time), but few data on this are available. robust analytical frameworks are urgently needed to define and measure health system surge capacity in order to inform analyses of resource gaps for emergency response scenarios. the effectiveness of resources such as antiviral drugs, ventilators, and general hospital care for improving the survival rates among severe influenza cases is also surrounded by considerable uncertainty and may vary between settings. our results show that, even if epidemiological parameters describing transmission and pathogenicity are kept constant, uncertainties in spare capacity and resource effectiveness lead to considerable uncertainties in estimates of avoidable mortalities rates. nevertheless, the distributions of model outputs from our multivariate uncertainty analysis still showed some significant differences when compared across countries and across resource types (figures 3 and 5) . furthermore, it seems likely that spare capacity and resource effectiveness would be higher in more resource-rich settings, which would only strengthen the findings of this study in terms of the geographic distribution of resource gaps and avoidable mortalities. estimates of resource gaps and avoidable mortality rates were also very sensitive to parameters describing pandemic severity, although similar patterns were observed across geographic areas for the range of values explored. however, the same cannot be said for the relative importance of gaps in different resource types. thus, although our results generally suggest that shortages of ventilators could be a major cause of avoidable deaths in low-and middle-income countries in southeast asia, investment in this resource should not necessarily be prioritized over other healthcare resources. a natural extension of this study would be to investigate the cost-effectiveness of investing different types of health systems resources for mitigating the burden of an influenza pandemic. however, more data on the effectiveness of different resources for managing severe influenza cases is needed before such assessments can be made. other limitations relate to the simplicity of the model structure. we assumed homogenous mixing and a constant basic reproduction number across all populations. in reality, heterogeneities in factors such as age-structure, geographic structure, population density, human behavior, and the underlying health of the population are all likely to play a role in transmission dynamics and burden of influenza. a previous modeling analysis, for example, has shown that higher levels of population heterogeneities, such as in age and spatial structuring of contacts, result in lower overall attack rates and peak prevalence for a given basic reproduction number [33] . however, there is a lack of data on such heterogeneities and how they might affect patterns of pandemic progression for our study region. ongoing studies, such as contact pattern surveys in asia similar to those undertaken in europe [34] , are attempting to rectify this. another limitation is that the resource data were collected between may and september 2009, which includes the first wave of the h1n1-2009 pandemic; thus some resource data (particularly for antiviral stockpiles) may be influenced by the time point within this period at which the data were recorded. given the above caveats, it is important to emphasize that we do not advocate these results to be accurate quantitative reflections of resources shortages or deaths that are likely to occur in any given pandemic scenario. rather, they highlight the scale of health system inequalities within and across countries in asia, and the considerable impact such inequalities could have on the pandemic disease burden. by indicating the relative disparities in resource availability within and across countries, and the potential consequences of resource shortages, these results could help guide investment decisions in scaling up resources to mitigate the burden figure 5 . estimated impact of resource mobilization/redistribution across provinces on avoidable mortality rates within each territory. data were calculated by estimating the number of avoidable deaths if available resources (including central stockpiles for oseltamivir) within each territory were geographically distributed in proportion to provincial population size, and comparing with the total number of avoidable deaths predicted given actual resource distribution. boxplots show medians, interquartile ranges, and 95 th percentile ranges derived from a multivariate uncertainty analysis. data are aggregated across provinces for cambodia, lao pdr, thailand, vietnam, and across counties for taiwan. data for indonesia are aggregated across districts of jakarta and bali only. doi:10.1371/journal.pone.0031800.g005 of future pandemics. as many of these resources have a generic healthcare function beyond pandemic influenza, they may also be useful to guide health system strengthening. figure s1 variation in estimated resource capacities across provinces within each territory for the modeled pandemic scenario. (docx) figure s2 geographical distribution of estimated avoidable deaths due to resource gaps for a modeled pandemic influenza scenario. modelling the impact of an influenza a/h1n1 pandemic on critical care demand from early pathogenicity data: the case for sentinel reporting the experiences of health care workers employed in an australian intensive care unit during the h1n1 influenza pandemic of 2009: a phenomenological study predicting spread of new pandemic swine-origin influenza a (h1n1) in local mid-size city: evaluation of hospital bed shortage and effectiveness of vaccination pandemic influenza-implications for critical care resources in australia and new zealand modeling the critical care demand and antibiotics resources needed during the fall 2009 wave of influenza a(h1n1) pandemic. plos curr influenza modelling the impact of an influenza pandemic on critical care services in england pandemic influenza and hospital resources modeling hospital response to mild and severe influenza pandemic scenarios under normal and expanded capacities ability of regional hospitals to meet projected avian flu pandemic surge capacity requirements effects of interventions on the demand for hospital services in an influenza pandemic: a sensitivity analysis flusurge-a tool to estimate demand for hospital services during the next pandemic influenza major issues and challenges of influenza pandemic preparedness in developing countries global trends in emerging infectious diseases estimation of potential global pandemic influenza mortality on the basis of vital registry data from the 1918-20 pandemic: a quantitative analysis preparing for the next pandemic strategies for containing an emerging influenza pandemic in southeast asia pandemic influenza preparedness and health systems challenges in asia: results from rapid analyses in 6 asian countries emerging infectious diseases in southeast asia: regional challenges to control responses to avian influenza and state of pandemic readiness capacity of thailand to contain an emerging influenza pandemic health system resource needs in se asia for pandemic influenza: systematic review and delphi consensus study health service resource needs for pandemic influenza in developing countries: a linked transmission dynamics, interventions and resource demand model estimated epidemiologic parameters and morbidity associated with pandemic h1n1 influenza the transmissibility and control of pandemic influenza a (h1n1) virus surge capacity associated with restrictions on nonurgent hospital utilization and expected admissions during an influenza pandemic: lessons from the toronto severe acute respiratory syndrome outbreak hospital emergency surge capacity: an empiric new york statewide study simecol: an object-oriented framework for ecological modeling in r international ministerial conference on animal and pandemic influenza: international financial and technical assistance (draft) concept of operations for triage of mechanical ventilation in an epidemic simple triage scoring system predicting death and the need for critical care resources for use during epidemics emergency response planning in hospitals capacity in thai public hospitals and the production of care for poor and nonpoor patients epidemic patch models applied to pandemic influenza: contact matrix, stochasticity, robustness of predictions social contacts and mixing patterns relevant to the spread of infectious diseases the asiaflucap project is coordinated by the london school of hygiene and tropical medicine with collaborators from the hamburg university of we are grateful to the many collaborators within this project consortium for their contribution towards resource characterization, data collection, and discussions at consortium meetings. these include: key: cord-000536-0mn1gbll authors: hu, le-le; chen, chen; huang, tao; cai, yu-dong; chou, kuo-chen title: predicting biological functions of compounds based on chemical-chemical interactions date: 2011-12-29 journal: plos one doi: 10.1371/journal.pone.0029491 sha: doc_id: 536 cord_uid: 0mn1gbll given a compound, how can we effectively predict its biological function? it is a fundamentally important problem because the information thus obtained may benefit the understanding of many basic biological processes and provide useful clues for drug design. in this study, based on the information of chemical-chemical interactions, a novel method was developed that can be used to identify which of the following eleven metabolic pathway classes a query compound may be involved with: (1) carbohydrate metabolism, (2) energy metabolism, (3) lipid metabolism, (4) nucleotide metabolism, (5) amino acid metabolism, (6) metabolism of other amino acids, (7) glycan biosynthesis and metabolism, (8) metabolism of cofactors and vitamins, (9) metabolism of terpenoids and polyketides, (10) biosynthesis of other secondary metabolites, (11) xenobiotics biodegradation and metabolism. it was observed that the overall success rate obtained by the method via the 5-fold cross-validation test on a benchmark dataset consisting of 3,137 compounds was 77.97%, which is much higher than 10.45%, the corresponding success rate obtained by the random guesses. besides, to deal with the situation that some compounds may be involved with more than one metabolic pathway class, the method presented here is featured by the capacity able to provide a series of potential metabolic pathway classes ranked according to the descending order of their likelihood for each of the query compounds concerned. furthermore, our method was also applied to predict 5,549 compounds whose metabolic pathway classes are unknown. interestingly, the results thus obtained are quite consistent with the deductions from the reports by other investigators. it is anticipated that, with the continuous increase of the chemical-chemical interaction data, the current method will be further enhanced in its power and accuracy, so as to become a useful complementary vehicle in annotating uncharacterized compounds for their biological functions. metabolism refers to a collection of chemical reactions in vivo, which keep an unceasing supply of matter and energy for living organisms to maintain life (e.g., growth and reproduction) [1] . these energy-using and energy-releasing chemical reactions catalyzed by enzymes are organized into many metabolic pathways. some compounds/small molecules play major roles in these pathways and are vital for many activities essential for life. for example, during the digestion, the energy rich molecules (i.e. carbohydrate) are broken apart to provide energy, which is then used by cells to build up complex molecules from simple molecules, such as utilizing amino acids to synthesize new proteins that the body needs. identifying the biological functions of compounds is an effective way to study the mechanisms of many basic biological processes [2] . on the other hand, small molecules are the cause, and the cure, for many diseases. for example, diabetes mellitus is a metabolic disease caused by insufficient or inefficient insulin secretary response and elevated blood glucose level [3] . compounds such as sulfonylureas [4] , acarbose [5] , biguanides, thiazolidinediones [5] , and sitagliptin [3] have been used as effective drugs for diabetic therapy. therefore, it is essential to annotate the bioactivities of compounds, which will benefit drug design and disease treatment. besides the conventional biochemical experiments, computational methods are alternative ways to annotate the biological functions of compounds. in recent years, various bioinformatics and structural bioinformatics [6] tools were developed to address this issue, such as quantitative structure activity relationship (qsar) [7, 8] , pharmacophore modeling [9] , molecular docking [10] , and monte carlo simulated annealing approach [11, 12] . different from these methods, lu et al. [1] and cai et al. [2] analyzed the biological functions of compounds by mapping them to the corresponding metabolic pathway classes, which are strongly associated with the biological functions of compounds. the functional group composition was used to represent the compounds, and the nearest neighbor algorithm and adaboost learner [13] were used to construct the prediction models by cai et al. [2] and lu et al. [1] , respectively. both the two prediction methods achieved quite promising results on their own datasets. however, none of their datasets contained the ''multi-function'' compounds that belong to two or more metabolic pathway classes. since these authors were only focused on addressing the singlelabel classification problem, their methods could not be used to deal with the ''multi-function'' compounds. actually, according to kegg [14] , among all the compounds with functional annotations, the ''multi-function'' compounds occupy about 8%. particularly, these multi-function compounds may play some unique role intriguing to both basic research and drug development and hence are worthy of our special attention. recently, the systems biology methods based on protein-protein interactions have been widely applied for predicting protein attributes [15, 16, 17, 18, 19] . these algorithms suggest that interactive proteins are likely to share the common biological functions [16, 17, 18, 19] , also more likely tending to have the same biological function than non-interactive ones [20, 21] . likewise, we can assume that the interactive compounds may tend to share the common biological functions. in this study, the chemical-chemical interactions were retrieved from stitch [22] (search tool for interactions of chemicals), where the interaction unit consists of two chemicals and their interaction weight. the interaction weight (confidence score) represents the probability that the interaction occurs between the two chemicals concerned. the interactive compounds can be classified into the following three categories: (i) ones that participate in the same reactions; (ii) ones that share the similar structures or activities; (iii) ones with the literature associations [22] . in a metabolism system, chemical reactions are organized into many metabolic pathways, thus the compounds involved in the same reactions are in the same metabolic pathways. similar structures or activity means that they share the similar functions, and hence they are likely to be in the same metabolic pathways. the co-occurrence of two compounds in many literatures suggests some kinds of direct or indirect relationships, indicating they have the potential to be in the same metabolic pathways. accordingly, it is rational to suppose that the interactive compounds tend to participate in the same metabolic pathways. in this study, we proposed a multi-target model based on chemical-chemical interactions for predicting the metabolic pathways where compounds participate in. our method sorts the possible metabolic pathways that are associated with the query chemical, providing a more comprehensive view of the biological effects of the compound. according to a recent comprehensive review [23] , to establish a really useful statistical predictor for a biological system, we need to consider the following procedures: (1) construct or select a valid benchmark dataset to train and test the predictor; (2) formulate the statistical samples with an effective mathematical expression that can truly reflect their intrinsic correlation with the attribute to be predicted; (3) introduce or develop a powerful algorithm (or engine) to operate the prediction; (4) properly perform cross-validation tests to objectively evaluate the anticipated accuracy of the predictor. below, let us describe how to deal with these steps. the compounds were retrieved from public available database kegg [14] (kyoto encyclopedia of genes and genomes) compound [ftp://ftp.genome.jp/pub/kegg/release/archive/ kegg/42/ligand.tar.gz] (release 42.0). subsequently, these compounds were mapped to the following 11 major metabolic pathway classes that are strongly associated with the biological functions of compounds (http://www.genome.jp/kegg/pathway. table 1 under the title of group-i). from the 4,366 compounds of group-i, 3,137 compounds were retrieved that can interact with any of the others as annotated by stitch database [22] (see table 1 under the title of group-ii). of the 4,366 compounds of group-i, 4,027 are involved in only one metabolic pathway class, 246 in two metabolic pathway classes, 54 in three metabolic pathway classes, 24 in four metabolic pathway classes, 9 in five metabolic pathway classes, 4 in six metabolic pathway classes, 2 in seven metabolic pathway classes, and none in eight or more metabolic pathway classes. of the 3,137 compounds of group-ii, 2,820 are involved in only one metabolic pathway class, 226 in two metabolic pathway classes, 53 in three metabolic pathway classes, 23 in four metabolic pathway classes, 9 in five metabolic pathway classes, 4 in six metabolic pathway classes, 2 in seven metabolic pathway classes, and none in eight or more metabolic pathway classes. note that since one compound may occur in more than one pathway class, the sum of the compounds over the 11 pathway classes in group-i turns out to be 4,860, which is greater than 4,366. likewise, the sum of the compounds over the 11 pathway classes in group-ii is 3,606, which is greater than 3,137. this is quite similar to the case of proteins with multiple location sites, as elaborated in [24, 25] . the chemicals interactions were retrieved from stitch [22] , a large database of known and predicted interactions of chemicals and proteins derived from experiments, literature, databases, and so on. as mentioned in introduction, there are three types of associations between two compounds in stitch: (i) cooccurrence in reactions, (ii) similar structures or activities, and (iii) literature associations. in the downloaded stitch chemicals interactions file: chemical_chemical.links.detailed.v2.0.tsv from http://stitch.embl.de/cgi/show_download_page.pl, there are 337,482 pairs of interactive compounds belonging solely to type i, 73,598 pairs solely in type ii, 2,152,508 pairs solely in type iii, 384 pairs in both type i and ii, 120,936 pairs in both type i and iii, 10,372 pairs in both type ii and iii, and 1,990 pairs in the three types, in total of 2,697,270 interactions. each of the interaction is quantified by the interaction confidence score, which represents the likelihood that the interaction occurs. in this study, the interactions with both interactive compounds occurring in the 4,366 compounds of group-i were extracted. as a result, 3,137 compounds with 75,949 interactions were collected to constitute the benchmark dataset of the current study (see table 1 under the title of group-ii). besides the 4,366 compounds (cf. table 1 under the title of group-i) with known metabolic pathway classes, there are 11,661 compounds without known metabolic pathway classes in kegg. among these compounds, 5,549 compounds that have annotated interactions with the compounds of the 4,366 compounds in stitch were collected. such 5,549 compounds are to form an independent dataset, being used to test our prediction method in hopes to acquire useful information for further investigation. as mentioned in introduction, the interactive compounds tend to participate in the same metabolic pathways. accordingly, for a query compound, the higher interaction confidence score with its interactive compound, the more likely they are to participate in the same metabolic pathway. the more its interactive compounds involving in a certain metabolic pathway, the more likely it is to participate in such metabolic pathway. based on these points, we should count not only the number of compounds interacting with the query compound, but also the corresponding interaction scores. thus, the desired predictor can be formulated via the following procedures. suppose the training dataset contains n compounds, which are denoted as fc 1 ,c 2 ,:::,c n g. the 11 metabolic pathway classes (cf. table 1 ) are expressed as fp 1 ,p 2 ,:::,p 11 g, where p 1 represents the 1 st metabolic pathway class (''carbohydrate metabolism''), p 2 the 2 nd metabolic pathway class (''energy metabolism''), p 3 the 3 rd metabolic pathway class (''lipid metabolism''), and so forth. thus, the descriptor of metabolic pathway classes to which the compound c i belongs to can be formulated as p(c i )~½p i,1 ,p i,2 ,:::,p i,j ,:::,p i,11 t (i~1,2,:::,n; j~1,2,:: where given a query compound c q , its interaction with the compounds in the training dataset can be defined as w (c q )~½w q,1 ,w q,2 ,:::,w q,i ,:: where w q,i represents the interaction confidence score between c q and c i . t is the transpose operator, and w q,i~0 if no interaction exists between them. here, we did not consider the selfinteraction, therefore w q,i~0 when q~i. accordingly, the likelihood that the query compound c q is involved in the j-th metabolic pathway class can be formulated by the following score which is the sum of the interaction confidence scores of c q with its interactive compounds in the training dataset by counting both the number of interactive compounds and the interaction confidence scores. obviously, the higher the score of eq. 4, the more likely c q is to be involved in the j-th metabolic pathway c j . thus, for a given query compound c q , we can use eq. 4 to calculate its 11 scores, with each associated with one of the 11 metabolic pathway classes. the class to which the compound c q most likely belongs should be the one with the highest score. in other words, the query compound c q will be predicted to belong to the mth metabolic pathway class if m~arg max j s(c q [j)jj~1,2,:::,11 where m is the argument of j that maximize the value of s(c q [j). since the problem in this study is of multi-label classification, we intend to provide flexible information by predicting some candidate metabolic pathway classes for the query compounds, rather than just the most likely metabolic pathway class. therefore, instead of eq. 5, let us consider the following equation containing 11 scores in a one-column vector: where d ; is a descending operator that sorts the 11 scores of eq. 4 for s(c q [j) according to the descending order (s 1 §s 2 § á á á §s j § á á á §s 11 ). if there is a tie among these scores, a random order will be made among those with a tie. consequently, the predicted metabolic pathway classes for the query compound can be derived according to the descending order of eq. 6; i.e., if s 1~s (p k [6), s 2~s (p k [1), s 3~s (p k [10) , then it follows that the query compound c q is involved in the 6 th metabolic pathway class (''metabolism of other amino acids'') will be ranked as the highest in the likelihood, that c q in the 1 st metabolic pathway class (''carbohydrate metabolism'') as the 2 nd , and that c q in the 10 th metabolic pathway class (''biosynthesis of other secondary metabolites'') as the 3 rd . the corresponding results thus obtained are, respectively, called the 1 st -order, 2 nd -order, and 3 rd -order predicted metabolic pathway classes. and so forth. in statistical prediction, the following three cross-validation methods are often used to examine a predictor for its effectiveness in practical application: independent dataset test, subsampling (such as 5-fold, 7-fold, or 10-fold cross-validation) test, and jackknife test [26] . in this study, the 5-fold cross-validation was employed to examine the performance of our method. the concrete procedures were that the training dataset were divided into five groups by splitting each of its subsets into five approximately equal-sized subgroups. each of these five groups was in turn used as a testing dataset and the rest used as training dataset, thereby generating five different success rates, with their average representing the success rate by the 5-fold cross-validation. for the j-th order prediction, the accuracy w j was calculated by where m j is the number of the compounds whose j-th order predicted metabolic pathway class is one of the true pathway classes that the compounds are involved with, and n is the total number of compounds in the dataset. such 11-order accuracies were used to evaluate our prediction method. it is obvious according to the definition of eq. 7 that, the higher the value of w j with a smaller value of j, or the lower the value of w j with a larger value of j, the better the prediction quality will be by our method. in the dataset, the average number of metabolic pathway class that each compound is involved in is calculated as where e i is the number of metabolic pathway classes that the compound c i is involved with. hence, another measurement -the likelihood that the first k order predicted metabolic pathway classes cover all the true metabolic pathway classes that the compound is involved in -can be formulated as usually, k is the smallest integer equal or greater than the average number of metabolic pathway classes (h). it is obvious from eq. 9 that the larger the value of l k , the better the prediction quality will be by our method. given a query compound, according to the information of its interactions with the 4,366 compounds in group-i ( table 1 ) whose metabolic pathway classes are known, the likelihood of its belonging to each of the 11 metabolic pathway classes can be easily calculated according to eq. 4. and the scores thus obtained were sorted according to a descending order (eq. 6) to yield the predicted metabolic pathway classes according to their different ranks or orders. in this study, our method was evaluated by the 5-fold crossvalidation on the benchmark dataset that contains 3,137 compounds in group-ii of table 1 . the 11-order prediction accuracies are shown in figure 1 . the first order (most likely) prediction accuracy is 77.97%, and the last order (least likely) prediction accuracy is 0.38%, which indicates a quite good performance of our method. the average number of metabolic pathway classes with which each compound is involved is 1.15 (cf. eq. 8), meaning that the average success rate by a random guess would be 1.15/ 11 = 10.45%, which is much lower than that by our method. accordingly, the parameter k in eq. 9 was set to (1.15+1) = 2; i.e., we may select the results of the first two orders of the predicted metabolic pathway classes for the query compounds. as we can see from figure 1 , the accuracies of both the 1 st and 2 nd order predictions are higher than that of the random guess. according to eq. 9 the metabolic pathway classes predicted by the 1 st and 2 nd orders have actually covered more than 80% of all the true metabolic pathway classes, suggesting that, of the results predicted by the 11 orders, more attention should be paid to those by the first two orders. listed in table 2 are the accuracies by each of the 11 prediction orders for the 3,137 compounds about their involvement in the 11 metabolic pathway classes using the 5-fold crossvalidation test. the highest accuracy achieved by the 1 st -order prediction was 80.96% for the 1 st metabolic pathway class (''carbohydrate metabolism''). and the results obtained by the 1 st and 2 nd prediction orders have covered 89.00% of the true metabolic pathway classes. the second highest accuracy by the 1 storder prediction was 78.77% for the 11 th metabolic pathway class (xenobiotics biodegradation and metabolism), while the results obtained by the 1 st and 2 nd prediction orders have covered 87.00% of the true metabolic pathway classes. both the two 1 st -order accuracies are higher than the overall 1 st -order prediction accuracy of 77.97%, and each of their combinations with the 2 nd -order predictions is also higher than the overall likelihood of 80.00%. as for the metabolic pathway classes with less compounds, such as ''glycan biosynthesis and metabolism'' class that contains only 68 compounds in group-i and 43 in group-ii (cf . table 1) , the predicted accuracies were relatively not as good as the others. it is anticipated that with more experimental data are available in future for the compounds in these classes, the corresponding prediction success rates will be improved. overall speaking, the aforementioned results are quite encouraging, indicating that our approach may become a useful tool to deal with this kind of very complicated systems. as stated in the method section, the interactive compounds derived from stitch tend to participate in the same metabolic pathways. for example, table 3 lists the interactions of dihydrouracil with other compounds. among the 32 interactive compounds, most of them appear in ''metabolism of cofactors and vitamins'' or ''metabolism of other amino acids'' or ''nucleotide metabolism'' pathway class (cf. table 1 ) just like dihydrouracil. dihydrouracil and uracil participate in pyrimidine metabolism pathway (belong to ''nucleotide metabolism''), where 5,6-dihydrouracil and nadp+ are catalyzed by dihydropyrimidine dehydrogenase (dpd) to form uracil and nadph+h+ [14, 27] . they are also co-mentioned in many pubmed abstracts such as [28, 29, 30, 31, 32, 33, 34, 35, 36, 37] . another two interactive compounds -dihydrouracil and dihydrothymine share a very similar structure, the only difference is that dihydrothymine has a methyl at the 5th position of the hexatomic ring while dihydrouracil has not [38] . according to the prediction criteria, when dihydrouracil was treated as a query compound, the first three order predicted metabolic pathways that it participates in are ''nucleotide metabolism'', ''metabolism of cofactors and vitamins'' and ''metabolism of other amino acids'', respectively, which are consistent with the true metabolic pathways that it is involved in. predicted results for the compounds with unknown metabolic pathway encouraged by the quite promising results obtained by the 5fold cross-validation test on the benchmark dataset of the 3,137 compounds, we applied the method to the 5,549 compounds whose metabolic pathways are unknown as mentioned in the materials and methods section. the predicted results thus obtained are given in table s1 . as discussed above, we selected the metabolic pathway classes obtained by the 1 st and 2 nd order predictions for these compounds, in hoping that the information thus obtained may provide useful clues for further investigations. actually, it is interesting to see that many of our predicted results have proved to be reasonable according to the reports from other investigators. for example, n-acetylgalactosamine 4-sulfate and its interactive compounds with pathway information are shown in table 4 . n-acetylgalactosamine 4-sulfate can bind to sulfate, glucuronic acid, galactose, xylose, fucose, na(+), glycerol, and phosphate to form complex to perform the biological function [39] . in pubmed abstracts, n-acetylgalactosamine 4-sulfate is comentioned with sulfate [40] , glucuronic acid [41] , galactose [42] , 39-phospho.pho. [43] , sugar-1-phosph. [44] , udp-glcnac [45] , indole-3-glyce. [46] , n-acetyl-d-glucosamine [47] , and gdpmannose [44] . besides, n-acetylgalactosamine 4-sulfate and nacetyl-d-glucosamine share a similar structure and the difference is that n-acetylgalactosamine 4-sulfate has a sulfate at the position 4 of the ring while n-acetyl-d-glucosamine has not [38] . from these evidences, n-acetylgalactosamine 4-sulfate is supposed to participate in the same metabolic pathways as its interactive compounds. it can be seen from table 4 that most of the interactive compounds of n-acetylgalactosamine 4-sulfate belong to the 1 st and 2 nd metabolic pathway classes. by considering all the interactions and the interaction confidence scores, it was predicted that carbohydrate metabolism (the 1 st class) and energy metabolism (the 2 nd class) would be the possible metabolic pathway classes that n-acetylgalactosamine 4-sulfate belongs to. actually, as a carbohydrate, n-acetylgalactosamine 4-sulfate reacts with chondroitin 4-sulfate to form hydrogen oxide and g12336 (i.e. (galnac) 2 (glca) 1 (s) 2 ), one kind of glycan which can participate in carbohydrate and energy metabolism. therefore, n-acetylgalactosamine 4-sulfate may also participate in carbohydrate and energy metabolism. another example is that cyclopropylamine in table 4 has 23 interactive compounds with known pathway information. cyclopropylamine, cyanuric acid, ammonia, n-cyclopropylammelide, c0761, hydroxyl radicals are in the same pathway -n-cyclopropylmelamine degradation [48, 49] , where n-cyclopropylmelamine first reacts with hydrogen oxide to form n-cyclopropylammeline and ammonia, and then n-cyclopropylammeline also reacts with hydrogen oxide to form ncyclopropylammelide and ammonia. after that, n-cyclopropylammelide reacts with hydrogen oxide to form cyanuric acid, cyclopropylamine and hydroxyl radicals. finally, cyanuric acid is transformed into hydrogen oxide and ammonia through cyanurate degradation. cyanuric acid, n-cyclopropylammelide, and c0761 are all in the 11 th pathway class. therefore, cyclopropylamine may also belong to the 11 th pathway class (xenobiotics biodegradation and metabolism). for other interactive compounds, they are comentioned with cyclopropylamine in pubmed abstracts, such as polyethylene [50] , 1-aminocyclopropane-1-carboxylic acid [51] , cyclopropanecarboxylic acid [52] , 3-hydroxyphenylacetic acid [53] , and acetophenone [54] . in table 4 , most of the interactive compounds of cyclopropylamine belong to the 11 th metabolic pathway classes. according to above analysis, cyclopropylamine is suggested to participate in the xenobiotics biodegradation metabolism, which was the 1 st -order predicted class for cyclopropylamine by our method. accordingly, it is quite reasonable to expect that our method may provide useful information for further investigating into biological functions of compounds from the viewpoint of system biology. as indicated by the above discussion and analysis, the results derived from the 1 st and 2 nd order predictions should be considered as the candidates for the metabolic pathway classes with which the query compound may be involved. in view of this, biochemical experiments should be conducted by mainly focusing on the targets predicted by the 1 st and 2 nd order predictions. the results obtained by the last five order predictions can be ignored due to their very low likelihood (,2%). consequently, the current prediction method can provide useful clues for further validation by experiments and expedite the research progress by prioritizing the targets concerned. it is instructive to note that for the 4,366 compounds in group-i of table 1 , there are still 1,229 compounds that can not be processed by the current method due to lack of the interaction information with other compounds within the dataset. it is expected that the problem can be solved by collecting as much chemical-chemical interaction information as possible from stitch, which is a large-scale and well-maintained resource in chemical biology, including the interactions information for over 2.5 million proteins and over 74,000 small molecules in 630 organisms. with the continuous increase of the interactions information, the performance of our method will be further improved. based on the chemical-chemical interactions information, a multi-target model was proposed for identifying the metabolic pathway classes with which a query compound is involved. since some compounds may be involved with more than one metabolic pathway class, our method is featured by the capacity able to provide a series of potential metabolic pathway classes for each of the query compounds investigated, instead of only one metabolic pathway class. it is anticipated that our method may become a useful tool in helping annotate the compound for their biological functions. table s1 each order predicted metabolic pathway class for the collected 5,549 compounds without known metabolic pathway classes. the predicted metabolic pathway class code corresponds to the code in table 1 . among the 11 predicted pathway classes, the first 2 order predicted metabolic pathway classes should be paid more attention to. 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enzymes and the biocyc collection of pathway/genome databases bacterial degradation of ncyclopropylmelamine. the steps to ring cleavage elongation changes of exploratory and root hair systems induced by aminocyclopropane carboxylic acid and aminoethoxyvinylglycine affect nitrate uptake and bnnrt2.1 and bnnrt1.1 transporter gene expression in oilseed rape aminocyclopropane carboxylic acid synthase is a regulated step in ethylenedependent induced conifer defense. full-length cdna cloning of a multigene family, differential constitutive, and wound-and insect-induced expression, and cellular and subcellular localization in spruce and douglas fir stereocontrolled synthesis of 3-(trans-2-aminocyclopropyl)alanine, a key component of belactosin a cytochrome p450-catalyzed oxidation of nbenzyl-n-cyclopropylamine generates both cyclopropanone hydrate and 3-hydroxypropionaldehyde via hydrogen abstraction, not single electron transfer effect of alpha-methylation on inactivation of monoamine oxidase by n-cyclopropylbenzylamine the authors are very much indebted to the two anonymous reviewers for their constructive comments, which were very helpful for strengthening the presentation of this paper. many thanks are also to kegg and stitch for providing data to support the current study. key: cord-001199-9khx93c0 authors: liu, fengchen; enanoria, wayne t. a.; ray, kathryn j.; coffee, megan p.; gordon, aubree; aragón, tomás j.; yu, guowei; cowling, benjamin j.; porco, travis c. title: effect of the one-child policy on influenza transmission in china: a stochastic transmission model date: 2014-02-06 journal: plos one doi: 10.1371/journal.pone.0084961 sha: doc_id: 1199 cord_uid: 9khx93c0 background: china's one-child-per-couple policy, introduced in 1979, led to profound demographic changes for nearly a quarter of the world's population. several decades later, the consequences include decreased fertility rates, population aging, decreased household sizes, changes in family structure, and imbalanced sex ratios. the epidemiology of communicable diseases may have been affected by these changes since the transmission dynamics of infectious diseases depend on demographic characteristics of the population. of particular interest is influenza because china and southeast asia lie at the center of a global transmission network of influenza. moreover, changes in household structure may affect influenza transmission. is it possible that the pronounced demographic changes that have occurred in china have affected influenza transmission? methods and findings: to address this question, we developed a continuous-time, stochastic, individual-based simulation model for influenza transmission. with this model, we simulated 30 years of influenza transmission and compared influenza transmission rates in populations with and without the one-child policy control. we found that the average annual attack rate is reduced by 6.08% (sd 2.21%) in the presence of the one-child policy compared to a population in which no demographic changes occurred. there was no discernible difference in the secondary attack rate, −0.15% (sd 1.85%), between the populations with and without a one-child policy. we also forecasted influenza transmission over a ten-year time period in a population with a two-child policy under a hypothesis that a two-child-per-couple policy will be carried out in 2015, and found a negligible difference in the average annual attack rate compared to the population with the one-child policy. conclusions: this study found that the average annual attack rate is slightly lowered in a population with a one-child policy, which may have resulted from a decrease in household size and the proportion of children in the population. the one-child-per-couple policy in china was introduced in 1979 in an effort to raise living standards by slowing population growth. subsequently, the policy reduced fertility rates [1, 2] and household sizes, with only one dependent child found in most households. the total birth rate dropped from 2.90, before the policy was introduced, to 1.94 among women over 35 years of age, and to 1.73 among women under 35 years old in 2001. women's preferences for smaller families have changed (35% prefer one child and 57% prefer two children according to a study in 2001) [3] . the total fertility rate decreased from 2.9 in 1979 to 1.7 in 2004, with a rate of 1.3 in urban areas and less than 2.0 in rural areas. this trend has created a distinct demographic pattern for nearly a quarter of the world's population, resulting in chinese urban families with predominantly one child and rural families with predominantly two children [4] . the spread of infectious diseases may depend on demographic characteristics, environmental changes, consumption behaviors (eating, drinking, culinary culture, etc.), other behaviors (sexual contacts, drug use, hospital procedures, etc.), and host conditions (malnutrition, diabetes, immune status, etc.) [5] . while the one child policy has had economic, demographic, and sociological ramifications far beyond the scope of infectious disease transmission, it is important to understand the consequences for influenza dynamics, in part because china and southeast asia lie at the center of a global transmission network of influenza [6] . demographic changes may affect influenza transmission dynamics because children have an increased susceptibility due to lower immunity. moreover, increased viral shedding and longer infectious periods in children lead to more influenza among susceptible populations [7] . demographic characteristics have been incorporated into many modeling studies [8, 9, 10] to help understand the effects on transmission of influenza or the socioeconomic impact of mitigation strategies [11, 12] . household composition is an important determinant of the transmission of respiratory pathogens including influenza [13, 14, 15, 16, 17, 18, 19] and remains an important feature of recent transmission models [20, 21, 22, 23, 24, 25] . this paper presents a study focusing on the indirect effects of demographic changes on influenza transmission. we developed a continuous-time, event-driven, individual-based stochastic simulation model for influenza transmission in a dynamic population. we used this model to simulate transmission while assuming different demographic control policies: the one-child policy, the absence of any control policy, and a strict one-child policy. the strict onechild policy was introduced to compare influenza transmission rates with a hypothetical one-child policy to rates with an actual one-child policy, since two or more children are often allowed in rural areas and for ethnic minorities [4] ; the existing census data do not reflect the effects of truly restricting families to one child. the model was used to simulate 30 years of influenza transmission in a dynamic population as follows: (1) we initialized the population using 1975 demographic data (four years before a one-child policy was fully launched in china); (2) we calibrated the population projections by fitting the simulated population with the one-child policy to the census and compared the simulated population without the one-child policy with projections from previous literature [26, 27] in which population growth was predicted under different demographic control policies; (3) we calibrated the influenza-specific parameters by fitting the annual attack rate and secondary attack rate from the reported literature [7, 28, 29, 30, 31, 32, 33] ; and (4) we compared the simulated annual attack rate and secondary attack rate in simulated populations with and without the one-child policy. in the plenary sessions of the 2011 chinese people's political consultative conference and the national people's congress, a two-child policy was proposed to start as early as 2015 [34] . experts suggested that the one-child policy may threaten china's economic growth due to the increase in the number of older people, a decrease in the number of younger workers, as well as a sex-ratio imbalance [34] . because a two-child policy was proposed to start as early as 2015 [34] , we also forecasted influenza transmission over a ten-year period (2015 to 2024) in a population with a two-child policy. our model has three main features: (1) influenza transmission, (2) population demographics, and (3) dynamic network structure. we used a susceptible-exposed-infectious-recovered (seir) model which included waning immunity and seasonality of influenza transmission. we used census data [35, 36, 37, 38, 39] (table 1) from china, to construct a population with demographic changes under a one-child policy. a simple dynamic network structure was used to group people with household links, school links and social links, allowing influenza to be transmitted along these links in the network while changing the state of each individual (s, e, i and r). the model structure is described in the section titled model structure (and in the text s1 and figure s1 ). influenza transmission parameters were calibrated using approximate bayesian computation (abc) [40, 41] as described in the calibration section (we chose parameters' ranges based on both the english and chinese literature [42] ). the computation section briefly discusses the implementation and computations based on calibrated parameters (table 2) ; a more detailed description can be found in the text s1. natural history of influenza. individuals infected with the influenza virus first pass through a latent period when they are asymptomatic and not infectious. we assumed that viral shedding does not take place during the latent period, and that the mean duration of the latent period is 1 to 2 days [43, 44, 45] . for influenza, the infectious period is assumed to begin about one day before the symptomatic period [44] . in general, individuals infected with influenza may be asymptomatic, and yet still shed the virus. the proportion of transmission by asymptomatic individuals is assumed to be one-third to one-half that of influenza-infected symptomatic individuals [46, 47, 48, 49] . the mean period during which a person may be asymptomatic but infectious is assumed to be 1 day [50] . individuals are assumed to become symptomatic and infectious with an average duration of 1.5 to 3.8 days [43, 44, 45, 51, 52] . mathematically, we represent the course of influenza according to the diagram shown in figure 1 (and figure s2 ). in this model, we classified influenza as being mild or not being mild; individuals in each severity type progress to different stages. mild cases and non-mild cases are classified as infected prior to all symptoms and infectiousness (e 0 1 and e 1 ), infectious but asymptomatic (i 0 1 and i 1 ), or recovered with strain specific immunity (r). the non-mild cases may be symptomatic and infectious as well (i 2 ), which occurs after asymptomatic infectiousness (i 1 ). table 2 lists the durations between stages. a recovered individual loses immunity with rate m, reverting to the uninfected susceptible (s) stage. we assume that individuals have age-specific death rate d, and a birth rate b; these dynamic population demographic features which are represented by the death and birth of each individual, will be described in the demographic description section given below. for a specific individual, we assume that the duration time between two stages is randomly chosen from an exponential distribution with a given rate. immune escape and seasonality. to model antigenic drift, our model is designed such that every individual has a maximum immunity level immediately following recovery from infection by a particular strain of influenza, but this immunity gradually wanes to zero over 3 to 8 years [28, 50] . following reinfection, the immunity level is restored to the maximum value and declines at the same rate thereafter. the underlying causes of influenza seasonality remain unclear [53, 54, 55] , despite many studies postulating possible causes. suggested causes have included changes in human mixing patterns or fluctuations in human immunity and environmental humidity [56] . the transmission of seasonal influenza tends to increase substantially from november to february in the northern hemisphere and from may to august in the southern hemisphere [57] . to incorporate seasonality of influenza transmission into our model, we modeled the transmission probability per contact as a sinusoidal function of time [57] according to p trans (t)~p base ze cos(2p(1z t{h d )), so that the transmission probability, p trans , varies during the course of the epidemic. here, p base is the baseline transmission probability, t is time, and e (where we assume 2p base ,e,p base ) characterizes the degree of seasonality (e = 0 corresponds to no seasonal variation at all). we let d denote the total duration of an epidemic season (for instance, 365 days in this model) and h (an offset from time 0) is the peak time of an epidemic season. our model adopted h as november 15, corresponding to northern china where influenza peaks in the winter [58] . in this model, the probability of infection for each individual depends on the immunity level, seasonality, and the contact rates (please see the text s1 for more details). china's demographic data. the demographic data were taken from the population statistics yearbooks for china, and from five censuses carried out in 1952, 1964, 1982, 1990 and 2000 [35,36,37,38,39] . some demographic data sources were extracted from previous articles [26, 27, 39, 59, 60] in which the population growth under different population control policies were predicted. key demographic parameters used in our simulation included age, household size, age-specific death rates, and age-specific fertility rates, as shown in table 1 . initially, we stochastically sampled age and household size from distributions fitted to the demographic data [26, 39, 60] . we used dynamic age-specific fertility rates and death rates from year 1975 to 2009 to simulate the population growth under conditions of the one-child policy; calibration details of the age-specific fertility rates can be found in text. population projections without the control of a one-child policy were implemented by assuming a static age-specific fertility rate (from 1975) and fixing the birth rate to the same value that it was in 1975 (which, of course, corresponds to an unrealistic population trajectory). we also analyzed the assumption of a very strict onechild policy that allows one female to have only one child in her life-this is stricter than the one-child policy as actually implemented. finally, we conducted a simple projection of the population with a proposed two-child policy (from 2015 to 2024), which allows one female to have two children. it was implemented by increasing the fertility rate for nulliparous females. to calibrate the population, we fit the age-specific population number of each year and the average household size of each year to the census data, then compared the population projections of our model with the census data and projections described in other studies [26, 39, 60] . dynamic network structure. we simulated the transmission of influenza using a simple dynamic network structure shown in figure 2 . specifically, we assumed that each individual is located in a household and links to other household members, and we assumed that each individual has several links to other individuals outside of his/her household. these links outside the household represent contacts in the community and an individual has a lower relative contact rate with outside links than with household links. for school-aged individuals, we assume that they are in primary and middle schools, and have school links to all of their schoolmates. the contact network of this model consists of each individual's household contacts, school contacts and casual contacts, and its dynamic is reflected by updating each individual's household, school and casual contacts which will be discussed in turn. household contacts. each individual in the model has household links that are initialized by grouping individuals into households based on the household size distribution data of china in 1975, and linking all household members of each household. during simulation, each individual's household links are updated dynamically (1) when the individual leaves his/her household between his/her age 14 and 18 years as a household with onemember, (2) when the single individual over 18 years of age has found (with a partnership searching rate per year) another single over 18 years of age to live with as a two-member household, (3) at the time the individual dies (with a dynamic age-dependent . individual b has two household members (c and d), two visible casual contacts (a and e), and three visible schoolmates (f, i and j), other social contacts and schoolmates of b are not shown in this small part of contact network. if b was an index case, the household contacts would be at highest risk of being infected due to the higher contact rates among household members than the casual and school contacts (for the contact rates of different link types, please see table 2 ). doi:10.1371/journal.pone.0084961.g002 effect of one-child policy on flu transmission plos one | www.plosone.org mortality rate), or (4) at the time the individual or one of the other family members gives birth to a baby (with a dynamic agedependent fertility rate). the dynamic age-dependent mortality rate and the dynamic age-dependent fertility rate are from the population data of china from 1975 to 2009. during the simulation, an individual's mortality rate and fertility rate depend on the current simulated year and the individual's current age. the partnership searching rate per year is calibrated to fit to the observed household size from 1975 to 2009. the dynamic agedependent fertility rates under the other three scenarios are assumed to be zero if the individual already has more than one child for the strict-one-child-policy, the same as the fertility rates in 1975 for the absence of one-child policy, and doubled from 2015 to 2024 for the two-child-policy. school contacts. each individual whose age is between the primary-school-age of 6 and 12 years or between the middleschool-age of 13 and 18 years has school links that are initialized using the primary and middle schools' statistical data of gansu province in china in 1975, and are updated annually by reassigning all individuals with school ages into primary or middle schools according to year-dependent average school size from 1976 to 2009, or are updated at the time the individual dies with the dynamic age-dependent mortality rate. casual contacts. each individual may have several random contacts per day with a daily contact rate contact casual = 16. once an individual becomes infectious, all of his/her casual contacts during the infectious period are randomly chosen from the population and their contacting times are predicted and scheduled using an exponential distribution with the casual contact rate per day, contact casual . transmission via the network. once an individual becomes infectious, an infectious period will be generated using an exponential distribution with recovery rate. during the individual's infectious period, the contact times between he/she and each of his/her household members are stochastically scheduled using an exponential distribution with the contact rate per household member per day, contact house = 10; transmission between the infectious individual and the susceptible household contacts will take place at the scheduled contact times. similarly, the casual contacts of the infectious individual during the infectious period are randomly chosen from the entire population, and the contact times between the infectious individual and his/ her casual contacts are scheduled using an exponential distribution with a casual contact rate per day, contact casual = 16. transmission between the infectious individuals and the susceptible casual contacts will be active at the scheduled times. in addition, the contacts between the infectious individual and his/her schoolmates during his/her infectious period are randomly picked from the individual's school links and are scheduled by an exponential distribution with school contact rate per day, contact school = 10. the transmission between the infectious individual and the susceptible school contacts will be active at the scheduled times. once a scheduled transmissible contact takes place between the infectious individual and one of his/her susceptible household members, schoolmates, or casual contacts, a successful transmission will be completed with a transmission opportunity which is a product of the seasonal transmission probability per contact, p trans , and the chance of immune escape, 1 -m i (t), where m i (t) (defined in the text s1) is a dynamic immunity level of a susceptible individual i at time t. the dynamic immunity level of an individual depends on his/her infection history, the immunity waning rate per year and the current time. the model is initialized with 10,000 individuals whose ages are generated from the age distribution of china in 1975. the household links for each individual are initialized with household size distribution of china in 1975, and the school links for each school age individual are initialized with the average school size of gansu province in china in 1975. casual contacts of each individual are randomly selected from the population with a casual contact rate per day contact casual = 16. five exogenous infectious cases with the same influenza strain are introduced into the population on november 15th in 1975 to start influenza transmissions via the contact networks of all individuals. at the beginning of the simulation, we assume that all individuals are completely susceptible. once an individual recovered from an infection, he/she will have a 100% immunity level which wanes with 10% immunity loss rate per year (m = 0.1). the demographydependent dynamic network of the population is reflected by updating household links and school links of each individual as stated above, which also depends on the scenario of population control policy for the current simulation. as a base scenario, we assume that the one-child policy is active, thus the mortality rates for ages 0 to 120 years and the fertility rates for ages 16 existing census data reflect those demographic changes caused by the one-child policy as actually implemented. in order to assess what would have occurred in the absence of such a policy or other demographic changes, we assumed a static fertility rate of that in 1975 for females. however, a strict one-child policy includes the assumption that there is no chance for a female who already has a child to give birth to a second child, an assumption that does not hold in practice. to calibrate the demographic component of our model, we first fitted the population projection with the available demographic data, as well as with other population projections [26, 27] in which they predicted population with a one-child policy and other control measures (see figure s3 (a)). then, we fitted the population age distribution of each year to demographic data in the years from 1975 to 2004, (see figure s3 (b)). finally, we required that the average household size (see figure s3 (c)) corresponded to the census data in 1964, 1982, 1990 and 2000, which reported average household sizes of 4.43, 4.42, 3.96 and 3.44 in these years, respectively [35] . we calibrated the model using eight influenza transmission parameters: (1) mean duration of the latent period, (2) mean duration of the asymptomatic infectious period, (3) mean duration of the symptomatic infectious period, (4) probability that a case will be mild, (5) immunity waning rate, (6) the degree of immunity following infection, (7) transmission probability per contact, and (8) contact rate between two household members ( table 2) . parameters (1), (2), (3) and (4) are age-dependent parameters with 5 age categories: 0 to 4, 5 to 9, 10 to 25, 26 to 49, and 50+ years. to calibrate these parameters, we chose parameter sets randomly from a uniform distribution with given upper and lower bounds (assuming independence among parameters). the annual attack rate (averaged over 30 years) and the simulated household secondary attack rate (averaged over 30 years, and the rate of each year was averaged over all households with index cases) were computed from each set of parameters. simulations yielding average annual attack rate (ar) within the range (0.1, 0.2) [28, 29, 30, 31] , and secondary attack rate (sar) inside the range (0.09,0.32) [7, 32, 33, 61, 62, 63] , were considered plausible; calibration was done by approximate bayesian computation [40, 41] . for details of the ar and sar we cited, please see tables s1 and s2. for each household with an index case, we calculated the secondary attack rate based on the proportion of household contacts who were infected by the index case in the household during the infectious period of the index case [62, 64] . the sar was averaged by using the secondary attack rates of all households with index cases. this calculation of the sar includes partially immune household contacts [7, 32, 33, 61, 62, 63] . simulations were run for 4000 sets of parameters, resulting in 646 parameter sets that fit the acceptable ar and sar ranges stated above. parameter sets having higher or lower values of ar or sar were excluded. finally, we used the 646 fitted (non-excluded) parameter sets and used them in the model to predict and study influenza transmission in the population under three scenarios: the one-child policy, the absence of a one-child policy, and the strict one-child policy. the individual-based model was implemented and programmed in c++ [65] and r [66] following our previously published agent-based transmission models [67, 68] . c++ was used for the main simulation program and r for the analysis of data generated by the main simulation program. to add scalability for simulations of large population sizes, we used an agent-based platform abm++ [69] which supports parallel and cluster computing. simulations were performed on the rti midas cluster, a cluster with 36 compute nodes with a total of 400 compute cores and 786 gb of distributed memory, running linux distribution of centos v5.5. the running time for a single run of the model varied with input parameters in tables 1 and 2 . given a fitted set of parameters with the one-child policy and an initial population size of 10000, it took about 500 to 800 seconds for a single run on one compute core with a speed of 2.30 ghz in the cluster. we simulated 30 years of influenza transmission in a representative population of initial size 10000 under three different scenarios: a population with a one-child policy (), a population without a one-child policy (), and a population with a strict onechild policy (), (following ''one-child policy'' represents ). under each of the scenarios, we used 646 fitted sets of parameters (described in the calibration section) to simulate influenza transmission. each scenario was simulated 100 times and the annual and secondary attack rates were averaged among 100 simulated ars and sars. we then computed the partial rank correlation coefficients (prcc) [67, 70] for each input parameter and the annual attack rate under the three different policy scenarios using the 646 sets. when the prcc is close to zero, the value of the parameter has little relation to the simulation output (see the text s1). the prcc values of key parameters are listed in table 3 . finally, we calculated the annual and secondary attack rates experienced by the population under the three policy scenarios. to explore the influenza transmission factors that are likely affected by the one-child policy, we estimated the average differences in the annual attack rate (dar) and the secondary attack rates (dsar) in the populations without and with the onechild policy control. we found that the population without the . ar and sar differences between populations without the one-child policy and with the one-child policy. (a) average difference in annual attack rate (dar: 6.08% (sd 2.21%)) between populations without the one-child policy and with the one-child policy, based on 646 calibrated parameter sets which yielded the annual attack rates between 10% and 20%, and secondary attack rates between 9% and 32%. for each parameter set, we simulated the influenza trajectories under two demographic control policies, and then computed the difference in average annual attack rates over 30 years between two policies. (b) difference in secondary attack rates (dsar: 20.15% (sd 1.85%)) between populations without one-child policy and with the child-policy, based on 646 calibrated parameter sets which yielded the annual attack rates between 10% and 20%, and the secondary attack rates between 9% and 32%. for each parameter set, we simulated the influenza trajectories under two demographic control policies, and then computed the difference in average secondary attack rates over 30 years between two policies. doi:10.1371/journal.pone.0084961.g003 one-child policy had an average annual attack rate that was slightly higher than the population with the one-child policy. the distribution of the difference of annual attack rates with a mean of 6.08% per year (with standard deviation (sd) 2.21%) using 646 fitted sets of parameters, in figure 3(a) , shows that all the values reflecting the dars between population without one-child policy and population with one-child policy are positive for all sets of parameters. here, each value of dar is the difference of the average annual attack rates over 30 years between two different policies. this supports the notion that the one-child policy gradually reduced the annual attack rate. the decrease in annual attack rates may be caused by the smaller household sizes and the decreased proportion of children in the population resulting from the one-child policy. the distribution of dsar, in figure 3 (b), shows that the expectation of the dsar is 20.15% per household per year (sd 1.85%) and there is no significant difference of secondary attack rates with the one-child policy introduced. however, the one-child policy had little to no discernible effect on the secondary attack rates. a larger population size gave similar results as stated above. we performed the same comparisons of the dar and dsar, comparing populations with the existing one-child policy with a hypothetical two-child policy. we assumed the two-child policy from 2015 to 2024; the simulations for a 10-year transmission period ( figure 4 ) did not show significant differences of dar and dsar (0.22% per year (sd 0.46%) and 20.02% per household per year (sd 0.81%), respectively). in addition, we conducted sensitivity analyses by increasing the contact rate per day within household and the immunity loss rate per year and varying their values from 12 to 20 for the contact rate and from 20% to 100% for the immunity loss rate in order to compare the difference in ar and the difference in sar between populations without and with the one-child policy ( figures 5 (a) and (b)). changes in household structure and the proportion of children in the population as a result of the one-child policy could have more effects on the ar, and the difference in ar could be as high as 60% under a scenario of very high immunity loss rate per year ( figures 5(a) ). however, the results showed that the difference in sar was not very sensitive to the contact rate in the household and the immunity loss rate (figures 5(b) ). the one-child policy has been applied in china for over 30 years, causing great changes in the demographic composition of the chinese population. to address the impact of demographic changes caused by the one-child policy (or similar changes which may have arisen for other reasons) on influenza transmission, we developed a continuous-time individual-based, stochastic, simulation model for influenza transmission in dynamic populations with the support of available demographic data. after calibrating the simulated population with available demographic data and published attack rates, we simulated 30 years of influenza transmission under three assumptions: a population with a onechild policy, a population without a one-child policy, and a population with a strict one-child policy. this study provides some evidence that demographic changes caused by demographic policy may slightly affect influenza transmission in populations. simulated results from this model show that populations without childbearing policies have slightly higher annual attack rates than populations with a one-child policy. we did not find significant differences in the secondary attack rates between populations with a one-child policy and populations without it. we predicted influenza transmission over 10 years (2015 to 2024) in a population with a hypothetical two-child policy, and found negligible differences of the average annual attack rates and secondary attack rates compared to the population with a onechild policy. one limitation of our findings is that it is impossible to know what would have happened in the absence of the one-child policy. because our goal was to highlight the role of household size and other related demographic changes, we simply assumed an extrapolation from 1970s trends. in reality, demographic changes may have occurred for other reasons in the absence of a one-child policy. moreover, this model did not distinguish contacts other than household and school (for example workplace [71, 72, 73] , or community [74] ). containment measures, such as different vaccine strategies [75, 76] and travel restrictions [77, 78] , were not considered in this model, allowing for a focus on the relationship between child policies and influenza transmission. we did not distinguish antigenic diversity [79] ; because aging populations have more cross-immunity for similar strains [8] . this limitation may underestimate an aging effect on influenza transmission. all parameters used in this model were defined from existing published literature. we did not assess the differences between pandemic years versus inter-pandemic years because of the assumption that there are no changes in influenza natural history parameters during the course of over 30 years. we did not use this model to answer an important question that whether or not the demographic changes affect pathogen emergence in china because of lacking sufficient data, and this question is beyond the scope of this paper. this study found that the average annual attack rate is slightly lower in a population with a one-child policy, which may result from a decreased household size (from 4.2 in 1979 to 3.5 in 2004 in the model) and the decreased proportion of children (who are more vulnerable to infection than adults) in the population because of the dramatically reduced fertility rates from 2.9 in 1979 to 1.3 in 2004. however there is no discernible difference in the sar. a possible reason for the absence of a discernible difference is that the decrease of average household size (from 4.2 to 3.5) might not be large and fast enough to obviously reflect the change in the secondary attack rate. we compared the results of this study with other recent studies [61, 80, 81, 82, 83] about the relation between household size and sar, household size and the overall attack rate. the lower annual attack rate with smaller household size is consistent with the results from fraser et al. [61] and kwok et al. [83] , but carcione et al. [81] found that individual risk was not associated with the household size. the absence of a discernible difference in the sar observed in this study is similar to the findings in [80] in which the sar remained stable as household size increased, while the sar increased with larger household size . ar and sar differences between one-child policy and two-child policy (10 years: 2015 to 2024). (a) dar (0.22% (sd 0.46%)) between one-child and two-child policies based on 646 calibrated parameter sets which yielded the annual attack rates between 10% and 20% and the secondary attack rates between 9% and 32%. for each parameter set, we simulated the influenza trajectories under two demographic control policies, and then computed the difference in average annual attack rates over 10 years (2015 to 2024) between two policies. (b) dsar (20.02% (sd 0.81%)) between one-child and two-child policies based on 646 calibrated parameter sets which yielded the annual attack rates between 10% and 20% and the secondary attack rates between 9% and 32%. for each parameter set, we simulated the influenza trajectories under two demographic control policies, and then computed the difference in average secondary attack rates over 10 years (2015 to 2024) between two policies. doi:10.1371/journal.pone.0084961.g004 in other studies [61, 82, 83] . the above comparisons included some studies in which the sar was measured empirically, though the relation between the simulated sar and household size may be controlled by the model structure. in this model, the sar was estimated by the proportion of household contacts of an index case who subsequently became infected [62, 64] , so that the simulated sar stands in relation to the simulated epidemic, which is in the same way the real-world empirical sar and its relation to the true unobserved epidemic. author contributions figure 5 . ar and sar differences under assumptions of different contact and immunity loss rates. (a) varying the value of contact rate per day between any two members in a household (from 12 to 20) and the value of immunity loss rate per year (from 20% to 100%) yielded that under the scenario of 12 of household contact rate and 100% of immunity loss per year, the ar in the population without the one-child policy could be 60% higher than the ar in the population with the one-child policy. (b) by varying the values of contact rate per day between any two members in a household (from 12 to 20) and the immunity loss rate per year (from 20% to 100%), the sar in the population without one-child policy could be 3% higher than the sar in the population without the one-child policy, when the contact rate per day in household is 12 and the immunity loss rate per year is 80%. doi:10.1371/journal.pone.0084961.g005 has china outgrown the one-child policy? demography. of population projections and projectiles family size, fertility preferences, and sex ratio in china in the era of the one child family policy: results from national family planning and reproductive health survey the effect of china's one-child family policy after 25 years environmental and social influences on emerging infectious diseases: past, present and future global migration dynamics underlie evolution and persistence of human influenza a (h3n2) risk factors of influenza transmission in households the shifting 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the c++ programming language software for data analysis: programming with r logistics of community smallpox control through contact tracing and ring vaccination: a stochastic network model costeffectiveness of tuberculosis evaluation and treatment of newly-arrived immigrants sensitivity and uncertainty analysis of complex-models of disease transmission -an hiv model, as an example school opening dates predict pandemic influenza a(h1n1) outbreaks in the united states school closure and mitigation of pandemic (h1n1) 2009, hong kong would school closure for the 2009 h1n1 influenza epidemic have been worth the cost?: a computational simulation of pennsylvania epidemic growth rate and household reproduction number in communities of households, schools and workplaces optimal h1n1 vaccination strategies based on self-interest versus group interest modeling targeted layered containment of an influenza pandemic in the united states human mobility networks, travel restrictions, and the global spread of 2009 h1n1 pandemic controlling pandemic flu: the value of international air travel restrictions predicting the epidemic sizes of influenza a/h1n1, a/h3n2, and b: a statistical method avian influenza h5n1 transmission in households secondary attack rate of pandemic influenza a(h1n1) 2009 in western australian households influenza transmission in a community during a seasonal influenza a(h3n2) outbreak (2010-2011) in mongolia: a community-based prospective cohort study modelling the proportion of influenza infections within households during pandemic and non-pandemic years serial intervals and the temporal distribution of secondary infections within households of 2009 pandemic influenza a (h1n1): implications for influenza control recommendations the transmissibility and control of pandemic influenza a (h1n1) virus key: cord-001117-llb4f74a authors: ji, wen-jie; ma, yong-qiang; zhou, xin; zhang, yi-dan; lu, rui-yi; guo, zhao-zeng; sun, hai-ying; hu, dao-chuan; yang, guo-hong; li, yu-ming; wei, lu-qing title: spironolactone attenuates bleomycin-induced pulmonary injury partially via modulating mononuclear phagocyte phenotype switching in circulating and alveolar compartments date: 2013-11-19 journal: plos one doi: 10.1371/journal.pone.0081090 sha: doc_id: 1117 cord_uid: llb4f74a background: recent experimental studies provide evidence indicating that manipulation of the mononuclear phagocyte phenotype could be a feasible approach to alter the severity and persistence of pulmonary injury and fibrosis. mineralocorticoid receptor (mr) has been reported as a target to regulate macrophage polarization. the present work was designed to investigate the therapeutic potential of mr antagonism in bleomycin-induced acute lung injury and fibrosis. methodology/principal findings: we first demonstrated the expression of mr in magnetic bead-purified ly6g-/cd11b+ circulating monocytes and in alveolar macrophages harvested in bronchoalveolar lavage fluid (balf) from c57bl/6 mice. then, a pharmacological intervention study using spironolactone (20mg/kg/day by oral gavage) revealed that mr antagonism led to decreased inflammatory cell infiltration, cytokine production (downregulated monocyte chemoattractant protein-1, transforming growth factor β1, and interleukin-1β at mrna and protein levels) and collagen deposition (decreased lung total hydroxyproline content and collagen positive area by masson’ trichrome staining) in bleomycin treated (2.5mg/kg, via oropharyngeal instillation) male c57bl/6 mice. moreover, serial flow cytometry analysis in blood, balf and enzymatically digested lung tissue, revealed that spironolactone could partially inhibit bleomycin-induced circulating ly6c(hi) monocyte expansion, and reduce alternative activation (f4/80+cd11c+cd206+) of mononuclear phagocyte in alveoli, whereas the phenotype of interstitial macrophage (f4/80+cd11c-) remained unaffected by spironolactone during investigation. conclusions/significance: the present work provides the experimental evidence that spironolactone could attenuate bleomycin-induced acute pulmonary injury and fibrosis, partially via inhibition of mr-mediated circulating monocyte and alveolar macrophage phenotype switching. idiopathic pulmonary fibrosis (ipf) is a chronic, progressive, interstitial fibrotic lung disease characterized by chronic lung inflammation, disruption of alveolar structure, interstitial fibroblast proliferation, and excessive extracellular matrix synthesis and deposition [1] [2] [3] . although evidence showed that the persistent inflammatory response is associated with progressive development of ipf, therapies currently used for ipf, namely anti-inflammatory or immunosuppressive drugs, are largely ineffective [4] . therefore, novel therapies capable of targeting inflammation without compromising body's immunity can still be a challenge in this area. macrophages in lung tissue play an important role in the clearance of pulmonary pathogens and steady-state homeostasis maintenance. emerging evidence suggests that there is a causal link between lung macrophage mediated inflammation and excessive tissue destruction elicited by variety of exogenous stimuli, i.e., silica and asbestos exposure, virus infection, etc., which will ultimately lead to a failure of inflammation resolution, a key feature that progressively promotes the development of lung fibrosis [5] [6] [7] [8] . on the other hand, macrophages are a cell population with high plasticity, and display functional diversity during different stage of inflammatory response [9, 10] . the activation state of macrophage can be generally characterized as classical activation (m1 polarization) that is associated with a th1 immune response, or alternative activation (m2 polarization) that is associated with th2 immune response [11] . in lung tissue, m1-like macrophages are the first line defense in acute lung injury and are later replaced by m2-like macrophages that contribute to tissue repair and fibrosis. it is generally believed during inflammation, myeloid ly6c hi monocytes contribute to lung macrophage replenishment [9, 12] . the results from recent basic studies indicate that manipulation of macrophage phenotype switch might be a potential target for many macrophage mediated disorders [13] [14] [15] . recently, usher and colleagues demonstrated that macrophages from mice lacking myeloid mineralocorticoid receptor (mr), exhibit a transcription profile that mimic alternatively activated macrophages, and are protected against angiotensin ii (angii) induced cardiac hypertrophy and fibrosis [16] . this work provides evidence indicating that mr in mononuclear phagocytes might be a potential target for therapeutic purpose. based on current evidence, we speculated that pharmacological inhibition of mr with clinically approved drug, may regulate lung macrophage phenotype switching, as well as their progenitors, bone marrow-derived circulating monocytes, and may confer novel therapeutic potential in a murine model of bleomycin-induced acute pulmonary injury and fibrosis. eight to ten weeks male c57bl/6 mice, weighing 16 to validate the mrna expression of mr in mouse circulating monocytes, circulating monocytes from c57bl/6 mice were purified from peripheral blood using a magnetic bead-based kit (easysep tm mouse monocyte enrichment kit, cat no. 19761, stemcell technologies, vancouver, bc, canada). the purity of enriched monocytes was confirmed by flow cytometry (see below). detailed methods for total rna isolation, reverse transcription, and real-time pcr analysis are shown below. to validate the protein expression of mr in circulating monocytes and alveolar macrophages, the purified monocytes and cells from bronchoalveolar lavage fluid (balf) were seeded on glass slides for immunohistological detection of mr. briefly, the cells were fixed with methanol, followed by permeabilization with 0.1% triton x-100. then, the cells were incubated with the primary anti-mouse mineralocorticoid receptor monoclonal antibody (1:200, ab41912, abcam, cambridge, ma, usa) at 4°c overnight. to ensure specificity, isotype control (igg2a) was prepared. for alveolar macrophages, the cells were further incubated with the primary anti-mouse f4/80 antibody (1:200, ab6640, abcam) at 37°c for 2 h. after washing with 0.01 m pbs, the cells were incubated with tetramethylrhodamine isothiocyanate (tritc)-conjugated goat anti-mouse secondary antibody [for alveolar macrophage, fluorescein isothiocyanate (fitc)-conjugated goat anti-rat secondary antibody was also added] in dark. then, cell nuclei were stained by 4,6-diamidino-2-phenylindole (dapi, sigma-aldrich, st. louis. mo, usa) with light protection. images were visualized by a fluorescence microscope (eclipse 80i, nikon, tokyo, japan). the unstained samples and samples stained with the secondary antibody without incubation with primary antibodies were used as negative controls and showed no signal during analysis. to induce pulmonary fibrosis, mice were lightly anesthetized by inhalation of ether. bleomycin a5 (2.5mg/kg body weight in 40μl saline) or saline was administered by oropharyngeal instillation as described previously [17] . animals were then randomly allocated into four treatment groups: 1) 0.9% normal saline (ns) only; 2) bleomycin (blm) only; 3) bleomycin plus 0.9% normal saline (blm+ns); 4) bleomycin plus 20mg/kg of spironolactone (blm+sp). from the day of the administration (day 0), vehicle (0.9% saline), sp (dissolved in 0.9% saline) were delivered by oral gavage once daily, and continued for 21 days. at 1, 3, 7, 14 or 21 days, animals were sacrificed by exsanguinations under sodium pentobarbital anesthesia (10 mice each time point). blood, balf and lung tissues were collected for the following assays. the balf was collected through an intratracheal cannula with three sequential 1 ml of 0.9% sterile saline and centrifuged at 300 g for 10 min at 4°c. the cell-free supernatant was stored at -80°c for analysis of cytokines. the cell pellet was resuspended in sterile 0.9% saline for total cell counts, differential cell counts, immunohistochemical staining, and flow cytometry analysis. the left lung (from which no balf was harvested) was fixed in 4% paraformaldehyde solution for 24h. after embedding in paraffin, 5 μm sections were prepared and stained with hematoxylin-eosin or masson's trichrome, and examined on a light microscope (e600pol, nikon, tokyo, japan). for detection of myofibroblasts, α smooth muscle actin (α-sma, 1:600, a2547, sigma-aldrich, st. louis. mo, usa) immunofluorescent staining was carried out as previously described [18] . for the evaluation of inflammatory response induced by bleomycin, semi-quantitative scoring criteria by szapiel and coworkers were used in a blinded fashion [19] . fibrosis and collagen was determined from 10 non-overlapping fields by using digital quantitative analysis (image pro plus software version 4.5, media cybernetics, silver spring, md, usa). the lung fibrosis index was defined as the sum of the total area of collagen in the entire visual field divided by the sum of total connective tissue area in the entire visual field. the collagen content in the whole left lung was determined by analysis of hydroxyproline as previously described [20] . in brief, lung lobes were homogenized in 1 ml of phosphate buffered saline (pbs, ph=7.4) and then hydrolyzed in 1 ml of 6 n hydrochloric acid for 16 hours at 110°c, and neutralized to ph 7.0 with naoh. chloramines t reagent (1 ml of 0.5 mol/l) was then added and the samples were left at room temperature for 20 minutes. then 20% p-dimethylaminobenzaldehyde solution (dissolved in 3.15 n perchloric acid) was added to each sample, and the mixture was incubated at 60°c for 15 minutes. absorbance was measured at 550 nm on a nanodrop 2000c spectrophotometer (thermo scientific, waltham, ma, usa). cells from blood, balf and lungs were subject flow cytometry analysis on a cytomics fc500 cytometer (beckman coulter, miami, fl, usa). all antibodies were obtained from biolegend (san diego, ca, usa). all data were analyzed with flowjo software (treestar, ashland, or, usa). for validation of the purity of magnetic bead-enriched circulating monocytes, anti-mouse cd11b-phycoerythrin (pe) (clone m1/70) and anti-mouse ly6g-percp-cy5.5 (clone 1a8) were used. for analysis of circulating monocyte subsets, ethylenediaminetetraacetic acid (edta) anti-coagulated whole blood was stained with anti-mouse cd11b-phycoerythrin (pe) (clone m1/70) and anti-mouse ly6c-fitc (clone hk1.4), incubated for 30 min at room temperature in the dark. following red cell lysis, samples were analyzed. for immunophenotypic analysis of alveolar macrophages (am), cells isolated from balf were first centrifuged (10 min at 400 g at room temperature), and the supernatant was discarded to remove dead cells. for each flow cytometry analysis, the cells were first suspended in 0.4% trypan blue in pbs, and the number of live and dead cells was measured using an automatic cell counter (counterstar tm , rui yu biotechnology co.,ltd, shanghai, china). by this method, the number of live cells in each sample is more than 95%. for subsequent flow cytometry analysis, the cells were incubated with anti-mouse f4/80-pe-cy5 (clone bm8), anti-mouse cd11c-pe-cy7 (clone n418) and anti-mouse cd206-pe (clone c068c2). following incubation, flow cytometry analysis was carried out. for immunophenotypic analysis of interstitial macrophages (ims), lung single-cell suspensions were prepared from lavaged lung (from which the balf was harvested) to reduce the contamination of am. in brief, the lower lobe of right lung were minced and incubated with 0.1 mg/ml collagenase solution (type i, sigma-aldrich) at 37°c for 60 min. after filtering through 40 μm nylon mesh, similar procedure to remove dead cells was carried out as did during sample preparation for am analysis, then the cell suspension was stained anti-mouse f4/80-fitc (clone bm8), anti-mouse cd11c-pe-cy7 (clone n418) and anti-mouse cd206-pe (clone c068c2). following incubation, samples were analyzed with flow cytometer. isotype antibodies (clone rtk2758 for f4/80; clone htk888 for cd11c; clone rtk2758 for cd206; clone rtk4530 for cd11b; clone rtk4174 for ly6c; clone rtk2758 for ly6g) were used to detect nonspecific binding. the gating strategies for analyzing am and im were according to previous report [21] . total rna from purified blood monocytes and lung tissue was isolated using trizol reagent (invitrogen, carlsbad, ca, usa) according to the manufacturer's instructions. total rna (2 μg) was reverse-transcribed into the cdna using a reverse transcription assay (promega, madison, wi, usa) in 25 μl of reaction volume according to the manufacturer's instructions. real-time pcr was performed with sybr green pcr master mix (roche diagnostics, indianapolis, in, usa) on an abi prism 7300 sequence detection system (applied biosystems, foster city, ca, usa) in triplicate and according to a two-step pcr protocol (5 min at 95°c, 40 cycles for 30 s at 95°c, 1 min at 60°c). the primer sequences are shown in table 1 . relative expression of real-time pcr products were normalized for expression of the β-actin and expressed as transcript fold change over ns mice using the 2 -△△ct method [22] . the levels of transforming growth factor β1 (tgf-β1), monocyte chemoattractant protein-1 (mcp-1)/chemokine (c-c motif) ligand 2 (ccl2), interleukin-4 (il-4), and interleukin-1β (il-1β) in the balf were measured by commercially available elisa kits (r&d systems, minneapolis, mn, usa), according to the manufacturer's instructions. all data are presented as the mean ± standard error of mean (sem). statistical analysis was performed using graphpad prism 5.0 software (graphpad, san diego, ca, usa). statistical comparison of multiple groups was performed by one-way anova with bonferroni post-hoc test or kruskal-wallis test followed by dunn's multiple comparisons (inflammation score and fibrosis index). a two-tailed p value less than 0.05 was considered statistically significant. by using magnetic bead-based monocyte enrichment method, more than 90% of the harvested cells were ly6g-cd11b+ ( figure 1a) . then we confirmed mr mrna expression in these cells by real-time pcr and pcr product electrophoresis ( figures 1b) . then, the mr protein expression of enriched monocytes was further validated by immunofluorescent staining (figure 1c) . using mouse balf, we also confirmed mr expression in alveolar f4/80+ macrophages ( figure 1d) . these results suggest that mr is expressed in mouse mononuclear phagocytes, which provides a basis for pharmacological intervention. figure 2 shows the detailed research protocol of in vivo pharmacological intervention study. figure 3 (a to h) shows the representative h.e. stained lung sections on day 7, which represents the peak magnitude of lung inflammatory response following bleomycin instillation. spironolactone treatment could significantly reduce the inflammatory response induced by bleomycin ( figure 3i) . panel j in figure 3 shows the results of differential cell counts from the balf that harvested on day 7. typically, the total fluid recovery was over 80% in all animals and the percentages of fluid recovered were not significantly different across all treatment groups. in agreement with histological findings, spironolactone treated lungs exhibited decreased total cell, macrophage, lymphocyte, neutrophil infiltration and esosinophils in alveoli. next, we measured the levels of inflammatory and profibrotic cytokines in the balf and determined related gene expression levels in lung tissue. as shown in figure 4 , compared with blm and blm+ns groups, spironolactone treatment was associated with downregulated ccl2/mcp-1, tgf-β1 and il-1β both at the mrna and the protein levels. in addition, markers for m2 polarization, such arginase-1 (arg-1) mrna level in lung tissue (figure 4g) , and il-4 protein content in balf ( figure 4f) were downregulated by spironolactone. figure 5 shows the profibrotic response using lung tissue that harvested on day 21. the histological analysis showed that mr antagonism was associated with reduced collagen deposition and α-sma positive cells (myofibroblasts). compared with ns group, the expression of type i and type iii collagen mrna in the lungs from blm and blm+ns groups were significantly upregulated, whereas spironolactone treatment could partially regress bleomycin-induced collagen expression upregulation, which was consistent with the histological findings. we next evaluated the effect of spironolactone treatment on circulating monocyte subset change. figure 6a shows the gating strategies for circulating monocyte subset analysis. as shown in figure 6b , compared with ns group, blm treated mice exhibited a significant increase of ly6c hi monocytes, starting from day 1, reaching the plateau level on day 3, then followed a gradual decrease till day 14. spironolactone treatment could significantly reduce bleomycin-induced the ly6c hi monocyte pool expansion on day 3 and thereafter. the reciprocal changes of ly6c lo monocyte subset is shown in figure 6c . using enzymatically digested lung tissue, we evaluated interstitial macrophage phenotype changes during drug intervention. as shown in figure 7b , one day after bleomycin challenge, the majority (more than 90%) of interstitial macrophages presented with a m1-like phenotype (f4/80+cd11c-cd206-), followed by a gradual decreasing trend of the proportion of m1-like macrophages, and this trend reached statistical difference on day 21. moreover, compared with blm and blm+ns groups, spironolactone has no obvious influence on interstitial macrophage phenotype switching induced by bleomycin. then we investigated the impact of spironolactone on alveolar macrophage phenotype changes. as shown in figure 8b , alveolar macrophages in ns group were mainly (more than 80%) presented with a m1-like phenotype (f4/80+cd11c +cd206-). after bleomycin challenge, there was a quick decrease of m1-like macrophage with a concomitant increase of m2-like phenotype (f4/80+cd11c+cd206+). whereas in spironolactone treated mice, this trend was partially normalized, indicating an inhibitory effect on alternative activation by mr antagonism. recent studies showed that the renin angiotensin aldosterone system (raas) plays an important role in the pathogenesis of lung injury [23] [24] [25] . in addition, the therapeutic efficacy of drug intervention targeting this system has been reported in bleomycin-induced lung injury models [26] [27] [28] [29] [30] [31] . zhao and coworker first demonstrated the therapeutical potential of spironolactone in ameliorating bleomycin-induced lung fibrosis [32] , which is also supported by a recent study [33] . a growing body of evidence suggests that manipulation of the mononuclear phagocyte phenotype switching could be a feasible approach to alter the severity and persistence of pulmonary injury and fibrosis in experimental models [34] [35] [36] . it has been demonstrated that mr plays an important role in regulating myeloid cell phenotype switching in different disease conditions [16, [37] [38] [39] [40] . to our knowledge, the role of mononuclear cell mr in mediating acute lung injury induced pulmonary fibrosis has not been addressed. the present work confirmed that mr antagonism by a clinically approved drug, spironolactone, could attenuate bleomycin-induced acute lung injury and fibrosis. specifically, mr inhibition partially attenuates ly6c hi monocyte expansion in circulating compartment and normalizes disturbed balance of macrophage polarization in alveolar compartment, leading to reduced alveolitis and collagen deposition in lung tissue. these findings highlight mononuclear phagocyte mr as a promising target for ameliorating acute lung injury and profibrotic response in lungs. the raas is a hormone system which acts on multiple physiologic pathways by regulating blood pressure and fluid balance. as the terminal effector of the raas cascade, the role of aldosterone/mr signaling has been recently implicated the pathogenesis of cardiovascular diseases, insulin resistance and diabetes, and chronic inflammation associated fibrosis [41] [42] [43] . these effects are supported by the fact that in addition to the kidney, there is a wide tissue distribution of mr, such as cardiomyocytes, endothelial cells, vascular smooth muscle cells, adipocytes and macrophages [44] . here, we demonstrated that mr is expressed both in purified murine circulating ly6g-/cd11b+ monocytes and in f4/80+ alveolar macrophages, providing a basis for mr regulation of monocyte/ macrophage phenotype switching. macrophages are professional phagocytic cells with different transcriptional profiles and functional capabilities depending on their origins from various organs [45] . broadly speaking, the lung tissue contains two tissue-resident macrophage compartments, i.e., alveolar macrophages and interstitial macrophages. the traditional belief that tissue-resident macrophages are derived from circulating monocyte progenitors has been challenged by recent fate mapping studies by showing that the steady-state turnover of alveolar macrophages is extremely low: 8 to 12 months after bone marrow transplantation, 70%-60% of alveolar macrophages are host derived [35, 46] . in addition, recent studies demonstrated that lung alveolar macrophages are established prior to birth and maintains themselves subsequently during adulthood independent of replenishment from circulating monocyte input in steady state [47, 48] . on the contrary, during acute lung inflammatory response, circulating monocytes have an important impact on the lung macrophage dynamics. in general, recent studies are in agreement with the notion that following injury, there is an increased accumulation of m2-like mononuclear cells in alveoli [10, [49] [50] [51] [52] . moreover, in patients with chronic obstructive pulmonary disease, a skewing of alveolar macrophages from an m1 to m2 phenotype has been observed [50, 53] . however, with regard to the origin of these m2-like cells, some controversy existed. in an endotoxin-induced lung inflammation model, maus and coworkers showed that despite a rapid recruitment of monocytes in lung tissue, the resident alveolar macrophage pool remained static throughout the duration of inflammation and the expansion of the lung macrophage pool was mainly mediated by an influx of the circulating monocytes, followed by their differentiation into tissue macrophages [46] . in agreement with this finding, recently osterholzer et al [54] , using a gene-targeted alveolar injury model, demonstrated an increased exudate macrophages and their progenitors, ly6c hi monocytes, both exhibiting m2 polarization in alveoli. in another study [36] , gibbons and colleagues adoptively transferred ly6c hi monocytes into bleomycin-treated mice during the progressive phase of lung fibrosis, which led to an exacerbation of disease progression and an increased accumulation of m2-like macrophage in the lung. surprisingly, these alternatively activated macrophages were host derived and not from the donor ly6c hi monocytes. as a corollary, regardless of their origins, our current knowledge points to a general scheme of their relationship: initially, acute lung injury induces a rapid expansion and infiltrating ly6c hi monocytes in lung tissue, which contributes to a paralleled increase of m2like macrophages (by direct differentiation or by paracrine effects) in alveolar compartment, and the severity and persistency of m2 polarization in alveolar macrophages would ultimately influence inflammation resolution and fibrosis. the above model highlights the circulating ly6c hi monocytes as a therapeutic target. although we did not use a monocytetargeted approach to suppress ly6c hi monocytosis, it is likely that spironolactone would also exert its major pharmacological effect on circulating monocyte pool since the efficacy of orally administered drug is significantly compromised by its inability to reach alveolar space at an appropriate concentration [55] . additionally, because evidence shown that monocyte infiltration would facilitates alveolar neutrophil emigration and determines the ongoing neutrophil influx in the persistent phase of acute lung injury [56] [57] [58] , suppression of ly6c hi monocytosis by spironolactone would concomitantly lead to a decreased tissue accumulation of neutrophils, which is also observed in our study. the present work has the following limitations. first, because spironolactone has anti-androgen effect, the observed effects of this work cannot be totally ascribed to mr antagonism. indeed, there is a sex discrepancy in bleomycin-induced lung fibrosis, and estrogen may have protective effect on this model [59, 60] . in this regard, mr knockout mice are preferred to address this issue. second, we did not observed significant changes in lung interstitial macrophages by spironolactone. previous study showed this population might have a role in limiting inflammation and fibrosis [61] . thus it remains unclear whether cd206 is an appropriate m2 marker for this population as recent study showed that the change of cd206 is modest after bleomycin challenge [51] , or this population is insensitive to mr inhibition, or due to enzymatic digestion-induced surface marker loss during sample preparation, a commonly encountered technical issue. third, due to the wide distribution of mr in the body, the mechanistical explanation of global mr antagonism is fairly complex. for example, aldosterone has been implicated in the pathogenesis of pulmonary hypertension [62] , and spironolactone has been shown to attenuate experimental pulmonary hypertension via mr inhibition in pulmonary artery smooth muscle cells [63] .admittedly, bleomycin is also a frequently used tool drug to induce pulmonary hypertension [64, 65] . the downregulation of α-sma by spironolactone observed in this study, also support an antifibrotic effect of spironolactone on fibroblasts. moreover, the functional expression of mr has been demonstrated in neutrophils [66] , which may also participate in spironolactone induced amelioration of lung fibrosis, as shown by reduced neutrophil count in balf. thus, in addition to its effect on mononuclear phagocytes, the mechanisms underlying therapeutic effect of systemic use of spironolactone on bleomycin-induced lung injury is multifactorial. forth, it seems obscure to interpret the effect of mr antagonism on alveolar macrophage polarization, since macrophages lacking myeloid mr exhibit alternative activation (m2 polarization), whereas our results showed that mr inhibition could reduce alveolar m2 polarization. it should be noted the long-established binary classification of macrophage in terms of classical (m1) and alternative activation (m2) is based on in vitro studies [67] . indeed, a recent study demonstrated eplerenone, another clinically approved mr antagonist, promotes alternative activation in human monocyte-derived macrophages [68] . however, macrophages in vivo maintain their plasticity and can alter their phenotype based on the microenvironment, including cytokine milieu among other factors [10] . as pointed early, drug administration via oral route, cannot reach alveolar space at an appropriate concentration. therefore, the alterations in alveolar macrophage polarization state cannot be ascribed to mr antagonist's direct effect. it is conceivable that suppression of inflammatory (ly6c hi subset) monocyte expansion should be the direct effect by spironolactone, which ameliorates lung injury via the "ly6c hi directed pulmonary alterative activation" mechanism [36] . thus, future monocyte-targeted approaches, as well as in vitro 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treatment of cardiovascular disease mineralocorticoid receptors, salt-sensitive hypertension, and metabolic syndrome extrarenal effects of aldosterone geneexpression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages resident alveolar macrophages are replaced by recruited monocytes in response to endotoxin-induced lung inflammation fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes a vicious circle of alveolar macrophages and fibroblasts perpetuates pulmonary fibrosis via ccl18 alternatively activated alveolar macrophages in pulmonary fibrosismediator production and intracellular signal transduction flow cytometric analysis of macrophages and dendritic cell subsets in the mouse lung low levels of insulin-like growth factor-1 contribute to alveolar macrophage dysfunction in cystic fibrosis smoking-dependent reprogramming of alveolar macrophage polarization: implication for pathogenesis of chronic obstructive pulmonary disease implicating exudate macrophages and ly-6c(high) monocytes in ccr2-dependent lung fibrosis following gene-targeted alveolar injury liposome-based drug delivery to alveolar macrophages monocytes are potent facilitators of alveolar neutrophil emigration during lung inflammation: role of the ccl2-ccr2 axis in vivo two-photon imaging reveals monocyte-dependent neutrophil extravasation during pulmonary inflammation monocytes control second-phase neutrophil emigration in established lipopolysaccharide-induced murine lung injury male sex hormones exacerbate lung function impairment after bleomycin-induced pulmonary fibrosis age and sex dimorphisms contribute to the severity of bleomycin-induced lung injury and fibrosis what is the clinical relevance of different lung compartments? aldosterone inactivates the endothelin-b receptor via a cysteinyl thiol redox switch to decrease pulmonary endothelial nitric oxide levels and modulate pulmonary arterial hypertension mineralocorticoid receptor antagonism attenuates experimental pulmonary hypertension lung extracellular superoxide dismutase overexpression lessens bleomycin-induced pulmonary hypertension and vascular remodeling therapeutic hypercapnia prevents bleomycin-induced pulmonary hypertension in neonatal rats by limiting macrophage-derived tumor necrosis factor-alpha aldosterone abrogates nuclear factor kappab-mediated tumor necrosis factor alpha production in human neutrophils via the mineralocorticoid receptor transcriptional regulation of macrophage polarization: enabling diversity with identity eplerenone promotes alternative activation in human monocyte-derived macrophages identification of myeloid cell subsets in murine lungs using flow cytometry key: cord-252347-vnn4135b authors: lee, wai-ming; kiesner, christin; pappas, tressa; lee, iris; grindle, kris; jartti, tuomas; jakiela, bogdan; lemanske, robert f.; shult, peter a.; gern, james e. title: a diverse group of previously unrecognized human rhinoviruses are common causes of respiratory illnesses in infants date: 2007-10-03 journal: plos one doi: 10.1371/journal.pone.0000966 sha: doc_id: 252347 cord_uid: vnn4135b background: human rhinoviruses (hrvs) are the most prevalent human pathogens, and consist of 101 serotypes that are classified into groups a and b according to sequence variations. hrv infections cause a wide spectrum of clinical outcomes ranging from asymptomatic infection to severe lower respiratory symptoms. defining the role of specific strains in various hrv illnesses has been difficult because traditional serology, which requires viral culture and neutralization tests using 101 serotype-specific antisera, is insensitive and laborious. methods and findings: to directly type hrvs in nasal secretions of infants with frequent respiratory illnesses, we developed a sensitive molecular typing assay based on phylogenetic comparisons of a 260-bp variable sequence in the 5' noncoding region with homologous sequences of the 101 known serotypes. nasal samples from 26 infants were first tested with a multiplex pcr assay for respiratory viruses, and hrv was the most common virus found (108 of 181 samples). typing was completed for 101 samples and 103 hrvs were identified. surprisingly, 54 (52.4%) hrvs did not match any of the known serotypes and had 12–35% nucleotide divergence from the nearest reference hrvs. of these novel viruses, 9 strains (17 hrvs) segregated from hrva, hrvb and human enterovirus into a distinct genetic group (“c”). none of these new strains could be cultured in traditional cell lines. conclusions: by molecular analysis, over 50% of hrv detected in sick infants were previously unrecognized strains, including 9 strains that may represent a new hrv group. these findings indicate that the number of hrv strains is considerably larger than the 101 serotypes identified with traditional diagnostic techniques, and provide evidence of a new hrv group. human rhinoviruses (hrvs), members of picornavirus family, are small nonenveloped viruses with a 7200-base mrna positive sense rna genome [1] . the first hrv was discovered in 1956 [2, 3] , and by 1987, 101 serotypes (1a and 1b to 100) were identified using susceptible cell cultures and specific antisera [4, 5, 6] . multiple epidemiologic studies of serotype circulation conducted between [1975] [1976] [1977] [1978] [1979] [1980] [1981] [1982] [1983] showed that .90% of field isolates could be identified with the 90 serotype-specific antisera prepared before 1973, and many serotypes identified earlier were still circulating [6, 7, 8] . these results suggested hrv serotypes are stable and do not undergo influenza virus-like antigenic drift [7] . hrvs are the most prevalent human respiratory pathogens [8, 9, 10, 11, 12] . annually, hrvs are responsible for .50% of all acute upper respiratory illness (common colds), the most frequent human illness. hrv infections occur year round worldwide and are epidemic in early fall and late spring in the temperate regions. hrv infections cause a wide range of clinical outcomes including asymptomatic infections, [13, 14, 15, 16, 17] upper respiratory illnesses, and in children, asthmatics, and other susceptible populations, lower respiratory symptoms. [18, 19, 20, 21, 22, 23] . defining the role of specific strains in various hrv illnesses has been difficult because traditional serology requires the isolation of hrv in susceptible cell cultures and neutralization tests against all 101 serotype-specific antisera [6] . this traditional serological method is insensitive, labor intensive and cumbersome [24] . more sensitive and faster molecular methods have been developed for serotyping enteroviruses, which are closely related to hrv [25] . in addition, molecular typing methods have been used to identify the links between illnesses and specific strains of pathogens such as dengue viruses, influenza viruses, human papillomaviruses, hepatitis c viruses, and hiv [26, 27] . molecular typing involves pcr amplification of a portion of the target viral genome, sequencing and phylogenetic analyses. in this report, we analyzed clinical specimens from sick infants with a new molecular method, and identified 26 new hrv strains including 9 that constitute a new hrv group. the length of the p1-p2 sequences (region between primer sites p1 and p2 in figure 1 ) varied only slightly between the 101 established serotypes, ranging from 261 to 273 bases. the maximum pairwise nucleotide divergence (%) between all 101 serotypes in this region was 45% ( figure 2 ). this result was similar to the maximum pairwise divergence of 101 vp4 sequences (46%) and slightly lower than that of vp1 sequences (54%) [28, 29] . furthermore, 97.5% of all the serotype pairs had .9% pairwise nucleotide divergence. the maximum pairwise divergences (%) of p1-p2 sequences among hrva and hrvb viruses were 33% and 27%, respectively. these results demonstrated the potential utility of this region for differentiating hrv serotypes. p1-p2 sequences of 101 hrv serotypes clustered into 2 previously defined genetic groups: hrva and hrvb phylogenetic tree reconstruction confirmed that the 101 p1-p2 sequences clustered into 2 genetic groups, a and b, (figure 3 ). the p1-p2 phylogenetic distribution of the serotypes into group was identical to that of published trees based on vp1 and vp4-vp2 sequences [28, 29, 30] , with the same 76 serotypes in the hrva group and 25 serotypes in hrvb group ( figure 3 ). the topology of the p1-p2 tree was similar to that of the vp1 and vp4-vp2 trees [28, 29, 30] . these results agreed with previous reports that the nucleotide phylogenies of hrvs are consistent across the whole genome, from 5'ncr through polyprotein to 3'ncr [31] . accurate typing of 79 clinical isolates by phylogenetic tree reconstruction of p1-p2 sequences to test whether p1-p2 sequences were suitable for hrv serotype identification, we compared the typing results of 79 culturable clinical isolates obtained by p1-p2 sequences with those by nim-1a sequences of vp1. the degenerate primers ev292 and ev222 for pcr amplification of nim-1a region were not sensitive enough for direct detection of small amount of hrv in original clinical samples (data not shown), and high titer infected cell lysates of cultured isolates were needed to produce enough pcr product for cloning and sequencing. the length of nim-1a and p1-p2 sequences ranged 329-356 bases and 263-271 bases, respectively. based on phylogenetic tree reconstruction of nim-1a sequences, the 79 clinical isolates were assigned to 24 different serotypes with highly significant bootstrap values (77-100%, table 1 ). identical assignment results were obtained with p1-p2 tree, although a number of assignments (hrv1b, hrv15, hrv85 and one of the 5 hrv89) had low but still significant bootstrap values (60%, 64%, 61% and 62%, respectively). interestingly, the nucleotide divergence between the clinical isolates and the respective reference strain was lower at the p1-p2 region (mean 3.5%, range 0-8%) than at the nim-1a region (mean 9.6%, range 5-13%) ( table 1) . this result agreed with previous reports that nucleotide sequences were more conserved at the ncrs than the coding region due to the preservation of conserved rna structure elements within the ncr [31, 32] . nasal lavage samples of 181 illnesses from 26 infants with frequent respiratory illnesses were analyzed by respiratory multicode assay [33] . hrv was detected in 108 samples (60%) ( table 2) . other viruses detected were enterovirus, rsv, adenovirus, coronavirus, influenza a virus, metapneumovirus and parainfluenza virus. among the 108 hrv-positive samples, 80 had only hrv and 28 had coinfection with at least one other respiratory virus. the identity of hrv in 101 of the 108 samples was determined by molecular typing. of the 7 samples that were not typed, 2 had no sample left and 5 did not yield p1-p2 fragments by semi-nested pcr. a total of 103 hrvs were identified in 101 samples. only 2 samples contained 2 different hrvs, indicating a low rate of infection with more than one strain. of the 103 hrvs, serotypes were assigned to 49 hrvs by phylogenetic tree reconstruction ( table 3 ). the assignments of 45 hrvs were strongly supported by highly significant bootstrap values (.74%). one assignment (hrv20) had a low but still significant bootstrap value (52%). the p1-p2 sequences of these 46 hrvs had 94-100% identity with the respective reference serotypes. three sequences clustered with hrv89 but had poor bootstrap values (,50%) although they had 99% identity with hrv89 reference sequences. this was probably because these 3 hrvs also matched well with hrv36, which has 97% identity with hrv89 in the p1-p2 region. these 46 hrvs and p3) and variable region between p1 and p2 (p1-p2 in red) at the 5'ncr and the pcr fragments used in this study. p1, p2 and p3 are located at bases 163-181, 443-463 and 535-551, respectively in hrv16 genome. pcr fragment a (about 900bps) was used to determine the 5'ncr sequences of all 101 hrv serotypes. it was amplified using pan-hrv pcr forward primer p1-1, which anneals to conserved region p1, and a serotype-specific reverse primer annealed to the 5' end of vp2 gene (between base# 1000 and 1100). pcr fragment b (about 390 bps) was generated with pan-hrv pcr forward primer p1-1 and reverse primer p3-1. pcr fragment c (about 300 bps) was generated with forward primer p1-1 and an equimolar mixture of reverse primers p2-1, p2-2 and p2-3. the variable sequences of p1-p2 were used for the molecular typing assay. doi:10.1371/journal.pone.0000966.g001 were grouped into 23 hrv serotypes; 42 (19 serotypes) were hrva and 4 (4 serotypes) were hrvb viruses ( table 3) . the p1-p2 sequences of 54 hrvs did not cluster with the homologous sequences of any of the 101 serotypes in phylogenetic tree reconstructions (figure 4 ). these 54 hrvs clustered into 26 new unique strains with high degree of nucleotide divergence from the respective nearest reference serotypes (mean 24.6%, range 12-35%) and between strains (mean 32.5%, range 10-46%) ( table 4 ). different new hrvs of the same strain had a high degree of identity among themselves (mean 98.4%, range 94-100%, table 4 ). seventeen of the new strains clustered with group a hrv ( figure 4 ) and had 12-35% pairwise nucleotide divergence from the nearest reference serotype (table 4 ). among them, 5 strains (w7, w15, w24, w28 and w36) clustered in a distant branch near serotypes 12, 45 and 78; 7 strains (w6, w10, w11, w12, w17, w23 and w25) in distant branch near serotypes 51, 65 and 71; strains w8, w9, w20 and w38 formed a new branch, and strain w33 was the lone member of a new branch. moreover, 9 strains (w1, w13, w18, w21, w26, w31, w32, w35 and w37) separated from both hrva and hrvb ( figure 4 ) and had 31-35% pairwise divergence from the nearest reference serotype (table 4 ). we propose that they represent a new hrv genetic group (hrvc). none of the new strains clustered with hrvb viruses. interestingly, none of the samples containing the new hrv strains produced cpe in standard wi-38 or mrc-5 cell cultures used for the detection and isolation of hrv (data not shown). hevs are closely related to hrvs [1] , and comprise .65 distinct serotypes that include polioviruses, coxsackieviruses a and b, echoviruses and the newer numbered evs. hevs are classified into 5 groups: poliovirus and human enterovirus a-d (hev-a-d), according to both biological and molecular properties. like hrvs, some hevs are upper respiratory pathogens. to determine the relationship of hrvc to hevs, phylogenetic tree reconstruction was performed with the p1-p2 sequences of 9 hrvc strains and respective sequences of all known hev (n = 74). the results indicate that hrvc viruses are distinct from hev: the pairwise divergence of p1-p2 sequences between a hrvc strain and the respective nearest hev ranged from 31% to 35% ( figure 5 ). in this report we demonstrate that the pool of circulating hrv strains is significantly larger than the collection of 101 known serotypes. less than half of the hrv detected in these young infants corresponded to previously recognized serotypes (table 3) , and 54 are previously unrecognized hrvs belonging to 26 new strains (table 4 ). moreover, 9 of the strains form a new genetic group "c" that is distinct from the previously defined groups hrva and hrvb and the human enteroviruses ( figure 5 ). the remaining 17 new strains cluster into existing distant or new branches in hrva group ( figure 4) . interestingly, none of the new viruses grew in standard tissue culture, which may explain why these viruses were previously undetected. these new hrv strains were detected with a sensitive molecular method to type hrv directly from the original clinical specimens. this new assay had 3 key components: sensitive pan-hrv primers and semi-nested pcr to amplify p1-p2 region from cdna prepared from original clinical specimens, a sequence database of 260-bp p1-p2 region of 5'ncr of all 101 hrv serotypes to serve as standard references for hrv identification, and phylogenetic tree reconstruction of the new p1-p2 sequences and the 101 homologous reference sequences. phylogenetic tree reconstruction has been shown to be more accurate than the other sequence analysis methods for molecular typing of enteroviruses [34] . interestingly, the 5'ncr is not suitable for typing of enterovirus. for example, the 5'ncr sequence of coxackieviruses do not correlate with serotype and vp1 sequence due to frequent recombination at the 5'ncr [35, 36] . in contrast, hrv maintains consistent phylogeny across its genome and has limited recombination [31] , and this characteristic enables 5'ncr sequence (p1-p2) to be used for strain identification. for enteroviruses, vp1 sequencing is commonly used for molecular serotyping because this region contains major antigenic sites that correlate well with serotype [25] . however, vp1 sequences of hrv are less conserved, and degenerate primer pairs such as ev292 and ev222 [24] that target this region were relatively insensitive for pcr amplification. the 101 established hrv serotypes (hrv1a to hrv100) were discovered and designated between 1956 and 1987. they were isolated using susceptible wi-38 cell cultures and then defined with serotype-specific antisera [4, 5, 6] . multiple epidemiologic studies of serotype circulation conducted between 1975-1983 showed that .90% of the field isolates could be identified with the 90 serotype-specific antisera (hrv1a through hrv89) prepared before 1973 and many serotypes identified earlier were still circulating [8, 11] [6, 7] . these results suggested that almost all hrv serotypes had already been identified and new serotypes were not evolving [6, 7] . in fact, only one possible new serotype, hrv-hanks, was reported in the next 20 years (1987 to 2006), and it was subsequently shown to be hrv21 by careful sequence analysis and neutralization testing [30] . recently, studies utilizing molecular techniques instead of culture-based diagnostics have provided evidence of additional strains of hrv [37, 38] . for example, lamson and colleagues identified 8 new hrvs in new york state (ny) by vp4 sequencing. they concluded that these hrvs represented a new genetic clade because they clustered in a branch at the root of the hrva phylogenetic tree [37] . in addition, mcerlean and colleagues obtained the complete genome sequence of a new hrv strain, hrv-qpm, in queensland, australia. they showed by phylogenetic analysis that hrv-qpm was a new member of hrva and belonged to a new genetic sub-lineage of hrva, hrv-a2, and the 8 new ny hrvs also belonged to hrv-a2 [38] . to compare the identities of these newly reported hrvs with our new strains, we performed phylogenetic tree reconstruction using p1-p2 sequences of our new strains, hrv-qpm, and the 101 established serotypes; and then a similar analysis using vp4 sequences of hrv-qpm, 8 ny hrvs, and the 101 established serotypes. the p1-p2 phylogenetic tree (not shown) revealed that hrv-qpm and one of our new hrva viruses, w24, were the same strain and thus supported mcerlean's conclusion that hrv-qpm was a group a virus. the vp4 tree (not shown) confirmed mcerlean's finding that hrv-qpm and the 8 ny hrvs belonged to the same cluster within hrva group. therefore the 8 ny hrvs and hrv-qpm were related to our 17 new hrva strains. in contrast, our 9 hrvc strains form a distinct group separated from both hrva and hrvb (figure 4) . analysis of 5'ncr of all hrv serotypes reveals that there are both highly conserved sequences (e.g. p1, p2 and p3 primer sites, figure 1 ) and also variable sequences between p1 and p2. the 260-bp p1-p2 region had up to 45% pairwise nucleotide divergence between serotypes, similar to that of vp4 (46%) and vp1 (54%). moreover, 97.5% of all the p1-p2 pairs from distinct serotypes had .9% pairwise nucleotide divergence. despite this inter-serotype variability, the p1-p2 sequence of 5'ncr was significantly more conserved between clinical isolates and the corresponding reference prototype strain (mean divergence 3.5%) compared to the nim-1a coding sequences of vp1 (mean divergence 9.6%, table 1 ). these data suggest that 5'ncr sequences may be under greater selective constraint, and it is known that this region contains rna structure elements that are critical for viral replication and translation [32] . in summary, more than half (52%) of the hrvs detected in these young infants were new hrv strains, and many of them clustered into a distinct genetic group c. these findings indicate that the number of hrv strains has been markedly underestimated by traditional viral culture and serotyping techniques, and also raise additional questions. first, how widespread are these this tree was generated as described in figure 3 . none of the new strains clustered with hrvb viruses. seventeen new strains (blue) belonged to hrva group, and 9 strains (red) cluster into a new group (''c'') that is separate from groups a and b. doi:10.1371/journal.pone.0000966.g004 putative ''group c'' hrv? our study population consisted of a group of infants who experienced frequent illnesses, and additional studies are needed to define the spectrum of hrv infections in unselected populations, and in subjects of other age groups. secondly, is the biology of these novel strains similar to that of other hrv? the inability to culture these viruses suggests that receptor utilization or other growth requirements are distinct. the development of molecular assays for the detection and analysis of respiratory viruses provide important tools for new epidemiologic and mechanistic studies to address these questions. a more complete understanding of the spectrum of respiratory viruses and their biology is essential for efforts directed at prevention and treatment of these common and clinically significant illnesses. this study was approved by the human subject committee of the school of medicine and public health, university of wisconsin -madison. written informed consent was obtained from the parents. for determining the standard reference sequences, infected cell lysates of prototype strains of 101 hrv serotypes were obtained from dr. fred hayden of u. virginia, charlottesville (serotype 1a, 1b, 2 to 10, 12 to 89, 91 to100 and hanks) and atcc (hrv11 and hrv90). hrv87 was excluded from the reference database because it has been reclassified as a human enterovirus [39, 40] . clinical samples were obtained from two sources. first, samples were obtained from infants ages 0-1 year participating in a prospective birth cohort study (childhood origins of asthma) in wisconsin to determine the role of viral and host factors in the pathogenesis of asthma [41] . of the 285 children who completed the first year of the study, 27 infants had frequent ($5) moderate to severe respiratory illnesses, and samples from 26 were available for further study. nasal lavage specimens were collected from 181 illnesses between spring of 1999 and spring of 2001. second, for validation of our molecular typing assay, infected cell lysates of 79 additional hrv clinical isolates were obtained from wisconsin state laboratory of hygiene (wslh). these clinical isolates were recovered from nasal lavages of infants in 1999 and 2000 using wi38 cell culture and identified by the characteristic cytopathic effect and acid lability of hrv [42] . pairwise sequence alignment, multiple sequence alignment, % identity calculation, distance (divergence) calculation, phylogenetic tree reconstruction and bootstrap analysis were performed using software clustal61.8.3 [43, 44] . in a typical analysis, the input sequences were first processed to produce a multiple alignment and matrixes of identity and distances (divergence) between all sequence pairs. the distance matrix was used by the neighbor joining method to produce an unrooted phylogenetic tree with branch length proportional to the divergence of the sequences. the confidence of the clustering of sequences was evaluated by bootstrapping (1000 replicates). bootstrap values of .700 (70%) indicate the highly significant clustering, whereas values ,500 (50%) indicate that the clustering is not statistically significant. the phylogenetic tree with bootstrap values was visualized using software njplot. preparation of cdna from nasal specimens cdna preparation was performed as described elsewhere [33] . briefly, nasal fluid (350 ml) was mixed with extraction carriers (glycogen and glycoblue) and 750 ml of trizol ls (invitrogen 10296), vortexed for 10 minutes, supplied with 230 ml of chloroform, vortexed again for 5 minutes and then microfuged for 5 minutes. the supernatant aqueous phase (,700 ml) was mixed with 600 ml isopropanol and this mixture was incubated at room temperature for 1 hr. the rna precipitant was pelleted by microfugation for 10 minutes, washed once with 75% ethanol, airdried and then dissolved in 20 ml water. to make cdna, 16 ml of rna solution was mixed with 24 ml of reaction solution containing promega amv-reverse transcriptase, amv-rt buffer, random primers, rnasin and dntps and then incubated at 25uc for 5 minutes, 42uc for 10 minutes, 50uc for 20 minutes, and 85uc for 5 minutes. rma is a new high-throughput, multiplex pcr-microsphere flow cytometry assay system for comprehensive detection of common respiratory viruses including rhinoviruses (hrv), enteroviruses, respiratory syncytial viruses (rsv), parainfluenza viruses, influenza viruses, metapneumoviruses, adenoviruses and coronaviruses. details of the rma assay have been previously described [33] . the output signal is expressed as mfi (median fluorescence intensity), and samples with an average signal .6 standard deviations of average negative control signals (typically 400 to 500 mfi) are regarded as positive. the rma is capable of distinguishing closely related hrv and enteroviruses [33] . selection of the target region to identify a genomic region suitable for molecular typing of hrv, we analyzed all published hrv sequences. these included complete genome sequences of 8 serotypes (1b, 2, 9, 14, 16, 39, 85 and 89) [45, 46, 47, 48, 49, 50, 51, 52] [28, 29, 30] , 3d (rna polymerase) sequences of 48 serotypes [53] and partial 5'ncr sequences of 37 serotypes [54, 55, 56] . careful alignment analysis of these sequences showed that only the 5'ncr region had highly conserved sequences (p1, p2 and p3 regions, figure 1 ) that could be used for making pan-hrv primers, and a long variable sequence (p1-p2, figure 1 ) suitable for serotype differentiation. however, 5'ncr sequences were available for only a fraction of 101 serotypes. cloning and sequencing of the 5'ncr of 101 reference hrv serotypes to establish a reference sequence database for molecular typing, we cloned and sequenced the 5'ncr of all 101 hrv serotypes. detailed viral rna preparation, rt-pcr, cloning and sequencing procedure have been described elsewhere [33] . briefly, total nucleic acids were prepared from 100 ml of infected cell lysate by phenol extraction and ethanol precipitation. rt (reverse transcription)-pcr was performed in a rt-pcr mix (invitrogen 11922-028) using the following conditions: 30 min at 50uc, 2 min at 94uc, 33 cycles of (30 sec at 94uc, 30 sec at 50uc, 60 sec at 68uc) and 5 min at 68uc. the pcr primer pairs were forward primer p1-1 (caagcacttctgtywcccc) for all serotypes and a serotype-specific reverse primer. primer p1-1 was designed within the conserved p1 region (figure 1 ). the reverse primer was selected for each of the 101 serotypes within a region corresponding to bases 1000-1100 of hrv16 near the 5' end of vp2 gene ( figure 1 ) according to published sequences [29] . the pcr product, a dna fragment of about 900 bp covering 75% of the 5'ncr, complete vp4 gene and about 200 bases of vp2 gene (pcr fragment a of figure 1 ), was isolated by agarose gel electrophoresis. after the agarose was removed by phenol extraction, pcr fragments were treated with kinase, ligated to a stui-linearized plamsid vector pmj3, and then transformed into e. coli. three plasmids with pcr fragment inserted were isolated for each serotype, amplified and purified. each viral dna fragment was completely sequenced (automated dna sequencing facility, u. wisconsin). the serotype identity of each sequence was verified by matching of its vp4/vp2 sequence to the respective published sequence [29] . semi-nested pcr amplification of p1-p2 region from original clinical specimens primer p1-1, which amplified the 5'ncr of all 101 serotypes, was chosen as the forward primer. for reverse primers, multiple candidates were designed within highly conserved p2 and p3 regions (figure 1 ). primers p3-1 (acgg-acacccaaagtag), p2-1 (ttagccacattcaggggc), p2-2 (ttagccacattcaggagcc) and p2-3 (ttagcc-gcattcagggg) were selected based on efficient hrv sequence amplification. for the first pcr, 2.5 ml of leftover cdna from the rma assay was added to a tube containing 23 ml platinum pcr supermix hf (invitrogen 12532-016), 1 ml of forward primer p1-1 (25 mm) and 1 ml of reverse primer p3-1 (25 mm). the reaction started with 2 min at 94uc, followed by a 'touchdown' cycle (the steps were 94uc for 20 s, 68uc down to 52uc (2uc intervals) for 30 s, and then 68uc for 40 s. there were two cycles for each annealing temperature down to 54uc followed by 12 cycles at 52uc, and then a final 5 min at 68uc. this reaction produces a pcr product of 390 bp (figure 1, fragment b) . for the second pcr, 5 ml of the first pcr product was transferred to a new pcr tube containing 50 ml platinum pcr supermix hf, 1 ml of forward primer p1-1 (25 mm) and 1 ml of each reverse primer p2-1 (25 mm), p2-2 (25 mm) and p2-3 (25 mm). the reaction conditions are 2 min at 94uc, 28 cycles of (20 sec at 94uc, 30 sec at 52uc, 40 sec at 68uc) and 3 min at 68uc. the final product is a 300 bp dna fragment ( figure 1, fragment c) . this semi-nested pcr protocol requires only 10 copies of cdna template per sample to produce sufficient product for cloning and does not produce nonspecific product from original clinical specimens (data not shown). fragment c was then purified, cloned, and 3 plasmids containing fragment c were isolated and sequenced for each sample. for 2 samples, 2 different p1-p2 sequences were found, indicating the presence of more than one serotype/strain, so 6 additional plasmids were analyzed. sequencing of the vp1 and 5'ncr of 79 hrv clinical isolates to determine whether the p1-p2 variable sequences within the 5'ncr were suitable for the differentiation and identification of hrv, we compared the typing results by p1-p2 sequences with those of nim-1a sequences of vp1. nim-1a is a dominant antigenic site of hrv first identified for hrv14 [1] , and the homologous sequences of this region correlate well with serotype of hrvs and enteroviruses [57, 58] . viral rna was isolated from virus-infected culture supernatant of 79 hrv clinical isolates with qiaamp viral rna mini kit (qiagen 52904) as described [24] . to obtain the vp1 sequences, viral rna was first amplified using primer pair ev292 and ev222 as described [24] with modified rt-pcr conditions: 10 min at 38uc, 40 min at 50uc, 3 min at 95uc, 40 cycles of (30 sec at 95uc, 45 sec at 42uc, 30 sec at 65uc) and 1 min at 70uc. the pcr product (,350 bp) was isolated by agarose gel electrophoresis and then sequenced [24] . to obtain the 5'ncr sequences, viral rna was amplified by rt-pcr using p1-1 and p3-1 primers, cloned and sequenced. assignment of serotypes and new strain a phylogenetic tree with bootstrap values and a matrix of % pairwise nucleotide divergence (distance6100%) were generated for each new sequence (nim-1a or p1-p2) and 101 homologous reference sequences using clustal6with default alignment parameters, which were selected after testing a range of parameters. next, a new isolate or detection was assigned the serotype to which it clustered with in the phylogenetic tree with a significant bootstrap value (.50%) [34] . in contrast, if the p1-p2 sequence of a new isolate or detection did not cluster with one of the 101 serotypes in the phylogenetic tree, and had .9% pairwise nucleotide divergence from the nearest reference serotype, it was designated as a new strain (prefixed with w). the threshold pairwise divergence value (9%) for assigning a new strain was determined after considering the pairwise divergence values between the 101 known serotypes (figure 2) , and the pairwise divergence values between reference serotypes and clinical isolates of the same serotype (table 1 , last column). two types of errors could be made in evaluating two sequences: a) deciding they were the same when they were actually different, and b) deciding they were different when they were actually the same. the probability of these errors was determined empirically using the data shown in figure 2 and table 1 , respectively. according to figure 2 , 99%, 98%, 97%, 95%, and 90% of the p1-p2 pairs of all 101 serotypes had .7%, .8%, .9%, .10% and .12% pairwise nucleotide divergence, respectively. as shown in table 1 (last column), the pairwise nucleotide divergence of all p1-p2 pairs between clinical isolates and the respective reference serotypes was consistently #8%. therefore, a threshold of 9% is associated with probabilities of ,3% and ,1% for errors a and b, respectively. the original p1-p2 sequences described in this report have been deposited in the genbank sequence database under accession no. eu126663 to eu126789. picornavirus structure and multiplication a cytopathogenic agent isolated from naval recruits with mild respiratory illnesses the isolation of a new virus associated with respiratory clinical disease in humans a collaborative report: rhinoviruses-extension of the numbering system rhinoviruses: a numbering system a collaborative report: rhinoviruses-extension of the numbering system from 89 to 100 rhinovirus infections in tecumseh, michigan: frequency of illness and number of serotypes the common cold rhinoviruses: important respiratory pathogens epidemiology of viral respiratory infections clinical virology a prospective, community-based study on virologic assessment among elderly people with and without symptoms of acute respiratory infection the september epidemic of asthma exacerbations in children: a search for etiology use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms predominance of rhinovirus in the nose of symptomatic and asymptomatic infants picornavirus infections in children diagnosed by rt-pcr during longitudinal surveillance with weekly sampling: association with symptomatic illness and effect of season respiratory illness caused by picornavirus infection: a review of clinical outcomes picornavirus infections: a primer for the practitioner rhinovirus: more than just a common cold virus rhinovirus respiratory infections and asthma rhinovirus infections: more than a common cold rhinovirus outbreak in a long term care facility for elderly persons associated with unusually high mortality improved molecular identification of enteroviruses by rt-pcr and amplicon sequencing typing of human enteroviruses by partial sequencing of vp1 the application of molecular phylogenetics to the analysis of viral genome diversity and evolution molecular epidemiology and evolution of emerging infectious diseases phylogenetic analysis of human rhinovirus capsid protein vp1 and 2a protease coding sequences confirms shared genus-like relationships with human enteroviruses genetic clustering of all 102 human rhinovirus prototype strains: serotype 87 is close to human enterovirus 70 vp1 sequencing of all human rhinovirus serotypes: insights into genus phylogeny and susceptibility to antiviral capsid-binding compounds genome-wide diversity and selective pressure in the human rhinovirus conserved rna secondary structures in picornaviridae genomes highthroughput, sensitive, and accurate multiplex pcr-microsphere flow cytometry system for large-scale comprehensive detection of respiratory viruses molecular identification of enterovirus by analyzing a partial vp1 genomic region with different methods molecular evolution of the human enteroviruses: correlation of serotype with vp1 sequence and application to picornavirus classification genotypic variation in coxsackievirus b5 isolates from three different outbreaks in the united states masstag polymerase-chain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like illness in new york state during characterisation of a newly identified human rhinovirus, hrv-qpm, discovered in infants with bronchiolitis enterovirus 68 is associated with respiratory illness and shares biological features with both the enteroviruses and the rhinoviruses human rhinovirus 87 and enterovirus 68 represent a unique serotype with rhinovirus and enterovirus features rhinovirus illnesses during infancy predict subsequent childhood wheezing relationships among specific viral pathogens, virus-induced interleukin-8, and respiratory symptoms in infancy multiple sequence alignment with the clustal series of programs the clustalx windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools amino acid changes in proteins 2b and 3a mediate rhinovirus type 39 growth in mouse cells molecular cloning and complete sequence determination of rna genome of human rhinovirus type 14 the complete nucleotide sequence of a common cold virus: human rhinovirus 14 the nucleotide sequence of human rhinovirus 1b: molecular relationships within the rhinovirus genus role of maturation cleavage in infectivity of picornaviruses: activation of an infectosome complete sequence of the rna genome of human rhinovirus 16, a clinically useful common cold virus belonging to the icam-1 receptor group evolutionary relationships within the human rhinovirus genus: comparison of serotypes 89, 2, and 14 human rhinovirus 2: complete nucleotide sequence and proteolytic processing signals in the capsid protein region sequence analysis of human rhinoviruses in the rna-dependent rna polymerase coding region reveals large within-species variation improved detection of rhinoviruses by nucleic acid sequence-based amplification after nucleotide sequence determination of the 5' noncoding regions of additional rhinovirus strains improved detection of rhinoviruses in clinical samples by using a newly developed nested reverse transcription-pcr assay amplicon sequencing and improved detection of human rhinovirus in respiratory samples sensitive, seminested pcr amplification of vp1 sequences for direct identification of all enterovirus serotypes from original clinical specimens comparison of classic and molecular approaches for the identification of untypeable enteroviruses we thank dr. fred hayden (u. virginia, charlottesville) for generously providing infected cell lysates of prototype hrvstrains. we also thank dr. key: cord-254117-2ttwaegh authors: priest, patricia c.; duncan, alasdair r.; jennings, lance c.; baker, michael g. title: thermal image scanning for influenza border screening: results of an airport screening study date: 2011-01-05 journal: plos one doi: 10.1371/journal.pone.0014490 sha: doc_id: 254117 cord_uid: 2ttwaegh background: infrared thermal image scanners (itis) appear an attractive option for the mass screening of travellers for influenza, but there are no published data on their performance in airports. methods: itis was used to measure cutaneous temperature in 1275 airline travellers who had agreed to tympanic temperature measurement and respiratory sampling. the prediction by itis of tympanic temperature (37.8°c and 37.5°c) and of influenza infection was assessed using receiver operating characteristic (roc) curves and estimated sensitivity, specificity and positive predictive value (ppv). findings: using front of face itis for prediction of tympanic temperature ≥37.8°c, the area under the roc curve was 0.86 (95%ci 0.75–0.97) and setting sensitivity at 86% gave specificity of 71%. the ppv in this population of travellers, of whom 0.5% were febrile using this definition, was 1.5%. we identified influenza virus infection in 30 travellers (3 type a and 27 type b). for itis prediction of influenza infection the area under the roc curve was 0.66 (0.56–0.75), a sensitivity of 87% gave specificity of 39%, and ppv of 2.8%. none of the 30 influenza-positive travellers had tympanic temperature ≥37.8°c at screening (95%ci 0% to 12%); three had no influenza symptoms. conclusion: itis performed moderately well in detecting fever but in this study, during a seasonal epidemic of predominantly influenza type b, the proportion of influenza-infected travellers who were febrile was low and itis were not much better than chance at identifying travellers likely to be influenza-infected. although febrile illness is more common in influenza a infections than influenza b infections, many influenza a infections are afebrile. our findings therefore suggest that itis is unlikely to be effective for entry screening of travellers to detect influenza infection with the intention of preventing entry of the virus into a country. rising concerns regarding influenza a (h5n1) and the pandemic of influenza a (h1n1) 2009 have led to the use of infrared thermal image scanners (itis) at some borders for the mass screening of travellers to detect those who might be infected with influenza [1] . itis measure body surface temperature rapidly, non-invasively, and with no contact, minimising the risk of contagion. they therefore have the potential to comply with the international health regulations' emphasis on containing the spread of disease in ways that avoid unnecessary interference with international traffic and trade [2] . evaluations of the use of itis in clinical settings have been conducted, and have reported sensitivities of 15% to 90% for confirmed fever depending on the cut-off used to define fever [3, 4, 5, 6] . however, these findings may not be applicable to border screening. itis measure body surface temperature, not body core temperature, and so itis temperature measurements are subject to the influence of a range of human and environmental factors. these include whether a person is sunburnt, has taken antipyretics or has circulatory problems, and also the ambient temperature and humidity. consequently it is important that the relationship between body surface temperature and body core temperature be evaluated within the environment in which itis are to be operated. in the airport setting, thermal scanning of arriving travellers has been used to screen for several different infectious diseases. during the outbreak of severe acute respiratory syndrome (sars) itis use was documented, however only the numbers of travellers triggering the scanner were reported, without stating the cut-off threshold used for fever or reporting on any subsequent method used to confirm febrile status [7, 8, 9] . a trial dengue fever screening programme found that among travellers arriving into cairns airport [10] 12% (118/963) of travellers who triggered the pre-set alarm threshold were confirmed to be febrile on tympanic temperature measurement. influenza screening in singapore found that only 12% of cases of pandemic (h1n1) 2009 infection with onset within 10 days of arrival were detected by itis on entry [11] . proper evaluation of a screening test requires that the 'gold standard' test is applied to both test positive and test negative participants in the study. to evaluate the use of itis in border screening for influenza, its performance in predicting both fever and also influenza infection is necessary. however to date no studies have been reported that tested itis negative travellers for either fever or influenza infection [12, 13] . we undertook both itis and tympanic temperature measurement on, and collected specimens for testing for influenza from, symptomatic and asymptomatic air travellers arriving into christchurch, new zealand during the southern hemisphere winter in 2008. this paper assesses the performance of itis in detection of fever and infection with seasonal influenza in these airline travellers. this evaluation of thermal image scanning was carried out as part of a larger study to measure the prevalence of seasonal influenza infection in arriving airline travellers and the effectiveness of a screening questionnaire for detecting those with influenza infection. the design followed closely a pilot study carried out in 2007 [14] . three airlines agreed to have their staff distribute a screening questionnaire to travellers (passengers and crew) during flights travelling from australian airports to christchurch, new zealand. the questionnaires were collected by research assistants following immigration processing on arrival in christchurch. 'symptomatic' travellers were defined as those who reported one or more of the following symptoms: cough, sore throat, sneezing, fever or chills, runny or blocked nose, muscle aches or pains, feeling generally unwell, chest discomfort or breathing difficulties. symptomatic travellers were all invited to have throat and nose swabs (copan italia spa, brescia, italy) taken and their temperature measured. in addition, half the questionnaires were marked and were randomly placed into the sets of questionnaires delivered to the flight crew (the sequence was determined by the rand function of microsoft excelß). arriving travellers carrying a marked questionnaire were also invited to have swabs and temperature taken. the nurse taking the swabs noted on the request form whether the traveller was symptomatic or asymptomatic. for the 23 working days from 21 august to 12 september 2008, cutaneous temperature from those travellers invited to participate who had given consent was measured using itis (therma-cam tm e45, flir systems, sweden) prior to swabs being taken. a focal plane array (1606120 pixels) was used on the front of the face and the side of the face (see figure 1 ) and the maximum temperature reading for each was recorded. after the swabs were taken, each participant's tympanic temperature was measured using an infrared tympanic thermometer (thermascan pro4000, braun, germany). the ambient temperature in the arrivals hall was a consistent 20.5uc at all times during data collection. all nasal and throat swab samples were analysed at canterbury health laboratories, christchurch. a multiplexed tandem polymerase chain reaction (mt-pcr) assay was employed to detect the presence of influenza a and b virus infection, as described by the manufacturer (easy-plex influenza a+b kit, cat. no. 3005.01, ausdiagnostics pty ltd, sydney, australia). stataß 10 was used to analyse the data. the cii command was used to calculate poisson exact confidence intervals around the proportion of influenza-infected travellers who were febrile. information about temperature measurements was collected on the swab consent form and linked to the symptom information on the questionnaire using a unique swab identifier. nine swab results were unable to be linked as their identifier had not been attached to any questionnaire. for these individuals the nurse's note of whether or not they were symptomatic was used to define their symptom status. analyses were performed to assess the accuracy of itis measurements in predicting two different tympanic temperature thresholds: 1. tympanic temperature $37.8uc (.100uf -the level used by the centers for disease control in defining 'influenza-like illness') [15] 2. tympanic temperature $37.5uc (the threshold used in the majority of reports) [12] . firstly, a receiver operating characteristic (roc) curve [16] was constructed. roc curves assess the ability of a test (in this case the itis measure) to discriminate between people who have, and who do not have, a condition (fever). the area under the roc curve for an uninformative test is 0.5. secondly, a level of itis temperature with sensitivity closest to 85% was chosen and the specificity calculated. finally, the positive predictive value (ppv) of the chosen level of itis temperature was estimated. the positive predictive value is the proportion of people who test positive (i.e. are 'positive' on itis) who actually have the condition of interest. it is not appropriate to calculate the ppv of itis measures directly in this sample since it was not a random sample of the population of travellers but was instead 'enriched' by including as many symptomatic travellers as were prepared to provide respiratory samples. therefore, the prevalence of fever (by each definition) in the holders of marked questionnaires was combined with the sensitivity and specificity of itis for detecting fever to estimate the ppv in the population of all travellers who arrived on the flights that took part in the study. to assess the utility of fever as a screening test for influenza infection (mt-pcr result), sensitivity, specificity, and population ppv for influenza were estimated for each tympanic temperature threshold, and the itis threshold used above. this study was approved by the new zealand health and disability multiregion ethics committee. written informed consent was obtained from all participants. in total, 5274 travellers returned a questionnaire during the study period, of whom 823 (15.6%) were symptomatic by our definition. figure 2 shows the pathway of potential participants through the study. accuracy of thermal scanning in predicting core temperature seven participants had a tympanic temperature of $37.8uc (2 reported no symptoms and 5 were symptomatic). five held marked questionnaires, giving a prevalence of fever by this definition of 0.5% (5/1063). half of the 38 participants with a tympanic temperature of $37.5uc were symptomatic. thirty-two of them held a marked questionnaire, so the prevalence of fever by this definition was 3.0% (32/1063). figure 3 is a roc curve showing the ability of itis front of face measurement to predict a tympanic temperature of $37.8uc. table 1 shows the test characteristics of itis as a predictor of tympanic temperature. for each definition of 'fever' (determined by tympanic temperature measurement), and for each site of itis measurement (front and side of face), the table shows: the itis threshold that gave a sensitivity closest to 85% in our data; the proportion of travellers with an itis measure above that threshold (i.e. who would have 'triggered' the itis during screening); the area under the roc curve; the actual sensitivity of that threshold; and its specificity. the prevalence of fever at each threshold in holders of marked questionnaires (as an estimate of the prevalence in this population of arriving travellers) is also shown, as well as the estimated ppv of itis for fever in this population. most (90%; 27/30) influenza-positive participants were symptomatic, but none (0%) had a measured tympanic temperature of $37.8uc (99%ci 0% to 18%), and only two (7%) had a measured tympanic temperature of $37.5uc(99%ci 0.3% to 31%). table 2 shows the ability of tympanic and itis temperatures to predict influenza infection in a population where the prevalence is 2% (the estimate of the prevalence of infection in this population of arriving travellers). with high sensitivity, specificity is very low. combined with the low prevalence of influenza infection in this population, ppv is also very low. influenza-positive participants reported that the first of their symptoms started between 12 hours and 24 days prior to answering the questionnaire, with symptom duration of 2 days or less in 11 participants, more than 2 and up to 5 days in 7 participants, and more than 5 days in 8 participants (3 were asymptomatic and 1 did not respond to this question). the greatest potential for the use of itis to screen incoming or departing travellers for infectious diseases such as a pandemic strain of influenza would be as the first stage of screening; that is, to identify and select out a high risk group for further assessment, for example by questionnaire, body core temperature measurement, and/or respiratory sample collection. this would require very high sensitivity for raised body temperature, as any travellers who 'slipped through' the screening process would enter the community and potentially spread infection. in addition, core temperature would need to be a good predictor of infection. can thermal scanning predict core temperature? this study shows that, among a group comprising both asymptomatic and symptomatic arriving international airline travellers, itis can have moderately high sensitivity and specificity for a high body core temperature of $37.8uc. however, the low prevalence of fever in arriving travellers means that the ppv is very low. measurement of the sensitivity of fever for influenza infection requires that afebrile as well as febrile people, from the same population, are tested for influenza infection. there are few studies that have done this, as symptoms of 'influenza-like illness', which include fever, are usually criteria for entry to studies of influenza [17, 18, 19] . such studies, with selected participants with a high prevalence of influenza infection, overestimate the sensitivity and dramatically overestimate the ppv of fever for influenza infection in unselected populations, such as airline travellers. a review of volunteer challenge studies [20] showed that not only were approximately 30% of influenza infections asymptomatic, but only 35% of those with symptoms had a measured fever .37.8uc. this study found a lower prevalence of fever among the participants infected with influenza b (7/101) than with influenza a h1n1(88/285; 31%) [20] . in this study, none of the 30 travellers subsequently identified as infected with influenza (most of whom had influenza b) had a temperature $37.8uc, and only two had a temperature $37.5uc. these results emphasise what is already known about fever as a symptom of influenza -while it clearly is one of the symptoms that can be experienced by people with influenza infection, it does not occur in all infected people [20] . the prevalence of fever is high in case series of patients with confirmed influenza infection [11, 21] , since often one of the criteria that is often used to determine whether testing takes place is the presence of fever. however, where fever is not used as a criterion for influenza testing, the prevalence of fever is by no means 100%, even among people with severe symptoms. for example, among 106 patients hospitalised with respiratory disease [22] , 39% of those with confirmed pandemic (h1n1) 2009 infection did not have a temperature of $37.8uc at any time during admission. in this study, the predominance of influenza b infection may partly explain the low prevalence of fever among infected participants (although the three with influenza a all had tympanic temperatures ,37.2uc ).even with more pyrexigenic strains, among travellers, who by definition are not severely unwell and in fact who are mostly not unwell at all, the proportion of influenza infected people who are afebrile can be expected to be much higher than among hospitalised patients [22] (because the sicker infected people don't travel), as shown in this study. it was a condition of conducting this study that we did not delay the transit of passengers through the airport by more than a few minutes and, therefore, measurements had to be made efficiently. we used a single measurement by an infrared tympanic thermometer as our 'gold standard' measure of core temperature. this approach may have introduced some random error into our results, but is unlikely to have caused systematic bias and is likely to be similar to the way that temperature would be confirmed in practice. in addition, our participants sat still at approximately 1m from the scanner for the itis measure and those who were wearing glasses were asked to remove them, steps likely to have provided greater accuracy than itis measures that are taken as numerous people walk past a fixed scanner in an arrivals hall. therefore our study provides an assessment of the best results that could be expected from the use of itis in border screening for influenza. in this study, no influenza-infected travellers had a measured tympanic temperature $37.8uc. we do not believe that this was because of systematic errors in tympanic temperature measurements, as these were measured by trained nurses using standard thermometers. we acknowledge that the number of infected travellers was relatively small at 30 but the probability is only 0.005 (0.5%) that the prevalence of fever among the population of infected travellers arriving from australia into christchurch at this time was greater than 18%; in other words the vast majority of infected travellers in this population were afebrile. among travellers, the proportion of influenza cases who are febrile may be low because those infected with influenza that is causing fever may feel too unwell to travel; 25% of travelassociated cases of pandemic (h1n1) 2009 infection with onset in singapore were symptomatic on embarkation but the proportion who were febrile was not reported [11] . in addition, it is possible that unwell infected travellers had used anti-pyretics prior to or during the flight, but this is a limitation of itis rather than of our study. the study assessed the performance of itis in the real world, which includes the fact that some unwell people take antipyretics. also, the flights that were part of this study were relatively short -3 to 4 hours -and it is possible that on longer flights some of the infected travellers might have become febrile. however, it remains unlikely that fever would occur in all, or even most, infected travellers arriving at any international airport [23] . good evidence on influenza virus transmissibility during the various phases of viral infection, (including afebrile infection and asymptomatic infection) is not available, but detection of viral rna on a respiratory sample does not necessarily mean that the infected person is, or will be, infectious. we were not able to perform culture for influenza virus in this study, so it is possible that some of the infected travellers were not shedding viable virus. although the approximately one third of participants whose symptoms were of 2 days' duration or less were likely to be in the early stages of their infection, those with longer duration of symptoms may not have been. unfortunately the symptoms of influenza are so non-specific that it is difficult to estimate the stage of influenza infection in a traveller with, for example, a cough that has been present for several weeks. nonetheless, it seems reasonable to conclude that at least a third, and probably more, of the infected (and afebrile) participants in this study were infectious on or after arrival into new zealand. influenza-infected arriving travellers include those who are symptomatic (with or without fever), those who become symptomatic during the flight, those who will develop symptoms following arrival, and those who will never have symptoms. it is not known whether the latter group are infectious, but clearly only the first two categories could potentially be detected by entry screening. most people who were infected but asymptomatic on boarding will still be asymptomatic on arrival at their destination [23] . however, in the absence of effective exit screening during the h1n1 2009 pandemic, some countries decided to use itis in entry screening with the hope that detecting travellers who were febrile on arrival would be worthwhile to reduce the probability of infected travellers entering the country, and that itis could detect them [1] . this study provides evidence to the contrary. the low ppv of itis measures for fever in this population means that the number of false positives who would require further investigation, presumably by taking a tympanic temperature, would be very high. in this study, using a front of face itis threshold of 35.4uc identifies 69% of travellers as requiring further investigation, of whom only 4.1% had a tympanic temperature $37.5uc. the ppv of any of the measures of temperature for influenza infection itself was lower, at less than 3%. however, the prevalence of disease is an important determinant of ppv, and the prevalence of influenza infection in this study, performed during the 'influenza season', was low at 1.9%. there are no other published estimates of the prevalence of influenza in arriving travellers, but it could be argued that the prevalence of infection would be higher during a pandemic, which typically infects a higher proportion of the population than seasonal influenza, than in this study. on the other hand, particularly if local containment strategies were in place in originating countries, the prevalence of infection in travellers might be lower during a pandemic. at the beginning of a pandemic, when effective entry screening would be most useful, the prevalence of infection among travellers and therefore the ppv will likely be much lower than the prevalence of seasonal influenza in this study. more importantly, raised temperature itself by any measurement technology is insufficiently sensitive for influenza infection for its measurement to be effective for mass screening in a pandemic situation. use of itis to identify travellers at high risk of fever, measuring the core temperature of itis-positive travellers, and then taking specimens from those with high core temperatures would have failed to identify all the influenza-infected travellers in this study. using a lower temperature threshold (however measured) for taking specimens could detect a high proportion of influenza-infected travellers only by taking specimens from what is likely to be an unfeasibly high proportion of travellers. governments may decide to implement entry screening, including itis, for reasons other than to actually detect most influenza-infected arrivals, for example to deter unwell people from travelling, or to demonstrate to their citizens that they are doing everything they can to protect population health. the risks associated with this approach include the potentially very large opportunity cost of further investigating itis 'positive' travellers, including quarantine of those febrile on tympanic temperature measurement pending specimen processing, and the potential for the loss of public confidence in the pandemic response when it becomes clear that many infected travellers were not detected by the screening and entered the country. in this study, during a seasonal epidemic of predominantly influenza type b, influenza-infected arriving travellers had a very low prevalence of fever. consequently, itis would not have identified influenza-infected travellers even though it performed moderately well at detecting febrile travellers. some aspects of this study may not generalise to a pandemic of influenza a. although febrile illness is more common in influenza a infections than influenza b infections, many influenza a infections are afebrile. our findings therefore suggest that itis is unlikely to be effective for entry screening of travellers to detect influenza infection with the intention of preventing entry of the virus into a country. entry screening to delay local transmission of 2009 pandemic influenza a (h1n1) revision of the international health regulations world health organisation screening for fever by remote-sensing infrared thermographic camera limitations of forehead infrared body temperature detection for fever screening for severe acute respiratory syndrome analysis of ir thermal imager for mass blind fever screening cutaneous infrared thermometry for detecting febrile patients world health organization working group on international and community transmission of sars (2004) public health interventions and sars spread thermal image scanners to detect fever in airline passengers, vancouver and toronto border screening for sars investigation of the optimal assessment of febrile passengers detected by infrared thermal scanning at an international airport epidemiology of travel-associated pandemic (h1n1) 2009 infection in 116 patients international travels and fever screening during epidemics: a literature review on the effectiveness and potential use of non-contact infrared thermometers physical interventions to interrupt or reduce the spread of respiratory viruses: systematic review screening for influenza infection in international airline travellers manual for the surveillance of vaccine-preventable diseases diagnostic tests 3: receiver operating characteristic plots clinical signs and symptoms predicting influenza infection predicting influenza infections during epidemics with use of a clinical case definition diagnosis of influenza in the community: relationship of clinical diagnosis to confirmed virological, serologic, or molecular detection of influenza time lines of infection and disease in human influenza: a review of volunteer challenge studies clinical features of the initial cases of 2009 pandemic influenza a (h1n1) virus infection in china clinical diagnostic criteria for isolating patients admitted to hospital with suspected pandemic influenza entry screening for severe acute respiratory syndrome (sars) or influenza: policy evaluation we thank the health emergency management branch, department of health and ageing, australia for lending us the scanner; christchurch international airport limited, new zealand customs service, and the participating airlines for their cooperation and assistance; and andrew strathdee for the laboratory testing.we are grateful to elisabeth wells, heath kelly, jonathan van-tam, and rebecca psutka for their helpful comments on drafts of this article, and to the anonymous reviewers for their helpful comments on the originally submitted version. key: cord-001249-awn9ayy6 authors: lasecka, lidia; baron, michael d. title: the nairovirus nairobi sheep disease virus/ganjam virus induces the translocation of protein disulphide isomerase-like oxidoreductases from the endoplasmic reticulum to the cell surface and the extracellular space date: 2014-04-08 journal: plos one doi: 10.1371/journal.pone.0094656 sha: doc_id: 1249 cord_uid: awn9ayy6 nairobi sheep disease virus (nsdv) of the genus nairovirus causes a haemorrhagic gastroenteritis in sheep and goats with mortality up to 90%; the virus is found in east and central africa, and in india, where the virus is called ganjam virus. nsdv is closely related to the human pathogen crimean-congo haemorrhagic fever virus, which also causes a haemorrhagic disease. as with other nairoviruses, replication of nsdv takes place in the cytoplasm and the new virus particles bud into the golgi apparatus; however, the effect of viral replication on cellular compartments has not been studied extensively. we have found that the overall structure of the endoplasmic reticulum (er), the er-golgi intermediate compartment and the golgi were unaffected by infection with nsdv. however, we observed that nsdv infection led to the loss of protein disulphide isomerase (pdi), an oxidoreductase present in the lumen of the endoplasmic reticulum (er) and which assists during protein folding, from the er. further investigation showed that nsdv-infected cells have high levels of pdi at their surface, and pdi is also secreted into the culture medium of infected cells. another chaperone from the pdi family, erp57, was found to be similarly affected. analysis of infected cells and expression of individual viral glycoproteins indicated that the nsdv pregn glycoprotein is involved in redistribution of these soluble er oxidoreductases. it has been suggested that extracellular pdi can activate integrins and tissue factor, which are involved respectively in pro-inflammatory responses and disseminated intravascular coagulation, both of which manifest in many viral haemorrhagic fevers. the discovery of enhanced pdi secretion from nsdv-infected cells may be an important finding for understanding the mechanisms underlying the pathogenicity of haemorrhagic nairoviruses. nairobi sheep disease virus (nsdv) belongs to genus nairovirus of the family bunyaviridae and causes a severe disease characterised by fever and haemorrhagic gastroenteritis in sheep and goats with a mortality rate up to 90% in a susceptible population [1, 2] . nairobi sheep disease was first reported in 1910 in nairobi, kenya and, in 1917 , nsdv was shown to be the causative agent of the disease by montgomery and colleagues [2] . the virus is endemic in east and central africa [2] [3] [4] [5] [6] [7] and an asian virus causing the same disease in india is called ganjam virus (gv) (reviewed in [8] ). based on genetic and serological studies, these viruses were identified as different isolates of the same virus [9, 10] . nsdv does not appear to be contagious and the virus needs to be transmitted by ticks in natural infection [2] . the virus is primarily transmitted by hard (ixodid) ticks, with rhipicephalus appendiculatus being the main vector in africa [4] and haemaphysalis intermedia the main vector in india [11] [12] [13] . sheep and goats are the only known mammalian reservoir for nsdv [3, 4] ; other livestock (e.g. cattle, horses) are refractory to the disease [2] . while the virus has a limited effect on animals bred in the enzootic areas due to the development of immunity by these animals while they are still protected by maternal antibodies, nsdv causes large economic losses during transport of animals through enzootic areas or during introduction of new livestock to these areas [2, [14] [15] [16] . currently there is no safe vaccine [17] . nsdv is closely related to a human pathogen, crimean-congo haemorrhagic fever virus (cchfv), which causes viral haemorrhagic fever with an average mortality rate of 30% (reviewed in [18, 19] ). after dengue virus (denv), cchfv is the second most widespread of the arboviruses pathogenic to humans [20] [21] [22] [23] [24] [25] [26] [27] [28] . the disease caused by cchfv in humans is similar to that caused in sheep and goats by nsdv infection [2, 29] , and is characterised by fever, myalgia, superficial and internal haemorrhage, abdominal pain and diarrhoea [30] [31] [32] [33] . while work on cchfv is limited to biosafety level (bsl) 4 laboratories and restricted by lack of a natural animal model to study the disease, nsdv may act as a suitable model to study haemorrhagic nairoviruses. nairoviruses are enveloped viruses which appear spherical in the electron microscope, with a diameter of approximately 100 nm [34] [35] [36] ; the viral genome consists of three negative-sense rna segments [37] [38] [39] which are encapsidated by the viral nucleoprotein forming, together with the viral rna, the ribonucleoprotein (rnp) [37, [40] [41] [42] [43] [44] [45] . the three rna segments are called small (s), medium (m) and large (l) and encode respectively the nucleoprotein (n) [37, 42, 46] , the viral structural (gn and gc) and non-structural glycoproteins [37, [47] [48] [49] [50] , and the rna-dependent rna polymerase (i.e. the l protein), which is associated with each rnp [17, [51] [52] [53] . replication of nairoviruses occurs in the cytoplasm, where the n and l proteins are also synthesised [54, 55] , while the viral glycoproteins are generated on endoplasmic reticulum (er)associated ribosomes [49, 50, [56] [57] [58] [59] . newly synthesised virions bud into the golgi and are transported, probably through the secretory pathway, to the plasma membrane, where the virions exit the infected cell after fusion of the carrier vesicle with the plasma membrane [35, 36, 60] . the m segment mrna is thought to be translated as a single polyprotein, which is further processed by the cellular signal protease [48, 50] to generate the glycoproteins pregn and pregc; in cchfv these precursors are further processed into mature glycoproteins gn and gc in a process which employs other cellular proteases [50, [57] [58] [59] . since the glycoproteins of nairoviruses mature in the er and golgi, and newly generated virions bud in the golgi, nairoviruses would be expected to affect the secretory pathway. however the effect of nairovirus replication on specific cellular compartments has not been studied; in this study we used specific antibodies in combination with laser scanning confocal microscopy to study the effects of nsdv on the secretory pathway of infected cells. we observed that while nsdv replication has no obvious effect on the structure of the er, the er-golgi intermediate compartment (ergic) or the golgi, it induces redistribution of luminal er oxidoreductases protein disulphide isomerase (pdi) and erp57, proteins which are involved in the formation and rearrangement of disulphide bonds during protein folding. in nsdv-infected cells, both pdi and erp57 are translocated from the er to the cell surface and to the medium surrounding the infected cells, a process which seems to depend on the nsdv glycoprotein precursor pregn. all cell culture was carried out as previously described [29] , with the exception of cje102 cells. these cells are a caprine endothelial cell line derived in 1999 from circulating endothelial cells isolated from a naïve creole goat that was part of a herd kept for research purposes by the french centre for agricultural research for development (cirad). the blood was taken for the purpose of isolating the cells. all animal sampling was conducted according to internationally approved oie standards, under personal and institutional authorizations set forth by the director of the veterinary services of guadeloupe on behalf of the prefect of guadeloupe (authorization number: a-971-18-01) and according to the regulations in the guide to the care and use of experimental animals provided by the french ministry of agriculture, which follow the relevant eu directive [61] . cell line cje102 was the kind gift of dr nathalie vachiéry, département emvt du cirad, domaine de duclos, prise d'eau, 97170 petit bourg, guadeloupe. the cells were cultured in glasgow modified eagle's medium (gmem) containing 10% foetal calf serum (fcs), penicillin (100 u/ml), streptomycin sulphate (100 mg/ml), 2 mm l-glutamine and 5% tryptose phosphate broth. the highly tissue culture passaged nsdv isolate from uganda (nsdvu) (nd66-pc9), which was obtained from dr piet van rijn, central veterinary institute of wageningen, netherlands, at the 75 th tissue culture passage, and the pathogenic gv isolate (nsdvi) (ig619, tvpii 236), which was obtained from professor robert b. tesh, the world reference center for emerging viruses and arboviruses at the galveston national laboratory, university of texas medical branch, galveston, texas, usa, were also already described by us [29] . all dna manipulation was done using standard methods; plasmids were grown in escherichia coli dh5a and dna was purified using cscl gradients. plasmids pcdna6-gv-n and pcdna6-gv-la plasmids have been previously described [61] . plasmid pcaggs-mcsii was the gift of professor adolfo garcia-sastre, mount sinai school of medicine, new york, usa. the pcaggs_mcsii_v5 construct was generated by insertion of v5 tag sequence into the multiple cloning site (mcs) of pcaggs-mcsii; pcaggs_mcsii_v5 was further used to generate pcaggs_mcsii_pregn_v5, pcaggs_mcsii_ns m _v5 and pcaggs_mcsii_pregc_v5. these constructs were generated by amplification of appropriate fragments of the gv (i. : ta-tcatatcgatggcaacctctttaactgcttttc) and pcdna-gv-m [62] as template. these fragments were then inserted upstream of, and in frame with, the v5-tag of pcaggs_mcsii_v5. all pcrs were performed using proofreading polymerase (kod; novagen). all inserts were sequenced completely. the generation of specific rabbit antisera to the amino-terminus of the gv (nsdvi) n protein will be described in detail elsewhere. briefly, bacterially expressed proteins consisting of the first 167 amino acids of the viral n protein or the first 169 amino acids of the viral l protein were used by a commercial company to immunise four rabbits and the resulting sera optimised for immunofluorescence microscopy. mouse monoclonal recognising the denatured form of the pregn glycoprotein from nsdvi, but not from nsdvu, was obtained from abmart. for detection of cellular markers the following antibodies were used: mouse monoclonal anti-pdi [clone 1d3] was obtained from bioquote, mouse monoclonal anti-pdi [clone rl90] and anti-calnexin [clone af18] antibodies were purchased from abcam; mouse monoclonal anti v5-tag:alexa fluor 488 was obtained from abdserotec, mouse monoclonal anti-a-tubulin from sigma, mouse monoclonal anti-gm130 from bd bioscience, mouse monoclonal anti-pcna (pc10) from santa cruz biotechnology. alexa fluor 488 goat anti-mouse igg (h+l), alexa fluor 568 goat anti-mouse igg (h+l), alexa fluor 488 goat anti-rat igg (h+l), alexa fluor 568 goat anti-rabbit igg (h+l) and zenon rabbit igg labelling kit were purchased from life technologies. rat anti-p102 antibody (clone 23c), was prepared by the institute of cancer research [63] and was kindly given to us by dr philippa hawes, pirbright institute; antibody to erp57 has been described previously [64] and was kindly given to us by dr christopher netherton, pirbright institute. vero cells or cje cells were plated at an initial seeding density of 4 x10 4 cells/well in 12-well plates. on the next day, cells were infected with nsdvi or nsdvu at a multiplicity of infection indicated for each experiment. the virus inoculum was removed after one hour, cells were washed once with phosphate buffered saline (pbs) and fresh medium was added. the infected cells were further incubated for the times indicated for each individual experiment. all transfections were carried out with transit-lt1 (mirus) according to the manufacturer's instructions. a ratio of 2 ml transit-lt1 reagent per 1 mg of dna was used; vero cells were plated as described above, usually 1 day prior to transfection. vero cells were plated as described above on 18 mm-diameter coverslips prior to infection or transfection. at the indicated times, cells were fixed with 3% paraformaldehyde (pfa) and then briefly permeabilised with ice-cold 100% methanol. the fixed and permeabilised cells were blocked with 0.2% porcine gelatine for at least 30 min before processing for antibody staining. for detection of surface proteins, vero cells were fixed with 3% pfa, blocked for 30 min with 0.2% porcine gelatine and incubated with primary antibody against pdi or erp57. after washing with pbs to remove unbound antibodies, the cells were again fixed with 3% pfa, treated briefly with ice-cold 100% methanol and again blocked with the 0.2% porcine gelatine. such permeabilised cells were incubated with anti-nsdv antibodies, and then proteins were visualised with fluorophore-conjugated secondary antibodies. initial confocal images were obtained using sequential scanning and dmire2 leica clsm tcs-sp2 confocal laser scanning microscope. the obtained tiff images were resized and overlays were prepared with adobe photoshop. statistical analysis of the effect of infection on distribution of host cell proteins was carried out using the chisq.test() function in r. nsdvi-infected cells were harvested with 0.4 ml of a lysis buffer (1% (v/v) nonidet p-40, 50 mm tris/cl ph 7.5, 150 mm nacl, 2 mm edta, 2.5 mm edta) containing protease inhibitor cocktail set iii (calbiochem) at a final dilution of 1/200. lysates were centrifuged at 10,000 g for 30 min at 4c and the supernatants were subjected to immunoextraction with 1 ml of an appropriate antibody and using protein g-agarose beads (millipore). after a series of washes (2x with 0.2% np40, 10 mm tris/cl ph 7.5, 150 mm nacl, 2 mm edta; 2x with 0.2% np40, 10 mm tris/cl ph 7.5, 500 mm nacl, 2 mm edta; and 1x with 10 mm tris/cl ph 7.5), the immunoprecipitated proteins were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds-page) and immunoblotting using specific antibodies. at the indicated times, infected or transfected cells were harvested and lysed with 100 ml of 1x sds sample buffer (new england biolabs). sds-page and western blots were carried out as previously described [65] . to analyse secreted proteins, cell were cultured in 12-well plates containing 0.5 ml of growth medium per well. the medium was collected, pre-cleared of cellular debris by centrifugation at 664 g for 10 min at 4uc, and supernatants were mixed with 5x sds-page loading buffer (4% sds, 250 mm tris/cl ph 6.8, 0.6 mg/ml of bromophenol blue, 7.5 mm dtt, 30% (v/v) glycerol in water). such prepared protein lysates were analysed by western blotting as described above. erp57 while the structure of the er, ergic and golgi remains unchanged two isolates of nsdv have been previously described by us [29] : a multiple-times passaged isolate of nsdv from uganda, which appeared attenuated upon infection of a susceptible animal, and an isolate of gv from india which had been passaged a limited number of times in mouse brain or bhk21 clone 13 cells and which caused haemorrhagic gastroenteritis in sheep upon experimental inoculation. since nsdv and gv have been shown to be the same virus [9, 10] , for simplicity we have referred to them both as nsdv in this paper, nsdvi for the pathogenic and nsdvu for the apathogenic isolate. we focussed on the pathogenic isolate as the best representative of a wild-type virus; this virus replicated reasonably well in vero cells, although it grew poorly in most of the other cell lines tested [29] . vero cells were infected with nsdvi at a low multiplicity of infection (moi), which allowed for direct comparison of infected and uninfected cells on a single slide and even in one focal view. after overnight infection, cells were fixed and stained with antibodies specific for ergic (ergic53), cisgolgi (gm130) or transgolgi (p102) markers. infected cells were detected using rabbit polyclonal antiserum against the viral n protein (which recognises both nsdvi and nsdvu isolates), and the distribution of the cellular proteins was compared in infected and uninfected cells by confocal microscopy. apart from a few infected cells where the n protein appeared to fill up the space occupied by the ergic, no obvious changes to the distribution or amount of ergic53 was observed ( figure 1 .a a-c). similar results were observed for the cis and transgolgi compartments, with no obvious changes to the distribution of gm130 and p102 (which are matrix proteins of the cis and transgolgi respectively [63, 66] ) when infected cells were compared to uninfected cells ( figure 1 .a d-i). nsdv replication did not appear to affect protein levels of these markers, as judged by fluorescence intensity. the same results were obtained for nsdvi-infected a549 cells, and for vero and a549 cells infected with nsdvu (data not shown). these results indicate that, despite budding into the golgi, often shedding large numbers of the virus particles [35] , nsdv does not appear to alter ergic and golgi structures (at least not sufficiently for the alteration to be detected by confocal microscopy). this suggests that the golgi and ergic remain functional during infection, thereby promoting viral egress. nsdv replication also had no apparent effect on microtubule and actin filament organisation ( figure s1 ); in addition, several nsdvinfected cells were found undergoing mitosis, showing a welldefined mitotic spindle as observed by a-tubulin staining, indicating that the centrioles likewise remained functional in nsdv-infected cells ( figure s1 ). a different picture was observed when the er of infected cells was observed. when staining cells with mouse anti-pdi antibody clone 1d3, which targets the c-terminal part of pdi, containing the kdel retention motif [67] , we discovered that in many infected cells the amount of pdi appeared to decrease, even disappearing completely from the er of about 1 in 3 infected cells (figure 2 a-c). to exclude the possibility that the viral glycoproteins mask the epitope recognised by the antibody clone 1d3, the experiment was repeated using mouse anti-pdi antibody clone rl90, which targets a different pdi epitope from that recognised by 1d3, since it inhibits the activity of pdi in vitro [68] and therefore recognises a region close to the enzymatic site of the protein. once again, many infected cells showed a reduced amount of pdi in the er ( figure 2 d-f), confirming that, in nsdv-infected cells, pdi shows a different staining pattern than in uninfected cells, and that many infected cells even appear to lose pdi entirely. the distribution of erp57, another soluble chaperone of the pdi-like family, was also studied in nsdvinfected cells; again many infected cells showed a similar redistribution or complete loss of erp57 from the er ( figure 2 g-i), indicating that other er-lumenal chaperones/proteins might also be affected. the fraction of infected cells in which pdi distribution was affected was quantified visually. the amount of pdi in the er of 150 infected and uninfected cells was investigated and approximately 33% of infected cells (53/150) were found to show either a drastically reduced level or total loss of intracellular pdi, while only 1 uninfected cell was seen with low/ absent pdi in the er (x 2 = 58.7, p,0.00001). in order to establish whether nsdv replication affects the structure of the entire er or just the distribution of specific proteins, we looked at the distribution of calnexin, an ermembrane chaperone, in infected and uninfected cells. no change to the cellular distribution of calnexin was observed (figure 2 j-l), indicating that, despite the disappearance of pdi and erp57, the overall structure of the er remains unchanged in nsdv-infected cells. to examine whether pdi and erp57 are selectively degraded during infection, vero cells were infected with nsdvi at a high moi such that all cells were infected, and total cellular levels of pdi and erp57 in these cells were analysed by western blotting. for comparison, we also determined the relative levels of calnexin, gm130 and p102 (proteins which showed no effect of infection on their distribution in individual cells by immunofluorescence); proliferating cell nuclear antigen (pcna) used as a loading control. surprisingly, given the confocal results, nsdvi infection appeared to have no effect on the total pdi and erp57 protein levels ( figure 3 ). in the case of very abundant proteins such as pdi, which represents around 1% of total cellular proteins (reviewed in [69] ), a small difference in protein level might not be detected by western blotting; however, during nsdv infection, 33% of the infected cells showed major or total loss of pdi (as estimated using immunofluorescence), therefore some change in the total pdi level would be expected to be seen if this protein was degraded in the infected cells. these data indicate that, although pdi and erp57 disappear from the er of infected cells, this is not a reflection of overall loss from the cell such as through degradation. export of er proteins in nairovirus-infected cells plos one | www.plosone.org pdi and erp57 appear at the surface of many nsdvinfected cells and are secreted from these cells the absence of pdi and erp57 degradation suggested that, although these proteins disappear from the er, they somehow remain associated with the nsdv-infected cells. one possibility was that these proteins are moving to the cell surface in infected cells, but are lost during fixation and washing, since pdi is known to be only weakly bound to the cell surface via non-covalent interactions with other molecules present at the cell surface [70] . this possibility was investigated using careful surface labelling of lightly fixed cells and confocal microscopy. to detect pdi and erp57 at the surface, vero cells infected with nsdvi at a low moi were fixed and then incubated with anti-pdi or anti-erp57 antibody without permeabilization. after washing away unbound antibodies, cells were again fixed with 3% pfa to ensure that any pdi/erp57 and bound antibodies remained attached to the cell surface throughout the staining procedure. to detect nsdv proteins, cells were then permeabilised with ice-cold methanol and incubated with rabbit anti-nsdv n protein serum or with mouse anti-nsdvi pregn (a monoclonal which recognises a sequence near the amino-terminus of the nsdvi pregn glycoprotein). we observed that many nsdv-infected cells showed significant amounts of cell surface pdi and erp57 (figure 4 .a). although small amounts of pdi and erp57 could be observed at the surface of even uninfected cells, nsdv infection led to a large increase in the amount of these proteins at the surface. approximately 1/3 rd of infected cells (calculated as a fraction (51/150) of viral proteinpositive cells with increased surface pdi/erp57 calculated over several focal views) showed this high level of surface pdi/erp57, and only 1/150 uninfected cells showed increased surface pdi/ erp57 (x 2 = 55.8, p,0.00001). we did observe a small fraction of cells the membranes of which were permeabilised by the initial fixation, leading to labelling of internal pdi or erp57; however, surface pdi/erp57 staining showed a completely different pattern to the resultant intracellular (er) staining. to ensure that the apparent surface pdi is not an artefact of the multi-step labelling procedure and that anti-pdi antibody does not re-associate with an internal epitope after opening of cells with methanol, we infected cells at a high moi and repeated the surface labelling of non-permeabilised cells using anti-pdi antibody as previously, but omitting the subsequent permeabilization step and staining for viral protein. enhanced surface pdi was detected in batches of infected but not in uninfected cells (figure 4 .b), confirming that surface pdi detected in nsdvinfected cells is not an artefact of the staining procedure. the disappearance of pdi/erp57 from the er and their appearance at the cell surface in infected cells suggests that nsdv replication and/or assembly induces movement of these proteins from the er to the cell surface. the effect of nsdv replication on pdi distribution in the cell was also tested in other cell types. while nsdvi grows relatively poorly in a caprine endothelial cell line (cje), it still induced redistribution of pdi from the er to the cell surface in these cells ( figure 5 ). similar changes to the distribution of pdi upon infection with nsdv (either nsdvi or nsdvu) were also observed in a549 cells and in a bovine foetal endothelial cell line (bfa) ( figure s2 ). this confirmed that redistribution of pdi during nsdv infection is not vero cell-specific, nor is it specific to a pathogenic or apathogenic virus type. nsdvi-infected cells were studied at different times post infection to determine when during infection changes to the distribution of pdi appear. vero cells were infected at high moi and fixed at 0, 1, 2, 4, 8, 12 and 16 hours post infection (hpi), after which pdi was labelled in the er or at the surface. the first changes to pdi distribution could be observed at 8 hpi, when a few cells showed a reduced amount of pdi in the er (observed visually as a clearly weaker fluorescence signal when compared to uninfected cells) and vesicle-like structures containing pdi could be noticed (figure 6 a-c) . at 12 hpi, several cells which had completely lost detectable pdi from the er were observed ( figure 6 d-f ) and at 16 hpi many cells lacking pdi were found ( figure 6 g-i) . the first surface pdi was only seen at 16 hpi ( figure 6 j-l); however, it is possible that a small increase of surface pdi would be missed at earlier time points due to the relatively low sensitivity of confocal microscopy. similar results were obtained for erp57 ( figure s3 ). interestingly, despite synchronising infection by washing away unbound virus 1 h post inoculation, it was observed that infection had not progressed to the same degree in all infected cells, so that, at 8 hpi, only a few cells showed detectable viral n protein, while at 12 hpi, at which time progeny virus can be detected [29] , cells with very different amounts of n were observed ( figure 6 ). this apparent variation in the extent of virus replication in individual cells from the same sample may be due to variation in the virions themselves (e.g. individual virus particles having different numbers of copies of each segment) or due to the virus replicating with different rates in different cells, possibly due to some effect of cell cycle, as the cell cycle of the cells used in this experiment was not synchronised. we also observed that some nsdvi-infected cells, despite containing large amount of n protein, showed very little or no pregn ( figure s4 ), which could explain why not all cells judged to be infected because of the presence of viral n protein had lost pdi from their er or showed surface pdi expression. differential expression of the n and pregn proteins in nsdv-infected cells may reflect the apparent non-equimolar amount of the three viral rna segments found previously in number of bunyaviruses [40, [71] [72] [73] . since pdi is a soluble protein which is thought to be retained at the cell surface through non-covalent interactions [70] , and the secretion of pdi has been observed from a number of cell types [70, 74] , we investigated whether pdi and erp57 not only appear at the surface of nsdv-infected cells but are also secreted from these cells. vero cells were again infected with nsdvi and culture supernatants (i.e. the cell medium) and the cells themselves were harvested, and proteins from supernatants and/or cell lysates were analysed by western blotting using specific antibodies. before harvesting, cells were checked by light microscopy for the appearance of any cytopathic effect (cpe), to ensure that only the medium from cells which appeared healthy was used in this study. after 48 h, in cells infected with nsdvi at a low moi, no cpe was observed in the culture, but a clear increase in levels of pdi and erp57 was detected in the medium. these proteins were found in the medium from infected cells but not from uninfected cells (figure 7) , while cellular proteins such as tubulin or calnexin were not found in the medium (figure 7) , indicating that the pdi, erp57 and nsdv n protein were not simply coming from ruptured cells. the nsdv glycoproteins mature in the er and golgi and their synthesis, like that of most glycoproteins, will involve the er chaperones. it is therefore possible that the viral glycoproteins may be involved in the redistribution of pdi/erp57 in infected cells. in order to visualise this directly, we labelled infected cells with rabbit anti-erp57 and mouse anti-pregn. it was observed that erp57 is redistributed in infected cells so that it is essentially all in the specific areas occupied by viral glycoproteins (figure 8.a) , suggesting that these chaperones may be recruited to the parts of the er involved in synthesising the viral glycoproteins. although we could not carry out a similar study of pdi/pregn colocalisation (no good rabbit anti-pdi was found), a similar association of pdi and viral glycoprotein is strongly suggested by studies in which we lysed infected cells and immunoextracted the lysates using mouse anti-pdi (clone rl90) antibody. we found pregn, but not n, among proteins co-precipitated by anti-pdi antibody from infected cells (figure 8 .b), indicating that pdi appears to interact, directly or indirectly, with pregn and possibly other of the viral glycoproteins in nsdv-infected cells (figure 8 .b) (note that reverse immunoprecipitation with anti-pregn was not figure 1 except that, after fixing with 3% pfa, cells were labelled with mouse anti-pdi (clone 1d3; a-c), mouse anti-pdi (clone rl90; d-f) or rabbit anti-erp57 (g-i). then cells were again fixed with 3% pfa, opened with ice-cold methanol, and stained with rabbit antiserum against the viral n protein (a-f) or with mouse anti-pregn antibody (g-i). proteins were visualised by co-staining with alexa fluor 488 goat anti-mouse igg (green) and alexa fluor 568 goat anti-rabbit igg (red). b. vero cells were infected with the nsdvi isolate at a moi of 6 tcid 50 or left uninfected. after 16 h, cells were fixed using 3% pfa and left nonpermeabilised. cells were incubated with mouse anti-pdi (clone 1d3) antibody followed by alexa fluor 488 goat anti-mouse igg (green). nuclei were counterstained using dapi (blue). bars correspond to 40 mm. doi:10.1371/journal.pone.0094656.g004 export of er proteins in nairovirus-infected cells plos one | www.plosone.org possible since the monoclonal antibody in question appears to recognise only denatured pregn). we constructed plasmids expressing nsdv glycoproteins pregn, ns m or pregc to determine which glycoprotein(s) were responsible for the redistribution of pdi/erp57. in silico analysis of the protein sequence encoded by the nsdv m segment suggested that the nsdv glycoproteins have a similar organisational arrangement to that described for cchfv [50] , although with some differences in the exact lengths of the different proteins. transmembrane (tm) regions and signalase cleavage sites were predicted using tmhmm server v. 2.0 and signalp 4.0 server [75] respectively; six hydrophobic regions tm 0 (n-terminal), tm 1 (631-653 aa), tm 2 (751-773 aa), tm 3 (786-803 aa), tm 4 (907-928 aa) and tm 5 (1524-1546 aa) were predicted with signalase cleavage sites just after the hydrophobic sequences of tm 0 (19q20 aa), tm 2 (770q771 aa) and tm 4 (927q928 aa). based on the nsdv m topology prediction, plasmids encoding pregn, ns m and pregc (corresponding respectively to aa 1-770, 750-927 and 907-1624 of the nsdvi m open reading frame) were constructed in which each protein was in frame with a carboxyterminal v5 epitope tag. expression of these constructs was tested in vero cells by western blotting and immunofluorescence to ensure that proteins of the expected size and localising to the expected compartments were expressed. the plasmid-expressed pregn-v5 was found in the er and cisgolgi, while the pregc-v5 was found only in the er, with no detectable colocalisation with the cisgolgi marker ( figure s5 a-f) . some of the plasmid-expressed ns m -v5 protein colocalised with the cisgolgi marker, however ns m -v5 also appeared in other compartments of the secretory pathway, one of which could be ergic ( figure s5 g-i) . vero cells were transfected with the pregn-v5, pregc-v5 and ns m -v5 expression constructs and their effect on pdi distribution in transfected cells was investigated by confocal microscopy. cells which expressed pregn-v5 lost pdi from the er (figure 9 a-c), while expression of pregc-v5 or ns m -v5 had no apparent effect on the pdi distribution (figure 9 d-i), suggesting that it is the (pre)gn but not (pre)gc or ns m which is primarily responsible for the translocation of pdi, and possibly erp57, to the cell surface. although in some cells over-accumulation of pregn-v5 appeared to change the morphology of the er, the cells which showed only moderate or low levels of pregn-v5 in the er also lost pdi from this compartment. in cells in which pregn-v5 was found only in the golgi (i.e. cells which expressed pregn at a very low level) no change to pdi was observed, suggesting that pregn needs to accumulate in the er, even at low levels, in order to affect the distribution of pdi. pregn-v5 had no effect on the distribution of calnexin (figure 9 j-l), indicating that the pregn-v5 does not affect the overall er structure when expressed at moderate levels, similarly to what was observed in nsdv-infected cells. as expected, expression of the viral n protein or the n-terminal half of the viral l protein (la) [62] had no effect on pdi distribution ( figure s6 ). taken together, these data suggest that nsdv infection causes the re-localisation of er soluble proteins, notably pdi and erp57, to the cell surface and ultimately the medium surrounding the infected cell, and this re-localisation appears to be due to association of these proteins with the nsdv (pre)gn glycoprotein. while both nsdv and cchfv are nairoviruses which cause a highly pathogenic haemorrhagic disease in their natural hosts, little is known about the mechanism of pathogenesis of these viruses; even the primary cellular targets are currently unknown. to better understand the replication of nairoviruses we have studied nsdv and its effects on the cellular compartments in infected cells, particularly concentrating on the host cell secretory pathway, which is used by nairoviruses for budding and egress of newly generated virions. despite a high throughput of viral glycoproteins processed through the secretory compartments, and viral budding in the golgi, the structure of the er and post-er compartments appeared to be unaffected in nsdv-infected cells when investigated using immunofluorescence. while what appear to be swollen golgi cisternae and vacuolisation of the cytoplasm has been observed by electron microscopy in nsdv-infected cells [35] , it is possible that changes to the golgi observed by electron microscopy are not extensive enough to be detected by confocal microscopy, or are not reflected in changes to the distribution of protein markers of those compartments. our findings agree with the absence of changes to the golgi observed in cells infected with cchfv or hantaan virus (htnv; a hantavirus) [76, 77] . in contrast, bunyamwera virus (bunv; an orthobunyavirus)-infected cells showed a condensed phenotype of the cis and transgolgi [78] [79] [80] , indicating that different bunyaviruses may have different effects on the golgi. while the ergic has been proposed as the first site of budding for phleboviruses, orthobunyaviruses and hantaviruses [76, [79] [80] [81] [82] [83] , no specific accumulation of the nsdv n colocalising with the ergic marker was observed in our studies. although some overlapping of n and ergic53 signal was noticed in some of the infected cells, such apparent colocalisation appeared to be due to the accumulation of very high levels of n protein in those cells. while the structure of the er remains unchanged, nsdv infection led to the redistribution of the soluble oxidoreductases pdi and erp57 from the er to the cell surface, and ultimately to the extracellular space (i.e. cell medium). redistribution of pdi has been observed also during replication of other viruses. for instance, pdi was redistributed from the peripheral er to figure 6 . time course of changes to pdi in nsdv-infected cells. vero cells were infected with the nsdvi isolate at a moi of 6 tcid 50 and fixed at 8, 12 and 16 hpi. for internal staining (a-i) cells were fixed using 3% pfa followed by ice cold methanol. cells were co-stained with mouse anti-pdi (clone 1d3) antibody and rabbit antiserum against the viral n protein. for surface pdi staining (j-l), cells were fixed with 3% pfa followed by staining with mouse anti-pdi (clone 1d3) antibody. then cells were again fixed with 3% pfa, opened with ice-cold methanol, and virus was detected by rabbit antiserum against the n protein. proteins were visualised with alexa fluor 488 goat anti-mouse igg (green) and alexa fluor 568 goat anti-rabbit igg (red). nuclei were counterstained using dapi (blue). bars correspond to 16 mm. doi:10.1371/journal.pone.0094656.g006 perinuclear membranous viral factories induced by kunjin virus (kunv; genus flavivirus) [84] . it is currently unknown whether kunv selectively redistributes pdi or also affects other er proteins; since the kunv glycoproteins e and m mature in the er [85] and kunv generates its viral factories by remodelling both the er and transgolgi [84] , it is possible that in kunvinfected cells the normal retrieval system of er proteins may not work. interestingly, despite changes to the er, no major effect on the golgi structure was found by immunofluorescence in kunvinfected cells [84] . another flavivirus, dengue virus (denv) (which, like nsdv, can cause a haemorrhagic fever), increased surface levels of pdi in infected endothelial cells [86] . as in our observations on nsdv-infected cells, total pdi levels were not affected in denv-infected cells [86] ; at present it is unknown whether denv replication affects the distribution of intracellular pdi. although some redistribution of pdi was found in bunvinfected vero cells, these changes most probably result from fragmentation of the er induced by the virus and this effect on pdi appears to be vero-specific, since these changes have not been observed in bunv-infected bhk21 cells [79] . no effect on pdi was detected in htnv-infected cells [76] . redistribution of soluble er proteins pdi, erp57 and colligin (a collagen-binding protein) has also been found in cells infected with african swine fever virus (asfv) [87, 88] , a dna virus which belongs to the family asfarviridae and which causes a severe haemorrhagic disease in domestic pigs. in asfv-infected cells, pdi, erp57 and colligin were redistributed from the er to the ergic (which contained the asfv protein pxp124l) or even entirely disappeared from the er, while the structure of the er was not affected [87] . total cellular levels of pdi, erp57, and colligin were not affected in asfv-infected cells and redistribution of these er soluble proteins was also observed in cells containing plasmid-expressed viral protein pxp124l, suggesting that these er proteins are not physically redistributed due to wrapping of the asfv progeny virions in the er but due to a specific effect of pxp124l. interestingly, all these very distinct viruses (a negativestrand rna virus, positive-strand rna viruses or a dna virus) use membranes of the secretory pathway to assemble their virions [35, 85, 89, 90] , and nsdv, denv and asfv all cause haemorrhagic disease [2, 91, 92] . the underlying mechanism for the redistribution of these soluble er proteins in nsdv infected cells remains to be determined. pdi and erp57, which contain an er retention motif at their carboxy-terminus, would normally be bound to kdel receptors in the ergic or cisgolgi and recycled back to the er, although it is known that this process is not totally efficient, and pdi at least has been observed to be released from some cells even under normal conditions [70, 74] . what is more, its release is stimulated in activated endothelial cells [93, 94] . as might be expected due to the generation of large amounts of viral glycoproteins in the er, nsdv induces the unfolded protein response (upr) in infected cells (unpublished observation); however, it has been reported that the upr appeared to diminish pdi secretion [74] , so it is unlikely that virus-induced upr is the cause of the pdi redistribution observed. none of the predicted glycoproteins of nsdv have a c-terminal sequence recognised by kdel receptors, so the viral proteins cannot be competing with the host cell proteins for kdel receptors, unlike the case of asfv pxp124l, the effects of which on er proteins is dependent on its own c-terminal kdel motif [87] . interaction of er chaperones (e.g. pdi, bip, calnexin and/or calreticulin) with viral glycoproteins during their folding in the er would be expected and has been shown for htnv and uukv [95] [96] [97] . it remains to be determined why the pdi is not recycled from the ergic or cisgolgi in this case, and why only the pregn protein appears to be involved in pdi distribution and not pregc. figure 9 . the effect of nsdv glycoprotein expression on pdi. vero cells were transfected with 1 mg of pcaggs_mcsii_pregn_v5 (a-c and j-l), pcaggs_mcsii_pregc_v5 (d-f) or pcaggs_mcsii_ns m _v5 (g-i). after 24 h, cells were fixed with 3% pfa followed by ice-cold methanol, and were stained using mouse anti-pdi (a-i) or anti-calnexin (j-l) antibodies followed by alexa fluor 568 goat anti-mouse igg (red). plasmid-expressed proteins were visualised with mouse anti-v5 antibody conjugated to alexa fluor 488 (green). nuclei were counterstained using dapi (blue). bars correspond to 40 mm. arrows in a-b and j-k indicate cells expressing pregn. doi:10.1371/journal.pone.0094656.g009 it is possible that bound pregn makes pdi not available for kdel receptor. while we have been unable to detect a separate mucin-like protein/gp38 protein, such as that cleaved from the cchfv pregn protein by an ski-1-like protease in the cisgolgi [57, 58] , in nsdv-infected cells, it may be that final folding of the nsdv (pre)gn occurs late in the exit pathway of the virion so that pdi is still required for protein maturation, even after virion assembly. further maturation of newly synthesised virions in post-transgolgi network has been observed in bunv-infected cells [79, 80] , supporting the possibility that pdi might play a role in late maturation of nsdv. late maturation, or even virion-bound pdi, may be required to prevent fusion of the viral membrane with the membrane of the secretory compartments during virus egress. fusion of the cchfv virion with the endosomal membrane is induced at ph 5.5 to 6.0 [98] , which is within the range of ph observed in the lumen of post-golgi compartments (reviewed in [99] ), so a system which prevented fusion of newly synthesised virions with intracellular membranes prior to virus egress would be of benefit. an alternative is that the virus benefits by inducing pdi release from cells because the pdi is required for virus entry. extracellular pdi, with its potential for rearranging disulphide bonds within viral glycoproteins, is thought to aid during entry of several viruses [86, [100] [101] [102] [103] [104] [105] ; free thiol groups in the f fusion protein of newcastle disease virus or the gp120 subunit of the human immunodeficiency virus env glycoprotein are critical for virus entry and their presence is thought to be mediated by cell surface pdi [100] [101] [102] [103] [104] [105] . recent data suggest that free thiol groups at the surface of hantaviruses might also play a role in infectivity of these viruses [106] . whether nsdv derives a selectable advantage from the redistribution of pdi and erp57 in infected cells or this redistribution is a bystander effect of the nsdv infection, extracellular pdi may contribute to the pathogenicity observed during nsdv infection. extracellular pdi can still reduce, oxidise and rearrange disulphide bonds and therefore activate molecules with which it interacts at the cell surface or in the extracellular space (reviewed in [107, 108] ). extracellular pdi has been shown to activate b1 and b3 subunit-containing integrins on endothelial cells and platelets [86, 93, 109] . integrins are known to take part in the cell-cell interactions promoting cell migration and vasodilation (reviewed in [110] ), processes which are enhanced during viral haemorrhagic fevers and which contribute to the haemorrhagic nature of the pathogenesis (reviewed in [111] ) e.g. denv-induced translocation of pdi to the cells surface has been shown to activate integrins on infected endothelial cells [86] . secreted pdi could also activate integrins at the surface of various uninfected cells, including endothelial cells and platelets, leading to increased vasodilatation and coagulopathy. extracellular pdi has been shown to activate tissue factor (tf) [112] , inducing intravascular coagulation, which may subsequently lead to disseminated intravascular coagulation (dic) through depletion of platelets and clotting factors, resulting in haemorrhaging [113] [114] [115] [116] ; dic has been observed in many cchf patients [31] [32] [33] 117] . finally, since erp57 (and probably pdi) takes part in the folding of the major histocompatibility complex (mhc) class i and therefore assists in facilitating immune surveillance and elimination of virus-infected cells [118,119,120,] viruses which translocate these er chaperones from the er may avoid adaptive immune responses, although down-regulation of mhc class i has not yet been described for nairovirus-infected cells. in this study we have demonstrated that nsdv induces the redistribution of soluble er oxidoreductases, specifically pdi and erp57, in infected cells, and that the viral pregn glycoprotein appears to be involved in this process. redistribution of pdi from the er of nsdv-infected cells to the cell surface and/or the extracellular environment may play a crucial role in the pathogenicity of nsdv. pdi may enhance virus infection or may contribute to the observed haemorrhagic outcome of the infection, although the exact importance of extracellular pdi and erp57 in natural infection remains to be determined. in recent years, pdi has become a popular potential drug target for treatment of thrombosis, neurodegenerative diseases and hiv infection [69, 121, 122] and it appears that pdi could also act as a target for treatment of at least some viral haemorrhagic fevers [86] . while a wide range of applications makes pdi inhibitors attractive agents, extracellular pdi appears to take part in a broad range of physiological functions, and therefore drug safety might become a limiting factor for the development of drugs which interfere with enzymatic activity of extracellular pdi. figure s1 effect of nsdv infection on the cellular cytoskeleton. vero cells were infected with nsdvi at a moi of 0.3. after 16 h, cells were fixed using 3% pfa and (a-f) stained using alexa fluor 488 phalloidin (green; actin) and rabbit anti-n, followed by alexa fluor 568 goat anti-rabbit igg (red); (g-o) cells were opened with ice-cold methanol and stained using mouse antia-tubulin antibody and rabbit anti-n, followed by alexa fluor 488 goat anti-mouse igg (green) and alexa fluor 568 goat anti-rabbit igg (red). nuclei were counterstained using dapi (blue). dashed boxes in a-c and g-i indicate the source of the enlarged areas shown in d-f and j-l, respectively. (tif) figure s2 effect of nsdv infection on pdi in human and bovine cell lines. a549 (human lung) cells (a-c) or bfa (bovine foetal aortic endothelial) cells (d-i) were infected with nsdvi at a moi of 0.3. after 16 h (a-c) or 72 h (d-f) cells were fixed and stained using specific antibodies. (a-f) cells were fixed using 3% pfa followed by ice-cold methanol, and then stained with mouse anti-pdi (clone 1d3) and rabbit anti-n. (g-i) cells fixed with 3% pfa only, labelled with mouse anti-pdi (clone 1d3), then again fixed with 3% pfa, opened with ice-cold methanol and stained with rabbit anti-n. proteins were visualised using alexa fluor 488 goat anti-mouse igg (green) and alexa fluor 568 goat anti-rabbit igg (red). nuclei were counterstained using dapi (blue). bars correspond to 40 mm. (tif) figure s3 time course of changes to erp57 in nsdvinfected cells. vero cells were infected with nsdvi at a moi of 6 and fixed at 8, 12 and 16 hpi. (a-i) cells were fixed using 3% pfa followed by ice cold methanol and stained with rabbit anti-erp57 antibody and mouse anti-pregn. (j-l) cells were fixed with 3% pfa, labelled with rabbit anti-erp57 antibody, again fixed with 3% pfa, opened with ice-cold methanol, and labelled using mouse anti-pregn. proteins were visualised with alexa fluor 488 goat anti-mouse igg (green) and alexa fluor 568 goat anti-rabbit igg (red). nuclei were counterstained using dapi (blue). bars correspond to 16 mm. (tif) figure s4 differences in the expression levels of n and pregn in nsdv-infected cells. samples were prepared as for figure 1 except that cells were stained with rabbit anti-n protein and mouse anti-pregn antibody. proteins were visualised using alexa fluor 488 goat anti-mouse igg (green) and alexa fluor 568 goat anti-rabbit igg (red). nuclei were counterstained using dapi (blue). bars correspond to 40 mm. arrows indicate cells where n but not pregn was detected. (tif) figure s5 localisation of plasmid-expressed nsdv glycoproteins. vero were transfected with 1 mg of pcaggs_mcsii_pregn_v5 (a-c), pcaggs_mcsii_pregc_v5 (d-f) or pcaggs_mcsii_nsm_v5 (g-i). after 24 h, cells were fixed with 3% pfa followed by ice cold methanol, and incubated with mouse anti-gm130 (cisgolgi) antibody, followed by alexa fluor 568 goat anti-mouse igg (red). then plasmid-expressed proteins were visualised with anti-v5 antibody conjugated to alexa fluor 488 (green). nuclei were counterstained using dapi (blue). bars correspond to 20 mm. (tif) figure s6 the effect of nsdv protein expression on pdi. vero cells were transfected with 1 mg of pcdna6-gv-n (ac) or pcdna6-gv-la (d-f). after 24 h, cells were fixed with 3% pfa followed by ice-cold methanol, and were stained using mouse anti-pdi (clone 1d3) and rabbit anti-n (a-c) or rabbit anti-l (d-f). proteins were visualised with alexa fluor 488 goat anti-mouse igg (green) and alexa fluor 568 goat anti-rabbit igg (red). nuclei were counterstained using dapi (blue). bars correspond to 40 mm. 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histocompatibility complex class i molecules by protein disulfide isomerase functions of erp57 in the folding and assembly of major histocompatibility complex class i molecules running the gauntlet: from peptide generation to antigen presentation by mhc class i protein disulfide isomerase inhibitors constitute a new class of antithrombotic agents inhibitors of protein disulfide isomerase suppress apoptosis induced by misfolded proteins we would like to thank professor robert b. tesh and dr piet van rijn for the gift of the gv and nsdv isolates; dr nathalie vachiery for the cje cells; professor adolfo garcia-sastre for pcaggs-mcsii plasmid; dr philippa hawes and dr christopher netherton of this institute for antibodies against gm130, p102, ergic53, pdi (clone 1d3) and erp57. key: cord-000937-8vk89i4h authors: law, john; jovel, juan; patterson, jordan; ford, glenn; o’keefe, sandra; wang, weiwei; meng, bo; song, deyong; zhang, yong; tian, zhijian; wasilenko, shawn t.; rahbari, mandana; mitchell, troy; jordan, tracy; carpenter, eric; mason, andrew l.; wong, gane ka-shu title: identification of hepatotropic viruses from plasma using deep sequencing: a next generation diagnostic tool date: 2013-04-17 journal: plos one doi: 10.1371/journal.pone.0060595 sha: doc_id: 937 cord_uid: 8vk89i4h we conducted an unbiased metagenomics survey using plasma from patients with chronic hepatitis b, chronic hepatitis c, autoimmune hepatitis (aih), non-alcoholic steatohepatitis (nash), and patients without liver disease (control). rna and dna libraries were sequenced from plasma filtrates enriched in viral particles to catalog virus populations. hepatitis viruses were readily detected at high coverage in patients with chronic viral hepatitis b and c, but only a limited number of sequences resembling other viruses were found. the exception was a library from a patient diagnosed with hepatitis c virus (hcv) infection that contained multiple sequences matching gb virus c (gbv-c). abundant gbv-c reads were also found in plasma from patients with aih, whereas torque teno virus (ttv) was found at high frequency in samples from patients with aih and nash. after taxonomic classification of sequences by blastn, a substantial fraction in each library, ranging from 35% to 76%, remained unclassified. these unknown sequences were assembled into scaffolds along with virus, phage and endogenous retrovirus sequences and then analyzed by blastx against the non-redundant protein database. nearly the full genome of a heretofore-unknown circovirus was assembled and many scaffolds that encoded proteins with similarity to plant, insect and mammalian viruses. the presence of this novel circovirus was confirmed by pcr. blastx also identified many polypeptides resembling nucleo-cytoplasmic large dna viruses (ncldv) proteins. we re-evaluated these alignments with a profile hidden markov method, hhblits, and observed inconsistencies in the target proteins reported by the different algorithms. this suggests that sequence alignments are insufficient to identify ncldv proteins, especially when these alignments are only to small portions of the target protein. nevertheless, we have now established a reliable protocol for the identification of viruses in plasma that can also be adapted to other patient samples such as urine, bile, saliva and other body fluids. traditionally, techniques such as cell culture, electron microscopy and serology have been instrumental in discovering unknown viruses. these procedures led to the identification of the viruses causing hepatitis a and b [1, 2] , acquired immune deficiency syndrome [3] , and acute respiratory syndrome [4] . classical microbiology and molecular biology methods also led to the somewhat serendipitous discovery of acanthamoeba polyphaga mimivirus [5] , a member of the nucleo-cytoplasmic large dna viruses (ncldv) that has been proposed to constitute a fourth domain of life [6] . in contrast, the discovery of hepatitis c virus (hcv) required a more targeted approach employing screening of bacteriophage display libraries with immune serum, thus heralding a new era of virus discovery [7] . more recently, an unbiased approach has been used to identify viruses by dnase treatment of serum, followed by amplification and sequencing of the nucleic acids protected in virions [8] . using this method, not only was hepatitis b virus (hbv) identified in the serum of an infant with acute hepatitis b, but so too were two species of bovine parvoviruses, possibly because bovine serum was used to dilute the experimental samples [8] . with the advent of next generation sequencing (ngs) [9] , the application of metagenomics to virus discovery has become more feasible. for instance, a metagenomic survey of the microflora of hives and royal jelly led to the identification of israeli acute paralysis virus in honeybee colonies exhibiting collapse disorder [10] . a similar approach was used to study the etiology of a human disease by sequencing cdna from a merkel cell cancer, where a viral transcript corresponding to a novel polyomavirus was identified and referred to as the merkel cell polyomavirus [11] . a metagenomic survey was also employed to examine infected tissues from three patients that had received liver or kidney transplantation from the same donor [12] . blastx alignments of 94,000 sequences yielded 14 hits that shared high similarity to lymphocytic choriomeningitis virus. the infectivity of the virus was subsequently confirmed combining cell culture, electron microscopy, and immunochemical assays [12] . additional contributions of metagenomics to clinical virology include the identification of a bunyavirus in patients with thrombocytopenia and leukopenia syndrome [13] . characterizations of the human gut virome have revealed the diversity and predominance of bacteriophages in this environment [14, 15] . recently, ngs was employed to determine the complexity of the human virome in febrile children with acute diarrhea [16, 17] . in addition, novel viruses from human and animal samples have been discovered, although their pathogenicity remains to be ascertained in most cases [18] [19] [20] [21] [22] [23] . ngs metagenomics has also been used in several environmental studies to discover new species of viruses [24, 25] . these studies estimated an abundance of 10 7 viruses per milliliter of water in marine environments [24] . others have identified a series of giant viruses in addition to the acanthamoeba polyphaga mimivirus [26] and characterized virophages as small viruses that infect larger viruses [27, 28] . detection of viruses in patients suffering from chronic disease adds an additional challenge because viral burden may be diminished with disease progression. nevertheless, ngs technologies are evolving at a rapid pace to discover unknown agents and an accompanying body of free software is currently available for data analysis. accordingly, our goal was to provide a detailed method for the construction of unbiased metagenomic libraries from body fluids, as well as a thorough bioinformatics pipeline for the analysis of sequencing data. in this report, we describe a viral metagenomic survey in plasma from patients affected by several hepatic disorders. dna and rna libraries enriched for viruses were sequenced using the illumina gaii platform. over 300 million high quality sequences were analyzed, enabling the identification of a series of viruses previously diagnosed by conventional assays as well as of several novel viral sequences. plasma samples were obtained from patients who read and signed the consent form according to protocols that were reviewed and approved by the human research ethics boards of the university of alberta. viral particles were enriched from 4-8 ml of plasma, from either single patients or pooled samples of several patients with the same diagnosis, depending upon sample availability. plasma was spun down at 2,6556g, for 5 min, at 4uc. the supernatant volume was brought up to 20 ml with tn buffer (0.1 m tris, ph 7.6; 0.1 m nacl), and the suspension run through a 0.2 mm midjet column (ge healthcare) at 4uc, to filter out bacterial and human cells. samples were concentrated in a final volume of 8 ml by tangential flow filtration, using 300 kda midjet columns at 4uc. half of the concentrated suspension was kept at 285uc. the remaining 4 ml were treated with 50 u/ml benzonase (sigma) in 2 mm mgcl 2 , at 21uc for 1.5 h. rna was extracted from 2 ml of concentrated suspension with the qiaamp viral rna mini kit (qiagen) according to manufacturer's instructions. as multiple columns were needed to process the 2 ml sample, all 60 ml eluates were pooled. the rna was then concentrated and treated with dnase i in a single column, using the qiagen rneasy microkit as recommended in the rna cleanup protocol, except that the dnase i amount was raised to 81 u. rna was finally eluted in 25 ml rnase-free water. dna was isolated from the other 2 ml fraction of concentrated suspension, using the dneasy blood and tissue kit (qiagen) as described in the purification from animal blood or cells protocol, including treatment with rnase a. dna was finally concentrated and eluted from all columns by sequentially passaging the same 80 ml buffer ae aliquot (,40 ml were recovered from the last column). seven rna libraries (aihp01, hbvp02, hcvp02, hcvp03, hcvp05, nshp01, norp01) and seven dna libraries (aihp01d, hbvp02d, hcvp02d, hcvp03d, hcvp05d, nshp01d, norp01d) were constructed from patients with autoimmune hepatitis (aih), hepatitis b virus (hbv) chronic infection, hepatitis c virus (hcv) chronic infection, non-alcoholic steatohepatitis (nash), and healthy subjects (nor). plasma from four patients attending an outpatient surgery ward without any known diagnosis of liver disease were pooled and used as control (sample norp01). plasma from three patients affected by aih or nash were also pooled in samples aihp01 and nshp01, respectively. the rest of samples derived from individual patients. for rna library preparation, first strand cdna was synthesized from half of the rna amount obtained during purification (usually below detection limit by spectrophotometry) in the presence of 3 mg of random hexamers (invitrogen), 0.5 mm dntps, 40 u of rnaseout (invitrogen), 100 u of reverse transcriptase superscript ii (invitrogen), and 1x first strand buffer (provided by the manufacturer), for 50 min at 42uc. for second strand synthesis, the first strand reaction was supplemented with 50 mm tris ph 7.8; 5 mm mgcl 2 , 0.3 mm dntps, 1 mm dtt, 2 u of rnase h and 50 u of dna pol i, and further incubated at 16uc for 2.5 h. for dna library preparation, purified dna was sheared in a covaris s2 instrument, using 6616 mm afa fiber snap-cap microtubes (covaris), and recommended conditions to generate a target peak of 200 nt (duty cycle: 10%, intensity: 5, cycles per burst: 200, treatment time: 180 sec, water bath temperature: 7uc). from this point on, rna and dna libraries were processed with a common protocol. dna protruding ends were blunted with the nebnext end repair module (neb). a 39 -da overhang was added with the nebnext da-tailing module (neb). finally, ligation-competent dna molecules were ligated to paired-end (pe) adapters harboring 39 -dt overhang (illumina) using the nebnext quick ligation module (neb), all according to protocols provided by manufacturers. samples were cleaned up using the qiaquick pcr purification kit (qiagen) after end repair or da-tailing and with the minelute pcr purification kit (qiagen) after ligation. elution volumes were adjusted as required for the next reaction. after ligation, samples were fractionated in a 2% agarose gel and a thin slice at ,300 nt was excised with 4.0 mm61.0 mm genecatcher disposable gel excision tips (gel company) and the dna was recovered with the qiaquick gel extraction kit (qiagen). libraries were then amplified with 50 u of pfxiii polymerase (invitrogen) in the presence of 1x reaction buffer (provided by the manufacturer), 1 mm primers [29] and 0.25 mm dntps. after 18 pcr cycles, amplification was titrated in a 2100 bioanalyzer (agilent) and additional pcr cycles were added as needed. when required, amplified adapter dimmers were removed with agencourt ampure magnetic beads (beckmann coulter). libraries were size selected on a 2% agarose gel. all libraries were sequenced at bgi-shenzhen (china), on a genome analyzer ii instrument (illumina). sequencing data is available from the sra repository under the following accession number: sra054231. low quality bases (quality score ''b'') were trimmed from the 39 end of each read end, and the remaining sequence was kept only when its high quality 59 moiety was longer than 29 nt. primer/ adapter sequences and low complexity regions were also trimmed out. soapaligner [30] was used to remove mitochondrial and ribosomal rnas, using the default parameters. repeatmasker [31] was then used with a database containing simple repeats, ribosomal and mitochondrial sequences, to further filter the reads with higher sensitivity. the remaining sequences were considered clean reads. to minimize cpu time, clean reads were aligned to the human, bacteria, and virus databases from ncbi, using soapaligner with the default parameters. remaining read ends were aligned with standalone blastn against the abovedescribed databases. an e-value of 1e-05 was used as the cutoff. in the specific case of the human database, 80% coverage and 80% identity were also required. when a query hit more than one taxon, hits were sorted by e-value and additional taxa were kept if their hits were within two nucleotides of the top hit. for example, if a query hit the human database with 55 identical nucleotides and the virus database with 53 identical nucleotides, the query sequence was then assigned to the ambiguous category humanvirus (hv). additionally, the taxonomy assigned to each end of a pair was compared and the ambiguity solved when both ends intersected the same taxon. for instance, for a given pair, if one end is classified as hv and its counterpart as h, this pair was reclassified as h-h. read ends that could not be classified were binned as unknown. non-human and non-bacteria (unknown, viral, phage, human endogenous retroviruses [herv] and ambiguous) read ends were subjected to de novo assembly with the soapdenovo-trans software (http://soap.genomics.org.cn/soapdenovo-trans.html), using the default parameters. we chose the transcriptome assembler soapdenovo-trans, instead of the older genome assembler soapdenovo, because it takes into account the problem of uneven coverage, which is present in both transcriptomic and metagenomic libraries. assembled scaffolds were aligned with blastx against the 'nr' taxonomy databases, including archaea, bacteria, herv, fungus, plant, human, invertebrate, mammal, phage, protist, vertebrate and virus entries. as before, an e-value cutoff of 1e-05 was used and the top hit was reported. in the specific case of phycodnavirus and mimivirus top hits, some scaffolds were reanalyzed with the hhblits algorithm, using the most recent version of the uniprot20 database [32] . hhblits utilizes profile hidden markov models to represent both query and database sequences; these profiles are then aligned using hhsearch [33] . total rna was isolated from 140 ml of plasma using the qiaamp viral rna kit (qiagen), followed by random-primed cdna synthesis using 1 mg of rna and superscriptii, as described above. pcr was performed using platinum taq high fidelity polymerase (invitrogen) in the presence of 0.5 mm hcvspecific primers (hcv-1: ctcccctgtgaggaac-tactgtct, hcv-2: ctcgcaagcaccctatcagg-cag), which anneals to conserved motifs in the 59 untranslated region of the hcv genome. likewise, dna was used as template to detect ttv with the following primers ttv-1: gtgggactttcacttgtcggtgtc, ttv-2: ga-caaatggcaagaagataaaggcc. a fragment of scaf-fold2306 was amplified using primers circo1: tgtcatagg-caaacctagcaccgt and circo2: tatgtggacccatccgaaagcctt. pcr conditions were as recommended by the polymerase manufacturer. the hcv and ttv primers bind to conserved sequences in many strains of the corresponding virus, while circovirus primers were deduced from the sequence of scaffold2306 (sequence available upon request). we defined the complexity of viruses present in the plasma of patients with chronic liver disease, using metagenomics. we chose plasma to filter out bacterial and human cells with the consequent enrichment of virus particles. in the context of virus identification and discovery using ngs, the removal of bacterial and human cells is highly desirable because their ribosomal and mitochondrial rna fractions are highly represented contaminants that often hinder detection of viral nucleic acids. our viral preparations were made from pooled plasma samples and split into two fractions for the preparation of rna and dna libraries using a protocol that minimizes handling of samples to preserve virome diversity. plasma samples were obtained from patients with autoimmune hepatitis (aih), chronic hepatitis b virus (hbv) infection, chronic hepatitis c virus (hcv) infection, non-alcoholic steatohepatitis (nash), and healthy subjects (nor); each rna library was named by diagnosis (e.g. aihp01) and a suffix 'd' was added for each dna library (e.g. aihp01d). sequences were analyzed using an in-house bioinformatics pipeline depicted in figure 1 (see materials and methods). we performed a taxonomic classification of reads into human, bacteria, phage, human endogenous retroviruses (herv), viruses, and unknown categories (supplemental tables s1-s14). a significant fraction of reads in each library could not be unambiguously assigned to a definitive category; these were therefore included into several ambiguous categories describing the combinations of taxa that were matched (supplemental tables s1-s14; figure 2 , in brackets). notably, the vast majority of reads in each library did not bear resemblance to any of the taxa available in the ncbi databases; these were assigned to the category ''unknown'' (figure 2; supplemental tables s1-s14). they represent a pool of sequences that can potentially be assembled into new genomes or segments thereof. although our filtration procedure was intended for enrichment of virus particles, some human, bacterial and phage nucleic acids escape tangential flow filtration -most likely when present in a cell-free form. however, our focus was directed to the analyses of virus populations and virus discovery. using viral preparations, the ratio of known viral to non-viral read ends was 1:229 ( figure 2 ). the density of viral read ends was highly variable and ranged from 70 to 14,401 read ends/million for the rna libraries and 30 to 16,655 read ends/million for the dna libraries. in most cases where a relatively high density of viral sequences was found, there was a single predominant virus (supplemental tables s15-s28). for instance, in all libraries with more than 100,000 viral sequences, a single virus and its quasispecies accounted for more than 75% of viral sequence abundance ( figure 2 ; bar graphs). it is notable that dna viruses were detected in rna as well as dna libraries, such as hbv and torque teno virus (ttv). however, it is unknown whether this finding represents the detection of viral transcripts or the inability to eradicate dna in the rna libraries. the relative abundance of hbv-and ttv-like sequences in rna libraries compared to dna libraries was 0.20% and 0.13%, respectively (hbv in sample hbvp02:30 vs 14,786 read ends/million; ttv in sample nshp01:22 vs 16,483 read ends/million). to a lesser extent (about one read per million), we also detected sequences resembling rna viruses in our dna libraries (supplemental tables s15-s28 ). this may represent alignment inaccuracies or stretches of unknown dna viruses that resemble rna viruses. retroviruses constitute a special case, as they are rna viruses that retro-transcribe into dna, and integrate into human chromosomes. on occasion, the proviral genome is found in the viral particle and therefore retroviral sequences may be observed in both dna and rna libraries. viruses from diverse families were found within each library ( figure 3) . anelloviruses, small circular single-stranded dna viruses, were predominant in aih and nash dna libraries, but poorly represented in the rest of samples. as expected, hepadnaviruses were found in a patient diagnosed with hbv and we found abundant flaviviruses in patients diagnosed with hcv. of note, the predominant virus detected in library hcvp02 was not hcv, but gb virus c (gbv-c), a flavivirus that was also detected in the aih library (figures 2 and 3) . in general, the expected hepatitis viruses were detected in plasma from patients with the appropriate clinical diagnosis, but in some cases the deep sequencing provided additional information. few hits to viral sequences were found in the libraries norp01 and norp01d used as a negative comparison group for hepatotropic viruses, (ratio to non-viral reads 1:13,689; figure 2 ). nearly complete viral genomes were detected at high coverage in the libraries from patients with chronic viral hepatitis. for example, the dna genome of hbv was covered several thousand times by reads in library hbvp02d ( figure 4a ). a single locus was not covered in the hbv sequence that we used as our reference (nc_003977.1), possibly related to polymorphisms in different genotypes. relatively good coverage was also found in hcv libraries hcvp05 ( figure 4a ) and hcvp03 (data not shown). we also observed nearly full coverage of gbv-c in sample aihp01 ( figure 4b ) and hcvp02 (not shown) as well as ttv in samples nshp01d ( figure 4b ) and aihp01d (data not shown). since the aihp01 and nshp01 samples contained pooled plasma from three patients, it was not possible to ascertain which of these patients were infected by ttv or gbv-c. we replicated the aih libraries with serum from a different patient, but neither ttv nor gbv-c were detected (data not shown) suggesting no relationship between ttv and/or gbv-c and disease. however, detection of such viruses in our libraries illustrates the sensitivity of our approach to conduct unbiased diagnoses of viral agents in plasma. of note, we observed 128 sequences that aligned to the hcv genotype 1 in a library derived from pooled plasma of three patients with nash (patients 66, 153 and 179; figure 4c ). this was unexpected because patients suffering from nash are usually tested for the presence of hcv prior to diagnosis [34] . therefore, we conducted rt-pcr on rna from each patient's plasma in duplicate, using hcv-specific primers. we found that the plasma from one patient in the pool used for library nashp01 was positive for hcv rna ( figure 4d ). similarly, we were able to confirm the presence of ttv dna in patients affected by nash and aih, but not in those affected by hepatitis c (figure 4b ). we detected ten reads mapping to ttv in library hcvp05d, out of ,31 million sequences, which explains our inability to detect this virus in pcr experiments. reads that were not classified as human or bacteria were subjected to de novo assembly using the soapdenovo-trans algorithm, and the resulting scaffolds were used to query the non-redundant protein databases. assembled scaffolds were much more abundant in dna libraries and most of the recognizable scaffolds hit to bacterial proteins during blastx runs. these data suggest that most of the reads that could not be classified during alignments with blastn belong to unknown bacteria. more specifically, 11% to 55% of scaffolds aligned to bacterial proteins. nonetheless, the vast majority of scaffolds (44% to 85%) did not resemble any protein in the 'nr' database, which likely reflects proteins that have not yet been discovered. assembly of viral sequences was also possible for all viruses shown in figure 2 as the most abundant virus in each library (data not shown). in addition, we were able to identify a number of viruses that were not previously detected by blastn on the unassembled read ends (table 1 and supplemental tables s29-s42 ). the most conspicuous were scaffolds that encoded polypeptides resembling proteins found in viruses with circular genomes and in nucleocytoplasmic large dna viruses (ncldvs). during blastx alignments, scaffolds from several libraries ranging in size from 600 to 2100 nt hit the replicase sequences from various circoviruses. the most striking example was scaffold2603 in library aihp01d ( figure 5a ), which was ,2100 nt long. after refining its sequence with gapcloser for soapdenovo, we found that the sequence encoded a polypeptide sharing ,65% identity with the replicase of a bat circovirus (btcv; ael87784.1; blastx alignments e-value 7.6e-75; 95% coverage) that was recently identified in china [35] . we then reassessed scaffold2603 and found that it encoded an additional protein with weak similarity to a putative protein from the circovirus-like genome rw-e [25] . interestingly, we also assembled scaffolds resembling circoviruses in library nshp01d and, at lower frequency, in library norp01d. when the 582 original read ends from scaffold2603 were aligned by blastn to the genomes of btcv or rw-e, the two closest relatives, only a small proportion showed any similarity to the circovirus genomes ( figure 5a ), supporting the genetic plasticity previously reported for this genus of viruses [25, 35, 36] . cloning and sequencing of pcr fragments amplified from sample aihp01 verified the existence of sequences assembled into scaffold2603. the replicase-like amino acid sequence encoded by scaffold2603 was used to construct a phylogenetic tree of circoviral replicases and the novel protein sequence formed a separated branch with the two related circoviruses, but was divergent from the rest of the circovirus replicases ( figure 5b ). ncldv constitute an especially challenging group of viruses to detect because they may be eliminated by size filtration and also because portions of their large genomes (.1 mb) resemble those figure 1 . schematic of bioinformatics pipeline used for processing of ngs libraries. high quality reads, excluding ribosomal and mitochondrial sequences, were aligned against the taxonomy databases of ncbi using blastn (taxonomic classification). unclassified or ambiguously classified reads, together with virus, phage, and herv sequences were assembled into scaffolds. scaffolds were used to query the non-redundant protein database of ncbi using blastx to identify viral proteins with similarity to predicted polypeptides in our scaffolds (finding novel viruses). given the large genomes of ncldvs, hits to this class of viruses were reanalyzed with the profile hidden markov model-based algorithm hhblits. pcr and sanger sequencing were used to confirm the presence of novel viral-like sequences in our samples. doi:10.1371/journal.pone.0060595.g001 of higher eukaryotes [37] . ncldv include the families poxviridae, phycodnaviridae, iridoviridae and mimiviridae [38] . we found hits to ncldv in all libraries included in this report (figure 3 and supplemental tables s29-s42). although five conserved ortholog gene clusters have been identified within the genomes of all ncldv [39] , none of these were found in our bioinformatics analyses. given that phycodnaviruses and mimiviruses (hereafter referred to as large viruses) have not been reported to infect humans, we re-evaluated a portion of the large virus blastx top hits using the hhblits algorithm [32] and the swissprot20 database. when blastx and hhblits results were compared, it was observed that both algorithms aligned remarkably similar fragments of the query sequences in the corresponding top hits ( figure s1 ). nonetheless, 19 out of 44 cases reported by blastx as large viruses proteins were re-classified as proteins from other taxa by hhblits, mainly bacterial proteins; 18 instances produced identical results (i.e. both algorithms reported the same target protein as top hit); and seven corresponded to other large viruses proteins (supplemental table s43 ). however, all were partial alignments ranging from 36 to 232 amino acids, not the entire target protein. such divergent results are a cautionary note on the intrinsic difficulty of identifying ncldv proteins from partial alignments. it is not possible to conclude that such sequences represent bona fide large viruses proteins until further experimental evidence is gathered. in this study, we demonstrated the utility of ngs to analyze the complexity of dna and rna viral populations in the plasma of patients with chronic liver diseases. we assembled scaffolds with nucleotide sequences unrelated to all viruses reported in the databases; these sequences encoded polypeptides with similarity to previously identified viral proteins, which is consistent with the identification of novel viral agents. in libraries that contained abundant viral hits, a predominant virus was found, which usually coincided with the clinical diagnosis, such as chronic hbv and hcv infection. the exception was sample hcvp02, where the predominant species was gbv-c, another flavivirus found to replicate in primary t and b lymphocytes [40] . so far, no pathogenic properties have been attributed to gbv-c [40, 41] . indeed, it has been suggested that gbv-c co-infection reduces the severity of hiv infection by mechanisms that remain poorly understood [40] [41] [42] [43] . although we found that one patient initially diagnosed with hcv harbored high levels of gbv-c but only small traces of hcv in plasma, it should be noted that gbv-c abundance is higher in blood than in liver while the opposite is true for hcv [42] . neither aih nor nash have been linked with a specific microbial infection. however, some have speculated that aih may be triggered by viruses [44] while others have suggested that fat may accumulate in the liver in response to virus infection [45] . accordingly, there may be some interest in further defining the virome in plasma from these patients. the sample from patients diagnosed with aih harbored abundant ttv and gbv-c. ttv was even more abundant in plasma from the nash patient, but absent in patients with hepatitis b or c. originally, ttv was isolated from a japanese patient suffering from cryptogenic hepatitis [46] , and subsequently found in serum from patients with hepatitis lacking diagnostic markers for viral infection [47] . as with gbv-c, no pathogenicity has been ascribed to ttv [34] ; however, it was proposed that ttv may play an indirect role on carcinogenesis by modulating t cells immunological responses [48] . also, increased ttv burden has been found in the plasma from immunocompromised subjects [49, 50] . several other disorders have also been linked to anelloviruses [51, 52] , but a proof for their role in pathogenesis is still lacking. nevertheless, ttv seems to reunite many of the idealized attributes of a well-adapted virus, including persistence, high levels of replication, prevalence in the population, and high transmissibility [53] . in three of our samples (aihp01d, nshp01d and norp01d), we assembled scaffolds that contained orfs encoding proteins similar to the replicase of a bat circovirus recently isolated in china [35] and a putative protein from another circovirus found in environmental samples [25] . members in the family circoviridae are ubiquitous, highly diverse and induce immuno-suppression in birds; at least one contributes to the development of postweaning multisystem wasting syndrome in pigs. however, no pathogenicity has been demonstrated in humans, although they are often found in human feces and plasma [18] [19] [20] [21] [22] 36, 54] . although we found circovirus sequences in patients with nash and aih, their presence does not imply any linkage with liver diseases, and indeed they were also found in our control libraries. identification of viruses in ngs libraries relies on alignments to reference genomes or segments thereof. this method is reliable for identical or highly similar sequences but has two obvious limitations. first, viruses with novel sequences may not be detected. second, sequences found in taxonomically divergent species may trigger spurious hits that are difficult to resolve by computational methods. in this regard, a group of viruses that deserve special attention are ncldv that include mimiviruses and phycodnaviruses [26] . the first of the so-called giant viruses reported was acanthamoeba polyphaga mimivirus; more recently, giant marseillevirus, cafeteria roenbergensis virus and megavirus chilensis were isolated [26, [55] [56] [57] . the genomes of those viruses may extend up to ,1.2 mb and their gene complements can be as large as 1000, which by far exceeds the number of protein coding genes in many bacteria [26] . however, these large viruses have not been reported in human samples and, based on our results, we are not prepared to say that we have seen them in our samples either. by combining viral preparations from plasma samples, deepsequencing and computational approaches, we have shown here the identification of a series of viruses from patients affected by chronic hepatobiliary disorders. the incorporation of a viral preparation step increased the ratio of virus to human sequences (1:229) by nearly two log-fold in comparison to a recent metagenomic study of febrile and afebrile children's nasopharyngeal swabs and plasma samples, where approximately 1:16,000 viral to human sequences were reported [16] . we also recovered viruses at high coverage that were previously diagnosed by traditional serological methods, while unsuspected viruses were detected in several other samples at high density. finally, we defined a minimal threshold of abundance of viral read ends, figure 2 . viral read ends represent only a small fraction of libraries from plasma. pie charts: classification of reads from each library into human, bacteria, virus, and unknown categories (herv and phage sequences are not included as they were found at low frequency, not visible at the scale of the pie charts). the number of clean reads (high-quality sequences excluding ribosomal, mitochondrial, and low complexity sequences; in black), ambiguous reads (in brackets), viral reads (in red), and the density of viral reads per million (in blue) are indicated. bar graphs: red bars depict total number of viral reads (normalized to 100%) and green bars represent the percentage thereof that corresponds to the most abundant virus in each library (n.i. not identified; indicates that no predominant virus was identified during blastn alignments). doi:10.1371/journal.pone.0060595.g002 which suffices to detect viruses present at low levels. namely, hcv was detected in patients affected by nash disease, at a density of approximately seven read ends per million. to make a clinical diagnosis of nash, other liver disease agents such as hcv should be excluded and the diagnosis of nash was therefore erroneous in the one case where hcv was detected in the metagenomics library and confirmed by pcr. we anticipate that, due to its high sensitivity and the capability to simultaneously detect a broad spectrum of microbes, metagetable 1 . representative viral proteins identified as high scoring hits during blastx alignments of assembled scaffolds. phylogenetic tree derived from the alignment of the replicase amino acid sequences from the circoviruses named on the tree branches (the genbank identifier is also indicated). the dendrogram was calculated using the neighbor joining method and the bootstraps option (1000 replications) of the nomics will be extensively implemented in clinical research and diagnostics in the near future. figure s1 aligned regions of the query sequences for top hits reported by blastx or hhblits. the names of the library and scaffold are included in the header of each alignment. green background indicates identical amino acids for the two sequences included in the alignment. in all cases, the two algorithms identified the same region of the query as being similar to the target, but one algorithm would occasionally align over a slightly larger region of the query, and in doing so a different target would sometimes be chosen as top hit. cases where blastx and hhblits reported different target proteins as top hits are indicated with an asterisk (*). cases where blastx and hhblits reported different ncldv proteins as top hits are indicated with two asterisks (**). supplemental tables s1-s14 breakdown of sequenced read ends according to their similarity to annotated sequences. high-quality reads were aligned to all mitochondrial and ribosomal databases available and the remnant is considered clean read ends. clean read ends were aligned to the taxonomy databases and assigned to taxa according to e-value and percentage similarity. reads that matched more than one taxon with similar identity (up to two divergent nucleotides) were binned as ambiguous. resolved ends: refers to read ends whose taxonomy was refined using the taxonomy of their corresponding paired end, as explained in materials and methods. notice that for a particular taxon (let's say taxon 1), 'resolved ends' could be greater than 'total ends' when read ends from a different taxon were reassigned to taxon 1. however, since it is a reclassification, the sum of read ends in all taxa should be the same for 'total ends' and 'resolved ends'. read ends that did not resemble any annotated sequence were binned as unknown. (xlsx) supplemental tables s15-s28 summary description of top hits to the virus database from single read ends alignments with blastn. the content of each column is as follows: count: number of read ends that aligned to the target sequence; target: target sequence id; target length (nt): length of target sequence in nucleotides; align coverage (nt): length of the region covered in the target sequence by the local blastn alignment; % align coverage: same as before, but expressed in percentage of the target length. supplemental tables s29-s42 summary description of top hits to the virus database from scaffolds alignments with blastx. the content of each column is as follows: scaffold: id of scaffold after assembly with soapdenovo-trans; length (nt): length of scaffold in nucleotides; v read ends: number of previously classified viral read ends included in the assembly of scaffold; amb read ends: number of previously classified ambiguous read ends included in the assembly of scaffold; u read ends: number of read ends previously binned as unknown included in the assembly of scaffold; target: target sequence id; target aa length: length of target sequence in amino acids; total aa align coverage: number of amino acids from the target included in the alignment; % align coverage: same as before, but expressed in percentage; e-val: e-value. identity %: percentage of amino acids from the alignments that are identical in the query and subject sequences. virus-like particles in serum of patients with australia-antigen-associated hepatitis hepatitis a: detection by immune electron microscopy of a viruslike antigen associated with acute illness isolation of a t-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (aids) coronavirus as a possible cause of severe acute respiratory syndrome a giant virus in amoebae reclassification of giant viruses composing a fourth domain of life in the new order megavirales isolation of a cdna clone derived from 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obesity of infectious origin a novel dna virus (ttv) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology tt-virus infection in north american blood donors, patients with fulminant hepatic failure, and cryptogenic cirrhosis tt viruses: oncogenic or tumorsuppressive properties? inverse relationship between the titre of tt virus dna and the cd4 cell count in patients infected with hiv tt virus infection: prevalence of elevated viraemia and arguments for the immune control of viral load new dna viruses identified in patients with acute viral infection syndrome tt virus in the nasal secretions of children with acute respiratory diseases: relations to viremia and disease severity tt virus infection: a novel virus-host relationship animal virus discovery: improving animal health, understanding zoonoses, and opportunities for vaccine development distant mimivirus relative with a larger genome highlights the fundamental features of megaviridae lausannevirus, a giant amoebal virus encoding histone doublets viruses with more than 1,000 genes: mamavirus, a new acanthamoeba polyphaga mimivirus strain, and reannotation of mimivirus genes we thank ling-hong hung and ram samudrala for their advice on hhblits. key: cord-253056-765rs3e7 authors: dionne, audrey; le, cathie-kim; poupart, steffany; autmizguine, julie; meloche-dumas, léamarie; turgeon, jean; fournier, anne; dahdah, nagib title: profile of resistance to ivig treatment in patients with kawasaki disease and concomitant infection date: 2018-10-17 journal: plos one doi: 10.1371/journal.pone.0206001 sha: doc_id: 253056 cord_uid: 765rs3e7 introduction: kawasaki disease (kd) can be associated with concomitant viral or bacterial infections. children with persistent or recurrent fever 36 hours after the end of intravenous immunoglobulin (ivig) are considered to be resistant to treatment and are at increased risk for coronary complications. although concomitant infection does not affect coronary outcome, it is unknown how it influences the response to ivig treatment. methodology: retrospective cohort study between 2008 and 2016 in a tertiary pediatric university hospital, including 154 children, of which 59 (38%) had concomitant infection. results: children with concomitant infection were more likely to have fever 48 hours after initial ivig treatment (36% vs 20%, p = 0.05) and to be treated with a second dose (33% vs 18%, p = 0.04). children with infection had higher c-reactive protein at the time of diagnosis (148 vs 112 mg/l, p = 0.04), and 48 hours after ivig administration (111 vs 59 mg/l, p = 0.003). nevertheless, there was no statistically significant difference in the prevalence of coronary complications (z-score > 2.5) between children with and without concomitant infection (36% vs 39%, p = 0.68). conclusion: children with kd and concomitant infection are more likely to have persistent fever and elevated inflammatory markers after treatment. this association increases the likelihood of receiving a second dose of ivig but not the risk of coronary complication. accordingly, prospective studies to distinguish true ivig resistance from infection induced persistent fever is warranted. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 kawasaki disease (kd) is an acute systemic vasculitis mostly affecting children younger than 5 years old. it is the most important cause of acquired heart disease in children in developed countries [1] . concomitant respiratory viral infections have been described in 8-42% of patients, and bacterial infections were found in 33% of patients [2] [3] [4] [5] [6] [7] [8] . the clinical presentation of patients with and without concomitant infection is similar [2, 8] . in one study, bacterial coinfection alluded to a trend towards a higher rate of resistance to intravenous immunoglobulin (ivig), without reaching statistical significance (26% vs 15%, odds ratio 1.77 (95% confidence interval 0.71-4.41)) [8] . the accepted definition of ivig resistance is based on the persistence or recrudescence of fever 36 hours after the end of ivig infusion [1] . however, it is unclear how concomitant infection influences the resolution of fever and response to ivig treatment. the aim of this study was to determine the impact of concurrent infection on the prevalence of ivig resistance and coronary outcome. this retrospective study included children with a diagnosis of kd between 2008 and 2016 followed at the sainte-justine's university hospital center (montreal, canada). inclusion criteria were a diagnosis of kd maintained at discharge based on current clinical practice and recommendations [1] ; and echocardiography measurements of the coronary artery (ca) at onset, 1-2 weeks and >3 months after diagnosis. the main outcome was resistance to ivig treatment in children with, versus those without concurrent infection. secondary outcomes included duration of fever, progress of inflammatory markers and coronary artery complications. this study was approved by the institutional research ethics committee of the chu sainte-justine. the institutional research ethics committee waived the requirement for informed consent. medical charts were reviewed for demographic characteristics, clinical course, laboratory values and infectious workup. clinical kd criteria were reviewed and children classified as complete or incomplete kd, in the presence of fever and �3 clinical criteria for the latter. delay in ivig administration was defined as > 10 days between onset of fever and ivig administration; and ivig resistance as persistent or recrudescent fever (> 38.5˚c) 36 hours after the end of ivig infusion. concurrent infection was defined as a clinical diagnosis and proven concurrent infection as patients with a positive microbiologic testing or imaging study in the presence of clinical symptoms. testing for concomitant infection was based on clinical symptoms. urinary tract infection was diagnosed according to current american academy of pediatrics guidelines [9] . gastroenteritis was defined as gastrointestinal symptoms with positive stool cultures for virus or pathogenic bacteria. children with respiratory symptoms and a positive respiratory virus multiplex reverse transcription polymerase chain reaction (influenza, parainfluenza, coronavirus, enterovirus, rhinovirus) were diagnosed with upper respiratory tract infection (urti). otitis media was diagnosed according to current american academy of pediatrics guidelines [10] . children with clinical signs of exudative pharyngitis in the presence of a positive group a streptococcus culture were diagnosed with concurrent infection. bacterial adenitis was diagnosed in the presence of clinical symptoms with signs of abscess formation and/or necrosis on ultrasound. children with positive serology for acute infection (igm) with epstein-barr virus (ebv), cytomegalovirus (cmv), measles, parvovirus and mycoplasma before ivig administration were considered to have concurrent infection. pneumonia was diagnosed when chest x-ray, interpreted by a pediatric radiologist, confirmed the presence of a consolidation, in the presence of respiratory symptoms. echocardiographic ca size was reviewed for all children at onset of the disease, with follow-up studies for a minimum of 3 months and up to one year after diagnosis of kd. ca zscores were calculated for the right coronary artery, left main coronary artery, left anterior descending artery and circumflex artery. ca aneurysm was defined as a localized dilatation of a portion with adjacent normal measurements, or an obvious saccular deformation of the ca. ca dilatation of non-aneurysmal segments was defined as a ca z-score > 2.5 [1] , calculated according to dallaire and dahdah [11] . early ca dilatation was defined as ca dilatation at onset up to one week following kd diagnosis, and late ca dilatation when persisting at 3 months' follow-up. quantitative variables were summarized as mean ± sd and categorical variables as frequencies and percentages. the shapiro-wilk test was used to test for normal distribution. comparison of clinical and laboratory data between patients with and without concurrent infection was performed using the student's t-test for continuous variables with normal distribution or the mann-whitney u test for continuous variables with non-normal distribution. anova for repeated measures was used to describe the variation of temperature and laboratory values over time. the fisher or the χ 2 tests were used for comparison of categorical variables. logistic regression was used to examine the association between concomitant infection and ca complications, controlling for confounder variables (ivig resistance). all analyses were performed with spss statistics version 23 (ibm, chicago, illinois). a two-tailed p value of <0.05 was deemed significant. the study included 154 children (101 male; 66%) aged 3.4±2.8 years. the median number of diagnostic criteria was 5 (range 2-6), with 59 (38%) children having incomplete kd criteria. ivig were administered to 150 (97%) patients, 6.7±2.6 days after onset of fever. delay in ivig administration occurred in 10 (7%) patients, and ivig resistance in 36 (23%). concurrent infections were diagnosed in 59 (38%) patients, of which 20 (34%) were viral and 39 (66%) bacterial. urti was diagnosed in 19 (32%) children, caused by respiratory syncytial virus (n = 3), parainfluenza (n = 3), rhinovirus (n = 4), influenza (n = 2), enterovirus (n = 2) and adenovirus (n = 4). three children had multiple viruses detected. two (7%) children were diagnosed with viral gastroenteritis. three (5%) children had a clinical and biological profile suggestive of acute cmv infection (positive igm and negative igg), and 2 (3%) of acute mycoplasma infection. otitis media was diagnosed in 7 (12%) of patients, and pneumonia in 8 (14%) children. group a streptococcus pharyngitis was diagnosed in 13 (22%). four (7%) patients had bacterial adenitis, complicated by retropharyngeal pharyngitis in one child. one child was diagnosed with perforated appendicitis and underwent surgery, with pathology confirming the diagnosis. one child had escherichia coli pyelonephritis. concomitant infection was proven by microbiologic testing and/or imaging in 48 (81%) of patients. characteristics of patients with viral versus bacterial infections are described in table 1 . during hospitalization, antibiotics were empirically initiated in 95 (62%) children, and completed in 36 (23%). antibiotics were initiated on average 2±5 days prior to ivig treatment, with antibiotics initiated prior to ivig treatment in the majority of cases (55, 67%). only a minority (4, 9%) were started on antibiotics after non-response to initial ivig treatment. age was similar between children with and without infection (3.5±2.7 versus 3.4±2.9 years, p = 0.94), with a similar proportion of male (34 (58%) versus 67 (71%) patients, p = 0.10). there was a similar proportion of children with incomplete clinical criteria among children with and without infection (20 (34%) versus 39 (41%), p = 0.38). there were 50 (58%) children with infection who received antibiotics compared to 45 (47%) without infection who received antibiotics (p<0.001). of the former, 29 (58%) completed antibiotic treatment versus 7 (16%) of the latter (p<0.001). most children with and without concurrent infection received ivig (57 (97%) and 93 (98%) patients; p = 0.63). delay in diagnosis and ivig administration occurred in 4 (7%) children with concurrent infection versus 6 (7%) without concurrent infection, p = 0.89 (table 2) . finally, there was no difference in delay of ivig administration whether patients presented with complete versus incomplete clinical criteria (7% and 7%; in general, basic laboratory values were similar between children with and without concurrent infections. c-reactive protein was however higher in cases with concurrent infections compared to cases without concurrent infection (148±96 versus 112±65 mg/l, p = 0.03) ( table 3) . similar results were found between patients with proven infection on microbiologic testing and/or imaging versus those without and results are presented in table 4 . overall, ivig resistance occurred in 36 (24%) patients. children with concurrent infection had higher rates of ivig resistance (19 (33%) versus 17 (18%) patients, p = 0.04), and higher temperature at 48 hours (fig 1) . they were also more likely to have fever > 38.5˚c at 48 hours, than those without concurrent infection (16 (36%) versus 15 (20%) patients, p = 0.05). this was accompanied with higher crp at time of diagnosis, remaining similarly higher in the first 72 hours after treatment (fig 2) . ivig resistance was higher in patients with proven infection on microbiologic testing and/ or imaging than those without (16 (35%) versus 20 (19%) patients, p = 0.04). there was no difference in response to treatment between patients who had a proven infection on microbiologic testing and/or imaging study versus those with clinical diagnosis of infection (16 (35%) versus 3 (27%) patients, p = 0.64). resistance to initial ivig treatment in patients with bacterial infection was nearly double that in patients with viral infection, although not reaching statistical significance (12 (46%) versus 4 (24%) patients, p = 0.12). there was no difference in response to treatment between patients with complete and incomplete clinical criteria (25 (28%) versus 10 (17%) patients, p = 0.12). patients who received antibiotics during their hospital course, independent of infection status, were at higher risk of ivig resistance than those who did not (27 (29%) versus 9 (16%) patients, p = 0.05). however, neither receiving antibiotics prior to ivig therapy (15 (27%) versus 11 (41%) patients, p = 0.22) nor completing antibiotic course (13 (36%) versus 14 (25%) patients, p = 0.25) were associated with response to treatment. there was no significant difference in coronary artery complications (fig 3) between patients with and without infection (21 (36%) versus 37 (39%), p = 0.68). this remained true even after adjusting for ivig resistance and number of ivig treatment (p = 0.47). resistance to ivig treatment was associated with an increased risk of ca complication both as a univariate (53% versus 34%, p = 0.037), and when adjusting for the presence of infection (p = 0.039). coronary artery dilation at time of diagnosis was found in 58 (38%) patients, similarly distributed according to the presence or absence of concurrent infection (20 (34%) versus 38 (40%) patients, respectively; p = 0.45), and persisted in 17 (11%) patients (in 5 (9%) versus 12 (13%) patients, respectively; p = 0.42). coronary aneurysms were diagnosed in 17 (11%) patients, without significant difference between patients with and without infection neither (7 (12%) versus 10 (11%) patients, p = 0.80). while the risk of coronary aneurysm was similar between patients with viral versus bacterial infection (5 (19%) versus 2 (12%) patients, p = 0.69), patients with bacterial infection were more likely to have coronary artery dilation (13 (48%) versus 3 (18%) patients, p = 0.04). in this retrospective series, the presence of a concomitant infection was associated with a higher rate of resistance to ivig treatment. patients with concomitant infection were more likely to have persistent fever and slower normalization of inflammatory markers after ivig treatment. the administration of antibiotics did not decrease this risk of resistance to ivig therapy. however, concomitant infection was not associated with an increased risk of coronary artery complications. more precisely, the higher likelihood of repeated ivig treatment when concurrent infection was present is defined as recrudescence of fever 36 hours after the end of ivig infusion based on expert opinion [1] . our results challenge this definition since fever may be maintained by an infection, either viral or bacterial, that is not likely to respond to ivig. inflammatory markers are often used to help guide the decision about the need for additional treatment in kd. in our series, patients with concurrent infection presented higher crp levels at baseline, as well as 48 hours after initial treatment. this is consistent with a retrospective study on the variability in response to ivig, which showed a higher rate of concomitant infection and higher crp levels in complete non-responders to ivig treatment compared to partial nonresponders and responders [12] . thus, both the persistence of fever after ivig treatment and elevated inflammatory markers contribute to the increased likelihood of "ivig resistance" in patients with concomitant infections. interestingly, non-response to ivig therapy was associated with the use of antibiotics in a prior single-center retrospective study [12] . one would have expected patients with concomitant infection, at least bacterial, to have lower rate of ivig resistance if the infection was controlled by antibiotics. both in prior reports and this series [12] , the use of antibiotic was found to be associated with non-response to ivig treatment, independent of whether or not an infection was confirmed. however, it is not clear if it is just reflecting the underlying infection, or kd that is resistant to initial treatment. another hypothesis would be that the presence of infection triggers an additional inflammatory response at the molecular levels that impact response to treatment and outcomes. il-1α and il-1β have been shown to be essential in the development of kd [13] . the il-1 pathways play a critical part of the host defense against microbial pathogens through activation of toll like receptors [14, 15] . moreover, inositol-triphosphate 3-kinase c (itpkc) has a critical role in mediating nlrp3 expression and intracellular calcium, which is then responsible for il-1β production [16] . genetic polymorphism in itpkc is associated with higher il-1β cytokines and treatment failure [16] . this raises the question if concomitant infection increases resistance to ivig treatment by increasing levels of il-1β cytokines. this could be very important as new treatment strategies are targeting il-1 blockade in recalcitrant kd. persistence of fever after ivig treatment is a strong risk factor for development of coronary aneurysms [1, 17] . in this series, patients with resistance to ivig treatment had a higher risk of developing ca complication. notwithstanding, the rate of ca complication was statistically independent of the presence of infection in our series and in previous series [8] . thus, the lack of increased risk of ca complication in the setting of persistent fever in patients with kd and concomitant infection argues for infection as the cause of fever as opposed to ivig resistance. it questions whether this "excessive" diagnosis of ivig resistance is well justified when there is a concomitant infection, and if the current definition of treatment resistance should be modified. retreatment with ivig or other immunosuppressive therapies may also have had a protective effect on the development of ca complication. however, the rate of ca complication was similar between patients with and without infection, even after accounting for the number of ivig treatment received. thus, other criteria need to be used to help with decision for retreatment of patients with persistent fever and concomitant infection. coronary artery dimensions may be a more useful marker for the need for additional treatment in this at risk population. in general, patients who are resistant to initial treatment receive a second dose of ivig (2 g/ kg) [18] . notwithstanding, at least 5% of children remain febrile despite multiple dose of ivig [18] . these particular patients are at even greater risk of ca complications, and additional therapies are usually administered [19] , including corticosteroids [20] [21] [22] , anti-tnf alpha agents [23, 24] , cyclosporine [25] , cyclophosphamide [26] and more recently anakinra (anti-interleukine1) [27, 28] . the use of these additional therapies is based on effectiveness in other vasculitis, with no prospective clinical trials to show effectiveness on coronary artery outcome. in patients with concerns for concurrent infection, the benefit of those additional therapies should be carefully balanced against the increased risk of infection. it is important to make this distinction, in order to 1) avoid using aggressive immunosuppressive therapies in patients with persistent fever due to uncontrolled infection and 2) avoid not treating aggressively a patient with kd that is resistant to initial treatment and at increased risk of serious coronary artery complications. however, this distinction can be clinically very difficult to answer, and caution should be exercise to prevent both cardiac complications and adverse side effects of therapies. there are limitations to this study essentially related to the retrospective methods, and the diagnosis of concomitant infection. on one hand, in the absence of systematic testing for infections there is a potential underestimation of the actual concurrent infection rate, as some infections could not be identified. on the other hand, inclusion of only children with infectious workup at time of initial kd diagnosis could have falsely increased the rate of concurrent infection. however, the rate of concomitant infection in this series was similar to those previously published [8] . moreover, positive testing for virus and/or throat culture in children could reflect a carrier status rather than actual concurrent infection. however, because infectious workup was performed based on children's symptoms, the positive results most likely represent an actual infection. serologies for different viral infections (cmv, ebv, mycoplasma) can be difficult to interpret in an acute inflammatory setting, with igm cross-reactivity. these infections could not be confirmed by pcr due to the retrospective nature. however, this could only have affected 3 children, and statistical analysis excluding these children did not affect the results. moreover, whereas the aha definition of resistance to ivig treatment is based on persistent fever 36 hours after the end of ivig infusion [1] , other factors are considered in the decision whether or not to retreat patients, including inflammatory markers and ca dimensions. thus, some patients were classified as resistant to treatment based on the definition, but were not retreated and had favorable evolution. small sample size of sub-groups analysis and secondary aims limit the statistical power, and results should be interpreted in light of these limitations. in this study, patients with concomitant infection had a higher rate of resistance to ivig treatment. patients with concomitant infection had longer duration of fever and slower normalization of inflammatory markers after initial treatment. however, the presence of infection was not associated with an increased risk of ca complication. accordingly, the persistence of fever in kd and the definition of resistance to ivig should be regarded speculatively when concurrent infection is present. decision to intensify treatment in patients with concurrent infection should not only be based on persistent fever. coronary artery dimensions may be a more useful indication for treatment in this patient population. prospective studies are needed to better refine which children truly require additional therapies. conceptualization: audrey dionne, nagib dahdah. diagnosis, treatment, and long-term management of kawasaki disease. a scientific statement for health professionals from the american heart association concurrent respiratory viruses and kawasaki disease influenza infection and kawasaki disease evaluation of the temporal association between kawasaki disease and viral infections in south korea detection rate and clinical impact of respiratory viruses in children with kawasaki disease concomitant respiratory viral infections in children with kawasaki disease viral infections associated with kawasaki disease infections and kawasaki disease: implications for coronary artery outcome urinary tract infection: clinical practice guideline for the diagnosis and management of the initial uti in febrile infants and children 2 to 24 months the diagnosis and management of acute otitis media new equations and a critical appraisal of coronary artery z-scores in healthy children variability in response to intravenous immunoglobulin in the treatment of kawasaki disease il-1 signaling is critically required in stromal cells in kawasaki disease vasculitis mouse model: role of both il-1α and il-1β inflammasome activation and il-1β and il-18 processing during infection the tlr and il-1 signaling network at a glance inositol-triphosphate 3-kinase c mediates inflammasome activation and treatment response in kawasaki disease predictive risk factors for coronary artery abnormalities in kawasaki disease intravenous gamma-globulin treatment and retreatment in kawasaki disease. us/canadian kawasaki syndrome study group treatment options for resistant kawasaki disease coronary artery complication in kawasaki disease and the importance of early intervention: a systematic review and meta-analysis effects of steroid pulse therapy on immunoglobulin-resistant kawasaki disease steroid pulse therapy for children with intravenous immunoglobulin therapy-resistant kawasaki disease: a prospective study infliximab treatment of intravenous immunoglobulin-resistant kawasaki disease infliximab for intravenous immunoglobulin resistance in kawasaki disease: a retrospective study cyclosporin a treatment for kawasaki disease refractory to initial and additional intravenous immunoglobulin initial intravenous gammaglobulin treatment failure in kawasaki disease rational and study design for a phage i/iia trial of anakinra in children with kawasaki disease and early coronary artery abnormalities (the anakd trial) usefulness and safety of anakinra in refractory kawasaki disease complicated by coronary artery aneurysm key: cord-001964-iy6qzq58 authors: muñoz-gonzález, sara; pérez-simó, marta; colom-cadena, andreu; cabezón, oscar; bohórquez, josé alejandro; rosell, rosa; pérez, lester josué; marco, ignasi; lavín, santiago; domingo, mariano; ganges, llilianne title: classical swine fever virus vs. classical swine fever virus: the superinfection exclusion phenomenon in experimentally infected wild boar date: 2016-02-26 journal: plos one doi: 10.1371/journal.pone.0149469 sha: doc_id: 1964 cord_uid: iy6qzq58 two groups with three wild boars each were used: group a (animals 1 to 3) served as the control, and group b (animals 4 to 6) was postnatally persistently infected with the cat01 strain of csfv (primary virus). the animals, six weeks old and clinically healthy, were inoculated with the virulent strain margarita (secondary virus). for exclusive detection of the margarita strain, a specific qrt-pcr assay was designed, which proved not to have cross-reactivity with the cat01 strain. the wild boars persistently infected with csfv were protected from superinfection by the virulent csfv margarita strain, as evidenced by the absence of clinical signs and the absence of margarita rna detection in serum, swabs and tissue samples. additionally, in pbmcs, a well-known target for csfv viral replication, only the primary infecting virus rna (cat01 strain) could be detected, even after the isolation in st cells, demonstrating sie at the tissue level in vivo. furthermore, the data analysis of the margarita qrt-pcr, by means of calculated δct values, supported that pbmcs from persistently infected animals were substantially protected from superinfection after in vitro inoculation with the margarita virus strain, while this virus was able to infect naive pbmcs efficiently. in parallel, ifn-α values were undetectable in the sera from animals in group b after inoculation with the csfv margarita strain. furthermore, these animals were unable to elicit adaptive humoral (no e2-specific or neutralising antibodies) or cellular immune responses (in terms of ifn-γ-producing cells) after inoculation with the second virus. finally, a sequence analysis could not detect csfv margarita rna in the samples tested from group b. our results suggested that the sie phenomenon might be involved in the evolution and phylogeny of the virus, as well as in csfv control by vaccination. to the best of our knowledge, this study was one of the first showing efficient suppression of superinfection in animals, especially in the absence of ifn-α, which might be associated with the lack of innate immune mechanisms. members of the pestivirus genus, within the flaviviridae family, account for a variety of diseases in farm animals, the most economically important of which are bovine viral diarrhoea virus (bvdv) and classical swine fever virus (csfv). classical swine fever virus (csfv) is the etiological agent of a highly contagious viral disease of swine affecting domestic pigs and wild boars [1] , which has caused major losses in stock farming [2, 3] . csfv is composed of a lipid envelope, a capsid and a single plus-strand rna genome carrying a single, large open reading frame (orf) flanked by two untranslated regions (utrs). the orf encodes a polyprotein of approximately 3900 amino acids, which are processed by cellular and viral proteases in the four structural proteins-c, e rns , e1, e2-and in the 8 non-structural proteins-n pro , p7, ns2, ns3, ns4a, ns4b, ns5a, and ns5b [4] . recently, it was proved that csfv can generate postnatal persistence by infecting both newborn piglets and wild boars with either low-and/or moderate-virulence strains, respectively. over the six weeks after postnatal infection, most of the infected animals remained clinically healthy, despite persistent high virus titres in the blood, organs and body secretions. importantly, these animals were unable to mount any detectable humoral or cellular immune responses. at necropsy, the most prominent gross pathological lesion was severe thymus atrophy. four weeks after infection, pbmcs from persistently infected seronegative piglets were unresponsive to both specific csfv and non-specific pha stimulation in terms of ifn-γ-producing cells. these results suggested the development of an immunosuppression state in these postnatally persistently infected pigs [5, 6] . in addition, it was shown that six-week-old, persistently csfv-infected pigs were unable to elicit specific immune responses following vaccination with a csfv lapinised c-strain vaccine (hclv) [7] . interestingly, the rna of the vaccinal c-strain was undetectable by specific rt-pcr [8] in any of the samples analysed after vaccination, including blood, nasal and rectal swabs, or organs throughout the experiment, suggesting a phenomenon of homologous interference, also known as superinfection exclusion (sie), between the high viral load generated by the primary persistent infection and the csfv vaccine strain. the sie phenomenon, defined as the ability of a primary virus infection to interfere with a secondary infection by the same or a closely related virus, has been described in a broad range of virus-host systems, including bacteria, plants, and animals, and in important pathogens of humans, such as rubella virus, human immunodeficiency virus (hiv), and hepatitis c virus (hcv), among others [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] . from an evolutionary standpoint, sie might be a conservative strategy, reducing the likelihood of recombination events between related strains [17, 21, 22] , thus determining the stability of viral sequences within the same cell. from a practical standpoint, sie has significant implications for the treatment or prevention of viral infections. in this regard, cross-protection of crops by purposeful infection with milder virus isolates is a widely accepted practice, and it is viewed as an effective and economical antiviral management strategy [23] . additionally, transplantation of hcv-infected liver grafts has been suggested as a treatment for already infected patients, given that the transplantation of a healthy organ would lead to rapid damage to the newly transplanted liver by the virus of the recipient patient [15, 24] . previous studies conducted in cell cultures with bvdv demonstrated that cells acutely infected with this virus were protected from a second infection by a homologous bvdv strain [17] . additionally, it was shown that csfv is generally noncytopathic, and it readily establishes persistent infections in cell culture. nevertheless, when persistently infected cultures were serially passaged more than 100 times, spontaneous generation of cytopathogenic (cp) csfv variants could occur. the few surviving cells of the cytopathic effect (cpe), although still infected, were also protected from the cpe after superinfection with cp csfv [25] . both studies supported the ability of pestiviruses to generate sie in cell cultures. thus, along with the availability of a persistent infection model of csfv, in the present study, we sought to assess sie against a highly virulent csfv strain at the organism level in six-week-old wild boars, rendered persistently csfv-infected at birth. our results showed that sie could occur at the systemic level in csfv-infected swine. pk-15 cells (atcc ccl 33) and sk6 cells [26] were cultured in dulbecco's modified eagle medium (dmem), supplemented with 10% foetal bovine serum (fbs), pestivirus-free, at 37°c in 5% co 2 . the cells were infected with 0.1 tcid 50 /cell in 2% fbs, and the virus was harvested 48 h later. additionally, st cells (atcc crl 1746) were cultured in dmem, supplemented with l-glutamine (2%) and 10% foetal bovine serum (fbs), pestivirus-free at 37°c in 5% co 2. peroxidase-linked assay (pla) [27] was used for viral titration following the statistical methods described by reed and muench [28] . the catalonia 01 (cat01) strain used in this study was isolated from the spanish csf epizootic in 2000-2001 [29] . this isolate belongs to the csfv 2.3 genogroup [30] . the course of infection by this strain was found to be mild [29, 31] . finally, the virulent margarita strain, which belongs to the csfv 1.4 genogroup [29, 32, 33] , was used. to elucidate the capacity of csfv to generate sie, two groups (a and b), with three male, sixweek-old wild boars in each, were used. these animals were acquired from gestion cinegetica integral sl farm (segovia, spain) and were housed in the experimental isolation facilities in the biosecurity level 3 laboratory of the centre de recerca en sanitat animal (cresa); they were fed a conventional piglet starter diet and pellets until the end of the trial (startrite 100, kwikstart, and prestarter; sca iberica s.a., zaragoza, spain) and were handled according to previous studies conducted in cresa [6] . group a (animals 1 to 3) was used as controls, and they tested pestivirus-free at the beginning of the study. the second group (group b), housed in an independent isolation unit at the bsl-3 facility of cresa, (animals 4 to 6), were postnatally persistently csfv-infected animals. these animals, which had been intranasally infected in the first 24 h after birth with the csfv cat01 strain, were viraemic and apparently healthy at six weeks old, although being immunosuppressed, they lacked csfv-specific cellular and humoral responses [5, 6] . both groups had an average weight of 6 kg per animal. after a five-day acclimation period, all of the animals were experimentally infected by i.m. injection in the neck [33] [34] [35] with 10 5 tcid 50 csfv margarita strain. in previous studies, this viral dose caused acute csf and often induced death at 10-15 days post-infection (dpi) [36] . sera and nasal and rectal swabs were collected at 0, 3, 7, 10 and 13 dpi. blood samples for the isolation of pbmcs were obtained at day 0 and at the time of euthanasia. a trained veterinarian recorded the clinical signs daily in a blinded manner [36] . the clinical signs compatible with csfv infection were anorexia, fever, conjunctivitis, diarrhoea, constipation, cyanosis of the skin, abdominal petechiae, dyspnoea, tremors, locomotive disturbances, reluctant walking, swaying movement of the hindquarters, posterior paresis, convulsions from mild to severe and prostration. particular stress was placed upon the registration of nervous symptoms [29, 33, 34, 36] . the clinical status of the animals was scored from 0 to 6 [29, 33, 34, 36] as follows: 0: no signs; 1: mild pyrexia; 2: pyrexia plus mild clinical signs; 3: mild-to-moderate clinical signs; 4: moderate clinical signs; 5: moderate-to-severe clinical signs; and 6: death. for ethical reasons, the animals were euthanised when the clinical score reached 5, when exhibiting a fall of the hindquarters, when there was inability to drink or feed, when prostration occurred or when exhibiting moderate nervous disorders. after euthanasia, an exhaustive necropsy was conducted, in which the presence of pathological symptoms in different organs and tissues was evaluated. surviving wild boars were euthanised at 13 dpi, and urine and tissues (spleen, liver, intestine, mesenteric lymph node, prescapular lymph node, bone marrow, medulla oblongata, lung, kidney, thymus and tonsil) were obtained at necropsy. euthanasia was performed according to european directive 2010/63/eu, using a pentobarbital overdose of 60-100 mg/kg administered via the anterior vena cava. the animal care and procedures were in accordance with the guidelines of the good experimental practices (gep), under the supervision of the ethical and animal welfare committee of the autonomous university of barcelona (uab), and they were approved under number 8804, according to the existing national and european regulations. additionally, the biosafety level of the viruses used in this study was stated as biosecurity level 3, as approved by the biosafety committee of the uab, with registration assignment ar-296-15. fifteen representative sequences of the three csfv genogroups were retrieved from genbank and aligned using bioedit [37] . two primers and probes were designed for specific detection of the margarita strain sequence (1.4 csfv genogroup) by targeting the 5´end of the e2 gene, as follows: forward primer (2333-2356), 5´-aagattacgaccacaatttacaac-3´; reverse primer (2411-2431), 5´-tcc tactgaccacattaagcg-3´and probe (2369-2389), 5´-ccatcaaggctatctgcacgg-3´. the nucleotide positions were based on the genome sequence of the margarita strain (genbank accession number aj704817). the probe was labelled with 6-fam at the 5´end and with bhq1 at the 3´end. the primers and probe were purified by reverse phase hplc. the one-step rt-pcr protocol was undertaken using the commercially available taqman1 one-step rt-pcr master mix reagents kit (applied biosystems roche). the real-time rt-pcr assay was optimised using a total volume of 25 μl. real-time qrt-pcr was performed using an applied biosystems1 7500 fast real-time pcr system. the temperature profile was 30 min at 50°c (reverse transcription), 15 min at 95°c (inactivation reverse transcriptase/activation taq polymerase), followed by 42 cycles of 15 s at 94°c (denaturation), 30 s at 57°c (annealing) and 30 s at 68°c (elongation). identical temperature profiles were used for all of the real-time rt-pcr runs, and fluorescence values were collected during the annealing step. twenty csfv rna preparations strains were used to determine the specificity and sensitivity of the assay (table 1) [30, 38] . to exclude the possibility of presence of csfv cat01 strain rna interfering with the assay sensitivity for the csfv margarita strain rna detection, mixtures from serial rna dilutions from both viral strains were analysed. in addition, mixtures from rna serum samples of group b (prior to the margarita strain inoculation), with samples from group a at 7 days post-infection with the margarita strain, were analysed. initial sample volume of 150 μl to obtain a final volume of 50 μl of rna, which was stored at -80°c. the presence of csfv rna in the serum and in nasal and rectal swabs, as well as in tissue samples, was analysed by a generic csfv qrt-pcr [39] . this test was used in our laboratory for inter-laboratory comparisons of csfv diagnoses, organised by the eu reference laboratory. positive results were considered for threshold cycle values (ct) equal to or less than 42. samples in which fluorescence was undetectable were considered negative. additionally, the qrt-pcr specific for the margarita strain, designed in this work (described above), was used to distinguish those samples infected with the margarita strain. serum samples were tested with neutralisation peroxidase-linked assay (npla) [40] , and the titres were expressed as the reciprocal dilution of serum that neutralised 100 tcid 50 of the cat01 or margarita strain in 50% of the culture replicates. the detection of e2-specific antibodies was performed using a commercial elisa kit (idexx); the samples were considered positive when the blocking percentage was 40%, following the manufacturer's recommendations. anti-ifn-α monoclonal antibodies (k9 and k17) and ifn-α recombinant protein (pbl biomedical laboratories, piscataway, new jersey, usa) were used in elisa to detect ifn-α in serum samples at 0, 3, 7 and 10 dpi [34, [41] [42] [43] . the cut-off value of the assay was calculated as the average of the optical density of negative controls (blank and negative sera before csfv infection) plus three standard deviations. cytokine concentrations in serum were determined using a regression line built with the optical densities of the cytokine standards used in the tests. elispot assay to detect csfv-specific ifn-γ cells was performed as previously described [34] , using pbmcs that were obtained at day 0 and at the time of euthanasia. briefly, plates (costar 3590, corning) were coated overnight with 5 μg/ml capture antibody (p2g10, pharmigen). detection was performed using a biotinylated antibody (p2c11, pharmigen). a total of 5x10 5 pbmcs/well were plated in triplicate at 0.1 multiplicity of infection (moi) of the cat01 and margarita csfv strains. moreover, the same samples were incubated in the presence of phytohaemagglutinin (pha) (10 μg/ml). the controls were incubated in the presence of mock-stimulated wells. the numbers of spots in the media for mock-stimulated wells were considered to constitute the baseline for the calculation of antigen-specific frequencies of ifn-γ-producing cells. samples from animal 1 (group a: margarita acutely infected wild boar; 10 dpi), animal 5 (group b: cat01 persistently infected wild boar and superinfected with csfv margarita strain; 13 dpi), and a pestivirus-free wild boar (animal 1 before infection), were used to assess sie in pbmcs (fig 1) . the pbmcs were isolated from whole blood by centrifugation on ficoll gradients (histopaque-1077; sigma). the number and viability of the pbmcs were determined by staining with trypan blue [33] . a total of 4x10 5 pbmcs/well from each animal were plated in quintuplicate at 37°c in 96-well plates with: (i) vehicle; (ii) the cat01 strain at a 0. was analysed by generic csfv qrt-pcr [39] and for the specific margarita strain by qrt-pcr detection assay (see above, section 2.3). for virus isolation, an established cell line sensitive for specific csfv proliferation, st cells, were cultured at 37°c in 96-well plates in triplicate in the presence of each of the collected cell suspensions. after 72 h, the supernatants were removed, and the collected st cells were washed twice and resuspended in 200 μl of sterile pbs. after two cycles of freeze-thaw at -80°c, the presence of csfv rna in the st cell samples was analysed by qrt-pcr for csfv [39] and the margarita strain (see above). in parallel, a st plate similarly inoculated with cell suspensions was used for confirmation by pla [27] . a delta ct (δct) for margarita strain rna detection was calculated as the differences between (i) the margarita ct value detected from the isolation of st from groups a or b and (ii) the ct value in st inoculated with margarita-infected naïve pbmc extract, being δct = ct (a) -ct (b) . the whole protocol was repeated twice, in st and also in sk6 cells using pbmcs from animals 1 (group a), 4 and 5 (group b) and cells from the naïve animal (number 1, group a), collected before margarita infection. the e2-gene fragment reported by lowings et al. [44] was amplified by end point rt-pcr [45] in sera, tonsil, lung and spleen from animals 1, 3 (group a), 4 and 5 (group b), collected at necropsy. additionally, the viral inoculums used in the experimental infections (cat01 and margarita strains) were evaluated. the amplification products were checked by electrophoresis on 2% agarose gel and were directly cleaned with a wizard1 pcr preps dna purification system (promega, madison, wisconsin, usa). sequencing reactions were conducted under bigdye tm terminator-cycling conditions using an abi 3130xl. forward and reverse sequences obtained from each amplicon were assembled using the contig express application in vector nti software, version 11 (invitrogen). the sequences from the e2-gene fragment obtained were aligned to analyse the sequence found in each sample. of the 20 csfv rna strains analysed, the assay detected only the csfv rna from the margarita strain (1.4 genogroup), while the other 19 csfv rna extractions were negative (table 1 ). this result indicated that the newly developed assay was highly specific for the detection of the csfv margarita strain, and there was no cross-reactivity with the other tested csfv strains from genogroup 2 (including the cat01 strain). the specificity of the assay was based primarily on mismatches in the probe-binding region but also to some extent on mismatches in primer-binding regions. the sensitivity of the assay was evaluated by testing 10-fold dilutions of the margarita strain rna. the analytical sensitivity was estimated to be as high as 0.4 tcid 50 . the assay had a reaction coefficient (r 2 ) of 0.994 (data not shown). positive results were considered for threshold cycle values (ct) equal to or less than 38. finally, the presence of cat01 rna strain in the sample containing the margarita strain rna did not affect the assay sensitivity (data not shown). animals persistently infected with the cat01 strain and inoculated with the virulent margarita strain (group b) showed neither clinical signs of disease nor fever at any time throughout the study, maintaining good health status (fig 2) . in contrast, animals from group a, infected with the margarita strain, presented mild clinical signs at 2 dpi that progressed to moderate within 48-72 h. at 7 dpi, animal 2 showed a clinical score value of 4; however, it was found dead at 8 dpi, with lesions of haemorrhagic diathesis. animals 1 and 3 progressed to dyspnoea, weight loss, swaying movement of the hindquarters, posterior paresis and high fever until 10 dpi, when euthanasia was performed. margarita rna was undetected in the sera from animals in group b, except for animal 4 at 13 dpi with a high ct value (ct 36.84), considered a low rna viral load (36, 39) (fig 3) . additionally, csfv margarita strain rna could not be detected in any of the nasal or rectal swabs collected from group b (data not shown). furthermore, in group b, csfv margarita rna was found only in the liver of animal 4 and also in the spleen of animals 4 and 5, with a low rna viral load. in contrast, all wild boars from group b (csfv persistently infected with cat01 strain) maintained during the whole trial a high and constant csfv rna load in serum, swabs and organs, when examined by generic csfv q-rt-pcr (table 2 ). in contrast, both qrt-pcrs (generic and specific for margarita strain) were positive in organs and samples collected from animals in group a ( table 2 ). the ct values were positive by the csfv generic qrt-pcr [39] , in both serum and swab samples, from 3 dpi onwards. ct values for the specific margarita assay were similar to those obtained by the csfv generic qrt-pcr. to evaluate the induction of csfv-specific antibodies, serum samples were analysed at different times after csfv margarita strain infection. the absence of antibody response, in terms of e2-specific antibodies and neutralising antibody titres, was found in both csfv acutely and persistently superinfected groups during the entire experiment (data not shown). previously, it was shown that csfv pi animals were unable to elicit an innate immune response, in terms of ifn-α production, against a csfv life-attenuated vaccine [7] . however, we wondered whether superinfection with a csfv virulent strain would trigger detectable levels of ifn-α in the csfv-superinfected wild boars (group b), given that ifn-α has been largely related to disease severity, as a hallmark of csfv acute infection [34, 46] . in the present work, we observed that progression of disease in group a was correlated with an increase in the levels of endogenous ifn-α after infection, as measured by elisa, with values that reached more than 240 u/ml in two of three animals at 7 dpi and 10 dpi (data not shown). in contrast, ifn-α was undetectable in all of the serum samples analysed both before (day 0) and after margarita inoculation of csfv catalonia persistently infected pigs (group b) (data not shown). pbmcs from all of the animals were analysed for virus-specific and non-specific ifn-γ responses by elispot assay at 0 and 13 dpi post-margarita strain inoculation. very few ifnγ-producing cells were found upon csfv and pha stimulation of pbmcs from all 3 of the csfv-superinfected animals (group b). these results supported our previous results showing that postnatal infection of piglets with csfv could result in virus persistence due to a lack of b-and t-cell responses (data not shown). it is well known that white blood cells, including the pbmcs, are targets for csfv replication [47, 48] . consequently, to examine whether the pbmcs collected from the csfv-superinfected animals (group b) and the acutely infected animals (group a), were permissive (or not) to csfv superinfection, we assayed in vitro inoculation of such samples, with either cat01 or margarita csfv strains. similarly, pbmc samples were mock-infected. additionally, pbmcs from a naïve animal were used as controls. as was expected, csfv-specific margarita rna was detected in the pbmcs from animals developing the csf acute disease (group a) in both mock and margarita-infected samples. furthermore, pbmcs from group b in vitro inoculated with margarita were also positive for csfv-specific margarita rna detection, but with a high ct value correlated with a lower rna load (table 3) . otherwise, pbmcs from group b in vitro mock-infected were negative for csfv-specific margarita rna detection (table 3) . following these findings, to decipher whether the detected rna load in group b might correspond to rna traces from the inocula or to the infecting virus, the previously analysed pbmc extracts were inoculated into a st cell line. consistently, the detected rna load notably increased in st after inoculation with the extract from margarita in vitro inoculated-naïve pbmcs; the obtained 7.76δ ct positive value confirmed the infectivity of the virus recovered from the pbmc samples. in contrast, margarita rna in group b in vitro mock-infected pbmcs remained undetectable even after st inoculation. furthermore, margarita rna load detection in group b in vitro margarita-infected pbmc samples decreased after inoculation of st cells, corresponding to higher ct values than those previously detected directly from pbmc extracts. remarkably, an 11.6 δct value was found in the st cells with margarita in vitro inoculated pbmcs from group b, relative to the value obtained in the st cell extracts from margaritainoculated naïve pbmcs (table 3 ). the whole protocol was repeated twice for animals 1 (group a), 4 and 5 (group b) in both sk6 and st cells, supporting the results with similar ct values (data not shown). similarly, the cells' positive infection was confirmed by pla testing, although this test cannot differentiate between cat01 and margarita csfv strains. to detect the presence of csfv rna of both viral strains (cat01 and/or margarita) in the sera, tonsil, and spleen of animals 1 and 3 (group a) and 4 and 5 (group b), the e2-gene fragment reported by lowings et al. [44] as a phylogenetic marker was amplified by end point rt-pcr [45] . in all of the samples analysed from animals that developed the csf acute form (group a), the sequence corresponding to the margarita strain (aj704817) used as the inoculum was detected. furthermore, the samples analysed from superinfected animals (group b: csfv catalonia 01 persistently infected inoculated with csfv margarita strain) only showed the sequence corresponding to the cat01 strain [30] (fig 4) . despite its significance, the mechanisms of mutual exclusion by viral variants are far from being completely understood, and the actual knowledge is basically derived from studies at the cellular level in established cell lines [14, 16, 19, 49] . very few reports have demonstrated the phenomenon of sie at the organism level, and, to our knowledge, these models have been limited to plant viruses, west nile virus (wnv) in mosquitoes, and peking duck hepatitis b virus (dhbv) [50] [51] [52] . in addition, it has not yet been demonstrated in a mammalian host at the systemic level. previous works have reported the capability of csfv to generate postnatally persistent infection in both domestic pigs and wild boars [5, 6] . subsequently, it was also shown that postnatally persistently infected pigs were unable to elicit a specific immune response to a csfv live attenuated vaccine and that the viral vaccine rna was undetectable in any of the samples analysed [7] . against this background, we assessed the capacity of csfv to generate sie in csfv persistently infected swine. for that purpose, csfv persistently infected wild boars were inoculated with a csfv strain that induce acute disease with a higher replication rate [29, 36] . because pestiviruses are immunologically and genetically closely related, accurate serological characterisation of csfv isolates is impeded by the extensive cross-reactions observed among pestivirus members and the limited availability of mabs capable of differentiating among different csfv isolates [27, 45, 53] . to differentiate the csfv margarita strain rna from the csfv cat01 strain rna in the samples from the present study, a specific qrt-pcr for margarita strain rna detection was developed. thus, alongside the model of infection with the margarita strain, the qrt-pcr assay developed allowed for clear discernment of whether there was actually a blockage that prevented susceptibility to infection by the second virus in both the absence of clinical signs and the absence of molecular detection of the superinfecting virus. notwithstanding the high infection rate of the cat01 strain in persistently infected animals from group b (primary virus infection), good health status was maintained after inoculation with the margarita csfv virulent strain (secondary infection) in the absence of viral detection in sera throughout the study, except in one animal at 13 dpi with a low margarita strain rna load (animal 4). despite the important role that neutralising antibodies play in csfv protection [29, 54] , complete absence of neutralising antibodies response was found after margarita strain infection in these animals. similarly, absence of an ifn-γ-producing cell response against csfv or pha was also observed. considering the role played by ifn-γ in the control of csfv infection [34, 55] and the lack of responsiveness to ifn-γ-producing cells after pha stimulation, the csfv-superinfected animals maintained a immunosuppression state similar to that previously described in postnatal persistent infection [5, 6] . previous work has proved how the failure to induce optimal levels of the humoral and cellular responses after csfv infection promoted the spread of the virus and its relationship with disease progression [29, 54] . in this regard, the implications of the cellular and neutralising antibody response in clinical protection against the acute form in the csfv-superinfected animals from this study are excluded. furthermore, no superinfecting virus excretion was detected in any of the animals from group b, whilst the high viral load generated by the strain that induced the persistent infection (cat01 strain or primary infection) was maintained until the end of the trial, supporting our previous results [7] . in contrast, the csfv margarita strain generated the acute form of the disease in animals from group a, with high margarita rna loads in all of the samples analysed. in addition, the failure of the humoral response in the pigs that developed acute csf was previously described [29] . in addition to the adaptive immune response, the innate immune response to the virus, as measured by type i ifn-α in the serum, also seemed to be impaired, in terms of ifn-α detection because ifn-α values were undetectable in the sera from postnatally persistently infected wild boars after csfv margarita strain inoculation. at the same time, the progression of the acute disease in group a was correlated with an increase in levels of endogenous ifn-α, as has been previously described [29, 46, 56, 57] . the absence of an ifn-α response in the cat01 persistently infected animals after margarita strain inoculation (secondary infection) probably was due to the almost complete lack of margarita strain replication in these animals. otherwise, specific csfv-blockade phenomena for ifn-α might be occurring. efficient viral strategies to escape the type i ifn-induced antiviral mechanisms have been described within pestivirus. in this regard, the viral rna triggers ifn synthesis, and the viral rnase e rns inhibits ifn expression induced by extracellular viral rna [58] . in addition, the viral protein n pro suppresses type i ifn (ifn-α/β) induction by mediating proteasomal degradation of ifn regulatory factor 3 (irf-3) [58] [59] [60] . for instance, in persistent infection, bvdv maintains "self-tolerance" by avoiding the induction of ifn, without compromising the ifn action against unrelated viruses ("nonself") [58] . in the case of csfv-infected pigs, it has been recently demonstrated that functional n pro significantly reduced local ifn-α mrna expression responses at local sites of virus replication [61] . these highly selective "self" models of evasion of the interferon defence system might be key elements in the success of persistent infections and could promote, in addition, the generation of sie phenomena. previous reports have suggested that the availability of mammalian models for sie in vivo is hampered by the interferon response generated against the infecting virus in these species [11, 24] . it is noteworthy that csfv postnatally persistently infected swine have shown an immunosuppression state comprising a reduction in interferon responses (types i and ii) [5] [6] [7] . this immunological status might promote the maintenance of a high and constant csfv load, as already described, preventing second viral entry [5] . nevertheless, further studies would be needed to clarify the molecular mechanisms involved in this phenomenon. at 13 dpi, low levels of margarita rna were detected only in some collected tissues from persistently and superinfected wild boars (group b), principally in animal 4, in which margarita rna was detected from the spleen and liver, as well as in the serum. however, the level of margarita rna detection was approximately fifteen times less than the acutely infected animals from group a ( table 2 ). the margarita rna levels found in the superinfected animals might be correlated with the low margarita strain viral loads in some macrophages in these tissues [62] . in contrast, despite pbmcs being a well-known target for csfv viral replication [62] , after in vitro assay, the presence of csfv margarita rna could not be detected in either the pbmcs or st cell extracts from group b. additionally, the in vitro superinfection of isolated pbmcs failed when they were derived from persistently infected piglets but were clearly positive for assays with cells from naïve animals, as demonstrated by means of calculated δct values, supporting that pbmcs from persistently infected animals were substantially protected from superinfection after in vitro inoculation with the margarita virus strain. these results suggest that sie still occurs at the tissue level (table 3 ). in contrast, the margarita strain rna could not be detected after the sequence analyses of the samples from persistently infected margarita-inoculated animals (group b) nor even in the tonsil, one of the main targets for csfv replication [3, 63] . nevertheless, next-generation sequence analyses would be of great interest to analyse these samples in detail, emphasising the spleen and liver tissues that were also positive for rna margarita strain detection after superinfection. altogether, although it is a very complex mechanism, if compared with the acutely infected group a, these results showed that a phenomenon of csfv sie occurred at the systemic level. nevertheless, the 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enhanced interferon-α response classical swine fever virus npro interacts with interferon regulatory factor 3 and induces its proteasomal degradation npro of classical swine fever virus contributes to pathogenicity in pigs by preventing type i interferon induction at local replication sites classical swine fever virus marker vaccine strain cp7_e2alf: shedding and dissemination studies in boars classical swine fever virus strain "c". how long is it detectable after oral vaccination? j vet med b infect dis vet public health we thank valentí rosell, iván cordón and david solanes for their help in the animal facilities. key: cord-003404-eqgc8v7y authors: may, win lai; kyaw, myat phone; blacksell, stuart d.; pukrittayakamee, sasithon; chotivanich, kesinee; hanboonkunupakarn, borimas; thein, khin nyo; lim, chae seung; thaipadungpanit, janjira; althaus, thomas; jittamala, podjanee title: impact of glucose-6-phosphate dehydrogenase deficiency on dengue infection in myanmar children date: 2019-01-02 journal: plos one doi: 10.1371/journal.pone.0209204 sha: doc_id: 3404 cord_uid: eqgc8v7y glucose-6-phosphate dehydrogenase (g6pd) deficiency may affect the clinical presentation of dengue due to the altered redox state in immune cells. we aimed to determine the association between g6pd deficiency and severity of dengue infection in paediatric patients in myanmar. a cross-sectional study was conducted among paediatric patients aged 2–13 years with dengue in yankin children hospital, myanmar. one hundred and ninety-six patients positive for dengue infection, as determined via pcr or elisa, were enrolled. dengue severity was determined according to the 2009 who classification guidelines. spectrophotometric assays determined g6pd levels. the adjusted median g6pd value of males in the study population was used to define various cut-off points according to the who classification guidelines. g6pd genotyping for mahidol, kaiping and mediterranean mutations was performed for 128 out of 196 samples by real-time multiplex pcr. 51 of 196 (26.0%) patients had severe dengue. the prevalence of g6pd phenotype deficiency (< 60% activity) in paediatric patients was 14.8% (29/196), specifically, 13.6% (14/103) in males and 16.2% (15/93) in females. severe deficiency (< 10% activity) accounted for 7.1% (14/196) of our cohort, occurring 11.7% (12/103) in males and 2.2% (2/93) in females. among 128 samples genotyped, the g6pd gene mutations were detected in 19.5% (25/128) of patients, with 20.3% (13/ 64) in males and 18.8% (12/64) in females. the g6pd mahidol mutation was 96.0% (24/25) while the g6pd kaiping mutation was 4.0% (1/25). severe dengue was not associated with g6pd enzyme deficiency or presence of the g6pd gene mutation. thus, no association between g6pd deficiency and dengue severity could be detected. trial registration: the study was registered following the who international clinical trials registry platform (who-ictrp) on thai clinical trials registry (tctr) website, registration number # tctr20180720001 introduction glucose-6-phosphate dehydrogenase (g6pd) is an enzyme presenting in the cytoplasm of human cells that participates in the pentose phosphate pathway and supplies reducing energy by maintaining levels of the co-enzyme nicotinamide adenine dinucleotide phosphate (nadph) [1] . g6pd deficiency is an x-linked inheritance. g6pd gene consists of 13 exons with approximately 18kb and is situated on the distal long arm of x chromosome (xq28). about 180 mutations of g6pd gene have been reported resulting in protein variants with different levels of enzyme activity. nadph is essential for both oxidant and antioxidant systems of cells. in the antioxidant pathway, nadph maintains the reduced form of glutathione to protect cells from oxidative damage [2] . this reduction in cells causes accumulation of redox oxidative species (ros) and leads to senescence [1] and haemolysis in red blood cell [3] . on the other hand, nadph is also involved in the oxidant pathway to produce ros. although the overproduction of these ros may adversely affect cell function, cells of immune system also need these reactive species to kill invading organisms. phagocytes of the immune system need these reactive species to kill invading pathogens as part of the innate immune system. in cases of severe g6pd deficiency, the lack of oxidative metabolism can cause a reduction in oxygen-dependent phagocytosis as observed in chronic granulomatous disease [4] and allows for viral replication [5] [6] [7] [8] . glucose-6-phosphate dehydrogenase (g6pd) deficiency affects more than 400 million people all over the world [9] and prevalence is approximately 35% in africa and ranges from 6.0 to 10.8% in southeast asia [10] . in myanmar, the prevalence is 11.1% and 4.2% in adult males and females respectively [11] , while it is 15.0% and 2.1% in healthy children males and females respectively [12] . to note, 11.8% of males and 21.0% of females possess the g6pd mutation [13] with the g6pd mahidol variant occurring in 91.3% of children in myanmar [14] . dengue virus infection is one of the leading causes of morbidity and mortality in children living in tropical and sub-tropical regions [15] . according to who, approximately 390 million people worldwide experience a dengue infection annually [16] . in southeast asia, dengue leads to over 5,900 deaths annually [17] . in myanmar, all four dengue serotypes are known to co-circulate, and children are at the highest risk for infection [18] . the reported case fatality rate was 7 per 1,000 dengue cases in 2014 [19] . most dengue patients present with undifferentiated febrile illness that some may progress to life threatening disease [20] . how patients' genetic background affects the development of severe infection has become an area of interest. g6pd deficiency is one of the reported genetic variants associated with infections [8, 21] . in vitro studies reported that monocytes from g6pd-deficient individuals had increased susceptibility to dengue virus serotype 2 infections along with higher viral replication [5, 6] . whether higher replication of dengue virus in g6pddeficient individuals increases the likelihood of disease severity remains unknown. herein, we investigated the association between g6pd deficiency and severity of dengue infection in paediatric patients in myanmar. a cross-sectional prospective study was conducted in yankin children hospital, yangon from august 2015 to august 2016. paediatric patients (age range: 2-13 years) with a fever for 1-7 days and clinically suspected of having dengue fever were screened. written informed consent and verbal assent were taken from caregivers and children, respectively. three millilitres of blood were taken, and sera were tested for the ns1 antigen as well as for igm and igg antibodies using the sd bioline dengue duo rapid diagnostic test (rdt) (standard diagnostics, korea). patients with a positive ns1 antigen and/or dengue igm result were enrolled. patients who had a diagnosis other than dengue, such as co-infections and immunocompromised status (e.g., steroid use, chemotherapy, and/or positive hiv status) were excluded. patients were followed 1-3 weeks after admission for convalescent serum sample collection. complete blood count (cbc), reticulocyte count (rc) and g6pd activity were measured within 24 hours after collection of acute sera. g6pd activity was assessed via spectrophotometry (randox laboratory, uk). the adjusted median value of g6pd activity for males in our cohort was used to define various cut-off points [22] . dengue severity was classified according to the 2009 who classification guidelines [23] . both acute and convalescent samples were stored at -80˚c prior to further testing. the g6pd activity, cbc and rc were re-checked at least one month after the first sample collection for patients at risk of being misdiagnosed for g6pd deficiency based on analyses of the first blood sample collected, such as patients with rc values > 2%, those who suffered or recovered from acute haemolytic anaemia, and those who had a blood transfusion before the first sample collection. the study protocol was approved by the ethics dengue, zika and chikungunya virus detection using a reverse transcriptase pcr assay. rna was isolated from acute patient serum samples (140 μl) using the qiaamp viral rna mini kit (qiagen, germany). rna extractions were performed according to the manufacturer's protocol. rna was stored at -80˚c until further use. one-step reverse transcriptase (rt) pcr was performed to detect dengue, zika and chikungunya viruses in a single assay using zdc multiples rt-pcr assay (bio-rad, usa). assays were performed according to the manufacturer's protocol, with an additional rnase p3 primer mix as an internal extraction control. for each rt-pcr assay, a positive control, provided from the kit; negative control (healthy donor sera); and no template control were included. all rna samples were performed in duplicate. a positive pcr was defined as when one or both duplicates had a signal above a fixed threshold of 200 for all the respective targets: fam for zika virus, hex for chikungunya virus, and texas red for dengue virus. g6pd activity. sample was stored at 4-8˚c, then transported to department of medical research (dmr) laboratory in yangon myanmar and processed within 24 hours. spectrophotometry was conducted using humalyser 3000 spectrophotometer (medsource ozone biomedicals, delhi, india) and kits from randox (cat no pd 410, randox laboratories ltd., crumlin, uk). the procedure provided in the manual with the kit was followed. normal and deficient g6pd controls (cat no pd 2617 for deficient and cat no pd 2618 for normal, randox laboratories ltd., crumlin, uk) were used. the results were considered valid if the measured activities of the controls were within the reference range. enzyme activity was determined by the spectrophotometer set at 37˚c to measure the rate of absorbance at 340 nm under ultraviolet light. enzyme activity was calculated and adjusted for the hb concentration value from complete blood count (cbc) recorded at the time of sample collection. the adjusted median value of g6pd activity for males in our cohort was used to define various cutoff points [22] . g6pd genotyping by real-time pcr. dna samples were extracted from 200 μl of whole blood using the qiaamp dna blood mini kit (qiagen, germany) according to the manufacturer's recommendation. three g6pd variants (g6pd mahidol c.487g>a; g6pd kaiping c.1388g>a; and g6pd mediterranean c.563c>t) previously reported in g6pd-deficient individuals in myanmar were detected by real-time pcr with the bio-rad cfx 96 real-time system and c1000 thermal cycler (bio-rad, usa). genotyping pcr, with melting curve analysis using dual-labelled, self-quenched probes, was performed using 25 μl reactions, containing 12.5 μl of iq multiplex power mix, 0.75 μl each of forward and reverse primers, 0.5 μl each of snp and wt probes, 9 μl sterile distilled water, and 1 μl genomic dna. the amplification conditions were 3 minutes at 95˚c for iq multiplex power mix activity, followed by 40 cycles of 15 seconds at 95˚c for denaturation, and 1 min at 70˚c for annealing and extension for g6pd genotyping. the samples were run together with the plasmid control for each targeted mutation; three separate pcr reactions were run in every sample for the three different mutations. fluorescence and ct values using the cfx manager software (bio-rad, usa) were used to determine genotypes. each sample's result was verified by examining the pcr curve generated to eliminate false-positive results due to aberrant light emission (s1 table) . acute dengue infection. laboratory confirmed acute dengue infection was defined by a positive qrt-pcr, ns1 antigen (ag) elisa, or igm elisa result. primary or secondary dengue infections were defined according to a negative or positive igg elisa result in the febrile phase (� 7 days), respectively [24] . dengue severity. the severity of dengue infection was classified according to the 2009 who guideline [23] . dengue patients were classified as non-severe or severe based on clinical and laboratory parameters. patients with non-severe dengue were classified into two groups based on the absence or presence of warning signs. non-severe dengue without warning signs was defined if the patient had fever with 2 of these following criteria: nausea/vomiting, rash, aches and pains, tourniquet test positive, leucopoenia without presentation of any warning signs. warning signs included abdominal pain or tenderness, persistent vomiting, clinical fluid accumulation, mucosal bleed, lethargy/restlessness, liver enlargement > 2cm and increase in haematocrit (� 44%) concurrent with rapid decrease in platelet count (<100 × 10 3 per μl). severe dengue was defined as: dengue shock syndrome (dss), fluid accumulation with respiratory distress, and/or severe bleeding that necessitated blood transfusion or involved an organ [23] . dss was diagnosed by the presence of hypotension or narrow pulse pressure (< 20 mmhg) [25] . g6pd deficiency. the who classification is based on the g6pd activity expressed as a percentage of the median g6pd value of a normal male population. class i-v are defined as: class i < 1% (associated with chronic non-spherocytic haemolytic anaemia), class ii 1-< 10%, class iii 10-<60%, class iv 60-150%, and class v > 150%. in this study, g6pd deficiency was classified as severe, moderate, or normal if enzyme activity was < 10%, 10-60%, or > 60%, respectively [26] . the adjusted median enzyme activity in males was determined as 100% activity of the study population [22] . only patients with laboratory confirmed acute dengue infection were included in the statistical analysis. data were evaluated using descriptive statistics, medians (interquartile ranges) for continuous variables and frequencies and percentages for categorical variables. chi-squared test or fisher's exact test (for categorical variables) and mann-whitney u test (for continuous variables) were used for comparative analyses between severe and non-severe dengue infections and between patients with or without g6pd deficiency. associations between g6pd status and dengue severity were evaluated using the binary logistic regression test with 95% confidence interval. data were analysed using ibm spss statistics (ibm, armonk, ny, usa) data manager software package. in total, 506 patients with clinically suspected dengue infection were screened by rdt. in our study, 294 (58.0%) patients were excluded (163 non-dengue and 131 with past dengue infection), and 212 patients were enrolled in the study (defined as positive ns1 ag and/or igm result) as indicated on fig 1. out of 212 enrolled patients, 16 were excluded (2 did not have glucose-6-phosphate dehydrogenase deficiency on dengue infection in children sufficient blood volume, 11 had a negative dengue result, and 3 lacked quantitative g6pd results). in total, 196 patients with laboratory confirmed acute dengue infection and with quantitative g6pd results were included in the analysis (fig 1) . among the 196 patients with a confirmed acute dengue infection, 51 (26.0%) were classified as severe dengue while 145 (74.0%) were classified as non-severe dengue. among the 145 patients with non-severe dengue, 115 (79.3%) presented with warning signs and 30 (20.7%) were without. all patients with severe dengue presented with dengue shock syndrome (dss). among these dss patients, one patient also had fluid overload with respiratory distress, and one patient had severe bleeding that necessitated blood transfusion. ten out of fifty-one (20.0%) of children with severe dengue were admitted to the icu, and 11 required a blood transfusion. patients with severe dengue were significantly younger (p = 0.001) and were hospitalised for a longer time (p <0.001) compared to non-severe dengue patients. the majority of patients were classified as secondary dengue (84.2%, 165/196), and only 15.8% (31/196) were classified as primary dengue. severe dengue was significantly higher in secondary dengue patients compared with the non-severe group (p = 0.007). nausea and vomiting were more frequent in patients with severe dengue (p = 0.001). however, a positive tourniquet test was less frequent in children with severe dengue (p = 0.007). other clinical warning signs, including tachycardia and hypotension, were significantly detected in patients with severe dengue. (p <0.001). patients with severe dengue also had significantly higher haematocrit, higher leukocyte counts, and lower platelet counts than nonsevere dengue patients (table 1) . the median g6pd activity of 196 dengue-confirmed patients was 5.4 u/g hb (0.1-11.5). the adjusted male median value of g6pd activity was determined by excluding 12 male samples with � 10% activity from the derived value [22] . the adjusted male median was 5.7 u/g hb (1.2-11.5) ( table 2 ). based on this value, g6pd activity < 0.057 u/g hb was defined as class i, activity between 0.057-0.57 u/g hb was class ii, 0.57-3.42 u/g hb was class iii, 3.42-8.55 u/g hb was class iv, and > 8.55 u/g hb was class v for this population ( table 3) . the prevalence of g6pd phenotypic deficiency, based on these cut-off reference values revealed that 14/196 (7.1%), including 12 (11.7%) males and 2 (2.2%) females were in class ii (severe g6pd deficiency) and 15/196 (7.7%), including 2 (1.9%) males and 13 (14.0%) females were in class iii (moderate g6pd deficiency) ( table 3) . real-time pcr for three known single nucleotide polymorphisms (snps) in the g6pd gene in myanmar, mahidol, kaiping and mediterranean, was performed in 128 of 196 dengue-confirmed samples (64 males and 64 females). among them, 25/128 (19.5%) had recognised snps, (13/64 (20.3%) were males while 12/64 (18.8%) were females). twenty-four patients had the mahidol mutation (96%), and one female (4%) had the kaiping mutation. the mediterranean genotype was not detected. samples from of 28/29 (96.5%) (14/ 14 males, 14/15 females) patients with g6pd deficient phenotypes were included in this genotypic study. among them, only 20/ 28 (71.4%) had mutations, while 9/ 28 (32.1%) (2 male and 7 female) did not have mutation. among the samples from patients without g6pd phenotype deficient, 6 / 100 (6%) samples (1 male and 5 female) had mutations (table 4) . among the 25 patients with a g6pd mutation, 14/25 (56.0%) were classified as who class ii and among them 13/14 (92.8%) were heterozygous male and one homozygous female. there was one male with the mahidol mutation in who class iv. this patient had a reticulocyte count < 2% while testing for g6pd activity. females with genotypic mutations have varying degrees of the g6pd deficient phenotype being classified as who class ii to who class iv (fig 2) . severe dengue was not associated with a g6pd deficiency phenotype nor genotype variants whether we used a cut off of < 30% (i.e. only including hemizygous males and homozygous females) or a cut off of < 60%, corresponding to classes i to iii of the who classification (table 5) . severe dengue was diagnosed in 5/29 (17.2%) children with < 60% enzyme activity and in 46/167 (28%) children with g6pd levels > 60% (p = 0.249). in addition, 6/25 (24%) study participants had a g6pd snp, and 25/103 (24%) did not have a detectable snp and severe dengue (p = 0.977). herein, we investigated if there is an association between dengue fever and g6pd deficiency in children in myanmar. the majority of children in this study, 84.2%, had secondary dengue infection possibly due to the four dengue serotypes circulating in this endemic area, which leads to subsequent infections [19] . a quarter of the children in the present study had a severe dengue fever, all of whom presented with dss. the frequency of secondary dengue infection was significantly higher in the severe dengue than in the non-severe dengue group, which is consistent with a meta-analysis of 40 studies in asia that described the association between dss and secondary dengue infection [27] . no association between severity of dengue infection and g6pd enzyme deficiency or g6pd mutation was detected. the result of this study is consistent with two earlier studies conducted in thailand [28, 29] . tanphaichitr and colleagues did not demonstrate a correlation between g6pd deficiency and dengue severity in 89 male paediatric patients (age range: 1-13 years) with dengue haemorrhagic fever age. in this study, 17 out of 72 patients had g6pd deficiency [28] . seridhoranakul et al. also did not demonstrate a significant relationship between g6pd deficiency and dhf in a study with 80 dhf patients, in which 9 patients were g6pddeficient, and 131 controls, in which 13 patients were g6pd-deficient [29] . tanphaichitr et al. reported a higher prevalence of g6pd deficiency in male dhf patients, which was 19.1% table 3 . prevalence of g6pd among confirmed dengue paediatric patients according to who classification. compared to 12% in bangkok, and suggested that g6pd-deficient males may suffer more from dhf [28] . the prevalence of g6pd deficiency in paediatric patients with dengue infection in the present study was lower than the prevalence of g6pd deficiency in children visiting the emergency department of yankin children hospital, ages: 1 month to 12 years old, who were screened for dengue via rdt (14.8% vs. 18.5%, respectively). in this study, g6pd deficiency glucose-6-phosphate dehydrogenase deficiency on dengue infection in children was diagnosed if enzyme activity was < 60% of the adjusted median value, which was slightly lower than the adjusted median value in our study (3.24 u/g hb vs. 3.42 u/g hb) [30] . when comparing the results of these two studies, conducted in the same setting, g6pd-deficient children may not be more susceptible to dengue infection compared to children with normal g6pd activity. two in vitro studies demonstrated that human monocytes from g6pd-deficient individuals had higher replication of dengue virus serotype 2 [5, 6] , and these studies suggested that the likelihood of severe dengue was likely to increase in g6pd-deficient individuals. the findings from our study did not support this hypothesis. however, viral load was not assessed in our study, thus, the association of viral load or viral replication to clinical outcomes was beyond the scope of this study. the different dengue serotype also affects clinical outcomes. although dengue serotypes were not assessed in this study, previous studies described that the prevalent serotype among patients with dhf in myanmar in recent years was dengue serotype 1 [31, 32] . additionally, all four serotypes participated in the 2015 outbreak [19] . the association between g6pd deficiency, viral load and different dengue serotypes warrant future studies. more than 180 different mutations in the g6pd gene have been identified, and these variants have different impacts on enzyme activity [33], which might result in different clinical manifestations. one study reported that patients with the g6pd mediterranean mutation showed a more severe clinical course during bacterial infection compared to patients with wild type g6pd [34] . most patients with g6pd mutation possessed the mahidol variant, which is consistent with previous studies in myanmar [10, 14] . genotypic mutation was not detected in 2 male and 7 female patients with phenotypic deficiency and may have been due to the limited number of genotypes that we sought; previous research in this region has identified other uncommon g6pd mutations for instance g6pd union, coimbra and canton [14, 35, 36] . bancone et al reported this molecular heterogeneity among this ethnicity along thai-myanmar border [35] . six patients (1 male and 5 females) who had genotypic mutation had normal g6pd activities; all had reticulocyte count < 2%, excluding a false negative phenotype due to haemolysis. g6pd mahidol is considered a moderately severe variant with varying g6pd activities which classifies it as who class ii/iii; almost all of male patients with mahidol variant had enzyme activities < 10% (i.e. were class ii). this finding is consistent with results of another study from the thai-myanmar border [35] . a significant association between g6pd mutation, in predominantly the mahidol variant, and dengue infection was not found. whether g6pd genotypes other than mahidol could affect dengue severity cannot be excluded based on our current study. thus, the effect of other g6pd gene mutations on dengue infection is unknown, and complementary studies should be performed in different populations. in this study, the demographic, clinical and haematological parameters of patients with severe and non-severe dengue were compared, and we found that young age is a risk factor significantly associated with severe dengue infection. data collected from 2011 to 2015 in myanmar also reported that the highest case fatality rate was seen in the infantile age group [19] . the clinical parameters of warning signs were more frequent in severe dengue patients, which was in agreement with previous studies; hepatomegaly was a risk factor of dss or severe dengue infection [37] [38] [39] [40] [41] [42] , abdominal pain was a prognostic factor for severe dengue [43, 44] , and lethargy was the best clinical sign to identify patients who may progress to severe dengue [45] . although there were no patients with persistent vomiting, those who had a history of vomiting were frequently more present in the severe dengue patient group. other studies reported that persistent vomiting was one of the factors associated with dss or high mortality [27, 37] . in the 2009 who classification for dengue, high haematocrit � 20% from baseline, concurrent with a rapidly decreasing platelet count, is listed as a warning sign [23] . in this study, patients with severe dengue had significantly higher haematocrit and lower platelet count than those with non-severe dengue. the findings in this study also reinforced the applicability of the warning signs outlined in the 2009 who guidelines to detect severe dengue infection in children in myanmar. the strength of this study was its prospective design. all acute dengue cases were confirmed by real-time pcr for the virus and elisa assays, and g6pd deficiency was confirmed by a gold standard quantitative spectrophotometric assay. although the cut-off level of g6pd activity did not represent all children in myanmar, it did reflect the g6pd activity of children with dengue infection seen at the yankin children hospital. there were limitations in our study. for example, dengue viral load and serotypes were not analysed; therefore, we were not able to detect the relationship among viral load, serotypes, dengue severity and g6pd deficiency. additionally, g6pd mutations were not assessed in all the participants, and the impact of rare g6pd variants previously reported in myanmar population could not be analysed, which may have impacted our statistical analysis. the parameters, e.g., chest x-rays, liver function tests and ultrasounds of chest and abdomen that may affect dengue severity classification in terms of major organ involvement or evidence of plasma leakage were not collected, and these factors may have affected our study's outcomes. we did not find an association between g6pd deficiency and dengue severity. most of our patients had g6pd mahidol, the most common variant in myanmar. this study proposes activity cut-off points of enzyme activity according to the who classification, as well as the prevalence of g6pd deficient and the predominant genetic mutation among paediatric patients. this study reconfirms the usefulness of the who 2009 dengue classification in paediatric dengue patients in myanmar. more research is needed to explore other factors related to dengue severity such as dengue virus viral load, dengue virus den serotypes and genotypes. supporting information s1 table. forward, reverse primers, snps and wt probes used in the study. 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deficiency and dengue hemorrhagic feve glucose-6-phosphate dehydrogenase deficiency among children visiting emergency department of yankin children hospital validation of the quantitative pointof-care carestart biosensor for assessment of g6pd activity in venous blood seven different glucose-6-phosphate dehydrogenase variants including a new variant distributed in lam dong province in southern vietnam association between clinical profiles and severe dengue infection in children in developing country dengue infection in children in ratchaburi, thailand: a cohort study. ii. clinical manifestations. plos neglected tropical diseases prognostic indicators for dengue infection severity disease severity and mortality caused by dengue in a dominican pediatric population. the american journal of tropical medicine and hygiene risk factors of dengue shock syndrome in children predictive symptoms and signs of severe dengue disease for patients with dengue fever: a meta-analysis clinical risk factors for dengue shock syndrome in children. paediatr indonesia predictive factors of dengue shock syndrome at the children hospital no clinical and laboratory signs associated to serious dengue disease in hospitalized children we would like to acknowledge ms. seungyeon kwak and mr. byeon mun sub from korea university guro hospital and ampai tanganuchitcharnchai from moru for their assistance. we also thank ms. atthanee jeeyapant for her data analysis expertise and ms. gaye proctor from moru for english language editing. we are also grateful to dr. kyaw zin thant, director general, department of medical research, for his help and support throughout the study. key: cord-255588-nh34lgdh authors: guo, fang; mead, jennifer; aliya, nishat; wang, lijuan; cuconati, andrea; wei, lai; li, kui; block, timothy m.; guo, ju-tao; chang, jinhong title: ro 90-7501 enhances tlr3 and rlr agonist induced antiviral response date: 2012-10-03 journal: plos one doi: 10.1371/journal.pone.0042583 sha: doc_id: 255588 cord_uid: nh34lgdh recognition of virus infection by innate pattern recognition receptors (prrs), including membrane-associated toll-like receptors (tlr) and cytoplasmic rig-i-like receptors (rlr), activates cascades of signal transduction pathways leading to production of type i interferons (ifn) and proinflammatory cytokines that orchestrate the elimination of the viruses. although it has been demonstrated that prr-mediated innate immunity plays an essential role in defending virus from infection, it also occasionally results in overwhelming production of proinflammatory cytokines that cause severe inflammation, blood vessel leakage and tissue damage. in our efforts to identify small molecules that selectively enhance prr-mediated antiviral, but not the detrimental inflammatory response, we discovered a compound, ro 90–7501 (‘2’-(4-aminophenyl)-[2,5′-bi-1h-benzimidazol]-5-amine), that significantly promoted both tlr3 and rlr ligand-induced ifn-β gene expression and antiviral response, most likely via selective activation of p38 mitogen-activated protein kinase (mapk) pathway. our results thus imply that pharmacological modulation of prr signal transduction pathways in favor of the induction of a beneficial antiviral response can be a novel therapeutic strategy. infection of viruses is promptly recognized by host innate pattern recognition receptors (prrs), including toll-like receptors (tlrs), rig-i-like receptors (rlrs), nod-like receptors and ctype lectins [1, 2] . this results in production of type i interferons (ifn), proinflammatory cytokines and chemokines that orchestrate the elimination of the pathogens. the essential role of the prrmediated innate immune response in defending against microorganism infection has been extensively demonstrated in vivo in murine models with knockout of the genes encoding either specific prrs or their key signaling components (reviewed in [3, 4] ). however, like adaptive immunity, the innate immune response can also be detrimental to hosts. indeed, in many occasions, it is not the viral replication itself, but the overwhelming production of proinflammatory cytokines that causes severe inflammation, tissue damage, blood vessel leakage and occasionally permeabilization of the blood brain barrier that leads to the penetration and infection of central nervous system by viruses [5, 6, 7] . in addition, because of the vital role of prrs in defending against virus infection, pharmacological activation of prr-mediated innate host response has been extensively explored as a broad-spectrum antiviral approach [8, 9, 10] . however, systematic administration of the prr agonists in doses necessary to achieve antiviral effects is usually associated with significant adverse reactions, due to the activation of a wide-spectrum of cellular responses and massive production of proinflammatory cytokines [11, 12, 13, 14] . tlrs and rlrs are two major types of prrs that recognize virus infection and induce innate immune response. interestingly, induction of type i ifns, the primary antiviral cytokines, and other proinflammatory cytokines upon activation of tlrs and rlrs is controlled by multiple overlapping, but distinct signal transduction pathways (reviewed in [15] ). while activation of nuclear factor kappa-light-chain-enhancer of activated b cells (nfkb) and distinct mitogen-activated protein kinase (mapk) pathways are critical for the production of many proinflammatory cytokines and chemokines, activation of the interferon regulatory factor 3 (irf3) (or irf7) pathway is only required for induction of type i ifns as well as a group of antiviral proteins, such as ifit1, guanylate binding protein 1 and zinc finger antiviral protein [16, 17, 18] . in addition, although the three map kinases, p38, erk and jnk, can be activated by tlr and rlr agonists and viral infection [19, 20] , each of the three mapks has been demonstrated to play distinct roles in regulating the expression of type i ifn and other proinflammatory genes [21, 22, 23] . for example, it has been shown recently that erk activation is required for tlr3-induced chemokine production in murine dendritic cells, whereas jnk activation has a negative regulatory effect on chemokine production [24] . it is, therefore, possible to pharmacologically modulate the virus-and/or prr-agonist-induced innate immune response by targeting distinct signal transduction pathways to selectively enhance the antiviral response, but alleviate the detrimental inflammatory response. it is conceivable that such a therapy should be effective to a broad spectrum of virus infections, either alone or in combination with prr agonists. in order to discover compounds with the expected pharmacological property, we set out to establish reporter cell lines for high throughput screening of small molecules that selectively enhance tlr3 ligand-induced ifn-b gene expression, but do not affect nfkb activation, which is a central player in the induction of proinflammatory cytokines, but plays a less prominent role in type i ifn gene expression [25] . our initial high throughput screening campaign has thus far identified a compound, ro 90-7501, that selectively enhances tlr3 and rlr ligand-induced ifn-b gene expression and antiviral response, most likely via activation of the p38 mapk pathway, but not the nfkb or irf3 pathway. our results have thus proved the concept that pharmacological modulation of tlr3 agonist-induced innate immune response can be a viable antiviral therapeutic strategy. type i ifns are the primary antiviral cytokines induced by the prr agonist-activated innate immune response, whereas nfkb is the key transcription factor for the induction of proinflammatory cytokines and chemokines, but plays a less important role in ifnb gene activation [25] . first, we set out to establish cell-based assays suitable for high throughput screening of compounds that selectively enhance tlr3 agonist-induced type i ifn production, without enhancing the activation of nfkb. two tlr3-expressing hek293 (293tlr3ha, invivogen)-derived stable reporter cell lines that express firefly luciferase under the control of a human ifn-b promoter [26] (designated 293tlr3/ifnbluc) or an artificial promoter containing consensus nfkb binding sites (clontech) (designated 293tlr3/nfkbluc) were established. as shown in fig. 1 , poly i:c treatment of both the reporter cell lines induced luciferase expression in a concentration dependent manner. as expected, poly i:c treatment of 293/ifnbluc cell line without tlr3 fails to induce luciferase expression (data not shown). moreover, as shown in figs. 2a and b, while the poly i:cinduced luciferase expression was dose-dependently inhibited by chloroquine (a lysosome acidification inhibitor) and ikk-2 inhibitor iv (an ikk-b inhibitor) in both 293tlr3/ifnbluc and 293tlr3/nfkbluc cells, ly294002, an inhibitor of phosphoatidylinositol 3-kinase (pi3k) that is required for activation of irf3 [27] , only inhibited poly i:c-induced luciferase expression in 293tlr3/ifnbluc, but not in 293tlr3/ nfkbluc cells (fig. 2c) . these results thus validated that the luciferase expression in 293tlr3/ifnbluc and 293tlr3/ nfkbluc cells was controlled by the functional ifn-b promoter and the nfkb responsive element, respectively. ro 90-7501 selectively enhances ifn-b, but not nfkb activation through screening of 1,280 small molecules in the library of pharmacologically active compounds (lopac) obtained from sigma, we were able to identify a compound, ro 90-7501 ('2'-(4-aminophenyl)-[2,59-bi-1h-benzimidazol]-5-amine) (fig. 3a) , that itself affected neither ifn-b nor nfkb promoter activity, but significantly enhanced poly i:c-induced ifn-b promoter activation and inhibited the activation of nfkb in a dose-dependent manner (figs. 3b and c). while its enhancement of ifnb promoter activation was statistically significant at doses ranging from 0.8 to 12.5 mm, its inhibition on nfkb activation was only statistically significant at the highest dose tested. no cytotoxicity was observed at concentrations up to 250 mm of ro 90-7501 by a mtt assay (fig. 3d ). ro 90-7501 enhances poly i:c-induced activation of ifnb promoter, but does not change the activation kinetics the observed increase of ifn-b promoter activity caused by ro 90-7501 may be due to the enhancement of poly i:c activation, or alternatively due to the disruption of a feedback inhibition pathway. in order to distinguish these two possibilities, a time-course study was performed. as shown in fig. 4a , the presence of ro 90-7501 increased the peak level of ifnb promoter activity at 10 h of poly i:c treatment, but the promoter activity declined with a similar kinetics since the peak time in the absence or presence of ro 90-7501. consistent with results shown in figs. 2b and c, ro 90-7501 inhibited the activation of nfkb (fig. 4b) . the results thus imply that ro 90-7501 most likely targets a molecular event in the activation, but not negative feedback regulation of tlr3 signal transduction pathway to prolong the activation of ifn-b promoter. ro 90-7501 modulates tlr3 signal transduction, but has no effect on cells that tlr3 signal transduction is not activated the differential regulation of ro 90-7501 on the activation of ifn-b promoter and nfkb induced by poly i:c suggests that the compound modulates tlr3 signal transduction, but does not target ligand rna or tlr3 itself. in agreement with this notion, we observed in a time-of-addition experiment that ro 90-7501 was able to efficiently enhance poly i:c activation of ifnb promoter when added 3 h before, at the same time or even 3 h after the addition of poly i:c into the culture media (fig. 5) . however, the compound did not enhance poly i:c-induced ifnb promoter activation when the cells were pretreated with the compound for 3 h, followed by treatment with poly i:c in the absence of ro 90-7501 for an additional 9 h (treatment schedule 6). these results indicate that ro 90-7501 has no effect on ifnb promoter activation in the absence of tlr3 ligand, suggesting the compound should be pharmacologically active only in virallyinfected, but not un-infected cells. to further confirm the observations made with the reporter cell lines, we measured the effects of ro 90-7501 on the tlr3 ligand-induced endogenous ifn-b and il-8 (a chemokine) expression in 293tlr3ha and thp-1 (a human monocytic cell line) cells. consistent with the results obtained with reporter assays, while treatment of the cells with ro 90-7501 alone failed to induce ifn-b gene expression, the compounds significantly increased the induction of ifn-bmrna in the presence of poly i:c in 293tlr3ha cells (fig. 6a) , and the secretion of type-i ifn in thp-1 cells (fig. 6b ), but not il-8 secretion (fig. 6c ). because the downstream signaling pathways leading to activation of ifn-b and inflammatory cytokine gene expression are largely shared by both tlr3 and cytoplasmic rlrs, we set out to test whether ro 90-7501 could also enhance the activation of the ifn-b promoter elicited by cytoplasmic delivery of double stranded rna, which activates both rig-i and mda5 [26, 28] , into 293/ifnbluc cells that do not express tlr3. as shown in fig. 7 , while ly294002, an inhibitor of pi3k that is essential for activation of irf3 by rig-i/mda5 [29] , dose-dependently inhibited ifn-b promoter-driven luciferase expression (fig. 7a) , ro 90-7501 significantly enhanced the ifn-b promoter activation by cytoplasmic-delivered poly i:c (fig. 7b) . hence, our results imply that ro 90-7501 most likely targets a shared ifnb induction pathway activated by both tlr3 and rlrs. ro 90-7501 enhances the antiviral response induced by poly i:c to investigate whether the enhanced ifn-b response by ro 90-7501 could result in an enhanced antiviral response, 293tlr3ha cells were mock treated, or treated with poly i:c, ro 90-7501, alone or in combination for 12 h. cells were then challenged with vesicular stomatitis virus (vsv). the virus yields were determined by a plaque assay. in agreement with the results presented above, treatment of the cells with ro 90-7501 alone did not demonstrate any detectable antiviral effect. however, the virus yields are significantly reduced in cells that received combination treatment than in those treated with poly i:c alone to determine the biological activity of ro 90-7501 on mapk pathways, 293tlr3ha cells were mock treated, or treated with poly i:c (2 mg/ml), ro 90-7501 (10 mm), alone or in combination, for 30 min. the levels of the total and phosphorylated p38, erk and jnk were determined by an immunoblot assay. as shown in fig. 9a , poly i:c treatment was able to activate all three mapk pathways tested. interestingly, treatment of the cells with ro 90-7501 alone was able to efficiently activate p38 makp and to a lesser extent, erk and jnk pathways. furthermore, ro 90-7501 was also able to enhance the activation of the three mapk by poly i:c. to determine the role of the three mapks in poly i:c-induced ifn-b promoter activation, inhibitors of the three mapks were applied to poly i:c treated 293tlr3/ifnbluc cells. as shown in fig. 9b , only the p38 inhibitor (sb202190), but not erk or jnk inhibitors (u0126 or sp600125), significantly inhibited poly i:c-induced ifn-b promoter activation. this observation, in conjunction with the facts that ro 90-7501 slightly suppresses poly i:c-induced nfkb activation (figs. 3 and 4) and does not affect poly i:c-induced irf3 nuclear translocation and activation of irf3-directed gene (ifit1 or isg56) expression (data not shown), implies that activation of p38 mapk pathway is most likely responsible for the observed enhancement of ifnb expression and antiviral response by ro 90-7501. tlrs and rlrs are the principal pattern recognition receptors that sense virus infection and mount a defense by activation of an innate immune response (reviewed in [3, 4] ). however, it has also been observed under many circumstances that the tlr-and/or rlr-mediated innate immune response plays a critical role in viral pathogenesis. for example, pneumonia caused by influenza virus or sars-coronavirus infections [30, 31, 32] , hemorrhagic fever caused by dengue virus and many other viruses [2, 33] and encephalitis caused by west nile virus infection [34] have all been demonstrated to be largely due to the detrimental effects of proinflammatory cytokines induced by the infection itself. moreover, the identification of tlrs and rlrs as key pathogen recognition receptors for innate immunity has sparked great interest in therapeutic manipulation of the innate immune system. tlr and rlr agonists are being developed for the treatment of cancer, allergies and viral infections, and as adjuvants for vaccines to prevent or treat cancer and infectious diseases [8, 35] . despite tremendous efforts, only imiquimod (aldara), a tlr7 agonist, has been approved by us fda for treatment of papillomavirusinduced genital warts and basal cell carcinoma via topical use [36] . due to the activation of a wide-spectrum of cellular responses, it is not surprising that systematic administration of the tlr agonists in doses necessary to achieve antiviral or anti-tumor effects is usually associated with significant adverse effects [11, 12, 13, 14] . studies of the signal transduction pathways elicited by tlr and rlr agonists have thus far revealed that induction of type i ifns and other proinflammatory cytokines is controlled by distinct signal transduction pathways [37, 38] . it is conceivable that pharmacological modulation of tlr and/or rlr signal transduction pathways may alter the cytokine profile induced by the prr agonists and thus differentially promote the beneficial innate immune response, but limit the detrimental effects. in our effort towards discovery of molecules with such a pharmacological property, we identified ro 90-7501, a compound that was originally reported as an inhibitor of the amyloid-beta peptide (abeta) assembly in vitro [39] . ro 90-7501 selectively enhances tlr3 agonist-induced type i ifn production in both human kidney epithelia and macrophage cell lines (fig. 7) , but does not promote nfkb activation and chemokine production. as expected, treatment of cells with ro 90-7501 significantly enhanced the antiviral activity of poly i:c (fig. 8) . molecular pathway analyses revealed that ro 90-7501 slightly suppressed poly i:c-induced nfkb activation, but did not apparently alter the tlr3 agonist-induced irf3 nuclear translocation and irf3 activated gene (isg56) expression (data no shown). interestingly, we showed that treatment of 293tlr3ha cells with ro 90-7501 was able to rapidly activate p38 mapk, and to a lesser extent, erk and jnk. although the molecular target and signaling pathways leading to the activation of the mapks by ro 90-7501 remain to be determined, it is plausible that ro 90-7501 activation of p38 mapk, which is essential for tlr3 agonist-induced ifn-b gene expression (fig. 9) , may be responsible for its enhancement of tlr3/rlr response. ironically, ro 90-7501 only enhances tlr3 response in the presence of its agonist, but fails in the condition that the cells were pretreated with the compound for 3 h, followed by treatment with poly i:c in its absence for an additional 9 h (fig. 5) . these results imply that besides p38mapk, an additional short-lived cellular response induced by ro 90-7501 may be also required for its innate immune enhancement activity. in corroboration with the work reported herein, conforti and colleagues recently showed that in vivo injection of poly(a:u), a tlr3 agonist, in combination with an immunochemotherapeutic regimen, was able to inhibit tlr3 positive breast tumor growth [40, 41] . interestingly, tlr3 agonists can elicit the production of a range of chemokines by tumor cells. while ccl5 blockade improved the efficacy of immunochemotherapy, cxcr3 blockade abolished its beneficial effects. therefore, this work shows that the opposing innate immune responses mediated by chemokines can be pharmacologically modulated to enhance the anticancer efficacy of tlr3 agonists. in summary, we and others have demonstrated that pharmacological modulation of tlr3 signaling or function of its downstream chemokines and/or cytokines can alter tlr3 agonist-induced innate immune response and enhance its antiviral and anticancer effects. regardless of whether ro 90-75401 or its derivatives can be developed as a therapeutic agent to enhance the antiviral response of tlr3 agonists, our study supports the concept that pharmacological modulation of tlr and rlr signal transduction pathways is an attractive approach for antiviral drug development. hek293 cells were cultured in dmem supplemented with 10% fbs, 2 mm l-glutamine, 1.5 g/l sodium bicarbonate, 50 u/ ml of penicillin and 50 mg/ml of streptomycin. 293tlr3ha, a hek293 cell-derived stable cell line expressing ha-tagged tlr3, was purchased from invivogen and cultured in complete dmem medium containing 30 mg/ml blasticidin. thp-1 cells were a kind gift of dr. r. phillips at immunotope inc. (originally purchased from atcc) and cultured in rmpi 1640 medium containing 10% fetal bovine serum and penicillin/streptomycin. hek-blue ifn-a/b cells (invivogen), expressing a secreted embryonic alkaline phosphatase (seap) under the control of the isg54 promoter, were cultured in complete dmem medium containing 100 mg/ml of zeocin and 30 mg/ml of blasticidin. vesicular stomatitis virus was propagated and titrated as described previously [42] . poly i:c was purchased from invivogen. ro 90-7501, chloroquin, sb202190 and u0126 were purchased from sigma. ly294002 and sp600125 were from cell signaling. ikk-2 inhibitor iv was purchased from santa cruz technology. 293tlr3ha cells were co-transfected with plasmids expressing firefly luciferase under the control of a human ifn-b promoter (pifnb-luc) [26] or an artificial promoter containing four repetitive consensus nfkb binding sites (clontech) and pcdna3 (invitrogen) at a molar ratio of 10 to 1. twenty-four hours post transfection, cells were re-seeded at a density of 10 4 cells per dish of 10-cm in diameter and cultured with medium containing 500 mg/ml of g-418. individual g-418-resistant cell colonies were picked at day 16 post transfection. the cell colonies demonstrating the highest levels of poly i:c-inducible luciferase expression (measured with steady-glo reagent, promega) were expanded into cell lines and designated as 293tlr3/ifnbluc or 293tlr3/ nfkbluc, respectively. in order to create similar cell lines for studying cytoplasmic rig-i-like receptor-meditated innate immune response, parental hek293 cells that are devoid of tlr3 were co-transfected with plasmids expressing firefly luciferase under the control of a human ifn-b promoter (pifnb-luc) [26] and pcdna3 at a molar ratio of 10 to 1. a g-418-resistant clone expressing a high level of luciferase in response to lipofectaminemediated transfection of poly i:c was identified and designated as 293/ifnbluc. 293tlr3/ifnbluc cells were seeded at a density of 5610 4 cells/well in black wall, flat-bottomed, clear 96-well plates (corning inc.). the cells were allowed to grow for 24 h before treatment. in the primary screening, the column 1 and 12 of each 96-well plate were mock treated or treated with 2 mg/ml of poly i:c, respectively. each of the remaining 80 wells were treated with 2 mg/ml of poly i:c and compound from the library of pharmacologically active compounds (lopac) (sigma) at a concentration of 5 mm. a sub-saturating concentration (2 mg/ml) of poly i:c was used to identify both positive and negative modulators of the tlr3 agonist-induced innate immune response. the firefly luciferase activities were measured after 18 h of dsrna addition by adding 100 ml/well steady-glo reagent (promega), followed by luminometry in a topcounter (perkin elmer). the raw luciferase activity data from each library compound-treated well were normalized as percentage activity relative to cells treated with poly i:c alone. the compounds that reduced or increased luciferase activity by more than 50% were considered as primary ''hits''. the hit compounds were subjected to further evaluation of cytotoxicity with mtt assay (promega) and confirmation of their ability to inhibit or enhance poly i:c activation of ifn-b promoter with the reporter assay described above at an expanded range of concentrations in triplicate. 293tlr3ha cells were mock treated or treated with 10 mm ro 90-7501, 2 mg/ml poly i:c, alone or in combination for 30 min and lysed with 1x laemmli buffer. a fraction of cell lysate was separated on sodium dodecyl sulfate-12% polyacrylamine gels and electrophoretically transferred onto pvdf membrane (bio-rad). membranes were blocked with pbs containing 5% nonfat dry milk and probed with antibodies against total, phophorylated p38, erk and jnk or b-actin (cell signal). bound antibodies were revealed by incubation with irdye secondary antibodies and imaging with li-cor odyssey system (li-cor biotechnology). 293tlr3ha cells were mock treated or treated with 10 mm ro 90-7501, 2 mg/ml poly i:c, alone or in combination. total rna was isolated with trizol (invitrogen) and cdna was synthesized using superscript iii cdna synthesis kit (invitrogen) and subjected to pcr amplification of ifn-b (forward primer sequence: 59-gcagctgcagcagttccagaa-39; reverse primer sequence: 59-gctaggagatcttcagtttcg-39) and b-actin (forward primer sequence: 59-gccctggcacc-cagcacaatg-39, reverse primer sequence: 59-ttaggttttgtcaagaaagggtg-39 ) using platinum taq polymerase (invitrogen). thp-1 cells were mock treated or treated with 10 mm ro 90-7501, 2 mg/ml poly i:c, alone or in combination for 6 h and culture media were harvested. hek-blue ifn-a/b cells (invivogen) were seeded at a density of 5610 4 cells per well in 96-well plates. one day post seeding, the cells were cultured with conditioned media harvested from the treated thp-1 cells for 20 h. the levels of seap in culture media were determined with quanti-blue assay (invivogen) by following the instruction of manufacturer (invivogen). il-8 in culture medium of 293tlr3ha cells treated with doses of poly i:c, 10 mm ro 90 7501, alone or in combination, was measured using a human il-8 elisa kit from invitrogen, following the manufacturer's instruction. 293/ifnbluc cells seeded into 96-well plates at a density of 4610 4 cells per well. twenty-four hours post seeding, cells were mock transfected (no rna) or transfected with 0.2 mg poly i:c per well with lipofectamine 2000 following the manufacturer's direction (invitrogen) and mock treated or treated with 10 mm of ro 90-7501 for 16 h. 293tlr3ha cells were seeded into 24-well plates at a density of 2610 5 cells per well. twenty-four hours post seeding, cells were either mock treated or treated with indicated concentrations of poly i:c in the absence or presence of 10 mm of ro 90-7501 for 12 h. cells were then infected with vsv at a moi of 0.001. culture media were harvested 16 h post infection and the virus yields were determined by a plaque assay [42] . to determine the cell viability, a mtt based assay was performed (sigma) as previously described [43] . 293tlr3/ ifnbluc cells were seeded at a density of 56104 cells per well in 96-well plates. one day post seeding, the cells were mock treated or treated with a serial dilution of ro 90-7501, ranging from 4 mm to 250 mm, for 16 h. the dose-dependent absorbance values at 570 nm, as indicator of cell viability, were then determined and plotted following manufacturer's instruction. activation of host pattern recognition receptors by viruses clec5a is critical for dengue-virus-induced lethal disease mda5/rig-i and virus recognition pattern recognition receptors and inflammation pathogenesis of viral hemorrhagic fever pathogenesis of west nile virus infection: a balance between virulence, innate and adaptive immunity, and viral evasion recent advances in deciphering viral and host determinants of dengue virus replication and pathogenesis therapeutic 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cord-012967-w1oc0wdd authors: eberle, jaelyn j.; von koenigswald, wighart; eberth, david a. title: using tooth enamel microstructure to identify mammalian fossils at an eocene arctic forest date: 2020-09-23 journal: plos one doi: 10.1371/journal.pone.0239073 sha: doc_id: 12967 cord_uid: w1oc0wdd lower eocene (wasatchian-aged) sediments of the margaret formation on ellesmere island in canada’s high arctic preserve evidence of a rainforest inhabited by alligators, turtles, and a diverse mammalian fauna. the mammalian fossils are fragmentary and often poorly preserved. here, we offer an alternative method for their identification. among the best preserved and extensive of the eocene arctic forests is the strathcona fiord fossil forest, which contains permineralized in situ tree stumps protruding from a prominent coal seam, but a paucity of vertebrate fossils. in 2010 and 2018, we recovered mammalian tooth fragments at the fossil forest, but they are so incomplete as to be undiagnostic by using their external morphology. we used a combination of light microscopy and sem analysis to study the enamel microstructure of two tooth fragments from the fossil forest—nufv2092b and 2092e. the results of our analysis indicate that nufv2092b and 2092e have coryphodon-enamel, which is characterized by vertical bodies that manifest as bands of nested chevrons or treelike structures visible in the tangential section under light microscopy. this enamel type is not found in other mammals known from the arctic. additionally, when studied under sem, the enamel of nufv2092b and 2092e has rounded prisms that open to one side and are surrounded by interprismatic matrix that is nearly parallel to the prisms, which also occurs in coryphodon enamel, based on prior studies. the tooth fragments reported here, along with some poorly preserved bone fragments, thus far are the only documented vertebrate fossils from the strathcona fiord fossil forest. however, fossils of coryphodon occur elsewhere in the margaret formation, so its presence at the fossil forest is not surprising. what is novel in our study is the way in which we identified the fossils using their enamel microstructure. lower eocene (wasatchian-aged) strata of the margaret formation, eureka sound group on ellesmere island, nunavut preserve evidence of lush mixed conifer-broadleaf rainforests inhabited by alligators, turtles, birds, and at least 25 mammalian genera [1] . first discovered in 1975 near the head of strathcona fiord [2] , the vertebrate fossils in the eureka sound group are few, fragmentary and weather-worn, which can make it challenging to identify them. by far the most abundant and diverse vertebrate fossils have come from the margaret formation cropping out on the matthew peninsula between bay and strathcona fiords [3] (fig 1) . nearly a century before the first discovery of eocene vertebrate fossils on ellesmere, eocene arctic forests were first documented when sergeant d.l. brainard, a survivor of the ill-fated greely expedition of 1881-1883, discovered petrified logs on northeastern ellesmere island [4] . among the best preserved, extensive, and photogenic of the eocene arctic fossil forests is the strathcona fiord fossil forest, which preserves permineralized in situ tree stumps protruding from a prominent coal seam (figs 1 and 2) . the tree stumps are large, with diameters ranging from 40 cm to over a meter, and closely spaced, indicating a dense forest comparable to today's cypress swamps in the southern united states [5] . eocene arctic paleotemperature estimates using multiple proxies suggest a mean annual temperature (mat) of 5-17˚c, with winters above freezing and summer temperatures above 20˚c [6] [7] [8] . further, the eocene arctic rainforests had high mean annual precipitation and humidity, comparable with today's temperate rainforests along the north american west coast [8] . in the summers of 2010 and 2018, je, de and team recovered tooth fragments belonging to a mammal and small, undiagnostic bone fragments at the strathcona fiord fossil forest. as far as we are aware, these are the first vertebrate fossils documented from this fossil forest. most fossil mammals can be identified to genus and often species by dental morphology. however, the tooth fragments from the strathcona fiord fossil forest are so incomplete as to be undiagnostic by using their external morphology. therefore, we analyzed the tooth enamel microstructure to assist us in identifying the fossils. mammalian prismatic enamel, the hardest and most resistant material in the body, consistently differs in its microstructure among clades of perissodactyla (odd-toed ungulates; [9] ), rodentia [10] , proboscidea [11] , and other groups. here, we demonstrate that the tooth fragments from the strathcona fiord fossil forest can be identified to genus based on their enamel microstructure. in so doing, we (1) document the first fossil mammal from the strathcona fiord fossil forest; (2) help refine the age and correlation of this site within the context of the margaret formation elsewhere on ellesmere island; and (3) underscore the utility of tooth enamel microstructure in identifying mammalian tooth fragments that cannot be identified by traditional paleontologic means. the tooth fragments described below were recovered from locality p201088-2(nu) on central ellesmere island, just south of 78˚40' n latitude along the southwestern coastal region of strathcona fiord (fig 1) . the general area, referred to as "strathcona fiord" [1] , includes the first two fossil vertebrate sites that were discovered in the canadian arctic (ellesmere island) by dawson and her colleagues in the 1970s [2] , approximately 10-12 km southeast and northeast of locality p201088-2(nu). the sharp-based sandstone that hosts locality p201088-2(nu) occurs less than a meter above the top of an approximately 2 m-thick coal that preserves an assemblage of in situ permineralized tree stumps and root balls coined the strathcona fiord fossil forest ( [5, 12] ; figs 2 and 3). vertebrate fossils in the strathcona fiord region produce from the eureka sound group, which consists of four formations in this area. in ascending order, these are the mount bell, mount lawson, mount moore, and margaret formations [13, 14] . the stratigraphic section that we examined and measured at strathcona fiord (fig 3) consists of the uppermost exposures of the fine-grained marine mount moore formation, overlain by non-coaly to coaly, paralic-to-non-marine deposits of the lower margaret formation [1, [13] [14] [15] . we place locality p201088-2(nu) approximately 316 m above the base of the multi-kilometer-thick margaret formation. based on the thickness data of [13] and [14] , we regard this position as occurring in the lower portion of the margaret formation. beds in this portion of the section are exposed along an extensive north-south trending ridge, and dip steeply (25˚-35˚) to the west. lastly, we note that during stratigraphic measurement and examination in our study area, we identified errors in formation identification on the geologic map [15] . specifically, these authors erroneously mapped the strathcona fiord fossil forest and its thick coal as part of the mount lawson formation, which instead is a paleocene-aged marine mudstone succession in the lower half of the eureka sound group [13] . strathcona fiord fossil vertebrate sites have previously been interpreted as early eocene (wasatchian) in age [1] [2] [3] 14] . based on these interpretations and stratigraphic patterns [14] , we suggest the age of the strathcona fiord fossil forest and locality p201088-2(nu) is also wasatchian, equivalent in time to the lower margaret formation interval at bay fiord (~25 kilometers to the north-northeast). locality p201088-2(nu) occurs just above the stratigraphic horizon where the margaret formation paralic succession (complexly and thinly interbedded marine to non-marine strata) transitions up-section into a 50 m thick succession of strictly non-marine, coastal-plain strata dominated by fine sandstones and minor coals. exposures of the lower margaret formation at bay fiord [14] record a similar pattern of overall regression and are characterized by an upsection transition from paralic to coaly coastal plain deposits. the host sandstone for locality p201088-2(nu) exhibits ripple laminae and abundant coalified root traces, the latter indicating that a stable and likely subaerial substrate was present for plant colonization subsequent to deposition of the sandstone. sparse occurrences of fossil vertebrate fragments are present among mudstone and ironstone intraclasts in the sandstone, suggesting that lower portions of the sandstone may have been deposited as a lag deposit during waning flow. angiosperm and gymnosperm leaf fragments, and more root traces are common in the uppermost portions of the host sandstone, again suggesting substrate stability between subsequent later-stage sediment accumulation events. no other intervals were observed lower in section that preserve well-developed associations of 'fossil forests,' ironstone and fossil vertebrate clasts, rooted horizons, fossil leaves, and an absence of marine indicators. furthermore, the presence of the fossil forest, multiple rooted horizons, leaf fossils, and ironstone and fossil bioclasts (the tooth fragments described below) all suggest forested conditions, exposed substrates, and incipient soil formation in a well-saturated setting subjected to episodic flooding. accordingly, we interpret this transitional stratigraphic interval as recording an up-section shift from frequently flooded paralic shoreline settings to a relatively more up-dip coastal-plain setting where non-marine conditions prevailed. this paleoenvironmental interpretation matches those previous hypothesized for the margaret formation that describe upward-coarsening cycles of interbedded cross-bedded sandstone, siltstone, mudstone, and coal. these are interpreted as proximal delta-front to delta-plain paleoenvironments characterized by abundant shoreline sands, alluvial to estuarine channels, coal swamps, lagoons and bays, and well-forested, low-gradient interfluves [13, 14, 16, 17] . nunavut fossil vertebrate (i.e., nufv) 2092b and 2092e (fig 4) , the tooth fragments analyzed in this study, were collected along with approximately 20 other tooth fragments and several small, weathered bone fragments by je, de, and team in july 2010 and by je in july 2018 at locality p201088-2(nu). the fossils were collected on class 2 nunavut territory palaeontologist permits 2010-003p and 2018-02p issued by the nunavut department of culture and heritage. detailed coordinates for locality p201088-2(nu) are on file at the nunavut department of culture and heritage in iqaluit and the canadian museum of nature (cmn) in ottawa, canada. given their similarity in thickness and external appearance, and the fact that they were recovered near one another, the tooth fragments probably represent the same taxon and individual. based upon their thickness, nufv 2092b and 2092e are from a relatively large mammal. however, their external morphology does not allow us to reliably identify the mammal to which they belong. therefore, we decided to study the enamel microstructure. to investigate the microstructure of nufv 2092b and 2092e, three traditional planes of section were studied -the horizontal (or transverse), vertical, and tangential sections (fig 5) , following [18] . the techniques for preparing and studying tooth enamel microstructure of large fossil mammals were described by [19] and are summarized here. first, nufv 2092b and 2092e were studied under a light microscope to orient the specimens and determine the direction of the occlusal surface. the specimens were subsequently embedded in epoxy resin, and oriented so that the desired sections (horizontal, vertical, or tangential) were placed parallel to the resin surface. the embedded specimens were left for 48 hours at room temperature under a fume hood to allow the epoxy to harden. nufv 2092b was cut using an isomet© low-speed saw with 0.3 mm blade thickness, to produce horizontal and vertical sections, whereas nufv 2092e was used for the tangential section. the specimens were ground in three steps. first, they were ground down to the enamel surface using a grinding wheel (grit 240). next, handgrinding was done on fine wet sandpaper (grit 800) placed over a glass sheet, and finally the specimens were ground with fine powder grit (grit 1000) mixed with water on a glass plate. between each of these steps, the specimens were analyzed under a light microscope to ensure that grinding was not too extensive. the ground surfaces were rinsed with water, cleaned in an ultrasonic cleaner, blown dry, and etched with 10% hydrochloric acid (2 normal hcl) for approximately three seconds. prior to scanning electron microscope (sem) analysis, the specimens were sputter-coated with gold or palladium. sem analysis was conducted on a camscan mv 2300 instrument in the palaeontology section of the institute of geosciences and a cambridge stereoscan 200 sem in the institut für biodiversität der pflanzen at the rheinische friedrich-wilhelms university in bonn, germany. in describing the enamel microstructure of nufv 2092b and 2092e, we follow the hierarchical system of classification of others [20, 21] . specifically, we define the units by their size and level of complexity. first, we describe the crystallites, the smallest units of enamel that are comprised of fine needles of hydroxyapatite with a diameter of less than 0.5 μm and length of more than 100 μm [22] . crystallites are visible under high magnification (1500x and higher). next, we describe the prisms that are made up of bundles of crystallites surrounded by a prism sheath. the size and shape of the prisms differ among clades of mammals. prisms form at the enamel-dentine junction (edj) and grow almost to the outer enamel surface (oes) [23] . they are typically arranged in groups or bands with the same prism orientation. their orientation defines the various enamel types [9, 24, 25] . the prismatic enamels of mammals often have two or more different enamel types. the most primitive enamel type among placental mammals is radial enamel, in which the prisms' long axes parallel one another and extend radially from near the edj towards the oes [18] . more complex enamel types occur in which bands of prisms change their orientations from the edj to the oes [20] . among the most often described enamel types are hunter-schreger bands (hsb), which are light and dark stripes often seen under a light microscope. hsb are an optical phenomenon caused by the different prism orientation in alternating bands, forming decussations [9, 24] . hsb occur in the enamel of most large mammals and often are arranged horizontally. however, a few taxa, including rhinocerotoids, have vertical hsb [9, 23, 26] . hsb function as a crack-stopping device [27, 28] . the level above the enamel type is the schmelzmuster, the three-dimensional distribution of enamel types within the enamel that has both biomechanical and phylogenetic controls. the number of possible combinations of enamel types or schmelzmusters is very large [25] . by studying the horizontal, vertical, and tangential sections of nufv 2092b and 2092e, we discovered a complex enamel microstructure that was challenging to interpret solely through sem analysis. consequently, we also studied nufv 2092b and 2092e at lower magnification under a light microscope using the light-guide effect described by [29] and more recently by [19] . if light hits an enamel prism approximately perpendicular to its axis, the light is reflected and the prism appears light in color. if, however, the light hits a prism parallel to its long axis, it disappears into the prism, and the prism appears dark. when the source of illumination is changed from one direction to another, bands that were dark in one will be light in the other, and vice versa. the light and dark bands, each comprised of many prisms with the same orientation, are the hsb. we used a combination of the light-guide effect and sem analysis to study the enamel microstructure of nufv 2092b and 2092e. based upon thickness, nuvf 2092b and 2092e belong to a relatively large mammal. the shiny outer surface of the enamel is covered by ridges and crenulations that extend vertically and diagonally at a steep angle (fig 4) . of the diverse mammalian fauna known from the early eocene arctic, the pantodont coryphodon is among the largest and best represented by fossils [30] , and the enamel on its teeth has vertical ridges and wrinkles [19] . however, these characters are not unique to coryphodon. many mammals, including brontotheres that also are known from the eocene arctic [14, 31, 32] , show varying amounts of rugosity and crenulations on the external surface of the enamel. others [19] have noted the appearance of vertical stripes in the tangential section of the enamel of coryphodon as well as ridges on the shearing crests of its molars, indicating the presence of vertical structures within the enamel. on the occlusal surface of nufv 2092b, there are weak ridges, and when the tangential section of the enamel is illuminated from one side, faint vertical light and dark stripes are visible. however, these characters also are not restricted to coryphodon, but occur in rhinocerotoids [23] and some extinct south american mammals including pyrotheres and astrapotheres [29, 33] . coryphodon, however, is characterized by a unique and complex enamel microstructure coined coryphodon-enamel [19] . nufv 2092b and 2092e were analyzed at low and high magnifications under both a light microscope and scanning electron microscope (sem) to determine whether coryphodon-enamel is present. below, we describe the crystallites, prisms, and schmelzmuster of the nufv specimens, and compare them to the enamel microstructure of coryphodon, rhinocerotoids and other large mammals known from the eocene arctic. the cross sections of prisms are best seen in the tangential section (fig 6a and 6b ). individual crystallites are visible at high magnification, where they appear as fine needles making up the prisms and interprismatic matrix (ipm) (fig 6b) . the crystallites in the prisms appear approximately parallel to those comprising the ipm, which is found in the enamel of coryphodon [19] . most of the prisms in nufv 2092e are rounded and have an open prism sheath, although there is some variability in shape, with some prisms being more oblong and narrowing towards the top. the prisms range in diameter from approximately 4-7 μm and the prism sheaths are open towards the base of the tooth or to one side. a horseshoe-shaped prism sheath was noted for coryphodon [19] , although it is not restricted to the genus. although close together, the prism sheaths do not appear to touch one another. rather, a thin (~1-2 μm) layer of ipm surrounds the prism sheaths and appears as a 'tail' below each prism that tapers towards the base of the tooth or to the side (fig 6b) . the combined interpretation of the transverse, vertical, and tangential sections of nufv 2092b and 2092e indicates that the enamel microstructure is dominated by elongate, steeply dipping or near vertical bodies of prisms that penetrate the enamel almost from the edj towards the oes, leaving thin inner and outermost zones. next to the edj, there is a thin inner zone of radial enamel in some places. however, in other areas next to the edj the prisms do not appear to run parallel to one another, and it is difficult to discern a pattern. the middle zone, which comprises the greatest thickness of enamel, is made up of a complex enamel wherein elongate bodies of variable thickness extend outward from the edj towards the oes (fig 7a) . at higher magnification, the elongate bodies are hsb-like in that they are comprised of steeply-dipping or rising prisms, and are separated from one another by transitional zones of horizontal (or nearly so) prisms (fig 7b) . the nearly vertical orientation of the elongate bodies becomes obvious in the tangential section (discussed below). however, in contrast to hsb that are identified in many large mammals [9] , the elongate bodies and intervening transitional zones are not of uniform thickness or spacing from one another in nufv 2092b. in the uncoated tangential section of nufv 2092e (fig 8) , the vertical bodies are clearly visible and complex. when light is illuminated from the bottom (fig 8a) , one sees a pattern of straight to slightly wavy, broad white lines that when magnified (fig 8b) , look like nested chevrons whose points are directed vertically or somewhat diagonally up towards the occlusal surface. these lines of nested chevrons or treelike structures extend upwards for a considerable distance, sometimes bifurcating, and are separated from one another by darker bands whose structure is the same, but point in the opposite direction. in the top one-third of the tooth, the light-colored bands become broader, more rounded, and lose their nested chevron appearance, and the darker bands in between are considerably narrower than the light-colored bands. the vertical structures illuminated in the tangential section (fig 8) appear to parallel the vertical crenulations and wrinkles on the outside surface of the enamel of nufv 2092e (fig 4) . further, just as the nested chevron structures transition into broader, more rounded bands towards the occlusal surface of nufv 2092e in the tangential section, so do the crenulations on the outside surface of the enamel (compare figs 4b with 8). nufv 2092e (figs 4b and 8) , probably a fragment of an anterior premolar, was ground the deepest in the lower third of the tooth, whereas the upper two-thirds of the specimen was more shallowly ground. therefore, the lower part of the tooth (where damage is evident by cracks and a small hole in the enamel) is hypothesized to capture the microstructure of the inner zone of enamel (nearest the edj), whereas much of the area above (external to) the edj (fig 8a) is likely correlative with the thick middle zone that is comprised of vertical, elongate bodies of prisms. the structures extend to the occlusal surface (top) of nufv 2092e, which is worn, so we hypothesize that the thin outer zone of radial enamel that occurs elsewhere next to the oes has been removed by occlusal wear. in the region of enamel along the left and right margins of nufv 2092e next to the oes (fig 8a) , however, the microstructure is indistinct and blurred in appearance; this may represent an outermost zone of radial enamel (discussed below). based on the tangential section (fig 8) , we predict that the nested chevrons should appear in cross section as vertical elongate bodies with variable thicknesses and branches, depending upon where the horizontal section transects them. further, we predict that they should be concentrated in the inner region of the middle zone of enamel. whereas, in the outer region of the middle zone (towards the oes) in horizontal section, we hypothesize that the elongate bodies should be broader in appearance, and the nested chevron structures should all but disappear in the outermost zone of enamel. in fact, the horizontal section (fig 7) appears to show just that-the outer region of the middle enamel zone shows broader bodies that transition in places into radial enamel near the oes. evident in vertical sections of nufv 2092b, an outer zone of radial enamel in which the prisms run parallel to each other lies near the oes (fig 9a) . in some areas, it may be up to 300 μm in thickness, whereas in other areas it is much thinner, and the microstructures of the thick middle zone extend nearly to the oes. in some areas adjacent to the oes, the prism sheath vanishes between the parallel-oriented prisms, so that these merge into a prismless outer enamel zone (plex; fig 9b) , a feature observed in a number of mammalian taxa [34] [35] [36] . in other areas, the enamel contains large vacuities, probably the result of diagenesis ( fig 9a, bottom left) . in summary, the analysis of nufv 2092b and 2092e in horizontal, tangential, and vertical sections at both low and high magnification indicates that the enamel is characterized by elongate, vertical bodies extending from near the edj towards the oes that are made up of steeply-angled prisms that decussate with adjacent bodies. the inner region of the middle enamel zone contains nested chevron or treelike structures that are evident in horizontal and tangential sections, whereas in the outer region, the vertical bands become broader and the nested chevron structures are lost. an outer zone of radial enamel or plex occurs next to the oes. the enamel microstructure that occurs in nufv 2092b and 2092e is comparable to that of the pantodont coryphodon described by [19] . coined "coryphodon-enamel" by these authors, this enamel type is characterized by vertical or oblique structures that in tangential section appear as light-colored bands of nested chevrons or treelike structures separated from one another by similar, though narrower, dark-colored bands. the difference in width between the light and dark stripes when light is reflected from one direction indicates that the prism orientation is not symmetrical [19] . also like coryphodon-enamel (although not restricted to it), nufv 2092b and 2092e have an outermost zone of radial enamel, as well as rounded prisms that open to one side and are surrounded by interprismatic matrix that is nearly parallel to the prisms. given the suite of characters shared with coryphodon-enamel, but predominantly the vertical bodies that manifest as lines of nested chevrons or treelike structures in tangential view, nufv 2092b and 2092e possess coryphodon-enamel. however, does coryphodonenamel occur in any of the other large mammals known from the arctic localities? in the canadian arctic, rhinocerotoids are known from early miocene sediments of the haughton formation on devon island (dawson 1990 ) and from the yukon [26] . rhinocerotoids have vertical hsb in their tooth enamel that look somewhat like the vertical elements in coryphodon-enamel [9, 23, 37] . however, the vertical bands in rhinocerotoid enamel contrast with those in coryphodon-enamel in that they are of consistent thickness and spacing from one another and separated by thin transitional zones of 2-3 prisms wide. in tangential section, rhinocerotoid enamel shows light and dark vertical lines that bifurcate in a regular pattern and lack the nested chevron or treelike structures in coryphodon-enamel (fig 9c and 9d) . although not nearly as regular in pattern as the vertical hsb in rhinocerotoids, coryphodon-enamel nevertheless cannot be considered irregular enamel. this enamel type, which occurs in proboscideans (elephants and their extinct relatives) and some rodents, is characterized by prisms that twist irregularly in bundles or as single prisms around each other, and bundles of prisms show a range of angles and attitudes with no consistent pattern [18, 38] . the brontotheres cf. eotitanops and palaeosyops are documented from early-middle eocene rocks of the margaret formation on ellesmere island [14, 32] , and tooth fragments from a larger, younger brontothere were recovered from middle eocene strata of the buchanan lake formation on nearby axel heiberg island [39] . however, brontothere tooth enamel differs from coryphodon-enamel in having u-shaped hsb, an intermediate condition between the horizontal hsb found in most large mammals and the vertical hsb of rhinocerotoids [23, 40] . tapiroids occur at early eocene localities in the margaret formation [14, 41] . however, tapiroids have horizontal to curved hsb [9, 38] . the nested chevron pattern seen in the tangential section of coryphodon-enamel is reminiscent of the zigzag hsb found in advanced carnivorans, particularly crocuta crocuta (hyaena) that correlate with ossiphagous (or bone-eating) habits [19, 42] . however, the enamel of c. crocuta differs from coryphodon-enamel in that the vertical structures show a symmetrical pattern when illuminated from opposing directions in tangential section, and the vertical light and dark bands are of similar thickness [19, 42] . several carnivoromorpha are known from the margaret formation [43] , but their enamel shows significant differences from coryphodon-enamel. miacis, cf. vulpavus, and viverravus are small-bodied members of carnivoromorpha whose tooth enamel is much thinner (and smoother) than that of nufv 2092b and 2092e. further, the tooth enamel of these early eocene carnivores contains undulating hsb, which are essentially horizontal, slightly wavy hsb [42] . the oxyaenid palaeonictis and mesonychid pachyaena are the largest carnivores in the margaret formation, although their fossils are rare, with each taxon represented by a single fossil in the arctic [43] . the enamel of these taxa has undulating hsb in the lower half of the tooth that transitions into zigzag hsb in the upper one-half to one-third of the tooth [44] . coryphodon-enamel altogether lacks undulating hsb. in addition to its namesake, coryphodonenamel is known to occur in middle eocene uintatherium and late eocene entelodon [19] , neither of which is known from the early eocene nor from the polar region. therefore, it is unlikely that the coryphodon-enamel reported here from the strathcona fiord fossil forest belongs to any other mammal besides coryphodon. the tooth enamel fragments reported here, along with some poorly preserved bone fragments, thus far are the only documented vertebrate fossils from the strathcona fiord fossil forest. however, coryphodon fossils occur elsewhere in the margaret formation on ellesmere island [30] , so its presence at the strathcona fiord fossil forest is not surprising. what is novel in our study is the way in which we identify the fossils nufv 2092b and 2092e to coryphodon by way of their enamel microstructure. complete mammalian teeth and jaws are morphologically diagnostic and readily identified to genus and even species. however, in the arctic, eocene vertebrate fossils are rare, and many are fragmentary. here, we provide an example of how enamel microstructure can be used to identify the mammal. the presence of coryphodon suggests that the strata containing the strathcona fiord fossil forest are temporally correlative with the early eocene (wasatchian) fossil-bearing strata of the margaret formation approximately 25 km further north at bay fiord, a conclusion reached independently by lithologic correlation. at bay fiord, perissodactyls, hyaenodontid creodonts, miacis, and cf. vulpavus, all of which first appear at mid-latitudes in the wasatchian, as well as the wasatchian index taxon pachyaena and the archaic ungulate anacodon, which last appears in the wasatchian, occur in the lower faunal level of the margaret formation [1, 2, 3, 14] . although coryphodon occurs at early middle eocene (bridgerian) localities at mid-latitudes, it is restricted to the early eocene (wasatchian) faunal assemblage in the arctic [30] . the strata containing the strathcona fiord fossil forest were initially mapped by harrison et al. (2009) as the late paleocene mount lawson formation of the eureka sound group. however, our study indicates that they should be re-mapped as the margaret formation on the basis of lithology, paleoenvironmental interpretations, and the presence of coryphodon. vertical elements such as those found in the enamel of the carnivore crocuta (hyaena) and entelodonts are hypothesized to be an adaptation to deal with high stresses during mastication [19, 44] . crocuta is carnivorous and ossiphagous, whereas entelodonts have been interpreted as omnivores and pig-like in their diet, ingesting a variety of food items, including plants, meat, and bones [45, 46] . in contrast, coryphodon, based on its transverse shearing lophs and carbon isotope values, is interpreted as an herbivore, and probably semiaquatic [47] . based on its oxygen and carbon isotope values, coryphodon is inferred to be a year-round resident above the arctic circle and therefore experienced months of darkness and the shutdown of photosynthesis during the polar winter [48] . seasonal isotopic variations, and specifically high winter δ 13 c values in the enamel of coryphodon teeth from ellesmere island, suggest a varied diet during the dark winter that probably included wood, leaf litter, and evergreen conifers [48] . the presence of vertical elements in the enamel microstructure of coryphodon may have pre-adapted these large mammals to ingesting tough, poorer quality food items during the long dark winters above the arctic circle. this could partly explain why coryphodon is the most abundant herbivore in the eocene arctic. life at the top of the greenhouse eocene world-a review of the eocene flora and vertebrate fauna from canada's high arctic paleogene terrestrial vertebrates: northernmost occurrence upper cretaceous and paleogene sedimentary rocks, eastern canadian arctic and related north atlantic areas new york: charles scribner's sons; 1886 a 50-million-year-old fossil forest from strathcona fiord warm 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canada-implications for age of the buchanan lake formation and brontothere paleobiology u-shaped orientation of hunter-schreger bands in the enamel of moropus (chalicotheriidae, mammalia) in comparison to some other perissodactyla a new 'tapir' from ellesmere island, arctic canada-implications for northern high latitude palaeobiogeography and tapir palaeobiology differentiations in hunter-schreger bands of carnivores carnivora, im vergleich zu vertretern der creodonta, arctocyonidae, mesonychidae, entelodontidae (placentalia), thylacoleodontidae, dasyuridae und thylacinidae (marsupialia) universität bonn a functional interpretation of the masticatory system and paleoecology of entelodonts evolution of tertiary mammals of north america stable isotopes in early eocene mammals as indicators of forest canopy structure and resource partitioning lower latitude mammals as year-round residents in eocene arctic forests we thank colleagues at the nunavut government (a. stubbing, s. leblanc) and the canadian museum of nature (k. shepherd, m. currie) for loan of the fossils. enamel microstructure analysis of the tooth fragments was carried out by j. eberle and w. v. koenigswald in the paleontology section of the institute of geosciences at the university of bonn in germany. we thank t. martin, chair of palaeontology at the institute of geosciences at the university of bonn, for allowing use of his enamel and imaging labs for preparation and imaging of the fossils. we also thank g. oleschinski (palaeontology, university of bonn) and h.-j. ensikat (institut für biodiversität der pflanzen, university of bonn) who worked with us to produce the sem images. careful reviews by k. rose, k.c. beard, an anonymous reviewer, and academic editor a. csank improved the manuscript. publication of this article was funded by the university of colorado boulder libraries open access fund. key: cord-002473-2kpxhzbe authors: das, jayanta kumar; pal choudhury, pabitra title: chemical property based sequence characterization of ppca and its homolog proteins ppcb-e: a mathematical approach date: 2017-03-31 journal: plos one doi: 10.1371/journal.pone.0175031 sha: doc_id: 2473 cord_uid: 2kpxhzbe periplasmic c7 type cytochrome a (ppca) protein is determined in geobacter sulfurreducens along with its other four homologs (ppcb-e). from the crystal structure viewpoint the observation emerges that ppca protein can bind with deoxycholate (dxca), while its other homologs do not. but it is yet to be established with certainty the reason behind this from primary protein sequence information. this study is primarily based on primary protein sequence analysis through the chemical basis of embedded amino acids. firstly, we look for the chemical group specific score of amino acids. along with this, we have developed a new methodology for the phylogenetic analysis based on chemical group dissimilarities of amino acids. this new methodology is applied to the cytochrome c7 family members and pinpoint how a particular sequence is differing with others. secondly, we build a graph theoretic model on using amino acid sequences which is also applied to the cytochrome c7 family members and some unique characteristics and their domains are highlighted. thirdly, we search for unique patterns as subsequences which are common among the group or specific individual member. in all the cases, we are able to show some distinct features of ppca that emerges ppca as an outstanding protein compared to its other homologs, resulting towards its binding with deoxycholate. similarly, some notable features for the structurally dissimilar protein ppcd compared to the other homologs are also brought out. further, the five members of cytochrome family being homolog proteins, they must have some common significant features which are also enumerated in this study. amino acids play the vital role for determining the protein structure and functions. but it is informative to know how the functionality of the group of proteins is changed while amino acid patterns are getting changed from one protein to another. it becomes quite harder and mostly time consuming to identify the uniqueness of proteins and their functionality from the wet lab experiments while working with complete sequence. in this regard, several techniques have been developed for the analysis of primary protein sequence that is helping the plos biochemist to work with only specific domain instead of the whole sequence which reduces the experiment time. geobacter sulfurreducens is one of the predominant metal and sulphur reducing bacteria [1] . the organism geobacter sulfurreducens is known to act as an electron donar and participate in redox reaction [2] . periplasmic c7 type cytochrome a (ppca) protein along with its four additional homologs (ppcb-e: ppcb, ppcc, ppcd, ppce) are identified in geobacter sulfurreducens genome [3] [4] [5] [6] . altogether, five proteins are highly conserved around "heme iv" but are not identical, and mostely differ in two hemes, "heme i" and "heme iii" [4] . these two regions are known to interact with its own redox partner. deoxycholic acid (conjugate base deoxycholate), also known as cholanoic acid, is one of the secondary bile acids, which are metabolic byproducts of intestinal bacteria used in medicinal field and for the isolation of membrane associated proteins [7, 8] . among the five members of cytochromes c7 family, only ppca can interact with deoxycholate (dxca) while its other homologs cannot. while interacting with dxca, it is observed that few residues are utilized [4, 6, 9] . it would be worthy if the reason of such an amazing difference towards recognizing a single compound can be found through the amino acids sequence viewpoints. further, one can also see the reason of the structural dissimilarity of ppcd compared to the other homologs [5] . in literature, in-silico techniques have been used to tackle the various problems through the analysis of dna, rna and protein sequences in bioinformatics field. specially, the authors are searching the protein blocks which are highly similar and conserved among the sub-group or entire family members [10] [11] [12] [13] . there are twenty standard naturally occurring amino acids which are diverse, arises complexity in the sequences, and have some group specific susceptibility. various reduced alphabet methods are established which can perform much better in certain conditions [14] [15] [16] [17] . sequence similarity is the most widely reliable strategy that has been used for characterizing the newly determined sequences [18] [19] [20] [21] . finding the functional/structural similarity from homolog sequences with low sequence similarity is a big challenging task in bioinformatics. to tackle this problem, several methods have been introduced that can identify homolog proteins which are distantly distributed in their evolutionary relationships [22] [23] [24] [25] . again, in microrna field the authors have developed a new identification technique of microrna precursors emphasizing on different data distributions of negative samples [26] . further, phylogenetic analysis are also studied from different viewpoints to find the evolutionary relationship among various species [27] [28] [29] . some authors have used the statistical tools for sequence alignment, alignment-free sequence comparison and phylogenetic tree [30] [31] [32] [33] . although every amino acid has individual activity, group specific function of amino acid is also obvious. methods have been introduced for the 2d graphical representation of dna/rna or protein sequences [34] [35] [36] [37] [38] [39] [40] where methods are based on individual score and position wise graphical representation. so, in this field establishment of a new methodology is always welcome with distinct findings. combining with various features for dna, rna and protein sequence a web server called pse-in-one (http:// bioinformatics.hitsz.edu.cn/pse-in-one/home/) is developed [41] which is user friendly and can be modified by users themselves. recently, the authors have classified the twenty standard amino acids into the eight chemical groups and have found some group and/or family specific conserved patterns which are involved in some functional role specially in motor protein family members [17] . in this study, the previously defined method [17] of reduced alphabets are used as an application into the cytochrome c7 family protein members. we introduced a new method of phylogenetic analysis based on chemical group dissimilarity of amino acids. in addition, we build the graph from primary protein sequence. in the designing of graph, we have designated the various chemical groups of amino acids as thevertices in the graph. the primary protein sequence is read as consecutive order pairs serially from first amino acid to the end of sequence, and each order pair is nothing but a connected edge between the two nodes where nodes in the graph are involved with different chemical groups of amino acids. the graph is drawn for every individual protein sequence and we look for various unique edges/ cycles among the entire family members. so any unique findings from the graph may be hypothesized as having a significant functional role in the primary protein sequence. because the variation in the graph is directly affected by the amino acid residues in some specific domain where a change of chemical group has taken place. we highlight all the significant points which are differing from one sequence to other. further, working with reduced alphabets and designing the graph require less complexity and easy visualization even if working with the larger sequences. order pair directed graph a directed graph g = (v, e) is a graph which consists of a set of vertices denoted by v = {v 1 , v 2 , . . ., v i }, and a set of connected edges denoted by e = {e 1,1 , e 1,2 , . . ., e i, j } where an edge e i, j exists if the corresponding two vertices v i and v j are connected and the direction of edge is from the vertex v i to the vertex v j . from the graph, various graph theoretic properties like edge connectivity, cycles, graph isomorphism etc. can be investigated to differentiate the graphs. given an arbitrary amino acids sequence, it is first transformed into the numerical sequence as described previously where amino acids are categorized into eight chemical groups according to the side chain/chemical nature of the amino acids [17] . the transformation is done using the following rules (eq 1) as per the classification. if a particular amino acid is read as a i , then the corresponding transformed group is g k and the numerical value k is defined by the following eq (1). : ifa i 2 fd; eg here, g 1 , g 2 , . . ., g 8 are the acidic, basic, aliphatic, aromatic, cyclic, sulfur containing, hydroxyl containing and acidic amide groups respectively [17] . the eight numerical values are considered as the vertices of the graph g i.e. v i 2 {1, 2, . . .8}. algorithm 1 is used to generate the directed graph from the primary protein sequence using matlab16b software. here, we obtain the graph which is the order pair digraph because an edge is constructed through the pair (source node, target node) which is obtained from the consecutive order pair list of amino acids in the primary protein sequence. so given an arbitrary amino acid sequence, we can find an order pair directed graph having at most eight vertices/nodes. output: an adjacency matrix and the corresponding order pair directed graph. define a null matrix (m) of size 8 by 8; define a 1-d array (t) of size l,; find x as the chamical group number of a i uisng eq (1); the phylogenetic tree is an acyclic graph showing the evolutionary relationship among the various biological species based on their genetic closeness. although various phylogenetic tree methods have already been studied, based on chemical nature of amino acids are not yet explored in the literature as per our knowledge. our method of phylogenetic tree formation used the dissimilarity matrix which is obtained for every pair of sequence on the basis of chemical group specific score of amino acids. so this method is completely alignment free and requires less computational complexity. firstly, we calculate the percentage of occurrence of amino acids from each chemical group using the following equation eq (2) . if there are n number of sequences which are denoted as s 1 , s 2 , . . .s n , then the corresponding length of the sequences are denoted as l 1 , l 2 , . . .l n . and a particular sequence s i is read as for the sequence s 1 , the first amino acid is read as s 1 1 , the second amino acid is read as s 2 1 and so on. for each g k group and a particular sequence s i , we count the total number of amino acids s i (t k ) and score per hundred s i (g k ) on using the following eqs (2) and (3) respectively. for example, if the primary protein sequence length is 80 aa, out of which 20 aa are from acidic group i.e. g 1 , then the score per hundred of the acidic group is 20 80 â 100 à á ¼ 25%. secondly, we measure the dissimilarity measure for every possible pair of sequence. the dissimilarity of two sequences s i and s j is denoted as d s i ; s j . for each group g k , we count the percentage of amino acid differences of the two sequences taking the mod value of the score obtained on using eq (4). this is done for all the respective eight chemical groups and all the values are added. finally, we get the dissimilarity matrix d of size n by n as shown below. dðn; nþ ¼ to draw the phylogenetic tree, we use the nearest distance (single linkage) method. the pair wise distances are the entities of the obtained dissimilarity matrix and the whole procedure is written in matlab 2016b software. five homologous triheme cytochromes (ppca-e) are identified in g. sulfurreducens periplasm and gene knockout studies revealed their involvement in fe(iii) and u(vi) extracellular reduction [1, 2] . cytochromes have been thoroughly studied for laboratory experiments because of their small size (about 90 amino acids). table 1 shows the gene name, accession number, protein name, length (#amino acids). the primary protein sequences are collected from http://www.uniprot.org/. sequence identity and the phylogenetic tree firstly, our analysis is directed to measure the primary protein sequence for every member. we obtain the percentage identity matrix of every pair of sequences ( sequence characterization of ppca and its homolog proteins ppcb-e: a mathematical approach exported from clustalw. it is observed that sequences are at least 47% similar. the maximum similarity is 76% which is found between ppca and ppcb. if we consider the ppca sequence which shows the minimum of 50% similarity with ppce and the maximum of 76% similarity with ppcb, we are not able to differentiate the ppca from other homologs on using the similarity percentage. secondly, we count rate of occurrence (frequancy of amino acids) of every individual amino acid of the respective five sequences which are shown in table 3 . then, we look for chemical group specific frequency for every sequence shown in table 4 using eq (3). now, we obtain the dissimilarity score of all possible two sequences (using eq (4)). say for an example, we compare the seq. no. 1 and seq. no. 2, we get the difference for acidic group is 2.1978 (10.9890-8.7912), basic group is 4.3956 (27.4725-23.0769) and so on (from table 4 ). total score after summing the eight groups is 17.5824 which measures the dissimilarity percentage of the said two sequences. similar results we get for all other pairs which are shown in table 5 . this table shows the biological distances between each pair of sequences. from this pair wise distance matrix, the phylogenetic tree is constructed as shown in fig 1, also discussed in method section. based on the phylogenetic tree of five members, we find that the ppca and ppcd, ppcb and ppce are mostly closed with regards to the frequency of amino acids of respective eight chemical groups. from fig 1 it is not obvious that ppca differs from other homologs, but if we go through the dissimilarity matrix (table 5) , we find some variations. here, it is observed that ppca differs by minimum of 16.5313% with ppcd, whereas for other homologs minimum dissimilarity sequence characterization of ppca and its homolog proteins ppcb-e: a mathematical approach is found for ppcd with ppcc which is 11.8535%. therefore among all the pairs, the high dissimilarity of ppca shows its uniqueness compared to its homologs. if we have a closer look into the list of amino acids, it is observed that the amino acids d, e, h, k, f, i, l, v, a, g, p, m, c, t are present among all the sequences. other amino acids are not common to all the member sequences. therefore, on the basis of chemical groups, all the amino acids from acidic, aliphatic, cyclic and hydroxyl containing groups are present. it is observed that the acidic, basic and hydroxyl containing groups percentage distinctly differ while compared ppca with other homologs. further, it is observed that only one proline(p) from cyclic group is present in ppcd while in other homologs, proline (p) is present at least 3 times. and another important observation is that the amino acid tryptophan (w) from aromatic group is present only in ppcd sequence. for every member of cytochrome c7 family, we draw a order pair directed graph using algorithm 1 which are shown in fig 2. there are maximum of eight possible nodes and the various directed edges among the nodes. we try to highlight the connected edges that show the uniqueness, specially in between the ppca and its homolog members and ppcd with other members separately as well as commonality to all members. details of the edge connectivity information for ppca and its homologs are shown in table 6 . we say two nodes (direction is from row to column) are connected or present if the cell symbol is 1, not present if the cell symbol is 0, and common to all the members if the cell symbol is ã . an edge between two nodes (in order) is basically a pattern https://doi.org/10.1371/journal.pone.0175031.g002 table 6 . existance of unique edges comparison between ppca and ppcb-e groups obtained from directed graph (fig 2) . ppcb-e node vs. node 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 * * * * * * (two distinct nodes or two distinct amino acids from two different chemical groups) of length 2. we find two particular edges, one edge (82) is present only in ppca sequence (approx. residues 41-42, s1 table) that is not found in other member sequences, and one edge (73) which is present in ppcb-e sequences (approx. residues 25-36, s1 table) , but this edge is not present in ppca sequence. while considering all the members, we find many edges which are common to all. further, ppcd is structurally dissimilar among the homologs [4] . while looking into the order pair directed graph, we find only one variation i.e. there is an edge (54) node 5 to node 4 among the ppca-c and ppce sequences which is not observed in ppcd (table 7) . this node transition where amino acid changes proline(p) to glycine (g) for ppca-c and ppce and for ppcd this transition is from glycine (g) to glycine (g), located in approximately residues 54-55 (s1 table) . again existence of edges between any two nodes either common to all or individual member specific have some significant role in the primary protein sequences. because node to node connectivity is the point of changes from one chemical group to the other in the primary protein sequence positions and this could be the effective characteristic for the structural or functional variation of proteins. although few residues are being responsible while interacting with dxca, the neighbouring residues of amino acids must be having a role for their unique characteristics. so the subdomain identification involving with different unique cycles would be worth mentioning in this regard. here, we have calculated the various cycles of length c l (3 c l 6) for group specific and individual member specific which are shown in in s2 table. say for an example, the cycle 7216457 of length 6 i.e. the directed edges are 7 ! 2 ! 1 ! 6 ! 4 ! 5 ! 7. for completing this cycle a particular subdomain is responsible. interestingly, we find various unique cycles for ppca, ppcd and ppcb-e. so there are some unique cycles which are distinctly present for ppca and its homolog proteins and vice versa. there are some unique cycles which are present in ppcd, but no unique cycle is present for ppca-c and ppce. highlighiting the sub-domain for some of the unique cycles of length 3, 4 and 5 are shown in fig 3(a) for ppca and fig 3(b) for ppcd. from fig 3, the cycle (2362) of length 3 whose sub-domain residues are within 13 to 48, that is the numerical sequence is 36. . .62 from fig 3(a) . one can see the corresponding amino acids residues from s1 table. for some cycles, there is a possibility of different sub domains because some edges are repeating more than once in the different positions of the sequence that can be counted for the same cycle. similarly, on varying the cycle length, we get different sub-domains or amino acid residues. these sub-domain findings might be of immense help to the bio-chemists for the understanding of physicochemical nature and the unique activity of various proteins. table 7 . existance of unique edges comparison between ppcd and ppca-c, ppce groups obtained from directed graph (fig 2) . ppca-c, ppce node vs. node 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 * * * * * * we take all the five sequences of ppca-e members, obtain the alignment sequence from clus-talw2. the alignment figure is shown fig 4. we mark the various blocks as r1, r2. . .r16 which are conserved. rectangular with highlighted regions are chemically conserved, and only highlighted regions are conserved based on individual amino acid. we find two highly conserved regions r13 and r22 which are having some variations. the first region (r13) is with 4 residues block (hkk/rh or 2222) among the members ppcb-e where all the amino acids from basic group, but in ppca this block is hkah or 2242 i.e. the 3rd position k/r is replaced by aliphatic amino acid alanine (a). the second region (r22) is gche/k or 4622/1 where 4th position amino acid is either from acidic or basic group i.e. both fall under charge group. if we look into the ppca sequence some dissimilarities are found in "heme i" region [3] [4] [5] . the two consecutive amino acids between regions r14 and r15 in ppca is kk (from basic group), but for ppcb-e only one amino acid is from basic group. previously it is observed that ppcd is structurally dissimilar [5] and the authors have shown that there is an addition of amino acid threonine (t) for ppcd sequence after the r15 region in fig 4. but, from figure we can see that another one amino acid valnine (v) insertion is viewed in region of r8 and r9. besides, various patterns which are common to ppca, but not in ppcb-e and vice versa shown in table 8 with bold color. for the pattern "624621" which is located with the combined regions of r21 and r22 ("heme iii" region), there is a change of amino acid threonine (t) for ppcd and lysine (k) for others. apart from these, we find an amino acid deletion both for the ppcd and ppce before the "heme iii" region. further, on combining the regions r6, r7 and sequence characterization of ppca and its homolog proteins ppcb-e: a mathematical approach r8 (pattern "44441"), the change for ppca sequence is phenylalanine (f) which is from arometic group whereas other sequences are from aliphatic group, and the change for ppcd sequence is histidine (h) which is from basic group whereas other sequences are also from aliphatic group. again the region between r17 and r18 ppcd contains the amino acid methionine (m) from the sulfur containing group while the other homologs contain phenylalanine (f) from aromatic group. altogether, group specific changes have significant role towards the binding with the dxca for ppca and the structural dissimilarity of ppcd. in this work, we have presented the sequence based characterization of cytochromes c7 family members. we specifically emphasize the distinguished features of ppca and ppcd compared to the other homologs. although the study suggests that percent identity among the five members varies between 46% and 75%, on the basis of chemical groups these are shown between 75% and 89%. we highlight some of the chemical groups and their percentage that can distinguish ppca and ppcd. the dissimilarity features of ppca may play significant role towards its binding with dxca. similar is the case that may happen for ppcd for its structural dissimilarity. our proposed graph theoretic model can easily show the instant change of amino acids from one group to the other in the sequences. further, the unique cycles for ppca and ppcd may expose their outstanding nature. and finally from the alignment graph, chemically conserved regions are highlighted. we observe some special patterns where amino acid(s) from some of the sequences are abruptly changed. all the cases will provide the features for ppca and ppcd that would explain their unique functionality and/or structural dissimilarity. it may be noted that there are some existing methodologies [11, 14, 16, 20, 22, 25, 30] which would reflect the sequence pattern information or key features of the observed sequence. many characteristics of the dna, rna and protein sequences can be found out from the web servers and standalone existing tools, one of the important web servers in this regard is defined in [41] . we look at the problem in a different manner, one dealing with embedded chemical properties of amino acids and various mathematical structures. in general, methodology defined in this article is very easy to implement to get the unique features of observed sequences. so, collectively our methodology will add to be combined for the machine learning algorithms to develop refined computational predictors. hence, the use of reduced alphabets (amino acids) technique involving mathematical basis with the embedded chemical properties of amino acids will be very much useful for the protein homology detection. supporting information s1 table. amino acids and transformed numerical sequence based on eight chemical groups for c7 five members. (pdf) s2 table. unique cycles for ppca-e, ppca, ppcb-e, ppcd. these cycles are involved in various sub-domains, some of which are shown in fig 3. 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sequence alignment as a prerequisitetto correct phylogenetic trees graph theory with applications to engineering and computer science protein flexibility predictions using graph theory dictionary of protein secondary structure: pattern recognition of hydrogenbonded and geometrical features use of information discrepancy measure to compare protein secondary structures 2-d graphical representation of protein sequences and its application to coronavirus phylogeny a 2d graphical representation of protein sequence and its numerical characterization similarity/dissimilarity analysis of protein sequences based on a new spectrum-like graphical representation pse-in-one: a web server for generating various modes of pseudo components of dna, rna, and protein sequences we thank dr. pokkuluri, phani raj (argonne lab, usa) for the initial discussions of the problem. key: cord-004068-d66lwylf authors: shimoda, tomoko; okubo, torahiko; enoeda, yoshiki; yano, rika; nakamura, shinji; thapa, jeewan; yamaguchi, hiroyuki title: effect of thermal control of dry fomites on regulating the survival of human pathogenic bacteria responsible for nosocomial infections date: 2019-12-27 journal: plos one doi: 10.1371/journal.pone.0226952 sha: doc_id: 4068 cord_uid: d66lwylf we monitored the survival of human pathogenic bacteria [escherichia coli (atcc), extended-spectrum β-lactamase-producing e. coli (clinical isolate), new delhi metallo-β-lactamase-producing e. coli (clinical isolate), staphylococcus aureus (atcc)] on dry materials (vinyl chloride, aluminum, plastic, stainless steel) at distinct temperatures (room temperature or 15°c–37°c). these bacteria favored a lower temperature for their prolonged survival on the dry fomites, regardless of the material type. interestingly, when mixed with s. aureus, e. coli survived for a longer time at a lower temperature. cardiolipin, which can promote the survival of s. aureus in harsh environments, had no effect on maintaining the survival of e. coli. although the trends remained unchanged, adjusting the humidity from 40% to 60% affected the survival of bacteria on dry surfaces. scanning electron microscopic analysis revealed no morphological differences in these bacteria immediately before or after one day of dry conditions. in addition, atp assessment, a method used to visualize high-touch surfaces in hospitals, was not effective at monitoring bacterial dynamics. a specialized handrail device fitted with a heater, which was maintained at normal human body core temperature, successfully prohibited the prolonged survival of bacteria [enterococcus faecalis (atcc), e. coli (atcc), pseudomonas aeruginosa (atcc), s. aureus (atcc), acinetobacter baumannii (clinical isolate), and serratia marcescens (clinical isolate)], with the exception of spore-forming bacillus subtilis (from our laboratory collection) and the yeast-like fungus candida albicans (from our laboratory collection)] on dry surfaces. taken together, we concluded that the tested bacteria favor lower temperatures for their survival in dry environments. therefore, the thermal control of dry fomites has the potential to control bacterial survival on high-touch surfaces in hospitals. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 strategies to ensure greater control of hospital-acquired infections are urgently needed. microbe-contaminated dry surfaces in hospitals that are frequently touched by hands, termed 'high-touch' surfaces, such as handrails, doorknobs, lockers, and the table surfaces of inpatients, present a high risk in terms of the spread of nosocomial infections [1] [2] [3] . it is well recognized that improved hand hygiene and cleaning of surfaces are critically important in controlling hospital-acquired infections [4] [5] [6] . while improved cleaning practices have led to a decrease in the transmission of hospital-acquired infections [7] [8] [9] , in general, hospital cleanliness is still not sufficient to control infections [10] [11] [12] . this is confirmed by the fact that outbreaks of hospital-acquired infections still occur worldwide [13] [14] [15] . it is therefore possible that there are other unknown factors responsible for bacterial survival, particularly on dry surfaces, in hospitals. we previously monitored hospital cleanliness on the surfaces of 752 sites in nurse and patient areas in three hospitals located in the central area of sapporo, japan, and revealed the presence of a wide range of organic contamination, including microbial contamination, that could be spread via hand touching [16] [17] [18] . considerable variability in bacterial detection was noted, even for the same surfaces, indicating that the ongoing cleanliness of dry surfaces in hospitals is insufficient [16] [17] [18] . furthermore, accumulating evidence indicates that various human pathogenic bacteria (such as pseudomonas, acinetobacter, enterococcus, staphylococcus, and enterobacteriaceae), including multidrug-resistant bacteria, can survive for prolonged periods on dry surfaces [19] [20] [21] [22] [23] [24] [25] . the reason for the variability in bacterial detection remains unclear, but changing physiological factors such as environmental temperature may affect bacterial survival. whether thermal control can regulate bacterial survival in dry environments remains unknown. in this study, we monitored the survival of human pathogenic bacteria [escherichia coli atcc, extended-spectrum β-lactamase (esbl)-producing e. coli, new delhi metallo-β-lactamase (ndm)-producing e. coli, and staphylococcus aureus atcc] on dry materials (vinyl chloride, aluminum, plastic, stainless steel) over a range of temperatures (room temperature to 37˚c). we found that bacteria favor lower temperatures for survival in dry environments. furthermore, we propose that fomites warmed to human body core temperature may help to control bacterial survival in dry environments, and that such a strategy may prohibit the emergence of human pathogenic bacteria in hospital environments, eventually reducing the need for antibiotics as well as disinfectants. the bacterial strains used in this study were as follows: e. coli atcc 25922, esbl-producing e. coli, ndm-producing e. coli [26] , and s. aureus atcc 29213. all of the bacteria were maintained on lb agar plates (nakalai tesque inc., kyoto, japan) with or without appropriate antibiotics at 37˚c. both the antimicrobial-resistant e. coli strains were isolated at hokkaido university hospital. the reference strains were purchased from the american type culture collection (atcc, manassas, va, usa). e. coli dh5α carrying gfp-expressing plasmid (pbbr122 encoding gfp) was also used for this study [27] . e. coli dh5α was maintained on lb agar with chloramphenicol (10 μg per ml) to maintain the plasmid. pseudomonas aeruginosa atcc29213 and enterococcus faecalis atcc29212 were also purchased from the atcc. acinetobacter baumannii and serattia marcescens were isolated from hokkaido university hospital. bacillus subtilis and the yeast-like fungus candida albicans from our laboratory collection were also used for this study. all bacterial strains (except for e. faecalis) and the yeast-like fungus were maintained on lb agar and pda agar, respectively. because of requiring more nutrients, e. faecalis was maintained on bhi agar. s. aureus atcc 29213 and e. coli atcc 25922 were separately adjusted in pbs at approximately 10 7 cfu per ml, and were either mixed or maintained separately in lb broth. then, 20 μl of each solution (5.0 × 10 5 cfu/spot) was spotted onto vinyl chloride material (3 × 3 cm) that is used commercially for flooring and roofing (thanks to toli corporation, osaka, japan and tajima roofing, tokyo, japan). the bacterial solution was left to dry and then incubated at room temperature (~20˚c) or in an incubator (30˚c and 37˚c) for up to 11 days. in some experiments, the bacterial suspensions were dried onto the material with cardiolipin (1 or 100 μm). after incubation, the dried spots were wiped with cotton swabs, and suspended in pbs. the bacteria were enumerated by spreading onto mannitol salt agar (for culturing of s. aureus) and macconkey agar (for culturing of e. coli), and bacterial numbers were expressed as cfus per spot. the spots of bacteria on the dry material were also visualized by sem observations (see below). the amounts of atp in each spot were monitored by a clean-trace luminometer 3m (saint paul, mn, usa), and the values were expressed as bioluminescence relative light units (rlus), according to a previously described protocol [17] . the survival of s. aureus atcc 29213 and e. coli atcc 25922 was also tested on other materials (10 × 10 cm; aluminum, plastic, stainless steel) at a range of temperatures (15˚c, 30˚c, 37˚c) for up to 11 days. bacterial numbers were evaluated as stated above. some experiments on plastic were adjusted to a constant humidity of 40%-60% by placing a 100-ml beaker filled with water into the incubator. however, unfortunately, we were unable to maintain a uniform humidity in this way. the humidity of the incubator without the beaker was less than 20%. humidity was measured with a general hygrometer. the dynamics of both esbl-and ndm-producing e. coli strains with or without s. aureus atcc 29213 on vinyl chloride material were also assessed. the bacteria were dried and then incubated at distinct temperatures (15˚c, 30˚c, 37˚c) for up to 11 days. bacterial numbers were evaluated as stated above. according to a previously reported method [17] , the vinyl chloride material (1 × 1 cm) spotted with bacteria was washed with saline, fixed with 2.5% (v/v) glutaraldehyde in phosphate-buffered saline (ph 7.4) for 2 h at room temperature, then treated with 2% (w/v) osmium tetroxide for 1 h at 4˚c. the samples were then dehydrated in ethanol, freeze-dried, and coated with osmium using a plasma osmium coater, for analysis using a scanning electron microscope (hitachi s-4800; hitachi, tokyo, japan). a stainless steel handrail pipe (diameter, 13 mm; thickness, 2 mm) equipped with a heater wire in the center of pipe to control the dry surface at body core temperature was constructed using an acrylic-processing assembly kit from fabcloud (tokyo, japan). bacterial dynamics on this surface were assessed as follows. in brief, the gfp-expressing e. coli dh5α were spotted at regular intervals (1 cm) onto the handrail device with or without the heater at approximately 10 6 cfu per spot (10 μl per spot). the handrail device (with or without heat control) was then incubated at room temperature for one day. the dried spots were wiped with cotton swabs, and suspended into pbs. the number of bacteria was evaluated by the gfp signals after spotting onto lb or macconkey agar plates containing chloramphenicol, and was expressed as cfus per spot. in addition, the surface temperature on the handrail pipe was monitored using a hand-held infrared sensor (ct-2000d; custom co., ltd., tokyo, japan) and a high-resolution infrared sensor (infrec r300; nec avio infrared technologies co., ltd., tokyo, japan) installed with appropriate software (infrec analyzer ns9500; nec avio infrared technologies co., ltd.). we also assessed whether the heated handrail device could work for the illumination of the other bacteria (s. aureus, p. aeruginosa, e. faecalis, a. baumannii, s. marcescens and b. subtilis) and the yeast-like fungus (c. albicans). as mentioned above, the survival of bacteria (p. aeruginosa, a. baumannii, and b. subtilis) and the yeast-like fungus after drying on the device was monitored on lb agar and pda agar, respectively. the survival of s. aureus and s. marcescens was also monitored on mannitol salt agar and macconkey agar, respectively. meanwhile, because of requiring more nutrients, e. faecalis was monitored on bhi agar. multiple comparisons of the data were assessed by bonferroni/dunn analysis. a p value of <0.05 was considered statistically significant. all calculations were conducted using excel for mac (2011) with statcel3c. to assess the survival of representative human pathogens under dry conditions, we monitored changes in bacterial numbers [e. coli (atcc 25922), s. aureus (atcc 29213), or a mixture of these bacteria (because it is possible that they are found together on 'high-touch' surfaces)] on vinyl chloride, a widely used material in everyday items, at distinct temperatures [room temperature (around 20˚c), 30˚c, 37˚c] for 11 days. bacterial numbers were assessed using the following indicators: the number of colony-forming units (cfus), the amount of atp, and morphological changes (as determined by sem analysis) ( fig 1a) . immediately (within 20 min) after drying, the cfu numbers of e. coli, but not s. aureus, showed a two-log reduction; however, both bacteria, either singularly or in a mixture, survived and retained the potential to be spread ( fig 1b) . the number of cfus decreased dramatically over time for both bacteria on dry surfaces, but this trend was amplified by an increase in temperature ( fig 1c) . interestingly, the presence of s. aureus appeared to enhance the survival of e. coli in the bacterial mixture compared with e. coli alone (fig 1c, see ' †'). sem observations revealed that the morphological changes in both bacteria, singularly or in a mixture, one day after drying was minimal, supporting the cfu assay results and indicating survival of both bacteria (fig 2) . atp assessment revealed no significant differences for either bacteria with varying temperature (s1 fig). also, cardiolipin, an agent that aids the survival of s. aureus in dry environments [28, 29] , did not support the survival of e. coli after drying, suggesting the presence of another mechanism independent of cardiolipin (s2 fig). in addition, although the trend observed with altered temperature did not change, humidity control (40-60%) had the opposite effect on the survival of microorganisms on dry surfaces (s3 fig). specifically, while the humidity control weakened the thermal effect on the survival of s. aureus on dry environments, a synergistic effect of inhibition on the survival of e. coli was observed. thus, our findings showed that these bacteria favor a lower temperature for their survival in dry environments, and humidity has a lesser effect. next, to confirm whether the effect of temperature on bacterial survival on dry surfaces is universal regardless of the material surface, we monitored changes in bacterial numbers [e. coli (atcc 25922), s. aureus (atcc 29213), or a mixture] on a range of common materials (vinyl chloride, aluminum, plastic, stainless steel) at distinct temperatures (15˚c, 30˚c, 37˚c) for 11 days, by assessing the cfu numbers ( fig 3a) . immediately (approximately 1 h) after drying, no differences in bacterial numbers were detected between the different materials ( fig 3b) . although the cfu numbers of both bacteria dramatically reduced over subsequent days on the dry surfaces, this temperature-dependent trend was unaffected by the type of material surface ( fig 3c) . thus, the material itself (vinyl chloride, aluminum, plastic, stainless steel) had no effect on the diminishing bacterial numbers under dry conditions. we also assessed whether the same trend was seen for multidrug-resistant human pathogenic bacteria using esbl-and ndm-producing e. coli isolated from hokkaido university hospital. the cfu assay confirmed increased survival of multidrug-resistant bacteria at lower temperature, as well as increased survival in the presence of s. aureus (fig 4) . taken together, we conclude that bacteria favoring a lower temperature for their survival in dry environments is a universal phenomenon, which is unaffected by the surface material or bacterial type. importantly, our data highlighted the potential for controlling the survival of bacteria on high-touch dry surfaces by warming dry fomites to human body core temperature. handrail device warmed to human body core temperature prohibited the prolonged survival of e. coli on this dry surface to test this possibility, we constructed a stainless steel handrail pipe (diameter, 13 mm; thickness, 2 mm) equipped with a heater wire in the center of the pipe to control the dry surface to body core temperature (s4a fig). assessment with a hand-held infrared sensor showed that the surface temperature on the handrail pipe was controlled to almost body core temperature, although the temperature on the device surface varied from the end to the center of the pipe (range: 30˚c-40˚c) (s4b fig). a heatmap generated with the high-resolution infrared sensor showed that in contrast to the stainless steel pipe without the heater, the surface temperature on the heated pipe was maintained at body core temperature (fig 5a) , indicating that it is an effective tool to monitor the influence of temperature on bacterial survival on a dry surface. we monitored the survival of gfp-expressing e. coli (dh5α) spotted onto the handrail surface at regular intervals (1 cm) compared with the gfp signals at equivalent spots on an lb agar plate, and assessed cfu numbers. gfp signals were dramatically decreased on the heated handrail compared with the unheated handrail even 6 h after drying, and the signals were virtually undetectable one day after drying (fig 5b) . consistent with this, the cfu numbers on the heated handrail were significantly reduced compared with those on the unheated handrail ( fig 5c) . in addition, we assessed whether the warming handrail could work on other bacteria. the results indicated that the prolonged survival of bacteria (e. faecalis, s. aureus, p. aeruginosa, a. baumannii, and s. marcescens) as well as e. coli, was significantly prohibited, although no effective elimination of the spore-forming bacterium (b. subtilis) was detected (fig 6) . we also confirmed that the yeast-like fungus (c. albicans) failed to survive immediately (within 6 h) after drying on the device (s5 fig). taken together, we concluded that the handrail device warmed to human body core temperature prohibited the prolonged survival of some bacteria and a yeast-like fungus responsible for nosocomial infections on a dry surface. currently, in the us, an estimated 2 million hospital-acquired infections are reported annually, associated with $5 billion of additional healthcare costs. the control of nosocomial infections is therefore a priority for healthcare providers worldwide [30] . here, we showed, for the first time, that a lower temperature is favored for the survival of both e. coli and s. aureus on dry fomites. we also demonstrated that a handrail device warmed to human body core temperature could prohibit the prolonged survival of several pathogens responsible for nosocomial infection (e. coli, s. aureus, e. faecalis, p. aeruginosa, a. baumannii and s. marcescens) and a yeast-like fungus (c. albicans) on this dry surface. innovations such as the development of thermal control of dry fomites and bacterial survival heated fomites, for example, heated handrails or doorknobs, may prevent the survival of bacteria responsible for nosocomial infections on dry hospital environments, without the need for disinfectants. it is well established that decreasing environmental temperature has a significant impact on the survival of pathogens in soil or water, sometimes leading to bacterial persistence [31] [32] [33] , but whether temperature influences bacterial survival on artificial materials, such as dry fomites in hospitals, remained unknown. other factors such as uv radiation, humidity, the presence of organic materials, and surface type are known to be associated with the ability of thermal control of dry fomites and bacterial survival bacteria responsible for hospital-acquired infections to survive on dry surfaces [34] [35] [36] ; however, temperature is a factor that can be easily and accurately controlled. furthermore, a temperature of 37˚c, which is the core body temperature, is a comfortable temperature that is not harmful to the touch [37] . for these reasons, we selected temperature as a critical factor to study its effects (in particular, body core temperature) on bacterial dynamics and survival on dry fomites. the numbers of cfus of both bacteria (e. coli and s. aureus), regardless of whether or not they carried multidrug-resistant genes, were dramatically reduced during the days after drying, and this decrease in bacterial numbers was significantly promoted by increased temperature. because water evaporation from bacterial cells can be accelerated on dry fomites at high temperature, we speculate that it is difficult for bacteria to maintain metabolic activity under such conditions; thus, their viability decreases. we found that gram-positive s. aureus could survive longer than gram-negative e. coli, potentially indicating that cell wall thickness plays a role in prolonging the survival of s. aureus on dry fomites. at lower temperature, e. coli survived for a few days whereas s. aureus survived for over 10 days. although the survival period for these bacteria is limited, particularly for e. coli, it is still sufficient time for these bacteria to transfer from person to person via high-touch surfaces. because of its ease of monitoring, atp bioluminescence has been employed as a visual indicator of general organic contamination of high-touch surfaces in hospitals [38] [39] [40] [41] [42] . however, our assessment of atp showed that, in contrast to the cfu assay, no significant differences in atp were associated with changes in temperature. evidence from our previous studies suggests that atp may be physically stable at a range of temperatures [16, 17] . furthermore, atp derived not only from bacteria but also from other sources such as fungi or dead cells may accumulate on high-touch surfaces. taken together, we concluded that atp is an unreliable measure for monitoring pathogenic bacteria on high-touch surfaces in hospitals. interestingly, the presence of s. aureus in the bacterial mixture promoted the survival of e. coli compared with e. coli alone. by contrast, cardiolipin, which is an effective factor promoting s. aureus survival in dry environments [28, 29] , had no effect on supporting the survival of e. coli after drying, suggesting the presence of another mechanism independent of cardiolipin. although this mechanism remains unknown, coexistence with cocci, which have a smaller surface area than bacilli and therefore retain water more effectively, may aid the survival of bacilli on dry surfaces. high-touch dry surfaces are invariably contaminated with s. aureus because this organism is a ubiquitous inhabitant of the human body, and it is therefore crucial to understand its dynamics on dry surfaces. cardiolipin is a membrane component that enhances stress tolerance, thereby maintaining bacterial viability [43] [44] [45] , and several studies have shown that cardiolipin is involved in biofilm formation [46, 47] . we therefore assessed whether the presence of cardiolipin affected the survival of e. coli on dry surfaces, but no impact was detected indicating that other factors may be involved. although bacterial numbers dramatically reduced each day on dry surfaces, the degree of this reduction varied slightly between experiments and the degree to which s. aureus promoted the survival of e. coli also varied between experiments. this indicated that there may be factors other than temperature that regulate bacterial survival on dry fomites. humidity, which can block water evaporation, has also been reported to have an impact on bacterial survival [34-36, 48, 49] . our experiments showed that constant humidity (40%-60%) affected the survival of bacteria on dry surfaces. whereas, after prolonged incubation without humidity control (less than 20% humidity) (see fig 1c) , variation in bacterial numbers appeared to be increased, suggesting that low humidity can favor the survival of small numbers of bacteria on dry materials. however, because of the difficulties in controlling humidity, we did not accurately adjust humidity in our experiments. therefore, further studies are needed in which humidity, as well as temperature, are more precisely controlled. as expected, we confirmed that the warming handrail adjusted to body core temperature prohibited the prolonged survival of several nosocomial-related pathogens including e. coli, s. aureus, e. faecalis, p. aeruginosa, a. baumannii, s. marcescens and c. albicans. the heated handrail device used in this study was constructed using an acrylic board processing equipment kit (see materials and methods), which cost 5,680 yen (jpy) (approximately $60). because this device was cheap and simple to construct, involving a stainless steel handrail pipe (diameter, 13 mm; thickness, 2 mm) equipped with a heater wire in the center of the pipe to control the dry surface at body core temperature, we believe that this technology may be applicable for the development of heated handrails for use in hospitals to control hospital-acquired infections. one limitation of this device was that the temperature varied from the end to the center of the pipe (range: 30˚c-40˚c), meaning that the temperature on the device surface was not constant, as indicated by the differences in bacterial numbers spotted onto the agar plates from each of the samples collected from the handrail. further improvements in the design of this device are therefore needed prior to practical use. in conclusion, we demonstrated that human pathogens (e. coli, s. aureus, e. faecalis, p. aeruginosa, a. baumannii, s. marcescens and c. albicans), responsible for nosocomial infections. favor a lower temperature for their survival in dry environments. based on this finding, we proposed a novel and simple strategy involving the warming of high-touch fomites in hospitals to body core temperature, to control the survival of human pathogenic bacteria responsible for nosocomial infections. however, this study had several limitations. as mentioned above, humidity, which can also influence bacterial survival, was not accurately controlled throughout our experiments [34-36, 48, 49] , and also the range of human pathogens used for this study were limited. further studies are needed to clarify the effect of humidity on bacterial survival under dry conditions and to confirm the reproducibility of our findings in other bacteria. further innovations are also needed to develop handrails or doorknobs with a uniform surface temperature equivalent to that of the core human body temperature before practical application in hospital environments. supporting information s1 fig. atp assessment of the bacterial spots on the vinyl chloride material showed that there were no significant differences corresponding to incubation times or temperature. the amount of atp was determined using a clean-trace luminometer (3m, usa), and the values were expressed as relative light units (rlus), according to a previously described protocol [17] . ec, e. coli atcc 25922. sa, s. aureus atcc 29213. rt, room temperature. 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heritable fitness advantage in subsequent dry exposure calcium and magnesium ions are membrane-active against stationary-phase staphylococcus aureus with high specificity rcs phosphorelay activation in cardiolipin-deficient escherichia coli reduces biofilm formation a cardiolipin-deficient mutant of rhodobacter sphaeroides has an altered cell shape and is impaired in biofilm formation bacterial contamination of inanimate surfaces and equipment in the intensive care unit indoor air humidity, air quality, and health-an overview we thank the edanz group (www.edanzediting.com/ac) for editing a draft of this manuscript. key: cord-001039-qocuprwb authors: hayasaka, daisuke; shirai, kenji; aoki, kotaro; nagata, noriyo; simantini, dash sima; kitaura, kazutaka; takamatsu, yuki; gould, ernest; suzuki, ryuji; morita, kouichi title: tnf-α acts as an immunoregulator in the mouse brain by reducing the incidence of severe disease following japanese encephalitis virus infection date: 2013-08-05 journal: plos one doi: 10.1371/journal.pone.0071643 sha: doc_id: 1039 cord_uid: qocuprwb japanese encephalitis virus (jev) causes acute central nervous system (cns) disease in humans, in whom the clinical symptoms vary from febrile illness to meningitis and encephalitis. however, the mechanism of severe encephalitis has not been fully elucidated. in this study, using a mouse model, we investigated the pathogenetic mechanisms that correlate with fatal jev infection. following extraneural infection with the jaoars982 strain of jev, infected mice exhibited clinical signs ranging from mild to fatal outcome. comparison of the pathogenetic response between severe and mild cases of jaoars982-infected mice revealed increased levels of tnf-α in the brains of severe cases. however, unexpectedly, the mortality rate of tnf-α ko mice was significantly increased compared with that of wt mice, indicating that tnf-α plays a protective role against fatal infection. interestingly, there were no significant differences of viral load in the cns between wt and tnf-α ko mice. however, exaggerated inflammatory responses were observed in the cns of tnf-α ko mice. although these observations were also obtained in il-10 ko mice, the mortality and enhanced inflammatory responses were more pronounced in tnf-α ko mice. our findings therefore provide the first evidence that tnf-α has an immunoregulatory effect on pro-inflammatory cytokines in the cns during jev infection and consequently protects the animals from fatal disease. thus, we propose that the increased level of tnf-α in severe cases was the result of severe disease, and secondly that immunopathological effects contribute to severe neuronal degeneration resulting in fatal disease. in future, further elucidation of the immunoregulatory mechanism of tnf-α will be an important priority to enable the development of effective treatment strategies for japanese encephalitis. japanese encephalitis virus (jev), which belongs to the genus flavivirus in the family flaviviridae, is a causative agent of acute central nervous system (cns) disease in humans and domestic animals [1] . pigs and birds are amplifiers or reservoir hosts of jev in the environment, providing a source of virus for blood feeding culex spp. mosquitoes [1] which may subsequently feed on and infect humans. japanese encephalitis (je) is a major public health issue in asia and the asia-pacific region [1, 2] . annually, 30,000-50,000 cases and 10,000-15,000 deaths are reported and more than 50% of survivors may suffer from neurological disability [2] . human infections are largely subclinical with a rate varying from 1:25 to 1:1000 [3, 4] . the clinical symptoms vary from mild to severe disease including a non-specific febrile illness, meningitis, encephalitis and meningoencephalitis, the latter being observed in the most severe cases [4] [5] [6] . . following an incubation period of 6-16 days, patients may develop fever, headache, vomiting, rigor and nausea [1, 4] . subsequently, encephalitic cases develop neurological symptoms including seizure, tremor, photophobia, decreased sensorium, generalized and localized paresis, movement disorder [4, 6] . signs of meningeal irritation such as neck stiffness may be present. these clinical features are not unique to je, thus, laboratory diagnosis is required to distinguish it from other neurological disorders. the variety of disease symptoms and the prognosis in je cases appears to be dependent on complex interactions between viral and host factors [4] . in particular, host factors appear to be important determinants of disease severity and a number of specific proinflammatory cytokines and chemokines are observed in severe je cases [7] . for example, it has been demonstrated that increased levels of tnf-α in cerebrospinal fluid (csf) and serum correlated with cases of severe disease [8] . however, how these biological cytokines and chemokines contribute to the severe disease has not been fully elucidated. therefore, understanding the mechanism of the specific host response in severe cases is an important priority to develop a specific treatment for the infectious disease. the laboratory mouse model is commonly employed to study the cns pathology induced by encephalitic flaviviruses [9] [10] [11] . in common with human cases, mice develop relatively similar neurological dysfunction and the pathologic changes in infected mouse brains are similar to those observed in human cases [6, 9] . although extraneural infection frequently does not result in detectable viremia or virus burden in mice, it is believed that initial virus replication occurs in dendritic cells (dcs) such as langerhans cells at the site of infection, and the infected dcs migrate to draining lymph nodes [6] . virus then invades the cns and hosts develop cns disease, although the mechanism by which the blood-brain-barrier is crossed is not completely understood [12] [13] [14] [15] [16] [17] [18] . cns pathology is the consequence of viral infection of the affected cells and the resulting inflammatory responses in the cns. flavivirus variants may induce different degrees of pathology, however, the host immune response is likely to be a more critical determinant of clinical outcome [19] . inflammatory responses mainly contribute to virus clearance and recovery from fatal disease. for example, cd8 + t cells are reported to have an important function in controlling virus infection [20] [21] [22] [23] [24] [25] , although one report showed only a subsidiary contribution of cd8 + t cells in jev infection [26] . on the other hand, in recent studies it was suggested that immunopathological mechanisms may contribute to the severity of outcome following some encephalitic flavivirus infections [19, [27] [28] [29] [30] . for example, it was reported that cd8 + t cell function enhances pathogenicity during wnv and mvev infections [29, 30] . furthermore, in tick-borne encephalitis virus (tbev)-infected mice the inflammatory response was reported to contribute significantly to the fatal outcome [28] . microglia are the resident macrophages in the brain and are activated in response to a number of different pathological states [31] . following jev infection, activated microglia play a significant role in the development of pathology by producing pro-inflammatory cytokines such as il-1, il-6 and tnf-α [32, 33] . although these pro-inflammatory cytokines have dual roles, acting both as protectors and degenerators of neurons [31] , tnf-α is believed to be one of the key factors that mediate immunopathology in the cns during encephalitic flavivirus infection. for example, it was suggested that tnf-α directly mediates neuronal apoptosis by the engagement of tnf receptor 1 (tnfr1), the tnfr-associated death domain (tradd) and neuronal death contributes to glial activation and subsequent neuroinflammation [31, 34, 35] . it was also shown that tnf-α and il-1β mediate rantes gene expression for the recruitment of immune cells and glutamate released by jev-infected microglia, involves tnf-α signaling and contributes to neuronal death [36, 37] . on the other hand, other studies have shown that tnf-α has a protective role against wnv infections and restricts wnv pathogenesis by promoting trafficking of mononuclear leukocytes into the cns [38, 39] . furthermore, neuronal tnf-α expression during wnv encephalitis may be an adaptive response to diminish cxcl10-induced death [40] . at this stage of our knowledge, therefore, the precise role of the tnf-α response in encephalitic flaviviral pathogenesis remains to be clarified. immunomodulatory cytokines also affect disease outcome of encephalitic flavivirus infection. il-10 is reported to have an effect on immunoregulation [41] . it was suggested that il-10 mediates protection from acute encephalitis and plays a central role in determining the clinical outcome of jev infection [42] . insufficient anti-inflammatory cytokine production of il-4 and il-10 in the brain is associated with increased tissue pathology [43] . il-10 displays a neuroprotective function during jev infection and regulates deleterious effects of proinflammatory cytokines [44] . furthermore, an experiment using il-10 ko mice showed that il-10 signaling plays a negative role in immunity against wnv infection and blockade of il-10 signaling helps to control viral infection [45] . thus, the precise role of the il-10 response following encephalitic flavivirus infection also remains to be resolved. in general, evaluation of virus pathogenicity and virulence in mouse models utilizes either the subcutaneous or intradermal route of infection. this is considered to be a reproducible model of natural human infections following the bite of an infected mosquito or tick and in the past, death was used as an index of pathogenesis [46] . however, it is known that peripheral infections with some strains of encephalitic flaviviruses do not exhibit normal dose response curves based on mortality. although this was first reported in the 1940's [47] , the reason for these apparent discrepancies were not fully understood. we previously showed that the oshima strain of tbev caused dose independent mortality and the fatality rate did not increase more than 50% with increasing virus challenge doses from 10 2 to 10 6 plaque forming unit (pfu) [48] . in our study of tbev, we suggested that the variation of fatal outcome in individual mice appeared to be due to variation in individual host responses [48] . the purpose of this study was to investigate the host factors that influence disease severity following jev infection in a mouse model. in particular, we focused on the variation of disease outcome in individual mice following extraneural infection with jev. we first compared the pathogenicity of two jev strains, which cause either dose-dependent or doseindependent mortality responses. we next compared severe or mild cases of mice infected with jev exhibiting doseindependent mortality and investigated the specific host responses such as tnf-α and il-10 expression in the cns. we also examined the roles of the specific cytokines observed in severe cases using appropriate knockout (ko) mice. the animal experiments were performed in accordance with the recommendations in the fundamental guidelines for proper conduct of animal experiment and related activities in academic research institutions under the jurisdiction of the ministry of education, culture, sports, science and technology. the experimental protocols were approved by the animal care and use committee of the nagasaki university (approval number: 091130-2-7/0912080807-7). the jath160 strain of jev was kindly provided by tomohiko takasaki, national institute of infectious disease, japan. stocks of jev jaoars982 and jath160 viruses were obtained from cell culture medium of baby hamster kidney (bhk) cells infected with viruses previously prepared in suckling mouse brains [49] . the bhk cells were maintained in eagle's minimal essential medium (emem; nissui pharmaceutical co.) containing 8% fetal calf serum (fcs) and antibiotics. c57bl/6j (b6) mice were purchased from japan slc corporation. b6 background il-10-/-mice were purchased from jackson laboratory, usa [50] . tnf -/-mice were kindly provided by yoichiro iwakura, research institute for biomedical sciences, tokyo university of science [51] . these mice were mated in the facility of nagasaki university. five to six week old mice were subcutaneously inoculated with a range of 10 0 -10 6 pfu of jev diluted in emem containing 2% fcs. mock infected mice were inoculated with emem from the supernatant medium of bhk cells. mice were weighed daily and observed for clinical signs including behavioral symptoms and signs of paralysis. thirteen days post infection (pi), dying and recovering mice were distinguished by the degree of weight ratio, and namely mice exhibiting more than 25% or less than 10% weight loss were recognized as dying or recovering mice, respectively. following subcutaneous inoculation with 10 4 pfu of jev, mice were euthanized and blood, spleen, brain and spinal cord were collected following perfusion with cold phosphate-buffered saline (pbs). brains were dissected to provide four separate fractions, ie the brain cortex, thalamus, cerebellum and brainstem. until they were used, these tissues were stored at -80° c. each tissue was homogenized in ten volumes of pbs containing 10% fcs and diluted with emem with 2% fcs. virus titers were determined by plaque forming assays using bhk cells and were expressed as pfu/g tissue [48] . mouse brains and spleens were collected after perfusion with cold pbs. freshly isolated brains and spleens were immediately immersed in rnalater (ambion). total rna was extracted using rneasy lipid tissue mini kit (qiagen) according to the manufacturer's instructions. the expression levels of cytokines were measured by real time-pcr as demonstrated previously [52] . the copy numbers were calculated as a ratio of the copy numbers of internal control glyceraldehyde-3-phosphate dehydrogenate. mice inoculated with jev were anesthetized and perfused with 10% phosphate-buffered formalin. fixed tissues were routinely embedded in paraffin, sectioned, and stained with hematoxylin and eosin. immunohistochemical detection of the jev antigens was performed as described previously [53] . rabbit polyclonal antibody against e protein was used to detect jev antigens. brains were collected from four mice in each group of dying and recovering mice at 13 days pi. the brains were homogenized and passaged in bhk cells for 2 days. viral rna was extracted from the supernatant medium of the bhk cells using qiaquick pcr purification kit (qiagen) according to manufacturer's protocol. reverse transcription was performed by using superscript iii reverse transcriptase (invitrogen) and random hexamers. pcr was performed to cover the whole genome sequence using takara ex taq dna polymerase (takara bio inc.). the cycle sequencing reaction was performed by using bigdye terminator v 3.1 cycle sequencing kit (life technologies) and the dna sequence was determined with applied biosystems 3730 dna analyzer (life technologies). serum samples were collected from infected mice. the levels in the serum were measured by using competitive enzyme immunoassay and sandwich enzyme-linked immunosorbent assay kits for corticosterone (assaypro), tnfα and il-10, il-12 (endogen) according to the manufacturer's instructions. recovery of leukocytes was performed by applying previously described methods [22, 54] . briefly, after perfusion with cold pbs, brains and thymus were removed and placed on ice in rpmi containing 5% fcs (nissui pharmaceutical co.). brains were strained and homogenized gently with a 70 µm cell strainer (bd biosciences). after washing with rpmi, the cell suspension was layered onto a 70% and 30% percoll gradient (ge healthcare bio-sciences ab) and centrifuged at 800 × g for 45 min at 23° c. the leukocytes were collected from between the 70% and 30% interface. thymocytes and splenocytes were also recovered from these mice. cells were strained with a 70 µm cell strainer (bd biosciences) and lysed with rbc lysis buffer (sigma-aldrich). after washing, cells were resuspended in rpmi medium. isolated cells were counted and kept on ice until the staining procedure. brain leukocytes were washed and blocked with rat anti-mouse cd16/32 (fc receptor) (beckman coulter) in facs buffer (pbs containing 0.1% bsa and 0.1% sodium azide). cells were stained with a mixture of different fluorescentlabeled antibodies directed at surface phenotypic markers, cd45-fitc, f4/80-pe, nk1.1-percp-cy5.5, cd4-pe-cy7, cd8-apc, cd19-alexa fluor 700, cd3e-efluor 450 (beckman coulter) and then fixed with 4% paraformaldehyde overnight. the stained cells were analyzed by galios™ flow cytometer (beckman coulter). leukocytes were recognized by characteristic size (forward scatter), granularity (side scatter) and cd45 expression. thymocytes were recognized by their characteristic size and cd4 + cd8 + double positive cells were recognized by the expression of cd4 + and cd8 + . kruskal-wallis test, and mann whitney test were used for statistical analysis to assess the significant differences of viral loads, expression levels of cytokines, and numbers of leukocytes. gehan-breslow-wilcoxon test was performed to assess the survival curves of jev-infected mice groups. p value <0.05 was considered statistically significant. in this study, we used inbred b6 mice to minimize the influence of the genetic background of individuals. subcutaneous infection with jaoars982 did not lead to a normal dose dependent curve of mortality ( figure 1a ). the mortality rate was not significantly increased when challenge doses ranged from 10 2 to 10 6 pfu per mouse, although the infectivity in the mice increased sequentially ( figure 1a ). on the other hand, jath160 infection exhibited a dose dependent mortality curve and the infectivity in the mice was consistent with the mortality ( figure 1a ). these observations indicate that individual jaoars982-infected mice exhibit a variable prognosis independent of virus challenge dose, whereas all jath160-infected mice died. because a virus challenge dose of 10 4 pfu of either jaoars982 or jath160 induced 100% infectivity ( figure 1a ), this dose was used for all further investigations to compare the pathogenesis. jaoars982-infected mice did not start to die until 13 days pi and the mean survival time (mst) was 15.5 ± 2.56 days ( figure 1b ). mice that died exhibited generalized clinical signs involving slowness in movement, ataxia, piloerection and anorexia. continuous weight loss was observed in mice that died, whereas survivors regained weight from 13 to 15 days pi onwards ( figure 1c ). on the other hand, jath160-infected mice started to die at 9 days pi and all mice had died by 15 days pi ( figure 1b ) following continuous weight loss ( figure 1c ). mst was 12.8 ± 0.89 days and was significantly shorter than that of jaoars982-infected mice (mann whitney test, p=0.0173). thus, we hypothesized that the cause of fatal disease was different between jaoars982and jath160-infected mice. infectious virus was detectable in the brain cortex and thalamus at 5 days pi in both jaoars982 and jath160infected mice without significant difference in titer ( figure 1d ). however, at 9 days pi viral loads of jath160-infected mice were significantly higher than those of jaoars982-infected mice in every region of the brain cortex, thalamus, brainstem, cerebellum and spinal cord ( figure 1d ). it is important to note that viral load in the brain cortex was higher than in other regions of the cns in both jaoars982 and jath160-infected mice ( figure 1d ), indicating that the brain cortex is the main target region for jev infection. thus, we next examined the cytokine levels in the brain cortex to compare the immune responses. the levels of tnf-α, ifn-γ, il-2 and il-10, but not il-4 and il-5 were significantly higher in jath160infected mice than in jaoars982-infected mice ( figure 1e , figure s1a ). the cytokine levels of tnf-α, ifn-γ, il-2 and il-10 were very low or undetectable in mock-infected mice and in infected mice at 5 days pi ( figure 1e ). corresponding to the viral loads, histopathological examination showed that a large number of neurons displayed jev antigens and severe cuffing was observed in the brain cortex of jath160-infected mice at 9 day pi ( figure 1f ). jaoars982-infected mice also exhibited jev antigen-positive neurons and cuffing, but at lower levels than those observed in jath160-infected mice ( figure 1f ). mock-infected mice showed no jev antigen-positive neurons or inflammatory reactions ( figure 1f ). these results confirm that during the early phase of infection, jath160-infected mice developed severe encephalitis with extensive neuronal infection which contrasts with the less extensive neuronal infection induced in jaoars982-infected mice. infectious virus was either not detectable or very limited in spleens (data not shown). interestingly, the levels of tnf-α and il-2 in spleens were up-regulated in jaoars982-infected mice at 5 and 9 days pi, however, they were not elevated in jath160-infected mice ( figure s1b ). the levels of ifn-γ, il-4, il-5 and il-10 were not significantly different between jaoars982 and jath160-infected mice ( figure s1b ). these observations suggest that i) inflammatory responses in peripheral organs were different from those in the cns, and ii) jath160 infection induced no significant expression of tnf-α in the spleen. in view of the observation that individual mice displayed different disease progress when infected with jaoars982 under identical conditions, we attempted to identify specific factors relating to disease severity outcome. initially, we mice were recorded for 21 days and no mice died after 21 days. infectivity was determined by anti-jev igg antibody seroconversion for more than 1:1000 of igg elisa titer. (b and c) b6 mice were subcutaneously infected with 10 4 pfu of jaoars982 (n=30) and jath160 (n=15). survival curves p: gehan-breslow-wilcoxon test. (c) the averages ratio of weight change of living mice at the time points compared with those of day 0 following subcutaneous infections with 10 4 pfu of jaoars982 (n=30) and jath160 (n=15). error bars represent the standard deviations. (d) viral loads in distinct regions of the cns following subcutaneous infections with 10 4 pfu of jaoars982 (day 5: n=5, day 9: n=15) and jath160 (day 5: n=5, day 9: n=8). p: mann whitney test. (e) mrna levels of tnf-α, ifnγ, il-2 and il-10 quantified by real-time pcr in the brain cortex of b6 mice infected with 10 4 pfu of jaoars982 (day 5: n=5, day 9: n=12), jath160 (day 5: n=5, day 9: n=5) and mock (n=8). p: mann whitney test. (f) histopathological features of brain cortex in b6 mice infected with 10 4 pfu of jaoars982 and jath160 at 9 days pi. jev antigens were detected using e-protein specific jev antibody (insets). each experiment represents six and four mice infected with jaoars982 and jath160, respectively. jaoars982infected mice showed slight inflammatory infiltration in meninges. in brain cortex, a few degenerated cells were presented (arrow) and were virus antigen-positive cells. in jath160-infected mice, severe inflammatory reactions were seen in meninges and perivascular area (asterisks). many virus antigen-positive cells were detected in degenerated neuronal cells of the cortex (arrows). doi: 10.1371/journal.pone.0071643.g001 attempted to discriminate severe and mild disease groups during the observation period by following the progression of weight change of individual mice. we discriminated dying and recovering mice based on whether they showed less than 0.75 or more than 0.90 of the weight ratio at 13 days pi ( figure 1c ). it was difficult to predict if mice would survive or die between 0.75 and 0.9 of the designated weight ratio, because within this window both dead mice and survivors were recorded. having established this defining parameter between severe and mild disease groups, we then attempted to examine specific factors relating to disease severity. we initially considered the possibility that the divergence of disease severity might be due to the selection of quasispecies variants from the jaoars982 virus population in the cns. accordingly, we compared the virus sequences recovered from the brains of dying and recovering mice (figure 2a) . however, no specific virus sequence differences were detected in viruses from either the severe or mild disease severity groups (dsg) (figure 2a) . furthermore, recovered viruses from either severe or mild dsg exhibited similar mortality patterns to those of the parent jaoars982 virus ( figure 2b) . noticeably, the viruses recovered from severe dsg mice did not induce 100% lethal infection in subsequent mouse virulence tests ( figure 2b ). these results indicate that quasispecies variants of jaoars982 did not contribute to the divergence of disease progression observed in all experiments with this virus; thus, other factors such as host response seem most likely to be the determinants of disease severity. we next compared the viral loads in the cns between severe and mild dsg in jaoars982-infected mice at 13 days pi. viral loads in brain cortex, thalamus and brainstem but not cerebellum and spinal cord were significantly higher in severe dsg mice than in mild dsg ( figure 3a ). however, in the brain cortex, the variance of virus titer in the mild dsg mice ranged from the minimal detection limit to 10 8 pfu/g of tissue, whereas all mice exhibited more than 10 6 pfu/g of tissue in the severe dsg ( figure 3a ). these results imply that 45.8% (11/24) of mice in the mild dsg produced high viral loads similar to those in the severe dsg. thus, it is likely that high viral infection alone is not a critical determinant of severe disease and additional factors contribute to the fatal encephalitis. therefore, to compare the specific immune responses in severe cases, we further subdivided the mice into three subgroups, severe group (sg), mild group with high viral load (>10 6 pfu/g of tissue) (mhg) or low viral load (<10 6 pfu/g of tissue) (mlg), and compared their cytokine levels in the brain cortex ( figure 3b ). all groups exhibited increased levels of inflammatory cytokines of tnf-α, il-10, ifn-γ and il-2, but not il-4 and il-5 in the brain cortex compared with the uninfected group (ug) ( figure 3b) . interestingly, the level of tnf-α in the sg was significantly increased when compared with those in the mhg and mlg ( figure 3b ). the level of il-10 in the sg was also significantly higher than in the mlg ( figure 3b ). although the difference was not significant, the level tended to be higher than that recorded in the mhg ( figure 3b ). on the other hand, ifn-γ did not show significant differences between the three groups, and il-2 levels in the sg were lower than in the mhg ( figure 3b ). histopathological examination revealed inflammatory infiltration with mononuclear cells in the brain cortex of both severe and mild cases of jaoars982-infected mice ( figure 4a to c). in severe cases, jev antigens were detected in neurons, and degenerated neurons were observed in a wide area of the brain cortex and medulla ( figure 4a ). on the other hand, in mild cases, there was variation of pathological features in some jaoars982-infected mice. other mild cases showed neuronal infections similar to those observed in severe cases but there was little neuronal degeneration in the brain cortex ( figure 4b ). other mice exhibited very limited evidence of neuronal infection and neuronal degeneration ( figure 4c ). mock-infected mice showed none of these pathological changes ( figure 4d ). in summary, both neuronal infection and cns pathology were associated with severe disease outcome. in particular, increased levels of tnf-α and il-10 in the brains appeared to be associated with severe disease, although it was not clear whether the increased levels were the cause or result of severe disease. interestingly, the levels of tnf-α in the spleens of sg and ug mice were similar and relatively low, whereas the levels of both mhg and mlg mice were significantly higher ( figure s2a ). il-10 levels in sg, mhg and mlg mice were high compared with those in ug mice. no significant differences were observed between the sg, mhg and mlg ( figure s2a ). on the other hand, the ifn-γ level in sg mice was lower than those recorded in the ug, mhg and mlg ( figure s2a ). there were no significant differences of il-2, il-4 and il-5 levels between ug, sg, mhg and mlg mice ( figure s2a ). some mice in the sg showed high levels of tnf-α in the serum, although no significant difference was observed when compared with other groups ( figure s2b ). il-10 in the serum of sg mice was significantly increased compared with ug and mhg mice ( figure s2b ). corticosterone levels in the serum were also significantly increased in sg mice compared with other groups ( figure s2b ). corticosterone, a major glucocorticoid hormone, is a strong immunosuppressant and is elevated under stress response conditions such as those preceding death [55, 56] . furthermore, severe cases resulting from infection with jaoars982 exhibited a significant reduction of cd4 + and cd8 + doubly-positive cells in the thymus ( figure s2c ). thymic depletion and body weight loss are the main features of the systemic stress response [55, 56] . these observations therefore suggest that sg mice exhibited a severe systemic stress response accompanied by immune suppression. thus, the roles of inflammatory cytokines appeared to be different in peripheral and cns tissues. to investigate in more detail, the role of tnf-α and il-10 during jev infection, we infected tnf-α ko and il-10 ko b6 comparison of viral genome sequences (nucleotide and corresponding amino acid -in parentheses) between recovered viruses from brains of severe (s14, s15, s18 and s19) and mild (m4, m10, m13 and m24) cases of jaoars982-infected b6 mice. (b) weight changes of b6 mice infected with 10 4 pfu of recovered viruses from severe (s14, s15, s18 and s19) and mild (m17 replaced to m4, m10, m13 and m24) cases. five mice in each group were inoculated subcutaneously and observed for 21 days. closed and open symbols identify mice that died or survived, respectively, during observation period. mice with jaoars982, and observed the disease courses compared with those of infected fully immunocompetent b6 mice. unexpectedly, the mortality rates of tnf-α ko and il-10 ko mice were increased compared with those of wt mice (77.3%, 43.2% and 26.7%, respectively) ( figure 5a ). msts of fatal cases in tnf-α ko and il-10 ko mice (12.6 ± 1.05 and 11.5 ± 0.80 days) were significantly shorter than those of wt mice (15.5 ± 2.14 days) (p=0.0087 and p=0.0039, respectively). consequently, these observations indicate that tnf-α and il-10 protect significant proportions of mice from fatal infection by pathogenic jaoars982 virus. importantly, tnf-α had a particularly pronounced protective effect. following inoculation with jaoars982 virus, there were no significant differences of infectious viral loads in the brain cortex between wt, il-10 ko and tnf-α ko at 5, 9 and 11 days pi ( figure 5b ). however within individual mice in each mouse group the range of viral infectivity varied from the lowest detection limit to 10 9 pfu/g of tissue at 9 and 11 days pi ( figure 5b) . it therefore appears that the increased mortality in il-10 ko and tnf-α ko mouse was not simply due to the increased viral loads in the brains, but other factors must also have contributed to the fatal disease in these ko mice. it was difficult to distinguish between dying and recovering mice on the basis of their clinical signs at 9 to 11 days pi. however, high viral loads in the brain cortex appeared to be necessary for fatal outcome. thus, we compared the inflammatory responses in the brain cortex of mice that contained high viral loads with more than 10 6 pfu per g of tissue ( figure 5b) . surprisingly, tnf-α ko mice exhibited significantly enhanced levels of ifn-γ, il-1β, il-2, il-4, il-5 and il-6 in the brain when compared with the wt and il-10 ko mice at 9 and 11 days pi ( figure 5c and figure s3a ). furthermore, at 5 days pi, the levels of il-4 and il-5 were higher in tnf-α ko ( figure s3b ). il-10 ko mice also exhibited the increased levels of ifnγ, il-1β, il-2, il-4 and il-6 compared with those of wt mice at 9 days pi, although the differences were smaller than those between tnf-α ko and wt mice ( figure 5c ). uninfected mice showed some significant differences of cytokine levels between the three groups, but the levels were very low compared with infected mice and were not significant ( figure s3c) . furthermore, the levels of perforin, granzyme b and fasl at 9 days pi, and granzyme a at 11 days pi were significantly increased in tnf-α ko mice compared with those of wt mice ( figure 5d and figure s3d ), whereas il-10 ko mice exhibited the increased level of perforin at 9 days pi ( figure 5d ). these cytokines are associated with immune-mediated neurodegeneration. these findings suggest that immunopathological effects in the cns contribute to the accelerated mortality in tnf-α ko mice infected with jaoars982. thus, il-10 and in particular tnf-α mediate immunomodulatory effects against such inflammatory responses. histopathological examination of tnf-α ko mice revealed severe neuronal loss in extensive areas of brain cortex when compared with wt mice (figure 6a and b) . however, the proportion of infiltrated cells involving leukocytes (cd45), t cells (cd3, cd4 or cd8), b cells (cd19), nk cells (nk1.1) and macrophages (f4/80) did not appear to differ significantly between tnf-α ko and wt mice ( figure 6c ). these observations suggest that the increased levels of cytokines in tnf-α ko mice were due to qualitative differences of their expression in inflammatory cells, rather than quantitative increases of infiltrating cytokine producing cells. in the spleens of mock-infected mice, there were no significant differences of ifn-γ levels between wt, il-10 and tnf-α ko mice ( figure s4a ). however, following jaoars982 infection the levels of ifn-γ in tnf-α ko mice were significantly increased compared with those of wt mice at 5 and 9 days pi ( figure s4b and c) . on the other hand, il-2 and il-4 levels in tnf-α ko mice were significantly higher than those of wt and il-10 ko mice during mock infection ( figure s4a ) and following jaoars982 infections ( figure s4b to d) . also, the level of il-5 in tnf-α ko was decreased compared with wt and il-10 ko mice at 5 days pi ( figure s4b) . these observations suggest that the patterns of cytokine levels observed in spleens were different from those of the brain and therefore that peripheral responses are unlikely to contribute to the increased disease severity in tnf-α ko mice. although the high virulence of jath160 is probably attributable to viral factors, we attempted to assess whether or not tnf-α might also contribute significantly to the pathogenicity observed following infection with jath160 virus. accordingly, mice were inoculated with jath160 virus at a challenge dose of 10 4 pfu per mouse, all wt, il-10 ko and tnf-α ko mouse groups died. however, tnf-α ko mice presented with significantly shorter survival times than b6 wt mice (9.57±1.19 days and 12.8±0.89 days, respectively, mann whitney test: p=0.0002) ( figure 7a ). it is important to note that viral loads in the brains were not significantly different for either wt, tnf-α ko or il-10 ko mice at 5 and 7 days pi ( figure 7b ). tnf-α ko mice exhibited significantly increased levels of ifn-γ and il-5 in the brains compared with wt and/or il-10 ko mice at 5 and 7 days pi following jath160 inoculation ( figure 7c and d) . however, levels of il-2 and il-4 in the brains were not increased when wt and tnf-α ko groups at 5 and or 7 days pi were compared ( figure 7c ). moreover, levels of perforin, granzyme a, granzyme b and fasl were not increased in tnf-α ko when compared with wt mice at 7 days pi. however, histopathological data showed that tnf-α ko mice presented with severe acute necrotic changes in the brain cortex compared which was not the case for wt and il-10 ko mice at 9 days pi ( figure 7d ). in the spleens, similar to the jaoars982 infection, the levels of ifn-γ, il-2 and il-4 in il-10 ko and tnf-α ko mice were significantly increased compared with those of wt mice at 7 days pi following jath160 infection, whereas the level of il-5 was decreased in tnf-α ko ( figure s5b) . these results suggest that the shorter survival time of jath160-infected tnf-α ko mice when compared with wt mice may be partially attributable to an immunopathological effect, whereas direct neuronal infection is likely to be the main cause of neurodegeneration in jath160-infected mice. this study provides the first evidence that tnf-α has an immunoregulatory effect on pro-inflammatory cytokines in the cns during jev infection and this results in protection from fatal disease due to infection with this virus. following jaoars982 virus infection, tnf-α ko mice exhibited significantly increased mortality rates when compared with wt mice. although it has been suggested that tnf-/-mice show developmental defects of the humoral immune system 10 4 pfu of jath160 at 9 days pi. jev antigens were detected using e-protein specific jev antibody (insets). each experiment represents four, five and six mice of wt, tnf-α ko and il-10 ko mice, respectively. the b6 wt and il-10 ko mice showed severe inflammatory reactions in the brain cortex. on other hand, the tnf-a ko mice exhibited acute necrotic changes with slight inflammatory reactions in the brain cortex. including a lack of primary b cell follicles [38, 57, 58] , tnf-α ko mice that we used in this study did not show significant depletion in the anti-jev igm response (data not shown) or in cytokine expression ( figure s4 ). in addition, no significant increases of viral propagation were observed in the peripheral and cns tissues. interestingly, there were no significant differences of viral load in the cns between wt and tnf-α ko mice. however, high inflammatory responses were observed in the cns of tnf-α ko mice. in particular, perforin, granzyme a, granzyme b and fasl, which are known to be associated with immune-mediated neurodegeneration, were significantly increased in the brains of tnf-α ko mice when compared with those of wt mice. these observations suggest that immunopathological effects contribute to the severe neuronal degeneration and fatal disease in tnf-α ko mice. il-10 ko mice also exhibited increased mortality and upregulated levels of inflammatory cytokines in the cns compared with wt mice and in common with tnf-α ko mice, there were no significant differences in viral loads. however, it is important to note that the levels of inflammatory cytokines of tnf-α ko mice and the resultant mortality were dramatically higher than those observed in il-10 ko mice. il-10 has an immunoregulatory function on various cells in the innate immune system including cytotoxic and helper t cells, nk cells and b cells [59] . il-10 signaling has a negative role in immunity against wnv infection [45] . it is also known that tnf-α is a critical regulatory cytokine exerting homeostatic and pathological effects in the csf [60] . therefore, our data imply that tnf-α mediates greater efficacy of immunoregulatory function during jev infection. in preparatory studies of jev infection, we attempted to inject tnf-α intravenously or intracerebrally after jev inoculation to examine whether or not this improved the disease outcome. however, there was no significant improvement in the condition of the mice or protection from death. administration of anti-mouse tnf-α antibody also showed no improvement of disease outcome. although we cannot totally exclude the possibility that failure of tnf-α administration to improve disease outcome, may have been the result of the technical design of the experiments, different responses of tnf-α in the cns when compared with peripheral tissues may partly explain our observations. therefore, further investigation of the immunoregulatory mechanism of tnf-α in vivo and in vitro will be required to understand the basis of the immunopathological effects observed during jev infection. in this study, we focused on the variation of disease severity in mice following jaoars982 infection to detect specific responses that may be associated with severe disease. thus, we discriminated severe and mild cases of mice by their weight ratio at 13 days pi. using this simple and effective approach, we had previously shown that specific immune responses were detected in severe disease cases when compared with mild cases following tbev infection [48] . loss of appetite probably caused the weight loss due to decreased food and water intake. however, undernourishment did not appear to be the simple cause of death, because our preliminary data showed that an infusion of glucose solution to compensate for weight loss did not prevent fatal disease. we first considered whether or not viral quasispecies could account for the diversity of disease outcome. our results did not support this possibility. we also identified specific immune responses including tnf-α and il-10 up-regulation in the brains of severe cases when compared with mild cases. in human cases, an increased level of tnf-α in the csf appears to correlate with je disease severity [8] . therefore, this jevinfected mouse model appears to be a reproducible model of severe je disease in human cases. furthermore, from the results of increased fatality in tnf-α ko mice, we propose that increased levels of tnf-α in the brains of severe cases in wt mice were probably produced in response to the disease severity, to alleviate the pathological impact of the encephalitis. it has been reported that immunopathological effects do contribute to flavivirus encephalitis [27] . cytolytc leukocytes such as cd8 + t cells induce cytopathology during some encephalitic flavivirus infections [28] [29] [30] and these leukocytes kill virus-infected cells using two distinct mechanisms viz., fas and granular exocytosis which involve perforin, granzyme a and b [61] [62] [63] [64] . in tnf-α ko mice, we showed that the increased levels of inflammatory cytokines including fas and the granular exocytosis correlated with severe encephalitis and fatal outcome. however, in wt mice, the apparent immunopathological features were not observed in dying mice 13 days pi. although it was difficult to identify dying and surviving mice before 13 days pi by their clinical signs, jaoars982-infected mice showed varying levels of fas and granular exocytosis in the brains at 11 days pi and some of them exhibited similar or higher levels compared with the tnfα ko mice ( figure s3d ). thus, fatal cases may exhibit severe encephalitis caused by immunopathological responses during the early phase of infection and thereafter severe clinical signs may appear in some mice. jaoars982-infected mice exhibited a variety of immune responses and different prognoses in individual mice. however, it was not clear how the immune response differentiated between dying and recovering mice. in order to explain these variable immune responses, we previously showed that specific t cell receptor (tcr) repertoires were present in dying mice during tbev infection [65] . furthermore, we also showed that specific tcr repertoires were detected in the dying mice compared with the recovering mice following jaoars982 infection (shirai, et al., unpublished results). these data raise the possibility that there may be a variety of specific t cell clones effecting either protective or pathogenetic functions in dying and recovering mice. dose independent mortality induced by encephalitic flaviviruses has been recognized but has been an unresolved problem since the 1940's [27, 47] . recently, it was suggested that induction of more vigorous innate immune responses might control early virus dissemination following increasing infectious challenge doses of virus [6, 26, 66] . we have also recently discovered that interferon alpha receptor knockout induces dose-dependent mortality following extraneural infection with jaoars982 (hayasaka, et al., unpublished results) . in addition, we previously reported that late death following tbev infection appears to be a key feature of dose independent mortality within the encephalitic flaviviruses [48] . in the current study, jaoars982-infected mice also displayed increased times to death and the variation of acquired immune responses which either showed protective or pathological effects, appeared to be correlated with severe disease. therefore, we propose that in addition to innate immune response, subsequent acquired immune responses, which varied contingently in individuals, appeared to be a determining factor associated with dose-independent mortality. interestingly, jath160-infected mice did not show increased levels of tnf-α in the spleen at 5 and 9 days pi. however, it is uncertain if the low level of tnf-α in the spleen directly contributed to the subsequent cns infection and the neuropathogenesis during jath160 infection. it is important to note that there were 17 amino acid differences in the genomic sequences of jath160 and jaoars982. therefore, it will be important to determine whether or not specific amino acid substitutions can influence tnf-α expression and thus contribute to the pathogenesis of the lethal process during jath160 infection. in conclusion, jath160-infected mice developed severe encephalitis and all mice died due to severe infections of the cns (figure 8 ). on the other hand, jaoars982-infected mice exhibited varying degrees of encephalitis and different prognoses (figure 8 ). we therefore propose that fatal outcome is attributable both to immunopathological changes in addition to high levels of cns infection. at this stage we cannot define the critical factors involved in the immunopathogenetic process ( figure 8 ). furthermore, up-regulation of tnf-α and il-10 in the brain appear to be important determinants of the pathogenetic response ( figure 8 ). clearly, further elucidation of the contribution of immunopathology and the suppressive impact of tnf-α, are important priorities to enable the development of effective treatment strategies for je. figure s1 . cytokine levels of spleen in b6 mice infected with jaoars982 and jath160. (a) mrna levels of il-4 and il-5 quantified by real-time pcr in the brain cortex of b6 mice infected with 10 4 pfu of jaoars982 (day 5: n=5, day 9: n=12), jath160 (day 5: n=5, day 9: n=5) and mock (n=8). p: mann whitney test. (b) mrna levels of tnf-α, ifnγ, il-2, il-10, il-4 and il-5 quantified by real-time pcr in the spleen of b6 mice infected with 10 4 pfu of jaoars982 (day 5: n=5, day 9: n=12), jath160 (day 5: n=5, day 9: n=5) and mock (n=8). p: mann whitney test. (tif) figure s2 . cytokine levels of spleen in severe and mild cases of jaoars982-infected mice. (a) mrna levels of tnf-α, il-10, ifnγ, il-2, il-4 and il-5 quantified by real-time pcr in the brain cortex of jaoars982-infected b6 mice at 13 days pi. uninfected group: u (n=8), severe group: s (n=8), mild group with high viral load of >10 6 pfu/g of brain tissue: mh (n=11), mild group with low viral load of <10 6 pfu/g of brain tissue: ml (n=13). p: kruskal-wallis test, p: mann whitney test. (b) the levels of il-10, tnf-α and corticosterone measured by enzyme-linked immunosorbent assay in the plasma of jaoars982-infected b6 mice at 13 days pi uninfected group (u group, n=6), severe group (s group, n=6), mild group with high viral load of >10 6 pfu/g of brain tissue (mh group, n=7), mild group with low viral load of <10 6 pfu/g of brain tissue (ml group, n=6). p: kruskal-wallis test, p: mann whitney test. (c) cd4 and cd8 expressions of thymocytes from mock, mild and severe cases of jaoars982-infected b6 mice at 13 days pi. each experiment represents four and fifteen mice of severe and mild cases, respectively. 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mice deficient for tumor necrosis factor and its 55-kda receptor interleukin-10 and the interleukin-10 receptor tumor necrosis factor-alpha and the roles it plays in homeostatic and degenerative processes within the central nervous system fas and perforin pathways as major mechanisms of t cell-mediated cytotoxicity two distinct pathways of specific killing revealed by perforin mutant cytotoxic t lymphocytes molecular mechanisms of lymphocyte-mediated cytotoxicity and their role in immunological protection and pathogenesis in vivo granzymes are the essential downstream effector molecules for the control of primary virus infections by cytolytic leukocytes tcell clones expressing different t-cell receptors accumulate in the brains of dying and surviving mice after peripheral infection with far eastern strain of tick-borne encephalitis virus chimeric live, attenuated vaccine against japanese encephalitis (chimerivax-je): phase 2 clinical trials for safety and immunogenicity, effect of vaccine dose and schedule, and memory response to challenge with inactivated japanese encephalitis antigen we thank tomohiko takasaki key: cord-003602-wtestt8i authors: jung, eunok; de los reyes v, aurelio a.; pumares, kurt jan a.; kim, yangjin title: strategies in regulating glioblastoma signaling pathways and anti-invasion therapy date: 2019-04-22 journal: plos one doi: 10.1371/journal.pone.0215547 sha: doc_id: 3602 cord_uid: wtestt8i glioblastoma multiforme is one of the most invasive type of glial tumors, which rapidly grows and commonly spreads into nearby brain tissue. it is a devastating brain cancer that often results in death within approximately 12 to 15 months after diagnosis. in this work, optimal control theory was applied to regulate intracellular signaling pathways of mir-451–ampk–mtor–cell cycle dynamics via glucose and drug intravenous administration infusions. glucose level is controlled to activate mir-451 in the up-stream pathway of the model. a potential drug blocking the inhibitory pathway of mtor by ampk complex is incorporated to explore regulation of the down-stream pathway to the cell cycle. both mir-451 and mtor levels are up-regulated inducing cell proliferation and reducing invasion in the neighboring tissues. concomitant and alternating glucose and drug infusions are explored under various circumstances to predict best clinical outcomes with least administration costs. glioblastoma multiforme (gbm) is the most common and the most aggressive type of brain cancer. the median length of survival time is approximately 12 to 15 months following diagnosis. gbm is characterized by anaplasia, nuclear atypia, cellular pleomorphism, mitotic activity, and more importantly, alternating phases of rapid proliferation and aggressive invasion into the surrounding brain tissue. this leads to an inevitably critical recurrence even after the surgical resection of the main tumor mass [1, 2] . the mainstay of treatment for gbm is surgery, followed by radiotherapy and chemotherapy. despite advances in these approaches, glioma cells can still invade the neighboring tissues beyond detection leading to tumor recurrence. high probability of main treatment failure also encourages researchers to investigate the use of innovative treatments when the first line of therapy has failed, in order to improve clinical outcomes [3] . in the tumor microenvironment (tme), glioma cells encounter many challenges including hypoxia, acidity, and limited nutrient availability. to maintain rapid growth, tumor cells need to adapt to these biochemical changes and modify their metabolic activity by increasing glycolysis even in the presence of oxygen. this process is called the warburg effect which requires consuming considerable amounts of glucose [4] . the tricarboxylic acid cycle, or krebs cycle, plays an important role in the breakdown of organic fuel molecules and the survival in non-a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 hypoxic normal differentiated cells. these molecules include glucose, fatty acids, and some amino acids. while differentiated cells favor this type of metabolism, which is very efficient in terms of atp production, tumor cells adopt the inefficient aerobic glycolysis producing relatively large amounts of waste product in the form of lactic acid [5] . this may provide cancer cells the advantage of not having to depend on oxygen as an energy source especially in a hostile tumor microenvironment, thus leading to longer survival [6] . inhibition of glycolysis may also prevent drug resistance thus a better understanding of this metabolic pathway may lead to better treatment options and clinical outcomes [7] . developing strategies of metabolic adaptation, angiogenesis, and migration is critical for cancer cells in order to survive metabolic stress and ensure enough nutrient supply as tumor mass accumulates where glucose supply may fluctuate due to heterogeneous biochemical and biophysical conditions [8] . therefore, adequate cellular responses to glucose withdrawal are critical for cancer cell survival. cancer cells then activate the 5 0 -adenosine monophosphate activated protein kinase (ampk) pathway under metabolic stress. it is the master cellular sensor of energy availability which enhances glucose uptake and conserve energy, thus avoiding cell death [9] . micrornas, also abbreviated as mirna, are approximately 22 nucleotide single-stranded non-coding ribonucleic acids (rnas) that are known to regulate gene expression [10] . dysregulation of microrna expression has been linked to oncogenic and tumor suppressor activities in several types of cancer, including gbm [11, 12] , where altered mirna expression contributes to tumorigenesis [13, 14] . godlewski et al. [8] identified an interesting mechanism of glioma cell migration and proliferation wherein a particular microrna, mir-451, and its counterpart, ampk complex (cab39/ lkb1/strad/ampk), determine whether the cell favors growth at the expense of invasion or conversely. moreover, they also identified a potential feedback loop between lkb1 and mir-451 allowing for a sustained and robust response to glucose withdrawal [15] . it was found out that (i) under high (normal) glucose conditions, up-regulation of mir-451 leads to the down-regulation of ampk complex, which then leads to elevated proliferation and decreased migration of glioma cells and (ii) glucose withdrawal induces down-regulation of mir-451 and up-regulation of ampk, which promotes cell migration with reduced proliferation. the mathematical models developed by kim et al. [14, 16, 17] describe the effects of the mir-451-ampk core control system on cell proliferation and migration in glioblastoma. it explains the response of mir-451 to high and low glucose levels as well as the mutual antagonism between mir-451 and ampk complex concentrations. kim et al. [18] then extended the model to include the dynamics of mammalian target of rapamycin (mtor), which is a protein kinase that links with other proteins as well as to the cell cycle dynamics, to form the mir-451-ampk-mtor core control system. this mutual antagonism between mir-451 and ampk, which was predicted in these mathematical models [14, [16] [17] [18] , was confirmed by recent experiments. for example, ansari et al. [19] recently found that (i) the mir-451 transcription in gbm cells is induced by unrestricted activity of its transcription factor oct1 (official gene symbol pou2f1) in the presence of abundant glucose, resulting in ampk inhibition through direct targeting of cab39 in the lkb1 complex; and (ii) the mir-451 level is inhibited through the phosphorylation and inactivation of oct1 at s335 by ampk in response to glucose depletion-induced metabolic stress, leading to a reciprocal negative feedback loop between mir-451 and ampk. in this case, suppression of mir-451 in turn leads to sustained ampk activities and a robust response to glucose withdrawal in gbm cells. the multiscale mathematical models [14, [16] [17] [18] also predict the growth-invasion cycling patterns of glioma cells in response to fluctuating glucose uptake in the tumor microenvironment. the core control system predicts bistability and hysteresis bifurcation when delayed down-regulation of mir-451 activities along certain molecular pathways would induce glioma cells to stay longer in the proliferative phase despite relatively low glucose concentrations, making this mechanism a therapeutic target. the cell cycle represents an integrated series of events that regulates complex processes including cell proliferation, cell division and dna replication, regulated by a complex hierarchy of genetic and metabolic networks which involves several transition states of varied lengths and checkpoints [20] . the stages of the cell cycle are as follows: (i) synthesis phase (s), a period where dna replication occurs; (ii) gap phase 2 (g2), during which proteins required for mitosis are produced; (iii) mitosis phase (m), a period where chromatin condensation, nuclear envelope breakdown (nebd), chromatid separation, and cytokinesis happens; (iv) gap phase 1 (g1), in which genes necessary for dna replication are activated and the protein agents of s phase progression are accumulated; and (v) resting phase (g0), a state in which cells can exit the cell cycle and enter a phase of quiescence or relative inactivity [21] . the progression of mammalian cell cycle is tightly regulated by coordinated activation of cyclin-dependent kinases (cdks) family [22] . the cdks are positively regulated by cyclins and negatively by cdk inhibitors (cdkis) such as the proteins p15, p16, p21 and p27. in cancer cells, cyclins are over-expressed while cdkis are under-expressed which results in the dysregulation of the cell cycle, and promoting uncontrolled cell growth [20] . tyson and novak [23, 24] identified that the transition between two stable steady states, g1 and s-g2-m cell-cycle phases, are described using the kinetic relations of the model that is controlled by changes in cell mass. a standard gbm treatment is surgery followed by chemotherapy and radiotherapy. however, even under best circumstances, the mean survival of this disease is about a year. poor outcomes of standard care treatments are due to the topographically diffuse nature of the disease [25] . by the time of diagnosis, typical gbm cells may have widely spread throughout the brain tissue [26] [27] [28] , increasing the potential of recurrence. thus, annihilation of distant tumor satellites is implausible despite surgically removing all the essential tumor seen on enhanced mri scan [29] . knowing the exact margins of a tumor mass in real patients is indeed a daunting task. in this study, it is assumed that a major tumour mass has been surgically removed and that the infiltrative tumor cells are near the surgical site. the objective is to prevent the glioma cells from further diffusing into the surrounding brain tissue. localization approach will be utilized, that is, glioma cell invasion will be blocked keeping them in a proliferative phase while also attempting to limit excessive growth before a second surgery [17] . our analytical tool is primarily based on the framework of optimal control theory, which has been successfully used to make informed decisions involving biological models such as optimal treatment strategies in human immunodeficiency virus (hiv) models [30] [31] [32] [33] , tuberculosis [34] [35] [36] , and cardiopulmonary resuscitation (cpr) techniques [37, 38] . optimal control theory is also applied to the mir-451-ampk core control system to determine the intravenous glucose and/or drug infusion protocols with least possible cost under various circumstances in de los reyes, et al. [39] . recently, kim et al. [40] developed an intracellular signaling pathway model that extends the mir-451-ampk-mtor core control system including the cell cycle dynamics. in this work, a potential control problem is formulated in order to maintain high levels of mir-451 and mtor (low levels of ampk) inducing cell proliferation prohibiting cell motility and invasion to the neighboring tissues. with glucose levels as a key regulator of mir-451 activity which also activates mtor in the downstream signaling pathway, glucose intravenous infusion is considered to up-regulate mir-451 and mtor concentrations above certain threshold values. in addition, a drug suppressing the inhibitory effect of mtor by the ampk complex is incorporated. this drug can be administered concomitantly or alternately with glucose as a secondary intravenous infusion. the controls are then given by dose rates of glucose and drug intravenous administrations regulating upstream and downstream signaling pathway, respectively. solution of the optimal control problem aims to determine infusion protocols with minimal glucose and drug amount, and least administration costs. hence, glucose and drug levels are regulated to prevent rapid tumour growth, hyperglycemia, and further drug complications. the results propose plausible glucose and drug intravenous infusion controls which indicate the time of administration, frequency, number of administrations, and dosages of glucose and drug per infusion. in the current study, we will first present the mir-451-ampk-mtor-cell cycle intracellular signalling pathway developed by kim et al. [40] . a drug module is incorporated to up-regulate mtor activities inducing cell proliferation. then an optimal control problem is formulated with the goal of activating mir-451 and mtor levels through glucose and drug intravenous infusions. two different infusion protocols will be explored, namely, concomitant and alternating glucose and drug intravenous administrations. optimal solutions for two strategies are presented and results on frequency, dosage per infusion, total glucose and drug amount, and relative cost incurred in the administrations are compared. the conclusion section discusses and summarizes the optimal control results, and provides outlook for future research directions. in this section, we present the basic components of intracellular signalling pathway of tumor cell containing the core control mir-451-ampk-mtor and cell cycle pathway developed in kim et al. [40] , incorporating a drug which blocks the inhibitory pathway of mtor by ampk complex. the core control model identifies a key mechanism which determines the molecular switches between the proliferative and migratory phases in response to fluctuating glucose and drug levels. the simplified signaling pathways consists of five key determinants of the intracellular structure, namely, glucose level g, mir-451 level m, ampk complex activity a, mtor concentration r, and drug level d. the intracellular cell cycle dynamics are developed by tyson and novak [23, 24] including only the essential interactions for its regulation and control. the model captures the kinetics of chemical processes within the cell such as production, destruction, and different molecule interactions. the transition between two main steady states, g1 and s-g2-m phases, of the cell cycle are described using the kinetic relations of the model that is controlled by changes in cell mass. . also included are the effects of the changes in oxygen dynamics at the macroscopic level through the activation and inactivation of hif-1 α. this results in changes in cell cycle length. in addition, the cell cycle inhibitory effect of p21 or p27 genes of hif-1 α is incorporated. kim et al. [40] proposed to link both the mir-451-ampk-mtor control system and the cell cycle dynamics to provide a mechanism driving the cell cycle to undergo the quiescent stage g0-phase depending on the concentration level of mtor. fig 1a depicts the detailed schematic diagram of the intracellular signalling networks including mir-451, ampk complex, mtor, and key players in the cell cycle module (cycb, cdh1, p55cdct, p55cdca, plk1). kinetic interpretation of arrows and hammerheads represent induction and inhibition in the signaling network, respectively. the dimensionless diagram of the core control mir-451, ampk complex, and mtor linking to the cell cycle is depicted in fig 1b. it should be noted that u g and u d are the sources of glucose and drug with decay rates μ g and μ d , respectively, which can be controlled exogenously. s 1 and s 2 are signaling sources to ampk complex and mtor, respectively, while α, β and γ are inhibition strengths, and ϕ denotes decay. it has been shown that high (normal) glucose concentration yields over expression of mir-451 levels (down-regulation of ampk complex and up-regulation of mtor) leading to elevated cell proliferation and reduced migration, while low glucose levels leads to down-regulation of mir-451 activities (up-regulation of ampk complex and down-regulation of mtor) reducing cell proliferation and inducing migration into the surrounding brain tissue [8, 15, 18, 40] . the effect of various glucose levels in the regulation of the core control is depicted in fig 2. kim et al. [14] developed a core control system of glioma cell migration and proliferation by using a regulatory network of key molecules (mir-451 (m), ampk (a)) as follows: as observed in experiments [8, 15, 19 ], the regulatory system in eq (1) includes a mutually antagonistic loop between mir-451 and ampk complex in response to high and low glucose levels (g). this genetic toggle switch induces a monostable and bistable system, characterizing proliferation and critical cell infiltration of gbm cells in brain [14] . in the follow-up studies [14, [16] [17] [18] including mtor (r) and cell cycle modules, this mutual antagonism and bistable system played a critical role in developing anti-invasion strategies. a mutually antagonistic feedback loop has been well-studied for its bistable properties by use of mathematical models ([42-45] and other references in [45] ). for instance, in a study on a genetic toggle switch in escherichia coli [42], a mutually inhibitory loop between repressor 1 and repressor 2 was in this study, we consider a drug d suppressing the inhibitory effect of mtor by ampk complex where the inhibition strength is given by z(d) = e −d . when d is large, γe −d is small which makes dr/dt to be large. thus, the presence of drug up-regulates mtor activity that could eventually lead to cell proliferation. fig 3 illustrates the mtor bifurcation curve. observe that increasing drug concentration shifts the hysteresis curve upwards keeping the same bistability window. hence, with higher drug levels for the same glucose concentrations, mtor is activated prompting elevated cell growth. in the mir-451-ampk-mtor core control system developed by kim and colleagues [14, [16] [17] [18] 40] , the bistability regime of main variables emerges in response to glucose levels. as it was shown in [40], the existence and size of the bistability window (|w b |) depend on other essential parameters and may disappear under perturbations of parameters. as it will be shown later (figs 4 and 5), some of key parameters are sensitive in creation or destroying the bistability while other parameters are not. after achieving the equilibrium, continuation of the curve is computed by varying the glucose concentration g and bifurcation points are detected labeled lp and cp for limit and cusp points, respectively. fig 6a illustrates the hysteresis diagram (g, r)−curves for different s 1 values. in order to obtain the cusp point (cp), fold continuation is computed starting at a limit point where two parameters g and s 1 are activated resulting to codim 2 bifurcations. both parameters (g, s 1 ) are varied along the curve where each point is a limit point for the equilibrium curve at the corresponding value of s 1 . this is depicted in fig 6b. the cusp point gives the threshold values th m , th a , and th r . a matlab software matcont was used for numerical continuation and bifurcation study of continuous and discrete dynamical systems [46] . the governing model equations for the dimensionless intracellular signaling dynamics are then described by the following ordinary differential equations d½plk1� dt ¼ k 9 ½mass s �½cycb�ð1 à ½plk1�þ à k 10 ½plk1�; strategies in regulating glioblastoma signaling pathways and anti-invasion therapy the cell cycle dynamics and the regulatory core control system are linked by a variable called pseudo-mass ([mass s ]) given by the oxygen dynamics through hif-1 α is described as and the growth rate μ is expressed as wherem is the probability density function with uniform distribution between −1 and 1. this growth rate formulation introduces cell cycle heterogeneity of length between 20 and 30 hrs to account for the natural variability between cell growth rates and to have a non-synchronous population [41] . the model parameters in system (2) are listed in tables 1 and 2 . in the current model formulation, mtor (r) is activated/inactivated in the same way as mir-451 (m). in addition, r links the core control system and the cell cycle dynamics via the pseudo-mass ([mass s ]) which influences the cell mass [mass] and the intracellular proteins (refer to eq (3)). therefore, when glucose supply is high, r is up-regulated (proliferative phase; up-regulated m, down-regulated a) and [mass s ] � [mass] yielding a typical cell cycle (see fig 7) . on the other hand, when glucose supply is low, r is down-regulated (migratory phase; down-regulated m; up-regulated a) and [mass s ] exceeds [mass] influencing the cells to enter into resting phase g0 [40] . let us assume that glucose (g) and drug (d) can be regulated through intravenous infusions and can be periodically administered. for illustrative purposes, consider 3h infusions every 12h with maximum dosage of 1 unit, and refer this as regular infusion. we then have the intracellular dynamics under regular infusion can be seen in fig 8. since glucose and drug levels oscillate, it follows that m and r also periodically fluctuates around the threshold (th m � 1.87, th r � 2.76) as can be seen in fig 8a. note that when m and r crosses the threshold value from above, respectively, peak in pseudo-mass is generated. it can thus be inferred that these peaks indicate cell migration since m and r levels are below their respective threshold values. strategies in regulating glioblastoma signaling pathways and anti-invasion therapy as shown in fig 8b, a trajectory of core control concentrations in mtor-mir-451-ampk space switches between proliferation and migration region. a closer look at the intracellular cell cycle proteins, mass, and mass s dynamics are illustrated in fig 8c. note that regular infusion significantly perturb the cell cycle dynamics stimulating several cell divisions (see mass profiles) and cell migration (see mass s ). indeed, fluctuating glucose levels in the microenvironment leads to the dichotomy of grow and go dynamics of glioblastoma cells yielding bigger tumor mass as reported in [14] , even in the presence of (fluctuating) drug concentrations to keep the cells in proliferation phase. in the current investigation, we aim to regulate the amount of glucose and drug infusions to up-regulate mir-451 and mtor above its threshold values inducing cell proliferation strategies in regulating glioblastoma signaling pathways and anti-invasion therapy avoiding migration to neighboring tissues. the modeling approach utilizes optimal control theory to identify infusion administration protocols. let u g (t) and u d (t) be the controls of the system representing dose rates of glucose and drug intravenous administrations, respectively. two different administration protocols are examined, namely, (1) concomitant, and (2) alternating glucose and drug infusions, with the following objective functionals respectively. here, m(t) and r(t) denote the level of mir-451 and mtor concentrations, respectively. parameters b 1 and b 2 are weight factors measuring the relative cost based on maximizing mir-451 m(t) and mtor r(t), and administering glucose and drug intravenous infusions over a specified time interval, respectively. the control costs are modeled by the linear combination of quadratic terms u 2 g ðtþ and u 2 d ðtþ. our objective is to find optimal infusion regimen for glucose and drug administrations, denoted by u � g ðtþ and u � d ðtþ, such that the objective functionals are satisfied, that is, where the bounds for the controls represent the limits on dose rates for glucose and drug administrations. assuming that mir-451, ampk, and mtor responses are regulated by glucose levels and influenced by drug levels, our control strategies deal with finding optimal control regimens for both glucose and drug intravenous infusions. we also note that the existence of optimal controls is guaranteed by standard results in control theory [51] . in this maximization problem, the necessary convexity of the integrand in the objective functional holds. therefore, we can proceed with applying pontryagin's maximum principle [52] . an iterative method is used for solving the optimality system which is a two-point boundary value problem having initial conditions for the state variables and terminal conditions for the adjoints. numerical simulations are obtained using a fourth-order iterative runge-kutta method. given the initial conditions and guess for the controls, state equations are solved using the forward scheme while the corresponding adjoint equations are solved using the backward scheme with the transversality conditions. the controls are updated by using a convex combination of the previous controls and the value from the characterizations. this is commonly referred as the forward-backward sweep method (fbsm) which is shown to be convergent [53] . further details on the optimal control under study can be found in the s1 appendix. (2)) was performed in order to determine which parameters have the most/least influence on the reference output (main variables). all the core control parameters were considered to assess their corresponding influence on the mir-451, ampk, and mtor activity. it was inferred that mir-451 activity will be enhanced by increasing glucose signal (g) and autocatalytic production rates (ℓ 1 , ℓ 2 ) of mir-451. these parameters were negatively correlated with ampk activity due to the mutual antagonistic mechanism between mir-451 and ampk complex. in addition, ampk activity will be up-regulated by an increase in signaling source s 1 and autocatalytic rates (ℓ 3 , ℓ 4 ) of the ampk complex in the model. it has been also noted that increasing s 1 and the inhibition strength of mtor by ampk (γ) down-regulates mtor level. in a similar fashion, s 2 is strongly positively correlated with mtor levels but little correlated with g, ℓ 1 , ℓ 2 , ℓ 3 , ℓ 4 , α, β, � 1 , � 2 at time 100. in this work sensitivity analysis is carried out to determine which model parameters have consequential effect in achieving or inhibiting bistability in the (g, r)−curve. as in [40], a method from [54] is adapted where a range for each parameter is selected and divided into n intervals of uniform length. for each parameter of interest, a partial rank the prcc values range between -1 and 1 with the sign determining whether a change in the parameter value will promote (+) or suppress (−) bistability. the following algorithm is performed: 1. assign a probability distribution d½m j;min ; m j;max � to each parameter μ j and let n be the number of samples to be selected. divide the interval [μ j,min , μ j,max ] into n equiprobable subintervals, and draw an independent sample from each subinterval. 2. assemble the lhs matrix l, wherein each row of l represents a unique combination of parameters sampled without replacement. 3. for each row of the lhs matrix l, solve for mtor r in terms of glucose g and check for bistability window w b . if bistability exists assign 1 to output variable w b in matrix y, else assign −1. 4. rank-transform the matrices l and y to obtain l r and y r . by rank-transform, we mean to replace the value by its rank when the data are sorted from lowest to highest, e.g. the smallest value is assigned a rank 1. tied values are assigned an average rank. strategies in regulating glioblastoma signaling pathways and anti-invasion therapy 5. fix a parameter μ j , which is encoded in the j th column in the matrix l r . form the following linear regression models using the data matrices l r and y r for μ j and y, respectively: compute ðm j àm j þ and ðy àŷþ, the residuals in the input parameter and the output after removing the linear effects of the other input parameters. 6. obtain the prcc of μ j using prcc m j ;y ¼ covðm j àm j ; y àŷþ ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi varðm j àm j þvarðy àŷþ 7. repeat steps 5 and 6 for the remaining parameters. it illustrates that a change in the mir-451 autocatalytic production rate (ℓ 1 ) or inhibition strength of ampk complex by mir-451 (β) enhances bistability. on the contrary, the hill-type coefficient ℓ 4 in the regulation of ampk is responsible for losing bistability. it should be noted that due to the model's structure, ℓ 1 up-regulates mir-451 which in turn increases mtor and suppress ampk activity. the parameter ℓ 4 promotes ampk complex and down-regulates mir-451 and mtor. however, other parameters ℓ 2 , α, ℓ 3 , s 1 , ℓ 5 , ℓ 6 , γ, s 2 are little sensitive in emergence or destruction of the bistability. the bistability of mir-451-ampk-mtor core control system depends on the geometric structure of its nullclines. in particular, bistability arises when m-and a-nullclines (i.e., dm dt ¼ 0 and da dt ¼ 0) intersect at three distinct points, producing one unstable and two stable steady states. the nullclines intersect three times due to their sigmoidicity influenced by catalytic rates ℓ 1 and ℓ 4 . these rates must be proportionate for a given glucose g level. otherwise, the nullclines will intersect only once. this bistability condition has been shown for a genetic toggle switch in e. coli [42] . fig 5a-5c depict the m-and a-nullclines under different g levels with various ℓ 1 and ℓ 4 values. the bifurcation curves for ℓ 1 , ℓ 4 and region of bistability for specific g levels are depicted in fig 5d. under given circumstances, ℓ 1 and ℓ 4 showed significant sensitivities in the bistability of the system. on the contrary, both ℓ 5 and ℓ 6 affect the r-nullcline only. it is illustrated in fig 5e that for several ℓ 5 and ℓ 6 values, r-and a-nullclines intersect at only one point, leading to a single steady state. in effect, m-and a-nullclines will also intersect once, producing monostability. hence, ℓ 5 and ℓ 6 show insignificant consequence in achieving bistability (see fig 4) . in the following numerical experiments, it is assumed that initially glioma cells are in growth phase (a probable occurrence of post primary tumour surgery) with m > th m , a < th a , and r > th r . it is also considered that glucose and drug can be administered exogenously as intravenous infusions. further, the weight parameters b 1 = 1 and b 2 = 1 are used as default values unless specified. in this strategy, glucose and drug infusions are administered simultaneously, in particular, both controls u g (t) and u d (t) are infused at the same. thus, this is referred to as concomitant control. here, it is assumed that the drug in consideration which blocks the inhibitory pathway of mtor by ampk complex had negligible side effects and had inconsequential chemical reactions with glucose. in order to determine an efficient strategy of concomitant infusion protocol, glucose and drug are administered concurrently at initial time t = 0 for 3 hours using the numerical scheme fbsm. infusion spontaneously increases glucose and drug concentrations as depicted in fig 9(a) and 9(b) . consequently, mir-451 and mtor levels are up-regulated while ampk complex is down-regulated as shown in fig 9c. a closer look at the control curves shows that both u g (t) and u d (t) decrease from 0 < t i < 3 suggesting that both glucose and drug dose rates should be decreased from time t i . this leads to the decrease of glucose and drug concentrations due to consumption and decay. accordingly, mir-451 and mtor levels decrease while ampk complex increases. before mir-451 crosses the threshold value th m , fbsm is again applied for the next 3 hours. this suggests a time for the next concomitant administrations in which mir-451 profiles are monitored subsequently. the procedures in tracking mir-451 profiles and applying fbsm for 3 hours are repeated over a specified time duration. thus, the number of concomitant administrations are determined. it is important to note that keeping mir-451 level above its threshold value consequently confine ampk and mtor levels, below and above its corresponding threshold values, respectively (see fig 9c) . suppose that concurrent glucose and drug administrations is not plausible due to unwanted chemical reactions. we propose another control strategy that administers glucose u g (t) and drug u d (t) infusions alternately. at initial time t = 0, glucose infusion is obtained by solving the optimal control problem using the numerical scheme fbsm for 3 hours. next, drug infusion is attained for the next 3 hours in a similar manner. hence, the controls u g (t) and u d (t) are applied one after the other. subsequently, mir-451 levels are then tracked and before it crosses the threshold value, glucose infusion u g (t) and drug infusion u d (t) are again administered alternately in a similar fashion above, over the specified duration of administration. thus, the time for the next alternating infusion is determined by tracking mir-451 levels. this strategy is referred simply as alternating control. this infusion protocol is shown in fig 11(a) and 11(b). note that glucose infusion increases mir-451 levels and drug infusion activates mtor activities keeping ampk down-regulated as depicted in fig 11c. the intracellular dynamics under alternating control is illustrated in fig 12. as shown in fig 12a, both concomitant and alternating control strategies are able to sustain elevated mir-451 and mtor levels above their threshold values and ampk levels below its threshold value. it was also shown that mtor-mir-451-ampk trajectory is restrained in the proliferation region prohibiting cell migration. further, number of cell divisions is reduced with slightly higher mass concentration before division compared to regular infusions but lower compared to constant (intermediate) high glucose concentrations. in this section, we compare the cost efficiency of the proposed strategies in terms of frequency of administration, dose per infusion, total glucose and drug amount, relative cost per infusion, and total cost incurred in the administrations. for the following results, the time for simulation duration is considered to be 168 hours (7days). recall that parameters b 1 and b 2 are the weight factors associated in our objective functional which represent the measure of costs involved in the administration of glucose u g (t) strategies in regulating glioblastoma signaling pathways and anti-invasion therapy and drug u d (t) infusions, respectively, which also includes dosage, type, brand, medical fee for administration, etc. fig 13a shows that as the cost of glucose administration becomes expensive (increasing b 1 values) with fixed drug administration cost (b 2 = 1.0), frequency of concomitant and alternating infusion increases. however, it should be observed that as frequency of administration increases, the optimal glucose dose per infusion decreases with drug dose per infusion increases, see fig 13b. increasing drug dosage compensates the decreasing glucose dosage in order to keep mtor activities up-regulated leading to cell proliferation. on the contrary, if drug administration cost increases (increasing b 2 values) with fixed glucose administration cost (b 1 = 1.0), frequency of administration remains almost constant. this is depicted in fig 13c. the glucose dose per infusion is almost the same but the drug dose per infusion decreases with increasing b 2 as illustrated in fig 13d. further, note that frequency of concomitant infusion control is always lower than that of alternating infusion control. in addition, drug (glucose) dose per infusion of concomitant control is always more (slightly less) than that of alternating control. for increasing glucose administration cost (increasing b 1 ) with fixed drug administration cost (b 2 = 1.0), total amount of glucose used for both control strategies generally decrease (except for high b 1 > 3.0 values), as depicted in fig 14a, while total amount of drug increases, see fig 14b. on the other hand, when drug administration becomes expensive (increasing b 2 strategies in regulating glioblastoma signaling pathways and anti-invasion therapy values) with fixed glucose administration cost (b 1 = 1.0), the total amount of glucose dosage is almost constant (just slightly increasing for concomitant control) as shown in fig 14c, but the total drug amount is decreasing as can be seen in fig 14d. as illustrated in fig 14, concomitant control use less total amount of glucose than that of alternating control. contrarily, concomitant administration consumes more total drug amount as compared to that of alternating control (except possibly for high glucose administration cost, b 1 > 3.0, see fig 14b) . figs 15 and 16 reflect the relative cost per infusion and total administration cost of glucose and drug infusions incurred under concomitant and alternating controls. with fixed drug administration cost (b 2 = 1.0), relative glucose cost per infusion and total administration cost increases as b 1 increases. note that alternating control cost for glucose is always higher than concomitant infusions, see fig 15(a) and 15(b) . the relative and total administration cost for concomitant infusion slightly increase as compared to alternating control as b 1 increases. again, alternating control incur more cost for higher glucose administration cost as illustrated in fig 15(c) and 15(d) . on the other hand, when b 1 = 1.0 and drug administration cost (b 2 ) increases, the relative glucose cost per infusion and total administration cost is almost constant. this can be seen in fig 16(a) and 16(b) . again, relative total cost for alternating control is higher than that of concomitant control. further, as b 2 increases, relative drug cost per infusion and total administration cost decreases (fig 16(c) and 16(d) ), since total drug dosage also decreases as shown in fig 14d. in this case, it can be seen that drug administration cost for alternating control is generally lower than that of concomitant control. observe that in fig 17, both concomitant (blue) and alternating (orange) control trajectories in glucose-mtor-drug space are restricted in a smaller region avoiding aggressive invasion, rapid proliferation, and unwanted drug complications. both strategies achieved the goal of keeping mtor (mir-451) up-regulated inducing cell proliferation and thus avoiding aggressive cell migration. it is important to note that under these proposed optimal control infusions, glucose and drug levels are regulated to prevent excessive cell division and tumor growth. as a consequence, these strategies suggest safer infusion administration preventing hyperglycemia for diabetic patients and risk of drug complications. table 3 provides the average frequency, dosage and relative cost of concomitant and alternating control strategies where b 1 = b 2 = 1.0 and simulation time is 168h (7d). the periodic switching behavior of glioblastoma cells between proliferation and invasion phases is highly influenced by fluctuating glucose levels [14, 17] . in response to high glucose supply, mir-451 and mtor are up-regulated and ampk complex is down-regulated inducing cell growth. on the contrary, low glucose level up-regulates ampk complex, down-regulating mir-451 and mtor, promoting cell migration [15] . the mutual antagonistic mechanism strategies in regulating glioblastoma signaling pathways and anti-invasion therapy between mir-451 (mtor) and ampk complex and the cell's strategic metabolic adaptation support the survival of cancer cells even in a nutrient-deprived microenvironment [14, 55] . in addition to rapid proliferation of glioblastoma cells, aggressive invasion to the surrounding tissue is a major cause of treatment failure. despite advances in medical imaging technology such as mri and pet, glioblastoma cells can spread beyond detection leading to tumor recurrence within 2 to 3 cm of the resection cavity even after surgical removal of a malignant glioblastoma [29] . these glioma cells are capable to deform cell membrane and nucleus for cell infiltration through a narrow gap between normal glial cells in brain tissue by upregulation of myosin ii along with actin bundles [26, 56] . while exact migratory patterns are still poorly understood, these invasive glioma cells prefer white matter and blood vessels [26, 57] with a wide range of speeds in the range of 5-80 μm/h [58] [59] [60] [61] showing sometimes saltatory patterns in the migration direction in brain [58] . prediction of tumour invasion directions in nearby tissue may help define exact boundaries of focal treatments (surgery or radiosurgery), preventing future growth and recurrence [57] . for example, medical doctors could determine specific locations for radiation target volumes, not just using a rough estimate of 2-3 cm in all directions as a guidance as commonly done today [57] . assuming that migratory cells are localized near the surgery site [16] , one possible approach is to keep the cells in its proliferative phase preventing them from invading brain tissue in a combination with transport of therapeutic drugs near blood vessels [18] . as a result, the tumor strategies in regulating glioblastoma signaling pathways and anti-invasion therapy mass will be visible for a succeeding surgery while killing proliferative tumor cells at the blood sites. in this study, we considered the intracellular dynamics of the mir-451-ampk-mtor-cell cycle signaling pathway model developed recently by kim et al. [40] . incorporated in the model is a drug component which blocks the inhibitory pathway of mtor by ampk complex. this drug targets up-regulation of mtor activities enhancing cell proliferation. the focus of the current work is to regulate up-stream signaling pathway via glucose infusion activating strategies in regulating glioblastoma signaling pathways and anti-invasion therapy mir-451, and control the down-stream pathway to cell cycle via drug infusion enhancing mtor activities. optimal control problem is formulated with the goal of keeping high levels of mir-451 and mtor to induce cell growth and reduce invasion to the surrounding tissues. in the framework of optimal control theory, two administration strategies are explored to achieve the goal with minimal cost incurred in glucose and drug administrations. the control strategies investigated in this study are (1) concomitant infusion control and, and (2) alternating glucose and drug infusion control. both strategies are able to switch on the proliferative mode of glioblastoma cells and turn off its migratory mode. cell cycle is regulated with fewer mass divisions restricting rapid growth. numerical results show that concomitant control had fewer infusions, lesser glucose dosage and cheaper administration cost. however, when glucose and drug poses unwanted chemical reactions during concurrent administration, alternating control would be beneficial with lower drug amount usage. the mathematical models of the mir-451-ampk-mtor core control [14, [16] [17] [18] 40 ] are based on a 'go-or-grow' hypothesis and supporting experiments in gbm cells [8, 15, 19] . however, the range of bistable behavior indicates a 'go-and-grow' program which has been proposed for other cancers too [62] . in the glioma cases, 'go-or-grow' hypothesis has been long suggested [29, 55, [63] [64] [65] [66] in addition to mir-451-induced 'go-or-grow' mechanism [8, 15, 19] . glioma consists of a bulky, proliferative core in the center and highly invasive individual cells in the outer rim [55, 63, 64, 67] and sequential transition between proliferative and invasive phenotypes characterizes tumor progression and may lead to faster growth [14] . at least at the microscopic level, these migration/proliferation phenotypes appear to be mutually exclusive characteristics at different time frames [64] , as suggested by in vivo imaging data of glioma cells migrating in a saltatory fashion [58] . glioma cells was shown to pause for a short period of time (�an hour) for cell division before the daughter cells begin to move again [58] . gal et al. [68] further experimentally observed that this phenotypic switch can happen under different extracellular environment: (i) proliferative type in soft agar via activation of myc signaling pathways in response to hepatocyte growth factor (hgf); and (ii) infiltrative type in matrigel through ras signaling pathways in response to the same hgf. recently, dhruv et al. [69] also affirmed the role of activation of key molecules in dichotomy between proliferation and invasion: c-myc and nfκb in the proliferative core and radially dispersed, invasive region of gbm tumors, respectively. glial cells can also interact with gbm tumor cells for phenotypic switch of gbm cells between cell migration and active proliferation [66] . although these experiment and mathematical modeling support the 'go-or-grow' hypothesis, it is not certain if alternative mechanism such as 'go-andgrow' occurs in such a heterogenous tme in real patients [64] . mansury et al. [70] for instance illustrated that individual glioma cell in a mixed group of proliferative and invasive phenotypes can depend on genotype of counter part and tumor microenvironment. in a similar conceptual studies on epithelial-to-mesenchymal transition (emt) and mesenchymalto-epithelial transition (met) [45] can give a hint on the complexities of this complex regulation of those two phenotypes and glioma in tme might not posses the simple 'go-or-grow' dogma. increasing number of evidences now suggest that the 'go-or-grow' model has similar molecular basis with emt [64] . for instance, hgf and tgfβ are major regulators of emt, and these also provide strong stimuli of gbm invasion [71, 72] and, upregulation of met and cd44 activities, as well as an activated nfκb signaling pathway, was reported in both mesenchymal gbm and metastatic cancer [73] [74] [75] [76] . all these experimental observations suggest that metastatic epithelial cancers and mesenchymal gbm drive common mechanisms that regulate the phenotypic transition between invasion and proliferation, suggesting the possibility of the 'go-and-grow' mechanism. the emt paradigm in gbm has not been extensively studied as relevant to the progression of the disease due to different origin and rare metastasis of glioma [77, 78] . further studies and experimentation need to be done for better understanding of the fundamental principles. in this paper, we did not take into account key microenvironmental factors such as endogenous immune dynamics including nk cells [79] and m1/m2 macrophages [72] , other major signaling networks [80, 81] such as e2f and myc [11, 82] , angiogenesis [80, 83] , biophysical interaction between tumor cells and blood vessels [80] , ecm remodeling for therapy [81, [84] [85] [86] , or growth factors [87, 88] such as epidermal growth factors [72, 89, 90] , fibroblast growth factors [91] , transforming growth factor-β [72, 92] , and csf-1 [72, 93, 94] , that may play critical roles in proliferation, progress, aggressive invasion of gliomas and development of anticancer strategies [95] . for example, endogenous nk cells may interfere oncolytic virus combination therapy in gbm while exogenous nk cells increase anti-tumor efficacy [79] , illustrating the complex nature of tumor microenvironment. recently, mtor was considered to be a master regulator of cell growth and recognized as a good therapeutic target for therapies in glioblastoma [96] . a recent study found that withaferin a and temozolomide can induce apoptosis and reduce drug resistance by g2/m cell cycle arrest through intrinsic and extrinsic apoptotic signaling pathways [97] . more detailed analysis and experiments on akt/mtor/pi3k are necessary. interestingly, radiation was shown to indirectly promote the export of lactate into the extracellular space and inhibition of ampk/nfκb signaling pathways were involved in radiation-induced invasion of cancer cells [98] . on the other hand, m2 microglia/macrophages induce matrix remodeling and glioma cell invasion [72, [99] [100] [101] . since pi3k signaling was shown to contribute to m2-polarization of these tumor associated macrophages (tams) [102] and pi3k binding to csf1r was shown to enhance spreading of macrophages, thus promoting glioma cell invasion [103] . therefore, better understanding of these pi3k-mtor-csf1r signaling networks in macrophages would lead to development of blocking aggressive infiltration of cancer cells. signaling pathways of apoptosis and necroptosis are important parts of oncolytic virus (ovs) therapy [104, 105] . tumor extracellular matrix (ecm) plays a significant role in regulation of glioma invasion in brain tissue as well as ov spread [106] . for example, cspgs, one of major tumor ecm in brain can characterize the invasive and non-invasive gliomas in a complex tme where microglia and astrocytes mechanically interact with cspgs and tumor cells [40, [107] [108] [109] . hybrid models [56, [110] [111] [112] and its associated optimal control strategies can be adapted to take into account this important intracellular signaling as well as cell population dynamics and tissue dynamics. better understanding of various roles of these components in tumor microenvironment may provide better anti-invasion strategies of glioma cells. however, our mathematical model in this work is a first step toward further experimental/ clinical investigation and more optimal anti-invasion strategies of gbm by incorporating these microenvironmental components. we will address these issues in future work. supporting information s1 appendix. optimal control problem. formulation of the optimal control problem for concomitant and alternating glucose and drug infusion. 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enhancement of tumor cell invasion bortezomib-induced unfolded protein response increases oncolytic hsv-1 replication resulting in synergistic antitumor effects bortezomib treatment sensitizes oncolytic hsv-1 treated tumors to nk cell immunotherapy choindroitinase abc i-mediated enhancement of oncolytic virus spread and anti-tumor efficacy: a mathematical model chondroitin sulfate proteoglycans potently inhibit invasion and serve as a central organizer of the brain tumor microenvironment contributions of chondroitin sulfate proteoglycans to neurodevelopment, injury, and cancer glycosaminoglycans and glioma invasion a hybrid model for tumor spheroid growth in vitro i: theoretical development and early results multiscale models of cell and tissue dynamics the role of the microenvironment in tumor growth and invasion this work is resulted from the konkuk university research support program (e.j.). this paper is also supported by the korea national research foundation (nrf) grant funded by the korean government (mest): nrf-2017r1a2b2004651 (e.j.) and the basic science research key: cord-002023-7zd5zhbz authors: hiremath, jagadish; kang, kyung-il; xia, ming; elaish, mohamed; binjawadagi, basavaraj; ouyang, kang; dhakal, santosh; arcos, jesus; torrelles, jordi b.; jiang, x.; lee, chang won; renukaradhya, gourapura j. title: entrapment of h1n1 influenza virus derived conserved peptides in plga nanoparticles enhances t cell response and vaccine efficacy in pigs date: 2016-04-19 journal: plos one doi: 10.1371/journal.pone.0151922 sha: doc_id: 2023 cord_uid: 7zd5zhbz pigs are believed to be one of the important sources of emerging human and swine influenza viruses (swiv). influenza virus conserved peptides have the potential to elicit cross-protective immune response, but without the help of potent adjuvant and delivery system they are poorly immunogenic. biodegradable polylactic-co-glycolic acid (plga) nanoparticle (plga-np) based vaccine delivery system enhances cross-presentation of antigens by the professional antigen presenting cells. in this study, norovirus p particle containing swiv m2e (extracellular domain of the matrix protein 2) chimera and highly conserved two each of h1n1 peptides of pandemic 2009 and classical human influenza viruses were entrapped in plga-nps. influenza antibody-free pigs were vaccinated with plga-nps peptides cocktail vaccine twice with or without an adjuvant, mycobacterium vaccae whole cell lysate, intranasally as mist. vaccinated pigs were challenged with a virulent heterologous zoonotic swiv h1n1, and one week later euthanized and the lung samples were analyzed for the specific immune response and viral load. clinically, pigs vaccinated with plga-np peptides vaccine had no fever and flu symptoms, and the replicating challenged swiv was undetectable in the bronchoalveolar lavage fluid. immunologically, plga-np peptides vaccination (without adjuvant) significantly increased the frequency of antigen-specific ifnγ secreting cd4 and cd8 t cells response in the lung lymphocytes, despite not boosting the antibody response both at preand post-challenge. in summary, our data indicated that nanoparticle-mediated delivery of conserved h1n1 influenza peptides induced the virus specific t cell response in the lungs and reduced the challenged heterologous virus load in the airways of pigs. swine influenza is a highly contagious acute respiratory viral disease of pigs, caused by h1n1, h1n2 and h3n2 subtypes of influenza a virus (iav). the disease is responsible for significant economic loss to the swine industry [1] . pigs also play a critical role in the emergence of new strains of influenza viruses by acting as a "mixing vessel" [2] . current swine flu vaccines are strain specific and they have been failed to induce cross-protection against genetically variant flu viruses [3] . moreover, intramuscularly delivered flu vaccine induces poor mucosal iga antibody and t cell responses [4] . the highly conserved influenza viral proteins across iav subtypes are matrix (m1 and m2), nucleocapsid (np) and stalk domain of hemagglutinin (ha). promising new generation flu vaccine platforms include use of highly conserved peptides in a vaccine formulation; because, recent developments in biotechnology tools have made the large scale production of antigenic peptides highly feasible at low cost. attempts were made to develop iav peptide vaccine by coexpressing conserved peptides of m protein 2 ectodomain (m2e) with hepatitis b capsid protein [5] , and also using cocktail of conserved t and b cells peptides [6] . but due to lack of identified effective vaccine delivery and potent adjuvant system, peptides based vaccine candidates have been unsuccessful to induce robust response in pigs. moreover, in intramuscularly vaccinated animals mucosal immune system is weakly activated. recently, chimeric construct that express m2e on the surface loop of norovirus p particle (m2e-pp) was shown to produce high levels of antibody response and protect mice from a lethal challenge [7] . in pigs, m2e-pp also induced specific immune response, but failed to provide protection from disease (unpublished data). potent vaccine delivery and adjuvant systems are essential to enhance immunogenicity of peptides vaccine [8] . one of the endeavors of 21 st century is delivery of vaccines and drugs through biocompatible and biodegradable polymer based nano or microparticles. plga (poly lactic-co-glycolic acid) is a nontoxic, fda and european medicines agency approved polymer, and widely used as a vehicle for drug and vaccine delivery [9, 10] . the properties of plga made it ideal to entrap even soluble vaccine ags in nanoparticles (np) (200-600 nm) to elicit strong immune response [11] . plga-np entrapped peptide ags are being protected from enzymatic or ionic degradation in vivo until they are uptaken by antigen presenting cells (apcs) [12] . particulate antigens are readily taken up by mucosal m cells and apcs in the nasal-associated lymphoid tissues in intranasally vaccinated animals [13] , which enhances antigen specific ifnγ secreting t cell response and production of high-affinity neutralizing antibodies in pigs [14, 15] . benefits of plga based intranasal vaccine delivery system with the inactivated porcine reproductive and respiratory syndrome virus (prrsv) in pigs and hepatitis b ags in rodents has been demonstrated [14] [15] [16] [17] . in this study, a cocktail of conserved two each of t and b cell peptides of human h1n1 iav and m2e-pp of swiv h1n1 were entrapped in plga-nps and characterized their vaccine properties in vitro. further, efficacy of the candidate vaccine was evaluated against a virulent heterologous zoonotic swiv h1n1 challenge in intranasally vaccinated pigs, coadministered with or without an adjuvant m. vaccae wcl. our results indicated induction of peptide specific t cell response, reduction in the lung viral load and clinical flu symptoms, but the specific antibody response was not boosted both in the pre-and post-challenged np based h1n1 peptides vaccinated pigs. dulbecco's minimum essential medium (dmem, lonza) supplemented with 10% fetal bovine serum (atlanta biologicals) at 37°c with 5% co 2 . for virus infection dmem containing tpck (l-1-tosylamido-2-phenylethyl chloromethyl ketone) trypsin (1 μg/ml) and without fetal bovine serum was used. for preparation of nanoparticles, polylactic-co-glycolic acid (plga) 75:25 (mw 40,000-75,000) (lakeshore biomaterials, al), poly-vinyl alcohol (mw 30,000-70,000) (sigma-aldrich) and dicholoro methane (acros organics) were used. micro bca (bicinchoninic acid) protein assay kit (pierce) was used for estimation of the peptides concentration. cytotoxic t lymphocyte specific peptides specific to sla-1 0401, np (a1): nsdtvgwsw; and pa (a2): ateyimkgvy of the pandemic 2009 h1n1 virus were used [20] . the sla-1 0401 and hla-i-a 0101 present the same antigenic peptides to cd8 t cells [20] and it is commonly expressed in five swine breeds [21] , indicating that it is a valuable sla i allele that has survived long-term evolutionary selection [20] . other two peptides used in the study were a7: nsengtcypgdfidyeelreqlssvssfekfeif of the ha1 domain of human a/south carolina/1/18 (h1n1) strain [genbank af117241] [6] ; and a8: npengtcypgyfadyeel reqlssvssferfeif of np protein chosen from human influenza h1n1 strain (a/new caledonia/20/99) [6, 22] . of these four peptides, a7 and a8 were evaluated as vaccine candidates against a swiv challenge in pigs [6] . m2e peptide, slltevetptrsewecrcsdssd, was from a swiv h1n1 consensus m2e sequence expressed as m2e-pp chimera, constructed using the p-particle expression vector [pgex-4t-1 containing p-domain sequence of norovirus va387, genogroup ii, cluster 4 (gii.4)] as described previously [7] . m2e-pp chimera was studied earlier in mice [7] . preparation of plga-nps entrapped conserved iav peptides plga-nps entrapped with four conserved h1n1 peptides of ha, np and pa, and m2e-pp were prepared using double emulsion method (water/oil/water) as described previously [23] . briefly, 1.2 mg each of the four peptides (total 4.8 mg) were suspended in 500 μl pbs, and mixed with 2% pva (w/v), 2% (w/v) sucrose and 2% (w/v) mg (oh) 2 and emulsified in 200 mg of plga dissolved in 5 ml dichloromethane using the probe sonicator (6 mm) for 30 sec at 30% power on ice. to the resulting water-in-oil (w/o) primary emulsion 23 ml of 2.5% w/v polyvinyl alcohol (pva) containing 1% polaxmer was added and divided equally into two tubes, and the emulsion was sonicated for 60 sec at 30% power on ice. the secondary emulsion was stirred overnight at 4°c to allow evaporation of the organic solvents. resulting plga-np were washed thrice with cold sterile milli q water and centrifuged at 10,000 rpm for 30 min. finally, the plga-np was resuspended in 5% sucrose in milli q water and lyophilized. similarly, 5 mg of m2e-pp chimera suspended in 500 μl of pbs was entrapped in plga-np. the peptides release profile from peptides entrapped in plga-np was determined by in vitro analysis over a period of 4 weeks as described previously [24] . briefly, 50 mg of plga np-peptide was suspended in 1 ml sterile pbs and centrifuged at 11,400 × g for 10 min at 4°c, and the supernatant was collected to estimate the burst release (~10 min after reconstitution). the pellet containing np-peptide was resuspended at every time point in 1 ml pbs and the harvested supernatant was collected at 0 (2 hr after reconstitution), 1, 3, 5, 10, 15, 20, 25 and 30 days and stored at -20°c. on the last day, np-peptide was hydrolyzed using the buffer containing 5% sds in 0.1 n naoh and recovered the remaining entrapped peptides; and the peptide concentration in all the samples were estimated using the micro bca protein assay kit [23] . the peptides released until day 30 and the amount released after hydrolysis were together considered to estimate the total peptide amount and the cumulative release was expressed in percentage. the amount of entrapped peptides in plga-nps was determined as described previously [23, 25] . briefly, freeze-dried nps (10 mg) were dissolved in 1 ml of freshly prepared buffer (5% sds in 0.1 n naoh) and incubated for 2 hr at 37°c with constant stirring. the mixture was centrifuged at 11,400 × g for 10 min, and the supernatant was collected and total protein concentration was quantified using the micro bca protein assay kit [23] . shape and surface morphology of plga-np was determined by scanning electron microscopy (hitachi s-3500 n) as described previously [15] . briefly, freeze-dried particles were dispersed in pbs at concentration of 1 mg/ml and was spread on to an adhesive stub, dried and then coated with gold/palladium under vacuum using an ion coater. the coated specimen was examined under the microscope at 10 kv. size distribution of the plga-nps was determined using nicomp 370 particle sizer (particle sizing systems, fl); and the zeta potential of the plga-nps was determined by zeta pals (brookhaven instruments corp, holtsville, ny, usa) as described previously [15] . m. vaccae (atcc#23027) was grown in endotoxin free 7h9 medium at 37°c in accordance to atcc instructions, and whole cell lysate (wcl) was prepared as previously described [26] . briefly, live m. vaccae culture was harvested, washed twice in sterile endotoxin free pbs (ph 7.4), and suspended in pbs containing 8 mm edta, proteinase inhibitors, dnase and rnase. bacteria were disrupted under endotoxin free condition using the bead beater until approximately 90% breakage was observed. lysates were centrifuged at 30,000 x g for 20 min to pellet the unbroken cells and insoluble cell wall components. the collected supernatant (wcl containing soluble components) was filter-sterilized using a 0.2 μm filter. protein content and endotoxin levels in the m. vaccae wcl was quantified by the bca protein assay kit [23, 26] , and lyophilized aliquots (5 mg protein) were stored at -80°c until used in the study. influenza specific antibodies are commonly found in majority of commercial pigs; hence, caesarean delivered colostrum-deprived 4 week old (n = 32) conventional large white-duroc crossbred pigs were used. pigs were randomly divided into five groups (n = 6 or 7 pigs per group). group 1: mock (mok); group 2: mock-challenge (mkc); group 3: peptides (pep); group 4: nanoparticle-entrapped peptides with adjuvant (npa) and; group 5: nanoparticleentrapped peptides (npp) ( table 1 ). in each vaccine dose of pep, npp, and npa, 50 μg each four peptides and 200 μg of m2e-pp chimera were included, and the vaccine was inoculated twice at 14 days interval as mist using a multidose aerosol device developed exclusively to use in pigs by prima tech (neogen corporation, usa). the adjuvant m. vaccae wcl (5 mg per pig) was mixed with plga-np entrapped peptide just before inoculation. pig groups 2, 3, 4 and 5 were challenged 14 days after the booster with the swiv h1n1 (sw/oh/24366/07) (6 x10 6 tcid 50 in 3ml), 50% volume delivered by intranasal and 50% by intratracheal route [19] . pigs were euthanized on day post-challenge (dpc) 7. mock-inoculated pigs were euthanized separately prior to killing of any infected animals. pigs were maintained, vaccinated, challenged and euthanized in our bsl-2 facility in accordance with the guideline and protocol approved by the institutional biosafety committee and institutional animal care and use committee. swiv h1n1 challenged pigs were observed daily for clinical signs of influenza at dpc 1 to 7 and the rectal temperature was recorded daily beginning 1 day before challenge. during necropsy the lungs were examined for gross pathological changes and scored for virus induced consolidation as described previously [27] . swiv titer in bal fluid collected at dpc 7 from euthanized pigs was quantified by qrt-pcr as previously described [28] . briefly, total rna was extracted from 50 μl of bal fluid using magmax™-96 rna multi-sample kit (ambion) according to the protocol provided by the manufacturer. the automated magmax™ express magnetic particle processor (life technology) was used for sample processing, and qpcr was performed to estimate viral rna load in samples. swiv rna load in test samples were calculated using the standard curve with y-axis being virus concentration in tcid 50 and x-axis was being the ct value. the replicating challenged swiv in the bal fluid was quantified by the indirect immunofluorescence assay (ifa) as described previously [15] . briefly, mdck cell monolayer cultured in 96-well plate was treated with ten-fold serially diluted bal fluid (100 μl) diluted in serum free dmem and supplemented with tpck trypsin (1 μg/ml), and after 24 hr cells were washed and fixed in 80% acetone in water. the air dried plates were immunostained using the influenza nucleoprotein specific mab (m058, calbioreagents, ca) followed by goat anti-mouse igg alexa 488 conjugated secondary antibody (invitrogen). the plates were washed and treated with pbs/glycerol (60:40) and the immunofluorescence was evaluated using an inverted fluorescent microscope (olympus ix51, center valley, pa). swiv specific iga and igg isotype antibodies in the bal and plasma samples were analyzed as described previously [29, 30] . briefly, 96-well plates were coated with pretitrated quantity of the challenge swiv (1x10 3 tcid 50 per well) in carbonate-bicarbonate buffer (ph 9.6) overnight at 4°c, and plates were washed and treated with blocking buffer (1% bsa and 0.1% tween 20 in pbs) for 2 hr at rt. bal fluid samples (1:25 diluted) were added to first wells and subsequently two-fold serially diluted and incubated for 2 hr at rt. bound virus specific isotype antibodies were detected using affinity purified goat anti-pig iga secondary antibody conjugated with hrp (bethyl laboratories inc. tx). finally, plates were developed using chromogen tmb (3,3 0 ,5,5 0 -tetramethylbenzidine) (kpl) and the reaction was stopped using 1 m phosphoric acid. plates were read using spectra max microplate reader at 450 nm. for bal fluid, the end point titer was determined based on the highest dilution of the sample in which the measured od value was greater than the mean od value of seven swiv negative pig bal fluid samples plus two standard deviations at the respective dilutions was used. similarly, swiv specific igg antibody end point titer from both bal fluid and plasma were determined by elisa. peptide specific iga level was also determined by coating peptides at the concentration of 2 μg/ ml and od values were compared between the experimental groups. swiv specific hemagglutination inhibition (hi) titer in pig bal fluid and plasma samples was determined in accordance with the recommendation of world organization for animal health (oie) [31] (world organization for animal health, 2014). swiv specific neutralizing antibody and hemagglutination inhibition titers in plasma and bal fluid were analyzed as described previously [32] . for determining the virus neutralizing (vn) antibody titers, plasma and bal fluid samples were heat inactivated (56°c for 30 min), two-fold diluted and incubated with an equal volume of 50 tcid 50 of the swiv per well for 2 hr at 37°c. the suspension was transferred into microtiter plates containing confluent monolayer of mdck cells and incubated for 24 hr at 37°c, followed by incubation with anti-swiv nucleoprotein specific mab (m058, cal bioreagents, ca), alexa-488 conjugated anti-mouse igg (h+l) secondary antibody and mounted with glycerol in pbs (6:4 ratio). vn titer is the reciprocal of the highest dilution of a test sample which inhibited greater than 95% of the virus infectivity. the peptide specific recall lymphocyte response was assayed as described previously [14, 33] with few modifications. briefly, lung mononuclear cells (lmncs) of all the pigs at dpc 7 were isolated by enzymatic digestion of lung tissues as described previously [14] . five million lmncs were restimulated with individual peptides (m2e, a1, a2, a7 and a8) (2 μg/ml), swiv (multiplicity of infection [moi] 1) or phytohaemagglutinin (10 μg/ml) for 72 hr at 37°c in enriched rpmi 1640 (e-rpmi) (10% fbs, gentamicin [100 μg/ml], ampicillin [20 μg/ml], 20 mm hepes, 2 mm lglutamine, 0.1 mm nonessential amino acids, 1 mm sodium pyruvate and 50 nm 2-me) in brefeldin a (b7651, sigma) were added during the last 6 hr of incubation of lmncs treated with or without the indicated stimulants. the surface immunostained cells were fixed with 1% paraformaldehyde and permeabilized with the cell-permeabilization buffer (85.9% deionized water, 11% pbs without ca 2+ or mg 2+ , 3% formaldehyde solution and 0.1% saponin) overnight at 4°c. the cells were washed and stained with fluorochrome-conjugated anti-pig ifnγ (clone p2g10) or its isotype control mab (bd biosciences) in 0.1% saponin containing fluorescence-activated cell-sorting (facs) buffer. immunostained cells were acquired using the facs aria ii (bd biosciences) flow cytometer and analyzed using flowjo (tree star, ashland, or, usa) software. all specific cell population frequencies were presented as the percent of total cd3 + lymphocytes. the culture supernatant of stimulated lmncs with swiv as described above was harvested after centrifuging the 48 well lmncs culture plates for 2 min at 3,500 rpm and subjected to ifnγ elisa as described previously [30] . background cytokine levels from each pig groups were subtracted from the specific stimulation. this study was carried out in strict accordance with the recommendations by public health service policy, united states department of agriculture regulations, the national research council's guide for the care and use of laboratory animals and the federation of animal science societies' guide for the care and use of agricultural animals in agricultural research and teaching, and all relevant institutional, state and federal regulations and policies regarding animal care and use at the ohio state university. the protocol was approved by the committee 48 well tissue culture plates. stimulated lmncs were immunostained and analyzed by flow cytometry as previously described [14, 30] . briefly, lmncs were first surface-labeled with pig lymphocyte specific purified, fluorochrome or biotin conjugated mabs [cd3έ (clone ppt3), cd4α (clone 74-12-4) and cd8α (clone 76-2-11)] (southernbiotech) followed by treatment with fluorochrome labeled streptavidin or anti-mouse isotype specific antibodies. for intracellular ifnγ staining, golgiplug™ (bd biosciences, san jose, ca, usa) and on the ethics of animal experiments of the ohio state university (protocol number: 2014a00000099). all the pigs were maintained, samples collected and euthanized, and all efforts were made to minimize the suffering of pigs. all the data were expressed as the mean of 6 or 7 pigs ± standard error of the mean (sem). statistical analyses were performed using one way analysis of variance (anova) followed by post-hoc tukey's test using graphpad instat prism (software version 5.0) to establish variations among mkc, pep, npp and npa pig groups. statistical significance was assessed at p<0.05 ( ã ), p<0.01 ( ãã ) and p<0.001 ( ããã ). in the plga particulate vaccine delivery system, optimum antigen loading efficiency, charge, stability and size of the np are important [34] . we entrapped m2e-pp and pooled four peptides separately in plga-np using the similar procedure. in vitro characterization of both peptides and m2e-pp entrapped nps showed comparable results and the data of m2e-pp is shown here. entrapment efficiency of m2e-pp in np was 50-54%. scanning electron microscopy (sem) imaging of np revealed them as spherical particles (fig 1a) . size distribution profile of the np was determined by dynamic light scattering technique and it was 227-316 nm with the majority of nps around 260 nm (fig 1b) . surface charge of the particles was -21.93±2.93 mv (an average value of ten runs) as measured by zeta potential analysis. in vitro protein release profile of m2e-pp from np-entrapped m2e-pp suspended in pbs overtime was quantified under physiological conditions. plga-np containing the vaccine cargo carry a small quantity of surface anchored ags and they rapidly release upon reconstitution in pbs ( 10 min) called the burst release; while the entrapped ags in np release over a period of 4-6 weeks [15] . we observed a burst release of 14.3%, cumulative release of 25% (including the burse release) after 2 hr (day 0) and the total release of 64% over a period of 4 weeks (fig 1c) . thus, our results indicated that plga-np release entrapped m2e-pp gradually, supporting the principle of depot-effect provided by plga-np under in vivo conditions. since pigs were vaccinated and challenged via nasal route, we quantified influenza ag specific t cell response in the lungs of pigs using lung mononuclear cells (lmncs). on the day of necropsy, isolated lmncs were either unstimulated or stimulated with the swiv or individual peptides and analyzed for the frequency of activated (ifnγ + ) t lymphocyte subsets. a representative graph depicting the gating pattern of restimulated porcine lymphocyte subsets secreting ifnγ belongs to swiv infected pigs is shown (fig 2a) . porcine immune system has a unique abundant population of cd4 and cd8 double positive t cell subset, which have combined t-helper, memory and cytotoxic t cell properties [35, 36] . our results detected both the virus and peptides (a1, a2, a7 and a8) specific increased frequency of cd4 + cd8α + ifnγ + t cells in lmncs of npp vaccinated pigs compared to all the other vaccine formulations received animals ( fig 2b) . this data indicated influenza ags specific activation of cd4 and cd8 double positive t cell subset in the lungs of npp vaccinated pigs. in pigs, naïve t-helper cells are identified based on the expression of combination of phenotypic markers, cd3 + cd4 + cd8α -. similarly, cd3 + cd4 -cd8α + markers bearing cells are either cytotoxic t cells (ctls) or γδ t cells; and cd3 + cd4 -cd8αβ + expressing cells are exclusively ctls [37] . frequency of ifnγ + t-helper cells and ctls/γδ t cells in lmncs were significantly higher in npp vaccinated pigs, irrespective of the cells unstimulated or stimulated with the viral ags or most of the peptides (fig 2c and 2d ). this data indicated the presence of activated other t cell subsets in npp vaccinated pigs, which continued to secrete ifnγ + when cultured ex vivo. further, to determine influenza ags specific recalled t cell response exclusively in npp vaccinated pigs, all the three t cell subsets which were ifnγ + in npp pig group were plotted separately ( fig 2e) . a white dotted line showed in the figure in each t cell subset separates the specific recalled t cell response over the control (c) unstimulated cells (fig 2e) . our results detected the presence of increased recalled cd4 + cd8α + ifnγ + t cell subset in both swiv and peptides a2, a7 and a8 stimulated lmncs (fig 2e, 1 st panel) . similarly, increased recalled t-helper cell population specific to peptides m2e, a1 and a2 (fig 2e, 2 nd panel) , and recalled specific cd3 +-cd4 -cd8α + cell response against the virus, m2e and a2 peptides (fig 2e, 3 rd panel) in npp vaccinated pigs were detected. this data analysis revealed that nanoparticle entrapped conserved influenza peptides induced the peptide specific t cell response in pigs. in addition, supernatants harvested from the cultured lmncs restimulated with the swiv had significantly higher levels of secreted ifnγ in npp compared to peptides-and mock-vaccinated pig groups (fig 2f) . to quantify humoral immune response in the vaccinated pigs, we performed igg antibody analysis in plasma samples collected at day pre-challenge 0 (dpc 0) using pretitrated amounts of h1n1 swiv and swiv m2e protein by elisa. our results showed very low levels of specific antibody response, and the data was not statistically significant among the vaccinated pig groups (fig 3a and 3b) . even in post-challenge plasma and bal fluid samples of vaccinated pigs collected at dpc 7, the virus specific iga and igg antibody, hemagglutination inhibition (hi) and virus neutralizing (vn) antibody titers were low and the data was not significantly different among the experimental pig groups (fig 3c-3h) . thus, our study indicated that antibody response in npp and npa vaccinated pigs at pre-challenge was weak or absent, and the detected antibody response at post-challenge was induced by the challenge virus. our results suggested the need of using additional b cell antigenic peptides or the inactivated virus gating pattern of lymphocytes of a pig infected with swiv h1n1 and restimulated ex vivo with the virus for 3 days and immunostained using fluorochrome conjugated pig specific cd3έ, cd4α and cd8α markers and intracellular ifnγ to identify the frequency of cd3 + cd4 + cd8α + ifnγ + , cd3 + cd4 -cd8α + ifnγ + and cd3 + cd4 + cd8α -ifnγ + cells is shown. lung mncs (lmncs) isolated on the day of necropsy from all the experimental pigs were either unstimulated (control, c) or stimulated with the swiv (v), conserved peptides (a1, a2, a7 and a8 peptides) or m2e (m) for 3 days and immunostained cells were gated for t cell subsets as described above for: (b) cd3 + cd4 + cd8α + ifnγ + ; (c) cd3 + cd4 -cd8α + ifnγ + ; and (d) cd3 + cd4 + cd8α -ifnγ + cells. (e) delineating the swiv specific t cell response in npp vaccinated pigs. three activated (ifnγ + ) t lymphocyte subsets in the lmncs of only npp vaccinated pigs either unstimulated (control, c) or stimulated with the swiv or indicated peptides are shown. a white dotted line drawn across each t cell subset separates the ags specific recalled t cell response over the control unstimulated cells of npp vaccinated pigs. (f) secreted ifnγ in the culture supernatant of virus restimulated lmncs of vaccinated and swiv challenged pigs was analyzed by elisa. background cytokine levels from each pig groups were subtracted from the specific stimulation. each bar indicates the average frequency of indicated t cell subset or cytokine from 6 or 7 pigs ± sem. asterisk denotes statistically significant difference at p<0.05 (*), p<0.01 (**) and p<0.001 (***) between the indicated two pig groups determined by one way anova followed by tukey post-hoc test for the significance between the indicated pig groups. in the mock and unentrapped peptides vaccinated and virulent swiv h1n1 challenged pigs, the rectal temperature was 1.5 to 2°f higher at dpc 1 to 3 compared to mock animals ( fig 4a) . the pigs which had fever were anorexic and lethargic during those initial days of viral challenge. similarly challenged, but npp and npa vaccinated pig groups did not suffer from hyperthermia ( fig 4a) . to associate the body temperature to viral load, we measured the viral rna copies in bal fluid and observed comparable rna load among all the virus-challenged pig groups (fig 4b) . but the replicating swiv was detected in ! 50% of mkc and pep pig groups, and it was undetectable in the npp and npa vaccinated animals (fig 4c) . the assay was repeated three times to confirm the live swiv titer in the bal fluid samples. nasal swabs collected at dpc 4 and 7 did not show any detectable replicating virus in any of the virus challenged pig groups (data not shown). in summary, clinical signs of flu and replicating detectable lung viral load were detected only in swiv challenged control (mck and pep), but not in plga-np peptides (npp and npa) vaccinated pig groups. control of influenza in pigs and humans is a challenge due to continuous genetic changes in the viral surface proteins. therefore, enhancing influenza virus specific immune response to highly conserved antigenic epitopes is important to achieve increased breadth of protective immunity. hence, to elicit strong specific response to influenza, we entrapped highly conserved h1n1 peptides in plga-nps and inoculated intranasally in the form of mist to pigs to efficiently reach the mucosal inductive sites in the respiratory tract. our results demonstrated a significantly reduced detectable infectious challenged virus in the airways of npp and npa vaccinated pigs, associated with augmented antigen-specific t cell response in the lung lymphocytes of npp inoculated animals. amounts of each of the four peptides entrapped in np-peptides vaccine is assumed to be the same, because in another independent study individual peptides entrapped in plga nps had comparable levels of entrapment efficiency. in addition, results of ex vivo restimulation of lmncs with all the four individual peptides elicited comparable peptide specific t cell response in npp vaccinated pigs. studies in mice demonstrated that intranasal administration of m2e-pp chimera induces high levels of m2e specific antibodies [7] . conserved a7 and a8 peptides elicit antibody response in pigs, but failed to reduce the flu symptoms [5, 6] . soluble antigens and peptides are poorly immunogenic, but when entrapped in nps elicit strong immune response [38, 39] . pulmonary immune response varies with the vaccine particle size and surface charge, because particles of 500 nm are readily phagocytized by dendritic cells and m cells at mucosal sites [40, 41] . consistent with that the size of our candidate np-peptide vaccine was approximately 300 nm. plga-np entrapped ags are stable for 4-6 weeks and facilitate activation and maturation of apcs [40, 42] . mechanism of induction of increased breadth of protective response by nps based vaccine delivery system is mediated through efficient internalization of particulate ags, followed by processing and cross-presentation of entrapped ags by dendritic cells and macrophages to naïve cd8 t cells [43] [44] [45] . this has been shown specifically using plga nps delivery system mediated by their ability to disrupt phagosomes and release of cargo into cytosol for mhc class i loading [44, 45] . based on our data with heightened peptide-specific cd4 cd8 double positive t cell response in lmncs of npp pig group, it is expected that both dendritic cells and macrophages in the lungs of pigs would have uptaken and efficiently cross-presented the plga-np delivered peptide ags to naïve t cells. pigs vaccinated with m2ehbc fusion proteins without np delivery system has failed to protect animals from a challenge virus induced flu symptoms [5] . -1 to 7) . swiv titer in bal fluid at dpc 7 was detected by (b) viral rna load by qrt-pcr, and (c) replicating infectious virus load using mdck cells by immunofluorescence assay. the ratio indicated above each bar represents the number of pigs positive for virus and contributed to the data out of 6 or 7 animals. the viral titer is expressed in tissue culture infective dose 50 (tcid 50 ) in each ml of the bal fluid. each data point or bar represents the average value of 6 to 7 pigs ± sem. asterisk denotes statistically significant difference at p<0.05 (*) between mkc and npp or npa pig groups determined by one way anova followed by tukey post-hoc test. in pigs, a significant upregulation of activated cd4 cd8 double positive t cells has been associated with protective response against classical swine fever, aujeszky's disease and prrs viruses [14, 15, 46, 47] . in particular in our study, npp but not npa vaccinated pigs elicited antigen-specific increased activated ifnγ secreting cd4 cd8 double positive t cell response in the lungs. interestingly, complete clearance of detectable infectious challenge virus was observed in the airways of both npp and npa vaccinated animals, despite the comparable low levels of humoral response. this data suggested the critical role played by nps entrapped peptides delivery system in pigs, as well as the role of adjuvant m. vaccae wcl in possibly skewing the response of npp towards the th2 or th1-th2 balanced state, which needs further investigation. earlier, we have shown strong virus specific antibody response in pigs vaccinated intranasally using plga-nps entrapped prrsv with a potent adjuvant m. tb wcl, which elicited a strong th1-th2 balanced response [14, 15] . in place of the adjuvant m. tb wcl, we used a nonpathogenic mycobacterium, m. vaccae derived wcl; because, m. vaccae has comparable biochemical and cellular components of m. tb [48] , and also it was shown to possess potent adjuvant effects in rodents [49, 50] . further, m. vaccae wcl was not entrapped in plga-np, because greater adjuvant effects by soluble compared to np entrapped m. tb wcl in intranasally vaccinated pigs was observed [14, 15] . overall, our study suggested that m. vaccae wcl is not a potent adjuvant to npp vaccine in pigs, indicating the need of including additional highly antigenic b cell peptides or inactivated swiv entrapped in nps and coadministered with a potent adjuvant to boost the specific antibody response in pigs. in conclusion, our study in pigs for the first time demonstrated that influenza h1n1 conserved peptides cocktail entrapped in biodegradable nanoparticles and delivered intranasally as mist induced epitope specific t cell response, despite not boosting the antibody response. therefore, our future studies are focused on including additional highly antigenic influenza virus b cell peptides and potent adjuvant/s to enhance specific mucosal and systemic antibody responses to nanoparticle flu vaccine candidate. this is important because, biodegradable nanoparticle vaccine delivery platform has translational value to improve cross-protective immunity against genetically variant influenza viruses. vaccine development for protecting swine against influenza virus cases of swine influenza in humans: a review of the literature swine influenza viruses a north american perspective cross-presentation in viral immunity and self-tolerance vaccination of pigs with a dna construct expressing an influenza virus m2-nucleoprotein fusion protein exacerbates disease after challenge with influenza a virus conserved synthetic peptides from the hemagglutinin of influenza viruses induce broad humoral and t-cell responses in a pig model a candidate dual vaccine against influenza and noroviruses more than one reason to rethink the use of peptides in vaccine design current advances in research and clinical applications of plga-based nanotechnology controlled-release vaccines-biodegradable polylactide/polyglycolide (pl/pg) microspheres as antigen vehicles nanotechnology for the biologist biodegradable nanoparticles for drug and gene delivery to cells and tissue intranasal administration of cpg oligonucleotides induces mucosal and systemic type 1 immune responses and adjuvant activity to porcine reproductive and respiratory syndrome killed virus vaccine in piglets in vivo an innovative approach to induce cross-protective immunity against porcine reproductive and respiratory syndrome virus in the lungs of pigs through adjuvanted nanotechnology-based vaccination adjuvanted poly(lacticco-glycolic) acid nanoparticle-entrapped inactivated porcine reproductive and respiratory syndrome virus vaccine elicits cross-protective immune response in pigs plga nanoparticle entrapped killed porcine reproductive and respiratory syndrome virus vaccine helps in viral clearance in pigs aerosolized pla and plga nanoparticles enhance humoral, mucosal and cytokine responses to hepatitis b vaccine establishment and characterization of a madin-darby canine kidney reporter cell line for influenza a virus assays characterization of triple reassortant h1n1 influenza a viruses from swine in ohio crystal structure of swine major histocompatibility complex class i sla-1 0401 and identification of 2009 pandemic swine-origin influenza a h1n1 virus cytotoxic t lymphocyte epitope peptides nomenclature for factors of the sla system, update genetic and antigenic analysis of the first a/new caledonia/20/99-like h1n1 influenza isolates reported in the biodegradable nanoparticle-entrapped vaccine induces cross-protective immune response against a virulent heterologous respiratory viral infection in pigs comparison of humoral and cell-mediated immune responses to cationic plga microspheres containing recombinant hepatitis b antigen intranasal m cell uptake of nanoparticles is independently influenced by targeting ligands and buffer ionic strength lipoglycans of mycobacterium tuberculosis: isolation, purification,and characterization altered pathogenesis of porcine respiratory coronavirus in pigs due to immunosuppressive effects of dexamethasone: implications for corticosteroid use in treatment of severe acute respiratory syndrome coronavirus application of real-time rt-pcr for the quantitation and competitive replication study of h5 and h7 subtype avian influenza virus protection of pigs against taenia solium cysticercosis using recombinant antigen or in combination with dna vaccine cross-protective immunity to porcine reproductive and respiratory syndrome virus by intranasal delivery of a live virus vaccine with a potent adjuvant serum and egg yolk antibody detection in chickens infected with low pathogenicity avian influenza virus swine influenza h1n1 virus induces acute inflammatory immune responses in pig lungs: a potential animal model for human h1n1 influenza virus development and antigen specificity of the lymphoproliferation responses of pigs to pseudorabies virus: dichotomy between secondary b-and t-cell responses factors affecting the loading efficiency of water-soluble drugs in plga microspheres functional and phenotypic analysis of porcine peripheral blood cd4/cd8 double-positive t cells perforin expression can define cd8 positive lymphocyte subsets in pigs allowing phenotypic and functional analysis of natural killer, cytotoxic t, natural killer t and mhc un-restricted cytotoxic t-cells phenotypic maturation of porcine nk-and t-cell subsets carbohydrate biopolymers enhance antibody responses to mucosally delivered vaccine antigens in situ gelling nasal inserts for influenza vaccine delivery size-dependent uptake of particles by pulmonary antigen-presenting cell populations and trafficking to regional lymph nodes mucosal immune responses following oral immunization with rotavirus antigens encapsulated in alginate microspheres differential effects of agarose and poly(lactic-co-glycolic acid) on dendritic cell maturation mucosal vaccine design and delivery antigen co-encapsulated with adjuvants efficiently drive protective t cell immunity cd8-dendritic cells and macrophages cross-present poly(d,l-lactate-co-glycolate) acid microsphere-encapsulated antigen in vivo cytolytic function for pseudorabies virusstimulated porcine cd4+ cd8dull+ lymphocytes t-helper cells from naive to committed cell walls of mycobacteria and related organisms; chemistry and immunostimulant properties orally administered mycobacterium vaccae modulates expression of immunoregulatory molecules in balb/c mice with pulmonary tuberculosis the ability of heat-killed mycobacterium vaccae to stimulate a cytotoxic t-cell response to an unrelated protein is associated with a 65 kilodalton heat-shock protein the authors would like to thank dr. juliet hanson and megan strother for assistance with animal studies. key: cord-260432-imslfm4l authors: marshall, jenika d.; courage, emily r.; elliott, ryan f.; fitzpatrick, madeline n.; kim, anne d.; lopez-clavijo, andrea f.; woolfrey, bronwyn a.; ouimet, mireille; wakelam, michael j. o.; brown, robert j. title: thp-1 macrophage cholesterol efflux is impaired by palmitoleate through akt activation date: 2020-05-21 journal: plos one doi: 10.1371/journal.pone.0233180 sha: doc_id: 260432 cord_uid: imslfm4l lipoprotein lipase (lpl) is upregulated in atherosclerotic lesions and it may promote the progression of atherosclerosis, but the mechanisms behind this process are not completely understood. we previously showed that the phosphorylation of akt within thp-1 macrophages is increased in response to the lipid hydrolysis products generated by lpl from total lipoproteins. notably, the free fatty acid (ffa) component was responsible for this effect. in the present study, we aimed to reveal more detail as to how the ffa component may affect akt signalling. we show that the phosphorylation of akt within thp-1 macrophages increases with total ffa concentration and that phosphorylation is elevated up to 18 hours. we further show that specifically the palmitoleate component of the total ffa affects akt phosphorylation. this is tied with changes to the levels of select molecular species of phosphoinositides. we further show that the total ffa component, and specifically palmitoleate, reduces apolipoprotein a-i-mediated cholesterol efflux, and that the reduction can be reversed in the presence of the akt inhibitor mk-2206. overall, our data support a negative role for the ffa component of lipoprotein hydrolysis products generated by lpl, by impairing macrophage cholesterol efflux via akt activation. lipoprotein lipase (lpl) is an extracellular enzyme required for the hydrolysis of triacylglycerols (tag), and to a much lesser extent phospholipids, from tag-rich lipoproteins within the bloodstream [1, 2] . lpl is anchored to the outside of the endothelial cells lining the luminal surface of capillaries in several tissues via heparan sulfate proteoglycans [3] [4] [5] , and as a headto-tail homodimer to glycerophosphatidylinositol high-density lipoprotein binding protein-1 [6] . lpl is expressed by several tissues, including adipose, skeletal, cardiac, breast, adrenal tissues, and also macrophages [3, 4] . in addition to its catalytic function, lpl also actively facilitates the uptake of lipoproteins and lipid hydrolysis products independently of catalytic activity, via a bridging function that brings the lipoproteins closer to the cell [7] . the expression of lpl is elevated in macrophages residing within atherosclerotic plaques, and its expression is positively correlated to the cholesterol content of the cells [8, 9] . in apolipoprotein e-null (apoe -/-) mice, the transgenic expression of human lpl accelerated atherosclerotic lesion formation [10] . conversely, when apoe -/mice were treated with mir-590 (which can inhibit macrophage lpl expression), lesion formation was prevented [11] . in thp-1 macrophages, the lentiviral overexpression of the long non-coding rna dapk1-it1 suppressed mir-590 expression, which in turn increased lpl expression and increased cholesterol accumulation [12] . though all of the mechanisms by which macrophage lpl contributes to the pathogenesis of atherosclerosis are not yet known, it has roles in the production of proinflammatory cytokines, smooth muscle cell recruitment, and it contributes to the lipid uptake of macrophages as part of their transition to lipid-laden foam cells [13] [14] [15] . previous work in our laboratory aimed to characterize the lipid species produced from the hydrolysis of total human lipoproteins by lpl, and it was demonstrated that the lpl hydrolysis products resulted in the phosphorylation of nine major signalling nodes and receptor tyrosine kinases within thp-1 human macrophages after 30 minutes [16] . the treatment of thp-1 macrophages with the total free fatty acid (ffa) component of the lpl hydrolysis products resulted in the phosphorylation of protein kinase b (also named akt), but none of the other signalling nodes and receptor tyrosine kinases [16] . it was postulated that one or more of the ffa liberated by lpl generated a molecular species of phosphatidylinositol (3, 4, 5) -trisphosphate (pip3) that preferentially activated akt [16] . akt is a serine/ threonine kinase that is downstream of phosphoinositide 3-kinase activity [17, 18] . the activation of akt involves two principal phosphorylation sites: ser-473 in the regulatory region and thr-308 in the active site [19] . akt itself is a kinase that can phosphorylate a variety of downstream proteins with a range of possible functions [18] . for example, akt phosphorylates tuberous sclerosis factor 2 (tsc2), which inactivates it and prevents it from inhibiting mammalian target of rapamycin complex 1; as a result, the anti-atherogenic process of cholesterol efflux is impaired [20] . we have also previously shown that the hydrolysis products liberated from total lipoproteins by lpl, and notably the total ffa component, impaired the gene expression of the cholesterol efflux transporters atp binding cassette transporter a1 (abca1), atp binding cassette transporter g1 (abcg1), and scavenger receptor bi (sr-bi) within thp-1 macrophages after 18 hours [21] ; in addition, cholesterol efflux to apolipoprotein a-i (apoa-i) was impaired [21] . because the total ffa component of lipoprotein hydrolysis products generated by lpl increases the levels of phosphorylated akt (pakt) [16] and it inhibits cholesterol efflux [21] , and the inhibition of akt improves cholesterol efflux [20] , we suspected that akt was a key intermediate in the mechanism by which lpl impairs cholesterol efflux. thus, we hypothesized that one or more specific fatty acids that exist within the total ffa component of lipoprotein hydrolysis products that are generated by lpl impair cholesterol efflux through the activation of akt. to test this hypothesis, using thp-1 macrophages, we examined the activation of akt in response to various ffa mixtures that contain the concentrations of ffa species that we previously reported to be found within lpl hydrolysis products from total lipoproteins [16] . we identified that palmitoleate significantly increased akt phosphorylation. we thus examined cholesterol efflux in response to incubations with palmitoleate and the akt inhibitor mk-2206. lastly, we examined the molecular species of phosphoinositides (pipx) of thp-1 macrophages treated with palmitoleate, to determine if there were changes to select pipx species that may contribute to a preferential activation of akt. we previously showed using antibody arrays that the hydrolysis products liberated by lpl from total lipoproteins (ρ<1.21 g/ml), as well as the reconstituted total ffa component matching that liberated by lpl at a physiological concentration of 0.68 mm, significantly increased the phosphorylation of akt after 30 minutes within thp-1 macrophages [16] . in a follow up to these previous observations, we first examined the phosphorylation of akt within thp-1 macrophages in response to the reconstituted total ffa component matching that liberated by lpl at 0.68 mm, over a time course between 10 minutes and 18 hours. relative to the vehicle controls for each time point, the percentage of phosphorylation of ser-473 on akt was higher, with maximal phosphorylation of 204% observed at 20 minutes (uncorrected p = 0.03) (fig 1a) . following a bonferroni-dunn correction on all points that were significant by multiple t-testing alone, only the 2 hour time point retained significance (with corrected p = 0.004). relative to the vehicle controls for each time point, the percentage of phosphorylation of thr-308 on akt reached a maximum of 226% at 15 minutes (uncorrected p = 0.02) (s1 fig) ; however, the phosphorylation returned to control levels by 30 minutes. following a bonferroni-dunn correction on all points that were significant by multiple t-testing alone, none of the time points retained significance. using the 2 hour time point because it retains significance following correction, we examined the phosphorylation of akt in response to different concentrations of the ffa mixture. relative to the vehicle control-treated cells, the percentage of akt phosphorylation at ser-473 increased in a dose-dependent fashion with ffa mixture concentrations of 0.17 mm, 0.34 mm, 0.68 mm, and 1.36 mm (fig 1b) . having clearly established that akt phosphorylation was affected by the reconstituted total ffa mixture that matched our previously reported concentrations of ffa species that are liberated by lpl from total lipoproteins, we sought to examine which class of fatty acid within the total ffa mixture might be responsible for the phosphorylation of akt. we prepared mixtures solely containing the saturated fatty acid (sfa), monounsaturated fatty acid (mufa), and polyunsaturated fatty acid (pufa) components of the total ffa mixture, we and tested their ability to phosphorylate akt within thp-1 macrophages. we chose a 2 hour incubation period because it retains significance following correction ( fig 1a) . our data show that only the mufa mixture significantly increased the phosphorylation of akt at ser-473 by 217% of control levels during a 2 hour period (p<0.05), which was comparable to the total ffa mixture (at 196% of control levels) (fig 2a) . we next tested the effect of oleate and palmitoleate, the two components of the mufa mixture, on the phosphorylation of akt; we show that only palmitoleate was able to significantly promote the phosphorylation of akt at ser-473, by 153% of control levels (p<0.05) ( fig 2b) . we previously postulated that the ffa component from lipoprotein hydrolysis products may be generating one or more specific species of pip3 that preferentially lead to the downstream activation of akt [16] . because the mufa component of the total ffa mixture, and specifically palmitoleate, resulted in the significant phosphorylation of akt, we examined if palmitoleate would indeed alter the levels of select species of pipx within thp-1 macrophages over an 18 hour period. we chose this time period because we previously showed it was a sufficient period for the total ffa to impair cholesterol efflux [21] . no significant changes were observed for all assessed species of phosphatidylinositol (pi)-(34:1, 36:0, 36:1, 36:2, 38:3, and 38:4) (fig 3a) , and for all assessed species of phosphatidylinositol monophosphate (pip)-(36:1, 36:2, 38:3, and 38:4) ( fig 3b) . however, there were notable changes in select phosphatidylinositol bisphosphate (pip2) and pip3 species. specifically, compared to control treatment, the treatment with palmitoleate increased the levels of the pip2 species 34:1 by 138% (p = 0.006) and 34:2 pip2 by 375% (p<0.0001) (fig 3c) , while the pip3 species 36:1 decreased a. thp-1 macrophages were incubated for 0, 10, 15, 20, 30, 120, 420, or 1080 minutes with either a vehicle control (control or c), or a 0.68 mm mixture of purified ffa (t) that matched the ratios observed post-hydrolysis of total human lipoprotein lipids by lpl. cell lysates were collected and proteins were subjected to immunoblot analyses. densitometry of akt and pakt were assessed; data are expressed as the ratio of pakt to total akt, as a percent of control. data are means 'se from three independent experiments, and statistical analysis was performed using multiple t-testing (uncorrected: †, p = 0.03; ‡, p = 0.008; §, p = 0.0003). following a bonferroni-dunn correction (with α = 0.05), only the 120 minute data retained significance (p = 0.004). inset, one complete set of immunoblot results from the three independent experiments. the complete set of uncropped immunoblots can be found in s8 fig. b . thp-1 macrophages were incubated for 2 hours with either a vehicle control (control), or a mixture of purified ffa that matched the ratios observed post-hydrolysis of total human lipoprotein lipids by lpl; concentrations of 0 mm, 0.17 mm, 0.34 mm, 0.68 mm, and 1.38 mm were tested. analyses of cell lysates were performed as in "a". data are from three independent experiments, and statistical analysis was performed using regression analysis (r 2 = 0.49, p = 0.01) and a t-test ( � , p = 0.02; �� , p = 0.005). inset, one complete set of immunoblot results from the three independent experiments. the complete set of uncropped immunoblots can be found in s9 fig. fig 2. analysis of ffa classes on akt phosphorylation. a. thp-1 macrophages were incubated for 2 hours with either a vehicle control (control or c), a 0.68 mm mixture of purified ffa (total or t) that matched the ratios observed post-hydrolysis of total human lipoprotein lipids by lpl, the sfa component of the total mixture (s), the mufa component of the total mixture (m), or the pufa component of the total mixture (p). cell lysates were collected and proteins were subjected to immunoblot analyses. densitometry of akt and pakt were assessed; data are expressed as the ratio of pakt to total akt, as a percent of control. data are from at least five independent experiments. statistical analysis was performed using a one-way anova with tukey's multiple comparisons testing. inset, one complete set of immunoblot results from the five independent experiments. the complete set of uncropped immunoblots can be found in s10 fig. b . thp-1 macrophages were incubated for 2 hours with either a vehicle control (control or c), 0.02 mm palmitoleate (16:1n-7 or 16:1), or 0.24 mm oleate (18:1n-9 or 18:1). cell lysates were collected and proteins were subjected to immunoblot analyses. analyses of cell lysates and data were performed as in "a". data are from five independent experiments. inset, one complete set of immunoblot results from the five independent experiments. the complete set of uncropped immunoblots can be found in s11 plos one levels to 55% of control (p = 0.006) but the pip3 species 34:2 levels increased by 192% (p = 0.005) ( fig 3d) . as we previously showed that the total ffa mixture impaired cholesterol efflux to apoa-i in thp-1 macrophages [21] , we sought to determine if impaired cholesterol efflux was in part due to the phosphorylation of akt. we examined cholesterol efflux to apoa-i in the absence or presence of the akt-specific inhibitor mk-2206, which abolishes akt phosphorylation within thp-1 macrophages at a concentration of 1 μm (s2 fig) . cholesterol efflux to apoa-i was assessed after an 18 hour incubation of cells with the total ffa mixture in the absence or presence of mk-2206, or a vehicle control. as expected, the total ffa treatment reduced the efflux of [ 3 h]cholesterol by 35% compared to control levels (p<0.001) (fig 4a) . cholesterol efflux was restored to control levels in the presence of mk-2206. lastly, we tested cholesterol efflux after an 18 hour incubation with each mufa component, oleate and palmitoleate, in the absence or presence of mk-2206, or a vehicle control. of note, oleate was at a 10.2-fold higher concentration versus palmitoleate. while oleate did not significantly affect cholesterol efflux (fig 4b) , palmitoleate significantly reduced cholesterol efflux by 31% compared to control levels (p<0.05); again, cholesterol efflux was restored to control levels in the presence of mk-2206. we also examined cholesterol efflux from thp-1 macrophages to hdl, and from acetylated ldl-treated thp-1 macrophages to both apoa-i and hdl, after an 18 hour incubation with palmitoleate; again, palmitoleate significantly reduced cholesterol efflux (s3 fig). to our surprise, although we previously showed that the total ffa mixture reduced the gene expression of the cholesterol transporters abca1, abcg1, and sr-bi [21] , immunoblot analyses showed that the treatment of cells with palmitoleate, or palmitoleate plus mk-2206, did not affect protein expression (s4 fig). in this study, we assessed the impact on akt phosphorylation and cholesterol efflux by the total ffa component that is derived from the hydrolysis of total lipoproteins by lpl. we show that the concentration of palmitoleate that is found within the total ffa component increased akt phosphorylation and reduced apoa-i-mediated cholesterol efflux (s5 fig)an effect that was reversed by inhibiting akt phosphorylation. the effect on cholesterol efflux to apoa-i and hdl was surprisingly independent of the protein expression for abca1, abcg1, and sr-bi. collectively, these data point toward an unknown mechanism that negatively influences the anti-atherogenic process of cholesterol efflux. akt is a 'master kinase' with over 100 unique downstream substrates and clearly defined roles in a variety of functions, such as cell growth and proliferation, insulin response, and angiogenesis [17] . as such, there are a myriad of pathways that could be influenced by a change in akt phosphorylation status, some of which have implications in atherogenic processes. for example, increased akt activation within human umbilical vein endothelial cells by tumor necrosis factor-α leads to the increased expression and secretion of monocyte chemoattractant protein-1, a protein involved in the recruitment of monocytes to a developing atherosclerotic lesion area [22] . akt also phosphorylates tsc2, which in turn prevents it from inhibiting mammalian target of rapamycin complex 1 and ultimately impairing cholesterol efflux; similar to our work, cholesterol efflux was improved in the presence of the akt inhibitor 10-[4'-(n,n-diethylamino)butyl]-2-chlorophenoxazine hydrochloride [20] . while in vitro studies exist to suggest that akt phosphorylation may be pro-atherogenic in nature, in vivo studies have not provided a clear role for akt in the development and progression of atherosclerosis. mice lacking both apolipoprotein e (apoe) and akt exhibit a more severe atherosclerotic phenotype versus mice lacking only apoe [23] . however, the absence of akt in lowdensity lipoprotein receptor-deficient mice did not further influence lesion development [24] . furthermore, mice lacking apoe display larger atherosclerotic plaque sizes and higher levels of phosphorylated akt versus mice lacking both apoe and the p110γ subunit of phosphoinositide 3-kinase (pi3k), which prevented akt phosphorylation [25] . it may be possible that akt and select downstream pathways play both protective and promotive roles in atherosclerosis, that may be dependent on the stage of development. this will require more careful future examination in vitro and in vivo. perhaps the most interesting finding from our study was that palmitoleate was sufficient to induce the activation of akt in the thp-1 macrophages and the inhibition of apoa-i-and hdl-mediated cholesterol efflux, while oleate at a 10.2-fold higher concentration failed to significantly affect these processes. we speculated that palmitoleate may reduce the expression of the cholesterol transporters abca1, abcg1, and sr-bi; however, immunoblot analyses showed no changes with transporter expression. future studies are necessary to examine why hours. cholesterol efflux was calculated as a percent of media [ 3 h]cholesterol per total cell and media [ 3 h]cholesterol; background efflux (in the absence of apoa-i) was subtracted from efflux data for apoa-i to obtain apoa-i specific efflux. data in "a" are from eight independent experiments, and data in "b" and "c" are from five independent experiments. statistical analysis was performed using a one-way anova with tukey's multiple comparisons testing. https://doi.org/10.1371/journal.pone.0233180.g004 hdl-mediated efflux was impaired. given that the mobilization of free cholesterol from lipid droplets via autophagy upstream of the abca1 transporter can be rate-limiting for cholesterol efflux to apoa-i [26, 27] , one likely possibility is that akt activation impairs this process. interestingly, akt inhibition was shown to increase abca1-mediated cholesterol efflux to apoa-i through supressing mammalian target of rapamycin complex 1 (mtorc1) [20] . conversely, akt activation by total ffa would be expected to increase mtorc1, which negatively regulates autophagy [28] , therefore leading to reduced autophagy-mediated lipid droplet-associated cholesteryl ester catabolism, reduced abca1-mediated cholesterol efflux to apoa-i, and cytosolic lipid droplet accumulation. this remains to be investigated. it is possible that other unsaturated fatty acids, such as arachidonate, docosahexaenoate, and eicosapentaenoate, inhibit cholesterol efflux through a similar mechanism, as these have been reported to impede apoa-i-mediated efflux [29] . we failed to show an effect with our pufa mixture on akt phosphorylation, but this may be due to differences in concentrations between the latter report and our current study, and potential differences in fatty acid metabolism between cell models. although there were no changes with protein expression, we previously reported that abca1, abcg1, and scarb1 mrna were reduced in response to the total ffa mixture [21] . however, an examination of the expression of the genes encoding these transporters, in the presence of the total ffa mixture without or with the akt inhibitor mk-2206, showed no difference of expression (s6 fig) . this is consistent with the findings of huang et al. [30] , who found that akt plays no role on the gene expression of cholesterol transporters in the hepg2 human hepatoma cell line. we also previously postulated that specific pip3 species may be generated to preferentially activate akt [16] . in the current study, an examination of thp-1 macrophage pipx in the absence or presence of palmitoleate ffa did reveal differences. although acyl chain fingerprinting was not performed, the 34:2 species of pip3 that is increased in thp-1 macrophages treated with palmitoleate may likely represent the incorporation of both palmitoleate and oleate. also reflecting this increase is the significant increase of the 34:2 pip2 species. we also observed a significant increase of the 34:1 pip2 species, which may represent the incorporation of stearate and palmitoleate; this is supported by the modest (but not significant) elevation of the 34:1 pi species. because the evaluation of pipx was on samples following an 18 hour incubation, it is possible that an earlier time point may have exhibited a more pronounced increase of select pipx species in response to palmitoleate, and also a likely elevation of 34:1 pip3, which we did not observe. however, while there remains more to elucidate about the mechanism of activation, these data provide clues to specific species of pipx that may preferentially influence the pi3k-akt pathway and cholesterol efflux-a focus of future investigation that is beyond the scope of the current study. in a 2009 meta-analysis evaluating the effects of certain types of fats on cardiovascular outcomes, it was suggested that replacing the sfa in the diet with pufa or mufa could improve outcomes [31] . the authors' hypothesis was initially that either mufa or pufa could improve cardiovascular disease risk when compared to sfa, but they surprisingly found no association with mufa. the authors indicated that the source of dietary mufa was primarily derived from animal fat, and they suggested that it was a possible confounding factor in their conclusions [31] . however, our data suggest that mufa incorporation into atherosclerotic plaques may actually be an accelerating factor by partially inhibiting cholesterol efflux. in fact, the rate of degradation of abca1 was shown to be increased in two different mouse macrophage models in the presence of unsaturated fatty acids [32] . what remains to be tested is how the impaired cholesterol efflux in response to a palmitoleate-enriched diet would translate into reverse cholesterol transport functionality in vivo. in conclusion, we have demonstrated that palmitoleate increases akt phosphorylation in thp-1 macrophages and reduces apoa-i-mediated cholesterol efflux through a mechanism mediated by akt. it is possible that other macrophage cell lines that do not require phorbol 12-myristate 13-acetate (pma) for differentiation exhibit the same phenomenon, but this remains to be examined. the likely accumulation of sterols in response to the total ffa mixture is further supported by work in our laboratory showing that the inhibition of pi3k reduces ffa-mediated neutral lipid accumulation in our model (s7 fig). of note, the total ffa mixture we used was derived from the concentration of ffa species that we previously reported are from total lipoproteins in the presence of lpl. these lipoproteins, and our subsequently tested ffa concentrations, were based on lipoproteins isolated from normolipidemic subjects. in the future, it would be of interest to examine the response of the pi3k-akt pathway using lipoproteins, and the ffa component liberated by lpl, from subjects with controlled and uncontrolled hyperlipidemia; samples from such subjects might exhibit unique lipoprotein lipidomes that may impair cholesterol efflux in response to lpl hydrolysis, in addition to harboring high-density lipoproteins with poor cholesterol efflux capacity. a total ffa mixture matching concentrations that we previously reported to be liberated by lpl during hydrolysis of total lipoproteins was prepared as previously described [16, 21] . briefly, myristate, palmitoleate, palmitate, linoleate, oleate, stearate, arachidonate, and docosahexaenoate (all purchased from nu-chek prep, elysian, mn, usa) were stored until needed at -20˚c under n 2(g) as 10 mg/ml solutions in liquid chromatography-mass spectrometry grade methanol (fisher scientific, ottawa, on, canada). prior to preparation of ffa mixtures, stock ffa were brought to room temperature. to prepare 1 ml media with 0.68 mm total ffa for cell culture, 18 thp-1 human monocytic leukemia cells (american type culture collection, manassas, va, usa) were cultured in t75 flasks with rpmi, which was further supplemented with 10% v/v fetal bovine serum (cat. #sh30396.03, fisher scientific) and 1% v/v a/a. the cells were incubated at 37˚c with 5% co 2(g) . to differentiate cells into macrophages, thp-1 cells were seeded into each well of a 6-well plate at a density of 3.86 x 10 5 cells/ml in rpmi containing 10% v/v fetal bovine serum, 1% v/v a/a, and 100 nm pma. after 48 hours, cells were washed three times with rpmi, then incubated with rpmi containing 0.2% w/v faf-bsa, 1% v/v a/a, and 100 nm pma. after 24 hours, cells were washed three times with rpmi, then incubated for 2 hours (unless otherwise stated) with 1 ml of media containing ffa (prepared as described above), or the vehicle control media. following incubation, cells were washed three times with 2 ml ice-cold phosphate-buffered saline (ph 7.0). cells were lysed on ice for 15 minutes with a commercial buffer (#98038, cell signaling technology, danvers, ma, usa), supplemented with 0.1% v/v protease/phosphatase inhibitor (#5872s, cell signaling technology); cells were collected and stored at -80˚c until needed. prior to use, the protein content of cell lysates was quantified using a bicinchonic acid protein assay kit (fisher scientific), according to manufacturer's instructions. proteins (5 μg) were separated by sds-page using 12% resolving gels and transferred to nitrocellulose membranes. for abca1, 60 μg of protein were separated by sds-page using 8% resolving gels. following transfer, the membranes were blocked overnight at 4˚c in trisbuffered saline (tbs) at ph 7.4, containing 5% w/v bovine serum albumin (bsa, millipore sigma), 0.05% v/v tween-20 (fisher scientific), and 0.05% w/v nan 3 (fisher scientific). after blocking, the membranes were incubated at 4˚c overnight with antibodies against human akt (1:1,000 dilution of cat. #9272, cell signaling technology), ser-473 phosphorylated human akt (1:2,000 dilution of cat. #4060, cell signaling technology), thr-308 phosphorylated human akt (1:2,000 dilution of cat. #2965s, cell signaling technology), human abca1 (1:500 dilution of cat. #nb400-105, novus biologicals, centennial, co, usa), human abcg1 (1:1,000 dilution of cat. #nb400-132, novus biologicals), human sr-bi (1:500 dilution of cat. #nb400-104, novus biologicals), or human β-actin (1:5,000 dilution of cat. #nb600-501, novus biologicals). all primary antibodies were diluted with tbs containing 5% bsa, 0.05% v/v tween-20, 0.05% w/v nan 3 . following incubation, membranes were washed four times with tbs containing 0.05% v/v tween-20, then incubated for 2 hours with horseradish peroxidase-conjugated antibodies against rabbit igg to detect akt, pakt, abca1, abcg1, and sr-bi (1:2,000 dilution of cat. #sa1-200, fisher scientific) or mouse igg to detect β-actin (1:2,000 dilution of cat. #sa1-100, fisher scientific). following incubation, membranes were washed four times with tbs, then visualized using an imagequant las 4000 system (ge healthcare, baie d'ufre, qc, canada) following development using the ecl™ prime western blotting detection reagent (ge healthcare), according to manufacturer's protocol. the densitometry of protein bands was quantified using imagej [33] . apoa-i (lee biosolutions, maryland heights, mo, usa) was salt-exchanged using a pd-10 desalting column (ge healthcare) equilibrated with phosphate-buffered saline, and the protein concentration was measured using the absorbance at 280 nm and the molar absorption coefficient of 1.23 ml/ mg�cm [34] . cholesterol efflux assays using thp-1 macrophages were carried out as previously described [21] . in brief, cells were cultured in 12-well plates and radiolabelled with 1 μci/ml pi, pip, pip2, and pip3 were analyzed as previously described [35] . in brief, lipids were extracted from cell pellets with acidified solvents prior to derivatization with diazomethyltimethylsilane. dry derivatized lipid extracts were dissolved in 100 μl methanol (80% aqueous solution) prior to liquid chromatography separation with mass spectrometry detection using an acquity uplc system system hyphenated to a qtrap 4000 mass spectrometer (waters, wilmslow, u.k). the mobile phase consisted of a linear gradient from 45% acetonitrile with 0.1% formic acid in water, changing over 20 minutes to 90% acetonitrile with 0.1% of formic acid in water. the column used was a waters beh 300 c4 (100 × 1.0 mm), 1.7 μm. the areas of the total ion current of each pip, pip2 and pip3 lipid molecular species were corrected for recovery to internal standard. the reported data were normalized to the recovery of the pi content. statistical analyses were performed using graphpad prism 8.2. an unpaired student's t test or one-way anova with tukey's multiple comparison post-test were used, unless otherwise stated. a p<0.05 was considered significantly different. unless otherwise stated, all data are given as mean ± sd. . cholesterol efflux in the absence or presence of 50 μg/ml apoa-i or 50 μg/ ml hdl was examined after 6 hours. cholesterol efflux was calculated as a percent of media [ 3 h]cholesterol per total cell and media [ 3 h]cholesterol; background efflux (in the absence of apoa-i or hdl) was subtracted from efflux data for apoa-i (or hdl) to obtain apoa-i (or hdl) specific efflux. data are means ± standard error, from a representative experiment with triplicate wells. statistical analysis was performed using a t-test. (pdf) s4 fig. protein expression of abca1, abcg1 , and sr-bi. in three independent experiments, thp-1 macrophages were incubated for 18 hours with either a vehicle control (control), 0.02 mm palmitoleate (16:1 or 16:1n-7), or 0.02 mm palmitoleate in the presence of 1 μm mk-2206 (+ mk). cell lysates were collected and proteins were subjected to immunoblot analyses. a. one complete set of immunoblot results. the complete set of uncropped immunoblots can be found in s12 fig. b . densitometry analysis of abca1, expressed as a percent of control. c. densitometry analysis of abcg1, expressed as a percent of control. d. densitometry analysis of abca1, expressed as a percent of control. note, data were normalized to densitometry data for β-actin. (pdf) in our model, macrophages take up the palmitoleate introduced to the cell medium through a fatty acid transporter (likely cd36). we suspect that palmitoleate can then be incorporated into phosphatidylinositide biosynthesis and leading to a pip3 species (via phosphatidylinositol 3-kinase (pi3k)) that preferentially activates both phosphoinositide-dependent kinase 1 (pdk1) and the mammalian target of rapamycin (mtor) complex 2 (mtorc2-which includes mtor, mammalian lethal with sec13 protein (mlst8), rictor, and stress-activated protein kinase-interacting protein 1 (sin1)). at the membrane interface, akt is then in turn phosphorylated by pdk1 at thr308 and by mtorc2 at ser473. the phosphorylation of akt renders it active, allowing it to phosphorylate other proteins. the akt-mediated pathway influencing cholesterol efflux remains to be determined. fig 1b. thp-1 macrophages were incubated for 2 hours with either a vehicle control (control), or a mixture of purified excitement for validated data, his firm believe in frontier research, and more importantly collaborating to push knowledge forward. he truly will be missed, andrea f. lopez-clavijo. evaluation of the roles of lipoprotein lipase and hepatic lipase in lipoprotein metabolism: in vivo and in vitro studies in man characterization of the lipolytic activity of endothelial lipase the tissue distribution of lipoprotein lipase determines where chylomicrons bind the tissue-specific expression of lipoprotein lipase: implications for energy and lipoprotein metabolism a disordered acidic domain in gpihbp1 harboring a sulfated tyrosine regulates lipoprotein lipase structure of the lipoprotein lipase-gpihbp1 complex that mediates plasma triglyceride hydrolysis heparan sulfate proteoglycans are involved in the lipoprotein lipase-mediated enhancement of the cellular binding of very low denisty and low density lipoproteins a proposal linking atherogenesis to the interaction of endothelial lipoprotein lipase with triglyceride-rich lipoproteins effect of cholesterol feeding on arterial lipolytic activity in the rabbit. atherosclerosis macrophage-specific expression of human lipoprotein lipase accelerates atherosclerosis in transgenic apolipoprotein e knockout mice but not in c57bl/6 mice microrna-590 inhibits lipoprotein lipase expression and prevents atherosclerosis in apoe knockout mice the lncrna dapk-it1 regulates cholesterol metabolism and inflammatory response in macrophages and promotes atherogenesis macrophage lipoprotein lipase promotes foam cell formation and atherosclerosis in vivo induction of tumor necrosis factor a gene experssion by lipoprotein lipase ldl receptor family-dependent and -independent pathways for the internalization and digestion of lipoprotein lipase-associated β-vldl by rat vascular smooth muscle cells hydrolysis products generated by lipoprotein lipase and endothelial lipase influence macrophage cell signalling pathways akt/pkb signaling: navigating downstream role of translocation in the activation and function of protein kinase b solution structure and backbone dynamics of the pleckstrin homology domain of the human protein kinase b (pkb/akt). interaction with inositol phosphates akt inhibition promotes abca1-mediated cholesterol efflux to apoa-i through supressing mtorc1 cholesterol efflux from thp-1 macrophages is impaired by the fatty acid component from lipoprotein hydrolysis by lipoprotein lipase tnf-α stimulation of mcp-1 expression is mediated by the akt/pkb signal transduction pathway in vascular endothelial cells loss of akt1 leads to severe atherosclerosis and occlusive coronary artery disease macrophage deficiency of akt2 reduces atherosclerosis in ldlr null mice deletion of the phosphoinositide 3-kinase p110γ gene attenuates murine atherosclerosis regulation of lipid droplet cholesterol efflux from macrophage foam cells microrna-33 regulates macrophage autophagy in atherosclerosis mtor regulation of autophagy eicosapentaenoic acid membrane incorporation impairs cholesterol efflux from cholesterol-loaded human macrophages by reducing the cholesteryl ester mobilization from lipid droplets mcp-1 impacts rct by repressing abca1, abcg1, and sr-bi through pi3k/akt posttranslational regulation in hepg2 cells major types of dietary fat and risk of coronary heart disease: a pooled analysis of 11 cohort studies unsaturated fatty acids inhibit cholesterol efflux from macrophages by increasing degradation of atp-binding cassette transporter 1 image to imagej: 25 years of image analysis apolipoproteinmediated plasma membrane microsolubilization: role of lipid affinity and membrane penetration in the efflux of cellular cholesterol and phospholipid quantification of ptdinsp3 molecular species in cells and tissues by mass spectrometry the authors with to thank greg west (the babraham institute) for his excellent technical assistance, and catherine wright (university of washington) for her recommended statistical analyses of select data. ffa that matched the ratios observed post-hydrolysis of total human lipoprotein lipids by lpl; concentrations of 0 mm, 0.17 mm, 0.34 mm, 0.68 mm, and 1.38 mm were tested. cell lysates were collected and proteins were subjected to immunoblot analyses. shown are one complete set of immunoblot results. a. pakt (of ser-473). b. akt. c. β-actin. during the review of the revised version of our manuscript, michael wakelam passed away on march 31, 2020 due to complications from a suspected covid-19 infection. it is not easy to say "michael wakelam was" because his legacy as a person, scientist, professor, parent, husband, colleague, and friend is still very much alive. it has been only a month of michael's passing away and it is impossible to imagine the babraham institute without him. michael's directorship as well as wisdom, dedication, loyalty, devotion to science and to the people, made the institute a lovely place to work in. michael's passion for lipidomics and mass spectrometry extended his scientific collaborations beyond the babraham institute to lots of groups in the united kingdom, and internationally. this publication reflects michael's key: cord-259771-653opx0h authors: dwivedi, varun; manickam, cordelia; binjawadagi, basavaraj; joyappa, dechamma; renukaradhya, gourapura j. title: biodegradable nanoparticle-entrapped vaccine induces cross-protective immune response against a virulent heterologous respiratory viral infection in pigs date: 2012-12-11 journal: plos one doi: 10.1371/journal.pone.0051794 sha: doc_id: 259771 cord_uid: 653opx0h biodegradable nanoparticle-based vaccine development research is unexplored in large animals and humans. in this study, we illustrated the efficacy of nanoparticle-entrapped uv-killed virus vaccine against an economically important respiratory viral disease of pigs called porcine reproductive and respiratory syndrome virus (prrsv). we entrapped plga [poly (lactide-co-glycolides)] nanoparticles with killed prrsv antigens (nano-kag) and detected its phagocytosis by pig alveolar macrophages. single doses of nano-kag vaccine administered intranasally to pigs upregulated innate and prrsv specific adaptive responses. in a virulent heterologous prrsv challenge study, nano-kag vaccine significantly reduced the lung pathology and viremia, and the viral load in the lungs. immunologically, enhanced innate and adaptive immune cell population and associated cytokines with decreased secretion of immunosuppressive mediators were observed at both mucosal sites and blood. in summary, we demonstrated the benefits of intranasal delivery of nanoparticle-based viral vaccine in eliciting cross-protective immune response in pigs, a potential large animal model. microencapsulation of vaccine agents and drugs in biodegradable polymer and its delivery in humans is an innovative approach to create more robust medications and vaccines in the 21 st century. the biocompatible and biodegradable polymer, plga [poly (lactide-co-glycolides)], is the us fda approved material in the development of nanoparticle-based controlled release delivery system [1] . plga slowly degrades and releases the vaccine over a long-period of time, thus avoiding the need of a booster dose [2] . being particulate in nature, the nanoparticle-mediated delivery promotes uptake of ags by professional antigen presenting cells (apcs). nanoparticle as a delivery vehicle to vaccines and drugs has been extensively evaluated in mouse models. however, there are limitations in the translation of novel rodent findings to improve human and food animal health [3] . therefore, pig may serve as a useful large animal model for such research. moreover, due to their physiological, anatomical, and immunological similarity to humans, pigs are already in use as an animal model system to study a few viral diseases [4] . although, the current study is focused on a respiratory pathogen which infects pigs, and the vaccine evaluation strategy may help to consider pig as a model system. among the swine diseases, porcine reproductive and respiratory syndrome (prrs) is highly devastating, causing an estimated economic loss of $664 million annually in the us [5] . this translates into $1.8 million losses per day annually. prrsv infects pigs of all ages and is caused by a highly mutating, positive sense, single stranded rna virus belongs to the family arteriviridae [6] . prrs in growing pigs causes anorexia, fever, respiratory distress, and enhanced susceptibility to secondary microbial infections; while in pregnant sows it is characterized by reproductive dysfunction and abortions [7] . primary prrsv permissive cells are alveolar macrophages (mws) [8] . prrsv rapidly modulates the host innate immune response, such as dampens the nk cell cytotoxicity and reduces the ifn-a production, and upregulates immunosuppressive mediators from as early as two days postinfection [9] ; which lead to poor adaptive immune response and delayed/weak virus neutralizing antibody response, resulting in prrsv persistence. nevertheless, due to high degree of constant genetic and antigenic variations, control of prrs remains a challenge to the swine industry worldwide. optimal mucosal immunization induces protective immune response at both mucosal and systemic sites compared to systemic immunization [10] . generation of protective iga response is essential to reduce and/or prevent the entry of pathogens whose principle port of entry is through mucosal sites [11] . mucosal immunization induces effective immune response at both local and distant effector sites. particulate antigen delivery system facilitates the passage of ags through mucosal barrier and leads to stimulation of the underlying mucosal immune cells [12] . biodegradable microspheres made of chitosan, plga, and liposome have been in use to deliver candidate vaccines to mucosal sites [13] . a study using nanoparticle-entrapped killed influenza virus vaccine administered with an adjuvant intranasally to mice, rabbits, and pigs elicited protective immune response, with better immunity induced in pigs by intranasal compared to intramuscular route of vaccination [14] . a single intranasal delivery of plga nanoparticle-entrapped schistosoma mansoni ags to mice elicited protective neutralizing antibody response in the lungs and blood [15] . since late 1990 s, modified live prrsv (prrs-mlv) and killed virus vaccines are in use to control prrs, but neither of them protects pigs completely against heterologous field viruses [16] . like the field virus, prrs-mlv also induces immunosuppression [17, 18] . moreover, there are reports of reversion of vaccine virus into virulence leading to severe disease outbreaks [19, 20] . although available killed prrsv vaccines are safe, they are poorly immunogenic [21] . thus, to control prrs outbreaks innovative vaccine strategies are required. in the current study, killed prrsv ags were encapsulated in plga nanoparticles and characterized the candidate vaccine in vitro and in a pre-and -postchallenge study in pigs. various immune correlates of protection were analyzed both at mucosal and systemic sites to show the evidences of cross-protective immunity. preparation of vaccine antigens and plga nanoparticlekilled prrsv vaccine (nano-kag) marc-145 cell-monolayer was infected with the prrsv vr2332 strain [22] at 0.001 moi (multiplicity of infection), and the harvested infected cell culture fluid was clarified and subjected to ultracentrifugation (20% sucrose overlay) at 100,0006g for 2 hr at 4uc. the semi-purified viral pellet was suspended in pbs; titrated, inactivated by uv irradiation (at 254 nm uv-irradiation [el series uv lamps, uvp, llc (ca); 8 watt/115v -60 hz/ 0.32 amps] for 1 hr), sonicated, and the protein content was estimated using the bca kit (biorad, ca). viral inactivation was confirmed by cell culture immunofluorescence assay in marc-145 cells. the viral pellet was aliquoted and stored at 270uc. control antigen was prepared similarly using uninfected marc-145 cells. sterile precautions were followed throughout the antigen preparation and processing procedures to avoid any bacterial contamination. nanoparticles were prepared using standard double emulsion solvent evaporation technique [23, 24] . briefly, 15% of plga 50/50 (750 mg) was dissolved in 5 mg of killed vr2332 proteins, homogenized at 6000 rpm for 90 seconds, then added to aqueous solution of 10% polyvinyl alcohol and homogenized. finally, the preparation was stirred overnight and the washed nanoparticles were freeze-dried and stored at 4uc. the amount of entrapped prrsv protein in the nanoparticles was determined as described previously [25] . the size and shape of nanoparticles was determined by scanning electron microscopy (hitachi s-3500n). bronchoalveolar lavage fluid (bal) collected from three 4-6 weeks old healthy spf pigs was processed to isolate mononu-clear cells (bal-mnc) [26] . bal-mnc (1610 6 cells per ml) were plated in a 24 well plate containing poly-l-lysine coated cover slips for 1 hr. non-adherent cells were aspirated and the adherent cells were treated with freeze-dried nano-kag containing different concentrations of prrsv proteins suspended in dmem containing 10% fbs. cells uninfected or infected with prrsv (vr2332 strain) at 0.1 moi for 12 hr was included as control. cells were fixed in 3% paraformaldehyde for 15 min, permeabilized (0.1% triton x-100) and blocked (pbs containing 5% bsa and 0.2% triton x-100) for 1 hr at room temperature (rt). subsequently, treated with anti-prrsv nucleocapsid specific mab sdow17 (rural technologies, inc.,) and early endosome specific goat polyclonal anti-early endosome antigen 1 (eea-1) igg (santa cruz biotechnology, santa cruz, ca), diluted in the dilution buffer (pbs containing 1% bsa and 0.1% triton x-100) for 1 hr at rt. followed by treatment with goat anti-mouse igg alexa flour488 and donkey anti-goat alexa flour 633 (invitrogen), and incubated for 1 hr at rt. cells were washed in between the treatment steps and treated with a mounting medium containing 2.5% dabco (sigma). stained coverslips were mounted on a clean glass slide using transparent nail polish and viewed under a leica confocal microscope. the acquired images were analyzed using leica confocal software. in vitro uptake of nano-kag by pig mws and determination of cd80/86 expression bal-mnc (1610 6 cells per ml) were seeded in a 24-well plate and untreated or treated with k-ag or nano-kag (2, 0.2, and 0.02 mg/ml of prrsv protein) and incubated for 3 hr at 37uc. cells uninfected or infected with prrsv (mn184 strain) at 0.1 moi for 12 hr was served as control. cells were treated with anti-prrsv n' mab followed by goat anti-mouse igg alexa flour488, washed, and fixed before analysis. to assess the expression of cd80/86 on professional antigen presenting cells (apcs), bal-mnc were treated as above for 16 hr at 37uc, washed and stained using biotinylated human ctla4-mouse immunoglobulin fusion protein (ancell, mn) and pe-conjugated cd172 (southern biotech) [27] , followed by streptavidin percpcy5.5. cells were fixed and analyzed using facs aria ii (bd biosciences) flow cytometer. conventional large white-duroc crossbred weaned specificpathogen-free (spf) pigs from three different litters at 3-4 wks of age were confirmed seronegative for prrsv, porcine respiratory corona virus, transmissible gastroenteritis virus, and porcine circo virus 2 antibodies. in a pre-challenge study, pigs (n = 9) were grouped randomly into three groups (n = 3 per group). group iunvaccinated (mock) pigs inoculated with dmem and pbs; group ii -inoculated with k-ag; group iii -inoculated with nano-kag. each vaccine (nano-kag and k-ag) dose has one mg of crude viral preparation containing ,5610 6 tcid 50 of inactivated virus. the vaccine was inoculated once, intranasally. all the pigs were euthanized on post-immunization day (pid) 15 and evaluated for innate and virus specific adaptive immune responses. in a post-challenge study, pigs (n = 12) were divided randomly into four groups (n = 3 per group). group i -mock pigs; group ii -inoculated with normal saline; group iii -inoculated with k-ag; group iv -inoculated with nano-kag. each vaccine dose had same amount of ags as described above. groups ii, iii, and iv were challenged with prrsv mn184 (0.5610 6 tcid 50 /ml , 2 ml per pig) on pid 21 and euthanized on day post-challenge (dpc or pc) 15. all the inoculations were performed once by intranasal route. the dose of nano-kag (1 mg per pig) was chosen based on the results of a dose-dependent response study performed earlier in pigs. mock-inoculated pigs were euthanized separately before sacrificing virus challenged animals. pigs received food and water ad libitum and maintained under the supervision of a veterinarian. all the pigs were maintained, samples collected, and euthanized as per the standard procedures with necessary efforts to minimize suffering of animals. the animal use protocol was approved by the committee on the ethics of animal experiments of the ohio state university. this study was carried out in strict accordance with the recommendations by public health service policy, united states department of agriculture regulations, the national research council's guide for the care and use of laboratory animals, and the federation of animal science societies' guide for the care and use of agricultural animals in agricultural research and teaching, and all relevant institutional, state, and federal regulations and policies regarding animal care and use at the ohio state university. the protocol was approved by the committee on the ethics of animal experiments of the ohio state university (protocol number: 08-ag028). all the pigs were maintained, samples collected, and euthanized, and all efforts were made to minimize suffering of animals. during necropsy the lungs and lymph nodes were examined grossly and histologically. macroscopic pulmonary lesions were given an estimated score based on the percentage of consolidated lesions in individual lobes as described previously [28] . the lung tissue samples collected from the caudal lobe was fixed in 10% neutral buffered formalin and sections (3 mm) made were stained for hematoxylin-and-eosin (h&e) as described previously [28] . frozen lung sections were immunostained as described previously [29] . briefly, sections were treated with prrsv nucleocapsid protein specific mab (sdow17) or isotype control mab followed by abc peroxidase staining kit (vectastain elite, vector labs) and the labeling was visualized by application of dab (3, 39diaminobenzidine) substrate (vector laboratories) and counterstained with hematoxylin. immunostained slides were examined by an unbiased certified veterinary pathologist to score the presence of prrsv ags. prrsv titer and virus neutralizing antibody titer in serum and in the lung homogenate was analyzed by indirect immunofluorescence assay (ifa) as previously described [30] . prrsv specific iga and igg antibodies in serum and lung lysate (homogenate) were analyzed by elisa. briefly, elisa plates were coated with pre-titrated semi-purified killed prrsv (mn184) ags (10 mg/ml) in carbonate-bicarbonate buffer (ph 9.6), washed and blocked (1% bsa+0.1% tween 20 in pbs). serum (1:100) and lung lysate (0.5 mg/ml, w/v) samples were added and incubated for 2 hr at rt. the bound virus specific isotype antibody was detected using anti-pig iga and igg secondary antibodies conjugated with hrp (kpl). plates were developed using the chromogen tmb and read at 450 nm. we also included non-prrsv antigen-coated plates as control and the od values obtained from experimental plate were subtracted from the control. five million pig pbmc, tbln (tracheobronchial lymph nodes) mnc, and lung mnc were subjected to ex vivo restimulation in the absence or presence of prrsv mn184 ags (50 mg/ml) as described previously [31] , and the harvested supernatant was analyzed to measure cytokines. cytokines secreted by immune cells cultured in the absence of prrsv ags was subtracted from the corresponding test value. serum samples, harvested culture supernatants, and lung lysates were analyzed for th1 (ifn-c and il-12), th2 (il-4), proinflammatory (il-6), and immunosuppressive (il-10 and tgf-b) cytokines by elisa [31] . amount of cytokines present in the lung lysate was normalized to picogram per gram of lung tissue. flow cytometry analysis was performed to determine the phenotype and the frequency of different immune cells by a multicolor immunoassay as described previously [31] . since mnc were isolated from different amounts (weights) of tissues (lungs and tbln) we did not assess the absolute cell numbers. in this study, we determined relative frequency of individual immune cell subset by immunostaining fixed number of mnc (one million) from each site of collection, and 50,000 events were acquired in bd facs aria ii (bd biosciences) and analyzed using flowjo software. all data were expressed as the mean of three pigs +/2 sem. statistical analyses were performed using one way analysis of variance (anova) followed by post-hoc tukey's test using graphpad instat (software version 5.0) to establish differences among unvaccinated, k-ag and nano-kag pig groups in postchallenge trial and between k-ag and nano-kag pig groups in pre-challenge trial. statistical significance was assessed as p,0.05. morphology of sham and prrsv ags entrapped plga nanoparticles was determined by scanning electron microscopy which revealed the size of particles as 200-600 nm (figure 1, a) . the average protein content in nanoparticles or core-loading was 0.50-0.55% (w/w), which represents an encapsulation efficiency of 50-55%. upon re-dispersion of the nano-kag in pbs, prrsv proteins were released slowly in the first 48 hr, later a gradual release profile was observed over the next 5-weeks (data not shown). uptake of nano-kag by apcs was studied using bal-mnc harvested from three healthy spf pigs. the confocal images of alveolar mws revealed preferential uptake of nano-kag but not unentrapped viral ags (k-ag), prrsv infected cells served as a positive control (figure 1, b) . engulfed nanoparticles delivered the prrsv ags to early endosomes and it was comparable to virusinfected control (figure 1, c & d) . further, nano-kag engulfed apcs underwent maturation as indicated by significantly increased expression of cd80/86 (figure 1, e) . in addition, 57% of bal-mnc treated with nano-kag was positive for prrsv protein comparable to virus infected cells (figure 1 , f ii & iv). in contrast, only 9% bal-mnc treated with k-ag were positive for viral protein (figure 1, f iii) . our results suggested that prrsv ags delivered in nanoparticles were phagocytosed by apcs and the released protein was found in the endosomes. in a pre-challenge study, intranasal delivery of nano-kag resulted in induction of innate immune response at both mucosal and systemic sites, indicated by a significant increase in the frequency of nk cells, dcs, and cd t cells in the lung mnc ( figure 2 , a-c); and cd t cells and dcs in the pbmc compared to k-ag vaccinated pigs (figure 2, h & i) . immune cells involved in adaptive arm of the immune response, such as cd4 + cd8 + t cells (th/memory) and cd8 + t cells were increased significantly in the lung mnc of nano-kag compared to k-ag vaccinated pigs (figure 2, d & e) . further, lung mnc and pbmc from nano-kag immunized pigs secreted significantly reduced levels of the cytokine, il-10, and higher amounts of il-6 in a recall response (figure 2, f, g, & j) . in addition, innate cytokine ifn-a was secreted at significantly higher levels in pigs vaccinated with nano-kag compared to both the control groups ( figure 2 , k). in a post-challenge study, nano-kag vaccinated mn184 challenged pigs were clinically healthy with no fever or respiratory distress. in contrast, both k-ag and unvaccinated, mn184 challenged pigs had irregular fever with reduced feed intake during the first two-week post-challenge. microscopic examination of h&e stained lung sections of unvaccinated and k-ag vaccinated, mn184 challenged pigs' revealed severe pneumonic lesions with massive infiltration of mononuclear cells with large consolidated area. in contrast, significantly reduced lung lesions were observed in nano-kag vaccinated virus challenged pigs (figure 3, a) . significantly reduced gross lung lesion scores in nano-kag immunized group compared to other two virus challenged groups was observed (figure 3, c) . immunohistochemistry analysis had revealed abundant prrsv antigen positive cells in the lung sections of unvaccinated and k-ag vaccinated, mn184 challenged pigs compared to nano-kag received pigs (figure 3, b & d) . prrsv titer in serum samples indicated a reduced viral load of greater than one-log at pc 7 with complete viral clearance by pc 15 in nano-kag vaccinated, compared to k-ag vaccinated and unvaccinated pigs (figure 3, e) . similarly, prrsv load in the lungs was also reduced (although not significant) in nano-kag compared to k-ag immunized, mn184 virus challenged pigs (figure 3, f) . also the prrsv titer (tcid 50 /ml) in both the serum and lung homogenate showed a lung homogenates of nano-kag immunized pigs contained significantly higher levels of virus specific iga and igg antibodies compared to unvaccinated and k-ag vaccinated, mn184 challenged pigs (figure 4, a & b) . in the serum samples of nano-kag vaccinated pigs increased iga antibody levels at pc 0 (samples collected on the same day as viral challenge but before inoculating the challenge virus) with a significant increase at pc 15 compared to either unvaccinated or k-ag vaccinated, mn184 challenged pigs was detected (figure 4, d) . the prrsv specific igg antibody levels in serum (figure 4 , e) and both iga and igg levels in the nasal swab (figure 4 , g & h) were significantly higher in nano-kag vaccinated, compared to unvaccinated and k-ag immunized pigs at dpc 15. significantly increased prrsv specific neutralizing antibody (vn) titers in serum of both k-ag and nano-kag vaccine received pig groups was observed at pc 7, which still remained high (although not significant) only in the nano-kag group at pc 15. in the lungs, a similar trend of increased (but not significant) titers of neutralizing antibodies in nano-kag immunized pigs was seen (figure 4, c & f) . nano-kag vaccine received mn184 virus challenged pigs had significantly increased innate ifn-a production in the lungs ( figure 5, a) . in k-ag vaccinated and unvaccinated pigs, a fourfold reduction in nk cell frequency compared to mock pigs was observed. in contrast, in nano-kag vaccinated pigs the nk cell frequency was significantly higher than the k-ag and unvaccinated virus challenged pigs ( figure 5, b) . further, lung nk cellcytotoxic function in unvaccinated and k-ag vaccinated, mn184 virus challenged pigs was completely suppressed; however, in nano-kag received pigs it was partially rescued (figure 5, c) . the frequency of cd t cells and cd4 + (but not cd8 + ) t cells in the lungs of nano-kag vaccinated animals were significantly increased compared to k-ag and unvaccinated, virus challenged pigs ( figure 5 , d, e & f). in the peripheral blood of nano-kag immunized pigs a significantly increased frequency of dcs, and in tbln significantly increased frequency of both dcs and cd t cells was observed ( table 1) . pigs vaccinated with nano-kag had significantly reduced foxp3 + t-regulatory cell (treg) population in the lungs, compared to unvaccinated and k-ag received pigs (figure 6, a) . immunosuppressive cytokines (il-10 and tgf-b) response in the lungs was significantly reduced in nano-kag vaccinated pigs; and also their decreased secretion was detected in lung mnc restimulated with killed mn184 ags (figure 6 , b, c, e & f). in contrast, a significantly increased ifn-c in the lung homogenate, and its secretion in antigen restimulated lung mnc was detected in nano-kag vaccinated pigs (figure 6, d & g) . in addition, pbmc secreted significantly reduced il-10 and tgf-b compared to k-ag vaccinated virus challenged pigs ( figure 6, h & i) , while in tbln-mnc, increased il-10 was seen compared to both virus challenged groups (figure 6, l) . increased ifn-c was secreted in both pbmc and tbln-mnc of nano-kag vaccine group compared to k-ag immunized pigs ( figure 6, j & m) . the level of proinflammatory cytokine, il-6, in a restimulation response was significantly reduced in tbln-mnc of nano-kag compared to both virus challenged pigs ( figure 6, k & n) . nanoparticle mediated vaccine delivery has shown a great promise in mouse models against influenza, parainfluenza, hepatitis b, plasmodium, and venezuelan equine encephalitis pathogens [13, 32, 33] . however, the knowledge related to crossprotective efficacy of such vaccines in a suitable large animal model is limited. our study has revealed the potency of nanoparticle-entrapped prrsv vaccine in pigs. prrs has been a dreadful disease causing huge economic loss in a majority of the swine producing countries in the world. nano-kag vaccine has upregulated the frequency of major innate immune players (nk cells, cd t cells, and dcs), and also enhanced the secretion of anti-viral cytokines (ifn-a and ifn-c) , which otherwise is suppressed by prrsv [34] . suggesting that in nano-kag vaccinated virulent heterologous prrs challenged pigs, these effectors played a pivotal role in the viral clearance. in contrast, increased viral load in the lungs of k-ag vaccinated pigs was perhaps due to antibody-mediated enhancement in the uptake lungs was determined by immunofluorescence assay. each bar represents average values from three pigs 6 sem. asterisk represents the statistical significant difference (p,0.05) between k-ag and nano-kag received pig groups, and q represents the statistical significant difference (p,0.05) between unvaccinated and nano-kag received pig groups. a similar trend in results was obtained in an independent second trial performed using same number of animals. doi:10.1371/journal.pone.0051794.g003 of prrsv by alveolar mws, in addition to increased immunosuppressive response. particulate ags has an inherent affinity for mucosal m cells and apcs and are phagocytosed passively by apcs [35] . nanoparticles protect the entrapped proteins from protease-mediated degradation at mucosal surfaces, and also aid in slow sustained release of the vaccine [36] . plga nanoparticle induces activation and maturation of human dcs by upregulating the expression of costimulatory and mhc class ii molecules, secretion of proinflammatory cytokines, and enhances the apcs allostimulatory property [37, 38] . consistent with the inherent adjuvant property of plga, pig apcs treated with plga nanoparticle vaccine (nano-kag) had increased expression of a costimulatory molecule, cd80/86. in our study, we did not include a pig group with empty pgla nanoparticles, as our primary goal was to augment the killed prrsv vaccine induced anti-prrsv cross-protective immunity with the help of plga, a well-known adjuvant and a delivery system to vaccines. thus, at present we do not have the information on how much of post-vaccination t cell immunity detected in nano-kag vaccinated pigs was due to the adjuvant effect of the pgla nanoparticles alone, which will be considered during our future investigations. however, a rapid uptake of nano-kag by lung apcs followed by translocation of viral ags into endosomal compartment was observed. suggesting that virus specific adaptive immune response could be elicited in the respiratory tract of pigs using plga nanoparticle-based killed prrsv vaccine. differential cell counts from bal fluid harvested from healthy mice, humans, and pigs have indicated that greater than 90% of cells are alveolar mws [39, 40] , suggesting that intranasally delivered nano-kag were phagocytosed by mws. earlier studies have demonstrated rapid uptake of chitosan nanoparticles by apcs followed by gradual release of antigen due to slow rate of degradation of chitosan by lysozymes [41] ; resulting in increased expression of costimulatory molecules, activation of dcs, and antigen presentation through mhc class i and ii molecules [42] . in our pre-challenge study, nano-kag vaccine significantly increased the frequency of cd8 + t cells, th/ memory cells, with concomitant increase in the secretion of innate (ifn-a), proinflammatory (il-6), and th1 (ifn-c) cytokines. immune potentiating ability of chitosan nanoparticles is mediated by the action of innate immune cells, in addition to enhanced production of il-6 and ifn-c [43] . phagocytosis of polystyrene latex microspheres by mws activate the signal transduction events in innate immune cells [44] . once activated, the apcs present the antigen through mhc class i and ii molecules to cd8+ and cd4+ t cells, respectively. in our post-challenge study using a virulent heterologous prrsv, nano-kag induced superior innate immune response was observed. studies have shown immune potentiating activity of nanoparticles in mice, pigs, and macaques; but the immune correlates were not evaluated in vaccinated virus challenged animals [45] . immunologically, prrsv modulates innate immune function of pigs by dampening the ifn-a production and nk cell frequency as well as its cytotoxicity, leading to weak/ delayed adaptive immune response [17, 34] . the nk cytotoxic function in nano-kag received pigs was partially rescued (not significant), suggesting the beginning of appearance of nk cell cytotoxic function with a complete rescue in their frequency (comparable to mock pigs). in pigs vaccinated with nano-kag, virus induced immunosuppressive responses were dampened along with a significant boost in both innate and virus specific adaptive immune response. the cd t cell is an important innate immune cell at mucosal sites and they possess non-mhc class i cytolytic activity. pigs possess relatively large population of cd t cells compared to other species and they secrete ifn-c [46] . cd t cell plays an important role in reducing the vaccinia virus load, and also in destruction of herpes simplex type 1 infected cells [47] . in nano-kag vaccinated pigs increased population of cd t cells, in addition to nk cells, cd4 + and cd8 + t cells, and increased secretion of ifn-c were detected at both mucosal and systemic sites. now and earlier we have demonstrated that both k-ag and mlv-prrs vaccinated and virus challenged, as well as unvaccinated prrsv infected pigs have immunosuppressed response mediated by increase in the population of tregs and secretion of il-10 and tgf-b, and reduced production of ifn-c [17, 31, 48] . in contrast, in nano-kag immunized pig lungs, tbln, and blood significantly reduced tregs frequency, associated with decreased il-10 and tgf-b, and increased ifn-c secretion, compared to unvaccinated and k-ag vaccinated virus challenged pigs was observed. in a pre-challenge study, at two-week post-vaccination increased secretion of proinflammatory cytokine, il-6, in nano-kag vaccinated pigs appears to be involved in initiation of adaptive immune response. diminished production of il-6 in post-challenged pigs at six-week post-vaccination was associated with reduced inflammatory lung pathology. mucosal immunization elicits production of iga antibodies and effector response at distant tissues [49] . the iga antibody is protective against various viral infections and they possess significant virus neutralization activity at both mucosal surfaces and blood [50] . in nano-kag immunized pigs increased levels of prrsv specific iga, igg in the lungs, blood, and nasal wash were observed. although, these prrsv specific antibodies did not significantly increase the neutralizing titers in the lungs, there was complete clearance of viremia at pc 15, which was associated with various other adaptive immune correlates. whereas in the lungs, the reduced viral titer was not significant, but a significant reduction of prrsv antigen by immunohistochemistry and reduced lung pathology implicates the presence of active anti-prrsv mucosal immune response elicited by nano-kag vaccine in the lungs (fig 3 & 4) . the most important finding from our study to swine farmers and researchers is that pigs vaccinated intranasally with nano-kag vaccine completely clear the prrsv viremia of a virulent heterologous virus by two-week post-challenge. further, in yet another nano-kag vaccine study we inoculated a booster dose of the vaccine and co-administered with a potent mucosal adjuvant, intranasally, showed the complete clearance of the replicating heterologous prrsv from the lungs and clearance of viremia earlier than the current study (binjawadagi et al., manuscript submitted). in conclusion, our study has suggested that innovative strategy of intranasal delivery of prrsv nano-kag vaccine has the potential to control prrs outbreaks, and it has the potential to reduce economic losses to swine producers. considering pig a useful large animal model, our study may serve as a useful impetus to undertake intranasal plga nanoparticle-based vaccine trials against human respiratory viral infections. each bar represents average values from three pigs 6 sem. asterisk represents the statistical significant difference (p,0.05) between nano-kag and k-ag received pig groups and q represents the statistical significant difference (p,0.05) between unvaccinated and nano-kag received pig groups. a similar trend in results was obtained in an independent second trial performed using same number of animals. doi:10.1371/journal.pone.0051794.g006 controlled-release vaccines-biodegradable polylactide/polyglycolide (pl/pg) microspheres as antigen vehicles degradation of poly(d,l-lactic acid) microspheres effect of molecular weight a road less travelled: large animal models in immunological research the porcine lung as a potential model for cystic fibrosis prrs costs industry $664 million annually nidovirales: a new order comprising coronaviridae and arteriviridae porcine reproductive and respiratory syndrome: clinical disease, pathology and immunosuppression immunohistochemical identification of porcine reproductive and respiratory syndrome virus (prrsv) antigen in the heart and lymphoid system of threeweek-old colostrum-deprived pigs evaluation of immune responses to porcine reproductive and respiratory syndrome virus in pigs during early stage of infection under farm conditions mucosal immunity: implications for vaccine development the mucosal immune system: from fundamental concepts to vaccine development routes of immunization and antigen delivery systems for optimal mucosal immune responses in humans pla and plga nanoparticles enhance humoral, mucosal and cytokine responses to hepatitis b vaccine a novel bioadhesive intranasal delivery system for inactivated influenza vaccines single-dose mucosal immunization with biodegradable microparticles containing a schistosoma mansoni antigen strain specificity of the immune response of pigs following vaccination with various strains of porcine reproductive and respiratory syndrome virus mucosal vaccines to prevent porcine reproductive and respiratory syndrome: a new perspective a modified live prrsv vaccine and the pathogenic parent strain induce regulatory t cells in pigs naturally infected with mycoplasma hyopneumoniae recombination in vaccine and circulating strains of porcine reproductive and respiratory syndrome viruses reversion of a live porcine reproductive and respiratory syndrome virus vaccine investigated by parallel mutations impact of immunizations with porcine reproductive and respiratory syndrome virus on lymphoproliferative recall responses of cd8+ t cells effect of genotypic and biotypic differences among prrs viruses on the serologic assessment of pigs for virus infection incorporation of microspheres into nerve guidance channels for drug delivery purposes. applied chemistry and chemical enginnering delivering neuroactive molecules from biodegradable microspheres for application in central nervous system disorders intranasal m cell uptake of nanoparticles is independently influenced by targeting ligands and buffer ionic strength swine influenza h1n1 virus induces acute inflammatory immune responses in pig lungs: a potential animal model for human h1n1 influenza virus porcine dendritic cells generated in vitro: morphological, phenotypic and functional properties altered pathogenesis of porcine respiratory coronavirus in pigs due to immunosuppressive effects of dexamethasone: implications for corticosteroid use in treatment of severe acute respiratory syndrome coronavirus an immune basis for lung parenchymal destruction in chronic obstructive pulmonary disease and emphysema detection and duration of porcine reproductive and respiratory syndrome virus in semen, serum, peripheral blood mononuclear cells, and tissues from yorkshire, hampshire, and landrace boars cross-protective immunity to porcine reproductive and respiratory syndrome virus by intranasal delivery of a live virus vaccine with a potent adjuvant induction of protective immune responses against venezuelan equine encephalitis (vee) virus aerosol challenge with microencapsulated vee virus vaccine immunogenicity of bovine parainfluenza type 3 virus proteins encapsulated in nanoparticle vaccines, following intranasal administration to mice interferon-alpha response to swine arterivirus (poav), the porcine reproductive and respiratory syndrome virus dendritic cell progenitors phagocytose particulates, including bacillus calmette-guerin organisms, and sensitize mice to mycobacterial antigens in vivo biodegradable polymer microspheres as vaccine adjuvants and delivery systems differential effects of agarose and poly(lactic-coglycolic acid) on dendritic cell maturation differential levels of dendritic cell maturation on different biomaterials used in combination products cellular variables in bronchoalveolar lavage fluids (balf) in selected healthy pigs metal-rich ambient particles (particulate matter 2.5) cause airway inflammation in healthy subjects studies on chitosan: 4. lysozymic hydrolysis of partially nacetylated chitosans antigen presentation and t cell stimulation by dendritic cells alveolar macrophage priming by intravenous administration of chitin particles, polymers of n-acetyl-d-glucosamine, in mice role of lipid rafts in innate immunity and phagocytosis of polystyrene latex microspheres induction of hiv-specific antibody response and protection against vaginal shiv transmission by intranasal immunization with inactivated shiv-capturing nanospheres in macaques gammadelta lymphocyte response to porcine reproductive and respiratory syndrome virus alpha beta and gamma delta t-cell networks and their roles in natural resistance to viral infections intranasal delivery of whole cell lysate of mycobacterium tuberculosis induces protective immune responses to a modified live porcine reproductive and respiratory syndrome virus vaccine in pigs distribution of poliovirus antibody in serum, nasopharynx and alimentary tract following segmental immunization of lower alimentary tract with poliovaccine cross-protection in mice infected with influenza a virus by the respiratory route is correlated with local iga antibody rather than serum antibody or cytotoxic t cell reactivity we would like to thank ms. ruthi patterson for her help in animal studies and tissue processing, and mathew weeman, drs. mahesh khatri and juliette hanson for their help in animal studies. drs. michael murtaugh and eric nelson had provided the prrsv reagents. conceived and designed the experiments: vd gjr. performed the experiments: vd cm bb dj gjr. analyzed the data: vd cm gjr. wrote the paper: vd gjr. key: cord-001435-ebl8yc92 authors: hoppe, sebastian; bier, frank f.; von nickisch-rosenegk, markus title: identification of antigenic proteins of the nosocomial pathogen klebsiella pneumoniae date: 2014-10-21 journal: plos one doi: 10.1371/journal.pone.0110703 sha: doc_id: 1435 cord_uid: ebl8yc92 the continuous expansion of nosocomial infections around the globe has become a precarious situation. key challenges include mounting dissemination of multiple resistances to antibiotics, the easy transmission and the growing mortality rates of hospital-acquired bacterial diseases. thus, new ways to rapidly detect these infections are vital. consequently, researchers around the globe pursue innovative approaches for point-of-care devices. in many cases the specific interaction of an antigen and a corresponding antibody is pivotal. however, the knowledge about suitable antigens is lacking. the aim of this study was to identify novel antigens as specific diagnostic markers. additionally, these proteins might be aptly used for the generation of vaccines to improve current treatment options. hence, a cdna-based expression library was constructed and screened via microarrays to detect novel antigens of klebsiella pneumoniae, a prominent agent of nosocomial infections well-known for its extensive antibiotics resistance, especially by extended-spectrum beta-lactamases (esbl). after screening 1536 clones, 14 previously unknown immunogenic proteins were identified. subsequently, each protein was expressed in full-length and its immunodominant character examined by elisa and microarray analyses. consequently, six proteins were selected for epitope mapping and three thereof possessed linear epitopes. after specificity analysis, homology survey and 3d structural modelling, one epitope sequence gavvalsttfa of kpn_00363, an ion channel protein, was identified harboring specificity for k. pneumoniae. the remaining epitopes showed ambiguous results regarding the specificity for k. pneumoniae. the approach adopted herein has been successfully utilized to discover novel antigens of campylobacter jejuni and salmonella enterica antigens before. now, we have transferred this knowledge to the key nosocomial agent, k. pneumoniae. by identifying several novel antigens and their linear epitope sites, we have paved the way for crucial future research and applications including the design of point-of-care devices, vaccine development and serological screenings for a highly relevant nosocomial pathogen. klebsiella pneumoniae is a gram-negative, facultative anaerobic rod-shaped bacterium belonging to the family of enterobacteriaceae. it is a non-motile, lactose fermenting organism, which has been known to cause severe lung damage if aspirated. other clinical symptoms common with klebsiella pneumoniae infections encompass urinary-tract-infections (uti) and wound infection potentially causing bacteremia and septicemia [1] . in recent years it has become one of the most persistent nosocomial agents, especially due to the increasing distribution of multiple resistances to antibiotics. the most prominent group of k. pneumoniae harboring a broad resistance spectrum incorporates the extendedspectrum beta-lactamase (esbl) expressing strains. due to their outstanding clinical relevance and occurrence as agents of nosocomial infections, it is highly desirable to rapidly detect the presence of these organisms and to find suitable measures to effectively counter any infection in the early stages [2] . while numerous dna-based typing methods exist [3] , these are often laborious and time-consuming. in contrast, user-friendly point-of-care devices applying antigen-antibody interactions would allow for a quick and reliable detection [4] . nevertheless, the knowledge about suitable antigens to be incorporated into such a device is scarce. thus, we have utilized a method to quickly assess novel immunogenic proteins of k. pneumoniae, which might serve as potential targets for a diagnostic tool. recently, we have successfully employed this approach to unveil immunogenic proteins for both campylobacter jejuni [5] and salmonella enterica [6] . concisely, prokaryotic cdna libraries are created, fusion proteins expressed and these constructs covalently attached to microarray surfaces via the use of a halotag (promega). subsequently, the microarrays are screened using polyclonal antibodies reactive to the donor species of the cdna. therefore, this approach enables a broad and reliable screening, while reducing cross-reactivity and background to a minimum [7] due to the highly selective and covalent binding of halotag to its specific ligand [8] . moreover, the high specificity of said interaction renders excessive protein purification steps normally encountered in microarray-based screening applications [9] obsolete. thus, it is a faster and more direct approach as spotting combines both the deposition of samples and enables the immediate purification by a simple washing step. in connection with the above screening approach, cdna derived expression libraries were generated to express a vast number of proteins from k. pneumoniae. these libraries offer the advantage of a smaller sample size as compared to genomic libraries. this is mainly due to genomic libraries encompassing highly truncated dna fragments as well as dna representing regions that do not encode proteins in the original organism. contrastingly, cdna libraries generated via the in-fusion smarter directional cdna library construction kit (clontech) have been known to lead to longer fragments and possess a high abundance of full-length clones [10] . thus, the overall number of clones required for screening is substantially reduced. still, prokaryotic cdna libraries display one main disadvantage. as bacterial mrna rarely contains a poly(a)-tail [11, 12] , isolation of the mrna from other rna species is tedious. while some methods exist to isolate the mrna prior to reverse transcription [13, 14] , we rather chose to normalize the cdna afterwards. consequently, the entire rna of k. pneumoniae was used for reverse transcription. next, normalization was performed using a duplex-specific nuclease [15] . this treatment has been shown to effectively reduce the highly abundant rrna derived cdna portions without implementing a bias, thus altering the overall composition of the cdna in favour of the mrna derived molecules [16] . in addition to this, ligation-independent cloning and electroporation were employed to enhance cloning efficiency [17] . in this work, we have screened 1536 clones to detect the presence of previously unknown immunogenic proteins. in summary, we identified 14 proteins that have not been described as immunogenic before. after further analyses and epitope mapping of several promising candidates, three proteins -a channel receptor, a putative transport protein and a hypothetical protein -revealed linear antigenic sites with varying specificity. our results offer the potential to be used for a wide array of applications including the generation of monoclonal antibodies that might be used in diagnostic examination. furthermore, several of the identified antigens or parts thereof might be suitable for vaccine development, either used in passive or active immunization. additionally, many virulence-associated factors harbor some immunogenic potential. thus, identification of novel immunogenic proteins might elucidate proteins involved in the pathogenicity and virulence of k. pneumoniae. consequently, this advances the understanding of this pathogen and illuminates new approaches to counter infections. the mean rin value of rna used for cdna generation and library construction was calculated to 7.361.3 (n = 5). after successful normalization, the cdna was cloned to create an expression library. of this library, 1536 different clones were screened using halolink slides. the known immunogenic proteins [18] , outer membrane protein 1a ompf (uniprot/swiss-prot: a6t721) and outer membrane protein 3a ompa (a6t751), were used as positive references, whereas both dihyroorotase pyrc (a6t7d6) and glyceraldehye-3-phosphate-dehydrogenase gapa (a6t8l2) served as negative references. after screening, the signal intensities of each sample were compared to the references and grouped accordingly. generally, three distinct groups were established. group i represents samples exceeding the intensities of both positive reference proteins, group ii encompasses samples ranging in between the different intensities of ompa and ompf, whereas group iii entails those samples that albeit showing higher intensities than the negative controls, are below both ompa and ompf. here, approximately 25% of samples belong to group i, while ii and iii contain 7% and 14% respectively. the remaining samples, 54%, fall into the same range as the negative protein references. consequently, 192 clones or 12.5% of the entire screening approach were selected for sequencing. these clones were all taken from group i. after sequencing, artificial fragments and known antigens were discarded to reveal potentially novel immunogenic proteins, see table s1 for a list of the initial identification via sequencing. some of the inserts were too heavily truncated to reliably identify the corresponding gene. moreover, in some cases subcloning the initially identified genes in full-length failed even after numerous attempts. therefore, those genes were removed from further characterization as the translated peptide fragments were too short to be significant. despite these limitations, 14 potentially novel immunogenic proteins were expressed in full length and used for further characterization. the 14 antigen candidates are summarized in table 1 including their locus tag, protein name, length and size in kda. the difference in immunodominant behaviour was assessed by ten independent microarray and elisa analyses employing two different antibodies. in summary, table 1 reveals the resulting mean q values and corresponding errors (n = 10). a q value above one represents higher intensity signals than ompa, the used positive reference. the highest mean q value was obtained for kpn_02199, a coa-linked acetaldehyde dehydrogenase and irondependent alcohol dehydrogenase; pyruvate-formate-lyase deactivase with 2.1660.39. however, as this protein is highly conserved within all bacteria, it was not considered for further analyses regarding specific antigenic sites. the same holds true for kpn_03668, the 50s ribosomal protein l11 methyltransferase. although a q value of 1.7060.38 was attained, the highly conserved nature of this protein renders it unsuitable within a diagnostic approach. contrastingly, kpn_00466 and kpn_01584, two hypothetical proteins, were selected for future investigations as little is known about the function of these proteins. their mean q values were 1.1560.22 and 1.9060. 36 respectively. moreover, kpn_01584 shows some homology to a known superantigen of yersinia pseudotuberculosis according to the ncbi protein cluster database [19] . thus, the potential utilization of these proteins within a diagnostic tool seems plausible. in addition, kpn_01100, a histidine triad protein, kpn_02202, glucose-1-phosphate uridylyltransferase, kpn_00 363, a nucleoside channel and receptor of phage t6 and colicin k and kpn_00459, a putative transport protein, were selected for further investigations via epitope mapping. while kpn_01100 and kpn_02202 revealed mean q values above one with 1.3160.27 and 1.6960.56, respectively, kpn_00459 and kpn_00363 failed to reach this level. rather, the q values attained were 0.8360.27 and 0.7760.14. while the q values of the latter two proteins are lower compared to some of the other proteins identified, their functional descriptions and membraneassociation render them highly attractive within a diagnostic question. hence, they might be more easily accessible in a whole cell detection approach than cytoplasmic proteins. epitope mapping revealed the potential presence of linear epitopes within three of the six proteins investigated, namely kpn_00363, kpn_00459 and kpn_00466. for kpn_00363 seven distinct regions were identified with intensities above 1000 a.u., see figure 1 . these comprised the peptides 2 and 3, 16-18, 29-30, 45-47, 50-51, 60-63 and 69-71. the highest mean value for these peptides was obtained for peptide 16 with more than 10000 a.u. the positive reference, i.e. rabbit igg, reached a mean value of more than 30000 a.u., whereas the negative control mbp showed intensities of less than 500 a.u. as the adjacent peptides are identical in all but 4 amino acids in sequence, a consensus can easily be derived from two or more neighboring peptides. as figure 2 reveals, only the first two peptides, llaagavvalsttfa and gavvalsttfaagaa showed some specificity during specificity control assays. here, the arrays were incubated with additional antibodies reactive to different bacterial species, namely campylobacter jejuni, staphylococcus aureus, e. coli and s. enterica. all remaining peptides display similar intensities when these antibodies were used as compared to the original k. pneumoniae antibodies. however, for the first two peptides a significant difference is observed. the mean value for peptide 2 was approximately 6500 a.u. with k. pneumoniae antibodies and dropped to less than 400 a.u. with the other antibodies. a similar trend is discernible for peptide 3, where a drop from 1100 a.u. to less than zero is visible. thus, the consensus sequence gavvalsttfa is likely a suitable linear epitope featuring specificity for k. pneumoniae. for the putative transport protein, kpn_00459, three regions scored intensities above 1000 a.u. including peptides 50 and 51, 59 and 60 as well as 79-81, see figure 3 . the positive reference again reached a mean value above 30000 a.u., while the negative control levelled out at less than 500 a.u. after specificity assays, the peptides 59 and 60 revealed predominantly specific binding by the k. pneumoniae antibody as the mean values dropped from approximately 5800 and 2800 a.u. respectively, to less than zero for all other antibodies tested, see figure 4 . thus, a consensus for this epitope can be derived to giafgavelfd. contrastingly, the remaining peptides revealed equal intensities independent of the antibody used. finally, for the third protein, kpn_00466, one pair of adjacent peptides, namely 11 and 12, achieved mean values of approximately 2000 a.u. within close proximity to the positive reference, as depicted in figure 5 . additionally, peptide 16 displayed the highest overall mean value with more than 7000 a.u., however neither of the two neighboring peptides (15 and 17) attained values of any significance. thus, the presence of a linear epitope in that region is rather unlikely. besides, as specificity assays illustrated, none of the three peptides showed specific interaction to the k. pneumoniae antibodies alone, see figure 6 . rather, signal intensities with antibodies reactive to c. jejuni fell into the same scope. therefore, nonspecific binding to these peptides is probable. the remaining three proteins under investigation failed to disclose any linear peptide region with significant signal intensities to assume linear epitopes to exist. for a better understanding of the suitability of the identified linear epitopes, structural modelling was employed using the swiss model automated mode. for kpn_00363 a model was constructed based on the crystal structure of the bacterial nucleoside transporter tsx of e. coli. however, the derived model only spans residues 31 to 294 and as such does not contain the derived consensus sequence of the linear epitope gav-valsttfa. nevertheless, the model displays a beta barrel structure typical of outer membrane-spanning transport proteins, see figure 7 for details. the first residues of the derived model, starting at position 31, are marked in orange and present a coiled region outside of the beta barrel. therefore, the likelihood of the gavvalsttfa region to reside within the barrel is slim. rather, an extension of the truncated coil seems plausible. consequently, the potential accessibility of the identified linear epitope appears high. furthermore, the predicted 3d structure of the first part of kpn_00459 is displayed in figure 8 . contrary to kpn_00363, two models were devised for kpn_00459. however, only one encompasses the identified linear epitope giafgavelfd, which is highlighted in orange and is situated as part of an alpha helix and an adjacent loop. as the sequence is not enclosed by other residues or structures, good accessibility ought to be provided. in order to predict the potential specificity of the epitopes, homology analyses were performed. whereas giafgavelfd of kpn_00459 exhibits a broad homology throughout with all residues identical in closely related species, see figure s3 : homology of kpn_00459, gavvalsttfa of kpn_00363 features four residues likely specific for k. pneumoniae within this particular sequence. the variance of the latter epitope's sequence considering closely related species to k. pneumoniae is summarized in figure 9 . specifically, the following residues have been replaced: valine by leucine (position four), both threonine residues by serine residues (position 8 and 9) as well as alanine by threonine (position 11). the influence of sequence variations on the binding capacity of the identified epitope gavvalsttfa was subsequently examined by performing an alanine scan. its results are summarized in figure 10 . the original consensus epitope sequence shows a mean intensity of approximately 1000 a.u. alterations of the first, second, fifth, eighth and ninth residue lead to a significant drop of signal intensities. consequently, signals of less than 300 a.u., in close proximity to the negative control mbp with 50 a.u. are obtained. contrastingly, by altering residues three or six of the consensus sequence gavvalsttfa, i.e. replacing the first valine or leucine by alanine, the resulting mean signal intensities are significantly increased to more than 1700 a.u. for replacing valine and surpassing 5600 a.u. after changing leucine to alanine. additionally, specificity assays showed no significant signal intensity for antibodies reactive to closely related species, i.e. e. coli and s. enterica. incubation with antibodies reactive to either of those two bacterial species resulted in signal intensities in the neighbourhood of the negative control independent of sequence alterations, see figure 11 . this was also true for gavlalsssft and saalaltssft. the screening of a cdna based expression library of k. pneumoniae has successfully identified a number of novel, previously unknown immunogenic proteins. this is well in accordance to previous achievements of this method, which we have employed for the identification of both c. jejuni [5] and s. enterica [6] antigens. consequently, in this current study we aimed to detect suitable proteins for the specific identification of k. pneumoniae. furthermore, identification of specific antigens might improve treatment opportunities including the development of suitable vaccines [20] . finally, detecting proteins with yet unknown functional and structural information to be antigenic might be an indication of their potential involvement in the pathogenic nature of an organism. thus, these proteins might be suitable access points for future investigations to improve the understanding of the underlying pathogenicity and virulence of k. pneumoniae. within the 14 proteins only a subset was selected for further analysis using epitope mapping. the rationale for these selections was based on three distinct features: the resulting q value, homology as well as functional and structural properties. while the q value mirrors a normalized intensity compared to a positive reference, it does not fully account for differences in expression levels, misfolding and other factors which might have had an influence on the binding reaction and thus on the overall intensity. still, it facilitates to rank the proteins and increases the likelihood of a given protein to be immunogenic if its q value is significantly high. however, proteins with outstanding q values were not chosen for epitope mapping by default. rather, the known homology and corresponding distribution of each protein was carefully evaluated. thus, proteins like kpn_02199 and kpn_03668 albeit scoring high q values were exempt from epitope mapping as they display a broad spectrum of homologous proteins across bacterial species. finally, the intrinsic properties of each protein, if available, were closely scrutinized. therefore, proteins with hypothetical character were predominantly chosen, as information on them is confined. in addition, all types of membrane-associated proteins deserved a better look, as these type of proteins offer a more direct route and accessibility, which might be of utmost importance in a future rapid point-ofcare device detecting whole organisms. after epitope mapping, three of the six proteins under investigation revealed sites with potential linear epitopes. still, the remaining proteins displayed no such sequences. this is, however, in accordance to the general prevalence of structural epitopes in comparison with linear epitopes in nature. in fact, approximately 90% of all epitopes are conformational rather than sequential [21, 22] . despite these potential shortcomings of the used linear epitope mapping method, a number of intriguing linear epitopes have been identified. notably, three proteins, kpn_00363, kpn_00459 and kpn_00466, harbored sites with potential linear epitopes. further careful examination, however, excluded kpn_00466 from future applications as the identified epitope sequence were nonspecific for k. pneumoniae, which might be due to the conserved character of the protein within the family enterobacteriaceae. still, kpn_00466 is a membrane protein and upcoming investigations might help to elucidate applications using this protein either for prevention of k. pneumoniae infections or detection thereof. in contrast, the other two proteins displaying linear epitopes, kpn_00363 and kpn_00459, indicated some specificity with the antibodies tested and two linear consensus sequences could be derived, gavvalsttfa and giafgavelfd, respectively. while the former sequence is present in kpn_00363, an ion channel protein tsx, which is conserved among enterobacteriaceae, the latter sequence is part of kpn_00459, a hypothetical protein with high similarity to a cation:proton antiport protein and conserved among proteobacteria. consequently, the homology of giafgavelfd (kpn_00459) is high throughout different bacterial species, which renders the sequence inapt for specific k. pneumoniae detection and other applications. in contrast, gavvalsttfa (kpn_00363) displays alterations within this sequence for bacterial species other than k. pneumoniae. as small as these alterations appear, they appear to have a crucial effect on the binding of antibodies to the target sequence and thus benefit specificity. this is well in line with the experimental results that indicate no binding by antibodies reactive to other bacteria to this sequence or any of its alterations. moreover, alanine scanning revealed a number of residues to be paramount for antibody binding. consequently, replacing the glycine (position 1), alanine (position 2), alanine (position 5) or either threonine (positions 7 and 8) leads to a significant loss of antibody binding observable by a dramatic reduction in signal intensity. on the contrary, removing either the first valine or leucine and inserting an alanine residue as a replacement results in a significant increase in signal intensity, potentially hinting at an improved antibody binding. the reason for these effects remains nebulous; however, it is not caused by a simple change in secondary structure. this is apparent as both aavvalsttfa and gavvaasttfa, two sequences at opposite sides regarding signal intensity, are predicted to consist solely of an alpha-helix as compared to the original sequence, which is predicted to contain a beta-strand (position 1 to 7) and a helical part (position 8 to 11) by emboss garnier. furthermore, as the original sequence is fully constructed of uncharged amino acids, alterations via alanine or glycine do not change the overall charge of the peptide. consequently, the differences in signal intensity cannot be deduced to implementation or removal of charged residues. finally, changes in hydrophobicity albeit present are minimal at best and may not suffice to explain the observed characteristics. still, one has to bear in mind that the accuracy of prediction based tools is often lacking, especially for emboss garnier with an accuracy in the range of 65%. consequently, some of the predicted secondary structures might differ. despite some uncertainty as to the mechanistic cause of the altered peptides to behave as they did, one fact remains obvious. none of the peptides showed any significant binding to antibodies not raised against k. pneumoniae. therefore, after alanine scanning, gavvalsttfa remains a suitable candidate for specific k. pneumoniae detection or treatment. still, accessibility of the epitope is paramount to a quick diagnostic tool. thus, modelling of the 3d structure of the protein was performed. unfortunately, the 3d modelling only succeeded for the part of the protein, which does not contain said linear epitope sequence. nevertheless, the pronounced beta barrel structure of the channel protein is visible and the linear epitope sequence, albeit absent, likely to be an extension of the freely accessible n-terminal region outside the barrel structure. thus, accessibility of the epitope by an antibody might be pronounced without the need to enter the cell or channel. membrane proteins harboring a beta barrel like structure have been shown in bacteria to be exclusively found in the outer membrane [23] . moreover, the mobile coils on the extracellular sides of these membrane proteins are pivotal for their function or interaction with other molecules. this renders the identified sequence an intriguing target for antibody-based detection. additionally, channel proteins harboring beta barrel like structures have been shown to be immunodominant in other bacterial species such as salmonella, haemophilus influenzae, e. coli, neisseria meningitides, shigella dysenteriae and chlamydia trachomatis [24] [25] [26] [27] [28] [29] . on the contrary, the model for kpn_00459 encompasses the identified consensus sequence of the linear epitope giafga-velfd, which is part of a loop between two alpha helices. the abundance of alpha helices suggests the protein to span the inner membrane [23] . in this topological design, helices are mostly located within the membrane, notably as transmembrane domains, whereas loops are located either on the cytoplasmic or periplasmic side of the cell. when considering prediction based methods, such as s_tmhmm for topological domains [30, 31] or emboss antigenic [32, 33] , part of giafgavelfd is assumed to be extracellular. combined with the high flexibility and degrees of freedom of random coil structures, the likelihood for good accessibility is high rendering the sequence a potentially attractive target for whole cell detection despite its lack of specificity. furthermore, these findings support the 3d model and underline the accuracy of the specified structure. another key aspect in determining the accuracy of the 3d model prediction is a so-called z-score [34] . the z-score for the model of kpn_00363 is 24.285 and 28.895 for kpn_00459 respectively. although the values are significantly below zero that does not inevitably indicate models of poor accuracy. in fact, low z-scores are often obtained if the protein under investigation is membraneassociated. this is mainly due to the inverse physicochemical properties of membrane proteins in comparison to soluble ones. hence, the low z-scores are more likely induced by this effect than caused by an insufficient accuracy of the models. in conclusion, we have successfully identified several novel antigens of k. pneumoniae and identified three proteins potentially harboring linear epitopes. subsequently, we achieved to identify two sequences displaying specificity during experimental investigations; however, one of these is doubtful as homology analysis has revealed it to be highly conserved among a broad spectrum of bacterial species. still, gavvalsttfa of kpn_00363 was identified to be specific both experimentally and has shown four residues within the eleven amino acid sequence to occur predominantly in k. pneumoniae only. thus, the likelihood for this linear epitope to be specific is high. this assumption was confirmed by alanine scanning revealing a number of pivotal residues for antibody binding. moreover, it was unearthed that neither e. coli nor s. enterica antibodies were able to bind to any of the sequences, original and modified. subsequent investigations might help to further nurture the insight into the suitability of this peptide for diagnostic and therapeutic applications. thus, monoclonal antibodies ought to be devised to be used for affinity investigations via biacore and to determine kinetics. furthermore, monoclonal antibodies could be used within a potential diagnostic tool and after validation ought to be tested with whole bacteria. if these antibodies are able to specifically detect intact k. pneumoniae cells, the resulting antibody might well be suited for integration into a point-of-care device. in a different approach, the identified epitope sequence could easily be produced in large quantity. this peptide might serve some role in serological screenings, especially if it proves to be immunodominant. consequently, antibodies against this epitope might be present in a plethora of patient sera. finally, all proteins identified here might be suitable candidates for vaccine development independent of the existence of a linear epitope, as structural epitopes might well be present and antigenicity ensured. nevertheless, additional in-depth analysis is required to determine a number of the key aspects of vaccine development prior to use. as a donor of rna the fully-sequenced strain k. pneumoniae mgh78578 was grown on solid trypticase-soy-agar (tsa) for 24 h at 37uc under aerobic conditions. for rna isolation a liquid culture was prepared by inoculating 10 ml of brain-heart-infusion broth (bhi) with a single colony and incubated overnight at 37uc, 140 rpm. this overnight culture was used to inoculate a flask containing 100 ml fresh bhi medium. the cells were harvested 6 h after inoculation. for initial screening a rabbit polyclonal igg antibody to k. pneumoniae (acris ap00792pu-n) was used. for further micro-array analyses of a subset of candidate proteins, elisa and epitope mapping this antibody was used as well as rabbit polyclonal igg antibody to k. pneumoniae (abcam ab20947). the antibodies were generated with k. pneumoniae atcc 43816 serving as an immunogen. specificity assays were performed employing rabbit polyclonal igg antibody to c. jejuni (acris ap24002pu-n), s. aureus (fitzgerald 20c-cr1274rp), e. coli (abcam ab137967) and s. enterica (abcam ab35156). detection was achieved by usage of secondary antibodies. goat polyclonal antibody to rabbit igg conjugated with chromeo-546 (abcam ab60317) for fluorescent and antibody conjugated with horseradish peroxidase (abcam ab6721) for a colorimetric readout were applied where appropriate. the cells were harvested by centrifugation (20006g, 10 min) and the resulting supernatant discarded. the pellets were resuspended in fresh bhi medium. for stabilisation of the rna, 1 ml of rnaprotect bacteria reagent (qiagen) was added to 0.5 ml of bacterial suspension and processed according to the manufacturer's instructions. lysis was performed with 200 ml of lysis buffer (30 mm tris-cl, 1 mm edta, 15 mg/ml lysozyme, .12 mau proteinase k) by pipetting and vortexing for 10 s. after incubation, 700 ml buffer rlt and 500 ml 96% ethanol were added and the lysate applied to rneasy bacteria mini kit spin columns (qiagen) for rna isolation following the manufacturer's instructions. during this procedure an on-column dnase digest was performed using rnase-free dnase i solution (qiagen) according to the manufacturer's instructions. the isolated total rna was eluted in 50 ml of rnase-free water and its concentration and purity analyzed by nanodrop (peqlab) measurements. the quality of isolated rna was assessed using the rna 6000 pico kit and bioanalyzer 2100 (agilent). the total rna was diluted to a working concentration of 200-500 pg/ml. the analysis was performed following manufacturer's instructions and the rna integrity number (rin) calculated by the 2100 expert software (agilent). the rin is defined to fall into a range of 0 to 10, with a higher score indicating a more intact rna, whereas lower numbers are associated with degraded rna molecules [35] . in order to use bacterial mrna as a substrate in cdna synthesis, polyadenylation was mandatory. the tailing was achieved using the poly(a) polymerase tailing kit (epicentre) following the alternate protocol offered by the manufacturer. briefly, up to 10 mg of total rna were combined with 2 ml poly(a) polymerase reaction buffer, 2 ml 10 mm atp, 0.5 ml riboguard rnase inhibitor and 1 ml poly(a) polymerase (4 u) in a total reaction volume of 20 ml. the reaction was incubated for 20 min at 37uc, terminated by the addition of 1 ml 0.5 m edta and purified by rneasy mini kit (qiagen) following manufacturer's instructions. yield and purity were determined by nanodrop measurements. for cdna synthesis the in-fusion smarter directional cdna library construction kit (clontech) was used according to manufacturer's instructions with slight modifications. 3.5 ml total, polyadenylated rna were mixed with 1 ml of 39 in-fusion smarter cds primer, heated first for 3 min at 72uc and then incubated for additional 2 min at 42uc. after addition of 5.5 ml mastermix (2 ml 5x first strand buffer, 0.25 ml 100 mm dtt, 1 ml 10 mm dntps, 1 ml 12 mm smarter v oligonucleotide, 0.25 ml rnase inhibitor and 1 ml smartscribe reverse transcriptase) the tubes were incubated for 90 min at 42uc. the reaction was terminated at 68uc for 10 min. for second strand cdna synthesis two 2 ml aliquots of first strand reaction were used in long distance pcr using phusion polymerase (finnzymes). each pcr reaction was comprised as follows: 2 ml first-strand reaction, 70 ml rnase-free water, 20 ml 5x phusion hf buffer and 2 ml each of dntp mix (10 mm), 59 figure 6 . specificity binding analysis of epitope peptides of kpn_00466. bar chart representing the mean relative fluorescence intensities (n = 10) of each peptide potentially harboring a linear epitope site after incubation with polyclonal antibodies reactive to k. pneumoniae (green) and c. jejuni (orange). none of the peptides shows a peculiar specific interaction; rather signal intensities are in the same vicinity for each peptide independent of the antibody used. this indicates mainly non-specific binding to occur. doi:10.1371/journal.pone.0110703.g006 pcr primer ii a (12 mm), 39 in-fusion smarter pcr primer (12 mm) and phusion polymerase with a total reaction volume of 100 ml. the pcr reactions were subjected to the cycling program with 98uc for 1 min as initial denaturation followed by 15 cycles of 10 s denaturation at 98uc, 30 s of primer annealing at 65uc and 6 min extension at 72uc. for improved pcr results optimization was performed as follows; 30 ml of the 15 cycle experimental tube were transferred to a separate pcr tube, cycling commenced and aliquots of 5 ml each were collected after 15, 18, 21, 24 and 27 cycles total. the different cycles were compared by gel electrophoresis and the experimental tubes subjected to additional cycles if necessary. finally, pcr reactions were purified using the qiaquick pcr purification kit (qiagen). the purity and yield of each reaction were analyzed by nanodrop measurements. normalization of double-stranded cdna was achieved with the trimmer-2 cdna normalization kit (evrogen) to reduce the number of cdna molecules derived from rrnas. briefly, 12 ml of cdna (approx. 100 ng/ml) were mixed with 4 ml of 4x hybridization buffer. for the trimming reaction 4 ml of this mixture were distributed to four different pcr tubes and overlaid with a drop of pcr-grade mineral oil. after centrifugation (130006g, 2 min), the tubes were incubated for 2 min at 98uc followed by 5 h at 68uc. next, pre-heated (68uc) duplex-specific nuclease (dsn) master buffer was added to each tube and incubation prolonged for 10 min. dsn was added to the first three tubes in decreasing concentrations -1 u/ml, 0.5 u/ml and 0.25 u/ml -with the fourth tube receiving dsn storage buffer and no enzyme as a control reaction. the incubation prolonged for 25 min at 68uc. after addition of 5 ml dsn stop solution and subsequent incubation for 5 min at 68uc, the tubes were placed on ice. the chilled reaction was diluted by addition of 25 ml sterile, rnase-free water. for amplification of normalized cdna, 1 ml of each reaction was used as template in pcr. each pcr reaction contained 1 ml of template from the normalization reaction, 33 ml nuclease-free water, 10 ml 5x phusion hf buffer, 1 ml 10 mm dntp mix (neb), 2 ml of each primer 59 pcr primer ii a (12 mm), 39 in-fusion smarter pcr primer (12 mm) and 1 ml phusion polymerase. the pcr was performed with initial denaturation at 98uc for 1 min and seven cycles of denaturation at 98uc for 10 s, primer annealing at 65uc for 30 s and extension at 72uc for 3 min, respectively. for optimization, the control tube was subjected to 7, 9, 11, and 13 cycles with 12 ml aliquots taken every two cycles. the optimization samples were analyzed by gel electrophoresis (1% agarose, tae, 100 v) and the optimal cycle number determined. the remaining three tubes were subjected to 9+ x cycles with x being the differential of the optimized cycles to the originally performed seven cycles. after the second pcr, the experimental reactions were compared to the optimal control reaction using gel electrophoresis as above. reactions showing a successful normalization were combined and used in a third pcr reaction. after the final pcr, the reactions were purified by qiaquick pcr purification kit. a major feature of the given model is the prominent beta barrel structure that originates from the abundance of beta strands. this is a typical feature of transport and channel proteins spanning the outer bacterial membrane. contrastingly, the identified linear epitope gavvalsttfa is located at the very beginning of the protein and thus not included in the given model. however, it is likely an extension of the truncated n-terminal region marked in orange. doi:10.1371/journal.pone.0110703.g007 figure 8 . 3d model of predicted structure of kpn_00459. the model was predicted using the automated mode of the swiss model application by expasy (university basel). as a template the crystal structure of a na(+)/h(+) antiporter nhaa of e. coli was used. the resulting model spans residues 12 to 390 of the full-length protein and was subsequently dyed using the chimera 1.7 software. coils are depicted in light green, beta strands in purple and alpha helices in blue. the potential linear epitope giafgavelfd is highlighted in orange. it comprises part of an alpha helix, a connective coil and the start of the next alpha helix. doi:10.1371/journal.pone.0110703.g008 in-fusion cloning and cloning vector for cloning pfn18a (promega) was used as a vector, as it features a n-terminal encoded halotag fusion protein, which allows for specific and covalent binding to a unique ligand, thus reducing background and minimizing cross-reactivity in immunoassays with halolink microarrays harboring the ligand on its surface. first, the vector needed to be linearized to be used with the in-fusion cloning technology. this was achieved by reverse pcr using ifs 18a for (59 ttgataccactgcttttc-catggcgatcgcgttatc 39) and ifs 18a rev (59 tctcatcgtaccccgtgtttaaacgaattcgggctcg 39). each reaction contained 2 ml each of 1:10 diluted pfn18a (10 ng/ml) and the two primers, 10 ml 5x phusion hf buffer, 1 ml 10 mm dntps, 0.5 ml phusion polymerase and 32.5 ml nuclease-free water to reach a total reaction volume of 50 ml. the pcr was run using a 25 cycle two-step program with 98uc denaturation for 10 s and 4 min extension at 72uc. after completion, 2 ml of dpni (20 u/ml) were added to the reaction and incubated at 37uc for 1 h. the presence of a single band was checked by gel electrophoresis and the remaining reaction purified by qiaquick pcr purification kit. cloning of normalized cdna and linearized pfn18a vector was performed following the manufacturer's instructions within the in-fusion smarter directional cdna library construction kit (clontech). electroporation 2 ml of the cloning reaction were mixed with 25 ml of electrocompetent acella e.coli cells (mobitec), a bl21 derivative, figure 9 . homology of linear epitope sequence gavvalstffa of kpn_00363. the sequence derived from k. pneumoniae mgh 78578 was used as a reference. identical residues are marked by dots, gaps by a horizontal dash and differences by the single letter amino acid code. seven of nine k. pneumoniae strains show identical epitope sequences, while two strains display changes in two residues. threonine replaces alanine at position 11, a change observed not only in these two strains but in almost all other bacteria within the list. additionally, threonine at position 9 is substituted by either serine or phenylalanine. bacteria other than k. pneumoniae show an additional number of amino acid substitutions, most prominently leucine for valine at position 4 and serine for threonine at position 8. in s.enterica the changes become more pronounced. glycine at position 1 is replaced by serine, valine at position 3 replaced by alanine and threonine inserted for serine at position 7. in some rare cases, other residues have also been substituted, e.g. valine replaces alanine at position 2 in shigella dysenteriae. doi:10.1371/journal.pone.0110703.g009 figure 10 . alanine scan of gavvalsttfa of kpn_00363. box-whisker plot (n = 12) of gavvalsttfa after alanine/glycine scanning. the box comprises 50% of the data, while the whiskers enclose 98%. the median is represented by a small horizontal line and the mean by a small rectangle. rabbit igg served as a positive reference, whereas mbp was used as a negative control. if alanine was present in the original present it was replaced by glycine, otherwise each amino acid was stepwise replaced by alanine. additionally, gavlalsssft and saalaltssft were included as they resemble sequences present in e. coli and s. enterica. switching glycine (position 1), alanine (positions 2 or 5), or threonine (positions 8 and 9) to alanine or glycine, results in a significant drop in signal intensities to levels below or at the negative control. in contrast, substituting valine (position 3) or leucine (position 6) by alanine, leads to an increase in signal intensities to 1700 a.u. and more than 5600 a.u., respectively. note the different axis scales prior and after axis break at 2000 a.u. doi:10.1371/journal.pone.0110703.g010 and electroporated in 1 mm cuvettes using the easyject plus electroporator (peqlab). conditions for electroporation were as follows: voltage = 1400 v, capacity = 25 mf, resistance = 200 v and a pulse duration of 5 ms. the electroporated cells were added to 970 ml of super optimal broth with catabolite expression (soc) and incubated at 37uc for 1 h with shaking at 250 rpm. afterwards, 150 ml of the transformation reaction were plated on lysogeny broth (lb) agar containing ampicillin. for each reaction at least two plates were prepared and incubated at 37uc for 16 h. a total number of 1536 clones were selected and transferred to 1.3 ml u96 deepwell plates (nunc) containing 0.8 ml lb-amp. the plates were incubated overnight at 37uc, 130 rpm. on the next day, the deepwell plates were centrifuged, the supernatant discarded and the pellets resuspended in 370 ml of lb-amp. a new set of u96 deepwell plates was prepared with 850 ml of fresh lbamp and inoculated with 100 ml each from the resuspended overnight cultures. the remaining 270 ml of resuspended overnight culture were mixed with 30 ml of sterile-filtered dmso and stored at 280uc. the newly inoculated plates were incubated for 6 h at 37uc, 130 rpm. afterwards, the temperature was reduced to 20uc, incubation continued for 1 h and protein expression induced by addition of 2 ml of 0.5 m b-d-1-thiogalactopyranoside (iptg). incubation persisted overnight at 20uc, 130 rpm. the cells were harvested by centrifugation (25006g, 10 min), the supernatant discarded and the pellets frozen at 220uc. after 15 min the pellets were resuspended in 180 ml of easylyse bacterial protein extraction solution (epicentre) and incubated for 5 min at room temperature. dnase i was mixed with dnase reaction buffer (10 mm tris-hcl, 2.5 mm mgcl 2 , 10 mm cacl 2 ), added to the reaction and incubation was carried on for 10 min at 37uc. the plates were centrifuged to collect cell debris for 3 min at 25006g. for each sample 10 ml of lysate were transferred to 384 microtiterplates (genetixx), which were used as reservoirs for the spotting procedure. the samples were spotted onto halolink slides (promega) using the qarray2 microarray spotter (molecular devices). 384 different samples were spotted per slide with three replicate slides per screening. in total 1536 samples were screened on 12 slides (n = 3). each sample was spotted as quadruplicates with controls in two identical sets of eighteen 10610 subarrays each (total number of spots per slide 3600). the controls used included ht-ompa and ht-ompf as positive reference proteins as these have been described as immunodominant before. as specificity controls ht-argc and ht-pyrc were used, representing proteins without known immunodominant behaviour, thus binding of the polyclonal antibodies is not expected. in addition two different e.coli strains -acella electrocompetent cells and krx single-step figure 11 . specificity assay of gavvalsttfa and derivatives. the bar chart represents the mean signal intensities (n = 12) of gavvalsttfa and several modified peptides with single amino acid replacements incubated with antibodies reactive to k. pneumoniae (green), e. coli (orange) or s. enterica (purple). the sequences on the left represent the original epitope and modified versions displaying an increase in signal intensity for k. pneumoniae antibodies. in contrast, sequences on the right harbor modifications causing a significant drop in intensity for k. pneumoniae. rabbit igg is used as a positive reference and mbp serves as a negative control. none of the sequences tested displayed any significant signal intensity above the negative control when incubated with either e. coli or s. enterica antibodies. doi:10.1371/journal.pone.0110703.g011 competent cells (promega) -were spotted as further controls. as those two lack proteins expressed with a halotag, they are used as negative controls. after spotting of the samples, the slides were incubated for 1 h at room temperature in a humidity chamber. next, slides were washed with pbs+0.05% igepal ca-630 (pbsi, sigma aldrich) and dried by nitrogen flow. the 2 well proplate module (grace biolabs) was attached to each slide. the top chamber was filled with 1.5 ml of rabbit-polyclonal antibody to k. pneumoniae (acris, 2 mg/ml) in pbs. the bottom chamber was incubated with pbs only. after 2 h of incubation at room temperature with gentle rocking, both chambers of each slide were washed three times with 2 ml of pbsi. secondary antibody (goat-polyclonal to rabbit igg conjugated with chromeo-546, abcam, 5 mg/ml) was subjected to each chamber in pbs and the slides were incubated at room temperature for 2 h in the dark under gentle rocking. finally, slides were washed three times with pbsi, the proplate modules removed and the slides dried by nitrogen flow. the slides were scanned on an axon genepix 4200a laser scanner (molecular devices) with the following settings: 532 nm laser, pmt gain 400, 40% laser power, lines to average 1, 10 mm resolution and standard green emission filter at 575 nm. the raw data sets of all the microarray analyses in this publication have been deposited in ncbi's gene expression omnibus [36] and are accessible through geo series accession numbers gse52536, gse52537, gse52538, and gse60588. the median fluorescence intensity of each spot corrected by the local background (median f532 -b532) was used. further, relative fluorescence intensity (rfi) was calculated by subtracting the signals of the bottom chamber from the raw data signals of the top chamber to account for non-specific binding of secondary antibodies. for screening of expression libraries we used the contrast method with either argc or pyrc as specificity control to determine the contrast via the formula: with rf f i control the median of all rfis of the control used. clones harboring strong signals in microarray screening were selected to be sequenced. sequencing was performed externally by lgc genomics using ht7f (59 acatcggcccgggtct-gaatc 39) and flxr (59 cttcctttcgggctttgttag 39) primers. after sequencing and identification of potentially immunodominant proteins, primers were designed to generate full-length clones for each identified gene, see table s2 for a list of the primers used. cloning was performed as mentioned above with slight modifications. the pfn18a vector was linearized using the following primer set; 18a if linear for (59 gtttaaac-gaattcgggctc 39) and 18a if linear rev (59 ggcgatcgcgttatcgctctg 39) with pcr conditions as mentioned before. protein expression, lysis, and spotting of fulllength proteins were performed as described above. the slides were incubated for 1 h at room temperature in a humidity chamber. for incubation with antibodies 3 well or 16 well proplate modules (grace biolabs) were attached to the halolink slides. processing of the slides was done similar to the original screening, however several different antibodies were used, see section antibodies. for testing of immunodominant characteristics with elisa, the crude lysate was first purified using halolink magnetic beads (promega) following the manufacturer's instructions. the proteins of interest were subsequently cleaved off by digestion with protev protease (promega) and concentration was determined by nanodrop measurements. the samples were diluted to a total protein content of 20 mg/ml in pbs and 50 ml of each sample was added to maxisorb plates (nunc). each sample was analyzed at least in triplicate. the elisa plate was covered with a lid and incubated overnight at 4uc in a humidity chamber. after five washing steps each with pbs+0.05% tween-20 (pbst), the plates were blocked using 200 ml 5% non fat dried milk in pbs per well for 2 h. afterwards, plates were washed three times with pbst. 100 ml of primary antibody solution (c = 4 mg/ml) in pbs containing 1% non fat dried milk were applied to each well using the respective desired antibody or pbs for controls. the plates were incubated for 2 h at room temperature and washed four times with pbst. next, 100 ml of conjugated secondary antibody (goat polyclonal to rabbit igg conjugated with horseradish peroxidase, abcam ab6721, c = 20 ng/ml) were added to each well and incubation carried on for 1 h. finally, plates were washed once again four times with pbst and 100 ml 3,39,5,59-tetramethylbenzidine (tmb, sigma-aldrich) was added to each well for detection. after 30 min of incubation at room temperature in the dark, the reaction was stopped by applying 100 ml of 2 m h 2 so 4 to each well. the optical density of each well was measured using the omega fluostar (bmg labtech) at a wavelength of 450 nm. primers were designed using primer3 [37] within geneious pro 5.6.5 [38] . the sequenced inserts were identified by blast [39] . peptide sequence secondary structures were predicted using the emboss garnier [40] algorithm and the transmembrane regions predicted by tmhmm2.0 [30, 31] . antigenic sites were predicted by emboss antigenic [32, 33] . data evaluation was performed by originpro 8 g (originlab) and microsoft excel. 3-dimensional structure predictions were performed using the swiss model automated mode [41] [42] [43] [44] [45] and pdb files were visualized and analyzed by the ucsf chimera package [46] .chimera is developed by the resource for biocomputing, visualization, and informatics at the university of california, san francisco (supported by nigms p41-gm103311). analysis of full-length proteins was achieved by combining the results from elisa and microarray data. hence, the rfi of each sample was calculated. next, a normalized rfi was generated by dividing the rfi of each sample by the median rfi of all the samples within an area of interest, i.e. incubation compartment. from these normalized rfis a median and standard deviation was calculated. if the median normalized rfi of the positive control was below the median normalized rfi of any of the negative references whilst taking the standard deviations into account, the test was rendered invalid. if the test passed the above criterion, the q values were calculated as follows: with rf f i sample the median of normalized rfis of the sample and rf f i pos:control the median of the normalized rfis of the positive control ompa respectively ompf. the resulting error was calculated by error propagation according to gauss. finally, incorporating all valid tests, the mean q value was determined along with its resulting error following error propagation by gauss, see table 1 . several proteins were chosen for epitope mapping. these were the proteins encoded by kpn_00363, kpn_00459, kpn_00466, kpn_01100, kpn_01584, and kpn_02202. the proteins were divided into 15-mer oligopeptides with an overlap of 11 amino acids in silico. the synthesis and coupling to microarray slides was performed externally by jpt peptide technologies gmbh. each peptide sequence was applied 9 times to one slide. the slides were used with proplate 3-well chamber system (grace) allowing for incubation with different antibodies. first, the slides were blocked with superblock blocking buffer (thermo fischer) for 2 h, washed five times with pbs+0.05% tween-20, primary antibodies applied, incubated overnight at 4uc with mild rocking, washed again, secondary antibodies applied for 2 h in the dark and after a final washing procedure, dried and scanned as above. two different primary antibodies to k.pneumoniae were tested. the bottom chamber was always used as a control chamber, incubated only with secondary antibody. the peptide gavvalsttfa and 11 modifications thereof created by substituting one amino acid by alanine/glycine were synthesized by jpt peptide technologies gmbh. these peptides in combination with two peptides showing closely related sequences, gavlalsssft and saalaltssfte, were applied 9 times to slides. incubation procedure was performed as described above for epitope mapping the expression of the desired halotag fusion proteins was checked by sds-page. after lysis of cells, 2 ml of each protein extract was mixed with 1 ml of 10 mm halotag alexa 488 ligand. after addition of 7 ml 1x tbs (100 mm tris, 150 mm nacl, ph 7.6) the reaction was incubated at room temperature for 30 minutes. 2 ml of each reaction were removed, mixed with 8 ml of 5x loading buffer (fermentas) and 1 ml dtt and heated for 5 min at 70uc. the separation was performed on a mini-protean tgx gel (biorad, any kd, 15 wells) in a protean ii xi cell chamber (biorad) for 30 min at 200 v. as a size reference benchmark fluorescent protein standard (life technologies) was used. fluorescence was measured in a fla-5100 (fujifilm) with excitation at 473 nm. figure s3 homology of giafgavelfd of kpn_00459. the sequence derived from k. pneumoniae mgh78578 was used as a reference. the 100 best matches after blast analysis are shown in the figure with dots indicating identical residues. for differentiation of the sequences the ncbi accession number of the parent protein is given followed by the strain designation, if available. only three e. coli strains in lines 63, 97 and 98 feature a valine residue at the second position instead of the consensus isoleucine. consequently, this sequence is highly conserved within the enterobacteriaceae including e. coli, klebsiella, salmonella, and enterobacter among others. (pdf) table s1 list of 192 sequenced clones after screening the clones were sequenced by lgc genomics using ht7f and flx primers. clones that were not successfully sequenced are indicated by ''-'', clones carrying inserts too short to be reliably mapped to a gene are marked as truncated. additionally, a few inserts were detected deriving from primer concatamers. these are displayed as ''artificial''. the remaining clones are indicated by the corresponding locus tag and protein name. several clones apparently carry identical inserts, especially obvious for kpn_01805 or kpn_02668. these were discarded from further analysis as the mapped inserts are very short and might have an artificial origin. inserts that were highly unlikely to garner new immunogenic proteins, antigens described previously, e.g. ompa, other molecules like trna and rrna were abolished from further analysis. table s2 primers used in this study. each primer is given with a name, its sequence in 59 to 39 direction and the target gene or vector. for each target f represents forward and r the reverse primer. the primers were used for cloning in the in-fusion smarter directional cdna library construction kit. (xls) manual of clinical microbiology clinical epidemiology of the global expansion of klebsiella pneumoniae carbapenemases extended-spectrum beta-lactamase producing klebsiella spp. in chicken meat and humans: a comparison of typing methods modern clinical microbiology: new challenges and solutions rapid identification of novel immunodominant proteins and characterization of a specific linear epitope of campylobacter jejuni application of a microarray-based immunoscreening for rapid identification of novel antigens of salmonella enteritidis microarray-based method for screening of immunogenic proteins from bacteria halotagbased purification of functional human kinases from mammalian cells severe acute respiratory syndrome diagnostics using a coronavirus protein microarray reverse transcriptase template switching: a smart approach for full-length cdna library construction polyadenylic acid sequences in e. coli messenger rna identification of the gene for an escherichia coli poly (a) polymerase a simple method to enrich mrna from total prokaryotic rna magnetic capturehybridization method for purification and probing of mrna for neutral protease of bacillus cereus dsn depletion is a simple method to remove selected transcripts from cdna populations duplex-specific nuclease efficiently removes rrna for prokaryotic rna-seq ligation-independent cloning of pcr products (licpcr) identification of vaccine candidate antigens of an esbl producing klebsiella pneumoniae clinical strain by immunoproteome analysis the national center for biotechnology information's protein clusters database protective efficacy of dna vaccines encoding outer membrane protein a and ompk36 of klebsiella pneumoniae in mice b-cell epitopes: fact and fiction x-ray crystallography of antibodies the structure of bacterial outer membrane proteins identification and characterization of ompl as a potential vaccine candidate for immune-protection against salmonellosis in mice the unique structure of haemophilus influenzae protein e reveals multiple binding sites for host factors directed evaluation of enterotoxigenic escherichia coli autotransporter proteins as putative vaccine candidates structure of the c-terminal domain of neisseria heparin binding antigen (nhba), one of the main antigens of a novel vaccine against neisseria meningitidis in vivo versus in vitro protein abundance analysis of shigella dysenteriae type 1 reveals changes in the expression of proteins involved in virulence, stress and energy metabolism surface expression, singlechannel analysis and membrane topology of recombinant chlamydia trachomatis major outer membrane protein predicting transmembrane protein topology with a hidden markov model: application to complete genomes a hidden markov model for predicting transmembrane helices in protein sequences a semi-empirical method for prediction of antigenic determinants on protein antigens new hydrophilicity scale derived from high-performance liquid chromatography peptide retention data: correlation of predicted surface residues with antigenicity and x-ray-derived accessible sites toward the estimation of the absolute quality of individual protein structure models the rin: an rna integrity number for assigning integrity values to rna measurements gene expression omnibus: ncbi gene expression and hybridization array data repository primer3 on the www for general users and for biologist programmers basic local alignment search tool analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins the swiss-model workspace: a web-based environment for protein structure homology modelling the swiss-model repository and associated resources swiss-model: an automated protein homology-modeling server swiss-model and the swiss-pdb viewer: an environment for comparative protein modeling protein modeling by email ucsf chimera-a visualization system for exploratory research and analysis the k. pneumoniae strain mgh 78578 was a kind gift of the group of s. bereswill (department of microbiology and hygiene, charitã© -university medicine berlin, berlin, germany). sh is greatly indebted to martina obry for her assistance during expression library construction and clone isolation. the authors would like to thank simone aubele for technical assistance. we also gratefully acknowledge michaela schellhase for microarray printing. conceived and designed the experiments: sh mvnr. performed the experiments: sh. analyzed the data: sh ffb mvnr. contributed reagents/materials/analysis tools: sh. wrote the paper: sh ffb mvnr. key: cord-010369-x9z8dg6a authors: saito, kyoko; fukasawa, masayoshi; shirasago, yoshitaka; suzuki, ryosuke; osada, naoki; yamaji, toshiyuki; wakita, takaji; konishi, eiji; hanada, kentaro title: comparative characterization of flavivirus production in two cell lines: human hepatoma-derived huh7.5.1-8 and african green monkey kidney-derived vero date: 2020-04-24 journal: plos one doi: 10.1371/journal.pone.0232274 sha: doc_id: 10369 cord_uid: x9z8dg6a the flaviviridae is a family of enveloped viruses with a positive-sense single-stranded rna genome. it contains many viruses that threaten human health, such as japanese encephalitis virus (jev) and yellow fever virus (yfv) of the genus flavivirus as well as hepatitis c virus of the genus hepacivirus. cell culture systems highly permissive for the flaviviridae viruses are very useful for their isolation, propagation, and diagnosis, an understanding of their biology, and the development of vaccines and antiviral agents. previously, we isolated a human hepatoma huh-7-derived cell clone, huh7.5.1–8, which is highly permissive to hepatitis c virus infection. here, we have characterized flavivirus infection in the huh7.5.1–8 cell line by comparing with that in the african green monkey kidney-derived vero cell line, which is permissive for a wide spectrum of viruses. upon infection with jev, huh7.5.1–8 cells produced a higher amount of virus particles early in infection and were more susceptible to virus-induced cell death than vero cells. similar outcomes were obtained when the cells were infected with another flavivirus, yfv (17d-204 strain). quantification of cellular and extracellular viral rna revealed that high jev production in huh7.5.1–8 cells can be attributed to rapid viral replication kinetics and efficient virus release early in infection. in a plaque assay, huh7.5.1–8 cells developed jev plaques more rapidly than vero cells. although this was not the case with yfv plaques, huh7.5.1–8 cells developed higher numbers of yfv plaques than vero cells. sequence analysis of cdna encoding an antiviral rna helicase, rig-i, showed that huh7.5.1–8 cells expressed not only a full-length rig-i mrna with a known dominant-negative missense mutation but also variants without the mutation. however, the latter mrnas lacked exon 5/6−12, indicating functional loss of rig-i in the cells. these characteristics of the huh7.5.1–8 cell line are helpful for flavivirus detection, titration, and propagation. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the huh-7 (no. jcrb0403) and vero (no. jcrb9013) cell lines were purchased from the japanese collection of research bioresources (jcrb) cell bank, national institute of biomedical innovation (osaka, japan). huh7.5.1-8, huh7.5.1, and huh-7 cells were maintained at 37˚c in an atmosphere of 5% co 2 in dulbecco's modified eagle's medium (dmem; wako pure chemical industries, osaka, japan; no. 044-29765) supplemented with 10% (v/v) fetal bovine serum (fbs; sigma-aldrich, st. louis, mo, usa; no. 172012-500ml), 0.1 mm nonessential amino acids (nacalai tesque, kyoto, japan; no. 06344-56), 100 u/ml penicillin g, and 100 μg/ ml streptomycin sulfate (nacalai tesque; no. 26253-84). vero cells were maintained at 37˚c in an atmosphere of 5% co 2 in eagle's minimal essential medium (emem; wako pure chemical industries; no. 051-07615) supplemented with 5% (v/v) heat-inactivated fbs, 100 u/ml penicillin g, and 100 μg/ml streptomycin sulfate. for comparative analysis, huh7.5.1-8 and vero cells were grown in the same dmem-based medium as above but with heat-inactivated fbs. before starting the experiments, vero cells were adapted to this medium through at least eight passages. jev production in vero cells was comparable between the dmem-based and emem-based media (s1a fig). the jev nakayama strain (isolated in japan in 1935) and jev rat strain [21] were propagated in vero cells. the yfv 17d-204 strain, a current vaccine strain derived from the 17d strain [22] , was kindly provided by dr. tomohiko takasaki (department of virology i, national institute of infectious diseases) and propagated in vero cells. the resultant culture supernatants were stored at −80˚c and used as virus stocks. these virus strains were chosen because they could be used in our biosafety level 2 laboratory. for virus titration, a plaque assay was performed on vero cells according to the manual provided on the website of the national institute of infectious diseases [23] with modifications. in brief, cell monolayers grown in 12-well plates (corning costar 1 ; no. 3513) were inoculated with virus samples in 0.4 ml per well of culture medium with reduced heat-inactivated fbs (2%) and were incubated at 37˚c for 2 h. after removal of the samples, the cells were incubated in 2 ml of overlay medium consisting of emem (nissui pharmaceuticals, tokyo, japan; no.05901), 0.22% (w/v) nahco 3 , 2% (v/v) heat-inactivated fbs, 2 mm l-glutamine (nacalai tesque; no. 16948-04), and 1% (w/v) methylcellulose (tokyo chemical industry, tokyo, japan; no. m0185) at 37˚c for 5 days. the cells were fixed with 10% (v/v) neutral buffered formalin (wako pure chemical industries; no. 133-10311) and stained with 0.0375% (w/v) methylene blue. wells with 10−100 plaques were used to calculate the virus titer in plaque formation units (pfu) . for the comparative plaque assay, cells were similarly grown and infected, but overlaid with 2.5 ml per well of dmem (nissui pharmaceuticals; no. 05919) supplemented with 0.2% (w/v) nahco 3, 2% (v/v) heat-inactivated fbs, 2 mm l-alanyl-l-glutamine (nacalai tesque; no. 04260-64), 0.1 mm nonessential amino acids, and 1.25% (w/v) methylcellulose. jev plaque formation in vero cells was not so different between the dmem overlay medium and the emem overlay medium (s1b fig). after fixation and staining, plates were scanned with a document scanner, and image data were obtained (s11 fig). to improve plaque visibility, an invert filter was applied to the image data, and highlights were adjusted using photoshop elements (version 14; adobe systems, san jose, ca, usa). total rna was extracted and purified from cells and culture supernatant using the blood/cultured cell total rna mini kit (favorgen biotech, pingtung city, taiwan; no. fabrk 001-2) and the viral nucleic acid extraction kit i (favorgen biotech; no. favnk 001-2), respectively, according to the manufacturer's instructions. to determine the copy numbers of jev rna (nakayama strain; genbank accession no. ef571853.1), the rna fraction was subjected to one-step qrt-pcr using the thunder-bird 1 probe one-step qrt-pcr kit (toyobo, osaka, japan; no. qrz-101) with primers #1 and 2 and a hydrolysis probe #3 (table 1 ). the following reactions were performed on the lightcycler 1 96 system (roche applied science): reverse transcription at 50˚c for 10 min and denaturation at 95˚c for 1 min, followed by 40 cycles of 95˚c for 15 s and 56˚c for 45 s. to normalize cellular jev rna levels, the copy numbers of mrna encoding glyceraldehyde-3-phosphate dehydrogenase (gapdh) (human, genbank accession no. nm_002046.3; chlorocebus sabaeus, genbank accession no. xm_007967342) in the same rna samples were determined with primers #6 and 7 and a hydrolysis probe #8 ( table 1 ; the sequences of #6−8 are homologous to both the human and monkey sequences) under the same qrt-pcr conditions, except that the annealing temperature was 60˚c. a standard curve was established using serial dilutions of plasmids encoding the jev e protein (bases 962 to 2491) or human gapdh. for construction of the plasmid encoding the jev e protein, the rna fraction prepared from a jev (nakayama strain) stock was reverse transcribed with primer #4 (table 1) total rna from cells was reverse transcribed with an oligo dt primer using the primescript™ ii 1st strand cdna synthesis kit (described above). the first strand cdna was used as a template to amplify a cdna fragment encoding rig-i (ddx58) (genbank accession no. (table 1) . upon sequence determination, nucleotide variations were excluded that were not consistent with the rna-seq data from huh7.5.1 (drr018792) and huh7.5.1-8 (drr018793) [20] or the whole-genome sequence data from huh-7 and huh7.5.1-8 (ddbj dra database under the project id prjdb7928) [25] . all the experiments were done with three biological replicates and repeated as described in fig comparison of jev production between huh7.5.1-8, huh7. first, the huh7.5.1-8 cell line was compared with the parental huh7.5.1 and the ancestral huh-7 cell lines regarding jev production. cells were infected with jev (nakayama strain, unless otherwise noted), and then the amount of infectious virus particles (titer) in culture supernatant and jev-induced cell death were monitored during 1−4 d pi. the relative jev titer of these cell lines increased and peaked at 2−3 d pi in a similar manner ( fig 1a) . under the same seeding conditions, doubling times of huh7.5.1-8, huh7.5.1, and huh-7 cells were not significantly different (after bonferroni correction, p > 0.0167) (s2b fig) . thus, jev production appeared to be comparable between the three cell lines and not enhanced in this lineage unlike for hcv production. in a parallel cell viability assay (fig 1b) , susceptibility to jevinduced cell death was not greatly different between the three cell lines. next, these cell lines were infected with jev, overlaid with methylcellulose-containing medium, and then compared regarding jev plaque formation. huh7.5.1-8 and huh7.5.1 cells similarly developed jev plaques, whereas huh-7 cells began to die at 3 d pi and detached from the surface of the well irrespective of infection at 5 d pi ( fig 1c) . these results indicated that the huh7.5.1-8 and huh7.5.1 cell lines are better than the huh-7 cell line for a jev plaque assay. although huh7.5.1-8 and huh7.5.1 cell lines were comparable in terms of jev infection, the huh7.5.1-8 cell line has the advantage of being phenotypically more stable than the huh7.5.1 cell line [20] . thus, we hereafter focused on the huh7.5.1-8 cell line and compared it with the vero cell line regarding jev production. jev production in huh7. for comparative analysis, we used the vero cell subline jcrb9013, in which jev production was almost equivalent to or slightly higher than that in other vero cell sublines (s4 fig). huh7.5.1-8 and vero cells under high and low confluency were infected with jev at moi 0.1, and then the virus titer in culture supernatant was monitored from 1 to 4 d pi (fig 2) . the culture supernatant of huh7.5.1-8 cells exhibited a higher relative virus titer than that of vero cells, particularly during the early infection times. the difference in the relative virus titer was remarkable in high-cell-density infection (fig 2a) compared with low-cell-density infection ( fig 2b) , implying that cell-cell contacts promote viral spread in huh7.5.1-8 cells. the difference in the titer was not accounted for by the growth rates of the two cell lines, because their doubling times under high confluency conditions were nearly equal (s2 fig). a parallel viability assay showed that huh7.5.1-8 cells lost viability faster than vero cells irrespective of cell density (fig 3) . the same trends were observed in jev production and cell viability at smaller moi (0.01), although high confluency appeared to be needed (s5 fig) . these results showed that jev production in the huh7.5.1-8 cell line is higher than that in the vero cell line during early infection times and that the huh7.5.1-8 cell line is more susceptible to jev-induced cell death than the vero cell line. the particle-to-pfu ratio was calculated for huh7.5.1-8-and vero-derived jev particles, and the ratio of the values of both particles was determined (fig 4) . both virus particles showed a similar particle-to-pfu ratio, ruling out the possibility that huh7.5.1-8-derived virus particles had a higher infectivity than vero-derived ones. the reason for large variations found at 2 and 3 d pi remains unknown, but that at 3 d pi may have been partly due to variable amounts of non-infectious viral rna leaking from dead huh7.5.1-8 cells (fig 3) . in a representative experiment (s6 fig) , the actual value of the particle-to-pfu ratio was minimally~600, which is roughly consistent with a previous report [26] . taken together with the results of fig 2, these results suggested that huh7.5.1-8 cells produce a higher amount of jev particles than vero cells during early infection times. next, we compared the jev replication kinetics between huh7.5.1-8 and vero cells by monitoring intracellular and extracellular viral rna levels during 0−73 h pi. an initial rise in intracellular viral rna level in huh7.5.1-8 cells was detected as early as 9 h pi, whereas the rise for vero cells was not remarkable during 0-12 h pi (fig 5a [a]) . similarly, the level of extracellular viral rna from huh7.5.1-8 cells rose earlier than that from vero cells (fig 5b [a] ). although the level of intracellular viral rna was not different between the two cell lines after 12 h pi (fig 5a [b] ), the level of extracellular viral rna was higher for huh7.5.1-8 cells than for vero cells during 12−48 h pi (fig 5b [b] ). furthermore, the relative amount of extracellular viral rna expressed as a percentage of the total (extracellular plus intracellular) amount of viral rna was about 2-fold higher in huh7.5.1-8 cells than in vero cells at 1 d pi (fig 6) . thereafter, the value for huh7.5.1-8 cells varied with each experiment, possibly due to variation in virus-induced cell death, but leading to no difference between the values for for both panels, bars with error bars represent the mean ± standard deviation (sd) of three independent experiments. statistical significance between cell lines was determined by a one-sample t test (for comparison with a hypothetical value,1) or an unpaired two-tailed t test using bonferroni correction and p values less than 0.0167 were considered statistically significant. ns, not significant. (c) cells were seeded at 4 x 10 5 cells per well of a 12-well plate one day before infection and then infected with jev at 40 pfu (determined with vero cells) per well. the cells were fixed and stained at 3−5 d pi. values given below the images are plaque numbers expressed as the mean ± sd of triplicates from one representative experiment. statistical significance was determined as described above. values in parentheses are p values, and those less than 0.0167 were considered statistically significant. similar results were obtained in two other independent experiments (s3 fig). https://doi.org/10.1371/journal.pone.0232274.g001 huh7.5.1-8 and vero cells. thus, huh7.5.1-8 cells may release jev more efficiently than vero cells at least at 1 d pi. collectively, these results suggested that rapid viral replication kinetics and efficient early virus release contribute to high jev production in huh7.5.1-8 cells. jev plaque formation (size and number) were compared between huh7.5.1-8 and vero cell lines at 3−5 d pi. as shown in fig 7a and s7a fig, clearly visible plaques were developed in huh7.5.1-8 cells at as early as 3 d pi, but not in vero cells at that time. in the course of the comparison of flavivirus production between huh7.5.1-8 and vero cell lines infection, plaques in huh7.5.1-8 cells grew more rapidly than those in vero cells. similar results were observed when the cells were infected with another jev strain, rat (fig 7b and s7b fig) . these results showed that the huh7.5.1-8 cell line supports more rapid jev plaque formation than the vero cell line. the rapid plaque formation in huh7.5.1-8 cells appears to reflect their high jev productivity and susceptibility to jev-induced cell death. the plaque numbers of the nakayama strain at 5 d pi were comparable between the two cell lines (fig 7a and s7a fig), whereas those of the rat strain tended to be higher in the huh7.5.1-8 cell line than in the vero cell line (fig 7b and s7b fig) . these results indicated that the difference in plaque numbers between the two cell lines varies by each virus strain. fig 2, and the culture supernatants were harvested at the indicated times. rna was extracted and purified from each culture supernatant, and viral rna copy number in the supernatant was determined by qrt-pcr and regarded as the number of virus particles. a virus titer in pfu of each culture supernatant was also determined by plaque assay, and particle-to-pfu ratio was calculated. each symbol represents the relative value calculated by dividing the value of huh7.5.1-8-derived jev by that of vero-derived jev obtained from the same experiment. bars with error bars represent the mean ± sd of the ratio from eight (1 and 2 d pi) or seven (3 d pi) independent experiments (the reason for the different sample sizes was that one experiment lacked data for 3 d pi). statistical significance was determined by a one-sample t test. values in parentheses indicate p values, and those less than 0.05 were considered statistically significant. (fig 8c and 8d) . the combined data showed that virus yield in huh7.5.1-8 cells was significantly higher than that in vero cells for high confluency at 2 d pi and low confluency at 1 and 2 d pi. furthermore, huh7.5.1-8 cells were more susceptible to yfv-induced cell death than vero cells (fig 8e and 8f ), as observed with jev. these results suggested that the huh7.5.1-8 cell line, compared with the vero cell line, has a higher virus productivity and susceptibility to virus-induced cell death upon flavivirus infection. next, we compared the yfv plaque formation between the two cell lines (fig 9 and s9 fig) . yfv plaques began to appear at 4 d pi in both cell lines. although plaques in huh7.5.1-8 cells were slightly smaller than those in vero cells, huh7.5.1-8 cells developed a several fold higher number of yfv plaques than vero cells. these results suggested that the high yfv productivity and susceptibility to yfv-induced cell death of huh7.5.1-8 cells can be mainly attributed to their high infection efficiency. although yfv plaques developed in vero cells at 4 d pi in fig 9, no cell death was observed with these cells at this time point in fig 8e and 8f . a possible explanation for this discrepancy in cell death may be the difference in culture conditions: the serum concentration was 10% for fig 8 and 2% for fig 9. in addition, the medium in the case of jev, the lower the serum concentration is, the more cell death occurs [27] . recently, codon 55 in the rig-i gene from the huh7.5.1-8 cell line was found to be heterozygous: mutant-type ata (ile) and wild-type aca (thr) [25] . to confirm whether the huh7.5.1-8 cell line is defective in rig-i, we amplified the rig-i cdna derived from total rna isolated from huh-7, huh7.5.1 and huh7.5.1-8 cells. an amplicon corresponding to the expected size for the rig-i sequence (~3 kbp) and another, with a smaller size (~2 kbp), were detected in the three cell lines (fig 10a) and directly sequenced (fig 10b) . the 3-kbp and 2-kbp cdnas from the huh-7 cell line exclusively contained the wild-type codon 55. in contrast, the 3-kbp cdnas from the huh 7.5.1 and huh7.5.1-8 cell lines contained the mutanttype codon 55, whereas the 2-kbp cdnas from these cell lines contained the wild-type codon 55. cloning and sequencing of the cdnas of huh-7 and huh7.5.1-8 cell lines revealed that the 3-kbp cdnas were full-length (fl1, 2), whereas the 2-kbp cdnas lacked exons 6−12 (sv1 found in both cell lines) or 5−12 (sv2 found in huh7.5.1-8) (fig 10c and s10 fig) . rig-i protein isoforms deduced from the splice variants (sv1, 2) completely lacked a helicase atpbinding domain, indicating that they are defective like rig-i protein with a mutated atpbinding site [28] . these results were consistent with the above findings [25] and indicated that huh7.5.1-8 is a rig-i null mutant cell line. although the huh7.5.1-8 cell line is more permissive to hcv infection than the huh7.5.1 cell line [20] , we found that this was not the case with jev infection: the huh7.5.1-8 and the parental huh7.5.1 cell lines were comparable in terms of jev productivity, susceptibility to jev-induced cell death, and jev plaque formation (fig 1) . even the ancestral huh-7 cell line was comparable except for plaque formation. these findings indicate that genetic factors contributing to the high permissiveness of the huh7.5.1-8 cell line to hcv infection are specific rather than broadly proviral. one such genetic factor might be involved in stable hcv receptor expression [20] . unlike the huh7.5.1-8 and huh7.5.1 cell lines, the huh-7 cell line was not maintained under our culture conditions for the plaque assay (fig 1c) , likely because our huh-7 cells had a lower fitness to cell culture than huh7.5.1-8 and huh7.5.1 cells. here, we found three differences in outputs of flavivirus infection between huh7.5.1-8 and vero cell lines. first, huh7.5.1-8 cells produced higher amounts of infectious jev and yfv than vero cells early in infection (figs 2 and 8a-8d ). in the case of jev, the high virus productivity of huh7.5.1-8 cells may be attributed not to the increase in infectivity of a virus particle (fig 4) , but to viral rapid replication kinetics and efficient virus release early in infection (figs 5 and 6) . therefore, huh7.5.1-8 cells are more suitable than vero cells for obtaining large amounts of flavivirus in a short period of time. recently, huh-7 cells were shown to produce higher amounts of zika virus than other cells including vero e6 cells (atcc crl-1586) early in infection [29] . considering that jev production in huh7.5.1-8 cells and the ancestral huh-7 cells were comparable (fig 1a) , high virus productivity early in flavivirus infection may be a common feature among the huh-7 lineage. in addition, a similar trend was observed when huh-7 cells were infected with middle east respiratory syndrome coronavirus [30] . thus, the feature may not be limited to flavivirus infection. second, we found that huh7.5.1-8 cells were more susceptible to jev/yfv-induced cell death than vero cells (figs 3, 8e and 8f ). these characteristics of the huh7.5.1-8 cell line may facilitate cell viability-based screenings of antiviral host factors and agents for flaviviruses. meanwhile, the potent cell death observed in huh7.5.1-8 cells appears to be a part of the reason for plateaued or reduced virus production late in infection. therefore, genetic engineering of the huh7.5.1-8 cell line to make it less susceptible to virus-induced cell death might yield a cell line with more flavivirus production. fig 2. (a, b) culture supernatants were harvested at 23, 47, 74, and 98 h pi, and virus titers in the supernatants were determined by plaque assay. each point represents the mean ± sd of triplicates from one representative experiment. some error bars are not visible due to their small size. similar results were obtained in two other independent experiments (s8 fig). (c, d) the titer ratio was calculated by dividing the titer value of huh7.5.1-8 cells by that of vero cells at each time point. bars with error bars represent the mean ± sd of three independent experiments (a, b and s8 fig). each symbol represents the mean from one experiment. (e, f) cell viability was determined at the indicated times. bars with error bars represent the mean ± sd of three independent experiments. statistical significance was determined by an unpaired two-tailed t test (a, b, e, f) or a one-sample t test (c, d). values in parentheses indicate p values, and those less than 0.05 were considered statistically significant. huh, huh7.5.1-8 cells. https://doi.org/10.1371/journal.pone.0232274.g008 comparison of flavivirus production between huh7.5.1-8 and vero cell lines third, we found that huh7.5.1-8 cells developed jev plaques rapidly (fig 7) and formed a larger number of yfv plaques (fig 9) compared with vero cells, though the difference in plaque numbers between the two cell lines varied by each virus. the jev plaque phenotype of huh7.5.1-8 cells appears to reflect their high virus productivity and susceptibility to virusinduced cell death. because a plaque assay is somewhat time-consuming, the plaque phenotype observed with jev infection in huh7.5.1-8 cells can be utilized to shorten the period of plaque assay. in addition, the high-number plaque phenotype observed with yfv infection in huh7.5.1-8 cells can be utilized to improve the sensitivity of detection and titration of yfv by comparison of flavivirus production between huh7.5.1-8 and vero cell lines plaque assay. vero cells may be more suitable to viruses requiring long-term cultivation to grow plaques, because they were more tolerant to our plaque assay conditions than huh7.5.1-8 cells. in addition, the higher tolerance of vero cells to flavivirus-induced cell death, compared with huh7.5.1-8 cells, may be a feature that makes the vero cell line advantageous for vaccine production. on top of these findings, we provided evidence that huh7.5.1-8 is a rig-i null mutant cell line. our results suggested that, in a huh7.5.1-8 cell, one allele of the rig-i gene generates full-length mrna with a t55i mutation that abolishes rig-i-mediated antiviral signaling, whereas the other allele generates defective splice variants without the mutation. one of the splice variants is also expressed in the parental huh-7 cell line. codon 55 of rig-i gene of huh7.5.1 cells was heterozygous as was that of huh7.5.1-8 cells, showing that the heterozygosity of the codon was stable at least during the time span for isolation of the huh7.5.1-8 clone from huh7.5.1 cells. at present, the underlying mechanisms that generate splice variants and their impacts on rig-i signaling remain to be studied. although jev has been restricted by rig-i in a mouse model [31] and mouse cell lines [32, 33] , jev production and susceptibility to jev-induced cell death were not dramatically altered in the huh7.5.1-8 and huh7.5.1 cell lines compared with the huh-7 cell line (fig 1a and 1b) , implying that the rig-i pathway may not work or may be counteracted by viral mechanisms [34] under our experimental conditions. in conclusion, our study highlighted the characteristics of the huh7.5.1-8 cell line that are helpful for improvement of flavivirus propagation, detection, and titration. further study is needed to investigate the potential versatility of the huh7. the culture supernatants were harvested at the indicated times. rna was extracted and purified from each culture supernatant, and viral rna copy number in the supernatant was determined by qrt-pcr. a virus titer in pfu of each culture supernatant was also determined by plaque assay, and particle-to-pfu ratio was calculated. each point represents the mean ± sd of triplicates from one representative experiment. statistical significance was determined by an unpaired two-tailed t test. values in parentheses indicate p values, and those less than 0.05 were considered statistically significant. huh, huh7. (sv1, 2) . alignment of nucleotide sequences was performed using genetyx version 14.0.0 (genetyx co., tokyo, japan). (pdf) s11 fig. unprocessed image data, related to figs 1c, 7, 9 , s1b, s3, s7 and s9. (pdf) s1 raw images. raw image data (fig 10a) . (pdf) estimated global incidence of japanese encephalitis: a systematic review fact sheets yellow fever studies on sv40 in tissue culture: preliminary step for cancer reserach in vitro studies on sv40 in tissue culture: preliminary step for cancer research in vitro biological characteristics and viral susceptibility of an african green monkey kidney cell line (vero) the genome landscape of the african green monkey kidney-derived vero cell line homozygous deletion of the alpha-and beta 1-interferon genes in human leukemia and derived cell lines guidelines for plaque-reduction neutralization testing of human antibodies to dengue viruses west nile virus infection and serologic response among persons previously vaccinated against yellow fever and japanese encephalitis viruses. vector borne zoonotic dis cell substrates for the production of viral vaccines the vero cell-derived, inactivated, sa14-14-2 strain-based vaccine (ixiaro) for prevention of japanese encephalitis growth of human hepatoma cells lines with differentiated functions in chemically defined medium highly permissive cell lines for subgenomic and genomic hepatitis c virus rna replication robust hepatitis c virus infection in vitro production of infectious hepatitis c virus in tissue culture from a cloned viral genome regulating intracellular antiviral defense and permissiveness to hepatitis c virus rna replication through a cellular rna helicase, rig-i roles of rig-i n-terminal tandem card and splice variant in trim25-mediated antiviral signal transduction viral rna detection by rig-i-like receptors rig-i in rna virus recognition isolation and characterization of an huh.7.5.1-derived cell clone highly permissive to hepatitis c virus characterization of the e-138 (glu/lys) mutation in japanese encephalitis virus by using a stable, full-length, infectious cdna clone the effect of prolonged cultivation in vitro upon the pathogenicity of yellow fever virus efficient trafficking of ceramide from the endoplasmic reticulum to the golgi apparatus requires a vamp-associated protein-interacting ffat motif of cert identification of characteristic genomic markers in human hepatoma huh7 and huh7.5.1-8 cell lines development of multiplex real-time reverse transcriptase pcr assays for detecting eight medically important flaviviruses in mosquitoes flavivirus activates phosphatidylinositol 3-kinase signaling to block caspasedependent apoptotic cell death at the early stage of virus infection regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp2 comparative analysis of different cell systems for zika virus (zikv) propagation and evaluation of anti-zikv compounds in vitro mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-alpha treatment differential roles of mda5 and rig-i helicases in the recognition of rna viruses rig-i mediates innate immune response in mouse neurons following japanese encephalitis virus infection roles of tlr3 and rig-i in mediating the inflammatory response in mouse microglia following japanese encephalitis virus infection japanese encephalitis virus ns5 inhibits type i interferon (ifn) production by blocking the nuclear translocation of ifn regulatory factor 3 and nf-kap-pab we thank dr. francis v. chisari (the scripps research institute, la jolla, ca, usa) for providing the huh7.5.1 cell line. we also thank dr. tomohiko takasaki (department of virology i, national institute of infectious diseases) for providing the yfv 17d-204 strain. key: cord-001843-ceatyj3o authors: huang, yong; xing, na; wang, zengguo; zhang, xiujuan; zhao, xiaomin; du, qian; chang, lingling; tong, dewen title: ultrasensitive detection of rna and dna viruses simultaneously using duplex undp-pcr assay date: 2015-11-06 journal: plos one doi: 10.1371/journal.pone.0141545 sha: doc_id: 1843 cord_uid: ceatyj3o mixed infection of multiple viruses is common in modern intensive pig rearing. however, there are no methods available to detect dna and rna viruses in the same reaction system in preclinical level. in this study, we aimed to develop a duplex ultrasensitive nanoparticle dna probe-based pcr assay (duplex undp-pcr) that was able to simultaneously detect dna and rna viruses in the same reaction system. pcv2 and tgev are selected as representatives of the two different types of viruses. pcv2 dna and tgev rna were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for tgev and pcv2 were added to form a sandwich-like complex with nucleic acids released from viruses. after magnetic separation, dna barcodes specific for pcv2 and tgev were eluted using dtt and characterized by specific pcr assay for specific dna barcodes subsequently. the duplex undp-pcr showed similar sensitivity as that of single undp-pcr and was able to detect 20 copies each of pcv2 and tgev in the serum, showing approximately 250-fold more sensitivity than conventional duplex pcr/rt-pcr assays. no cross-reaction was observed with other viruses. the positive detection rate of single mmpsand duplex mmps-based duplex undp-pcr was identical, with 29.6% for pcv2, 9.3% for tgev and 3.7% for pcv2 and tgev mixed infection. this duplex undp-pcr assay could detect tgev (rna virus) and pcv2 (dna virus) from large-scale serum samples simultaneously without the need for dna/rna extraction, purification and reverse transcription of rna, and showed a significantly increased positive detection rate for pcv2 (29%) and tgev (11.7%) preclinical infection than conventional duplex pcr/rt-pcr. therefore, the established duplex undp-pcr is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility. along with the development of large-scale and intensive swine production, mixed infections of multiple pathogens are increasingly becoming common in swine farms. porcine reproductive and respiratory syndrome virus (prrsv), porcine circovirus type 2 (pcv2), classical swine fever virus (csfv), porcine pseudorabies virus (prv), transmissible gastroenteritis virus (tgev), porcine parvovirus (ppv) and porcine epidemic diarrhea virus (pedv) are major pathogens causing heavy economic losses in swine industry [1] [2] [3] [4] [5] [6] [7] [8] . on the basis of clinical signs, it is difficult to determine whether sick pigs are infected by single or multiple viruses [9] . therefore, it is imperative to establish an effective and rapid method to detect multiple dna and rna viruses simultaneously in single sample. traditional diagnostic methods of dna and rna viruses are mainly dependent on detection of viral proteins and nucleic acids. currently, common methods for detecting viral antigen in solution is enzyme-linked immunosorbent assay (elisa), but elisa shows some shortcomings that are difficult to overcome. for example, elisa requires at least one highly specific antibody for a particular viral antigen, but a specific antibody is not able to detect all viral strains of a kind of virus due to genotype difference; a elisa kit is usually for one kind of virus, leading to that elisa detection are complicated, time-consuming and expensive, and it is difficult to achieve scale detection when we need to detect a variety of viruses at the same time. in addition, it is difficult to find viral infection by elisa when viral load is below a certain level because the sensitivity base line of elisa is relatively higher [10, 11] . the nucleic acids-based detection methods include conventional pcr, rt-pcr, loop-mediated isothermal amplification (lamp) and real-time pcr, which have been used in the diagnosis of virus infection [12] [13] [14] [15] [16] . although lamp and real-time pcr are more sensitive than conventional pcr or rt-pcr, lamp can only detect one pathogen at a time and lamp products are difficult to identify, while real-time pcr requires special or expensive instruments and easily shows false positive results [17, 18] . however, more critical is that all the existing pcr-based assays need rna/dna extraction. it is known that the extraction and detection procedures of dna and rna are different from each other. rna is easily degradable as compared to dna, so in pcrbased methods of detecting rna viruses, viral genomic rna extracted from field samples should be utilized to synthesize complementary dna (cdna) first, which are time-consuming and labor-intensive. undp-pcr is an ultrasensitive nanoparticle dna probe-based pcr method, in which magnetic microparticles (mmps) coated with virus specific dna probes and gold nanoparticles (aunps) coated with virus specific oligonucleotides are used to enrich virus genomes from samples and form an aunp-rna/dna-mmp complex. then the specific oligonucleotides are released and characterized by pcr after the complex is washed. in the previous study, we established this method to detect dna virus pcv2, which exhibited a detection limit of 2 copies of pcv2 genomic dna and 10 copies of pcv2 in serum that is 500-fold more sensitive than conventional pcr [19] . however, it is still needed to test whether this method can be used in the detection of rna virus. in this study, we aimed to develop a method for simultaneous detection of preclinical dna and rna virus mixed infection in the same reaction system based on undp-pcr method. tgev and pcv2, as the representatives of rna and dna viruses respectively chosen from a variety of viruses related to porcine diseases, were used to establish duplex undp-pcr assays. pcv2, a dna virus with a circular genome of 1.7 kb, has been reported to cause wide infection throughout the world and serious damage to pig producers, while tgev, an enveloped virus with a positive-stranded rna genome, has been recognized as a principal causative agent of enteric disease [20, 21] . firstly, the undp-pcr assay for tgev was developed and identified. then, single mmps-based or duplex mmps-based duplex undp-pcr assays for both pcv2 and tgev was developed. mmps coated with specific dna probes for either tgev or pcv2 (single mmps), or for both tgev and pcv2 (duplex mmps) were used to enrich tgev and pcv2 viral genomes from serum samples, and aunps coated with optimal oligonucleotides (oligo) specific for either tgev or pcv2 were used to magnify weak signals from very low level of tgev/pcv2 virus enriched by mmps from serum samples. the duplex undp-pcr assay is suitable for simultaneous detection of rna and dna viruses in early viral infection, providing an effective approach for diagnosis of swine diseases. viruses and cells tgev strain (genbank no. hq462571), pcv2 strain (genbank no. eu366323), ppv yl strain (genbank no. jn860197), pedv strain (genbank no. af353511) and prrsv shaanxi strain (genbank no. hq401282) used in this study were isolated and purified previously by our team and stocked in our laboratory [22] [23] [24] [25] [26] . the csfv shimen strain (genbank no. ay775178) was provided kindly by professor yanming zhang [27] . these virus strains were maintained at -80°c and used as standard viruses for this study. tgev, pcv2 and ppv were propagated in pk-15 cells. csfv was propagated in st cells. pedv was propagated in vero cells. prrsv was propagated in marc-145 cells. four types of cells (pk-15, st, vero, and marc-145) were maintained in dulbecco's modified eagle medium (dmem) (gibco, gaithersburg, md, usa) supplemented with 10% heat inactivated fetal calf serum (gibco). during the period from nov, 2014 to jun, 2015, 162 serum samples were collected from healthy pigs in pig-producing farms near xianyang and baoji of shannxi province, china. the pigs were humanely euthanized by injecting with 15 mg/kg of ketamine in the jugular vein, then 3-5 ml of blood samples were collected from each pig by jugular venipuncture. the serum samples were tested using the conventional pcr/rt-pcr assay and single or duplex undp-pcr. the experiment was approved by the ethical committee for animal experiments of the northwest a&f university and performed according to the animal ethics procedures and guidelines of the people's republic of china. no other specific permissions were required for this study. purification of viral dna or rna viral rna/dna kit (omega, usa) was used to extract and purify viral genomic dna or rna from virus-infected cell cultures or serum samples according to the manufacture's protocol. then the extracted viral rna was reverse transcribed into the complementary dna (cdna) using the reverse transcriptase kit (takara corp., japan) according to manufacturer's instructions. in this study, specific tgev dna probes and oligonucleotides used in undp-pcr were designed via multiple sequence alignment of complete genomes of orf1a from various tgev strains published on national center for biotechnology information using vector nti 9 and dnastar software package. primers for conventional rt-pcr method of detecting tgev and pedv were designed using primer-blast software on the basis of tgev and pedv highly conserved region of orf1a. primers for conventional pcr/rt-pcr detection of pcv2, ppv, prrsv and csfv were designed as described in previous studies [14, 19] . all the primers, probes and oligonucleotides designed for this experiment owned higher specificity to make sure diagnosis more precise. the primers, probes and oligonucleotides presented in table 1 were synthesized by sangon (shanghai, china). preparation of mmps coated with tgev and / or pcv2 specific probes for undp-pcr assay in the presence of water-soluble n-ethyln9[3-dimethylaminopropyl] carbodiimide hydrochloride (edc), tgev and/ or pcv2 specific oligonucleotide probes modified with 5' amino (nh2) were bond to the carboxylated-modified myone dynabeads to form a peptide bond. the detailed steps of the assay were according to manufacturer's instruction [28] . then the functionalized mmps were resuspended in tris-edta (te) buffer to a concentration of 10 mg/ml and stored at 4°c until required. preparation of aunps coated with tgev or pcv2 specific oligos aunps coated with tgev or pcv2 specific oligo were prepared as described in previous study [19] , briefly, 1 ml of 15 nm-diameter aunps (10 nmol/l) were washed and resuspended in 100 μl sterile deionized water. then, the 5' sulfydryl (sh)-modified tgev/pcv2 specific oligonucleotides were added and mixed with aunps to establish covalent au-s bond. in the binding process of thiolated aunps and sh-modified oligonucleotides, a final concentration of 0.01 m pbs (0.1 m nacl in 0.01 m of phosphate buffer, ph 7.0) was developed by adding 0.1 m pbs to the reaction tube three times to incubate for more than 48 hours at room temperature. then, unbound dna probes were removed by washing twice with 0.01 m pbs. finally, the prepared functionalized aunps were stored in 0.01 m pbs at 4°c until used. viral serum samples were mixed with same volume of lysis buffer (0.01 m tris-hcl, 1 mm edta, 0.015 m nacl, 0.5% sds, ph 8.0), and then boiled for 15 minutes to release viral rna or dna. the products were mixed with 2 μl of probe-coated mmps and 5× hybridization buffer (5×ssc, 0.1% tween-20 and 2% sds in h 2 o). the mixture of these components was incubated for 30 minutes by stirring. subsequently, 2 μl of functionalized aunps were added, followed by incubation at 50°c for 40 minutes. the sandwich-like aunp-rna/dna-mmp complexes in the tube were separated using magnetic wells and washed twice with 1 ml of hybridization buffer and twice with 1 ml te buffer to remove remaining hybridization buffer and unbound probes-functionalized mmps and oligos-functionalized aunps. the oligonucleotides on the surface of gold nanoparticles were eluted using 100 μl of elution buffer (0.5 m dtt, 10 mm tris-hcl, 1 mm edta, ph 7.5) for 10 minutes at room temperature. then, the eluted oligonucleotides were precipitated with naac and absolute alcohol. the precipitated oligonucleotides were mixed with specific capture ssdna and detected by pcr using specific detect-pcr primers for pcv2 and/or tgev in table 1 . the pcr assay was performed as described in the previous study [19] . the pcr products were separated by electrophoresis through 1.5% agarose gel stained with ethidium bromide and were photographed under uv light. the sensitivity, specificity, and reproducibility of the undp-pcr assay for tgev tgev genomic rna was extracted using rna kit and reverse transcribed to synthesize cdna. then, the part of tgev orf1a gene was amplified from cdna using the primers (5'-cgtaatggtgacagccgat-3'/5'-agcagcatcacggaaaccat-3'). the 435 bp amplified products were cloned into pmd-19t simple vector (takara, japan) and sequenced. the concentration of the plasmid was measured by nanodrop 2000 spectrophotometer (thermo scientific, usa). the formula: amount (copies/μl) = 6.02×10 23 (copies/mol) × concentration (g/μl)/mw (g/mol) was used to calculate the plasmid copy number. the viral copy number in samples per ml was tested by real-time pcr with serially diluted plasmid from 10 3 to 10 9 copies/ml. to test the sensitivity, the quantitative serum samples were diluted serially from 10 to 10 4 copies/ml, and then detected by undp-pcr and conventional rt-pcr. the specificity of undp-pcr was tested through comparing with pcv2, ppv, prrsv, pedv and csfv. inter-assay and intra-assay were performed in three replicates by testing 3 different concentrations of diluted serum samples (5×10 3 copies/ml, 5×10 2 copies/ml, 50 copies/ml) for three consecutive days to test reproducibility of the undp-pcr assay for tgev. to test the sensitivity of duplex undp-pcr assay for tgev and pcv2, the serum samples containing tgev or pcv2 were diluted serially from 10 4 to 1 copies/ml respectively. the diluted samples containing same viral copy numbers of tgev and pcv2 per ml were mixed, and then tested by single mmps-based and duplex mmps-based duplex undp-pcr, or conventional pcr/rt-pcr. the specificity and reproducibility of duplex undp-pcr assay for dna and rna viruses in the study of evaluating the specificity of duplex undp-pcr, ppv, pedv, prrsv and csfv were tested by the established method. the inter-assay and intra-assay tests were carried out in triplicate by detecting three different concentrations of mixed serum containing serial diluted tgev and pcv2 (2000, 200, 20 copies/ml) to evaluate the reproducibility of this assay. to find a rapid and ultrasensitive diagnosis method for preclinical mixed infection of dna and rna viruses, a series of related experiments were performed to establish two kinds of protocol for duplex undp-pcr assay as schematically depicted in fig 1. in the previous study, we have optimized and established a undp-pcr method for dna virus pcv2, which can detect 10 copies/ml of pcv2 serum sample and exhibited high specificity and reproducibility [19] . in the present study, pcv2 and tgev are selected as representative dna and rna viruses, respectively. therefore, a undp-pcr method for rna virus tgev needs to be established first. tgev, an enveloped virus of the coronaviridae family, contains a single-stranded positivesense rna genome of 28.5 kb. the first two-thirds of the viral genome encodes two replicases and only exists in genomic rna, while the last third exists in both genomic rnas (grna) and subgenomic mrnas (sgmrnas) to encode structural and nonstructural proteins of virus [21, 29, 30] . in this assay, we first quantified viral number using primers targeted to tgev grna and different sgmrnas and found that viral numbers were significantly different when the primers were targeted to tgev grna and different sgmrnas (data not shown). therefore, we designed probes targeted to replicase protein-encoding region orf1a to quantify the amount of tgev grna. six targeted regions were selected from the 5' end, middle and 3' end of the orf1a for designing probes 1 to 6 ( table 1 ). all the targeted sequences of these probes are highly conserved in variety of tgev strains. probes 1 to 6 were coated to magnetic microparticles to prepare functional magnetic beads mmp-p1, -p2, -p3, -p4, -p5 and -p6, respectively. to select the optimal probes for capturing of tgev genomic rna, capture efficiency of these designed probes were determined by conventional rt-pcr assay. the results showed that all six probes could capture tgev rna, however mmp-p1 and mmp-p2 exhibited higher capture ability for tgev rna (fig 2a) . next, we assessed capture efficiency of functionalized gold nanoparticles coated with oligonucleotides (oligo) 1 to 6 that shared same targeted sequence with probe 1 to 6. the prepared functionalized au-nps were precipitated by centrifugation and were detected by rt-pcr assay. the results showed that oligo 1 and 2-coated au-np possessed higher binding ability with tgev nucleic acid (fig 2b) . therefore, in the undp-pcr assay for tgev, probe 1-functionalized mmps and oligo 2-functionalized au-nps are optimal for capture of tgev rna and formation of sandwich complex. serial 10-fold dilutions of tgev in serum samples were tested to assess the sensitivity of undp-pcr for rna virus. tgev genomic rna was released by boiling with lysis buffer with rnase inhibitors and was used to form aunp-rna-mmp complexes, followed by magnetic separation and oligonucleotide elution. the oligonucleotides were then purified and detected by undp-pcr. as shown in fig 3a, visible targeted bands around 501 bp could be seen in simultaneous detection of dna and rna virus by duplex undp-pcr lanes representing serum samples with viral concentrations ranging from 10 3 copies/ml to 20 copies/ml respectively, but it could not be detected in the negative control serum without tgev and the tgev serum sample below 20 copies/ml, indicating that the detection limit of undp-pcr assay for tgev was 20 copies/ml in serum sample. however, at least 5000 copies/ ml of tgev serum sample was able to be detected by conventional rt-pcr assay (fig 3b) , suggesting that the sensitivity of undp-pcr specific for tgev was 250-fold that of the conventional rt-pcr for tgev. the reproducibility of undp-pcr for tgev was estimated by three independent runs for three consecutive days, with triplicates of each concentration (5×10 3 copies/ml, 5×10 2 copies/ ml, 50 copies/ml). consistent results of undp-pcr were obtained in the inter-assay and intra-assay test (fig 4a) . ppv, pcv2, prrsv, csfv, pedv, tgev and the serum collected from healthy pigs were used to evaluate the specificity of the established undp-pcr for tgev. in the fig 4b, the undp-pcr detection only appeared to be positive with tgev, whereas no specific pcr products of 501 bp were obtained from the assay using pcv2, csfv, prrsv, pedv, ppv or healthy pigs serum as pending samples, which indicated that undp-pcr assay had high specificity for tgev and did not show cross-reactivity with pcv2, csfv, prrsv, pedv and ppv. to compare the sensitivity of single mmps-based and duplex mmps-based duplex undp-pcr assay for simultaneous detection of tgev and pcv2 in same reaction assay system, qualified serum samples of tgev and pcv2 were diluted serially with the range from 1 to 10 3 copies/ml. then the diluted samples containing tgev and pcv2 were mixed as the template for testing the sensitivity of single mmps-based and duplex mmps-based duplex undp-pcr assay. as shown in fig 5a and 5b , the detection limits of single mmps-based and duplex mmps-based duplex undp-pcr were identical with 20 copies/ml for tgev and pcv2 in serum. however, at least 5000 copies/ml of pcv2 and tgev serum sample was able to be detected by conventional duplex pcr/rt-pcr assay (fig 5c) . these results suggested that the sensitivity of duplex undp-pcr specific for pcv2 and tgev was 250-fold that of simultaneous detection of dna and rna virus by duplex undp-pcr the conventional duplex pcr/rt-pcr for these two viruses, and that mmps coated with one virus probe or two virus probes did not affect the capture efficiency of functionalized mmps and the sensitivity of undp-pcr assay. the specificity and reproducibility of duplex undp-pcr for both dna and rna viruses to evaluate the specificity of single mmps-based and duplex mmps-based duplex undp-pcr for both dna and rna viruses, magnetic beads coated with specific probes for tgev and pcv2 alone or together and gold nanoparticles coated with specific oligos for tgev and pcv2 alone were added to one single reaction tube. as shown in fig 6a and 6b , both assays were specific to the target viruses, producing specific amplified products (501 bp for tgev and 268 bp for pcv2). no amplicons were yielded with ppv, prrsv, csfv, pedv and samples from health pigs. three independent replicates assay of both assays gave highly consistent results respectively (fig 6c and 6d ). pre-clinical serum samples from epidemic farms without diseased pigs were detected for tgev and pcv2 using duplex undp-pcr assay and conventional duplex pcr/rt-pcr. conventional duplex pcr/rt-pcr detection showed that among 162 samples, 7 samples were pcv2 positive, 2 samples were tgev positive, but none of samples were found to be positive for both pcv2 and tgev. whereas duplex undp-pcr assay showed that 48 samples were pcv2 positive, 15 samples were tgev positive, 6 samples were positive for both pcv2 and tgev ( table 2 ). the positive samples detected by the duplex undp-pcr method included all of the samples found to be positive by the conventional duplex pcr/rt-pcr. the positive detection rate of duplex undp-pcr was 29.6% for pcv2, 9.3% for tgev and 3.7% for pcv2 and tgev mixed infection, which increased 29% and 11.7% for pcv2 and tgev preclinical infection than that of conventional duplex pcr/rt-pcr (p < 0.01). as shown in table 2 , the results were consistent when single mmps-based and duplex mmps-based duplex undp-pcr were used to detect these samples, suggesting two kinds of duplex undp-pcr possessed same detection efficiency. next, the relative band intensities of the unknown samples were compared with that of standard virus samples (50, 500 and 5000 copies/ml pcv2 and tgev) to evaluate the viral loads of pcv2 and tgev in all of positive samples. among 48 pcv2 positive samples, 7 samples were over 5000 copies/ml, 15 samples were between 5000 and 500 copies/ml, 23 samples were between 500 and 50 copies/ml, and 3 samples were below 50 copies/ml in viral load (data not shown); among 15 tgev positive samples, 2 samples were over 5000 copies/ml, 5 samples were between 5000 and 500 copies/ml, 6 samples were between 500 and 50 copies/ml, and 2 samples were below 50 copies/ml (fig 7a) . among 6 pcv2 and tgev double positive samples, the pcv2 viral loads of 4 samples were between 5000 and 500 copies/ml, 1 sample was between 500 and 50 copies/ml, and 1 sample was below 50 copies/ml, the tgev viral loads of 3 samples were between 5000 and 500 copies/ml, 2 sample were between 500 and 50 copies/ml, and 1 sample was below 50 copies/ml (fig 7b) . taken together, this undp-pcr assay was found to be the best method for detecting dna and rna viruses in preclinical samples simultaneously. all the results indicated that undp-pcr-based assay was a rapid and economical approach with high specificity and high sensitivity. to date, traditional approaches to detect mixed infection of rna and dna viruses present some limitations, such as complex virus genome isolation procedures, limited sensitivity, narrow detection range and lack of specific antibodies for different viruses, which lacks the [9, 31] . in addition, low virus titer in early stage of infection is not able to be detected by conventional pcr or rt-pcr, failing in timely diagnosis of infection. viruses are more likely to spread across piggeries and cause more sickness and death in piglets, which will bring huge threat to pig industry [32] . with the development of nanotechnology, the undp-pcr based on nanoparticle and dna probe makes it possible to detect dna and rna viruses simultaneously especially in subclinical infection without the need for nucleic acid extraction separately [33] . duplex undp-pcr assay gains more merits over established traditional approaches for rna/dna virus diagnosis. the first advantage that should be mentioned is that duplex undp-pcr is more time-saving and cost-effective than other routine pcr-based methods. in this study, field samples collected from pig-producing farms were boiled with lysis buffer for 15 min to release viral genome, so there is no need to extract nucleic acid using dna/rna extraction kits. particularly, unlike other pcr-based assays, extracted and purified rna need to be reverse transcribed into cdna. hence, the whole detection process could be completed in short period of time. secondly, this assay could detect dna and/or rna virus in serum samples with an extreme low concentration in preclinical infection. accordingly, functionalized magnetic beads and gold nanoparticles which were coated with tgev and/or pcv2 specific probes and oligonucleotides targeting two distinct virus genomic sequences were added to form a sandwich complex. in each binding events, the gold nanoparticles carried with large number of oligonucleotides, which could be used as templates for subsequent pcr assay. thus, weak signals from extreme low concentration of samples were highly amplified. in addition, the duplex undp-pcr could detect dna and rna virus from large-scale serum samples simultaneously. in the reaction system, magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for tgev and pcv2 were added to capture and enrich viral nucleic acid, so this approach could detect infection of tgev and pcv2 alone or together in a single reaction tube. the results showed that duplex undp-pcr (single mmps-based and duplex mmps-based) developed in the study was able to detect 20 copies for pcv2 and tgev. the sensitivity of duplex undp-pcr was approximately 250-fold that of conventional duplex pcr/rt-pcr. in terms of specificity, specific probes and oligonucleotides for tgev and pcv2 were assessed through testing capture efficiency and specificity of different probes-or oligonucleotidescoated magnetic beads or gold nanoparticles. as a consequence, duplex undp-pcr showed high specificity with tgev and pcv2, yielding different size of pcr products (501 bp and 268 bp respectively). the results of detection for preclinical samples indicated that duplex undp-pcr assay (29.6% for pcv2, 9.3% for tgev and 3.7% for pcv2 and tgev mixed infection) described here was more sensitive than conventional detection methods (4.3% for pcv2, 1.2% for tgev and 0% for pcv2 and tgev mixed infection). the duplex undp-pcr assay developed in this study provided a useful tool for simultaneous detection of rna (tgev) and dna viruses (pcv2) without the need for viral nucleic acid extraction, purification and reverse transcription. this assay could increase positive detection rates of virus infection and is useful to evaluate the viral loads in pre-clinically infected samples. in summary, the duplex undp-pcr assay is an economical and rapid detection approach with high specificity and sensitivity. preliminary study on prevalence, risk factor and genetic homogeneity of porcine reproductive and respiratory syndrome virus in registered pig farms in heilongjiang, china. transboundary and emerging diseases journal of veterinary diagnostic investigation: official publication of the american association of veterinary laboratory diagnosticians classical swine fever in china: a minireview molecular epidemiology of outbreak-associated pseudorabies virus (prv) strains in central china prevalence of emerging porcine parvoviruses and their co-infections with porcine circovirus type 2 in china molecular epidemiology of porcine epidemic diarrhea virus in china occurrence and 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immunohistochemistry, and in situ hybridization for the detection of porcine epidemic diarrhea virus in pigs. canadian journal of veterinary research = revue canadienne de recherche veterinaire development of multiplex pcr for simultaneous detection of six swine dna and rna viruses reverse transcription loop-mediated isothermal amplification for rapid detection of transmissible gastroenteritis virus a real-time taqman rt-pcr assay with an internal amplification control for rapid detection of transmissible gastroenteritis virus in swine fecal samples rapid and sensitive detection of porcine epidemic diarrhea virus by reverse transcription loop-mediated isothermal amplification combined with a vertical flow visualization strip. molecular and cellular probes development of a novel real-time rt-pcr assay with lux primer for the detection of swine transmissible gastroenteritis virus preclinical detection of porcine circovirus type 2 infection using an ultrasensitive nanoparticle dna probe-based pcr assay molecular analysis of porcine circovirus type 2 strains from uruguay: evidence for natural occurring recombination. infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases coronavirus genome structure and replication. current topics in microbiology and immunology isolation and identification of porcine transmissible gastroenteritis virus shaanxi strain and sequence analysis of its n gene evidence for different patterns of natural intergenotype recombination between two pcv2 parental strains in the field sequence analysis of porcine parvovirus yl strain and prokaryotic expression of vp2 porcine epidemic diarrhea virus n protein prolongs s-phase cell cycle, induces endoplasmic reticulum stress, and up-regulates interleukin-8 expression. veterinary microbiology cloning, sequence analysis and prokaryotic expression of orf5 gene of prrsv sx strain in vitro inhibition of classical swine fever virus replication by sirnas targeting npro and ns5b genes a method for the high efficiency of water-soluble carbodiimide-mediated amidation the structure and functions of coronavirus genomic 3' and 5' ends. virus research structure and functional relevance of a transcription-regulating sequence involved in coronavirus discontinuous rna synthesis establishment and application of a multiplex pcr for rapid and simultaneous detection of six viruses in swine a transmissible gastroenteritis in pigs nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins we thank professor yanming zhang (northwest a&f university) for providing csfv. we also would like to thank benxiang and hengda for providing preclinical samples. conceived and designed the experiments: yh nx dt. performed the experiments: nx zw xz qd xz lc. analyzed the data: yh nx. contributed reagents/materials/analysis tools: yh dt. wrote the paper: yh nx. key: cord-003125-iptisi1m authors: machablishvili, ann; chakhunashvili, giorgi; zakhashvili, khatuna; karseladze, irakli; tarkhan-mouravi, olgha; gavashelidze, mari; jashiashvili, tamar; sabadze, lela; imnadze, paata; daniels, rodney s.; ermetal, burcu; mccauley, john w. title: overview of three influenza seasons in georgia, 2014–2017 date: 2018-07-27 journal: plos one doi: 10.1371/journal.pone.0201207 sha: doc_id: 3125 cord_uid: iptisi1m background: influenza epidemiological and virologic data from georgia are limited. we aimed to present influenza like illness (ili) and severe acute respiratory infection (sari) surveillance data and characterize influenza viruses circulating in the country over three influenza seasons. methods: we analyzed sentinel site ili and sari data for the 2014–2017 seasons in georgia. patients’ samples were screened by real-time rt-pcr and influenza viruses isolated were characterized antigenically by haemagglutination inhibition assay and genetically by sequencing of ha and na genes. results: 32% (397/1248) of ili and 29% (581/1997) of sari patients tested were positive for influenza viruses. in 2014–2015 the median week of influenza detection was week 7/2015 with b/yamagata lineage viruses dominating (79%); in 2015–2016—week 5/2016 was the median with a/h1n1pdm09 viruses prevailing (83%); and in 2016–2017 a bimodal distribution of influenza activity was observed—the first wave was caused by a/h3n2 (55%) with median week 51/2016 and the second by b/victoria lineage viruses (45%) with median week 9/2017. for ili, influenza virus detection was highest in children aged 5–14 years while for sari patients most were aged >15 years and 27 (4.6%) of 581 sari cases died during the three seasons. persons aged 30–64 years had the highest risk of fatal outcome, notably those infected with a/h1n1pdm09 (or 11.41, ci 3.94–33.04, p<0.001). a/h1n1pdm09 viruses analyzed by gene sequencing fell into genetic groups 6b and 6b.1; a/h3n2 viruses belonged to genetic subclades 3c.3b, 3c.3a, 3c.2a and 3c.2a1; b/yamagata lineage viruses were of clade 3 and b/victoria lineage viruses fell in clade1a. conclusion: in georgia influenza virus activity occurred mainly from december through march in all seasons, with varying peak weeks and predominating viruses. around one third of ili/ sari cases were associated with influenza caused by antigenically and genetically distinct influenza viruses over the course of the three seasons. 32% (397/1248) of ili and 29% (581/1997) of sari patients tested were positive for influenza viruses. in 2014-2015 the median week of influenza detection was week 7/2015 with b/yamagata lineage viruses dominating (79%); in 2015-2016-week 5/2016 was the median with a/h1n1pdm09 viruses prevailing (83%); and in 2016-2017 a bimodal distribution of influenza activity was observed-the first wave was caused by a/h3n2 (55%) with median week 51/2016 and the second by b/victoria lineage viruses (45%) with median week 9/2017. for ili, influenza virus detection was highest in children aged 5-14 years while for sari patients most were aged >15 years and 27 (4.6%) of 581 sari cases died during the three seasons. persons aged 30-64 years had the highest risk of fatal outcome, notably those infected with a/h1n1pdm09 (or 11.41, ci 3.94-33.04, p<0.001). a/ h1n1pdm09 viruses analyzed by gene sequencing fell into genetic groups 6b and 6b.1; a/ h3n2 viruses belonged to genetic subclades 3c.3b, 3c.3a, 3c.2a and 3c.2a1; b/yamagata lineage viruses were of clade 3 and b/victoria lineage viruses fell in clade1a. plos influenza viruses affect people of all ages and cause mild to severe disease sometimes leading to fatal outcomes [1] [2] [3] [4] . surveillance of influenza like illness (ili) and severe acute respiratory infection (sari) represent an important tool for tracking trends of virus spread and changes in globally circulating influenza viruses [5] . the first pandemic of the 21 st century caused by influenza virus a/h1n1pdm09 demonstrated the importance of influenza surveillance worldwide. the monitoring of changes in seasonal influenza viruses plays a critical role in defining influenza vaccine composition [6, 7] . moreover, data obtained from surveillance systems enables healthcare authorities to better understand timings of influenza activity and consequent mobilization of resources (e.g. vaccines, antivirals), notably prioritized vaccination of risk groups among other measures. however, limited data are available on influenza morbidity and mortality in georgia [8] [9] [10] and none of these publications describe antigenic and genetic characteristics of seasonal influenza viruses circulating in the country. georgia is located in the caucasus region and covers a territory of 69700 km 2 with a population of around 3.7 million people. prior to 2006, a nationwide population-based surveillance system for influenza and upper respiratory tract infection provided influenza data based only on clinical diagnosis without laboratory confirmation. in 2007, in collaboration with us cdc and other international stakeholders, sentinel surveillance of ili and sari was initiated. the national influenza center (nic) at the national center for disease control and public health, georgia (ncdc&ph) now screens specimens collected at ili/sari surveillance sites all year round to monitor activity of influenza viruses. in this study we describe epidemiological and virologic data for three consecutive influenza seasons obtained from ili/sari surveillance systems. the objectives were to: (1) define periods of influenza activity in georgia; (2) assess the proportions of influenza infections among ili and sari cases; (3) determine most affected age groups; (4) describe epidemiological characteristics of influenza-associated fatal cases; and (5) determine antigenic and genetic profiles of influenza viruses circulating in the country. ili surveillance was carried out in one outpatient clinic with a predetermined catchment of approximately 60,000 residents in the capital city tbilisi (5% of 1.2 million population). the children's central hospital, the largest children's clinic of the country also in tbilisi, and four hospitals in kutaisi (the biggest city in western georgia) were selected as sari surveillance sites (s1 fig). medical staff and epidemiologists at each site were trained on case definitions, specimen collection, storage and transportation, completion of individual patient questionnaires and ili/sari aggregated forms. and influenza viruses from various countries, were downloaded from the epiflu database of the global initiative on sharing all influenza data (gisaid) (http://www.gisaid.org). nucleotide and deduced amino acid sequences were aligned and analyzed using bioedit (http://www. mbio.ncsu.edu/bioedit/bioedit.html) and maximum likelihood phylogenetic trees were estimated using raxml v8.2x with a gtrgamma substitution model (https://sco.h-its.org/ exelixis/software.html), followed by annotation with amino acid substitutions defining nodes and individual virus gene products using treesub (https://github.com/tamuri/treesub/blob/ master/readme.md). phylogenies were based on full-length open-reading-frames with the signal peptide component being removed from ha genes and stop codons removed from all genes: h1-ha 1647, h3-ha 1650, n1-na 1407, n2-na 1407, b/vic-ha 1710, b/yam-ha 1707, b/vic-na 1398 and b/yam-na 1398 nucleotides, respectively. trees were visualized using figtree (http://tree.bio.ed.ac.uk/software/figtree/) and highlighted using adobe illustrator cc 2015.3 (http://www.adobe.com/uk/products/illustrator/features.html). sequence accession numbers for all reference and test viruses are given in s1 table. ethical considerations written informed consent was not required for this study. verbal consent from patients and guardians in the case of children was sufficient for sample and data collection as the study was carried out under georgia's routine public health surveillance practice. fisher's exact test was used for statistical analysis of data with chi squared estimation of significance. p values of 0.05 were considered as statistically significant. data analysis was performed using epi info (version 7). median, range, and interquartile range (iqr) were calculated to assess continuous variables. as georgia is in the northern hemisphere, an influenza season is defined as the time period from week 40 of each year to week 20 of the following year, when the vast majority of influenza detections are made, and only data for these periods were analyzed. ili consultation rates per 100 000 population and sari vs total hospital admissions were used for evaluating season dynamics. the seasonal threshold was calculated using who guidelines and methodology [5] . a total of 12,912 ili and 10,218 sari patients met the ili/sari case definitions at sentinel sites during three consecutive influenza seasons 2014-2017; of these, 1,248 (9.7%) ili and 1,997 (19.5%) sari patients were sampled and tested for influenza. overall 30% (978/3,245) specimens were positive: 397 (32%) ili and 581 (29%) sari (including four specimens: three ili and one sari-positive for both influenza a and b viruses). of the 397 influenza virus-positive ili samples over three seasons (2014-2017) 132 (33%) were a/h1n1pdm09, 121 (30%) were a/h3n2 and 147 (37%), influenza b (three were positive for both influenza a and b viruses). during influenza activity periods weekly outpatient visits for ili rose in parallel with laboratory-confirmed influenza virus detection (fig 2) . based on our data seasonal threshold was determined as an ili referral of 231/100,000. the 2014-2015 influenza season had an unusually high referral of ili cases which was considerably earlier than laboratory-confirmed influenza virus detection, presumably due to circulation of other respiratory viruses. only 18 of 1248 swabbed ili participants had been vaccinated during the three seasons; of these, eight persons became ill with influenza a/h3n2 and two with a/h1n1pdm09. out of 581 specimens testing positive for influenza viruses (table 1) , 241 (41%) were a/ h1n1pdm09, 138 (24%) a/h3n2 and 203 (35%) influenza b (one was positive for both influenza a/h3n2 and b). during weeks when influenza activity was high (fig 1) sari admissions rose above an approximate 10% threshold: in 2014-2015 sari cases made up between 10% and 17% of weekly hospital admissions; in 2015-2016 and 2016-2017 seasons these rates were higher, ranging from 11% to 39% and from 11% to 25%, respectively (fig 3) . influenza virus detection rates among hospitalized patients were highest, almost equally, in the age groups 30-64 years (55%, or = 3.48, ci 2.58-4.69, p<0.001) and 15-29 years (53%, or = 3.03, ci 2.14-4.28, p<0.001) ( table 1) among 1,997 sampled sari cases, only 25 were vaccinated against influenza; of these, three tested positive for a/h1n1pdm09 and three for a/h3n2. during the three influenza seasons, 27 (4.6%) patients out of 581 laboratory-confirmed influenza sari cases died, all of whom were patients in kutaisi sari sentinel hospitals. of the fatal cases 19 (70%) were male and 8 (30%) female. influenza a/h1n1pdm09 was associated with overview of three influenza seasons in georgia, 2014-2017 patients received antiviral treatment within 48 hours after the onset of clinical signs, the median time between disease onset and antiviral prescription was 6 days (range 1-13, iqr 4-7 days). the median time between symptomatic illness onset and influenza-associated death was 6 days (range 3-44, iqr 1-13 days). only one patient was vaccinated against influenza, a 73 year old male resident of a disabled persons center suffering with mental and kidney health problems, who subsequently died. a/h1n1pdm09. eighteen (86%) of 21 viruses were successfully recovered by who cc london and studied by hi assay. all but two viruses were well recognized in hi assays, within 2-fold of the homologous titer, by antiserum raised against the vaccine virus, a/california/7/ 2009; a/georgia/567/2015 and a/georgia/791/2016 were recognized at titers 4-and 16-fold lower respectively. both these viruses also showed reduced recognition, compared to the respective homologous titers, by antisera raised against several reference viruses. while a/ georgia/567/2015 carried an ha1 substitution (n156s; fig 4) in a position known to affect antigenicity [13] we present an analysis of data obtained from ili and sari sentinel surveillance sites and virus characterization to provide an overview of three consecutive influenza seasons in georgia. the majority of laboratory-confirmed influenza detections were seen from december through to march, but seasonal peaks occurred in different weeks and coincided with those observed in the european region [14] [15] [16] . influenza virus dominance was similar to that in europe but for the 2014-2015 season when influenza b predominated in georgia and the ukraine, a/h3n2 viruses predominated in other european countries or co-dominated with influenza b [17] [18] [19] [20] . during a season approximately one third of both ili and sari cases sampled by sentinel sites in georgia were associated with influenza virus infection. increased ili consultation rates and vaccine viruses against which post-infection ferret antisera were raised for use in hi assays are in bold type. the scale bar represents nucleotide substitutions per site. https://doi.org/10.1371/journal.pone.0201207.g005 overview of three influenza seasons in georgia, 2014 georgia, -2017 at outpatient clinics correlated with raised numbers of laboratory-confirmed influenza infections. the same trend was observed for sari hospital admissions: the proportion of sari cases among total hospitalizations doubled during peak weeks of influenza activity compared to weeks with low or no influenza detections. the number of sari hospitalizations was lowest in 2014-2015, an influenza b season, and highest in 2015-2016 when a/h1n1pdm09 viruses predominated. children under 5 years of age were influenza virus-positive less frequently compared to older patients, reflecting the reports of a recent egyptian study [21] . in our study the influenza confirmation rate was highest among ili patients aged 5-14 years while the proportions of influenza-associated sari cases were highest in the age groups 15-29 and 30-64 years. such an age distribution might be influenced by the fact that during weeks of no or low influenza activity referral and/or sampling of children, notably those under 5 years of age, was higher compared to older patients; the rate of influenza detections in children could be lowered by this increased referral compared to adults. among ili patients those aged 5-14 years were most often infected with influenza a/h3n2 or b viruses, while a/h1n1pdm09 infection was most commonly observed in individuals aged 30-64 years; similar observations have been described in bulgaria [22] . the proportion of sari cases testing positive for influenza a/h3n2 was highest in the age group 30-64 years, influenza b related hospitalization was mainly observed in the age groups 5-14 and 15-29 years but the majority of viruses were not ascribed to a lineage, while a/h1n1pdm09 viruses were detected most frequently in adults aged 15-29 and 30-64 years. of influenza-confirmed sari cases in georgia approximately 5% died while the percentage of fatal outcomes among hospitalized influenza-confirmed cases has been reported to vary from 0 to 4% in other countries [21, [23] [24] [25] . a fatal outcome was predominantly associated with a/h1n1pdm09 infection and patients in the age group 30-64 years had the highest probability of a fatal outcome. our data are consistent with findings from other countries showing that adults were at higher risk of death than younger patients when infected with a (h1n10pdm09 viruses [25] [26] [27] [28] . the vast majority of fatal cases suffered with at least one chronic condition, with cardiovascular and neurological disorders being the leading comorbidities. in a previous study based on data collected during the 2009 pandemic and post-pandemic seasons in georgia, the major risk factors for a fatal outcome were lung disease, heart disease and pregnancy [10] . antigenic and genetic characterization of influenza viruses from georgia mostly showed similarities to strains circulating worldwide. since their emergence a/h1n1pdm09 viruses have evolved forming eight genetic groups [29] . viruses of recent years mainly fall into genetic group 6 which has three sub-divisions 6a, 6b and 6c. as observed globally, georgian viruses from 2014-2015 and the early part of 2015-2016 seasons belonged to the 6b clade while those later in the 2015-2016 season fell into subgroup 6b.1 [29] . the 6b and 6b.1 viruses carried several mutations in ha and na genes compared to the a/california/7/2009 vaccine virus. five ha amino acid substitutions were located in four antigenic sites (sa, sb, ca1, ca2) while substitution s185t (190 loop) and residue 222 polymorphism in one virus were in the vicinity of the receptor-binding site; such substitutions can affect the antigenic properties of a/ h1n1pdm09 viruses [30] . however, the majority of a/h1n1pdm09 viruses from georgia, and those detected worldwide, remained antigenically similar to the a/california/7/2009 vaccine virus as assessed with post-infection ferret antisera. over the period of this study a/h3n2 viruses evolved rapidly genetically and showed some antigenic change, antigenic drift, such that three vaccine viruses were recommended: who.int/influenza/vaccines/virus/recommendations/en/). in 2014-2015, viruses from georgia fell in genetic clades 3c.2a and 3c.3b while the majority of a/h3n2 viruses detected in the northern hemisphere belonged to clades 3c.2a and 3c.3a and the vaccine was reported to have low effectiveness [31, 32] . similar clade mismatching was observed in 2015-2016 (3c.2a1 and 3c.3a) and 2016-2017 (3c.2a1) in georgia, with 3c.2a1 subclade viruses predominating in some other parts of the world during both seasons [33] . worldwide multiple mutations were detected in a/h3n2 viruses with many encoding clade defining ha amino acid substitutions, some of which were located in antigenic sites a (7), b (7), d (2) and e (4) and/or altered ha glycosylation patterns; modifications known to cause antigenic drift [34, 35] . however, the great majority of 3c.3a, 3c.2a and 3c.2a1 genetic group viruses yielded poor/no agglutination of rbcs, making antigenic characterization by hi impossible. based on those that could be analyzed by hi [37] . genetic studies of the b/victoria lineage viruses revealed several substitutions in the ha and na. circulating viruses did not share the egg-adaptive substitution n197k in ha1, observed in egg-propagated b/brisbane/60/2008 vaccine virus, but had the substitutions i117v and n129d in ha1. there were a greater number of amino acid substitutions in the na but the significance, if any, of these substitutions is unknown. our study had several limitations that could influence the results of analyses performed. false negative laboratory results may have been obtained due to the late referral of patients to clinics. individual questionnaires were filled out during sampling and subsequent data regarding course of disease were not collected so we were unable to assess disease severity characteristics (developing pneumonia, need for icu, etc.). for the same reason, we did not evaluate chronic conditions among non-fatal ili/sari cases to determine risk factors associated with any severe courses of disease. despite limitations, this study contributes to a much better understanding of the epidemiological and virologic characteristics of influenza in georgia. influenza virus activity in the country was mainly observed from december through march each season with varying peak weeks and predominant viruses. on average one third of patients with ili/sari screened in each season had influenza virus infection. among ili cases influenza detection was highest in patients aged 5-14 years while the highest proportions of influenza-confirmed sari cases were among adults. persons aged 30-64 years had a higher risk of a fatal outcome. the observed circulation of antigenically and genetically variable viruses in georgia and selection of a/georgia/532/ 2015 as a reference virus by who cc, london illustrates the need for routine surveillance to contribute to the work of the who global influenza surveillance and response system. world health organization. influenza fact sheet excess all-cause and influenza-attributable mortality in europe estimating influenza and respiratory syncytial virus-associated mortality in western kenya using health and demographic surveillance system data a joint analysis of influenza-associated hospitalizations and mortality in hong kong world health organization. global epidemiological surveillance standards for 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hemagglutinin of a/fujian/411/ 02-like influenza viruses are responsible for antigenic drift from a/panama/2007/99 crystal structure of unliganded influenza b virus hemagglutinin trivalent and quadrivalent influenza vaccination effectiveness in australia and south africa: results from a modelling study. influenza and other respiratory viruses the authors wish to thank: (i) cdc, atlanta, usa for financial support of influenza surveillance in georgia; (ii) wrair, usa for providing sequencing primers; (iii) all sentinel clinic staff for sample and data collection; (iv) all wic staff involved in virus characterization studies. key: cord-254340-e1x0z3rh authors: cruz, christian joy pattawi; ganly, rachel; li, zilin; gietel-basten, stuart title: exploring the young demographic profile of covid-19 cases in hong kong: evidence from migration and travel history data date: 2020-06-26 journal: plos one doi: 10.1371/journal.pone.0235306 sha: doc_id: 254340 cord_uid: e1x0z3rh this paper investigates the profile of covid-19 cases in hong kong, highlighting the unique age structure of confirmed cases compared to other territories. while the majority of cases in most territories around the world have fitted an older age profile, our analysis shows that positive cases in hong kong have been concentrated among younger age groups, with the largest incidence of cases reported in the 15–24 age group. this is despite the population’s rapidly aging structure and extremely high levels of population density. using detailed case data from hong kong’s centre for health department and immigration department, we analyze the sex and age distribution of the confirmed cases along with their recent travel histories and immigration flows for the period january to april 2020. our analysis highlights hong kong’s high proportion of imported cases and large overseas student population in developing covid-19 hotspot areas such as the united kingdom. combined with community action and targeted and aggressive early policy measures taken to contain the virus, these factors may have contributed to the uniquely younger age structure of covid-19 cases in the city. consequently, this young profile of confirmed cases may have prevented fatalities in the territory. recent research has highlighted the importance of a demographic approach to understanding covid-19 transmission and fatality rates. the experience in hong kong shows that while an older population age structure may be important for understanding covid-19 fatality, it is not a given. from a social science perspective at least, there is ‘no easy answer’ to why one area should experience covid-19 differently from another. the hong kong special administrative region of the people's republic of china (hereafter hong kong) is a city and special administrative region of china in the eastern pearl river delta by the south china sea. in 2019, hong kong had roughly 7.5 million people in a 1,104-square-kilometre (426 square miles) territory while in kowloon, where more than three in ten residents reside, population density is at 48,930 persons per square kilometer. this makes it one of the most densely populated territories in the world [1] . apart from being a rapidly aging society [2, 3] , hong kong is also a migration destination. for the period 2015 to 2020, the net migration number was 147,000 or a net of four in-migrants per 1,000 of the population [1] . hong kong faces the challenge of infectious diseases as a consequence of various factors, including high population density, increasing environmental pollution, high migration inflows and outflows, the emergence of new infections as well as the changing lifestyle and behavior of its residents [4, 5] . the most recent significant outbreak was the severe acute respiratory syndrome (sars) outbreak which reached hong kong in march 2003 [5] . based on world health organization (who) data until july 11, 2003 , a total of 1,755 sars cases had been identified in the territory, of which 298 people died of the disease [6] . at that point, it was largely believed that hong kong was unprepared to be one of the epicenters of the sars epidemic. from this recent experience, did hong kong learn from the hard lessons of the past? the who officially declared the novel coronavirus infections (hereafter covid-19) outbreak as a pandemic on march 11, 2020, after it had spread to more than 100 countries and resulted in tens of thousands of cases within a few months. in hong kong, however, the first case was reported on january 23, 2020. in early february, the government was strongly criticized for policy responses related to a variety of issues, including the legality of, and access to face masks; border closure; medical fees and quarantine policy [7] . during this early period, unfavorable comparisons were made with other regional governments (especially macau and singapore) who appeared to be managing the crisis more effectively [8, 9] . however, by the time of writing in june 2020, new cases being reported in hong kong-especially local transmissions-are very rare [10] . that this has occurred without the general lockdown policies seen in other parts of the world is even more remarkable. our study includes an examination of the age and sex distribution of the covid-19 confirmed cases in hong kong and an exploration of how the different measures to combat this outbreak resulted in a relatively low number of cases and deaths. specifically, as demographers, we wished to explore the extent to which insights from demographic science could assist in explaining the nature of the hong kong experience of covid-19. in this paper, we highlight the potential impact of the young profile of the confirmed cases on the total number of mortalities and the effect of early, aggressive policy measures including travel bans, enforced quarantines and contact-tracing imposed by the hong kong government as early as january 27, 2020 in containing the spread of the covid-19. data on confirmed covid-19 cases were taken from the centre for health protection (chp) of the hong kong department of health. we assessed the age and sex distribution of the confirmed cases, discharge status (discharged, hospitalized or died) and type of transmission. we obtained the data regarding cumulative cases by age group from january 23 to april 16, 2020, which allowed us to determine the age group that registers the highest number of confirmed cases over time. daily migrant inflows and outflows data was retrieved from the hong kong immigration department showing arrivals at each border checkpoint into hong kong broken down by citizenship status. in addition, we retrieved detailed archived datasets pertaining to the travel histories of confirmed covid-19 cases, which are updated on an almost daily basis by the chp. we excluded travel histories pertaining to domestic travel (buses, trains and ferries) and a small number of journeys pertaining to outbound from hong kong. we linked travel histories to confirmed case identifications (ids) in order to examine the age structure and timing of cases where the apparent source of infection was not in hong kong (i.e. not a community infection). these include cases with a travel history from countries with widespread infection or where infection from a confirmed case who traveled occurred. by doing so, we highlight the importance of returnee hong kong residents from overseas hotspots on the relatively young age structure of confirmed cases during the second wave in march 2020. we also gathered data on policy measures implemented by the hong kong government in order to highlight the possible impact of major border closures and quarantine arrangements imposed by the hong kong government in reducing further numbers of imported covid-19 infections. we also utilized secondary data for comparative demographic analyses from the hong kong census and statistics department, united nations population division and the chinese center for disease control and prevention. compared with the aging hong kong population [1] , the covid-19 confirmed cases have an entirely different distribution. fig 1 shows [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] . less than a tenth (7.9%) are aged 65 and over. this age distribution of the local covid-19 confirmed cases does not fit the general profile of other territories wherein the infections are more concentrated among those in the older age groups. although, of course, this may be related to testing patterns by age, rather than true differences in incidence. the who coronavirus disease 2019 situation report 89 shows that as of april 13, 2020, there were a total of 716,570 confirmed cases with reported information on age and sex coming from 113 countries, territories and areas; of these confirmed cases, the median age for males is 52 (interquartile range, iqr: 35-64) years and for females 50 (iqr 35-64) years [11] . examples of territories with an older profile of confirmed cases are mainland china where the majority of confirmed cases (54.0%) as of february 11, 2020 belong to the older age groups, at least 50 years old [12] , and the philippines wherein as of march 25, 2020, the majority of the confirmed cases are in the age group 50 and older [13] . there is also an imbalanced sex ratio among the covid-19 confirmed cases in hong kong with a higher proportion of male cases, representing 54% of the total. the number of male cases outnumbered females in almost all age groups, except in the 25-34 age group where they made up only 47% of cases. for all other age groups, male cases were between 50 to 60% of the total; an exception was for those aged under 15 of which 68% of cases were male, however, the total number of cases at this age was extremely small (25 out of the total 1,017 cases). this sex profile with a higher prevalence of male cases is similar to the outbreaks in other territories [12, 13] the youngest confirmed cases in hong kong were two males under one-year-old while the oldest confirmed case is a 96-year-old female. nearly half of the 1,017 confirmed cases (48.0%) had already been discharged from the hospital as of april 16, 2020. in short, covid-19 confirmed cases in hong kong were found primarily among the working and school-age groups, and to a certain degree, among men. by april 16, 2020, almost three months after the first reported case on january 23, 2020, four deaths among confirmed covid-19 cases were reported in hong kong. three such deaths were over 70 years old (two males and one female) while the fourth was the case of a 39-year old male. it is possible that this young age structure of confirmed cases in hong kong contributed to the extremely low numbers of fatalities compared to other territories. a large proportion of hong kong's confirmed cases were 'imported'. three in five confirmed cases (61.0%) have travel history from countries with widespread infection or were directly infected by a confirmed case who travelled; hence, they are considered cases with imported transmission (see fig 2) . two-fifths of these imported confirmed cases (41.1%) are young adults in the age group 15-24 years old. the proportion of imported versus local cases differs significantly based on age group. nine in ten of the confirmed cases in the 15-24 age group (91.4%), the group with the largest incidence of covid-19, were in fact imported. in contrast, 59.7% of cases aged 55-64 and less than half of those in other age groups are considered imported. most of hong kong's cases in the younger age groups occurred in a second wave of infections during march. before march 18, 2020, the age group with the highest frequency of covid-19 cases was 55-64 followed closely by the 65 and over age group (see fig 3) . however, after march 18, 2020 the number of new cases in hong kong sharply increased, most of these being imported cases in younger age groups. this timing of these imported cases is similar to that experienced by china wherein the imported cases more than doubled starting from march 18, 2020, mostly coming from countries with high outbreak of covid-19 infections like the united kingdom (uk), united states of america (usa) and spain [14] . however, age-specific data about imported cases is not available in china. analysis of the travel histories of hong kong cases suggests that the majority of imported cases in the 15-24 age group may be hong kong residents who are studying or working abroad. the travel history data collected and archived by the chp over the study period was matched to case numbers from the case data to study the travel history and age profile of all known imported cases. the data collected by the chp contains the travel histories of all confirmed covid-19 cases up to fourteen days before the case was reported and formed part of attempts of the government to track and trace the movements of all newly reported cases. this is done in order to contact and isolate anyone with whom they had close contact in the days before the onset of symptoms, including those whom they may have sat close to on flights. of the 508 covid-19 cases with an overseas travel history tracked by the chp, 47.4% were imported from the uk, 9.1% from the usa and 3.9% each from qatar, canada and switzerland. by far the largest age group with a travel history was those aged 15-24 with 205 cases, of which the majority were from the uk (62.4%), 8.3% from the usa, and 4.4% to 5.4% each from switzerland, qatar and the netherlands. the next largest group with travel histories was those aged 25-34 with 86 cases, nearly half of these coming from the uk (48.8%). we are not able to ascertain the employment or other characteristics of these cases. hong kong has a large population of mobile residents who hold citizenship or permanent residency but work, study or are retired overseas. according to the 2016 by-census, just under 220,000 permanent residents of hong kong were deemed 'mobile', meaning they had spent between one to three months in the city of the previous six [15] . in 2016, the largest population of mobile residents was those aged 15-24, with a total of 47,938 most of whom were registered as students. we were not able to obtain the country of temporary overseas residency from the census data. the next largest population of mobile residents were those aged 65 and over (42, 299 ) who were mostly registered as retired, while a further 40,926 were aged 55 to 64 mostly employees or retirees. in addition, according to the 2016 by-census hong kong is home to a sizable number of non-permanent residents; many of these residents may return to their home countries on a regular basis for study, work, or family visits. the largest groups of non-chinese nationals were filipinos (186,000) and indonesians (159,901), most of whom work as domestic workers on temporary foreign-worker permits. hong kong was also home to 35,069 british citizens, almost 15,000 americans and 66,690 citizens from south asian countries (india, nepal and pakistan), while a further 121,775 hong kong chinese were registered as being domiciled overseas in 2016 [15] . the large increases in newly confirmed cases in the younger age groups, the majority of which were imported, occurred from march 10, 2020 onward (fig 4) . this was in tandem with a large increase in confirmed cases and deaths throughout multiple countries in europe, usa and canada. on march 13, 2020, the hong kong security bureau had announced a 'red outbound travel alert' ('red ota') for the schengen area, announcing a mandatory 14-day home quarantine for all arrivals from the schengen area. this was followed by a 'red ota' for ireland, united kingdom and the usa on march 15, 2020 with the announcement of home quarantine arrangements for all travelers from the three states to begin four days later (see s1 table for a full timeline of policy events). fig 4 shows the arrivals into hong kong over the period from january 24 to april 16, 2020. most arrivals were hong kong residents travelling via the airport. on february 4, 2020, all land and sea border points were closed except for two control points-the shenzhen bay control point and hong kong-zhuhai-macao bridge. daily arrivals into the city fell dramatically after the end of the chinese lunar new year holidays at the end of january 2020, and took another dramatic fall after the home quarantine arrangements for all arrivals from china were put into place on february 8, 2020. arrival numbers followed a relatively steady pattern at under 25,000 per day until dropping off sharply from march 19, 2020, when the new 14-day home quarantine arrangements were put in place for all arrivals into the city, regardless of whether or not they were residents. from march 25, 2020, all non-residents were barred from entering the city except for nationals of macau, taiwan or mainland china. these border closures and sharply lower inbound-travel movements together with hong kong's aggressive policy of testing, contact-tracing and quarantine of confirmed cases and their close contacts (see s1 table) undoubtedly contributed to the sharp decline in newly confirmed cases during the month of april [16] . on april 20, 2020, hong kong recorded its first day without a confirmed case, from a peak of more than 60 cases per day in late march. social distancing and rapid population behavioral changes also likely played a role, with measures such as wearing masks, working from home and school closures leading to an estimated 44.0% reduction in seasonal influenza incidence [16] . the containment of a severe local outbreak of covid-19 in hong kong thus far, and the very high incidence of confirmed cases in younger age groups mostly among hong kong residents returning to the city from overseas hotspot areas, have surely contributed to the very low number of fatalities in the city-state, with only four deaths reported by april 23, 2020 out of 1,030 confirmed cases. one death occurred in the 80 years and over age group, out of twelve total cases. in italy, china and south korea, vastly higher case fatality rates were recorded for those in the older age groups; as at march 31, 2020 case fatality rates for those above 80 years old were at 27.7% in italy and 18.3% in south korea [17, 18] . this paper takes a social scientific approach to illustrate the particularities of covid-19 outcomes in hong kong up until now. the paper is a simple, descriptive analysis of the available data on hand. a recent paper highlighted the importance of a demographic approach to understanding covid-19 transmission and fatality rates [18] . the paper suggested that 'the age structure of a population may help explain differences in fatality rates across countries and how transmission unfolds.' fig 5 shows the full population pyramid for hong kong in 2016. observing the discrepancy in the shapes of the pyramids in figs 5 and 1, it is clear the experience from hong kong suggests that age structure alone is not a universal factor in shaping transmission and fatality rates. despite having more than 18.0% of the population 65 and over and an extremely high population density, a package of policies designed to contain the virus spreading from younger imported cases and becoming a sustained local outbreak ensured that case and mortality numbers stayed low. a further difference between hong kong and other settings characterized by higher rates of infection (and fatality) is the lower levels of distribution of residential care and support among older persons. there is strong evidence that people living in residential/nursing homes are particularly vulnerable to not only infection and its rapid spread, but to severe covid-19 infection and fatality [19] [20] [21] ; so much so that the who referred to care home infection and fatality rates as an "unimaginable human tragedy" [22] . in a study of official data in ten countries, deaths in care homes account for between 19-72% of all deaths [21] -although international comparison is difficult because of differences in cause of death reporting as testing procedures. in common with other parts of the world, the hong kong government has issued guidelines to support residential care homes in preventing infection [23] , as well as offering other support in terms of provision of personal protective equipment (ppes) and infection protection services [24] and a switch to online care support for those who would ordinarily visit day care centers [25] . there may thus be institutional reasons for the low levels of transmission among older people. the size and percentage of the older population resident in care homes differs widely around the world [26] . in the uk, where a high percentage of infections and fatalities have occurred in care homes [19, 20] , it is estimated that around 5.3% of the population aged over 70 is resident in care homes [27, 28] . in addition, a further 6.9% of that population receive care support in their own homes, including from carers making multiple home visits in a day-potentially another area of risk for infection [27, 28] . in contemporary hong kong, meanwhile, the proportion of over 70s in residential/nursing homes is estimated to be around 3.6% [1, 29] . these lower rates of care home residence may have contributed to lower overall transmission and fatality numbers among the elderly by creating lower opportunities for sustained local spread within the elderly population. low-income migrant workers, an already neglected group in terms of health and other support mechanisms during this pandemic [30] , represent a further group often characterized by communal living. highly elevated transmission rates have been seen in some settings where such migrant workers often live in cramped, unsanitary conditions [31] . in singapore, for example, over half of the purpose-built and factory-converted dormitories have been affected [32] ; a factor held primarily responsible for the 'second wave' of infections in the city-state. it has been estimated that some 80% of all cases have been linked to such dormitories. compared to singapore, such 'dormitories' are rare in hong kong. perhaps the primary, related housing issue in hong kong is 'subdivided housing'; home to up to 209,000 poor, urban individuals [33] and sometimes referred to as 'coffin houses' because of their very small sizes [34] . while often characterized by poor hygiene, low environmental [35] and safety standards [36] , it appears that policy measures in hong kong, which succeeded in stemming local transmission chains, meant these quasi-communal units were not left exposed to rapid covid-19 transmission. a final possible factor mentioned by dowd et al. [18] concerns the possibility that 'intergenerational interactions, co-residence, and commuting may have accelerated the outbreak in italy through social networks that increased the proximity of elderly to initial cases.' this would be derived from a mechanism whereby the younger population most susceptible to initial infection [37] transmit to the elder population through such contact. in hong kong, multi-generational residence is common [38] , where around 50% of those aged 65 and over live with their adult children [39] -much higher than in italy (or, indeed, any other setting in europe or north america characterized by high transmission rates) [40] . the extremely high population density of hong kong coupled with short distances and highly efficient transport systems means that there is a high degree of residential proximity as well regular contact between older parents and their children. a further dimension of intergenerational, intrahousehold interaction involves migrant domestic workers, who increasingly operate a major means of care support within the household. there is little evidence in hong kong that such workers were responsible for transmission within the household. as such, the hypothesis suggested for italy appears inconsistent with the hong kong case at least. the general quality of population health data in hong kong is generally accepted to be high, not least because of the centralized healthcare system in the territory. furthermore, in this case a very high degree of health surveillance and monitoring was in force throughout the period. this analysis relied heavily on secondary data for both the information on the confirmed cases and travel histories. given this, there are possible concerns for under diagnosis and under reporting especially during the start of the outbreak until before its first peak as have also been observed in other countries [41] . the unique age and sex distribution of the cases in hong kong may also be affected by testing patterns. there may, of course, have been unreported and/or asymptomatic cases in hong kong which will have escaped our analysis. however, there is no current consensus on such rates of asymptomatic infection and, therefore, how one might either estimate or correct in the hong kong case [42] . finally, the challenge of ascertaining primary cause of death and the role played by covid-19 interacting with other factors [43] [44] [45] is not a significant issue in hong kong given the very small number of deaths. a major limitation is that the spread of covid19 has not yet ended. any observations of a 'case in progress' are prone to future, unexpected changes which will change the narrative already described. of course, by the time this paper is published it is eminently possible that events have taken a turn for the worse and a third wave of infections occur. despite this, hong kong has clearly demonstrated a capacity to control both the transmission and fatality of covid-19 until this point. as of june 11, 2020 there have been no covid-19 ascribed deaths since march 14, 2020. furthermore, as of april 29, 2020, hong kong has reported no new cases for the sixth time in ten days [10] , and there were just five cases of local transmission in the month of may. from the start of may, public facilities began to reopen, some border restrictions were lifted, and civil servants returned to work in their offices. as of mid-june, public facilities such as beaches, libraries, museums, swimming pools have largely reopened, as have the last group of commercial leisure/entertainment facilities (karaoke lounges, nightclubs, bathhouses and party venues). clearly, much more research is required to concretely establish both the epidemiological and social factors contributing to the hong kong experience. the data which will allow such analysis will only come on stream in the future. at this point more complex statistical analysis will be able to be performed. furthermore, it will then become possible to link data on transmission and fatality through to other clinical and vital records (including the planned census in 2021). such data will also need to be complimented by survey data and qualitative research to provide a broader sense of the hong kong context. policy interventions, institutional systems, household and living arrangements each played a role in a complex, interwoven way. however, we must not overlook the role played by the community itself who, through behavioral change and increased vigilance, appear to have been equally instrumental in shaping hong kong's covid-19 experience. it may be in this way that the greatest lessons of sars have been learned [5] . supporting information s1 table. timeline world population prospects: the 2019 revision the population problem in pacific asia remeasuring ageing in hong kong sar; or "keeping the demographic window open prevention and control of communicable diseases in hong kong. government printer the sars epidemic in hong kong: what lessons have we learned? who. cumulative number of reported probable cases of sars. in: who [internet 7 reasons hongkongers are angry about the gov't response to the coronavirus macau's last covid-19 patient recovers, with no new cases for a month why did singapore have more coronavirus cases than hong kong? in: south china morning post another day of no new covid-19 cases in hong kong situation report-89. world health organization the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19)-china demographic research and development foundation, inc. (drdf). covid-19 and the older filipino population: how many are at risk? uppi/ drdf research brief weekly assessment of the covid-19 pandemic and risk of importation-china population by-census impact assessment of non-pharmaceutical interventions against coronavirus disease 2019 and influenza in hong kong: an observational study. lancet public health the epidemiological characteristics of 2019 novel coronavirus diseases (covid-19 demographic science aids in understanding the spread and fatality rates of covid-19 death toll in uk care homes from coronavirus may be 6,000, study estimates. financial times number of deaths in care homes notified to the care quality commission, england. in: office for national statistics mortality associated with covid-19 outbreaks in care homes: early international evidence. international longterm care policy network unimaginable human tragedy" in europe's care homes, says who. japan times guidelines for residential care homes for the elderly or persons with disabilities for the prevention of coronavirus disease (covid-19) (interim) online day care keeping elderly hongkongers active during isolation nursing homes in 10 nations: a comparison between countries and settings how big is the problem in care homes? bbc office for national statistics kong ngo shows care homes they can stop keeping elderly in restraints. south china morning post the neglected health of international migrant workers in the covid-19 epidemic migrant workers in cramped gulf dorms fear infection current situation on migrant workers in dormitories-home thematic report: persons living in subdivided units tiny affordable housing in hong kong air and hygiene quality in crowded housing environments-a case study of subdivided units in hong kong a brief discussion on fire safety issues of subdivided housing units in hong kong social contacts and mixing patterns relevant to the spread of infectious diseases solidarity, ambivalence and multigenerational co-residence in hong kong. contemporary grandparenting: changing family relationships in global contexts living arrangements and older people's labor force participation in hong kong intergenerational co-residence during later life in europe and china level of underreporting including underdiagnosis before the first peak of covid-19 in various countries: preliminary retrospective results based on wavelets and estimating the extent of asymptomatic covid-19 and its potential for community transmission: systematic review and meta-analysis. medrxiv pathological findings of covid-19 associated with acute respiratory distress syndrome autopsy in suspected covid-19 cases case-fatality rate and characteristics of patients dying in relation to covid-19 in italy we would like to thank the academic editor and reviewers for their helpful and constructive suggestions. we would also like to thank professor stéphane helleringer for his comments on an earlier draft. the authors have declared that no competing interests exist. key: cord-000267-xroo7z7g authors: xiao, xiaodong; zhu, zhongyu; dankmeyer, jennifer l.; wormald, michael m.; fast, randy l.; worsham, patricia l.; cote, christopher k.; amemiya, kei; dimitrov, dimiter s. title: human anti-plague monoclonal antibodies protect mice from yersinia pestis in a bubonic plague model date: 2010-10-13 journal: plos one doi: 10.1371/journal.pone.0013047 sha: doc_id: 267 cord_uid: xroo7z7g yersinia pestis is the etiologic agent of plague that has killed more than 200 million people throughout the recorded history of mankind. antibiotics may provide little immediate relief to patients who have a high bacteremia or to patients infected with an antibiotic resistant strain of plague. two virulent factors of y. pestis are the capsid f1 protein and the low-calcium response (lcr) v-protein or v-antigen that have been proven to be the targets for both active and passive immunization. there are mouse monoclonal antibodies (mabs) against the f1and v-antigens that can passively protect mice in a murine model of plague; however, there are no anti-yersinia pestis monoclonal antibodies available for prophylactic or therapeutic treatment in humans. we identified one anti-f1-specific human mab (m252) and two anti-v-specific human mab (m253, m254) by panning a naïve phage-displayed fab library against the f1and v-antigens. the fabs were converted to igg1s and their binding and protective activities were evaluated. m252 bound weakly to peptides located at the f1 n-terminus where a protective mouse anti-f1 mab also binds. m253 bound strongly to a v-antigen peptide indicating a linear epitope; m254 did not bind to any peptide from a panel of 53 peptides suggesting that its epitope may be conformational. m252 showed better protection than m253 and m254 against a y, pestis challenge in a plague mouse model. a synergistic effect was observed when the three antibodies were combined. incomplete to complete protection was achieved when m252 was given at different times post-challenge. these antibodies can be further studied to determine their potential as therapeutics or prophylactics in y. pestis infection in humans. yersinia pestis (y. pestis) is the causative agent of plague that has killed over an estimated 200 million people in previous pandemics [1] . the current incidence of plague is low but the animal reservoirs for y. pestis exist worldwide. sporadic cases have been reported recently with an average case number of 2,500 worldwide [2] . y. pestis can be rendered airborne and its potential use as a bioweapon is recognized [3] as a category a agent on the niaid list of biodefense-related pathogens. current treatment for plague consists of antibiotics, while a live attenuated vaccine against plague is used in the former soviet union for prevention [4] . nevertheless, these live attenuated whole-cell vaccines or killed whole-cell vaccines have adverse effects to varying degrees [4] . though both types of treatment are efficacious, there is a need for an alternative treatment for plague [5] . a multiple-antibiotic-resistant isolate of y. pestis has been isolated, and drug resistance was shown to be mediated by a self-transferable plasmid [6, 7] . a subunit vaccine, which consists of two virulent factors, the f1 protein and v-antigen, is currently in human clinical trials [8] [9] [10] . studies involving the vaccine antigens in various formats have provided the proof-of-concept data that humoral response can be efficient in protection against y. pestis [11, 12] . there are multiple reports that mouse anti-plague monoclonal antibodies (mabs) against a y. pestis challenge can passively protect a mouse against plague [13] [14] [15] . therefore, mab therapy may be an attractive alternative to the existing treatments for plague. despite the promising possibilities, there remains a major hurdle in the treatment against plague and that is the possible immune response of humans to the mouse mabs that are currently available. one possibility to ameliorate the immune response against the mouse mab is to humanize the mab for use in humans, or another alternative is to develop new and fully human anti-plague monoclonal antibodies for clinical usage [16] . we describe here the isolation of three mabs from a large naive human phage-displayed fab library. one, designated as m252, is against the f1-antigen and the other two (m253, m254) are against the v-antigen. when used alone, m252 displayed good protective effects, whereas m253 and m254 did not. however, a clear synergistic effect was found when they were used together. maximum protection by m252 alone could be achieved by altering the antibody administration schedule. this is the first report describing the isolation of fully human anti-plague mabs that show efficacy in a mouse model of plague. these antibodies represent a significant breakthrough toward possible adjunctive therapeutic treatment of y. pestis infection in humans. with the f1 antigen, only the plate format yielded positive fab clones after four rounds of selection with the f1 antigen. sequencing of the clones confirmed that they were identical and designated as m252. with the v-antigen, the plate and bead format each yielded two positive fab clones after four rounds of selection with each format. one clone from each format, designated as m253 and m254, respectively, was selected for further analysis. sequence analysis revealed that m252 has heavy and light chains originated from germlines ighv1-2*02 and igkv1-16*01 respectively. m253 originated from ighv1-18*01 and igkv1-9*01, while m254 was from ighv3-43*01 and igkv1-27*01. the mutational rate ranged from zero to less than 10%. this is typical for antibodies isolated from naïve human libraries by panning against viruses causing acute infection in contrast to neutralizing antibodies selected from immune human libraries by panning against hiv-1, which causes a chronic infection [17] . each of the clones was then transformed into hb2152 cells and the respective fab was expressed and purified ( figure 1a ). after conversion to igg1 expressing clones, the three antibody clones were transiently transfected into freestyle hek 293f cells, and the expressed igg1s were purified ( figure 1c ). to determine both the specificity and affinity of the selected antibodies, elisa with both fab and igg formats were conducted as described in the methods. all fabs and iggs bound to their respective antigens specifically without cross-reaction to other antigens tested (figure 1b and d) . anti-f1 fab and igg have apparent affinities in the low and sub-nm range, respectively. both m253 and m254 fabs have apparent affinities of approximately 100 nm (figure 1b and d) . their iggs however have sub-nm apparent affinities (avidities). the avidity effect is very pronounced for all three antibodies. low level of competition between the human anti-f1 and anti-v fabs and mouse anti-f1 and anti-v mabs the three human anti-plague fabs were used in competition-elisas against a panel of mouse anti-plague mabs. the mouse anti-plague mabs included the anti-f1 mab f1-04-a-g1, and five anti-v mabs, which included the anti-v mab 7.3 m that was highly protective. we found no apparent competition between the human anti-v m253 fab and the mouse anti-v mabs (figure 2a ). however, we observed some weak competition between the human anti-v m254 fab antibody and some of the mouse anti-v mabs (7.3 m, 10-1 m, and 74-1 m) that we did not see with the human anti-v m253 fab antibody (figure 2b ). the competition between the human anti-f1 m252 fab antibody and the mouse anti-f1 mab was also minimal (figure 2c ). we ran two controls in the competition studies with the human and mouse mabs. for one control we did not add a primary antibody (labeled nc, figure 2a -2c), and for a second control we used a nonspecific mouse isotype igg1 mab in the competition assay (labeled bm, for a burkholderia mallei igg1 mab). there was also a lack of competition between the human anti-v m253 and m254 fab antibodies, suggesting that these two human anti-v fabs recognize different epitopes (one which may be conformational) on the v-antigen (figure 2d ). of note, however, is that when the competition-elisa was performed in a different fashion, namely when the human anti-f1 or anti-v mabs were allowed to bind to the respective antigens before adding the mouse anti-f1 or anti-v mabs, moderate competition was detected between the human anti-f1 m252 and the mouse anti-f1 mabs, as well as between the human anti-v m253 and mouse anti-v 84-1 mabs (data not shown). to characterize the binding of the human anti-f1 and anti-v mabs to the f1-and v-antigens, respectively, more closely, we examined the binding of the human mabs to two separate panels of overlapping peptides. one panel covered the full-length of the f1 antigen (27 peptides) and the other -the v antigen (53 peptides). for the human anti-f1 m252 mab, there was a weakmoderate binding signal with peptides 1 and 2, which are located at the n-terminus of the f1-antigen (figure 3a ). this suggests that the m252 mab may also recognize a conformational region that involves peptides 1 and 2. the binding by human anti-v m253 mab resulted in a strong signal with peptide 2 and a weak signal with peptide 1 (figure 3b ). the anti-v m254 mab, did not bind to any of the peptides, suggesting that its epitope may be conformational ( figure 3c ). in initial studies with m254 we saw some weak binding to peptides 36 and 42, but when we repeated the binding studies with the human anti-v mabs we did not see binding to peptides 36 and 42. this might explain its weak competition with mouse antibodies that recognize diverse epitopes on the v-antigen ( figure 2b ). the positive signals seen with the m253 and m254 mabs with v-antigen peptides (numbers 19, 20, 27, and 28) are nonspecific signals that are generated by the secondary antibody (amemiya et al. unpublished). the epitopes of the mouse antibodies have also been determined by the peptide binding assay (amemiya, et al. unpublished). the data is consistent with the competition-elisa presented in this study. to test if the human anti-f1 and anti-v iggs bind their respective targets on bacterial cells, we performed flow cytometry analysis. both the mouse anti-f1 f1m mab and human anti-f1 m252 mab bound specifically to y. pestis grown at 37uc and not to the same strain grown at 26uc. neither did they bind to a control e. coli strain grown at 37uc (figure 4 , bottom panel). this is consistent with previous reports that the expression of the f1-antigen is regulated by temperature (37uc), and it is not expressed at rt. the human anti-v m254 mab showed minor binding to the y. pestis grown at 37uc, although we do not normally see binding with the human anti-v m253 or mouse anti-v mabs, which included 7.3 m, 74-1 m, and 141-1 m (amemiya, unpublished). a mouse igg1 isotype control mab, which did not show any binding to the wholecells, was included to show binding by the mouse and human anti-f1 mabs was antibody specific. to further confirm the binding data we used immunofluorescence technique. the results were consistent with the flow cytometry data, where only the mouse and human anti-f1 mab bound to y. pestis whole-cells ( figure 5 ). neither the human anti-v m254 mab ( figure 5d ) nor mouse anti-v mabs, like 7.3 m (data not shown) nor control samples ( figure 5a , nonspecific mouse igg1 isotype; figure 5e , no primary mab) showed significant binding to y. pestis. human anti-f1 and v mabs protect synergistically against a y. pestis challenge in a bubonic plague model the ability of the human anti-f1 and anti-v mabs to passively protect mice against a y. pestis infection was evaluated in a bubonic plague model. the human anti-f1 and anti-v mabs were used either separately or together in different combinations. when mabs m252 and m253 and m254 were given to mice separately before challenge with y. pestis co92, only the human anti-f1(m252) mab showed some efficacy. the mean-time-to-death (mtd) in the m252 mab-treated mice was shifted to 13.0 days (1/ 6 survivors) when compared with mice given normal mouse serum (nms), which had a mtd of 7.0 days (0/6 survivors) ( figure 6a ). unlike m252, however, the human anti-v mabs, m253 and m254, did not show any significant protection [mean-time-to-death (mtd) of 6.7 days (0/6 survivors) and 7.3 days (0/6 survivors), respectively] when compared to the nms -treated mice. the mouse anti-f1 (f1m) and anti-v (7.3 m) control mabs both passively protected all (6/6) mice under the same challenge conditions (mtd of 21 days). when both human anti-v mabs were given to mice passively ( figure 6b ), no improvement in protection after challenge was observed (mtd of 6.8 days, 0/6 survivors), which was similar to that seen with the nms-treated mice (mtd of 7.3 days, 0/6 survivors). however, when the human anti-f1 m252 mab (mtd of 11.3 days, 2/6 survivors) was given together with the two human anti-v m253 and m254 mabs, a greater number of mice were passively protected (mtd 14.0, 5/ 6 survivors) than when the antibodies were used separately, suggesting a synergistic effect. although we saw some protection with the human m252 mab, and a synergistic protective effect when the human m252 mabs was given with the human m253 and m254 mabs in the bubonic plague model, we wondered if there was any effect (positive or negative) with a nonspecific human igg1 mab by itself or when combined with the human anti-f1 m252 mab in the number of survivors in the mouse model of plague. to answer this question, we injected five groups of mice with the following mabs: one group of mice with only a nonspecific human igg1 mab (hu-igg1, 1500 mg); another group of mice with the mouse anti-f1 mab (f1m, 500 mg); another group of mice with the human anti-f1 mab (m252, 500 mg). two other groups of mice were included, that were given either f1m (500 mg) or m252 (500 mg), with the nonspecific hu-igg1 mab (1000 mg) one day before challenge with y. pestis co92 ( figure 7 ). all mice in the group that received only the nonspecific hu-igg1 mab died by day 8, which was similar to the control antibody mouse groups in figure 6a and 6b. the same number of survivors was obtained (6/6) whether mice were given only mouse f1m mab or f1m combined with the nonspecific hu-igg1. as we have seen previously ( figure 6a ), only 1/6 mice survived in the group that received only human m252 mab. when the human m252 mab was combined with the nonspecific hu-igg1 mab, we obtained one more survivior (2/6) than we obtained without the nonspecific hu-igg1 mab. this variation in the number of survivors was not different than we saw previously ( figure 6b ). in addition, the mtd was not affected by the presence of the nonspecific hu-igg1 (15.8 days) when given with the hu-antif1 mab (15.8 days). these results suggest that the nonspecific hu-igg1 mab had little effect on the survival of mice given the human m252 mab or mouse f1m mab. delaying time of delivery of human anti-plague mabs provided better protection against a plague challenge one possible reason we observed less protection with the human anti-f1 and anti-v mabs in the mouse plague model was that the level of the human iggs may not have been sustained in the mouse over time compared to the mouse igg mabs, we tested this hypothesis in two separate studies. we first examined the concentration of the human antibody in mice directly, by measuring the level of m252 (anti-f1) and m253 (anti-v) in serum after they were given the human mabs by i.p. injection. mouse sera were collected at different time points after the initial dosing and human igg levels were monitored by direct elisa. as seen in figure 8a , the anti-f1 m252 mab appeared to have a half-life of approximately 8 days, and the half-life of m253 mab was approximately 10 days. after 21 days, the levels of these two human antibodies were undetectable. in contrast, the level of both the mouse anti-f1 (f1m) and anti-v (7.3 m) mabs may have decreased initially like the human anti-plague mabs, but after 21 days, the levels were still approximately 40-50% of the initial concentration ( figure 8b ). the half-live of human igg mabs in mice reported here is similar to what was found in another study where human mabs were used against another biothreat agent [18] . in contrast, a human igg molecule would have an average serum half -life of 21 days in a human [19] . because of these findings we then administered the anti-f1 m252 mab at different time points relative to the time of challenge. while the original regimen provided consistently modest protection, administration of the human m252 mab 24 and 48 hours postchallenge provided increasing protection with the 48 hours schedule provided complete protection ( figure 9 ). antibody administration at even later time points was not performed since mice began to die 3-4 days after challenge without any treatment. however, we did evaluate the effect of a second dose of antibody at a later time point. in this group, mice first received an initial dose of human anti-f1 m252 mab 24 hour before challenge as was done with the earlier protocols. these mice then received a second dose of the human anti-f1 m252 mab 5 days after challenge. there was an increase in both the number of survivors (5/6) and mtd (20 . epitope mapping of the human anti-f1 and anti-v antibodies by peptide binding assay. a. each of twenty-seven peptides that covered the full length of the f1-antigen were used to coat an elisa plate (0.05 ml of a 25 mg/ml solution of each peptide), and binding by the human anti-f1 m252 mab (0.05 ml of a 10 mg/ml solution) was analyzed. the sample labeled f1 was the full-length antigen used to coat the plate as the positive control (0.05 ml of a 2 mg/ml solution), and the sample labeled media was the negative control with no primary antibody added. b and c. each of fifty-two peptides that covered the full length of the v-antigen was used to coat an elisa plate (0.05 ml of a 25 mg/ml solution), and the human anti-v m253 (b) and m254 (c) mabs were used (0.05 ml of a 10 mg/ml solution), respectively, to analyze for binding. the sample labeled v was the positive control (0.05 ml of a 2 mg/ml solution), and the sample labeled media was the negative control without the primary antibody. doi:10.1371/journal.pone.0013047.g003 days) approaching the efficacy displayed by a single dose administered 48 hour after challenge. these data suggest that the optimum serum concentration of the human igg1s was critically dependent on the time of administration, and that the optimum concentration of the human anti-plague igg1s in turn determined the outcome of the treatment protocol. antibiotics have been at the forefront of combating bacterial infection for decades with great success. however, the develop-ment of new antibiotics is struggling to keep pace with the emergence of drug resistant bacterial strains, for example as in y. pestis [6] . there has been an intense interest in developing antibody-based therapies as an alternative method of treatment [5] . initially, antibody-based therapy was mostly limited to treating cancer or immune disorders. however, because of a better understanding of the pathogenesis of infectious agents, and the advancement in the development of protective or neutralizing antibodies, the use of antibody-based therapy against infectious agents has become more frequent [20] . in this report we described the first isolation of fully human mabs against the y. pestis figure 6 . human anti-f1and anti-v mabs show synergistic protection when used together in the murine bubonic plague model. the human and mouse anti-f1 and anti-v mabs were given i.p. to mice 24 hrs before parenteral challenge with y. pestis co92, and the number of surviving mice for each treatment group was monitored for 21 days after challenge. 6a. the following antibodies and amounts were used: normal mouse serum (nms, 500 mg); mouse anti-f1 (f1m, 500 mg); mouse anti-v (7.3 m, 100 mg); human anti-f1 (m252, 500 mg); human anti-v (m253 and m254, 500 mg each). 6b. the following mabs and amounts were used: nms, 500 mg; f1m, 500 mg; m252, 500 mg; m253+ m254, 500 mg each; m252+ m253+ m254, 500 mg each. doi:10.1371/journal.pone.0013047.g006 virulence factors f1-and v-antigens. previous studies have shown that mouse mabs can be effective in protecting mice against y. pestis ( [13] [14] [15] . however, because they are mouse mabs they are not safe to use in their present form in humans [21] . of particular concern is the immune reaction against mouse primary antibody sequences in human system. this may lead to severe adverse effects and at the same time reduce the potential benefits. it is highly desirable to have fully human antibodies for these reasons. the fully human anti-f1 (m252) reported here displayed moderate to good protection against a bubonic plague challenge with y. pestis co92. on the other hand, the two anti-v mabs (m253, m254) when used separately did not show any efficacy, but when they were used together with m252, the combination of the human anti-plague mabs resulted in better protection overall, suggesting a synergistic effect between the antibodies. a similar effect was reported in studies using mouse anti-f1 and anti-v mabs in a mouse model of plague [15] . further in our case with the human anti-f1 mabs, when we gave mice the human anti-f1 mab 1-2 days after challenge, we saw a greater protection against a plague challenge could be achieved. this suggested that the maintenance of serum concentration of the human mabs in the mouse was possibly one critical factor for better protection. kinetic studies revealed that indeed the serum concentration of the human antibodies dropped further than the mouse anti-plague mabs over the course of the study. it is also plausible that the human antiplague mabs might bind to other mouse antigens nonspecifically, thus decreasing the amount of free circulating human anti-plague antibody in the mouse. figure 7 . a nonspecific human igg1 antibody (hu-igg1) had little effect on the mean-time-to-death (mtd) and number of surviving mice that received the human anti-f1 mab. the nonspecific human igg1mab, and the human and mouse anti-f1 and anti-v mabs were given i.p. to mice 24 hrs before parenteral challenge with y. pestis co92, and the number of surviving mice for each treatment group was monitored for 21 days after challenge. mouse or human mabs and their amounts were given to the following groups of mice: nonspecific human igg1 (hu-igg1, 1,500 mg); f1m (f1m, 500 mg); f1m (500 mg) + hu-igg1(1,000 mg); m252, 500 mg; m252 (500 mg) + hu-igg1 (1,000 mg). doi:10.1371/journal.pone.0013047.g007 another important underlying factor for efficient protection by antibodies is the epitopes the antibodies recognize. although the human anti-f1 (m252) mab appeared to bind to the same region as the mouse antif1, which was at the amino-terminal end of the f1antigen (amemiya et al., unpublished), we could not demonstrate direct competition between these two antibody species. this observation may be the result of the nature of the antibody binding site or epitope, because several mouse mabs isolated independently recognized the same region or epitope on the f1-antigen, behaved in the same manner (amemiya et al., unpublished). it may be that once these mabs bound to the amino-terminal end of the f1antigen, they may not readily come off the protein or may dissociate very slowly. whether this is because the binding site involved both linear and conformational sites is not known, but both the mouse and human anti-f1 mabs bound to the whole anti-f1 antigen very well, but only weakly -moderately to the 59-peptides. nevertheless, the human m252 mab was as protective as the mouse anti-f1 mab when the human mab was given after challenge. it has also been reported that a neutralizing epitope on the v-antigen was located in a region spanning amino acids 135 to 275, and a possible minor, secondary neutralizing epitope exists near the amino-terminal region of the v-antigen [14] . neither of our human anti-v antibodies reported here competed with the mouse anti-v antibodies efficiently. the minor competition between the human anti-v fab antibody and the mouse anti-v antibodies suggests that the recognition site of the human anti-v antibodies is slightly different or they may partially share conformational binding site. these differences might be one reason for their inability to protect as efficiently as the human anti-f1 m252 mab. exactly how the human anti-f1 252 m mab is able to protect mice may be directly related to the presence of f1 antigen on the surface of the plague organism. the f1-antigen has been reported to be anti-phagocytic [22, 23] . the ability of macrophages to take up the plague organism is directly related to the lack of the f1antigen, and resistance to phagocytosis is related to the presence of the f1-antigen. the binding of the human anti-f1 252 m mab to the surface of the plague bacilli or opsonization may trigger phagocytosis of encapsulated bacilli into macrophages, thereby allowing phagocytic cells to clear the host of the pathogen. the exact mechanism by how the v-antigen exerts it virulence is not completely known. there are reports showing that vantigen is secreted into the growth medium and the secretion is important for virulence [24, 25] . the secretion of v-antigen in the medium has been described to be dependent on contact with the host cell, and could also be directed into the host cell by a yersinia outer proteins (yops) dependent secretion (ysc) type iii system (ttss) [26] [27] [28] . it also has been suggested that free v-antigen may enter the cell by endocytosis besides being injected into the cell by the ysc ttss [29] . once inside the host-cell, we do not know exactly what host proteins interact with the intracellular vantigen [29] . nevertheless, there is some evidence that anti-v antibodies enhance phagocytosis through possibly the fc receptor, and thereby block yop delivery into the host cell, and thus preventing ysc dependent ttss injection of v-antigen into the host cell [30, 31] . in this study, however, we were not able to detect binding of the human anti-v mabs on the surface of y. pestis cells by flow cytometry or fluorescent microscopy, suggesting that the vantigen was not on the bacterial cell surface under the conditions used in our studies or the expression level of v-antigen was below the level of detection. as has been discussed previously, however, the presence of v-antigen on the surface of the cell may be dependent on contact with the eukaryotic host cell. the highly specific region of the neutralizing epitope (s) on the v-antigen suggests a possible ligand-receptor interaction between the vantigen and a cellular factor. this indicates that perhaps the vantigen exerts its biological effect through mechanisms other than mediating the ttss pathway. in conclusion, the human anti-plague antibodies reported here represent perhaps the ones that are closest to practical clinical use. they may be safer and more efficient in the human system due to their fully human nature, and a likely longer half-life in humans. also, intravenous application of the antibodies in humans may be a rapid delivery system that may augment antibiotics treatment in plague-exposed individuals. finally, the affinity of all three antibodies can be further increased using readily available techniques, may reduce the dose required for efficient protection. the successful development of these three human anti-plague figure 9 . post-challenge administration of the human anti-f1 m252 mab conferred better protection. the human anti-f1 m252 mab was administered before or after y. pestis challenge, and mice were monitored for 21 days after challenge. the mouse anti-v 7.3m mab (100 mg) was used as a positive control mab. normal human serum (nhs, 500 mg), which was used as a negative control, had only 5 mice per group. the numbers behind each antibody represent the time in days in which the antibody was administered (500 mg) to mice relative to the day of challenge (day 0). the two numbers after the human anti-f1 m252 (21 and +5) represent two different days when the mab (500 mg) was added to the same group of mice relative to the day of challenge. doi:10.1371/journal.pone.0013047.g009 antibodies in this model suggests that new and more potent anti-v antibodies can be potentially developed using the same approach but with restricted v-antigen subunit fragments containing the critical neutralizing epitopes, and perhaps other virulent factors. the y. pestis co92 strain used in the challenge studies was originally obtained from t. quan, centers for disease control and prevention, fort collins, co. it was isolated from the sputum of a human case of pneumonic plague [32] . the y. pestis co92 was grown and inoculum prepared for challenges essentially as described previously [33] . a y. pestis pgmstrain, which was originally isolated from y. pestis co92, and used in the antibody binding studies described below was obtained from susan l. welkos (usamriid, frederick, md). y. pestis purified f1,v, and f1-v [34] protein antigens were obtained from brad powell (usamriid, fort detrick, frederick, md). the 27-peptide array that covered the f1-antigen were 14to 17-mers with 11 amino acid overlaps; they were obtained from the biodefense and emerging infections research resources repository (bei)(manassas, va). the 53-peptide array that covered the v-antigen were 15-to 17-mers with 11 or 12 amino acid overlaps and were obtained from bei. the anti-f1 mouse mab f1-04-a-g1 (or mf1) was provided by george anderson (usamriid) [13] , and anti-v mouse mabs 10-1 m, 74-1 m, 84-1 m, and 141-1 m as well as the control igg1 mouse anti-burkholderia mallei (bm) antibody were obtained from sylvia trevino (usamriid) and anti-v mouse mab 7.3 (7.3 m) was obtained from jim hill (porton down, wiltshire, uk) [14] and used in competition elisas and as positive controls in mouse passive protection experiments. all mice mabs were igg1 isotypes. the human igg1 control mab used in the passive protection study was an anti-human igfii mab. purified f1-and v-proteins were either coated directly to maxisorp plates (nunc, denmark) in pbs buffer at 4uc, overnight for plate format panning or were biotin-labeled first with ez-link sulfo-nhs-lc-biotin (pierce, rockford, il) for streptavidinconjugated magnetic bead format panning. the labeling was performed according to the manufacture's recommended protocol. for the plate format, approximately 10 12 fabs displayed on the surface of phage amplified from a large naive library [35] were suspended in pbs with 2% dry milk and applied to wells coated with the f1-or v-proteins. after incubating for 2 hours at room temperature, each well was washed 5 times for the first round and 10 times for the subsequent four rounds before the phage were rescued with tg1 cells at the exponential growth phase. for the bead format, biotin-labeled f1-and v-antigens were first incubated with the same amount of phage as in the plate format in 1 ml of pbs+2% dry milk suspension at room temperature for 2 hour. fifteen ml of dynabeads myone streptavidin t1(invitrogen dynal as, oslo, norway) pre-blocked with pbs+2% dry milk was then added to the antigen/phage mixture for one hour at room temperature. the beads were then washed 5 times with pbs for the first round and 10 times for the subsequent four rounds of selection. phage were then rescued with tg1 cells. a total of four rounds were performed for each antigen with each format. monoclonal elisa was then performed to select for positive clones. one hundred clones were screened for each antigen from each format. only clones displaying an od405.2.0 were selected for plasmid preparation and sequencing. expression, purification, conversion to igg1, and generation of stable clones clones selected as described above were transformed into e.coli strain hb2151 for expression [36] . briefly, a single clone was inoculated into 2yt supplemented with 100 units of ampicillin and 0.2% glucose and incubated at 37uc with shaking. when the od600 reached 0.6-0.9, iptg was added to achieve a final concentration of 1 mm and the culture was shifted to 30uc with shaking and incubated overnight. cells were then collected, and lysed with polymyxin b (sigma, st louis) in pbs, and mixture subjected to ni-nta agarose bead (qiagen, hilden, germany) purification. for igg1 production, the heavy and light chains of the respective fabs were cloned into the bi-cistronic expression vector pdr12 kindly provided by dennis burton (scripps research institute, la jolla, ca). for small scale igg1 production, transient transfection and expression in freestyle hek 293f cells (invitrogen, carlsbad, ca) were used. for large scale production, stable clones were generated using cho-k1 (atcc, manassas, va) cells. briefly, the heavy and light chains of the three human anti-plague igg1s were cloned into pdr12 vectors and transfected into cho-k1 cells. one day after transfection, the cells were replated and subjected to selection in gmem medium supplemented with 25 mm msx. two weeks later, the msx resistant clones were amplified further. the clones were tested for the expression of respective igg1s and then adapted to growth in serum-free medium hyqsfm4cho (hyclone, logan, ut) supplemented with 30 mm msx. the serum-free growth medium was then collected and passed through a protein a-sepharose resin column for igg1 purification. an elisa assay was used to assess the binding ability of the fabs and igg1s. briefly, f1-and v-antigens were coated to a costar high binding 96-well plate (corning, corning, ny) and incubated overnight at 4uc. the next day, the plate was blocked with 2% dry milk in pbs before serial dilution of fabs or iggs were applied to the plate. after an incubation of the plate at 37uc for one hour, anti-his-horse radish peroxidase (hrp) for fab detection or anti-human-fc-hrp (for igg detection) in pbs+2% dry milk was added to each plate and incubated for another hour at 37uc. the plates were then washed four times, and the abts substrate (roche, mannheim, germany) was added. after approximately 10 min at room temperature, the od405 was taken. for competition studies between the mouse mabs and human fabs the antigen at 2 mg/ml (f1-protein or v-antigen) was used to coat 96-well plates (immulon 2hb, thermo electron, milford, ma), and the plates were incubated overnight at 4uc. after washing the plates, a blocking solution (1% bovine serum albumin with 0.05% tween 20 in pbs) was added to the plates, and plates incubated for 1 hr at 37uc. the flag-labeled human fabs, and biotinylated-mouse mab were allowed to bind to the antigen simultaneously for 1 hr at 37uc. to detect the presence of the human fab, an anti-flag-m2-peroxidase conjugate was used, and to detect the amount of biotinylated mouse mab present, a streptavidin-conjugated hrp was added for 1 hr at 37uc, before adding a hydrogen peroxidase-3,39,5,59-tetramethylbenzidine solution. the color reaction was allowed to develop at room temperature for 15 min and read at 450 nm. for analysis of igg1 binding to f1-or v-antigen peptides, the peptides were added to the plates in 0.05 ml per well at 25 mg/ml. and the plates incubated overnight at 4uc in immunlon 2hb 96well plates. after the washing and blocking steps as described above, binding of the human and mouse mabs iggs was detected as described above. the binding of all mabs to the peptides was evaluated at 10 mg/ml. flow cytometry was used to analyze the binding of the human anti-f1 and anti-v antigen igg1s to y. pestis pgmwhole-cells. y. pestis pgmwas first streaked onto a sheep-blood agar plate and grown at room temperature (rt) for 3 to 4 days until colonies were readily visible. a single colony was picked and inoculated into 10 ml of heart infusion broth (remel, lenexa, ks) containing 0.2% xylose and 2.5 mm cacl 2 , and cells grown overnight (o/n) at rt with rigorous shaking. the next day, an aliquot of the bacteria culture was shifted to 37uc, and the other remained at rt, and the cultures were allowed to continue for another 3 hours before the bacteria were collected and suspended in pbs. ten ml of bacterial cells was mixed with 90 ml of fc block solution [pbs with 1% fcs and 10 mg/ml fcblock (bd bioscience)] to achieve a final density of 1610 7 cells/ml. human or mouse iggs were added to the bacterial cell suspension to a final concentration of 10 mg/ml. after incubating at 4uc for 30 min, the bacterial cells were collect by centrifugation and suspended in 1 ml of pbs, and cells washed twice. the bacterial cells were then suspended in the same fc block solution, and secondary antibodies, which included either goat anti-mouse igg-fitc (pierce, rockford, il) or goat anti-human igg-fitc (southern biotech, birmingham, al) were added to the cells at a dilution of 1:100. after 30 min at 4uc, the cells were then washed three times with pbs and subjected immediately to facs analysis using a facscalibur (bd bioscience, san diego, ca), after the cells were fixed with 4% paraformaldehyde. for detection of antibody binding to whole-cells by immunofluorescent microscopy, the growth of y. pestis pgmand the sample preparation for binding by human or mouse anti-f1 or anti-v mabs was identical as that for the facs analysis, except a nikon t-2000 fluorescent microscope (nikon instruments inc., melville, ny) was used for detection. passive protection by human or mouse anti-f1 or anti-v mabs and y. pestis challenge studies antibodies to be evaluated for their efficacy against a plague challenge were given intraperitoneal (i.p.)(500 mg per mouse, except when stated differently in the figure legend) to 6-10 week old balb/c mice 24 h before they were challenged or at time points post-challenge as indicated in the figure legend. the challenge dose was prepared from frozen stocks of y. pestis co92 that were streaked on tryptose blood agar slants and incubated at 28uc for 48 h. after the incubation period, the slants were rinsed with 10 mm potassium phosphate buffer, ph 7.0, and cell density adjusted to the required density with the same buffer. mice were given the challenge dose of ld 50 ,25-40, subcutaneously in 0.2 ml, where 1 ld 50 is equal to 1.9 cfu [37] and observed for at least 21 days. research was conducted in compliance with the animal welfare act and other federal statues and regulations relating to animals and experiments involving animals and adheres to principles stated in the guide for the care and use of laboratory animals, national research council, 1996. the facility where this research was conducted is fully accredited by the association for assessment and accreditation of laboratory animal care international. the studies involving mice were approved by the iacuc at the u.s. army medical research institute of infectious diseases, animal protocol number ap-07-040. half-life of human or mouse anti-f1 and anti-v mabs given passively to mice an elisa as described above was used to detect the amount of human or mouse anti-f1 and anti-v mabs present in mice over time. briefly, f1-v protein (2 mg/ml in 0.2 m carbonate buffer, ph 9.4)) was used to coat a 96-well plate (immulon 2hb) overnight at 4uc before washing and blocking. two-fold dilutions of mouse serum taken retro-orbitally after i.p. administration of the mab were made in 1x pbs with 1% bsa and 0.05% tween-20, added to plates and incubated for 1 hr at 37uc before washing. the amount of human or mouse anti-f1 or anti-v mab binding to the antigens was detected by the addition of goat-anti-human or goat-anti-mouse igg conjugated to hrp (southern biotechnology). the results from 3 mice at each time point for each mab was performed in triplicate and were reported as the mean of the reciprocal of the highest dilution giving a mean od of at least 0.1, which is at least twice the standard deviation (sd). yersinia pestis -etiologic agent of plague the anti-plague system and the soviet biological warfare program yersinia pestis (plague) vaccines passive antibody administration (immediate immunity) as a specific defense against biological weapons multidrug resistance in yersinia pestis mediated by a transferable plasmid multiple antimicrobial resistance in plague: an emerging public health risk protection against experimental bubonic and pneumonic plague by a recombinant capsular f1-v antigen fusion protein vaccine a new improved subunit vaccine for plague -the basis of protection human immune response to a plague vaccine comprising recombinant f1 and v antigens shortand long-term efficacy of single-dose subunit vaccines against yersinia pestis in mice an igg1 titre to the f1 and v antigens correlates with protection against plague in the mouse model protection of mice from fatal bubonic and pneumonic plague by passive immunization with monoclonal antibodies against the f1 protein of yersinia pestis regions of yersinia pestis v antigen that contribute to protection against plague identified by passive and active immunization synergistic protection of mice against plague with monoclonal antibodies specific for the f1 and v antigens of yersinia pestis drug discovery and designs germline-like predecessors of broadly neutralizing antibodies lack measurable binding to hiv-1 envelope glycoproteins: implications for evasion of immune responses and design of vaccine immunogens human monoclonal antibodies against anthrax lethal factor and protective antigen act independently to protect against bacillus anthracis infection and enhance endogenous immunity to anthrax antibodies monoclonal antibodies against viruses and bacteria: a survey of patents immunogenicity of engineered antibodies the role of multiplication of pasteurella pestis in mononuclear phagocytes in the pathogenesis of flea-borne plague role of fraction 1 antigen of yersinia pestis in inhibition of phagocytosis biosynthesis and purification of v and w antigen in pasterurella pestis diminished lcrv secretion attenuates yersinia pseudotuberculosis virulence the vantigen of yersinia forms a distinct structure at the tip of injectisome needles the v-antigen of yersinia is surface exposed before target cell contact and involved in virulence protein translocation lcrv of yersinia pestis enters infected eukaryotic cells by a virulence plasmid-independent mechanism in vitro intracellular trafficking of virulence antigen during infection by yersinia pestis anti-v antigen antibody protects macrophages from yersinia pestis-induced cell death and promotes phagocytosis anti-lcrv antibody inhibits delivery of yops by yersinia pestis kim5 by directly promoting phagocytosis cattransmitted fatal pneumonic plague in a person who traveled from colorado to arizona recombinant v antigen protects mice against pneumonic and bubonic plague caused by f1-capsule-positive and -negative strains of yersinia pestis design and testing for a nontagged f1-v fusion protein as vaccine antigen against bubonic and pneumonic plague potent crossreactive neutralization of sars coronavirus isolates by human monoclonal antibodies overview: amplification of antibody genes studies on the contribution of the f1 capsule-associated plasmid pfra to the virulence of yersinia pestis we thank anthony bassett and wilson ribot for their technical expertise and members of our groups for helpful discussions. key: cord-263453-7v4y02j6 authors: nishiura, hiroshi; klinkenberg, don; roberts, mick; heesterbeek, johan a. p. title: early epidemiological assessment of the virulence of emerging infectious diseases: a case study of an influenza pandemic date: 2009-08-31 journal: plos one doi: 10.1371/journal.pone.0006852 sha: doc_id: 263453 cord_uid: 7v4y02j6 background: the case fatality ratio (cfr), the ratio of deaths from an infectious disease to the number of cases, provides an assessment of virulence. calculation of the ratio of the cumulative number of deaths to cases during the course of an epidemic tends to result in a biased cfr. the present study develops a simple method to obtain an unbiased estimate of confirmed cfr (ccfr), using only the confirmed cases as the denominator, at an early stage of epidemic, even when there have been only a few deaths. methodology/principal findings: our method adjusts the biased ccfr by a factor of underestimation which is informed by the time from symptom onset to death. we first examine the approach by analyzing an outbreak of severe acute respiratory syndrome in hong kong (2003) with known unbiased ccfr estimate, and then investigate published epidemiological datasets of novel swine-origin influenza a (h1n1) virus infection in the usa and canada (2009). because observation of a few deaths alone does not permit estimating the distribution of the time from onset to death, the uncertainty is addressed by means of sensitivity analysis. the maximum likelihood estimate of the unbiased ccfr for influenza may lie in the range of 0.16–4.48% within the assumed parameter space for a factor of underestimation. the estimates for influenza suggest that the virulence is comparable to the early estimate in mexico. even when there have been no deaths, our model permits estimating a conservative upper bound of the ccfr. conclusions: although one has to keep in mind that the ccfr for an entire population is vulnerable to its variations among sub-populations and underdiagnosis, our method is useful for assessing virulence at the early stage of an epidemic and for informing policy makers and the public. when an emerging influenza virus appears in humans, an early concern is whether the virus has the potential to cause a devastating pandemic, i.e., the global spread of an infection killing a substantial number of people. to assess the pandemic potential, two critical aspects need to be studied: the transmission potential and the clinical severity of the infection [1] [2] [3] . it is widely known in epidemiology that the former aspect, the transmission potential, can be quantified by the reproduction number, i.e., the average number of secondary cases generated by a single primary case [1, 4] , by characterizing the heterogeneous patterns of transmission (e.g. age-specificity) [5] , and by measuring other epidemiological quantities such as household secondary attack rate. there are two different approaches to assessing the latter aspect of a pandemic, the virulence of infection. one is to explore specific genetic markers of the virus that are known to be associated with severe influenza (e.g. the pb1 gene) [6] , although the absence of a known marker, as was for example the case in a novel swine-origin influenza a (h1n1) virus (s-oiv), does not necessarily indicate that the virus is benign [7] . another is an epidemiological approach to quantification of the case fatality ratio (cfr), the conditional probability of death given infection (or disease; see below). the cfr in general is vaguely defined as the ratio of deaths to cases, whose denominator should ideally be the total number of infections, but is frequently taken to be only the diagnosed cases due to the impossibility of counting all infected individuals. because in the early phase of an outbreak information is often limited to confirmed cases, we concentrate on confirmed cases only, and refer to the cfr as the confirmed cfr (ccfr) for clarity. as the world has experienced a global spread of s-oiv since april 2009, methods have been sought for the real-time assessment of virulence by measuring the ccfr which is a representative of the epidemiological measurements of virulence [2, 3] . nevertheless, a much-used crude estimate of the ccfr, i.e. the ratio of the cumulative number of deaths to cases at calendar time t, tends to yield a biased (and mostly underestimated) ccfr due to the time-delay from onset to death [8] ; similar estimates of such a biased ccfr for severe acute respiratory syndrome (sars) have shown how such estimates can vary substantially as an epidemic progresses, stabilizing only in the later stages of the outbreak [8, 9] . in the following we will use the terms biased and unbiased ccfr when we refer to this particular source of bias. improving an early epidemiological assessment of an unbiased ccfr is therefore crucial for the initial determination of virulence, shaping the level and choices of public health intervention, and providing advice to the general public [10] . to obtain an estimate of the ccfr, the lesson from the sars outbreak is that a statistical technique is required that corrects the underestimation, e.g. a technique addressing censoring [8, 11, 12] . nevertheless, in the case of novel s-oiv, an early unbiased estimation of the ccfr has appeared particularly challenging. initial reports from the government of mexico suggested a virulent infection, whereas in other countries the same virus was perceived as mild [13] . in the usa and canada there were no deaths attributed to the virus in the first 10 days following a declaration of a public health emergency by the world health organization. even under similar circumstances at the early stage of the global pandemic, public health officials, policy makers and the general public want to know the virulence of an emerging infectious agent. that is, a simple method for assessing ccfr is called for, even when only a few deaths have been reported, or even when there has been no report of deaths. except for another unbiased ccfr estimate in mexico (0.4%, range 0.3-1.5%) [1] , this early assessment has been missing. in the usa, a technical discussion has taken place on the crude measurement of the biased ccfr using the cumulative numbers of deaths and confirmed cases so far [10] . in line with this, an epidemiological method and its practical guide for early assessment of virulence are called for. the present study aims at developing a simple method to assess the virulence of an emerging influenza virus at the early stage of the epidemic, even when there have been only a few deaths or none at all. the method takes into account the time from the onset of symptoms to death, while differing from previously published statistical methods which employ censoring techniques [8, 11] . as an example, we give an early prediction of the ccfr of s-oiv infection in the usa and canada, and show that the unbiased ccfr, as estimated by our method at the early stage of the epidemic in these countries, was in fact comparable to that estimated for mexico [1] . our unbiased estimation of the ccfr does not address all sources of error in data (e.g. underdiagnosis of infected individuals) and we summarize the relevant issues in the discussion. we assess the virulence of s-oiv by measuring the risk of death, expressed as the ccfr. the ccfr is interpreted as the conditional probability of death given confirmed diagnosis [14] . since the data of s-oiv infection we use in the present study are only confirmed cases, we have replaced ''infection'' in the denominator of cfr by confirmed diagnosis of infection (see discussion). accordingly, an unbiased estimator of ccfr would be the proportion of deaths among confirmed cases at the end of an epidemic. although one could instead assess the virulence by measuring the proportion of hospitalized cases among a total number of confirmed cases, criteria for hospital admission are not universal, being influenced by isolation policies and in some regions by cultural and social differences. in the following, the notation used to represent the three different statistical measurements of ccfr is: (i) b t , which is a crude, biased estimate of the ccfr calculated at time t; (ii) p, which is an unbiased ccfr to be estimated in the present study, and is the unknown parameter that governed the outbreaks; and (iii) p t , a random variable, which yields an estimator of p (see below) and is regarded as the realized value in one particular outbreak. first, b t , a crude and biased estimate of ccfr, calculated at time t, is given by the ratio of the cumulative number of deaths d t to the cumulative number of confirmed cases c t : during the outbreak of severe acute respiratory syndrome (sars) in 2002-03, it was shown that this estimator, b t , considerably underestimates the ccfr [8] . this is easily demonstrated by relating c t and d t to the incidence function c t (i.e. the number of new confirmed cases on day t), and the conditional probability density function f s of the time from onset to death, given death. first, c t is the cumulative number of confirmed cases up to time t: second, d t is the cumulative number of deaths up to time t: as we mentioned above, p t is the realized proportion of confirmed cases to die from the infection, and is a random variable, which would be an unbiased estimator for p. therefore, b t can be rewritten as as can be observed in equation (4), the estimator b t is smaller than the realized p t , because the time delay from onset to death, expressed in the double summation in the numerator, results in the numerator being smaller than the denominator (note that f s is a probability distribution). therefore we refer to b t as the biased estimator of the ccfr: it gives a biased estimate, calculated on day t, of the ccfr [8, 11] . when we observe the entire course of an epidemic (i.e. tr'), b t tends to p t and becomes an unbiased estimator. the aim is to obtain an unbiased estimator ''well before'' observing the entire course of the outbreak. an adjustment of the estimator b t by a factor of underestimation is achieved by rearranging equation (4): we use p t as the unbiased estimator of p, which is informed by three pieces of information: the cumulative number of deaths d t ; the incidence c t ; and the distribution of the time from onset to death f s . the former two are observed during the course of an epidemic. when there are a few deaths or none at all, an assumption has to be made for f s , e.g. from literature based on previous outbreaks (see below for detailed descriptions of f s ). we call the multiplicative factor in equation (4) the factor of underestimation, u t , defined by the estimator p t can be written as p t = b t /u t . figure 1 depicts the concept of the sampling scheme. the cumulative number of cases c t is regarded as the total population size. of these, only a proportion u t has been at risk for dying by time t, whereas the outcome for the remaining proportion 1 -u t is still unobserved. among the u t c t cases that have been at risk, d t have died and u t c t -d t have survived the infection. this is a sample from a binomial distribution with sample size u t c t and probability p: an alternative way of deriving this probability is by first considering the total number, y, of people in the sample c t that will ultimately die from infection, which is binomially distributed with sample size n = c t and probability p. however, because of the time delay from onset to death, we do not observe this outcome by time t: only for a proportion u t is the outcome observed. hence our observation is a hypergeometric sample from a population of size c t , with sample size u t c t , and number of deaths y [15, 16] : which is equivalent to equation (7). we can use equation (7) as a likelihood function to obtain the maximum likelihood estimate of p t : the 95% confidence interval of p t is derived from the profile likelihood. further technical details, especially where an exponential growth of incidence is observed, are given in the supporting information s1. for calculation of the factor of underestimation u t , two pieces of information are needed: the incidence function c t and the distribution of time from onset to death f s . for c t , we use the published dates of onset among confirmed cases, while f s is assumed known. we analyze empirical datasets of two different infectious diseases: sars in hong kong (2003) and s-oiv infection in the usa and canada (2009). first, we examine a simplified version of our method by using only deaths and cases from an early stage of the sars epidemic, and compare our estimate against the eventual stable estimate at the end of the epidemic. for simplicity, we employ an exponential distribution for the distribution of the time from onset to death, f(s), with a mean of 35.9 days [11] , and f s is subsequently calculated as the daily increase in f(s), i.e., f s = f(s)2f(s21). second, we use the most recent published datasets of s-oiv epidemics in which the dates of illness onset for confirmed cases are known [17, 18] . the latest such reports for the usa and canada were at may 1 and june 10, 2009, respectively. in the usa, there were 399 confirmed cases by may 1, with 394 known dates of onset ( figure 2a ). among 399 confirmed cases, 2 cases resulted in death by may 1. in canada, there were 2978 confirmed cases, with 2004 known dates of onset by june 10, among which 4 cases died by june 10 ( figure 2b ). the biased ccfr estimates, b t in these countries were 0.50% ( = 2/399) and 0.13% ( = 4/2978), respectively. the six deaths are insufficient to determine the distribution of time from onset to death for these countries. we therefore employ a gamma distribution for f(s) (to calculate f s ), with reference to historical data for h1n1 [19] , with a mean length of 9 days and a variance of 39.7 days 2 (coefficient of variation 70%, shape parameter 2.04) [20] . to address the uncertainty, we examine the sensitivity of our unbiased ccfr estimate to different means (6-14 days) and variances (9-159 days 2 ). see supporting information s2 for further technical details. for the unbiased ccfr, we use 399 and 2978 cases, respectively, as our c t in equation (9) for the usa and canada. the population and sampling process for estimating the unbiased confirmed case fatality ratio during the course of an outbreak. at time t we know the cumulative number of confirmed cases and deaths, c t and d t , and wish to estimate the unbiased case fatality ratio p, by way of the factor of underestimation u t . if we knew u t we could specify the size of the population no longer at risk (u t c t , shaded), although we do not know which surviving individuals belong to this group. a proportion p of those in the group still at risk (size (1-u t )c t , unshaded) is expected to die. because each case no longer at risk had an independent probability of dying, p, the number of deaths, d t , is a sample from a binomial distribution with n = u t c t , and p t = p. doi:10.1371/journal.pone.0006852.g001 similarly, d t is 2 and 4 deaths, respectively. nevertheless, since the adjustment of underestimation requires dates of symptom onset, we use 394 and 2004 cases for computing u t . although this has little impact on the estimate for the usa, the ccfr in canada is likely to be underestimated by our estimator, because the majority of the 974 cases whose dates of onset have yet to be clarified, may have experienced their symptom onset close to the latest time point of observation. we subsequently compare ccfr estimates between the usa and canada by means of fisher's exact test. for the hypothesis testing, the number of deaths, d t , as well as the number of those survived, calculated as u t c t 2d t , is compared between two countries. the factor of underestimation u during the exponential growth phase is independent of time t and given by where m(-r) is the moment generating-function of f(s), given the exponential growth rate r which is estimated via a pure birth process (see supporting information s3). that is, when f(s) is the density of an exponential distribution with mean t, we have u = m(2r) = 1/(1+rt). figures 3a and 3b show the cumulative numbers of cases and deaths of sars, and figure 3c the observed (biased) ccfr estimates as a function of time, i.e. the ratio of the cumulative number of cases to deaths at time t. due to the delay from onset of symptoms to death, the biased estimate of ccfr at time t underestimates the realized ccfr at the end of an outbreak (i.e. 302/1755 = 17.2 %). nevertheless, even by only using the observed data for the period 19 march to 2 april, equation (10) yields an appropriate prediction (figure 3d ), e.g. the unbiased ccfr at 27 mar is 18.1 % (95% ci: 10.5, 28.1). an overestimation is seen in the very early stages of the epidemic, but the 95% confidence limits in the later stages include the realized ccfr (i.e. 17.2 %). when only a few deaths have been reported at the early stage of an epidemic, the unbiased ccfr estimate is given by minimizing the negative logarithm of the likelihood (see equation (9)). given 2 and 4 deaths in the usa and canada, respectively, and employing a gamma-distributed time from onset-to-death, the unbiased estimates of the ccfr are 1.23% (95% confidence interval (ci): 0.21, 3.76 %) and 0.18% (95% ci: 0.05, 0.41%) in the usa and canada, respectively. the estimate in the usa appears significantly higher than that in canada (fisher's exact test; p,0.01). the uncertainty bounds on the unbiased ccfr estimates in both countries overlap with that estimated for mexico [1] . sensitivity analysis suggests that the expected values may lie in the range of 0.81-4.48% and 0.16-0.22% in the usa and canada, respectively ( figure 4 ). even when there has been no observation of death by time t, it would be useful for policy makers to understand the implication of no deaths for interpreting virulence in a conservative way. when d t = 0 equation (7) simplifies to: which would result in an unbiased ccfr estimate of 0. because sampling a finite number of cases during the course of an outbreak cannot prove that infection never results in death, a more useful result would be the maximum ccfr with a certain level of confidence if no deaths are observed after c t cases. to obtain this result, we rearrange equation (11) to obtain where p max is the maximum ccfr given c t cases and no deaths, at a confidence level of 1-a, e.g. 95% if a = 0.05. equation (12) is useful for obtaining a conservative estimate of virulence (i.e. upper bound of possible ccfr estimates) when no deaths have been reported by time t. in particular, during the early exponential growth phase the factor of underestimation, u, is independent of t. assuming that the exponential growth phase of influenza continued until april 21 and 24, 2009, respectively, in the usa and canada, r in these countries is estimated at 0.183 (95% ci: 0.133, 0.245) per day and 0.300 (95% ci: 0.241, 0.367) per day, it should be noted that confirmed cases include substantial numbers of imported cases from abroad. in canada, a few cases whose dates of onset were unable to be traced are also included according to their dates when a specimen was collected (the exact number of such cases is not known). assuming that their impact on our estimation procedure is negligibly small, we regard all cases in b as representing the dates of onset. doi:10.1371/journal.pone.0006852.g002 respectively (see supporting information s3). the resulting p max in the usa and canada (based on 42 and 91 cases and no deaths) is shown in figure 5 . these upper bounds are examined for confidence levels at 95% and 99%. if the mean and variance of the time from onset to death are 9 days and 39.7 days 2 , and we employ a gamma distribution, p max is estimated at 21.2% and 30.7% at a = 0.05 and 0.01 in the usa. similarly, p max in canada is estimated at 16.8% and 24.6% at a = 0.05 and 0.01, respectively. we propose a new epidemiological method for assessing the virulence of an emerging infectious disease at the early stage of an epidemic. the results with the hong kong sars dataset prove the usefulness of this method that corrects the biased ccfr estimator which is simply the ratio of cumulative deaths to cases. early in the epidemic, the ultimately realized ccfr is within the confidence interval obtained by our method. the proposed method is particularly useful when an epidemic curve of confirmed cases is the only data available (i.e. when individual data from onset to death are not available, especially, during the early stage of the epidemic). our estimates suggest that the virulence of s-oiv h1n1 infection is comparable to the virulence observed in past influenza pandemics of the 20th century (,2.0 % for the 1918-19 pandemic and,0.5 % for the 1957-58 pandemic [21] ). although our estimates may not be as high as 2.0%, and even though the unbiased ccfr estimate for the usa is a likely overestimation (see below), we should emphasize that antiviral treatment and other medical interventions have been instituted from the beginning of this pandemic. our results show that the few observations of death in the usa and canada give us no reason to believe that the unbiased ccfr, and therefore the virulence of the novel pandemic strain, is smaller in the usa and canada than in mexico. nevertheless, given that the cfr of seasonal influenza is equal to or less than 0.1% [10] , our estimates (with the lower bound of ccfr close to the 0.1%) do not offer conclusive results to indicate that the s-oiv is more virulent than seasonal influenza, but do point in that direction. it should be noted that our method only adjusts underestimation due to time delay from onset to death, and other epidemiological characteristics associated with unbiased estimation of the ccfr have yet to be addressed. in the present study, we estimated the ccfr as the proportion of deaths among confirmed cases. this definition was chosen, because of our aim to use the minimally available data, and so we were not able to estimate the proportion of deaths among all symptomatic cases, and not able to estimate the proportion of deaths among all those infected (symptomatic and asymptomatic). the issue of defining the correct denominator population can never be completely resolved, but it is essential to realize how the obtained estimate relates to other situations [8] . by only using confirmed cases, it is clear that all cases will be missed that do not seek medical treatment or are not notified, as well as all cases that are asymptomatic. this means that our ccfr estimate is higher than the proportion of deaths among infecteds, and may be considered an overestimate. however, when relating our estimate to previous pandemics, it should also be realized that the current pandemic is the first where many confirmatory diagnoses of influenza have been recorded using rt-pcr techniques, allowing improved precision of ccfr estimates over those for previous influenza epidemics. whereas the use of rt-pcr in the current pandemic may yield a smaller denominator (and thus an overestimate of cfr compared to previous pandemics), other pandemics could have involved substantial numbers of falsepositive cases in the denominator. developing a method which permits comparable assessment of virulence is ongoing. the comparisons between the realized ccfr (horizontal grey line), the unbiased ccfrs based on observations by calendar time t, and the biased ccfr estimates, b t , given by the ratio of deaths to cases. each prediction was obtained by using the exponential growth rate r up to time t and the cumulative numbers of deaths and cases at time t, and the mean time from onset-to-death of 35.9 days [11] which is assumed to follow an exponential distribution. overestimation is seen in the early stages of the epidemic, but the 95% confidence limits in the later stages include the realized ccfr. doi:10.1371/journal.pone.0006852.g003 figure 6 shows the time course of biased ccfr estimates in the usa and canada based on the reporting date of confirmed cases and deaths to the world health organization. note that the estimates in figure 6c are different from our b t due to unavailability of the date of onset, although they give an approximate indication of the time-course of the biased ccfr. it is striking to see that the biased ccfr during the very early stage (i.e. from late april to mid-may) showed a declining trend following a single spike. the biased ccfr estimates at later time points show a slight increase as a function of time, which is consistent with our knowledge of underestimation of the ccfr [8] . the early spike may be explained by a time-varying coverage of confirmed diagnoses which could have increased as a function of time (i.e. cases in the very beginning of the epidemic were less likely to be confirmed). other plausible explanations include (1) demographic stochasticity, (2) effective treatment, and (3) heterogeneous risk of death among subpopulations. as for (1), because the number of deaths in the usa and canada was very small during the early stage, the spike may reflect (unpredictable) probabilistic variations in the number of deaths among a small number of confirmed cases. if that is the case, our unbiased ccfr estimate for the usa (with data until may 1) may be too high, not because of a systematic bias but just by chance. in relation to factor (2), it is plausible that cases diagnosed in later stages of the epidemic receive treatment at an early stage of illness (or even before symptom onset). with respect to (3), the risk of dying is likely to be different for different subpopulations [8, 10, 22, 23] . it should be noted that the composition of sub-populations (e.g. agegroups and those with a specific underlying disease) is likely to vary as a function of time, and a ccfr estimate for the entire population, such as ours, is influenced by this variation. these points need to be addressed in future studies. to fully clarify the virulence and its epidemiological characteristics (e.g. variable risks by age and underlying diseases), two lessons for surveillance and data sharing should be noted. first, rather than updating the data based on date of reporting, it is critically important to summarize the data according to the date of onset both at local and global levels. knowing the date of symptom onset is a key to applying our proposed estimation framework to empirical observation. second, epidemiological data should be updated in a precise reporting interval at least during the early stage of an epidemic (so that the data permit estimation of the unbiased ccfr). given that mean time from onset to death is around 9 days, weekly data do not enable us to make our explicit adjustment. optimal reporting for the early ccfr estimation may be incorporated into official pandemic response plans. moreover, in addition to using death as an outcome of virulence, the usefulness of other epidemiological measurements of severe manifestation (e.g. the number of admissions to intensive care unit) needs to be explored. despite a need to further clarify heterogeneous risks of death for the s-oiv pandemic, early assessment of virulence by means of our unbiased ccfr estimator is useful for informing policy makers and the general public about the potential severity of an infectious disease (of course, one needs to ensure an understanding of the above mentioned bias among non-experts). we have shown that underestimation can be adjusted in a very simple manner, and our approach enabled us to obtain an unbiased ccfr estimate by only minimizing a binomial deviance. these methods are particularly useful when there have been only a few deaths or even no death at all by time t during the course of an epidemic. uncertainties surrounding the unbiased estimate of ccfr based on a few deaths can partly be addressed by sensitivity analysis of the estimate to different lengths of time from onset to death. an observation of zero deaths in a given country (or a specific setting) should not be deemed a signature of a ''benign'' virus without observing a substantial number of cases. we have shown that a conservative upper bound of ccfr is a more useful interpretation of the observed number of cases without death. in this way, given that we have some prior knowledge or a few observations of death which permit us to assume f(s) is known, epidemiologists and biostatisticians in each country or locality can directly apply our method to assess the virulence of an infection at the early stage of any emerging infectious disease. during the final stages of revision, it came to our attention that an epidemiological study on ccfr of s-oiv with similar techniques and statistical philosophy has been published online [24] , indicating that the preliminary estimate of ccfr for a combination of the usa, canada and mexico is 0.5% and emphasizing a need to accurately capture the cases for the denominator. supporting information s1 given by the ratio of deaths per confirmed cases. the data were extracted from irregular situation updates of the world health organization [25] , and the horizontal axis (time) corresponds to the date of reporting. therefore, it should be noted that the estimate suffers reporting delay, and in this sense, the calculated biased ccfr is different from our b t (based on date of onset) in the main text. the most recent report was made on june 19. since the interval of update has been irregular, the cumulative number of cases and deaths is kept the same as the latest report when there was no update on the corresponding date. doi:10.1371/journal.pone.0006852.g006 pandemic potential of a strain of influenza a (h1n1): early findings how severe will the flu outbreak be? swine flu goes global mathematical epidemiology of infectious diseases: model building, analysis and interpretation transmission potential of the new influenza a(h1n1) virus and its age-specificity in japan pathogenicity of highly pathogenic avian influenza virus in mammals epidemic science in real time methods for estimating the case fatality ratio for a novel, emerging infectious disease severe acute respiratory syndrome: temporal stability and geographic variation in case-fatality rates and doubling times managing and reducing uncertainty in an emerging influenza pandemic epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong non-parametric estimation of the case fatality ratio with competing risks data: an application to severe acute respiratory syndrome (sars) mexico's mystery: why is swine flu deadlier there? case fatality proportion strategies and statistics of sampling for rare individuals a new probability formula for surveys to substantiate freedom from disease emergence of a novel swine-origin influenza a (h1n1) virus in humans available at: www deaths from bacterial pneumonia during 1918-19 influenza pandemic time variations in the transmissibility of pandemic influenza in prussia textbook of influenza update: novel influenza a (h1n1) virus infections -worldwide serum cross-reactivity antibody response to a novel influenza a (h1n1) virus after vaccination with seasonal influenza vaccine assessing the severity of the novel influenza a/h1n1 pandemic epidemic and pandemic alert and response (epr), world health organization (2009) influenza a(h1n1) key: cord-262345-hti1jjpn authors: eddy, lucy h.; bingham, daniel d.; crossley, kirsty l.; shahid, nishaat f.; ellingham-khan, marsha; otteslev, ava; figueredo, natalie s.; mon-williams, mark; hill, liam j. b. title: the validity and reliability of observational assessment tools available to measure fundamental movement skills in school-age children: a systematic review date: 2020-08-25 journal: plos one doi: 10.1371/journal.pone.0237919 sha: doc_id: 262345 cord_uid: hti1jjpn background: fundamental movement skills (fms) play a critical role in ontogenesis. many children have insufficient fms, highlighting the need for universal screening in schools. there are many observational fms assessment tools, but their psychometric properties are not readily accessible. a systematic review was therefore undertaken to compile evidence of the validity and reliability of observational fms assessments, to evaluate their suitability for screening. methods: a pre-search of ‘fundamental movement skills’ or ‘fundamental motor skills’ in seven online databases (pubmed, ovid medline, ovid embase, ebsco cinahl, ebsco sportdiscus, ovid psycinfo and web of science) identified 24 assessment tools for school-aged children that: (i) assess fms; (ii) measure actual motor competence and (iii) evaluate performance on a standard battery of tasks. studies were subsequently identified that: (a) used these tools; (b) quantified validity or reliability and (c) sampled school-aged children. study quality was assessed using consensus-based standards for the selection of health measurement instruments (cosmin) checklists. results: ninety studies were included following the screening of 1863 articles. twenty-one assessment tools had limited or no evidence to support their psychometric properties. the test of gross motor development (tgmd, n = 34) and the movement assessment battery for children (mabc, n = 37) were the most researched tools. studies consistently reported good evidence for validity, reliability for the tgmd, whilst only 64% of studies reported similarly promising results for the mabc. twelve studies found good evidence for the reliability and validity of the bruininks-oseretsky test of motor proficiency but poor study quality appeared to inflate results. considering all assessment tools, those with promising psychometric properties often measured limited aspects of validity/reliability, and/or had limited feasibility for large scale deployment in a school-setting. conclusion: there is insufficient evidence to justify the use of any observational fms assessment tools for universal screening in schools, in their current form. introduction primary care facilities [21] . it follows that children from a lower ses are less likely be identified as needing extra support with fms development under current service provision, and therefore less likely to be offered intervention (at least within the uk). universal fms screening in primary schools would provide a more equitable approach to identifying those children in greatest need of support. there are currently a large number of assessment tools used to measure fms both clinically, and for research purposes. a large proportion of these assessment tools rely on an assessor observing children perform fms on a battery of standardised tasks. standardised observational measures are considered a useful way to assess children's fms in schools [22] as they are reasonably low cost (relative to objective wearable sensors), have minimal data entry and analysis requirements for schools, and are also less susceptible to bias than proxy reports [23] . there are a large number of observational assessment methods being marketed to schools [22] . the saturation of such measures makes it difficult for teachers, practitioners, and researchers to know which assessment is best suited to identify accurately children who are struggling with fms development. this evaluation is particularly challenging as there is a lack of clarity in the literature regarding the validity and reliability of the available observational measures. a systematic review was required to document the psychometric properties of the observational assessment tools being promoted as measures of fms to allow schools and health practitioners to make informed decisions about fms assessment tools. this systematic review aims to: (i) establish a comprehensive summary of the observational tools currently used to measure fms that have been subjected to scientific peer-review; (ii) examine and report the validity and reliability of such assessments. methods for this systematic review were registered on prospero (crd42019121029). a preliminary search was conducted to identify assessment tools that were identified in peerreview published research as measures of fms in school-aged children. this pre-search was conducted in the seven electronic databases (pubmed, medline, embase, cinahl, sportdiscus, psycinfo and web of science) in december 2018, and was subsequently updated in may 2020, using the search terms 'fundamental movement skills' or 'fundamental motor skills'. assessment tools identified in this pre-search were included in the subsequent review if they were confirmed to: (i) assess fundamental movement skills, including locomotor, object control and/or stability skills [24] ; (ii) observationally measure actual fms competence (i.e. physical, observable abilities); (iii) assess children on a standard battery of tasks which were completed in the presence of an assessor. proxy reports and assessments that measured perceived motor competence were therefore excluded from the review. no restrictions were placed on the health/ development of included participants, as schools are faced with these issues, so any assessment tool that is going to be used in an educational setting would need to be appropriate for use with children both with and without developmental difficulties. the titles and abstracts of the results of this pre-search were screened by the lead reviewer (lhe) to identify assessment tools mentioned within them that were being used to assess fms. any studies stating they were assessing fms but omitting mention of the specific assessment tool in the title or abstract underwent a further full text review. the search strategy developed (see s1 table) was applied in seven electronic databases (pubmed, medline, embase, cinahl, sportdiscus, psycinfo and web of science) in january 2019, and was then updated in may 2020. conference abstracts identified were followed up by searching for the full articles or contacting authors to clarify whether the work had been published. for the initial search (dec 2018), titles and abstracts were screened in their entirety by one reviewer (lhe), and two reviewers (nfs & klc) independently assessed half of these studies each. the same process was followed for full text screening to identify eligible studies. reviewers were not blind to author or journal information and disagreement between reviewers was resolved through consultation with a fourth reviewer (ddb). for the update, the same process was repeated with two different reviewers (me-k & nsf, in place of nfs & klc). three reviewers each extracted information from a third of the studies in the review in both the initial search (lhe, klc & nfs) and the update (me-k, ao & nsf). data extraction and an assessment of the methodological quality of each study were completed using the consensusbased standards for the selection of health measurement instruments (cosmin) checklist [25] , which outlines guidance for the reporting of the psychometric properties of health-related assessment tools. information was extracted on: (i) author details and publication date; (ii) sample size and demographic information related to the sample; (iii) the assessment tool(s) used; (iv) the types of psychometric properties measured by each study; (v) the statistical analyses used to quantify validity or reliability and whether they were measured using classical test theory (ctt) or item-response theory (irt); (vi) the statistical findings. methodological quality ratings for each study were recorded as the percentage of the standards met for the included psychometric properties and generalisability. when an irt method was used, a second quality percentage was calculated, based on the cosmin guidelines for irt models [25] . the lead reviewer (lhe) and a second reviewer (ao) each evaluated half of the studies for methodological quality, with a 10% cross-over to ensure agreement. agreement was 100%, so no arbitration was necessary. many studies used different terminologies to describe the same type of validity or reliability, so it was necessary to set a definition for each psychometric property and categorise study outcomes in accordance to the cosmin checklist [25] (see table 1 ). interpretability and face validity (sub-section of content validity) were not included as these could not be quantified using statistical techniques. responsiveness was not included, as this is recognised as being separate to validity or reliability within the cosmin guidance. due to a large variation in the statistical tests used to assess validity and reliability, a meta-analysis was not possible. to enable ease of interpretation of studies that utilised statistical analyses, a traffic light system was used (poor, moderate, good and excellent; see table 2 ), which allowed such results to be grouped into different bands according to thresholds for these statistical values suggested in previous research. the results of all outcomes which utilised other statistical tests are described in the text. for the studies that included multiple metrics for each psychometric property, the traffic light colour used to represent each type of validity or reliability in subsequent tables is a reflection of the mean value of specific fms related task scores, or subtest scores, as appropriate. a full breakdown of results for each study can be found in s2 table. the pre-search identified 33 possible fms assessment tools of which three were removed for not meeting criteria 1. inter-rater reliability the level of agreement between different assessors' scores of children on an assessment tool. how consistent an assessor is at scoring children using an assessment tool. test-retest reliability the stability of the children's scores on an assessment tool over a minimum of two time points. internal consistency the level of agreement between items within an assessment tool. the extent to which an assessment is representative of the components/facets it was designed to measure. structural validity the degree to which an assessment tool measures what it was designed to measure. the degree to which an assessment tool and its' normative data can be used to assess fms in countries other than the one it was designed in. the degree to which scores on assessments are consistent with hypotheses made by authors (e.g. internal relationships between subscales, relationships to scores of other assessment tools or differences between relevant groups. the level of agreement between two assessment tools. the degree to which performance on an assessment tool can be used to predict performance on another measure, tested at a later date. https://doi.org/10.1371/journal.pone.0237919.t001 no information could be found explaining the assessment tool, and authors either did not respond to queries, or no contact information could be found for the author. this left 24 assessment tools for inclusion in the systematic review, which reviewed studies if they: (i) used assessment tool(s) identified in the pre-search; (ii) measured validity or reliability quantitatively; (iii) sampled children old enough to be in compulsory education within their country. studies were not excluded based on sample health or motor competence. concurrent validity was only examined between the 24 assessment tools identified in the pre-search. electronic searches initially identified 3749 articles for review. fig 1 demonstrates the review process which resulted in 90 studies being selected (for study table see s2 table) . included articles explored the validity and/or reliability of sixteen of the assessment tools identified in the pre-search. the search did not identify any articles for the remaining eight assessment tools (see table 3 ), so the reliability and validity of these measures could not be evaluated in this review. only nine of the assessment tools identified in the pre-search assess all three components of fms: locomotion, object control and balance the included studies recruited a total of 51,408 participants aged between three and seventeen years of age, with sample sizes that ranged from 9 to 5210 ( [58] . eight defined themselves as sampling children with learning and/or attentional problems [54, [59] [60] [61] [62] [63] [64] [65] , three studies recruited children with visual impairments [66] [67] [68] , and the sample of one study included children with a disability or chronic health condition [69] . information regarding socioeconomic status (ses) was included in one article which stated they sampled from low ses [70] , while two studies recruited samples from indigenous populations (in australia and canada, respectively) [44, 71], the latter of which focused on the recruitment of children whose mothers drank alcohol during pregnancy [71] . cosmin quality assessment fig 3 shows the results of the generalisability subscale of the quality assessment for the included studies. the cosmin checklist [25] revealed multiple issues with reporting in the included studies, with 85% of studies not providing enough information to make a judgement about missing responses, and 76% of studies failing to report the language with which the assessment tool was conducted. additionally, over a third of the studies included in this review did not adequately describe the method of recruiting participants, the age of participants, or the setting in which testing was conducted. observational assessment methods were defined categorically as either assessing fms using a "process" or "product-oriented" methodology [97] . process-oriented measures require decisions to be made as to whether children are meeting specific performance criteria whilst completing skills (e.g. when running, is the non-support leg is bent at a ninety degree angle?). product-oriented assessments focus on the outcome of movements (e.g. how quickly can a child can complete a movement?). given these two different approaches to measuring fms, which can used for different purposes in the literature, they were distinguished for this review. of the 24 assessment tools identified, nine were product-oriented, thirteen were process-oriented, and two assessment tools included both process and product methodologies (see table 3 ). despite the pre-search identifying nine product-oriented assessments in the fms literature, the systematic review only identified research on the validity and reliability of six of these measures (described below twenty-three studies evaluated the validity and/or reliability of the mabc or mabc-2. all of the ten cosmin categories this review focused on (see table 1 ), were evaluated for the mabc. overall there was strong evidence for inter-rater reliability for these assessments (table 4 ). however, there were more mixed results for other aspects of validity and reliability, with the weakest evidence being found in support for internal consistency. intra-rater reliability was only looked at in two studies [83, 120] with poor intra-rater reliability (icc = .49 for both the balance and aiming and catching subtest) demonstrated in the study exploring this construct in norwegian children [120] . there was good evidence for test-retest reliability, with only one out of five studies in a sample of teenagers [121] finding moderate correlations (mean icc for fms skills = .74). an adapted version of the mabc-2 was also tested (e.g. increasing the colour contrast on the ball), with results showing that the modified version was a reliable assessment tool for use with children with low vision (inter-rater reliability-icc = .97; test-retest reliability-icc = .96; internal consistency-cronbach's alpha ranged from 0.790 to 0.868) [66] . strong evidence for content validity was found for both the brazilian [83] and the chinese [122] versions of the assessment tool, with concordance rates amongst experts ranging from 71.8%-99.2%. additionally, one study found that children with asperger syndrome perform worse on all three subtests of the mabc than typically developing children, as hypothesised [57]. cross-cultural validity was studied in four papers, looking at swedish, spanish, italian, dutch and japanese samples in comparison to us or uk norms [88, [127] [128] [129] . results showed that uk norms were not suitable for use to evaluate the performance of italian children, as significant differences were found for eleven of the twenty seven items on the mabc-2 [129] . differences were also found between the performance of uk children and dutch children, however these differences were not statistically significant. the us standardised sample was found to be valid for a swedish sample [127] , but not for a spanish sample, for which us norms left a large proportion of the sample below the 15 th percentile [128] . structural validity was assessed by ten studies, with six finding evidence for a three factor (manual dexterity, aiming & catching and balance) model [78, 122, 126, [129] [130] [131] . one study darsaklis et al. [96] holm et al. [120] hua et al. [122] jaikaew et al. [125] kita et al. [126] valentini et al. the psychometric properties of observational assessments of fundamental movement skills for school children found a four factor solution, with a general factor for age band 1, four factors with balance split into static and dynamic for age band 2, and a 3 factor correlated model for age band 3 [132] . similarly, another study found evidence for a bifactor model with one general factor, and three sub-factors for age band one [81] . evidence was also found for a five factor solution, with balance and manual dexterity each split into two factors [124] . an adolescent study found a two factor model (manual dexterity and aiming and catching) was more appropriate as ceiling effects were evident on balance tasks [133] . the results of the cosmin quality assessment of mabc studies show that two studies which found excellent results, had the lowest quality ratings, in which they met 13% and 29% of generalisability and inter-rater reliability criteria respectively [96, 125] . additionally, the singular study which found mabc normative data to be valid in another country only had a quality rating of 39% [127] . the mabc study with the best quality rating (81% of criteria met), only found moderate results for internal consistency [126] , and the single study which found that mabc norms data are cross-culturally valid, only had a quality rating of 39%. when considering cosmin quality ratings alongside the results of these studies, it would suggest that caution should be taken when interpreting the results of studies exploring the psychometric properties of the mabc. twelve studies stated that they explored the validity and reliability of the bot, bot-2 or bot-2 short form (sf), of which six reported results that could be quantified into poor, moderate, good and excellent evidence, which are detailed in table 5 . three studies looked at the inter-rater reliability of the bot, all of which found good evidence in support of this aspect of reliability [54, 71, 96] , however one of these studies provided no information about the sample, including size and demographic information [96] . the results for test-retest reliability were more mixed than for the mabc, with the two studies finding low correlations on scores between tests sampling from children with cerebral palsy (icc = .4) [52] and children living in aboriginal communities in australia (mean icc for fms = .097) [71] . one study did show evidence of the bot being a reliable measure of fms in children with intellectual deficits [65] . one study explored the cross-cultural validity of the bot-2 norm scores with a large brazilian sample (n = 931) and found mixed results [79] . results showed that brazilian children outperformed the bot normative data on bilateral coordination, balance, upper-limb coordination, and running speed and agility subtests, but similar percentile curves were found for both populations on upper limb coordination and balance subtests [79] . five studies explored the structural validity of the bot. the bot-2 sf was also found to have good structural validity once mis-fitting items were removed for children aged 6-8 years, but ceiling effects were found for older children (aged 9-11 years) [134] . two studies exploring lucas et al. structural validity found good evidence utilising rasch analysis, with results indicative of unidimensionality, with the overarching factor accounting for 99.8% [64] and 82.9% [73] of the variance in test scores for children with intellectual deficits (bot), and typically developing children (bot-bf), respectively. similarly to the results of the rasch studies, one additional study found that the four subscales were correlated, so a bifactor model, with an overarching motor skill factor, and four correlated sub-factors [81] . when the subscales and composite scales were evaluated separately using rasch analysis, one study found multiple issues with fine motor integration, bilateral coordination, balance and body coordination which limit the justification of their use including multi-dimensional scales, items working differently for males and females, disordered item difficulty ratings, and/or the ability of the subscale/ composite score to differentiate between abilities [135] . the quality of the studies evaluating the validity and reliability of the bot may have influenced the results though, as the study with the greatest quality rating (83%) found good results for inter-rater reliability [71] , but two studies with lower ratings (13% [96] and 53% [54]) reported excellent results for this psychometric property, suggesting that reliability scores may have been inflated by poorer quality studies. additionally, the reviewed bot studies only evaluated seven of the ten cosmin categories (see table 3 ). other product-oriented assessment tools. three studies evaluated the validity and reliability of the körperkoordinationstest für kinder (ktk) [77, 80, 136] . two studies looked at the structural validity of the ktk, and found adequate evidence to support a one factor structure, interpreted as representing "body coordination" [77, 80]. the internal consistency of the ktk was consistently found to be good across samples in finland, portugal and belgium (α ranged from .78 -.83), however, as hypothesised there were significant differences between groups, in which children from portugal and belgium performed worse than finnish participants [136] . additionally, there was evidence of high inter-rater reliability (94% agreement) [77] . two studies evaluated the validity and reliability of the athletic skills track (ast) [98, 137] . the results of both studies suggest that the ast has good test-retest reliability with intraclass correlations ranging from .8 [137] to .88 [98] . cronbach's alpha was used in one of these studies to examine internal consistency, with results ranging from .7-.76 for the three versions of the ast [137] . it is, however, important to note that only two psychometric properties from the cosmin checklist [25] were evaluated, and the quality ratings for these studies were lower than 60%. the psychometric properties of the fms polygon were tested in one study [138] , finding strong evidence for intra-rater reliability (icc = .98). factor analysis also explored the structure of the assessment tool, revealing four factors: object control (tossing and catching a volleyball), surmounting obstacles (running across obstacles), resistance overcoming obstacles (carrying a medicine ball) and space covering skills (straight running). these psychometric properties of the fms polygon, should however, be interpreted with caution, as the above study only had a quality rating of 43% [138] . the structural validity of the mot 4-6 was evaluated by one study with a high quality rating (79%) using rasch analysis, which established four of the items had disordered thresholds and needed to be removed from the assessment (grasping a tissue with a toe, catching a tennis ring, rolling sideways over the floor and twist jump in/out of a hoop). results also showed that with one additional item removed (jumping on one leg into a hoop), there was an acceptable global model fit for the mot 4-6 [139] . thirteen process-oriented assessment tools were identified by the pre-search as measuring fms. of these, seven had been evaluated for validity and reliability (described below). no research was found evaluating the psychometric properties of the: children's motor skills protocol (cmsp) [99] , instrument for the evaluation of fundamental movement patterns [36], objectives-based motor-skill assessment instrument [109] , ohio state university scale for intra-gross motor assessment (osu-sigma) [110] , preschooler gross motor quality scale (pgmq) [46] and smart start [115] . test of gross motor development (tgmd). the results of twenty-one studies which evaluated the psychometric properties of various versions of tgmd can be found in table 6 . nine out of ten cosmin psychometric properties were evaluated by tgmd studies. consistently good evidence for inter-rater and intra-rater reliability was observed, with only one study finding less than 'good' (moderate) correlations when testing sessions were video recorded [140] . one study evaluated these aspects of reliability using a content validity index (cvi) and found good evidence for both inter and intra-rater reliability when testing chilean children, with cvis ranging from .86 to .91 [141] . an additional study evaluated the inter and intra-rater reliability of the tgmd second and third editions using percentage agreement [69] . results showed agreement for inter-rater reliability was 88% and 87% for the tgmd-2 and tgmd-3 respectively, and for intra-rater reliability the percentage agreement was 98% for the tgmd-2 and 95% for the tgmd-3 [69] . fewer studies examined the test-retest reliability of the tgmd, but those that did demonstrated that for the tgmd-2 [ barnett et al. [72] capio et al. [59] garn & webster [147] houwen et al. [68] issartel et al. [142] kim et al. [143] lopes et al. [146] simons et al. [63] valentini et al. [82] ward et al. [148] valentini et al. [84] brian et al. [67] estevan et al. [149] maeng et al. [150] magistro et al. [151] rintala et al. [140] valentini et al. [85] wagner et al. [144] webster & ulrich [145] nb the psychometric properties of observational assessments of fundamental movement skills for school children retest reliability was evidenced with a cvi of .88 [141] and bland altmann plots found 95% confidence intervals were within one standard deviation [77], with .96 agreement ratio [146] . evidence for internal consistency was more mixed, but there was strong evidence that all items in the tgmd-3, once modified for children with asd and visual impairments could still measure fms as an overarching construct [56, 67] . evidence for good internal consistency of the tgmd was also found when testing children with intellectual deficits [59] . sixteen studies evaluated the structure of the items within various editions of the tgmd, consistently finding a two factor model (locomotion and object control) for the tgmd [152] , tgmd-2 [59, 63, 68, 77, 82, 142, 143, 146, 147] , tgmd-2 sf [84] and tgmd-3 [85, 144, 145, 149, 151] , as predicted by multiple studies [59, 146, 149, 152] . it is, however, important to note that some of these models enabled cross-loading of items [e.g. 147], some models were hierarchical in nature [77] and in one case a two factor model, whilst best fit, explained only 50% of the total variance [142] . evidence was however found to suggest that the structural validity of the tgmd is stable across countries, with the data from populations in greece, brazil, germany, the usa, south korea and portugal all evidencing a two factor model [67, 82, 143, 144, 146, 152] . the content validity of the brazilian translation of the tgmd-2 and tgmd-3 was evaluated by two studies, with stronger evidence for the validity of the tgmd-2 (cvi = .93 for clarity and .91 for pertinence) than the tgmd-3 for which the cvi for the clarity of the instructions only reached .78 [82, 85] . the spanish translation of the tgmd-2 was also tested for clarity and pertinence, with results finding a cvi of .83 [141] . cross cultural validity was investigated in one study that compared flemish children with intellectual deficits to us normative data [63] , which found significant differences, with large effect sizes (1.22-1.57), indicating us standardised data was inappropriate for use as a comparison within this population. additionally, a large study based in belgium hypothesised that belgian children would perform similarly to us norms on locomotor scores, but that belgian children would score lower on object control tasks, however, belgian children had significantly worse gmq, locomotor and object control scores, thus showing that us normative data was not appropriate for this sample [153] . the cosmin quality rating of tgmd studies did not appear to effect results, as the relative quality ratings of all studies that found excellent results only varied by 16% [56, 59, 61, 63, 68, 72, 82, 84, 85, 144] (54-70%). however, predictive validity was not explored by the included tgmd studies. other process-oriented assessment tools. the psychometric properties of the fg-compass [102] were evaluated in one study, in which expert scores were compared to undergraduate student scores [154] . results showed kappa values ranging from .51-.89, with moderate levels of agreement on average (m = .71). playbasic was found to have good inter-rater reliability (mean icc = .86), and moderate internal consistency (mean α = .605) in one study [44] . two studies evaluated playfun, finding good to excellent inter-rater reliability (icc ranged from .78 -.98) and good internal consistency (average α = .78) [44, 91] . additionally, hypotheses testing validity and structural validity were assessed, with performance increasing with age as hypothesised, and an acceptable model fit for the proposed five factor structure [91] . despite the quality ratings of these studies varying, (43% and 76%), the higher quality study found the more promising results [91] . one study evaluated the psychometric properties of the teen risk screen [48], with results demonstrating good evidence for the internal consistency (mean α = .75) and test-retest reliability (mean r = .64) of subscales. confirmatory factor analysis (cfa) was used to evaluate the structural validity of the teen risk screen, however, the analysis was not completed on the model they proposed (6 subscales). authors claimed that due to small sample sizes, only three of the six subscales were evaluated separately, and the final three were grouped together. as this analysis did not measure the intended model, results are not detailed in this review. get skilled get active (gsga), the peabody developmental motor scales (pdms-2) and the victorian fms assessment were all used in concurrent validity studies, however, no articles were found evaluating any other aspects of validity and reliability of these measures. two assessment tools from the pre-search measure both product-and process-orientated aspects of movement: canadian agility and movement skill assessment (camsa) [92] and pe metrics [113, 114] . there is limited evidence for the reliability of the camsa with one study finding moderate effect sizes for inter-rater, intra-rater and test-retest reliability, as well as internal consistency [92] . one other study found strong evidence for the test-retest reliability of the camsa [74], however that study had a lower quality rating (49% compared to 77%). one study evaluated the structural validity of pe metrics using rasch analysis and found good evidence that all of the items were measuring the same overarching set of motor skills [155] . it is, however, necessary to interpret this result with caution, as the cosmin quality rating for this study was only 43%. limited evidence was found for concurrent validity across the 23 assessment tools included in the review (see table 7 ). a large proportion of the studies exploring this aspect of validity did so against either the mabc (15 studies) or the tgmd (10 studies). between product-oriented. the findings of studies exploring the concurrent validity of product-oriented assessment tools mostly yielded good results, with only three out of thirteen studies finding less than good evidence for correlations between measures. of these three studies, one found a poor correlation (kappa = .43) between the mabc and the bot [60] , and two studies found moderate correlations between the mabc and the short form of the bot [93] , as well the ast and the ktk, as hypothesised [137] . two studies evaluated the concurrent validity of the bot-2 complete form, and the bot-2 short form [62, 156] . one found poor correlations between subtests (r ranged from .08 -.45) [156] , and the other reported moderate correlations between tasks in a sample of children with adhd (r ranged from .12 -.98) [62] . a modified version of the ktk (with hopping for height removed) was also compared to the standard ktk, which was found to have high levels of validity [89]. one study used pearson table 7 . concurrent validity of assessment tools. the psychometric properties of observational assessments of fundamental movement skills for school children correlations to evaluate the concurrent validity between the mot 4-6 and the ktk, with results showing moderate correlations for children aged 5-6 (mean r = .63), as was hypothesised prior to testing (r >. 6 ). in addition to the results detailed in table 6 , one study looked at the concurrent validity of assessing children using the mabc in person and via tele-rehabilitation software, with results showing no significant difference between scores, as hypothesised [76] . as well as this, the mabc and the bot-sf had a positive predictive value of .88, with twenty one out of twenty four children testing positively for motor coordination problems also scoring below the fifteenth percentile on the mabc [90]. between process-oriented. one study utilised the tgmd to explore the concurrent validity of the gsga assessment tool [97] . significant differences were found between the number of children who were classified as mastering fms versus those who had not, in which gsga was more sensitive and classified a greater number of children as exhibiting non-mastery [97] . three studies also explored the relationship between multiple versions of the tgmd. results revealed that children with asd perform better on the tgmd-3 with visual aids compared to the standard assessments [56] . similarly, modified versions of the tgmd-2 and tgmd-3 were both found to be valid for use in children with visual deficits [67] . additionally, one study showed significant differences between subtest scores on the second and third editions of the tgmd across year groups and gender, in which participants performed better on the tgmd-2 [69] . between product-and process-orientated. the results comparing process and productoriented assessment tools against each other were also mixed, particularly with regards to the concurrent validity between the mabc and the tgmd, for which correlations ranged from .27-. 65 [53, 68, 82, 83, 157] . study quality did not appear to have an effect on the size of the correlation between the mabc and the tgmd. two studies also reported significant differences in level of agreement on percentile ranks [53, 157] . the ktk and the tgmd-2 also differed significantly in terms of their classifications of children into percentile ranks [70] . the concurrent validity of the camsa and both the playbasic and playfun assessment tools were assessed by one study, which found moderate correlations between camsa and both play assessment tools, smaller than was hypothesised [44] . lastly, good cross-product/process concurrent validity was reported between the mabc and the pdms [122] , as well as the camsa and the victorian fms assessment tool [74] and the tgmd and the fms polygon, as hypothesised [138] . the aim of the review was to evaluate the psychometric properties of observational fms assessment tools for school-age children. there were no studies evaluating the validity or reliability of eight (33%) of the available measures (from 24 identified tools). of the remaining sixteen, nine (38%) assessment tools only had a single study examining their psychometric properties. multiple papers evaluating various aspects of validity and reliability were only found for the: mabc (37studies), tgmd (35 studies), bot (22 studies), ktk (10 studies), camsa (3 studies), the mot 4-6 (4 studies) and playfun (2 studies). the tgmd was the assessment tool with the most consistently positive evidence in favour of validity and reliability. however, it is important to consider the suitability of observational assessment tools for use in schools, alongside the evidence for the psychometric properties of measures [158] . recent research by klingberg et al. established a framework to evaluate the feasibility of implementing fms assessments in schools [22] . one of the criteria for feasibility detailed in the report was the type of assessment, in which it was stated that product-oriented measures were preferable because they require less training, and are less prone to error. so despite the tgmd being the assessment tool with the greatest evidence for validity and reliability, it is arguably less feasible to implement in schools settings because it is process-orientated [22] . notably, despite the strong evidence for the psychometric properties of the tgmd, this assessment tool does not measure balance. recent research has established that balance is an important aspect of fms [24] so it is important to recognise the limitations of using tools which do not measure such skills. it seems reasonable to suggest that exploration of the fms proficiency of children in schools should involve an assessment tool which encompasses locomotor skill, object control and balance to enable insights into the skills which underpin a child's ability to participate in physical activity [5] . the systematic review found nine product-oriented assessment tools. the product-oriented measure with the most promising feasibility in klingberg et al.'s review [22] , which was also included in this review, was the ast [98] . there is, however, insufficient evidence on the psychometric properties of this assessment tool to allow confidence in its use, as only two of the ten forms of validity and reliability specified by the cosmin checklist [25] were evaluated in the studies we reviewed [98, 137] . moreover, the ast assesses how quickly a child can perform a range of fms, rather than how well each child can perform these movements, arguably limiting the value of the results obtained by the assessment because it focuses solely on speed of movement. additionally, this assessment, again, does not include a measure of balance. thus, it would also not provide a school with a comprehensive picture of pupils' fms. only three of the product-oriented assessment tools in this review measure locomotion, object control and balance. the measure with the largest number of psychometric properties evaluated from these three tools was the mabc. however, the evidence for the validity and reliability of this assessment tool was very mixed, and the quality of the studies that found strong evidence for its psychometric properties was questionable. moreover, the mabc requires specialist equipment such as mats, which contribute to making the measure expensive to buy (approximately £1000). this may not be feasible with increasing pressure on school budgets [159] . the mabc also takes an extended period of time to administer (30-60 minutes), and must be delivered 1-to-1 by a trained professional. these time and resource constraints makes it difficult to recommend to schools as a feasible screening measure, despite it being advocated as the current 'gold standard' for detecting motor skill deficits in europe [160] . the bot was the next most explored product-oriented assessment tool that measures all three aspects of fms, and whilst it was not considered in the klingberg et al. evaluation of the feasibility of assessments [22] it is again, notably costly to purchase and takes between 45-60 minutes to assess each child. thus, with teachers feeling increasingly concerned about the time they have available to cover the 'core' assessed curriculum [161] , it appears unlikely that schools would be willing to invest the time required to universally assess fms all pupils using this tool. the final product-oriented assessment tool which assesses all three aspects of fms is 'stay in step' [47] . there were, however, no studies found that evaluate the psychometric properties of this assessment tool. this is particularly problematic as it is already being used within schools in australia. it is crucial that assessment tools are developed using a rigorous process which ensures they have strong psychometric properties. schools have limited capacity for new initiatives, so it is important that assessment tools being marketed to them are not only feasible for use, but can also accurately measure fms and identify children that need additional support, otherwise the assessment becomes redundant, and a waste of already stretched resources. in summary, this review offers a guide to help researchers, clinicians and teachers make an informed decision on available observational fms assessment tools. however, as discussed, there are a number of limitations with regard to all available assessments which need to be considered. there is an appetite amongst health practitioners to use schools as settings for motor skill assessments [19] but currently available measures have limited utility within such environments. the majority of existing assessments are commercial products creating significant financial implications for schools that wish to deploy these tests at scale. moreover, a lot of these tests require a substantial investment of time as they are designed to be conducted with a single child, with children tested in a serial manner. meanwhile, the tests that do exist without some of these limitations (e.g. ast and ktk) have limited evidence for their validity and reliability, and/or do not measure all three aspects of fms [24] , which limits the justification of their use within evidence-based health and educational practice. either, assessment tools with strong evidence for validity and reliability (e.g. tgmd) need to be modified to be feasible for use in schools, or feasible tests (e.g. ast) need more research to be done to establish psychometric properties. currently, schools would have to choose an assessment tool based on either feasibility or strong psychometric evidence alone, however, it is known from educational research that there needs to be a trade-off between the two for school-based initiatives to be implemented consistently, and effective [158] . this review reveals that there are a large number of novel observational assessment tools that have been and are continuing to be developed to measure fms proficiency in school-age children. we would argue that authors must consider from the outset how to make such tools feasible for use in schools. the results also showed that not enough fms assessment tools being developed include all three aspects of fms. in particular, balance has been neglected despite research establishing it as a crucial addition to this group of motor skills [24] . in addition, it is important that the evaluation of the psychometric properties of these new tools is comprehensive, spanning all psychometric properties outlined by the cosmin guidelines [25] . one of the main limitations of the studies included in this review was the tendency for the authors to be selective about which aspects of validity and reliability were tested. all aspects of validity/ reliability in the cosmin guidelines evaluated by this review were measured by at least one study, however, no single aspect was measured than more by half of the studies. the most commonly measured aspects of validity and reliability were inter-rater reliability (45% of studies) and structural validity (42% of studies). future research should consider evaluating predictive validity (1% of studies) and cross-cultural validity (7% of studies) using normative data more often, as these were the most neglected psychometric properties. the lack of consistency for measuring psychometric properties makes it difficult to draw any conclusions about the quality of the tools advertised, particularly when the reports involve the testing of specially selected samples (e.g. children with asd) where there are fewer studies undertaken. it is clear from the published literature there is insufficient evidence to justify the use of current fms assessment tools for screening in schools. it follows that: (i) researchers, teachers, and clinicians should be cautious when selecting existing measures of fms for use in these settings; (ii) there is a need to develop low 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the test of gross motor development-third edition in a large sample of italian children construct validity of the test of gross motor development: a crossvalidation approach assessing fundamental motor skills in belgian children aged 3-8 years highlights differences to us reference sample the reliability of classification decisions for the furtado-gallagher computerized observational movement pattern assessment system-fg-compass. research quarterly for exercise and sport development and calibration of an item bank for pe metrics assessments: standard 1. measurement in physical education and exercise science test of motor proficiency second edition (bot-2): compatibility of the complete and short form and its usefulness for middle-age school children. frontiers in pediatrics the comparison of school-age children's performance on two motor assessments: the test of gross motor development and the movement assessment battery for children. physical education and sport pedagogy what exactly do rct findings tell us in education research? stretched too thin? the relationship between insufficient resource allocation and physical education instructional time and assessment practices. teaching and teacher education european academy for childhood disability (eacd): recommendations on the definition, diagnosis and intervention of developmental coordination disorder (long version) teacher perceptions on the delivery and implementation of movement integration strategies: the class pal (physically active learning) programme key: cord-010368-plpghewn authors: kenmoe, sebastien; kengne-nde, cyprien; modiyinji, abdou fatawou; bigna, jean joel; njouom, richard title: association of early viral lower respiratory infections and subsequent development of atopy, a systematic review and meta-analysis of cohort studies date: 2020-04-24 journal: plos one doi: 10.1371/journal.pone.0231816 sha: doc_id: 10368 cord_uid: plpghewn introduction: existing evidence on the relationship between childhood lower respiratory tract infections (lrti) and the subsequent atopy development is controversial. we aimed to investigate an association between viral lrti at <5 years and the development of atopy at > 2 years. methods: we conducted a search at embase, pubmed, web of science, and global index medicus. we collected data from the included articles. we estimated the odds ratio and the 95% confidence intervals with a random effect model. we determined factors associated with atopy development after childhood lrti using univariate and multivariate meta-regression analyses. we recorded this systematic review at prospero with the number crd42018116955. results: we included 24 studies. there was no relationship between viral lrti at <5 years and skin prick test-diagnosed-atopy (or = 1.2, [95% ci = 0.7–2.0]), unknown diagnosed-atopy (or = 0.7, [95% ci = 0.4–1.3]), atopic dermatitis (or = 1.2, [95% ci = 0.9–1.6]), hyperreactivity to pollen (or = 0.8, [95% ci = 0.3–2.7]), food (or = 0.8, [95% ci = 0.3–2.5]), or house dust mite (or = 1.1, [95% ci = 0.6–2.2]). although not confirmed in all studies with a symmetric distribution of the 23 confounding factors investigated, the overall analyses showed that there was a relationship between childhood viral lrti at < 5 years and serum test diagnosed-atopy (or = 2.0, [95% ci = 1.0–4.1]), allergic rhinoconjunctivitis (or = 1.7, [95% ci = 1.1–2.9]), hyperreactivity diagnosed by serum tests with food (or = 5.3, [1.7–16.7]) or inhaled allergens (or = 4.2, [95% ci = 2.1–8.5]), or furred animals (or = 0.6, [95% ci = 0.5–0.9]). conclusion: these results suggest that there is no association between viral lrti at < 5 years and the majority of categories of atopy studied during this work. these results, however, are not confirmed for the remaining categories of atopy and more particularly those diagnosed by serum tests. there is a real need to develop more accurate atopy diagnostic tools. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 atopy is a genetic predisposition to the development of allergic diseases such as atopic dermatitis, atopic eczema, atopic asthma, atopic conjunctivitis or allergic rhinitis [1] . atopy also includes increased hypersensitivity to inhaled or food allergens, with the development of ige mediated by th2 cells [2] . atopic diseases is associated to a significant morbidity and a very important economic burden for society [3] . atopic disease prevalence has experienced in recent decades an exponential increase in the world [4, 5] . common viruses associated with lower respiratory tract infections (lrti) include influenza, rhinovirus, respiratory syncytial virus (hrsv), metapneumovirus, parainfluenzavirus, enterovirus, adenovirus, bocavirus, and coronavirus [6, 7] . data have showed the association between hrsv lrti and subsequent wheezing or asthma [8] [9] [10] [11] . with the advent of molecular assays, the description of childhood infections caused by non-hrsv has further demonstrated the implication of these diseases in long-term sequelae [12, 13] . a meta-analysis by liu et al., have demonstrated the association between childhood rv infections and the subsequent development of asthma [13] . a systematic review showed that pneumonia mainly due to adenovirus was linked to sequelae including obstructive pulmonary disease or chronic bronchitis [12] . the subgroup analyses in this latter however found that the 3 included studies with mycoplasma pneumoniae pneumonia were not associated with long-term sequelae [12] . studies on the prevalence of atopy among people presenting with viral lrti in childhood have shown divergent results [14] [15] [16] . some studies have reported an increased risk of allergic sensitization after viral lrti in childhood [14, 17] . protection against allergic sensitization through the stimulation of th-1 cytokine production has been suggested by other studies [16] . maximum confusion has been demonstrated by other studies that have shown no influence of childhood viral lrti in the development risk of subsequent atopy [15, 18, 19] . the resolution of the question of the association between viral lrti in childhood and the subsequent development of atopy could serve as a basis for preventive measures and management of atopic diseases [20] [21] [22] . the purpose of this systematic review and meta-analysis of long-term sequelae of lower respiratory tract infections in early childhood (a learned study) was to investigate the association between viral lrti at <5 years and the atopy development at > 2 years. dissemination guidelines [23] and reported according to the prisma (preferred reporting items for systematic review and meta-analysis) guidelines (s1 table) [24] . study participants were children with a history of laboratory confirmed viral lrti before 5 years. viral lrti was considered using the definition proposed by the authors of the included studies. children with viral lrti, as reference cases, were compared to control children who had no history of lrti in childhood. studies including only participants with medical conditions (premature birth, immunodeficiency or other comorbidities) were excluded. the exposure in this systematic review was a viral lrti in children at <5 years. the outcome of this systematic review was the development of atopy at> 2 years including atopic diseases and sensitization to food and atmospheric allergens. atopic status was determined by skin prick tests (spt) and total or allergen-specific serum ige antibody assessed by immunoassays. included studies were prospective and retrospective cohorts with a minimum follow-up duration of one year. atopic diseases were diagnosed clinically. we considered atopy category whose data on outcomes were available in three or more studies. the pubmed, excerpta medica database, web of science, and global index medicus databases were queried for articles published from inception through july 15, 2019. all languages and geographic areas were considered for this systematic review. the combination of terms used for the bibliographic search is listed in table 1 . we manually screened the included studies and relevant review reference lists to locate additional articles. two independent investigators (ks and afm) reviewed the titles and abstracts of the articles found by the electronic and manual search [25] . we summarized the selection process of potentially relevant articles on a prisma flowchart. the disagreements between the two investigators were resolved by discussion and consensus. two researchers (ks and afm) independently extracted data from full text of included articles. the following data was collected (title, first author, year of publication, time of data collection, country, participants interview period, lrti type, lrti rank, lrti period, age at lrti, virus associated with the lrti, control age, control gender, total number of cases and controls, numbers with atopy at follow up, and data on confounders). the discrepancies were resolved by discussion and a consultation of a third arbitrator (rn) if necessary. in accordance with newcastle-ottawa scale (nos) criteria including patient selection, comparability of groups, and outcome evaluation, two independent researchers (ks and afm) assessed the quality of all included studies [26] (s2 table) . to reach consensus, all the differences were discussed between the two researchers. we estimated odds ratio (or) as a measure of the association between childhood viral lrti and subsequent atopy development. we evaluated the publication bias by visual inspection of funnel diagram and egger test. we estimated heterogeneity between studies by the q test and the i 2 statistic [27, 28] . we considered heterogeneity as significant between studies for p-value <0.1 or i 2 > 50%. we conducted sensitivity analyses with studies with a low risk of bias, studies including only inpatients or the first episode of viral lrti. we performed subgroup analyses on the basis of the type of lrti, who region, age at lrti, age at follow up, and type of virus detected in lrti. we applied a multivariate metaregression with a stepwise manual selection procedure to identify factors associated with the variation of overall risk of atopy. we successively removed from the model variables by considering the signification of the p-value and information criteria for the model like log-likelihood, deviance, akaike information criterion (aic), bayesian information criterion (bic), and corrected akaike information criterion (aicc). we reported the explained heterogeneity (r2) by variables included in models. a variable with p value < 0.05 was considered statistically significant in the final model. we assessed the influence of confounding factors by conducting a sensitivity study that included only equitably distributed studies for each risk factor between reference cases and controls. for each potential confounding factor, we assessed the distribution between reference cases and controls by recalculating the p values using the exact tests of fisher and chi-2. the two p values> 0.05 of fisher's exact test and chi-2 indicated a symmetric distribution of the confounding factor between reference cases and controls. we synthesized the study selection process in fig 1. the electronic (4634 articles) and manual (23 articles) searches identified 4657 articles. the first selection by titles and abstracts resulted in the exclusion of 4249 irrelevant articles. we read and fully reviewed the complete texts of the remaining 330 articles. we excluded a total of 309 articles for multiple reasons including mismatch of the study population (no control group, inclusion of non-viral lrti and non-lrti infections, and inclusion of only patients with underlying medical conditions), the type of study not appropriate (case report, comment on study, editorial, and review), lack of data on outcomes, conference abstract or complete texts not found, and irrelevance of the article-supplementary. we finally included 22 articles (24 studies) in the qualitative and quantitative synthesis of this systematic review . we showed the individual characteristics of the publications included in s3 table. most studies were conducted in europe, detected hrsv, had a low risk of bias, included hospitalized children under 1 year of age with their first episode of bronchiolitis, had followed children between 5-10 years old, and were prospective. children with a history of viral lrti in childhood were recruited between 1960 and 2014 and articles were published between 1981 and 2017.the individual nos score from the included studies are presented in s4 table. comparison of reference cases with controls infant post viral lrti atopy rhinoconjunctivitis was significantly more frequently reported in reference cases than in controls (or = 1.7, 95% ci = 1.0-2.9). positivity for furred animals was significantly more frequent in controls compared to reference cases (or = 0.6, 95% ci = 0.5-0.9). the overall effect remained unchanged for the majority of our results when assessed by sensitivity analyses of the impact of lrti rank, hospitalization, and study quality ( table 2 ). the effect observed in the main analysis of the positive serum test was lost for studies reporting the first episodes of lrti (or = 2.0, 95% ci = 0.6-6.7) and hospitalized children (or = 2.5, 95% ci = 0.9-6.5). in contrast to the association observed between lrti history and development of allergic rhinoconjunctivitis (or = 1.7, 95% ci = 1.1-2.9), no effect was observed for studies reporting the first episode of lrti (or = 1.4, 95% ci = 0.5-3.6). the significant preponderance of furred animal positivity in controls compared to reference cases was lost in studies including only the first lrti episodes (or = 0.5, 95% ci = 0.3-1.0). in the subgroup analyses (s5 table) , a statistically significant association between childhood viral lrti and subsequent atopy was observed only in the bronchiolitis subgroup for the catepositivity to atopy by serum tests was not significantly associated only for patients 5 to 10 years (or = 1.0, 95% ci = 0.5-2.1). positivity to house dust mite allergen was only associated with patients aged 15 to 20 years (or = 2.8, 95% ci = 1.0-7.6). the positivity to food allergens by serum test was inversely proportional to the age of the patients and the association was lost between 5 and 10 years (or = 2.0, 95% ci = 0.7-5.3). there was no statistically significant difference by who region and viruses screened subgroups. in metaregression analyses, only the type of lrti was admitted in the best multivariate model for the type of lrti in the atopy diagnosed by spt, atopy diagnosed by serum test, and allergic rhinoconjunctivitis (s6 table) . follow-up delay of participants was positively associated with house dust mite and negatively associated with atopy diagnosed by serum test for food. a total of 84.8% (89/105) of the 23 confounding factors collected in the included studies had a symmetric distribution between reference cases and control participants (s7 table) . we conducted a sensitivity analysis that included only studies with symmetric distribution for these confounding factors for atopy categories with a significantly different distribution between reference cases and controls (atopy diagnosed by serum tests, positivity to food and inhalant allergens by serum tests, allergic rhinoconjunctivitis, and furred animals). the significant difference observed in the overall analysis was lost in the majority of these categories of atopy (s8 table) . there was no heterogeneity in overall and sensitivity analyses for atopy with unknown or not reported diagnosis method, atopic dermatitis, food allergy, furred animals, and positive serum tests for inhalants ( table 2 ). the analyses of atopy diagnosed by spt showed a publication bias (p egger = 0.085). the funnel diagrams of the main analysis are presented in the s2-s12 figs. our results highlight that there is no relationship between a history of lrti at < 5 years and atopy diagnosed by spt, atopy diagnosed unknown/not reported, atopic dermatitis, and hyperresponsiveness to common allergens including pollen, food allergens or house dust mites. our results on atopy diagnosed by serum tests, allergic rhinoconjunctivitis, and positivity by serum tests to food or inhaled allergens cannot be definite with an increased risk observed in the global analysis and not confirmed by the analyses in studies reporting confounding factors. the increased risks of developing atopy observed in some subgroup analyses were more frequent in case of bronchiolitis due to hrsv between 9-12 months and in prospective studies conducted in europe. our results are consistent with the quantitative analysis by knyber et al. who concluded that there was no relationship between hospitalization for hrsv bronchiolitis at < 1 year and subsequent allergic sensitization [10] . similar to the findings of this review, kneyber et al. also concluded that hrsv infection in childhood was associated with allergic sensitization to food or inhaled allergens tested with serum tests. however, we have no definitive conclusion on this point since our analyses, taking into account studies with confounders, such as family history of atopy [51] , did not confirm this finding. similar to the findings of the present work, several studies have also shown divergent results between serum and skin tests [52] [53] [54] [55] . there are many hypothetical reasons that may explain these observed differences between serum and skin test results. first, differences in the composition and/or concentration of skin and serum tests targets may lead to differences in the results of both tests [52] . in a context of immune immaturity, for example, insufficient migration of mast cells to the epidermis could lead to positive results for serum tests and false negative for skin tests. serum tests also involve false positive results due to nonspecific binding with the antibodies used [56] . technical differences in the handling of skin and serum tests may also be involved in the differences observed between the two methods [57] . the systematic review by fauroux et al. reported for studies conducted between 1995 and 2015 in industrialized countries the controversial nature of the results on the association between infantile hospitalizations for hrsv lrti and subsequent atopy [58] . pérez-yarza in a systematic review including children younger than 3 years with hrsv respiratory infection from 1985 to 2006 also suggested controversial findings about the subsequent risk of allergic sensitization development defined by positive skin or serum tests specific for common allergens [11] . while this systematic review may help clarify the relationship between lrti in childhood and subsequent atopy, the weaknesses of the work must be emphasized. more than three quarters of the included studies in this systematic review were from europe. this suggests an important problem in the external validity of our results on a global scale, with the absence of america, south east asia and eastern mediterranean. this systematic review is the only one to date to address this topic with a strict atopy definition with the consideration of 11 different categories depending on the type of diagnosis used, allergic diseases and sensitization to common allergens. this systematic review includes a multitude of sensitivity analyses with studies reporting their first episode of lrti, studies reporting children hospitalized for lrti, and quality of studies. other special strengths of this systematic review include the large size of the participants included, 5294 reference cases and 27091 controls, the long follow-up period of more than half a century of children from birth to about 30 years old and with several points of follow-up including all age groups. the data was carefully extracted from a structured questionnaire and we used an appropriate data analysis to consider 23 important confounding variables. no relationship was found in this systematic review between viral lrti at <5 years and the subsequent development of a spt-diagnosed atopy, sensitization to common allergens or the development of atopic dermatitis. this conclusion was not confirmed for the association between viral lrti at < 5 years and the subsequent development of serum test diagnosed atopy, serum test positive for food or inhaled allergens, allergic rhinoconjunctivitis, and sensitization to furred animals. thus, more longitudinal investigations adjusted to confounding factors are important to elucidate the implication of childhood lrti in the development of atopy or allergy to food or inhalant assessed by serum tests, allergic rhinoconjunctivitis, and sensitization to furred animals. these findings should encourage research on the long-term burden of viral lrti in childhood in non-european regions and non-hrsv viruses. prospective randomized studies including intervention against the development of the lrti would be ideal to rule out the residual confusion about the causal relationship between infantile lrtis and the development of subsequent atopy. the imminent arrival of the vaccine against hrsv on the market or the prophylactic means such as palivizumab could be a way to carry out these interventional studies. to reduce the burden of atopy, there a real need of more accurate diagnosis tools and efforts should focus on other major risk factors including genetic predisposition, diet habits, air pollution, family size, and the use of vaccines or antibiotics. supporting information s1 allergy and allergic diseases. first of two parts t-cell subsets (th1 versus th2) economic burden of adult patients with moderate to severe atopic dermatitis indicated for systemic treatment investigating international time trends in the incidence and prevalence of atopic eczema 1990-2010: a 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sequelae from childhood pneumonia; systematic review and meta-analysis association between rhinovirus wheezing illness and the development of childhood asthma: a meta-analysis allergic predisposition among infants with bronchiolitis acute bronchiolitis: predisposing factors and characterization of infants at risk atopic versus infectious diseases in childhood: a question of balance? patterns of allergic respiratory disease in children with a past history of bronchiolitis respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years symptoms, atopy, and bronchial reactivity after lower respiratory infection in infancy effect of palivizumab prophylaxis on subsequent recurrent wheezing in preterm infants early ribavarin treatment of bronchiolitis: effect on long-term respiratory morbidity interventions to prevent long-term consequences of centers for reviews and dissemination. crd's guidance for undertaking reviews in 301 healthcare: centers for reviews and dissemination preferred reporting items for systematic reviews and meta-analyses: the prisma statement rayyan-a web and mobile app for systematic reviews assessing risk of bias in prevalence studies: modification of an existing tool and evidence of interrater agreement quantifying heterogeneity in a meta-analysis the combination of estimates from different experiments acute bronchiolitis in infancy as risk factor for wheezing and reduced pulmonary function by seven years in akershus county human metapneumovirus bronchiolitis in infancy is an important risk factor for asthma at age 5 hospitalization for rsv bronchiolitis before 12 months of age and subsequent asthma, atopy and wheeze: a longitudinal birth cohort study association of an early respiratory syncytial virus infection and atopic allergy respiratory morbidity 20 years after rsv infection in infancy respiratory status and allergy after bronchiolitis risk factors for virusinduced acute respiratory tract infections in children younger than 3 years and recurrent wheezing at 36 months follow-up after discharge: the pediatric infectious disease journal exhaled nitric oxide in acute phase of bronchiolitis and its relation with episodes of subsequent wheezing in children of preschool age causal direction between respiratory syncytial virus bronchiolitis and asthma studied in monozygotic twins long-term consequences of respiratory syncytial virus acute lower respiratory tract infection in early wheezing, asthma, and pulmonary dysfunction 10 years after infection with respiratory syncytial virus in infancy adolescent asthma after rhinovirus and respiratory syncytial virus bronchiolitis rsv bronchiolitis and risk of wheeze and allergic sensitisation in the first year of life asthma and immunoglobulin e antibodies after respiratory syncytial virus bronchiolitis: a prospective cohort study with matched controls respiratory syncytial virus bronchiolitis in infancy is an important risk factor for asthma and allergy at age 7 severe respiratory syncytial virus bronchiolitis in infancy and asthma and allergy at age 13 asthma and allergy patterns over 18 years after severe rsv bronchiolitis in the first year of life atopy does not predispose to rsv bronchiolitis or postbronchiolitic wheezing factors predisposing to abnormal pulmonary function after adenovirus type 7 pneumonia association between pronounced iga response in rsv bronchiolitis and development of allergic sensitization respiratory morbidity in adulthood after respiratory syncytial virus hospitalization in infancy the outcome after severe bronchiolitis is related to gender and virus development of allergy in children. i. association with virus infections discordance between aeroallergen specific serum ige and skin testing in children < 4 years of age comparison of skin-prick test and specific serum ige determination for the diagnosis of latex allergy comparison of serum-specific ige (immunocap) and skin-prick test results for 53 inhalant allergens in patients with chronic rhinitis skin prick test and specific serum ige in the diagnostic evaluation of suspected cow's milk and hen's egg allergy in children: does one replace the other? allergic rhinitis and its impact on asthma (aria) 2008 update (in collaboration with the world health organization, ga(2)len and allergen) comparison of the multi-test ii and comforten allergy skin test devices the burden and long-term respiratory morbidity associated with respiratory syncytial virus infection in early childhood key: cord-000981-6vloa2w3 authors: bálint, zoltán; zabini, diana; konya, viktoria; nagaraj, chandran; végh, attila g.; váró, györgy; wilhelm, imola; fazakas, csilla; krizbai, istván a.; heinemann, akos; olschewski, horst; olschewski, andrea title: double-stranded rna attenuates the barrier function of human pulmonary artery endothelial cells date: 2013-06-03 journal: plos one doi: 10.1371/journal.pone.0063776 sha: doc_id: 981 cord_uid: 6vloa2w3 circulating rna may result from excessive cell damage or acute viral infection and can interact with vascular endothelial cells. despite the obvious clinical implications associated with the presence of circulating rna, its pathological effects on endothelial cells and the governing molecular mechanisms are still not fully elucidated. we analyzed the effects of double stranded rna on primary human pulmonary artery endothelial cells (hpaecs). the effect of natural and synthetic double-stranded rna (dsrna) on hpaecs was investigated using trans-endothelial electric resistance, molecule trafficking, calcium (ca(2+)) homeostasis, gene expression and proliferation studies. furthermore, the morphology and mechanical changes of the cells caused by synthetic dsrna was followed by in-situ atomic force microscopy, by vascular-endothelial cadherin and f-actin staining. our results indicated that exposure of hpaecs to synthetic dsrna led to functional deficits. this was reflected by morphological and mechanical changes and an increase in the permeability of the endothelial monolayer. hpaecs treated with synthetic dsrna accumulated in the g1 phase of the cell cycle. additionally, the proliferation rate of the cells in the presence of synthetic dsrna was significantly decreased. furthermore, we found that natural and synthetic dsrna modulated ca(2+) signaling in hpaecs by inhibiting the sarco-endoplasmic ca(2+)-atpase (serca) which is involved in the regulation of the intracellular ca(2+) homeostasis and thus cell growth. even upon synthetic dsrna stimulation silencing of serca3 preserved the endothelial monolayer integrity. our data identify novel mechanisms by which dsrna can disrupt endothelial barrier function and these may be relevant in inflammatory processes. endothelial function is essential for vascular integrity. the endothelium provides a barrier, regulates vascular tension, and is involved in angiogenesis and haemostasis. local and systemic inflammation, however, can impair endothelial function and can lead to cellular damage increasing endothelial permeability and loss of epithelial barrier function [1, 2] . endogenous rna release and circulating rna like virus-associated double stranded rna (dsrna) may contribute to the development of endothelial dysfunction. endothelial cells express toll-like receptor 3 (tlr3) [3] which is activated by dsrna [4, 5] . the activation of tlr3 affects cell homeostasis [6, 7] and causes changes at functional [8, 9] , as well as inflammatory gene expression level [10] . at cellular level, dsrna induces a signaling cascade [11, 12] leading to tlr3 receptor upregulation [4, 13] . at organ level, repeated and long-term administration of synthetic dsrna causes inflammation [14, 15] and leads to impairment of lung function in mice [16] [17] [18] . however, the biological activity of circulating extracellular rna is poorly understood. recently, an extracellular rna-induced activation of vegf has been shown, leading to increased permeability of cerebral endothelial cells, which are the main components of the blood brain barrier [19] . this hyperpermeability can occur due to exposure of the cells to total rna [8] or the synthetic analogue of dsrna, polyinosinic-polycytidylic acid (poly i:c) [20] and can lead to disintegration of adherens junctions [21] . endothelial permeability regulation [22] and function [23, 24] is a ca 2+dependent process [1, 25] . a rise in intracellular ca 2+ in the ecs occurs through ca 2+ influx or by release from the sarcoendoplasmic reticulum (ser) resulting in plasma membranelocated ca 2+ channel activation [26, 27] . to maintain the ca 2+ homeostasis of the cell, the ca 2+ stores are refilled by the sermembrane-located sarco/endoplasmic reticulum ca 2+ atpase (serca) [28] . serca is encoded by three homologous genes: serca1, serca2 and serca3 [29] , out of these in human pulmonary artery endothelial cells (hpaecs) only serca2 and serca3 isoforms are expressed [30, 31] . however, serca plays an important role not only in the ca 2+ homeostasis [24, 30, 32] , but it is vital for cell cycle control [33] , proliferation and regulation of cellular permeability as well. in the present study we investigated alternative pathways of dsrna on primary hpaecs. changes in cell morphology, permeability, cellular junctions, mechanical properties and ca 2+ homeostasis were characterized. furthermore, we assessed the effects of natural and synthetic dsrna on gene expression, proliferation of hpaecs and on serca. human pulmonary artery endothelial cells (hpaecs) were obtained from lonza (allendale, new jersey) and they were cultured according to the manufacturer's instructions. the endothelial specific media (vasculife, lifeline) was changed every third day. cells in passages 5-9 were used for the experiments and the endothelial phenotype was regularly checked for von willebrand factor expression. poly i:c was purchased from amersham pharmacia, l-dna from fermentas (sd0021), ly-294002 and 2,5-di-t-butyl-1,4benzohydroquinone (bhq) was received from sigma. doublestranded rna (sense: 59-uac-acc-guu-agc-aga-cac-cdtdt-39, antisense: 59-ggu-guc-ugc-uaa-cgg-ugu-adtdt-3) was from qiagen. total cellular rna was isolated from hpaecs with the rneasy mini kit (qiagen). phosphoakt and akt antibodies were obtained from cell signalling technology inc. all chemicals were dissolved and diluted to the desired concentration in experimental solution containing in mm: 145 nacl, 5.5 kcl, 1.8 cacl 2 , 1 mgcl 2 , 10 glucose and 10 hepes. the ph was set to 7.4. all the solutions were freshly prepared on the day of the experiment and stored at 4uc until they were used. the experiments were carried out at room temperature unless otherwise stated. for measuring the cellular barrier properties hpaecs were grown until confluence on semi-permeable inserts. the teer was measured using an endohm chamber connected to an evom resistance meter (world precision instruments, sarasota, usa) and teer values were recorded every 30 min for the first 5 hours and additionally at 18, 20, 22 and 24 hour timepoints after treatment with 25 mg/ml poly i:c, 25 mg/ml dsrna, 2.5 mg/ ml totalrna, 2.5 mg/ml l-dna or control solution. the measurements were carried out at 37uc. the resistance of a blank filter insert filled with the control media was subtracted as background value from the total resistance of each culture insert [34] . to assess the permeability changes of the endothelial monolayer, hpaecs were grown until confluence on semi-permeable inserts and incubated for 24 h with 25 mg/ml poly i:c, 25 mg/ ml dsrna, 2.5 mg/ml totalrna, 2.5 mg/ml l-dna, 25 mm ly-294002, 30 mm bhq or control solution (vehicle control). after treatment, fitc-labelled dextran was added to the upper compartment of the insert (ecm640 in vitro vascular permeability assay kit, chemicon). the fluorescence of the solution from the bottom well was measured with a fluorescent plate reader (lex = 485 nm, lem = 525 nm; optima, bmg labtech). for quantitative rt-pcr and as a stimulation agent, total cellular rna from hpaecs was isolated with the rneasy mini kit from qiagen. the protocol for purification of total rna from cells using spin technology was followed (cat. no./id: 74104). additionally, dnase digestion during rna isolation was carried out with the rnase-free dnase-set from qiagen (cat. no./id: 79254). the agilent 2001 bioanalyzer and agilent rna 6000 nano assay protocol were used to quantify the concentration and the purity of the isolated total rna. the total cellular rna isolated from endothelial cells was converted to cdna using a revertaid h minus first strand cdna synthesis kit (fermentas). pcr amplifications were performed by ab7900 (applied biosystems) using the following primers: hs_gapdh_2_sg (qt01192646) -gapdh, hs_atp2a3_1_sg (qt00087220) -serca3; hs_atp2a2_1_sg (qt00077231) -serca2; and the sybr green i master mix (qiagen). amplifications were performed in triplicates and the mean threshold cycle (ct) reading was used. gene expressions were quantified relative to the housekeeping gene (gapdh) and normalized to the expression level of untreated control samples (delta-delta ct method). hpaecs were seeded on chamber slides and were grown until confluence then they were treated for 24 h with 25 mg/ml poly i:c, 25 mg/ml dsrna, 2.5 mg/ml totalrna, 2.5 mg/ml l-dna, 25 mm ly-294002, 30 mm bhq or kept untreated (control). the cells were fixed with a buffer containing 100 ml phosphate buffered saline solution (pbs) ph 7, 650 mg na 2 hpo 4 , 400 mg nah 2 po 4 , 1.5 ml methanol, 10 ml 4% formaldehyde. the slides were washed with pre-warmed hepes buffered saline solution (hbss) for 20 min at room temperature (rt). after washing the cells for 5 min in pbs, they were covered for 30 min with 100 nm glycerol (rt). 365 min washing steps with pbs were performed and followed by permeabilization with 0.1% triton-x-100 for 30 min at rt. afterwards 365 min pbs washing steps were performed. the cells were blocked for 30 min with 3% bsa in pbs at rt, than they were washed again for 5 min. the primary antibody (ve-cadherin, 1:200 dilution, abcam; zo-1, 1:100 dilution, zymed) was added to the cells for 30 min (rt). after washing for 365 min, the cells were incubated at rt for 30 min with goat anti-mouse antibody conjugated with af-594 (1:500 dilution, molecular probes) in dark. finally, the cells were counterstained with dapi to identify the nuclear dna. duplicates processed without primary antibodies served as negative controls. fluorescence was imaged using a zeiss 200 m inverted epifluorescent microscope. for phalloidin staining the cells were fixed with 4% formaldehyde for 30 min at 4uc, permeabilized using acetone at 220uc for 10 min. after blocking with 3% bovine serum albumin for 30 min, the coverslips were incubated with alexa488-phalloidin (molecular probes). mounting was performed in anti-fading embedding medium (biomeda) and the distribution of the signal was imaged using a nikon eclipse te2000u photomicroscope with epifluorescent capabilities connected to a digital camera (spot rt ke). protein extracts were prepared from hpaecs in ripa buffer containing protease-inhibitor and phosphatase-inhibitor tablet (roche, vienna, austria). equivalent amounts of protein were resolved on 10% sds polyacrylamide gels and proteins were transferred to the nitrocellulose membrane. nonspecific antibody binding was blocked by incubation in 5% (m/v) non-fat dry milk powder in tbst (20 mm tris-cl, ph 7.5, 150 mm nacl, 0.1% (v/v) tween 20) at room temperature for 1 h. afterwards, the blots were incubated overnight with a 1:1000 diluted primary antibody at 4uc. after washing the membranes in tbst buffer and incubating with 1:2000 diluted horseradish-peroxidase conjugated anti-igg secondary antibody for 1 h at room temperature, specific immunoreactive signals were detected by enhanced chemiluminescence (ecl, amersham, freiburg, germany). the cells were cultured on gelatine coated, 25 mm glass cover slips. after reaching confluence, they were treated for 24 h with 25 mg/ml poly i:c, 25 mg/ml dsrna, 2.5 mg/ml l-dna, 2.5 mg/ml total rna or left untreated (control). the cover slips were loaded with fura-2/am (2 mmol/l) in dark for 45 min followed by a washing step in experimental solution as previously described [35] . after 15 min, the single glass cover slip was mounted on the stage of a zeiss 200 m inverted epifluorescence microscope coupled to a polychrome v monochromator (till photonics, germany) light source in a sealed temperaturecontrolled rc-21b imaging chamber (warner instruments, usa) and perfused with prewarmed solution (30uc). fluorescence images were obtained with alternate excitation at 340 and 380 nm. the emitted light was collected at 510 nm by an aircooled andor ixon camera (andor technology, ireland). measurements were made every 3 s. background fluorescence was recorded from each cover slip and subtracted before calculation. the acquired images were stored and processed offline with tillvision software (till photonics, germany). [ca 2+ ] i was calculated as described by grynkiewicz et al. [36] . maximal and minimal ratio values were determined at the end of each experiment by first treating the cells with 1 mmol/l ionomycin (maximal ratio) and then chelating all free ca 2+ with 10 mmol/l egta (minimal ratio). cells that did not respond to ionomycin were discarded. the cells were stimulated with 100 mm histamine or 15 mm 2,5-di-t-butyl-1,4-benzohydroquinone (bhq: selective serca blocker) in the presence and absence of extracellular ca 2+ , after incubation for 24 h with 25 mg/ml poly i:c, 25 mg/ml dsrna, 2.5 mg/ml l-dna, 2.5 mg/ml total rna or control solution. the measurements were performed with an asylum mfp-3d head and molecular force probe controller (asylum research, santa barbara, ca, usa). the driver program mfp xop was written in igor pro software (version 5.0.3, wavemetrics, lake oswego, or, usa). the mfp-3d head was mounted on a zeiss axiovert 200 invert optical microscope. the cells were cultured on gelatine-coated 15 mm diameter glass cover slips until confluence. the sample was mounted on the stage of the microscope and contact mode image and force curve acquisition was performed. afterwards the cells were treated with 25 mg/ml poly i:c or left untreated (control). the image and force curve acquisition was repeated every 30 min for 3 hours. for imaging in solution, gold coated, silicon nitride, rectangular cantilevers were used (bio-lever, bl-rc150 vb-c1, olympus optical co. ltd., tokyo, japan). the imaging in solution and the determination of the young's moduli were made as previously reported [37, 38] . cells were plated at a density of 10 4 cells/well in 2%fbs/ medium in 96-well plates and were allowed to adhere overnight. the concentration-dependent effect of 24 h incubation with poly i:c, dsrna, totalrna, l-dna, ly-294002 or bhq on growth and proliferation of hpaecs was determined by [ 3 h]thymidine incorporation as an index of dna synthesis. [ 3 h]thymidine (0.2 mci/well) was added for the last 16 h. after 24 h incubation the cells were harvested and transferred to filter-plates. the radioactivity was measured using a beta-counter. to measure the cell-cycle dependent amount of dna per cell, propidium-iodide staining was performed on methanol-fixed hpaecs after 24 h incubation with poly i:c or control solution. the cells were trypsinized, centrifuged for 5 min at 2206g at 4uc, and then washed with 10 ml pbs. the pellet was re-suspended and again centrifuged for 5 min at 2206g at 4uc and dissolved in 500 ml pbs. for 10 6 cells 5 ml of 70% ice cold methanol was added for 10 min at 4uc. for data analysis the fca3.3 dna subg1 protocol was used. small interfering rnas (sirna) against serca3 were commercially synthesized (siserca3, sc-41295, santa cruz biotechnology). as negative control, non-silencing rna (sictl, control sirna-a, sc-37007, santa cruz biotechnology) which does not target any human gene product was used. either 200 nm sictl or 200 nm siserca3 was mixed with 500.000 hpaecs and 100 ml of basic nucleofector solution from basic endothelial cells nucleofector kit (lonza). this mixture was electroporated according to the manufacturers' instruction. knock-down was confirmed by quantitative rt-pcr and western blot with specific primers and antibody. permeability measurement with fluorescently labelled dextran and ve-cadherin staining were performed 48-56 h post transfection. numerical values are given as means 6 sd of n cells. intergroup differences were assessed by factorial analysis of variance with post hoc fisher's least significant difference test or student's t-test (p values ,0.05 were considered significant). for the live cell ca 2+ imaging data analysis, the basal level of ca 2+ was determined as an average value of the first 50 seconds of the curve. afterwards, the histamine-induced ca 2+ peak height after subtracting the baseline as well as the plateau duration of the ca 2+ response were quantified. the plateau duration is the time from the maximum ca 2+ peak height until the intracellular ca 2+ declines to the basal level. to determine the blocking potency of poly i:c on hpaecs proliferation, concentration-inhibition curves were constructed from the fcs-induced hpaecs proliferation in the presence of different drug concentrations in the media. the proliferation values are given in the presence of poly i:c as a fraction of the fcs-induced proliferation in the absence of poly i:c. the normalised values were fitted by means of a nonlinear leastsquares method with the equation: 1(1+c(ic50)-1)h)-1, where c was the drug concentration, ic50 was the concentration giving a half-maximum effect and h was the hill coefficient. because the hill coefficient was less than 1, it was set to one, accounting for a 1:1 binding stoichiometry. double-stranded rna (dsrna) or its synthetic analogue (poly i:c) significantly decreased the electric resistance and increased the permeability of the confluent human pulmonary artery endothelial (hpaec) monolayer as shown on figure 1 . primary hpaecs were incubated with dsrna or poly i:c up to 24 hours and the transendothelial electric resistance (teer) was measured ( figure 1a) . l-dna, which is not a toll-like receptor 3 ligand [8] , was used as a control. significant decrease in teer was observed already after 18 hours of treatment suggesting that both, dsrna and poly i:c disrupted the integrity of the endothelial monolayer. in contrast, the incubation with l-dna had no significant effect. consistently, the trafficking of fitc-labeled dextran molecules through the confluent endothelial monolayer was significantly increased after 24 h dsrna, poly i:c or total rna application ( figure 1b, 131630%, 140623% and 152645% of control, respectively). 24 h incubation with l-dna had no significant effect (98615% of control). an elongation of the poly i:c treated cells and the appearance of rearranged fiber structures was observed on the actin staining (figure 2a , b) and more prominently in the live cell atomic force microscopy images ( figure 2c, d) . the representative amplitude image of the control hpaecs showed a regular cell shape as seen in figure 2c , whereas elongated cells were detected on the images taken from the 24 h poly i:c treated culture ( figure 2d ). the poly i:c treatment was also associated with a time dependent stiffening of the cells ( figure 2e ). the young's moduli of the hpaecs was determined by direct force measurements on 5 distinct points above the nuclear region of the numbered cells shown in figure 2c and d. a significant increase in the young's moduli from 0.2560.15 kpa to 2.1561.5 kpa upon 24 h poly i:c treatment was observed ( figure 2e ). the integrity of the intercellular junctions was further investigated by vascular endothelial-cadherin (ve-cadherin) and zonula occludens 1 (zo-1) staining. the continuous membrane staining for both ve-cadherin and zo-1 showed colocalization of these two proteins ( figure 3a ). 24 h of poly i:c treatment caused disruption of the endothelial barrier integrity. the ve-cadherin and zo-1 membrane staining either disappeared or became discontinuous ( figure 3b ). next, we investigated the tlr3 -phosphatidylinositol 3-kinase (pi3-kinase) driven regulation of endothelial permeability and proliferation. poly i:c treatment resulted in a 35-fold upregulation of tlr-3 in hpaecs ( figure s1 , methods s1). however, both ve-cadherin and zo-1 stainings ( figure 3a) showed a cumulative effect of poly i:c and ly-294002 (pi3-kinase blocker) with a total disappearance of both proteins from the cell membranes. consistently, the fitc-labelled dextran traffic was significantly increased by 24 hours of ly-294002 (152.10624.18%) and this effect was additive with the poly i:c effect, leading to a 501.8068.27% increase in the amount of transmitted fluorescent dextran molecules ( figure 3b) . additionally, the proliferation of the hpaecs was blocked to 50.73610.32% by ly-294002, similar to the poly i:c effect (44.74615.92%) and further inhibited to 24.9567.44% when both compounds were administered together ( figure 3c ). (table 1 ) and on the 100 mm histamine-induced peak ca 2+ concentration of the cells (table 2 ). in contrast, 24 h dsrna incubation significantly prolonged the 100 mm histamine-induced ca 2+ signal in the absence ( figure 5a ) or presence of extracellular calcium ( figure 5c ). similar results were observed with poly i:c, whereas l-dna incubation had no effect ( figure 5 ). this prolonged ca 2+ signal ( figure 5b , d) suggested that dsrna treatment caused a delay in the clearance of cytoplasmic ca 2+ , which is necessary for replication. to further investigate the effect of dsrna on the sarcoendoplasmic reticulum ca-atpase (serca), gene expression analysis was performed. both serca isoforms (serca2b and 3) present in hpaecs showed a significant decrease in mrna expression after 24 h of poly i:c treatment ( figure 5e next, the effect of poly i:c and dsrna treatment on the phosphorylation of phospholamban, an endogenous inhibitor of serca was assessed. after 24 h treatment a significant decrease in the phospholamban phosphorylation was observed ( figure 5g , h). while an inhibitory effect of dsrna on the serca pump was expected, we investigated the serca blocker (bhq) induced dose-dependent inhibition of the hpaec proliferation ( figure 6a ). the effect was similar to that of dsrna or poly i:c ( figure 4a ). bhq treatment led to disruption of the ve-cadherin staining similar to poly i:c or ly-294002 ( figure 6c ). this effect was accompanied by increase in the endothelial permeability ( figure 6b ) pointing to decreased endothelial barrier function. next, we investigated the effect of poly i:c after sirna silencing of serca3 on hpaecs (figure 7) . the observed increase in permeability upon 25 mg/ml poly i:c treatment on sictl hpaecs, was significantly reduced with the silencing of serca3 ( figure 7a ). parallel, confocal microscopic images revealed that knock down of serca3 caused less ve-cadherin signal loss upon 24 h poly i:c stimulation as compared to sictl ( figure 7c ). the silencing of serca3 confirmed the previous findings obtained with the serca blocker, bhq. altogether, these data suggest that synthetic dsrna treatment alters the function of serca, which inhibits cell proliferation by inducing g1 arrest in the hpaecs and contributing to endothelial dysfunction. the major function of the vessel-forming endothelial cells is to maintain the blood-tissue barrier. viral rna has been identified as a pathogenic factor in different pulmonary diseases [39, 40] . circulating rna may lead to pulmonary endothelial dysfunction and thus to increased endothelial permeability [8] . the precise mechanism by which circulating rna induces endothelial barrier disruption in the lung has not been completely understood yet. the present study addressed novel pathways of dsrna effect on primary human pulmonary artery endothelial cells (hpaecs). our data showed that natural dsrna treatment significantly decreased electric resistance indicating disruption in the hpaec monolayer integrity. a similar effect could be observed with the widely used synthetic dsrna-analog, poly i:c. incubation with l-dna did not cause significant permeability changes indicating the specificity of the dsrna effect. as dsrna and poly i:c treatment mimics viral infection, our results are in line with previous observations on bluetongue virus-infected human primary microvascular endothelial cells [41] . the ec monolayer disrupting effect of dsrna was further confirmed by using fluorescently labeled dextran trafficking, showing that stimulation with either poly i:c, total rna or dsrna resulted in an increase in transferred dextran amount suggesting a significant loss in endothelial barrier function. the tight, isolating monolayer of the endothelial cells is accomplished by junctional structures, like adherens and tight junctions. the major component for the maintenance of monolayer integrity belongs to the transmembrane protein family, the cadherins [21] . the vascular endothelial cells express in their membrane the vascular endothelial cadherin (ve-cadherin) member of this family. poly i:c incubation decreased ve-cadherin signal, pointing to disrupted cell-cell contacts in the monolayer. a similar decrease was reported as a result of dengue-2 virus infection in human umbilical vein endothelial cells [21] . we observed a similar decrease in the zo-1 protein upon poly i:c treatment. furthermore, our results indicate that not only the junctional proteins contribute to dissociation of intercellular contacts, but also the actin reorganization. the poly i:c treated cells presented with an intense peripheral actin staining accompanied by cellular shape changes visible on the immunofluorescent pictures. similar cytoskeletal rearrangement was shown to be induced by 6 h bluetongue virus infection in primary human microvascular endothelial cells [41] . although, the authors report on a recovery of both electrical resistance and actin/ve-cadherin staining after 24 h, this was not the case in either dsrna, or poly i:c treatment in our experiments. this could be explained by the differences between the human lung microvascular and arterial endothelial cells. to investigate the possible involvement of the pi3-kinase pathway in the poly i:c induced permeability and structural changes, we tested the effect of a pi3-kinase blocker (ly-294002) on permeability, proliferation and junctional staining. it has been shown that the tlr3 -pi3-kinase pathway could be involved in the dsrna induced signalling [42] . indeed, we observed an upregulation of the tlr3 receptor upon poly i:c treatment. the block of pi3-kinase resulted in increased hpaec permeability, decreased proliferation and disruption of ve-cadherin and zo-1 staining. on primary hpaecs, the blocking was additive to the effect of poly i:c, pointing to a pi3-kinase independent mechanism of action. however, others report that pi3-kinase is involved in dsrna signalling [42] . in their study, a mammalian heterologuos expression system (hek293) was used with a higher concentration of poly i:c. we could confirm the inhibitory effect of pi3-kinase on serca expression, as it was previously reported with a pi3-kinase inhibitor on serca dependent calcium handling [43] . however, the effect on downregulation of both isoforms was additive pointing to a pi3-kinase independent mechanism of action, similar to the effects we observed on hpaecs permeability and proliferation. structural changes caused by inflammatory stimuli may lead to dissociation of cell-cell junctions leading to a widened intercellular space that facilitates transendothelial flux [1] . we observed similar structural rearrangement in light microscopy: the poly i:c treated cells presented a morphological change with an elongated shape. in-situ atomic force microscopy (afm) imaging and force measurements detected structural changes in the endothelial cells at single cell level. endothelial barrier-disruptive agents have been reported to cause changes in cytoskeletal mechanics of hpaecs [44] . in our afm images, cells treated with poly i:c showed an elongated morphological shape. furthermore, the poly i:c treatment also led to an approximately 10-fold stiffening of the cells. this is in line with the observations of the effect of other barrier-disruptive agents on hpaecs [44, 45] . the change in the mechanical properties of the cells can lead to disturbed or disrupted cell-cell contacts and may contribute to endothelial dysfunction. a critical factor for the regular endothelial function is the ca 2+ homeostasis [25] . our data suggest that, beside structural changes, poly i:c and dsrna also affects endothelial ca 2+ homeostasis. acute administration of either poly i:c, or dsrna did not result in any ca 2+ response, but 24 hours of treatment led to a significantly prolonged histamine-induced ca 2+ response, without affecting the basal ca 2+ level. further, we focused on intracellular ca 2+ mobilization. the key player of the ca 2+ store refilling is the sarcoendoplasmic reticulum ca-atpase (serca) [28] and its proper regulation is vital for normal cell function [33] . an inhibitory effect of poly i:c on serca is suggested by the increased duration of the histamine-induced signal, observed in the presence and absence of extracellullar ca 2+ . we observed a decrease in the phospholamban phosphorylation upon poly i:c or dsrna treatment, which contributes to enhanced block of serca function. phospholamban is a negative regulator of serca function [46] . phospholamban phosphorylation causes its dissociation from serca and removes inhibition [47] . rt-pcr showed a decrease in the expression of both isoenzymes of serca upon poly i:c treatment. however, upon dsrna treatment no changes in the gene expression could be observed. thus, our data indicate, that dsrna does not regulate serca expression like its synthetic analogue (poly i:c). nonetheless, both caused decrease in the phospholamban phosphorylation. furthermore, the serca blocker (bhq) induced a dose-dependent inhibition of the hpaec proliferation, a decrease in the ve-cadherin staining and increase in the transendothelial transport similar to the effect of dsrna or poly i:c. the sirna silencing of serca3 on hpaecs confirmed the figure 5 . prolonged histamine-induced ca 2+ plateau in hpaecs after poly i:c incubation accompanied by serca downregulation and phospholamban dephosphorylation. representative traces of 100 mm histamine induced intracellular ca 2+ rise under ca 2+ -free (a) and ca 2+ (c) conditions showed a prolonged decay of intracellular ca 2+ level in hpaecs after 24 h poly i:c (red line), dsrna (green line) or total rna (orange line) treatment. as a control, l-dna had no effect (blue line). arrow indicates the application of histamine (his). (b) the histamine induced transient ca 2+ plateau duration was significantly longer in the case of treatment (*p,0.05, **p,0.01, ***p,0.001 compared to control, untreated sample) in the absence (b) and presence (d) of 1.8 mm extracellular ca 2+ . bar graphs show expression of serca3 (e) and serca2b (f) isoform of the sarcoendoplasmic reticulum ca 2+ atpase pump. results are from 3 independent experiments each performed in triplicates (**p,0.01, ***p,0.001 compared to vehicle control). (g) phospholamban phosphorylation upon 24 h poly i:c and dsrna treatment (p-plb -phosphorylated phospholamban). (h) bar graph represents p-plb/a-tubulin ratio from 3 independent western blot experiments (***p,0.001 compared to vehicle control). doi:10.1371/journal.pone.0063776.g005 involvement of serca in the poly i:c induced endothelial dysfunction. our data indicate that poly i:c induces g1 arrest in the hpaecs by inhibiting the function of serca, which is vital for cell cycle control. in rat aortic endothelial cells, a decrease in serca activity resulted in a delayed g1 to s phase transition in the cell cycle [33] . serca dysfunction has been also reported following exposure of pancreatic beta cells to cytokines [48] . however, in our experiments not only the function, but also the expression of serca decreased upon poly i:c treatment, which could result in disturbance of cell cycle regulation [33] . exposure of hpaecs to poly i:c in fact disturbed the regular cell cycle. furthermore, a dose and time-dependent accumulation of hpaecs in the g1-phase was observed. this increase was accompanied by decrease in the number of cells both, in s and in g2/m phase, indicating that the cell cycle is arrested. in addition, poly i:c treatment resulted in inhibition of proliferation, with no increase in apoptosis ( figure s2 , methods s1). as the ca 2+ homeostasis of the cells was altered through affecting the serca pump but without change in apoptosis, we conclude that poly i:c causes ca 2+ mishandling, due to serca downregulation and functional inhibition, thereby leading to g1 arrest and finally to inhibition of hpaec proliferation. in conclusion, our data suggest that exposure to synthetic double-stranded rna modulates ca 2+ signaling in human pulmonary artery endothelial cells by inhibiting their ca 2+ extruding pump, the sarco-endoplasmic ca 2+ atpase. the cell cycle and the cell monolayer integrity are affected resulting in an accumulation of the hpaecs in g1 phase of the cell cycle. the circulating dsrna due to reducing phospholamban phosphorylation may alter intracellular ca 2+ homeostasis and thus cell growth, leading to endothelial cell dysfunction. it is tempting to hypothesize that circulating extracellular rna (e.g. in viral infection) acts as a barrier disrupting agent and contributes to the development of human pulmonary vascular dysfunction. figure s1 bar graphs represent the tlr3 gene fold change compared to untreated control after 24 hours of poly i:c or l-dna stimulation. (tif) figure s2 bar graphs represent percentage of apoptotic cells as measured by pi and annexin v staining upon 24 hours of stimulation (stauro -staurosporin; ***p,0.001 as compared to control). methods s1 quantitative rt-pcr of tlr3 and apoptosis measurements in hpaecs. (docx) molecular mechanisms of endothelial hyperpermeability: implications in inflammation mechanisms of disease -atherosclerosis -an inflammatory disease key differences in tlr3/poly i : c signaling and cytokine induction by human primary cells: a phenomenon absent from murine cell systems a second binding site for double-stranded rna in tlr3 and consequences for interferon activation structural basis of toll-like receptor 3 signaling with double-stranded rna when two strands are better than one: the mediators and modulators of the cellular responses to double-stranded rna a key role for toll-like receptor-3 in disrupting the hemostasis balance on endothelial cells extracellular rna mediates endothelial-cell permeability via vascular endothelial growth factor activation of endothelial toll-like receptor 3 impairs endothelial function modulation of doublestranded rna-mediated gene induction by interferon in human umbilical vein endothelial cells toll-like receptor signaling tlr3 in antiviral immunity: key player or bystander? ifn-alpha enhances tlr3-mediated antiviral cytokine expression in human endothelial and epithelial cells by up-regulating tlr3 expression cxcr2/cxcr2 ligand biological axis impairs alveologenesis during dsrnainduced lung inflammation in mice tlr3 activation stimulates cytokine secretion without altering agonist-induced human small airway contraction or relaxation long-term activation of tlr3 by poly(i:c) induces inflammation and impairs lung function in mice doublestranded rna exacerbates pulmonary allergic reaction through tlr3: implication of airway epithelium and dendritic cells double-stranded rna induces similar pulmonary dysfunction to respiratory syncytial virus in balb/c mice signaling mechanism of extracellular rna in endothelial cells extracellular rna constitutes a natural procoagulant cofactor in blood coagulation peripheral blood mononuclear cells increase the permeability of dengue virus-infected endothelial cells in association with downregulation of vascular endothelial cadherin ca2+ signaling, trp channels, and endothelial permeability sustained ca2+ transfer across mitochondria is essential for mitochondrial ca2+ buffering, store-operated ca2+ entry, and ca2+ store refilling ion channels and their functional role in vascular endothelium store-operated calcium entry promotes shape change in pulmonary endothelial cells expressing trp1 functional role of trpc channels in the regulation of endothelial permeability requirement of the inositol trisphosphate receptor for activation of storeoperated ca2+ channels ca2+ refilling of the endoplasmic reticulum is largely preserved albeit reduced ca2+ entry in endothelial cells the sarco/endoplasmic reticulum calcium-atpase 2b is an endoplasmic reticulum stress-inducible protein ca2+-pumps and na+-ca2+-exchangers in coronary artery endothelium versus smooth muscle serca pump isoform expression in endothelium of veins and arteries: every endothelium is not the same modulation of b-cell endoplasmic reticulum calcium homeostasis by epstein-barr virus latent membrane protein-1 rid e-8501-2011 rid b-2880-2009 the exit from g(0) into the cell cycle requires and is controlled by sarco(endo)plasmic reticulum ca2+ pump endothelium-derived prostaglandin i-2 controls the migration of eosinophils alterations in the ankyrin domain of trpv4 cause congenital distal sma, scapuloperoneal sma and hmsn2c a new generation of ca-2+ indicators with greatly improved fluorescence properties changes induced by hyperosmotic mannitol in cerebral endothelial cells: an atomic force microscopic study regulation of cerebral endothelial cell morphology by extracellular calcium a novel coronavirus associated with severe acute respiratory syndrome protein kinase c modulates pulmonary endothelial permeability: a paradigm for acute lung injury bluetongue virus and double-stranded rna increase human vascular permeability: role of p38 mapk novel roles of tlr3 tyrosine phosphorylation and pi3 kinase in double-stranded rna signaling phosphatidylinositol 3-kinase regulates ca2+ signaling in pancreatic acinar cells through inhibition of sarco(endo)plasmic reticulum ca2+-atpase endothelial permeability is controlled by spatially defined cytoskeletal mechanics: atomic force microscopy force mapping of pulmonary endothelial monolayer stiffness and heterogeneity of the pulmonary endothelial glycocalyx measured by atomic force microscopy phospholamban is present in endothelial cells and modulates endothelium-dependent relaxationevidence from phospholamban gene-ablated mice ca2+-related signaling and protein phosphorylation abnormalities play central roles in a new experimental model of electrical storm cytokines downregulate the sarcoendoplasmic reticulum pump ca2+ atpase 2b and deplete endoplasmic reticulum ca2+, leading to induction of endoplasmic reticulum stress in pancreatic beta-cells the excellent technical assistance of maria schloffer, alexandra hof and elisabeth pöllitzer is greatly appreciated. the authors are thankful for the valuable suggestions and helpful discussions given by dr. andelko hrzenjak, prof. wolfgang f. graier and prof. klaus t. preissner and especially to prof. kenneth e. weir for carefully reading the manuscript. key: cord-261908-olcuq6tm authors: lai, ka-man; bottomley, christian; mcnerney, ruth title: propagation of respiratory aerosols by the vuvuzela date: 2011-05-23 journal: plos one doi: 10.1371/journal.pone.0020086 sha: doc_id: 261908 cord_uid: olcuq6tm vuvuzelas, the plastic blowing horns used by sports fans, recently achieved international recognition during the fifa world cup soccer tournament in south africa. we hypothesised that vuvuzelas might facilitate the generation and dissemination of respiratory aerosols. to investigate the quantity and size of aerosols emitted when the instrument is played, eight healthy volunteers were asked to blow a vuvuzela. for each individual the concentration of particles in expelled air was measured using a six channel laser particle counter and the duration of blowing and velocity of air leaving the vuvuzela were recorded. to allow comparison with other activities undertaken at sports events each individual was also asked to shout and the measurements were repeated while using a paper cone to confine the exhaled air. triplicate measurements were taken for each individual. the mean peak particle counts were 658×10(3) per litre for the vuvuzela and 3.7×10(3) per litre for shouting, representing a mean log(10) difference of 2.20 (95% ci: 2.03,2.36; p<0.001). the majority (>97%) of particles captured from either the vuvuzela or shouting were between 0.5 and 5 microns in diameter. mean peak airflows recorded for the vuvuzela and shouting were 6.1 and 1.8 litres per second respectively. we conclude that plastic blowing horns (vuvuzelas) have the capacity to propel extremely large numbers of aerosols into the atmosphere of a size able to penetrate the lower lung. some respiratory pathogens are spread via contaminated aerosols emitted by infected persons. further investigation is required to assess the potential of the vuvuzela to contribute to the transmission of aerosol borne diseases. we recommend, as a precautionary measure, that people with respiratory infections should be advised not to blow their vuvuzela in enclosed spaces and where there is a risk of infecting others. aerosols play an important role in the spread of communicable diseases [1, 2] . aerosol transmission can be airborne, where contaminated droplet nuclei exhaled by an infected individual are inhaled by a susceptible individual. a second route of infection is when deposited droplets are carried to the mouth or nose through physical contact, often by hand [3] . airborne aerosol transmission is believed to make a major contribution to the spread of diseases such as tuberculosis and measles [4, 5] . aerosols have also been implicated in the transmission of diseases such as the common cold, chickenpox, rubella, influenza, pneumococcal disease and severe acute respiratory syndrome (sars) [3, 6, 7, 8, 9, 10] , although their contribution is less clear cut as non aerosolized respiratory secretions also contribute to the spread of these diseases. some airborne pathogens are extremely contagious; in the usa an outbreak of measles was traced to a sporting event where transmission was found to have occurred between an athlete in the arena and spectators in the stadium, with no evidence of close contact [11] . spread of respiratory disease is of particular concern in large crowds and at international gatherings [12, 13] . this includes the annual muslim pilgrimage to mecca and associated sites, during which respiratory infections are the most common cause of hospitalization [14] . the reported infections include tuberculosis and influenza and the saudi ministry of health recommends wearing of protective face masks by those attending the hajj [13, 15] . the emergence of epidemic strains of flu have also caused concern; in 2009 fears around the spread of influenza h1n1 resulted in a temporary ban on public events in some countries [16] . aerosols are created and expelled into the atmosphere during coughing, sneezing, singing or talking. if the person has a respiratory infection a proportion of the aerosols may carry pathogenic particles [17, 18, 19] . the size of a contaminated aerosol droplet is crucial in determining its ability to transmit disease. whereas large drops (.100 microns diameter) will rapidly fall to the ground smaller droplets may remain suspended in the air where evaporation can occur resulting in the formation of tiny 'droplet nuclei' that can stay airborne for hours or days [20, 21] . these particles can be breathed in by susceptible individuals who may then become infected. the fate of the droplet nuclei on inhalation also depends on their size; particles greater than five microns are likely to remain in the upper airways but smaller particles are more likely to deposit in the alveoli and so may transmit infections of the lower respiratory tract such as tuberculosis [22, 23] . the vuvuzela is a plastic blowing horn that has been adopted by sports fans to provide audible support for their team. it is used in several countries in asia and africa and is particularly popular in south africa where it figured prominently during the recent fédération internationale de football association (fifa) world cup. the instrument is typically 60 cm in length, tapering from a bell end 11.5 cm in diameter to a mouthpiece of 2.5 cm. vuvuzela playing requires forceful and sustained blowing. air from the lungs is expelled from the mouth through vibrating lips held against the plastic mouthpiece and out through the instrument. we speculated that the mode of action may facilitate the propagation and dissemination of aerosols from the respiratory tract of the person blowing the instrument. the instrument is frequently used in crowded situations and it was therefore important to determine the extent of aerosol production to assess whether blowing the vuvuzela might assist in the spread of aerosol borne diseases. to assess the number of aerosols propagated during blowing the vuvuzela eight volunteers (4 male and 4 female) were each given an instrument and asked to blow enthusiastically, as if they were attending a football match. to enable comparison with other activities undertaken at sporting events each volunteer also shouted into a paper cone constructed to have the same 115 mm diameter exhale opening as the vuvuzela ( figure 1 ). particles exiting the vuvuzela or shouting cone were assessed using a laser particle counter and enumerated in six categories according to their diameter. the velocity of air as it exited the devices was measured with a hot-wire anemometer and peak airflows were recorded. the duration of playing was recorded with a stopwatch. triplicate experiments were undertaken for each individual tested. airborne particles exiting the instrument were measured every second throughout the experiment and reported as particles per litre. the mean concentration of particles recorded from playing the vuvuzela and shouting were 658610 3 and 3.7610 3 per litre respectively. to compare the number of particles emitted by an individual when shouting to the number emitted when playing the vuvuzela, the data were log 10 transformed and the difference was calculated for each individual. the mean log 10 difference was 2.20 (95% ci: 2.03,2.36; p,0.001). men expelled particles at a higher mean concentration than the women when playing the vuvuzela (741610 3 vs 575610 3 per litre) although this was not statistically significant (p = 0.69).when shouting there was no difference in the numbers of particles captured (male:female; 3.4610 3 vs 3.9610 3 per litre, p = 0.89). aerosols were enumerated in six size categories according to the diameter of the particle: 0.5-0.7 mm; 0.7-1.0 mm; 1.0-3.0 mm; 3.0-5.0 mm; 5.0-10.0 mm and .10.0 mm. the distribution of particles by size category is presented in figure 2 . the great majority (97%) of particles captured from both the vuvuzela and the shouting cone were between 0.5 and 5 microns in diameter and small enough to enter the lower respiratory tract. the geometric mean (gm) particle diameter was calculated for each experiment and is presented in table 1 . slightly larger particles were emitted when playing the vuvuzela compared to shouting the mean duration for vuvuzela playing events was 2.1 sec (range: 1.25-3.90 sec) and the shouting lasted for an average of 2.2 sec (range: 0.96-4.72 sec). the peak velocity of air exiting the vuvuzelas was higher than from the shouting cone with a mean of 0.59 ms 21 (range:0.12-1.80 ms 21 ;) compared to a shouting mean of 0.18 ms 21 (range: 0.07-0.32 ms 21 ) and this difference was statistically significant (p = 0.03). this was equivalent to airflow of 6.1 and 1.8 ls 21 respectively, for the vuvuzela and shouting. although the duration in playing vuvuzelas between females and males were similar (2.1 sec), the mean peak airflow was nearly double in males compared to females, 7.9 compared to 4.3 ls 21 (this difference was not statistically significant p = 0.19). the difference between females and males in shouting was not as apparent, although males also had a higher peak airflow compared to females, 2.1 compared to 1.6 ls 21 (p = 0.37). we have estimated the numbers of aerosols exiting the vuvuzela when blown by male and female adults. in triplicate experiments from eight individuals the mean concentration of particles exiting the vuvuzela was 658,000 per litre. the mean peak volume of air exiting the instrument was 6.1 litres per second. thus we estimate that approximately 4 million particles per second were being disseminated from the vuvuzela at peak blowing times. for shouting we estimated a peak aerosol concentration of 3,700 per litre or 7,000 particles per second (assuming peak flow volume of 1.8 ls 21 ). the data we obtained for shouting is in broad agreement with a recent study of particles exhaled by healthy adults during normal to deep breathing (tidal volume range: 20-80%) where between 5 and 5,000 droplets per litre were recorded [24] . the differences we observed between male and female volunteers might be explained by differences in their lung capacities, however this was not measured [24] . our results suggest that the vuvuzela is an efficient means of propagating large numbers of aerosols. the great majority of particles measured were of a size that could remain suspended in the air as droplet nuclei and would be capable of entering the alveolar airspaces of the lung. during normal (resting) breathing an adult inhales approximately 7 litres of air each minute, of which 5 litres reaches the respiratory bronchioles [25] . when attending a sporting event and surrounded by vuvuzela players a spectator could expect to inhale large numbers of respiratory aerosols over the course of the event. actual exposure would be affected by the proximity of the vuvuzelas and ambient ventilation which would serve to dilute the stream of particles. the large number of aerosols emitted by the vuvuzela raises the possibility that, if used by persons with an infection of the respiratory tract, they could act a conduit for the spread of infectious particles. for ethical and safety reasons we only examined healthy volunteers during this study; assessment of pathogenicity of aerosols disseminated by the vuvuzela will require further study using patients with known respiratory infections. aerosols can be created at various locations within the respiratory tract [26] and carriage of pathogens by exhaled aerosols depends on the site of infection and the quantity pathogenic particles in the airways [18] . we speculate that aerosols propagated while blowing the vuvuzela may originate in either the lower or upper respiratory tract, or the mouth. to obtain the desired trumpet sound when blowing the vuvuzela air is forced through the lips into the opening of the instrument which may serve to create further aerosols, or alter the size of droplets produced elsewhere in the respiratory tract. in addition to the manner in which the instrument was blown the number of contaminated particles expelled will vary according to the pathogen, the site of infection and the extent of disease. some infections may result in inflammation and physiological changes within the respiratory tract that would affect the person's capacity to blow the vuvuzela [27] . in addition, some conditions are associated with changes in the rheology of respiratory secretions that might affect aerosol formation [28, 29] . studies of cough aerosols from pulmonary tuberculosis patients and cystic fibrosis patients with bacterial infections found that the concentration of infectious particles varied widely between patients [30, 31] . to attain an accurate assessment of the vuvuzela's potential to disseminate infected aerosols, sample sizes will need to be increased to include individuals having a range of upper and lower respiratory tract infections. symptomatic and non symptomatic carriers should be assessed. in addition to counting the number and size of particles, the viability of infectious particles should also be assessed. for bacterial infections this might be achieved by modification of a cough aerosol sampling system previously used to assess tuberculosis patients [30] . coughing, sneezing, singing and talking can all produce aerosols capable of transmitting airborne respiratory diseases [17, 18, 30, 32] . reports from earlier investigators suggest that coughs may produce up to 5,000 droplet nuclei and a sneeze may generate as many as 900,000 particles [21, 33] . the data we present suggests that blowing the vuvuzela for even a short time period has the potential to create more droplet particles than either coughing or sneezing. there were some limitations to this study that may have had an impact on the results. the particle counter used to assess the concentration of particles recorded measurements at one second intervals and it is possible that the peak values recorded were not the maximum level of particle produced. as it was not possible to assess variation in flow rates over the blowing period the total number of particles expelled during a blowing or shouting event could not be estimated. the performance of individuals and production of aerosols may have been influenced by their respective lung capacities [24] , this factor was not assessed in the experiment. the use of a paper cone to assess the droplets from shouting was not ideal as the surface areas and shape of the paper cone may increase the chance that particles attach to the surface rather than remain in the airstream, affecting the number and size of particles reaching the counter. as exhaled air cools and mixes with ambient air condensation droplets may form. although ambient air temperature and humidity remained similar in all experiments, the difference in shape between the cone and the vuvuzela may have affected the mixing and rate of formation of table 1 . exhale duration, peak air velocity, particle concentration and mean particle diameter recorded during playing the vuvuzela and shouting by four male and four female volunteers. these transient droplets. a further consideration is that only healthy individuals were recruited for this study, and as described above, it is possible that people with respiratory illness with impaired lung function would perform differently when blowing the vuvuzela. nonetheless we have demonstrated that these plastic trumpets provide an excellent means of propagating respiratory aerosols, exceeding both sneezing and coughing as a means of disseminating droplet nuclei and we conclude that their potential to spread respiratory diseases requires further investigation. the frequency, duration, and vigor of vuvuzela playing will vary considerably from person to person, depending on the occasion and their expertise at blowing and we are unable to comment on the number of aerosols produced during an entire sporting event. a further factor is the environment in which they are used; open situations with a strong draft or breeze will serve to rapidly dilute the aerosols produced but transmission risks may be considerably higher in enclosed arenas. a further risk factor for disease transmission will be the density of vuvuzela players and the prevalence of respiratory infections in the population. as far as we are aware this is the first report in the scientific press regarding the issue of aerosol dissemination by the vuvuzela and no epidemiological data regarding impact of the instrument on disease transmission have been reported. similarly there have been no reports of disease transmission from sharing vuvuzelas, or from transfer of non aerosolized respiratory secretions that collect inside the instruments. the vuvuzela has become popular in south africa, a country with the highest urban prevalence of tuberculosis in the world and that recently experienced a measles epidemic [34] . it has been used at domestic soccer games for the past decade and was adopted by many visiting fans during the 2010 fifa world cup competition. the tournament was held during late june and early july and coincided with the annual flu season. surveillance reports show an increase in the proportion of influenza b compared to previous years, but evidence to link this to the presence of visiting spectators is not presented [35] . similarly a number of measles cases were confirmed amongst visitors from other countries but evidence as to the source of their infections is not available [36] . the plastic vuvuzela is believed to have emerged as a child's toy, before being adopted by sports fans in africa and parts of asia, where it is a multi-million dollar industry. in africa it has become a symbol of the soccer industry but vuvuzelas are also blown by fans of cricket and rugby football. they have been banned from a number of sports grounds due to the volume of noise emitted and safety concerns arising from their ability to nullify public address systems. studies have previously suggested that vuvuzela playing poses a risk of noise induced hearing loss [37, 38] . we recommend that consideration is taken of their propensity to disseminate respiratory aerosols and that persons with respiratory infections be advised not to blow their vuvuzela in places where they risk infecting others. this should include enclosed spaces and crowded venues such as large sporting events. we also recommend that research be commissioned to determine the risks to public health posed by the vuvuzela. this study was undertaken at the healthy infrastructure research centre at university college london. ethical approval was obtained from university college london research ethics committee and informed consent was obtained in writing from all participants. eight healthy volunteers, 4 males and 4 females working in the research centre participated. the experiments were conducted in a closed room free from drafts. the study subjects were in the age range 20 to 45 years and all self reported as being free from illness. to avoid cross contamination a new vuvuzela instrument was provided for each participant (boogie blast co, johannesburg, sa). the velocity of air leaving the instrument was measured using a hot-wire anemometer (testo, ukflow) and duration of playing recorded with a stopwatch. our initial measurements showed that the average time of playing the vuvuzela was about 2 sec. to enable comparison with other activities undertaken at sporting events each individual tested was requested to also shout into a paper cone constructed to have the same diameter exhale opening as the vuvuzela (figure 1 ). subjects were requested to hold their shout for about 2 sec (not compulsory) and to shout the word ''go''. particles exiting the vuvuzela or the shouting cone were measured using a six channel laser particle counter (lighthouse 5016, uk). particles were enumerated in six categories according to their diameter: 0.5-0.7 mm; 0.7-1.0 mm; 1.0-3.0 mm; 3.0-5.0 mm; 5.0-10.0 mm and .10.0 mm. a 0.047 litre sample of air was tested every second and the number of particles recorded. analyses are based on either the average or peak concentration observed during the vuvuzela or shouting event. triplicate experiments were undertaken for each individual tested. volume of the airflow was estimated by multiplying the peak air velocity recorded in the anemometer with the duration of playing and shouting and the surface area of the exhale opening of the vuvuzela and paper cone. in each experiment (in which an individual either shouted or blew on a vuvuzela) data were collected on particle concentration every second. these data were summarized in one of two ways: i) as the concentration observed in the 2 nd second after the start of the experiment, which usually corresponded to the peak concentration or ii) as the average concentration over the length of the shout or vuvuzela blow. all analyses were carried out using both peak and average concentrations. however, since both yielded similar results, we restrict our presentation to the analysis of peak concentrations. the geometric mean size (gm) of particles was calculated for each experiment by fitting a log normal distribution to the particle size data at peak concentration. estimates were obtained by maximum likelihood, allowing for interval-censoring (particle sizes were recorded using the categories 0.5-0.7 mm; 0.7-1.0 mm; 1.0-3.0 mm; 3.0-5.0 mm; 5.0 -10.0 mm and .10.0 mm). each individual shouted and blew the vuvuzela three times. statistical analysis of particle concentrations and gm particle size were based on the means of these triplicate measurements; this was done to eliminate dependence in the data arising from repeat measurements on the same individual. to compare the number of particles emitted when shouting to the number emitted when playing the vuvuzela, we logtransformed each individual's average concentration (averaged over the three measurements) when shouting and when playing the vuvuzela and calculated the difference between these logtransformed values. the confidence interval for the mean difference and p-value were based on a paired (one-sample) t-test. differences in the average (gm) particle size between vuvuzela and shouting were similarly assessed using a paired t-test (although the data were not log-transformed in this instance). comparisons between men and women of particle concentration and airflow were made using permutation tests (based on the wicoxon rank sum statistic) rather than t-tests owing to the small numbers (4 men and 4 women). the mechanism of transmission of pathogenic organisms affecting the respiratory tract 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over response to influenza a outbreak singing and the dissemination of tuberculosis expulsion of pathogenic organisms from respiratory tract airborne infection on air-borne infection study ii. droplets and droplet nuclei the size and the duration of air-carriage of respiratory droplets and droplet-nuclei distribution and deposition of inhaled particles in respiratory tract on the mechanics of droplet nuclei infection. ii quantitative experimental air-borne tuberculosis in rabbits characterization of exhaled particles from the healthy human lung-a systematic analysis in relation to pulmonary function variables respiratory physiology upate in anaethesia: world federation of societies of anaesthesiologists size distribution and sites of origin of droplets expelled from the human respiratory tract during expiratory activities pulmonary function during and after common respiratory infections macrorheology of cystic fibrosis, chronic obstructive pulmonary disease & normal sputum airborne infectious disease and the suppression of pulmonary bioaerosols cough-generated aerosols of mycobacterium tuberculosis: a new method to study infectiousness cough-generated aerosols of pseudomonas aeruginosa and other gram-negative bacteria from patients with cystic fibrosis epidemiology of primary tuberculosis in an industrial school characterization of expiration air jets and droplet size distributions immediately at the mouth opening global tuberculosis control:who severe acute respiratory illness (sari) surveillance: influenza report fifa soccer would cup, south africa: communicable disease risks and surveillance. pretoria: national institute for communicable diseases vuvuzela sound measurements vuvuzela -good for your team, bad for your ears we would like to thank hector altamirano-medina for providing technical support. we are grateful to the volunteers who gave their breath in this study. key: cord-273764-itu39mln authors: li, taisheng; xie, jing; he, yuxian; fan, hongwei; baril, laurence; qiu, zhifeng; han, yang; xu, wenbing; zhang, weihong; you, hui; zuo, yanling; fang, qing; yu, jian; chen, zhiwei; zhang, linqi title: long-term persistence of robust antibody and cytotoxic t cell responses in recovered patients infected with sars coronavirus date: 2006-12-20 journal: plos one doi: 10.1371/journal.pone.0000024 sha: doc_id: 273764 cord_uid: itu39mln most of the individuals infected with sars coronavirus (sars-cov) spontaneously recovered without clinical intervention. however, the immunological correlates associated with patients' recovery are currently unknown. in this report, we have sequentially monitored 30 recovered patients over a two-year period to characterize temporal changes in sars-cov-specific antibody responses as well as cytotoxic t cell (ctl) responses. we have found persistence of robust antibody and ctl responses in all of the study subjects throughout the study period, with a moderate decline one year after the onset of symptoms. we have also identified two potential major ctl epitopes in n proteins based on elispot analysis of pooled peptides. however, despite the potent immune responses and clinical recovery, peripheral lymphocyte counts in the recovered patients have not yet been restored to normal levels. in summary, our study has, for the first time, characterized the temporal and dynamic changes of humoral and ctl responses in the natural history of sars-recovered individuals, and strongly supports the notion that high and sustainable levels of immune responses correlate strongly with the disease outcome. our findings have direct implications for future design and development of effective therapeutic agents and vaccines against sars-cov infection. sars, or severe acute respiratory syndrome, is a serious respiratory illness caused by a novel variant of coronavirus (sarsassociated coronavirus, sars-cov) [1] [2] [3] [4] [5] [6] [7] [8] . others and we have previously demonstrated that the persistent and high levels of n protein-specific and s glycoprotein-specific neutralizing antibody (nab) responses during the first several weeks of infection are correlated with the disease outcomes [9] [10] [11] [12] [13] [14] [15] . however, little is known about magnitude and longevity of the both humoral and ctl responses after prolonged recovery. studying the long-term changes in humoral and ctl responses in recovered patients will not only verify the earlier findings from short-term follow-ups but also to further establish correlates of protection to be generated by future vaccine candidates. starting in march 2003, we have enrolled and sequentially followed up 30 patients who were diagnosed and recovered from sars-cov infection according to clinical criteria released by the world health organization (http://www.who.int/csr/sars/casedefinition/en). sequential blood samples were collected at 1, 3, 6, 12 and 24 months after the onset of symptoms from the enrolled patients at the department of infectious diseases, peking union medical college hospital in beijing under the guidelines of the ethical review committee at hospital. recovered patients were defined as those free from the acute illness (high body temperature, dry cough or light-white sputum, shortness of breath, hypoxia, and air-space consolidation in lungs) approximately 1 month after the onset of symptom with definitive sero-positivity against sars-cov lysates at least two consecutive occasions. clinically, these recovered patients regain their normal body temperature, experience no cough or chest pain, and have normal chest radiograph and normal pulmonary function. the average age of these patients were 37611 with 13 are being male and 17 female. all the participating patients were antibody and antigen negative for hiv-1, cytomegalovirus (cmv), and epstein-barr virus (ebv). for purposes of comparison, blood samples were also obtained from 70 normal healthy age matched individuals. the average age for these individuals is 39610 with 36 are being male and 34 female. using flow cytometry, we first studied the sequential changes in the absolute numbers of total lymphocytes, cd3, cd4, cd8 t lymphocytes, b lymphocytes and natural killer (nk) cells over the two years follow-ups and compared with that from normal healthy controls. as show in fig. 1 , recovered patients clearly experienced two distinct phases of cell restoration in the peripheral blood; an initial rapid phase for all the cell populations studied in the first 3 months after the onset of symptoms followed by a significant slower phase during the subsequent months. during the first 3 months, the average increase for the cell populations studied was as high as 22% per month. the mean absolute total lymphocytes, cd3, cd4, and cd8 t lymphocytes, b lymphocytes and nk cells has increased from 1349 to 1870 cells/mm 3 , 1130 to 1268 cells/ mm 3 , 511 to 591 cells/mm 3 , 440 to 547 cells/mm 3 , 120 to 152 cells/mm 3 , and 103 to 254 cells/mm 3 , respectively. the rapid phase for lymphocyte recovery is reminescinet of what had reported through the cross-sectional studies on the recovered sars patients during the first few weeks of onset of symptom [2, 7, 8, 16, 17] . as we and others shown previously, the initial rapid phase in peripheral lymphocyte recovery usually coincided with the improving clinical condition of sars patients [2, 7, 8, 16, 17] . after the first 3 months, however, the percent of increase dropped to 0.07% per month and, in most cases, remained unchanged or slightly decreased from the previous time points (fig. 1) . such distinct rate of lymphocyte recovery in the two phases is likely reflective of different mechanisms in lymphocyte regeneration, proliferation and distribution in vivo. furthermore, with the exception for b lymphocytes, the mean absolute numbers for total lymphocytes, cd3, cd4, cd8 t lymphocytes, and nk cells at 24 months after the onset of symptom remained statistically different from that in normal healthy age-matched controls. this finding suggests that complete restoration of peripheral lymphocyte may require a longer period or peripheral lymphocyte reduction in sars patients is permanent despite of recovery from clinical manifestation of sars-cov infection. longer follow-ups of these patients will be needed to address these two possibilities. to study the sequential changes in humoral responses against sars-cov, we used our previously published elisa-based and pseudotyped retrovirus-based neutralization systems [11] . we first analyzed the temporal changes in total serum igg specific for sars-cov, using a commercially available elisa assay (no s20030004, huada comp, beijing, china) based on purified whole virus lysates. as shown in fig. 2a , there was an initial increase in total igg from month 1 to 3 after onset of symptom followed by a gradual decrease over the ensuing period. in fact, much of the decrease was observed during month 3 to 18 after onset of symptom with no significant changes were found afterwards ( fig. 2a ). in addition, we also characterized temporal changes in n protein-specific antibody and s glycoprotein-specific neutralizing antibody (nab) responses in these patients. our experiments were conducted with serum samples in two different dilutions (1/100 and 1/900) and fig. 2b depicts the temporal changes in n protein-specific antibodies over the 24-month followup based on n protein-based elisa. we have found that antibodies against n protein were detectable throughout the entire period of study. within the first 6 month after onset of symptom, n protein-specific antibody remained relatively high although there is a clear trend of decrease over time, with more significant drop in titers between month 6 and 12 after onset of symptom and no dramatic changes afterwards (fig. 2b) . the same trend of changes in n protein-specific antibodies was also observed for experiments conducted with 1/900 dilution (fig. 2b) . in regard to s glycoprotein-specific nab, we used our previously published pseudotyped retroviral system with s glycoprotein on the surface of virion and hiv-1 proteins encapsulated within [11] . consistent with what have been observed for n-protein specific antibodies, high and sustainable levels of s glycoprotein-specific nab were detected throughout the entire phase of study (fig. 2c) . at 1/100 dilution, all samples had potent neutralizing activities capable of neutralizing at least 60% of input virion, with median level activities larger than 95% (fig. 2c ). there is also of trend of decrease in s glycoprotein-specific nab titer over time, but no dramatic drop was found between month 6 and 12 as was found for the n protein-specific antibodies. lastly, the same trend of changes in s glycoprotein-specific nab was also observed for experiments conducted with 1/900 dilution (fig. 2c) . it remains to be seen whether the levels of sars-cov-specific antibodies will remain unchanged or continuously decline after 24 months of follow-up. to study the sequential changes in ctl responses against sars-cov, we used elispot-based technique to quantify the number of inf-c releasing cells in the peripheral blood against peptide pools covering the entire n protein derived from the urbani strain [3] . the peptide pools were made of 57 peptides which are 15-18 amino acid residues in length overlapping by 10 amino acid residues. the peptide pools were obtained through the biodefense and emerging infections resources repository at national institute of health (nih) in the united states (http:// www.beiresources.org/listing/index.cfm). we first studied the sequential changes in total number of inf-c releasing cells, expressed as spot forming cell (sfc) per million pbmc, in the peripheral blood over the 18 months after onset of symptom. fig. 3a shows the average number of inf-c releasing cells detected at month 3, 12 and 18 after onset of symptom. as observed for antibody responses, detectable levels of ctl responses were found throughout the study period, although there is a clear trend of decrease over time (fig. 3a) . to further identify the major epitopes recognized by the ctl, the 57 peptides were further divided into 15 peptide pools in a cross-broad fashion (nx1-7 vs. ny1-8) (fig. 4, upper panel) . the intensity and magnitude of ctl responses against the nx1-7 and ny1-8 peptide pools were presented as either the total number of sfc per million pbmc or the percent of samples having detectable levels of inf-c releasing cells (fig. 3b) . fig. 3b summarizes results for all the samples tested and four peptide pools, namely nx4, nx6, ny6, and ny7, were found to be preferentially recognized by recovered sars patients (fig. 3b ). through the cross-broad analysis, we have found that there are potential two major ctl epitopes in the n protein. one is located within the region covered by peptides nc9568 and nc9569, and the other is covered by peptides nc9584 and nc9585 (fig. 4, upper panel) . peptides nc9568 and nc9569 correspond to amino acid sequence masgggetalalllldrlnqleskv between positions 211 and 235 in the n protein, whereas peptides nc9584 and nc9585 match the sequence of twltyhgaiklddkdpqfkdnvill between positions 330 and 354 (fig. 4, lower panel) . serotyping of mhc class i and ii have found that a2, 3, 11 and 24, b51 and 60, and dr4, 9, 12, and 15, and dq5, 6, 7, 8, and 9 are the predominant among our study population (table 1) . future work would be required to further fine mapping the actual ctl epitopes in these two regions and their association with particular serotypes of mhc class i. in this report, we have extended our early study by sequentially monitoring 30 recovered sars patients over a 2-year period to characterize temporal changes in humoral and ctl responses against sars-cov. we have shown for the first time that recovered patients have persistent and robust binding as well as neutralizing antibody and ctl responses throughout the study period with a moderate decline one year after the onset of symptoms. in particular, s glycoprotein-specific nab responses are persistently high in the recovered patients even 24 months after onset of symptom. such high and sustainable levels of sars-covspecific immune responses in these patients are clear distinction from that found in patients who succumbed to the disease [11] , suggesting that antibody responses likely play an important role in determining the ultimate disease outcome of sars-cov infected patients. in addition, we have also identified two potential major ctl epitopes in n protein based on elispot analysis of 57 peptides. future work will be required to identify the actual epitopes in greater details. however, despite the potent immune responses and clinical recovery, peripheral lymphocyte counts in the recovered patients have not yet been restored to normal levels. this finding suggest that complete restoration of peripheral lymphocyte may require longer period of time, or the lymphocyte destruction during sars-cov infection is permanent and can only be repaired partially. in summary, our study has for the first time characterized the temporal and dynamic changes of humoral and ctl responses in the natural history of sars-recovered individuals and strongly support the notion that high and sustainable levels of immune responses correlated strongly with the disease outcome. we strongly believe that our findings have direct implications for the design and development of therapeutics and vaccines against sars-cov infection and replication. thirty sars patients were enrolled in march 2003 and have continuously followed up since then. they were diagnosed and recovered from sars-cov infection according to clinical criteria released by the world health organization (http://www.who.int/ csr/sars/casedefinition/en). sequential blood samples were collected with their informed consent and approval from the ethical review committee at the department of infectious diseases, peking union medical college hospital in beijing. the average age of these patients were 37611 with 13 are being male and 17 female. for purposes of comparison, blood samples were also obtained from 70 normal healthy age matched individuals. the average age for these individuals is 39610 with 36 are being male and 34 female. for flow cytometric analyses of various lymphocyte populations in the peripheral blood, we used our previously published protocols [17] . analysis of binding antibodies against whole sars-cov lysates or n protein, and s glycoprotein-specific neutralizing antibody (nab) were conducted as previously reported [11] . elispot assays were performed using a commercially available kit (diaclone, france) according to manufacture's introduction. the peptide pools covering the entire n protein of the urbani strain were used to stimulate patients' peripheral blood mononuclear cells. the peptide pools were made of 57 peptides which are 15-18 amino acid residues in length overlapping by 10 amino acid residues. these peptides were obtained through the biodefense and emerging infections resources repository at national institute of health (nih) in the united states (http:// www.beiresources.org/listing/index.cfm). student's t test analysis was used to determine the significance of the results. values of p#0.05 indicated statistical significance. identification of a novel coronavirus in patients with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus a novel coronavirus associated with severe acute respiratory syndrome newly discovered coronavirus as the primary cause of severe acute respiratory syndrome a cluster of cases of severe acute respiratory syndrome in hong kong identification of severe acute respiratory syndrome in canada longitudinally profiling neutralizing antibody response to sars coronavirus with pseudotypes neutralizing antibodies in patients with severe acute respiratory syndrome-associated coronavirus infection antibody responses against sars coronavirus are correlated with disease outcome of infected individuals antibody response of patients with severe acute respiratory syndrome (sars) targets the viral nucleocapsid antibody responses against sars-coronavirus and its nucleocaspid in sars patients longitudinal profile of immunoglobulin g (igg), igm, and iga antibodies against the severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein in patients with pneumonia due to the sars coronavirus detection of specific antibodies to severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein for serodiagnosis of sars coronavirus pneumonia clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study significant changes of peripheral t lymphocyte subsets in patients with severe acute respiratory syndrome we feel in debt to patients' willingness to participate our study. conceived and designed the experiments: zc lz tl jx yh lb. key: cord-013333-7jx4t0ol authors: palloni, alberto; mceniry, mary; huangfu, yiyue; beltran-sanchez, hiram title: impacts of the 1918 flu on survivors' nutritional status: a double quasi-natural experiment date: 2020-10-20 journal: plos one doi: 10.1371/journal.pone.0232805 sha: doc_id: 13333 cord_uid: 7jx4t0ol robust empirical evidence supports the idea that embryonic and, more generally, intrauterine disruptions induced by the 1918-flu pandemic had long-term consequences on adult health status and other conditions. in this paper we assess the 1918-flu long-term effects not just of in utero exposure but also during infancy and early childhood. a unique set of events that took place in puerto rico during 1918–1919 generated conditions of a “double quasi-natural experiment”. we exploit these conditions to empirically identify effects of exposure to the 1918 flu pandemic and those of the devastation left by an earthquake-tsunami that struck the island in 1918. because the earthquake-tsunami affected mostly the western coast of the island whereas early (in utero and postnatal) exposure to the flu was restricted to those born in the interval 1917–1920, we use geographic variation to identify the effects of the quake and timing of birth variation to identify those of the flu. we benefit from availability of information on markers of nutritional status in a nationally representative sample of individuals aged 75 and older in 2002. we make two contributions. first, unlike most fetal-origins research that singles out early nutritional status as a determinant of adult health, we hypothesize that the 1918 flu damaged the nutritional status of adult survivors who, at the time of the flu, were in utero or infants. second, we target markers of nutritional status largely set when the adult survivors were infants and young children. estimates of effects of the pandemic are quite large mostly among females and those who were exposed to the earthquake-tsunami. impacts of the flu in areas less affected by the earthquake are smaller but do vary by area flu severity. these findings constitute empirical evidence supporting the conjecture that effects of the 1918 flu and/or the earthquake are associated not just with disruption experienced during the fetal period but also postnatally. the spanish flu virus of 1918-19 is an example of a perfect storm: like hiv and unlike seasonal influenza, it was highly lethal but, unlike hiv and like other influenza, it was rapidly and efficiently spread [1] [2] [3] [4] [5] . the combination of these two traits made the pandemic one of the deadliest in human history [6, 7] . as in the rest of the world, the a/h1n1 influenza in puerto rico was characterized by its unique temporal sequence, peculiar age pattern, and case morbidity and lethality [8] [9] [10] . jointly, the age pattern of incidence and morbidity and mortality levels, created unfavorable conditions for all but especially for women of childbearing ages and those who were pregnant at the time or who had recently given birth [11, 12] . these conditions may have compromised not just fetal growth but also infant and young children' health, both highly dependent on maternal health status and parental care. as if the onslaught of the flu had not been enough, on october of 1918, precisely when the pandemic was gathering force during its second, most lethal wave, a strong earthquake (the san fermin earthquake) struck the western part of the island. this was immediately followed by a tsunami, two major aftershocks in a two-month interval following the earthquake, and multiple smaller ones spread over the subsequent year or so [13] . research on the lasting effects of the 1918 pandemic are strengthened by the fact that the event can be considered a quasi-experiment: it was unexpected, difficult to avoid and, in most cases, there were no contemporaneous exogenous events that could have produced similar outcomes [8, 11] . the puerto rican earthquake was also unexpected, hard to avoid in areas struck by it and, asides from the flu pandemic, unaccompanied by other major events that could have scarred even more an already vulnerable population. thus, in one stroke, an unlikely combination of two events handed us conditions of a unique double quasi experiment. this paper departs somewhat from others on effects of 1918 flu pandemic. first, it seeks to shed light on a rather unexplored dimension of the 1918 pandemic, namely, its effects on markers of nutritional status of individuals who were exposed to it in utero or during infancy. with the exception of one study [12] , we know of no other attempt to investigate such an association. analyses of impacts on the nutritional status of 1918 flu survivors require a focus on mechanisms that could disturb physiological growth and developmental processes during infancy, early childhood and even early adolescence, not just those that operate in utero. it is known that embryonic and, more generally, intrauterine disruptions influence neural development (brain tissue), metabolic balance (pancreas, liver), nephron growth (kidneys and blood pressure regulation) or lung and heart functioning [14] [15] [16] [17] . in addition, embryonic and fetal development is also about growth of cartilage, bone, and muscle tissue, all of which are implicated in subsequent postnatal physical development [18] . furthermore, impairment of growth processes that occur during the fetal period can be aggravated if postnatal conditions deteriorate. thus, fetal growth could be compromised when pregnant mothers experience illnesses and are exposed to extreme stress. in addition to these pre-natal mechanisms, the flu and the earthquake separately and jointly must have triggered conditions that, in all likelihood, disrupted processes of growth and development that take place during the first year of life and even later, during early childhood. with the benefit of hindsight that the covid19 pandemic permits, we know that this must also have been the case in 1918 as infant and child malnutrition in low-to middle -income countries today are expected to be severely affected with lasting future consequences [19] . the social and economic aftermath of the 1918 flu must have borne close resemblance to the devastation being caused by covid19 and the interventions implemented to halt the spread of the virus: sharp declines in household income, increases in poverty and destitution, lack of access to social, health and other protective services, and massive disruption of food supplies and other resources. but there is more: when, due to illnesses or death, mothers cannot breastfeed normally, are unable to provide adequate maternal care, proper nutrition, grooming, and hygiene, early growth and development could go astray. furthermore, when infants and children are exposed to subsequent, lasting adverse environmental and material conditions, catch-up growth may be a non-starter [20, 21] . if, in addition, an earthquake-tsunami struck some areas, the destruction there could only have been made worse. for these reasons, it is important to assess not just the 1918 flu 's impacts of in in-utero exposure, but also those generated by adverse postnatal conditions. second, we build the case on a unique quasi-experimental research design, a product of the occurrence of two simultaneous events, one involving timing of exposure (to the flu and the earthquake) and the other geography of exposure (areas with different flu severity and differentially affected by the earthquake-tsunami). we aim to show that the flu pandemic and the earthquake-tsunami combine to generate impacts that neither of these events could have produced separately and are strongly associated with both gestational and postnatal exposures. human physical growth depends on early embryonic and fetal events, maternal exposures (including stressors), maternal health status, and parental effects, including maternal capacity to nourish during the fetal and postnatal stages [22] [23] [24] . of particular importance is the length and intensity of breastfeeding [25] [26] [27] , protection from infections and parasitic diseases [28] , recovery from illness [29, 30] and reduction of environmental stressors [31] . these parental effects are strongly associated with maternal (and paternal) health status, household (family) environments and access to resources. embryonic and fetal growth. by and large, fetal nutrition depends on maternal diet and placental capacity to deliver nutrients (including oxygen, fat, proteins, hormones, scfa) [32] . it is well-known that maternal nutritional status influences the entire process of fetal development and can have strong impacts of the infant's subsequent growth [33] . it is also known that poor maternal health status can derail the normal course of a pregnancy and complicate delivery. in particular, maternal infections during pregnancy could compromise normal fetal development and the ultimate effects depend on both the timing of infections, their intensity, and duration. these effects are associated with inflammatory responses induced by the infections themselves. in addition to the potentially fetal organogenic damage associated with the flurelated cytokine storms [2, 34] , bouts of hyperthemia induced by the inflammation can also lead to deleterious outcomes, including miscarriages, premature labor, stillbirths, congenital anomalies, and growth restrictions [35] [36] [37] . the latter are a result of irregularities of the physiology of bone and muscle tissues formation. as is known, bone develops from embryonic mesoderm and proceeds by ossification of cartilage tissue formed from mesenchyme. in addition to bone formation, hyperthermia can also affect limb myogenesis as it disrupts and delays the involvement of several crucial regulatory factors. jointly, dysregulation of bone and muscle tissue formation can compromise normal physical growth [37] . early and late infant development. because of mother's milk properties, intensity and length of breastfeeding is of crucial importance for an infant's early growth, particularly during the first 6 months of life [38, 39] . aside from its beneficial nutritional properties [40] , breastmilk contains important compounds that strengthen infants' immune response and act as a shield to reduce risks of viral, bacterial, and parasitic diseases [41] . most infant infections and parasitic diseases reduce appetite, limit food intake and/or impair the child's nutrient absorption capabilities [42] . thus, the combination of illnesses and breastfeeding interruption, cessation, or irregularities during the first 6 months can compromise not only the quality and quantity of nutrients available for early growth but also reduce absorption and metabolization of those available [43] . these disruptions compromise the ability of an organism to satisfy energetic demands to sustain rapid cell division and organ growth and formation during critical periods [31] . although early growth faltering can be offset by subsequent catch-up growth phase, this will not take place in the absence of material conditions that can sustain rapid growth and maturation [20, 44] . in populations with widespread poverty and vulnerable maternal health status, the process of catch-up growth may never get off the ground and children who could have benefitted from it will fail to attain physical growth milestones [45] . gender differentials in early growth and development. there are important differences between female and male physical growth and development trajectories [46] . should one expect that responses to insults are also different? why should this be the case? there are three sets of factors that can cause gender differentials, one promoting stronger effects among females and the other generating stronger effects among males. first, male embryos are known to be more vulnerable than female embryos [18] . this implies that an early sieve operates to remove some of the vulnerable male embryos even in the absence of exogenous shocks. if, in addition, conditions under which growth and development take place turn negative during critical periods, male embryos and fetuses that could have experienced more serious post-natal developmental problems will never be born. furthermore, it is well-known that male infants experience higher mortality than female infants [47] . excess infant mortality can operate as a second sieve disproportionally selecting out more frail male births. thus, it is quite possible that selection forces alone result in stronger effects among females who are, on average, subject to weaker early selection pressures. however, a second set of factors could amplify effects among males and attenuate them among females. in many low-to middle-income countries, in the past and even more recently, there was pervasive male child preferences that, despite not resulting in outright child neglect, biased the flow of scarce parental resources toward male children [48, 49] . it is plausible to conjecture that under added sources of stress induced by exogenous shocks, male child preferences could have more dire consequences among females than during normal periods, particularly among girls who are more vulnerable to begin with. this is a source of selection that should attenuate effects of early insults among females since those who survive them are of lower than average frailty. finally, an alternative explanation that does not rely on selection arguments has to do with gender differentials in growth and development. it has been known for quite some time [50] that male physical growth stretches over a longer period of time than female physical growth. this increases males' window of opportunity for replenishment and recovery from past interruptions of development and growth. if this is so, males who survive to adulthood could have benefited from these added opportunities and be less likely to carry through life scars associated with very early insults. given the nature of our data, it is impossible to sort out between these various forces that combine to produce observed gender differential in responses to the flu and earthquake. long lasting effects of the flu. the above considerations lead us to hypothesize that exposure to the flu during two critical periods, e.g in utero and/or during infancy, must have had non-negligible influences on early nutritional status and should be reflected in poor adult markers of physical growth. by the same token, exposure to stresses and material deprivation brought about by the earthquake-tsunami could have disrupted embryonic, fetal and postnatal growth and, as consequence, facilitated growth faltering and attainment of substandard markers of physical growth. furthermore, as did happen in other populations, the effects of the flu are likely to be stronger among those who experienced the pandemic in areas more severely affected by it [8, 11] . finally, both in utero and postnatal vulnerability to the flu was likely augmented by conditions associated with the earthquake [44] . if so, we should find that the impact of the flu among individuals exposed to the pandemic in utero or during the first year of life) and individuals exposed later in childhood or adolescence) is larger among those born in areas struck by the earthquake-tsunami (e.g. exposed to the earthquake) than among those born elsewhere in the island. to test these hypotheses, we use a study design that relies on geographic identification of local areas (municipio of birth) classified by flu and earthquake severity. we use dates of birth to assess exposure during critical periods. table 1 is a representation of the design. we use prehco (puerto rican elderly health conditions) data base [51] . prehco is a twowave panel of the non-institutionalized puerto rican population aged 60 table 2 . puerto rican birth cohorts born before 1927, were exposed to mortality experiences similar to those in latin american countries. their infant and early child mortality levels correspond to life tables with life expectancies at birth not exceeding 35 years and are in the range of 180-220 per 1,000 births. up until 1950 and during their late adolescence and early adulthood, these birth cohorts' mortality conditions are embedded in life tables with life expectancy at birth of around 45-50 years. mortality improvements relative to early infancy and early childhood largely originate in eradication of parasitic and vector born infectious diseases (period 1910-1930) as well as to increasing standards of living that marked the period 1900-1940. finally, their post-1950 mortality experience is heavily influenced by the introduction of (ii) the severity of the earthquake is gauged by distance to epicenter and we classify municipios as affected (high severity of earthquake) and non-affected (low severity of earthquake) according to distance from epicenter (see methods section). (iii) the exposure contrast between cells a-d, on one hand, and cells e-h, on the other, is associated with a cohort' s timing of birth and the conditions to which they were exposed during two critical period (in utero and infancy). birth cohorts belonging to these cells are at risk of developing growth problems whereas those belonging to cells e-h are not. the contrast between cells a and b, on one hand, and c and d, on the other, is associated with severity of the event, namely, severity of the 1918 flu and "severity" of earthquake (municipios that suffer the earthquake vs municipios that were less affected or not affected at all). there are also contrasts between birth cohorts that belong to mixtures of municipios of type a or b and c or d. thus, those exposed individuals belonging to cohorts born in municipios that are simultaneously of type b and d are expected to suffer the most. we expect no such contrasts across birth cohorts born in municipios of type e-h. https://doi.org/10.1371/journal.pone.0232805.t001 chemotherapy (antibiotics, sulfa) and vaccinations as well as by increased prevalence of modern chronic illnesses, including type ii diabetes, heart disease and cancer [52] life expectancy at birth between the years 2000 and 2005, the period during which prehco was fielded, was of the order of 78-79. these cohorts' life expectancy at ages 60 is of the order of 18-20 years. in summary, the birth cohorts we study experienced highly heterogeneous mortality conditions throughout their lifetime, ranging from severe during their early childhood years to mild and lenient in later life. flu exposure. in contrast to other studies of the 1918 flu, we identify a wider period during which exposure is assumed to have taken place and, in addition to the fetal period, include time intervals during which post-natal care may have been disrupted as a consequence of the epidemic and/or the earthquake. if, as it is most likely, the post-natal mechanisms are also relevant for outcomes other than physical growth markers (adult health, mortality, cognition, educational attainment, etc. . .), studies that ignore them will underestimate the total effects when using a restrictive definition of exposure for some "treated" cases will be assumed to be "controls". to capture the extended exposure including gestational and post-natal exposures we define a dummy variable attaining the value 1 if an individual's birth is reported to have taken place during 1918 or during the first six months of 1919 (see appendix for alternative definitions of exposure). this indicator is a compromise between preservation of the ability to assign effects to fetal and post-natal exposure according to timing and duration and sample size constraints. flu severity. because we lack information on the incidence and case fatality of the pandemic in puerto rico, we follow past research and create a proxy indicator of flu severity using the excess total mortality registered during the flu period [1] . to construct an index of severity we consider total mortality during the two years period 1918-1919 for each of the 76 municipios, the smallest administrative units in puerto rico, estimate expected deaths using age-specific mortality rates during 1918 in the us, and then compute the ratio of mortality rate observed in a municipio to the observed rate [53] . the resulting quantity is an indirectly standardized mortality ratio, a conventional index computed when information of age specific death rates is absent. we classify as high severity all municipios above the 90th centile of the severity index distribution. those between the 50th and 90th centiles are classified as intermediate severity and the remaining as low severity (see s1 fig) . earthquake-tsunami exposure. exposure to earthquake-tsunami is assessed according to the timing of birth of a cohort and municipio of birth. we consider that a birth cohort's early conditions may have been affected by the earthquake if the timing of birth falls within the same window defined as critical for the flu (see above). thus, to capture extended exposure including gestational and post-natal exposures we use the same dummy defined to flag flu exposure. earthquake severity. we classify municipios of birth into three groups depending on the severity of the earthquake: (i) most severe (all municipios in the west coast of puerto rico, (ii) mild (all municipios to the east and immediately adjacent to those classified as severe and (iii) not severe, (the remaining municipios) (see s4e table for municipio membership by classes in what follows we use a 0/1 dummy variable to flag municipios in group (i). municipio poverty levels. to assess poverty levels in municipios of birth we adopt the classification constructed by clark [54] . municipios were grouped into three classes according to their population size, assessed value, and government income. a total of 25 municipios are plos one either in the wealthiest or an intermediate class and the remaining ones are in the poorest category. in this paper we use a 0/1 binary indicator to contrast the poorest and the remaining municipios. when using this indicator in models with no fixed effects we verify that the indicator behaves as expected, namely, it is strongly and negatively correlated with both knee height and height. the models we discuss in this paper, however, only rely on a fixed effects formulation and the impact on municipio of birth poverty levels is absorbed by the fixed effects. thus, an important set of confounders associated with early poverty are neutralized in the fixed effect model. markers of early nutritional status: knee height and adjusted height. we use pre-hco's anthropometry module for the assessment of height and knee height [55] . to attenuate biases due to skeletal compression, we adjust height measures using estimates of compression by gender and age observed in a sample of individuals who were followed for a long period of time (see s2 fig) [56] . the magnitude of the adjustments is considerable and, if anything, they will lead to overcorrection and downward biases of effects on height. on the other hand, knee height is also a marker of early nutritional status and is unaffected by skeletal compression. other outcomes are frequently studied in the literature on the 1918 pandemic. among them is bmi. we do not examine these here since our interest is on markers of early nutritional status and neither bmi nor any of many indicators available in the survey are suitable. we use ols regressions with municipio fixed effects and treat adjusted height and knee height as continuous variables. we also estimated sur, models to account for correlation between knee height and height. results from these models are available upon request. although there are some differences between sur and ols estimates (standard errors of regression coefficients), the differences are minor and do not affect inferences. we also estimated biprobit models using binary indicators for knee height and height. the drawback of these models is that they depend on arbitrary cut points. in all cases we use our preferred measures of exposures, namely, exposure = 1 to flag birth cohorts born during the critical period defined before and to distinguish them from birth cohorts born before and after such critical period (exposure = 0). we consider severity of flu using two alternative indicators. the first is a single dummy variable (severity_flu) that distinguishes between municipios where the flu is considered to be severe from those in which it was mild. the second consists of two dummies that identify municipios with severe and intermediate severity. a dummy variable, severity_earth, serves to distinguish municipios which were severely affected by the earthquake from all others. in addition, the model contains first, second and third order interaction effects. the specification for outcome k (k = 1 for knee height and k = 2 for height) is where z ki is the outcome k for individual i. c i is a vector of control variables that includes years of education, year of birth, and a dummy variable for gender. this regression formulation is a difference-in-difference model that seeks to identify (i) differences in the impact of the flu by timing of exposure between high and low flu severity areas and (ii) differences in the impact of the earthquake by timing of exposure between areas affected by high and low severity of the earthquake-tsunami and, finally, differences in the effect of exposure between areas affected by both high severity of flu an earthquake and those only impacted by one of these. exposure i is a 0/1 variable for exposure to flu/earthquake. severity_flu i is a 0/1 variable for flu severity in the municipio of birth; in some models we used a more fine-tuned classification and employed two dummies to distinguish municipios with high, moderate(intermediate) and low severity. severity_earth i is a 0/1 variable for earthquake severity in the municipio of birth, and ε ki is an idiosyncratic error. in turn, the parameters for the equation of outcome j are a constant, α k , and a vector of effects associated with controls, β k . the effect of exposure, γ k , reflects the combined impacts of flu and earthquake exposure for cohorts born during the critical period. the parameter θ k measures the difference of effects of flu exposure between those born in high and low severity municipios whereas the difference of effects of exposure between those born in municipios affected by the earthquake and those born in the remaining municipios is, δ k. finally, μ k is a measure of the excess impact of exposure during the critical period among individuals born in municipios with high severity flu and affected by the earthquake relative to those also exposed but in areas of low flu severity and not affected by the earthquake. to assess differentials by gender we also introduce interaction terms involving the 0/1 binary variable for gender (female = 1 for females), timing of exposure, and flu and earthquake severity. a few remarks are important. first, our models do not include a control for age as there is no relation between markers of nutritional status and age. second, additional controls were tried but discarded since they did not change results. third, all our initial models included a linear term for birth year to account for secular trends in height and knee height as well as to purge out effects of other time variant factors related to birth cohorts. since estimates from models that did include these covariates are no different from those that did not, we only discuss results from the latter. fourth, we estimate models with municipio fixed effects. this implies that the main effects of earthquake and flu severity, as well as other variables at the municipio level, are absorbed by the fixed effects. finally, we estimate models with adjusted variance-covariance matrices of errors to relax assumption of independence of observations and requiring only. model justification and interpretation. estimates from the above model can be interpreted causally only if some conditions are satisfied. first, other than the earthquake and flu, there are no concurrent phenomena that could influence outcomes. second, the occurrence of earthquake and flu are uncorrelated. third, unmeasured cohort conditions that influence the outcomes of interest vary continuously between cohorts. finally, selection due to differential survival among those exposed and non-exposed is minor. the first condition is likely to be met since, to our knowledge at least, other than being very marginally affected by military mobilization and disruptions caused by wwi, puerto rico was not affected by other simultaneous large-scale disruption. the second condition is also likely to be met. however, our assessment of flu severity relies on estimated excess deaths during the flu period. because the earthquake also contributed to excess deaths, at least in areas severely affected by it, the severity indicator could introduce a correlation between the two events. since in most of our analysis we rely on a coarse binary indicator for severity, this correlation is unlikely to influence results. the third condition refers to the possibility than unmeasured conditions associated with the nutritional status of cohorts born at the time of the flu and earthquake, changed abruptly as a consequence of these shocks themselves and/or some concurrent event. for example, if draftees to wwi armies were disproportionately drawn from middle income classes, the composition of births by class origin during that period suddenly changed. since social class of origin is strongly associated with nutritional status, these birth cohorts would be composed by individuals who are at higher risk of manifesting deficits in adult markers, independently of the flu and the earthquake. but, the population of puerto rican wwi draftees from middle to high classes in us armies must have been insignificant since the total number of puerto rican recruits was in the hundreds, not thousands. the final condition is that there is no selection due to differential survival of individual exposed and non-exposed to exogenous shocks. because conditions that determine poor early nutritional status also increase child and adult mortality risks, it is quite likely that selection in our sample of older adults will induce to downward biases on estimates of effects of exposure to flu and earthquake. in s1 file we provide a rough assessment of the potential magnitude of these biases. we first discuss results from baseline models for knee height and height that only include exposure during the critical period (and years of education as a control variable). we then examine whether there are differences in the effects of exposure across areas with contrasting flu severity. this is followed by a review of models that also include a dummy variable for earthquake and higher order interaction effects. these models enable us to assess whether the pandemic and the earthquake combine to induce impacts of larger magnitude than either event separately. individual regression coefficients are displayed in tables 3-6 (table 7 is a compact summarizes of estimates of total (net) effects.) the flu: models for knee height and height including exposure during critical period and flu severity baseline models. table 3 displays estimates of coefficients for models with knee height and height as dependent variables, control variables, and the additive and interaction effect of gender. as assessed by the overall value of r-square, the model for height fits much better than the one for knee height (.42 vs .15). the reason for this is that gender is a much stronger predictor of height than of knee height. in any case, the goodness of fit of these models is quite good for this kind of individual level data. note that the coefficients for the single control variable, years of education, is very large and in the expected direction. this result is reassuring because it confirms that educational attainment reflects early conditions that influence markers of nutritional status. note that because individual adult educational attainment itself could be influenced by the pandemic, the relation between education and markers of nutritional status could also be spurious. second, the effects of exposure during the critical period (exposure) are in the expected direction although it is only statistically significant in the model for knee height where the magnitude of the coefficient is close to twice the standard error. the reduction in knee height among those exposed during the critical window is about 1.24 cms or 4 percent of the mean value in the sample and the corresponding regression coefficient is marginally significant (-1.24 (.61)). the magnitude of this effect is slightly smaller than a third of the total effect of gender (-3.49), the covariate that has the largest effect in the model. it should be kept in mind that this estimate mixes the impact of exposure to both flu and earthquake. the total reduction in knee height among exposed females is 1.33 (-1.24+(-.09)) and an f-test reveals this is significant at the .04 level. third, the model for height reveals a gender differential that penalizes females: the reduction in height among exposed females is of the order of 2.65 cms, about 2 percent of the mean value in the sample, with a coefficient about twice its standard error. an f-test confirms that the total effect exposure on females' height is different from 0 (pr f o > f � = 10.4 is .0020). thus, exposure during critical period leads to losses of knee height and height but gender differentials in these losses are only manifested in adjusted height. models with flu severity. table 4 displays only estimates of parameters of interest from models that include the interaction term between exposure and flu severity as well as those associated with the gender variable. as in the previous case, the model does not control for earthquake and the effect associated with the variable exposure absorbs both the impact of the flu and the earthquake. three results stand out. the additive effect of exposure on knee height is properly signed and slightly larger than in the baseline model (-1.02 (.67)) whereas the added impact on females is close to zero (.14 (1.04)). the effect of exposure is larger in the hardest hit regions and leads to a decrease of about .86 cms but the regression coefficient does not pass a standard statistical test (-.86 (1.46). third, the impact among exposed females in high severity area is weak (-.67 (2.11)) and statistically insignificant. the model for height leads to somewhat different inferences, however. as in the previous case the additive effect of exposure is small but, unlike the model for knee height, there is an important gender differential as exposed females lose about 3 cms (about 3 percent of the mean) and the regression coefficient estimate is more than twice its standard error (-2.87 (1.42)). in fact, an f-test reveals that the total impact among females (the additive effect of exposure and the interaction between exposure and gender) is statistically different from 0 (pr f o > f � = 9.87 is .0025). however, there is no evidence of a contrast between impact of exposure in the total population (-1.30 (2.58)) nor among females (1.11 (3.80)). fig 2a and 2b display box-plots of predicted values of knee height and height for exposed and non-exposed individuals born in high and low flu severity municipios using coefficients from table 4 . the gradient between exposed and non-exposed and between those born in different flu severity municipios is apparent for knee height and of the order of 2 to 4 cms. the contrasts are in the expected direction for height but are of smaller magnitudes. because we find important differentials by gender, we also estimated a model with the female subsample only. although we lose some statistical power, we gain somewhat by reducing the confounding impact of heterogeneity of effects by gender. furthermore, to fine-tune the indicator for flu severity we use all three classes of severity (small, moderate and severe). table 5 displays the new models' estimates. these suggest somewhat stronger inferences than allowed by the previous model. in fact, although the additive effects of exposure on knee height is correctly signed but small, the interaction effect associated with the most severely affected municipios are quite large and more than twice their standard errors (-1.84(.77)). exposed females in areas of high severity lost a total of nearly 2.64 cms in knee height or about 5 percent of the mean in the sample. the f-test indicates that the total effect of flu exposure is statistically significant (prf o >f � = 79.13 is .0002). the results for height are even stronger: exposed females in high severity areas lost a total of 6.05 cms (-2.81+ (-3.24)) or about 14 percent of the mean in the sample and both the additive and interaction coefficients are statistically significant. as in the case of knee height, the f test for the total impact on height confirms that the overall impact is important (prf o > f � = 5.38 is .024). fig 3a and 3b display plots of predicted values of knee height and height by flu severity using coefficients from table 5 . the gradients are sharp and suggest losses of about 1.5 to 2 cms of knee height and 3 to 4 cms of height among those exposed in high flu severity areas (relative to those born in low flu severity areas). contrasts are large (between 3 and 4 cms of knee height and 5 and 6 cms in height) between those exposed and not exposed. the observed patterns identified above can be summarized as follows: had we been interested only on the impact of the flu and ignored the concurrent earthquake-tsunami, we would have stopped the analysis here. our conclusion would have been that females bore the brunt of the flu pandemic and that those born in municipios hardest hit by the flu experienced damage in both phenotypes of interest as they lose as much as 3 to 4 cms of knee height and 5-6 cms of height. however, during the period that the pandemic prevailed in the island, and coinciding with its peak strength, a strong earthquake-tsunami castigated the west costal and proximate interior region of the island. it is then possible that inferences from models that ignore the earthquake attribute to the flu (in high and low severity areas) impacts that might be attributable to the earthquake. this is an example where the joint occurrence of two exogenous shocks, the flu and earthquake, violates one of the prerequisites needed to justify treating the pandemic as a quasi-natural experiment. the only escape from this trap is to estimate models that account for earthquake severity. could the estimated influenza effects be confounded with impacts of the earthquake? were the effects of the flu magnified in areas affected by the earthquake or those of the earthquake magnified in areas of high flu severity? to answer these questions, we estimated models including the additive and interactive effects of the earthquake. we seek to determine whether (i) there are independent effects of the flu and the earthquake and their relative magnitudes, (ii) there are important synergies between the two events, and (iii) females were singularly affected. table 6 shows results of the full models that include exposure, flu severity, and earthquake severity as well as interaction effects associated with gender. because the introduction of a new dummy for earthquake and associated interaction effects leads to substantial losses of observations in some cells of the data, we return to use the binary variable for severity of earthquake as identified in table. the results for knee height are somewhat mixed but they shed light on an intriguing story and are summarized below. the results for height, on the other hand, are weaker although they too reveal important regularities. knee height. does exposure during critical windows make a difference? the additive effect of exposure in the model for knee height is properly signed but significant only at .10 level (-.95 (.65)). in contrast, exposure during critical windows among individuals born in municipios more severely affected by the earthquake translates into sizeable and significant effects independently of gender. the estimate of the corresponding regression coefficient implies knee height reductions of the order of 3.75 cms and the associated regression coefficient is significant at the .004 level. however, exposure in high flu severity areas does not appear to lead to important reductions in knee height as the estimated effect is negative, as expected, but does not attain statistical significance (-1.26 (2.23)). similarly, exposure during critical periods in municipios of high flu severity and also affected by the earthquake does not result in particularly large negative impact. in fact, the opposite appears to be the case as the additive effect of exposure during critical periods in areas affected by the earthquake and with high flu severity is positive albeit with a large standard error (4.72(2.95)). the final problem we investigate is whether the above patterns are different for females. exposed females in the general population are not affected (2.17 (3.09)). nor are those exposed and born in earthquake municipios (3.72 (3.53)) or exposed and born in high flu severity areas 17 (3.09) ). however, exposed females born in areas struck by the earthquake and of high flu severity are singularly affected. in fact, we find a sizeable and significant impact among females exposed during the critical period who were born on earthquake and high flu severity areas. the estimate of effects for these females implies a reduction of knee height of about -10.28 cms, a figure that is statistically significant despite a large standard error. because the additive effect of exposure in areas of high flu severity and affected by the earthquake implies an average increase in knee height of about 4.72, albeit with a large standard error (2.95), the total effect among females is negative and leads to a loss of 5.68 cms. to check the importance of these effects we employ two f-tests. the first corresponds to the null hypothesis that the magnitude of the overall effects of the earthquake and the flu among females is zero, that is, the sum of the effects females' experience by virtue of being exposed during critical periods, being born in areas affected by the earthquake, and with high flu severity is not significantly different from 0. the observed total or net impact (5.68) yields an f = 24.03 (prf o > 24.03 is .0001). the second test seeks to verify the null hypothesis that the difference between effects of females exposed during critical windows and those not exposed is 0. the observed value of the test statistic is equal to -6.41 among females exposed and -.73 among those not exposed. the difference translate into an f statistic (21.78) that would be observed in a population where the difference is zero with a probability of less that .0001. these two tests offer empirical evidence confirming that females whose exposure during critical windows took place in areas of high flu severity and affected by the earthquake are most damaged by these events. fig 4a and 4b are box plots of predicted values of knee height for selected categories of exposed and non-exposed individuals who experienced combinations of the worst conditions. although there is important within-category variability (particularly in the worst one, namely, among individuals exposed in high earthquake and flu severity areas) the between category gradients are in accordance with the conjectures. height. the results for height are weaker than those for knee height even for females. exposure during critical periods by itself does not influence height (.24 (1.36)) nor does it whether it is combined with the earthquake (.96 (1.80)) or high flu severity areas (-4.84 (4.20)) or both (5.12 (4.64)). the last two regression coefficients are large but very noisy, possibly due to small number of cases. as was the case for knee height, exposed females are more affected as they lose 2.74 cms and the corresponding regression coefficient is marginally significant. females who were exposed in earthquake areas experience reductions in height but the estimate is too noisy (-3.24 (3.93)). the effect on females who were exposed in high severity areas appear to gain in height but, here again, the regression coefficient is subject to quite a bit of uncertainty (or in high flu severity municipios (5.08 (6.19))). finally, and unlike the case of knee height, height reduction among exposed females born in earthquake and high severity areas is large but only half its standard error (-4.39 (7.33)). as in the case of knee height, however, the total impact on exposed females is equivalent to a total loss of about 8.92 cms, and statistically significant (prf o >f = .05). the difference between impacts on females exposed and those not exposed is 5.93 cms (prf o > f = .0018) the above results suggest four inferences. first, the flu pandemic alone had important impacts on knee height and height but only among females born in high severity flu areas. second, the earthquake-tsunami and the flu pandemic combined to induce damage but mostly on females' knee height and somewhat less on females' height. the net effects on knee height are sizeable, translate into reductions of the order of 5.69 cms or about 10 percent of the mean value of knee height in the sample, and are statistically significant from 0. the net effects on height are also statistically significant and equivalent to losses of 10 cms representing about 6 percent of the mean value in the sample. the total effects of female exposure on knee height are two to three times larger among those who were born in municipios with high flu severity and affected by the earthquake. table 7 is a synthetic representation of key results for the female subpopulation. the table includes a brief summary of main findings (columns 3 and 4) and estimates of total effects and associated f-statistics (columns 5 and 6). yellow cells represent findings consistent with initial conjectures. green cells are associated with weaker evidence and grey cells with little or no empirical support. an examination of yellow cells suggests that female exposure during the critical period led to total losses in knee height and height of the order 1.33 and 2.65 cms respectively (first row of and last two columns of table). exposure within high flu severity areas increases these losses to 2.63 a 3.44 cms (fifth row and last two columns of the table). finally, when exposure takes place in areas hit hard by both the earthquake and the flu losses increase to about 5.7 and 9.9 cms respectively (next to last row and last two columns). a cautionary note on interpretation. the impacts in table 7 are not trivial and, given the definition of exposure we use here, they are consistent with the idea that it is the combination of perturbations during the fetal and the postnatal period that mediate the impacts of the two external shocks that struck this population. these effects are larger than those one gets when using an exposure variable that constrains the critical window to the fetal period only. but, how strongly can we lean on this interpretation of findings? although the empirical analysis presented above is suggestive and consistent with the initial conjecture there are alternative interpretations. human markers of physical growth and development, other than those assessed at birth, are shaped by a multiplicity of factors, from genetic make-up to pre-pregnancy maternal health status to exposures in utero, to conditions encountered in infancy, early childhood and adolescence, and to adult and older adult experiences. even though the two markers of nutritional status we use here, height and knee height, measure phenotypes whose growth and development dynamics are subject to important constraints that are set early in life, they are also shaped by conditions experienced after the narrow window we flagged here as critical. furthermore, these conditions are directly or indirectly related to the two exogenous events of interest. fig 5 is a rendition of the main relations involved. the figure is designed to emphasize that the way to secure evidence supporting a strong interpretation that attributes effects of external events on adverse early conditions only is to control for "other factors" also influenced by the events. although the effects of the latter could very well be accounted for by individual measures (education) and contextual indicators (municipio poverty index), all of which are included in our models, they may be insufficient to purge effects represented by the path in the lower part of fig 5. childhood stunting induced by a protracted period of poverty following the earthquake may derail physical growth permanently. if so, the effects we estimate cannot be solely imputed to exposure during the early critical window we focus on here. but, by the same token, catch-up growth in early adolescence, years after the event of interest took place, could conceal effects of exposures in the critical window. these two sets of effects will offset each other, and we will not be able to rank them in importance. for this reason, we argue that it is inappropriate to downplay the mediating role of early conditions. the two markers of physical growth, but less so knee height than height, are influenced by heterogeneous conditions that are experienced in very different windows of time during fetal development (particularly during the last three months of pregnancy), infancy, early childhood and adolescence. because they involve biological processes that unfold at different stages, they are not perfectly correlated. in fact, the correlation between these two measures ranges from .3 to .6 [57] [58] [59] . also, it is known that birthweight is correlated with adult height and that this association is partially explained by intrauterine conditions and genetic factors [60] . although we know of no study confirming this, it must be the case that knee height is also highly correlated with birth weight and, as height, is influenced by very early conditions, e.g. before birth, as well. to capture these processes a more complex model is needed, one that includes measures of exposure in multiple critical periods thus allowing room for an assessment of time-varying effects on single metrics of nutritional status as well as effects heterogeneity across metrics. we conclude that even if we cannot attribute in toto the estimated effects to mediating paths only involving very early conditions, the evidence gathered suggests that these conditions are important contributors to markers of nutritional status. robustness of findings: systematic errors, small sample sizes, the role of chance and selection. although estimates of effects on knee height are, by conventional standards, "statistically significant" (at levels p < .01 or less), we should provide additional empirical evidence to support our inferences. we do this for three related reasons. the first is that the estimates could be contaminated by systematic measurement of errors. the second is that they are based on a small sample from which the model we estimate demand quite a lot. the third is that the results may be due to chance. systematic errors. we conducted two tests to assess possibility of systematic underestimation of knee height among those exposed to the flu and born in areas impacted by the earthquake. first, the distribution of knee height shows no deviant extreme cases and the smallest values in earthquake areas are within 1.5 of a standard deviation. second, in a more radical test we ignored the lowest values of knee height and re-estimated models. to turn estimated effects from worthy of note (p < [.02-.05]) to mundane (p>.05) we need to exclude observations of knee height below the first quartile of the distribution, a rather implausible surgery. the role of chance. most epidemiological and population health research highlights findings on the basis of classic-fisher criteria, that is, based on a priori chosen significance level (say α < .025 in a two-tailed test). the analysis we carried out was couched on this model as we highlighted results that would pass standard statistical tests with α < .025 in two-tailed test. we are saying nothing new when we point out that this type of criterion can be misleading. this is of concern as extreme values of a statistic can be obtained just by chance and are more likely to occur with small samples and demanding models such as ours. to assess this possibility, we pursue two routes: (a) perform a permutation test [61] and (b) compute bounds for false discovery rates [62] . (a) permutation test: we implement permutation tests on estimates of effects obtained in the most complex model that includes third order interaction effects (female exposure x severity x earthquake). the objective is to add some strength to inferences drawn from conventional hypothesis testing, namely, that the effects on knee height (and height) of the size we observe occur with small probabilities, ideally below .05. this exercise suggests that in a permutation repeated 1000 times the coefficient of the most extreme of exposure (in high flu severity and earthquake) would be lower than what we observe between 4 and 9 percent of the time. this is a bit less stringent and provides weaker support than the conventional test statistic (see s3 fig) . (b) false discovery rate: this is the conditional probability that if the null hypothesis is rejected, it is erroneously rejected. this quantity is usually quite different from the conventional α as it is a function of α, power, and the true magnitude of effects. alternative values of these parameters lead to the graph s4 fig. given our p-values ([.01-. 02]) and approximate power (.50-.60), we conclude that the probability of uncovering effects only by chance is between .10 and .30, hardly a comforting range but quite common in clinical and population studies [62] . selection effects. it is highly likely that the sample of survivors to age 70 is highly selected and that selection could have been a function of nutritional status and, therefore, a partial result of the events of interest. prehco is not unlike other survey of elderly people, all of which confront this problem. we argued before that these biases are in all likelihood attenuating estimates of effects. in s1 file we develop simple expressions to evaluate the potential magnitude of these biases and conclude that, under conditions prevailing in puerto rico, we might be underestimating effects by as much as 15 to 20 percent. is the magnitude of the 1918 flu effects relevant? while empirical findings confirm that flu exposure, in the broadest sense defined here, is associated with markers of early nutritional status, it is unclear whether their magnitude is substantively meaningful. to shed some light on this issue we proceed indirectly and compare predicted changes in individual stature associated with flu exposure with changes in stature throughout the period of mortality decline in western europe. because knee height appears to be the metric most sensitive to exposure, an ideal test would have been to use the estimated changes in knee height we observe here as a result of exposure with those experienced by other populations. because there are no historical records of knee height, we cannot perform this test. however, we can exploit the fact that knee height is moderately associated with adjusted height (r-squared~.40 in our sample) and still draw tentative inferences by predicting changes in height from estimated changes in knee height. we find that the reduction in height implied by the estimated reduction in knee height due to flu exposure (between 1.5 and 6.0 cms for an average of .89 of the sample's knee height standard deviation) is associated with a proportionate adjusted height reduction of about .0243 (based on a log-log regression of adjusted height on knee height). note that it took forty years, between 1860 and 1900, for the mean height of the dutch population to experience proportionate gains that are half as large as the losses induced by the flu and earthquake. a similar inference follows from a comparative assessment of the average reduction in height associated with exposure during critical periods in high flu and earthquake severity areas. as an added piece of evidence consider this: an increase of 1 cm in knee height in our sample translates into a decrease in mortality risks above age 75 of the order of 4%. this, in turn, translates into an increase in life expectancy at that age from 5.75 to 6.14, about 7 percent (calculated using life tables for females in model west of the coale-demeny female life table system using levels 7 and 9). is the evidence retrieved here strong enough to support the argument the impact of the flu and earthquake may operate outside the narrow window of fetal exposure? we suggested that past research on the long-run effects of the 1918 influenza may be somewhat limited by a preoccupation with fetal exposure. this is justified since there is strong evidence supporting the idea that embryonic and, more generally, intrauterine disruptions are influential. however, fetal development is also about growth of cartilage, bone and muscle tissue, all of which are implicated in subsequent postnatal physical development. furthermore, impairments in the fetal period can only be aggravated if post-natal conditions are unfavorable, as may happen as a consequence of maternal or paternal illness. this justifies our claim that the flu pandemic could have also perturbed the post-natal period and through both, fetal and postnatal exposures, affected children's nutritional status. our estimates, particularly those for females born in high severity areas and/or in areas impacted by the earthquake-tsunami, are large, statistically "relevant", and robust to checks. this evidence does not imply that fetal exposure is irrelevant but that it, together with postnatal conditions, may combine in a highly poisonous cocktail that impedes attainment of physical growth landmarks. the paper has shortcomings. first, an ideal test of our hypotheses requires to contrast effects in multiple windows of exposure (fetal, infancy, and early adolescence). although we did define alternative critical windows (see s1-s4 tables) model estimation using more than one critical window became quite difficult due data sparsity. second, while both nutritional status markers are sensitive to the exogenous shocks, one is more so than the other. unlike knee height, adjusted height is more likely to be influenced by measurement errors and by events that take place in windows of time more removed from infancy and early childhood. it is difficult to tell whether the differential responses of these markers is as a result of errors or due to real differences in physiological processes that underpin development of different parts of the human body. third, the sample is small and vulnerable to produce effects where there are weak ones. unlike other research, we are not dealing with observations in the tens of thousands but with an effective sample size orders of magnitude below that. buffering against this possibility with permutation tests and computations of false discovery rates can only suggest but not prove that our results are immune to false discovery. fourth, for the most part our results only offer support for effects on females, not on the entire population. although we propose conjectures that could explain gender differentials, we have no way of testing them with the data at hand. finally, and on a different note, puerto rico is a tiny dot in a world map, with a population size that has always been, then and now, an infinitesimal fraction of the total world population. why would anybody bother with all of this? it turns out that the unlikely collusion of two simultaneous natural disasters and the accidental availability of empirical records of survivors, generates a unique opportunity to identify effects of broadly defined early exposures to shocks. in particular, we uncovered some evidence suggesting that past research on the impacts of the 1918 flu pandemic may have missed something important: the influence of the combined disruption of fetal and postnatal life on the ultimate fate of subsequent physical growth. we are not so much trumpeting a new finding as we are identifying a relation that deserves a second look in future research on the 1918 pandemic or, importantly, on the longlasting effects of the covid19 pandemic. supporting information s1 the long-lasting influenza: the impact of fetal stress during the 1918 influenza pandemic on socioeconomic attainment and health in sweden from two mutations, an important clue about the spanish flu death patterns during the 1918 influenza pandemic in chile estimating reproduction numbers for the 1889-90 and 1918-20 influenza pandemics in the city of madrid effects of the spanish influenza pandemic of 1918-19 on later life mortality of norwegian cohorts born about 1900 mortality patterns associated with the 1918 influenza pandemic in mexico: evidence for a spring herald wave and lack of preexisting immunity in older populations updating the accounts: global mortality of the 1918-1920 "spanish" influenza pandemic the long-lasting influenza: the impact of fetal stress during the 1918 influenza pandemic on socioeconomic attainment and health in sweden the long-term consequences of the global 1918 influenza pandemic: a systematic analysis of 117 ipums international census data sets spanish flu and early 20th-century expansion of a coronary heart disease-prone subpopulation is the 1918 influenza pandemic over? long-term effects of in utero influenza exposure in the post-1940 lingering prenatal effects of the 1918 influenza pandemic on cardiovascular disease government printing office fetal origins of adult diease: strength of effects and biological basis mothers, babies, and health in later life the fetal matrix: evolution, development and disease: cambridge development and evolution: cambridge impacts of covid-19 on childhood malnutrition and nutrition-related mortality. the lancet short stature associated with intrauterine growth retardation: final height of untreated and growth hormone-treated children patterns of catch-up growth the develpmental origins of health and disease: cambridge the developmental origins of health and diseases (dohad) concept: past, present, and future london the intrauterine and early postnatal origins of cardiovascular disease and chronic bronchitis the nature of child malnutrition and its long-term implications community and program influences on the mortality of malaysian infants effects of inter-birth intervals and breastfeeding on infant and early childhood mortality synergism of nutrition, infection, and immunity: an overview resistance and life expectancy: disease and death during the economic development of japan an analytical framework for the study of child survival in developing countries. population and development review maternal care, gene expression, and the transmission of individual differencesin stress reactivity across generations building muscle: molecular regulation of myogenesis. cold spring harbor perspective in biology maternal nutrition and fetal development the cytokine storm of severe influenza and development of immunomodulatory therapy viral infections during pregnancy fever in pregnancy and its maternal and fetal outcomes pax3 induces differentiation of juvenile skeletal muscle stem cells without transcriptional upregulation of canonical myogenic regulatory factors the optimal duration of exclusive breastfeeding: a systematic review human milk composition: nutritents and bioactive factors the immunological components of human milk and their effect on immune developmenty in infants nutrition and health from womb to tomb the interaction between nutrition and infection: world health organization the developing human: clinically oriented embryology ( 8th ed): philadelphia: sanuders maternal and child undernutrition: global and regional exposures and health consequences long-run consequences of exposure to natural disasters boys live dangerously in the womb regional model life tables and stable populations the care of children: the influence of medical innovation and medical institutions on infant mortality 1750-1914 diffusion of ideas about personal hygiene and contamination in poor countries: evidence from guatemala. social science & medicine sex differences in human development inter-university consortium for political and social research [distributor adult mortality in latin america and the caribbean observations on mortality during the 1918 influenza pandemic porto rico and its problems prehco protocol: an exercise to examine interviewer measurement error for anthropometric measures. unpublished report decline of height with age in adults in a general population sample: estimating maximum height and distinguishing birth cohort effects from actual loss of stature with aging stature estimation in critically ill patients equations for predicting stature in white and black elderly individuals stature predictions equations for elderly hispanics in latin america by sex and ethnic background associations between birth size and later height from infancy through adulthood: an individual based pooled analysis of 28 twin cohorts participating in the coda twins project the design of experiments an investigation of the false discovery rate and the misinterpretation of p values we thank berty lumey for very helpful comments to the first draft of the manuscript. conceptualization: alberto palloni. key: cord-266797-uglsx7se authors: anastassopoulou, cleo; russo, lucia; tsakris, athanasios; siettos, constantinos title: data-based analysis, modelling and forecasting of the covid-19 outbreak date: 2020-03-31 journal: plos one doi: 10.1371/journal.pone.0230405 sha: doc_id: 266797 cord_uid: uglsx7se since the first suspected case of coronavirus disease-2019 (covid-19) on december 1st, 2019, in wuhan, hubei province, china, a total of 40,235 confirmed cases and 909 deaths have been reported in china up to february 10, 2020, evoking fear locally and internationally. here, based on the publicly available epidemiological data for hubei, china from january 11 to february 10, 2020, we provide estimates of the main epidemiological parameters. in particular, we provide an estimation of the case fatality and case recovery ratios, along with their 90% confidence intervals as the outbreak evolves. on the basis of a susceptible-infectious-recovered-dead (sidr) model, we provide estimations of the basic reproduction number (r(0)), and the per day infection mortality and recovery rates. by calibrating the parameters of the sird model to the reported data, we also attempt to forecast the evolution of the outbreak at the epicenter three weeks ahead, i.e. until february 29. as the number of infected individuals, especially of those with asymptomatic or mild courses, is suspected to be much higher than the official numbers, which can be considered only as a subset of the actual numbers of infected and recovered cases in the total population, we have repeated the calculations under a second scenario that considers twenty times the number of confirmed infected cases and forty times the number of recovered, leaving the number of deaths unchanged. based on the reported data, the expected value of r(0) as computed considering the period from the 11th of january until the 18th of january, using the official counts of confirmed cases was found to be ∼4.6, while the one computed under the second scenario was found to be ∼3.2. thus, based on the sird simulations, the estimated average value of r(0) was found to be ∼2.6 based on confirmed cases and ∼2 based on the second scenario. our forecasting flashes a note of caution for the presently unfolding outbreak in china. based on the official counts for confirmed cases, the simulations suggest that the cumulative number of infected could reach 180,000 (with a lower bound of 45,000) by february 29. regarding the number of deaths, simulations forecast that on the basis of the up to the 10th of february reported data, the death toll might exceed 2,700 (as a lower bound) by february 29. our analysis further reveals a significant decline of the case fatality ratio from january 26 to which various factors may have contributed, such as the severe control measures taken in hubei, china (e.g. quarantine and hospitalization of infected individuals), but mainly because of the fact that the actual cumulative numbers of infected and recovered cases in the population most likely are much higher than the reported ones. thus, in a scenario where we have taken twenty times the confirmed number of infected and forty times the confirmed number of recovered cases, the case fatality ratio is around ∼0.15% in the total population. importantly, based on this scenario, simulations suggest a slow down of the outbreak in hubei at the end of february. introduction an outbreak of "pneumonia of unknown etiology" in wuhan, hubei province, china in early december 2019 has spiraled into an epidemic that is ravaging china and threatening to reach a pandemic state [1] . the causative agent soon proved to be a new betacoronavirus related to the middle east respiratory syndrome virus (mers-cov) and the severe acute respiratory syndrome virus (sars-cov). the novel coronavirus sars-cov-2 disease has been named "covid-19" by the world health organization (who) and on january 30, the covid-19 outbreak was declared to constitute a public health emergency of international concern by the who director-general [2] . despite the lockdown of wuhan and the suspension of all public transport, flights and trains on january 23, a total of 40,235 confirmed cases, including 6,484 (16.1%) with severe illness, and 909 deaths (2.2%) had been reported in china by the national health commission up to february 10, 2020; meanwhile, 319 cases and one death were reported outside of china, in 24 countries [3] . the origin of covid19 has not yet been determined although preliminary investigations are suggestive of a zoonotic, possibly of bat, origin [4, 5] . similarly to sars-cov and mers-cov, the novel virus is transmitted from person to person principally by respiratory droplets, causing such symptoms as fever, cough, and shortness of breath after a period believed to range from 2 to 14 days following infection, according to the centers for disease control and prevention (cdc) [1, 6, 7] . preliminary data suggest that older males with comorbidities may be at higher risk for severe illness from covid-19 [6, 8, 9] . however, the precise virologic and epidemiologic characteristics, including transmissibility and mortality, of this third zoonotic human coronavirus are still unknown. using the serial intervals (si) of the two other well-known coronavirus diseases, mers and sars, as approximations for the true unknown si, zhao et al. estimated the mean basic reproduction number (r 0 ) of sars-cov-2 to range between 2.24 (95% ci: 1.96-2.55) and 3.58 (95% ci: 2.89-4.39) in the early phase of the outbreak [10] . very similar estimates, 2.2 (95% ci: 1.4-3.9), were obtained for r 0 at the early stages of the epidemic by imai et al. 2.6 (95% ci: 1.5-3.5) [11] , as well as by li et al., who also reported a doubling in size every 7.4 days [1] . wu et al. estimated the r 0 at 2.68 (95% ci: 2.47-2.86) with a doubling time every 6.4 days (95% ci: 5.8-7.1) and the epidemic growing exponentially in multiple major chinese cities with a lag time behind the wuhan outbreak of about 1-2 weeks [12] . amidst such an important ongoing public health crisis that also has severe economic repercussions, we reverted to mathematical modelling that can shed light to essential epidemiologic parameters that determine the fate of the epidemic [13] . here, we present the results of the analysis of time series of epidemiological data available in the public domain [14] [15] [16] (who, cdc, ecdc, nhc and dxy) from january 11 to february 10, 2020, and attempt a threeweek forecast of the spreading dynamics of the emerged coronavirus epidemic in the epicenter in mainland china. our analysis was based on the publicly available data of the new confirmed daily cases reported for the hubei province from the 11th of january until the 10th of february [14] [15] [16] . based on the released data, we attempted to estimate the mean values of the main epidemiological parameters, i.e. the basic reproduction number r 0 , the case fatality (ĝ) and case recovery (b) ratios, along with their 90% confidence intervals. however, as suggested [17] , the number of infectious, and consequently the number of recovered, people is likely to be much higher. thus, in a second scenario, we have also derived results by taking twenty times the number of reported cases for the infectious and forty times the number for the recovered cases, while keeping constant the number of deaths that is more likely to be closer to the real number. furthermore, by calibrating the parameters of the sird model to fit the reported data, we also provide tentative forecasts until the 29th of february. the basic reproduction number (r 0 ) is one of the key values that can predict whether the infectious disease will spread into a population or die out. r 0 represents the average number of secondary cases that result from the introduction of a single infectious case in a totally susceptible population during the infectiousness period. based on the reported data of confirmed cases, we provide estimations of the r 0 from the 16th up to the 20th of january in order to satisfy as much as possible the hypothesis of s � n that is a necessary condition for the computation of r 0 . we also provide estimations of the case fatality (ĝ) and case recovery (b) ratios over the entire period using a rolling window of one day from the 11th of january to the 16th of january to provide the very first estimations. furthermore, we calibrated the parameters of the sird model to fit the reported data. we first provide a coarse estimation of the recovery (β) and mortality rates (γ) of the sird model using the first period of the outbreak. then, an estimation of the infection rate α is accomplished by "wrapping" around the sird simulator an optimization algorithm to fit the reported data from the 11th of january to the 10th of february. we have started our simulations with one infected person on the 16th of november, which has been suggested as a starting date of the epidemic and run the sir model until the 10th of february. below, we describe analytically our approach. let us start by denoting with s(t), i(t), r(t), d(t), the number of susceptible, infected, recovered and dead persons respectively at time t in the population of size n. for our analysis, we assume that the total number of the population remains constant. based on the demographic data for the province of hubei n = 59m. thus, the discrete sird model reads: rðtþ ¼ rðt à 1þ þ biðt à 1þ ð3þ the above system is defined in discrete time points t = 1, 2, . . ., with the corresponding initial condition at the very start of the epidemic: s(0) = n − 1, i(0) = 1, r(0) = d(0) = 0. here, β and γ denote the "effective/apparent" per day recovery and fatality rates. note that these parameters do not correspond to the actual per day recovery and mortality rates as the new cases of recovered and deaths come from infected cases several days back in time. however, one can attempt to provide some coarse estimations of the "effective/apparent" values of these epidemiological parameters based on the reported confirmed cases using an assumption and approach described in the next section. let us first start with the estimation of r 0 . initially, when the spread of the epidemic starts, all the population is considered to be susceptible, i.e. s � n. based on this assumption, by eqs (2), (3) and (4), the basic reproduction number can be estimated by the parameters of the sird model as: let us denote with , the reported new cases of infectious, recovered and dead at time t, with cδi(t), cδr(t), cδd(t) the cumulative numbers of confirmed cases at time t. thus: where, x = i, r, d. starting with the estimation of r 0 , we note that as the province of hubei has a population of 59m, one can reasonably assume that for any practical means, at least at the beginning of the outbreak, s � n. by making this assumption, one can then provide an approximation of the expected value of r 0 using eqs (5), (2), (3) and (4). in particular, substituting in eq (2), the terms βi(t − 1) and γi(t − 1) with δr(t) = r(t) − r(t − 1) from eq (3), and δd(t) = d(t) − d(t − 1) from eq (4) and bringing them into the left-hand side of eq (2), we get: adding eqs (3) and (4), we get: finally, assuming that for any practical means at the beginning of the spread that s(t − 1) � n and dividing eq (7) by eq (8) we get: note that one can use directly eq (9) to compute r 0 with regression, without the need to compute first the other parameters, i.e. β, γ and α. at this point, the regression can be done either by using the differences per se, or by using the corresponding cumulative functions (instead of the differences for the calculation of r 0 using eq (9)). indeed, it is easy to prove that by summing up both sides of eqs (7) and (8) over time and then dividing them, we get the following equivalent expression for the calculation of here, we used eq (10) to estimate r 0 in order to reduce the noise included in the differences. note that the above expression is a valid approximation only at the beginning of the spread of the disease. thus, based on the above, a coarse estimation of r 0 and its corresponding confidence intervals can be provided by solving a linear regression problem using least-squares problem as: estimation of the case fatality and case recovery ratios for the period january11-february 10 here, we denote byĝ the case fatality and byb the case recovery ratios. several approaches have been proposed for the calculation of the case fatality ratio (see for example the formula used by the national health commission (nhc) of the people's republic of china [18] for estimating the mortality ratio for the covid-19 and also the discussion in [19] ). here, we adopt the one used also by the nhc which defines the case mortality ratio as the proportion of the total cases of infected cases, that die from the disease. thus, a coarse estimation of the case fatality and recovery ratios for the period under study can be calculated using the reported cumulative infected, recovered and dead cases, by solving a linear regression problem, which for the case fatality ratio reads: accordingly, in an analogy to the above, the case recovery ratio reads: as the reported data are just a subset of the actual number of infected and recovered cases including the asymptomatic and/or mild ones, we have repeated the above calculations considering twenty times the reported number of infected and forty times the reported number of recovered in the toal population, while leaving the reported number of dead the same given that their cataloguing is close to the actual number of deaths due to covid-19. here we note that the new cases of recovered and deaths at each time time t appear with a time delay with respect to the actual number of infected cases. this time delay is generally unknown but an estimate can be given by clinical studies. however, one could also attempt to provide a coarse estimation of these parameters based only on the reported data by considering the first period of the outbreak and in particular the period from the 11th of january to the 16th of january where the number of infected cases appear to be constant. thus, based on eqs (3) and (4), and the above assumption, the "effective" per day recovery rate β and the "effective" per day mortality rate γ were computed by solving the least squares problems (see eqs (2) and (4): as noted, these values do not correspond to the actual per day mortality and recovery rates as these would demand the exact knowledge of the corresponding time delays. having provided an estimation of the above "effective" approximate values of the parameters β and γ, an approximation of the "effective" infected rate α, that is not biased by the assumption of s = n, can be obtained by using the sird simulator. in particular, in the sird model, the values of the β and γ parameters were set equal to the ones found using the reported data solving the corresponding least squares problems given by eqs (14) and (15) . as initial conditions we have set one infected person on the 16th of november and ran the simulator until the last date for which there are available data (here up to the 10th of february). then, the optimal value of the infection rate α that fits the reported data was found by "wrapping" around the sird simulator an optimization algorithm (such as a nonlinear least-squares solver) to solve the problem: where f t ða; b; gþ ¼ cdi sird ðtþ à cdiðtþ; where, cδx sird (t), (x = i, r, d) are the cumulative cases resulting from the sird simulator at time t; w 1 , w 2 , w 3 correspond to scalars serving in the general case as weights to the relevant functions. for the solution of the above optimization problem we used the function "lsqnonlin" of matlab [20] using the levenberg-marquard algorithm. as discussed, we have derived results using two different scenarios (see in methodology). for each scenario, we first present the results for the basic reproduction number as well as the case fatality and case recovery ratios as obtained by solving the least squares problem using a rolling window of an one-day step. for their computation, we used the first six days i.e. from the 11th up to the 16th of january to provide the very first estimations. we then proceeded with the calculations by adding one day in the rolling window as described in the methodology until the 10th of february. we also report the corresponding 90% confidence intervals instead of the more standard 95% because of the small size of the data. for each window, we also report the corresponding coefficients of determination (r 2 ) representing the proportion of the variance in the dependent variable that is predictable from the independent variables, and the root mean square of error (rmse). the estimation of r 0 was based on the data until january 20, in order to satisfy as much as possible the hypothesis underlying its calculation by eq (9). then, as described above, we provide coarse estimations of the "effective" per day recovery and mortality rates of the sird model based on the reported data by solving the corresponding least squares problems. then, an estimation of the infection rate α was obtained by "wrapping" around the sird simulator an optimization algorithm as described in the previous section. finally, we provide tentative forecasts for the evolution of the outbreak based on both scenarios until the end of february. scenario i: results obtained using the exact numbers of the reported confirmed cases confidence intervals. as more data are taken into account, this variation is significantly reduced. thus, using all the available data from the 11th of january until the 10th of february, the estimated value of the case fatality ratioĝ is � 2.94% (90% ci: 2.9%-3%) and that of the case recovery ratiob is � 0.05 (90% ci: 0.046-0.055). it is interesting to note that as the available data become more, the estimated case recovery ratio increases significantly from the 31th of january (see fig 2) . in figs 3, 4 and 5, we show the coefficients of determination (r 2 ) and the root of mean squared errors (rmse) forr 0 ,b andĝ, respectively. the computed approximate values of the "effective" per day mortality and recovery rates of the sird model were γ � 0.01 and β � 0.064 (corresponding to a recovery period of � 15 d). note that because of the extremely small number of the data used, the confidence intervals have been disregarded. instead, for our calculations, we have considered intervals of 20% around the expected least squares solutions. hence, for γ, we have taken the interval (0.008 and 0.012) and for β, we have taken the interval between (0.05 and 0.077) corresponding to recovery periods from 13 to 20 days. as described in the methodology, we have also used the sird simulator to provide an estimation of the "effective" infection rate α by optimization with w 1 = 1, w 2 = 2, w 3 = 2. thus, we performed the simulations by setting β = 0.064 and γ = 0.01, and as initial conditions one infected, zero recovered and zero dead on november 16th 2019, and ran until the 10th of february. the optimal, with respect to the reported confirmed cases from the 11th of january to the 10th of february, value of the infected rate (α) was � 0.191 (90% ci: 0. 19-0.192) . this corresponds to a mean value of the basic reproduction numberr 0 � 2:6. note that this value is lower compared to the value that was estimated using solely the reported data. finally, using the derived values of the parameters α, β, γ, we performed simulations until the end of february. the results of the simulations are given in figs 6, 7 and 8. solid lines depict the evolution, when using the expected (mean) estimations and dashed lines illustrate the corresponding lower and upper bounds as computed at the limits of the confidence intervals of the estimated parameters. as figs 6 and 7 suggest, the forecast of the outbreak at the end of february, through the sird model is characterized by high uncertainty. in particular, simulations result in an expected number of � 180,000 infected cases but with a high variation: the lower bound is at � 45,000 infected cases while the upper bound is at � 760,000 cases. similarly for the recovered population, simulations result in an expected number of � 60,000, while the lower and upper bounds are at � 22,000 and � 170,000, respectively. finally, regarding the deaths, simulations result in an average number of � 9,000, with lower and upper bounds, � 2,700 and � 34,000, respectively. thus, the expected trends of the simulations suggest that the mortality rate is lower than the estimated with the current data and thus the death toll is expected to be significantly less compared with the expected trends of the predictions. as this paper was revised, the reported number of deaths on the 22th february was 2,344, while the expected number of the forecast was �4300 with a lower bound of �1,300. regarding the number of infected and recovered cases by february 20, the cumulative numbers of confirmed reported cases were 64,084 infected and 15,299 recovered, while the expected trends of the forecasts were �83,000 for the infected and �28,000 for the recovered cases. hence, based on this estimation, the evolution of the epidemic was well within the bounds of our forecasting. for our illustrations, we assumed that the number of infected is twenty times the number of the confirmed infected and forty times the number of the confirmed recovered people. based on this scenario, fig 9 depicts an estimation of r 0 for the period january 16-january 20. using the first six days from the 11th of january to the 16th of january,r 0 results in 3.2 (90% ci: 2.44.0); using the data until january 17,r 0 results in 3.1 (90% ci: 2.5-3.7); using the data until january 18,r 0 results in 3.4 (90% ci: 2.9-3.9); using the data until january 19,r 0 results in 3.9 (90% ci: 3.3-4.5) and using the data until january 20,r 0 results in 4.5 (90% ci: 3.8-5.3). it is interesting to note that the above estimation of r 0 is close enough to the one reported in other studies (see in the introduction for a review). note that the large variation in the estimated values ofb andĝ should be attributed to the small size of the data and data uncertainty. this is also reflected in the corresponding confidence intervals. as more data are taken into account, this variation is significantly reduced. thus,using all the (scaled) data from the 11th of january until the 10th of february, the estimated value of the case fatality ratioĝ now drops to � 0.147% (90% ci: 0.144%-0.15%) while that of the case recovery ratio is � 0.1 (90% ci: 0.091-0.11). it is interesting also to note that as the available data become more, the estimated case recovery ratio increases slightly (see fig 10) , while the case fatality ratio (in the total population) seems to be stabilized at a rate of � 0.15%. in figs 11, 12 and 13, we show the coefficients of determination (r 2 ) and the root of mean squared errors (rmse), forr 0 ,b andĝ, respectively. the computed values of the "effective" per day mortality and recovery rates of the sird model were γ � 0.0005 and β �0.16d −1 (corresponding to a recovery period of � 6 d). note that because of the extremely small number of the data used, the confidence intervals have been disregarded. instead, for calculating the corresponding lower and upper bounds in our simulations, we have taken intervals of 20% around the expected least squares solutions. hence, for γ we have taken the interval (0.0004 and 0.0006) and for β, we have taken the interval between (0.13 and 0.19) corresponding to an interval of recovery periods from 5 to 8 days. again, we used the sird simulator to provide estimation of the infection rate by optimization setting w 1 = 1, w 2 = 400, w 3 = 1 to balance the residuals of deaths with the scaled numbers of the infected and recovered cases. thus, to find the optimal infection transmission rate, we used the sird simulations with β = 0.16d −1 , and γ = 0.0005 and as initial conditions one infected, zero recovered, zero deaths on november 16th 2019, and ran until the 10th of february. the optimal, with respect to the reported confirmed cases from the 11th of january to the 10th of february value of the infected rate (α) was found to be � 0.319(90% ci: 0.318-0.32). this corresponds to a mean value of the basic reproduction numberr 0 � 2. 29, simulations result in an expected actual number of �8m infected cases (corresponding to ã 13% of the total population) with a lower bound at �720,000 and an upper bound at �37m cases. similarly, for the recovered population, simulations result in an expected actual number of �4.5m (corresponding to a 8% of the total population), while the lower and upper bounds are at �430,000 and �23m, respectively. finally, regarding the deaths, simulations under this scenario result in an average number of �14,000, with lower and upper bounds at �900 and �100,000. importantly, under this scenario, the simulations shown in fig 14 suggest a decline of the outbreak at the end of february. table 1 summarizes the above results for both scenarios. we note that the results derived under scenario ii seem to predict a slowdown of the outbreak in hubei after the end of february. we have proposed a methodology for the estimation of the key epidemiological parameters as well as the modelling and forecasting of the spread of the covid-19 epidemic in hubei, china by considering publicly available data from the 11th of january 2020 to the 10th of february 2020. by the time of the acceptance of our paper, according to the official data released on the 29th of february, the cumulative number of confirmed infected cases in hubei was �67,000, that of recovered was �31,300 and the death toll was �2,800. these numbers are within the lower bounds and expected trends of our forecasts from the 10th of february that are based on scenario i. importantly, by assuming a 20-fold scaling of the confirmed cumulative number of the infected cases and a 40-fold scaling of the confirmed number of the recovered cases in the total population, forecasts show a decline of the outbreak in hubei at the end of february. based on this scenario the case fatality rate in the total population is of the order of �0.15%. at this point we should note that our sird modelling approach did not take into account many factors that play an important role in the dynamics of the disease such as the effect of the incubation period in the transmission dynamics, the heterogeneous contact transmission network, the effect of the measures already taken to combat the epidemic, the characteristics of the population (e.g. the effect of the age, people who had already health problems). also the estimation of the model parameters is based on an assumption, considering just the first period in which the first cases were confirmed and reported. of note, covid-19, which is thought to be principally transmitted from person to person by respiratory droplets and fomites without excluding the possibility of the fecal-oral route [21] had been spreading for at least over a month and a half before the imposed lockdown and quarantine of wuhan on january 23, having thus infected unknown numbers of people. the number of asymptomatic and mild cases with subclinical manifestations that probably did not present to hospitals for treatment may be substantial; these cases, which possibly represent the bulk of the covid-19 infections, remain unrecognized, especially during the influenza season [22] . this highly likely gross under-detection and underreporting of mild or asymptomatic cases inevitably throws severe disease courses calculations and death rates out of context, distorting epidemiologic reality. another important factor that should be taken into consideration pertains to the diagnostic criteria used to determine infection status and confirm cases. a positive pcr test was required to be considered a confirmed case by china's novel coronavirus pneumonia diagnosis and treatment program in the early phase of the outbreak [14] . however, the sensitivity of nucleic acid testing for this novel viral pathogen may only be 30-50%, thereby often resulting in false negatives, particularly early in the course of illness. to complicate matters further, the guidance changed in the recently-released fourth edition of the program on february 6 to allow for diagnosis based on clinical presentation, but only in hubei province [14] . the swiftly growing epidemic seems to be overwhelming even for the highly efficient chinese logistics that did manage to build two new hospitals in record time to treat infected patients. supportive care with extracorporeal membrane oxygenation (ecmo) in intensive care units (icus) is critical for severe respiratory disease. large-scale capacities for such level of medical care in hubei province, or elsewhere in the world for that matter, amidst this public health emergency may prove particularly challenging. we hope that the results of our analysis contribute to the elucidation of critical aspects of this outbreak so as to contain the in the digital and globalized world of today, new data and information on the novel coronavirus and the evolution of the outbreak become available at an unprecedented pace. still, crucial questions remain unanswered and accurate answers for predicting the dynamics of the outbreak simply cannot be obtained at this stage. we emphatically underline the uncertainty of available official data, particularly pertaining to the true baseline number of infected (cases), that may lead to ambiguous results and inaccurate forecasts by orders of magnitude, as also pointed out by other investigators [1, 17, 22] . supporting information s1 conceptualization: cleo anastassopoulou, constantinos siettos. early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia who statement regarding cluster of pneumonia cases in wuhan, china novel coronavirus(2019-ncov) genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. the lancet a pneumonia outbreak associated with a new coronavirus of probable bat origin epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study. the lancet initial public health response and interim clinical guidance for the 2019 novel coronavirus outbreak-united states clinical features of patients infected with 2019 novel coronavirus in wuhan clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china preliminary estimation of the basic reproduction number of novel coronavirus (2019-ncov) in china, from 2019 to 2020: a data-driven analysis in the early phase of the outbreak report 3: transmissibility of 2019-ncov nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study. the lancet mathematical modeling of infectious disease dynamics the johns hopkins center for health security. daily updates on the emerging novel coronavirus from the johns hopkins center for health security the johns hopkins center for health security. coronavirus covid-19 global cases by johns hopkins csse an interactive web-based dashboard to track covid-19 in real time. the lancet infectious diseases real-time tentative assessment of the epidemiological characteristics of novel coronavirus infections in wuhan, china, as at 22 -national health commission (nhc) of the people's republic of china methods for estimating the case fatality ratio for a novel, emerging infectious disease coronavirus may transmit along fecal-oral route, xinhua reports 2019-novel coronavirus (2019-ncov): estimating the case fatality rate-a word of caution formal analysis: lucia russo, constantinos siettos. methodology: lucia russo, constantinos siettos. writing -review & editing: cleo anastassopoulou, athanasios tsakris, constantinos siettos. key: cord-001368-ymp1pj3r authors: zhang, chao; he, xinlong; gu, yaping; zhou, huayun; cao, jun; gao, qi title: recombinant scorpine produced using sumo fusion partner in escherichia coli has the activities against clinically isolated bacteria and inhibits the plasmodium falciparum parasitemia in vitro date: 2014-07-28 journal: plos one doi: 10.1371/journal.pone.0103456 sha: doc_id: 1368 cord_uid: ymp1pj3r scorpine, a small cationic peptide from the venom of pandinus imperator, which has been shown to have anti-bacterial and anti-plasmodial activities, has potential important applications in the pharmaceutical industries. however, the isolation of scorpine from natural sources is inefficient and time-consuming. here, we first report the expression and purification of recombinant scorpine in escherichia coli, using small ubiquitin-related modifier (sumo) fusion partner. the fusion protein was expressed in soluble form in e. coli, and expression was verified by sds-page and western blotting analysis. the fusion protein was purified to 90% purity by nickel–nitrilotriacetic acid (ni(2+)–nta) resin chromatography. after the sumo-scorpine fusion protein was cleaved by the sumo protease, the cleaved sample was reapplied to a ni(2+)–nta column. tricine/sds-page gel results indicated that scorpine had been purified successfully to more than 95% purity. the recombinantly expressed scorpine showed anti-bacterial activity against two standard bacteria including staphylococcus aureus atcc 29213 and acinetobacter baumannii atcc 19606, and clinically isolated bacteria including s. aureus s, s. aureus r, a. baumannii s, and a. baumannii r. it also produced 100% reduction in plasmodium falciparum parasitemia in vitro. thus, the expression strategy presented in this study allowed convenient high yield and easy purification of recombinant scorpine for pharmaceutical applications in the future. the oldest known scorpions lived around 430 million years ago in the silurian period, on the bottom of shallow tropical seas, hence regarded as the oldest terrestrial arthropods [1] . scorpions use venoms for immobilization of prey and protection against predators. scorpion venoms consist of a complex of several toxins that exhibit a wide range of biological properties and actions, as well as chemical compositions, toxicity, pharmacokinetic, and pharmacodynamic characteristics [2] . scorpions are a rich source of antimicrobial peptides: (1) androctonin isolated from the hemolymph of androctonus australis, shows marked sequence similarity to tachyplesins, polyphemusins and gomesin [3, 4] ; (2) hadrurin from the venom of hadrurus aztecus, which is hemolytic [5] ; (3) opistoporin-1, which possesses hemolytic activity, and opistoporin-2, both from the venom of the south african scorpion opistophtalmus carinatus [6] ; (4) scorpine, which is the subject of this study, arising from the venom of pandinus imperator, was shown to have anti-bacterial and anti-plasmodial activity in vitro [7] , and has shown a potent toxic effect on sexual and asexual stages of plasmodium berghei and plasmodium falciparum, respectively, and also a strong inhibition of dengue 2 virus (denv-2) infection [8] . native scorpine purified from venom glands has a molecular mass of 8, 350 da. it has a peculiar structure compared to other known amps. its n-terminal amino acid sequence is similar to cecropins, whereas its c-terminal region has several disulfide bridges, similar to the structure of defensins [7] . scorpine's wide range of activities provides the possibility that scorpine can be used as an antimicrobial and anti-plasmodial agent in the future. for pharmaceutical applications, a large quantity of antimicrobial peptides need to be produced economically. isolation of antibacterial peptides from natural sources is inefficient and timeconsuming. the biosynthesis of recombinant antimicrobial peptides in vivo is currently the most popular technique of preparing polypeptides. the escherichia coli expression system is still the most commonly used because of its high level of expression, the relative simplicity of the dna manipulations, and the short time required to produce product. because of their natural destructive behavior toward microorganisms and relative sensitivity to proteolytic degradation, antimicrobial peptides are often produced by being fused to a fusion partner in heterologous hosts to neutralize their innate toxic activity and increase their expression levels [9] . small ubiquitin-related modifier (sumo) is an ubiquitin-related protein that functions by covalent attachment to other proteins. it is known that the sumo, fused at the n-terminus with other proteins, can fold and protect the protein by its chaperoning properties, making it a useful tag for heterologous expression. these advantages include the manner in which protein expression is enhanced, proteolytic degradation of the target protein is decreased, protein folding and solubility are increased, and purification and detection are simplified [10] [11] [12] . in this study, we provide the first report of the procedures for obtaining a recombinant scorpine by using sumo fusion partner and investigate its anti-bacterial and anti-plasmodial activities. bacterial strains, vectors, and enzymes e. coli dh5a (maintained in our laboratory) was used for subcloning and plasmid amplification. e. coli bl21 (de3) (novagen, usa) was used as the expression host. the linearized psumo vector with bsa i and xho i restriction sites and t7 promoter and kanamycin resistance and 66his sequence was purchased from lifesensors (lifesensors, malvern, pa, usa). sumo protease containing a histidine-tag was also the product of lifesensors (malvern, pa, usa). all the restriction enzymes and t4 dna ligase were purchased from takara biotech co. ltd (dalian, china). strains of staphylococcus aureus atcc 29213 and acinetobacter baumannii atcc 19606 were purchased from china general microbiological culture collection center (cgmcc, china) and cultured according to the method provided by cgmcc. strains of s. aureus s, s. aureus r, a. baumannii s, and a. baumannii r were isolated in clinc in the third people's hospital of wuxi. the bacteria sampling was obtained in the burn wounds from burn patients, and was crossed to cultivate on blood agar for 24 hours to obtain monoclonal colony, and then monoclonal bacteria were multiplication cultured in liquid medium for 24 h. the identification of proliferative monoclonal bacteria was first carried out with using vitek r 2 -compact automatic bacteria identification device (biomerieux) and furthermore, the isolated bacteria were confirmed through polymerase chain reaction amplification of conserved region of nuc gene for s. aureus (fw: 59-gcgattgatggtgatacggti-39, rv: 59-agccaagccttgacgaactaaagc-39) [13] , and multiplex amplification of 16 s-23 s ribo-somal dna intergenic spacer region and conserved region of the reca gene for a. baumannii (fw: 59-cattatcacggtaattagtg-39, rv: 59-agag-cactgtgcacttaag-39 for 16 s-23 s ribosomal dna intergenic spacer region; fw: 59-cctgaatcttctgg-taaaac-39, rv: 59-gtttctgggctgccaaacattac-39 for reca gene) [14] . all pcr products were sequenced to confirm in genscript corporation (nanjing, china). the antibiotic susceptibility was determined according to the clinical laboratory standards institute (clsi) procedure [15] . the minimum inhibitory concentrations (mics) of selected antibiotics were shown in table 1 . the mics were determined at concentrations, at which there was no visible growth. the susceptible (s), and resistant (r) strains were defined based on mic values of ,4 mg/ml, and more than 16 mg/ml, respectively [16] . the written informed consents were obtained from the patients in the third people's hospital of wuxi; human red blood cells were obtained from wuxi red cross blood center (wuxi, china). this study was approved by the ethics committees of the third people's hospital of wuxi and jiangsu institute of parasitic diseases. from the metarhizium fungus with scorpine gene of interest, which was a gift from fang's laboratory [17] , scorpine gene was amplified by pcr using primers (fw: 59-taggtctctagg-tatgggctggattaacgaggagaag-39 and rv: 59-at-tactcgagttagtaggagagaggggtgcc-39).the pcr fragments were separated using 1.0% gel electrophoresis, purified with a dna gel extraction kit (takara, china). the resulting pcr product was digested with bsa i and xho i, and ligated into the psumo plasmid at the corresponding restriction sites [18] . the ligation mixture was transformed into e. coli dh5a cells for verification by sequencing (nanjing genscript bio. co. ltd). the psumo/scorpine plasmid that had been constructed, was transformed into competent e. coli bl21 (de3). three colonies were picked and cultured in 4 ml sterilized luria-bertani (lb) medium with vigorous shaking (220 rpm) at 37uc, to an optical density (od 600 nm ) of 0.6. isopropyl-b-d-1-thiogalactopyranoside (iptg) (0.5 mm) was then added to induce the expression of the recombinant protein at 28uc for 8 h. the sds-page analysis was performed according to laemmli [19] using 12% polyacrylamide gels. the total expression protein samples from cell lysates after induction were mixed with equivalent sample buffer (125 mm tris-hcl, ph 6.8, 20% glycerol, 4% sds, 0.005% bromophenol blue, and 10% 2mercaptoethanol). gels were stained with coomassie brilliant blue r-250. the tricine/sds-page analysis for the resolution of proteins smaller than 30 kda was performed according to the reference [20] using 16.5% polyacrylamide gels. gels were stained with coomassie brilliant blue r-250. for western blotting, the same protein sample was separated on a 12% polyacrylamide gel under reducing conditions and then transferred to a polyvinylidene difluoride (pvdf) membrane (roche applied science). the western blotting was performed as described [21] . the rabbit igg secondary antibody was used against his-tag primary antibody (novagen). the blots were developed using tmb immunoblotting system. bl21(de3)-psumo/scorpine strains were cultured in 200 ml sterilized lb medium with vigorous shaking (220 rpm) at 37uc to an optical density (od 600 nm ) of 0.6. isopropyl-b-d-1-thiogalacto-pyranoside (iptg) (0.5 mm) was then added to induce the expression of the recombinant protein at 28uc for 8 h. cultures were collected by centrifugation at 12,0006g, at 4uc for 10 min and the cell pellet frozen at 280uc. the pellet from 200 ml culture was then resuspended in 20 ml binding buffer (20 mm tris, 500 mm nacl, 20 mm imidazole, and 10 mm phenylmethylsulfonyl fluoride, ph 8.0) and lysed on ice by sonication at 400 w for 100 cycles (4 s working, 8 s free). the supernatant of the cell lysate resulting from centrifugation at 12,0006g at 4uc for 20 min was applied to a nickel-nitrilotriacetic acid (ni 2+ -nta, novagen) affinity chromatography column according to the manufacturer's instruction. after extensive washing with binding buffer (10 column volumes), the fusion protein was eluted with five column volumes of elution buffer (20 mm tris, 500 mm nacl, and 250 mm imidazole, ph 8.0). the peak fractions with high-uv values at 280 nm, which were detected by lpdataview (biorad), and containing the fusion protein were pooled and dialyzed overnight at 4uc against phosphate buffered saline (pbs, ph 8.0). the dialyzed fusion protein was reacted with 1 u sumo protease per 50 mg fusion protein at 30uc for 1 h. since both sumo and sumo protease had 66his tags, but scorpine did not, the cleaved sumo fusion samples could be re-applied to the nickel column to obtain the purified scorpine by subtracting the 66his-tagged proteins. briefly, after the sumo fusions were cleaved by the sumo protease, the sample was loaded onto a nickel column with ni 2+ -nta resin. most of scorpine without 66his tags was eluted (five column volumes) in the flow-through (unbound) fractions, and the rest was recovered by washing the resin with binding buffer (10 column volumes). the eluted and washed proteins appearing in fractions with high-uv values at 280 nm were pooled as the final purified sample. the purified proteins were checked on sds-page. purified scorpine was filtered through a 0.22 mm filter membrane and stored at 280uc for activity assays. to measure effects of recombinant scorpine on the growth of bacteria planktonic cell, the bacteria were transferred into 96-well flat-bottomed polystyrene microtiter plates (bd falcon, sanjose, ca, usa) at approximately 10 5 cfu/ml in tryptic soy broth (tsb) in the presence of recombinant scorpine (5 and 10 mm) and cultured at 37uc for 24 h under static conditions, aerobically. after cultivation, the supernatant was 10-fold serially diluted with 0.1% sterile buffered peptone water (bpw), and the dilutions were pour-plated with tryptic soy agar (tsa). the bacteria numbers were determined and calculated by the counts on agar plates. to measure effects of recombinant scorpine on the biofilm formation ability of bacteria, the crystal violet (cv) method was used [22] . briefly, the intact bacteria remainder in each well of the plate after the supernatant transfer was rinsed three times with 0.1% sterile bpw to remove planktonic and loosely attached cells, and the washed wells were air dried at 55uc for 1 h. the dried attached cells were stained with 1% cv solution at 37uc for 30 min, washed twice with sterile distilled water and then air-dried at 55uc for 1 h. the stained biofilm cells were destained with 95% ethanol and measured at 570 nm (od) (cv biofilm ). the negative control (cv control ) was used to reduce the background staining from the cv-stained biofilm cells. the biofilm formation ability was expressed by biofilm formation index (bfi = [cv biofilm -cv control ]/od planktonic ). anti-plasmodial assay the p. falciparum fcc1/hn strain was used for the assay. the fcc1/hn line was isolated from hainan island, china [23] . the strain was cultured in human erythrocytes type a+ at 5% hematocrit. the strain was cultured in rpmi (gibco) medium supplemented with hepes 25 mm, glutamine 2 mm, glucose 2 g/l, nahco 3 2 g/l, hypoxanthine 29.25 mg/l, gentamicin 60 mg/l and albumax 1.6% at ph 7.4. cultures were kept in 96% nitrogen, 3% co 2 and 1% oxygen atmosphere at 37uc, and fresh medium was added every 24 h [24] . synchronization of parasite cultures with 5% sorbitol (sigma) was performed as previously described [25] . briefly, parasites were taken when they were mostly at the ring stage, and then spinned down at 1000 rpm to a pellet; 4 ml of 5% sorbitol (in distilled water) were added onto the pellet and incubated for 45 min at room temperature and at the same time shaken with 2 or 3 times, then centrifuged at 1000 rpm and washed with 3 times in malaria culture medium, and finally, the synchronized parasites at the ring stage were cultured in human erythrocytes type a+ at 5% hematocrit. about 24 h after the synchronized parasites were cultured, the majority trophozoite stage parasites were diluted to 1.5% parasitemia and exposed to recombinant scorpine (5 and 10 mm) for 24, 48 and 72 h. the medium was discharged, and fresh medium with the appropriate recombinant scorpine concentration was added every 24 h. the percentage of infected red blood cells was determined by two experienced microscopists using the microscopic examination of thin blood films stained with giemsa. a total of 5,000 cells from several fields were counted for every thin blood film. each recombinant scorpine concentration was tested at least triplicate. controls with recombinant scorpine solvent (pbs) and non-treated infected erythrocytes were included [8] . all experiments were conducted in duplicate for at least three replicates. results were expressed as means6s.d. statistical analysis was performed according to student's t-test by one-way analysis of variance. significant difference was taken as p,0.05. the construct for scorpine expression, containing a his-tag for affinity purification, was depicted in fig. 1 . the recombinant plasmid psumo/scorpine sequence was verified by dna sequencing (nanjing genscript bio. co. ltd). the correct construct was transformed into the expression host e. coli bl21 (de3). as shown in fig. 2a and b , there was an obvious protein band after iptg induction, which could be detected using the western blotting method with anti-66his tag antibody. the apparent molecular weight of the sumo fusion protein was about 18 kda (the scorpine gene encoded a protein of 75 amino acids, with 8 kda; and the molecular weight of sumo is about 10 kda). as described above, ni-nta resin was used for fusion protein purification. most of the proteins without 66his tags were removed from the ni 2+ -nta resin using washing buffer containing 20 mm imidazole, and the 66his-tagged sumo/ scorpine was eluted with more than 90% purity using elution buffer containing 250 mm imidazole (fig. 2a) . the purity was estimated through sds-page gels stained using coomassie blue. the sumo/scorpine protein was competently cleaved after incubation with sumo protease at 30uc for 1 h, according to the method provided by lifesensors (lifesensors, malvern, pa, usa). after the cleaved sample was re-applied to a ni 2+ -nta column to remove his 6 -tagged sumo and sumo protease, finally purified scorpine was obtained, and 16.5% tricine/sds-page gel results indicated that scorpine had been purified successfully to more than 95% purity (fig. 2c ). it was very important to demonstrate that recombinant scorpine was capable of producing inhibition of bacteria growth similar to that originally shown for native scorpine as purified from p. imperator venom [7] . as shown in fig. 3 , recombinant scorpine was able to inhibit the growth of two standard bacteria including s. aureus atcc 29213 and a. baumannii atcc 19606 from china general microbiological culture collection center, and four clinically isolated bacteria including s. aureus s, s. aureus r, a. baumannii s, and a. baumannii r. in natural habitats, the biofilm formation is an important survival strategy for bacteria that can be embedded within a selfproduced extracellular polymeric matrix of polysaccharides, proteins, lipids and nucleic acids [26, 27] . we investigated effects of recombinant scorpine on the biofilm formation ability of two standard bacteria including s. aureus atcc 29213 and a. baumannii atcc 19606, and clinically isolated bacteria including s. aureus s, s. aureus r, a. baumannii s, and a. baumannii r. as shown in fig. 4 , recombinant scorpine was able to significantly inhibit the biofilm formation of bacteria, including (a) s. aureus atcc 29213 (b) s. aureus s (c) s. aureus r (d) a. baumannii atcc 19606 (e) a. baumannii s, except that inhibition of recombinant scorpine against the biofilm formation of (f) a. baumannii r did not appear to be significant. the trophozoite stage cultures of plasmodium falciparum were exposed to recombinant scorpine. as shown in fig. 5 and fig. s1 , the tested concentrations (5 and 10 mm) of recombinant scorpine reduced plasmodium falciparum parasitemia over the time course of the experiment in relation to controls in vitro. and, a critical dependence on exposure time was also observed in recombinant scorpine treated cultures. at 48 h of exposure, no parasites were observed at recombinant scorpine 5 mm or above. in addition, no infected erythrocytes were detected after 72 h, at all tested concentrations. during the process of the anti-plasmodial assay in our study, we did not observe that scorpine was hemolytic, at least for recombinant scorpine in escherichia coli. this further suggest that recombinant scorpine had anti-plasmodial activities. scorpion venoms are rich sources of peptides with a variety of pharmacological functions, special those that interact with membrane permeability for na + , k + , ca 2+ and clof excitable and non-excitable cells [28] . several antimicrobial peptides have been described from scorpions [3] [4] [5] . among the peptides isolated from the venom of the african scorpion pandinus imperator, a molecule, named scorpine, was identified [7] , showing antibacterial and anti-plasmodial activities [7, 8] , with the possibility of being used as an anti-microbial and anti-plasmodial agent in the future. in this study, we have shown that the expression and purification of recombinant scorpine in escherichia coli, using the small ubiquitin-related modifier (sumo) fusion partner. small ubiquitin-related modifer (sumo) is an ubiquitin-related protein that functions by covalent attachment to other proteins. sumo has 18% sequence identity with ubiquitin [29] . the yeast saccharomyces cerevisiae has only a single sumo gene (smt3) that is essential for viability [30] . in contrast to yeast smt3, three members of sumo have been described in vertebrates: sumo-1, sumo-2, and sumo-3. human sumo-1, a 101 amino acid polypeptide, shares 50% sequence identity with human sumo-2/ sumo-3 [31] , which are close homologues. it is known that sumo, fused at the n-terminus with other proteins, can fold and protect the protein by its chaperoning properties, making it a useful tag for heterologous expression [12, 32] . all sumo genes encode precursor proteins with a short c-terminal sequence that extends from the conserved c-terminal gly-gly motif. sumo proteases remove sumo from proteins, by cleaving the c-termini of sumo (-ggaty) in yeast to the mature form (-gg) or deconjugating it from lysine side chains [33, 34] . the former activity (protease) is useful for removal of sumo as an expression tag. in this study, we expressed scorpine as sumo fusions in e. coli to evaluate its anti-bacterial and anti-plasmodial activities. the sumo fusion protein was successfully expressed in e. coli. the sumo/scorpine fusion protein could be completely cleaved using sumo protease, as shown in fig. 2c . the scorpine, which is about 8 kda, was recovered with 95% purity using nickel affinity chromatography again. similar to that originally shown for native scorpine as purified from p. imperator venom [7] , the recombinant scorpine was able to inhibit the growth of not only standard bacteria from china general microbiological culture collection center, but also clinically isolated bacteria, suggesting potential important clinical applications (fig. 3) . to further confirm the potential clinical values of scorpine, we investigated effects of recombinant scorpine on biofilm formation ability of bacteria, using the cv method [22] . as shown in fig. 4 , the recombinant scorpine was able to inhibit the biofilm formation of bacteria. the biofilm formation as a bacterial survival strategy leads to increased resistance to heat, acid, preservatives, and antibiotics [35, 36] . bacterial infections can mainly occur after consumption of contaminated foods. the ingested bacteria are exposed to acidic stress and bile salt under oxygen-limited conditions during transit through the stomach, the small intestine, and the colon. these stress conditions can influence antibiotic resistance patterns, biofilm-forming abilities, and virulence properties [37] . moreover, antibiotic-resistant bacteria can possibly reside in biofilms and lead to enhanced tolerance to adverse environmental conditions, causing serious infectious diseases [38] [39] [40] . our data showed that the recombinant scorpine was able to inhibit not only the growth of bacteria but aslo the biofilm formation of bacteria, suggest that the potential clinical use of scorpine in the future. malaria, caused by plasmodium infection, is one of the most debilitating parasitic diseases of humans, with an estimated 225 million clinical cases and 781,000 deaths per year [41] . scorpine was shown to have anti-plasmodial activities [7, 8] . so, we investigated effects of recombinant scorpine on p. falciparum. as shown in fig. 5 , the tested concentrations reduced plasmodium falciparum parasitemia over the time course of the experiment, using p. falciparum trophozoite stage cultures that were exposed to recombinant scorpine. these data suggest that similar to that originally shown for native scorpine, recombinant scorpine in e. coli was able to inhibit the infection of plasmodium spp. in summary, the sumo fusion partner and customized expression and purification protocol described here have further improved the efficiency and lowered the costs of scorpine production. the sumo fusion partner could also be widely applied to the industrial production of a variety of enzyme proteins in e. coli. figure s1 the representative microscopic images of the parasites treated with recombinant scorpine for 0 h, 48 h, and 72 h, using the microscopic examination of thin blood films stained with giemsa (1006, oil immersion). (tif) oxygen transport proteins: i. structure and organization of hemocyanin from scorpion (buthus sindicus) scorpion venoms as a potential source of novel cancer therapeutic compounds recombinant scorpine with cleavage of his-sumo tag using sumo protease, was added to plasmodium falciparum fcc1/hn cultures at different concentrations (10 and 5 mm) for 24, 48 and 72 h. the number of infected erythrocytes was counted as described in the ''materials and methods'' section. asterisks indicate significant difference (*p,0.05; comparision with the solvent control). (b) the representative microscopic images of the parasites treated with recombinant scorpine for 24 h, using the microscopic examination of thin blood films stained with giemsa (1006, oil immersion characterization of novel cysteine-rich antimicrobial peptides from scorpion blood androctonin, a hydrophilic disulphide-bridged nonhaemolytic anti-microbial peptide: a plausible mode of action hadrurin, a new antimicrobial peptide from the venom of the scorpion hadrurus aztecus antibacterial and antifungal properties of alpha-helical, cationic peptides in the venom of scorpions from southern africa scorpine, an antimalaria and anti-bacterial agent purified from scorpion venom recombinant scorpine: a multifunctional antimicrobial peptide with activity against different pathogens trxa mediating fusion expression of antimicrobial peptide cm4 from multiple joined genes in escherichia coli sumo fusion technology for difficult-to-express proteins expression and purification of human urodilatin by small ubiquitin-related modifier fusion in escherichia coli production of bioactive c-glutamyl transpeptidase in escherichia coli using sumo fusion partner and application of the recombinant enzyme to l-theanine synthesis detection of staphylococcus aureus by polymerase chain reaction amplification of the nuc gene polymerase chain reaction assay for the detection of acinetobacter baumannii in endotracheal aspirates from patients in the intensive care unit methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically activity of glycylcyclines cl 329998 and cl 331002 against minocycline-resistant and other strains of methicillin resistant staphylococcus aureus development of transgenic fungi that kill human malaria parasites in mosquitoes molecular cloning. in: a laboratory manual cleavage of structural proteins during the assembly of the head of bacteriophage t4 human glutathione stransferase p1-1 interacts with traf2 and regulates traf2-ask1 signals colorimetric method for identifying plant essential oil components that affect biofilm formation and structure fusion of two malaria vaccine candidate antigens enhances product yield, immunogenicity, and antibody-mediated inhibition of parasite growth in vitro human malaria parasites in continuous culture synchronization of plasmodium falciparum erythrocytic stages in culture biofilms: survival mechanisms of clinically relevant microorganisms the biofilm matrix scorpion toxins specific for na + -channels ubiquitin-like proteins: new wines in new bottles ubiquitin and its kin: how close are the family ties? functional heterogeneity of small ubiquitin-related protein modiwers sumo-1 versus sumo-2/3 expression and puriwcation of sars coronavirus proteins using sumo-fusions a new protease required for cell-cycle progression in yeast the yeast ulp2 (smt4) gene encodes a novel protease speciwc for the ubiquitin-like smt3 protein biofilm formation and control in food processing facilities biofilm formation and the food industry, a focus on the bacterial outer surface role of the acid tolerance response in virulence of salmonella typhimurium cross-resistance to antibiotics of escherichia coli adapted to benzalkonium chloride or exposed to stress-inducers characterization of biofilmforming abilities of antibiotic-resistant salmonella typhimurium dt104 on hydrophobic abiotic surfaces biofilm formation by multidrug-resistant salmonella enterica serotype typhimurium phage type dt104 and other pathogens a research agenda to underpin malaria eradication we thank the anonymous reviewers, who provided constructive criticisms and helpful suggestions. key: cord-262759-ec2c25q3 authors: hsieh, yi-ting; lin, mei-hui; ho, hung-yao; chen, lei-chin; chen, chien-cheng; shu, jwu-ching title: glucose-6-phosphate dehydrogenase (g6pd)-deficient epithelial cells are less tolerant to infection by staphylococcus aureus date: 2013-11-04 journal: plos one doi: 10.1371/journal.pone.0079566 sha: doc_id: 262759 cord_uid: ec2c25q3 glucose-6-phosphate dehydrogenase (g6pd) is a key enzyme in the pentose phosphate pathway and provides reducing energy to all cells by maintaining redox balance. the most common clinical manifestations in patients with g6pd deficiency are neonatal jaundice and acute hemolytic anemia. the effects of microbial infection in patients with g6pd deficiency primarily relate to the hemolytic anemia caused by plasmodium or viral infections and the subsequent medication that is required. we are interested in studying the impact of bacterial infection in g6pd-deficient cells. g6pd knock down a549 lung carcinoma cells, together with the common pathogen staphylococcus aureus, were employed in our cell infection model. here, we demonstrate that a lower cell viability was observed among g6pd-deficient cells when compared to scramble controls upon bacterial infection using the mtt assay. a significant increase in the intracellular ros was detected among s. aureus-infected g6pd-deficient cells by observing dichlorofluorescein (dcf) intensity within cells under a fluorescence microscope and quantifying this signal using flow cytometry. the impairment of ros removal is predicted to enhance apoptotic activity in g6pd-deficient cells, and this enhanced apoptosis was observed by annexin v/pi staining under a confocal fluorescence microscope and quantified by flow cytometry. a higher expression level of the intrinsic apoptotic initiator caspase-9, as well as the downstream effector caspase-3, was detected by western blotting analysis of g6pd-deficient cells following bacterial infection. in conclusion, we propose that bacterial infection, perhaps the secreted s. aureus α-hemolysin in this case, promotes the accumulation of intracellular ros in g6pd-deficient cells. this would trigger a stronger apoptotic activity through the intrinsic pathway thereby reducing cell viability when compared to wild type cells. glucose-6-phosphate dehydrogenase (g6pd) is the key enzyme that catalyzes the first reaction, the oxidation of glucose-6-phosphate to 6-phosphogluconolactone, in the pentose phosphate pathway, thereby providing reducing energy to all cells by maintaining the level of the reduced coenzyme nicotinamide adenine dinucleotide phosphate (nadph). nadph plays an important role in maintaining the supply of reduced glutathione to counterbalance oxidantinduced oxidative stress [1] . redox imbalance may induce cell apoptosis and necrosis, thus highlighting the role of g6pd in defending against oxidative damage [2, 3] . g6pd deficiency is the most prevalent enzyme defect in humans and affects an estimated 400 million people worldwide, especially in populations historically exposed to endemic malaria [4] . the most common clinical manifestations are neonatal jaundice and acute hemolytic anemia, which is caused by the impairment of the erythrocyte's ability to remove harmful oxidative stress triggered by exogenous agents such as drugs, infection, or fava bean ingestion [1, 4] . hemolytic anemia caused by infection and subsequent medication is a clinically important concern in patients with g6pd deficiency. this issue has been a primary focus for many decades in relation to efforts to understand the impact of plasmodium infection (malaria) and antimalarial drugs [5, 6] . antimicrobial drug-induced hemolysis is considered the most common adverse clinical consequence of g6pd deficiency [7] . it has also been demonstrated that infections caused by certain viruses, such as hepatitis viruses (a, b, and e) and cytomegalovirus, were associated with hemolytic anemia in patients with g6pd deficiency [8, 9] . recently, it has been shown that infection by particular viruses, such as enterovirus 71, dengue virus, and coronavirus, was enhanced in g6pddeficient cells [10] [11] [12] . however, the impact of bacterial infection on patients with g6pd deficiency still remains to be clarified. most studies have focused on investigating the antibiotic-induced hemolysis after treatment for bacterial infection [7] . in addition, a case report showed that infection by clostridium difficile may have triggered hemolysis and led to severe jaundice in a g6pddeficient neonate, while another case report described hemolysis caused by acinetobacter baumannii infection [13, 14] . wilmanski and colleagues demonstrated that hyperinflammation (increasing cytokine levels) caused by acute endotoxemia (induced by the injection of escherichia coli lipopolysaccharide) resulted in increased mortality in g6pddeficient mice [15] . several studies also indicated that g6pd deficiency in leukocytes can result in chronic granulomatous disease (cgd) and possibly alter the host defense mechanisms for bacterial infections [16] [17] [18] . thus far, the impact of bacterial infection on patients with g6pd deficiency has been found to primarily affect the blood cells, leading to hemolysis or immune weakness based upon the above studies. bacterial infection or treatment with septic plasma might induce mitochondrial dysfunction by the accumulation of reactive oxygen species (ros) and nitric oxide radical (no ． ) in lymphocytes or epithelial cells leading to cell apoptosis [19] [20] [21] . therefore, we propose that cells with g6pd deficiency may be less tolerant to the oxidative stress caused by bacterial infection. in the present study, we investigate the direct impact of bacterial infection on g6pd-deficient epithelial cells using staphylococcus aureus as a model pathogen. s. aureus, which has long been recognized as a major cause of healthcareassociated infections, can result in sepsis and septic shock leading to vascular damage and multiple organ failure. vancomycin is one of the primary choices to treat infections resulting from multidrug-resistant s. aureus, including methicillin-resistant s. aureus (mrsa). our previous study demonstrated that the vancomycin-treated vancomycinresistant s. aureus (vrsa) strain did enhance cytotoxicity through the activation of σ b and alternation of virulence expression [22] . whether such enhancement is even stronger in g6pd-deficient cells was also investigated in this study. the vancomycin-resistant s. aureus strain sjc1200 was generated by introducing a vancomycin resistance-carrying plasmid (pg1546) into strain atcc 12598 as described previously [23] . briefly, the p r vanrsp h haxp y vanyp z vanz gene cluster (the van operon within tn1546) in e. faecalis hip12467 was amplified and then cloned into pghl6 from which the luxab gene was removed to generate pg1546. all bacterial strains were routinely cultured at 37°c with the specific required antibiotics (sigma) in bhi broth or on agar plates. a lung carcinoma cell line (a549) obtained from the american type culture collection (atcc) and derivatives of this cell line were used in the present study. the stable g6pdknockdown cell line, a549-5.20 , and the control cell line transfected with pci-neo vector only, a549-5s-5, were generated, approved and kindly given by dr. hung-yao ho [11] . briefly, g6pd-rnai plasmids were generated by the ligation of complementary oligonucleotides into the pci-neo mammalian expression vector followed by transfection into a549 cells to generate a549-5.20. the cells were cultured in dmem (gibco brl) supplemented with 10% fetal calf serum, penicillin (100 u/ml), and streptomycin (100 u/ml) at 37°c in a humidified atmosphere of air and 5% co 2 . before infection, overnight culture of the s. aureus strain sjc1200 was diluted back to an o.d. a600 of 0.2 and subcultured in 10 ml bhi broth without any antibiotic at 37°c until the o.d. a600 =0.6. bacterial infection of cells was performed by inoculating with sjc1200 (at a multiplicity of infection of 100; moi=100) in the absence or presence of vancomycin (32 μg/ ml). under specified circumstances, the function of α-hemolysin secreted by s. aureus was inhibited by adding oroxylin a (2 μg/ml; sigma) [24] , and the infected cells were cultured at 37°c in a humidified atmosphere of air and 5% co 2 for further use. for samples that were to be observed using a microscope, sterile glass cover slips were inserted into each of the wells in advance. the vrsa strain sjc1200 was grown overnight at 37°c with shaking in bhi broth. the next day, the bacterial culture was diluted (1/200) in fresh bhi broth with or without vancomycin (32 μg/ml) and then incubated at 37°c with shaking. at 0, 3, 6, 9, 12, and 24 h, the bacteria were plated on agar for enumeration of the surviving cfus. time-kill curves were then constructed by plotting the mean colony counts (log 10 cfu/ml) versus time from three experiments. cell viability tests were performed using the mtt assay with the cell proliferation reagent mtt (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide; sigma), as described previously [25] . the mtt tetrazolium ring is cleaved only by active mitochondria, yielding purple formazan crystals whose amount directly correlates with the viable cell count. at 16 h post-infection, 10 μl of a 5 mg/ml mtt solution was added into each well, and the plates were incubated at 37°c for 2.5 h. the purple formazan crystals were dissolved by adding 100 μl of mtt solubilization solution (sigma), and the absorbance at a 570 was spectrophotometrically measured with a reference wavelength of a 690 . the results were expressed as the percent absorbance of each experimental well versus the well containing untreated cells. three wells per experimental condition were counted in three independent experiments. the formation of intracellular ros was visualized by detecting dichlorofluorescein (dcf) derived from the oxidation of dihydrodichlorofluorescein (h 2 dcf) as previously described [12] . at 12 h post-infection, 5 μm dichlorofluorescein diacetate (h 2 dcfda; invitrogen) was added to each well at 37°c for 20 min and examined under a fluorescence microscope. quantification of ros formation was performed by flow cytometry. cells were treated as described above followed by two washes in pbs and trypsin treatment. flow cytometric and data analyses were performed as described elsewhere [12] . the mean fluorescence intensity (mfi) of the dcf channel was determined using cellquest pro software (becton dickinson). the results were expressed as the fold change of the percentage increase in dcf channel. cell apoptosis/necrosis was visualized by annexin v-fitc/ propidium iodide (pi) staining with a commercial apoptosis detection kit (biovision) by microscopy according to the manufacturer's instructions, as well as analysis by flow cytometry. phosphatidylserine translocation in apoptotic cells was stained with annexin v-fitc showing green fluorescence, whereas the nucleic acids in dead (necrosis) cells was stained with pi emitting red fluorescence. dapi was employed to stain the cell nucleus, producing blue fluorescence. at 16 h postinfection, cells were washed three times with pbs. for microscopy, 1× binding buffer was added to each well together with dapi, annexin v-fitc, and pi (5 μl of each) for 5 min. cover slips were removed and observed by confocal microscopy. for flow cytometry analysis, cells were trypsinized following a wash in pbs and resuspended in 1× binding buffer. five min after the addition of annexin v-fitc and pi (5 μl of each), flow cytometry analysis was performed using a facscalilbur (bd bioscience). the cells were excited with the 488 nm laser and green apoptotic cells were detected at 530 nm, while red necrosis cells were detected at 620 nm. no fluorescence could be detected in live cells, and cells showing both green and red fluorescence were considered to be in late apoptosis. biochemical markers of apoptosis were also detected by western blotting analysis. the cells were lysed in lysis buffer described elsewhere and the protein concentration of the lysate was determined by the bradford method [26] . after resolving the protein lysates by sds-page, the samples were transferred to nitrocellulose membranes and blocked with 5% milk in tbs containing 0.1% tween 20 (tbst). the membranes were then probed with 1:1000 primary antibody (mouse multiclonal; sigma) caspase-3, caspase-8, caspase-9 or gapdh at 4°c overnight. after washing in tbst, membranes were probed with 1:5000 secondary horse antimouse hrp-linked antibody (sigma) for 1 h and developed using ecl reagents (bioman). the blots were photographed using an x-omat 2000 (kodak) film processor. a student's t-test and non-parametric tests were used to analyze the experimental data and to compare means. pvalues of less than 0.05 were considered statistically significant. to understand whether g6pd-deficient epithelial cells were less tolerant than control cells to bacterial infection, the mtt assay was employed to evaluate cell viability upon s. aureus infection. the results shown in figure 1 indicate that cell viability of both a549-5s-5 (the scramble control) and a549-5.20 (g6pd-deficient) cell lines was decreased at 16 h post-infection. however, a549-5.20 cell viability (50%) was significantly less (p < 0.05) than that of a549-5s-5 cells (69%). we previously demonstrated that vancomycin could activate σ b in vancomycin-resistant staphylococcus aureus resulting in the enhancement of cytotoxicity [22] . the effect of vancomycintreated vrsa infection on a549 cells and their g6pd-deficient counterparts was investigated. no additional effect was observed upon vancomycin treatment in either the scramble control or g6pd-deficient cells (figure 1 ). we hypothesized that g6pd-deficient cells are less tolerant to oxidative stress upon bacterial infection, leading to the accumulation of more intracellular ros when compared to the control scramble cells. this was confirmed by directly observing dcf intensity within cells by fluorescence microscopy. as shown in figure 2a , the average level of green fluorescence intensity was higher in vrsa-infected cells than in untreated cells of both the a549-5s-5 and a549-5.20 cell lines. an even stronger fluorescence intensity was observed among vrsa-infected a549-5.20 cells than among a549-5s-5 cells. treatment with vancomycin showed no obvious effect on the fluorescence intensity of either cell line upon vrsa infection, but a slight increase in ros production was observed in vancomycin-treated control cells when compared to vancomycin-free cells of both cell lines. the production of ros was then further quantified by determining the mfi of the dcf channel. infection by vrsa resulted in a 2.6-and 5.0-fold increase in mfi among a549-5s-5 and a549-5.20 cells, respectively ( figure 2b and 2c). a more significant accumulation of ros production in g6pd-deficient cells than in control scramble cells (p < 0.05) was observed upon bacterial infection. vancomycin treatment of vrsa-infected cells also significantly reduced ros accumulation in a549-5s-5 and a549-5.20 cells, and vancomycin treatment alone showed a slight but non-significant increase in ros production in both cell lines ( figure 2b and 2c). to rule out reduced ros accumulation that might be due to increased bacterial killing upon vancomycin treatment, a bacterial killing kinetic assay was performed. slower bacterial growth was observed in the presence than in the absence of vancomycin for the first 9 hours, and no significant difference was seen for the duration of the experiment (24 h; figure s1 ). in addition, at 12 h post-infection, aliquots of bacteria-infected cell culture media were removed and plated on agar, but no significant difference between the cfus of the different experimental conditions was evident (data not shown). the van operon-carrying plasmid (pg1546) was also found in all of the colonies, indicating that the vancomycin resistance construct was stable in sjc1200 (data not shown). whether the accumulation of more intracellular ros resulted in stronger apoptotic activity in g6pd-deficient cells was investigated by fluorescence microscopy. the results shown in figure 3 indicate that most of the a549-5.20 cells showed apoptosis, necrosis or both phenotypes upon vrsa infection, whereas apoptotic activity was weaker in a549-5s-5 cells under the same condition. vancomycin treatment in vrsainfected cells apparently reduced apoptotic activity in both a549-5s-5 and a549-5.20 cells, as detected by the proportion of pi-stained cells. vancomycin treatment alone had no significant effect on apoptotic activity in either cell line. the quantification assay for apoptotic activity was evaluated by flow cytometry. the percentage of apoptotic and necrotic cells increased to 15.39% in vrsa-infected a549-5s-5 cells at 16 h post-infection. the percentage was significantly higher in a549-5.20 cells (30.22%; p < 0.05) than in control scramble cells (figure 3 , bottom row). as observed microscopically, vancomycin treatment of vrsa-infected cells significantly reduced the percentage of apoptotic and necrotic cells to 12.31% and 21.76% in a549-5s-5 and a549-5.20 cells, respectively. we detect a slight and non-significant increase in apoptotic activity upon vancomycin treatment alone in both a549-5s-5 and a549-5.20 cells (figure 3) . in addition to morphological markers, biochemical markers of apoptosis were detected by western blotting analysis. because caspases-8, -9 and -3 are located at key junctions in apoptosis pathways, their expression in a549-5s-5 cells was compared to that in a549-5.20 cells upon different treatments [27, 28] . the results shown in figure 4 indicate that a similar level of the cleaved caspase-8 (33 and 10 kda) was observed upon vrsa/ vancomycin-treated vrsa infection in both a549-5s-5 and a549-5.20 cells, suggesting a similar signal strength to trigger the extrinsic apoptotic pathway. a weak cleaved caspase-8 expression was found in bacteria-free a549-5.20 cells, implying that the g6pd-deficient cells were less tolerant to extrinsic death ligands during culture. on the other hand, vrsa infection obviously triggered higher expression of the precursor (37 kda) and cleaved forms (22 and 10 kda) of caspase-9, thereafter leading to higher expression levels of pro-caspase-3 (35 kda) and cleaved caspase-3 (20 and 11 kda), which is the effector caspase, in a549-5.20 cells than in scrambled control cells (figure 4) . the above results suggest that a stronger intrinsically apoptotic signal was triggered in g6pd-deficient cells upon bacterial infection. in addition, vancomycin treatment of vrsa-infected cells reduced the expression levels of cleaved caspase-9 and -3 in both cell lines (figure 4) . trace or non expression of caspase-9 and -3 was observed in bacteriafree cell cultures. we previously showed that a decrease in hla (encoding αhemolysin) expression and hemolytic activity was observed in strain sjc1200 upon vancomycin treatment [22] . to determine whether the reduced ros accumulation and apoptotic activity, particularly in g6pd-deficient cells, was due to deceased αhemolysin expression upon vrsa infection in the presence of vancomycin, the production of intracellular ros and cell apoptosis when the α-hemolysin inhibitor oroxylin a was added to the media was quantified by flow cytometry. hemolysis was inhibited when sjc 1200 cells were inoculated on blood agar plates containing either oroxylin a (2 μg/ml) or vancomycin (32 μg/ml) ( figure s2 ). treatment with oroxylin a or vancomycin, significantly reduced the intracellular ros and apoptotic activity of both cell lines in the presence of infection. oroxylin a treatment alone showed no significant effect on the accumulation of ros or cell apoptosis ( figure 5 ). in the present study, we demonstrate that g6pd-deficient epithelial cells were less tolerant to s. aureus infection through the accumulation of more ros leading to stronger apoptotic activity. studies on patients with g6pd deficiency have mostly focused on severe hemolytic anemia, extreme hyperbilirubinemia (jaundice), and bilirubin encephalopathy, especially in neonates [1] . as described, the effect of infection on patients with g6pd deficiency was studied mainly with regard to hemolytic anemia caused by plasmodium infection, antimicrobial drugs, and viral and bacterial infections. in addition to hemolytic anemia, the enhancement of both viral infection and immune weakness (such as neutrophil dysfunction) were also addressed. our study demonstrates a direct impact of bacterial infection on g6pd-deficient epithelial cells, a result that is in contrast to that found in blood cells. the lack of ros (such as o 2 -) production in phagocytes (neutrophils and monocytes) may cause chronic granulomatous disease-like clinical symptoms in patients with g6pd deficiency. such phagocyte dysfunction may lead to recurrent infections by catalase-positive microorganisms (such as staphylococci) because those bacteria could be phagocytosed but not digested [29] . on the other hand, it has been demonstrated that bacterial infection may induce oxidative stress in lymphocytes, in mammary epithelial cells, or in yeast [30] [31] [32] . consistent with the above findings, our results indicated that intracellular ros were generated in a549 lung carcinoma cells upon s. aureus infection (figure 2) . we further propose that the capacity for ros removal is impaired in g6pd-deficient cells following infection-induced oxidative stress. as expected, the accumulation of ros was significantly higher in g6pd-deficient cells than in control scramble cells following exposure to vrsa (figure 2) . therefore, the impact of bacterial infection on patients with g6pd deficiency is twofold. first is the immune weakness due to the dysfunction of phagocytes. second is the impairment of ros removal upon bacterial infection, as we have demonstrated in epithelial cells. it has been well demonstrated that apoptosis signalregulated kinase 1 (ask1), a member in the mitogen-activated protein kinase (mapk) cascades, is activated upon oxidative stress, leading to cell apoptosis [33] . we demonstrated that the impairment of ros removal in g6pd-deficient cells resulted in a higher apoptotic activity than in control scramble cells ( figure 3) . the caspases, a family of cysteine proteases, play essential roles in apoptosis. the classical apoptotic machinery involves the activation of initiator caspases such as caspase-8 and -9 upon death signals, thereby cleaving the inactive pro-forms of effector caspases such as caspase-3 to activate them [27, 28] .the commencement of the extrinsic apoptotic pathway depends on the activation of caspase-8 through the binding of extracellular death ligands to transmembrane death receptors. in contrast, the intrinsic pathway is triggered by the release of cytochrome c from mitochondria, thereby activating caspase-9. both pathways can lead to the activation of caspase-3 and other effector caspases [34] . our results presented here indicate that expression of active caspase-9, as well as the downstream caspase-3, was much higher in g6pd-deficient cells than in control scramble cells upon vrsa infection, suggesting that mitochondrial dysfunction may be the major cause of the increase in cell apoptosis (figure 4) . a recent study demonstrated that the incubation of epithelial cells with enterotoxin isolated from aeromonas veronii increased the accumulation of intracellular ros leading to a loss of mitochondrial membrane potential and thereafter undergoing apoptosis through the mitochondrial pathway [20] . the s. aureus strain sjc1200 used in this study was constructed under the atcc 12598 strain genetic background which is known to lack a variety of exotoxins except for α-hemolysin [35] . therefore, we may propose that α-hemolysin could cause mitochondria dysfunction similar to that found in a. veronii. we further highlight a much worse outcome in g6pd-deficient cells upon bacterial infection. unlike the lack of mitochondria in peripheral red blood cells (rbcs), the impact of bacterial infection on rbcs (causing hemolysis) and other cells is different in patients with g6pd deficiency. our previous study demonstrated that antibiotic treatment on a drug-resistant s. aureus strain would appear to be an environmental stress that activates σ b , a stress response transcription factor [22] . in that study, vancomycin induced σ b activity led to the alternation of downstream virulence genes in vrsa strains, as well as the increase in cytotoxicity to the human bronchial epithelial cells (beas-2b). the increased expression of bacterial binding capacity, but not the decreased expression of α-hemolysin, associated with increased σ b activity, might be the key contributor to the cytotoxicity. however, similar results were not observed in this study when the a549 cell line was used, neither in g6pd knock down or in control scramble cells. it has been reported that α-hemolysin was an important mediator of cytotoxicity in a549 cells whereas it was tolerated by beas-2b cells [22, 36] . as proposed above, α-hemolysin should be the major cause of mitochondria dysfunction through the accumulation of ros in a549 cells and derivatives. because the expression of α-hemolysin was decreased in sjc1200 upon vancomycin treatment, the impact of vancomycin treatment on the cytotoxicity of vrsa-infected a549 cells was not obvious in either g6pd knock down or control scramble cells (figure 1 ). in fact, vancomycin treatment alleviated the vrsa infection-enhanced ros accumulation and apoptosis in both cells even though cell survival was not improved (figures 1-4) . this phenomenon may be a result of the increased binding affinity of vrsa to the cells upon vancomycin treatment, which leads to stronger cytotoxicity and possibly bypasses the mitochondria dysfunction pathway, as we reported previously [22] . therefore, the net impact of vancomycin treatment on a549 cell survival in the presence of infection was similar to the vancomycin-free condition. nevertheless, we cannot rule out that the misuse of antibiotics in other drug-resistant strains will trigger the expression of other exotoxins that can cause a stronger oxidative stress in g6pd-deficient cells. although the susceptibility of different cell types to different bacterial virulence factors is variable, we suppose patients with g6pd deficiency are less tolerant to bacterial infection. in conclusion, we demonstrate a cell damage mechanism in g6pd-deficient epithelial cells upon bacterial infection. most of the cellular ros are generated from the mitochondria, where there are a variety of mechanisms to reduce the accumulation of ros or protect against oxidative stress [37] . the loss of mitochondrial membrane potential due to bacterial infection, possibly via s. aureus α-hemolysin in this case, leads to an increase in ros production. in g6pd-deficient cells, the impaired reconversion of glutathione disulphide to reduced glutathione caused by the imbalanced nadph/nadp + redox state renders the cell unable to remove ros effectively. therefore, the accumulation of excess ros results in stronger cell apoptosis through the mitochondrial pathway. one of the most important clinical concerns in treating bacterial infection in patients with g6pd deficiency is to avoid the usage of antibiotics which can cause hemolytic anemia [7] . vancomycin treatment did not further enhance vrsa cytotoxicity toward g6pd-deficient and control scramble a549 cells, as opposed to what we had previously observed in beas-2b cells [22] . figure s1 . time-kill curve of the vrsa strain sjc1200. strain sjc1200 was incubated without (open diamond) or with vancomycin (32 μg/ml; solid circle), and the results are presented as the means±sd of the log 10 cfu/ml from three separate experiments. (tif) figure s2 . the effects of oroxylin a or vancomycin treatment on the hemolytic activity of sjc1200 cells. sjc1200 cells (2×10 4 cfu) were inoculated onto a (a) blank blood agar plate or (b) plate containing vancomycin (32 μg/ml) or (c) oroxylin a (2 μg/ml) and incubated at 37°c overnight. 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spratt, donald e. title: an angelman syndrome substitution in the hect e3 ubiquitin ligase c-terminal lobe of e6ap affects protein stability and activity date: 2020-07-08 journal: plos one doi: 10.1371/journal.pone.0235925 sha: doc_id: 11857 cord_uid: brbqgbpz angelman syndrome (as) is a rare neurodevelopmental disorder characterized by speech impairment, intellectual disability, ataxia, and epilepsy. as is caused by mutations in the maternal copy of ube3a located on chromosome 15q11-13. ube3a codes for e6ap (e6 associated protein), a prominent member of the hect (homologous to e6ap c-terminus) e3 ubiquitin ligase family. e6ap catalyzes the posttranslational attachment of ubiquitin via its hect domain onto various intracellular target proteins to regulate dna repair and cell cycle progression. the hect domain consists of an n-lobe, required for e2~ubiquitin recruitment, while the c-lobe contains the conserved catalytic cysteine required for ubiquitin transfer. previous genetic studies of as patients have identified point mutations in ube3a that result in amino acid substitutions or premature termination during translation. an as transversion mutation (codon change from ata to aaa) within the region of the gene that codes for the catalytic hect domain of e6ap has been annotated (i827k), but the molecular basis for this loss of function substitution remained elusive. here, we demonstrate that the i827k substitution destabilizes the 3d fold causing protein aggregation of the c-terminal lobe of e6ap using a combination of spectropolarimetry and nuclear magnetic resonance (nmr) spectroscopy. our fluorescent ubiquitin activity assays with e6ap-i827k show decreased ubiquitin thiolester formation and ubiquitin discharge. using 3d models in combination with our biochemical and biophysical results, we rationalize why the i827k disrupts e6ap-dependent ubiquitylation. this work provides new insight into the e6ap mechanism and how its malfunction can be linked to the as phenotype. angelman syndrome (as) is a neuro-genetic disorder that effects 1 in 15,000 people characterized by symptoms such as developmental delay, speech impairment, intellectual disability, walking and balance disorders, and epilepsy [1] [2] [3] . individuals also demonstrate a unique behavioral pattern that typically includes a happy demeanor, easily provoked laughter, short attention span, sleep disturbance, and an affinity for water [2, 4] . as is caused by the loss of gene function of the maternal copy of ube3a on chromosome 15 . ube3a is paternally imprinted in neurons resulting in expression of the maternal allele alone, whereas other tissues retain normal biallelic expression patterns [2, [5] [6] [7] [8] . the human gene mutation database (hgmd) currently lists 161 different genetic mutations of ube3a [9] . of the genetic mechanisms that have been described as the cause of as, an estimated 70-80% of cases contain deletions in the maternal chromosome 15q11-q13 [2, 3] . another 10-20% of affected individuals harbor mutations in their maternally inherited ube3a gene, 3-5% of cases are due to two paternal copies of the chromosome, and lastly 3-5% of affected individuals have the paternal imprint of the maternal chromosome leaving no functioning copy of the ube3a gene [2, 3] . ube3a codes for the e3 ubiquitin ligase e6-associated protein (e6ap). this protein is a member of the homologous to e6ap carboxy-terminus (hect) family of e3 ubiquitin ligases and plays an important role in the ubiquitylation-signaling pathway. ubiquitylation is a post-translational modification of targeted proteins that initiates processes such as protein degradation, intracellular trafficking, and other signaling events [10, 11] . ubiquitin is transferred onto a substrate through a cascade of three enzymes (e1, e2, e3), with the combinatorial effect of the approximately 40 e2 and over 600 e3 enzymes ultimately determining substrate specificity [12] [13] [14] . the fate of the ubiquitylated substrate is determined by the ubiquitin linkage chain type by the e2/e3 combination [10, 11, 15] . for example, k29 and k48 linkages target a substrate protein for proteasomal degradation, while k63 linkages are involved in dna repair mechanisms and intracellular targeting [10, 11, 16] . the dysregulation of any of these components involved in the ubiquitylation process leads to a myriad of different diseases including various cancers, developmental disorders, and neurodevelopmental disorders including as [8, [16] [17] [18] . e6ap was first identified as an e3 ubiquitin ligase through its ability to target the tumor suppressor protein p53 for degradation in conjunction with the human papilloma virus protein e6 [19, 20] . e6ap is a 100 kda protein that catalyzes the covalent attachment of ubiquitin to its various substrates through the use of its c-terminal hect domain (residues 518-875). the hect domain consists of an n-terminal lobe (n-lobe) and a c-terminal lobe (c-lobe) connected by a flexible three-residue hinge [21] . a broad cleft at the interface of the two lobes contains the catalytic cysteine required for ubiquitylation [21] . e6ap selectively builds k48-polyubiquitination chains, consistent with its ability to target substrates for proteasomal degradation, and this ubiquitin chain-linkage specificity is located in the c-lobe of the e6ap hect domain [22] . e6ap has been shown to interact with numerous cellular proteins and can regulate a number of different homeostatic cellular processes [23] . prime examples of e6ap-regulated processes include cell cycle control through centrosomal regulation [24] , dna repair through its interaction with uv excision repair protein rad23 homolog a (hhr23a) [25] , targeting tuberin (tsc2) for proteosomal degradation [26] , signal transduction by binding to multiple src family tyrosine kinases [27] , breast cell proliferation through calmodulin/ca 2+ mediated proteosomal degradation of the estrogen receptor (er) [28] , and coordinating the inflammatory response in conjunction with annexin a1 [29] . while the genetic link between mutations in ube3a and as is well established in the literature [1-4, 6, 7] , the effect that each specific as mutation has on the translated protein product have not been fully characterized or well understood. in this study, we show that the as i827k substitution (also described as i804k based on an alternate open reading frame start codon [30] [31] [32] [33] ) partially disrupts the overall 3d fold of the hect c-terminal lobe of e6ap leading to its aggregation and diminished e6ap-ubiquitylation activity in vitro. we unambiguously demonstrate that the as i827k mutation in ube3a is a loss of function mutation. this biophysical study clarifies how the i827k substitution in the c-terminal lobe domain of e6ap contributes to the angelman syndrome phenotype. the original dna construct for the human hect c-lobe of e6ap (e6ap c-lobe ; uniprot q05086, residues 761-875) was codon optimized and synthesized by atum (newark, ca, usa) [34] [35] [36] . the gene was cloned into an ampicillin-resistant t7-inducible plasmid with an n-terminal his 6 affinity tag followed by a tev protease cleavage site (enlyfq/gs). the resulting his 6 -tev-e6ap c-lobe vector was subsequently used as the template to insert the i827k mutation using the sprinp protocol [37] . the catalytic cysteine was changed to an alanine (c843a) using the same protocol. due to subsequent precipitation issues of the i827k substituted construct and to increase solubility, the e6ap c-lobe -i827k open reading frame was subcloned into an expression vector with an n-terminal his 6 -sumo fusion tag using compatible 5' bamhi and 3' xhoi restriction sites. the hect domain of e6ap (residues 518-875) was pcr amplified from a plasmid coding for the full-length e6ap purchased from addgene (plasmid #8655; watertown, ma, usa) [27] and subcloned into the his 6 -sumo vector using compatible 5' bamhi and 3' xhoi sites. all plasmids (phis 6 -tev-e6ap c-lobe , phis 6 -sumo-e6ap c-lobe -i827k, phis 6 -sumo-e6ap hect and phis 6 -sumo-e6ap hect -i827k) were isolated using the monarch plasmid miniprep kit (new england biolabs, ipswich, ma, usa), quantified by a 280 using a nanodrop one c uv-vis spectrophotometer (thermo-fisher, waltham, ma, usa), and verified by dna sequencing (macrogen, cambridge, ma, usa). the phis 6 -tev-e6ap c-lobe , phis 6 -sumo-e6ap c-lobe -i827k, phis 6 -sumo-e6ap hect and phis 6 -sumo-e6ap hect -i827k expression plasmids were transformed into e. coli bl21 (de3) ril+ competent cells and grown at 37˚c in luria-bertani media supplemented with ampicillin 100 mg/l and chloramphenicol 34 mg/l. the e6ap proteins grown for heteronuclear nmr analysis were grown in minimal m9 media (2 x 1l) supplemented with 1 g/l of 15 nh 4 cl and 2 g/l of 13 c-glucose as the sole nitrogen and carbon sources. when the cultures reached an od 600 of 0.6-0.8, protein expression was induced with the addition of 0.5 mm iptg for 20 hours at 16˚c. the cells were harvested by centrifugation 6000 x g for 10 minutes at 4˚c using a sorvall lynx 4000 superspeed centrifuge with a fiberlite f10-4x1000 lex carbon fiber rotor (thermo-fisher) and resuspended in cold wash buffer (50 mm na 2 hpo 4 ph 8.0, 300 mm nacl, 10 mm imidazole) supplemented with problock gold bacterial protease inhibitor cocktail (goldbio, st. louis, mo, usa). the cells were then lysed using an avestin emulsiflex-c5 homogenizer (avestin, ottawa, on, canada) and clarified by ultracentrifugation using an optima l-80 xp ultracentrifuge with a ti 70.1 rotor (beckman-coulter) for 40 minutes at 41,000 rpm at 4˚c. the clarified supernatant containing the desired his 6 -tagged e6ap protein was then isolated using 5 ml of hispur ni-nta resin (thermo-fisher) and eluted with elution buffer (50 mm na 2 hpo 4 ph 8.0, 300 mm nacl, 250 mm imidazole). fractions containing e6ap protein were pooled and incubated at 25˚c for one hour in the presence of tev or sumo protease to cleave the n-terminal his 6 or his 6 -sumo tag, followed by overnight dialysis at 4˚c against wash buffer to remove excess imidazole. to separate the cleaved his 6 -or his 6 -sumo tag and his 6 -tagged protease from the desired e6ap protein, the cleaved sample was passed through the hispur ni-nta resin column a second time and the flow-through containing the desired tag-free e6ap protein was collected. the e6ap protein was then concentrated using a 10 mwco amicon ultra-15 centrifugal filter (millipore) to about 1 ml, and run through a superdex-75 gel filtration column using an akta pure 25l fast performance liquid chromatography (fplc) system with gel filtration buffer (50 mm hepes, 100 mm nacl, 1 mm dtt, ph 7.5 at 4˚c) at a flow rate of 1 ml/min. due to inherent insolubility issues and after numerous attempts, the e6ap hect and e6ap c-lobe -i827k proteins were unable to be passed through the size exclusion column. circular dichroism spectroscopy was performed on the e6ap c-lobe and e6ap c-lobe -i827k substituted protein using a jasco j-815 cd spectropolarimeter. the proteins were prepared by buffer exchange into low salt (10 mm na 2 hpo 4 ph 7.4, 30 mm nacl) using a 10 mwco slide-a-lyzer dialysis cassette (thermo-fisher). the samples were diluted to 70 μm and loaded into a quartz cuvette with a 1 mm pathlength. wavelength scans were averaged from six trials recorded from 260 to 195 nm at 10˚c using 1 nm increments and an averaging time of 1 second. the same sample was used afterwards to obtain a melting curve of the mutant monitoring changes in α-helical content at 222 nm over a temperature range of 5-90˚c, temperature slope 1˚c/min, data pitch 0.3, response 4 seconds, bandwidth 1 nm, sensitivity standard 100 mdeg, and voltage of 600 mv. ubiquitination assays were conducted with 10 μm alexa fluor 647 n-terminally labeled ubiquitin, 10 μm e1 activating enzyme ube1 (uba1), 15 μm e2 conjugating enzyme ube2l3 (ubch7), and e3 ligase (35 μm e6ap c-lobe or e6ap hect , as well as their variants), 2 μm dtt, 20 mm atp, 40 mm mgcl 2 in 50 mm hepes ph 7.5, 100 mm nacl. each reaction was incubated at 37˚c in a water bath for 30 minutes. to determine the presence of ubiquitin~thioester intermediates, appropriate samples were supplemented with 10 mm dtt. reactions were terminated by adding gel loading dye and heating at 95˚c in a dry bath for one minute. the samples were then loaded onto a bis-tris gel at ph 6.4 and run for 1 hour at 120 v. the gels were removed from the apparatus and immediately visualized on an ibright fl1000 imaging system (thermo-fisher) using the fluorescent gel imaging setting for alexa fluor 647. the 1 h-15 n heteronuclear single quantum correlation (hsqc) spectra [38] of 15 n-labeled e6ap c-lobe (3.5 mm) and e6ap c-lobe i827k (188 μm) were collected at 25˚c in a varian inova 600 mhz 4-channel solution-state nmr spectrometer equipped with a 5-mm pfg triple-resonance probe housed and maintained in the carlson school of chemistry and biochemistry at clark university. the samples were prepared to 600 μl in nmr buffer (20 mm na 2 hpo 4 ph 7.0, 100 mm nacl, 2 mm tcep, 1 mm edta), 10% d 2 o. the spectra were referenced to the methyl peaks of 2 mm 4,4-dimethyl-4-silapentane-1-sulfonic acid (dss) set at 0 ppm and 2 mm imidazole was added as an internal ph indicator [39] . the backbone resonances were sequentially assigned using standard 2d and 3d experiments from the varian biopack including 1 h-15 n-hsqc, hncacb [40] , cbca(co)nh [41] , hnca [42] [43] [44] , hn(co)ca [44, 45] , hn(ca)co [46] , and hnco [42] [43] [44] . side chain assignments were determined using c (co)nh [47] , h(cco)nh [47, 48] , as well as both aliphatic and aromatic [49] , hcch-tocsy [50, 51] and 1 h-15 n and 1 h-13 c-noesy experiments [52] [53] [54] . all data were processed using nmrpipe and nmrdraw [55] and the spectra were analyzed using nmrviewj [56, 57] . all chemical shift assignments and experiments were deposited into the biological magnetic resonance databank (http://www.bmrb.wisc.edu) under accession code 50084. wild-type e6ap c-lobe and e6ap c-lobe -i827k angelman syndrome substituted protein showed similar secondary structural content by circular dichroism, with minima at 208 and 222 indicating both proteins are predominantly α-helical at 10˚c (fig 1a) . to test the thermal stability of wild-type e6ap c-lobe and e6ap c-lobe -i827k, melting curves were obtained for each protein from 5-90˚c. the melting curve showed a marked decrease in thermal stability of the e6ap clobe -i827k (t m of 47.7˚c) when compared to e6ap c-lobe wild-type (t m of 57.7˚c) demonstrating that the angelman syndrome i827k substitution appears to partially destabilize the 3d fold of the protein (fig 1b) . the angelman mutant appears to start unfolding around 40˚c, close to the physiological temperature of 37˚c, whereas the e6ap c-lobe wild-type begins to unfold around 55˚c. these results indicate that the as substitution has a similar global fold at cooler temperatures similar to the wild-type, whereas the melting curve suggests that the i827k as substitution is detrimental to e6ap c-lobe stability. the transfer of ubiquitin through the ubiquitylation pathway onto the catalytic cysteine of the e6ap was assessed using a fluorescent ubiquitin activity assay. using this assay, we are able to observe the sequential transfer of fluorescent ubiquitin from the e1 activating enzyme ube1 (aka uba1), to the e2 conjugating enzyme ube2l3 (aka ubch7), and finally onto the hect e3 ubiquitin ligase. this assay allowed for the direct comparison of wild-type to the i827k angelman substituted e6ap hect and e6ap c-lobe activities. as shown in fig 2, the progression of the ubiquitylation cascade as ubiquitin is transferred sequentially onto the e1 activating enzyme ube1 in an atp-dependent manner, followed by a transfer onto the e2 conjugating enzyme ube2l3. furthermore, the labile nature of the thiolester ube1~ubiquitin and ube2l3~ubiquitin complexes was demonstrated by the addition of the reducing agent dtt (fig 2) . polyubiquitin chains formation by wild-type e6ap hect decreased in the presence of dtt. it is noteworthy that the addition of dtt did not result in a complete reduction of the e3~ubiquitin thioester bond in the e6ap hect , as was expected, possibly due to non-specific autoubiquitylation of the e6ap hect fig 1. circular dichroism spectra for e6ap c-lobe (wild-type-�, i827k substitution-◆) . a) wavelength scans of the e6ap c-lobe constructs show similar secondary structure content at 10˚c. b) melting curves show the e6ap c-lobe -i827k substitution has a lower the tm (47.7˚c) than wild-type e6ap c-lobe (57.7˚c). https://doi.org/10.1371/journal.pone.0235925.g001 during the reaction. this is consistent with previous reports of the isolated e6ap hect being able to autoubiquitylate itself in the absence of substrate [58, 59] . interestingly, the i827k substitution on the other hand did not result in any polyubiquitin chain formation, demonstrating that the as substitution decreases ubiquitylation activity. with regards to the e6ap c-lobe , the as i827k mutation also showed diminished activity compared to the wild-type e6ap c-lobe . polyubiquitin chains were not formed in either of the reactions, indicating that the n-lobe is required for the efficient catalysis of chain formation, consistent with activity assays for e6ap c-lobe [59] and other isolated hect e3 ubiquitin ligase c-lobes for huwe1, smurf2, and ubr5 [60, 61] . interestingly, the as i827k substituted e6ap c-lobe was still able to be monoubiquitinated, albeit to a lesser extent that wild-type e6ap c-lobe . this observation could possibly be due to the structural disruption of the i827k mutation, which prevents the proper presentation of the e6ap c-lobe catalytic cysteine to e2~ubiquitin complex in the absence of the e6ap n-lobe. mutating the catalytic cysteine to an alanine (c843a) resulted in the complete loss of ubiquitylation activity, as would be expected due to the requirement of e6ap c843 in thiolester bond formation with ubiquitin. the 1 h-15 n heteronuclear single quantum correlation (hsqc) spectra for 15 n-labeled wildtype e6ap c-lobe and the e6ap c-lobe -i827k substituted protein were used to analyze possible structural changes caused by the as mutation. the wild type e6ap c-lobe spectrum showed a well-folded protein, with well dispersed peaks, indicating that each amino acid was located in its own unique chemical environment (fig 3a) . in contrast, the spectrum of the as i827k substituted e6ap c-lobe showed many collapsed amide peaks that were mostly localized to the center of the spectrum (fig 3b) indicating that the protein was partially unfolded. if the protein was completely unfolded protein would have peaks collapse to the center of the spectrum as solvent exposed peaks no longer experience shielding effects of neighboring amino acids. the disappearance of many peaks can be explained by the decreased signal/noise ratio due to the lower protein concentration as well as the line broadening effects caused by protein aggregation [62] . this is consistent with our inability to purify e6ap c-lobe i827k protein after multiple attempts using gel filtration chromatography due to the protein aggregating and eluting in the void volume. furthermore, the concentration used for i827k substituted protein in nmr was similar to the concentrations used in our activity assays and cd experiments, where we did not observe any protein precipitation issues. based on the known e6ap crystal structure (pdb 1d5f) [21] , we hypothesize that the as i827k substitution leads to a structural disruption of the e6ap c-lobe due to a hydrophobic residue being switched to a residue with a positively charged sidechain terminus that would disrupt the hydrophobic network in the core of the domain. the i827 sidechain is not solvent accessible and would not readily accommodate the polar terminus of the lysine sidechain. this is corroborated by the noesy data that shows the i827 amino acid side chain methyl groups make numerous noe contacts to several different surrounding hydrophobic side chains including t774, y776, i787, f790, w791, l824, and m825 (fig 4) . the same rationale would apply to the newly published structure of the domain-swapped e6ap dimer (pdb 6tgk), as i827 is still found embedded in the monomeric hydrophobic core and is not involved in the dimerization interface [63] . this structural disruption likely induces an allosteric change that reduces the catalytic activity and leads to its subsequent aggregation. this is supported by the cd data showing the presence secondary structure at 37˚c as well as our observation of the probability plot was made by inputting the experimentally determined resonance assignments for e6ap c-lobe into the online webserver csi 3.0 [64] . the propensity to form an α-helix or β-strand are denoted in red and blue, respectively. the position of the i827 residue is marked with a star. https://doi.org/10.1371/journal.pone.0235925.g005 aggregated protein eluted in the void volume using gel filtration chromatography. the loss of structural integrity due to the as i827k substitution correlates with the loss of ubiquitylation activity for e6ap (fig 2) . we are confident that our nmr resonance assignments for the e6ap c-lobe (residues 761-875) are correct and complete as a chemical shift index analysis, which predicts the secondary structure elements based upon chemical shift deviations of the backbone atoms (ca, c', cβ, n, ha, and nh) [64] , are in good agreement with the known tertiary structure of c-terminal lobe of e6ap (fig 5) . this study provides a structural and biophysical rationale for the e6ap ubiquitin ligase activity loss due to the i827k substitution in as. this is in good agreement with a previous report that showed the as i827k substitution resulted in decreased ubiquitylation of the e6ap substrate hhr23a and instability in vivo [31] . continued studies on the e6ap structure and function will help to understand how as genetic mutations in ube3a result in enzymatic insufficiency and may lead to potential treatments to alleviate the angelman syndrome phenotype. 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assignments of isotopicallyenriched proteins resolution enhancement and spectral editing of uniformly 13c-enriched proteins by homonuclear broadband 13c decoupling 1h-1h correlation via isotropic mixing of 13c magnetization, a new three-dimensional approach for assigning 1h and 13c spectra of 13c-enriched proteins a 4d hcch-tocsy experiment for assigning the side chain 1h and 13c resonances of proteins overcoming the overlap problem in the assignment of 1h nmr spectra of larger proteins by use of three-dimensional heteronuclear 1h-15n hartmann-hahn-multiple quantum coherence and nuclear overhauser-multiple quantum coherence spectroscopy: application to interleukin 1 beta three-dimensional heteronuclear nmr of nitrogen-15 labeled proteins heteronuclear three-dimensional nmr spectroscopy of the inflammatory protein c5a nmrpipe: a multidimensional spectral processing system based on unix pipes using nmrview to visualize and analyze the nmr spectra of macromolecules nmr view: a computer program for the visualization and analysis of nmr data e6ap/ube3a ubiquitin ligase harbors two e2~ubiquitin binding sites analysis of ubiquitin recognition by the hect ligase e6ap provides insight into its linkage specificity beta-sheet augmentation is a conserved mechanism of priming hect e3 ligases for ubiquitin ligation structure of the hect c-lobe of the ubr5 e3 ubiquitin ligase using nuclear magnetic resonance spectroscopy to study molten globule states of proteins crystal structure of the catalytic c-lobe of the hect-type ubiquitin ligase e6ap csi 3.0: a web server for identifying secondary and super-secondary structure in proteins using nmr chemical shifts the authors thank dr. guoxing lin for maintaining the 600 mhz nmr spectrometer housed in the carlson school of chemistry and biochemistry at clark university. key: cord-048360-n9sih438 authors: villard, viviane; agak, george w.; frank, géraldine; jafarshad, ali; servis, catherine; nébié, issa; sirima, sodiomon b.; felger, ingrid; arevalo-herrera, myriam; herrera, socrates; heitz, frederic; bäcker, volker; druilhe, pierre; kajava, andrey v.; corradin, giampietro title: rapid identification of malaria vaccine candidates based on α-helical coiled coil protein motif date: 2007-07-25 journal: plos one doi: 10.1371/journal.pone.0000645 sha: doc_id: 48360 cord_uid: n9sih438 to identify malaria antigens for vaccine development, we selected α-helical coiled coil domains of proteins predicted to be present in the parasite erythrocytic stage. the corresponding synthetic peptides are expected to mimic structurally “native” epitopes. indeed the 95 chemically synthesized peptides were all specifically recognized by human immune sera, though at various prevalence. peptide specific antibodies were obtained both by affinity-purification from malaria immune sera and by immunization of mice. these antibodies did not show significant cross reactions, i.e., they were specific for the original peptide, reacted with native parasite proteins in infected erythrocytes and several were active in inhibiting in vitro parasite growth. circular dichroism studies indicated that the selected peptides assumed partial or high α-helical content. thus, we demonstrate that the bioinformatics/chemical synthesis approach described here can lead to the rapid identification of molecules which target biologically active antibodies, thus identifying suitable vaccine candidates. this strategy can be, in principle, extended to vaccine discovery in a wide range of other pathogens. human plasmodium falciparum (pf) infection is a dramatic public health problem. today approximately forty percent of the world's population is at risk of malaria. malaria causes more than 300 million acute clinical cases, and at least one million deaths annually. ninety percent of malaria deaths occur in sub-saharan african countries mostly among young children and pregnant women (http://rbm.who.int). thus, there is an urgent need of a malaria vaccine. however, vaccine discovery, in general and particularly in malaria, is still a very empirical process. in fact, protective antigens do not bear any structural, physico-chemical or sequence-related characteristics that would allow their identification. for protective humoral responses, the only recognized characteristics of antigens are their antigenicity/immunogenicity and accessibility. in addition, the lack of surrogate markers of protection renders the vaccine discovery process difficult and time consuming. hence, in spite of a constant and impressive progress in molecular biology techniques and antigen identification and expression [1, 2] , vaccine discovery is still labor-intensive, making the approach fastidious, costly and poorly adapted to highthroughput screening. proper protein folding and solubility remain a limitation in numerous cases. thus, overcoming the bottlenecks of manufacturing and identification of fragments/proteins, as possible targets of a protective immune response still constitute a scientific and technical challenge. we addressed this challenge by combining bioinformatics, chemical peptide synthesis and functional protection assays. we focused on the search for a-helical coiled coil motifs that, in general, do not exhibit a folding problem, and are a target of effective antibodies for several current malaria vaccine candidates (eg. lsa-1 [3] , lsa-3 [4] , msp-3 [5] , and msp-6 [6] and other pathogens [7] . msp-1, another leading malaria vaccine [8] , also contains predicted a-helical coiled coil regions (unpublished results). our choice was based on the following considerations. first, the a-helical coiled coil motif bears a characteristic seven amino acid residue repeat (abcdefg) n with hydrophobic residues located in a and d positions and hydrophilic residues generally elsewhere. this motif can be easily identified by bioinformatic analysis. secondly, an important known characteristic of the ahelical coiled coil domains is that, taken separately from the whole protein, they frequently and readily fold into the same stable oligomeric structure [9] . thirdly, for this reason, the a-helical coiled coil fragments are frequently recognized by conformational dependent antibodies, and can similarly elicit antibodies reactive with structurally ''native'' epitopes. in addition, these domains are short (about 40 residues) and can be rapidly produced by chemical synthesis. furthermore, when the antibodies have an anti-parasite biological activity, this designates the corresponding proteins/ fragments as potential, novel vaccine candidates to be further developed and assessed. here, we focus on the pf parasite erythrocytic stage, a target of protective antibodies and describe a straightforward, rapid procedure based on bioinformatic analysis of a-helical coiled-coil motifs and peptide synthesis. the screening of the pf genome [10] using generalized sequence profiles [11] identified several hundred proteins containing putative a-helical coiled coil motifs. through proteome and transcriptome data [12] [13] [14] we assessed which of these molecules are expressed in the pf parasite erythrocytic stage. the combined analysis/assessment identified over 100 segments associated with this stage and displaying the putative a-helical coiled coil motifs with high probability score (table s1) . out of these a-helical coiled coil fragments, in general 30-40 amino acids long, present either in the same protein or in different ones, 95 were chemically synthesized and hplc purified. among them, longer peptides (up to 70 amino acids), which contained one or more a-helical coiled coil domains, were also synthesized (antigens 1, 12 and 83; table s1 ). the selected antigens were then tested in elisa assays for reactivity with three panels of sera obtained from adult donors from burkina faso, tanzania and colombia, respectively. to our surprise, all of the a-helical coiled coil fragments were antigenic, though the prevalence of responders varied greatly (tables 1 and s1 ). in this manner, 71 proteins were identified whose lengths varied from 200 to 10,000 amino acids. twenty-one peptides with the highest prevalence of responders and elisa mean od value were selected for further studies. variation in recognition among the three panels of sera may be due to differences in the genetic background of the hosts, of the parasites and, most likely, to distinct malaria transmission conditions in the three regions. the high level of recognition of the a-helical coiled coil motifs may be explained by the fact that taken separately from the whole protein these fragments readily fold into the same stable structure in aqueous solution. indeed, circular dichroism (cd) studies of selected peptides associated with biological activities (tables 1 and 2) indicate that they predominantly assume an a-helical conformation in water. peptides 14, 27 and 45 ( figure s1a ) exhibit a cd pattern characteristic of a high a-helical content, whereas the remaining peptides show cd profiles similar to that shown for peptide 12 ( figure s1b ) or intermediate between those shown in figures s1a and s1b characteristic of a partial a-helical organization. when analyzed by size exclusion chromatography on fplc columns, peptides presented elution profiles between those exhibited by chymotrypsin and ribonuclease (mw 24 and 13kda, respectively). the cd and size exclusion chromatography results suggest that peptides adopt an a-helical coiled-coil structure, which need to be unambiguously ascertained by nmr and ultra-centrifugation studies. to test the biological activity of peptide-specific antibodies, the latter were purified by affinity chromatography using three serum pools obtained from papua new guinean adults. the 3 serum pools were first tested in elisa assays against 21 peptides that were the most antigenic (table 1) ; from these, 18 peptide-specific antibodies were purified from the most positive serum pool and tested again in elisa. these 18 antibodies all reacted with parasite native proteins in infected red blood cells as shown by ifat ( figure 1a ; table 2 ). reactivity was restricted to blood stages, since the antibodies did not react with sporozoites stages (data not shown), and this reactivity was also peptide-specific as shown by ifat competition assays with the corresponding peptide ( figure 1a ). the specificity of the antibodies obtained was investigated in detail, particularly since several peptides contain glutamic acid (glu)-rich sequences which are known to generate cross reactivity among several malarial glu-rich proteins [15] . cross-reactions were systematically investigated using each of the 18 affinitypurified antibodies on each of the 18 peptides. results show thatwith few exceptions-each antibody preferentially recognizes the peptide against which antibodies were affinity-purified, i.e. they are specific for the corresponding peptide (table s2) . to determine if non-specific antibody binding to solid phase-adsorbed antigens could be responsible for the rare cross-reactivities detected, elisa competition assays were performed. to this end, binding of antibodies to the solid phase-adsorbed antigen was competed against increasing concentrations of the homologous or cross-reacting peptides. only homologous peptides competed best whereas peptides having sequence similarity did not (figures 2a, 2b , and s2b), or at a much higher concentration ( figure s2a ) including the shorter glu-rich peptides derived from the cterminus of peptide 27 and 45 (figures 2a and 2b) . finally, the pattern of recognition of peptides by the various sera tested, which differ markedly from one to the other (data not shown), confirms the above results i.e., specificity of antibodies to the corresponding peptide. antibodies corresponding to the 18 selected peptides were tested for direct and cell-mediated anti-parasite activity. clinical experiments have shown that the antibody-dependent cellmediated inhibition (adci) of p. falciparum malaria represents one of the mechanisms controlling parasitemia and thereby clinical manifestations in humans [16] . twelve peptide-specific antibodies proved able to induce a strong (more than 40%) and intermediate (lower than 40%) monocyte-dependent parasite killing (table 1) , whereas, in the absence of monocytes, no direct effect of antibodies on parasite growth was observed. the effects were in the range observed with antibodies from african adults who have the highest natural protection known against malaria. therefore peptidespecific, human affinity-purified antibodies were functionally effective as shown by their ability to react with parasite proteins and to inhibit parasite growth. thus, in vitro functional assays show that peptide-specific antibodies elicited by natural exposure to the parasite can induce protective mechanisms effective against malaria. sixteen peptides -twelve targeted by adci positive antibodies and four controls-were used to immunize cb6f1 mice ( table 2) . eleven of them elicited an intermediate or high antibody response, four of which also recognized the parasite protein in infected erythrocytes as determined by ifat ( figure 1b ; table 2 ). as seen before for human antibodies, recognition was restricted to blood stages since sporozoites were negative in ifat assays (data not shown) and by ifat competition assays with the corresponding peptide ( figure 1b ). anti-peptide 27 mouse antibodies, which are positive in ifat, are also specific for the homologous peptide 27 but not for the sequence related peptides 9, 12 and 45 (table s2) . thus, peptides, which were chosen for their propensity to form ahelical coiled coil, can induce the production of antibodies that recognize epitopes present in the native protein. improvement of the immunogenicity and structural specificity of the remaining peptides might be achieved in the future by a) a short elongation at the n-and c-terminal ends, b) stabilizing the a-helix as suggested by cooper et al. and lu and hodges [17, 18] and/or c) use of other adjuvants. genetic polymorphism in current vaccine candidates is a major limitation to vaccine development. however available genotyping studies of our peptide sequences in parasite isolates of worldwide origin indicate very limited polymorphism (plasmodb 5.2, and unpublished sequencing data). a few peptide dna sequences show deletion of one entire heptad repeat so that the shorter region still preserves its potential for the a-helical coiled coil formation. with regard to the structural features and cellular location prediction of the proteins corresponding to the peptides selected for adci assays ( table 1) , 15 of the proteins contain a pentapeptide conforming to the pexel consensus [19, 20; 21, 22] , but that none of these have a position within the amino acid sequence that conforms to the location of known active pexel motifs (see materials and methods and membrane segments, and none of them has a gpi anchor. only one protein contains a signal sequence. fourteen proteins are predicted to be in the cytoplasm, one in the nucleus, one in the mitochondria, and one in the peroxysomes (table s3 ). the prediction of the sub-cellular localization of these proteins should be taken with caution because gene annotation is being constantly updated and/or protein trafficking of the parasite is complex and not fully elucidated [23] . further investigations will be required to determine the actual localization of the corresponding antigens. the predicted localization is a priori surprising for molecules able to trigger an adci activity. however, recent studies have shown that in addition to merozoite surface proteins, soluble proteins released at the time of schizont rupture were equally effective at triggering adci provided they defined at least two epitopes [24] , which is the case for a-helical coiled coil heptad repeats. therefore, molecules expressing a trans-membrane domain that can be exported to the parasite or host cell membrane, as well as molecules present in the cytoplasm of maturing schizonts and released by bursting schizonts can trigger antibodies to cross-link fc-c receptors on monocytes to achieve pf parasite killing. in conclusion, an approach combining a genome-wide search by bioinformatics of a-helical coiled coil protein motifs and chemical synthesis can lead to the rapid identification and development of new malaria vaccine candidates. in fact, this approach is straightforward and easy to scale up; vaccine formulations may comprise mixtures of peptides or single constructs made up of several epitopes. in principle, this strategy can be extended to the discovery of proteins and vaccine candidates in other complex pathogens. the pf 3d7 genome [10] was used for the bioinformatics analysis. the generalized sequence profile method and the pftools package [11] were used to search for the short a-helical coiled coil domains. the coiled coil profiles were constructed using an alignment of several amino acid sequences corresponding to the known coiled coil domain. two profiles containing four and five heptad repeats were used for the analysis. the cut-off levels of the profiles were chosen by tests performed against sequence database of proteins with the known 3d structures. subsequently, the coiled coil figure 1 . immunofluorescence microscopy analysis of pf 3d7 parasites with peptide specific antibodies. acetone/methanol-fixed schizonts and merozoites were reacted with a: human peptide specific, affinity purified antibodies obtained with peptides 12 and 14 (table 1) and b: sera from mice immunized with peptide 27 (table 1) . grey: bright field images; blue staining: indicates dapi nuclear staining of schizont stage parasites; red staining shows labeling of peptide specific antibodies by cy3-conjugated anti-human or anti-mouse igg specific antibody. merge picture is an overlay of the blue and red fluorescence channel. doi:10.1371/journal.pone.0000645.g001 regions selected by this approach were tested manually for the presence of the characteristic heptad repeats. these proteins were also analyzed by the coils program [25] . the selected a-helical coiled coil containing proteins were further tested on their possible surface location and gpi anchoring by using the following programs: identification of potential signal peptides, secretomep and signalp (http://www. cbs.dtu.dk/services/) [26] ; transmembrane spanning regions (tmpred http://www.ch.embnet.org/software/tmpred_ form.html and tmhmm http://www.cbs.dtu.dk/services/ tmhmm; [27, 28] ), and gpi-anchored proteins (http://mendel. imp.univie.ac.at/sat/gpi/gpi_server.html; [29] ) and prediction of sub-cellular localization (ptarget http://bioinformatics. albany.edu/,ptarget; [30] ). to identify the pexel-like motifs in sequences of the selected proteins we used the following pattern [ [deq] that represents a combination of the pexel patterns indicated in recent papers [21, 22] . the presence of the identified proteins in the asexual erythrocytic stages was also checked using the published data on the transcriptome and proteome of this stage of development of p. falciparum (www.plasmodb.org; [31] ). peptides were synthesized on the advanced chemtech (hatley st george, uk) ac t348 omega multi channel synthesizer and the applied biosystem synthesizer 431a and 433a (foster city, ca) using solid-phase fmoc chemistry. crude peptides were purified by rp-hplc (c18 preparative column) and analyzed by mass spectrometry (maldi-tof; applied biosystem, foster city, ca). chemicals and solvents used for peptide synthesis were purchased from fluka (buchs, switzerland) and novabiochem (laufelfinger, switzerland). circular dichroism (cd) spectra of peptides were recorded on a jasco j-810 spectrometer (jasco corporation, tokyo, japan) equipped with a temperature controller and a 0.1 cm path length cuvette. the measurements were made in water at ph 7.3 and 22uc and at a peptide concentration of 0.2 mg/ml. the sera from burkina faso were collected in the village of goundry located in the central mossi plateau, between 15 and 50 km north of the capital ouagadougou, in the province of oubritenga. the climate is characteristic of areas of sudanese savannah, with a dry season from november to may and a rainy season from june to october. malaria transmission is very high during the rainy season and markedly seasonal. ethical clearance was obtained from the ministry of health, burkina faso. after obtaining informed consent from parents and caretakers, heparinized venous blood samples were collected during a crosssectional survey during the malaria low transmission season 1998. the tanzanian sera came from a large-scale community based study undertaken in kikwalila village, kilombero district, morogoro region from 1982 to 1984. blood samples from adults (.15 years) were taken by finger prick and the serum was kept at -70uc until use. research and ethical clearance for the study was obtained by the tanzanian commission for science & technology. the colombian sera were collected in buenaventura the main port on the colombian pacific coast after human informed consent, during a cross sectional survey carried out from february to may 2002 within the framework of a project supported by the colombian research council, colciencias. the area has unstable transmission of both p. falciparum and p. vivax malaria. ethical clearance to draw blood from human volunteers was human purified antibodies were used at 5 mg/ml; ifat was not performed on ring stages. valle. blood was taken by venipuncture into tubes containing edta and sera fractionated and stored frozen until use. the sera from adults from papua new guinea (png) pooled for affinity purification were collected in the maprik district of the east sepik province, during a cross sectional survey in july 1992 within the framework of the malaria vaccine epidemiology and evaluation project (mveep) supported by the united states agency for international development [32] . the area is highly endemic for malaria. ethical clearance for mveep was obtained from the png medical research advisory committee. blood was taken by venipuncture into tubes containing edta. the pool of immune african globulins (piag) used for adci was prepared from immune individuals living in endemic areas and negative control igg (n-igg) was obtained from a pool of more than 1000 french adult donors with no history of malaria. briefly, the igg fractions from both positive and negative controls were purified using a size exclusion trisacrylh gf05m (pall bioseprah; pall life sciences, ny) column followed by an ionic exchange deae ceramic hyperdh f column (pall bioseprah). purified igg were then extensively dialyzed against rpmi and kept at 4uc until use. cb6f1 mice were injected 3 times with 20 mg of the indicated peptide in montanide isa 720 at the base of the tail on day 1, 22 and 78. bleeding was performed 10 days after the second and third immunization. elisa was performed according to lopez et al. [33] and anti human igg-or anti mouse igg conjugated to alkaline phosphatase was used (sigma, st louis, mo) as second antibody. individual human sera from 37, 42 and 39 adults donors from burkina faso, tanzania and colombia respectively were used at 1:200 dilution. serum was considered positive if the optical density (od) reading was higher than the mean od value+3 standard deviation (sd) of the negative controls (individual serum samples from 8 to 11 naïve swiss donors) or if the od ratio of the mean of duplicate experimental values to the mean od of the negative control was higher than 2. for mouse sera, the end point value was determined as the last dilution of the mean od value+3 standard deviation (sd) of the negative control (non immune sera). elisa competition assays were performed by incubating either each of the 18 selected human affinity-purified antibodies, or antibodies elicited in mice, together with each of the 18 antigens over the indicated range of concentrations for 30 minutes at room temperature prior to addition to the elisa peptide-coated plate wells. antigen-sepharose conjugate preparation: 5 mg of antigen was dissolved in 1 ml of coupling buffer (0.1 m nahco 3 containing 0.5 m nacl, ph 8.0). the cnbr-sepharose 4b (amersham bioscience ab, uppsala, sweden) was activated by swelling in 1 mm hcl and then washed with coupling buffer. the antigen solution was added to the gel and the mixture was stirred for 1h at rt. after the coupling reaction, excess antigen was washed away with coupling buffer. the unreacted activated groups were blocked by treatment with ethanolamine (0.25 m; ph 8.0) for 30 min at rt. the gel was then washed with sodium acetate buffer (0.1 m; ph 4.0), followed by coupling buffer. the antigensepharose beads were either used or stored at 4uc in pbs (1x) containing 1 mm azide. isolation of specific antibody: pooled human serum was diluted five times with pbs (1x) containing 0.5 m sodium chloride and mixed with antigen-sepharose conjugate. this mixture was then stirred gently on a wheel o/n at 4uc. after centrifugation the supernatant was collected and stored at 220uc for further use. the antigen-sepharose beads were then washed with 5 ml of trizma base tris (20 mm containing 0.5 m nacl, ph 8.0) then with 5 ml of tris (20 mm, ph 8.0). the elution of bound antibody was achieved with glycine (0.1 m, ph 2.5). the fractions obtained were instantly neutralized with tris (1 m, ph 8.0), dialyzed against phosphate buffer (0.1m, ph 7.0) and the antibody concentration was determined by the absorbance of the solution at 280 nm. slides coated with pf sporozoites were dried at rt for 30 minutes, fixed with 100% acetone at 4uc for 10 minutes, washed 2 times in pbs-0.05% tween 20, dried carefully and blocked with 20 ml/ well of pbs-3% bovine serum albumin (bsa) for 30 minutes at rt. slides coated with pf merozoites were fixed with 100% acetone at 220uc for 15 minutes and dried o/n at rt. the appropriate antibody or serum dilutions prepared in pbs-3% bsa were distributed (10 ml/well) and incubated for 1h at rt in a humid chamber. after washing with pbs-0.05% tween-20, goat anti-human or goat anti-mouse polyvalent immunoglobulins conjugated to cy3 (molecular probes) diluted 1/500 in pbs-3% bsa or anti-human igg (fc specific) fitc conjugate (sigma) diluted 1/50 in evans blue solution (1/50000) was added (400 ml/slide) and incubated for 1h at rt in a humid chamber in the dark. slides were washed as above, covered with 50 % glycerol, sealed and read using a fluorescence microscope (leica dmirb dc200). the uganda palo alto strain (fup/c) was cultured in rpmi-1640 supplemented with 0.5% albumax i (gibcobrl-invitrogen, san diego, ca). for adci assays, blood stage parasite cultures were synchronized by at least two successive sorbitol treatments followed, after maturation over 24 h, by floatation on 1% porcine skin gelatin type a (sigma). blood monocytes (mn) were prepared from cytapheresis samples obtained from healthy blood donors with no previous history of malaria (lecourbe blood bank, paris, france). peripheral blood mononuclear cells (pbmc) were separated on ficoll density gradients j prep (techgen, les ulis, france) and washed in ca 2+ and mg 2+ free hbss buffered with 10 mm hepes (both from gibcobrl-invitrogen). cells were then distributed on polystyrene 96-well flat-bottomed culture plates (tpp, trasadingen, switzerland) and adherent mn were selected by incubation for 2 h at 37uc, in a humidified 5% co 2 atmosphere. more than 90% of the adherent cells obtained in this manner were mn as estimated by the non-specific esterase test (a-naphtyl acetate esterase; sigma). mn from each donor were tested prior to adci assays and only those without direct inhibitory effect were used in assays. to wells containing 2610 5 mn purified as described above, 50 ml of an asynchronous parasite culture at 0.5 % parasitemia and 4 % hematocrit were added. wells were then supplemented with test or control antibodies (ab) and the total volume adjusted to 100 ml with culture medium. after 48 h and 72 h, 50 ml of culture medium were added to each well and after 96 h the adci assay was stopped and the final parasitemia was determined by light microscopy on giemsa-stained smears by counting $50,000 red blood cells. for each ab tested, duplicate wells included the following controls 1) non-specific monocytic inhibition, both mn+parasite, and mn+n-igg+parasites and 2) direct inhibition by control or test igg, both n-igg+parasites, and test abs+parasites. piag and n-igg were used at a final concentration of 1 mg/ml as positive and negative controls respectively. immunopurified tests abs were used at 15 mg/ml. the specific growth inhibitory index (sgi) which considers the parasite growth inhibition due to the effect of test abs cooperating with mn was calculated as follows: sgi = 1006[12(% parasitemia with mn and test abs/% parasitemia test abs)/(% parasitemia with mn and n-igg/% parasitemia n-igg)]. figure s1 cd spectra of the peptides 45 (s1a) and 12 (s1b) found at: doi:10.1371/journal.pone.0000645.s001 (12.32 mb tif) figure s2 elisa inhibition assay using anti-human peptide specific antibodies. binding of peptide specific antibodies to peptides 76 (s2a) and 9 (s2b) absorbed on elisa plates was inhibited by incubating specific antibodies (1-2 mg/ml) with peptides 14, 76 and 81 (s2a) and peptides 8 and 9 (s2b), respectively (see material and methods). peptides 14, 76 and 79 share nnm or mnn as sequence similarity while peptides 8 and 9 do not exhibit any apparent sequence similarity. table s3 structural feature and cellular location prediction of the proteins containing the peptides whose specific antibodies were tested in adci (table 1) . found at: doi:10.1371/journal.pone.0000645.s005 (0.05 mb doc) identification of vaccine candidates against serogroup b meningococcus by whole-genome sequencing reverse vaccinology and genomics plasmodium falciparum liver stage antigen-1 is well conserved and contains potent b and t cell determinants protection against plasmodium falciparum malaria in chimpanzees by immunization with the conserved pre-erythrocytic liver-stage antigen 3 phase i malaria vaccine trial with a long synthetic peptide derived from the merozoite surface protein 3 antigen plasmodium falciparum merozoite surface protein 6 displays multiple targets for naturally occurring antibodies that mediate monocyte-dependent parasite killing template-based coiled-coil antigens elicit neutralizing antibodies to the sars-coronavirus phase 1 randomized double-blind safety and immunogenicity trial of plasmodium falciparum malaria merozoite surface protein fmp1 vaccine de novo design of alpha-helical proteins: basic research to medical applications genome sequence of the human malaria parasite plasmodium falciparum a flexible motif search technique based on generalized profiles transcriptomics and proteomics: tools for the identification of novel drug targets and vaccine candidates for tuberculosis a proteomic view of the plasmodium falciparum life cycle the transcriptome of the intraerythrocytic developmental cycle of plasmodium falciparum crossreactive antigens between life cycle stages of plasmodium falciparum antibodies that protect humans against plasmodium falciparum blood stages do not on their own inhibit parasite growth and invasion in vitro, but act in cooperation with monocytes mapping of conformational b cell epitopes within alpha-helical coiled coil proteins a de novo designed template for generating conformation-specific antibodies that recognize alpha-helices in proteins targeting malaria virulence and remodeling proteins to the host erythrocyte a host-targeting signal in virulence proteins reveals a secretome in malarial infection proteomic analysis identifies novel proteins of the maurer's clefts, a secretory compartment delivering plasmodium falciparum proteins to the surface of its host cell multi-character population study of the vir subtelomeric multigene superfamily of plasmodium vivax, a major human malaria parasite a maurer's cleft-associated protein is essential for expression of the major malaria virulence antigen on the surface of infected red blood cells a novel antibody-dependent cellular cytotoxicity mechanism involved in defense against malaria requires costimulation of monocytes fcgammarii and fcgammariii predicting coiled coils from protein sequences improved prediction of signal peptides: signalp 3.0 tmbase-a database of membrane spanning proteins segments predicting transmembrane protein topology with a hidden markov model: application to complete genomes prediction of potential gpimodification sites in proprotein sequences ptarget [corrected] a new method for predicting protein subcellular localization in eukaryotes plasmodb: the plasmodium genome resource. a database integrating experimental and computational data the malaria vaccine epidemiology and evaluation project of papua new guinea: rationale and baseline studies a synthetic malaria vaccine elicits a potent cd8(+) and cd4(+) t lymphocyte immune response in humans. implications for vaccination strategies the authors wish to thank luis rodrigues and florela penea for the synthesis and purification of peptides and thomas smith for discussion and all of the blood donors. key: cord-262846-1mhimfsf authors: gray, nicholas; calleja, dominic; wimbush, alexander; miralles-dolz, enrique; gray, ander; de angelis, marco; derrer-merk, elfriede; oparaji, bright uchenna; stepanov, vladimir; clearkin, louis; ferson, scott title: is “no test is better than a bad test”? impact of diagnostic uncertainty in mass testing on the spread of covid-19 date: 2020-10-21 journal: plos one doi: 10.1371/journal.pone.0240775 sha: doc_id: 262846 cord_uid: 1mhimfsf testing is viewed as a critical aspect of any strategy to tackle epidemics. much of the dialogue around testing has concentrated on how countries can scale up capacity, but the uncertainty in testing has not received nearly as much attention beyond asking if a test is accurate enough to be used. even for highly accurate tests, false positives and false negatives will accumulate as mass testing strategies are employed under pressure, and these misdiagnoses could have major implications on the ability of governments to suppress the virus. the present analysis uses a modified sir model to understand the implication and magnitude of misdiagnosis in the context of ending lockdown measures. the results indicate that increased testing capacity alone will not provide a solution to lockdown measures. the progression of the epidemic and peak infections is shown to depend heavily on test characteristics, test targeting, and prevalence of the infection. antibody based immunity passports are rejected as a solution to ending lockdown, as they can put the population at risk if poorly targeted. similarly, mass screening for active viral infection may only be beneficial if it can be sufficiently well targeted, otherwise reliance on this approach for protection of the population can again put them at risk. a well targeted active viral test combined with a slow release rate is a viable strategy for continuous suppression of the virus. during the early stages of the united kingdoms sars-cov-2 epidemic, the british government's covid-19 epidemic management strategy was been influenced by epidemiological modelling conducted by a number of research groups [1, 2] . the analysis of the relative impact of different mitigation and suppression strategies concluded that the "only viable strategy at the current time" is to suppress the epidemic with all available measures, including the lockdown of the population with schools closed [3, 4] . similar analysis in other countries lead to over half the world population being in some form of lockdown by april 2020 and over 90% of global schools closed [5, 6] . these analyses have highlighted from the beginning that the eventual relaxation of lockdown measures would be problematic [3] . without a considered cessation of the suppression strategies the risk of a second wave becomes significant, possibly of greater magnitude than the first as the sars-cov-2 virus is now endemic in the population [7, 8] . although much attention was focused on the number of tests being conducted and the effect that testing could have in supressing the disease [9] [10] [11] . not enough attention has been given to the issues of imperfect testing, beyond matt hancock, uk secretary of state for health and social care, stating in a press conference on 2nd april 2020 that "no test is better than a bad test" [12] . in this paper we will explore the validity of this claim. the failure to detect the virus in infected patients can be a significant problem in highthroughput settings operating under severe pressure, with evidence suggesting that this is indeed the case [13] [14] [15] [16] [17] . the public are rapidly becoming aware of the difference between the 'have you got it?' tests for detecting active cases, and the 'have you had it?' tests for the presence of antibodies, which imply some immunity to covid-19. what may be less obvious is that these different tests need to maximise different test characteristics. to be useful in ending lockdown measures, active viral tests need to maximise the sensitivity. high sensitivity reduces the chance of missing people who have the virus who may go on to infect others. there is an additional risk that an infected person who has been incorrectly told they do not have the disease, when in fact they do, may behave in a more reckless manner than if their disease status were uncertain. the second testing approach, seeking to detect the presence of antibodies to identify those who have had the disease would be used in a different strategy. this strategy would involve detecting those who have successfully overcome the virus, and are likely to have some level of immunity (or at least reduced susceptibility to more serious illness if they are infected again), so are relatively safe to relax their personal lockdown measures. this strategy would require a high test specificity, aiming to minimise how often the test tells someone they have had the disease when they haven't [18] . a false positive tells people they have immunity when they don't, which may be worse than if people are uncertain about their viral history. the successes of south korea, singapore, taiwan and hong kong in limiting the impact of the sars-cov-2 virus has been attributed to their ability to deploy widespread testing, with digital surveillance, and impose targeted quarantines in some cases [13] . this testing has predominantly been based on the use of reverse transcription polymerase chain reaction (rt-pcr) testing. during the 2009 h1n1 pandemic the rapid development of high sensitivity pcr assay were employed early with some success in that global pandemic [19] . these tests, when well targeted, clearly provide a useful tool for managing and tracking pandemics. these tests form the basis of much of the research into the incidence, dynamics and comorbidities of sars-cov-2, but few, if any, of these studies give consideration to the impact of false test results [20] [21] [22] [23] [24] . increasing reliance on lower-sensitivity tests to address capacity concerns is likely to make available data on confirmed cases more difficult to accurately utilise [19] . it may be the case that false test results contribute to some of the counter-intuitive disease dynamics observed [25] . there is evidence that both active infection [26] [27] [28] [29] [30] and antibody [31] [32] [33] tests lack perfect sensitivity and specificity even in best-case scenarios. alternative screening methods such as chest x-rays may be found to have high sensitivity based on biased data [34] or may simply perform poorly even compared to imperfect rt-pcr tests [29] . the foundation for innovative in order to answer this question there are a number of important statistics: • sensitivity σ-out of those who actually have the disease, that fraction that received a positive test result. • specificity τ-out of these who did not have the disease, the fraction that received a negative test result. the statistics that characterise the performance of the test are computed from a confusion matrix (table 1) . we test n infected people who have covid-19, and n healthy people who do not have covid-19. in the first group, a people correctly test positive and c falsely test negative. among healthy people, b will falsely test positive, and d will correctly test negative. from this confusion matrix the sensitivity is given by (1) and the specificity by (2). s ¼ a n infected ð1þ sensitivity is the ratio of correct positive tests to the total number of infected people involved in the study characterising the test. the specificity is the ratio of the correct negative tests to the total number of healthy people. importantly, these statistics depend only on the test itself and do not depend on the population the test is intended to be used upon. when the test is used for diagnostic purposes, the characteristics of the population being tested become important for interpreting the test results. to interpret the diagnostic value of a positive or negative test result the following statistics must be used: • prevalence p-the proportion of people in the target population that have the disease tested for. • positive predictive value ppv-how likely one is to have the disease given a positive test result. • negative predictive value npv-how likely one is to not have the disease, given a negative test result. the ppv and npv depend on the prevalence, and hence depend on the population you are focused on. this may an entire nation or region, a sub-population with covid-19 compatible symptoms, or any other population you may wish to target. the ppv and npv can be calculated using bayes' rule: to illustrate the impact of prevalence on ppv, for a test with σ = τ = 0.95, if prevalence p = 0.05, then the ppv = 0.5. therefore, a positive result only indicates a 50% chance that an individual will have the disease given that they have tested positive, even though the test is highly accurate. fig 1 shows why, for 1000 test subjects there will be similar numbers of true and false positives even with high sensitivity and specificity of 95%. in contrast, using the same tests on a sample with a higher prevalence p = 0.5 we find the ppv = 0.95, see fig 2. similarly, the npv is lower when the prevalence is higher. sir models offer one approach to explore infection dynamics, and the prevalence of a communicable disease. in the generic sir model, there are s people susceptible to the illness, i people is "no test is better than a bad test"? infected, and r people who are recovered with immunity. the infected people are able to infect susceptible people at rate β and they recover from the disease at rate γ [38] , fig 3 shows how people move between the different states of an sir model. once infected persons have recovered from the disease they are unable to become infected again or infect others. this may be because they now have immunity to the disease or because they have unfortunately died. to explore the effect of imperfect testing on the disease dynamics when strategies testing regimes are employed to relax lockdown measures, three new classes were added to the model. the first is a quarantined susceptible state, q s , the second is a quarantined infected state, q i , and the third is people who have recovered but are in quarantine, q r , as shown in is "no test is better than a bad test"? the present model is similar to other sir models that take into account the effect of quarantining regimes on disease dynamics, such as lipsitch et al. (2003) [39] or giordano et al. (2020) [23] . lipsitch et al. implement quarantine in their model but do not incorporate the effects on the dynamics from imperfect testing, nor do they consider how the quality and scale of an available test affect the spread of a disease. diagnostic uncertainty plays no part in the model they present. likewise, giordano et al reduce population based diagnostic strategies to two parameters which confound test capacity, test targeting, and diagnostic uncertainty. again, they do not investigate the role that diagnostic uncertainty plays in the spread of a disease. the intent of this model is not to create a more sophisticated sir model, but to investigate how diagnostic uncertainty affects the dynamics of an epidemic. the model evaluates each day's population-level state transitions. there are two possible tests that can be performed: • an active virus infection test that is able to determine whether or not someone is currently infectious. this test is performed on some proportion of the un-quarantined population (s + i + r). it has a sensitivity of σ a and a specificity of τ a . • an antibody test that determines whether or not someone has had the infection in the past. this is used on the fraction of the population that is currently in quarantine but not infected (q s + q r ) to test whether they have had the disease or not. this test has a sensitivity of σ b and a specificity of τ b . each test is defined by a number of parameters. testing each day is limited by the test capacity c, the maximum number of tests that can be performed each day. each day a population n will be submitted for testing. the targeting capability of the test, t indicates the probability that an individual submitted for testing is positive, this is effectively the ppv of the initial screening effort. this results in a number of individuals m being considered for screening who are negative, of which k will be tested. targeting must be imperfect, as if it were perfect there would be no need for testing. unless otherwise stated, scenarios consider a default targeting of t = 0.8, representing an extremely effective screening capability that is nonetheless imperfect. if daily testing targets are a goal regardless of the prevalence of the illness, t can be overruled to ensure n � c for example. this condition is referred to as strict capacity and is denoted with boolean parameter g, defaulting to true for all scenarios. tests can also be conducted periodically by changing the test interval parameter d. these default to 1, i.e. daily testing. each test has unique parameters, so for example test a (active virus infection test) has a targeting parameter t a whilst test b (antibody test) has t b . the parameters σ, τ, t, c, g and d define a test. a person in any category who tests positive in an active virus test transitions into the corresponding quarantine state, where they are unable to infect anyone else. a person, in q s or q r , who tests positive in an antibody test transitions to s and r respectively. any person within i or q i who recovers transitions to r, on the assumption that the end of the illness is clear and they will know when they have recovered. for this parameterisation the impact of being in the susceptible quarantined state, q s , makes an individual insusceptible to being infected. similarly, being in the infected quarantined state, q i , individuals are unable to infect anyone else. in practicality there is always leaking, no quarantine is entirely effective, but for the sake of exploring the impact of testing uncertainty these effects are neglected from the model. other situations may require including this effect. the sir model used in this paper uses discrete-time binomial sampling for calculating movements of individuals between states. for a defined testing strategy these rates are defined as follows: in eq 7, bin(n, p) refers to a binomial distribution with count n and rate p, h(n, k, m) refers to a hypergeometric distribution with populations n and k and a sample size m. the model must be initialised with a defined population split between the six states. at each time step t, the model calculates the number of persons moving between each state in the order defined above. the use of binomial and hypergeometric sampling was prompted by a desire to incorporate aleatory uncertainty in each movement. the current approach does not account for epistemic uncertainty, fixing the model parameters σ, τ, c, t and d. a discrete time model was selected to allow for comparisons against available published data detailing recorded cases and recoveries on a day-by-day basis. if the tests were almost perfect, then we can imagine how the epidemic would die out very quickly by either widespread infection or antibody testing with a coherent management strategy. a positive test on the former and the person is removed from the population, and positive test on the latter and the person, unlikely to contract the disease again, can join the population. more interesting are the effects of incorrect test results on the disease dynamics. if someone falsely tests positive in the antibody test, they enter the susceptible state. similarly, if an infected person receives a false negative for the disease they remain active in the infected state and hence can continue the disease propagation and infect further people. in order to explore the possible impact of testing strategies on the relaxation of lockdown measures several scenarios have been analysed. these scenarios are illustrative of the type of impact, and the likely efficacy of a range of different testing configurations. • immediate end to lockdown scenario: this baseline scenario is characterised by a sudden relaxation of lockdown measures. • immunity passports scenario: a policy that has been discussed in the media [40] [41] [42] . analogous to the international certificate of vaccination and prophylaxis, antibody based testing would be used to identify those who have some level of natural immunity. • incremental relaxation scenario: a phased relaxation of lockdown is the most likely policy that will be employed. to understand the implications of such an approach this scenario has explored the effect of testing capacity and test performance on the possible disease dynamics under this type of policy. under the model parameterisation this analysis has applied an incremental transition rate from the q s state to the s state, and q r to r. whilst the authors are sensitive to the sociological and ethical concerns of any of these approaches, the analysis presented is purely on the question of efficacy. for the purpose of the analysis we have selected a population similar in size to the united kingdom, 6.7 × 10 7 people, β and γ were set to 0.32 and 0.1 respectively, this was ensure that r 0 value of the model was broadly in line with other models [43, 44] . under the baseline scenario, characterised by the sudden and complete cessation of lockdown measures, we explored the impact of infection testing. under this formulation the initial conditions of the model in this scenario is that the all of the population in q s transition to s in the first iteration. the impact of infection testing under this scenario was analysed in fig 5 using the parameters shown in table 2 . these scenarios consider the impact of attempts to control the disease through increased testing capacity and a more sensitive test. a test capacity range between 1 × 10 5 and 2 × 10 5 was considered as representative of the capabilities of a country such as the uk. to illustrate the sensitivity of the model to testing scenarios an evaluation was conducted with a range of infection test sensitivities, from 50% (i.e of no diagnostic value) to 98%. the specificity of these tests has a negligible impact on the disease dynamics in these scenarios. a false positive would mean people are unnecessarily removed from the susceptible population, but the benefit of a reduction in susceptible population is negligibly small. as would be expected the model indicates a second wave is an inevitability and as many as 20 million people could become infected within 30 days. a high-sensitivity test has little impact table 2 . https://doi.org/10.1371/journal.pone.0240775.g005 is "no test is better than a bad test"? beyond quarantining a slightly higher percentage of the population if capacities are low. at higher capacities this patterns remains, though peak infection counts are marginally reduced. overall it is clear that reliance on infection testing, even with a highly sensitive test and high capacities, is not enough to prevent widespread infection. the immunity passport is an idiom describing an approach to the relaxation of lockdown measures that focuses heavily on antibody testing. wide-scale screening for antibodies in the general population promises significant scientific value, and targeted antibody testing is likely to have value for reducing risks to nhs and care-sector staff, and other key workers who will need to have close contact with covid-19 sufferers. the authors appreciate these other motivations for the development and roll-out of accurate antibody tests. this analysis however focuses on the appropriateness of this approach to relaxing lockdown measures by mass testing the general population. antibody testing has been described as a 'game-changer' [45] . some commentators believe this could have a significant impact on the relaxation of lockdown measures [41] , but others note that there are severe ethical, logistical and medical concerns which need to be resolved before antibody testing could support a strategy such as this [46] . much of the discussion around antibody testing in the media has focused on the performance and number of these tests. the efficacy of this strategy however is far more dependent on the prevalence of antibodies (seroprevalence) in the general population. without widescale antibody screening it is impossible to know the seroprevalence in the general population, so there is scientific value in such an endeavour. however, the seroprevalence is the dominant factor to determine how efficacious antibody screening would be for relaxing lockdown measures. presumably, only people who test positive for antibodies would be allowed to leave quarantine. the more people in the population with antibodies, the more people will get a true positive, so more people would be correctly allowed to leave quarantine (under the paradigms of an immunity passport). the danger of such an approach are false positives. we demonstrate the impact of people reentering the susceptible population who have no immunity. we assume their propensity to contract the infection is the same as those without the false sense of security a positive test may engender. on an individual basis, and even at the population level, behavioural differences between those with false security from a positive antibody test, versus those who are uncertain about their viral history could be significant. the model parametrisation here does not include this additional confounding effect. to simulate the seroprevalence in the general population the model is preconditioned with different proportions of the population in the q s and q r states. this is analogous to the proportion of people that are currently in quarantine who have either had the virus and developed some immunity, and the proportion of the population who have not contracted the virus and is "no test is better than a bad test"? have no immunity. of course the individuals in these groups do not really know their viral history, and hence would not know which state they begin in. the model evaluations explore a range of sensitivity and specificities for the antibody testing. these sensitivity and specificities, along with the capacity for testing, govern the transition of individuals from q r to r (true positive tests), and from q s to s (false positive tests). each of the plots in figs 6 and 7 show the effect of different seroprevalence in the population. to be clear, this is the proportion of the population that has contracted the virus and recovered but are in quarantine. the analysis has explored a range of seroprevalence from 0.1% to 50%. fig 6 explores the impact of a variation in sensitivity, from a test with 50% sensitivity to tests with a high sensitivity of 98%. it can be seen, considering the top row of fig 6, that the sensitivity of the test has no discernible impact on the number of infections. the seroprevalence entirely dominates. this is possibly counter intuitive, but as was discussed above, even a highly accurate test produces a very large number of false positives when seroprevalence is low. in this case that would mean a large number of people are allowed to re-enter the population, placing them at risk, with a false sense of security that they have immunity. the bottom row of fig 6 shows the proportion of the entire population leaving quarantine over a year of employing this policy. at low seroprevalence there is no benefit to better table 3 . performing tests. this again may seem obscure to many readers. if you consider the highest seroprevalence simulation, where 50% of the population have immunity, higher sensitivity tests are of course effective at identifying those who are immune, and gets them back into the community much faster. a more concerning story can be seen when considering the graphs in fig 7 . now we consider a range of antibody test specificities. going from 50% to 98%. low specificities (τ < 0.9) table 4 . https://doi.org/10.1371/journal.pone.0240775.g007 lead to extreme second peaks, and could possibly lead to more. this is due to the progressive release of false-positives from the quarantined population, which eventually swells the susceptible population to a size where the infection count can resume exponential growth. high specificities avoid this at the cost of a prolonged lockdown, which is naturally limited by the lower false-positive rate. clearly some means of release beyond immunity passports would be required to avoid this scenario. notably, a reasonably specific test (τ b = 0.9) is capable of restraining a second peak to reasonably low levels regardless of seroprevalence. this may allow for other means of reducing lockdown measures, though with very low seroprevalence this could still be a potentially risky strategy. the dangers of neglecting uncertainties in medical diagnostic testing are pertinent to this decision [47] . considering the above, some form of incremental relaxation of lockdown seems appropriate. this could take many forms, it could be an incremental restoration of certain activities such as school openings, permission for the reopening of some businesses, the relaxation of stay-athome messaging, etc. under the parameterisation chosen for this analysis the model is not sensitive to any particular policy change. we consider a variety of rates of phased relaxations to quarantine. to model these rates we consider a weekly incremental transition rate from q s to s, and q r to r. in fig 8, three weekly transition rates have been applied: 1%, 5% and 10% of the quarantined population. whilst in practice the rate is unlikely to be uniform as decision makers would have the ability to update their timetable as the impact of relaxations becomes apparent, it is useful to illustrate the interaction of testing capacity and release rate. the model simulates these rates of transition for a year, with a sensitivity and specificity of 90% for active virus tests. the specifics of all the runs are detailed in table 5 . fig 8 shows five analyses, with increasing capacity for the active virus tests. in each, the 3 incremental transition rates are applied with a range of targeting capabilities. the value of 0.8 used previously represents an unrealistically extreme case of effective targeting. the ppv, as discussed above, has a greater dependence on the prevalence (at lower values) in the tested population than it does on the sensitivity of the tests, the same is true of the specificity and the npv. it is important to notice that higher test capacities cause a higher peak of infections for higher release rates. this has a counterintuitive explanation. when there is the sharpest rise in the susceptible population (i.e., high rate of transition), the virus rapidly infects a large number of people. when these people recover after around two weeks they become immune and thus cannot continue the spread of the virus. however, when the infection testing is conducted with a higher capacity up to 150,000 units per day, these tests transition some active viral carriers into quarantine, so the peak is slightly delayed providing more opportunity for those released from quarantine later to be infected, leading to higher peak infections. this continues until the model reaches effective herd immunity after which the number of infected in the population decays very quickly. having higher testing capacities delays but actually has the potential to worsen the peak number of infections. at 10% release rate, up to a capacity of testing of 150,000 these outcomes are insensitive to the prevalence of the disease in the tested population. this analysis indicates that the relatively fast cessation of lockdown measures and stay-home advice would lead to a large resurgence of the virus. testing capacity of the magnitude stated as the goal of the uk government would not be sufficient to flatten the curve in this scenario. the 1% release rate scenario indicates that a slow release by itself is sufficient to lower peak infections, but potentially extends the duration of elevated infections. the first graph of the top row in fig 8 shows that the slow release rate causes a plateau at a significantly lower number of infections compared to the other release rates. poorly targeted tests at capacities less than 100,000 show similar consistent levels of infections. however, with a targeted test having a prevalence of 30% or more, the 1% release rate indicates that even with 50,000 tests per day continuous suppression of the infection may be possible. the per-day testing capacity is varied across the five columns of graphs. rate, the percentage of the initial quarantined population being released each week is varied among rows. the prevalence of infections in the tested population is varied among different colours. to facilitate comparison within each column of graphs, the gray curves show the results observed for other rates and prevalences with the same testing intensity. model parameters are shown in table 5 . https://doi.org/10.1371/journal.pone.0240775.g008 at the rate of 5% of the population in lock-down released incrementally each week the infection peak is suppressed compared to the 10% rate. the number of infections would remain around this level for a significantly longer period of time, up to 6 months. there is negligible impact of testing below a capacity of 100,000 tests. however, with a test capacity of 150,000 tests the duration of the elevated levels of infections could be reduced if the test is extremely well targeted (t a = 0.7), reducing the length of necessary wide-scale lockdown. if this level of targeting is not achieved, increasing capacity may again increase peak infections, so care must be taken to ensure a highly targeted testing strategy. this analysis does support the assertion that a bad test is potentially worse than no tests, but a good test is only effective in a carefully designed strategy. more is not necessarily better and over estimation of the test accuracy could be extremely detrimental. this analysis is not a prediction; the numbers used in this analysis are estimates and the sirq model used is unlikely to be detailed enough to inform policy decisions. as such, the authors are not drawing firm conclusions about the absolute necessary capacity of tests. nor do they wish to make specific statements about the necessary sensitivity or specificity of tests or the recommended rate of release from quarantine. the authors do, however, propose some conclusions that would broadly apply when testing and quarantining regimes are used to suppress epidemics, and therefore believe they should be considered by policy makers when designing strategies to tackle covid-19. • diagnostic uncertainty can have a large effect on the dynamics of an epidemic. and, sensitivity, specificity, and the capacity for testing alone are not sufficient to design effective testing procedures. policy makers need to be aware of the accuracy of the tests, the prevelence of the disease at increased granularity and the characteristics of the target population, when deciding on testing strategies. • caution should be exercised in the use of antibody testing. assuming that the prevalence of antibodies is low, it is unlikely antibody testing at any scale will support the end of lockdown measures. and, un-targeted antibody screening at the population level could cause more harm than good. • antibody testing, with a high specificity may be useful on an individual basis, it has scientific value, and could reduce risk for key workers. but any belief that these tests would be useful to relax lockdown measures for the majority of the population is misguided. • the incremental relaxation to lockdown measures, with all else equal, would significantly dampen the increase in peak infections, by 1 order of magnitude with a faster relaxation, and 2 orders of magnitude with a slower relaxation. • as the prevelence of the disease is suppressed in different regions, it may be the case that small spikes in cases could be the result of false positives. this problem is potentially exacerbated by increased testing in localities in response to small increases in positive tests. policy decisions that depend on small changes in the number of positive tests may, therefore, be flawed. • for infection screening to be used to relax quarantine measures the capacity needs to be sufficiently large but also well targeted to be effective. for example this could be achieved through effective contact tracing. untargeted mass screening at any capacity would be ineffectual and may prolong the necessary implementation of lockdown measures. epidemiological models used for policy making in real time will need to take into account the impact of diagnostic uncertainty of testing, as well as the dynamical behaviour and sensitivity analyses of modelled parameters in an appropriately complex model that may need to include quarantining, contact tracing and other surveillance strategies, test availability and targeting, and multiple subpopulations of susceptible, infected and recovered categories. covid-19: government announces moving out of contain phase and into delay phase scaling up our testing programmes. department of health and socal care impact 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the viral shedding of 2019-ncov infections abstract interim guidelines for collecting, handling, and testing clinical specimens from persons for coronavirus disease 2019 (covid-19) correlation of chest ct and rt-pcr testing in coronavirus disease evaluation of novel antigen-based rapid detection test for the diagnosis of sars-cov-2 in respiratory samples. available at ssrn 3569871 potential false-negative nucleic acid testing results for severe acute respiratory syndrome coronavirus 2 from thermal inactivation of samples with low viral loads performance of vivadiag covid-19 igm/igg rapid test is inadequate for diagnosis of covid-19 in acute patients referring to emergency room department rapid point-of-care testing for sars-cov-2 in a community screening setting shows low sensitivity prediction models for diagnosis and prognosis of covid-19 infection: systematic review and critical appraisal sars-cov-2 diagnostics: performance data point-of-care versus lab-based testing: striking a balance molecular and antibody point-of-care tests to support the screening, diagnosis and monitoring of covid-19. oxford covid-19 evidence service transmission dynamics and control of severe acute respiratory syndrome why it's too early to start giving out immunity passports' could speed up return to work after covid-19 britain has millions of coronavirus antibody tests, but they don't work the reproductive number of covid-19 is higher compared to sars coronavirus euro surveillance: bulletin europeen sur les maladies transmissibles = european communicable disease bulletin boris johnson and donald trump talk up potential'game-changer' scientific advances on coronavirus developing a national strategy for serology (antibody testing) in the united states calculated risks: how to know when numbers deceive you key: cord-251979-j3mme15e authors: kandeel, amr; dawson, patrick; labib, manal; said, mayar; el-refai, samir; el-gohari, amani; talaat, maha title: morbidity, mortality, and seasonality of influenza hospitalizations in egypt, november 2007-november 2014 date: 2016-09-08 journal: plos one doi: 10.1371/journal.pone.0161301 sha: doc_id: 251979 cord_uid: j3mme15e background: influenza typically comprises a substantial portion of acute respiratory infections, a leading cause of mortality worldwide. however, influenza epidemiology data are lacking in egypt. we describe seven years of egypt’s influenza hospitalizations from a multi-site influenza surveillance system. methods: syndromic case definitions identified individuals with severe acute respiratory infection (sari) admitted to eight hospitals in egypt. standardized demographic and clinical data were collected. nasopharyngeal and oropharyngeal swabs were tested for influenza using real-time reverse transcription polymerase chain reaction and typed as influenza a or b, and influenza a specimens subtyped. results: from november 2007–november 2014, 2,936/17,441 (17%) sari cases were influenza-positive. influenza-positive patients were more likely to be older, female, pregnant, and have chronic condition(s) (all p<0.05). among them, 53 (2%) died, and death was associated with older age, five or more days from symptom onset to hospitalization, chronic condition(s), and influenza a (all p<0.05). an annual seasonal influenza pattern occurred from july–june. each season, the proportion of the season’s influenza-positive cases peaked during november–may (19–41%). conclusions: in egypt, influenza causes considerable morbidity and mortality and influenza sari hospitalization patterns mirror those of the northern hemisphere. additional assessment of influenza epidemiology in egypt may better guide disease control activities and vaccine policy. from november 2007-november 2014, 2,936/17,441 (17%) sari cases were influenzapositive. influenza-positive patients were more likely to be older, female, pregnant, and have chronic condition(s) (all p<0.05). among them, 53 (2%) died, and death was associated with older age, five or more days from symptom onset to hospitalization, chronic condition(s), and influenza a (all p<0.05). an annual seasonal influenza pattern occurred from july-june. each season, the proportion of the season's influenza-positive cases peaked during november-may (19-41%) . in egypt, influenza causes considerable morbidity and mortality and influenza sari hospitalization patterns mirror those of the northern hemisphere. additional assessment of influenza epidemiology in egypt may better guide disease control activities and vaccine policy. globally, acute respiratory infections (ari) are the fourth leading cause of death [1] and second highest cause of years of life lost [2] , and cause over 4.25 million deaths each year [3] . previous studies have indicated that influenza comprises a substantial portion of ari morbidity and mortality [4] [5] , with one estimate that 18% of global ari deaths were due to influenza infection in 2010 [6] and another that influenza causes 250,000-500,000 deaths each year [7] . however, little is known about influenza epidemiology in many parts of the developing world, particularly in the eastern mediterranean region (emr). the arab republic of egypt, the second most populous country of the emr, is a medium human-development country [8] located at the junction of northeastern africa and southwestern asia. egypt has a desert climate with hot, arid summers and moderate winters. the population is over 86.8 million, and 32% are under 15 years old [9] . in egypt, studies have evaluated limited aspects of human infection with avian influenza and the advent of the 2009 influenza pandemic [10] [11] [12] [13] [14] ; however, nationwide data on influenza morbidity, mortality, and seasonality are limited. resolving knowledge gaps in influenza epidemiology in egypt serves several purposes. first, a seasonal influenza vaccine is available each year that can prevent infections. recent u.s. estimates of the effectiveness of northern hemisphere influenza vaccines ranged from 19-60% [15] . even a moderately effective vaccine could reduce influenza-associated hospitalizations and lost worker productivity. understanding whether influenza strains match those in either northern or southern hemisphere circulation will elucidate whether northern or southern hemisphere seasonal influenza vaccine is a viable policy. additionally, data on whether demographic subpopulations are disproportionately affected by influenza may lead to designation of priority groups if vaccine availability is low. second, improved understanding of the timing of influenza circulation may result in a more cost-efficient timing of resource allocation (e.g., antivirals) for hospitals and other clinical settings. this could reduce financial burdens on hospitals during times of elevated influenza transmission. finally, enhanced data on annual influenza activity may provide a baseline metric to evaluate future activity. therefore, influenza epidemics may be detected earlier allowing public health officials to respond faster and have more opportunity to interrupt transmission. in 2007, the eastern mediterranean acute respiratory infection surveillance (emaris) network was established to initiate sentinel surveillance for severe acute respiratory infections (sari) to provide a better understanding of the epidemiology and viral etiologies of sari in the emr. the emaris network was formed through a collaboration of participating countries' ministries of health, u.s. centers for disease control and prevention (cdc), u.s. naval medical research unit no. 3 (namru-3), and world health organization (who) eastern mediterranean regional office. we describe seven years of influenza epidemiology from the emaris-egypt multi-site sentinel sari surveillance system. the aims of this study were to (1) assess the proportion of sari cases having influenza infection in egypt; (2) examine the types and subtypes of detected influenza viruses in egypt; (3) compare demographic and clinical characteristics of influenza-positive sari cases to those of influenza-negative sari cases in egypt; (4) quantify influenza deaths and assess influenza mortality risk factors in egypt; and (5) establish a defined period of influenza seasonality in egypt. a hospital surveillance team conducted sari surveillance at each site. these teams included a surveillance coordinator, internist, pediatrician, and laboratory focal person, each of whom was trained on case definitions, enrollment procedures, specimen collection, and data recording. teams screened all hospitalized patients with the sari case definition. all hospitalized patients (including weekend admissions) who met the case definition and were admitted between november 1, 2007 and november 30, 2014 were eligible for enrollment, and those who provided informed consent were enrolled and assigned a unique study identification (id) number. the case definition for identifying eligible patients was standardized across the emaris network. emaris participants reviewed the case definition each year, and it changed several times due to partner input and updated who guidance on sari: from 2007-2009, the 2006 who sari case definition was used [16] ; from 2010-2011, the case definition became any hospitalized patient 31 days or older meeting the 2006 who definition, or any patient meeting the cdc international emerging infection program pneumonia case definition [17] , or any patient suspected to have sari; from 2012-2014, the 2011 revised who sari case definition was used (any hospitalized patient with a history of fever (38°c) and cough in the past seven days) [18] , although clinicians could still enroll patients suspected to have sari; and finally, in 2014, the symptom history was extended to the past ten days to align with the 2014 revised who sari case definition [19] . each enrolled patient had an oropharyngeal and nasopharyngeal swab taken with a sterile tip flocked with nylon fiber swab applicator within 24 hours of hospital admission. all collected swabs from a patient were placed in a 15 ml tube with 2 ml viral transport medium and agitated vigorously for 10 seconds in a vortex mixer. the resulting supernatant was split into two cryovials pre-labeled with the patient's study id number and stored in -70°c freezers before testing. real-time reverse transcription polymerase chain reaction (rtrt-pcr) was conducted to detect influenza virus, types, and influenza a subtypes (determination of influenza b lineage was not done) [20] . specimens were first tested at the hospital or central public health laboratory (cphl) of the egypt ministry of health (moh), a recognized who national influenza center, and then at namru-3, a who regional reference laboratory, for quality assurance. namru-3 results were recorded as the gold standard final result by study id into microsoft excel (redmond, wa, usa). laboratory results were recorded as influenza negative, a/h1n1, a/h1n1pdm09, a/h3n2, a/h5n1, a/unsubtyped, or b. for analytical purposes, specimens positive for two or more influenza types were coded according to the first specimen listed of: a/h5n1, a/h1n1pdm09, a/h3n2, a/h1n1, b, a/unsubtyped. hospital surveillance teams completed a case report form including the unique study id and demographic and clinical data for each enrolled patient. no other identifying information was recorded. the form was standardized across the emaris network. teams followed each patient prospectively until discharge, transfer to another hospital, or death, and collected data including age, sex, reported history of fever and cough, date of symptom onset, date of hospitalization, and date of last follow-up (i.e., the date of discharge, transfer to another hospital, or death). sari cases and associated laboratory results were dated as the patient's hospital admission date. teams also abstracted data from medical records, such as chronic medical conditions and pregnancy status. each form was entered into microsoft access at moh and sent to a central database at namru-3. data managers performed double data entry and resolved any identified discrepancies amongst each other. the surveillance case report form database was merged with the laboratory result database, and the resulting database was cleaned and imported into sas 9.4 (sas institute, cary, nc, usa) for analysis. bivariate analyses of demographic and clinical data were conducted with the influenza laboratory result (positive versus negative) to generate counts and percentages and compare categorical levels by pearson's chi squared test. the odds of death among influenza-positive cases were modeled with logistic regression using different explanatory variables (age group: pediatric <15 years old versus adult 15 years old; sex: male versus female; days from symptom onset to hospitalization: 0-2 versus 3-4 versus 5; chronic conditions: at least one versus none; and influenza type: a versus b). statistical significance was set at an alpha level of 0.05. continuous variables were assessed individually to generate the median, range, and interquartile range (iqr). monthly influenza positivity rate was calculated as the proportion of specimens positive for influenza out of the total specimens tested for influenza each month. influenza seasonality was evaluated by examining the monthly average number and proportion of influenza-positive specimens. the influenza season was defined as beginning on the month having the lowest average influenza activity over the entire period and ending one year later. the month having the highest proportion of a season's influenza-positive specimens was considered that season's peak while the month having the lowest proportion was considered that season's nadir. namru-3 institutional review board (irb), cdc institutional review board, and egypt moh institutional review board approved the sari surveillance protocol in 2007 and maintained approval for the duration of the surveillance period. written informed consent was not required by the irbs due to the minimal risk faced by patients and because sentinel sari surveillance is part of egypt's national routine respiratory infection surveillance system of the egypt moh (the requirement was waived), but verbal informed consent was required. hospital surveillance teams provided consent information to patients both written and verbally in arabic, allowing adequate time for consideration. verbal informed consent was also obtained from the parents, caretakers, or guardians on behalf of all children/minors enrolled in the study. verbal informed consent was documented by the enrolling physician signing the consent form if the patient approved. the informed consent process for this study was approved by the three aforementioned institutional ethical review boards. from november 2007-november 2014, 17,441 patients met the sari case definition and had a specimen tested for influenza by rtrt-pcr (s1 file). of these, 2,936 (17%) were influenzapositive. a total of 2,013 (12%) were influenza a-positive and 923 (5%) were influenza b-positive. of the 2,013 influenza a-positive specimens, 115 (6%) were subtyped as seasonal a/ h1n1, 1,200 (60%) as a/h1n1pdm09, 674 (33%) as a/h3n2, 16 (1%) as a/h5n1, and 8 (<1%) as a/unsubtyped. demographic and clinical characteristics of sari patients by viral influenza result are shown in table 1 . influenza-positive patients differed from influenza-negative patients by age group (p<0.0001). among those with available data on age, 30% of influenza-negative patients were under five years old compared to 16% of influenza-positive patients. the median age of influenza-positive patients was higher than that of influenza-negative patients: 30 years (range 1 month-90 years, iqr 12-50 years) compared to 27 years (range 1 month-95 years, iqr 3-50 years). including missing data, influenza-positive cases were more likely to be female (and if female, be pregnant), have a preexisting chronic condition, require ventilation, be admitted to an intensive care unit (icu), and die (all p<0.0001). when missing data were excluded, statistically significant (p<0.05) differences persisted except for ventilation, icu admission, and death. the median duration from symptom onset to hospitalization among all patients was 3 days (iqr 2-4 among influenza-positive and 2-5 among influenza-negative), and this did not change by sex, age group, or both sex and age group. the median duration from hospitalization to end of follow-up among all patients was 5 days (iqr 4-6 among influenza-positive and 4-7 among influenza-negative), and this did not change by sex, age group, or both sex and age group. of the 2,936 influenza-positive cases, 53 (2%) died. influenza-positive deaths comprised 19% of sari deaths. among the 53 influenza deaths, 83% were 15-64 years old, 53% were male, and 55% had a preexisting chronic condition. twelve percent (3/25) of females who had influenza and died were pregnant. influenza a accounted for 83% of influenza-positive deaths, and of the 44 influenza a deaths, 70% were subtyped as a/h1n1pdm09, 18% as a/h5n1, 9% as a/ h3n2, and 2% as a/unsubtyped. the sari case fatality rates by influenza virus type were: 50% (8/16) a/h5n1, 13% (1/8) a/unsubtyped, 3% (31/1,200) a/h1n1pdm09, 1% (9/923) b, 1% (4/ 674) a/h3n2, and 0% (0/115) seasonal a/h1n1. the median duration from symptom onset to hospitalization and the median duration from hospitalization to end of follow-up were both 4 days among influenza-positive cases who died. factors associated with death among influenza-positive cases are displayed in table 2 . adjusting for all covariates, the odds of death among influenza-positive cases were significantly greater for adult versus pediatric patients, patients having symptoms for five or more days before hospitalization versus two or less days, patients with a preexisting chronic condition, and patients having influenza a versus b (all p<0.05). the calendar month with the lowest average number and proportion of influenza-positive cases was july, making july the influenza activity nadir. the influenza season in egypt was therefore defined as beginning in july and concluding in june. the calendar month having the highest average number and proportion of influenza-positive cases was december, making december the influenza activity peak. seasonal influenza activity peaks (fig 1) discussion this is the first study to examine national influenza epidemiology and seasonality in egypt. data from egypt's sari surveillance system were used to describe influenza hospitalizations at eight geographically-representative egyptian hospitals across a seven-year period. we found that approximately one of every six sari patients had an influenza infection, and during months of peak influenza activity, this was as high as one of every two. influenza-positive patients were different from influenza-negative patients in a number of ways, though differences regarding ventilation, icu admission, and death were inconclusive. death among influenza-positive patients was associated with four characteristics. we also found marked influenza seasonality beginning in july each year and ending in june. seasonal peaks occurred from november to may, and the peak month contained an average of 27% of the entire season's influenza infections. our findings indicate that influenza leads to substantial morbidity and mortality in egypt among people of all ages and both sexes. from 2007-2014, nearly 3,000 influenza hospitalizations were detected by the eight sentinel hospitals, approximately 17% of sari hospitalizations. this figure is nearly double those recently found in other emaris network countries: 8% in oman and 9% in jordan [21] [22] , and larger than estimates obtained in multiple african [5, 23] and asian countries [24] [25] . furthermore, influenza accounted for 19% of sari deaths in egypt. this mirrors the recent worldwide estimate of 18% [6] . comparison of demographic characteristics revealed that some individuals have increased odds of severe influenza infection in egypt. among the age groups encompassing 5-65 years, patients were more likely to have influenza. more influenza-negative patients were under five years old, possibly due to pediatric respiratory syncytial virus (rsv) infection which has been shown to be more prevalent among infants and young children in egypt [13, [26] [27] . a similar proportion of influenza-positive and influenza-negative patients were 65 years or older, possibly reflecting that elderly adults are simply predisposed to aris of various etiologies. other factors associated with influenza among those hospitalized with sari from this study include pregnancy and chronic conditions. influenza-positive women were more likely to be pregnant, and numerous other studies have identified pregnancy as a risk factor for influenza and influenza complications [28] [29] [30] [31] [32] . chronic conditions were also associated with influenza hospitalization, as 27% of influenza-positive patients had at least one. previous studies have shown that chronic disease is a risk factor for influenza and complications due to influenza [33] [34] [35] [36] . although 2% of patients with influenza died, there was no apparent difference in mortality between influenza-positive and negative patients. regardless, patients with influenza infection comprised 19% of total sari deaths. analysis of influenza mortality among hospitalized patients with influenza found four potential risk factors: older age, chronic conditions, a longer duration from symptom onset to hospitalization, and influenza a virus infection. overall, the number of deaths was greater for those with influenza a than influenza b, which aligns with previous research [37] [38] . the influenza subtype with the highest mortality (50%) was a/ h5n1 avian influenza. as of march 3, 2015, the worldwide a/h5n1 case fatality rate was 55% [39] , similar to our finding. furthermore, though 13% of patients with influenza a/unsubtyped died, there were only eight such infections. additionally, a longer duration from symptom onset to hospitalization increased the odds of death among influenza-positive patients. this supports previous research that found a longer duration from influenza symptom onset to hospitalization increased the odds of death [40] [41] , which may be due to individuals' healthcareseeking behavior, i.e., avoiding a hospital visit until it is too late for medical intervention. in addition to influenza hospitalization morbidity and mortality in egypt, we were able to assess seasonality over seven years. we observed an annual seasonal pattern beginning in july and ending the following june. overall influenza activity peaked in november-may, with the peak month having 19-41% of that entire season's influenza infections and accounting for 30-50% of all sari cases that month. these seasonal peaks were largely driven by influenza a, and in some seasons, there were bimodal influenza peaks corresponding to an earlier influenza a peak followed by a later influenza b peak. climate has been shown to correlate with influenza activity worldwide, particularly lower temperatures and lower relative humidity [42] [43] [44] . although egypt has a desert climate with year-round high temperatures [9], we observed annual northern hemisphere influenza seasonality with activity peaks in the winter and spring, perhaps due to lower temperatures during those months. based on these data, seasonal influenza vaccination in egypt may be a useful recommendation. the system identified predominant influenza strains each season [45] , indicating some seasonal influenza transmission could have been interrupted by vaccination. however, future studies are needed to antigenically characterize the predominant influenza strains in egypt to assess their similarity to strains included in the corresponding season's vaccine. possible priority groups for vaccination in egypt include pregnant women, individuals with chronic conditions, and older adults based on aforementioned findings. influenza seasonality in egypt suggests an ideal timing for resource allocation and provides a baseline metric on which to compare future activity. these data indicate that egyptian hospitals should prepare for peak influenza activity from november-may. stockpiling antiviral medication, increasing staff capacity, and reserving hospital beds could begin in october and be scaled back in may. also, moh and partners could use these data to determine average weekly influenza hospitalization activity (smoothed with moving averages) for evaluation of future activity. this will allow for earlier discovery of unusual influenza activity, including crossing of epidemic thresholds, allowing for a faster public health intervention. there were limitations to these data. first, the system's detection of influenza relied on the case definition's sensitivity and specificity, and the definition changed over time based on who guidance. it is possible the system missed some hospitalized individuals with influenza. however, the case definition was standardized across the network and the system detected a higher proportion of influenza sari hospitalizations than other countries. second, more comprehensive demographic and clinical data were unavailable, and thus more nuanced analyses of risk factors were not possible. this is a reality of surveillance as only basic data are collected to reduce staff burden. third, data were missing for a sizeable number of individuals for some variables. therefore, statistical comparisons were presented both including and excluding missing data, and some comparisons yielded divergent results (implying statistical significance was driven by the extent of missing data for some variables). work to maintain the quality and integrity of surveillance data through the emaris network is ongoing, including improving motivation among hospital surveillance teams. strengths of this study stem from its scale, length of observation, and capabilities to detect influenza virus. the use of eight sentinel hospitals representatively distributed throughout egypt allowed for adequate generalizability of findings to the national level. furthermore, with over 17,000 patients enrolled, there was ample power to detect meaningful differences in demographic, clinical, and virological characteristics. the seven-year period of surveillance afforded the opportunity to examine influenza seasonality over time, which is only possible with several years of data. finally, egypt's cphl and namru-3 enabled sensitive and specific detection of influenza viruses and types via rtrt-pcr for all 17,441 sari patients. this is the first documented examination of national influenza epidemiology in egypt. given the dearth of influenza knowledge in the emr, our study helps fill a critical epidemiologic gap for the second most populous country in the region. our findings indicate that influenza is an important cause of morbidity and mortality. furthermore, it appears to have northern hemisphere seasonality, though not as pronounced as in temperate climate countries. therefore, seasonal influenza vaccination may be a viable policy. ultimately, the emaris network provided numerous benefits, namely increased epidemiologic and laboratory capacity, enhanced international health regulations (2005) compliance, and a sustainable sari surveillance system. such a system is a crucial component of modern disease control, as evidenced by the recent appearance of middle east respiratory syndrome coronavirus [46] . it may also be adapted for other novel aris, which is imperative given growing concern over the emergence of new influenza viruses with pandemic potential [47] [48] . continued surveillance for influenza and further assessment of its molecular epidemiology will better inform decision-makers about disease control activities in egypt and the greater emr. supporting information s1 file. raw data. data file (sas format) for the severe acute respiratory infection (sari) sentinel surveillance system in egypt. (sas7bdat) the findings and conclusions in this reports are those of the authors and do not necessarily reflect the official policy or position of the department of the navy, department of defense, the centers for disease control and prevention, u.s. government, nor egypt ministry of health. patrick dawson and maha talaat are contractors of the u.s. government. mayar said is an employee of the u.s. government. this work was prepared as part of their official duties. title 17 uscx105 provides that "copyright protection under this title is not available for any work of the united states government." title 17 usc x 101 defines u.s. government work as work prepared by a military service member or employee of the u.s. government as part of that person's official duties. the study protocol was approved by the naval medical research center institutional review board in compliance with all applicable federal regulations governing the protection of human subjects. conceptualization: ak mt. data 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laboratory-confirmed influenza in kinshasa, democratic republic of congo incidence of influenza-like illness and severe acute respiratory infection during three influenza seasons in bangladesh estimating influenza outpatients' and inpatients' incidences from 2009 to 2011 in a tropical urban setting in the philippines viral etiologies of lower respiratory tract infections among egyptian children under five years of age incidence and clinical features of respiratory syncytial virus infections in a population-based surveillance site in the nile delta region risk of acute respiratory disease among pregnant women during influenza a epidemics fatal swine influenza pneumonia during late pregnancy hospitalizations with respiratory illness among pregnant women during influenza season severity of 2009 pandemic influenza a (h1n1) virus infection in pregnant women pregnant women infected with pandemic influenza a(h1n1)pdm09 virus showed differential immune response correlated with disease severity impact of epidemic type a influenza in a defined adult population survey of underlying conditions of persons hospitalized with acute respiratory disease during influenza epidemics in houston impact of respiratory virus infections on persons with chronic underlying conditions the burden of influenza illness in children with asthma and other chronic medical conditions the impact of influenza epidemics on mortality: introducing a severity index mortality associated with influenza and respiratory syncytial virus in the united states world health ogranization. cumulative number of confirmed human cases for avian influenza a(h5n1) reported to who infection and death from influenza a h1n1 virus in mexico: a retrospective analysis outcomes of adults hospitalised with severe influenza association of influenza epidemics with global climate variability influenza virus transmission is dependent on relative humidity and temperature seasonality, timing, and climate drivers of influenza activity worldwide available candidate vaccine viruses and potency testing reagents world health organization. interim surveillance recommendations for human infection with middle east respiratory syndrome coronavirus pandemic threat posed by avian influenza a viruses epidemiology, ecology and gene pool of influenza a virus in egypt: will egypt be the epicentre of the next influenza pandemic? the authors wish to thank all egypt ministry of health and central public health laboratory staff and hospital surveillance coordinators for their participation and continued excellence in sari surveillance. key: cord-280482-o887a7q9 authors: xu, suming; wang, xu; wang, yaoqin; lutgendorf, susan; bradley, catherine; schrepf, andrew; kreder, karl; o'donnell, michael; luo, yi title: transgenic mice expressing mcp-1 by the urothelium demonstrate bladder hypersensitivity, pelvic pain and voiding dysfunction: a multidisciplinary approach to the study of chronic pelvic pain research network animal model study date: 2016-09-29 journal: plos one doi: 10.1371/journal.pone.0163829 sha: doc_id: 280482 cord_uid: o887a7q9 monocyte chemoattractant protein-1 (mcp-1) is one of the key chemokines that play important roles in diverse inflammatory and chronic pain conditions. interstitial cystitis/bladder pain syndrome (ic/bps) is a chronic and debilitating inflammatory condition of the urinary bladder characterized by the hallmark symptoms of pelvic pain and voiding dysfunction. to facilitate ic/bps research, we used transgenic technology to develop a novel urothelial mcp-1 secretion mouse model (uro-mcp-1). a transgene consisting of the uroplakin ii gene promoter and the mouse mcp-1 coding sequence with a secretory element was constructed and microinjected. uro-mcp-1 mice were found to express mcp-1 mrna in the bladder epithelium and mcp-1 protein in the urine, and developed bladder inflammation 24 hours after intravesical administration of a single sub-noxious dose of lipopolysaccharide (lps). the inflamed bladders of uro-mcp-1 mice exhibited elevated mrnas for interleukin (il)-1ß, il-6, substance p precursor, and nerve growth factor as well as increased macrophage infiltration. in parallel with these phenotypic changes, uro-mcp-1 mice manifested significant functional changes at days 1 and 3 after cystitis induction. these functional changes included pelvic pain as measured by von frey filament stimulation and voiding dysfunction (increased urinary frequency, reduced average volume voided per micturition, and reduced maximum volume voided per micturition) as measured by micturition cages. micturition changes remained evident at day 7 after cystitis induction, although these changes were not statistically significant. control wild-type c57bl/6 mice manifested no clear changes in histological, biochemical and behavioral features after similar cystitis induction with lps. taken together, our results indicate that uro-mcp-1 mice are hypersensitive to bladder irritants such as lps and develop pelvic pain and voiding dysfunction upon cystitis induction, providing a novel model for ic/bps research. aberrant overexpression of monocyte chemoattractant protein-1 (mcp-1; also named ccl2) has been observed in diverse inflammatory and chronic pain conditions [1] . mcp-1 is produced by multiple cell types including bladder epithelial cells [1, 2] and plays an important role in recruiting monocytes/macrophages as well as other leukocytes to sites during an inflammatory process. the fundamental importance of mcp-1 and its cognate receptor ccr2 are underscored by studies using genetic knockout mice lacking either of these two proteins. these genetically deficient mice display decreased macrophage recruitment and activation, increased susceptibility to mucosal infection, and reduced t cell responses [3] [4] [5] . moreover, transgenic models of tissue specific mcp-1 expression have recapitulated many inflammatory disorders such as insulitis, pneumonitis and encephalitis but typically only after an additional inflammatory stimulus is introduced [6] [7] [8] [9] . in humans, elevated mcp-1 has been observed in various inflammation and autoimmune associated diseases such as inflammatory bowel disease, multiple sclerosis, diabetes, rheumatoid arthritis, and allergic asthma [1] . elevated mcp-1 has also been reported for overactive bladder (oab) [10, 11] and chronic prostatitis/chronic pelvic pain syndrome (cp/cpps) [12] . similarly, animal studies have demonstrated elevated mcp-1 in bladder outlet and ureteral obstruction, which correlate with bladder and renal pathology, respectively [13, 14] . in addition, elevated mcp-1 in the bladder has been observed to associate with bladder inflammation, reduced bladder capacity, and pelvic pain in a cyclophosphamide (cyp)-induced cystitis model [15] . moreover, one study demonstrated that elevated mcp-1 in the bladder promoted histamine release from mast cells in a protamine sulfate and lipopolysaccharide (lps)-induced cystitis model [16] . another study demonstrated that mast cell associated mcp-1 mediated bladder inflammation and chronic pelvic pain in a uroplakin peptide-induced autoimmune cystitis model [17] . all these findings suggest that aberrant overexpression of mcp-1 may contribute to pelvic pain and voiding dysfunction in interstitial cystitis/bladder pain syndrome (ic/bps), a chronic and debilitating inflammatory condition of the urinary bladder characterized by the hallmark symptoms of pelvic pain and voiding dysfunction [18] . here we report the development of a transgenic mouse model (uro-mcp-1) that secretes mcp-1 by the bladder epithelium and develops bladder inflammation, pelvic pain and voiding dysfunction upon intravesical administration of a single sub-noxious dose of lps. the uro-mcp-1 model demonstrates the hallmark symptoms of ic/bps and provides a novel model for ic/ bps research. all animal experiments were approved by university of iowa animal care and use committee (permit number: 1308153) and performed according to the guide for the care and use of laboratory animals of the national institutes of health. the schematic structure of the transgenic mcp-1 construct is shown in fig 1a. a plasmid containing the uroplakin ii (upii) gene promoter was kindly provided by dr. t. sun at the new york university school of medicine [19] . the 3.6 kb fragment containing the upii gene promoter was excised and placed upstream to the mouse mcp-1 coding sequence (0.52 kb). an intron sequence was inserted between the upii gene promoter and the mcp-1 transgene to facilitate discriminating mcp-1 mrna from its genomic dna in rt-pcr analysis [20] . the 4.9 kb kpnl-draiii dna fragment consisting of the above-mentioned sequences and a poly a additional site was microinjected into fertilizing eggs (b6/sjl background; the jackson laboratory, bar harbor, maine). transgenic founders were backcrossed with c57bl/6 mice (charles river laboratories, wilmington, ma) for 10 generations to generate c57bl/6 congenic uro-mcp-1 mice. each generation was confirmed for the presence of the mcp-1 transgene by pcr genotyping using a sequence-specific primer pair (5'-cgaggtcgactgcagaag-3' and 5'-tgaggtggttgtggaaaagg-3'; 690 bp). pcr was performed for 40 cycles at 94°c for 30 seconds, 55°c for 30 seconds, and 72°c for one minute. the pcr products were analyzed by 1% agarose gel electrophoresis. rt-pcr was performed on various tissues from an uro-mcp-1 mouse. mcp-1 mrna product is indicated by a red arrow. gapdh was used as an internal control. pupii-mcp-1, a plasmid containing the transgenic mcp-1 dna sequence (a control for mcp-1 genomic dna). c57bl/6 bladder, a c57bl/6 mouse bladder (a negative control). (c) uro-mcp-1 mice express mcp-1 mrna in the bladder epithelium. the bladder epithelium from an uro-mcp-1 mouse was processed for rt-pcr. mcp-1 mrna product is indicated by a red arrow. gapdh was used as an internal control. pupii-mcp-1 served as a control for mcp-1 genomic dna. c57bl/6 bladder epithelium served as a negative control. (d) uro-mcp-1 mice express mcp-1 protein in the urine. urine was collected from both c57bl/6 (n = 5) and uro-mcp-1 mice (n = 12) and analyzed for mcp-1 by elisa. data are shown as mean ± s.d. *p<0.001 as compared to the urine of c57bl/6 mice. female mice (6-8 weeks old) were used due to a higher incidence of ic/bps in females than males in humans and the easier feasibility of intravesical procedures in females. mice were anesthetized by intraperitoneal (i.p.) injection with 100 μl of a mixture solution of ketamine (87.5 mg/kg) and xylazine (12.5 mg/kg). the bladder was then catheterized urethrally with a 24 gauge 3/4" long plastic intravenous catheter (smiths medical, southington, ct), instilled with 1 μg of lps (e. coli 055:b5, sigma-aldrich, st. louis, mo) in 100 μl phosphate-buffered saline (pbs), and retained for 1 hour. mice instilled with 100 μl pbs in the bladders served as controls. bladders were collected and processed for formalin fixation, paraffin embedment, section preparation, hematoxylin and eosin (h&e) staining, and photography as described previously [21] . bladder inflammation was scored in a blinded manner based on infiltration of inflammatory cells in the lamina propria and the presence of interstitial edema as described previously: 1+ (mild infiltration with no or mild edema), 2+ (moderate infiltration with moderate edema), and 3+ (moderate to severe infiltration with severe edema) [21] . bladder immunohistochemistry was performed as described previously [21] . briefly, the bladders were fixed in 10% neutral formalin, embedded in paraffin, and cut into 5 μm sections. after antigen retrieval and blocking, slides were incubated with biotinylated rat anti-mouse f4/ 80 antibody (biolegend, san diego, ca; clone: ci:a3-1; rat igg2b) or control biotinylated rat igg2b (biolegend, san diego, ca; clone: rtk4530;) overnight. slides were developed conventionally using streptavidin-horseradish peroxidase complex (sav-hrp) and diaminobenzidine (dab) substrate solution (bd pharmingen). after rinsing, slides were counterstained with hematoxylin solution and photographed using an olympus bx-51 microscope. total rnas were extracted from the bladder and bladder epithelium using qiagen rneasy mini kit (qiagen, valencia, ca) as described previously [21] . cdna was synthesized using invitrogen superscript iii reverse transcriptase (invitrogen, carlsbad, ca) and oligo dt. pcr amplification was performed on cdna products using taq dna polymerase (new england biolabs, ipswich, ma) and sequence-specific primer pairs for mcp-1 (5'-cgaggtcgactgc agaag-3' and 5'-tgaggtggttgtggaaaagg-3'; 560 bp), il-1ß (5'-gcccatcc tctgtgactcat-3' and 5'-aggccacaggtattttgtcg-3'; 230 bp), il-6 (5'-gttctctgggaaatcgtgga-3'and 5'-ggaaattggggtaggaagga-3'; 339 bp), tachykinin-1 (substance p precursor) (5'-gccaatgcagaactacgaaa-3' and 5'-gcttggacagctccttcatc-3'; 280 bp), nerve growth factor (ngf) (5'-ctgtggaccccagactgttt-3' and 5'-cactgagaactcccccatgt-3'; 194 bp), and glyceraldehyde-3-phosphate dehydrogenase (gapdh) (5'-gttc cagtatgactccact-3' and 5'-gtgcaggatgcattgctg-3'; 321 bp). gapdh was amplified for 25 cycles and other molecules were amplified for 40 cycles. the pcr products were run on a 2% agarose gel, stained with ethidium bromide, and imaged by gel doc ez imager (bio-rad laboratories, hercules, ca). urine was collected using micturition cages (see below) and levels of urinary mcp-1 were measured using elisa with paired capture and detecting antibodies (r&d systems, minneapolis, mn). mice were placed in individual micturition cages (columbus instruments, columbus, oh) for 24-hour real time recording of voiding habits with 12-hour light and 12-hour dark cycles as described previously [22] . the cages consist of a fluid receptacle that funnels the captured urine droplets into a specimen freezer to prevent urine evaporation and chemical changes. a balance beneath the specimen freezer measures the weight of the collected urine. mice had free access to drinking water but were restrained from solid food to prevent feces from interfering with measurement of urine output. the entire system was computer interfaced for automated data acquisition in 2-minute intervals using oxymax software (columbus instruments). urinary frequency, voided volume per micturition, and total urine volume were recorded. pelvic pain was assessed by quantifying referred tactile allodynia of the lower abdominal region in response to applied force with a series of calibrated von frey filaments as described previously [22] . mice were kept in individual plexiglas chambers (6 x 10 x 12 cm) with a stainless steel wire grid floor and allowed to acclimate for 20 minutes before testing. five individual filaments (stoelting co., wood dale, il) with forces of 0.04, 0.16, 0.4, 1 and 4 grams were used in ascending order of force. the filament was applied perpendicularly to the skin for 1-2 seconds with intervals of 5 seconds between each stimulus for a total of 10 applications. stimulation was confined to the lower abdominal area in the general vicinity of the bladder. a positive response to filament stimulation was considered when mice showed sharp abdominal retraction, instant licking or scratching of the stimulated area, or jumping. response frequency was calculated as the percentage of positive response to each filament. tactile sensitivity of the plantar region of the hind paw was assessed using the same calibrated von frey filaments. the 50% withdrawal threshold was calculated and presented. a positive response to hind paw stimulation was defined as either a sharp withdrawal or licking of the tested paw. results were analyzed using statistics package for social sciences (spss 13.0, chicago, il), and presented as mean ± s.d. for urinary mcp-1 levels and mean ± sem for both voiding habit and pelvic pain changes. data was compared using student's t-test (two groups) or anova followed by lsd post hoc tests (multiple groups). a value of p<0.05 was considered statistically significant. uro-mcp-1 mice were generated through microinjection of a 4.9 kb dna construct consisting of the upii gene promoter fused to the mouse mcp-1 coding sequence with a secretory element (fig 1a) . the upii gene promoter is an urothelium-specific promoter and facilitates the expression of mcp-1 by the bladder epithelium [19] . uro-mcp-1 mice were found to express mcp-1 mrna in the bladder but not in other organs tested (fig 1b) . further analysis indicated the expression of mcp-1 mrna by the bladder epithelium of uro-mcp-1 mice but not that of wild-type c57bl/6 mice (fig 1c) . urine from uro-mcp-1 mice contained a significantly higher level of mcp-1 protein (5.39 ± 0.93 ng/ml) compared to urine from wild-type c57bl/6 mice (0.022 ± 0.016 ng/ml) (fig 1d) . uro-mcp-1 mice appear healthy and do not spontaneously develop bladder inflammation and symptoms during their lifespan. uro-mcp-1 mice exhibit bladder hypersensitivity and develop bladder inflammation upon intravesical administration of a single sub-noxious dose of lps the bladders of uro-mcp-1 mice show a hypersensitive response to intravesical lps. compared to wild-type c57bl/6 mice (n = 7; score: 0-+), uro-mcp-1 mice developed clear histological bladder inflammation (n = 8; score: ++-+++) 24 hours after intravesical instillation of a single sub-noxious dose of lps (1 μg of lps in 100 μl pbs) (fig 2a, table 1 ). intravesical instillation of pbs (100 μl) did not induce bladder histological changes in either strain of mice. the lps-treated bladders of uro-mcp-1 mice exhibited severe interstitial edema, mucosal hyperemia, and cellular infiltration in the lamina propria. in parallel with bladder histopathology, the inflamed bladders expressed elevated levels of mrnas for il-1ß, il-6, substance p precursor (pre-sp), and ngf as detected by rt-pcr (fig 2b) . immunohistochemistry revealed increased f4/80 positive cell (macrophages) infiltration in the inflamed bladders bladder inflammation is associated with voiding dysfunction in uro-mcp-1 mice uro-mcp-1 mice were evaluated for voiding habits using micturition cages before (baseline) and 1, 3 and 7 days after intravesical pbs or lps treatment (table 2, s1 table) . for comparison, wild-type c57bl/6 mice were evaluated in parallel for voiding habits (s2 and s3 tables). there were no significant changes in voiding habits after intravesical pbs treatment compared to baseline voiding habits for both uro-mcp-1 (s1 table) and c57bl/6 mice (s3 table) . our prior analysis also showed no significant differences in baseline voiding habits between c57bl/ 6 and uro-mcp-1 mice (s4 table) . while a single sub-noxious dose of lps failed to induce clear changes in voiding habits in c57bl/6 mice (s2 table) , the lps treatment induced significant changes in voiding habits in uro-mcp-1 mice at days 1 and 3 ( table 2) both wild-type c57bl/6 and uro-mcp-1 mice were evaluated for pelvic pain using von frey filament stimulation before (baseline) and 1, 3 and 7 days after intravesical pbs or lps treatment (fig 4a) . there were no significant differences in baseline pelvic responses between c57bl/6 and uro-mcp-1 mice or in pelvic response changes after pbs treatment in both mouse strains. intravesical lps at a single sub-noxious dose induced no clear changes in pelvic response in c57bl/6 mice. however, the same lps treatment induced significant changes in pelvic response in uro-mcp-1 mice at days 1 (0. and uro-mcp-1 mice (right panels) were treated intravesically with 100 μl pbs or 1 μg of lps in 100 μl pbs and evaluated for voiding habits using micturition cages at 1, 3 and 7 days after intravesical treatment (see table 2 and s2 table) . the baseline voiding habits were included for comparison (see s1 and s3 tables). the results are representative of 5-7 mice for each of baseline, pbs-treated (day 1), and lps-treated (day 1) groups in both mouse strains. (50% threshold) of the plantar region of the hind paw before (baseline) and 1, 3 and 7 days after intravesical pbs or lps treatment in both wild-type c57bl/6 and uro-mcp-mice ( fig 4b) , suggesting that the pain developed in intravesical lps-treated uro-mcp-1 mice was restricted to the pelvis. ic/bps is one of the most refractory diseases in urology today and the effort to develop animal models that can reproduce the clinical correlates of the human disease is greatly needed for ic/bps research. since the etiology of ic/bps remains elusive and many factors appear to be causative for the disease, animal models with diverse pathological pathways have been developed [22] . it is now generally agreed that a valid ic/bps animal model must present, at minimum, pelvic or bladder nociception and/or voiding dysfunction such as urinary frequency [22] . in this study we created a novel transgenic cystitis model (uro-mcp-1) that secretes mcp-1 by the bladder epithelium and develops bladder inflammation upon intravesical instillation of a single sub-noxious dose of lps. besides bladder histopathology, the uro-mcp-1 model demonstrates both qualitative and quantitative changes in bladder functions such as increased pelvic pain sensitivity, increased urinary frequency, reduced average volume voided per micturition (urgency), and reduced maximum volume voided per micturition (bladder capacity). because of the genetic stability of the incorporated transgene, the uro-mcp-1 model is stable and reproducible and provides a unique translational model for ic/bps research. uro-mcp-1 mice do not spontaneously develop bladder inflammation, pelvic pain and voiding dysfunction in the unmanipulated state, suggesting that the urothelial expression of mcp-1 alone is not sufficient to cause bladder inflammation and functional changes in these mice. however, uro-mcp-1 mice readily develop phenotypical and functional changes upon intravesical administration of a single sub-noxious dose of lps. our observation is similar to those observed in other mcp-1 transgenic models. gunn and associates reported that transgenic mice expressing mcp-1 by type ii alveolar epithelial cells showed no morphologic evidence of inflammation in the lung but exhibited enhanced inflammatory response upon treatment with either intraperitoneal lps or intravenous yeast wall glucan [7] . huang and associates reported that transgenic mice expressing mcp-1 by astrocytes only manifested neurological impairment and encephalopathy after treatment with intravenous pertussis toxin plus subcutaneous complete freund's adjuvant [8] . similarly, trujillo and associates reported that transgenic mice expressing mcp-1 by oligodendrocytes predisposed mice to a defective immune response to a minimally lethal neurotropic coronavirus and developed encephalitis following intracranial infection by the virus [9] . these observations indicated that development of inflammatory disorders is dependent on both genetic and environmental factors in the mcp-1 transgenic models. constitutive expression of mcp-1 by the bladder epithelium renders the bladders of uro-mcp-1 mice hypersensitive to otherwise sub-noxious irritative stimuli such as lps. we have observed that uro-mcp-1 mice exhibit a much lower threshold trigger for producing exaggerated responses to a single sub-noxious dose of lps as compared to wild-type c57bl/6 mice. intravesical administration of lps at 1 μg in 100 μl pbs efficiently induced profound bladder inflammation in uro-mcp-1 mice but not in wild-type c57bl/6 mice. this dose of lps used for cystitis induction in uro-mcp-1 mice was only one-tenth of the dose commonly used for cystitis induction in wild-type c57bl/6 mice [23, 24] . this feature of the uro-mcp-1 model is clinically relevant, as subclinical infection can be a causative factor for ic/bps and patients with ic/bps are often found to be hypersensitive to minor bladder irritants [25] . inflammation plays a central role in the pathogenesis of ic/bps and may directly affect bladder function in ic/bps patients [25] . our recent studies supported the fundamental role of inflammation in ic/bps, as the production of toll-like receptor 4 (tlr4) mediated proinflammatory cytokines il-1ß and il-6 was significantly associated with multiple ic/bps pain indicators [26] [27] [28] . as a clinically relevant model, the uro-mcp-1 model develops bladder inflammation and functional changes such as pelvic pain, urinary frequency and urgency. in addition to bladder histopathology, the inflamed bladders expressed elevated mrnas for proinflammatory cytokines il-1ß and il-6. moreover, the inflamed bladders also expressed elevated mrnas for ngf (a neurotrophic factor) and substance p precursor (a neurotransmitter), suggesting the presence of a strong neuro-immune interaction in the animal model. multiple cell types including urothelial cells, macrophages, mast cells, and neurons are known to express these inflammatory factors, reflecting an ongoing local inflammatory response in the bladders of uro-mcp-1 mice. all these inflammatory factors have been detected in the urine and/or bladder tissues of ic/bps patients [29] . the uro-mcp-1 model described in this study represents an acute cystitis model. future studies will focus on extending this model to a chronic cystitis model to more closely mimic human ic/bps. the other limitation of the present study is the lack of direct evaluation of bladder nociception. although our data indicate the presence of increased pelvic pain sensitivity, we will continue demonstrating bladder pain by using more specific methods such as the bladder distention-evoked visceromotor response (vmr) method [22] . future studies are also warranted to investigate the role of tlr4 in the uro-mcp-1 model and use this model for therapeutic development. the uro-mcp-1 model demonstrates bladder hypersensitivity, pelvic pain, and voiding dysfunction, providing a novel model for ic/bps research. supporting information s1 monocyte chemoattractant protein-1 (mcp-1): an overview mycobacterium bovis bacillus calmette-gué rin (bcg) induces human cc-and cxc-chemokines in vitro and in vivo wound healing in mip-1alpha (-/-) and mcp-1(-/-) mice mice lacking mcp-1 have enhanced susceptibility to an interstitial polymicrobial infection due to impaired monocyte recruitment impaired monocyte migration and reduced type 1 (th1) c-c chemokine receptor 2 knockout mice increased expression of ccl2 in insulin-producing cells of transgenic mice promotes mobilization of myeloid cells from the bone marrow, marked insulitis, and diabetes monocyte chemoattractant protein-1 is sufficient for the chemotaxis of monocytes and lymphocytes in transgenic mice but requires an additional stimulus for inflammatory activation pertussis toxin-induced reversible encephalopathy dependent on monocyte chemoattractant protein-1 overexpression in mice transgenic ccl2 expression in the central nervous system results in a dysregulated immune response and enhanced lethality after coronavirus infection urine cytokines suggest an inflammatory response in the overactive bladder: a pilot study differential profile analysis of urinary cytokines in patients with overactive bladder monocyte chemoattractant protein-1 and macrophage inflammatory protein-1α as possible biomarkers for the chronic pelvic pain syndrome recruitment of bone marrow derived cells to the bladder after bladder outlet obstruction urinary concentration and tissue messenger rna expression of monocyte chemoattractant protein-1 as an indicator of the degree of hydronephrotic atrophy in partial ureteral obstruction expression and function of ccl2/ccr2 in rat micturition reflexes and somatic sensitivity with urinary bladder inflammation mcp-1-induced histamine release from mast cells is associated with development of interstitial cystitis/bladder pain syndrome in rat models chronic pelvic allodynia is mediated by ccl2 through mast cells in an experimental autoimmune cystitis model diagnosis and treatment of interstitial cystitis/bladder pain syndrome: aua guideline amendment a tissue-specific promoter that can drive a foreign gene to express in the suprabasal urothelial cells of transgenic mice vectors for high-level expression of cdnas controlled by tissuespecific promoters in transgenic mice urinary bladder epithelium antigen induces cd8+ t cell tolerance, activation, and autoimmune response animal models of urologic chronic pelvic pain syndromes: findings from the multidisciplinary approach to the study of chronic pelvic pain research network gene expression profiling of mouse bladder inflammatory responses to lps, substance p, and antigen-stimulation modulating bladder neuro-inflammation: rdp58, a novel anti-inflammatory peptide, decreases inflammation and nerve growth factor production in experimental cystitis role of inflammation in bladder function and interstitial cystitis inflammation and inflammatory control in interstitial cystitis/bladder pain syndrome: associations with painful symptoms toll-like receptor 4 and comorbid pain in interstitial cystitis/bladder pain syndrome: a multidisciplinary approach to the study of chronic pelvic pain research network study inflammation and symptom change in interstitial cystitis or bladder pain syndrome: a multidisciplinary approach to the study of chronic pelvic pain research network study potential urine and serum biomarkers for patients with bladder pain syndrome/interstitial cystitis we thank dr. tung-tien sun for providing the upii gene promoter-containing plasmid, dr. timothy l. ratliff for providing the micturition cages, and ms. kris greiner for editorial review of the manuscript. this work was supported in part by grants uo1dk082344 and ro1dk100891 from the national institute of diabetes digestive and kidney diseases.transgenic mice were generated at the university of iowa genome editing core facility directed by william paradee, phd and supported in part by grants from the nih and from the roy j. and lucille a. carver college of medicine. we wish to thank norma sinclair, patricia yarolem and joanne schwarting for their technical expertise in generating transgenic mice. key: cord-048339-nzh87aux authors: caley, peter; becker, niels g.; philp, david j. title: the waiting time for inter-country spread of pandemic influenza date: 2007-01-03 journal: plos one doi: 10.1371/journal.pone.0000143 sha: doc_id: 48339 cord_uid: nzh87aux background: the time delay between the start of an influenza pandemic and its subsequent initiation in other countries is highly relevant to preparedness planning. we quantify the distribution of this random time in terms of the separate components of this delay, and assess how the delay may be extended by non-pharmaceutical interventions. methods and findings: the model constructed for this time delay accounts for: (i) epidemic growth in the source region, (ii) the delay until an infected individual from the source region seeks to travel to an at-risk country, (iii) the chance that infected travelers are detected by screening at exit and entry borders, (iv) the possibility of in-flight transmission, (v) the chance that an infected arrival might not initiate an epidemic, and (vi) the delay until infection in the at-risk country gathers momentum. efforts that reduce the disease reproduction number in the source region below two and severe travel restrictions are most effective for delaying a local epidemic, and under favourable circumstances, could add several months to the delay. on the other hand, the model predicts that border screening for symptomatic infection, wearing a protective mask during travel, promoting early presentation of cases arising among arriving passengers and moderate reduction in travel volumes increase the delay only by a matter of days or weeks. elevated in-flight transmission reduces the delay only minimally. conclusions: the delay until an epidemic of pandemic strain influenza is imported into an at-risk country is largely determined by the course of the epidemic in the source region and the number of travelers attempting to enter the at-risk country, and is little affected by non-pharmaceutical interventions targeting these travelers. short of preventing international travel altogether, eradicating a nascent pandemic in the source region appears to be the only reliable method of preventing country-to-country spread of a pandemic strain of influenza. the emergence of a pandemic strain of influenza is considered inevitable [1] . provided the emerged strain is not too virulent, it may be possible to eliminate a nascent influenza pandemic in the source region via various combinations of targeted antiviral prophylaxis, pre-vaccination, social distancing and quarantine [2, 3] . if early elimination in the source region is not achieved, then any delay in a local epidemic that a country can effect will be highly valued. to this end, countries may consider introducing non-pharmaceutical interventions such as border screening, promoting early presentation of cases among arriving passengers, requiring the use of personal protective equipment during travels (e.g. the wearing of masks), and reducing traveler numbers. while the case for believing that measures such as these can not stop the importation of an epidemic from overseas has been argued strongly, whether it be sars or influenza [4] [5] [6] , the extent to which such interventions delay a local epidemic is currently not well quantified, and hence of considerable interest. in this paper we demonstrate how the delay to importation of an epidemic of pandemic strain influenza may be quantified in terms of the growing infection incidence in the source region, traveler volumes, border screening measures, travel duration, inflight transmission and the delay until an infected arrival initiates a chain of transmission that gathers momentum. we also investigate how the delay is affected by the reproduction number of the emerged strain, early presentation of cases among arriving passengers, and reducing traveler numbers. as noted in previous simulation modeling [7] , many aspects of this delay have a significant chance component, making the delay a random variable. therefore, the way to quantify the delay is to specify its probability distribution, which we call the delay-distribution. some issues of the delay distribution, such as the natural delay arising in the absence of intervention and the effect that reducing traveler numbers has on this delay has been studied previously [6] [7] [8] . specifically, if the originating source is not specified, and homogeneous mixing of the worlds population is assumed, then the most likely time to the initial cases arising in the united states is about 50 days assuming r 0 = 2.0 [7] . the additional delay arising from travel restrictions appears minimal until a.99% reduction in traveler numbers [6] [7] [8] . this paper adds to previous work [5] [6] [7] [8] by simultaneously including a wider range of epidemiological factors and possible interventions, such as elevated in-flight transmission, flight duration, the effect of wearing of mask during flight, early presentation of cases among travelers, and quarantining all passengers from a flight with a detected case at arrival. consider a region in which a new pandemic strain of influenza has emerged, and a region currently free from the infection. we refer to these as the source region and the at-risk country, respectively. travel between these countries is predominantly via commercial air travel and/or rapid transport which could potentially be subject to border screening and other interventions. we restrict our discussion to air travel. the aim is to assess the effects that a variety of non-pharmaceutical border control measures have, individually and in combination, on the time it takes before the epidemic takes off in the at-risk country. an epidemic is said to have ''taken off'' when it reaches 20 current infectious cases, after which its growth is highly predictable (i.e. nearly deterministic) and the probability of fade-out by chance is very low, if intervention is not enhanced. the source country of origin will undoubtedly have a large impact on the natural delay until importation of an epidemic, although this is difficult to quantify [7] . an alternative is to fix the originating city, for example a highly connected city such as hong kong [6] , with the obvious effect that results are highly dependent on the choice. we adopt no specific source region, but assume that the number of international travelers originating from it is reasonably small (see methods), suggestive of a rural or semirural source region [2] . it is further assumed that the current heightened surveillance for pandemic influenza is continued and that a nascent pandemic with human-to-human transmission is identified and the pandemic is declared when there are 10 concurrent cases in the source region. for an epidemic to take off in an at-risk country, a series of events need to occur. first, the epidemic needs to get underway in the source region. second, an intending traveler needs to be infected shortly before departure. third, the infected traveler must actually travel and successfully disembark in the at-risk country. fourth, the infected traveler, or fellow travelers infected during the flight, must initiate an epidemic in the at-risk country with the infectiousness that remains upon arrival. finally, the epidemic needs to reach a sufficient number of cases to begin predictable exponential growth. international spread of the emerged pandemic strain of influenza may occur when a recently infected person travels. by 'recently infected' we mean that their travel is scheduled to occur within ten days of being infected. we assume that the number of individuals traveling from the source region to the at-risk country each day is known. the probability that a randomly selected traveler is a recently-infected person is taken to be equal to the prevalence of recently-infected people in the source region on that day. the incidence of infection in the source region is assumed to grow exponentially initially, with the rate of exponential growth determined by the disease reproduction number (the mean number of cases a single infective generates by direct contact) and the serial interval (the average interval from infection of one individual to when their contacts are infected) ( figure 1a) . the time since infection of a recently-infected traveler is a key component of the calculations, because it affects the chance of positive border screening, the chance of in-flight transmission and the infectivity remaining upon arrival in the at-risk country. the time since infection at the time of scheduled departure is random and the dependence of its probability distribution on the exponential growth rate of infection is illustrated by figure 1b (see also supporting information). the higher the epidemic growth rate in the source region, the greater the probability than an infected traveler will have been infected more recently. it is assumed that individuals detected by departure screening are prevented from traveling. to be detected by screening an infected traveler must be symptomatic and positively screened. an individual is assumed to become symptomatic 48 hours after being infected (cf. [3] who use 1.9 days). the probability of being symptomatic when presenting for departure screening is computed from the curve in figure 1b . the distribution of the time since infection immediately after departure screening, given that the infected traveler was not detected, is given by the curve in figure 1c . it contains an adjustment for the probability of being detected at departure. the instantaneous rate at which susceptible contacts are infected depends on the time since infection, and is described by an infectiousness function ( [9] , page 45). we use a peaked infectiousness function, motivated by viral shedding and household transmission data [2] , which has a serial interval of 2.6 days. the basic reproduction number (r 0 ), namely the reproduction number when there is no intervention in place and every contacted individual is susceptible, is given by the area under the infectiousness function. however, our concern is with the effective reproduction number r that holds when various interventions are in place. we obtain any r by simply multiplying the infectiousness function by the appropriate constant (to make the area under the curve equal to r). this keeps the serial interval the same. in the absence of suitable data we assume for most scenarios that the aircrafts ventilation and filtration systems are functioning properly, and that infected travelers transmit the infection at the same rate during a flight as they would while mixing in the community. we examine the sensitivity of this assumption by increasing the inflight transmission by as much as 10-fold (as could potentially happen if air-circulation and filtration systems malfunction, e.g. see [10] ). the in-flight transmission rate is set to zero under the optimistic scenario that all travelers wear 100% effective masks during transit. in terms of a sensitivity analysis this illustrates what would be achievable in a best-case scenario. the number of offspring that an infected traveler infects during a flight is a random variable, taken to have a poisson distribution with a mean equal to the area under the infectiousness function over to the flight duration. travelers infected during flights of less than 12 hours duration are asymptomatic at arrival and will not be detected by screening. the probability that an arriving traveler who was infected in the source region is detected on arrival is computed from the distribution of the time since infection on arrival. this distribution is obtained from the curve in figure 1c by shifting it to the right by an amount equal to the duration of the flight. the distribution of the time since infection for an individual infected in the source region, who passes through arrival screening undetected has a further adjustment for the chance of being detected at arrival ( figure 1d ). this curve shows that an infected traveler who escapes detection at departure and arrival is highly likely to enter the at-risk country with most, or all, of their infectious period remaining. authorities are assumed to implement one of two control options when detecting an infected traveler by arrival screening. under option one (individual-based removal), all passengers who test negative are released immediately and only passengers who test positive are isolated. under the second option (flight-based quarantining), authorities prevent all passengers from dispersing into the community until the last person has been screened from that flight. should any one passenger be detected as infected then all passengers will be quarantined, as previously recommended [5] . transmission chains can be initiated in the at-risk country by infected travelers who mix within the community upon arrival. suppose now that a flight arrives with one, or more, infected passengers who mix within the community. we classify these infected arrivals into those who are 'pre-symptomatic' and those who are 'symptomatic' at entry. it is assumed that the 'symptomatic' infected arrivals do not recognize their symptoms as pandemic influenza and will not present to medical authorities. in other words, they spend the remainder of their infectious period mixing in the community. on the other hand, the 'presymptomatic' infected arrivals, including all individuals infected during flight, are assumed to mix freely in the community only from entry until they present to medical authorities after some delay following the onset of symptoms. not all infected travelers entering the community initiate a 'major' epidemic, even when the reproduction number (r) exceeds one. quite generally, the distribution of the size of an epidemic initiated by an infected arrival is bimodal, with distinct peaks corresponding to a major epidemic and a minor outbreak ( figure 1e ). in the latter event the outbreak simply fades out by chance despite there being ample susceptibles in the population for ongoing transin (b), the step illustrates the probabilistic removal of travelers who have completed their incubation period. in (d), the distribution of time since infection in (c) will have shifted to the right by an amount equal to the flight duration, and cases incubated in-flight may be detected by symptomatic screening, as will those symptomatic cases that were not detected previously. screening sensitivity for this illustration is 60% on both departure and arrival. (e) upon entering the community undetected, an infected traveler may initiate a minor (inconsequential) or major epidemic, depending on the characteristics of the disease and public health policy. doi:10.1371/journal.pone.0000143.g001 mission [11] . the number of cases in an outbreak that fades out is typically very small compared to an epidemic. the probability that a typical infective generates a local epidemic is computed by using a branching process approximation [12] for the initial stages of the epidemic, and equating 'epidemic' with the event that the branching process does not become extinct. this calculation is well known (e.g. [13] , page 473), but is modified here to allow for the fact that the process is initiated by a random number of infected arrivals and some of them have spent a random part of their infectious period before arriving in the at-risk country. the distribution for the random number of individuals infected by an infected individual when all their contacts are with susceptible individuals is needed for the calculation. the lack of data prevents a definitive conclusion for the most appropriate offspring distribution for influenza transmission [14] , and we use a poisson distribution with a mean equal to r, discounted for individuals who spent only some of their infectious period mixing in the at-risk country. a poisson offspring distribution is appropriate when the area under the infectiousness function is non-random (i.e. all individuals have the same infection 'potential'). we assume that r is the same in the source region and the at-risk country. for an undetected infected traveler and all their in-flight offspring to fail to initiate an epidemic on arrival, all of the chains of transmission they initiate must fail to become large epidemics (see supporting information). we calculate the probability distribution of d, the total delay until an epidemic gathers momentum by noting that it is given by d = d 1 +d 2 , where d 1 is the time until an epidemic is first initiated and d 2 is the time from initiation until the local epidemic gathers momentum. for an epidemic to be first initiated in the at-risk country on day d, it must have not been initiated on all previous days. hence the probability distribution of the time delay (d 1 ) until the epidemic is first initiated in the at-risk country following identification in the source region is described by: pr (d 1~d )~ p p 1 p p 2 p p 3 ::: p p d{1 p d where p d denotes the probability that the epidemic is initiated on day d , and p p d~1 {p d denotes the probability that the epidemic is not initiated on day d (see supporting information for calculation of p d ). once successfully initiated, an epidemic may initially hover around a handful of cases before reaching a sufficient number of cases for its growth to become essentially predictable. as mentioned, 20 concurrent cases is our criterion for an epidemic to have gathered momentum. we determine the distribution of d 2 , the time to this occurrence, from 10,000 stochastic simulations and approximate this empirical distribution by a shifted gamma distribution. our criterion of 20 concurrent cases is conservatively high, as results from the theory of branching processes shows that the probability of a minor epidemic (and hence no take-off) starting from 20 concurrent cases is about 3610 28 when r = 1.5, and even smaller for higher values of r. finally, the distribution of the total delay (d = d 1 +d 2 ) from the pandemic being identified in the source region until 20 cases in the at-risk country was calculated by the convolution of the distributions of d 1 and d 2 . for the illustrative purposes, we chose values of 1.5, 2.5 and 3.5 for r, which encompass estimates proposed for previous pandemics [2, 3, 15] . the number of people within the infected source region was assumed reasonably small (5 million), and there was one flight per day traveling from the source region to the at-risk country carrying 400, 100 or 10 passengers. a higher number of travelers affects the delay only marginally, assuming the epidemic takes off in the source region (see results). we assume a typical travel duration between attempted departure and possible arrival of 12 hours, but also examine the effect of varying this from 0-48 hours. the time to presentation following symptom onset is varied from 'immediately' to 'never presenting', with a time of 6 hours considered likely in the presence of an education campaign. the sensitivity of symptomatic screening is varied from 0-100%, with results presented for 0, 50 and 100% sensitivity. the probability that a recently infected traveler evades screening is substantial even if screening reliably detects symptomatic travelers (figure 2a) , because the typical travel duration is shorter than the 2-day incubation period. in addition, during the early stages of the epidemic a high r in the source region acts to increase the probability that an infected traveler has been infected quite recently and hence will escape detection due to being asymptomatic during their travels (figure 2a ). for example, assuming 100% sensitivity for detecting symptomatic infection, we calculate that during the early stages of the epidemic the proportion of infected travelers that evade both departure and arrival screening after 12 hours of travel is 0.26, 0.45 and 0.59 for disease reproduction numbers 1.5, 2.5 and 3.5, respectively. as the duration of travel approaches the disease incubation period, effective symptomatic screening substantially reduces the likelihood that a traveler evades screening and initiates an epidemic ( figure 2b ). reducing the time from the onset of symptoms to presentation (and subsequent isolation) for each infected arrival also reduces the probability that a major epidemic is initiated, however the best case scenario of infected travelers and all their in-flight offspring presenting immediately following the onset of symptoms still poses a substantial risk of epidemic initiation arising from pre-symptomatic transmission ( figure 1c ). the delay contains a fairly substantial natural component, primarily due to the time it takes to increase the number of infectives in the source region sufficiently to make the chance of a recently infected traveler appreciable ( figure 3a ), and the time (d 2 ) it takes for a local epidemic in the at-risk country to gather momentum following successful seeding ( figure 4a ). in the absence of any interventions, the number of infected individuals who successfully enter the community of the at-risk country initially increases exponentially ( figure 3a ). with individual-based removal of infected travelers, the number of individuals entering the at-risk country undetected by screening is proportionately reduced over the course of the epidemic ( figure 3a ). with flightbased quarantining, the number of infected individuals entering the at-risk country undetected is dramatically reduced over the course of the epidemic, even for relatively insensitive screening ( figure 3a ). with flight-based quarantining, the number of infected passengers slipping through undetected is bimodal, with the first peak occurring when the number of infected travelers attempting to travel is still in single figures. without screening, the daily probability that an epidemic is initiated (p d ) increases, and becomes near certain once the number of infected travelers arriving undetected exceeds about 10 ( figure 3b, solid line) . with screening and individual-based removal of infected individuals, p d follows a similar pattern only reduced somewhat. with screening in combination with flightbased quarantining, this probability is changed dramatically. after an initial rise it dips, to become essentially zero during the height of the epidemic in the source region ( figure 3b , dotted line). this arises because once a flight has several infected travelers, the probability that at least one is detected approaches one (even if screening is imperfect), and all passengers on such a flight are quarantined. once the epidemic starts to wane in the source region (assuming the unlikely event of the pandemic strain is restricted to the source region), the probability of initiation rises once again. the corresponding distribution of d 1 , the delay until the effects of r and the time from symptom onset to presentation on the probability that an infected traveler, having entered the wider community following arrival, will initiate an epidemic. there is no screening. doi:10.1371/journal.pone.0000143.g002 the epidemic is first initiated in the at-risk country, is bi-modal in the presence of screening ( figure 3c) . although flight-based quarantining is effective in preventing the entry of infected travelers during the height of the epidemic, a substantial cumulative risk of initiation has already occurred before this from the handful of infectives that have slipped through undetected ( figure 3b ). hence, whilst the effect of border screening, particularly in conjunction with flight-based quarantining, on the daily probability of initiation is dramatic, its effect on the delay to initiation is much less pronounced ( figure 3c ). border screening, even with perfect sensitivity for detecting symptomatic cases, tends to increase d 1 , the time to an epidemic being initiated, by a matter of days to weeks. the time (d 2 ) from initiation (the arrival of the index case) to an epidemic reaching 20 concurrent cases within the at-risk country is adequately modeled using a shifted gamma distribution ( figure 4a ). the convolution of this right-skewed gamma distribution with the left-skewed delaydistribution of d 1 (figure 3c ) yields the distribution for d, the total delay until the epidemic reaches 20 cases in the at-risk country ( figure 4b ). the distribution of d is approximately symmetrical. the effect of border screening on the total delay d is quite modest, though sensitive to how screening is implemented. for example, with r = 1.5 and 400 travelers per day, 100% sensitive screening with individual-based removal increases the median delay from 57 to 60 days ( figure 4b ). flight-based quarantining would extend the median delay to 70 days. in general, the added delay arising from flight-based quarantining is about four-fold that arising from individual-based removal. the natural component of the delay is highly sensitive to the disease reproduction number ( figure 5a ). for example, with 400 passengers per day departing the source country and in the absence of any interventions, the median delay ranges from a low of 17 days for r = 3.5 to 57 days for r = 1.5 ( table 1 ). the delay is less sensitive to the number of intending travelers, with little appreciable increase in the median delay occurring until traveler numbers become very low ( figure 5b ). for example, if r = 1.5, with no other border control measures, decreasing the number of intending travelers departing the source region from 400 to 100 per day increases the median total delay d from 57 to 66 days. a further decrease in the number of intending travelers to 10 per day increases the median delay to 83 days ( table 1) . the delay is quite insensitive to the rate of transmission in-flight. for example, with r = 1.5, a 12-hour flight, 400 travelers per day and no other interventions, preventing in-flight transmission altogether increases the median delay from 57 to 58 days. conversely, doubling the rate of in-flight transmission reduces the median delay from 57 to 56 days. a 10-fold increase in the rate of transmission in-flight only decreases the median delay from 57 to 53 days. encouraging the early presentation of cases among travelers following the onset of symptoms has a limited effect on the delay distribution ( figure 5c ). for example, for r = 1.5, 400 intending travelers per day and no other interventions, reducing the time to presentation from 'never presenting' to 6 hours increases the median delay from 57 to 61 days. immediate presentation at symptom onset only increases the median delay a further day in this scenario. in general, the additional delay achieved by introducing nonpharmaceutical border control measures is generally small in comparison with the natural delay ( figure 5d ). for the scenario with r = 1.5 and 400 intending travelers per day, a combination of 100% flight-based quarantining, 100% compliance with mask wearing during travel and immediate presentation at symptom onset extends the estimated median delay from 57 to 79 days ( figure 5d ). this added delay diminishes in absolute terms as r increases. for example, if the same interventions are applied with r = 3.5, the median delay is extended from 17 to just 20 days ( figure 5d ). the one exception to this generalisation is when travel numbers are reduced dramatically. the added delay achieved when a drastic reduction in travel numbers is combined with other border control measures appears to be greater than adding the delays each achieves on its own. for example, if r = 1.5, and we reduce the number of intending travelers from 400 to 10 per day, implement 100% flight-based quarantining, implement compulsory mask wearing during travel and presentation at 6 hours following symptom onset then there is a substantial probability (0.74) that the pandemic strain will never be imported (assuming the epidemic is confined to the source country). the estimated quartile delay (the median in this case is undefined) to the start of a major epidemic in an at-risk country is extended from 50 to 125 days. again, the added delay decreases rapidly as r increases, and if the above interventions were applied with r = 3.5, the estimated median delay is extended from 17 to 26 days, and the importation of the epidemic is certain ( figure 5d ). we have formulated a model of the importation of an infectious disease from a source region to an at-risk country that permits a comprehensive analysis of the effect of border control measures. our results are most relevant to the early stage of a pandemic when most cases are contained within a single source region. once the pandemic has spread to several countries, models with greater complexity and ability to more realistically model global mixing patterns [6] [7] [8] are required. our model is developed with a pandemic-strain of influenza in mind, but could apply to any emerging infectious disease that is transmitted from person to person. we have assumed a poisson distribution for the number of secondary infections, which a natural choice when each infected individual has the same infectivity profile. a distribution with a larger variance is appropriate when individuals vary substantially in their infectiousness. our results are conservative in the sense that they give an upper bound for the probability that an infected traveler manages to initiate an epidemic, compared to an offspring distribution with a greater variance but the same reproduction number [14] . the nature of the next pandemic influenza virus, and particularly its reproduction number, is uncertain. if its reproduction number is low (r,2.0), our results indicate that at-risk countries receiving a reasonably small number of travelers (say 400 per day) from the infected source region can expect a natural delay until importing an epidemic of the order of 2 months. this is quite variable and under favourable conditions it could be 4 months. however, the natural delay decreases rapidly as r increases. the additional delay from isolating individuals detected by border screening is merely a few days under most plausible scenarios, even if both departure and arrival screening is introduced and screening detects every symptomatic traveler. while the extra delay is more than quadrupled if flights with a detected case(s) are quarantined, the effect remains modest (weeks at most) and it is questionable whether the extra delay achieved warrants the disruption created by such a large number of quarantined passengers. in-flight transmission is a commonly raised concern in discussions about the importation of an infection, so inclusion of in-flight transmission is an attractive feature of our model. events of substantial in-flight transmission of influenza have been documented [10, 16] and modeling of indoor airborne infection risks in the absence of air filtration predicts that in-flight transmission risks are elevated [17] . however, it difficult to estimate the infectiousness of influenza in a confined cabin space, as there is undoubtedly substantial under-reporting of influenza cases who travel and fail to generate any offspring during flight. provided the aircraft ventilation system (including filtration) is operational, it is considered that the actual risk of in-flight transmission is much lower than the perceived risk [18] . our results indicate that the delay is relatively insensitive to the rate of in-flight transmission, making in-flight transmission less of an issue than commonly believed. a highly elevated transmission rate inflight will hasten the importation of an epidemic only marginally. consistent with this, eliminating in-flight transmission by wearing protective masks increases the delay only marginally. early presentation by infected arrivals not detected at the borders was found to add only a few days to the delay. to some extent this arises due to our assumption that pre-symptomatic transmission can occur, for which there is some evidence. in contrast, ferguson et al. [2] assume that the incubation and latent periods are equal, with a mean of 1.5 days. in their model presymptomatic transmission is excluded and infectiousness is estimated to spike dramatically immediately following symptom onset and declining rapidly soon afterwards. under their model assumptions, immediate presentation at onset of symptoms would reduce transmission effectively. however, as presentation occurs some time after onset of symptoms and the bulk of infectivity occurs immediately after onset of symptoms the results on the effect of early presentation of cases are likely, in practical terms, to be similar to those found here. given the variable nature of influenza symptoms, there is likely to be a difference between the onset of the first symptoms as measured in a clinical trial (e.g. [19] ) and the time that a person in the field first suspects that they may be infected with influenza virus. to fully resolve the issue of how effective very early presentation of infected travelers is in delaying a local epidemic we need better knowledge about the infectiousness of individuals before and just after the onset of symptoms. it is assumed that the pandemic is identified and declared when there are 10 concurrent cases in the source region attributed to human-to-human transmission, and that screening is applied at both departure and arrival. the time between screening events is assumed to be 12 hours and infected travelers are not isolated following the onset of symptoms. of the border control measures available, reducing traveler numbers has the biggest effect on the delay and even then it is necessary to get the number of travelers down to a very low number. an equivalent control measure is to quarantine all arriving passengers with near perfect compliance. our results indicate that short of virtually eliminating international travel, border control measures add little to avoiding, or delaying, a local epidemic if an influenza pandemic takes off in a source region. all forms of border control are eventually overwhelmed by the cumulative number of infected travelers that attempt to enter the country. the only way to prevent a local epidemic is to rapidly implement local control measures that bring the effective reproduction number in the local area down below 1, or to achieve rapid elimination in the source region, in agreement with other recent studies [6] [7] [8] . preventing the exponential growth phase of an epidemic in the source region appears to be the only method able to prevent a nascent influenza pandemic reaching atrisk countries. text s1 estimating the daily probability of epidemic initiation found at: doi:10.1371/journal.pone.0000143.s001 (0.08 mb pdf) mitigation strategies for pandemic influenza in the united states strategies for containing an emerging influenza pandemic in southeast asia containing pandemic influenza at the source border screening for sars entry screening for severe acute respiratory syndrome (sars) or influenza: policy evaluation delaying the international spread of pandemic influenza strategies for mitigating an influenza pandemic will travel restrictions control the international spread of pandemic influenza analysis of infectious disease data an outbreak of influenza aboard a commercial airline should be expect population thresholds for wildlife disease? the theory of branching processes matrix population models: construction, analysis, and interpretation superspreading and the effect of individual variation on disease emergence transmissibility of 1918 pandemic influenza influenza outbreak related to air travel a probabilistic transmission dynamic model to assess indoor airborne infection risks transmission of infectious diseases during commercial air travel use of the oral neuraminidase inhibitor oseltamivir in experimental human influenza we thank james wood, katie glass and belinda barnes and an anonymous reviewer for helpful comments. conceived and designed the experiments: nb pc. performed the experiments: pc dp. analyzed the data: nb pc dp. contributed reagents/materials/analysis tools: pc dp. wrote the paper: nb pc. key: cord-000248-zueoyesj authors: berretta, regina; moscato, pablo title: cancer biomarker discovery: the entropic hallmark date: 2010-08-18 journal: plos one doi: 10.1371/journal.pone.0012262 sha: doc_id: 248 cord_uid: zueoyesj background: it is a commonly accepted belief that cancer cells modify their transcriptional state during the progression of the disease. we propose that the progression of cancer cells towards malignant phenotypes can be efficiently tracked using high-throughput technologies that follow the gradual changes observed in the gene expression profiles by employing shannon's mathematical theory of communication. methods based on information theory can then quantify the divergence of cancer cells' transcriptional profiles from those of normally appearing cells of the originating tissues. the relevance of the proposed methods can be evaluated using microarray datasets available in the public domain but the method is in principle applicable to other high-throughput methods. methodology/principal findings: using melanoma and prostate cancer datasets we illustrate how it is possible to employ shannon entropy and the jensen-shannon divergence to trace the transcriptional changes progression of the disease. we establish how the variations of these two measures correlate with established biomarkers of cancer progression. the information theory measures allow us to identify novel biomarkers for both progressive and relatively more sudden transcriptional changes leading to malignant phenotypes. at the same time, the methodology was able to validate a large number of genes and processes that seem to be implicated in the progression of melanoma and prostate cancer. conclusions/significance: we thus present a quantitative guiding rule, a new unifying hallmark of cancer: the cancer cell's transcriptome changes lead to measurable observed transitions of normalized shannon entropy values (as measured by high-througput technologies). at the same time, tumor cells increment their divergence from the normal tissue profile increasing their disorder via creation of states that we might not directly measure. this unifying hallmark allows, via the the jensen-shannon divergence, to identify the arrow of time of the processes from the gene expression profiles, and helps to map the phenotypical and molecular hallmarks of specific cancer subtypes. the deep mathematical basis of the approach allows us to suggest that this principle is, hopefully, of general applicability for other diseases. in a seminal review paper published nine years ago, hanahan and weinberg [1] introduced the ''hallmarks of cancer''. they are six essential alterations of cell physiology that generally occur in cancer cells independently of the originating tissue type. they listed: ''self-sufficiency in growth signals, insensitivity to growth-inhibitory signals, evasion of the normal programmed-cell mechanisms (apoptosis), limitless replicative potential, sustained angiogenesis, and finally, tissue invasion and metastasis''. more recently, several researchers have advocated including ''stemness'' as the seventh hallmark of cancer cells. this conclusion has been reached from the outcomes of the analysis of high-throughput gene expression datasets [2, 3] . the new role of stemness as a hallmark change of cancer cells is also supported by the observation that histologically poorly differentiated tumors show transcriptional profiles on which there is an overexpression of genes normally enriched in embryonic stem cells. for example, in breast cancer the activation targets of the pluripotency markers like nanog, oct4, sox2 and c-myc have been shown to be overexpressed in poorly differentiated tumors in marked contrast with their expression in welldifferentiated tumors [4] . other authors suggest different hallmarks, with many papers pointing alternative processes as their primary focus of their research. the difference may stem from the fact that these authors prefer to cite as ''key hallmarks'' physiological changes which occur at a ''lower level'' scale closer to the molecular events. these authors cite, for example, ''mitochondrial dysfunction'' [5, 6] (including, but not limited to ''glucose avidity'' [7] and ''a shift in glucosemetabolism from oxidative phosphorylation to glycolysis'' [6, 8] , ''altered glycolysis'' [9] , ''altered bioenergetic function of mitochondria'' [10] ), ''dysregulation of cell cycle and defective genome-integrity checkpoints'' [11] , ''aberrant dna methylation'' [12] (''promoter hypermethylation of hallmark cancer genes'' [13] and ''cpg island hypermethylation and global genomic hypomethylation'' [14] ), ''shift in cellular metabolism'' [15, 16, 17] , ''regional hypoxia'' [18] , ''microenviroment acidosis'' [19] , ''abnormal microrna regulation'' [20, 21] , ''aneuploidy'' and ''chromosome aberrations'' [22, 23, 24, 25, 26] , ''disruption of cellular junctions'' [27] , ''avoidance of the immune response'' [28] , ''pre-existing chronic inflammatory conditions'' [29, 30] , ''cancerrelated inflammation'' [29] , ''disabled autophagy'' [28] , ''impaired cellular senescence'' [31] , ''altered nf-kappab signalling'' [32] , ''altered growth patterns, not altered growth per se'' [33] , ''disregulated dna methylation and histone modifications'' [34] , ''tissue dedifferentiation'' [35, 36] , and ''somatically heritable molecular alterations'' [37] . this research enriches the list of the most important cancer hallmarks. however, these physiological changes occur at a ''lower'' molecular level they are likely related sub events of the orginial seven instead of newly discovered ''key hallmarks''. more recently, luo et al attempted a ''stress-based'' description of some of the hallmarks in terms of ''stresses'' (''dna damage/replication stress, proteotoxic stress, mitotic stress, metabolic stress, and oxidative stress'') [38] . while this is an interesting descriptive grouping, it is still a phenotypical characterization. what is needed is a higher level unifying genotypical characterization, from which individual disregulated processes can be identified in a quantitative way using the existing high-throughput data capture methodologies. it is clear that a unifying hallmark is needed if we aim at quantifying the cell's progression. it is then evident for us that a unifying mathematical formalism is necessary to uncover the cell transcriptome's progression from a normal to a more malignant phenotype. we start our quest assuming an implicit working hypothesis common to many research groups around the world: the macroscopic physiological changes (i.e. hanahan and weinberg's ''hallmarks'') must also correlate with global alterations of the molecular profiles of gene transcription. it is also assumed that the ''hallmark changes'' occur along a certain timeline, but that some of the sub-processes discussed before are concurrent. these processes may start in a slow incremental way with some of the major changes being early events while others (e.g. tissue invasion and metastasis) are likely later processes triggered by new events during cancer progression. the timeline is not explicit and it is also likely that cancer subtypes progress to similar timelines. in some cases the sequence of events are better understood (e.g. some leukaemia subtypes [39] ). the elicitation and regulation of molecular events is likely to be an ongoing quest during this century for many types of cancer. it is not to be assumed that some of the transitions of the transcriptome are gradual. that is a hypothesis that is unnecessary in this study. we envision that the progression of cancer may have ''switches'', with a number of concurrent converging events leading to macroscopic observable changes in the gene expression profile resulting in dramatic variations of expression patterns. for instance, these molecular switches could not be characterized by an ''oncogene'' but by a large number of the genes that have changed its transcriptional state. these abrupt changes may be triggered by the confluence of several non-linear interactions, and are likely to be related to the physiological hallmarks we refer to above. the presence of macroscopic observable changes that are computable from a large number of relatively smaller changes mean that it may be possible to find an objective mathematical formalism to infer the turning point at which these radical changes occur. it is then evident that computing the jensen-shannon divergences, the normalized shannon entropy, and the statistical complexity of samples reveal different global transcriptional changes. it is, however, not easy to infer if these changes would correlate with a gradual progression or sudden changes. however, one valid mathematical possibility is that the most important ''hallmark of cancer'', a unifying principle above all, is the existence of a measurable gradual ''progression'' from a well-differentiated gene expression profile (corresponding to a healthy tissue). this would reveal the timeline of a higher level process that is observable and measurable via a change of normalized shannon entropy and an increment of jensen-shannon divergences from the originating tissue type. if this is the case, by correlating the changes in information theory quantifiers with the expression of the genes we would be able to not only uncover useful biomarkers to track this progression but to explain the ''hallmarks'' in an ordered timeline. the timeline also yields clinical and translational important outcomes. such analytical methodology will naturally produce ''a continuous staging'' of the cancer samples, based on a solid foundations of information theory, based on the knowledge of transcriptional profile of healthy cells as reference to measure divergences. in addition, as a mathematical methodology, it can be applied to other high-throughput technologies for which a probability distribution function of observed abundances has been computed. with these ideas in mind, we provide a ''transcriptomic-driven'' method revealing important biomarkers for cancer progression a direction of time for which they are presented. the method, however, is generalizable to other type of high-throughtput techonologies (e.g. proteomic studies). we have chosen two types of cancers to study which are almost at the antipodes in terms of progression rates: prostate cancer and melanoma. prostate cancer progresses very slowly. pathological samples are common in autopsies of men as young as 20 years old. by the age of 70 more than 80% of men have these alterations, a fact that already shows a relationship of this cancer type with increasing age. the clinical management of prostate cancer requires the identification of the so-called gleason patterns in the biopsies [40] , which after almost fifty years is still ''the sole prostatic carcinoma grading system recommended by the world health organization''. however, undergrading, underdiagnosis, interobserver reproducibility and variable trends in grading have been observed as major problems [41, 42] . melanoma, on the other hand, differs from prostate cancer in its rapid progression [43] and it is considered one of the most aggressive types of cancer. one of melanoma's usual markers of progression and concern (i.e thickness) is measured in millimetres, which gives a rough idea of how devastatingly fast the disease can spread. we will present our results starting with one prostate cancer dataset, followed by another in melanoma, to come back to the prostate cancer discussion using another highly relevant dataset. this is a departure from the alternative approach in which each disease is discussed in separate sections. however, after considering several possibilities, we are convinced that our approach is the most appropriate to showcase the technique and its power. details on the datasets and methods used are given in the 'materials and methods' section of this paper. we also refer to the original studies and manuscripts associated to the three datasets we analysed. and available at the web address given above). after imputation of missing values, we first calculated the normalized shannon entropy and the mpr-statistical complexity for the each sample. the flowing section explains the context in which our results were generated (refer to the 'materials and methods' section for detail on how our quantities are computed). the normalized shannon entropy measure is widely used in ecosystem modelling to quantify species diversity, where it is acknowledge as having great sensitivity to relative abundances of species in an ecosystem [45] . we utilise the same sensitivity to differentiate a samples in cancer datasets. figure 1 shows that the normalized shannon entropy of prostate cancer tumor samples do not differ much from normal samples. this is in contrast to lymph node metastasis samples that appear to have smaller values of normalized shannon entropy. a mathematical interpretation of this result is that the samples from lymph node metastases have cells that not only varied their transcriptomic profile, they have also ''peaked'' the distribution of expression values with significant fold increases on a smaller number of probes. this explains the reduction in normalized shannon entropy. we note that there are several mechanisms that can explain a macroscopically observable global reduction of transcription. for instance, this may indicate that a relatively large number of genes have reduced their expression levels by genome damage, changes in gene regulation, or other silencing processes. it is reassuring to observe that the changes of the most prototypical quantitative measure we can draw from information theory, the normalized shannon entropy correlate well with the transition between normal samples with to ones with metastases. however, it is also evident from that normal samples do not differentiate much from the tumor group (the normalized shannon entropy values do not differ much). it is then not the number of genes with high expression values, but the change in the distribution of expression levels on the molecular profile, that can provide the other measure that could distinguish these other samples. this must be handled by the other statistical complexity measures to be discussed next. several statistical complexity measures can be defined which aim to clarify our argument. we will first discuss the results of computing the mpr-statistical complexity measure (in the previous figure the y-coordinates correspond to the mpr-statistical complexity values of each sample). the mpr-statistical complexity is proportional to both the normalized shannon entropy associated to the transcription profile and the jensen-shannon's divergence between that probability density function and the uniform probability distribution. again, we refer the reader to the 'materials and methods' section for an explanation of how these magnitudes are computed. although the results of using the mpr-statistical complexity might not seem particularly impressive, there are a few reasons why we introduce them at this stage. we want to illustrate a fact that can already be observed when we employ this measure on this dataset. in this dataset, for a given entropy value interval, normal tissue samples tend to have relatively lower mpr-statistical complexity values than tumor and lymph node metastasis. this means that both prostate cancer and metastases samples diverge from a ''more uniform'' distribution indicating that the distribution ''peaks'' in fewer active genes. it also means that, in terms of jensen-shannon's divergence, the transcriptional profile of a normal prostate cell sample is ''closer'' to a uniform distribution than to the one that is observed in a prostate cancer cell sample. the reader will readily argue, and with reason, that the transcriptional profile of a normal cell is tissue-specific and that it hardly resembles that of a uniform distribution of expression values. that is correct and this observation motivates the introduction of two new statistical complexity measures. we generically call these two variants as 'm-complexities' (with 'm' standing for ''modified''). they have the same functional form as the mpr-statistical complexity, but instead of computing the jensen-shannon's divergence from a uniform probability distribution we compute it against an ad hoc probability distribution functions derived from the data. in this sense, these measures are more supervised then the mpr-statistical complexity is. another perspective is that the mpr-statistical complexity is a special case of this measure in which the ad hoc probability distribution function of reference is the equiprobability distribution. the relevance of this measure derives from being a general definition that allows [44] . metastatic samples have typically lower values of normalized shannon entropy than normal samples and prostate cancer primary tumors. the reduction in normalized shannon entropy indicates that there exists a significant reduction on the expression of a large number of genes, or that the gene profile of metastatic samples has a more ''peaked'' distribution (due to the upregulation of a selected subset of genes). both possibilities just cited are not mutually exclusive. we also note that neither the normalized shannon entropy, nor the mpr-statistical complexity (as a single unsupervised quantifier), can help differentiate between tumor and normal samples, indicating that other information theory quantifiers are required for this discrimination. doi:10.1371/journal.pone.0012262.g001 accommodating several different reference states. we will use it to measure divergences to the ''initial'' and ''final'' transcriptomic states (two states of reference). taken as computed averages over normal samples, and respectively metastatic ones, these measures will allow tracking the processes of differentiation of a cancer cell from a particular tissue type. for example, using lapointe et al.'s dataset, the m-normal statistical complexity quantifier first requires the computation of the probability distribution function of the average gene expression profile of all normal prostate samples. afterwards, the normalized shannon entropy and the jensen-shannon's divergence of any sample profile will be computed using the divergence to that averaged normal distribution. analogously, we compute the m-metastases statistical complexity quantifier by first calculating the average profile of the metastases samples, and then generating the corresponding probability distribution function, finally computing the jensen-shannon's divergence with that profile. we refer to the 'materials and methods' section for details of the calculations. the results can be observed in figure 2 . on the x-axis, the lymph node metastases have the largest values of m-normal indicating a divergence from the normal profile. in addition, the m-metastases values of normal samples tend to be higher than most of the metastasis samples (with the exception of only one). figure 2 shows a gradual progression of the samples positions on this plane from a well-differentiated tissue type specific profile, first to a more heterogeneous primary tumor cluster, and finally to an even less differentiated metastatic profile. the result presented in figure 2 shows that the prostate cancer samples, which are not metastases and therefore could have been scattered anywhere on the plane, are clustered on a particular confined area between the two other groups. we understand that there are reasons to be sceptical about this result being not just a simple consequence of the gene selection process used by lapointe et al. for example, if we assume that the 5,153 probes singled out by lapointe et al. in their figure one of ref. [44] (and that constitute our original data) have been selected with a supervised method that try to distinguish between normal and metastases, then the relative position of normal and metastases samples is perhaps something to be expected. however, even under that assumption, what is not expected is the position of all primary tumor prostate cancer samples, linking the normal cluster of samples with the metastases one. note that the definition of both the m-normal and m-metastases measures do not use any information from the primary tumor prostate cancer samples, so the location of these samples between the normal cluster and the metastases, bridging them naturally is something to highlight. together with figure 1 , it gives evidence that supports the working hypothesis that a gradual ''progression'' occurs, from the normal tissue specific profile to the metastasis one. indeed, following our line of argument, figure 2 has even more relevance when we highlight the fact that the 5,153 probes have not been selected with a supervised method. the authors say that the only selection criteria was to single out the 5,153 cdnas whose expression varied most across samples. in the supplementary notes of their paper the authors say: ''we included for subsequent analysis only well measured genes whose expression varied, as determined by (1) signal intensity over background .1.5-fold in both test and reference channels in at least 75% of samples, and (2) 3-fold ratio variation from the mean in at least two samples; 5,153 genes met these criteria.'' as a consequence, figure 2 has been generated without class selection bias only using the genes that have the most varied expression pattern. we now turn to another aspect of the statistical complexity and entropy analysis. we note that figure 2 shows that the metastases samples have a clear reduction on normalized shannon entropy in comparison with the values observed for the normal samples. at the same time, metastases samples, as expected, have higher mnormal complexity than the normal samples ( figure 2 ). it is then interesting to evaluate the value of the jensen-shannon divergence of these samples and to identify the genes that most correlate with the variations of jensen-shannon divergence to quantify one of the factors that is related to the statistical complexity changes. we have computed the correlation of the gene expression profile corresponding to each of the 5,123 probes. for each of the 5,123 probes, we computed both the pearson correlation (x-axis of figure 3 ) and the spearman correlation (y-axis of figure 3 ) of each probe profile with the jensen-shannon divergence having as probability distribution of reference that of a metastasis profile (these values are called jsm2-pearson and jsm2-spearman in the accompanying excel file provided). with this data, we have produced figure 3 , a scatter plot of the values associated to each probe. in this figure, there are two probes that are immediately recognizable by any cancer researcher, and in particular for those in prostate cancer: klk3/psa (prostate specific antigen) and fos. the interpretation of these scatter plots is not immediate and needs an introductory explanation. each dot corresponds to one probe of the array. for example, a dot that is very close to the origin of coordinates (0,0) indicates a probe such that its pattern of gene expression (across all samples) is not correlated with the jensen-shannon divergence to the average profile of a metastasis pattern. it is, in essence, a probe which is highly uninteresting in this regard. probes that have a high correlation, across all samples, either positive or negative with the jensen-shannon divergence to the average profile of a metastasis pattern are highly informative. they ''co-express'' with this measure. although we provide in the supplementary material the information corresponding to all probes, we will discuss just a few of them. this will allow the reader to understand these plots and will put our results in the perspective with current research in prostate cancer. we particularly highlight the position of klk3/ psa, fos and ccl2. to our surprise, we have found which is perhaps the most famous biomarker in prostate cancer klk3/ psa (kallikrein-related peptidase 3), probe g_914588 (correlations of 20.9312 and 20.9000 respectively). fos and klk3/ psa are the second and the fourth most negatively correlated probes in this ranking of all the genes in the microarray. with opposite signs for correlations are cdkn2d, foxm1, and brca2. the following is a discussion of a selection of probes (highlighted in figure 3 ) in the context of prostate cancer. cdkn2d (cyclin-dependent kinase inhibitor 2d, p19, inhibits cdk4). one of the genes that has strong positive correlations is cdkn2d, (cyclin-dependent kinase inhibitor 2d, p19, inhibits cdk4) (pearson correlation of 0.7543, spearman correlation 0.6833), probe g_145503. a gene that shows a positive correlation with the divergence of a metastasis profile indicates a gene that has a putative reduced expression on these samples. cdkn2d is a known regulator of cell growth regulator and controls cell cycle g1 progression [46, 47] . loss of cdkn2d in cancer cells is one event which is generally associated to a more malignant phenotype. foxm1. another probe that presents positive correlations is foxm1 (forkhead box m1), with pearson correlation of 0.7039 and spearman correlation 0.7500), probe g_564803. it has been recently shown that the depletion of foxm1 still allows cells to enter mitosis but they are unable to complete cell division. as a consequence this leads to mitotic catastrophe or endoreduplication [48] . foxm1 is considered a key regulator of a transcriptional cluster which is that is essential for proper execution of the mitotic program and the control of chromosomal stability [49] . brca2 -(breast cancer 2, early onset). another gene with positive correlations is brca2 (breast cancer 2, early onset), probe g_193736, with pearson correlation of 0.8161 and spearman correlation 0.7333). while the loss of brca2 function and its consequences in prostate cancer is being reconsidered [50, 51, 52, 53] , brca2 is generally regarded as a ''tumor suppressor'', with an established role in maintaining genomic stability via its function in the homologous recombination pathway for double-strand dna repair. this result is supporting its proposed function. loss of brca2 function is thus a warning sign of the existence of error prone cell processes. in prostate cancer brca2 has been associated to promotion of invasion through upregulation of mmp9 [54] . brca2 loss of function due to mutations is linked to poor survival in prostate cancer [55] and rare germline mutations have been associated with early-onset of prostate cancer [56] . ccl2/mcp-1 (chemokine (c-c motif) ligand 2). bone is one of the most common sites of prostate cancer metastasis; close to 85% of men who die of prostate cancer have bone metastasis [57] . the successful metastatic process to bone follows from the activation of osteoclasts with bone resorption, which in turns leads to the release of different growth factors from the bone matrix [58] . ccl2 has been previously reported as expressed in human bone marrow endothelial cells; the ccl2 stimulation promotes prostate cancer cell migration and proliferation [57, 59] and it has been proposed as a paracrine and autocrine factor for invasion and growth of prostate cancer [60] . as a consequence of this central role in the tumor microenvironment, ccl2 is being the object of several studies and is included in the list of potential targets for novel therapies [60, 61, 62, 63, 64, 65, 66, 67, 68, 69] . fos (v-fos fbj murine osteosarcoma viral oncogene homolog). a probe for fos (g_811015; correlations of 20.9380 and 20.9500 computed with pearson and spearman) has a similar correlation than klk3/psa. the high rank of fos was unexpected, but perhaps it is less of a surprise for some experienced researchers in prostate cancer as its role has been highlighted in the past [70, 71, 72] . amplification of members of the mapk pathway was associated with androgen independent prostate cancer, and co-expression of raf1, erbb2/her2 and c-fos would lead to this phenotype [73] . we will not discuss in depth the known relationships between fos, lamin a/c and prostate cancer. we leave this discussion for later, as lamin a/c will also appear in our study of the other prostate cancer dataset studied in this paper. lamin a/c appears as a member of a set of genes with reduced expression for higher grade primary prostate cancer samples (note that the current analysis that gave fos as a biomarker is on lymph node metastatic samples like here). however, we would like to point out a connection that is currently hypothesized between lamin a/c and fos, the gene we have just discussed. ivorra et al. have recently proposed that ''lamin a overexpression causes growth arrest, and ectopic c-fos partially overcomes lamin a/c-induced cell cycle alterations. we propose lamin a/c-mediated c-fos sequestration at the nuclear envelope as a novel mechanism of transcriptional and cell cycle control'' [74] . in addition: ''c-fos accumulation within the extraction-resistant nuclear fraction (ernf) and its interaction with lamin a are reduced and enhanced by gain-of and lossof erk1/2 activity, respectively.'' [75] . these novel interactions between lmna and fos, their putative role in prostate cancer metastasis and their seemingly different behaviours in prostate cancer lymph node metastases warrant further investigation. sox9 (sry (sex determining region y)-box 9). this transcription factor has been recently identified as having an importat role during embryogenesis and in the early stages of prostate development [76, 77] and in testis determination [78] , processes that link sox9 upregulation to cancer development [79] . basal epithelial cells do express sox9 in a normal prostate. while there exists no detectable expression in lumina epithelial cells, sox9 has already been reported as ''expressed in primary prostate cancer in vivo, at a higher frequency in recurrent prostate cancer and in prostate cancer cell lines (lncap, cwr22, pc3, and du145)'' [80] . wang et al., also in [80] add that: ''significantly, down-regulation of sox9 by sirna in prostate cancer cells reduced endogenous ar protein levels, and cell growth indicating that sox9 contributes to ar regulation and decreased cellular proliferation. these results indicate that sox9 in prostate basal cells supports the development and maintenance of the luminal epithelium and that a subset of prostate cancer cells may escape basal cell requirements through sox9 expression.'' an increased value of sox9 expression in advanced prostate cancer has been associated to tumor progression and the epithelial-mesenchymal transition [81] . sox9 expression has been associated with a putative subgroup of prostate cancer [82] , associated to lymph-node metastasis (as seems to be the case in this dataset) and has a know role in chondrogenic differentiation processes [83] . klk3/psa -(kallikrein-related peptidase 3)/prostate specific antigen. to finalize our initial discussion on this dataset, we address klk3. the high ranking of klk3/psa in our list is perhaps one of the most remarkable retrodictive outcomes of our approach. klk3/psa (also known as prostate specific antigen) is a conspiquous member of our top rank list. it is perhaps the best blood biomarker for prostate cancer screening. its relevance and popularity as a target of studies is so wide that it makes unfeasible any serious attempt to uncover its relevance in the prostate cancer literature. a search using pubmed using the keyword 'klk3' (and the other alias names of this gene) reveals a total of 11,429 published papers. of course, many of these publications relate to its role for early screening, but in this study we are uncovering its role as a tissue biomarker. our results echoes a recent contribution by s. miyano's and his collaborators [84] on a massive meta-analysis of microarray datasets. it is also in line with results from clinical studies that indicate that a 5-year psa value is useful for predicting prostate cancer recurrence. [85] . certainly the dynamics of psa, now perhaps with fos and sox9 added to the set of biomarkers of interest, warrant further investigation for patient population stratification after initial treatment. the biomarkers discussed in this section warrant further investigation in prediction of lymph-node metastasis and clinical management of prostate cancer [86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109] . we refer the reader to the supplementary material to have a complete list of probes and their correlations with the information theory quantifiers. the following sections present the results that we obtained with a melanoma dataset. our aim is to observe if variations of the normalized shannon entropy and the statistical complexity measures, mpr-complexity and the modified forms m-normal and m-metastases, provide interesting results in a different disease and experimental setting. in this case we have selected a gene expression dataset from haqq et al. [110] containing information of 14,772 cdnas in 37 samples (figure two from the [110] ). the 37 samples include 3 normal skin, 9 nevi, 6 primary melanoma and 19 melanoma metastases. this datasets has more phenotypical characteristics for the group of samples. after an initial process of data cleaning, we removed 35 probes which had an unsually high expression value on only a few samples, in some cases on a single one. the dataset we work with from original contributed by haqq et al.consists of 14,737 probes. first, we computed the normalized shannon entropy and the mpr-statistical complexity for each sample (refer to the 'materials and methods' section for a detailed presentation of these calculations). figure 4 shows the values of these quantifiers for each sample. we first observe an important difference between figure 1 and figure 4 . in this melanoma dataset, neither the use of the normalized shannon entropy nor the mpr-complexity helps to discriminate between normal skin, nevi, primary and metastastic melanomas. nevertheless, we decided to present this figure for methodological reasons. we envision that some researchers will calculate the normalized shannon entropy and mpr-complexity using all the probes. we note that in figure one of haqq et al's original paper, the whole probe set was previously filtered by selecting those which vary across samples, thus indicating that they may have information about disease subtypes (although the phenotypic types were not biasing the selection). in this case we want to illustrate both the normalized shannon entropy and mpr-complexity calculated using all the probes does not give the expected benefits. we will now see the benefits of using the m-complexities. as we did for prostate cancer (see figure 2 ), we aim at identifying if the use of the modified forms of the statistical complexity (the m-complexities) could give some insight where the normalized shannon entropy and mpr-complexity measures fail. to compute the m-normal measure, we need to define the average gene expression profile for a normal cell (which we call p ave ). we thus resort to the three normal skin profiles and we produce the average based on these profiles (details for computing the average profiles are given in the 'materials and methods' section). we call m-skin the resulting measure that relies on this profile. analogously, we need to compute a pattern for m-metastasis, and we proceed to calculate the p ave profile averaging over the 19 metastases samples. the result is encouraging, as samples plotted in the (m-skin, m-metastasis)-plane cluster in groups, showing an important m-skin complexity transition between normal skin cells and nevi. most importantly, this method naturally shows that some of the metastatic samples have a large value of m-skin complexity, so we present the results of another experiment, aimed at clarifying this fact. in their original publication, haqq et al. classified the melanoma metastases in two groups due to their molecular profiles: five samples were classified as 'type i' and fourteen as 'type 2' based on a hierarchical clustering approach. our result reinforced the view that the type ii melanomas metastasis is a pretty homogeneous group, we will present the results on the (mskin, m-metastasis i)-plane. this means that now the p ave profile will not be obtained by averaging over the 19 metastases samples, but instead using only the 14 samples which have been labelled as 'type ii'. as such, we aim at revealing if type i samples are indeed different in this plane, and if other clusters are also present. figure 5 presents the results. the first fact worth commenting is the pronounced gap between normal skin samples and the nevi, primary, and metastatic melanoma samples as revealed by the mskin measure. note also that the m-skin is based on the average profile that of the normal samples, which indicates that no information about the profiles of metastasis are used, yet m-skin reveals that increasing values of this measure may be linked with a 'progression' from nevi to primary and metastasis melanoma profiles. we now introduce another useful technique to identify genes which correlate with the transitions. the challenge is to find genes which are related with the progression towards metastases profiles, even when we recognize that there the group of metastasis samples is heterogeneous (containing at least two groups). since the final outcome of figure 4 and figure 5 is that the normalized shannon entropy does not help much in this experimental scenario, we will concentrate only on one of the multiplicative factors of the mcomplexities, the jensen-shannon divergence. we compute two p ave profiles, one with the normal skin samples only, and the other with all the metastasis samples (regardless their type). we will call the two divergences jsm0 and jsm5 respectively. we then compute the spearman correlation of the profile of all gene probes in the array across the 37 samples to both jsm0 and jsm5. we have listed all probes according to the absolute value of the difference of these correlations, i.e. abs. diff. (probe) = |jsm0(probe)2jsm5 (probe)| in decreasing order. the results are provided as haqq-plosone-supfile.xls, in the sheet labelled 'results-correlation'. the rationale is to identify those probes which are highly correlated (both positively or negatively) with the jensen-shannon divergence of the normal tissue profile and that ''reverse signs''. for instance, a probe for the tp63 gene (tumor protein p63, keratinocyte transcription factor ket), aa455929, is ranked in the third position. its correlation with the jensen-shannon divergence of the normal skin type is relatively high and negative (jsm0 = 20.63632) while at the same time is has a positive correlation with the jensen-shannon divergence of the metastasis profile (jsm5 = 0.62138). in the ranking, the first probe that presents the opposite behaviour is one for ada (adenosine deaminase), aa683578. figure 6 helps to understand the relationship of these correlations with expression. not only are these genes well correlated with the divergences, they also seem to be good markers of the progression from one tissue type profile to the metastasis profile. we will now discuss three of these genes in the context of current biological knowledge on melanoma drivers and metastatic progression. we provide many references for one of them, spp1 (secreted phosphoprotein 1 or osteopontin). the discussion on this gene will be left for later, when we will discuss specifc oncosystems related to cell proliferation, chemotaxis and responses to external simulus. figure 7 shows the expression of ada (adenosine deaminase, aa683578) as a function of tp63 (keratinocyte transcription factor ket, aa455929). all normal skin samples, as well as nevi and a couple of primary melanomas have relatively low values of ada but they express tp63. there is a change of roles in metastatic and some primary melanomas, which have reduced tp63 expression but increased values of expression of ada. as we will later see, these events correlate with other major transcriptional modifications which involve dozens of genes and that we have been able to map thanks to functional genomics bioinformatics tools. the role of spp1 will be discussed in that context after some references to tp63, ada, and plk1 which follow. tp63. the product of this gene [111, 112] belongs to the same protein family of its more famous relative, tp53, a gene that is often mutated in human cancers [113] and highly regarded as a key ''tumor suppressor''. tp63's product, p63, is a homologous protein to p53, which is considered to be phylogenetically newer [114] and also regarded as an important apoptotic and cell-cycle arrest protein. mice that lack tp53 are born alive with a propensity for developing tumours; mice that lack tp63 do not appear to be tumour prone, although, new results are partially contradicting earlier findings [115] . it appears that the diverse roles of the isoforms of the p63 family reveal that there exists a crosstalk with the different isoforms of the p53 family that needs to be systematically investigated [116] . it has recently been shown that p63 is a key regulator of the development of stratified epithelial tissues [113] and that its deletion results in loss of stratified epithelial and of all keratinocytes [117] . melanocytes also express two isoforms of p63 [118] , but p63 expression is not reported in 57 out of 59 tumors in a tissue microarray study performed by brinck et al. [119] . it is clear that the the role of loss of expression of tp63 in melanoma warrants further investigation. ada -(adenosine deaminase) and dpp4/cd26 (dipeptidylpeptidase 4, cd26, adenosine deaminase complexing protein 2). a link between tp63 and ada has already been reported in the literature. ada is a gene involved in cell division and proliferatation [120] and it has been suggested to have a regulatory role in dendritic cell innate immune responses [121] .translational modification is also a function of p63. sbisa et al. have proved that ada is a direct target of isoforms of p63, which is an important discovery as ada has two tp53 binding sites, leading to a complex metabolic balance due to the different relationships between this trio and p21 yet to be completely elicitated [120, 122] . several studies indicate elevation of adenosine deaminase levels in sera of breast [123] , head and neck [124] , colorectal [125] , acute lymphoblastic leukaemia [126] and laryngeal cancers [127] . we observe a marked increase of expression of a probe for ada with melanoma progression while at the same time we observe a loss of expression of a probe corresponding to dpp4/cd26 (dipeptidylpeptidase 4, cd26, adenosine deaminase complexing protein 2), a membrane-bound, proline-specific serine protease [128] that has been attributed tumor suppressor functions [129] . it has been previously reported that loss of dpp4 immunostaining helps to discriminate malignant melanomas from deep penetrating nevi, a variant of benign melanocytic nevus [130] and early reports of their absence in metastatic melanomas exist [131, 132] . as deep penetrating nevi can mimic the vertical growth phase of nodular malignant melanoma, and ada could potentially be downregulat-ing dpp4 [133, 134] we believe that the elicitation of the complementary role of these two biomarkers to distinguish these two entities is necessary and also warrants further clinical studies. plk1 (polo-like kinase 1 (drosophila)). another probe for gene that ranks high as a positive marker of metastasis is plk1, polo-like kinase 1, serine/threonine protein kinase 13 (aa629262). plk1 is a centrosomal kinase [135] which is figure 7 . scatter plot showing the expression of the probe corresponding to ada (adenosine deaminase), aa683578 (y-axis) and tp63 (tumor protein p63), aa455929 (x-axis). all the samples that have tp63 expression are normal or nevi, with two primary melanomas still preserving tp63 expression but with higher ada. the trend reverses for the rest of the primary melanoma samples and the metastatic ones, which all express ada but not tp63. doi:10.1371/journal.pone.0012262.g007 analogously, we compute the jensen-shannon divergence of each sample with the average metastastic profile and we also compute the correlation of each probe with this measure (y-axis). the position of one probe corresponding to the tp63 gene (tumor protein p63, keratinocyte transcription factor ket), aa455929, is highlighted. the expression of this probe has a relatively high negative correlation with the jensen-shannon divergence of the normal skin type (jsm0-spearman = 20.63632) while at the same time is has a positive correlation with the jensen-shannon divergence of the metastasis profile (jsm5 = 0.62138). the first probe that presents an opposite behaviour is one for ada (adenosine deaminase), aa683578. probes for spp1 (secreted phosphoprotein 1 or osteopontin) and plk1 (polo-like kinase 1 or drosophila) are also highlighted. while plk1 is currently less recognized as a biomarker in melanoma research, the importance of spp1 in cutaneous pathology [315, 318, 320, 321] and in particular in melanoma [208, 209, 210, 211, 212, 214, 215, 216, 217, 218, 219, 222, 226, 264, 314, 315, 316, 317, 319, 322, 323, 324, 325, 326, 327, 328, 804, 805, 806, 807, 808, 809] is increasing. using a 5-biomarker panel that included spp1, kashani-sabet et al. used tissue microarrays on 693 melanocytic neoplasms to show that spp1 expression collaborates significantly improving the detection of high percentage of melanomas arising in a nevus, spitz nevi, dysplastic nevi and misdiagnosed lesions [253] . like in the case of prostate cancer ( figure 3 , in which klk3/psa -prostate specific antigen was highlighted), our method allows the detection of important biomarkers with a high degree of concordance with current biological understanding of metastatic processes. doi:10.1371/journal.pone.0012262.g006 regarded as being linked to centrosome maturation and spindle assembly [135] . plk1 expression has also been singled out as a biomarker of a ''death-from-cancer'' signature, sharing with others the function of being an activator of mitotic spindle check point proteins. with other proteins it would has a stem cell-like expression profile phenotypically characterized by enabling metastasis with anoikis resistance and disregulated cell-cycle control [136] . plk1 inhibition could be a common target for gastric adenocarcinoma [137] , bladder cancer [138] , colon cancer [139, 140] , hepatocellular carcinoma [141] , medullary thyroid carcinoma [142] , esophageal cancer [143] , pancreatic cancer [144] and in some types of non-hodgkin lymphomas [145] and breast cancer [146] . plk1's spearman correlation with the values of the jensen-shannon divergence of samples with the normal skin profile is relatively high (0.5863). plk1 also has a high value of (negative) spearman correlation with the values of the jensen-shannon divergence of samples with the average metastatic profile (20.44571 in the comparison, it was found that metastatic malignant melanomas with expressed plk1 at markedly elevated levels (median, 60.00% vs. 37.98%; p-value,0.000053), concluding that plk1 is a reliable biomarker for patients at high risk of metastases, even when the most important prognostic clinical factor (breslow's maximum thickness of the primary malignant melanoma) indicates the contrary [147] . we consider this an important finding as plk1 silencing is already part of an integrated oncolytic adenovirus approach currently being studied in mice models of orthotopic gastric carcinoma [148] and has promise due to the lack of a reported measurable immune response of sirna-based therapeutics [149] . another positive note is the less sensitivity to plk1 depletion of cells with a functional p53 [150, 151] , and can help to sensitize cells to chemotherapy (as observed in lung cancer [152] ). this constraint of aneuploid cancer cells to plk1 expression, particularly in cells with inactivated p53 [153] , could be exploited by lentivirus-based rna interference [154] . correlation analysis with jensen-shannon divergences reveals biomarkers for loss of cell adhesion, cell-cell communication, impairment of tight junction mechanisms and dysregulation of epithelial cell polarity. as discussed before, the probe for ada (adenosine deaminase) is the first that has a different trend. since we put all metastasis samples together in the same group when we calculated the average probability profile (and we have a heterogeneous group) we have on our ranking 58 probes that appear before ada (we refer to the supplementary file haqq-plosone-supfile.xls). an analysis using gather (http://gather.genome.duke.edu/) [155] to interpret the collective influence of the lack of expression of all these genes in the metastasis samples reveals an interesting new perspective. using gene ontology, we found that six of the 44 genes identified by gather are related to epidermis development (cdsn, dsp, evpl, gjb5, krt13, krt5), p-value ,0.0001, bayes factor 16, and eight genes are related to cell adhesion (cdsn, cldn1, dsg1, dst, lgals7, lrig3, pcdh21, pkp1), p-value,0.0001, bayes factor 7. ank1 (ankyrin 1, erythrocytic), aa464755 was also singled out as by our gene ontology analysis as related to the maintenance of epithelial cell polarity (p-value = 0.002, bayes factor 3). the use of another profiler of genome signatures (g:profiler, [156] ) also reinforces the view that many genes that have lost expression are related to 'epidermis development' (col17a1, dsp, evpl, gjb5, krt13, krt5, lce1c, mafg, tgm3) with p-value = 7.78e-11. thirteen are associated with gene ontology function of cell communication (ank1, cdsn, cldn1, dsg1, dst, gchfr, gjb5, gpr115, lgals7, lrig3, pcdh21, pkp1, ptger3), albeit with a pvalue of only 0.02. gchfr is also involved in nitric oxide metabolism. if we add to the list of 44 genes already recognized by gather the other 77 probes that after ada in this ranking have also loss of expression (until we found pdxp (pyridoxal (pyridoxine, vitamin b6) phosphatase), the evidence is stronger, now col7a1, gjb5, klk4, and krt1 also is in this group (the bayes factor of this association returned by gather is now 21 for the go term 'epidermis development'). 'cell adhesion' has now 13 genes, cdsn, cldn1, col7a1, dsc2, dsg1, dst, jup, lgals7, lrig3, pcdh21, pkp1, slit3 thbs3 (p-value,0.001, bayes factor 10). these results are considered statistically very relevant as identifiers of a particular process which seems to be undermined by this collective loss of expression. if we put all this information together, we clearly observe a pattern of downregulation of gene expression that is associated with an impairment of epidermis development and the maintainance of its structure ( figure 8 and table 1 ). this is, perhaps, an instantiation of one of the ''extended hallmarks of cancer'' (that of ''tissue dedifferentiation''). this process includes the loss of function of genes that are essential for the maitainance of tight junction and epithelial cell-cell communication. while loss of epithelial structure is related to these genes, we observe that those that increase expression are associated to other developmental processes, not necessarily concerted in this panel. instead they show a pattern of increasing cell motility, chemotaxis and positive regulation of cell proliferation. we will first discuss the processes related to the loss of adhesion, which could be linked to an increased probability of metastatic potential of these cells. the loss of expression of plakophilin 1, junction plakoglobin, desmoplakin and desmoglein 1 indicate deficiencies in desmosome processes. in general, this panel is composed of a number of genes that are losing expression during progression and that have gene ontology annotations related to tight junctions, gap junctions, adherens junctions and desmosomes, and an impaired set of processes that link, via intercellular channels and bridges, the cells of the epidermis. mutations in these genes are linked to a number of skin genetic diseases [157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170] the desmosome are cell-cell adhesive junctions which provide a mechanical coupling between cells. these junctions are found in several epithelial tissues and the decreased assembly of the desmosome has been shown to be a common feature of many epithelial cancers [171, 172] . plakoglobin helps to connect transmembrane elements to the cytoskeleton [173] . plakophilin 1 [174] (pkp1, one of the genes in our panel above) is a desmosomal plaque component [175] that stabilizes desmosomal proteins at the plasma membrane [176, 177] and, with desmoplakin [178] , recruits filaments to sites of cell-cell contacts [179] . as a consequence, it has been proposed that the lack of pkp1 increases keratinocyte migration [180] and loss of pkp1 expression in head and neck squamous cell carcinoma and in esophageal squamous cell carcinoma may contribute to an invasive phenotypic behaviour [171] , perhaps as a consequence of the impaired recruitment of desmoplakin. the desmoglein-specific cytoplasmic region (dscr) is the site of caspase cleavage during apopotosis and is a conserved region of yet undefined function and unknown structure, but it specifies the function of the desmoglein family of cell adhesion molecules (of which dsg 1 is a member). it has been recently shown that the dscr has a weak interaction with pkp1, plakophilin 1 (ectodermal dysplasia/skin fragility syndrome) and the cytoplasmic domain of desmocollin 1 [181] . plakoglobin is cleaved by caspase 3 during apoptosis [182] . in addition, kami et al. in ref [181] also report and conclude that: ''desmoglein 1 membrane proximal region also interacts with all four dscr ligands, strongly with plakoglobin and plakophilin and more weakly with desmoplakin and desmocollin 1. thus, the dscr is an intrinsically disordered functional domain with an inducible structure that, along with the membrane proximal region, forms a flexible scaffold for cytoplasmic assembly at the desmosome''. as previously discussed, all these genes progress towards a loss of expression, and they are highly correlated. figure 9 shows the average expression of pkp1/plakophilin 1 (ectodermal dysplasia/ skin fragility syndrome), (nm_000299) and jup, junction plakoglobin, (bx648177) on the x-axis against that of dsp, desmoplakin (nm_004415 hs.519873) on the y-axis. again, we see a clear pattern of progressive reduction of expression from normal skin and nevi (green and yellow, respectively), primary melanomas (in orange) and melanoma metastases (red). joint loss of expression of claudin 1 and members of the aquaporin family are also linked to a transition to a more malignant phenotype we note however, the gene ontology annotation is not the only way that we can make sense of this information. a detailed analysis of that list of 58 genes reveals other proteins involved in tight junction, like aquaporin 3 (aqp3). probes for aqp3 and claudin 1 (cldn1) have reduced expression with the progression of the disease as shown in figure 10 . aqp3 (gill blood group) is a member of the aquaporin family of proteins, and currently is recognized as an 'aquaglyceroporin' [183] of great importance to maintain skin hydration of mammals epidermis [184] . three proteins of this family (aqp1, aqp3, and aqp9) have probes that seem correlated with melanoma progression, all losing their expression in the process of going from normal skin to metastatic melanoma. aqp3 water channels have been pointed out as an essential pathway for volume-regulatory water transport in human epithelial cells [185] . aqp3 is also selective for the passage of glycerol and urea and it has been suggested that osmotic stress up-regulates aqp3 gene expression in cultured keratinocytes [186] . aqp3 was found to be the predominant aquaporin in human skin which increased expression and altered cellular distribution of aqp3 in eczema thus contributing to water loss [187] . the putative involvement of aquaporins in the progression of melanoma, uncovered by our method in our results, warrants further investigation as it has been recently shown that another member of this family (aqp8) also facilitates hydrogen peroxide diffusion across membranes [188] . it is suspected that aqp3 has other functions with a suggestion that it is involved in ultraviolet radiation induced skin dehydration [189] . there is no probe for aqp8 in haqq et al.'s dataset that we could scrutinize from its trend with progression but we note that a novel strategy for drug development for melanoma (i.e. elesclomol) works by inducing apoptosis via a mechanism of elevation of reactive oxygen species (of course, including hydrogen peroxide in cancer cells) thus exploiting the ''achilles hell of cancer metabolism'' [190] . claudin 1, cldn1 [191] , a gene which is reported to be ''normally expressed in all the living layers of the epidermis'' [192] , in concert with aqp3, is a key component of the tight junction complexes of the epidermis. low cldn1 gene expression was correlated with shorter overall survival in lung adenocarcinoma. overexpression of cldn1 was correlated with suppression of cancer cell migration, invasion and metastasis [193] . hoevel et al. report that re-expression of cldn1, in breast tumor spheroids, induces apoptosis and they conclude: ''these findings support a potential role of the tight junction protein cldn1 in restricting nutrient and growth factor supplies in breast cancer cells, and they indicate that the loss of the cell membrane localization of the tight junction protein cldn1 in carcinomas may be a crucial step during tumor progression'' [194] . tokes et al.also report that malignant invasive breast tumors are negative table 1 . gene names and probe accession number of the 27 probes with genes annotated with functions on cell adhesion, cell-cell communication, tight junction mechanisms and epithelial cell polarity shown in the heat map in figure 8 . for cldn1 [195] . as in breast cancer [196] , in which reduced expression correlated with recurrence status, the low expression of cldn1 and other tight junction proteins seems to contribute to cellular detachment. the complementary set of correlations with the jensen-shannon divergences unveils biomarkers for cell proliferation, chemotaxis, and responses to external simulus. if the use of gene ontology has produced very peculiar results, helping us to link the loss of expression of 44 genes with a significant change in epithelial structure and development. a natural question arises: ''which is the significance of another set, now arbitrarily chosen to be also of the same cardinality (i.e 44 genes) with the complementary behavioural pattern?'' we have now listed all the probes according to diff. (probe) = jsm0(probe)2jsm5(probe) in decreasing order. the results are provided as haqq-plosone-supfile.xls ('results-correlation' sheet). this now gives ada as the first ranked gene. again using gather [155] on the first 44 genes recognized by the software, and again using gene ontology, we observe as most important common function that of cell motility (ccl3, cxcl10, fprl1, sema6a, spp1), p-value = 0.0002, bayes factor 5, and chemotaxis (ccl3, cklfsf7, cxcl10, fprl1, spp1), p-value,0.0001, bayes factor 7. the genes cxcl10, spp1, and wars, together with another gene that has been annotated as related to positive regulation of mitosis (sch1), have also been annotated as regulators of cell proliferation (pvalue = 0.007, bayes factor 2). using the g:profiler software [156] , we obtain a complementary information. sixteen genes (including spp1, sema6a, lef1 [197] , cd230, als2cr2, dkk1, cyfip2, shc1, ankrd7, ifi6, cited1, and mid1) have been associated to the gene ontology term of 'developmental process'. spp1 -secreted phosphoprotein 1 (osteopontin). spp1 is one of the most conspicuous melanoma biomarkers [198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222] (see also the references cited in figure 6 and note its eminent position in this scatter plot). in 1990, craig et al. reported that spp1 may work as an autocrine adhesion factor for tumor cells (see also [204, 223, 224] ). they observed that ''spp1 mrna, which is barely detectable in normal mouse epidermis, was expressed at moderate-to-high levels in 2 of 3 epidermal papillomas and at consistently high levels in 7 of 7 squamous-cell carcinomas induced by an initiation-promotion regimen'' [225] . the evidence is being constantly expanded on the role of spp1 as a molecular prognostic biomarker in melanoma [226] . activation of spp1 may be an important event that allows the transformed melanocytes to invade the dermis as proposed by geissinger et al. in 2002 [208] . this causes spp1 to avoid the apoptotic stimulus, one of the ''hallmarks of cancer'', which invasive cells will be receiving from this new tissue. if we extend the literature-based search so that we now include the first 200 gene probes recognized by gather then we have 27 gene probes associated with the gene ontology in terms of ''cell proliferation'' (p-value = 0.0002, bayes factor 5), and 'regulation of cell proliferation', p-value = 0.003, bayes factor 3). however, other partners of plk1 appear and their function in 'mitotic cell cycle' (pvalue = 0.0003, bayes factor 5) is increasingly present (in particular, the m phase of the mitotic cell cycle). the details of the gene ontology terms which are significant and the genes associated to them are listed in table 2 . the analysis using g:profiler largely coincides with the analysis using gather, however, it retrieves 12 genes associated with the m phase of mitotic cell cycle, namely: aurka and aurkb [227, 228, 229] , bub1 [230, 231] , cdca5a/sororin/p35 [232] , cdc7 [233, 234] , chek1 [235] , kif23/mklp-1 [227, 236, 237] , map9/asap [238, 239] , ncapd3, ncapg2 [240] , nek6 [241, 242, 243, 244] , plk1 [147, 245, 246] , pttg1/securin [247] , shc1/p66 [248, 249, 250] (discussed in the context of shc4 signalling), and tfdp1/dp-1 [251] . these are a significant finding by g:profiler (p-value = 4.03e-07). we have listed above some of the genes gene associated to the m phase of mitotic cell cycle and associated references which are either to current research in melanoma and/or its biological function. we now list other genes which have been associated with the term 'cell proliferation' by gather. these genes are: arpc1b [252] , arpc2 (which, together with spp1, is also in the novel 5-biomarker panel of kashani-sabet et al. [253] ), bccip (brca2 and cdkn1a-interacting protein)/p21-and cdkfigure 10 . expression of a probe for cldn1 (claudin 1) (y-axis) as a function of a probe for aquaporin 3 (x-axis). other members of the aquaporin family of proteins have a similar behaviour. aqp3, together with cldn1 are key components of the tight junction complexes of the epidermis and their joint loss of expression seem to be related to a transition to a more malignant phenotype. we use the same color coding as figure 9 . doi:10.1371/journal.pone.0012262.g010 associated protein 1) [254] , bst2/bone marrow stromal antigen 2/tetherin [255] , ccl3/mip-1alpha [256, 257, 258] , cct4, cdca5/sororin [259, 260, 261, 262, 263] , cenpf/mitosin [264] , cxcl1/chemokine (c-x-c motif) ligand 1 (melanoma growth stimulating activity, alpha) [265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285] (in uveal melanoma see [286] ), cxcl10 [256] , flt1/vegfr1 [287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299] , fth1/ferritin heavy chain [300, 301, 302] (which may indicate a necessary condition for the mainainance of iron sequestration and suppression of reactive oxygen species accumulation [303] ), fprl1, lig3/dna ligase 3 [304] (which, together with xpa and ercc5 is associated to dna repair in ionizing radition studies [305] ), mcmdc1, psen2, nrp2/neuropilin 2/vascular endothelial cell growth factor 165 receptor 2 [306, 307, 308] , sema6a (a member of the semaphorin family, of increasing importance in cancer research [309, 310, 311] and in particular due to its observed upregulation in undifferentiated embryonic stem cells [312] ), slamf1/cd150 (a marker associated with hematopoietic stem cells [313] ), spp1/osteopontin (which, together with arpc2, is also in the novel 5-biomarker panel of kashani-sabet et al. [253] ) [206, 207, 208, 209, 210, 211, 212] [206,207,208,209,210, 211,212,214,215,216,217,218,219,220,221,222,226,314,315,316,-317,318,319,320,321,322,323,324,325,326,327,328,329] , stk6 [230, 330] , and wars/tryptophanyl-trna synthetise [331] . figure 11 shows a heat map of discussed gene probes annotated with functions on cell proliferation. the references provided next to each gene help to related these upregulated genes in the context of current research in melanoma or with the m phase of mitotic cell cycle, showing a high degree of correlation between our results and with published literature. another microarray dataset we have selected to evaluate for the relevance of transitions of normalized shannon entropy and statistical complexity was contributed by true et al. [332] in 2006. the original goal of true et al. was to identify a molecular correlate for gleason patterns 3 and, if possible, the clinically most worrisome patterns 4 and 5. they partially succeeded by linking the expression of only 86 genes with gleason pattern 3 [332] using a standard statistical analysis. in this study, we eliminated sample 02-209c since data was acquired using a different platform and would not be useful for our analysis. the remaining thirty one (31) samples were assayed with the gpl3834 (fhcrc human prostate pedb cdna array v4) platform using 15,488 probes. we also eliminated all the probes with missing values, remaining 13,188 probes. we have first plotted the samples on the (normalized shannon entropy, mpr-statistical complexity) plane ( figure 12 ). it was interesting to observe that there exists a high correlation between the two measures. samples that are entirely composed of gleason pattern 3 tend to have a greater value of normalized shannon entropy than 0.985. we can also identify a cluster of samples that present gleason patterns which are either 4 or 5. note that there seems to be two outliers (02_003e and 03_063) to the generic trend of the other 29 samples. the two outliers are samples that correspond to samples labelled as having gleason 3 patterns and both have unusually low values of normalized shannon entropy that are well below the values of the rest of the group. this raised a suspicion about the true nature of this phenomenon. if the labelling is correct, this may indicate a subsampled group of prostate cancer that has gleason 3 pattern characteristics but very low entropy. alternatively, it may indicate an experimental bias for reasons we can not explain with the available clinical information. in order to clarify the situation, and see if we can declare these two samples as outliers of the other group, we performed another experiment. we have now computed two modified complexities, which we will call mgleason 3 and m-gleason 5 ( figure 13 ). the names are probably selfexplanatory, but a brief reminder follows. to calculate the mpr-complexity, by definition, we have used the equiprobable distribution as our probability distribution of reference (for the computation of the jensen-shannon divergence of the gene expression profile to this distribution). in the case of the m-gleason 3, the probability distribution of the reference is obtained averaging all the probability distributions of the samples that have been labelled as gleason 3 (analogously, we calculated mgleason 5) . samples that have gleason pattern 3 and 5 appear as separate clusters in the (m-gleason 3, m-gleason 5) plane with the two putative outliers of the general trend far apart (even if they have been used to calculate the average probability distribution function of the gleason 3 pattern). even samples with gleason 4 pattern are located closer to samples of gleason patterns 3, and 5, indicating that, perhaps, there exists a subsampled subtype of prostate cancer or there might be another experimental bias or factor that at present we can not resolve with the information we have for these samples. consequently, we have decided to eliminate both samples (02-003e and pna_03-063a) from further calculations. with these considerations, we now have a dataset with 13,188 probes and 29 samples as our dataset for further analysis. figure 14 shows the distribution of the samples using the normalized shannon entropy and the mpr-complexity. by definition, the positions of the 29 samples in the plane do not change (this figure is basically ''zooming in'' one region of figure 12 that contains these samples). we note again, however, that the 29 samples seem to be separating in three different clusters. whether we can argue about the existence or not of these gaps in normalized shannon entropy, it is clear that there seems to be a progression as we have seen with lapointe et al's dataset. there is a group of three samples with gleason pattern 3 that seem to have the the largest normalized shannon entropy values. there is also a cluster that only contains samples of either gleason pattern 4 and 5, all with normalized shannon entropy values smaller than 0.985. there is also very little variation (see figure 15 ) of the positions of the 29 samples on the (m-gleason 3, m-gleason 5)-plane, indicating a degree of robustness that the computation of these modified complexities have, even in the presence of some outliers. after observing that figure 14 shows a correlation of gleason pattern score with normalized shannon entropy, we asked ourselves: 'which are the genes that most positively and negatively correlate with the transitions of normalized shannon entropy?' we have plotted spearman versus pearson correlation values of probe expressions to attempt to find those that best correlate, either positively or negatively, with the normalized shannon entropy values of the samples. the results have revealed some of the most relevant biomarkers of progression, and some unexpected newcomers. figure 16 shows the pearson and spearman correlations of all the 13,188 probes in the dataset with the normalized shannon entropy values of the samples. we have highlighted some particular genes that are discussed below. cdkn2c (cyclin-dependent kinase inhibitor 2c (p18, inhibits cdk4). when we compute the correlations of the probes expressions with the normalized shannon entropy values of the samples, the gene that has the most negative correlations is cdkn2c (cyclin-dependent kinase inhibitor 2c -p18, inhibits cdk4 -nm_078626), which has been previously associated with the transition from prostatic intraepithelial neoplasia (pin) to prostate cancer [68] figure 12 . we plot the values of two modified statistical complexities, which we will call m-gleason 3 and mgleason 5. instead of using the equiprobable distribution as our probability distribution of reference (for the computation of the jensen-shannon divergence of the gene expression profile to this distribution), as required for the mpr-statistical complexity calculation, we used a different one. for the m-gleason 3, the probability distribution of the reference is obtained averaging all the probability distributions of the samples that have been labelled as gleason 3 (analogously, we calculated mgleason 5) . this is analogous to our approach in melanoma ( figure 5 ) in which we used normal and metastatic samples as reference sets for a modified statistical complexity. we observe that, even in this case, 02_003e and 03_063 continue to appear as outliers. in addition to the evidence, we have observed that the deletion of these two samples did not significantly alter the identification of biomarkers. doi:10.1371/journal.pone.0012262.g013 samples 02_003e and 03_063 seem to be outliers to this trend, and in the case of 03_063 the sample is not even close to a hypothetical linear fit which seems to be the norm for all the samples. figure 13 will provide further evidence that may indicate that these two samples are outliers or not to the overall trend. doi:10.1371/journal.pone.0012262.g012 with the dedifferentiation process, with preoperative psa levels and the percent of gleason 4 and 5 cancers [338] . amacr, cyclin g2, cdk4 and cdk7. other probes that also have high negative correlations with the shannon normalized entropy correspond to ccng2 (cyclin g2) cr598707, cdk4 (cyclin-dependent kinase 4), cdk7 (cyclin-dependent kinase 7, tfiih basal transcription factor complex kinase subunit) [339] , and amacr (alpha-methylacyl-coa racemase), an ''obscure metabolic enzyme (that has taken) centre stage'' [340] as judged by the extraordinary convergence to this biomarker in prostate. we believe that our result is an important finding. amacr was not judged of importance according to the methodology used in [332] and it was barely cited in that manuscript. here we present results, from an unifying biological and informational principle, which allows (using ref. [332] 's own data) the identification of the most central current biomarker with a truly compelling body of support in independent studies [ [490, 491] . knockdown of brca1 results in the accumulation of multinucleated cells, indicating that brca1 regulates gene expression of an orderly progression during mitosis [492] , preserving chromosomal stability [490] . brca1 showed decreased expression in a study involving immortalized prostate epithelial cells before and after their conversion to tumorigenicity [493] . lack of brca1 function may impair activation of stat3 [494] . inactivation of tp53 by somatic mutations is also associated to the panel of disruptions which are common for this ''tumor suppressor'' [113] . one possible mechanism for gene silencing is cpg island methylation. rabiau et al.show in [495] that brca1, rassf1, gstp1 and ephb2 promoter methylation is common in prostate biopsy samples. mannicia et al. suggest that the mitochondrial localization of brca1 proteins may be a significant factor in regulating the mitochondrial dna damage [5] . sfpq -(polypyrimidine tract-binding protein-associated splicing factor). the most positively correlated gene with the loss of normalized shannon entropy is sfpq/psf (polypyrimidine tract-binding protein-associated splicing factor) (spearman correlation of 0.7902), a multifaceted nuclear factor [496, 497] which is also a putative regulator of growth factor-stimulated gene expression [498] . this is extremely interesting as it has been recently shown that the ar/psf complex interacts with human psa gene and that psf inhibits ar transcriptional activity [499] . the loss of expression of sfpq and other proteins that together regulate androgen receptor-mediated gene transcription [500] (see also [501, 502] ) may indicate they have a role not only as a biomarker of the progression and well as transitions of the disease to androgen independence. in a study of human labor, dong et al., also showed that sfpq acts as a progesterone receptor corepressor, thus putatively contributing to the functional withdrawal of progesterone [503] . we will return to this particular gene later on the 'discussion' section as new evidence of its role in nuclear organization has been documented. cd40 -(tnfrsf5, b-cell surface antigen cd40). the loss of normalized shannon entropy gives us several markers that indicate a de-differentiation from a epithelial basal phenotype and an increasing loss of control of cell cycle regulation (due to uncoordinated upregulation of cdk4, cdk7, ccng2 with their functional partners). this poses the question: what can we observe while looking at the genes that most positively correlate with the loss of normalized shannon entropy? we observe, second on the ranking of all samples, a probe for cd40 (tnfrsf5, b-cell surface antigen cd40), bx381481 with a spearman correlation of 0.7616. loss of cd40 expression has been previously reported in prostate cancer and it is the object of a study that attempts to establish dendritic cell gene therapies [504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522] . we will continue discussing cd40 in the following subsection in concert with other genes. another natural question can be asked: which is the extra information that we can obtain the by analysing the correlations with the mpr-statistical complexity in this case? as we have discussed before, and can be appreciated from figure 14 , there is a strong correlation between the mpr-statistical complexity and the value of the normalized shannon entropy. it appears in prostate cancer, as in this gene expression dataset, the reduction of entropy is not the major factor responsible for the increase in mpr-statistical complexity. again, it is perhaps better to now look at one of the multiplicative factors of the statistical complexity measure, the jensen-shannon divergence to the equiprobability distribution, as this is increasing the mpr-complexity. cd40. we present more evidence of the case of cd40 as a biomarker, since a probe for cd40 (bx381481) ranks 6 th (the spearman correlation of the probe expression with the jensen-shannon divergence from the equiprobability distribution is 20.5764). cd40 is a member of the tnf receptor superfamily. notably, in 56 out of 57 archival prostate cancer samples palmer et al. have reported no cd40 expression [518] . however, cd40 expression was present in normal prostatic acini, so they proposed that ''invasive prostate cancer is a cd40-negative tumour'' (see the previous results of moghaddami et. al. [514] ). matching our observations, they proposed that cd40 provides ''insight into progression of cancer from normal epithelium''; our proposed methodology is revealing this fact as well. depletion of cd40 in the tumour microenvironment may be central in avoiding the action of the immune system [506] , as prostate cancer induces a progressive suppression of the dendritic cell system [520] . it is perhaps a central piece which should be put together in the context of other pieces of information coming from immunotherapy [508, 512, 513, 516] and pharmacological studies [507] that warrant serious investigation towards the design of new and improved clinical studies [508, 517] . cd59 molecule, complement regulatory protein. four probes for protectin [335, 523, 524] , cd59, with spearman correlations with the jensen-shannon divergence from the equiprobable distribution, ranging from 20.61823 to 20.5089, rank between the 1 st and 39 th position (when we rank genes according to this correlation in ascending order). cd59 is an interesting gene as ''a comprehensive investigation of cd59 expression in prostate cancer has not been conducted yet'' [524] . like lmna (which is ranked third and will be discussed later) the rank of cd59/ protectin means that these genes progressively loose expression of these probes. cd59 is expressed in the prostatic epithelium [525] and in prostasomes [526] ; secretory granules which are produced, stored and released by the glandular epithelial cells of the prostate [527] . babiker et al. concluded in [335] that prostasomes (via expression cd59) contribute to the protection of malignant cells from complement attack. we now investigate if the ratio of deltacatenin to cd59 can is a more robust biomarker for non-invasive prostate cancer detection, particularly after the results presented in [528] . we also note that cd59 may be also relevant to reveal the heterogeneous nature of prostate cancer. its correlation was good, but is not lower than 20.62, which in our experience, indicates that we may be dealing with at least two types tumors in this dataset. indeed, xu et al. obtained cd59 mrna levels were determined by real-time pcr in matched (tumor/normal) microdissected tissues from 26 cases and they found that: ''high rates of cd59 expression were noted in 36% of prostate cancer cases and were significantly associated with tumor pt stage (p = 0.043), gleason grade (p = 0.013) and earlier biochemical (psa) relapse in kaplan-meier analysis (p = 0.0013). on rna level, we found an upregulation in 19.2% (five cases), although the general rate of cd59 transcript was significantly lower in tumor tissue (p = 0.03)'' [524] . they concluded that: ''cd59 protein is strongly expressed in 36% of adenocarcinomas of the prostate and and is associated with disease progression and adverse patient prognosis'' [524] . jarvis et al. have previously hypothesized that cd59 expression, in some cancer cells, may help to regulate the immunological response, protecting them from the cytolytic activity of complement [523] (see also [529, 530] ). lmna (lamin a/c). the third probe in the ranking corresponds to a lmna (lamin a/c), ay528714. mutations on lmna have been linked at 10 different human diseases [531, 532] . lmna, due to its functions, could be involved in important cell fate decisions as lamins are involved in the organization of the functional state (and position) of interphase chromosome [531] . lamins are ''scaffolders'' for the function of nuclear processes such as chromatin organization, dna replication, cellular integrity and transcription [532] . as a consequence lamins are involved in several clinical syndromes [533, 534, 535] . among the recent functions attributed to lmna is as an intrinsic modulator of ageing within adult stem cells via a mechanism where lmna act as signalling receptors in the nucleus. these observations correspond to pekovic and hutchinson who observed that dysfunction of lmna leads to inappropriate activation of self-renewal pathways and initiation of stress-induced senescense [536] . in lmna-deficient mouse embryonic fibroblasts (lmna(2/2) mefs), the loss of lmna''dramatically affects the micromechanical properties of the cytoplasm'', since ''both the elasticity (stretchiness) and the viscosity (propensity of a material to flow) of the cytoplasm in lmna(2/2) mefs are significantly reduced'' [537] . using ballistic intracellular nanorheology to evaluate the micromechanical properties of the cytoplasm of these cells, lee et al. conclude: ''together these results show that both the mechanical properties of the cytoskeleton and cytoskeleton-based processes, including cell motility, coupled mtoc and nucleus dynamics, and cell polarization, depend critically on the integrity of the nuclear lamina, which suggest the existence of a functional mechanical connection between the nucleus and the cytoskeleton. these results also suggest that cell polarization during cell migration requires tight mechanical coupling between mtoc and nucleus, which is mediated by lamin a/c'' [537] (see also [538, 539] ). in addition to these very interesting findings, a functional association of lmna and the retinoblastoma protein (prb) exists. nitta et al. have shown that prb needs to be stabilized by lmna for ink4a-mediated cell cycle arrest and that somatic mutations in lmna may also have a role in tumor progression [540] . in mammalian cells, lmna a) colocalizes with c-fos at the nuclear envelope, b) suppresses ap-1 through a direct interaction with c-fos and, in lmna-null cells perinuclear localization of c-fos is absent (but it is restored when it is overexpressed, c) lmna-null cells have enhanced proliferation [74] . these results obtained by ivorra et al. are giving the indication that of yet another mechanism of cell cycle and transcriptional control mediated by lmna [74] (see also [541] ). lmna has also been proposed as an inhibitor of adipocyte differentiation [542] . hutchingson et al. have proposed the alias of ''guardian of the soma'' for lamins a and c as they seem to have ''essential functions in protecting cells from physical damage, as well as in maintaining the function of transcription factors required for the differentiation of adult stem cells'' [543] . from our results, we can not completely establish if the downregulation of cd40 and cd59 are enough to pinpoint an impaired or abnormal immune response. if we continue the inspection of the list, the first 20 probes give us more supporting evidence. the 20 probes correspond to 13 different genes. five of these 13 genes have genome ontology information annotated as ''defense response'', the above mentioned cd59 and cd40 as well as il4r (interleukin 4 receptor, cr616481), xbp1 (x-box binding protein 1, ak093842) and hla-a (major histocompatibility complex class i hla-a29.1, bu075230). takahashi et al. [544] report an inverse correlation between xbp1 expression and histological differentiation in a series of prostate cancers without hormonal therapy, the expression of xbp1 was localized in epithelial and adenocarcinoma cells of the prostate and the majority of refractory cancer cases exhibited weak xbp1 expression), mst1/stk4 (along with mst2/stk3) act as inhibitors of endogenous akt1, a mediator of cell growth and survival [545] . we can not yet know what reason is behind their joint downregulation, but another interesting common denominator is that 12 out of 13 genes share a regulatory motif for nf-kappab (according to transfac, v$nfkb_q6_01). a putative role for nf-kappab in prostate cancer has been reported based on the observation of the centrality of nfkb on two up-and downregulated networks compairing prostate tumors and healthy tissue [546] and in a larger study by mcdonnel et al. [547] (255 core prostate cancer tissue microarrays from 47 prostatectomy specimens). several other researchers are currently investigating different roles of the nfkb family in prostate cancer [548, 549, 550, 551, 552, 553, 554, 555] and it could be a promising target for intervention [555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571 ]. if we include other genes following the ranking order, the first 38 genes in the ranking include 33 that have the regulatory motif v$nfkb_q6_01 (gather reports for this list a p-value of 0.0006). even when we double the list to the probes that correspond to the first 76 different genes recognized by gather, 58 of them have the regulatory motif v$nfkb_q6_01, with p-value = 0.003 (atp6ap2, bcat1, btg2 [572, 573, 574, 575, 576, 577, 578] , c14orf123, c18orf45, ccl2, cd302, cd40 (already discussed), cd59 (already discussed), chi3l1, col16a1, commd6, crabp2, csrp1, ctbp2, ctgf (connective tissue growth factor, [579, 580, 581, 582] ), des, dmn, dnajb1, egf, emp1, fhl2 [583, 584, 585, 586, 587, 588] , gripap1, gstm1 [589, 590] , hbegf, il4r, itga3, itga7, junb [591, 592] , kiaa0152, kiaa1191, kiaa1324, klf6, lamb2, lmna (already discussed), nfatc1, nfkb2, nudc [593] , p4hb, pdk2, pim1, pisd, pxn, rap1b, rnf40, sara1, sec61a1, sgta [594] , slc12a2, srd5a2, stat6 [595, 596] , tacstd2, tbx1, tmed3, vps39, wdfy3, xbp1 [544] , zak). this result indicates that our results support the importance of nfkappa-b and the huge amount of research effort to understand the role of the nfkappa-b activity and its potential as a target for intervention in prostate cancer (file s4). the group of 58 biomarkers contains one of particular interest, stat6. this gene is considered a survival factor in prostate cancer and a key regulator of the genetic transcriptional program responsible for progression [595] . stat6 has been recently linked to hpn as one of the most robust pair of biomarkers for prostate cancer using an integrative approach that linked several microarray datasets [596] . analysis using gather of this group reveals that six of these 58 genes are in kegg pathway path:hsa04510, focal adhesion (egf, itga3, itga7, lamb2, pxn, rap1b, p-value,0.0007) and from these there are three in pathway:hsa045122, ecm-receptor interaction (itga3, itga7, lamb2, p-value,0.005) while four of these six are also in path:hsa04810: regulation of actin cytoskeleton, (egf, itga3, itga7, pxn, p-value,0.01). lamb2. alterations of the gene profile of lamb2 and cdkn2c/p18(ink4c), a cdk4 inhibitor, have been reported on the transition from prostatic intraepithelial neoplasia (pin) to prostate cancer [597] (see also [333] ). 3). the contribution of the loss of these integrins and the subsequent derived impairment on cell adhesion has been reported in several tumours. ren et al. in [598] report that ''focal or no integrin alpha 7 eexpression in human prostate cancer and soft tissue leiomyosarcoma was associated with a reduction of metastasis-free survival (for example, for prostate cancer with focal or no expression, 5-year metastasis-free survival was 32%, 95% ci = 24.4% to 40.3%, and for prostate cancer with at least weak expression, it was 85%, 95% ci = 79% to 91%; p-value,.001)''. ''any method involving the notion of entropy, the very existence of which depends on the second law of thermodynamics, will doubtless seem to many far-fetched, and may repel beginners as obscure and difficult of comprehension.'' willard gibbs, graphical methods in the thermodynamics of fluids, (1873) the changes of the normalized shannon entropy and statistical complexity of the gene expression profile of a cancer cell are associated with the gradual deterioration of genome transcriptional information content due to the modification of its structural and functional integrity during disease progression. our results clearly suggest that we can track the cancer cell's progression by following observable changes in the shannon entropy and, in particular, by employing the jensen-shannon divergence of the gene expression profile of a sample to the normal expression profile. we have also shown if an average expression profile of some state of interest can be properly defined (i.e. distant metastasis) then the jensen-shannon divergence can help us to identify which probes best correlate with these measures resulting in useful biomarkers. before any thermodynamical consideration could be discussed, we note that there is a clear and objective informational perspective that our study delivers. in this study we have chosen to position ourselves as the 'receivers' of a 'transcriptional message'. in this experimental perspective the tumor tissue is the 'sender' (the source of information) and the high-throughput technology (gene expression microarrays in this case) can be regarded as the transmission medium (providing noise and distortion). as we explain in the 'materials and methods' section, the shannon entropy of a gene expression profile is the average expected surprisal of that profile understood as a message. the normalized shannon entropy makes this surprisal an intensive measure and the correlation of the gene expression patterns across samples with this measure can deliver useful biomarkers to track the progression of transcriptional change. after normalization, we have a measure that does not depend of the number of probes of the high-throughput technology, although, it obviously does depend on the type of probes used. we believe that the readers may have already noticed an apparent paradox. while some researchers understand cancer progression as a mechanism that increases entropy, we actually observe a reduction of normalized shannon entropy in this work. this means that our normalized average expected surprisal, as receivers of the transcriptional message, is smaller. we must then discuss the physical meaning of thermodynamic entropy, its current use in systems biology and cancer research genetics and the informational measure we use in this paper to clarify these notions in this context. in biomedical research there exists a certain consensus among cancer researchers that genetic instability or ''mutability'' is a major critical force of cancer progression, but it is not the only one to consider. it is clear that the mutational damage of key genes (like tp53, tert, brca1, rb1, etc.), and the collective damage inflicted on key dna repair mechanisms (like nucleotide-excision repair and base-excision repair) collaborate for an increasing acceleration of the number of genomic changes. sub-microscopic alterations of the genome accumulate in cancer progression in an irreversible way and ''are compounded by the widespread scrambling of the chromosome structure, and thus the karyotype, found in cells from the great majority of solid tumours'' [599] . in weinberg's own words [599] : ''we learned that this chromosomal chaos also contributes this progression forward''. this ''chromosomal chaos'' [600] or ''cancer as a chromosomal disease'' perspective is viewed by some researchers not as just a side consequence of mutational damage, but as the main core theme to understand a number of unexplained issues in cancer progression. ''in sum, cancer is caused by chromosomal disorganization, which increases karyotypic entropy'' [601] . regarding the cancer types studied in this paper, one particular ''measure of disorder of a system'', aneuploidy, has been observed in poorly-differentiated prostate cancer cells and it is often associated with a more agreessive phenotype [602, 603] , increased psa levels [604, 605] , and correlate with gleason score [606, 607, 608] . gene fusions and chromosomal rearrangements are other source of increase in the ''disorder'' of the genome organization and they are increasingly being recognized as a major player in prostate cancer progression [609] . the increase in ''karyotypic complexity'' and ''extended aneuploidy and heteroploidy'' may be already enough to develop a malignant melanoma phenotype, as the report of gagos et al. indicate [610] . the observed finding of aneuploidy in melanoma (also including uveal melanoma) is also increasingly important due to a number of different independent observations [247, 611, 612, 613, 614, 615, 616, 617, 618] . it is in this context that the word 'entropy' has been used. the magnitude of the ''chromosomal chaos'' is also evident from comparative genomic hybridization (cgh) studies which show significant variations in the copy number of individual chromosomal segments. 'chaos' is really a very appropriate word to describe what we observe from cgh data. the genomic changes are not distributed uniformly at random. 'chaos' has been described by some researchers as ''a kind of order without any periodicity''. some common changes seem to consistently appear in several independently arising tumours of the same type, and sometimes the researchers suggest common links [619] . our work has addressed, in part, this question: ''can we quantify the chaos observed in the genome from the increasingly available transcriptional data and relate it to tumour progression?'' if no commonalities were observed, we would not have found interesting biomarkers that seem that strongly correlate with the divergences from normal tissue types. we know from our results that these commonalities do occur. we need to go back to basics to explain these evolving concepts and resolve this apparent paradox. the phrase ''karyotypic entropy'' has been used in the past to define what is actually a divergence from the normal chromosome structure and it genomic organization. this denomination has also been employed by several authors, notably [601] , but it has also been used in at least two other publications [620, 621] . these works have in common the use of this term to refer to a ''disorder'', fuelled by the undergraduate textbooks indoctrination of associating increase of entropy in natural spontaneous processes with the increase of ''observed disorder'' in the system. we propose that the use of a natural measure of divergence, the jensen-shannon divergence, could not only be a more formal, but also more appropriate modelling approach. as such, we propose to introduce the term 'karyotypic divergence' or 'karyotypic jensen-shannon divergence' to replace this concept and to avoid a subjective approach. why is it the case that we observe the normalized shannon entropy of the transcriptional profile decreasing with cancer progression when intuitively our average expected surprisal (shannon entropy) should increase with progression? arieh ben-naim in his recent book ''a farewell to entropy: statistical thermodynamics based on information'' [622] comments:''it is interesting to note that landsberg (1978) not only contended that disorder is an ill-defined concept, but actually made the assertion that 'it is reasonable to expect 'disorder' to be an intensive variable'''. ben-naim also states: ''in my view, it does not make any difference if you refer to information or to disorder, as subjective or objective. what matters is that order and disorder are not well-defined scientific concepts. on the other hand, information is a well-defined scientific quantity, as much as a point or a line are scientific in geometry, or mass or charge of a particle are scientific in physics.'' however, in a manuscript entitled ''can entropy and 'order' increase together ?'' landberg defines (in an attempt to decouple the notions of order and entropy), for a thermodynamical system that can be on n states the 'disorder' d(n) to be the normalized entropy (which is a function of n) divided by boltzmann's constant [623] . 'disorder' then is an intensive magnitude bounded by 0 and 1, and 'order' is defined as 1-d(n) . while landberg's decoupling argument between order and entropy [623] may still be controversial in physics, the question is pertinent for our apparent paradox (the question that motivates this subsection). borrowing from the title of his paper we could now state the central question as ''can shannon entropy increase while the normalized shannon entropy decrease?'' the solution of this apparent paradox is a trick of escapologism, perhaps also paralleled by what a cancer cell may be experiencing (or ''reacting'' in response to increased sources of stresses), and it is worth discussing in this context. let h[x] be shannon entropy for an ensamble x with n different values. we will now assume, and here is the trick, that n is not a constant, but a function of time n(t). let d(x (n(t) )) be the normalized shannon entropy. by definition d(x(n(t))) = h(x(n(t)))/log 2 (n(t)). then, just by taking the time derivatives it can be shown that the time variation of d(x(n(t))) can be negative, although the time rate of h[x] can be positive. where k is a constant. the escape to our paradox is ''achieved'' via making explicit the time variability of n(t). landberg explicitly mentions that biological systems are examples where growth processes increase n(t), and perhaps the increased diversity in the transcriptome of a cancer cell during progression is one of such examples. this discussion somehow resolves the apparent disassociations due to language barriers that may exist between the different disciplines (physics, information theory, molecular biology and oncology). a biologist may regard a cancer cell as an entity that, during progression, may ''spread'' its transcriptomic profile, including the generation of a large number of novel molecular species (due to adquired characteristics during its ''devolution'' from the normal type). in our informational perspective, this would be analogous to a situation in which the sender of a message, after some time, decides to increase the size of the alphabet of transmitted symbols. clearly, it is intuitive to think that the receiver would be in a situation of increased shannon entropy. however, if the receiver is not aware of the new symbols (or is not able to detect them) and some of the symbols of the previous alphabet are no longer used, the receiver would now perceive a reduction of normalized shannon entropy, observing an increasing order. we now borrow an illustrative example from landberg [623] , but we add a twist to this argument for the purpose of illustrating this discussion. suppose we have a sender transmitting only two possible symbols (n = 2), and we will assume that we have the same probability, let's denote this as (1/2, 1/2). then the average expected surprisal (shannon entropy), is h(x) = 1, and the normalized shannon entropy is also equal to one. assume now that now our sender starts to transmit using another symbol, so that we now have theoretical probabilities of (0.5, 0.25, 0.25). then n = 3, and the average expected surprisal increases to h(x9) = 1.5 the normalized shannon entropy is now 1.5/log 2 (3) = 0.946â�¦ (a reduction). this 'third symbol' could actually represent a new ''molecular species'' or a protein isoform that would not be normally expressed in that tissue type [624] , or even something entirely new, product of a mutational/deletional event. if our hypothetical high-throughput technology can only be detecting the first two symbols, and following the conventions we established in the 'materials and methods' section, we would be ''observing'' frequencies of (2/3,1/ 3) since the other events would not be detected with our equipment. as a consequence, the both the log 2 (2) = 1, shannon entropy and the normalized shannon entropy are both reduced to 0.918293. obviously, we can not count what we can not observe. as a consequence, a degenerating transcriptional profile that produces novel molecular species, and at the same time reduces those which we can not measure with a particular technology, would look increasingly more ordered. we envision that physicists may find here a fertile ground to explore new ideas and attempt novel mathematical formalisms for cancer progression from the realm of finite-state thermodynamics [625] and in particular endorevesible processes [626] and endoreversible thermodynamics [627] . some molecular alterations would then be part of the set of revesible processes that could occur in a cancer cell, while other processes like aneuploidy or gene fusions could be truly ''irreversible genetic switches'' associated with cancer progression [628] . if we assume that the process is slow (i.e. the times required for significant variations of the transcriptome's profile is large in comparison with the cell's processes time scales), and follwing the results of spirkl and reis [626] , it may be possible that we have a constant entropy production rate exists during cancer progression leading to hauptmann's ''entropic devolution'' [629] . hauptmann sees a malignant tumour as ''a dissipative structure arising within the thermodynamical open system of the human body'' that starts when ''a localized surplus of energy exists and there is no possibility to export entropy. an energetic overload in most malignant cells is indicated by their abnormally high phosphorylation state.'' his perspective, preceeded in part by dimitrov [630] , klimek [631, 632] and marinescu and viculetz [633] might then fit well an endoreversible thermodynamic formalism. hauptmann says in [629] ''i believe that cancer is a special kind of adaptation to energetic overload, characterized by multiplication and mutation of genomic dna (generation of new biomolecules which enhance the probability of survival under harmful conditions), and by chiral alterations (reduction of entropy by entrapping energy) leading to abnormal configurated biomolecules. in this regard the genetic alterations are probably secondary changes. cancer serves to dissipate energy in a type of developmental process but one in which the results are harmful to the whole organism: an entropic devolution.'' this thermodynamical perspective is now worth exploring and we will discuss it in this context. assuming that a cancer cell is in a state of ''energy overload'', without ''the possibility of exporting entropy'', could it lead to some type of ''genetic alterations''? which key mechanisms might be impaired? what consequences is this ''system'' delivering? could this be another hallmark for oncosystems indentification? in 1871, in this book called ''theory of heat'', maxwell speculated the idea of ''a being, who can see the individual molecules'' and who has enough reactive intelligence to open and close a unique small hole existing between two communicating vessels (called 'a' and 'b'). an ideal gas filled both vessels, so that starting at uniform temperature the intelligent being could observe the molecules and close and open the hole accordingly to a mission: ''to allow only the swifter molecules to pass from a to b, and only the slower ones pass from b to a.'' the being, ''without expenditure of work raise the temperature of b and lower that of a in contradiction to the second law of thermodynamics.'' the ability of the ''being'' to use observable information about the system to lower the thermodynamical entropy has motivated many articles in physics and fuelled the imagination of many since it was originally introduced by mawell, and named as ''demon'' by thomson three years later [622] . an excellent collection of articles until 1990 [634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644] was edited by leff and rex [645] . the maxwell ''demon'', far from being ''exorcised'' from physics, still inspires interesting new perspectives [634,635,636,637,638,639,640,641,642,643,644,646,647 ]. in a letter to peter guthrie tait, maxwell writes about the ''demons'': ''is the production of an inequality of temperature their only occupation? no, for less intelligent demons can produce a difference in pressure as well as temperature by merely allowing all particles going in one direction while stopping all those going the other way. this reduces the demon to a valve. as such value him. call him no more a demon but a valve like that of the hydraulic ram, suppose.'' (from [645] , p. 6). maxwell gives again here a sign of his brilliant mind, ''degrading'' the demon to a valve, but also offering an inspiring perspective to oncosystems research. which types of mechanisms exist in biological systems, and particularly in individual cells, to control these differential values in key parameters? could changes of key physical parameters for metabolic processes of the cytoplasm and cell's organelles like temperature, volume, ph or electrochemical potentials be also implicated in cancer progression? the influence of temperature may be giving an interesting working hypothesis for further research. what are the consequences if cancer cells are a different type of open system which also operates at a different temperature than a normal cell? butler et al. have studied p53 and they argue that at temperatures above 37 degrees centigrades wild-type p53 spontaneously loses dna binding activity. while folding kinetics do not show important changes in a range from 5 to 35 degrees c, the unfolding rates accelerate 10,000-fold. this leads to a somewhat unexpected mechanism of p53 inactivation. it could be the case that a fraction of p53 molecules become trapped in misfolded conformations with each folding-unfolding cycle due to the increased frequency of cycling. the occurrence of misfolded p53 proteins can lead to aggregation and subsequent ubiquitination in the cell, leading to p53 inactivation [648, 649] . if a key ''guardian of the genome integrity'' [650, 651] and its remarkable conformational flexibility [652] is challenged by an increase of temperature [653] , its role in genotoxic damage and adaptive response (like that of the skin to uvb damage [654] ) may be impaired. the same may occur for other members of the dna damage response. an increment in temperature has already been linked to skin carcinogenesis. boukamp et al. report in that [655] ''exposure of immortal human hacat skin keratinocytes (possessing uv-type p53 mutations) to 40 degrees c reproducibly resulted in tumorigenic conversion and tumorigenicity was stably maintained after recultivation of the tumors.'' on the other hand, natural gradients on physical biochemical properties can also be challenged in a cancer cell. this in turn derives in metabolic processes running under abnormal parametric circumstances. it is well-known that compartimentalization, in biological systems, naturally require the existence of mechanisms that would keep some key state variables relatively constant, or within bounds, for normal operation of the metabolic processes. one example is very illustrative and a case in point. instead of demons, holes, or valves, the cell requires pores in its membranes to allow osmotic regulatory processes, yet it should preclude the conduction of protons. this is a nanotechnological design problem not faced by maxwell, but certainly solved by biological systems without the need of an ''intelligent being'' as mawell cleverly pointed to tait in his letter. this discussion brings us to one of the gene families we have already discussed in this paper, the aquaporins [184, 656, 657, 658, 659, 660, 661] . they are considered the primary water channels of cell membranes [662, 663, 664, 665] . the specific functions of each member of this family are now being slowly mapped by several research labs around the world [666] . their clinical role in cancer [667, 668, 669, 670, 671, 672, 673, 674, 675] ,obesity [676] , malaria [677, 678] and other diseases is emerging [657, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689] . in [690] , our group observed the dowregulation of aqp3 in all melanoma cell lines studied of the nci-60 dataset of ross et al.; this dowregulation was also observed for the cns and renal cell lines. aqp3 was relatively upregulated for leukaemia and colon cell-lines (we refer the reader to the supplementary material of [690] for details). inhibition of aqp3 in prostate cancer cells was already proposed as a mechanism that increases the sensitivity to cryotherapy treatment [691] . the aquaporins are not ''an intelligent being'' in any real sense, yet they are so formidable selective that they could easily parallel maxwell demon's efficiency in creating the right conditions for the cell. wu et al. give us some clues on the role of point mutations in the aqp1 and how their effective electrostatic proton barrier can be impaired [692] . the elicitation of the detailed mechanistic explanation of this extraordinary selectivity is under intense investigation with a number of techniques, including sophisticated molecular dyanamics simulations, for an overview of this field see [665, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708] . one less known feature of aquaporins is that they may not only channel water, but also carbon dioxide and ammonia [709, 710, 711] , glycerol [712] and urea and other small solutes [713] and, very relevant for cancer research, hydrogen peroxide [188] . at least two of members of this family have been observed in the inner mitochondrial membrane in different tissues. this in turn may indicate mitochondrial roles for aquapotins in osmotic swelling induced by apoptotic stimuli [714] . could it be possible that we can track cancer progression by looking at some of these ''maxwell demons''? we have seen in figure 10 , that aqp3 has a reduced expression with increased progression in our melanoma dataset. cao et al., reported that ultraviolet radiation induced aqp3 down-regulation in human karatinocytes; thus aqp3 has become a strong and plausible link between uv radiation, skin dehydration [186, 715] and photoaging [189] . this may indicate an impared function on skin hydration [184, 185, 716, 717, 718, 719] . the expression of aqp3, as well as aqp1, aqp5, and aqp9 seem to be correlated with melanoma progression, indicating a common pattern of downregulation from the higher values in normal skin and benign nevi (see figure 17 ). does a similar pattern of aquaporin downregulation exist in prostate cancer? wang et al. have looked at the expression and localization of aqp3 in human prostate using cell lines as well as patient samples. they have observed aqp3 mrna ''in both normal and cancerous epithelia of human prostate tissues, but not in the mesenchyme. in the normal epithelia of the prostate, localization was limited to cell membranes, particularly the basolateral membranes. however, the expression of aqp3 protein in the cancer epithelia was not observed on the cell membranes.'' this finding seems to implicate the subcellular localization of aqp3 as a possible indicator of a transition to a more malignant phenotype. lapointe's dataset allows us to see the downregulation of aqp3 and aqp1. a large subgroup of primary prostate tumors has reduced levels of aqp3 and aqp1 as most of the lymph node metastasis samples [ figure 18 ]. one critique that we are aware we could receive is that the current manuscript presents a novel methodology and an underlying unifying theory based on retrodictions or postdictions. indeed we have shown that the use of the normalized shannon entropy and the information theory quantifiers (the m-complexities and the jensen-shannon divergence) allow to monitor cancer progression and to identify the best biomarkers that correlate with the transcriptomic changes. our approach works in a retrodiction way in that it looks at data already obtained by other studies, but gives a unifying framework to track cancer progression. for instance, on true et al's dataset, our unifying hallmark of cancer gives not only maoa, which was already identified in the original publication, but also amacr, cd40, cdk4, etc. are very important biomarkers for prostate cancer. analogously, the identification of klk3/psa in lapointe's dataset is another important retrodiction which shows the power of the method. in some sense our approach also works in a postdiction way, as it helps to evaluate the speculation that cancer cells have ''an entropic devolution''. our results show that the variations of normalized shannon entropy and jensen-shannon divergences indeed give measurable changes, and that these changes are related to important biomarkers in the two types of cancer studied in this work. in addition, we remark that we are literally making hundreds, or even thousands of predictions. the results in the 'supplementary material' provide this information for the detailed scrutiny of our peers. we believe that other probes with gene expression patterns in high correlation with the probes discussed in this paper, and perhaps less studied by immunohistochemistry and other methods in the two cancer types studied here, are worth exploring as a group of biomarkers. these predictions can be tested with further studies on staging and patient stratification. a very recent study by ballal et al. have linked brca1 to telomere length and maintenance and its loss from the telomere in response to dna damage [720] (see also [721] ). we have previously mentioned that brca1 is a conspiquous biomarker arising from the analysis of true et al.'s dataset using our methods. we found this to correlate with a preivous study that showed that brca1 has a reduced expression in immortalized prostate epithelial cells before and after their conversion to tumorigenicity [493] . we also mentioned that the knockdown of brca1 leads to anaccumulation of multinucleated cells [492] , preserving chromosomal stability [490] . ballal et al. telomeric chip assays to detect brca1 at the telomere and reported time-dependent loss of brca1 from the telomere following dna damage. due to the role of telomeres in maintaining chromosomal stability [722] and the inverse correlation of telomere length and divergent karyotypes in prostate cancer cell lines [723, 724] (as well as the recognized role of telomere dysfunction in the induction of apoptosis or senescence in vivo [725, 726, 727, 728, 729, 730] , increase of mutation rates [731] , dna fragmentation [732] , and their relation with dna damage signalling [733] ), we checked for other probes of genes involved in telomeric function. from those which we were able to identify in true et al's dataset, we have found a strong high correlation of the expression of brca1 with terf2/trf2 (telomeric repeat binding factor 2) [734] and a negative correlation with the expression pattern of terf2ip (telomeric repeat binding factor 2, interacting protein) [ figure 19 ]. finally, one particular type of probes has also caught our attention, and we would like to refer to them before concluding this section. with the denomination of 'non-coding rna' we identify those rna molecules which are functional but that are not translated into proteins. many microarray chips contain probes that are annotated as 'non-protein coding', indicating that there might be some valuable expression data that we can also mine for information. we note that our method, although employing transcriptomic data, does not limit its application to proteincoding information, and that the combined use of protein-coding and non-coding protein probe expression would allow a more comprehensive view of the transcriptional state of the cell. among non-protein coding, micrornas [735] are gaining acceptance as key players in several cancers [736, 737, 738] (including prostate cancer [739, 740] ), but the so-called ''long noncoding rnas'' [741] are also gaining a place in the scenario of cancer biomarkers (see [742] , and [743, 744, 745] ). we thus turned our attention to these probes that have been annotated as ''nonprotein coding'' and we highlight some of them that have very high correlation values with the normalized shannon entropy in true et al's prostate cancer dataset. in particular, the probes for malat1/ malat-1 [742, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758] have a very conspiquous position (see figure 20) . they located very closely to other protein coding biomarkers that have also lost expression and have been discussed in this work like sfpq, cd40, brca1, and tp53 (see figure 16 ). malat1 has been recently pointed as a biomarker in primary human lobular breast cancer as a result of an analysis of over 132,000 roche 454 highconfidence deep sequencing reads [749] . an international team, searching on thousands of novel non-coding transcripts of the breast cancer transcriptome, has been able to identify more than three hundred reads corresponding to malat1 [749] . this is a noncoding rna which was identified in 2003 in non-small cell lung cancer, was shown to be highly expressed (relative to gapdh) in lung, pancreas and prostate, but not in other tissues including muscle, skin, stomach, bone marrow, saliva, thyroid and adrenal glands, uterus and fetal liver [758] . malat-1, also known as neat2, is considered to be ''extraordinarily conseved for a noncoding rna, more so than even xist'' [754] . our results indicate that the reduction of expression of some non-coding rnas, in particular of malat-1, and snora60 with respect to their normal expression in prostate, as well as the upregulation of snhg8 and snhg1 should be monitored as useful biomarkers to track disease progression. we will now address another non-coding rna called neat1 which, like neat2, is also conserved in the mammalian lingeage. before we move onto neat1, we will first recall a previous result. we have noted before the conspiquous position of sfpq/psf (polypyrimidine tract-binding protein-associated splicing factor) in figure 16 . the expression of a probe for spqf has the highest correlation with the values of the normalized shannon entropy. we highlighted before that sfpq/psf is a putative regulator of growth factor-stimulated gene expression [498] . the loss of sfpq expression during the progression of prostate cancer may be an important key to understand this disease or one of its subtypes. we have also mentioned that the ar/psf complex interacts with the psa gene (perhaps the most well-established prostate cancer biomarker) and that sfpq/psf inhibits ar transcriptional activity [499] . kuwahara et al. showed that sfpq together with nono (non-pou-domain-containing, octamer binding protein) and pspc1 (paraspeckle protein 1 alpha isoform, formerly known as psp1) are expressed in mouse sertoli cells of the testis and form complexes that function as coregulators of androgen receptormediated transcription [500] . while new research results [759] link sfpq and nono/p54nrb with the rad51 family of proteins (largely regarded as another key protector of chromosome integrity as being involved in homologous recombination dna repair), it is perhaps sfpq and nono's co-localization in paraspeckles that make this group also remarkable [760] . paraspeckles [760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777] are a novel nuclear compartment, of approximately 0.2-1 mm in size, discovered in 2002, by fox et al. in dundee scotland, following the identification of the protein pspc1 (af448795) in the nucleolar proteomics project at lamond's lab which is described well by fox et al. [777] . three years later, fox, bond and lamond showed that nono and pspc1 form a heterodimer that localizes to paraspeckles in an rna-dependent manner [773] . paraspeckles are dynamic structures, observed in numbers that vary between 10 and 20, that seem to control gene expression via retention of rna in the nucleus [772] . a long noncoding rna called neat1/men epsilon/beta [754, 760, 762, 764, 778] , that colocalizes with paraspeckles, seems to be integral to their structure. depletion of neat1 erradicates paraspeckles and a biochemical analysis by clemson et al indicates that the neat1 binds with paraspeckle proteins sfpq/psf, p54nrb/nono and pspc1. neat1 is also known as tncrna (trophoblast-derived noncoding rna) [754, 779, 780, 781, 782, 783, 784, 785, 786] and probes for tncrna exist on this dataset, we have observed in true et al.'s dataset that there exists a high correlation between the normalized shannon entropy with the expression of sfpq/psf, p54nrb/nono, and tncrna. overall, this implies that the disruption of the function of the paraspeckles is correlated with the increasing signs of deterioration of normal transcriptomic state of the cells. while a causal relationship still needs to be proved, we admire the mathematical elegance of the normalized shannon entropy of the samples, a global measure of the average expected surprisal of the transcriptome, which in turn has lead us to consider the dysfunction of the smallest nuclear body as a putative biomarker of disease progression. the role of sfpq/psf in the control of tumorigenesis is under investigation [787] and the information coming from these studies would need to be integrated with their role, together with p54nrb/nono and tncrna, in paraspeckles if we want to achieve a better understanding of these mechanisms. in this contribution we have shown that for the melanoma and prostate cancer datasets studied, the quantitative changes of information theory measures, normalized shannon entropy, jensenfigure 19 . the stacked average gene expression of probes corresponding to brca1 and terf2 (telomeric repeat binding factor 2) in true et al's prostate cancer dataset. the first group of samples (1 to 9 in green) correspond to gleason 3 pattern, indicating that most of the samples in this group have no significantly reduced expression of this pair of genes. the second group of columns (10 to 21 in yellow) correspond to gleason 4 patterns and the last 8 columns (22 to 29 in red) correspond to gleason 5 samples. a very recent study by ballal et al. have linked brca1, to telomere length and maintenance and its loss from the telomere in response to dna damage [720] (see also [721] ). there is an increasing trend of dowregulation, so it would be interesting to evaluate if indeed this pair of proteins could be an early marker of dowregulation useful to evaluate samples with gleason pattern 2, or if may constitute a biomarker useful to distinguish a prostate cancer subtype. doi:10.1371/journal.pone.0012262.g019 shannon divergence and the novel statistical complexity quantifiers defined here are in high correlation with gene expression changes of well-established biomarkers associated to cancer progression. in addition, variations of the basic technique (i.e. a modified form of statistical complexity) which allows us to better understand the phenotypic changes observed in these samples which are associated with the progression and the transitions of the gene expression profiles. for instance, in a properly defined statistical complexity vs. entropy plane, on a melanoma dataset first studied in ref. [110] , samples appear in well differentiated ''clusters''. these clusters correlate well with the phonotypic characteristics of normal skin, nevi, primary and metastatic melanoma. in this ''complexity vs. entropy'' plane, primary melanomas samples appear ''bridging'' benign nevi and metastatic melanoma samples. our results may also suggest that the evolution of metastatic melanoma leads to at least two different subtypes. the normalized shannon entropy of a transcriptional sample profile is calculated associating the measured expression values of a gene with the relatively probability of being expressed. we have observed that, in general, the transcriptomes of tumour progressing cells tend to have lower values of normalized shannon entropy than normal ones. given a population of normal cells of a given tissue type it is then possible to compute useful measure of divergence of cancer cell profiles from the normal expression average profile, in terms of information theory quantifiers, the shannon eveness normalized entropy and generalized statistical complexity [788, 789, 790] . in addition, our observation of the correlation of the statistical complexity of tumours with its natural progression allows an unprecedented way of finding biomarkers that links with the gradual deterioration of the genome integrity. the proposed methodology uncovered, for the first time, evidence of the putative role of impared centrosome cohesion in melanoma progression. statistical complexity has then been able to pinpoint otherwise unrecognized biomarkers in concert with existing ones, reinforcing the view that ''chromosomal chaos'' and ''cancer as a chromosomal disease'' can be a useful guiding principle to understand the molecular biology of cancer and uncover the timeline of its progression. this is a powerful method to uncover ''oncosystems'' instead of ''oncogenes''. ''oncosystems'' are a highly differentially disregulated set of genes that, if linked with the molecular ''hallmarks of cancer'' described in the introduction, and existing databases with putative common functional genomic annotations, can help to understand the biological progression pathways that drive the disease. on one of the prostate cancer dataset studied (obtained from a previous published study, [44] ), we observe a gradual pattern of reduction of normalized shannon entropy from three well characterized tissue types: normal prostate, primary prostate tumours and lymph node metastases. on a different dataset on prostate cancer (from ref [332] ), we observe that a group of samples having gleason figure 16 is that we have now highlighted the position of s ome probes which have been annotated as corresponding to ''non-coding rnas''. in particular, we highlight those of malat1 (metastasis associated lung adenocarcinoma transcript 1, (non-protein coding)), snora60 (small nucleolar rna, h/aca box 60); both increasingly downregulated, snhg1 (small nucleolar rna host gene 1 (non-protein coding)) and snhg8 (small nucleolar rna host gene 8 (non-protein coding)). the probes for malat1/malat-1 [742, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758] have a very conspiquous position, which we could judge a priori to be equivalent in relevance to those of the previously discussed roles of sfpq, cd40, brca1, and tp53 (see figure 16 ). malat1 has been recently pointed as a biomarker in primary human lobular breast cancer as a result of an analysis of over 132,000 roche 454 high-confidence deep sequencing reads. within the thousands of novel non-coding transcripts of the breast cancer transcriptome, guffanti al., identified more than three hundred reads corresponding to malat1 [749] . this non-coding rna, first identified in 2003 in non-small cell lung cancer, was shown to be highly expressed (relative to gapdh) in lung, pancreas and prostate, but not in other tissues including muscle, skin, stomach, bone marrow, saliva, thyroid and adrenal glands, uterus and fetal liver (see figure four of ref. [758] ). our results indicate that the reduction of expression of some non-coding rnas, in particular of malat-1, and snora60 with respect to their normal expression in prostate, as well as the upregulation of snhg8 and snhg1 should be monitored as useful biomarkers to track disease staging and progression to a more malignant phenotype. interestingly enough, a study published in 2006 by nadminty et al. has shown that klk3/psa modulates several genes, reporting a 16.5 fold downregulation of malat1 [810] . while these results have been obtained using the human osteosarcoma cell line saos-2, our results indicate that malat1 expression in the normal prostate and in cancer cells could also be considered as a relevant biomarkers to be tested in the future. doi:10.1371/journal.pone.0012262.g020 patterns 4 and 5 (two patterns which are typically associated to an aggressive phenotype) have lower normalized shannon entropy values than a subset of gleason pattern 3 (a pattern which is normally associated to a less aggressive phenotype but which nevertheless is still of clinical concern). however, a group of samples having gleason patterns 3, 4, and 5 is revealed; this mixed cluster has a mid-range entropy. this is an interesting fact which correlates with the limitations observed in ref. [332] . we note the authors' comment: ''we were unable to identify a cohort of genes that could distinguish between pattern 4 and 5 cancers with sufficiently high accuracy to be useful, suggesting a high degree of similarity between these cancer histologies or substantial molecular heterogeneity in one or both of these groups.'' our results provide a conciliatory middle ground that explains the perceived clinical usefulness of gleason pattern classification, widely used around the world, while at the same time reveals the reason for the difficulties of obtaining a good transcriptional signature for the other two patterns [791] . we have seen, through a detailed discussion of several biomarkers in three different datasets, that the variation of the gene expression distributional profile can be characterized via information theory quantifiers. our study also showed that current established biomarkers of the two diseases studied seem to correlate with those that best co-variate with these quantifiers. for instance, amacr, in our second prostate cancer dataset studied, naturally appears as one of the most correlated genes (in both the pearson and the spearman sense) with the pattern of variation of entropy of the samples. together with maoa, which is the highlighted gene in true et al.'s [332] original publication, amacr is now being recognized as one of the best biomarkers in primary prostate cancer with approximately 180 publications dedicated to it in the past five years. we have also shown that many gene probes that best correlate with the divergence of the normal tissue profile have been identified as useful biomarkers (via other accepted validation methods). this said, the use of other sources of information, like pathway or gene ontology databases has lead as to the identification of other cell processes that may be altered. we have presented a unifying hallmark of cancer, the cancer cell's transcriptome changes its normalized shannon entropy (as measured by high-througput technologies), while it increments its physical entropy (via creation of states we might not measure with our devices). this hallmark allows, via the use of the jensen-shannon divergence, to identify the arrow of time of the process, and helps to map the phenotypical and molecular hallmarks of cancer as major converging trends of the transcriptome. the methodology has produced remarkable postdictions and retrodictions that show that it can predictively guide biomarker discovery. we refer the reader to the original publications for details of methods for data collection, but we highlight here some aspects that are important to understand the data generation process for the purpose of our analysis. samples were obtrained from radical prostatectomy surgical procedures. samples are labelled as ''tumors'' if they contain at least 90% of cancerous epithelial cells, and they were considered as ''non-tumor'' if they contain no tumor epithelium and are from the noncancerous region of the prostate. the later samples were labelled ''normals'' although the authors alert that some may contain dysplasia. in this dataset, lapointe et al. have performed a gene expression profiling by using cdna microarrays containing 26,260 different human genes (unigene clusters). using 50 mg of total rna from prostate samples cy5-labeled cdna was prepared and cy3-labeled cdna used 1.5 mg of mrna common reference, pooled from 11 human cell lines (see ref. [792] ). the fluorescence ratios were subsequently normalized by mean centering genes for each array, a relatively standard procedure. in addition, to minimize potential print run specific bias, lapointe et al. report that ratios were then mean centered for each gene across all arrays according to ref. [793] . we have only used the genes that the authors report in their first figure, 5,153 genes that have been well measured and have significan variation in some of the samples. for the other details of their matrials and methods we refer the readers to the supporting notes and the materials and methods section of their original publication [44] . samples were obtained from nevus volunteers and melanoma patients and only those samples that have more than 90% of tumor cells were profiled. the 20,862 cdnas used (research genetics, huntsville, al) represent 19,740 independent loci. (unigene build 166).median of ratio values from the experiment were subjected to linear normalization in nomad (which can be accessed at http://derisilab.ucsf.edu), log-transformed (base 2), and filtered for genes where data were present in 80% of experiments, and where the absolute value of at least one measurement was .1. in this dataset, samples have information of 15,488 spots per array, with a total of 7,700 unique cdnas represented. the samples were obtained from frozen tissue blocks from 29 radical prostatectomies accessioned and selected to represent gleason grades 3, 4, and 5. the samples are ''treatment naã¯ve'', meaning that they were also selected such that their gene expression profile is also and the absence of any bias that the treatment before prostatectomy. the frozen sections (8 mm) were cut from optimal cutting temperature medium blocks and immediately fixed in cold 95% ethanol. around 5,000 epithelial cells from both histologically benign glands and cancer glands were separately laser-capture microdissected (lcm). the authors of the study have also been very careful to include only one gleason pattern in each laser-captured cancer sample, following a process in which the patterns were assessed independently by two investigators.the matched benign epithelium was captured for each cancer sample for a total of 121 samples. an important characteristic of this dataset is the normalization procedure. for each spot and in each channel (cy3 and cy5), true et al. substracted the median background intensity from the median foreground intensity, and subsequently the log ratios of cancer expression to benign expression were computed. these ratios were obtained by first dividing the background-subtracted intensities (prostate cancer/benign) and then taking the logarithm base 2. in the case that the median background intensity was greater than the median foreground intensity, the spot was considered missing. we refer to the original publication for the other aspects of imputation, spot quality and filtering, but, like in lapointe et al's study, they also filter to keep informative (expression ratios of benign versus cancer should at least be 1.5-fold or greater in at least half of one of the gleason groups as one of the selection criteria). shannon entropy. in many circumstances, experimental measurements are associated with the accumulation of individual results which, ultimately, qualitatively and quantitatively characterized our experimental observations. the presence (or absence) of a particular result of an individual experimental measure is called an event. an event which can take one of several possible values is called a random variable. analogously, a random event is an event that can either fail to happen, or happens, as a result of an experiment. an event is certain if it can not fail to happen and it is said to be impossible if it can never happen. following andreyev [794] , we will define the probability p(x) of an event x, as the theoretical frequency of the event x about which the actual frequency occurrence of the event shows a tendency to fluctuate as the experiment is repeated many times. the shannon information content of an event x (or the surprisal of an event x, [795] ), is defined as h(x)~log 2 1 p(x) following mckay [796] , an ensamble x is a triple (x,a x ,p x ), where x is the value of a random variable, which takes on one of a set of possible values, a x~f a 1 ,a 2 ,:::,a i ,:::,a n g, having probabilities p x~f p 1 ,p 2 ,:::,p n g, with p(x~a i )~p i , p i â§0 and x a i [a x p(x~a i )~1. the shannon entropy of an ensemble x (also known as the uncertainty of x), denoted as h[x], is defined to be the average shannon information content. it is the average expected surprisal for an infinitely long series of experiments. we use the theoretical frequencies to compute this average, and then we have suppose that we have a fair dice, the theoretical frequency of an event 'the dice shows a three' is 1/6, (if the dice is assumed fair, the theoretical frequency is the same for any number from 1 to 6). in that case a hypothetical experimentalist guessing will have an average expected surprise of h[x] = log 2 (6) . we note the two natural bounds that the entropy can have. the shannon entropy of an ensemble x is always greater or equal to zero. it can only be zero if p(x~a i )~1 for only one of the n elements of a x~f a 1 ,a 2 ,:::,a i ,:::,a n g. on the other hand, the shannon entropy is maximized in the case that p(x~a i )~1=n. this is the so-called ''equiprobable distribution'', a uniform probability distribution over the finite set. transcriptional shannon entropy. let f (j) i the expression value of probe i (i = 1,â�¦, n) on sample j (j = 1, â�¦, m). for each sample j we first normalize the expression values. we interpret them as the theoretical frequency of a single hybridization event. we then define a probability distribution function (pdf) over a finite set as: the uniform (equiprobably) distribution is defined as i let h e~h â½p e ~log 2 n, then in this paper we always use the normalized shannon entropy, defined as: i h e , j~1, . . . ,m the jensen-shannon divergence and the statistical complexity measures given a probability distribution function over a discrete finite set, is then straightforward to calculate its normalized shannon entropy if we have the theoretical frequencies. several measures of ''complexity'' of a probability distribution function have been proposed. in this work we have used statistical complexity measures. all the complexity measures used in this work are the product of a normalized shannon entropy of the probability distribution function, and a divergence measure to a reference probability distribution function. we follow earlier proposals by lã³pez-ruiz, mancini and calbet who first introduced a statistical complexity measure based on such a product in [797] . the lmc-statistical complexity is the product of the normalized shannon entropy, h[p], times the disequilibrium, q[p]; the latter given by the euclidean distance from p to p e , the uniform probability distribution over the ensemble. in this paper we used a later modification which we refer as the mpr-statistical complexity [43] which replaces the euclidean distance between p to p e by the jensen-shannon divergence [788, 798] . the jensen-shannon divergence is linked in physics to the thermodynamic length [799, 800, 801, 802] . we define the mpr-statistical complexity [790] as: where q p (j) ,p e ã� ã� q 0 j s p (j) ,p e ã� ã� , q 0 is a normalization factor, and j s p (1) ,p (2) ã� ã� is the jensen-shannon's divergence between two probability density functions p (1) and p (2) , which in turn is defined as j s p (1) ,p (2) ã� ã� h p (1) zp (2) 2 in this work, in many cases we compute the jensen-shannon divergences of a probability with a probability of reference which is not the uniform probability distribution over the ensemble. in general, it is the average over a subset of probability distribution functions which are consider to be either the ''initial'' of ''final'' states of interest. let p ave be such an average, then the m-statistical complexity of a probability distribution function p (j) , given a p ave of reference, is given by c (m) p (j) ã� ã�~h p (j) ã� ã� : j s p (j) ,p ave ã� ã� an illutrative example. in order to discuss a relatively simple example that can intuitively provide a grasp of the basic mathematical principles of information theory we present a hypothetical ''gene expression'' dataset involving four samples each with the expression of five unique probes corresponding to five genes (not necessarily different) as follows in table 3 . one of the quantifiers that we use in this contribution describes a measure of order for a sample: the normalized shannon entropy also known as shannon evenness index [803] . this section focuses on this quantifiers use and importance (refer to the 'materials and methods' section to see how this measure is calculated). in sample 4 all probes have the same expression therefore it has the highest achievable value of normalized shannon entropy (h = 1). the normalized shannon entropy values for samples 1 and 2 are the same (h = 0.82). sample 3, which tends to be less peaked and has the two most significantly expressed genes with the same value, has a higher value of normalized shannon entropy (h = 0.92) (see figure 21 ). this simple example shows that the normalized shannon entropy variations of the gene expression profile convey information about global transcriptomic changes; however, this measure alone is not enough to characterize the deviations from normal tissue profiles. for example, assume that sample 1 is the normal profile of a particular tissue type. assume that sample 3 is the profile of a cancer cell that originated from that tissue type, the variation of normalized shannon entropy can be related to this malignant change. however, as sample 2 illustrates, normalized shannon entropy is not enough to let us to measure the variation from a profile and at least another information theory quantifier is needed. we resort to statistical complexity quantifiers, which in turn use the jensen-shannon divergence [798] to provide this complementary dimension [800] (refer to the 'materials and methods' section for a mathematical definition of the jensen-shannon divergence). figure 21 shows how the jensen-shannon divergence helps us to evaluate the variation between profiles. samples 1 and 2, as perhaps intuitively expected, have the largest divergence between them, their jensen shannon divergence is 0.286636 (js(1,2) = table 3 . an example dataset to illustrate the principles of shannon entropy and the information theory quantifiers used in this work. table 3 . sample 4 has the largest attainable value since the expression of all probes is the same. samples 1 and 2, which have the same set of expression values, although in different probes, have the same value of normalized shannon entropy. as a consequence, there is a need for another quantifier of gene expression to address the permutational indistinguishability of these two expression profiles. the jensen-shannon divergence provides a natural alternative (see table 4 ). doi:10.1371/journal.pone.0012262.g021 table 4 . jensen-shannon divergence values using the example introduced in table 3 . table 4 . let h p (j) ã� ã� be the normalized shannon entropy of a transcriptional sample profile, then the mpr-statistical complexity c (mpr) p (j) ã� ã� is defined as being proportional to the product of the normalized shannon entropy times the jensen-shannon divergence of the profile with the equiprobable distribution (in the example above the equiprobable distribution is that of sample 4). then we have where q 0 is a normalization factor. once again, we refer to the 'materials and methods' sections for the accompanying formal mathematical presentation. as a consequence, we can plot the mpr-statistical complexity of the samples of our example as a function of the normalized shannon entropy as can be seen in figure 22 . annotated genes. a full list of gene references in this paper along with their descriptions from ihop (http://www.ihop-net. org/unipub/ihop/) can be found in supplementary material reference file s5. stemness, cancer and cancer stem cells stemness'' genomics law governs clinical behavior of human cancer: implications for decision making in disease management an embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors mitochondrial localization, elk-1 transcriptional regulation and growth inhibitory functions of brca1, brca1a, and brca1b proteins mitochondria and cancer glucose avidity of carcinomas mitochondrial dna instability and metabolic shift in human cancers roles of p53, myc and hif-1 in regulating glycolysis -the seventh hallmark of cancer alteration of the bioenergetic phenotype of 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inflammation ck2-site phosphorylation of p53 is induced in deltanp63 expressing basal stem cells in uvb irradiated human skin adenosine deaminase activity in the serum and malignant tumors of breast cancer: the assessment of isoenzyme ada1 and ada2 activities adenosine deaminase, xanthine oxidase, superoxide dismutase, glutathione peroxidase activities and malondialdehyde levels in the sera of patients with head and neck carcinoma activities of adenosine deaminase and 59-nucleotidase in cancerous and noncancerous human colorectal tissues serum adenosine deaminase and arylsulphatase a as an index of early infiltration of central nervous system in acute lymphoblastic leukemia serum adenosine deaminase and total superoxide dismutase activities before and after surgical removal of cancerous laryngeal tissue dipeptide proline diphenyl phosphonates are potent, irreversible inhibitors of seprase (fapalpha) dipeptidyl peptidase inhibits malignant phenotype of prostate cancer cells by blocking basic fibroblast growth factor signaling pathway loss of dipeptidyl peptidase iv immunostaining discriminates malignant melanomas from deep penetrating nevi a marker for neoplastic progression of human melanocytes is a cell surface ectopeptidase expression of cd26/dipeptidyl-peptidase iv in benign and malignant pigment-cell lesions of the skin adenosine downregulates dppiv on ht-29 colon cancer cells by stimulating protein tyrosine phosphatases and reducing erk1/2 activity via a novel pathway adenosine down-regulates the surface expression of dipeptidyl peptidase iv on ht-29 human colorectal carcinoma cells: implications for cancer cell behavior differential regulation of centrosome integrity by dna damage response proteins genomic models of metastatic cancer: functional analysis of death-from-cancer signature genes reveals aneuploid, anoikis-resistant, metastasis-enabling phenotype with altered cell cycle control and activated polycomb group (pcg) protein chromatin silencing pathway expression patterns of polo-like kinase 1 in human gastric cancer overexpression of polo-like kinase 1 (plk1) and chromosomal instability in bladder cancer polo-like kinase 1 expression is a prognostic factor in human colon cancer polo-like kinase 1 (plk1) is overexpressed in primary colorectal cancers aberrant polo-like kinase 1-cdc25a pathway in metastatic hepatocellular carcinoma polo-like kinase 1 expression in medullary carcinoma of the thyroid: its relationship with clinicopathological features silencing of polo-like kinase (plk) 1 via sirna causes inhibition of growth and induction of apoptosis in human esophageal cancer cells identification of human polo-like kinase 1 as a potential therapeutic target in pancreatic cancer robust diagnosis of non-hodgkin lymphoma phenotypes validated on gene expression data from different laboratories polo-like kinase isoforms in breast cancer: expression patterns and prognostic implications expression of polo-like kinase (plk1) in thin melanomas: a novel marker of metastatic disease biodistribution and kinetics of the novel selective oncolytic adenovirus m1 after systemic administration confirming the rnai-mediated mechanism of action of sirna-based cancer therapeutics in mice normal cells, but not cancer cells, survive severe plk1 depletion polo-like kinase (plk)1 depletion induces apoptosis in cancer cells effect of antisense rna targeting polo-like kinase 1 on cell growth in a549 lung cancer cells a panel of isogenic human cancer cells suggests a therapeutic approach for cancers with inactivated p53 plk1 depletion in nontransformed diploid cells activates the dna-damage checkpoint gather: a systems approach to interpreting genomic signatures profiler-a webbased toolset for functional profiling of gene lists from large-scale experiments cryptic splicing at a non-consensus splice-donor in a patient with a novel mutation in the plakophilin-1 gene genetic diseases of junctions ectodermal dysplasia-skin fragility syndrome resulting from a new homozygous mutation, 888delc, in the desmosomal protein plakophilin 1 clinical and molecular significance of splice site mutations in the plakophilin 1 gene in patients with ectodermal dysplasiaskin fragility syndrome inherited disorders of desmosomes hereditary mucoepithelial dysplasia: clinical, ultrastructural and genetic study of eight patients and literature review homozygous splice site mutations in pkp1 result in loss of epidermal plakophilin 1 expression and underlie ectodermal dysplasia/skin fragility syndrome in two consanguineous families assessment of splice variant-specific functions of desmocollin 1 in the skin desmosomes: structure and function in normal and diseased epidermis genomic amplification of the human plakophilin 1 gene and detection of a new mutation in ectodermal dysplasia/skin fragility syndrome genomic organization and amplification of the human plakoglobin gene (jup) skin fragility and hypohidrotic ectodermal dysplasia resulting from ablation of plakophilin 1 hereditary diseases of desmosomes a novel genodermatosis caused by mutations in plakophilin 1, a structural component of desmosomes decreased plakophilin-1 expression promotes increased motility in head and neck squamous cell carcinoma cells the distribution of the desmosomal protein, plakophilin 1, in human skin and skin tumors plakoglobin is required for effective intermediate filament anchorage to desmosomes plakophilins-hard work in the desmosome, recreation in the nucleus? the function of plakophilin 1 in desmosome assembly and actin filament organization plakophilins: multifunctional proteins or just regulators of desmosomal adhesion? comparative analysis of armadillo family proteins in the regulation of a431 epithelial cell junction assembly, adhesion and migration desmoplakin assembly dynamics in four dimensions: multiple phases differentially regulated by intermediate filaments and actin a role for plakophilin-1 in the initiation of desmosome assembly lack of plakophilin 1 increases keratinocyte migration and reduces desmosome stability the desmoglein-specific cytoplasmic region is intrinsically disordered in solution and interacts with multiple desmosomal protein partners the fate of desmosomal proteins in apoptotic cells aquaporin water channels in mammals skin aquaporins: function in hydration, wound healing, and skin epidermis homeostasis roles of aquaporin-3 water channels in volume-regulatory water flow in a human epithelial cell line osmotic stress up-regulates aquaporin-3 gene expression in cultured human keratinocytes increased expression of aquaporin 3 in atopic eczema specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes all-trans retinoic acid attenuates ultraviolet radiation-induced down-regulation of aquaporin-3 and water permeability in human keratinocytes reactive oxygen species: a breath of life or death? role of claudins in tumorigenesis changes in the distribution pattern of claudin tight junction proteins during the progression of mouse skin tumorigenesis claudin-1 is a metastasis suppressor and correlates with clinical outcome in lung adenocarcinoma reexpression of the tj protein cldn1 induces apoptosis in breast tumor spheroids claudin-1, -3 and -4 proteins and mrna expression in benign and malignant breast lesions: a research study decreased expression of claudin-1 correlates with recurrence status in breast cancer constitutive activation of wnt/beta-catenin signaling pathway in migrationactive melanoma cells: role of lef-1 in melanoma with increased metastatic potential osteopontin expression and distribution in human carcinomas functional analysis of the osteopontin molecule osteopontin activation of c-src in human melanoma cells requires the cytoplasmic domain of the integrin alpha v-subunit osteopontin n-terminal domain contains a cryptic adhesive sequence recognized by alpha9beta1 integrin an examination of the effects of hypoxia, acidosis, and glucose starvation on the expression of metastasis-associated genes in murine tumor cells structural requirements for alpha 9 beta 1-mediated adhesion and migration to thrombin-cleaved osteopontin chimeric peptides of statherin and osteopontin that bind hydroxyapatite and mediate cell adhesion natural metabolites of 1alpha, 25-dihydroxyvitamin d(3) retain biologic activity mediated through the vitamin d receptor osteopontin deficiency reduces experimental tumor cell metastasis to bone and soft tissues osteopontin stimulates tumor growth and activation of promatrix metalloproteinase-2 through nuclear factor-kappa b-mediated induction of membrane type 1 matrix metalloproteinase in murine melanoma cells autocrine stimulation by osteopontin contributes to antiapoptotic signalling of melanocytes in dermal collagen cooperative role of osteopontin with type i collagen on the metastasis of murine melanoma cells osteopontin induces nuclear factor kappa bmediated promatrix metalloproteinase-2 activation through i kappa b alpha/ ikk signaling pathways, and curcumin (diferulolylmethane) down-regulates these pathways osteopontin-deficiency suppresses growth of b16 melanoma cells implanted in bone and osteoclastogenesis in co-cultures nuclear factor-inducing kinase plays a crucial role in osteopontin-induced mapk/ikappabalpha kinasedependent nuclear factor kappab-mediated promatrix metalloproteinase-9 activation gene expression analysis to identify mrna markers of cardiac myxoma osteopontin: it's role in regulation of cell motility and nuclear factor kappa b-mediated urokinase type plasminogen activator expression osteopontin expression correlates with melanoma invasion osteopontin in melanocytic lesions-a first step towards invasion? osteopontin expression correlates with melanoma invasion osteopontin expression and serum levels in metastatic uveal melanoma: a pilot study osteopontin is a downstream effector of the pi3-kinase pathway in melanomas that is inversely correlated with functional pten alpha-v-dependent outside-in signaling is required for the regulation of cd44 surface expression, mmp-2 secretion, and cell migration by osteopontin in human melanoma cells regulation of mesenchymal stem cell and chondrocyte differentiation by mia systematic search for the best gene expression markers for melanoma micrometastasis detection cd44 and the adhesion of neoplastic cells non-rgd domains of osteopontin promote cell adhesion without involving alpha v integrins secreted phosphoprotein mrna is induced during multi-stage carcinogenesis in mouse skin and correlates with the metastatic potential of murine fibroblasts osteopontin as a molecular prognostic marker for melanoma aurora b-mediated abscission checkpoint protects against tetraploidization sns-314, a pan-aurora kinase inhibitor, shows potent anti-tumor activity and dosing flexibility in vivo map kinase meets mitosis: a role for raf kinase inhibitory protein in spindle checkpoint regulation molecular classification of melanoma using real-time quantitative reverse transcriptasepolymerase chain reaction the gene expression profiles of primary and metastatic melanoma yields a transition point of tumor progression and metastasis defective cell cycle checkpoint functions in melanoma are associated with altered patterns of gene expression cdc7 expression in melanomas, spitz tumors and melanocytic nevi global analysis of gene expression changes during retinoic acid-induced growth arrest and differentiation of melanoma: comparison to differentially expressed genes in melanocytes vs melanoma cell cycle regulation of central spindle assembly relocation of aurora b from centromeres to the central spindle at the metaphase to anaphase transition requires mklp2 asap is a novel substrate of the oncogenic mitotic kinase aurora-a: phosphorylation on ser625 is essential to spindle formation and mitosis asap, a human microtubule-associated protein required for bipolar spindle assembly and cytokinesis comprehensive expression profiling of tumor cell lines identifies molecular signatures of melanoma progression the nek6 and nek7 protein kinases are required for robust mitotic spindle formation and cytokinesis the nimafamily kinase nek6 phosphorylates the kinesin eg5 at a novel site necessary for mitotic spindle formation integrative approach for differentially overexpressed genes in gastric cancer by combining large-scale gene expression profiling and network analysis nek6 is involved in g2/m phase cell cycle arrest through dna damage-induced phosphorylation polo-like kinase 1 (plk1) in non-melanoma skin cancers targeted depletion of polo-like kinase (plk) 1 through lentiviral shrna or a small-molecule inhibitor causes mitotic catastrophe and induction of apoptosis in human melanoma cells expression and possible role of hpttg1/securin in cutaneous malignant melanoma antagonism of p66shc by melanoma inhibitory activity ralp, a new member of the src homology and collagen family, regulates cell migration and tumor growth of metastatic melanomas melanoma: targeting signaling pathways and ralp deregulated e2f transcriptional activity in autonomously growing melanoma cells arpc1b gene is a candidate prediction marker for choroidal malignant melanomas sensitive to radiotherapy a multi-marker assay to distinguish malignant melanomas from benign nevi bccip functions through p53 to regulate the expression of p21waf1/cip1 identification of genes expressed in malignant cells that promote invasion chemokine expression in melanoma metastases associated with cd8+ t-cell recruitment a potential immune escape mechanism by melanoma cells through the activation of chemokine-induced t cell death multiplex analysis of serum cytokines in melanoma patients treated with interferon-alpha2b regulation of centromeric cohesion by sororin independently of the apc/c sororin is required for stable binding of cohesin to chromatin and for sister chromatid cohesion in interphase sororin, the cell cycle and sister chromatid cohesion sororin, a substrate of the anaphase-promoting complex, is required for sister chromatid cohesion in vertebrates cooperative roles of c-abl and cdk5 in regulation of p53 in response to oxidative stress gene expression signatures for tumor progression, tumor subtype, and tumor thickness in laser-microdissected melanoma tissues a receptor for the malarial parasite plasmodium vivax: the erythrocyte chemokine receptor mgsa/gro transcription is differentially regulated in normal retinal pigment epithelial and melanoma cells nuclear factor-kappa b activation by the cxc chemokine melanoma growth-stimulatory activity/growth-regulated protein involves the mekk1/p38 mitogen-activated protein kinase pathway endothelin-1 induces cxcl1 and cxcl8 secretion in human melanoma cells gene expression profiling reveals cross-talk between melanoma and fibroblasts: implications for host-tumor interactions in metastasis threedimensional culture of melanoma cells profoundly affects gene expression profile: a high density oligonucleotide array study evidence of involvement of cxc-chemokines in proliferation of cultivated human melanocytes role of cxcl1 in tumorigenesis of melanoma melanoma growth stimulatory activity in primary malignant melanoma: prognostic significance a novel nf-kappa b-inducing kinase-mapk signaling pathway up-regulates nf-kappa b activity in melanoma cells induction of melanoma in murine macrophage inflammatory protein 2 transgenic mice heterozygous for inhibitor of kinase/alternate reading frame constitutive ikappab kinase activity correlates with nuclear factor-kappab activation in human melanoma cells mgsa/gromediated melanocyte transformation involves induction of ras expression the tumorigenic and angiogenic effects of mgsa/gro proteins in melanoma autocrine and paracrine roles for growth factors in melanoma localization of mgsa/gro protein in cutaneous lesions melanoma growth stimulatory activity: isolation from human melanoma tumors and characterization of tissue distribution immunoaffinity purification of melanoma growth stimulatory activity characterization of the role of melanoma growth stimulatory activity (mgsa) in the growth of normal melanocytes, nevocytes, and malignant melanocytes enhanced tumor-forming capacity for immortalized melanocytes expressing melanoma growth stimulatory activity/growth-regulated cytokine beta and gamma proteins expression and migratory analysis of 5 human uveal melanoma cell lines for cxcl12, cxcl8, cxcl1, and hgf inhibition of multiple vascular endothelial growth factor receptors (vegfr) blocks lymph node metastases but inhibition of vegfr-2 is sufficient to sensitize tumor cells to platinum-based chemotherapeutics pilot study on the interaction between b16 melanoma cell-line and bone-marrow derived mesenchymal stem cells circulating serum levels of angiogenic factors and vascular endothelial growth factor receptors 1 and 2 in melanoma patients analysis of the tyrosine kinome in melanoma reveals recurrent mutations in erbb4 the role of vascular endothelial growth factors and their receptors in malignant melanomas the role of circulating angiogenic factors in patients operated on for localized malignant melanoma simultaneous blockade of vegfr-1 and vegfr-2 activation is necessary to efficiently inhibit experimental melanoma growth and metastasis formation flt-1 intraceptor induces the unfolded protein response, apoptotic factors, and regression of murine injury-induced corneal neovascularization an autocrine loop directed by the vascular endothelial growth factor promotes invasiveness of human melanoma cells overproduction of vegf concomitantly expressed with its receptors promotes growth and survival of melanoma cells through mapk and pi3k signaling mmp9 induction by vascular endothelial growth factor receptor-1 is involved in lungspecific metastasis release and complex formation of soluble vegfr-1 from endothelial cells and biological fluids angiogenesis and vascular growth factor receptor expression in malignant melanoma association of increased levels of heavychain ferritin with increased cd4+ cd25+ regulatory t-cell levels in patients with melanoma heavy chain ferritin activates regulatory t cells by induction of changes in dendritic cells immunosuppressive effects of melanoma-derived heavy-chain ferritin are dependent on stimulation of il-10 production ferritin heavy chain upregulation by nf-kappab inhibits tnfalpha-induced apoptosis by suppressing reactive oxygen species sealing of chromosomal dna nicks during nucleotide excision repair requires xrcc1 and dna ligase iii alpha in a cell-cycle-specific manner gene expression profiles in radiation workers occupationally exposed to ionizing radiation human melanoma cells secrete and respond to placenta growth factor and vascular endothelial growth factor expression patterns of placenta growth factor in human melanocytic cell lines neuropilins in tumor biology semaphorin signaling in cancer cells and in cells of the tumor microenvironment-two sides of a coin from plasma membrane to cytoskeleton: a novel function for semaphorin 6a semaphorins at the interface of development and cancer comparative integromics on non-canonical wnt or planar cell polarity signaling molecules: transcriptional mechanism of ptk7 in colorectal cancer and that of sema6a in undifferentiated es cells deciphering developmental stages of adult myelopoiesis downregulation of ccn3 expression as a potential mechanism for melanoma progression osteopontin expression in normal skin and non-melanoma skin tumors osteopontin stimulates melanoma growth and lung metastasis through nik/mekk1-dependent mmp-9 activation pathways osteonectin downregulates ecadherin, induces osteopontin and focal adhesion kinase activity stimulating an invasive melanoma phenotype osteopontin expression in spitz nevi serum markers to detect metastatic uveal melanoma osteopontin and the skin: multiple emerging roles in cutaneous biology and pathology the hedgehog pathway transcription factor gli1 promotes malignant behavior of cancer cells by up-regulating osteopontin expression of phosphorylated-stat3 and osteopontin and their correlation in melanoma matricellular proteins produced by melanocytes and melanomas: in search for functions osteopontin and 'melanoma inhibitory activity': comparison of two serological tumor markers in metastatic uveal melanoma patients extracellular and intracellular mechanisms that mediate the metastatic activity of exogenous osteopontin endothelin signaling axis activates osteopontin expression through pi3 kinase pathway in a375 melanoma cells a highthroughput study in melanoma identifies epithelial-mesenchymal transition as a major determinant of metastasis serum osteopontin, an enhancer of tumor metastasis to bone, promotes b16 melanoma cell migration gene-specific fluorescence in-situ hybridization analysis on tissue microarray to refine the region of chromosome 20q amplification in melanoma identification of proteins regulated by interferon-alpha in resistant and sensitive malignant melanoma cell lines a molecular correlate to the gleason grading system for prostate adenocarcinoma ink4c and pten constrain a positive regulatory loop between cell growth and cell cycle control survey of differentially methylated promoters in prostate cancer cell lines transfer of functional prostasomal cd59 of metastatic prostatic cancer cell origin protects cells against complement attack differentially expressed genes in two lncap prostate cancer cell lines reflecting changes during prostate cancer progression amine oxidase activities in chemically-induced mammary cancer in the rat the significance of monoamine oxidase-a expression in high grade prostate cancer from androgen receptor to the general transcription factor tfiih. identification of cdk activating kinase (cak) as an androgen receptor nh(2)-terminal associated coactivator alphamethylacyl-coa racemase-an 'obscure' metabolic enzyme takes centre stage biopsy tissue microarray study of ki-67 expression in untreated, localized prostate cancer managed by active surveillance alpha-methylacyl-coa racemase (p504s) expression in evolving carcinomas within benign prostatic hyperplasia and in cancers of the transition zone expression of alpha-methylacyl-coa racemase (p504s) in various malignant neoplasms and normal tissues: astudy of 761 cases using an amacr (p504s)/34betae12/p63 cocktail for the detection of small focal prostate carcinoma in needle biopsy specimens diagnostic utility of alpha-methylacyl coa racemase (p504s) on prostate needle biopsy discovery and clinical application of a novel prostate cancer marker: alpha-methylacyl coa racemase (p504s) alphamethylacyl-coa racemase: a multi-institutional study of a new prostate cancer marker quantitative immunohistochemical detection of the molecular expression patterns in proliferative inflammatory atrophy the importance of determining the aggressiveness of prostate cancer using serum and tissue molecular markers golph2 protein expression as a novel tissue biomarker for prostate cancer: implications for tissue-based diagnostics optimization of laser capture microdissection and rna amplification for gene expression profiling of prostate cancer ) alpha-methylacyl-coa racemase: expression levels of this novel cancer biomarker depend on tumor differentiation elevated alpha-methylacyl-coa racemase enzymatic activity in prostate cancer comparison of monoclonal antibody (p504s) and polyclonal antibody to alpha methylacyl-coa racemase (amacr) in the work-up of prostate cancer immunohistochemical detection of carcinoma in radical prostatectomy specimens following hormone therapy basal cell subpopulation as putative human prostate carcinoma stem cells a statistical method for identifying differential gene-gene co-expression patterns alphamethylacyl-coa racemase (amacr/p504s) protein expression in urothelial carcinoma of the upper urinary tract correlates with tumour progression sequence variation in alpha-methylacyl-coa racemase and risk of early-onset and familial prostate cancer malignant transformation of human benign prostate epithelial cells by high linear energy transfer alpha-particles expression of alpha-methylacylcoenzyme a racemase in dysplastic barrett's epithelium quantitative analysis of a panel of gene expression in prostate cancer-with emphasis on npy expression analysis the value of using an amacr/34betae12/p63 cocktail double staining for diagnosis of prostate carcinoma decreased gene expression of steroid 5 alpha-reductase 2 in human prostate cancer: implications for finasteride therapy of prostate carcinoma alpha-methylacyl-coa racemase: a new molecular marker for prostate cancer alpha-methylacyl-coa racemase: a variably sensitive immunohistochemical marker for the diagnosis of small prostate cancer foci on needle biopsy neoadjuvant docetaxel treatment for locally advanced prostate cancer: a clinicopathologic study biomarkers for prostate cancer method for quantification of a prostate cancer biomarker in urine without sample preparation effects of the dual 5 alpha-reductase inhibitor dutasteride on apoptosis in primary cultures of prostate cancer epithelial cells and cell lines routine immunohistochemical staining for high-molecular weight cytokeratin 34-beta and alpha-methylacyl coa racemase (p504s) in postirradiation prostate biopsies search for residual prostate cancer on pt0 radical prostatectomy after positive biopsy branched fatty acids in dairy and beef products markedly enhance alpha-methylacyl-coa racemase expression in prostate cancer cells in vitro alpha-methyl coa racemase expression in renal cell carcinomas diagnostic utility of a p63/alpha-methyl-coa-racemase (p504s) cocktail in atypical foci in the prostate value of new prostate cancer markers: alpha methylacyl coa racemase (p504s) and p63 evaluation of p63 and p504s markers for the diagnosis of prostate cancer a preliminary study of the baboon prostate pathophysiology alternative spliced variants of the alpha-methylacyl-coa racemase gene and their expression in prostate cancer a variant of the alpha-methyl-acyl-coa racemase gene created by a deletion in exon 5 and its expression in prostate cancer utility of alpha-methylacyl coenzyme a racemase (p504s antibody) as a diagnostic immunohistochemical marker for cancer alpha-methylacyl-coa racemase as a marker in the differential diagnosis of metanephric adenoma frequent overexpression of ets-related gene-1 (erg1) in prostate cancer transcriptome human telomerase and alpha-methylacyl-coenzyme a racemase in prostatic carcinoma. a comparative immunohistochemical study prostate cancer detection on urinalysis for alpha methylacyl coenzyme a racemase protein decreased alpha-methylacyl coa racemase expression in localized prostate cancer is associated with an increased rate of biochemical recurrence and cancer-specific death quantitative determination of expression of the prostate cancer protein alphamethylacyl-coa racemase using automated quantitative analysis (aqua): a novel paradigm for automated and continuous biomarker measurements ) alpha-methylacyl coenzyme a racemase as a tissue biomarker for prostate cancer prostatic ductal adenocarcinoma presenting as a urethral polyp: a clinicopathological study of eight cases of a lesion with the potential to be misdiagnosed as a benign prostatic urethral polyp ) alpha-methylacyl coenzyme a racemase, ki-67, and topoisomerase iialpha in cystoprostatectomies with incidental prostate cancer emerging biomarkers for the diagnosis and prognosis of prostate cancer extraction and processing of high quality rna from impalpable and macroscopically invisible prostate cancer for microarray gene expression analysis kinetic fluorescence reverse transcriptase-polymerase chain reaction for alphamethylacyl coa racemase distinguishes prostate cancer from benign lesions expression profiling identifies a novel alpha-methylacyl-coa racemase exon with fumarate hydratase homology alphamethylacyl coa racemase in pulmonary adenocarcinoma, squamous cell carcinoma, and neuroendocrine tumors: expression and survival analysis isolation of human prostatic epithelial plasma membranes for proteomics using mirror image tissue banking of radical prostatectomy specimens expression of alphamethylacyl-coa racemase (p504s) in nephrogenic adenoma: a significant immunohistochemical pitfall compounding the differential diagnosis with prostatic adenocarcinoma oct4a is expressed by a subpopulation of prostate neuroendocrine cells humoral immune response to alpha-methylacyl-coa racemase and prostate cancer comparison of annexin ii, p63 and alpha-methylacyl-coa racemase immunoreactivity in prostatic tissue: a tissue microarray study ancillary alpha-methylacyl-coa racemase immunocytochemistry in the diagnosis of adenocarcinoma of the prostate in urinary cytology: a case report alpha-methylacyl-coa racemase (p504s)/34betae12/p63 triple cocktail stain in prostatic adenocarcinoma after hormonal therapy differences between latent and clinical prostate carcinomas: lower cell proliferation activity in latent cases variation of alpha-methylacyl-coa racemase expression in prostate adenocarcinoma cases receiving hormonal therapy phytanic acid, amacr and prostate cancer risk transcriptome analysis of human colon caco-2 cells exposed to sulforaphane expression of alpha-methylacyl-coa racemase in papillary renal cell carcinoma alpha-methylacyl-coa racemase expression is upregulated in gastric adenocarcinoma: a study of 249 cases sensitivity of p504s/alpha-methylacyl-coa racemase (amacr) immunohistochemistry for the detection of prostate carcinoma on stored needle biopsies diagnostic utility of immunohistochemistry in morphologically difficult prostate cancer: review of current literature the prostate-specific g-protein coupled receptors psgr and psgr2 are prostate cancer biomarkers that are complementary to alpha-methylacyl-coa racemase partial atrophy on prostate needle biopsy cores: a morphologic and immunohistochemical study abundant expression of amacr in many distinct tumour types peroxisomal disorders affecting phytanic acid alphaoxidation: a review alpha-methylacyl-coa racemase protein expression is associated with the degree of differentiation in breast cancer using quantitative image analysis analysis of alpha-methylacyl-coa racemase (p504s) expression in high-grade prostatic intraepithelial neoplasia serum levels of phytanic acid are associated with prostate cancer risk detection of alpha-methylacyl-coenzyme a racemase in postradiation prostatic adenocarcinoma tumor suppressor activity of glucocorticoid receptor in the prostate detection of alpha-methylacyl-coenzyme-a racemase transcripts in blood and urine samples of prostate cancer patients alphamethylacyl-coa racemase as an androgen-independent growth modifier in prostate cancer peroxisomal branched chain fatty acid beta-oxidation pathway is upregulated in prostate cancer a nonclassic ccaat enhancer element binding protein binding site contributes to alpha-methylacyl-coa racemase expression in prostate cancer sequence variants of alpha-methylacyl-coa racemase are associated with prostate cancer risk how often does alphamethylacyl-coa-racemase contribute to resolving an atypical diagnosis on prostate needle biopsy beyond that provided by basal cell markers? alpha-methylacyl-coa racemase: a novel tumor marker over-expressed in several human cancers and their precursor lesions expression and diagnostic utility of alphamethylacyl-coa-racemase (p504s) in foamy gland and pseudohyperplastic prostate cancer a novel diagnostic test for prostate cancer emerges from the determination of alphamethylacyl-coenzyme a racemase in prostatic secretions deletion hotspots in amacr promoter cpg island are cis-regulatory elements controlling the gene expression in the colon gene expression profiles in the pc-3 human prostate cancer cells induced by nkx3.1 usefulness of cytokeratin 5/6 and amacr applied as double sequential immunostains for diagnostic assessment of problematic prostate specimens conversion of prostate cancer from hormone independency to dependency due to amacr inhibition: involvement of increased ar expression and decreased igf1 expression oct4a is expressed by a subpopulation of prostate neuroendocrine cells a duplex quantitative polymerase chain reaction assay based on quantification of alphamethylacyl-coa racemase transcripts and prostate cancer antigen 3 in urine sediments improved diagnostic accuracy for prostate cancer a continuous assay for alpha-methylacylcoenzyme a racemase using circular dichroism group iia phospholipase a as a prognostic marker in prostate cancer: relevance to clinicopathological variables and disease-specific mortality biomarkers for prostate cancer immunohistochemical algorithms in prostate diagnostics: what's new?]. pathologe alpha-methylacyl-coa racemase (amacr) in fine-needle aspiration specimens of prostate lesions biopsy tissue microarray study of ki-67 expression in untreated, localized prostate cancer managed by active surveillance urine markers in monitoring for prostate cancer expression of alpha-methylacyl-coa racemase (p504s) in sebaceous neoplasms expression of prostatic acid phosphatase (psap) in transurethral resection specimens of the prostate is predictive of histopathologic tumor stage in subsequent radical prostatectomies prostate carcinogenesis induced by n-methyl-n-nitrosourea (mnu) in gerbils: histopathological diagnosis and potential invasiveness mediated by extracellular matrix components synthesis and use of isotope-labelled substrates for a mechanistic study on human alpha-methylacyl-coa racemase 1a (amacr; p504s) s-100a1 is a reliable marker in distinguishing nephrogenic adenoma from prostatic adenocarcinoma molecular cloning and preliminary analysis of the human alpha-methylacyl-coa racemase promoter autopsy evaluation of a prostate cancer case treated with brachytherapy loss of heterozygosity (loh), malignancy grade and clonality in microdissected prostate cancer allelic loss and microsatellite instability in prostate cancers in japan tumour suppressor gene mutations in benign prostatic hyperplasia and prostate cancer mutations in brca1 and brca2 and predisposition to prostate cancer brca1a has antitumor activity in tn breast, ovarian and prostate cancers coexpression of the mutated brca1 mrna and p53 mrna and its association in chinese prostate cancer exploiting the achilles heel of cancer: the therapeutic potential of poly(adp-ribose) polymerase inhibitors in brca2-defective cancer prostate cancer patients with brca2 mutation face poor survival brca1 mutations and prostate cancer in poland common variation in the brca1 gene and prostate cancer risk brca1 in special populations male brca1 and brca2 mutation carriers: a pilot study investigating medical characteristics of patients participating in a prostate cancer prevention clinic truncating brca1 mutations are uncommon in a cohort of hereditary prostate cancer families with evidence of linkage to 17q markers unravelling the genetics of prostate cancer prostate-cancer screening targets men with brca mutations brca1 and prostate cancer gene mutational rate, gleason score, and bk virus infection in prostate adenocarcinoma: is there a correlation tp53 mutation in prostate needle biopsies-comparison with patients follow-up shared tp53 gene mutation in morphologically and phenotypically distinct concurrent primary small cell neuroendocrine carcinoma and adenocarcinoma of the prostate unique substitution of chek2 and tp53 mutations implicated in primary prostate tumors and cancer cell lines molecular biology in prostate cancer a microdissection approach to detect molecular markers during progression of prostate cancer molecular biology of prostate cancer progression molecular heterogeneity in prostate cancer: can tp53 mutation unravel tumorigenesis abnormalities in primary prostate cancer: single-strand conformation polymorphism analysis of complementary dna in comparison with genomic dna. the cooperative prostate network status and prognosis of locally advanced prostatic adenocarcinoma: a study based on rtog 8610 localization of prostate cancer metastasis-suppressor activity on human chromosome 17 correlation of genetic and immunodetection of tp53 mutations in malignant and benign prostate tissues molecular markers for predicting prostate cancer stage and survival role in tumorigenesis of silent mutations in the tp53 gene chromosomal imbalances, loss of heterozygosity, and immunohistochemical expression of tp53, rb1, and pten in intraductal cancer, intraepithelial neoplasia, and invasive adenocarcinoma of the prostate increase of androgen-induced cell death and androgen receptor transactivation by brca1 in prostate cancer cells androgen regulation of the androgen receptor coregulators breast cancer susceptibility gene 1 (brcai) is a coactivator of the androgen receptor common mutations in brca1 and brca2 do not contribute to early prostate cancer in jewish men brca1 in hormonal carcinogenesis: basic and clinical research role of direct interaction in brca1 inhibition of estrogen receptor activity brca1 regulates gene expression for orderly mitotic progression the consequences of chromosomal aneuploidy on gene expression profiles in a cell line model for prostate carcinogenesis constitutive activation of jak-stat3 signaling by brca1 in human prostate cancer cells methylation analysis of brca1, rassf1, gstp1 and ephb2 promoters in prostate biopsies according to different degrees of malignancy psf and p54(nrb)/nono-multi-functional nuclear proteins the psf.p54nrb complex is a novel mnk substrate that binds the mrna for tumor necrosis factor alpha ptb-associated splicing factor regulates growth 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bone metastasis analysis of gene expression in the tumor-associated macrophage nuclear factor inducing kinase: a key regulator in osteopontin-induced mapk/ikappab kinase dependent nf-kappab-mediated promatrix metalloproteinase-9 activation prostate-specific antigen modulates genes involved in bone remodeling and induces osteoblast differentiation of human osteosarcoma cell line saos-2 the authors would like to thank three research associates of our centre (osvaldo rosso, carlos riveros and john marsden) for discussions on this topic. we thank the first two, in particular to osvaldo, for their collaboration on data cleaning and the computation of the entropy and jensen-shannon divergences. we thank marsden for his advice on editing the final draft. pm would also like to thank two stimulating discussions (decades apart) with dr. carlos reigosa and prof. elizabeth blackburn, as well as the invisible hand of prof. yaser abu-mostafa. key: cord-000224-2lz03oqb authors: porter, kristen a.; kelley, lauren n.; george, annette; harton, jonathan a.; duus, karen m. title: class ii transactivator (ciita) enhances cytoplasmic processing of hiv-1 pr55gag date: 2010-06-24 journal: plos one doi: 10.1371/journal.pone.0011304 sha: doc_id: 224 cord_uid: 2lz03oqb background: the pr55(gag) (gag) polyprotein of hiv serves as a scaffold for virion assembly and is thus essential for progeny virion budding and maturation. gag localizes to the plasma membrane (pm) and membranes of late endosomes, allowing for release of infectious virus directly from the cell membrane and/or upon exocytosis. the host factors involved in gag trafficking to these sites are largely unknown. upon activation, cd4+ t cells, the primary target of hiv infection, express the class ii transcriptional activator (ciita) and therefore the mhc class ii isotype, hla-dr. similar to gag, hla-dr localizes to the pm and at the membranes of endosomes and specialized vesicular mhc class ii compartments (miics). in hiv producer cells, transient hla-dr expression induces intracellular gag accumulation and impairs virus release. methodology/principal findings: here we demonstrate that both stable and transient expression of ciita in hiv producer cells does not induce hla-dr-associated intracellular retention of gag, but does increase the infectivity of virions. however, neither of these phenomena is due to recapitulation of the class ii antigen presentation pathway or ciita-mediated transcriptional activation of virus genes. interestingly, we demonstrate that ciita, apart from its transcriptional effects, acts cytoplasmically to enhance pr160(gag-pol) (gag-pol) levels and thereby the viral protease and gag processing, accounting for the increased infectivity of virions from ciita-expressing cells. conclusions/significance: this study demonstrates that ciita enhances hiv gag processing, and provides the first evidence of a novel, post-transcriptional, cytoplasmic function for a well-known transactivator. hiv polyprotein gag serves as a scaffold to promote assembly of progeny virions at cellular membranes [1] and recruits components of the vesicular protein sorting pathway to facilitate virus budding [2, 3, 4] . concomitantly, the virally encoded protease begins to cleave gag, which is required for complete virion maturation and infectivity [5, 6, 7] . gag proteins can be detected at both the pm and the membranes of endosomes among different cell types, suggesting that budding is not limited to one cell-type specific locale [8, 9, 10, 11, 12, 13, 14, 15, 16] . further, host factors which participate in targeting gag trafficking to particular membranes are largely unknown. as gag and infectious virus can originate from two cellular locations, two models for gag trafficking have emerged. the first model proposes that following synthesis, gag traffics to endosomal membranes, and upon exocytosis is deposited on the pm, where it serves as the site for productive virus assembly [14, 17] . the second model proposes that gag is first trafficked to the pm, where virus assembly occurs, and then excess gag is internalized to intracellular compartments [14, 18, 19, 20] , that serve as sites of productive virus assembly [15, 21] . mhc class ii heterodimers follow a similar trafficking route, appearing at both the pm and specialized multivesicular bodies (mvbs) called mhc class ii containing compartments (miics) [22] . mhc class ii is utilized by antigen presenting cells (apcs) to present exogenous processed antigen to cd4+ t cells [22, 23, 24] . mhc class ii genes, including: hla-dr, -dp and -dq and the accessory molecules, invariant chain (ii) and hla-dm, are transcriptionally activated by the class ii transactivator (ciita), the global regulator of coordinate class ii mhc gene expression [25, 26] . as ciita is induced in cd4+t cells upon activation, these cells express mhc class ii [27, 28] . upon synthesis, hla-dr heterodimers are assembled in the er and the immature complex (hla-dr+ ii) travels through the secretory pathway to miics, where the specialized hla-dm chaperone loads the hla-dr heterodimer with peptide [22, 29, 30] . interestingly, both immature and mature forms of hla-dr can be found at the pm and can be subsequently internalized to miics due to a di-leucine motif in the cytoplasmic tail of ii (immature hla-dr) and a dileucine motif and/or ubiquitination of conserved lysine residues within the hla-dr b chain (mature hla-dr), respectively [22, 29, 31, 32, 33, 34, 35, 36] . therefore, a connection between hla-dr and gag trafficking would not be surprising as both have an alternative route to intracellular compartments by way of the pm. indeed, expression of hiv-1 nef, vpu and gag have been shown to alter hla-dr trafficking [37, 38, 39, 40] . in addition, hla-dr is preferentially acquired on the viral envelope of budding virions, which enhances virion infectivity and may play a role in bystander apoptosis of t lymphocytes [41, 42, 43] . therefore, hla-dr localization at virus assembly sites is not unexpected. finzi et al. (2006) addressed the contribution of mhc class iiinduced miic formation to pr55gag trafficking by monitoring virus budding in the presence of transiently expressed hla-dr heterodimers [8] . in hek-293t cells expressing hla-dr, there was a marked redistribution of gag to intracellular compartments, and reduced virus release at the pm; mature virus budded into hla-dr containing multivesicular bodies and was retained [8] . we hypothesized that recapitulating endogenous expression of the entire class ii antigen presentation pathway in producer cells via expression of ciita would restore infectious virus release and provide a more physiologically relevant model for hiv-1 assembly studies. as expected, stable or transient expression of ciita did not induce intracellular retention of gag. however, hla-dr induced gag retention could not be alleviated by transient co-expression with hla-dm and/or ii, or ciita-mediated upregulation of the recycling endosome gtpase, rab4b [44] . further, mutating ubiquitinatible lysine residues or complete truncation of the cytoplasmic tails of the hla-dr a and b chains did not restore virus release. rather, limiting the amount of hla-dr dna transfected into cells restored virus release and alleviated gag retention. curiously, ciita expressing cells produced virus that was significantly more infectious than ciita deficient cells, and this was independent of the class ii antigen presentation pathway. ciita enhances infectivity of virions from producer cells through a novel function, by improving maturation through an increase in viral protease-dependent gag processing. using a panel of ciita mutants, we demonstrate that cytoplasmic ciita increases gag-pol polyprotein levels. overall, our work reveals a novel cytoplasmic, post-transcriptional function of ciita, which is expressed upon t cell activation and is constitutively expressed in macrophages and dendritic cells, all known targets of hiv infection. transient expression of the class ii heterodimer, hla-dr, induces intracellular accumulation of gag and a marked reduction in extracellular virus release from producer cells [8] . this previous study focused on hla-dr in the absence of ii and hla-dm, critical components of the antigen processing and presentation pathway which influence mhc class ii trafficking. to determine if expression of the complete mhc class ii pathway could overcome gag and virus retention we used ciita to coordinately express these genes. cells were either transiently (pcciita) or stably (a293t-ciita) transfected with ciita ( figure s1 ) and hiv-1 nl4-3 dna, and gag localization was monitored by immunofluorescence and compared to cells transiently expressing the hla-dr a and b heterodimers (pcdrab1b5), or vector-only (pcdna3.1). as expected, a weak but uniform gag signal was present in vector-only transfected cells (figure 1aa ) and transient hla-dr expression induced a marked redistribution of gag as indicated by a dense, punctuate gag staining pattern (figure 1ad ). conversely, gag accumulation was not observed when ciita was either transiently or stably expressed (figure 1ab or c, respectively). therefore, transient expression of hla-dr in producer cells does indeed lead to gag retention, which is not observed in the presence of ciita. however, recapitulation of the mhc class ii pathway in trans (pcdrab1b5+ii+hla-dmab), also resulted in dense accumulation of gag signal (figure 1ae ), suggesting that ciita-mediated coordinate activation of hla-dr, -dm and ii expression is insufficient to overcome gag retention. flow cytometric analysis confirmed these findings, as cells transfected with hla-dr heterodimers and/or co-transfected with some or all of the components of the class ii antigen presentation pathway stained as gag hi , indicating gag accumulation ( figure 1b and c). however, this population was absent in cells transiently or stably expressing ciita ( figure 1b and c) . therefore, absence of gag accumulation in ciita expressing cells is likely not due to its transactivation of the mhc class ii antigen presentation pathway. to assess whether ciita-mediated alleviation of gag retention in class ii expressing cells correlated with restored virus production, virus particle and infectious virus release were measured by p24 elisa and ghost cell infectivity assays, respectively. virus release, both infectious and particle titers) were reduced when cells were transfected with either hla-dr or other components of the mhc class ii antigen presentation pathway ( figure s2 ), confirming a correlation between gag retention and reduced virus titers in the presence of hla-dr, as previously demonstrated [8] . these assays also demonstrate a dramatic increase in infectious virus release from ciita-expressing cells as compared to cells expressing vector only (figure 2a & s2), despite significantly fewer virus particles being released from a ciitaexpressing cell (as measured by pg/ml of p24 in the culture supernatant, figure 2b & s2). therefore, while fewer virus particles are released from a ciita-expressing cell, those particles are significantly more infectious ( figure 2c ). previously, hla-dr incorporation into the envelope of budding virions was demonstrated to enhance virion infectivity [45, 46] . to determine if ciita-enhancement of virion infectivity was due to hla-dr or other components of the class ii antigen presentation pathway, we measured virion infectivity from cells expressing these proteins. however, virions from these cells were just as infectious as virions released from vector only controls, yet not as infectious as virions from cells either stably or transientlyexpressing ciita ( figure 2d ). therefore, ciita enhancement of virion infectivity is independent of the mhc class ii antigen presentation pathway. together, these data suggest ciita has two effects on the hiv replicative cycle in producer cells, both of which are independent of the mhc ii antigen processing pathway; i) it does not induce hla-dr, mediated intracellular retention of gag and ii) it increases the infectivity of hiv virions. ciita has been shown to upregulate expression of rab4b, a small gtpase involved in endocytic recycling [44, 47] . therefore, we hypothesized that ciita via the action of rab4b, could increase gag recycling back to the pm, thereby alleviating hla-dr-mediated gag retention. commercially available rab4 antibodies cannot distinguish between rab4a and 4b. however, krawczyk et al. (2007) demonstrated by chromatin immunoprecipitation that ciita specifically associates with the rab4b and not the rab4a promoter [44] . immunoblotting demonstrated that there is an increase in rab4 in ciita-expressing cells ( figure 3a ), therefore it is likely that this is in the form of rab4b. however, when rab4b and hla-dr were co-expressed in producer cells with the hiv-1 nl4-3 plasmid, it did not rescue intracellular retention of gag ( figure 3b ). in fact, infectious virus release from cells transiently co-expressing rab4b and hla-dr was further reduced, suggesting that rab4b does not alleviate hla-drmediated retention of gag ( figure 3c ). up until this point, we had not co-expressed ciita with hla-dr in producer cells to determine if ciita could alleviate retention of gag and/or restore infectious virus release as would be expected. interestingly, when ciita was transiently expressed with the hla-dr heterodimers in producer cells, gag still accumulated intracellularly and infectious virus production remained reduced ( figure 3b and c, respectively), suggesting that expression of ciita is not sufficient to compensate for hla-drmediated gag retention. however, when hla-dr is endogenously expressed under the control of ciita, retention is alleviated. [8] . ubiquitination of a conserved lysine residue on the hla-dr b chain induces intracellular accumulation of class ii heterodimers in mhc class ii compartments (miics) [35, 36] . thus, the ubiquitination state of hla-dr, might influence gag retention. to address this idea, lys225arg (pcdrabk225r) and lys222/ 225arg (pcdrabk222/225r) point mutations were made in the hla-dr beta chain. despite these mutations, hla-dr expression on the surface of a293t cells was not significantly altered (flow cytometric data not shown), nor was intracellular retention of gag alleviated or infectious virus release restored ( figure 4a and b, respectively). therefore, we attempted to alleviate gag retention and restore infectious virus release, as demonstrated by finzi et al. (2006) [8] , by complete truncation of the cytoplasmic tails of both hla-dr a and b (pcdradcyto and pcdrbdcyto, respectively). however, loss of either cytoplasmic tail (pcdradcytob and pcdrabdcyto), or both cytoplasmic tails (pcdradcy-tobdcyto), from the heterodimer did not alleviate gag retention and even further reduced infectious virus release from these cells ( figure 4a and b, respectively). our results may differ from finzi's because of the differences in cell type, or hla-dr gene alleles, nevertheless they strongly suggest that transient hla-dr expression in producer cells induces gag retention. further, flow cytometry to monitor surface hla-dr expression demonstrates that transient transfection of hla-dr induces an approximate half-log to log-fold increase in hla-dr as compared to cells stably or transiently expressing ciita, respectively ( figure s1 ). therefore, it is possible that hla-dr induced gag retention is a consequence of hla-dr overexpression in this transient system to test this possibility, virus producer cells were transfected with increasing amounts of hla-dr in the presence of hiv-1 nl4-3 dna, and infectious virus release was monitored. as hla-dr expression increased, the level of infectious virus release decreased ( figure 4c ). further, while not statistically significant, there was a positive correlation (r = 0.9954) between gag accumulation (gag hi ) and increasing hla-dr expression ( figure 4d ), suggesting gag retention is likely related to hla-dr overexpression. similarly, when hla-dm, which is structurally similar to hla-dr, was overexpressed in producer cells, gag retention was also induced ( figure 4d ) and infectious virus release reduced (data not shown). as endogenous expression of the class ii antigen presentation pathway by ciita does not induce gag retention and limiting expression of hla-dr or -dm likewise has a limited effect on gag retention in producer cells, these data collectively suggest that overexpression of the class ii pathway alters gag trafficking in producer cells leading to reduced infectious virus release. however, these data do not provide an explanation for the increased infectivity of virions released from ciita-expressing cells. viral protease cleavage of the gag and gag-pol polyproteins is required for virion maturation and infectivity [5, 6] . figure 5a ) [13, 48, 49, 50, 51] . as there is an increase in virion infectivity from ciita-expressing cells, we hypothesized that the processing of gag polyproteins may be enhanced in these cells. as suspected, analysis of cell lysates from equal numbers of hiv-1 transfected a293t-ciita and a293t cells demonstrated a higher ratio of mature cap24 to pr55gag in ciita-expressing cells ( figure 5 b&c), demonstrating that gag processing in the presence of ciita is enhanced. additionally, increased levels of processing intermediates are present in a293t cell lysates. specifically, the p41 (ma-ca-sp1) and p25 (ca+p2) products were increased (figure 5 b&c) in these cells, indicating that gag processing in the presence of ciita is more efficient. gag processing is also enhanced in cell-free virions from cells either transiently or stably expressing ciita, as demonstrated by the increased levels of cap24, and the loss of the p41 and p25 intermediate products relative to pr55gag ( figure 5d&e ). collectively, these results suggest that ciita increases gag polyprotein processing, leading to enhanced production of infectious virions. hla-dr is incorporated into the hiv virion envelope [52] and is transcriptionally activated by ciita, therefore its contribution to gag processing was assessed. virions from hla-dr-expressing cells exhibited less gag processing than virions from ciitaexpressing cells ( figure 5f , bottom panel). when virions from both cell lines were affinity-purified on hla-dr binding columns, gag processing was reduced in those produced in the hla-drexpressing cells as compared to virions from cells which express ciita ( figure 5f , upper panel and 5g), suggesting that hla-dr alone does not contribute to increased gag processing. similar to our previous results, there are fewer processing intermediates present in virions from ciita-expressing cells, suggesting that ciita expression enhances virion infectivity by increasing maturation through more complete gag processing. cleavage of the gag and gag-pol polyprotein is mediated specifically by the virally-encoded protease [53, 54, 55] . we considered that ciita might increase viral protease processing of gag; thus, we examined gag processing and infectious virus release from ciita-expressing hiv producer cells in the presence of a protease inhibitor. if ciita is increasing protease processing, such an enhancement should be overcome by lower doses of the hiv protease inhibitor, lopinavir. as expected, between concentrations of 0.6 and 1.25 nm of lopinavir infectious virus release from ciita-expressing cells was reduced to that of a293t cells treated with vehicle only ( figure 6a ). to determine if the reduced gag processing correlated with decreased infectious virus release, we monitored gag cleavage by western blotting and as expected, between 0.6 and 1.25 nm of liponavir, gag processing in virions from ciita-expressing cells was returned to that of vehicle treated, vector-only control cells ( figure s3 ). these results directly demonstrate that ciita-mediated enhancement of gag processing and infectious virus release is through the hiv protease. the only known function of ciita is as a transcriptional coactivator; therefore, we thought it likely that it drives the expression of a gene which enhances viral protease-mediated gag processing. therefore, we reasoned that expression of ciita mutants which fail to localize to the nucleus should not mediate increased gag processing or enhanced infectious virus release. to test this idea, a panel of ciita mutants (gtp2, a magnesium ion coordination site mutant and gtp3, a guanine ring-binding domain mutant and l1035p, a point mutant in the c-terminal leucine rich repeat domain, which are all defective for nuclear localization and fail to activate hla-dra transcription [56, 57, 58] ), were individually co-expressed with hiv-1 nl4-3 dna, and infectious virus release and gag processing were monitored. interestingly, producer cells expressing these mutants also produced more infectious virus as compared to vector-only control ( figure 7a ) and had increased gag processing in both cell-bound virions and cell-free virions ( figure 7b and figure s4 , respectively). loss of ciita nuclear localization (and therefore coactivation potential), did not hinder increased gag processing and infectious virus release, strongly suggesting that ciita acts cytoplasmically to increase hiv virus maturation independent of its transactivation function. we further evaluated ciita transactivation potential as a mechanism of enhanced virus release utilizing a different model system. nih 3t3 balb/c cells (expressing human p32 to allow for virus like particle production [59] ) expressing ciita and transfected with an ltr-deficient hiv genome construct (pchiv pal),under control of a cmv promoter had a dramatic increase in virus-like particle production as compared to nih 3t3 cells in the absence of ciita ( figure s5 ). this result provides further evidence that ciita enhancement of virus production is independent of its transactivation potential on the hiv ltr. to determine the mechanism by which ciita mediates protease activity in these cells, we analyzed the expression of the gag and gag-pol in ciita-expressing cells. expression of the hiv protease is extremely toxic to cells, and is thus very tightly regulated during virus replication [60, 61, 62] . the viral protease arises from the alternative gag translation product ( figure 5a ), gag-pol which, accounts for only 2-10% of gag polyproteins and results from ribosomal frameshifting at a conserved heptamer ''slippery sequence'' and a secondary stem-loop structure on gag-pol transcripts [51, 63] . therefore, increased viral protease-mediated processing of the gag polyproteins from ciita-expressing cells may be due to an increase in overall viral protease levels, due to increased gag-pol synthesis. to establish the ability of ciita to mediate the enhanced production of hiv protease, cells were transiently co-transfected with either ciita or pcdna and hiv-1 nl4-3 dna, and were treated with 80 nm of lopinavir to inhibit the majority of gag polyprotein processing. gag-pol levels relative to gag were then determined by western blotting. while 1-2% of all gag polyprotein in cell lysates of vector-only cells was in the form of gag-pol, expression of ciita increased this level to almost 5% ( figure 7c and d) , indicating that ciita increases gag-pol levels in virus producer cells. we also measured the potential of the cytoplasmic ciita mutants to increase gag-pol levels relative to gag and, interestingly, gag-pol made up approximately 13, 20 and 15% of all gag polyproteins in gtp2, gtp3 and l1035p -expressing cells, respectively. this result further demonstrates a novel transcription-independent role for cytoplasmic ciita during hiv infection, where ciita enhances gag-pol protease expression, which subsequently enhances virus maturation and infectivity. [16] . we speculated that complete expression of the antigen presentation pathway, through expression of ciita, would alleviate this phenotype in virus producer cells. interestingly, we noticed two phenomena: i) ciita did not induce intracellular retention of gag or impair virus release in producer cells despite expression of hla-dr and ii) the virus released from cells expressing ciita was significantly more infectious. upon investigation of ciita-mediated alleviation of hla-dr-induced gag retention, we found that this effect was not due to the lack of ii or hla-dm (key components of antigen presentation), as had been expected, nor was it a consequence of rab4b expression in these cells. further, when lysine residues in the hla-dr chain were mutated or both of the hla-dr chain cytoplasmic tails were truncated, gag was still retained and virus release inhibited. in addition, when ciita was expressed in conjunction with hla-dr this effect could not be alleviated, suggesting that gag retention is a consequence of hla-dr overexpression. indeed, retention of gag is directly correlated to levels of hla-dr expression. further, we did not observe gag retention when class ii antigen presentation pathway genes were expressed at endogenous levels via ciita expression. overexpression of hla-dm, which is structurally similar to hla-dr also induced gag retention, suggesting that retention of gag is a likely consequence of altered trafficking of overexpressed class ii antigen presentation pathway components rather than a physiologically relevant phenomenon. despite observing similar hla-dr and gag retention/reduced virus release effects as finzi et al.(2006) , our results differed in that we did not observe the requirement for intact hla-dr a and b-chain cytoplasmic tails for the induction of gag retention. in our hands, when the ubiquitinatible lysine residues in the hla-dr b-chain were mutated or the cytoplasmic tails of the hla-dr dimer were completely removed, there was no alleviation of gag retention or virus release. beyond the obvious differences between the previous work [8] and our own (i.e. provirus construct and cell type), our mutant data may differ from theirs because arginine residues were substituted for lysine 215 and tyrosine 220 residues in the hla-dr a and b chain, respectively, in order to ensure stabilization of the truncated hla-dr molecule at cellular membranes. these differences in mutant constructs may potentially explain discrepancies in our results. we also observed that expression of hla-dm was sufficient to induce gag retention and impede virus release from cells. however, finzi et al.(2006) did not observe retention in the presence of hla-dm or hla-do [8] . this difference may be explained by their use of a bicistronic construct for expression of the hla-dm heterodimer; however, this strategy was also used to express hla-dr, which still induced retention [8] . irrespective of these differences, gag retention and loss of virus release correlates with increasing hla-dr expression. these results do not exclude the possibility that hla-dr and gag trafficking may be linked. indeed, we have demonstrated that gag co-immunoprecipitates with hla-dr from ciita expressing cells and this interaction is independent of the cytoplasmic tails of hla-dr (data not shown). further, other hiv proteins may be linked to class ii trafficking. stumptner-cuvelette et al. (2003) and chaudhry et al.(2009) , have independently demonstrated that hiv nef induces internalization of surface class ii in epithelial and monocytic cell lines, respectively [37, 64] . however, we did not observe downregulation of hla-dr from the cell surface, following transfection of the hiv genome into ciita-expressing epithelial cells (data not shown) or hiv infection of ciita-expressing cd4+ t cells (unpublished data). further, gluschankof and suzan demonstrated that expression of gag-pol restored hla-dr presence at the cell surface in the h78-c10.0 line, a t cell clone deficient for surface hla-dr expression [40] . collectively, our work and that of others suggests a link between hla-dr and gag trafficking, where localization may be cell-type dependent. one of the more interesting findings of this study is that while overall viral titers from ciita-expressing cells decrease the infectivity of these particles is significantly enhanced. increased infectivity was due to improved virion maturation in a viral protease-dependent manner. not only was processing to capsid p24 more complete in ciita expressing cells, but vector-only control cells and those only expressing hla-dr contained increased levels of processing intermediates. the presence of higher levels of processing intermediates in virus produced in these cells may help explain the reduced infectivity, as studies in both mlv [65] and hiv [7] demonstrate that partially cleaved gag products act transdominantly to reduce virion infectivity through reduced reverse transcription of viral rna, despite the presence of functional reverse transcription polymerase [7] . next we demonstrated that ciita increased gag processing through the viral protease and evaluated whether this enhancement was a consequence of ciita-mediated transcriptional activation. the ciita l1035p mutant, which fails to translocate to the nucleus [58] , demonstrated increased gag processing and virus release compared to vector-only control, suggesting a cytoplasmic role for ciita in the later stages of the hiv infection cycle. this does not preclude the possibility that an undetectable level of ciita l1035p might translocate to the nucleus. however, no l1035p was detected in the nucleus after a 24 hour treatment with the nuclear export inhibitor, leptomycin b [58] . further, the predominantly cytoplasmic gtp-binding ciita mutants, gtp2 and gtp3, had a similar increase in infectious virus release versus vector-only expressing cells. previously, it was demonstrated that increased gag-pol levels severely impair virion infectivity through disruption of rna genome dimerization [62] . further, hiv protease is known to be toxic to cells as it leads to the production of the novel procaspase 8 cleavage product, casp8p41, which induces apoptosis through loss of mitochondrial membrane potential [66] . therefore, we would expect that virions from ciita-expressing cells would be reduced in titer and infectivity, as there is increased gag-pol and protease. however, while overall viral particle numbers (as measured by p24 elisa) from ciita expressing cells were decreased, the infectivity of these virions was significantly increased when calculated by infectious units per pg p24. interestingly, at 0.6 nm lopinavir, the infectivity of virions from ciita-expressing cells increased over vehicle-treated controls, despite reduced gag processing. further, the mutants of ciita, which produced the highest level of gag-pol, did not demonstrate a linear increase in gag processing or virion infectivity, which may be explained by previous work demonstrateing that increased gag-pol levels impairs viral infectivity [61, 62] . therefore it is likely that ciita is capable of increasing gag-pol levels to a point which can impede virus maturation, albeit not enough to reduce it to levels observed from vector-only expressing cells. therefore, overall infectivity of virions is increased from cells expressing ciita despite an altered gag to gag-pol ratio. future studies should focus on how ciita can increase this ratio without severely impairing virus release as well the novel mechanism by which ciita increases gag-pol levels. preliminary studies in this laboratory suggest that ciita enhancement gag-pol may be due to increased ribosomal frameshifting (data not shown). finally, it is tempting to speculate that ciita, which is expressed upon cd4+ t cell activation and increases viral protease levels, may also contribute to casp8p41-mediated apoptosis, which may link ciita to the decimation of t cells in the galt during primary viremia [67] . thymic epithelial cells [68] , cervical epithelial cells [69] , human colonic epithelial cells [70] and oral keratinocytes (normal human oral epithelial cells) [71] have all demonstrated in vitro the capability of being productively infected with hiv. infection of epithelial cells provides potential in vivo significance to this study, especially considering that thymic epithelial cells constitutively express mhc class ii (and thus ciita) [28] . in addition, most other cells can be stimulated to express ciita in the presence of ifn-c (i.e. a pro-inflammatory environment), which is induced during hiv infection of the galt [72] . overall, this study demonstrates that the function of ciita may be more broad than previously thought. given this previously undescribed role in enhancement of virion maturation, the precise consequences of ciita expression during the hiv replicative cycle may provide rationale for the development of novel antiviral therapeutics. a293t cells [73] were maintained as previously described [74] and ciita-stable a293t cell lines were generated by cotransfecting pvu i linearized pdna3.flag.ciita8 [75] and pcmv4his, a mammalian selection vector which encodes the histidinol dehydrogenase gene under control of the cmv promoter into the cells. stable clones were selected in dmem medium containing 5 mm l-histidinol (sigma-aldrich., st. louis, mo) and cloned by limiting dilution assay. ghost cell medium was supplemented with 0.2 mg/ml g418, 0.1 mg/ml hygromycin b and 1 mg/ml puromycin (sigma) as previously described [76] . using cdna reverse transcribed from a293t-ciita rna, we amplified hla-dra, hla-drb1 and hla-drb5 sequences (genbank accession nos. nm019111, nm002121, and nm002125, respectively), using primers containing forward restriction site xbai and the reverse restriction site hindiii (table s1 ). the haplotyping of hla-dr isotypes expressed by a293t cells was determined by the transplantation immunology lab, albany medical college. primers were designed to include an intact kozak sequence upstream of the translation start site. hla-dra and b5 plasmids were then used for site-directed mutagenesis using primers indicated (table s1 ). transient transfections of all plasmids, including: pdna3.flag.ciita8 [75] , ciita-gtp2 and -gtp3 [56] and ciita -l1035p [58] , p33-143 (coding for the p33 and p35 isoforms of invariant chain-a kind gift provided by dr. eric o. long, niaid), hla-dma and b (pmcfr-pac and pdmb/mcfrkindly provided by dr. lisa k. denzin, sloan-kettering cancer center), egfp-rab4b (kindly provided by dr. marci scidmore, cornell university), and hiv gag-igfp [77] (kindly provided by dr. benjamin chen, mount siani school of medicine) were performed using a 3:1 ratio fugene hd transfection reagent to dna in serum-free media as suggested by the manufacturer (roche, indianapolis, in). virus plasmid dna provided half of total dna used in transfection reactions. nih 3t3 balb/c cells are transfected with 1.5 mg ciita and 4.5 ml fugene hd (7:2 ratio of fugene to dna for optimal cells growth) and selected for with l-histidionol for two weeks and analyzed for mhc ii expressing using fluorescence-activated cell sorter (facs) with pe-conjugated mouse igg2a (cedar lane) against i-a d and clones were selected by limiting dilution. these cells were co-transfected with p32cdna and pchiv pal (which contains all hiv genes except the ltr sequences) at a 2:1 ratio. the p32cdna was donated by the peterlin laboratory [59] . both mhc ii expressing vlps and mhc ii-negative vlps were produced and concentrated 10-fold in a 100 k molecular weight cut-off filter tube (millipore) for 15 minutes at 4000 rpm prior to titering via hiv-1 p24 ca antigen capture assay kit were performed following the manufacturer's instructions (nih aids research and reference reagent program). cytoplasmic rna from 48 hr cultures of each cell type was collected using the rneasy mini kit according to manufacturer's instructions (qiagen, inc., valencia, ca). 1ug of dna-free rna was run out on a non-denaturing gel to ensure equal concentrations of each sample followed by reverse transcription of 1 mg of each rna sample using the iscript cdna synthesis kit, following manufacturer's instructions (biorad, hercules, ca). 50 ng of cdna was used to amplify hla-dm, ciita, gapdh, and ii from each cell type to confirm expression. forward and reverse primers sequences used in rt-pcr experiments indicated in table s1 . cells were analyzed for hla-dr expression as previously described [56] . briefly, cells were incubated with murine clone l243 [78] . densitometric analysis was performed as previously described [80] and the percentage of total hiv-1 a ca p24 (183-h12-5c) [79] reactive bands was used as a measure of gag processing [81] . intracellular gag staining 10 6 cells were permeabilized with intraprep permeabilization reagent (beckman-coulter, fullerton, ca), following manufacturer's instructions. gag was stained with fitc-conjugated fh190-1-1 (beckman-coulter) for approximately 15 m prior to analysis and data interpretation as performed above, with the exception of the percentage of gag+ cells was determined after gating on hla-dr+ cells. virions were purified using the mmacs streptavidin kit (miltenyi biotec inc., auburn, ca.), according to the manufacturer's instructions using biotinylated-l243 (biolegend). ghost assay [82] to determine infectious virus production and p24 elisas to determine virus particle release using the hiv-1 p24 ca antigen capture assay kit were performed following the manufacturer's instructions (nih aids research and reference reagent program). cells were transfected with either pcciita or empty pcdna vector 4.5 hours prior to lopinavir (nih aids research and reference reagent program) or dmso (vehicle-only) treatment at indicated concentrations. virus and cell supernatants were collected approximately 17 hours post-treatment. for determina-tion of p160gag-pol to p55gag ratios, producer cells were transfected with ciita constructs (pccitia, gtp2, gtp3, l1035p) or vector only control (pcdna) and then transfected with pnl4-3 16 hours later, prior to being treated with 80 nm lopinavir. cell lysates were used to monitor the levels of pr55gag to pr160pol via western blotting with hiv-1 a ca p24 (183-h12-5c) after 17 hours. table s1 primers used in this study. hiv-1 gag proteins: diverse functions in the virus life cycle tsg101: hiv-1's ticket to ride tsg101 and the vacuolar protein sorting pathway are essential for hiv-1 budding hiv gag mimics the tsg101-recruiting activity of the human hrs protein the activity of the protease of human immunodeficiency virus type 1 is initiated at the membrane of infected cells before the release of viral proteins and is required for release to occur with maximum efficiency partial inhibition of the human immunodeficiency virus type 1 protease results in aberrant virus assembly and the formation of noninfectious particles human immunodeficiency virus (hiv-1) gag processing intermediates trans-dominantly interfere with hiv-1 infectivity major histocompatibility complex class ii molecules promote human immunodeficiency virus type 1 assembly and budding to late endosomal/multivesicular body compartments assembly of infectious hiv-1 in human epithelial and t-lymphoblastic cell lines infectious hiv-1 assembles in late endosomes in primary macrophages more than one door -budding of enveloped viruses through cellular membranes retrovirus budding productive human immunodeficiency virus type 1 assembly takes place at the plasma membrane evidence that productive human immunodeficiency virus type 1 assembly can occur in an intracellular compartment the cell biology of hiv-1 virion genesis endosomes, exosomes and trojan viruses intracellular destinies: degradation, targeting, assembly, and endocytosis of hiv gag vpu and tsg101 regulate intracellular targeting of the human immunodeficiency virus type 1 core protein precursor pr55gag plasma membrane is the site of productive hiv-1 particle assembly endosomal trafficking of hiv-1 gag and genomic rnas regulates viral egress mhc class ii transport at a glance regulation of mhc class ii expression, a unique regulatory system identified by the study of a primary immunodeficiency disease genetic control of mhc class ii expression minireview: specificity and expression of ciita, the master regulator of mhc class ii genes genetic control of mhc class ii expression epigenetic control of ciita expression in leukemic t cells function and regulation of mhc class ii molecules in t-lymphocytes: of mice and men trafficking of mhc class ii molecules in the late secretory pathway mhc class ii molecules on the move for successful antigen presentation achieving stability through editing and chaperoning: regulation of mhc class ii peptide binding and expression identification of the hla-dm/hla-dr interface hla-dm induces clip dissociation from mhc class ii [alpha][beta] dimers and facilitates peptide loading the invariant chain is required for intracellular transport and function of major histocompatibility complex class ii molecules surface expression of mhc class ii in dendritic cells is controlled by regulated ubiquitination dendritic cells regulate exposure of mhc class ii at their plasma membrane by oligoubiquitination human immunodeficiency virus-1 nef expression induces intracellular accumulation of multivesicular bodies and major histocompatibility complex class ii complexes: potential role of phosphatidylinositol 3-kinase human immunodeficiency virus type 1 vpu protein interacts with cd74 and modulates major histocompatibility complex class ii presentation down-modulation of mature major histocompatibility complex class ii and up-regulation of invariant chain cell surface expression are well-conserved functions of human and simian immunodeficiency virus nef alleles hiv-1 gag polyprotein rescues hla-dr intracellular transport in a human cd4+ cell line differential incorporation of cd45, cd80 (b7-1), cd86 (b7-2), and major histocompatibility complex class i and ii molecules into human immunodeficiency virus type 1 virions and microvesicles: implications for viral pathogenesis and immune regulation distinct mechanisms of cd4+ and cd8+ t-cell activation and bystander apoptosis induced by human immunodeficiency virus type 1 virions mhc class ii and hiv pathogenesis: lots of data, few conclusions expression of rab4b, a protein governing endocytic recycling, is co-regulated with mhc class ii genes the presence of hostderived hla-dr1 on human immunodeficiency virus type 1 increases viral infectivity the acquisition of host-derived major histocompatibility complex class ii glycoproteins by human immunodeficiency virus type 1 accelerates the process of virus entry and infection in human t-lymphoid cells identification of ciita regulated genetic module dedicated for antigen presentation endosomal trafficking of hiv-1 gag and genomic rnas regulates viral egress vivoprocessing of pr160gag-polfrom human immunodeficiency virus type 1 (hiv) in acutely infected, cultured human t-lymphocytes comparison of human immunodeficiency virus type 1 pr55(gag) and pr160(gag-pol) processing intermediates that accumulate in primary and transformed cells treated with peptidic and nonpeptidic protease inhibitors programmed ribosomal frameshifting in hiv-1 and the sars-cov cellular proteins bound to immunodeficiency viruses: implications for pathogenesis and vaccines hiv-1 protease specificity of peptide cleavage is sufficient for processing of gag and pol polyproteins the regulation of sequential processing of hiv-1 gag by the viral protease complete mutagenesis of the hiv-1 protease gtp binding by class ii transactivator: role in nuclear import gtp-dependent recruitment of ciita to the class ii major histocompatibility complex promoter leucine-rich repeats of the class ii transactivator control its rate of nuclear accumulation human p32 protein relieves a posttranscriptional block to hiv replication in murine cells cell killing by hiv-1 protease overexpression of the gag-pol precursor from human immunodeficiency virus type 1 proviral genomes results in efficient proteolytic processing in the absence of virion production maintenance of the gag/gag-pol ratio is important for human immunodeficiency virus type 1 rna dimerization and viral infectivity the human immunodeficiency virus type 1 ribosomal frameshifting site is an invariant sequence determinant and an important target for antiviral therapy nef promotes endocytosis of cell surface mhc class ii molecules via a constitutive pathway mutant murine leukemia virus gag proteins lacking proline at the n-terminus of the capsid domain block infectivity in virions containing wild-type gag hiv-1 protease processes procaspase 8 to cause mitochondrial release of cytochrome c, caspase cleavage and nuclear fragmentation the gastrointestinal tract is critical to the pathogenesis of acute hiv-1 infection immunopathogenic mechanisms of hiv infection productive infection of a cervical epithelial cell line with human immunodeficiency virus: implications for sexual transmission galactosyl ceramide (or a closely related molecule) is the receptor for human immunodeficiency virus type 1 on human colon epithelial ht29 cells human immunodeficiency virus type 1 infection and replication in normal human oral keratinocytes hiv infection and gut mucosal immune function: updates on pathogenesis with implications for management and intervention characteristics of a human cell line transformed by dna from human adenovirus type 5 separation of human immunodeficiency virus type 1 replication from nef-mediated pathogenesis in the human thymus importance of acidic, proline/serine/ threonine-rich, and gtp-binding regions in the major histocompatibility complex class ii transactivator: generation of transdominant-negative mutants primary human immunodeficiency virus type 2 (hiv-2) isolates, like hiv-1 isolates, frequently use ccr5 but show promiscuity in coreceptor usage predominant mode of human immunodeficiency virus transfer between t cells is mediated by sustained env-dependent neutralization-resistant virological synapses hla-dr alpha chain residues located on the outer loops are involved in nonpolymorphic and polymorphic antibody-binding epitopes macrophage-tropic human immunodeficiency virus isolates from different patients exhibit unusual v3 envelope sequence homogeneity in comparison with t-cell-tropic isolates: definition of critical amino acids involved in cell tropism functional analysis of glucan binding protein b from streptococcus mutans human immunodeficiency virus (hiv-1) gag processing intermediates trans-dominantly interfere with hiv-1 infectivity characterization of a thymus-tropic hiv-1 isolate from a rapid progressor: role of the envelope key: cord-262748-v4xue7ha authors: xu, yongtao; yu, shui; zou, jian-wei; hu, guixiang; rahman, noorsaadah a. b. d.; othman, rozana binti; tao, xia; huang, meilan title: identification of peptide inhibitors of enveloped viruses using support vector machine date: 2015-12-04 journal: plos one doi: 10.1371/journal.pone.0144171 sha: doc_id: 262748 cord_uid: v4xue7ha the peptides derived from envelope proteins have been shown to inhibit the protein-protein interactions in the virus membrane fusion process and thus have a great potential to be developed into effective antiviral therapies. there are three types of envelope proteins each exhibiting distinct structure folds. although the exact fusion mechanism remains elusive, it was suggested that the three classes of viral fusion proteins share a similar mechanism of membrane fusion. the common mechanism of action makes it possible to correlate the properties of self-derived peptide inhibitors with their activities. here we developed a support vector machine model using sequence-based statistical scores of self-derived peptide inhibitors as input features to correlate with their activities. the model displayed 92% prediction accuracy with the matthew’s correlation coefficient of 0.84, obviously superior to those using physicochemical properties and amino acid decomposition as input. the predictive support vector machine model for selfderived peptides of envelope proteins would be useful in development of antiviral peptide inhibitors targeting the virus fusion process. fusion process is the initial step of viral infection, therefore targeting the fusion process represents a promising strategy in design of antiviral therapy [1] . the entry step involves fusion of the viral and the cellular receptor membranes, which is mediated by the viral envelope (e) proteins. there are three classes of envelope proteins [2] : class i e proteins include influenza virus (ifv) hemagglutinin and retrovirus human immunodeficiency virus 1 (hiv-1) gp41; class ii e proteins include a number of important human flavivirus pathogens such as dengue virus (denv), japanese encephalitis virus (jev), yellow fever virus (yfv), west nile virus (wnv), hepatitis c virus (hcv) and togaviridae virus such as alphavirus semliki forest virus (sfv); class iii e proteins include vesicular stomatitis virus (vsv), herpes simplex virus-1 (hsv-1) and human cytomegalovirus (hcmv). although the exact fusion mechanism remains elusive and the three classes of viral fusion proteins exhibit distinct structural folds, they may share a similar mechanism of membrane fusion [3] . a peptide derived from a protein-protein interface would inhibit the formation of that interface by mimicking the interactions with its partner proteins, and therefore may serve as a promising lead in drug discovery [4] . enfuvirtide (t20), a peptide that mimicks the hr2 region of class i hiv-1 gp41, is the first fda-approved hiv-1 fusion drug that inhibits the entry process of virus infection [5] [6] [7] . then peptides mimicking extended regions of the hiv-1 gp41 were also demonstrated as effective entry inhibitors [8, 9] . furthermore, peptides derived from a distinct region of gb virus c e2 protein were found to interfere with the very early events of the hiv-1 replication cycle [10] . other successful examples of class i peptide inhibitors include peptide inhibitors derived from sars-cov spike glycoprotein [11] [12] [13] and from pichinde virus (picv) envelope protein [14] . recently, a peptide derived from the fusion initiation region of the glycoprotein hemagglutinin (ha) in ifv, flufirvitide-3 (ff-3) has progressed into clinical trial [15] . the success of developing the class i peptide inhibitors into clinical use has triggered the interests in the design of inhibitors of the class ii and class iii e proteins. e.g. several hydrophobic peptides derived from the class ii denv and wnv e proteins exhibited potent inhibitory activities [16] [17] [18] [19] [20] . in addition, a potent peptide inhibitor derived from the domain iii of jev glycoprotein and a peptide inhibitor derived from the stem region of rift valley fever virus (rvfv) glycoprotein were reported [21, 22] . examples of the class ii peptide inhibitors of enveloped virus also include those derived from hcv e2 protein [23, 24] and from claudin-1, a critical host factor in hcv entry [25] . moreover, peptides derived from the class iii hsv-1 gb also exhibited antiviral activities [26] [27] [28] [29] [30] [31] , as well as those derived from hcmv gb [32] . computational informatics plays an important role in predicting the activities of the peptides generated from combinatorial libraries. in silico methods such as data mining, generic algorithm and vector-like analysis were reported to predict the antimicrobial activities of peptides [33] [34] [35] . in addition, quantitative structure-activity relationships (qsar) [36] [37] [38] [39] [40] and artificial neural networks (ann) were applied to predict the activities of peptides [41, 42] . recently, a support vector machine (svm) algorithm was employed to predict the antivirus activities using the physicochemical properties of general antiviral peptides [43] . however, the mechanism of action of antiviral peptides is different from antimicrobial peptides; in fact, various protein targets are involved in the virus infection. e.g. hiv-1 virus infection involves virus fusion, integration, reverse transcription and maturation, etc. thus it is difficult to retrieve the common features from general antiviral peptides to represent their antiviral activities. virus fusion is mediated by e proteins. although e proteins are highly divergent in sequence and structure, they share a common pathway of membrane fusion dynamics. i.e. e proteins experience significant conformational change to form a-trimer-of-hairpin, which drives the fusion of viral membrane and host membrane [44] . the antiviral peptides derived from enveloped proteins function by in situ binding to their respective accessory proteins, disrupting forming of the trimer-of-hairpin and membrane fusion, and therefore inhibiting the virus infection. in view of the important role of e proteins in virus fusion process and common mechanism of action of self-derived peptides, we developed a svm model to predict the antiviral activities of self-derived peptides using sequence-based statistical scores as input features. the sequencebased properties were calculated by a conditional probability discriminatory function which indicates the propensity of each amino acid for being active at a specific position. our model exhibited remarkably higher accuracy in predicting the activities of self-derived peptides, compared to the previous models developed for general antiviral peptides using classical physicochemical properties as descriptors [43] . the method would be useful in identification of entry inhibitors as a new generation of antiviral therapies. 202 peptide virus entry inhibitors of enveloped viruses were collected, among them, 101 are active peptides and 101 are non-active peptides. these peptides comprised the 75p+75n training set of svm models. the remaining 26 active peptides and 26 non-active peptides inhibitors were used as the test set. amino acid composition. amino acid composition is the fraction of each amino acid in a peptide. the fraction of the 20 amino acids was calculated using the following equation: fraction of amino acid x ¼ total number of x = peptide length five physicochemical properties were used in svm models. isoelectric point (pi), molecular weight (mw) and grand average of hydropathicity (gravy) [45] were calculated using the protparam tool implemented in expasy web server. solvent accessibility and secondary structure features were calculated using sspro and accpro packages implemented in the scratch protein predictor server [46] . sequence-based statistical scoring function. the knowledge-based statistical function is developed from the concept of residue-specific all-atom probability discriminatory function (rapdf) [47] . rapdf is a structure-based statistical scoring function. it is based on the assumption that averaging over different atom types in experimental conformations is an adequate representation of the random arrangements of these atom types in any compact conformation. here we developed a sequence-based statistical scoring function, where we presume that averaging over different amino acid sequences with experimental validated inhibitive activities is an adequate representation of the random amino acid sequences with any inhibitory activity. the basis of this assumption is that the peptides share a common mechanism of action, i.e. the peptides derived from e proteins bind competitively to their partner proteins, disrupt the forming of a-trimer-of-hairpin, and therefore inhibit the virus membrane fusion. the sequence-based scoring function is described in the following form: sðfq i a gþ ¼ àln here, q i a 2 factiveg. pðq i a jcþ is the probability of observing amino acid i in an active peptide sequence; pðq i a þ is the probability of observing amino acid i in any peptide sequence, active or nonactive. they are approximately estimated using the following forms: similarly, we employed a dataset of experimentally verified non-active peptides in developing the statistical function, where q i a 2 finactiveg. for a given amino acid sequence, 20 columns of input are generated, corresponding to the occurrence of twenty natural amino acids at each position. each column is assigned a value of n ã (−log-likelihood), where n is the number of amino acid and −log-likelihood is derived from the statistical function score. each of the features thus combines the propensity of the amino acid for being active or non-active with the corresponding amino acid composition. below is an example of calculating the statistical scores for a given peptide sequence: the amino acid order for svm input features is set as: acdefghiklmnpqrstvwy. if the amino acid sequence of an active peptide inhibitor is: svm models combined with radial basis function (rbf) kernel parameters were developed using the c-svc module in libsvm (version 3.1) [48, 49] and executed under the matlab interface. the performance of svm depends on two parameters, gamma -g and cost-c [50] . the default value is 1 for -c and 1/k for -g, where k is the number of input entries. various pairs of (c, g) values were converted to exponential values (i.e. 2 x ;2 y ) and optimized using cross-validation and the pair with the best cross-validation accuracy was selected. 5-fold cross validation was performed to evaluate the performance of svm models. in the evaluation process, dataset was partitioned randomly into five equally sized subsets. the training and testing were carried out five times, each time four distinct subsets being used as training sets and the remaining subset as test set. the results were averaged over all five rounds of validation. the following equations were used to evaluate the prediction quality of the svm models [48, 51] : in the above equations, tp is the number of true positives, tn is the number of true negatives, fp is the number of false positives and fn is the number of false negatives. matthew's correlation coefficient (mcc) reflects the performance of the model. it ranges between -1 to 1 and a larger mcc value indicates a better prediction. svm learning algorithm is a powerful machine learning method that has been widely used in pattern recognition and classification. svm trains a dataset of experimentally validated positive and negative samples and generates a classifier to classify unknown samples into two distinct categories (positive or negative). we performed an exhaustive literature search on self-derived peptide inhibitors of enveloped proteins and collected experimentally validated peptides derived from the three classes of e proteins. for those peptides with overlapping segments, only one peptide sequence was kept. 202 peptides were found, among them, 101 are active peptides and 101 are non-active peptides ( table 1) . 75 active peptide inhibitors and 75 non-active peptides (75p+75n) of e proteins were used as the training dataset in svm learning; the remaining 26 active and 26 non-active peptides (26p+26n) were used as the test set. svm input features. three svm models were developed using different features as input descriptors, namely physicochemical properties (denoted as eapphysico), amino acid composition (eapcompo) and statistical scoring function amino acid composition (eapscoring). knowledge-based statistical functions are rooted in the bayesian (conditional) probability formalism and derived directly from properties observed in the known folded proteins [52] [53] [54] . in knowledge-based scoring function, it was presumed that averaging over different atom types in experimental conformations is an adequate representation of the random arrangements of these atom types in any compact conformation [55] . because the three classes of e proteins have different structural folds, it is difficult to retrieve a structure-based feature that is relevant to their antiviral activities. generally speaking, any property associated with folded proteins can be converted into an energy function [56] . since amino acid sequence determines the structural folds and properties of proteins/peptides, we presumed that a sequence-based statistical scoring function averaging over different amino acid sequences exhibiting inhibitive activities is an adequate representation of the random combinations of all twenty amino acid exhibiting any activity. in this approach, a peptide sequence derived from e protein is represented by twenty features each corresponding to the propensity of observing each of the twenty natural amino acids to be either active or non-active. a vector space of twenty sequence-based statistical scores was used as the eapscoring input entries in the svm learning. we also built a svm model using physicochemical properties as input features. because of the feature of membrane fusion process, it was suggested that functional regions in glycoproteins need to be solvent accessible, hydrophobic and flexible [57] . actually the majority of known peptide entry inhibitors share a common physicochemical property of being hydrophobic and amphipathic with a propensity for binding to lipid membranes [58] . therefore, here the properties of e peptide inhibitors were described by five physicochemical parameters: pi, mw, gravy index (positive and negative gravy values indicate hydrophobic and hydrophilic peptides, respectively), solvent accessibility (exposed or buried) and secondary structure features (propensity for adopting α-helix, β-sheet or turn structure). these physicochemical features were calculated for each of the peptides and used as the eapphysico input entries in the svm learning. a third svm model eapcompo was also built where the fractions of amino acids in a peptide were used as input features in the machine learning process. svm training. the svm models were trained using the experimentally validated 75p+ 75n data sets. during 5-fold cross validation, the training set was randomly partitioned into four subsets with equal size of (15p+15n) and a remaining subset (15p+15n). three svm models were built using sequence-based statistical scores, physicochemical properties and amino acid composition, respectively. the performances of the three models are shown in table 2 . it can be seen that the eapscoring model performed best among the three models during 5-fold cross validation. a "grid-search" combined with cross-validation was adopted to search for the optimal parameters -c and -g in svm models [49] . the result of the grid search is shown in the support information (s1 file). it is shown that the performances of three eap models during 5-fold cross validation have been improved significantly using the optimized parameters ( table 2) . the performance of the svm models was evaluated using an independent dataset of experimentally validated peptides that were not contained in the learning dataset (table 1 ). in the eapphysico model where physicochemical properties of peptides were used as input features, an accuracy of 65% with a mcc value of 0.31 was observed (table 3 ). in the eapcompo model where amino acid composition features were used, the predictive accuracy and the mcc value are slightly higher. when the sequence-based statistical function scores were used as input in the eapscoring model, a remarkable accuracy of 92% was achieved with a mcc value of 0.84. thus the sequence-based statistical scores developed in the present research are predominantly superior to the conventional physicochemical properties or amino acid decomposition features in identifying active peptides derived from enveloped proteins. avppred is a web server for prediction of the activities of general antiviral peptides (avps) based on a number of experimentally validated positive and negative data sets [43] . the peptide inhibitors employed in avppred target a variety of biological targets involved in virus infection. in contrast, the self-derived peptides of enveloped proteins being studied in the present research competitively bind to e proteins so as to mediate the virus fusion process. because the self-derived peptides share similar mechanism of action, it is feasible to retrieve common features from them to build predictive svm models. in order to evaluate the performance in predicting peptide inhibitors of the enveloped virus, we compared the avppred models with our eappred models using an independent 26p+26n dataset as test set. the results are shown in table 3 . four different features were employed in the avppred models, namely conserved motif search using meme/mast, amino acid composition, sequence alignment using blast and physicochemical parameters including secondary structure, charge, size, hydrophobicity and amphiphilic character [43] . when the avpmotif model was used to predict the activities of the self-derived peptide inhibitors, it performed rather poorly with accuracy of 52% and mcc of 0.14. this is not surprising because avpmotif was developed based on 20 general antiviral peptide motifs. however, the self-derived peptide inhibitors may not share a conserved motif with the general antiviral peptides since the latter interact with various biological targets with different mechanisms of action. in the avpalign model, the peptide sequences were classified into active and non-active databases and the query peptide sequences were matched against the active and non-active databases using the blast program. compared with avpcompo and avpphysico, avpalign performed better with a predictive accuracy of 73% and mcc value of 0.52. fusion mechanism is highly conserved among related viruses and entry of viruses into host cells has been inhibited by peptides derived from various regions of envelope glycoproteins [59] . self-derived peptides would inhibit interactions of their original domain by mimicking its mode of binding to partner proteins [4] . because similar sequences are often associated with similar structure and function, the sequence-based property avpalign would account for the activities of the self-derived peptide inhibitors which regulate the virus fusion by mimicking the binding to e proteins. in the avpphysico model, 25 best performing physicochemical properties were selected out of the 544 properties to build the svm model [43] . antiviral peptide inhibitors are generally amphiphilic [60] and the activities of peptide entry inhibitors are dependent on their interfacial hydrophobicity [58] . therefore we only employed five physicochemical properties reflecting hydrophobicity, solvent accessibility and secondary structure features as svm input features. it was demonstrated that the accuracy and mcc of eapphysico is comparable to that of avpphysico model, indicating the five properties used in current modeling building are critical for their activities. the mcc value of the avpcompo models is 0.20, indicating that the antiviral activities of the peptides are related to amino acid composition. when the amino acid composition was used as input, the predictive accuracy of the eapcompo model was higher than that of the avpcompo model, indicating the peptide inhibitors of e proteins employed in the training set is sufficient to represent the contribution of amino acid composition to their inhibitive activities. in the eapcompo model, the preference of the amino acid composition was ranked as: p, r, q, d, f, w, e, l, t, i, n, h, y, c, a, s, m, v, k, g (fig 1) . the role of arginine-arginine pairing and its contribution to protein-protein interactions has been investigated by computational approaches [61] . the higher abundance of r at protein-protein interfaces compared to k may be attributed to the formation of cation-π-interactions and the greater capacity of the guanidinium group in r to form hydrogen bonds (compared to k) [62] [63] [64] . furthermore, it was suggested that the interface regions are enriched in aliphatic (l, v, i, m) and aromatic (h, f, y, w) residues and depleted in charged residues (d, e, k) with the exception of arginine [62, [65] [66] [67] [68] [69] . this is in agreement with our amino acid composition analysis, where higher population of aliphatic leu residue as well as aromatic residues trp and phe was observed, whereas positively charged lys was hardly observed. the predominant occurrence of proline and glutamine residues is characteristic for the unique protein-protein interactions for e proteins. e.g. a conserved proline-rich motif was suggested to be engaged in monomer-monomer interactions in dengue e proteins [70] . a conserved glutamine-rich layer is involved in the extensive hbond network in hiv-1 gp41 e proteins [71] . thus the preference of the amino acid composition identified from the eapcompo model is generally in accordance with the predominant residues involved in protein-protein interactions, manifesting the amino acid composition of the self-derived peptide inhibitors are closely related to their potential activities in mediating the protein-protein interactions in the virus fusion process. because the antiviral activities of peptides are dependent on amino acid composition, we presume amino acid composition discriminated by the propensity of their activities would be an intrinsic feature in the self-derived peptide inhibitors which share a common mechanism of action. when statistical function scores were employed in the svm model (eapscoring), a remarkable predictive accuracy of 92% with an ideal mcc value of 0.84 was achieved, significantly better than any avp models. the logarithm form of the discriminatory function (eq 1) can be deemed as the pseudo energy of the system. in our previous study, we suggested that the stability of proteins is related to their in situ binding potential to the partner regions [72] . the prominent performance of eapscoring model indicates the sequence-based stability feature of self-derived peptides may reflect their potential of binding to e proteins so as to regulate the virus entry process. we developed three svm models using physicochemical properties, amino acid composition and statistical discriminative function as input features. the prediction accuracy and the mcc value of the eapphysico model where five physicochemical properties were employed are comparable with the previous avpphysico model where 25 physicochemical properties were used. the avpcompo and eapcompo models demonstrated that the activities of antiviral peptides are dependent on amino acid composition. a sequence-based scoring function was developed for the self-derived peptide inhibitors of e proteins. the outperformance of the eapscoring models supports our hypothesis that an intrinsic feature, represented by the propensity of each amino acid for being active in self-derived peptides, is responsible for the activities of the peptides to regulate virus fusion by mimicking the binding to their accessory proteins. the sequence-based statistical scoring function would be useful in development of novel antiviral therapies to target the initial step of viral infection. supporting information s1 file. parameters optimization by grid-research combined with 5-fold cross validation. x-axis is log2 g , y is log2 c and z-axis represents accuracy(%) ( figure a targeting cell entry of enveloped viruses as an antiviral strategy. molecules class iii viral membrane fusion proteins virus membrane-fusion proteins: more than one way to make a hairpin can self-inhibitory peptides be derived from the interfaces of globular protein-protein interactions? characterization of a putative cellular receptor for hiv-1 transmembrane glycoprotein using synthetic peptides propensity for a leucine zipperlike domain of human immunodeficiency virus type 1 gp41 to form oligomers correlates with a role in virus-induced fusion rather than assembly of the glycoprotein complex a synthetic peptide from hiv-1 gp41 is a potent inhibitor of virusmediated cell-cell fusion hiv gp41 c-terminal heptad repeat contains multifunctional domains. relation to mechanisms of action of anti-hiv peptides inhibition of human immunodeficiency virus type 1 entry in cells expressing gp41-derived peptides peptides derived from a distinct region of gb virus c glycoprotein e2 mediate strain-specific hiv-1 entry inhibition inhibition of severe acute respiratory syndrome-associated coronavirus (sars-cov) infectivity by peptides analogous to the viral spike protein synthetic peptides outside the spike protein heptad repeat regions as potent inhibitors of sars-associated coronavirus suppression of sars-cov entry by peptides corresponding to heptad regions on spike glycoprotein design and characterization of glycoprotein-derived peptide inhibitors of arena virus infection autoimmune technologies, safety, tolerability, and pk of escalating doses of flufirvitide-3 dry powder for inhalation in healthy subjects peptide inhibitors of dengue virus and west nile virus infectivity structural optimization and de novo design of dengue virus entry inhibitory peptides. plos neglected tropical diseases antiviral peptides targeting the west nile virus envelope protein peptide inhibitors of dengue virus entry target a late-stage fusion intermediate inhibition of dengue virus entry into target cells using synthetic antiviral peptides inhibition of japanese encephalitis virus entry into the cells by the envelope glycoprotein domain iii (ediii) and the loop3 peptide derived from ediii a fusion-inhibiting peptide against rift valley fever virus inhibits multiple, diverse viruses a peptide derived from hepatitis c virus e2 envelope protein inhibits a post-binding step in hcv entry early events in hepatitis c virus infection: an interplay of viral entry human claudin-1-derived peptide inhibits hepatitis c virus entry peptides containing membraneinteracting motifs inhibit herpes simplex virus type 1 infectivity evidence for a role of the membrane-proximal region of herpes simplex virus type 1 glycoprotein h in membrane fusion and virus inhibition the identification and characterization of fusogenic domains in herpes virus glycoprotein b molecules analysis of synthetic peptides from heptad-repeat domains of herpes simplex virus type 1 glycoproteins h and b conformational modifications of gb from herpes simplex virus type 1 analyzed by synthetic peptides multiple peptides homologous to herpes simplex virus type 1 glycoprotein b inhibit viral infection peptide inhibition of human cytomegalovirus infection a theoretical approach to spot active regions in antimicrobial proteins optimization of antibacterial peptides by genetic algorithms and cheminformatics antibp2: improved version of antibacterial peptide prediction application of 'inductive' qsar descriptors forquantification of antibacterial activity of cationic polypeptides qsar analysis of antimicrobial and haemolytic effects of cyclic cationic antimicrobial peptides derived from protegrin-1 design of novispirin antimicrobial peptides by quantitative structure-activity relationship qsar modeling and computer-aided design of antimicrobial peptides identification of novel antibacterial peptides by chemoinformatics and machine learning de novo design of potent antimicrobial peptides evaluating different descriptors for model design of antimicrobial peptides with enhanced activity toward p. aeruginosa avppred: collection and prediction of highly effective antiviral peptides structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme a simple method for displaying the hydropathic character of a protein scratch: a protein structure and structural feature prediction server an all-atom distance-dependent conditional probability discriminatory function for protein structure prediction support-vector networks working set selection using second order information for training svm a practical guide to support vector classification. initial version comparison of the predicted and observed secondary structure of t4 phage lysozyme distance-scaled, finite ideal-gas reference state improves structure-derived potentials of mean force for structure selection and stability prediction a distance-dependent atomic knowledge-based potential for improved protein structure selection statistical potential for assessment and prediction of protein structures comparison of database potentials and molecular mechanics force fields scoring functions for de novo protein structure prediction revisited peptide inhibitors against herpes simplex virus infections peptide entry inhibitors of enveloped viruses: the importance of interfacial hydrophobicity structure-based design of inhibitors of protein-protein interactions: mimicking peptide binding epitopes broad-spectrum antivirals against viral fusion the molecular origin of like-charge arginine -arginine pairing in water residue frequencies and pairing preferences at proteinprotein interfaces dissection of specific and non-specific protein-protein interfaces dissecting protein-protein recognition sites principles of protein-protein interactions studies of protein-protein interfaces: a statistical analysis of the hydrophobic effect the atomic structure of protein-protein recognition sites genome-wide studies of protein-protein interaction the interface of protein-protein complexes: analysis of contacts and prediction of interactions prediction of protein-protein interactions in dengue virus coat proteins guided by low resolution cryoem structures the fusion activity of hiv-1 gp41 depends on interhelical interactions computational identification of self-inhibitory peptides from envelope proteins the authors are grateful for the computing resources from qub high performance computing centre. the authors declare no conflict of interest. key: cord-268816-nth3o6ot authors: roy, satyaki; ghosh, preetam title: factors affecting covid-19 infected and death rates inform lockdown-related policymaking date: 2020-10-23 journal: plos one doi: 10.1371/journal.pone.0241165 sha: doc_id: 268816 cord_uid: nth3o6ot background: after claiming nearly five hundred thousand lives globally, the covid-19 pandemic is showing no signs of slowing down. while the uk, usa, brazil and parts of asia are bracing themselves for the second wave—or the extension of the first wave—it is imperative to identify the primary social, economic, environmental, demographic, ethnic, cultural and health factors contributing towards covid-19 infection and mortality numbers to facilitate mitigation and control measures. methods: we process several open-access datasets on us states to create an integrated dataset of potential factors leading to the pandemic spread. we then apply several supervised machine learning approaches to reach a consensus as well as rank the key factors. we carry out regression analysis to pinpoint the key pre-lockdown factors that affect post-lockdown infection and mortality, informing future lockdown-related policy making. findings: population density, testing numbers and airport traffic emerge as the most discriminatory factors, followed by higher age groups (above 40 and specifically 60+). post-lockdown infected and death rates are highly influenced by their pre-lockdown counterparts, followed by population density and airport traffic. while healthcare index seems uncorrelated with mortality rate, principal component analysis on the key features show two groups: states (1) forming early epicenters and (2) experiencing strong second wave or peaking late in rate of infection and death. finally, a small case study on new york city shows that days-to-peak for infection of neighboring boroughs correlate better with inter-zone mobility than the inter-zone distance. interpretation: states forming the early hotspots are regions with high airport or road traffic resulting in human interaction. us states with high population density and testing tend to exhibit consistently high infected and death numbers. mortality rate seems to be driven by individual physiology, preexisting condition, age etc., rather than gender, healthcare facility or ethnic predisposition. finally, policymaking on the timing of lockdowns should primarily consider the pre-lockdown infected numbers along with population density and airport traffic. we process several open-access datasets on us states to create an integrated dataset of potential factors leading to the pandemic spread. we then apply several supervised machine learning approaches to reach a consensus as well as rank the key factors. we carry out regression analysis to pinpoint the key pre-lockdown factors that affect post-lockdown infection and mortality, informing future lockdown-related policy making. population density, testing numbers and airport traffic emerge as the most discriminatory factors, followed by higher age groups (above 40 and specifically 60+). post-lockdown infected and death rates are highly influenced by their pre-lockdown counterparts, followed by population density and airport traffic. while healthcare index seems uncorrelated with mortality rate, principal component analysis on the key features show two groups: states (1) forming early epicenters and (2) experiencing strong second wave or peaking late in rate of infection and death. finally, a small case study on new york city shows that days-to-peak for infection of neighboring boroughs correlate better with inter-zone mobility than the interzone distance. states forming the early hotspots are regions with high airport or road traffic resulting in human interaction. us states with high population density and testing tend to exhibit during pre-and post-covid periods to show that the odds of mortality of whites and blacks are statistically equivalent [23] . myers et al. analyzed the covid-19 positive patients in california to investigate its prognosis in the higher age groups and individuals with preexisting conditions [24] . zoabi et al. applied ml on 51,831 covid-19 positive patients to understand the effect of gender, age and contact to show that close social interaction is a strong feature for covid-19 transmissibility [25] . khan et al. applied regression tree, cluster analysis and principal component analysis on worldometer infection count data to study the variability and effect of testing in prediction of confirmed cases [26] . finally, pan et al. studied the effects of the myriad public health interventions (such as lockdown, traffic restriction, social distancing, home quarantine, centralized quarantine, etc.) on 32,583 covid-19 patients, with respect to their age, sex, residential location, occupation, and severity [27] . contributions: while it is evident that factors such as gender, race, age, testing, social contact and distancing have been analyzed in a piecemeal manner, there is no comprehensive study that combines the demographic, economic, and epidemiological, ethnic and health indicators for infection and mortality from covid-19. to address this gap, we carry out a machine learning-based analysis with the following three objectives. 1. we curate a dataset of diverse features (detailed in sec. 2.1) from 50 states of usa. this dataset is somewhat unique, since, in addition to the above features, it includes factors such as airport traffic, homeless and variations in lockdown dates. also, note that the lockdown was enforced on the us states at around the same time, when each state was at a different stage of the covid-19 infection cycle. 2. we analyze the variation of covid-19 infection spread and mortality rates using a set of standard supervised ml methods. we rank the key discriminatory factors based on the importance score calculated from randomized decision trees. we combine the findings to identify the most vulnerable age groups and us states. we also show the effect of testing and lockdowns on the infection spread dynamics. 3. we utilize multiple linear regression to gauge the extent to which the key pre-lockdown factors affect the post-lockdown infected and death numbers. this study assigns weights to features and drive mitigation efforts and large scale policymaking. our data-driven experiments using supervised methods demonstrate that population density, testing [28] and airport traffic [29] are key factors contributing to infection and mortality rates. furthermore, high age group (40 and beyond, and specifically exceeding 60) population are more vulnerable. principal component analysis on the key features show two groups: highly affected us states (1) forming early epicenters and (2) showing consistent or newly peaking rate of infection and death. multiple regression analysis shows that the postlockdown numbers are most influenced by the pre-lockdown infected and death numbers followed by population density and airport activity, while overall healthcare index of a state does not seem to play a part in the overall death count. similarly, the race of individuals did not play any significant role in the infection or mortality numbers. despite increased testing rates, the fraction of individuals tested positive drop approximately three weeks into the lockdown, suggesting that the social distance measures has had an impact on curbing spread. finally, we discuss the role of mobility and distance in infection spread. in the absence of large-scale inter-state mobility data, our case study on the boroughs of new york city show that peaks of infection correlate better with inter-zone mobility than the interzone distance. all the experiments have been performed using scikit-learn, which is a popular machine learning library in python [30] . let us discuss the details of the two datasets used in this work. 2.1.1 data from us states. our dataset has been carefully curated from several open sources to examine the possible factors that may affect the covid-19 related infection and death numbers in the 50 states of usa. the individual open-access data sources as well as the integrated (curated) dataset has been shared on github (https://github.com/satunr/covid-19/tree/master/us-covid-dataset). below, we discuss a summary of the features and output labels of the integrated dataset. • gross domestic product (in terms of million us dollars) for us states [31] (filename: source/ gdp.xlsx, feature name: gdp). • distance from one state to another (is not measured in miles but the euclidean distance between their latitude-longitude coordinates between the pair of states [32] ) (filename: source/data_distance.xlsx, feature name: d(state1, state2)). • gender feature(s) is a fraction of total population representing the male and female individuals [33] (filename: source/data_gender.csv, feature name: male, female). • ethnicity feature(s) are the fraction of total population representing white, black, hispanic and asian individuals (we leave out other smaller ethnic groups) [34] (filename: source/ data_ethnic.csv, feature name: white, black, hispanic and asian). • healthcare index is measured by agency for healthcare research and quality (ahrq) on the basis of (1) type of care (like preventive, chronic), (2) setting of care (like nursing homes, hospitals), and (3) clinical areas (like care for patients with cancer, diabetes) [35] (filename: source/data_health.xlsx, feature name: health). • homeless feature is the number of homeless individuals of a state [36] (filename: source/ data_homeless.xlsx, feature name: homeless). the normalized homeless population of each state is the ratio between its homeless and total population. • total cases (and deaths) of covid-19 is the number of individuals tested positive and dead [37] (filename: source/data_covid_total.xlsx, feature name: total cases and total death). the normalized infected/death is the ratio between the infected/death count to total population of the given state. • infected score and death score is obtained by rounding normalized total cases and deaths to discrete value between 0-6 (feature name: infected score, death score). • death-to-infected is a feature measuring impact of death in terms of the difference between death and infected scores. it is calculated as max(death score -infected score, 0). • lockdown type is a feature capturing the type of lockdown (shelter in place: 1 and stay at home: 2) in a given state [37, 38] (filename: source/data_lockdown.csv, feature name: lockdown). • day of lockdown captures the difference in days between 1st january 2020 to the date of imposition of lockdown in a region [39] (filename: source/data_lockdown.csv, feature name: day lockdown). • population density is the ratio between the population and area of a region [40] (filename: source/data_population.csv, feature name: population, area, population density). • traffic/activity of airport measures the passenger traffic (also normalized by the total traffic across all the states of usa [41] (filename: source/data_airport.xlsx, feature name: busy airport score, normalized busy airport). • age groups (0-80+) in brackets of 4 year (also normalized by total population) [40] (filename: source/data_age.xlsx, feature name: age_to_, norm_to_, e.g. age4to8); we later group them in brackets of 20 for the purposes of analysis. • peak infected (and peak death) measures the duration between first date of infection and date of daily infected (and death) peaks [40] (feature name: peak infected, peak death). • testing measures the number of individuals tested for covid-19 (total number, before and after imposition of lockdown) [38, 42] (filename: source/data_testing.xlsx, feature name: testing, pre-lockdown testing, post-lockdown testing). • pre-and post-infected and death count measures the number of individuals infected and dead before and after lockdown dates (feature name: testing, pre-infected count, pre-death count, post-infected count, post-death count). • days between first infected and lockdown date (feature name: first-inf-lockdown). the above features, their abbreviations and summary statistics (i.e., mean, standard deviation, maximum and minimum) are enlisted in table 1 . note that, for gender and ethnicity we report the fraction of the total state population falling in each category. the new york city (nyc) datasets (https://github.com/ satunr/covid-19/blob/master/us-covid-dataset/nyc_dist_mob.xlsx) show the inter-borough distance and mobility as well as covid-19 infected (https://github.com/satunr/covid-19/blob/master/us-covid-dataset/nyc-inf.xlsx) and death counts (https://github.com/ satunr/covid-19/blob/master/us-covid-dataset/nyc-dth.xlsx) for the 5 boroughs of nyc, namely, manhattan, queens, brooklyn, bronx and staten island. table 1 . summary of features and their statistics (i.e., mean, standard deviation (dev.), maximum (max.) and minimum (min.)). the features in the order shown under "feature name" are: gdp, inter-state distance based on lat-long coordinates, gender, ethnicity, quality of health care facility, number of homeless people, total infected and death, population density, airport passenger traffic, age group, days for infection and death to peak, number of people tested for covid-19, days elapsed between first reported infection and the imposition of lockdown measures at a given state. factors affecting covid-19 infected and death rates inform lockdown-related policymaking • mobility data (based on traffic volume counts collected by dot for new york metropolitan transportation council (nymtc) [43] ) shows the number of trips from one borough to another. • covid-19 data shows the number of covid-19 infected and death counts for each borough [44] . we acquire the daily infected and testing counts across us from january-july, 2020 [45] . this dataset is part of the covid tracking project that collect covid-19 statistics on the numbers on tests, cases, hospitalizations, and patient outcomes from every us state and territory by voluntary public participation. we use the scikit-learn library kbinsdiscretizer to group the continuous feature values into discrete values by creating balanced clusters using the quantile strategy [46] . 2.1.5 supervised learning methods. supervised machine learning algorithms learn a function that maps the input training data (i.e., features) to some output labels [47] . in this work, we consider the following supervised learning techniques. (refer [48] [49] [50] [51] [52] [53] [54] for the details on these ml approaches.) • support vector machine (svm) is used for classification and regression problems that maps the inputs to high-dimensional feature spaces. svm operates on hyperplanes-decision boundaries that help classify the data points. the objective is to maximize the separation between the data points and the hyperplane. svm is memory efficient and effective for datasets with fewer data samples [55] . • stochastic gradient descent (sgd) is an iterative approach that fits the data to an objective function [56] . as the name suggests, it is a stochastic variant of the popular gradient descent (gd) optimization model [57] . in gd, the optimizer starts at a random point in the search space and reaches the lowest point of the function by traversing along the slope. unlike gd that requires calculating the partial derivative for each feature at each data point, sgd achieves computational efficiency by computing derivatives on randomly chosen data points. • nearest centroid (nc) is a simple classification model that represents each class by the centroid of its members. subsequently, it assigns each data point to the cluster whose centroid is the closest to it. nc is particularly effective for non-convex classes and does not suffer from any additional dependencies on model parameters [58] . • decision trees (dts) are a classification and regression technique that assigns target labels based on decision rules inferred from data features [59] . dt maintains the decision rules using a tree. a data point is assigned to a class by repeatedly comparing the tree root with the data point value to branch off to a new root. • gaussian naive bayes (nb) are a class of fast, probabilistic learning techniques that apply the bayes' theorem to assign labels to the data points [60] . while supervised ml approaches generally yield reliable prediction accuracy, they often suffer from overfitting or convergence issues [47, 61] . each of the above approaches has its own advantages and disadvantages. svm works well when the underlying distribution of the data is not known. however, it is prone to overfitting when the number of features is much greater than the number of samples. sgd needs low convergence time for a large dataset, but it may require to fit a number of hyperparameters. conversely, dt involves almost no hyperparameters, but often entails slightly higher training time. unlike dt, nb requires less training time but works on the implicit assumption that all the attributes are mutually independent. finally, nc is a fast method but is not robust to outliers or missing data. in the context of our work, we intuit that the discriminatory feature(s) will yield a high accuracy irrespective of the underlying supervised ml algorithm used. • accuracy function measures the fraction of matches between the predicted and actual labels in a multi-label classification, i.e., the ratio of correctly predicted observations to the total observations. it can be calculated as: in the above equation, tp, tn, fp, fn denote true positive, true negative, false positive and false negative, respectively. • extra trees classifier is an estimator that fits randomized decision trees (called extra-trees) on data samples. the memory and computation overhead of this approach can be controlled by regulating the size of the extra trees. the nodes in the tree are split into sub-trees resulting in high accuracy (i.e., drop in impurity). thus, feature importance is measured as total reduction in impurity affected by that feature [62] . • multiple regression (mr) is a statistical tool to capture the linear relationship between the independent and the dependent variables x and y of a function y = g(x). in our context, mr generates a linear relationshipŷ where b fi is the coefficient that captures the contribution of feature f i towards the dependent variable y, while β 0 and � are the intercept and error terms, respectively. given any pair of vectors v andv (jvj ¼ jvj ¼ n), we apply the following standard statistical operations: • mean centering subtracts the mean μ from each element of a vector v, i.e., v 0 = v − μ(v). this standardization adjusts the scales of magnitude by making the new mean 0 and helps compare data from varied sources or having different datatypes. • mean squared error (mse) is calculated as 1 • pearson correlation coefficient (pcc) between v andv measures the strength of a linear association between two variables, where the value pcc = 1 is a perfect positive correlation and −1 is perfect negative correlation. • positivity rate ρ is the ratio between the number of individuals tested positive to the number of tests performed daily [63] . this section is classified into the following three subsections: (1) and (2) table 2 . unless otherwise stated, the feature set comprises gdp, gender, ethnicity, health care, homeless, lockdown type, population density, airport activity, and age groups, whereas the output labels consist of infected and death scores on a scale of 0-6. we apply supervised machine learning (ml) approaches to identify the key factors affecting covid-19 infected and death counts. for each supervised ml technique, we perform an exhaustive search of all possible combinations of any 5 features and identify the feature subset (s) with the highest accuracy (discussed in sec. 2.2) as the most important features. fig 1 shows the scores for different supervised methods. although proposing a machine learning algorithm that works best on covid-19 data is not the purpose of this study, it is worth reporting that decision tree classifier (dt) slightly outperforms the other algorithms for both cases of infected and death scores. we create a pool of all features participating in at least one combination for output labels of infected and death scores. fig 2 shows a heatmap of the importance i for all such features against each supervised technique. for infected score as output label (top figure), homeless (home), population density (pd), airport activity (air), testing (test), white (wht), etc. have the highest i. for death score as output label, pd, air, test and age groups above 50 years (age50_54 and age80_84) exhibit the highest importance. we apply the extra trees classifier to generate the impurity-based rank for the features (discussed in sec. 2.2). fig 3a shows the top 5 important features corresponding to the infected and death scores, respectively. it is interesting that for both cases, the same set of features, namely, population density, days to peak, airport traffic, testing and high age groups, are identified. also note that the same features exhibit a very high participation in the 5-feature combinations shown in fig 2. next, as a validation exercise, we apply dimension reduction on the factors affecting covid-19 infected and death rates inform lockdown-related policymaking table 1 we discussed in sec. 2.1, that our initial dataset groups ages into brackets of 4 (0-4, 4-8, and so on). our results from supervised learning (sec. 3.1) and extra trees (sec. 3.2) suggest that high age groups are important factors affecting the infected and death scores of covid-19. to understand the effect of covid-19 infected and death scores on low and high age groups, we create two feature sets for population of age �40 and >40. fig 4a shows that for both cases of infected and death, the accuracy (acc) is higher for higher age groups. we explore this by repeating the above experiment, this time, with a feature set of groups 40-60 and >60. fig 4b depicts that acc for age group 60+ is marginally higher, suggesting that the elderly are amongst the most vulnerable, however the difference in mortality rates in this case was not statistically significant. we carry out a study to identify the pre-lockdown factors of any region (us states in our case) that contribute to the overall post-lockdown infection and death numbers. we partition the total infected and death numbers for each state into pre-and post-lockdown infected and death counts. we then create a feature set consisting of population density, airport business, pre-lockdown infected, pre-lockdown death, days between first infected to lockdown and age group above 80. the features represent the set of observable factors for the administrative and health bodies and were already shown to possess high feature significance in the previous factors affecting covid-19 infected and death rates inform lockdown-related policymaking section. the output labels are the post-lockdown infected and post-lockdown death numbers. we perform the following experiments: 3.4.1 identification of discriminating features. we carry out a simple preprocessing step to convert each feature entry to percentile (with respect to the feature vector) and rank the us states in the decreasing order of infected and death scores (fig 5) . we calculate the weighted average percentile of features for the top and bottom k = 10 us states using the formula where p(f i ) and ρ(f i ) are the percentile and rank of the i th feature value, while r is the number of us states (equal to maximum rank). we intuit that the feature exhibiting the maximum difference in weighted average percentile for top and bottom k covid-19 affected us states are the discriminating ones. fig 6a shows the percentile difference suggesting that airport and population density are the most significant, while days between first infected to lockdown and age group of 80+ are the least discriminating. we apply multiple regression (mr) (see sec. 2.2) to measure the weightage of each of the above features in the observed post-lockdown infected (post_inf) and post-death numbers (post_dth). we eliminate the days between first infected to lockdown (fst-lock) and age group 80+, which are the least discriminating features from the percentile analysis (see fig 6a) . as a prerequisite for mr, we need to eliminate features that are mutually correlated. fig 6b shows that pre-inf and pre-dth are highly correlated, and hence we run two separate batches of mr: (1) population density, airport business, pre-lockdown infected and (2) population density, airport business, pre-lockdown death. we explore the effect of testing and lockdown on infection spread. we utilize positivity ratio ρ (defined in sec. 2.3) to gauge how widespread the infection spread is [63] . we acquire the daily infected and testing count in us (see sec. 2.1.3) and plot the mean daily ρ across all states over the period of february-july 2020. fig 7a shows that the testing increased over a period time, while the positivity ratio dropped post lockdown (shown in red dotted line). while, testing (and, by extension, positivity ratio) is an effective epidemiological indicator, it cannot curb infection spread by itself. however, fig 7a shows that the ρ has dropped approximately three weeks into the lockdown, suggesting that the latter had an impact on curbing spread by minimizing social contact. table 3 shows that pre-infected and pre-death with high coefficients contribute highly towards factors affecting covid-19 infected and death rates inform lockdown-related policymaking the post-lockdown infected and death numbers, followed by population density and airport traffic. this finding is further supported by the p values reported for the respective features. note that the r 2 scores for all the four cases are >0.8, suggesting that the output features capture a high proportion of the variance in the input features. overall, pre-infected count has higher coefficient and r 2 score and emerges as a marginally better discriminating feature of post-lockdown effects than the pre-death count. factors affecting covid-19 infected and death rates inform lockdown-related policymaking in sec. 3.2, we perform pca on the feature set of the key factors to show that states with high infection and death numbers stand out of the cluster of other states. these states include some erstwhile hotspots forming group 1 (such as new york city, new jersey, massachusetts, connecticut, rhode island) as well as states experiencing a steady infection and death count and also a strong second wave forming group 2 (such as texas, washington, california, georgia, arkansas, utah and colorado) (fig 3b) . in the pca analysis, pc1 and pc2 account for 41% and 21% variance, respectively. we explore how each feature influences each component to show that pc1 is driven by factors such as airport activity and high age groups (70 and beyond), while pc2 is dominated by population density, airport, age (80+) and testing. notice in fig 3b, though both groups 1 and 2 exhibit high spread across pc1, group 2 forms a slightly denser cluster than group 1, implying that it exhibits an even mix of pc1 and pc2 features. we intuit that the early peaking in infection in group 1 states is due to high road and airport mobility leading to high mixing and infection spread that is manifested in the elderly population. group 2 shows enduring infection spread due to high population density and testing, in addition to airport activity and populations with higher age group. we study how demographics affect covid-19 numbers to show that states with higher age groups (particularly 60 and beyond) numbers are the most vulnerable. finally, we split the infected and death numbers on the pre-and post-lockdown epochs and apply multiple linear regression to show that pre-lockdown infected and death, population density and airport contribute highly to the post-lockdown numbers. this analysis can be particularly effective in pinpointing the most vulnerable states and recommending lockdown policies on starting dates and duration to curb pandemic spread. note that our present study pertains to the identification of the discriminatory features with respect to the date of lockdown. there exists several unanswered questions regarding the impact of length, scheduling strategies, lockdown types and extent of lockdowns on pandemic spread that need to be answered. such an analysis requires a richer feature set as well as a sound understanding of the dynamics of infection spread in terms of healthcare, distance, mobility, etc. as a preliminary study, we first explore whether there is any relationship between the health care index (health) of a us state and the number of transitions from infected to death (dth/inf) in this state. the pearson's correlation coefficient (see sec. 2.3) between the two factors is 0.11, suggesting that the overall mortality numbers is largely unrelated to the healthcare facility and may solely depend on the infected individual's attributes, such as age, comorbidities, infection severity, etc. second, since proximity plays a role in infection spread, neighboring regions should peak at nearly the same time. we posit that mobility may play an even greater role in the spread, than a static measure like distance between a pair of regions. in the absence of a inter-state mobility dataset, we create two feature sets for the nyc boroughs dataset (see sec. 2.1): (1) inter-borough distance and (2) inter-borough mobility. each borough b has a distance and mobility vector d b = {d b1 , d b2 � � �} and m b = {m b1 , m b2 � � �} where d bi and m bi are the probabilistic measure of distance and mobility between a borough b with borough i. we calculate the correlation of the mean squared error (see sec. 2.3) of the distance/mobility vectors of any pair of boroughs b 1 and b 2 against the absolute difference of their peak to infected or peak-to-death features. fig 7b suggests that mobility yields a higher correlation (0.44) than distance (0.22) suggesting that mobility is a slightly more informative feature to analyze infection spread. we are currently working towards broadening the scope of this study in different directions. first, this work attempted to apply ml analysis on a wide range of features, making the the states of united states the ideal choice, specifically from the standpoint of data availability. in future we would like to extend this work by running these experiments on epidemiological, demographic and economic data of different countries. it would be interesting to report the variation in the discriminatory features identified for different countries. second, we identify population density, testing, airport activity and pre-lockdown infected count as key features driving the post-lockdown infection and death numbers. we plan to utilize these findings to design policies on the timing, duration and stringency of lockdown for future pandemics. third, all the input features discussed in this work are static or time invariant. it is imperative to analyze the evolution of dynamic features (such as gdp and unemployment rates) from the pre-covid to the post-covid timelines to uncover the long-term economic effects of covid-19. machine learning is emerging as an important tool to predict the dynamics of spread of covid-19 and identify the key factors driving infection and mortality rates. while existing works study the effects of gender, race, age, testing, social contact and distancing separately, we present an unified analysis of the demographic, economic, and epidemiological, ethnic and health indicators for infection and mortality rates from covid-19. we curate a dataset of us states comprising features (from varying sources discussed in sec. 2.1) that may potentially impact infection and death rates of covid-19. we run several supervised machine learning techniques to identify and rank the key factors correlating with infection and fatality counts. population density, testing rate, airport traffic, high age groups emerge as significant, while ethnicity, gender, healthcare index, homeless and gdp have little or no impact on pandemic spread and mortality. coronavirus: what have been the worst pandemics and epidemics in history coronavirus world map: which 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hundreds test positive for covid-19 at tyson foods plant in arkansas covid-19 cases rise as hospitalizations remain low in colorado utah confirms 394 new coronavirus cases; 3 more deaths on sunday the authors would like to acknowledge the editor/reviewers for critically assessing the materials and providing suggestions that significantly improved the presentation of the paper. furthermore, they acknowledge the department of computer science, virginia commonwealth university for its computational resources. validation: satyaki roy.visualization: satyaki roy. writing -review & editing: preetam ghosh. key: cord-003377-9vkhptas authors: wu, tong; perrings, charles title: the live poultry trade and the spread of highly pathogenic avian influenza: regional differences between europe, west africa, and southeast asia date: 2018-12-19 journal: plos one doi: 10.1371/journal.pone.0208197 sha: doc_id: 3377 cord_uid: 9vkhptas in the past two decades, avian influenzas have posed an increasing international threat to human and livestock health. in particular, highly pathogenic avian influenza h5n1 has spread across asia, africa, and europe, leading to the deaths of millions of poultry and hundreds of people. the two main means of international spread are through migratory birds and the live poultry trade. we focus on the role played by the live poultry trade in the spread of h5n1 across three regions widely infected by the disease, which also correspond to three major trade blocs: the european union (eu), the economic community of west african states (ecowas), and the association of southeast asian nations (asean). across all three regions, we found per-capita gdp (a proxy for modernization, general biosecurity, and value-at-risk) to be risk reducing. a more specific biosecurity measure–general surveillance–was also found to be mitigating at the all-regions level. however, there were important inter-regional differences. for the eu and asean, intra-bloc live poultry imports were risk reducing while extra-bloc imports were risk increasing; for ecowas the reverse was true. this is likely due to the fact that while the eu and asean have long-standing biosecurity standards and stringent enforcement (pursuant to the world trade organization’s agreement on the application of sanitary and phytosanitary measures), ecowas suffered from a lack of uniform standards and lax enforcement. highly pathogenic avian influenzas have become a major threat to human and livestock health in the last two decades. the h5n1panzootic (2004 ongoing) has been one the most geographically widespread and costly, resulting in the loss of hundreds of millions of poultry in 68 countries [1] and over 450 human deaths worldwide-a mortality rate of 60 percent [2, 3] . for h5n1, and other h5 subtypes, most countries reporting poultry outbreaks also report evidence of the disease in wild bird populations, and the mechanisms for the spread of h5n1 have been identified as a combination of wild bird transmission and the live poultry trade [4, 5] . plos risk factors. our primary interest is in the role of live poultry imports as a source of traderelated avian influenza risk at the regional level. we note that other poultry products, such as packaged meat and eggs, do pose a risk, but it is significantly lower. although avian influenza can persist in frozen meat, contact with that meat is unlikely to cause infection [30] . furthermore, since hpais are lethal to egg embryos, eggs are not a potential source of transmission [31] . the data comprise an unbalanced panel covering 53 countries over 13 years; the lack of balance is due to the fact that membership of the eu changed over the timeframe. the response variable in all models estimated was a log transformation of the number of h5n1 poultry outbreaks in a given country in a given year, obtained from the emergency prevention system for animal health (empres), a joint project of the fao and oie [32] . the log transformation was applied to account for the wide disparities in the numbers of the outbreaks across countries. in 2010, for example, indonesia recorded 1206 outbreaks while romania, the only eu country to be infected that year, had only 2. in addition to reflecting the differing directions and intensities of risk factors, this also reflects differences in reporting conventions for h5n1 at the international level [33] . a series of outbreaks may be reported separately in one country, but be treated as a single event in another. data on trade in live poultry were obtained from the united nations' comtrade database (comtrade.un.org) and resourcetrade.earth, a project of the royal institute of international affairs (www.chathamhouse.org). these report the total imports of live poultry into a given country in a given year by weight (kg). the data on trade in live poultry did not distinguish between different types of domestic birds, such as chickens, duck, and geese, but grouped them under a single commodity category of "live poultry." with respect to wild bird migration as a pathway for h5n1 spread, we used the density of wild bird habitat as a proxy for the presence and scale of migratory bird populations, and the likelihood that wild and domestic birds will mix. lakes, wetlands, and (irrigated) agricultural areas have been consistently identified as wintering and breeding grounds for migratory birds, and as places where wild birds may come into contact with free-ranging poultry [34] [35] [36] [37] [38] [39] [40] [41] . the indicator for wild bird habitat used in this study was the set of "important bird and biodiversity areas" (ibas) for "migratory and congregatory waterbirds" identified by birdlife the live poultry trade poses different avian influenza risks in different regions of the world table 1 . the distribution of h5n1 poultry outbreaks between 2004-2016 across the member states of asean, ecowas, and eu. "-" signifies that the country was not a member of its associated trade bloc in that given year . 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 brunei 0 0 0 0 0 0 0 0 0 0 0 0 0 cambodia 23 1 5 1 1 1 3 4 1 8 5 2 1 indonesia 1 3 20 0 20 1503 1206 1155 308 260 310 107 269 laos 19 0 1 7 13 6 1 international (datazone.birdlife.org). in their 2006 analysis of h5n1 spread, kilpatrick, chmura (4) also identified ibas as a proxy for migratory birds and the infection risks they pose. country-level statistics on socioeconomic and agro-ecological conditions were taken from the united nations' food and agriculture organization (www.fao.org/faostat/en/) and the world bank (data.worldbank.org). agricultural land cover was reported as a percentage of total land area of the country. per-capita gdp was reported in purchasing power parity terms as current international dollars. data for 2016 for these two variables were missing for certain countries. in these cases, the gaps were filled by extrapolating the missing data as a linear trend of the preceding 11 years. we assume that agricultural land-where free-ranging chickens, ducks, and geese are commonly raised in all three regions-also acts as a relevant proxy for susceptible poultry. data on the biosecurity measures targeting avian influenza undertaken by each country were obtained from the world organisation for animal health (oie) (www.oie.int). these report a standardized series of biosecurity controls targeting wildlife and livestock diseases, including those related to surveillance, vaccination, border checks, and management of wild disease reservoirs, and whether or not a given country undertook them in a given year. we chose a subset of these biosecurity measures we considered most relevant to h5n1 avian influenza risks for inclusion in our model. additionally, in any given year, there were 1 to 4 countries that did not provide a report of biosecurity measures to the oie; we assumed that this indicates an absence of action, and the dataset records these cases as zeroes. our modeling approach relied on generalized linear models (glm) to analyze a panel of data on disease outbreaks and associated risk factors. in this we follow others who have sought to predict the spread of h5n1 at both national and international levels [42] [43] [44] or h7n9 [45] [46] [47] . glms are well suited to epidemiological studies because of their flexibility regarding data type and the distribution of response variables, their simplicity of application, and their frequency of use [33] . our identification strategy involved the selection of three specifications for each of two estimators. we adopted both random and fixed effects estimators. hausman tests conducted at the all-regions level favored a random effects estimator, as the p-value exceeded the 5% threshold below which fixed-effects regression is conventionally considered necessary. some factors that influence the likelihood and number of outbreaks in a given country or region are not likely to change significantly over the course of several years, or even a decade. in our dataset, netherlands for example, the amount of land covered by wild bird habitat is time-variant, while agricultural land and even per-capita gdp for many countries experienced relatively modest variations over the timeframe of the study. in this case, and as the hausman diagnostics indicate, a random effects estimator is more appropriate. nevertheless, since we wished to control for timeinvariant characteristics of regions and countries we also implemented fixed effects estimators at both the aggregate and trading bloc levels, implicitly assuming no changes in the trade or biosecurity environment at the bloc level that we are unable to control for. our first specification (model 1) included a number of factors related to disease risk but excluded both live poultry imports and biosecurity measures. included predictors were land area, human population, per-capita gdp in purchasing power terms, agricultural area, wild bird habitat area, and the live chicken population. our second specification (model 2) added intra-regional trade bloc and extra-bloc imports of live poultry. our third specification (model 3) added four main biosecurity measures: border precautions, general surveillance, vaccination prohibition, and wild disease reservoir management. all are categories of oie-reported biosecurity measures taken against avian influenza. the general forms of the estimated random and fixed effects models were: where y it denotes the number of poultry outbreaks in country i in year t, x includes the predictors for model 1, z includes the additional predictors for model 2, u includes the additional predictors for model 3, ecowas and asean are dummy variables for the two titular regional trade blocs (the eu is the reference group), and u it and ε it are the "between" and "within" errors respectively. to account for heteroskedasticity, we used robust standard errors. finally, since the data used in this analysis are reported annually, and h5n1 has been a conspicuous and fast-moving epidemic (meaning the effects of an outbreak are unlikely to persist over a long period of time) among poultry, we did not use a lag structure in our statistical analysis. therefore, we assumed that the factors driving an outbreak in a given year are contemporaneous with it (e.g., an outbreak that occurred in 2012 were modelled using trade volumes from 2012). we were also constrained by data availability in our use of annual increments: although monthly data exist for outbreaks, they do not for important predictor variables such as percapita gdp, human and poultry populations, the volume of live poultry traded, and biosecurity. regressions results from all models, including both random and fixed effects, are reported in tables 2-5 . at the all-regions level, the results for the random-and fixed-effects models were very similar, with the same set of predictor variables being statistically significant (i.e., p-values below the 5% or 10%) and the same direction of impact on the response variable. this set of predictors was human population (positive direction), per-capita gdp (negative direction), intra-trade bloc live poultry imports (negative direction), extra-trade bloc live poultry imports (positive direction), and the biosecurity measure of surveillance (negative direction). additionally, although the coefficient values for the same predictor differed between the two estimators, all pairs were within the same order of magnitude. the only exception to this was migratory waterbird habitat variable-the percent of land area covered by ibas for migratory and congregatory waterbirds. this was statistically significant and negative (i.e., had a mitigating impact on h5n1 poultry outbreaks) for the fixed-effects model but was not significant for the random-effects model. the overall r-squared for the random-effects model was significantly higher than that for the fixed-effects model (0.451 vs. 0.0181). the "between rsquared" value was particularly high (0.682) in the random effects model, signaling the importance of variation among countries (as opposed to "within r-squared," which measures the variation within countries over time). as we had expected, we found significant differences across trade regions. in the randomeffects model, ecowas diverged from all-regions conditions and from asean with respect to per-capita gdp and extra-bloc imports: while the two predictors were, respectively, riskdecreasing and risk-increasing at the all-regions level and in asean, they had the opposite impacts in ecowas. furthermore, ecowas differed from the all-regions level and from the eu in terms of intra-bloc imports: while this was risk-decreasing for the former two, it was risk-increasing for ecowas. finally, there were predictors that were statistically insignificant at the all-regions level but had a significant effect within different regions. for asean, agricultural land cover was a mitigating factor for outbreaks while wild disease reservoir management showed a strong positive relation with outbreaks. for ecowas, wild waterbird habitats and border precautions had a mitigating effect on outbreaks while vaccination prohibition and wild reservoir management had a positive effect. in the eu, the population of live chickens had a strong negative relation table 2 . results from the regression models of h5n1 outbreak risk factors for member states in all three regions; regressor coefficients are reported and statistically-significant factors are marked by asterisks. a blank space signifies that the variable was not included in the given model. units model 1 model 2 model 3 with outbreaks, while vaccination prohibition, similar to the case with ecowas, was positively related. following liang, xu (5), there is a perception that the long distance transmission of highly pathogenic avian influenza h5n1 was largely due to wild bird migration, with the live poultry trade playing a minor and more localized role in some cases. our concern here has been to identify the nature of the risk posed by the live poultry trade in different regions of the world, and the conditions affecting that risk. our measure of development status, per-capita gdp, is simultaneously a proxy for modernization, biosecurity, consumption, and value-at-risk. as a proxy for modernization, it reflects risk-reducing differences in production methods. industrial livestock production methods typically include on-farm biosecurity measures that protect poultry from contact with disease-carrying wild birds. unlike traditional methods of free-range or "backyard" husbandry, factory production minimizes the likelihood of poultry intermingling with wild birds or being exposed to environmental pathogen pollution. for all its epidemiological, ecological, and ethical problems, industrial livestock production allows for more timely and widespread disease surveillance and vaccination, and for greater compliance with animal health regulations [48] . at the same time, per-capita gdp growth is also associated with risk-increasing changes in meat consumption, and hence poultry production. indeed, the highest income elasticity of demand for meat and fish has been found in the poorest households and the poorest countries [49] . in developing countries, 71% of the additions to meat consumption are from pork and poultry, with poultry dominating pork [50] . absent changes in on-farm biosecurity, increased table 3 . results from the regression models of h5n1 poultry outbreak risk factors for the association of southeast asian nations (asean); regressor coefficients are reported and statistically-significant factors are marked by. a blank space signifies that the variable was not included in the given model. units model 1 model 2 model 3 production implies increased risk. across all regions, the net effect of income growth is to reduce risk, dominating risk-increasing changes. in the ecowas region-the lowest income region-the effect is the opposite. the risk-increasing effects of income growth dominate the risk reducing effects (table 3) . amongst the landscape variables-land area, the proportion in agriculture, and the proportion in ibas-our results reveal no uniform relation to h5n1 outbreaks. at the all-regions level we found a weakly negative relation between outbreaks and the proportion of the land area in ibas (table 1 ). this was driven by the european union, which includes the highest proportion of land area in ibas, but also the most industrialized forms of poultry production. the degree to which poultry production is industrialized also shows up in the coefficients on poultry numbers, which are negative and significant only for the eu (table 4 ). while spatial heterogeneity at the landscape scale is important in terms of avian ecology, we were unable to take explicit account of these more detailed considerations in a country-scale analysis. the impacts of regional differences in biophysical conditions that are not directly controlled for are, however, included in bloc-level fixed effects. our primary concern is with the role of the live poultry trade, and how that differs between regions. across all regions we find that live poultry imports into a trade bloc are risk increasing. this is consistent with past studies that have shown that extra-bloc live poultry imports may be a significant source of additional avian influenza risk where they do not meet bloc sanitary and phytosanitary standards. the eu's common market and the asean free trade regime in particular have long-standing and standardized protocols, in accordance with the world trade organization's agreement on the application of sanitary and phytosanitary measures. but the two blocs have quite different exposures to external risk. a study of highly pathogenic avian influenza introductions to vietnam, for example, found that extra-asean imports of table 4 . results from the regression models of h5n1 poultry outbreak risk factors for the economic community of west african states (ecowas); regressor coefficients are reported and statistically-significant factors are marked by asterisks. a blank space signifies that the variable was not included in the given model. units model 1 model 2 model 3 population # people 6.05x10 -9 3.85x10 -8�� 7.34x10 -9 4.21x10 -8�� 1. live poultry increased the risk of introduction [51] . this is also what our study finds for the asean region (table 2) . we do not see an equivalent effect for the eu (table 4 ), reflecting differences in both import volumes and the biosecurity measures applied to imports. the eu imports less and applies stricter biosecurity measures to those imports. the ecowas story is different. extra-bloc live poultry imports are risk reducing, not risk increasing (table 3 ). it is likely that imports from outside the bloc reduce avian influenza risk in the region in part because they meet biosecurity standards that are more stringent than the standards applied in the region. the effects of intra-bloc trade in live poultry mirror the effects of extra-bloc trade. in the eu and asean, intra-bloc trade is risk reducing (tables 2 and 4 ). this may reflect a "substitution effect" in which imports of safer intra-bloc poultry crowds out riskier extra-bloc imports. other studies have come to similar conclusions. eu-derived live poultry imports to spain, for example, were found to pose no threat of avian influenza introduction [52] . once again, eco-was is the exception. extra-ecowas imports of live poultry are risk reducing while intrabloc imports are risk increasing (table 2 ). this is likely due to poor internal biosecurity, such as lax standards and inconsistent execution of inspections. regulatory standards within the ecowas trade bloc have been weak for the whole of the study period [53] . while harmonized sanitary and phytosanitary standards for the 15 member states of ecowas were in principle adopted in 2010, most ecowas states had yet to submit legislation for international certification by 2017 [54] . failure to adopt and enforce unified standards may be partly due to income constraints in ecowas countries. in ppp terms, the bloc's per-capita gdp in 2016 was less than half that of asean and approximately 1/8 th that of the eu, meaning it had less resources available for biosecurity policies and institutions. political instability may be another important obstacle: a table 5 . results from the regression models of h5n1 poultry outbreak risk factors for the european union (eu); regressor coefficients are reported and statistically-significant factors are marked by asterisks. a blank space signifies that the variable was not included in the given model. units model 1 model 2 model 3 the live poultry trade poses different avian influenza risks in different regions of the world number of ecowas member states, including nigeria, niger, sierra leone, mali, liberia, and cote d'ivoire have suffered from civil wars and armed insurgencies over the past two decades. such fraught geopolitical conditions are not conducive to the establishment and enforcement of cross-border regulations. it goes without saying, though, that certification of sanitary and phytosanitary legislation in ecowas states, and the establishment of enforcement agencies to bring states into compliance with the sps agreement and codex alimentarius is a necessary condition of improving regional trade-related biosecurity. in terms of biosecurity measures more specifically, we did not have direct measures of onfarm biosecurity (but conjecture that biosecurity is increasing in per-capita gdp), but we did have measures of four biosecurity policies at the national level. these include: (1) border precautions (measures applied at airports, ports, railway stations or road check-points open to international movement of animal, animal products and other related commodities, where import inspections are performed to prevent the introduction of the disease, infection or infestation); (2) general surveillance (surveillance not targeted at a specific disease, infection or infestation); (3) prohibition of vaccination (prohibition of the use of a vaccine to control or prevent the infection or infestation); and (4) management of wildlife reservoirs (measures to reduce the potential for wildlife to transmit the disease to domestic animals and human beings). the management of wild disease reservoirs differs widely across countries, but techniques include vaccination, treatment of infections with drugs, isolation of infected populations, population translocation, reproduction reduction, culling, and control (draining, flooding, or burning) of wild disease reservoir habitat [55] . of these measures, only general surveillance was significant at the all-regions level, while at the bloc level the effects of the different measures were frequently ambiguous. in the eu, for example, only the prohibition of vaccination was significant, and then in positive relation to outbreaks. for poultry, vaccination may be prohibited because the practice makes it difficult to distinguish infected from vaccinated flocks. this makes it a concomitant of policies centered on livestock culling as the primary response to outbreak risk [56] . no other biosecurity policy was found to have a statistically significant relation to outbreaks in the region. the same set of policies had opposite effects in asean and ecowas. the prohibition of vaccination and the management of wild reservoirs were positively related to outbreaks in ecowas but negatively related to outbreaks in asean, while border protection measures were negatively related to outbreaks in ecowas but positively related to outbreaks in asean. this may reflect regional disparities in the quality of implementation not captured in the data. but it may also reflect the greater importance of trade in the transmission of the disease in ecowas. in their survey of the international spread of h5n1 in the early years of the global epidemic, kilpatrick, chmura (4) found that transmission into europe was by wild birds, that transmission into southeast asia was by the poultry trade, and transmission into africa by a balance of both. our results suggest that after introduction, inter-country spread had differing dynamics in each region. while intra-bloc trade facilitated h5n1 spread among west african countries, it did not in either europe or southeast asia. in these areas, greater risk was posed by out-ofregion live poultry imports. in recent decades, avian influenzas have emerged as a major threat to human and animal health across the world. in particular, hpai h5n1, which was first isolated in 1996, has been the most widespread and among the most devastating in terms of livestock and human mortality. it has inflicted severe losses to poultry stocks and caused hundreds of human deaths. even today, as other avian influenzas have become epidemic, h5n1 remains in circulation among wildlife and livestock. identifying and quantifying the mechanisms of its international spread can help lay the groundwork for prediction and mitigation. it may also provide an instructive framework for the management of other avian influenzas. in this study, we considered the risk posed by the international trade in live poultry and the effects of associated biosecurity measures. differing agro-ecological and socioeconomic conditions across the trade regions were shown to influence epidemic dynamics in different ways, with certain factors being risk-enhancing or risk-decreasing in one region but having the opposite effect, or no significant effect, in another. in policy terms, there is no one-size-fits-all solution to mitigating avian influenza spread. the particular conditions, including those related to the trade agreements and associated regulatory standards, of a given region need to be carefully considered. but overall, biosecurity measures are potentially effective at controlling h5n1 risks, and should be undertaken as a means to forestall spread-in general, mitigation of epidemics is significantly more cost-efficient than suppression [57] . on-farm and other forms of domestic biosecurity may be more important than trade-related measures, but where the protection of trade pathways is weak, the risk of avian influenza spread is clearly higher. supporting information s1 file. detailed information on data sources. the public sources of the data used in this study, and how they were acquired, are described. 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optimization of a global strategy to address the pandemic threat we would like to thank ann kinzig, jim collins, ben minteer, and peter daszak for their insightful comments and discussions on the research presented here. conceptualization: tong wu, charles perrings. key: cord-048358-z5klydpi authors: catic, andré; fiebiger, edda; korbel, gregory a.; blom, daniël; galardy, paul j.; ploegh, hidde l. title: screen for isg15-crossreactive deubiquitinases date: 2007-07-25 journal: plos one doi: 10.1371/journal.pone.0000679 sha: doc_id: 48358 cord_uid: z5klydpi background: the family of ubiquitin-like molecules (ubls) comprises several members, each of which has sequence, structural, or functional similarity to ubiquitin. isg15 is a homolog of ubiquitin in vertebrates and is strongly upregulated following induction by type i interferon. isg15 can be covalently attached to proteins, analogous to ubiquitination and with actual support of ubiquitin conjugating factors. specific proteases are able to reverse modification with ubiquitin or ubls by hydrolyzing the covalent bond between their c-termini and substrate proteins. the tail regions of ubiquitin and isg15 are identical and we therefore hypothesized that promiscuous deubiquitinating proteases (dubs) might exist, capable of recognizing both ubiquitin and isg15. results: we have cloned and expressed 22 human dubs, representing the major clades of the usp protease family. utilizing suicide inhibitors based on ubiquitin and isg15, we have identified usp2, usp5 (isot1), usp13 (isot3), and usp14 as isg15-reactive proteases, in addition to the bona fide isg15-specific protease usp18 (ubp43). usp14 is a proteasome-associated dub, and its isg15 isopeptidase activity increases when complexed with the proteasome. conclusions: by evolutionary standards, isg15 is a newcomer among the ubls and it apparently not only utilizes the conjugating but also the deconjugating machinery of its more established relative ubiquitin. functional overlap between these two posttranslational modifiers might therefore be more extensive than previously appreciated and explain the rather innocuous phenotype of isg15 null mice. posttranslational modification by ubiquitin regulates processes such as proteasomal degradation, intracellular trafficking, and transcription. ubiquitin is attached to substrates in covalent isopeptide linkage or as an n-terminal fusion [1] [2] [3] . ubiquitination, however, is reversible: the ubiquitin moiety can be released from substrates through the action of deubiquitinating proteases, which may rescue ubiquitinated substrates from their degradative fate [4] . in contrast, proteasome-associated dubs enhance the rate of proteasomal degradation by removing bulky poly-ubiquitin chains from substrate proteins prior to proteolysis. such dubs enhance the processivity of the proteasome toward target proteins, and also recycle ubiquitin, a modifier that itself turns over slowly [5, 6] . dubs are furthermore required to hydrolyze the ubiquitin precursor and generate the active ubiquitin monomer. inspection of mammalian genomes shows the presence of more than 100 genes that encode putative dubs, consistent with their specific and diverse regulatory functions. ubiquitin-specific proteases (usps) are the dominant family among dubs [7] . ubiquitin-like molecules show sequence and structural similarity to ubiquitin. unlike ubiquitination, modification by ubls usually does not target proteins for destruction by the proteasome. a notable exception may be fat10, a modifier that serves as a ubiquitin-independent signal for proteasomal degradation [8] . the conjugation of ubls to target proteins follows reaction pathways similar to those involved in ubiquitination [9] . the enzymes that attach or cleave ubls are generally distinct from the ligases or proteases of the ubiquitin pathway. a closely related homolog of ubiquitin in vertebrates is the ubl polypeptide isg15, an interferon-inducible gene product that is strongly upregulated following viral or bacterial infection [10] . however, the molecular and regulatory consequences of isgylation remain unknown [11] . isg15 consists of two ubiquitin domains in a tandem arrangement, similar to fat10. unlike other members of the ubl family, isg15 co-opts at least one of ubiquitin's conjugating enzymes, ubc8 [12, 13] and the ubiquitin ligase herc5 [14] [15] [16] [17] . usp18 constitutes the only presently appreciated isopeptidase specific for isg15, and its absence has profound effects on innate immunity, leading to increased resistance to certain viral infections [18, 19] . notably, these effects appear not to be contingent upon proteolytic activity of usp18 [20, 21] . apart from usp18, additional proteases for isg15 must exist, since the isg15 precursor protein is cleaved properly in usp18 knockout mice [19] . the c-terminal six amino acids of ubiquitin and isg15 are identical. this tail region is required for specific recognition of ubiquitin by conjugating enzymes, and also for recognition of ubiquitin adducts by isopeptidases [22, 23] . the overlap in conjugation between ubiquitin and isg15, as well as their cterminal similarity, imply the existence of promiscuous dubs, capable of removing both ubiquitin and isg15 from substrate proteins. here, we report on the identification of new isg15specific proteases measured by reactivity toward active-site directed probes and isopeptide-linked substrates [24] [25] [26] . activity-based profiling of dubs figure 1 shows a consensus phylogram based on the alignment of catalytic core sequences of dubs, including the majority of known human usp homologs. in this tree, the isg15-protease usp18 clusters close to usp5 (isot1) and its isoform usp13 (isot3). previous work had identified usp5 as a protease with affinity for both ubiquitin [27] and isg15, as shown by its reaction with an electrophilic isg15 derivative, isg15-vinyl sulfone (isg15vs) [28] . to probe for additional isg15-reactive proteases, we have cloned and expressed a total of 22 human dub homologs from different clades of this phylogram (indicated with arrows), 17 of which reacted with a ubiquitin-based probe and/or an isg15based probe (see below). the screen was based on in vitro transcription and translation (ivt) of cloned cdnas, which affords a rapid method to generate radiochemically pure proteins. this technique allows the generation of dubs that cannot be readily expressed in bacterial systems, or that are sequestered in subcellular compartments or in multimolecular complexes when expressed in cell lines. to profile for dub specificity, we used recombinantly expressed ubiquitin, sumo1 and isg15, and installed an electrophilic trap at their c-terminus to obtain the active-site probes ubiquitin-vinylmethyl ester (ubvme), su-mo1vme and isg15vs, respectively [28] . dubs generated by ivt were incubated with each of these three probes (figure 2 ), followed by direct analysis of the reaction mixture by sds-page. we have determined by x-ray crystallography that probes of this type form a covalent adduct with the catalytic cysteine residue of active dubs to yield a thioether-linked adduct between enzyme and probe [26] . when unmodified ivt products are run adjacent to samples incubated with these activity-based probes, the adduct is readily detected through a shift in apparent molecular mass. usp2, usp5, usp13 and usp14 reacted with isg15vs ( figure 2b , c, d, e). the following observations confirm the validity of our assay. first, the bona fide isg15-isopeptidase usp18 displayed reactivity only towards isg15vs (figure 2a ), whereas most of the dubs reacted only with ubvme. an example is shown with cgi-77 ( figure 1 , 2f), a previously uncharacterized otubain-homolog. second, as a negative control, the sumo protease senp2 formed an adduct exclusively with sumo1vme ( figure 2g ). we found no evidence for any of the dubs evaluated here to display reactivity toward the sumo1 probe (not shown). the presence of the reactive group alone is clearly not sufficient for binding to the active-site cysteine of a protease and specificity of a dub thus depends on the peptide moiety of the probe, containing either ubiquitin, sumo1 or isg15. lastly, all covalent modifications of dubs by active-site directed probes were blocked by pretreatment of the translated polypeptides with the sulfhydryl alkylating agent n-ethylmaleimide (nem) (figure 2 ), confirming the cysteinedependency of adduct formation. in accordance with previous results [28] , we observed a non-linear decrease in electrophoretic mobility of the isg15vs adducts. the isg15 probe has a mass of 17.4 kda, whereas the size increase observed for each of the dubs investigated here is in the order of 25-110 kda, when bound to isg15vs. the correlation between the abnormal shift and the initial mass of the unmodified dub suggests that the decrease in gel mobility is based on steric properties of the branched adduct, and is not caused by covalent modification of a single dub by multiple isg15vs molecules. in fact, in our sample set, the observed size increase of the isg15vs-dub adducts based on sds-page very closely matched a logarithmic equation ( figure 3a and see methods). to further verify that dubs are modified by only a single isg15vs probe per molecule, we replaced the catalytic cysteine at position 114 in usp14 with a serine residue. as expected, this mutation abolished all labeling ( figure 3b ). our assay was conducted in ivt lysate and the size increase of the isg15vs adduct could potentially reflect modification of usp14 by additional factors. however, even usp14 that was recombinantly expressed in bacteria and .95% pure showed the same abnormal electrophoretic mobility for its isg15vs adduct ( figure 3c ). mass spectrometry confirmed the monovalent modification of purified usp14 by isg15vs and excluded covalent binding of additional factors to the complex ( figure 3d ). collectively, these experiments establish that the observed shift in apparent molecular mass of the isg15vs-dub adducts is solely a consequence of its unusual electrophoretic behavior. it also underscores the uncertainties in estimating the degree of modification of a target protein with ubls by sds-page alone. usp14 reacts more efficiently with isg15vs in its proteasome-associated form usp14 and its yeast counterpart ubp6 show significantly higher activity when bound to the 26s proteasome [29] . this activity may in fact be strictly dependent on association with the proteasome, as shown for ubp6 [30] . as further evidence for a physiological role of the interaction of usp14 with isg15, we investigated whether the allosteric activation of usp14 also influences its reactivity toward isg15vs. we examined labeling of usp14 with the ubiquitin-and the isg15-based probes as a function of the concentration of added purified proteasomes. as a negative control, we evaluated usp5, a dub that is not a known interaction partner of the 26s complex in vivo. as anticipated, the inclusion of purified proteasomes had no effect on the isg15vs-or ubvme-reactivity of usp5 ( figure 4b ). in contrast, we observed a dose-dependent increase in isg15vs adduct formation of usp14 with increasing proteasome concentration, indicating enhanced activity of this dub. the effect was similar in magnitude to that seen for the ubiquitin probe ( figure 4a , c). while recombinant usp14 in its purified form bound to electrophilic probes ( figure 3c ), we did not detect robust hydrolytic activity against ubiquitin-amc or against ubiquitinor isg15-linked isopeptide fusion proteins (data not shown). however, using sequential ultracentrifugation to obtain a cytosolic fraction that is enriched in 26s proteasomes [29] , we could show that proteases in this fraction efficiently and specifically cleave an isg15-isopeptide linked substrate ( figure 5a , b). the absence of proteolytic intermediates suggests specific cleavage of the isopeptide bond. in addition, the same bait peptide linked to sumo1 was stable and not hydrolyzed, even upon prolonged incubation for over 24 hours with the proteasome fraction. proteolysis of the isg15-linked peptide substrate was inhibited by inclusion of nem, indicating cleavage by cysteine proteases. analysis by reaction with isg15vs supports that usp14 is the only active red arrows depict dubs that bound to neither probe (ubvme, isg15vs, or sumo1vs), whereas black arrows indicate dubs that formed covalent adducts with the indicated probes. our screen represents the first biochemical proof for protease activity of usp13 and the otubainhomolog cgi-77 (dub homologs without publication record regarding biochemical function are marked with an asterisk). otubain1 (otu1) is an exception in that it binds to alkylhalide-or aldehyde-based probes, but not to the michael acceptors employed in this study (data not shown). doi:10.1371/journal.pone.0000679.g001 isg15-specific protease in the proteasome-enriched fraction ( figure 5c ). while we cannot formally exclude the possibility of a yet undefined enzyme binding to isg15vs, we consider this unlikely: such a protease would have to display a mass highly similar to that of usp14 and, furthermore, it would have to sediment after centrifugation for 5 hours at 100,000 g. however, only few deubiquitinating enzymes are sedimentable, none at a level comparable to usp14 [29, 31] . the wealth of usps found in the human proteome likely reflects substrate specificity, but potentially also complementation in terms of expression profiles and subcellular distribution. we therefore sought to analyze the intracellular distribution pattern of a subset of our crossreactive dubs, using confocal microscopy. the analysis of a genome-wide set of c-terminal gfp fusion proteins for yeast had shown remarkably few with altered function or subcellular distribution (,5%), validating the choice of such cterminal modifications [32] . using anti-g/yfp antibodies, we also utilized this tag to assay for activity of dubs in cell lysate. we cloned and transiently expressed five usp-eyfp constructs in 293t cells: usp5, usp13, usp14, usp3, and usp36 ( figure 6a ). lysate of usp14 eyfp transfected cells was incubated with the ubiquitin and the isg15 probe, and assayed by anti-yfp immunoblot analysis ( figure 6b ). whereas the usp14 eyfp construct reacted with both probes, the respective c114s mutant mutation of the catalytic cysteine residue to serine (c114s) in usp14 abolishes its reactivity toward ubvme and isg15vs. when stored for longer periods at room temperature, the probes polymerize covalently, presumably by formation of secondary amine bonds between internal lysine residues and the reactive michael acceptor at the c-terminus, thus resembling isopeptide-linked polyubiquitin. such polymeric probes of ubvme likely caused the additional high-molecular mass adducts observed for usp14. note that the smallest version of these adducts (a ubvme dimer) has a maximum electrophoretic mobility similar to that of the diubiquitin-like isg15vs when complexed to usp14. the absence of any adducts in the c114s mutant of usp14 excludes the possibility of multiple binding sites for the probes. (c) purified recombinant usp14 labels with ubvme and isg15vs and results in the same abnormal mobility shift for isg15vs-usp14 as seen in the ivt labeling experiments. (d) maldi-tof mass spectroscopic analysis of usp14 after incubation with isg15vs. as described above for ubvme, isg15vs also engages in internal polymerization. molecular masses consistent with tri-and tetrameric isg15vs are marked in this spectrogram by the numbers in superscript. monovalently modified usp14 results in an adduct of predicted size, indicated with a red arrow. this complex is unique to the mixture containing both usp14 and isg15vs, and is absent in the mass spectra of either component alone (data not shown). doi:10.1371/journal.pone.0000679.g003 did not, in agreement with the results of our ivt screen. with respect to subcellular distribution, we were particularly interested in the expression pattern of usp5 and usp13, given that these two isoforms displayed different specificity in ubvme and isg15vs labeling experiments. usp5 eyfp was found throughout the cell ( figure 6c, upper left panel) , similar to usp18 [33] . in contrast, its close relative usp13 eyfp was expressed mainly in the nucleus in a speckled pattern ( figure 6c, upper right panel) . consistent with the in vivo interaction between usp14 and the proteasome [5] , we observed usp14 eyfp predominantly in the cytoplasm, though we also noticed fluorescence in the nucleus ( figure 6c, lower left panel) . to demonstrate that the presence of the c-terminal yfp fusion does not interfere with the endogenous distribution of these enzymes, we analyzed usp3 eyfp ( figure 6c , lower right panel) and usp36 eyfp ( figure 6c , lower right panel). usp3 is predicted to be a nuclear protein [34] and usp36 was . reactivity of usp14 toward isg15vs is augmented by proteasomal association. usp14 and usp5 were generated by ivt. their activity toward isg15vs and ubvme was analyzed in the presence of increasing concentrations of purified human 26s proteasomes. (a) activity of usp14 toward ubvme and isg15vs increases as a function of the concentration of added purified 26s proteasomes (in mg/ml). (b) the activity of usp5 remains unaffected. (c) quantification of the radioactive signal of covalently modified usp5 and usp14. binding affinity is depicted on the y-axis as percent in labeling intensity, determined by the ratio of labeled versus unlabeled usp5 or usp14. the ratio in the absence of exogenous proteasomes is defined as 100%. doi:10.1371/journal.pone.0000679.g004 figure 5 . proteasome-associated usp14 has isg15-specific isopeptidase activity. (a) scheme depicting the ubl-peptide conjugate used to assay isopeptidase activity. the biotinylated peptide heptamer is attached to either isg15 or sumo1 in isopeptide-linkage. upon hydrolysis of the isopeptide bond by a specific dub, the heptamer is released and the biotin signal lost. (b) incubation of proteasomeenriched fraction (''5 hr pellet'') with ubl-peptide conjugate. after overnight incubation, the isg15-peptide conjugate is completely cleaved, resulting in loss of the biotin-signal (significant proteolysis occurs already after one hour, data not shown). this activity is sensitive to nem. hydrolysis is not observed for the sumo1-peptide conjugate. (c) anti-ha immunoblot of ha-isg15vs treated subcellular fractions. based on previous identification [28] and on electrophoretic mobility, usp5 is the dominant isg15-reactive dub in the five-hour supernatant, which is enriched for uncomplexed proteins of light and moderate size (red asterisk). the five-hour pellet represents heavy cytosolic complexes, in particular the 26s proteasome, and contains usp14 as the only isg15reactive dub (blue asterisk). doi:10.1371/journal.pone.0000679.g005 identified as a nucleolar protein [35] . both proteins were detected in the expected subcellular compartment. combined, these results indicate that isg15-specific proteases are expressed throughout the cell -a feature also proposed for dubs. this observation supports the notion that unlike sumoylation, which is believed to mostly occur in the nucleus [36] , isg15-modification affects many cellular compartments. our data show the existence of multiple isg15-reactive dubs, a finding that further strengthens the similarities between ubiquitin and isg15. usp2 is a highly active protease [37] and it represents one of only few mammalian dubs with a known target. usp2 exhibits oncogenic potential in prostate cancer by stabilizing its substrate fatty acid synthase (fas) [38] , and fas has indeed been identified as a target of isg15 modification [39] . furthermore, usp2 has been implicated in the regulation of the p53 pathway [40] . the recently solved structures of usp2 and usp14 [41, 42] show that both proteases accommodate the ubiquitin molecule in a shallow pan-like protrusion. based on the orientation of the ubiquitin protein in both structures, isg15 easily fits into the catalytic domain of usp2, usp14, and usp5 (data not shown) without apparent steric clashes (figure 7 ) [23, 43] . stimulation by interferons alters the composition of the proteolytic proteasome core [44] , tailoring its activity toward generation of peptide-mhc complexes for inspection by the immune system. interferon treatment also results in enhanced modification of proteins by isg15 -a factor that evolved in the vertebrate lineage, and whose origin thus coincides with that of the adaptive immune system. interestingly, inhibition of the proteasome leads to rampant accumulation of isg15-modified substrates [45] . while nothing is known about the molecular functions of isg15, its structural relative fat10 is a modifier that destines proteins for degradation by the proteasome [8] . we now have demonstrated that usp14 exhibits proteasome-associated isopeptidase activity toward isg15. could therefore isg15 be a (co-)modifier of proteins destined for proteasomal degradation? we have found no evidence that usp14 markedly changes the amount of isg15-modified substrates in cells (data not shown). however, usp14 is not a vital protease [30, 46] and its low catalytic turnover does not affect overall ubiquitin conjugation either [47] . a recent study suggests that usp14 might have a more complex role, by inhibiting the proteasome in addition to acting as a deubiquitinase [48] . the close sequence relationship between usp5 and usp13 (61.4% identity, 26.9% similarity) is contrasted by the functional differences and localization of these two proteases. these enzymes provide a unique opportunity to investigate the structural features that may contribute to ubiquitin versus ubiquitin-like specificity. a characteristic of usp5 and usp13 is the tandem occurrence of a uba domain, which has been implicated in the binding of ubiquitin [49] . our results raise the possibility that uba domains in general interact not only with ubiquitin, but also with isg15. alternatively, isg15 with its multiple lysines could act as a ubiquitination anchor, and usp5 may be a protease responsible for depolymerization of such chains [50, 51] . moreover, the cterminal hydrolase that processes the isg15 precursor has not been identified yet, and any of the novel isg15-specific proteases described here are potential candidates. the ubiquitin gene is prone to duplications and insertions, leading to the formation of new fusion proteins [52, 53] . isg15 likely emerged approximately 400-600 million years ago, when a nucleotide stretch from the polyubiquitin precursor gene, encoding a ubiquitin-dimer, was accidentally inserted in an area of the genome that was or that came under control of an interferon promoter. from an agnostic point of view, one could argue that isg15 simply has no relevant function. the moderate or absent phenotype of the isg15 knockout in mice [11] , the fact that isg15 has not (yet) established its very own family of conjugating and deconjugating enzymes, and isg15's relatively low degree of conservation between species would all support this view. yet, isg15's massive expression upon interferon challenge [54] likely reflects a role in anti-microbial or anti-viral defense [55] [56] [57] [58] [59] . and adaptation to the specific needs of host immunity often demands polymorphism. as a result, some genes most critical to the immune response are paradoxically least conserved. for example, cytokines and cytokine receptors substantially differ between species [60] and it is interesting to note that isg15 and ubiquitin were initially reported to be cytokines [61, 62] . similar observations were made for the ubiquitin-like modifier fubi (also known as fau or mnsfb) [63, 64] . if true, how do these factors gain access to the secretory pathway? it may pay to approach isg15 from a less conventional perspective and from this vantage point, we might uncover new functions of ubiquitin as well. usp2 and usp18 cdnas were cloned from a human kidney cdna library (biochain institute, inc.). the cdnas encoding the other human dubs were obtained from atcc. all cdnas encoding fulllength dubs were subcloned into pcdna3. the protein sequences of human dubs were obtained from the national library of medicine and the core domains were aligned with the clustalw algorithm (ebi server) [65] and manually edited with genedoc (http://www.psc.edu/biomed/genedoc/) by k.b. nicholas & hb nicholas jr., using the putative active-site cysteine as an alignment anchor. the phylogram represents a consensus tree based on 100 bootstrap iterations, calculated by the minimum evolution method under default parameters [66] . ivt was performed using the ''tnt-t7 quick reticulocyte lysate system'' kit (promega) for 30 to 45 min (0.25-1 mg dna per reaction). then, aliquots of the reaction mix were treated with rnase b (1 mg/ml, sigma) for 10 min and incubated with the probes as described below. sds-page followed by fluorography was performed as described [67] . the synthesis of human ubiquitin and ubl probes has been described [24, 28] . ivt products were incubated with saturating amounts of the individual probes (0.2-0.4 mg/10 ml ivt lysate). preincubation with nem was performed for 10 min at room temperature at a final concentration of 10 mm, after rnase b treatment. autoradiograms were subjected to quantification of the optical density with nih image software (version 1.32j) as ratio of labeled versus unlabeled ivt products. purified human 26s proteasomes for the experiments in figure 4 were purchased from biomol international and inhibited with mg132 (50 mm). e. coliexpressed human recombinant usp14 for the experiments shown in figure 3c to 50 ml of conjugation buffer (100 mm tris, ph 7.4, 5 mm mgcl 2 , 20 mm dtt, 40 mm atp) was added isg15 (boston biochem, 9.4 mm final) or gst-sumo1 (boston biochem, 10.4 mm final) and biotinylated peptide 7-mer (biotin-vkakiqd-oh, 250 mm final). the solution was mixed thoroughly and to this was added isg15 activating enzyme (boston biochem, isg15 e1, 50 nm final) and ubch8 (boston biochem, 250 nm final), or sumo activating enzyme (sae1/sae2 heterodimer, 50 nm final) and ubch9 (250 nm final). the solution was mixed and incubated for 15 hours at 37uc. the reaction mixture was transferred to a microcentrifuge membrane filter (vivascience, 5000 da mwco), diluted to 600 ml total volume with 50 mm tris, ph 7.4 and concentrated at 4uc to 50 ml. this dilution/concentration procedure was repeated six times. the products were transferred to a clean tube and diluted with 50 mm tris, ph 7.4 to a final volume of 100 ml. the sumo1-and isg15-linked biotinylated isopeptide was detected after transfer to a pvdf membrane (perkin elmer) with streptavidin-hrp (amersham). el4 cells were lysed with glassbeads and the proteasome-enriched fraction was retrieved by consecutive ultracentrifugation steps as previously described [29] . proteasome activity was inhibited with mg132 (50 mm). 10 mg of fraction protein were incubated with 0.2 mg of n-terminally ha-tagged isg15vs in a total volume of 10 ml for two hours at room temperature to detect isg15vsreactive proteases by anti-ha immunoblotting. the cleavage assay for isg15-or sumo1-branched peptides was performed at 37uc using 20 mg of total protein from the proteasome-enriched fraction and 5 ml of branched peptides in a total volume of 10 ml. ha-isg15vs treated subcellular fractions were analyzed with a monoclonal anti-ha antibody (12ca5). immunoblotting was performed as published [67] . 293t cells were maintained in dme medium as described [67] . various constructs were expressed by transient transfection, using a liposome-mediated transfection protocol (5-10 mg of dna/ 20 ml of lipofectamine-2000 per 10 cm dish; invitrogen) as described [67] . cells were analyzed between 24 and 48 h after transfection. c-terminal eyfp fusion proteins of dubs were generated by subcloning from pcdna3.1 into pegfp-n1 (clontech). np40 lysates of usp14 eyfp and usp14 c114s-eyfp transfected 293t cells were prepared and incubated with activesite probes as described [24] . due to the high similarity with gfp, eyfp fusion proteins can be detected with a polyclonal anti-gfp rabbit serum [68] . immunoblotting was performed as published [67] . immunofluorescence experiments were performed as described [67] with minor modifications. cells were allowed to attach to slides overnight. after fixation with 3.7% paraformaldehyde for 20 min at room temperature, subcellular localization of eyfp fusion proteins was analyzed with a perkin elmer spinning disk confocal microscope ultraview rs system. the microscope used was a nikon te2000-u inverted unit with a nikon 1006 1.4na dic lens. the imaging medium was nikon type a immersion oil. the ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction a ubiquitin ligase complex assembles linear polyubiquitin chains mechanisms underlying ubiquitination mechanism and function of deubiquitinating enzymes deubiquitinating enzymes are in/(trinsic to proteasome function) the doa4 deubiquitinating enzyme is required for ubiquitin homeostasis in yeast a genomic and functional inventory of deubiquitinating enzymes fat10, a ubiquitin-independent signal for proteasomal degradation a superfamily of protein tags: ubiquitin, sumo and related modifiers isg15: the immunological kin of ubiquitin isg15, an interferon-stimulated ubiquitin-like protein, is not essential for stat1 signaling and responses against vesicular stomatitis and lymphocytic choriomeningitis virus the ubch8 ubiquitin e2 enzyme is also the e2 enzyme for isg15, an ifn-alpha/ beta-induced ubiquitin-like protein interferoninducible ubiquitin e2, ubc8, is a conjugating enzyme for protein isgylation herc5, an interferon-induced hect e3 enzyme, is required for conjugation of isg15 in human cells herc5, a hect e3 ubiquitin ligase tightly regulated in lps activated endothelial cells identification and herc5-mediated isgylation of novel target proteins herc5 is an ifn-induced hecttype e3 protein ligase that mediates type i ifn-induced isgylation of protein targets ubp43 (usp18) specifically removes isg15 from conjugated proteins role of isg15 protease ubp43 (usp18) in innate immunity to viral infection ubp43 is a novel regulator of interferon signaling independent of its isg15 isopeptidase activity reexamination of the role of ubiquitin-like modifier isg15 in the phenotype of ubp43-deficient mice small-molecule inhibitors and probes for ubiquitin-and ubiquitin-like-specific proteases the ubiquitin binding domain znf ubp recognizes the c-terminal diglycine motif of unanchored ubiquitin chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family chemistry-based functional proteomics: mechanism-based activity-profiling tools for ubiquitin and ubiquitin-like specific proteases structure of the ubiquitin hydrolase uch-l3 complexed with a suicide substrate zinc is required for the catalytic activity of the human deubiquitinating isopeptidase t specific and covalent targeting of conjugating and deconjugating enzymes of ubiquitin-like proteins a novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of usp14 multiple associated proteins regulate proteasome structure and function pathways accessory to proteasomal proteolysis are less efficient in major histocompatibility complex class i antigen production global analysis of protein localization in budding yeast cloning and characterization of a novel human ubiquitin-specific protease, a homologue of murine ubp43 (usp18) psort: a program for detecting sorting signals in proteins and predicting their subcellular localization nucleolar proteome dynamics sumo and ubiquitin in the nucleus: different functions, similar mechanisms? ubiquitin-specific protease 2 as a tool for quantification of total ubiquitin levels in biological specimens the isopeptidase usp2a regulates the stability of fatty acid synthase in prostate cancer human isg15 conjugation targets both ifn-induced and constitutively expressed proteins functioning in diverse cellular pathways the deubiquitinating enzyme usp2a regulates the p53 pathway by targeting mdm2 structural basis of ubiquitin recognition by the deubiquitinating protease usp2 structure and mechanisms of the proteasome-associated deubiquitinating enzyme usp14 crystal structure of the interferon-induced ubiquitin-like protein isg15 ifn-beta mediates coordinate expression of antigen-processing genes in rsv-infected pulmonary epithelial cells proteasomes modulate conjugation to the ubiquitin-like protein, isg15 synaptic defects in ataxia mice result from a mutation in usp14, encoding a ubiquitin-specific protease transgenic rescue of ataxia mice with neuronal-specific expression of ubiquitinspecific protease 14 deubiquitinating enzyme ubp6 functions noncatalytically to delay proteasomal degradation the uba domain: a sequence motif present in multiple enzyme classes of the ubiquitination pathway metabolism of the polyubiquitin degradation signal: structure, mechanism, and role of isopeptidase t deubiquitinating enzymes: their functions and substrate specificity concerted and birth-and-death evolution of multigene families ubiquitin-conserved protein or selfish gene? identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays from the cover: ifn-stimulated gene 15 functions as a critical antiviral molecule against influenza, herpes, and sindbis viruses identification of interferon-stimulated gene 15 as an antiviral molecule during sindbis virus infection in vivo the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme influenza b virus ns1 protein inhibits conjugation of the interferon (ifn)-induced ubiquitin-like isg15 protein innate antiviral response targets hiv-1 release by the induction of ubiquitin-like protein isg15 genetic susceptibility to infectious disease ifn-induced 15-kda protein is released from human lymphocytes and monocytes differentiation of t cells in nude mice ubiquitin-like moiety of the monoclonal nonspecific suppressor factor beta is responsible for its activity ubiquitin-like polypeptide conjugates to acceptor proteins in concanavalin a-and interferon gamma-stimulated t-cells using clustal for multiple sequence alignments mega3: integrated software for molecular evolutionary genetics analysis and sequence alignment cotranslational endoplasmic reticulum assembly of fc{varepsilon}ri controls the formation of functional ige-binding receptors visualization of the er-tocytosol dislocation reaction of a type i membrane protein swiss-model and the swiss-pdbviewer: an environment for comparative protein modeling we thank a. di bacco from the gill lab for the senp2/pet28b construct, and d. finley for helpful comments (harvard medical school). we are also grateful to c. dahl for the maldi-tof analysis of the usp14-isg15vs complex (harvard biopolymer facility). we would like to thank d.e.zhang for donating the constructs encoding ube1l and ubc8 (scripps research institute), and h.w. virgin for sharing anti-isg15 antibodies (washington university). the structures of usp2 and usp14 in complex with ubiquitin and the structure of isg15 were obtained from the pdb website (2hd5, 2ayo, 1z2m) and isg15 was manually modeled onto usp2 and usp14 with the spdb-viewer [69] . other: devised the screening strategy: ac ef. conducted the biochemical and cellular experiments, the microscopy analysis and the bioinformatics: ac. cloned dub encoding constructs: ef. supported the experimental approach: ef. helped draft the manuscript: ef. provided isopeptidelinked bait peptides: gk. supported the dub cloning: db pg. cosupervised the project: pg hp. drafted the manuscript: ac. key: cord-048364-yfn8sy1m authors: fraser, christophe title: estimating individual and household reproduction numbers in an emerging epidemic date: 2007-08-22 journal: plos one doi: 10.1371/journal.pone.0000758 sha: doc_id: 48364 cord_uid: yfn8sy1m reproduction numbers, defined as averages of the number of people infected by a typical case, play a central role in tracking infectious disease outbreaks. the aim of this paper is to develop methods for estimating reproduction numbers which are simple enough that they could be applied with limited data or in real time during an outbreak. i present a new estimator for the individual reproduction number, which describes the state of the epidemic at a point in time rather than tracking individuals over time, and discuss some potential benefits. then, to capture more of the detail that micro-simulations have shown is important in outbreak dynamics, i analyse a model of transmission within and between households, and develop a method to estimate the household reproduction number, defined as the number of households infected by each infected household. this method is validated by numerical simulations of the spread of influenza and measles using historical data, and estimates are obtained for would-be emerging epidemics of these viruses. i argue that the household reproduction number is useful in assessing the impact of measures that target the household for isolation, quarantine, vaccination or prophylactic treatment, and measures such as social distancing and school or workplace closures which limit between-household transmission, all of which play a key role in current thinking on future infectious disease mitigation. the household is a fundamental unit of transmission for many directly transmitted infections. in addition, the household provides a ''laboratory'' within which key measures of transmission such as infectiousness, generation time and the effect of immunity or vaccination can be studied [1] . in recent years considerable effort has gone into understanding the dynamics of transmission within populations organised into households using mathematical models [2, 3, 4, 5, 6] . most effort has gone into analysing the asymptotic behaviour of these models, elucidating the threshold levels of transmission required for infection to be self-sustaining, calculating final epidemic sizes, or predicting the impact of generalised or targeted interventions designed to reduce or eliminate transmission. in parallel, methods have been derived to estimate the parameters which govern transmission within the household from detailed case reports [7, 8, 9, 10] . however, scant effort appears to have been paid to how to apply household structured models to the analysis of epidemics, either retrospectively or in real time. concurrently, mathematical models have played an ever greater role in interpreting and responding to emerging pathogens. these models have typically been either of the ''simple but tractable'' variety which ignore or average over demographic structure and social mixing patterns [11, 12] or the ''complex computer simulation'' variety that capture many details of demographic structure and dynamics, but of whom the behaviour can only be determined by intensive numerical analysis [13, 14, 15] . the aim of this study is to develop methods of a perhaps ''slightly less simple but still tractable'' variety that capture some of the detail that micro-simulations have shown is important, but which can be rapidly applied (say on a daily basis) in an emerging outbreak situation, to inform policy. more specifically, the aim is to arrive at a method to estimate the key transmission and control parameters for a model of transmission within and between households from as few detailed observations as are likely to be gathered in the heat of a major outbreak. the resulting analysis will still be based on major simplifications in respect to all the spatial and other social constructs that govern disease transmission, but less so than those based on the very simplest assumption of free, homogeneous mixing. in this context, it should be stated that even in the best, most robustly parameterised microsimulations, gross approximations are made in describing the fabulously complex web of human behaviour, and even they are only attempts to characterise the statistical properties of the system as a whole. extensive effort is, and should continue to be, spent on identifying the conditions where different types of simplification (household models, static network models, spatial metapopulation models…) can and can't be justified, and in developing analytical approximations to describe disease transmission within such simplified structures. individual based simulations of influenza and smallpox pandemic spread and control, incorporating detailed information on population density, age structure, commuting patterns, workplace sizes and long-distance travel have highlighted the particular importance of the household as a fundamental unit of transmission [13, 14, 16, 17, 18] (and reviewed in [19] ). pure household models have been used fruitfully to explore detailed policy options in a city-wide response to an influenza pandemic [20] . it thus seems a priori that household models are a natural starting point in terms of extending theory previously developed for the simplest assumption of homogeneous mixing. the analysis presented here will focus on deriving new estimators for individual and household reproduction numbers, denoted r (t ) and r * (t ) respectively. the individual reproduction number r (t ) is defined roughly as the average number of people someone infected at time t can infect over their entire infectious lifespan; as i will show below, there are several ways of defining this more precisely. the household reproduction number r * (t ) is defined here as the average number of households a household infected at time t can infect [3, 6] . the individual reproduction number r (t ) rightly plays a privileged role in epidemiology, as it is a meaningful measure within any contact network. however, of the possible summary measures of epidemic progress, it is not necessarily the most useful. for example, for an emerging directly transmitted pathogen, such as pandemic influenza virus, public health interventions may target the household rather than the individual, enforcing household quarantine as well as offering antivirals to the household to limit transmission within the household. in such a situation, the household reproduction number r * (t ) is more directly related to the parameters which characterize the intervention, and is thus a better measure of the effect of these interventions. these quantities (r (t ) and r * (t )) share the two essential properties of reproduction numbers, namely that they increase when infectiousness increases and decrease when infectiousness decreases (monotonicity), and that they mark a threshold that separates exponentially growing epidemics (when r (t ).1 or equivalently r * (t ).1) from exponentially declining epidemics (when r (t ),1 or equivalently r * (t ),1) [3, 6] . the structure of the paper focuses first on deriving estimators for individual reproduction numbers, then on household reproduction numbers and finally on examples of pandemic influenza dynamics and measles. though less well known than their compartmental counterparts (sir, sis, etc…), time-since-infection models offer a more intuitive starting point for modelling infectious disease transmission, and importantly for this application, they provide two other major advantages. first, it is typically easier to identify their key parameters, and second they more readily adapt to describe multi-level transmission (by multi-level, i mean here withinhousehold and between household). a disadvantage is that it can be harder to include heterogeneities. nomenclature is confusing, since both types of model have their origin in the same classic paper of kermack and mckendrick [21] , and both the sir model and the simplest time-since-infection model are known as ''the kermack-mckendrick model''. the model, in the formalism chosen here, predicts the changing incidence rate i (t) as a function of calendar time t in terms of the transmissibility, denoted b (t, t ), an arbitrary function of calendar time t and time since infection t. b (t, t) typically reflects pathogen load, or perhaps more precisely pathogen shedding. it is commonly a single peaked function reflecting pathogen growth followed by immune suppression, or host death, but can be more exotic such as the double peaked profile associated with early and late transmission of hiv [22] , or the repeated peaks of malaria [23] . b (t, t) also reflects the effective contact rate between infectious and susceptible individuals, which can change during the course of a single infection, increasing for example if a person coughs or sneezes due to respiratory disease, or decreasing if a person takes to bed with illness, and during the course of the epidemic as public health measures are implemented. more discussions of the components (infectiousness and contact) of b (t, t) can be found in [24] . because i am interested in outbreaks of emerging infections, i will not describe explicitly reductions in the susceptible population caused by the epidemic. formally this corresponds to working in the infinite population limit. this assumption is not essential for this section however, since b (t, t) could also be thought of as incorporating the proportion of cases that are susceptible; the assumption becomes more important in the later sections on household models. mathematically, transmission is defined by a poisson infection process such that the probability that, between time t and t+d, someone infected a time t ago successfully infects someone else is b (t, t)d, where d is a very small time interval. this assumption then results in a prediction that the mean incidence i (t ) at time t follows the so-called renewal equation this equation states that the number of newly infected individuals is proportional to the number of prevalent cases multiplied by their infectiousness. it may often be convenient (and realistic) to truncate the function b (t, t) at a time t m such that b (t, t) = 0 for all t.t m . the asymptotic behaviour of incidence i (t ) is determined by reproduction numbers [21, 25] . two intuitively defined reproduction numbers are the case reproduction number, which i denote r c (t ), and the instantaneous reproduction number, which i denote r (t ). the case reproduction number r c (t ) is a property of individuals infected at time t, and is the average number of people someone infected at time t can expect to infect. for a person infected at time t it is the total infection hazard from time t onwards, i.e. while the case reproduction number has been widely used, it may also be worth considering a quantity which i call the instantaneous reproduction number r (t ), a property of the epidemic at time t. it is the average number of people someone infected at time t could expect to infect should conditions remain unchanged. it is given by to illustrate the distinction between r c (t ) and r (t ), consider a situation where the transmission rate is abruptly reduced at a time t = t i . the instantaneous reproduction number r (t ), which estimates how many people one case would infect if circumstances were to remain fixed, would abruptly switch from a high to a low value at time t i . the case reproduction number r c (t ), on the other hand, estimates how many people each case actually infects. it will thus account for the fact that someone infected at time t,t i may spend part of their infectious period before and after the reduction in transmission which occurs at time t i and thus r c (t ) will smoothly transition from higher to lower values. to derive simple estimating equations for r (t ), i consider the case where this function is separable, which corresponds to saying that the relative progression of infectiousness as a function of time since infection is independent of calendar time. in this case b (t, t) can be written as the product of two functions w 1 (t ) and w 2 (t), i.e. b t,t ð þ~w 1 t ð þw 2 t ð þ ð4þ a counter-example might be when reactive patient isolation is introduced and acts to reduce infectiousness in late stage infection, in which case b (t, t) can't be decomposed in this way. for this type of situation, it may be reasonable to assume the b (t, t) can be decomposed separately in different stages of the epidemic, pre-and post-implementation of isolation measures, for example. since b (t, t) is a product, i can arbitrary normalise one or other of the functions w 1 (t) and w 2 (t), so without loss of generality, i choose w 2 (t) to have total integral 1, i.e. ð ? 0 w 2 t ð þdt:1. the function w 1 (t) is equal to the instantaneous reproduction number r (t). the function w 2 (t) is then the distribution of how these infection events are distributed as a function of time since infection t. this is an idealised definition of the generation time distribution, which i denote w (t). thus, infectiousness can be decomposed as the product of the instantaneous reproduction number and the generation time distribution, i.e. the relationship between the idealised generation time distribution w (t) and the distribution of observed generation times can be rather complex for a number of reasons. first, infections are rarely observed, and thus must be either backcalculated or the generation times must be based on a surrogate such as the appearance of symptoms [1, 12] . second, right censoring can cause the observed generation times to be shorter or longer than expected for a growing or declining epidemic, respectively [26] . third, as apparent here, if the reproduction number r (t) changes due to depletion of susceptibles, changes in contact rates or public health measures, then this will also change the observed generation times for infectious individuals during that period of change. thus the distribution w (t) is really intended as a measure of infectiousness which will correspond to generation times for an index case in an ideal large closed setting where contact rates are constant. it can be inferred from data on the timing of cases, as in [10, 13] . inserting (6) into (1) yields a novel estimator for instantaneous reproduction number by substituting the decomposition (6) into equation (2) , a relation between the instantaneous and case reproduction number is obtained: i.e. the case reproduction number is a smoothed function of the instantaneous reproduction number. usually, incidence is reported as a discrete time series of the form i i incident cases reported between time t i and time t i +1, in which case the generation time distribution should be appropriately discretised into a form w i such that p n i~0 w i~1 . the estimators for the reproduction numbers become and equation (10) was proposed by [12, 27] as a real time estimator of the reproduction number, while equation (9) was first used for analysing polio transmission in india [28] (based on the work presented in this manuscript). while the case reproduction number is an intuitively appealing quantity, the instantaneous reproduction number estimated by equation (9) should also be considered for practical applications as it may suffer fewer problems of right censoring in an incompletely observed epidemic. right censoring is a real problem in using the case reproduction number to track an epidemic in real time, since the estimator for r c (t) at time t is seen in equation (10) to rely on knowing the incidence at future time-points. an algorithm to deal with this issue was proposed by [29] , but switching instead to the instantaneous reproduction number estimated by equation (9) may be a simpler solution. right censoring is not however the only complication associated with estimating reproduction numbers in practice, and is not completely absent from (7) due to the delay in detecting infections. left censoring may also arise due to not knowing the baseline number infected if an epidemic has been unfolding for some time before observations are recorded. finally, estimating the generation time distribution may not be straightforward. several strategies are possible to deal with the fact that one never observes infections, but rather as a time series of cases of the form c i , where case definitions could be based on symptoms, hospitalisation or seroconversion. one strategy, used in [12] , is simply to ignore this and use cases as surrogates of infection for estimation of both the generation time and the reproduction numbers. often though, it may be possible to characterise a distribution of the time from infection to becoming a case, say j i where p n i~0 j i~1 . if a case is defined by symptoms then this would be the incubation period distribution. one can then backcalculate incidence as follows a drawback of this approach is that the estimated incidence time series iˆi will tend to be over-smoothed relative to the original time series i i . it also makes clear that there is still a problem of right censoring in an incompletely observed epidemic in the estimator of equation (9), though less than in equation (10) . statistical properties of these estimators are straightforward [12, 28] . one previously noted point [12, 28] is that because these estimators are essentially ratios of incidences, they can be used in cases where only a fraction of cases are observed, such as for polio where only a tiny fraction of infections lead to disease (of the order of 1 in 200), though the confidence intervals will change. a special case applicable to many cases where surveillance is poor is when only the epidemic growth/decline rate r is known. in this case the incidence takes the form i (t) = i (0)exp (rt) and both estimators (7) and (8) for the reproduction number become where the reproduction numbers are now expressed as a function of the exponential rate of change r. this is likely a useful formula, presented and studied in detail in [30] , where the links to earlier ecological and demographic modelling were also highlighted. much of the subsequent analysis will concern itself with deriving an equation equivalent to (12) for the household reproduction number r * (r). the model defined above assumes that the function b (t, t) describes the ''natural history'' of infection in each infected individual. before specialising to the model of household transmission, it is first worth considering the case where different individuals experience different ''natural histories'', defined here by the susceptibility to infection, and infectiousness after infection. i denote a vector of random variables x = {x1, x2, …} to describe factors which influence susceptibility or infectiousness. for example for the standard seir model of infection the random variables would be the durations of the latent period (l) and the infectious period (d), i.e. x = {l,d}. let f (x ) denote the probability distribution of these random variables amongst new infections (taking into account differences in susceptibility), defined such that where the integral is taken over the domain of the random variables. in other words, f (x ) is the proportion of new infections that have state x. let b (x, t, t) denote the infectiousness profile of an individual with state x. assuming that all individuals mix homogeneously, then the transmission model defined earlier by equation (1) is generalised to where i (x , t) is the incidence of infections with state x. i define the function k(t) to denote the integral which clearly depends only on time t and not state x. the total incidence at time t is defined by the integral by substituting equation (14), which can be rewritten as i (x, t ) = f (x ) k (t ), into equation (16), i obtain that k (t ) = i tot (t ) and thus that i can now substitute (17) into (14) to obtain dividing both sides of this equation by f(x)yields an equation for the total incidence if i define the average infectiousness as follows then equation (19) can now be seen to be the standard kermack-mckendrick model of equation (1), i.e. in other words, in this model of an emerging infectious disease epidemic with heterogeneities in susceptibility and infectiousness, the dynamics of mean total incidence of infection is exactly equivalent to the basic model where the infectiousness is appropriately averaged using equation (20) . once an expression is derived for the average infectiousness b (t, t), the results such as equations (9) or (12) can be used without further consideration of the heterogeneities in infectiousness or susceptibility. heterogeneities which are transmitted or preserved from one infection to the next, for example due to non-random mixing between different risk groups, a situation not considered here, lead to a more complex result. some public health interventions such as isolation and contact tracing can induce such heritability even if it is not a basic property of the transmission process [31, 32] . a useful exercise in applying this formalism (not elaborated here) is the derivation of standard formulae for the basic reproduction number as a function of the exponential growth rate r for the seir model [30] . one approach to estimating household reproduction numbers is simply to switch perspective from individual to household, directly estimate the generation time distribution (times taken for one household to infect another) and incidence of infection of households, and apply the results of equations (9) or (12) to estimate reproduction number as a function of time, r * (t), or exponential growth rate, r * (r). because, as i have shown, the linearised kermack-mckendrick model is applicable even when susceptibility and infectiousness are heterogeneous, this method is acceptable despite the fact that households may be quite heterogeneous in size and in the number of people infected. one analogous situation where this approach has been used is in estimating farm-to-farm reproduction numbers in the 2001 uk foot-and-mouth virus epidemic [27] . however, unless specifically tailored to this task, it is unlikely the data will be collected in the requisite form for this approach to be used in the human household situation. thus, in this section i explore the alternative approach of explicitly modelling transmission within and between households. homogeneous transmission models can be interpreted as twolevel hierarchical models, where the processes which guide the natural history of infection within the host are considered separate from those which drive transmission between hosts. the link between the two can be thought of as the function b (t, t) which translates the impact of changing processes within the host into changing infectiousness as a function of time since infection. the approach taken here to modelling household transmission is to study a three-level hierarchical model of transmission. the three levels are within-host, within-household, and between households. the natural history of infection is described by the individual infectiousness function b (t, t). i assume in this section that individuals are homogenous in infectiousness and susceptibility. i then use this to predict the course of epidemics within households, and derive a function b * (t, t * ) which describes the average infectiousness of a household towards other households as a function of the time since the household was infected, t * (from here-on, i use the starred symbols to denote properties of households, and un-starred symbols to denote properties of individuals). the basic idea behind this analysis is illustrated in fig 1. to simplify the notation, and because the main aim of this section is to study the case of an epidemic growing exponentially, i consider the situation where infectiousness is independent of calendar time t. this could be relaxed, though only if variation in time is somewhat slower than the typical duration of infection within a household. more specifically, the model assumptions are that: n individuals are distributed into households, and mix randomly and homogeneously outside of their household; n within a small time interval d, an individual who has been infected a time t ago infects a person at random in the population with probability b g (t )d; n within this same time interval he or she infects each susceptible individual in his or her household with probability b l (t, n)d (this is allowed to depend on the household size n, since empirical evidence suggests such variation may occur [10] ); n the population is large, and the disease has low prevalence, so that the probability of a household being repeatedly infected is negligible; n the functions b g (t ) and b l (t, n ) are proportional to each other as functions of the time since infection t. as a result of the last assumption and of the discussion around equation (6), the infectiousness functions can be decomposed as b g (t) = r g w (t) and b n (n, t) = r n w (t), where r g is the average number of people each infected individual infects through random (non-household) contacts, w (t) is the generation time distribution for between household transmission, and r n is a parameter describing infection within the household whose interpretation will be clarified below. i start by analysing the process of transmission within a single infected household of size n in terms of the functions r n and w (t). consider first a household of size 2, where one individual is infected at time t * = 0. given the poisson process described by the assumptions listed above, the probability that the second individual the probability that the second person is never infected is the distribution of times of infection of the second individual, conditional on infection, is then where 2l q 2 (t * )/lt * is the rate of change of the cumulative probability of not being infected, i.e. the probability density of being infected at time t * , and the normalising factor 1-q 2 is the total probability of being infected. the difference between w 2 (t * ) and the standard generation time distribution w (t) is a saturation effect, so that the second case tends to get infected earlier as the infectiousness of the index case (r 2 ) is increased. the infectiousness of the second individual towards other nonhousehold members of the population, conditional on his or her infection, and described as a function of the time t * since the infection of the household is thus the convolution of w 2 (t * ) and b g (t), so that the total infectiousness of the household is generalising this exact result to larger households involves some complications. consider for example a household of size 3, where one individual is infected at time t * = 0. the probability that neither of the other two individuals is infected by the first individual at time t * is q 3 t ã ð þ~exp {r 3 ð tã 0 w s ð þds à á directly analogous to the situation for households of size 2. however this is somewhat greater than the actual probability that they are not infected at all, since once one of these two is infected, they can also infect the other, and thus the probability that they each escape infection is somewhat less than q 3 :q 3 ? ð þ~exp {r 3 ð þ. to progress further with analysing this system, i propose to approximate the process by assuming that infections within a household can be approximately described by a discrete generation reed-frost model, i.e. where the probability of not being infected in each generation is (q n ) m where m individuals are infected in the previous generation and q n u exp (2r n ). q n is the escape probability of each infectious-susceptible pair of individuals considered in isolation. in the formalism proposed by ludwig, this corresponds to using infectious rank as a surrogate for infectious generation [33] . dynamics are recovered by assuming the times between generations are described by the standard generation time distribution w (t). the ordering of infection events has no influence on the final number of individuals infected [33] , and therefore this approximation will produce exact results for the final number of people infected in each household. because of the possibility of ''later'' generations preceding ''earlier'' ones, as noted in the case of households of size 3 above, and because of ignoring the saturation effect present in equation (22) in terms of the actual generation times within households, this approximation will overestimate the time taken for individuals to become infected in the household. because of the general form of the relation between generation time and reproduction number seen in equation (12) , this will result in over-estimates of the household reproduction number r * (r). to provide a counter-balancing under-estimate of r * (r), i also consider an alternative approximation obtained by assuming the same total number of cases as predicted by this reed-frost model, but where all cases are assumed to be infected by the first index case. this is not a formal lower bound, since in the limit of infinite infectiousness within the household, all members of the household will be infected simultaneously upon introduction of the infection into the household. i find however that even for the example of highly infectious measles virus (below), the under-approximation is sufficient to provide a practical lower bound. the probability of different chains of infection within households can easily be computed from the assumed reed-frost model [2] . i denote pr( {m 1 , m 2 , …, m n }|n) the probability of a chain of infection occurring in a household of size n where m 1 index cases infects m 2 , who in turn infect m 3 tertiary cases and so on, up to a maximum of n generations of infection. it is an assumption of the where pr m iz1 j m 1 , . . . ,m i f g ,n ð þbinomial the second approximation is that the time taken for one infected to infect the next is distributed according to the standard generation time distribution w (t). the time at which someone in the (i+1) th generation of infection is infected is as a result drawn from the i th auto-convolution of this distribution, denoted here w [i] (t * ) and defined by the recursive convolution equation which satisfies . consider now an individual in the i th generation of infection in the household, and consider this household at a time t * after the first index case was infected. this individual must have been infected at some earlier time s ( t * distributed according to the distribution w [i-1] (s). his or her infectiousness to others outside of the household will be given by b g (t * -s). thus, by averaging over all possible values of s, the average infectiousness of such an individual in the i th generation is thus having averaged over all possible times of infection in the chain of transmission events in the household, infectious households are stratified by their size and by the number of cases in each generation. using the notation defined earlier, i define the state vector x = {n, m 1 , …, m n } of variables which define the infectiousness and susceptibility of the infected household, where n is the household size and m i is the number of infected individuals in the i th generation of infection in the household. the infectiousness of a household with this state x towards other households, mediated by random mixing of individuals between households, is the sum of the infectiousness of all the individuals each given by equation (27) given that this infection process involves random mixing of individuals outside their household, the distribution of sizes of households which get infected is the so-called size-biased household distribution. this is the distribution of sizes one obtains by sampling individuals at random in the population and recording the size of their household, as opposed to the more commonly recorded household size distribution which is obtained by sampling households at random. if k n denotes the household size distribution, then is the size-biased household size distribution. given a household of size n gets infected, the probability of a chain of infections is given by the reed-frost probabilities pr ({m 1 , …, m n }|n). the distribution of the random variables x = {n, m 1 , …, m n } at infection is thus f x ð þ~k n : pr m 1 , . . . ,m n f g j n ð þ ð 30þ the mean infectiousness of a household is let m = s i m i be the average total number of cases in an infected household. the household reproduction number takes an intuitive and well known form derived in [3, 6] , expressed in terms of the parameter r g as follows: i.e. the household reproduction number is the product of the expected number of infections in a household multiplied by the number of people each individual infects out of their household. the mean household generation time distribution (time for one household infecting the next) is the mean generation time for households, t g * , can be expressed in terms of the individual generation time t g as the generation time distribution w * (t * ) can be used for the previously defined estimators of reproduction numbers (7)-(12) using household incidence data or just exponential growth rates. the exponential growth rate r for an exponentially growing epidemic is the same whether measured for individual or household incidence. for an exponentially growing or declining epidemic, one obtains the estimator now consider the integration where the first equality uses the definition of the auto-convolution, the second is a re-ordering of integrals, the third involves changing variables to u = t * -s, the fourth is a factorisation and the fifth arises by induction. the sixth uses the definition of the individual reproduction number r (r ) one obtains ignoring household structure from equation (12) . the household reproduction number can be expressed in terms of the individual reproduction number r (r ) as examination of equation (33) immediately reveals that the estimate for the number of people each person infects out of the household is i have thus derived a simple analytic relation between the individual and household reproduction numbers. both are approximations, ignoring the effects of local saturation on the generation time, which will tend to produce overestimates of the reproduction number. an alternative approximating to the household reproduction number, which provides an underestimate, is found when all secondary household cases are assumed to arise in the second generation, i.e. using equation (38) there are two reasons for considering household structure in analysing the pandemic influenza situation. first, influenza transmission is known to be concentrated within the household, and thus parameter estimates which ignore this heterogeneity are likely to be frail. second, many public health policies for future pandemics are likely to be organised around the household. the net effect of social distance measures such as school and workplace closures and cancellation of social gatherings is effectively to reduce transmission out of households (and perhaps inadvertently to increase transmission within them). furthermore, antiviral treatment and prophylaxis and quarantine measures are likely to be targeted at whole households rather than individuals (though restricting families with one suspect case to stay together without any other support is possibly undesirable) [16, 17, 20] . a number of studies have identified the parameters needed to estimate the household reproduction number for influenza [8, 10, 11, 17] . it is important to bear in mind that these parameters could be quite different in future pandemics, and thus that robust methodology may be more useful in responding to new outbreaks than numerical estimates obtained for past outbreaks. while it would be straightforward to use demographic data and exponential growth rates from earlier pandemics combined with interpandemic data on the transmissibility of influenza within households to obtain estimates of r * for historical pandemics, it has not been shown that the within household transmission parameters for inter-pandemic influenza adequately describe the pandemic situation, so i focus instead on providing illustrative examples using current demographic data (on the household size distribution from the uk) [34], and recent data on the transmissibility of influenza in modern households [10] . the household size data from 2001is truncated to size 6, and i assume that all households of size 6 or greater have size exactly 6. the data are k 1 = 29% (i.e. 29% of households are single person households), k 2 = 35%, k 3 = 16%, k 4 = 14%, k 5 = 5% and k 6 = 2%. the size of the mean household is thus 2.38 (average size of households where households are sampled at random), while the household of the mean individual has size 3.06 (average size of household to which individuals belong, where individuals are sampled at random). from the french influenza study [10] , i obtain maximum likelihood estimates of the within household transmission parameter of r n = 1.35/n 1.0 (which is consistent with the best fit to the tecumseh data [8] of r n = 1.27/n 0.97 ). the former study followed seronegative households for a two week winter outbreak of seasonal influenza. the corresponding escape probabilities are q 2 = 50.9% (i.e. the probability of not being infected by the other household member in a household of size two is 50.9%), q 3 = 63.8%, q 4 = 71.4%, q 5 = 76.4% and q 6 = 79.9%. on the scale of other infections, this places influenza as being approximately as infectious as mumps, but a lot less infectious than either varicella-zoster or measles [1] . by applying the reed-frost model to these data with this distribution of households, i obtain estimates of the average number of infections in each generation of infection of m 1 u 1, m 2 = 0.64 (i.e. the first index case directly infects an average of 0.64 people in his or her household), m 3 = 0.19, m 4 = 0.036, m 5 = 0.0037 and m 6 = 0.00021, and thus the estimate for the total expected number of cases in an infected household is m = s 6 i = 1 m i = 1.87, to be compared to the mean size of 3.06. these calculations are performed in microsoft excel 2007 using equation (25) . there is not yet a consensus on the generation time of influenza [13, 14, 16, 30, 35] , with estimates ranging from 2.6 days in [13] to 5.3 days in [14] . i use a gamma distribution with mean t g = 2.85 days and standard deviation 0.93 days, as reported in [30] . based on these data, i compare the predicted and simulated infectiousness of households in fig. 2 , which shows the average over all households sizes and compares this to the final analytical approximation given by equation (31) for b * (t * ), and also the alternate approximation which considers all secondary infections to arise in the second generation of infection ; the simulations and the first approximation are clearly in good agreement. individual based stochastic simulations were programmed using berkeley madonna, and are described in appendix s1. for the case of an exponentially growing epidemic, the estimates of the individual and household reproduction numbers, r and r * respectively, are shown in figure 3 , along with the estimate of the number of people one person infects outside their household, r g . for r * , both the under-and over-estimating approximations are shown, along with estimates obtained from the simulated generation time distribution. as expected for this low-infectiousness scenario, the simulated values are closer to the overestimating approximation. the range between these approximations which bracket the true value is rather narrow, indicating that the method is predictive. for the 1918 ''spanish flu'' h1n1 pandemic, the median growth rate in large us cities was r = 0.20 per day [30, 35] , with comparable estimates in the uk [17] . this value also serves as an upper estimate for the spread of the h2n2 pandemic virus in 1957 [17] . based on this growth rate, the estimated individual reproduction number is r = 1.74, while the estimated household reproduction number is r * = 2.26, and thus the out-of-household reproduction number is r g = 1.21. of course, households were bigger in 1918 than now, so that the actual value of r * was likely higher than this. these estimates would imply that a proportion 121/r * = 56% of between household transmission would need to be blocked to prevent epidemic spread. figure 3 could provide a rough guide to the likely values of r * and r g for a new influenza pandemic where the rate of exponential growth can reliably be determined. consider someone who the index case in their household; they would be expected to infect r g = 1.21 people out of their household and m 2 = 0.64 within their household. this validates assumed proportions of transmission within and between households from earlier simulation studies [17, 20] . the sum of these is greater than r since the reproduction number r is an average over different generations of infection within the household. for this value, the estimate of r which takes into account local saturation effects was determined numerically to be r = 1.79. fig 3 shows that for all values of r, numerically estimated values for r (r ) are close to the curve estimated from application of equation (12) which ignores local saturation effects. as a final check of the method, epidemics within a community of 2,000 households were simulated using an individual based stochastic model (see appendix s1). i choose r g = 1.21 as inferred from an epidemic growth rate of r = 0.20 per day, and the other parameters as described above. the exponential rate of growth was then re-estimated directly from the simulated incidence timeseries to be r = 0.19 (figure 4) , close to the predicted value of r = 0.20. this provides further support for the validity of this method, especially since no restrictions were placed on re-infection of households within this small simulated community. as noted above, influenza is relatively uninfectious compared to other common viruses. for a contrasting application of the method, i now focus on measles which was the most infectious of the pathogens studied in [1] . measles also has a more peaked generation time distribution, so that generations of infection are more distinct, and to make the contrast with the influenza estimates yet greater, i also use demographic data on household size chosen from the national census in 1961, when household sizes were greater than they are now. this analysis is perhaps a little artificial when applied to measles, since a large proportion of the population will have immunity either due to past infection or vaccination with the live mmr vaccine. the principal motivation is to further test and illustrate the methods in a case where good data on the transmission dynamics within households are available. stratification by household of the recent outbreaks of measles caused by decreasing uptake of the mmr vaccine could reveal whether household heterogeneities should have be accounted for in estimating the changing reproduction number of measles [36] . the household size data from 1961 is truncated to size 6, and i assume that all households of size 6 or greater have size exactly 6. the data are k 1 = 14% (i.e. 14% of households are single person households), k 2 = 30%, k 3 = 23%, k 4 = 18%, k 5 = 9% and k 6 = 7%. the size of the mean household is thus 2.99 (average size of households where households are sampled at random), while the household of the mean individual has size 3.66 (average size of household to which individuals belong, where individuals are sampled at random). hope-simpson reported susceptible-infectious escape probabilities of q = 69.9% for mumps, q = 39% for varicella, and q = 24.4% for measles in under 15s [1] . the results were reported independent of household size, and were regarded as unreliable in over-15s. based on applying the reed-frost model to the measles (t) ) is shown, as is the infectiousness of the typical infected household (denoted b * (t * )). this latter curve is obtained by simulating over 10,000 epidemics of transmission within households starting from one infected case. the two analytical approximations described in the text are also shown. ''approx 1'' is the main approximation described, while ''approx 2'' is the one obtained by assuming that all infections occur in the second generation of infection within the household. parameters are as described in the main text, and the curves are arbitrarily scaled such that each individual infects on average one person outside of the household (i.e. r g = 1). doi:10.1371/journal.pone.0000758.g002 estimate with this distribution of households, i obtain estimates of the average number of infections in each generation of infection of m 1 u 1, m 2 = 2.01 (i.e. the first index case directly infects an average of 2.01 people in his or her household), m 3 = 0.50, m 4 = 0.020, m 5 = 0.00036 and m 6 = 0.0000031, and thus the estimate for the total expected number of cases in an infected household is m = s 6 i = 1 m i = 3.54, to be compared to the mean size of 3.66. hope-simpson also reported the intervals between linked cases in households using different case definitions [1] ; the intervals for what he regarded as the most reliable case definition, ''maximum rash''. these data is well described by a gamma distribution (not shown). the maximum likelihood estimate of the generation time is t g = 10.5 days with standard deviation 2.4 days. based on these data, i repeat the simulations of the previous section on influenza but with parameters for measles in figs 2, 3 and 4. figure 2b shows that, as expected, the average infectiousness of a household is less well approximated by either approximation than for the much less infectious case of influenza. in this case, multiple peaks of infectiousness corresponding to generations of infection within the household can be clearly distinguished, and there are more cases in the second generation of infection than in the first. in terms of the predicting of the household reproduction number r * , the method is still found to be strongly predictive (as evidence by the small gap between upper and lower estimate) and reliable (compared to numerical estimates). while in influenza, the simulations were close to the upper approximation, here they are closer to the lower approximation, as expected for the more infectious situation of measles transmission. simulations of transmission within a community of households were again found in figure 4b to validate the approach. the difference in the shape of the epidemic curve with influenza reflects the different shape of the generation time distribution, though the exponential growth rate is the same. new methods were presented to estimate both the individual and household reproduction number during an epidemic. the new method presented for estimating the individual reproduction number relates closely to earlier work [12, 27, 30] , but provides an alternative and possibly simpler solution to the problem of incomplete observations during an unfolding epidemic [29] . it also provides an alternative and perhaps more satisfying solution than the incidence-to-prevalence ratio method [37, 38] to the problem of long generation time distribution infections such as hiv, where epidemiological circumstances can change substantially within the course of a single infection, and thus the case reproduction number represents too much of an average to convey secular changes in behaviour and transmission. nothing in this study challenges the central role of the individual reproduction number as an epidemiological measure; because the empirical measures of reproduction number proposed here and in [12, 27, 29] use incident observed cases as the base, all of the complication in defining the 'typical' or 'eigen' case for structured models discussed most clearly in [24] are neatly sidestepped. what this study does highlight is that much complexity is hidden in effectively defining and estimating the generation time distribution for a structured population. in the case studied here, generation times between individuals are shorter for within household transmission than between household transmission, particularly for more infectious pathogens, and this resulted in systematic biases associated with estimating the reproduction number while ignoring this effect, which were quite substantial in the case of highly infectious measles virus. the methods presented for the estimation of household reproduction numbers were not affected by this problem in the same way. analytical approximation were derived which bracketed estimates between a lower and upper bound, and numerical simulations showed the range within these brackets to be narrow. these approximations were shown to be robust, but it is worth noting that assumptions are made about the population mixing randomly out of their households and results are only valid in the scenario of an emerging pathogen where overall prevalence is low. the usefulness of these methods is likely to be found in predicting and understanding the impact of household targeted infection control measures in an emerging epidemic. this actually covers a wide class of interventions since the household is a central living and administrative unit in most populations. decisions regarding isolation, quarantine, vaccination and prophylaxis may often be made for entire households. similarly school and workplace closures as well as restrictions on leisure activity can be thought of as trying to reduce between household transmission. analytical approaches are also invaluable in calibrating and providing independent checks on more detailed individual based micro-simulations, such as [13, 17, 20] . some control interventions require more subtle analyses; for example it has been shown that vaccinating whole households is not the most effective strategy for a given vaccine coverage rate, and that alternative strategies such as preferentially vaccinating larger households could be considered [39] . further avenues of research include studying the statistical properties of these estimators for different situations. the assumption made here, that individuals mix nearly homogeneously out of their household may be an appropriate approximation for describing transmission within a neighbourhood or even a city [20] , but ultimately one should also consider developing the estimators for more complex demographic situations such as a hierarchy of organisations (household, to village, to region, to country, etc…) or a more complex overlap of households, workplaces and regular social spaces. also of interest is the study of intervention measures, particularly those that respond to the presence of a symptomatic cases; the measures of pre-symptomatic transmission presented in [25] clearly generalise to a household, but analytical results on the efficacy of isolation and quarantine are not evidently obtainable. the estimators of the household reproduction number have been shown here to be robust on their own terms, but i have not addressed the issue of model misspecification, for example to inaccurate determination of the generation time distribution or to individual heterogeneity in infectiousness or susceptibility within households. further scenarios could be explored both to test the method with different infections and to address the issue of model misspecification. there are many cases where it may be desirable to quantify household transmission, but where a degree of natural or vaccineinduced immunity may be present in the population, a problem not addressed here. in considering these more complex situations, while it may not be possible to obtain analytic forms for the infectiousness of a household, numerical forms can usually be obtained quickly and still offer benefits over full individual based micro-simulations in easily exploring a wide range of parameters. finally, the likely practical benefits of estimating household transmission parameters in an emerging epidemic need to be clearly established and communicated, and the most effective ways to enhance data collection protocols to allow their rapid estimation need to be identified. appendix s1 description of the simulations found at: doi:10.1371/journal.pone.0000758.s001 (0.08 mb pdf) infectiousness of communicable diseases in the household (measles, chickenpox and mumps) the mathematical theory of epidemics the effect of household distribution on transmission and control of highly infectious diseases a general model for stochastic sir epidemics with two levels of mixing preventing epidemics in a community of households epidemics with two levels of mixing household and community transmission parameters from final distributions of infections in households estimating household and community transmission parameters for influenza analyses of infectious disease data from household outbreaks by markov chain monte carlo methods a bayesian mcmc approach to study transmission of influenza: application to household longitudinal data transmission dynamics and control of severe acute respiratory syndrome different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures strategies for containing an emerging influenza pandemic in southeast asia containing pandemic influenza at the source transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions mitigation strategies for pandemic influenza in the united states strategies for mitigating an influenza pandemic smallpox transmission and control: spatial dynamics in great britain large-scale spatial-transmission models of infectious disease reducing the impact of the next influenza pandemic using household-based public health interventions a contribution to the mathematical theory of rates of hiv-1 transmission per coital act, by stage of hiv-1 infection population dynamics of untreated plasmodium falciparum malaria within the adult human host during the expansion phase of the infection mathematical epidemiology of infectious diseases: model building, analysis and interpretation factors that make an infectious disease outbreak controllable a note on generation times in epidemic models transmission intensity and impact of control policies on the foot and mouth epidemic in great britain new strategies for the elimination of polio from india estimating in real time the efficacy of measures to control emerging communicable diseases how generation intervals shape the relationship between growth rates and reproductive numbers superspreading and the effect of individual variation on disease emergence the effectiveness of contact tracing in emerging epidemics final size distributions for epidemics transmissibility of 1918 pandemic influenza measles outbreaks in a population with declining vaccine uptake definition and estimation of an actual reproduction number describing past infectious disease transmission: application to hiv epidemics among homosexual men in denmark is hiv out of control in the uk? an example of analysing patterns of hiv spreading using incidence-to-prevalence ratios optimal vaccination schemes for epidemics among a population of households, with application to variola minor in brazil to check the method for consistency, i simulate ten epidemics of influenza (a) and measles (b) within a fully susceptible community of 2,000 households. i use parameters estimated for an epidemic growth rate r = 0.20 per day, and condition on nonextinction of the epidemic. in c and d, the natural logarithm of the incidence is compared to the fixed slope curve r = 0.20 predicted by the model (thick line). linear regression through these data yields the estimate r = 0.19 in both cases. doi:10.1371/journal.pone.0000758.g004 key: cord-264880-0tmd9knh authors: li, zhao; liu, yong; wei, qingquan; liu, yuanjie; liu, wenwen; zhang, xuelian; yu, yude title: picoliter well array chip-based digital recombinase polymerase amplification for absolute quantification of nucleic acids date: 2016-04-13 journal: plos one doi: 10.1371/journal.pone.0153359 sha: doc_id: 264880 cord_uid: 0tmd9knh absolute, precise quantification methods expand the scope of nucleic acids research and have many practical applications. digital polymerase chain reaction (dpcr) is a powerful method for nucleic acid detection and absolute quantification. however, it requires thermal cycling and accurate temperature control, which are difficult in resource-limited conditions. accordingly, isothermal methods, such as recombinase polymerase amplification (rpa), are more attractive. we developed a picoliter well array (pwa) chip with 27,000 consistently sized picoliter reactions (314 pl) for isothermal dna quantification using digital rpa (drpa) at 39°c. sample loading using a scraping liquid blade was simple, fast, and required small reagent volumes (i.e., <20 μl). passivating the chip surface using a methoxy-peg-silane agent effectively eliminated cross-contamination during drpa. our creative optical design enabled wide-field fluorescence imaging in situ and both end-point and real-time analyses of picoliter wells in a 6-cm(2) area. it was not necessary to use scan shooting and stitch serial small images together. using this method, we quantified serial dilutions of a listeria monocytogenes gdna stock solution from 9 × 10(-1) to 4 × 10(-3) copies per well with an average error of less than 11% (n = 15). overall drpa-on-chip processing required less than 30 min, which was a 4-fold decrease compared to dpcr, requiring approximately 2 h. drpa on the pwa chip provides a simple and highly sensitive method to quantify nucleic acids without thermal cycling or precise micropump/microvalve control. it has applications in fast field analysis and critical clinical diagnostics under resource-limited settings. digital polymerase chain reaction (dpcr) is a powerful method for nucleic acid detection and absolute quantification, in which the diluted sample and reaction components are partitioned into hundreds, or even millions, of individual, parallel reaction chambers so that each contains one or no copy of the templates [1] [2] [3] [4] . after endpoint amplification, the template concentration in the original sample is determined by a poisson statistical analysis of the number of "positive" partitions (in which the amplified target is detected) versus "negative" partitions (in which it is not). dpcr offers many advantages over quantitative pcr (qpcr) [5] , such as absolute quantification without dependence on cycle thresholds or external references, and much higher accuracy and sensitivity [6] [7] [8] [9] . applications of dpcr span many areas of biology, including liquid biopsy [10] [11] [12] , copy number variation analysis [13, 14] , rare sequence detection [15, 16] , gene expression analysis [5, 17, 18] , single-cell genomics analysis [19] [20] [21] , pathogen detection and microbiome analysis [2, 22, 23] , as well as calibration for next-generation sequencing [24] . dpcr has been successfully performed in a variety of formats, such as multiwell plates [1, 3] , an emulsion pcr [25] [26] [27] , microdroplets [19, [28] [29] [30] [31] , microfluidic chambers [32] , a spinning disk platform [33] , and a slipchip [34, 35] , and these can be summarized as chip-based or droplet-based dpcr [4] . however, most of the reported microfluidic chips still require a complex fabrication process, many tubes for liquid transportation, syringe pumps for pressuredriven flow, and a pneumatic system for microvalve control [36] . current droplet-based approaches also require pumping equipment and an external rapid readout device. to ensure a homogeneous droplet size distribution, the flow rate of droplet production by t-junctions [37, 38] or flow focusing [39] [40] [41] must be precisely controlled. moreover, all of these dpcr methods still require thermal cycling and accurate temperature control. to avoid thermal cycling, different isothermal amplification methods have been developed that rapidly amplify nucleic acids to detectable levels at a single temperature [42, 43] , such as loop-mediated amplification (lamp) [44] , rolling circle amplification (rca) [45] , helicasedependent amplification (hda) [46] , nucleic acid sequence-based amplification (nasba) [47] , recombinase polymerase amplification (rpa) [48] , transcription-mediated amplification (tma) [49] , multiple displacement amplification (mda) [50] , and strand-displacement amplification (sda) [51] . in contrast to other isothermal amplification techniques, rpa offers several important advantages for point-of-care applications: it requires a lower amplification temperature of between 25°c and 42°c, is tolerant to impure samples, amplifies targets to detectable levels more rapidly (15-30 min) , and uses lyophilized enzymes without cold chain storage and transport [48] . recently, a number of reports have proposed rpa-based strategies for pathogen detection. the detection formats include fluorescence detection in real time or endpoint detection via a lateral flow strip [52] [53] [54] [55] . reverse transcription rpa can be also used to detect rna targets from foot-and-mouth disease virus or middle east respiratory syndrome coronavirus [56, 57] . additionally, rpa is highly sensitive for the detection of hiv proviral dna and mycobacterium tuberculosis dna [52, 55, 58] . instrument-free and electricity-free rpa can be performed using body heat or heating achieved using sodium acetate trihydrate for nucleic acid diagnostic tests in low-resource settings that lack expensive thermal cycling pcr equipment or even electric power [59, 60] . in contrast to the common dpcr, the developments of digital isothermal amplification techniques are still insufficient. to the best of our knowledge, only lamp, mda, sda, rca, and rpa have their corresponding digital amplification techniques, which are almost performed in droplets or microfluidic chips [61] [62] [63] [64] [65] . digital rpa (drpa) has been implemented only on the slipchip and in droplets produced by centrifugal step emulsification [66, 67] . the method requires extensive development to enable applications to other simple platforms, without any complicated and precise control. in this work, we developed a picoliter well array (pwa) chip to perform real-time drpa for absolute quantification of nucleic acids. the pwa chip was manufactured from a silicon substrate and etched with 27,000 consistently sized picoliter wells. we demonstrated that sample loading using a scraping liquid blade is suitable for the pwa chip, which was simpler, faster, and resulted in less reaction dropout than droplet-based approaches. additionally, unlike other microfluidic chips, it did not require a complex fabrication process or a pneumatic control system. passivating the pwa chip surface using a methoxy-peg-silane agent eliminated cross-contamination. for wide-field fluorescence imaging in situ, we creatively placed an optical cube between the chip and object lens to reduce the loss of light intensity in a conventional microscope. not only did this ensure that the light intensity was able to excite the fluorescence signal, but it also avoided the need to obtain images by scan shooting and stitching serial small images together. finally, we sealed the pwa chip in a homemade copper chamber filled with oil and successfully performed real-time drpa on an isothermal incubation setup for the absolute quantification of serial dilutions of a listeria monocytogenes gdna stock solution. the substrate for the pwa chip was an n-type (100) single crystalline silicon wafer, with a thickness of 500 μm and a diameter of 100 mm. the silicon wafer was chemically cleaned by sequential immersion in acetone, ethanol, and deionized (di) water for 15 min, with sonication. then, it was immersed in a piranha solution (h 2 so 4 :h 2 o 2 = 3:1) at 180°c for 30 min, rinsed thoroughly with di water, and dried with nitrogen. positive photoresist az 6130 (thickness, 5 μm) was spin-coated on the silicon wafer for subsequent lithography (ma6; suss microtec, garching, germany) with a photolithography mask. under the protection of the photoresist, a picoliter well array was formed using deep reactive-ion etching (plasmalab system 100; oxford instruments, concord, ma, usa) and the remaining photoresist was removed with acetone. after cleaning, a 200-nm-thick silicon dioxide (sio 2 ) layer was added to the wafer in a thermal oxidation furnace at 1050°c. to covalently couple methoxy-peg-silane (2-[methoxy(polyethyleneoxy)propyl]-trimethoxysilane; j&k scientific, beijing, china) to the pwa chip surface, the bare chips were treated in an oxygen plasma for 30 s to clean the surfaces (mercator control systems, inc., santa ana, ca, usa). then, they were placed in 1 g of methoxy-peg-silane dissolved in 100 ml of anhydrous toluene with 1% triethylamine as a catalyst overnight at room temperature [68, 69] . before the application of the pwa chip, the physically adsorbed methoxy-peg-silane moieties were removed by rinsing the chip in ethanol and di water, sequentially. finally, the pwa chip was dried with nitrogen and used immediately. the l. monocytogenes gdna stock solution was prepared by rehydrating the dried dna powder with dnase/rnase-free water. the final concentration was 40 pg μl −1 , which was verified using a nanodrop spectrophotometer (thermo fisher scientific). one picogram of l. monocytogenes gdna contained approximately 315 copies. the volume of reaction solution in each microwell was 314 pl, and for a gdna sample concentration of 40 pg μl -1 , approximately 9 × 10 -1 copies per well was expected. the detailed description of the calculating method is shown in s1 fig. the sample loading instrument included a scraping liquid blade and a chip carrier. detailed operation steps are shown in fig 1. the scraping liquid blade was composed of a hard glass slide and a piece of soft silica gel (thickness, 3 mm), which were pasted together at an end; the chip carrier was composed of another glass slide and a 3m adhesive tape, which prevented the chip from sliding in the process of scraping (fig 1a) . the prepared 20-μl digital rpa reagents were carefully transferred onto one end of the pwa chip by a pipette. depressing the pipette to the second stop was unsuitable for reducing the introduction of air bubbles to the chip. the blade was held at a 40-60°angle relative to the chip carrier so that the edge of silica gel was placed at the end of pwa chip. the angle of the blade was adjusted slightly until visual confirmation that rpa reagents were wetting the chip. then, in one smooth motion, the blade was dragged slowly across the chip, while a slight downward pressure was applied to dispense the reagent ( and 1b). the scraping time was approximately 5 s. the pwa chip sat at room temperature for 20 s for residual liquid evaporation. the chip was then gently overlaid with excess mineral oil (m8410; sigma-aldrich, st. louis, mo, usa) via a disposable pipette until the entire surface was fully covered (fig 1b) . mineral oil was used because it is lighter than water (0.82-0.88 g ml −1 ) and its solubility in water is extremely low [9] . it makes the rpa reaction independent of each other. therefore, the subsequent packaging operation can not affect the accuracy of drpa results, even at room temperature. the copper chamber was filled with mineral oil prior to transferring and packaging the finished pwa chip after sample loading (fig 1c and 1d) . finally, a piece of 2.5-mm-thick quartz glass cover-plate was fixed on the copper chamber by four screws (fig 1e and 1f) . the whole process avoided air bubbles. to strengthen the air tightness between the glass and the copper chamber, a rubber o-ring was added around the chamber. the bright images of the sample loading and chip packaging process can be found in s2 fig. the isothermal incubation setup for drpa-on-chip was achieved using a 40 mm × 40 mm thermoelectric cooler (tec), which was controlled by a pid temperature controller ai-518 (yudian automation technology, xiamen, china) with labview software (national instruments, austin, tx, usa) (fig 2a) . temperature feedback was accomplished by inserting a 1-mm-thick pt100 thermistor between the chip packaging device and the tec unit. a 12 v × 3 a fan and a custom-fabricated aluminium block were placed beneath the tec unit to dissipate waste heat. after sample loading and chip sealing, the picoliter droplets were heated to 39°c for 20 min. the ability to perform on-chip isothermal heating was necessary for realtime observations of the entire pwa chip during rpa amplification. a wide-field, high-resolution imaging setup was required to adequately resolve all 27,000 picoliter wells. as illustrated in fig 2a, large field of view images were captured using an olympus microscope szx7 (olympus, tokyo, japan) and a monochrome cooled camera mc21 (microshot technology, guangzhou, china) equipped with a 1.4 million pixel high-resolution ccd sensor (sony, tokyo, japan). a resolution of 16 pixels per microwell can be achieved for the whole pwa chip. these were not sufficient to generate greater than 20 mw cm -2 light intensity on the entire chip area, which is necessary to excite the fluorescence signal for detection by the ccd camera. to address this problem, an optical cube was placed between the chip and object lens to shorten the path of excitation light to reach the fluorescent material. this improvement could reduce the loss of light intensity using a conventional microscope. the cube filters (chroma technology corp., bellows fall, vt, usa) were composed of a 505-nm dichroscope, a filter set of 480/30 nm for fluorescence excitation and 535/40 nm for emission collection of the fam dye, and were positioned 10 mm above the chip packaging device. the light source, a high-power 3 w blue led bead (micro-shot technology, guangzhou, china), was coupled to the cube in a horizontal direction. imagej (nih, bethesda, md, usa) and custom matlab code were used to systematically detect and quantify fluorescent "positive" microwells and to analyze their density and averaged fluorescence intensity [35] . background subtractions and contrast enhancement were performed for quantification of the drpa results. the image acquired at the beginning (0 min) was regarded as the background noise of the experiment; other images acquired subsequently subtract it in situ for contrast enhancement. to enhance the visual contrast, grayscale images were converted to green. the quantitative results were then compared to the expected number of positive microwells predicted from poisson statistics for serial dilutions of the known sample concentration. silanization of the pwa chip to avoid cross-contamination the manufactured finished pwa chip was 20.8 mm × 16 mm, with 27,000 closely packed microwells. the height and diameter of the completed wells were 40 μm and 100 μm, respectively, with edge-to-edge spacing of 20 μm and volumes of 314 pl. a scanning electron microscope (sem) image of the silicon picoliter well array is shown in fig 2b. native silicon is an inhibitor of pcr [70] ; accordingly, the chip surface was coated with a compact 200-nm-thick silicon dioxide (sio 2 ) layer by thermal oxidation. however, the chip surface still absorbed polymerase protein and other reaction reagents, primarily owing to the great increase in the surface-to-volume ratio in the micro-scale environment, thus decreasing the dna amplification efficiency. the conventional strategy involves treating the reaction chambers with 0.2% bovine serum albumin (bsa) solution at 80°c for 30 min before loading the pcr reaction mix [71] or adding 1 μl of 50 mg ml −1 bsa solution to 20 μl of pcr master mixture as a blocking protein to occupy the adsorption sites and reduce the loss of polymerase [9] . the "bsa strategy" can similarly ensure the success of rpa reactions in the picoliter wells, but it cannot eliminate cross-contamination that may occur between adjacent microwells owing to the edge-to-edge spacing of only 20 μm. there may be a thin liquid film on the chip surface after scraping pra reagents into the picoliter wells. although it will evaporate quickly, i.e., in 10-30 s, before oil sealing, the precise evaporation time is unknown because it is related to environmental humidity and temperature, which vary among experiments. the dna amplification products enter adjacent microwells by residual liquid passage; thus, many clusters of positive rpa wells appear gradually from 5 min to 10 min with cross-contamination on the non-silanization chip (fig 3a) . passivating the pwa chip surface by a silanizing agent may effectively eliminate cross-contamination during drpa. methoxy-peg-silane could produce a compact monomolecular layer on the sio 2 surface by covalent binding (fig 2c) , which can protect pcr enzymes from being adsorbed and does not interfere with pcr reactions [72] . additionally, it is highly compatible with mineral oil for sealing and fixes water molecules by hydrogen bond interactions. accordingly, even when minor residual liquid remains after evaporation for 20 s, it is unable to flow on the methoxy-peg-silane surface and connect adjacent microwells. there is no cross-contamination on the silanization chip; positive wells with a single dna template amplify independently and the fluorescence intensity for adjacent negative wells does not change (fig 3b) . moreover, the silanization of the pwa chip is very easy to perform, unlike the complicated modifications required for openarray, with a hydrophilic interior surface and hydrophobic exterior surface [72] . we developed a simple and effective scraping liquid blade to partition a sample into as many as 27,000 independent reaction microwells. we scraped liquid with a certain strength to make the soft silica gel contact closely with the chip surface. thus, after dispensing the reagent effectively, there was lesser residual liquid on the surface for reducing the opportunity of crosscontamination [73] . to produce a large number of monodispersed droplets, the scraping time (~5 s) is much shorter than that of other microdroplet techniques, in which droplets are commonly produced sequentially by t-junctions or flow [37] [38] [39] [40] [41] . current digital droplet systems take considerable time for droplet production, which leads to amplification in the bulk solution. thus, they cannot be used to perform drpa. in contrast, the platform presented here can be applied to many dna amplification techniques, such as rpa, pcr, rca, and lamp. this sample loading style does not require pumping equipment or precise microvalve control [36] . in the scraping process, a relatively slow operation is suitable to fill microwells with the liquid reagent; otherwise, the wells may contain air bubbles in the bottom. once heated, the bubbles rush out, diffuse, and cause reaction failure on the pwa chip. in contrast to previously performed drpa reactions on the slipchip, the pwa chip works without precise "slip" control [66] . if the slipchip reduces the microwell volume and increases the density rapidly for a high-throughput detection like the pwa chip, the complexity and precision of the "slip" operation would increase rapidly, too. in contrast to previously performed drpa reactions in droplets produced by centrifugal step emulsification, the droplets of the pwa chip are more consistently sized, avoiding the uneven droplets volumes affecting the quantification results, and their volumes are smaller, i.e. picoliter-sized [67] . reagent consumption for each experiment on the pwa chip was less than 20 μl. a small reaction volume not only contributes to powerful and high-throughput detection, but also generates much less reagent waste and reduces the cost of each reaction. chip packaging to seal the pwa is another key technology. the picoliter droplets evaporate easily in the incubation, even when sealed with mineral oil. when the reagent concentration increases to a certain degree owing to water evaporation, the rpa reaction is not carried out in the picoliter wells, and the more demanding drpa cannot be performed. thus, we designed a chip chamber to seal the finished pwa chip after sample loading. the glass cover-plate serves to create a vapor barrier to reduce oil and rpa reagents from evaporating during incubation [9] ; and the rubber o-ring strengthens the air tightness between the glass and the copper chamber when the screws are tightened at the four corners. conventional dpcr can be also performed successfully on the pwa chip with the packaging device, for a thermocycling temperature and reaction time of up to 95°c and 2 h, respectively (unpublished results). as an isothermal amplification technique, rpa cannot be simply triggered using a "hot-start," which is established in conventional pcr. instead, the rpa reaction mixture (i.e., the oligo mix, buffer, template, and enzymes) is prepared without the addition of mg 2+ to prevent the reaction from starting. after mg 2+ is added, the reaction proceeds, sometimes at room temperature. to disable amplification between the addition of the mgac solution to the rpa reaction mixture and sample loading, reagents were pre-cooled to 0°c during preparation. after the mgac solution was added to the rpa reaction mixture, the sample loading operation was completed in less than 15 s, allowing very little time for the solution to heat or for amplification to begin. additionally, no thermal cycling is necessary for rpa, thus enabling very easy and cheap digital amplification. we modified a commercial fluorescence microscope to minimize the distance between the optical cube and the pwa chip, which ensures sufficient light intensity to fully excite the fluorescence signal to obtain wide-field, high-resolution images. this method affords several significant advantages over previous approaches: (a) the macro-fluorescence imaging setup is capable of viewing 6-cm 2 areas in a single snapshot at 0.8× magnification, without the need to obtain images by scan shooting and stitching serial small images together [34] . thus, image acquisition and processing are rapid. (b) this approach allows the excitation light to vertically illuminate the chip through the optical cube and avoids flatfield corrections due to oblique beam illumination [74] . an uneven light field affects the quantification results. (c) the widefield images are captured in situ, enabling real-time fluorescence detection. real-time fluorescence imaging of rpa amplification is demonstrated in fig 4 for a 20 -min period at 5-min increments (these images are enlarged sections from the wide-field fluorescence images for enhanced visualization). instead of monitoring only the end-point fluorescent intensity, which is done in many single dna molecular application experiments, our real-time fluorescence detector is useful for investigating amplification uniformity and for optimizing the total time required for incubation [74] . as shown in fig 4, the rpa amplification is rapid, such that the number of positive points no longer increases after 15 min. overall drpa-on-chip processing requires less than 30 min, which is a 4-fold decrease compared to dpcr, with a processing time of approximately 2 h. the reduced time to results is especially important for fast field analysis and critical clinical applications, such as in cases of sepsis. furthermore, real-time analysis improves detection sensitivity because it allows the use of a reference frame to subtract background noise, which cannot be achieved with a single end-point frame. temporal information also helps to differentiate between amplified signals and noise because the rate at which the intensity changes can be expected to increase gradually from frame to frame, providing another parameter by which to detect positive droplets. a plot of fluorescence intensity vs. reaction time is shown in s3 fig. we characterized the performance of the pwa chip for digital rpa using serial dilutions of a l. monocytogenes gdna stock solution (fig 5) . the expected concentration of the gdna template was estimated as the number of copies per well (cpw), and the concentration of the original gdna stock solution was verified spectrophotometrically. the detailed method for calculating the expected cpw is presented in the preparation of drpa reagents section. as the gdna template was diluted, the expected cpw values were 9 × 10 -1 , 1.8 × 10 -1 , 3.6 × 10 -2 , 1.2 × 10 -2 , and 4 × 10 -3 . after incubation, the number of positive rpa wells on the pwa chip decreased proportionally (fig 5a-5e) . no evidence of contamination was observed as no false positives were observed in the negative control (fig 5f) . we repeated the experiments three times at each gdna concentration to test the robustness and reproducibility of digital rpa on the pwa chip. a poisson statistical analysis of the drpa results was performed as previously described [35, 66] . as shown in fig 6, the measured cpw with drpa was highly concordant with the expected cpw, with an average error rate of less than 11% (n = 15). accurate qpcr experiments can estimate original dna concentrations with variation of less than 1 cycle number, resulting in 150-200% errors in original concentration estimates [75] ; accordingly, this drpa method had better performance. furthermore, it is noteworthy that potential pipetting errors during dilution and potential losses of dna during sample preparation could increase the deviation between two estimates. we developed a pwa chip with 27,000 consistently sized wells (314 pl) to perform isothermal dna quantification using drpa at 39°c for 20 min on a homemade isothermal heater. sample loading using a scraping liquid blade was simple, fast, and consumed minimal reagents (< 20 μl). passivating the pwa chip surface by a methoxy-peg-silane agent eliminated crosscontamination among wells. our optical design enabled wide-field fluorescence imaging in situ, with both end-point and real-time analysis of picoliter wells in 6-cm 2 areas during drpa. we applied the pwa chip to accurately quantify serial dilutions of a l. monocytogenes gdna stock solution. the drpa reaction was robust and free of cross-contamination on the pwa chip, but the specificity of the device and strategies for multiplex detection remain to be examined. the digital pwa chip can be readily applied to other nucleic acid amplification techniques, such as pcr, rca, elisa, and lamp. fig. (a) bright-field image of the sample loading instrument containing a chip carrier and a scraping liquid blade, displayed on a ruler to show scale. the scraping liquid blade is composed of a glass slide and a piece of silica gel (thickness, 3 mm), which are pasted together at an end; the chip carrier is composed of another glass slide and a 3m adhesive tape, which prevents the chip from sliding in the process of scraping. (b) after sample loading by scraping the rpa reagents into the picoliter wells array, let the pwa chip sit quietly in room temperature for 20 seconds for the little residual liquid evaporating. then sealing the chip with excess mineral oil via a disposable pipette until the entire surface was fully covered. (c-e) transferring the sample loading finished pwa chip from the chip carrier to the copper chamber filled with mineral oil. (f) fixing the glass cover-plate on the copper chamber with screws. the rubber o-ring is added around the chamber to strengthen the air tightness. the whole process avoids air bubbles. table. the sequences of the primers and probe for drpa-on-chip (all in 5 0 ! 3 0 direction). 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pluripotent stem cells droplet digital pcr measurement of her2 copy number alteration in formalin-fixed paraffin-embedded breast carcinoma tissue the applicability of digital pcr for the assessment of detection limits in gmo analysis digital pcr analysis of maternal plasma for noninvasive detection of sickle cell anemia circulating micrornas as stable blood-based markers for cancer detection multiplexed target detection using dna-binding dye chemistry in droplet digital pcr transcription factor profiling in individual hematopoietic progenitors by digital rt-pcr single-cell genomics microfluidic single-cell real-time pcr for comparative analysis of gene expression patterns highly precise measurement of hiv dna by droplet digital pcr probing individual environmental bacteria for viruses by using microfluidic digital pcr digital pcr provides sensitive and absolute calibration for high throughput sequencing beaming: single-molecule pcr on microparticles in water-in-oil emulsions beaming up for detection and quantification of rare sequence variants transforming single dna molecules into fluorescent magnetic particles for detection and enumeration of genetic variations microdroplet-based pcr enrichment for large-scale targeted sequencing on-chip, real-time, single-copy polymerase chain reaction in picoliter droplets high-throughput single copy dna amplification and cell analysis in engineered nanoliter droplets droplet-based microfluidic systems for high-throughput single dna molecule isothermal amplification and analysis digital pcr on an integrated self-priming compartmentalization chip spinning disk platform for microfluidic digital polymerase chain reaction nanoliter multiplex pcr arrays on a slipchip megapixel digital pcr correlations of droplet formation in t-junction microfluidic devices: from squeezing to dripping dynamics of droplet formation at t-shaped nozzles with elastic feed lines microfluidic flow focusing: drop size and scaling in pressure versus flow-rate-driven pumping emulsification in a microfluidic flowfocusing device: effect of the viscosities of the liquids droplet formation and breakup dynamics in microfluidic flow-focusing devices: from dripping to jetting isothermal amplified detection of dna and rna isothermal amplification methods for the detection of nucleic acids in microfluidic devices loop-mediated isothermal amplification of dna mutation detection and singlemolecule counting using isothermal rolling-circle amplification helicase-dependent isothermal dna amplification nucleic acid sequence-based amplification dna detection using recombination proteins direct detection of microorganisms in clinical specimens using the gen-probe transcription mediated amplification system comprehensive human genome amplification using multiple displacement amplification strand displacement amplification-an isothermal, in vitro dna amplification technique quantification of hiv-1 dna using real-time recombinase polymerase amplification real-time microfluidic recombinase polymerase amplification for the toxin b gene of clostridium difficile on a slipchip platform isothermal recombinase polymerase amplification (rpa) of schistosoma haematobium dna and oligochromatographic lateral flow detection a paper and plastic device for performing recombinase polymerase amplification of hiv dna a portable reverse transcription recombinase polymerase amplification assay for rapid detection of foot-and-mouth disease virus reverse transcription recombinase polymerase amplification assay for the detection of middle east respiratory syndrome coronavirus rapid detection of mycobacterium tuberculosis by recombinase polymerase amplification equipment-free incubation of recombinase polymerase amplification reactions using body heat non-instrumented incubation of a recombinase polymerase amplification assay for the rapid and sensitive detection of proviral hiv-1 dna self-priming compartmentalization digital lamp for point-of-care microfluidic continuous flow digital loop-mediated isothermal amplification (lamp). lab chip digital mda for enumeration of total nucleic acid contamination isothermal circular-strand-displacement polymerization of dna and microrna in digital microfluidic devices digital quantification using amplified single-molecule detection digital isothermal quantification of nucleic acids via simultaneous chemical initiation of recombinase polymerase amplification reactions on slipchip centrifugal step emulsification applied for absolute quantification of nucleic acids by digital droplet rpa patterning functional proteins with high selectivity for biosensor applications spatially selective surface platforms for binding fibrinogen prepared by particle lithography with organosilanes. interface focus surface passivation of microfabricated silicon-glass chips for pcr a novel miniaturized pcr multi-reactor array fabricated using flip-chip bonding techniques nanoliter high throughput quantitative pcr cell confinement in patterned nanoliter droplets in a microwell array by wiping 1-million droplet array with widefield fluorescence imaging for digital pcr novel real-time quantitative pcr test for trisomy 21 this work was supported by grants from the instrument developing project of the chinese academy of sciences (grant no. yz201301) and the state key program of national natural science of china (grant no. 61334008). we are grateful to fengying miao and yuchen feng for their technical assistance in the development of pwa chip-based drpa. we furthermore want to thank liwen luo from twistdx china for helpful discussion and providing the sequences of the rpa oligos. key: cord-276763-x3dqi0ym authors: lowery-north, douglas w.; hertzberg, vicki stover; elon, lisa; cotsonis, george; hilton, sarah a.; vaughns, christopher f.; hill, eric; shrestha, alok; jo, alexandria; adams, nathan title: measuring social contacts in the emergency department date: 2013-08-21 journal: plos one doi: 10.1371/journal.pone.0070854 sha: doc_id: 276763 cord_uid: x3dqi0ym background: infectious individuals in an emergency department (ed) bring substantial risks of cross infection. data about the complex social and spatial structure of interpersonal contacts in the ed will aid construction of biologically plausible transmission risk models that can guide cross infection control. methods and findings: we sought to determine the number and duration of contacts among patients and staff in a large, busy ed. this prospective study was conducted between 1 july 2009 and 30 june 2010. two 12-hour shifts per week were randomly selected for study. the study was conducted in the ed of an urban hospital. there were 81 shifts in the planned random sample of 104 (78%) with usable contact data, during which there were 9183 patient encounters. of these, 6062 (66%) were approached to participate, of which 4732 (78%) agreed. over the course of the year, 88 staff members participated (84%). a radiofrequency identification (rfid) system was installed and the ed divided into 89 distinct zones structured so copresence of two individuals in any zone implied a very high probability of contact <1 meter apart in space. during study observation periods, patients and staff were given rfid tags to wear. contact events were recorded. these were further broken down with respect to the nature of the contacts, i.e., patient with patient, patient with staff, and staff with staff. 293,171 contact events were recorded, with a median of 22 contact events and 9 contacts with distinct individuals per participant per shift. staff-staff interactions were more numerous and longer than patient-patient or patient-staff interactions. conclusions: we used rfid to quantify contacts between patients and staff in a busy ed. these results are useful for studies of the spread of infections. by understanding contact patterns most important in potential transmission, more effective prevention strategies may be implemented. presentation of an infectious patient to an emergency department (ed) brings a substantial risk of cross infection. ed cross infection risk was demonstrated dramatically during the 2003 severe acute respiratory syndrome coronavirus (sars co-v) epidemic. the son of the first index case arriving in toronto fell ill after caring for his mother [1] . he visited a crowded ed and waited hours for a hospital bed assignment. subsequently, 126 nosocomial sars infections among patients and staff were traced to direct or indirect exposure to this patient; several of these victims died. since this incident, ed crowding has worsened, [2] increasing commingling of acutely infected patients with other susceptible and high-risk patients, thereby increasing the risk of ed cross infection. this sars outbreak is not an isolated incident. ed visits have previously been shown to be a significant risk factor for subsequent infection in the pediatric population, [3, 4] as well as in the elderly [5] . cross infection of patients in the ed is also an important concern to patients and staff in other hospital areas, since more than 40% of hospitalized patients originate from the ed [6] . annually, a global epidemic of influenza results in significant morbidity and mortality [7, 8] . although evidence suggests influenza may be transmitted via airborne and contact routes, [9] most authorities agree influenza is transmitted primarily in droplets passing between people, as in the sars co-v outbreak [10] . droplet-mediated cross infection typically occurs within a one (1) meter (m) radius between source and exposed, as the droplets are of adequate size that gravity pulls them to the ground before they can travel laterally [9] . until recently, many mathematical models of the cross infection process had assumed that individuals are mixing and coming into contact with each other randomly [11] . recent research has begun to show that humans do not come into contact with each other according to a uniformly random process; human interaction is highly influenced by other external factors [12] [13] [14] [15] . knowledge of the social and spatial structure of interpersonal contacts in the ed will provide information useful for building biologically plausible mathematical models of cross infection risk, [16] which can guide development of cross infection control measures. advances in technology have made the automated tracking of individuals possible and increasingly affordable using a variety of types of real time location sensing systems, such as radiofrequency identification (rfid) and motes. while the manufacturing and retail sectors of our economy have been making use of such technology to track goods for years, declining costs have enabled researchers to deploy such systems to investigate human motion in a variety of settings. although investigations of human movements and resulting contacts have been conducted in a variety of settings, such as schools [17, 18] and academic conferences, [14, 19] there has been relatively limited deployment for research purposes in the health care setting. notably there have been four such studies in settings such as a pediatric emergency department, [20] two hospital wards with airborne precautions, [21] a general pediatric ward, [22] and a medical intensive care unit (micu) [23] . we report here the results of a year-long deployment of a rfid system covering all areas of an adult ed, describing the contacts between and among patients and staff. the goal of this study was to determine contact characteristics among patients and staff in the ed of a busy urban hospital. the number and duration of contacts between individuals is described overall and by patient-patient (pp), patient-staff (ps), and staff-staff (ss) type contacts. ethics statement: the emory university institutional review board (irb) granted waiver of all elements of informed consent and waiver of hipaa authorization. this is a prospective study. the study was conducted in the ed of emory university hospital midtown in the midtown area of atlanta, ga. the ed occupies 25,000 square feet, and includes triage, fast track, acute care, and observation functions. it has an annual census of 57,000 visits. contact was defined as any two individuals located within 1 m of each other in contiguous two-dimensional space, measured by radiofrequency identification (rfid). an active rfid proximity detection system was installed in 2008 and activated in early 2009 (radianse corp., amherst, ma). the ed was divided into 89 twodimensional zones, and these zones were configured around hard and soft architectural features such that when two individuals were in the same zone simultaneously, they were within 1 m of one another with a very high probability. the floor plan of the ed as divided into zones is given in figure 1 . all areas of the ed were covered by the system. rfid tags transmitted their unique identifier every ten seconds. the system was dispersed such that rfid tag signals would be detected by at least three receivers. receivers relayed information back to a server, where a proprietary algorithm determined tag location and assigned a corresponding zone identifier. to facilitate line-of-sight determination in the presence of many walls (i.e., two subjects on either side of a wall but otherwise within 1 m would not be considered as in contact), the ed was divided into zones. data were retrieved using mysql database (oracle corp., redwood shores, ca) queries and stored in microsoft excel (microsoft corp., redmond, wa). a representative sample of 104 12-hours shifts over one year were randomly selected for direct observation. two 12-hour periods (one 7am-7pm (day shift) and one 7pm-7am (night shift)) were randomly selected for study from each week between july 1 2009 and june 30 2010. this selection was made in order to get a sense of seasonal variability and to ensure that day and night shifts were equally represented throughout the year. week was chosen as a blocking factor for the design since there is a distinct rhythm to patient-and work-flow in the ed within a week. the study budget was designed to support the study of no more than two shifts per week. staff. rfid tags were issued to assenting ed staff members to wear. patients. immediately prior to each study period, the research team placed rfid tags on assenting patients already enrolled in the process of care in the ed. research team members placed tags on newly arriving assenting patients during the study period. psychiatric patients, patients in police custody, and patients who were expected to be discharged within a short time were not approached for rfid tagging, due to either irb concerns (first two groups) or study staff limitations (last group). patients in this last group were generally waiting in an exam room and had very few contacts. ed staff removed patient tags at the earliest of time of patient discharge (for those discharged), time of hospital admission (for those admitted), or time of study period conclusion. data were collected from three different sources. the electronic health record (ehr) (cerner millennium electronic health record, cerner corp., kansas city, mo) provided standard clinical and demographic characteristics for patients. the tag identification database tracked tag information. the radianse database provided time-stamped information about tag zones. race was abstracted from the ehr as entered by hospital registration staff, who as part of routine operations ask patients to identify their race and ethnicity. this variable is reported since social mixing may vary culturally and thus it may impact the ultimate generalizability of our study. contacts were described in 2 ways. a unique pair of individuals could make contact multiple times over the course of a shift. ''contact pairs'' defined multiple contacts as one pair and the duration of these contacts were cumulated. in contrast, ''discrete contact events'' treated multiple discontinuous instances as multiple contacts of one contact pair. figure 2 gives a schematic representation of these definitions. for each second of each shift we created adjacency matrices in which every participant in that shift was cross-listed against every other participant. by aligning these matrices along the dimension of time, we created a 3-dimensional time-resolved adjacency object (trao) for each shift in the study. we used data from the radianse system to determine if any two participants were in contact at a given instance, assigning 19s in the appropriate cells of the relevant adjacency matrix for that instance. otherwise 09s were assigned if participants were not in contact at that time or if neither participant was present in the ed at that time. by summing in the time dimension over these objects we could determine the number of contact pairs formed among participants and the total duration of contact for each pair. we then examined the contact sequence for each contact pair in the time dimension as represented by a sequence of 19s and 09s. each distinct sequence of 19s that was interrupted by 09s was called a contact event. this is also represented in figure 2 . selection bias. to reduce selection bias, study periods were randomly selected. we aimed to study every assenting patient and ed-based staff member present in the ed during these periods. measurement bias. to reduce measurement bias, ed staff were advised to utilize standard operating procedures when performing data entry into the clinical information system. no subjects were asked to record study-specific information. the research team alone performed entry of linkage data for patients and rfid tag data. classification bias. to reduce classification bias, the rfid system determined subject locations independently of the clinical information systems. off-the-shelf software was utilized to determine contacts. the system was calibrated for accuracy and reliability prior to the start of the study. initial calibration of the system verified that a radio signal emitted anywhere within the ed footprint would be captured by at least three and typically four receivers. further verification was completed prior to study initiation to demonstrate that a radio signal received from any location would be accurately mapped to the appropriate zone. no ongoing systematic analysis was performed after the initial setup since hardware and software location and configuration were static. however, the system was utilized during routine, nonresearch operations, 24/7 during the entire study year, to locate both human and equipment resources in real time. no disparities were noted nor reported by ed staff during this extensive observation. furthermore, during the twelve-hour study periods, research assistants interacted with the system in real time to locate patients, staff, and equipment. again, no location disparities were noted. funding constraints limited our ability to record each instance research assistants interrogated the system for a specific badge location and then found the badge at that location, although research staff were trained to record any operational aberrations. this descriptive study was designed so sufficient data would be available to adequately characterize temporal aspects of contact variability and simulate epidemic spread at seasonally appropriate times of year. simple descriptive statistics were utilized to estimate number, duration, and nature of contacts (pp, ps, and ss) during the study period, using sas v9.3 for windows 7 enterprise (sas institute, cary nc). in particular, data were summarized over a shift, and these summary values were further aggregated. data were highly skewed, thus we present summary percentile values. friedman's test [24] along with tukey's posthoc procedure [25] was used to test if the contact characteristics were the same for pp, ps, and ss using the medians from each shift. from 730 potential twelve-hour sampling periods, we randomly selected 104 for observation. usable data were obtained from 81 shifts (78%). the remaining shifts in the sample were excluded due to equipment problems (n = 9; 8%) and insufficient study staffing (due to illness, inclement weather, etc., n = 14; 13%). over the course of the study year 57,514 distinct patient admissions occurred (table 1) . of these, 9183 (16%) occurred in the 81 shifts studied. among these admissions, the research team did not approach 3121 (34%). of the remaining 6062, 941 (16%) were excluded for patient-related reasons (patient refused assent (38%), patient could not assent (13%), nurses recommended exclusion (15%), imminent discharge (21%), other (13%)). another 389 (6%) were excluded for technical reasons, leaving 4732 patientadmissions with usable data. the median percent of patients approached for each shift was 65% and the median percent participating was 85%. as the study progressed, the flux of patients increasingly exceeded the capacity of the research team to approach all patients (figure 3 ), although the percentage of patients participating of those approached remained constant. a median of 61 patients /shift were tagged (iqr 46-68). demographic and clinical characteristics of the patient admission data are shown in table 1 , summarized for the population over the year, all patients in our 81 sampled shifts, and patient study participants only. there are no clinically meaningful differences between participants and the general population with respect to age, sex, and race. however, participants tended to present with greater acuity. there were parallel increases in length of stay (los) and percent admitted to the hospital as the overall patient population narrows to participants. there were 105 staff eligible, of which 88 (84%) agreed to participate. the median number of staff participants per shift was 28 (iqr 19-36). although the staffing pattern provided for a maximum of 31 staff per shift, occasional staff meetings, skill seminars, and double coverage caused the number of staff to exceed this maximum in some shifts. as staff are a vulnerable population we did not collect demographic information in order to preserve anonymity and thus maximize participation. staff participants were observed in shifts numbering between 1 and 77, with a median of 23. we observed a median of 86 participants per shift (interquartile range (iqr) 73-104). the median of the maximum number of participants in the ed simultaneously was 56 for day and 48 for night shifts. contact characteristics (number per shift and duration) are summarized over 81 shifts (table 2 ). median and iqr values for quantity and duration of contacts within shift and by type of contact (i.e., patient-patient, patient-staff, staff-staff) are shown. numbers of contact events are described by shift, by participant, and by contact pair. numbers of contact pairs are described by shift and by participant. duration of contact is described by total per shift, per contact event, per contact pair, and per participant. we observed a total of 293,181 contact events across all shifts. in the typical observation period, 2084 (median) contact events occurred, with most of type ss. a similar pattern was seen in contact events per participant and per pair. we observed 478 (median) contact pairs per shift, with pp and ss contacts having equally large frequency (180 pp, 170 ss) while ps contacts (108) were less numerous. the number of distinct participants with which any other participant came into contact was largest for ss (13.5) as compared to pp (5) and patient -staff interactions of both types (2) . a typical patient participant came into contact with 2 staff participants, while a typical staff participant came into contact with 4 patient participants. in a typical observation period, 426 (median) hours of contact were observed, with ss hours being an order of magnitude greater than pp or ps. similar patterns were seen with hours of contact per hour of shift. most contact events were short (,3 minutes for pp and ps, ,10 minutes for ss). total minutes per pair were similarly short for pp and ps (2.6 and 2.4 minutes respectively) but longer by a factor of more than 10 for ss (40.2 minutes). a similar pattern was observed for total duration in contact for participants. a typical patient participant had 8.9 minutes of contact with staff participants, while a typical staff participant had 25.1 minutes of contact with patient participants. the distributions of the total duration of contacts per participant by contact type (pp, p with s, s with p, and ss) are given in figure 4 . note that the distribution of total contact duration of staff with other staff is much less right-skewed than for the other contact types. the cumulative distributions of the number of contacts per participant (degree) by contact type are given in figure 5 . note that these distributions are not consistent with a power law distribution. an off-the-shelf, commercially available, rfid system was used to measure contacts in an ed. the data described here illustrate the complexity of interpersonal interactions in this setting. we found substantial differences in contact characteristics of the three mixing subgroups. in general, ss interactions were more numerous and longer than pp or ps interactions. therefore, given a susceptible population of patients and staff, the biological gradient created by individuals in contact favors cross infection among staff. this finding is consistent with previous studies of contacts in the healthcare environment which highlight that table 2 . summary of contact characteristics per shift among patients and staff in an emergency department, over 81 shifts 1 . there were a total of 185 individuals in 81 shifts that did not make a contact while under surveillance. they are not included in these calculations. 2 the median and quartiles of each shift were calculated and the median of these values are reported. the median of all types will not be the sum of the 3 subtype medians. 3 all comparisons across groups types were significant by friedman's test at p,0.0001, except for contact pairs/shift, which was significant at p = 0.004. tukey's post hoc procedure was used to determine which groups were different and the ordering. 4 a contact event is defined as any two people being within 1 meter of each other; multiple discontinuous instances between the same two individuals are here counted as multiple contacts. 5 one contact pair is defined as any two people who have at least one instance of being within 1 meter of each other ( = an edge or link); multiple discontinuous instances are here counted as a single contact. 6 total hours/shift is the sum of all instances of contact. nb: shift duration ranged from 5 to 12 hours. doi:10.1371/journal.pone.0070854.t002 the validity of this study for the general healthcare population is limited by several factors. the study period coincided with the novel h1n1 influenza outbreak. the methods of this study made it impossible to know how the novel influenza outbreak or its associated publicity impacts generalizability of our results to the general population of ed's. informal observations of interpersonal behavior made by research study staff suggest no change in number or duration of interpersonal contacts. sources of bias. despite our countermeasures, several types of bias were present in the study, which might result in biased estimates of number and duration of contacts among all mixing groups. several factors led to the presence of selection bias. first, the study was performed in a busy, urban ed with unique facility footprint, staffing pattern, and patient demographics. therefore these results may not be applicable to all ed's or even to other similar, non-ed healthcare environments. study criteria excluded visitors and non-ed based hospital staff present in the ed (e.g., cleaning staff, hospital chaplains) as well as prehospital personnel. moreover, only 88 staff participated, although 105 staff were employed during the study year. irb approval was contingent on anonymization of staff beyond job category (physician, nurse, other patient care, administrative). therefore, number and total duration of staff contacts are underestimated. second, over time we observed fewer rfid signals from staff participant tags. while it is reasonable to conclude that there was a decline in staff participation over the course of the year, there is also high probability that some (and perhaps most) of the decline could be attributed to battery failure in the permanent tags. the battery half-life was one year, and these badges were activated six months prior to the commencement of our year of official study observation in order to test and calibrate the system. indeed, the fall-off in staff participation is consistent with exponential failure time with half-life of one year. a spurious system setting prevented the receipt of planned alerts regarding weakening batteries. after the conclusion of the study period, we found a substantial number of staff tags with dead batteries. thus the fall-off in staff participation is likely due to battery failure rather than staff selecting out of the study. regardless of the reason, the effect of this decline is underestimation of the number and duration of contacts. lastly, despite utilizing randomly selected observation periods and waiver of all elements of informed consent, patient participants tended towards higher acuity triage score, higher likelihood of hospital admission, and longer ed los than the ed population at large. the study may also be affected by measurement bias. study staffing was inadequate to provide tagging of patients at the instant of ed arrival. thus actual los was longer than studied los, so number, degree, and duration of contacts have likely been underestimated. moreover we made a protocol decision not to tag patients who were awaiting discharge. in this case, the number and duration of contacts have also likely been underestimated. on the other hand, we assumed that discrete contact events occurred when the adjacency matrix elements of the trao for subjects i and j along the time dimension were two strings of 19s separated by as few as three 09s. such an occurrence could reflect, for instance, a staff member seeing a patient in an exam room (series of 19s), stepping out of the room for as little as three seconds (0, 0, 0), then returning to the room (series of 19s). since we could not separate these very real possibilities from those due to poor signal quality, we elected to leave these as separate events. in this case, we may have overestimated the number of contact events while underestimating event duration. another place where measurement bias may have occurred was in the waiting room. our focus on getting greater separation between patient exam rooms came at the expense of less separation in the waiting room, a large (,2500 square foot) open square-shaped area that was divided for system purposes into two zones. all participants that the system located to one waiting room zone were counted as in contact. given the hard and soft architectural features of this space, it is highly likely that individuals colocated within the space would have a high probability of being in close personal contact. however, we could have overestimated the number and duration of contacts for patients in one waiting room zone that were actually more than 1 m apart, but we also could have underestimated the number and duration of contacts for patients in two zones that in reality were seated next to each other. the waiting room population is very dynamic. there are a number of factors that contribute to this. the waiting room lies between the main entrance for walk-in patients and the registration area. thus upon first entry patients and/or their accompanying visitors must walk through the thick of other patients to sign into the system. second, the front-end process of care requires patients to make multiple trips to and from the waiting room: sign-in, triage, hospital registration, care initiation, emergency radiology, and, in some cases, to see a mid-level provider. also, to enhance patient satisfaction with the ed visit experience, the waiting room contains many diversions to address patient comfort, for example, a coffee station, telephones, reading materials, vending machines, restrooms, and, during the time of the study, a designated smoking area. all of these factors contribute to high mobility of patients in the waiting room during their visit, resulting in more brief contacts than might initially be expected. in the case of a highly transmissible virus, such patterns of contacts may be sufficient for cross infection. however, for a less transmissible virus, many brief contacts may not generate sufficient exposure to the index case that would result in cross infection. several factors contributed to classification bias. software requirements mandated division of the ed footprint into mutually exclusive zones, for which any two individuals simultaneously in a zone would likely (but not certainly) be in contact with one another. the net effect of this procedure should result in neither overestimation nor underestimation of study results. challenges were encountered among staff compliance with badges. hospital staff members typically work in designated areas, and thus perceive themselves to be in constant visual contact with their team members. therefore, to the staff, benefits to be obtained by tracking potential cross infection exposures are outweighed by costs -for instance, annoyance due to dropping of tag when affixed to pocket, tangling with stethoscope when carried on lanyards around the neck, or interfering with hospital staff id tags. we attempted to improve staff compliance by issuing permanent tags for staff to wear and providing periodic education sessions. staff did not have to activate their tags nor record their use, therefore we had a completely passive inclusion system for staff but not for patients. for staff, we considered the shift start time as the time that the tag was first located outside staff only areas or the observation period begin time, whichever occurred last. similarly the shift end time was the time that the tag was last located outside staff only areas, or the observation period end time, whichever occurred first. this was done in order to account for tags that might be stored in a locker or left on a sweater draped over a desk chair when the staff member was not working. most importantly the number and duration of staff-staff contacts demonstrate the dangers of ill or infectious staff members at work. this finding is consistent with simulations conducted by others [22, 23] . if transmission is related to a biological gradient of exposure as defined by magnitude and duration of contacts, then pathogens transmissible by droplets appear to have a higher likelihood of cross infection from working contagious staff (i.e. ''presentees'') to susceptible peers. in the case of annual influenza outbreaks, this finding underscores the importance of vaccination of healthcare employees assigned to the ed in order to prevent health care service interruption due to widespread staffing illness (i.e. absentees) since infectious humans may be shedding virus hours or days prior to developing symptoms. in the case of novel infectious diseases transmissible by droplets, it also underscores the risk for staff cross infection once one staff member has been infected. healthcare systems might consider keeping staff in droplet precautions even when not with symptomatic patients, as well as other efforts to reduce the likelihood of cross infection among staff in general. ed staff tend to look at their patients as high risk, while viewing other staff as ''safe'' unless symptomatic. however, implementation of infection-control measures is more difficult in the ed than in other hospital areas, since patients' conditions have not been identified upon arrival. ed staff are at higher risk for cross infection than personnel in other hospital areas [27] . interestingly, the number of contacts per participant is not consistent with a power law distribution, indicating that our networks are not scale-free. this finding was unexpected a priori, since this property has not been found for other common types of networks, although it has been found by gundlapalli et al. in a study of contacts in a pediatric ed [20] . as gundlapalli and colleagues noted, patients and staff in particular do not associate in a manner that would be appropriate for a preferential attachment model that would give rise to a power law distribution -newly arriving patients are assigned to staff as staff discharge their current patients. importantly, mathematical models of cross infection in the ed that assume contact networks that are scalefree in nature may not describe the cross infection process correctly. five other studies are directly comparable to ours. polgreen and colleagues report results of shadow observations of staff over 40hour observation periods during one year throughout a large, academic, rural hospital [26] . the authors found 71% of staff contact events were with other staff. our results are compatible with this observation. isella et al. report the results of one continuous week of rfid determination of contacts between staff, patients, and visitors in a general pediatric hospital in rome, italy [22] . there are too many substantial differences in study design (pediatric vs adult populations, observation continuous over one week vs 12 hour shifts throughout one year, general hospital ward vs emergency department) to compare their findings to ours. lucet and colleagues implemented an rfid system in two french hospital wards (one infectious disease, one pulmonology) over a three-month period in order to determine contacts between staff and patients under respiratory precautions due to tuberculosis [21] . implicit in these precautions is severe curtailment of the possibility of pp contacts, precluding direct comparison with our results. most recently hornbeck and colleagues report on the use of a mote-based sensor system to characterize the locations of staff in a 20-bed micu [23] . data were collected for only seven days, and reported for only two days. only staff were given wearable badges, whereas fixed beacons were placed in patient rooms and in other commonly shared patient care areas (e.g., hallways, nurses' station). no patient-specific data were collected. there were 16 staff present, on average, in each shift, in comparison to our observation of a median of 28 staff per shift. in the four shifts reported, the typical staff had approximately 50 (median) contacts, with sp contacts less than ss contacts and for both day and night shifts. contacts were short, typically less than 1 minute (median), for both sp and ss contacts and for both day and night shifts. these were much fewer and much shorter than the contacts we observed. other than the difference in the purpose of the unit, another factor that might account for the differences between our observations and theirs might be the area of the unit footprint, which we could not determine. in the study with the most comparable setting and methods, gundlapalli and colleagues report data on contacts between 1261 patients and 87 staff in a pediatric hospital ed collected over the course of a randomly chosen month [20] . they constructed networks from existing clinical informatics resources, notable a proprietary patient flow management system as well as a locator system for which staff were given ir badges to wear. in this case, locations of staff were zoned, then merged with the patient flow system data to create a dataset describing ps interactions. the system did not cover the waiting room, in contrast to ours. each staff had contact, on average, with 6 patients, while each patient had contact on average with 3 staff. they also delimited these data for one day, and the resulting network described interactions among 21 staff and 40 patients. the average contact duration per pair was 20.16 seconds, and the average number of contacts per participant was 4.86. for both analyses, ss and pp interactions were not considered. the degree distribution was not consistent with a power law distribution, as we also observed. coupled with our observation, there may be some other forces at work here, which may play out in the cross infection process. certainly, as these authors note, there is not a preferential attachment model working for staff-patient assignments, as at least the initial contacts have more to do with the length of the queue of patients for which the staff are already caring. although the two hospital-based rfid studies are not directly comparable to ours, both studies report issues with participation and with the system similar to our experience. in the italian study there was excellent participation (96.6%), but data from approximately half of the patients (39/76) and visitors (30/61) had to be excluded for technical reasons [22] . among 79 days of observation in the french study, less than half could be used in the analysis due to reasons similar to ours (i.e. failure of staff to carry tag, technical issues) [21] . rfid systems and other remote location sensor technologies will find greater applications in health care systems in the future, with increasing technical capabilities. we have demonstrated that social contacts in the ed can be quantified over long periods. this paper has barely tapped this rich data resource. in future papers we will explore the dynamics of the social networks we have characterized by relating our contact metrics with staff and patient characteristics as well as with the stages of patient care. our findings will also inform mathematical models and simulation studies to determine the potential risks of cross infection and the likelihood of the infection spreading to the rest of the hospital through an admitted patient cross infected in the ed. considering patient and staff interactions in the frame of a network opens up possibilities for major improvements not only in infection control, but also in facilities design and in work-and patient-flow management. investigation of a nosocomial outbreak of severe acute respiratory syndrome (sars) in toronto, canada hospital emergency departments: crowding continues to occur, and some patients wait longer than recommended time frames pediatric emergency room visits: a risk factor for acquiring measles measles transmission in health facilities during outbreaks risk of infection 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interactions between healthcare workers and patients under airborne precautions close encounters in a pediatric ward: measuring face-to-face proximity and mixing patterns with wearable sensors using sensor networks to study the effect of peripatetic healthcare workers on the spread of hospital-associated infections the use of ranks to avoid the assumption of normality implicit in the analysis of variance nonparametric and distribution-free methods for the social sciences prioritizing healthcare worker vaccinations on the basis of social network analysis responding to the severe acute respiratory syndrome (sars) outbreak: lessons learned in a toronto emergency department drs. lowery-north and hertzberg had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. the authors would like to thank the staff of the emergency department of emory university hospital midtown. we would also like to thank the staff of emory university technology services and the rollins school of public health office of information services. key: cord-048367-yya6w976 authors: jónsson, stefán r.; larue, rebecca s.; stenglein, mark d.; fahrenkrug, scott c.; andrésdóttir, valgerdur; harris, reuben s. title: the restriction of zoonotic perv transmission by human apobec3g date: 2007-09-12 journal: plos one doi: 10.1371/journal.pone.0000893 sha: doc_id: 48367 cord_uid: yya6w976 the human apobec3g protein is an innate anti-viral factor that can dominantly inhibit the replication of some endogenous and exogenous retroviruses. the prospects of purposefully harnessing such an anti-viral defense are under investigation. here, long-term co-culture experiments were used to show that porcine endogenous retrovirus (perv) transmission from pig to human cells is reduced to nearly undetectable levels by expressing human apobec3g in virus-producing pig kidney cells. inhibition occurred by a deamination-independent mechanism, likely after particle production but before the virus could immortalize by integration into human genomic dna. perv inhibition did not require the dna cytosine deaminase activity of apobec3g and, correspondingly, apobec3g-attributable hypermutations were not detected. in contrast, over-expression of the sole endogenous apobec3 protein of pigs failed to interfere significantly with perv transmission. together, these data constitute the first proof-of-principle demonstration that apobec3 proteins can be used to fortify the innate anti-viral defenses of cells to prevent the zoonotic transmission of an endogenous retrovirus. these studies suggest that human apobec3g-transgenic pigs will provide safer, perv-less xenotransplantation resources and that analogous cross-species apobec3-dependent restriction strategies may be useful for thwarting other endogenous as well as exogenous retrovirus infections. , bats [2] and chimpanzees [3] . domesticated animals can also function as zoonotic intermediates (e.g., [4, 5] ). additional and unprecedented opportunities for zoonoses occur when live cells, tissues or organs are transplanted from one species to another [6] . however, despite risks and technical and immunological challenges, several xenotransplantation procedures have shown preclinical promise for treating diabetes, heart, kidney and other human diseases (e.g., [7] [8] [9] ). pigs are favourable xenotransplantation sources because of their human-like physiology, large litters, short gestation period and genetic malleability [10] . however, pig to human virus transmission has been a concern since it was shown that porcine endogenous retroviruses (pervs) could infect human cells in culture [6, [11] [12] [13] . although perv transmission has yet to be documented in xenotransplantation patients, significant concerns still exist regarding perv and other potentially pathogenic viruses [14, 15] . strategies to reduce the likelihood of perv transmission have been proposed, such as selective breeding for lower levels of perv, rnai transgenesis to knock-down perv expression or systematic deletion of active perv copies (e.g., [15, 16] ). the first two are unlikely to be completely effective or risk-free and the third, albeit theoretically feasible, may be overly technical and prohibitively expensive. therefore, alternative, robust and costeffective methods to reduce perv transmission and possible xenozoonotic infections are desirable. apobec3g is a single-strand dna cytosine deaminase best understood as a potent inhibitor of hiv-1 replication [17] [18] [19] [20] [21] [22] [23] . it can however also inhibit a variety of other exogenous and endogenous retroviruses/elements (e.g., [17, 18, [24] [25] [26] [27] [28] [29] ). apo-bec3g engages an assembling retrovirus particle, accesses the rna genome-containing virus core and, upon reverse transcription, deaminates cdna cytosines to uracils (c-to-u). catastrophic levels of uracil either directly inactivate the coding capacity of the virus or trigger the degradation of the viral dna. the former manifests as genomic strand-specific guanine to adenine (g-to-a) hypermutations (cdna strand c-to-t transitions). however, in several instances, it is noteworthy that the deaminase activity of apobec3g or other apobec3 proteins is partly or even completely dispensable (e.g., hiv-1, hepatitis b virus, l1 and alu [27] [28] [29] [30] [31] [32] ). interestingly, throughout evolution, the retroviruses of many mammals appear to have become largely immune to apobec3g or to the apobec3g-like proteins of their hosts. hiv-1 expresses an accessory protein, vif, which neutralizes apobec3g through ubiquitination and degradation [33] [34] [35] [36] . simian immunodeficiency virus (siv) uses a similar vif-dependent mechanism [17, 26] . foamy viruses employ an unrelated viral protein called bet, for which the precise neutralization mechanism is currently unclear [37, 38] . murine leukaemia virus (mlv) and human t-lymphotrophic virus-1 (htlv-1) may simply avoid apobec3 proteins by preventing encapsidation [26, 39, 40] . however, cell-based studies have indicated that an apobec3 protein from a mammal to which the virus has not yet adapted may provide an effective strategy for thwarting species-specific viral counter-defenses. for instance, human apobec3g can potently inhibit the replication of siv (except isolates such as siv cpz which encode a vif protein closely related to that of hiv-1), feline foamy virus and mlv (e.g., [17] [18] [19] [20] 25, 26, 39, 41, 42] ). similarly, mouse apobec3 can potently block hiv-1 replication (regardless of vif), although it is completely unable to impede the replication of mlv [26, 41] . one of the most dramatic examples to date used human and mouse apobec3 proteins to inhibit the mobilization of the yeast retrotransposons ty1 and ty2 [43, 44] . thus, it is reasonable to hypothesize that cross-species expression of an apobec3 protein may be used to create a powerful barrier to impede or perhaps even block retrovirus infection. here, this rationale is applied to the specific question of whether human apobec3g expression can inhibit the transmission of perv from pig to human cells. the results demonstrate that perv transmission can be strongly inhibited by apobec3g. to determine whether expression of human apobec3g would inhibit the transfer of perv from pig to human cells it was first necessary to establish a long-term co-culture system. a trans-well assay was set up to monitor perv transmission from pig kidney pk-15 fibroblasts to recipient human embryonic kidney 293t cells ( figure 1a ). these two cell types were used because transmission from pk-15 to 293 cells had been reported previously [11] . the trans-well system enabled co-cultures to be sustained for several weeks, and it facilitated the recovery of each cell type for downstream analyses. an additional benefit of this co-culture system (not provided by transient assays) is that it enables the simultaneous analysis of multiple, endogenous perv elements, which are precisely the targets one would want to monitor and ideally inhibit in (xeno)transplantation procedures. at each co-culture passage point, surplus human 293t cells were used to prepare genomic dna. perv transmission was monitored by subjecting these samples to quantitative (q)-pcr. perv-specific pol gene pcr products could be detected in the human genomic dna samples after approximately two weeks of continuous co-culture ( figure 1b) . from the point of first detection onward, the total number of perv transmissions continued to increase, averaging 190 new events per day per 50,000 cells (10 5 beta-actin copies; sem = 62; n = 5 experiments). importantly, pig cells did not breach the 293t cell compartment because q-pcr analyses of the same human genomic dna samples failed to detect a concomitant transfer of pig genomic dna ( figure 1b ; also see online figure s1 for perv pol gene q-pcr standard curves, representative perv-specific datasets and human beta-actin controls). moreover, perv copy number did not increase over a two week interval when infected 293t cells were grown in isolation, indicating that perv was not replicating in the human cells and that the majority of the observed transmission events were derived from the pk-15 cells (i.e., new events). these results combine to indicate that the trans-well assay provided a robust system for monitoring bona fide zoonotic perv transmissions. the second key step in addressing our experimental question was isolating pk-15 clones that stably expressed human apobec3g. clones expressing human apobec3g cdna or an empty vector control were established in parallel ( figure 2a ). immunoblotting identified clones with apobec3g levels similar to those in known apobec3g-expressing human t cell lines cem and h9, which are non-permissive for growth of vif-deficient hiv-1 [19] . although it was impossible to achieve a physiologic expression level, the comparative immunoblot at least ensured that the levels of apobec3g were equal or lower than those present in wellstudied, non-permissive human t cell lines. co-culture experiments were set up to compare perv transmission from two independently derived pk-15 clones expressing human apobec3g and two vector expressing controls. remarkably, the human apobec3g expressing pk-15 clones showed levels of perv transmission that were lower than the q-pcr detection threshold of approximately 10 copies ( figure 2b ; . this graph summarizes data for two independently derived pk-15 clones, v1 (squares) and v2 (diamonds). all data points were calculated using results from duplicate q-pcr reactions of genomic dna from three parallel (but independent) co-cultures. the error bars indicate the sem. see the materials and methods and online figure s1 for additional details, representative raw data and controls. doi:10.1371/journal.pone.0000893.g001 online figure s1 ). in contrast, the control clones showed high levels of perv transfer by co-culture day 17 and transmission events continued to accumulate through the duration of the experiment. the kinetics of perv transmission were similar to those reported in figure 1b (these results also contributed to the aforementioned transfer rate calculations). these data were further corroborated by additional experiments where perv transmission was monitored simultaneously by q-pcr and by reverse-transcriptase elisa assays (online figure s2 ). an ultimate application of the technology described here raises the potential problem that human apobec3g may not be subjected to (proper) post-translational regulation in pig cells and it may therefore promote carcinogenesis. expression of human apobec3g in a heterologous system has been shown to trigger elevated levels of genomic c/g-to-t/a transition mutation [44] . therefore, to help mitigate this risk (in addition to establishing clones that expressed relatively modest apobec3g levels; above), we asked whether the predominantly cytoplasmic localization pattern of human apo-bec3g would be maintained in pk-15 and in a swine testes cell line, st-iowa ( figure 3 ; compare with other apobec3g reports [18, 35, 41] ). unlike some other apobec3 proteins such as human apobec3b, which is mostly nuclear, both human apobec3g and pig apobec3f appeared predominantly cytoplasmic in either the human or the pig cell lines (figure 3 ; [28, 31, 41] ). both proteins also appeared to concentrate in cytoplasmic punctae, which varied in number and were apparent in some of the cells regardless of species of origin (described previously for apobec3g; e.g., [45, 46] ). overall, these near-identical localization patterns suggested that human apobec3g is not aberrantly regulated in pig cells and, interestingly, that these proteins might be subjected to the same cellular regulatory mechanism(s). during the course of these experiments, we reported some of the activities of pig apobec3f [41] . it could strongly inhibit the replication of hiv (regardless of vif) and modestly inhibit the replication of mlv, a gamma-retrovirus phylogenetically related to perv. therefore, we wondered whether pig apobec3f was expressed in pk-15 and, if so, whether perv resists this cellular defense. to begin to address this possibility, rt-pcr was used to test pk-15 cells for pig apobec3f expression. pig apobec3f mrna was detected readily (online figure s3a ). full cdna sequencing revealed that the predicted apobec3f protein of pk-15 cells was 98% identical to the variant we reported previously [41] . eight amino acid differences were found, but both the pk15 and the previously reported apobec3f sequences were represented in pig genomic dna sequences suggesting that these may be breed-specific polymorphisms (r.s.l. and r.s.h., manuscript in preparation). these observations indicated that either perv resists the endogenous apobec3f protein of its host or that the level of suppression by pig apobec3f is not sufficient to inhibit perv transmission. to begin to distinguish between these two hypotheses, pk-15 clones over-expressing pig apobec3f were established and used . pk-15 and 293t cell lysates were used as negative controls. cem and h9 were used as positive controls for apobec3g expression. a non-specific (but pan-species) band is shown as a protein loading control (marked by an asterisk). (b) q-pcr data using genomic dna prepared from 293t cells co-cultured with two independently derived apobec3g expressing pk-15 clones (g1 and g2, circles and triangles, respectively) or two vector control clones (v1 and v2, diamonds and squares, respectively). the experimental parameters are identical to those used in figure 1b in transmission experiments. the former hypothesis was favored because pig apobec3f over-expression did not significantly interfere with perv transmission (online figure s3b ). these results were further supported by pcr experiments showing that perv could be amplified readily from 293t cells that had been co-cultured with pk-15 over-expressing pig apobec3f (unlike the apobec3g scenario; below). we further noted that it is highly unlikely that another resident apobec3 protein contributes to perv restriction, because genomic dna sequencing showed that pigs have only one apobec3 gene, apobec3f (r.s.l. and r.s.h., manuscript in preparation). these observations combined to indicate that perv is resistant to the endogenous apobec3 protein of its host. in hindsight, this was not particularly surprising given the emerging trend that (successful) retroviruses are selected in part by their ability to evade the apobec3 proteins of their host species (see introduction). the hallmark of apobec3g-dependent retrovirus restriction is plus-strand g-to-a hypermutation, which is caused by the deamination of minus-strand cdna c-to-u during reverse transcription [17, 18, 20, 24, 25] . the deamination of cytosines within singlestrand dna requires glutamate 259 (e259) of apobec3g [47] [48] [49] . based on homology to structurally defined deaminases, e259 likely functions by helping position the water molecule that ultimately initiates the deamination reaction by attacking the cytosine ring (as a hydroxide; reviewed by [17, 23, 50, 51] ). to determine whether dna deamination is required the apobec3g-dependent inhibition of perv transmission, we established a new set of pk-15 clones expressing apobec3g, apobec3g e259q or a vector control (figure 4, inset) . surprisingly, both apobec3g and the e259q derivative diminished perv transmission to near background levels ( figure 4) . these data demonstrated that the mechanism of inhibition does not require the dna deaminase activity of apobec3g. these data were further supported by the fact that plus strand g-to-a hypermutations were not apparent in the dna of the rare transmission events that occurred in the presence of human apobec3g (below). to begin to genotype the infectious pervs and to further probe the mechanism of perv restriction by human apobec3g, the perv pol gene dna was amplified from human 293t cells, cloned and sequenced ( figure 5a ; online figure s4 ). twenty-nine and twentytwo sequences were analysed from apobec3g and control experiments, respectively. to minimize possible pcr biases, any sequence that was recovered multiple times was considered one event, unless it arose from independent experiments. these dna sequence analyses revealed several important points. first, in contrast to vector control and pig apobec3f overexpressing co-cultures, perv pol gene dna was difficult to amplify from the genomic dna of 293t cells that had been cocultured with apobec3g-expressing pk-15 cells ( figure 5b and every significant sampling point in our q-pcr experiments). taking this together with the observation that apobec3g does not effect pk-15 virus production (similar rt levels were observed in cell-free supernatants in the presence or absence of apo-bec3g; data not shown), we infer that apobec3g restricts perv transmission after virus production but before provirus integration (i.e., between entry and integration). apobec3g may restrict perv at an early reverse transcription stage, possibly by interfering with primer binding, dna synthesis and/or integration is deamination-independent. perv-specific q-pcr data using genomic dna prepared from 293t cells co-cultured with pk-15 clones expressing apobec3g (g; triangles), apobec3g-e259q (ge259q; circles) or empty vector (v; squares). two datasets, each with an independent pk-15 clone in three replica co-culture wells, were collected in parallel and averaged for each data point. one standard error of the mean is shown. the experimental parameters are identical to those used in figure 1b as shown recently for apobec3g and hiv-1 substrates (e.g., [30, [52] [53] [54] [55] ). second, control co-culture perv transmission events were exceptionally diverse, as 11 unique pol sequences were detected and only 4 were found multiple times (online figure s4) . these data suggested that pk-15 cells have at least 11 active pervs capable of infecting human 293t cells, a number consistent with previous studies that reported the existence of approximately 17-50 perv copies in total (with only a fraction being replicationcompetent; [11, 12] ). in contrast, the rare perv sequences derived from the apobec3g co-culture experiments showed a much lower genetic complexity. only three unique sequences were recovered, each differing by a single nucleotide (online figure s4) . in parallel experiments with hiv-based viruses, this apobec3g expression construct caused approximately 30 g-to-a hypermutations per 1000 bases analyzed (e.g., [41] ). thus, approximately 12 g-to-a transitions should have been recovered in these perv dna analyses (nearly 90 if multiply recovered sequences would have been considered). the absence of hypermutated perv proviral dna provided further support for a deaminase-independent mechanism of restriction, which may share features with other instances described previously (e.g., [27] [28] [29] [30] ). we have established a quantitative assay to monitor the zoonotic transmission of perv to human 293t cells. expression of human apobec3g in the pig pk-15 cell line strongly inhibited perv zoonoses, while the endogenous apobec3f protein of pigs appeared considerably less effective. these data are the first to show that human apobec3g can inhibit perv and the first to demonstrate that apobec3 proteins can be used purposefully to reduce if not prevent zoonotic retroviral infections. these results were not anticipated because human apobec3g has a relatively weak effect against the perv-related gamma-retrovirus mlv [26, 39] . our data indicate that the engineering of pigs to express human apobec3g (or an equally potent non-porcine apobec3) may result in animals whose cells and tissues are much less likely to disseminate functional perv. the deamination-independence of the restriction mechanism suggests that a catalytically inert apobec3g protein, such as e259q, may be equally potent and simultaneously reduce the risk of cancer-promoting mutagenesis. apobec3g or apobec3g-e295q expressing pigs may therefore constitute safer source animals for pig-to-human xenotransplantation procedures. in contrast to knockdown, knockout (by gene targeting or selective breeding) or most chemical-based anti-viral approaches to neutralize perv [15] , the apobec3 antiviral defense system has several advantages including a potentially broad neutralizing activity (effective against perv and likely several other endogenous and exogenous viruses) and an applicability to situations where many copies of a virus are already present in a genome. analogous transgenic applications can be envisaged, such as using cross-species apobec3 expression to purposefully impede known viruses (e.g., the aids virus hiv-1 or the hepatitis b virus hbv). moreover, for humans and other mammals with multiple apobec3 proteins, our data encourage the development of methods to induce/up-regulate endogenous apobec3 proteins, which have the capacity but may not normally restrict a particular virus (e.g., human apobec3b and hiv-1). the porcine kidney pk-15 fibroblast cell line and the swine testes st-iowa cell line were obtained from the atcc and cultured in dulbecco's modified eagle's medium (invitrogen) supplemented with 10% fetal bovine serum (gemini), and 25 units/ml penicillin and 25 mg/ml streptomycin at 37uc and 5% co 2 . human embryonic kidney 293t and hela (a. bielinsky, university of minnesota) cell lines were grown under the same conditions. the t cell lines h9 and cem (m. malim, kings college london) were cultured in rpmi-1640 supplemented with 10% fetal bovine serum (gemini), and 25 units/ml penicillin and 25 mg/ml streptomycin at 37 uc and 5% co 2 . plasmids encoding human apobec3g, human apobec3g-e259q and porcine apo-bec3f were described previously [41] . the human apobec3g and porcine apobec3f cdna sequences used here are identical to genbank accession numbers, nm_021822 and nm_001097446, respectively. stable apobec3g-or vector control-expressing pk-15 cell lines were constructed by transfection using fugene6 according to the manufacturer's protocol (roche) or by electroporation (biorad, 250v, 950 mfa). clones were selected using growth medium containing 1 mg/ml g418 (roche), and apobec3g expressing clones were identified by immunoblotting using a polyclonal antibody toward human apobec3g (j. lingappa, university of washington). all pk-15 clones were maintained in growth medium supplemented with 250 mg/ml g418 to ensure stable expression. long-term co-culture assays were performed in 6 well tissue culture plates with inserts (transwellh, corning inc.). this system uses a membrane with 0.4 mm diameter pores, which keeps the two cell types separated physically but simultaneously allows diffusion of nutrients and small molecules including virus particles of approximately 0.1 mm (including perv). each experiment was initiated with 75,000 pk-15 cells (insert) and 75,000 293t cells (well) as illustrated ( figure 1a ). at 72 hr intervals, each cell type was washed with pbs, subjected to mild trypsinization and diluted into 4 parts fresh growth medium. excess 293t cells were used to prepare genomic dna (qiagen dneasy kit). the rate of perv transfer was calculated using the pol gene levels from the last two data points (usually spanning a 3 day period). the difference between these levels represents perv pol gene dna that has accumulated per 100,000 human beta-actin gene copies (50,000 cells assuming that the 293t cell line has two beta-actin copies) per time period. individual rates from 5 independent experiments were averaged to determine the overall transmission rate (190+/262 events per day per 50,000 cells). data from figures 1b, 2b and s2a contributed to rate calculations. genomic dna was isolated from human 293t cells using the dneasy kit (qiagen). duplicate 25 ml pcr reactions consisting of 10 ng of 293t genomic dna, 100 nm primers and 26 iq sybr green super mix (biorad) were run on an icycler iq multicolor real-time pcr detection system (biorad). the thermocycler conditions consisted of an initial denaturation of 95uc for 5 min and 50 cycles of denaturation (95uc for 15 sec) and annealing (58uc for 30 sec). after the 50 cycles, a melting curve analysis (55uc to 95uc) was performed to confirm product specificity. the cycle threshold (c t ) was generated using biorad software and it was used to calculate the amount of target dna (perv pol or human beta-actin). a standard curve was generated using the method of dorak [56] and a dilution series (10 to 10 7 copies) of a linearized plasmid containing the relevant 193 bp perv pol gene fragment. the equation generated from the standard curve (slope and y intercept) was used to determine the efficiency of the pcr reaction and to quantify the number of perv pol gene or human beta-actin copies in the q-pcr reactions. perv copy numbers were normalized to those of beta-actin using the method of [56] . the primer sets used in this study were: perv pol (193 bp): 59-aac cct tta ccc ttt atg tgg at and 59-aaa gtc aat ttg tca gcg tcc tt; standard reverse transcription (rt)-pcr reactions were performed using rna prepared from pk-15 cells (trizol protocol, invitrogen), m-mlv reverse transcriptase was used for cdna synthesis using an oligo dt primer (ambion) and taq polymerase was used for pcr (roche). the primers specific to pig apobec3f were 59-tgg tca cag agc tga agc ag and 59-ttg ttt tgg aag cag cct tt (175 bp). the semi-nested primer set used to detect plasmid-expressed pig apobec3f was 59-cca agg agc tgg ttg att tc (exon 6, reaction 1), 59-ctg gag caa tac agc gag ag (exon 7, reaction 2) and 59-tag aag gca cag tcg agg, with the latter being vector specific (319 bp and 190 bp products, respectively). the mammalian beta-actin primers were 59-cct tca att cca tca tga agt g and 59-cca cat ctg ctg gaa ggt (236 bp). these primers amplify equally well a 236 bp beta-actin fragment from all mammals tested, including pigs and humans (e.g., online figure s3 ). the human apobec3g-gfp, pig apobec3f-gfp and gfp expression constructs were described previously [28, 41] . the pig and human cell lines were maintained as above. one day prior to transfection, 5,000-20,000 cells were seeded onto labtek chambered coverglasses (nunc). after 24 hrs incubation, these cells were transfected with 250 ng of the relevant plasmid construct. after 24 hrs of additional incubation, images of the live cells were acquired using a zeiss axiovert 200 microscope at 4006 total magnification. images were analyzed using image j software (http://rsb.info.nih.gov/ij). whole cell protein extracts were prepared from 293t cells by suspending 500,000 cells in pbs, sonicating twice for 5 seconds and clarifying the lysates by centrifugation. soluble protein levels were quantified using a biorad bradford assay. 10 mg of cell lysate was tested for reverse transcriptase activity using a c-type-rt activity assay (cavidi tech) following the manufacturers' instructions. cell-free pk-15 supernatants (perv-containing) were assayed directly using the cavidi tech elisa assay. human 293t cell genomic dna was prepared from terminal cocultures and 50 ng was used for high fidelity, perv pol genespecific pcr reactions (phusion polymerase; finnzymes). 193 bp products were cloned using the zero blunt topo pcr cloning kit (invitrogen) and sequenced (university of minnesota advanced genetic analysis facility). sequence comparisons were performed using sequencher software (gene codes corp.) and publicly available clustal w alignment algorithms (http://align.genome. jp/). figure s1 quantitative real-time pcr analyses. (a) standard curves depicting q-pcr data obtained using dilutions of a linearized perv pol gene plasmid alone (squares) or diluted plasmid plus 10 ng of 293t cell genomic dna (diamonds). under both conditions, all template amounts (10 to 10 7 copies) amplified efficiently (the log-linear slope equations are shown). the correlation co-efficiency value (r 2 ), which reports the technical accuracy of the assay, is also indicated. the standard curve data points were the average of 2 independent reactions with deviations smaller than the symbols (i.e., c t errors for each point ranged from 0 to 0.4). (b) two representative control q-pcr datasets showing the amplification of perv pol gene dna from pig pk-15 cell genomic dna (circles). two additional control q-pcr datasets showing that the perv-specific primers fail to amplify product from uninfected human 293t cell genomic dna (squares). the reaction threshold, 10 times the mean standard deviation of the background fluorescence level (biorad), is indicated. (c) representative co-culture q-pcr amplification curves of perv pol gene dna. template genomic dna isolated from human 293t cells co-cultured with vector expressing pk-15 cells (diamonds) or human apobec3g-expressing pk-15 cells (triangles) was used. (d) representative q-pcr amplification curves of the 293t cell beta-actin gene, which served as an internal standard for quantifying the real-time pcr data. raw q-pcr data will be made available on request. found at: doi:10.1371/journal.pone.0000893.s001 (9.93 mb tif) figure s2 apobec3g inhibits perv transmission. (a) a graph showing the accumulation of perv pol gene-specific pcr products in 293t cells co-cultured with a control cell line (v3) but not with an apobec3g-expressing cell line (g1). the data points were an average of two q-pcr runs and the difference between each run was smaller than the plotted symbol. the experimental parameters were identical to those used in the experiments shown in figures 1b and 2b. (b) relative levels of reverse transcriptase(rt)-activity detected in soluble extracts of day 28 co-cultured 293t cells, which were used to generate the q-pcr data shown in figure s2a . uninfected 293t cell lysates had a relatively high endogenous rt activity. therefore, to help with the presentation of these data, this level was normalized to one and all of the other data were calculated relative to this value. the level of rt activity in pk-15 extracts was much higher than that of 293t cell extracts (+/2perv) and it had reached saturation (out of range) when these data were collected. found at: doi:10.1371/journal.pone.0000893.s002 (4.76 mb tif) figure s3 pig apobec3f is expressed in pk-15 cells and its over-expression does not markedly inhibit perv transmission. (a) an image of an ethidium bromide-stained agarose gel showing the results of an rt-pcr amplification experiment using pk-15 cellular rna and appropriate controls. the top panel shows that pk-15 and representative pk-15 derived clones all expressed pig apobec3f, as indicated by the specific 175 bp pig apobec3f pcr product (confirmed by dna sequencing). 293t cell mrna and a diluted pig apobec3f expression plasmid were used as negative and positive controls, respectively. a larger, non-specific band was apparent only in the 293t cell rt-pcr reactions. the bottom panel shows that a conserved, 236 bp beta-actin gene fragment could be amplified from both pk-15 cells and human 293t cells (but not from diluted plasmid dna). note that this primer set differs from the human-specific set used in the q-pcr experiments. the sizes of the marker (m) dna bands are shown. (b) an image of an ethidium bromide-stained agarose gel showing expression of plasmid-derived pig apobec3f in pk-15 cells after 26 days of continuous co-culture. non-transfected (nt) cells and diluted apobec3f plasmid dna (pdna) provided negative and positive controls, respectively. the larger 319 bp (far right lane only) and smaller 190 bp bands are the specific pcr products of the first and second rounds of semi-nested pcr, respectively (confirmed by dna sequencing). (c) a histogram summarizing the level of perv transmission that was observed after 23 days of co-culturing human 293t cells with pk-15 cells expressing a vector control or over-expressing pig apobec3f. two datasets, each with an independent pk-15 clone in three replica co-culture wells, were collected in parallel and averaged for each histogram bar. one standard error of the mean is shown. the experimental parameters are identical to those used in figure 1b . found at: doi:10.1371/journal.pone.0000893.s003 (8.52 mb tif) figure s4 genetic variation in zoonosed perv pol gene sequences. (a) sequences of the perv pol gene fragments cloned from 293t cells co-cultured with control vector-expressing pk-15 cells. the number of times that each sequence was recovered is shown (n). experiments 1 and 2 used genomic dna prepared from the 293t cells used to generate the data shown in online figure s2 (day 28 samples) and figure 2b (day 23), respectively. the most frequently detected 147 bp perv pol gene sequence is shown in its entirety (which together with pcr primers makes up the 193 bp product shown in figure 5 ). identical nucleotides in other sequences are represented by dashes and non-identical nucleotides by the indicated dna bases. genbank accession numbers are shown for pol gene fragments with 100% identity to previously reported sequences. (b) sequences of the perv pol gene fragments cloned from 293t cells co-cultured with control apobec3g-expressing pk-15 cells. parameters are identical to those described above. found at: doi:10.1371/journal.pone.0000893.s004 (0.05 mb doc) genomic analysis of increased host immune and cell death responses induced by 1918 influenza virus bats are natural reservoirs of sars-like coronaviruses origin of hiv-1 in the chimpanzee pan troglodytes troglodytes isolation and molecular identification of nipah virus from pigs influenza: emergence and control xenografts and retroviruses prolonged diabetes reversal after intraportal xenotransplantation of wild-type porcine islets in immunosuppressed nonhuman primates heart transplantation in baboons using alpha1,3-galactosyltransferase gene-knockout pigs as donors: initial experience marked prolongation of porcine renal xenograft survival in baboons through the use of alpha1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue will the pig solve the transplantation backlog? infection of human cells by an endogenous retrovirus of pigs two sets of human-tropic pig retrovirus productive infection of human primary cells and cell lines with porcine endogenous retroviruses search for cross-species transmission of porcine endogenous retrovirus in patients treated with living pig tissue. the xen 111 study group the potential hazards of xenotransplantation: an overview inhibition of porcine endogenous retroviruses (pervs) in primary porcine cells by rna interference using lentiviral vectors role and mechanism of action of the apobec3 family of antiretroviral resistance factors broad antiretroviral defence by human apobec3g through lethal editing of nascent reverse transcripts isolation of a human gene that inhibits hiv-1 infection and is suppressed by the viral vif protein the cytidine deaminase cem15 induces hypermutation in newly synthesized hiv-1 dna apobec-mediated viral restriction: not simply editing? intrinsic immunity: a front-line defense against viral attack retroviral restriction by apobec proteins apobec3g cytidine deaminase inhibits retrotransposition of endogenous retroviruses dna deamination mediates innate immunity to retroviral infection speciesspecific exclusion of apobec3g from hiv-1 virions by vif apobec3g targets human t-cell leukemia virus type 1 apobec3b and apobec3f inhibit l1 retrotransposition by a dna deamination-independent mechanism inhibition of hepatitis b virus replication by apobec3g antiviral potency of apobec proteins does not correlate with cytidine deamination cellular inhibitors of long interspersed element 1 and alu retrotransposition apobec3 proteins inhibit human line-1 retrotransposition induction of apobec3g ubiquitination and degradation by an hiv-1 vif-cul5-scf complex the vif protein of hiv triggers degradation of the human antiretroviral dna deaminase apobec3g hiv-1 vif protein binds the editing enzyme apobec3g and induces its degradation the antiretroviral enzyme apobec3g is degraded by the proteasome in response to hiv-1 vif foamy virus bet proteins function as novel inhibitors of the apobec3 family of innate antiretroviral defense factors the antiretroviral activity of apobec3 is inhibited by the foamy virus accessory bet protein murine retrovirus escapes from murine apobec3 via two distinct novel mechanisms resistance of human t cell leukemia virus type 1 to apobec3g restriction is mediated by elements in nucleocapsid evolutionarily conserved and non-conserved retrovirus restriction activities of artiodactyl apobec3f proteins ancient adaptive evolution of the primate antiviral dna-editing enzyme apobec3g inhibition of a yeast ltr retrotransposon by human apobec3 cytidine deaminases apobec3g hypermutates genomic dna and inhibits ty1 retrotransposition in yeast human retroviral host restriction factors apobec3g and apobec3f localize to mrna processing bodies antiviral protein apobec3g localizes to ribonucleoprotein complexes found in p bodies and stress granules the retroviral hypermutation specificity of apobec3f and apobec3g is governed by the c-terminal dna cytosine deaminase domain complementary function of the two catalytic domains of apobec3g antiviral function of apobec3g can be dissociated from cytidine deaminase activity cytidine deamination and resistance to retroviral infection: towards a structural understanding of the apobec proteins multifaceted antiviral actions of apobec3 cytidine deaminases apobec3f can inhibit the accumulation of hiv-1 reverse transcription products in the absence of hypermutation. comparisons with apobec3g inhibition of formulaprimed reverse transcription by human apobec3g during human immunodeficiency virus type 1 replication cytidine deaminases apobec3g and apobec3f interact with human immunodeficiency virus type 1 integrase and inhibit proviral dna formation human immunodeficiency virus type 1 cdnas produced in the presence of apobec3g exhibit defects in plus-strand dna transfer and integration real-time pcr construction and use of retroviral vectors encoding the toxic gene barnase key: cord-269690-6r2bfydw authors: de lorenzo, rebecca; conte, caterina; lanzani, chiara; benedetti, francesco; roveri, luisa; mazza, mario g.; brioni, elena; giacalone, giacomo; canti, valentina; sofia, valentina; d’amico, marta; di napoli, davide; ambrosio, alberto; scarpellini, paolo; castagna, antonella; landoni, giovanni; zangrillo, alberto; bosi, emanuele; tresoldi, moreno; ciceri, fabio; rovere-querini, patrizia title: residual clinical damage after covid-19: a retrospective and prospective observational cohort study date: 2020-10-14 journal: plos one doi: 10.1371/journal.pone.0239570 sha: doc_id: 269690 cord_uid: 6r2bfydw data on residual clinical damage after coronavirus disease-2019 (covid-19) are lacking. the aims of this study were to investigate whether covid-19 leaves behind residual dysfunction, and identify patients who might benefit from post-discharge monitoring. all patients aged ≥18 years admitted to the emergency department (ed) for covid-19, and evaluated at post-discharge follow-up between 7 april and 7 may, 2020, were enrolled. primary outcome was need of follow-up, defined as the presence at follow-up of at least one among: respiratory rate (rr) >20 breaths/min, uncontrolled blood pressure (bp) requiring therapeutic change, moderate to very severe dyspnoea, malnutrition, or new-onset cognitive impairment, according to validated scores. post-traumatic stress disorder (ptsd) served as secondary outcome. 185 patients were included. median [interquartile range] time from hospital discharge to follow-up was 23 [20–29] days. 109 (58.9%) patients needed follow-up. at follow-up evaluation, 58 (31.3%) patients were dyspnoeic, 41 (22.2%) tachypnoeic, 10 (5.4%) malnourished, 106 (57.3%) at risk for malnutrition. forty (21.6%) patients had uncontrolled bp requiring therapeutic change, and 47 (25.4%) new-onset cognitive impairment. ptsd was observed in 41 (22.2%) patients. at regression tree analysis, the ratio of arterial oxygen partial pressure to fractional inspired oxygen (pao(2)/fio(2)) and body mass index (bmi) at ed presentation, and age emerged as independent predictors of the need of follow-up. patients with pao(2)/fio(2) <324 and bmi ≥33 kg/m(2) had the highest odds to require follow-up. among hospitalised patients, age ≥63 years, or age <63 plus non-invasive ventilation or diabetes identified those with the highest probability to need follow-up. ptsd was independently predicted by female gender and hospitalisation, the latter being protective (odds ratio, or, 4.03, 95% confidence interval, ci, 1.76 to 9.47, p 0.0011; or 0.37, 95% ci 0.14 to 0.92, p 0.033, respectively). covid-19 leaves behind physical and psychological dysfunctions. follow-up programmes should be implemented for selected patients. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 since the identification of severe acute respiratory syndrome-coronavirus-2 (sars-cov-2) as the causative agent of coronavirus disease-2019 (covid19) , more than four million cases were reported worldwide, mortality reaching 6.68% as of the 20 th of may 2020 [1]. the majority of affected patients manage to overcome the acute phase of the disease and appear to achieve clinical recovery [2] . knowledge of early disease characteristics accumulates rapidly. however, sequelae of covid-19 remain unexplored. it seems, therefore, reasonable to question whether it is safe to lower the guard. monitoring recovered patients over time might be revelatory of what comes next, maximizing preparedness and optimizing medical care. persistent radiological lung abnormalities and breathing difficulties were reported in patients recovered from previous coronavirus diseases [3, 4] . the applicability of these observations to sars-cov-2-infected patients is unknown [5, 6] . inflammation is a recognized promoter of tissue fibrosis [7] . as such, the burden of pulmonary dysfunction after covid-19 recovery may be substantial. the suggested neurotropism of sars-cov-2 might entail neurocognitive sequelae of covid-19 [8] , and the persistence of other disease features cannot be excluded [9, 10] . psychological health in convalescent patients is also a matter of concern. fear of infection-associated complications, prohibition of human contact, and uncertainty about reacceptance in society may jeopardize mental well-being and influence quality of life, prompting to the need of adequate mental counselling. alertness and awareness of what to expect are crucial not to underestimate health problems and to guarantee timely interventions. this would aid in preventing national health care systems from being overwhelmed by the sudden surge of conditions requiring medical assistance. with the belief that hospital discharge is far from being the endpoint of monitoring and precautionary measures, we set up a covid-19 follow-up outpatient clinic to longitudinally follow patients recovered from covid-19. here, we report a first assessment of the information gathered on covid-19 sequelae and propose strategies to identify patients who may benefit from continued monitoring. the covid-biob study protocol conforms to the declaration of helsinki, was approved by the hospital ethics committee, namely comitato etico ospedale san raffaele (ce-osr, protocol no. 34/int/2020), and registered on clinicaltrials.gov (nct04318366). for patients able to provide a signed informed consent (ic) at the time of hospital admission, written ic was obtained prior to data collection. otherwise, patients were consented as soon as they were able to sign. this study is reported in compliance with the strobe statement [11] . a comprehensive evaluation of physical, neurological, cognitive and mental health was performed by a multidisciplinary team consisting of internists, nutritionists, neurologists, and psychiatrists (fig 1) . data about the initial presentation of covid-19 and the disease course were retrospectively scrutinized from medical records in the presence of the patient during follow-up evaluation and collected. complete physical examination and vital sign assessment were integrated with detailed patient medical history. the modified medical research council (mmrc) scale for dyspnoea was used to quantify residual shortness of breath [12] , and a visuo-analog scale (vas) for self-rated health status [13] . percent of body weight change and the mini nutritional assessment (mna) screening tool served as indicators of nutritional status [14] . the mna screening tool was initially developed for detecting undernutrition in the elderly, but it has subsequently been adopted in several clinical settings and patient populations [15] [16] [17] . the tool identifies individuals at risk of malnutrition or malnourished based on the presence of reduction in food intake (due to loss of appetite, digestive problems, chewing or swallowing difficulties), disease burden (psychological stress or acute disease in the previous 3 months or presence of neuropsychological problems), weight loss, body mass index or reduced mobility. an mna value �7 indicates malnutrition and a score between 8 and 11 identifies patients at risk of malnutrition [14] . complete neurological examination was performed to investigate neurological sequelae. cognitive function was assessed through the montreal cognitive assessment (moca) score [18] , and cognitive impairment was defined by a score <24 in the absence of known history of neurocognitive disease. psychiatric unstructured clinical interview was conducted to investigate the presence of a current major psychiatric disorder (depressive disorders, bipolar and related disorders, anxiety disorders, psychotic disorders, eating disorders, and trauma-related disorders) according to the diagnostic and statistical manual of mental disorders (dsm-5). validated self-report questionnaires were used to assess quality of life through the world health organization quality of life (whoqol-bref), post-traumatic stress disorder (ptsd) through the impact of events scale-revised (ies-r), anxiety through the state-trait anxiety inventory form y (stai-y), and insomnia through the women's health initiative insomnia rating scale (whiirs) [19] [20] [21] [22] . demographical data (i.e. age, gender, and ethnicity), comorbidities (i.e. hypertension, htn, coronary artery disease, cad, diabetes mellitus, dm, chronic obstructive pulmonary disease, copd, chronic kidney disease, ckd, active cancer, and current psychiatric disorder according to dsm-5), as well as body mass index (bmi), axillary body temperature, and laboratory values (i.e. the ratio of arterial oxygen partial pressure, pao 2 in mmhg, to fractional inspired oxygen, fio 2 , expressed as a fraction, pao 2 /fio 2 , white blood cell count, wbc, neutrophil to lymphocyte ratio, nlr, liver enzymes, lactate dehydrogenase, ldh, c-reactive protein, crp, estimated glomerular filtration rate, egfr using the ckd-epi equation) at ed presentation were extracted for all patients. acute respiratory distress syndrome (ards) was defined as pao 2 /fio 2 <300 [23] . for hospitalised patients, length of stay (los), transfer to the intensive care unit (icu), and non-invasive ventilation (niv) administration were also recorded. data collected at the follow-up visit included vital parameters, percent of body weight change from hospital admission, mmrc for dyspnoea, mna, vas and whoqol scores, and the presence of cognitive impairment, ptsd, anxiety, and insomnia according to the generally accepted cut-off scores (moca <24, ies-r �33, stai-state �40, stai-trait �40, and whiirs �9, respectively). previous need for psychiatric interventions and previous or current intake of psychotropic drugs were also collected. tachypnoea was defined as respiratory rate (rr) >20 breaths/min [24] , measured by counting respiratory chest movements of over a period of 60 seconds. prior to analysis, data were cross-checked with medical charts and verified by data managers and clinicians for accuracy. to investigate the relevance of the follow-up visit, we created a composite dichotomous variable, i.e. need of follow-up, which identified patients requiring medical advice after covid-19 recovery. accordingly, the need of follow-up was defined by the presence of at least one of the following: i) tachypnoea, ii) mmrc for dyspnoea score �2, iii) uncontrolled blood pressure requiring a change in therapy (increase in dose or new prescription of at least one anti-hypertensive drug, i.e. diuretics, calcium channel blockers, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and beta blockers), iv) mna score �7, v) presence of cognitive impairment. the need of follow-up variable represented the primary outcome. psychiatric disturbances were not included in the primary outcome for the purpose of the analysis, and ptsd was used as secondary outcome. descriptive statistical analyses were performed for all variables. dichotomous variables were expressed as absolute frequencies (percentage), and continuous variables as medians [iqr] . group comparisons were performed using the χ2 test or fisher's exact test for categorical variables, and the mann-whitney u test for continuous variables. to investigate the impact of individual variables on the need of follow-up, we performed univariable and multivariable logistic regression analyses both for the entire cohort and for hospitalised patients only. we subsequently employed a regression tree (rt) algorithm to identify risk groups based on the need of follow-up, within the entire cohort (rt 1) and the hospitalised population (rt 2). the rt algorithm uses recursive partitioning to sequentially split a cluster of patients into increasingly homogeneous sub-groups based on several independent variables, selecting the optimal sequence of classifications as defined by a hierarchy of prognostic factors and associated cut-points [25] . demographical data, comorbidities, bmi, clinical and laboratory features at ed presentation, and hospitalisation due to covid-19 were included as predictors in rt 1. los, niv administration, and transfer to icu, together with demographical data and comorbidities, were used as covariates in rt 2. the results of these analyses were graphically represented. the area under the receiver operating characteristic (roc) curve (roc auc ) was used as a quality metric of the regression trees. univariable and multivariable logistic regression analyses were employed to identify predictors of ptsd among age, gender, bmi at ed presentation, comorbidities, hospitalisation, and ards. missing data was not imputed. all statistical analyses were performed using r statistical package (version 4.0.0, r foundation for statistical computing, vienna, austria), with a two-sided significance level set at p <0.05. as the study addresses an urgent unmet clinical need in response to a global public health emergency, patients and members of the public were not directly involved in the design, conduct, or reporting of this research. from 7 april to 7 may 2020, a total of 195 covid-19 patients were evaluated at the covid-19 follow-up outpatient clinic of san raffaele university hospital. of these, 10 had been admitted for reasons other than covid-19 and were therefore excluded for the present analysis. all patients included (n = 185) had a positive sars-cov-2 rt-pcr test result from a nasopharyngeal and/or throat swab. characteristics at disease onset and follow-up assessment measures of the cohort are reported in tables 1 and 2, respectively. of the 185 patients included in the analysis, 68.1% had been hospitalised, while the rest were discharged from the ed. most inpatients received hydroxychloroquine in conjunction with lopinavir/ritonavir, which was the standard therapy for covid-19 at our institution at the time patients included in the study were admitted to hospital. additional treatments were prescribed based on the severity of the clinical picture. patients managed at home were prescribed symptomatic treatments. patients were assessed after a median [interquartile range, iqr] time from hospital discharge of 23 [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] days. hospitalised patients were older than patients discharged from the ed, more commonly males and white. the two populations did not differ in terms of bmi at hospital admission and medical history, with the exception of htn, which was more frequent in hospitalised patients. laboratory findings at ed presentation of hospitalised and non-hospitalised patients are presented in table 1 . at follow-up evaluation, 54 (29.2%) patients had shortness of breath or were tachypnoeic. 116 (62.7%) patients were malnourished or at risk for malnutrition, and approximately one quarter of patients achieved moca scores compatible with cognitive impairment, despite no known history of cognitive disorders. psychiatric disturbances including anxiety, insomnia, or ptsd were observed in 83 (44.9%) patients (table 2) . hospitalised patients had a tendency towards a more important weight loss during disease and towards higher rr values, compared with patients discharged from the ed. conversely, patients discharged from the ed had lower whoqol scores, reflecting a decreased quality of life, especially in the psychological domain. anxiety and ptsd were more frequent among patients discharged from the ed ( table 2) . the need of follow-up, defined as the presence at follow-up evaluation of at least one among rr >20 breaths/min, uncontrolled blood pressure requiring therapeutic change, moderate to very severe dyspnoea, malnutrition, or new-onset cognitive impairment, was present in 109 (58.9%) patients (fig 2) . this number rose to 126 (68.1%) when including ptsd. no significant difference in the need of follow-up was found between hospitalised patients (75 of 126, 59.5%) and patients discharged from the ed (34 of 59, 57.6%). age predicted the need of follow-up at regression analyses in the entire cohort. specifically, for each additional year of age, the odds of requiring post-discharge monitoring increased by 4% (table 3) . univariable and multivariable regression analyses predicting the need of follow-up within the hospitalised population are described in table 4 . rt analysis identified three variables, namely pao 2 /fio 2 and bmi at ed presentation, and age, that robustly classified patients into risk groups for the need of follow-up after discharge, nlr, neutrophil to lymphocyte ratio; ast, aspartate aminotransferase; alt, alanine aminotransferase; ldh, lactic dehydrogenase; crp, c-reactive protein; egfr, estimated glomerular filtration rate. and indicated the cut-offs that maximized the separation among the resulting patient clusters (rt 1, fig 3) . the three groups were: low probability of need (pao 2 /fio 2 �324 and age <63 years), intermediate probability of need (pao 2 /fio 2 <324 and bmi lower than 33 kg/m 2 or pao 2 /fio 2 �324 and age �63 years), and high probability of need (pao 2 /fio 2 <324 and bmi �33 kg/m 2 ). the roc auc for rt 1 was 0.85. most patients in the low probability of need group (65.5%) were discharged from the ed. the fraction of patients that had been hospitalised was higher in the other groups, reaching the totality of patients in the high probability of need group. the prevalence of hospitalisation in the obtained groups is depicted. age, gender, ethnicity, history of hypertension, coronary artery disease, chronic kidney disease, diabetes mellitus, body mass index (bmi), axillary body temperature, ratio of arterial oxygen partial pressure to fractional inspired oxygen (pao 2 /fio 2 ), aspartate transaminase, lactic dehydrogenase, and c-reactive protein at emergency department presentation, and hospitalisation were included in rt 1 analysis. pao 2 / fio 2 was available for 155 patients. bmi was available for 160 patients. pts, patients. pao 2 /fio 2 , ratio of arterial oxygen partial pressure to fractional inspired oxygen. bmi, body mass index. https://doi.org/10.1371/journal.pone.0239570.g003 when rt analysis was restricted to hospitalised patients (n = 126), four variables emerged as strong predictors of the need of follow-up (rt 2, fig 4) . age, niv administration, history of dm, and los stratified patients into three groups: low probability of need (age <63 years, no niv administration, no history of dm and los <8 days), intermediate probability of need (age <63 years, no niv administration, no history of dm and los �8 days), and high probability of need (age <63 years plus niv or history of dm, or age �63 years). the roc auc for rt 2 was 0.69. decreasing age, female gender and positive psychiatric history were significantly associated with the risk of developing ptsd after covid-19. hospitalisation, instead, emerged as protective (table 5) . at multivariable analysis, only female gender and hospitalisation survived as independent predictors of ptsd occurrence. no significant impact was observed for bmi or other comorbidities on ptsd development (table 5) . here, we present an early analysis of a multidisciplinary follow-up of patients recovered from covid-19. between 7 april and 7 may, 2020, 185 patients previously referred to our institution for covid-19 were evaluated. patient characteristics at disease onset were faithful to previously described data [26] [27] [28] . our analysis reveals that many patients, despite apparent clinical recovery at discharge, had clinically relevant medical problems when evaluated after approximately 3 to 4 weeks. for example, one third of them complained of dyspnoea, and 22.2% had a rr >20 breaths/min. radiological signs of interstitial pneumonia have been described in covid-19 [29] . whether these alterations will persist remains to be established. indeed, viral eradication does not preclude progression to parenchymal fibrosis, and data on pulmonary function after clinical recovery are urgently needed. uncontrolled htn was also highly prevalent in our cohort. this is consistent with the hypothesis that sars-cov-2 infection may be associated with chronic cardiovascular damage [30] , and highlights the need of cardiovascular care in the management of covid-19 patients. as high as 68.3% of hospitalised patients were malnourished or at risk of malnutrition, as were 51.0% of patients managed at home. malnutrition has been reported in hospitalised covid-19 patients [31] , and is likely due to systemic inflammation-related hypercatabolism [32] . ards survivors lose lean body mass during acute illness but gain fat mass in the first year after recovery, which may adversely affect functional outcomes [33] . nutritional assessment and counselling are crucial to these patients. the finding that even patients managed at home were at risk of malnutrition is novel and warrants further investigation. gastrointestinal symptoms [34] and smell and taste disturbances [35] associated with sars-cov-2 infection are possible mechanisms underlying this phenomenon. we observed cognitive impairment in a quarter of our patients, despite no history of cognitive disorder. cognitive sequelae of covid-19 might be due to direct viral pathogenicity or immune-mediated mechanisms [8] . in line with a previous study [36] , 22.2% of patients developed ptsd. independent predictors were female gender, in agreement with the prevalence of the disorder in the general population [37] , and hospitalisation, which had a protective effect. this might be due to psychosocial stressors such as lockdown and isolation at home, secluded from caregivers, and to a higher vulnerability to inflammation-induced mood and behavioural changes in women [38] . covid-19 follow-up cannot be separated from an accurate cognitive and psychological monitoring [39] . to set up a follow-up outpatient clinic in times of emergency may be arduous. apart from logistic difficulties, careful monitoring programs are energy-and time-consuming, and selection of patients who most likely benefit from follow-up programmes may be necessary. we found that older age is a strong predictor of the need of follow-up in both patients who were hospitalised and those who were discharged from the ed. through recursive partitioning analysis, we identified a hierarchy of independent predictors able to estimate the odds of requiring follow-up after covid-19. accordingly, within the entire patient cohort, in addition to older age, lower pao 2 /fio 2 values at ed presentation and obesity discriminated patients not to be lost at hospital discharge. among hospitalised patients, priority should be given to patients older than 63 years, or to younger patients receiving niv or with a history of dm, the latter being a known predictor of severity in viral infections, including covid-19 [40] . in line with our results, age emerged as being an independent predictor of ards development in a previous report on severe acute respiratory syndrome (sars) patients [41] . likewise, metabolic syndrome-related conditions including obesity and diabetes were found to increase the risk of developing severe illness in patients with middle east respiratory syndrome coronavirus (mers-cov) infection [42, 43] . although proving causality may be challenging, our findings reinforce the hypothesis that systemic metabolic derangement may precipitate coronavirus diseases, owing to the need of post-recovery monitoring. potential mechanisms may include endothelial dysfunction, the proinflammatory state, as well as the dysfunctional innate immune response common to both metabolic and viral disorders [43] [44] [45] [46] . a main limitation of our study is that instrumental exams were not included in patient monitoring. nevertheless, clinical measures may be informative surrogates in times of crisis. our follow-up covid-19 outpatient clinic was recently upgraded by adding spirometry, electrocardiography, and lung ultrasound in routine evaluations. patients will be subsequently evaluated at 3 and 6 months from hospital discharge [47] . another potential limitation is the lack of external validation of our regression tree models. on the other hand, the inclusion of a well characterized population monitored using uniform standards of care, and with the same healthcare access, minimizes the risk of ascertainment bias. although information on treatment received during the acute phase was not available for all covid-19 survivors, treatments in the outpatient setting were quite homogenous, whereas in the inpatient setting treatments other than those specifically used for covid-19 were driven by illness severity, which in our analysis was accounted for by including variables such as administration of non-invasive ventilation, length of stay, and transfer to intensive care unit. our study suggests that covid-19 may leave behind physical and psychological dysfunctions, whose underestimation may be costly in terms of long-term morbidity and mortality. multidisciplinary follow-up of these patients is therefore crucial to avoid a second wave of late health problems associated with this pandemic. in this sense, selected patient subgroups should be prioritised. supporting information s1 dataset. the dataset employed for this manuscript. 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humans, saudi arabia prevalence of comorbidities in the middle east respiratory syndrome coronavirus (mers-cov): a systematic review and meta-analysis microvascular covid-19 lung vessels obstructive thromboinflammatory syndrome (microclots): an atypical acute respiratory distress syndrome working hypothesis is diabetes a risk factor for a severe clinical presentation of dengue?-review and meta-analysis connecting type 1 and type 2 diabetes through innate immunity. cold spring harb perspect med post-covid-19 follow-up clinic: depicting chronicity of a new disease the authors wish to thank the covid19 key: cord-268524-lr51ubz5 authors: droit-volet, sylvie; gil, sandrine; martinelli, natalia; andant, nicolas; clinchamps, maélys; parreira, lénise; rouffiac, karine; dambrun, michael; huguet, pascal; dubuis, benoît; pereira, bruno; bouillon, jean-baptiste; dutheil, frédéric title: time and covid-19 stress in the lockdown situation: time free, «dying» of boredom and sadness date: 2020-08-10 journal: plos one doi: 10.1371/journal.pone.0236465 sha: doc_id: 268524 cord_uid: lr51ubz5 a lockdown of people has been used as an efficient public health measure to fight against the exponential spread of the coronavirus disease (covid-19) and allows the health system to manage the number of patients. the aim of this study (clinicaltrials.gov nct 0430818) was to evaluate the impact of both perceived stress aroused by covid-19 and of emotions triggered by the lockdown situation on the individual experience of time. a large sample of the french population responded to a survey on their experience of the passage of time during the lockdown compared to before the lockdown. the perceived stress resulting from covid-19 and stress at work and home were also assessed, as were the emotions felt. the results showed that people have experienced a slowing down of time during the lockdown. this time experience was not explained by the levels of perceived stress or anxiety, although these were considerable, but rather by the increase in boredom and sadness felt in the lockdown situation. the increased anger and fear of death only explained a small part of variance in the time judgment. the conscious experience of time therefore reflected the psychological difficulties experienced during lockdown and was not related to their perceived level of stress or anxiety. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 in 2020, faced with a virus that is uncontrollable because of its unknown [1] and virulent nature (sars-cov-2), the governments of different countries of the european union, as well as of the whole world, found themselves obliged to impose a lockdown on their citizens. this unprecedented public measure is thought to allow the health system to manage the number of patients in hospital and ensure that they receive proper care in the context of the covid-19 outbreak. in france, confinement was officially imposed in the month of march (on march 17 th at 12:00 noon). this lockdown, which requires a large number of people to stay at home, thus depriving them of their liberty, is a situation never previously encountered and its psychological consequences in the short and medium term are not yet known. researchers into time perception can nevertheless easily imagine that this life in lockdown completely changes individuals' relationship to time, i.e. their experience of time. however, to our knowledge, no studies have as yet investigated this question. very recent scale surveys or survey projects on covid-19 conducted all around the word (e.g., china, korea, iran and united kingdom) suggest that the lockdown situation generates new or heightened emotional states in the form of an increase in psychological distress [2] [3] [4] [5] [6] . nonetheless, in the different distress scales used, the different dimensions of emotion (valence and arousal) were not dissociated, and no survey has examined their relationships to time experience, even though emotion and the experience of time are known to be intrinsically linked. the aim of the present study was thus to conduct a scale survey on a large sample of an as yet untested population-french people-in order to assess not only the perceived stress related to covid-19 but also the emotions (happiness, boredom, arousal) felt during as compared to before the lockdown and their links to the subjective experience of time. the experience of time corresponds to one's feeling about time, i.e., the conscious judgment of the speed of the passage of time [7, 8] . this has received relatively little attention by researchers in the field when compared to research into individuals' abilities to perceive short durations (< 1 minute). this is probably due to the challenge of objectively examining just what makes up the experience of each individual, and therefore the role of higher-level cognitive mechanisms (e.g., consciousness, memory, self-awareness) [9] [10] [11] . indeed, the judgment of the passage of time can be seen as a mirror of the subjective experience of one's internal state [12] [13] [14] . for example, contrary to the generally held belief that time seems to pass faster as we get older, some studies have demonstrated that the feeling of the passage of time in the immediate moment is not directly related to age (young adult vs. older adult), but to people's subjective emotional experience and lived activities [10, 15, 16] . the passage of time is in fact a sensitive index of emotional experience felt in the present moment and of its variations as a function of life conditions. it is thus important to investigate individuals' judgments about how fast time seems to pass in the exceptional situation of lockdown and the factors explaining these. from a general standpoint, the literature provides evidence of the role of emotional experience as a critical factor in the experience of time. nevertheless, the famous expression "time flies when you feel good; time drags when you feel bad" is not straightforward to explain, as negative feelings are diverse and may involve varying mechanisms. more precisely, the emotional experience can be divided into two fundamental dimensions, valence (pleasure vs. displeasure) and activation (calmness vs. excitement/alertness) [17, 18] . these two dimensions interact in the characterization of any given emotion. for example, while the emotions of sadness and fear are both negative, the former is weakly activating (or even deactivating) while the latter is strongly activating. accordingly, the level of felt arousal has been shown to be a prominent factor in temporal mechanisms: the more individuals report being in a state of arousal, the faster time is reported to pass. several studies have shown a lengthening of estimates of short temporal intervals in situations of acute stress, for example when participants are faced with unpleasant stimuli [19] [20] [21] or when they imminently expect a very unpleasant event, e.g., electric shock [22, 23] . however, few studies have examined the effect of chronic stress on time judgments, such as that experienced by people with the covid-19 virus or subjected to lockdown. in the context of chronic stress, i.e. when stress is extended over several days or weeks as in the case of hospital nurses, cocenas-silva et al. [24] showed that duration judgments were no longer altered by physiological stress as measured by physiological markers, but rather by subjective psychological stress as assessed by a self-reported scale. in addition, one can assume that different mechanisms are at work in the case of an emotion, such as fear (an immediate and ephemeral negative state directed towards a specific event), compared to a more diffuse affective state, like anxiety or perceived stress (a prolonged negative state whose origin is not necessarily identified) [25] . the covid-19 pandemic, i.e., the risk that you or your loved ones will be affected by the disease as well as uncertainty about this disease, could produce chronic stress that has consequences for mental and physical health. it is well known that chronic stress affects the immune system, suppressing protective and increasing pathological immune responses [26] . there is thus a risk in this period of pandemic that the chronic stress related to covid-19 and its corollaries (anxiety, fear of death) are particularly high and therefore impact the subjective experience of time by speeding up the perceived passage of time. consequently, we hypothesized a significant relationship between stress and time experience during the lockdown imposed by the covid-19 pandemic. furthermore, in this covid-19 period, it is critical to consider not only the disease-related perceived stress but also the consequences for life of being locked down at home, as well as the direct and indirect effects on daily psychological and social functioning. as a recent survey highlighted, confining people increases their sense of boredom [2] . boredom corresponds to "the aversive state of wanting, but being unable, to engage in satisfying activity" and involves, in particular, low arousal, negative affects [27, p 483 ]. in particular, some studies have shown that boredom produces a feeling of the slowing down of time rather than a speeding up [14, 28] . an alternative hypothesis was thus that boredom would prevail over stress in the experience of time. since boredom is associated with negative emotion of low level of arousal, we thus expected participants to experience of slowing down of time with the boredom experienced during the lockdown. it was not possible a priori to identify which hypothesis would be valid, i.e., which are the factors related to and influencing the experience of time in a lockdown situation, the perceived stress in the stressful situation of covid-19 and/or-by contrast-other affective states characterized by a decrease in arousal such as boredom. indeed, on one hand, the fear and distress generated by the morbid nature of the crisis and its repercussions (fear for one's health and for that of one's family and friends) or by inappropriate housing quality (stress at home) or working conditions (job stress) could increase people's sense of alertness, and therefore lead to a speeding of the passage of time. on the other, confinement at home and social distancing could result in an increased sense of sadness (i.e., less happiness) and boredom, and thus in the feeling that the passage of time slows down. here, a large sample of french people were asked to answer a scale survey during the lockdown period. this consisted of a series of questions, i.e., demographic questions but also questions on the stress perceived (covid-19 stress, home stress, job stress, anxiety), the emotions (happiness, arousal, boredom) felt during compared to before the lockdown and the experience of time. the participants were asked to assess their experience of the passage of time according to three periods of the lockdown: in the immediate moment, during the day, during the last week, as well as before the lockdown for comparison purposes. the sample consisted of 4364 french participants, 3436 women and 928 men (mean age = 41.5, sd = 12.81, min = 16, maxi = 89, n 16-17 years = 11). the participants completed the questionnaire at home (72.5%) or at work (27.5%). the study was reviewed and approved by the human ethics committees sud est vi, france (clinicaltrials.gov nct 04308187). all participants were volunteers and were informed of the objective of the survey and that their data would be processed anonymously and be used for research purposes. the ethics committee waived the need for written consent considering that if people respond to the questionnaires by going to the website, they are giving their consent. furthermore, they can withdraw it at any time. the few minors who completed the questionnaire did so with the consent of their parents who sent them the survey. the responses to the demographic questions allowed us to characterize the surveyed population. 71.8% of participants were married or equivalent (civil partner, etc.) and 27.2% were single (1% other). their distribution as a function of education level was: 1.5% certificate of general education, 21.9% high school vocational certificate, 0% high school diploma, 40.6% bachelor's degree, 24.5% master's degree and 11% doctoral degree. the percentage of participants per professional category was: jobseekers: 4.4%; students: 6.2%; farmers: 0.3%; craftsmen/shopkeepers/business executives: 5.7%; white-collar workers: 30%; manual workers: 8.9%; intermediate professions, 35.7%; retired: 6.3% (2.5% no response). we implemented an open epidemiological, observational, descriptive study by administering a self-reported questionnaire proposed to volunteers using redcap 1 software available through the covistress.org website. the redcap 1 questionnaire was hosted by the university hospital of clermont-ferrand. the questions analyzed in this manuscript were therefore specific questions included in a large questionnaire composed of different thematic sections of questions (s1 questions). the thematic sections were presented in random order after the demographic questions. the online questionnaire was distributed several times through mailing lists held by institutions and french social groups. there were no exclusion criteria. the data that we analyzed were obtained for the period of lockdown from march 31 th to april 12 th , 2020, whereas the french lockdown was ordered on march 17 th at 12:00 noon. the time taken to complete the survey lasted between 5 and 20 minutes on average, depending on sub-items. for the main outcomes, we used a visual analog scale (vas), i.e., a non-calibrated line of 100 mm, ranging from 0 to 100 [29, 30] . the subjective experience of time was thus assessed using this vas, which went from very slowly (0) to very fast (100). the question was "what are your feelings about the speed of the passage of time". there were four time questions, one for the passage of time before the lockdown, and three for during the lockdown: now, for the day, and for the week. the stress resulting from covid-19 as well as job stress and home stress, health-related and financial concerns and anxiety were assessed using the same vas. the emotional dimensions tested were also assessed with the vas for the period before the lockdown and during the lockdown (now): fear of death (not at all vs. at lot), arousal (calm vs. excited), happiness (sad vs. happy), anger (peaceful vs. angry), boredom (occupied vs. bored). the quality of sleep and level of fatigue were also examined in the survey using the vas. as explained above, these different questions were presented in different thematic sections presented in a random order (s1 questions). we performed analyses of variance on the subjective experience of time. we also examined correlations and ran a linear regression model on all the measures of interest by using the standardized data. we used the variance inflation factor (vif) to examine the multicollinearity in the regression analysis [31] . finally, to examine the results of the linear regression model in more detail, we also performed an analysis of mediation. the analyses were performed with spss and the bonferroni correction was systematically applied when necessary. a preliminary analysis of variance performed on the subjective experience of time showed a marked difference between the experience of time before and during the lockdown (fig 1) . that time passed faster when a longer period of time was considered, i.e., a week compared to a day or the present moment (bonferroni comparisons, p < 0.01). to simplify the results, the subsequent statistical analyses are based on the difference in time ratings for the question on the period before the lockdown and that for the present moment (during the lockdown). indeed, the meaning of temporal judgment during the lockdown is relative to that before the lockdown. in addition, the results were similar when the analyses were only performed on the ratings for the present moment. a positive value of our temporal difference index therefore indicates that the individuals experience a slowing down of time during the lockdown, a negative value a speeding up of time and a null value no difference. the anova performed on this temporal difference index, with level of education, professional category and whether the individuals were at work or home as factors, did not show any significant effect (all f < 1). there was indeed no significant difference in time experience before the lockdown situation as a function of these factors. only a small effect of professional category was observed in the present time judgment during the lockdown, f the anova on the temporal index with sex and marital status (single vs. not single) as factors showed a significant main effect of sex, f(1, 4084) = 14.77, p < 0.001, η 2 p = .004, and status, f(1, 4084) = 11.74, p < 0.001, η 2 p = .003, with no sex x status interaction (p > 0.10). this suggests that the single people in our sample tended to experience a greater difference in the flow of time during the lockdown when compared to before (29.19 vs. 24.19) . indeed, in the lockdown situation, time in the present was judged to pass slower by the single people (m = 47.12, sd = 30.12) than by the others (m = 53.93, sd = 29.15). the women also tended to feel a greater slowing down of time than the men (29.41 vs. 23.89) during as compared to before the lockdown, but time passed faster for the women than for the men before the lockdown (80.51 vs. 74.37), f(1, 4084) = 71.11, p < 0.001, η 2 p = .02. nevertheless, their responses to the stress questions indicated that they tended to be more stressed than the men, even though the sex difference only explained a very small proportion of variance ( table 1 shows the correlation matrix (s1 table) between the subjective experience of time (difference in the judgment of the passage of time between before the lockdown and the present moment, i.e., during the lockdown) and the different tested factors. an examination of table 1 reveals that several dimensions were associated with the slowing down of time during as compared to before the lockdown. with regard to stress, the participants experienced that time passed slower-rather than faster-with an increase in the level of perceived stress, i.e., the perceived stress related to covid-19 (r = .18) as well as the stress at home (r = .23) and at work (r = .08). a slowing down of time was therefore observed as the stress level increased. this deceleration of subjective time was observed even if the stress value reported on the vas was high, and higher for covid-19-related stress than for home and job stress (covid-19 stress, m = 61.50, sd = 28.87; job stress, m = 57.94, sd = 32.65; home stress, m = 46.97, sd = 32.65, f(2, 7466) = 342.78, p < 0.001, η 2 p = .08 (all bonferroni tests, p < 0.001). the rating for each type of stress was indeed significantly different from zero (t(4196) = 138.18, t(4184) = 93.06, t(3892) = 10.13, respectively, all p < 0.001). finally, the stress resulting from covid-19 was more closely associated with anxiety (r = .75, p < 0.001), the fear of death (r = -.42, p < 0.001) than it was with the experienced time per se. inconsistently with our first hypothesis, the level of correlation between the experience of time and covid-19-related stress was therefore very low, and this was also the case for stress in the other contexts (home, work). as suggests table 1 , the experience of time was more correlated with boredom (r = -.48, p < 0.001) and decreased happiness (r = .39, p < .0001) than with the level of perceived stress. therefore, the participants experienced a slowing down of time as boredom increased and happiness decreased during the lockdown. as the time judgment was significantly correlated with several dimensions, to identify the best predictor of the subjective experience of time we performed a regression analysis on the time judgments with the different significant dimensions entered into the same model ( table 2 ). the examination of multicollinearity in the regression analysis using the vif indicated no problematic presence of multicollinearity (all vif < 3) [31] . the results of this regression analysis indicated that the perceived stress resulting from covid-19 and its spread was not a table 1 . correlations between the passage of time (difference between before the lockdown and for the present, i.e., during the lockdown) and the different tested factors (z-scores). participants were in the lockdown situation, the more they experienced a slowing down of time. indeed, time was experienced as passing increasingly slowly in the present moment compared to before the lockdown as the level of boredom rose (fig 2) . it also seemed to slow down as happiness decreased, i.e., as sadness increased (fig 3) . increasing boredom and decreasing happiness were therefore the two main predictors of the experience of the passage of time during the lockdown. since these two dimensions are related, we conducted statistical analyses to estimate whether the boredom mediated the effect of emotion on the experience of time and, conversely, whether emotion mediated the effect of the boredom of the experience of time. the mediation analyses indicated that boredom contributes to explaining the effect of emotion on the experience of the passage of time, with a significant indirect effect of 0.159 (β), se = .01, 95% ci (.138; .1812), z = 14.7, p < 0.001, 34.4% of mediation) (fig 4) . however, the direct effect of emotion (sadness) on the time experience remained significant (β = . the results of our survey showed that the stress felt by a broad cross-section of the french population during the lockdown was high, in particular with regard to stress relating to the covid-19 pandemic, as is indicated by the rating of 61.50 (+/-28.87) on a 100-mm vas. the level of perceived stress linked to covid-19 was even higher than the stress at work and at home. covid-19 stress was, in fact, related to the participants' anxiety and their fear of death. the more anxious and frightened they were about death, the more stressed they were in the face of this disease. these results are entirely consistent with the initial results of surveys on covid-19 conducted, in particular, in china [5, 6] and iran [4] , which have shown an increase in psychological distress as a result of the covid-19 pandemic. however, as reported by qui et al. [5] , it is noteworthy that people's distress does not reach a pathological level (m = 23.65), with only 5% of the population suffering from severe distress and 29% from mild or moderate distress. in addition, the proportion of individuals presenting psychological distress disorders before the covid-19 is unknown. however, the chinese suffer less psychological distress and have greater life satisfaction when working in the office than at home, whereas the opposite seems to be the case in the french population, as suggested by the significantly lower level of stress at home than at work. this suggests that there are some differences in culture or living conditions between people in different countries with regard to stress management in similar social isolation situations. the originality of our results is to show that, although the level of stress was quite high, it had little impact on the current subjective experience of time. indeed, the participants did not feel a speeding up of time related to the increase in their stress level. this is contrary to the results of studies on timing which have described a lengthening of duration estimates and the experience of a faster passage of time when the levels of stress and anxiety are high [21, 32, 33] . however, these findings were obtained in intense and concisely emotional situations, when the subjects were faced or expecting a forthcoming threatening event, or in individuals with high-anxiety traits. in the situation of lockdown at home, the current level of stress was therefore not high enough to affect the sense of time. indeed, the level of arousal remained low, although it increased slightly between the period before and during the lockdown. to conclude, one might nevertheless think that it would have been more convincing to record the physiological markers of stress. however, this was not possible in the lockdown situation which was rapidly decided on by the public authorities [34, 35] . in addition, cocenas et al. [24] recently showed that perceived stress was a better predictor of changes in time estimates than physiological stress per se in the case of prolonged stressful situations, for example in the case of hospital nurses at work. in addition, the likelihood of encountering a series of intensely stressful events may be reduced in the present isolation situation. family life involving the care of children can obviously be a source of stress. our study did indeed indicate that women were more stressed at home than men, but were even more so when they were single than part of a family, and that the number of children only slightly increased the stress level at home (r = .08, p < 0.001). rather than covid-19-related stress or home and job stress, our study showed that it was the emotional experience of everyday life during the lockdown that influenced the sense of time. indeed, the participants clearly reported experiencing a slowing down of the passage of time during in comparison to before the lockdown. and the most reliable predictors of this slowing down were the feelings of boredom and sadness. our results are consistent with those of recent studies on time judgments that have pointed out the critical role of emotion in human beings' sense of time [for a review 35] and of boredom [14, 28, 36] . these studies have indeed found a slowing down of time as both sadness and boredom increase. in line with theoretical models of boredom [27] , the present study found that the degree of boredom experienced was related not only to arousal but mostly to negative emotional experience: the more bored people were in lockdown, the sadder they were. the boredom is known to be linked to depression [37, 38] , and depressed people feel a slowing down of time [39] . consequently, the experience of boredom in the lockdown and the judgment of a slower passage of time have increased sadness and could lead to pathological depression. however, in the lockdown situation, the level of boredom explained a proportion, but not all, of the effect of sadness on the experience of the passage of time. other factors that we need to examine in a future study could also help to explain sadness and time experience in the lockdown, such as social withdrawal. the changes in the sense of time in lockdown were therefore due to the significant increase in both boredom and sadness. the literature on boredom suggests that it is involved in a multitude of behaviors and psychological dimensions and that it has a negative side, as in the sadness observed in our study, as well as a positive side. indeed, trait boredom is associated with psychological difficulties (e.g., drug abuse, depression, anxiety, binge eating) [40, 41] . however, some recent functional approaches have also suggested that boredom constitutes a key signal to change behavior by orientating humans to try to find a more satisfying situation [42] . in the context of lockdown, one may therefore wonder what influence this feeling of boredom has on the development of pro-social behaviors or on compliance with the containment situation in the short or longer term (does it only result in bad things or also in good things?). in the lockdown situation, people may have more time. however, they "die" of boredom and sadness and time slows down, drags on. the sense of the passage of time is, ultimately, a phenomenological time that is closely related to the self and the sense of existence [13] . as stated by jean-paul sartre, human beings are defined by their acts and their effects on others. however, when they have more time but are isolated and cannot act-they have nothing to do-they are overwhelmed by sadness and boredom. it would seem important for future surveys to examine whether this feeling is valid in all cultures and for all people. it also seems to be important to identify whether other factors specific to individual characteristics or living conditions, to representations/beliefs toward covid-19 or government policies contribute to changes in the sense of time in the lockdown situation. some authors nevertheless defend the benefits of boredom. however, this raises the question of individual abilities to cope with the feeling of boredom in industrial societies. individual differences in coping with boredom can potentially predict psychological difficulties, health problems and increased vulnerability to psychopathologies such as depression [43] . it is thus a serious problem and one which has to be taken into account. in conclusion, the changes in the sense of time in the lockdown situation, imposed as an efficient solution to the covid-19 pandemic, reflect the major psychological difficulties that people are experiencing during the lockdown. 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measures the covistress network is headed by pr. frédéric dutheil (frederic.dutheil@uca.fr) chu key: cord-270408-4qqyb8sd authors: pane, masdalina; imari, sholah; alwi, qomariah; nyoman kandun, i; cook, alex r.; samaan, gina title: causes of mortality for indonesian hajj pilgrims: comparison between routine death certificate and verbal autopsy findings date: 2013-08-21 journal: plos one doi: 10.1371/journal.pone.0073243 sha: doc_id: 270408 cord_uid: 4qqyb8sd background: indonesia provides the largest single source of pilgrims for the hajj (10%). in the last two decades, mortality rates for indonesian pilgrims ranged between 200–380 deaths per 100,000 pilgrims over the 10-week hajj period. reasons for high mortality are not well understood. in 2008, verbal autopsy was introduced to complement routine death certificates to explore cause of death diagnoses. this study presents the patterns and causes of death for indonesian pilgrims, and compares routine death certificates to verbal autopsy findings. methods: public health surveillance was conducted by indonesian public health authorities accompanying pilgrims to saudi arabia, with daily reporting of hospitalizations and deaths. surveillance data from 2008 were analyzed for timing, geographic location and site of death. percentages for each cause of death category from death certificates were compared to that from verbal autopsy. results: in 2008, 206,831 indonesian undertook the hajj. there were 446 deaths, equivalent to 1,968 deaths per 100,000 pilgrim years. most pilgrims died in mecca (68%) and medinah (24%). there was no statistically discernible difference in the total mortality risk for the two pilgrimage routes (mecca or medinah first), but the number of deaths peaked earlier for those traveling to mecca first (p=0.002). most deaths were due to cardiovascular (66%) and respiratory (28%) diseases. a greater proportion of deaths were attributed to cardiovascular disease by death certificate compared to the verbal autopsy method (p<0.001). significantly more deaths had ill-defined cause based on verbal autopsy method (p<0.001). conclusions: despite pre-departure health screening and other medical services, indonesian pilgrim mortality rates were very high. correct classification of cause of death is critical for the development of risk mitigation strategies. since verbal autopsy classified causes of death differently to death certificates, further studies are needed to assess the method’s utility in this setting. each year, muslims from all over the world undertake the hajj pilgrimage to and in saudi arabia [1] . in recent years, over 2 million people from 140 countries undertook the hajj annually, including over 200,000 people from indonesia, the world's most populous muslim majority country. performance of the hajj and its rites is physically demanding [2] . extreme physical stressors such as sun exposure, heat (37°c during the day and 20°c at night), thirst, crowding, steep inclines and traffic congestions over a prolonged period of time (40 days per pilgrim over the 70-day hajj season) increase health risks. since pilgrims tend to be older and many have medical comorbidities [3] , these factors exacerbate existing risk for ischemic and congestive cardiovascular disease, fluid and electrolyte abnormalities, and respiratory and other infectious diseases including emerging diseases such as middle east respiratory syndrome coronavirus [4] . in the last two decades, the mortality rate of indonesian pilgrims, excluding years in which disasters such as stampedes occurred, fluctuated between 200-380 deaths per 100,000 persons during the ten-week hajj period [5] . few countries have published pilgrim mortality rates, but compared to where they are available, the indonesian rate is much higher [6] . for example, in 1998, the hajj mortality rate amongst isfahani pilgrims from iran was 13 per 100,000 pilgrimages [7] . in 2004, the mortality rate for all iranian pilgrims was 47 per 100,000 pilgrimages, and in 2005, 24 per 100,000 [6] . even compared to the yearly mortality rate in indonesia, the mortality rate for hajj pilgrims ranged between 1,765 and 3,353 per 100,000 per year; by comparison, the indonesian estimated national crude death rate was 700 per 100,000 in 2003 [8] . since 1950, indonesian authorities have provided medical services to hajj pilgrims. pre-departure health screening and vaccination as well as temporary medical clinics staffed by indonesian doctors in saudi arabia were introduced progressively. mortality surveillance was also established to reduce mortality and aid in administrative processes for life insurance claims. prior to 2008, the patterns, causes of death and factors associated with mortality were not well understood for indonesian pilgrims. data collected were limited to basic demographic characteristics and general cause of death. cause of death could only be obtained from the hospital death certificate or the flight doctor's records if a death occurred in the community. these were based on clinical examination and any available laboratory tests as documented in the patient medical record, but they lacked detailed information about the cause of death or the patient's pre-existing conditions. given the hugely elevated mortality risk relative to the general indonesian population, information on cause of death and underlying health conditions is critical to enhance pilgrim health management and to prevent excess mortality. in 2008, the ministry of health introduced an additional surveillance tool -verbal autopsy -to better understand the causes of pilgrim mortality. verbal autopsy allows for the systematic investigation of probable causes of death through structured questionnaires [9] . the world health organization (who) advocates the use of verbal autopsy in situations where only a fraction of deaths occur in hospitals or in absence of vital registration systems [10] . previous studies have shown that verbal autopsy improves diagnosis of cause of mortality and the method continues to be used in various countries [11] [12] [13] . this study describes the findings from the mortality surveillance conducted during the hajj in 2008; the year in which verbal autopsy was introduced. we explore both personspecific and site-specific factors associated with mortality, and compare the cause of death from the routine death certificate to the newly introduced verbal autopsy method. ethics approval for the study was obtained from the ministry of health national institute of health research and development ethics review committee with approval number lb.03.04/ke/4687/2008. written consent was not obtained from the patients involved, but this was waived by the ethics review committee as mortality data were analysed anonymously. in 2008, 206,831 indonesians undertook pilgrimage during hajj. the majority were from java (58%) and sumatra (24%) islands, with small proportions from kalimantan (6%), sulawesi (4%) and other (8%) islands. the majority of pilgrims, anecdotally reported to be 95%, joined one of the governmentsponsored hajj pilgrimage travel services that include 40 days of travel. the remainder joined private more expensive pilgrimage travel services that are of shorter duration (25 days) and provide better accommodation and services. a number of preventive and curative healthcare services were available to all hajj pilgrims prior to and during their travels in saudi arabia. pre-departure, each indonesian pilgrim was required to visit a government healthcare facility for a medical check-up and to receive a pocketbook outlining their health conditions, medications and vaccination status. pilgrims were mandated to receive meningococcal vaccine and were advised to receive influenza vaccine before their departure. flights were chartered by the indonesian government to accommodate 300-450 pilgrims. each flight had one doctor and two nurses to accompany the pilgrims. these healthcare workers treated, triaged and conducted health surveillance for pilgrims. hospitalizations and incidental reporting of any deaths occurring outside a healthcare facility were notified daily to the indonesian public health team based in saudi arabia during the hajj. saudi authorities made first aid posts, health centres and hospitals available. in mecca, six hospitals were set up during pilgrimage, while in medinah, pilgrims could access established hospitals. in addition to the saudi facilities, indonesian health authorities set up field hospitals in mecca and medinah specifically for indonesian pilgrims. indonesia also sent 306 specialist doctors including internists, pulmonologists, cardiologists and psychiatrists, public health workers, nurses, pharmacists and sanitarians to support the pilgrimage. all deaths were reported from the flight doctor or the saudi hospital to the indonesian public health team located in saudi arabia during the hajj. mortality data collected by the indonesian public health team were entered into a database for data analysis. standard variables in the database included name, age, sex, home address, employment, flight group, time and place of death, date of arrival into saudi arabia, cause of death as per the hospital or flight doctor death certificate. in 2008, the indonesian ministry of health mandated a verbal autopsy form to be filled out by the flight doctors accompanying pilgrims. the verbal autopsy form was developed based on the standards established by who and adapted to hajj pilgrimage needs [14] . the verbal autopsy form obtained detailed information about medical history, signs and symptoms, and other circumstances regarding the death from family or friends who travelled with the deceased. in most cases, interviews were conducted with a combination of the treating physician, the deceased person's spouse or pilgrims in the same flight group. the form aimed to increase the specificity of the cause of death by elucidating medical history and recent events. the flight doctors were trained in administering the verbal autopsy form prior to departure from indonesia and were required to complete it within the week of a pilgrim's death. once the form was completed, the indonesian public health team stationed in saudi arabia sent it to the ministry of health in indonesia for analysis and determination of cause of death. the form was analysed separately by two trained staff at the ministry of health to determine the cause of death. if discordant, the staff compared their analyses to achieve consensus. the cause of death based on this verbal autopsy method was then recorded in the database and compared to that reported by the hospital or flight doctor death certificate. we used counts and proportions to describe demographic characteristics of indonesian pilgrims and fatalities. the chi-square test and chi-square test for trend were used for the analysis of categorical data. to compare the two methods for establishing cause of death (verbal autopsy and death certificate), we compared the percentages for each cause of death category using mcnemar's test. all 206,831 indonesian pilgrims were aged 18 years or more, where the majority (59%) were aged between 41 and 59 years ( table 1) . most pilgrims were female (55%, table 1 ). according to the pre-embarkation medical assessment, 28% of pilgrims were classified as high risk due to underlying health conditions such as diabetes, hypertension, other chronic diseases or if they were 60 years or older. pilgrims in 2008, 446 indonesian pilgrims died during the hajj in saudi arabia. the overall mortality rate was 216 deaths per 100,000 pilgrimages. mortality rates were highest in those ≥60 years of age (722 per 100,000 pilgrims), and rates significantly increased with increasing age (p<0.01, table 2 ). mortality rates were higher in males (296 deaths per 100,000 pilgrims) compared to females (150 deaths per 100,000 pilgrims, p<0.01, table 2 ). for the 4 deaths in 18-40 year old age-group, 2 were male and 2 were female. according to the death certificates, three died due to cardiac arrest and one due to asphyxia in a patient with active tuberculosis. most deaths occurred in mecca (n=305, 68%), followed by medinah (n=106, 24%) and jeddah (n=35, 8%). the majority (57%) of deaths occurred in hospital, but a large proportion of deaths also occurred in pilgrims' apartments/sleeping areas (36%). most (77%) deaths occurred during pilgrims' active hours between 5am and 9pm: mortality rates were higher in the afternoon (possibly due to the heat) and early morning ( figure 2a ). weekly mortality rates increased in week 6, exceeding the expected crude mortality rate (cmr) of 5 per 100,000 per day [15] , and remained high until the end of the 10-week hajj period (figure 2b ). in week 8, the number of deaths started to decrease. however, since the overall number of pilgrims also decreased, the mortality rates remained very high since some of the indonesian pilgrims who were hospitalized died in the later weeks of the hajj. for pilgrims on route 1, the number of deaths increased sharply four weeks after arrival into saudi arabia by which time the pilgrims had already reached mecca (figure 2b ). for pilgrims on route 2, the number of deaths peaked in the third week after their arrival into saudi arabia, at which stage the pilgrims were in mecca (figure 2c ). the number of deaths remained large thereafter until the end of the hajj for those undertaking route 2. there was no statistical difference in the number of deaths occurring for pilgrims undertaking route 1 (200 out of 93,357 pilgrims) compared to those on route 2 (231 out of 113,474, p=0.72). however, the trends from week to week during the hajj period were significantly different between pilgrims on route 1 compared to those on route 2 (χ 2 for trend=9.23, p=0.002). most deaths were due to cardiovascular diseases and respiratory diseases (table 3) . a greater proportion of deaths were attributed to cardiovascular disease by the flight doctor or hospital death certificate (66%) compared to the cause of death ascertained using the verbal autopsy method (49%, p<0.001). significantly more deaths had unspecified cause based on the verbal autopsy method (10%, versus 0, p<0.001). as part of the demographic data collected for each pilgrim, height and body mass were recorded. based on the hospital/flight doctor death certificate, 38 of the 446 (9%) pilgrims who died had body mass index ≥27.5 indicating obesity for asian body types [16] . of these, 26 died due to cardiovascular disturbances, 7 due to respiratory illness, 2 due to metabolic disturbances, 2 had undefined sudden death and 1 due to trauma (neck fracture). this study describes the demographics, patterns and causes of mortality in indonesian pilgrims in 2008. nearly one third of pilgrims undertaking the hajj in 2008 were considered high risk due to underlying health conditions or their age. mortality rates were found to be greatest in males and in those aged ≥60 years, in whom most deaths were attributed to cardiovascular and respiratory diseases. studies from other countries with pilgrims found similar trends, including a preponderance in male mortality [4, 17] . these findings highlight the special characteristics of the hajj compared to other mass gathering events. many muslims wait decades for the opportunity to perform the hajj, and by the time they receive the chance, they may have a multitude of age-related health concerns [18] . correct classification of deaths is critical to target preventive interventions and provide health services [14] . this study compared cause of death according to the flight doctor and death certificate records to the newly introduced verbal autopsy method. fewer deaths were attributed to cardiovascular diseases using verbal autopsy but this method resulted in a greater number of deaths having ill-defined cause of death. verbal autopsy method may have reduced misclassification by removing pressure from clinicians having to extrapolate cause of death in situations where it may have been ill-defined or unclear. however, this hypothesis warrants further investigation. since the verbal autopsy method is dependent on the skills of the field personnel collecting the data, the timing to limit recall bias and the method is most suited to diseases with specific symptoms and presentation [10, 19] , the use of verbal autopsy for hajj mortality surveillance should be further evaluated. based on both the death certificates and verbal autopsy categories, cardiovascular disease was the leading cause of indonesian pilgrim mortality in 2008. performance of obligatory rites during the hajj constitutes stressful exercise which is not generally recommended by doctors for those with ischemic heart disease, hypertension or heart failure as such exercise may increase the risk of heart attacks [20] . this risk may be further elevated in the heat, where dehydration may lead to increase in body temperature and heart rate, and a decrease in cardiac output [21] . an iranian study showed that when patients with severe cardiovascular disease were prohibited from attending the hajj and other patients with cardiovascular disease were provided with appropriate medications and monitoring during pilgrimage, mortality rates were significantly lower than those for other pilgrims [7] . this supports the need for careful pre-departure health screening, exclusion of the severely ill, provision of appropriate drug therapies or increased physical exercise prior to departure, and monitoring during pilgrimage to reduce mortality. one-third of indonesian pilgrim mortality was attributed to respiratory diseases. pneumonia is a common illness that is life-threatening to the elderly, especially those with comorbidities such as diabetes or hypertension [4] . a number of studies have shown that pneumonia is the primary cause of critical illness during the hajj and that etiologies include gramnegative organisms, streptococcus pneumoniae, and mycobacterium tuberculosis [2, 18, 22] . for tuberculosis patients, the physical stressors of the hajj may increase the risk of severe illness and mortality, and the intense crowding during the pilgrimage may increase the risk of disease transmission. a recent study found that 10% of malaysian pilgrims had a significant increase in immune response to quantiferon tuberculosis assay antigen post-hajj compared to pre-hajj [23] . since indonesia is a high-burden country for tuberculosis [24] , pre-departure screening should continue to exclude those with active disease. other potential public health measures to reduce mortality due to respiratory diseases include increasing coverage of influenza vaccine and pneumococcal vaccine. such measures were applied by iranian public health authorities in 2005, which halved the incidence of respiratory diseases and decreased the mortality rates from 47 per 100,000 in 2004 to 24 per 100,000 in 2005 [6] . deaths amongst indonesian pilgrims traveling on route 2 who went to mecca first peaked earlier than those traveling on route 1. most deaths among indonesian pilgrims occurred in the middle-latter weeks of the hajj period during the stay in mecca and afterwards in arafah-mina. obligatory rites conducted at holy sites in mecca and arafah-mina are known to involve intense physical activity [7] . these may have been too strenuous for some pilgrims, especially older or relatively sedentary pilgrims. surprisingly, 36% of deaths occurred in the accommodation provided to pilgrims during the hajj. this highlights that despite the presence of an accompanying health team, not all patients were referred to hospital prior to critical stages of illness. one limitation of this study is that further details about deaths occurring in accommodation were not available for analysis. lack of data limited other important analyses including deaths by health risk status and type of travel service (government or private) used. the rates of mortality as well as causes of death may differ based on these categories and may impact recommendations for intervention. pattern of diseases among visitors to mina health centers during the hajj season, 1429 h (2008 g) hajj: health lessons for mass gatherings common health hazards in french pilgrims during the hajj of 2007: a prospective cohort study the epidemiology of hajj-related critical illness: lessons for deployment of temporary critical care services* comparison of mortality and morbidity rates among iranian pilgrims in hajj how to reduce cardiovascular mortality and morbidity among hajj pilgrims: a multiphasic screening, intervention and assessment verbal autopsy: current practices and challenges potential and limits of verbal autopsies factors associated with place of death in addis ababa applying verbal autopsy to determine cause of death in rural vietnam accuracy of who verbal autopsy tool in determining major causes of neonatal deaths in india verbal autopsy standards: ascertaining and attributing causes of death famine-affected, refugee, and displaced populations: recommendations for public health issues appropriate body-mass index for asian populations and its implications for policy and intervention strategies causes of admission to intensive care units in the hajj period of the islamic year 1424 clinical and temporal patterns of severe pneumonia causing critical illness during hajj a review of data-derived methods for assigning causes of death from verbal autopsy data physical activity and stroke in british middle aged men influence of graded dehydration on hyperthermia and cardiovascular drift during exercise tuberculosis is the commonest cause of pneumonia requiring hospitalization during hajj (pilgrimage to makkah) high risk of mycobacterium tuberculosis infection during the hajj pilgrimage country profile -indonesia indonesian pilgrims suffer high mortality rates despite predeparture screenings, accompanying medical teams and the availability of specialized health services during the hajj. this study highlights the importance of surveillance during the hajj to understand the health risks and strengthen the evidencebase on which policy can be developed [2] . further studies are needed to assess verbal autopsy's utility in this setting. the role of the accompanying health teams as first responders needs to be reviewed to determine how they can best reduce indonesian pilgrim mortality. an evaluation of the current mortality surveillance system is also warranted to ensure that the data collected appropriately serves the public health purpose of reducing pilgrim mortality. lastly, lessons need to be learnt from other countries including their hajj mortality patterns and risk mitigation strategies. key: cord-285546-5tjhdczt authors: green, manfred s.; peer, victoria; schwartz, naama; nitzan, dorit title: the confounded crude case-fatality rates (cfr) for covid-19 hide more than they reveal—a comparison of age-specific and age-adjusted cfrs between seven countries date: 2020-10-21 journal: plos one doi: 10.1371/journal.pone.0241031 sha: doc_id: 285546 cord_uid: 5tjhdczt background: crude case-fatality rates (cfrs) for covid-19 vary widely between countries. there are serious limitations in the cfrs when making comparisons. we examined how the age distribution of the cases is responsible for the covid-19 cfr differences between countries. methods: covid-19 cases and deaths, by ten-year age-groups, were available from the reports of seven countries. the overall and age-specific cfrs were computed for each country. the age-adjusted cfrs were computed by the direct method, using the combined number of cases in all seven countries in each age group as the standard population. a meta-analytic approach was used to obtain pooled age-specific cfrs. findings: the crude overall cfrs varied between 0.82% and 14.2% in the seven countries and the variation in the age-specific cfrs were much smaller. there was wide variation in the age distribution of the cases between countries. the ratio of the crude cfr for the country with the highest cfr to that with the lowest (6.28) was much lower for the age-adjusted cfrs rates (2.57). conclusions: the age structure of the cases explains much of differences in the crude cfrs between countries and adjusting for age substantially reduces this variation. other factors such as the definition of cases, coding of deaths and the standard of healthcare are likely to account for much of the residual variation. it is misleading to compare the crude covid-19 cfrs between countries and should be avoided. at the very least, age-specific and age-adjusted cfrs should be used for comparisons. , who declared covid-19 as a pandemic. by october, 2020, almost all countries had been affected, and globally there were reports of more than 37 million cases and more than a million deaths. early estimates indicated that the average proportion of deaths among the diagnosed cases, defined as the case-fatality rate (cfr), was around 2.3% [2] . however, subsequently, the reported crude covid-19 cfrs varied widely between countries [3, 4] . the limitations of comparing crude cfrs in general, has been evaluated previously [5] . the strong positive association between the covid-19 cfr and age has been demonstrated both in observational studies [6] and in a model-based analysis [7] , particularly over the age of 40. the interpretation of the cfr depends on the context in which it is used. in a single cohort of patients, it can indicate the severity of the disease at a single point in time. it can also be used to assess trends in the impact of changes in health care over time. in the current context of the covid-19 pandemic, cfrs are commonly compared between countries and that may lead to speculation on differences in healthcare. for example, it may be concluded that the cfrs somehow reflect the successes and failures of the different countries in the treatment of serious cases. in general a number of factors could impact on the both the numerator and denominator of the cfr. this is especially important for the cfr for covid-19. there could be misclassification of causes of death and there are likely to be variations in the definition of cases in the denominator. substantial confounding by age could impact on cfr comparisons between different groups. a related concept is the infection fatality rate (ifr) which includes asymptomatic cases in the denominator, which need to be identified by screening tests. since the ifr is rarely available for covid-19, in this paper we consider only the cfr. in this paper, we examined the contribution of the age distribution of the cases when comparing the covid-19 cfrs between seven countries, with widely varying cfrs. we studied published crude and age-specific cfrs in cohorts of cases of covid-19 in seven countries, with varying periods of follow-up. the countries chosen were based on the accessibility of the data. the first case covid-19 in israel was confirmed on 21 february 2020. the first case in south korea was announced on 20 january 2020.the covid-19 spread from hubei province, china, after december 2019.sars-cov-2 was confirmed to have reached spain, sweden, italy, and canada on end of january 2020. the data on cases and deaths by age group were available from china on february 11, 2020. for south korea cases and deaths were updated to the end of april. for spain, the cases and deaths were updated to mid-may 2020. the information for israel, italy, canada and sweden were updated during august 2020. the data for each country were not necessarily updated to the time of the study, and the cfr's may have changed over time. age-specific data on the cases and deaths by ten-year age groups (0-9, 10-19, . . ...80+), were available for seven countries: italy [8] , spain [9], sweden [10] , china [11] , s korea [12] , israel (ministry of health, personal communication) and canada [13] . the outcome variable was defined as the crude cfr defined as the number of deaths divided by the number of reported cases. the exposure variable was the individual country. age-group was considered as a confounding variable. age-adjustment was carried by the direct method, using the distribution of the combined cases of all six countries by age group as the standard population. 95% confidence intervals were computed for each age-adjusted rate using winpepi [version 11.65, aug, 2016]. open access aggregative and anonymous data were used and there was no need for ethics committee approval. the age-specific number of cases, number of deaths and the crude cfrs by country are given in table 1 . the distributions of the cases vary markedly between the countries. for example, israel and south korea are heavily weighted in the 20-39 age group, china has a more balanced distribution and italy, sweden and canada are heavily weighted in the over 70 age groups. the distributions of the cases for each country are shown in fig 1. it is clear that distributions vary widely and are not necessarily related to the age distribution of the population of the country. for example, for south korea, there is a relatively large number of cases in the age group 20-29, due to an outbreak affecting that age group in particular. the age-specific cfrs are shown in fig 2. while there are differences in the age-specific cfrs between countries, the trend of steeply increasing cfrs in the oldest age groups is evident. age groups 0-9 and 10-19 were excluded from the figure, since there were almost no deaths. the crude (%) and age-adjusted cfrs (%) are compared in table 2 . the crude cfrs varied from 0.82% for israel to 14.20% for italy and the ratios of the crude cfrs compared with lowest cfr, varied between 0.36 and 6.28. the age-adjusted cfrs varied between 4.20% for china to 10.80% for italy and the ratios of the age-adjusted cfrs for each country compared with the lowest cfr adjusted varied between 1.00 and 2.57. fig 3 shows graphically the marked reduction in the differences between the crude (%) and age-adjusted cfrs (%) for the seven countries. we used meta-analytic methods to obtain weighted pooled estimates of the cfr's by age group. the results of the meta-analysis are presented in the forest plot in fig 4, in our study, we examined crude, age-specific and age-adjusted cfrs for covid-19 in seven countries, with widely varying crude cfrs. the trends in the age-specific cfrs were remarkably similar in the seven countries, with the cfr's increasing steeply in those over 70. after adjusting for age, the marked differences in the crude cfrs were substantially reduced. these findings demonstrate the importance of accounting for age when comparing rates in general and cfrs in particular. the results of this study are strengthened by the use of national data or large datasets from a number of countries, with considerable differences in the extent of the pandemic in each country. it should be stressed that the age distribution of the cases was used to compute the age-adjusted cfrs and not the age distribution of the total population in each country. in addition to the age distribution of the cases, the use of the cfr for comparisons between countries has other important limitations. selection bias is clearly present when calculating the denominator on the basis of reported cases. as mentioned, the cfr must be distinguished from the ifr, which includes asymptomatic cases identified by deliberate or incidental screening with diagnostic tests. in addition, if only those with more severe symptoms are tested this will affect the denominator of the cfr and will depend on the testing strategy of each country. if more mild cases are identified, this is likely to reduce the cfr. there is a lag time between the reporting of the case and the death which can occur up to weeks later. in the country reports, cases and deaths are usually reported at the same time, so the cases in the denominator are usually an overestimate of the true denominator which should be the number of cases reported sometime earlier [14, 15] . this will have a more dramatic effect when the number of cases are rising rapidly. selection bias may also affect the numerator if only deaths occurring in hospital are reported. information bias can be present in both the numerator and denominator of the cfr. the definition of the cases may be biased due to the variability of the sensitivity and specificity of the diagnostic tests for covid-19. information bias in the numerator can occur when the cause of death is coded. this could be particularly problematic in elderly people with multiple co-morbidities. the purpose of this paper is to demonstrate the dramatic effect of confounding by the age distribution of the cases when using crude overall cfrs for country comparisons. this was shown in an earlier paper when comparing six countries, and we have extended it to a comparison of seven countries with widely different cfrs. the age structures of the population of the seven countries used in this study vary markedly. the percentage of the population age 65 and over is 12% in israel, 23% in italy 23%, 9.3% in s korea, 19.6% in spain, 20% in sweden, 11% in china and 17.6% in canada [16] . however, the main impact of confounding by age was due to the differences in the age distribution of the cases. this was largely due to the specific circumstances of exposure. for example, most of the cases in italy occurred in an area of a particularly old population [3] . in some countries, many of the cases were medical personnel, a large number of whom were relatively young women [17] . in south korea, a large percentage of the cases were young women associated with a specific religious group [18] . in germany, many of the cases were relatively young people returning from skiing holidays in austria and italy [19] . in israel, the largest outbreaks occurred in the ultra-orthodox jewish community, where the number of children per family is much higher than in the general population. other factors affecting the age distribution of the cases, depended on the frequency of outbreaks in homes for the elderly [20] . the results of this study once again demonstrate the pitfalls of comparing unadjusted rates. the assumption that differences between countries in testing policies or standard of treatment accounted for the wide discrepancies in cfrs, is not well-founded. this does not mean that there are no differences. for example, it is possible that where the health services were overloaded, younger patients were more likely to be admitted to intensive care units with better chances of survival. clearly, the data are incomplete and other factors affecting cfrs such as case definitions, use of different denominators, underlying health conditions and the standard of health services are likely to play important roles. in order to assess the impact of these factors, age-specific and age-adjusted cfrs must be used. in addition to the selection and information biases inherent in computing cfrs, the age structure of the cases dramatically impacts on the differences in the crude cfrs between countries. failure to account for this source of confounding markedly distorts the country comparisons. the substantial reduction in the differences in the age-adjusted cfrs suggest that differences in the standard of healthcare between these countries may not play as important a role in affecting the death rates, as some have hypothesized. crude covid-19 cfrs have no real use for between country comparisons and should be avoided. in general, for comparisons between groups and countries, age-adjusted cfrs can be used, but age-specific covid-19 cfrs are generally far more meaningful. similarity in case fatality rates (cfr) of covid-19/sars-cov-2 in italy and china case-fatality rate and characteristics of patients dying in relation to covid-19 in italy early estimation of the case fatality rate of covid-19 in mainland china: a data-driven analysis potential biases in estimating absolute and relative case-fatality risks during outbreaks covid-19: death rate is 0.66% and increases with age, study estimates estimates of the severity of coronavirus disease 2019: a model-based analysis aggiornamento nazionale the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19)-china, 2020. the novel coronavirus pneumonia emergency response epidemiology team a30501000000&bid = 0031&list_no = 367045&act = view coronavirus disease 2019 (covid-19): epidemiology update". public health agency of canada real estimates of mortality following covid-19 infection estimating case fatality rates of covid-19 impact on mental health and perceptions of psychological care among medical and nursing staff in wuhan during the 2019 novel coronavirus disease outbreak: a cross-sectional study korean society of infectious diseases; korean society of pediatric infectious diseases report on the epidemiological features of coronavirus disease 2019 (covid-19) outbreak in the republic of korea from a german exception? why the country's coronavirus death rate is low we express our appreciation to the official institutions of all countries for the providing their data on covid-19. conceptualization: manfred s. green, dorit nitzan. key: cord-255013-njpuc475 authors: he, xiaocui; korytář, tomáš; zhu, yaqing; pikula, jiří; bandouchova, hana; zukal, jan; köllner, bernd title: establishment of myotis myotis cell lines model for investigation of host-pathogen interaction in a natural host for emerging viruses date: 2014-10-08 journal: plos one doi: 10.1371/journal.pone.0109795 sha: doc_id: 255013 cord_uid: njpuc475 bats are found to be the natural reservoirs for many emerging viruses. in most cases, severe clinical signs caused by such virus infections are normally not seen in bats. this indicates differences in the virus-host interactions and underlines the necessity to develop natural host related models to study these phenomena. due to the strict protection of european bat species, immortalized cell lines are the only alternative to investigate the innate anti-virus immune mechanisms. here, we report about the establishment and functional characterization of myotis myotis derived cell lines from different tissues: brain (mmbr), tonsil (mmto), peritoneal cavity (mmpca), nasal epithelium (mmnep) and nervus olfactorius (mmnol) after immortalization by sv 40 large t antigen. the usefulness of these cell lines to study antiviral responses has been confirmed by analysis of their susceptibility to lyssavirus infection and the mrna patterns of immune-relevant genes after poly i:c stimulation. performed experiments indicated varying susceptibility to lyssavirus infection with mmbr being considerably less susceptible than the other cell lines. further investigation demonstrated a strong activation of interferon mediated antiviral response in mmbr contributing to its resistance. the pattern recognition receptors: rig-i and mda5 were highly up-regulated during rabies virus infection in mmbr, suggesting their involvement in promotion of antiviral responses. the presence of cd14 and cd68 in mmbr suggested mmbr cells are microglia-like cells which play a key role in host defense against infections in the central nervous system (cns). thus the expression pattern of mmbr combined with the observed limitation of lyssavirus replication underpin a protective mechanism of the cns controlling the lyssavirus infection. overall, the established cell lines are important tools to analyze antiviral innate immunity in m. myotis against neurotropic virus infections and present a valuable tool for a broad spectrum of future investigations in cellular biology of m. myotis. bats belong to one of the most abundant, diverse and widely distributed mammalian groups. in the order of chiroptera which is divided into two suborders megachiroptera and microchiroptera, a total of 1,240 species have been yet described [1] . bats evolved early and changed very little over the past 52 million years [2] . their wide distribution and migratory behaviour favour bats as vectors for viruses and raise concerns over their role in zoonotic diseases [3] [4] [5] . among the large number of viruses detected in bats, some like hendra virus, nipah virus, severe acute respiratory syndrome coronavirus (sars), ebola virus, west nile virus were reported to be zoonotic [4, [6] [7] [8] [9] [10] [11] . also 13 of the 15 lyssaviruses, except mokola virus and ikoma lyssavirus, were detected in bats. in north america bats host rabv, whereas in europe european bat lyssavirus type 1 and 2 (eblv-1 and eblv-2) are found in different bat species [12, 13] . annually, there are approximately 55,000 human deaths caused by rabies, especially in the developing countries of asia and africa [14] . despite most human rabies deaths are associated with dog rabies, some of them can be directly linked to the contact with bats, such as 8 out of 226 human rabies cases were of bat origin in the americas in 1983 and a few human cases caused by eblvs were reported in europe to date [15] [16] [17] [18] . although bat associated viruses can cause severe diseases in various mammals, they seem to be less pathogenic for bats [3, [19] [20] [21] [22] [23] [24] [25] . after experimental infection with hendra or nipah virus, bats showed no clinical disease, while guinea pigs succumbed to the same dose of virus [21, 22] . similar situation was also observed in hendra virus infection in horses and bats [24] . lyssaviruses are the only viruses that were reported to cause clinical disease in bats [26] . however, only a small proportion of bats develop clinical symptoms after experimental infection [25, 27] . this indicates a critical difference in the development of viral disease between bats and other mammals and requires profound investigation of bat immunology and host-virus interactions. since all of 52 identified european bats species are endangered and strictly protected, the use of animal trials for the investigation of immune mechanisms in bats is not possible. thus, development of stable cell lines for in vitro studies derived from european bat species is desirable. so far, several bat cell lines were reported in previous studies, but most of them were established from non-european bats, like tb1-lu from tadarida brasiliensis, mvi/it from myotis velifer incautus, and several primary immortalized cell lines from pteropus alecto, carollia perspicillata, eidolon helvum and rousettus aegyptiacus [28] [29] [30] [31] . viral infection studies have been carried out in the fruit bat cell lines to investigate the susceptibility, infection kinetics of henipavirus as well as the host innate immunity [28, 32] . however, the susceptibility to lyssavirus has not yet been examined in these cell lines. additionally, except for a brain cell line from eptesicus serotinus employed to investigate the type i interferon (ifn) response after lyssavirus infection [33] , the use of a bat cell line as a tool for studies into lyssavirus infection in its natural reservoir host is rare. a broader variety of bat cell lines, particularly european bat cell lines from tissues of immune relevance, is therefore urgently in demand for lyssavirus-host studies. in this study, we established different cell lines from the european bat m. myotis, evaluated their susceptibility to eblv-1, eblv-2 and rabv infection and investigated innate immune gene responses after the polyinosinic:polycytidylic acid (poly i:c) stimulation. the established m. myotis cell lines present a valuable in vitro model to study the interactions between lyssaviruses and their natural host, and to shed light on the mechanisms of resistance in bat's central nervous system (cns). ethical approval for all of the capturing and sampling were confirmed by the competent authorities in the respective federal republic of germany and czech republic. the czech academy of sciences ethics committee reviewed and approved the animal use protocol no. 169/2011 in compliance with law no. 312/ a single m. myotis male was captured in sloupsko-sosuvske caves of the moravian karst (czech republic, coordinates 49u 249 40.880 and 16u 449 20.540). the bat was kept to minimize stress and handling between capture and euthanasia in a clean plastic box with soft mesh to enable roosting under temperature of hibernation torpor of 6uc and transferred to our laboratory at veterinary and pharmaceutical sciences brno (czech republic) within a day. it was anesthetized to insensitiveness using isofluranum (isofluran, piramal healthcare, uk), and then transcription pcr (rt-pcr) using sv40t specific primers [35] . the protein expression was controlled by the immunofluorescence and western blot as described below. briefly, cells were first fixed with 3% paraformaldehyde and permeabilized with 0.5% triton x. after washing with pbs, cells were stained with mouse anti-sv40t monoclonal antibody (santa cruz biotechnology) and goat anti-mouse igg alexa fluor (invitrogen) as second antibody and visualized by fluorescence microscope. for western blot, the same mouse antibody was used as primary antibody and bound antibody was detected with goat anti-mouse igg peroxidase (sigma). images were developed using the ecl kit (thermo scientific pierce) according to the manufacturer's instructions. to confirm the identity of the established m. myotis cell lines derived from brain (cerebrum) (designated mmbr), tonsil (mmto), peritoneal cavity (mmpca), nasal epithelium (mmnep) and nervus olfactorius (mmnol), a m. myotis-specific pcr was developed. an nadh dehydrogenase subunit 1 (nd1) gene (genbank accession number: dq915043) from m. myotis was used as a species specific molecular marker. the genomic dna from different cell lines was isolated by dneasy blood & tissue kit (qiagen). the concentration and purity of genomic dna were determined by nanodrop (thermo). pcr was performed using a specific primer pair nd1-f and nd1-r (table 1) and genomic dna as a template by gotaq flexi dna polymerase (promega) to get the nd1 fragments. pcr products were cloned into pcr2.1 vector (invitrogen) and transformed into e. coli competent cells. plasmids were extracted from positive clones and sequenced by applied biosystems 3130 genetic analyzer (life technologies) at the friedrich-loeffler-institute, germany. to evaluate the ifn response of m. myotis cell lines and the induction of ifn mediated signaling, poly i:c was used to stimulate the cells. different cell lines were seeded in 24-well plates at a density ranging from 1.2 to 2610 5 cells/well, and cultured as described above. around 20 hours after seeding, cells were transfected with poly i:c (sigma) at a concentration of 10 mg/ ml by lipofectamine 2000 (invitrogen) following the manufacturer's instructions. twenty four hours post stimulation, cells were harvested into rlt buffer (qiagen) for rna extraction by an rneasy mini kit (qiagen). early after immortalization, the third passage immortalized cell lines were used to check the infectivity of rabv. cells were infected with rabv (european fox isolate, fused with green fluorescent protein, gfp) at a moi of 10. twenty four hours post infection (hpi), infected cells were fixed and permeabilized as described above and visualized by fluorescence microscope. mmbr and mmto cells that were infected with a serial moi of 0.01, 0.1, and 1.0 were harvested at 24 hpi and used for rna extraction. to confirm the infectivity in later passaged cells, different immortalized cell lines of more than 15 passages were infected with lyssaviruses rabv, eblv-1 (e. serotinus isolate) and eblv-2 (m. daubentonii isolate) at a moi of 0.1. the infected cells were cultured as described above. cells were collected for rna extraction at 24 hpi and quantitative real-time pcr (qrt-pcr) was performed on the cfx96 touchdetection system (bio-rad) using sensifast sybr one-step kit (bioline) according to the protocol. immunofluorescence analysis was performed on fixed cells using fitc conjugated anti-rabies monoclonal antibody (sifin) at 72 hpi as described before [36] . to further confirm the susceptibility, mmbr and mmnol cell lines were infected with eblv-1 at a moi of 0.01 to set the sensitivity at a ct value of 22 for the inoculation dose. the viral supernatant was either changed or not changed with fresh medium at 1 hpi, and viral replication levels were measured by qrt-pcr over 72 hpi. qrt-pcr was introduced to measure the mrna expression levels of immune related molecules in response to poly i:c stimulation and virus infection. the selected molecules include ifn induced genes: ifn stimulated gene 56 (isg56), isg43, myxovirus resistance 1 (mx1) and ifn induced protein with tetratricopeptide repeats 3 (ifit3), and pattern recognition receptors (prrs): toll-like receptor 3 (tlr3), retinoic acidinducible gene 1 (rig-1) and melanoma differentiation-associated protein 5 (mda+5). all of these primers were designed based the sequence resources from our own un-published sequence database and public databases of bat species. the softwares for primers design include primer premier 5, online tools: http://bioinfo.ut. ee/primer3-0.4.0/ and http://www.ncbi.nlm.nih.gov/tools/ primer-blast/. primers of target genes and internal control bactin were listed in table 1 . qrt-pcr was performed on the cfx96 touchdetection system (bio-rad) using sensifast sybr one-step kit (bioline) according to the protocol. to assess the specificity of the pcr amplification, a melting curve analysis was performed at the end of the reaction. the relative expression levels of targets were calculated by 2 2ddct method [37] . molecular characterization of the mmbrbecause the mmbr is derived from the cns, the target of fatal infections by lyssaviruses, a further characterization of cell type of mmbr is desired to improve the understanding of the antiviral defense in the cns. the expressions of cluster of differentiation (cd) 68, a marker for cells of macrophage lineage [38] , and cd14, a marker expressed in activated microglia [39] , were investigated by rt-pcr in different cell lines. specific primers for cd14, cd68 and internal control b-actin were listed in table 1 . the rt-pcr was prepared according to the instructions of the one-step rt-pcr kit (qiagen). all data were presented as means 6 s.d. statistical significant differences were analysed by one-way anova using the spss software package. five m. myotis cell lines brain (mmbr), tonsil (mmto), peritoneal cavity (mmpca), nasal epithelium (mmnep) and nervus olfactorius (mmnol) were successfully established by transformation with sv40t gene integrating into the chromosomal dna. varying cell morphologies were observed in the cell lines, with mmbr, mmto, mmnep and mmnol being fibroblastic-like, and mmpca being epithelial-like (fig. 1a) . the mrna expression of sv40t antigen was detected in all cell lines (fig. 1b) . protein level expression was confirmed in four of the five cell lines by immunofluorescence microscopy and western blot, respectively ( fig. 1c and d) . in mmto, the protein level of sv40 t antigen was under detectable because the transcriptional level is significantly low determined by rt-pcr (fig. 1b) . after immortalization, all five cell lines grew for more than 30 passages. the identity of the cell lines was validated by a m. myotis specific pcr using nd1 gene as a molecular marker. a predicted 515-bp fragment was obtained from genomic dna of each cell line, and further confirmed by sequencing. as the first step towards the characterization of the innate immune competence of different cell lines, the permanent or inducible expression of molecules involved in cell autonomous responses was examined. the prrs: tlr3, rig-1 and mda5, display a various distribution pattern in different cell lines (fig. 2) . of note, mmbr has the lowest levels of tlr3, rig-1 and mda5 (fig. 2) . for tlr3, about 10-fold higher mrna levels (p,0.05) were observed in mmto, mmpca, mmnep and mmnol compared to mmbr, respectively, while for mda5 about 30-fold (mmto; mmnep) or about 6-fold (mmpca; mmnol) (p,0.05) higher expression levels were measured (fig. 2) . additionally, more than 200 times higher expression levels of rig-1 were shown in other cell lines compared to mmbr (p,0.05) (fig. 2) . further investigation focused on the expression of tlr3, isg56, isg43 and mx1 induced by the poly i:c stimulation (fig. 3) . the obtained results indicate a 4-fold in mmbr, mmnep and mmnol and 8-fold in mmpca (p,0.05) increase in the tlr3 expression, whilst no change in mmto (fig. 3a) . all of the ifn induced genes were up-regulated to different extents in different cell lines (fig. 3b, c, d and e) . in detail, isg56 expression increased from 19-fold in mmbr to as high as more than 9000-fold in mmnol (p,0.05) (fig. 3b) . the expression of isg43 ranged from 10 to 145 times more and mx1 from 2 to 100 times more in mmto and mmbr, respectively ( fig. 3c and d) . ifit3 was upregulated from 12 to 420 times more in mmto and mmnol, respectively (p,0.05) (fig. 3e ). being a natural reservoir species, the main advantage of the permanent m. myotis cell lines is their susceptibility to lyssavirus infection. at an early stage of immortalization, cell lines displayed a significant susceptibility to rabv (moi of 10 at 24 hpi) as demonstrated by the infection with gfp fused rabv. notably, mmbr exhibited considerably lower viral load compared to the other cell lines (fig. 4) . later, all passaged immortalized cell lines showed susceptibility to eblv-1, eblv-2 and rabv in a different extent (fig. 5a and b) . generally, the mmbr cell line presented lower sensitivity to all three lyssaviruses (moi of 0.1) than the other four cell lines measured by qrt-pcr at 24 hpi (fig. 5a) , and monitored by immunofluorescence at 72 hpi (fig. 5b) . thus, the susceptibility could be ordered as mmnol and mmnep fully susceptible with a very high replication rate, mmpca and mmto susceptible with a much less viral replication of eblv-1 and 2, mmbr susceptible for eblv-1 and rabv with a very low viral replication and just single infected cells after eblv-2 infection (fig. 5b) . the different susceptibility of the cell lines to lyssavirus infection was further confirmed by the growth kinetics of eblv-1 in two representative models: mmbr, much less susceptible and mmnol, highly susceptible (fig. 5c ). to further evaluate the cell line models for study of the different susceptibility between mmbr and other cell lines, mrna expressions of prrs and ifn induced genes were investigated in mmto and mmbr after rabv infection (moi 0.01 to 1.0). the expression of all three prrs remained mostly unchanged in mmto, while it was significantly regulated in mmbr with 2-fold increased expression of tlr3, about 25-fold increased expression of rig-1 and mda5 at moi of 1.0 (p,0.05) (fig. 6a) . a comparable expression pattern was observed for the isg56, isg43, mx1 and ifit3, which was nearly not up-regulated in mmto but displayed a dose dependent increase in mmbr along with the increase of moi, especially for isg56 and ifit3 (fig. 6b ). isg56 mrna level increased from 6 to 513 times, ifit3 from 2 to 85 times in the infected mmbr (p,0.05). microglia are macrophage-like cells that are resident immune effector cells in the cns [39] . they are activated in response to infection or injury and play a central role in immune surveillance and host defense [39] . the rt-pcr results showed that cd14 and cd68 are expressed only in mmbr but not in the other four cell lines (fig. 7) . this suggested mmbr is a microglia-derived cell line. cell autonomous and innate immune mechanisms are the first line defenses against viral infections. this is mediated mainly by the prrs and the machinery of the ifns and ifn induced effector molecules [40] [41] [42] . viral pathogens like lyssaviruses developed evasive strategies to escape these host defenses by counteracting the ifn mediated immune responses [43] . co-evolution of the lyssaviral evading and bat's protective mechanisms resulted in an optimal balance, which protect bats as the 'natural host' from severe clinical symptoms or death. bats, which changed very little over past 52 million years, illustrate this phenomenon very well by the resistance to lethal diseases caused by viruses in other mammals [11, [21] [22] [23] [24] .to understand the specificity of hostpathogen interactions in 'natural host' like bats, studies in bats have to be performed. however, due to the strict protection of the endangered european bat species, in vitro models have to be used. in this study, we successfully established five m. myotis cell lines derived from neural and immune related tissues. to ensure the suitability of these cell lines to analyze virus-host cell interaction, the susceptibility to the infection as well as the presence of corresponding defensive pathways have to be confirmed. first, the existence of the viral sensors tlr3, rig-1 and mda5 in these permanent cell lines suggests a capacity of these cell lines to sense a broad range of rna viruses. the increased expression of dsrna receptor tlr3 and ifn induced genes isg56, isg43, mx1 and ifit3 after stimulation with poly i:c mimicking a viral infection indicates that these cell lines can be used as effective in vitro models to study the bat's innate immune responses to virus infection [32, 44] . furthermore, to serve as valuable models would be a varying susceptibility of such cell lines to infection by lyssaviruses. in the present study, different susceptibility observed in different m. myotis cell lines using eblv-1, eblv-2 and rabv might be related to the different capacity of the cell lines to produce antiviral mediators and control the infection. moreover, the strong difference in the susceptibility to rabv infection between mmbr and other cell lines provides a unique opportunity for comparative investigations of cell autonomous and innate immune mechanisms in a reservoir host. in addition to the lyssaviruses, the other member from the rhabdoviridae family, like vesicular stomatitis virus (vsv) can also be investigated by using these models in the future studies. preliminary results indicate a correlation between the observed varying susceptibility and the ability to up-regulate the prrs and the ifn induced genes. emerging evidences have shown that prrs play pivotal roles in antiviral immunity in the cns [45] . in the brain derived cell line mmbr, the high up-regulations of rig-1 and mda5 revealed activation of rig-i-like receptor pathway during rabv infection. as previously reported, rig-1 is a major prr to induce ifn in the rabv infected cells, and mda5 may function to sustain the ifn induction [46] . the increased expressions of ifn induced genes: isg56, isg43, mx1 and ifit3 in mmbr indicate that the production of ifn was induced by activated rig-1 and mda5. in contrast, the low expression level of tlr3 implies a vague involvement of tlr3 in anti-rabv infection immunity or resistance. it was shown that tlr3 participated in and benefited the rabv pathogenesis in human neuron cells [47] . however, the roles of tlr3 during rabv infection in bats need further investigations. importantly, the significant expression patterns of prrs observed in presented cell line models provide an access to this issue in vitro. to reach a successful infection, the viruses must overcome the barriers of innate immune system. it was reported that ifn production and signaling pathways were antagonized in p. alecto cell lines under henipavirus infection [32] . similarly, a recent study showed limited expressions of type i ifns and ifn induced genes during lyssaviruses infection in an e. serotinus brain cell line [33] . a correlation between the low viral load and high expression levels of ifn induced genes in mmbr contrasts to the high viral load and a silent expression pattern of antiviral effectors in mmto, providing an evidence of a countermeasure to ifn system by lyssavirus in the peripheral tissue versus a protective mechanism to infection in the brain tissue of bats. microglial cells are one of the major cell populations in the brain tissue. additionally, comparing to neurons, they can be infected by different rabv strains to a lesser extent [48, 49] . the presence of cd14 and cd68 as well as the anti-lyssavirus responses in mmbr support a microglia-like feature of mmbr in the cns. it was reported that a mouse microglia cell line can activate strong innate immunity during rabv infection [50] . the robust immune responses of the microglia-like mmbr demonstrated a critical role of microglia in the anti-rabies defense in bat's cns. in addition to the function of microglia, the clearance of infected viruses in the cns requires systematical responses through the complex interactions of different brain resident cells. herein, the establishment and identification of a microglia-like cell model is a first step towards understanding of the complex reactions of cns in response to lyssavirus infection in the reservoir species. overall, this preliminary study using established cell lines implies that immune mechanisms that control the virus replication are present in the cns of bats. it seems that the ability to control the pathogenic rabv replication via ifn system in the cns contributes to the asymptomatic outcome in bats. in conclusion, the established immortalized cell lines from the european bat m. myotis displaying a variable susceptibility to different lyssaviruses will serve as a useful model to study virus-host interactions and antiviral resistance mechanisms 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and human microglia and astrocytes global gene expression changes in bv2 microglial cell line during rabies virus infection we would like to thank dr. thomas müller and dr. conrad m. freuling for the eblv-1 and eblv-2 strains, dr. stefan finke for the rabv strain, matthias lenk for the prsvag1 plasmid, and dr. miroslav kovarik from the administration of the moravian karst protected landscape area (nature conservation agency of the czech republic) for cooperation in obtaining the m. myotis specimen. key: cord-262832-5iejckwx authors: yen, muh-yong; wu, tsung-shu joseph; chiu, allen wen-hsiang; wong, wing-wai; wang, po-en; chan, ta-chien; king, chwan-chuen title: taipei's use of a multi-channel mass risk communication program to rapidly reverse an epidemic of highly communicable disease date: 2009-11-23 journal: plos one doi: 10.1371/journal.pone.0007962 sha: doc_id: 262832 cord_uid: 5iejckwx background: in september 2007, an outbreak of acute hemorrhagic conjunctivitis (ahc) occurred in keelung city and spread to taipei city. in response to the epidemic, a new crisis management program was implemented and tested in taipei. methodology and principal findings: having noticed that transmission surged on weekends during the keelung epidemic, taipei city launched a multi-channel mass risk communications program that included short message service (sms) messages sent directly to approximately 2.2 million taipei residents on friday, october 12th, 2007. the public was told to keep symptomatic students from schools and was provided guidelines for preventing the spread of the disease at home. epidemiological characteristics of taipei's outbreak were analyzed from 461 sampled ahc cases. median time from exposure to onset of the disease was 1 day. this was significantly shorter for cases occurring in family clusters than in class clusters (mean±sd: 2.6±3.2 vs. 4.39±4.82 days, p = 0.03), as well as for cases occurring in larger family clusters as opposed to smaller ones (1.2±1.7 days vs. 3.9±4.0 days, p<0.01). taipei's program had a significant impact on patient compliance. home confinement of symptomatic children increased from 10% to 60% (p<0.05) and helped curb the spread of ahc. taipei experienced a rapid decrease in ahc cases between the friday of the sms announcement and the following monday, october 15, (0.70% vs. 0.36%). by october 26, ahc cases reduced to 0.01%. the success of this risk communication program in taipei (as compared to keelung) is further reflected through rapid improvements in three epidemic indicators: (1) significantly lower crude attack rates (1.95% vs. 14.92%, p<0.001), (2) a short epidemic period of ahc (13 vs. 34 days), and (3) a quick drop in risk level (1∼2 weeks) in taipei districts that border keelung (the original domestic epicenter). conclusions and significance: the timely launch of this systematic, communication-based intervention proved effective at preventing a dangerous spike in ahc and was able to bring this high-risk disease under control. we recommend that public health officials incorporate similar methods into existing guidelines for preventing pandemic influenza and other emerging infectious diseases. the viral illness known as acute hemorrhagic conjunctivitis (ahc) is frequently accompanied by a highly transmissible acute eye infection. this infection is most often caused by the adenovirus, enterovirus 70, and coxsackie's virus. coxsackie a24 infection was first reported in 1969 in ghana and has since appeared around the world [1, 2, 3, 4, 5] . since late 2002, several ahc epidemics have occurred in asian countries such as korea, malaysia, and singapore [6, 7, 8] . in september 2007, southern china's coxsackie a24 ahc epidemic spread to hong kong after first appearing on the mainland in early summer. on september 18th, a cluster of cases with ahc-like symptoms was first unofficially reported by the media in keelung, a harbor city bordering taipei (816 km away from hong kong), in northern taiwan. because ahc was not on taiwan's list of reported communicable diseases at the time, it was difficult for public health officials to collect adequate epidemiological data until october 4th, when a dramatic spike in ahc cases was reported by the mass media. at that time, the keelung department of health reported 2722 cases of pink eye disease among public school students. although general control measures were taken, case numbers continued to increase rapidly in keelung, particularly during weekends. also on oct. 4th, taipei city department of health was alerted to its first ahc cluster (20 cases from a primary school) in neihu, a taipei district neighboring keelung (figure 1 ). taipei was more prepared for the ahc outbreak as its alertness was raised by media coverage of outbreaks in nearby keelung. the local department of health was also able to identify clusters quickly because it had made school reporting of influenza-like illness clusters (or other unusual clinical presentations) a mandatory practice since 2003. taipei's department of health had reorganized its disease control and prevention system against emerging infectious diseases (eid) after 2003's severe acute respiratory syndrome (sars) epidemic but had not yet tested the system under real life circumstances. in launching countermeasures to bring the ahc outbreak under control, the taipei department of health was also able to test its system and fine-tune its public health response for future eids. to evaluate the effectiveness of these intervention measures, daily surveillance was conducted to analyze the incidence rate and temporal-spatial distribution of new ahc cases. in addition, students' parents were sent questionnaires to capture their experience with these preventative measures. taipei city, the largest city in taiwan, is located in northern taiwan and directly borders keelung city ( figure 1 ). in 2007, taipei, a city of 2.6 million people occupying 271.8 km 2 , had a student population of 277,159 (10.53%). taipei city has a larger population size and density than neighboring keelung city (table 1) , and is, thus, exceedingly vulnerable to microbial transmission and eid outbreaks. this study was approved by the advisory committee for infectious diseases control of taipei city's department of health, taipei city government. informed consent was obtained from the parents of participating schoolchildren in writing before they were asked to complete the questionnaire (file s1). in response to a large outbreak of sars in 2003, taipei's department of heath established a new crisis management system with the goals of detecting eids early, implementing appropriate and timely public health responses, and administering effective risk management [9, 10] . at the start of taiwan's 2007 ahc outbreak, local health departments in all of taiwan's cities were engaged in a passive surveillance system in which schools reported cases as they occurred. on october 4th, taipei's department of health urged schools and kindergartens with clusters of more than three ahc cases to actively report cases to the local department of health twice weekly. agespecific incidence rates of ahc among school-aged children during the outbreak were also calculated using the collected surveillance data. when total ahc cases in taipei reached close to 500 on october 11, 2007 , an active school-based surveillance system was launched by the city to monitor trends in the spread of ahc and evaluate the effectiveness of intervention measures. schools were required to make daily reports to taipei's department of education, regardless of whether there were any new cases on a given day. as the incidence rate of the last reported ahc cases in keelung (on october 22) was 0.14%, we set this rate as a cut-off point for comparing the effectiveness of the countermeasures used by the two cities. tailing data, i.e., data that fell below the 0.14% incidence rate was excluded from our analysis. we used this cut-off to define the duration of the ahc epidemics for both cities. according to our definition, the ahc epidemics for each city began from the date of the first reported cluster and ended on the date that rates of new case incidence dropped to 0.14% or below. epidemiological design and data analysis 1. study subjects. we analyzed 461 reported ahc cases from ten taipei schools (totaling 18,134 students). students from six of the ten schools, three elementary schools and three junior high schools, made up most of the reported ahc case numbers. two elementary schools and two junior high schools that were also included in the research reported continuous ahc occurrences during the week that our field epidemiological study was conducted. for each of the ten schools, we randomly selected one class that had had occurrences of ahc and one class where there had been no occurrence of ahc as our case and control groups, respectively. data collected from these individual school groupings were gathered and divided into one study and one control group for data analysis. 2. case definition of ahc and clusters (family, school). the ahc cases in this study all involved an acute conjunctiva inflammation that included eye redness (pink eye) accompanied with pain, swelling, tearing, or discharge from one or both eyes. family clusters were defined as two or more cases of ahc occurring in one family within 14 days. class clusters were defined as three or more ahc cases in one class of students within 7 days. both family clusters and class clusters were charted through epidemiological investigation. 3. tempo-spatial data analysis. district-specific ahc attack rates were calculated by dividing the number of reported cases for each district by the total number of schoolchildren under its supervision. the kriging method [11, 12] , a statistical mapping technique utilizing data collected at each location, was used to interpolate each grid cell over a spatial domain. in this study, the centroid of each grid designated the attack rate in each district. in order to observe spatio-temporal spreading, kriging assessed the spatio-temporal interactions in a diffusion map. we made a surface plot of daily time series as a gradient interpolated between adjacent days and district data points. distance in the map symbolizes relative geographic relationship rather than actual distance. townships were ordered from east to west and from north to south. to identify the possible etiologic agent of the outbreak, health care professionals were required to administer eye swabs and conduct laboratory testing. local health workers were then able to match the epidemiological characteristics for the confirmed agent, coxsackie a24, with appropriate and specific prevention and control measures. government officials of the taipei city department of health informed the media of the outbreak through press releases and used a variety of health education methods to reach the public. beginning on october 5, public service messages were delivered to kindergartens, primary schools, middle schools, and high schools to encourage children to avoid touching their eyes, wash their hands routinely, and participate in disinfecting the school environment. within schools, health education programming during daily morning assemblies provided updates to students and teachers regarding the current status of the epidemic and additional measures that were needed to reduce infections. a special telephone hotline was also established to improve case reporting and provide up-to-date disease counseling from health care institutions. these measures, though helpful, had also been used in keelung and had, thus far, proven inadequate at containing the epidemic. in early october 2007, taipei city government decided to adopt a more aggressive campaign against the epidemic by implementing an ''incident management system.'' this system required various administrative agencies to follow an integrated disaster response plan. although school closures and class cancellation were not required for schools with reported ahc cases, schools were encouraged to persuade symptomatic students to stay home and provide guidance on how to prevent the spread of the infection in the home environment. in addition, schools were authorized to keep symptomatic students from entering the school in case parents insisted on their attendance. if infected students were able to gain entry to school premises, the school was authorized to prevent them from joining public activities (such as swimming). school absenteeism was also reported and recorded daily. the taipei city department of health devised backup plans should the above-mentioned measures not succeed. these plans included separate care facilities for ahc patients at ophthalmic clinics, more intensive segregation of symptomatic students from classmates (in the classroom, at public washbasins, and during outdoor student activities), and quarantines that required symptomatic students to stay at home for seven days. monday incidence reports ( figure 2 ) exhibited tremendous increases in ahc case incidence during weekends in keelung. mindful of these weekend spikes, taipei implemented a multichannel risk communication prevention program during the weekend of friday, october 12 (2,253 new cases of ahc were reported in taipei on that date). this risk communication program focused on communicating directly to the public through three routes: (1) schools delivered a taipei department of health letter signed by the mayor (that detailed ahc information and prevention methods) for students to take home to their parents, (2) the mayor held a press conference to discuss the epidemic and offer guidance to citizens for preventing the spread of the disease, and (3) over 2.2 million short message services (sms) messages, a communication tool for exchanging short text messages between mobile telephonic devices, were delivered to all taipei mobile phone numbers. the messages briefed taipei residents on the current status of the epidemic and recommended citizen-level control measures. all communications suggested that symptomatic students stay at home, apart from other members of the family, and recommended household disinfection. on october 31st, all taipei students involved in the study were asked to give their parents a questionnaire devised to collect epidemiological data and assess their opinions regarding taipei city's infection control measures. the questionnaire asked about the clinical symptoms/signs of children with ahc, school attendance, and inquired on parents' sources of disease prevention information. parents were also asked to comment on their degree of satisfaction with taipei city's public health efforts and provide suggestions for improving future sms alerts. we evaluated the effectiveness of the control measures based on the duration of the epidemic and the attack rate of the disease among school students in both taipei and keelung. as keelung was without a risk communications program, and had only applied the general control measures recommended by taiwan's center for disease control (taiwan cdc), the effectiveness of the special measures taken by taipei city could be readily calculated through direct comparison. because this study utilized risk communication methods used to minimize the public health threat of an unusual outbreak, we briefly describe the 2007 ahc epidemic below and analyze the effectiveness of the chosen methods using epidemiological measures. 1. ahc attack rates in taipei and keelung cities. at the beginning of the epidemic, the etiologic agent of this ahc outbreak was unknown. on october 12th, based on culture and sequencing analysis performed at taiwan cdc, the pathogen was identified as the a24 variant of the coxsackie virus [13] . the epidemic lasted 13 days in taipei (5,414 cases), and 34 days in keelung (6,154 cases) ( table 1) . keelung city applied general control measures and keelung cdc monitored daily reported new cases as suggested by taiwan's cdc. however, there was no further assessment by keelung on the effectiveness of its control measures. the crude attack rate of ahc in keelung was significantly higher than in taipei (14.92% vs. 1.95%; p,0.001). after the risk communication program was implemented in taipei the overall incidence in the city decreased significantly (0.093% before vs. 0.056% after; p,0.001) while the incidence rates in keelung continued to increase almost every weekend ( figure 2 ). the greatest number of ahc cases in taipei city occurred on a friday (october 12), signaling an upcoming weekend spike in infections. however, on that day, taipei city government launched the multi-channel risk communication program, greatly reducing the incidence rate and, in effect, causing a sharp weekend drop in new cases. because of such measures, the epidemic ebbed much earlier in taipei than in keelung ( figure 3 ). 2. epidemiological characteristics. the outbreaks began in keelung in september of 2007. due to the high frequency of transportation from keelung to taipei, the epidemic gradually reached taipei. districts in taipei city closest to keelung city began to see an increase in their attack rates on october 8th (figure 3 ). the wave of new infections moved steadily from the northeast districts to the southwest districts of taipei. in both cities, the disease spread citywide within a short period (4 days), as shown in figure 1 , and the median time between exposure and onset of disease was as short as 1 day [mean 6 standard deviation (sd): 2.663.2 days, range 0 to 16 days] [ figure 4 ]. the mean time from exposure to onset of ahc was significantly shorter in family clusters than in school cluster cases (mean 6 sd: 2.663.2 vs. 4.3964.82 days, p = 0.03). the mean and range of time between exposure and disease onset was also significantly shorter in larger family clusters (.3 ahc cases per family) than in smaller ones (, = = 3 ahc cases) (1.261.7 days, range 0 to 6 days vs. 3.964.0 days, range 0 to 16 days, respectively) (p,0.01). in terms of risk factors (table 2) , boys were at significantly higher risk for ahc-related pink eye than girls (or = 2.14, p,0.001), and older children (over 10 years old) were generally at higher risk for pink eye both at home (or: 2.56, p = 0.008) and at school (or 3.51, p,0.001). in general, children at northeastern taipei schools, located closest to keelung, were at greater risk for pink eye (or = 2.26, p-value = 0.003) both at home (or = 3.93, p-value ,0.001) and at school (or = 2.46, p-value = 0.001), than those at schools located in districts further away (figure 1 ). in addition, school children were identified as the index cases in 75.5% of fifty-three family clusters (40/53), demonstrating their high risk for transmitting the disease to other family members. 1. home confinement of symptomatic school children. on friday, october 12th (a day marked for its dramatic spike in new cases) the multi-channel mass risk communication program was launched in taipei just prior to the weekend. on that day, three containment measures were urgently implemented: (1) the mayor addressed the press about the outbreak, (2) a letter written by the taipei city department of health and signed by the mayor was given to students to take home to their parents, and (3) sms messages were sent to all taipei citizens with mobile phones. the collective message of all three channels emphasized the severity of the outbreak and outlined preventive measures, including home confinement. according to results obtained from the questionnaire (table 3) , home confinement of symptomatic students increased from a rate of 10% prior to the implementation of the risk communication program to 60% afterwards (p,0.05). 2. disease containment. as mentioned above, taipei launched its mass risk communication program on october 12th. this was also the day that the epidemic reached its highest peak. as demonstrated in figure 2 , the pre-weekend case surge was reduced by almost half by october 15th (following the mass risk communication campaign in taipei). in keelung, where no such risk communication program was launched, the epidemic lasted for thirty-four days and had fourteen days of high attack rates within that period. in contrast, the epidemic lasted thirteen days in taipei and had less than five days of high attack rates following the implementation of the risk communication program. the diffusion map in figure 3 further illustrates the limited scope and short duration of high ahc attack rates (labeled in red) in taipei districts. by october 17, attack rates in all taipei districts reduced to moderate levels (labeled in yellow) and then further reduced to low levels (labeled in green) on october 26 (figure 3) . according to taipei city's parents' responses to the questionnaire, primary sources for ahc information included tv news broadcasting (54.45%), daily school health education programs during morning assemblies (34.92%), and the taipei city department of health letter signed by the mayor (29.28%) ( table 4 ). approximately fourteen percent (14.32%) of parents surveyed identified the short message service (sms) issued by the taipei city department of health as their primary source of ahc information. based on a five-point scale, parents who received the sms communication felt more satisfied with this method as a means of public health communication than those who did not receive sms messages (3.89 vs. 3.01; p,0.05). prompt and effective prevention and control measures to combat eids are imperative to maintaining public health and safety. in this study, we found that the multi-channel risk communication program launched midway through the 2007 ahc epidemic in taipei city increased the number of students confined to their homes, reduced total duration and affected areas of the epidemic, and decreased the number of dangerous, high attack days. by interrupting the prevailing transmission route between school and home, the program effectively inhibited the spread of this highly communicable disease in the community. ahc has been identified as a highly contagious disease, capable of far-reaching, epidemic spread, since its first reported case in 1970 [2, 14] the coxsackie virus a24 (cv-a24) variant in particular has been the causing agent of several difficult outbreaks. it has also been a significant challenge to sensitivity and timeliness efforts in disease surveillance systems [13, 15] . in addition, the virus's eye-related symptoms [16, 17, 18] are not easily differentiated from other infections (i.e. the human strain of netherland's 2003 avian influenza, h7n7) [19, 20, 21, 22] . during the 2007 ahc epidemic in taiwan, schools were found to be epicenters of transmission. by focusing disease control efforts in the school system, taipei was more effective than neighboring keelung (which relied on traditional control strategies) at interrupting the school-family-community cascade of transmission. because ahc was not initially listed as a reportable disease when this outbreak first occurred, accessible data was initially unavailable for cases before october 4, 2007. several characteristics of the 2007 outbreak suggest a foreign source for taiwan's ahc epidemic. the introduction of the outbreak in keelung, a port city, and the genotype ii status of the 2007 cv-24 virus (all cv-24 viruses isolated in taiwanese outbreaks prior to 2007 had belonged to genotype iii [13] ) strongly suggest that the virus was imported in the summer of 2007. the spread worsened in taiwan after schools returned to session in september. although health officials in keelung had advocated for hand washing, eye protection, and disinfection, new cases continued to rise after october 8. the cv-24 virus, spread primarily through person-toperson contact and contact with infected fomites, may also have spread through contact with respiratory droplets and fecal-oral routes, resulting in rapid and widespread transmission [13, 15] . older children were more likely to be members of case clusters at home and at school. the reasons for this increased likelihood is unknown, though it may be related to higher rates of participation in team sports and/or less compliance with rules of hygiene. research may be needed to fully understand this phenomenon. in essence, school children serve as index cases in around seventy-five percent of family clusters (table 2) and are, indisputably, the largest transmitters of the virus. the findings of the study illustrate that schools serve as epicenters of cv-24 transmission and are effective intervention targets for containing the virus before its introduction to the general population. the large weekend increases of cv-24 infection that occurred in keelung may have resulted from a lack of preventive measures at home, e.g. ineffectiveness at keeping symptomatic children separate from other family members and/or improper disinfection of the home environment. because the median time from exposure to onset is short (median 1 day in figure 4 ), ahc is able to spread quickly from one index case to other family members, many of whom might take it back to schools and larger communities over the weekend. this creates a highly efficient family-schoolcommunity transmission cascade that is capable of spreading quickly throughout a city. therefore, measures that effectively reduce and isolate infections at home and at school would contribute greatly to breaking the transmission cycle. in the face of an unexpected outbreak of eid, risk communication and management are essential for keeping infection and mortality rates under control. the large disparity between mortality rates of the 1918 influenza pandemic in philadelphia and st. louis in the united states was due to distinct differences in the preparedness and timeliness of their respective local public health responses [23, 24] . this example serves to illustrate the importance of preventing and containing eids in a systematic and timely manner. as the capital of taiwan, taipei city has a much larger population size and density than keelung city (table 1 ). in addition, taipei is a highly dynamic city with frequent economic, cultural, and social change. this inconstancy, coupled with its status as a large international travel hub, makes the city particularly vulnerable to eid. much of taipei's preparedness was the result of the city's experience as taiwan's epicenter of the sars outbreak in 2003. after the sars outbreak, taipei city government initiated a new public health plan using an integrated infection control system against eid. this new system integrated early detection of outbreaks (particularly in hospitals and schools), epidemiological investigation, and epidemiologically based public health prevention and control policies. the renovated division of disease control and prevention (taipei's cdc) also became the core operational unit for implementing crisis management procedures and facilitating policy. these systematic upgrades allowed for quick enactment of multi-channel risk communication measures during the 2007 outbreak of conjunctivitis. the 2007 ahc outbreak provided the perfect opportunity for taipei health authorities to test the effectiveness of the newly renovated system. measures that were used to contain this epidemic may also, in turn, serve as a practice model for dealing with future influenza pandemics. there are many similarities between ahc and novel influenza: (1) there is currently no available vaccine for the circulating virus strain and anti-viral treatment is limited, (2) time from exposure to onset is very short, (3) the disease is highly contagious and easily contracted through contact with contaminated aerosols/droplets or fomites, and (4) schools serve as epicenters for the spread of the virus to households and, eventually, to the larger community. it has been previously demonstrated that home confinement of symptomatic children can dramatically limit the spread of ahc and influenza in the community [25, 26, 27] . the timely launch of our multi-channel risk communication program to all taipei citizens on friday, october 12, allowed us to provide clear instructions to families on how to prevent the infection at home over the course of weekend, when infection rates would normally increase. these direct communication methods successfully convinced parents to keep their symptomatic children at home (from 10% to 60% in table 3 ). with contagious students confined to their homes, school transmissions decreased dramatically and public health officials were able to contain the taipei outbreak quickly. the effectiveness of these innovative methods in the heavily populated metropolitan area of taipei city was evidenced by how quickly the epidemic, which had infected 5414 students, subsided after 2 weeks. this can be compared with the two months that were needed to control taipei's previous cv-24 epidemic in 1987 [28] . the importance of appropriate risk communication in response to disasters has often been overlooked by public health officials in taiwan. traditionally, the mass media has helped disseminate epidemic information and increase awareness on how health risks may be reduced. however, the modern mass media has, at times, had a negative impact on public health efforts by encouraging public indifference or sensationalizing incomplete and inaccurate information. the media's reporting of the sars outbreak contributed to unnecessary chaos in the early phases of the epidemic [29] . rather than rely on the media alone to convey productive messages regarding the epidemic, taipei's cdc was able to implement its multi-channel mass risk communication program to reach the public directly during critical points in the epidemic. we believe the sms messaging component of the program was integral to the success of the 2007 ahc intervention and will continue to explore the use of this tool. we know from our questionnaire that fourteen percent of parents who confined their affected children at home during 2007's ahc outbreak reported that their decision to comply with this preventive measure was based on the sms messages they received. although the effects of the cell phone method cannot be fully isolated from the multichannel risk communication system in this study, we believe that future interventions that utilize sms exclusively will provide more insight on the effectiveness of this method. taipei's cdc has since conducted a sms campaign on chinese valentine's day in 2009 to reach high-risk groups for human immunodeficiency virus (hiv). the message asked the public to answer an aids-related trivia question and also provided information on free, anonymous hiv testing. after this exploratory campaign, anonymous hiv screening rates went up 20% from the same time the year before. as sms messaging is still an emerging approach to wide-scale information dissemination, we believe that further examination into the effectiveness of cell phone-based risk communication methods will need to be done at the local, national, and international level. taipei's ability to launch such a large-scale sms campaign was a direct result of taiwan's communicable disease act (2006). this act allowed government officials to override the people's right to privacy when responding to epidemic disasters. in this case, the taipei city government held a contract with taiwan's six major mobile phone companies and committed all of them to allowing six free public service messages (per year) to be sent to their users if deemed necessary by the proper authorities. the sms message in response to the ahc epidemic was sent to 2.2 million registered mobile phone users in taipei city. public satisfaction with the sms campaign was high, especially amongst parents of school children. while this mass communication method has been advocated for use in many asian countries, taiwan's large-scale employment of sms technology was, to the best of our knowledge, the first such attempt in the world. while it was, overall, an effective means of communication, the efficacy of this method was limited by several factors. first, the sudden influx of over two million messages put a large burden on the network system and resulted in a long delay (many received their message around midnight). second, many taipei mobile phones users are registered in other cities and did not receive the messages. third, consumer weariness may have caused some people to ignore the long, unsolicited message before they read it. to reduce technical limitations, we suggest that sms surge capacity be increased and tested at both non-epidemic and pre-pandemic stages. other limitations to the sms tool and alternative mass communication prevention methods can be fine-tuned with more experience, frequently updated guidelines, an efficient system for risk management, an integrated public health plan, and extra training for public health personnel [30, 31] . another important challenge is reaching diverse populations, particularly those with low socio-economic status that may not be able to afford mobile phone service (and reside in less affluent, high-risk areas) [32] . this may be less of problem in taiwan than in other countries. this study had several limitations, including a lack of early data on pink-eye cases in keelung. after the conclusion of the outbreak, there was also no further assessment on the effectiveness of control measures in keelung. this study was non-randomized and missing epidemiological information for calculating secondary attack rates, age-specific asymptomatic ratios, and geographical diffusion of ahc cases outside taipei and keelung. seroepidemiological studies in the future should be able to obtain more accurate secondary attack data with the use of comprehensive infection and disease exposure data. in conclusion, the timely launch of the multi-channel risk communication program described in this study greatly reduced the duration and number of cases of taiwan's 2007 ahc epidemic. these efforts effectively avoided a potentially large-scale epidemic of ahc in taipei city. in encountering challenges such as outbreaks of influenza pandemic, or other eids with short incubation periods, public health officials need to prepare an integrated and timely administrative public health response. urgent intervention and education must reach the community directly through multi-module channels, like sms, for rapid communication. geographical variations in epidemiological characteristics, as described in our comparison analysis of the ahc epidemic in keelung versus taipei, are similar to variations in swine-origin h1n1 outbreaks in mexico versus united states. such similarities support this intervention's potential applicability to the prevention and control of other eids. based on the findings of this study, we believe that the success of this risk communication method is dependent on: (1) the timeliness of the communication, (2) simplicity and consistency of the message, (3) appropriateness of the channels of dissemination, (4) transparency of the information, and (5) public faith in the communicator; in this instance the mayor of taipei. while we found that the use of sms significantly contributed to the effectiveness of the risk communication program, more sms-based campaigns and research, like the hiv testing campaign mentioned in our manuscript, are needed to fully evaluate its effectiveness at communicating public health messages to the public. file s1 informed consent and questionnaire. written informed consent was obtained before parents of the participated schoolchildren responded to the questionnaire. found at: doi:10.1371/journal.pone.0007962.s001 (0.12 mb pdf) unusual type of epidemic conjunctivitis in 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epidemic intensity during the 1918 influenza pandemic acute hemorrhagic conjunctivitis investigation of a large-scale community outbreak in dade county preparedness for the spread of influenza: prohibition of traffic, school closure, and vaccination of children in the commuter towns of tokyo outbreaks of influenza and influenza-like illness in schools in england and wales outbreak of acute hemorrhagic conjunctivitis due to coxsackie a24 variant-taiwan sars wars: an examination of the quantity and construction of health information in the news media infectious diseases and governance of global risks through public communication and participation ethical decision making in a crisis: a case study of ethics in public health emergencies infectious diseases and governance of global risks through public communication and participation we greatly appreciate local public health professionals who helped coordinate and implement efforts for controlling the 2007 ahc outbreak. we are also grateful to the taiwan cdc for their laboratory support. the authors would also like to thank two english editors, mr. james steed and ms. peggy lee, for their assistance in the final edits of this manuscript. conducted geographical diffusion data analysis using a geographical information system: tcc. responsible for designing the epidemiological study: cck. formulated questionnaire questions: cck. selecting focus areas for data analysis: cck. revised this manuscript: cck. key: cord-253436-dz84icdc authors: wille, michelle; muradrasoli, shaman; nilsson, anna; järhult, josef d. title: high prevalence and putative lineage maintenance of avian coronaviruses in scandinavian waterfowl date: 2016-03-03 journal: plos one doi: 10.1371/journal.pone.0150198 sha: doc_id: 253436 cord_uid: dz84icdc coronaviruses (covs) are found in a wide variety of wild and domestic animals, and constitute a risk for zoonotic and emerging infectious disease. in poultry, the genetic diversity, evolution, distribution and taxonomy of some coronaviruses have been well described, but little is known about the features of covs in wild birds. in this study we screened 764 samples from 22 avian species of the orders anseriformes and charadriiformes in sweden collected in 2006/2007 for cov, with an overall cov prevalence of 18.7%, which is higher than many other wild bird surveys. the highest prevalence was found in the diving ducks—mainly greater scaup (aythya marila; 51.5%)—and the dabbling duck mallard (anas platyrhynchos; 19.2%). sequences from two of the greater scaup cov fell into an infrequently detected lineage, shared only with a tufted duck (aythya fuligula) cov. coronavirus sequences from mallards in this study were highly similar to cov sequences from the sample species and location in 2011, suggesting long-term maintenance in this population. a single black-headed gull represented the only positive sample from the order charadriiformes. globally, anas species represent the largest fraction of avian cov sequences, and there seems to be no host species, geographical or temporal structure. to better understand the eitiology, epidemiology and ecology of these viruses more systematic surveillance of wild birds and subsequent sequencing of detected cov is imperative. coronaviruses (covs) are found in a wide variety of animals in which they can cause respiratory, enteric, hepatic, and neurological disease of varying severity. in humans, covs cause a large fraction of common colds [1] , as well as more rare, but serious disease such as the outbreak of severe acute respiratory syndrome (sars, caused by sars-cov) in 2003, and the polymerase (rdrp) were generated using methods described in [14] or [15] , and sequences generated in this study have been deposited in genbank under accession numbers kt882615-28. resulting sequences were aligned using the mafft algorithm [16] implemented in geneious r7 (biomatters, new zealand). the nucleotide substitution model was determined in mega 5.2 [17] and maximum likelihood trees were constructed using phyml [18] with alrt branch support in seaview [19] . trees were projected using figtree v1.4 [20] . a total of 764 birds from 11 species of anserifomes (ducks, geese, swans, n = 691) and 11 species of charadriiformes (gulls, terns, shorebirds, n = 143) were sampled in this study. overall, the prevalence of cov was high, at 18.7%, but species, groups and orders were unevenly represented. diving ducks had the highest prevalence (39%), driven by high prevalence in greater scaup (aythya marila; 51.5%), however the sample size was small (n = 37). dabbling ducks of the genus anas also had high prevalence; mallard, in particular, had a prevalence of 19.2%. while anseriformes had the highest prevalence, in the order charadriiformes, cov was detected in a black-headed gull (chroicocephalus ridibundus), but absent in tern and wader species sampled (table 1) . eleven sequenced mallard cov and three sequenced scaup cov were gammacoronaviruses. putative maintenance, despite no global spatial, temporal, host species patterns a global phylogeny of all sequenced avian gammacoronaviruses (not ibv-like) rdrp show no spatial, temporal or host species differentiation between clades. that is, all clades contain sequences from multiple geographic locations, different years of sampling, and different host species (fig 1a, s1 and s2 figs) . indeed, viruses sequenced in this study were placed in a clade containing sequences from hong kong, china, beringia, the united states of america and sweden (fig 1b and s2 table) . however, despite limited global patterns of clade differentiation, 8/10 sequences from mallards in 2007 were most closely related (within 98-99% identity) to cov sequences from mallards at ottenby in 2011 (fig 1b and s1 table) , suggesting local circulation of these rdrp. mallard cov sequence 69998 had the highest pairwise identity to viruses from hong kong, but is nested in the broad and highly similar clade containing the largest number of sequences from ottenby in 2011 ( fig 1b) . a single mallard cov sequence (69740) was in a differentiated clade, most similar to sequences from hong kong and more distantly china (fig 1b and s2 table and s2 fig) . sequences from scaup cov were not similar to mallard cov; scaup 67699 was similar to sequences from beringia and china (within 98% identity; s1 table) . two scaup cov sequences (67693 and 67703) were highly similar to only one sequence in genbank, which was collected from a tufted duck (aythya fuligula) in hong kong (jn788847). all other sequences were less than 92% similar, indicating a differentiated lineage that is poorly sampled (fig 1b and s1 in this study we aimed to further contribute to the natural history of avian coronaviruses by screening an array of waterbirds for these viruses. we found a prevalence of 18.7% cov, which is higher than the 0-15% reported previously in wild bird studies [11, 14, 15, [21] [22] [23] [24] [25] [26] [27] . this may be due to the high proportion of dabbling ducks, particularly mallard, in our study, and the temporal and spatial features of the dabbling duck sampling. more specifically, sampling of dabbling ducks occurred at a migratory stopover site, and thus high prevalence could be driven by migrants which not only import viruses through infected individuals, but also replenish the pool of susceptible individuals across the migratory period, resulting in high prevalence at these locations [28, 29] . directly comparing prevalence estimates to previous studies, however, is challenging due to the array of different methods utilized, ranging from conventional pcr to multiplex real-time pcr, in addition to species distributions and seasonal variations. regardless, this study corroborates previous studies suggesting the importance of dabbling ducks (anas sp) as hosts of avian coronaviruses [11] [12] [13] [14] [15] [21] [22] [23] [24] [25] , but also highlighting the importance of diving ducks. the high diving duck prevalence in this study was driven by high prevalence in greater scaup, which could be due to congregation in very large flocks facilitating transmission, but could also be due to other factors such as a local variation in disease prevalence or reflect outbreak dynamics wherein the virus rapidly expands across the susceptible population. overall, diving ducks have only been sampled in this study and by chu et al. (2011) , so it is unclear whether this pattern of high prevalence is present globally. we found a low prevalence in waders, which is in contrast to [14] and [21] , which found a very high prevalence (>20%) in waders, but corroborates findings in [22, 25, 26] . this may be due to differences in congregation and feeding patterns of waders between the locations, but has to be interpreted with caution, as numbers of waders sampled in our study, and others, were small. finally, while assessment of gulls is limited, this study corroborates findings with muradrasoli et al (2010) indicating that this group requires further scrutiny. in order to better assess cov dynamics, resampling the same site across time is imperative, and this is the first study to do so. coronaviruses from mallards were previously assessed using samples collected at ottenby in 2011 [15] and we detected high similarity between viruses from 2007 and those from 2011. indeed, 8/10 cov were most closely related to sequences from 2011, which strongly suggests maintenance and/or location transmission of these rdrp lineages at this location. this is unlikely to be a coincidence as swedish mallard cov make up less than 1/3 of those available sequences, and all current rdrp clades are comprised of sequences from asia. the waterfowl host appears important in these samples, however assessing host species biases is challenging due to the overwhelming number of sequences from anas species. however, coronaviruses from diving ducks were rather different from existing diversity as demonstrated by the similarity between two of three scaup cov sequences from this study and tufted duck cov sequence from hong kong. the limited number of sequences in this clade could be due to sampling shortcomings, or that currently developed primers do not amplify this clade effectively. despite few studies, small samples sizes and differences in prevalence, what is clear, is that in the northern hemisphere waterfowl species, especially dabbling and diving ducks are important in the epidemiology of avian covs. it is interesting to note that these patterns are very similar to those found in low pathogenic influenza a viruses: high prevalence in waterfowl and gulls in the northern hemisphere [30] , and little host species and temporal structuring within waterfowl derived viruses in the conserved polymerase genes (such as pb2, pb1) [31] . further, detection in fecal or cloacal samples suggest these viruses may also replicate in the gastrointestinal tract, which is the true for turkey coronavirus [9] . as demonstrated by years of influenza sampling [32] , to better understand the eitiology, epidemiology and ecology of these viruses more systematic surveillance needs to be undertaken and more viruses need to be sequenced, particularly full genomes. given the importance of coronaviruses as human pathogens, as exemplified by sars and mers-cov and the potential for wild birds as reservoirs, spreaders and mixing vessels, further studies of coronaviruses in wild birds are warranted. human coronaviruses: what do they cause? antivir ther coronaviruses: important emerging human pathogens bats are natural reservoirs of sars-like coronaviruses middle east respiratory syndrome (mers): a new zoonotic viral pneumonia. virulence virus taxonomy: classification and nomenclture of viruses: ninth report of the internaltional committee on taxonomy of viruses interspecies transmission and emergence of novel viruses: lessons from bats and birds discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavius and avian coronavirus as the gene source of gammacoronavirus and deltacoronavirus evolutionary insights into the ecology of coronaviruses turkey coronavirus is more closely related to avian infectious bronchitis virus than to mammalian coronaviruses: a review pathogenicity of turkey coronavirus in turkeys and chickens avian coronavirus in wild aquatic birds genomic analysis and surveillance of the coronavirus dominant in ducks in china broadly targeted multiprobe qpcr for detection of coronaviruses: coronavirus is common among mallard ducks (anas platyrhynchos) prevalence and phylogeny of coronaviruses in wild birds from the bering strait area (beringia) temporal dynamics, diversity, and interplay in three components of the viriodiversity of a mallard population: influenza a virus, avian paramyxovirus and avian coronavirus multiple alignment of dna sequences with mafft molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods estimating maximum likelihood phylogenies with phyml seaview version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building figtree v1.1.1: tree figure drawing tool diverse gammacoronaviruses detected in wild birds from madagascar detection and molecular characterization of infectious bronchitis-like viruses in wild bird populations genetically diverse coronaviruses in wild bird populations of northern england molecular identification and characterization of novel coronaviruses infecting graylag geese (anser anser), feral pigeons (columbia livia) and mallards (anas platyrhynchos) identification of avian coronavirus in wild aquatic birds of the central and eastern usa surveillance of avian coronaviruses in wild bird populations of korea absence of coronaviruses, paramyxoviruses, and influenza a viruses in seabirds in the southwestern indian ocean animal migration and infectious disease risk juveniles and migrants as drivers for seasonal epizootics of avian influenza virus global patterns of influenza a virus in wild birds the evolutionary genetics and emergence of avian influenza a viruses in wild birds spatial, temporal, and species variation in prevalence of influenza a viruses in wild migratory birds we wish to thank and the duck trappers at ottenby bird observatory and jonas waldenström for collecting and providing samples used in this study, jonas blomberg for kindly providing sequence, mallard cov sequences generated in this study are indicated with a filled circle and scaup cov sequences with an asterisk. scale bar represents number of substitutions per site. traditional projection of panel a with support values is presented in s2 fig. key: cord-281124-4nhy35xn authors: soowannayan, chumporn; cowley, jeff a.; michalski, wojtek p.; walker, peter j. title: rna-binding domain in the nucleocapsid protein of gill-associated nidovirus of penaeid shrimp date: 2011-08-03 journal: plos one doi: 10.1371/journal.pone.0022156 sha: doc_id: 281124 cord_uid: 4nhy35xn gill-associated virus (gav) infects penaeus monodon shrimp and is the type species okavirus in the roniviridae, the only invertebrate nidoviruses known currently. electrophoretic mobility shift assays (emsas) using his(6)-tagged full-length and truncated proteins were employed to examine the nucleic acid binding properties of the gav nucleocapsid (n) protein in vitro. the emsas showed full-length n protein to bind to all synthetic single-stranded (ss)rnas tested independent of their sequence. the ssrnas included (+) and (−) sense regions of the gav genome as well as a (+) sense region of the m rna segment of mourilyan virus, a crustacean bunya-like virus. gav n protein also bound to double-stranded (ds)rnas prepared to gav orf1b gene regions and to bacteriophage m13 genomic ssdna. emsas using the five n protein constructs with variable-length n-terminal and/or c-terminal truncations localized the rna binding domain to a 50 amino acid (aa) n-terminal sequence spanning met(11) to arg(60). similarly to other rna binding proteins, the first 16 aa portion of this sequence was proline/arginine rich. to examine this domain in more detail, the 18 aa peptide (m(11)pvrrplppqpprnarli(29)) encompassing this sequence was synthesized and found to bind nucleic acids similarly to the full-length n protein in emsas. the data indicate a fundamental role for the gav n protein proline/arginine-rich domain in nucleating genomic ssrna to form nucleocapsids. moreover, as the synthetic peptide formed higher-order complexes in the presence of rna, the domain might also play some role in protein/protein interactions stabilizing the helical structure of gav nucleocapsids. gill-associated virus (gav) infects penaeus monodon shrimp and is the type species of the genus okavirus in the roniviridae, the only currently known invertebrate nidoviruses. in all other nidoviruses, the nucleocapsid (n) protein is encoded by a gene located near to the genome 39-terminus and downstream of genes encoding other virion structural proteins [1] . however, in gav and other genotypic variants in the yellow head virus (yhv) complex, the n protein is encoded in the orf2 gene which resides immediately downstream of the 20 kb 59-terminal orf1a/1b replicase gene [2, 3] . the deduced molecular masses of the 144-146 amino acid (aa) n proteins of gav and yhv (16.0-16.3 kda) are lower than those estimated by sds-page (20) (21) (22) , which for yhv has been reported to be due to a c-terminal cluster of acidic residues [3, 4] . immuno-electron microscopy has confirmed that the n protein is the primary structural protein component of okavirus nucleocapsids [2, 5] . amongst strains of genotypes 1 (yhv), 2 (gav), 3, 4 and 5 in the yhv complex [6] , most amino acid variations (up to 17.2%) in the deduced n protein sequence occur in the highly charged nand c-terminal domains [3, 6] . nonetheless, the n proteins of gav and yhv share common antigenic sites as evidenced by their cross-reactivity for a yhv n protein monoclonal antibody [5, 7] and polyclonal antiserum to a synthetic peptide designed to a c-terminal sequence of the gav n protein [2] . as with the n proteins of coronaviruses and toroviruses [8, 9] , the n proteins of gav and yhv lack cysteine residues and are highly basic [2, 3] . it also possesses proline-rich and basic residue-rich domains likely to facilitate rna binding as hypothesized for similar sequences in the n protein of toroviruses [2, 10] . the n protein length in okaviruses is intermediate to the corresponding proteins of arteriviruses (110-128 aa) and toroviruses (160-167 aa), and much shorter than the n protein of coronaviruses (377-454 aa) [2, 11, 12, 13, 14] . the process by which n proteins encapsidate genomic rna to form nucleocapsids has been examined in many rna viruses [8, 15, 16, 17] and in coronaviruses, as an example, the n protein interaction with rna shows no preference for sequence, indicating that the specific nucleation of viral rna likely requires additional factors [8, 18] . here we have examined recombinant gav n protein constructs in electrophoretic mobility shift assays (emsas) to identify its nucleic acid binding specificities in vitro, and its rna binding domain, as initial steps to understanding the process by which nucleocapsids form in okaviruses. the n protein was found to bind ssrna, dsrna as well as ssdna in a sequence independent manner and the rna binding site was localized to an 18 aa proline/arginine-rich sequence near to its n-terminus. a full-length orf2 gene and five constructs designed to produce n proteins with n-and/or c-terminal truncations (fig. 1a) were cloned into pqe10 and used to express recombinant his 6 -n fusion proteins in e. coli. the purity of the expressed proteins following ni 2+ -nta affinity chromatography and assessed by sds-page and cbb staining is shown in fig. 1b . migration of the full-length his 6 -n protein (qe10-n2; 144 aa, 16.0 kda deduced mass) trailed the 20.1 kda marker protein slightly, consistent with an elevated mass estimated previously [4] plus the additional ,1 kda mass of the n-terminal his 6 sequence. the his 6 -n protein construct qe10-n1 (134 aa, 14.8 kda) with a 10 aa n-terminal truncation migrated slightly below qe10-n2 and constructs qe10-n14 (84 aa, 9.3 kda) and qe10-n38 (51 aa, 5.6 kda) with 60 aa and 93 aa n-terminal truncations, respectively, migrated at lower masses of ,16.5 and ,15.5 kda, respectively. construct qe10-n22 (93 aa, 10.4 kda) with a 51 aa c-terminal truncation migrated at ,15 kda, and protein qe10-n25 (50 aa, 5.52 kda) with 10 aa and 60 aa n-and c-terminal truncations, respectively, migrated at ,10 kda. in addition to using cbb-stained gels (fig. 1b) , the fidelity of the recombinant his 6 -n protein constructs was confirmed in western blots using horseradish peroxidase (hrp) labeled-ni 2+ to detect the his 6 -tag on each protein (fig. 1c) . except for qe10-n14, all his 6tagged proteins were detected and were estimated to be .90% pure, with single bands detected for all but qe10-n1 and qe10-n38 by both methods. the qe10-n1 protein preparation contained additional minor bands of ,22 kda and ,29 kda. the qe10-n38 preparation contained a minor band (,28 kda) about twice the size of the primary protein suggesting that it might be a protein dimer rather than a contaminant. the reason why the recombinant qe10-n14 protein with a 61 aa n-terminal truncation failed to bind the ni 2+ -hrp probe following sds-page and transfer to a nitrocellulose membrane is not known, but possibly it was due to some refolding of the protein that rendered the nterminal his 6 -tag inaccessible [19] . yields of the purified proteins were estimated to be in the order of 10-30 mg/ml of bacterial culture. moreover, the identity of each recombinant n protein was confirmed by matrix-assisted laser desorption ionization time-offlight (maldi-tof) mass spectrometry analyses of peptides generated following tryptic digestion (data not shown). to assess the ability of the recombinant gav n proteins to bind rna in vitro, a total of 15 (+) and (2) sense ssrnas ( fig. 2a) synthesized to different regions of the gav genome (supplementary table s1 ) were examined in electrophoretic mobility shift assays (emsas). gav genome regions selected for analysis included the 59-and 39-terminal sequences, intergenic regions, and sequences in orf1a, orf1b, orf2, orf3 and orf4. of the 15 synthetic ssrnas, all but one (ssrna-4) resolved as a single band following electrophoresis in a non-denaturing agarose gel. emsa binding reactions (20 ml) were conducted at ph 8.0 in the presence of 100 mm nacl and contained 300 ng each synthetic ssrna and 1 mg full-length gav his 6 -n protein (qe10-n2) equivalent to rna:protein molar ratios ranging from 1:17 to 1:71 for the longest to shortest ssrnas. using these binding conditions, emsas showed that the gav n protein could bind to all ssrnas examined irrespective of sequence or polarity (fig. 2b) . migration shifts were smaller with the shorter ssrnas (i.e., ssrna-12, -13, -14 and -15), with protein-bound ssrna complexes migrating as smears slightly behind free ssrna. the likely reason for this was the rna:n protein molar ratios of the shorter ssrnas (approximately 1:17 for ssrna-12 and -13, and 1:27 for ssrna-14 and -15), which were considerably lower than for the longer ssrnas. in the presence of the full-length gav n protein, migration of a 849 nt ssrna synthesized to the mourilyan virus (mov) m rna segment was retarded similarly to equivalent-sized gav ssrnas (fig. 3a) . gav n protein binding to two dsrnas was also assessed (fig. 3b) . for dsrnas prepared from (+) ssrna-1 and (2) ssrna-3 to the genome 59-terminus and from (+) ssrna-6 and (2) ssrna-8 to the orf2 gene, migration in the presence of the full-length n protein was retarded as evidenced by a trailing smear above the primary dsrna band suggestive of variability in n protein binding amounts. gav n protein binding to dsdna and ssdna was also assessed (figs. 3c and 3d). using dsdnas comprising pcr products used as templates to synthesize gav ssrna-1 and ssrna-2 and the same reaction conditions used to assess ssrna binding, no evidence was obtained for the migration of either dsdna being retarded, although some smeared material that migrated more slowly than the primary band was observed (fig. 3c) . for the 6.4 kb genomic ssdna of bacteriophage m13, migration was clearly retarded in the presence of full-length n protein (fig. 3d) . to investigate interactions between the gav n protein and ssrna in more detail, emsas were undertaken using varied molar ratios of full-length n protein and ssrna-1 (fig. 4) . using a constant amount of ssrna in the presence of increasing amounts of n protein (ssrna:protein molar ratios of 1:10 to 1:100), ssrna migration was clearly retarded at a ratio of 1:20 and at ratios of 1:30 or greater, little if any ssrna with unaltered migration was evident (fig. 4a ). starting with a ssrna:protein ratio of 1:100, ssrna amounts were then increased progressively to return the ssrna:protein molar ratio to 1:10, and ssrna migration was observed to be retarded less as ssrna amounts increased (fig. 4b) . from either data set, it was evident the ssrna migration was retarded to similar extents at comparable ssrna:protein molar ratios (figs. 4a and 4b). to identify the gav n protein domain responsible for rna binding, emsas were undertaken using his 6 -n protein constructs with n-or c-terminal deletions or both (fig. 1a) and gav ssrna-1, -2 and -6 (supplementary table s3 ). as shown in fig. 5 , the migration of all three ssrnas was retarded in the presence of the full-length n protein and the constructs qe10-n1 and qe10-n22 containing 10 aa n-terminal and 51 aa c-terminal deletions, respectively. in the presence of n protein construct qe10-n25 containing both a 10 aa n-terminal and a 84 aa cterminal deletion, the amounts of ssrna-1 and -2 detected in the gel were low and ssrna-6 failed to enter the gel altogether. however, the migration of ssrna-1 and -2 that entered the gel was clearly retarded. migration of none of the three ssrnas was retarded in the presence of the n protein constructs qe10-n14 and qe10-n38 containing n-terminal truncations of 60 aa and 95 aa, respectively. as these data indicated that the n-terminus of the gav n protein was essential for ssrna binding, an 18 aa synthetic peptide m 11 pvrrplppqpprnarli 29 corresponding to a proline/arginine-rich domain in this region was tested in emsas for its ability to bind to various ssrnas (fig. 6a) . using ssrna-1, -2, -3, -6, -7 and -8 at ssrna:peptide molar ratios of approximately 1:319, 1:277, 1:319, 1:268, 1:221 and 1:268, respectively, the migration of each ssrna was retarded substantially. trailing ladder-like patterns were also evident particularly for ssrna-1 and ssrna-2, possibly due to the ssrna-peptide complexes aggregating or forming multimers. for ssrna-3 and ssrna-8, ssrnas were so aggregated in the presence of peptide that little ssrna was able to enter the gel. similarly when peptide binding to bacteriophage m13 ssdna was assessed, ssdnapeptide complexes or aggregates formed that precluded any ssdna from migrating into the gel (fig. 6b) . the same effect was observed when dsdna pcr products used to prepare ssrna-1 and ssrna-2 were incubated with the peptide, although small quantities of non-aggregated dsdna with unaffected migration were also evident in the gel (fig. 6b ). here we have characterized the rna binding properties and identified the rna binding domain of the gav n protein using agarose gel emsa analysis of various synthetic rnas reacted with various recombinant n protein constructs expressed in bacteria as well as a synthetic peptide. purified full-length gav n protein bound readily to synthetic (+) and (2) sense ssrnas prepared to various gav genome regions. the n protein also bound to a (+) sense ssrna prepared to the m rna segment of mourilyan virus (mov), a shrimp bunya-like virus, to dsrnas prepared from (+) and (2) sense gav ssrnas, and to genomic ssdna of bacteriophage m13 but not to dsdna amplified by rt-pcr from two gav genome regions. the ability to bind rna was expected due to the structural role of the n protein in okavirus nucleocapsids [2, 3, 5, 20] . the fact that rnas were bound irrespective of their sequence or polarity indicates that specific nucleation sequences, rna folding structures or other factors might be needed to direct the gav n protein to encapsidate genomic (+) ssrna, as with the n proteins of many viruses, including coronaviruses [8, 21] , which share the ability to bind rna in non-sequence specific manner. this may be due to the basic charge properties of these n proteins. as the gav n protein possesses more basic amino acids (20/144 = 13.9%, pi 9.84) than acidic amino acids (13/144 = 9.0%) [2] , its net positive charge is likely to promote association with polyanions such as rna soon after its synthesis. however, the processes directing specific binding of okavirus n protein to genomic rna to form nucleocapsids in the potential presence of other rnas also capable of binding clearly need to be investigated further, and experiments to examine this would be assisted greatly by shrimp cell culture systems capable supporting virus replication becoming available. to localize the nucleic acid binding domain, recombinant his 6tagged gav n proteins were expressed in e. coli from various plasmid constructs containing the full-length orf2 gene or five gene fragments designed to generate variable-length n-and/or cterminal truncations. the presence of the his 6 -tag allowed the proteins to be purified by ni 2+ -affinity chromatography and in sds-page analyses, all six purified recombinant n proteins possessed masses equivalent to those predicted from the length of truncations, the addition of the n-terminal his 6 -sequence, and knowledge that the masses of the gav and yhv n proteins estimated by sds-page are higher than those deduced from their amino acid sequences [3, 4, 22] . emsa data generated using three different ssrnas reacted with these truncated gav n protein constructs localized the rna binding domain to a 50 aa n-terminal sequence spanning met 11 -arg 60 . in this region, the gav n protein sequence is quite basic (theoretical pi = 11.72) due to the presence of eight positively-charged residues and only two negatively-charged residues. database searches undertaken using the interproscan program (http://www.ebi.ac.uk/interproscan/) [23] were unable to identify sequences close in similarity to other better characterized rna-binding or rna-recognition motifs. however, some general sequence similarities, mainly relating to the relatively high proportion of arginine residues, were noted with the short arginine-rich motifs (arms) that mediate rna binding of the rev and tat proteins of human immunodeficiency virus (hiv) [24] , and of the anti-terminator n protein of bacteriophages q21 and p22 [25] . (table 1) the propensity of the met 11 -arg 60 region of the gav n protein to bind nucleic acids was confirmed in emsa analyses of aproline-and arginine-rich peptide sequence (m 11 pvrrplppqpprnarli 29 ) synthesized to the first 18 aa portion of region, and which was found to bind strongly to ssrna as well as ssdna and dsdna. proline-rich protein sequences are known to primarily form helices rather than b-sheets, and in the p33 replicase protein of tomato bushy stunt virus (tbsv), for example, an arginine/proline-rich rprrrp motif has been shown to bind ssrna in preference to dsrna or dsdna [26] . however, as the n protein peptide displayed little obvious binding preference for ssrna compared to dsdna, additional analyses using variant peptide sequences will be needed to identify which amino acids are responsible for conferring this broad nucleic acid binding capability. the n protein of the coronavirus mouse hepatitis virus (mhv) has also been found to possess an ability to bind both genomic and sub-genomic viral rnas as well as cellular rnas to form ribonucleoprotein (rnp) complexes [27] . however, during the encapsidation process, association of the viral membrane (m) protein confers selective binding of genomic-length (+) ssrna directed by a 190 nt packaging signal positioned toward the 39-end of the orf1b gene [27] . in a preliminary attempt to identify an rna packaging signal in the gav genome, emsas were performed using ssrnas synthesized to various genome regions including (i) an orf1b gene 39-region spanning the relative position to the genome packaging signal identified in mhv [28] , (ii) a 39-terminal genome region corresponding in position to the region in the infectious bronchitis virus (ibv) genome reported to contain an rna binding domain [29] and (iii) the 59-genomic rna terminus which, in coronaviruses, has also been reported to interact specifically with n protein [30] . however, recombinant gav n protein bound all synthetic ssrnas tested and whilst it is possible that its binding affinity and/or interaction kinetics varied amongst the rnas, these could not be quantified using the emsa method. thus, based on this uniformity of binding, other experimental approaches will be needed to identify what sequence, if any, in the gav genome acts as a packaging signal or confers specificity for n protein binding. as in mhv, the identification of such rna packaging signals will likely require establishment of cell culture systems to allow the characterization of sequences of mutant defective-interfering genomic rnas packaged into virions [28, 31] . in the emsas, increasing the molar ratio of n protein retarded ssrna migration more significantly, suggesting the binding of multiple n protein molecules to each rna and/or interactions occurring between n protein-ssrna complexes. based on molar ratios examined using a 626 nt ssrna, migration was visibly retarded more when the estimated molar n protein:ssrna ratio was increased from 20:1 to 30:1, and whilst no additional retardation was evident at a ratio of 50:1, it increased again at a ratio of 100:1. this apparent biphasic n protein ssrna interaction suggests that there is some point at which n protein binding becomes saturated, but once present in vast excess, either more than one complex type can form or unit complexes aggregate. a similar phenomenon occurs in prunus necrotic ringspot virus, a plant ilarvirus containing a tripartite (+)ssrna genome, in which two different complex types can form in a nonsequence-specific manner between the 32 kda movement protein (mp) and ssrna4 [32] . one ssrna complex forming in the presence of high mp amounts (mp:ssrna molar ratio 400:1) was observed to enter an emsa gel, whereas another complex type that formed at a lower molar ratio (120:1) did not. moreover, urea denaturation had little in any effect on the type of ssrna:mp complex formed at higher mp amounts but disaggregated the complex type formed in lower mp amounts, suggested that the latter might comprise rod-like structures restricting gel entry and that excess mp might promote increased protein-protein interactions resulted in a more compact globular ssrna-mp complex capable of entering the gel matrix [32] . although something similar might be the reason for the biphasic interaction of the gav n protein with ssrna, delineation of the nature of the various complexes that can form will require further investigation. based on its structural role in nucleocapsids [4, 5] , it was expected that the gav n protein would be capable of binding ssrna. indeed such ssrna binding activity was confirmed in emsa analyses of various rnas and recombinant n protein constructs. moreover, analyses with variably truncated n protein constructs as well as a synthetic peptide showed that this activity was not dictated by any specific sequence constraints and was localized to a short, highly-charged n-terminal motif rich in arginines and prolines. whilst these findings advance our understanding of the rna binding capabilities of the n protein of the crustacean okaviruses, additional studies are now needed to determine the mechanism by which genomic ssrna is nucleated either specifically or preferentially. cloning and expression of full-length and truncated gav his 6 -tagged n proteins the gav orf2 gene and sequences with variable 59-and/or 39-terminal truncations (fig. 1a) were amplified by pcr using various primers containing bam hi sites (supplementary table s2 ) to allow in-frame insertion with the n-terminal his 6 -tag of pqe10 (qiagen). a plasmid containing the entire orf2 gene [2] was used as template for all pcrs. amplified dna products were digested with bam hi and ligated into pqe10. plasmid dna was transformed into competent dh5a or m13[prep4] e. coli host strains and clones were grown on luria broth (lb) agar plates containing 100 mg/ml ampicillin or 100 mg/ml ampicillin plus 25 mg/ml kanamycin. to screen clones for orf2 inserts in the correct orientation, rapid colony pcrs were performed using the pqe-specific primer 59-cccgaaaagtgccacctg-39 (qia-gen) in combination with various orf2 gene-specific anti-sense primers (supplementary table s2 ). plasmid dna prepared to each clone selected for analysis was sequenced using the pqespecific primer and clone specific reverse primers to confirm the fidelity of the dna insert. two plasmid clones containing the inserts were each inoculated into 2 ml lb medium containing 100 mg/ml ampicillin (plus 25 mg/ml kanamycin for m13[prep4] cells) and grown overnight at 37uc in a shaking incubator. a portion (500 ml) of each culture was inoculated into 10 ml pre-warmed lb medium containing antibiotics and the culture was shaken vigorously at 37uc until the optical density (od) at 600 nm had reached 0.5. protein expression was induced by the addition of 0.1 to 1.0 mm final concentration of isopropyl-b-d-1-thiogalactopyranoside (iptg) and incubation at 37uc was continued overnight. cells were collected by centrifugation (8,0006g, 10 min, 4uc), disrupted by sonication in lysis buffer (300 mm nacl, 10 mm imidazole, 50 mm nah 2 po 4 ph 8.0) for non-denatured protein purification and the orf2 his 6 -fusion proteins were purified using ni +2 -nta agarose beads as described in the qiaexpressionist tm instruction manual (qiagen). the proteins were stored at 280uc until used in emsas. for some orf2 his 6 -fusion proteins, e. coli cells were lysed in 8 m urea and protein was purified under denaturing conditions as described in the qiaexpressionist tm manual. purified denatured proteins (500 ml each) were refolded by dialysis overnight at 4uc in 200 ml refolding buffer (3 m urea, 500 mm nacl, 10 mm reduced glutathione, 1 mm oxidized glutathione, 1 mm edta, 20 mm tris-hcl ph 7.1). the dialysis buffer was then replaced with 200 ml pbs and dialysis was continued for 24 h at 4uc. purified his 6 -orf2 fusion proteins were concentrated by centrifugation using either a ym3 or ym10 centricon tm membrane concentrator (millipore) and protein yields were quantified using a bca protein assay kit (pierce) and also estimated visually in comparison to protein standards resolved by sds-page and stained with coomassie brilliant blue (cbb) r250. yields of purified proteins were in the order of ,4 mg each. an aliquot of each purified recombinant his 6 -orf2 protein was separated by sds-page using a 15% polyacrylamide gel run at 120 v for 90 min. a pair of identically loaded gels was separated simultaneously. proteins in one gel were stained with cbb ( fig 1b) and proteins in the other were electro-transferred onto a hybond tm -c nitrocellulose membrane (amersham) using towbin transfer buffer (25 mm tris, 192 mm glycine, 20% methanol and 0.1% sds) and a hoefer semi-dry protein transfer system run at 225 ma for 90 min. the membrane was blocked with 5% skim milk powder in tris-buffered saline (tbs; 0.9% nacl in 100 mm tris-hcl ph 7.5) for 15 min. the blocked membrane was washed with three changes of tbs for 10 min each before being incubated for 35 min in ni 2+ -hrp (sigma-aldrich) diluted 1:1,000 in tbs. the membrane was washed with three changes of tbs for 10 min each and incubated in developing solution (30 mg 4-chloronaphtol, 2.5 ml ice-cold methanol and 20 ml hydrogen peroxide in tbs, 50 ml) for 30 s to 3 min to detect color signal. once a positive signal was observed, the reaction was stopped by incubation in tbs for 10 min. images on membranes (fig. 1c) and cbb-stained gels were captured using an epson perfection v200 photograph scanner. the synthetic peptide (mpvrrplppqpprnarli) was purchased from shanghai science peptide biological technology co. ltd., china. the lyophilized peptide, reported by the manufacturer to be 99.2% pure as determined by high performance liquid chromatography (hplc), was dissolved to appropriate concentration in double-distilled water. plus-and minus-sense ssrnas were synthesized from pcr products amplified from plasmids containing cdna inserts corresponding to various regions of the gav genome ( fig. 2a) . the plasmids included: (i) pgav22, containing a 623 nt orf1a gene sequence extending to the 59-terminus of the genome; (ii) pgav16, containing a 4282 nt sequence beginning 509 nt upstream of the orf1b gene 39-terminus and extending to a position 3773 nt downstream of the orf3 gene 59-terminus; and (iii) pgav12.1, containing a ,2.7 kb insert extending from a region within the orf3 gene to the gav genome 39-polya tail (supplementary table s1 ). sequences of pcr primers used to amplify dna products are shown in supplementary table s3 . in each pcr, either the sense or the anti-sense gav-specific primer included a 59-terminal t7 rna polymerase promoter sequence. each pcr contained ,100 ng plasmid dna, 25 pmol of each primer, 5 ml 106pcr buffer (670 mm tris-hcl ph 8.8), 3 ml 25 mm mgcl 2 , 1 ml 10 mm dntp mix, 0.5 ml 5.5 u/ml taq dna polymerase (promega) and dnase/rnase-free water was added to final volume of 50 l. thermal cycling conditions used in the pcrs were 95uc for 4 min, 35 cycles of 95uc for 30 s, 56uc for 30 s, and 72uc for 120 min, followed by 72uc for 7 min. pcr products were purified using a qiaquick tm gel purification kit (qiagen) according to the manufacturer's instructions. purified dna products were treated with klenow dna polymerase to remove 39-adenosine overhangs and end-filled using taq dna polymerase prior to rna synthesis. to synthesize rna, 1 mg purified dna was added to a 20 ml reaction prepared using the megascript tm high yield in vitro rna transcription kit (ambion), and rna synthesis and dna removal were performed according to the kit instructions. synthetic rna was purified using a megaclear tm rna purification column (ambion), eluted in 100 ml elution buffer (10 mm tris-hcl ph 8.0) and stored at 280uc. mourilyan virus (mov) rna was synthesized using 1 mg pst i-linearized pmov4.1 dna containing a 849 nt cdna corresponding to the m (membrane glycoproteins g1/g2) ssrna genome segment [33, 34] as a template for in vitro t7 rna transcription. double-stranded (ds)rnas corresponding to the gav genome 59-terminus and a sequence including the intergenic region downstream of orf1b to the end of orf2 were prepared by adding together equimolar amounts of plus-and minus-sense ssrnas in megaclear tm (ambion) elution buffer, heating at 95uc for 10 min and then annealing at room temperature for 30 min. the formation of dsrna was confirmed by comparing its migration to each ssrna in a 1% agarose-tae (40 mm trisacetate ph 8.0, 1 mm edta) gel. three pcr products selected randomly were used to assess nprotein binding to dsdna in emsas. single-stranded dna extracted from bacteriophage m13 using phenol chloroform extraction was kindly provided by dr andrew mcdevitt, csiro livestock industries, australia. an electrophoretic mobility shift assay (emsa) was used to identify n-protein binding to nucleic acid [32, 35] . in each assay, 300 ng synthetic ssrna/ssdna/dsdna in megaclear tm elution buffer (ambion) was heated at 85uc for 5 min and cooled at room temperature for 15 min before addition of gav his 6 -n fusion protein or synthetic peptide (1 mg each), 10 ml binding buffer (100 mm nacl, 50% glycerol in 10 mm tris-hcl ph 8.0) and 2 units of ribolock tm rnase inhibitor (fermentas). the reaction volume was adjusted to 20 ml with diethyl pyrocarbonate (depc)-treated water and it was incubated at room temperature for 30 min. following the addition of 2 ml 1% bromophenol blue tracking dye, the rna-protein sample was separated by electrophoresis in a 1% agarose-tae gel containing 0.5 mg/ml ethidium bromide. the relative migration of free nucleic acid and nucleic acid-n protein complexes was visualized using a uv transilluminator and gel images were recorded using a gel-doc documentation system (biorad). the molar ratio of ssrna-1 to full-length his 6 -n protein (pqe10-n2) in the reaction was 1:42. to study the effect of relative rna and n protein concentrations on binding, varying molar ratios of rna:n protein (1:0, 1:10, 1:20, 1:25, 1:30, 1:50, 1:100) were also tested. table s1 details of each gav synthetic ssrna examined including positions in gav genome, polarities and length and the plasmid dna used as template for pcr. (doc) table s2 pcr primers used to amplify full-length and truncated orf2 gene coding sequences to construct pqe10 gav n protein expression vectors and also used in colony pcrs to determine insert orientations following cloning. (doc) nidovirales: evolving the largest rna virus genome the gene encoding the nucleocapsid protein of gill-associated nidovirus of penaeus monodon prawns is located upstream of the glycoprotein gene structural and antigenic analysis of the yellow head virus nucleocapsid protein p20 multiplex rt-nested pcr differentiation of gill-associated virus (australia) from yellow head virus (thailand) of penaeus monodon detection and differentiation of yellow head complex viruses using monoclonal antibodies genetic diversity in the yellow head nidovirus complex monoclonal antibodies specific to yellow-head virus (yhv) of penaeus monodon background paper. functions of the coronavirus nucleocapsid protein identification and primary structure of the gene encoding the berne virus nucleocapsid protein identification and characterization of a porcine torovirus antigenic structure of the nucleocapsid protein of porcine reproductive and respiratory syndrome virus bovine torovirus: sequencing of the structural genes and expression of the nucleocapsid protein of breda virus sequence analysis of the bovine coronavirus nucleocapsid and matrix protein genes comparative analyses of the nucleocapsid genes of several strains of infectious bronchitis virus and other coronaviruses interactions amongst rabies virus nucleoprotein, phosphoprotein and genomic rna in virus-infected and transfected cells rabies virus chaperone: identification of the phosphoprotein peptide that keeps nucleoprotein soluble and free from non-specific rna localization of an rna-binding domain in the nucleocapsid protein of the coronavirus mouse hepatitis virus coronavirus nucleocapsid protein is an rna chaperone gene expression response to misfolded protein as a screen for soluble recombinant protein gill-associated virus of penaeus monodon prawns -molecular evidencefor the first invertebrate nidovirus. nidoviruses (coronaviruses and arteriviruses) in vitro assembly of an infectious cdna clone of infectious bronchitis virus and its application as a gene transfer vector yellow-head virus: a rhabdovirus-like pathogen of penaeid shrimp interproscan -an integration platform for the signature-recognition methods in interpro structural variety of arginine-rich rna-binding peptides rna binding specificity of hnrnp a1: significance of hnrnp a1 high-affinity binding sites in pre-mrna splicing characterization of the rna-binding domains in the replicase proteins of tomato bushy stunt virus cooperation of an rna packaging signal and a viral envelope protein in coronavirus rna packaging analysis of efficiently packaged defective interfering rnas of murine coronavirus -localization of a possible rna-packaging signal the infectious bronchitis virus nucleocapsid protein binds rna sequences in the 39 terminus of the genome specific interaction between coronavirus leader rna and nucleocapsid protein identification and characterization of a coronavirus packaging signal rna-binding properties and mapping of the rna-binding domain from the movement protein of prunus necrotic ringspot virus rt-nested pcr detection of mourilyan virus in australian penaeus monodon and its tissue distribution in healthy and moribund prawns preliminary molecular and biological characterisation of mourilyan virus (mov): a new bunya-related virus of penaeid prawns gel retardation conserved structures and diversity of functions of rna-binding proteins the authors would like to thank dr. andrew mcdevitt of csiro livestock industries, australia for providing bacteriophage m13 dna. key: cord-278018-3qemb0x3 authors: li, li; qiao, dan; fu, xiaoying; lao, suihua; zhang, xianlan; wu, changyou title: identification of m.tuberculosis-specific th1 cells expressing cd69 generated in vivo in pleural fluid cells from patients with tuberculous pleurisy date: 2011-08-22 journal: plos one doi: 10.1371/journal.pone.0023700 sha: doc_id: 278018 cord_uid: 3qemb0x3 th1 cell-mediated immune responses at the site of active infection are important to restrict the growth of m.tuberculosis (mtb) and for the spontaneous resolution of patients with tuberculous pleurisy (tbp). in the present study, we found that without any stimulation, cd4(+) t cells in pleural fluid cells (pfcs) from patients with tbp expressed significantly higher levels of cd69 than pbmcs from patients with tuberculosis (tb) or healthy donors. cd4(+)cd69(+) t cells expressed t-bet and il-12rβ2. after stimulation with mtb-specific antigens, cd4(+)cd69(+) t cells expressed significantly higher levels of ifn-γ, il-2 and tnf-α than cd4(+)cd69(−) t cells, demonstrating that cd4(+)cd69(+) t cells were mtb-specific th1 cells. in addition, cd4(+)cd69(+) t cells were mostly polyfunctional th1 cells that simultaneously produced ifn-γ, il-2, tnf-α and displayed an effector or effector memory phenotype (cd45ra(−)ccr7(−)cd62l(−)cd27(−)). moreover, the percentages of cd4(+)cd69(+) t cells were significantly and positively correlated with polyfunctional t cells. interestingly, sorted cd4(+)cd69(+) but not cd4(+)cd69(−) fractions by flow cytometry produced ifn-γ, il-2 and tnf-α that were significantly regulated by cd4(+)cd25(+) treg cells. taken together, based on the expression of cd69, we found a direct quantitative and qualitative method to detect and evaluate the in vivo generated mtb-specific polyfunctional cd4(+) t cells in pfcs from patients with tbp. this method can be used for the potential diagnosis and enrichment or isolation of mtb-specific th1 cells in the investigations. tuberculous pleurisy (tbp) is characterized by an intense chronic accumulation of inflammatory cells at the disease site, including the activation of th1 cells and their preferential recruitment to the affected areas [1, 2] . however, tbp remains difficult to identify despite numerous diagnostic tools [3] . currently, the definite diagnosis of tuberculosis (tb) pleural effusions depends on the demonstration of acid-fast bacilli in the sputum, pleural fluid, or pleural biopsy specimens [4] . moreover, high levels of ifn-c are detected in tuberculosis pleural fluid [5, 6] . therefore, measurement of ifn-c in the pleural fluid has also gained wide acceptance in the diagnosis of tb pleural effusions [7] . however, these assays did not provide information regarding antigen-responsive cells. an abundance of immunocompetent cells in the pleural effusion provides investigators with a good model to study the correlates of a protective immune response at the site of infection. the enrichment of th1 cells in pleural fluid has been previously documented [8] . however, detailed phenotypic and functional characterizations for these cells are still unknown. the bcg vaccine is an attenuated strain of mycobacterium bovis and includes loss of the esx 1 locus, which encodes two family members, culture filtrate protein of 10 kda (cfp-10) and early secretory antigenic target 6 (esat-6). these antigens have an important diagnostic role in distinguishing bcg vaccination from mtb infection [9] [10] [11] [12] [13] . although esat-6 and cfp-10 contain numerous potential t cell epitopes, the immune response during infection is often focused toward a few immunodominant epitopes. in the present study, we selected the six most dominant peptides identified in esat-6 and cfp-10, as reported to be frequently recognized by subjects in indian, european, cambodian, and middle eastern cohorts with both latent and active tb [14] [15] [16] [17] [18] [19] . it has already been demonstrated that t-lymphocytes in the lungs, both in normal individuals and in those with granulomatous disease, are almost entirely cd45ro + memory t cells and express both early-and late-activation markers, such as cd69 and cd29 respectively [20] . previous data have demonstrated that almost all of the ccr5 + and ccr3 + cd4 + t cells recruited to the lungs of patients with active tb express the memory t-cell phenotype and that the majority may have been recently activated [21] . cd69 is a membrane molecule transiently expressed on activated lymphocytes, and its selective expression in inflammatory infiltrates probably suggests that it plays a role in the pathogenesis of inflammatory diseases [22] . previous studies have also demonstrated the usefulness of the determination of cd69 on cd4 + t cells after in vitro stimulation with tuberculin as a rapid indicator of immune sensitization against mtb [23, 24] . however, these studies did not directly assess the function of cd69 + cells. the present study was initially designed to analyze the discrepancy of phenotypes among pleural fluid cells (pfcs) from patients with tbp, pbmcs from tb patients and normal donors. we found a significant increase in cd69 expression on cd4 + t cells in pfcs. importantly, cd4 + cd69 + cells were mostly th1 cells specific for esat-6 and cfp-10 peptides. the phenotypic and functional analysis of cd69-expressing cells strongly suggested that cd69 could be a useful marker for the identification or enrichment of antigen specific th1 cells at local sites following mtb infection. cd69 may be useful for diagnostic procedures, including specific immune monitoring during tb infection or after vaccination, as well as for therapeutic applications such as targeted adoptive th1 cell therapies. significantly higher expression of cd69 on cd4 + t cells in pfcs from patients with tbp than in pbmcs from tb patients and healthy donors without any stimulation our initial experiments assessed the surface expression of cd69 on cd4 + t cells from pfcs of patients with tbp and pbmcs from tb patients (ptb) and healthy donors (hd). as expected, without any stimulation, a significantly higher level of cd69 expression was observed on cd4 + t cells from pfcs of tbp patients than on pbmcs from either ptb or hd ( fig. 1a and b, p,0.001). moreover, higher levels of cd69 expression (11.60%611.93%, mean 6 sd) on cd4 + t cells were observed in 78% of the pfcs from individuals with tbp than on the pbmcs of ptb or hd (1.48%61.60% for healthy donors and 1.38%61.68% for tb patients, mean 6 sd). no significant difference in cd69 expression was observed on pbmcs from ptb and from hd ( fig 1b, p = ns) . in addition, we also demonstrated that cd4 + cd69 + t cells expressed significantly higher levels of hla-dr than cd4 + cd69 2 t cells, a molecule which is usually induced on cd4 + t cells after activation (fig 1c and d , p,0.001). unexpectedly, no significant difference was observed in the expression of cd25, another activation marker, which is usually temporally expressed on activated t cells or constitutively expressed on regulatory t cells (fig 1c and d , p = ns). significantly higher expression of t-bet and il-12rb2 by cd4 + cd69 + t cells than by cd4 + cd69 2 t cells in order to confirm whether cd4 + cd69 + t cells in pfcs were th1 cells, we further evaluated the expression of t-bet, an important transcription factor specific for th1 cells. the results showed that more than 90% of cd4 + cd69 + t cells and about 57.24% of cd4 + cd69 2 t cells expressed t-bet (fig 2a) . the expression of t-bet on cd4 + cd69 + t cells was significantly higher than that on cd4 + cd69 2 t cells (fig 2b, p,0 .01). we next examined the expression of il-12rb2, another marker highly specific for differentiated th1 cells. similar to the results obtained for t-bet expression, the majority of cd4 + cd69 + t cells constitutively expressed il-12rb2 ( fig. 2a) , and the expression was significantly higher than that observed on cd4 + cd69 2 t cells (fig 2c, p,0 .05 ), further confirming that the cd4 + cd69 + t cell subset was highly enriched for th1 cells. esat-6 and cfp-10 peptides-specific th1 cells were preferentially enriched within cd4 + cd69 + t cells because cd4 + cd69 + t cells were predominantly th1 cells, we aimed to examine whether direct ex vivo evaluation of surface expression of cd69 without in vitro upregulation permits direct assessment of mtb-specific th1 cells. our initial experiments indicated that stimulation of pfcs with esat-6 and cfp-10 peptides resulted in increased surface cd69 expression that is difficult to elucidate (data not shown). short-term stimulation of these cells includes incubation with secretion inhibitory brefeldin a (bfa), which can block the transport of newly synthesized cd69 molecules to the cell surface and thus allows for the direct assessment of the relationship between in vivo cd69 expression and cytokine production. after culturing pfcs with esat-6/ cfp-10 peptides and bfa, we analyzed the production of th1 cytokines by intracellular cytokine staining and polychromatic flow cytometry. as shown in fig 3a and b , cd4 + cd69 + t cells produced greater amounts of ifn-c, il-2 and tnf-a than did cd4 + cd69 2 t cells. statistical results demonstrated that the percentages of ifn-c, il-2 or tnf-a-producing cd4 + cd69 + t cells were significantly higher than those for cd4 + cd69 2 t cells (p,0.001, fig 3c) . in order to further confirm that the responses of t cells were specific for mtb, we also stimulated pfcs from tuberculous pleurisy patients with other irrelevant antigens such as sars-cov s peptides (stffstfkcygvsatkl) and (nfsqilpdplkptk rsfi) as well as hepatitis b virus surface antigen (hbsag). we found that these irrelevant antigens could not induce the production of ifn-c, il-2 and tnf-a by neither cd4 + cd69 + nor cd4 + cd69 2 t cells. however, following stimulation with mtb-specific peptides of esat-6/cfp-10, cd4 + cd69 + t cells from the same patients expressed high levels of ifn-c, il-2 and tnf-a (data not shown). it has been suggested that t cells simultaneously producing ifn-c, il-2 and tnf-a are associated with protective immunity and concomitant beneficial outcomes, at least in chronic viral infections such as hiv. we therefore compared the expression of ifn-c, il-2 and tnf-a in cd4 + cd69 + , cd4 + cd69 2 and total cd4 + t cells after short-term in vitro stimulation with esat-6/ cfp-10 peptides. we showed that cd4 + cd69 + and cd4 + cd69 2 t cells concurrently and individually produced ifn-c, il-2 and tnf-a (fig 4a, b ). this assay also identified cells that expressed cd69 but lacked expression of any cytokine measured (as evidenced by a subset of cd4 + cd69 + ifn-c 2 il-2 2 tnf-a 2 cells). these cells probably contained other effector functions, for example, the secretion of mip-1b or other cytokines. the total antigen-specific responses were defined as the percentages of t cells expressing any combination of ifn-c, il-2 or tnf-a, and thus, seven distinct functional populations could be delineated (ifn-c/il-2/tnf-a triple expressers; ifn-c/il-2, ifn-c/tnf-a or tnf-a/il-2 double expressers; or ifn-c, il-2 or tnf-a single expressers). interestingly, polyfunctional t cells (ifn-c/il-2/ tnf-a triple expressers) dominated the cd4 + cd69 + t cell response. however, frequencies of t cells producing only tnf-a were the highest when analyzing the distribution of the seven subsets within either the cd4 + cd69 2 population or all cd4 + t cells (fig 4c, d) . the percentages of double expressers were similar within cd4 + cd69 + , cd4 + cd69 2 and total cd4 + t cells. our results demonstrated that total mtb-specific cd4 + t cells were not dominated by polyfunctional t cells. however, polyfunctional t cells were primarily enriched within cd4 + cd69 + t cells. we further analyzed the correlation between the percentages of cd69-expressing cd4 + t cells and polyfunctional t cells. interestingly, significant positive correlation between cd4 + cd69 + t cells and polyfunctional t cells was observed ( fig 4e, cd4 + cd69 + t cells were mostly effector memory t cells we next investigated the phenotype of cd4 + cd69 + t cells relating to naïve/memory markers. as shown in fig 5a, cd4 + cd69 + t cells were predominantly cd45ro + cd45ra 2 , thereby exhibiting a memory cell phenotype. in contrast, cd4 + cd69 2 t cells contained significantly higher percentages of cd45ra + and lower percentages of cd45ro + cells than did cd4 + cd69 + t cells. moreover, we also examined the expression of cd127 and cd27 and found that cd4 + cd69 + t cells had significantly lower expression of both markers ( fig 5b) . to further validate whether mtb-specific cd4 + cd69 + t cells were memory cells, we tested the phenotype of ifn-c-secreting cd4 + cd69 + t cells by assessing typical memory t cell markers. as expected, the majority of cd4 + cd69 + ifn-c + t cells displayed a cd45ra 2 ccr7 2 cd62l 2 cd27 2 effector memory cell phenotype (fig 5c) , suggesting that these cells were capable of producing multiple cytokines quickly at local sites. we further investigated whether purified cd4 + cd69 + t cells, stained by fluorescently labeled monoclonal antibody and sorted by flow cytometry, would be compatible with assays that required live cells. we first purified cd4 + t cells and cd14 + cells by positive selection with microbeads. thereafter, cd4 + cd69 + and cd4 + cd69 2 t cell fractions were obtained by flow cytometrybased cell sorting (fig 6a) . we then cocultured cd4 + cd69 + or cd4 + cd69 2 t cells with cd14 + cells (as antigen-presenting cells) in the presence of esat-6/cfp-10 peptides (p1-p6) and measured the production of th1 cytokines. notably, considerable levels of ifn-c and il-2 were produced by cd4 + cd69 + t cells, whereas little was observed from the cd4 + cd69 2 fractions. however, tnf-a production was readily detected in unstimulated cells, and significantly higher tnf-a was measured following stimulation of the cd4 + cd69 + fractions ( fig 6b) . moreover, significantly higher levels of ifn-c, il-2 and tnf-a were produced by cd4 + cd69 + cells than by cd4 + cd69 2 t cells (p,0.001, fig 6b) . we also assessed percentages of th1 cytokineproducing cells by flow cytometry. as expected, cd4 + cd69 + t cells were highly enriched for ifn-c, il-2 and tnf-a production compared with cd4 + cd69 2 cells (fig 6c-e) . thus, cd69 expression provides a means to identify the vast majority of antigen-specific cd4 + t cells, regardless of the type of cytokine measured. importantly, our assay conditions based on the cd69 coculture assay retained the ability of cells to secrete multiple cytokines. cd4 + cd25 + t cells inhibit production of th1 cytokines by cd4 + cd69 + t cells in order to examine whether cd4 + cd25 + treg cells could directly inhibit cytokine production by cd4 + cd69 + t cells, we sorted cd4 + cd69 + and cd4 + cd25 + t cells by flow cytometry (fig 7a) . in order to further confirm that cd4 + cd25 + t cells were regulatory t cells, we found that the majority of cd4 + cd25 + t cells (.95%) expressed foxp3(data not shown), the specific marker for this subset. we further confirmed this observation by incubation of sorted cd4 + cd69 + or cd4 + cd25 + t cells with cd14 + t cells, respectively. the result demonstrated that neither ifn-c nor tnf-a was produced by the cd4 + cd25 + t cells, whereas cd4 + cd69 + t cells produced large amounts of ifn-c and tnf-a upon coculture (fig 7b) , confirming that cd4 + cd25 + t cells were hyporesponsive and represented regulatory t cells. we then cocultured cd4 + cd69 + with cd4 + cd25 + t cells at different ratios in the presence of cd14 + cells. cd4 + cd69 + cells stimulated with esat-6/cfp-10 peptides (p1-p6) produced considerably greater amounts of th1 cytokines compared with unstimulated cells. the addition of cd4 + cd25 + t cells into cultures resulted in the inhibition of ifn-c, il-2 and tnf-a production in a dose-dependent manner (fig 7c-e) . were gated on cd4 + cd69 + ifn-c + and cd4 + cd69 + ifn-c 2 cells and analyzed for the expression of memory markers cd45ra, ccr7, cd62l and cd27, as indicated. upper panel represents the control. lower panel represents the expression of each marker. cd4 + cd69 + ifn-c + t cells are shown in black, whereas cd4 + cd69 + ifn-c 2 t cells are depicted in gray. the expression of each marker within cd4 + cd69 + ifn-c + t cells was overlaid with cd4 + cd69 + ifn-c 2 t cells. the numbers in the quadrants indicate the percentage of cells among cd4 + cd69 + ifn-c + t cells. one representative result from ten independent experiments with similar results is shown. doi:10.1371/journal.pone.0023700.g005 overall, our assay conditions showed that cd4 + cd25 + cells could inhibit th1 cytokine production by cd4 + cd69 + t cells. without treatment, tuberculous pleurisy (tbp) usually resolves spontaneously [25] and thus is thought to be a useful model to study the correlates of protective immune responses at the site of infection [26, 27] . meanwhile, the diagnosis of tbp is difficult and continues to pose clinical challenges. currently, the two major advances in the diagnosis of tbp have been pleural biopsies [4] and adenosine deaminase (ada) assessment in pleural fluid [28, 29] . the free (unstimulated) pleural fluid ifn-c assay is another method established for the diagnosis of tbp [30] . however, both ada and ifn-c are non-specific markers of inflammation [30] . the detection of t cell responses specific for defined antigens is essential for the evaluation of pathogenic or protective immunity induced by infectious pathogens or vaccines. cd4 + t cell responses are commonly determined by the expression of various cytokines, such as ifn-c, il-2 and tnf-a for th1 cells, after short-term stimulation with specific antigens. the development of ifn-c release assays (igras) has emerged as an attractive alternative to the tuberculin skin test for detection of latent tb infections and might also contribute to assisting in the diagnosis of tbp [31] . igras are based on the principle that t cells of individuals sensitized with tb antigens produce ifn-c when they re-encounter highly mtb-specific antigens such as esat-6 and cfp-10. however, this assay precludes further analysis because it is lethal to cells. moreover, the igras are not recommended on either the blood or the pleural fluid for the diagnosis of tbp [31] . consistent with other studies, we found that the expression of cd69 was significantly increased on cd4 + t cells in pfcs from patients with tbp [32] . we further demonstrated that the majority of cd4 + cd69 + cells were th1 cells by analyzing expression of the th1 markers t-bet and il-12rb2. in order to figure 7 . cd4 + cd25 + cells inhibit the production of th1 cytokines by sorted cd4 + cd69 + t cells. (a) cd4 + cd25 + and cd4 + cd69 + cells were sorted from purified cd4 + t cells. (b) production of ifn-c and tnf-a by sorted cd4 + cd69 + and cd4 + cd25 + cells in the presence of purified cd14 + t cells following stimulation with p1-p6. (c-e) sorted cd4 + cd25 + and cd4 + cd69 + cells were cultured at different ratios and cocultured with purified cd14 + cells in the presence or absence of p1-p6. the supernatants were collected, and cytokine production was assessed by elisa. one representative result from three independent experiments with similar results is shown. significant differences compared with incubation with p1-p6 in the absence of tregs are indicated (*, not significant;**, p,0.05;***, p,0.001). doi:10.1371/journal.pone.0023700.g007 figure 6 . isolation of viable cd4 + cd69 + and cd4 + cd69 2 t cells. (a) cd4 + cd69 + and cd4 + cd69 2 cells were sorted from magnetic beadpurified cd4 + t cells. (b) sorted cd4 + cd69 + and cd4 + cd69 2 cells were cocultured with purified cd14 + cells in the presence or absence of bcg or p1-p6. supernatants were collected and cytokine production was assessed by elisa. significant differences compared with cd4 + cd69 2 fractions are indicated (***, p,0.001). (c, d) after stimulation for 8 hours, cd4 + t cells were gated and the expression of th1 cytokines by cd4 + cd69 + and cd4 + cd69 2 cells was analyzed. numbers in the quadrants indicate percentages of cells among the cd4 + cd69 + or cd4 + cd69 2 populations. (e) the frequencies of ifn-c, il-2 or tnf-a-producing cd4 + cd69 + (closed circles) or cd4 + cd69 2 t cells (open circles, n = 3-5) are demonstrated. horizontal lines represent mean value. doi:10.1371/journal.pone.0023700.g006 further confirm that cd4 + cd69 + cells were mostly mtb-specific t cells, we performed intracellular cytokine staining. we noted that stimulation of pfcs resulted in increased surface cd69 expression. we overcame this limitation by incubation with bfa during short-term stimulation. using this method, we were able to directly assess the relationship between in vivo cd69 expression and cytokine production. using polychromatic flow cytometry, we demonstrated for the first time that the majority of mtb specific th1 cytokine-producing cells were enriched within the cd4 + cd69 + subset, whereas cd4 + cd69 2 cells scarcely produced cytokines. in addition, pfcs from tuberculous pleurisy patients were stimulated with sars-cov s peptides (stffstfkcygvsatkl) and (nfsqilpdplkptkrsfi) as well as hepatitis b virus surface antigen (hbsag). these irrelevant antigens could not induce the production of ifn-c, il-2 and tnfa by neither cd4 + cd69 + nor cd4 + cd69 2 t cells. however, following stimulation with mtb-specific peptides of esat-6/ cfp-10, cd4 + cd69 + t cells from the same patients with tuberculous pleurisy expressed high levels of ifn-c, il-2 and tnf-a, indicating that the response was mtb-specific. most importantly, the detection of cd69 is useful for the direct identification of mtb-specific th1 cells in vivo without further stimulation. t cells that produce multiple factors simultaneously are termed polyfunctional t cells and have been shown to provide vaccineinduced immunity and protection against disease progression in hiv-1 infection [33, 34] . in the present study, analysis of total cd4 + th1 subpopulations indicated that the majority of th1 cells produced only one cytokine. however, polyfunctional cd4 + t cells simultaneously producing ifn-c, il-2 and tnf-a dominated the cd4 + cd69 + t cell population, but the percentage of tnf-a single expressers was higher in both the cd4 + cd69 2 and the total cd4 + t cell subsets compared with cd4 + cd69 2 cells. our results indicated that detection or isolation of cd69 + t cells is a good method for the identification or enrichment of polyfunctional t cells. interestingly, significant positive correlation was observed between the percentages of cd69-expressing cd4 + t cells and polyfunctional t cells. our results demonstrated for the first time that the expression of cd69 may be a useful marker for the definition of mtb-specific polyfunctional t cells in vivo. a number of studies in humans suggested that polyfunctional t cells may indeed be involved in mediating protection in tb [34] [35] [36] [37] [38] [39] . however, it has also been indicated in the recent studies that polyfunctional cd4 + t cells may be a useful biomarker of active tb and that polyfunctional responses are not associated with protection against tb [40] . in the present study, we hypothesized that the highly increased frequencies of cd69-expressing cd4 + t cells (polyfunctional t cells) in pleural fluid may be associated with protection against tb and thus lead to the self-resolution tendency of tbp. a previous study has indicated that cd4 + cd69 + cd25 2 cells represented a new subset of regulatory t cells in the tumor model [41] . these cd4 + cd69 + cd25 2 cells are hyporesponsive and can suppress proliferation of cd4 + t cells in a cell-to-cell contact manner. in our study, however, cd4 + cd69 + t cells represented effector or effector memory cells and quickly produced large amounts of cytokines following short-term stimulation. moreover, we also assessed other markers (foxp3 and ctla-4) essential for the suppressive function of regulatory t cells and found that these molecules were not differentially expressed by cd4 + cd69 + and cd4 + cd69 2 t cells, further confirming that cd4 + cd69 + t cells were not regulatory t cells (data not shown). these differing results were probably associated with discrepancies in local circumstances, including cytokines, chemokines and antigen presenting cells that differ between tumor and inflammation contexts. moreover, it remains to be determined if cd69 is induced by the pleural fluid environment or if cd4 + cd69 + t cells are selectively recruited from the circulation. previous studies have indicated that cd4 + cd25 + t cells can inhibit antigen-specific t cell responses in tb patients [42] [43] [44] [45] [46] [47] . however, none of these studies has isolated mtb-specific cd4 + t cells directly. in the present study, we purified viable cd4 + cd69 + and cd4 + cd25 + treg cells directly and found that cd4 + cd25 + treg cells could inhibit cytokine production by cd4 + cd69 + t cells. in addition, our results clearly demonstrated that cd4 + cd25 + t cells were hyporesponsive and scarcely produced cytokines following short-term stimulation, further confirming that these cells were regulatory cells. in our previous studies, we have demonstrated that the suppressive effect of treg cells on ifn-c production by cd4 + cd25 + t cells [43] and cd t cells [48] was mainly mediated via the release of il-10 but not tgf-b. in summary, we demonstrated that cd69 as a useful marker for mtb-specific th1 cells in pfcs from patients with tbp enabled a direct ex vivo estimation of the quantity, as well as the quality, of mtb-specific th1 responses. the majority of cd4 + cd69 + t cells but not total cd4 + t cells, were dominated by polyfunctional t cells. importantly, significantly positive correlation was observed between cd69-expressing cd4 + t cells and polyfunctional t cells, suggesting that cd69 is a good marker for the identification or enrichment of polyfunctional t cells in pfcs. moreover, isolation of live mtb-specific th1 cells according to surface cd69 expression without any stimulation offers an opportunity for further investigation. written informed consent was obtained from all patients and healthy donors. ethics approval for the present study was obtained from the ethics committee of the zhongshan school of medicine, sun yat-sen university (guangzhou, china) and the chest hospital of guangzhou (guangzhou, china). a total of fifty patients with tuberculous pleurisy (15 females and 35 males, range 18-71 years of age) were recruited from the chest hospital of guangzhou, china. diagnosis of pleural effusion from tb etiology was based on one of the following criteria: (i) demonstration of mtb on pleural fluid smear (by the ziehl-neelsen method); (ii) pleural fluid or pleural biopsy specimens growing m. tuberculosis on lowenstein-jensen medium; or (iii) histological evidence of caseating granuloma on biopsy specimens of pleural tissue with positive staining for m. tuberculosis. twentyone patients (10 females and 11 males, range 20 to 50 years of age) with active pulmonary tuberculosis (culture-confirmed pulmonary tb) were also included in this study. patients who had been previously diagnosed with hiv, hbv, or hcv or with a history of autoimmune diseases were excluded from the study. none of the patients was receiving mtb-related treatments at the time of the study. twenty-two healthy volunteers between the ages of 20 and 54 were recruited from sun yat-sen university. for the detection of mtb-specific immune responses, we selected six highly immunogenic and largely hla-dr-restricted peptides. four peptides were derived from esat-6, and two were derived from the cfp-10 protein of mtb. synthetic peptides of 20 amino acids (aa) in length were obtained from shenzhen hanyu pfcs were isolated by lysing erythrocytes using ammonium chloride solution and resuspended to a final concentration of 2610 6 cells/ml in complete rpmi 1640 medium (invitrogen, grand island, ny) supplemented with 10% heat-inactivated fetal calf serum (fcs; hyclone, logan, ut), 100 u/ml penicillin, 100 mg/ml streptomycin, 2 mm l-glutamine, and 50 mm 2mercaptoethanol. peripheral blood mononuclear cells (pbmcs) were isolated by ficoll-hypaque gradient centrifugation of heparinized venous blood obtained from healthy individuals or tb patients. for the detection of intracellular cytokines, cells were incubated at a concentration of 2610 6 cells/ml with 1 mg/ml peptides plus 1 mg/ml anti-cd28 and 1 mg/ml anti-cd49d for 8 h in the presence of brefeldin a (bfa, 10 mg/ml; sigma-aldrich, st louis, mo). for the detection of t-bet, surface markers were first stained and cells were then fixed in 2% formaldehyde, permeabilized in 90% methanol and labeled with anti t-bet mab. the expression of il-12rb2 was detected by indirect immunofluorescence analysis. briefly, cells were incubated with mab for il-12rb2, followed by biotin-sp-conjugated anti-rat igg f(ab') 2 fragments, and were finally incubated with streptavidin-pe. all incubations were performed at 4uc and cells were washed twice between incubations. flow cytometry was performed using bd facscalibur (bd biosciences) or facsaria ii (bd biosciences) and the data were analyzed using flowjo software (treestar, san carlos, ca). cd4 + and cd14 + cells were isolated from pfcs by positive selection with anti-cd4 or anti-cd14 microbeads (miltenyi biotec, bergisch gladbach, germany). for isolation of cd4 + cd69 + , cd4 + cd69 2 and cd4 + cd25 + cells, purified cd4 + t cells were stained with pe-conjugated cd69 and fitc-conjugated anti-cd25 and then sorted on a facsaria ii high-speed cell sorter. the purity of cells, as assessed by flow cytometry, was 95-97% for each cell subset. purified cd4 + cd69 + , cd4 + cd69 2 or cd4 + t cells were cocultured with purified cd14 + cells (at ratio of 1:2 ) in the presence or absence of bcg, esat-6 and cfp-10 peptides. after 48 hours, cell-free supernatants were collected and assessed for ifn-c, il-2 and tnf-a production by elisa kits (bd bioscience pharmingen). for the detection of intracellular cytokines, cells were cultured for 8 hours and then subjected to flow cytometry analysis. to examine the role of cd4 + cd25 + cells in the regulation of cytokine production by sorted cd4 + cd69 + t cells, purified cd4 + cd69 + (teff), cd14 + cells and cd4 + cd25 + cells (treg) were co-cultured in various ratios (teff:treg) in the presence of esat-6 and cfp-10 peptides. cell-free supernatants were collected and assessed for ifn-c, il-2 and tnf-a by elisa. the wilcoxon matched pairs test (two-tailed) was performed to determine statistical differences between the groups using graphpad prism software version 5. inhibition of cytokine production by cd4 + cd25 + t cells was analyzed using the paired student's t test. a value of p,0.05 was considered statistically significant. conceived and designed the experiments: cyw. performed the experiments: ll. analyzed the data: ll. contributed reagents/materials/ analysis tools: dq xyf shl xlz. wrote the paper: ll. some problems concerning local cellular immunity in tuberculosis production of the rantes chemokine in delayed-type hypersensitivity reactions: involvement of macrophages and endothelial cells can pleural tuberculosis be diagnosed using interferongamma release assays? diagnosis and treatment of tuberculous pleural effusion in compartmentalization of pro-inflammatory cytokines in tuberculous pleurisy cytokines in pleural liquid for diagnosis of tuberculous pleurisy update on tuberculous pleural effusion cytokine polarization in miliary and pleural tuberculosis mycobacterium tuberculosis and the host response development of new vaccines and diagnostic reagents against tuberculosis effect of bcg vaccination on risk of mycobacterium tuberculosis infection in children with household tuberculosis contact: a prospective community-based study the development and impact of tuberculosis vaccines recent findings in immunology give tuberculosis vaccines a new boost direct ex vivo analysis of antigen-specific ifn-gamma-secreting cd4 t cells in mycobacterium tuberculosis-infected individuals: associations with clinical disease state and effect of treatment enumeration of t cells specific for rd1-encoded antigens suggests a high prevalence of latent mycobacterium tuberculosis infection in healthy urban indians characterization of a mycobacterium tuberculosis peptide that is recognized by human cd4+ and cd8+ t cells in the context of multiple hla alleles differential t cell responses to mycobacterium tuberculosis esat6 in tuberculosis patients and healthy donors aspartic acid homozygosity at codon 57 of hla-dq beta is associated with susceptibility to pulmonary tuberculosis in cambodia human th1 cell lines recognize the mycobacterium tuberculosis esat-6 antigen and its peptides in association with frequently expressed hla class ii molecules in situ activation of helper t cells in the lung expansion of ccr5+ cd4+ t-lymphocytes in the course of active pulmonary tuberculosis cd69 controls the pathogenesis of allergic airway inflammation cd69 expression on cd4+ t lymphocytes after in vitro stimulation with tuberculin is an indicator of immune sensitization against mycobacterium tuberculosis antigens restoration of cellular immunity against tuberculosis in patients coinfected with hiv-1 and tuberculosis with effective antiretroviral therapy: assessment by determination of cd69 expression on t cells after tuberculin stimulation pleural tuberculosis: an update cytokine production at the site of disease in human tuberculosis correlates of protective immune response in tuberculous pleuritis diagnostic accuracy of adenosine deaminase in tuberculous pleurisy: a meta-analysis adenosine deaminase activity is a sensitive marker for the diagnosis of tuberculous pleuritis in patients with very low cd4 counts novel tests for diagnosing tuberculous pleural effusion: what works and what does not? interferon-gamma release assays for the diagnosis of tb pleural effusions: hype or real hope? increased susceptibility to apoptosis of cd56dimcd16+ nk cells induces the enrichment of ifn-gamma-producing cd56bright cells in tuberculous pleurisy hiv nonprogressors preferentially maintain highly functional hiv-specific cd8+ t cells the novel tuberculosis vaccine, aeras-402, induces robust and polyfunctional cd4+ and cd8+ t cells in adults immunisation with bcg and recombinant mva85a induces long-lasting, polyfunctional mycobacterium tuberculosis-specific cd4+ memory t lymphocyte populations modified vaccinia ankara-expressing ag85a, a novel tuberculosis vaccine, is safe in adolescents and children, and induces polyfunctional cd4+ t cells bacillus calmette-guerin vaccination of human newborns induces t cells with complex cytokine and phenotypic profiles detection of polyfunctional mycobacterium tuberculosis-specific t cells and association with viral load in hiv-1-infected persons dynamic relationship between ifngamma and il-2 profile of mycobacterium tuberculosis-specific t cells and antigen load multifunctional cd4+ t cells correllate with active mycobacterium tuberculosis infection cd69+ cd4+ cd25-t cells, a new subset of regulatory t cells, suppress t cell proliferation through membrane-bound tgf-beta 1 regulatory t cells are expanded in blood and disease sites in patients with tuberculosis increased frequency of cd4(+)cd25(high) treg cells inhibit bcg-specific induction of ifn-gamma by cd4(+) t cells from tb patients cd4(+)cd25(+)foxp3(+) regulatory t cells suppress mycobacterium tuberculosis immunity in patients with active disease regulatory t cells depress immune responses to protective antigens in active tuberculosis foxp3+ regulatory t cells suppress effector t-cell function at pathologic site in miliary tuberculosis a role for cd4+cd25+ t cells in regulation of the immune response during human tuberculosis cd4+ cd25+ treg cells inhibit human memory gammadelta t cells to produce ifn-gamma in response to m tuberculosis antigen esat-6 key: cord-003484-ylpa702c authors: blázquez, elena; rodríguez, carmen; ródenas, jesús; navarro, núria; riquelme, cristina; rosell, rosa; campbell, joy; crenshaw, joe; segalés, joaquim; pujols, joan; polo, javier title: evaluation of the effectiveness of the surepure turbulator ultraviolet-c irradiation equipment on inactivation of different enveloped and non-enveloped viruses inoculated in commercially collected liquid animal plasma date: 2019-02-21 journal: plos one doi: 10.1371/journal.pone.0212332 sha: doc_id: 3484 cord_uid: ylpa702c the objective of this study was to evaluate the effectiveness of the surepure turbulator ultraviolet-c (uv-c, 254 nm wavelength) irradiation equipment on inactivation of different enveloped and non-enveloped viruses in commercially collected liquid animal plasma. specifically, pseudorabies virus (prv), porcine reproductive and respiratory syndrome virus (prrsv), porcine epidemic diarrhea virus (pedv), bovine viral diarrhea virus (bvdv), classical swine fever virus (csfv), swine influenza virus (siv) as enveloped viruses and porcine parvovirus (ppv), swine vesicular disease virus (svdv), porcine circovirus type 2 (pcv-2) and senecavirus a (sva) as non-enveloped viruses, were inoculated in bovine or porcine plasma and subjected to different uv-c irradiation doses (0, 750, 1500, 3000, 6000 and 9000 j/l) using an uv-c device developed for opaque liquid working under turbulent flow. the enveloped viruses tested were inactivated at < 3000 j/l of uv-c, being the dose needed to inactivate 4 log tcid(50) (4d) of 1612 j/l for prv,1004 j/l for prrsv, 1953 j/l for pedv, 1639 j/l for siv, 1641 j/l for csfv and 1943 j/l for bvdv. the non-enveloped viruses tended to have higher 4d values: 2161 j/l for ppv, 3223 j/l for sva and 3708 j/l for svdv. because the initial viral concentration was <4.0 log for pcv-2, it was not possible to calculate the 4d value for this virus. in conclusion, these results demonstrated that the surepure turbulator uv-c treatment system is capable of inactivating significant levels of swine viruses inoculated in commercially collected porcine or bovine plasma. it was concluded that irradiation with uv-c can provide an additional redundant biosafety feature in the manufacturing process of spray-dried animal plasma. introduction spray-dried animal plasma (sdap) is a protein source widely used in pig feed due to its functional components able to improve post-weaning performance and survival [1, 2] . the manufacturing process of sdap involves the collection of blood from healthy animals at a commercial abattoir that have been inspected by veterinary officials and passed as fit for slaughter for human consumption. immediately after collection, the blood is treated with an anticoagulant, chilled, transported to the manufacturing plant and centrifuged to obtain plasma and blood cell fractions. alternatively, the blood can be centrifuged at the abattoir and the plasma fraction is chilled and transported to the manufacturing plant. at the manufacturing plant, the plasma is concentrated by membrane filtration and spray-dried, achieving 80˚c throughout its substance [2] . the process for the production of sdap has been validated to effectively inactivate many viral and bacterial pathogens [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] . these data demonstrate that commercially produced sdap is a safe feed ingredient. however, it is appropriate to evaluate new technology that could be included into the manufacturing process representing added redundant microbial inactivation steps. typical plasma manufacturing plants are capable of processing between 25,000 mt to 450,000 mt of liquid plasma annually. many different techniques have been used to inactivate or remove viruses from human plasma products, i.e. intravenous immunoglobulin (ivig). for example, solvent/detergent mixtures have been effectively used to inactivate lipid enveloped viruses [15] . however, this process requires significant volume of solvent/detergent mixtures that must be removed and disposed. enveloped viruses can be inactivated by the addition of unsaturated fatty acids [16] , or caprylic acid in combination with low ph and temperature [17, 18] . while many of these methods are very effective for plasma fractions (purified immunoglobulins or albumin) these methods are not appropriate for the production of sdap because these processes result in many denatured plasma proteins. as a rule, non-enveloped viruses are more resistant to inactivation than enveloped viruses [19] . an alternative to inactivation is removal of the virus from plasma fractions by nanofiltration [20] . however, this is not a practical option for industrial production of sdap since the volume of product to be processed is very large and most proteins are retained in the retentate. similarly, other removal procedures used in the human plasma industry, like precipitation or chromatography are impractical due to the large volumes used by sdap manufacturing industry. ultraviolet-c (uv-c) radiation represents an alternative to chemical inactivation methods [21] . such methodology uses a shortwave electromagnetic radiation with a wavelength of 254 nm (range of 250 and 270 nm), which induces damage in the nucleic acids. uv-c radiation disrupts dna and rna [22] , and is therefore effective at inactivating a wide range of microorganisms. in fact, it has been extensively used for disinfection of water, surfaces and food products [23, 24] . the inability of uv-c radiation to penetrate opaque liquids can be overcome by introducing adequate turbulence insuring that all of the liquid is exposed to the surface of the uv-c light [25] [26] [27] [28] [29] [30] . uv-c treatment has been shown to reduce bacterial contamination while not inactivating protein functionality or growth enhancing properties of sdap [31] . surepure has designed a uv-c treatment system that is able to reduce the pathogen burden in milk, fruit juices, etc. [26, 28, 29] . the objective of this study was to assess inactivation efficiency of the surepure turbulator uv-c irradiation system with selected swine enveloped viruses pseudorabies virus (prv), porcine reproductive and respiratory syndrome virus (prrsv), porcine epidemic diarrhea virus (pedv), bovine viral diarrhea virus (bvdv), swine influenza a virus (siv) and classical swine fever virus (csfv) and non-enveloped viruses porcine parvovirus (ppv), swine vesicular disease virus (svdv), porcine circovirus 2 (pcv-2) and senecavirus a (sva) inoculated in liquid bovine or porcine plasma. these viruses were selected because of their economic impact in livestock production and as models of viruses from different families with various genome type and size. pseudorabies virus. pseudorabies virus strain nia3 [32] , kindly supplied by joan plana (fort dodge veterinaria, vall de bianya, spain), was propagated in the pk-15 cell line (provided by the institute of virology (ue and oie reference laboratory for csfv, hannover), using a standard growth media (sgm) containing minimum essential medium eagle (mem-e; thermofisher, waltham, ma, usa) supplemented with 1% penicillin 10,000 u/ml and streptomycin 10 mg/ml (thermofisher), 0.5% nystatin 10,000 iu/ml (sigma-aldrich), 1% l-glutamine 200 mm (thermofisher) and 5% of heat inactivated fetal bovine serum tested free for virus and antibodies against pestiviruses (fbs; biowest, miami, fl, usa). pk-15 cells were grown in 175-cm 2 flasks (corning, corning, ny, usa), and when cells were confluent, the medium was discarded and adsorption was performed at a moi 0.01. virus stock was produced in the same cells to obtain 84 ml with a viral titer of 10 8.95 tcid 50 /ml that was used to inoculate 24 l of bovine plasma, achieving a final viral titer of 10 6.5 tcid 50 /ml. porcine reproductive and respiratory syndrome virus. porcine reproductive and respiratory syndrome virus vp21 strain [33] was propagated in the marc-145 cell line (atcc clr12231) grown in sgm with 5% fbs. cells were cultured in 75-cm 2 flasks. when cells were confluent, the media was discarded and the adsorption was done using the virus at a moi 0.01. after 1.5 hours at 37˚c, inoculum was removed and 30 ml of medium were added. this procedure was repeated until achieving 1,800 ml of viral suspension with a titer of 10 5.57 tcid 50 /ml that was used to inoculate 24 l of bovine plasma achieving a final viral titer of 10 4.42 tcid 50 /ml. porcine epidemic diarrhea virus. porcine epidemic diarrhea virus cv777 strain [34] , kindly provided by dr. hans nauwynck (university of ghent, belgium), was propagated in vero cells (atcc ccl-81) grown in sgm with 10% fbs. cells were cultured in 175-cm 2 flasks and when they were confluent, the media was removed and cells were rinsed twice with pbs. finally, inoculum was added at moi 0.001 and adsorption was done for 1 hour at 37˚c. subsequently, the inoculum was discarded, flasks were rinsed twice with pbs and mem-e was supplemented with 1% penicillin 10,000 u/ml and streptomycin 10 mg/ml (thermofisher), 0.5% nystatin (10,000 u/ml), 1% l-glutamine (200 mm), 0.05% trypsin and 0.3% tryptose. the viral stock was produced in the same cells to obtain 2,110 ml of suspension with a viral titer of 10 5.42 tcid 50 /ml that was used to inoculate 24 l of bovine plasma achieving a final viral titer of 10 4.7 tcid 50 /ml. bovine viral diarrhea virus. bovine viral diarrhea virus nadl was provided by the institute of virology (ue and oie reference laboratory for csfv, hannover) and was propagated in mdbk cells (provided by the institute of virology (ue and oie reference laboratory for csfv, hannover), grown in sgm with 5% fbs tested free of pestivirus antibodies. cells were cultured in 175-cm 2 flasks. media was discarded and the adsorption was done on confluent cells using bvdv strain at a moi 0.01. after 1.5 hours at 37˚c, inoculum was removed and 50 ml of medium were added. this procedure was repeated until getting 255.3 ml of a suspension with a titer of 10 7.87 tcid 50 /ml that was used to inoculate 24 l of porcine plasma achieving a final viral titer of 10 4.5 tcid 50 /ml. classical swine fever virus. classical swine fever virus strain alfort 187, provided by the institute of virology (ue and oie reference laboratory for csfv, hannover), was propagated in the pk-15 cell line (provided by the same institute of virology, hannover), grown in sgm supplemented with 5% of fbs free from pestivirus antibodies. a total of 160 ml of virus stock solution was produced in the same cells at a moi 2, for a virus titer of 10 7.36 tcid 50 /ml that was used to inoculate 24 l of bovine plasma achieving a final viral titer of 10 5.19 tcid 50 /ml. swine influenza virus. swine influenza virus strain h1n1 a/swine/spain/sf11131/2017 [35] was propagated in mdck cell line (atcc ccl-34) grown in dmem (thermofisher, waltham, ma, usa) supplemented with 1% penicillin (10,000 u/ml), 1% streptomycin (10mg/ml; thermofisher), 0.5% nystatin (10,000 u/ml; sigma-aldrich), 1% l-glutamine 200mm; thermofisher) and 5% fbs. cells were cultured in 175-cm 2 flasks. when cells were confluent, the media was discarded and the adsorption was done at moi 0.1. after 1 hour at 37˚c, inoculum was removed and 30 ml of medium were added. with this procedure, 300 ml of viral suspension with a final virus titer of 10 7.2 tcid 50 /ml that was used to inoculate 24 l of bovine plasma achieving a final viral titer of 10 5.3 tcid 50 /ml. senecavirus a. senecavirus a isolate bra/uel-pr/15 was kindly provided by dr. amauri alfieri (universidade estadual de londrina, londrina, brazil) and was propagated in pk-15 cell line (provided by the institute of virology (ue and oie reference laboratory for csfv, hannover), grown in sgm with 10% fbs. viral infection was done in confluent 175 cm 2 flasks at a moi 0.001. after 1 hour of absorption, 50 ml of sgm were added. cpe was visible at 72 h and the flasks were frozen. following this procedure 1,100 ml of sva with a titer of 10 6.76 tcid 50/ ml was produced, that was used to inoculate 24 l of bovine plasma achieving a final viral titer of 10 5.43 tcid 50/ ml. swine vesicular disease virus. swine vesicular disease virus, strain uk-72, was provided by david k.j. mackay (european community reference laboratory for foot and mouth disease, institute for animal health. pirbright, uk) to laboratorio de sanidad animal (barcelona). svdv was propagated in sk-rst cell line (atcc crl-2842), grown in sgm supplemented with 5% fbs. a virus stock was produced using the same cell line at moi 0.001 to obtain 24 ml of a virus stock solution with a titer of 10 7.38 tcid 50 /ml that was used to inoculate 24 l of bovine plasma achieving a final viral titer of 10 6.00 tcid 50 /ml. porcine circovirus 2. porcine circovirus 2 genotype b isolate sp-10-7-54-13 [36] was cultured in the pk-15 cell line (provided by the institute of virology (ue and oie reference laboratory for csfv, hannover), grown in sgm with 10% fbs. a mix of 6 ml of virus stock and 7 x 10 6 pk-15 cells resuspended in 50 ml of mem-e (moi 0.1) were added in 175 and 25 cm 2 flasks. at 24 hours cells were treated with glucosamine to facilitate the virus infection. fortyeight hours later, viral infection was checked by ipma [37] in the 25 cm 2 flask. if more than 25 positive cells were counted in a microscope field, the 175 cm 2 flask was trypsinized and the cells were transferred to 3 new 175 cm 2 flasks. the process was repeated until 2,316 ml of virus stock with a titer of 10 4.79 tcid 50 /ml that was used to inoculate 24 l of bovine plasma achieving a final viral titer of 10 3.76 tcid 50% /ml. porcine parvovirus. porcine parvovirus strain nadl-2 was kindly provided by dr albert bosch (department of genetics, microbiology and statistics school of biology, university of barcelona, spain). it was propagated in sk-rst cells (atcc crl-2842), grown in sgm supplemented with 5% fbs. one ml of virus stock and 9 ml of mem-e supplemented with 1% pyruvate (merck kgaa, darmstadt, germany) were added to a conical tube with 16 x 10 6 sk-6 cells and shaken for 30 minutes at 104 rpm and 37˚c. after that time, the contents of the tube were transferred to a 175 cm 2 flask, in which 40 ml of mem-e supplemented with 1% pyruvate were added. this procedure was repeated until obtaining 811 ml of viral suspension with a titer of 10 6.91 tcid 50% /ml that was used to inoculate 24 l of bovine plasma achieving a final viral titer of 10 5.44 tcid 50 /ml. genomic data and virion size of each virus used in this study are displayed in table 1 . bovine and porcine blood was obtained from eu inspected slaughter facilities from animals inspected and approved for slaughter for human consumption. blood was collected in stainless steel containers with sodium phosphate as anticoagulant. blood was refrigerated and transported to the apc europe laboratory (apc-europe s.l.u., granollers, spain) and plasma was separated by centrifugation. plasma was frozen at -20˚c. prior to virus inoculation and uv-c irradiation, plasma was thawed and filtered to eliminate potential cryoprecipitate. before virus inoculation, a 100-ml sample of plasma was stored at -80˚c to determine absence of the test virus and absence of neutralizing antibodies to the test virus. for all viruses tested, after virus inoculation to plasma, the 24 l mixture was divided into three equal 8 l aliquots. each aliquot was subjected to uv-c irradiation to provide triplicate analytical results. because antibodies against porcine viruses are not expected to be found in bovine blood, bovine plasma was inoculated with prv, prrsv, pedv, csfv, siv, sva, svdv, ppv and pcv-2. subsequent test confirmed the bovine plasma was negative for specific antibodies against the test virus. similarly, porcine plasma was tested for bvdv antibodies by neutralizing peroxidase monolayer assay. the uv-c reactor system surepure turbulator used for the study was designed for opaque liquids, manufactured by surepure operation ag (zug, switzerland) and is described elsewhere [30] . briefly, the system consists of a reactor with a stainless-steel inlet and outlet chamber with a stainless steel corrugated spiral tube between the chambers. inside the spiral tube is an uv-c 254 nm wavelength germicidal lamp of 100-watt (w) output (30 w uv-c output) protected by a quartz sleeve. the liquid flows between the corrugated spiral tube and the quartz sleeve. the tangential inlet of the reactor creates a high velocity and turbulence in the inlet chamber and brings the liquid close to contact with the surface of the uv-c light source. the liquid is pumped at a minimum flow rate of 4000 l/h with a reynolds value in excess of 7500 (reynolds number > 2,600 indicates a turbulent flow). the uv-c dosage is expressed as j/l. the operation time of the uv-c treatment is based on the quantity of product to be treated and the flow rate of the product feed. at a flow rate of 4000 l/h, 9 s are required for 10 l of product to pass through the reactor once; thus, one turn of the product through the system is equivalent to a uv-c dose of 22.95 j/l. the uv dosage per l of liquid treated for one reactor with continuous flow was calculated as follows: dosage = total uv-c output per unit (w) / flow rate (l/s) = 25.50 w / 1.11 l/s = (25.50 j/s)/ (1.11 l /s) = 22.95 j/l. using an ammeter, the input current to the machine and its consumption were checked and it was established that they were appropriate according to the technical specifications and the data sheet of the uv-c lamp provided by surepure. a standard 'cleaning in place' (cip) process as described by keyser et al. [38] based on a treatment with naoh 5%, was implemented prior to and following each uv-c treatment. plasma flow was stabilized at 4000 l/h with the uv lamp switched off. after 5 minutes of stable flow, a positive control (0 j/l) sample was collected into sterile container. then, the uv-c lamp was switched on and irradiation started. 175 ml of treated plasma were collected into sterile containers at different uv-c doses (750, 1500, 3000, 6000, and 9000 j/l). infectivity of samples was determined in target cell cultures using the microtiter assay procedure [39] . the microtiter assay was done in a 96-well plate for all tested viruses, except for pedv. titration of virus was done using the whole plate for every dilution, from -1 to -5 dilutions, to amplify the detection capability of the test. final titer was expressed as log tcid 50 / ml. once all samples were titrated and the first negative dose was found, further steps were developed to ensure full inactivation of the samples. first, 50 ml of this negative dose sample were inoculated into 10 175-cm 2 culture flasks and incubated at 37˚c for 5 days. the flasks were frozen and thawed three times. the liquid was then centrifuged at 3000 rpm and 25 ml were inoculated into 5 175-cm 2 flasks. the flasks were incubated 5 days at 37˚c. the procedure was repeated again and, finally, only one flask was inoculated with 5 ml and incubated at 37˚c for 5 days. if the final flask was negative, it was considered that no viral particles were present in the initial sample; if the final flask was found positive, a titration of the plasma was repeated again on ten 96-well plates to analyze a total volume of 50 ml. due to the cytotoxic effect of the plasma, strong washes to eliminate serum used to propagate the cell culture and trypsin addition, pedv was titrated by means of pfu/ml. a 12-well culture plate with 100% confluency was inoculated with diluted plasma in mem supplemented with 0.05% trypsin and 0.3% tryptose, but without fbs. whole plates were used for each dilution, from -1 to -5, to amplify the detection capability of the test. conversion to tcid 50 /ml was done multiplying pfu x 0.7 (https://www.lgcstandardsatcc.org/support/faqs/48802/ converting+tcid50+to+plaque+forming+units+pfu-124.aspx?geo_country=es). negative samples were inoculated to 175-cm 2 culture bottles to analyze a total volume of 50 ml (10 ml by bottle), and passaged three times before being discarded as negative. if a sample was found positive, a titration of pure plasma was repeated on 10 12-well plates to analyze the same total volume of 50 ml. this procedure was intended to increase 10 times the initial analyzed volume. microbial inactivation due to thermal and non-thermal processes can be represented by eight possible curves [40] . the ginafit software was used to test linear and non-linear survival curves [40] , using the biphasic [41] ,weibull [42] , weibull plus tail [43] and biphasic plus shoulder [40] models. the ginafit software has been used to test survival kinetics of different bacteria and viruses when submitted to heat treatment or uv-c irradiation [44] . the biphasic model [40, 41] uses the eq (1): where n0 is the initial bacterial concentration; t is time; f is the fraction of the initial population in a major subpopulation, (1-f) is the fraction of the initial population in a minor subpopulation, and k max1 and k max2 are the specific inactivation rates of the two populations, respectively. the weibull model [42] shows the eq (2): where δ is a scale parameter denoted as the time for the first decimal reduction, and p is the shape parameter that describes concavity or convexity of the curve. if p>1 the curve shows convexity and if p<1 the curve is concave. the weibull plus tail model [43] uses the eq (3): where n res is the number of resistant microorganism subpopulation. the biphasic plus shoulder model [40] follows the eq (4): where n0 is the initial bacterial concentration; t is time; k max1 and k max2 are the specific inactivation rates of the two populations and s1 are the degrees of freedom used for the parameter estimation by ginafit the log linear model [40] presents the eq (5): where n represents de microbial cell density, n0 the initial microbial cell density, t is time; k max is the first order inactivation constant and ln(10) represents de decimal reduction time. the log-linear plus tail model [40, 45] uses the eq (6): logn ¼ logðð10 log n0 à 10 logn res þþ à e ðk max dþ þ 10 logn res ð6þ where k max is the inactivation rate of the log linear part of the curve and n res is the number of resistant microorganism subpopulation. the log-linear plus shoulder model [40, 45] shows the eq (7): where n0 is the initial bacterial concentration; t is time; k max is the inactivation rate and s1 are the degrees of freedom. using these equations, the software automatically calculates the 4d value (the dose needed to inactivate 4 log of viral load). in the case of bvdv, since the titer expressed as log tcid 50 / ml did not achieve 4 log, the 4d value was calculated for log tcid 50 in 10 ml of tested sample. in the case of pcv2, the 4d value could not be calculated due to the process did not achieve 4 log of inactivation. data were expressed as the mean as log10 tcid 50 with standard deviations of three independent replicates. mean, standard deviations, anova and f-test for comparisons were calculated with excel 2007 (microsoft office). the tukey test was done to determine significant differences between uv-c radiation doses. mean square error (mse), goodness of fit in terms of root mean square error (rmse), correlation coefficient (r 2 ) and adjusted correlation coefficient (adj-r 2 ) values were calculated with the ginafit software [40] . the inactivation model with the best fit corresponded to the model with the smallest rmse [40] . bovine plasma used in the studies was negative for viral contamination or neutralizing antibodies against tested viruses (prv, prrsv, pedv, csfv, siv, sva, svdv, ppv and pcv-2). in addition, porcine plasma was free from bvdv and bvdv antibodies. the results of uv-c viral inactivation are summarized in table 2 and in figs 1, 2 and 3. in general, all enveloped viruses tested were inactivated at <3000 j/l and showed non-linear inactivation kinetics, biphasic or weibull distributions, with low rmse values resulting in 4d values under 3000 j/l (figs 1 and 2 and table 2 ). the two best non-linear inactivation kinetics with the smallest rmse are included in table 2 . greater ranges of stability were found for non-enveloped viruses. ppv was inactivated at <3000 j/l, but other non-enveloped viruses such as sva, svdv and pcv-2 required a greater uv-c dose to be inactivated ( table 2 ; fig 3) . for example, all three samples of svdv at 6000 j/l were negative by the microtiter assay. however, the subsequent 3 blind passages performed for each 6000 j/l sample were positive. afterwards, a titration of 25 ml was done by duplicate, obtaining one positive out of 960 wells, so, it was concluded that there were 0.02 particles/ml (0.014 tcid50%) in the 6000 j/l samples (1 particle/50 ml assayed volume). to further confirm the dose required to inactivate svdv, three additional blind passages were performed for the three samples irradiated at 9000 j/l, and one was found positive. titration of the original sample of 9000 j/l (25 ml) was done; one out of 480 inoculated wells was positive. it was calculated that 0.04 particles per milliliter (0.028 tcid50%) were still present in the only positive sample of 9000 j/l (1 particle / 25 ml assayed volume). the most resistant virus was pcv-2, since the total log10 reduction achieved was 2.71 log10 at 9000 j/l. the efficacy of microbial reduction by uv-c treatment of liquids depends on different factors, including the microorganism used, the opaqueness of the liquid, the presence of suspended particles, and the microbial contamination level at the starting point [46] . animal plasma from slaughterhouse is an opaque liquid; therefore, uv-c radiation does not penetrate this liquid. this problem has been overcome by introducing turbulent flow achieving that the whole volume of liquid passes close to the uv-c radiation source [25] [26] [27] [28] [29] [30] . the uv-c system used in this work can be expanded to efficiently process large volumes of liquid compatible with commercial production of fruit juices, wine [26, 38] and milk [47] . it is important to differentiate this unique design of the surepure turbulator system for treatment of high volume of opaque liquids from other uv systems developed for treatment of human plasma fractions [25, 27] that are designed for treatment of small plasma fraction bags under gentle agitation but cannot be scalable to the volumes used by the commercial sdap industry. a preliminary step to implement industrial plasma irradiation with uv-c consisted of demonstrating such treatment does not affect the functionality of proteins present in sdap. similar growth and health performance of pigs fed diets with uv-sdap versus non-irradiated sdap has been reported [31, 48] . the next step was to investigate the inactivation capacity of the system on possible contaminating viruses in the plasma. for this purpose, selected viruses representing enveloped and non-enveloped, dna and rna, single-stranded and double-stranded viruses of importance for porcine industry were spiked into sdap and irradiated. furthermore, the different viruses table 2 tested may be considered surrogates for other untested viruses belonging to the same family or genus, although it is always recommended to test specifically each particular virus [49] . in the present study, uv-c radiation was more effective inactivating tested enveloped viruses compared to the non-enveloped viruses. enveloped viruses could not be detected when the uv-c dose was >3000 j/l. the best fit lines describing inactivation kinetics of the ultraviolet-c inactivation of enveloped and non-enveloped viruses inoculated in liquid animal plasma enveloped viruses were curvilinear biphasic equations. this could be due to lack of more points of collection; in many cases the last data point(s) appeared to be beyond the minimum uv-c radiation required to inactivate the entire virus in the sample, creating an apparent curvilinear shape. cutler et al, [50] reported biphasic inactivation kinetics of prrsv, bvdv and siv using a different uv-c delivery system and with the virus suspended in culture media rather than blood plasma. although almost a lineal response was observed in the present study, the best model for bvdv was weibull, and biphasic plus shoulder for siv. nevertheless, in both studies, despite using two different irradiation equipment (here versus the used by cutler et al. [49] ), the relative sensitivity of the virus to uv-c inactivation was similar with prrsv being more sensitive than bvdv or siv. a total inactivation (3.16 log) of bvdv was achieved at 3000 j/l. the inactivation kinetics obtained with bvdv is very similar to the one observed in the other tested pestivirus, csfv, which could indicate that members of the same genus could respond similarly to uv-c treatment. prv titer decreased more than 4 log at uv-c irradiation treatment < 3000 j/l, which represented a better inactivation ratio than that reported by another experiment using riboflavin-uv photochemical based technology [49] . the degree of inactivation by uv-c irradiation may depend on the technology used [51] . to the authors' knowledge, this is the first study showing effective results in the inactivation of pedv by means of uv-c. the non-enveloped viruses that were tested appeared to be more resistant to inactivation by uv-c radiation. the inactivation curve for ppv was consistent with previously published data [31] , with apparent linear inactivation in response to increasing uv-c dose. likewise, the inactivation curve for sva appeared to be linear except for the highest uv-c dose. the inactivation curve for svdv showed minimal inactivation at low uv-c dose, suggesting that a minimal uv-c dose was necessary before virus was inactivated. this has been reported by others [40] who suggested that for some virus inactivation does not occur until a minimum uv-c dose is achieved. however, in this experiment appears to be another shoulder in the inactivation curve at intermediate uv-c dosage (1500 and 3000 j/l). furthermore, while the highest doses appeared to inactivate all the virus, subsequent passages detected very low residual viral infectivity. the inactivation curve for pcv-2 demonstrated that uv-c radiation inactivated this virus; however, even at the highest dose (9000 j/l), residual levels of pcv-2 were recovered. the presence of a resistant viral subpopulation has been attributed to the ability of some viruses to take advantage of host cell repair mechanisms [52] , or in some cases they can code for their own repair machinery [53] . it has also been argued that viral particle clumping, particularly with other cells or debris, may shield some virus from uv-c radiation [50] . in the present study, a difference between the theoretical titer in the spiked plasma and the measured titer at time zero was observed. the difference was greater with enveloped viruses than with non-enveloped viruses. generally, enveloped viruses are more sensitive to environmental conditions such as salts, temperature changes, freezing and thawing or presence of anticoagulants [19] and could explain the reduction in titer before irradiation compared to the theoretical titer [4] . there was not a clear relationship between rate of inactivation and genome type or size. theoretically, dna is more sensitive to uv-c due to the presence of thymine [54] . in contrast, dna repair can reduce the uv-c effect, especially for dsdna viruses [55] . in the present study, prv (a dsdna virus) had 4d reduction estimated at 1612 j/l, which was not apparently different from the 4d reduction values obtained for other tested rna viruses (pedv and siv). obtained results with viruses evaluated in this study suggest that uv-c inactivation susceptibility of both types of viral genome was similar. it has been suggested that the longer the genome, the higher the susceptibility to uv-c damage [56] . however, based on the present results, increased genome size did not appear to increase virus susceptibility to uv-c radiation when spiked into commercially collected animal plasma and exposed to uv-c. only a weak linear trend (r 2 = 0.43) was found in the current data when comparing genome size to the 4d of viruses (without outlier's results pedv and prv). curiously, wang et al. [25] found that viruses with large genomes (adenovirus and reovirus) were inactivated with higher uv-c doses than viruses with shorter genomes. these authors indicated that their unexpected survival curves compared with other uv-c irradiation studies was difficult to explain, and suggested differences in the shape, size and lamp geometry of uv-irradiation systems as well as in protein concentrations and composition of process streams to explain their results. also, genome configuration may affect the virus sensitivity to uv-c radiation. for example, both ppv and pcv-2 have a relatively small genome size (5.07 and 1.77 kb, respectively). however, estimated 4d for ppv was 2161 j/l while that for pcv-2 was >9000 j/l. it is possible that the linear configuration of ppv genome could be more easily damaged, whereas the circular genome of pcv-2 may provide resistance to the formation of thymidine dimers by uv-c radiation. in any case, the present study should be considered exploratory in nature regarding the relationship between size and type of the genome and susceptibility to inactivation by uv-c irradiation. in addition to the photochemical damage of uv-c irradiation on nucleic acids, uv-c also induces reactive oxygen species that may interact with the external lipid bilayer membrane of enveloped viruses [57] , causing lipid peroxidation [58] . this effect may further explain why enveloped viruses were very susceptible to uv-c irradiation. in fact, in the present study, irradiation doses used were between 30-375 times higher than those described in the literature [59] . these differences could be due to the presence of proteins and other molecules in solution, to the possibility of virus clumping with these molecules, to the greater opacity of the liquid against uv, to the higher basal absorbance and/or to the darker color of the liquid plasma solution compared to other more transparent solution like water or other buffers like pbs. nevertheless, our results are closer to irradiation values found in other opaque raw materials like milk or juices [26, 60] . overall, the surepure uv-c turbulator design was effective in inactivating a wide variety of virus spiked into commercially collected liquid animal plasma. data from the present study indicated that the uv-c irradiation of liquid plasma is a technology that could provide an additional inactivation step to the industrial production process for sdap. exposure to uv-c is extensively used for the disinfection of liquid media and surfaces due to its germicidal activity [23, 24] . therefore, uv-c irradiation of liquid plasma during the manufacturing process for sdap has a potential application for inactivation of microbial contaminants without causing negative effects on the nutritional or physical qualities of the treated material [51, 61] . furthermore, the uv-c mechanism of inactivation (dna or rna damage) is different than the thermal inactivation provided by the spray-drying step. in consequence, the uv-c step can be considered independent from the spray-drying process and may be a synergistic process as both procedures have different inactivation targets. in conclusion, obtained results demonstrate uv-c as a suitable technology to be applied in the manufacturing process of sdap as a redundant biosafety step for the inactivation of viruses of concern for the livestock industry. supporting information s1 spray dried animal plasma as an alternative to antibiotics in weanling pigs. asian-australasian spray dried plasma as an alternative to antibiotics in piglet feeds, mode of action and biosafety. porc heal manag. porcine health management bactericidal effect of the spray-drying system for animal plasma on two different e. coli animal strains. icomst-rome, 25 efficacy of spray-drying to reduce infectivity of pseudorabies and porcine reproductive and respiratory syndrome (prrs) viruses and seroconversion in pigs fed diets containing spray-dried animal plasma commercially produced spray-dried porcine plasma contains increased concentrations of porcine circovirus type 2 dna but does not transmit porcine circovirus type 2 when fed to naive pigs effect of spray-dried plasma form and duration of feeding on broiler performance during natural necrotic enteritis exposure efficacy of spray-dried bovine serum on health and performance of turkeys challenged with pasteurella multocida porcine epidemic diarrhea virus rna present in commercial spray-dried porcine plasma is not infectious to naïve pigs inactivation of swine vesicular disease virus in porcine plasma by spray-drying lack of transmission of porcine circovirus type 2 to weanling pigs by feeding them spray-dried porcine plasma commercial spray-dried porcine plasma does not transmit porcine circovirus type 2 in weaned pigs challenged with porcine reproductive and respiratory syndrome virus no transmission of hepatitis e virus in pigs fed diets containing commercial spray-dried porcine plasma: a retrospective study of samples from several swine trials survivability of porcine epidemic diarrhea virus (pedv) in bovine plasma submitted to spray drying processing and held at different time by temperature storage conditions the spray-drying process is sufficient to inactivate infectious porcine epidemic diarrhea virus in plasma inactivation of viruses in labile blood derivatives. i. disruption of lipid-enveloped viruses by tri(n-butyl)phosphate detergent combinations inactivation of lipid-enveloped viruses in labile blood derivatives by unsaturated fatty acids inactivation of lipid-enveloped viruses in proteins by caprylate enveloped virus inactivation by caprylate: a robust alternative to solvent-detergent treatment in plasma derived intermediates annex 4 guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products nanofiltration of plasma-derived biopharmaceutical products advances in ultraviolet light technology for non-thermal processing of liquid foods introduction to research in ultraviolet photobiology advantages and limitations on processing foods by uv light monitoring and control of uv and uv-tio2 disinfections for municipal wastewater reclamation using artificial neural networks virus inactivation and protein recovery in a novel ultraviolet-c reactor efficacy of ultraviolet radiation as an alternative technology to inactivate microorganisms in grape juices and wines uvc irradiation for pathogen reduction of platelet concentrates and plasma development of a hydrodynamic model for the uv-c treatment of turbid food fluids in a novel "surepure turbulator" swirl-tube reactor validation of hydrodynamic and microbial inactivation models for uv-c treatment of milk in a swirl-tube "surepure turbulator™ ultraviolet (uv-c) inactivation of enterococcus faecium, salmonella choleraesuis and salmonella typhimurium in porcine plasma ultraviolet light (uv) inactivation of porcine parvovirus in liquid plasma and effect of uv irradiated spray dried porcine plasma on performance of weaned pigs experimental studies in weaned pigs with three vaccines against aujeszky's disease immune responses of pigs after experimental infection with a european strain of porcine reproductive and respiratory syndrome virus experimental infection of pigs with a new porcine enteric coronavirus, cv 777 genetic characterization of influenza a viruses circulating in pigs and isolated in north-east spain during the period porcine circovirus type 2 (pcv2) cap and rep proteins are involved in the development of cell-mediated immunity upon pcv2 infection dynamics of porcine circovirus type 2 infection in a herd of pigs with postweaning multisystemic wasting syndrome ultraviolet radiation as a non-thermal treatment for the inactivation of microorganisms in fruit juice establishment, viral susceptibility and biological characteristics of a swine kidney cell line sk-6 ginafit, a freeware tool to assess non-log-linear microbial survivor curves tailing of survival curves of bacterial spores on calculating sterility in thermal preservation methods: application of the weibull frequency distribution model a modified weibull model for bacterial inactivation surrogate organisms for pathogenic o157:h7 and non-o157 escherichia coli strains for apple juice treatments by uv-c light at three monochromatic wavelengths structural model requirements to describe microbial inactivation during a mild heat treatment the use of ultraviolet radiation as a non-thermal treatment for the inactivation of alicyclobacillus acidoterrestris spores in water, wash water from fruit processing plant and grape juice concentrate inactivation of mycobacterium avium ssp. paratuberculosis in milk by uv treatment ultraviolet irradiation of spray-dried porcine plasma does not affect the growth performance of nursery pigs when compared with non irradiated bovine plasma inactivation of viruses in platelet and plasma products using a riboflavin-and-uv-based photochemical treatment kinetics of uv254 inactivation of selected viral pathogens in a static system inactivation credit of uv radiation for viruses, bacteria and protozoan (oo)cysts in water: a review effect of ultraviolet radiation on the bacillus subtilis phages spo2, spp1 and phi 29 and their dnas fowlpox virus encodes a novel dna repair enzyme, cpd-photolyase, that restores infectivity of uv light-damaged virus the physical state of viral nucleic acid and the sensitivity of various to ultraviolet light host-cell reactivation in mammalian cells. ii. survival of herpes simplex virus and vaccinia virus in normal human and xeroderma pigmentosum cells predicted inactivation of viruses of relevance to biodefense by solar radiation proteomic analysis of uvc irradiation-induced damage of plasma proteins: serum amyloid p component as a major target of photolysis dose dependent variance in uv-c radiation induced effects on carbon and nitrogen metabolism in the cyanobacterium nostoc muscorum meg1 evaluating ultraviolet sensitivity of adventitious agents in biopharmaceutical manufacturing efficacy of ultraviolet (uv-c) light in a thin-film turbulent flow for the reduction of milkborne pathogens an evaluation of ultraviolet light (uv 254) as a means to inactivate porcine reproductive and respiratory syndrome virus on common farm surfaces and materials we want to thank the secretaria de universitats i recerca del departament d'economia i coneixement de la generalitat de catalunya (2014 di 066) for its colaboration. key: cord-267973-uvz7kavu authors: do, lien anh ha; bryant, juliet e.; tran, anh tuan; nguyen, bach hue; tran, thi thu loan; tran, quynh huong; vo, quoc bao; tran dac, nguyen anh; trinh, hong nhien; nguyen, thi thanh hai; le binh, bao tinh; le, khanh; nguyen, minh tien; thai, quang tung; vo, thanh vu; ngo, ngoc quang minh; dang, thi kim huyen; cao, ngoc huong; tran, thu van; ho, lu viet; farrar, jeremy; de jong, menno; van doorn, h. rogier title: respiratory syncytial virus and other viral infections among children under two years old in southern vietnam 2009-2010: clinical characteristics and disease severity date: 2016-08-08 journal: plos one doi: 10.1371/journal.pone.0160606 sha: doc_id: 267973 cord_uid: uvz7kavu background: despite a high burden of respiratory syncytial virus (rsv) infections among children, data on demographic and clinical characteristics of rsv are scarce in low and middle income countries. this study aims to describe the viral etiologies, the demographic, epidemiological, and clinical characteristics of children under two years of age who were hospitalized with a lower respiratory tract infections (lrti), focusing on rsv (prevalence, seasonality, subgroups, viral load) and its association with disease severity. methods: a prospective study among children under two years of age, hospitalized with lrti was conducted in two referral pediatric hospitals in ho chi minh city, vietnam, from may 2009 to december 2010. socio-demographic, clinical data and nasopharyngeal swabs were collected on enrolment and discharge. multiplex real-time rt-pcr (13 viruses) and quantitative rsv rt-pcr were used to identify viral pathogens, rsv load and subgroups. results: among 632 cases, 48% were rsv positive. rsv infections occurred at younger age than three other leading viral infections i.e rhinovirus (rv), metapneumovirus (mpv), parainfluenza virus (piv-3) and were significantly more frequent in the first 6 months of life. clinical severity score of rsv infection was significantly higher than piv-3 but not for rv or mpv. in multivariate analysis, rv infection was significantly associated with severity while rsv infection was not. among rsv infections, neither viral load nor viral co-infections were significantly associated with severity. young age and having fever at admission were significantly associated with both rsv and lrti severity. a shift in rsv subgroup predominance was observed during two consecutive rainy seasons but was not associated with severity. conclusion: we report etiologies, the epidemiological and clinical characteristics of lrti among hospitalized children under two years of age and risk factors of rsv and lrti severity. respiratory syncytial virus (rsv) is the leading cause of lower respiratory tract infections (lrtis) in young children. 50% of children are infected by rsv during their first year of life, and by 3 years of age, 100% have experienced at least one infection [1] . previous studies [2] [3] [4] have shown the importance of rsv in hospitalized children in vietnam. hospital records from the two main paediatric referral centers in ho chi minh city show that 77% of hospitalized children with lrti are under 2 years old (unpublished data). in addition, severe disease from rsv infection seems to exclusively occur in this population [3, 5] . however, information on detailed clinical, epidemiological features and virological characteristics of rsv infections (e.g. disease burden, demographics, seasonal variations of rsv and other viral infections, circulating genotypes and subgroups, viral load) or on the frequency / impact of other respiratory viruses among vietnamese children under two years old are limited [6] . here, we aimed to describe the viral etiologies and the demographic, epidemiological, and clinical characteristics of children under two years of age who were hospitalized with a lrti, focusing on rsv (prevalence, seasonality, subgroups, viral load) and its association with disease severity. the study was conducted from may 2009 to december 2010 (to cover two rsv seasons, that normally coincide with the rainy season) at children's hospital 1 (ch1) and children's hospital 2 (ch2), the two largest pediatric referral centers for southern vietnam, located in ho chi minh city. children from the respiratory wards (rw), emergency care units (ecu) and intensive care units (icu) were eligible for inclusion in the study. patients admitted to the rw are typically those that initially present to the outpatient clinics and were subsequently indicated for admission, while those admitted to ecu or icu typically presented with more severe symptoms. inclusion criteria were age between 1 month to 2 years, cough, a clinical diagnosis of lrti based on who criteria [7] , and onset of symptoms 4 days prior to hospital admission. exclusion criteria were patients with known non-respiratory or non-infectious respiratory diseases such as asthma. were collected on the day of enrolment (day 1) and on day 7 or discharge (if patients were discharged before day 7). edta blood were used for whole blood cells counting, and liver enzyme measurement by ch1 and ch2 laboratory. swabs were placed in viral transport medium [8] and kept at 4°c for a maximum of 24h, then aliquoted and stored at -80°c until further processing for molecular diagnostics [9, 10] . the study was approved by the institutional review board of children's hospitals 1 and 2, the scientific and ethics committee of the hospital for tropical diseases, ho chi minh city, vietnam and by the oxford university tropical research ethics committee (oxtrec), oxford, uk. written informed consent was obtained from parents or legal guardians of children prior to enrolment into the study. rna extraction from nasopharyngeal swabs was done as described previously [11] . rna was analyzed by multiplex real-time rt-pcr on a roche light cycler 480 ii thermocycler (roche diagnostics, penzberg, germany) [9, 10] and rsv locked nucleic acid (lna) real-time rt-pcr (lna assay) on a dna engine peltier thermocycler and chromo 4 real-time pcr system detector (bio-rad, hercules (ca), usa) [11] . the multiplex real-time pcr detects 13 other human respiratory viruses: influenza virus a (flu a); influenza virus b (flu b); adenovirus (adv), enterovirus (env), human metapneumovirus (mpv), human coronaviruses (cov-229e, oc43, hku1, sars cov & nl63), human rhinovirus (rv a, b and c), parainfluenza virus 1, 2 and 3, 4 (piv1, 2, 3, 4), parechovirus (pev) and human bocavirus (bov) [9, 10] . the lna assay assesses viral load (log10 copies/ml) of rsv subgroups a and b (rsv a, rsv b) [11] . for every rsv positive patient, the second sample (at discharge or day 7) was also assessed by lna assay. definitions. severe disease on admission was defined as having an oxygen saturation of spo2<92 or requiring supplemental oxygen or ventilatory support (by nasal cannula, nasal continuous positive airway pressure (ncpap), mask cpap or mechanical ventilation/intubation) or clinical cyanosis. a clinical severity score (adapted from [12] [13] [14] [15] ) was introduced to grade the clinical status of patients at enrolment (table 1) . only patients from whom all components in the score table were available were given a clinical severity score. long hospitalization was defined as longer than 7 days. statistical analysis. associations between categorical and continuous variables were analyzed using the mann-whitney-u test or kruskal-wallis test for continuous variables, and the fisher exact test for dichotomous variables. spearman's rank correlation coefficient was used to assess a general monotonic trend between two continuous variables. a simple linear regression model was used to measure linear associations between rsv viral load and age or day of illness or number of leucocytes in blood. multivariable logistic regression analysis was performed to assess risk factors for severe disease or long hospitalization. the following variables were considered for the models: age, sex, premature birth, previous hospitalization for respiratory illness, other household members sick at home, living with smoker(s), number of members at home, fever, rsv infection (rsv positivity, viral load, and subgroup) and rv infection (rv positivity as single infection or co-infection with rsv). the models' predictive ability was investigated by calculating the area under the receiver operating characteristic (roc) curve of the model (auc), i.e the higher the auc the better prediction power the model has. the hosmer-lemeshow goodness-of-fit test was done to assess model adequacy. the hosmer-lemeshow test of goodness-of-fit tests the null hypothesis that there is no difference between the observed and predicted values of the response variables. therefore, when the test is not significant (p>0.05) the null hypothesis cannot be rejected and this means that the model fits the data well. risk factors for disease severity or long hospitalization were further assessed using odds ratios (or) and 95% confidence intervals (95% cis). all statistical tests were performed as two-tailed tests at 5% significance in either r version 2.13.1 (r foundation for statistical computing, vienna, austria) or intercooled stata version 9.2 (college station, tx, usa). a total of 632 children aged 1-24 months (median 7, iqr 4-12 months) were enrolled into the study between may 2009 and december 2010. 10/632 (2%) patients were admitted to icu, 36/ 632 (6%) to the ecu and 586/632 (92%) to the respiratory ward. the monthly distribution of children hospitalized for lrti and enrolled in this study during the study period are shown in fig 1. diagnoses on admission were based on clinical symptoms, routine laboratory tests and chest radiography; physicians were unaware of viral diagnostic laboratory results. 438/632 (69%) patients were diagnosed with bronchiolitis; 164/632 (26%) with pneumonia, 22/632 (3%) with combined bronchiolitis and pneumonia, 3/632 (0.4%) with laryngotracheitis, and 5/ 632 (0.8%) with undifferentiated lrti. in addition to respiratory symptoms, 21/632 (3%) patients had diarrhoea on admission, and 4/632(0.6%) had congenital heart disease (ventricular or atrial septum defects). only 18/598 (3%) had blood culture test which was part of standard clinical care at hospitals and only 5/18 were positive (1 staphylococcus aureus, 1 streptococcus pneumoniae, 1 haemophilus influenzae, 1 pseudomonas aeruginosa, 1 klebsiella spp). 535/632 (85%) children received antibiotics, 100% children with clinical diagnosis of pneumonia received antibiotics. high alt** 1 high ast** 1 discharge information was available for 596/632 (94%) cases: 363/596 (61%) of patients fully recovered; 226/596 (38%) had incomplete recovery at the time of discharge; 4/596 (1%) went home without permission (mostly due to economic reasons, patients could not pay hospitalization fee) or formal hospital discharge; and 3 patients (1%) died in the hospital. one fatal case (8 months old) was diagnosed with septicemia and severe pneumonia (s. pneumoniae was recovered from blood culture; no respiratory viruses were detected in nasopharyngeal swabs) and died on the second day of admission. the two other fatal cases were 6 and 3 months old and did not have any severe indications on admission but deteriorated quickly after 5 days of hospitalization. no organisms were recovered from blood culture and virology results yielded a single rv infection and a triple infection (rv, piv-3 and bov), respectively. viral etiologies were identified in the vast majority (91%, 574/632) of patients; single viral infections accounted for 375/632 (59%) of cases and co-infections with multiple viruses were found in 199/632 (31%) ( table 2) . rsv was the most frequently detected: overall (302/632, 48%) and in single infections, while rv was most frequently detected in co-infections (table 2) . a significantly higher proportion of rv, env, bov, adv, piv-2, piv-3, cov and pev were detected among co-infections when compared to the single infections (table 2 ). for mpv, rv, piv-2, piv-3 and piv-4, the relative viral load in single infections was significantly higher than in co-infections (table 2) . rsv-rv co-infection was the most common double infection, identified in 147/632 (23%) cases. triple infections were identified in 47/632 (7%) and in 5/632 cases, 4 different viruses were detected (rsv-rv-piv3-cov, env-rv-cov-bov, adv-env-rv-piv2; rsv-adv-env-rv). (2) median log copies/ml rsva (iqr) 7.5 (6.7-8.2) 7.7 (6.7-8.1) 7.4 (6.3-8.2) 0.2 (1) rsv b, n(%) 139 (22) 93 (25) 46 (23) 0.7 (2) median log copies/ml rsvb (iqr) 7.6 (6.9-8.2) 7.7 (7.1-8.2) 7.5 (6.6-8.0) 0.2 (1) flu, n(%) median cp-value flu b (iqr) 31 (29-33) 32 (29) (30) (31) (32) (33) 30 (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) 0.8 (1) adv, n(%) 37 (6) 3 (1) 34 (17) 0.001 (2) median mpv, n(%) 30 (5) 17 (5) 13 (7) 0.3 (2) median (1) piv-3, n(%) 47 (7) 18 (5) (1) pev, n(%) 12 (2) 2 (1) 10 (5) 0.001 (2) median cp-value pev (iqr) 28 (26) (27) (28) (29) 28 (26) (27) (28) (29) (30) (31) 28 (27) (28) (29) 0.7 (1) bov, n(%) 42 (7) 7 (2) 35 (18) 0.001 (2) median cp-value bov (iqr) 29 (25-33) 23 (22) (23) (24) (25) (26) (27) (28) (29) (30) 30 (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) (35) 0.1 (1) p-value was calculated between single infections and co-infections groups, (1) mann-whitney test, in the univariate analysis, lrti severity was associated with younger age (median age in months [iqr] = 5 [3] [4] [5] [6] [7] [8] [9] [10] in severe cases versus 7 [4] [5] [6] [7] [8] [9] [10] [11] [12] in non-severe cases, mann-whitney test pvalue = 0.001 -s1 fig) , presence of fever (72%, 76/106 in severe cases versus 47%, 247/526 in non-severe cases, fisher exact p-value = 0.001), living in a household with a high number of people (median household members [iqr] = 5 [4] [5] [6] in severe cases versus 4 [3] [4] [5] in nonsevere cases, mann-whitney test p-value = 0.03), or having other household members sick at home (50%, 52/103 in severe cases versus 37%, 193/528 in non-severe cases, fisher exact pvalue = 0.001). co-infections were more frequent among the non-severe cases, and the clinical severity score was inversely correlated to the number of viruses detected (spearman cor = -0.09, p = 0.04). among the single infections, viral load was not associated with severity. for rv, single infections were significantly more common (21%, 22/106) in severe cases versus in non-severe cases (12%, 65/526) (fisher's exact test p-value = 0.02). the relationship between rv and severity was also observed in increased odds ratios for elevated (worse) clinical score (or = 1.86, 95% ci (1.08-3.17), p = 0.02). similar relationships were not observed for rsv single infection cases. in multivariate analyses, significant predictors of severity within the entire study population were younger age, presence of fever, and rv single infection ( table 3 ). the hosmerlemeshow goodness-of-fit test result was χ 2 = 5.94, p-value = 0.65) and the auc for the prediction model was 0.74. similarly, predictors of long hospitalization were younger age, presence of fever, and previous hospitalization with respiratory illness were significant ( table 3 ). the hosmer-lemeshow goodness-of-fit test result was χ 2 = 7.38, p-value = 0.5) and the area under the roc curve for the prediction model of long hospitalization was 0.66. rsv infection was not correlated to disease severity, or to duration of hospitalization (table 3) . rsv infections occurred at a younger age than the 3 other leading single viral infections, i.e rv, mpv, piv-3 (tables 4 and 5) , and were significantly more frequent in the first 6 months of life (fisher exact test p-value = 0.001). in contrast, none of the other viruses exhibited significant age group distributions (tables 4 and 5). a significant inverse correlation was observed between rsv load at enrolment and patient age. indeed, on average rsv viral loads decreased by 0.03 log copies/ml per month increase in age (t = -2.37, p-value = 0.02). a higher clinical severity score was found only among rsv single infection cases compared to those of piv-3 single infection cases (mann-withney test p-value = 0.02, table 5 ), but not in any of the other pair-wise comparisons. while rsv single infection had a significantly shorter duration of hospitalization, a higher prevalence of elevated ast and a lower leucocyte count than rv single infection cases (table 5 , mann-withney test p-value = 0.02, 0.02 and 0.001, respectively). we also observed a significantly lower number of leucocytes in rsv single infection versus those in rsv co-infection (mann-withney p-value = 0.001), and an inverse (2) 12 (71) 0.9 (2) 10 (56) 0.3 (2) median number of household members (iqr) 4 (4-6) 4 (4-6) 0.7 (1) 4 (3) (4) (5) 0.3 (1) 4 (3-6) 0.4 (1) living with smokers, n (%) 102/196 (52) 51/86 (59) 0.3 (2) 5 (29) 0.07 (2) 12 (67) 0.2 (2) medical story (2) 16 (94) 0.07 (2) 13 (72) 0.8 (2) premature birth, n(%) 20/199 (10) 14/85 (16) 0.1 (2) 0/16 (0) n.a 1/17 (6) 0.5 (2) daycare, n (%) 25/197 (13) 10/84 (12) 0.9 (2) 2/16 (13) 1.0 (2) 5/18 (28) 0.08 (2) previous hospitalization with respiratory illness, n (%) 22 (11) 23 (26) 0.001 (2) 2 (12) 0.9 (2) 3 (17) 0.5 (2) other household members sick at home, n (%) 76/197 (39) 36/85 (42) 0.6 (2) 5 (29) 0.5 (2) 5 (28) 0.4 (2) clinical characteristics fast breathing, n(%) 166 (83) 75 (86) 0.4 (2) 14 (82) 1.0 (2) 14 (78) 0.6 (2) cyanosis, n(%) 6 (3) chest indrawings, n(%) 193 (96) 81 (94) 0.3 (2) 15 (88) 0.1 (2) 15 (83) 0.01 (2) stridor, n(%) wheezing, n(%) 192 (96) 76 (88) 0.01 (2) 16 (94) 0.8 (2) 17 (94) 0.8 (2) fever (>37.5°c), n(%) 112 (56) 50 (57) 0.8 (2) 7 (41) 0.2 (2) 6 (33) 0.07 (2) fever ( 38.5°c), n(%) 46 (23) 13 (15) 0.1 (2) 5 (29) 0.5 (2) 2 (11) 0.2 (2) rash, n(%) 2 (1) 3 (3) 0.1 (2) 0 (0) n.a 1 (6) 0.1 (2) runny nose, n(%) 181 (91) 70 (81) 0.03 (2) 15 (88) 0.8 (2) 17 (94) 0.5 (2) low spo 2 , n(%) 19/140 (14) 6/60 (10) 0.5 (2) 1/14 (7) 0.5 (2) 1/17 (6) 0.4 (2) median of duration of hospitalization (iqr) 6 (5-7) 6 (5-8) 0.02 (1) 6 (4-7) 0.9 (1) 6 (5-7) 0.9 (1) duration of hospitalization 7 days, n(%) 77 (38) 43 (49) 0.08 (2) 7 (41) 0.8 (2) 7 (39) 1.0 (2) high alt, n(%) 54 (27) 16 (18) 0.1 (2) 2 (12) 0.2 (2) 3 (17) 0.3 (2) high ast, n(%) 44 (22) 9 (10) 0.02 (2) 4 (23) 0.9 (2) 3 (17) 0.6 (2) median of number of white cells in blood (k/mm 3 ) (iqr) 10 (8-14) 13 (11) (12) (13) (14) (15) (16) (17) 0.001 (1) 11 (9-13) 0.8 (1) 11 (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) 0.6 (1) severe cases, n(%) 36 (18) 22 (25) 0.2 (2) 3 (18) 1.0 (2) 3 (17) 1.0 (2) median of clinical score (iqr) 6 (4-7) (n = 186) 5 (4-7) (n = 80) 0.5 (1) 5 (4-6) 0.4 (1) 4.5 (4-6) 0.02 (1) diagnosis bronchiolitis, n(%) 162 (81) 65 (75) 0.3 (2) 10 (59) 0.06 (2) 9 (50) 0.005 (2) treatments antimicrobial agents, n(%) 169 (84) 62 (71) 0.01 (2) 17 (100) 0.07 (2) 17 (94) 0.2 (2) corticosteroids, n(%) 9 (4) 6 (7) 0.4 (2) 1 (6) 0.8 (2) bronchodilators, n(%) 168 (84) 67 (77) 0.2 (2) 9 (52) 0.002 (2) 10 (56) 0.004 (2) supplemental oxygen, n(%) 31 (15) 21 (24) 0.08 (2) 2 (12) 0.7 (2) 2 (11) 0.6 (2) outcomes full recovery, n(%) 119/195 (61) 46/81 (57) 0.5 (2) 11 (73) 0.8 (2) 11 (65) 0.9 (2) severe cases, n(%) 36 (18) 22 (25) 0.2 (2) 3 (18) 1.0 (2) 3 (17) 0.9 (2) death, n(%) p-values were based on comparison with children having rsv single infections. (1) mann-withney test, (2) fisher's exact test, n.a: not applicable correlation between rsv load in nasopharyngeal swabs and leucocyte count in blood collected at enrolment (coeff = -0.56, 95%ci = -0.98 to -0.14, p-value = 0.009). among 302 rsv positive cases, 156/302 (52%) were rsv a, 139/302 (46%) were rsv b, and 7/302 (2%) were both rsv a and b. there was no difference in proportion of a and b subgroups between single and co-infections, and no difference in viral load among subgroups at enrolment (table 2) . strong seasonal variations of rsv prevalence with peaks during the rainy season from may to october were observed over the two seasons of the study. rsv b was dominant during the first season and rsv a during the second season (fig 2) . overall case numbers for mpv and influenza were insufficient to detect patterns of seasonality. the most frequently detected viruses in rsv co-infection were rv, env and adv (s1 table) . in the univariate analysis, the number of viruses co-detected with rsv or the rsv viral load was not associated with rsv severity, or with clinical severity score (cor = 0.06, p = 0.1 and cor = -0.3, p = 0.2, respectively). rsv patients with long duration of hospitalization had a significantly higher rsv viral load than others (median of rsv load (iqr) 8 (7-8) versus 7 (7) (8) mann-withney test p-value = 0.004, respectively). no associations with rsv subgroups and severity or duration of hospitalization were found. in multivariate analyses for the rsv-infected population, the logistic regression analysis of ten selected predictors identified age (or = 0.88, 95%ci:0.81-0.95, p-value = 0.001), fever (or = 4.80, 95%ci:2.26-10.19, p-value = 0.001), and having another household member sick at home (or = 2.04, 95%ci: 1.03-4.02, p-value = 0.04) as independent variables associated with disease severity in rsv-infected children ( table 3 ). the hosmer-lemeshow goodnessof-fit test result was χ 2 = 5.73, p-value = 0.68) and the area under the roc curve for the prediction model was 0.76. for risk factors of long hospitalization among rsv patients, only fever (or = 2.67, 95%ci: 1.59-4.48, p-value = 0.001) was a significant independent predictor ( table 3 ). the hosmer-lemeshow goodness-of-fit test result was χ 2 = 9.35, p-value = 0.31 and the auc for the prediction model of long duration hospitalization was 0.67. again, rsv subgroups or rsv viral load were not associated with disease severity or long hospitalization in multivariate analyses. over the last decade, multiplex molecular diagnostics have revolutionized the diagnostics of respiratory infections and greatly expanded the available data on viral etiologies and coinfection [6, [16] [17] [18] [19] [20] [21] [22] . here, using a previously described multiplex assay to detect 13 different respiratory viruses, viruses were identified in 91% of enrolled patients, and viral co-infection was found in 31%. the high prevalence of viral infections among lrti cases, the predominance of rsv and rv infections, and the co-infection rates between rsv and rv, env and adv in our study were similar to other published work in comparable populations [21, 23] . in these previous studies, among hospitalized children, the proportion of viral causes ranged from 93-97%; rsv and rv were the leading causes, ranging from 64 to 73% and from 30-34%, respectively [21, 23] . our study confirms the importance of rsv infection in children under two as shown by many other studies [17, 21, [23] [24] [25] and is the first study to examine the demographic, clinical and virological characteristics of rsv infections in south vietnam. our results confirm and extend previous observations regarding associations of rsv infections with young age compared to the 3 other leading viral infections (rhinovirus (rv), metapneumovirus, parainfluenza virus (piv-3), wheezing, runny nose, leucopenia (among rsv single versus rsv co-infection or rv single infection) and risk factors (premature birth) [17, 21, [23] [24] [25] [26] . in addition, we observed a significant negative correlation between rsv load in nasopharyngeal swabs and leucocyte count in blood collected at enrolment (coeff = -0.56, 95%ci = -0.98 to -0.14, pvalue = 0.009). these findings are consistent with previous reports [26, 27] and it could be hypothesized that leukocytes in rsv infection are being recruited at specific sites away from the circulating blood [28, 29] . one of our aims was to determine risk factors for severity and long hospitalization among all study patients and among rsv-infected patients. we observed that rsv load at enrolment was significantly related to long hospitalization in univariate analysis, but this was not confirmed in the multivariate analysis; and rsv infection, rsv viral load, rsv subgroups and rsv slope (i.e rsv load dynamic between two time points: admission and discharge, it was calculated by dividing the difference of viral load at admission and at discharge by the number of days between these two time points-data not shown), did not correlate to disease severity or long hospitalization. only young age and fever were independent predictors for disease severity in both populations (study population and rsv population). reports about the relation between rsv infection [30, 31] or rsv load [12, 26, 32, 33] or rsv subgroups [34] [35] [36] [37] [38] and disease severity have been contradicting. marguet et al. reported that rsv infection caused more severe disease than rv infection among children under 1 year of age. in a study by tran et al. conducted in children hospital 2, ho chi minh city, targeting hospitalized children under 15 years, rsv positive children had significantly higher clinical severity scores compared to rsvnegatives. however, this comparison was based on univariate analysis without considering other confounding factors such as age [6] . papadopoulos et al. found, among children less than 18 months, that the presence of rv increases the risk for severe disease significantly, by approximately five-fold. rsv infection was also correlated with severity, however, this did not reach significance [30, 31] . rsv loads at the second collection had a significant negative correlation (correlation = -0.33, p-value = 0.009) with day of illness, while this correlation was not found for the enrolment samples which were collected on any day during the first days of illness (s2 fig). this rsv load trend during rsv infection was also observed in other studies [39] and confirmed the study samples were collected on the early day of rsv illness. thus far, observations regarding the relation between viral co-infections and disease severity have been contradictory and biased by different study designs and viral diagnostic tools used [24, 40] . similar to other studies, we observed a significantly lower proportion of co-infections in severe cases compared to non-severe cases [21, 41] . however, among rsv infected cases, we did not observe significant differences in disease severity between single and co-infected cases. in addition, there were significant differences between rsv-rv coinfections (3/45, 6.6%) versus rsv-env (9/27, 75%)-fisher's exact p-value = 0.006 but no significant versus rsv-adv co-infections (2/18, 11%). interestingly, and in agreement with papadopoulos et al. [31] , although rv was detected mostly as a coinfection, rv single infection was identified as an independent risk factor of severe disease. this finding should be interpreted with caution because the role of rv in the pathogenesis of severe lrti remains a topic of debate [30, 31, [42] [43] [44] [45] [46] [47] . cross-reactivity in the pcr detection of env and rv using 5'utr primers is a known problem, particularly for ev-d68 which has a rhinoviral 5' utr sequence, and this complicates ascertainment of env and rv diagnoses [48] . despite the fact that the study population from our current study and our previous study [3] and from other studies [2, 6] consisted of hospitalized children and is therefore not representative of vietnamese children in general, a consistent rsv seasonal pattern with peaks during the rainy season (between may and october) was observed from 2005 to 2010. the clear shift of rsv subgroups was not observed during the period 2005 to 2007 [3] but we observed this in our current study and tran et al. also confirmed the dominance of rsv a during the same period [6] . in many studies, rsv has been reported as a highly seasonal infection and rsv outbreaks are frequently associated with the rainy season in areas with tropical and subtropical climate [5, 49] . possible explanations for this include meteorological effects such as humidity and uvb radiation on the environmental stability of rsv viruses. the stability of rsv in aerosols was shown to increase with higher humidity [50] . moreover, population behaviors as staying indoors during cold or rainy seasons may facilitate transmission of rsv. from this cohort, we have recently reported analysis of rsv whole genomes [51] and are currently analysing host expression profiles of blood and nasopharyngeal swabs at two time points (manuscript in preparation). there are a number of limitations to our study. firstly, few bacterial diagnostic results were obtained from patients. the role of bacteria as a cause of lrti or as cause of superinfection, especially in rsv and influenza virus infection [52] , is important for antibiotic intervention strategies. lower airway secretions (sputum for example) are considered the optimal specimen type for detecting bacterial (co-) infection, yet are often difficult to obtain from young children, and can only be readily obtained from intubated children. secondly, only a limited number of patients were enrolled as compared to the total number of lrti hospitalizations in two hospitals during the study period (632/45134, 1%) although the number of enrolled patients followed a similar pattern as the total number of patients (fig 1) . in summary, our study has contributed detailed clinical and virological data on rsv and other viruses in respiratory infections among children under 2 years old, the most vulnerable age group, in a lower middle income setting in asia: vietnam. the data on seasonality of viruses are crucial for health care management, such as 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infections in children less than 5 years of age in indonesia the relationship of meteorological conditions to the epidemic activity of respiratory syncytial virus direct whole-genome deepsequencing of human respiratory syncytial virus a and b from vietnamese children identifies distinct patterns of inter-and intra-host evolution increased susceptibility to bacterial superinfection as a consequence of innate antiviral responses we acknowledge study nurses hoang thi minh tu, nguyen thi hong ngoc, nguyen viet truong, nguyen thi ngoc ha, nguyen thi thanh nha, le thi kim loan, ho huynh nhu, tran thi tuyet nhung, huynh thi phuong thao, for sample collection; staff in data management at oucru for entering data; vo nhi ha and nguyen thi thanh thuy and the clinical trials unit at oucru and children's hospital 1 and 2 for coordinating the trial.this study was funded by the wellcome trust of great britain (077078/z/05/z). the funder had no role in study design, data collection or analysis, the decision to publish, or preparation of the manuscript. wrote the paper: lahd jeb hrvd mdj. key: cord-048477-ze511t38 authors: patel, mahomed s.; phillips, christine b.; pearce, christopher; kljakovic, marjan; dugdale, paul; glasgow, nicholas title: general practice and pandemic influenza: a framework for planning and comparison of plans in five countries date: 2008-05-28 journal: plos one doi: 10.1371/journal.pone.0002269 sha: doc_id: 48477 cord_uid: ze511t38 background: although primary health care, and in particular, general practice will be at the frontline in the response to pandemic influenza, there are no frameworks to guide systematic planning for this task or to appraise available plans for their relevance to general practice. we aimed to develop a framework that will facilitate planning for general practice, and used it to appraise pandemic plans from australia, england, usa, new zealand and canada. methodology/principal findings: we adapted the haddon matrix to develop the framework, populating its cells through a multi-method study that incorporated the peer-reviewed and grey literature, interviews with general practitioners, practice nurses and senior decision-makers, and desktop simulation exercises. we used the framework to analyse 89 publicly-available jurisdictional plans at similar managerial levels in the five countries. the framework identifies four functional domains: clinical care for influenza and other needs, public health responsibilities, the internal environment and the macro-environment of general practice. no plan addressed all four domains. most plans either ignored or were sketchy about non-influenza clinical needs, and about the contribution of general practice to public health beyond surveillance. collaborations between general practices were addressed in few plans, and inter-relationships with the broader health system, even less frequently. conclusions: this is the first study to provide a framework to guide general practice planning for pandemic influenza. the framework helped identify critical shortcomings in available plans. engaging general practice effectively in planning is challenging, particularly where governance structures for primary health care are weak. we identify implications for practice and for research. primary health care, and in particular general practice, will be at the frontline in the response to pandemic influenza. preparedness planning for this sector has lagged behind public health planning, despite evidence from sars [1, 2] and influenza epidemics [3] of the important role played by general practice. preparedness may be defined as the capacity to respond to a range of public health threats including natural disasters and infectious disease outbreaks, human-caused accidents and intentional attacks [4] . there is an increasing recognition of the need for an 'allhazards' approach to planning that integrates acute clinical care, public health, and emergency management systems [4] . since september 2001, the us government has invested about $5 billion to upgrade preparedness plans for emergency management systems [5, 6] . there are three challenges for pandemic planning by general practice. first, there is no systematic framework for planning this sector's response. preparing for health threats and emergencies is an essential function of public health, but is not core business for general practice. second, the way in which ambulatory health services will interact with each other and with the broader health system response to a pandemic is unclear. general practitioners (gps) in canada [7] , australia [8] and the uk [9] have expressed uncertainty about how to participate in such a response. third, planning and implementing changes for pandemic influenza across the health system is complex. although there is little evidence linking specific preparedness activities to effective system-wide responses to pandemic influenza [5, 6] , change management theories point to a need for dynamic partnerships between general practices and other ambulatory care services, hospitals and public health departments [10] . the strength and structure of these linkages vary around the world, depending on decentralisation processes, the regulatory and legal system, and financing within health systems [11, 12] . although general practice, or family medicine, is organised differently in different countries, there is considerable potential for transferable learning at the meso-level of management planning [11] . we aimed to develop a framework that will facilitate systematic planning for the general practice response to pandemic influenza and used it to appraise coverage of key elements in publicly available pandemic plans from australia, england, usa, new zealand and canada. to guide planning and to appraise available plans, we adapted the haddon matrix, a planning tool developed in the field of injury research and intervention [13] , and more recently applied to the public health response to bioterrorism, sars [14] , and pandemic influenza [15] . the matrix consists of a grid of columns of four factors (human, agent, and physical and organisational environment) impacting upon the event [15] . pandemic influenza may be perceived as a form of injury on a mass scale and the matrix helps us understand the multi-dimensional nature of epidemics and of the associated challenges that could be expected by general practice. the framework can be readily shared with public health units and other parts of the health system, as it identifies the general practice contributions to primary health care services and to public health surveillance and control. because all disasters are local, the matrix is flexible enough to allow a focused analysis of the smallest unit of study, such as an individual, or group of general practitioners. the methods used to construct the cells of the modified haddon matrix have been detailed elsewhere [16] . in brief, a team with expertise in social science, public health and general practice reviewed objectives and strategies in who guidelines for preparing and responding to a pandemic [17] to define the context and potential contributions of general practice. next, we undertook a narrative review of the peer-reviewed and grey literature on pandemic influenza to identify papers that elaborated strategies relevant for general practice. a search of the peerreviewed literature through pubmed using the terms 'general practice', 'family physician', 'family medicine' and various combinations of the terms 'influenza', 'epidemic', 'preparedness' and 'pandemic' yielded 24 eligible papers from 157 search results . the process of constructing the framework and populating the cells was informed by organisational theories that emphasise multilevel approaches to change from the individual to the broader health system [10, 18] , and by methods for measuring [5] and improving the quality [6] of public health emergency preparedness. we tested our framework through interviews with a purposive sample of health professionals engaged in pandemic planning. nineteen general practitioners and practice nurses with expertise in pandemic planning were nominated by the two participating divisions of general practice, each of which was a national leader in disaster preparedness and response. eight general practice policy leaders were identified by representative organisations (australian medical association, royal australian college of general practitioners, australian general practice network). group interviews were held with 14 state and territory public health leaders attending a national pandemic preparedness meeting. we held two workshops, attended by representatives of state and territory health services, commonwealth policymakers, non-government organisations, and general practice organisations. in addition, we conducted two focus groups of gps and nurses working in aged care in two cities. finally, we undertook four desktop exercises [19] attended by 25 gps, 11 practice nurses and 10 administrative staff. the five countries in this study had national response plans. contextualised detail about health-sector responses is contained in plans at the level of administrative decentralisation where decisions are made about patient-service groupings including general practice. in practice, this level was the state or provincial health departments in federal systems where those jurisdictions have responsibility for health service management and planning (usa, canada, and australia). in england, the managerial level for health services is located at the primary care trust (pct), while in new zealand it occurs at the level of the district health board. although these are not identical loci of health service governance, they were sufficiently similar in the planning aims for comparisons to be drawn. plans were obtained from websites of health departments of states or provinces (usa, australia, canada), district health boards (new zealand) and pcts (england) ( figure s1 ). for new zealand and england, publicly available records of board meetings were also examined. consumer information and isolated sub plans (e.g. for infection control) were excluded. plans for 95 jurisdictions were identified; six were excluded as they addressed isolated aspects such as only the distribution of medications, or communication with the public, leaving 89 plans suitable for analysis. of the five countries, canada exhibits the most variation between provinces in health system coordination. we examined the websites of canada's 84 provincial regional health authorities (rhas, 14 plans identified) and ontario's 36 public health units (26 plans identified) and 14 local health integration networks (no pandemic plans identified). we excluded the rha and public health unit plans from inter-country quantitative analysis, as their level of devolution and/or responsibilities for health management differed from those examined in the other four countries, but have included descriptive details from some of the rha plans where they illustrate innovative approaches. all plans were examined by two clinicians, and searched for the following terms: primary care, primary health, ambulatory, general practice, general practitioner, gp, family practice, family physician. the roles of general practice/family practice in the plans were assessed across the four domains of general practice identified in the first part of this project. no attempt was made to quantify the extent of coverage of general practice in the plans as this rarely extended beyond a few sentences. where there was detailed coverage of an issue, we analysed the text and the health system context. the study was approved by the australian national university human research ethics committee and the national research and evaluation ethics committee of the royal australian college of general practitioners. written informed consent was obtained from participants. a conceptual framework of the general practice response to pandemic influenza is shown in table 1 . the framework identifies four domains of practice: clinical services, public health responsibilities of general practice, internal (physical and organisational) environment of the general practice unit, and the macro-environment of general practice. in each domain, we list the key challenges to be anticipated by general practice during an influenza pandemic, and the type of responses that need to be addressed in the plan. table 2 summarises the organisational levels in the five countries, the proportion of jurisdictions with accessible pandemic plans, and coverage of general practice in these plans. while almost all plans from us jurisdictions were accessible, three quarters of australian states/territories and one third of new zealand's district health boards had accessible plans. only 13% (20/152) of england's pcts had pandemic plans available in the public domain. figure s1 shows the jurisdictions and health management systems whose plans were included in this study; they comprise 49 jurisdictions from the usa, 20 from england, 8 from canada, and 6 each from australia and new zealand. table 3 shows the number and rates of coverage of each of the four domains of the general practice response in jurisdictional plans of the five countries. the domain covered most frequently was influenza-related clinical care (in all plans from england and canada). overall less than half the plans mentioned non-influenza clinical care, with the exception being england, where 90% of pct plans mentioned non-influenza clinical care. public health surveillance was addressed in all plans from canada and new zealand and infection control in general practice in almost all plans from england and canada. functional linkages of general practice with other parts of the health system were addressed in almost all the english plans, but a smaller proportion of other plans. clinical care essential planning elements. this domain includes two sets of clinical care needs. the first, prevention and treatment of influenza, includes care for the surge in patients with acute respiratory illness, and for people at high risk of exposure to, or complications from, influenza. these aspects are discussed extensively in the literature [20] [21] [22] [23] . most people with influenza can be managed in the community, protecting hospitals by delaying or avoiding admission and facilitating early discharge. the second clinical care need is for non-influenza-related care. general practitioners provide most chronic disease care, though there is inter-country variation in their capacities to do this efficiently [24, 25] . while activities like cervical screening may cease in a pandemic, chronic illnesses like diabetes or cardiac disease will still need management. some acute care usually undertaken in hospitals, like acute asthma or injuries, may be transferred to the community. in an earlier paper, we advanced a range of models of practice to balance clinical services for influenza and non-influenza care [16] . in the recovery phase, the clinical needs of patients are for psychological care and chronic illness management. if the pandemic occurs in waves, as in 1918-19, recovery activities may need to be tempered by preparations for the next wave. coverage of essential elements in plans. all canadian and english plans outlined a role for general practice in clinical care for influenza. while only 41% of plans from the usa addressed clinical care for influenza by primary care practitioners (table 3) , every us plan included guidelines on influenza management by hospital physicians. some plans articulated a surge in demand for influenza care as a threat to general practice's survival, and proposed assessment and treatment clinics as a way of protecting them [26, 27] . in other plans [28] [29] [30] the response to a surge was to support general practices to become more resilient by collaborating and changing their work practices. in two us state plans, the failure of the ambulatory care sector in the face of a surge was assumed. the planning challenge became to find ways to redeploy workers into other health care sectors [31, 32] . most plans were sketchy on systems to maintain non-influenzarelated clinical care, with the exception of some pct plans, which included activities like triage, extended prescribing, identifying deferrable reasons for presentation, and management of more acute problems to protect hospitals [29, [33] [34] [35] [36] . the main non-influenza clinical area was mental health care, mentioned in six plans from the usa [37] [38] [39] [40] [41] [42] (reflecting a focus in the national plan [43] ) and one canadian plan [44] . coverage of the needs of vulnerable populations-the elderly, homeless, prisoners and the psychologically unwell -was most detailed in plans from canada and england. essential planning elements. this domain includes surveillance of influenza-like illness and influenza virology, and control of influenza in the general practice and the community. surveillance includes early diagnosis and notification, and specimen collection to confirm clinical diagnosis and to monitor viral characteristics and resistance to antiviral drugs. gps and private specialists are currently central to surveillance activities [45] [46] [47] [48] . in the early stages of the pandemic, it is likely that public health authorities will undertake contact tracing to facilitate containment, but their capacity to sustain this approach as the epidemic continues will be limited. general practice may then be expected to include contact tracing, and monitoring and support of people in quarantine or home isolation. other responsibilities may include prescribing and dispensing antiviral drugs and participating in mass immunisations against the pandemic strain of the virus. coverage of essential elements in plans. surveillance in general practice was mentioned in 53% of us plans and in only 33% of english plans, in all canadian and new zealand plans, and all but one australian plan ( table 3 ). the low rates of coverage of surveillance in pct plans are not in accord with the uk plan which imputes to general practice a role in surveillance, and recommends that pcts operationalise this recommendation [49] . the college of family physicians in canada is a partner in fluwatch, recruiting sentinel physicians to undertake surveillance, so this role is well understood within the canadian health sector. the role of general practice in contact tracing, in monitoring people in home isolation, and in distributing antiviral drugs is unclear in most plans. home care by gps for people in quarantine is mentioned in two us plans [50, 51] , and one english plan [36] , though the recently released guidelines for pcts anticipate a role for general practices in home care [52] . in all country plans, dispensing antiviral medications was generally performed by public health units. only 22% of pct plans and 40% of us plans mention a role for primary care in dispensing antiviral medications. none of the canadian plans, and only one nz and two australian state plans, mentioned antiviral dispensing by primary care. the only plan to set out contingencies when decisions about dispensing may change was one canadian rha plan [27] . although immunisation was mentioned most frequently after surveillance as a public health activity by general practices, in most plans the immunisations were against pneumococcal disease and seasonal influenza, but not mass immunisations against pandemic influenza. essential planning elements. this domain includes the physical environment of the general practice and its practice-level organisation. the risk of transmission of infections within the surgery could be minimised through separate waiting rooms and entrances, triage and personal protective equipment and handwashing facilities. hogg has outlined infections control procedures in the practice and the associated financial costs [53] . some general practices (for example, those with small waiting rooms, or only one consulting room) may be deemed too much of a transmission risk to continue providing face-to-face services. the practice needs to develop strategies to maintain reliable and efficient access to essential drugs and equipment and influenza and pneumococcal vaccines. it also needs to strengthen the capacity of its communication technologies with patients and the broader health system, including telephones, faxes, internet, work-from-home technologies for staff, compatible software for sharing electronic medical records, and recall and reminder systems for patients. preparation at the organisational level relates mainly to business continuity plans. these plans should include leadership delegations, staffing contingencies, safe and flexible working hours and family care plans for staff, criteria for considering clinic closure, recruiting and training ancillary staff, early psycho-social support, support for making difficult clinical decisions, record keeping to ensure accountability for actions and 'inactions', use of antiviral medications, and plans for simulation exercises to complement training, and to evaluate and refine local practice plans. tools [54, 55] and desktop simulation exercises [19] are available to help gps plan for continuity. coverage of essential elements in plans. infection control strategies were well covered in plans from canada and england, but were mentioned in only 39% of us plans ( table 3) . none of the plans provided an inventory of fixed features, such as size and layout of waiting room, or a single entrance, which could compromise infection control. business continuity was a focus of the english plans, which frequently referenced resources available on the uk resilience website [56] . this aspect of preparedness was enhanced after the exercise winter willow simulation in february 2007, and new pct guidelines addressing workforce planning [52] . some pct plans addressed the need for general practice resilience in the face of workforce sicknesses [33] , increased aggression from patients, and threatened loss of capacity in single doctor practices [57] . few plans from other countries discussed business continuity for primary care in such detail. this may be because such issues are felt to be outside the normal purview of state or provinces, and to be the responsibilities of the businesses themselves or corporate interests. essential planning elements. this domain includes the overall organisation of, and interactions with, the health system that will facilitate or impede effective functioning of general practice services during a pandemic, including adaptation of relevant regulatory and financing systems. the health system requires a plan that adopts the 'all-hazards approach' and integrates roles, responsibilities and actions for acute clinical care, public health, and emergency management systems [4] . this calls for coordination across general practices and other ambulatory care services to ensure primary health care needs within the community are effectively monitored and addressed; with hospitals to avoid/delay hospitalisation and facilitate early discharge; and with public health units to share responsibilities for contact tracing, monitoring and treating people in home isolation or quarantine, dispensing of anti-viral medications, and participation in mass immunisations against pandemic strains of the virus (when these become available). neighbouring general practices and other ambulatory care services will need local leadership with strategic approaches to collaborate and maintain services through a pandemic. england's pcts and new zealand's primary health organisations (phos) represent two ways of linking general practices under the governance of regional boards. these networks are consolidated by financial relationships between the pct or the pho and general practices. the links between australia's divisions of general practices and gps are purely voluntary. in the usa, managed care systems function as another way of linking ambulatory and hospital services. communication infrastructure between canada's family practitioners, 25% of whom are solo practitioners [58], is still being developed, as is the incorporation of general practice into canada's pan-canadian public health network [59] . the regulatory environment includes accreditation of retired medical practitioners and allied health professionals, laws and regulations which support or hinder the flow of qualified personnel across a jurisdiction's health facilities [48] , and ensuring an appropriate medicolegal framework to support clinical decisions on prioritising medical care during a pandemic, for example, modifying clinical standards, deferring treatment, and restricting access to certain treatments. funding mechanisms for general practice may impact upon the capacity to provide extra services. in countries with fee-for-service payment systems, general practices may profit from a surge in attendances, but may equally run into business difficulties if they are short-staffed for prolonged periods. gps funded through a capitated system may have more freedom to alter their practice to provide different service mixes. in the post-event phase, patients and gps may require support for psychological recovery. it may be necessary to provide some formal relief through a system of locum gps from areas less affected by the pandemic. organisational partnerships at this stage may need to be with social services and mental health support services. coverage of essential elements in plans. countries with mechanisms for linking general practices with other sectors were more likely to address networking in their plans. ninety five per cent of english plans addressed systems to support collaboration between general practices (table 3) . these plans addressed buddy systems, practice networks, and contingency plans for communities of practice. four of the six new zealand plans also addressed collaboration, though only one in significant detail; this plan outlined a distinction between key practices, and other practices which might decide to partner one another [55] . of the three canadian provincial plans that addressed collaboration, the most comprehensive was from quebec, which identified a need to bridge the gap between salaried practitioners and independent physicians. the plan of the montreal regional authority [60] operationalises this by setting up a system of active and sustained outreach by the public health department to independent physicians. the absence of plans for networking between general practice and public health is most marked in the usa. with the exception of louisiana [61] , us plans which mentioned networking did so in one line, generally advocating partnership between private and public services without indicating how this might occur. louisiana's strategic approach built a participatory structure for rural practitioners through a partnership between the state public health department and the bureau of primary rural health care. the canadian national pandemic plan [62] is framed around a set of ethical precepts incorporated into pandemic planning at the provincial and regional health level. the uk has recently released an ethical framework for policy and planning, though this has not yet been incorporated into planning documents [63] . the regulatory framework most mentioned was in relation to credentialing for retired gps and other volunteers [33, 64, 65] , and less frequently, indemnity [36] . although most plans include coverage of the relevant public health legislation, no country's plan included an inventory of legislation relevant to general practice that might need to be amended. only one plan [66] and the pct guidelines [52] , canvas the potential of recompense for financial loss to a general practice. the only country in which the planning level coincided with the level that made decisions about funding of health care was canada. one regional health authority plan provided an outline of specific issues likely to affect physicians, and raised the possibility of reviewing funding mechanisms in a pandemic [67] . there appear to be no ancillary plans addressing principles of altered funding for private physicians in a pandemic. this is the first study to provide a framework that brings together multiple functions, structural relationships and the responsiveness of general practice to prepare for pandemic influenza. the framework provides clarity of purpose and a structure to guide planning through four functional domains: clinical care, public health responsibilities, and the internal and macro environments of general practice. the domains have been structured as integral components of a complex system that can respond to uncertainty [68] and be adapted for a given local setting and health system context. we draw three conclusions regarding general practice from our analysis. first, none of the 89 jurisdictional plans addressed all domains of the general practice response during a pandemic. second, while many aspects of the first three domains are included in plans for general practice, there are critical gaps and inconsistencies in the fourth domain (macro-environment) that render some elements of the jurisdictional plan ungrounded or unrealistic. third, few plans addressed the broader ambulatory care context, including the need to engage private specialists and other allied health professionals [48] . planning and implementing change across the health system is complex. targeting individual sectors for change (e.g. public health departments, hospitals or general practices) without securing reciprocal changes and strengthening inter-relationships across the health system, is unlikely to succeed [10, 18] . planners must consider how connectivity across the health system might be strengthened to enable optimal use of general practice resources for planning [68] . while this may be challenging, particularly in countries with weak governance structures for primary health care, omitting general practice input into the planning process may be considered unethical [69] and counterproductive. limitations of the study: our findings are exploratory rather than definitive, and indicate directions for further planning and research. like any new tool, the framework and its application in a given context needs testing and refinement through simulation exercises targeting ambulatory care services as well as the broader health system. planning is an evolving activity that reflects a 'map' rather than a 'destination', and our findings provide a snapshot of the plans accessible in late 2007. the scope and content of the plans will change over time, as seen in two countries that adjusted their plans after simulation exercises, exercise cumpston in australia [70] and winter willow in the uk [71] . interestingly, the former identified specific weaknesses in the involvement of the primary health care sector and made recommendations to better integrate primary health care providers into planning at the national and jurisdictional levels [70] . national and sub-national pandemic plans may be intended to provide a strategic focus and not to elaborate on operational activities; it is possible the latter may have been addressed, but were not accessible at the time of our study. another potential limitation of our study is that the gaps we identified in many plans were grounded in theories about the ways to enhance the quality and outcomes of clinical care [10, 18] or of public health preparedness planning [6] . the science of preparedness planning is still maturing [4] [5] [6] and there is relatively little systematic evidence for linking specific preparedness structures to the ability to implement efficient and effective responses [5, 6] . two important limitations to the implementation of preparedness activities are uncertainties in knowing how much preparedness is enough [5] and in having a measurable assessment of the outcomes of preparedness activities. it may be more meaningful to perceive of the activities as a 'preparedness production system' in which a variety of processes and activities have been completed to prepare for an optimal response [6] . we are unable to comment on the extent to which these preparedness plans have been implemented, except in the case of those jurisdictions which have held pandemic exercises [70, 71] . general practice response is rarely tested in pandemic exercises, which tend to focus on hospital and public health responses. a notable exception is operation sparrowhawk in singapore, where the feasibility of general practice influenza clinics was tested [72] the haddon matrix is not a final check-list for preparedness planning but a problem-solving tool used as a starting framework for planning. the contents of each cell of the matrix help identify a particular problem or challenge that needs to be addressed. we recognise that the challenges will be neither static over time, nor uniform across general practices; responses will have to be modified in the context of the general practice setting as the pandemic evolves and as other parts of health system, particularly hospitals and public health units respond to the epidemic. implications of our study for primary health care in developing countries: endemic and epidemic infectious diseases inflict high levels of morbidity and mortality in developing countries because of a combination of poor living conditions, effects of multiple concurrent illnesses particularly in children, fragile national health systems, overburdened and overstressed health workers, and negative work environments [73] . although our study targeted general practice in developed countries, the conceptual framework we developed (table 1) can be used by primary health care services in developing countries to deconstruct the multidimensional challenges posed by pandemic influenza. identifying possible solutions and apportioning responsibilities across components of the health system is more complex. operational guidelines have been developed for the detection and rapid containment of a potentially pandemic strain of influenza to the epicentre of the outbreak [74] , for example, if this were to occur in a south east asian country. however, because of the immense global implications of such an event, this intensive strategy will need to be supported by extraordinary resources from the global community, an action not sustainable once the pandemic strain spreads beyond the initial epicentre. in an analysis of pandemic influenza plans in asia-pacific countries in 2006, coker found that although all countries recognised the importance of pandemic planning, operational responsibility particularly at the local level, remained unclear; most plans relied on specialised flu hospitals, while few developed the possibility of caring for patients at home [75] . (the study made no reference to primary health care or the private practice sector). in his analysis of public health emergencies in developing countries, quarantelli identified relatively poor adaptive capabilities to be the key barrier to effective responses at the central and local levels [76] . possible reasons included poorer public health infrastructures and human and financial resources, organisational structures that functioned mainly in a top-down manner with a strong emphasis on structures more than functions, and lack of planning initiatives the further away one moved from central level [76] . many poor countries already have a health crisis, and need massive international investments, including mobilisation and strengthening of human resources to build sustainable health systems, strong leadership and political commitment [73] . in the face of the pandemic threat, primary health care in developing countries will need resources to develop a suite of policies, including: clarification of what essential primary health care will continue through a pandemic, developing health workforce plans that may entail diverting clinicians from other areas of the health workforce, establishing non-hierarchical links between primary health care, hospitals and public health, and injecting funds into hospital and primary care preparedness simultaneously. it may be argued that the absence of general practice elements from pandemic plans is not problematic, that it is outside the responsibility of public health departments that do not have a governance role for general practice. we argue instead that the general practice sector, which is characterised by loose networks between ambulatory care services, and often lacks the appropriate organisational structure and mandate, cannot spearhead many elements of planning for primary care. this calls for actions by health departments as well as by general practices. actions by health departments. ensuring that the community receives appropriate health care during public health emergencies is a government responsibility. consequently, health departments must emphasise in national and sub-national plans, the critical need for all levels of the health system to integrate the general practice sector in the planning process. this should include appropriate general practice representation in high level planning and decision-making committees, in incident-commandcontrol structures and in the management of community-based specialised clinics such as 'fever clinics' or 'community information and assessment centres'. good planning must focus on the planning process rather than the production of a written document [76] . the process includes collaborative activities such as meetings, drills, exercises, simulations, developing techniques for training, knowledge transfer, identifying and obtaining resource materials, and continually updating materials and strategies. these planning activities are important not only because they inform, but because they also foster collaborative learning and problem-solving, and generate an atmosphere of mutual trust and solidarity among people who will be affected by a pandemic and whose collaboration will be essential in the response. the willing general practitioner sector [7, 8] is an essential resource for extending the surge capacity of health departments. health departments should harness and support interactions and networking among general practices, and between them and ambulatory health care providers, hospitals and public health units. the role of general practice in contact tracing, monitoring and treating people in home isolation or quarantine, dispensing antiviral drugs and participating in mass vaccinations -omitted in most plans -needs to be clarified. in addition, health departments should modify or adopt where appropriate, legislation and financing mechanisms to enable general practices to function optimally during the pandemic. action to support planning by general practice. while the diversity of the general practice sector means that there will not be guidelines to cover all scenarios and contexts, a coherent approach would enable multi-actor accountability and more efficient, contextual planning by jurisdictions. the guidelines for pcts [52] are an example of such an approach, designed for a particular health system. they could act as a useful point of departure for planning integrated general practice plans by other health systems. there is a need for a system of sharing innovations and exemplary solutions to challenges for pandemic planning by general practice, analogous to those targeting mainly hospitals and public health departments [77] . given the diversity in organisation of general practice systems, a web presence comparing exemplary approaches from different health systems would be a useful resource for planners. an important challenge will be ensuring collaboration and coordination across the health sector during a pandemic. research is needed to identify the prevailing barriers and facilitators to effective collaboration across the health sector, how these may change under the stressor of a pandemic, and how this information could be used to optimise the response. the regulatory environment is founded on a set of ethical principles, often unarticulated. since there is likely to be some dispute between utilitarian philosophical approaches used in public health and deontological or virtue ethical approaches used in clinical medicine [78] , there is a need for some preparatory work with general practitioners clarifying ethics of clinical behaviour, restriction of liberty under quarantine orders, and resource allocation and distribution. in an established pandemic, it is likely that there will be shortfalls in the gp workforce, due to illness among gps, caring duties or closure of small practices. non-hospital clinical specialists, retired general practitioners, allied health professionals and medical students could be trained to fill the gap in services. research is needed to define the clinical work that can be done by other health personnel in general practice, eligibility criteria and accreditation processes for this cadre of workers, and optimal training processes. all public health problems have a clinical dimension, and all clinical problems have a public health dimension. at present, the plans in the five countries provide more detail on the public health dimension of the pandemic. there are intercountry differences in the emphases provided to different domains of the general practice response. some of this reflects the emphasis on particular elements contained within the relevant national plan. some of the differences are due to the ways in which general practice is structured in a country, and the strengths of its linkages to other components of the health sector. there is an urgent need to incorporate general practice and the broader primary care sector into pandemic planning activities, and to undertake the preparedness activities that would make this sector, which provides the majority of health care work, a true partner in pandemic response. figure s1 jurisdictions or health management organizations whose plans were included in the study. found at: doi:10.1371/journal.pone.0002269.s001 (0.04 mb doc) outbreak of severe acute respiratory syndrome in hong kong special administrative region: case report a study on sars awareness and health-seeking behaviour -findings from a sampled population attending national healthcare group polyclinics assessing the burden of influenza and other respiratory infections in england and wales public health preparedness: a systems-level approach assessing public health emergency preparedness: concepts, tools, and challenges quality improvement in public health emergency preparedness enhancing public health response to respiratory epidemics: are family physicians ready and willing to help? the gp's response to pandemic influenza: a qualitative study a survey of the preparedness for an influenza pandemic of general practitioners in the west midlands planning and studying improvement in patient care: the use of theoretical perspectives the management of new primary care organizations: an international perspective is decentralisation a real solution? a three country study using the haddon matrix: introducing the third dimension the application of the haddon matrix to public health readiness and response planning a systematic analytic approach to pandemic influenza preparedness planning australian general practice and pandemic influenza: models of clinical practice in an established pandemic global influenza preparedness plan. the role of who and recommendations for national measures before and during pandemics improving the quality of health care in the united kingdom and the united states: a framework for change pandemic influenza in general practice simulation exercise general practice: professional preparation for a pandemic the pandemic influenza threat: a review from the primary care perspective a general practice perspective of pandemic influenza pandemic influenza: how it would progress and what it would require of you care of patients with chronic disease: the challenge for general practice on the front lines of care: primary care doctors' office systems, experiences, and views in seven countries nsw health interim pandemic action plan vancouver island health authority (2006) pandemic influenza plan pandemic influenza contingency plan bath & north east somerset major incident and contingency plan -pandemic influenza supplement available: www.greenwichpct.nhs.uk/publications/file.aspx?int_ version_id = 2443 via the internet. accessed arizona influenza pandemic response plan keep camden working: managing an influenza pandemic: camden's integrated influenza pandemic management plan for the health and social care community community infection control pandemic influenza policy pandemic%20flu%20policy%20amend%207%20june%2006.pdf via the internet. accessed 13 pandemic influenza operational response plan influenza pandemic contingency plan march06.doc via the internet. accessed 13 arkansas responds: arkansas pandemic influenza response plan pandemic influenza plan influenza pandemic preparedness plan pandemic influenza plan public health pandemic influenza plan virginia emergency operations plan attachment pandemic influenza hhs pandemic influenza plan ontario health plan for an influenza pandemic surveillance of influenza-like illness in england and wales during 1966-2006 establishing thresholds for influenza surveillance in victoria surveillance for influenza-united states, 1997-98, 1998-99, and 1999-00 seasons the health care response to pandemic influenza: position paper. american college of physicians pandemic influenza contingency plan pandemic influenza preparedness and response plan pandemic influenza preparedness and response plan pandemic influenza: guidance for primary care trusts amd primary care professionals on the provision of healthcare in a community setting in england the costs of preventing the spread of respiratory infection in family physician offices: a threshold analysis a gp work plan for pandemic flu practice pandemic planning resource uk resilience: human flu pandemic pandemic influenza plan executive summary college of family physicians of canada canada's public health system: building support for frontline physicians agence de la santã© et des services sociaux de montrã©al (montreal agency of health and social services) (2007) montreal pandemic influenza plan-health mission statewide draft pandemic influenza plan the canadian pandemic influenza plan for the health sector responding to pandemic influenza: the ethical framework for planning and policy district of columbia department of health (2005) pandemic influenza preparedness plan pandemic influenza contingency plan tasmanian health action plan for pandemic influenza pandemic influenza response plan complexity science: the challenge of complexity in health care preparing for an influenza pandemic: ethical issues national pandemic influenza exercise: exercise cumpston 06 report exercise winter willow -lessons identified roundup: exercise promotes singapore's preparedness for flu pandemic human resources for health: overcoming the crisis interim protocol: rapid operations to contain the initial emergence of pandemic influenza pandemic influenza preparedness in the asia-pacific region quarantelli e major criteria for judging disaster planning and managing and their applicability in developing societies promising practices: pandemic preparedness tools raising the profile of public health ethics in australia: time for debate we are grateful to our colleagues from general practices, public health units and general practice organizations who have contributed to this study, and to ms sally hall, ms marianne shearer, ms hannah walker, dr jonathon anderson, dr ron mccoy, dr chris hogan, dr kathryn antioch, and ms monika thompson. key: cord-002973-bkr4ndl2 authors: seifi, morteza; walter, michael a. title: accurate prediction of functional, structural, and stability changes in pitx2 mutations using in silico bioinformatics algorithms date: 2018-04-17 journal: plos one doi: 10.1371/journal.pone.0195971 sha: doc_id: 2973 cord_uid: bkr4ndl2 mutations in pitx2 have been implicated in several genetic disorders, particularly axenfeld-rieger syndrome. in order to determine the most reliable bioinformatics tools to assess the likely pathogenicity of pitx2 variants, the results of bioinformatics predictions were compared to the impact of variants on pitx2 structure and function. the mutpred, provean, and pmut bioinformatic tools were found to have the highest performance in predicting the pathogenicity effects of all 18 characterized missense variants in pitx2, all with sensitivity and specificity >93%. applying these three programs to assess the likely pathogenicity of 13 previously uncharacterized pitx2 missense variants predicted 12/13 variants as deleterious, except a30v which was predicted as benign variant for all programs. molecular modeling of the pitx2 homoedomain predicts that of the 31 known pitx2 variants, l54q, f58l, v83f, v83l, w86c, w86s, and r91p alter pitx2’s structure. in contrast, the remaining 24 variants are not predicted to change pitx2’s structure. the results of molecular modeling, performed on all the pitx2 missense mutations located in the homeodomain, were compared with the findings of eight protein stability programs. cupsat was found to be the most reliable in predicting the effect of missense mutations on pitx2 stability. our results showed that for pitx2, and likely other members of this homeodomain transcription factor family, mutpred, provean, pmut, molecular modeling, and cupsat can reliably be used to predict pitx2 missense variants pathogenicity. paired-like homeodomain transcription factor 2 (pitx2, refseq nm 000325.5, mim# 601542) is located at 4q25 and is expressed in the developing eye, brain, pituitary, lungs, heart, and gut [1] . mutations in human pitx2 or the forkhead box transcription factor c1 (foxc1; 6p25, refseq nm 001453.2, mim# 601090) underlie the autosomal dominant disorder called axenfeld-rieger syndrome (ars; mim# 602482) [2] [3] [4] [5] . ars is a full penetrant, but clinically and genetically heterogeneous disorder characterized by developmental anomalies involving both ocular and non-ocular structures [6] . to date, 87 identified including deletions, insertions, splice-site mutations, and coding region frameshift, nonsense and missense mutations [7] [8] [9] [10] [11] [12] [13] . identifying new disease-associated variants is becoming increasingly important for genetic testing and it is leading to a significant change in the scale and sensitivity of molecular genetic analysis [14] . one of the most frequent approaches for detecting novel variants in target genes is using direct gene sequencing. however, due to increasing number of newly identified missense variants, it is often difficult to interpret the pathogenicity of these variants as not all the mutations alter protein function, and the ones that do may also have different functional impacts in disease [15, 16] . thus, prior to detailed analyses, novel variants cannot be easily classified as either deleterious or neutral, because of their unknown functional and phenotypic consequences. therefore, further research should be conducted to validate the genetic diagnosis when a novel missense variant is discovered. preferably, in vitro characterization of novel variants should be undertaken; however, due to facility limitation, it is often not practicable to experimentally verify the impact of large number of mutations on protein function [17] . another robust approach to substantiate the pathogenicity is using animal models by generating the homologous mutation that recapitulates the human phenotype; but, similar to in vitro studies, these are time-consuming, labor-intensive, difficult and expensive, making this approach unfeasible to experimentally determine the pathogenicity effects of all novel identified variants [18] . to circumvent the above mentioned limitations and to provide fast and efficient methods for predicting the functional effect of nonsynonymous variants on protein stability, structure, and function, several computational tools have been developed [19] [20] [21] . protein stability and structure are key factors affecting function, activity, and regulation of proteins. conformational changes are necessary for many proteins' function and disease-causing variants can impair protein folding and stability. missense variants are also capable of impairing protein structure, likely by affecting protein folding, protein-protein interaction, solubility or stability of protein molecules. the structural effect of mutational changes can be examined in silico on the basis of three-dimensional structure, multiple alignments of homologous sequences, and molecular dynamics [22] [23] [24] . therefore, analysing sequence data in silico first and detecting a small number of predicted deleterious mutations for further experimental characterization is a key factor in today's genetic and genomic studies. in general, bioinformatics prediction methods obtain information on amino acid conservation through alignment with homologous and distantly related sequences. the most common criteria considered in many bioinformatics programs for predicting the functional effect of an amino acid substitution are amino acid sequence conservation across multiple species, physicochemical properties of the amino acids involved, database annotations, and potential protein structural changes [23, 25, 26] . as mentioned above, resources for in vitro and in vivo functional analysis of novel variants are constrained in most clinical laboratories. therefore, identifying and reporting novel variants that are likely to be pathogenic often requires accurate prediction using computational tools. in a previous study, we examined the effect of foxc1 variants on protein structure and function by combining laboratory experiments and in silico techniques. our results showed that integration of different algorithms with in vitro functional characterization serves as a reliable means of prioritizing, and then functional analyzing, candidate foxc1 variants [27] . unlike most previous studies that focused on using only polyphen and sift to predict the pathogenicity of missense mutations, here, we investigated the predictive value of sift, poly-phen and nine other prediction tools by comparing their predictions to in vitro functional data for pitx2 variants. the bioinformatics programs found to be most reliable were then used to predict the likely consequences of 13 functionally-uncharacterized pitx2 variants. we also performed molecular modeling on all the pitx2 missense mutations located in the homeodomain and compared the results with the findings of protein stability algorithms to identify the most reliable tools in predicting the effect of missense mutations on pitx2 stability. to the best of our knowledge, this is the first study that incorporates the results of functional studies in conjunction with bioinformatics approaches for predicting the pathogenicity of mutations in pitx2 gene. lists of pitx2 missense variants were assembled from the previous literature and a search using the clinvar [28] , human gene mutation database (hgmd) [29], the genome aggregation database (gnomad), and the single nucleotide polymorphism database (dbsnp). this study found 47 pitx2 missense variants; 31 of which were described in the literature as being associated with ars or coronary artery disease (cad), while the remaining 16 variants, were considered as benign variants (fig 1) . eighteen of the 31 variants were classified as pathogenic based on functional studies utilizing site-directed mutagenesis, expression studies, and other functional analysis ( table 1) . thirteen of 31 variants were described as associated with ars and cad in the absence of functional analyses on pitx2 structure or function. sixteen snps, with population allele frequencies > 0.0005 were identified from the gnomad and the clin-var. based upon the allele frequency (approximately 10-fold greater than the disease frequency of ars) these have been considered benign polymorphisms. nucleotide numbering of the mutations herein indicates cdna numbering with +1 as the a of the atg translation initiation codon in the ncbi reference sequence nm_000325.5, while the amino positions are based on the corresponding ncbi reference sequence np_000316.2. this study is a retrospective case report that does not require ethics committee approval at our institution. all patients' mutations and phenotypes were obtained from previously published studies. these programs were used to analyse 18 functionally characterised pitx2 missense variants plus 13 additional, functionally uncharacterized pitx2 missense variants. sift program provides functional predictions for coding variants, based on the degree of conservation of amino acid residues in sequence alignments derived from closely related sequences, collected by psi-blast algorithm [60]. the polyphen-2 (polymorphism phenotyping-2) server predicts possible effect of an amino acid change on the structure and function of a protein using several sources of information such as straightforward physical and comparative considerations [61] . panther-psep is a new application that analyses the length of time a given amino acid has been conserved in the lineage leading to the protein of interest. there is a direct association between the conservation time and the likelihood of functional impact [62] . mutpred is a free web-based application that utilizes a random forest algorithm with data based upon the probabilities of loss or gain of properties relating to many protein structures and dynamics, predicted functional properties, and amino acid sequence and evolutionary information [52] . mutationtaster is a tool that combines information derived from various biomedical databases and uses established analysis programs. unlike sift or poly-phen-2 which work on dna level, mutationtaster processes substitutions of single amino table 2 for more information on the prediction tools used in this study. the nmr structure of the homeodomain of pitx2 complexed with a taatcc dna binding site (pdb: 2lkx) were analyzed by the swiss-model server (http://www.expasy.org/spdbv/ ; provided in the public domain by the swiss institute of bioinformatics, geneva, switzerland). model structures of wild-type and mutants were created in swiss-pdb viewer and investigated using the anolea server (http://melolab.org/anolea). for structure predictions of pitx2, sequence in fasta format was obtained from ncbi database (np_001191327.1). eight different protein stability programs (duet, sdm, mcsm i-mutant3.0, mupro, iptree-stab, cupsat, and istable) were used to predict the effects of missense mutations on the stability of pitx2 protein. duet is a web server that uses integrated computational approach to predict effect of missense mutations on protein stability [66] . duet calculation is based on complementary data regarding the mutation including secondary structure [67] and a pharmacophore vector [68] . sdm, a computational method, has been demonstrated as the most appropriate method to use along with many other programs. sdm assesses the amino acid substitution occurring at specific structural environment that are tolerated within the family of homologous proteins of defined three dimensional structures and change them into substitution probability tables [69] . mcsm relies on graph-based signature concept and predicts not only the effect of single-point mutations on protein stability, but also protein-protein and protein-nucleic acid binding [70] . i-mutant3.0 is a neural-network-based web server that predicts automatically protein stability changes upon single point protein mutations based on either protein sequence or protein structure. i-mutant3.0 can predict the severity effect of a mutation on the stability of the folded protein [71] . mupro is a set of machine learning programs that accurately calculates protein stability alterations based on primary sequence information particularly where the tertiary structure is unrevealed, overcoming a major restriction of previous methods which are based on the tertiary structure [72] . iptree-stab is a web service and mainly provides two function modules of services including discriminating the stability of a protein upon single amino acid substitutions and predicting their numerical stability values [73] . cupsat uses protein environment specific mean force potentials (through solvent accessibility and secondary structure specificity) to analyse and predict protein stability changes upon point mutations [74] . istable, a combined predictor, was designed by using sequence information and prediction data from various element predictors. istable is available with two different input types: structural and sequential [75] . please see table 3 for more information on the stability predictors used in this study. previous analyses of missense variations in different human diseases predicted that the stability margin without any immediate effect on protein fitness is 1-3 kcal mol -1 [77] [78] [79] . mutations that reduce the protein stability by >2 kcal mol -1 contribute to severe disease phenotypes [80, 81] . therefore, in this study, all variations were classified as predicted to be neutral (-1.5 < δδg < 1.5), stabilizing (δδg > 1.5) or destabilizing (δδg < -1.5). the protein sequence and/or protein structure with mutational position and amino acid residue of 18 previously functionally characterized pathogenic pitx2 missense variants, plus 16 snps with a population frequency of higher than 0.05% (thus considered benign polymorphisms), were used to test the predictive value of eleven common bioinformatics prediction programs; sift, polyphen-2, panther-psep, mutpred, mutationtaster, provean, pmut, fathmm, nssnpanalyzer, align gv-gd, and revel (table 4 and table 5 ). to evaluate the performances of the programs, seven measures (sensitivity, specificity, accuracy, precision, positive predictive value (ppv), negative predictive value (npv), and matthews correlation coefficient (mcc)) were calculated by comparing the results of all programs with previously generated functional data. for pitx2, mutpred, provean, and pmut were the most reliable of the bioinformatics tools in predicting the pathogenicity effects of all 18 functionally characterized missense variants in pitx2, with sensitivity and specificity of > 93% (fig 2) . then, revel tool showed high sensitivity and specificity, 94.44% and 87.50%, respectively. analysis of the sensitivity and specificity sift showed that this program had good sensitivity (72.22%) but low specificity (43.75%). although polyphen-2, mutationtaster, panther-psep, fathmm, and align gv-gd exhibited over 83% sensitivity, they were unable to identify the benign polymorphisms, showing specificity of 37.50%, 6.25%, 43.75%, 6.25%, and 6.25%, respectively. the predictive value of nssnpanalayzer was similar to that of sift program, with sensitivity and specificity of 66.67% and 43.75%, respectively. the most reliable programs found in this study's analyses (mutpred, provean, and pmut) were then used to predict the likely pathogenicity of 13 pitx2 missense variants for which functional testing has not been performed (table 6) . interestingly, the a30v variant unanimously was predicted as benign by all three programs. the remaining 12 pitx2 variants were predicted to be disease-associated mutations by all programs. molecular models for the homeodomain of wild-type and variant-containing pitx2 proteins were designed using threading algorithms to assess impairment of pitx2 structure by missense variants. three functionally characterised variants, n100d, l105v, and n108t, were excluded from these molecular modeling analyses since they are not located in the homeodomain, which is the only portion of pitx2 with a known structure. wild-type amino acids were changed to variant residues to determine putative structural effects of the remaining 15 functionally analysed pitx2 variants through anolea mean force potential calculations. the molecular modeling identified three mutations as high-risk (l54q, v83l, and r91p) to change the structure of pitx2, particularly in the h1, h2, and h3 subdomains (fig 3) . the r91p variant was predicted to grossly disrupt the non-local amino acid side chain contacts. similar, although less profound, effects were predicted when l54 and v83 were altered to glutamine and leucine, respectively. in contrast, the remaining twelve amino acid variants showed no predicted substantially altered pairwise interactions, indicating that these missense variants are predicted to have minor or no effects on pitx2's structure (s1 fig). molecular modeling was also performed on the nine functionally uncharacterised pitx2 missense mutations located in the homeodomain. four mutations (f58l, v83f, w86c, w86s) were predicted to change the structure of pitx2 (fig 4) , while, the remaining five variants (r62h, p64l, p64r, r69c, and r90p) were predicted to have minor or no impact on pitx2's structure (s2 fig). to assess the performance of eight different stability predictor programs (duet, sdm, mcsm, i-mutant3.0, mupro, iptree-stab, cupsat, and istable) in predicting the effect of missense mutations on pitx2 protein stability, the change in protein stability (δδg) were computed for all 24 pitx2 homeodomain variants (15 functionally characterised and 9 functionally uncharacterised mutations) (table 7) . of these eight programs, cupsat was the most consistent with the results of our molecular modeling, by identifying 5 of 7 destabilizing mutations that were also predicted to be destabilizing by molecular modeling (v83l, v83f, w86s, w86c, and r91p). computational analysis of pitx2 mutations sdm also showed high consistency with the results of our molecular modeling, by detecting 4 of 7 destabilizing mutations that were also predicted to be destabilizing by molecular modeling (l54q, r91p, w86s, and w86c). duet, mcsm, and i-mutant3.0 identified 3 and iptree-stab detected 2 of 7 destabilizing mutations detected by molecular modeling. mupro and istable were unable to identify any of the 7 destabilizing mutations predicted by molecular modeling. although in silico programs are not a substitute for wet-lab experiments, they can provide a supportive role in the experimental validation of disease-associated alleles and can help further diagnostic strategies by prioritizing the most likely pathogenic novel variants. while many tools are available for assessing the functional significance of variants, determining the reliability of prediction results is challenging. in this context, the current study investigated the combination of experimental findings, molecular modeling, in silico mutation prediction programs, and stability prediction software to assess the pathogenicity of pitx2 missense variants. in silico methods that correctly identify deleterious variants do not always inevitably work well for benign predictions. the methods determined by this study to be preferred for analyses of pitx2 variants were those best able to distinguish both pathogenic and benign variants, thus yielding the highest accuracy. our results showed that mutpred, provean, and pmut tools were the most accurate in predicting pathogenicity of pitx2 missense variants (fig 2) . the sensitivity and specificity of these three tools in recognizing pitx2 disease-causing variants were over 93%, indicating the strong performance of these programs in identifying as pathogenic only pitx2 variants with significant functional defects. after these three tools, revel showed highest sensitivity and specificity, 94.44% and 87.50%, respectively. sift showed good sensitivity (72.22%) but low specificity (43.75%). polyphen-2, mutationtaster and panther-psep, fathmm, and align gv-gd demonstrated > 83% sensitivity, but, they were unable to identify the benign polymorphisms, showing the specificity of 37.50%, 6.25%, 43.75%, 6.25%, and 6.25%, respectively. the predictive value of nssnpanalayzer was similar to that of sift program, with sensitivity and specificity of 66.67% and 43.75%, respectively. our results showed, therefore, that computational analysis of pitx2 mutations mutpred, provean, and pmut can be utilized with high confidence to test whether or not a pitx2 missense variant is likely to be deleterious. interestingly, mutpred was the only in silico program that ranked in the top three programs in identifying both pathogenic and benign pitx2 and foxc1 variants [27] . a likely explanation for mutpred's high ranking is that it evaluates the most factors in making assessments. however, since the number of variants available for testing in this study were small, a larger dataset would confirm that our results are reproducible and generally applicable. the three programs that were found to be the most reliable (mutpred, provean, and pmut) were then used to assess the likely pathogenicity of thirteen pitx2 missense variants for which functional analyses have not been performed, but which have been associated with ars or cad (table 6 ). our results showed that mutpred, provean, and pmut predicted as pathogenetic 12/13 of the variants. the a30v variant was scored as non-pathogenetic/benign by all three programs. while it is possible that a30v is an example of a false negative for all three programs, it is likely that this variant is instead benign. functional testing of the a30v variant is needed to determine which of these possibilities is accurate. various intramolecular interactions are involve in stabilizing and folded state of protein, including hydrophobic, electrostatic, and hydrogen-bonding [95] [96] [97] [98] . the stability state of a protein is key factor in its proper functionality. in fact, up to 80% of mendelian disease-causing mutations in protein coding regions are predicted to be caused by altering protein stability [99] . in recent years, due to the availability of high-throughput array-based genotyping methods [100] and next generation sequencing platforms [101, 102] , a large number of snps has been reported. however, the association of missense variants with protein stability has often been difficult to predict. fortunately, recent advances in computational prediction of protein stability offers potential insight into this question. we used two parallel prediction methods to investigate the possible effects on pitx2 protein structure and stability of missense variants. knowledge of a protein's 3d structure can be used to predict the functionality of protein and the possible impact of variants on protein conformation and structure. we thus first used molecular modelling analyses to assess and compared the total energy difference between native and mutated modeled structure of pitx2 proteins. the results predicted that while most pitx2 variants did not dramatically affect the protein tertiary structure, seven variants (l54q, f58l, v83f, v83l, w86c, w86s, and r91p) altered the total energy level in comparison with the native structure, suggesting that these amino acid substitutions changed the structure of the pitx2 protein. molecular modeling of the pitx2 homeodomain predicted that these variants impair the required energy to maintain the proper folding of helix 1-3 and cause global destabilization of the structure of pitx2. these seven amino acids are either invariant (e.g., w86) or highly conserved in the approximately 300 homeobox proteins analyzed, consistent with a pivotal role of these residues in the homeodomain [103] [104] [105] . these seven amino acids are tightly packed hydrophobic amino acids responsible for holding helices of the pitx2 homeodomain together, supporting our molecular modeling predicting that mutations of these amino acids disrupt pitx2 structure. for f58l, v83f, and v83l, the native wild-type residues and the introduced mutant residues differ in size, probably causing loss of hydrophobic interactions in the core of the protein, particularly involving helix 1-3. for l54q, w86c, w86s, and r91p, the wild-type residues and the mutant residues are different in both size and charge, likely disturb the local structure of protein thereby altering protein structure and function. residues v83, w86, and r91 are located within the third helix which is specifically responsible for binding with the major groove of the dna [106] . thus, the prediction that these mutations impair the capacity of this helix to interact with dna is consistent with this knowledge and with previous functional characterizations that showed reduced dna-binding capacities of the v83l and r91p mutant pitx2 proteins [5, 107] . consistent with bioinformatics predictions of deleterious affects of mutation of w86, mutations of the neighboring amino acids (r84w and k88e) have been shown to decrease the ability of the mutant proteins to interact with dna [39, 108] . residues l54 and f58 are located in helix 1 of the homeodomain, responsible for contacting with the minor groove of the dna. molecular modeling of l54q is consistent with the computational analysis of pitx2 mutations suggestion that mutations in these highly-conserved residues in helix 1 of the homeodomain might disturb the dna-protein binding affinity. our prediction is supported by the fact that changing the leucine to a glutamine (l54q) disrupts dna-protein complex, indicating the necessity of leucine at position 54 for pitx2 binding ability [109] . thus, consistent with our recent studies on foxc1 protein [110] , the results of molecular modeling of pitx2 are strongly consistent with the functional characterization of pitx2 missense variants. the results from our molecular modeling analysis were also compared to the predictions of eight stability predictor methods (duet, sdm, mcsm, i-mutant3.0, mupro, iptree-stab, cupsat, and istable). based on our analyses, it appears that cupsat performs the best of the seven methods evaluated here in predicting the effect of missense mutations on pitx2 protein stability, with sdm, duet, mcsm, and i-mutant3.0, performing weaker, consistent with the results of previous studies [111, 112] . our results indicate that further studies are required to improve δδg predictions, especially for buried amino acids. in this study, for the first time, we evaluated the impact of missense variants on pitx2 stability, structure and function by integrating stability prediction algorithms, bioinformatics mutation prediction tools, and molecular modeling. our results showed that mutpred, provean, pmut, molecular modeling, and cupsat are reliable methods to assess pitx family missense variants in the absence of laboratory experiments. however, for our analyses, it must be noted that we used sixteen snps as non-pathogenetic control variants to investigate the performance of prediction programs. although we considered snps with a population frequency of >0.05% as benign, we cannot formally exclude that these snps might have un-documented pathogenic effects on pitx2. in addition, while the prediction methods used in this study are not gene-specific, generalization of the performance of these programs to other human genes may be inappropriate without additional study. when assessing the pathogenicity of missense variants, it is necessary to be cautious on depending merely on in silico programs without 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of foxc1 mutations in patients with axenfeld-rieger syndrome screening of mutations affecting protein stability and dynamics of fgfr1-a simulation analysis performance of protein stability predictors the authors would like to thank the members of the walter laboratory for critical reading of the manuscript and for helpful comments. key: cord-260653-5qwtvm9x authors: chikhlikar, priya; de arruda, luciana barros; maciel, milton; silvera, peter; lewis, mark g.; august, j. thomas; marques, ernesto t.a. title: dna encoding an hiv-1 gag/human lysosome-associated membrane protein-1 chimera elicits a broad cellular and humoral immune response in rhesus macaques date: 2006-12-27 journal: plos one doi: 10.1371/journal.pone.0000135 sha: doc_id: 260653 cord_uid: 5qwtvm9x previous studies of hiv-1 p55gag immunization of mice have demonstrated the usefulness of targeting antigens to the cellular compartment containing the major histocompatibility complex type ii (mhc ii) complex molecules by use of a dna antigen formulation encoding gag as a chimera with the mouse lysosome-associated membrane protein (mlamp/gag). in the present study, we have analyzed the magnitude and breadth of gag-specific t-lymphocyte and antibody responses elicited in rhesus macaques after immunization with dna encoding a human lamp/gag (hlamp/gag) chimera. elispot analyses indicated that the average gag-specific ifn-γ response elicited by the hlamp/gag chimera was detectable after only two or three naked dna immunizations in all five immunized macaques and reached an average of 1000 spot-forming cells (sfc)/10(6) pbmcs. high ifn-γ elispot responses were detected in cd8(+)-depleted cells, indicating that cd4(+) t-cells play a major role in these responses. the t-cell responses of four of the macaques were also tested by use of elispot to 12 overlapping 15-amino acids (aa) peptide pools containing ten peptides each, encompassing the complete gag protein sequence. the two mamu 08 immunized macaques responded to eight and twelve of the pools, the mamu b01 to six, and the other macaque to five pools indicating that the hlamp/gag dna antigen formulation elicits a broad t-cell response against gag. additionally, there was a strong hiv-1-specific igg response. the igg antibody titers increased after each dna injection, indicating a strong amnestic b-cell response, and were highly elevated in all the macaques after three immunizations. moreover, the serum of each macaque recognized 13 of the 49 peptides of a 20-aa peptide library covering the complete gag amino acid sequence. in addition, hiv-1-specific iga antibodies were present in the plasma and external secretions, including nasal washes. these data support the findings of increased immunogenicity of genetic vaccines encoded as lamp chimeras, including the response to dna vaccines by non-human primates. several human immunodeficiency virus type 1 (hiv-1) antigenic formulations have been tested in animal models and clinical trials, but the immunogenicity and protection achieved to date are still far from the desired goals for an hiv-1 vaccine. while the correlates of immune protection are still only vaguely defined, it is now generally recognized that an effective hiv-1 vaccine must elicit strong t-cell responses as well as neutralizing antibody [1] [2] [3] [4] . it has been postulated that the immunogenicity of a protein antigen can be enhanced by targeting the antigen to the major histocompatibility complex type ii (mhc ii) processing compartment of professional antigen-presenting cells (apcs). one of the approaches is the use of the lysosomal associated membrane protein-1 (lamp) as a lamp/antigen chimera to target endogenous antigens to the mhc ii processing compartment [5] [6] [7] [8] [9] [10] . many laboratories have reported that lamp targeting can greatly enhance the immune responses against a number of antigens, including hpv-16-e7 and e6 proteins; human telomerase reverse transcriptase (htert); west nile virus prem-e; the thyroid hormone receptor (tshr); hiv-1 gag, env gp120, env gp160, and nef; human melanosomal antigen (mage-3); dengue 2 prem-e; listeriolysin o; and sars coronavirus n protein [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] . several approaches have been used to design dna vaccines encoding lamp/antigen chimeras. one frequently used construct contains the transmembrane and cytoplasmic (tmcy) domains of lamp added to the c-terminus of the protein antigen. more recently, we have found that some antigens, including hiv-1 gag, must be incorporated into the entire lamp molecule in order to effect enhanced antigen expression and trafficking to the lysosomal academic editor: douglas nixon, university of california, san francisco, united states of america compartment. several lamp/antigen chimeras, including proteins of west nile, dengue, sars cov, hpv and hiv-1, have been shown to co-localize in vitro with mhc ii, lamp-1, lamp-2, and h-2m in multiple cell types by confocal imunofluorescense microscopy, and/or by immunogold electron microscopy [11] [12] [13] [14] [15] [16] 24] . the immunological benefits of lamp-targeted antigens have been demonstrated in several mouse strains and, most importantly, also in humans [25] [26] . lamp/antigen chimeras have been shown to induce increased cd4 + responses to the antigens in several assay systems, producing increased secretion of il-2, il-4, il-5 and ifn-c cytokines; increased proliferative responses; a greater number of spot-forming cells (sfcs) in elispot assays; intracellular cytokine staining (ics); higher precursor frequencies; increased functional avidity and a broader response repertoire [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] . the increased cd4 + -mediated responses produced by the lamp/chimeras are thought to play an important role in modulating b-cell, cd8 + responses and the development of immune memory [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] . when compared to the non-targeted molecules, lamp/antigens elicited greatly increased antibody titers, viral neutralization, antibody affinity and numbers of b-cell epitopes recognized. cd8 + responses were also enhanced in several lamp/antigen chimeric systems, as assessed by tetramer staining, ifn-c elispot, chromium release, and the functional avidities and t-cell response repertoires of cd8 + cells. the longevity of the immunological memory of b cells and cd8 + cells is also increased in animals immunized with lamp/chimeras. previous studies of hiv-1 dna antigen formulations have described an hiv-1 p55gag dna vaccine that elicited strong, broad and poly-functional cellular and humoral immune responses in immunized mice when the gag sequence was incorporated into the complete lamp cdna sequence and the coding sequences were bracketed by the inverted terminal repeat (itr) sequences of adeno-associated virus (aav) [14] [15] [16] . these observations in mice led us to investigate the immunogenicity of a naked dna encoding the hiv-1 lamp/gag antigen formulation in rhesus macaques, a relevant animal model for testing hiv vaccines for potential use in humans. we found that immunization of these primates with a human lamp/gag (hlamp/gag) dna vaccine promoted a humoral and cellular immune response, which was associated with a sustained activation of b-lymphocytes as well as cd4 + and cd8 + t cells. furthermore, gag-specific t cells were detected after only two immunizations and were further expanded by booster injections of hlamp/gag chimera. these results substantiate the usefulness of the lamp vaccination strategy as a means of effectively eliciting t-and b-cell responses to naked dna immunization of rhesus macaques. the mouse lamp/gag (mlamp/gag) plasmid was constructed as described previously [16] : nucleotides 1-1503 of the hiv-1 hxb2 p55gag gene (genbank tm accession number k03455; hiv sequence database, los alamos national laboratory theoretical biology and biophysics, los alamos, nm, usa) was inserted into the pitr vector [27] , which contains the aav-itr flanking the expression elements (cytomegalovirus promoter and bovine growth hormone polyadenylation signal). the p55gag sequence was inserted between the luminal domain and the tmcy domain of mouse lamp (mlamp) (genbank tm accession number j03881), as described previously [16] . in the present study, the complete mlamp cdna described above was replaced by the human lamp-1 (hlamp) cdna (genbank tm accession number nm005561). the control plasmids, consisting of mlamp and hlamp, were constructed using the respective complete lamp sequences without gag. the hlamp/gag plasmid used for vaccination of rhesus macaques was produced by quality biological inc. (gaithersburg, md, usa), with a dna purity of 96% and endotoxin level of 0.33 eu/mg. human 293 cells were plated in 6-well plates (2610 6 cells/well) and transfected with plasmid dna (4 mg) using the fugene tm 6 (roche applied science, indianapolis, in, usa) transfection reagents according to the manufacturer's instructions. the western blot analysis was done as previously described [15] . evaluation of hlamp/gag targeting by confocal microscopy mhc ii (i-ek)-expressing cells (dcek.icam.hi7, a gift of dr. susan swain, the trudeau institute, saranac lake, ny, usa) [28] were plated onto poly-lysine-coated coverslips in 6-well plates (2610 6 cells/well) and incubated overnight. the cells were transfected with the dna plasmids using lipofectamine tm 2000 transfection reagent (invitrogen life technologies, carlsbad, ca, usa), and 24-48 h later they were stained to evaluate cellular localization. coverslips were fixed in 2% paraformaldehyde in phosphate-buffered saline (pbs) for 5 min and washed with pbs, then blocked and permeabilized with pbs containing 4% normal goat serum and 0.1% saponin. for detection of gag expression, the cells were incubated for 1 h with mouse anti-gag monoclonal antibody (provided by dr. james hildreth, johns hopkins school of medicine, baltimore, md, usa) at a 1:50 dilution. after three washes with 0.1% saponin in pbs, the cells were incubated for 1 h with texas red-labeled goat anti-mouse igg (bd pharmingen, san diego, ca, usa) at a 1:500 dilution. co-localization of the hlamp/gag chimera with mhc ii of the transfected dcek cells was performed by double immunostaining, first for the hlamp/ gag chimera proteins as described above and followed by anti-mhc ii, by incubating the cells for 1 h with fitc-labeled goat antimouse i-ek (14-4-4s) (bd pharmingen) at a 1:75 dilution. the cells were then washed three times with pbs, and the coverslips were mounted onto glass slides using prolong antifade reagent (molecular probes, eugene, or, usa). confocal microscopy was performed using a wallac confocal laser scanning microscope. colocalization of the proteins with mhc ii was thus determined by merging fluorophore images individually captured and digitally colored by use of adobe photoshop 7.0 (adobe system corp., san jose, ca, usa). immunization of mice female balb/c mice, 6-8 weeks of age, were obtained from charles river (kingston, ny, usa). mice in groups of eight were each immunized twice (21 days apart), intramuscularly (i.m.), with 50 mg of the indicated plasmid and sacrificed 7 days later. for cd8 + experiments, the mice were challenged 21 days after the first immunization with a vdk-1 vaccinia virus plasmid containing the native gag sequence (10 7 plaque-forming units; nih aids research and reference reagent program, rockville, md, usa). antibody responses mouse serum was obtained from the tail vein 7 days after the second immunization. serum igg levels were measured by enzyme-linked immunosorbent assay (elisa) as described previously [16] . preparation of splenocytes for assaying the t-cellmediated immune responses of immunized mice singlecell suspensions depleted of red blood cells were prepared from freshly isolated mouse splenocytes in culture medium (rpmi medium 1640 supplemented with 5% v/v fbs, 100 u/ml penicillin/streptomycin, 2 mm glutamine, 50 mm 2mercaptoethanol, and 0.01 m hepes buffer). splenocytes were counted and resuspended at 10610 6 cells/ml in culture medium for t cell-mediated assays. t-cell responses of immunized mice were measured as described previously [15] . rhesus macaques five healthy 4-to 8-kg male rhesus macaques were maintained in the non-human primate facility of southern research institute, frederick, md, usa. animal care and treatment were in accordance with standards approved by the institutional animal care and use committee, according to the principles set forth in the guide for the care and use of laboratory animals, national research council, national academy press, 1996. immunization of macaques each animal was immunized intramuscularly (i.m.) five times at weeks 0, 4, 14, 22 and 38 with 5 mg of hlamp/gag dna plasmid using a biojector, and 18 blood samples were drawn over the 42 week experiment period. mucosal samples including mouth swabs, nasal and rectal washes were collected after four dna immunizations. blood and mucosal samples of non-immunized macaques were supplied periodically as controls. isolation of peripheral blood mononuclear cells (pbmcs) pbmcs were isolated by centrifugation (4006g, 40 min) on a ficoll-hypaque gradient (amersham biosciences piscataway, nj, usa). mononuclear cells were collected from the interface and washed three times in cold phosphate-buffered saline with ca 2 + and mg 2 + (gibco, grand island, ny, usa). residual red blood cells were removed with ack lysing buffer (quality biological inc., gaithersburg, md, usa). pbmcs to be used for cytokine analysis or elispot assay were resuspended in supplemented culture medium (rpmi 1640 medium containing 1% fbs, 100 u/ml penicillin/streptomycin, and 2 mm lglutamine). cd8 + lymphocyte separation positive selection of cells expressing cd8 + antigen was performed with monoclonal antihuman cd8 + conjugated to r-phycoerythrin (pe) and microbeads conjugated to monoclonal anti-pe antibodies (cd8 microbead kit, miltenyi biotec, bergisch gladbach, germany) as described by the manufacturer. the cd8 + cells were .98% pure as assessed by flow cytometry. fractionated cells were suspended in rpmi-10 medium and used on the same day for elispot assay. hiv gag-specific ifn-c elispot assay the frequency of ifn-c-producing cd4 + or cd8 + t cells from immunized rhesus macaques was measured by elispot assays, using the ifn-c elispot set from bd-biosciences pharmingen according to the manufacturer's protocol. initially, elispot plates were coated with anti-human ifn-c ig at 5 mg/ml and incubated at 4uc overnight. after blocking with rpmi-1640 containing 10% fbs for 2 h at room temperature (rt), total pbmcs (3610 5 cells/well) were cultured with hybridoma serum-free medium (gibco) supplemented with 1% fbs, 100 u/ml penicillin/streptomycin and 2 mm l-glutamine, in the presence of 10 mg/ml of hivsf2 p55 gag recombinant protein (rgag); or hiv gag 15-amino acids (aa) peptides overlapping by 11-aa; or 20-aa overlapping by 10-aa. a negative control included in each assay consisted of medium lacking hiv antigen. phytohemagglutinin (pha) (sigma, st. louis, mo, usa) at 5 ng/ml with 500 ng/ml ionomycin (sigma) was included as a positive control for each rhesus macaque. after 16 h of culture, the plates were washed and incubated with biotinylated anti-human ifn-c for 2 h at rt, followed by hrpconjugated avidin for 1 h at rt. the reaction was developed with 3-amino-9-ethylcarbozole substrate (calbiochem-novabiochem corporation, san diego, ca, usa). analysis of the ifn-c levels was performed using an immunospot image analyzer (cellular technology limited, cleveland, oh, usa). background spots obtained with medium alone were subtracted from each experimental value. all results were expressed as mean number of sfc per 10 6 pbmcs. for each antigen, a responder rhesus macaque was defined as one exhibiting a significant number ifn-c sfc cells over the background value or value for non-immunized macaques, at any time point over the immunization course. analysis of il-6 secretion by capture elisa total pbmcs (5610 5 cells) from immunized macaques were cultured in a 96well plate (nunc, roskilde, denmark) containing recombinant human il-2 (500 u/well). except for the negative and positive control wells containing culture medium alone and 5 ng/ml pha (sigma) containing 500 ng/ml ionomycin, respectively, pbmcs were stimulated with 10 mg/ml hivsf2 p55 rgag. after a 48-h incubation at 37uc in 5% co 2 , the supernatants were collected for detection of secreted cytokines and stored at 220uc until further use. il-6 concentrations were determined using a monkey il-6 opteia tm set (bd pharmingen). hiv gag-specific humoral and mucosal immune responses serum samples were tested throughout the experimental period for the presence of binding antibody to hiv gag. nunc plates were coated with 5 mg/ml hiviiib lysate in sodium carbonate-bicarbonate buffer, ph 9.4 (pierce, rockford, il, usa). after overnight incubation at 4uc, the solution was removed, and the plates were washed six times with pbs containing 0.05% tween-20 (pbs-t) wash buffer. the plates were then incubated for 2 h at 37uc with blocking buffer (pbs-t with 5% fbs) and then washed three times with pbs-t. igg responses were measured with four serial 1:3 dilutions, starting at 1:100, in blocking buffer. iga responses in serum, nasal, mouth and rectal washes were measured with 100 ml of 10-fold diluted sample added to the blocked plate in duplicate. the plates were incubated overnight at 4uc. after six washes, 100 ml of horseradish peroxidase-conjugated rabbit anti-monkey igg (sigma) diluted 1:5000 in blocking buffer was added to each well. for the detection of iga antibodies, 100 ml of goat antimonkey iga (nordic immunology, tilburg, netherlands) diluted 1:5000 in blocking buffer was added to each well. the plates were incubated for 2 h at 37uc and washed eight times with washing buffer. turbo tmb substrate solution (bd pharmingen) was then added to each well and incubated for 15 min at rt. the reaction was stopped by adding 100 ml of 1 m sulfuric acid, and absorbance at 450 nm was measured in a bio-rad model 3550 microplate reader. b-cell epitope mapping hiv-1 gag 20-aa peptides, spanning residues 1 to 490 with a 10-aa overlap (nih aids research and reference reagent program), were diluted in 0.1 m sodium carbonate-bicarbonate buffer, ph 9.4, to give a concentration of 5 mg/ml, and 50 ml of the individual peptide was added to each well of a 96-well plate, in duplicate. after overnight incubation at 4uc, the solution was removed, and the plates were washed six times with pbs-t. they were then incubated with blocking buffer for 2 h at 37uc and washed three times. serum samples (100 ml of a 1:300 dilution in blocking buffer) were added to each well, and the plates were incubated overnight at 4uc. after the plates were washed six times, 100 ml of hrp-conjugated rabbit anti-monkey igg (sigma), diluted 1:5000 in blocking buffer, was added to each well. the plates were incubated for 2 h at 37uc and then washed. turbo tmb substrate solution (bd pharmingen) was added to each well and incubated for 15 min at rt. the reaction was stopped by adding 100 ml of 1 m sulfuric acid, and absorbance at 450 nm was measured in a bio-rad model 3550 microplate reader. statistical analyses all the graphs were made using graphpad prism version 4.0a for macintosh (graphpad software, san diego, ca, usa). lamp/gag expression and cellular trafficking to the cellular mhc ii compartment has been repeatedly demonstrated in studies with murine lamp chimeras [11] [12] [13] [14] [15] [16] . the present study confirmed the similar expression and trafficking of the human lamp/gag protein chimera. western blotting with anti-gag antibody showed the presence of ,200 kda lamp/gag protein at comparable levels in 293 cells transfected with either mouse or human lamp/gag ( figure 1a ). there appeared to be increased degradation of gag in the hlamp/gag transfected cells; however, the significance of this is not known as the results of other similar studies of mlamp/gag transfected cells have shown variable levels of gag degradation products [14] [15] [16] . co-localization of the hlamp/gag chimera transgene product with the cellular mhc ii was confirmed with transfected dcek cells stained with anti-gag and anti-mhc ii monoclonal antibodies ( figure 1b) . verification of the in vivo immune responses to the hlamp/gag chimera and a comparison of the responses to mlamp/gag were examined with mice immunized on days 1 and 21 with 50 mg dna of the pitr plasmid vectors encoding the mlamp/gag and hlamp/gag chimeras. total anti-gag igg responses were assayed with blood collected on day 28 (figure 2a ). the response of mice immunized with the hlamp/gag construct was considerably greater than the response to the mlamp/gag. splenocytes of mice sacrificed after the two immunizations were assayed for t-cell ifn-c responses. the mhc ii-targeted mouse and human lamp/gag plasmids elicited comparable ifn-c + responses (8000 pg/ml and 6000 pg/ml, respectively) ( figure 2b) . assays of cd8 + t-cell responses were carried out with mice immunized once with 50 mg of the plasmid dna, followed 21 days later by in vivo expansion of gag-specific t cells through inoculation with recombinant vaccinia-gag-pol (rvvgag-pol). five days later, the mice were injected with an immunodominant h-2kdrestricted gag peptide epitope and sacrificed after 2 h for the ex vivo assay. mice immunized with both the mouse and human lamp/gag chimeras uniformly developed significantly strong cd8 + responses as measured by epitope-specific cd8 + tetramer binding, intracellular ifn-c staining, and ctl lysis of peptidepulsed target cells ( figure 2c ). the larger fraction of cd8 + t cells expressing ifn-c (.20%) following inoculation with recombinant vaccinia virus encoding gag, in comparison with the proportion of tetramer positive cells, is attributed to the massive in vivo expansion of gag-activated cd8 + t cells reactive against several other gag mhc i epitopes, besides the immunodominant h-2kd-amqmlketi peptide-tetramer epitope complex. the magnitude of the vaccine-induced t-cell responses varied from macaque to macaque; however, in every case, ifn-c secretion was detected after the second immunization at week 8, reached the highest level of 600 to 1200 sfc/10 6 cells at week 18 following the third immunization, and declined gradually until further immunizations were given at weeks 22 and 38 ( figure 4) . these studies included an analysis of the relative efficiency of the 15-or 20-aa peptides in stimulating t-cell responses in vitro, and we observed that pbmcs of all the five immunized macaques stimulated with the 15-and 20-aa gag peptide pools separately, showed no significant difference in the ifn-c response ( figure 5 ). cd4 + -specific responses were also studied by elispot analyses with cd8 + -depleted pbmc stimulated with rgag. all five macaques showed a response in the range of 200-1000 ifn-c + sfc/10 6 cd8 + -depleted cells, indicating a potent cd4 + -mediated response ( figure 6 ). the repertoire of peptide-specific responses elicited by lysosomal targeting in balb/c mice has been shown to include the same immunodominant epitopes as those elicited by the native antigen; however, the chimeric antigen commonly elicits enhanced and additional t-cell responses that were not detected with the native antigen [24] . in an analysis of the potential effect of lamp targeting on the breadth of the t-cell repertoire in macaques, pbmcs from the immunized macaques were tested at week 20, after 3 dna immunizations, for t-cell responses to 12 different pools consisting of ten peptides each, of the 15-aa gag peptide set (figure 7) . t-cell responses to specific peptides were detected with each of the four immunized macaques that were included in this study. three of the immunized macaques showed responses to five to eight of the peptide pools. one of the animals, macaque #3162, showed activation of ifn-c + cells in response to all of the peptide pools. collectively, these data suggest that hlamp/gag can prime a broad t-cell response in primates. one remarkable feature of the dna-encoded lamp-targeted antigens has been the dramatic increase in antibody-mediated responses of immunized mice. similar findings have been obtained with the five immunized macaques, each of which showed strong humoral immune responses (figure 8) . the strength of these responses by the individual macaques followed the same pattern as the t-cell responses. high anti-gag igg antibody titers were present in serum after immunization with hlamp/gag, indicating effective priming. four of the five vaccinated macaques (#3119, 3162, 3159 and 3117) developed antibodies against hiv-1 after the second immunization and high antibody titers were achieved by these four macaques following the third dna immunization at week 14. the remaining macaque (#3125), which showed the weakest t-cell response, also elicited a relatively weak antibody response as compared to the other 4 macaques ( figure 8a ). the third dna immunization worked as a booster for all the macaques, and antibody titers reached their highest values after the 5th injection (at week 38). the end-dilution titers of the igg antibodies achieved after four hlamp/gag immunizations ranged from 2,700 to 24,300 ( figure 8b ). serum samples of the immunized macaques were also assayed for iga production, significant levels were detected in three (#3119, 3159 and 3162) of the immunized macaques ( figure 9a ). il-6 is a key cytokine for terminal differentiation of b-cells into iga-secreting plasma cells in both the mouse and human systems [29] [30] . interestingly, il-6 production was seen in the same three macaques ( figure 9b) , correlating with the plasma iga response. the presence of gag-specific iga in external secretions, including mouth swabs and nasal and rectal washes collected after four dna immunizations, was also assayed ( figure 9c ). the levels of iga antibodies detected in mouth swabs and nasal and rectal washes varied remarkably among the individual macaques; however, comparatively higher iga antibodies were found in nasal washes of all the five macaques. serum samples collected from the five macaques at week 24, after four dna immunizations, were analyzed individually in elisa assays against each of the 20-aa gag peptides, overlapping by 10-aa ( figure 10 ). the repertoires of peptides recognized by each of the macaque sera were very similar; all of the animals reacted with the same 13 of the 49 gag 20-aa peptides tested. three peptides containing b-cell determinants were located at the aminoterminal, whereas, most of the peptides recognized were located in the carboxy-terminal region of the gag protein. these results from immunized macaques support the conclusion that vaccination with an hlamp/gag chimera can prime a broad b-cell response [24] . this study demonstrates that rhesus macaques immunized with a dna plasmid vaccine-encoding gag as an hlamp/gag chimera develops strong antigen-specific humoral responses as well as cd4 + and cd8 + t-cell responses. previous studies by others with candidate dna vaccines have shown protection of small animals against pathogenic challenges [31] ; however, their performance in primates has generally been disappointing. most naked dna vaccines have produced sub-optimal immune responses, even with repeated boosting, with only moderate t-cell responses when compared to live-attenuated virus or recombinant virus vaccines and very low or no humoral responses. in contrast, hlamp/gag dna immunization elicited potent cd4 + t-cell as well as gagspecific cd8 + t-cell responses in macaques after only a few dna immunizations. the responses included highly significant igg antibody titers after two dna immunizations in four of the five immunized macaques and iga responses in serum and nasal washes. all of the macaques showed a high igg antibody titer after three dna immunizations, with a rapid and enhanced immune memory response with the fourth and fifth immunization. the breadth of the t and b cell repertoire elicited by dna hiv vaccines is thought to be important for pathogen control, perhaps by preventing the selection of escape mutants. we have previously reported that immunization of balb/c mice with lamp-targeted gag increases the breadth of b-and t-cell responses. moreover, others have shown that human dendritic cells transfected with lamp-targeted hiv nef are able to induce the activation of an increased repertoire of t-cells isolated from infected patients [26] . in this study, each of the four macaques tested, produced gag-specific ifn-c + responses to many gag 15aa peptide pools, indicating a broad range of t-cell responses. a question that remains to be investigated is whether this result reflects a true increase in the number of new epitopes being recognized as a result of hlamp/gag administration or whether the ability of hlamp/gag to increase the overall magnitude of the cellular immune response simply enhances our ability to detect epitopes that would otherwise have been below the limit of detection. we also mapped b-cell epitopes of linear, 20-aa gag peptides with serum samples obtained from the macaques after three dna immunizations with the hlamp/gag chimera. the results of these analyses indicated that some of the peptides behaved as dominant epitopes and induced strong b-cell activation in all five macaques. these include two dominant peptides located in the aminoterminal portion of the molecule, and 10 peptides located in the carboxy-terminal portion of the gag protein. the amino-terminal region is known for its antigenic properties [32] [33] [34] and has been reported to contain both b and t-cell epitopes and to be relatively conserved among european hiv-1 isolates [33, 35] . the carboxyterminal region is also known to bind antibodies developed during natural hiv-1 infection in humans [36] . our studies also showed that iga antibodies were present in the serum and external secretions of the hlamp/gag-immunized macaques. furthermore, il-6 production was detected in three of the five immunized macaques. il-6 is known to play a key role in the terminal differentiation of iga-committing b cells into igasecreting plasma cells in both mice and humans [29] [30] . in this study, the increased production of il-6 by gag-specific cd4 + t cells is consistent with the induction of hiv-specific iga b-cell responses in addition to systemic igg antibody responses. iga and igg antibodies may function as a first line of defense, preventing hiv/siv adherence to the mucosal surface or interfering with viral replication through secretory iga [37] . although t-cell immune responses induced by dna immunization are generally moderate, previous studies have demonstrated that dna prime is very important in heterologous prime-boost immunization regimens with viral vectors. the prime-boost formulations have been shown to induce strong cellular immune responses and protection in malaria [38] [39] and sivmac [40] [41] models. moreover, the dna prime and viral boost approach has been shown to be important in increasing the breadth of the response repertoire of viral vector vaccines [42] , thereby contributing to the overall immunological protection. there is growing consensus that plasmid dna represents a particularly good prime immunogen. therefore, considerable work is now being done to explore the use of bimodal vaccine regimens in which the plasmid dna is used to prime the immune response and a live recombinant vector is used to boost that immunity [43] . these studies collectively suggest that more work is needed before a dna approach alone or a dna prime followed by a viral vector boost is able to completely control the pathogenic challenge in these model systems. a recent report has shown consistent and strong ctl responses to gag in macaques immunized with dna_crl 1005-adjuvant gag plasmids and boosted with rad5 encoding gag [44] . the studies have established that a significant percentage of humans respond to both the dna and adenovirus approaches, and more evaluation of further potent dna vaccines is clearly warranted. the current study was limited by the small number of animals and the lack of an hiv challenge system to assess the protective efficacy afforded by the hiv hlamp/gag dna vaccine. a further goal is to test the effect of lamp targeting on additional relevant antigens of the siv challenge model, in collaboration with the g. pavlakis and b. felber group (human retrovirus section, basic research laboratory, national cancer institute, frederick, md, usa). the rational design of an aids vaccine correlates of immune protection in hiv-1 infection: what we know, what we don't know, what we should know vaccination preserves cd4 memory t cells during acute simian immunodeficiency virus challenge preserved cd4+ central memory t cells and survival in vaccinated siv-challenged monkeys the motif tyr-x-x-hydrophobic residue mediates lysosomal membrane targeting of lysosome-associated membrane protein 1 major histocompatibility complex class ii compartments in human and mouse b lymphoblasts represent conventional endocytic compartments segregation of mhc class ii molecules from mhc class i molecules in the golgi complex for transport to lysosomal compartments the role of endosomes and lysosomes in mhc class ii functioning involvement of miic-like late endosomes in b cell receptor-mediated antigen processing in murine b cells transport of peptide-mhc class ii complexes in developing dendritic cells west nile premembrane-envelope genetic vaccine encoded as a chimera containing the transmembrane and cytoplasmic domains of a lysosome-associated membrane protein: increased cellular concentration of the transgene product, targeting to the mhc ii compartment, and enhanced neutralizing antibody response /lamp chimera targeted to the mhc class ii compartment elicits longlasting neutralizing antibodies sars coronavirus nucleocapsid immunodominant t-cell epitope cluster is common to both exogenous recombinant and endogenous dna-encoded immunogens dna vaccine encoding human immunodeficiency virus-1 gag, targeted to the major histocompatibility complex ii compartment by lysosomal-associated membrane protein, elicits enhanced long-term memory response inverted terminal repeat sequences of adeno-associated virus enhance the antibody and cd8(+) responses to a hiv-1 p55gag/lamp dna vaccine chimera hiv-1 p55gag encoded in the lysosome-associated membrane protein-1 as a dna plasmid vaccine chimera is highly expressed, traffics to the major histocompatibility class ii compartment, and elicits enhanced immune responses targeting antigen in mature dendritic cells for simultaneous stimulation of cd4+ and cd8+ t cells induction of primary carcinoembryonic antigen (cea)-specific cytotoxic t lymphocytes in vitro using human dendritic cells transfected with rna synergistic neutralizing antibody response to a dengue virus type 2 dna vaccine by incorporation of lysosome-associated membrane protein sequences and use of plasmid expressing gm-csf lysosome-associated membrane protein-1-mediated targeting of the hiv-1 envelope protein to an endosomal/lysosomal compartment enhances its presentation to mhc class ii-restricted t cells the enhanced immune response to the hiv gp160/lamp chimeric gene product targeted to the lysosome membrane protein trafficking pathway enhanced induction of telomerase-specific cd4(+) t cells using dendritic cells transfected with rna encoding a chimeric gene product engineering an intracellular pathway for major histocompatibility complex class ii presentation of antigens dendritic cell-lysosomal-associated membrane protein (lamp) and lamp-1-hiv-1 gag chimeras have distinct cellular trafficking pathways and prime t and b cell responses to a diverse repertoire of epitopes telomerase mrna-transfected dendritic cells stimulate antigen-specific cd8+ and cd4+ t cell responses in patients with metastatic prostate cancer expansion of hiv-specific cd4+ and cd8+ t cells by dendritic cells transfected with mrna encoding cytoplasm-or lysosome-targeted nef gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein costimulatory requirements of naive cd4+ t cells. icam-1 or b7-1 can costimulate naive cd4 t cell activation but both are required for optimum response human and murine interleukin 6 induce high rate iga secretion in iga-committed b cells human appendix b cells naturally express receptors for and respond to interleukin 6 with selective iga1 and iga2 synthesis heterologous protection against influenza by injection of dna encoding a viral protein monoclonal antibodies to conserved regions of the major core protein (gag24) of hiv-1 and hiv-2 characterization of murine monoclonal antibodies directed against the core proteins of human immunodeficiency virus types 1 and 2 highly conserved epitope domain in major core protein p24 is structurally similar among human, simian and feline immunodeficiency viruses human immunodeficiency virus type 1 p24 production and antigenic variation in tissue culture of isolates with various growth characteristics mapping of igg subclass and t-cell epitopes on hiv proteins by synthetic peptides comparison of iga versus igg monoclonal antibodies for passive immunization of the murine respiratory tract enhanced immunogenicity for cd8+ t cell induction and complete protective efficacy of malaria dna vaccination by boosting with modified vaccinia virus ankara boosting with recombinant vaccinia increases immunogenicity and protective efficacy of malaria dna vaccine effective induction of simian immunodeficiency virus-specific cytotoxic t lymphocytes in macaques by using a multiepitope gene and dna primemodified vaccinia virus ankara boost vaccination regimen simian immunodeficiency virus dna vaccine trial in macaques enhanced breadth of cd4 t-cell immunity by dna prime and adenovirus boost immunization to human immunodeficiency virus env and gag immunogens control of a mucosal challenge and prevention of aids by a multiprotein dna/mva vaccine vaccineinduced immunity in baboons by using dna and replication-incompetent adenovirus type 5 vectors expressing a human immunodeficiency virus type 1 gag gene we thank dr. susan swain, from the trudeau institute, saranac lake, ny, for the fibroblast cell line dcek.i-cam.hi7; dr. deborah mcclellan for editorial assistance; dr. james hildreth, for providing the mouse anti-gag monoclonal antibody; and betty hart and delores henson for their excellent technical assistance. several reagents were obtained through the aids research reagents program, division of aids, niaid, national institute of health: 15-and 20-aa gag peptides and purified p55 gag protein. key: cord-262876-civfvk45 authors: su, tong; han, xue; chen, fei; du, yan; zhang, hongwei; yin, jianhua; tan, xiaojie; chang, wenjun; ding, yibo; han, yifang; cao, guangwen title: knowledge levels and training needs of disaster medicine among health professionals, medical students, and local residents in shanghai, china date: 2013-06-24 journal: plos one doi: 10.1371/journal.pone.0067041 sha: doc_id: 262876 cord_uid: civfvk45 background: disaster is a serious public health issue. health professionals and community residents are main players in disaster responses but their knowledge levels of disaster medicine are not readily available. this study aimed to evaluate knowledge levels and training needs of disaster medicine among potential disaster responders and presented a necessity to popularize disaster medicine education. methods: a self-reporting questionnaire survey on knowledge level and training needs of disaster medicine was conducted in shanghai, china, in 2012. a total of randomly selected 547 health professionals, 456 medical students, and 1,526 local residents provided intact information. the total response rate was 93.7%. results: overall, 1.3% of these participants have received systematic disaster medicine training. news media (87.1%) was the most common channel to acquire disaster medicine knowledge. although health professionals were more knowledgeable than community residents, their knowledge structure of disaster medicine was not intact. medical teachers were more knowledgeable than medical practitioners and health administrators (p = 0.002). clinicians performed better than public health physicians (p<0.001), whereas public health students performed better than clinical medical students (p<0.001). in community residents, education background significantly affected the knowledge level on disaster medicine (p<0.001). training needs of disaster medicine were generally high among the surveyed. ‘lecture’ and ‘practical training’ were preferred teaching methods. the selected key and interested contents on disaster medicine training were similar between health professionals and medical students, while the priorities chosen by local residents were quite different from health professionals and medical students (p<0.001). conclusions: traditional clinical-oriented medical education might lead to a huge gap between the knowledge level on disaster medicine and the current needs of disaster preparedness. continuing medical education and public education plans on disaster medicine via media should be practice-oriented, and selectively applied to different populations and take the knowledge levels and training needs into consideration. over the past decade, the intensity and frequency of natural and man-made disasters have been noticeably increasing all over the world. hurricane, earthquake, flood, outbreaks of infectious diseases, nuclear leakage, oil spills, and other disasters in recent years have caused huge economic losses, serious environmental disruption and lasting psychological impairment to the survivors [1] [2] [3] [4] [5] . community residents are the very ones directly affected by disasters. therefore, self-rescue and mutual-aid are essential to form the first defense line. disaster relief and assistance are mainly carried out by medical rescue teams, which are constituted of health professionals from on-call health agencies such as military medical systems and centers for disease control and prevention (cdc) [6] . hence, community residents and health professionals as the key components of first responders should be sufficiently trained to perform timely and effective medical rescue [7] . disaster medicine training, an integrated part of efficient disaster preparedness, is vital for community residents to perform timely self-rescue and mutual-aid and also for health professionals to develop comprehensive skills [8] . since the '9.119 terrorist attack, many countries have put emphasis on disaster medicine training and have sponsored various researches focusing on a wide range of disaster medicine, including description and assessment of the current disaster medicine training programs in order to improve the efficiency of disaster rescue. however, these researches on disaster medicine have mainly been conducted in developed countries, while data from developing countries are scarce [9] [10] [11] [12] [13] [14] . from a global perspective, disaster frequently attacks developing countries with weak public health infrastructure and often results in severe consequences. in china, the severe acute respiratory syndrome (sars) in 2002-2003 resulted in 5,327 cases and 343 deaths [15] . the devastating earthquake in sichuan, china, in 2008 caused more than 69,000 deaths, 18,341 missing and 374,176 wounded persons [16] . however, disaster medicine has not been included either in the undergraduate curriculum of medical schools or in the continuing medical education in china. in the past decades, chinese medical education system has experienced flexuous reforms [17] [18] [19] . traditional medical education and assessment criteria have been largely clinically oriented, while disaster medicine has been long neglected [20] . recently, efforts have been made to implement disaster medicine education in china. the current program of disaster medicine education focuses on developing particular small scale training programs, such as short-term training course of disaster nursing for undergraduates, psychosocial training program for mental health workers, and emergency preparedness training program for public health staff [21] [22] [23] [24] . however, current knowledge status and training needs of main players on disaster medicine were unknown. to the best of our knowledge, only one study surveyed the disaster medicine education needs of health professionals who participated in the earthquake rescue, but their related knowledge was not evaluated [20] . in this study, we evaluated the knowledge levels and training needs in populations that are most likely to be involved in disaster rescue. these data are essential in developing proper medical training programs of disaster medicine. three groups of participants in shanghai, china, were enrolled in this cross-sectional epidemiological study: health professionals, medical students, and community residents. a stratified cluster random sampling strategy was used to select health professionals and medical students. a total of 600 health professionals were composed of medical practitioners, medical teachers, and health administrators. the medical practitioners were clinicians, public health physicians, nurses, and medical technicians from two comprehensive tertiary hospitals and three cdcs. the medical teachers and 500 medical students were selected from 2 medical schools. health administrators were from the municipal health bureau and district health bureaus. a multi-stage sampling method was used to select 1,600 community residents. we first randomly selected 5 communities in the yangpu district. in each community, we randomly selected 27, 80, 65, 58, 58 and 32 residents (320 residents per community) at the age of ,20 years, 20-30 years, 30-40 years, 40-50 years, 50-60 years and .60 years, respectively, according to the 2010 census data of age composition in shanghai. a structured questionnaire for health professionals/medical students was designed by three investigators (ts, hz, and gc) based on the university examination data bank of emergency medicine, preventive medicine, and health management, as well as published literatures [11, 20] . after two rounds of discussion among the investigators and three rounds of discussion with external experts, final version of the questionnaire was made of three sections. the first section included demographic information such as age, gender, educational level, medical profession, and disaster rescue experience. the second section contained 16 multiple-choice questions (q1-q16) as a knowledge test covering various aspects of disaster medicine. in this section, participants could get one score for each correctly answered question and zero for an incorrect answer. the full score was 16. the third section had 5 multiple-choice questions regarding the training needs of disaster medicine. the first questionnaire is presented as questionnaire s1. based on this questionnaire, we designed the second questionnaire for community residents (questionnaire s2). the second questionnaire had 11 multiple-choice questions (q1-q11) as a knowledge test. eight questions were included in both questionnaires due to their importance in disaster medicine (table s1) . before each survey, trained research assistants would give detailed instructions. the participants were then asked to finish the questionnaire independently. informed consent was initially distributed to every candidate study subjects to help them make a fully voluntary decision on participating or declining. participants who provided their written informed consent were included in this study. the study protocol conformed to the 1975 declaration of helsinki and was approved by the ethics committee of second military medical university. descriptive statistics were conducted for demographic characteristics. differences in categorical variables were determined using the chi-square test. analysis of variance (anova) was used to compare the total scores on average among different participants. student-newman-keuls (snk) test was used to correct for multiple comparisons. multivariate linear regression was used to analyze the factors contributing independently to the knowledge score. a beta coefficient was calculated to indicate the effect of each independent variable on the score. all tests were two-sided and conducted using spss version 16.0 (spss, chicago, il). a p value of ,0.05 was defined as statistically significant. a total of 547 (91.2%) health professionals, 456 (91.2%) medical students, and 1,526 (95.4%) community residents provided complete information. of the 2,529 participants, 1,315 (52.0%) were men and 2,093 (82.8%) were younger than 50 years. table 1 shows the demographic characteristics. most of the health professionals had a bachelor's degree or higher in contrast to community residents (74.6% vs. 23.4%). health professionals were composed of 380 (69.5%) medical practitioners, 65 (11.9%) medical teachers, and 102 (18.6%) health administrators. of the 380 medical practitioners, 147 were clinicians, 134 were public health physicians, 77 were nurses and the remaining 22 were medical technicians. the professional titles of health professionals were research assistant (14.0%), senior research assistant (45.3%), assistant professor (33.1%), associate professor (5.1%), and full professor (1.8%). of medical students, 236 (62.7%) majored in clinical medicine and 170 (37.3%) majored in public health. among community residents, 52.0% had no stable employment or retired. of all participants, 197 (7.8%) had disaster relief experience and 33 (1.3%) had ever received systematic training of disaster medicine. for all 2,529 participants, most of them (87.1%) had low or moderate self-estimated knowledge concerning disaster medicine, and media (newspaper, magazine, internet, and tv/radio) was the most common channel to acquire knowledge on disaster medicine. table 2 depicts the correct answer rates to the 16 questions (q1-q16) in the knowledge test using the first questionnaire. the questions were correctly answered by .50% of the professionals and students except q14, q15, and q16. average total score of the knowledge test was 11.00 (95% ci = 10.80-11.21) for health professionals and 11.07 (10.86-11.27) for medical students (p = 0.661) ( figure 1a ). although the score of the two populations was not significantly different, there were significant differences in correctly answering individual questions: q3, q4, q9, q12, and q13 ( figure 2a ). in health professionals, the score was 10.97 (10.73-11.21), 11.89 (11.33-12.45), and 10.57 (10.10-11.04) for medical practitioners, medical teachers, and health administrators, respectively (p = 0.002 for the comparison of three groups) ( figure 1b ). for pairwise comparison, snk test showed that medical teachers' average score was significantly higher than medical practitioners' (p = 0.010) and health administrators' (p = 0.001), while there was no statistically significant difference between medical practitioners and health administrators (p.0.05). the rates of correctly answering 9 questions (q2, q4, q5, q9, q10, q11, q12, q13, and q16) were significantly different among medical practitioners, medical teachers, and health administrators (p,0.05) ( table 2) . for example, in answering q5, medical teachers did better than medical practitioners (p = 0.004) and health administrators (p,0.001). moreover, the knowledge level was also significantly different among clinicians, public health physicians, nurses, and medical technicians, especially in correctly answering 5 questions (table s2) . clinicians performed better than public health physicians (p,0.001) ( figure s1 ). in medical students, the score in public health students (11.54, 95% ci = 11.28-11.80) was higher than that in clinical medicine students (10.79, 10.51-11.07) (p,0.001) ( figure 1c ). the rates of correctly answering 8 questions (q3, q4, q5, q6, q7, q8, q13, and q15) were significantly different between the students of 2 majors (p,0.05) ( table 2) . table 3 shows the rate of right responses to the 11 questions (q1-q11) in community residents. the questions were correctly answered by .50% of community residents except q4 and q5. after stratified by educational level, the score of well-educated (bachelor or higher) group (7.42, 7.20-7.65) was significantly higher than that of poor-educated (junior college or lower) group (6.91, 6.80-7.03) (p,0.001) ( figure 1d ). the rates of correct answers to 7 questions (q2, q4, q5, q7, q8, q9, and q10) were significantly different between the two groups (p,0.05). we compared the rates of correctly answering the 8 common questions in both questionnaires (table s1 ) between health professionals and community residents. the rates were generally figure 1 . comparison of the total score on average of disaster medicine knowledge test. a. health professionals and medical students: no significant difference (p = 0.661); b. three groups of health professionals: total score on average of medical teachers was significantly higher than that of medical practitioners (p = 0.010) and health administrators (p = 0.001); c. medical students of two majors: total score on average of public health students was significantly higher than clinical medicine students (p,0.001). d. community residents of different educational levels: total score on average of those with high education background was significantly higher than those without (p,0.001). doi:10.1371/journal.pone.0067041.g001 lower in community residents than in health professionals (57.8% vs. 72.4%) except q5 ( figure 2b ). multivariate linear regression analysis indicated that educational level (b = 0.204, p,0.001) and professional title (b = 0.142, p = 0.008) were significantly associated with an increased knowledge score, whereas age was inversely related to the score (b = 20.193, p,0.001), in health professionals. educational level was the unique factor significantly associated with an increased score in community residents (b = 0.214, p = 0.001). public health major was the factor significantly associated with an increased score in medical students (b = 0.661, p = 0.002). a. health professionals vs. medical students: p,0.05 for q3 'self-rescue measures in an earthquake', q4 'triage and treatment priority', q9 'concept of first aid abc', q12 'tourniquet hemostasis', and q13 'skills of psychological assistance in post-disaster relief'; b. health professionals vs. community residents: p,0.001 for q4 'cardiopulmonary resuscitation procedure', q5 'difference between remote and urban rescue', q8 'self-rescue measures in an earthquake', q9 'location of temporary toilets during disaster rescue', q10 'skills of psychological assistance in post-disaster relief', and q11 'epidemic prevention strategies after a disaster' and p,0.05 for q6 'fracture fixation and transport' and q7 'self-rescue measures in a high-rise fire'. doi:10.1371/journal.pone.0067041.g002 table 4 depicts the training needs of health professionals and medical students. the overall opinions on teaching method, course arrangement, and teaching material were consistent among the two groups. more than half of these participants selected 'lecture', 'practical training', and 'disaster movies or videos' as preferred teaching methods. most participants chose 'required course for public health professional' as the major training course, and preferred using 'national unified textbook' as standard teaching material. however, medical teachers considered that 'practical training' and 'disaster movies or videos' were not appropriate for teaching disaster medicine, in contrast to medical practitioners and health administrators. most health administrators believed that disaster medicine training should be a required training subject not only for public health professionals but also for clinicians. table 5 shows disaster medicine training needs of community residents. the majority (88.5%) selected 'need to learn disaster medicine' and 'need of disaster medicine course for children'. about half of community residents selected 'lecture' and 'practical training' as preferred teaching methods. more than 70% of community residents selected 'willing to participate in disaster simulation drill regularly' and believed that 'community volunteer team for disaster relief should be set up and willing to participate volunteer team'. compared to community residents with lower educational level, those with higher education background considered that 'systemic study' was more appropriate for teaching (54.3% vs. 43.6%, p,0.001). figure 3 presents the key contents concerning disaster medicine training prioritized by health professionals, medical students, and community residents. more than 50% of health professionals and medical students selected the contents of 'first aid skills', 'epidemic prevention and control', 'psychological problems in post-disaster relief', and 'principles of disaster disposal' as important contents; while most community residents chose 'first aid skills' and 'basic concepts of disaster medicine' as important contents. significant differences existed among subgroups within each group of participants. for example, compared to medical practitioners, medical teachers considered that 'triage and evacuation' was less important (32.2% vs. 50.8%, p = 0.022) (table s3) . twenty-five items covering most aspects of disaster medicine were provided for the selection of interested training contents (table s4 ). figure 4 presents the most interested contents of disaster medicine training prioritized by health professionals, medical students, and community residents. health professionals selected 'basic principles of disaster rescue' (74.0%), 'treatment principles and first-aid skills' (69.8%), and 'psychological relief' (64.4%) as the most interested contents, while community residents selected 'basic principles of disaster rescue' (47.9%) and specific disaster events such as 'earthquakes' (40.9%) and 'fire disaster' (40.8%). in this study, we evaluated the current knowledge levels and training needs of disaster medicine among health professionals, medical students, and community residents in shanghai, china. in general, our results reflected a high vulnerability of our populations when facing disaster. the knowledge level of disaster medicine was not satisfactory in health professionals except medical teachers. although the majority of the health professionals received formal medical education, few of them have ever received systematic training of disaster medicine (table 1) . for health professionals and medical students, less accurate responses to q14, q15, and q16 (table 2) indicate the low levels of knowledge on disaster psychology and disaster administration. the two components have been long neglected and should be added to disaster medicine training and specially addressed to these involved in psychological relief and administrative tasks. lack of knowledge regarding ptsd is an issue needs to be particularly addressed. because of the cultural perception in the chinese society, psychological health hasn't been widely accepted as a critical component in traditional medical and public health education. even though there is a rising awareness of its indispensible importance in recent years [1] , relevant educational program and public health campaign are still lagging behind. in health professionals, the significant differences among different professions ( figure 1) were mainly presented in their answers to the 9 questions covering 4 aspects: self-help and first-aid skills, triage and evacuation, psychological relief, and population vulnerability assessment. health administrators did not show their proficiency in disaster administration and disaster rescue organization, for they poorly answered the related questions such as q5. leadership training programs could effectively improve the emergencyhandling capability of health administrators who might be involved in disaster rescue [25, 26] . moreover, there were significant differences in knowledge levels among 4 specialties (clinicians, public health physicians, nurses, and medical technicians) of medical practitioners. clinicians showed higher knowltable 5 . training needs of community residents and their differences between the 2 educational level groups (number, %). edge level than other specialties, even on the aspect of epidemic prevention and control (table s2 and figure s1 ), which is one of the major tasks of public health physicians. the differences in the knowledge level indicate that the medical education in china had been largely clinically oriented; and little attention has been paid to public health preparedness, especially disaster preparedness. future training plans should clearly define the roles of public health physicians and health administrators in disaster rescue and enhance their capabilities to meet up-to-date requirements [27] . the main reason of the lack of disaster medicine knowledge for health professionals might be that disaster medicine has rarely been included in medical school curriculum and continuing medical education, and no appropriate public health programs focusing on disaster preparedness. surprisingly, public health students showed a higher knowledge level than clinical medicine students (p,0.001) ( figure 1c ). after the sars outbreak, the importance of public health preparedness has been emphasized with a curriculum restructure for public health major students. in addition to the traditional courses such as epidemiology, training programs for public health preparedness such as health management has been added as the main courses for public health major in some medical schools. however, disaster medicine is being developed as a training course in only a couple of medical schools in china. our results indicate that future public health physicians are expected to perform better in disaster rescue. interestingly, the knowledge level of health professionals was inversely related to age, which is in contrast to the general belief that older professionals have more experiences and therefore more knowledgeable. one possible explanation is that the young are more likely to have frequent access to modern media such as the internet and thus gain 'exposure' to updated information on disaster medicine. community residents displayed very poor knowledge and skills of disaster medicine. not surprisingly, community residents generally lacked specialty knowledge such as 'cardiopulmonary resuscitation procedure' and 'difference between remote and urban rescue' (table 3 and figure 2b ). an important finding is that community residents with higher education background had higher knowledge level of disaster medicine than those without ( figure 1d ). thus, it is urgent to tailor community training programs for the residents with different education background and popularize disaster medicine education via modern media. this study also pointed out the training needs of disaster medicine. most participants selected 'lecture' and 'practical training' as preferred teaching methods. most health professionals and medical students suggested that disaster medicine should be a 'required course for public health professional' and asked for a 'national unified textbook' as standardized teaching material. most community residents believed 'need to learn disaster medicine' and 'need of disaster medicine course for children', and selected 'willing to participate in disaster simulation drill regularly' and 'community volunteer team for disaster relief should be set up, and willing to participate volunteer team' (table 4, table 5 ). these results indicate that the training needs of disaster medicine is very high in chinese society and disaster medicine trainings should be executed as indispensable courses for health professionals, medical students, and community residents. meanwhile, the three groups of participants selected some different key and interested contents for disaster medicine training (figure 3 and figure 4 ). this reflects that distinct perception of disaster determines the different needs of disaster medicine training in different populations. similar differences in several items of the training needs were also presented among the subgroups of study participants. training programs such as disaster simulation and disaster exercise have proven to be effective and can rapidly deliver core elements of disaster medicine and improve the knowledge level and ability of disaster response [10, 28, 29] . therefore, future continuing disaster medicine education should focus on developing practice-oriented and core elements-highlighted training courses. except the high-level interests in 'basic principles of disaster relief', there were some differences of interested contents among different populations, indicating future training program design should consider both core elements and interests, and customize to different needs. as medical teachers were more knowledgeable in disaster medicine than other populations surveyed ( figure 1b) , they should play a leading role in disaster medicine training. based on these data, we suggest a diagram flow of disaster medicine training as the shanghai model in figure s2 . the present survey was conducted in shanghai, one of the areas with well developed economy and affluent medical resources in china. after further evaluation, the shanghai model of disaster medicine training suggested in this study should be validated and generalizable to other developing areas where the problem of unmatched economic development and disaster medicine education also exist. these data also provide useful evidence to help developing disaster medicine training plans in other developing world. the current study had limitations. our community participants were from one district (yangpu) in shanghai chosen by cluster sampling. sample sizes may influence results if comparing subgroups within clusters. furthermore, other groups of disaster first responders such as firefighters and military personnel were not included in the current survey. future studies focusing on these special groups will provide valuable information for disaster preparedness. in conclusion, this large epidemiological study provided important data concerning knowledge level and training needs among the populations that would be involved in disaster rescue or affected by disasters. from a health education perspective, disaster training programs are urgently needed, with specific emphasis on certain contents, such as psychological relief and administrative skills. our study enables a more comprehensive evaluation of current disaster preparedness situation and facilitates designing future disaster medicine training programs in china and other developing countries. figure s1 comparisons of the total scores on average and rates of correctly answering 5 important questions among clinicians, public health physicians, nurses, and medical technicians. a. comparison of average scores; b. comparison of correct answer rates. (tif) figure s2 suggested diagram of disaster medicine training (shanghai model). table s1 list of the same questions in two questionnaires. (doc) questionnaire s1 questionnaire for health professionals and medical students. questionnaire s2 questionnaire for community residents. (doc) mental health problems among the survivors in the hard-hit areas of the yushu earthquake resilience in the face of disaster: prevalence and longitudinal course of mental disorders following hurricane ike relationships between traumatic symptoms and environmental damage conditions among children 8 months after the 2011 japan earthquake and tsunami impact of the deepwater horizon oil spill on a deep-water coral community in the gulf of mexico incidences, types, and influencing factors of snow disaster-associated injuries in ningbo, china how china responded to the may 2008 earthquake during the emergency and rescue period disaster medicine training in family medicine: a review of the evidence healthcare worker competencies for disaster training factors influencing collaborative activities between non-professional disaster volunteers and victims of earthquake disasters disaster 101: a novel approach to disaster medicine training for health professionals perspectives of future physicians on disaster medicine and public health preparedness: challenges of building a capable and sustainable auxiliary medical workforce medical student disaster medicine education: the development of an educational resource a disaster medicine curriculum for medical students european survey on training objectives in disaster medicine the sars epidemic in mainland china: bringing together all epidemiological data analysis of injuries and treatment of 3,401 inpatients in 2008 wenchuan earthquake-based on chinese trauma databank current perspectives on medical education in china medical education in china for the 21st century medical education and medical education research and development activities in modern china need for continual education about disaster medicine for health professionals in china-a pilot study development and evaluation of an undergraduate training course for developing international council of nurses disaster nursing competencies in china pilot training program for developing disaster nursing competencies among undergraduate students in china china-australia training on psychosocial crisis intervention: response to the earthquake disaster in sichuan evaluating the effectiveness of an emergency preparedness training programme for public health staff in china development and evaluation of a leadership training program for public health emergency response: results from a chinese study does leadership training make a difference? the cdc/uc public health leadership institute: 1991-1999 an estimation of canada's public health physician workforce a simulation-based biodefense and disaster preparedness curriculum for internal medicine residents what a disaster?! assessing utility of simulated disaster exercise and educational process for improving hospital preparedness key: cord-260572-vd65ygtm authors: kim, curi; ahmed, jamal a.; eidex, rachel b.; nyoka, raymond; waiboci, lilian w.; erdman, dean; tepo, adan; mahamud, abdirahman s.; kabura, wamburu; nguhi, margaret; muthoka, philip; burton, wagacha; breiman, robert f.; njenga, m. kariuki; katz, mark a. title: comparison of nasopharyngeal and oropharyngeal swabs for the diagnosis of eight respiratory viruses by real-time reverse transcription-pcr assays date: 2011-06-30 journal: plos one doi: 10.1371/journal.pone.0021610 sha: doc_id: 260572 cord_uid: vd65ygtm background: many acute respiratory illness surveillance systems collect and test nasopharyngeal (np) and/or oropharyngeal (op) swab specimens, yet there are few studies assessing the relative measures of performance for np versus op specimens. methods: we collected paired np and op swabs separately from pediatric and adult patients with influenza-like illness or severe acute respiratory illness at two respiratory surveillance sites in kenya. the specimens were tested for eight respiratory viruses by real-time reverse transcription-polymerase chain reaction (qrt-pcr). positivity for a specific virus was defined as detection of viral nucleic acid in either swab. results: of 2,331 paired np/op specimens, 1,402 (60.1%) were positive for at least one virus, and 393 (16.9%) were positive for more than one virus. overall, op swabs were significantly more sensitive than np swabs for adenovirus (72.4% vs. 57.6%, p<0.01) and 2009 pandemic influenza a (h1n1) virus (91.2% vs. 70.4%, p<0.01). np specimens were more sensitive for influenza b virus (83.3% vs. 61.5%, p = 0.02), parainfluenza virus 2 (85.7%, vs. 39.3%, p<0.01), and parainfluenza virus 3 (83.9% vs. 67.4%, p<0.01). the two methods did not differ significantly for human metapneumovirus, influenza a (h3n2) virus, parainfluenza virus 1, or respiratory syncytial virus. conclusions: the sensitivities were variable among the eight viruses tested; neither specimen was consistently more effective than the other. for respiratory disease surveillance programs using qrt-pcr that aim to maximize sensitivity for a large number of viruses, collecting combined np and op specimens would be the most effective approach. acute respiratory illness (ari) is a significant cause of mortality and morbidity worldwide, especially in young children [1] . viruses play an important role in ari, accounting for up to 90% of lower respiratory tract infections in children , 5 years [2] . a variety of sample collection techniques and specimen sources can be used to detect respiratory etiologies, including nasopharyngeal (np) swabs, oropharyngeal (op) swabs, nasopharyngeal aspirates (npas), nasal swabs, nasal washes, sputa, and saliva specimens. although npas may be the most sensitive specimens, especially when conventional diagnostic methods such as immunofluorescence or culture are used [3] , obtaining an npa is more difficult than obtaining a swab, and collecting npas in an outpatient or field setting may not always be feasible [4, 5] . molecular methods like reverse transcription-polymerase chain reaction (rt-pcr) are becoming widely used for identification of respiratory etiologies [6] . because molecular tests are more sensitive than conventional methods, less invasive specimen collection techniques than npa may now approach comparable yields [5, 7] . depending upon patient characteristics, especially age, obtaining either -or both -np and op swabs can be quite physically challenging. using only one type of swab would be easier logistically, cheaper, and would enable comparisons across surveillance systems. to evaluate the comparative yields of np and op swabs in detecting key respiratory viruses by real-time rt-pcr (qrt-pcr), we conducted a prospective study using paired np and op specimens from patients at two respiratory disease surveillance sites in kenya. ethical approval for the surveillance activities for influenza and other respiratory viruses was obtained from the kenya medical research institute (kemri) ethical review committee (protocol number 1161). after formal human subjects determination, u.s. centers for disease control and prevention (cdc) determined this surveillance activity to be nonresearch and therefore approval was not required from the cdc institutional review board. written informed consent was obtained from adults and from the parents or guardians of minors. the study population consisted of pediatric and adult patients visiting two health-care sites from june 9, 2009 to august 31, 2010, whose illness met the case definition for influenza-like illness (ili) or severe acute respiratory illness (sari). the case definitions for ili and sari (table 1) were adapted from those of the world health organization [8, 9] . the maximum number of eligible ili patients was limited to three per day for each site; there was no limit to the number of sari patients tested. the health-care sites are in the north eastern and rift valley provinces of kenya and are part of a wider national influenza sentinel surveillance system run jointly by the kenya ministry of public health and sanitation and kemri/centers for disease control and prevention-kenya (cdc-k). np and op swabs were separately collected from patients with ili or sari by trained surveillance officers. for the np swab, a polyester-tipped flexible aluminum-shafted applicator (25-801d, puritan, guilford, maine, usa) was inserted into one of the nostrils until resistance was felt at the nasopharynx, then rotated 180 degrees and withdrawn. for the op swab, a nylon flocked plastic-shafted applicator (503cs01, copan diagnostics, murrieta, ca, usa) was used to sample the posterior oropharyngeal mucosal membrane. after swabbing, the swab applicator was cut off, and each absorbent swab was placed into a vial containing 1 ml of viral transport media (vtm). vtm was prepared at the kemri/cdc-k laboratory using standard who protocol [10] . vials were stored at 4uc for up to 72 hours until before shipment to the kemri/cdc-k laboratory in nairobi, where they were stored at 280uc until testing. the specimens were vortexed, and a 100-ml volume was used for total nucleic acid extraction using the qiaamp viral rna mini kit (qiagen gmbh, hilden, germany) according to the manufacturer's instructions. one step qrt-pcr was performed by using the agpath-id one-step rt-pcr kit (applied biosystems, carlsbad, california, usa). np and op specimens from each patient were separately tested by singleplex qrt-pcr for eight viral pathogens: adenovirus, influenza a virus, influenza b virus, human metapneumovirus (hmpv), parainfluenza viruses (piv) 1-3, and respiratory syncytial virus (rsv). the primers, probes and positive controls for all viruses were provided by cdc-atlanta. sequences for the primers and probes are shown in table 2 [11] . we tested for influenza a virus with the conserved matrix gene-base qrt-pcr; positive influenza a samples were also subtyped as 2009 pandemic influenza a (h1n1) virus (2009 h1n1), influenza a (h3n2) virus (h3n2), and seasonal influenza a (h1n1) virus (h1n1) [12] . fluorescence was read at the combined annealing-extension step at 57uc and recorded as threshold cycle (c t ) values. a c t value #39.9 was regarded as positive; c t values $40.0 were regarded as negative. the qrt-pcr test did not discriminate between viral mrna and genomic rna. specimens were not tested if the following conditions existed when the specimen arrived at the lab: there was no swab, the volume was less than 600 ml, the specimen was at room temperature, patient identification was absent or inadequate, or the patient questionnaire was absent. in addition, the test results were discarded for any specimen whose internal control (human ribonuclease p gene) was negative. agreement of the results between the paired np and op specimens was assessed by using the kappa coefficient. we used the following nomenclature to describe the relative strength of agreement associated with kappa statistics: , 0 = poor; 0-0.2 = slight; 0.21-0.4 = fair; 0.41-0.6 = moderate; 0.61-0.8 = substantial; and 0.81-1 = almost perfect [13] . the assessment was carried out separately for each respiratory virus and for each of the influenza a subtypes. similar to previous studies [5, 7] , we assessed the sensitivity for each sampling method by considering any positive from either of the specimens as a true positive. we compared the sensitivities using the mcnemar's test to account for the correlated binary all ages (all of the following): 2. cough or sore throat 3. does not meet criteria for sari for infants ages 1 week to , 2 months (any of the following): n respiratory rate of .60 per minute n severe chest indrawing n nasal flaring (when an infant breathes in) n grunting (when an infant breathes out) for children ages 2 months to ,5 years: outcomes from paired data. to avoid bounds above 100%, we used the exact binomial confidence limits. statistical significance was set at a p-value ,0.05. data were analyzed with sas software version 9.2 (sas institute, cary, nc, usa). during the study period, 2,374 paired np and op swabs were collected. forty-three specimens were rejected because of poor quality. of the 2,331 paired specimens included in the analysis, 754 (32.3%) were from patients with ili and 1,577 (67.7%) were from patients with sari. overall, 1,402 (60.1%) paired specimens were positive for at least one virus, including 393 (16.9%) specimens that were positive for more than one virus. the median number of days from onset of symptoms to specimen collection was 2 days, and 95.9% of samples were collected within 5 days from onset of symptoms (range 0-30 days). the median age of patients was 1 year (range 1 month to 70 years), and 81.6% of patients (n = 1,902) were less than 5 years old (table 3) . fewer than half (46.1%) of the patients were female (n = 1,074). adenovirus was the most commonly identified virus, detected in $1 specimens from 679 (29.1%) patients (table 4) . for all patients combined (ili and sari), the relative sensitivities of the swab types varied by virus ( for all sari patients (n = 1,577), op swabs were significantly more sensitive than np swabs for adenovirus and 2009 h1n1 virus, but np swabs were more sensitive than op swabs for influenza b virus, piv 2, and piv 3. for all ili patients (n = 754), op swabs were more sensitive than np swabs for adenovirus and overall influenza a virus; np swabs were not more sensitive than op swabs for any of the viruses in this illness category ( table 5 ). the relative sensitivities of np and op swabs from children , 5 years old (n = 1,902) mirrored the overall results. when results in this age group were stratified by sari and ili, the comparative sensitivities for np and op swabs reached statistical significance in the same pattern as that for all sari and ili patients. however, for patients aged 5-17 years (n = 344) and for patients 18 years and older (n = 85), neither swab was significantly more sensitive for any of the viruses, including when results were stratified by sari and ili status. for male patients (n = 1,257), specimen performance for all viruses mirrored the overall results. for female patients (n = 1,074), as with the overall results, op swabs were significantly more sensitive than np swabs for influenza a virus and adenovirus, while np swabs were significantly more sensitive than op swabs for piv 2; however, there was no statistical difference by swab type for 2009 h1n1 virus, influenza b virus, and piv 3. there was a substantial agreement (k.0.60) between the np and the op swabs for all viruses except adenovirus, unsubtypable influenza viruses, piv 1, and piv 2 ( table 4) . piv 1 had a moderate strength of agreement (k = 0.59) between swabs, but adenovirus and piv 2 had only a fair strength of agreement (k = 0.33 and 0.39, respectively). unsubtypable influenza viruses had poor strength of agreement (k = 20.68). if only the more table 2 . primers and probes used in this study. (table 4 ). to our knowledge, this is the largest study to use qrt-pcr to compare np and op swabs for a range of respiratory viruses. we found that the relative performance of specimen type varied by virus. neither specimen performed uniformly better: np swabs were more sensitive for some viruses (influenza b virus, piv 2, and piv 3), op swabs were more sensitive for others (overall influenza a virus, 2009 h1n1 virus, and adenovirus), and there was no difference for the rest of the viruses. the large number of patients in this study allowed us to perform comparative statistical analysis with a relatively high degree of precision. for adenovirus, op swabs were more sensitive than np swabs, a finding that was statistically significant in both ili and sari patients. this difference may reflect the fact that the major site of initial replication of adenoviruses is the non-ciliated respiratory epithelium of the oropharynx [14] . the kappa value between np and op swabs for adenovirus was low (k = 0.33). this result is consistent with findings of lambert et al., in which adenovirus accounted for the highest proportion of discordant paired npa and nasal-throat swab specimens from children [7] . adenoviruses include over 50 serotypes, and the low concordance between np and op specimens for these viruses may reflect different cell tropisms of the adenovirus serotypes for different parts of the respiratory tract [15] . although we did not conduct serotyping, future studies that evaluate specimen performance for specific adenovirus serotypes could test this hypothesis. for influenza viruses, sensitivities of np and op swabs differed by both type and subtype: np swabs were more sensitive than op swabs for influenza b virus, while op swabs were more sensitive than np swabs for overall influenza a and 2009 h1n1 virus; there was no significant difference between swabs for h3n2 virus or the unsubtypable influenza a viruses. the sensitivities of np and op swabs for unsubtypable influenza a viruses were low, and the strength of agreement between the two swabs was poor. however, unsubtypable influenza a viruses were likely a mix of 2009 h1n1, seasonal h1n1, and h3n2 viruses, making it difficult to interpret this finding. because half the influenza a specimens were 2009 h1n1, the overall influenza a results were biased towards the 2009 h1n1 findings. previous studies evaluating np and op swabs in detecting influenza viruses found np swabs to be more sensitive than op swabs, but these studies used combined outcomes for influenza a and b viruses and did not analyze by influenza a subtypes [6, 16, 17] . the difference in sensitivities of the two swabs in our study may reflect different affinities of influenza types and subtypes for different locations in the respiratory tract. while all influenza viruses infect the respiratory epithelium from the nasopharynx to the bronchioles, 2009 h1n1 virus (and h5n1 virus, which we did not find in our study) can infect lower parts of the respiratory tract, including the alveoli, more commonly than seasonal influenza [18] . this difference could account for the better sensitivity of op swabs, which reach deeper into the respiratory tract than np swabs, for 2009 h1n1 virus. of note, op swabs have been shown to have superior yield over np swabs for human cases of avian influenza a (h5n1) [19, 20] . the op swab sensitivities and kappa values of the parainfluenza viruses were relatively low, with the sensitivity of the op swab for piv 2 being the lowest of any virus in our study. this preference for np swabs is consistent with reports that nasal washes and nasal aspirates have yielded the highest rates of viral recovery for piv [21] . this study had several limitations. first, we compared only np and op swabs; although many routine surveillance systems for the percentage of cases that would have been missed if only the more sensitive swab had been used for viruses which had significant differences in sensitivities between swabs. influenza and respiratory diseases collect np and/or op swabs, other surveillance systems collect npas, np washes, or nasal swabs [22] . while recent studies have used pcr to compare the relative yield of npas with those of nose-throat swabs or nasal swabs for respiratory viruses, no studies have evaluated more than three sampling techniques [5, 7, 23, 24] . head-to-head studies in the future comparing np swab, op swab, npa, np wash, nose-throat swab, nasal swab, and nasal wash specimen types would provide important information for decisions about which specimens to use for respiratory disease surveillance systems. second, the np and op swabs consisted of different kinds of swab material and used different designs. we used conventional polyester swabs for sampling the nasopharynx and flocked nylon swabs for the oropharynx. although flocked swabs are superior to conventional swabs for cell recovery, a study comparing different swab material and design (rayon versus nylon flocked swabs) in both the nasopharynx and oropharynx found that the difference in the cycle threshold values between sampling sites was much greater than the difference between swab material and design [25] . because neither specimen type was consistently more sensitive than the other, we think it is unlikely that the difference in swab material and design substantially affected our results. additionally, while we tested for eight viruses, we did not include some common viruses, such as coronaviruses and rhinoviruses, and we did not test for bacteria. finally, the number of adults (n = 85) in this study was relatively small, accounting for just 3.6% of all patients, and as a result there was limited power to compare the sensitivities of np and op swabs for specific viruses in this population. in summary, np and op specimens collected from patients with respiratory illness had variable sensitivities by qrt-pcr for eight viruses. neither specimen was consistently more sensitive than the other. collecting both swabs had a complementary effect; even when there was higher sensitivity for one technique over the other, the lower-sensitivity technique still identified a considerable number of cases not identified by the higher-sensitivity one. for respiratory disease surveillance programs using qrt-pcr that aim to maximize sensitivity for a large number of viruses, collecting combined np and op specimens would be the ideal approach. however, the enhanced sensitivity of using both swabs comes at a higher cost; this includes not only the expense of the second swab, but further patient discomfort as well as additional time and effort from the person taking the sample. thus, for surveillance systems with limited resources, a single-swab approach, whether np or op, would be the most logistically simple and maintain moderate sensitivity for many of the pathogens we tested in our study. the percentage of cases that would have been missed if only the more sensitive swab had been used for viruses which had significant differences in sensitivities between swabs. estimates of world-wide distribution of child deaths from acute respiratory infections viral etiology of acute respiratory tract infections in children presenting to hospital: role of polymerase chain reaction and demonstration of multiple infections optimal sampling sites and methods for detection of pathogens possibly causing community-acquired lower respiratory tract infections comparison of nasopharyngeal flocked swabs and aspirates for rapid diagnosis of respiratory viruses in children comparison of combined nose-throat swabs with nasopharyngeal aspirates for detection of pandemic influenza a/h1n1 2009 virus by real-time reverse transcriptase pcr identification of respiratory viruses in adults: nasopharyngeal versus oropharyngeal sampling comparing nose-throat swabs and nasopharyngeal aspirates collected from children with symptoms for respiratory virus identification using real-time polymerase chain reaction handbook: imci integrated management of childhood illness who regional office for europe guidance for influenza surveillance in humans collecting, preserving and shipping specimens for the diagnosis of avian influenza a(h5n1) virus infection. guide for field operations application of taqmanh low density arrays for simultaneous detection of multiple respiratory pathogens cdc protocol of realtime rtpcr for influenza a(h1n1) the measurement of observer agreement for categorical data adenoviruses adenoviruses comparison of four clinical specimen types for detection of influenza a and b viruses by optical immunoassay (flu oia test) and cell culture methods use of throat swab or saliva specimens for detection of respiratory viruses in children comparison of the pathology caused by h1n1, h5n1, and h3n2 influenza viruses three indonesian clusters of h5n1 virus infection in 2005 avian influenza a (h5n1) infection in humans parainfluenza viruses a practical guide to harmonizing virological and epidemiological influenza surveillance detection of multiple respiratory pathogens during primary respiratory infection: nasal swab versus nasopharyngeal aspirate using real-time polymerase chain reaction comparative study of nasopharyngeal aspirate and nasal swab specimens for diagnosis of acute viral respiratory infection swabbing for respiratory viral infections in older patients: a comparison of rayon and nylon flocked swabs the authors wish to acknowledge the contributions of the field surveillance officers and the kemri/cdc laboratory personnel without whom this work would not have been possible. we especially want to thank dennis odhiambo and hashim m. haji. we acknowledge steven lindstrom and alexander klimov from the influenza division of cdc-atlanta for contributing the primers and probes and the pcr testing protocol. we also thank drs vincent kahi, james ndirangu and millhia abul kader of the international rescue committee, kenya. this manuscript is published with permission of the director of the kenya medical research institute. the findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the centers for disease control and prevention. key: cord-276372-q1fzxt3r authors: conde, dalia a.; colchero, fernando; gusset, markus; pearce-kelly, paul; byers, onnie; flesness, nate; browne, robert k.; jones, owen r. title: zoos through the lens of the iucn red list: a global metapopulation approach to support conservation breeding programs date: 2013-12-11 journal: plos one doi: 10.1371/journal.pone.0080311 sha: doc_id: 276372 cord_uid: q1fzxt3r given current extinction trends, the number of species requiring conservation breeding programs (cbps) is likely to increase dramatically. to inform cbp policies for threatened terrestrial vertebrates, we evaluated the number and representation of threatened vertebrate species on the iucn red list held in the isis zoo network and estimated the complexity of their management as metapopulations. our results show that 695 of the 3,955 (23%) terrestrial vertebrate species in isis zoos are threatened. only two of the 59 taxonomic orders show a higher proportion of threatened species in isis zoos than would be expected if species were selected at random. in addition, for most taxa, the management of a zoo metapopulation of more than 250 individuals will require the coordination of a cluster of 11 to 24 isis zoos within a radius of 2,000 km. thus, in the zoo network, the representation of species that may require cbps is currently low and the spatial distribution of these zoo populations makes management difficult. although the zoo community may have the will and the logistical potential to contribute to conservation actions, including cbps, to do so will require greater collaboration between zoos and other institutions, alongside the development of international agreements that facilitate cross-border movement of zoo animals. to maximize the effectiveness of integrated conservation actions that include cbps, it is fundamental that the non-zoo conservation community acknowledges and integrates the expertise and facilities of zoos where it can be helpful. the conservation status of known biodiversity has undergone a worrying decline in the last few decades [1, 2] . if the present trends continue, the conservation community will be challenged with a large number of species for which there is no viable conservation outcome. as a result, conservation breeding programs (cbps) may offer the only feasible option to avoid the extinction of particular species until appropriate habitat can be found or restored [3] [4] [5] . the need for well-managed cbps, including those supported by the zoo community, has never been greater, but the space and logistical demands for managing cbps could exceed the current capacity of the zoo community [1, 5] . although we know that 15% of threatened vertebrate species are represented in zoos [3] , we still do not have an overview of the number and distribution of these species within the global zoo network. therefore, to establish effective responses to species' cbps, it is important to determine the current representation of threatened species within zoos and have a general understanding of the complexity of managing them within the global zoo network. the development of cbps is challenging and has received criticism due to problems including hybridization risk, high costs and the diversion of resources away from habitat protection [6] [7] [8] [9] . because of these factors, cbps have usually been considered only as a last resort and, as a result, are normally only implemented when populations dwindle to fewer than 20 individuals (e.g. whooping crane, grus americana [10] ). still, cbps have played a major role in 13 of the 68 species that have shown a status improvement in iucn red list reassessments [11, 12] . moreover, for mammals, captive breeding together with reintroduction programs and hunting restriction have been the most effective conservation actions, since ensuring protected areas alone has not been enough [13] . as a result, in most cases effective conservation plans require the integration of a range of management options where cbps could be necessary. for example, martin et al. [14] compared the failure of conservation intervention for the recently extinct christmas island pipistrelle (pipistrellus murrayi) with the potentially successful intervention in the case of the orange-bellied parrot (neophema chrysogaster), where conservation actions, including cbps, were implemented in a timely manner and coordinated among several institutions. as threats to biodiversity increase, the management of at-risk species requires a spectrum of interventions that can range from habitat protection to the establishment of cbps with the eventual aim of reintroduction into the wild [3, 4] . therefore, differentiating between in situ and ex situ management is becoming irrelevant [4, 15] . instead, a continuum of management practices exists, which ranges from truly wild and self-sustaining populations to managed populations dependent on a certain level of human care for persistence. for example, the iucn ssc conservation breeding specialist group has recently launched an ambitious conservation framework called the one plan approach, which promotes participation between different institutions and stakeholders with the aim to consider all populations of a particular species, both inside and outside their natural range, under a unified management plan [16] . the zoo community is in an ideal position to support and further contribute to develop such conservation programs because they are involved in both captive breeding and field conservation programs. for example, zoos have extensive knowledge of the husbandry, behavior and veterinary procedures required to develop cbps [17] , and members of the world association of zoos and aquariums (waza) are, collectively, the third largest financial supporter of species conservation in their natural habitats (providing us$350 million/year), while also being actively involved in many of those projects [18] . to understand the space devoted to threatened species and the potential complexity of managing cbps as zoo-held metapopulations, we evaluated the representation of the iucn red list threatened species held within the zoo network. we used data published from conde et al. [3] , processed from the international species information system (isis) organization. isis is a network of more than 800 zoos and aquariums that shares information about ,2.5 million individuals among the member institutions. this information system makes it possible to analyze the amount of space devoted to threatened species and its spatial distribution. therefore, isis is a key institution to assess the potential of zoos to develop cbps. to understand if the number of threatened species in zoos is the result of a sound prioritization or if it has been more an opportunistic process, we analyzed whether the representation of threatened species per taxonomic order is significantly higher to what would be expected if species were randomly collected (i.e. without collection planning). in addition, to appraise the approximate complexity of managing zoo-held species as metapopulations, we estimated the distance between zoos clusters at which the probability of reaching an average metapopulation size of 50, 100 and 250 individuals is maximized. with these analyses we can better infer how complex it is to reach increasing population sizes between isis zoos, assuming that cbps could be more successful when population sizes are larger and clustered in zoos at closer proximity. since the clustering of zoos could facilitate special treaties to move individuals across borders. based on our results, we discuss the potential of zoos to contribute towards cbps for terrestrial vertebrates and make some policy recommendations. to determine whether the number of threatened species in isis zoos is different from what would result from a random sample, we carried out the following analyses. for every taxonomic order i of terrestrial vertebrates there is a given number n i of species worldwide, out of which a fraction m i is known to be threatened. similarly, we know the number of species for every order z i that are represented in isis zoos and the subset w i of those that are threatened. we developed a monte carlo algorithm to understand which orders have a number of threatened species in isis zoos significantly different from a random sample of species in the wild. this algorithm was based on an iterative procedure that, at every step s, randomly sampled, without replacement, z i species within every order from the worldwide list. from these randomly sampled species, the algorithm counted the number of speciesŵ w i,s classified as threatened. the algorithm then calculated the indicator y i, j,s that assigned 1 if a number j of threatened species were sampled and 0 otherwise, such that j = 0, …, min(z i , m i ). the probability of randomly finding j threatened species is calculated as where s is the total number of iterations. since the algorithm ran for several thousand iterations, we were able to construct a distribution (i.e. empirical probability mass function, pmf) of the number of threatened species per order that could have been found in isis zoos if they had sampled species at random from the wild. the probability in the pmf that corresponds to the real number of threatened species in zoos ( p i,j : w i~j ) was used as an analogue to the p-value. those orders that had a number of threatened species that matched the lower bounds of the pmf (i.e. p-value of 0.05) were classified as being significantly underrepresented. similarly, those that matched upper bounds of the pmf (i.e. above the 0.95 quantile) were catalogued as being overrepresented. the remaining species could not be distinguished from a random sample. to understand the complexity of maintaining cbps of threatened species across the isis zoo network, we developed a second algorithm to find the optimal radial distance from any given zoo at which the probability of finding a metapopulation size of at least 50, 100 or 250 recruited living individuals was highest relative to that radial distance. the algorithm was based on a monte carlo procedure that, at each step s, a zoo j was selected at random, then found all zoos z j,r located at a distance r from zoo j, as well as the number of zoos z i j,r within that radial distance that held species i. we labeled the zoos included within that radius as a cluster, k j,r . the algorithm then counted how many individuals of a given threatened species i were included in the cluster, such that where n i,k is the population size for species i in zoo k. at each cluster, we assigned the indicator where m is a pre-established metapopulation threshold (i.e. 50, 100 or 250 individuals). we chose 50, 100 and 250 individual metapopulation thresholds because a population size of 50 has been historically considered as a minimum viable population [19] , and 250 individuals is the threshold defined by the iucn red list as a ''very small wild population'' in the critically endangered category [20] . moreover, very few threatened species in isis zoos have populations for which a higher threshold is possible. for instance, only 44 species (6.3%) have more than 1,000 individuals (see below). we considered 100 individuals as an arbitrary intermediate between these two values. this procedure was repeated for 2,000 iterations and then the algorithm was repeated, increasing the radial distance by 100 km until a maximum radius of 10,000 km was reached. from the 2,000 iterations for each radius, the algorithm calculated the probability of finding at least m individuals for species i and radius r as where s is the total number of steps (2,000) in the monte carlo procedure. here, we replaced the subscript j with s to indicate that zoos were chosen at random with replacement. similarly, we calculated the average number of zoos for each species at each distance interval as z z r~1 s x s s~1 z s,r : we repeated this procedure for radii ranging from 0 to 10,000 km, using 100 km increments. we excluded species for which the minimum population size was never reached, even when including all isis zoos. for each class c and metapopulation threshold m, we calculated the average ratio between probability p i,r,m and distance d r for radius r as where i c is the total number of threatened species in class c that was included in the analysis. finally, we found the optimal cluster, this is the optimal radius dã c,m and the optimal number of zoos zã c,m , where the ratio r c,r,m was highest for each class and population threshold m. these optimal clusters imply that, relative to that radius, the probability of reaching a metapopulation of at least m individuals is highest. for simplicity, we present the results pooled by taxonomic class. collectively, in 2011, the 837 isis zoos held 3,955 species of non-domestic terrestrial vertebrates (table 1) . of these, more than half (58%) were birds, one quarter (25%) were mammals, 11% were reptiles and 6% were amphibians ( figure 1 ). twenty-three percent (691/3,955) of the species in isis zoos belong to a threatened category (table 2) . within each class, the percentage of threatened species varies widely among each order, ranging between 8% for birds (195/2,308) and 27% for mammals (262/ 978). isis zoos held a total of 455,317 individuals of non-domestic terrestrial vertebrates, of which 22% (91,063/455,317) belong to a threatened species (table s1 ). our analysis of the number of threatened species in isis zoos, broken down by order, shows that most collections are not distinguishable from what would be expected if the species were selected at random (figure 2 , tables s2, s3, s4, s5). exceptions occur in mammals in the order dasyuromorphia (australian carnivorous marsupials) and in reptiles for testudines (turtles): isis zoos hold 50% and 79%, respectively, of the order's threatened species. on the other hand, threatened species were under-represented in zoos for the mammalian orders eulipotyphyla (insectivores) and rodentia (rodents). for birds, threatened species were under-represented in nine of the 25 orders held in zoos, whereas for amphibians this was only the case for caudata (salamanders). for the threatened amphibian species in isis zoos, 27% reach a metapopulation size threshold of .50 individuals. almost half of the threatened species of mammals, birds and reptiles in isis zoos reach the same threshold (44%, 47% and 43%, respectively), and 18% of the threatened mammals reach a threshold above 250 individuals (table 3) . however, many of those species are distributed among numerous zoos, thus if those species are not managed as a metapopulation, their conservation potential will be greatly reduced. as expected, the optimal distance radii and the number of zoos required to maximize the probability of reaching a given population increase with the metapopulation size threshold (.50, .100 and .250 individuals) ( figure 3) . however, the magnitude of the increase varies among classes; for birds and reptiles, the difference between optimal distances is just 200 km and 300 km, respectively, while for mammals and amphibians, the differences are up to 900 km (table 4) . furthermore, at the optimal radial distances, the probability that a cluster of zoos has more than 50, 100 or 250 individuals of a given threatened species ranges from 0.16 to 0.42 (table 4) . however, only 3.5% to 10.2% of zoos within an optimized cluster have individuals of the species. moreover, these percentages vary considerably among the four classes of terrestrial vertebrates. birds and mammals make up the largest proportion of terrestrial vertebrates held in zoos. although the majority of zoo collection plans were not originally focused on holding threatened species [21] , 23% of their collections are currently devoted to them. however, for most of the taxonomic orders, our results show that representation of threatened species is not different from what would be expected if species were selected at random. broken down by class, it is clear that threatened birds make up the lowest proportion in the zoo network. this may be explained by factors including: i) some species are difficult to breed in captivity (e.g. seabirds), ii) some species have specialized dietary requirements (e.g. insectivores), and iii) import/export restrictions, such as those added in the wake of the sars (severe acute respiratory syndrome) epidemic, which make it challenging to manage birds across borders and to import new species. mammalian and reptilian zoo collections include the highest proportion of threatened species; however, only the orders dasyromorphia and testudines are significantly over-represented. the dasyromorphia are a particular focus of cbp efforts in australian zoos [22] . however, despite the interest in this group, six threatened dasyromorphia species are not yet represented in zoos. the high representation of threatened testudines is partly because many zoos serve as rescue centers for confiscated individuals. the turtle survival alliance has played a key role in promoting the conservation of turtles and has been working on linking zoos and governmental institutions to ensure the rescue of animals from the illegal trade [23] . however, since many of those individuals come from the illegal trade, it is hard to include them as part of cbps because, in many cases, their origins are uncertain [23] . for amphibians, the relatively low proportion of threatened caudata in zoos may reflect their cryptic behavior and small size [24] , which makes them difficult to display. in general, the small number of amphibian species may also be due to practical issues such as the difficulty in obtaining permits to transport individuals. although amphibian collections in zoos and zoo-supported centers have significantly increased in the last 10 years [12, 25] , zoos still only hold 3% of the world's threatened amphibians [3] . this emphasizes the need for zoos to increase their contribution towards amphibian cbps either as part of their collections or by further contributing to the development of breeding centers in their local areas [26] . with this in mind, the amphibian ark emerged with a strong zoo component with the mission of ensuring the global survival of amphibians, focusing on those that cannot currently be safeguarded in nature and where zoos can play a key role [27] . zoos hold ,15% of the world's threatened terrestrial vertebrates [3] . however, in their collections, 23% of isis zoos species (691/3,955) belong to a threatened category. nevertheless, most of their populations are small and are distributed across the zoo network. therefore, for the zoo community, one of the main challenges of managing their threatened species in cbps is the complexity of moving individuals across borders and the coordination of conservation efforts among zoos and other institutions at a global level. the enormity of this task is clear from our results. for example, the optimal radius for finding a metapopulation size of more than 250 individuals for a given threatened species is 1,700 km for reptiles and 2,200 km for mammals. within these optimized clusters the number of zoos that hold a given threatened species is low compared with the number of zoos available within the clusters. for example, on average only 13% (17/227) of all the zoos within a cluster hold a given threatened reptile species. this implies that, under current conditions, to manage a metapopulation above 250 individuals requires an optimized cluster of ,20 zoos that could be up to 4,000 km apart. given this complexity, it is not surprising that most zoo populations of threatened species are not managed as metapopulations [28] , nor that most are not yet sustainable in the long term [29] . we found that, on average, for threatened species in the isis network, fewer than 10% of zoos within optimized clusters hold a particular species. therefore, it would be possible to improve the network within an optimized cluster by increasing the number of zoos that contain individuals of a focal species, managing these collections as a single metapopulation and potentially reducing the distances between zoos. this level of organization could result in zoos focusing on particular cbps for fewer taxa, rather than having a small number of individuals of many threatened species. this is particularly important since specialization has been shown to increase breeding success [5] . this observation does not mean that zoos should shift their entire collections towards one or a few at-risk species, since responsible zoos have other conservation goals such being centers for education and research [21] . rather, it means that zoos within a particular region can most efficiently increase their conservation contribution by developing collectively managed cbps devoted towards a smaller number of focal species. the proportion of threatened species that exceed a threshold metapopulation size of 250 individuals is rather low, ranging from only 9% for birds to 18% for mammals. however, the percentages of species reaching the threshold of .50 individuals range from 27% for amphibians to ,45% for the other three classes. isis zoos have only a small number of threatened species for which population sizes are above 1,000 individuals. although this number has been suggested as an appropriate threshold over which genetic diversity should be maintained [30] , most of the species that have been recovered from cbps come from populations below 30 to 20 individuals (see [9] ). in this sense, it is important not to underestimate the potential of some of these isis populations. nonetheless, the zoo community should aim at providing populations to cbps that can ensure genetic and demographic sustainability [31] . additionally, cbps should not be implemented only when species have reached dramatically low numbers, at which point their chances of success are lower [3] . our results stress that, for many species, appropriate management and coordination within an optimized cluster of zoos can potentially increase their numbers to at least 250 individuals. in table 3 . number of species in isis zoos with population sizes within specific thresholds for the different red list categories (see table 2 for red list categories definition). addition, it is expected that these population sizes will be larger if we include non-isis member institutions. however, it is important to stress that the successful management of cbps as metapopulations requires the collaboration and coordination of zoos within a global network such as isis. 1. the zoo community should identify potential zoo clusters for the conservation of prioritized species. particularly for smallbodied species, zoos could potentially hold a large number of individuals within a particular region. although the clusters should ideally be in close proximity to the species' natural habitat, most isis zoos are currently located away from major biodiversity hotspots [3] and therefore, zoos' support of breeding centers within the native range may be an option. 2. cluster-level integrated management plans should be implemented to ensure the coordination of cbps with habitat conservation and other in situ efforts (one plan approach [15, 16] . for this, the development of greater coordination among zoos using networks such as isis, together with conservation ngos, academic and governmental institutions, will be essential (see [4] ). 3. species-specific clusters should ideally be replicated to minimize the potential impact of catastrophic events. 4. cross-border management policies for zoos should be modified. in this sense, the management of cbps by clusters can facilitate treaties for the management of particular focal species. to ensure sustainable metapopulations it will require more than the coordination and will of the zoo and conservation community. although we acknowledge the need for public health management and vigilance against illegal trade, the development of cross-border management policies will be key to achieving successful cbps. for example, cites could award special permits to facilitate movement of targeted individuals within cbp clusters. , average number of zoos where the species is found as a function of the radial distance (middle row) and ratio between the probability of finding the metapopulation and the radial distance (lower row) for each taxonomic class of terrestrial vertebrates. when the ratio is highest, we obtain the optimal radial distance between zoos and the optimal probability of finding a metapopulation size above the threshold. this is, at that ratio the probability of finding the metapopulation is highest with respect to the zoo cluster radial distance. for display purposes all ratios were multiplied by 1,000. doi:10.1371/journal.pone.0080311.g003 global change represents an unprecedented challenge for the maintenance of biodiversity [32] [33] [34] . it is expected that even under the most optimistic impact and adaptation scenarios, a great number of species may require the integration of a suite of conservation actions, including cbps. furthermore, species that have no likelihood of in situ persistence for the foreseeable future represent an additional conservation challenge. for example, under current global warming trends, most polar and some montane species are likely to fall into this category [35] , in addition to species whose habitat will be lost by urbanization [36] . as a result, deciding which species could be part of successful cbps and which institutions should modify their collections to become part of a particular cluster needs to be the result of a sound prioritization approach. furthermore, simply holding these species, even in numbers above the minimum 50 individuals, is not sufficient [31] , and cbps need to be integrated with other aspects of species conservation, such as habitat protection and restoration, eradication of invasive species and population management [3] [4] [5] 15, 18] . our results show that, in the zoo network, the representation of species that may require cbps is currently low for most taxa and the spatial distribution of these zoo populations makes management difficult. however, the zoo network already devotes 23% of its collections to threatened species; for mammals, 18% of those reach population sizes above 250 individuals. zoos in collaboration with other institutions have already saved a number of species from extinction, but it has been mostly opportunistic rather than strategic. if zoos collectively focus on their strength as a global network, they have the potential for the development of integrated conservation programs that include cbps. to maximize effectiveness, the collaboration of the global zoo network with governmental institutions, regional and international trade authorities, ngos and academia should be fostered. such collaborations are already underway, and termed a one plan approach [16] . however, it is essential to strengthen these institutions' contributions, include special international treaties and collaborations to help slow down current extinction trends. thanks to the iucn ssc conservation breeding specialist group for facilitating the discussions that set the direction of this paper; to isis member institutions for making their basic data available in the isis portal; to alexander scheuerlein and zjef pereboom for useful comments on the manuscript; and to robert wiese, william van lint, jenny gray and jeffrey bonner for sharing their perspectives and expertise on zoo collections. additionally, we would like to thank the editor and two external reviewers for helpful suggestions and detailed comments that significantly improved our manuscript. optimal radial distance between zoos, average probability of reaching the metapopulation size threshold within that radial distance, average number of zoos within radial distance and average number of zoos within radial distance that hold the threatened species. the optimal radial distance represents the distance needed to optimize the probability of reaching an average metapopulation size within the shortest possible distance between zoos (the metapopulation thresholds m are .50, .100 and .250 individuals). the column for ''number of species in cluster'' indicates the number of threatened species for which all isis zoos have at least m individuals. doi:10.1371/journal.pone.0080311.t004 monitoring change in vertebrate abundance: the living planet index global biodiversity: indicators of recent declines an emerging role of zoos to conserve biodiversity bring the captive closer to the wild: redefining the role of ex situ conservation buying time for wild animals with zoos recent captive-breeding proposals and the return of the ark concept to global species conservation parks or arks: where to conserve threatened mammals? limitations of captive breeding in endangered species recovery captive breeding -a useful tool in the preservation of biodiversity? why we should aim for zero extinction the impact of conservation on the status of the world's vertebrates zoos and captive breeding -response using the iucn red list to determine effective conservation strategies acting fast helps avoid extinction integrating the captive and the wild the one plan approach: the philosophy and implementation of cbsg's approach to integrated species conservation planning research in zoos: a growth area in conservation building a future for wildlife?'' evaluating the contribution of the world zoo and aquarium community to in situ conservation minimum population sizes for species conservation conservation biology the application of zoos victoria's ''fighting extinction'' commitment to the conservation of leadbeater's possum gymnobelideus leadbeateri zoo-based amphibian research and conservation breeding programs the amphibian ark: a global community for ex situ conservation of amphibians identifying gaps and opportunities for inter-regional ex situ species management sustaining the ark: the challenges faced by zoos in maintaining viable populations minimum viable population size: a meta-analysis of 30 years of published estimates achieving true sustainability of zoo populations synergies among extinction drivers under global change extinction risk from climate change impacts of climate change on the future of biodiversity climate change in the eastern himalayas: observed trends and model projections global forecasts of urban expansion to 2030 and direct impacts on biodiversity and carbon pools key: cord-002953-4rqoenhr authors: arruda, andréia gonçalves; vilalta, carles; puig, pere; perez, andres; alba, anna title: time-series analysis for porcine reproductive and respiratory syndrome in the united states date: 2018-04-03 journal: plos one doi: 10.1371/journal.pone.0195282 sha: doc_id: 2953 cord_uid: 4rqoenhr industry-driven voluntary disease control programs for swine diseases emerged in north america in the early 2000’s, and, since then, those programs have been used for monitoring diseases of economic importance to swine producers. one example of such initiatives is dr. morrison’s swine health monitoring project, a nation-wide monitoring program for swine diseases including the porcine reproductive and respiratory syndrome (prrs). prrs has been extensively reported as a seasonal disease in the u.s., with predictable peaks that start in fall and are extended through the winter season. however, formal time series analysis stratified by geographic region has never been conducted for this important disease across the u.s. the main objective of this study was to use approximately seven years of prrs incidence data in breeding swine herds to conduct time-series analysis in order to describe the temporal patterns of prrs outbreaks at the farm level for five major swine-producing states across the u.s. including the states of minnesota, iowa, north carolina, nebraska and illinois. data was aggregated retrospectively at the week level for the number of herds containing animals actively shedding prrs virus. basic descriptive statistics were conducted followed by autoregressive integrated moving average (arima) modelling, conducted separately for each of the above-mentioned states. results showed that there was a difference in the nature of prrs seasonality among states. of note, when comparing states, the typical seasonal pattern previously described for prrs could only be detected for farms located in the states of minnesota, north carolina and nebraska. for the other two states, seasonal peaks every six months were detected within a year. in conclusion, we showed that epidemic patterns are not homogeneous across the u.s, with major peaks of disease occurring through the year. these findings highlight the importance of coordinating alternative control strategies in different regions considering the prevailing epidemiological patterns. introduction industry-driven voluntary swine disease control programs emerged in north america in the early 2000's. since then, those programs have been widely used for monitoring and surveillance of diseases of economic importance to swine producers. even though these control programs have been relatively common for prrs in north america [1] [2] [3] , data emerging from those programs have rarely been assessed for research purposes. one example of such initiatives is the dr. morrison's swine health monitoring project (mshmp), a nation-wide monitoring program for swine diseases that affect and are economically important for the u.s. swine industry, including the porcine reproductive and respiratory syndrome (prrs), the porcine epidemic diarrhea (ped), and senecavirus a. the university of minnesota led this initiative starting in 2011, in collaboration with the american association of swine veterinarians (aasv), the national pork board, and the swine health information center. since then, the mshmp has served as a means to capture the occurrence of infectious diseases for situations in which there is an absence of a regulatory framework [4] . at the time of writing of this manuscript, in december 2017, the program represented a considerable portion of breeding herds in the country (approximately 45%; [5] ) and collected disease incidence data on a weekly basis. porcine reproductive and respiratory syndrome has been previously reported as a seasonal disease with predictable peaks starting during mid-october [6] ; therefore, it is not uncommon that farm-level prevention efforts are intensified during this time. however, previous research conducted by our research group suggested that prrs virus transmissibility as captured by the time-dependent reproduction number is incredibly region-and system-dependent [7] , for reasons that could potentially include (but are not limited to) demographic and environmental factors [8, 9] . even though the swine industry commonly reports prrs as a seasonal disease, systematic disaggregation of data into regions and systems combined with formal and full assessment and comparison of seasonality prrs patterns across different us regions has never been published in the peer-reviewed literature. one of the reasons for the lack of such information is that consistent collection of longitudinal disease data over time has been traditionally uncommon for the swine industry in north america. however, after years of data collection, the mshmp database offered a unique opportunity for conducting, for the first time, temporal analysis such as the one described here. time-series analysis is a field in development, and, even though methods are currently available; these have not been largely explored for modelling of infectious diseases in food animal production systems. one of the few studies published in the peer-reviewed literature used cattle mortality data from over nine years from a region in spain to apply autoregressive integrated moving average (arima) modelling and hierarchical time series to determine basal patterns of bovine fallen stock at different scales (region, province, county and municipality) [10] . the researchers concluded that the time series approaches were useful as tools for comparing mortality patterns across different populations over time [10] . the main objective of this study was to use time-series analysis to describe the temporal patterns of prrs at the farm level for five major swine-producing states across the u.s. specifically, our aim was to investigate whether yearly patterns commonly described for prrs were in fact conserved across different u.s. states. our main hypothesis was that there was one yearly seasonal peak would be found for prrs for all examined states and systems (winter season). evaluating that hypothesis will help to understand the epidemiological dynamics of prrs in the u.s. ultimately, that information will help to design and implement surveillance and control programs for one of the most financially devastating diseases of swine in the country. the source population for this project corresponded to breeding herds (herds that contain sows) that participated in the dr. morrison's swine health monitoring project from july 2009 (starting on the 26 th week of 2009) to october 2016 (finishing on the 41 st week of 2016). even though the mshmp source population includes over 1,000 participating sow farms, only swine herds that contributed with complete data from those previously mentioned years were enrolled in this study, so that a closed population could be examined. data were aggregated retrospectively at the week level for the number of herds that were considered as having an active outbreak each week. farms would be allocated to that category as soon as outbreaks were identified and reported by herd veterinarians and based on a number of features. those features included one or a combination of the conditions of presence of prrs-consistent clinical signs and identification of the virus via diagnostic testing, and/or isolation of a different prrs virus strain as compared to previous existing strains in the herd; as described elsewhere [7] . once herds reported an outbreak, they were considered 'status 1' or 'positive unstable' following aasv prrs classification guidelines [11] until there was absence of clinical signs and no detectable viremia in weaned piglets for a minimum of 90 days (tested at least every 30 days) [11] . descriptive analysis was conducted using r v.3.2.3 (r core team, 2013). first, we characterized the number of sites per region, the mean (standard deviation [sd]) number of animals in the herds and the mean number of farms containing animals actively shedding prrs virus (considering a closed population) over time. second, descriptive analysis of the time series was conducted by plotting the raw counts of sites classified as prrs 'status 1' for all the source population and for the subpopulations according to geographical region. statistics such as mean, median, minimum, maximum, variance and autocovariance functions were analyzed for each time series separately to determine basic patterns in the data and to aid in the decision of whether an alternative model for low counts [12] would be needed during time series analysis. the level of resolution used in this study was weeks because this was the finer level of resolution available from the mshmp and avoided possible weekend or holiday effects. counts of swine farms containing animals actively shedding prrs virus was used as the main outcome of interest. data from a total of 388 weeks were available for analysis (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) . data were stratified at the state level and separately analyzed. the five states examined included minnesota (mn), iowa (ia), north carolina (nc), nebraska (ne), and illinois (il). autoregressive integrated moving average (arima) modelling was conducted as described elsewhere [10] . in brief, the following steps were taken for construction of models: first, data were decomposed and plotted to visually assess the presence and characteristic of the trend and the seasonality, as well as the autocorrelation (acf) and the partial autocorrelation (pacf) plots. second, autoregressive moving average modelling was performed, including trend and seasonality terms as covariates. here, the seasonality was represented by including trigonometric covariates (or fourier terms). seasonal cycles of one year and six, four and three months were tested by multivariate linear regression using the least square method [10] . the selection of the most appropriate model for each time series of the study was based on three criteria: assessment of the akaike information criterion (aic), statistical significance of the parameters of the model at a reasonable significance level of 0.05, the box-pierce test, and the evaluation of the behaviour of the residuals checking its autocorrelation and partial autocorrelation function. for series in which the counts contained a considerable number of zeros, a generalized linear autoregressive moving average model with a poisson structure was also attempted ('glarma' r package; [13] ), with model selection again being prioritized as described above. there were 268 farms included in this study; the majority of participating farms were located in the swine dense regions of minnesota and iowa. fig 1 shows table 1 . fig 2 depicts the prevalence, upper and lower 95% confidence intervals of farms containing animals actively shedding prrs virus over the weeks examined in this study. there was a difference in the nature of prrs seasonality among states. a fall/ winter seasonal pattern was only detected for farms located in the states of minnesota, north carolina and nebraska. for the other two states (iowa and illinois), two seasonal peaks were detected within a year: one every 6 months (biannual). figs 3 and 4 shows the fit and diagnostics for all final models, and covariate estimates, standard errors and aic values are provided on table 2 . all models appeared to fit well considering the graphs and all p-values from the box-pierce test were p > 0.05. autoregressive integrated moving average (arima) modelling was used here to describe seasonal patterns of prrs outbreaks in swine farms across five states of the united states. this stratification allowed for the comparison across different subpopulations of farms and identification of important differences in disease prevalence over time, which, prior to the study here, were believed to be homogeneous in the country. the main finding of this study was that prrs seasonality varies according to geographical region, and the commonly referred "prrs season" is not necessarily the only time of increase in disease incidence. even though prrs is anecdotally considered a seasonal disease, we showed that, for some regions among the u.s., there are other considerable peaks of disease during the year. sporadic cases have anecdotally been referred to as "summer outbreaks". even though it has been reported that cold temperatures (winter season) and low relative humidity favor prrs virus survival [14] , some factors might help explain these off-winter incidence of prrs cases. first, during summer months there is larger commingling of animal producers in state fairs or animal shows [15] , which could facilitate disease transmission in the absence of adequate biosecurity practices [16, 17] . second, lack of awareness of occurrence of other prrs cases outside the neighborhood and system could contribute to a relaxed attitude towards some biosecurity measures during these months (e.g. less truck washing, drying and disinfecting events), allowing for virus spread [18] . lastly, considering the volume of activities that occur in the current structure of the swine industry [19] , it is not surprising that there are a lot of opportunities for the pathogens to spread via non-obvious manner such as through feed trucks, fomite and personnel, therefore; even though the virus might be easily transmitted during winter months, the amount of activity is enough for these sporadic events to occur in considerable amounts. another interesting finding from this study was the presence of an alternating trend for all examined states within of the u.s., except for the state of iowa, the largest pork producing states in the country (approximately 31.4% of the total us hog and pig inventory, [20] ), which had an increasing linear trend over the examined years. the period during which this changing point occurred was between may 2013 and late march 2014, which, not surprisingly, coincides with the calendar year in which the u.s. had a porcine epidemic diarrhea outbreak [21, 22] . it has been previously reported that during 2013-2014, there were significantly fewer prrs cases for reasons that are still hypothesized [22] . due to the fact that an unpredictable table 1 event occurred in the population examined here, our data were not considered robust enough for division into testing and training and potential prediction. such analysis may be, however, conducted in the future, when enough data would be gathered over the years. strengths of the study here are the large sample size, the geographic representation of the major pig-producing u.s. states, and the potential impact of the research, given that this is time-series analysis for porcine reproductive and respiratory syndrome in the united states the disease that cause the most far-reaching impact to the swine industry of the country. furthermore, data available included over nine years of weekly disease data classified in a standardized manner, which is somewhat rare for an industry-based swine project. arima models were used because they are powerful tools that can be directly fitted using standard packages in r. however the use of families of continuous-time arma processes [23] , or integer-valued ar processes [12] could be also worth exploring in further research. an important limitation of the study is that solely breeding herds were included as these were the only production types reporting disease status to the mshmp; therefore, extrapolation of findings to growing pig populations should be made with caution. however, the most valuable animals in the value chain of the industry were included in the assessment, supporting the importance and impact of the findings. we also acknowledge misclassification bias is possible for the cases in which farms were not correctly classified by the veterinarian as positive shedding. this would be especially a problem for herds that have some kind of underlying immunity (e.g. vaccinated or live-inoculated herds). we realize that, the utilized scheme of classification does not fully detect farms that have low prevalence of animals that are shedding the virus. however, this was the best available method for classifying swine herds in regards to prrs at the present time, and we the advantage relies on the fact that a standardized method was used throughout the study period, therefore; if this misclassification occurred, it would be consistent across farms, which we believe would dramatically change our conclusions. finally, emergence of ped at a given point during the study period prevented researchers to develop predictive models at this instance, but the current work will serve as basis for establishing a disease baseline by region, as well as to bring awareness that prrs is a seasonal disease, and that there may be more than one peak in prrs cases during the course of an year. farm and region-level variables were not available to us, but could be collected to investigate potential drivers of the state and system-level differences reported herein. in conclusion, we showed that prrs seasonal patterns are not homogeneous across the u.s., with some important pork producing states having biannual prrs peaks instead of the previously reported winter peak. findings from this study 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america comparison between the 2013-2014 and 2009-2012 annual porcine reproductive and respiratory syndrome virus epidemics in a cohort of sow herds in the united states a construction of continuous-time arma models by iterations of ornstein-uhlenbeck processes the authors would like to acknowledge the mshmp participants for sharing information. key: cord-048471-7jszm1nd authors: salim, omar; clarke, ian n.; lambden, paul r. title: functional analysis of the 5′ genomic sequence of a bovine norovirus date: 2008-05-14 journal: plos one doi: 10.1371/journal.pone.0002169 sha: doc_id: 48471 cord_uid: 7jszm1nd background: jena virus (jv), a bovine norovirus, causes enteric disease in cattle and represents a potential model for the study of enteric norovirus infection and pathogenesis. the positive sense rna genome of jv is organised into orf1 (non-structural proteins), orf2 (major capsid protein) and orf3 (minor capsid protein). the lack of a cell culture system for studying jv replication has meant that work to date has relied upon in vitro systems to study non-structural protein synthesis and processing. principal findings: only two of the three major orf1 proteins were identified (p110 and 2c) following in vitro translation of jv rna, the n-term protein was not detected. the n-term encoding genomic sequence (5′gs) was tested for ires-like function in a bi-cistronic system and displayed no evidence of ires-like activity. the site of translation initiation in jv was determined to be at the predicted nucleotide 22. following the insertion of an epitope within the 5′gs the jv n-term protein was identified in vitro and within rna transfected cells. conclusions: the in vitro transcription/translation system is currently the best system for analysing protein synthesis and processing in jv. unlike similarly studied human noroviruses jv initially did not appear to express the n-terminal protein, presenting the possibility that the encoding rna sequence had a regulatory function, most likely involved in translation initiation in an ires-like manner. this was not the case and, following determination of the site of translation initiation the n-term protein was detected using an epitope tag, both in vitro and in vivo. although slightly larger than predicted the n-term protein was detected in a processed form in vivo, thus not only demonstrating initial translation of the orf1 polyprotein but also activity of the viral protease. these findings indicate that the block to noroviral replication in cultured cells lies elsewhere. jena virus, a bovine norovirus, is a member of the caliciviridae family of positive sense rna viruses and was first isolated from the diarrhoeic stools of newborn calves [1, 2] . jv is a type i genogroup iii (giii) norovirus which is closely related to the type ii giii bovine noroviruses newbury agent 2 and dumfries [3, 4] . the giii noroviruses are responsible for causing enteric disease in cattle [2, 5] and, thus, likely share a similar tissue tropism to the human-associated enteric noroviruses. like human noroviruses [6] bovine noroviruses have a high seroprevalence [4] . jv is therefore a potentially useful model for studying the molecular biology of enteric norovirus pathogenesis and replication. the 7.3 kb polyadenylated rna genome of jv has been characterised previously [7] and, like other noroviruses, is organised into 3 open reading frames (orfs). orf1 encodes the non-structural proteins in the form of a large 185 kda polyprotein, which is subsequently cleaved into functional replication proteins by the viral encoded 3c-like protease. orf2 encodes the structural capsid protein (56 kda) and orf3 encodes a small basic protein, which has been shown to function as a minor capsid component [8] . jv orf1 is consistent with other caliciviruses in that it encodes a 39 kda 2c-like nucleoside triphosphatase (ntpase), a 3c-like protease and a 56 kda 3d-like rna-dependent rna polymerase [7, [9] [10] [11] [12] [13] . however, the genomic sequence within the 59 region of jv orf1 (59gs) displays a high level of divergence. this divergence is mainly attributed to the presence of several proline-encoding polypyrimidine tracts within the region predicted to encode a 35 kda nterminal protein [7] . the predicted size of n-terminal proteins relative to the size of the respective 2c proteins differs within the norovirus genus. within the gi noroviruses, such as southampton virus, the n-terminal protein (44.8 kda) is larger in size compared to the 2c protein (39.6 kda). this is in contrast to the gii noroviruses, such as lordsdale virus and camberwell virus, in that the n-terminal protein is smaller in size compared to the 2c protein [11, 14] . this is also the case for jena virus in which the predicted jv n-terminal protein (35 kda) is smaller than the jv 2c protein (39 kda) [7] . the norovirus n-terminal protein varies in relative size across the genus, and the encoding sequence bears no similarity to other cellular or viral proteins. alignment of the n-term protein sequences of various noroviruses indicates little similarity between genogroups within the first 180 residues, however towards the cterminal end of the protein similarity between the amino acid residues increases. recent studies investigating the functions of the norwalk virus n-terminal protein have successfully demonstrated association with the golgi apparatus in transfected cells [15] . in addition this study also identified a picornaviral 2b like region within the n-terminal protein, suggesting that the protein is involved with host cell membrane interactions, reinforcing other findings that have suggested that the norwalk virus n-terminal protein disrupts intracellular protein trafficking, including proteins destined for the host cell membrane [16] . a 3c protease-mediated cleavage event within the n-terminal protein (37 kda) was described for camberwell virus, a genogroup 2 norovirus, yielding proteins of 22 kda and 15 kda [17] . based on these observations and location within the genome it was hypothesised that the nterminal protein of noroviruses corresponds to the 2ab region in picornaviruses. another possibility is that the n-term encoding rna itself serves to function as a translational enhancer by interacting with cellular proteins involved in translation. indeed, this phenomenon has been previously reported for norwalk virus, within which a double stem loop structure has been predicted at the 59 end of the genomic rna [18] . it was subsequently demonstrated that elements within the 59 end of norwalk virus bind specifically with cellular proteins such as la, ptb and pcbp2 [19] which have all been implicated in ires-mediated cap-independent translation in the closely related picornaviruses [20] [21] [22] [23] . in this study the role of the jv 59gs was investigated, including its potential to direct capindependent translation initiation. the precise location of translation initiation in jv was also investigated. previous studies of norovirus polyprotein processing have yielded three major products following in vitro transcription and translation, representing the uncleaved 3abcd, n-term and 2c proteins. however, initial analysis of jv polyprotein processing indicated that only two major proteins are synthesised initially which, based on molecular weight predictions, are the 3abcd (110 kda) and the 2c (39 kda). the lack of an n-terminal protein encoded by the jv 59gs, predicted to be 35.3 kda, is unique among the noroviruses that have been studied in this way. the in vitro transcription and translation profile for jv was therefore studied in more detail. as initial experiments had analysed tnth reactions following a 1 hr incubation, reaction aliquots were harvested at time points before and after the recommended 1 hr incubation. the results in figure 1 show that there are no major reaction products synthesised prior to the 1 hr time point, at which time the 3abcd/p110 and 2c/p39 proteins are clearly visible. extended incubation past the 1 hr point resulted in further proteolytic cleavage of p110 that coincided with the appearance of proteins of the following sizes: 86 kda, 55 kda, and 51 kda. in addition proteins of 29 kda, 22 kda and 20 kda were also visible at the 24 hr time point (figure 1, lane 7) . the only protein that was consistently visible following the 1 hr time point was the 2c/p39 protein. despite prolonged incubation there was no indication that the n-terminal/p35 protein was synthesised. a comprehensive study of polyprotein processing within the murine norovirus (mnv) suggests likely identities for the equivalent proteins in the similar profile for jv [12] . using region specific antisera the authors were able to identify p110 as the 3abcd uncleaved precursor, p90 as the 3bcd, p57.5 as the 3d-like polymerase, p52 as a 3abc precursor and p40 as the 2clike ntpase, which was determined by mutagenesis and microsequencing experiments. the 19 kda protein was identified as the 3c-like protease. the antisera used to detect the mnv nterm protein recognised 3 products; one was the predicted molecular weight at 39 kda and the other two bands migrated as a 45 kda doublet. the 59gs region of jv is highly divergent compared to other noroviruses, mainly due to the relatively high cytosine content (32%), which contributes to an overall g/c content of 58%. there are many polypyrimidine tracts within the sequence, potentially yielding a relatively high degree of rna secondary structure. previous studies have described potential secondary rna structure and interaction with proteins involved with iresmediated translation within the 59 genomic region of norwalk virus [18, 24] . it was of interest therefore, based on these findings, to ascertain whether or not the 59gs of jv possessed ires-like properties within the context of a 'bi-cistronic' expression system, independently of other viral proteins, including the vpg which, in other caliciviruses, has been shown to be associated with translation initiation factors [25, 26] . traditionally the bi-cistronic vector system has been used to define potential ires-like sequences from a variety of viral and cellular mrnas, and is recognized as being the standard test for this function [27] . a bi-cistronic vector is comprised of a 59 and 39 cistron; translation of the 59 cistron being cap-dependent and translation of the 39 cistron regulated by the putative ires-like sequence. thus, if the 39 cistron is translated in addition to the 59 cistron then the sequence of interest is said to have ires-like properties, as translation is initiating internally. to test for ires-like function in jv, bi-cistronic constructs were made with a cap dependent 59 egfp cistron and a 39 lacz cistron under the translational control of either the jv 59gs (pegfp-c1/ jv59gs/lacz) or an authentic emcv ires (pegfp-c1/ires/ lacz). crfk cells were transfected with the bi-cistronic constructs and, following incubation, were assayed for egfp and lacz expression. both constructs were able to direct translation of the egfp cistron effectively as expected (figure 2a and figure2d). the use of an authentic emcv ires to direct translation of the lacz cistron was also effective (figure 2e), with levels of b-galactosidase activity comparable to those of the b-galactosidase reporter ( figure 2f ). however, no b-galactosidase activity was detected from cells transfected with the pegfp-c1/jv59gs/lacz construct (figure 2b ), demonstrating that the jv 59gs was unable to initiate translation, and therefore, in this context, did not possess any ires-like functions. as it was clear that the jv 59gs did not posses any ires-like functions it was necessary to determine the location of translation initiation within orf1. this was predicted be the atg encoding methionine at nucleotide position 22, as it is situated in a favourable context for translation initiation [7] . to investigate this multiple translation termination codons (polystop) were inserted into the jv genome within the 3b-encoding region, downstream of the 59gs, to halt translation at a defined point. in vitro transcription and translation of this construct would, in theory, yield a product whose size would relate to the initiation codon used within the 59gs (figure 3 ). to address the unlikely event of translation read-through or re-initiation downstream of the polystop, which would result in subsequent translation of the 3c protease and cleavage of the truncated orf1 polyprotein, a mutation was made within the active site encoding region of the 3c protease within jv orf1, to prevent any viral mediated cleavage of orf1 translation products (jv 3c mut /polystop). a point mutation of the critical cysteine residue within the highly conserved gdcg motif to a glycine residue was performed, and this approach has been described for the successful inactivation of other norovirus' 3c activity [28] . in vitro transcription and translation analysis was performed on jv wild type ( figure 4 within the 3c region of jv successfully inactivated the 3c protease, thus a large, .200kda uncleaved polyprotein is yielded following tnth. the major product generated by jv 3c mut / polystop was calculated to be 103kda in size. based on computer predictions this is in agreement with the initiation of translation occurring at nucleotide 22, which demonstrates that the jv n-term protein is translated in full in vitro. at this time it is not possible to determine whether translation of intracellular vpgbound viral rna initiates at nucleotide 22, although it is likely given the favourable context in which the initiation codon is situated. as the jv n-term was found to be translated in vitro attempts were made to express and purify the protein in bacteria for immunisation so that the protein could be identified by radioimmune precipitation assay (ripa), as it was possible that the nterm protein was migrating on gels aberrantly and possibly comigrating with 2c. attempts to express the protein in bacteria were unsuccessful due to toxicity. therefore, the 14aa v5 epitope encoding sequence was cloned in frame into the jv cdna construct at nucleotide position 123 (jv v5). the v5 epitope originates from the p and v proteins of the sv5 paramyxovirus [29] , for which a commercially available monoclonal antibody is used for detection. following in vitro transcription and translation of jv v5 a new product, approximately 42 kda in size, was visible ( figure 5 , lane 2). this product was not observed in any prior analyses of jv. to confirm that this protein was v5/n-term associated the tnth reaction was subjected to ripa using the anti-v5 antibody ( figure 5, lane 3) . this confirmed expression of the n-term protein in vitro. to confirm expression of the v5/n-term protein in cell culture capped rna was synthesised from the jv v5 t7 cdna construct, which was used to transfect crfk cells. as there is currently no host cell line in which to propagate jv the crfk cell line was used as it has been shown to support the replication of feline calicivirus [30] . confocal immunofluorescence of transfected cells using the anti-v5 antibody demonstrated expression of the v5/nterm protein in cultured cells ( figure 6 ). expression of the v5/nterm protein was diffuse and did not co-localise with the golgi/ er/plasma membrane marker wheat germ agglutinin (wga) and therefore displays a different pattern of cellular expression compared to norwalk virus [15] . cells transfected with the wild type full length jv rna were negative for fluorescence (data not shown). lysates of cells transfected with wild type jv and jv/v5 rna were subjected to western blot using the anti-v5 antibody ( figure 7) . no product was present for cells transfected with wild type jv rna, but a protein of approximately 42 kda in size was visible in cells that had been transfected with jv/v5 rna, confirming n-term expression and size as seen in the in vitro system. in addition, this important observation also confirms for the first time that the jv 3c protease was active in cells transfected with capped rna as the size of the v5/n-term indicated successful cleavage of the protein from the orf1 polyprotein. to address the issue of potential rapid degradation of the jv nterm protein crfk cells were transfected with jv v5 rna and were harvested at designated time points following the addition of the protein synthesis inhibitor cycloheximide. cell lysates were analysed by western blot using the anti-v5 antibody (figure 8 ). the consistent appearance of the n-term/v5 protein suggested that it is stable and insensitive to degradation by viral and host cell proteases. the predicted molecular weight of the jv n-term is 35.3 kda, based on the site of initiation of translation and location of conserved cleavage sites. the appearance, therefore, of a previously unseen 42 kda protein in the in vitro transcription and translation profile was unexpected but this protein does represent a translation product for the jv 59gs. to date, it has not been possible to explain the difference in the predicted and observed sizes for the jv n-term, and the addition of the 14 amino acid v5 epitope within jv n-term does not account for this apparent large shift in molecular weight. however, a recent study described a similar anomaly when investigating proteolytic processing in the murine norovirus mnv-1 [12] . the predicted molecular weight for the mnv-1 n-term protein was 38.3 kda. the authors successfully generated antisera against the mnv-1 n-term and used it to immunoprecipitate the protein from in vitro transcription and translation reactions and observed that the n-term existed as a 45 kda doublet, in addition to the predicted size of 38 kda. however, when mnv-1 n-term antisera was used to probe mnv-1-infected cell lysates only the 43-45 kda doublet and a large 115 kda precursor could be detected, suggesting that the predicted 38 kda form of the n-term is not generated in cell culture. again, it was not possible to conclusively determine the cause of this discrepancy, but it was speculated that the n-term protein may migrate abnormally in sds-page, or may be proteolytically processed at a previously unknown cleavage site downstream of the protein's predicted c-terminus. it is also possible that the n-term protein might be modified in some way leading to a shift in observed molecular weight. at this time the same conclusions would seem appropriate for the jv n-term. in addition, it is not known why the jv n-term was previously not detected in in vitro transcription and translation studies prior to the insertion of the v5 epitope. it cannot be ruled out, however, that the wild type jv n-term aberrantly co-migrates with the 39 kda jv 2c protein in sds-page. indeed, the appearance of the v5/ n-term product from transfected cell lysates would appear to be one of a doublet (figure 8) , also analogous to the observed appearance of the mnv n-term protein in infected cells, suggesting the likelihood of a further cleavage site within the jv n-term protein which has yet to be elucidated. nevertheless, these studies clearly demonstrate that a protein representative of the 59gs of jv is translated both in vitro and in vivo and is proteolytically processed from the orf1 polyprotein following translation initiation at nucleotide 22. human norovirus infection has been shown to be the leading cause of non-bacterial gastroenteritis [31] , however there is currently no cell culture system available to facilitate viral replication and ethical considerations have hindered progress in establishing a permissive human organ culture system. the study of jena virus offers a potential animal model of enteric noroviral infection. however, until a permissive bovine cell and/or organ culture systems is established analysis of the molecular mechanisms underpinning viral replication and pathogenesis rely upon in vitro systems, most notably polyprotein synthesis and processing. unlike similarly studied human noroviruses jv initially did not appear to express the n-terminal protein, presenting the possibility that the encoding rna sequence had a regulatory function itself, most likely involved in translation initiation in an ires-like manner. this was shown not to be the case and, following determination of the site of translation initiation at the predicted nucleotide 22 the n-term protein was detected following the insertion of an epitope tag, both in vitro and in vivo. although slightly larger than predicted the n-term protein was detected in a processed form in vivo, thus not only demonstrating initial translation of the orf1 polyprotein but also activity of the viral encoded protease. these important findings indicate that the block to replication of enteric norovirus in cultured cells cannot be attributed to a failure to synthesise and process the non-structural proteins. the detection of processed and active orf1 proteins in transfected cultured cells, however, highlights the potential for the development of cell and bovine organ based systems to facilitate the replication of jena virus. the pegfp-c1 vector (clontech) comprises of an egfp coding sequence under the control of a cmv promoter and a kozak translation initiation site. downstream of the egfp sequence is the multiple cloning site containing unique bglii, saci, hindiii and apai restriction sites. contruction of pegfp-c1/jv 59 gs/lacz was as follows; the jv 59 gs sequence was amplified from the jv full length cdna clone [7] using bio-x-act dna polymerase (bioline) with the primers 59 gs f (59-aactgca-gatcttaataagtgaatgaagactttgacgat-39), containing the bglii restriction site (bold) and two in-frame translation termination codons (underlined) to ensure that translation of the egfp sequence did not carry over to the 59 gs, and 59 gs r (59-aactgcaagcttctgcaggacacaatgagg-39), containing thehindiii restriction site. the jv 59 gs amplicon was ligated to the pegfp-c1 vector, following restriction enzyme digestion of both amplicon and vector with bglii and hindiii restriction enzymes, and the ligated dna used to transform e.coli top10 (invitrogen). this intermediate construct was named pegfp-c1/ jv 59 gs. the lacz coding sequence was amplified from the psvb-gal reporter vector (promega) using bio-x-act dna polymerase and the primers lacz f (59-aactgcaagcttga-tatgggggatcccgtcgttttacaacg-39), containing the hindiii restriction site (bold) and a kozak translation initiation site (underlined), and lacz r (59-aactgcgggcccttat-tatttttgacaccagacca-39) containing the apai restriction site (bold) and translation termination codons (underlined). the lacz amplicon was ligated to the pegfp-c1/jv 59 gs vector following restriction enzyme digest of both amplicon and vector with hindiii and apai restriction enzymes, and the ligated dna used to transform e.coli top10. the construct was verified by sequencing. construction of pegfp-c1/ires/lacz was as follows; the emcv ires sequence was amplified from the pires2-egfp vector (clontech) using bio-x-act dna polymerase and the primers ires bgl f (59-actcgaagatcttaa-tagagcttcgaattctgcagtcga-39), containing the bglii restriction site (bold) and translation termination codons (underlined) to prevent carry over translation as before, and ires sac r (59-actcgagagctctgtggccatattatcatc-gtg-39), containing the saci restriction site (bold). the ires amplicon was ligated to the pegfp-c1 vector follwing restriction whole cell lysate was collected at the following time points following chx treatment: 0 hr, 1 hr, 3 hr, 6 hr, 12 hr, 24 hr. bradford analysis was performed on the lysates to ensure equal loading. following western analysis the ecl treated membrane was exposed to film for 1 min. molecular weight marker is represented in lane 1. doi:10.1371/journal.pone.0002169.g008 enzyme digest of both amplicon and vector with bgiii and saci restriction enzymes, and the ligated dna used to transform e.coli top10. the lacz amplicon described previously was ligated to the intermediate pegfp-c1/ires vector following restriction enzyme digest of both amplicon and vector with hindiii and apai restriction enzymes, and the ligated dna used to transform e.coli top10. the jv 3c protease mutant was created by point mutation of the critical tgt encoded cysteine residue, within the gdcg active site motif, to a ggt encoded glycine residue by mutagenic overlap pcr using bio-x-act dna polymerase. three rounds of amplification using the jv full length cdna clone as template were used to generate the final mutant protease cassette. round 1 used the primers jv f1 (59-cgtctcagggttgatact-39) and jv mut 1 (59-gcaaccaccgtcaccag-39), yielding a 222 bp amplicon (point mutation nucleotide shown in bold). round 2 used the primers jv mut 2 (59-ctggtgacggt-ggttgc-39) and jv r2 (59-ttcctgggaggaacaagtt-39), yielding a 651 bp amplicon. amplicons generated in rounds 1 and 2 were pooled to serve as template for round 3 using the primers jv nf (59-atgtcaaccaccaccagc -39) and jv nr (59-aagggctccggtgaagg-39). this cassette contained two bcli restriction sites flanking the 3cprotease active site, as also found in the wild-type full length clone. restriction digest using bcli was used to remove the appropriate wild-type cassette from the jv full length clone. the mutant cassette was also digested with bcli prior to ligation to the bcli-digested jv full length clone. the ligated dna was used to transform e.coli top10, and was designated jv 3c mut . construction of jv 3c mut /polystop was as follows: complementary oligonucleotides with three translation termination codons (underlined) in each reading frame in sense and anti-sense orientations were desgined in such a way that upon annealing the duplex would contain blunt termini. the oligonucleotides were termed pstop top (59-ctaggtaagtaaacgcgtctact-cactcac-39) and pstop comp (59-gtgagtgagta-gacgcgtttacttcaatag-39). each oligo (1 mg) was incubated with t4 polynucleotide kinase and atp to phosphorylate the 59 termini, pooled and heated to 75uc for 15 min, and left to cool to room temperature to anneal the oligos. following purification the polystop duplex was ligated to eco47iii digested jv 3c mut , and ligated dna was used to transform e.coli top10. the duplex contained the unique restriction site mlui (shown in bold) to assist screening of recombinant clones. the v5 epitope (n-gly-lys-pro-ile-pro-asn-pro-leu-leu-gly-leu-asp-ser-thr-c) is recognized by the anti-v5 monoclonal antibody (invitrogen). complementary oligonucleotides encoding the v5 epitope were designed in such a way as to generate sacii compatible termini following annealing (bold), and to preserve the reading frame when inserted into the sacii restriction site at nucleotide 123 within the 59 gs of the jv genome (underlined). the oligos were termed v5 top (59-ggtaagcctatccc-taaccctctcctcggtctcgattctacgagc-39) and v5 comp (59-tcgtagaatcgagaccgaggagagggt-tagggataggcttaccgc-39). the oligos were phosphorylated and annealed as described previously and the duplex ligated to the sacii digested jv full length clone. ligated dna was used to transform e.coli top10. in vitro coupled transcription and translation was performed using the tnth coupled reticulocyte lysate system (promega) as per the manufacturer's instructions. reactions were incubated at 30uc for 1-2 hr. for non-radiolabelled reactions the 35 s-methionine was replaced with 1 mm unlabelled methionine (2 ml). reaction products (1-2 ml) were analysed by sds-page. gels were stained and prepared for autoradiography by incubating for 30 min in a solution containing 32 g sodium salicylate, 100 ml methanol and 100 ml dh 2 o. gels were dried under vacuum and the reaction products were detected by exposure to kodak x-omat scientific imaging film (sigma) at 270uc for 16 hr followed by developing using a kodak automated developer. specific v5-tagged proteins synthesised by tnth were precipitated from 5-10 ml of reaction product using the anti-v5 monoclonal antibody (invitrogen) at the recommended dilution in 600 ml of 16 ripa buffer (diluted from 10x stock: 10 mm tris-hcl (ph 7.5), 1 mm edta, 0.15 mm nacl, 0.1% sds, 0.5% empigen bb, 0.1 mm phenylmethylsulphonylfluoride) for 1 hr at 37uc. this was followed by a second incubation of tube for 2 hr rotating at room temperature with goat anti-mouse immunoglobulin g agarose beads (sigma) to absorb the immune complexes. the beads were washed three times with 500 ml 16 ripa buffer and once with 500 ml pbs. the beads were resuspended in sample buffer for analysis by sds-page and autoradiography as before. endotoxin-free preparations of plasmid dna were prepared using the genelute tm endotoxin free plasmid midi prep kit (sigma). crandall-reese feline kidney cells (crfks) were seeded into a 12 well tray at approximately 40-50% confluence. crfk cells were transfected with no dna (negative control), psv-b-gal (control for b-galactosidase activity), pegfp-c1/jv 59 gs/lacz and pegfp-c1/ires/lacz (control for ires activity) using the superfect tm transfection reagent (qiagen) as per the manufacturer's recommendations. following a 16 hour incubation the cells were observed for egfp expression using a leica leitz dmrb fluorescence microscope. the cells were washed in pbs and fixed using a 0.5% solution of glutaraldehyde for 30 min at room temperature. the cells were incubated with an x-gal stain solution: 5 mm k 3 fe(cn) 6 , 5 mm k 4 fe(cn) 6 , 2 mm mgcl 2 , 1x x-gal (sigma) for 4 hours at 37uc and were observed for b-glactosidase activity by light microscopy. the experiment was performed more than once to confirm the results. jv v5 and jv flc t7 cdna plasmid constructs were linearised using ndei (invitrogen). capped rna was synthesised using the mmessage mmachineh capped rna transcription kit (ambion) according to the manufacturer's instructions. crfk cells were seeded into 6 well trays at approximately 50% confluence and were transfected with 2 mg purified rna per well using transmessenger transfection reagent (qiagen) according to the manufacturer's instructions. for immunofluorescence crfk cells were seeded onto 19 mm coverslips in 6 well trays and were transfected with rna as described. following a 24 hr incubation the coverslips were washed with pbs and fixed in 4% formaldehyde for 15 min at room temperature. cells were permeabilised and blocked in saponin buffer, also used as staining buffer, (0.1% saponin, 10% foetal calf serum, 0.1% sodium azide) for 1 hr at 4uc. cells were stained using an anti-v5 monoclonal antibody (invitrogen) followed by an anti-mouse alexafluor 488 conjugated secondary antibody (molecular probes) at the recommended dilution in staining buffer for 30 min in the dark. cells were then stained for 30 min in the dark with a wheat germ agglutinin alexafluor 594 nm conjugate (molcular probes) to allow identification of plasma and golgi membranes. coverslips were washed and mounted onto slides using vectashield containing dapi (vector labs). microscopy was performed using an inverted leica tcs-nt confocal laser scanning microscope. the anti-v5 antibody was also used to detect v5-tagged protein by western blot. cell lysates were prepared following transfection using lysis buffer (0.15 m sodium chloride, 0.5% (v/v) sodium deoxycholate, 0.1% (w/v) sds, 50 mm tris-cl ph 8.0) and protease inhibitor cocktail (sigma). lysates were incubated for 15 min on ice followed by sonication to shear genomic dna. following bradford analysis equal protein content from jv v5 and jv flc lysates were run on a 10% sds-page gel and subsequently transferred onto immobilon-p pvdf membrane (millipore) according to the manufacturer's recommendations. the membrane was probed using the anti-v5 monoclonal antibody at the manufacturer's recommended dilution, followed by an anti-mouse hrp-copnjugated secondary antibody (santa cruz) at the recommended diltution. the ecl western blotting reagents kit (g.e. healthcare) was used to detect antibody bound protein, which was visualised by exposure to biomax light film (kodak). crfk cells were seeded into 6 well trays and transfected with capped jv v5 rna as described. following a 24 hr incubation cycloheximide (sigma) was added to the cells at a final concentration of 50 mg/ml. cells were harvested at indicated times for the preparation of lysates for v5 western analysis as described above. bradford reagent (sigma) was used to ensure equal loading of lysates according to the manufacturer's recommendations. studies into diarrhoea of young calves. sixth communication: detection and determination of pathogenicity of a bovine corona virus and an undefined icosahedric virus. archives of experimental veterinary medicine studies into diarrhoea of young calves-seventh communication: ''zackenvirus'' (jena-agens 117/80)-a new diarrhoea pathogen to calf. archives of experimental veterinary medicine complete genomic characterization and antigenic relatedness of genogroup iii, genotype 2 bovine noroviruses genotype 1 and genotype 2 bovine noroviruses are antigenically distinct but share a cross-reactive epitope with human noroviruses characterization of a calici-like virus (newbury agent) found in association with astrovirus in bovine diarrhea the seroepidemiology of genogroup 1 and genogroup 2 norwalk-like viruses in italy molecular characterization of a bovine enteric calicivirus: relationship to the norwalk-like viruses two nonoverlapping domains on the norwalk virus open reading frame 3 (orf3) protein are involved in the formation of the phosphorylated 35k protein and in orf3-capsid protein interactions processing map and essential cleavage sites of the nonstructural polyprotein encoded by orf1 of the feline calicivirus genome rabbit hemorrhagic disease virus: genome organisation and polyprotein processing of a calicivirus studied after transient expression of cdna constructs organisation and expression of calicivirus genes cleavage map and proteolytic processing of the murine norovirus nonstructural polyprotein in infected cells norovirus protein structure and function open reading frame 1 of the norwalklike virus camberwell: completion of sequence and expression in mammalian cells norwalk virus n-terminal nonstructural protein is associated with disassembly of the golgi complex in transfected cells norwalk virus nonstructural protein p48 forms a complex with the snare regulator vap-a and prevents cell surface expression of vesicular stomatitis virus g protein activity of the norovirus camberwell proteinase and cleavage of the n-terminal protein encoded by orf1 sequence and genomic organization of norwalk virus la, ptb, and pab proteins bind to the 39 untranslated region of norwalk virus genomic rna requirement of poly(rc) binding protein 2 for translation of poliovirus rna stem-loop structure synergy in binding cellular proteins to the 59 noncoding region of poliovirus rna direct evidence that polypyrimidine tract binding protein (ptb) is essential for internal initiation of translation of encephalomyocarditis virus rna sonenberg n (1993) la autoantigen enhances and corrects aberrant translation of poliovirus rna in reticulocyte lysate interaction of cellular proteins with the 59 end of norwalk virus genomic rna the genomelinked protein vpg of the norwalk virus binds eif3, suggesting its role in translation initiation complex recruitment vpg of murine norovirus binds translation initiation factors in infected cells new ways of initiating translation in eukaryotes? polyprotein processing in southampton virus: cleavage in trans by the 3c-like protease identification of an epitope on the p-proteins and v-proteins of simian-virus 5 that distinguishes between 2 isolates with different biological characteristics electron-microscopic observation of feline kidney-cells infected with a feline calicivirus viral gastroenteritis outbreaks in europe key: cord-000347-gdra8xhj authors: gibbons, henry s.; broomall, stacey m.; mcnew, lauren a.; daligault, hajnalka; chapman, carol; bruce, david; karavis, mark; krepps, michael; mcgregor, paul a.; hong, charles; park, kyong h.; akmal, arya; feldman, andrew; lin, jeffrey s.; chang, wenling e.; higgs, brandon w.; demirev, plamen; lindquist, john; liem, alvin; fochler, ed; read, timothy d.; tapia, roxanne; johnson, shannon; bishop-lilly, kimberly a.; detter, chris; han, cliff; sozhamannan, shanmuga; rosenzweig, c. nicole; skowronski, evan w. title: genomic signatures of strain selection and enhancement in bacillus atrophaeus var. globigii, a historical biowarfare simulant date: 2011-03-25 journal: plos one doi: 10.1371/journal.pone.0017836 sha: doc_id: 347 cord_uid: gdra8xhj background: despite the decades-long use of bacillus atrophaeus var. globigii (bg) as a simulant for biological warfare (bw) agents, knowledge of its genome composition is limited. furthermore, the ability to differentiate signatures of deliberate adaptation and selection from natural variation is lacking for most bacterial agents. we characterized a lineage of bgwith a long history of use as a simulant for bw operations, focusing on classical bacteriological markers, metabolic profiling and whole-genome shotgun sequencing (wgs). results: archival strains and two “present day” type strains were compared to simulant strains on different laboratory media. several of the samples produced multiple colony morphotypes that differed from that of an archival isolate. to trace the microevolutionary history of these isolates, we obtained wgs data for several archival and present-day strains and morphotypes. bacillus-wide phylogenetic analysis identified b. subtilis as the nearest neighbor to b. atrophaeus. the genome of b. atrophaeus is, on average, 86% identical to b. subtilis on the nucleotide level. wgs of variants revealed that several strains were mixed but highly related populations and uncovered a progressive accumulation of mutations among the “military” isolates. metabolic profiling and microscopic examination of bacterial cultures revealed enhanced growth of “military” isolates on lactate-containing media, and showed that the “military” strains exhibited a hypersporulating phenotype. conclusions: our analysis revealed the genomic and phenotypic signatures of strain adaptation and deliberate selection for traits that were desirable in a simulant organism. together, these results demonstrate the power of whole-genome and modern systems-level approaches to characterize microbial lineages to develop and validate forensic markers for strain discrimination and reveal signatures of deliberate adaptation. bacillus atrophaeus is a soil-dwelling, non-pathogenic, aerobic spore-forming bacillus related to b. subtilis. for more than six decades, this organism has played an integral role in the biodefense community as a simulant for biological warfare and bioterrorism events (bw) and is commonly referred to by its military two-letter designation ''bg'' [1, 2] . b. atrophaeus has served in studies of agent dispersal [3] , decontamination simulations [4, 5] and large-scale process development [6] . in addition to its historical use as a bw simulant, it is currently in widespread commercial use as a surrogate for spore-forming bacteria [5, 7] and is the basis of numerous assays for spore inactivation [8, 9] . in addition to its role as a simulant, the organism plays an important role in the biotechnology industry as a source of restriction endonucleases and of the glycosylation inhibitor nojirimycin [10] . the taxonomic placement of b. atrophaeus has changed dramatically over the years. originally isolated as b. globigii in 1900 (migula) as a variant of b. subtilis, it was originally distinguished from b. subtilis by the formation of a black-tinted pigment on nutrient agar and by low rates of heterologous gene transfer from b. subtilis [11] . it has been alternately known as b. subtilis var. niger, b. niger, and has been confused with b. licheniformis [12] . other than the formation of the dark pigment, it is virtually indistinguishable from b. subtilis by conventional phenotypic analysis [13] , and the lack of distinguishing metabolic or phenotypic features has contributed to the confusionin the taxonomic placement of this organism. low interspecies dna transfer frequencies suggested substantial divergence [11] . based onanalysis of comparative dna hybridization, phenotypicand biochemical tests, nakamura advocated that pigment-producing b. subtilis-like isolates should be classified as a distinct species termed b. atrophaeus [13] . recently, more sensitive typing methods such as amplified fragment length polymorphism analysis showed that b. atrophaeus strains could be classified into two major biovars: var. globigii encompassing the classical, commonly used bg isolates, and var. atrophaeus encompassing other closely related yet genetically distinct strains [14] . here we report the definitive molecular typing of several bgstrains using whole-genome sequences, and develop a plausible microevolutionary history of a commonly used lineage based on the accumulation of mutations over time and during transfer between laboratories.the selected strains span more than six decades of development, use, and transfer of bgbetween various institutions and laboratories and offer an unparalleled opportunity to investigate mutation under selection and drift over time. phenotypic analysis revealed substantial heterogeneity both between and within strains, even in type strains, while highthroughput metabolic profiling revealed metabolic ''enhancements'' to a population that had returned to the university of wisconsin (uw) from camp detrick in 1952. whole-genome comparisons of single-nucleotide polymorphisms (snps), small insertion/deletion motifs (indels), and large-scale genomic architecture analysis by optical maps are combined to generate a plausible history of acquisition and use of operationally relevant strains by the american type culture collection (atcc) and by several laboratories within the biodefense community. finally, our analysisof mutation profiles revealed potential signatures of the deliberate selection of strains with properties of enhanced growth and spore yields, properties that were deemed desirable in a simulant [6] . we also report genetic differences between strains in use in the biodefense community and the commercial sector that argue for adoption of a more uniform standard for b. atrophaeus as a simulant. strains and growth conditions b. atrophaeus strains and their sources are indicated in table 1 . archival strains were maintained as spores in sterile soil at the university of wisconsin (figure 1 ). the 1013 lineage, originally founded from the 1942 strain, was extensively passaged by serial transfer every 12-18 months on agar slants for 30 years. unless otherwise indicated, strains were grown using lb agar plates, lb agar brothor tryptic soy agar containing 5% sheep's blood (sba, healthlink) at 37uc. spores were germinated by plating on lb media at 37uc. plates were examined by stereomicroscopy using indirect lighting and imaged usinga nikon smz1500 with a total magnification of 166. colonies exhibiting distinct morphologies were repeatedly streaked to confirm stability of the phenotype. genomic dna was prepared from all isolates using the blood and cell culture dna midi kit for bacteria (qiagen) from 10 ml overnight cultures in lb. baci051-n was sequenced at the naval medical research center, while all other isolates were sequenced to .25-fold coverage at the us army edgewood chemical biological center by massively parallel pyrosequencing on the roche/454 gs-flx using the titanium reagent package. draft genome sequences of all isolates were assembled de novo using newbler [15] (roche) and analyzed using both newbler and lasergene (dnastar, madison, wi). the 1942 vogel isolate was designated as the reference strain and was brought to completion using standard finishing techniques. the draft genome of bacillus atrophaeus var.globigii was finished at the department of energyjoint genome institute (jgi) using a combination of illumina [16] and 454 datasets [15] . for this genome, we constructed and sequenced an illumina gaii shotgun library which generated 15120217 reads totaling 544 mb, which was combined with 454 titanium standard library which generated 387327 reads totaling 137 mb of 454 data. all general aspects of library construction and sequencing performed at the jgi can be found at http://www.jgi.doe.gov/. the initial draft assembly contained 25contigs in 25scaffolds. the 454 titanium standard data were assembled with newbler, version 2.3. the newbler consensus sequences were computationally shredded into 2 kb overlapping fake reads (shreds). illumina sequencing data wereassembled with vel-vet, version 0.7.63 [17] , and the consensus sequences were computationally shredded into 1.5 kb overlapping fake reads (shreds). we integrated the 454 newbler consensus shreds, the illumina velvet consensus shreds and using parallel phrap, version sps -4.24 (high performance software, llc). the software consed [18, 19, 20] was used in the following finishing process. illumina data was used to correct potential base errors and increase consensus quality using the software polisher developed at jgi (alla lapidus, unpublished). possible mis-assemblies were corrected using gapresolution (cliff han, unpublished), dupfinisher [21] , or sequencing cloned bridging pcr fragments with subcloning. gaps between contigs were closed by editing in consed, by pcr and by bubble pcr (j-f cheng, unpublished) primer walks. a total of 79additional reactions and 10shatter libraries were necessary to close gaps and to raise the quality of the finished sequence. the total size of the genome is 4 168 266 bp and the final assembly is based on 137 mb of 454 draft data which provides an average 33.46 coverage of the genome and 544 mb of illumina draft data which provides an average 1336 coverage of the genome. the complete sequence and wgs were deposited at ddbj/ embl/genbank under accession numbers listed in table 2 . the wgs versions described in this paper are the first versions, e.g. aefm01000000. templated assembly of the remaining strains were mapped to the 1942 finished sequence using the gsmapper tool in newbler (roche). high-confidence mutations were selected from newbler ''hcdiffs'' calls (table s1 ) by applying additional selection criteria that mandated high quality scores in both reference and templated assemblies with .80% of the sequencing reads differing from the reference, elimination of mutation calls associated with homopolymer tracts (with the exception of tracts that were formed by a deletion -see below), and a minimum coverage depth of 56 with bidirectional sequence reads. finally, the raw 454 reads from the 1942 isolate were mapped to the finished sequence to assess error bias in the 454 process and to correct for residual sequencing errors in the finished sequence. accession numbers of the relevant wholegenome shotgun sequences are found in table 3 . phylogeny was calculated using paup 4.0b10. fifty-eightnucleotide positions were used with gaps being treated as a ''5th base'' and all characters assuming equal weight. one thousandbootstrap replicates were computed using a heuristic search with the optimal criterion set to ''parsimony''. the tree was created using stepwise addition. nineteen loci in which putative mutations were identified from the 454 dataset were re-sequenced from pcr products by standard sanger dye-terminator methods. no false-negatives or false-positives were identified among the re-sequenced loci; however resequencing of the apparent mutation at position 1486408revealed mixed genotypesin several isolates that are artifacts of a large duplication in the 1942 chromosome. therefore, this signalcannot be considered a true snp. annotation, comparative genomic analysis, and multiple alignments preliminary annotations were generated using a combination of the rast [22] algorithm (rast.nmpdr.org). loci containing mutations were used to query the non-redundant (nr) databases and refseq protein databases at ncbi using directed blastx and blastp. the comparative blast tool from rast was utilized for genome-wide protein sequence comparisons to b. subtilis. results were filtered for bidirectional hits. multiple alignments were generated by megalign from the lasergene software package using the custalw algorithm. genomic dna was prepared from live bacteria on agar slants to maximize the yield of extremely high-molecular weight dna. optical maps were generated by digestion with ncoi of dna arrayed linearly on glass slides and the resulting maps were aligned and compared with the mapsolver software package (opgen, inc., gaithersburg md). using an information-based method for genomic classification [23] , the sequence contigs from bg isolates 1942, 1013-2 and 49822 were analyzed in order to map the phylogenetic relationships of these isolates to other bacillus species. in this method, genomic content is characterized by the frequencies of occurrence of short n-mers contained within each sequence (n typically from 3 to 16). these n-mers are then rank ordered by genome. the pair-wise comparison of the rank of n-mers within two different genomes is then used to compute an informationbased genetic distance (ibgd), where the sum of the differences in rank for all possible n-mers is weighted by an entropy factor that depends on the frequencies of occurrence of the respective n-mers in the two genomes. the pair-wise ibgd values are then used to construct a phylogenetic network [24] . bacilli genomes were obtained from genbank. this method for phylogenetic characterization enables computation even with the unassembled reads, and it can be applied to draft or partial genome sequence data, which was the case for the three b. atrophaeus genomes studied here. the first seven bgstrains listed in table 1 were streaked for single colonies on bhi plates and incubated at 33uc overnight, followed by subculturing a second time under the same conditions. subsequently, cell suspensions were prepared according to biolog specifications, with od readings ranging between 0.35-0.45 at 600 nm. biolog phenotypic microarray plates pm1 through pm20, were inoculated according to the manufacturer's specifications, and incubated at 37uc for 72 hours. readings were taken every 15 minutes, and data processed by omnilog phenotype microarray file management/kinetic plot and parametric modules. two biological replicates of the experiment were conducted for each strain. pm1-10 contain single wells for each growth condition whereas pm11-20 contain quadruplicate wells for each condition. the area under the curve (auc) values were computed by adding all omnilog values at all time points for each of the 1200 distinct phenotypes produced from the omnilog software. the auc values from the two different biological replicates for each unique phenotype were averaged. the ratio for each auc was calculated between the 6 query strains (detrick-1, detrick-2, detrick-3, 1013-1, 1013-2, and dugway) and reference parent strain (1942) . for the purpose of visualization, 1920 phenotypes were included in the heatmap (i.e. this better represents the locations of the phenotypes which correspond to different modes of action categories). the same ratios were used for the phenotypes that have replicates. the ratio values were formatted as pm1 to pm20 for each strain across the columns and wells a i to h i , where i = 1 to 12 for the rows. the results were plotted in a heatmap using r [25] . positive growth wells are represented by greenblocks while negative growth wells are represented by red blocks. catalase activitywas assayed by spotting drops of hydrogen peroxide (3%) onto isolated colonies on lb agar plates. colonies were monitored for bubble formation, signifying the release of water and oxygen. a colony was considered to be catalase positive by observation of bubbles. streaks of detrick 1, detrick 2, and 1013 strains were grown for two days on tsa plates containing sba.bacterial cell mass was scraped using an inoculating loop (1 ml) from the streak and resuspended in pbs. sporulation was evaluated by bright field phase-contrast microscopy. phase-bright free sporesand phasedark vegetative cellswere counted. five representative viewing fields were counted from each strain for each experiment. this experiment was completed in triplicate by repeating once per day over the course of three consecutive days. in order to compare the percent sporulation between detrick 1 and detrick 2, and detrick 1 and 1013, a mixed analysis of variance (anova) was used to complete the analysis. strain and viewing field were evaluated as fixed factors, and replicate was included as a random factor. the natural log of the percent sporulation was taken to obtain a normal distribution of the residual error. tukey's method was applied to compare the difference between the mean log percent sporulation. we traceda potential provenance of the commonly used bgstrains through an exhaustive search of the open literature and the archives of the university of wisconsin,which suggested a possible lineage from which the ''military'' bgstrains were derived. the original source of the strains were the collections at the university of wisconsin during the 1930s and 1940s, from which the strains were transferred to camp detrick at the initiation of the us army's bw program at the beginning of the second world war [6, 26] . at camp detrick, bg was used as a non-pathogenic surrogate in process development for sporeforming bacteria it is tempting to speculate that the university of wisconsin supplied bg to porton down: a note found in the archive of dr. baldwin's papers, dated february 19, 1943 , contained an order from dr. fildes (presumably sir paul fildes, a noted bacteriologist active in the british bw program at the time), for a batch of b. subtilis spores. it is not clear whether bg or b. subtilis subsp. subtilis was supplied, or whether this material was actually delivered. unfortunately, original records describing in detail the maintenance of the strains during the period 1942-1955 were destroyed as per us army policy at the time (dr. mark wolcott, usamriid; personal communication), and the personnel who had first-hand knowledge of the strain passage histories and methods are deceased. therefore, the actual source of the camp detrick isolates must be inferred from published work [6] , limited available documentation (e.g. atcc 9372) and the genome sequences presented herefrom camp detrick the isolates were eventually transferred to atcc as b. subtilis var. niger ''red strain.'' the desire to maintain a phenotypically and genotypically uniform simulant throughout the biodefense communityprompted us to elucidate whether significant phenotypic and/or genomic differences had accumulated in any of the commonly used isolates during the growth and transfer of strains to different institutions and to compare the isolates in broad use today to the so-called ''mil-spec'' strain (atcc 9372).in contrast, the origin of atcc 49822 prior to acquisition f. young's laboratory (the depositor) is unclear. we obtained isolates from archival spore suspensions in sterile soilfrom the university of wisconsin with legible labels dating back as far as 1942 ( figure 1 ; table 1 ). these isolates included an archival stock dated 1942 that likely predated the transfer to camp detrick, as well as material that had been returned to the university of wisconsin from camp detrick in 1952. a derivative of the 1942 strain that had been repeatedly passaged in vitro on agar slants over a period ofapproximately 30 years allowed us to compare the genomic signatures of deliberate selection with the effects of long-term in vitro passage. in addition, a sample of strain nrs-356 [13] , which is mentioned as a possible parent strain in correspondence between various academic laboratories and camp detrick, was also obtained from the same source as the 1942 ''vogel'' strain. these isolates were subsampled, germinated on lb plates, screened for colony morphology variation (see below). genomic dna was prepared from these isolates for sequencing. upon initial plating of the archival and modern-daybg stocks, we noted distinct colony morphotypes for many of the strains, with some strains containing multiple variants ( figure 2 , table 2 ). some of these morphotypes were consistent with those observed by hayward et al. [6] whooriginally described the emergence of colony variants in ''b. globigii.'' as in the earlier report, individual morphotypes were stable and did not interconvert with high frequency (data not shown), suggesting that these morphotypes were the result of relatively rare chromosomal mutations, although 1013-1 occasionally threw off papillae in heavier streaks (not shown). multiple morphotypes were noted for atcc9372, atcc 49822, detrick, and 1013, while the archival 1942 isolate, the isolate obtained from dugway proving ground (dugway) and baci051 appeared to be pure populations on lb. all strains tested positive for bgusing real time-pcr primers specific to the recf gene (methods s1) [27] . the appearance of multiple colony morphotypes even within single ''strains'' strongly suggested an asyet undescribed level of genetic diversity within these samples that likely affected the expression of cell-surface components and/or sporulation. the intra-strain colony morphology variation was particularly dramatic in the in vitro passaged 1013 and atcc9372 isolates, in which one variant of each lineage had lost the production of color on lb orsbaplates (figure 2 ), suggesting more dramatic alterations to the genome. draft genome sequences were generated from several bgstrains in our collection. a summary of the results from the sequenced isolates is indicated in table 3 . all of the ''military'' isolates (detrick clones1through 3, baci051, dugway) were extremely closely related to each other and to both atcc9372 variants. the atcc isolates possessed additional mutations that were absent in the ''military'' isolates. the size of the finished and closed genome of b. atrophaeus var. globigii 1942 was 4,168,266 bp, and annotation using rast [22] revealed 4433 features, including 4343 proteincoding sequencesand 90 rna molecules [28] . the preliminary annotations derived from rast are available as genbank .gbk files in the supplementary material. on average, the genome of b. atrophaeus is approximately 86% identical to b. subtilis on the nucleotide level,supporting its delineation as a distinct species and agreeing well with previous estimates [29] . analysis of the ibgd using whole-genome sequences (n-mer length .4) supported the identification of b. subtilis 168 as the closest relative among sequenced bacterial genomes ( figure 3 ). for this particular case, n = 5 (i.e., there were 4 5 = 1024 total 5-mers used to compute the ibgd). the ibgd values were relatively insensitive to the choice of n over the range of 4-8. thethree bggenomes analyzed grouped closely together, and our analysis of the bacillus-wide phylogeny using ibgd revealed the phylogenetic distance of that b. subtilis/b. atrophaeus species from b. anthracis, supporting the inferences published elsewhere from rrna sequence analysis ( figure 3 ) [30] . primary amino acid sequences of rast-annotated proteins are on average 72% (median 83%) identical between b. atrophaeus and b. subtilis. when only the proteins that yielded bidirectional blast hits in rast are examined, the predicted proteome of b. atrophaeus is, on average, 83% identical (86% median) to b. subtilis. we utilized the finished sequence of the 1942 isolate as a reference strain for templated assembly of the remaining bg draft sequences. two additional atcc isolates of b. atrophaeus (49337 and 6537) were distinguishable from var. globigii on the basis of very high snp/indel counts, lower coverage ofand percentage of reads mapping to the 1942 reference, and unique genomic features which supported their proposed classification as var.atrophaeus [14] . the distinguishing genomic features of var.atrophaeus strains and the delineation of the b. atrophaeus clade from b. subtilis will be published elsewhere. optical restriction mapping [31, 32, 33] was used to compare the overall genomic structure of selected isolates. no differencesin overall genome architecture between the ''military'' bg isolates, the archival 1942 isolate, or 1013-1 were observed (figure 4 , data not shown), suggesting that the global architecture of these isolates is relatively stable, even over 30 years of serial in vitro passage.however, the optical maps and sequence coverage analysis of 1013-2 and 9372-1revealed substantial deletions of approximately 72,727and 23,678 bases, respectively, of genomic materialspanning from positions 3,992,613 to 4,065,341 (1013-2) or 4,022,138-4,045,817 (atcc 9372-1) (figure 4 ; table s2 ). the genes within this deleted region are listed in table s3 but notably contain genes encoding for nitrite reduction, germination (gerkabc), and biosynthesis of the lipopeptide surfactin (srfcab) [34, 35] . a defect in surfactin production is a particularly intriguing candidate for the morphology and pigmentation variations in 1013-2 and atcc 9372-1, since disruption of srfa has been shown to have dramatic effects on spreading motility on semisolid media, on biofilm formation [34, 36] , and low-grade hemolytic activity. using the de novo assembled draft sequence from the 1942 isolate as a template for subsequent analysis of snps and small indels in the other ''military'' isolates, we generated a list of highconfidence, discriminatorymutations that differentiate the strains ( figure 5a ). the nature and annotation of the mutations are found in table 4 and can be assigned an approximate temporal order in which they occurred ( figure 5b ). based on this analysis, 1942 is the most likely parental strain for all of the isolates in this study, with the 1013 lineage diverging earliest, followed by 49822, then the ''military'' lineage prior to the transfer to camp detrick. this conclusion is based on the observation that 49822 shares three snps with detrick-1. the latter is the most likely progenitor of the other ''military'' isolates, since it has the fewest mutations relative to strain 1942. detrick-1 can be differentiated from other ''military'' isolates by possessing the parental allele of spo0f rather than the h101r allele (position 3231470) that is characteristic of all of the other ''military'' bgisolatesand the atcc9372 strains. the two colony morphology variants of atcc9372 each exhibited distinct mutation profiles indicating that the reference strain is in fact a mixed population of at least two genetically distinct substrains. the 72 kb deletion in 1013-2 included the structural genes for biosynthesis of surfactin, a cyclic lipopeptide with a mild hemolytic activity [34] . to test whether the ''military'' and in vitro passaged strains possessed low-grade hemolytic activity, we streaked these variants on rich agar media containing 5% sheep's blood and looked for hemolysis. to our surprise, all strains exhibited striking variation in their coloration (figure 2a) , with the 1942, 9372-1 and detrick-1 isolates considerably darker on blood agar than the other ''military'' and in vitro passaged isolates. in addition, on lb the 1013-2 and 9372-1 isolates appeared white and off-white, respectively. pigmentation of b. subtilis colonies is associated with production of a melanin-like pigment by the cota protein, a major component of the spore coat [37] . in addition to the variations in pigmentation, streaks of the 1942 and detrick-1 isolates were consistently translucent under transillumination ( figure 2b ). these zones of translucency are suggestive of weak b-hemolysis, which has previously been observed in b. subtilis strains that produce high levels of surfactin [34, 35, 38] . the other strains exhibited either weak a-hemolysis or none at all, with the exception of the strongly hemolytic 49822-1 variant. at least in the ''military'' lineage, the quasi-hemolytic phenotype and darkbrown colony pigmentation correlated with the presence of a wildtype spo0f allele, suggesting that the ability of b. atrophaeus to lyse red blood cells may be regulated in part by spo0f. however this was not universally the case; the baci051 strain had two discernible variants on sba (not shown), one of which appeared to have recovered partial hemolytic activity ( figure 2b ). to gain insight into the effects of genetic divergence of the adapted isolates on their metabolic capacity, the detrick isolates and the separate 1013 isolates were compared by multiphenotype analysis using the omnilog system, which allows the highthroughput comparison of 96620 discrete growth conditions, including carbon, nitrogen, phosphorus, sulfate, nutrient supplements, ph, osmolytes as well as a broad class of growth inhibitors. the growth of the 1942 strain was used as a reference for determining relative growth rates of the other strains. the results of mutations exhibiting high quality scores in both reference and query sequences and with differences from the template exhibited in .85% of the individual sequencing reads are indicated as a blackened box. in one case (position 259001 in atcc 9372-1) an initial false-negative due to the formation of a homopolymeric tract was found by direct inspection of the assemblies. the genes whose functions are altered by the given mutation are indicated in table 4 these experiments are summarized in figure 6 and table s4 . in general, growth of the 1013 isolates was significantly diminished relative to the 1942 in many different growth conditions, most notably in the ability to use amino acids and peptides as carbon and nitrogen sources, to withstand osmotic stress, and to grow under reduced ph. in addition, the strains had developed sensitivity to beta-lactams, quinolones, and membrane-disrupting activities. these results suggested broad combined effects of several mutations on the phenotype of the strains. in addition to the spo0f(a98p) allele, which is a likely candidate for highly pleiotropic effects on the decision to sporulate under many different conditions, both strains contain substitutions in the yetf and yqge genes that may be contributing to the phenotypes observed. the more pronounced defect in 1013-2 may be attributable to defects in the gerab and gerac genes and the large 72 kb deletion which contains several genes involved in germination. by contrast, the detrick isolates in general grew more robustly than the 1942 strain under multiple growth conditions. increased relative growth rates were particularly pronounced for acidic conditions and media containing osmolytes, but particularly for wells containing sodium lactate [6] . another isolate in the ''military'' lineage, dugway, is clearly derived from the detrick lineage by snp/indel profiling yet has a metabolic profile that is much closer to the parental strain. like the detrick isolates, the dugway strain grows better at low ph, but many of the other conditions do not promote elevated growth relative to 1942. only one mutation differentiates that isolate from the detrick-2 isolate -a 2-bp insertion in the yojo gene encoding a putative activator of nitric oxide (no) synthesis. again, the physiological role of this mutation is unclear, although nitric oxide synthesis plays a critical role in modulating antibiotic resistance in bacillus spp. [39] . in addition to its role in promoting resistance to antibacterial drugs, no is known to modulate b. subtilis genes involved in nitrate respiration when oxygen is limited [40] ; thus the lowered growth in this strain may reflect the inability to grow to higher densities and overcome the resulting lower oxygen tension. an additional isolate, baci051 is clearly derived from dugway, yet two variants have accumulated additional mutations in sigh (spo0h), hpr/scoc, and ebrb. notably, the phenotype of baci051-e on plates more closely resembles the 1942 strain ( figure 2b ). sequencing of the ''military'' isolates revealed a frameshift mutation in the kata gene encoding the major vegetative catalase [41] . the absence of catalase activity in ''military'' isolates was confirmed by adding a solution of 3% h 2 o 2 to smears of various strains. in contrast to the 1942 strain, which exhibited immediate and robust catalase activity, the strains containing the frameshift lacked this activity. a small amount of bubbling could be seen, probably due to the presence of a second catalase normally packaged in spores [42] . to test whether the phenotype observed on blood agar was associated with differences in sporulation, selected strains were grown for two days as patches on blood agar, resuspended in pbs annotations are a combination of rast and directed tblastn and blastp searches vs bacillus databases. 4 forms part of a large polypeptide synthase containing highly homologous regions. 5 also shared with strain atcc 49822. 6 the conserved start codon of the rada gene (bg3899) of b. atrophaeus and b. subtilis falls within the bg3898 orf. therefore bg3898 as called by rast is not deemed likely to be a protein-coding gene. 7 in putative transmembrane region. doi:10.1371/journal.pone.0017836.t004 table 4 . cont. and counted directly. strain detrick-2exhibited significantly higher percentages of phase-bright spores than the detrick-1 strain (figure 7 , mean +/2 standard error of the mean). similar results were observed for the 1942 and dugway strains (not shown). the 1013-1 strain exhibited an even higher degree of sporulation than the detrick-1 strain under identical conditions ( figure 7 ). bacillus atrophaeus has historically been grouped with b. subtilis, and is usually described as a black-pigmented variant (var. niger) because of its many phenotypic similarities to the bettercharacterized b. subtilis. both organisms are soil-dwelling, nonpathogenic saprophytes, but have been differentiated by the ability to produce pigment on nutrient media containing an organic nitrogen source [13] . the orange pigmentation of b. atrophaeus var.globigii spores made it an attractive simulant for b. anthracis, facilitating the detection of dispersed spores in complex environmental samples. recently, more sensitive phylogenetic approaches using aflp have delineated b. atrophaeus as a separate species [13, 14] . the taxonomic confusion has arisen due to inadequately sensitive typing methods, and has led to misattribution of pathogenic qualities associated with some b. licheniformis strains to the b. atrophaeus strains currently in use as simulants [12] , for which no direct evidence of pathogenicity exists. this report defines the genomic composition of b. atrophaeus var.globigii and clearly separates the species by wholegenome phylogenetic analysis. in this study, we generated a high-quality, closed reference genome for the 1942 isolate using a combination of 454, illumina, and directed sanger sequencing. we expect the final genome to have an error rate of ,1 in 50,000 basepairs. when we mapped the 454 datasets for all of the isolates back to the finished sequence that was generated using the same dna, we noted several putative snps that were common to all datasets (table 4) . we believe these represent errors introduced during generation of the final consensus sequence, as they did not appear when the isolates were mapped against draft sequence generated exclusively using the 454 platform; these are currently being verified and the final sequence will be updated. our sequences of multiple, closely related strains of this organism allow us to trace the derivation of the ''military'' bg isolates currently in use to a culture present at camp detrick during the 1940s and 1950s. the origin of atcc 49822 is not as clear, but a publication from that era suggests a possible common origin at the university of wisconsin [43] . while that strain is unlikely to be nrs-356 itself, given the presence of several strain-specific snps in our sequence, the snps common to both 49822 and the ''military'' lineage suggest a common ancestor that is not represented among the strains sequenced for this study. given the lack of original records, it is unclear whether the nrs-356 variant in this study might have passed through camp detrick and been returned to the university of wisconsin. however, given the date on the label and the general secrecy of operations at camp detrick during the second world war [26] we consider this possibility unlikely. during development of bgas a simulant for b. anthracis, strains were selected that exhibited the most desirable characteristics, those being rapid growth, high spore yield, and experimental reproducibility. without being aware of the nature of the genetic alterations in their ''optimized'' strains, bw workers at camp detrick selected a mutant that provided dramatically higher total and relative spore yields, and generated consistent experimental results [6] . these strains were adopted into the inventories of numerous biodefense laboratories and have been used for many figure 7 . the spo0f(h101r) and spo0f(a98p) alleles are associated with hypersporulation. phase-contrast microscopy of bg strains after two days of growth on sba. vegetative cells appear as phase-dark rods, while spores appear as round, phase-bright globules. the mean percentage sporulation of each strain in a representative experiment is given 6sem. the experiment was repeated on three consecutive days; representative results of a single experiment are shown. statistical significance was determined by mixed anova (tukey's method, p,0.05). doi:10.1371/journal.pone.0017836.g007 figure 6 . omnilog phenotypic arrays of b. atrophaeus subsp. globigii strains. six strains were each inoculated into twenty 96-well omnilog plates and grown at 37uc. reduction of tetrazolium dye by respiring cells was measured every 15 minutes by optical density. dye reduction relative to the 1942 strain is shown; the red ratio values indicate less respiration while the green ratio values indicate more respiration as compared to the 1942 strain. individual arrays or strains are displayed in each of the six major columns labeled detrick 1, detrick 2, detrick 3, 1013-1, 1013-2, and dugway. a) heat map of all conditions for each strain. each of the twenty plates for each strain is represented by the notation pm01-pm20 (left-toright for each strain) along the x-axis. the rows represent the well position, and are denoted as a i to h i (i = 1 to 12) from the bottom to the top of the plot in each array along the y-axis. each cell ratio value represents the average of two biological replicates for each strain. plates pm01-pm10 contains single wells for each growth condition, while plates pm11-pm20 contain quadruplicate wells for each growth condition. solid circle indicates wells containing sodium lactate; dotted circle indicates well containing l-serine at ph 4.5. the details of the 1920 growth conditions can be found in the first worksheet labeled ''all strain auc data'' in table s4 . b) most significant phenotypes for each of the six test strains as compared to the 1942 strain. the phenotypes with statistically significant increases and/or the decreases in ratio values for each of the six strains are presented. for the 1013 isolates only the conditions giving the five largest changes are presented. the number in each color block indicates the ratio for the test strain relative to the parent strain for the phenotype specified. the details of all significant phenotypes for each test strain can be obtained in table s4 . bold italic font indicates p,0.05. doi:10.1371/journal.pone.0017836.g006 decades in simulations of decontamination and dispersal [12] . by applying a combination of genomic and biochemical profiling techniques, our data demonstrate that the bg isolates were ''enhanced'' by researchers at camp detrick during the development of the organism as a simulant. the selection of a strain with the desired properties appears to have occurred in at least two discrete steps, as shown by the genome sequences and metabolic profiles. the initial step appears to have been the adaptation of a strain to growth in corn steep liquor, an acidic medium rich in protein and lactate [44] . the robust growth of the detrick strains relative to 1942 in low-ph medium containing high lactate levels is likely due to mutations in mmgd (2-methylcitrate synthase, position 2029530), or a short-chain 3-oxoacyl-[acyl-carrierprotein] reductase (position 3437350), or both. the most likely candidate for a mutation in the detrick isolates that increases growth is the frameshift in mmgdthat occurred following the divergence from the 49822 lineage and results in an altered c-terminus ( figure s1 ). the mmgd geneencodes a 2-methylcitrate synthase that is expressed in the mother cell at the intermediate stages of sporulation [45] . a null mutation in mmgd had no perceptible effect on sporulation, although other tca-cycle enzymes when mutated led to a loss of sporulation [45] . the effects of the frameshift mutation on sporulation and cellular physiology on the function of the enzyme are not clear at this time. we speculate that the frameshift mutation alters the substrate specificity of mmgd in favor of citrate, thus increasing the flux of lactate-derived intermediates through the tricarboxylic acid cycle. evidence for this possibility includes the observations that 2-methylcitrate synthases can have partial citrate synthase activity [45] and that the b. subtilis mmgd gene can complement a glta (citrate synthase) mutant of e. coli [46] . alternatively, alteration of function of mmgd may have predisposed the lactate-adapted strain to acquisition of a hypersporulating phenotype, which is not readily isolated or stable in b. subtilis (see below); however the presence of a hypersporulating phenotype in an independently evolved lineage (1013) of bg indicates that the species may have an intrinsic predisposition to evolving such a phenotype in vitro. the ''military'' strains also grow more readily on media containing d,l-diaminopimelic acid (meso-dap), a major component of bacterial peptidoglycan. corn steep liquor is derived from the incubation of corn in water at 42-55uc, during which a lactic fermentation by a community of wild organisms including numerous uncharacterized bacillus spp. occurs. total bacterial counts at the conclusion of csl production can be quite high [44] , thus the availability of such compounds for growth is not surprising. another potential source of meso-dap could be bacterial autolysis during sporulation. the relative roles of each of the alleles in growth on lactate and/or meso-dap is the subject of current investigation in our laboratory. the second step in the development of bg as a simulant appears to have been the deliberate selection of a hypersporulating variant [6, 47] . importantly, the selection of a strain optimized for spore yield resulted in the fixation of a new spo0f allele that has no counterpart among the available spo0f sequences (figure 8 ). the sole spo0fsequence that differs at position 101 is that of b. clausii, in which tyrosine replaces histidine. notably, the spo0f(h101r) mutation is distinct from a separate spo0f(a98p) mutation present in the in vitro passaged 1013 isolates. given that the amino acid sequence of b. atrophaeus spo0f is identical to that of b. subtilis but for two conservative substitutions, it is likely to have very similar if not identical biochemical properties. detrick-1 and 1942 likely represent one of the two r colony morphotypes described by hayward et al. [6] , whereas the hypersporulating f morphotypes likely arose due to the emergence of the spo0f(h101r) mutation. however, the possibility that detrick-1 represents a reversion mutant at this locus from detrick-2 cannot formally be excluded, but since it represented the dominant morphotype in the 1952 detrick vial we believe this is unlikely. the presence of the spo0f(h101r) allele in the atcc 9372 strains suggests that these strains were acquired by atcc after this mutation appeared within the detrick lineage. experiments to verify the roles of each allele in modulating sporulation are currently in progress. preliminary results indicate that transformation of b. subtilis dspo0f with b. atrophaeus dna and selection of spo+ cells dramatically alters colony morphology independently of the spo0f allele introduced; additional studies to verify the effects of each allele are currently in progress (james hoch, personal communication). the h101r and a98p allelesare likely to alter the response to signals promoting sporulation. aspo0f(h101a) allele results in a sporulation-proficient strain that throws off sporulation-deficient papillae [48] , and the same mutation has been shown to suppress the spo 2 phenotype of a strain containing a defective kina allele. h101 has been proposed as a potential metal-binding site with particular affinity for cu 2+ [49] . binding of cu 2+ (or another divalent metal) at this site may modulate interaction with one or more sensor kinases that promote sporulation. substitution of positively charged arginine at this position could potentially mimic the binding of a metal cation in the loop containing h101, resulting in altered sporulation of the strains due to a change in the interaction with the kinases governing sporulation. it is unclear why, given the proposed role of divalent cu 2+ in suppressing sporulation, h101r would result in a hypersporulation phenotype. the mechanistic relationship between spo0f(h101r) and the hypersporulation phenotype will be tested in future experiments. both variants in the 1013 lineage possess an a98p allele in spo0f. although the presence of several other mutations within this lineage confounds the attribution of the hypersporulating phenotype to this allele at this time, the presence of a mutation in the same gene as another hypersporulating mutant is highly suggestive. the effect of proline substitution at position 98 on spo0f functionis not immediately obvious, but the relatively inflexible proline residue can disrupt alpha-helices in protein structures. the 1013-1 lineage exhibits a hypersporulating phenotype even more pronounced than spo0f(h101r) strains in the ''military'' lineage. the observation that hypersporulating phenotypes have emerged during cultivationof two independent b. atrophaeus lineages point to the possibility that certain in vitro selection pressures may actually favor hypersporulating variants. the selection pressures acting on the sporulation pathwayare highlighted by the sheer number of mutations discovered within the entire data set that occur in proteins known to play roles in sporulation. nine of the 38 mutations (23%) found in all lineages were in genes that directly or indirectly regulate either entry into stationary phase or sporulation; this number exceeds the number that would be expected if mutations were to occur by chance, since less than 5% of b. subtilis genes are dedicated to regulatory processes of any kind [50, 51] . in addition to the mutations found within the ''military'' lineage, the two variants of atcc 49822 shown in figure 2 differ by mutations in rpob (table s5) which also plays a role in entry into sporulation [52] . null mutations in spo0f resulting in asporogenous phenotypes contribute to colony morphology variation in b. anthracis, b. thuringiensis and b. subtilis [53, 54, 55] . enhanced in vitro ''fitness'' is also a likely driver behind the recovery of asporogenic b. anthracis mutants that were discovered during the investigation into the b. anthracis attacks of 2001 [56] . because the process of sporulation is highly energy-intensive and irreversible once commenced, mutants that delay sporulation (or fail to sporulate altogether) to take advantage of remaining nutrients would out-compete wild-type cells during repeated passage in vitro in the absence of other selection pressures, as has been demonstrated in extended in vitro evolution studies with b. subtilis under relaxed sporulation conditions [57] . this may not be universally the case, since gain-of-function mutations in sporulation such as those observed in this studymay compete favorably with wild-type cells if cannibalism of vegetative cells by sporulating bacteria is the dominant selective pressure [58] . finally, horizontally transferred genetic elements can have dramatic effects on sporulation: for example, recent studies of phage lysogeny in b. anthracis have revealed the ability of several integrated phages to positively affect the kinetics of sporulation upon lysogeny of commonly used b. anthracis strains [59] . this study identifies the spo0f(h101r) allele as the signature of a deliberate selection during the development of b. atrophaeus as a simulant. however, without the knowledge of the history and the analysis of the phenotypes of the strains originating from ''camp detrick'' as published in the open literature, attribution of this genotype to a deliberate selection event would not have been definitive, since a similar phenotype is observed in the 1013 lineage which to our knowledge was not deliberately selected for any specific trait. any study designed to determine genomic ''signatures'' of deliberate enhancement or selection is likely to require an analysis of the baseline likelihood that mutations conferring a similar phenotype would emerge and become fixed by natural processes within an evolutionary timeframe consistent with a known time interval or number of passages. available evidence suggests that hypersporulation is not easily evolved in vitro. maughan and coworkers attempted to evolve populations of a laboratory strain of b. subtilis with a hypersporulating phenotype by repeatedly heat-shocking cultures. while their efforts to enrich for hypersporulators failed, other studies revealed that asporogenous mutants evolved readily [60, 61] , confirming many early studies ( [62] and references therein). with the exception of the studies by maughan et al., most ofthese investigators applied selections intended to inhibit sporulation rather than to enrich for strains with elevated sporulation rates. the 1013 lineage was never heat-shocked during its many transfers; thus the adaptations seen in this work are the result of balancing sporulation versus vegetative growth for prolonged periods on agar slants. however, because undomesticated isolates were observed to sporulate to 98-100% [60] , we cannot formally exclude the possibility that in vitro culture of the 1942 strain following its isolation for an unknown period by the university of wisconsin might have selected for a hyposporulating variant. in this scenario, the h101rand a98p mutations would represent suppressor mutations. we consider this possibility unlikely, given the phenotypic similarity of two environmental isolates in the uw collection (1942 and nrs-356). furthermore, a progression toward darker pigmentation and greater hemolysisis evident in the ''military'' lineage ( figure 2b ). these phenotypic changes are associated with the accumulation of additional mutations including a p145l substitution mutation in sigh, a positive regulator of sporulation [63, 64] and an a13p mutation in scoc, a negative regulator of sporulation [65] . together, the strains analyzed in this study suggest strong selective pressures on the genes in the sporulation pathway, and more carefully controlled studies should be carried out to determine the dynamics of in vitro evolution and adaptation of spore-forming organisms, as has been done extensively in e. coli [66, 67, 68, 69, 70] . unexpectedly, the ''military'' lineages were also marked by the loss of catalase activity, whose presence is an identifying feature of both b. subtilis and b. atrophaeus [13] . this activity was present in a separate lineage of in vitro passaged organisms, so it is not immediately clear why ''military'' isolates, i.e. those subjected to selection within the early days of the development of bg as a simulant organism, would have lost the catalase activity characteristic of the parental isolate. because the kata gene product is not found in spores [41, 71] , we consider it unlikely that the absence of this activity would impact the resistance of spores to decontamination reagents, and thus any antioxidant resistance phenotype exhibited by spores of ''military'' isolates would likely have gone unnoticed. however, direct comparisons of the ''military'' b. atrophaeus lineages to the progenitor strains have not been done, and pleiotropic effects of a spo0f mutation on spore physiology cannot currently be excluded. whole-genome approaches are becoming critical components of microbial forensics. the snps and indels identified in the analysis of evidentiary materials currently become the basis for higherthroughput assays to screen large numbers of samples [56, 72] . decreasing costs of whole-genome sequencing, and the comprehensive nature of the analysis, may make this the preferred method of forensic analysis of microbial samples in the future. with recently developed techniques of allele quantitation within populations by mass spectrometry [73] , real-time pcr [74] , and census-bysequencing [68, 75] , it may be possible to quantitate accurately rare alleles within any given microbial population. we are particularly intrigued by the possibility that, given a mixture of different variants and sufficient sequencing power, ultra-high coverage sequencing may prove to be a more quantitative means of enumerating the relative populations in a sample even before the presence of variants has been established. the results from sequencing two strains of baci051 in this study provide evidence of such hidden diversity. the genomic basis of interlaboratory strain variation is only beginning to become evident, with recent studies tracing the histories of commonly used lab strains of b. subtilis 168, e. coli, salmonella enterica serovar typhimurium 14028s, pseudomonas aerugi-nosapa01 and mycobacterium tuberculosis h37rv [76, 77, 78, 79, 80, 81] . these have revealed significant divergence of putatively identical strains from one laboratory to another, largely arising from mutations that accumulate during serial passage. like the earlier work, our study highlights the utility of approaches based on wholegenome sequencing for the discrimination of closely related strains, especially when investigating the provenance for a given isolate. tragically, at least 13 institutions are known to have destroyed archival collections of select agents [82] following the implementation of mandatory monitoring and reporting requirements, representing an incalculable loss of phenotypic and genomic diversity. this report underscores the importance of maintaining the genetic heritage preserved in the culture collections of individual investigators and institutions. discovery of phage display peptide ligands for speciesspecific detection of bacillus spores a miniature biochip system for detection of aerosolized bacillus globigii spores anthrax letters: personal exposure, building contamination, and effectiveness of immediate mitigation measures the sterilizing action of gaseous ethylene oxide; sterilization of contaminated objects with ethylene oxide and related compounds; time, concentration and temperature relationships virulent spores of bacillus anthracis and other bacillus species deposited on solid surfaces have similar sensitivity to chemical decontaminants strain variation as a factor in the sporulating properties of the so-called bacillus globigii difference between the spore sizes of bacillus anthracis and other bacillus species comparative sterilization effectiveness of plasma in o 2 -h 2 o 2 mixtures and ethylene oxide treatment biological indicator for ethylene oxide sterilization, paper strip. the united states pharmacopeia/the national formulary. rockvillemd: us pharmacopeia characterization of bacillus subtilis dsm704 and its production of 1-deoxynojirimycin interspecies transformation in bacillus: sequence heterology as the major barrier advisory panel for the study of long-term health effects of participation in project shad (2007) long-term health effects of participation in project shad (shipboard hazard and defense) taxonomic relationship of black-pigmented bacillus subtilis strains and a proposal for bacillus atrophaeus sp. nov detection of molecular diversity in bacillus atrophaeus by amplified fragment length polymorphism analysis genome sequencing in microfabricated high-density picolitre reactors velvet: algorithms for de novo short read assembly using de bruijn graphs base-calling of automated sequencer traces using phred. ii. error probabilities base-calling of automated sequencer traces using phred. i. accuracy assessment consed: a graphical tool for sequence finishing finishing repeat regions automatically with dupfinisher the rast server: rapid annotations using subsystems technology genomic classification using an information-based similarity index: application to the sars coronavirus application of phylogenetic networks in evolutionary studies r: a language and environment for statistical computing the biology of doom: the history of america's secret germ warfare project evaluation of the biological sampling kit (biskit) for large-area surface sampling the national microbial pathogen database resource (nmpdr): a genomics platform based on subsystem annotation reclassification of bioindicator strains bacillus subtilis dsm 675 and bacillus subtilis dsm 2277 as bacillus atrophaeus identifying experimental surrogates for bacillus anthracis spores: a review optical mapping as a routine tool for bacterial genome sequence finishing optical mapping and 454 sequencing of escherichia coli o157 : h7 isolates linked to the us 2006 spinach-associated outbreak optical maps distinguish individual strains of escherichia coli o157 : h7 identification of a genetic locus required for biosynthesis of the lipopeptide antibiotic surfactin in bacillus subtilis srfa is an operon required for surfactin production, competence development, and efficient sporulation in bacillus subtilis rapid surface motility in bacillus subtilis is dependent on extracellular surfactin and potassium ion cota of bacillus subtilis is a copper-dependent laccase cloning and characterization of srfb, a regulatory gene involved in surfactin production and competence in bacillus subtilis endogenous nitric oxide protects bacteria against a 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anthrax agent bacillus anthracis: bacteriophage-mediated ecological adaptations stochastic processes influence stationaryphase decisions in bacillus subtilis the population genetics of phenotypic deterioration in experimental populations of bacillus subtilis studies on bacterial spores: iii. a contribution to the physiology of spore production in bacillus mycoides temporal regulation of the bacillus subtilis early sporulation gene spo0f bacillus subtilis early sporulation genes kina, spo0f, and spo0a are transcribed by the rna polymerase containing sigma h sequence analysis and regulation of the hpr locus, a regulatory gene for protease production and sporulation in bacillus subtilis experimental evolution with e. coli in diverse resource environments. i. fluctuating environments promote divergence of replicate populations genome evolution and adaptation in a long-term experiment with escherichia coli genome-wide mutational diversity in an evolving population of escherichia coli the spread of a beneficial mutation in experimental bacterial populations: the influence of the environment and genotype on the fixation of rpos mutations clonal adaptive radiation in a constant environment formation and composition of the bacillus anthracis endospore strain-specific single-nucleotide polymorphism assays for the bacillus anthracis ames strain high-throughput oncogene mutation profiling in human cancer rapid quantification of single-nucleotide mutations in mixed influenza a viral populations using allele-specific mixture analysis detecting snps and estimating allele frequencies in clonal bacterial populations by sequencing pooled dna high-precision, whole-genome sequencing of laboratory strains facilitates genetic studies tracing ancestors and relatives of escherichia coli b, and the derivation of b strains rel606 and bl21(de3) genome diversity of pseudomonas aeruginosa pao1 laboratory strains genomic sequencing reveals regulatory mutations and recombinational events in the widely used mc4100 lineage of escherichia coli k-12 short-term signatures of evolutionary change in the salmonella enterica serovar typhimurium 14028 variation among genome sequences of h37rv strains of m. tuberculosis from multiple laboratories destruction of microbial collections in response to select agent and toxin list regulations we thank dr. kevin p. o'connell for helpful discussions and insights into the manuscript and for facilitating the collaboration with the university of wisconsin. we also thank kristin willner and amy butanifor assistance with sequencing. we thank gary ouellette for help with phylogenetic analysis and drs. mark wolcott (usamriid) and james hoch (scripps) for helpful discussions. the opinions presented here are those of the authors and are not necessarily those of the u.s. government or any of its agencies. information in this report is unclassified and cleared for public release. key: cord-004091-gex0zvoa authors: abdulkareem, shaheen a.; augustijn, ellen-wien; filatova, tatiana; musial, katarzyna; mustafa, yaseen t. title: risk perception and behavioral change during epidemics: comparing models of individual and collective learning date: 2020-01-06 journal: plos one doi: 10.1371/journal.pone.0226483 sha: doc_id: 4091 cord_uid: gex0zvoa modern societies are exposed to a myriad of risks ranging from disease to natural hazards and technological disruptions. exploring how the awareness of risk spreads and how it triggers a diffusion of coping strategies is prominent in the research agenda of various domains. it requires a deep understanding of how individuals perceive risks and communicate about the effectiveness of protective measures, highlighting learning and social interaction as the core mechanisms driving such processes. methodological approaches that range from purely physics-based diffusion models to data-driven environmental methods rely on agent-based modeling to accommodate context-dependent learning and social interactions in a diffusion process. mixing agent-based modeling with data-driven machine learning has become popularity. however, little attention has been paid to the role of intelligent learning in risk appraisal and protective decisions, whether used in an individual or a collective process. the differences between collective learning and individual learning have not been sufficiently explored in diffusion modeling in general and in agent-based models of socio-environmental systems in particular. to address this research gap, we explored the implications of intelligent learning on the gradient from individual to collective learning, using an agent-based model enhanced by machine learning. our simulation experiments showed that individual intelligent judgement about risks and the selection of coping strategies by groups with majority votes were outperformed by leader-based groups and even individuals deciding alone. social interactions appeared essential for both individual learning and group learning. the choice of how to represent social learning in an agent-based model could be driven by existing cultural and social norms prevalent in a modeled society. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 when facing risks, people go through a complex process of collecting information, deciding what to do, and communicating with others about the effectiveness of their actions. social influence may interfere with personal experiences, making peer groups and group interactions important factors. this is especially important in understanding disease diffusion and the emergence of epidemics, as these phenomena annually take thousands of lives worldwide [1] . hence, good responsive and preventive strategies at both the individual and government levels are vital for saving lives. a choice of strategy depends on behavioral aspects, complex interactions among people [2] , and the information available about a disease [3] . perceiving the risk of an infectious disease may trigger behavioral change, as during the 2003 sars epidemic [4] . gathering information and experience through multiple sources is essential for increasing disease risk awareness about the disease and taking protective measures [5] . to help prevent epidemics, we need advanced tools that identify the factors that help spread of information about life-threatening diseases and that change individual behavior to curbs the diffusion of disease. various scientific approaches have been developed to tackle this challenge. network science is prominent in studying how epidemics propagate and how different awareness mechanisms can help to prevent the outbreak of disease. some researchers propose a framework with different mechanisms for spreading awareness about a disease as an additional contagion process [6] . others model populations as multiplex networks where the disease spreads over one layer and awareness spreads over another [7] . the influence of the perception of risk on the probability of infection also has been studied [8] . several recent studies have shown how information spreads in complex networks [9, 10] . however, a different approach is needed to account for individual heterogeneity (such as income and education levels), the richness of the information on social and spatial distance or media influence. here, a combination of modeling with data-driven machine learning becomes particularly attractive. simulation tools are commonly used to assess the effects of policy impacts in the health domain [3, 11, 12] . among the models for policy-making, agent-based modeling (abm) is recommended as the most promising modeling approach [13] . abm studies the dynamics of complex systems by simulating an array of heterogeneous individuals that make decisions, interact with each other, and learn from their experiences and the environment. the method is widely used to analyze epidemics [14] [15] [16] [17] . its advantage is in analyzing the factors that influence the spread of infectious diseases and the actions of individual actors [18] . as a bottom-up method, abm integrates micro-macro relationships while accommodating agents' heterogeneity and their adaptive behavior. it ensures that the interaction between the spatial environment and the behavior agents can integrate a variety of data inputs including aggregated, disaggregated and qualitative data [19] [20] [21] [22] . two processes are essential in representing agents' health behavior and disease dynamics, the evolution of risk perception, and selection of a coping strategy. hence, the core of a disease abm lies in defining the learning methods that steer these two processes. sensing of information (global, from the environment, and social, i.e., from other agents), exchanging information (i.e., interactions between agents), and processing of information (i.e., decision making) are critical. machine learning (ml) techniques can support these three elements and offer a more realistic way to adjust agents' behavior in abm [23] [24] [25] [26] . as more data become available in the analysis of the spread of disease, supporting abm with data-driven approaches becomes a prominent research direction. ml has the potential to enhance abm performance, especially when the number of agents is large (e.g., pandemics) and the decision-making process is complex (e.g., depending on both past experience and new information from the environment and peers). ml approaches in abm can provide agents with the ability to learn by adapting their decision-making process in line with new information. people make decisions both as individuals and as members of a group who imitate the decisions taken by the group or its leader [27] . information about social networks is becoming increasingly available, e.g., through social media analysis. it may reveal collective behavior in various domains, including health [28] . for example, people are not entirely rational and imitate others in their views about vaccines [29] . many abms rely solely on the decisions of individuals, paying little attention to group behavior [30] . yet, mirroring emotions, beliefs, and intentions in an abm with the collective decision making of crowds affects social contagion in abms [31] . agents-individuals and groups-may learn in isolation or through interactions with others, such as their neighbors [32] . in isolated learning, agents learn independently, requiring no interaction with other agents. in interactive learning, several agents are engaged in sensing and processing information and communicating and cooperating to learn effectively. interactive learning can be done in multiple ways, i.e., based on different social learning strategies [33] . agents might be represented as members of local groups, learning together and mimicking behavior from other group members (i.e., collective learning) [34] . yet, the impact of different types of interactive learning in groups compared to learning by an individual is an under-explored domain in the development of abms of socio-environmental systems. this article examines the influence of individual vs group learning on a decision-making process in abms enhanced with ml. to illustrate the implications of individual and collective intelligence in abms, we used a spatially explicit disease model of cholera diffusion [35] as a case study. bayesian networks (bns) steer agents' behavior when judging on risk perception (rp) and coping appraisal (ca). we quantitatively tested the influence of agents' ability to learn-individually or in a group-on the dynamics of disease. the main goal is, therefore, methodological: to introduce ml into a spatial abm with a focus on comparing individual learning to collective learning. the added value of the analysis of alternative implementations of learning in abms goes beyond the domain of disease modeling. it illustrates the effects of individuals learning and collective learning on the field of abms of socio-environmental systems as a whole. therefore, our main objectives are to (1) simulate the learning processes of agents on a gradient of learning from individual to collective, and (2) understand how these learning processes reveal the dynamics of social interactions and their emergent features during an epidemic. to address these objectives, the article aims to answer the following research questions: (rq1) what is the impact of social interactions on the perceptions and decisions of intelligent individuals facing a risk? (rq2) how do different implementations of group learning-deciding by majority voting vs by leaders-impact the diffusion process? (rq3) what are the implications of implementing collective learning for risk assessment combined with individual coping strategies? by answering these methodological questions for our case study, we reveal whether individuals perform better than groups at perceiving risks and at coping during epidemics. to explore the implications of intelligent learning on the gradient from individual to collective, we advance the existing cholera abm (cabm) originally developed to study cholera diffusion [35] . in cabm, mls steer agents' behavior [23, 35, 36] , helping them to adjust risk perception and coping during an epidemic outbreak. for this study, we ran eight abms to test various combinations of individual and group learning, using different information sources-with or without interactions among agents-as factors in the bns. we investigate the extent to which the epidemic spreads, depending on these different learning approaches regarding risk perception and coping decisions. s1 appendix provides a technical description of the model and the mls. below we briefly outline the processes in cabm essential to understand the performed simulation experiments. nowadays, 69 countries worldwide are labeled as cholera-endemic, with 2.8 million cases each year leading to 91,000 deaths [37] . people in urban slums and refugee camps are at high risk of cholera because of limited or no access to clean water and adequate sanitation. cabm is an empirically and theoretically grounded model developed to study the 2005 cholera outbreak in kumasi, ghana [35] . the open-source code for the model code is available online. cabm is grounded in the protection motivation theory (pmt) in psychology [23, 38] . the empirically-driven bns model a two-stage decision process of people facing a disease risk: learning to update risk perceptions (threat appraisal, bn1 in fig 1) and making decisions about how to adapt their behavior during the epidemic (coping appraisal, bn2 in fig 1) . according to pmt, threat appraisal depends on individual perceptions of the severity of the disease (evaluating the state of the environment and observing what happens to others) and one's own susceptibility. the coping appraisal is driven by the perceived response efficacy (the belief that the recommended behavior will protect) and one's own self-efficacy (the ability to perform the recommended behavior). cabm simulates individuals who are spatially located in a city. these agents differ by income and education level. individual agents form households and neighborhood groups and are susceptible to cholera at the beginning of the simulation. cabm implements an adjusted seir model [39] as explained in fig 2 below . instead of going directly from susceptible to exposure, we introduced an awareness component in which agents can assess their risk. options included: no risk perception in which the agent will be exposed (arrow 1, fig 2) ; no risk perception yet no exposure (arrow 2, fig 2) ; and risk perception leading agents to the coping phase (arrow 3, fig 2) . exposure to cholera takes place through the use of unsafe river water. agents can influence their exposure by selecting alternative water sources. these alternative water sources can either reduce their exposure to zero (arrow 5, fig 2) or have no effect on their infection risk (arrow 4, fig 2) . their actions are contingent on income and education levels, as well as on the information that they retrieve from their own experience, information received from others, or observations of the environment. it is not possible to judge by sight whether surface water is infected with cholera, but the agents use other types of visual pollution, e.g., floating garbage, as a proxy. when household agents find the visual pollution level too high, they may decide on an alternative. household agents with high incomes do not take a risk and will buy safe water. in cabm, the risk perception was updated using bn1, which depends on the agent memory (me), the visual pollution at the water fetching point (vp), and the evidence of the severity of the epidemic based on communication from the media (m) and potentially with neighbor households (cnh). media broadcast news about cholera starting on day 21 onward (see: ghana news archive). during a simulation, household agents may also interact with their neighbors zero to seven times a day (applied randomly) [40] . when interactive learning was activated, social interactions among household agents helped to share information on cholera cases that occurred in their communities and on the effectiveness of coping decisions. if risk perception was positive (bn1 returns a value above 0.5), household agents activate bn2 to decide which action (d1 -d4, fig 1) to take given their income (i) and education (e) level, the experience of their own household with cholera (oe), and possibly their neighbors' experiences with cholera (ne) [22] . s1 appendix provides further details on how the bns are implemented, together with tables of the parameters. sensitivity analysis of the aggregated model dynamics on the bns inputs and training alternatives can be found in [23, 36] . a feeling of risk among individuals is fueled by the type of information, the amount of information communicated, and the attention to specific information that may trigger fear and stimulate a learning process regarding a new response strategy [41] . gained information helps individuals (i) to estimate the severity of the emerging event, (ii) to assess the probability of being exposed to infection, and (iii) to evaluate the efficiency of their coping responses. we used a complex network approach to illustrate the gradual processes from individual to collective learning in cabm (fig 3) . each stage is presented as a single network over which a given learning process spreads. each network in fig 3 had the same set of nodes and connections to show how different processes can lead to different outcomes in the same network structure when different information is used to make decisions. in individual learning (fig 3, process 1a and process 1b), agents depend on their prior knowledge (memory, experience, and/or the perceived risk of the environment, such as visual pollution). such learning is the process of gaining skills or knowledge, which an agent pursues individually to support a task [42] . group learning is the process of acquiring new skills or knowledge that is undertaken collectively in a group of several individual agents and driven by a common goal [32] . group learning can be realized by making all group members use their own ml algorithms to gather information to perform a specific sub-task (decentralized), and then pool their opinions collectively by making one decision for the entire group (fig 3, process 2a and process 2b). here, we adopt a "majority vote" as the resolution mechanism in the decentralized group decision-making. however, group learning can also be realized by introducing a single agent (leader) who uses ml to learn for the whole group to help it accomplish its group task (centralized). in the centralized group learning, agents in the group copy the decisions of their leader. in both cases, all agents that belong to a group share the same decision, but the information on which this decision is based on varies considerably (fig 3, process 3a and process 3b). both individuals and groups may learn by either by taking information from their social networks (i.e., have it as an additional source of information in their ml algorithms) or not. when individual agents are isolated learners (fig 3, process 1a) , they do not have a social network but use only their own information to make a decision in an isolated environment using the information they possess. when individuals learn in an interactively (fig 3, process 1b) , https://doi.org/10.1371/journal.pone.0226483.g003 they gain new skills or knowledge by perceiving information, experience, and the performance of other agents through their social network. like individual agents, groups can also learn in isolation or interactively. in isolated learning, agents learn independently within their groups, without exchanging any information with each other or with their neighbors (fig 3, process 2a and process 3a). in interactive learning, agents communicate with their neighbors to learn effectively within their groups (fig 3 process 2b and process 3b). neighbors could be members of the same group or belong to other income/education groups but live in the same community and share the water collection points. therefore, there might be communication across the groups (fig 3, process 2b and process 3b). groups can be defined in different ways and at different hierarchical levels. this model uses three levels of an organization, the individual agent, groups of agents and communities that comprise several groups. in cabm, household agents living in the same community are grouped based on their income and education level since their coping behavior depends on these factors. agents' behavior in the disease abm also is contingent on their geographic location. hence, all neighbors that share the same water fetching point may contact and exchange information between their groups in cabm. the size and compilation of the groups impact the results of the different learning strategies. when applying interactive learning, a group's decision can be influenced by information retrieved from neighbors inside the group and neighbors outside the group but inside the community. for interactive groups, process 2b (fig 3) shows a situation in which individual household agents make decisions that account for interactions in their social networks (as in process 1b). then each household conducts a majority vote, allowing it to proceed with the option chosen by the majority of its members. process 3b (fig 3) shows a situation in which the leaders of each group make decisions based on their interactions with others (nodes a, b, and c are leaders of groups g1, g2, and g3 respectively in fig 3) . the decisions of group leaders are adopted by the household agent of the group. we designed eight simulation scenarios to answer the research questions about the influence of isolated vs interactive individual learning (rq1); centralized vs decentralized learning in processes-during both the risk perception (rp, bn1) and coping appraisal (ca, bn2) processes (rq2); and collective learning about risk perception combined with individual coping appraisal (rq3) on the dynamics of the epidemic and the performance of the model (table 1) . we systematically vary cabm settings following the steps in fig 4 to change the gradient of intelligent learning (steps 2 and 3) in different cognitive stages corresponding to our decisions of interest: risk and coping appraisal (step 1). table 1 shows the setup of the eight scenarios that reflects the three stages shown in fig 4. the area of the case study captured in cabm is 19.2 km 2 and comprises of 21 communities. we assumed that high-income households bought water, so they were excluded from intelligent learning. communities can have up to four groups based on their income and education levels. ten to fifteen percent of the household agents in the case study area usually fetch water from the river. two communities in our dataset (#11 and #20) hosted only high-income households, so they were excluded from the intelligent learning. hence, we simulated 76 groups spread over 19 communities. each simulation was run for 90 days with a time step equal to one hour. given the inherent randomness of abms, we ran each model for 100 times, generating a new synthetic population every 10 runs. besides the extensive gis data and aggregated data on disease dynamics, we ran a survey via a massive open online course (mooc) geohealth in two rounds (2016 and 2017) to gain data on individual behavior. the participants-primarily students from developing countrieswere introduced to the problem of cholera disease, saw pictures of water, and were asked if they would use the water as it is (d1 in fig 1) , walk to a cleaner water point (d2), or use the water after boiling it (d3). the survey data were used to construct and train our bns [36] . we also used these data to evaluate the results of expert-driven bns in cabm [43] . table 2 shows that trust in boiled water was much higher than trust in un-boiled water. agents also changed their behavior and began boiling water in the model. to evaluate the impact of individual and social intelligence on agents' learning processes regarding risk perception and coping appraisal and the resulting patterns of disease spread, we used four output measures: disease diffusion, risk perception, spatial patterns, and model performance. these aspects are described in more detail in the odd protocol (s1 appendix). we also measured the performance of models m1 -m8 in terms of run time and the number of intelligent decision steps, i.e., when agents called their bn1 and/or bn2. given the stochastic nature of abms, we ran each of the eight models 100 times. the average and standard deviations of the results of these runs for each output measure were listed in table 3 . behavioral changes can lead to different duration times of the epidemic and reduce the number of infected cases [44] . this was shown by running cabm with the eight models. models m5, m6, m7, and m8 recorded a longer duration of active infection during the epidemic (75-79 days, table 3 ). these results are closer to the real duration of the epidemic in 2005 (75 days, table 3 ). m5, m6, and m8 applied centralized learning, while m7 applied decentralized learning, but only for the risk perception stage. m2, which is individual learning with social interactions, also recorded a shorter duration when compared to the real data of 2005 (68 days in m2). however, isolated learning and decentralized learning for both risk perception and coping appraisals recorded shorter epidemic duration, with an average difference of -25% compared to the empirical data (table 3) . all eight scenarios generated more infected cases than the empirical data. this was because infection with cholera bacteria leads to a clinical spectrum that ranges from asymptomatic cases to symptomatic cholera cases. asymptomatic cases are not reported, although they represent roughly half of all cases [45] . in our simulations, we did not differentiate between symptomatic and asymptomatic cases; we considered all infected cases are considered to be symptomatic cases. therefore, following [45] , in table 3 , we reported that 57% of the total infected cases occurred when running the eight models. m8, which uses centralized learning for risk perception and individual interactive learning for coping appraisal, reported the fewest infected cases (2,107 against 1,621 in reality). this was followed by m2 (individual social learning) with 2,279 cases and m1 (individual isolated learning) with 2,457 occurrences. these three values reflect the fact that when household agents learned to cope and make decisions individually, they were more efficient than when they were in groups. when these decisions were combined with social interactions, they lead to better protection (m2 and m8). in general, group behavior had a negative effect, although centralized groups had a less negative impact compared to decentralized ones. finally, in m7, where household agents learned risk perception in decentralized groups and learned to cope individually, 2,911 infected cases were recorded (table 3) . hence, cabm household agents' engagement in decentralized groups for appraising disease risk hindered the perception of risk, lowering agents' motivation to change their behavior to more protective alternatives. the spatial distribution of infected cases (spi) of m8 reported the closest spi over the communities (0.75) compared to 1 in the empirical data. this was followed by m5, with 0.7 ( table 3 ). the spatial patterns of the two collective learning models (m8 and m5) reflected their similarity to the spatial patterns in the empirical data. the correlation between the peak of the epidemic and the peak of risk perception reflects the responsiveness of the household agents' risk perception of the epidemic. scenarios m2, m5, m6, and m8 were more responsive. that is, the peak of risk perception in m2 came three days after its epidemic peak, and the peaks in m5, m6, and m8 came seven days after their epidemic peaks (table 3) . m1, m3, m4 and m7 showed peaks for risk perception near the end of the simulation time. individuals in m1 were isolated, along with individuals in m3; therefore, they kept following their usual behavior of fetching water and using it as it is. in m4 and m7, household agents depended on majority votes in their groups to make their decisions on risk and to change behavior. more explanations are represented visually in the next sections. table 4 shows the number of steps and the time required to run one simulation of each model. the number of agents that were supposed to go for risk perception daily was 15% of the total number of household agents (which totaled 8,500). this percentage was derived from national statistical data from ghana statistical services [35] . over the 90 days of the epidemic, 114,750 agents appraised their risk perception (use their bn1). table 4 also shows the number of steps, during which agents perceived the risk of disease (i.e., risk perception equals 1). notably, in m3 -m8, if a group at large assessed the risk perception as zero, then none of its members did the coping appraisal, i.e., the number of steps when bn2 was activated is zero. in such cases, only the total number of steps with activated bn1 assessing risk perceptions was included in table 4 . models with centralized learning required the shortest computation times (table 4) . for example, m5, where only the isolated leaders with the centralized learning consult their bns, had the best performance with the shortest runtime. moreover, m5 and m6 recorded the fewest steps across all models. although the average number of agents with risk perception per simulated day was high (410 agents), there were only 6,840 steps in risk perception and the same number of steps when coping appraisal mls were activated. that is, only leaders activated their bn1 and bn2. this is only 22% compared to what it would be if agents decided individually. without voting, only one agent per group assessed the situation and made decisions. this made m5 and m6 time-efficient. on the opposite end, m4 recorded the highest computational time because of the intensive calculations required in the individual agents' network and the decentralized group network. among all models, m4 recorded the longest process time. agents individually perceived risk (bn1) before going back to their groups to negotiate a final decision on risk perception and then repeating the same individual-group sequence for the coping appraisal. in models m5, m6, and m8 only one agent per group-a total of 76 leaders-assessed risk perception daily, leading to 6,840 steps over the 90-day epidemic. in m5 and m6 only the 76 leaders also went for coping appraisal, while in m8, the group members individually assessed the coping appraisal (26,370 steps in m8 vs 6,840 steps in m5 and m6). calibration of the original model was conducted in two steps: first the hydrological submodel was calibrated, followed by a calibration of the complete model [35] . after the calibration, a stability check was performed [35] . for the current work, the objective of this epidemic model is not to reproduce the real data. it is focusing on the impact of social interactions (present or not, on the level of individual or groups) on both risk perception and coping appraisal of the individual agent. to calibrate a scenario further, one would need risk perception data for that area for the duration of the epidemic. however, such data are very scarce, not only for kumasi but worldwide. hence, risk perception was randomized at initialization. therefore, the eight models cannot be calibrated individually because they need to be comparable at initialization. s2 appendix shows the statistical analysis that was performed on the output data of the eight models to show and analyze the distribution of the obtained results. when household agents evaluated the risks of getting cholera and made coping decisions individually (m1), they relied only on their own experience. that is, each had individual bn1 and bn2 and did not communicate with neighbors. scenario m2 extends this stylized isolated benchmark case by assuming that while agents continued to make decisions individually, they table 4 risk perception and behavioural change during epidemics did share information with neighbors about the perception of risk and protective behavior. that is, both bn1 and bn2 included neighbors' experiences among the information input nodes. fig 5 shows the epidemic curves and the dynamics of risk perception for all scenarios. in the absence of social interactions, more agents became infected with cholera. the peak of the epidemic curve in m1 (in-i) is higher than m2 (in-n), leading to 11% more cases of disease ( fig 5 and table 3 ). overlaying risk perception and epidemic curves suggests that when agents made decisions in isolation (m1: in-i), the dynamics of risk perception were hardly realistic (fig 5a) . namely, when the epidemic was at its peak, household agents in m1 responded very slowly, with bn1 delivering a wrong evaluation of risk perception (fig 5a) . they became aware of the risks very late, so when the epidemic vanished, the number of agents with risk perception = 1 kept increasing. in the absence of communication and experience sharing among peers (in-i), the information about disease spread slowly and there was a significant time-lag between the occurrence of the disease and people's awareness. the small stepwise increase, around day 21, was because the media started to broadcast information about the epidemic on that day. in m2, household agents behaved according to the expected pattern: risk perception became amplified by media coverage and social interactions and then vanished as disease cases became rare (fig 5b) . only those who experienced cholera infection in their households remained alert. household agents in m2 after day 21 had more responses to the media's news compared to isolated agents. media supported the agents' social interactions with their risk perception and behavioural change during epidemics neighbors, which led to more agents perceiving risk, especially when the number of infected cases reached their peak (fig 5b) . even in m2, there were limitations of making decisions about risk perceptions individually: risk perception fell too quickly, implying that people stopped worrying about the epidemics although they continued. since household agents in m1 did not have interactions with other agents, running this model required less time than m2 (creating a 10% increase in performance, table 3 ). the interaction between household agents required time to process the information exchanged between agents. in addition, (m1: in-i) and (m2: in-n) were approximately the same in terms of the realistic spatial distribution of infected cases over the communities, with values of 0.65 and 0.66, respectively (table 3) . fig 6 presents the spatial distribution of decision types over the study area in both m1 (in-i) and m2 (in-n). the household agents in isolated learning were not aware of the cholera-infected cases in their neighbors' household. household agents in m1 took an unsecured decision and trusted more in using the water fetched from the river as it is (d1 in fig 6a) . household agents in m2 were more rational and mostly boiled the water that they fetched from the river (d3 in fig 6b) . in decentralized learning, groups of household agents vote for risk perception and coping appraisal. the final decision of the group is the output of the majority votes. thus, all group members follow the final decision of the group. these groups represent the democratic system, which depends very much on the composition of the group. the decentralized groups with a majority vote can lead to a negative perception of risk. besides, a coping appraisal that depends on a majority vote can lead to inappropriate decisions regarding protection from cholera. when individuals are engaged in social groups, their behaviors are not independent anymore https://doi.org/10.1371/journal.pone.0226483.g006 [46] . this leads to an increase in the randomization of decentralized learning models (m3 and m4). these two models had higher standard deviations in all measures ( table 3) . the qualitative patterns of the three scenarios (m3, m4, and m7) were the same regardless of the social interactions that added new information to ml (fig 5) . for the development of the disease, the voting mechanisms seemed to overwrite individual judgments. the m3 scenario assumes that household agents were isolated when performing risk perception and coping appraisals. in contrast, m4 and m7 allowed household agents to communicate with neighbors during the process of risk perception and before making a coping decision. as a result, m4 and m7 generated greater risk perception than m3 (fig 5c, 5d and 5e ). this suggests that the social interactions still amplify both the awareness of risks and the diffusion of preventive actions. given approximately the same peak heights, the epidemic curves in the three majority voting scenarios reported more infected cases than the other models. among the majority votes, m7 reported the fewest infected cases, since household agents in their coping appraisal relied on themselves rather than their decentralized groups. overall, it seems that all three models-m3, m4, and m7 -got the process of disease risk evaluation wrong. in those cases, risk perception slowly grew in the days when the epidemic was peaking (fig 5c, 5d and 5e) and did not react to the peak in any way, which is unrealistic. moreover, risk perception in the three models continued to grow when the epidemics were almost over. risk perception peaked when there was no longer a risk, i.e., in the last days of the simulation, as shown in table 3 . hence, group voting on risk perception operated with a major time lag: household agents ignored early signals of disease that occurred in just a few households. then they increased their awareness about risk only when most of them were already infected, and they continue to be falsely alerted when the epidemic was over. in m3, the small stepwise increase in risk perception represents the response to media, and it is similar to m1 (in-i) in its development (fig 5c) . the household agents in their decentralized groups did not have contact with neighbors, therefore, no cases were reported to them from their neighborhoods. as such, they were disconnected from what is happening around them. in m4 and m7, which included social interactions, the development of risk perception seems more responsive, especially after the activation of media on day 21. nevertheless, their response time was still slow (fig 5d and 5e ). in these models, the group decisions were very much dependent on the composition of the group members' opinions. these varied from one another and had different information sources for the final decisions about risk perception (in both m4 and m7) and coping appraisal (in m4). thus, majority voting led to unsecured decisions. groups in these models were heterogeneous in that household agents had different levels of exposure to the group members with which they voted. decentralized groups with isolated input information (m3) led household agents to vote to use the water fetched from the river (d1) most of the time (fig 7, map a) . because of their lack of communication with neighbors, household agents missed the opportunity to get information about the infection in their neighborhoods. this explains the higher numbers of infected cases in the majority vote models. social interactions in both m4 and m7 helped agents make better decisions, although following the majority still biased their choices. for instance, in m4 high-income communities (upper communities in maps b and c, fig 7) , household agents mostly used the river water as it was even though they were rich enough to boil it before using it (d3) or to buy bottled water (d4). the opposite also occurred when a majority vote forced low-income households to buy bottled water, which is an expensive decision for them. the group voting on the coping appraisal in m4 might have made individual members uncomfortable when they followed the decisions of their groups even though they might not protect. in reality, household agents sought a balance between preventive behavior and their capability to implement it. moreover, there is always the possibility of routinely changing one's mind based on daily updates of information regarding the epidemic and updates from neighbors. as in m4, the household agents in m7 relied on their decentralized groups for risk perception. this, often led to risk ignorance (fig 5e) . however, since the agents in m7 decided on coping appraisals individually, more agents adopted d1 (fig 7c) . when they perceived risk during the last days of the epidemic, household agents in the middle-income level switched to boiling water or buying bottled water (d3, d4 in fig 7c) . those in the low-income level walked to another water fetching point (d2). in centralized groups, one household agent is randomly selected to be the group leader. the leader is responsible for risk perception and the coping appraisal of the group. group members copy the risk perception and disease preventive decisions of their leaders. it is argued that group leaders may improve their group's performance if they model the responses to the situation the group faces [47] . in this article, we considered two types of leaders: a dictator making top-down decision about risk perception and coping strategy (m5 and m6), and an opinion leader evaluating risk perception top-down but giving group members the freedom to pursue their own disease coping behavior (m8). the qualitative trends of all three models coincided with what is expected: peaks caused by amplification of risk perception followed by a gradual decrease when epidemics plateau (fig 5f, 5g and 5h) . the centralized group learning on average represented the processes well, as the leader alerted the group members about the disease. however, since no real data are available on risk perception dynamics or the actual coping behaviors that people pursued during the epidemic, we cannot determine which of the models m5, m6, and m8 is the best. the following subsections compare models with a leader-dictator (m5, and m6) to one with an opinion leader (m8). a dictator-leader decides on behalf of his group regarding disease risk and coping strategies, and both decisions are adopted top-down. a dictator leader learns either in isolation (m5) or in interaction with her/his neighbors (m6). isolated dictators in m5 are overestimated disease risks (fig 5f) . for example, if such a leader had his/ her own bad experience with cholera, s/he would keep warning the group. with social interactions (m6), there is less uncertainty in the process of updating the risk perception than in m5. for example, compare risk perception assessments around the epidemic peak (fig 5g) . fig 8 illustrates the impact of social interactions on the dictator's decisions regarding coping appraisal. isolated leaders guided their groups to various types of decisions (fig 8a) , which were sometimes less secure decisions (e.g., d1). with social interactions, leaders relied on their neighbors and decided more often to walk to a point along the river where the water was cleaner (d2). very few dictators directed their groups to boil the fetched water (d3) or buy bottled water (d4) (fig 8b) . this shows how centralized decisions making undermines heterogeneity in individual circumstances, such as disease exposure or coping capacity. in m8, the leaders in the centralized groups were responsible for evaluating disease risks for their groups, but they interacted with neighbors during the risk perception process. for the coping appraisal, the group members made their own decisions, using the information from their social networks. as a result of this combination of centralized speed alertness about risk perception and individual coping strategies, m8 generated the fewest infections. the shape of the epidemic curve (except for its height) is very close to the empirical data of 2005, (fig 5h) . as in m6, the uncertainty in the process of risk perception in m8, is lower than in m5 (fig 5h) . the risk perception curve developed around the epidemic peak followed the dynamics of the epidemic (fig 5g) . when group members relied on social interaction to learn about the effectiveness of various coping strategies but eventually chose one themselves (m8), there was a diversity of coping strategies. fig 8c shows the spatial distribution of different types of decisions during the simulation. more household agents went for d3 and d4, which were considered to be the most protective decisions. consequently, communities pursued at least three types of decisions, reflecting the disease coping diversity so important for resilience. the goal of this paper is to perform a systematic comparison of individual vs group learning. the methodological advancements showed that different implementations of individual and collective decision-making in agents' behavior led to different model outcomes. in particular, the stepwise approach of testing how learning (on a gradient from individual learning-without any interactions-to collective-with social networks) affects an abm's dynamics is generic and can be used for other models. to illustrate the subtle difference in implementing learning in abms, we used the example of the spatial empirical abm of cholera diffusion with intelligent agents that employ ml to assess disease risk and decide on protective strategies, which define the dynamics of the epidemic. interactive learning, which assumes that agents share information about risks and potential protective actions, outperformed isolated learning both for individuals and in groups. this underlines the fact that social learning in the decision-making process is very important in abms. while we used disease modeling as a case study, the results may be contingent on the endogenous dynamics of this particular cholera abm. notably, simulation results may differ for abms with other underlying dynamics. this calls for further scrutiny in testing and reporting cases of intelligent social and individual learning in other models. the results indicate that decentralized groups with majority votes are less successful than groups with leaders, whether dictators or opinion leaders. when evaluating current disease risks, majority voting appears to be the worst mechanism for group decisions, often arriving at a wrong decision because of time lags compared to the dynamics of objective disease risks. perceiving risk is a very personal decision-making process [48] . in contrast, when leaders develop risk perception and propose it to the group, such groups perform better in terms of risk appraisal. moreover, opinion leaders are very effective in helping their group members be alert about disease while giving them the freedom to make coping decisions that accommodate heterogeneity in their socio-economic status and geographical locations. in contrast, dictatorleaders and majority votes that impose a decision that all group members must follow are less effective in reducing the incidence of disease. in our simulation experiments, the structure of the groups is simple and is formed based on the spatial and socio-demographic characteristics of the agents. as grouping seems to have an impact on the spatio-temporal diffusion of the disease, the importance of disease modeling stresses the fact that for this type of model a careful evaluation of the social structures in the case study area should be conducted, to generate trustworthy results. future research should focus on constructing groups based on different variables (family ties, religion, tribes). also, in our abm the leaders had no particular knowledge but were randomly selected and assigned to groups. in reality, this may not be the case. leaders may have access to better information or have already earned the group's trust and respect. in addition, decentralized groups can be improved by giving greater weights to more trusted partners to make wise decisions. the model's performance can be a strong argument when the number of agents is massive, e.g., when simulating a pandemic or epidemics within a very large population is needed to detect a worldwide diffusion mechanism. in that case, social group learning, as described in model m5, is a very good alternative to individual interactive behavior. moreover, m5 shortens the computation time by 73% while maintaining a good quality model output. the number of contacts each household agent has when they are in their collective learning may impact the diffusion of cholera. however, running a fat tail distribution of the number of contacts would be an interesting topic for future study. different considerations steer the ultimate decision on which type of social behavior to use. besides the technical model performance metrics discussed here, the choice of a particular type of social behavior can also be based on the society that is being modeled. different political systems, the presence of tribes, and different ethnic groups or religious leaders require careful considerations of the social interactions in a model. one should make sure that the actual situation regarding social learning represents the cultural and social norms of the society being modeled. in this article, it was not possible to define, which implementation (m1 -m8) represented the situation in kumasi most closely. to validate the risk perception-behavior, one would need risk perception data for that area for the duration of the epidemic. however, such data are very scarce, not only for kumasi but worldwide. as we illustrated in this study, many different implementations of social behavior using ml are technically possible, but data are needed to validate alternative implementations. yet, research on risk perception during epidemics is often conducted too late (when the peak is over) or at distance (not in the area where the disease spreads). hence, researches provide little empirical proof of people's behavior and risk perception. more research on risk perception during epidemics, including other variables such as cultural aspects and group behavior, can be very helpful in generating a model that represents a specific society realistically. on a technical note, agent-based modeling software does not always include ml toolkits and libraries. this complicates the implementation of different types of social intelligence. hence, better integration of abm and ml in one software package or linkable libraries could eliminate this problem in the future. finally, an important direction of future research is to implement other ml techniques besides bns, such as decision trees and genetic algorithms. in addition, modeling groups with different ml algorithms may lead to different results since groups will be heterogeneous in terms of members' learning algorithms. several developments in health research drew our attention to the implementation of learning in disease models. one is the 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of household contacts of patients with cholera in behavioral modeling and simulation risk perception and human behaviors in epidemics risk perception it's personal. environmental health perspectives. national institute of environmental health science key: cord-285433-ehnu83qe authors: sun, hongliu; qi, cai; niu, yu; kang, tengfei; wei, yongxin; jin, gang; dong, xianzhi; wang, chunhua; zhu, wei title: detection of cytomegalovirus antibodies using a biosensor based on imaging ellipsometry date: 2015-08-21 journal: plos one doi: 10.1371/journal.pone.0136253 sha: doc_id: 285433 cord_uid: ehnu83qe background: cytomegalovirus (cmv) is the most common infectious cause of mental disability in newborns in developed countries. there is an urgent need to establish an early detection and high-throughput screening method for cmv infection using portable detection devices. methods: an antibody analysis method is reported for the detection and identification of cmv antibodies in serum using a biosensor based on high spatial resolution imaging ellipsometry (bie). cmv antigen (cmv-3a) was immobilized on silicon wafers and used to capture cmv antibodies in serum. an antibody against human immunoglobulin g (anti-igg) was used to confirm the igg antibody against cmv captured by the cmv-3a. results: our results show that this assay is rapid and specific for the identification of igg antibody against cmv. further, patient serum was quantitatively assessed using the standard curve method, and the quantitative results were in agreement with the enzyme-linked immunosorbent assay. the cmv antibody detection sensitivity of bie reached 0.01 iu/ml. conclusions: this novel biosensor may be a valuable diagnostic tool for analysis of igg antibody against cmv during cmv infection screening. cmv is the most common infectious cause of mental disability in newborns in developed countries [1] . detection of cmv antibodies is effective for systematic screening for cmv infection [2] . for instance, the cmv immunoglobulin g (igg) avidity assay can help to distinguish primary from non-primary human cmv infections [3] [4] [5] . the key immune methods used for cmv antibody detection are: enzyme-linked immunosorbent assay (elisa) [6] , elecsys [7] , electrochemiluminescence immunoassay (eclia) [8] , immunofluorescence assay (ifa) [9] , flow cytometry (fcm) [10] and immunoblots [11] . additionally, new immune methods for cmv antibody detection have been developed, such as the chemiluminesent microparticle immunoassay (cmia) [12] and protein microarrays [13, 14] . however, these methods suffer from inherent limitations, such as length of testing time, the needs for expensive equipment, specialist skills, low sensitivity, and complicated sample preparation processes. for example, conventional elisa is still the main diagnostic test for cmv, and commercial cmv elisa kits are available. although elisa is often used as a comparison method for cmv antibody detection [13] , obvious shortcomings include the need of tracer label, plate washing, the indirect format of detection, and the length of time necessary for testing. thus, a rapid, simple, direct, and high-throughput method for cmv antibody detection is urgently needed. the first biosensor based on imaging ellipsometry (bie) was developed in 1995 [15, 16] . compared to the methods above, the advantages of bie are evident, e.g., high-throughput multiplexed analysis and quantitative, label-free rapid testing. previous applications of bie mainly focus on the biomedical fields [17, 18] , such as high-throughput disease diagnosis of hepatitis b virus (hbv) marker [19, 20] , the detection of avian influenza virus (aiv) [21] and antibodies against severe acute respiratory syndrome (sars) detection [22] . thus, the biosensor technology offers important tools for disease diagnosis. as such, new applications have recently been developed, including those for the analysis of the interaction between tropomyosin allergens and antibodies [23] , and the interaction between soluble n-ethylmaleimide-sensitive factor attachment receptor (snare) proteins [24] . to date, however, bie has not been applied for the detection of cmv antibody, particularly for the identification of the cmv igg and igm antibodies. the purpose of this study was to detect antibodies against cmv-3a in patient serum using a bie microarray with cmv-3a, as well as to specifically identify captured igg antibody against cmv. antibodies against cmv qualitatively were detected using bie, and then, goat antibody against human igg (anti-igg) was added to the area with the captured cmv antibody to confirm igg antibody against cmv. a standard curve representing different concentration gradients was also established for the quantitative detection of cmv antibodies. as such, the concentration of cmv antibody in serum was quantitatively detected by bie and then compared using elisas. silicon wafers were purchased from the general research institute for nonferrous metals (china). n-hy-droxysuccinimide (nhs) and 1-(3-dimethyla-minopropyl)-3-ethylcarbodiimide hydrochloride (edc), tween-20, bovine serum albumin (bsa), igg, and blocking buffer (10×, b6429) were purchased by sigma. anti-igg and a goat antibody against human igm (anti-igm) were purchased from beijing bo sheng bio-technology co. ltd. cmv-3a was purchased from galaxybio. the cmv-3a was a fusion of three segments of pp150, gp52 and pp65, which have strong antigenicity epitopes. fusion of the multi-epitope both enhances the sensitivity/specificity and reduces false negatives. thus, cmv-3a was used as the ligand in the bie assay. purified cmv antibody (pp65, c-term, rabbit, 1 mg/ml) was purchased from antibodies-online.com. patient serum samples were purchased from qilu hospital of shandong university and clinical information is listed in table a in s2 file. torch elisa kits used to analyze serum samples were purchased from medson inc. the detection process and analysis were executed according to the manufacturer's instructions. microplates were coated with native cmv antigens, highly purified by sucrose gradient centrifugation and inactivated. the solid phase was first treated with the diluted sample, and igg molecules to cmv were then captured, if present, by the antigens. after washing out all of the other sample components, bound anti-cmv igg molecules were detected by the addition of specific polyclonal anti-h-igg antibodies labelled with peroxidase (hrp) in the second incubation. the enzyme captured on the solid phase, acting on the substrate/chromogen mixture, generates an optical signal that is proportional to the amount of anti-cmv igg antibodies present in the sample. a calibration curve, calibrated against the first w.h.o international standard, makes possible a quantitative determination of the igg antibody in the patient. ne solution was prepared with nhs (0.05 m) and edc (0.2 m) in deionized water (18.3 mocm) from a milli-q plus system (millipore, bedford, ma). phosphate-buffered saline (pbs) was prepared with 140 mm nacl, 2.7 mm kcl, 10 mm na 2 hpo 4 , and 1.8 mm kh 2 po 4 (ph 7.3) in deionized water. pbst buffer was prepared with 1% tween-20 in pbs. cmv-3a (0.1 mg/ml) was prepared with pbst. bsa (10 mg/ml) was prepared with pbst. the blocking reagent was a 1:1 (vol:vol) mixture of 10 mg/ml bsa and 10× blocking buffer. purified cmv antibody, igg, anti-igg, and anti-igm were diluted to a concentration of 0.1 mg/ml with pbst. serum samples were diluted as indicated with pbst for qualitative or quantitative detection. the bie combines high spatial resolution imaging ellipsometry (figa in s1 file) with a microfluidic system (figb in s1 file) to analyze biomolecular interaction [25] . the microfluidic system is used for surface patterning and array production, as well as for solution delivery, ligand immobilization and target capture [26] . the microfluidic system has four main parts: a sample plate, multi-cell array, micro-channels and pumps. a multi-cell array was formed when a polydimethyl-siloxane (pdms) pattern mold contacted the surface of a silicon substrate, each cell having an inlet and an outlet for solution passage. the physical size of each cell was~1.5×1×0.5 mm 3 . the inlet micro-channels were placed into sample plate, and the outlet micro-channels were connected with pumps (ism939, ismatec, switzerland. www.ismatec.com) offering negative pressure. the simple channel junctions can be used in serial or parallel formats to simultaneously analyze single or multiple samples. imaging ellipsometry is a display technique for ultrathin film and surface characterization [27] , which is used to read and analyze the protein arrays made by microfluidic systems. the incident wave of polarized light irradiates the sample as a probe beam and is thereby modified resulting in a reflective or transmission beam having the ability to carry sample information, such as protein layer thickness. when imaging ellipsometry is used to detect layer thickness, the reflection intensity is represented in grayscale, and the variation in layer thickness leads to changes in the grayscale value. if the refractive index is invariant, the grayscale value is directly proportional to the thickness of the protein layer within the range of 0~30 nm layer thickness, i.e., i = kd, where i is the light intensity and d is the layer thickness [28] . under these conditions k is a constant and can be determined from a protein layer with known grayscale values and known thickness [29] . there is also a relationship between surface concentration and film thickness: surface concentration (μg/cm 2 )k×d, where k = 0.12 [27] . thus, the grayscale value directly reflects layer thickness and surface concentration. the higher the grayscale value, the thicker the layer and the higher the surface concentration. silicon wafers were cut into 20×10 mm 2 rectangles and rinsed with deionized water. after soaking in piranha solution (30% h 2 o 2 :98% h 2 so 4 = 1:3, vol/vol) for 30 min to increase the number of silanol groups on the wafer surface, and rinsing with deionized water and ethanol, the wafers were soaked in a mixture of 3-aminopropyltriethoxy-silane (aptes) and absolute ethanol (aptes:absolute ethanol = 1:10, vol/vol) and incubated for 2 h with gentle agitation. the reaction of aptes with the surface silanol groups resulted in covalent immobilization of-o-si(oh) 2 -(ch 2 ) 3 -nh 2 , forming a layer of densely packed amino groups on the surface. after rinsing three times with absolute ethanol, the wafers were incubated in a saturated solution of succinic anhydride in ethanol for 3 h with gentle agitation. the ch 2 ch 2 cooco-group of succinic anhydride reacted with the -nh 2 of -o-si(oh) 2 -(ch 2 ) 3 -nh 2 group immobilized on the surface, generating carboxyl groups (-(ch 2 ) 3 nh-co-(ch 2 ) 2 -cooh). once prepared, the wafers were stored in ethanol. the above-modified silicon wafers were used as the substrate. a piece of silicon wafer was placed on the microfluidic mold of the microfluidic system so that surfaces of the wafers were patterned to form small, regular cells in an array format. then, the carboxyl groups were activated by pumping 10 μl of ne into each cell at a flow rate of 5 μl/min and passed through the surface of the wafer. in the presence of nhs, edc transfers carboxyl groups to the sulfo-nhs ester, which reacts with the amino groups of proteins to immobilize the proteins on surface. next, the ligand was immobilized: cmv-3a as ligand or probe was pumped into each cell (10 μl per cell at 1 μl/min). third, blocking occurred by pumping the blocking reagent into each cell (50 μl per cell at 1 μl/min) and passing it through each ligand area. thus, a sensing array surface with cmv-3a was formed to catch cmv antibodies. fourth, targets were detected. pbst was used as blank control (50 μl per cell at 1 μl/min) and purified cmv antibody as a positive control (50 μl per cell at 1 μl/min) by pumping them into several of the individual cells. simultaneously, patient serum samples were also pumped into remaining cells (50 μl per cell at 1 μl/min). the cmv antibodies in the serum were captured when they interacted with the cmv-3a on the sensing surface. all cells were rinsed with pbst (20 μl per cell at 20 μl/min) between every two consecutive operation steps. finally, the microarray wafers were removed from the microfluidic system. after being rinsed with deionized water and dried with nitrogen, the wafers were analyzed using imaging ellipsometry. if the cmv antibodies in the solution or serum interacted with the cmv-3a on the surface and formed a complex, the layer in that area became thicker. the experimental results were recorded as images in grayscale, and binding of cmv antibodies resulted in a significant increase in grayscale value. to detect the specificity of antibodies against cmv, cmv-3a as ligand was immobilized on two columns (fig 1) . pbst buffer was added as blank control to two areas on the first row. simultaneously, purified cmv antibody (0.1 mg/ml) was added as positive control to two areas on the second row. normal serum without cmv antibodies was added as negative control to two areas on the third row. three patient serum samples (no. 956, 933, and 978; see table a in s2 file for sample information) were analyzed on the following rows, respectively. the increase in light reflection density at each area revealed that cmv antibodies in the samples interacted with the cmv-3a immobilized on the chip. we further analyzed the types of cmv antibodies captured by the cmv-3a (fig 2) . in the first step, igg was immobilized as ligand on the first and second columns. simultaneously, cmv-3a was immobilized on the third, fourth, fifth, and sixth columns. in the second step, sample no. 948 was added to the third and fourth columns, and sample no. 940 was added to the fifth and sixth columns. pbst buffer was added as blank control to remaining areas. in the third step, anti-igg and anti-igm were added to third and fourth rows to identify and confirm in the first step, igg was immobilized as ligand on the first and second columns. cmv-3a was immobilized on the third, fourth, fifth, and sixth columns. in the second step, pbst buffer was added as blank control to the corresponding areas in the image. sample no. 948 was added to the third and fourth columns, and sample no. 940 was added to the fifth and sixth columns. in the third step, pbst buffer was added as blank control to the first areas in every column. anti-igg and anti-igm were added to the third and fourth rows, respectively. if the antibody captured by the ligand was igg or igm. pbst buffer was added as blank control to remaining areas. to establish a calibration curve, a serum sample (no. 942, 21.8 iu/ml, see table a in s2 file) was used, and five levels of serial dilution containing 0.011, 0.043, 0.170, 0.681, and 2.725 iu/ ml of cmv antibody were prepared in pbst. cmv-3a was immobilized as ligand on two columns (fig 3) . after blocking, pbst buffer, cmv antibody and normal serum without cmv antibody were added as blank, positive, and negative controls (respectively) to the first, second, and third rows, respectively. the 0.011-, 0.043-, 0.170-, 0.681-and 2.725-iu/ml samples were added from the fourth to the eighth rows, respectively. concentration plotted on the x-axis and the variation of the grayscale value compared to blank control was plotted on the y-axis to generate the calibration curve (fig 3) . after acquiring the calibration curve, the concentrations of antibodies in samples of unknown concentration could be determined on the curve according to their grayscale values. measurements of every sample were repeated in two areas on one chip (fig 4) . commercial elisa cmv antibody kits were used as controls according to the manufacturer's instructions. for statistical analyses, the corresponding p-values were calculated with single factor analysis of variance in microsoft office excel according to the quantity of areas in images and patients' serum samples in table a in s2 file [30] . in qualitative and quantitative detection experiments, significant changes in grayscale value detection areas compared to the blank control areas and the detection areas were deemed positive signals if p-value was < 0.05. if the p was 0.05, the detection areas were deemed negative signals. in comparison of the elisa and bie data, the results of the two methods were deemed in agreement if p was 0.05. if p was < 0.05, the two methods were deemed in disagreement. the correlation coefficient (r-value) of bie and elisa was calculated with analysis of correlation coefficient in microsoft office excel (table b in s2 file). compared to blank controls areas, the purified cmv antibody and patient serum sample detection areas had markedly thicker films, with the average grayscale value displaying significant increases, while negative control areas did not (fig 1) . the mean grayscale value of blank controls was measured at 117.45 ± 0.92, and the value of the purified cmv antibody was measured at 176.8 ± 3.39. thus, the mean grayscale value of purified cmv antibody minus the mean grayscale value of blank control was 59.4 (p = 0.002). the value of the negative control was 125.9 ± 3.18. the mean grayscale value of the negative control minus the mean grayscale value of the blank control was 8.4 (p = 0.07). this indicated that there were specific interactions between the purified cmv antibody and the cmv-3a. therefore, cmv antibodies in patient samples could be captured by the cmv-3a immobilized on the substrate. indeed, the average grayscale value of the serum samples analyzed significantly increased relative to the controls (fig 1) . the mean serum value was 253.58 ± 0.49, and the mean grayscale value of serum minus the mean grayscale value of the blank control was 136.1 (p = 2.8×10 −5 ), indicating that cmv antibodies were abundant in the serum samples. this was consistent with the elisa results (table a in s2 file). when purified human igg was used as the ligand, the average grayscale value of the anti-igg detection areas significantly increased, while the anti-igm detection areas did not (left two columns in fig 2) . the mean grayscale value of the anti-igg minus the mean grayscale value of the blank control was 96.2 (p = 0.003). the mean value of the anti-igm minus the mean grayscale value of the blank control was 8.15 (p = 0.29). these data are indicative of the specific interactions between the anti-igg and the human igg immobilized on the chip, which could be used as references to determine whether the antibodies in samples are igg. compared to the cmv antibody areas, the anti-igg detection areas displayed significant increases, while the anti-igm detection areas did not (fig 2) . for example, the value of sample 948 was 156.2 ± 2.6, while the value of the anti-igg was 189.6 ± 4.4, for a difference of 33.4 (p = 0.02). however, the value of the anti-igm was 154.25 ± 0.25, and the difference was not obvious (p = 0.53). this indicated that igg cmv antibodies were captured by the chip. for sample no. 940 detection, the value of the anti-igg was 237.7 ± 11, and the increase was 77.3 (p = 0.01). the result also indicated that the content of igg in samples no. 948 and 940 was different. five concentration gradients of a serum sample (no. 942, 21.8 iu/ml, see table a in s2 file) measured in serially diluted samples were used to determine the sensitivity of the bie assay (fig 3) . we found that the change in signal intensity was consistent with the increase in cmv antibody concentration. the cmv antibody detection sensitivity levels reached 0.01 iu/ml. the grayscale values of the blank control were 124.7 ± 0.5, and the grayscale values of the dilutions as low as 0.01 iu/ml were 132.3 ± 0.1, i.e.,~6.1% greater than that of the controls (p = 0.03). repeating these tests > 10 times, our results demonstrated that the sensitivity of the assay reached 0.01 iu/ml or less. each variation in grayscale value was linked to a corresponding concentration of the cmv antibody over the range of 0.011-2.725 iu/ml, and the in the first step, cmv-3a was immobilized as the ligand on two columns. in the second step, pbst buffer was added as a blank control to two areas on the first row. simultaneously, purified cmv antibody was added as a positive control to two areas on the second row. negative serum was added as a negative control to two areas on the third row. the serial dilutions of cmv antibodies were added as analytical samples on the following rows. the same concentration was measured in two duplicate areas. quantitative detection of cmv antibodies in clinical serum 41 cmv patients (table a in s2 file) with quantitative results by elisa were tested with bie (fig 4) . for different serum samples, the changes in bie signal intensity were different. the comparison of results between the two methods is shown in fig 5. single factor analysis and correlation coefficient analysis revealed that the results were in agreement between elisa and bie (f = 1.38<f 0.05 = 3.96; p = 0.24>0.05, r = 0.7) ( table b in s2 file). high-throughput detection is suitable for mass cmv screening in women of childbearing age. when coupled with microfluidic technologies, bie can greatly improve the throughput for cmv detection on one chip. the microarrays developed here have multiple cells immobilized with different ligands for different cmv antibody subtypes. imaging ellipsometry is also suitable to high-throughput analysis. it can be used to visualize the variation in signal from all units of the microarray with high spatial resolution. patient sample analysis results from one microarray can simultaneously be identified, and the data can be obtained in several seconds using an imaging ellipsometer. presently, our method can provide simultaneous 48 reaction areas. with its enhanced throughput and lower cost, bie may be used in clinical mass cmv screenings for healthy births in the future. antibody detection is widely available for the clinical diagnosis of cmv infection. however, the identification of antibody types may help to determine the course of disease, and bie may be used as a clinical primary screening tool for cmv patients. igm is produced in large amounts early in infection (reaching a peak in the first month), and is followed by igg production [7, 31] . levels of igm decline in the months following the onset of infection, whereas igg levels persist for the rest of the patient's life [32, 33] . determining igg avidity can provide additional guidance on infection status, and low avidity igg is initially present but increases over time [34] . a positive result for igm combined with low-to-moderate igg suggests avidity a primary cmv infection within the past 3 to 4 months [35] . the visualized bie image (fig 2) could reflect types via difference in brightness, so this technology can achieve rapid screening. to date, our work is simply a demonstration for igg type antibody detection with bie. igm and other antibodies needed to screen for specific antigen can be incorporated in the future for further practical applications. presently, most methods mention the relative sensitivity (%) of detection for cmv antibodies [8, 36] , but little in provided about absolute sensitivity. compared other methods, the absolute sensitivity of bie can reach 0.01 iu/ml, and it has good resolution in the range of 0.1-1.0 iu/ml on the calibration curve for quantitative detection. the reference range for detection of cmv antibodies has previously been published. for example, elecsys (roche diagnostics) immunoassays have an equivocal range (0.5-1.0 iu/ml) of cmv igg [12] . in our experiment, elisa as a control method had a reference range of 0.4-0.6 iu/ml. therefore, the sensitivity of our bie biosensor is below the reference range and has already reached clinical standards. however, there is a substantial discordance between the elisa and the bie in some patients (i.e., 940, 956, 964, 984, and 980). samples from these patients displayed stronger signal using elisa than bie. in elisas, anti-cmv igg molecules are detected by the addition of polyclonal specific anti-h-igg antibodies labelled with horseradish peroxidase (hrp). in contrast, the bie biosensor directly identified the variation of the surface anti-cmv igg concentration after capturing target without a secondary label. thus, this label-free method may avoid some detection of cmv antibodies in patient serum using bie. in the first step, cmv-3a was immobilized as the ligand on six columns. in the second step, pbst buffer was added as a blank control to six areas on the first row. simultaneously, purified cmv antibody was added as a positive control to the last two areas on the second row. patient serum samples were added as analytical samples on the following areas, respectively. the same serum sample was measured in two duplicate areas (no.940, 959,938 no sample, p15-9, and pbst control are underlined). interference factor. in addition, the grayscale images offered uniform areas, helping to avoid false positive signals. when the correlation coefficient (r-value) and p-value were calculated after excluding the number of severe mutations (940, 956, 964, and 984), r-value = 0.93 and p = 0.8 the statistical results showed more agreement between elisa and bie. in conclusion, we have developed a label-free and multiplex screening cmv igg biosensor method. the high-throughput detection is suitable for mass cmv screening of women of childbearing age. further, our results demonstrate that the sensitivity of bie has already reached clinical diagnose standards for anti-cmv igg. thus, on the basis of anti-cmv igg detection, bie can be used for qualitative and quantitative detection of more types of cmv antibodies using a simple and fast procedure. supporting information s1 file. contains fig a. imaging ellipsometry. (a) imaging principle [23] ; and (b) laboratory prototype. fig b. prevention of maternal-fetal transmission of cytomegalovirus maternal igg avidity, igm and ultrasound abnormalities: combined method to detect congenital cytomegalovirus infection with sequelae diagnosis and management of human cytomegalovirus infection in the mother, fetus, and newborn infant new advances in the diagnosis of congenital cytomegalovirus infection maternal igg avidity, igm and ultrasound abnormalities: combined method to detect congenital cytomegalovirus infection with sequelae a simple method to quantitate ip-10 in dried blood and plasma spots clinical evaluation of new automated cytomegalovirus igm and igg assays for the elecsys analyser platform value of cmv-igm detection by electrochemiluminescence immunoassay in diag-nosis of cmv infection in pregnant women. maternal and child health care of china microtubule network facilitates nuclear targeting of human cytomegalovirus capsid the use of flow cytometry for the detection of cmv-specific antigen (pp65) in leukocytes of kidney recipients improving diagnosis of primary cytomegalovirus infection in pregnant women using immunoblots detection of human cytomegalovirus igg antibody using automated immunoassay with recombinant antigens gives uniform results to established assays using whole virus antigens multiplexed infectious protein microarray immunoassay suitable for the study of the specificity of monoclonal immunoglobulins development of recombinant antigen array for simultaneous detection of viral antibodies a biosensor concept based on imaging ellipsometry for visualization of biomolecular interactions imaging ellipsometry revisited: developments for visualization of thin transparent layers on silicon substrates development of biosensor based on imaging ellipsometry and biomedical applications biosensors for health detection of hepatitis b markers using biosensor based on imaging ellipsometry evaluation of a new ca15-3 protein assay method: optical protein-chip system for clinical application detection of avian influenza virus subtype h5 using a biosensor based on imaging ellipsometry investigation of interaction between two neutralizing monoclonal antibodies and sars virus using biosensor based on imaging ellipsometry analysis of interaction between tropomyosin allergen and antibody using a biosensor based on imaging ellipsometry analysis of interactions between snare proteins using imaging ellipsometer coupled with microfluidic array feasibility of protein a for the oriented immobilization of immunoglobulin on silicon surface for a biosensor with imaging ellipsometry a label-free protein microfluidic array for parallel immunoassays the use of the isoscope ellipsometer in the study of adsorbed proteins and biospecific binding reactions optimization of off-null ellipsometry for air/solid interfaces off-null ellipsometry revisited: basic considerations for measuring surface concentrations at solid/liquid interfaces maternal, fetal and neonatal diagnosis of congenital human cytomegalovirus infection cytomegalovirus strain diversity in seropositive women cytomegalovirus reinfections in healthy seroimmune women avidity of immunoglobulin g directed against human cytomegalovirus during primary and secondary infections in immunocompetent and immunocompromised subjects value of cytomegalovirus (cmv) igg avidity index for the diagnosis of primary cmv infection in pregnant women human cytomegalovirus detection by real-time pcr and pp65-antigen test in hematopoietic stem cell transplant recipients: a challenge in low and middle-income countries we thank qilu hospital of shandong university for clinical cmv patient samples. key: cord-003062-qm8kalyt authors: chowdhury, fazle rabbi; ibrahim, quazi shihab uddin; bari, md. shafiqul; alam, m. m. jahangir; dunachie, susanna j.; rodriguez-morales, alfonso j.; patwary, md. ismail title: the association between temperature, rainfall and humidity with common climate-sensitive infectious diseases in bangladesh date: 2018-06-21 journal: plos one doi: 10.1371/journal.pone.0199579 sha: doc_id: 3062 cord_uid: qm8kalyt bangladesh is one of the world’s most vulnerable countries for climate change. this observational study examined the association of temperature, humidity and rainfall with six common climate-sensitive infectious diseases in adults (malaria, diarrheal disease, enteric fever, encephalitis, pneumonia and bacterial meningitis) in northeastern bangladesh. subjects admitted to the adult medicine ward of a tertiary referral hospital in sylhet, bangladesh from 2008 to 2012 with a diagnosis of one of the six chosen climate-sensitive infectious diseases were enrolled in the study. climate-related data were collected from the bangladesh meteorological institute. disease incidence was then analyzed against mean temperature, humidity and average rainfall for the sylhet region. statistical significance was determined using mann-whitney test, chi-square test and anova testing. 5033 patients were enrolled (58% male, 42% female, ratio 1.3:1). all six diseases showed highly significant (p = 0.01) rises in incidence between the study years 2008 (540 cases) and 2012 (1330 cases), compared with no significant rise in overall all-cause hospital admissions in the same period (p = 0.19). the highest number of malaria (135), diarrhea (266) and pneumonia (371) cases occurred during the rainy season. on the other hand, the maximum number of enteric fever (408), encephalitis (183) and meningitis (151) cases occurred during autumn, which follows the rainy season. a positive (p = 0.01) correlation was observed between increased temperature and the incidence of malaria, enteric fever and diarrhea, and a negative correlation with encephalitis, meningitis and pneumonia. higher humidity correlated (p = 0.01) with a higher number of cases of malaria and diarrhea, but inversely correlated with meningitis and encephalitis. higher incidences of encephalitis and meningitis occurred while there was low rainfall. incidences of diarrhea, malaria and enteric fever, increased with rainfall, and then gradually decreased. the findings support a relationship between weather patterns and disease incidence, and provide essential baseline data for future large prospective studies. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 global warming is not a myth, rather a reality [1, 2] , and the impact of climate change is multidimensional on health. the world health organization (who) recently reported an estimated 12.6 million deaths each year due to unhealthy environments, particularly climate change and pollution [3] . the report also identifies diarrheal diseases, respiratory infections and malaria in the top ten causes of environment related deaths [3] . climate change influences the emergence and re-emergence of many infectious diseases [1, 2, 4, 5] . dengue fever, tickborne diseases, diarrheal disease, enteric fever, viral encephalitis, respiratory tract infections, and meningitis are much increased in recent times [1] [2] [3] [4] 6] . in addition, emerging and reemerging diseases such as chikungunya and zika, have also been linked to climate change influences, and have been proposed as partially responsible for autochnous transmission in places not traditionally endemic for such diseases [7, 8] . analysis of global temperature data led the intergovernmental panel for climate change (ipcc) to the conclusion that the average global temperature over land and ocean surfaces has risen by 0.85˚c in the period from 1880 to 2012 [1] . they predicted that global surface temperature will increase by 1.5˚c compared to the year 1850 by the end of the 21st century (2081-2100) [1] . climate change is predicted to directly influence zoonotic infectious disease transmission by changing the geographic range of a vector [9] . altered climatic conditions may increase vector biting rate and the reproduction rate of the vector and shorten the pathogen incubation period [9] [10] [11] [12] . climate-related increases in sea surface temperature and sea levels can lead to higher incidence of waterborne infectious diseases [9, 13, 14] . as a low-income country, bangladesh itself plays very little role in the process of global warming, but becomes one of the most seriously affected victims of climate change. bangladesh is the biggest delta and contains the second largest river basin in the world [15] . the majority of the land is low and flat, and only 10% lies over one meter above the mean sea surface [15] . because of monsoon weather and the presence of the bay of bengal in the south, extreme weather events like flood and cyclone are common [15, 16] . this unique geographic and topographic location makes it reportedly the most vulnerable country to climate change effects [16] . lack of resilience and adaptive capacity, dense population and poverty make the situation worse [16] . unfortunately, very few studies on the relationship between various environmental variables and trends of infectious disease incidence have been performed so far in bangladesh, although there are reports of some infections increasing sporadically in different regions of the country [17] [18] [19] . climate change and health related studies are so far mainly reported from developed countries, but studies from vulnerable countries are still meagre [20, 21] . furthermore, published studies typically only focus on a single disease. this study examined six infectious diseases based on clinical syndromes and laboratory support (malaria, enteric fever, encephalitis, diarrheal disease, pneumonia and meningitis) to offer a broader scope on the trend of these infectious diseases and their possible relation to climate change in bangladesh. we chose these six diseases based on the reports of ipcc (2014) and who (2016) where they were listed as climate-sensitive infectious diseases important for asia [1] [2] [3] . the main objective of the study was to see the burden of the six climate sensitive diseases over five years and to analyze the possible relationship of them with common climatic variables. the findings will be of interest to public health experts and policy makers to stimulate effective measures to combat infectious diseases and related epidemics in bangladesh and in other vulnerable countries. sylhet divison in northeast bangladesh, an area of approximately 12,298 km 2 with a population of around 10 million. all case files of the adult medicine ward from 2008 to 2012 were enrolled from the hospital archive. the diagnosis of the six studied diseases were confirmed based on the combination of clinical and relevant laboratory diagnosis. incomplete files, patients discharged on risk bonds, patients who died within 24 hours (due to insufficient time to reach a diagnosis) and absconded patients were excluded from the study. square p<0.001) whereas encephalitis occurred more frequently in females in females (53% vs. 47%, chi square p<0.001) ( table 1 ). the highest percentage (27%; 1373) of disease over the five years studied occurred in the 4 th year (2011). there was a trend towards increasing number of cases of the six infectious diseases studied over the five years ( fig 1a) and there is a significant difference (p = 0.01) between the total number of cases in 2008 (540) and 2012 (1330). all six infectious diseases individually increased in number over the five years, especially pneumonia which rose from 131 cases in 2008 to 411 cases in 2012. this increase does not appear to be due to increased numbers of patients using the hospital overall. in comparison, the number of admissions for all causes to the hospital and to the medicine department over three of the years of study from 2010 to 2012 (data unavailable for 2008 and 2009) did not change (mann-whitney u test p = 0.191, fig 1b) . the highest number of cases occurred in autumn (1436; 29%) followed by rainy season (1423; 28%), winter (1204; 24%) and summer (971; 19%). pneumonia (1352; 27%) was the most common disease of the six, and malaria (443; 9%) was the least prevalent disease. individually, the highest number of malaria (135), diarrhea (266) and pneumonia (371) cases occurred in rainy season. on the other hand, the maximum number of enteric fever (408), encephalitis (183) and meningitis (151) cases occurred during autumn (fig 2) . the mean temperature in our study region during 2008-12 was 25.0˚c, and there was no significant difference in the number of cases occurring below or above that level (table 2 ). in 2012, the average temperature was more than 25˚c in eight out of twelve months (fig 3) . there was a significant positive correlation between increasing temperature and the incidence of malaria (p = 0.0001), and a trend for positive correlation between increasing temperature and the incidence of diarrhea (p = 0.081). there was a significant negative correlation between increasing temperature and incidence of encephalitis (p = 0.001), meningitis (p = 0.001) and pneumonia (p = 0.017). table 3 shows the association of the six diseases with the average humidity of the studied period of 2008 to 2012. we analysed the incidence of each disease in each of three groups of humidity recordings by month: less than 76.5%, 76.5-77.5% and more than 77.5% based on the mean humidity of the study site. less than 76.5 percent humidity was associated with the highest percentage (2458; 49%; p = <0.01) of all the six studied diseases (table 3) . average humidity of 76.5-77.5% and > 77.5% was associated with 27% (1373) and 24% (1203) disease respectively. however, in 2012, the average humidity was ! 76.5% in nine out of twelve months (fig 4) . higher humidity was correlated with a higher number of cases of malaria (p = 0.0001), enteric fever (p = 0.0001) and diarrhea (p = 0.0001), but inversely correlated with meningitis (p = 0.0001), encephalitis (p = 0.0001) and pneumonia (p = 0.0001). the average rainfall by month was divided into three groups, less than 275 mm, 275-375 mm and more than 375 mm in table 4 . the highest percentage (2458; 48.8%) of all the six diseases studied were related with less than 275 mm of average rainfall. 45% (201) malaria, 43% (539) enteric fever, 48% (413) diarrhea, 52% (312) encephalitis, 52% (708) pneumonia and 53% (285) meningitis fell under this category (chi-square p = 0.010). average rainfall of 275-375 mm and > 375mm was associated with 24% (1203) and 27% (1373) of the total disease burden respectively. analyzing the data of 2012, in six out of twelve months, there were 375 mm average rainfall (fig 5) . higher incidences of encephalitis (p = 0.001) and meningitis (p = 0.001) happened while there was low rainfall. incidences of diarrhea (p = 0.002), malaria (p = 0.001), pneumonia (p = 0.002) and enteric fever (p = 0.002) increased with rainfall, and then gradually decreased. (2008-12) . the highest number of malaria, diarrhea and pneumonia cases occurred in rainy season, whilst the maximum number of enteric fever, encephalitis and meningitis recorded during autumn. student t test was applied to obtain the level of significance. https://doi.org/10.1371/journal.pone.0199579.g002 table 2 . association of disease with the temperature of five years (2008-2012). temperature previous studies in bangladesh have either focused on a single disease or relied on people's perception of climate change and infectious disease. to our knowledge, this study is the first observational study which specifically focuses on individual infectious diseases and explores the relationship between each disease and three common weather variables. the male to female ratio in this study was 1.3: 1. although there are no published data, it is generally believed that more males come into hospital in bangladesh because traditionally females wait at home with their disease until complications develop [22, 23] . it is likely that women get less opportunity to come into hospital and have less access to medical care. there was no significant difference by gender in the frequency of each individual disease except pneumonia and encephalitis, which significantly affected a greater proportion of the male and female population respectively. the increase in pneumonia cases seen in males may be because men have more occupational exposure to conditions that increase the chance of receiving a diagnosis of pneumonia such as dust, fumes, smoking etc. all six diseases showed a significant (p = 0.01) rise in incidence between the study years 2008 and 2012. the number of cases more than doubled for all diseases except enteric fever, which also showed significant increases. in contrast, during this same period the total number of admissions to the medical ward and to in the hospital showed no significant rise. we propose that the rise in frequency of the studied diseases could be explained by the influence of weather changes. it is important to note that during this period no epidemics of the studied diseases happened in the sylhet region. the total impression is consistent with previous reports. in a review of the literature, the incidence of pyogenic meningitis, encephalitis and dengue was predicted to be greatly influenced and increased by global warming in the coming years [24] . w.h.o reported dengue, viral encephalitis, diarrheal disease, enteric fever, pneumonia and meningitis as most sensitive to climate factors, and predicted a huge rise of cases in tropical countries [25] . climate change cell, bangladesh reported that, from 1984 to 1993 there were 301,651cases of malaria in bangladesh, but from 1994 to 2003 it increased to 507,485 (68% increased incidence) [26] . although after the introduction of artemisinin treatment and government and other partner organization lead massive drive for malaria elimination, the cases decreased to 1.4/1000 population in bangladesh [27] . the same report revealed an increasing trend for diarrheal diseases, kala-azar and skin diseases in three districts (drought-prone rajshahi, flood-prone manikganj and salinity-dense satkhira) of bangladesh between 1999 to 2005 [26] . the report also described a positive correlation between rainfall and diarrheal and skin disease in rajshahi and satkhira, and a negative correlation of diarrheal disease with temperature [26] . another focus group discussion (fgd) based study reported an increased number of diarrheal diseases, typhoid and skin problems after the cyclone sidr and aila in southern part (barguna and khulna) of the country [28] . we found the highest number of malaria (135), diarrhea (266) and pneumonia (371) cases occurred during the rainy season. the findings are consistent with other national and international studies. highest cases of falciparum malaria were found in north-eastern india during higher humidity was correlated with a higher number of cases of malaria, enteric fever and diarrhea, but inversely correlated with meningitis, encephalitis and pneumonia. two-way anova test was applied to obtain the level of significance. https://doi.org/10.1371/journal.pone.0199579.g004 temperature, rainfall, humidity and climate-sensitive infectious diseases in bangladesh the rainy season [29] . in the chittagong hill tract districts of bangladesh, where malaria is most endemic, the frequency of cases was highest in rainy season [30] . an increased incidence of malaria in north-west of india has been suggested through computational modelling [31] . studies in africa revealed mixed results, with the highest number of malaria cases during the rainy season in mali, but most cases during autumn in northern ghana [32, 33] . according to the ipcc report, respiratory infections also follow a seasonal pattern [2] . in tropical settings, where most deaths due to pneumonia occur, the incidence of lower respiratory tract illness in children is generally increased during rainy season and it supports our findings [34] . a thai study of viral pneumonia reported the highest number of cases in rainy season [35] , in line with our findings. this pattern of increased pneumonia cases during rainy season in tropical countries contrasts with the well described increase in pneumonia seen during colder months in temperate climates [36, 37] . for diarrheal disease, our findings are supported by previous reports of increased incidence during the rainy season in taiwan (24) and bangladesh (25) . we found a large number of enteric fever cases (759; 61%) occurred in rainy season and autumn. this agrees with earlier studies in both dhaka, bangladesh (26) where the highest number (45%) of enteric fever occurred during monsoon period [38] ,and a cambodian study [39] but no relationship between incidence of enteric fever and season was seen in kenya [40] . in our study, most meningitis (293; 55%) and encephalitis (355; 59%) occurred during autumn and winter. this finding is partly consistent with other studies done in africa. meningitis epidemics in west africa occurred during the coolest season [41] . a recent time series analysis over 66 countries found that bacterial meningitis season peaks during the winter months, [42] , similar to our findings. a major causative organism of meningitis (neisseria meningitides) was found to be high and active during dry periods in the presence of dust and were then washed away with rainfall, so as the case frequency fell down [43, 44] . however, in our study, we found an almost equal number of meningitis cases in rainy season compared to other seasons. regional findings of seasonality in terms of encephalitis are also supportive of our findings. highest number of encephalitis cases in rainy period (august-september) were seen in nepal [45] . in india, the incidence of japanese encephalitis was also highest during august to november (rainy and early winter) with a peak in october [46] . another study from china also reported similar findings [47] . the current rate of increase of the annual minimum temperature (by 0.1˚c) is higher than that of the annual maximum temperature (by 0.09˚c) in bangladesh [48] . the annual average rainfall is increasing by 10.6 mm per decade outside the usual rainy period, while rainfall during the season is decreasing by 7.6 mm per decade [49, 50] . in our study, we found a positive correlation between the incidences of malaria, enteric fever and diarrhea with increasing temperature. these results are similar to other national and international findings. a previous study in chittagong, bangladesh showed increased malaria cases with increasing temperature table 4 . association of disease with the rainfall of study years (2008-2012). temperature, rainfall, humidity and climate-sensitive infectious diseases in bangladesh [30] . studies have shown a positive relationship between increased malaria and increasing temperature [51, 52] . studies in south asia and south america (venezuela and columbia) have documented the association between malaria outbreaks and the el nino southern oscillation (enso) cycle [53] . a significant increase in malaria cases with increasing temperature was seen in rwanda and uganda [54, 55] , but this was not seen in studies done in the african highland region [56, 57] . increased temperature allows faster replication of mosquito incidences of diarrhea, malaria, pneumonia and enteric fever increased with rainfall. two-way anova test was applied to obtain the level of significance. https://doi.org/10.1371/journal.pone.0199579.g005 temperature, rainfall, humidity and climate-sensitive infectious diseases in bangladesh populations [47, 58] . higher temperatures also change human behavior, for example more outdoor activities may be undertaken which further increases the risk of exposure [47] . diarrheal cases including dysentery have been found to be higher in high temperature in bangladesh, taiwan and china [59] [60] [61] . a study in dhaka, bangladesh showed an increase number of enteric fever cases with an increase temperature which supports our findings [38] , as does a similar study from southern australia [62] . furthermore, a strong linear association has been noted between temperature and notification of salmonellosis globally [53] . temperature affects the transmission of food-borne disease in various ways. the temperature directly affects the rate of replication of bacterial and protozoan pathogens and the survival of enteroviruses in the environment [61, 63] . in addition, these variations may also have a significant impact on the environmental reservoirs of infection as well as human behavior [63] . moreover, both salmonella and cholera bacteria, for example, proliferate more rapidly at higher temperatures; salmonella in animal gut and food, cholera in water [13, 53] . our study shows an inverse relationship between encephalitis, meningitis and pneumonia with temperature. the pneumonia findings are consistent with a study done in china, but not with another study conducted in spain [36, 64] . for encephalitis and meningitis our findings differ to other studies done in india, china and niger where they found increased evidence of cases and vector during hot environment [47, 65, 66] .viral encephalitis cases in sweden have reportedly increased in response to a succession of warmer winters over the past two decades [53] . this inverse relationship with temperature in our study merits further exploration. in recent decades, more heatwaves have been reported in south asia, which also bring a change in humidity [67] . eighteen heatwaves were reported in india between 1980 and1998 [67] . our study found a higher trend of malaria, enteric fever and diarrhea cases with higher humidity. the findings are further supported by studies done in chittagong, bangladesh and china with similar trend [30, 61] . relative humidity influences biological and feeding behavior of mosquitoes [47] . at higher humidity, mosquitoes generally survive for longer and disperse further [47, 58] . higher humidity also affects the rate of replication of bacterial and protozoan pathogens and their survival in the environment [63] . we did not find any influence of humidity on enteric fever and pneumonia, in line with other studies [34, 36] . the incidence of pneumonia, encephalitis and meningitis is inversely related to humidity in this study. in india an inverse association was seen between the number of culex mosquitoes (the vector for japanese encephalitis) and humidity [66] , although in china, higher cases of encephalitis was seen with higher humidity [47] . these mixed findings may reflect differences in etiology of encephalitis and bacterial meningitis, as well as differences in case definitions between study sites. these could also explained by diagnostic limitations, and in some cases the meningoencephalitis syndrome may be caused by bacteria favoring dry and dusty weather. heavy rainfall can have a diverse range of effects on disease. for example, in tropical and subtropical regions with crowding and poverty, heavy rainfall and flooding may trigger behavioral changes such as increased contact between people and distribution of pathogens in floodwater, leading to outbreaks of diarrhea [58] . we found that the incidence of diarrhea, malaria, enteric fever and pneumonia increased with rainfall. for diarrhea, our findings are consistent with other national and international studies [59] [60] [61] . a positive relationship between increased rainfall and the incidence of enteric fever was also found in dhaka, bangladesh and southern australia [38, 62] . one possible explanation is, heavy rainfall may affect the frequency and level of contamination of drinking water and hence the spread waterborne infections [13, 68] . area with existing high burdens of infectious disease and poor sanitary infrastructure often experience increased rates of diarrheal diseases after heavy rainfall [58] . our findings in terms of malaria are also similar to other national and regional studies [30, 31] . rainfall plays an important role in the transmission of malaria, as mosquitoes need water to support the larval and pupal stages of development [31, 58] . a study also found higher deaths from acute lower respiratory tract illness during rainy season in a pediatric population [34] . in 2011-12, the highest incidences of encephalitis and meningitis occurred while there was very low rainfall. our findings are supported by other reports in the case of meningitis [44, 65] . nevertheless, researchers found the opposite for japanese encephalitis, where the vector replicates and transmits more disease with high rainfall [45, 66] . we acknowledge a limitation of this study is the relatively short timeframe. extending our study duration to ten years would have allowed greater power to detect differences, but we did not have the resources to do this. another limitation is that due to the retrospective design of the study with recruitment from the hospital archive, we relied on the diagnosis in the patient notes, which in some instances may be wrong. we tried to overcome this limitation by setting case definitions for enrollment in the study and included only the cases who survived for more than 24 hours, where there was more clinical information to make an informed diagnosis. the death rate in the first 24 hours due to these infections (bacterial meningitis-0.92%; encephalitis 2.32% and all-cause mortality 3.62%) during that period was low and unlikely to influence our results [69] . due to limitations of funding and time, we conducted this study in only one hospital of northeastern region of bangladesh. further studies in other regions of bangladesh are highly desirable to represent the situation across the country. this study reported the influence of temperature, humidity and rainfall on six climate sensitive infectious diseases in the northeastern region of bangladesh. weather and climate extremes affect all sectors of the health, economy and development. the findings can be used as a baseline to launch a large cohort study throughout the country in future. this study is pivotal in giving direction to our public health experts, clinicians and other policy makers to update and change future strategies to combat the burden of climate-related health events in bangladesh and other countries. climate change 2014 synthesis report summary for policy makers climate change 2014: impacts, adaptation and vulnerability cambridge: inter governmental panel on climate change an estimated 12.6 million deaths each year are attributable to unhealthy environments world health organization impact of climate change on health the socio-ecology of zoonotic infections. clinical microbiology and infection: the official publication of the european society of clinical microbiology and infectious diseases health and climate change: a roadmap for applied research el nino and climate change-contributing factors in the dispersal of zika virus in the americas? autochthonous chikungunya transmission and extreme climate events in southern france climate changes, environment and infection: facts, scenarios and growing awareness from the public health community within europe climate change: present and future risks to health, and necessary responses global climate change and emerging infectious diseases potential impact of climatic variability on the epidemiology of dengue in risaralda, colombia pandemics, pathogenicity and changing molecular epidemiology of cholera in the era of global warming predicting endemic cholera: the role of climate variability and disease dynamics usa: climate link: a global knowledge portal for climate change & development practitioners climate change induced natural disasters impact on the health of the coastal people in bangladesh climate and health. 8th international congress of bangladesh society of medicine climate change and health in bangladesh: a baseline cross-sectional survey climate changeimpacts and responses in bangladesh. brussels: european parliament awareness of and attitudes towards heat waves within the context of climate change among a cohort of residents in adelaide climate change and local public health in the united states: preparedness, programs and perceptions of local public health department directors gender difference in treatment seeking behaviors of tuberculosis cases in rural communities of bangladesh. the southeast asian journal of tropical medicine and public health rural women's access to health care in bangladesh: swimming against the tide? social work in public health reducing the impact of climate change using climate to predict disease outbreaks: a review reduction in malaria prevalence and increase in malaria awareness in endemic districts of bangladesh climate change impact: the experience of the coastal areas of bangladesh affected by cyclones sidr and aila rainfall and malaria transmission in north-eastern india relationship between climate change and incidence of malaria in chittagong hill tracts forcing versus feedback: epidemic malaria and monsoon rains in northwest india season, fever prevalence and pyrogenic threshold for malaria disease definition in an endemic area of mali seasonal profiles of malaria infection, anaemia, and bednet use among age groups and communities in northern ghana childhood pneumonia: a neglected, climate-sensitive disease? incidence, seasonality and mortality associated with influenza pneumonia in thailand do seasonal changes and climate influence the etiology of community acquired pneumonia? what is the seasonal distribution of community acquired pneumonia over time? a systematic review typhoid fever and its association with environmental factors in the dhaka metropolitan area of bangladesh: a spatial and time-series approach the molecular and spatial epidemiology of typhoid fever in rural cambodia population-based incidence of typhoid fever in an urban informal settlement and a rural area in kenya: implications for typhoid vaccine use in africa climate drives the meningitis epidemics onset in west africa seasonal dynamics of bacterial meningitis: a timeseries analysis environmental risk and meningitis epidemics in africa dust clouds and spread of infection japanese encephalitis in hill and mountain districts a long-term study on vector abundance & seasonal prevalence in relation to the occurrence of japanese encephalitis in gorakhpur district weather variables and japanese encephalitis in the metropolitan area of jinan city temperature and precipitation projections over bangladesh and the upstream ganges, brahmaputra and meghna systems knowledge and perception about climate change and human health: findings from a baseline survey among vulnerable communities in bangladesh trends in extreme rainfall events of bangladesh theoretical and applied climatology how malaria models relate temperature to malaria transmission optimal temperature for malaria transmission is dramatically lower than previously predicted climate change and human health: present and future risks highland malaria in uganda: prospective analysis of an epidemic associated with el nino climatic warming and increased malaria incidence in rwanda climate change and the resurgence of malaria in the east african highlands climatic suitability for malaria transmission in africa, 1911-1995 climate anomalies and epidemics in south america at the end of the colonial period modeling the impact of climate variability on diarrhea-associated diseases in taiwan association between climate variability and hospital visits for non-cholera diarrhoea in bangladesh: effects and vulnerable groups climate variations and bacillary dysentery in northern and southern cities of china climate variations and salmonellosis transmission in adelaide, south australia: a comparison between regression models weather and the transmission of bacillary dysentery in jinan, northern china: a time-series analysis weather: driving force behind the transmission of severe acute respiratory syndrome in china? the effects of climatic factors on the distribution and abundance of japanese encephalitis vectors in kurnool district of andhra pradesh, india severe heat waves over the indian subcontinent in 1998, in perspective of global climate current science potential impact of macroclimatic variability on the epidemiology of giardiasis in three provinces of cuba ministry of health and family welfare (mohfw): sylhet m. a.g.osmani medical college hospital we thank the staff of the hospital archive and administration departments of somch for their cooperation. we are also grateful to the nurses and doctors of all the units of medicine department for their support. key: cord-013265-qrfi6e5c authors: isono, toshihito; domon, hisanori; nagai, kosuke; maekawa, tomoki; tamura, hikaru; hiyoshi, takumi; yanagihara, katsunori; kunitomo, eiji; takenaka, shoji; noiri, yuichiro; terao, yutaka title: treatment of severe pneumonia by hinokitiol in a murine antimicrobial-resistant pneumococcal pneumonia model date: 2020-10-15 journal: plos one doi: 10.1371/journal.pone.0240329 sha: doc_id: 13265 cord_uid: qrfi6e5c streptococcus pneumoniae is often isolated from patients with community-acquired pneumonia. antibiotics are the primary line of treatment for pneumococcal pneumonia; however, rising antimicrobial resistance is becoming more prevalent. hinokitiol, which is isolated from trees in the cypress family, has been demonstrated to exert antibacterial activity against s. pneumoniae in vitro regardless of antimicrobial resistance. in this study, the efficacy of hinokitiol was investigated in a mouse pneumonia model. male 8-week-old balb/c mice were intratracheally infected with s. pneumoniae strains d39 (antimicrobial susceptible) and nu4471 (macrolide resistant). after 1 h, hinokitiol was injected via the tracheal route. hinokitiol significantly decreased the number of s. pneumoniae in the bronchoalveolar lavage fluid (balf) and the concentration of pneumococcal dna in the serum, regardless of whether bacteria were resistant or susceptible to macrolides. in addition, hinokitiol decreased the infiltration of neutrophils in the lungs, as well as the concentration of inflammatory cytokines in the balf and serum. repeated hinokitiol injection at 18 h intervals showed downward trend in the number of s. pneumoniae in the balf and the concentration of s. pneumoniae dna in the serum with the number of hinokitiol administrations. these findings suggest that hinokitiol reduced bacterial load and suppressed excessive host immune response in the pneumonia mouse model. accordingly, hinokitiol warrants further exploration as a potential candidate for the treatment of pneumococcal pneumonia. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 pneumonia represents a major threat in a globalized society as it spreads easily by droplets. there are very few therapeutic agents that can tackle infection by drug-resistant bacteria or emerging viruses [1] . furthermore, self-organ damage owing to excessive host immunity can also occur in severe pneumonia [2] . therefore, treatment of severe pneumonia requires both elimination of drug-resistant microorganisms and suppression of hyperimmunity that damage the host lungs. hinokitiol, a tropolone-related compound has been isolated from chamaecyparis obtusa and thuja plicata donn ex d. don, which are trees in the cypress family, as β-thujaplicin [3, 4] . previous studies have shown antitumor, anti-inflammatory, antioxidant, and antibacterial activities of hinokitiol [5] [6] [7] [8] [9] . we recently showed that hinokitiol was effective against streptococcus pneumoniae in vitro, regardless of antimicrobial resistance [10] . at present, no studies have examined the effect of hinokitiol on pneumococcal infection in vivo. s. pneumoniae is a gram-positive diplococcus resident in the human upper respiratory tract [11] . this bacterium causes not only pneumonia but also otitis media, sinusitis, bronchitis, and empyema [12] , as well as invasive pneumococcal diseases, including meningitis, bacteremia, and septicemia [13] . the primary treatment of pneumococcal diseases relies on the use of effective antibiotics. in the early 2000s, macrolides were strongly recommended for pneumococcal pneumonia [14] [15] [16] ; however, their widespread use has augmented the risk of infection with macrolide-resistant s. pneumoniae [17, 18] . our previous study showed that more than 80% of s. pneumoniae isolated in municipal hospitals were non-susceptible to macrolides [19] . in addition to the appropriate prescription and consumption of antibiotics, new therapeutic agents need to be developed. in this study, we investigated the effect of hinokitiol on bacterial loads and analyzed the suitable administration route of hinokitiol using a murine pneumococcal infection model. furthermore, we also examined the effect of hinokitiol on inflammation in a severe pneumonia murine model. antimicrobial-susceptible s. pneumoniae strain d39 (serotype 2) was purchased from the national collection of type cultures (salisbury, uk). macrolide-resistant s. pneumoniae clinical strain nu4471 (serotype 19, azithromycin minimum inhibitory concentration (mic) � 1 mg/ml) was isolated from patients with respiratory tract infections [20] . all strains were grown in tryptic soy broth (becton dickinson, franklin lakes, nj, usa) at 37˚c as described previously [21] . male 8-10-week-old balb/c mice were obtained from nihon clea tokyo, japan. mice were maintained under standard conditions in accordance with our institutional guidelines. all animal experiments were approved by the institutional animal care and use committee of niigata university (sa00223). hinokitiol was purchased from fujifilm wako pure chemical industries (osaka, japan) and solubilized in phosphate-buffered saline (pbs) containing 10% ethanol. thiazolyl blue tetrazolium bromide (mtt) was purchased from sigma-aldrich (st. louis, mo, usa). the anesthetic mixture consisted of medetomidine hydrochloride (domitol; meiji seika pharma co., ltd., tokyo, japan), midazolam (dormicum; astellas pharma inc., tokyo, japan), and butorphanol (vetorphale; meiji seika pharma co., ltd.) mixed at 0.15, 2.0, and 2.5 mg/kg body weight with normal saline (otsuka pharmaceutical factory, inc. tokyo, japan) [22] . balb/c mice were injected intraperitoneally once a day with 0, 10, 20, 30, 50, and 100 mg/kg hinokitiol for 7 days. body weight was monitored every day. the change in body weight was considered an indicator of systemic toxicity of hinokitiol. the to evaluate the toxicity of intratracheally administered hinokitiol, mice were injected 500 μg/ml hinokitiol in 50 μl pbs. human alveolar epithelial cell line a549 (atcc ccl-185) was obtained from riken cell bank (ibaraki, japan). cells were seeded in a 96-well plate (becton dickinson) and grown to 90% confluence in dulbecco's modified eagle medium (fujifilm wako pure chemical industries) supplemented with 10% fetal bovine serum (japan bio serum, hiroshima, japan) and 100 u/ml penicillin and 100 μg/ml streptomycin (both fujifilm wako pure chemical industries) at 37˚c in 5% co 2 . a549 cells were treated with various concentrations (10-1000 μg/ml) of hinokitiol or 0.5% triton x-100 for 3 h. mtt assays were performed to determine cell viability as described previously [23] . mice (n = 8 per group) were anesthetized with a mixture of medetomidine hydrochloride, midazolam, and butorphanol and were intratracheally infected with s. pneumoniae strain d39 or strain nu4471 [1.0 × 10 9 colony forming units (cfu) in 50 μl pbs] using the microsprayer aerosolizer (penn-century inc., philadelphia, pa, usa). uninfected control mice (n = 5 per group) were intratracheally injected with 50 μl pbs (uninfected-pbs group) or 500 μg/ml hinokitiol (uninfected-hinokitiol group). at 1 h after infection, all infected mice were anesthetized again and 500 μg/ml hinokitiol (infected-hinokitiol group) was administered intratracheally to infected mice, while control mice were administered 50 μl pbs (infected-pbs group). to evaluate the efficacy of repeated hinokitiol administration in a pneumonia mouse model, all mice were intratracheally infected with s. pneumoniae strain nu4471 (3.0 × 10 8 cfu/mouse). after 1 h, 50 μl pbs was injected into the respiratory tract (untreated group; n = 5), followed by intratracheal injection of pbs every 24 h. in the hinokitiol injection group, all mice were initially intratracheally administered with hinokitiol 1 h after infection. the single administration group (n = 5) was injected with pbs after initial hinokitiol administration at 24 h intervals. in the double administration group (n = 5), the second administration of hinokitiol was performed at 24 h after infection, and pbs was administrated after 48 h from infection. in the three-dose group (n = 5), hinokitiol was intratracheally administered every 24 h after the first administration. all mice were sacrificed 72 h after from pneumococcal infection. at 18 h after infection, balf and serum samples were collected from five mice. lung tissue samples were collected from three mice. balf samples were plated onto blood-agar plates and cultured aerobically to count cfu. to determine the concentration of pneumococcal dna in murine serum, dna was isolated from 50 μl of serum obtained from the infected mice using qiaamp spin columns (qiagen, hilden, germany). then, absolute quantification by realtime pcr was performed using the steponeplus real-time pcr system via the sybr green detection protocol (thermo fisher scientific, waltham, ma, usa). the primers used for realtime pcr targeted a fragment of the pneumolysin-encoding gene of s. pneumoniae [24] . the forward primer sequence was 5 0 -agcgatagctttctccaagtgg-3 0 and the reverse primer sequence was 5 0 -cttagccaacaaatcgtttaccg-3 0 . for pathological examinations, lung tissue samples were fixed with 4% paraformaldehyde (fujifilm wako pure chemical industries). paraffin-embedded sections were made by biopathology institute co., ltd (oita, japan). lung sections were stained with hematoxylin and eosin (sakura finetek japan co., ltd., tokyo, japan). they were observed under a biorevo bz-9000 microscope (keyence, osaka, japan). to detect neutrophil elastase (ne) in lung tissue, paraffin-embedded sections were treated with rabbit anti-mouse ne antibody (abcam, cambridge, uk) in blocking solution (thermo fisher scientific). after overnight incubation at 4˚c, the secondary alexafluor 488-conjugated goat anti-rabbit igg antibody (thermo fisher scientific) in blocking buffer was added, followed by a 2 h incubation in the dark. the samples were then washed with pbs and mounted with nuclear staining mounting medium (prolong tm diamond antifade mountant with dapi; thermo fisher scientific). finally, samples were observed with a confocal laser-scanning microscope (carl zeiss, jena, germany). the number of neutrophils in balf was counted by flow cytometry. prior to staining cells in balf, their surfaces were blocked with anti-cd16/32 antibody (thermo fisher scientific) at 4˚c for 5 min [25] . then, cells were stained with anti-cd11b-allophycocyanin and ly-6gphycoerythrin antibody (thermo fisher scientific) in pbs containing 1% bovine serum albumin (sigma-aldrich) at 4˚c for 40 min in the dark [26] . samples were washed and fixed with 4% paraformaldehyde in pbs. flow cytometry analysis was performed on a novocyte flow cytometer with novoexpress software (acea biosciences, san diego, ca, usa) [26] . the cells were gated by their forward and side scatter. neutrophils were identified by the expression of both ly-6g and cd11b [26] . ne activity in balf was determined using the specific substrate n-methoxysuccinyl ala-ala-pro-val p-nitroanilide (merck millipore, billerica, ma, usa) as described previously [27] . briefly, samples were incubated in 0.1 m tris-hcl (ph 8.0) containing 0.5 m nacl and 1 mm substrate at 37˚c for 24 h, after which absorbance at 405 nm was measured. for cytokine and chemokine measurements, balf samples were centrifuged at 2000 × g at 4˚c for 15 min. then, enzyme-linked immunosorbent assay was performed to determine the concentrations of (i) c-x-c motif chemokine ligand l (cxcl-1; proteintech group, chicago, il, usa), (ii) interleukin (il)-1β, il-6, and tumor necrosis factor (tnf) (biolegend inc., san diego, ca, usa) in balf and serum according to the manufacturers' instructions. data were analyzed statistically by analysis of variance with tukey's multiple-comparison test. where appropriate (comparison of two groups only), unpaired t-tests were conducted. all statistical analyses were performed using graphpad prism software version 8.03 (graphpad software, inc., la jolla, ca, usa). values of p < 0.05 were considered significant. we first evaluated the systemic toxicity of hinokitiol. balb/c mice were intraperitoneally injected with hinokitiol once a day for 7 days as specified in the supplementary information. as shown in s1 fig, administration of �20 mg/kg hinokitiol significantly decreased body weight 3 days after the first administration (p < 0.05). hence, we lowered the maximum hinokitiol concentration for systemic administration in mice to 10 mg/kg. we used this dosage to test whether intraperitoneal administration of hinokitiol ameliorated pneumonia in a mouse intratracheal pneumococcal infection model. there was no significant decrease on pneumococcal cfu in the bronchoalveolar lavage fluid (balf) (s2 fig; p > 0 .05) and little effect on the transcriptional levels of various cytokine genes, such as cxcl1, il1b, il6, and tnf, in lung tissue (s3 fig; p > 0.05 ). the negative results from systemic administration of hinokitiol prompted us to test whether intratracheal administration of hinokitiol represented a better route for therapeutic intervention in pneumococcal pneumonia. evaluation of hinokitiol cytotoxicity against a549 alveolar epithelial cells in vitro revealed no significant decrease in viability except at 1000 μg/ml, whereby cell viability was reduced by 54% (fig 1a; p < 0.05). furthermore, compared with pbs injection, intratracheal hinokitiol administration caused negligible morphological changes in lung tissues in mice (fig 1b) . our previous in vitro study showed that 0.3 μg/ml hinokitiol exerted antibacterial effect against s. pneumoniae regardless of antimicrobial resistance [10] . here, compared with pbs injection, intratracheal administration of 500 μg/ml hinokitiol in a pneumococcal pneumonia mouse model decreased inflammatory cell migration and prevented destruction of alveolar tissue (fig 2) . in addition, intratracheal administration of hinokitiol significantly decreased cfu in balf of both antimicrobial-susceptible strain d39 and macrolide-resistant strain nu4471 (fig 3a; p < 0.05). because the severity of pneumococcal pneumonia is related to bacteremic pneumonia [28] , next, we investigated whether hinokitiol treatment inhibited s. pneumoniae. fig 3b shows that compared with pbs injection in mice, hinokitiol administration significantly decreased s. pneumoniae serum dna concentration (p < 0.05). previous studies have shown that excessive activation of neutrophils causes the release of ne, which contributes to severe lung injury [28] . therefore, controlling the excessive accumulation of neutrophils may be an essential therapeutic strategy to combat severe pneumonia [28, 29] . fig 4c) . similarly, ne activity in balf was significantly decreased in the hinokitiol administrated group which infected with pneumococcus compared to pbs injected group (fig 4d; p < 0.05). excessive production of inflammatory cytokines or chemokines is a hallmark of tissue injury and severe pneumonia [30, 31] . the concentration of cxcl-1, il-1β, il-6, and tnf was significantly lower in the balf from the infected-hinokitiol group compared with that from the infected-pbs group (fig 5; p < 0.05) . serum il-6 and tnf levels are associated with mortality of patients with pneumonia [32] . our previous study revealed that pneumococcal bacteremia induced an increase in serum cytokine levels [33] . here, the concentration of cxcl-1, il-1β, and il-6, but not tnf, was significantly decreased in the serum of the infected-hinokitiol group compared with that in the infected-pbs group (fig 6; p < 0.05) . meanwhile the chemokine and cytokines concentrations in balf and serum of uninfected control groups were either below (il-6) or just above the limit of detection (cxcl-1, il-1β and tnf). taken together, these findings suggested that the antibacterial action of hinokitiol in the lungs of pneumonia mice reduced the production of cytokines or chemokines in balf and serum. the above results suggested that intratracheal hinokitiol administration was effective in inhibiting s. pneumoniae in mouse lung. however, in clinical settings, antimicrobial agents are administrated several times for the treatment of pneumococcal pneumonia, and thus, we next sought to investigate whether repeated hinokitiol injection decreased the bacterial load in (fig 7; p < 0.05) . meanwhile, no significant differences were observed in balf and serum bacterial loads at 72 hours post-infection in the group between the untreated group and the groups which administrated hinokitiol once or twice. these results suggest that repeated administration of hinokitiol is required for the effective treatment of pneumococcal pneumonia. the present study showed that intratracheal administration of hinokitiol decreased the bacterial load in a mouse pneumonia model regardless of macrolide resistance of s. pneumoniae. as a result, the production of inflammatory cytokines and release of ne also decreased, eventually preventing the disruption of lung tissue. these results suggest that the antibacterial activity of hinokitiol has potential as a treatment against pneumococcal pneumonia. an important aspect when selecting a drug is to evaluate and compare its therapeutic index [34] . although hinokitiol exhibits antibacterial effect against various pathogenic organisms [10] , dosages above 12.7 and 14.8 mg/kg/day in male and female rats, respectively, have been shown to produce systemic side effects such as chronic toxicity [35] . based on our observations and these values, intraperitoneal injection of 10 mg/kg hinokitiol would be the maximum concentration for systemic administration in mice. however, this amount did not decrease bacterial load in our mouse pneumonia model, suggesting that this concentration is inadequate to achieve mic against s. pneumoniae in the lungs. topical administration of antibacterial agents has been successfully evaluated in the clinic for the prevention as well as therapeutic treatment of invasive pulmonary infections. our in vitro findings indicate that hinokitiol exhibited little cytotoxicity against pulmonary alveolar epithelial cells. aminoglycoside formulations for inhalation were developed to maximize drug delivery to the respiratory tract and minimize toxicity [36] [37] [38] . opting for an alternative route such as intratracheal administration of hinokitiol achieved the mic against s. pneumoniae, reducing disruption of lung tissue and suppressing bacteremia without systemic side effects. intratracheal administration demonstrated the efficacy of hinokitiol in a mouse pneumonia model; however, this may not necessarily indicate successful and complete treatment of pneumonia. clinically, treatment of infectious diseases requires thorough bactericidal effect until the infectious inflammation is resolved [39, 40] . therefore, a repeated dose of antibiotics is often prescribed to maintain an adequate concentration of the drug at the site of infection [41] [42] [43] . our repeated administration experiment showed that although single or two-time hinokitiol administration did not significantly lower the bacterial load 4 days after infection, repeating the procedure three times successfully decreased the bacterial load in the mouse pneumonia model. this finding suggests that hinokitiol administrated intratracheally does not exhibit antibacterial activity for an extended period and repeated administration may be required to treat pneumococcal pneumonia. effective use of hinokitiol will demand further research to optimize the administration interval and treatment period. hinokitiol intratracheal injection: neutrophil elastase (ne: green), dapi (blue). magnification: 40×; scale bar: 50 μm. (d) ne activity in balf of mice infected with either antimicrobial-susceptible s. pneumoniae strain d39 or macrolide-resistant strain nu4471 and then treated with pbs or hinokitiol. data represents the mean ± sem (n = 5) and was evaluated using one-way analysis of variance with tukey's multiple-comparisons test. � significantly different from the infected control group at p < 0.05. nd: not-detected, ns: not-significant; � p < 0.05. https://doi.org/10.1371/journal.pone.0240329.g004 antimicrobial resistance is a global problem [44] ; one way to deal with it is by developing alternative antimicrobials [45] , including from plant essential oils [46] . hinokitiol has been shown to inhibit the growth of antimicrobial-resistant organisms in vitro, such as methicillinresistant staphylococcus aureus and multidrug-resistant s. pneumoniae [10, 27, 47] . in this study, intratracheal administration of hinokitiol suppressed bacterial pneumonia induced by macrolide-resistant pneumococci. thus, hinokitiol could represent a new weapon against the growing class of macrolide-resistant s. pneumoniae. neutrophils are rapidly recruited to the lungs of pneumonia patients [48] . previous studies have shown that s. pneumoniae induced the release of ne from neutrophils, leading to the disruption of alveolar epithelial cells [28] . moreover, several animal studies have reported that the inhibition of ne activity suppressed lung injury in pneumococcal pneumonia [49, 50] . in this study, intratracheal hinokitiol administration reduced neutrophil infiltration and elastase release in the lungs of pneumonia mice, consequently diminishing lung injury and pneumococcal dna detection in serum. excessive production of inflammatory mediators induces a severe systemic response in pneumonia [51] . although antibacterial treatment is an important approach against pneumonia, regulating excessive inflammation is equally essential [52, 53] . hinokitiol was previously shown to reduce the transcription of inflammatory cytokines from epithelial cells in vitro [6, 54] . in this study, intratracheal administration of hinokitiol decreased the concentrations of cytokines and chemokines in balf and serum. these findings suggest that the anti-inflammatory property of hinokitiol, in addition to its antibacterial activity, might ameliorate the severity of pneumonia by downregulating the production of inflammatory mediators. certain limitaitions are noted in the current study. first of all, the administration of hinokitiol 1 h after bacterial inoculation is not representative of the clinical scenario, where antibiotics and other therapies are often initiated days after infection onset. additionally, most mouse experiments were only evaluated at a single time point in this study as obtaining balf and lung tissues requires the mice to be sacrificed. finally, the study does not include any conventional antibiotic comparators. the results of multiple doses indicate the limit of the antibacterial effect of hinokitiol and are expected to play a role as an adjunct to other antibacterial drugs. therefore, further research is needed to apply hinokitiol to the treatment of pneumonia, including hinokitiol alone as well as in combination with existing antibacterial drugs and hinokitiol. in conclusion, the present study showed that intratracheal hinokitiol administration suppresses acute pneumococcal pneumonia in vivo, regardless of whether bacteria are susceptible to macrolides. in case of severe pneumonia caused by antimicrobial-resistant pneumococci, a systemic dose of existing antimicrobials alone may not be successful [55, 56] . adjunctive use of hinokitiol may facilitate the treatment of pneumococcal pneumonia and reduce the use of existing antimicrobials. supporting information s1 fig. changes in body weight in mice intraperitoneally injected with hinokitiol. male 8-week-old balb/c mice were intraperitoneally injected once a day with 0, 10, 20, 30, 50, and 100 mg/ kg hinokitiol in pbs containing 10% ethanol buffer for 7 days. body weight was monitored every day. data represent the mean ± sem (n = 4) and were analyzed using two-way anova with dunnett's multiple comparison test; � p < 0.05. (tif) male 8-week-old balb/c mice were intratracheally infected with s. pneumoniae strain d39 (1.0 × 10 9 cfu in 50 μl pbs) and, 1 h after infection, they (n = 5) were intraperitoneally injected with hinokitiol (10 mg/kg, n = 5) or pbs (containing 10% ethanol, n = 5). after 18 h, balf samples were plated on blood-agar plates and cultured aerobically to count cfu. data represent the mean ± sem (n = 5) and were analyzed using student's t-test; � p < 0.05. (tif) s3 fig. hinokitiol does not affect the transcription of chemokine and cytokine genes in mice. real-time pcr was performed to quantify the transcription of cxcl1, il1b, il6, and tnf in mouse lung tissue. total rna was extracted from mouse lung tissue using tri reagent (molecular research center, inc., cincinnati, oh, usa), and quality was assessed spectrophotometrically at 260 and 280 nm. the rna was reverse-transcribed using superscript vilo master mix (thermo fisher scientific, waltham, ma, usa) and the cdna was quantified using the steponeplus real-time pcr system according to the manufacturer's protocol. values were normalized to those of gapdh mrna and are presented as fold change relative to the mrna transcript levels of the control group. data represent the mean ± sem (n = 5) and were analyzed using student's t-test; � p < 0.05. (tif) all mice were intratracheally infected with s. pneumoniae strain nu4471 (3.0 × 10 8 cfu/mouse). pbs injection or hinokitiol (500 μg/ml in pbs) injection via the tracheal route was started after 1 h from infection. pbs or hinokitiol injection into the air tract was performed at 24 h intervals. all mice were sacrificed and samples were collected after 72 h from infection. 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polymorphonuclear leukocyte effects of specific neutrophil elastase inhibitor, sivelestat sodium hydrate, in murine model of severe pneumococcal pneumonia inhibition of neutrophil elastase reduces lung injury and bacterial count in hamsters proinflammatory and anti-inflammatory cytokines as mediators in the pathogenesis of septic shock impact of macrolide therapy on mortality for patients with severe sepsis due to pneumonia addition of a macrolide to a beta-lactam-based empirical antibiotic regimen is associated with lower in-hospital mortality for patients with bacteremic pneumococcal pneumonia anti-inflammatory effects of hinokitiol on human corneal epithelial cells: an in vitro study a review of clinical failures associated with macrolide-resistant streptococcus pneumoniae resistance among streptococcus pneumoniae: implications for drug selection we would like to thank editage (www.editage.com) for english language editing. key: cord-277548-hgmmtew3 authors: lou, emil; teoh, deanna; brown, katherine; blaes, anne; holtan, shernan g.; jewett, patricia; parsons, helen; mburu, e. waruiru; thomaier, lauren; hui, jane yuet ching; nelson, heather h.; vogel, rachel i. title: perspectives of cancer patients and their health during the covid-19 pandemic date: 2020-10-30 journal: plos one doi: 10.1371/journal.pone.0241741 sha: doc_id: 277548 cord_uid: hgmmtew3 introduction: the immunosuppressive nature of some cancers and many cancer-directed treatments may increase the risk of infection with and severe sequelae from coronavirus disease 2019 (covid-19). the objective of this study was to compare concerns about covid-19 among individuals undergoing cancer treatment to those with a history of cancer not currently receiving therapy and to those without a cancer history. methods: we conducted a cross-sectional anonymous online survey study of adults currently residing in the united states. participants were recruited over a one-week period (april 3–11, 2020) using promoted advertisements on facebook and twitter. groups were compared using chi-squared tests, fisher’s exact tests, and t-tests. results: 543 respondents from 47 states provided information on their cancer history and were included in analyses. participants receiving active treatment reported greater concern about infection from the sars-cov-2 coronavirus (p<0.001), higher levels of family distress caused by the covid-19 pandemic (p = 0.004), and greater concern that the general public does not adequately understand the seriousness of covid-19 (p = 0.04). those with metastatic disease were more likely to indicate that covid-19 had negatively affected their cancer care compared to patients with non-metastatic cancer (50.8% vs. 31.0%; p = 0.02). the most commonly reported treatment modifications included chemotherapy delays. conclusions: patients undergoing active treatment for cancer were most concerned about the short-term effects of the covid-19 pandemic on the logistics as well as potential efficacy of ongoing cancer treatment, longer term effects, and overarching societal concerns that the population at large is not as concerned about the public health implications of sars-cov-2 infection. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the coronavirus disease 2019 (covid-19) pandemic has led to sudden shifts in healthcare, including re-categorization of "essential" care [1] . the population of patients with a current or previous history of cancer represents a unique subset that as a whole may be more susceptible to sars-cov-2 coronavirus infection and sequelae of covid-19 [2] . this is thought to be due to the immunosuppressive nature of some cancers and many cancer-directed treatments, and possibly due to more frequent clinical visits for patients undergoing active treatment and for those in active surveillance following treatment compared to patients without cancer [3, 4] . under usual circumstances, a cancer diagnosis elicits anxiety regarding scheduling logistics, outcomes, and side-effects of cancer-directed treatments [5] . the recent changes in cancer care due to the covid-19 pandemic [6] , including treatment delays, cancellation of procedures deemed not essential, and a transition into virtual rather than in-person clinic visits, have likely added to these usual uncertainties and fears associated with having cancer. we conducted a cross-sectional survey study to examine emotional well-being and health care decision-making in individuals with and without a history of cancer. we hypothesized that while distress and anxiety are increased in the general population related to the pandemic, these emotions would be higher in patients with a diagnosis of cancer. the primary objective of this survey study was to compare emotional well-being and decision-making among cancer patients undergoing therapy during the covid-19 health crisis to two control groups: 1) cancer survivors who are not currently undergoing treatment and 2) those without a history of cancer. this anonymous cross-sectional online survey study was approved by the university of minnesota institutional review board. to be eligible, participants had to be 18 years of age or older, currently reside in the united states, and be able to read and write in english. individuals were recruited over a one-week period (april 3-11, 2020) using promoted ads on the social media apps facebook and twitter, with separate ads specifically targeting those with a history of cancer. invitations to participate in the survey were also posted by the american cancer society. survey data were collected and stored using redcap, a webbased data collection tool [7] . survey items included demographics, personal concerns about covid-19, and among those currently receiving cancer therapy, perceived effects of the covid-19 pandemic on cancer treatment. validated measures were used or modified as appropriate when possible. symptoms of generalized anxiety and depression were measured using the general anxiety disorder-7 (gad-7) [8] and patient health questionnaire (phq-8) [9] , respectively; and potentially clinically relevant cutoffs of 10 or greater were used for each. the number of covid-19 cases in each state was determined using data from the centers of disease control and prevention (cdc) as of april 3, 2020 [10] , and categorized as �1000 versus >1000 for analyses. we used chi-squared tests and analysis of variance (anova) to compare demographic characteristics between the three groups of interest: 1) cancer patients currently undergoing therapy, 2) individuals with a current or past diagnosis of cancer not currently undergoing therapy, 3) individuals with no history of cancer. the following demographic variables were categorized for analyses: gender (male, female, non-binary), race (white, black or african american, asian, other), annual household income (<$50,000, $50,000-$99,999, �$100,000), education (no college degree, at least college degree), partner status (single, married/partnered), employment status (not working, working part or full time, retired). descriptive statistics summarized rates of treatment plan changes among cancer patients currently receiving therapy, and compared those with and without metastatic disease using chi-squared and fisher's exact tests. data were analyzed using sas 9.4 (cary, nc), and p-values <0.05 were considered statistically significant. among the 839 individuals who opened the survey, 815 (97%) were eligible for the study. a total of 763 answered at least one question and 543 participants provided sufficient information on their cancer history to be included in this analysis. among eligible participants, 25.6% were undergoing active cancer treatment, 29.8% reported a history of cancer but no current active treatment, and 44.6% reported no history of cancer. forty-seven states were represented; there were no respondents from alaska, vermont or wyoming. minnesota had the highest percentage of respondents (24.0%). only two respondents reported being diagnosed with sars-cov-2. the average age of participants was 55.5 years (sd = 14.4), and the majority was female (82.8%) and non-hispanic white (95.0%), with 23.9% being responsible for children under 18 years old, and 15.2% caring for other adult(s). those with a history of cancer were older (p = 0.001), more likely to be retired (p = 0.003), and less likely to have a child under 18 years old living with them (p = 0.006; table 1 ). participants with cancer currently undergoing treatment were more likely to report their general health as fair or poor (p<0.001). all other demographic characteristics were balanced between the groups, including the number of covid-19 cases in state of residence. among those ever diagnosed with cancer, the three most common cancers were breast (40.5%), colon (16.6%), and non-melanoma skin cancer (10%). approximately one-quarter (25.3%) of those with a cancer diagnosis reported stage iv cancer. rates of generalized anxiety and depression were similar across groups (table 2 ). however, those in the active treatment group were more likely to report high levels of concern about getting infected by sars-cov-2 (71.9%), which was significantly higher than those who had completed cancer treatment (47.9%), or had no history of cancer (51.9%; p<0.001). in contrast, there was a gradient across groups when asked about their risk for a severe manifestation of the infection (p<0.001); only 33.8% of those with no cancer history felt they were at high risk for severe disease compared with 58.0% of those who had completed cancer treatment, and 90.7% of those who were in active treatment. most respondents in the three groups reported that they regarded covid-19 as a "moderately" or "very" serious threat, but patients with a history of previously treated cancer or cancer undergoing active treatment were more likely to report practicing complete social/physical distancing in the previous week as compared to individuals without cancer (p<0.001). despite this difference, a majority of all three groups (>80%) reported high concern about close family members or friends becoming infected. patients undergoing active treatment reported the highest level of family distress caused by the covid-19 pandemic (p = 0.004), as well as concern that some others did not adequately understand the seriousness of covid-19 and its implications (p = 0.04). among those undergoing therapy, 20.7% overall reported no contact with their oncologists about treatment plans since the pandemic began. those with metastatic cancer were significantly more likely to have had contact with their oncologist than those with non-metastatic disease (86.8% vs. 70.9%; p = 0.03) ( table 3) . a higher proportion of participants with metastatic cancer reported that they "strongly" or "somewhat strongly" agreed with the statement that covid-19 had negatively affected their cancer care, compared to participants with nonmetastatic cancer (50.8% vs. 31.0%; p = 0.02). some (n = 26, 17.9%) of those currently receiving cancer therapy stated the pandemic changed their ongoing treatment plans. the most commonly specified form of treatment modification was delay or stopping of chemotherapy infusion (n = 14, 53.8%), followed by delayed surgery (n = 5, 19.2% ). concern about risk of exposure to covid-19 was the most commonly cited reason for any changes (80.8%); other reasons included hospital or clinic policies (38.5%) or strict visitor policies (30.8%). those who reported having treatment plans changed were more likely to report clinically relevant symptoms of anxiety (52.0% vs. 17.0%, p<0.001) and depression (56.0% vs. 20.0%, p<0.001) than those who did not report a treatment plan change. almost all participants noted a switch to at least some telehealth from in-person clinical visits. more than half of participants stated that decision-making was shared between the patient and physician; 7.2% reported that the decision was made "with little or no input from my doctor", and conversely 19.2% reported that the decision was made "with little or no input from me." patients with cancer undergoing active treatment had the highest level of concern of getting infected by sars-cov-2. more than 90% in active treatment feared having a severe manifestation of the infection. this same population reported the highest level of family distress caused by the covid-19 pandemic, indicating that related anxiety extended beyond just individual concern. however, the increased extent of anxiety was focused on covid-19 and potential complications related to this infection. as the self-reported prevalence of generalized anxiety and depression did not differ significantly between patients actively being treated for cancer vs. respondents with no history of cancer, it is possible that the societal level of anxiety overall was high during the onset of the pandemic, independent of cancer diagnosis, and this may have accounted for that lack of difference. nearly 20% of participants reported that treatment changes during the pandemic were done "with little or no input" from them, but rather the logistical changes of cancer care were initiated exclusively or nearly exclusively by their treatment team. the full impact of the covid-19 pandemic on cancer patients may not be known for years, if not decades. in the immediate short-term, the current situation has rapidly altered the landscape of acceptable modifications to treatment in order to most effectively balance safety with efficacy of treatment. what we learned from this study is that, whether in fact or by patient perception, the extent of engagement of cancer-treating teams did not match the height of anxiety that many patients with cancer had regarding their own health and also the health of their family members during the onset and peak of this pandemic. we can use this information to better educate the medical community that patients need firm guidance and communication early and often during such times of crisis; this point is especially important as we now anticipate additional 'waves' of peaks of the covid-19 pandemic, and cannot rule out future pandemics from other causes. initial efforts to quickly adapt cancer care to the new environment prioritizing safety focused mostly on discussions among medical experts; these results shine a spotlight on the equally important need to prioritize community engagement with the patient advocacy community and support groups to disseminate needed reassurance, in addition to conveying these changes to patients directly. individual partnerships between patient and physician are integral and crucial to achieving this goal. the major concerns noted by respondents in our survey align with those reported on april 17, 2020 in the american cancer society's (acs) findings summary from its recent advocacy survey on the pandemic's impact on cancer patients and survivors [11] . key findings from that survey included 50% of cancer patients and survivors reporting impact on their healthcare during this time, with 27% of all patients who had been undergoing active treatment reporting some delay. similar to our survey, 40% of acs respondents undergoing active treatment expressed acute concerns about effects on their cancer-directed therapy plans [11] . our survey identifies some important gaps in knowledge. specifically, what risks are posed to those that have a history of cancer but have completed treatment and are in less frequent contact with their care team? this group perceived their risk for contracting sars-cov-2 as being notably lower than those on active treatment. however, they also were concerned that they were at higher risk for developing severe disease. how susceptibility and disease pathogenesis are impacted by prior cancer therapy is a pressing research question, given the large number of survivors who have completed treatment, and accelerated aging and increased prevalence of cardiovascular disease that often accompany chemotherapeutic treatments. one strength of this study was harnessing social media to quickly disseminate a research study in a short period of time. however, limitations of this method led to disproportionate representation of cancer patients residing in minnesota and the midwest, thus limiting generalizability of study results to all cancer patients and the general population. the majority of respondents was skewed toward a specific (>90% white and >80% female) demographic that also represented a technologically savvy population familiar and at ease with use of social media and associated technology. a more diverse population with regards to race and sex would have been more representative of the population of patients with cancer in the u.s. and may have provided different results. furthermore, the heterogeneity of cancer types represented, with inherent differences in treatment strategies and effect of treatment modifications and delays, impacts the ability to report specific alterations in treatment plans and the effect of specific management alterations on patient emotional state. another limitation is the inability to discern between perception or misperception of patients on whether their cancer care was negatively affected by early changes to routine administration of treatment, in efforts to maximize patient safety. some general examples of such changes included modifications to dosages and frequency of administration of systemic chemotherapies at a time when the duration and severity of covid-19 were not immediately clear. recent and future studies objectively examining changes in patterns of cancer diagnoses and outcomes in the years to come will help to clarify this issue. in summary, patient respondents undergoing active treatment for cancer harbored concerns about short-term effects of the covid-19 pandemic on the logistics and potential efficacy of ongoing cancer treatment, about longer term effects, and that the population at large did not take the public health implications of the sars-cov-2 virus seriously enough. the cancer care landscape is quickly evolving to meet the needs of patients by attempting to balance safety with treatment efficacy. over the long term, some of these adaptations, including virtual visits, may be permanently adopted by patients and healthcare systems as integral to overall cancer care. regardless of which approaches will be taken, partnership and adequate levels of communication between cancer care teams and patients with cancer are and will continue to remain critical during and for a long time following the current pandemic. supporting information s1 file. covid-19 perceptions of health survey. (pdf) funding acquisition: rachel i writing -original draft taking the longer view of covid-19 clinical characteristics of covid-19-infected cancer patients: a retrospective case study in three hospitals within wuhan coronavirus cancer monitoring project team. the uk coronavirus cancer monitoring project: protecting patients with cancer in the era of covid-19 the impact of the covid-19 pandemic on cancer patients logistic toxicity, an unmeasured burden of healthcare. forbes magazine ethics and resource scarcity: asco recommendations for the oncology community during the covid19 pandemic research electronic data capture (redcap)-a metadata-driven methodology and workflow process for providing translational research informatics support a brief measure for assessing generalized anxiety disorder: the gad-7 the phq-8 as a measure of current depression in the general population covid-19) cases in u covid-19 pandemic impact on cancer patients and survivors: survey findings summary key: cord-288502-qqg41daz authors: martini, katharina; blüthgen, christian; walter, joan elias; nguyen-kim, thi dan linh; thienemann, friedrich; frauenfelder, thomas title: patterns of organizing pneumonia and microinfarcts as surrogate for endothelial disruption and microangiopathic thromboembolic events in patients with coronavirus disease 2019 date: 2020-10-05 journal: plos one doi: 10.1371/journal.pone.0240078 sha: doc_id: 288502 cord_uid: qqg41daz background: to evaluate chest-computed-tomography (ct) scans in coronavirus-disease-2019 (covid-19) patients for signs of organizing pneumonia (op) and microinfarction as surrogate for microscopic thromboembolic events. methods: real-time polymerase-chain-reaction (rt-pcr)-confirmed covid-19 patients undergoing chest-ct (non-enhanced, enhanced, pulmonary-angiography [ct-pa]) from march-april 2020 were retrospectively included (covid-19-cohort). as control-groups served 175 patients from 2020 (cohort-2020) and 157 patients from 2019 (cohort-2019) undergoing ct-pa for pulmonary embolism (pe) during the respective time frame at our institution. two independent readers assessed for presence and location of pe in all three cohorts. in covid-19 patients additionally parenchymal changes typical of covid-19 pneumonia, infarct pneumonia and op were assessed. inter-reader agreement and prevalence of pe in different cohorts were calculated. results: from 68 covid-19 patients (42 female [61.8%], median age 59 years [range 32–89]) undergoing chest-ct 38 obtained ct-pa. inter-reader-agreement was good (k = 0.781). on ct-pa, 13.2% of covid-19 patients presented with pe whereas in the control-groups prevalence of pe was 9.1% and 8.9%, respectively (p = 0.452). up to 50% of covid-19 patients showed changes typical for op. 21.1% of covid-19 patients suspected with pe showed subpleural wedge-shaped consolidation resembling infarct pneumonia, while only 13.2% showed visible filling defects of the pulmonary artery branches on ct-pa. conclusion: despite the reported hypercoagulability in critically ill patients with covid-19, we did not encounter higher prevalence of pe in our patient cohort compared to the control cohorts. however, patients with suspected pe showed a higher prevalence of lung changes, resembling patterns of infarct pneumonia or op and ct-signs of pulmonary-artery hypertension. introduction recent observations suggest that respiratory failure in novel coronavirus disease 2019 (covid19) is not driven by the development of acute respiratory distress syndrome alone, but that microvascular thrombotic processes may play a role [1] . this may have important consequences for the diagnostic and therapeutic management of these patients. there is a strong association between d-dimer levels, disease progression and chest ct features suggesting venous thrombosis. in addition, various studies in patients with covid-19 have shown a very strong association between increased d-dimer levels and severe disease/poor prognosis [1] . different studies have shown that covid-19 is characterized by hypercoagulability and endothelial dysfunction [2, 3] . this, together with the commonly observed higher occurrence of altered coagulopathy in sepsis [4] , makes covid-19 patients prone to thromboembolic events. in fact, some trials demonstrated higher prevalence of cardiac, cerebrovascular, and pulmonary vascular events in covid-19 patients [2, [5] [6] [7] . however, since detection of typical lung imaging features of covid-19 does not require intravenous contrast agent, patients with covid-19 pneumonia are generally imaged with non-contrast chest ct. one hypothesis is, that the high mortality observed among covid-19 patients may be partly due to undiagnosed pulmonary embolism (pe) and pulmonary in situ thrombosis, outlining the possible role of ct pulmonary angiography (ct-pa) in patients with rapid clinical worsening [2, 8] for appropriate management. micro-embolism caused by microscopic endothelial disruption, however, are too small to be captured on ct. the only surrogate for the underlying pathophysiologic process might be secondary lung changes such as peripheral wedge-shaped consolidation. the pathophysiology might be comparable to that of organizing pneumonia (op), where alveolar epithelial injury leads to an activation of the coagulation cascade and fibrin deposition within the alveoli causing the typical parenchymal changes on high resolution ct (hrct) of the lungs [9] . to date, studies showed a significantly higher percentage of macroscopic pe in covid-19 patients [2, 5] . however, the dark figure of unreported microscopic thromboembolic events in the lungs remain unclear. therefore, the purpose of the study was to investigate possible typical parenchymal lung changes resembling patterns of infarct pneumonia or op as surrogate for microscopic thromboembolic events in covid-19 patients. in this retrospective cohort study, we included data of consecutively admitted adults with covid-19 to the isolation wards and intensive care units at the university hospital zurich, one of the largest tertiary care centres in switzerland. covid-19 was confirmed in all cases using real-time polymerase chain reaction (rt-pcr) for severe acute respiratory syndrome virus 2 (sars-cov-2). clinical information and diagnostic results were extracted from our hospital electronic medical records. the swiss ethics committee (swissethics, section zurich) approved the study and written informed consent was waived due to the retrospective nature of the study. patients undergoing ct-pa for suspected pe at our institution from march to april 2020 (cohort 2020 -tested negative for covid-19 infection) and march to april 2019 (cohort 2019 -no test available; but acquired before the outbreak of covid-19) in the same time frame served as two control cohorts. chest x-ray was performed in all patients with covid-19 as baseline investigation on admission. in case of a normal chest x-ray and clinical suspected pneumonia, a ct chest without contrast was performed. ct-pa was performed if at least one of the following criteria was present: 1) clinical signs and symptoms of deep vein thrombosis, 2) tachypnoea, 3) decreased oxygen saturation, or 4) high oxygen demand. single-energy ct was performed in all patients on third-generation ct scanner (somatom force, somatom definition as, or somatom definition flash; siemens healthcare, forchheim, germany) equipped with an integrated high-resolution detector (stellar technology; siemens). scanning parameters were as follows: ct was performed at 100 kvp with quality reference current-time product of 80 mas, a pitch of 1.2, gantry rotation time 0.5 s, slice acquisition of 192 x 0.6 mm by means of a z-flying focal spot. the onsite ct technician explained the breathing instructions to the patient. for ct-pa a double-syringe power injector (ct exprés, bracco-former swiss medical care, switzerland) infused iv contrast via an antecubital, subclavian or internal jugular venous access. 80 ml of iv contrast (iopromide, ultravist, 300 mg j/ml, bayer healthcare, germany) was followed by 50ml saline bolus, both at a flow rate of 4 ml/s. bolus tracking was performed with a threshold at 100 hu (at 100 kvp) in the main pulmonary artery, with a trigger delay of 10 seconds. all images were reconstructed with advanced modelled iterative reconstruction (admire, siemens healthcare, forchheim, germany) at a strength level of 3, using a slice thickness of 1.5 mm, an increment of 1 mm, and a tissue convolution kernel (bl34). the image matrix was 512 x 512 pixels. the images were presented to two independent readers (tf and tdlnk, both attending radiologists with 20 and 11 years of experience and blinded to clinical information. evaluation of le specific findings. both readers independently assessed the images for the presence and location (central vs. segmental vs. sub-segmental) of pe and presence of peripheral wedge-shaped consolidation as signs of infarct pneumonia. furthermore, readers measured the right/left ventricular ratio (rv/lv ratio), the width of the pulmonary artery and the ratio between pulmonary artery and aorta (p/a ratio) in order to capture indirect signs for raised pulmonary arterial pressure and right heart failure. according to ende-verhaar et al. [10] rv/lv ratio was calculated by dividing the maximal distance between the ventricular endocardium and the interventricular septum, perpendicular to the long axis of the heart measured on standard axial views. the maximum dimensions for both ventricles were used for measurements. according to lee et al. [11] the pulmonary artery and ascending aorta were assessed on a transverse image at the level of the pulmonary artery bifurcation. vessel diameters were obtained by measuring the widest diameter vertical to the long axis of the main pulmonary artery (fig 1) . while pa diameter and p/a ratio was performed in the entire datasets, rv/lv ratio could only be measured in contrast-enhanced ct scans. evaluation of lung parenchymal changes. further parameters for covid-19 pneumonia and op were assessed (for details see table 1 ). images were assessed at a random order over a time period of one week. readers were allowed to modify the window width and level after the initial presentation with a mediastinal and lung window. we chose to not use one of the several proposed scores for covid-19, since we wanted to focus on specific parenchymal changes, which might get lost when an overall scoring system is used. analyses were performed using the picture archiving and communication system (pacs) of our hospital (impax, version 6.5.5.1033; agfa-gevaert, mortsel, belgium) on a high-definition liquid crystal display monitor (barco; medical imaging systems, kortrijk, belgium). statistical analyses were conducted using commercially available software (spss, release 26.0; spss, chicago, il, usa). continuous variables were expressed as mean +/-standard deviation (sd) while categorical variables were expressed as frequencies or percentages. cohen's kappa (κ) was used to assess inter-reader agreement for presence of pe. -results were stratified qualitatively by score (slight agreement 0.01-0.20; fair agreement 0.21-0.40; moderate agreement 0.41-0.60; good agreement 0.61-0.80; excellent agreement 0.81-0.99 [12] . prevalence of pe was calculated for each cohort. man-whitney-u-test and a two-sided t-test for unpaired variables was used to test for statistical significance. a two-sided p-value below 0.05 was defined to indicate statistical significance. table 1 . imaging findings in the covid-19-cohort-lung parenchyma changes. computed tomography pulmonary angiography (ct-pa), number of cases (n), predominant (pred.), ground-glass opacification (ggo), cryptogenic organizing pneumonia (cop), bronchocentric pattern: conspicuous cuffs around larger bundles with extension to lung periphery, band like pattern: thick radial bands (�8mm width), sometime containing a small air bronchogram, which distinguishes it from linear atelectasis, crazy paving: ggo and superimposed interlobular septal thickening, pulmonary artery (pa), diameter (dia.), pulmonary artery aorta ratio (p/a ratio), right/left ventricular ratio (rv/lv ratio). fig 2) . mean time from onset of clinical symptoms was of 7.2 days (sd ±8.9) on date of ct. the following comorbidities were present: cardiovascular disease (26.5%), arterial hypertension (51.5%), diabetes (35.3%), chronic renal failure (26.5%), and chronic pulmonary disease (19.1%). 23.5% of patients (n = 16) were receiving anticoagulative therapy at time point of ct, which mostly was performed already on admission. mean value of d-dimer was of 2.7 mg/l (sd±2.7) (non-ct-pa group 1.8 mg/l ±1.2 vs. ct-pa group 3.1 mg/l ±3.1; p = 0.269). detailed information on clinical findings can be found in table 3 . mean inter-reader agreement for imaging findings was good (k = 0.781). female, n (%) 20 covid specific findings. predominant findings in covid-19 patients where ground glass opacities (ggo, 57.4%) with a basal, subpleural distribution, followed by consolidations (41.2%) with a similar distribution pattern ( table 1 ). the majority of patients showed bilateral lung changes (94.1% vs 6.9%; respectively). additional findings such as lymphadenopathy (defined as enlarged lymph nodes > 1cm short axis) or pleural effusion were relatively rare (11.8% and 25.0%, respectively). pe specific findings. in the covid-19 cohort, five patients presented with pe (13.2%), while in cohort 2019 14 patients (8.9%) and cohort 2020 16 patients (9.1%) were diagnosed with pe (p = 452) ( table 2 and figs 1 and 3) . covid-19 patients suspected with pe tended to show more triangular or wedge shaped peripheral ggo compared to covid-19 patients without suspected pe (21.1% vs. 13.3% respectively, p = 0.465). half of covid-19 patients with patterns resembling infarct pneumonia on ct showed signs of right heart failure and pulmonary hypertension on ct (increased diameter of the pulmonary artery, lowered rv/lv ratio or increase in p/a ratio). patients who underwent ct-pa and patients, where ct showed peripherally wedge-shaped consolidation resembling patterns of infarct pneumonia had significantly higher p/a ratio compared to their counterparts (p/a ratio 0.98 vs 0.91, p = 0.022 and p/a ratio 0.93 vs 0.88, p = 0.024; respectively). median rv/lv ratio tended to be higher in patients showed ct sign of infarct pneumonia compared to patients without this pattern (rv/lv 0.88 vs 0.94, respectively; p = 0.289 (tables 1 and 4) . op specific findings. covid-19 patients showed parenchymal changes typical of op, namely rhomboid shape (18.2%), bronchocentric pattern (50.0%), band like pattern (36.8%), and crazy paving (36.8%). the nodular reversed halo sign was only seen in 8.8% of covid-19 patients (fig 4) . hypercoagulability is present in severe infection [13] [14] [15] and has also been reported in covid-19: recent studies showed, that covid-19 patients have increased d-dimers, fibrin degradation product (fdp), and fibrinogen levels compared to healthy volunteers [16] . these alterations in the state of coagulation reportedly lead to a higher incidence of thromboembolic events [2] . in contrary to this, we could not show a higher prevalence of pe in our covid-19 cohort compared to control groups from 2019 and 2020. however, we encountered lung parenchyma changes, mimicking those of infarct pneumonia and op. normally, the annual incidence of venous thromboembolism is reported to be between 20-70 cases per 100.000 [17, 18] . one third of those patients have acute pe, while two thirds remain isolated deep vein thrombosis [19] . the current approach to patients with suspected pe is based on a clinical adjudication of patients into a high (>15%) and a non-high risk group of early pe-related death and ct-pa is the first-choice imaging modality in the former [17] . a hypothesis is, that the high mortality observed among covid-19 patients may be partly due to unrecognized pe and pulmonary in situ thrombosis. however, current guidelines advocate the use of non-contrast chest ct for the diagnosis, severity assessment, and monitoring of covid-19 [20] . although the mortality of covid-19 infection seems relatively low, patients with severe or critical disease are at high risk of developing acute respiratory distress syndrome (ards) and to be admitted to the intensive care unit (icu). therefore, accurate diagnosis and monitoring of disease progression from the early stages is essential to improve the otherwise unfavourable clinical outcomes [2, 8] . higher prevalence of pe would outline the possible role of ct-pa in patients with covid-19 infection and rapid clinical worsening [2, 8] to appropriately diagnose and manage those patients. in line with our results lodigiani et al. reported a prevalence of 10% pe in patients undergoing ct-pa (the cumulative rate of arterial and venous thromboembolic events was of 21%) [5] . in our study this percentage does not significantly differ from the covid-19 negative control groups during the same period in 2019 and 2020 and also not from the numbers reported in the literature [19] . this would stay in contrast to the previously believed higher incidence of thromboembolic events in covid-19 patients [16] . however, more than 20% of our patients suspected with pe, showed signs of infarct pneumonia, despite the lack of visible filling defects in the branches of the pulmonary artery. we hypothesize, that the altered coagulation function in covid-19 results from microscopic endothelial disruption, which leads to the formation of micro-embolism, too small to be detected on ct-pa. this is in line with a previous report from our hospital on three post-mortem case studies. findings suggest an endothelial infection with sars-cov-2 leading to endotheliitis in covid-19 of most organs including the lungs with consecutive table 4 . imaging findings in the covid-19-cohort-ct signs for right heart failure/pulmonary artery hypertension. peripheral wedge-shaped consolidation (pwsc) as imaging biomarker for microinfarction, number of cases (n), pulmonary artery (pa), diameter (dia.), pulmonary artery aorta ratio (p/a ratio), right/left ventricular ratio (rv/lv ratio). microthrombosis [3] . this leads to the conclusion, that the only the surrogate for the underlying pathophysiologic process during covid-19 are the secondary lung changes which become visible as peripheral wedge-shaped consolidation, as typical signs of infarct pneumonia. further, in more than half of our patients (regardless if with suspected pe or not) we encountered patterns normally seen in op. this may be due to a similar histopathological mechanism: alveolar epithelial injury is accompanied by damage to the basement membrane; consecutively, plasma proteins and inflammatory cells can leak into the airspace and activate the coagulation cascade leading to fibrin deposition. fibrin organizes into intra-alveolar fibroinflammatory buds involving whorls of myofibroblasts in a connective tissue matrix. the final step is resorption of the inflammatory process from the centre, resulting the typical image of the reversed halo sign [9] . additionally, in recent studies d-dimer and fdp were found to be especially predictive of disease progression and higher levels of d-dimer and fdp correlated with increased disease severity [16] ; hence, patients with increased levels of d-dimers and fdp (and therefore more prone to develop thromboembolic disease) show progressive lung changes, potentially "misinterpreted" by the radiologist as an increase in infective consolidation but in reality related to microinfarction. our hypothesis is further strengthened by the higher presence of ct signs for pulmonary artery hypertension and right heart failure in patients undergoing ct-pa or with ct patterns resembling infarct pneumonia. finally, we hypothesize, that the vascular pathology in covid-19 is microscopic, and thus not per se visible on ct-pa due to the restricted resolution of ct. however, what we are able to see on ct, is the response of the lung as surrogate for the microangiopathic disease, showing typical changes of lung infarction and organizing pneumonia. different studies have shown that covid-19 is characterized by hypercoagulability and endothelial dysfunction [2, 3] . hypercoagulability and endothelial dysfunction lead to a nonnegligible number of arterial and venous thrombosis in different organ systems [21, 22] . this urges the need for adequate screening procedures and antithrombotic strategies to manage thromboembolic events [23] . our study has the following limitations: first, we do not have histopathologic confirmation of infarct pneumonia or op of our patients. however, ethical concerns precluded the performance of lung biopsies in these patients. however preliminary histopathologic findings showed endothelial cell infection and endotheliitis which leads to impaired systemic microcirculatory function and there sequalae [3] . further studies involving also histopathologic examination (also in form of post-mortem studies) would be needed to confirm our hypothesis. second, the evaluated covid-19 cohort is relatively small. third, dual energy (de) ct would have been useful in the quantitative assessment of pulmonary perfusion and would have further strengthen our hypothesis of microinfarction in covid-19 patients. however, since current guidelines advocate the use of non-contrast ct chest for the diagnosis, severity assessment, and monitoring of covid-19 (20) we only performed non-contrast enhanced single energy ct for diagnosis and follow-up of lung consolidation in covid-19 patients. only if clinicians suspected pe and criteria to undergo ct-pa were present, the single energy ct-pa was performed, according to the standard procedure of our institution. however, the findings of our study would stress the need of de-ct in covid-19 patients to capture altered pulmonary perfusion in cases of suspected microembolic disease. in conclusion, despite the disturbance in blood coagulation function reported in patients with covid-19, we did not encounter higher prevalence of pe in our covid-19 cohort compared to control the cohort 2019 or cohort 2020. however, our covid-19 cohort showed lung changes resembling those of infarct pneumonia and op as well as ct-signs of pulmonary-artery hypertension. this may imply, that the vascular pathology in covid-19 patients is of microangiopathic nature and hence generally too small to be captured directly by ct. visible lung changes in ct might be a surrogate for the underlying pathology caused by sars-cov-2 unveiling the invisible endothelial changes within the lungs. an increased p/a ratio may be a hint to the underling pathology and warrant further investigation. diagnosis, prevention, and treatment of thromboembolic complications in covid-19: report of the national institute for public health of the netherlands clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study endothelial cell infection and endotheliitis in covid-19. the lancet clinical characteristics of coronavirus disease 2019 in china venous and arterial thromboembolic complications in covid-19 patients admitted to an academic hospital in coronavirus disease 2019 (covid-19) and cardiovascular disease cardiovascular disease as a biomarker for an increased risk of covid-19 infection and related poor prognosis abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia organizing pneumonia: a kaleidoscope of concepts and morphologies accuracy and reproducibility of ct right-to-left ventricular diameter measurement in patients with acute pulmonary embolism comparison of ct-determined pulmonary artery diameter, aortic diameter, and their ratio in healthy and diverse clinical conditions the measurement of observer agreement for categorical data blood coagulation: a powerful bactericidal mechanism of human innate immunity coagulation and innate immune responses: can we view them separately? blood involvement of the contact phase and intrinsic pathway in herpes simplex virus-initiated plasma coagulation prominent changes in blood coagulation of patients with sars-cov-2 infection clinical management of acute and chronic disease a population-based perspective of the hospital incidence and case-fatality rates of deep vein thrombosis and pulmonary embolism. the worcester dvt study the epidemiology of venous thromboembolism. circulation acr recommendations for the use of chest radiography and computed tomography (ct) for suspected covid-19 infection immune complement and coagulation dysfunction in adverse outcomes of sars-cov-2 infection left gonadal vein thrombosis in a patient with covid-19-associated coagulopathy covid-19 and venous thromboembolism: a meta-analysis of literature studies dan linh nguyen-kim, friedrich thienemann, thomas frauenfelder. key: cord-265812-1hcp36cw authors: de jong, cornelis n.; saes, lotte; klerk, clara p. w.; van der klift, marjolein; cornelissen, jan j.; broers, annoek e. c. title: etanercept for steroid-refractory acute graft-versus-host disease: a single center experience date: 2017-10-26 journal: plos one doi: 10.1371/journal.pone.0187184 sha: doc_id: 265812 cord_uid: 1hcp36cw background: acute graft-versus-host disease (agvhd) is an important complication of allogeneic stem cell transplantation (allosct). high dose glucocorticosteroids, are currently recommended as first-line treatment for grade ii-iv agvhd resulting in overall complete responses (cr) in 40%-50% of patients. no standard second-line regimen has been established. different options have been reported, including anti-tnfα antibodies. methods: we retrospectively reviewed the outcome of 15 patients with steroid-refractory (sr) agvhd treated with etanercept at our institution. patients were transplanted for a hematological malignancy and received either a myeloablative or a non-myeloablative conditioning regimen. prophylaxis of gvhd consisted of cyclosporin a and mycophenolic acid. results: acute gvhd was diagnosed at a median of 61 days post-transplantation. all patients had grade iii agvhd of the gut. second-line treatment with etanercept was started at a median of 13 days after initiation of first-line therapy. overall response rate was 53%, with cr in 3 patients and pr in 5 patients. median overall survival after initiation of treatment with etanercept was 66 days (range 5–267) for the entire group. median overall survival was 99 days (range 47–267 days) for responders and 17 days (range 5–66 days) for non-responders (p<0.01). nevertheless, all patients died. causes of death were progressive gvhd in 7 patients (47%), infection in 6 patients (40%), cardiac death in 1 patient (6.7%) and relapse in 1 patient (6,7%). conclusion: second-line treatment with etanercept does induce responses in sr-agvhd of the gut but appears to be associated with poor long-term survival even in responding patients. introduction allogeneic stem cell transplantation (allosct) has been established as an important treatment modality for patients with hematological malignancies, aplastic anemia, and inborn errors of hematopoietic progenitor cells. nevertheless, major lethal and non-lethal complications still prohibit a broader application of allosct. acute graft-versus-host disease (agvhd) is a major cause of morbidity and mortality after allosct [1] . high dose systemic glucocorticosteroids (steroids) are currently recommended as first-line treatment for grade ii-iv agvhd resulting in overall complete responses (cr) in 40%-50% of patients [2, 3] . however, the likelihood to respond to treatment decreases with increasing severity of the disease. patients with grade ii agvhd at diagnosis are significantly more likely to achieve a cr to initial treatment with high dose steroids as compared to patients with more advanced agvhd [4, 5] . the prognosis of patients with agvhd who fail to respond to high dose steroids is poor [2] . currently there is no standard second-line treatment for steroid-refractory agvhd (sr-agvhd). numerous strategies to treat sr-agvhd have been reported, but results have been disappointing with varying response rates and long term overall survival (os) of only 20-30% [2] . tumor necrosis factor alpha (tnfα) is involved in the pathophysiology of agvhd by activating antigen-presenting cells, recruiting effector cells and causing tissue damage [6] . etanercept is a recombinant human tumor necrosis receptor fusion protein which binds tnfα with high affinity and as a consequence inhibits the biological activity of tnfα. studies that have investigated the use of anti-tnfα as primary as well as secondary treatment in agvhd have shown promising response rates. first-line combination with methylprednisolone yielded a cr rate of 69% [7] and second-line treatment resulted in an overall response rate (orr) of 46% [8] . second-line therapy combining etanercept with daclizumab showed an orr of 67% [9] . treatment with multi-agent combination therapy including etanercept has been reported to induce responses in 81% of patients [10] . based on these data we implemented the use of etanercept as second-line treatment for sr-agvhd. here we report the results of a retrospective analysis of patients with sr-agvhd treated with etanercept in our center. we retrospectively studied 15 allosct patients treated with etanercept for sr-agvhd at the erasmus mc cancer institute between january 2009 and april 2013. the institutional review board approved the transplantation protocols, and all patients provided informed consent for stem cell transplantation and data collection. none of the transplant donors were form a vulnerable population and all donors or next of kin provided written informed consent that was freely given. according to underlying disease and age, patients received either myeloablative, non-myeloblative or reduced-intensity conditioning regimens (conditioning intensities according to bacigalupo [11] ). transplants were unmanipulated grafts form hla-identical siblings (sib) or at least 7/8 (a, b, c, drb1) hla-matched unrelated donors (mud). patients without a suitable sib or mud received an umbilical cord blood transplantation. in case of umbilical cord blood (ucb), a double ucb transplantation was performed using two at least 4/6 (a, b, drb1) matched cord blood units. acute gvhd: prophylaxis, diagnosis, treatment and response criteria standard agvhd prophylaxis for all allograft recipients included cyclosporin a (csa) and mycophenolic acid (mpa) or its prodrug mycophenolate mofetil (mmf). in case of a 7/8 hla matched mud, anti-thymocyte globulin (atg) was added to the conditioning regimen. levels of csa were measured by high performance liquid chromatography on whole blood samples and aimed at trough levels of 250-350 μg/l. acute gvhd was graded according to the modified glucksberg criteria [12] , and the diagnosis of agvhd was preferably confirmed by histology of involved tissues. first-line treatment for grade ii to iv agvhd consisted of prednisolone 2mg/kg/day and csa or mpa/mmf. grade i agvhd was treated with topical steroids. steroid refractoriness (sr) was defined as either progressive disease (pd) or mixed response (mr) after 7-14 days of first-line treatment, no response/stable disease (sd) after 10 days, or progression of initial partial response (pr) after 10 days of treatment. once sr-agvhd was established, second-line treatment with etanercept was initiated, in addition to first-line therapy. etanercept (enbrel, pfizer) was administered subcutaneously twice weekly at a dose of 25 mg for an intended period of eight weeks. prophylaxis for all patients included (val)acyclovir (herpes viruses) and cotrimoxazole (pneumocystis jirovecii) until at least one year after transplantation or prolonged in case of gvhd. patients with sr-agvhd treated with etanercept received additional anti-aspergillus prophylaxis with voriconazole. furthermore, since august 2012, patients with agvhd of the lower gastrointestinal tract (irrespective of steroid responsiveness) with complaints of bloody diarrhea or large mucosal defects at endoscopy, received levofloxacin. patients at risk for cytomegalovirus (cmv) and epstein-barr virus (ebv) reactivations were monitored by quantitative polymerase chain reaction (pcr) of plasma and treated preemptively according to local protocols. cr was defined as resolution of all signs and symptoms of agvhd. pr was defined as improvement of 1 stage in 1 or more organs without progression in other organs. mr was defined as improvement in at least one organ with progression in at least one other organ. sd was defined as absence of improvement. pd was defined as progression in one organ with no improvement in any other organ. the primary endpoint of the study was the best overall response rate (cr and pr) after start of second-line treatment with etanercept. secondary endpoints were os and toxicity. os was calculated using the actuarial method of kaplan and meier from the time of starting second-line treatment with etanercept until death. causes of death were classified as treatment-related mortality (trm) or relapse-related mortality. the characteristics of all fifteen patients treated with etanercept for sr-agvhd are shown in table 1 . median age was 54 years (range 22-65 years), eleven patients (73%) were male. one patient received a myeloablative conditioning (ma) regimen consisting of cyclophosphamide (2 x 60 mg/kg) and 2 x 6 gray (gy) total body irradiation (tbi). all other patients either received a reduced intensity conditioning (ric) with cyclophosphamide (1 x 60 mg/kg), fludarabin (4 x 40 mg/m2) and tbi (2 x 2 gy) or a non-myeloablative conditioning (nma) with fludarabin (3 x 30 mg/m2) and tbi (1 x 2 gy). eleven patients received peripheral blood stem cells either from hla identical sibling donors (n = 3) or from hla matched unrelated donors (n = 8). four patients received cord blood (cb) derived stem cells. one patient developed late-onset agvhd after discontinuation of immunosuppressants, at day 393 post-transplant. all other patients were on gvhd prophylaxis at the onset of agvhd. acute gvhd developed at a median of 61 days after transplantation (range 23-393 days). all patients had grade iii gvhd of the gut, the majority had involvement of a second organ. first-line treatment consisted of csa and prednisolone 2/mg/kg/day in fourteen patients; one patient was treated with high dose steroids combined with mmf, because impaired renal function prohibited the use of calcineurin inhibitors. initiation of second-line treatment with etanercept. second-line treatment with etanercept was initiated at a median of 13 days (range 5-36 days) after start of first-line treatment with high-dose steroids. grades and organ involvement of agvhd target organs at start of etanercept are shown in table 2 . high-dose prednisolone was maintained until achievement of objective gvhd response, with the exception of four cases in which severe infectious complications prompted earlier tapering. the median number of etanercept doses was 16 (range table 3 . clinically significant infectious complications (ctc-ae grade !3 or grade 2 requiring systemic treatment) occurred in 13 patients (87%), including bacterial sepsis in 9 patients (60%), viral infections in 8 patients (53%; including cmv and ebv reactivation, bk virus, rhinovirus, and coronavirus), and invasive pulmonary aspergillosis in 3 patients (20%). one patient experienced a seizure, which was attributed to csa, rather than etanercept. infectious complications are shown in table 4 . median os after initiation of second-line treatment with etanercept was 66 days (range 5-267) for the entire group. as shown in fig 1, median os was 99 days (range 47-267 days) for causes of death were progressive agvhd in 7 patients (47%), infection in 6 patients (40%), relapsed acute myeloid leukemia in one patient (6.7%), and cardiac arrest in 1 patient (6.7%). in half of the patients who succumbed to an infection, ongoing gvhd was considered to be a contributing factor. acute gvhd is an important cause of morbidity and mortality following allosct. steroidrefractory acute gvhd in particular is associated with a poor prognosis with a long term overall survival of only 20-30%. studies evaluating second-line treatment with the anti-tfnα agent etanercept have shown promising results with overall response rates of 50-80% [8] [9] [10] . we performed a retrospective analysis in a small cohort of patients treated with etanercept for grade iii sr-agvhd of the gut. second-line treatment with etanercept resulted in an encouraging overall response rate of 53%. nevertheless, eventually all treated patients died, most importantly due to progression of gvhd and opportunistic infections. our results compare well to a recent prospective study published by van groningen et al. concerning 21 patients with sr-agvhd treated with a combination of etanercept and inolimomab, an il-2 receptor antibody [13] . this cohort was strikingly similar to ours with respect to age, agvhd severity and proportion of patients with gut involvement. despite an orr of 48%, os was only 10% at a median of 55 days, due to progressive gvhd, serious infections and relapse of underlying disease. the pattern of a promising initial response in about 50% of patients, followed by non-relapse mortality (nrm) in patients due to either progressive gvhd or the occurrence of serious infections, in particular, compares well to our findings. wolff et al. prospectively studied the use of etanercept (16mg/m 2 ) in combination with dacluzimab (1mg/kg), an il-2 receptor antibody, in 21 patients with sr-agvhd (9). this cohort was younger with a median age of 44 years, and included fewer patients with high-stage involvement of the gut. although a promising orr of 67% was observed, survival was disappointing as only 4 out of 21 patients survived. some studies, however, report better overall response and survival rates as compared to our cohort of patients. kennedy et al. retrospectively studied the outcome of 16 patients, median age 42 years, with sr-agvhd treated with a combination of atg, tacrolimus and etanercept with or without mmf and reported an significantly higher orr of 81% and os of 50% [10] . in this analysis patients with less severe agvhd were also included, with severity of gut gvhd ranging from stage 0 to 4. xhaard et al. compared survival and infection rates in patients receiving mmf (56%), inolimomab (22%), or etanercept (23%) in addition to steroids and calcineurin inhibitors in sr-agvhd [14] . the etanercept treated patients were younger than our cohort with a median age of 46 years (range 10-60 years). most of them had high-stage gut involvement. treatment response rate was 28% in etanercept treated patients. two-year survival was 30% (95% ci: 22-41) and was not significantly different among the groups. busca et al. reported on the use of etanercept in 13 sr-agvhd patients [8] . this cohort compares well to ours with respect to age (median 52 yeara, range 26-70 years), but is more heterogeneous with respect to agvhd severity and comprises fewer patients with stage 3 to 4 gut involvement. overall response rate was 46% and 69% of patients were alive at a median followup of 429 days (range 71-1007 days). in the publications reporting on etanercept as salvage therapy for sr-agvhd, different approaches to tapering of high-dose steroids are being described, such as no tapering [13] to tapering starting at 14 days after start of etanercept [14] . wolff et al. reported to start tapering from the onset of response, like we did. the difference in orr and survival rate as observed in these studies as compared to the present study might be explained by the type and grade of gvhd involved. our study included a relatively high proportion of patients with severe stage iii and iv agvhd with bowel involvement, which is a well-known adverse prognostic factor for response to therapy [4] . in addition, the combination of different agents with etanercept in some studies and variable definitions of sr-agvhd limit direct comparison of results. moreover, interpretation of clinical response may be difficult, especially in case of gvhd of the gut. irrespective of response and survival rates, the rate of significant infectious complications is high in all reported studies. we report infectious complications in 81.3% of the patients. in 40% of patients, death was attributable to an infectious complication. the risk of opportunistic infections is known to be high in sr-agvhd, due to the strong immunosuppressive regimen imposed on a frail, recovering post-transplant immune system. anti-tnfα agents, in particular, are associated with a high incidence of opportunistic infections [15] . moreover, the use of cb derived stem cells in four patients might have contributed, as transplantation of cb derived stem cells, in particular, is associated with delayed immune reconstitution [16] . therefore, adequate monitoring and prophylaxis of infections is important. in our study, invasive aspergillosis was the cause of death in 50% of patients that died of an infection. susceptibility to aspergillus is known to be strongly increased by gvhd and immunosuppressive therapy [17] . adequate prophylaxis by hospitalizing sr-agvhd patients in high-efficiency particulate arrestance (hepa)-filtered rooms if necessary and treatment with anti-fungal medication for the duration of immunosuppressive therapy is warranted. in the present study, all but one patient received antifungal prophylaxis at the time of initiation of etanercept. thirteen patients received voriconazole and one patient was treated with amphotericin inhalations as elevated liver enzymes impeded the use of voriconazole. despite prophylaxis, three patients developed invasive mould infections. the first patient developed an invasive aspergillosis despite amphotericin inhalations. the second patient proved to have a voriconazole-resistant aspergillus, and the third patient developed a double infection of voriconazole-resistant aspergillosis and zygomycosis. viral infections were observed in 9 out of 15 patients, with reactivation of cmv in 4 patients and ebv in 3 patients. in one patient a rapid rise in ebv viral load was accompanied by lymphadenopathy and a monoclonal b-cell population in the bone marrow. this ebv-lymphoproliferative disease was successfully treated with rituximab in combination with reduction of immunosuppression. cmv was monitored by pcr in patients at risk and treated pre-emptively with (val)ganciclovir. nevertheless, one patient developed cmv-related colitis under pre-emptive treatment, probably related to the severe immunosuppressive state of our patients due to the gvhd itself and the immunosuppressive agents used. episodes of septicemia were observed in 9 patients including four due to gram-negative bacteria. all episodes of gramnegative septicemia occurred either before the introduction of levofloxacin or were caused by less susceptible strains. in conclusion, although second-line treatment of sr-agvhd of the gut with etanercept was associated with a promising initial response rate, overall survival appeared very poor, mainly due to progression of gvhd and opportunistic infections. alternative strategies to prevent and treat sr-agvhd are urgently needed and prospective studies should be prioritized to improve the grim prognosis of sr-agvhd. graft-versus-host disease how i treat refractory acute gvhd first-and second-line systemic treatment of acute graft-versus-host disease: recommendations of the american society of blood and marrow transplantation response of 443 patients to steroids as primary therapy for acute graft-versus-host disease: comparison of grading systems treatment of moderate/severe acute graft-versus-host disease after allogeneic bone marrow transplantation: an analysis of clinical risk features and outcome role of tumor necrosis factor-alpha in graft-versus-host disease and graft-versus-leukemia responses etanercept plus methylprednisolone as initial therapy for acute graft-versus-host disease recombinant human soluble tumor necrosis factor receptor fusion protein as treatment for steroid refractory graft-versus-host disease following allogeneic hematopoietic stem cell transplantation treatment of steroid-resistant acute graft-versus-host disease with daclizumab and etanercept combination antithymocyte globulin and soluble tnfalpha inhibitor (etanercept) +/-mycophenolate mofetil for treatment of steroid refractory acute graft-versus-host disease defining the intensity of conditioning regimens: working definitions consensus conference on acute gvhd grading combination therapy with inolimomab and etanercept for severe steroid-refractory acute graft-versus-host disease steroid-refractory acute gvhd: lack of long-term improved survival using new generation anticytokine treatment infection risk associated with anti-tnf-alpha agents: a review immune reconstitution after double umbilical cord blood stem cell transplantation: comparison with unrelated peripheral blood stem cell transplantation incidence and outcome of invasive fungal diseases after allogeneic stem cell transplantation: a prospective study of the gruppo italiano trapianto midollo osseo (gitmo) conceptualization: jan j. cornelissen, annoek e. c. broers. annoek e. c. broers. key: cord-258172-p54j4zzo authors: barker, harlan; parkkila, seppo title: bioinformatic characterization of angiotensin-converting enzyme 2, the entry receptor for sars-cov-2 date: 2020-10-28 journal: plos one doi: 10.1371/journal.pone.0240647 sha: doc_id: 258172 cord_uid: p54j4zzo the world health organization declared the covid-19 epidemic a public health emergency of international concern on march 11th, 2020, and the pandemic is rapidly spreading worldwide. covid-19 is caused by a novel coronavirus sars-cov-2, which enters human target cells via angiotensin converting enzyme 2 (ace2). we used a number of bioinformatics tools to computationally characterize ace2 by determining its cell-specific expression in trachea, lung, and small intestine, derive its putative functions, and predict transcriptional regulation. the small intestine expressed higher levels of ace2 mrna than any other organ. by immunohistochemistry, duodenum, kidney and testis showed strong signals, whereas the signal was weak in the respiratory tract. single cell rna-seq data from trachea indicated positive signals along the respiratory tract in key protective cell types including club, goblet, proliferating, and ciliary epithelial cells; while in lung the ratio of ace2-expressing cells was low in all cell types (<2.6%), but was highest in vascular endothelial and goblet cells. gene ontology analysis suggested that, besides its classical role in the renin-angiotensin system, ace2 may be functionally associated with angiogenesis/blood vessel morphogenesis. using a novel tool for the prediction of transcription factor binding sites we identified several putative binding sites within two tissue-specific promoters of the ace2 gene as well as a new putative short form of ace2. these include several interferon-stimulated response elements sites for stat1, irf8, and irf9. our results also confirmed that age and gender play no significant role in the regulation of ace2 mrna expression in the lung. a zinc metalloenzyme, angiotensin-converting enzyme (ace) was discovered 64 years ago and first named as a hypertension-converting enzyme [1] . classically, ace is well known for its roles in the regulation of arterial pressure through conversion of angiotensin i to active angiotensin ii and cleavage of bradykinin and neurotensin [2] . as a zinc metalloenzyme, ace belongs to a large cluster of zinc-binding proteins. the first zinc metalloenzyme, carbonic anhydrase was discovered in 1932 by meldrum and roughton [3] and thereafter thousands of such metalloenzymes have been reported in different species of all phyla [4, 5] . angiotensin-converting enzyme 2 (ace2) was first discovered in 2000 when a novel homologue of ace was cloned [2, 6, 7] . although ace and ace2 share significant sequence similarity in their catalytic domains, they appear to act on different peptide substrates of angiotensins [8, 9] . previous studies identified ace2 as a functional receptor for severe acute respiratory syndrome corona virus 1 (sars-cov-1) which led to an outbreak of sars infection in 2003 [10] . ace2 is also a crucial receptor for the novel corona virus (sars-cov-2), which has caused a large global outbreak of covid-19 infection with rapidly growing numbers of patients (32, 968 ,853 confirmed cases as of september 28 th , 2020, https://www.who.int/ emergencies/diseases/novel-coronavirus-2019). a recent report suggested that soluble ace2 fused to the fc portion of immunoglobulin can neutralize sars-cov-2 in vitro [11] . this result was further confirmed by showing that human recombinant soluble ace2 reduced sars-cov-2 infection on cultured vero-e6 cells in a dose dependent manner [12] . therefore, ace2 also holds promise for treating patients with coronavirus infection. the structural key for target cell infection by coronavirus is the viral spike (s) protein of sars-cov. ace2 acts as a locking device for the virus, whereby the binding of the surface unit s1 facilitates viral attachment to the surface of target cells [13] . the cellular serine protease (tmprss2) promotes sars-cov entry via a dual mechanism. it cleaves both the sars-cov s protein and the virus receptor, ace2, promoting both the viral uptake and the viral and cellular membrane fusion events [13] [14] [15] . the critical residues contributing to the receptor-spike protein interaction were first determined for sars-cov-1 [16] and recently in three independent studies for sars-cov-2 [17] [18] [19] . it has been proposed by biolayer interferometry studies that the receptor-binding domains of sars-cov-1 and sars-cov-2 s proteins bind with comparable affinities to human ace2 [20] . in contrast, a modelling study suggested that binding of sars-cov-2 is stronger [21] , which was convincingly confirmed by structural and biochemical data [17, 18] . the clinical characteristics of covid-19 infection have recently been described based on data from 1,099 patients from mainland china [22] . it was found that the clinical characteristics of covid-19 mimic those of sars-cov-1 infection. the most dominant symptoms include fever, cough, fatigue, and sputum production, whereas gastrointestinal symptoms are less common. in laboratory parameters, lymphopenia was detected in 83.2% of patients on admission. according to another recent survey of 278 patients with pneumonia caused by sars-cov-2, fever was the most common symptom, followed by cough [23] . bilateral pneumonia has been detected by computed tomography scans in 67.0% of patients [24] . a recent study from wuhan, china listed the most common clinical complications determined in critically ill covid-19 patients [25] . the complications during clinical worsening included acute respiratory distress syndrome and respiratory failure, sepsis, acute cardiac injury, and heart failure. data on the localization of virus receptors can provide insight into mechanisms of virus entry, tissue tropism, and pathogenesis of the disease. therefore, it is of particular interest to correlate covid-19 symptoms with the distribution pattern of ace2. the first studies performed by northern blotting indicated that ace2 is located in the human heart, kidney, and testis [2] . quantitative polymerase chain reaction (qpcr) showed the highest expression levels in the human cardiovascular system, testis, kidney, and intestine [26] . by immunohistochemistry, the expression of the ace2 protein was identified in the human lung alveolar epithelial cells (type i and ii pneumocytes), enterocytes of the small intestine, the brush border of the renal proximal tubules, the endothelial cells of arteries and veins, and arterial smooth muscle cells in several organs [27] . it was proposed that this distribution pattern of ace2 could explain the tissue tropism of sars-cov-1 for the lung, small intestine, and kidney [28] . on the other hand, the symptoms of covid-19, in contrast to sars-cov-1 infection, are not associated to the same extent with the gastrointestinal tract in spite of the high expression of ace2 in the intestinal enterocytes [29] . in covid-19, diarrhea has been reported in just 3.8% of patients, in contrast to 40-70% in sars-cov-1 infection [22, 30] . a recent report indicated diarrhea in 18.1% of 254 covid-19 patients [31] . there are conflicting reports on the expression of ace2 in the upper respiratory tract [30] . hamming and coworkers found that only the basal layer of nonkeratinized airway squamous epithelium shows positive signal [27] , whereas sims and colleagues demonstrated ace2 expression on the luminal surface of ciliated cells in freshly excised human nasal and tracheobronchial tissue [32] . ren and coworkers showed weak ace2-positive signal in the epithelial cells of trachea and main bronchus [33] . although lymphopenia is a typical feature of sars [22, 30] , ace2 is not highly expressed on t or b cells or macrophages in the spleen or lymphoid organs [27] . it is known that both sars-cov and sars-cov-2 infections lead to worse outcome in the elderly [30, 34] . recent studies have also indicated higher case fatality rates in males than females [35] . therefore, one aim of the present study was to investigate whether age or gender could contribute to the regulation of ace2 expression. we also decided to explore the transcriptional regulation of ace2 gene expression using a novel computational tool recently developed by the first author of this article. notably, data on ace2 distribution is still conflicting, and thus we aimed to get a more comprehensive view of the cell types expressing the receptor of sars-cov-2. finally, we studied the coexpression of ace2 with other genes and explored its putative functions using a gene ontology enrichment analysis. from the fantom5 project [36] , cap analysis of gene expression (cage) sequencing of cdna has been performed in 1,839 human samples from 875 different primary cells, tissues, and cell lines (description of all public datasets used presented as s1 table) . expression of transcription start sites (tsss) was extracted and combined for all genes in all samples as tags per million (tpm). from this compiled set, ace2 gene expression was extracted and presented as barplot using the matplotlib [37] and seaborn [38] python libraries. similarly, human gene expression data (as tpm) was extracted from the gtex database, which is an ongoing largescale project to identify human variation, regulation, and gene expression [39] , along with metadata on the samples. ace2 gene expression values were separated by tissue and compared among 10-year interval age groups to determine if the values showed any differences throughout the lifecycle. boxplots for tissues of relevance were generated using matplotlib and seaborn libraries. in each of the tissues present in the gtex dataset, expression values for ace2 were compared with expression of all other genes by spearman correlation analysis using the scipy [40] python library to identify those genes with concordant expression patterns. bonferroni correction was used to derive an adjusted p-value threshold of 9.158e-07. for each tissue, those genes which both satisfied the bonferroni-adjusted p-value threshold and had a correlation of expression of 0.50 or greater were analyzed using the gprofiler gene ontology (go) enrichment analysis [41] python library to identify possible enriched terms in biological process (bp), molecular function (mf), cellular component (cc), human phenotype (hp), kegg pathway, and wikipathways (wp) ontologies. immunohistochemical localization of human ace2 was evaluated from immunostained specimens provided by protein expression atlas (https://www.proteinatlas.org/) [42] . the dataset included three specimens of duodenum, three specimens of kidney, three specimens of testis, three specimens of lung, and two specimens of nasopharynx. the images of the fig 2 represent duodenum from 77-years-old female, kidney from 36-years-old male, testis from 38-years-old male, lung from 61-years-old female, and nasopharyngeal mucosa from 78-years-old female. according to protein expression atlas the immunostainings were performed with the rabbit anti-human polyclonal antibody (hpa000288; sigma aldrich, st. louis, mo) raised against 111 n-terminal amino acids of ace2 and diluted 1:250 for the staining. analysis of ace2 promoter regions was performed using the tfbsfootprinter tool (https:// github.com/thirtysix/tfbs_footprinting) which uses transcription-relevant data from several major databases to enhance prediction of putative tfbss, including: all cell types aggregated and merged human atac-seq data from encode [43] , transcription start sites and expression data from fantom5 [44] , expression quantitative trail loci from gtex [39] , tfbs metacluster data from gtrd [45] , tfbs binding profile data from jaspar [46] , and sequence and conservation data from ensembl [47] . detailed description of this novel tool is under preparation [48] . previous studies identified two distinct tissue-specific transcription start sites (tss) for intestine and lung expression [49] , which correspond to primary protein-coding ensembl transcripts enst00000252519 and enst00000427411, respectively. these two transcripts were targeted for transcription factor binding site (tfbs) analysis; first with a scan for all 575 jaspar tfs and input parameters of 1,000 base pairs (bp) upstream and 200 bp downstream (relative to the tss); secondly with a limited set of 15 interferon-stimulated tf genes and a broader area of 1,500 bp upstream and 500 bp downstream. likewise, an analysis of the promoter region of a putative new short form of ace2 was performed. single-cell expression datasets were identified for relevant tissues/cells of lung (human) [50] , trachea (mouse) [51] , and small intestine (mouse) [52] . using a modified workflow described previously in [53] , for each dataset the samples were filtered by gaussian fit of read count, expressed gene count, and number of cells in which a gene is expressed. counts were normalized by cell, log transformed, principle component analysis performed with 15 components, and k-nearest neighbors computed using scanpy [54] , and then the full dataset normalized with r package 'scran' [55] . batch correction by individual and sample region was performed with scanpy using the combat function. the top 1,000 genes with highly differential expression were identified for cluster analysis which was performed with uniform manifold approximation and projection (umap) and force directed graph models. the top 100 marker genes were identified as those with higher expression unique to each cluster by welch t-test in scanpy. expression of the ace2 gene was mapped onto cluster figures to determine overlap with previously identified cell types or cell type marker genes identified in the literature. cell type was mapped by expression of known marker genes of cell types expressed in the lung and small intestine, as defined by de novo prediction in the original articles. comparisons of ace2 expression values in different tissues and between groups delineated by age or sex, were carried out by one-way anova using the stats package in the scipy [40] python library. only groups with 20 or more observations and a 2-sided chi squared probability of normality of < = 0.1 (due to the robustness of anova to non-normal distributions) were used for comparison. correlation of gene expression values was calculated by two-sided spearman rankorder analysis, where a bonferroni-corrected p-value threshold was computed using α = 0.05/ number of comparisons. gene ontology enrichment analyses performed using the gprofiler tool utilize a custom algorithm for multiple testing of dependent results, which corresponds to an experiment-wide threshold of α = 0.05. tfbsfootprinter analysis of the ace2 promoter limits results for individual tfbss whose score satisfies a genome-wide threshold of α = 0.01. the first aim of our study was to investigate different human tissues using publicly available datasets for the distribution of ace2 mrna and protein. in the fantom5 dataset, the highest values for ace2 mrna, ranked according to signal intensity, were seen for the small intestine, dura mater, colon, testis, thalamus, and rectum (fig 1) . fig 2 shows the expression of ace2 protein in selected human tissues. representative example images of the ace2 immunostaining were prepared from tissue specimens of the human protein atlas database (https://www.proteinatlas.org/). the results indicate a strong signal for ace2 protein in the brush border of small intestinal enterocytes. in the kidney, strong immunostaining reactions were present in the epithelial cells of proximal convoluted tubules and bowman´s capsule. the seminiferous tubules and interstitial cells of testis also demonstrated strong immunostaining. no immunoreactions for ace2 were observed in the lung specimens. very weak signal, associated with apical membranes, was detected in sporadic ciliary cells of a nasopharyngeal mucosa sample. although the evaluation of immunostaining reaction is generally considered semiquantitative at most, the results seem to correlate fairly well with the corresponding mrna expression levels. the respiratory tract is the main target region that is affected by covid-19 infection. bulk rna-seq data from lung specimens showed low expression levels for ace2 (fig 1) . therefore, we performed an analysis of single cell rna-seq using both human lung and mouse trachea datasets, representing the breadth of the lower respiratory tract. figs 3 and 4 show the expression of ace2 mrna in identified cell types of lung and trachea, respectively. in lung, ace2 expressing cells are generally uncommon with no cell type having a ratio of ace2-expressing cells greater than 2.6%. the cell types with the greatest proportion of ace2 expression are those of arterial vascular endothelial cells (2.55%), goblet cells (2.02%), and venous vascular endothelial cells (1.33%). in trachea, the highest ratio of ace2-expressing cells included the club cells (16.62%), goblet cells (13.84%), and ciliary epithelial cells (6.63%). since both the airways and intestine contain goblet cells, sars-cov-1 affects gastrointestinal tract, and bulk rna-seq data shows high expression in small intestine and colon, we decided to analyze another single cell rna-seq dataset covering mouse intestinal epithelial cells. since both age and gender may contribute to onset and severity of covid-19 symptoms we aimed to investigate the effect of these variables on the expression levels of ace2 mrna. fig 6 indicates that some tissues showed a slight trend to lower expression in older age categories. among all tested tissues, statistically significant differences between the age categories were immunohistochemical localization of ace2 protein in selected human tissues. in the duodenum (a), the protein is most strongly localized to the apical plasma membrane of absorptive enterocytes (arrows). the goblet cells (arrowheads) show weaker apical staining. intracellular staining is confined to the absorptive enterocytes. in the kidney (b), ace2 shows strong apical staining in the epithelial cells of the proximal convoluted tubules (arrows) and bowman´s capsule epithelium (arrowheads). the distal convoluted tubules are negative (asterisk). the testis specimen (c) shows strong immunostaining in the seminiferous tubules (arrows) and interstitial cells (arrowheads). the lung sample (d) is negative. in the nasopharyngeal mucosa (e), ace2 signal is very weak and only occasional epithelial cells show weak signals (arrows). immunostained specimens were taken from the protein expression atlas (https://www. proteinatlas.org/). https://doi.org/10.1371/journal.pone.0240647.g002 seen in the tibial nerve (p = 8.58 x 10 −6 ), minor salivary gland (p = 0.002), aorta (p = 0.003), whole blood (p = 0.005), transverse colon (p = 0.010), hypothalamus (p = 0.039), and sun exposed skin (p = 0.046). importantly, the lung specimens showed no significant difference of ace2 mrna expression as normalized, batch-corrected counts is shown for comparison in upper panel. the force directed layout plot was computed and visualized in scanpy [54] . for each cell type the ratio of cells expressing ace2 is presented in addition to a stacked barplot of the relative cell type frequencies in the whole dataset. alveolar type i (ati), alveolar type ii (atii), pulmonary neuroendocrine cells (pnec), smooth muscle cells (smc), vascular endothelia (ve). https://doi.org/10.1371/journal.pone.0240647.g003 ace2 mrna expression between different age categories (p = 0.681). complete data on ace2 mrna expression levels in different age categories are shown in s2 table. to make a binary comparison of expression by age, samples were divided into groups of <50 and �50 years of age. in comparison of these younger and older age groups, significant differences in expression were found in tibial nerve (p = 2.47 x 10 −7 ), whole blood (p = 3.21 x 10 −4 ), minor salivary gland (p = 4.89 x 10 −4 ), sun exposed skin (p = 0.003), transverse colon (p = 0.022), testis (p = 0.025), esophageal muscle layer (p = 0.040), and subcutaneous adipose tissue (p = 0.045). additionally, with the same age groups, comparisons were made for both males and females (s3 table) . in males, ace2 expression was lower in the �50 age group for tibial nerve , sigmoid colon (p = 0.017), testis (p = 0.025), visceral adipose tissue of omentum (p = 0.026), sun exposed skin (p = 0.027), whole blood (p = 0.032), and bladder (p = 0.042); while increased in coronary artery (p = 0.015). in females, ace2 expression was lower in the �50 age group in whole blood (p = 0.005) and sun exposed skin (p = 0.049); and higher in esophagus (p = 0.007) and terminal ileum (p = 0.022). ace2 mrna levels largely overlapped between male and female sexes as shown in fig 7. in the lung, no statistically significant difference was observed in the expression levels between the male and female subjects (p = 0.908). statistically significant differences were observed in the adipose tissue (p = 0.0001), whole blood (p = 0.0002), amygdala (p = 0.0006), transverse colon (p = 0.0008), data is from geo dataset gse92332 [52] . ace2 mrna expression as normalized, batch-corrected counts is shown for comparison in upper panel. the force directed layout plot was computed and visualized in scanpy [54] . for each cell type the ratio of cells expressing ace2 is presented in addition to a stacked barplot of the relative cell type frequencies in the whole dataset. https://doi.org/10.1371/journal.pone.0240647.g005 plos one muscle layer of esophagus (p = 0.002), left ventricle of heart (p = 0.005), epstein-barr virus-transformed lymphocytes (p = 0.015), and esophagus-gastroesophageal junction (p = 0.024). notably, there was no clear sex-specific trend pointing to one direction in all these cases. ace2 mrna expression levels in all studied tissues sorted according to subjects´gender are shown in s4 table. proximal promoter contains putative tfbss for ileum, colon, and kidney expression tfbs analysis of the ace2 intestinal transcript promoter (enst00000252519) revealed several candidate binding sites which occur in a cluster extending from 400 bp upstream of the effect of age on ace2 mrna expression levels. data is extracted from the gtex dataset as tpm. in these organs, anova revealed significant differences between age categories in tibial nerve (p = 8.58 x 10 −6 ), minor salivary gland (p = 0.002), and whole blood (p = 0.005). in other tissues, the differences did not reach statistical significance. the highest tpm values are seen in the small intestine, testis, and kidney. https://doi.org/10.1371/journal.pone.0240647.g006 transcription start site; cdx2, hnf1a, foxa1, sox4, tp63, hnf4a, dux4, foxa2, nr2f6, and sox11 ( fig 8a) . these predicted sites overlap an evolutionarily conserved region in mammals and are proximal to several atac-seq peaks. in several tissues these tfs are found to be highly positively correlated (>0.7) with expression of ace2, as determined using rna-seq gene expression values from the gtex dataset: cdx2 (colon, terminal ileum), hnf1a (colon, kidney, terminal ileum), foxa1 (cervix, colon, terminal ileum), hnf4a (colon, terminal ileum), foxa2 (colon, kidney), nr2f6 (colon, kidney, terminal ileum), and sox11 (kidney). in addition, two of the tfs are highly negatively correlated with ace2 expression dux4 (kidney) and foxa1 (kidney). full prediction results are included in s5 table and tf correlations by tissue are present in s6 table. an expanded analysis for binding sites of interferon-stimulation mediating tf genes in the region 1,500 bp upstream to 500 bp downstream of the ace2 intestinal transcript's tss revealed putative binding sites for stat1 (3x), stat3 (4x), stat5a:stat5b dimer (3x), stat4, stat1:stat2 dimer (2x), irf2, irf3, irf4 (2x), irf5 (2x), irf8 (3x), and irf9 (s1 fig) . each of these predictions satisfied a pwm p-value threshold of <0.0001, while only the stat1, irf9, and irf8 sites also satisfied a combined affinity score p-value of <0.05. analysis of the ace2 lung transcript promoter (enst00000427411) produced putative tfbs predictions for esrra, hnf4a, cdx2, cebpa, esrrb, mef2b, tcf7, tcf7l2, jun, and lef1 (fig 8b) . full prediction results are included in s5 table. the predicted tfbss clustered within 200 base pairs of the tss, and overlap with evolutionarily conserved regions, tfbs metaclusters, and atac-seq peaks. the tfs corresponding to predicted tfbss, which are positively correlated (>0.7) with ace2 expression, are esrra (terminal ileum, colon), hnf4a (terminal ileum, colon), cdx2 (colon, terminal ileum), cebpa (colon, terminal ileum), esrrb (cervix), tcf7l2 (testis). those tfbss with tfs which strongly (<-0.7) negatively correlate with ace2 are esrra (kidney) and tcfl72 (kidney). the lung-specific transcript tss aligns with the p3@ace2 fantom5 dataset cage peak, which indicates that the expression of this transcript is much lower than the intestinal transcript, which corresponds with p1@ace2 and p2@ace2 fantom5 cage peaks. common between the two tissue-specific transcripts, are predictions for cdx2 and hnf-family transcription factors. an expanded analysis for binding sites of interferon-stimulation mediating tf genes in the region 1,500 bp upstream to 500 bp downstream of the lung transcript's tss revealed putative binding sites for stat1:stat2 dimer, stat1 (2x), stat3, stat4, stat6 (2x), and irf8 (s1 fig) . each of these predictions satisfied a pwm p-value threshold of <0.0001, while only the irf8 site also satisfied a combined affinity score p-value of <0.05. tfbs analysis of the recently identified putative short form ace2 transcript [56] [57] [58] , with a tss between exons 9 and 10 of the canonical gene, produced predictions for irf9, irf8, jund, fosl1, gata1, junb, irf4, jun, and fos, among others (s5 table) . within the first -56 to -31 bp upstream of the short-form ace2 tss, are overlapping binding sites for several irf tfs and a stat1:stat2 dimer, while further upstream are binding sites for several stat tfs at -662 to -647 bp and -911 to -897 (fig 9) . each of these predictions satisfied a pwm p-value threshold of <0.001, while only the irf9, irf8, and irf4 sites also satisfied a combined affinity score p-value of <0.05. coexpression analysis identified numerous genes in ileum, testis, colon, and kidney which are highly correlated (>0.8) with ace2 (tables 1 and s7 ). in particular, in the ileum there are a number of genes with correlation values greater than 0.95. in contrast, analysis of the lung shows a maximum correlation of expression of 0.6275. the genes with which ace2 mrna expression shows the highest levels of coexpression code for metalloprotease and transporter proteins. selected tissue-specific ace2-correlated genes, determined with bulk rna-seq data, are presented with their expression levels within the scrna-seq trachea and intestinal datasets in go enrichment analysis of ace2 mrna expression in all tissues produced 22 terms which were enriched in bp, cc, hp, kegg, and wp ontologies ( the predominant pathological features of covid-19 infection largely mimic those previously reported for sars-cov-1 infection. they include dry cough, persistent fever, progressive dyspnea, and in some cases acute exacerbation of lung function with bilateral pneumonia [32] . major lung lesions include several pathological signs, such as diffuse alveolar damage, inflammatory exudation in the alveoli and interstitial tissue, hyperplasia of fibrous tissue, and eventually lung fibrosis [59] [60] [61] . it has been shown by fluorescence in situ hybridization technique that sars-cov-1 rna locates to the alveolar pneumocytes and alveolar space [62, 63] . mossel and colleagues demonstrated that sars-cov-1 replicates in type 2 (at2) pneumocytes, but [36] and has been identified as potentially relevant for interferon-mediated transcription of this new ace2 transcript [56] ; it is displayed in the cage peaks track along with an arrow indicating the tss. the region analyzed represents -1,000 bp to +200 bp relative to the putative tss, while nucleotide positions at bottom are given relative to the ace2 full length transcript. https://doi.org/10.1371/journal.pone.0240647.g009 table 1 . genes associated with ace2 mrna expression in selected human tissues. derived from gtex bulk rna-seq data [39] . not in type 1 (at1) cells [64] . considering all of these facts, it is not surprising that most histopathological analyses have been focused on distal parts of the respiratory airways, while the regions other than the alveolus have been less systematically studied. to understand better the pathogenesis of covid-19 we need to know where ace2, the receptor for sars-cov, is located within the human respiratory tract and elsewhere. overall, different studies including ours have convincingly shown that several organs, such as the small intestine, colon, kidney, and testis, express higher levels of ace2 than the lung and other parts of the respiratory tract. our analysis of ace2 expression in the human lung show low levels of expression in all cell types, with arterial vascular endothelial cells achieving the highest overall ratio of just~2.5%. the present results based on mouse tracheal dataset suggested that ace2 mrna is predominantly expressed in the club cells, goblet cells, and ciliated epithelial cells, and at significantly higher frequency than found in the lung. the mouse dataset used in our study contained no secretory3 cells, which lukassen and colleagues recently reported to trachea expression data is taken from gse103354 [51] and intestinal epithelia data is derived from gse92332 [52] . visualized in scanpy [54] . https://doi.org/10.1371/journal.pone.0240647.g010 express the highest levels of ace2 mrna along the human respiratory tract [65] . another study reported positive expression in the type at2 pneumocytes [66] , which is in line with the results of lukassen et al. [65] , but only a few cells appeared positive. a third study based on single cell expression data demonstrated the strongest positive signal in the lung at2 cells, while other cells including at1 cells, club cells, ciliated cells, and macrophages showed weaker expression [67] . a fourth single cell expression analysis using gene expression omnibus (geo) database recently demonstrated ace2-positive signal in 1% of at2 cells and in 2% of respiratory tract epithelial cells [68] . this correlates with our own findings for lung. for comparison about 30% of ileal epithelial cells were ace2-positive, and 44% of enterocytes in small intestine of mouse. immunohistochemical analysis of mouse tissues has shown positive signal in the club cells, at2 cells, endothelial cells, and smooth muscle cells [69] . in spite of the obvious discrepancies between different datasets, that highlights the need for large numbers of thoroughly characterized cells for single cell rna-seq analyses, we can now make some conclusions of the expression of ace2 mrna in the respiratory tract. first, ace2 is positively though weakly expressed in the at2 cells of the lung and less so in at1 cells. second, ace2 also shows a weak positive signal, but at significantly higher proportions of cells, in several other cell types of the trachea, including goblet cells, club cells, and ciliated cells. third, based on the findings of lukassen et al. [65] secretory3 cells, a transient cell type of the bronchial tree, may express the highest levels of ace2. these ace2-positive cell types may represent the main host cells for sars-cov-2 along the whole respiratory tract. however, the median percentage of ace-expressing secretory3 cells in the study was less than 6%, significantly less than that observed in club (16.62%), goblet (13.84%), and ciliated (6.63%) cells of trachea we have identified in the gse103354 dataset. goblet cells, ciliated epithelial cells, and club cells are considered important cell types for the protection of airway mucosa. lukassen and coworkers [65] described secretory3 cells as intermediate cells between goblet, ciliated, and club cells. if sars-coronaviruses predominantly attack these cells, locating along the airway segments including the trachea, bronchi, and bronchioles until the last segment that is the respiratory bronchioles, it would be obvious that physiological protective mechanisms are severely affected. defective mucosal protection and inefficient removal of pathogens due to viral infection may contribute to onset of severe bilateral pneumonia that is common for sars-diseases [70] . this pathogenic mechanism is supported by previous findings, showing that early disease is manifested as a bronchiolar disease with respiratory epithelial cell necrosis, loss of cilia, squamous cell metaplasia, and intrabronchiolar fibrin deposits [32] . in fact, it has been suggested that early diffuse damage as a result of sars-cov-1 infection may actually initiate at the level of the respiratory bronchioles [71, 72] . our findings confirm that the respiratory tract tissues have quite limited expression levels of ace2 compared to several other tissues that show much more prominent signal. because ace2 is highly expressed in the intestine [29] , as also confirmed by our bioinformatics study, it would be obvious to predict that both sars-cov-1 and -2 infections cause significant gastrointestinal pathology and symptoms including diarrhea. interestingly, the patients with covid-19 have reported less gastrointestinal symptoms than the sars-cov-1-infected patients [22, 30] . the pathophysiological basis for this phenomenon is not understood at this point, and thus further investigations on this topic are warranted. when we initiated the present study, we hypothesized that understanding better the transcriptional regulation of the ace2 gene might help to explain the peculiar distribution pattern of ace2 in tissues. since upregulation of ace2 would reflect an increased number of sarscoronavirus receptors on cell surfaces, it could possibly help us to understand the mechanisms why certain patients (males more than females, old more than young, smokers more than non-smokers) are more susceptible to the most detrimental effects of the covid-19 infection. in our study, the signals for ace2 mrna in the lung specimens did not vary much in different age groups nor did they show significant differences between males and females, which is in line with previous findings [65] . therefore, different expression levels of lung ace2 may not explain the variable outcome of the disease concerning age groups and genders. importantly, our studies on this aspect were performed using whole tissue rna-seq values, and at least one other analysis using single-cell rna-seq data has identified changes in ace2 expression associated age, sex, and smoking status for various cell types [73] . specifically, they have found ace2 expression to increase with age in basal and multiciliated cells, and higher expression for males in airway secretory cells and alveolar at2 cells. additionally, a study of ace2 expression in nasal epithelium (not included in gtex dataset) showed lowest levels in young children (<10) with increasing values in later age groups [74] . it has been recently discussed that different innate and adaptive immune responses related to both age and gender may contribute to variable outcome of severe viral diseases [35] . it is clearly one major research area to be followed regarding covid-19 infection. to investigate the transcriptional regulation of the ace2 gene we made predictions for the binding sites of transcription factors within the proximal promoter region of the intestine-specific and lung-specific human ace2 transcript promoters. our findings introduced several putative binding sites in the ace2 promoter for known transcription factors, which showed high levels of coexpression with ace2 in several tissues including the ileum, colon, and kidney. the identified transcription factors could represent potential candidate target molecules which regulate ace2 expression. two of our predictions, for hnf1a and hnf1b, have been previously identified experimentally to drive ace2 expression in pancreatic islet cells and insulinoma cells, respectively [49] . later work by the same group has shown that our prediction of foxa binding sites in the ace2 promoter are also likely correct [75] . it is of interest that ace2 might be regulated by oxygen status. zhang and coworkers previously demonstrated that ace2 mrna and protein levels increased during the early stages of hypoxia and decreased to near-baseline levels at later stages after hypoxia inducible factor (hif)-1α accumulation [76] . based on these findings ace2 has been listed as a hif1α-target gene [77] , although it does not follow the typical hif1α regulated expression pattern, nor is there any predicted hif1α binding site in our analyses. however, hnf1b has been identified as upregulated in hypoxia in kidney, independent of hif1α [78] , and in hypoxic embryonic stem cells hif1α has been shown to increase expression of transcription factors tcf7/lef1 (predicted to bind the promoter of the lung-specific ace2 transcript) through wnt/β-catenin signaling [79] . recent work has shown that ace2 expression is stimulated by interferon alpha (ifn-α) in vitro and computationally identified evidence for stat1, stat3, irf8, and irf1 tfbss in the ace2 promoter [80] . another recent study has identified correlations of expression between ace2 and other interferon stimulated genes [81] . our analysis produced several putative binding sites for interferon-stimulation mediating tf genes, proximal to the tsss of the intestine-specific (stat1, irf8, and irf9) and lung-specific transcript (irf8). the findings of these studies, and our own, potentially reveal a scenario where sars-cov-2 infection itself may induce expression of ace2 and thus provide a self-perpetuating route of increased cellular infection. this could explain how such low overall ace2 expression in normal lungs translates into a fatal disease state. however, a series of recent pre-print articles have identified a previously unreported novel ace2 transcript with a transcription start site occurring between exons 9 and 10 of the canonical ace2 gene. alternatively, this truncated transcript has been named ltr16a1-ace2 [56] , delta-ace2 [57] , or simply 'short ace2' [58] . in all cases the authors have concluded that this new transcript is more strongly stimulated by interferon, though their results differ on whether transcription of the long form is [58] or is not [56, 57] interferon-related. importantly, a protein resulting from this transcript would lack the 356 amino acids of the first 9 exons, and thus not contain the known sars-cov-2 binding domains, and all three studies have hypothesized that the short form ace2 would not likely be a point of viral entry. one study was unsuccessful in attempting to produce a viable protein from the short transcript [56] , while others [57, 58] identified peptides indicative of the short ace2 protein in data from mass-spec analysis of cancer samples (ovary, colon, breast) from the cancer genome atlas (tcga). in addition, blume and coworkers were able to use western blotting of an ace2 antibody targeting the c-terminal domain to identify appropriately sized bands (~50kda), expected of the short form, in nasal epithelial cells and bronchial epithelial cells [58] . all three studies found that the ratio of expression of the short form to long form is highest in nose and mouth and reduces as you progress down the airways and digestive tracts. ng et al. in [56] have identified a long terminal repeat (ltr) (ltr16a1) occurring in intron 9 of ace2, comprising most of the first exon of short-form ace2, which they posit may be related to the observed interferon reactivity. our tfbs analysis of ltr16a1 and the promoter of the short ace2 transcript revealed putative binding sites for multiple irf proteins and a stat1:stat2 dimer immediately upstream (<50 bp) of the newfound tss and ltr16a1 (fig 9) , and stat protein binding sites further upstream. while all three studies promote this new short form of ace2 as more strongly upregulated by interferon, two [57, 58] also contain results that show that long form ace2 is itself also upregulated, to a lesser extent, in presence of interferon or infection. these three manuscripts represent the leading edge of, and first forays into, our understanding of ace2 expression in response to interferon. accordingly, while there are broad strokes of agreement, there is not consensus on all points. peer-review of the results needs to be first performed, and subsequent study is needed to validate and expand these new findings. in summary, at present, there is evidence that both short and long forms of ace2 are upregulated by interferon, however the short form appears to be more strongly upregulated, appears to be expressed at higher levels in nose and mouth, and lacks the domains currently understood to bind the sars-cov-2 spike protein. our results show significant predictions for interferon response elements in the proximal promoters of all three ace2 transcripts. further deepening the importance of understanding the role of interferon in covid-19 is a new study showing that the presence of auto-antibodies against either or both of interferon alpha (ifnα) and interferon omega (ifnω) were rare in healthy, asymptomatic, or mild sars-cov-2 infection, but over-represented among patients with life-threatening covid-19 pneumonia [82] . the full story of the regulation of ace2 expression remains an enigma and there appear to be many factors involved. one limitation of our study is that it is focused on mrna expression and transcriptional regulation only. there may exist factors which function at posttranscriptional level. indeed, srivastava and colleagues recently demonstrated that sars-cov-2 infection induces alterations in the post-transcriptional regulatory networks in human tissues through the function of rna binding proteins and micro-rnas [83] . there has been clinical concern that the use of ace inhibitors and angiotensin receptor blockers could increase the expression of ace2 and increase patient susceptibility to viral host cell entry [84, 85] . previous studies have suggested that both ace inhibitor and angiotensin ii receptor type i antagonist therapies increase ace2 mrna expression in rat heart [86] . there has also been some evidence in humans showing increased expression of ace2 in the heart, brain, and even in urine after treatment with angiotensin receptor blockers [84] . since these drugs are widely used for treatment of hypertension and heart failure, it would be important to determine in covid-19 patients whether these medications have any significant effects on symptoms or outcome of the disease. gene ontology investigations revealed interesting novel data on potential physiological roles of ace2. the five most significant gene ontology terms included angiogenesis, blood vessel morphogenesis, vasculature development, cardiovascular system development, and blood vessel development. angiotensin-(1-7) is a direct product of ace2, and through binding with the mas receptor has been shown to advance angiogenesis in injured cardiac tissue (myocardial infarction), by increasing expression of vegf-d and mmp-9 [87] , and in stroke [88] . other studies have suggested that ace2, either by reducing angiotensin ii or through activities of the ace2/angiotensin-(1-7)/masr axis, may be negatively associated with angiogenesis in various cancers [81, [89] [90] [91] . it also appears to play a role in angiogenesis in uterus during pregnancy [92] . our study of scrna-seq data from human lung showed that the arterial and venous vascular endothelial cell types had the highest ratios of ace2-expressing cells, first and third highest, respectively. in another study, ace2 expression was detected in blood vessels [27] , while a recent study showed that sars-cov-2 is capable of directly infecting blood vessel cells [12] . endothelial ace2 expression may be linked to clotting and multi-organ dysfunction reported in many patients with covid-19 [93] . our go analysis provided evidence that ace2 is involved in the kegg pathway 'complement and coagulation cascades'. indeed, patients with severe covid-19 often present with coagulation abnormalities that mimic other known systemic coagulopathies, such as disseminated intravascular coagulation (dic) or thrombotic microangiopathy, but covid-19 has its own distinct features [94] . based on the present finding, angiogenesis/blood vessel morphogenesis may be considered another putative function for ace2 in addition to its classical role as the key angiotensin-(1-7) forming enzyme [95] . our bioinformatics study confirmed the low expression of ace2 in the respiratory tract. in lung it was lowest of all, while significantly higher in the trachea. bulk rna-seq analyses indicated the highest expression levels in the small intestine, colon, testis, and kidney. in the human lung scrna-seq dataset, the strongest positive signals for ace2 mrna were observed in vascular endothelial cells, goblet cells, ciliated cells, and at2 and at1 pneumocytes. in the mouse trachea dataset, positive signals were most common in club cells, goblet cells and ciliated epithelial cells. the results suggest that sars-cov infection may target the cell types that are important for the protection of airway mucosa and their damage may lead to deterioration of epithelial cell function, finally leading to a more severe lung disease with accumulation of alveolar exudate and inflammatory cells and lung edema, the signs of pneumonia recently described in the lung specimens of two patients with covid-19 infection [96] . gene ontology analysis based on expression in all tissues suggested that ace2 is involved in angiogenesis/ blood vessel morphogenesis processes in addition to its classical function in renin-angiotensin system. many findings reported here have not yet been verified in vivo or in vitro. therefore, the validity of the bioinformatics results needs to be verified by future experimental research. the preparation and function of the hypertensin-converting enzyme a novel 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posttranscriptional regulatory networks across human tissues by sponging rna binding proteins and micro-rnas covid-19 and angiotensin-converting enzyme inhibitors and angiotensin receptor blockers: what is the evidence? jama. 2020:in press focus on receptors for coronaviruses with special reference to angiotensin-converting enzyme 2 as a potential drug target-a perspective effect of angiotensinconverting enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensin-converting enzyme 2 angiotensin 1-7 promotes cardiac angiogenesis following infarction angiotensin-converting enzyme 2 priming enhances the function of endothelial progenitor cells and their therapeutic efficacy angiotensin 1-7 inhibits angiotensin iistimulated head and neck cancer progression ace2 inhibits breast cancer angiogenesis via suppressing the vegfa/vegfr2/erk pathway the angiotensin-converting enzyme 2 in tumor growth and tumor-associated angiogenesis in non-small cell lung cancer ace2 and ang-(1-7) in the rat uterus during early and late gestation dysfunctional coagulation in covid-19: from cell to bedside coagulation abnormalities and thrombosis in patients with covid-19 the ace2/angiotensin pulmonary pathology of early-phase 2019 novel coronavirus (covid-19) pneumonia in two patients with lung cancer key: cord-278224-sq7tokbx authors: protopopova, alexandra; hall, nathaniel j.; brown, kelsea m.; andrukonis, allison s.; hekman, jessica p. title: behavioral predictors of subsequent respiratory illness signs in dogs admitted to an animal shelter date: 2019-10-23 journal: plos one doi: 10.1371/journal.pone.0224252 sha: doc_id: 278224 cord_uid: sq7tokbx individual variability is evident in behavior and physiology of animals. determining whether behavior at intake may predict subsequent illness in the animal shelter may influence the management of dogs housed at animal shelters and reduce overall disease. while normally associated with mild disease and low mortality rates, respiratory disease nevertheless poses significant challenges to the management of dogs in the stressful environment of animal shelters due to its highly infectious nature. therefore, the aim of the study was to explore whether behavior at intake can predict subsequent occurrence and progression of upper respiratory disease in dogs at animal shelters. in a correlational study, 84 dogs were assessed throughout their stay at a city animal shelter. the dogs were subjected to a behavioral assessment, 1 min in-kennel behavioral observations across two observation periods, and the collection of urinary cortisol:creatinine (c:c) ratio. the occurrence and progression of upper respiratory disease was monitored through repeated clinical exams (rectal temperature and the occurrence of nasal and ocular discharge, and presence of coughing and sneezing). a basic pls path regression model revealed that time in the shelter (estimate = .53, p < .001), and sociability (estimate = .24, p < .001) and curiosity scores (estimate = .09, p = .026) were associated with increased illness. activity and anxiety scores, however, were not associated with illness. urinary c:c, taken on the first full day, did not predict subsequent illness when accounting for time. limitations included attrition of dogs, a small percentage receiving vaccinations, and continuous and non-systematic rotation of dogs in the kennels. understanding if behavior can predict subsequent illness may improve shelter management practices, and in turn, result in improved live-release outcomes. respiratory disease, while normally associated with mild symptoms and low mortality rates, poses significant challenges to the management of dogs in the highly stressful environment of plos animal shelters due to its highly infectious nature [1] . canine infectious respiratory disease complex (cirdc), also known as kennel cough complex, is composed both of bacterial and viral agents associated with low mortality but high prevalence (canine adenovirus-2, canine parainfluenza virus, canine respiratory coronavirus, canine herpesvirus, canine influenza, mycoplasma bronchiseptica, and mycoplasma cynos) and agents associated with high mortality though much lower prevalence (canine distemper virus and streptococcus equi subsp. zooepidemicus [1] [2] [3] ). cirdc signs include ocular and nasal discharge and cough [1] . the highly infectious nature of the complex has implications for animal shelters due to the need to isolate symptomatic animals, inability to neuter infected animals, and the reluctance of potential adopters to bring infectious animals into their homes, where they may have other dogs. associated veterinary care for symptomatic animals also taxes limited shelter resources [4] . finally, canine distemper and strep zoo, though less prevalent than the others in the complex, can be associated with significant loss of life in a crowded shelter [5] . the outcome of these challenges is that many shelter dogs exhibiting signs of cirdc may be euthanized rather than placed for adoption if there are budget and/or time constraints [6, 7] . not only does the shelter provide an ideal setting for disease transmission, with dogs housed in close proximity to each other, but the stress of the shelter environment likely reduces the immune system's ability to respond to microbial challenge [1, 8] . differential susceptibility to various pathogens has been well established in young dogs and pregnant females [9] as well as individuals who are immunocompromised due to an already established disease (e.g., feline immunodeficiency virus [10] ). however, other less obvious factors, such as increased stress, may increase susceptibility in the shelter. the shelter environment presents an array of psychosocial stressors for dogs, resulting in increased activity of the hypothalamic-pituitary-adrenal (hpa) axis, as indicated by elevated cortisol levels in animals in that environment compared to animals in pet homes, at least in the first few days after admittance [11] . the hpa axis is the pathway that is responsible for the activation of the stress response in animals. an environmental stressor triggers the hypothalamus to release corticotropin-releasing hormone (crh) and arginine vasopressin, which, in turn, stimulate the production of adrenocorticotropin hormone (acth) in the anterior pituitary. the release of the latter hormone stimulates the release of corticosterone or cortisol from the adrenal cortex into the blood stream, depending on the species [12, 13] . the high levels of cortisol (or corticosterone) inhibit further production of the crh and acth resulting in a negative feedback loop (see [14] for a discussion in shelter dogs). cortisol levels negatively correlate with the levels of secretory immunoglobulin a (s-iga) in dogs [15] . yet s-iga plays a central role in the mucosal immune system, the body's primary defense against infection of the respiratory system [16] . individual variability is evident in behavior and physiology of human and non-human animals; individuals tend to cope with stressors in systematic and consistent ways [17] . correlations between behavior and physiological ability to cope with environmental stressors, such as disease and parasite infection, have been demonstrated in a wide variety of species. capitanio, medoza, and baroncelli [18] found that rhesus macaques that were high in the sociability trait showed a more rapid decrease in plasma cortisol concentrations, a higher igg response, and a lower viral load when challenged with a simian immunodeficiency virus. pigs that spent more time struggling when flipped over on their backs for a brief amount of time, have been found to have a higher concentration of cortisol and lower immune function [19] . free-roaming tom cats that were higher in aggression had a higher viral load of feline immunodeficiency virus [20] . trapped norway rats, who had a higher presence of wounds (as an indicator of aggression), also showed a higher level of hantavirus infection, and in turn, higher infection status males showed elevated serum testosterone and corticosterone concentrations, among other differences in neurotransmitters [21] . the activity-exploration profiles of siberian chipmunks, as measured by a standardized test, predicted the numbers of ticks present on the animals; tick load increased with space use [22] . more recently, the predictive effect of behavior on immune function under chronic stress conditions has been extensively explored in cattle [23, 24] . temperamental cattle, those that display shorter latency to exit and higher velocity when exiting their enclosure, have been shown to have a higher cortisol concentration and a weaker immune response to pathogens [23] . the predictive nature of behavior on immune function or disease status in dogs has not received much attention. however, early prediction of illness in a shelter environment may lead to higher life-saving through improvements in population management. to decrease overall disease within an animal shelter, experts recommend removing sick animals from the population as well as differential treatment of those that are more likely to succumb to disease [25] . thus, characterizing dogs on intake into high and low-risk categories may decrease overall incidence of disease in animal shelters as well as protect those that are more susceptible. recently, corsetti et al. [26] have suggested that dogs displaying a bold personality were less susceptible to diseases in the animal shelter. the researchers assessed 28 dogs for one month at the shelter. the behavior of the dogs was assessed using standardized personality tests, including a t-maze and a novel object test. the complex "boldness" trait was statistically derived from scores from several other tested traits (e.g., activity, attentiveness, dominance, and sociability). the aim of the study was to extend the work of corsetti et al. by exploring whether behavior at intake can predict the subsequent development of cirdc in dogs surrendered to animal shelters. adult dogs of unknown breed (n = 84; those that appeared to be approximately 1 year of age and older; 61% male) housed at the lubbock animal shelter, a city shelter in lubbock, texas, were enrolled in the study from february through november, 2016. the shelter is an openadmission shelter, which admits owner-surrendered, stray, and confiscated animals. the dogs which are available for adoption are placed into a separate kennel area, with the remaining dogs in a different area. for this study, only dogs that were not in the adoptable area were enrolled. dogs were excluded from the study due to the presence of sign of illness (e.g., nasal discharge, coughing, etc.) on day 1, pronounced aggression towards the experimenters, obvious injury, and mothers with litters. dogs were group housed, with some exceptions, in 1.6m x 1.2m x 1.9m steel kennels with cement siding and floors. a guillotine door divided the two parts of each kennel. occasionally the guillotine door was raised and the dogs were given access to both of the kennels. dogs who were dog-aggressive, as evidenced by fighting when group housed, were placed into the kennel alone. the kennels contained a water and food bowl. staff cleaned the kennels and fed the dogs daily. dogs were not routinely taken out of their kennels. occasionally the kennels had a towel or blanket in them. the front two rows of kennels were for male dogs and the middle two rows were for female dogs. the last row of kennels was used for aggressive, pregnant/lactating or injured dogs. there were drainage grates directly in front of the kennels and a cement walkway in between rows of kennels. eleven dogs (13%) received vaccinations (combined canine distemper virus, hepatitis, parvovirus, and parainfluenza administered subcutaneously, and bordetella administered intranasally) after intake (one dog each on days 1 and 2, two on day 3 , one each on days 6, 7, 8, 9, and 10, and two on day 11) . however, no systematic programs to vaccinate dogs on intake were present at the time of the study in the shelter, with an approximate >80% unvaccinated (at intake) dogs present at the shelter during the study. the new dogs were placed into kennels with existing unvaccinated dogs, thus, it is very likely that the animals were already exposed to pathogens prior to developing immunity even for the few dogs that were vaccinated on day 1. moreover, efficacy of vaccination against cirdc is variable, and the most significant predictive factor in whether a dog contracts cirdc may be the number of days in the shelter, rather than vaccination status [5, 27] . while the housing practices, husbandry, and outcome decisions were beyond our control and strictly at the shelter staff's discretion, our study procedures were approved by the texas tech university institutional animal care and use committee (#15064-09). dogs were handled gently, with care and respect, and we tried to be the best part of their otherwise stressful day. withholding vaccination from all dogs would have made our results easier to interpret. however, dogs are routinely vaccinated in shelters with multivalent vaccines that include highly effective protection against lethal diseases such as distemper virus and parvovirus. for this reason, withholding vaccination for the purposes of this study was not deemed ethical. at the time of the study, the animal shelter had poor disease management practices, including poor sanitation, poor medical care, no vaccination at-intake, overcrowding, and continuous rotation of dogs in the kennels. since the time of our study, welfare improvements have been made, including intake vaccinations, improved medical care and sanitation practices. dogs were enrolled in the study the day after they arrived at the shelter (arrival day was coded as day 0, and data collection began on day 1) . the dogs' intake id number, intake date, and kennel tag number were recorded. there were two cohorts of dogs: those for whom data collection occurred on monday, wednesday, friday and those for whom data collection occurred on tuesday, thursday, and saturday. on day 1 to day 14 (a total of seven observations), the dogs were videotaped in their kennel for 1 min using a kodak pixpro spz1, while one or two experimenters stood passively in front of the kennel. the behaviors during the in-kennel videos were coded at a later time. this short observation period was previously used to detect behavioral differences across kenneled dogs in the animal shelter environment [28, 29] . on days 1 through 14, using a slip lead, the dogs were led outside into a 34m x 26m fenced yard and the dog's health was assessed. the yard contained synthetic grass and concrete with a large window looking into the yard from inside the shelter. on day 3, prior to the health assessment, a behavioral assessment was conducted and saliva and urine were collected. if insufficient volumes of these samples were obtained, the sample collection was repeated for day 5 or 6. saliva samples were collected prior to the health exam using inert polymer swabs (salivabio children's swab, salimetrics, carlsbad, ca, usa) held in the dog's mouth for 60 s, but were lost due to human error during analysis; therefore, no data are reported. health observation. at least two researchers were present to conduct the health assessment. the presence of coughing and sneezing, and nasal and ocular discharge were noted during approximately 1 min observations of the dog in the kennel (these signs were noted as important through conversations with several experienced shelter veterinarians). the operational definitions of these categories are listed in table 1 . the dog's body condition score (purina body condition system [30] ) with a range of 1-9 was recorded. on few occasions, the dog was too sick or distraught to exit their kennel; in that case, the health assessment occurred inside the kennel. while one experimenter briefly and gently restrained the dog (when necessary), the second obtained a rectal temperature twice. if the two values differed by more than 0.1˚c, the temperature was taken a third time. the dogs were fed dog treats (pup-peroni1 dog snacks, big heart pet inc., san francisco, ca, usa) throughout the health assessment as a distraction. dogs that consistently refused to allow for the collection of rectal data were excluded from further procedures and data analysis; three dogs refused several times during their stay, resulting in ten missing time points (out of 403 time points total). on 22.6% of observations, the two observers collected data independently in order to calculate inter-observer agreement. cohen's kappa (κ) was calculated to determine agreement between the two observers in their score determination for the condition of the nose, the eyes, and the presence of coughing and sneezing. there was high agreement for all four measures, κ nose condition = .84 (95% ci, .78 to .89), κ eye condition = .73 (95% ci, .63 to .80), κ coughing = .88 (95% ci, .65 to 1), κ sneezing = .86 (95% ci, .75 to .98). behavioral assessment. following the collection of 1 min video clips of in-kennel behavior, the videos were coded on all behaviors listed in table 2 . videos were coded using a partialinterval coding procedure with 5 s time bins, in which an occurrence or non-occurrence of each behavior was noted. the behavioral dependent variables were derived by taking the proportion of the time bins in which a behavior occurred. a portion of the videos (24%), selected at random, were double coded. inter-observer agreement was calculated by adding the number of agreements by interval, dividing by the total number of intervals, and multiplying by 100. the inter-observer agreement was calculated for each behavior independently by summing all agreements of whether or not a behavior occurred in that interval, dividing by the sum of agreements and disagreements, and multiplying by 100. the average agreement across behaviors was 99% (sd = 0.01%; min: 95% for "leaning on wall," max: 100% for "out of sight," "chasing tail," "lying down," "cowering," "tucking tail," "growling," "howling," and "leg lift"). the behavioral assessment was modified from hennessy et al. [31] to contain three components to measure activity, sociability, and boldness/curiosity. a 1m x 1m square was marked off using adhesive measuring tape in the outdoor yard. the dogs were first allowed a few minutes to habituate to the area as well as to toilet (urine was collected at this time; see below for details). the first component of the test consisted of the researcher allowing the dog to remain alone, unrestrained, in the outdoor yard. the researcher videotaped the dog through the window for 2 min. the second component of the test included the researcher kneeling in the center of the 1m x 1m square for 3 min. if the dog had two or more paws inside the square, the researcher would pet and praise the dog using the hand closest to the body of the researcher to allow for the dog to escape at any moment. a second researcher videotaped the interaction through the window. the third part of the test involved the placement of a clear plastic tub, with a remote-control car inside, placed within a 1m x 1m square on the side of the play yard. one researcher stood approximately 1.5 m away from the car and controlled it using a remote table 2 . operational definitions of all of the behaviors that were observed during the in-kennel observation period. front of kennel located between front of cage, and up to and including the midpoint of the visible kennel located between back wall of kennel, and up to, but not including, midpoint of the visible kennel. not visible from the front of the cage, behavior cannot be defined facing forward head is oriented such that the observer is able to see more than the side profile of face likely eye contact with the eyes of the observer head is oriented such that the observer is not able to see more than the side profile of face moving forward distance between the dog and the observer is decreased distance between the dog and the observer is increased both front paws make contact with the cage door that does not include lunging orients towards tail repeatedly (more than 3 times) and continuously repeatedly (more than 3 steps) locomoting around kennel in fixed route supported upright with all four legs to continuously and erratically drive the car inside the tub. the other researchers stood on the opposite side of the pen and videotaped the interaction for 2 min. these videos were coded on the behaviors listed in table 3 and additionally on the corresponding behaviors listed in table 4 . the videos were coded using the partial-interval coding procedure with 5 s time bins, in which an occurrence or non-occurrence of the behavior was noted. the behavioral variables were derived by taking the proportion of the time bins in which a behavior occurred. a portion of the videos (29%), selected at random, were double coded. inter-observer agreement was calculated by adding the number of agreements by interval, dividing by the total number of intervals, and multiplying by 100. inter-observer agreement was calculated for each behavior independently. the average agreement across behaviors was 99% (sd = 0.01%; min: 97.3% for "walking," max: 100% for "jumping on fence," "howling," "tail tucked," "cowering," "cowering," "body trembling," "grab car," and "play bow"). urinary cortisol:creatinine ratio. the urine was collected using a clean plastic vial. immediately following collection, urine was labeled and placed in a cooler with ice packs. following the collection of the urine for the day, the samples were taken to a secured freezer in the texas tech university animal and food sciences building. twenty dogs did not urinate when taken outside, resulting in 64 of dogs containing urinary c:c data. the urine was shipped, in dry ice, to an independent texas a&m veterinary medical diagnostic laboratory (college station, tx, usa) for analysis. a cortisol radioimmunoassay from mp biomedicals (mp biomedicals, llc, santa ana, ca, usa) was utilized. urine was extracted with dichloromethane using 0.5 ml of urine with 1 ml of solvent. the procedure involved first mixing for 5 min followed by 5 min of rest. following rest, the sample was dried down under nitrogen (50 μl per tube) and 25 μl of stripped canine serum was used to the quantification standard of the assay kit (25 μl of standard, control, and target serum). health coding. the health observations (table 1) were coded to provide a quantifiable severity of the observation as an "illness score". the coding scheme for translating observations to a numerical score is presented in the same table. the median rectal temperature was coded as a numerical value. behavior coding and filtering. when considering each behavioral variable for each observation period, 254 behavioral variables were scored across the study period. we implemented a variable quality selection procedure. first, we removed all variables in which fewer than 8 dogs (~10% of all dogs) exhibited across the study period. this filter retained 201 variables. second, we removed all variables that showed little variability across dogs. variables with a standard deviation of less than .01 were removed, leaving a total of 141 behaviors. because the in-kennel behaviors were evaluated at multiple time points, we further restricted our analysis to use the behavioral variables from the first two observation days to predict health observations across the 14 study days. all data are available in the supplementary materials; however, for the aims of this study to predict health outcomes, we restricted our analysis on the first two observation periods (day 1 and day 3). only 56 behavioral variables remained for exploration. path analysis. to identify whether temperament influenced overall health, we conducted a path analysis using the plspm package in r [32] . to test the hypothesis that curiosity, sociability, anxiety, and activity may impact illness risk, the coded behaviors were categorized into latent variables by the first author based on previous research by hennessy et al. (see table 5 [31, [33] [34] [35] [36] ). each behavioral indicator variable was included as a reflective indicator of the latent variable. for the classification of behaviors not listed in the previous study, we attempted to group similar behaviors into established categories. for example, "gazing" and "proximity" to car were grouped with "approach" to car. in-kennel behaviors were interpreted taking into account previous research that showed that some behaviors correlated strongly together and were emitted by dogs when a person was actively interacting with them through the kennel [29] . previous research has shown that "back of kennel" is highly correlated with "front of kennel" and can be considered in unison; the same phenomenon occurs for "facing forward" and "facing backwards". combined, these can be labeled "back and forth facing or motion" as was done in protopopova et al. an example of this phenomenon can be demonstrated by observing a dog pacing back and forth in the kennel. because "gazing", "jumping on cage", "barking", "whining", and "wagging tail" increased in previous research when a person actively solicited attention, these may be considered as part of sociability [29] . hekman and colleagues previously found that panting and lip licking were positively correlated with salivary cortisol concentrations, indicating stress [37] . thus, we included "panting" and "licking self" into the "anxiety" latent variable. "leaning on the wall" has previously been found to correlate with a long length of stay at the shelter [29] , which may indicate some form of distress. therefore, we elected to place this behavior into the "anxiety" latent variable; however, we recognize that this table 4 . operational definitions of the additional behaviors that were observed in the sociability and the curiosity components during the behavioral assessment. was a subjective decision. "barking" and "proximity to the experimenter" during the boldness/ shyness test were logically grouped into the "anxiety" latent variable as they may have indicated distress of the dog as a result of the toy car; again, we recognize that this was a subjective decision. all activity-related behaviors were grouped into the "activity" latent variable. an initial model was fit and the loadings, cross loadings, cronbach's alpha, and dillon-goldstein's rho were checked to evaluate the unidimensionality of the temperament and health variables. indicator variables that were poorly correlated with their respective latent variable (cronbach's alpha & dillon-goldstein's rho < .4) were removed from consideration. the model was re-evaluated and temperament and health variables were assessed for unidimensionality with a raised criterion of .5 for cronbach's alpha and dillon-goldstein's rho. the remaining indicator variables were deemed acceptable for inclusion. to evaluate whether the latent temperament variables were associated with health, we proposed a basic structural model in which curiosity, sociability, anxiety, activity, and time in the shelter independently predicted illness (fig 1) . details of this model are described in the results. table 6 shows the attrition from the study. dogs were euthanized (83%), sent to the adoption floor (7%), returned to owner (6%), or died in their kennel (3.5%). most dogs were available for the behavioral assessments through the first 3-4 days; however, only 19 dogs remained by day 14. during this time, health became progressively worse. fig 2 shows the change in the health observations across time. health steadily worsened as indicated by increases in the severity of the illness observation scores. in addition, temperature increased indicative of fever. fig 3 shows the mean rectal temperature across time as well as the 95% confidence interval (boot-strapped confidence intervals obtained via packages ggplot2) [38] . the dotted line indicates the threshold for fever (39˚c). at study initiation, the 95% confidence interval was well below the fever threshold. however, by day 8 through the end of the study, the 95% confidence interval overlapped with a fever threshold. together, these results clearly indicate the development of illness and systematic increase in severity across the study period. through our established exclusion criterion for relatedness of a behavioral variable to the latent variable, 32 variables were with cronbach's alpha and dillon-goldstein's rho < .4. in a table 7 and table 8 shows latent variable loadings. to evaluate whether activity, sociability, anxiety, curiosity, and time in the shelter were related to the illness score, we conducted a basic pls path regression model in which our 5 latent variables were tested for association with illness. fig 4 shows the hypothesized path model with regression coefficients. table 9 shows the model estimates and statistical significance. overall, sociability, curiosity and time in the shelter were significantly associated with illness. as expected, as time in the shelter increased, illness scores did also (estimate = .53, p < .001). increases in sociability scores (estimate = .24, p < .001; fig 5) and curiosity (estimate = .09, p = .026) were associated with increased illness. activity and anxiety, however, were not associated with illness. the mean c:c ratio was 18.4 x 10 −6 (sd = 11.2 x 10 −6 ). due to missing c:c ratio data (19/83; 23% missing), c:c ratio data were excluded from the pls path regression. to evaluate whether table 6 . https://doi.org/10.1371/journal.pone.0224252.g002 the c:c ratio was associated with illness, a linear mixed model with dog id as a random effect and c:c ratio and time in the shelter as fixed effects indicated that the c:c ratio was not associated the illness score, although time in the shelter was (see table 10 ). to further explore in a reduced sample, whether the c:c ratio was associated with the latent behavioral variables, we computed a cross-correlation matrix between c:c ratio and our latent variable scores from our pls path model. c:c ratio was slightly negatively correlated with sociability (r = -.22), indicating that more sociable dogs had lower c:c ratios. however, due to the large number of correlations and reduced sample size for this analysis, we did not compute p-value to interpret statistical significance. lastly, the cross-correlations indicate sociability and curiosity were positively correlated (r = .42), suggesting these variables may be related and perhaps a more complex path analysis may be worth exploring in future studies with a larger sample size. a correlation matrix was constructed with c:c ratio, time at the shelter, standardized illness, sociability, and curiosity scores. time in the shelter and the standardized illness score had a moderate correlation of .52. sociability and curiosity scores had a moderate correlation of .42. sociability had a moderate correlation with the standardized illness score of .35. standardized curiosity and illness scores had a lower correlation of .20. c:c ratio had a lower negative correlation of -.23. no correlation was present between sociability and time in the shelter, curiosity and time in the shelter, and c:c ratio and illness score (table 11 ). in support of previous research, we found that time in the shelter was positively associated with the incidence of illness symptoms. across time, each sign of upper respiratory illness (coughing, sneezing, ocular and nasal discharge, and fever) became more severe. whereas increases in all signs of illness were already evident as early as the third day in the shelter, by two weeks, the average dog in this animal shelter had a fever, colored nasal discharge, clear ocular discharge, and half of dogs were coughing and/or sneezing. this data supports previous research that found that the risk of coughing increased by 3% each day [39] . out of the four behavioral components, only sociability and, to a much lesser extent, curiosity, but not activity or anxiety was associated with illness. dogs who had higher standardized scores in both sociability and curiosity in the first few days after intake, were more likely to have higher illness scores. sociability consisted of tail wagging and jumping on the cage when a person came up to the kennel and jumping, leaning on, and staying in proximity to the person during the sociability test. curiosity consisted of paying attention to the remote-controlled car (approaching, retreating from, gazing at, and staying in proximity to the car) during the boldness test. the decision to label these behaviors as "curiosity" rather than "boldness" was arbitrary and was informed by subjective opinion by the authors that the dogs' behavior was more closely in line with the human concept of curiosity (i.e., information seeking) rather than boldness (e.g., a willingness to take risks). furthermore, in our study, we did not assess for repeatability and thus are limited in the interpretations of our data in terms of personality or temperament literature. according to visual analysis of the data, a clear positive linear relationship was evident between the standardized sociability and illness scores. however, the relationship appeared less clear between standardized curiosity and illness scores, with some potential outliers driving the positive correlation. it is also noteworthy that sociability and curiosity were moderately correlated, suggesting a potential underlying trait or that a more complex path model might be suggested for future larger studies. previous research has suggested that the various behavioral components may be part of a greater whole. for example, svartberg and forkman [36] suggested that various traits, such as sociability and exploration, among others, may be related to a single higher-order dimension. corsetti and colleagues [26] have also combined the individual traits of activity, attentiveness, dominance, and sociability to differentiate dogs into proactive and reactive coping styles. however, they found that dogs displaying the proactive style (higher boldness, higher sociability) had a lower incidence of illness; our current results seem to be contrary to this previous data. however, corsetti et al. [26] did not find any statistically significant predictors when assessing individual traits, such as boldness, activity, sociability, or anxiety; the lack of statistically significant correlations among individual traits to illness ). urinary c:c ratio, taken on the first full day, was not associated with subsequent illness in the animal shelter. previous research suggests that coping style has a link with the responsiveness of the hpa axis. the proactive coping style has previously been found to correlate with low cortisol responses in dogs [40, 41] . and in fact, we did find that cortisol had a low negative correlation with the sociability component. interestingly, the same negative correlation of cortisol and sociability (but to conspecifics) was found in rhesus macaques [18] . however, instead of the proactive temperament protecting the dogs from illness (through the reduction of cortisol), we found a positive association between sociability and illness. as the correlation between cortisol and sociability was low, this finding may be a type i error, and no true relationship may exist between the two in this population; alternatively, the relationship between proactive coping style and hpa reactivity may differ in this species or in this environment. our data may fit the risk-of-parasitism (rop) hypothesis, which suggests that animals that exhibit bold or exploratory behavior encounter more parasites or pathogens (e.g., [42] ). the probability of encountering a parasite increases mechanically as the animal engages in exploratory behavior. for example, pumpkinseed sunfish that exhibited a bold temperament were more likely to have a higher parasite load [42] . tom cats who exhibited a more dominant and bold temperament were also more likely to be infected with feline immunodeficiency virus [20] . norway rats with higher testosterone were more likely to engage in fighting and more likely to have a hantavirus infection [21] . wood mice that were infected by nematode exhibited more locomotion [43] . similarly, in our study, dogs that were more curious and social may have encountered more infected surfaces, thus were more exposed to pathogens than dogs that were not curious nor social. however, during the time of the study, the animals were typically group housed in relatively small kennels with continuous rotation of animals and no sanitation prior to new arrivals. therefore, the already very high risk of transmission in this particular shelter reduces the likelihood that the rop hypothesis accounts for the entirety of these results. nevertheless, this hypothesis remains a viable candidate for the explanation of the found phenomenon, and more data and experiments are required. an additional hypothesis has been put forth to explain whole-animal differences in immune function, the pace-of-life-syndrome (pols) hypothesis. the pols hypothesis has originally been used to differentiate different species by their "pace of life," or metabolic and reproductive evolutionary strategy [44, 45] . for example, some species may prioritize reproduction but not immune function or longevity. this strategy may be regarded as "live fast, die young." in contrast, some species may prioritize longevity and immune function instead of reproduction -the "live slow, die old" strategy [45, 46] . recently, the pols hypothesis has been utilized to explain whole animal differences within a single species [47] . in fact, such differences have been previously suggested in dogs [48] . thus, it is possible that intra-species differences in dogs may also follow these two evolutionary strategies, with one strategy prioritizing immune function and the other prioritizing reproduction. perhaps dogs that are curious and social are utilizing the "live fast, die young" strategy, and are thus not prioritizing immune function. in fact, due to dogs' reliance on human influence, perhaps human-directed sociability is a strategy for dogs to ensure medical care; thus, by putting more resources into sociability, fewer resources are needed for immune system function. chersini, hall, and wynne [49] suggested that dogs may utilize human intervention for their survival; people rate pups at weaning as most desirable, and this is also the time when dams leave their pups to fend for themselves. with pup survival being only around 70% in free-ranging situations, human involvement becomes crucial to the dog [50] . while intriguing, our current data are not adequate for supporting or refuting this hypothesis. in order to provide support, future data need to show that social and curious dogs also have higher litter size, higher basal metabolic function, and shorter lives. in addition, future data would need to show that people will spend more money or effort on healthcare of social dogs. according to the path analysis model, the outcome illness variable consisted of rectal temperature, the presence of nasal discharge, and coughing. sneezing and ocular discharge did not load well onto the illness factor; however, it is noteworthy that both also increased with time, suggesting that they may be associated with the later part of disease progression, or with a distinct disease process. another important consideration is the potential paradoxical effect in which a positive correlation maybe observed across individuals but a negative correlation observed within individuals [51] . thus, although we observed positive relationships with sociability and curiosity, the correlation at the individual level maybe negative, such that when a dog is more sociable than its typical average, it may be less likely to develop an illness. the present study, however, is limited in its ability to detect such effects due to our limit of only two data points from the first two observations per dog. had we taken more longitudinal data for dogs that stayed for longer periods, this would have been an interesting analysis. several limitations were present in the current study. due to human error, we were not able to assess the immune function of the dogs directly. future research may need to verify the effects of temperament on immune function itself, rather than relying on the indirect measure of subsequent illness. however, in the shelter environment, the predictive nature of temperament remains to be meaningful, regardless of the underlying mechanism. a second limitation was that 12 (14%) of the animals received vaccinations against pathogens that contribute to cirdc, the target disease complex. vaccination on intake can reduce disease incidence in shelters, probably through stimulation of innate immunity. only a subset of these dogs (n = 9; 10%), however, received them during the data collection phase. of the 9 dogs that were vaccinated during the data collection phase, 6 showed signs of illness post-vaccination (s1 fig). it is likely that even the vaccinated dogs were still exposed to pathogens prior to vaccination. nevertheless, future research may circumvent this issue by administering a vaccine challenge to all and measuring the immune response directly. this might also circumvent the problem of animals having unknown prior vaccination histories. a third limitation was that dogs were continuously rotated through the kennels, thus resulting in different groups of dogs per kennel at the different observation times. this shelter procedure made it difficult to assess the risk-of-parasitism hypothesis as well as generally made it difficult to account for the effect of conspecifics during kenneling. finally, we also found high rates of attrition from the study. this limits the longitudinal sample size. to try to limit the effects of attrition, we focused our analysis on early prediction of illness and therefore utilized predictors obtained from the first three days of entering the study only, allowing us to have information on the predictors for all dogs. further, our predictor of interest was health, and unfortunately, many dogs were becoming sick early on, with many dogs showing illness in the first week, while we still retained many of the dogs. nonetheless, it's unclear to what degree the data maybe censored due to euthanasia before developing an illness or going up for an adoption. expanding on the current sample size would be an important follow-up study. regardless of the biological and/or evolutionary mechanism by which dogs with certain temperaments were more susceptible to illness, these data are important for the establishment of predictive models in the applied animal shelter environment. providing knowledge about which dogs are more susceptible to illness, would allow shelter staff to manage the dog population more effectively. a suggestion may be to treat highly social animals as immunocompromised and manage these individuals similarly to nursing moms and puppies. current best-practices suggest housing immunocompromised individuals in a different location away from the general population and taking additional care with disease transmission in these rooms [52] . however, further applied research is needed in order to develop behavioral screening assessments that would adequately predict subsequent illness. to summarize, we found a positive relationship between some temperament traits of dogs, namely sociability and curiosity, and subsequent signs of cirdc illness in the animal shelter environment. due to significant dropout of participants, however, we were not able to observe whether individual variability had a similar relationship. further, we did not find any effect of urinary c:c ratio on subsequent illness. these data are contrary to previous pilot data that suggest that proactive temperaments may protect dogs from subsequent illness. explanations for our data may include the risk-of-parasitism and pace-of-life syndrome hypotheses. future research is needed to differentiate between these two hypotheses as well as develop predictive models for use in animal sheltering. 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continuum in pumpkinseed sunfish (lepomis gibbosus): an ecological study of a psychological trait apodemus sylvaticus infected with heligmosomoides polygyrus (nematoda) in an arable ecosystem: epidemiology and effects of infection on the movements of male mice performance, personality, and energetics: correlation, causation, and mechanism integrating animal temperament within ecology and evolution. biological reviews personality and the pace-of-life syndrome: variation and selection on exploration, metabolism and locomotor performances slow pace of life in tropical sedentary birds: a common-garden experiment on four stonechat populations from different latitudes the pace of life under artificial selection: personality, energy expenditure, and longevity are correlated in domestic dogs dog pups' attractiveness to humans peaks at weaning age. anthrozoos comparative social ecology of feral dogs and wolves a simple method for distinguishing within-versus between-subject effects using mixed models strategies for management of infectious diseases in a shelter we thank the lubbock animal shelter staff for allowing us to conduct the study. we also thank the following undergraduate students, without whom we could not have conducted the study: kerbey jacobs, francine camara kaercher, sadie bowling, sarah huerta, julianna maynard, jocelyn banschbach, morgan rowland, and priscilla rubio. we thank the dogs, especially who have died during this study; they are not forgotten. writing -review & editing: alexandra protopopova, nathaniel j. hall, kelsea m. brown, jessica p. hekman. key: cord-267519-a0bcmjkn authors: bravi, francesca; flacco, maria elena; carradori, tiziano; volta, carlo alberto; cosenza, giuseppe; de togni, aldo; acuti martellucci, cecilia; parruti, giustino; mantovani, lorenzo; manzoli, lamberto title: predictors of severe or lethal covid-19, including angiotensin converting enzyme inhibitors and angiotensin ii receptor blockers, in a sample of infected italian citizens date: 2020-06-24 journal: plos one doi: 10.1371/journal.pone.0235248 sha: doc_id: 267519 cord_uid: a0bcmjkn aims: this retrospective case-control study was aimed at identifying potential independent predictors of severe/lethal covid-19, including the treatment with angiotensin-converting enzyme inhibitors (acei) and/or angiotensin ii receptor blockers (arbs). methods and results: all adults with sars-cov-2 infection in two italian provinces were followed for a median of 24 days. arbs and/or acei treatments, and hypertension, diabetes, cancer, copd, renal and major cardiovascular diseases (cvd) were extracted from clinical charts and electronic health records, up to two years before infection. the sample consisted of 1603 subjects (mean age 58.0y; 47.3% males): 454 (28.3%) had severe symptoms, 192 (12.0%) very severe or lethal disease (154 deaths; mean age 79.3 years; 70.8% hypertensive, 42.2% with cvd). the youngest deceased person aged 44 years. among hypertensive subjects (n = 543), the proportion of those treated with arbs or acei were 88.4%, 78.7% and 80.6% among patients with mild, severe and very severe/lethal disease, respectively. at multivariate analysis, no association was observed between therapy and disease severity (adjusted or for very severe/lethal covid-19: 0.87; 95% ci: 0.50–1.49). significant predictors of severe disease were older age (with aors largely increasing after 70 years of age), male gender (aor: 1.76; 1.40–2.23), diabetes (aor: 1.52; 1.05–2.18), cvd (aor: 1.88; 1.32–2.70) and copd (aor: 1.88; 1.11–3.20). only gender, age and diabetes also predicted very severe/lethal disease. conclusion: no association was found between covid-19 severity and treatment with arbs and/or acei, supporting the recommendation to continue medication for all patients unless otherwise advised by their physicians. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 novel coronavirus disease (covid-19) is spreading worldwide, and has caused over 250,000 deaths so far [1] . the mortality rate varies widely by age and across individuals, ranging from 0.2% among healthy, young-adults, to >10% among older persons with pre-existing conditions [1] . although the pharmacological treatment was not assessed, the first observational studies on patients with severe disease reported a high prevalence of comorbidities that are often treated with angiotensin converting enzyme (ace) inhibitors, such as cerebrovascular diseases, coronary heart disease, hypertension and diabetes [2] [3] [4] . observing that human pathogenic coronaviruses bind their target cells through angiotensin-converting enzyme 2 (ace2) [5] [6] [7] [8] , and that a few studies reported an increase in ace2 expression mediated by angiotensin ii type-i receptor blockers (arbs) and ace inhibitors (more consistently on animals than in humans) [9] [10] [11] [12] [13] [14] [15] [16] , some hypothesized that the increased expression of ace2 would facilitate infection with severe acute respiratory syndrome coronavirus 2 (sars-cov-2), thus the hypertension treatment with ace2-stimulating drugs, as well as ace2 polymorphisms, might increase the risk of developing severe covid-19 [17] [18] [19] . consequently, this would lead to a serious conflict regarding treatment, because ace2 reduces inflammation and has been suggested as a potential new therapy for inflammatory lung diseases, cancer, diabetes, and hypertension [17, [20] [21] [22] [23] . in the wake of two preliminary cohort studies reporting a lower [24] or similar [25] covid-19 mortality among inpatients hypertensive subjects treated with arbs and ace inhibitors, the potential predictors of covid-19 and of disease severity, including anti-hypertensive medications, were recently analyzed by a few observational studies [26] [27] [28] . with one exception [27] , no increased risk emerged from the use of arbs or ace inhibitors; however, the role of other potentially linked predictors, including age and cardiovascular comorbidities [17, 29, 30] , differed across the population analyzed, and still requires confirmation. we have performed a case-control study on all sars-cov-2 infected subjects diagnosed in two italian provinces, retrieving admission and pharmacological data up to two years before infection, in order to confirm the potential independent predictors of severe/lethal covid-19, including treatment with ace inhibitors and/or arbs. this case-control, retrospective study compared the proportion of subjects treated with arbs and/or ace inhibitors among three groups of subjects with sars-cov-2 infection: a. asymptomatic infection or mild disease, defined as fever or malaise plus at least one of the followings: sore throat, muscle pain, shortness of breath, dry cough, headache, conjunctivitis, and diarrhea [31] , with no hospital admission; b. severe disease, requiring hospital admission, not in an intensive care unit; c. very severe or lethal disease, requiring admission in an intensive care unit and/or causing death. the sample includes all subjects with diagnosis of infection made in the province of ferrara, up to april 2, and the province of pescara, italy, up to april 24, 2020, by the central laboratory of the university hospital of ferrara or the central laboratory of the pescara hospital (and confirmed by the national institute of health). all diagnoses were made using (real time) reverse transcription polymerase chain reaction (rrt-pcr) on oropharingeal specimens. the assays were those originally proposed by the charité-universitätsmedizin berlin institute of virology [32] , and then endorsed by the who [33] . the data on background pharmacological treatment up to the previous two years (january 1, 2018) were obtained from the national database of drug prescription, and integrated with clinical chart information for hospitalized subjects. data have been collected on the following drugs: ace inhibitors (atc classes: c09a and c09b), arbs (c09c and c09d), and insulin or other anti-diabetic drugs (a10). information on age, gender, and pre-existing conditions of all subjects were obtained through data-linkage with hospital discharge abstracts (italian sdo), which have been queried from the day of the diagnosis until january 1st, 2015. all admission data have been revised manually by two physicians (lm and mef) and the following conditions have been included in the analyses: malignant tumors, major cardiovascular diseases (heart failure, myocardial infarction and stroke-cvd), type ii diabetes, renal disease and chronic obstructive pulmonary diseases (copd, bronchitis, pneumonia, asthma, and emphysema). the study complies with the declaration of helsinki, the research protocol was approved by the ethics committee of the emilia-romagna region (code 287, approved on march 24, 2020), and the requirement for informed consent was waived because of the retrospective and pseudo-anonymized nature of the data. first, the differences across groups with mild, severe or very severe/lethal disease were evaluated using two-way anova with post-hoc tukey hsd test for continuous variables, and mantel-haenzsel chi-squared test for categorical ones. a sample restricted to hypertensive subjects was used to compare the subjects treated and untreated with ace inhibitors or arbs. multivariate logistic regression was used to investigate the potential independent predictors of severe or very severe/lethal covid-19. four models were built, two were restricted to hypertensive subjects (a and b), and two included the total sample (models c and d) and. models a and c were fit to predict severe or very severe disease (grouped together), while models b and d to predict very severe/lethal disease only (and repeated to predict death, with similar results, which have not been shown to avoid redundancy). all recorded variables (age, gender, cvd, diabetes, renal disease, cancer and copd) were included a priori in all models, with the exception of treatments with ace inhibitors and arbs, that were excluded from models c and d because of multicollinearity with hypertension. standard diagnostic procedures were adopted to check all models validity: influential observation analysis (dbeta, change in pearson chi-square and similar), multicollinearity, interaction terms, hosmer-lemeshow test for the goodness of fit and c statistic (area under the receiving operator curve). statistical significance was defined as a two-sided p-value<0.05, and all analyses were carried out using stata, version 13.1 (stata corp., college station, tx, 2014). our sample of 129 hypertensive subjects with severe/lethal covid-19, and 414 hypertensive subjects with mild disease or asymptomatic infection, had 80% statistical power to detect a difference of 20% or higher (corresponding to a relative risk �1.20) in the risk of severe/lethal disease between users of arbs or ace inhibitors (exposed group), and non users (controls). the sample consisted of 1603 subjects (mean age 58.0y; 47.3% males): 454 (28.3%) had severe symptoms, 192 (12.0%) very severe or lethal disease ( table 1 ). the sample of infected subjects, compared with the available estimates of the general population of the two provinces [34] , showed higher prevalence of diabetes (12.1% in the sample versus ffi5% in the general population); hypertension (33.9% versus ffi17%), copd (6.0% versus ffi3%) and cvd (16.1% versus ffi3%). 154 subjects deceased (mean age 79.3 years; 54.6% males); of them, 70.8% were hypertensive, 42.2% were diagnosed a cvd; 27.9% diabetics. twenty subjects with very severe disease were younger than 60 years; the youngest being a male aged 33 years. of those deceased, eight were younger than 60 years, and the youngest was a female aged 44 years. at univariate analysis, as compared to the subjects with mild disease, those with severe or very severe/lethal disease were significantly more likely to be older, diabetics, hypertensive, diagnosed with copd, cvd, and renal disease, and treated with arbs and/or ace inhibitors (all p<0.05). among hypertensive subjects (n = 543), however, the proportion of those treated with arbs or ace inhibitors (whose medication was not discontinued during the follow-up) 11-3.20) , and older age, which showed an exponential increase after 70 years: compared with the subjects younger than 50 years, the aors of those aged 70-79 and �80 years were 5.72 (3.81-8.58) and 9.06 (6.04-13.6), respectively (model c; table 3 ). only male gender, older age and diabetes also predicted very severe/lethal disease (model d; table 3 ). the main findings of this retrospective, observational study, are the following: first, it is confirmed that, among hypertensive subjects, the use of ace inhibitors or arbs up to two years preceding sars-cov-2 infection did not affect the severity of covid-19. second, older age, male gender, diabetes, and the presence of copd or cvd were independent predictors of severe disease, with a sharp increase of risk among subjects older than 70 years. third, only older age, male gender and diabetes were associated with a higher likelihood of very severe/ lethal disease. several hypotheses have been made on the association between covid-19 progression and treatment with ace inhibitors and arbs [20-22, 35, 36] . on one side, some asked whether the therapy should be discontinued during sars-cov-2 pandemic [17, 37] because covid-19 was strongly associated with hypertension, which is frequently treated with arbs and ace inhibitors [2] [3] [4] . it was indeed hypothesized that: (a) ace2 up-regulation mediated by arbs (and, to a lesser extent, by ace inhibitors) might increase patients' susceptibility to sars--cov-2 entry into host cells and further viral propagation [18, 19] , (b) virus binding to ace2 might reduce its activity, thus leading to increased levels of angiotensin ii and consequent pulmonary vasoconstriction, inflammation and oxidative organ damage, and increased risk of acute lung injury [20] . on the opposite side, other scientists suggested that, other than being harmful, arbs and ace inhibitors use in patients with cardiovascular risk factors and known or suspected covid-19 may even exert a beneficial effect, as ace2 up-regulation could increase the conversion of angiotensin ii to angiotensin-(1-7), a peptide with potentially protective anti-inflammatory properties [35, 36] . recently, a few large observational studies based upon in-and outpatient electronic health records [26] [27] [28] examined the association between antihypertensive medications and the risk of covid-19 and/or a severe/lethal disease: our results are in line with most of the previous findings on an absence of risk with ace inhibitors and/or arbs use. in brief, the studies by mancia et al [26] , and reynolds et al [28] reported no difference in covid-19 severity or death between treated and untreated subjects, for both arbs and ace inhibitors. instead, the results by mehta et al [27] were partially discordant: the study found no association between treatment with arbs and death, but those treated with ace inhibitors showed a significantly higher risk of icu admission. in our sample, in order to further investigate the potential beneficial effects of ace inhibitors, the impact of which might be larger in patients with diabetes, copd, or cardiovascular diseases, we performed additional analyses, stratified by comorbidities. we found however no significant differences in the risk of severe/lethal covid-19 among treated and untreated patients with either cvd, or diabetes and copd (data not shown). besides sample size and provenance, there were few differences between this and previous studies: all studies included all infected subjects, hospitalized or not, evaluated disease progression beyond mortality, and retrieved medications and admissions from electronic health records (with the exception of mehta et al, who assessed the medications exclusively at the time of testing for sars-cov-2 [27] ). overall, the present and previous findings confirm those from preliminary chinese cohorts [24, 31] , and although confirmation from randomized studies is required [38] , they strongly support the statements of several experts [39, 40] and scientific societies, including the european medicines agency [41] , the european society of cardiology [42] , and the american heart association [43] , who recommend continuation of arbs or ace inhibitors medication for all patients, unless otherwise advised by their physicians. with regard to the role of the other risk factors that have been suggested for severe covid-19, including age, male gender, hypertension, diabetes, copd, and major cardiovascular diseases, it has been correctly argued that, so far, available data were unadjusted, thus the relative importance of underlying health conditions was unclear [21, 29] . in this study, we found support for a potential role of gender, diabetes, copd and cvd, beyond age, in covid-19 progression to a severe disease, whereas only gender and diabetes significantly increased the risk of a lethal or very severe outcome. thus, the present study confirms prior findings on the independent relationship of older age and male gender with death [44] , and of copd with progression towards severe disease [26] . instead, at least two issues may have influenced the conflicting results on the role of cvd and copd in predicting very severe/ lethal disease (an association showed in some prior populations [44] -but lacking in the present as well as in other recent findings [26] ): first, the relatively small sample of the present study; second, a marked difference in the population here enrolled, as compared to previous studies which included randomly selected sars-cov2 negative subjects as controls [26] . given the present scenario, further population-based cohort studies, with longer follow-up are clearly needed [29] to clarify these findings. in addition to a relatively small sample, a limitation of the present study is the lack of tobacco smoking and body mass index among the variables that have been recorded, because we could not extract such information for half of the deceased subjects, as well as for many of those that were not hospitalized. other limitations are the lack of an evaluation of the severity of the underlying cardiovascular diseases, and the absence of data on other antihypertensive medications. however, their potential role in altering the relationship between arbs or ace inhibitors and risk of severe/lethal covid-19 remains unclear: none of the previous studies on the topic assessed cardiovascular diseases severity [26] [27] [28] , and the two studies that included the use of other antihypertensive drugs into multivariable analyses did not find substantial differences between the adjusted and unadjusted relative risks of death [26, 28] . in conclusions, the present study did not find any association between covid-19 severity and treatment with arbs, ace inhibitors, or both, and confirms previous findings in supporting the recommendation of several scientific societies to continue arbs or ace inhibitors medication for all patients, unless otherwise advised by their physicians, who should thus be reassured. the adjusted analyses substantially confirm prior reports, indicating that the risk of severe or lethal covid-19 largely and significantly increases among the elderly, males, diabetics, and those with copd or major cardiovascular diseases. covid-19 coronavirus outbreak clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study clinical characteristics of coronavirus disease 2019 in china clinical characteristics of 140 patients infected with sars-cov-2 in wuhan sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus the sars-cov s glycoprotein: expression and functional characterization urinary angiotensin-converting enzyme 2 in hypertensive patients may be increased by olmesartan, an angiotensin ii receptor blocker effects of captopril related to increased levels of prostacyclin and angiotensin-(1-7) in essential hypertension increased angiotensin-(1-7)-forming activity in failing human heart ventricles: evidence for upregulation of the angiotensin-converting enzyme homologue ace2 effect of angiotensinconverting enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensin-converting enzyme 2 map kinase/phosphatase pathway mediates the regulation of ace2 by angiotensin peptides angiotensin ii at1 receptors regulate ace2 and angiotensin-(1-7) expression in the aorta of spontaneously hypertensive rats upregulation of angiotensin-converting enzyme 2 after myocardial infarction by blockade of angiotensin ii receptors. hypertension localization of ace2 in the renal vasculature: amplification by angiotensin ii type 1 receptor blockade using telmisartan are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? can angiotensin receptor-blocking drugs perhaps be harmful in the covid-19 pandemic? preventing a covid-19 pandemic: ace inhibitors as a potential risk factor for fatal covid-19 coronavirus disease 2019 (covid-19) infection and renin angiotensin system blockers covid-19 and angiotensin-converting enzyme inhibitors and angiotensin receptor blockers: what is the evidence? drugs and the renin-angiotensin system in covid-19 renin-angiotensin-aldosterone system inhibitors in patients with covid-19 association of inpatient use of angiotensin converting enzyme inhibitors and angiotensin ii receptor blockers with mortality among patients with hypertension hospitalized with covid-19 association of renin-angiotensin system inhibitors with severity or risk of death in patients with hypertension hospitalized for coronavirus disease 2019 (covid-19) infection in wuhan renin-angiotensin-aldosterone system blockers and the risk of covid-19 association of use of angiotensin-converting enzyme inhibitors and angiotensin ii receptor blockers with testing positive for coronavirus disease renin-angiotensin-aldosterone system inhibitors and risk of covid-19 covid-19: risk factors for severe disease and death case-fatality rate and characteristics of patients dying in relation to covid-19 in italy evaluation and treatment coronavirus detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr world health organization. laboratory testing for coronavirus disease (covid-19) in suspected human cases-interim guidance aspects of daily life-health status-regions and cities [aspetti della vita quotidiana: stato di salute-regioni e tipo di comune renin-angiotensin-aldosterone system inhibitors in patients with covid-19 renin-angiotensin system blockers and the covid-19 pandemic: at present there is no evidence to abandon renin-angiotensin system blockers covid-19 and the cardiovascular system inhibitors of the renin-angiotensin-aldosterone system and covid-19 switching antihypertensive therapy in times of covid-19: why we should wait for the evidence sars-cov2: should inhibitors of the renin-angiotensin system be withdrawn in patients with covid-19? european heart journal ema advises continued use of medicines for hypertension, heart or kidney disease during covid-19 european society of c. position statement of the esc council on hypertension on ace-inhibitors and angiotensin receptor blockers patients taking ace-i and arbs who contract covid-19 should continue treatment, unless otherwise advised by their physician2020 opensafely: factors associated with covid-19-related hospital death in the linked electronic health records of 17 million adult nhs patients key: cord-013099-j816c3tw authors: blease, charlotte; kharko, anna; locher, cosima; desroches, catherine m.; mandl, kenneth d. title: us primary care in 2029: a delphi survey on the impact of machine learning date: 2020-10-08 journal: plos one doi: 10.1371/journal.pone.0239947 sha: doc_id: 13099 cord_uid: j816c3tw objective: to solicit leading health informaticians’ predictions about the impact of ai/ml on primary care in the us in 2029. design: a three-round online modified delphi poll. participants: twenty-nine leading health informaticians. methods: in september 2019, health informatics experts were selected by the research team, and invited to participate the delphi poll. participation in each round was anonymous, and panelists were given between 4–8 weeks to respond to each round. in round 1 open-ended questions solicited forecasts on the impact of ai/ml on: (1) patient care, (2) access to care, (3) the primary care workforce, (4) technological breakthroughs, and (5) the long-future for primary care physicians. responses were coded to produce itemized statements. in round 2, participants were invited to rate their agreement with each item along 7-point likert scales. responses were analyzed for consensus which was set at a predetermined interquartile range of ≤ 1. in round 3 items that did not reach consensus were redistributed. results: a total of 16 experts participated in round 1 (16/29, 55%). of these experts 13/16 (response rate, 81%), and 13/13 (response rate, 100%), responded to rounds 2 and 3, respectively. as a result of developments in ai/ml by 2029 experts anticipated workplace changes including incursions into the disintermediation of physician expertise, and increased ai/ml training requirements for medical students. informaticians also forecast that by 2029 ai/ml will increase diagnostic accuracy especially among those with limited access to experts, minorities and those with rare diseases. expert panelists also predicted that ai/ml-tools would improve access to expert doctor knowledge. conclusions: this study presents timely information on informaticians’ consensus views about the impact of ai/ml on us primary care in 2029. preparation for the near-future of primary care will require improved levels of digital health literacy among patients and physicians. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 attention in medicine and related fields has increasingly focused on the potential of big data, artificial intelligence (ai), and machine learning (ml) to change the delivery of healthcare [1] [2] [3] [4] . much of this debate has focused on the promise of ai/ml to augment or even disintermediate the clinical roles of physicians in gathering and monitoring patient health information, and to undertake core tasks such as diagnostics, prognostics, and the formulation of personalized patient healthcare plans [1, [4] [5] [6] [7] [8] [9] . differentiating the hype from hope in the discourse about ai/ml in medicine is crucial to better understand the scope for the computerization of medicine. although broad predictions of the impact of ai/ml on healthcare are ubiquitous, credible short-term predictions are necessary to address questions about resource allocation, and the adequacy of medical education and training. recently a number of surveys have explored medical students', and physicians' views about the impact of ai/ml on the future of medical practice [10] [11] [12] [13] [14] [15] [16] . currently, there is scarce exploration of consensus views among informaticians [17] ; in particular, on how ai/ml might meaningfully influence medical care in the short-term [18] . to address this research gap, we designed a delphi survey to explore leading health informaticians' predictions about the impact of machine learning on primary care in the us in 2029. to our knowledge, this is the first investigation of experts' opinions about the impact of ai/ml on the future of the near future of general medical practice. the delphi method, developed by the rand corporation in the 1950s [19, 20] is designed to pool the opinions of a purposive sample of identified experts in a given field to establish consensus predictions [21, 22] . delphi polls rely on non-probability sampling techniques to identify a panel of experts: since participants are not randomly selected, representativeness is neither intended nor assured [23] . the selected panel of experts is invited to answer a series of questions anonymously [23] . participants are next asked to reassess their initial judgments in light of group trends until consensus is obtained [24] . this anonymous, iterative technique carries distinctive advantages over focus groups by avoiding the influences of individual dominant personalities, group-think, and helps to keep participants 'on topic' [19, 23] . delphi surveys are particularly well suited to exploring consensus views related to new lines of inquiry, and for establishing goal-setting, and needs assessments in policy-making [19, 23] . since delphi polls provide more accurate predictions than other forecasting methodologies, the approach is often used as a policy and practice heuristic for health care management, and resource allocation [25] [26] [27] . we used a modified delphi technique which is structured into three discrete rounds [19, 23, 28, 29] . in round one, questions are open-ended, requiring free-text answers. responses are aggregated and coded into a series of statements. in round two, experts are provided with this list of statements, and requested to provide their level of agreement with each item. depending on the survey items, round 2 and 3 questionnaires requested 'yes' or 'no' responses, or participants' level of agreement with statements on 7-point likert scales: 1 = greatly decrease, 2 = moderately decrease, 3 = slightly decrease, 4 = remain the same, 5 = slightly increase, 6 = moderately increase, 7 = greatly increase; 1 = very unlikely, 2 = moderately unlikely, 3 = slightly unlikely, 4 = uncertain, 5 = slightly likely, 6 = moderately likely, 7 = very likely; or 1 = strongly disagree, 2 = moderately disagree, 3 = slightly disagree, 4 = neutral, 5 = slightly agree, 6 = moderately agree, 7 = strongly increase. those statements that reach a predefined level of agreement are omitted to reduce participant survey fatigue, and items that lack consensus are re-circulated via a final anonymous poll. in the third and final round, panelists are reminded of their own response to the remaining statements as well as the median response of other experts, and are invited to preserve or revise their answer. a key aim of delphi methodology is to maintain as high a response rate as possible rounds [23, 30, 31] , and the accuracy of forecasts has been demonstrated to improve between each round [32] . although there is no universally agreed sample size for delphi polls [23] , our aim was to balance the size of the panel with a high response rate between the three rounds. we therefore aimed to achieve a panel of around 12-15 individuals who would agree to share their expertise, and be committed to giving their time to respond to each round. using purposive sampling methodology, the research team compiled a list of 27 highly trained and knowledgeable individuals with context-specific knowledge about health informatics and primary care in the us. addressing the question about how to identify domain-specific 'experts', our goal was to prioritize panelists for their recognized competence in the field of health informatics. we defined expertise to mean a person who had published significant contributions within the field of health informatics, and/or individuals who were currently appointed as research leaders, or as health information officers. acknowledging that heterogeneous panels have been shown to result in more accurate estimates [33] , and that what counts as an expert can be influenced by goals, values, and the manner in which knowledge is generated, we aimed to recruit diverse participants from across academia, healthcare, non-profit organizations, and industry; and to strive for panelists with a varied complementarity of interests within health informatics. measures were also taken to ensure demographic diversity among invited participants along the lines of gender, age, nationality, and race/ethnicity. this study was deemed exempt research by beth israel deaconess medical center institutional review board and granted ethical approval by the university of plymouth, uk. prospective panelists were contacted via email in september 2019, with an invitation and internet link to the survey. individuals were informed that we desired a commitment on the part of experts to respond to all three rounds, that adequate response time would be given to answer each round of the survey, participation was voluntary and unpaid, and that participants could withdraw at any time. prospective participants were also informed that they would remain anonymous to other participants, their individual responses would not be shared with other panelists, and their contribution would be confidential. respondents' names were also replaced with a study id number by ak in order to preserve participant anonymity among other team members in data analysis. we created an electronic questionnaire on jisc online surveys hosted at the university of plymouth, uk (https://www.onlinesurveys.ac.uk/). the poll incorporated a three-step modified delphi method which took place between september 2019 and january 2020. participants were sent 3 reminders after each round of the survey, and given 4-6 weeks to respond to rounds 1 and 2, and 8 weeks to respond to round 3 which fell over the new year period. in the first round, the delphi survey requested demographic information; this was followed by 5 sections, with 7 open-ended questions, on the impact of machine learning on primary care by 2029 (see s1 appendix; table 1 we also included a final comment-box for feedback on the survey. responses to round 1 were collated and coded into lists of statements. coding was conducted by cb and independently reviewed by cl and ak, and subsequent revisions were made. comments that were unrelated to the themes, or were deemed redundant were eliminated. similar statements were grouped together and translated into concise items; whenever possible, replication of exact phrasing by participants was employed. these items were circulated in round 2, and an online survey was sent to each individual member of the panel. participants were requested to respond to categorical variables by selecting a 'yes' or 'no' response, and to questions with continuous variables by using predefined 7-point likert scales (see s2 appendix). prior to consensus analysis of responses in rounds 2 and 3, for categorical variables consensus was set at � 75%, and for continuous variables consensus along 7-point semantic differential scales was set at an interquartile range of � 1 [19, 34] . after analysis of round 2 results, items that did not reach consensus were redistributed for round 3. in round 3, each participant received a personalized survey link. panelists were reminded of their response to items in round 2, and provided with the median collated response of the other participants. we obtained 17/29 response for round 1 (see table 2 ). one invited expert circulated the online survey to another respondent who was later excluded bringing the total to 16/29 (55%) (see fig 1) . round 1 comprised of 4 (25%) female and 12 (75%) male participants. respondents differed from invited non-respondents in terms of gender: initial invitations were extended to 11 (38%) females and 18 (62%) male experts. all 16 panelists in round 1 held an md (n = 11, 69%), phd (n = 9, 56%), or both (n = 1, 6%) (also see: acknowledgments). responses to round 1 were translated into itemized lists of statements. as a result of this process, the was survey was expanded into 57 items arranged into 7 sections: (i) diagnostic accuracy (10 items), (ii) healthcare disparities (5 items), (iii) empathic care of patients (8 items), (iv) access to care (9 items), (v) primary care workforce (7 items), (vi) technological advancements in primary care (10 items), and (vii) the long-term future of the profession (8 items) (see s2 appendix). the panel was repeatedly prompted to forecast changes to primary care by 2029, and questions emphasized that predictions should be restricted to the us context. throughout the survey experts were reminded to "predict what you believe will happen and not what you personally would like to see happen". after completing each section, participants were also invited to provide free text comments, and following completion of the survey, offered to provide any additional feedback. in round 2, 13/16 experts participated in the online survey (response rate of 81%) [see s3 appendix, for round two raw data]. in round 3, 13/13 experts responded (response rate of 100%). in rounds 2 and 3, participants included 4 (31%) females, and 9 (69%) male participants (see table 2 for demographic information). table 3 presents the item means and standard deviations for item responses, and also indicates the items that reached consensus in round 2, those that obtained consensus in round 3, and items that failed to secure expert consensus. as described in the methods, and as indicated in table 3 , items reflect three different 7-point likert scales. to undertake interpretation of panelists' predictions, these items were divided into three rational, a priori categories. for the scale identified as 'i', responses were bounded into items that experts expected to increase (item mean of 4.5 and greater), remain about the same (item mean of 3.5-4.4), and to decrease (item mean of 3.4 and less). for the scale identified as 'l', responses were differentiated into items that the experts predicted to be likely (item mean of 4.5 and greater), items that they were uncertain about (item mean of 3.5-4.4), and those they predicted to be unlikely (item mean of 3.4 and less). finally, for the scale identified as 'a', responses were bounded into items about which the panel agreed (item mean of 4.5 and greater), those that they were neutral on (item mean of 3.5-4.4), and those items about which they disagreed (item mean of 3.4 and less). computer science / informatics 6 (38%) 5 (39%) academia 13 (81%) 10 (77%) n-count, m-average value per sample, %-percentage of the sample, rounded to the nearest whole value, sd-standard deviation. average value and sd were calculated only for age. 1 round 1 calculations exclude the one non-eligible respondent. 2 questions for which some participants selected more than one option. https://doi.org/10.1371/journal.pone.0239947.t002 themes primary care workforce. informaticians disagreed with the prediction that in the us "there is a 90% chance primary care doctors will be obsolete 100 years from now". panelists also agreed that primary care in the us would be one of the last specialties to be replaced by ai/ml. in the short-term, by 2029 in the us, experts forecast that advancements in ai/ml will incur a number of workforce changes in primary care (see fig 2) . table 3 . iqr, mean, and sd for round 2 & 3. access to care dependent on educational background some contrasting predictions emerged (see table 4 ). beyond 2029, experts without a medical degree (md) considered it likely that primary care doctors would always be needed to deliver empathic aspects of care-a prediction strongly agree. 2 while there is some ambiguity between how to interpret the difference between these statements, we retained them to preserve the predictions as submitted by our experts. 3 count and percentage of 'yes' responses. 4 these items were later omitted since, on further reflection it as unclear what might be meant by the term "required". values in bold indicate consensus statements. iqr-interquartile range, m-mean, sd-standard deviation. https://doi.org/10.1371/journal.pone.0239947.t003 that did not engender consensus among panelists with a medical degree. similarly, panelists without a medical education strongly agreed that the adoption of ai/ml tools in us healthcare will be slow, by 2029, due to the culture of medicine while those with a medical education did not reach consensus on this item. conversely, experts with a medical degree forecast that by 2029 us doctors will transition from the role of dispensers of knowledge to managing teams and information systems; however, there was no consensus on this item among participants without an md. diagnostic accuracy. overall, our experts forecast that by 2029 ai/ml will increase rates of diagnostic accuracy especially for conditions where the markers of illness are relatively homogenous (see fig 3) . among their predictions, panelists envisaged that by ai/ml tools will improve diagnostic accuracy among persons with limited access to human experts, individuals identifying as from minority groups, or for those with rare conditions. access to care. as a result of the disintermediation of physicians' expertise, our experts predicted that by 2029, ai/ml will increase access to primary care in the us (see fig 4) . comparisons of ratings between participants with and without a medical education resulted in some divergence. respondents without an md predicted that, by 2029 as a result of ai/ml, patient access to medical care in the us will lag behind other developed countries; participants with an md did not reach consensus on this item (see table 4 ). empathic care of patients. experts envisaged that by 2029 in the us, the availability of ai/ml tools will help to augment levels of empathic care (see fig 5) . panelists were divided on whether, by 2029, ai will offer direct resources for delivering empathic care to patients (see table 4 ). participants with an md considered this unlikely, while others failed to reach consensus on this item. the collective forecasts of medical informaticians have been missing from discussions about how al/ml will influence the short-term future of primary care (see box 1) . in this delphi poll there was consensus that in the next decade in the us, ai/ml will engender training and primary care work forces changes, improve rates of diagnostic accuracy, and increase access to primary care. economists forecast that in the coming decades, ai/ml will revolutionize the workplace [35, 36] . taking a long view, informaticians in this delphi poll predicted that 100 years from now it is unlikely that primary care doctors will be obsolete. panelists further envisaged that primary care will be one of the last medical specialties to be displaced by technology. however, in the short term, by 2029, our experts did foresee workforce and training changes in us primary care as a result of ai/ml. experts were collectively uncertain about whether ai/ml tools would enable lower-level clinicians to do higher level jobs, though it was not clear whether this prediction was driven by technological or regulatory considerations. panelists anticipated a shift towards computing and engineering in the educational background of students entering medical school in 2029, and increased training demands on medical students to work with ai/ ml in healthcare. however, the survey did not reveal whether experts perceived there to be risks of physicians using ml/al tools without a computing and engineering background, or indeed, an ethics or evidence-based perspective, on these techniques. the panel's predictions on education trends should also be observed against the currently limited debate about the need for curricular changes in medical education [37] [38] [39] . for many reasons, including financial, social, and geographical, timely access to primary care in the us remains a considerable problem. compounding matters, with fewer medical students entering primary care, inefficiencies, and demographic changes-an ageing population, and more people suffering from chronic conditions for longer-it is widely envisaged that ambulatory medicine will become increasingly strained [40, 41] . the results of this delphi poll suggest that ai/ml tools may help to address some of these challenges. experts envisaged that by 2029 there would be increased access to care via ai/ml-enabled tools for medical triage and routine patient self-diagnosis, and with the growth of telemedicine. the panel also predicted increasing medical precision. by 2029, experts envisaged that the use of ai/ml-enabled tools among patients will help to reduce diagnostic errors both for diseases with homogenous symptoms, and for more difficult medical cases. perhaps contributing to these reductions, experts anticipated that advancements in ai/ml will engender revisions in disease classifications. these positive predictions should be viewed against current evidence that diagnostic error is both common and harmful. in the us, recent estimates suggest a diagnostic error rate of 13-15% affecting the lives of around 12 million americans annually, contributing to 10% of all deaths, and the highest proportion of medical malpractice claims [42] [43] [44] . patients from racial and ethnic minorities, and those on low-incomes, are at higher risk of diagnostic error [45] . our experts predicted that diagnostic accuracy will increase for individuals with limited access to care, minorities, or patients with rare conditions. while there are currently considerable concerns about the potential for algorithmic biases to be baked into ai/ml tools, driven in part by the underrepresentation of underprivileged demographic groups in training phases of machine learning [46] , there was consensus among our delphi panel that data collection in 2029 will be more representative of minority populations. this prediction may help to explain why the panel anticipated improved diagnostic accuracy for minorities. nonetheless, experts were less optimistic that ai/ml will narrow health disparities in the us by 2029. current findings point to a "digital divide" in healthcare. many factors drive current differential usage of digital health innovations including costs, lack of broadband access, and lower levels of digital and health literacy among underprivileged populations [47, 48] . research also suggests that in the us, health app usage is more common among people who are younger, better educated, on a higher income, or in better health [49] . our panel predicted that us healthcare will become increasingly productized. although the poll provided no causal explanations for this prediction, in a growing health app economy, experts may have anticipated that disadvantaged patients will continue to be less likely to adopt ehealth tools. in addition, there was consensus that private hospitals will have greater access to ai/ml-enabled resources to improve diagnostic accuracy than public hospitals. existing structural disparities in care may also have been perceived to be a factor that will perpetuate inequities in ehealth. our delphi poll provided nuanced forecasts on the theme of physician empathy. there was collective consensus that, by 2029, ai/ml would not free up more time with patients in us primary care; however, experts did forecast that levels of empathy in primary care would increase in this time period. the survey did not fully illuminate the reasons for this but there was consensus that ai/ml-enabled tools will assist physicians in shared decision-making, and help provide information on patients' lifestyles and the social determinants of individuals' health. conceivably, the panel may have envisaged that such data might enhance physicians' personal knowledge about patients thereby fostering more empathic care. again, these views appear to differ subtly from those of physicians. in qualitative research a common prediction among physicians is that, by liberating health professionals from administrative tasks, ai/ml will indirectly facilitate more time with patients thereby enhancing levels of empathy [11, 15] survey research also indicates skepticism among physicians that ai/ml will be able to directly substitute for, or augment clinicians, in the provision of empathic care [10] [11] [12] 15] . in terms of physicians' responsibilities, experts did not envisage that ai/ml will help to reduce documentation burdens by 2029 [10, 12] . this prediction contrasts with the more optimistic opinions of surveyed physicians. for example, in 2019, a global survey of psychiatrists found that the majority (83%, 657/791) judged it likely that future technology will fully replace physicians in the task of documentation with 84% (552/657) of these respondents predicting that this will happen in the next 10 years [12] . similarly, in 2018, survey research conducted among primary care physicians in the uk revealed comparable results: most uk general practitioners (80%, 578/720) anticipated that future technology will fully replace humans in the task of documentation with 79% (458/578) of these respondents believing that this will happen in the next decade [10] . finally, experts in this delphi poll did not weigh in on specific policy, legal, or ethical issues in relation to the impact of ai/ml on primary care. however, there was consensus that by 2029 regulatory issues will pose greater challenges than technical problems. to our knowledge this is the first delphi poll to explore experts' predictions about the shortterm effects of ai/ml on a medical specialism. major strengths of the survey were the high response rates between rounds, and the diversity of participants. although only around one third of round 3 panelists were female (31%), currently around 25% of health it leaders in the us are women [50] . the expert panel comprised leading health informaticians around half of whom also had a medical background. panelists were drawn from diverse backgrounds, nationalities, and ethnicities including 3 participants in round 1 who do not reside in the us but who are knowledgeable about the us healthcare system. we also note that the majority of experts primarily held allegiances to academia, and medicine, rather than industry; nonetheless, this may have been a strength rather than a limitation, resulting in more modest predictions. this survey has several limitations. as with all delphi polls, there is no guarantee of accuracy in forecasts. no standardized guidelines exist for identifying, excluding, or selecting suitable experts from the field of interest [23, 27] . reliability of predictions is dependent on the specialist knowledge of the participants which can be influenced by norms and values, motivational biases, and stakeholder interests [51, 52] . although there was strong consensus among our panel of experts, we noted some divergence in opinions between participants with and without a medical degree. conceivably, professional medical allegiances may have affected predictions; overall, however, we cannot speculate on how the composition of our panel strengthened or diminished the quality of predictions. whilst participant retention rates between rounds were high, the number of panelists was limited, and more participants in the first round may have resulted in different consensus opinions [53, 54] . importantly, two events arising in the immediate period after data collection-one global and one in the us context-may affect the reliability of the delphi poll. the coronavirus pandemic has (and currently is) exerting a significant impact on the delivery of primary care in the us. driven by this crisis, current evidence shows a substantial uptick in demand for telemedicine consultations, and in the use of ai/ml-driven triage tools [55, 56] . although it is too early to predict with certainty whether increase in these applications will persist after the pressure on frontline medicine has abated, it seems possible that our experts' forecasts on the influence of ai/ml on access to care may be especially well supported. second, the survey was administered prior to the finalized ruling, in march 2020, by the national coordinator of health information technology (onc) on the 21 st century cures act [57] . designed to maximize innovation in healthcare by creating a competitive health app economy, this federal ruling sets out technical standards about how data must be shared, mandating patients' right to access their digital medical records. while the final ruling may have been anticipated by some of our experts in the months preceding the announcement, we cannot be certain about whether or how its publication might otherwise have influenced consensus predictions of our participants. however, we suggest that uncertainty prior to the ruling may have fostered more cautious predictions about the impact of ai/ml on primary care among our experts. a good hockey player plays where the puck is. a great hockey player plays where the puck is going to be. this delphi poll provides the consensus predictions of leading health informaticians on the impact of ai/ml on primary care in the us. the panel forecast that, in the long-term (100 years from now) primary care doctors will not be obsolete, and furthermore, that general medicine will be one of the last medical specialties to be displaced by technology. by 2029 in the us, however, experts did forecast that ai/ml will exert an impact on the delivery and quality of primary care. specifically, the panel predicted increased rates of diagnostic accuracy including for the most disadvantaged patient populations, greater access to primary care, and enhanced levels of empathic patient care. against the panel's forecast that healthcare in the us would be increasingly productized, there was consensus that regulatory issues will pose greater challenges than technical ones in improving diagnostic accuracy. experts were also less optimistic about the prospects of ai/ml to precipitate other desirable short-term changes in medicine. by 2029 in the us, the panel predicted that ai/ml would not narrow healthcare disparities, reduce documentation burdens on primary care physicians, or increase the total time spent with patients. in the next decade, experts forecast increased ai/ml training requirements for medical students. the central goal of delphi polls is expert prediction. however, forecasts can also help us to exert control over the future by facilitating forward planning, and focusing attention on where, and how, relevant actors might intervene to create preferable outcomes. innovations in digital care pose myriad practical, ethical, and regulatory issues including (but by no means limited to): the creations of standards for assessing the reliability and approval of medical algorithms and apps, questions about patient privacy, and the security of patients' online health information [58, 59] . in reviewing these findings we are struck by the contrastive predictions of our experts with those of surveyed physicians [10, 11] . as others have noted, medical schools have been slow to adapt curricula and offer courses aimed at promoting ai/ml literacy among students [37] [38] [39] . we conclude that to empower both physicians and patients, and to rise to the challenges of the next decade, it is incumbent on the medical community, health and medical educators, and policy-makers to take action to improve digital literacy both among patients and our current and future health professionals [59] . the fate of medicine in the time of ai a glimpse of the next 100 years in medicine virtual care for improved global health can mobile health technologies transform health care? deep learning-a technology with the potential to transform health care machine learning and the profession of medicine the evolution of patient diagnosis: from art to digital data-driven 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misdiagnosis-related harms in malpractice claims: the "big three"-vascular events, infections, and cancers unequal treatment: confronting racial and ethnic disparities in healthcare. national academies press genetic misdiagnoses and the potential for health disparities association between patient portal use and broadband access: a national evaluation are patients electronically accessing their medical records? evidence from national hospital data differences in access to and preferences for using patient portals and other ehealth technologies based on race, ethnicity, and age: a database and survey study of seniors in a large health plan what 3 charts say about women in health it leadership roles, salary and the rise of chief nursing officers trusting judgements: how to get the best out of experts rethinking expertise a general framework for analysing diversity in science, technology and society evidence for a collective intelligence factor in the performance of human groups telemedicine gets a boost from coronavirus pandemic: medicare patients get more flexibility in seeking remote treatment. the wall street journal covid-19 transforms health care through telemedicine: evidence from the field office of the national coordinator for health information technology (onc), department of health and human services (hhs). 21st century cures act: interoperability, information blocking, and the onc health it certification program: final rule machine learning in medicine: addressing ethical challenges the age of surveillance capitalism: the fight for a human future at the new frontier of power: barack obama's books of 2019 it is standard practice in delphi polls to acknowledge the panel of experts who shared their valuable insights. we are indebted to them for giving up their time. the following participants conceptualization: charlotte blease. key: cord-284501-5i0w74q4 authors: armesto, maria; cavanagh, dave; britton, paul title: the replicase gene of avian coronavirus infectious bronchitis virus is a determinant of pathogenicity date: 2009-10-09 journal: plos one doi: 10.1371/journal.pone.0007384 sha: doc_id: 284501 cord_uid: 5i0w74q4 we have previously demonstrated that the replacement of the s gene from an avirulent strain (beaudette) of infectious bronchitis virus (ibv) with an s gene from a virulent strain (m41) resulted in a recombinant virus (beaur-m41(s)) with the in vitro cell tropism of the virulent virus but that was still avirulent. in order to investigate whether any of the other structural or accessory genes played a role in pathogenicity we have now replaced these from the beaudette strain with those from m41. the recombinant ibv was in effect a chimaeric virus with the replicase gene derived from beaudette and the rest of the genome from m41. this demonstrated that it is possible to exchange a large region of the ibv genome, approximately 8.4 kb, using our transient dominant selection method. recovery of a viable recombinant ibv also demonstrated that it is possible to interchange a complete replicase gene as we had in effect replaced the m41 replicase gene with the beaudette derived gene. analysis of the chimaeric virus showed that it was avirulent indicating that none of the structural or accessory genes derived from a virulent isolate of ibv were able to restore virulence and that therefore, the loss of virulence associated with the beaudette strain resides in the replicase gene. avian infectious bronchitis virus (ibv) is a member of the genus coronavirus, family coronaviridae, order nidovirales [1, 2] . together with the genetically closely related turkey coronavirus [3] [4] [5] [6] , pheasant coronavirus [7] , and recently identified coronaviruses from several species of wild birds [8, 9] , a beluga whale [10] and an asian leopard cat [11] form the group 3 coronaviruses. ibv is a highly infectious pathogen of domestic fowl that replicates primarily in the respiratory tract but also in epithelial cells of other organs, including the gut, kidney and oviduct [12] [13] [14] , and is the causative agent of infectious bronchitis, a disease that is responsible for economic losses in the poultry industry throughout the world [15] . coronaviruses are enveloped viruses that replicate in the cell cytoplasm and contain a single-stranded, positive-sense rna genome of 28 to 32 kb [16] . ibv has a 27.6 kb rna genome and like all coronaviruses contains the four structural proteins; spike glycoprotein (s), small membrane protein (e), integral membrane protein (m) and nucleocapsid protein (n) which interacts with the genomic rna. all coronaviruses also encode a set of accessory protein genes of unknown function that are not required for replication in vitro [17] [18] [19] [20] [21] [22] [23] , but may play a role in pathogenesis [19, 22] . ibv encodes two accessory genes, genes 3 and 5, which both express two accessory proteins 3a, 3b and 5a, 5b, respectively. in addition to the structural and accessory genes, two-thirds of a coronavirus genome comprises the replicase gene, which expresses two polyproteins, pp1a and pp1ab, in which pp1ab is an extension product of pp1a as a result of a -1 ribosomal shift mechanism. the two polyproteins are cleaved by two types of virus-encoded proteinases usually resulting in 16 non-structural proteins (nsp1-16); ibv lacks nsp1 thereby encoding nsp216. we have previously shown that the ibv accessory genes are not required for replication in vitro [17, 18] ; however, we could not determine any role of the ibv accessory proteins in pathogenicity as our reverse genetics system is based on the avirulent beaudette strain. replacement of the beaudette s gene with the corresponding m41 s gene sequence altered the tropism of the ribv but did not result in a change in virulence [24] [25] [26] . this implied that although the ibv s gene may play a role in virulence, associated with tropism, expression of an s gene from a virulent strain alone was not sufficient to alter the avirulent phenotype associated with beaudette. infectious bronchitis is mainly controlled by the use of live attenuated vaccines derived from virulent viruses by multiple serial passages, usually greater than 50 passages, in 10-11-day-old embryonated chicken eggs [13, [27] [28] [29] . as a consequence of this process the virus becomes more adapted for the embryo, reflected by more efficient replication and higher pathogenicity for the embryo, with concomitant attenuation for chickens and in some cases loss of immunogenicity. however, the mutations associated with attenuation of pathogenicity for the chicken are unknown and variable leading to differing efficacies associated with different vaccines. our previous studies have shown that replacement of the s gene from the avirulent beaudette isolate with that from a virulent virus (m41) did not restore virulence but did alter the tropism of the ribv and restore immunogenicity for subsequent challenge with m41. previously beaudette had been considered to be poorly immunogenic and never used as a vaccine strain [30] . in this study, we describe the generation of recombinant ibvs that consisted of the replicase gene from the avirulent beaudette strain and the structural and accessory genes from the virulent m41 isolate of ibv, to determine whether the replicase or the combination of the structural and accessory genes of ibv play a role in pathogenesis. the growth of ibv in chick kidney (ck) cells was as described previously [31] [32] [33] . the ibv isolates used were: (1) beaudette-ck (beau-ck; [34] ), a virus adapted for growth in ck cells that can grow on but has not been adapted for growth in vero cells, an african green monkey cell line; (2) beau-r, a recombinant ibv (ribv) produced from a full-length cdna of beau-ck using our ibv reverse genetics system [35] ; and (3) m41-ck, an isolate derived from m41 [36] following adaption to growth on ck cells. both the beaudette and m41 strains of ibv belong to the same, massachusetts, serotype. all ibv strains were titrated in ck cells. vaccinia viruses were grown and titrated on vero cells and large stocks for dna isolation were grown in bhk-21 cells [37] . all nucleotide and amino acid residue numbers refer to the positions in ibv beau-r [35] accession n o aj311317. the region of the ibv m41 genome corresponding to the structural, accessory genes and the 39-utr was ligated onto the last 1416 nt of the beau-r replicase gene and inserted into hindiii and sali digested pgpt-neb193 [25] . the resulting plasmid, pgpt-beaur-rep-m41-struct-3utr, consisted of the 39-end of the replicase gene of beau-r, and the region of the m41-ck genome encoding the structural and accessory genes terminated by the m41-ck derived 39utr (fig. 1 ). the ibv cdna within pgpt-beaur-rep-m41-struct-3utr was introduced, by homologous recombination using the transient dominant selection (tds) ( [25, 37] ), into the ibv beaudette cdna within the vaccinia virus genome in rvv-beaur-rep-dstruct containing beau-r-derived sequence corresponding to the replicase gene followed by the first 376 nt of the s gene, part of the n gene and the 39-utr (fig. 1) . briefly, 50% confluent monolayers of vero cells were infected with rvv-beaur-rep-dstruct, containing the beau-r cdna sequence, at a moi of 0.2 and transfected 2 h later with 5 mg of pgpt-beaur-rep-m41-struct-3utr in lipofectin (invitrogen). resultant phenotypically gpt + rvvs were selected by three rounds of plaque purification using vero cells in the presence of 25 mg/ml mycophenolic acid (mpa), 250 mg/ml xanthine and 15 mg/ml hypoxanthine. randomly selected mpa resistant rvvs were grown and plaque purified three times using vero cells in the absence of selection medium. this resulted in a second recombination event (see fig. 1 ) involving the loss of the gpt gene from the rvvs, and either generation of an ibv cdna corresponding to the sequence in rvv-beaur-rep-dstruct or a full-length ibv cdna consisting of a beau-r replicase gene and the rest of the ibv genome derived from ibv m41-ck within the vv genome (fig. 1) . pcr was used to confirm the absence of the gpt gene in resulting rvvs, which were further screened by pcr amplification of the ibv 39-utr, using oligonucleotides bg56 (59-26941 caacagcgcccaaagaag 26958 ) and 93/100 (59-27607 gctctaactctatactagcct 27587 -39), part of the replicase gene, using oligonucleotides bg40 (59-18941 atc-taatttgctcgttca 18958 -39 and bg128 (59-19720 cgccac-tcctttgtcgcttc 19739 -39, and the junction of the replicase and s genes using oligonucleotides bg40 (59-18941 atctaatt-tgctcgttca 18958 -39 and bg134 (59-21398 agcaatt-gaaactgaaagtg 21417 -39. oligonucleotides bg-56 and 93/100 were used to discriminate between the 39-utrs from beau-r and m41 derived sequences; the 39-utr from beau-r results in a 667 bp product but a 483 bp pcr from m41-ck, due to a 184 nt deletion in the m41 39-utr. a rvv, rvv-beaur-rep-m41-struct, that contained a full-length ibv cdna consisting of the replicase gene from beau-r and the rest of the genome derived from ibv m41-ck was identified and used for further work. recombinant vaccinia virus dna from rvv-beaur-rep-m41-struct containing the beaur-rep-m41-struct chimaeric fulllength ibv cdna was purified and used for the rescue of ribvs in ck cells using rfpv/t7 [38] for the generation of infectious ibv rna ( [25, 35, 37] ). resultant chimaeric ribvs were passed three times in ck cells before being used in subsequent experiments. total cellular rna was extracted from ibv-infected ck cells using the rneasy method (qiagen) and rt-pcr (ready-to-go tm rt-pcr beads) for amplification of the 39-utr of the ribv-derived rna, using oligonucleotides bg 56 and 93/100, to confirm the identity of the ribv. confluent monolayers of ck cells in 6-well plates were infected with viruses at a moi of 0.1 pfu in triplicate for each time point. following attachment, for 1 h at 37uc, the cells were washed twice with phosphate-buffered saline (pbs) to remove residual virus and incubated at 37uc. samples of media were collected at 24, 48, 72 and 96 h post-infection and assayed in triplicate for progeny virus by plaque assay using ck cells. chicken tracheal organ cultures (tocs) were prepared from 19-day-old specific pathogen free (spf) rhode island red chicken embryos [39] . groups of five tocs, in triplicate, were inoculated with 0.5 ml of medium containing 5.4610 4 pfu/ml of each virus. after incubation at 37uc for 1 h, the inoculum was removed and the tocs were washed three times with pbs, 1 ml of medium was added and the tocs were incubated at 37uc until the samples were taken. at the selected time points medium from the tocs was removed and analysed for progeny virus by plaque titration on ck cells. all experiments were carried out in accordance with the uk home office guidelines using spf rhode island red chickens obtained from the poultry production unit of the institute for animal health. four groups (n = 12) of 1-day-old spf rhode island red chickens were inoculated via the conjunctival (eye drop) and intranasal routes with 10 6 pfu/ml of each virus in a total of 0.1 ml serum-free bes (n,n-bis(2-hydroxyethyl)-2aminoethanesulphonic acid) medium. the chickens were housed in positive-pressure, hepa-filtered isolation rooms, and each group was housed in a separate room. the birds in the mockinfected group were inoculated with serum-free bes medium. the ibv-associated clinical signs used to determine pathogenicity were snicking, tracheal rales (a sound emanating from the bronchi, also detected by vibrations when holding a chick), wheezing (dyspoena), nasal discharge, watery eyes and ciliary activity of the trachea [26] . chicks were observed daily for clinical signs; snicks (a sound similar to a sneeze) were counted by two persons over 2 minutes. birds were checked individually for the presence of tracheal rales, nasal discharge, watery eyes and wheezing. tracheas were removed from three randomly selected chickens from each group at 4 and 7 days post-inoculation for assessment of ciliary activity. ten 1 mm sections were cut from three different regions of each trachea and the level of ciliostasis of each tracheal section was determined. the remaining regions of the tracheas from the infected birds were cut longitudinally and the epithelial cells scraped from the tracheas and transferred to 1 ml pbs. the samples were analysed for the presence of viable ibv by titration in tocs or used for rna extraction using the rneasy method and analysed by rtthe m41-ck-derived cdna, representing the m41 structural and accessory genes and the m41 39-utr, within pgpt-beaur-rep-m41-struct-3utr was fused to the beau-r replicase gene in the rvv by a homologous recombination event between the beau-r replicase sequence common to both constructs. a potential rvv, rvv-beaur-rep-m41-struct, containing a full-length ibv cdna with the replicase gene from beau-r and the rest of the genome from m41-ck was isolated following the tds process. the complete plasmid dna was fused to the truncated beau-r cdna by a singlestep homologous recombination event; via the beau-r replicase sequence common to both sequences. the initial resultant rvvs had a gpt + phenotype allowing selection in the presence of mycophenolic acid. removal of mycophenolic acid resulted in two types of spontaneous intramolecular recombination events, due to the instability of the ibv cdna with tandem repeats of similar sequences, resulting in either rvv-beaur-rep-d-struct (no modification) or in rvv-beaur-rep-m41-struct; the desired rvv. both recombination events resulted in the loss of the gpt gene. the ibv genes representing the structural and accessory genes are shown along with the 39-utrs and igr sequences, a potential recombination event is indicated between the 1416 nt of the beau-r replicase gene sequence common to both constructs. doi:10.1371/journal.pone.0007384.g001 pcr using oligonucleotides bg56 and 93/100 to determine the identity of the 39-utr. confluent monolayers of ck cells were used for serial passage of rbeaur-rep-m41-struct 25 times. briefly, cells were infected with the ribv and 24 h post-infection medium was collected, diluted 1:10 and used to infect a new monolayer of ck cells. this process was repeated until passage 25 (p 25 ). total rna was extracted from the p 25 infected ck cells and rt-pcr was used to generate a series of overlapping pcr products covering the complete genome of the p 25 ribv. the rt-pcr products were sequenced using a variety of oligonucleotides, derived from the beau-ck sequence [40] . assembly of the sequences was performed using gap4 of the staden sequence software programs [41] . generation of chimaeric ribvs with the replicase gene from beaudette and the rest of the genome m41-ck a full-length ibv-derived cdna, within the vaccinia virus genome, was generated by homologous recombination using the tds method and consisted of the replicase gene from the apathogenic ibv strain beau-r and the structural and accessory genes plus the 39-utr from the pathogenic m41 strain of ibv. this was achieved using a beau-r-based receiver sequence consisting of the complete replicase gene, followed by the first 376 nt of the s gene fused to the n gene and 39-utr (fig. 1) . the donor sequence consisted of the last 1246 nt of the beau-r replicase gene fused to m41-ck-derived cdna from the s gene to the poly(a) tail ( fig. 1 ). following tds, dna was extracted from 20 rvvs, potentially containing the chimaeric ibv full-length cdna. analysis by pcr, using gpt specific primers to confirm the loss of the e. coli gpt gene, on six of the rvv dnas confirmed the loss of the gpt gene following the second tds recombination event (fig. 1 ). the ibv cdnas within these six rvv dnas were analysed (1) for the presence of the m41-ck-derived 39-utr sequence, which is 184 nt shorter than the beau-r 39-utr [42] , (2) the junction between the replicase and s gene using oligonucleotides bg40 and bg134 and (3) for the presence of the m41-ck-derived m gene using oligonucleotides bg52 (59-24945 gaatggtgttctt-tattg 24962 -39) and bg146 (59-25549 tctaacactc-taagttgag 25567 -39); to confirm that the second tds recombination event had not resulted in generation of the starting receiver sequence (fig. 1 ). two rvvs, rvv-beaur-rep-m41-struct-2 and rvv-beaur-rep-m41-struct-12, that did contain the m41-ck-derived 39-utr and m gene were further screened by spot sequence analysis of the ibv cdna to confirm that the region downstream of the beau-r replicase gene had been replaced with the corresponding sequence from m41-ck; the sequences were as expected for the required chimaeric ibv sequence. two infectious ribvs, rbeaur-rep-m41-struct-2 and rbeaur-rep-m41-struct-12, were recovered from dna extracted from rvv-beaur-rep-m41-struct-2 and rvv-beaur-rep-m41-struct-12, respectively, using ck cells, previously infected with rfpv/t7, to provide t7 rna polymerase, and co-transfected with the rvv dna and pci-nuc [35] . the transfected ck cells (p 0 ) were incubated until they showed a cytopathic effect (cpe), the medium was filtered to remove any rfpv/t7 and any potential ribv passaged three times more on ck cells (p 3 ). total rna from the infected p 3 ck cells was extracted and analysed by rt-pcr and spot sequence analysis. sequence analysis showed that rbeaur-rep-m41-struct-12 contained an extra adenosine nucleotide at position 25317 in a six base polyadenosine repeat sequence within the m41 intergenic region (igr) between the end of the m gene and start of gene 5. consequently only rbeaur-rep-m41-struct-2 from the p 3 ck cells was titrated in ck cells and used for further characterisation. sequence analysis of the m41-ck-derived sequence in rbeaur-rep-m41-struct-2 revealed three nucleotide changes when compared to the sequence in rvv-beaur-rep-m41-struct-2 ( table 1 ). the nucleotide changes resulted in one amino acid change in the s protein, the other was a silent mutation and a single amino acid change in the n gene. comparison of the sequence indicated that the changes arose during rescue or during the first three passages of the rescued viruses. twelve one-day-old spf chickens, in four groups, were inoculated with 0.1 ml of 10 6 pfu/ml of rbeaur-rep-m41struct-2, m41-ck or beau-r or using 0.1 ml serum-free medium (mock infection) by eye-drop and intranasally. the birds were observed for clinical signs up to 11 days post-inoculation. at 4 and 7 days post-infection the tracheas of three randomly selected chickens from each group were examined for ciliary activity and presence of ibv. observations for clinical signs, snicking, wheezing and nasal discharge, of an ibv infection were carried out daily on each group from three days post-infection. only chickens inoculated with m41-ck showed any clinical signs of an ibv infection. as can be seen from figure 3, m41-ck induced high rates of snicking, which peaked by day 5 post-inoculation, whereas beau-r and rbeaur-rep-m41-struct-2 induced a much lower rate of snicking, similar to those observed for the mockinoculated chickens (fig. 3a) . similarly, only chickens (11-16%) inoculated with m41-ck showed any nasal discharge three, four and six days post-inoculation (fig. 3b) . again, wheezing was only observed in chickens inoculated with m41-ck, in which 90% of the infected chickens demonstrated wheezing by 7 days postinoculation (fig. 3c) . analysis of the tracheas isolated from the chickens showed that the mock, beau-r and rbeaur-rep-m41-struct-2 infected chickens had .95% ciliary activity whereas the chickens inoculated with m41-ck showed 0% ciliary activity (100% ciliostasis; fig. 3d ). in summary, our observations of the parameters used to assess pathogenicity demonstrated that rbeaur-rep-m41-struct-2 was not pathogenic and that it had the characteristics associated with beau-r rather than m41-ck. in order to determine whether there was any virus in the tracheas of the infected chickens, the epithelial cells were scraped from the tracheas taken from three chickens from each group four days post-infection and any virus present titrated in tocs. the average titre of m41-ck, isolated from the tracheas of the three chickens, was 4.1 log 10 cd 50 /ml, in contrast no detectable virus was isolated from the tracheas taken from the chickens infected with either beau-r or rbeaur-rep-m41-struct-2. this result indicated that if any virus was present in the tracheas of the chickens infected with beau-r or rbeaur-rep-m41-struct-2 the amounts present were at least 4 log 10 cd 50 /ml lower than the m41-ck detected from the tracheas of the chickens infected with m41-ck. analysis of the tracheal epithelial cells isolated from the infected chickens, for the presence of ibv by titration on tocs, had indicated that either there was no beau-r or rbeaur-rep-m41-struct-2 present or that the levels of both viruses were below detection. our previous results had shown that beau-r was not reliably detected in the tracheas of beau-r in infected chickens whereas m41-ck is detectable [26, 43] . to check for the presence of m41-ck-, beau-r-or rbeaur-rep-m41-struct-2-derived rna in the tracheal epithelial cells from the infected chickens, total rna was isolated from the epithelial cells and analysed by rt-pcr using oligonucleotides bg56 and 93/100, corresponding to the 39-utr of the ibv genomes. as can be seen from fig. 4 , the tracheal epithelial cells isolated from chickens infected with m41-ck were positive for the presence of m41-ck-derived rna. in contrast, no ibv-derived rna was detected in the tracheal epithelial cells of chickens infected with either beau-r or rbeaur-rep-m41-struct-2 supporting our virus isolation data. furthermore, sequence analysis of the ibv-derived rt-pcr product amplified from the tracheal epithelial cells of chickens infected with m41-ck confirmed that it was derived from ibv m41-ck. this was consistent with previous results suggesting that beau-r probably does not reach the tracheas of chickens infected by the eye-drop and intranasal routes and that similarly rbeaur-rep-m41-struct-2 did not appear to reach the epithelial cells of the tracheas of infected chickens. a ribv, beaur-m41(s) [24] , which consisted of the beau-r genome but with an m41-derived s gene, had the tropism of m41 in vitro but the characteristics of beau-r in vivo indicating, together with our rbeaur-rep-m41-struct-2 result, that the inability of beau-r-derived viruses to reach the tracheas of infected chickens resides within the replicase gene. infection of tracheal epithelial cells ex vivo is a method of growing ibv strains that have not been adapted for growth either in embryonated eggs or primary cell cultures and results in cessation of ciliary activity (ciliostasis) of the epithelial cells. previous work demonstrated that our ribvs, beau-r and beaur-m41(s), caused ciliostasis of infected tocs with concomitant production of progeny virus [26] . analysis of tracheal epithelial cells from chickens infected with ribv-beaur-rep-m41-struct-2 had shown that there was no virus present. therefore, we decided to investigate the replication of rbeaur-rep-m41-struct-2, for comparison with beau-r and m41-ck, ex vivo in tocs. progeny viruses in toc medium taken from each group of infected tocs at the specific time points were titrated by plaque assay on ck cells to investigate the growth kinetics of the three ibvs in tocs. as can be seen from fig. 5 all three viruses replicated in the tocs. although the viruses reached maximum titres by 24 h postinfection, the titre of rbeaur-rep-m41-struct-2 was between 1-2 log 10 units lower than the titres observed for m41-ck and beau-r, respectively, at this and most later time points. interestingly, although rbeaur-rep-m41-struct-2 replicated and produced following rescue of rbeaur-rep-m41-struct-2 all growth experiments were carried out using virus that had been passaged three times (p 3 ) in ck cells. we routinely use the p 3 -derived ribvs so that there is a lower probability that changes will have occurred within the virus genome, as with other positive strand rna viruses multiple passage will result in changes in both nucleotides and amino acids; for example the acquisition of the extra adenosine residue in rbeaur-rep-m41-struct-12. the ribv, rbeaur-rep-m41-struct-2, showed slightly reduced growth properties when compared with both parental viruses, beau-r and m41-ck. surprisingly, although rbeaur-rep-m41-struct-2 grew in tocs it did not cause ciliostasis, which is induced by both parental viruses. due to the chimaeric nature of the ribv the 59-utr, including the leader sequence, corresponds to beau-r, with the 39-utr derived from m41-ck. there are known nucleotide differences in these regions of the ibv genome between the two viruses ( [33, 42] . this introduces the possibility that if the two utrs interact this could affect the growth properties of the the growth characteristics of rbeaur-rep-m41-struct-2-p 25 were initially examined in ck cells and compared to rbeaur-rep-m41-struct-2-p 3 . as can be seen from fig. 6a the peak titre of rbeaur-rep-m41-struct-2 -p 25 was higher, approximately 0.5 log 10 unit, than for rbeaur-rep-m41-struct-2 -p 3 at 24 h post-infection and remained higher throughout the time course. subsequently, the growth properties of rbeaur-rep-m41-struct-2-p 25 were then studied in tocs and as can be seen from fig. 6b rbeaur-rep-m41-struct-2-p 25 ribv grew to a higher titre than rbeaur-rep-m41-struct-2-p 3 , reaching a 1 log 10 unit difference by 72 h postinfection, although the two viruses had a similar titre by 96 h postinfection. however, analysis of tocs infected with rbeaur-rep-m41-struct-2-p 25 also showed that this virus did not cause ciliostasis. in order to determine whether any nucleotide changes may have been responsible for the observed phenotypic changes of the p 25 ribv we decided to initially sequence the 59-and 39-utrs of rbeaur-rep-m41-struct-2-p 25 for comparison to the corresponding sequences of rbeaur-rep-m41-struct-2-p 3 and the two parental viruses beau-r and m41-ck. no nucleotide changes were found within the 59-and 39-utr sequences of the rbeaur-rep-m41-struct-2-p 25 virus indicating that the change in growth pattern was not due to one of the two utrs changing to the other parental sequence. as a result of this finding we sequenced the complete ribv rbeaur-rep-m41-struct-2-p 25 genomic sequence and identified only three other nucleotide changes (table 1 ) in addition to those identified within rbeaur-rep-m41-struct-2-p 3 . these new nucleotide differences corresponded to a single amino acid change in the s gene and two nucleotide changes within orf 5b of gene 5 when comparing the m41-ck-derived sequences within rbeaur-rep-m41-struct-2-p 3 and ribv rbeaur-rep-m41-struct-2-p 25 sequences; indicating that these changes arose on further passage of rbeaur-rep-m41-struct-2-p 3 . the two nucleotide changes within the orf 5b sequence, nucleotides 25815 and 25816 (uuraa), resulted in the introduction of a premature stop codon in orf 5b and a modification of the n gene transcription regulatory sequence (trs) from uucuuaa-caa to aacuuaacaa, the latter sequence being the more predominant ibv trs. sequence analysis of the rbeaur-rep-m41-struct-2 viruses between passages p 3 and p 25 (fig. 7) indicated that at p 5 , the aa mutation was more prominent than the parental (uu) sequence and that in subsequent viruses, derived from passages p 10 , p 15 , p 20 and p 25 , the original parental sequence gradually decreased and disappeared from detection. our previous work on the swapping of the s gene from the avirulent beaudette (beau-r) strain of ibv with an s gene derived from the pathogenic m41-ck isolate of ibv showed that although the s gene was responsible for tropism it was not responsible for virulence [24] [25] [26] . although our results showed swapping of the s gene did not result in virulence we could not rule out the fact that other ibv structural (e, m and n) and accessory (3a, 3b, 5a and 5b) genes may also play a role and therefore be required for the acquisition of virulence. we therefore decided to exchange the region of the beau-r genome from the end of the replicase gene to the poly(a) tail with the corresponding sequence from m41-ck to investigate whether exchange of the structural and accessory genes was sufficient to confer pathogenicity to a resultant chimaeric ribv, rbeaur-rep-m41-struct. such an approach would also allow us to determine whether or not the replicase gene plays a role in the pathogenicity of ibv. ibv beaudette is a well known apathogenic lab strain of ibv that was attenuated by multiple, several hundred, passages in 11-day-old embryonic chicks [30] , therefore loss of virulence could have resulted in multiple changes throughout the genome. ibv beau-r is a molecular clone of beaudette-ck [35] ) that is also avirulent in chickens [26] . the objective of this study was to determine whether replacing the beau-r structural and accessory genes with those from virulent m41-ck would result in a ribv that was virulent when compared to beau-r. we have used our ibv reverse genetics system [24, 25, 35] to produce chimaeric ibv, rbeaur-rep-m41-struct, consisting of the replicase gene from beaudette (beau-r) and the s, 3a, 3b, e, m, 5a, 5b, n and the 39-utr from m41-ck. the m41-ck-derived structural and accessory gene sequence was fused to the beau-r replicase by homologous recombination using the tds method [25, 37] and ribvs were rescued in ckcs. the m41-ck-derived region, in addition to the structural and accessory genes in common with beau-r, also contains an untranslated region, the intergenic untranslated region (igr), between the m and gene 5, which is 305 nt in beaudette and 350 nt in m41-ck. the m41-ck igr, like some other strains of ibv, contains a potential open reading frame (orf) of 285 nt potentially encoding a 94 amino acid product of 11 kd with the initiation codon immediately downstream of the m gene stop codon. a similar orf has been identified at a similar position in the genome of turkey coronavirus (tcov) [5, 6] , the only trs identified for tcov is 288 nt upstream of the potential orf, within the m gene, but with low identity to the tcov canonical trs. no sg mrna for this potential orf has been identified in tcov or ibv, including m41-ck, infected cells. the lack of a sg mrna, the long distance between the initiation codon and a potential trs and the loss of the potential orf, as a result of several deletions, in some strains of ibv indicates that the orf is probably a pseudogene. two ribvs, rbeaur-rep-m41-struct-2 and rbeaur-rep-m41-struct-12, were rescued from the rvvs, rvv-beaur-rep-m41-struct-2 and rvv-beaur-rep-m41-struct-12, respectively, and analysed for the presence of the m41-ck-derived sequence. analysis of rbeaur-rep-m41-struct-12 identified an extra adenosine nucleotide at position 25317 in a six base polyadenosine repeat sequence within the potential 11.5 kd orf in the m41-ck igr, which had the potential for inactivating this potential gene product. to rule out the possibility that the loss of this potential gene product could affect any pathogenicity of this virus we decided to proceed with rbeaur-rep-m41-struct-2, which had the correct sequence, for subsequent experiments. we have characterised rbeaur-rep-m41-struct-2-p 3 in vitro, ex vivo and tested its pathogenicity in vivo using one-day-old-chickens. the overall growth pattern of rbeaur-rep-m41-struct-2 in ckcs was more similar to that of beau-r (fig. 2) except it grew to a titre of about 1 log 10 less throughout the growth cycle. the main objective of this study was to determine whether swapping of the structural and accessory genes would restore virulence. to this effect we used rbeaur-rep-m41-struct-2 to infect one-day-old chicks, and used clinical signs and measurement of the ciliary activity of the epithelial cells lining the trachea of the infected chickens to assess any pathogenicity associated with the virus. no clinical signs, snicking, wheezing and nasal discharge, associated with an ibv infection were observed in the chickens infected with either rbeaur-rep-m41-struct-2 or beau-r; the latter as in previous experiments did not show any clinical signs [26] . in contrast, chickens infected with m41-ck did show clinical signs of an ibv infection (fig. 3) ; as shown previously [26] . these observations demonstrated that rbeaur-rep-m41-struct-2 was not pathogenic indicating that replacement of the structural and accessory genes did not restore virulence. analysis of epithelial cells, removed from the tracheas of the infected chickens, for the presence of infectious ibv using tocs failed to detect virus from chickens infected with either beau-r or rbeaur-rep-m41-struct-2. in contrast, epithelial cells from chickens infected with m41-ck showed the presence of virus with a titre of 4 log 10 cd 50 in tocs, demonstrating that m41-ck was present in the tracheas of the infected chickens. analysis of the epithelial cells for the presence of any ibv-derived rna by rt-pcr also indicated that no beau-r or rbeaur-rep-m41-struct-2 was present or was below detection in the epithelial cells examined; this was in contrast to the cells examined from chickens infected with m41-ck, in which virus-derived rna was detected by rt-pcr (fig. 4) . subsequent analysis of rbeaur-rep-m41-struct-2 ex vivo using tocs surprisingly showed that the ribv did not cause ciliostasis when directly used to infect tocs, in contrast to beau-r and m41-ck which caused ciliostasis. this observation raised the possibility that the lack of ciliostasis and detection of rbeaur-rep-m41-struct-2 in the tracheal epithelial cells of chickens infected by the virus using tocs for our pathogenicity experiments may have been interpreted incorrectly as the read out for these tests was ciliostasis. however, it should be noted no clinical signs were observed nor was any rbeaur-rep-m41-struct-2-derived rna detected. growth analysis of rbeaur-rep-m41-struct-2 in tocs showed that the lack of ciliostasis observed with rbeaur-rep-m41-struct-2 was not due to the inability of the virus to replicate in tocs (fig. 5) . the ribv rbeaur-rep-m41-struct-2 was able to replicate in tocs, though the amount of virus produced was about 1 log 10 less than for the growth of beau-r and m41-ck, somewhat analogous to the growth pattern observed for the virus on ckcs. ibv rbeaur-rep-m41-struct-2 consisted of the beau-r replicase and m41 structural and accessory genes and as a consequence had a 59-utr derived from beau-r and a 39-utr from m41. there are known differences between ibv 39 utrs, in fact the m41-ck 39-utr is 184 nt smaller than the beau-r 39-utr, the remaining part of the m41-ck 39-utr representing the conserved region of the ibv 39-utr and contains the predicted rna secondary structures believed to be involved in replication [44] . there are 7 and 3 nucleotide differences between the 59-and the conserved region of the ibv 39-utrs, respectively, of beau-r and m41-ck ( [33, 42] , raising the possibility that if the two utrs interact during replication their heterologous nature could be responsible for the observed decrease in growth for rbeaur-rep-m41-struct-2 in both ckcs and tocs. impairment in growth on tocs may also have been responsible for the loss of ciliostasis. we hypothesised that an increase in growth would be a selective advantage to the virus and therefore decided to serially passage rbeaur-rep-m41-struct-2 25 times on ckcs to see whether any adaption could result in a higher growth rate and a virus that could cause ciliostasis in tocs. for example, due to the very few differences in the 59-and 39-utrs, between the two parental viruses, a nucleotide substitution in either utr that may result in a more homogenous interaction, as seen with either parental virus, may result in an increase growth rate. analysis of the passaged virus, rbeaur-rep-m41-struct-2-p 25 , on ckcs and tocs showed an altered growth rate in comparison to rbeaur-rep-m41-struct-2-p 3 (fig. 6) . however, rbeaur-rep-m41-struct-2-p 25 still did not cause ciliostasis in tocs. sequence analysis of the 59-and 39-utrs of rbeaur-rep-m41-struct-2-p 25 showed that there were no nucleotide substitutions within these regions when compared to rbeaur-rep-m41-struct-2-p 3 , indicating that the change in the growth characteristics was not associated with changes in the utrs. we therefore sequenced the entire genome of rbeaur-rep-m41-struct-2-p 25 for comparison with the sequence in rvv-beaur-rep-m41-struct, used for generating rbeaur-rep-m41-struct-2-p 3 , in order to identify any potential changes that may have been responsible for the phenotypic change in growth. we identified three nucleotide changes between the p 3 and p 25 viruses, one resulted in an amino acid change, pherleu, in the s1 region of the s gene and two adjacent nucleotides in the orf-5b sequence that had two potential effects, (1) the introduction of a premature stop codon in orf-5b and (2) modification the n gene trs. these observations indicate that the increase in growth associated with rbeaur-rep-m41-struct-2-p 25 could have arisen from the amino acid change in the s protein, the loss of orf-5b or a potential change in the expression levels of sg mrna 6, which is responsible for the expression of the n protein. overall, the main conclusion from our results is the fact that rbeaur-rep-m41-struct-2 was nonpathogenic; indicating that the loss of virulence associated with beaudette is not determined by the structural and the accessory genes of ibv but resides within the replicase gene. our results demonstrating that the ibv replicase gene is a determinant of pathogenicity differs from the conclusion by navas-martin et al. [45] who reported that the differences in pathogenicity between two strains of the murine coronavirus mouse hepatitis virus (mhv), mhv-jhm and mhv-a59, mapped to the mhv genome encoding the structural and accessory genes downstream of the haemagglutinin esterase (he) gene. the replicase gene was interchangeable between the two virus genomes but the differences in pathogenicities associated with two viruses did not appear to correlate with the replicase genes of the rmhvs generated. however, a major difference between the two mhv isolates and the two ibv viruses we used was that the two mhvs were pathogenic but demonstrated different pathogenicities, neurovirulence and hepatitis, respectively, whereas the difference between the two ibv isolates used in this work was that one causes disease and the other does not. it is clear from the mhv work that both replicase sequences are of a ''virulent'' phenotype and that the pathogenicity phenotype of the recombinant viruses is associated with the structural and accessory genes. in contrast our results demonstrated that loss of virulence per se can be determined by some attenuating modification to one or more of the coronavirus replicase components. recent work involving the attenuation of a nephropathogenic strain of ibv by serial passage in embryonated chicken eggs, reported that the virus became fully attenuated by passage p 110 [46] . sequence analysis of the 39-7 kb (s gene to poly(a) tail) region of the genome from the p 110 virus identified several amino acid substitutions and a 109 nt deletion within the 39 utr when compared to p 0 virus. the authors reported that the changes identified were potentially responsible for attenuation; they did not report any changes within the replicase gene of the p 110 virus and indicated that other changes, apart from those identified in the 39-7 kb region, within the genome may also be involved in attenuation. other workers attenuated the virulent ark dpi 11 strain of ibv following 101 passages in embryonated eggs (ark dpi 101) and compared the complete genomes of both viruses; identifying 21 nucleotide changes corresponding to 17 amino acid changes [47] . the nucleotide changes resulted in eight amino acid changes in nsp2 (1), nsp3 (3), nsp6 (2), nsp10 (1) and nsp13 (1) of the replicase protein, eight amino acid changes in the s protein and one amino acid in orf 5a and the n protein. the authors were unable to confirm which amino acid changes were responsible for loss of pathogenicity. taking into consideration our results that loss of virulence associated with the beaudette strain resides in the replicase and the results of ammayappan et al. [47] , it is possible that very few amino acid substitutions within the replicase gene can result in attenuation following serial passage in embryonated eggs. the replicase gene of ibv encodes 15 nsps, some of them with known enzymatic functions [48] . how these proteins function in the context of pathogenesis is still not well understood, however, some of the nsps for other coronaviruses have been linked to loss of virulence. for example, the loss of nsp1 from mhv did not affect replication in tissue culture but severely attenuated the rmhv in vivo [49] ; inactivation of the mhv adp-ribose-10phosphatase activity in nsp3 caused a reduction in virus replication in the livers of infected mice but did not induce liver disease [50] and a single amino acid change in the mhv nsp14 did not alter the replication of the rmhv in tissue culture but resulted in attenuation in mice [51] . our previous spike swapping results demonstrated that introduction of an s protein from a virulent isolate of ibv did not confer virulence on beau-r indicating that loss of virulence was not receptor-mediated. results from work described here has shown that replacing all the beaudette structural and accessory proteins with those from a virulent isolate of ibv did not restore virulence. generation of the ribv produced in this work can either be viewed as replacing the structural and accessory genes of an avirulent virus with those of a virulent virus or replacing the replicase gene of a virulent isolate (m41-ck) with one from an avirulent virus (beau-r). in either scenario our results indicate that loss of virulence associated with ibv beaudette resides within one or more of the 15 ibv replicase proteins comprising the ibv replicase gene. virus taxonomy classification and nomenclature of viruses a comparative sequence analysis to revise the current taxonomy of the family coronaviridae turkey coronavirus is more closely related to avian infectious bronchitis virus than to mammalian 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sequence of the genome of the coronavirus avian infectious bronchitis virus a new dna sequence assembly program sequences of the nucleocapsid genes from two strains of avian infectious bronchitis virus open reading frames 3a and 3b of infectious bronchitis virus cis-acting sequences required for coronavirus infectious bronchitis virus defective-rna replication and packaging replicase genes of murine coronavirus strains a59 and jhm are interchangeable: differences in pathogenesis map to the 39 one-third of the genome altered pathogenicity, immunogenicity, tissue tropism and 39-7 kb region sequence of an avian infectious coronavirus strain after serial passage in embryos. vaccine identification of sequence changes responsible for the attenuation of avian infectious bronchitis virus strain arkansas dpi coronavirus replicative proteins coronavirus non-structural protein 1 is a major pathogenicity factor: implications for the rational design of coronavirus vaccines mouse hepatitis virus liver pathology is dependent on adp-ribose-10phosphatase, a viral function conserved in the alpha-like supergroup singleamino-acid substitutions in open reading frame (orf) 1b-nsp14 and orf 2a proteins of the coronavirus mouse hepatitis virus are attenuating in mice we thank drs. francesca culver and abu-bakr abu-median for help with removal, processing and extraction of rna from the tracheas control chickens or chickens infected with ibv. key: cord-290034-4b0mshqa authors: le, yen h.; nguyen, khanh c.; coleman, kristen k.; nguyen, tham t.; than, son t.; phan, hai h.; nguyen, manh d.; ngu, nghia d.; phan, dan t.; hoang, phuong v. m.; trieu, long p.; bailey, emily s.; warkentien, tyler e.; gray, gregory c. title: virus detections among patients with severe acute respiratory illness, northern vietnam date: 2020-05-12 journal: plos one doi: 10.1371/journal.pone.0233117 sha: doc_id: 290034 cord_uid: 4b0mshqa severe acute respiratory illness (sari) is a major cause of death and morbidity in lowand middle-income countries, however, the etiologic agents are often undetermined due to the lack of molecular diagnostics in hospitals and clinics. to examine evidence for select viral infections among patients with sari in northern vietnam, we studied 348 nasopharyngeal samples from military and civilian patients admitted to 4 hospitals in the greater hanoi area from 2017–2019. initial screening for human respiratory viral pathogens was performed in hanoi, vietnam at the national institute of hygiene and epidemiology (nihe) or the military institute of preventative medicine (mipm), and an aliquot was shipped to duke-nus medical school in singapore for validation. patient demographics were recorded and used to epidemiologically describe the infections. among military and civilian cases of sari, 184 (52.9%) tested positive for one or more respiratory viruses. influenza a virus was the most prevalent virus detected (64.7%), followed by influenza b virus (29.3%), enterovirus (3.8%), adenovirus (1.1%), and coronavirus (1.1%). risk factor analyses demonstrated an increased risk of influenza a virus detection among military hospital patients (adjusted or, 2.0; 95% ci, 1.2–3.2), and an increased risk of influenza b virus detection among patients enrolled in year 2017 (adjusted or, 7.9; 95% ci, 2.7–22.9). as influenza a and b viruses were commonly associated with sari and are treatable, sari patients entering these hospitals would benefit if the hospitals were able to adapt onsite molecular diagnostics. although severe acute respiratory illness (sari) is a major cause of death and morbidity in low-and middle-income countries, the etiology of sari is often unconfirmed due to the lack of molecular testing capabilities in under-resourced regions. hospitals and clinics often rely on broad nationwide influenza surveillance data to describe pneumonia and sari outbreaks, a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 neglecting onsite molecular diagnosis of influenza and non-influenza respiratory viruses as potential etiologic agents. however, efforts beyond utilizing influenza surveillance to describe pneumonia and sari in vietnam have been demonstrated as feasible and efficient by the vietnam ministry of health [1] . in addition to influenza viruses, non-influenza respiratory viruses with zoonotic potential are a serious emerging threat to nations where intermixing of large populations of humans and animals occurs [2] . for example, coronaviruses are prone to cross-species transmission [3] resulting in global outbreaks of human respiratory diseases such as severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and most recently, coronavirus disease 2019 (covid-19) [4] . we also see potential for adenoviruses and enteroviruses to jump species [2, 5] , and community-acquired pneumonia treatment guidelines highlight the increasingly recognized presence of viruses as lower respiratory pathogens [6] . furthermore, our recent study describing pneumonia infections in east malaysia revealed a high prevalence of influenza a and respiratory syncytial viruses in the region [7] , suggesting that underresourced hospitals and clinics in southeast asia could benefit from more robust surveillance of respiratory viruses causing pneumonia and sari. in an effort to examine evidence for select viral infections among military and civilian hospital patients with sari in northern vietnam, we conducted a prospective study of such patients seen at four hospitals in the hanoi area. we also sought to epidemiologically describe these infections using patient demographics (gender and age-group). we focused upon detecting high-impact respiratory viruses [8] often associated with human epidemics [2] . informed written consent was used to gain permission to collect nasopharyngeal (np) swab specimens and clinical questionnaire data from patients with sari at four hospitals in vietnam: hai phong general hospital and military hospitals 103, 108 and 354. for minors included in the study, written assent was obtained, as well as written consent from their parents or guardians. study subjects included any patient admitted to the hospital study site having a respiratory illness with acute onset in the previous 10 days, fever of 38˚c or higher, and cough. patients excluded from the study included those admitted to the hospital more than 48 hours prior to examination by the study physician, persons less than 5 years of age, persons with known hiv/aids or active tuberculosis, and persons who underwent a surgical procedure within 14 days of examination by the study physician. upon collection, clinical specimens were transported on ice to either the military institute of preventive medicine (mipm) or the national institute of hygiene and epidemiology (nihe), both in hanoi, vietnam, and preserved at -80˚c until screened for a panel of human respiratory viruses (see molecular methods below). residual original specimens were again preserved at -80˚c and shipped on dry ice to the duke-nus laboratory of one health research in singapore. laboratory staff at duke-nus were blind to the results of the assays at nihe and mipm. results from the two efforts were then compared, and the etiologic agent was confirmed. the few discordant were retested and the results agreed upon. blood samples from a portion of sari patients at hai phong general hospital were cultured in bd bactec medium (bd vietnam co. ltd, ho chi minh, vietnam) and tested for bacteremia using analytical profile index. rna and dna extraction, rt-pcr/pcr, and dna sequencing. rna or dna extractions were performed using the qiamp minelute virus spin kit (qiagen, inc., valencia, ca). real-time rt-pcr/pcr was conducted on a biorad cfx96 c1000 touch thermal cycler real-time system at nihe, mipm, and duke-nus in singapore. conventional rt-pcr was conducted at duke-nus using superscript iii one-step rt-pcr system with platinum taq dna polymerase. following common standard operating procedures and previously published pcr/rt-pcr conditions [9] [10] [11] [12] , np swabs were examined for molecular evidence of influenza a and b virus (iav, ibv) [9] , adenovirus [10] , enterovirus (ev) [11] , and coronavirus (cov) [12] . primer and probe sequences for targeted pathogens are recorded in s1 table. a positive and a no-template control were included in each run. coronavirus-positive samples were sent to bio basic asia pacific pte. ltd. (singapore) for sanger sequencing. study data were imported into stata version 15.0 (statacorp, college station, tx, usa) for statistical analyses. pearson's chi-squared or fisher's exact tests were used to examine for differences in viral detections by gender, age group, year of study enrollment, and hospital (military vs. civilian). logistic regression was used to calculate odds ratios (or) and 95% confidence intervals (ci) for risk factors associated with prevalent viral detections. to further examine these risk factors, stepwise, unconditional logistic regression using a saturated model and backward elimination (exclusion p > 0.05) was used. a total of 348 sari patients were enrolled at the four hospitals from january 2017 to january 2019, with 151 (43.4%) of the patients enrolled at one of the three military hospitals (military hospital 103, 108 and 354) and 197 (56.6%) enrolled at hai phong general hospital ( table 1 ). the majority of study patients (62.4%) were female and all study patients were aged 15 years or older. all patients studied at military hospitals 103, 108 and 354 were enrolled in 2017, and due to multiple administrative requirements and a large dengue epidemic, study enrollments at hai phong general hospital were not uniformly made over time (s1 fig). among the 348 sari patients, 184 (52.9%) tested positive for one or more respiratory viruses. fifty-three of the sari patients from hai phong general hospital received blood culture tests for bacterial infections, and one of those patients tested positive for pseudomonas aeruginosa. influenza a virus was the most prevalent virus detected (64.7%), followed by influenza b virus (29.3%), enterovirus (3.8%), adenovirus (1.1%), and coronavirus (1.1%), ( age-group and gender were not predictors for specific viral detections. however, pearson's chi-squared and fisher's exact tests demonstrated statistically significant differences in influenza a and b virus by year of study enrollment and between hospitals (military vs. civilian). table 4 ). the first case of influenza a virus in this study was detected from a military hospital patient enrolled in february 2017, followed by detections throughout the study, with the highest number of detections in april 2017 and november 2018 (fig 1) . the first case of influenza b virus in this study was detected from a civilian hospital patient in march 2017, followed by detections throughout the study until february 2018, with the highest number of detections in april and may 2017. influenza b virus was not detected from march 2018 through january 2019. there were no confirmed cases of co-infection with influenza a and b viruses in our study. cases of influenza a and b virus were found among all study age quartiles and genders ( table 2 ). there was one case of co-infection with enterovirus and coronavirus 229e including a female military hospital patient 28 years of age. enterovirus cases were found among all genders and age quartiles except for patients � 57 years of age. the 2 coronavirus cases were found among female patients aged 25-34 years. the 2 adenovirus cases were found among female military hospital patients aged 15-34 years. viruses identified in our study are known to sometimes cause severe respiratory disease in humans. similar to pneumonia etiology studies in vietnam and central china [1, 13, 14] , influenza a virus was the most common virus detected among patients in our study. notably, influenza b virus was not detected during the final 10 months of the study (march 2018 through january 2019), which reflects the near absence of influenza b virus cases reported in vietnam during that time [15] . however, our study enrollments paused from march 2018 to july 2018 due to administrative requirements and therefore the number of influenza cases at our hospital study sites during that time is unknown. the increased risk of influenza a virus infection among military hospital patients in our study was expected, as military trainees often seek care at these hospitals and rates of respiratory illness among military trainees tends to exceed those found in civilian populations [16] . enterovirus is a widespread pathogen among children and is often overlooked in adults [17, 18] . in our study, 6 of the 7 enterovirus infections were among young adults, which could have been attributable to their probable contact with young children. additionally, 2 pairs of the enterovirus infections occurred within 1-2 weeks, suggesting that these could have been clusters. lastly, 5 of the enterovirus infections were detected from late march to june 2017, which reflects seasonal patterns of epidemics of enterovirus 71 in some asian countries [19] . our study was limited in that we did not subtype the enterovirus-positive patient samples. it is also possible that the enterovirus assays used in our study could have detected rhinoviruses, as cross-reactivity between rhinoviruses and enteroviruses has previously been demonstrated [20] . adenovirus is an important pathogen among military and civilian populations [21, 22] . adenovirus infections among civilian populations are increasingly reported in southeast asia [23] [24] [25] [26] , therefore it is interesting that no adenovirus cases were confirmed among the civilian hospital patients enrolled in our study. this observation suggests that adenovirus might be largely restricted to the military population and military hospitals in northern vietnam. however, it is important to note that adenovirus infections are often found among pediatric populations, and patients < 15 years of age were not enrolled in our study. although only 2 cases of adenovirus infection were confirmed in our study, our approach was limited in that we did viral causes of sari in northern vietnam not subtype the adenovirus-positive samples, which has been demonstrated as a useful tool in identifying emerging and virulent adenoviruses among populations [26, 27] . in contrast to the enterovirus-and adenovirus-positive samples in our study, we subtyped the coronavirus-positive samples. coronavirus hku1 and 229e were identified, which are both common causes of mild respiratory illness. however, both study patients with coronavirus were admitted to the intensive care unit (icu) with symptoms of respiratory failure requiring oxygen therapy. the study patient with coronavirus 229e had a confirmed co-infection with enterovirus. we suspect that the severity of respiratory illness among the 2 young adult patients infected with coronavirus could be due to co-infection, although the effect of viral coinfections on disease severity are unclear for some common respiratory viruses [28] and the study patient with coronavirus hku1 did not have a confirmed co-infection. however, we screened patients only for the respiratory viruses mentioned in our study, and therefore we could not rule out co-infections with other viral or non-viral pathogens. microbiological testing for bacterial infections among sari and pneumonia patients is not routine procedure at the study hospitals and therefore only 53 of the sari study patients received blood culture tests, and only one of those patients tested positive for p. aeruginosa bacteremia. this patient was a 30-year-old female from the civilian hospital who also tested positive for influenza a virus. our study was limited in that we also did not have the capacity to test patient samples for human parainfluenza, human metapneumovirus, or rsv. however, these diseases are primarily found in patients outside of this study's recruited age range, such as infants, young children, and older adults. nevertheless, human parainfluenza viruses are members of high-impact viral groups with pandemic potential [8] , and surveilling for these viruses among sari and pneumonia patients in our study's age range could be valuable. with an overall goal to reduce sari in vietnam, this study aimed to examine evidence for select viral infections among sari patients in northern vietnam. we chose to focus on highimpact respiratory viruses [8] that are often associated with human epidemics [2] . as influenza viruses were commonly associated with sari and are treatable, the hospitals in this study could benefit from onsite molecular diagnostic capability. additionally, our results display enterovirus, adenovirus and coronavirus infections among the sari cases, suggesting that cities in northern vietnam could benefit also from local surveillance of non-influenza respiratory viruses. expanding severe acute respiratory infection (sari) surveillance beyond influenza: the process and data from 1 year of implementation in vietnam. influenza other respir viruses a mini review of the zoonotic threat potential of influenza viruses, coronaviruses, adenoviruses, and enteroviruses. front public health coronaviruses: important emerging human pathogens three emerging coronaviruses in two decadesthe story of sars, mers, and now covid-19 a systematic review of evidence that enteroviruses may be zoonotic. emerging microbes & infections diagnosis and treatment of adults with community-acquired pneumonia. an official clinical practice guideline of the high prevalence of viral infections among hospitalized pneumonia patients in equatorial sarawak, malaysia. open forum infectious diseases preparedness for a high-impact respiratory pathogen pandemic us cdc protocol for real-time rt-pcr detection of influenza viruses improved real-time pcr assay for detection and quantification of all 54 known types of human adenoviruses in clinical samples. medical science monitor: international medical journal of experimental and clinical research characterizing the picornavirus landscape among synanthropic nonhuman primates in bangladesh detection of coronaviruses in bats of various species in italy the substantial hospitalization burden of influenza in central china: surveillance for severe, acute respiratory infection, and influenza viruses the incidence and aetiology of hospitalised community-acquired pneumonia among vietnamese adults: a prospective surveillance in central vietnam world health organization (who) global influenza surveillance and response system (gisrs) respiratory diseases among us military personnel: countering emerging threats. emerging infectious diseases etiology and clinical outcomes of acute respiratory virus infection in hospitalized adults enterovirus infection in adults presenting with nonspecific febrile illness during summer evolution and spatiotemporal dynamics of enterovirus a71 subgenogroups in vietnam. the journal of infectious diseases epidemic 2014 enterovirus d68 cross-reacts with human rhinovirus on a respiratory molecular diagnostic platform vaccine-preventable adenoviral respiratory illness in us military recruits adenovirus type 4 respiratory infections among civilian adults, northeastern united states severe pediatric adenovirus 7 disease in singapore linked to recent outbreaks across asia epidemiology, clinical presentation and respiratory sequelae of adenovirus pneumonia in children in kuala lumpur, malaysia a report of adult human adenovirus infections in a tertiary hospital. open forum infectious diseases adenoviral infections in singapore: should new antiviral therapies and vaccines be adopted? the journal of infectious diseases molecular typing of clinical adenovirus specimens by an algorithm which permits detection of adenovirus coinfections and intermediate adenovirus strains co-infections with influenza and other respiratory viruses. respiratory regulation-the molecular approach we thank the numerous non-author personnel at the military institute of preventive medicine (mipm), the national institute of hygiene and epidemiology (nihe) and hai phong provincial preventive medicine center for recruiting patients and performing molecular analyses. key: cord-276577-06boh550 authors: schanzer, dena l.; garner, michael j.; hatchette, todd f.; langley, joanne m.; aziz, samina; tam, theresa w. s. title: estimating sensitivity of laboratory testing for influenza in canada through modelling date: 2009-08-18 journal: plos one doi: 10.1371/journal.pone.0006681 sha: doc_id: 276577 cord_uid: 06boh550 background: the weekly proportion of laboratory tests that are positive for influenza is used in public health surveillance systems to identify periods of influenza activity. we aimed to estimate the sensitivity of influenza testing in canada based on results of a national respiratory virus surveillance system. methods and findings: the weekly number of influenza-negative tests from 1999 to 2006 was modelled as a function of laboratory-confirmed positive tests for influenza, respiratory syncytial virus (rsv), adenovirus and parainfluenza viruses, seasonality, and trend using poisson regression. sensitivity was calculated as the number of influenza positive tests divided by the number of influenza positive tests plus the model-estimated number of false negative tests. the sensitivity of influenza testing was estimated to be 33% (95%ci 32–34%), varying from 30–40% depending on the season and region. conclusions: the estimated sensitivity of influenza tests reported to this national laboratory surveillance system is considerably less than reported test characteristics for most laboratory tests. a number of factors may explain this difference, including sample quality and specimen procurement issues as well as test characteristics. improved diagnosis would permit better estimation of the burden of influenza. although influenza virus infection is associated with considerable morbidity and mortality [1] [2] [3] , laboratory confirmation of clinical illness is the exception rather than the rule. clinicians do not routinely seek laboratory confirmation for several reasons: diagnosis will often not alter patient management, a paucity of real-time, accurate, inexpensive testing methods [4] and because influenza is not recognized as the etiology of the clinical presentation [5] . accurate diagnosis of influenza-like illness, however, could improve clinical care through reduced use of antibiotics and ancillary testing, and more appropriate use of antiviral therapy [6] . although rapid influenza tests such as pointof-care tests are purported to generate results in a timely fashion to influence clinical care, the performance characteristics of the currently available tests are sub-optimal [7] . new technologies with improved sensitivity such as reverse-transcriptase polymerase chain reaction (rt-pcr) [8] as well as the use of more effective collection systems such as the flocked nasopharyngeal swab compared to traditional rayon wound swabs, and the recommendation to collect more ideal specimens, such as nasopharyngeal swabs rather than throat swabs are likely to improve diagnostic sensitivity [9] [10] [11] [12] . the performance characteristics of currently available tests for influenza vary considerably and the overall sensitivities of these tests when used in routine practice are also dependent on the type of specimen collected, the age of the patient and point in their illness in which they are sampled [4, 9, [13] [14] [15] . we sought to estimate the sensitivity of influenza testing based on results of a national respiratory virus surveillance system using a model-based method [1, 2, [16] [17] [18] . weekly respiratory virus identifications from september 1999 to august 2006 were obtained from the respiratory virus detection surveillance system (rvdss), public health agency of canada [19, 20] . the rvdss collects, collates, and reports weekly data from participating laboratories on the number of tests performed and the number of specimens confirmed positive for influenza, respiratory syncytial virus (rsv), para-influenza virus (piv), and adenovirus. specimens are generally submitted to laboratories by clinicians in the course of clinical care, and by clinicians participating in one of our national influenza surveillance programs, (fluwatch [20] ). indicators of influenza activity are reported year round on a weekly basis to the fluwatch program. the rvdss is supplemented by case reports of influenza positive cases [19, 21] . from the case reports, influenza a was confirmed in all age groups and sporadic cases were confirmed in the off-season months of june through september. infants and children under the age of 5 years accounted for 25% of the influenza a positive tests, and persons over the age 65 years another 35%. unfortunately, fluwatch surveillance data does not provide the total number of tests by age. testing practices are known to be varied [22, 23] . the predominant testing methods used for influenza detection varied considerably by province or laboratory and over time. for the 2005/06 season a survey of laboratory techniques in current use indicated that culture accounted for 44% of the diagnostic tests with rt-pcr, rapid antigen tests and direct fluorescent-antibody assay (dfa) accounting for 21%, 19%, and 16% respectively [23] . the weekly number of tests negative for influenza was modelled, using poisson regression, as a function of viral identifications for influenza, rsv, adenovirus and piv as well as a baseline consisting of seasonality, trend and holiday variables. the estimated baseline implicitly accounts for influenza tests on specimens taken from patients with respiratory infections due to respiratory pathogens other than the four viruses captured in the rvdss, as long as both the testing behaviour of clinicians and respiratory illnesses caused by other respiratory pathogens follow a consistent seasonal pattern as prescribed by the model (see below, the poisson regression model with a linear link function was estimated using sas [24] proc genmod: coefficients b 5 to b 9 are multipliers. the weekly number of influenza negative tests estimated to be falsely negative is given by b 5 infla w +b 6 inflb w . the weekly number of influenza negative tests attributed to rsv is given by b 7 rsvp w. , and similarly for adenovirus and piv. for each positive influenza a test, an additional b 5 tests above baseline were performed and found to be negative. by specifying a linear link, a value of 0.33, say, for coefficient b 5 , means that for every test for which influenza a was confirmed, 0.33 additional tests, on average, were performed on truly influenza a positive specimens and found to be negativewhich corresponds to a sensitivity of 75%. sensitivity was calculated as the number of influenza positive tests divided by the number of influenza positive tests plus the model-estimated number of false negative tests, or equivalently, the estimates of sensitivity for influenza a and b are given by 1/ (1+b 5 ) and 1/(1+b 6 ) respectively. the false negative rate is 1 minus sensitivity. while the null value for b 5 is zero, which indicates no statistical association between the number of influenza positive tests and the number of influenza negative tests, the corresponding null value for sensitivity is 1. for each test confirmed positive for rsv, on average b 7 tests were performed for influenza and found to be negative for influenza. these b 7 tests are attributed to an rsv infection, however the number of influenza-negative tests that actually tested positive for rsv is unknown. if all specimens had been tested for the same viruses (panel tests), 1/b 7 would correspond to the sensitivity for rsv testing, and the sensitivity for adenovirus and piv given by 1/b 8 and 1/b 9 respectively. some laboratories are known to test for viruses sequentially [22] , and so 1/b 7 -1/b 9 were not interpreted as estimates of the sensitivity for other viruses. sequential testing may occur if a rapid test for influenza is negative and the laboratory then performs pcr or culture testing. similarly in young children with a respiratory illness in the winter, rapid tests for rsv infection may be performed first, and only specimens with negative results submitted for subsequent testing for influenza or other respiratory viruses [25] . by contrast, many laboratories conduct panel tests for multiple viruses for ease of handling, decreased patient sampling, and recognition that coinfection can occur. either form of sequential testing would not bias the estimate of sensitivity applicable to test results reported to rvdss, though significant use of rapid antigen tests in the laboratories reporting to rvdss would reduce the overall sensitivity. as a single specimen may undergo multiple tests, the false-negative rate applicable to a specimen that has undergone multiple tests would be expected to be much lower than the system average for individual tests. parameters b 1. to b 4 account for trends and the seasonality of truly negative specimens (patients presenting with other acute respiratory infections). over 50,000 tests for influenza were reported to the rvdss each year, peaking in 2004/05 at 101,000. overall 10% of the influenza tests were positive for influenza, ranging from 4% to 13% depending on the season. the proportion positive for rsv, parainfluenza and adenovirus averaged 9%, 3% and 2% respectively. as seen in figure 1 , no virus was identified in 75% of specimens submitted for testing (white area under the curve). even for the winter months of december through april, one of these 4 viruses was identified on average in no more than 30% of the specimens. the strong and consistent synchronization of negative tests with influenza positive tests, as seen in figure 1 , is suggestive that false negative results contributed to the large number of negative tests during periods of influenza activity. the sensitivity for influenza a testing averaged 33.7% (with model-estimated 95% confidence intervals of 33.3-34.1) for the 1999/2000-2005/06 period. influenza b testing had a similar estimated sensitivity at 34.7 (95% ci 33.4-36.1). estimated sensitivities varied somewhat from season to season, generally ranging from 30%-40% (table 1) , and provincial level estimates, as well, were within a similar range. stratifying by province or season produced similar estimates for the sensitivity of influenza a testing: 32% (95% ci 30-34) and 36% (95% ci 33-41) respectively. estimates of sensitivity based on test results reported to the rvdss for individual laboratories with sufficient data to fit the model showed significant variation, with estimates of sensitivity ranging from 25-65%. as expected, laboratories using primarily rapid antigen tests had lower estimated sensitivities, and laboratories that used pcr methods had higher sensitivity estimates. however, information on testing procedures is limited primarily to the 2005/06 survey. as well, additional irregularities were noticed in the laboratory data and not all laboratories provided sufficient data to fit the model. figure 2 illustrates a good model fit where the weekly number of influenza negative tests is well explained by the model covariates, with a few exceptions. firstly, it is evident that additional specimens were tested during the sars period, as indicated by the period where the number of weekly influenza negative tests exceeded the expected number, or equivalently, a period of successive positive residuals. residuals typically capture random variation; hence represent tests that can not be allocated based on the specified model. in addition to the sars period, testing appears to have been elevated for a number of weeks in january 2000 during the peak of the 1999/2000 a/ sydney/05/97 (h3n2) season in which respiratory admissions were unusually elevated [26, 27] , and in december 2003, when an elevated risk of paediatric deaths associated with the a/fujian/411/02 (h3n2) strain [28] was identified in the us. as these periods corresponded to a period of heightened public awareness due to severe influenza outbreaks, parameter estimation was repeated without these data points. exclusion of these data points did not alter the sensitivity estimate for influenza. the attribution of influenza negative test results to influenza and other viruses is illustrated in figure 3 . the baseline curve is the model estimate of the number of tests that were likely truly negative for all four viruses tested. a reduction in specimen collection and testing, primarily for viruses other than influenza, is also evident over the christmas period ( figure 3) . the weekly proportion of tests confirmed positive for influenza peaked each season at 15 to 30%. accounting for the model estimated false negative rate suggests that during periods of peak influenza activity, 40-90% of tests were performed on specimens taken from persons recently infected with influenza. influenza was confirmed in only 14% of specimens sent for testing over the winter period, whereas the sensitivity estimate would imply that up to 40% of influenza tests could be attributed to an influenza infection. the corresponding figures for the whole year indicate that 10% of specimens were confirmed positive for influenza and 30% of influenza tests could be model-attributed to an influenza infection annually. despite a relatively large number of tests in the off-season, the number of influenza positive tests was almost negligible; suggesting that the false positive rate applicable to rvdss influenza testing is minimal. the model estimated sensitivity based on influenza test results reported to the rvdss of 30-40% is much lower than the standard assay sensitivities documented in the literature. standard sensitivities for diagnostic procedures used by participating laboratories ranged from 64% for rapid antigen tests to 95% for rt-pcr tests, averaging 75% for the study period [23] . as performance characteristics of specific tests are generally based on high quality specimens, the difference of approximately 40% is likely linked to any one of many operational procedures that affects the quality of the specimen and its procurement. unlike validation studies, our samples are taken from a variety of clinical settings and processed with a variety of procedures across the country. as well, variation in the indications for diagnostic testing may vary across the country. as there are many other respiratory pathogens that are not routinely tested for, or reported to the rvdss, including human metapneumovirus (hmpv), coronaviruses, and rhinoviruses for which patients may seek medical care and present with influenza like illness [29] [30] [31] [32] , a large proportion of negative test results was expected. the overall model fit, and the general consistency of the sensitivity estimates, suggests that these many respiratory viruses were reasonably accounted for by the seasonal baseline and that the strong association between the number of influenza positive and influenza negative tests on a weekly basis is indicative of a significant number of false negative results, rather than the activity of another virus or viruses exactly synchronous with influenza. the latter would bias the estimated sensitivity of the system downwards. however, to significantly and consistently bias the estimate, the degree of synchronization would have to be fairly strong, persist over the whole study period, and occur in all provinces. synchronization was not observed among the rvdss viruses (influenza a, influenza b, rsv, adenovirus and piv), and elsewhere other viruses such as rhinovirus, coronavirus and hmpv accounted for only a small proportion of the viral identifications and were not found to be synchronized with influenza [33] . as well, patients may present for care due to a secondary bacterial infection. while any specimen would likely test negative as the virus, at this point, is likely not detectable, the model would statistically attribute a negative test in this case to the primary infection; one of the four rvdss viruses or to the seasonal baseline that represents other respiratory infections, depending on the level of viral activity at the time of the test. this is not considered a source of bias. the large variation in false negative rates estimated for individual laboratories reporting to the rvdss suggests that standardization of sample procurement, testing and reporting procedures would likely reduce the overall false negative rate. the accuracy of diagnostic tests is known to be affected by the quality of the specimen [10, 11] , its handling, the timing of collection after symptom onset, and the age of the patient [14, 15] . even with the most sensitive molecular methodologies, yield was shown to be strongly related to the time since onset of symptoms [9, 14] , with a 3-fold decline in proportion positive within 3 to 5 days after onset of symptoms for both rt-pcr and culture procedures. for most laboratory tests, specimen procurement within 72 hours of from the onset of symptoms is recommended [6] , yet patients often present much later in the course of illness. estimates of the median time since onset of symptoms suggest a delay of 3 and 5 days for outpatient and inpatients respectively [15] , however these estimates are limited to patients with laboratory confirmed influenza. in addition, there are inherent differences in the performance characteristics of the currently used diagnostic tests [4, 6, 8, [34] [35] [36] [37] [38] . lack of standardization between diagnostic tests and algorithms used in different laboratories reporting to the rvdss adds to this complexity. the routine use of rt-pcr testing has only recently become available in canada (only 20% of tests used rt-pcr methods as of 2005/06 [23] ), but increased use of this modality is expected to improve accuracy. population or system level sensitivity estimates that include the effects of sample quality are limited. grijalva and colleagues [39] estimated the diagnostic sensitivity in a capture recapture study of children hospitalized for respiratory complications at 69% for a rt-pcr based system and 39% for a clinical-laboratory based system (passive surveillance of tests performed during clinical practice, and using a variety of commercially available tests). though the expected proportion of influenza tests that were due to influenza infections is unknown and variable, our model estimate of 30% appears plausible. cooper and colleagues [33] attributed 22% of telephone health calls for cold/flu to influenza over two relatively mild years, and elsewhere 20% of admissions for acute respiratory infections (including influenza) in adults aged 20-64 years were attributed to influenza, and 42% for seniors [1] . while there are limitations with this approach, there are no other simple alternatives to assist in the interpretation of the rvdss data. it would have been helpful to analyze data based on each specimen sent for testing. with only the number of weekly tests and number of positive results, we were unable to calculate the number of specimens that were actually found to be negative for all four viruses, or to estimate the extent of co-infection. coinfection, which was not accounted for in our model, could result in an under-estimation of the number of falsely negative tests, as the attribution of an influenza negative test that was actually coinfected with influenza and another respiratory virus would have to be split between the viruses. with auxiliary information associated with each specimen, model estimates of false negative rates based on, for example, test type, time since onset of symptoms, age of the patient, or clinical presentation would have allowed us to explore the reasons for the high false negative rates. as the false negative rate appears to be laboratory dependant (data not shown), this estimated range is applicable only to the rvdss for the study period. a significant reduction in the false negative rate is anticipated as methods become standardized and with the uptake of the new rt-pcr methods. as positive results, particularly for culture, are often obtained a week or more after the specimen was received, some positive results may have been reported in a different week than the test. multiple test results for a single specimen may have also contributed to reporting irregularities. these irregularities would tend to bias the estimated parameter towards zero, and hence the estimated sensitivity towards 1. considering the overall model fit and the relative severity of influenza [1] , we conclude that our estimate of sensitivity may be slightly over-estimated (number of false negatives under-estimated). poor test sensitivity contributes to the chronic underestimation of the burden of influenza in the general population. since estimates of the burden of illness drive planning for preventive and therapeutic interventions, it is important to improve all aspects leading to improved diagnostic accuracy. we have illustrated a simple method that uses the surveillance data itself to estimate the system wide sensitivity associated with the weekly proportion of tests confirmed positive. although our estimate of sensitivity is only applicable to the interpretation of the rvdss data over the study period, similar estimates for specific cohorts or laboratory procedures may help guide further investigation into the reasons for the large number of false negative test results. the capacity for improved diagnostic accuracy will ultimately improve our understanding of the epidemiology of influenza. role of influenza and other respiratory viruses in admissions of adults to canadian hospitals co-morbidities associated with influenza-attributed mortality influenzaattributable deaths: canada 1990-1999 sensitivity of diagnostic tests for influenza varies with the circulating strains accuracy and interpretation of rapid influenza tests in children role of the laboratory in diagnosis of influenza during seasonal epidemics and potential pandemics the limitations of point 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immunosorbent assay for diagnosis of influenza a virus infection in different age groups hospitalization attributable to influenza and other viral respiratory illnesses in canadian children influenza-attributed hospitalization rates among pregnant women the modelled attribution of the weekly number of specimens tested for influenza to influenza (a and b), and adenovirus, parainfluenza virus, and rsv combined is shown along with the numbers confirmed positive. the total is the number of weekly tests for influenza (most were likely panel tests). the baseline accounts for routine tests in the hypothetical absence of influenza, rvs, adenovirus and parainfluenza activity, and corresponds to the model estimate of the number of tests that were truly negative for all tested viruses. the blue area (light plus dark) corresponds to tests attributed to influenza, with the light blue area corresponding to tests confirmed positive for influenza. the purple area (light plus dark) corresponds to tests attributed to rsv, adenovirus or parainfluenza influenza-associated hospitalizations in the united states influenza in canada: 2005-2006 season influenza in canada: 2003-2004 season antiviral therapy and outcomes of influenza requiring hospitalization in ontario impact of changing laboratory diagnostics on influenza surveillance sas/stath 9 user's guide strategy for efficient detection of respiratory viruses in pediatric clinical specimens prescription for excellence: how innovation is saving canada's health care system emergency department overcrowding: ambulance diversion and the legal duty to care influenza-associated deaths among children in the united states characterization of viral agents causing acute respiratory infection in a san francisco university medical center clinic during the influenza season human metapneumovirus infections in adults: another piece of the puzzle human metapneumovirus infection in adults human metapneumovirus infection in the canadian population the contribution of respiratory pathogens to the seasonality of nhs direct calls superiority of reverse-transcription polymerase chain reaction to conventional viral culture in the diagnosis of acute respiratory tract infections in children real-time pcr in clinical microbiology: applications for routine laboratory testing evaluation of three immunoassay kits for rapid detection of influenza virus a and b performance of six influenza rapid tests in detecting human influenza in clinical specimens comparison of the directigen flu a+b test, the quickvue influenza test, and clinical case definition to viral culture and reverse transcription-pcr for rapid diagnosis of influenza virus infection estimating the undetected burden of influenza hospitalizations in children the authors acknowledge the support of the national fluwatch network and all those involved in the collection and compilation of this data. special thanks to the anonymous reviewers for valuable comments. key: cord-289285-aof7xy13 authors: michaelis, martin; geiler, janina; naczk, patrizia; sithisarn, patchima; leutz, anke; doerr, hans wilhelm; cinatl, jindrich title: glycyrrhizin exerts antioxidative effects in h5n1 influenza a virus-infected cells and inhibits virus replication and pro-inflammatory gene expression date: 2011-05-17 journal: plos one doi: 10.1371/journal.pone.0019705 sha: doc_id: 289285 cord_uid: aof7xy13 glycyrrhizin is known to exert antiviral and anti-inflammatory effects. here, the effects of an approved parenteral glycyrrhizin preparation (stronger neo-minophafen c) were investigated on highly pathogenic influenza a h5n1 virus replication, h5n1-induced apoptosis, and h5n1-induced pro-inflammatory responses in lung epithelial (a549) cells. therapeutic glycyrrhizin concentrations substantially inhibited h5n1-induced expression of the pro-inflammatory molecules cxcl10, interleukin 6, ccl2, and ccl5 (effective glycyrrhizin concentrations 25 to 50 µg/ml) but interfered with h5n1 replication and h5n1-induced apoptosis to a lesser extent (effective glycyrrhizin concentrations 100 µg/ml or higher). glycyrrhizin also diminished monocyte migration towards supernatants of h5n1-infected a549 cells. the mechanism by which glycyrrhizin interferes with h5n1 replication and h5n1-induced pro-inflammatory gene expression includes inhibition of h5n1-induced formation of reactive oxygen species and (in turn) reduced activation of nfκb, jnk, and p38, redox-sensitive signalling events known to be relevant for influenza a virus replication. therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of h5n1 disease. highly pathogenic h5n1 influenza a viruses are considered to be potential influenza pandemic progenitors [1] [2] [3] [4] [5] [6] . at least for the first wave of an h5n1 pandemic, no sufficient amounts of adequate vaccines will be available [1] [2] [3] [4] [6] [7] [8] . therefore, antiviral therapy for influenza a viruses including highly pathogenic h5n1 virus strains remains of great importance for the first line defense against the virus [1] [2] [3] [4] 6, 9] . the neuraminidase inhibitors oseltamivir and zanamivir as well as the adamantanes amantadin and rimantadin that interfere with the influenza m2 protein are licensed for the treament of influenza [1] [2] [3] [4] 6] . however, the use of both drug classes is limited by the emergence of resistant virus strains. in seasonal influenza strains, the majority of h3n2 viruses and a great proportion of h1n1 viruses in humans are now considered to be amantadine-and rimantadine-resistant [10] [11] [12] [13] . moreover, a drastic increase in oseltamivir-resistant h1n1 viruses has been reported during the 2007/2008 influenza season in the northern hemisphere [14] [15] [16] [17] . preliminary data from the united states predict a further rise for the 2008/2009 season, possibly resulting in more than 90% of the circulating h1n1 strains to be oseltamivir resistant [14] . h5n1 virus strains appear to be generally less sensitive to antiviral treatment than seasonal influenza a virus strains and treatment-resistant h5n1 strains emerge [1] [2] [3] [4] 6, [18] [19] [20] [21] . more-over, parenteral agents for the treatment of seriously ill patients are missing. glycyrrhizin, a triterpene saponine, is a constituent of licorice root. it has been found to interfere with replication and/or cytopathogenic effect (cpe) induction of many viruses including respiratory viruses such as respiratory syncytial virus, sars coronavirus, hiv, and influenza viruses [22] [23] [24] [25] [26] [27] [28] . moreover, antiinflammatory and immunomodulatory properties were attributed to glycyrrhizin [26] . the severity of human h5n1 disease has been associated with hypercytokinaemia (''cytokine storm'') [29, 30] . delayed antiviral plus immunomodulator treatment reduced h5n1-induced mortality in mice [31] . therefore, antiinflammatory and immunomodulatory effects exerted by glycyrrhizin may be beneficial for treatment of h5n1. also, glycyrrhizin is a known antioxidant [26] and antioxidants were already shown to interfere with influenza a virus replication and virus-induced pro-inflammatory responses [32] [33] [34] . stronger neo-minophagen c (snmc) is a glycyrrhizin preparation (available as tablets or parenteral formulation) that is approved in japan for the treatment of chronic hepatic diseases and is marketed in japan, china, korea, taiwan, indonesia, india, and mongolia. here, we investigated the influence of snmc on h5n1 replication, on h5n1-induced cytokine expression, on h5n1-induced cellular oxidative stress, and on critical h5n1-induced cellular signalling events in human pneumocytes (a549 cell line). glycyrrhizin (stronger neo minophagen c) was obtained from minophagen pharmaceuticals co., ltd. (tokyo, japan). the influenza strain a/vietnam/1203/04 (h5n1) was received from the who influenza centre (national institute for medical research, london, uk). the h5n1 influenza strain a/thailand/ 1(kan-1)/04 was obtained from prof. pilaipan puthavathana (mahidol university, bangkok, thailand). virus stocks were prepared by infecting vero cells (african green monkey kidney; atcc, manassas, va) and aliquots were stored at 280uc. virus titres were determined as 50% tissue culture infectious dose (tcid 50 /ml) in confluent vero cells in 96-well microtiter plates. a549 cells (human lung carcinoma; atcc: ccl-185, obtained from lgc standards gmbh, wesel, germany) were grown at 37uc in minimal essential medium (mem) supplemented with 10% fbs, 100 iu/ml of penicillin and 100 mg/ml streptomycin. human monocytes were isolated from buffy coats of healthy donors, obtained from institute of transfusion medicine and immune haematology, german red cross blood donor center, johann wolfgang goethe-university, frankfurt am main. after centrifugation on ficoll (biocoll)-hypaque density gradient (biochrom ag, berlin, germany), mononuclear cells were collected from the interface and washed with pbs. then, monocytes were isolated using magnetically labeled cd14 microbeads (miltenyi biotec gmbh, bergisch gladbach, germany) following the manufacturer's instructions. monocytes were cultivated in imdm supplemented with 10% pooled human serum, 100 iu/ml of penicillin, and 100 mg/ml streptomycin. the cellular viability was assessed on confluent cell layers with celltiter-gloh luminescent cell viability assay (promega gmbh, mannheim, germany) according to the manufacturers' protocol. cell viability was expressed as percentage of non-treated control. to determine intracellular np localisation, h5n1-infected a549 were fixed 8 hours p.i. for 15 min with ice-cold acetone/ methanol (40:60, mallinckrodt baker b.v., deventer, the netherlands) and stained with a mouse monoclonal antibody (1 h incubation, 1:1000 in pbs) directed against the influenza a virus nucleoprotein (np) (millipore, molsheim, france). an alexa fluor 488 goat anti-mouse igg (h&l) (invitrogen, eugene, oregon, usa) was used (1 h incubation, 1:1000 in pbs) as secondary antibody. nuclei were stained using 49,6-diamidino-2phenylindole (dapi) (sigma-aldrich chemie gmbh, munich, germany). fluorescence was visualised using olympus ix 1 fluorescence microscope (olympus, planegg, germany). for flow cytometric analysis, the same antibodies were used. the cytopathogenic effect (cpe) reduction assay was performed as described before [34] . confluent a549 cell monolayers grown in 96-well microtitre plates were infected with influenza a strains at the indicated multiplicities of infection (mois). after a one hour adsorption period, cells were washed to remove non-detached virus. the virus-induced cpe was recorded at 24 h post infection (p.i.). unless otherwise stated, a549 cells were continuously treated with glycyrrhizin starting with a 1 h pre-incubation period. for time-ofaddition experiments, glycyrrhizin was added exclusively during the 1 h pre-incubation period, exclusively during the 1 h adsorption period, or after exclusively after the wash-out of input virus. total rna was isolated from cell cultures using tri reagent (sigma-aldrich, munich, germany). real time pcr for h5 was performed using described methods [35] . the following primers were used: sense 59 acg tat gac tac ccg cag tat tca g 39; antisense 59 aga cca gcy acc atg att gc 39; probe 6-fam-tca aca gtg gcg agt tcc cta gca-tamra. the fraction of cells with fractional dna content (''sub-g1'' cell subpopulation) indicates cytotoxicity. sub-g1 cells are considered to be dead (usually apoptotic) cells. cells were fixed with 70% ethanol for two hours at 220uc. the cellular dna was stained using propidium iodide (20 mg/ml) and analysed by flow cytometry (facscalibur, bd biosciences, heidelberg, germany). caspase activation was measured using the caspase-glo 8, 9, or 3/7 assays (promega, mannheim, germany) following the manufacturer's instructions. cell culture supernatants were collected and frozen at 280uc. cytokines/chemokines were quantified by specific elisa duo sets (r&d systems gmbh, wiesbaden, germany) following the manufacturer's instructions. nfkb activity was investigated in h5n1 (moi 0.01)-infected cells by quantification of the nfkb subunits rel a (p65) and nfkb1 (p50) from nuclear extracts using the transam tm transcription factor dna-binding elisas (active motif, rixensart, belgium). nuclear extract were prepared using the nuclear extract kit (active motif, carlsbad, ca, usa) following the manufacturer's instruction. cell culture supernatants were investigated for chemotactic activity by measurement of the activity to induce monocyte migration through membrane inserts in 24-well plates (pore size 8 mm; bd biosciences, heidelberg, germany). monocytes (1610 6 in 100 ml of imdm with 10% pooled human serum) were added into the cell culture inserts (upper chamber) and cell culture supernatants (300 ml), were added to the lower chamber of the well. after a 48 h incubation period, cells were fixed with 4% paraformaldehyde and permeabilised with pbs containing 0.3% tritron x-100. then, nuclei were stained with 49,6-diamidino-2phenylindole (dapi). the upper side of the membrane was wiped with a wet swab to remove the cells, while the lower side of the membrane was rinsed with pbs. the number of cells at the lower side of each membrane was quantified by counting of cells from three randomly chosen sections (3.7 mm 2 ) using an olympus ix 1 fluorescence microscope (olympus, planegg, germany). cells were lysed in triton x-sample buffer and separated by sds-page. nuclear extract were prepared using the nuclear extract kit (active motif, carlsbad, ca, usa) following the manufacturer's instruction. proteins were detected using specific antibodies against bactin (sigma-aldrich chemie gmbh, munich, germany), jnk, phosphorylated jnk, p38, or phosphorylated p38, (all purchased from new england biolabs gmbh, frankfurt am main, germany) and were visualised by enhanced chemiluminescence using a commercially available kit (amersham, freiburg, germany). reactive oxygen species (ros) were detected using the image-it live green reactive oxygen species kit (molecular probes, distributed by invitrogen, karlsruhe, germany). two groups were compared by t-test. more groups were compared by anova with subsequent student-newman-keuls test. the a549 cell line, derived from a human pulmonary adenocarcinoma, is an established model for type ii pneumocytes [36] , and commonly used for the investigation of the effect of influenza viruses on this cell type [see e.g. 6,37,38]. if not otherwise stated, glycyrrhizin was continuously present in cell culture media starting with a 1 h preinfection period. glycyrrhizin 200 mg/ml (the maximum tested concentration) did not affect a549 cell viability (data not shown) but clearly decreased cpe formation in a549 cells infected with the h5n1 influenza strain a/thailand/1(kan-1)/04 at mois of 0.01, 0.1 or 1 ( figure 1a ). similar results were obtained in a549 cells infected with strain a/vietnam/1203/04 (h5n1) (suppl. figure 1a) . staining of a549 cells for influenza a nucleoprotein 24 h after infection with strain h5n1 a/thailand/1(kan-1)/04 indicated that glycyrrhizin 200 mg/ml significantly reduces the number of influenza a nucleoprotein positive cells ( figure 1b) . to examine the influence of glycyrrhizin on virus progeny, a549 cells were infected with the h5n1 influenza strain a/ thailand/1(kan-1)/04 at moi 0.01 or moi 1 and infectious virus titres were determined 24 h post infection ( figure 1c ). while glycyrrhizin in concentrations up to 50 mg/ml did not affect h5n1 replication, moderate effects were exerted by glycyrrhizin 100 mg/ ml and more pronounced effects by glycyrrhizin 200 mg/ml (moi 0.01: 13-fold reduction, moi 1: 10-fold reduction). next, influence of glycyrrhizin on h5n1 replication was confirmed by the detection of viral (h5) rna using quantitative pcr. only glycyrrhizin concentrations $100 mg/ml significantly reduced figure 1b) or h5n1 a/vietnam/1203/04-infected (suppl. figure 1c ) a549 cells (moi 0.01) 24 h post infection. time-of-addition experiments revealed that maximal effects were achieved when glycyrrhizin was continuously present starting with a 1 h pre-incubation period ( figure 1d ). addition of glycyrrhizin post infection showed reduced antiviral effects while pre-incubation alone or glycyrrhizin addition during the adsorption period did not significantly affect h5n1 replication. for investigation of h5n1-induced cytokine expression, five pro-inflammatory genes were chosen that had been correlated to severity of influenza disease: cxcl10 (also known as interferon-cinducible protein 10, ip-10), interleukin 6 (il6), interleukin 8, (il8; also known as cxcl8), ccl2 (also known as monocyte chemoattractant protein 1, mcp-1), and ccl5 (also known as rantes). a549 cells were infected with h5n1 a/thailand/ 1(kan-1)/04 or h5n1 a/vietnam/1203/04 at moi 0.01, 0.1, or 1. glycyrrhizin treatment was performed with 25, 50, 100, or 200 mg/ml. cytokine expression was detected 24 h post infection by elisa. glycyrrhizin did not affect cytokine expression of noninfected cells (data not shown) but inhibited expression of all cytokines investigated in h5n1-infected cells in a dose-dependent manner (figure 2, figure 3a ). effects were more pronounced at lower mois. notably, expression of all cytokines except il8 was significantly inhibited after treatment with glycyrrhizin 50 mg/ml figure 3a ) although these glycyrrhizin concentrations had no effect on h5n1 replication in a549 cells (figure 1, figure s1 ). cytokine expression by influenza a virus-infected respiratory cells causes recruitment of peripheral blood monocytes into the lungs of patients where they differentiate to macrophages which are thought to contribute to influenza a virus pathogenicity [5, 39] . in a chemotaxis assay, the influence of glycyrrhizin was investigated on migration of monocytes towards supernatants of h5n1 a/thailand/1(kan-1)/04 (moi 0.1)-infected a549 cells through 8 mm filters. monocyte migration towards supernatants of h5n1-infected cells was strongly increased relative to migration towards supernatants of non-infected cells. treatment of h5n1infected cells with glycyrrhizin 100 mg/ml clearly suppressed chemoattraction activity of supernatants ( figure 3b ). influenza viruses including h5n1 have been shown to induce caspase-dependent apoptosis in airway cells and this apoptosis has been correlated to the virus pathogenicity [40, 41] . glycyrrhizin concentrations up to 200 mg/ml did not affect caspase activation in non-infected cells ( figure 4a-c) . glycyrrhizin concentrations $100 mg/ml inhibited h5n1 a/thailand/1(kan-1)/04 (moi 0.01)-induced activation of the initiator caspases 8 and 9 as well as of the effector caspases 3/7 in a549 cells as determined 24 h post infection ( figure 4a-c) . lower glycyrrhizin concentrations did not affect h5n1-induced apoptosis. the detection of cells in sub-g1 phase resulted in similar findings ( figure 4d ). substances that inhibit h5n1-induced caspase 3 activation including caspase 3 inhibitors cause nuclear retention of rnp complexes [34, 42] . in accordance, glycyrrhizin also interfered with nuclear export rnp at moi 1 ( figure s2 ). similar results were obtained in moi 0.01 h5n1 a/thailand/1(kan-1)/04infected cells ( figure s3 ). influence of glycyrrhizin on h5n1-induced activation of nuclear factor kb (nfkb), p38, and on h5n1-induced cellular reactive oxygen species (ros) formation activation of nfkb, p38, and jnk have been associated with influenza a virus replication and virus-induced pro-inflammatory gene expression [34, [43] [44] [45] [46] [47] . while glycyrrhizin did not influence nfkb activity in non-infected a549 cells in the tested concentra-tions (data not shown), glycyrrhizin inhibited nfkb activation in h5n1-infected cells ( figure 5a ). moreover, glycyrrhizin inhibited h5n1-induced phosphorylation of the mapks p38 and jnk ( figure 5b ). in addition to their roles during influenza a virus replication and virus-induced cytokine/chemokine expression, nfkb, p38, and jnk are constituents of redox-sensitive signalling pathways [48] [49] [50] [51] . antioxidants had been already found to interfere with influenza a virus-induced signalling through nfkb, p38, and jnk, with influenza a virus replication, and with influenza a virus-induced pro-inflammatory gene expression [32] [33] [34] . since glycyrrhizin is known to exert antioxidative effects [26] we speculated that glycyrrhizin may interfere with h5n1-induced ros formation. indeed glycyrrhizin exerted clear antioxidative effects in h5n1 (moi 0.01)-infected cells ( figure 5c ) causing significant reduction of ros formation already at a concentration of 25 mg/ml ( figure 5d ). here, we show that glycyrrhizin inhibits the replication of highly pathogenic h5n1 influenza a virus, h5n1-induced apoptosis, and h5n1-induced expression of pro-inflammatory cytokines in lung-derived a549 cells. after intravenous administration, achievable plasma concentrations of glycyrrhizin have been described to be about 100 mg/ml [52] . therefore, the glycyrrhizin concentrations found to interfere with h5n1 replication and h5n1-induced pro-inflammatory gene expression in the present report are in the range of therapeutic plasma levels. notably, although higher glycyrrhizin concentrations were needed to interfere with sars coronavirus replication [22] than with h5n1 replication, beneficial results were reported in glycyrrhizin (snmc)-treated sars patients in comparison to sars patients who did not receive glycyrrhizin [23] . notably, investigation of different glycyrrhizin derivatives against sars coronavirus led to the identification of compounds with enhanced antiviral activity [53] . therefore, glycyrrhizin might also serve as lead structure for the development of novel anti-influenza drugs. experimental results suggested that glycyrrhizin might be able to affect seasonal influenza a virus disease by antiviral and immunomodulatory effects [26, 27] . mice were prevented from lethal h2n2 infection by glycyrrhizin although no influence on virus replication was detected. the mechanism was suggested to be induction of interferon-c in t-cells by glycyrrhizin [54] . moreover, glycyrrhizin was shown to influence seasonal influenza a virus replication through interaction with the cell membrane [25, 28] . however, these effects were observed only in concentrations $200 mg/ml when glycyrrhizin was added during the virus adsorption period. since glycyrrhizin addition during the adsorption period did not influence h5n1 replication in our experiments it appears not likely that membrane effects contribute to anti-h5n1 effects detected here in lower concentrations. our results rather suggest that glycyrrhizin interferes with h5n1-induced oxidative stress. influenza a virus (including h5n1) infection induces ros formation. antioxidants were found to inhibit influenza a virus replication and influenza a virus-induced pro-inflammatory gene expression [32] [33] [34] and glycyrrhizin is known to exert antioxidative effects [26] . here, glycyrrhizin interfered with h5n1-induced activation of nfkb, p38, and jnk representing redox-sensitive signalling events [48] [49] [50] [51] involved in influenza a virus replication and influenza a virusinduced cellular cytokine/chemokine production [34, [43] [44] [45] [46] 55] . glycyrrhizin 50 mg/ml significantly reduced h5n1-induced activation of nfkb. in addition, glycyrrhizin concentrations as low as 25 mg/ml effectively interfered with h5n1-induced ros formation and with phosphorylation of the redox-sensitive mapks p38 and jnk. in our model, activation of p38 appears to be critical for h5n1-associated redox signalling since p38 inhibition had been shown before to mimick effects of the antioxidant n-acetyl-cysteine (nac) [34] . interestingly and in contrast to glycyrrhizin, nac failed to inhibit h5n1 replication or h5n1-induced cytokine/chemokine expression in therapeutically relevant concentrations. glycyrrhizin diminished h5n1-induced cellular cytokine/ chemokine production in concentrations (#50 mg/ml) that did not interfere with h5n1 replication although redox-sensitive signalling pathways have been described to be involved in both processes. therefore, h5n1-induced proinflammatory gene expression appears to be more sensitive to inhibition of ros formation than h5n1 replication. indeed, influenza viruses had been shown to induce cellular pathways through replicationdependent and -independent events [56] . in a previous report, we could show that similar glycyrrhizin concentrations like those investigated here interfered with h5n1-induced pro-inflammatory gene expression but not with h5n1 replication in human monocyte-derived macrophages [57] . in addition, other immunomodulatory treatment regimens that did not influence h5n1 replication reduced mortality in h5n1-infected mice [31, 58] . therefore, glycyrrhizin represents a potential additional treatment option that interfers with both h5n1 replication and h5n1induced expression of pro-inflammatory cytokines in lung cells. interference with immune responses may also result in the loss of control of virus replication by cytotoxic immune cells including natural killer cells and cytotoxic cd8 + t-lymphocytes. global immunosuppressants like corticosteroids failed to protect from lethal influenza virus infection [59] . moreover, antiviral drugs may interfere with cytotoxic cells that control virus replication as demonstrated for ribavirin that was shown to hamper nk cell cytolytic activity [60] . in this context, glycyrrhizin had already been shown not to affect natural killer cell activity in the concentrations used here [57] . in conclusion, we show in this report that therapeutic concentrations of glycyrrhizin (used as clinically approved parenteral preparation snmc) interfere with highly pathogenic h5n1 influenza a virus replication and h5n1-induced proinflammatory gene expression at least in part through interference with h5n1-induced ros formation and in turn reduced activation of p38, jnk, and nfkb in lung cells. since we used the clinical formulation snmc effects of other ingredients like glycin or cystein cannot be excluded. vaccines and antiviral agents will fail to meet global needs at least at the beginning of a severe influenza a virus pandemic [61] . anti-inflammatory and immunomodulatory agents are considered to be important candidates as constituents of anti-influenza treatment strategies that may save lives in an influenza pandemic situation [61] . therefore, glycyrrhizin may complement the arsenal of potential drugs for the 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the authors thank mrs. kerstin euler, mrs. gesa meincke, and mrs. christina matreux for technical support. key: cord-003498-4ct0ywnw authors: bdeir, najat; arora, prerna; gärtner, sabine; hoffmann, markus; reichl, udo; pöhlmann, stefan; winkler, michael title: a system for production of defective interfering particles in the absence of infectious influenza a virus date: 2019-03-01 journal: plos one doi: 10.1371/journal.pone.0212757 sha: doc_id: 3498 cord_uid: 4ct0ywnw influenza a virus (iav) infection poses a serious health threat and novel antiviral strategies are needed. defective interfering particles (dips) can be generated in iav infected cells due to errors of the viral polymerase and may suppress spread of wild type (wt) virus. the antiviral activity of dips is exerted by a di genomic rna segment that usually contains a large deletion and suppresses amplification of wt segments, potentially by competing for cellular and viral resources. di-244 is a naturally occurring prototypic segment 1-derived di rna in which most of the pb2 open reading frame has been deleted and which is currently developed for antiviral therapy. at present, coinfection with wt virus is required for production of di-244 particles which raises concerns regarding biosafety and may complicate interpretation of research results. here, we show that cocultures of 293t and mdck cell lines stably expressing codon optimized pb2 allow production of di-244 particles solely from plasmids and in the absence of helper virus. moreover, we demonstrate that infectivity of these particles can be quantified using mdck-pb2 cells. finally, we report that the di-244 particles produced in this novel system exert potent antiviral activity against h1n1 and h3n2 iav but not against the unrelated vesicular stomatitis virus. this is the first report of dip production in the absence of infectious iav and may spur efforts to develop dips for antiviral therapy. influenza a virus infection is responsible for annual influenza epidemics and intermittent pandemics that are associated with significant morbidity and mortality [1] . the ability of iav to constantly change in response to immune pressure or antiviral treatment limits the effectiveness of currently used antiviral interventions. thus, vaccines against seasonal influenza need to be annually reformulated and will provide little if any protection against pandemic influenza [1] . moreover, the effectiveness of antivirals targeting the viral proteins m2 and neuraminidase a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 is compromised by the frequent emergence and transmission of resistance mutations [1, 2] . therefore, novel approaches to combat influenza are urgently needed. iavs are enveloped and harbor eight segments of genomic viral rna. defective interfering (di) genomic segments can be generated in iav infected cells due to errors of the viral polymerase [3, 4] . di segments usually harbor a large deletion which inactivates the open reading frame encoded by the segment [3, 4] . the di segments can interfere with amplification of wild type (wt) segments, potentially by competing for viral and cellular resources required for segment replication. moreover, di rnas can be packaged into progeny virions, termed defective interfering particles (dips), and coinfection of target cells with dips and iav will result in preferential amplification of dips and suppression of iav spread [3, 4] . this effect has been observed in cell culture [5] [6] [7] [8] and in experimentally infected animals [5, [9] [10] [11] [12] [13] [14] [15] and may extend to unrelated viruses [14, 16] , due to the activation of the interferon system [15, 16] . moreover, dip application in a therapeutic or preventive setting prevents or ameliorates influenza in animal models [3] [4] [5] [10] [11] [12] [13] [14] [15] [16] . in sum, dips can be considered natural antivirals produced in the context of infection with iav and many other viruses and may provide a basis for the development of new strategies for antiviral intervention. at present, amplification of dips requires coinfection of cells with dips and wt virus, termed standard or helper virus, which subsequently needs to be inactivated by uv light [3, 4, 17, 18] . the presence of standard virus poses a safety concern when products for animal and human use are generated and complicates the interpretation of experimental data. plasmid systems encoding for wt and di segments along with cell lines expressing the iav proteins for which the genomic information has been lost upon di rna formation might circumvent this issue [4, 19] . however, expression of the viral polymerase subunit pb2 in trans was found to be insufficient for robust amplification of iav variants harboring temperature sensitive mutations [20, 21] and it has been speculated that similar limitations might apply to the production of dips [4] . moreover, it has been suggested that pb2 expression might be toxic to cells [4] . therefore, it is currently unknown whether the strategy outlined above might allow for production of segment 1-derived dips and at present no system for generation of dips in the absence of standard virus has been reported. di-244 is a naturally occurring di-rna found in hen's eggs [22] . di-244 is derived from segment 1, which encodes pb2, and harbors a 1,946 nucleotides comprising deletion [4, 22] . this deletion removes most of the pb2 orf but leaves the 3' 244 nucleotides and 5' 151 nucleotides of segment 1 intact which are sufficient for segment replication and packaging [4, 22] . here, we investigated whether coexpression of wt segments 2-8, pb2 protein and di-244 rna allows for production of dips. employing a novel di-244 variant encoding mscarlet-i, we show that di-244-based dips are efficiently produced in cells expressing a codon optimized version of pb2 and that these dips exert potent antiviral activity. plasmids for rescue of the a/pr/8/34 (h1n1) strain, phw191-phw198, were used throughout this study and have been previously described [23] . to generate a retroviral vector encoding pb2, the pb2 open reading frame was amplified from phw191 using primers pb2-qcx ip-5n (5-ccgcggccgcaccatggaaagaataaaagaactac-3) and pb2-3xbgl (5-gg agatctcgagctaattgatggccatccgaat-3) and cloned into the retroviral vector pqcxip-mcs using noti and xhoi [24] . this self-inactivating vector allows constitutive expression of pb2 and puromycin resistance genes coupled by an internal ribosome entry site (ires). an optimized sequence of pb2 was generated by hand to maximize sequence deviation from pb2 and optimizing codon usage for influenza a virus and humans (s1 fig). this sequence was synthesized and cloned by geneart (regensburg, germany) and subcloned using noti and xhoi sites into pqcxip-mcs. a plasmid for di-244 rescue was generated by splice overlap pcr, using phw191 as template and primer pairs flua aari-pb2-1g (5-cga tcacctgctcgagggagcgaaagcaggtc-3)/iavseg1-di244rep-rev (5-aatgaggaa tcccctcagttaagcggccgctgcggtaccagatctcttctcctgtcttcctga-3) and iavseg1-di244rep-for (5-tcaggaagacaggagaagagatctggtaccgcagcggccgct taactgaggggattcctcatt-3)/flua aari-pb1-2341r (5-cgatcacctgc tctctat tagtagaaacaaggcattt-3). the product of the splice overlap pcr was then purified and amplified with the segment specific primer pair flua aari-pb2-1g/flua aari-pb1-2341r and cloned into phw2000-ggaari, using golden gate cloning, generating phw2000-di244-mcs [25] . in addition, a construct containing a multiple cloning site (mcs) was generated for later insertion of reporter genes. for this, the pcr fragments were amplified using phw191 as template and primer pairs flua aari-pb2-1g/iavseg1-di244rep-rev (5-aatgaggaatcccct cagttaagcggccgctgcggtaccagatctcttctcctgtcttcc tga-3) and iavseg1-di244rep-for (5-tcaggaagacaggagaagagatctggtaccgca gcggccgcttaactg aggggattcctcatt-3)/ flua aari-pb1-2341r followed by splice overlap joining and golden gate cloning. as reporter gene, mscarlet-i without internal sali and noti sites and fused to the porcine teschovirus-1 (ptv1) 2a sequence (gatnfsllkqagdveenpgp) was cloned into the mcs as a bglii/noti fragment. in this way, a pb2 (aa 1-41)-2a-mscarlet-i orf was generated, which allows the detection of the presence of di-244 via mscarlet-i fluorescence. the template for mscarlet-i, pmscarlet-i_c1, was a gift from dorus gadella (addgene plasmid # 85044) [26] . the integrity of pcr-amplified, cloned sequences was verified by sequence analysis. all cells were cultured at 37˚c and 5% co 2 . 293t human embryonic kidney cells and vero cells were maintained in dulbecco's modified eagle medium (dmem; gibco) containing 10% fetal bovine serum (fbs, gibco), penicillin (pen, 100 iu/ml) and streptomycin (strep, 100 μg/ ml). 293t cell lines stably expressing pb2 were grown in the presence of 1 μg/ml puromycin. madin-darby canine kidney cells (mdck) were cultured in glasgow's mem (gmem) with 10% fetal bovine serum (fbs, gibco) and pen/strep. all cell lines were obtained from collaborators and were regularly checked for mycoplasma contamination. mdck cells stably expressing pb2 or pb2opt were cultivated in the presence of 1.5 μg/ml puromycin. influenza a viruses a/panama/2007/99 (h3n2) [24] and a/pr/8/34 (h1n1) produced in embryonated chicken eggs were used to assess the antiviral activity of dips. we further employed a recombinant vesicular stomatitis virus (vsv) that expresses a dual reporter consisting of egfp and firefly luciferase from an additional transcription unit located between the open reading frames for the viral glycoprotein and polymerase [27] . the production of mlv particles for transduction of cells followed an established protocol [25, 28] . briefly, 293t cells seeded in t25 flasks were transfected with 6 μg of retroviral vector (e.g. pqcxip-pb2), 3 μg mlv-gag-pol plasmid and 3 μg vsv-g expression plasmid, employing the calcium phosphate transfection method. the culture medium was exchanged at 8 h after transfection. after 48 h, mlv particle-containing supernatant was harvested, cleared by passing through a 0.45 μm filter, aliquoted and then stored at -80˚c. for retroviral transduction, cells were seeded in 96-well plates at 5,000 (mdck) or 10,000 (293t) cells/well in 50 μl cell culture medium. on the next day, 50 μl of supernatant containing mlv particles was added per well followed by spinoculation at 4,000 × g for 30 min for enhancement of transduction [29] . two days after transduction, the cells were detached and transferred into 24-well plates containing cell culture medium supplemented with 1 μg/ml (293t) and 1.5 μg/ml (mdck) puromycin. in parallel, non-transduced cells were treated similarly to control for effective cell killing by the antibiotics. 293t were seeded at a cell density of 2 × 10 5 cells/well in 12-well plates. the following day, the cells were transfected using the calcium phosphate method. the concentrations of plasmids to be transfected were largely adapted from published work [30] : 10 ng of pcaggs plasmids encoding viral rna polymerase proteins (pb2, pb, pa) and 100 ng of plasmid encoding np were cotransfected with 50 ng of plasmid ppoli-luc, which encodes the firefly luciferase reporter gene flanked by the noncoding regions of segment 8 of a/wsn/33. empty plasmid was used to ensure that all transfections were conducted with the same total amount of plasmid dna. for analysis of functionality of pb2 in 293t cells stably expressing this protein, transfection was carried out as described above but the plasmid encoding pb2 was omitted. as control, the plasmid encoding pb1 was omitted. the cells were washed at 6-8 h after transfection and harvested at 24 h post transfection. luciferase activities in cell lysates were measured using the plate chameleon v plate reader (hidex) and microwin 2000 software. for analysis of pb2 expression in 293t and mdck cells, the cells were seeded in 6-well plates, incubated for 24 h, harvested and lysed in 200 μl of laemmli sds-page sample buffer (5% glycerine, 1% sds, 2.5% ß-mercaptoethanol, 0.5% bromophenol blue, 0.5 mm edta,0.5m tris ph 6.8). samples were heated to 95˚c for 10 min and separated via sds-page using 12.5% polyacrylamide gels. proteins were then transferred onto a nitrocellulose membrane (ge health care) using a mini-protean tetra cell (biorad) powered at 110 v for 90 minutes. membranes were blocked with 5% skimmed milk diluted in pbs-tween and incubated with primary rabbit polyclonal antibodies against pb2 (1:1,000, gentex, irvine, usa) overnight at 4˚c. subsequently, membranes were washed and incubated with anti-rabbit hrp (horseradish peroxidase)-conjugated secondary antibodies (1:10,000, dianova) for one hour. finally, chemiluminescent substrate hrp juice plus (p.j.k.) was added onto the membrane and bands were visualized using a chemocam imager (intas). in order to detect ß-actin, the membrane was subsequently stripped using stripping buffer (62.5 mm tris hcl ph 6.8, 2% sds, 100 mm ß-mercaptoethanol) for 30 min at 5˚c, washed three times with pbs-tween, and incubated with anti ß-actin mouse (1:500 sigma-aldrich) overnight. the membrane was then washed and incubated with antimouse hrp-conjugated secondary antibody (1:10,000, dianova) for one hour. hrp juice plus was added and bands were visualized as previously described. quantification of pb2 and pb2opt expression was carried out using the program imagej (fiji distribution) [31] . in order to normalize data, signals measured for pb2/pb2opt were divided by those measured for beta-actin. for dip production, a coculture of 200,000 mdck cells and 700,000 293t cells stably expressing pb2 was seeded in t25 flasks. the next day, cells were cotransfected via the calcium phosphate method with 1 μg each of plasmids encoding di-244-mscarlet-i and wt iav genomic segments 2-8. culture medium was changed at 8 h post transfection. at 48 h post transfection, cells were washed with phosphate buffered saline (pbs) without calcium and magnesium and dmem medium supplemented with 0.2% bsa (macs bsa), 0.5 μg/ml tosyl-phenylalanychloromethyl-ketone (tpck)-trypsin (sigma), penicillin (100 iu/ml) and streptomycin (100 μg/ ml) was added. as negative control, transfection of parental mdck and 293t cells was analyzed. supernatants were harvested from all cultures at 4, 6, 8 and 10 days post transfection, cleared by centrifugation at 4,000 rpm for 10 min to remove debris, aliquoted and stored at -80˚c. infectivity of supernatants was analyzed by focus formation assay as described [25, 32] but using mdck cells expressing pb2 or pb2opt as targets. in brief, mdck-pb2/pb2opt cells seeded in 96-well plates were washed and incubated for 1 h with serial dilutions of dip-containing supernatants. thereafter, supernatants were removed and infection medium (gmem with 0.2% bsa and pen/ strep) supplemented with 0.5% methylcellulose and 0.5 μg/ml tpck-trypsin was added. plates were incubated for 72 h and then stained using anti iav polyclonal antibody (millipore). images were taken on a zeiss lsm800 equipped with a 10x/0.45 plan-apochromat objective, 488 nm and 561 nm diode lasers and zen imaging software (zeiss). fluorescent signals (red channel, 561 nm laser) were detected with gaasp detector employing the same sensitivity for all images of a series, while bright field signals were recorded with an esid detector (photodiode) with individually adjusted sensitivity. to test antiviral activity of dips against iav and unrelated vsv, we performed infection experiments in the presence of dip-containing or dip-free supernatants and subsequently compared viral titers in the culture supernatants. for this, mdck cells were seeded in 96-well plates at a density of 10,000 cells/well. on the next day, dip-containing supernatants or dip-free control supernatants were 10-fold serially diluted. subsequently, mdck cells were washed twice with pbs and 50 μl of the respective supernatants were mixed with 50 μl of virus and the mixture inoculated onto the mdck cells. after a 1 h incubation, 100 μl of fresh infection medium supplemented with 0.5 μg/ml tpck-trypsin was added and the cells were further incubated for 24 h (vsv) or 72 h (iav) before viral titers in the culture supernatants were determined. virus titration was performed on confluent monolayers of mdck (iav) or vero (vsv) cells that were grown in 96-well plates. after aspiration of the culture medium, cells were washed twice with pbs and inoculated with 50 μl of 10-fold serial dilutions of the culture supernatants of iav or vsv infected mdck cells. after 1 h of incubation with iav containing supernatants, the medium was removed and 100 μl infection medium supplemented with 1% avicel and 0.5 μg/ml tpck-trypsin (iav/mdck) was added per well. after 1h incubation with vsv-containing supernatants, 200 μl infection medium supplemented with 0.5% methylcellulose (vsv/vero) were added on top, and the cells were further incubated for 24 h. iav titers were quantified by antibody staining, using the focus formation assay as previously described [25, 32] . in order to quantify vsv titers, egfp-positive foci were counted under the fluorescence microscope. all titers are given as focus forming units per ml (ffu/ml). we sought to determine whether di-244 particles can be amplified in the absence of standard virus if producer cells are engineered to express pb2. for this, we first used retroviral transduction and selection antibiotics to generate 293t and mdck cell lines stably expressing pb2. immunoblot revealed that the cell lines obtained by selection expressed robust levels of pb2 (fig 1a and fig 1b) . in order to analyze whether pb2 is functional in these cells, we employed a mini-replicon system, which measures the amplification of a firefly luciferase encoding iav reporter segment upon coexpression of pb2, pb1, pa and np [30] . we found that transfection of 293t-pb2 cells with a plasmid encoding the reporter segment alone yielded luciferase activity in the background range while cotransfection of pb2, pb1, pa and np expression plasmids increased luciferase activity more than 1,000-fold (fig 1c) . importantly, this increase was not observed when the pb1 plasmid was omitted while omission of the pb2 plasmid had no impact on reporter activity (fig 1c) . thus, the pb2 protein stably expressed in 293t cells was functional. unfortunately, similar studies in mdck cells were not feasible due to the low transfectability of these cells. we next investigated whether the 293t-pb2 and mdck-pb2 cells allowed the generation of di-244 particles, using the experimental setup depicted in fig 2a. in order to be able to visually inspect di-244 production and spread, we generated a di-244 variant that encodes for mscarlet-i, a red fluorescent protein [26] . transfection of a mixture of 293t/mdck cells with plasmids encoding iav wt segments 2-8 jointly with a plasmid encoding di-244-mscarlet-i resulted in occasional and moderate red fluorescence (fig 3a) . in contrast, frequent and prominent red fluorescence was observed in 293t-pb2/mdck-pb2 cocultures (fig 3a) , indicating that the stably expressed pb2 promoted amplification of the di-244-mscarlet-i di rna. in order to examine whether amplification of the di-244-mscarlet-i di rna resulted in the production of infectious dips, the supernatants of the transfected 293t-pb2/mdck-pb2 cells were inoculated onto mdck-pb2 cells ( fig 2b) . as controls, the supernatants were also added to mdck wt cells. inoculation of mdck-pb2 cells with supernatants from 293t-pb2/ mdck-pb2 cells resulted in infection of the target cells, as determined by expression of mscarlet-i (fig 3b) . the number of mscarlet-i-positive cells was concentration dependent and supernatants taken at 6 days post transfection from dip producing cells contained the highest amount of infectivity ( fig 3b) . finally, no cells with prominent red fluorescence were detected under control conditions, indicating that dips were only infectious for mdck-pb2 but not mdck wt cells. we next asked whether di-244 production could be quantified by focus formation assay, which is based on detection of iav antigens by antibody staining and is frequently employed to measure iav infectivity. moreover, we examined whether results obtained in the focus formation assay would match those obtained upon counting of foci based upon red fluorescence. foci were observed in mdck-pb2 but not in mdck control cells, confirming that dip infectivity requires pb2 expression in target cells. quantification of dip infectivity by focus formation assay revealed that maximum titers of roughly 1 x 10 3 dips per ml were obtained and counting red fluorescent foci yielded roughly comparable results (fig 3b and fig 3c) . thus, expression of pb2 is sufficient for di-244 production in the absence of helper virus but production efficiency is moderate. dip titers of 1 x 10 3 particles per ml are low and may limit experimentation. therefore, we next asked whether alteration of codon usage for pb2 expression might increase pb2 expression efficiency and dip production. for this, we modified the codons in the pb2 expression plasmid (s1 fig) to reflect codon preferences of human genes and iav. as a second criterion for codon choice, we opted for maximal sequence difference between the a/pr/8/ 34-based sequence previously used for pb2 expression and the newly generated, optimized pb2 sequence (pb2opt), in order to prevent potential recombination events. 293t and mdck cells were engineered to stably express pb2opt and immunoblot revealed that expression levels of pb2opt in mdck but not 293t cells were higher than those obtained upon expression of non-codon-optimized pb2 (fig 1a and fig 1b) . moreover, growth of pb2opt cells was comparable to that of control cells and pb2opt expression was readily detectable after multiple passages, suggesting that expression was not associated with overt cytotoxicity. finally, analysis of the average of five experiments conducted as described for panel a and quantified via the imagej program is shown. signals measured for pb2 or pb2opt were normalized against those measured for beta-actin. error bars indicate standard error of the mean (sem). two tailed paired students t-test was used to assess statistical significance. (c) 293t cells stably expressing pb2 were cotransfected with plasmids encoding an iav luciferase reporter segment and the indicated iav proteins. luciferase activities in cell lysates were determined at 24 h post transfection. the results of a representative experiment carried out with triplicate samples are shown. error bars indicate standard deviation. two tailed paired students t-test was used to assess statistical significance. similar results were obtained in three separate experiments. c.p.s., counts per second. https://doi.org/10.1371/journal.pone.0212757.g001 new system for production of defective interfering particles 293t-pb2opt cells in the mini-replicon assay showed that pb2opt supported iav segment replication (fig 4) . next, we examined whether pb2opt supports dip production with higher efficiency than unmodified pb2. efficient di-244-mscarlet-i di rna amplification was observed in transfected pb2opt cells (not shown) and supernatants obtained from these cells were highly infectious for mdck-pb2opt cells even when diluted 1:1,000 (fig 5a) . in contrast, the supernatants were not infectious for mdck cells (fig 5a) . moreover, a direct comparison of 293t-pb2/mdck-pb2 and 293t-pb2opt/mdck-pb2opt cells for production of infectious dips and for dip amplification upon infection revealed that the pb2opt cells were more efficient. thus, more red fluorescent cells were observed when supernatants from pb2 expressing cells were added to mdck-pb2opt as compared to mdck-pb2 cells (fig 5b) . similarly, supernatants from pb2opt cells were more infectious for target mdck-pb2opt cells as compared to mdck-pb2 cells. in keeping with this observation, quantification of production of infectious dips by focus formation assay and counting of red fluorescent cells revealed that at least 80% of foci (identified by antibody staining) were positive for mscarlet-i, as expected, and that pb2opt cells produced up to 4 x 10 6 infectious dips per ml and thereby exceeded titers obtained with pb2 cells (2,5 x 10 3 ) by~1,500-fold (fig 5c and fig 5d) . di-244 can inhibit spread of diverse iavs and, likely via induction of interferon (ifn), may also inhibit spread of unrelated viruses [3, 4] . in order to investigate the antiviral activity of di-244-mscarlet-i, we first analyzed whether di-244-mscarlet-i produced in pb2opt cells interfered with the spread of a homologous iav, a/pr/8/34, in mdck cells (fig 2c) . for this, mdck cells were coinfected with the indicated dilutions of di-244 containing supernatants and a/pr/8/34 at an moi of 0.1, 0.01 and 0.001 (fig 6a) . this resulted in iav/dip ratios of approximately 1:10 (undiluted dip containing supernatants, iav at moi 0.1), 1:100 (undiluted dip containing supernatants, iav at moi 0.01) and 1:1,000 (undiluted dip containing supernatants, iav at moi 0.001), respectively. the supernatants from 293t/mdck wt cells transfected with plasmids for di-244 production were used as negative control. the control supernatants did not appreciably interfere with a/pr/8/34 infection while supernatants from pb2opt cells efficiently blocked iav infection in a concentration dependent manner, with highest antiviral activity observed at an iav/dip ratio of 1:1,000 (fig 6a) . specifically, infection efficiency relative to untreated virus (set as 100%) was 1 ± 0.5% in the presence of dip containing supernatants at a dilution of 10 0 and 93 ± 13% in the presence of control supernatants (average of six independent experiments). moreover, di-244 containing supernatants also inhibited infection by a/panama/2007/99 (h3n2) in a concentration dependent manner (fig 6b) , in keeping with the concept that di-244 exerts broad anti-iav activity [3, 4] . finally, di-244 containing supernatants did not inhibit vsv infection (fig 6c) , indicating that di-244 neither interfered with vsv genome replication nor altered viral control by a potential ifn response in mdck cells. these results show that di-244 produced in pb2opt expressing cells exerts potent anti-iav activity. for production of dips (di-244-mscarlet-i), a coculture of 293t-pb2 and mdck-pb2 cells was cotransfected with plasmids harboring di-244-mscarlet-i and the wt iav genomic segments two to eight. subsequently, trypsin was added for ha activation and supernatants were harvested at the indicated time points. (b) for quantification of dip production, mdck-pb2 cells were inoculated with dip containing supernatants and the number of red cells was counted or the number of foci was determined using focus formation assay. (c) for analysis of antiviral activity of dips, mdck cells were coinfected with iav wt and dips followed by focus formation assay. https://doi.org/10.1371/journal.pone.0212757.g002 the generation of dips in iav infected cells has been recognized by von magnus several decades ago [33] and dips hold promise as novel antiviral agents [3, 4] . however, exploitation of dips for antiviral therapy requires efficient production systems that do not depend on the presence of standard virus. here, we report a di-244 variant encoding a fluorescent protein that permits monitoring of dip production. moreover, we demonstrate that cells expressing microscopy. (c) the number of infected cells (as determined by red fluorescence) in panel b was quantified. in parallel, infection of cells was analyzed by focus formation assay and the number of foci quantified. the results of a representative experiment are shown in panels a-c and were confirmed in two separate experiments. https://doi.org/10.1371/journal.pone.0212757.g003 two tailed paired students t-test was used to assess statistical significance. c.p.s., counts per second. https://doi.org/10.1371/journal.pone.0212757.g004 new system for production of defective interfering particles pb2 allow generation of infectious di-244 particles solely from plasmids and in the absence of standard virus. finally, our study shows that dips produced in this system suppress spread of different iav subtypes but not vsv in cell culture. di-244 particles and other dips have so far been amplified in cell culture or hen's eggs in the presence of standard virus [3, 4, 17] . in addition, production of di-244 particles from a plasmid system has been described [34, 35] . this approach relies on the transfection of plasmids for production of infectious iav in conjunction with a plasmid containing the di-244 segment and results in the co-production of dips and standard virus [34, 35] . before dip preparations produced in these systems can be used for experimentation, the remaining standard virus needs to be inactivated by uv light [18] . this approach builds on the preferential inactivation of standard virus relative to dips. thus, a mutation in a gene essential for viral spread will abrogate infectivity of standard virus but may have no effect on dip infectivity since the missing proteins will be complemented in trans in cells coinfected with dips and standard virus. however, controlling the efficiency of uv inactivation of standard virus is technically challenging. moreover, the effect of uv light on dip infectivity is difficult to determine and both issues may complicate large scale production of dips as well as interpretation of experimental data and animal trials. thus, establishment of novel cell culture systems for dip production in the absence of standard virus is an important task. our results show that cell lines expressing pb2 allow production and quantification of di-244 particles solely from plasmids and in the absence of standard virus. this finding was not expected given that several reports indicate that pb2 expression alone is insufficient to allow robust spread of iav variants with temperature sensitive mutations in the pb2 gene at nonpermissive temperatures [20, 21] . moreover, it has been suggested that pb2 expression might be associated with unwanted cytotoxic effects [4] . the present study suggests that up to 4 x 10 6 di-244 particles/ml can be produced in cells expressing codon optimized pb2, which roughly translates into production of 10 infectious dips per cell, and it can be speculated that efficiency of dip production can be further increased by employing cell lines stably coexpressing pb1, pb2 and pa. occasionally, weak fluorescence has been observed in dip inoculated control cells. this is most likely attributable to low levels of di-244 mrna expression facilitated by pb2 protein associated with di-244 vrna present in the infecting dips. in contrast, no evidence for production of infectious iav due to recombination between the di-244 rna and the rna encoding for pb2 was obtained, as judged by bright field microscopy, immunofluorescence, focus formation assay and rt-pcr analysis, indicating that the dip production system reported here is safe. quantification of dip production so far relied on quantitative rt-pcr and hemagglutination assay [4, 8, 17] , which do not provide information on particle infectivity. this limitation has been overcome by the present study which demonstrates that infectivity of di-244 particles can be quantified using a standard technique, focus formation assay. the availability of this new system for production of defective interfering particles method should help comparing results obtained with different di-244 preparations or other segment 1 dips and should thus advance the development of dips as antiviral agents. in this context, it is noteworthy that a iav/dip ratio of 1:1,000 resulted in the most prominent antiviral activity in our hands and a very similar ratio, 1:3,400 (as determined by estimations based on quantitative rt-pcr (dip) and infectious units (iav)), was previously reported to be the average of three (moi 0.1, moi 0.01) and six (moi 0.001), respectively, independent experiments is shown. infection in the absence of supernatants was set as 100%. error bars indicate standard error of the mean (sem). two tailed paired students t-test was used to assess statistical significance. (b) the experiment was carried out as described for panel a but a/panama/2007/99 (h3n2) was used for infection. the average of three independent experiments is shown. infection in the absence of supernatants was set as 100%. error bars indicate sem. two tailed paired students t-test was used to assess statistical significance. (c) the experiment was carried out as for panel a but cells were infected with gfp-encoding vsv and supernatants were harvested for titration at 24 h post infection. the results of a single representative experiment conducted with triplicate samples are shown and were confirmed in two separate experiments. https://doi.org/10.1371/journal.pone.0212757.g006 minimally required to protect mice from severe influenza [4] . thus, our study confirms and extends published work indicating that dips have to be provided in vast excess to exert antiviral activity. whether sufficient numbers of dips can be delivered to the human respiratory tract and remain stable to provide protection against influenza for a prolonged time remains to be determined. in this context, one can speculate that an iav:dip ratio of less than 1:1,000 might be sufficient for antiviral activity in humans, since dips might exert direct antiviral activity by inhibiting iav genome replication and induce the ifn system. moreover, dips were reported to have a long residence time in the respiratory tract of mice and dip-treated animals were found to still be protected at one week after treatment [4, 35] . thus, dip stability in the respiratory tract might not pose a major hurdle to the use of dips for influenza prevention and therapy in humans. finally, it should be stated that reassortment of dips with iav in coinfected cells is likely to occur. however, if dips based on the low pathogenic a/pr/8/34 or related viruses are used (like in the present study), such reassortment events should not result in viruses with increased transmissibility or virulence as compared to the wt virus. it is believed that di-244 can interfere with spread of diverse iav in cell culture due to genome competition [3, 4] . indeed, di-244 produced in pb2opt cells exerted comparable antiviral activity against h1n1 and h3n2 iav (no statistically significant differences), in keeping with h3n2 polymerase complexes being fully functional on h1n1 genomic segments [36] . this matches data published for di-244 generated by use of standard virus [35] and demonstrates that dips produced in pb2 expressing cells are fully functional, although the activity of purified dips remains to be examined. di-244 can also interfere with the spread of influenza b virus (ibv) and unrelated respiratory viruses in the infected host and this is thought to be due to induction of innate immune responses, particularly the ifn response [14, 16] . in contrast, dip-mediated inhibition of ibv infection in cell culture is not observed, due to absence of genome competition [13, 14] . the absence of antiviral activity of dips against vsv confirms lack of genome competition. moreover, it suggests that dips might not have modulated a potential ifn response in mdck cells, although it should be noted that such a response might have been impeded due to the presence of trypsin in the culture medium [37] . collectively, we report, to our knowledge, the first experimental system for production of dips without standard virus and for quantification of dip infectivity, which should promote efforts to develop dips for antiviral therapy. winkler. drug resistance in influenza a virus: the epidemiology and management defective interfering influenza virus rnas: time to reevaluate their clinical potential as broad-spectrum antivirals? cloned defective interfering influenza rna and a possible pan-specific treatment of respiratory virus diseases dual-functional peptide with defective interfering genes effectively protects mice against avian and seasonal influenza a defective interfering influenza rna inhibits infectious influenza virus replication in human respiratory tract cells: a potential new human antiviral attempts to detect homologous autointerference in vivo with influenza virus and vesicular stomatitis virus continuous influenza virus production in cell culture shows a periodic accumulation of defective interfering particles cloned defective interfering influenza virus protects ferrets from pandemic 2009 influenza a virus and allows protective immunity to be established the influence of defective-interfering particles of the pr-8 strain of influenza a virus on the pathogenesis of pulmonary infection in mice interfering vaccine (defective interfering influenza a virus) protects ferrets from influenza, and allows them to develop solid immunity to reinfection in vivo antiviral activity: defective interfering virus protects better against virulent influenza a virus than avirulent virus defective interfering virus protects elderly mice from influenza a novel broad-spectrum treatment for respiratory virus infections: influenza-based defective interfering virus provides protection against pneumovirus infection in vivo defective interfering influenza virus confers only short-lived protection against influenza virus disease: evidence for a role for adaptive immunity in di virus-mediated protection in vivo defective interfering influenza a virus protects in vivo against disease caused by a heterologous influenza b virus cell culture-based production of defective interfering particles for influenza antiviral therapy homologous interference mediated by defective interfering influenza virus derived from a temperature-sensitive mutant of influenza virus replication-incompetent influenza a viruses that stably express a foreign gene expression of a functional influenza viral cap-recognizing protein by using a bovine papilloma virus vector expression of the three influenza virus polymerase proteins in a single cell allows growth complementation of viral mutants characterization of putative defective interfering (di) a/wsn rnas isolated from the lungs of mice protected from an otherwise lethal respiratory infection with influenza virus a/ wsn (h1n1): a subset of the inoculum di rnas eight-plasmid system for rapid generation of influenza virus vaccines tetherin sensitivity of influenza a viruses is strain specific: role of hemagglutinin and neuraminidase influenza a virus encoding secreted gaussia luciferase as useful tool to analyze viral replication and its inhibition by antiviral compounds and cellular proteins mscarlet: a bright monomeric red fluorescent protein for cellular imaging a gxxxa motif in the transmembrane domain of the ebola virus glycoprotein is required for tetherin antagonism ifitm proteins inhibit entry driven by the mers-coronavirus spike protein: evidence for cholesterol-independent mechanisms human immunodeficiency virus type 1 spinoculation enhances infection through virus binding the viral nucleoprotein determines mx sensitivity of influenza a viruses fiji: an open-source platform for biological-image analysis influenza a virus does not encode a tetherin antagonist with vpu-like activity and induces ifn-dependent tetherin expression in infected cells incomplete forms of influenza virus defective segment 1 rnas that interfere with production of infectious influenza a virus require at least 150 nucleotides of 5' sequence: evidence from a plasmid-driven system influenza virus protecting rna: an effective prophylactic and therapeutic antiviral seasonal h3n2 and 2009 pandemic h1n1 influenza a viruses reassort efficiently but produce attenuated progeny trypsin promotes efficient influenza vaccine production in mdck cells by interfering with the antiviral host response we thank robert webster for the 8-plasmid system for pr8 (phw191-phw198) and georg kochs and martin schwemmle for plasmids for the replicon assay and defense advanced research projects agency (darpa, intercept program) for support. conceptualization: stefan pöhlmann, michael winkler. key: cord-280846-bbv6f5gf authors: greninger, alexander l.; chen, eunice c.; sittler, taylor; scheinerman, alex; roubinian, nareg; yu, guixia; kim, edward; pillai, dylan r.; guyard, cyril; mazzulli, tony; isa, pavel; arias, carlos f.; hackett, john; schochetman, gerald; miller, steve; tang, patrick; chiu, charles y. title: a metagenomic analysis of pandemic influenza a (2009 h1n1) infection in patients from north america date: 2010-10-18 journal: plos one doi: 10.1371/journal.pone.0013381 sha: doc_id: 280846 cord_uid: bbv6f5gf although metagenomics has been previously employed for pathogen discovery, its cost and complexity have prevented its use as a practical front-line diagnostic for unknown infectious diseases. here we demonstrate the utility of two metagenomics-based strategies, a pan-viral microarray (virochip) and deep sequencing, for the identification and characterization of 2009 pandemic h1n1 influenza a virus. using nasopharyngeal swabs collected during the earliest stages of the pandemic in mexico, canada, and the united states (n = 17), the virochip was able to detect a novel virus most closely related to swine influenza viruses without a priori information. deep sequencing yielded reads corresponding to 2009 h1n1 influenza in each sample (percentage of aligned sequences corresponding to 2009 h1n1 ranging from 0.0011% to 10.9%), with up to 97% coverage of the influenza genome in one sample. detection of 2009 h1n1 by deep sequencing was possible even at titers near the limits of detection for specific rt-pcr, and the percentage of sequence reads was linearly correlated with virus titer. deep sequencing also provided insights into the upper respiratory microbiota and host gene expression in response to 2009 h1n1 infection. an unbiased analysis combining sequence data from all 17 outbreak samples revealed that 90% of the 2009 h1n1 genome could be assembled de novo without the use of any reference sequence, including assembly of several near full-length genomic segments. these results indicate that a streamlined metagenomics detection strategy can potentially replace the multiple conventional diagnostic tests required to investigate an outbreak of a novel pathogen, and provide a blueprint for comprehensive diagnosis of unexplained acute illnesses or outbreaks in clinical and public health settings. the 2009 h1n1 influenza a virus (2009 h1n1) is a novel reassortant virus comprised of genomic segments originating from swine, avian, and human influenza strains [1, 2] . after the initial identification of a swine-origin h1n1 by the cdc in april 2009, the novel virus rapidly achieved sustained human-to-human transmission worldwide, prompting a who declaration of a level 6 pandemic and rapid development of a vaccine [3] . as of august 6th, 2010, there have been at least 50 million cases and 18,449 confirmed deaths worldwide from the 2009 h1n1 pandemic (http://www.who.int/csr/don/2010_08_06/en/index.html). the emergence of 2009 h1n1 was marked by diagnostic difficulties that hampered early efforts at detection. rapid point-of-care diagnostics were insensitive for diagnosing the novel virus, and while existing molecular methods such as rt-pcr were able to detect influenza, they could not differentiate 2009 h1n1 from seasonal h3n2 or h1n1 infection [4] . initial efforts to characterize the virus relied on traditional sanger sequencing and the use of pcr primers to highly conserved regions in the influenza genome, methods that would likely have failed had the virus been more genetically divergent [1, 2] . no clinical or laboratory test was initially available to identify 2009 h1n1 with high sensitivity and specificity, and the virus may have circulated undetected in the human population for months [5] . thus, there is a clear need for the introduction of broad-range viral detection methodologies into clinical and public health settings to deal with emerging threats such as 2009 h1n1 influenza. metagenomic approaches, including microarrays and deep sequencing, have proven increasingly successful in recent years for the diagnosis of infectious diseases by novel viral pathogens [6] . the virochip is a pan-viral microarray that is designed to simultaneously detect all known viruses [7, 8] . novel viral species or strains can also be detected on the basis of conserved sequence homology to known viruses, as demonstrated by the discovery of the sars coronavirus by virochip in 2003 [9, 10] . other novel viruses previously identified by virochip include a new clade of rhinoviruses [11] , a human cardiovirus associated with respiratory and diarrheal illness [12] , and avian bornavirus, the etiologic agent of outbreaks of proventricular dilation disease (pdd) in birds [13, 14] . for detection of known respiratory viruses, the virochip microarray was found to have a sensitivity and specificity comparable to or superior to conventional diagnostic testing [11, 15] . deep sequencing is a complementary approach capable of detecting viruses that are too divergent from known viruses to be detected by either pcr or microarray techniques [16, 17, 18, 19, 20, 21, 22] ; reviewed in [6] . de novo pyrosequencing has enabled the discovery of two novel arenaviruses, an arenavirus associated with a fatal cluster of cases in solid-organ transplant patients (dandenong virus) [22] , and a hemorrhagic feverassociated arenavirus from south africa (lujo virus) [21] . metagenomic approaches are especially attractive in the study of influenza given the constant threat of antigenic drift and shift [23] . microarrays can rapidly type the origins of different segments to guard against the threat of novel reassortants and to detect coinfections with other pathogens [8, 15, 24, 25, 26, 27, 28] , while pyrosequencing or the use of resequencing microarrays can monitor the emergence of mutations that confer virulence or resistance to antiviral drugs [29, 30, 31] . deep sequencing strategies have been recently employed to detect seasonal influenza viruses in three clinical samples [32] , as well as identify quasispecies of 2009 h1n1 in a single autopsy lung sample [33] . here we analyzed a group of 17 early cases from the 2009 h1n1 pandemic from the united states (california), canada, and mexico by virochip and deep sequencing. we show that the virochip, in the absence of a priori information, was capable of rapid characterization of 2009 h1n1 as an influenza a (h1n1) virus most closely related to swine influenza viruses. we also demonstrate the utility of deep sequencing of clinical samples to identify and characterize not only the novel pathogen but also the microbiota and host response to infection. nasopharyngeal swab samples collected from 17 patients in north america from april to june of 2009 were included in this study (table s1 ; fig. 1a ). study patients were children or young adults, 59% (10 of 17) male, with an average age of 22 years (range 1 to 41 years). although the majority of cases (65%, 11 of 17) were upper respiratory infections in the outpatient setting, 35% (7 of 17) of patients were hospitalized for presumed h1n1 infection. all three study patients from california were hospitalized in the intensive care unit (icu) with h1n1-associated pneumonia. two of the three california patients were pregnant women, a group previously reported at high risk for severe complications from h1n1 infection [34] . to determine whether a pan-viral microarray assay was capable of identifying novel 2009 h1n1 in the absence of a priori sequence information, we used the virochip to comprehensively screen for viruses in 29 nasopharyngeal swab samples from individuals with influenza-like illness. seventeen samples from mexico (n = 5), canada (n = 9), and the united states (california) (n = 3) were suspected or pcr-confirmed cases of h1n1 infection. the remaining control samples, all from california, were positive for h1n1 seasonal influenza (n = 2), h3n2 seasonal influenza (n = 3), another respiratory virus (n = 6), or negative by all diagnostic testing (n = 1). of note, the probes on the virochip were designed prior to the emergence of pandemic 2009 h1n1. virochip hybridization patterns corresponding to 15 of 17 samples suspected or confirmed positive for 2009 h1n1 grouped together by hierarchical cluster analysis, and were clearly distinguishable from patterns corresponding to seasonal h3n2 influenza virus ( fig. 2a) . two of the 17 samples, mex-1225 and mex-730, exhibited very weak microarray probe intensities on virochip analysis and were grouped with the negative control samples. the finding of weak virochip intensities for these two samples was reproducible, and likely secondary to low viral titers and/or high background from host nucleic acid which can interfere with the random pcr amplification step of virochip analysis [8, 15] . the average normalized microarray intensities for the 2009 h1n1 samples were more pronounced with h1n1 probes derived from swine influenza (a/swine/wisconsin/464/98) than with probes derived from human influenza (a/human/puerto rico/8/34); the difference was statistically significant for 13 of 17 samples by t-test analysis (fig. 2b) . microarrays corresponding to seasonal h1n1 influenza displayed the opposite pattern, with h1n1 probes derived from human influenza stronger in intensity than those derived from swine influenza. minimal cross-hybridization in influenza a probes was observed with samples either negative or harboring other respiratory viruses. the virochip microarray was also analyzed using z-score analysis and e-predict, an automated method for viral species identification in microarrays [7, 35] . both methods incorporate information from all of the oligonucleotide probes on the virochip simultaneously, including probes to the full range of influenza strains, and revealed that a virus most similar to swine influenza a (a/swine/wisconsin/464/98) was present in these samples (data not shown). thus, the virochip detected the presence of a virus most closely related to swine influenza a/ h1n1 in samples from individuals infected with 2009 h1n1. to further characterize the metagenomics of 2009 h1n1 infection in humans, we labeled the 17 influenza samples positive for 2009 h1n1 by virochip with distinct molecular barcodes and analyzed them by paired-end deep sequencing on three lanes of an illumina genome analyzer iix. after trimming reads to remove barcodes and exclude low-complexity or primer sequences, 11,427,212 high-quality 60-bp sequence reads were subjected to an iterative blastn analysis pipeline (fig. 1b) . from the initial set of reads, a total of 9,819,120 (85.9%) reads were alignable (word size = 11, e-value = 1610 210 or 1610 25 ) to sequences obtained from the ncbi non-redundant nucleotide (nt) database (march 2010 build). the distribution of reads aligning to human, bacterial, and viral sequences varied greatly depending upon the laboratory preparation method for the clinical sample prior to sequencing ( figure 3 ). samples from mexico were not treated with dnase post-extraction, resulting in most reads from those samples (75 to 98%) aligning to human genomic dna. in contrast, samples that were treated with dnase post-extraction (united states and canada) contained many reads aligning to abundant, non-coding rnas, including human and bacterial ribosomal rna (rrna). despite dnase treatment, reads aligning to the human mitochondrial genome remained abundant for several samples. the overall percentages of bacterial (4.65%), viral (1.95%), other (1.02%) and nonaligning (14.1%) reads were low, with some notable individual exceptions. approximately 85% of reads from sample bc-59 aligned to bacterial sequences, mostly corresponding to the streptococcus genus. two samples from british columbia had a disproportionately large number of reads that aligned to viruses due to the addition of ms2 phage as an internal positive control for the luminex respiratory virus panel (rvp) assay (luminex, austin, tx). to determine if alterations in the upper respiratory tract microbiome were present in samples from patients infected with h1n1 virus, we further analyzed the deep sequencing reads aligning to bacteria. interestingly, all bacterial reads aligned to rrna sequences, likely due to the high relative abundance of rrna transcripts in bacteria [36] , and no bacterial mrna reads were observed. given the high degree of conservation of rrna sequences in bacteria, each read could only be unambiguously classified at the genus level (or at the family level in the case of enterobactericeae) (fig. 4) . in general, a high level of diversity of bacterial families was observed in different samples, reflecting the known diversity of bacterial flora in the nasopharynx [37] . reads to the top three bacterial families comprised anywhere from 22 to 97% of all bacterial reads, depending on the sample (fig. 4a ). moraxellaceae or enterobactericeae were the most common bacterial families found, accounting for one of the three most common bacterial families in 14 of 17 samples (fig. 4b ). significant numbers of reads aligning to the streptococcus genus were only seen in 5 of 17 samples, while reads to unculturable agents (''environmental samples'') accounted for between 1 to 48% of sequences aligning to bacteria. the majority of viral reads (n = 122,487, 55.1%) aligned to members of the orthomyxoviridae (influenza) family (table 1) . phages comprised the second most common group of viruses, including known controls (e.g. ms2 control phages), trace reagent contaminants (e.g. bacteriophage m13), and environmental phages. reads aligning to plant viruses were observed in two samples (bc-76 and mex-1225). sixteen reads to murine leukemia viruses (mlvs) were detected in 4 of 17 samples. in general, these mlv reads aligned slightly better to moloney murine leukemia virus (mmlv) than to the recently described human xenotropic murine leukemia-related virus (xmrv) [38, 39, 40] , although, for some reads, the alignments were identical. interestingly, a single overlapping 97-nt paired-end read from bc-22 aligned with 98% identity to a filovirus, ebola-sudan (97% query coverage, e-value = 9610 239 ) and mapped to a gene coding for the ebola viral glycoprotein. this bc-22 read also aligned to other filoviruses (69-98% identity) but did not align to any other viral family. we were able to reproduce detection of this single filovirus read by specific rt-pcr, but, despite the use of multiple sets of primers, we were unable to recover additional filovirus sequence from this sample (data not shown). aside from influenza, the only other respiratory viral pathogen detected was human adenovirus 4 in one canadian sample (bc-22, 26 reads). influenza reads aligning to orthomyxoviridae were found in all specimens, ranging from 6 to 53,671 reads, or 0.00011% to 10.9% of all aligning reads. after stratifying by originating location and corresponding method of sample processing (pre-dnase and/or post-dnase treatment), the percentage of total reads aligning to influenza was linearly correlated with calculated viral titers by realtime quantitative rt-pcr for sites in the united states (california) and canada but not in mexico (fig. 5a ). treatment with dnase twice yielded the best percentage recovery of however, despite very low viral titers, h1n1 influenza was still detectable by deep sequencing, with 270 and 18 reads aligning to influenza for cal-uc12 and bc-59, respectively (table 1) . influenza reads from all 8 segments were found in 7 of 17 samples, while reads from at least two different segments were found in every sample ( fig. 6 ; figs. s1 and s2). interestingly, reads to the ns (nonstructural) segment were significantly less likely to be found than reads to other segments (p,0.001 by chi-square analysis), with ns reads detected in only 8 of 17 samples. in contrast, reads to pb2 were found in all 17 samples. samples from mexico had the worst overall coverage, likely due to the high percentage of human genomic background sequences as a result of not treating samples with dnase post-extraction. in the sample with the best overall coverage (bc-22), 97.0% of the 2009 h1n1 genome had at least 1x coverage, while 91.5% of the genome had at least 3x coverage. we sought to assess the accuracy of deep sequencing relative to traditional sanger sequencing as well as search for single nucleotide polymorphisms (snps) in the assembled deep sequencing reads that may represent heterogeneous populations of influenza. deep sequencing reads from the two samples in the study with the highest coverage of the 2009 h1n1 genome, bc-22 (97.1% coverage at 1x) and cal-uc2 (92.6% coverage at 1x), were mapped to their full-genome scaffolds obtained by sanger sequencing. overall, there was a high degree of concordance (.99.9%) with deep sequencing and traditional sanger sequencing for both bc-22 and cal-uc2 (table s2) . next, to identify potential mutations at key sites in the genome that mediate drug resistance and virulence, we compared the genomes of bc-22 and cal-uc2 with the full-genome reference sequence a/california/06/2009(h1n1) ( table s2 ). in total, there were 30 nonambiguous nucleotide differences between bc-22 and the original california strain and 39 nonambiguous nucleotide differences between cal-uc2 and the original strain, of which 80% and 85% were detected by sanger sequencing and 77% and 72% by deep sequencing alone, respectively. these nucleotide differences corresponded to 12 and 18 nonsynonymous mutations in the bc-22 and cal-uc2 genomes, respectively. the genome segment with the most amino acid changes was the ha gene (27 of 69 nucleotide differences, or 39.1%). for both bc-22 and cal-uc2, there were no differences from the reference sequence a/california/06/2009(h1n1) at key sites in the genome corresponding to known mutations for antiviral resistance, enhanced transmission, or increased virulence. database in these 12 samples were considered to arise from mrna transcripts. since no rna-seq data corresponding to nasopharyngeal samples from healthy individuals can be found in the ncbi sequence read archive (http://www.ncbi.nlm.inh.gov/sra), we used a set of nasopharyngeal swab samples collected from 11 kleine-levin syndrome (kls) patients with influenza-like illness but testing negative for influenza as background controls. genes associated with immunity and interferon were significantly upregulated (p,0.05) in four of the 12 influenza samples as compared to the control samples, and from two study sites with different specimen processing protocols (fig. 7a ). the interferon-related genes that were preferentially up-regulated in 2009 h1n1 samples relative to controls coded for chemokine ligands, 29-59 oligoadenylated synthetases, ifn-inducible proteins, and stat proteins, proteins previously shown to play essential roles in the interferonmediated host immune response to viral infection [41] . upregulation of interferon and immunity genes was seen in the four samples with the highest 1x coverage of the influenza genome but not necessarily in samples with a high number of total transcriptome reads (fig. 7b ). genes associated with cell structure and motility as well as protein metabolism and intracellular protein trafficking were also upregulated in 2009 h1n1 samples relative to control samples (fig. 7a ). we hypothesized that the unprecedented depth of coverage obtainable by deep sequencing could enable detection and even de novo assembly of novel viral pathogens in the absence of any reference genome. to demonstrate the feasibility of this approach in elucidating the cause of outbreaks from hitherto novel or unknown viruses, we generated a modified nt database from which all sequences corresponding to the orthomyxoviridae family had been removed (fig. 8a) . pure de novo assemblies of paired-end reads pooled from all 17 2009 h1n1 samples which did not align to the modified nt database were then constructed using geneious software (geneious, auckland, nz). of the 582 final contigs (contiguous sequences) of length greater than 100 bp generated from the de novo assembly, 21 contigs mapped onto a reference sequence of 2009 h1n1 influenza, with an n50 contig size of 1,167 bp and coverage of 89.5% of the genome (fig. 8b) . the four longest contigs all corresponded to 2009 h1n1 and included the nearly full-length sequences of the pb1 (polymerase), np (nucleoprotein) and na (neuraminidase) segments (fig. 8c) . a cursory analysis of the remaining 561 contigs that did not map to the influenza genome revealed that ,90% of these sequences were low-complexity sequencing artifacts. from a similar analysis performed on the best single sample (bc-22), de novo assembly of 90.3% of the genome was possible. no misassemblies were observed even though the percentage of orthomyxoviridae reads from all 17 h1n1 samples or bc-22 alone comprised only 7.6% or 2.6% of the corresponding unaligned reads to the modified nt database, respectively. in this study, we demonstrate the utility of a metagenomicsbased strategy combining a rapid, broad-spectrum diagnostic assay (the virochip microarray) with comprehensive deep sequencing to identify and characterize a novel outbreak pathogen, using pandemic 2009 h1n1 influenza as a case example. the virochip was capable of differentiating seasonal h3n2 and h1n1 influenza from the novel 2009 pandemic strain by the use of multiple probes designed a priori, and accurately characterized the novel virus as closely related to swine influenza viruses. complementary deep sequencing of 17 clinical samples collected early in the course of the 2009 h1n1 pandemic enabled detection of reads to the novel strain in every sample, as well as de novo assembly of ,90% of the genome amidst an enormous background of unaligned sequence reads. as with the sars coronavirus, the lack of a broad-based diagnostic test that was sufficiently sensitive and specific likely contributed to the delayed identification of 2009 h1n1 as the cause of an initial outbreak of influenza-like illness in mexico. our approach -using the virochip microarray for rapid screening and deep sequencing for de novo assembly and more detailed characterization -is robust, comprehensive, and directly applicable for the investigation of future outbreaks from novel viral pathogens, especially those which do not grow in culture. nearly all previous studies of metagenomic sequencing of viruses from human clinical material for detection and discovery have employed pyrosequencing [6, 16, 17, 18, 19, 20, 21, 22, 32, 42, 43] . the longer reads from pyrosequencing (250-450 bp) facilitate the assembly of individual reads into contigs, which assists in the classification of reads by homology-based blast alignments. our results suggest, for example, that longer-read sequencing methods such as pyrosequencing may be better suited than illumina sequencing for identification of bacteria. accurate discrimination of bacteria at the species (or even genus) level was not possible with short 60-bp illumina reads (fig. 4b) , as rrna sequences corresponding to related bacteria were found to be essentially 100% identical over a 60-bp range. we did not de novo assemble the short illumina reads aligning to bacterial rrna into longer, more easily identifiable contigs given the known presence of multiple related bacterial species in the nasopharynx and the very high (and likely) risk of producing misassemblies. on the other hand, a key advantage of illumina sequencing is the significant increase in read depth (,100-fold) relative to pyrosequencing. the greater depth of sequencing provides better and more accurate genome coverage as well as increased capability to multiplex clinical samples. in our study of 2009 h1n1 influenza by deep sequencing, illumina read lengths of 60 bp were sufficiently long to accurately classify the vast majority of individual reads, thus making de novo contig assembly prior to alignment unnecessary. in addition, we were limited by the computational resources required for de novo assembly of ,11.5 million sequences in a reasonable amount of time. our results demonstrate that a computational pipeline consisting of sequential alignments of individual reads using highstringency cutoffs (fig. 1) , followed by de novo assembly of the remaining unaligned reads and detailed analysis of the resulting contigs (fig. 8) , is an efficient strategy for analyzing the large datasets generated by illumina sequencing of clinical samples and for pathogen discovery. in particular, the final de novo assembly step is critical in identifying divergent sequences that may correspond to novel pathogens. the assembly of longer contigs also eases the burden of computationally intensive amino acid comparison algorithms such as blastx, tblastx and phi-blast by reducing the number of sequences that need to be analyzed. although the costs of deep sequencing (,$200-$1000 usd/ sample) are still much greater than the reagent costs for diagnostic rt-pcr assays (,$10 usd), deep sequencing is able to generate much more useful information including data on the microbiome and host gene expression as well as whole-genome sequencing of pathogens which would require, for example, at least $1,200 usd per influenza genome via traditional methods. in addition, unbiased deep sequencing allows broad-based detection of novel viruses that may elude diagnosis by specific rt-pcr assays. with paired-end read lengths achievable on the illumina platform now approaching 300 bp, the use of deep sequencing for identification of even highly divergent pathogens at exceedingly low titers becomes feasible. the number of reads corresponding to 2009 h1n1 and the ability to map sequences onto the genome were highly dependent on the method of sample preparation. the fewest viral reads were detected in the five samples from mexico that were not treated with dnase after nucleic acid extraction. these samples contained the highest percentage of human genomic sequence, with over 90% of sequences aligning to the human genome, as well as the fewest reads to bacteria. nakamura et al. also found .90% host genomic material in nasopharyngeal aspirates from influenzainfected individuals, and detected a similarly low percentage of reads to influenza, despite the use of centrifugation to remove bacterial and cellular material [32] . our data indicate that one of the most important steps in viral detection by deep sequencing may be the removal of host genomic dna by post-extraction treatment with dnase -a relatively straightforward procedure for most clinical laboratories. as post-extraction dnase treatment was not 100% efficient (with the observation of residual dna mitochondrial reads in several samples), it is true that additional pre-processing by filtration, ultracentrifugation, or density-gradient purification would likely have resulted in even better enrichment of viral sequences [6] . however, for this metagenomics study, we sought to mimic as closely as possible a clinical/public health laboratory setting, and such enrichment strategies may be labor-intensive and neither conducive to the demands of clinical laboratories nor practical given the limited amount of clinical sample available. in addition, an important drawback of directed viral purification methods is that by biasing the sample towards virus detection, they can severely hinder the ability to detect potential non-viral pathogens or investigate patterns of human gene expression in response to viral infection, as we have done here. it has been suggested that co-infections with streptococcus pneumoniae may impact the severity of the clinical presentation in patients infected with 2009 h1n1 [44] . remarkably, very few copathogens, either viral or bacterial, were found among the 17 samples chosen for analysis. deep sequencing revealed that only five samples had a significant number of bacterial reads to the streptococcus genus, which may either represent potential pathogens (e.g. streptococcus pneumoniae), or simply normal human nasopharyngeal viral flora (e.g. streptococcus oralis, sanguis, and/or mitis). one sample from a canadian patient with an upper respiratory infection from 2009 h1n1 (bc-22) did contain a small number of reads from adenovirus type 4 (n = 26). these results imply that copathogens did not play a significant role in infection by 2009 h1n1 influenza in the 17 samples analyzed. further study of a much larger sample set will be needed to fully ascertain the role of co-pathogens in 2009 h1n1 infection. although 4 of 17 samples contained reads to murine leukemia viruses, it appears more probable on the basis of sequence alignments that the source of these reads is trace mmlv vector contamination of the reverse transcriptase enzyme used in preparing libraries for deep sequencing rather than infection by xmrv, a gammaretrovirus associated with prostate cancer and chronic fatigue syndrome in humans [39, 40] and recently detected in the respiratory tract [38] . in addition, one sample (bc-22) contained a paired-end read of 97 nt that was a 98% match to the filovirus ebola-sudan. we considered this most likely a laboratory contaminant, since we did not identify any other reads to filoviruses and were unable to amplify additional filovirus sequence from bc-22. however, the origin of this sequence ultimately remains unknown, as we could not subsequently detect it in any sample or reagent by specific rt-pcr, and none of the laboratories involved in the analysis works with ebola virus. we also found reads corresponding to plant viruses in two samples (bc-76 and mex-1225). detection of plant viruses in nasal swab samples is not unexpected given that the nasopharynx is contiguous with the oropharynx, and that an abundance of plant viruses of presumed dietary origin can be found in the human digestive tract [45] . our discovery of unexpected viral reads corresponding to murine leukemia viruses, filoviruses, and plant viruses in the deep sequencing data underscores the challenges of analyzing rare or unique sequences that may correspond to colonizing flora, reagent/sample contamination, or even true pathogens. using deep sequencing, we were able to recover and assemble near full-length genome sequences of the 2009 h1n1 virus from two individual patient specimens. since the molecular determinants of influenza pathogenesis have been well-studied, we were able to analyze the genomic sequence for single nucleotide polymorphisms (snps) that specifically correlate with antiviral resistance, enhanced transmission or increased virulence. for a novel virus, where such genotypic findings would not yet be correlated with phenotypic data, the overall sequence and structure of the genome may still yield valuable insights into viral transmission and mechanisms of pathogenesis. analysis of transcriptome reads demonstrated that immunity and interferon genes were significantly up-regulated in 4 nasopharyngeal swab samples from 2009 h1n1-infected patients as compared to background controls. interestingly, these 4 samples did not necessarily have a high number of transcriptome reads, but did have the highest 1x coverage of the influenza genome (fig. 7b) . coverage of the influenza genome may be the best proxy measure for influenza activity and the associated host response in deep sequencing analysis, as it is relatively independent of pcr duplication artifacts that can be introduced during sample preparation for deep sequencing [46] . our finding of increased expression of interferon-associated host genes in clinical samples from patients with 2009 h1n1 infection is intriguing and consistent with very recent data showing that the ns1 protein of 2009 h1n1 lacks the ability to block general host gene expression in both human and swine cells in vitro [47] . further studies are needed to assess the clinical relevance of increased interferon-associated host gene expression in 2009 h1n1 infection in vivo. nevertheless, this finding highlights the power of an unbiased deep sequencing approach by not only facilitating detection of the viral pathogen but also enabling elements of the host response to be simultaneously interrogated, thus generating new hypotheses for analysis of host-pathogen interactions. our study is the first to explore the relationship between viral titer and depth of sequencing required to detect a candidate viral pathogen. in samples that are treated with dnase after nucleic acid extraction (''post-dnase''), there is a linear correlation between percentage of viral reads in the deep sequencing data and viral titers as estimated by quantitative rt-pcr (fig. 5a ). there is no such observed correlation if samples do not undergo post-dnase treatment, likely secondary to high residual host genomic background, and thus these findings may only apply to rna viral targets. however, it is encouraging that sequence reads corresponding to 2009 h1n1 were detectable in all 17 samples, even in those samples with very low viral titers approaching the analytical limits of detection for rt-pcr assays (i.e. cal-uc12 and bc-59) (fig. 5b) . more studies are clearly needed on the metagenomics of different viral agents (e.g. rna vs. dna viruses; lysogenic vs. lytic, etc.) across different stages of infection (e.g. pre-symptomatic, acute, or chronic) and in different types of clinical samples (e.g. respiratory secretions, stool, blood, cerebrospinal fluid, tissue etc.). in addition, although the virochip microarray has a 12 to 24 hour turnaround time and can be rapidly deployed as a comprehensive screen for viral pathogens in the setting of outbreaks, the complementary deep sequencing approach is currently limited by the time necessary to perform a full pairedend sequencing run on an illumina genetic analyzer iix (,1 week) and to analyze the data on a highly parallel computational platform running 32 cores simultaneously (,1 week). nevertheless, the data that can be obtained in days to weeks by microarrays and deep sequencing would take months to years using conventional methods. furthermore, third-generation sequencing technologies which can generate deep sequencing data within hours are now available [48, 49] . the results described here provide a blueprint for the eventual use of microarrays and sequencing as routine diagnostic tools for pathogens in clinical and public health laboratories. cases of influenza-like illness in canada and mexico that were confirmed or suspected positive for 2009 h1n1 infection were identified by each individual state public health agency. informed consent was not obtained as the analysis of samples for pathogens is part of the mandate of clinical testing for each individual agency, and samples as well as demographic, clinical, and laboratory data were de-identified prior to analysis. cases in california were enrolled in a viral diagnostics study approved by the university of california, san francisco (ucsf) institutional review board (irb) (protocol #h9187-32565), and written informed consent was obtained from all study participants and/or their legal guardians. for all cases, collected samples were analyzed under protocols approved by the ucsf irb (protocol #h49187-32368). cases of influenza-like illness in canada and mexico were reported by providers and hospitals to the british columbia centre for disease control (bc-cdc), the university of toronto/ ontario agency for health protection and promotion (oahpp), and the veracruz ministry of health. suspected or confirmed h1n1 cases were identified through routine laboratory surveillance under protocols approved by each individual state public health agency. for each case, non-identifying demographic and clinical data were reported on standardized forms. cases in california were identified by infectious disease physicians at university of california at san francisco (ucsf), and demographic/clinical data were abstracted from the medical record. nasopharyngeal swab samples collected in viral transport media from 17 patients with either laboratory-confirmed or suspected cases of 2009 h1n1 influenza virus were analyzed. twelve additional nasopharyngeal swab samples from california were included as negative controls for the virochip. rna extractions were performed differently using established protocols specific to individual laboratories. for mexico, 200 ml of sample with linear polyacrylamide (ambion, inc., austin, tx) added as an rna carrier was treated with turbo dnase (ambion, inc., austin, tx)) for 30 minutes at 37uc (''pre-dnase'' treatment); nucleic acid was then extracted using the purelink rna-dna kit (invitrogen, carlsbad, ca) according to the manufacturer's instructions. for british columbia and ontario, 200 ml and 250 ml, respectively, of sample were extracted using the nuclisens easymag automated extraction system and then treated with turbo dnase (''post-dnase'' treatment). for california, 200 ml of sample was treated with turbo dnase, nucleic acid was extracted with the zymo viral rna kit (zymo research, orange, ca), and then the extracted nucleic acid was treated again with turbo dnase (''pre-/post-dnase'' treatment). total rna was reverse-transcribed to cdna, amplified by a modified round a/b random pcr method, and labeled with cy3 or cy5 fluorescent dye as previously described [7, 8] . the labeled samples were normalized to 10 pmol of incorporated dye and hybridized overnight using the agilent gene expression hybridization kit according to the manufacturer's protocol (agilent technologies, santa clara, california). the microarray slides used were custom-designed 8615k or 8660k arrays synthesized by agilent technologies, each containing 11,956 virochip 70-mer oligonucleotide probes in common. these 11,956 common probes include conserved as well as specific probes representing all viral species in genbank, and were derived from a previous 2008 virochip design [12] . the virochip is not yet commercially available, but is currently implemented as a core diagnostic service of the ucsf viral diagnostics and discovery center (http:// vddc.ucsf.edu). slides were scanned at 2 mm resolution in xdr (extended dynamic range) mode using an agilent dna microarray scanner. virochip microarrays were analyzed with hierarchical cluster analysis, e-predict, and z-score single oligonucleotide analysis as previously described [7, 11, 35] . average normalized microarray intensities were analyzed using a twosample, two-tailed t-test for unequal variances. all virochip microarrays used in this study were submitted to the ncbi geo database (study accession number gse24034; microarray accession numbers gsm591597-591641; microarray design accession numbers gpl10897 for the 8615k array and gpl10896 for the 8660k array). sequences corresponding to each influenza segment were obtained by one-step rt-pcr using either a qiagen one-step rt-pcr kit (qiagen, valencia, ca) or an invitrogen super-scriptiii one-step rt-pcr kit with high fidelity platinum taq (invitrogen, carlsbad, ca), with primers designed based on conserved terminal and central regions of each segment (table s3 ). reactions run with the qiagen kit were done in duplicate to account for potential polymerase errors. pcr products were run on a 1.5% agarose gel, and bands of the correct size were cut and extracted. fragments were cloned into a pcr2.1 vector (invitrogen, carlsbad, ca) and sanger sequenced (elim biopharmaceuticals, hayward, ca) with m13f and m13r primers as well as the original conserved pcr primers. at least three-fold redundancy was obtained for each segment. the whole-genome sequences of 2009 h1n1 samples bc-22 and cal-uc2, including all 8 segments, have been submitted to genbank (genbank accession numbers cy073781-cy073788 for bc-22 and cy073789-cy073796 for cal-uc2). quantitative rt-pcr for influenza was performed with primers based on conserved regions in the ha segment, h1n1-ha-275f (59-gggaaatccagagtgtgaatcact-39) and h1n1-ha-375r (gctctcttagctcctcataatcgatg-39) using a stratagene mx3005p real-time pcr system. the pcr was performed using a qiagen one-step rt-pcr kit (qiagen, valencia, ca) in a 12.5 ml reaction containing 3.25 ml h 2 o, 2.5 ml 5x pcr buffer, 2.5 ml q solution, 0.5 ml dntp, 0.5 ml rt/taq enzyme mix, 0.75 ml of each primer, 0.25 ml sybr green and 1.5 ml total rna. conditions for the pcr were 50uc for 30 min, 95uc for 15 min; 40 cycles of 95uc for 30 s, 50uc for 30 s, and 72uc for 1 min; and a final extension at 72uc for 7 min. the viral titer was estimated using a standard curve derived from an in vitro-transcribed and quantified influenza mrna standard (data not shown). for the canadian samples, viral titers as determined through ct values were compared with real-time rt-pcr data obtained independently from reference laboratories in vancouver (bc-cdc) and toronto (oahpp) to check for accuracy (data not shown). libraries for deep sequencing were prepared from amplified cdna libraries using previously published protocols [50] . briefly, libraries were cleaved with type iis restriction endonucleases (gsui), and truncated adapters containing unique 3 or 6 bp molecular barcodes were ligated on the resulting strand ends. fulllength adapters were subsequently added via an additional 15 to 25 cycles of pcr. libraries were size-selected on a 4% polyacrylamide gel at approximately 350 bp average length, and then loaded at a final concentration of 10 pm on three lanes of a second-generation genome analyzer iix (illumina, san diego, ca). paired-end reads were sequenced for 67 cycles in each direction. fluorescent images were analyzed using the illumina basecalling pipeline 1.5.0 to obtain paired-end sequencing data. paired-end reads were subsequently classified by strict barcodes, filtered to eliminate low-complexity sequences with an lzw compression ratio cutoff of 0.4, split into individual reads, and stripped of any remaining primer sequences using blastn alignments (word size = 11, e-value = 1610 210 ). blastn alignments to publicly available sequence databases were performed using four extralarge high cpu-instance amazon elastic cloud computing (ec2) servers comprising a total of 32 core processors, with an approximate computational time of 24 hours per sequencing lane. sequence reads that aligned to human rrna and mitochondrial genome by blastn (word size = 11, e-value = 1610 10 ) were initially removed. remaining reads were then classified by successive blastn alignments to the refseq human transcriptome database (word size = 11, e-value = 1610 210 , september 2009 build [release 37]) and ncbi non-redundant nucleotide (nt) database (word size = 11, e-value = 1610 25 , march 2010 build). all reads that aligned to nt were sorted by their best hit into their respective taxa for analysis. reads that aligned to nt but that did not align to human, bacterial, or viral sequences were categorized as ''other'', and consisted solely of environmental sequences or plasmids/artificial constructs. the presence of murine leukemia virus and ebola virus in the deep sequencing libraries was independently confirmed by specific rt-pcr and direct sequencing (data not shown). we also separately performed directed blastn alignments of the original set of unclassified deep sequencing reads to an influenza sequence database (word size = 11, e-value = 1610 25 ) to verify that no influenza reads were incorrectly classified as human or other nonviral sequence (data not shown). all high-quality sequence reads have been submitted to the ncbi sequence read archive (http://www.ncbi.nlm.nih.gov/sra) (accession number sra023755). reads corresponding to the human mrna transcriptome were counted by gene and compared to transcriptome reads from nasopharyngeal swab samples from kleine-levin syndrome (kls) patients with influenza-like illness. kls is a neuropsychological disorder marked by hypersomnolence, hyperphagia, and hypersexuality usually found in adolescent boys [51] . an ongoing metagenomic analysis of samples from 11 patients with kls (''kls samples'') by deep sequencing found only 24 reads to a single rhinovirus a isolate in one sample and no reads to influenza in any sample; in addition, all kls samples were negative for both pandemic and seasonal influenza by specific rt-pcr (data not shown). as such, it was thought that the kls samples could serve as appropriate background controls from individuals with influenza-like illness (but testing negative for influenza) for the purposes of transcriptomic analysis. in particular, the kls samples were derived from a similar demographic as the h1n1 samples (children and young adults) and were processed identically for deep sequencing. the transcriptomic analysis was performed as follows. first, we selected all genes in refseq that were significantly overexpressed in 2009 h1n1 nasopharyngeal swab samples relative to kls background samples (p,0.05, by bonferroni-corrected fisher's exact test). to identify overrepresented categories of genes, we inputted the overexpressed refseq genes into the panther (protein analysis through evolutionary relations) database (http://panther.appliedbiosystems.com) and compared them to a human reference list of transcribed genes [52] . categories of genes significantly up-regulated in the influenza data relative to the human reference list (p,0.05, by bonferroni-corrected fisher's exact test) were reported. snp analysis of sequence reads mapping to influenza was performed using the ssaha2 software package [53] . both pairedend read information and fastq-formatted quality scores were incorporated in the calling of snps. snp calling was performed at very high stringency to ensure accuracy. specifically, the illumina deep sequencing data had a calculated overall error rate of ,1% by analysis of the phix control lane (illumina inc., hayward, ca), and at least 5-fold coverage and a quality score of 30 were required for mapping of deep sequencing reads at any given position in the genome. a minority snp was defined as present if occurring $25% of the time at a given nucleotide position. paired-end de novo assembly of the influenza genome was performed using geneious version 5.0.1 software [54] , with strict parameters (word length = 18; 0% mismatch/gap tolerance; only paired-end reads included in assembly; expected paired-end mate distance = 200) to avoid misassembly, the de novo assembly algorithm used by geneious is a greedy algorithm similar to that used in multiple sequence alignment programs such as clustalw (matt kearse, geneious inc., personal communication) [55] . initial generated contigs of size $100 bp were further assembled de novo in geneious at high sensitivity with ''fine tuning'' of gaps and 3-nt trimming of contig ends. final de novo assembled contigs were then mapped to the full-genome reference sequence a/california/06/2009(h1n1) (genbank accession numbers fj966960 -fj966965 and fj971074-fj971075). out of the remaining 561 contigs that did not map to influenza, ,90% of the contigs corresponded to low-complexity sequence that was identified as such 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high-throughput sequencing approach characterization of quasispecies of pandemic 2009 influenza a virus (a/h1n1/ 2009) by de novo sequencing using a next-generation dna sequencer california pandemic working g (2010) severe 2009 h1n1 influenza in pregnant and postpartum women in california e-predict: a computational strategy for species identification based on observed dna microarray hybridization patterns development and quantitative analyses of a universal rrna-subtraction protocol for microbial metatranscriptomics the nih human microbiome project xenotropic murine leukemia virus-related gammaretrovirus in respiratory tract identification of a novel gammaretrovirus in prostate tumors of patients homozygous for r462q rnasel variant detection of an infectious retrovirus, xmrv, in blood cells of patients with chronic fatigue syndrome antiviral actions of interferons clonal integration of a polyomavirus in human merkel cell carcinoma metagenomic analysis of respiratory tract dna viral communities in cystic fibrosis and non-cystic fibrosis individuals streptococcus pneumoniae coinfection is correlated with the severity of h1n1 pandemic influenza rna viral community in human feces: prevalence of plant pathogenic viruses substantial biases in ultra-short read data sets from high-throughput dna sequencing inefficient control of host gene expression by the 2009 pandemic h1n1 influenza a virus ns1 protein real-time dna sequencing from single polymerase molecules the next generation becomes the now generation the long march: a sample preparation technique that enhances contig length and coverage by high-throughput short-read sequencing kleine-levin syndrome: a systematic study of 108 patients panther pathway: an ontology-based pathway database coupled with data analysis tools ssaha: a fast search method for large dna databases multiple sequence alignment using clustalw and clustalx we thank aurora parã­s at the ministry of health bsl-3 state laboratory in veracruz for providing 2009 h1n1 samples from mexico. we thank david schnurr, sharon messenger, and shigeo yagi at the california department of public health for providing two seasonal h1n1 samples for virochip analysis. we thank emmanuel mignot at stanford university for providing respiratory samples from kls patients as negative controls. we thank catherine liu, peter chin-hong, and brian schwartz for their help in identifying patients infected with 2009 h1n1 influenza at ucsf. we also thank clement chu, peter skewes-cox, and marã­a soto del rã­o for expert technical assistance. key: cord-254313-g2oc32dm authors: klink, thomas; rankin, danielle a.; piya, bhinnata; spieker, andrew j.; faouri, samir; shehabi, asem; williams, john v.; khuri-bulos, najwa; halasa, natasha b. title: evaluating the diagnostic accuracy of the who severe acute respiratory infection (sari) criteria in middle eastern children under two years over three respiratory seasons date: 2020-04-30 journal: plos one doi: 10.1371/journal.pone.0232188 sha: doc_id: 254313 cord_uid: g2oc32dm objective: the world health organization created the severe acute respiratory infection (sari) criteria in 2011 to monitor influenza (flu)-related hospitalization. many studies have since used the sari case definition as inclusion criteria for surveillance studies. we sought to determine the sensitivity, specificity, positive predictive value, and negative predictive value of the sari criteria for detecting ten different respiratory viruses in a middle eastern pediatric cohort. materials and methods: the data for this study comes from a prospective acute respiratory surveillance study of hospitalized children <2 years in amman, jordan from march 16, 2010 to march 31, 2013. participants were recruited if they had a fever and/or respiratory symptoms. nasal and throat swabs were obtained and tested by real-time rt-pcr for eleven viruses. subjects meeting sari criteria were determined post-hoc. sensitivity, specificity, positive predictive value, and negative predictive value of the sari case definition for detecting ten different viruses were calculated and results were stratified by age. results: of the 3,175 patients enrolled, 3,164 were eligible for this study, with a median age of 3.5 months, 60.4% male, and 82% virus-positive (44% rsv and 3.8% flu). the sensitivity and specificity of the sari criteria for detecting virus-positive patients were 44% and 77.9%, respectively. sensitivity of sari criteria for any virus was lowest in children <3 months at 22.4%. removing fever as a criterion improved the sensitivity by 65.3% for detecting rsv in children <3 months; whereas when cough was removed, the sensitivity improved by 45.5% for detecting flu in same age group. conclusions: the sari criteria have poor sensitivity for detecting rsv, flu, and other respiratory viruses—particularly in children <3 months. researchers and policy makers should use caution if using the criteria to estimate burden of disease in children. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 respiratory infections are the second leading cause of global years of life lost in all ages, and the leading cause of mortality in children under five years [1] . in 2011, the world health organization (who) created a case definition for severe acute respiratory infection (sari) in an attempt to standardize global surveillance of hospitalization related to influenza (flu)-allowing national health authorities to interpret their data in an international context [2] . flu is particularly difficult to surveil because its clinical presentation is often indistinguishable from other respiratory viruses [3] . with that in mind, the who designed the sari criteria to strike a balance between sensitivity and specificity, while also noting that the case definition is not necessarily intended to capture all cases but to describe trends over time [4] . several studies, including at least nine in the eastern mediterranean region, have been published since 2011 using the sari case definition as inclusion criteria to report a combination of clinical characteristics, risk factors, viral burden, or outcomes in adult and pediatric populations for flu and other respiratory viruses [5] [6] [7] [8] [9] [10] [11] [12] [13] . only a handful of studies have evaluated the effectiveness of the criteria by including both sari-positive and sari-negative patients, allowing them to calculate the diagnostic accuracy of the criteria for detecting flu and respiratory syncytial virus (rsv) [14] [15] [16] [17] [18] [19] . the diagnostic accuracy of the criteria for other individual respiratory viruses remains unknown. additionally, only one of these studies stratified age to include a group of children less than three months old, and studies of this type from the eastern mediterranean region are lacking [17] . moreover, those studies that have included both sari-positive and sari-negative pediatric patients have indicated that the sari criteria are less sensitive in younger pediatric patients, but only one such study stratified by the youngest children [14, [16] [17] [18] . this means that studies using the sari case definition as inclusion criteria may underestimate the burden of disease in this age group [5] . therefore, we sought to evaluate the diagnostic accuracy of the sari criteria for eleven respiratory viruses in a large hospitalized pediatric cohort in amman, jordan in children less than two years old who presented with fever and/or respiratory symptoms. we conducted a prospective active surveillance study of acute respiratory infections in hospitalized children <2 years in amman, jordan. participants were recruited over a three-year period (march 16, 2010-march 31, 2013) within 48 hours of hospital admission for fever and/ or respiratory symptoms with one of the following admission diagnoses: ari, apnea, asthma exacerbation, bronchiolitis, bronchopneumonia, croup, cystic fibrosis exacerbation, febrile seizure, fever without localizing signs, respiratory distress, pneumonia, pneumonitis, pertussis, pertussis-like cough, rule out sepsis, upper respiratory infection (uri) , or other. children were excluded only if they had chemotherapy-associated neutropenia and/or were newborns who had never been discharged [20] . trained local staff obtained written informed consent from parents or guardians of all participants. the study was approved by the institutional review boards of the university of jordan, the jordanian ministry of health, and vanderbilt university. al-bashir hospital is a 185 pediatric bed (120 pediatric and 65 neonatal intensive care unit) government-run hospital that serves the jordanian capital, amman, a city with >2 million inhabitants. the catchment area is densely populated, low-income, and includes a palestinian refugee camp. due to government policy, children <6 years are provided free care at al-bashir regardless of insurance status. during the study period, there were 11,230 hospitalizations of children <2 years. throat and nasal swabs from all participants were obtained by trained research staff. demographic, social, and medical histories were obtained by standardized questionnaires, which were a component of the interview portion of the case report form. all interviews were conducted in arabic to parents and recorded in english. following patient discharge, clinical outcome data and antibiotic use during the hospitalization data were systematically collected from the medical record. all data were entered into a secure redcap tm (research electronic data capture, vanderbilt university, nashville, tn, usa) database. extracted information is explained in detail in a previous publication [20] . throat and nasal swabs were combined in transport medium (m4rt 1 , remel, usa) aliquoted into magmax tm lysis/binding solution concentrate (life technologies, usa), snap frozen, stored at -80˚c, and shipped to nashville, tn. original and lysis buffers were tested by real-time rt-pcr for eleven respiratory viruses: rsv, human metapneumovirus (hmpv), human rhinovirus (hrv), flu a, b, and c, parainfluenza virus (piv) 1, 2, and 3, adenovirus, and middle east respiratory syndrome coronavirus (mers-cov) [21] . cycle threshold (ct) values were determined, with lower ct corresponding with higher viral load. the who sari case definition includes three elements: hospitalization with ari with symptom onset in the past 10 days, history of fever or measured fever �38 c˚, and cough [4] . in order to capture this definition in our subjects, we used information obtained from the questionnaire and extracted from the medical chart. days of symptoms were captured in the questionnaire. fever was captured with at least one of the following: an admission or discharge diagnosis of fever, measured temperature �38 c˚at admission, or reported history of fever as a symptom of current illness. cough was recorded if it was reported as a symptom or was captured as an admission or discharge diagnosis. descriptive statistics were reported as frequencies or mean, median and interquartile range (iqr) where appropriate. odds ratios for covariates contributing to sari status were calculated using a series of simple logistic regressions, followed by multiple logistic regressions, controlling for age and sex. holm-bonferroni adjustments were made to account for multiple analyses with the same dependent variable. covariates included age, sex, birthweight, prematurity, medical history, smoke exposure [nargila (smoking pipe) or cigarettes], vitamin d level, breastfeeding, length of illness, admission diagnoses of pneumonia, bronchopneumonia, bronchiolitis, sepsis, or febrile seizure, viral detection, and markers of illness severity including antibiotics before or after hospitalization, length of stay, icu admission, oxygen use, mechanical ventilation, and death. all analyses were completed using stata version 15.1. sensitivity, specificity, positive predictive value (ppv), and negative predictive value (npv) of the sari criteria and modifications excluding either fever or cough were calculated for each respiratory virus tested. parainfluenza viruses 1, 2, and 3 were combined into one category, as was flu a, b, and c. this was done for simplicity and relatively low numbers of detections of each subtype. for npv and ppv, we assumed the prevalence found in our study to be accurate for hospitalized children with fever or respiratory symptoms. for each virus, the prevalence of cough and fever was also calculated. these data were then stratified by age into three groups: less than 3 months, 3-5 months, and 6-23 months. these groups were chosen to compare results to a previous study; and were based off a visual analysis of the age of virus-positive patients meeting sari criteria [17] . between march 16, 2010, and march 31, 2013, there were 3,793 eligible hospitalized infants and 3,175 (83.7%) were enrolled as previously described [20] . seven of these were excluded: four admitted with the diagnosis of meningitis and three who were older than two years. of the remaining 3,168 subjects, four were excluded for the purpose of this analysis because they did not have information on the sari criterion of illness duration. therefore, 3,164 subjects (83.4% of eligible patients) are included in our analyses. the median age for enrolled subjects was 3.5 months (table 1) . most were male (60.4%), had household exposure to smoke or nargila (76.6%), and reported exclusive breastfeeding (60.6%). viral pathogens were detected in 2,581 (81.5%) subjects, and 315 (10.1%) had at least one underlying medical condition. of the 3,164 subjects included, 1,261 (39.9%) met sari criteria ( table 1) . when compared to their sari-negative counterparts, sari-positive subjects tended to be older, were less likely to have a history of premature birth, and had a shorter duration of illness prior to hospitalization. no significant differences were detected in sex, underlying medical conditions, smoke exposure, vitamin d levels, birth weight, or breastfeeding history. sari-positive patients were more likely to be diagnosed with pneumonia (or: 1.77; 95% ci: 1.41-2.22; p-value: <0.001) and bronchopneumonia (or: 3.83; 95% ci: 3.21-4.55; p-value: <0.001). alternatively, sari-negative subjects were more likely to be diagnosed with rule out sepsis (or: 0.37; 95% ci: 0.20-0.47; p-value: <0.001) and febrile seizure (or: 0.07; 95% ci: 0.03-0.14; p-value: <0.001). no significance was found in the prevalence of bronchiolitis between the two groups (or: 0.96; 95% ci: 0.79-1.17; p-value: 0.687). sari-positive subjects were more likely to receive antibiotics both before and during hospitalization. no differences were detected between length of stay, icu admission, oxygen use, mechanical ventilation, or death (table 1) . virus detection was more common in sari-positive patients compared to sari-negative subjects (or: 2.84; 95% ci: 2.27-3.56; p-value:<0.001). including co-detections, sari-positive diagnostic accuracy of the who severe acute respiratory infection (sari) criteria in the middle east subjects were more likely to have rsv, and hmpv (table 1) . co-detections were also more prevalent in sari-positive patients. an analysis of single virus detections only yielded one significant difference with the analysis that included co-detections: sari-negative patients were more likely to have a single virus infection of hrv, whereas there was no significant difference between groups when co-detections were included following holm-bonferroni adjustment ( table 1 ). there were no differences seen in ct values by sari status. mers-cov was not detected in any sample. overall, the sensitivity of the sari criteria including co-detections for detecting virus-positive patients was 44%, with a specificity of 78%, ppv of 89.8%, and npv of 24% (figs 1 and 2) . the difference in sensitivity and specificity between single and co-detections for a particular virus was within 10%, but differences were larger changes for ppv and npv depending on the prevalence of the virus in the study. sari criteria showed the highest sensitivity for single hmpv detections (64.8%). the sensitivity of the sari criteria did not reach above 60% for any other virus. specificity of the criteria was greatest for rsv detections including rsv and co-detections (66.9%). specificity was between 58.2% and 63.2% for all other viruses. ppv was greatest for rsv detections at 53.6%, including co-detections, and lowest for single-flu detections at 2.2%. conversely, npv was greatest for single-flu detections at 98.8% and lowest for rhinovirus detection, including codetections, at 59%. the prevalence of fever was highest in patients with single-flu detection at 90%; whereas those with rsv, including co-detections, had a fever rate of 52.4%. the cough rate was highest in those with single-hmpv and single-rsv detections at 96.1% and 96% respectively. the cough rate was lowest in those with single-adenovirus detections at 56.2%. modifying the sari criteria by removing fever or cough changed the diagnostic accuracy for each virus (fig 2) . sensitivity of the sari-nof and sari-noc criteria was greater for detecting each virus than the original sari criteria. sari-nof had the greatest gains in sensitivity with an increase of 35�1% in detecting any of the viruses, including a 44.1% increase in rsv detection. conversely, sari-noc had a 13.2% increase in sensitivity for detecting any of the viruses and a 2.7% increase for rsv detection. specificity for detecting all viruses was decreased for both sari-nof and sari-noc criteria compared to the original criteria. the specificity of the sari-nof criteria for detecting any of the viruses was decreased by 17.8%. the largest decrease was for adenovirus with a change of -32.4%. the specificity for the sari-noc criteria decreased by 43.3% for detecting any of the viruses. the changes in ppv were more modest for the modified criteria. the ppv of the sari-nof criteria for any of the viruses decreased by only 0.1%. the ppv for the sari-noc criteria for any of the viruses decreased by 10.4%. the npv of the sari-nof criteria increased by 15.5% for any of the viruses; whereas, the sari-noc criteria saw a decrease of 8.5% in the same category. the proportion of virus-positive patients meeting sari criteria by their age yielded a clear trend, with the youngest patients being less likely to meet criteria (figs 1 and 3a) . data were stratified into three age groups (<3 months, 3-5 months, 6-23 months) to compare the sari criteria to the modified criteria in virus-positive patients (fig 3a) , showing that the sari-nof criteria had the highest sensitivity in all age groups. differences were also observed with regards to the presence of fever in virus-positive patients across the groups. virus-positive patients had a reported or measured fever in 44.4%, 60.6%, and 75.5% in the less than 3 months, 3-5 months, 6-23 months groups, respectively (fig 3b) . fever was more common in the virus-negative patients for each age group ( table 2 ). the sensitivity, specificity, ppv, and npv of both the original and modified criteria for each virus was also stratified by age group (figs 4-6) . the sari criteria had the lowest sensitivity in the youngest age group at 22.4% for detecting any of the viruses. its specificity in this category, diagnostic accuracy of the who severe acute respiratory infection (sari) criteria in the middle east however, was 90.6%. the sari criteria had the highest sensitivity in the oldest age group at 64.3% for detecting any of the viruses. the specificity in this age category was 57.7%. the sari-nof criteria saw the greatest gains in sensitivity amongst the youngest age groups compared to the original criteria with an increase of 46.6% for detecting virus-positive patients (figs 4-6 ). our study found that for ari surveillance, the sari criteria fails to capture more than 50% of children with respiratory virus infections (rvi), and it performs even more poorly for children <3 months. therefore, it is important to be cautious when using the sari criteria for surveillance studies, especially if trying to determine the burden of illness to select viruses to influence policy decisions (e.g. vaccine implementation and/or effectiveness). if understanding the true burden of disease is desired, a higher sensitivity is important. while the sari case definition was initially intended for flu surveillance, several studies have since used it for surveillance of other respiratory viruses [16] [17] [18] [22] [23] [24] . our study provides researchers and public health officials the data on diagnostic accuracy of the criteria for detecting ten different respiratory viruses and would caution sari use for true rvi burden in children. during our study period, the sari criteria correctly identified a little over half of the subjects who were flu-positive. comparisons to previous studies are nuanced due to differing age stratifications, but this result is similar to the 52% sensitivity reported by the amini (2017) diagnostic accuracy of the who severe acute respiratory infection (sari) criteria in the middle east study in quebec for children <1 year; however, it is lower than the 79.2% found in that same study for its one to four-year-old age group [14] . similarly, two studies in kenya that reported diagnostic accuracy of the who severe acute respiratory infection (sari) criteria in the middle east results for participants aged two months to four years found sensitivities of 84.3% and 89.6% [15, 24] . the specificity of the criteria for detecting flu in our study (60.6%) also differs from what was found in the aforementioned studies (13.1%-29.5%). the age distribution differences likely explain these discrepancies as our cohort consists only of children under two years, and the sari criteria were the least sensitive (15.9%) and most specific (80.5%) for detecting flu in our patients under three months, who make up 45.4% of our study population. the sensitivities and specificities of the criteria for our patients aged 3-5 months (72.2%, 50.4%) and 6-23 months (75.4%, 39.9%) are more closely aligned with the findings of the previous studies. therefore, if policy makers are deciding if flu vaccine should be administered to pregnant women or infants based on the sari definition, they would be underestimating the true burden of flu illness. our findings for the diagnostic accuracy of the criteria for detecting rsv also have some differences from previous studies, including one study that included similar age group stratifications-allowing for a more direct comparison [17] . for children <3 months, our study showed a sensitivity of 25.2%, lower than the 55% found by rha (2018) in south africa [17] . however, our 74% sensitivity in the 6-23 months age group more closely matched their groups of 6-11 and 12-23 months with sensitivities of 77% and 81%, respectively. the latter result is also in line with the findings of nyawanda (2016), which reported sensitivities for children <1 year at 79.4%, and one to four years at 86.2%. our finding for the specificity of the criteria was greater than these two previous studies across all age groups but differed most from rha for children less than three months (85% vs 54%). similar to rha, however, the specificity of the criteria in our study for our oldest age group (6-23 months, 47.7%) was nearly half that of our diagnostic accuracy of the who severe acute respiratory infection (sari) criteria in the middle east youngest age group (85.1%). one potential explanation for differences between our studies is the use of singleplex rt-pcr in our study versus the multiplex rt-pcr that was used in rha, with singleplex historically being more sensitive. given that rsv vaccines are in the pipeline, it is important to also capture all cases of rsv to determine the true burden of illness to document if the vaccine is effective at impacting disease. thus, if the sari definition is used for ari surveillance studies, it will once again underestimate the burden of rsv illness, and therefore altering the inclusion criteria to include either fever and/or cough may be more accurate. the differences in diagnostic accuracy of the sari criteria by age is the most significant finding of this study. the sari criteria performed poorly with regards to sensitivity for the detection of any respiratory virus in children under three months. only 22.4% of children in this age group with a respiratory virus were captured by the case definition. this increased to 64.3% in children aged 6-23 months. conversely, the criteria are very specific in the youngest age group for detecting any respiratory virus compared to the oldest age group (90.6% vs. 64.3%). we further dissected by symptoms and found that having the combination of both fever and cough was the reason for low sensitivities and having either one or the other could capture more rvi cases. specifically, the lower rates of fever-and to a lesser extent, cough-in patients <3 months who are respiratory virus-positive account for the poor sensitivity of the sari criteria in this age group. only 44.4% of the virus-positive patients <3 months had a reported or measured fever, compared to 75.5% of their 6-23 months counterparts. consistent with what has been found in previous studies [25] , removing fever from the sari criteria greatly increased the sensitivity of detecting any respiratory virus in children <3 months, particularly for rsv, which then increased by 65.3% [18] . notably, both the ppv and npv for detecting rsv also increased with this change. in general, for this youngest age group, removing fever as a criterion increases sensitivity more than it decreases specificity for each virus tested, and it results in minimal changes to ppv and npv. this result would seem to advise caution when mandating that fever must be included as a criterion for this age group. our study has several strengths. it is the first to report the diagnostic accuracy of the who sari criteria for detecting multiple viruses, including but not limited to flu and rsv. it is also the first study of this kind in the middle east. our prospective study took place over three years, included very young children, and included both fever and/or respiratory criteria, which allowed us to compare sari-positive to sari-negative children. our study also has a number of limitations. it only includes hospitalized children <2 years who had ari symptoms from one large hospital in amman, jordan. this limits its generalizability to other locations, or to study populations that include all hospitalized patients. additionally, the 2011 sari criteria were developed during our study period. while our study design allowed us to capture all the necessary elements to determine which patients met sari criteria, it involved combining together the data as opposed to a straightforward data collection tool. part of the data used in this study also included a parental survey of symptoms-introducing a source of reporting bias. the sari criteria were initially developed by the who in 2011 as part of its recommendations for global flu surveillance. the ultimate goal of such surveillance is "to minimize the impact of diagnostic accuracy of the who severe acute respiratory infection (sari) criteria in the middle east the disease by providing useful information to public health authorities so they may better plan appropriate control and intervention measures, allocate health resources, and make case management recommendations" [4] . however, our study found that more than half of the rvi cases would have been missed using the sari criteria. therefore, it is critical to know how well these criteria perform in different patient populations and geographic settings. our study shows that use of the criteria in children <3 months significantly underestimates the burden of disease for rsv, flu, and other viruses. therefore, removing fever as a mandatory criterion in this age group, particularly for rsv, would greatly increase sensitivity with an acceptable decrease in specificity. overall, more studies need to be conducted in the middle east to gather more information on this finding; however, it is advisable that policymakers are cautious when using the sari criteria. diagnostic accuracy of the who severe acute respiratory infection (sari) criteria in the middle east global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the global burden of disease study revision of clinical case definitions: flu-like illness and severe acute respiratory infection clinical signs and symptoms predicting flu infection world health organization. global epidemiological surveillance standards for flu flu hospitalization epidemiology from a severe acute respiratory infection surveillance system in jordan the burden of fluassociated hospitalizations in oman circulation of respiratory syncytial virus in morocco during 2014-2016: findings from a sentinel-based virological surveillance system for flu estimation of flu and severe acute respiratory illness incidence (burden) in three provinces of the islamic republic of iran viral etiology, seasonality and severity of hospitalized patients with severe acute respiratory infections in the eastern mediterranean region morbidity, mortality, and seasonality of flu hospitalizations in egypt incidence of flu virus-associated severe acute respiratory infection in damanhour district flu-associated severe acute respiratory infections in 2 sentinel sites in lebanon characteristics of severe acute respiratory infection associated hospitalization in yemen evaluation of the new world health organization case definition of severe acute respiratory infection for flu surveillance during the peak weeks of two flu seasons in quebec comparison of severe acute respiratory illness (sari) and clinical pneumonia case definitions for the detection of flu virus infections among hospitalized patients, western kenya evaluation of case definitions to detect respiratory syncytial virus infection in hospitalized children below 5 years in rural western kenya performance of surveillance case definitions in detecting respiratory syncytial virus infection among young children hospitalized with severe respiratory illness-south africa evaluation of case definitions for estimation of respiratory syncytial virus associated hospitalizations among children in a rural community of northern india what are the most sensitive and specific sign and symptom combinations for flu in patients hospitalized with acute respiratory illness? results from western kenya natural history and epidemiology of respiratory syncytial virus infection in the middle east: hospital surveillance for children under age two in jordan human metapneumovirus in hospitalized patients in amman, jordan human metapneumovirusassociated severe acute respiratory illness hospitalisation in hiv-infected and hiv-uninfected south african children and adults detection of non-flu viruses in acute respiratory infections in children under five-year-old in etiology and incidence of viral acute respiratory infections among refugees aged 5 years and older in hagadera camp performance of case definitions used for flu surveillance among hospitalized patients in a rural area of india key: cord-287739-58fth3xl authors: huang, yhu-chering; lien, rey-in; su, lin-hui; chou, yi-hong; lin, tzou-yien title: successful control of methicillin-resistant staphylococcus aureus in endemic neonatal intensive care units—a 7-year campaign date: 2011-08-12 journal: plos one doi: 10.1371/journal.pone.0023001 sha: doc_id: 287739 cord_uid: 58fth3xl background: methicillin-resistant staphylococcus aureus (mrsa) is among the most important nosocomial pathogens in the intensive care unit (icu) worldwide, including taiwan. since 1997, our neonatal icus (nicus) had become endemic for mrsa. methodology/principal findings: to control mrsa spread in our nicus, we implemented a series of infection control measures stepwise, including reinforcement of hand hygiene since january 2000, augmentation of aseptic care over the insertion site of central venous catheter since july 2001, introduction of alcohol-based handrubs since april 2003, surveillance culture for mrsa and cohort care for the colonized patients between march 2003 and february 2004, and surveillance culture with subsequent decolonization of mrsa between august 2005 and july 2006. after implementation of these measures, mrsa healthcare-associated infection (hai) density reduced by 92%, from 5.47 episodes per 1000 patient-days in 1999 to 0.45 episodes per 1000 patient-days in 2006; mrsa bloodstream infection reduced from 40 cases in 1999 to only one case in 2006. compared to those obtained during the period of surveillance culture without decolonization, both rates of mrsa colonization (8.6% vs. 41%, p<0.001) and infection (1.1% vs. 12%, p<0.001) decreased significantly during the period of surveillance and decolonization. molecular analysis of the clinical isolates during the study period showed that the endemic clone, which dominated between 1998 and 2005, almost disappeared in 2006, while the community clones increased significantly in 2006–2007. conclusion/significance: through infection control measures, mrsa hais can be successfully controlled, even in areas with high levels of endemic mrsa infections such as our nicus. methicillin-resistant staphylococcus aureus (mrsa) is among the most important pathogens of bacteremia in the intensive care units (icu). nowadays, mrsa becomes endemic in most hospitals around the world [1, 2] and accounts for 40-60% of all healthcareassociated s. aureus infections. colonized patients are the major reservoirs of mrsa in hospitals. colonizing strains may serve as endogenous sources for overt clinical infections or may spread to other patients [3] [4] [5] [6] [7] [8] [9] [10] [11] . to reduce and control healthcare-associated infections (hais) caused by mrsa, a ''search and destroy'' strategy, which first detects the patients with mrsa colonization and then decolonizes the mrsa with certain antimicrobial agents, was recently proposed and implemented in some hospitals of different countries, with inconsistent effects [12] [13] [14] [15] [16] . in taiwan, mrsa was first documented in early 1980s and rapidly increased in 1990s [17] . in 2000, methicillin resistance had been identified in 53-83% of all s. aureus isolates in 12 major hospitals of taiwan [18] . in our neonatal icus (nicus), s. aureus is the leading pathogen of hais and mrsa represented majority of all the s. aureus isolates since 1997. between 1997 and 1999, the prevalence of s. aureus among the hais increased significantly from 32.4% in 1997, 43 .6% in 1998, to 52.6% in 1999. the percentage of mrsa among s. aureus isolates also rose significantly from 87.2% in 1997, 92.1% in 1998, to 95.1% in 1999 [19] . apparently, our nicus were endemic for mrsa. we then implemented a series of infection control interventions stepwise in our nicus to try to reduce hais caused by mrsa. after a 7-year campaign, mrsa hais was successfully controlled temporarily with the implementation of the strategy of ''search and destroy''. here, we report our experiences for mrsa control in the nicus. chang gung children's hospital is a university-affiliated teaching hospital, situated in northern taiwan, that provides a range of care, from primary to tertiary care, and is a part of chang gung memorial hospital (cgmh). there are three nicus, distributed on 2 floors, in this children's hospital. currently, there are 17 and 20 beds in nicu-1, and nicu-2, respectively. nicu-3 included two areas, 12 level -iii beds in area 1 and 45 non-level-iii beds in area 2 (special care nurseries). all the healthcareassociated infections (hais) in three nicus from 1999 to 2007 were prospectively collected and recorded according to the standard definition of hais [20] . the study included the institution's healthcare infection data, which were routinely collected and reported by the institution's infection control committee, and was also among the institution's quality-improvement programs proposed by the institution's infection control committee. since active surveillance for mrsa control is considered to be quality improvement, irb approval was not required to be included when application for the grants and thus the study was not reviewed by the institutional review board (irb) of chang gung memorial hospital at that time and informed consent could be waived [21] . since 2000, a series of infection control interventions were implemented stepwise in our nicus to try to reduce healthcareassociated infections caused by mrsa (table 1) . we firstly reenforced hand washing before and after contact with the infants hospitalized in nicus since january 2000 by increasing infection control education of, increasing infection control practitioner's audits of, and feedback of hais data to the health care workers (hcws) working in nicus. from a case-control study conducted in 2001, we found that the presence of skin infection at onset was one of the risk factors for mrsa bacteremia in these infants [22] . standardized operation procedures for the insertion and the continuous care of peripherally inserted central venous catheter (picc) were revised, aiming to accelerate the placement process (by a designated team) and to improve the aseptic care over the insertion site. briefly, after successful insertion, 10% povidineiodine containing alcohol (75%) was applied to the insertion site, normal saline used to decolorize, and the area was covered by a transparent dressing (''tegaderm''). nurses checked the insertion site frequently and changed the dressing every 3 days. the picc lines were not impregnated with antibacterial or antiseptic agents and antibiotic lock prophylaxis was not used. the strategy commenced in july 2001. from march 2003 to february 2004, screening for mrsa carriage among the hospitalized infants at nicu-1 and -2 was conducted [23] , which was supported by the research grants. during the nicus stay, specimens from the nares, postauricular areas, axillae, and umbilicus were obtained weekly and sent for detection of mrsa. the infants with mrsa colonization, if identified, were separated from non-colonized infants and placed in a segregated area of the units, and cohort care by designated nurses was implemented. almost at he same time, the outbreak of severe acute respiratory syndrome (sars) occurred in taiwan, and alcohol-based handrubs were introduced into the hospital in april 2003 and were used in these nicus thereafter. from august 2005 to july 2006, we implemented the ''search and destroy'' strategy into nicu-1 and -2, which was also supported by the research grants. from the previous surveillance study [22] , we learned that nearly 90% of the colonized infants are detected within the first 2 weeks of admission and sampling of both nares and umbilicus is adequate for surveillance cultures in this population. hence, during this period, only specimens from both nares and umbilicus were obtained, within 24 hours of admission and then weekly for two weeks (3 times in total). in addition to placing the colonizing infants in a segregated area and cohort care, decolonization procedures with topical mupirocin ointment application to nares and umbilical area were administered twice daily for five consecutive days if they still stayed in the nicus. if an infant with mrsa colonization had mrsa clinical isolates, the clinical isolates as well as the colonized isolates were genotyped and compared. mrsa isolates recovered from clinical diagnostic samples (beyond surveillance culture specimens) submitted to the clinical microbiologic laboratory were regarded as clinical isolates. in accordance with the standard definition of hais [20] , any infant with clinical isolates of mrsa who was receiving antimicrobial therapy was categorized as experiencing an episode of infection. however, from august 2006 to october 2007, no active surveillance for mrsa was conducted in these nicus since no research grants supported. surveillance cultures for health care workers (hcws) were performed, during surveillance periods, and specimens were obtained from the nares of hcws working in both units. intranasal mupirocin treatment was applied to the nares of each hcw with mrsa colonization. specimens for surveillance culture were obtained with a cotton swab, placed in a transport medium (venturi transystem), and then processed in the microbiology laboratory within 4 hours. identification of mrsa was confirmed according to national committee for clinical laboratory standards guidelines [24] . except for year 2002, mrsa clinical isolates from the hospitalized infants at these nicus between 1998 and 2007 were collected and selected for genotyping analysis. for those years with more than 40 clinical mrsa isolates collected (129 isolates in [23, 25] , which are also displayed in this study. colonized isolates from the infants and hcws were also molecularly characterized. the molecular methods included pulsed-field gel electrophoresis (pfge) with smai digestion, staphylococcal chromosomal cassette (sccmec) typing, and multilocus sequence type (mlst). in addition, the presence of panton-valentine leukocidin (pvl) genes was also examined. all the procedures were described previously [25] [26] [27] [28] [29] [30] . the genotypes of pfge were designated, as in our previous studies [25] [26] [27] [28] [29] , in alphabetical order; any new type, if identified, was designated consecutively. pfge patterns with ,4-band differences from an existing genotype were defined as subtypes of that genotype and were labeled with arabic number suffixes. two isolates were considered to be indistinguishable, related, or distinct if they had the same subtype, the same genotype, or a different type, respectively. we compared mrsa colonization and subsequent infection between the infants with and without topical mupirocin traetment by means of x 2 (continuity-adjusted) or student's t tests. relative risk and/or odds ratios (ors) were calculated with 95% confidence intervals (cis). healthcare-associated infection density and mrsa hai density from 1999 to 2007 were analyzed by mantel-haenszel chi-square test. statistical analyses of the data were performed with epiinfo, version 6 (centers for disease control and prevention, atlanta, ga) and sas for windows, version 6.11 (sas institute, cary, nc). through these infection control measures, hais in these 3 nicus caused by mrsa as well as by all bacterial pathogens decreased gradually and significantly from 1999 to 2007 ( the results of surveillance culture without decolonization were published previously [22] and are summarized in table 3 . briefly, mrsa colonization was detected for 41% of 783 infants surveyed during their nicu stay and was noted for 91% of 92 infants with mrsa infections. previous colonization was detected in 68 episodes (81%) of mrsa infections; colonized and clinical isolates were indistinguishable in 63 episodes (93%). during the period of surveillance and decolonization, mrsa colonization was detected for 8.6% of 452 infants surveyed. of the 39 infants with colonization, intranasal mupirocin ointment was administered to 26 infants who still stayed in the nicus. followup cultures were obtained from 18 infants and showed positive in two infants. second course of intranasal mupirocin ointment was administered in both infants and subsequently eradicated mrsa in both cases. one of them developed mrsa sepsis before the second course of therapy was commenced. all 4 isolates (2 clinical and 2 colonized isolates) from this case were genetically indistinguishably. in addition to this case, 4 additional mrsa infected cases were identified, without previous colonization. five (5.9%) of 85 health care workers were colonized with mrsa. the comparison between the two periods is shown in table 3 and significant difference was noted in terms of rates of infection, colonization and colonization with subsequent infection. however, no significant difference was noted in terms of rates of mrsa non-colonized but with infection and colonization of health care workers. the detailed molecular characteristics of mrsa isolates are shown in table 4 . from the 429 clinical isolates analyzed, a total of 7 pulsotypes were identified. there were two major clones and characterized as sequence type (st) 239 (or its single locus variant)/pulsotype a (hungary clone)/sccmec iii or iiia/pvlnegative, accounting for 62% of the isolates, and st59/pulsotype c/sccmec iv/pvl-negative, accounting for 26%. the former clone was dominant (.50% of the isolates) from 1998 to 2004, became weakened (39% of the isolates) in 2005, reached zero in 2006, and then resurged in 2007 (18% of the isolates). the latter clone remained steady (around 30%) during the study period, (2005) (2006) belonged to the linage of st59. in contrast, most colonized isolates from hcws, regardless of during which period, belonged to the clone of st59/pulsotype c/sccmec iv/pvl-negative. the present study demonstrates that through infection control measures, hais caused by mrsa can be successfully controlled temporarily, even in high level mrsa endemic neonatal intensive care units. in the current study, mrsa hai density was reduced by 92%; ha bloodstream infection caused by mrsa was reduced from 40 cases per year to only one case per year. with the reduction of hai caused by mrsa, the ha infection density decreased proportionally and significantly. it appears that zero ha mrsa bacteremia in nicus is not infeasible, even in mrsa endemic units like ours, if effective infection control measures are implemented and executed strictly. during the study period, neither the manpower of nursing staff nor the bed occupation rate (more than 95%) in our nicus was changed. the reduction of mrsa infection was gradual and significantly; it appeared to occur prominently for two specific time periods, from 2001 to 2002 and from 2004 to 2005. for the first time period, we revised the standardized operation procedure for insertion and continuous care of peripherally inserted central venous catheter (picc). this strategy was based on our findings from the case-control study that the presence of skin infection at onset was associated with mrsa bacteremia in these infants. augmentation of aseptic procedure and care of the insertion of central venous catheter (cvc) has been documented to be able to reduce the incidence of catheter-related bacteremia [31] and seemed to be somewhat effective in our nicus. conducting the strategy of surveillance culture with subsequent decolonization of mrsa carriage in two of three nicus was the major change of infection control measures in the second time point in the current study. compared to those during the period of surveillance culture without decolonization, both mrsa colonization rate and mrsa infection rate among the nicu infants decreased significantly. in contrast, the rate of mrsa infection among the non-colonized infants was similar during both periods; even, mrsa ha infection rate increased slightly in 2007 when the strategy of ''search and destroy'' was discontinued since the supported grant was due. (afterwards, another project with a cross-over design was granted and conducted since november 2007) altogether, these findings seemed to suggest that topical mupirocin treatment may effectively decolonize mrsa carriage in these infants and reduce the subsequent mrsa infection. though the susceptibility test of mrsa isolates to mupirocin was not performed in this series, we believe that most isolates were susceptible to mupirocin since this medication, though licensed, had not been used in taiwan for years. however, since no control group (only historical control) was included and not every infant with mrsa colonization received topical mupirocin therapy in the current study, further studies are needed to elucidate this issue [32] [33] [34] [35] . compared to adult icus, patients admitted to nicus are relatively ''simple''. most infants hospitalize immediately after birth and a substantial proportion of the infants are inborn. in addition, vertical transmission of mrsa is infrequently seen in newborns. therefore, most infants do not have mrsa colonization on admission to nicus. then, if the hcws working in and the environmental objects in nicus are free form mrsa, the hospitalized infants would not acquire mrsa colonization and/or infection during their nicu stay. in the current study, between 2003 and 2006, nasal mrsa carriage rate among hcws working in our nicus ranged from 4.8% to 13% for 4 surveys [23] . but no survey for environmental objects was conducted during the study period. however, the condition would not change, even though mrsa may be introduced into the units anytime, if each hcw can perform hand hygiene exactly and strictly. these may partly explain why the ''search and destroy'' strategy can be effective in our nicus but not so effective reported from adult icus otherwise [14] . from the molecular analysis of mrsa isolates, we found that the epidemic as well as endemic clone, st239/pulsotype a (hungary or brazilian clone), dominated between 1998 and 2005 in our nicus, even accounting for 90% of the clinical and colonized isolates in certain years. this clone was almost eradicated, not identified from any clinical isolate, from our nicus in 2006; however, it still accounted for 13% of the colonized isolates during 2005-2006. this clone returned and identified from clinical isolates again in 2007 and persisted in 2008 (data not shown here) but accounted for less than 20% of the clinical isolates. the clone, st239/pulsotype a, had prevailed in our hospital (cgmh) and even the whole island between 1992 and 2001, accounting for 54% to 93% of clinical isolates from different hospitals islandwide [26, [36] [37] [38] [39] . however, the predominance was decreasing during 2004-2005 in our hospital (cgmh) [38] , so was in our nicus in the current study almost at the same time. in contrast, the clone of st59/pulsotype c/sccmec iv/ pvl-negative accounted for one-fourth of the clinical isolates and remained relatively steady throughout the study period. the clone accounted for most colonized mrsa isolates from healthy children [27] as well as a substantial proportion of communityassociated mrsa infection in taiwan [28, 29, [40] [41] [42] and thus was categorized as a community strain recently. more than 70% of colonized isolates from hcws working in these nicus also belonged to this clone during the study period, suggesting that they might acquire mrsa colonization in the community rather than in the hospital. in addition, two other community clones, also belonging to st59 linage, emerged and increased markedly during 2006-2007. it has been reported that community-associated mrsa strains spread into the hospital and even successfully replaced the original hospital strains [43, 44] . the changing molecular epidemiology needs more surveillance and its clinical implication and significance needs more observations. results from the surveillance culture, we reported previously [23] , indicated that preceding or concurrent colonization was detected for .80% of the infants with mrsa infection, and the clinical isolates were indistinguishable with the colonized isolates in .90% of the episodes, on the basis of molecular evidence. these strongly suggest the association between mrsa colonization and subsequent infection and indirectly suggest that to reduce mrsa infection in the infants hospitalized in nicus, active surveillance with subsequent decolonization of mrsa is mandatory. as the issue which disinfectants or antimicrobials, topical or systemic administration, are effective deserve further studies. methicillin-resistant staphylococcus aureus change in the epidemiology of methicillin-resistant staphylococcus aureus in intensive care units in us hospitals risk and outcome of nosocomial staphylococcus aureus bacteraemia in nasal carriers versus non-carriers methicillinresistant staphylococcus aureus (mrsa) nares colonization at hospital admission and its effect on subsequent mrsa infection natural 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infection with topical intranasal mupirocin: an evidencebased review randomized, placebo-controlled, double-blind trial to evaluate the efficacy of mupirocin for eradicating carriage of methicillin-resistant staphylococcus aureus mupirocinbased decolonization of staphylococcus aureus carrier in residents of 2 long-term care facilities: a randomized, double-blind, placebo-controlled trial intranasal mupirocin to prevent postoperative staphylococcus aureus infections longitudinal analysis of methicillin-resistant staphylococcus aureus isolates at a teaching hospital in taiwan frequent recovery of a single clonal type of multidrug-resistant staphylococcus aureus from patients in two hospitals in taiwan and china changing molecular epidemiology of methicillin-resistant staphylococcus aureus bloodstream isolates from a teaching hospital in northern taiwan change in the molecular epidemiology of methicillin-resistant staphylococcus aureus bloodstream infections in taiwan epidemiological typing of community-acquired methicillin-resistant staphylococcus aureus isolates from children in taiwan successful multiresistant community-associated methicillin-resistant staphylococcus aureus lineage from taipei, taiwan, that carries either the novel staphylococcal chromosome cassette mec (sccmec) type v t or sccmec type iv comparison of both clinical features and mortality risk associated with bacteremia due to methicillin-resistant staphylococcus aureus and methicillin-sensitive s. aureus hospital transmission of community-acquired methicillin-resistant staphylococcus aureus among postpartum women community strain of methicillin-resistant staphylococcus aureus involved in a hospital outbreak the authors thank all the colleagues working in the neonatal intensive care units for their support and cooperation and the infection control team for their data collection and cooperation. key: cord-261410-kb91eagd authors: park, ji young; kim, bong-joon; lee, eun jung; park, kwi sung; park, hee sun; jung, sung soo; kim, ju ock title: clinical features and courses of adenovirus pneumonia in healthy young adults during an outbreak among korean military personnel date: 2017-01-23 journal: plos one doi: 10.1371/journal.pone.0170592 sha: doc_id: 261410 cord_uid: kb91eagd background: the number of pneumonia patients increased suddenly in korean military hospitals in late december 2014, indicating the urgent need for an epidemic outbreak investigation. methods: we conducted a prospective study of pneumonia etiology among immunocompetent young adults admitted to daejeon armed forces hospital. patient blood and sputum samples were subjected to conventional culture, serology, and polymerase chain reaction tests for respiratory viruses and atypical pathogens. results: from january to may 2015, we enrolled 191 (189 male) adults with pneumonia; the mean age was 20.1 ± 1.3 years. five patients had severe pneumonia, and one died. pathogenic human adenoviruses were most common (hadv, 153/191 [80.1%]), indicating a hadv pneumonia outbreak. genotyping of 35 isolates indicated that 34 matched hadv-55 and one matched hadv-2. hadv pneumonia infected recruit trainees most frequently. high and prolonged fever, nasal congestion, sore throat, and pharyngeal inflammation were significantly more common in the hadv pneumonia group, compared to patients with other or unknown causes of pneumonia. only 12% of hadv pneumonia patients displayed leukocytosis, whereas febrile leukopenia (62.7%) and thrombocytopenia (41%) were commonly observed. hadv pneumonia patient chest ct scans displayed ground glass opacity (with or without septal thickness) with consolidation in 50.0% of patients. conclusions: an outbreak of hadv respiratory infection occurred at the korean military training center. hadv pneumonia exhibited specific laboratory and clinical features, and although most patients were cured without complication, some progressed to respiratory failure and fatality. therefore, hadv vaccine should be provided to military trainees in korea. introduction viral respiratory infection is particularly important in military populations who experience overexertion, psychological stress, and crowding within confined spaces [1] . there are many reports of respiratory virus outbreaks in the military [1] [2] [3] . in many settings, human adenovirus (hadv) was the main causative pathogen and occasionally led to death [3, 4] . hadvs are also important in the pathogenesis of community acquired pneumonia (cap) among both immunocompetent and immunocompromised individuals [5, 6] . although despite reports of a low prevalence in previous studies (1.4-4%), adenovirus-related cap was recently ranked in the top 10 etiologies of cap by larger studies [7, 8] . moreover, hadv is easily transmittable and can be highly contagious [9] . the clinical features of respiratory adenoviral infection among military personnel were described previously; however, hadv pneumonia in immunocompetent individuals and risk factors of disease progression to severe pneumonia or acute respiratory failure have not been well studied. in december 2014, a sudden increase in patients with febrile respiratory illness and pneumonia occurred among military hospitals of the south korean army, including our institution. medical staff noted that the rates of hadv-positive respiratory specimens had also increased. therefore, we deduced an emergent hadv outbreak and aimed primarily to identify the pathogenic agent(s) causing the sudden increase in pneumonia cases. we also aimed to describe the clinical features and radiological findings of hadv pneumonia in immunocompetent individuals. we conducted a prospective study of cap in immunocompetent military trainees or active duty soldiers admitted for pneumonia to daejeon armed forces hospital, south korea from january to may 2015. patients are referred to this 500-bed hospital by basic and advanced military training centers and other military hospitals. in south korea, military service is mandatory for all healthy men !18 years old. trainees spend 6 weeks on basic military training, and then proceed to advanced training centers or active duty [10] . all military trainees or active duty members, but not officers, were eligible for enrollment if they were !18 years old and had been admitted to the study hospital for pneumonia, defined by acute respiratory symptoms (fever, cough, sputum, dyspnea, and pleuritic chest pain) and pulmonary infiltrates on chest x-rays or computed tomography (ct) scans. patients diagnosed with other pulmonary diseases were excluded. command (afmc 15016-irb-15-011), and informed consent was obtained from all patients. consent was verbal in nature because an etiological evaluation of pneumonia is considered routine care. the verbal consent procedure was approved by the ethics committee of the armed forces medical command. the study medical officer maintained a register of patients who consented verbally to participate in the cohort. patient sputum samples for conventional culture and polymerase chain reaction (pcr) tests and blood samples for culture and serologic tests were collected before prescribing medications, which were chosen at the physicians' discretion. outpatient clinic or emergency room investigating physicians collected clinical information. sputum specimens were collected from all patients at enrollment. these were acceptable for culture if they satisfied murray-washington classification degrees iv or v [11] . sputum specimens were cultured routinely and tested by pcr. severe pneumonia was defined by one or more of the following criteria: 1) invasive mechanical ventilation, 2) use of vasopressors, 3) >50% of lung parenchymal involvement. all cases were scored according to the pneumonia severity index (psi) and curb-65 [12, 13] . we performed multiplex pcr for human respiratory viruses using the advansure™ rv realtime pcr kit (lg life sciences, korea; supplementary methods). this assay targets 12 types of pathogenic rna viruses: rhinoviruses a/b/c, influenza viruses a/b, coronaviruses 229e/ nl63/oc43, respiratory syncytial viruses a/b, parainfluenza viruses 1/2/3, and metapneumovirus; and two types of dna viruses: adenovirus and bocavirus [14] . we performed multiplex pcr for the respiratory bacterial pathogens mycoplasma pneumoniae, chlamydophila pneumoniae, legionella pneumophila, and bordetella pertussis, using the seeplex 1 pneumobacter ace detection assay (seegene, seoul, korea; supplementary methods). streptococcus pneumoniae and haemophilus influenzae were not analyzed because we could not differentiate true infection from colonization of these pathogens [15] . we performed nested pcr in the hypervariable region of the hexon gene for genotyping using previously described nested pcr conditions and primer sequences for hexon gene amplification [16] . pcr products were purified with the qia quick pcr purification kit (qiagen, valencia, ca, usa) prior to their use as nucleotide sequencing templates. the genotype of each isolate was determined according to the serotype of the highest scoring strain in genbank, using the basic local alignment search tool (blast). categorical variables were analyzed by the chi-squared or fisher's exact test. continuous variables were compared by the student's t-test or mann-whitney u-test. a p-value <0.05 was considered statistically significant. all analyses were performed using spss for windows ver. 18 table 2 ). typical bacteria (klebsiella pneumoniae) were cultured by conventional culture in only one patient. cases involving co-infection with multiple pathogens are listed in s1 table. hadv pneumonia outbreak epidemiology the number of weekly hadv-positive cases was plotted (fig 2a) . hadv cases peaked at week 10 (early march 2015). a secondary peak occurred at week 17. hadv pneumonia cases occurred throughout the training period but were most common around the end of basic training (week 6, fig 2b) . most hadv pneumonia patients were basic military trainees or personnel who had recently completed training; active duty service personnel were not usually affected, even during outbreak peaks. there were no significant differences in the mean age, smoking status, and influenza vaccination rate ( table 1 ). the median military service period (including training period) was shorter in the hadv-positive pneumonia group than in the negative group (7.0 vs. 8.6, p = 0.032). we compared clinical characteristics according to the hadv pcr test results (tables 1 and 3) . fever, high fever (!39.0˚c), nasal congestion, sore throat, throat clearing, headache, and pharyngeal inflammation were more common among hadv pneumonia patients than in others. many hematologic findings of the hadv pneumonia group differed significantly from those of the negative group (table 3) , including leukocytosis, febrile leukopenia, thrombocytopenia, and hematocrit. the mean serum levels of c-reactive protein (crp) and procalcitonin were not significantly different between the two groups. pleural fluid analysis was available for three hadv pneumonia patients, and their samples were found to be lymphocyte dominant (mean, 76.1%) with a mean adenosine deaminase level was 61.9 iu/l. the mean psi score was higher among the hadv-positive group than the negative group (29.8 vs. 24.8 p = 0.005) and psi class rates tended to be higher among the hadv-positive group (p = 0.046, table 4 ). chest ct images were obtained from all patients except one. ground-glass opacity (ggo; an area of increased opacity without obscuration of the underlying vessels) and septal thickening were more common in the hadv-positive group than in the negative group (p = 0.014 and p = 0.001, respectively). nodules and bronchial wall thickening were less common in the hadv pneumonia group. the parenchymal opacities in hadv-positive pneumonia patients among hadv pneumonias, severe pneumonia was observed in five patients. severe pneumonia patients and others did not differ significantly with respect to demographic characteristics and most symptoms (s3 table) . however, high fever, dyspnea, and chest discomfort were more frequent and febrile periods were significantly longer among patients with severe hadv pneumonia, compared to others (8.6 ± 1.9 vs. 6.3 ± 1.6 days; p = 0.002). the time from fever onset to the greatest radiologic aggravation (increased opacity on follow-up chest x-ray) was also longer in the severe group (9.0 ± 2.7 vs. 6.3 ± 1.6, p = 0.001). severe hadv pneumonia was associated with a lower white blood cell count (wbc) and platelet count on admission day (p <0.001 and p = 0.042, respectively). although the mean crp value was higher in the severe group (p = 0.002), the mean serum procalcitonin concentration did not differ significantly between patients with severe pneumonia and others (p = 0.102; s4 table) . there were some complications among hadv pneumonia patients. acute heart failure occurred in two patients. delirium occurred in one patient. one patient developed upperextremity deep vein thrombosis, possibly resulting from a central line catheter. two patients required mechanical ventilation. of these latter patients, one expired from respiratory and heart failure ( table 4 ). our results show that an outbreak of hadv pneumonia occurred in korean military training centers and indicate that emergent-type hadv-55 infections might have caused the outbreak. in this outbreak, hadv pneumonia was associated with specific clinical symptoms, laboratory results, and chest ct scan findings. recently, multiple outbreaks of acute respiratory disease have been associated with an emergent variant, hadv-55 (formerly named hadv-11a). whole-genome sequencing of this variant indicates potential hexon gene recombination between the hadv-11 and hadv-14 strains [17] . such outbreaks occurred in the military forces of turkey in 2004 [18] and singapore in 2005 [19] . in china, several outbreaks of hadv-55 respiratory disease have been reported not only in the military, but also in the community (e.g., a senior high school in 2006 [20] , beijing in 2011 [21] , military in 2012 [1] , and a physical training facility in 2013 [22] ). since 2012, hadv-55 has also been identified among severe pneumonia patients at a korean military hospital [23] . most hadv pneumonia patients were basic trainees or personnel who finished their training recently. these results were similar to those of previous studies [24] . it is well documented that the most significant factor leading to hadv infection and disease is a lack of preexisting, type-specific immunity against hadv-4 and hadv-7. moreover, anti-hadv4 immunity provided 60% protection from adenovirus-related hospitalization and 98% protection from infection [1, 25] . in the korean military, it is not certain why active duty service personnel were infected less frequently than trainees. one possibility is that the former group may have had preexisting hadv-55 antibodies from previous exposure. another possibility is that the former group had other cross-protective hadv antibodies. in 2006, hadv-7 was the dominant type of hadv in the korea military [26] . hadv-7 is in the same species group as hadv-55 (hadv-7/11/14/55 are in species group b) [27] . when a hadv-14 outbreak occurred at u.s. military training facilities, preexisting hadv-7 neutralizing antibodies provided cross-protection against hadv-14 in recruits [28] . other predisposing factors that might be associated with selective infection of newly recruited trainees include overcrowding and physical or emotional stress [29, 30] . hadv pneumonia diagnoses began in the 2nd week of training and peaked at week 6, in close accordance with previous u.s. army studies. kolavic-gray et al. [25] found that hadv-4-related acute respiratory diseases among military trainees peaked during week 5 of training, and tate et al. [28] reported that hadv-14-associated febrile respiratory illness peaked during weeks 4-6. one possible reason for this timing pattern is that sufficient numbers of infected or colonized trainees may be required to cause an outbreak [28] . moreover, the 6-week training period is epidemiologically important because trainees usually move to advanced training centers or into active duty after completing basic training, possibly creating a secondary outbreak. our prospective analysis demonstrated that this hadv pneumonia was associated with specific clinical features that differed from other pneumonias. although the mean fever duration in hadv pneumonia patients was similar to those in previous studies (6-9 days) [21, 31, 32] , patients with hadv pneumonia had higher and longer fevers than the hadv-negative group. moreover, the fever duration associated with severe hadv pneumonia was prolonged relative to that of mild-moderate pneumonia, again corroborating previous reports [32] . patients with hadv pneumonia also had more upper respiratory tract symptoms than those in the negative group, suggesting that hadv infected or colonized the upper respiratory tract first, then progressed to a lower respiratory tract infection. the most typical laboratory findings of hadv pneumonia were febrile leukopenia and thrombocytopenia. in agreement with our results, vento et al. [33] demonstrated lower wbc and platelet counts in a hadv-14 pneumonia among u.s. military trainees. patients with severe pneumonia had fewer wbc and lower platelet counts than those with mild to moderate pneumonia. moreover, the mean times from fever onset to the nadir of wbc and to the greatest radiologic aggravation were similar and correlated (r = 0.513, p <0.001). these findings suggest that hematologic parameters might be used to monitor hadv pneumonia and to index risk factors for disease progression. finally, although the crp levels were moderately elevated in our study, the mean procalcitonin level was only 0.35 μg/l, confirming that a bacterial infection was unlikely [34] . in fact, bacterial coinfection was rare. among the 153/191 adenovirus-positive patients, only 3.9% (6/153) exhibited evidence of bacterial infection (typical: 1; atypical: 5). in contrast, among the 38/191 adenovirus-negative patients, 47.4% (18/38) had bacterial infections (p <0.001). this further confirms the hadv infection findings in our cohort. very few case studies of the ct findings of hadv pneumonia exist in the literature [35] . the most common pattern in our study was ggo with or without septal thickness and central consolidation, a finding specific to hadv pneumonia that was not observed with hadv-negative pneumonia. our ct scans also demonstrated that single lobe involvement was more common than multi-lobe involvement, in contrast to a previous case report that suggested such cases usually manifested as bilateral involvement [35] . these observations might be useful during the differential diagnosis of young adults presenting with pneumonia symptoms during an outbreak; however, further radiological studies are needed. no antiviral agents have been approved to treat hadv pneumonia, and only limited data on the clinical responses of immunocompromised patients to cidofovir are available. however, kim et al. [23] , in another study of severe hadv infection in the korean military, suggested that early treatment with cidofovir should be considered for respiratory failure due to hadv pneumonia. most patients in our study were treated empirically with antibiotics, and most hadv pneumonia cases were self-limiting without an antiviral agent. however, cidofovir was administered at a 5-mg/kg weekly dose to two severe hadv pneumonia patients. one patient recovered without sequelae, whereas the other patient died. it would be unsafe to offer cidofovir to all hadv pneumonia patients because of the risk of renal toxicity. moreover, this drug is expensive and is not stocked regularly in hospitals. in this respect, the early identification of patients who might develop severe pneumonia is most important when selecting cases that will receive antiviral treatment [23] . interestingly, the psi scores and curb-65 values were higher in the severe pneumonia group, whereas the absolute scores in this group did not indicate high risk. notably, age is an important factor in these pneumonia severity score systems; accordingly, the lower scores obtained in our study might be partly attributable to the young ages of our adult patients. in addition, that these scoring systems were validated in communities where the main pathogens were bacterial, rather than viral [36] . similar results were observed in pandemic h1n1 influenza pneumonia studies in which psi and curb-65 failed to predict admissions to intensive care or the need for mechanical ventilation [37, 38] . our study has one limitation. because pneumonia increased abruptly in our patients, we were uncertain of which pathogen(s) caused the outbreak and did not plan to conduct hadv genotyping at the study outset. we only sent partially banked sputum specimens for hadv genotyping confirmation after realizing that there a hadv outbreak was occurring in the korean military. it is uncertain whether vaccines against types 4 and 7, which are currently only available in the u.s. army, will effectively protect against hadv type 55. therefore, new, more suitable vaccines should be developed for subtypes responsible for current outbreaks. in conclusion, this hadv pneumonia exhibited specific clinical and laboratory features and chest ct findings. although the pneumonia severity varied, this infection can induce morbidity and fatality. a respiratory virus surveillance system and epidemiological expertise are urgently needed and hadv vaccination should be considered in korean military training centers. the current smoking rate was 32.3%, and 94.8% of patients had received the seasonal (2014-15) influenza vaccine. excepting one patient who was incidentally diagnosed with gitelman's syndrome, none had systemic disease respiratory infections in the u.s. military: recent experience and control outbreak of acute respiratory disease caused by human adenovirus type 7 in a military training camp in shaanxi vaccine-preventable adenoviral 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serotype 14 infection viral pneumonia adenovirus pneumonia in adults: radiographic and high-resolution ct findings in five patients the pneumonia severity index: a decade after the initial derivation and validation severity assessment tools in icu patients with 2009 influenza a (h1n1) pneumonia severity of influenza a 2009 (h1n1) pneumonia is underestimated by routine prediction rules. results from a prospective, population-based study the authors thank bong joon kim conceptualization: jyp jok. key: cord-013263-xw611i8k authors: dederichs, melina; weber, jeannette; muth, thomas; angerer, peter; loerbroks, adrian title: students’ perspectives on interventions to reduce stress in medical school: a qualitative study date: 2020-10-15 journal: plos one doi: 10.1371/journal.pone.0240587 sha: doc_id: 13263 cord_uid: xw611i8k the mental health of medical students remains to be a matter of concern. numerous setting-based and individual-based interventions for student mental health have been proposed in the literature. however, the student perspective on those interventions has been largely neglected. this study aims to explore how medical students perceive different interventions and if they desire any additional changes with regard to their studies. eight focus groups with 71 participants were conducted at a large german medical school. focus groups were recorded, transcribed and content-analyzed using maxqda 18. we found that medical students prefer setting-based interventions. most proposed interventions were on a setting-based level. for instance, students asked for more information on the university’s psychosocial counseling services and for better information management regarding contact persons. interventions proposed in the literature received mixed reactions: several participants did not favour a pass/fail grading system. students considered a peer-to-peer mentoring program for freshmen very helpful. students had diverse attitudes towards balint groups. they approved of several self-management courses, most of them being related to time or stress management. interestingly, the most urgently wanted interventions appear to be rather easy to implement (e.g. a mentoring program). this study explored the medical student perspective on student mental health interventions. additionally, our study illustrates the benefit and feasibility of involving students early on in the conception of interventions. further research with a representative sample is needed to obtain broader information on the acceptance of the suggested interventions. medical students show a high prevalence of mental illnesses worldwide, such as high levels of depression and depressive symptoms [1] .to address medical students' poor mental health, setting-based and individual-based interventions have been proposed [2] [3] [4] [5] . setting-based interventions aim to improve health by modifying environmental factors, e.g. curricular changes in medical schools [3, 6, 7] . individual-based approaches, by contrast, seek a change in the individual, e.g., by providing skills that help to cope with stress [8, 9] . although some interventions different perspectives. we therefore conducted a qualitative study with focus groups to gather insights and specific explanations for why students deemed specific interventions useful or not. our study aims to answer the following questions: 1. what interventions do medical students themselves suggest and wish for? 2. how do medical students perceive interventions that have been described in the literature (pass/fail grading, a peer-to-peer mentoring program, balint groups, and self-management courses)? participants were recruited through social media or advertisement on campus of heinrich-heine-university of düsseldorf in germany. the only criteria for participation were to be currently enrolled as medical student and not having participated at our previous focus groups [29] . participants of one focus group were recruited through a stress-management seminar. participation was explicitly on a voluntary basis. all participants gave their informed consent. students were compensated for their participation with two cinema tickets and one cinema discount card. focus groups were conducted until data saturation was reached. students were grouped into focus groups according to their period of study. this maximised the likelihood that participants could relate to each other in terms of experience regarding classes and exams. four groups included students in the preclinical part of their studies (year 1 to 3) and four groups included students both from the clinical part (year 4 to 5) as well as students in their clinical internship year (after the second state examination). the focus groups took place in june and july 2019 at the medical faculty of heinrich-heine-university düsseldorf. in 2013, the medical faculty introduced a new curriculum. traditionally, medical students take one final exam organised by the state (state-examination) for each of the three periods of study: the first after the preclinical phase (after year 3), the second after the clinical phase (after year 5) and the last one after one year of practical internship at a hospital. with the new curriculum, the first state examination takes place after the first two years. permission to conduct this study was given by the ethics committee of the medical faculty of heinrich-heine-university of düsseldorf. after provision of written informed consent, participants were asked to fill out a questionnaire covering demographic data. the focus groups were conducted by tm, a faculty member and psychologist by training, while md, a junior researcher and trained psychologist, took notes. the focus groups followed a topic guide developed by md and tm (s1 file). both facilitators provide lectures for medical students and are involved in research on medical students' wellbeing. participants were informed about the facilitators' research focus. first, participants were asked about their experience in medical school so far and in which situations they had encountered obstacles or experienced stress. secondly, students were asked about potential changes and interventions that could contribute to stress reduction in medical school. previous research at our faculty already pointed out high stress levels and specific stressors of our medical students [29, 30] . to answer the second research question (how the students perceived literature-based interventions), we discussed interventions proposed in the literature with medical students. the topic guide contained a list of the interventions (see introduction section), which were explained by the facilitator and then discussed by the students one after the other. if necessary, the facilitator asked follow-up questions to further explore students' views. every single participant was encouraged to contribute to discussions at an early stage. all focus groups were audio-recorded, transcribed and content analyzed by md and jw according to mayring [31] using maxqda 18. jw is experienced with occupational health and qualitative research [29] . research questions were transformed into deductive main categories (students' suggestions, interventions from literature). during analysis these categories were further broken down into sub-categories by inductive category formation. after md completed the first coding round of 25% of the data, al, an experienced qualitative researcher [29, 30, 32, 33] , reviewed the coding scheme. the coding scheme was then adapted and a second coding round was performed by md. jw coded the data of four groups independently. both versions were compared and discrepancies resolved. after that, md recoded the remaining four groups and transferred changes made to the coding system in the previous discussion with jw. finally, jw and md discussed the focus groups once more. there was no need for further recoding since there were only minor differences between the analyses of md and jw. due to logistic constraints, corrections and feedback on transcripts and research findings were not obtained from study participants. the completed checklist of consolidated criteria for reporting qualitative research (coreq; [34] ) can be found in s2 file. eight focus groups consisting of 5 to 16 medical students were conducted. focus groups lasted between 90 and 130 minutes. in total, 71 medical students (56 women) participated. with n = 2816 medical students being enrolled at the medical faculty in the summer semester of 2020, we included 2.5% of the population in our focus groups. participant age ranged between 19 and 39 years (mean = 23.71; sd = 3.50). all participants engaged in a comprehensive discussion about what they would like to change in medical school to improve student wellbeing. in the following, we will present their proposed starting points for interventions. regulations for absence of lecturers. students reported that especially in the clinical study phase, many lecturers did not show up for teaching. students asked for a substitution or at least a designated contact person. attendance. attendance rules were perceived to be too strict. according to the study regulations, an attendance of at least 85% presence per subject is required. however, if one subject consists of three appointments for example, all three seminars have to be attended. by attending only two of them, the presence would be 67%, thus below 85%. students therefore requested that if one misses class due to sickness or death of a family member, an exception should be granted. according to the participating students, there should also be the option to complete a compensatory task, for instance, holding a presentation on the subject that was missed. as another solution for not missing a mandatory event, participants suggested switching groups with their peers for the required event only. due to the high number of mandatory classes during the preclinical phase, students wished for less mandatory attendance during this phase. coordination of practical and theoretical phases. the düsseldorf medical curriculum combines phases of theoretical studying (lectures) and practical experience (clinical traineeship). students wished that the curriculum is adapted so that theoretical and practical lectures as well as corresponding tutorials are better aligned and deal with the same subjects. this was believed to increase their learning effect. students noted that the workload between theoretical and practical phases fluctuates significantly with high workload during theoretical phases and low workload during practical phases. therefore, they wished for shorter practical phases to distribute the workload more evenly. often, the lecture-free week takes place after the clinical traineeship. however, students would rather have a week off for studying after completing the theoretical lectures when they find to have more content to repeat and memorize. counseling. participants pointed out that they would benefit from a broader psychological support system. regarding the on-campus counseling service, students wished for longer opening hours as well as opening hours that are compatible with their schedules. free of cost confidential counseling services were perceived as useful for reaching out to students to address mental health issues. overall, students wished for more information regarding mental health problems and interventions. possible solutions students brought up were for instance a mandatory lecture about stress related to medical school, coping strategies and support contacts. additionally, students suggested that a newsletter should be emailed close to the first state examination to present information on emergency contacts and counseling services. registration processes for elective subjects. students explained that when they want to register for a subject, they have to log in to an online portal. there they would have to wait until midnight until the registration process opens. they further explained that registrations are allocated on a first come, first serve basis. therefore, students stated that without access to a fast and reliable internet connection at that time they would not be able to take the class they prefer. students would like this procedure be replaced by a selection based on preference. in this procedure, students could indicate their preference within a certain period. then they would be matched according to their choice. if there is no personnel to do this task, some students suggested that at least the first come-first serve procedure was opened during daytime. information management. students reported a lack of information management in medical school. they requested a comprehensive online portal containing information on classes and exams. moreover, students expressed that they would benefit from an introductory lecture for each new topic block that contains all relevant administrative information, e.g. on how to register for an exam. a faq that addresses most urgent questions concerning the first state examination was considered useful. online lectures. many students raised the topic of online lectures. they perceived online lectures as an efficient measure to reduce stress and improve flexibility. especially commuters were thought to profit significantly from it. online lectures were thought to be a great learning tool and aid with the preparation of future seminars. one student requested a contact person in case any questions arise during a session. "i think it would be great to have this opportunity. i personally learn at night. in the morning, i don't pick up much, but at night i am really diligent. and it were a lot easier to combine family, job and university. that would be a huge advantage." refresher courses. students stated that they would benefit from refresher courses before the second state examination. "i believe it would be pretty fair, for instance, to have courses that prepare you for the state examination in practical terms during the semester. that would be, i think, not bad." examinations. students reported that their exams cover several subjects at once. students asked for a restricted number of subjects within the same exam. they reported that this could be achieved if exams included more questions on the same subject per exam instead of the same subject posing only a few questions per exam over several exams. "now for instance [ they also suggested grades acquired in seminars should be added to the exam score and act as a buffer. it was discussed whether more open-ended questions instead of multiple-choice questions would be beneficial. some students stated that they could explain their knowledge better in open-ended questions. moreover, it would motivate them to learn more details. others preferred the existing multiple-choice format. teaching content. participants made a range of suggestions to improve the quality of teaching. for instance, students wished for a contact person to report verbal harassment of students like depreciative comments by teaching staff. exact teaching guidelines containing learning goals for every subject were considered potentially helpful. they also wished for more covering of content relevant for the second state examination. a perceived lack of interest and skill of teaching staff was thought to be solved through employing senior students or external referents. they asked for more interactive and clinically orientated teaching. elective subjects. in total, 14 elective subjects have to be completed over the course of five years at the medical school in düsseldorf. some of the subjects are graded. participants raised the idea to reduce the total number of required courses to reduce stress and provide more time for state exam preparation. "it is good that we have elective subjects. but there are too many. and it is stressful, to have eight elective subjects here and another six elective subjects there. it would be great if we had less." alternatively, additional credit for scientific projects, clinical lectures or extracurricular activities was suggested. clinical traineeship. students asked to move the clinical traineeships towards the end of the medical curriculum, because students felt that they are often confronted with medical conditions for which they are not prepared yet and often clinicians presuppose content they have not dealt with yet. students reported frequent difficulties in their clinical traineeships. for instance, clinicians that are supposed to teach them are often not available and, if they are present, students felt that they do not take time to teach them adequately. more available contact persons or a central complaints office that assists them were mentioned as a possible solution to this issue. preparation for the second state examination. students wished for lecture free and exam free time for preparation before the second state examination. they requested to have at least 100 days to dedicate completely to studying. this way they stated they could follow the learning schedule they wanted to use (so called "100-day study plan" which is well-known and widely used by medical students in germany). "it is a question of planning. then, if you have these one hundred days, i think that would help a lot. and it would be nice if it wasn't exactly one hundred days but a couple of days more because maybe you don't study seven days a week." participants had many ideas on how to provide more time to study. participants proposed shortening clinical trainings, or to move them towards the end of the medical curriculum to avoid that students have to pass other exams right before the second state examination. students were willing to complete their clinical training during semester break or to begin the semester early to provide more preparation time for the second state examination. measures proposed in the literature pass-fail system. there was no consensus among the students in our focus groups related to the usefulness of pass-fail systems. some were in favour of eliminating grades because it would reduce pressure and stop demotivation through bad grades. others in favour of the pass-fail system argued grades did not have any informative value regarding their ability as a physician anyway and were thus of no importance. students opposing the pass-fail system reported to appreciate the incentive they received from good grades and to appreciate the feedback to better understand whether they had learned enough. students depending on a scholarship or on receiving particularly good grades raised concerns regarding their ability to compete and prove their performance. scholarship holders among the students reported that in order to keep their scholarship, they had to prove to belong to the top students in their semester. without grades, this might be difficult to prove and students from other universities might have an advantage. in düsseldorf, the final grade for the first state examination is calculated as a cumulative sum score of grades from the first to the third year and of the performance on the day of the state examination. students argued that this way they did not solely depend on one's day performance and therefore the stress on the day of the first state examination was reduced. some preferred keeping grades to maintain this system. however, students suggested alterations of the pass-fail system. they were in favour of eliminating grades from some subjects to reduce stress but maintain the current system of collecting grades for the first state examination. mentoring. almost all participants were in favour of a mentoring program. they appreciated it for its easy access and voluntary nature. the idea of having a permanent contact person that provides not only informational and emotional support but also further networking opportunities to higher semesters was welcomed. the personal experience and organizational knowledge of a senior student was thought to reduce stress significantly. "i would have liked having someone who told me 'everything is fine, this is normal. i experienced it too.' especially at the beginning of medical school. . . i had just moved here. . . and everything was new. having someone who can answer questions related to studying or simply help you." many students in senior semesters expressed the desire to become a mentor themselves. those who did not want a mentoring program stated they already established such a contact to a senior student by themselves. there were many suggestions regarding the design of a mentoring program. a pool with volunteers, possibly on an online-based platform was proposed. students further proposed that mentors should receive some form of compensation, either monetary or in a non-monetary form, like an honorary position. students argued that their willingness to participate in a mentoring program also depended on the mentor's personality. they feared that overly performance-driven students might be a bad role model in terms of stress management. students who would not want to participate in a balint group stated that they preferred being mentored by their peers or supervising physicians. they reported to rely on their own social networks for emotional support. some students expressed fear of stigmatisation due to participation in a balint group. they worried about talking freely about unpleasant experiences and being stigmatized by their peers. therefore, they would not want to participate. self-management courses. students were interested in learning self-management strategies or competences to strengthen their resilience. in this context, they wished for subjects on stress-management and relaxation techniques. moreover, students wished for a time-management course and more capacities in an existing elective subject deals with mind-body-medicine. participants stated they would benefit from courses that covered efficient learning strategies and presentation skills. there was little consensus among participants regarding the question when and how those classes should be implemented in their curriculum. in general, students perceived lack of time as a barrier to participate in a non-obligatory class. some students suggested that it should be an ongoing offer over a long period of time where students could decide for themselves every week whether they wanted to participate or not. students preferred to schedule such courses at the beginning of medical school and shortly before the first state examination. general dissatisfaction with already existing self-management courses on campus was expressed. students either perceived lack of information about those classes, lack of quality of available classes or limited capacity to enroll all interested students. the aim of the present study was to explore which interventions students suggest to improve their mental health and to discuss interventions suggested in the literature. in our eight focus groups, students suggested specific solutions to their perceived obstacles in medical school. interventions that students proposed most frequently pertained to setting-based aspects such as curricular changes, improved information management and new regulations concerning absence of teaching staff and students. many of the suggested interventions, such as online lectures, appear rather easy to implement. due to the current covid-19 pandemic (time of publication; [35] ), our faculty addressed the wish for more online lectures by creating new digital learning opportunities and structures. eventually these structures will persist after the university switches to conventional lectures once again. it is also striking that many of the suggested interventions might not only reduce stress, but also improve several aspects of teaching (e.g. the coordination of practical and theoretical phases). this suggests that improvement of well-being and improvement of academic outcomes go into the same direction. in contrast to students' wish for setting-based interventions, most interventions being proposed in the literature focus on the individual [8] . however, such interventions are believed to not tackle the root of the problem [36] . it is argued that instead of teaching students how to cope with stress, the causes for stress need to be addressed [36] . some individual-based interventions (e.g. stress-management courses) might even have the opposite effect by being an addition to existing classes and workload. it is further of interest that we observed that a significant number of our participants did not favor the interventions suggested by the literature. for instance, several students did not prefer a switch to a pass/fail grading system. these students perceived grades as helpful, were concerned that the absence of grades would have a negative impact on their academic performance, or would cause increased stress at the first state examination. however, this latter concern is not supported by the data [12, 37] . it is possible that the idea of a pass/fail grading system elicits discomfort among our students because it is rather uncommon in germany. when the pass/fail system was first introduced in the us similar concerns were raised [38] . a shift to a pass/fail system was perceived as a disadvantage for students and students coming from a university with pass/fail grading were thought to experience difficulties finding a residency placement. therefore, the authors did not recommend such a transition [38] . however, a recent study showed that a pass/fail grading does not curtail career prospects [29] . specifically, there were no differences in residency placement (receiving a placement at all and percentage of students that receive top specialty choices) and overall academic performance between pass/fail and tiered grading system cohorts [29] . besides pass/fail grading being uncommon in germany, it would also require a change of the persisting score calculation for the first state examination. additionally, it would require a cultural shift towards a non-competitive environment among all students in order to be successful. therefore, instead of fully switching to pass/fail grading only, a mixed approach might be more feasible in which pass/fail grading could be adopted in some subjects while other subject are still graded. in this approach, grades still contribute to the result of the state examination. this would address medical students' preference to accumulate marks and thereby reduce stress during the first state examination without a prompt change that requires time for adaptation. possible mentoring programs by senior students were well received by the participants in our study. more advanced students were believed to provide invaluable informational and emotional support. since such a program has relatively low costs and was also favored by potential mentors, we propose starting a peer-to-peer mentoring program at our university as recommended by others [39] . balint groups received mixed reactions. this is in line with previous research suggesting indifferent to mildly positive student attitudes towards balint groups [21, 22] . a qualitative study from finland reported a more positive evaluation of balint groups [23] . here, students reported being satisfied with their participation and benefiting from the groups [23] . in our present study, especially those students who had already encountered a difficult situation with a patient stated that they would be grateful for this kind of opportunity and support. therefore, the implementation of a balint group with voluntary participation on request as suggested by students could be considered. it could build on already established peer-support-programs such as the programs at brigham and women's hospital in boston, which has repeatedly served as a model for peer-support-programs [40] . students themselves requested a variety of self-management courses. most were interested in learning relaxation techniques and how to deal with stress. they stated that existing offers, like a time management class, are less attractive because their study curriculum does not allow for the implementation of taught strategies in this class. overall, they expressed the wish that classes to improve resilience should be accessible to everyone. some stress management courses (e.g. "stress management" or "mind-body-medicine") are only offered as elective subjects and are therefore only available for a fraction of the students. we suggest that all students receive a basic stress-management training and psychoeducation, preferably at the beginning of the curriculum. at some universities, such as the monash university medical school in australia, mindfulness and stress management have been part of the curriculum for many years [41] . since some studies found reliable short-term effects [8, 42] , students could benefit from it in most stressful times without the concern of losing time to study. for the latter reason we also suggest that more classes that address mental health are offered. in addition to face-toface training, e-mental-health-solutions could be made accessible for students for quick support. students' wish for e-learning opportunities suggests that they are open to digital formats that grant them more independence in terms of time and location of use. furthermore, information on specific counseling opportunities should be made more accessible. it is possible that raising awareness of medical students' mental health will reduce fear of stigmatization by peers which was mentioned as a concern in the context of balint groups. overall, we find that students proposed more setting-based interventions than individualbased interventions. this is in sharp contrast with the persistent emphasis on individual-based interventions in medical schools [8] [9] [10] 43] . importantly, while setting-based interventions are sometimes considered expensive or difficult to implement [7] , most ideas in the focus groups (e.g. attendance rules, a new course selection procedure, teaching guidelines) seem easily feasible and resource-friendly and will not only improve wellbeing, but also academic performance. a strength of this study is its rich data, which were collected among as much as 71 students from a broad range of semesters in eight focus groups until data saturation was reached. however, the generalizability of our findings may be limited since only students from the medical school at the university of düsseldorf were included in this study. our findings might be specific to issues and obstacles encountered at universities in germany and therefore only somewhat transferable to universities in other countries. when we asked the participants about their opinions on a pass/fail grading system, we did not clarify in detail whether they would still like feedback on their performance if a pass/fail grading system was implemented. thus, we do not know to what extent students perceive a pass/fail grading system and feedback on their performances mutually exclusive. further, we cannot rule out selection bias. one might argue that only those students suffering from stress or those who are especially dissatisfied with medical school attended the focus groups. on the other hand, one might assume that those students experiencing a high level of stress choose not to participate because of the additional workload. the fact that focus group facilitators were members of the teaching staff and that one focus group was held within a class on stress management could also have affected the observations. md's and tm's position as a teaching staff member might have elicited a social desirability bias and reduced willingness to share sensitive topics. however, also in this seminar, we felt that students spoke very openly about their issues. the contents of this focus group did not differ thematically from the other focus groups. this study explored which type of interventions students consider acceptable and useful. based on our data we are however unable to evaluate the feasibility and effectiveness of proposed interventions. however, we believe that only interventions that are favored by students and address their specific needs will be successful. in contrast to interventions to improve medical students' mental health that are proposed in the literature, we find that medical students mostly proposed interventions on a setting level rather than on an individual level. importantly, many interventions suggested by the students are low-cost and easy to implement. we believe that considering the student perspective is a key factor in designing mental health interventions. further research with a representative sample is needed to obtain more generalizable information on the acceptance of the proposed interventions and to test them in terms of feasibility and effectiveness by using both qualitative and quantitative approaches. prevalence of 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physician awareness group and the first year of training on hematology-oncology fellows stressors perceived by the para-clinical undergraduate medical students teaching the clinical encounter in psychiatry: a trial of balint groups for medical students reflecting on our practice: an evaluation of balint groups for medical students in psychiatry a qualitative analysis of student balint groups in medical education: contexts and triggers of case presentations and discussion themes. patient education and counseling self-management education: history, definition, outcomes, and mechanisms. annals of behavioral medicine user involvement: a review of the benefits and challenges. behaviour & information technology acceptability of healthcare interventions: an overview of reviews and development of a theoretical framework. bmc health services research helping medical students develop lifelong strategies to cope with stress a comprehensive medical student wellness program-design and implementation at vanderbilt school of medicine. academic medicine: journal of the association of american medical colleges stressors and resources related to academic studies and improvements suggested by medical students: a qualitative study medical students' perceptions of stress due to academic studies and its interrelationships with other domains of life: a qualitative study qualitative content analysis: theoretical foundation, basic procedures and software solution what are the perceived influences on asthma self-management at the workplace? a qualitative study psychosocial working conditions and diabetes self-management at work: a qualitative study consolidated criteria for reporting qualitative research (coreq): a 32-item checklist for interviews and focus groups meeting between federal chancellor merkel and the minister-presidents of the lä nder to discuss the coronavirus-press release 2020 medical student perspective on stress: tackling the problem at the root evaluating a grading change at ucsd school of medicine: pass/fail grading is associated with decreased performance on preclinical exams but unchanged performance on usmle step 1 scores pass fail grading-a disadvantage for students applying for residency stressors and starting points for health-promoting interventions in medical school from the students' perspective: a qualitative study peer support for clinicians: a programmatic approach the health enhancement program at monash university medical school enhancing the health of medical students: outcomes of an integrated mindfulness and lifestyle program academic psychiatry: the journal of the american association of directors of psychiatric residency training and the association for academic psychiatry we would like to thank johanna schwerdt for her invaluable help understanding the medical school curriculum and putting the students' perspectives into the right context. loerbroks. key: cord-255384-tljyx6ua authors: decaro, nicola; pinto, pierfrancesco; mari, viviana; elia, gabriella; larocca, vittorio; camero, michele; terio, valentina; losurdo, michele; martella, vito; buonavoglia, canio title: full-genome analysis of a canine pneumovirus causing acute respiratory disease in dogs, italy date: 2014-01-06 journal: plos one doi: 10.1371/journal.pone.0085220 sha: doc_id: 255384 cord_uid: tljyx6ua an outbreak of canine infectious respiratory disease (cird) associated to canine pneumovirus (cnpnv) infection is reported. the outbreak occurred in a shelter of the apulia region and involved 37 out of 350 dogs that displayed cough and/or nasal discharge with no evidence of fever. the full-genomic characterisation showed that the causative agent (strain bari/100-12) was closely related to cnpnvs that have been recently isolated in the usa, as well as to murine pneumovirus, which is responsible for respiratory disease in mice. the present study represents a useful contribution to the knowledge of the pathogenic potential of cnpnv and its association with cird in dogs. further studies will elucidate the pathogenicity and epidemiology of this novel pneumovirus, thus addressing the eventual need for specific vaccines. canine infectious respiratory disease (cird) is a multifactorial disease affecting dogs of all ages, which is typically induced by simultaneous viral and bacterial infections [1] . apart from wellknown canine respiratory pathogens, such as canine adenovirus type 2, canine herpesvirus [2] , canine distemper virus [3] , and canine parainfluenza virus [4] , novel viruses are being continuously associated with cird occurrence in dogs. these include canine influenza virus [5] , canine respiratory coronavirus [6] , canine pantropic coronavirus [7] [8] , canine bocaviruses [9] , and canine hepacivirus [10] . pneumoviruses (family paramyxoviridae, subfamily pneumovirinae, genus pneumovirus) are enveloped, single-strand negative-sense rna viruses that are associated with respiratory disease in mammals and birds. apart from the prototype species human respiratory syncytial virus (hrsv) and its ruminant relative bovine respiratory syncytial virus (brsv), a murine pneumovirus (mpv), also known as pneumonia virus of mice, is included in the genus pneumovirus [11] . this virus, which is only distantly related to human and ruminant rsvs, is a natural rodent pathogen circulating among research and commercial rodent colonies [12] . recently, a pneumovirus was associated to respiratory disease in canine breeding colonies in the united states [13] [14] . the virus, designated as canine pneumovirus (cnpnv), was found to be very closely related to mpv, displaying 95% nucleotide identity with the mpv prototype isolate j3666 [14] . experimental infection of mice with the canine isolate demonstrated that cnpnv is able to replicate in the mouse lung tissue inducing pneumonia [15] . although the virus was discovered more than 4 years ago, to date there is no complete genomic sequence, which prevents a comprehensive comparative study with other members of the pneumovirinae subfamily. the aim of the present manuscript is to report the detection and molecular characterisation of this emerging virus in dogs with respiratory disease in italy. the full-length genome of a prototype strain was determined and analysed in comparison with american strains and other pneumoviruses. the study did not involve any animal experiment. only sample collection from naturally infected dogs was carried out, consisting of a single nasal swab per dog. this was needed for the laboratory analyses and did not involve any suffering of the sampled animals. in 2012, an outbreak of cird occurred in a canine shelter of the apulia region, southern italy, involving 37 out of 350 housed animals. all the dogs involved were mixed bred and included animals aged from 2.5 months to 12 years; neither age nor gender predisposition was evident for the cird occurrence. pest control was carried out only sporadically in the shelter, but there were no systematic measures against rodent and insect populations. housed dogs were routinely vaccinated against canine parvovirus (cpv), canine distemper (cdv), canine adenoviruses (cadvs) and leptospira spp., while no specific vaccine for prophylaxis of cird was employed. respiratory signs were generally mild consisting of cough and/or nasal discharge with no evidence of fever. haematological parameters were not evaluated. nasal and pharyngeal swabs were collected from two cird-affected mixed-breed dogs, a 7-month-old male and a 3-year-old female. swabs were immersed in 1.5 ml viral transport medium consisting of dulbecco's modified eagle's medium (dmem) supplemented with 5% fetal calf serum (fcs), 1000 iu/ml penicillin, 1000 mg/ml streptomycin and 10 mg/ml amphotericin b. aliquots of the nasal and pharyngeal swab extracts were combined and subsequently clarified by centrifuging at 2,5006g for 10 min. one-hundred-forty microliters of the supernatants were then used for rna extraction by means of qiaamph viral rna mini kit (qiagen s.p.a., milan, italy), following the manufacturer's protocol and the rna templates were stored at -70uc until their use. all rna extracts were subjected to a previously-established rt-pcr assay for detection of cnpnv rna [14] , with minor modifications. briefly, a one-step method was adopted using superscript tm one-step rt-pcr for long templates (invitrogen srl, milan, italy), according to the manufacturer's instructions, and primers sh1f/sh187r that amplify a 208-bp of the small hydrophobic (sh) protein gene ( table 1 ). the following thermal protocol was used: reverse transcription at 50uc for 30 min, inactivation of superscript ii rt at 94uc for 2 min, 40 cycles of 94uc for 30 s, 54uc for 30 s, 68uc for 60 s, with a final extension at 68uc for 10 min. the pcr products were detected by electrophoresis through a 1.5% agarose gel and visualisation under uv light after ethidium bromide staining. in addition to the gel-based rt-pcr, a real-time rt-pcr assay based on the taqman technology was developed for the rapid detection and quantification of the cnpnv rna in all clinical samples. reactions were carried out using platinumh quantitative pcr supermix-udg (invitrogen srl) in a 50-ml mixture containing 25 ml of master mix, 300 nm of primers cnpnv-for and cnpnv-rev, 200 nm of probe cnpnv-pb (table 1 ) and 10 ml of template rna. duplicates of log 10 dilutions of standard rna were analyzed simultaneously in order to obtain a standard curve for absolute quantification. the thermal profile consisted of incubation with udg at 50uc for 2 min and activation of platinum taq dna polymerase at 95uc for 2 min, followed by 45 cycles of denaturation at 95uc for 15 s, annealing at 48uc for 30 s and extension at 60uc for 30 s. cnpnv positive samples were inoculated into semiconfluent canine fibroma (a-72) cells, as previously described [14] . inoculated cells were maintained in d-mem supplemented with 5% fcs and monitored daily for the occurrence of cytopathic effect (cpe). after 6 days of incubation, the monolayers were tested for cnpnv antigen by an immunofluorescence (if) assay using a monoclonal antibody targeting hrsv (monosanh, sanbio bv, uden, the netherlands). the cells were sub-cultured every 6-8 days for 5 consecutive passages. the rt-pcr products obtained with primer pair sh1f/ sh187r were subjected to direct sequencing at the baseclear b.v. (leiden, the netherlands). the sequences were manually edited and analyzed using the bioedit software package [16] and the ncbi's (htttp://www.ncbi.nlm.nih.gov) and embl's (http:// www.ebi.ac.uk) analysis tools. in order to obtain new insights into the genetic diversity of cnpnv, the italian prototype strain dog/bari/100-12/ita/2012 was submitted to rt-pcr amplification and subsequent sequence analysis of the full-length genome, using oligonucleotide retrieved from previous studies [13] [14] . additional rt-pcr assays with the enzyme mix superscripth ii rt/platinumh taq hi fi (invitrogen srl) and subsequent sequencing attempts were performed to close gaps between assembled contigs and to sequence unresolved genomic regions using primers designed on the alignment of the reference cnpnv strains with the closely related mpv. all the pcr amplicons were cloned and consensus sequences were elaborated using at least three clones per fragment. to determine the sequence of the 39-leader and of the 59-trailer ends (genome sense), viral rna was reverse transcribed using ns2 negative-sense and l positive-sense primers, respectively, and the resulting cdnas were amplified using a 59 race kit (59 race system for rapid amplification of cdna ends, invitrogen srl), following the manufacturer's instructions. sequence analyses were conducted as for the sh fragment. phylogenetic and molecular evolutionary analyses were conducted using mega4.1 beta [17] . phylogenetic trees based on the 8,598 nucleotide (nt) fragment available for extant cnpnvs and on the amino acid (aa) sequences of nucleocapsid (n) and fusion (f) proteins were elaborated using both parsimony and neighborjoining methods, supplying a statistical support with bootstrapping over 1000 replicates. the following pneumovirus reference strains were used for phylogeny (genbank accession numbers are indicated in parentheses): cnpnv strains dog/brne17/usa/ 2008 (gu247050) and dog/ane4/usa/2008 (hq734815); mpv strains 15 (ay729016) and j3666 (nc006579); hrsv strain b1 (nc_001781); brsv strain atue51908 (nc_001989). the distantly-related metapneumovirus human metapneumovirus (hmpv) can97-83 (nc_004148) was used as outgroup. the full-length genome of the cnpnv strain dog/bari/100-12/ ita/2012 was deposited in genbank under accession number kf015281. respiratory specimens were submitted to molecular detection of other respiratory pathogens of dogs, such as canine parainfluenza virus (cpiv) [18] , reoviruses [19] , cadvs [20] , cdv [21] , canine respiratory coronavirus (crcov) [22] , canine pantropic coronavirus [23] [24] , canine minute virus [25] , canid herpesvirus type 1 [26] , canine influenza virus [27] , canine hepacivirus [28] , canine bocaviruses [9] , bordetelella bronchiseptica [29] , streptococcus equi subsp. zooepidemicus [30] , mycoplasma cynos and mycoplasma canis [31] . standardised procedures were carried out for in vitro isolation of other cird-associated bacteria. samples were plated out on 5% sheep blood agar and cultured aerobically at 37uc for 24 h for the detection of aerobic pathogens. bacteria were identified by standard biochemical procedures and analytical profile index (api, biomérieux italia s.p.a., rome, italy). both sampled dogs tested positive by rt-pcr targeting the sh gene of cnpnv. by sequence analysis of the pcr product, a 100% nucleotide (nt) identity was found between the two detected cnpnv strains. by means of real-time rt-pcr, the dogs were confirmed to be infected by cnpnv, displaying discrete viral titers, which were 2.06610 5 (dog 100/12) and 1.59610 4 (dog 101/12) rna copies ml 21 of template. no other respiratory pathogens were detected in the analyzed samples. all attempts to isolate either cnpnv strains were unsuccessful, as shown by the absence of cpe and by negative if testing. the virus detected in one dog (dog/bari/100-12/ita/2012) was assumed as prototype of the cnpnv strains circulating in the shelter and submitted to full-genomic sequence analysis. this showed that the genome of strain dog/bari/100-12/ita/2012 (bari/100-12) was 14,884 nucleotides (nt) in length and displayed the same organization as mpv with 10 genes that encode for 12 putative proteins. the same genomic organization was found as in mpv with the gene order 39-ns1-ns2-n-p-m-sh-g-f-m2-l-59 and with the coding regions being flanked by leader and trailer regions at the 39 and 59 ends (genome sense), respectively (fig. 1a) . the genome size was 1 and 3 nt shorter than that of mpv isolates j3666 and 15, respectively, whereas no comparison was possible with extant cnpnvs whose full-length genomes are not available [14] . the full-length genome of the italian cnpnv strain displayed the highest nt identity (95.7-95.8%) to mpv, whereas the genetic relatedness to hrsv and brsv was less than 50% (table 2) . when the analysis was restricted to the 8,600 and 8,598 nt available for reference cnpnv strains brne17 and ane4, respectively, that span from the very 39 end of the l gene to the 59end of the leader region (genome sense), an overall sequence identity of 96.5-96.6% was found against the canine strains. this identity was only slightly higher than that displayed against mpvs (94.8-95%, table 2 ). in the negative genome sense, the leader region consisted of a 42 nt u-rich sequence as for that of strain mpv/j3666, whereas it was 1, 2 and 3 nt shorter than the same sequence of mpv/15, hrsv and brsv (fig. 1b) . the nt identity to other members of the genus pnuemovirus ranged from 68.2% (hrsv) to 83.3% (mpv/j3666). only the 59-end 25 nt had been sequenced for the reference cnpnv strains showing a 100% identity to the same stretch of the leader region of strain bari/100-12. the 14 nt at the 39 end were conserved among pneumoviruses. the a-rich trailer sequence at the 59 end (genome sense) was 91 nt long as that of mpv strains 15 and j3666, whereas the corresponding sequences of hrsv and brsv were 154 and 161 nt long (fig. 1c) . the nt identity was very high to mpv isolates (94.5-95.6%), being markedly lower to hrsv (52.7%) and brsv (47.3%). no comparison was possible with the analogous sequences of cnpnv strains brne17 and ane4 (that had not been determined), whereas a conserved 59-end stretch of 12 nt was found among all pneumoviruses analysed with the exception of the fourth position where hrsv and brsv exhibited the substitution g to a (fig. 1c) . non-translated regions were found to be located between the protein coding regions, including gene start (gs) and gene end (ge) sequences that define the transcriptional boundaries on the negative strand template and short intergenic regions (igr) lying between the ge of one gene and the gs of the following coding region (data not shown). nucleotide and amino acid identities of the italian cnpnv to other pneumoviruses in the different genomic regions are shown in table 2 and table 3 , respectively, whereas table 4 shows the aa substitutions encountered in the encoded proteins in comparison to extant cnpnvs and mpvs. strain bari/100-12 displayed intact genes for non-structural proteins ns1 (410 nt) and ns2 (571 nt) with respect to extant cnpnvs and mpv/15, whereas the same genes were shorter for mpv/j3666 (406 and 566 nt, respectively). hrsv and brsv had longer ns1 (532 and 527 nt, respectively) and shorter ns2 sequences (502 and 494 nt, respectively). in these genomic regions gene strain bari/100-12 was closely related to other cnpnvs (94.7% of nt identity), as well as to mpv (94.2% of nt identity). the encoded proteins ns1 and ns2 had the same length (113 and 156 aa, respectively) in all cnpnvs and mpvs, with the bari/100-12 ns1 and ns2 products being more closely related to the latter viruses (99.1% and 95.5% of aa identity, respectively). while the former protein showed only 1 aa variation in comparison to extant cnpnvs, 10 substitutions were encountered in the ns2 polypeptide. the n protein gene was 1,220 nt long, as for extant cnpnvs, which is 1 and 5 nt longer than the same region of mpvs 15 and j3666, respectively. the nt identity in this region ranged from 60% for brsv to 97.2% for cnpnv/brne17. the relatedness to other cnpnvs (98.5% of aa identity) was confirmed in the putative n protein (393 aa), which displayed 6 substitutions among canine viruses. the phosphoprotein (p) gene had the same length (907 nt) as for reference cnpnvs, which was the longest among analysed pneumoviruses. the nt identity was 97.4% against other cnpnvs and 94.8-95.6% against mpvs. two different products, 295 and 137 aa long respectively, are encoded by this gene. the major polypeptide of strain bari/100-12 displays the best aa identity (99.7%) against other canine isolates and only 1 aa change over other cnpnvs. the matrix (m) protein gene was 932 nt long as for cnpnvs brne17 and ane4, but it was 1 and 5 nt longer than in mpvs 15 and j3666, respectively. the genic relatedness against other canine and murine isolates was 96.8-96.9% and 95.3-95.4% of nt identity, respectively. the putative m protein was 257 aa long and exhibited approximately the same aa identity (98.1-98.5%) with canine and murine strains, with 4 mutations with respect to american strains. the small hydrophobic (sh) protein gene displayed the same length as cnpnv/ane4 and mpvs, whereas in strain brne17 this sequence was 1 nt shorter. the genetic distance to mpvs was higher in the sh gene than in other regions, with only 89.4-90% nt identity in comparison to an identity of 95.5% showed against extant canine strains. however, in the encoded protein (92 aa long), while the aa identity to strain mpv/j3666 was again high (97.8%), mpv/15 showed a lower relatedness with 92.4% of aa identity. four changes were detected among the sh products of the different canine viruses. the attachment (g) protein gene had the same length (1,334 nt) in all cnpnv strains, which was 1 and 5 nt longer that that of murine strains 15 and j3666, respectively. the analogous gene of hrsv and brsv was 412 and 494 nt shorter, respectively. the genetic relatedness to other canine isolates (96.4-96.6% nt identity) was slightly higher with respect to mpvs (94.1-94.4%). g sequences are available for additional 10 carnivore pneumoviruses, two of which are of feline origin [32] . when these sequences were included in the analyses, the overall nt identity to italian cnpnvs ranged from 91.6% to 98.2%. the encoded g protein of canine and murine pneumoviruses had different length ranging from 121 aa (strain cnpnv/brne17) to 414 aa (strains cnpnv bari/100-12 and ane4). thus, the aa identities to strain cnpnv-bari/100-12 ranged from 91.9% (mpv/j3666) to 97.1% (cnpnv/ane4). however, when recent cnpnvs were included, the aa identities varied from 89.6% to 95.9%. apart from the 293-aa deletion observed in cnpnv/ brne17, 12 mutations were evident in this product, 8 of which were also present in the g sequences of carnivore pneumovirus recently analysed (table 4 ). an opposite situation was found in the f protein gene, were hrsv and brsv displayed sequences that were 239-240 nt longer than those of canine pneumoviruses (1,663 nt) and 240-246 nt longer than those of murine strains (1,657-1,662 nt) . also in this region the nt identities to other cnpnvs were slightly higher than to mpvs (97.4% against 96.8-96.9%), whereas in the encoded protein (537 aa) the aa identities were approximately equal for canine and murine strains (98-98.3%). the f protein displayed 9 aa changes among cnpnvs. the mpv m2 gene had been suggested to be 875 nt long [33] , whereas renshaw et al. [14] reported a 926-nt m2 sequence for cnpnvs based on the presence of the gs sequence upstream the location previously detected for mpv [34] . consequently, the m2 gene length was re-determined as 928 and 921 nt for mpv isolates 15 and j3666, respectively. in strain bari/100-12, this gene was 926 nt long and shared a 96.4% and 95.1% nt identity with other cnpnvs and mpvs, respectively. interestingly, the genetic relatedness was higher to hpmv (46.2% nt identity) than to hrsv/brsv (44.6-45%). the pneumovirus m2 gene is usually translated into two proteins, m2-1 and m2-1, from alternative reading frames. in both proteins that were 176 and 98 aa in length, respectively, the italian cnpnv was more closely related to extant canine viruses than to mpvs (aa identities of 98.9% and 99% in the m2-1 and m2-2 products, respectively). the m2-1 protein showed 2 aa substitutions, whereas the m2-2 product was conserved among cnpnvs. comparison of the bari/100-12 large polymerase (l) gene with that of other canine strains was not possible since those sequences have not been determined for cnpnvs. the l gene of cnpnv-bari/100-12 was 6,333 long showing similar length in comparison with mpv isolates 15 (6,332 nt) and j3666 (6,326 nt) . in this region, the nt identity against murine strains was 96.6-96.7%. interestingly, hmpv was more closely related to strain bari/100-12 (56.3% nt identity) than were hrsv and brsv (both displaying 54% nt identity). however, when the aa sequence was analysed, the genetic relatedness to hrsv/brsv (49.1-49.9% aa identity) was higher than that with hpmv (48.6% aa identity). in order to include in the analysis the nt sequences available for extant cnpnvs, phylogeny was first constructed on the 8,598-nt fragment spanning from the very 39 end of the l gene to the 59end of the leader region (genome sense) [14] . in the maximum parsimony tree elaborated using these sequences, strain cnpnv/ bari/100-12 clustered with other cnpnv isolates and with the murine strain j3666, whereas mpv/15 was more distantly related ( fig. 2a) . the italian cnpnv was tightly intermingled with extant canine isolates also in the tree constructed on the n protein (fig. 2b) , whereas that obtained from the f protein revealed an unexpected subclustering with murine strains (fig. 2c) . the same tree topologies were obtained through phylogenetic analysis using the neighbor-joining method (data not shown). in recent years, novel viruses have been recognized as causative agents of cird, including influenza viruses [5] , hepaciviruses [28] , and bocaviruses [9] . cnpnv was first detected in 2 animal shelters with cird in the northeastern united states during 2008-2009 [13] . although cnpnv was discovered at least 4 years ago, only two thirds of the viral genome have been analysed so far [14] . in this paper, the identification of cnpnv in italy is reported together with a further evidence of its association to cird. the full-length genome of the italian prototype strain was determined, which confirmed the close genetic relatedness of strain bari/100-12 to the american isolates and of cnpnv to mpv. although cnpnv has been very recently detected in uk [35] , no sequences are yet available from those isolates, thus preventing any sequence comparison among european cnpnvs. although mpv was first identified in the 1940s, to date most reports of virus detection are from laboratory mouse and rat colonies. a 10-year survey in french laboratory rodents showed that 13.9% and 15.9% of the tested rat and mouse colonies, respectively, had antibodies against mpv [36] . serosurveys conducted in wild rodents in uk gave inconsistent results, with antibodies being detected in certain species of wild voles and mice [37] and in wild grey squirrels (sciurus carolinensis) [38] , but not in wild house mice (mus musculus) [39] . in italy there are no recent data about the mpv circulation in laboratory or wild rodents. however, if the mouse virus circulates in the field, there are several chances for its species jump to the canine host. in this scenario, kenneled dogs should be highly exposed to the cross-species transmission, considering that shelters usually attract large populations of rodents and pest control measures are not carried out on a regular basis. apart from kenneled dogs, hunting dogs could be also exposed to mpv infection through occasional contacts with wild rodents during hunting activities. introduction of mpv-like viruses in italy may have occurred through migration of infected wild rodents or importation of infected dogs from other countries. in addition, with the limited data available so far, whether the murine viruses are ancestors of the canine strains or the two groups of viruses co-evolved independently from a common ancestor could not be assessed. further studies providing additional canine and murine viral sequences are needed in order to date back the emergence of cnpnv. despite the presence of discrete viral titres in the clinical samples, as calculated by a newly established real-time rt-pcr assay, attempts to isolate in vitro the italian cnpnv were unsuccessful. this finding could be explained with virus inactivation during sample transportation or storage or, alternatively, with poor sensitivity of the canine cells employed for virus isolation. in fact, even if a-72 cells have been reported to be highly permissive for cnpnv replication [14] , virus adaptation to the in-vitro growth may vary based on different clones of the same cell line due to different levels of expression of cell receptors. currently, the pathogenic potential of cnpnv is not completely known. however, recent reports of natural infections in dogs [13] and experimental infections in mice [15, 32] have suggested its potential role in the occurrence of cird. additional evidence has been very recently obtained during an epidemiological survey carried out in the united kingdom [35] . in that study, cnpnv was detected in tracheal tissues of 29/205 kenneled dogs, most of which had suffered from mild to moderate respiratory disease. the role of cnpnv in the canine respiratory disease is also corroborated by the findings reported in the present paper where cnpnv strain bari/100-12 was associated to acute respiratory disease in a shelter. although the laboratory investigations failed to detect additional agents of cird, other, less common causes cannot be definitively ruled out. kennels and shelters are likely to be mostly exposed since the poor hygienic conditions and the overcrowding may facilitate the widespread circulation and increased virulence of the multiple pathogens responsible for the respiratory disease [1] . further studies will elucidate the pathogenicity and epidemiology of this tables 2 and 3 . the distantlyrelated metapneumovirus human metapneumovirus (hmpv) can97-83 (nc_004148) was used as outgroup. a statistical support was provided by bootstrapping over 1,000 replicates. the scale bars indicate 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british field rodents serological evidence of murine pathogens in wild grey squirrels (sciurus carolinensis) in north wales serological survey of virus infection among wild house mice (mus domesticus) in the uk key: cord-048483-umvrwgaw authors: van der sande, marianne; teunis, peter; sabel, rob title: professional and home-made face masks reduce exposure to respiratory infections among the general population date: 2008-07-09 journal: plos one doi: 10.1371/journal.pone.0002618 sha: doc_id: 48483 cord_uid: umvrwgaw background: governments are preparing for a potential influenza pandemic. therefore they need data to assess the possible impact of interventions. face-masks worn by the general population could be an accessible and affordable intervention, if effective when worn under routine circumstances. methodology: we assessed transmission reduction potential provided by personal respirators, surgical masks and home-made masks when worn during a variety of activities by healthy volunteers and a simulated patient. principal findings: all types of masks reduced aerosol exposure, relatively stable over time, unaffected by duration of wear or type of activity, but with a high degree of individual variation. personal respirators were more efficient than surgical masks, which were more efficient than home-made masks. regardless of mask type, children were less well protected. outward protection (mask wearing by a mechanical head) was less effective than inward protection (mask wearing by healthy volunteers). conclusions/significance: any type of general mask use is likely to decrease viral exposure and infection risk on a population level, in spite of imperfect fit and imperfect adherence, personal respirators providing most protection. masks worn by patients may not offer as great a degree of protection against aerosol transmission. with a potential influenza pandemic looming, governments need to decide how they can best use available resources to protect their people against severe illness and death, and to mitigate health and social effects for society as a whole. much research is being devoted to develop optimal strategies for the use of (pre)pandemic vaccines and of anti-virals. there are only limited data to assess the potential effectiveness of non-pharmaceutical interventions to reduce the risk of transmission, including the effect of different kinds of face-masks worn by the general public or by patients. respiratory infections such as influenza are transmitted through infectious particles, small enough to be suspended in air [1] . influenza transmission can occur via large droplets, which only remain suspended in the air for a short period of time thus requiring close contact, and can occur via small airborne particles, which remain suspended in air for considerable longer periods of time, and can thus be transmitted over larger distances [2] . furthermore, some transmission may occur via direct contact with respiratory secretions such as on hands and surfaces [2] . interruption of transmission may allow containment of major outbreaks, like pandemic influenza. opportunistic data collected during the sars epidemic in asia suggested that population-wide use of face masks may significantly decrease transmission of not only sars but also influenza [3, 4, 5, 6, 7] . as part of pandemic preparedness, many are contemplating the contribution wide-spread use of masks could have [8, 9] . as this has major implications for resource allocation and for communication, there is great need for data to guide such decisions and make them evidence-based. protective effects of face masks have been studied extensively, but usually this involved personal respirators for professionals under idealized conditions, because of specific applications, for instance in military or occupational uses, involving protection of specifically trained personnel. this is different from deployment of masks in the general population during an outbreak of an infectious disease, where anyone may encounter the infectious micro-organism, implying much greater heterogeneity, in training levels (experience and understanding), goodness of fit of a mask, and activities interfering with mask use and thus reducing potential reduction of transmission. the protective effect of masks is created through a combined effect of the transmission blocking potential of the material, the fit and related air leakage of the mask, and the degree of adherence to proper wearing and disposal of masks. personal respirators such as those worn by staff attending tb patients, are used primarily to protect the wearer, and are designed to fit to the face with as tight a seal as possible. their efficiency is graded on the degree of protection the material offers, assuming a perfect fit and optimal compliance. in contrast, surgical masks, as commonly worn in the operating theatre, are primarily used to protect the environment from the respiratory droplets produced by the wearer. with these masks, facial fit is much looser. the fit of home made masks, which could be e.g. made of a tea cloth or other comparable material available in the home, is likely to be even looser. thus personal respirators confer a higher degree of protection than surgical masks, and these are again likely to give a higher degree of protection than home-made masks. in professional situations, ample time might be available prior to use to ensure a perfect fit and to give extensive counselling on adherence, but it is unlikely this will apply to the general population in case of a pandemic. it is possible that the discomfort in wearing associated with a certain type of masks will lead to reduced adherence and thus to a loss in overall protectiveness [10, 11] . indeed a review among health care workers could not determine whether personal respirators conferred better protection for the health care workers than surgical masks [10] . to investigate the levels of protection, and their variation, wearing of face masks could convey to untrained subjects we designed a study in which healthy volunteers would be wearing different types of professional and home-made masks during a selection of activities, in different conditions (inward protection). we also assessed the protection different types of masks could convey when worn by a simulated infectious patient (outward protection). resulting quantitative descriptions of distributions of protection factors may be used for assessing the importance of mask use in respiratory disease transmission. three different experiments were undertaken to assess 1) shortterm protection for different types of masks worn during 10-15 minutes by the same volunteer following a standardized protocol, 2) long-term protection of a specific mask worn continuously by a volunteer for 3 hours during regular activities, and 3) effectiveness of different types of mask in preventing outgoing transmission by a simulated infectious subject. inward protection was defined as the effect of mask wearing to protect the wearer from the environment; outward protection was defined as the effect of a mask on protecting the environment from the generation of airborne particles by a patient (or in this case a mechanical head). in the first short-term experiment, 28 healthy adult volunteers were recruited, as well as 11 children between 5 and 11 years of age. each volunteer followed the same protocol wearing a filtering facepiece against particles (ffp)-2 mask 1872vh (3m); which is the european equivalent of a n95 mask, a surgical mask (1818 tie-onh, 3m; with a filtering efficiency of around 95% for particles of sizes between 0.02 mm to 1 mm; http://jada.ada.org/ cgi/content/full/136/7/877) and a home-made mask (made of td cerise multih teacloths, blokker). in this standard protocol, the volunteer was asked to perform five successive tasks in a fixed sequence 1.5 minute of duration each: no activity-sit still, nod head (''yes''), shake head (''no''), read aloud a standard text, stationary walk. in this sequence of activities, the respiratory rate is gradually increased. throughout this exercise, the concentration of particles was measured on both sides of the mask through a receptor fixed on the facial and on the external side. these were connected to a portable counter of all free floating particles in the air via an electrostatic particle classifier and counter, the portacounth. the portacounth can register particles floating in the air with sizes between 0.02 mm to 1 mm, covering most of the size range of infectious respiratory aerosols [12] . total inward leakage (til) percentage was calculated by dividing the concentrations on the outside and on the inside (til = (concentration inside/concentration outside)6100); the calculated quantitative protection factor was the inverse of the leakage (pf = (til/ 100) 21 ). to ensure small numbers of particles produced by the volunteers would not affect measurements, we checked that at least 10,000 particles per cm 3 particles of this size class (0.02 mm-1 mm) were present in the room which were produced by a number of lit candles. (figure 1 ) in the second long-term experiment, 22 volunteers, all adults, 10 men, 12 women, were divided into 3 groups. each group wore a single type of mask for a period of three hours, being either a ffp2 mask (4 males, 4 females), a surgical mask (3 males, 4 females) or a home-made mask (3 males, 4 females), similar to the masks used in the short-term experiment described above. at the beginning and end of each three-hour period, full series of measurements were taken using the standardised protocol as described for the short-term experiment, and during the three hour period while wearing the masks, participants reported back at regular intervals for a short measurement during rest (absence of activity). for the remainder of the period, participants carried on with their usual daily activities. during regular activities in between measurements, the probes of the masks were plugged which did not involve dislodging of the masks. in the final experiment, we assessed the effectiveness of different types of masks in reducing outgoing transmission from an infectious subject shedding aerosolised particles. this was simulated by fitting the different types of masks to an artificial test head, which was connected to pc-driven respirator (bacouh lama amp, modelref 1520307). breathing frequency was varied to mimic different respiratory rates (15, 25 and 40/minute). only expiration was simulated; twice for each mask at each respiratory rate. the breathing flow was defined as (respiratory rate/minute x volume per breath (2 litres)) resulting in a breathing flow of 30, 50 and 80 litres per minute, which correlates with light (walking), medium (marching with backpack) and strenuous (running) activities [13] . concentrations of particles were measured as described above by a tsi portacount respirator fit tester, model 8020, measuring outward protection, rather than inward protection. all volunteers received written information prior to the experiments and gave oral informed consent. for the children also a parent gave oral informed consent, and a parent remained present during the experiments. the dutch central committee on research involving human subjects (ccmo) informed us in writing that this project did not need to be assessed by an ethics committee. protection factors (pf) calculated from measurements of particle concentration by portacounth devices were reported as the ratio of particle concentrations outside and inside the mask. this is a similar concept to the fit factor as used by the us occupational safety and health administration (http://www.osha.gov/pls/ oshaweb/owadisp.show_document). therefore, a higher pf is better and pf = 1 means complete absence of protection. for statistical analysis, the following transformation was used: the inverse of the pf (1/pf) can be interpreted as a probability (that any particle succeeds in moving through the barrier the mask provides). the logit transformation is a standard transformation to transform the probability scale (0,1) to the real axis (-infinity, +infinity) to allow standard regression techniques (including anova) to test the effects of co-variables (mask type, age class, sex, activity, duration of use) on transformed pfs in a linear model, using the statistical application r (version 2.5.0). the p-values are based on testing the ratio of mean squares for a factor (like 'mask') and the mean square of errors (random fluctuations), assuming that ratio is f-distributed. whenever the p-value (the probability of a greater value of the tested ratio) is greater than 0.05, the ratio is considered significantly different from 1 ( = indifference) at the 95% level. all masks provided protection against transmission by reducing exposure during all types of activities, for both children and adults (table 1) . within each category of masks, the degree of protection varied by age category and to a lesser extent by activity. we observed no difference between men and women. surgical masks provided about twice as much protection as home made masks, the difference a bit more marked among adults. ffp2 masks provided adults with about 50 times as much protection as home made masks, and 25 times as much protection as surgical masks. the increase in protection for children was less marked, about 10 times as much protection by ffp2 versus home-made masks and 6 times as much protection as surgical masks. in these short term experiments, adjusting for covariates, face mask type had a strongly significant independent effect on protection (p,0.001). children were significantly less protected than adults (p,0.001). there was no significant impact of activity on protection. as in the short term experiment, mask type was a strong determinant of protection (table 2 ). protection factors for each type of mask were similar to the protection factors measured in the short term experiments for adults. there was considerable variability between volunteers. the median protection factors measured over a 3 hour period increased for those wearing homemade masks, decreased for those wearing ffp2 masks, and did not show a consistent pattern for those wearing a surgical mask (figure 2 ), but overall protection factors calculated per type of mask were stable over time, and did not change statistically significant with prolonged wearing. overall, protection factors were relatively stable over time for each individual (anova p = 0.4). males and females did not have significantly different protection factors (anova p = 0.9). as in the short term experiment, protection conferred by surgical masks was higher than protection given by a home-made mask, and protection provided by a ffp2 masks was again markedly higher than protection provided by a surgical mask. as in the short term experiment, more strenuous activities (reading and walking) tended to increase the protection of the home-made mask and to a lesser extent of the surgical mask, and decreased the protection by the ffp2 mask, but there was no overall significant effect of type of activity on pf (anova p = 0.1). outward protection experiment in a final experiment, retention of particles expelled inside the masks was studied. here again, mask type was strongly correlated with (transformed) protection factors. protection factors for all type of masks were considerably lower than those observed for inward protection. the home-made masks only provided marginal protection, while protection offered by a surgical mask and an ffp2 mask did not differ ( figure 3) . the simulated breathing frequency did not significantly affect the measured protection factors. adjusting for covariates, mask type and particle concentration, but not flow rate, were significant factors for protection in the reverse flow experiment. in our experiments, the main determinant of the magnitude of protection factors measured by masks was the type of mask, which can be seen as a proxy for potential reduction in infectious disease transmission. the duration of wear and the type of activity did not have a significant impact on exposure reduction. thus, the expected superior protection conferred by a professional ffp2 mask compared to a surgical mask or a home-made mask was maintained when these ffp2 masks were worn by healthy lay people in spite of the increased risk of a poor fit and significant behavioural leakage. children were significantly less protected from exposure than adults, which might be related to an inferior fit of the masks on their smaller faces. although we observed a high degree of individual variability in the degree of protection conferred as reflected in the wide interquartile ranges of the measured pfs, no systematic difference was found between men and women, suggesting a poorer fit only has a noticeable impact on protection when the mismatch between face and mask is considerable. all types of masks provided a much higher degree of exposure protection against inward transmission of particles, then in preventing outward transmission by a mechanical head as a proxy for an infected patient exposing the environment. data from professional users suggest a decrease in protection over time due to a reduction in fibre charges [13] . in our data, this effect was not significantly present, although a tendency towards reduced protection over time was seen for the ffp2 masks. also, our study showed a high degree of individual variation in exposure protection. this is important as it reflects the presence of many different sources of variation, behavioural as well as anatomical, which can also be expected to be present if the general population would be requested to wear face masks in case of a pandemic. furthermore, we do not know from these experiments whether reduced exposure has a linear or non-linear relationship to the reduction of infection risk. although this could imply that individual subjects may not always be optimally protected, from a public health point of view, any type of general face mask usage can still decrease viral transmission. also, it is important not to focus on a single intervention in case of a pandemic, but to integrate all effective interventions for optimal protection. surprisingly, the protection conferred by each of the masks appeared stable over time and was not dependent on activity. this suggests that leakage associated with suboptimal fit and compliance was stable over time. the tendency towards improved protection of the poorer fitting masks with increased activities such as reading, might be attributable to reduced leakage when breathing through the mouth rather than the nose, which could give some overpressure and thus reduce inward leakage. we had assumed that compliance would decrease during the three hours of continuous wearing, in particular with more strenuous activities. indeed, among professionals like cullers, there have been some anecdotal reports that ffp3 masks were associated with poorer compliance than ffp2 masks in wearing. where a reduction in protection was found with the ffp2 mask, the reverse was seen for the home-made mask. it is possible that the experimental situation, sufficient motivation to endure a relatively limited time of discomfort, and the absence of physically challenging activities, has provided more stable protection than might be found in reallife situations. however, overall these experiments show that significant protection against influenza transmission upon exposure can be conveyed also for lay people, including children, in spite of imperfect fit and imperfect adherence. it is also clear that home-made masks such as teacloths may still confer a significant degree of protection, albeit less strong than surgical masks or ffp2 masks. home made masks however would not suffer from limited supplies, and would not need additional resources to provide at large scale. home made masks, and to a lesser degree surgical masks, are unlikely to confer much protection against transmission of small particles like droplet nuclei, but as the reproduction number of influenza may not be very high [14] a small reduction in transmissibility of the virus may be sufficient for reducing the reproduction number to a value smaller than 1 and thus extinguishing the epidemic [15] . greater reduction in transmissibility may be achieved if transmission is predominantly carried by larger droplets. in a typical human cough half of the droplets may be small (,10 mm), but these comprise only a small fraction (2.5*10 26 ) of the expelled volume [12] . smaller droplets may however more easily penetrate the smaller bronchi and be more effective in transmission [1] . a more detailed analysis of aerosol and droplet inoculation and infectivity may provide better insight into the impact of either transmission mode on population spread. the difference in measured protection against inward and outward protection is remarkable, and cannot be explained from the available data as we only measured the overall effect. a differential effect on the amount of leakage seems most plausible. at the same time, we cannot exclude that wearing of face masks, even ffp2 or surgical masks by patients might still significantly reduce transmission. however, the observed limited particle retention in our experiments may still be an overestimate of protection, as it may for instance be challenging to enforce adherence to mask wearing by a patient who is short of breath. wearing of masks by caregivers might be more feasible and more effective, in particular where additional preventive measures are in place as well for caregivers. furthermore, we should bear in mind that this is an experimental study, with relatively small numbers of volunteers, which limits the generalisability of some of our findings. e.g., for masks to have any impact during an actual pandemic, people may need to be wearing masks during several weeks with many shorter or longer mask-free periods. furthermore, the pfs may be an over-or underestimation of the actual protection conferred. and although our simulated patient varied its breathing frequency, we have not assessed the impact of e.g. coughing or sneezing on outward transmission through a mask. a recent analysis of the 1918 epidemic, noted that cities where strict interventions were implemented early on to prevent transmission, were overall worse-off than cities where some degree of transmission occurred early on [16] . given the need for the population to acquire sufficient natural immunity over time, it can not be excluded that the amount of protection conferred by home made masks might sufficiently reduce viral exposure to impact on transmission during the early waves, while allowing people enough exposure to start mounting an efficient immune response. further field studies are needed to assess acceptability and effectiveness of masks worn by people from the general population. also, experimental data are needed to develop dose-response models which may improve understanding of determinants of transmission. a cost-effectiveness analysis might give further insights in the relative benefits of home made masks. review of aerosol transmission of influenza a virus transmission of influenza a in human beings sars transmission, risk factors and prevention in hong kong respiratory infections during sars outbreak risk of respiratory infections in health care workers: lesson on infection control emerge from the sars outbreak risk factors for sars among persons without known contact with sars patients factors influencing the wearing of facemasks to prevent the severe acute respiratory syndrome among adult chinese in hong kong nonpharmaceutical interventions for pandemic influenza world health organisation writing group (2006) non-pharmaceutical interventions for pandemic influenza, national and community measures protecting health care workers from sars and other respiratory pathogens: a review of the infection control literature modelling control strategies of respiratory pathogens towards understanding the risk of secondary airborne infection: emission of respirable pathogens do n95 respirators provide 95% protection level against airborne viruses, and how adequate are surgical masks? transmissibility of 1918 pandemic influenza mathematical epidemiology of infectious diseases. model building, analysis and interpretation the effect of public health measures on the 1918 influenza pandemic in us cities we thank nicole brienen for preparatory literature searches, rob schimmel and tineke albers for support in conducting the experiments, and all volunteers. volunteers in figure 1 gave written permission for reproduction. key: cord-268977-hcg2rrhl authors: feikin, daniel r.; njenga, m. kariuki; bigogo, godfrey; aura, barrack; aol, george; audi, allan; jagero, geoffrey; muluare, peter ochieng; gikunju, stella; nderitu, leonard; balish, amanda; winchell, jonas; schneider, eileen; erdman, dean; oberste, m. steven; katz, mark a.; breiman, robert f. title: etiology and incidence of viral and bacterial acute respiratory illness among older children and adults in rural western kenya, 2007–2010 date: 2012-08-24 journal: plos one doi: 10.1371/journal.pone.0043656 sha: doc_id: 268977 cord_uid: hcg2rrhl background: few comprehensive data exist on disease incidence for specific etiologies of acute respiratory illness (ari) in older children and adults in africa. methodology/principal findings: from march 1, 2007, to february 28, 2010, among a surveillance population of 21,420 persons >5 years old in rural western kenya, we collected blood for culture and malaria smears, nasopharyngeal and oropharyngeal swabs for quantitative real-time pcr for ten viruses and three atypical bacteria, and urine for pneumococcal antigen testing on outpatients and inpatients meeting a ari case definition (cough or difficulty breathing or chest pain and temperature >38.0°c or oxygen saturation <90% or hospitalization). we also collected swabs from asymptomatic controls, from which we calculated pathogen-attributable fractions, adjusting for age, season, and hiv-status, in logistic regression. we calculated incidence by pathogen, adjusting for health-seeking for ari and pathogen-attributable fractions. among 3,406 ari patients >5 years old (adjusted annual incidence 12.0 per 100 person-years), influenza a virus was the most common virus (22% overall; 11% inpatients, 27% outpatients) and streptococcus pneumoniae was the most common bacteria (16% overall; 23% inpatients, 14% outpatients), yielding annual incidences of 2.6 and 1.7 episodes per 100 person-years, respectively. influenza a virus, influenza b virus, respiratory syncytial virus (rsv) and human metapneumovirus were more prevalent in swabs among cases (22%, 6%, 8% and 5%, respectively) than controls. adenovirus, parainfluenza viruses, rhinovirus/enterovirus, parechovirus, and mycoplasma pneumoniae were not more prevalent among cases than controls. pneumococcus and non-typhi salmonella were more prevalent among hiv-infected adults, but prevalence of viruses was similar among hiv-infected and hiv-negative individuals. ari incidence was highest during peak malaria season. conclusions/signficance: vaccination against influenza and pneumococcus (by potential herd immunity from childhood vaccination or of hiv-infected adults) might prevent much of the substantial ari incidence among persons >5 years old in similar rural african settings. compared with other regions, the mortality rate among older children and adults remains several-fold higher in sub-saharan africa, where acute respiratory infections (ari) are a leading cause of this high mortality, as well as associated morbidity [1] . however, data on etiologies and rates of ari among persons $5 years old in africa have been principally focused on few geographic areas and pathogens (e.g., pneumococcus, tuberculosis). few studies have comprehensively examined the etiologies of ari among africans $5 years of age. more importantly, there is even less available data on disease incidence for specific etiologies, which can inform public health policies regarding treatment and prevention. from population-based surveillance in rural western kenya undertaken from 2007-2010, we report bacterial and viral etiologies of ari by age group, hospitalization status, hivinfection status and season. we also provide incidence by etiology, adjusted for health-care seeking and presence of pathogens in asymptomatic controls. written informed consent was obtained for data and specimen collection at the clinics and households. for children ,13 years of age, written informed consent was obtained from parents or guardians for specimen collection. for minors aged 13-17 years of age, written informed consent was obtained from parents or guardians and written assent from the minor him/herself for specimen collection. the protocol and consent forms were reviewed and approved by the institutional review boards of kemri (#932) and cdc (#4566). cdc's international emerging infections program and the kenya medical research institute (kemri) have conducted population-based, infectious disease surveillance since late 2005 in asembo, nyanza province, in rural western kenya [2, 3] . all households in 33 villages within 5 kilometers of the referral facility, st. elizabeth lwak mission hospital (lwak hospital), were offered enrollment -78% were enrolled. the surveillance population on july 1, 2007, included 21,420 persons $5 years old, who had resided permanently in the area for at least 4 calendar months [4, 5] . enrollment was continuous since the project's beginning. malaria transmission was holoendemic [5, 6] . hiv prevalence was high (17% in adults $18 years in 2008) [7] . from march 1, 2007 , to february 28, 2010 , all enrolled participants received free medical care by kemri/cdc-trained nurses and clinical officers at lwak hospital for acute illnesses [2, 3] . lwak hospital has 40 inpatient beds and manages most hospitalizations, only referring complicated patients. no chest radiographs were taken during the study period. ari was defined, using a variation on the definition of severe acute respiratory illness suggested for influenza surveillance, as cough or difficulty breathing or chest pain and documented axillary temperature $38.0 o c or oxygen saturation ,90% or hospitalization [2, 8] . for ari patients, blood, nasopharyngeal and oropharyngeal specimens using polyester-tipped swabs, and urine were collected. giemsa-stained malaria blood smears were performed on patients with history of fever or documented temperature $38.0uc. community interviewers visited enrolled households every two weeks and questioned participants using a standardized questionnaire, in the local language, about all illness episodes in the past two weeks, including symptoms and health-seeking [3] . for this analysis, we defined ari from the household visits as cough, difficulty breathing or chest pain and reported fever. the ari cases from the household visit were only used to assess healthseeking patterns in the area and not directly included in the incidence calculations. no specimens were collected at household visits. from january 1, 2009, asymptomatic controls were enrolled from lwak hospital. eligible controls were those who presented with non-severe illness (i.e., not requiring hospitalization), for immunizations, or for medicine refills. eligible controls could not have had fever, any respiratory symptoms or diarrhea in the past two weeks. each month we attempted to enroll a targeted number of controls, frequency-matched to cases by age and hiv status. the monthly target was 23 controls aged $5 years old (six hivinfected), yielding power to detect a significant difference in detection of a pathogen between 12% of cases and 5% of controls. nasopharyngeal and oropharyngeal swabs were collected on controls. clinic nurses attempted to collect five to ten ml of blood for culture, which was inoculated into commercially-produced blood culture bottles and bacterial growth identified using standard methodology, which we have described previously (bactec tm aerobic plus tm , becton dickinson, belgium) [9, 10] . naso-and oropharyngeal swabs were placed together in 1 ml viral transport media without antibiotics and transported the same day at 2uc-8uc to kemri/cdc laboratories near kisumu, approximately 60 km from lwak hospital, where each specimen was divided into four aliquots and stored at 270uc. in monthly batches, a frozen aliquot was transported on dry ice to the kemri/cdc laboratory in nairobi, where lab technicians, blinded to case-control status, tested it after one freeze-thaw cycle, using quantitative real-time reverse transcription polymerase chain reaction (qrt-pcr). nucleic acid was extracted from 100 ml aliquots of each sample using qiagen's qiaamp viral rna minikit (qiagen inc, california), according to manufacturer's instructions. prior to august 2008, taqmanh universal pcr master mix (applied biosystems, california) was used for qrt-pcr; later qrt-pcr was carried out using agpath-id tm onestep rt-pcr reagents (applied biosystems). samples were tested for the presence of adenovirus, respiratory syncytial virus (rsv), human metapneumovirus (hmpv), influenza a and b viruses, and parainfluenza virus (piv) types 1-3 using qrt-pcr assays using previously published assays [11, 12] . each clinical specimen was also tested for the human ribonuclease p gene to measure nucleic acid integrity and to confirm sample adequacy. a qrt-pcr test result was considered positive if an exponential fluorescence curve was produced that crossed the assigned threshold at c t ,40.0 [12] . between january 1, 2009, and february 28, 2010, enhanced testing was done, which included testing respiratory swabs for three additional viruses (rhinovirus, enterovirus, and parechovirus) and atypical bacteria, and testing of asymptomatic controls. for testing of swabs for these additional pathogens, total nucleic acid extracts were prepared from 100ml of specimen per aliquot using magmax viral rna isolation kit (applied biosystems) in nairobi and transported to the viral respiratory diseases laboratory at cdc atlanta on dry ice. singleplex qrt-pcr for rhinovirus, enterovirus and parechovirus were performed using previously published methodologies [13, 14, 15] . the rhino-and enterovirus assays targeted the 59 noncoding region of an area of high sequence similarity between human rhinovirus and enterovirus and exhibit some cross-reactivity (s. oberste, personal communication). therefore, positive rhinovirus and/or enterovirus qrt-pcr were reported together as rhino/enterovirus positive. for atypical bacteria, multiplex qrt-pcr was performed at kemri/cdc laboratories in nairobi for mycoplasma pneumoniae, chlamydia pneumoniae and pan-legionella species using published assays [12, 16] . urine specimens were tested for pneumococcal urine antigen using the binaxnowh kit (binax inc., maine) starting in may 2007; kits were not consistently available throughout the entire study period. sputum specimens were not collected by protocol, but only based on the clinician's suspicion of pulmonary tuberculosis, and microscopy was done at lwak hospital using ziehl-neelsen staining. hiv testing was performed as part of a home-based testing initiative during 2008 when all persons $13 years in the surveillance area were offered hiv testing (two parallel rapid hiv tests), as described previously [7] . seventy-eight percent of eligible adults agreed to be tested. we assumed that a person's hiv status during home-based testing was the same throughout the study period. hiv-testing was not performed routinely in the clinic on most patients during this period. structured questionnaires on scannable paper forms detailing the current illness were completed for all sick visits at lwak hospital. (teleformh software, cardiff tm , vista, ca). at the household visits, data were collected using personal digital assistants (pdas) [3] . analysis was performed using sas (version 9.2, cary, nc). proportions were compared using chi-square test or fisher's exact test, and medians by wilcoxon rank sum test. rate ratios and 95% confidence intervals were calculated using fisher's method (computer programs for epidemiologists, pepi, version 4.0x) for crude rates and the delta method for adjusted rates [17] . correlation between monthly rates of ari and percent positive for each pathogen were tested by pearson's or spearman's correlation coefficient, depending on normality of the data. we compared detection of each virus by qrt-pcr between cases and asymptomatic controls. odds ratios (or) and 95% confidence intervals were calculated using unconditional logistic regression, adjusting for age group, season when the swab was taken (december-february, hot and dry; march-may, long rains; june-august, cooler and dry; september-november, short rains) and hiv status (positive, negative, unknown). we used the or to calculate pathogen-attributable fractions, which estimated the proportion of cases positive for each virus in which the virus was the likely cause [18, 19] . the pathogen-attributable fractions were calculated as (or-1)/or; pathogen-attributable fractions were only calculated for viruses with ors that were statistically significant (p,0.05). for purposes of this analysis, we assumed the pathogen-attributable fraction was 1.0 for s. pneumoniae based on the negligible probability of detection of s. pneumoniae in blood or urine of asymptomatic controls [20, 21] . ari incidence was calculated as the number of ari clinic visits per 100 person-years of observation. revisits for the same illness were not counted as separate episodes. permanent residence status in the surveillance area was used to determine person-time contribution. adjusted rates of clinic visitation were calculated accounting for the percentage of all clinic visits made for ari that went to lwak hospital as opposed to other area clinics, as determined from the household visits [3] . etiology-specific incidence was calculated by applying the proportions of each etiology to the adjusted incidence of ari ( figure 1 ). inpatients were over-represented among ari patients tested at lwak hospital (35% inpatients), compared to ari patients who visited a health facility in the community (5% inpatients), as determined by the household surveillance. therefore, the inpatient and outpatient etiologic results from lwak hospital for each pathogen were weighted using a 19:1 outpatient:inpatient ratio. after adjusting for the outpatient/inpatient distribution, the pathogenattributable fraction for each pathogen was applied to adjust the etiology-specific proportions. for those pathogens found to have an or that included 1.0, no incidences were calculated because the role of the pathogen as a cause of ari was not supported by our data. during the study period, 3,406 ari patients $5 years old were seen at lwak hospital, of whom 2,212 (65%) were outpatients and 1,194 (35%) were hospitalized. among ari patients, 2,978 (87%) had cough, 980 (29%) had difficulty breathing and 1,209 (35%) had chest pain; 2,602 (76%) had documented temperature $38.0uc, 188 (6%) had oxygen saturation ,90% without fever (among a total of 242 with oxygen saturation ,90%), and 616 (18%) were hospitalized without documented fever or low oxygen saturation. the majority (59%) was aged 5-17 years old and only 10% were $50 years old. fifty-eight percent were female. overall, 73 (2%) ari patients died within 30 days of their clinic visit (0.2% among outpatients, 6% among inpatients). among ari patients $18 years old, 893 (65%) had hiv test results available, of whom 439 (49%) were hiv-infected, compared to 17% hiv-positivity among the entire surveillance population $18 years old [7] . among 3,406 ari patients, 8.6% reported receiving one or more antibiotics before presentation (4.7% cotrimoxazole, 2.3% penicillin or amoxicillin, 2.0% other). epidemiologic characteristics were similar between patients who did (n = 1,677, 49%) and did not (n = 1,729, 51%) have blood cultures taken ( table 1 ). the median blood volume for culture was 4.0 ml. four percent of blood cultures grew contaminants ( table 2 ). among uncontaminated cultures, the most common bacteria isolated were streptococcus pneumoniae (3%) and non-typhi salmonella (3%). the percentage of patients with s. pneumoniae identified increased to 16% when adding urine antigen results to blood cultures (23% inpatients versus 14% outpatients, p = 0.003). among the 131 patients who had s. pneumoniae detected, 10 (8%) were positive by blood culture only, 114 (87%) by urine antigen only, and 7 (5%) by both tests. the case-fatality ratio was higher among those with positive blood cultures for s. pneumoniae (7%) compared with those with only urine antigen detected (0.8%, p = 0.049). s. pneumoniae were detected more frequently among hiv-positive patients (28%) than hiv-negative patients (13%, p = 0.002). mycoplasma pneumonia was detected in 0.7% of patients; no legionella or chlamydia pneumoniae were detected (table 2) . among 47 sputum samples tested by smear for mycobacterium tuberculosis, 7 (15%) were read as positive. epidemiologic characteristics were similar between patients who did (n = 1,216, 36%) and did not (n = 2,190, 64%) have naso/ oropharyngeal swabs taken (table 1) . overall during the time period of full viral testing, 68% of swabs were positive for at least 1 virus, detection being higher among outpatients (71%) than inpatients (58%, p = 0.008, table 2 ). the detection of at least one virus decreased with increasing age (p,0.001). the most commonly detected virus was rhino/enterovirus (33%) followed by influenza a virus (22%). detection of other viruses ranged from ,1% to 9%. influenza a virus was more often detected in outpatients (27%) than inpatients (11%, p,0.001) and among those ,35 years old (23%) than those $35 years old (11%, p,0.001). influenza b virus was also more common among outpatients (8%) than inpatients (2%, p,0.001). all other viruses were similarly detected among inpatients and outpatients. there were no significant differences in viral detection between hivpositive and hiv-negative patients. of 569 patients with full pathogen testing, 29% had .1 pathogen identified (table 3 ). detecting .1 pathogen was more common among hiv-positive patients (40%) than hiv-negative patients (25%, p = 0.04), but in similar proportions among inpatients (24%) and outpatients (31%, p = 0.21). the coinfection prevalence among those who had influenza (41%) was lower than that among those with other pathogens (66%, p,0.001). when limited to only those pathogens significantly more common in cases than controls, 18% of ari patients had .1 pathogen identified; still no differences between inpatients (15%) and outpatients (19%) existed for coinfection (p = 0.51). cases (n = 766) had a younger age distribution than controls (n = 273), with 68% and 42% aged 5-17 years old, respectively (p,0.01, table 4 ). more cases (44%) than controls (33%) were enrolled in the hot, dry season (p,0.01). among those with hivtesting results available, 33% of cases and 39% of controls were hiv-positive (p = 0.28). after adjustment for age, season, and hiv status, detection of influenza a virus, influenza b virus, rsv and hmpv in the naso/ oropharynx was associated with being a case (table 4 ). influenza a virus and hmpv were more strongly associated with outpatients than inpatients, although the confidence intervals overlapped. in contrast, rsv was associated with being an inpatient, but not an outpatient. piv2 and piv3 were also more common among cases, but did not reach statistical significance. rhino/enteroviruses and adenovirus were equally common among cases (33% and 11%, respectively) and controls (24% and 12%, respectively). the number of ari cases tended to peak in june and july each year, with a smaller peak in october-december ( figure 2 ). there was a trend towards increasing ari cases during the three year period (p = 0.002, linear regression). the number of ari cases was highest in january-february 2010 when pandemic h1n1 influenza a virus circulated widely in asembo [22] . of the pathogens associated with case status in the case-control study, the monthly rate of ari was correlated with the percentage positive for influenza a virus (p = 0.018), influenza b virus (p = 0.026), and hmpv (p = 0.012), but not for s. pneumoniae and rsv. of note, when we limited the analysis to the 32 months prior to november 2009, when pandemic h1n1 virus first appeared, the monthly correlation between ari case rates and viral respiratory pathogens was no longer significant. the percentage of malaria blood smears positive among ari patients ranged from (13) 1041 (10) 341 (8) 3406 (11) -1194 (62) 2212 (8) 489 (14) 802 (7) cfr a for ari cases (5) 15 (4) 73 (2) -69 (6) 4 (0.2) 20 (4) 6 (1) blood cultures 965 (48) 556 (53) 156 (46) 1677 (49) -613 (51) 1082 (49) 257 (53) 365 (46) without contaminant 924 (96) 544 (98) 149 (96) 1617 (96) -582 (95) 1035 (96) 249 (41) 353 (59) s. pneumoniae (4) 10 (7) 41 (3) 3 (7) 27 (5) 14 (1) 15 (6) 8 (2) positive urine pneumococcal antigen c 68 (13) 42 (17) 12 (20) 122 (15) 1 (0.8) 37 (19) 85 (13) 26 (23) 21 (12) s. pneumoniae by blood culture or urine antigen c 68 (14) 47 (19) 16 (27) 131 (16) 2 (2) 45 (23) 86 (14) 30 (28) 22 (13) h. influenzae (3) 25 (2) 14 (6) 6 (2) other pathogenic bacteria d 5(0.5) 8 (1) 2 (1) 15 (1) 1 (7) 9 (2) 6 (1) 2 (1) viruses and atypical bacteria naso/orophangeal specimens 716 (35) 386 (37) 114 (33) 1216 (36) -396 (33) 820 (37) 179 (37) 255 (32) influenza a virus 169 (24) 69 (18) 11 (10) 249 (20) 0 29 (7) 22 (27) 27 (15) 53 (21) influenza b virus 45 (6) 21 (5) 4 (4) 70 (6) 0 8 (2) 62 (8) 9 (5) 20 (8) rsv 57 (8) 28 (7) 7 (6) 92 (8) 3 (3) 33 (8) 59 (7) 17 (9) 13 (5) adenovirus 78 (11) 28 (7) 7 (6) 113 (9) 0 29 (7) 84 (10) 19 (11) 16 (6) parainfluenza 1 (1) 34 (3) 1 (3) 6 (2) 28 (3) 4 (2) 5 (2) parainfluenza virus 3 46 (6) 17 (4) 6 (5) 69 (6) 1 (1) 14 (4) 55 (7) 9 (5) 13 (5) human metapneumo 42 (6) 13 (3) 5 (4) 60 (5) 0 10 (3) 50 (6) 10 (6) 11 (4) rhino/enterovirus e 109 (38) 29 (24) 9 (34) 147 (33) 3 (2) 39 (35) 108 (33) 13 (24) 8 to 69% by month, and tended to be higher in the rainy months ( figure 2 ). the monthly ari rate was correlated with the malaria prevalence throughout the 36 months (p = 0.015), as well as in the 32 months prior to pandemic h1n1 (p = 0.007). the adjusted overall incidence of ari resulting in a clinic visit was 12.0 per 100 person-years for persons $5 years of age and 8.4 per 100 person-years for persons $18 years of age (table 5) . among hiv-positive persons $18 years old, the rate of ari was 21.4 per 100 person-years, compared to 5.6 for hiv-negative persons (rr 3.8, 95% ci 3.5-4.2). the highest pathogen-specific incidence among persons $5 years old was influenza a virus at 2.6 per 100 person-years, followed by pneumococcus at 1.7 per 100 person-years ( table 6 ). rates of all pathogens were higher among hiv-positive than hiv-negative persons. our study was unique in several respects. first, we compared etiologies in inpatient and outpatient settings, whereas most previous adult etiology studies in africa have focused on severely ill, hospitalized patients [23, 24, 25, 26] . we demonstrated different etiologic predominance for the same broadly defined ari syndrome based on hospitalization status, with pneumococcus more common among inpatients and influenza viruses and hmpv among outpatients. second, we enrolled a group of asymptomatic controls. as has been noted elsewhere, a positive result from pcr is not necessarily conclusive as to etiology because of its high analytic sensitivity; pcr may detect small amounts of nucleic acid due to past or asymptomatic infections [27, 28, 29, 30] . by enrolling asymptomatic controls, we were able to calculate a pathogenattributable risk, which suggested that several viruses commonly found among ari cases (i.e., rhino/enterovirus and adenovirus), were not associated with illness. third, because we embedded our etiologic study in ongoing population-based surveillance, we were able to calculate incidence of etiology-specific ari. the accuracy of our rate calculation was likely improved by adjusting for several factors that are often neglected, namely health-seeking patterns in the population and the attributable fraction of illness for each pathogen. using the calculated incidence, we estimated that approximately 3% and 2% of adults in nyanza province (approximately 4.5 million persons $5 years, 2009), where lwak is located, will have an episode of ari from influenza viruses and pneumococcus each year, respectively, which translates to approximately 135,000 and 90,000 illnesses, respectively, in the province [31] . as expected, pneumococcus was the most common bacterial cause of serious ari in this population, and was particularly prevalent among hiv-infected persons [2, 25, 32, 33, 34, 35, 36] . there was more than a 5-fold increase in the frequency of positive results for pneumococcus when incorporating the urine antigen test, supporting previous evidence that most pneumococcal pneumonia is non-bacteremic [25, 37] . non-typhi salmonella (nts) was the second most common bacterium identified in ari patients. nts bacteremia commonly presents with respiratory symptoms in children, although its role in causing pneumonia based on lung aspiration studies is doubtful [25, 38] . alternatively, patients could have a mixed infection where nts bacteremia is accompanied by another pathogen causing ari, particularly among hiv-infected persons who are at increased risk for both [23, 25, 38, 39] . the role of atypical bacteria as a cause of ari in african adults is unclear. in south africa, c. pneumoniae and legionella pneumophila table 3 . coinfection among ari cases aged $5 years of age, limited to those that had full testing -nasopharyngeal/oropharyngeal specimens, blood culture and urine antigen testing for pneumococcus. were detected by seroconversion in 21% and 9% of adults with pneumonia [40] . in botswana, 17% of mostly hiv-infected adults had evidence for m. pneumoniae infection [24] . apart from these studies, other studies of adults in less developed african countries showed low prevalence of atypical bacteria, a finding supported by our study [25, 34, 41, 42] . influenza a virus was the most commonly detected virus. a prominent role of influenza viruses as a cause of ari in africa is becoming clear as more surveillance data becomes available [22, 43] . we found influenza viruses more often in outpatients than inpatients. we have previously speculated that even when detected among inpatients with ari, influenza viruses might not be the primary reason for the admission [44] . several other viruses besides influenza virus were associated with ari. we found rsv associated with ari among inpatients, but not outpatients. rsv has been shown to be the leading cause of hospitalized ari in african children [45] . rsv has a less clear etiologic role among african adults. one study among south african adults did not find an excess in hospitalizations or mortality during rsv seasons [46] . in the u.s., several studies have postulated that rsv can cause pneumonia in adults, especially those with underlying disease or the institutionalized elderly, although none of these studies evaluated asymptomatic controls [47, 48] . in contrast to rsv, we found hmpv associated with ari among outpatients, but not inpatients. the only study of hmpv among adults on the african continent was in egypt and found hmpv in 14% of hospitalized adults with lower respiratory tract infections, but no asymptomatic controls were included [49] . coinfection was common, with almost one-third of ari patients having .1 pathogen identified. coinfection could represent a true biologic phenomenon where infectious cofactors are necessary for pathogenesis. alternatively, the frequency of coinfection could reflect the fact that certain pathogens, such as rhino/enteroviruses and adenoviruses, are commonly detected in the upper airways, with unclear clinical significance. of note, ari patients with influenza viruses detected had less coinfection, which might be related to the high pathogen attributable risk of influenza viruses and the lack of need for a coinfection to cause illness. our study cannot be considered a comprehensive assessment of the etiologies of ari. additional specimens likely would have increased the yield of pathogens identified. lung aspirates have been shown to have high diagnostic yield in african children, and one study in african adults, as they sample material directly from the affected sections of the lung [25, 50, 51] . however, lung aspirates are rarely performed in african adults with pneumonia due to the potential risk of pneumothorax in populations with high risk of pneumocystis jiroveci pneumonia (pcp). bronchoscopy with bronchoalveolar lavage has been used in a few hospitals in africa, and has improved diagnostic yield, although its use is usually reserved for severely ill, hypoxic patients in tertiary hospital settings [23, 25, 26, 34, 36] . while good-quality sputum specimens might have a role in diagnosis of some etiologies, if strict criteria for assigning a predominant organism are used, sputum is generally considered too prone to contamination with upper respiratory tract secretions to be useful diagnostically [30, 37, 52, 53] . lack of these additional specimens limited our detection of pneumocystis jiroveci and mycobacteria tuberculosis, two important causes of severe respiratory infection in high hiv prevalence populations in africa [23, 25, 26, 34, 36, 54, 55, 56] . moreover, we did not test for some viruses, such as coronaviruses and bocavirus, which might play a role in pneumonia, although their pathogenicity, particularly in adults, is still debated [11, 34, 57, 58] . while we did not observe discrete seasonality of ari in western kenya as previously shown in temperate climates, there tended to be yearly peaks coincident with the peak malaria season, which usually occur 1-2 months after the rainy season begins. there could be several explanations for this. malaria infection could augment the risk for ari. an association between malaria and bacteremia in african children has been shown, although no direct associations between malaria and pneumonia in children or adults has been proven [59] . second, similar climactic conditions could favor both malaria and pneumonia. we have shown previously that influenza incidence tends to be highest in kenya during a broad wave that mostly corresponds with the southern hemisphere winter, from june to october, a time period that also encompasses peak malaria season [5, 22] . lastly, malaria might cause symptoms that meet the nonspecific ari case definition we used. the overlap in symptoms between malaria and pneumonia in children is well-documented [60] . although similar evidence does not exist for older persons, the non-specificity of the ari definition we used suggests that symptomatic malaria could have resulted in an illness that met the ari case definition. our study had several other limitations. first, not all ari patients were sampled. reasons for not collecting swabs included high patient volume, after hours clinic attendance, and rare refusals (,10%). while we showed similar demographic characteristics and case-fatality ratios between sampled and non-sampled patients, undetected sampling bias might still have occurred that could influence the pathogens detected. second, we used a broad ari definition that likely captured a range of illnesses from influenza-like illness to pneumonia. without further diagnostic procedures, such as chest radiographs, we could not confirm the anatomic location of the respiratory infection. third, although the controls were by definition asymptomatic in the two weeks prior to enrollment, they might have been in the incubation period of a viral infection and subsequently developed symptoms. if this were true, a small number of controls might have been inaccurately classified. fourth, unlike in developed countries, we found lower rates of ari among the elderly, which we speculate is due to lower healthcare utilization by the elderly in rural kenya [3, 61, 62] . fifth, we assumed that a person's hiv status during home-based testing in 2008 was unchanged throughout the study period, which might have led to some misclassification of hiv status. however, we feel that the number of misclassified individuals is small because the annual incidence of hiv among adults in the area has been estimated at 1% (kemri/cdc unpublished data), which at the most would have resulted in 39 individuals among the 1,291 ari patients with hiv results having misclassified hiv status for some period of the three year surveillance period. lastly, despite the lack of a statistical association with ari at the population level for some viruses, an etiologic role of a virus detected in any given individual case cannot be ruled out. a study in thailand showed that rhinovirus was associated with hospitalized respiratory illness among adults, particularly among the elderly [63] . outbreaks of rhinovirus-associated pneumonia have been suspected among the elderly in developed countries [64] . adenovirus has on occasion been implicated as a cause of pneumonia in african adults, but without a control group for comparison [65] . our study demonstrated several opportunities to make an impact on the incidence of ari among older children and adults in africa. the high incidence among hiv-infected persons suggests that expanded testing and access to cotrimoxazole prophylaxis and anti-retroviral drugs will likely decrease the number of ari cases, as has been shown before for pneumococcal infections [66, 67] . vaccines could also decrease the incidence. pneumococcal conjugate vaccine was introduced for infants in kenya in early 2010 and if herd immunity occurs, as it has in developed countries, then decreases in adult pneumococcal disease incidence are expected [68] . moreover, targeted vaccination among high-risk groups, such as hiv-infected adults, might be applicable for both influenza and pneumococcus [22, 32] . participation with kemri/cdc. we thank brett whitaker for assistance in virologic testing at cdc, atlanta. we thank sonja olsen for her critical review of the manuscript. this paper is published with the approval of the director of kemri. the findings and conclusions are those of the authors and do not necessarily represent the views of the centers for disease control and prevention. the pathogen-attributable fraction (paf) is calculated as (or-1)/or using viral prevalence data from cases and controls. for pneumococcus, afe was assumed to be 1. b the percentage of ari was adjusted for the paf and then adjusted for the prevalence among inpatients and outpatients for each pathogen at lwak hospital and the percentage of ari cases in the community who were seen in clinics as inpatients and outpatients (see methods). c only persons with complete testing for s. pneumoniae including blood culture and urine antigen testing were used to calculate the percent of ari due to s. pneumoniae. doi:10.1371/journal.pone.0043656.t006 high rate of pneumococcal bacteremia in a prospective cohort of older children and adults in an area of high hiv prevalence in rural western kenya the burden of common infectious disease syndromes at the clinic and household level from population-based surveillance in rural and urban kenya health and demographic surveillance in rural western kenya: a platform for evaluating interventions to reduce 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on the witwatersrand. a 7-year study the impact of antiretroviral treatment on the burden of invasive pneumococcal disease in south african children: a time series analysis changes in invasive pneumococcal disease among hiv-infected adults living in the era of childhood pneumococcal immunization sustained reductions in invasive pneumococcal disease in the era of conjugate vaccine we thank oliver morgan for assistance with control enrollment, joe oundo for establishing blood cultures, and joy chebet for graphical assistance. we thank the management of st. elizabeth lwak mission hospital and the people of asembo for their continued support and august 2012 | volume 7 | issue 8 | e43656 key: cord-258366-fu9b446y authors: couto, carla r.; pannuti, cláudio s.; paz, josé p.; fink, maria c. d.; machado, alessandra a.; de marchi, michela; machado, clarisse m. title: fighting misconceptions to improve compliance with influenza vaccination among health care workers: an educational project date: 2012-02-06 journal: plos one doi: 10.1371/journal.pone.0030670 sha: doc_id: 258366 cord_uid: fu9b446y the compliance with influenza vaccination is poor among health care workers (hcws) due to misconceptions about safety and effectiveness of influenza vaccine. we proposed an educational prospective study to demonstrate to hcws that influenza vaccine is safe and that other respiratory viruses (rv) are the cause of respiratory symptoms in the months following influenza vaccination. 398 hcws were surveyed for adverse events (ae) occurring within 48 h of vaccination. ae were reported by 30% of the hcws. no severe ae was observed. a subset of 337 hcws was followed up during four months, twice a week, for the detection of respiratory symptoms. rv was diagnosed by direct immunofluorescent assay (dfa) and real time pcr in symptomatic hcws. influenza a was detected in five episodes of respiratory symptoms (5.3%) and other rv in 26 (27.9%) episodes. the incidence density of influenza and other rv was 4.3 and 10.8 episodes per 100 hcw-month, respectively. the educational nature of the present study may persuade hcws to develop a more positive attitude to influenza vaccination. the compliance with influenza vaccination has been historically poor among health care workers (hcws) varying from 2 to 36% around the world [1] [2] [3] [4] . a recent review of relevant predictor studies of self-reported reasons for accepting or rejecting influenza vaccination showed that the two major reasons for rejecting are misconceptions or lack of knowledge about influenza infection; and a lack of convenient access to vaccine. on the other hand, hcws compliant to seasonal vaccination are generally older, believe in vaccine efficacy, take the vaccine for self-protection, and have been previously vaccinated [5] . at hospital das clinicas, university of sã o paulo school of medical sciences, a previous study showed a 34% compliance with influenza vaccination among hcws. in the mentioned study, the main reasons for non-compliance were the perception of vaccine inefficacy and the fear of adverse reactions [4] . respiratory symptoms occurring after vaccination are frequently misinterpreted as vaccine failure which reinforces the hcw's skepticism on vaccine efficacy. to overcome these false beliefs, we proposed a prospective study in a cohort of hcws to demonstrate that influenza vaccine is safe and other respiratory viruses (but not influenza) are generally the cause of respiratory symptoms in the months following influenza vaccination. this study was conducted at hospital das clinicas, university of são paulo school of medical sciences (hc-fmusp) from may to october 2006. the hospital das clinicas is a 2,000-bed tertiary teaching hospital consisting of 5 buildings attached to the university of são paulo. the main building has approximately 900 beds and contains most of the surgical and clinical wards and 12 intensive care units. hospital das clinicas has an estimated 15,000 hcws, including permanent and casual staff, employees, students, and volunteers. since 1999, annual influenza vaccination has been offered free of charge to all hcws. vaccination usually takes place at the hospital's immunization center during working hours, from monday to friday. in 2006, as a strategy to increase compliance with influenza vaccination, vaccine was offered at places of easy access during expanded hours, as suggested by 61% of the interviewed in previous survey [4] . in addition, an educational campaign was carried out emphasizing the safety and importance of influenza vaccination. detailed information about the 2006 educational and vaccination campaign have been published elsewhere [6] . during vaccination campaign, hcws were invited to participate in the present study which had two steps: 1) evaluation of vaccine safety and, 2) cohort study to evaluate which respiratory viruses were more frequently detected in hcws presenting respiratory symptoms in the four-month period following vaccination. sample size was estimated taking into account an expected frequency of 10% of adverse events in adult population. considering an acceptable frequency rate of up to 13%, we estimated to enroll at least 377 hcws (epiinfo version 6). three hundred and ninety eight vaccinated hcws were surveyed for adverse events occurring within the first 48 h after influenza vaccination. a subset of 337 hcws participated in the follow-up phase of the study. to assure that all hospital sectors were represented, the cohort was defined during the assessment of adverse events which was performed by a hospital epidemiologist nurse, through visits in all hospital floors and sectors. afterward, these hcws were actively surveyed twice a week, at work place, during four months, to check for the occurrence of respiratory symptoms and nasal wash sampling which was done in 93 of the participants. figure 1 shows the algorithm of the study. a followup time of four months was proposed taking into account the period when serum antibodies elicited by influenza vaccine are expected to maintain protective levels. the following adverse events were actively surveyed: fever, headache, malaise, myalgia, local pain, local edema and allergic reaction. other adverse events spontaneously reported were also registered. during follow-up visits, participants were asked about the presence of the following symptoms: fever, coryza, blocked nose, sneeze, cough, watery eyes, headache, myalgia, sore throat, hoarseness, sibilance, and dyspnea. allergy was ruled out in those with sneezing as the only symptom. influenza like illness (ili) was defined by the presence of fever and cough and/or sore throat according to the cdc definition [7] . in the presence of any of the above mentioned symptoms, a nasal wash sample was taken according to englund et al, kept at 4uc to 8uc and processed at the virology laboratory within four hours from sampling [8] . nasal washes were taken from hcws who consent with sampling and whose duration of symptoms did not exceed three days. respiratory syncytial virus (rsv), influenza (inf) a and b, adenovirus (adv) and parainfluenza virus (piv) were diagnosed by direct immunofluorescent assay (dfa) according to the manufacturer's instructions (imagenh dako, cambridgeshire, uk). aliquots of nw samples were stored at 280uc for later pcr and real time pcr processing. pcr was used to detect coronavirus and picornavirus. rt-pcr products for picornavirus were subsequently sequenced to differentiate rhinovirus from enteroviruses. real time pcr (taqman assay) was used to diagnose human metapneumovirus (hmpv). to increase the sensitivity of influenza diagnosis, a real time pcr (taqman assay), was added to the diagnostic tools. similarly, a nested adenovirus pcr was used along with dfa due to the low sensitivity of the latter in diagnosing adv. the pcr protocols used in the present study have been published elsewhere [9] [10] [11] [12] . hcws were informed about the results of the dfa up to 48 h after sampling. spss 15 .0 (spss inc., chicago, il) was used with the x 2 test or fisher's exact test for discrete variables, and student's t test or the mann-whitney u test for continuous variables. tests of significance were two sided, and p,0.05 was considered to be statistically significant. incidence density (id) of respiratory symptoms, influenza virus infections and other respiratory virus infections were calculated by the formula described below. id results were expressed per 100 hcw-month [13] . the interviews for adverse events occurring within 48 h of vaccination were made from one to 10 days after vaccination. the majority of the participants (81.4%) were surveyed within the first week of vaccination. all hospital sectors were represented (table 1) . one hundred and twenty of the 398 hcws (30.2%) reported at least one adverse event (ae). table 1 shows the occurrence of adverse events according to the demographic characteristics of the vaccinees. sector of work was the only variable associated with the presence of adverse events (p = 0.017). the sectors with highest frequency of adverse events were the virology laboratory (87.5% of the subjects), burn unit (54.5%), nephrology (52.4%) and pneumology (45.5%). in the remaining sectors, ae were reported by less than 40% of the subjects. those surveyed in the first five days after vaccination were more likely to report such events [95 (79.2%) versus 25 (20.8%); p,0.0001]. local ae were reported by 18.3% of the participants, systemic ae by 71.6%, and 10% of the participants reported both local and systemic aes. headache, myalgia and malaise were more frequently reported (50%, 45.8% and 45%, respectively). local pain and local edema was reported by 17.5% and 5% of the hcws. no severe adverse event was observed. during the 4-month period following influenza vaccination, respiratory symptoms were evaluated in 337 hcws. a total of 4,182 follow-up visits were performed (median 12 per hcw, ranging from one to 25 visits). one hundred and twenty-one hcws (36%) developed 192 episodes of respiratory symptoms. coryza, cough, sore throat and myalgia were reported by 36.3%, 25%, 17.4% and 13.7% of the participants, respectively. seventy-one of them (58.7%) presented more than one episode suggestive of upper respiratory infection (uri). ili was observed in 17 of the 192 episodes (8.8%). mean time to the occurrence of respiratory symptoms was 2.9 (0.7 to 5.2) months. the incidence density of respiratory symptoms was 12.4 episodes per 100 hcw-month. nasal washes were taken in 93 of the 192 episodes of uri. in 61 episodes (66.3%) no respiratory virus was found, even though 82% had coryza, 53% had cough and 49% had sore throat. the frequency of ili was similar among hcws who agreed with sampling and those who did not agree (58.8% versus 41.2%, p = 0.37) ( misconceptions about influenza vaccine, be it about its safety or its effectiveness, have been identified in all studies included in a recent review of attitudes and predictors of influenza vaccination among hcws, highlighting the importance of education efforts [5] . initial symptoms of rv infections are often unspecific such as fever, malaise, or myalgia. as influenza vaccine is offered when other rv are circulating (e.g., rsv), vaccinated hcws developing symptoms within 48 h of vaccination misinterpret those signs as vaccine adverse events. in addition, the occurrence of respiratory symptoms in the months following vaccination is mistaken as vaccine failure. other respiratory infections as the cause of such symptoms are hardly ever considered. to diminish the arguments of fear of adverse events or perception of vaccine inefficacy, this prospective study was conducted to demonstrate to a subset of hcws from our hospital, that severe adverse events following influenza vaccination are rare and the episodes of respiratory symptoms occurring in the first four months after vaccination are generally caused by other respiratory viruses and not by influenza virus. as expected, no severe adverse event was observed in the present study, and the events more frequently reported, such as headache, myalgia and malaise could be related to influenza vaccine itself as well as to other causes, given their unspecificity. in adults, the adverse event more frequently reported after intramuscular administration of inactivate vaccines is local pain, affecting 10% to 64% of the vaccinated [14] [15] [16] . in the present study, 17.5% of the participants reported local pain. systemic reactions like fever, malaise and myalgia can also occur after inactivate vaccines. in the present study, the frequency of systemic aes (over 70%) was higher than reported in previous studies. recent publications have shown rates of systemic adverse events ranging from 30% to 59% in hwcs [17, 18] . ideally, the subjects should have been surveyed within the first four days of vaccination. as we preferred to apply the questionnaire personally, rather than by mail or phone calls, only 49.7% of the participants were interrogated up to the fourth day, due to the great number of interviews. thus, the high rate of systemic adverse events observed in the present series may be either an overestimation by the subjects or a consequence of the survey method applied. a recent study evaluating vaccine coverage in korea has demonstrated that interview surveys provide more reliable information than telephone surveys, showing lower missing rates and 100% of agreement with the immunization registry record [18] . anaphylaxis and neurological reactions are rare [15, 19] . the frequency of adverse events observed in the present study may be overestimated taking into account the subjectiveness of selfreported unspecific symptoms. as hcws are aware of vaccine adverse events and fear its consequences, it is comprehensible that these events will be more frequently reported by them than by general population. another study conducted in the same hospital demonstrated that hcws reported significantly more adverse events (52.9%) than the elderly (25.3%) [20] . the higher frequency of adverse events reported by hcws surveyed in the first five days of vaccination, as compared with those surveyed after the fifth day, may suggest that people may be more predisposed to remember any symptom possibly associated with the vaccine if inquired within the first days of vaccination. on the other hand, we believe that if the adverse events were severe or important, they would not be missed if inquired after 6 to 10 days. interestingly, we observed that some sectors showed significantly higher rates of ae than others, supporting the subjectivity of the information. also, this data may suggest a mouth to mouth effect among sector coworkers influencing the self-report of ae. among hcws, the belief that coworkers take influenza vaccine influences the vaccine uptake. thus, it is possible that the same occurs concerning to adverse events. continued education of health professionals is essential to highlight not only the epidemiological importance of the vaccine, but also its safety and the low risk of severe adverse events. our study also demonstrated that the respiratory symptoms occurring in the months following influenza vaccination were more frequently caused by other respiratory viruses and generally do not mean vaccine failures. one limitation of our study is that in only 93 of the 192 episodes of respiratory symptoms (48.4%) the subjects agreed with nw sampling. nw sampling is a simple but uncomfortable procedure and this fact may explain why some hcws preferred not to get tested during working hours. one could argue that influenza cases could be missed among those not tested. however, we believe that this loss has not affected our results as the frequency of ili was similar between those who agreed with sampling and those who did not ( table 2 , p = 0.37). the incidence density of other respiratory viruses was 2.4 times greater than incidence density of influenza. probably, this difference would be even greater if real time pcr was also performed to increase the sensitivity of the diagnosis of other respiratory viruses as well. in addition, more cases of other rv infections would be diagnosed if a larger number of professionals were tested, increasing the difference between the incidence density of influenza and other rv. influenza infection is characterized by the abrupt occurrence of fever, headache, myalgia, and dry cough. during influenza season, the presence of these symptoms is highly predictive of influenza infection and summarizes the case definition of influenza-like illness (ili), which has been used worldwide for influenza surveillance purposes. however, the sensitivity and positive predictive value of such definition can vary greatly depending on the co-circulation of other respiratory viruses in the community [21] . indeed, bellei et al. have recently reported that 70% of ili cases in the city of são paulo were caused by other agents, mainly rhinovirus, which peaks along with influenza [22] . similar results have been previously published by other authors [21] . in our series, influenza cases in vaccinated hcws were mild and occurred significantly earlier following vaccination in comparison to other respiratory viruses. this finding may be explained by the marked seasonality of influenza in são paulo city as reported previously [23, 24] , peaking in early winter and coinciding with the initial period of the study. the effectiveness of influenza vaccines is related predominantly to the age and immune competence of the vaccinee and the degree of similarity between the viruses in the vaccine and those in circulation. vaccine effectiveness in preventing laboratory-confirmed influenza illness when the vaccine strains are well matched to circulating strains is 70-90% in randomized, placebo-controlled trials conducted among children and young healthy adults, but is lower among elderly or immunocompromised persons [25] . in adults $65 years old, the efficacy of influenza inactivate vaccine varies from 30% to 40% [26] . trials that measure laboratory-confirmed influenza virus infections as the outcome are the most persuasive evidence of vaccine efficacy [25] . in the present study, only five of the 337 vaccinated hcws (1.5%) acquired influenza. in accordance with the educational nature of our study, we considered all cases of influenza as vaccine failures, since vaccinated health personnel look forward to be protected against influenza. molecular characterization of influenza cases was not performed to check for possible mismatches between circulating viruses and vaccine strains, which could possibly justify those failures. our study demonstrated that the fear of severe adverse events seems unjustified as well as the perception of vaccine inefficacy. uri following influenza vaccination were generally caused by other respiratory viruses and not by influenza. in times of pandemic influenza a h1n1 and widespread vaccination, healthcare and emergency medical services personnel are among the priority groups recommended to receive the h1n1 influenza vaccine. it is time to overcome definitively the misconceptions about the vaccine as well as the fear of adverse events. so far, the vast majority (93%) of adverse events reported to vaers after receiving the trivalent 2010-2011 influenza vaccine, were classified as ''non serious'', e.g., soreness at the vaccine injection site [27] . we believe that the educational nature of the present study may persuade hcws to develop a more positive attitude to influenza vaccination. influenza vaccination among medical residents in a teaching hospital influenza vaccination rates and motivators among healthcare worker groups influenza immunisation: attitudes and beliefs of uk healthcare workers attitudes of health care workers to influenza vaccination: why are they not vaccinated? influenza vaccination of health care workers in hospitals-a review of studies on attitudes and predictors intervention to increase influenza vaccination rates among healthcare workers in a tertiary teaching hospital in brazil * update: influenza activity-united states rapid diagnosis of respiratory syncytial virus infections in immunocompromised adults detection of human rhinovirus rna in nasal washings by pcr polymerase chain reaction for rapid diagnosis of respiratory adenovirus infection frequency of human metapneumovirus infection in hematopoietic sct recipients during 3 consecutive years simultaneous detection of influenza viruses a and b using real-time quantitative pcr measures of disease frequency effectiveness and cost-benefit of influenza vaccination of healthy working adults: a randomized controlled trial frequency of adverse reactions after influenza vaccination the effectiveness of vaccination against influenza in healthy, working adults immunogenicity of a monovalent pandemic influenza a h1n1 vaccine in health-care workers of a university hospital in japan adverse events associated with the 2009 h1n1 influenza vaccination and the vaccination coverage rate in health care workers adverse reactions to influenza vaccine in the elderly occurrence of early adverse events after vaccination against influenza at a brazilian reference center predicting influenza infections during epidemics with use of a clinical case definition acute respiratory infection and influenza-like illness viral etiologies in brazilian adults low mortality rates related to respiratory virus infections after bone marrow transplantation use of oseltamivir to control influenza complications after bone marrow transplantation prevention and control of seasonal influenza with vaccines: recommendations of the advisory committee on immunization practices (acip) the efficacy of influenza vaccine in elderly persons. a meta-analysis and review of the literature summary of 2010-2011 trivalent influenza vaccine data from the u.s. vaccine adverse event reporting system the authors thank the infection control group of hospital das clínicas, school of medical sciences, university of são paulo. key: cord-267042-nvwnbp2j authors: gaspard, philippe; mosnier, anne; simon, loic; ali-brandmeyer, olivia; rabaud, christian; larocca, sabrina; heck, béatrice; aho-glélé, serge; pothier, pierre; ambert-balay, katia title: gastroenteritis and respiratory infection outbreaks in french nursing homes from 2007 to 2018: morbidity and all-cause lethality according to the individual characteristics of residents date: 2019-09-24 journal: plos one doi: 10.1371/journal.pone.0222321 sha: doc_id: 267042 cord_uid: nvwnbp2j background: gastroenteritis (ge) and respiratory tract infection (rti) outbreaks are a significant issue in nursing homes. this study aimed to describe ge and rti outbreaks with infection and all-cause lethality rates according to the individual characteristics of nursing home residents. methods: clinical and virological surveillance were conducted (2007 to 2018). virus stratifications for the analysis were: outbreaks with positive norovirus or influenza identifications (respectively nov+ or flu+), episodes with no nov or influenza identification or testing (respectively novor flu-). associations between individual variables (sex, age, length of stay (los), autonomy status) and infection and lethality rates were tested with univariate and mantel-haenszel (mh) methods. results: 61 ge outbreaks and 76 rti oubreaks (total 137 outbreaks) were recorded involving respectively 4309 and 5862 residents. in univariate analysis, higher infection rates and age were associated in nov+, nov-, and flu+ contexts, and lower infection rates were associated with longer stays (nov+ and nov-). in mh stratified analysis (virus, sex (female/male)) adjusted for los (<4 or ≥4 years), the odds of being infected remained significant among older residents (≥86 years): nov+/male (odds ratio (or(mh)): 1.64, 95% confidence interval (ci): 1.16–2.30) and flu+/female and male (respectively or(mh): 1.50, ci: 1.27–1.79 and 1.73, ci: 1.28–2.33). in univariate analysis, lower autonomy status (nov+, flu+ and flu-) and increased age (flu+) were associated with higher lethality. in mh adjusted analysis, significant or(age) adjusted for autonomy was: flu+/ ≥86 years compared with <86 years, 1.97 (1.19–3.25) and or(autonomy) adjusted for age for the more autonomous group (compared with the less autonomous group) was: flu+, 0.41 (0.24–0.69); flu-, 0.42 (0.20, 0.90). conclusion: the residents of nursing homes are increasingly elderly and dependent. the specific infection and lethality risks according to these two factors indicate that surveillance and infection control measures are essential and of high priority. introduction gastroenteritis (ge) and respiratory tract infection (rti) outbreaks represent a significant burden of illness in nursing homes. viruses cause the majority of these outbreaks, and noroviruses and influenza viruses are the most common pathogens [1, 2] . previous studies have suggested that viral respiratory infections and norovirus outbreaks are a common cause of hospitalization or death, particularly among elderly individuals [3] [4] [5] . the impact of outbreaks has been described in terms of both frequency and epidemiology, but little is known about infection rates and all-cause lethality in ge and rti nursing home outbreaks in relation to the individual characteristics of the residents [6] . the residents of these institutions are increasingly elderly and dependent, and the impact of this trend on the seasonal outbreak burden requires in-depth investigation. the results of studies focused on this issue could yield valuable information for nursing homes, allowing them to adapt their infection control strategies, in particular for improved assessment of infection risk. our objective was to describe ge and rti infection and all-cause lethality rates according to the individual characteristics of nursing home residents (sex, age, length of stay, autonomy status), and to identify specific susceptibility patterns related to these types of viral outbreaks in these facilities. the present study explored outbreaks in 14 sites (28 units with geriatric nursing home activities for a total of 1121 beds) caring for dependent people in southern alsace (an area in northeastern france). data were collected between september 2007 and august 2018 [7, 8] . each site was geographically independent and autonomous for social and care management. units were located within the larger sites and were defined as a place having a dedicated team at one location. during outbreaks at one site, only the residents in the units with confirmed cases were included. outbreak inclusion depended on institutional alert to the hygiene team. surveillance was done in each unit independently, and the members of staff had to inform a physician or charge nurse when two or more potential related cases of pneumonia or ge were observed within four days and when three or more cases were observed for other rti. units also had to inform the hygiene team when these threshold values were exceeded. for influenza, the first suspected case led to a local alert and the hygiene team was contacted. a practitioner from the hygiene team collected the information and evaluated the clinical signs, the virology information and the epidemiological context with the physician in the affected unit. the detected cluster was only put under surveillance if the hygiene team considered that there was a potential outbreak phenomenon. the duration of 4 days was in relation with the national protocol with alert to the authorities when 5 cases occurred within 4 days [9, 10] . on a local level and in addition to the clusters reported to the authorities, clusters with at least 3 cases within a period of seven days in one unit could be recorded if they were reported to the hygiene team. because several outbreaks could potentially occur in the same unit during the surveillance period, a resident could be included repeatedly in different clusters. as a result, the observed patterns reflected the characteristics of an institutional population with longitudinal and pluriannual exposures. personal information and clinical information was collected by a practitioner from the hygiene team directly from the residents' health care records. personal information was collected for all those present the first day of the outbreak. the collected information included: month and year of birth, sex, date of arrival at the nursing home and autonomy status. the autonomy status of residents in french nursing homes is assessed using the aggir scale (autonomy gerontology groups iso-resources), which is the legal instrument for evaluating dependency in the elderly and whose primary purpose is the allocation of means and resources [11] . with the aggir scale, autonomy is classified into 6 iso-resource groups (gir): gir 1 (bedridden or armchair-bound persons, mental functions seriously altered and requiring continuous presence), gir 2 (bedridden or armchair-bound persons, mental functions not totally altered and requiring assistance in most activities of daily living, or mental functions altered with preserved ability to get around), gir 3 (preserved mental autonomy with partially preserved motor autonomy and assistance several times a day for physical autonomy), gir 4 (moves around the home and sometimes assistance for washing, dressing, physical activities or eating), gir 5 (only occasional assistance for washing, meal preparation, and housework), gir 6 (autonomy for essential tasks of daily living). in outbreaks where the influenza virus was identified, influenza vaccination status and oseltamivir prescriptions were recorded as well. a file is transmitted in the supporting information with all previous data (s1 data). ge was defined as the sudden onset of vomiting and/or diarrhea over a 24 h period: (i) diarrhea �3 episodes, (ii) and/or vomiting �3 episodes, (iii) or diarrhea or vomiting <3 episodes with two or more other symptoms (diarrhea, vomiting, stomach ache, abdominal cramps, nausea, fever, mucus in stools) [1] . rti presentation in older adults may be atypical, like for other acute illnesses in this age group [12] . we used the recommended definitions for rti surveillance in geriatric units, divided in 3 subcategories: (i) common cold syndromes or pharyngitis (at least two of the following criteria: runny nose or sneezing, stuffy nose (i.e. congestion), sore throat or hoarseness or difficulty swallowing, dry cough, swollen or tender glands in the neck (cervical lymphadenopathy)), (ii) influenza-like illness (both the following criteria must be met: fever and at least three other symptoms (chills, new headache or eye pain, myalgia or body aches, malaise or loss of appetite, sore throat, new or increased dry cough)) and (iii) lower respiratory tract infection (both of the following criteria must be met: at least two respiratory signs or symptoms (new or increased cough, new/increased sputum production, o 2 saturation <94% or reduced >3% from baseline, abnormal lung examination (new or changed), pleuritic chest pain, respiratory rate �25 breaths/min and one or more constitutional signs/symptoms (fever, leukocytosis, confusion, acute functional decline)) [13] [14] [15] [16] . infection corresponding to one of these three subcategories was included in this study and classified as rti. for both infection types, the practitioner from the hygiene team obtained clinical information from the patient's health care records, and members of the health care team were consulted if necessary to complete any missing information. at the end of the episode (within seven days after the last identified case), case inclusion as exposed and not infected (eni) or exposed and infected (ei) was determined with a resident physician. in order to study the lethality, the presence of each infected resident was evaluated once at least 56 days after the last case of each outbreak. each resident was followed up retrospectively during eighth 7-day interval (i n, n = 1 to 8, total 56 days, between the date of onset of symptoms and the fifty-sixth day of the studied period) with three different possibilities: present (alive and officially residing in the institution), lost to follow-up (alive at the date of departure but no longer residing in the institution (return home, transfer to another institution)) or death (death recorded in the health care record). the dates of death and lost to follow-up were recorded. testing for the virus was not systematic and was decided by the physicians in each institution in the presence of clinical signs. for ge, stool samples were sent to the national reference centre for gastroenteritis viruses in dijon for laboratory testing, as previously described [8] . for rti surveillance, rapid tests were used to identify the influenza virus. the rapid immunoassay diagnosis tests used for influenza detection were: clearview1 exact influenza a and b (inverness medical, cologne, germany) from 2007 to 2014 and influenzatop1 (alldiag, strasbourg, france) from 2014 to 2018. given the low sensitivity of influenza rapid tests, they were no longer used once the control measures had been implemented and the influenza outbreak was under control. samples were also occasionally sent to hospital laboratories or to the national reference centre for influenza viruses to detect viruses with real-time rt-pcr [17] . most testing targeted the norovirus (nov) and influenza virus, but other tests were occasionally performed by the national reference centre for influenza viruses (rhinovirus, respiratory syncytial virus, human metapneumovirus, parainfluenza 1, 2, 3 and 4, and coronavirus) and the national reference centre for enteric viruses (rotavirus, astrovirus, and adenovirus). because testing for the viruses was variable (from one institution/physician to another, not used in some outbreaks, types of virus sought) and considering the poor sensitivity of the rapid influenza tests, these two sources of data were used to define the epidemiological context of confirmed outbreaks. consequently, individual cases were included consistently in all episodes according to clinical signs and medical evaluation. when virus testing was negative, the clinical signs were recorded and medical evaluation was used as previously to classify the included residents as infected or not infected. the epidemiological context of each outbreak was defined according to whether the virus had been identified or not. one or more positive samples led to the qualification of a nov (nov+) or flu (flu+) context. the other episodes were qualified as flu or nov outbreaks with no specific identification or testing (nov-and flu-). flu and nov contexts did not eliminate other potential enteric or respiratory pathogens. sex, age, length of stay (los, in years) and autonomy status were described for all exposed residents. influenza vaccination and oseltamivir administration rates were calculated for confirmed influenza outbreaks. dichotomous or categorical variables were expressed as percentages. in univariate analysis, the categories were specific in order to obtain a precise description of the age and los variables. class intervals were 5 years for age and one year for los. the residents classified as gir 4 to 6 (sometimes, occasional and no assistance) were grouped together because they were few. for the multi-level analysis with 2x2 tables, a median value was used to define the two-level age categories. for los, assessing the longest stays was necessary to identify the effect of longer exposure in a nursing home. consequently, a four-year cutoff was chosen to create the two categories. for autonomy, the two most dependent categories (gir � 2) were grouped together and compared with the more autonomous categories (gir � 3). the outbreak epidemiological contexts were used with the four categories: nov+, flu+, nov-and flu-. other ge or rti viruses were occasionally identified, but the number of results was too limited to develop separate analyses. however, all the results are available in the tables about the virus investigations along with nov and influenza identifications. for the different categories, infection rate (ei/exposed residents (er), in percentage) was calculated according to sex, age group, los and autonomy status. to investigate all-cause lethality and define the appropriate period for the 56-day monitoring (d 1 to 56 ), the all-cause lethality rate per 7-day interval (lr n /i n, n = 1 to 8 ) was calculated: (number of deaths during interval i n /(ei alive the first day of n th studied interval minus lost to follow-up ei during the interval i n ) � 100). seeing as successive clusters could occur within the same site, potentially influencing allcause lethality, the serial interval in days (si d ) was calculated. the si d was the time period between the onset of symptoms of the last case in initial outbreak (n) and the onset of symptoms of the first case in the following outbreak (n+1). investigations were performed when si d was shorter or equal to the length of the previous d 1 to 56 and the following parameters were evaluated for these specific situations: number of episodes, residents infected in both outbreaks, and death among the identified individuals. finally, according to the death rate and the impact of successive outbreaks, the number of 7-day intervals (n.i n ) to take into account was defined, and the all-cause lethality rate was analyzed during these periods (i 1 to n th .i n or d 1 to 7 � n ). all-cause lethality rates were calculated with the following formula: (number of deaths from d 1 to 7 � n /(ei number at d 1 minus lost to follow-up among ei during the period d 1 to 7 � n ) � 100). estimation of the turnover rate per 7-day interval among the infected residents was calculated on the base of the los (median in years) with the following formula: (proportion of discharged residents: 50.0% in the case of the median)/[(median los � 365)/7)]. the average rate of residents discharged per 7-day period was calculated: [(number of lost to follow up during the period d 1 to 7 � n /number of exposed and infected residents at d 1 )/n 7-day interval] � 100. as some residents were included in several outbreaks during the surveillance, the observations were not completely independent; non-parametric tests were used as a result. in univariate analysis, chi-square or fisher exact tests (expected number of frequencies fewer than 5) were used to compare infection and lethality rates according to the studied parameters and the odds ratio was calculated by median-unbiased estimation. the kruskal-wallis test was used to compare median values. confidence intervals for medians were calculated with bootstrap methods. covariate adjusted analyses were performed with two tables (2x2). the respective impact of each individual factor was tested with mantel-haenszel chi-squared tests. the equality of the stratum odds ratios was tested with the woolf test of homogeneity. finally, for each virus context, multiple tables (2x2) were generated and tested with confounding variables, effect modifiers or covariables. statistical analyses were done using r for mas os x version r 3.4.1 software with rstudio version 1.0.153. a file is transmitted in the supporting information with all r codes and the packages used (s1 r codes). differences were considered significant at p � 0.05. the french data protection authority approved data collection and analysis (de-2013-074) and the local ethics committee (espace local de réflexion ethique, centre hospitalier de rouffach) approved the study protocol (erle-32). according to the french law for biomedical research and human experimentation, individual written consent was not required from the patients or their relatives for data collection. each year, the referring local practitioner of the study coordinated with the doctors working in the nursing home. at the beginning of the surveillance period, information regarding participation in the study was displayed in the family vising area, including a document about their right to access and rectify personal data. after collection, data were rendered anonymous. no specific authorization was needed to retrospectively analyze anonymous data collected during routine care in the context of routine surveillance. a total of 137 outbreaks were recorded in the 14 sites. rti outbreaks were more frequent than ge outbreaks (76 outbreaks and 5862 exposed residents vs. 61 outbreaks and 4309 exposed residents, respectively). overall, 7643 of the exposed residents were women and 2528 were men. the median age was 86.7 years old (interquartile range: 81.1-91.0 years). virus investigations (respectively 389 samples for rti and 143 for ge with all the detailed results in s1-s4 tables) confirmed a considerable number of norovirus-related ge outbreaks (34/61) and influenza-related rti outbreaks (46/76). for ge outbreaks, 2524 residents were in a nov+ context versus 1785 in a nov-context, and for rti outbreaks, 3479 residents were in a flu+ context versus 2383 in a flu-context. for ge surveillance in the nov+ context, there were 1093 ei residents versus 1431 eni residents, whereas in the nov-context, there were 583 ei residents versus 1202 eni residents. therefore, the infection rate was higher in the nov+ context (43.3%) than in the nov-context (32.7%, (odds ratio (or): 0.63, 95% confidence interval (ci): 0.56-0.72), p < 0.001). for rti surveillance, the rates of infection were similar with and without confirmed influenza: 31.5% (n = 1095 ei residents /3479 exposed residents) vs. 30.5% (n = 728 ei residents/ 2383 exposed residents, or: 0.96, ci: 0.85-1.07, p = 0.47). moreover, infection rate in the nov + context was higher than the three other contexts: nov-(or: 0.63, ci: 0.56-0.72), flu+ (or: 0.60, ci:0.54-0.67) and flu-(or: 0.58, ci: 0.51-0.65). in univariate analysis, certain individual characteristics were associated with significant variations in the infection rate (s5 table) . the infection rate increased with age (except in the flucontext) and, decreased with los during ge outbreaks. the covariate adjusted analysis revealed specific significant effect modification according to sex (nov+) and los (nov-) (s6 table) . in analyses stratified according to virus and sex, age adjusted for los remained significant for flu+ and nov+ outbreaks (males). in nov-context, the effect modification of los remained significant (table 1) . finally, when autonomy was included and adjusted for age (virus, sex, los stratification), the less autonomous residents (female/los<4 years/age<86/ gir 1-2) were affected more severely by flu+ outbreaks with specific effect modification according to age (s7 table) . the study of lethality rates in infected residents over the 56 days after onset indicated that there were significant variations for rti but no change for ge (table 2 ). significant differences appeared after 28 days in the context of flu+ outbreaks and other rti outbreaks. the analysis of successive or simultaneous clusters in the same institutions was performed when the time period between the onset of symptoms of the last case in outbreak n and the onset of symptoms of the first case in outbreak n+1 was �56 days (s8 table) . 44 of the 137 outbreaks (32.12%) were identified, and 194 of the 3499 exposed and infected residents contracted multiple infections. the percentage of exposed and infected residents implicated in more than one virus stratification was 11.09% ((194 � 2)/3499). moreover, two deceased residents were included in the nov-na and flu lethality analyses because death occurred within 56 days for both infections. the analysis of virus stratification of the 44 outbreaks showed the absence of successive clusters for the same category. the same analysis for the first four 7-day intervals (days 1 to 28) showed the respective values: 26 outbreaks (18.98%), 117 residents ((6.69% ((117 � 2)/3499)), one dead resident. finally, according to the higher lethality impact during the first four 7-day intervals and to limit the impact of successive clusters in the same site, all cause lethality rates were studied according to individual parameters for the four 7 days intervals with the respective number of according to the surveillance type (ge or rti), the lethality rates differed significantly: 1.6% versus 3.4% (respectively nov+ and nov-contexts, or: 2.24, ci: 1.16-4.39, p = 0.02) and 8.3% versus 5.6% (respectively flu+ and flu-, or: 0.67, ci: 0.45-0.97, p = 0.04). in univariate analysis (s9 table) , low autonomy status in the nov+, flu+ and flu-contexts was most significantly associated with increased all-cause lethality, and age was associated with higher lethality in the flu+ context. in the adjusted analysis, no significant statistical differences were identified in ge outbreaks. for rti episodes, the adjusted analysis showed that autonomy had a significant impact when adjusted for sex, age or los (flu+ and flu-na) and that age had a significant impact when adjusted for sex, autonomy or los (flu+) (s10 table) . in table 3 , the specific effects of age or autonomy were tested. significant or age adjusted for autonomy were: flu+/age �86 years (compared with the <86 group), 1.97 (1.19-3.25). or autonomy adjusted for age were for gir 3-6 (compared with gir 1-2): flu+, 0.41 (0.24-0.69); flu-, 0.42 (0.20, 0.90). finally, despite the low number of residents and deaths per category, and consequently the limited robustness of the results, autonomy adjusted for age with stratification according to virus, sex and los showed that the effects were higher among subgroups of less autonomous residents (female or male/los<4 years/gir 1-2) in flu+ outbreaks, and there was also higher mortality in the small subgroup of autonomous men with los � 4 years (higher mortality) (s11 table) . in the flu+ context, data regarding vaccination status and oseltamivir prescriptions were available but not used in this study. in the present study, surveillance data obtained during ge and rti outbreaks in nursing homes were used to construct stratified analyses and to identify specific infection and all-cause lethality rates according to the residents' individual characteristics. the infection rates observed here were similar to those found in previous studies of nov and influenza outbreaks (odds of being infected during a flu+ outbreak were around 40% less than during a nov+ outbreak). reported infection rates were close to 30.0% in influenza outbreaks and 40.0% in nov outbreaks [18] [19] [20] . older age appeared to increase the likelihood of ge and influenza infection, with increasing rates among older residents. age is a well-known factor for influenza and norovirus severity in the elderly and in nursing homes [21, 22] . for nov, the highest incidence estimates (5-year age strata) was found in the �85 year-category (approximately 800 men and for 1,400 women per 100,000 inhabitants). in our study, univariate analysis (nov+) showed that the odds of being infected were 1.5 to 1.6 times higher if a resident was older than 85. moreover, an adjusted analysis of ge outbreaks highlighted different effects among subgroups of residents according to sex and los. indeed, multiple and/or repeated exposure to ge viruses while institutionalized may lead to susceptibility or possible increased immunity in some residents [23] . for the sex variable, two factors could explain the effect: a possible selection bias with men reporting mild infections less than women (particularly in the <86 years subgroup) or that male susceptibility was different (age, los, immunity,. . .). a german study from 2013 also reported a greater impact in women [21] . moreover, when age analysis was stratified by sex and los, no epidemic impacts in nursing homes significant impact was observed in women; the only significant differences were fewer infections in men in the <86-subgroup (except in the nov-with los <4 years). for the rti outbreaks, sex and los variables did not have a significant effect. in residents older than 86, the odds of being infected in flu+ context were 1.5 times higher for women and 1.7 for men. in univariate analysis, contrary to the other virus contexts where odds ratios were rarely above 2, residents over 95 years old had increased odds of infection of � 2.8 compared with the 70-year-old category, and for the 100 year-old group the odds were approximately 3.8. in the flu+ context, autonomy adjusted for age (virus, sex and los stratification) revealed a possible increase in infection rates among less autonomous residents. a previous study found that when elderly residents were exposed to the a(h3n2) virus, there were higher rates of infection and reinfection, and more significant effects on the institution than with other influenza types/subtypes. in the community, the relative illness ratio (rir) in the [22, 24] . the incidence of influenza infection and the associated risks were well described by age group, but the specific impact according to age was not studied. the results of this work highlighted the specific age distribution of influenza illnesses among the nursing home residents and the more significant impact among the older residents. this specific susceptibility could be a critical factor in the institutional exposure and dissemination of influenza and could partly explain the high infection impact in the elderly institutional population. in this work, autonomy status was not the main factor associated with infection (no significant impact in ge and in flu-contexts). however, in flu+ outbreaks, a high level of dependency was associated with a higher risk of falling ill. this observation implies that staff could play a role in the spread of infection (highly dependent and less mobile residents are less likely to contaminate themselves) or that the more active residents may be less fragile and/or have a greater involvement in the recommended infection control measures. finally, improving compliance with personal hygiene measures both for nursing staff and residents might be expected to have a beneficial effect on infection rates. previous studies identified higher nov infection rates in highly dependent individuals, but the results were not adjusted for age and los to take into account the potential correlation with the autonomy status [20] . lethality is difficult to assess in nursing homes because death is frequent. our ge and rti episodes occurred during the winter seasons, and there are possible interactions between outbreaks and increased mortality at this time of the year [25] . the all-cause lethality rate of the infected residents in our study reflected global mortality including ge and rti outbreaks and the global epidemiological context. not surprisingly, a higher all-cause lethality rate was observed in the influenza contexts, as reported in previous studies [25] [26] [27] . age and autonomy had similar effects in the different contexts, but in ge and to a lesser degree in flu-outbreaks, the relatively small number of deaths could have limited the power of the statistical tests. in nursing homes, residents are generally discharged due to death. the number of residents lost to follow up was low (0.14% in the first 28 days), so the 7-day interval turnover rate calculated on the base of the median los provides a good indication of the average case fatality rate. the lethality rate for nov+ outbreaks was similar to the estimated 7-day interval turnover rate (1.6%) indicating that this context had a limited impact on the death rate. the all-cause lethality rate was most affected by age and autonomy. both individual characteristics were significant in the flu+ outbreaks, and autonomy adjusted for age was significant in the flu-episodes. the influence of age on mortality in a context of influenza has already been described: a very high mortality rate (831/100,000 inhabitants) was reported in persons 90 years of age and older compared with those aged 65-69 years (23/100,000 inhabitants) [28] . in our univariate analysis, the higher risk was observed in the �90 group whose risk of death was at least 2.6 higher than the <70 group. when adjusted for autonomy, the impact of age was not significant in more autonomous residents in the flu+ context and not at all in the flucontext. the opposite analysis (autonomy adjusted for age) showed higher global impact in the less autonomous group (flu-) or only in the �86 age group (flu+). age and autonomy are a reflection of resident's level of frailty. clinical frailty scores were not used in this study, but in a previous study of patients with critical illness, they were associated with greater mortality, regardless of age [29] . this suggests that in addition to age, autonomy can be a valuable indicator for the assessment of outbreak impact in outbreak surveillance. other studies have suggested that age and certain comorbidities are independent risk factors for the influenza mortality rate or that mortality increase according to the number of risk factors [28, 30] . comorbidities and underlying diseases of various severities could reflect overall frailty and consequently the risk of death. in nursing homes, information about autonomy and age are easier to collect and interpret than data on comorbidities. these various approaches should be evaluated and compared in the goal of optimizing risk assessment among nursing home residents. the present work has two main limitations. first, the virus information was incomplete (limited identification, mainly influenza rapid tests for the rti). consequently, some episodes in the levels with no available identification may also have been associated with influenza or norovirus, and multiple contaminations could have been underestimated or not taken into account. moreover, vaccination and oseltamivir prescriptions were recorded but not included because the influenza genotype was not determined and identification was limited. secondly, the deaths of uninfected residents were not recorded in this protocol even though such data would have provided valuable information about the global epidemiological context. in conclusion, specific susceptibility patterns were observed among exposed residents. in this cohort of nursing homes, infection rates varied according to virus, sex, length of stay and age, and there were major differences in lethality depending on virus, age and autonomy score. the collected data were easy to record and could be used to improve the characterization of seasonal outbreaks in nursing homes, whose residents are particularly vulnerable. finally, as the average age and dependency level of residents continues to increase, subsequently increasing the risk of infection and death, health care staff will have to be increasingly vigilant during seasonal outbreaks and targeted interventions should be implemented. supporting information s1 data. (csv) s1 r codes. (r) s1 table. (xlsx) s10 table. (xlsx) s11 table. 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nursing homes and risk factors for spread: a systematic review and meta-analysis of observational studies epidemiology of norovirus gastroenteritis in germany 2001-2009: eight seasons of routine surveillance age distribution of influenza like illness cases during post-pandemic a(h3n2): comparison with the twelve previous seasons, in france pathogenesis of noroviruses, emerging rna viruses comparative age distribution of influenza morbidity and mortality during seasonal influenza epidemics and the 2009 h1n1 pandemic effects of cold weather on mortality: results from 15 european cities within the phewe project mortality due to influenza in the united states-an annualized regression approach using multiple-cause mortality data effectiveness of influenza vaccines in preventing severe influenza illness among adults: a systematic review and meta-analysis of test-negative design case-control studies co-morbidities associated with influenza-attributed mortality frailty and subsequent disability and mortality among patients with critical illness pneumonia and influenza deaths during epidemics: implications for prevention all the medical, nursing, management teams in the institutions for their commitment to this key: cord-254825-c5d0wul9 authors: kim, sei won; jo, sung jin; lee, heayon; oh, jung hwan; lim, jihyang; lee, sang haak; choi, jung hyun; lee, jehoon title: containment of a healthcare-associated covid-19 outbreak in a university hospital in seoul, korea: a single-center experience date: 2020-08-14 journal: plos one doi: 10.1371/journal.pone.0237692 sha: doc_id: 254825 cord_uid: c5d0wul9 background: our hospital experienced the first healthcare-associated covid-19 outbreak in seoul at the time the first covid-19 cases were confirmed in korea. the first confirmed covid-19 patient was a hospital personnel who was in charge of transferring patients inside our hospital. to contain the virus spread, we shutdown our hospital, and tested all inpatients, medical staff members, and employees. methods: we retrospectively analyzed the results of sars-cov-2 rt-pcr testing according to the contact history, occupation, and presence of respiratory symptoms. closed-circuit television (cctv) was reviewed in the presence of an epidemiologist to identify individuals who came into contact with confirmed covid-19 patients. results: a total of 3,091 respiratory samples from 2,924 individuals were obtained. among 2,924 individuals, two inpatients, and one caregiver tested positive (positivity rate, 0.1%). although all confirmed cases were linked to a general ward designated for pulmonology patients, no medical staff members, medical support personnel, or employees working at the same ward were infected. contact with confirmed covid-19 cases was frequent among inpatients and medical support personnel. the most common contact area was the general ward for pulmonology patients and medical support areas, including clinical and imaging examination rooms. finally, the total number of hospital-associated infections was 14, consisting of four diagnosed at our hospital and ten diagnosed outside the hospital. conclusions: the robust control of the covid-19 outbreak further minimized the transmission of sars-cov-2 in the hospital and local communities. however, there was also a debate over the appropriate period of hospital shutdown and testing of all hospital staff and patients. future studies are required to refine and establish the in-hospital quarantine and de-isolation guidelines based on the epidemiological and clinical settings. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 in december 2019, a novel coronavirus disease was first reported in wuhan, hubei province, china [1, 2] . since then, human-to-human transmission of the novel coronavirus has been confirmed [3] and has caused serious illness and death [4] . following the covid-19 outbreak in china, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the virus responsible for covid-19, has spread in many countries across the world, including korea, iran, european countries, and the usa [5] . in korea, the number of covid-19 patients who were confirmed with real-time reversetranscription polymerase chain reaction (rt-pcr) assay was 10,564, while 222 deaths were reported until april 14, 2020 [6] . among the covid-19 patients, 14 patients were linked to a single university hospital. these were the first covid-19 cases resulting from healthcare-associated infections (hai) in seoul. hence, the seoul government announced the closure of the hospital. moreover, outpatient admission was temporarily stopped for approximately two weeks. inpatients who had come into contact with confirmed covid-19 patients were isolated inside the hospital. a total of 2,924 people, including hospitalized patients, medical/paramedical staff, employees, and caregivers, were all tested for sars-cov-2 using rt-pcr assay. in this study, we report our experience from our covid-19 cases, as well as discuss the necessity of widespread sars-cov-2 rt-pcr testing and hospital shut down, particularly in major hospitals, to prevent hai. our hospital (eunpyeong st. mary's hospital, the catholic university of korea) is an 808-bed university hospital located in eunpyeong district, northwest seoul, korea. the hospital opened in april 2019. the main building of the hospital has 17 floors above ground and seven floors below ground. the hospital receives approximately 2000 to 3000 outpatient visits and 150 to 200 patient visits to the emergency room daily. the hospital has nine floors for inpatients, which contains two general wards. each general ward has nine rooms with four beds each, and two rooms with a single bed. the 4-bed rooms have 2 m space between beds, which are separated by curtains for privacy (fig 1) . in this study, we retrospectively analyzed the results of sars-cov-2 rt-pcr testing, contact history, and presence of respiratory symptoms in a single center with a healthcare-associated covid-19 outbreak. we also reviewed data from epidemiological surveys, from the korea centers for disease control and prevention (kcdc) and the infection control unit of our hospital. the covid-19 prevention measures before and after the healthcare-associated outbreak were also reviewed. this study was approved by the institutional review board of eunpyeong st. mary's hospital (pc20rasi0040). we reviewed the history of patients to assess whether they visited china or other high-risk countries within two weeks prior to the outbreak of healthcare-associated covid-19, or if they came into contact with confirmed covid-19 cases. after the initial assessment, patients without fever or respiratory symptoms had no specific restrictions inside the hospital. patients with fever (>37.5˚c) or respiratory symptoms were transferred to the triage room, which is located in a different part of the hospital to that of the infectious diseases department and the emergency room. after obtaining samples for rt-pcr testing, patients were sent home for self-isolation until the results were made available. if the results were positive, patients were referred to government-designated hospitals. medical staff in the triage room used level-d personal protective equipment (ppe) and everyone in the hospital was encouraged to wear masks and follow hand hygiene practices. the first confirmed covid-19 case was a 35-year-old man who was in charge of transferring patients inside our hospital between february 2 and 17. on february 17, 2020, he visited the clinic due to a one-week history of fever, cough, and myalgia. although he had no history of travel or close contact with confirmed covid-19 cases, chest x-ray showed ground-glass opacities in both lower lobes. considering the possibility of covid-19, rt-pcr confirmation testing was performed on february 20, 2020. after sars-cov-2 infection was confirmed, the seoul city government announced the closure of the hospital on february 21, 2020, to prevent a healthcare-associated outbreak. outpatient clinics and the emergency room were also closed, and new patient admissions were stopped. individuals inside the hospital who had contact history, fever, or respiratory symptoms, were closely monitored. in total, 3,091 respiratory samples from 2,924 people were obtained from february 21 to february 28. two thousand one hundred seventy-one samples were processed and tested in the department of laboratory medicine of our hospital, while 920 samples were analyzed by a commercial laboratory (samkwang medical laboratories, korea). sputum and combined nasopharyngeal (np) and oropharyngeal (op) swabs were collected from patients with acute respiratory symptoms and purulent manifestations. combined np and op swabs were also collected from asymptomatic individuals. np and op swabs were collected in the t-swab transport™ utm (noble biosciences, korea) while sputum specimens were collected in 50cc falcon tubes. sputum samples were prepared in phosphate buffer solution. qiaamp dsp viral rna mini kit (qiagen gmbh, hilden, germany) with qiacube system (qiagen), as well as nx-48 viral na kit (genolution, korea) with nextractor nx-48 system (genolution) were used for rna extraction. nucleic acid was extracted according to the manufacturer's instructions. sars-cov-2 nucleic acid was amplified by real-time rt-pcr using the powerchecktm 2019-ncov real-time pcr kit (kogenebiotech, korea). abi 7500 (applied biosystems, usa) real-time pcr system was used for the amplification of the results of sars-cov-2 rt-pcr were analyzed according to occupation, presence of the respiratory symptoms, and contact history. the admission department, floor, and room location were also assessed. contact was defined as presence in the same room with covid-19 confirmed patients, or in the same outpatient clinic or examination room, 30 minutes before and after covid-19 confirmed patients. moreover, closed-circuit television (cctv) was reviewed in the presence of an epidemiologist, and people who had been within 2 m of confirmed patients were considered individuals with contact history. after the hospital staff member responsible for transporting patients was confirmed as the first covid-19 case, people with contact history, fever, or respiratory symptoms were tested for sars-cov-2 infection with rt-pcr (fig 2) . among the admitted patients, patient with pneumonia who was receiving treatment was confirmed as the 2 nd covid-19 patient (fig 3) . chest ct showed that the patient had multiple, ground-glass opacities in both lungs. this patient was not previously tested for covid-19, as the patient had no history travel to china or other high-risk countries, and had no close contact with confirmed patients. due to a contact history with this inpatient, the first diagnosed medical staff was considered as nosocomial covid-19 infection. additionally, one caregiver who was in the same room with the 2 nd covid-19 patient also tested positive for sars-cov-2. the caregiver confirmed as covid-19 was also considered as nosocomial covid-19 infection. as the nosocomial covid-19 infection continued to be discovered, the hospital started complete enumeration survey of all patients (fig 2) . the fourth covid-19 patient was also an inpatient with pneumonia such as the second covid-19 patient. on february 25, the hospital decided to perform a complete enumeration survey of all medical staff and employees to help control the healthcare-associated covid-19 outbreak. as this was the first major healthcare-associated transmission cluster reported in seoul, the city government imposed strict measures to control the spread of the virus. the entire outpatient clinic and emergency rooms were temporarily closed for two weeks, as per the guidelines set during the 2015 middle east respiratory syndrome (mers) outbreak [7] . the entire hospital was thoroughly cleaned and disinfected. inpatients who had had no contact with confirmed covid-19 patients and had no symptoms were discharged, while inpatients who had had contact with confirmed covid-19 patients were quarantined in single rooms for two weeks. a complete enumeration survey was conducted from january 23 to january 28 to prevent further spread of the healthcare-associated infection by eliminating the possibility of asymptomatic transmission [8] . two thousand nine hundred twenty-four inpatients and employees (213 doctors, 901 nurses, 271 medical support staff, 952 hospital employees, 494 inpatients, 87 guardians and caregivers, and 11 volunteers) had undergone sars-cov-2 testing by real-time rt-pcr ( table 1 ). the employees who had contact with confirmed covid-19 patients selfisolated for two weeks. two patients and one caregiver tested positive for sars-cov-2. the overall covid-19 positivity rate in the complete enumeration survey (100 × positive tests/ total number of tests conducted) was 0.1%. fifty-one individuals were re-tested more than twice to monitor the progression of respiratory symptoms. after the first case was reported, epidemiologists from kcdc and the infection control unit of our hospital reviewed electronic medical charts, cctv, and personal movements to identify individuals with potential contact with confirmed covid-19 patients. an additional three confirmed cases were identified, and the contact list was updated. the number of people who underwent sars-cov-2 testing with real-time rt-pcr and had contact with covid-19 confirmed patients is shown in table 2 . the overall proportion of people who came into contact with confirmed covid-19 patients was 9.9%. most of these individuals were inpatients (28.2%) who had stayed on the same ward with confirmed covid-19 patients. contact with confirmed covid-19 cases was also frequent among medical support personnel (12.9%), including staff from the radiologic department, rehabilitation unit, and phlebotomists. three confirmed covid-19 patients (2 nd , 3 rd , 4 th patients) stayed on 9gw. four caregivers, 40 healthcare workers, and 12 inpatients came into contact with confirmed covid-19 patients on 9gw. the most common area of contact with confirmed covid-19 patients was the 9gw and medical support area, such as the clinical or imaging examination room ( table 3) . discharged patients, caregivers, and healthcare workers who had left before the hospital closed were grouped according to the possibility of covid-19 exposure, based on electronic medical charts, cctv, and personal movements. four hundred and seventy-nine people were determined as high-risk, including 58 caregivers, 11 healthcare workers, and 410 discharged patients, all of whom were notified by phone. they were also examined for respiratory symptoms and advised to stay home and avoid contact with other people even if they had no respiratory symptoms. two discharged patients, one caregiver and one visitor were later diagnosed with covid-19 at a different hospital or community health center (fig 3) . as additional patients were confirmed, the number of exposed people increased to 1,215; these individuals received advice as per the kcdc covid-19 guidelines. finally, the total number of hospitalassociated infections was 14, consisting of four diagnosed at our hospital and ten diagnosed outside the hospital. since the report of the first covid-19 cases in wuhan, china, 1,812,734 confirmed cases and 113,675 deaths have been reported worldwide until april 14, 2020 [9] . severe symptoms develop in approximately 14% of covid-19 patients, and the overall mortality is around 2% of confirmed covid-19 cases [10] . advanced age, development of severe symptoms, and comorbidities are the primary risk factors associated with covid-19 mortality [11, 12] . as there are no effective treatments for covid-19, prevention of further virus spread is the only way to control the covid-19 pandemic [13] . prevention measures are particularly important in hospitals, where numerous elderly patients and individuals with comorbidities are found. in 2015, nosocomial mers outbreaks were reported in korea, caused by contacts with infected outpatients and inpatients [14] . although strict precaution and prevention measures were followed according to the kcdc guidelines, 14 healthcare-associated covid-19 cases were reported in our hospital, including 4 people diagnosed in-hospital and 10 people diagnosed outside of the hospital. compared to small hospitals or care units, the impact of healthcare-associated infections can be detrimental in large university hospitals with numerous visitors and severely ill patients. the first confirmed covid-19 patient in our hospital was a hospital personnel responsible for transporting patients. however, the only person who came into contact with him and was later confirmed with covid-19 was his father. the second confirmed covid-19 patient (inpatient with pneumonia) showed high viral rna load (ct values in rt-pcr: e gene, 17.19; rdrp gene, 17.64). despite the high viral load, only two direct transmissions could be identified. asymptomatic transmission of covid-19 has also been reported [15, 16] , and viral loads can be high as symptomatic carriers [17] . therefore, to control the spread of the virus, we tested all people in the hospital to identify asymptomatic and undiagnosed individuals. despite extensive exposure to four confirmed patients, only a few hai were detected. several reasons could explain the minimal spread of sars-cov-2 in our hospital. importantly, korea experienced an outbreak of mers in 2015; hence, nearly all hospitals have implemented infection prevention guidelines [7] . immediately after the outbreak of covid-19 in china, the korean government imposed prevention measures [18] , and the infection control unit of our hospital restricted hospital visitors with respiratory symptoms or travel history. as per the kcdc recommendations [19] , masks were recommended for all patients and medical staff, and the importance of hand-washing was emphasized. furthermore, our hospital was newly built with good ventilation facilities. distance between the beds in multi-bed rooms was over 2 m. we know from the sars outbreak that distance between beds � 1m was a significant risk factor associated with healthcare-associated sars transmission [20] . curtains were installed between the beds, most of which were kept closed for privacy. this could have greatly contributed to the containment of the virus spread [21] . additionally, the maximum number of beds in a room was four, which is lower than other university hospitals in seoul. hospital closure, inpatient isolation in single rooms, self-isolation of individuals with contact history for two weeks, and complete enumeration survey of all inpatients, medical staff, and employees regardless of contact history or development of respiratory symptoms, further minimized the transmission of sars-cov-2 in the hospital and local communities. the robust control of the nosocomial covid-19 outbreak reassured the remaining inpatients and local community that the risk of in-hospital transmission was low. however, the measures taken were time-consuming, laborious, and expensive. complete enumeration could be achieved due to the rapid installation of new molecular equipment. before the nosocomial outbreak, one abi 7500 (applied biosystems) real-time pcr system and two qiacube (qiagen) preparation systems were available in the department of laboratory medicine to test for sars-cov-2 and other pathogens. to increase sars-cov-2 rt-pcr testing capacities during the nosocomial outbreak, we installed new viral rna preparation systems and one abi 7500 system on february 24th. comparison between nextractor nx-48 systems and qiacube systems, as well as between two abi systems, were performed. the test capacity of sars-cov-2 rt-pcr testing increased to approximately 800 tests per day after the installation of the new equipment. including the commercial laboratory tests, we could test approximately 1,000 samples in 24 hours. in addition to the introduction of new equipment, the working hours and shifts of laboratory medical technicians were adjusted to allow for 24-hour testing. the infection control unit and every department of our hospital continuously monitored inpatients and employees for fever and respiratory symptoms. fifty-one individuals underwent sars-cov-2 rt-pcr testing more than twice to monitor the progress of respiratory symptoms. moreover, for some individuals, several respiratory specimens were tested to account for the incubation period of sars-cov-2, which is estimated to be 5.1 days [22] . moreover, sars-cov-2 rt-pcr testing was repeated prior to patient de-isolation; however, considering the cost, this may not be possible in all hospitals [23] . our hospital was reopened on march 9th, 17 days after the closure and, until now, no additional covid-19 cases have been confirmed. the duration of the hospital closure was based on the seoul government guidelines established during the mers outbreak [7] . according to "management of medical center with covid-19 confirmed patients" guidelines established by the central disaster and safety countermeasures headquarters of korea, hospitals should close when the risk of transmission is high [24] . on march 9th, the korean medical association announced that 17 days of closure would be a laborious administrative process and suggested that the old guidelines based on the mers outbreak should be revised. during the hospital closure, the existing patients could not receive treatments, and some were denied care elsewhere. due to the complete hospital shutdown, patients with scheduled chemotherapy, radiation therapy, hemodialysis, emergency operation, or scheduled births faced health challenges. in conclusion, the robust control of the covid-19 outbreak further minimized the transmission of sars-cov-2 in the hospital and local communities. however, there was also a debate over the appropriate period of hospital shutdown and testing of all hospital staff and patients. future studies are required to refine and establish the in-hospital quarantine and deisolation guidelines based on the epidemiological and clinical settings. the continuing 2019-ncov epidemic threat of novel coronaviruses to global health-the latest 2019 novel coronavirus outbreak in wuhan, china clinical features of patients infected with 2019 novel coronavirus in wuhan, china. the lancet a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster. the lancet coronavirus infections-more than just the common cold coronavirus disease 2019 (covid-19) situation report-49: world health organization the updates on covid-19 in korea as of 14 april middle east respiratory syndrome infection control and prevention guideline for healthcare facilities transmission of 2019-ncov infection from an asymptomatic contact in germany world health organization. who health emergency dashboard; coronavirus (covid-19) severe sars-cov-2 infections: practical considerations and management strategy for intensivists epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study. the lancet respiratory medicine asymptomatic carrier state, acute respiratory disease, and pneumonia due to severe acute respiratory syndrome coronavirus 2 (sars-cov-2): facts and myths preliminary epidemiological assessment of mers-cov outbreak in south korea estimating the asymptomatic proportion of coronavirus disease 2019 (covid-19) cases on board the diamond princess cruise ship clinical characteristics of 24 asymptomatic infections with covid-19 screened among close contacts in nanjing, china. sci china life sci sars-cov-2 viral load in upper respiratory specimens of infected patients early epidemiological and clinical characteristics of 28 cases of coronavirus disease in south korea why did outbreaks of severe acute respiratory syndrome occur in some hospital wards but not in others? relationship between hospital ward design and healthcareassociated infection rates: a systematic review and meta-analysis the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application feasibility of controlling covid-19 outbreaks by isolation of cases and contacts central disaster and safety countermeasures headquarters of korea. management of medical center with covid-19 confirmed patients we greatly appreciate the support from soon-yong kwon, seung-hye choi, rev. fabian park chang yeob, jae-taek hong, seung eun jung, infection control unit and emergency headquaters of eunpyeong st. mary's hospital. we also thank all the members of the eunpyeong st. mary's hospital for their efforts and devotion during the crisis of covid-19. key: cord-003548-zuwt7gk5 authors: cai, haiming; deng, jinbo; li, jiaoqing; ma, miaopeng; huang, chaoyuan; zhao, peijing; ming, feiping; liang, qianyi; jia, junhao; zhang, shuxia; zeng, min; zhang, linghua title: modulating the 3’ end-dna and the fermentation process for enhanced production and biological activity of porcine interferon-gamma date: 2019-03-26 journal: plos one doi: 10.1371/journal.pone.0214319 sha: doc_id: 3548 cord_uid: zuwt7gk5 porcine gamma interferon is a cytokine produced by activated t cells and nk cells with broad-spectrum antiviral activity and immunomodulatory function. however, pifn-γ is a secretory protein that has a short half-life in organisms and is easily inactivated, making it difficult to apply widely in clinics. therefore, we tried to optimize the expression of pifn-γ in pichia pastoris to obtain a large amount of highly active, easily purified pifn-γ protein in vitro. through c-terminal sequence analysis, we found a signal sequence (ekreaeae) that was easily enzymolysed by a signal peptide enzyme, resulting in degradation and inactivation of the pifn-γ protein. in this study, we optimized the pifn-γ gene recombination sequence and mutated the 3' end of the pifn-γ gene, resulting in a higher expression level and stronger biological activity, as well as a significant upregulation in the expression of the interferon-stimulated genes mx1 and oas1 in ipec-j2 jejunal epithelial cells. our data also showed that the fermentation process could significantly improve productivity. a recombinant pichia pastoris strain with the optimized pifn-γ gene could obtain a high yield of pifn-γ protein, up to 9536 mg/l, after staged incubation for 0–24 h at 28°c, ph 6.0, and 50% dissolved oxygen (do), followed by incubation for 24–72 h at 25°c, ph 6.0 and 30% do. these data demonstrated, for the first time, that the expression level of pifn-γ in pichia pastoris was improved significantly by gene optimization with 3' end mutation and a fermentation process that maintained good biological activity, which is beneficial to the application of pifn-γ in animal husbandry. interferon gamma (ifn-γ) is a cytokine with antiviral and immunomodulatory functions produced by activated t cells and nk cells [1] [2] [3] . the total porcine interferon gamma ifn-γ (pifn-γ) gene is 501 bases and encodes 166 amino acids. twenty-three amino acids compose a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the signal peptide, and 143 amino acids of pifn-γ function in the activity of the mature protein [4] . pifn-γ mainly participates in the immune regulation effect by activating the body's own immunity against viral attack [5] . the antiviral effect of interferon does not directly kill the virus but rather binds to cell surface receptors [6] to induce cells to produce enzyme-active antiviral proteins adar, pkr, mx, oas, and rnasel, which degrade viral rna, inhibit viral replication and translation, and inhibit viral shell formation to achieve antiviral effects [7] [8] [9] . studies have shown that pifn-γ can significantly inhibit the proliferation of reproductive and respiratory syndrome virus (prrsv) [6] and plays an important role in anti-swine fever virus (csfv) cellular immunity [10] . a study also showed that pifn-γ could enhance the immune response of piglets to dysentery antigens and played an important role in anti-salmonella infection. pifn-γ has broad application prospects due to its highly effective antiviral and immunomodulatory effects. the researchers also used different expression systems to express pifn-γ, but most of them have a problem with low protein expression or activity, which limits the further application of pifn-γ [11] . under normal circumstances, the interferon species in animals is highly specific, has low expression levels and is difficult to extract and purify, wang li et ., al studies found that e.coli, baculovirus can improve the co-expression level of interferon, while the expression products need to undergo degeneration, complex re-purification, which were seriously affecting the biological activity of interferon [11] [12] [13] , so research and development of a biological system for efficient production of interferon has aroused the attention of scholars globally. pifn-γ was first expressed in escherichia coli (e. coli) by genetic engineering, and with the improvement of extraction process, the pifn-γ protein extraction rate reached 95%~97%, while the other 3% 5% was e. coli protein, which could easily cause substantial side effects such as fever, allergy and other adverse reactions. to express recombinant pifn-γ with high efficiency, low cost and few side effects, in recent years, research on the expression system of methanolic yeast has progressed rapidly [14] . this system is regulated by the methanol-regulated alcohol oxidase 1 (aox1) promoter, which strictly controls the expression of exogenous genes [15] and has high stability and high secretion. the host bacterium pichia pastoris has little endogenous protein secretion but highly active exogenous protein secretion, which is beneficial for downstream separation and purification operations and large-scale fermentation production; therefore, pichia pastoris is a suitable eukaryotic expression system for exogenous genes [16] . in our previous studies, we found that interferon could not be purified by nickel-column affinity chromatography. by analysis of the 3' end of the gene, we found that the amino acids in positions 126-132 of the c-terminus may be similar to the cleavage site of the secreted α signal peptide before its secretion. c-terminal peptides would be cleaved, and pifn-γ protein with a complete c-terminus would fail to be secreted. after the optimization of the 3'-peptide chain, a large increase in biological activity was observed in yeast. in addition, it is well known that the fermentation process, especially the fermentation tank technology, is very important for the production of foreign proteins secreted by pichia pastoris. pichia pastoris does not affect normal growth and metabolism due to the accumulation of fermentation products. it is also easy to expand from shake flask culture to high density fermentation without affecting the expression level of exogenous genes, so it has great potential for high density fermentation; however, the expression system of pichia pastoris has a great difference in the expression of different exogenous proteins, reaching up to 12 g/l or as low as only 1 mg/l, a 10,000-fold difference [17] . this difference in expression of the foreign protein stems, on the one hand, from the characteristics of the foreign gene itself, and on the other hand, from the fermentation conditions, with the latter having an important influence. the effect of temperature on the activity of yeast fermentation products, the physical properties of fermentation broth and the direction of biosynthesis, as well as the do in the fermentation matrix, are very important to the growth of aerobic microorganisms. therefore, in this study, we used the pichia pastoris eukaryotic expression system, regulating the 3' end-dna and fermentation process for enhanced production and biological activity of pifn-γ in recombinant pichia pastoris, to ensure the high-level expression of the protein at the transcription and translation levels, which laid a foundation for the construction of high-yield strains of pifn-γ pichia pastoris. the use of secretory expression greatly simplifies the subsequent purification steps and costs of the target protein and facilitates its industrial production. strains p. pastoris x33, e. coli dh5α, and ppiczαa were purchased from invitrogen (ca, usa). the ipec-j2 cell line (small intestinal epithelial cell) was a gift from deng yiqun, south china agricultural university. to improve the production of recombinant pifn-γ in p. pastoris, we used a novel deterministic computational algorithm costar for codon optimization [18] . the optimal synthetic sequence, pifn-γ 1 (genbank: mh513659), had the following properties: enhanced codon usage bias of the heterologous gene; no unwanted cleavage sites for restriction enzymes and negative cis-acting elements; a reduced number of highly repetitive nucleotide sequences; adaptive g+c content; and inhibited local formation of the transcribed mrna secondary structure. we designed a cloning approach based on synthetic overlapping primers and a pcr assembly strategy to construct a synthetic pifn-γ fragment without the signal peptide sequence (genbank: nm_213948). the pifn-γ cdna (genbank: np_999113) was used as a template for optimization to generate a set of twelve overlapping oligonucleotides. a mixture of the oligonucleotides was used in the first-round pcr procedure consisting of ten cycles, using kod-fx polymerase to generate the coding sequence. then, the region for the xhoⅰ restriction site and kex2 cleavage site of the α-factor signal sequence of p. pastoris were added to the 5' terminus of the synthesized fragment in the second-round pcr using the primer pifn-γ-forward. at the same time, the region for the xbaⅰ restriction site was linked to the 3' terminus using the primer pifn-γ-reverse, which was inserted into the expression vector ppiczαa to construct the plasmid ppiczαa-pifn-γ 1 (s1 fig) . the other region for the xbaⅰ restriction site was linked to the 3' terminus using the primer pifn-γ-reverse with six histidine (his) tags. the complete artificial dna was inserted into the expression vector ppiczαa to construct the plasmid ppiczαa-pifn-γ 1 -his (s2 fig) . the arginine (r) at positions 129 and 131 of the pifn-γ mature peptide chain was mutated to a histidine (h); that is, the amino acid sequence of the peptide chain from 126 to 132 was mutated from lrkrkrs to rkhkh, and accordingly, the corresponding codon was mutated from aga and cgt to cat (s3 fig). then, we used a novel deterministic computational algorithm, costar, for codon optimization of the pifn-γ 1 ' gene (genbank: mh513659) [18] . through comprehensive analysis and optimization, the full-length pifn-γ gene with signal peptide mutation was obtained. the obtained fragment was inserted into the ppiczαa vector to construct two mutant secretory expression vectors ppiczαa-pifn-γ 1 ' and ppiczαa-pifn-γ 1 '-his tag. e. coli dh5α was used for the construction of the recombinant expression plasmid and standard plasmid. all plasmids were confirmed by dna sequencing, and the plasmids used in this study are shown in s1 transformation of p. pastoris and screening of transformants p. pastoris x33 electrocompetent cells were transformed with saci-linearized ppiczαa-pifnγ 1 , ppiczαa-pifn-γ 1 ', ppiczαa-pifn-γ 1 -his tag, or ppiczαa-pifn-γ 1 '-his tag. positive transformants were screened on ypds containing 500 μg/ml zeocin. transformants confirmed by pcr were grown overnight in bmgy in preparation for use as competent cells, and they were used for a second round of electroporation. the transformation mix was spread on bmgy plates containing 500 or 1000 μg/ml zeocin for direct selection of potential multicopy recombinants. colonies of transformants were cultivated in bmgy culture medium containing 100 mm potassium phosphate (ph 6.0), 1.34% yeast nitrogen base without amino acids, 4 × 10 −5 % biotin, and 1% glycerol (bmgy). cultures were grown in 500 ml flasks containing 50 ml of bmgy and were incubated at 28˚c on a rotary shaker at 230 rpm. when the od 600 reached 5-6, the cells were centrifuged at 3500 rpm for 5 min, and the cell pellet was resuspended in 50 ml of bmmy. to maintain the expression of the product, 100% methanol was added to a final concentration of 0.5% (v/v) every 24 h. after 48 h of induction, 20 μl of culture supernatant was analyzed by sds-page. to purify the pifn-γ protein in the supernatant, the culture supernatant was dialyzed against 50 mm tris-hcl, ph 7.5. the cation exchanger cm-sephadex was pre-equilibrated with 50 mm tris-hcl, ph 7.5, and then the culture supernatant was packed in a column and washed with the same buffer until the absorbance reached <0.1 at 280 nm. after this step, pifn-γ protein was eluted with a stepwise gradient of nacl (0.1, 0.4, 0.6, 0.8, and 1 m nacl prepared in 50 mm tris-hcl, ph 7.5). the eluted fractions containing pifn-γ were pooled and dialyzed against 5 mm tris-hcl (ph 7.5) with 150 mm nacl, 5 μm znso 4 , and 5% glycerol [19] for further studies. protein extractions from cytoplasmic and membrane-associated fractions were performed according to shen et al. [20] . briefly, cells were harvested with 1.5 × 10 8 cells washed in pbs ph 7.4 and resuspended in 300 μl of yeast breaking buffer. an equal volume of acid-washed glass beads was added, and cells were disrupted by vortexing ten times for 1 min with 1-min intervals on ice. the lysate was centrifuged at 10,000×g for 30 min at 4˚c, and the supernatant was collected. the pellet was further resuspended in 100 μl of yeast breaking buffer plus 2% sds. after centrifugation at 4000×g for 5 min at 4˚c, the supernatants containing the membraneassociated proteins were collected. twenty micrograms of cytoplasmic proteins or membraneassociated proteins determined by bca protein assay was analyzed by sds-page and western blot. equal amounts of extracted proteins and 20 μl of culture medium were analyzed by 15% sds polyacrylamide gel electrophoresis (page); after electrophoresis, gels were stained with coomassie blue r-250. for the western blot assay, the proteins were transferred to pvdf membrane, and the membrane was incubated at 37˚c with 5% nonfat milk. afterward, the membrane was incubated with a 1:2000 dilution of goat anti-pglyrp-1 polyclonal antibody (santa cruz, usa) overnight at 4˚c and incubated with hrp conjugated rabbit polyclonal anti-goat igg (cwbio, china) at a dilution of 1:4000. immunoreactive bands were visualized with the enhanced hrp-dab chromogenic substrate kit (tiangen, china). shake flask fermentation at different temperatures. a positive clone of a single colony was scraped from the bmgy plate and placed in 300 ml shake flasks containing 50 ml of seed medium (ypd) at 28˚c, ph 6.0, and 200 rpm. the strains were cultured for 16-24 h, and then the seeds were transferred to shake flasks and fermented at 15˚c, 20˚c, 25˚c and 30˚c at ph 6.0 for the next 48 h [21] . five liter fermenter with different do levels. the liquid culture volume was 60% (vv-1), the inoculation volume was 10% (vv-1) [22] , and the stirring speed was 150 rpm, with 50% do at 28˚c for 24 h. then, the fermentation temperature was reduced to 25˚c and the culture was continued from 24 h to 72 h (based on data of shake flask fermentation at different temperatures), with the do at 20%, 30%, 40% and 50% [23] . for determination of the recombinant pifn-γ protein concentration, the supernatant was obtained by centrifugation of the fermentation broth at 10,000 rpm for 10 min and subjected to sds-page gel electrophoresis. the electrophoretic gel was scanned by gel imaging and analyzed by bandscan 5.0 software to calculate the protein concentration. the wet weight of yeast was determined by removing 10 ml of fermentation liquid, centrifuging twice at 1000 rpm for 5 min, and weighing the yeast. the ipec-j2 cell line, a gift from deng yiqun, south china agricultural university, was mainprior to the treatment, ipec-j2 cells were resuspended by trypsinization with 0.25% trypsin-edta and seeded into 24-well plates at 2×10 5 cells/well without antibiotics. the concentration of the cell suspension was 5×10 4 /ml, and 100 μl of cell culture medium was added to each well. the cells were inoculated into 6 wells and cultured for 24 h at 5% co 2 at 37˚c. after cell adherence, pifn-γ 1 , pifn-γ 1 -his, pifn-γ 1 ', and pifn-γ 1 '-his were added to a final concentration of 10 μg/3 ml. then, the cells were collected, and total rna was extracted from the cells. total rna was extracted from ipec-j2 cells using the trizol method. rna integrity was checked on 1% agarose gels and quantified using nanodrop (thermo scientific, usa). after heating at 85˚c for 30 min to denature rna, 500 ng of total rna was subjected to reverse transcription using the revertra ace quantitative real-time pcr (qpcr) rt kit (toyobo, japan). expression of mrna was quantified with qpcr using a commercial reagent kit. for each of the targeted genes, a pair of oligonucleotide primers was designed using primer premier 5.0 software based on the sequences registered in the genbank database (genbank accession number: oas1: xm_021073680.1, mx1: nm_214061.2, pkr: ku212868, and actin: af216956). values for each target gene were normalized using actin. expression values were calculated using the 2 -44ct method. the copy numbers of the oas1, mx1, and pkr genes in each strain were estimated according to the published method with modifications. the actin sequence was used as an endogenous gene, while oas1, mx1, and pkr sequences were used as target genes. data were analyzed using the statistical analysis system (sas 9.1.3). differences in expression levels were investigated using one-way analysis of variance (anova). means of the values were compared using duncan's multiple comparison tests. a p value of <0.01 was considered significant. construction of expression plasmids. ppiczαa-pifn-γ 1 and ppiczαa-pifn-γ 1 -his tag in p. pastoris. costar software was used to optimize the encoding sequence of pifn-γ, and the optimized pifn-γ 1 gene (genbank: mh513659) was obtained by overlapping pcr (fig 1a) with a molecular weight of 500 bp. the optimized results were that the base sequence similarity between the optimized sequence and the original sequence was 70.35%, and the g/c value was 43.0%. the full-length pifn-γ 1 gene and pifn-γ 1 -his tag were obtained by overlapping pcr, and the plasmids were synthesized by sangon biotechnology. nucleotide sequencing analysis confirmed that the plasmids contained the correct gene. then, the recombinant strains containing pifn-γ 1 and pifn-γ 1 -his were generated by transformation of p. pastoris x33 with the linearized vector ppiczαa (fig 1b) . the molecular weight of the carrier was approximately 1000 bp. construction of 3' c-terminal signal peptide mutation expression plasmids ppiczαa-pifn-γ 1 ' and ppiczαa-pifn-γ 1 '-his tag in p. pastoris. the 129th and 131st arginine (r) of the previously optimized pifn-γ 1 gene signaling peptide were mutated to histidine (h), the peptide chain from positions 126 to 132 of the amino acid sequence was mutated from lrkrkrs to pkhkh, and the corresponding codon was mutated from aga and cgt to cat. two mutant secretory expression vectors were constructed, named ppiczαa-pifn-γ 1 ' and ppiczαa-pifn-γ 1 '-his tag. the plasmids were synthesized by sangon biotechnology. nucleotide sequencing analysis confirmed that the plasmids contained the correct gene. then, the recombinant strains containing pifn-γ 1 ' and pifn-γ 1 '-his tag were generated by transformation of p. pastoris x33 with the linearized vector ppiczαa (fig 1c) . the molecular weight of the carrier was approximately 1000 bp. the recombinant strains containing pifn-γ 1 , pifn-γ 1 -his tag, pifn-γ 1 ' and pifn-γ 1 '-his tag in p. pastoris x33 were induced in bmgy (48 h, 28˚c, 230 rpm), and the bacteria were collected. the expression of pifn-γ protein was detected by sds-page. data are shown in fig 2. the recombinant strains of pichia pastoris x33 containing pifn-γ 1 , pifn-γ 1 -his tag, pifn-γ 1 ' and pifn-γ 1 '-his tag all expressed pifn-γ. after pifn-γ gene optimization, the expression level of protein in pichia pastoris increased significantly compared with unoptimized pifn-γ (p<0.05), especially after the 3' c-terminal mutation optimized pifn-γ 1 (p<0.01). from the electrophoresis results (fig 2) , the molecular weight relationships of expressed pifn-γ proteins were as follows: pifn-γ 1 '-his tag> pifn-γ 1 '> pifn-γ 1 -his tag�pifn-γ 1 �pifn-γ (16.8 kda). the results showed that pifn-γ 1 -his tag could secrete pifn-γ protein in pichia pastoris, while western blot electrophoresis and nickel column purification could not detect the target protein (fig 3a and 3b) . however, western blot analysis successfully detected the complete pifnγ peptide chain secreted by pifn-γ 1 '-his tag in pichia pastoris (fig 3c) , which had significant differences compared with the pifn-γ 1 -his tag (p<0.01). purification on a ni-column after expression of the x33-pifn-γ 1 '-his tag yeast strain fermented with 1 mg/ml zeocin (fig 3d) was successful in obtaining the target protein pifn-γ, with a content of up to 536.4 mg/ml. from the above results, the expression of the pifn-γ gene in pichia pastoris was significantly increased after gene optimization and signaling peptide-amino acid mutation. shake flask fermentation at different temperatures in pichia pastoris. the strains were inoculated on bmgy containing 0.5% methanol, and the bacteria were collected at 30˚c, 25˚c, 20˚c and 15˚c. cells were lysed, the expression of pifn-γ protein was detected by sds-page, and the content of pifn-γ protein was detected by bca protein assay (fig 4a) . the effect of different temperature fermentations on the expression of pifn-γ in pichia pastoris showed that the expression of pifn-γ protein at 25˚c was significantly higher than at 15˚c and 20˚c (p<0.05), especially for pifn-γ 1 ' and pifn-γ 1 '-his tag. after 25˚c fermentation, the pifn-γ protein content of the recombinant yeast was up to 773.4 mg/l and 848.2 mg/ l, which was significantly higher than the protein produced by other fermentation temperatures (p<0.01). therefore, the recombinant pichia pastoris containing the optimized pifn-γ high fidelity pcr after optimization. m 3452 wide-type protein standard molecular weight marker, lane 1-2 for optimized pifn-γ 1 gene. (b) transformation of pifn-γ 1 and pifn-γ 1 -his tag in p. pastoris. m 3452 wide-type protein standard molecular weight marker, lane1-3 for ppiczαa-pifn-γ 1 -his tag, lane 4-6 for ppiczαa-pifn-γ 1 . (c) transformation of ppiczαa-pifn-γ 1 'and ppiczαa-pifn-γ 1 '-his tag in pichia pastoris. m for dl2000 marker, lane 1-2 for ppiczαa-pifn-γ 1 '-his tag, lane 3-5 for ppiczαa-pifn-γ 1 '. (pifn-γ 1 : a modified porcine gamma by yeastcodon preference; pifn-γ1-his tag:a modified porcine gamma (pifn-γ 1 )with adding histidine label; pifn-γ 1 ':a modified pifn-γ (pifn-γ 1 )with c-terminal amino acid mutation; pifn-γ 1 '-his tag:a modified pifn-γ (pifn-γ 1 )with c-terminal amino acid mutation and adding histidine label). https://doi.org/10.1371/journal.pone.0214319.g001 fig 2. protein expression of pifn-γ in p. pastoris. m 3452 wide-type protein standard molecular weight marker, lane 0 for pifn-γ, lane 1 for x33 strains, lane 2-3 for pifn-γ-his tag, lane 4-5 for pifn-γ protein in p. pastoris, lane 6-7, pifn-γ 1 ' protein in p. pastoris, lane 8-9, pifn-γ 1 '-his tag protein in p. pastoris. (pifn-γ: for unoptimized pifn-γ; pifn-γ 1 : a modified porcine gamma by yeastcodon preference; pifn-γ 1 -his tag: a modified pifn-γby yeast codon preference(pifn-γ 1 )with adding histidine label; pifn-γ 1 ': a modified pifn-γ (pifn-γ 1 )with c-terminal amino acid mutation; pifn-γ 1 '-his tag: a modified pifn-γ (pifn-γ 1 )with c-terminal amino acid mutation and adding histidine label). https://doi.org/10.1371/journal.pone.0214319.g002 enhance the production and biological activity of porcine interferon-γ gene with the 3' end signal peptide mutation obtained a higher yield of pifn-γ protein (cultivation for 0-24 h at 28˚c and then 24 h-72 h at 25˚c). fermentation with different do concentrations in pichia pastoris. the effect of different do concentrations on the expression of pifn-γ in pichia pastoris was increased with the increase in do concentration in a certain range, but when the concentration of do exceeded 40%, the expression was significantly reduced (p<0.01). the expression of pifn-γ protein was maximal when the concentration of do in the substrate was 30%, especially for the recombinant pichia pastoris with the optimized pifn-γ gene and the 3' signaling peptide mutation, which had significantly higher expression than that of the other treatment groups (p<0.05). after 25˚c fermentation, the content of protein of pifn-γ1' and pifn-γ1'-his tag were up to 773.4 mg/l and 848.2 mg/l in shaker fermentation tank, when the concentration of do in the substrate was 30%, the content of pifn-γ1' and pifn-γ1'-his tag protein reached 8912 mg/l and 9557 mg/l in fermentation tank. from the above results, it can be seen that with fermentation tank fermentation, compared with shaker fermentation, yeast secretion of the porcine interferon increased significantly, while for the shaker fermentation, secretion increased 10 times and yeast wet weight also increased approximately 30%. the best fermentation conditions were 28˚c, ph 6.0, and do 50% from 0 to 24 h followed by 25˚c, ph 6.0, and do 30%, 24 h-72 h. purified pifn-γ 1 , pifn-γ 1 -his, pifn-γ 1 ', and pifn-γ 1 '-his protein were added to the ipec-j2 cell culture solution at a final concentration of 10 μg/3 ml. the cells were cultured for 24 h at 5% co2 and 37˚c. then, the cells were collected, and total rna was extracted from the cells. oas1, mx1 and pkr gene expression was detected in ipec-j2 by qpcr after reverse transcription of the rna. the difference in oas1 expression levels after stimulation of different protein samples in ipec-j2 cells. the expression level of oas1 in the cells of each treatment group was significantly improved (p<0.05) (fig 5a) , especially in the pifn-γ 1 ' and pifn-γ 1 '-his tag treatment groups, which had 78.17 times and 71.74 times increased expression levels of oas1, respectively (p<0.01). meanwhile, pifn-γ 1 ', pifn-γ 1 '-his tag, pifn-γ 1 and pifn-γ 1 -his tag treatment groups had significantly higher expression of oas1 in cells than the control group (p<0.01). it was also found that the expression level of oas1 in the treatment groups, pifnγ 1 ' and pifn-γ 1 '-his tag, was significantly higher than that of pifn-γ 1 and pifn-γ 1 -his tag in the processing group by as much as two times (p<0.05), but the difference in the corresponding group was not obvious. the pifn-γ expressed in each optimized treatment group increased the expression of oas1 after 80˚c treatment for 30 min, which was significantly enhance the production and biological activity of porcine interferon-γ higher than that of the control group (p<0.05) but was significantly lower than that of the group without heat treatment. the difference in mx1 expression levels after stimulation of different protein samples in ipec-j2 cells. the expression level of mx1 in the cells of each treatment group was significantly improved (p<0.05) (fig 5b) , especially in the pifn-γ 1 ' and pifn-γ 1 '-his tag treatment groups, which had 61.39 times and 64.13 times increased expression levels of mx1, respectively (p<0.01). meanwhile, the pifn-γ 1 ', pifn-γ 1 '-his tag, pifn-γ 1 and pifn-γ 1 -his tag treatment groups had significantly higher mx1 expression than the group with 80˚c heating for 30 min (p<0.01). it was also found that the expression level of mx1 in pifn-γ 1 ' and pifn-γ 1 '-his of the tag treatment group was significantly higher than that of pifn-γ 1 and pifn-γ 1 -his in the tag processing group (p<0.05), but the difference in the corresponding group was not obvious. fig 5c, all recombinant expression of the pifn-γ protein was increased compared to the blank control, and the expression levels of pkr in the eight treatment groups were increased significantly compared to the blank control group (p<0.05), but there was no significant difference between each experimental group and its corresponding heat-treatment group. tianyuan et al., studies have found that when the pig interferon gene was transferred to the prokaryotic carrier, the expressed protein station is 40%, mainly in the form of inclusion body, and the purification activity is affected to a certain extent [24] . the pichia pastoris expression system is an excellent eukaryotic expression system developed in recent years: it has a strong aox promoter, an exogenous protein that has a certain function in translation modification, and the advantages of primitive nuclear biological molecular genetics and simple operation. to date, more than 1000 exogenous proteins [25] have been successfully expressed by the pichia pastoris expression system. however, there are some defects in the expression system of pichia pastoris: some exogenous proteins are easily degraded by pichia pastoris, there are still some exogenous proteins in the system that cannot be expressed or have low expression, and some exogenous proteins may have excessive glycosylation modification. a large number of studies have shown that the appropriate and targeted optimization of exogenous genes is of great significance for improving their expression level [26] [27] . sreekrishna et al. [28] found that the expression level of human serum albumin mrna was increased more than 50 times after it was optimized, similar to that of aox1 mrna. xu et al. [29] found that the xylanase gene optimized by a yeast-preferred codon could significantly improve its (a) effect of temperature on expression of pifn-γ protein in p. pastoris by shake flask fermentation. recombinant pifn-γ pichia pastoris, at 28˚c, ph 6.0, cultured 24 h, then 15˚c, 20˚c, 25˚c, 30˚c, ph 6 for 48 h, the results of the independent experiments were indicated as mean ±sd. � p<0.05 �� p<0.01 indicated significant difference vs. corresponding group (see "materials and methods"). (b) effect of do content of pifn-γ protein in p. pastoris by fermentation of 5l fermentation tank. recombinant pifn-γ pichia pastoris, at 28˚c, ph 6.0, 50% do cultured 24 h, then 25˚c, ph 6, 30% do, for the next 48 h, the results of the independent experiments were indicated as mean ±sd. � p<0.05 �� p<0.01 indicated significant difference vs. corresponding group (as shown in"materials and methods"). (c) effect of do content of pifn-γ protein in p. pastoris by fermentation of 5l fermentation tank (at 28˚c, ph 6.0, cultured 24 h, then 15˚c, 20˚c, 25˚c, 30˚c, ph 6 for 48 h). the figure showed the change of wet weight of yeast cells after different do fermentation. the results of the independent experiments were indicated as mean ±sd. � p<0.05 �� p<0.01 indicated significant difference vs. corresponding group(as shown in"materials and methods").(pifn-γ 1 : a modified porcine gamma by yeast codon preference; pifn-γ 1 -his tag: a modified porcine gamma by yeast codon preference (pifn-γ 1 )with adding histidine label; pifn-γ 1 ': pifn-γ 1 with c-terminal amino acid mutation; pifn-γ 1 '-his tag: pifn-γ 1 with c-terminal amino acid mutation and adding histidine label). https://doi.org/10.1371/journal.pone.0214319.g004 enhance the production and biological activity of porcine interferon-γ expression level. hu et al. [30] studied the expression of t-cell immune toxin by pichia pastoris and found that the cdna sequence was rich in a/t bases, which led to the early termination of transcription. after the a/t content and distribution were adjusted by codon optimization, the target protein was successfully expressed. in this study, the gene sequence optimization software developed by the state key laboratory of pest control and resource utilization at sun yat-sen university, the costar optimization tool, was combined with the optimization software eugene, the mrna local energy and the pichia pastoris codon optimization table to comprehensively reconstruct the pifn-γ coding sequence. there was a certain amount of exogenous protease expression inside and outside of the cells of the host bacterium pichia pastoris, so that most exogenous proteins were subject to degradation during both intracellular expression and secretory expression, which was also an important factor affecting the expression. degradation led to a decrease in the yield of the target protein, and the fragments formed by degradation also caused great difficulty in separation and purification, especially for the degradation products with small differences in the molecular weight of the target protein. because of their physical and chemical properties, the conventional separation method was very difficult to use for their separation, resulting in greatly reduced product yield, low product purity, and decreased activity of the protein [31] . the intracellular protease mainly involves the degradation of protein precursors to produce active proteins, and the removal of protein signaling peptides after the transfer to the membrane leads to protein inactivation. by analyzing the c-terminal sequence of the pifn-γ protein, it was found that the lrkrkrs sequence of the c-terminal amino acid is similar to the signal peptide sequence (ekreaeae) and is easily enzymolysed by the signal peptide enzyme, resulting in degradation and inactivation of the pifn-γ protein. therefore, we mutated the arginine at positions 129 and 131 of the c-terminal end of the pifn-γ protein to histidine (lrkrkrs mutation to rkhkh), thereby allowing the pifn-γ protein to be secreted without degradation, and we added a histidine tag at the end of the c-terminus for easy purification and further research. the temperature had an effect on the activity of yeast fermentation product, the physical properties of fermentation broth and the direction of biosynthesis [32] . the culture temperature of pichia pastoris is suitable between 28~30˚c, but 28˚c is the optimum temperature for yeast body growth. it was found that the proper temperature reduction of pichia pastoris is beneficial to the secretion of exogenous proteins, which may be due to the high temperature formation of disulfide bonds, which altered the structure of target proteins and seriously affected protein activity and yield [23] . at the same time, dissolved oxygen in the fermentation substrate was very important for the growth of aerobic microorganisms. in the process of fermentation, especially for pichia pastoris, ensuring that the supply of oxygen met the demand for oxygen was an important factor for improving the fermentative yield. in earlier studies, the the expression level of pkr in blank control cells was set as a reference. the results of the independent experiments were indicated as mean ±sd. � p<0.05 �� p<0.01 indicated significant difference vs. control group (as shown in "materials and methods"). (pifn-γ 1 : a modified porcine gamma by yeast codon preference; pifn-γ 1 (h): pifn-γ 1 with 80˚c heating treatment for 30 min; pifn-γ 1 -his tag: a modified porcine gamma by yeast codon preference (pifn-γ 1 ) with adding histidine label; pifn-γ 1 (h)-his tag: pifn-γ 1 -his tag with 80˚c heating treatment for 30 min pifn-γ 1 ': pifn-γ1 with c-terminal amino acid mutation; pifn-γ 1 '(h): pifn-γ 1 ' with 80˚c heating treatment for 30 min; pifnγ 1 '-his tag: pifn-γ 1 with c-terminal amino acid mutation and adding histidine label; pifn-γ 1 '-his tag(h): pifn-γ 1 '-his tag with 80˚c heating treatment for 30 min). https://doi.org/10.1371/journal.pone.0214319.g005 enhance the production and biological activity of porcine interferon-γ expression level of pifn-γ in pichia pastoris was 108 mg/l. compared with that, the expression level of pifn-γ in this study was up to 9557 mg/l [33] [34] . by using a fermentation tank for large-scale fermentation, the expression level of exogenous protein could be increased 10~100 times that of a common shaker because the ventilation of flask fermentation was not ideal, and the density of fungal growth and the expression of exogenous protein were strongly affected. it was found that in the process of large-scale fermentation of pichia pastoris, if the do is less than 20%, it will affect growth and metabolism. if the do is more than 60%, the cells will have oxygen poisoning. therefore, the effective control of do in large-scale fermentation is very important for the expression of exogenous proteins [21] . through our study on the fermentation conditions of gradient temperature and gradient do, it was found that the recombinant pichia pastoris strain could obtain a high yield of pifn-γ protein after stepwise variable temperature fermentation (0-12 h incubation at 28˚c and 50% do and 24 h-72 h of incubation at 25˚c, ph 6.0 and 30% dissolved oxygen, up to 9536 mg/l). meanwhile, analysis of porcine interferon activity by qpcr showed that the expression of the oas1 and mx1 genes in ipec-j2 cells could be significantly improved by the pifn-γ gene c-terminal mutation in 4 strains. the target proteins upregulated by the c-terminal peptide-specific amino acid mutations are more likely to be stimulated by the cells. we speculated that the integrity of the c-terminus of the pifn-γ peptide chain is important for the formation or maintenance of the pifn-γ spatial two-polymer structure. the structure of the c-terminal end was not complete, which resulted in the decreased activity of pifn-γ. after the c-terminal mutation of the pifnγ sequence, the secreted expression of pifn-γ protein contained a complete peptide chain structure and maintained high biological activity. meanwhile, we also found that the pifn-γ protein expressed by gene optimization was still active after high temperature treatment, indicating that the c-terminal mutation strategy is beneficial to the maintenance of the stability and biological activity of the pifn-γ protein structure. in conclusion, in this study, pichia pastoris was used as the expression system, the optimized pifn-γ gene sequence was used as an inserting fragment, and high copy secretory expression strains of pifn-γ 1 , pifn-γ 1 -his tag and pifn-γ 1 ' and pifn-γ 1 '-his tag pichia pastoris were constructed successfully. the functional differences between 4 different proteins were compared at the cell level to analyze their stimulating activity, and the processing mechanism and optimal expression strategies of exogenous proteins secreted in pichia pastoris were studied. the results showed that the c-terminal signaling peptide gene mutation and the addition of a histidine tag in pifn-γ resulted in a highly expressed pifn-γ protein that could be obtained in pichia pastoris with higher bioactivity. it was also shown that when recombinant pichia pastoris pifn-γ was cultured for 0-24 h at 28˚c and 50% do, and then for 24-72 h at 25˚c and 30% do, higher secretion (up to 10 times compared to shake flask fermentation) was achieved while maintaining good living conditions. this article does not contain any studies with human participants or animals performed by any of the authors. bats are natural reservoirs of sars-like coronaviruses anti-human immunodeficiency virus activity of tau interferon in human macrophages: involvement of cellular factors and betachemokines the nature of the principal type 1 interferon-producing cells in human blood identification and sequence of an accessory factor required for activation of the human interferon gamma receptor comparison of antiviral activities of porcine interferon type i and type ii the role of ifn-gamma in immune responses to viral infections of the central nervous system mx proteins: gtpases with antiviral activity interferon-induced mx proteins: dynamin-like gtpases with antiviral activity the eif2alpha kinases: their structures and functions research progress of porcine interferon. swine industry molecular 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bioanalysis sheng wu gong cheng xue bao = chinese journal of biotechnology fusion expression and bioactivity comparison of porcine beta-defensin-2 and porcine interferon-gamma in pichia pastoris sheng wu gong cheng xue bao = chinese journal of biotechnology sheng wu gong cheng xue bao = chinese journal of biotechnology key: cord-255576-738khdwv authors: van duyne, rachel; guendel, irene; klase, zachary; narayanan, aarthi; coley, william; jaworski, elizabeth; roman, jessica; popratiloff, anastas; mahieux, renaud; kehn-hall, kylene; kashanchi, fatah title: localization and sub-cellular shuttling of htlv-1 tax with the mirna machinery date: 2012-07-10 journal: plos one doi: 10.1371/journal.pone.0040662 sha: doc_id: 255576 cord_uid: 738khdwv the innate ability of the human cell to silence endogenous retroviruses through rna sequences encoding micrornas, suggests that the cellular rnai machinery is a major means by which the host mounts a defense response against present day retroviruses. indeed, cellular mirnas target and hybridize to specific sequences of both htlv-1 and hiv-1 viral transcripts. however, much like the variety of host immune responses to retroviral infection, the virus itself contains mechanisms that assist in the evasion of viral inhibition through control of the cellular rnai pathway. retroviruses can hijack both the enzymatic and catalytic components of the rnai pathway, in some cases to produce novel viral mirnas that can either assist in active viral infection or promote a latent state. here, we show that htlv-1 tax contributes to the dysregulation of the rnai pathway by altering the expression of key components of this pathway. a survey of uninfected and htlv-1 infected cells revealed that drosha protein is present at lower levels in all htlv-1 infected cell lines and in infected primary cells, while other components such as dgcr8 were not dramatically altered. we show colocalization of tax and drosha in the nucleus in vitro as well as coimmunoprecipitation in the presence of proteasome inhibitors, indicating that tax interacts with drosha and may target it to specific areas of the cell, namely, the proteasome. in the presence of tax we observed a prevention of primary mirna cleavage by drosha. finally, the changes in cellular mirna expression in htlv-1 infected cells can be mimicked by the add back of drosha or the addition of antagomirs against the cellular mirnas which are downregulated by the virus. human t-lymphotropic virus type 1 (htlv-1) was originally discovered in 1980, identified as the first human retrovirus, and currently infects more than 20 million people worldwide [1] [2] [3] [4] [5] . htlv-1 is the etiologic agent of adult t-cell leukemia/lymphoma (atll) and htlv-1-associated myelopathy/tropical spastic paraparesis (ham/tsp) in infected individuals. oncogenesis is due primarily to the viral transactivator protein, tax, a 40-kda phosphoprotein that regulates not only viral transcription, but acts to manipulate host cellular functions such as cell cycle progression, apoptosis, chromatin remodeling, and other signal transduction pathways [6] [7] [8] . recently, much interest has developed in elucidating the cross-talk between tumor development and htlv-1 infection as it relates to the innate host response, in particular the small rna regulatory network. human microrna (mirna) sequences derived from the genome have the ability to silence cellular genes and are currently considered a primary host immune defense against cellular invaders such as pathogens and viruses. in a host cell, mirnas are the product of the rna interference (rnai) pathway, a regulatory and innate defense mechanism that is conserved in eukaryotes. this pathway utilizes short non-coding rna sequences of 18-21 nucleotides to bind to mrna sequences with complementary homology, subsequently restricting the translation of these transcripts [9] . following rna pol ii transcription of a gene, the pri-mirna consists of a series of rna hairpins protruding from an rna message with a 59cap and poly-a tail. this pri-mirna is cleaved by a microprocessor complex of nuclear proteins, drosha, an rnase ii endonuclease, and dcgr8 (pasha), an rna-binding protein, to form a stem and loop rna-structure called pre-mirna. this pre-mirna is shuttled out of the nucleus into the cytoplasm via exportin5 and is further processed by an additional rnase iii enzyme, dicer, which cleaves the hairpin into a short mirna duplex. these mirna then associate with the rna induced silencing complex (risc), composed of ago2 and trbp proteins, which then aids in mirna-mediated target recognition. this guide effector protein complex assists in either degrading the targeted message or preventing its translation [10] . the dysregulation of this pathway is highly evident across a variety of cancers and viruses, including hiv-1, htlv-1, influenza, hcv, ebola, vaccinia, pfv-1, lacv, adenovirus, and sars-cov although the mechanisms of action for most of these viruses remains to be determined [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] . indeed, cellular mirnas are able to silence endogenous retroviruses, sequences which typically share a high degree of homology to present day retroviruses, such as htlv-1 and hiv-1. htlv-1 tax acts to transactivate the viral long terminal repeat (ltr) through tax-responsive elements (tres) in the u3 region. this occurs through transcriptional induction of tres, posttranslational modifications of tre-binding factors, and binding with transcription factors. tax is known to interact with the transcription factors creb, serum-responsive factor (srf), and nf-kb as well as with the cell cycle related proteins cyclin d2 and d3, mitotic checkpoint regulators (mad1), cyclin-dependent kinases (cdks), cdk inhibitors p16 ink4a and p21/waf1, and p53 [5, [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] . phosphorylation of tax is necessary for tax localization in nuclear bodies as well as activation of cellular gene expression through the nf-kb pathway. tax activates htlv-1 transcription through creb and three cre enhancer sequences on the ltr. tax also interacts with cbp/p300, which is involved in the formation of the preinitiation complex as well as the p300/cbp-associated factor, p/caf, which also plays a role in active htlv-1 gene transcription [37, [44] [45] [46] . taken together, htlv-1 tax is heavily involved in direct interactions with critical cellular proteins that control not only viral gene expression but also cellular genes involved in tumor formation. we have previously shown that tax interacts directly with the cellular rb (retinoblastoma) protein and targets rb for degradation via the proteasome pathway, resulting in a decrease in rb protein expression in htlv-1 infected cells and a dysregulation of the cell cycle [47] . due to the nature of tax to manipulate and control cellular proteins such as rb and overall cell cycle progression, we became interested in determining the effect of htlv-1 infection and tax expression on other cellular proteins regulating oncogenesis, including the rnai pathway. htlv-1 encodes an additional regulatory protein, called rex, which has recently been implicated in having rna silencing suppressor activity. specifically, rex interacts with both rna and dicer, suppressing dicer's enzymatic activity [15] . recently, tax has been shown to specifically mediate the downregulation of cellular mirnas which are associated with the regulation of chromatin remodeling factors [48] . specifically, tax downregulated mir-149 and mir-873, both of which directly target p300 and p/caf [48] . this suggests that htlv-1 infection and its viral transactivators may play a role in dysregulation of the rnai pathway. here we show that htlv-1 infection in the presence of tax, significantly downregulates drosha protein expression. we show the in vitro interaction between tax and drosha, both in the nucleus and in complex in the presence of proteasome inhibitors. we observe a downregulation of drosha functionality in the presence of tax as measured by primary mirna cleavage. finally, the changes in cellular mirna expression in htlv-1 infected cells can be mimicked in uninfected cells supplementing excess functional drosha or the addition of antagomirs against the cellular mirnas which are downregulated by the virus. tax acts as the htlv-1 viral transactivator protein by interacting with various cellular and viral proteins including transcription factors, kinases, and chromatin remodeling proteins, ultimately altering cellular pathways such as apoptosis and the cell cycle to control viral replication and oncogenesis [6] [7] [8] . recently, retroviruses have been shown to control the cellular rnai pathway in order to evade viral inhibition and also to hijack both the enzymatic and catalytic components of the mirna machinery. this viral response assists in promoting an active viral infection, or a latent state of infection [11, [48] [49] . indeed, other retroviruses such as hiv-1 have been shown to impart a specific suppression of dicer expression in monocyte-derived-macrophages as compared to t-cells [50] . in order to examine the role htlv-1 infection has on possible control of the rnai pathway, we initially screened for the protein levels of core components of the rnai machinery in htlv-1 infected cells as compared to uninfected cells. total protein levels of drosha, dgcr8, dicer, ago2, and b-actin were detected in three uninfected t-cell lines, h9, jurkat, and cem, as well as in three htlv-1 infected, taxpositive t-cell lines, c81, mt2, and mt4 ( figure 1a , lanes 1-3 and lanes 4-6). drosha protein levels were significantly downregulated in all of the htlv-1 infected, tax-positive cells tested when compared to the uninfected cells. specifically, drosha is decreased by 62.6% in c81 cells, 97.3% in mt2 cells, and 91.5% in mt4 cells when compared to cem cells (normalized to b-actin). dicer protein was also slightly downregulated in c81 cells, but more so in mt2 cells when compared to cem cells (normalized to b-actin). conversely, dgcr8 and ago2 showed no significant changes in protein level amongst all cell lines screened, suggesting that the downregulation seen for drosha and dicer is specific and not a consequence of the entire pathway being inhibited. interestingly, the protein levels of the core components of the rnai machinery were also detected in two htlv-1 infected, tax-negative cell lines ( figure 1a, lanes 7, 8) . drosha was downregulated by 77.1% in mt1 cells and 51.3% in ed-cells as compared to cem cells (normalized to b-actin). we titrated both mt1 and ed-cells in order to determine if this downregulation was an artifact and indeed, with increasing concentrations of protein extract (50, 75 , and 100 mg), drosha levels also increase ( figure 1b) , suggesting that the relative decrease in drosha is less in htlv-1 infected, tax-negative cells as compared to tax-positive cells. we reproduced these results in htlv-1 infected cd4 + primary t-cells ( figure 1c) , where drosha levels are dramatically downregulated as compared to uninfected cells (lane 2 compared to lane 1). in order to rule out the possibility that the loss of drosha protein is due to a decrease in transcript levels, we performed an rt-pcr of total mrna isolated from cem, mt2, ed-, and mt1 cells for drosha transcripts. in figure 1d , we show that the drosha transcript levels are unchanged as compared to gapdh across all four of these cell lines. next, we attempted to mimic this downregulation of drosha and dicer by introducing both tax and htlv-1 into uninfected 293t cells and screening for core rnai machinery components. 293t cells were transfected with three concentrations (0.1, 1.0, 10 mg) of plasmids expressing pach (full-length htlv-1 clone), pctax, pach.m22 (mutated tax gene), or an empty vector control. the pach.m22 plasmid contains a double amino acid substitution at positions 130 and 131 and eliminates the ability of tax to transactivate the nf-kb pathway [51] [52] [53] [54] . cells were collected 48 hours post-transfection and were western blotted for the presence of drosha, dicer, ago2, ago1, tax and b-actin. figure 1e shows endogenous protein levels of core rnai machinery components in 293t cells transfected with pcdna and other vectors. comparing drosha proteins levels in cell expressing pach, pctax, and pach.m22 to the pcdna control (lanes 6, 7, 8 and 9, 10, 11 and 12, 13, 14 to lanes 2, 3, 4, respectively) results in a 27.5% decrease in pach (10 mg) expressing cells (lane 8), a 67.3% decrease in pctax (10 mg) expressing cells (lane 11) and only a 10.4% decrease in pach.m22 (10 mg) expressing cells. these results imply that in the pach.m22 mutant tax expressing cells, we observe close to endogenous levels of drosha, indicating the downregulation of drosha may be the result of a functional tax. overall, relative expression levels of dicer, ago2, and ago1 reproduce the data from figure 1a . the tax western blot serves as a positive control for tax expression (lanes 2 and 6-14). the b-actin western serves as a positive loading control. again we confirmed that this decrease in drosha protein levels is due to tax expression and not due to an effect on total transcript levels ( figure 1f ). collectively, these data indicate that htlv-1 infection in the presence of tax downregulates the rnai enzyme drosha in chronically infected cell lines, transfected cells, and primary cells infected with htlv-1 virus. the viral protein tax has been well documented as interacting directly with multiple critical cellular proteins that control oncogenesis. we sought to determine whether or not tax interacts with drosha in order to downregulate and/or signal the drosha protein for possible degradation. to investigate the potential interaction of tax and drosha, we transfected pctax into hela cells and stained the cells 48 hours post-transfection for confocal imaging. we labeled both untransfected and tax-transfected hela cells with antibodies against brg1, drosha, rb, git2, and tax. brg1 and rb serve as positive tax-interacting proteins as we have previously shown their intracellular interaction [1, 47] . brg1 is constitutively expressed in hela cells in the absence of tax, however post-tax transfection, brg1 is found almost exclusively in the nucleus and colocalizes with tax ( figure 2a) . similarly, rb is heterogeneously expressed in hela cells, however colocalizes with tax in the nucleus of tax-expressing cells ( figure 2d ). interestingly, in tax expressing cells, drosha is almost exclusively found in the nucleus at specific foci colocalizing with tax ( figures 2b, c) . in order to address possible nonspecific shuttling between the nucleus and the cytoplasm due to transfection conditions or overexpression of tax, we utilized git2 to serve as a negative control for a protein that does not shuttle to the nucleus or colocalize in the presence of tax ( figure 2e ). therefore, our imaging data show that tax and drosha colocalize in the nucleus of tax-expressing cells. the full length tax protein contains a variety of domains that impart specific protein-protein interactions as well as interactions with sub-cellular components. the n-terminus of tax contains both a nuclear localization signal (nls) as well as a zinc-finger domain which is involved with interactions with cellular transcription factors [31, 49, [55] [56] [57] . specifically, this domain of tax is responsible for tax-mediated creb transactivation. the c-terminus of tax is responsible for nuclear-cytoplasmic shuttling and contains a golgi localization motif, a secretion motif, an lxcxe-like motif, and a pdz-binding domain [5, 47, [58] [59] [60] [61] [62] [63] [64] . additionally, the region of tax between the n-and c-termini includes two leucine zipper motifs used for dimerization as well as activation of nf-kb [5, [58] [59] [60] [61] . we have previously shown that tax is capable of binding to cellular rb via its lxcxe-like motif [47] . to identify the domain of tax that binds to drosha, we performed an in vitro binding assay using full-length tax and various fragments covering the entire tax protein fused to gst. figure 3a shows the results of a gst-tax pulldown from hela whole cell extracts, followed by a western blot for the presence of drosha. drosha bound to wild type tax, but much stronger binding was observed at the n-terminal domain that contained sequence 1-244. this is partly because the c-terminus may fold back and contain inhibitory activity in an in vitro binding assay. we have also confirmed the interaction of tax and drosha in vitro with an immunoprecipitation (ip). we transfected 293t cells with the htlv-1 clone pach.tax (5 mg) or pach.m22 (5 mg) mutant tax in the presence of the proteasome inhibitor psi (10 mm). our rational for using a proteasome inhibitor was to be able to isolate complexes that were available for biochemical analysis prior to the targeted degradation via the proteosome pathway. we collected cells 48 hours post transfection and lysed whole cell extracts (,1 mg) for an overnight ip with a-tax, a-drosha, or a-igg antibodies. the following day, protein a + g beads were incubated with the extracts for 2 hours, the beads were washed with low salt buffer, proteins eluted in laemmli buffer, and were subsequently western blotted for the presence of drosha. as shown in figure 3b , drosha can be successfully iped out of cells expressing wild type tax with a-tax in the presence of a proteasome inhibitor (lane 5). 293t cells alone serve as a positive control for the drosha western blot (lane 1) as well as a positive control for the drosha ip/western blot (lane 3). interestingly, the mutant ach.m22 tax is unable to interact with drosha (lane 7). this tax mutant is only faintly ubiquitinylated and can't interact with the proteasome, therefore indicating that the interaction of tax and drosha and the subsequent degradation requires interaction with the proteasome [65] . the a-drosha antibody used for the western blot recognizes all three isoforms of drosha, corresponding to the molecular weights of 170, 156, and 115 kda, which accounts for the multiple drosha bands. however, the strongest tax binding is to the 170 kda form. a ubiquitin western tax-negative (ed-, mt1 ) t-cell lines were screened for endogenous protein levels of drosha, dgcr8, dicer, and ago2. seventy-five micrograms of total lysates were used for western blots. multiple bands for dgcr8 are indicative of isoforms at 86 and 65 kda. b-actin serves as a loading control. b) total lysates of htlv-1 infected, tax-negative cell lines ed-and mt1 were titrated (50, 75 and 100 mg) and screened for endogenous protein levels of drosha, dgcr8, dicer, and ago2. multiple bands for dgcr8 are indicative of isoforms at 86 and 65 kda. b-actin serves as a loading control. c) whole cell lysates (50 mg) from htlv-1 infected primary cd4 + t-cells were run on a 10% tris-glycine sds-page gel and western blotted for drosha. data is representative of three independent experiments. b-actin serves as a loading control. d) total rna was isolated with trizol from cem, mt2, ed-, and mt1 cell lines. rt-pcr was performed for cellular drosha transcript levels. gapdh serves as a housekeeping gene loading control. e) uninfected 293t cells were transfected with pcdna (0.1, 1.0, and 10 mg), pach (0.1, 1.0, and 10 mg), pctax (0.1, 1.0, and 10 mg), or pach.m22 (0.1, 1.0, and 10 mg) and were screened for protein levels of drosha, dicer, ago2, ago1, and tax. seventy-five micrograms of total lysates was used for western blots. b-actin serves as a loading control. f) total rna was isolated with trizol from the transfected cells in panel e. rt-pcr was performed for cellular drosha transcript levels. gapdh serves as a housekeeping gene loading control. doi:10.1371/journal.pone.0040662.g001 blot is included as an indication that drosha is being ubiquitinated in the presence of tax, however, not in the presence of a tax mutant (compare lane 5 to lane 7). a summary of relative drosha binding affinity to tax constructs from multiple experiments is depicted in figure 3c . based on these data, tax binds to drosha via the n-terminus (aa 1-244), given that the strongest binding was shown to occur within this region, and can also be identified in complex with drosha. collectively, these data indicate that tax and drosha interact and can be found in complex in vitro. previously, we have shown that the binding of tax and rb results in a targeted degradation of rb via the proteasome pathway [47] . to investigate whether tax utilizes a similar mechanism to decrease drosha protein levels, we designed experiments using proteasome inhibitors. we treated htlv-1 infected c81 cells with proteasome inhibitors psi and alln (0.1, 1.0, 10 mm). at 48 hours post-treatment, the cells were collected and western blotted for the presence of drosha. figure 3d shows the western blot of drosha normalized to b-actin. both psi and alln treated cells showed an increase in drosha protein levels concurrent with increasing concentration of proteasome inhibitor. indeed, the 10 mm-treated cells show almost a 30% increase in drosha protein levels compared to c81 cells alone. all treated cells exhibited no toxicity across three independent experiments (data not shown). this data further indicates that drosha protein levels can be rescued from suppression by htlv-1 infection with proteasome inhibitors, suggesting that drosha is targeted by tax to be degraded via the proteasomal pathway. collectively, these data point to the regulation of drosha by direct binding to the nterminus of tax and further degradation by the proteasomal pathway. we have shown that tax and drosha interact in vitro and that this complex results in a downregulation/degradation of the cellular drosha protein. this suppression of drosha is not completely efficient however, as there is still some endogenous drosha remaining in htlv-1 infected cells. here we question whether the remaining drosha is functional in tax expressing cells. we utilized a system where we quantify levels of cellular mir326 in tax transfected cells normalized to a u6 control. we then calculate drosha functional efficiency as a measure of the relative level of mirna present versus the relative level of primary transcript, where 100% is the rate of production in the control. to this end, we transfected 293t cells with expression vectors (1 mg) for wildtype tax or the following iterative tax deletion mutants: td1 (d1-37), td55 (d55-92), td99 (d99-142), td150 (d150-198), td254 (d254-287), or td319 (d319-353). we collected the cells 48 hours post-transfection and isolated total rna by trizol extraction. the expression of mir326 was determined by the quantimir pcr kit and is shown as a fold change in figure 4a normalized to u6 and relative to mock transfected control. this data indicates that the tax d1 mutant and the taxd55 mutant increase mir326 expression 1.5 and 4 fold, respectively, above basal levels. interestingly, the tax mutants spanning from residue 99 to 353 have no functional implications for mir326 expression. these mutants are as functional as the tax wt, indicating that these regions of deletion are not critical for drosha function. the significance of the tax d1 and d55 mutants therefore implies that the regions of tax deleted in these mutants are necessary for drosha interaction as well as suppression of function. in order to compare the increase in expression of mir326 to the overall transcript levels in the cell, therefore ruling out non-specificity, we detected primary transcripts encoding mir326 against a region upstream of the mirna hairpin. these values were normalized to gapdh and are shown in figure 4b as a ratio of the relative level of mirna present over the relative level of primary transcript. the mock transfected cells were set to 100% efficiency. this data indicates an increase in drosha efficiency of 100% over the mock transfected cells with the tax d1 mutant. the tax wt vector decreased drosha functionality by 25% as compared to the control. it is interesting that the tax d1 mutant increased the efficiency of processing of drosha 100%, however, only increased the relative mir326 expression 1.5 fold. these two values are not necessarily proportional as the extrapolation of an active, functional drosha in the presence of this mutant, does not necessarily have to result in a direct effect on mir326. in figure 4c we represent a more detailed schematic of the binding domains and motifs of tax. we depict important binding and interactive regions of tax with brackets, including the numbered residues. we also depict the cellular proteins with which tax interacts below each sequence of interest. we propose that drosha is binding to the n-terminus of tax, however, more specifically to the region of tax from 1-92, depicted as the grey shaded area. we have previously confirmed that tax interacts with the proteasome at residues 23-62, which is also the site of the nls, zn finger, as well as the domain involved in creb activation [47] . this data agrees with the interactions of tax and drosha in figure 3 , where the n-terminus of tax is responsible for drosha binding. collectively, these data indicate that the drosha in tax-containing and htlv-1 infected cells is mostly functionally inactive and the functional suppression of drosha is dependent on its interaction with a small region of the n-terminus of tax. we have shown above that drosha is downregulated, degraded, and mostly inactive in htlv-1 infected cells, however, it was not clear what effect this dysregulation of drosha would have on viral replication. here we investigated whether loss of cellular drosha by sirna knockdown could affect viral replication. in doing this, it is important to distinguish between a drosha-specific dysregulation as opposed to an overall disruption of the rnai pathway, therefore we used sirna against other rnai components as a control. we transfected 293t cells with htlv-1 infectious pach clone (5 mg), and 24-hours post transfection, with sirnas against luciferase (150 nm), drosha (50, 150, 300 nm), dgcr8 (50, 150, 300 nm), and ago2 (50, 150, 300 nm). at 72hours posttransfection, we collected cell culture supernatants and assayed for the presence of virus using an rt (reverse transcriptase) assay. results of such an experiment are shown in figure 5a . here we observed that pach clone was able to replicate well with the highest concentration of sidrosha and even more dramatic results were obtained with sidgcr8. interestingly, the loss of dgcr8 across all three concentrations of sirna resulted in a consistent activation of viral replication. a knockdown of ago2 did not result in a significant increase in viral replication. western blot confirmation of the sirna knockdowns are shown in figure 5b . in order to determine whether or not the inhibition of ago2 would result in a decrease in viral fitness and an inhibition of replication, we adopted an alternative approach and treated 293t cells transfected with pach with the risc/ago2 inhibitor acriflavine (acf) (0.1, 1.0, and 2.5 mm) [66] . at 72 hours post transfection, we collected supernatants and assayed for the presence of virus via rt assay. we observed that that the efficient, drug inhibition of ago2 function decreases viral replication at levels comparable to siago2 transfection. collectively, these data indicate that the loss of drosha and dgcr8 in htlv-1 infected cells results in an increase in viral replication and its release from the cell. we were next interested in the regulation and downstream effects of the rnai pathway in htlv-1 infected cells. htlv-1 infection results in a dramatic up (i.e. mir-130b, mir-18a, mir-20b) and downregulation (i.e. let-7i, mir-132, mir-199a) of many host cellular mirnas [67] . the modulation of these host mirnas has an effect on the expression of cellular proteins such as p300, nf-kb, and brm, to name a few, which are all recruited by tax and play a key role in activating htlv-1 gene transcription [5, [30] [31] [32] [33] [34] [35] [36] [38] [39] [40] [44] [45] 56, 60, [68] [69] [70] [71] . to better define the functional significance of drosha downregulation by tax, we transfected 293t cells with pach.tax (5 mg) and 24 hours later added back pgfp-drosha (10 mg). the cells were collected 72 hours later and were lysed (50 mg) for western blots of the corresponding downstream proteins, ikk-b, brm, and b-actin. here the rationale was that tax would decrease drosha levels, resulting in downregulation of mirna, such as let7i, mir-199a-3p and mir-132. downregulation of these mirnas would in turn effect the translation of mrnas for genes such as ikk-b and brm (possibly regulated by mir-199a-3p) and other proteins important for htlv-1 gene expression. data in figure 6a shows that transfection of tax downregulates both ikk-b and brm as compared to the control drosha add back experiment (compare lanes 2 and 3). on the other hand, the evidence of drosha downregulation in the presence of tax implies the downregulation of cellular mirnas in response to htlv-1 infection and subsequent increase in target protein expression. of particular interest are a set of cellular mirnas, such as let-7i, mir-199a-3p, and mir-132 that are downregulated after infection, resulting in an upregulation of cellular proteins that are known to be recruited and utilized by the htlv-1 promoter. in order to mimic the effect tax has on cells by sequestering the endogenous mir, therefore allowing for the basal, unchecked expression of these cellular proteins, we transfected 293t cells with either tax or an antagomir against endogenous cellular mirs which are downregulated with htlv-1 infection. antagomirs used include antihsa-let-7i to target p50 and p65 mirna, anti-hsa-mir-132 to target gsk-3b mirna, and anti-hsa-mir199a-3p to target ikkb and brm mirna. cells were collected 72 hours post transfection and western blots were performed for ikk-b, p65, p50, gsk-3b, and b-actin. results in figure 6b are shown as densitometry counts of western blots for each indicated protein normalized to b-actin, plotted as a percent change (measured as arbitrary counts) between antagomir treated cells and 293t cells alone. these results indicate that there was a dramatic increase of protein expression of ikk-b, and p65 of approximately 300 and 250%, respectively. there was an approximate 10% increase in protein levels of p50, gsk-3b and brm upon antagomir treatment as compared to cells alone. collectively, these data indicate that proteins, such as ikk-b, among others, may directly be regulated by the tax/drosha interaction in htlv infected cells. htlv-1 is an oncogenic retrovirus which targets and manipulates common cellular tumor suppressors and signaling pathways in order to establish and maintain a productive infection. recently, the role of mirnas in both tumor development and viral infections has provided evidence that the rnai pathway itself is also targeted by this virus. indeed, all dna tumor viruses, with the exception of human papillomaviruses, encode viral mirnas which modulate tumorigenesis, therefore indicating that the cellular rnai regulatory pathway is important for viral pathogenesis and control. for example, epstein barr virus (ebv) encodes 25 viral mirna precursors which are expressed differentially amongst various stages of latency and act to suppress chemokines, inhibit the viral dna polymerase balf5, and downregulate the cellular protein puma (p53 upregulated modulator of apoptosis) [72] [73] [74] [75] . this complex interaction network between viruses and the rnai pathway can be best described as reciprocal due to host cell mirnas targeting both cellular and viral transcripts as well as viral mirnas targeting both cellular and viral transcripts. additionally, viruses can produce rna molecules and express proteins which disrupt the cellular rnai pathway, resulting in either induction or suppression. ebv induces the expression of the cellular mir-155 which assists in the expansion of infected b-cells and promotes their transformation whereas hiv-1 generates both rna sequences and proteins which suppresses the rnai pathway. indeed, hiv-1 has been shown to not only encode its own tar (trans-activation response region)-derived mirna which acts as an rnai decoy, but it also utilizes the viral transactivator tat as an rnai suppressor [14, [21] [22] [76] [77] [78] [79] [80] . in this study, we characterize the effect htlv-1 infection has on the cellular rnai pathway. we have demonstrated that drosha is downregulated in htlv-1 infected cell lines, htlv-1 transfected cells, and infected primary cells. we have also shown that dicer is downregulated in htlv-1 infected cell lines only, suggesting that this loss of dicer could be due to a cell line specific phenomenon and not so much htlv-1 infection or early stage replication. (d319-353) . a) forty-eight hours post transfection, total rna was isolated by trizol extraction and expression of mir326 was determined by quantimir pcr kit using primers specific to mir326. values shown are normalized to u6 and shown relative to control. b) primary transcripts encoding mir326 were detected by rt-qpcr against a region upstream of the mirna hairpin. quantities were normalized to gapdh and efficiency of drosha processing was determined by charting the expression of mature mirna over the expression of the primary transcript. data is shown with the efficiency in the control cells set to 100%. c) a graphical depiction of the tax mutant constructs td1 and td55 with deleted regions and the corresponding domains and motifs of full length tax. the function of each domain is indicated in brackets and arrows, as well as the associated interacting cellular proteins below each region. the proposed drosha binding region is indicated by the grey shaded box. doi:10.1371/journal.pone.0040662.g004 htlv-1 gene expression is regulated not only by tax, but also by the viral protein rex. rex induces the expression of htlv-1 structural proteins post-transcriptionally, by binding to the rxre (rex-responsive element) on the u3 region of the htlv-1 ltr (long-terminal repeat) [81] [82] [83] . recently, rex has been shown to suppress the rnai pathway by interacting with cellular dicer, inhibiting the conversion of shrna to sirna [15] . as we have shown that drosha is downregulated not only with htlv-1 infection, but specifically in the presence of tax, therefore we hypothesized that tax could be interacting directly with drosha. tax-transfected hela cells show distinct, punctate foci in the nucleus when stained with antibodies against both tax and drosha in independent experiments. we observed that tax and drosha colocalize distinctly, however it is important to note that this htlv-1-dependent downregulation of drosha is not completely efficient, and that there is still some endogenous drosha remaining in these cells. this is analogous and similar to the colocalization of cellular rb and tax in cells ( figure 2d) as we have previously shown that tax binds directly to rb and is a necessary interaction for the subsequent degradation of rb in vitro [47] . we next demonstrated that drosha can be found in complex with tax at the n-terminus, specifically at the region spanning aa 1-244. within this n-terminal region of tax, we observed two potential motifs that could be utilized for this interaction, the zinc finger motif and the leucine-zipper-like region. the n-terminus of tax is most commonly known for the creb (camp-responsive element-binding)-transactivation domain where the transcription factors p300/cbp, and p/caf (p300/cbp-associated factor) bind and promote viral transcription from the ltr [56] . we have specifically shown the interaction of drosha and tax with an immunoprecipitation of drosha from tax-expressing cells in the presence of proteasome inhibitors. we indicate that this interaction is not seen when a cell is expressing the tax mutant, ach.m22. interestingly, the n-terminus portion of tax is also known to interact with the proteasome, therefore this dual binding allows tax to bring drosha in close proximity with the proteasome complex, resulting in the degradation of drosha. this proteasomal degradation is confirmed by a 50% recovery of total drosha protein levels in htlv-1 infected cells when treated with proteasome inhibitors psi (cbz-ile-glu-(o-t-bu)-ala-leucinal) and alln (n-acetyl-leu-leu-norleucinal). these proteasome inhibitors have also been well documented as anticancer and antiagiogenic therapeutics. furthermore, a number of colleagues in the field have shown that proteasome inhibitors such as ps-341 or bortezomib can in fact downregulate htlv-1 levels, resulting in a decrease of t-cell leukemia cells [84] [85] [86] [87] [88] . this is consistent with our sidrosha and proteasome inhibitor data, where a drug/ peptide inhibitor can regulate mirna machinery by increasing proteins such as drosha resulting in an increased host cellular rnai response against htlv-1 activity. three large studies have recently examined the dysregulation of cellular mirnas in both htlv-1 infected cell lines and atll samples, resulting in surprisingly little overlap [67, [89] [90] [91] . these studies do however provide a large pool of upregulated and downregulated cellular mirnas from which to pull functional data in the context of htlv-1 infection. we show here, that in the presence of tax, endogenous drosha is less functional and not as efficient in processing mirnas. drosha efficiency, as measured by the processing of endogenous mir326 as compared to overall levels of transcript, was highest in the presence of n-terminal tax deletion mutants from residues 1-92. this again indicates that the interaction between tax, drosha, and the proteasome occurs on the n-terminal region of tax. we also show that in the absence of drosha, htlv-1 viral replication increases by at least two-fold. the knockdown of dgcr8 also resulted in a significant increase in htlv-1 replication, suggesting a dysregulation of the microprocessing complex. the downregulation of htlv-1 replication with ago2 knockdown was validated with a comparable downregulation when cells were treated with the risc inhibitor acf. we suggest that htlv-1 dysregulates the rnai pathway, including up-and down-regulation of cellular mirnas by inhibiting the function of and degrading drosha, resulting in a modulation of tumor and viral suppressing cellular functions. one mirna upregulated in htlv-1 infection, mir-146a, can be activated by tax through an nf-kb-dependent pathway. this mirna, however, has been experimentally shown to both interfere with htlv-1 infected cell growth, as well as stimulate infected cell growth upon overexpression [90] . overexpression of mir146a appears to favor the proliferation of an htlv-1 infected cell population, however this effect is not always reproducible [67] . due to the nature of the control of mirna production in htlv-1 infection, it is not surprising to observe an increase in viral replication in the absence of drosha, due likely to the downregulation of a cellular mirna that would otherwise suppress viral replication. in order to investigate the control of mirna production in htlv-1 cells, we analyzed the pool of published downregulated mirnas for those that target cellular proteins known to interact with tax and regulate transcription. our rationale here was that if htlv-1 infection downregulates a particular mirna which targets a cellular protein necessary for tax transactivation, we could mimic this interaction by introducing an antagomir into uninfected cells, therefore disabling the endogenous mirna and increasing protein expression. based on three recent studies which examined expression changes of cellular mirnas in htlv-1 infected cell lines, we selected the downregulated mirnas, mir-199a, mir-132, and mir-let7i for further analysis [67, [89] [90] 92] . in ovarian cancer cells, mir-199a-3p has been shown to regulate ikk-b to affect nf-kb activity as well as targeting the swi/snf subunit brm in a variety of human cancers [93] [94] . upon kshv (kaposi's sarcoma-associated herpesvirus) infection, as well as hsv-1 (herpes simplex virus-1) and hcmv (human cytomegalovirus) infection, mir-132 is upregulated and regulates the transcriptional co-activator p300 [95] . following microbioal infection of cryptosporidium parvum, mir-let7i forms an inducible silencing complex with the nf-kb p50 subunit [96] . confirming this rationale in the scope of htlv-1 infection, we observed a increase in ikk-b and brm protein levels in the presence of tax and an opposite effect upon overexpression of functional drosha. we observed an increase in cellular levels of ikk-b, p65, p50, gsk-3b, and brm upon treatment with antagomirs against the above downregulated mirnas as compared to cells alone, indicating that the antagomirs mimic the cellular protein expression seen in tax-expressing cells. tax plays a major role in the activation of cellular nf-kb, however, htlv-1 infected cells also develop taxindependent activation of nf-kb. this delicate balance between activation and suppression of these cellular genes via the manipulation of the rnai pathway by htlv-1 infection strongly supports the notion that dysregulation of drosha and other core components of the rnai machinery control the rate of infection and t-cell transformation. antibodies used for confocal microscopy are as follows: a-brg1 (sc-10768), a-rb (sc-74562), and a-git2 (sc-5416) were obtained from santa cruz biotechnology, inc. (santa cruz, ca), a-drosha (ab12286) was obtained from abcam (cambridge, ma), a-tax was generated in house, and alexafluor 488 goat-anti-rabbit (a11008), alexafluor 660 donkey-anti-rabbit (a21083), and alexafluor 568 goat-anti-mouse (a11004) were obtained from invitrogen (carlsbad, ca). antibodies used for western blotting are as follows: a-drosha (ab12286), a-dgcr8 (ab82876), a-dicer (ab14601), a-ago2 (ab57113), and a-ago1 (ab5070-100) from abcam (cambridge, ma), a-tax was generated in house, a-actin (sc-1615), a-ubiquitin (sc-9133), a-p65 (sc-7151), a-p300 (sc-585), and a-ikk-b (sc-7329) were obtained from santa cruz biotechnology, inc. (santa cruz, ca), and a-gsk-3b (27c10) was obtained from cell signaling (beverly, ma). sirna against luciferase (d-002050-01-20) was obtained from thermo scientific (rockford, il) and sirna against sidrosha (gs29102), sidgcr8 (gs54487), and siago2 (gs27161) were obtained from qiagen (valencia, ca). 293t cells were transfected with plasmid dna according to attractene lipid reagent recommended protocols (qiagen, valencia, ca). briefly, 293t cells at 2.5610 5 cells were plated in dmem +/+/+ (500 ml, with serum, with l-glutamine, with penicillin/streptomycin) in a 24-well plate. a reaction mixture containing pcdna, pach, pctax, or pach.m22 (0.1, 1.0, or 10 mg) plasmid dna was incubated with attractene reagent (3 ml) in dmem 2/2/2 (100 ml, without serum, without l-glutamine, without penicillin/streptomycin). the reaction mixture was incubated for 20 min at 25uc and then added dropwise onto the cells. cells were collected 48 hours post-transfection. lipid transfections of pctax, pach, and sirna were performed according to attractene and lipofectamine rnaimax (invitrogen, carlsbad, ca) lipid reagent recommended protocols (qiagen, valencia, ca). briefly, 2.5610 5 cells were plated in dmem +/+/+ (500 ml) in a 24-well plate. a reaction mixture containing ach dna (5 mg) was incubated with attractene reagent (3 ml) in dmem 2/2/2 (100 ml). the reaction mixture was incubated for 20 min at 25uc and then added dropwise onto the cells. at 24 hours post-transfection, a reaction mixture containing siluc (150 nm), sidrosha (50, 150, 300 nm), sidgcr8 (50, 150, 300 nm) or siago2 (50, 300 nm), was incubated with rnaimax reagent (1.5 ml). hela cells were grown on coverslips for 48 hrs post pctax transfection. cells were fixed with 4% paraformaldehyde for one hour. cells were permeabilized with triton x-100 in pbs (0.5%) for 20 min and washed with pbs without ca 2+ and mg 2+ . cells were blocked at 25uc for 10 min with goat serum (3%) in pbs without ca 2+ and mg 2+ . cells were incubated with primary antibody (1:200) in goat serum (3%) for one hour, in the dark, at 37uc. cells were washed 36 with pbs without ca 2+ and mg 2+ and secondary antibodies were added (1:50) in goat serum (3%) for one hour, in the dark, at 37u. cells were washed 36 with pbs without ca 2+ and mg 2+ and a sufficient volume of dapi working solution was added to completely cover the sample, incubating for 2-10 min, in the dark, at 25uc. coverslips were washed to remove excess dapi and mounted onto a slide with fluoromount g (10 ml, southern biotech, birmingham, al). cells were imaged with a zeiss lsm 710 confocal system (george washington university, center for microscopy and image analysis). cells were collected from culture and spun at 1,800 rpm, for 5 min, at 4uc, and pellets were washed twice with phosphate buffered saline (pbs) without ca2+ and mg2+ (quality biological). cell pellets were resuspended in lysis buffer (50 mm tris-hcl, ph 7.5, 120 mm nacl, 5 mm edta, 0.5% np-40, 50 mm naf, 0.2 mm na3vo4, 1 mm dtt, one complete protease cocktail tablet/50 ml) and incubated on ice for 20 min, with gentle vortexing every 5 min. cell lysates were centrifuged at 10,000 rpm for 10 min. protein concentrations were determined using bradford protein assay (bio-rad, hercules, ca). cell extracts were loaded (50-75 mg) on a 4-20% sds-page gel, run at 200 v, and transferred onto nitrocellulose membranes. membranes were blocked with pbs containing 0.1% tween-20 and milk (5%), and incubated overnight at 4uc with the appropriate primary antibody. membranes were incubated with the appropriate secondary antibody and developed next day. whole cell extracts from cd4 + primary t-cells were prepared using m-per mammalian protein extraction reagent (pierce, usa). cell debris was cleared from the lysates by centrifugation (5 min, 14,000 g). protein concentrations were determined using bradford protein assay (bio-rad, hercules, ca). cell lysates were resolved by sds-page and transferred to a pvdf membrane. the blots were blocked for 1 h with odyssey blocking buffer (licor, lincoln, ne) and rinsed with pbst (pbs +0.05% tween-20), once for 10 min and twice for 5 min. the membranes were then incubated with either a-drosha (1:1000) or a-actin (1:2000) monoclonal antibodies (abcam, cambridge, ma), for 2 hours at room temperature, rinsed with pbst (once for 10 min and twice for 5 min), and incubated for 1 hour with an a-rabbit and a-mouse secondary antibody (licor, lincoln, usa) conjugated to irdy800 and irdy680 respectively. signals were detected by licor pearl pulse imager. densitometry was performed using imagej software (wayne rasband, nih, usa). c81 htlv-1 infected cells were plated at a density of 1610 6 cells/well in a 12-well plate. cells were treated with three concentrations (0.1, 1.0, and 10 mm) of the proteasome inhibitors alln (208750), and psi (539160) obtained from emd4biosciences (san diego, ca). cells were collected 48 hours post treatment, washed, lysed, and western blotted for drosha and b-actin. alternatively, 293t cells were treated with 10 mm of psi and 24 hours later were transfected with the indicated expression vectors (pach.tax or pach.m22). cells were collected, washed, lysed, and prepared for immunoprecipitation. total rna was isolated from the indicated cell types using the trizol protocol (invitrogen, carlsbad, ca). from total rna, 2 ml of rna was used for the cdna synthesis reaction using iscript (bio-rad, hercules, ca). the newly synthesized cdna was used as the template for a pcr reaction for drosha (tgcaactgg-tagccacagag, acactgctgaagctgggatt) and gapdh (ggaaggtgaaggtcggagtcaa, ccttgacggtgccatggaat). expression of mir326 was determined by the quantimir pcr kit (sbi, mountainview, ca) using the mir326 detection (sense) primer: cctctgggcccttcctccag. primary transcripts encoding mir326 were detected by rt-qpcr (caccaacatcc-tagcccaaacg, aagtgagaaccgggaagcaag). cell extracts from 293t cells alone, cells transfected with pach.tax (5 mg), or pach.m22 (5 mg) were collected as described above. whole cell extracts (,1 mg) were incubated overnight, rotating, at 4uc with a-igg, a-drosha, or a-tax. the next day, 50 ml of a 30% slurry of protein a+g beads (calbiochem, rockland, ma) was added to the ips and incubated for two hrs, rotating, at 4uc. the ips were spun briefly and beads were washed 16 with tne 150 +0.1% np-40, followed by a 16 wash with tne 50 +0.1% np-40. proteins were eluted off of the beads with laemmli buffer, run on a gel, and western blotted for both drosha and ubiquitin. supernatants were collected at indicated time points to test for the presence of virus. viral supernatants (10 ml) were incubated in a 96-well plate with a rt reaction mixture containing 16 rt buffer (50 mm tris-hcl, 1 mm dtt, 5 mm mgcl 2 , and 20 mm kcl), 0.1% triton-x100, poly(a) (1 u/ml), poly d(t) (1 u/ml), and [ 3 h]ttp. the mixture was incubated overnight at 37uc, 5 ml of the reaction mix was spotted on a deae filtermat paper, washed four times with 5% na 2 hpo 4 , three times with water, and then dried completely. rt activity was measured in a betaplate counter (wallac, gaithersburg, md). data is shown as cpms (counts per minute). hela whole cell extracts (1 mg) were prepared and incubated with 10 ug of the following gst-tax constructs bound to glutathione-sepharose beads: gst-tax wildtype, gst-tax 1-244 (n-terminus), gst-tax 244-336 (c-terminus), and gst-tax 288-353. extracts were brought up to 500 ml in tne 50 +0.1% np-40 (100 mm tris-hcl, ph 7.5; 50 mm nacl; 1 mm edta; 0.1% np-40) buffer and incubated overnight at 4uc with rotation. the following day, samples were spun and washed twice with tne 150 +0.1% np-40 (100 mm tris, ph 8.0; 300 mm nacl; 1 mm edta, 0.1% np-40) buffer and 16 with tne 50 +0.1% np-40 to remove nonspecifically bound proteins. samples were loaded and run on a 4-20% tris-glycine sds-page gel and subjected to western blotting for the presence of drosha. antagomirs were obtained from qiagen as follows: miscript mirna inhibitor anti-hsa-let-7i (catalog # min0000415), antihsa-mir-199a-3p (catalog # min0000232), and anti-hsa-mir-132 (catalog #min0000426). antagomirs were transfected into 293t cells using attractene as described above. briefly, 293t cells were plated at 1610 5 cells/well in a 6 well plate. antagomirs were incubated in dmem 2/2/2 (100 ml, 100 nm) with attractene (1.5 ml) at 25uc for 20 min. the transfection mix was added to the cells dropwise. cells were collected at 72 hrs post transfection, lysed, and extracts (50 mg) were loaded onto a 4-20% sds-page gel. western blots were performed using antibodies against p65, p300, ikk-b, and b-actin. densitometry was performed using imagej software (wayne rasband, nih, usa). chromatin dynamics associated with hiv-1 tat-activated transcription history of the discoveries of the first human retroviruses: htlv-1 and htlv-2 detection and isolation of type c retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous t-cell lymphoma t-cell lines established from human t-lymphocytic neoplasias by direct response to t-cell growth factor htlv-i tax protein binds to mekk1 to stimulate ikappab kinase activity and nf-kappab activation molecular mechanisms of cellular transformation by htlv-1 tax localization of human t-cell leukemia virus type 1 tax to subnuclear compartments that overlap with interchromatin speckles htlv-1 and apoptosis: role in cellular transformation and recent advances in therapeutic approaches the microrna world: small is mighty biogenesis of small rnas in animals micrornas and human retroviruses the ebola virus vp35 protein is a suppressor of rna silencing hepatitis c virus core protein is a potent inhibitor of rna silencing-based antiviral response evidence that hiv-1 encodes an sirna and a suppressor of rna silencing interaction of human t-cell lymphotropic virus type i rex protein with dicer suppresses rnai silencing adenovirus va1 noncoding rna can inhibit small interfering rna and microrna biogenesis a cellular microrna mediates antiviral defense in human cells human influenza virus ns1 protein enhances viral pathogenicity and acts as an rna silencing suppressor in plants hiv-1 tat rna silencing suppressor activity is conserved across kingdoms and counteracts translational repression of hiv-1 the ns3 protein of rice hoja blanca virus complements the rnai suppressor function of hiv-1 tat differential rna silencing suppression activity of ns1 proteins from different influenza a virus strains hiv-1 tar rna subverts rna interference in transfected cells through sequestration of tar rna-binding protein ebolavirus proteins suppress the effects of small interfering rna by direct interaction with the mammalian rna interference pathway the 7a accessory protein of severe acute respiratory syndrome coronavirus acts as an rna silencing suppressor suppression of short interfering rna-mediated gene silencing by the structural proteins of hepatitis c virus la crosse virus nonstructural protein nss counteracts the effects of short interfering rna interferon antagonist proteins of influenza and vaccinia viruses are suppressors of rna silencing physical interaction of human t-cell leukemia virus type 1 tax with cyclin-dependent kinase 4 stimulates the phosphorylation of retinoblastoma protein human t cell leukemia virus type 1 oncoprotein tax targets the human mitotic checkpoint protein mad1 tax protein of htlv-1 interacts with the rel homology domain of nf-kappa b p65 and c-rel proteins bound to the nfkappa b binding site and activates transcription functional and biochemical interaction of the htlv-i tax1 transactivator with tbp interaction of the human t-cell lymphotropic virus type 1 tax transactivator with transcription factor iia the human t-cell leukemia virus type 1 oncoprotein tax inhibits the transcriptional activity of c-myb through competition for the creb binding protein molecular interactions involved in the transactivation of the human t-cell leukemia virus type 1 promoter mediated by tax and creb-2 (atf-4) an exposed kidlike domain in human t-cell lymphotropic virus type 1 tax is responsible for the recruitment of coactivators cbp/p300 transcriptional and post-transcriptional gene regulation of htlv-1 the coactivator cbp stimulates human t-cell lymphotrophic virus type i tax transactivation in vitro htlv-i tax transrepresses the human c-myb promoter independently of its interaction with cbp or p300 retroviral oncoprotein tax induces processing of nf-kappab2/p100 in t cells: evidence for the involvement of ikkalpha protein domains involved in both in vivo and in vitro interactions between human t-cell leukemia virus type i tax and creb complementation cloning of nemo, a component of the ikappab kinase complex essential for nf-kappab activation htlv-1 tax protein interacts with cyclin-dependent kinase inhibitor p16ink4a and counteracts its inhibitory activity towards cdk4 htlv-i tax induces a novel interaction between p65/rela and p53 that results in inhibition of p53 transcriptional activity anchoring of creb binding protein to the human t-cell leukemia virus type 1 promoter: a molecular mechanism of tax transactivation control of camp-regulated enhancers by the viral transactivator tax through creb and the co-activator cbp protein profile of tax-associated complexes the htlv-i tax oncoprotein targets the retinoblastoma protein for proteasomal degradation htlv-1 tax mediated downregulation of mirnas associated with chromatin remodeling factors in t cells with stably integrated viral promoter viral oncogenes, noncoding rnas, and rna splicing in human tumor viruses absence of dicer in monocytes and its regulation by hiv-1 immortalization of cd4(+) and cd8(+) t lymphocytes by human t-cell leukemia virus type 1 tax mutants expressed in a functional molecular clone mutational analysis of conserved n-linked glycosylation sites of human immunodeficiency virus type 1 gp41 identification of htlv-i tax trans-activator mutants exhibiting novel transcriptional phenotypes constitutive activation of nf-kappa b is essential for transformation of rat fibroblasts by the human t-cell leukemia virus type i tax protein the amino terminus of tax is required for interaction with the cyclic amp response element binding protein and p300/ camp-responsive element-binding protein associated factor interact with human t-cell lymphotropic virus type-1 tax in a multi-histone acetyltransferase/ activator-enhancer complex characterization of a novel nuclear localization signal in the htlv-i tax transactivator protein activation of ikkalpha and ikkbeta through their fusion with htlv-i tax protein the central region of human t-cell leukemia virus type 1 tax protein contains distinct domains involved in subunit dimerization genotoxic stress and cellular stress alter the subcellular distribution of human t-cell leukemia virus type 1 tax through a crm1-dependent mechanism human t-lymphotropic virus type i tax activates i-kappa b kinase by inhibiting i-kappa b kinaseassociated serine/threonine protein phosphatase 2a definition of a minimal activation domain in human t-cell leukemia virus type i tax htlv-1 tax nucleocytoplasmic shuttling, interaction with the secretory pathway, extracellular signaling, and implications for neurologic disease the c-terminus of the htlv-1 tax oncoprotein mediates interaction with the pdz domain of cellular proteins stable ubiquitination of human t-cell leukemia virus type 1 tax is required for proteasome binding identification of small molecules that suppress microrna function and reverse tumorigenesis role of micrornas in htlv-1 infection and transformation effect of triangle ventricular pacing on haemodynamics and dyssynchrony in patients with advanced heart failure: a comparison study with conventional bi-ventricular pacing therapy pcaf interacts with tax and stimulates tax transactivation in a histone acetyltransferase-independent manner transcription regulatory complexes bind the human t-cell leukemia virus 59 and 39 long terminal repeats to control gene expression human t-cell leukemia virus type 1 tax requires direct access to dna for recruitment of creb binding protein to the viral promoter ebv micrornas in primary lymphomas and targeting of cxcl-11 by ebv-mir-bhrf1-3 epstein-barr virus-encoded microrna mir-bart2 down-regulates the viral dna polymerase balf5 an epstein-barr virus-encoded microrna targets puma to promote host cell survival modulation of lmp1 protein expression by ebv-encoded micrornas human cellular restriction factors that target hiv-1 replication hiv-1 tat interaction with dicer: requirement for rna micrornas in human immunodeficiency virus-1 infection rna interference and hiv-1: hits and misses rna interference against viruses: strike and counterstrike functional analysis of human t-cell leukemia virus type i rex-response element: direct rna binding of rex protein correlates with in vivo activity human t-cell leukemia virus type 2 rex protein increases stability and promotes nuclear to cytoplasmic transport of gag/pol and env rnas phosphorylation of two serine residues regulates human t-cell leukemia virus type 2 rex function proteasome inhibitor, bortezomib, potently inhibits the growth of adult t-cell leukemia cells both in vivo and in vitro effects of the proteasome inhibitor ps-341 on tumor growth in htlv-1 tax transgenic mice and tax tumor transplants ps-341 or a combination of arsenic trioxide and interferon-alpha inhibit growth and induce caspase-dependent apoptosis in kshv/hhv-8-infected primary effusion lymphoma cells efficacy and mechanism of action of the proteasome inhibitor ps-341 in t-cell lymphomas and htlv-i associated adult t-cell leukemia/lymphoma p53 facilitates degradation of human t-cell leukaemia virus type i tax-binding protein through a proteasome-dependent pathway deregulation of microrna involved in hematopoiesis and the immune response in htlv-i adult t-cell leukemia microrna mir-146a and further oncogenesis-related cellular micrornas are dysregulated in htlv-1-transformed t lymphocytes microrna profile changes in human immunodeficiency virus type 1 (hiv-1) seropositive individuals pyrosequencing of small non-coding rnas in hiv-1 infected cells: evidence for the processing of a viral-cellular double-stranded rna hybrid regulation of ikkbeta by mir-199a affects nf-kappab activity in ovarian cancer cells micrornas mir-199a-5p and -3p target the brm subunit of swi/snf to generate a double-negative feedback loop in a variety of human cancers mir-132 regulates antiviral innate immunity through suppression of the p300 transcriptional co-activator nfkappab p50-ccaat/enhancer-binding protein beta (c/ebpbeta)-mediated transcriptional repression of microrna let-7i following microbial infection rachel van duyne is a predoctoral student in the microbiology and immunology program of the institute for biomedical sciences at the george washington university. this work is from a dissertation to be presented to the above program in partial fulfillment of the requirements for the ph.d. degree. we would like to thank dr. kt jeang at nih for the risc inhibitor acriflavine (acf) as well as the reagents and resources provided from dr. zachary klase. dr. klase is a postdoc under supervision of dr. jeang. we would also like to thank dr. lee ratner for the use of the pach clone. we would also like to thank dr. pooja jain for the use of the htlv-1 infected primary cd4+ t-cells. key: cord-002939-6a3ga6v9 authors: ribeiro, ana freitas; pellini, alessandra cristina guedes; kitagawa, beatriz yuko; marques, daniel; madalosso, geraldine; fred, joao; albernaz, ricardo kerti mangabeira; carvalhanas, telma regina marques pinto; zanetta, dirce maria trevisan title: severe influenza a(h1n1)pdm09 in pregnant women and neonatal outcomes, state of sao paulo, brazil, 2009 date: 2018-03-26 journal: plos one doi: 10.1371/journal.pone.0194392 sha: doc_id: 2939 cord_uid: 6a3ga6v9 to investigate the factors associated with death and describe the gestational outcomes in pregnant women with influenza a(h1n1)pdm09, we conducted a case-control study (deaths and recovered) in hospitalized pregnant women with laboratory-confirmed influenza a(h1n1)pdm09 with severe acute respiratory illness (sari) in the state of são paulo from june 9 to december 1, 2009. all cases were evaluated, and four controls that were matched by the epidemiological week of hospitalization of the case were randomly selected for each case. cases and controls were selected from the national disease notification system-sinan influenza-web. the hospital records from 126 hospitals were evaluated, and home interviews were conducted using standardized forms. a total of 48 cases and 185 controls were investigated. having had a previous health visit to a healthcare provider for an influenza episode before hospital admission was a risk factor for death (adjusted or (or(adj)) of 7.93, 95% ci 2.19–28.69). although not significant in the multiple analysis (or(adj) of 2.13, 95% ci 0.91–5.00), the 3(rd) trimester deserves attention, with an or = 2.22, 95% ci 1.13–4.37 in the univariate analysis. antiviral treatment was a protective factor when administered within 48 hours of symptom onset (or(adj) = 0.16, 95% ci 0.05–0.50) and from 48 to 72 hours (or(adj) = 0.09, 95% ci 0.01–0.87). there was a higher proportion of fetal deaths and preterm births among cases (p = 0.001) and live births with low weight (p = 0.019), compared to control subjects who gave birth during hospitalization. after discharge, control subjects had a favorable neonatal outcome. early antiviral treatment during the presence of a flu-like illness is an important factor in reducing mortality from influenza in pregnant women and unfavorable neonatal outcomes. it is important to monitor pregnant women, particularly in the 3(rd) trimester of gestation, with influenza illness for diagnosis and early treatment. introduction pregnancy constitutes an important risk for the development of influenza-related complications and hospitalization. the 1918 and 1957 influenza pandemics showed increased mortality in pregnant women [1, 2] . the reasons for the increased risk during pregnancy probably derive from a combination of immunological and physiological factors. an increased susceptibility to certain intracellular pathogens has been described [3] . the identification of a new viral subtype, influenza a(h1n1)pdm09, in mexico and the united states in april 2009 and its worldwide dissemination led the who to announce the beginning of a pandemic in june 2009 [4] . a study developed during the first month of the outbreak in united states estimated that the rate of admission for pandemic h1n1 influenza in pregnant women was higher than in the general population (0.32 per 100,000 pregnant women, 95% ci 0.13-0.52 vs 0.076 per 100,000 population at risk, 95% ci 0.07-0.09) [5] . since 2012, the world health organization (who) has defined this group as the highest priority for vaccination [6] . a study conducted in california among hospitalized pregnant women and women of reproductive age with influenza a(h1n1)pdm09 showed that pregnant women in the 2 nd and 3 rd trimesters who delayed treatment(! 48 hours) were more likely to undergo admission to the icu or death [7] . most studies evaluate risk factors for increased severity in pregnant women [8, 9, 10, 11] and few studies have analyzed the risk factors for death in pregnant women from influenza a(h1n1)pdm09. a systematic review study showed a higher risk of hospitalization in pregnant women with influenza a(h1n1)pdm09, with relative risks ranging from 4.3 to 7.2. however, only one study showed a higher risk of death in pregnant women with influenza a (h1n1)pdm09 (rr 10.2) and seven studies presented no significant risks (0.3 to 1.3) [12] . meta-analysis also showed no higher risk of death in pregnant women with pandemic influenza, or 0.99 (95% ci 0.67 to 1.46) [8] . a higher risk of fetal abnormalities in pregnant women with both seasonal influenza virus infection and a(h1n1)pdm09 infection was reported. in addition, women with a diagnosis of a(h1n1)pdm09 infection had a higher risk of placental problems, antepartum haemorrhage, and antepartum complications [13] . during the pandemic, there were a significant number of deaths in pregnant women from influenza a(h1n1)pdm09 in são paulo, which justifies this study. the objective of this study was to analyze factors associated with death in pregnant women with influenza a(h1n1) pdm09 and severe acute respiratory illness (sari) and describe the gestational and neonatal outcomes. the state of são paulo has a population of more than 41 million inhabitants, with 598,473 live births in 2009, according to data from the information department of the unified public health system (datasus) of the ministry of health. a case-control study was conducted that evaluated pregnant women living in são paulo with confirmed infection of influenza a(h1n1)pdm09 and hospitalized with sari, defined as: fever and cough and dyspnea or pneumonia or respiratory failure or tachypnea or radiological alterations consistent with pneumonia or oxygen therapy or mechanical ventilation. the definition of sari has been adapted from the one stated by the who to increase sensitivity in the detection of cases and controls [14] . in 2009, the ministry of health of brazil established the compulsory notification of any hospitalized case of influenza associated with sari and the inclusion of an epidemiological investigation into the influenza-web database of the national disease notification system (sinan). all hospitalized pregnant women who were notified with influenza associated with sari and eventually died during the epidemic period from july 9 th to december 1 st 2009 in são paulo were included in the analysis. for each death (case), four controls were randomly selected from those who recovered. the cases and controls were identified from the sinan, using the following variables for selection: rt-pcr (positive for influenza a/h1n1pdm09), final classification (confirmed), evolution (recovered or death), hospitalization (yes), date of hospitalization, hospital and residence in the são paulo state. the controls were matched by epidemiological week of admission date of the case to adjust for possible variations in access to treatment and clinical protocols. all pregnant women had a laboratory confirmation of influenza a(h1n1)pdm09 from a sample of respiratory secretions using the rt-pcr method, performed at the adolfo lutz institute, the public health laboratory [15] . for data collection, trained health professionals used two standardized forms: one to collect hospital record information and the second was used for home interviews. for the cases, the interviews were conducted with close family members, and for the controls, the interviews were conducted with the patients themselves. the hospital form included the following variables: pathological history, health care, symptoms on admission, admission to the intensive care unit, antiviral treatment (the ministry of health released the antiviral oseltamivir), use of antibiotics, complications, laboratory tests, radiological examinations, evolution, gestational and neonatal outcomes. the home form included the following variables: sociodemographics, history of a previous health visit to a healthcare provider for the influenza episode that resulted in hospitalization (after the onset of symptoms and before hospital admission date), vaccination history and gestational and neonatal outcome. education level was classified as low (no schooling or incomplete primary), medium (complete primary or incomplete high school) or high (complete high school or university). occupations were grouped using the occupational risk pyramid for the pandemic in the occupational safety and health act of the national institute for occupational safety and health-cdc, which classified the occupation risk for influenza infection as low (professional managers and other university and technical professionals without close contact with the population), medium (professionals in the areas of education, trade, service and administration with close contact with the population), or high and very high risk (doctors, nurses, other health professionals and support staff in the health services) [16] . a pre-test with 10 patients with influenza a(h1n1)pdm09 was performed to identify and correct any errors. the questionnaires are presented in s1 data collection form and the complete data used in this study are shown in s1 database. the study was started during the epidemic to support the actions of epidemiological surveillance, and the use of the data was approved by the ethics committee of the school of public health of usp (protocol 2283, of.coep/312/11). data collection complied with the recommendations of the national health council for research in human beings, including the signing of a consent form. clinical and demographic variables are presented as medians and interquartile ranges or percentages, mann-whitney u or chi-square tests were used for comparisons, as appropriate. odds ratios and 95% confidence intervals (95% ci) were calculated to evaluate factors associated with death. for the multiple logistic regression, variables were selected with a p of <0.20 in the univariate analysis and those considered important for the adjustment. the initial model included: health plan; previous visit to a healthcare provider; the presence of at least one of the high-risk medical conditions for developing influenza-related complications (adapted from the centers for disease control and prevention): asthma, neurological and developmental disorders, heart disease, kidney disease, liver disease, hemoglobinopathies, endocrine disorders, immunosuppressive diseases and obesity, with the absence of these conditions as reference; the use of an antiviral [no use (as reference), 48 hours from the first symptoms, > 48 and 72 hours, > 72 hours], pregnancy trimester [1 st and 2 nd trimesters (as reference) and 3 rd trimester]. the data were controlled for education level and age. the wald and hosmer-lemeshow tests were used to evaluate the significance of the variables and test the fit of the model, respectively. analyses were performed using spss (ibm, armonk, ny, usa), version 17.0. p values of <0.05 were considered significant. in são paulo, in 2009, 51 pregnant women with confirmed influenza a(h1n1)pdm09 infection notified in sinan eventually died. a total of 204 controls were randomly selected among the 525 pregnant women hospitalized with confirmed influenza a(h1n1)pdm09 infection notified in sinan who recovered. the investigation was conducted in 126 hospitals where all cases and controls were hospitalized. with regard to the 51 cases and 204 controls initially identified in sinan for the study, 22 files were missing or did not meet the case or control requirements, resulting in 48 cases and 185 controls reviewed. two pregnant women who died were identified in another study [13] , and included in the present. the home interviews were performed for 42 cases (87.5%) and 165 controls (89.2%), as shown in fig 1 . table 1 presents the socio-demographic characteristics of the pregnant women. there were no significant differences in socio-demographic distribution between cases and controls, and median age, family income, education level, smoking history, occupational risk for influenza infection, previous pregnancy and existence of private health insurance were not associated with death. table 2 shows the distribution of cases and controls according to clinical variables and visits to a healthcare provider. a total of 92.7% of cases sought medical care for the influenza episode prior to hospitalization. this proportion was higher than that of the controls. when evaluating the preconditions for admission, the presence of other risk conditions for developing complications related to influenza did not differ in cases and controls. asthma and obesity were the most common conditions, among cases (8.3% each) and controls (8.9% vs. 3.2%, respectively). the use of an antiviral during hospitalization was an important protective factor against death, with proportions of treatment at 77.1% and 91.4% in cases and controls, respectively. the proportions of women who received antiviral within the first 48 hours of symptoms were 27% and 63.4% in cases and controls, respectively. the protective effect was also observed for treatment starting within 48 to 72 hours (5.4% and 13% in cases and controls, respectively). the initiation of treatment with an antiviral more than 72 hours after the onset of symptoms did not present significant protection. the median number of days between the date of the first symptoms and hospitalization was four for the cases and two for the controls (p = 0.003). treatment with antibiotics was used in 100% of cases and 64.9% of controls. the average number of antibiotics used was 3.9 for cases and 1.1 for controls. among the cases, there was a higher proportion of patients admitted to the intensive care unit (95.8% vs. 16.2%), and a higher proportion of cases underwent mechanical ventilation compared to controls (100% vs. 13%). the cases exhibited higher rates of complications than the controls (100% vs. 10.3%), predominantly: respiratory distress syndrome (72.9% vs. 4.9%), shock (75.0% vs. 0.5%), sepsis (64.6% vs. 3.2%), infections (35.4% vs. 3.8%) and renal alterations (35.4% vs. 2.7%). there were three episodes of pre-eclampsia, which evolved to death (cases) and one in the control group. none of them fulfilled the definition of hellp syndrome, named for three features of the disease (hemolysis, elevated liver enzyme levels, and low platelet levels). all cases that evolve to death had influenza a(h1n1)pdm09 as the main cause in the death certificate. co-infection with other infectious agents occurred in 20.4% of cases and 1.6% of controls, with the following pathogens found: acinetobacter baumannii (n = 4), klebsiella pneumoniae (n = 3), staphylococcus aureus (n = 3), pseudomonas aeruginosa (n = 2), enterococcus spp. (n = 2), candida spp (n = 2), candida albicans (n = 1), klebsiella spp. (n = 1) and streptococcus pneumoniae (n = 1). the results of chest radiology were assessed in 93.8% of cases and in 80% of controls. among the cases, 91.6% presented with alterations, with a consolidation pattern evident in 50% of cases. among the controls, 59.5% presented alterations, with a consolidation pattern evident in 14.4% of cases. table 3 shows the findings from the laboratory examinations at the time of hospital admission. the cases presented lower median platelet, hemoglobin and hematocrit counts and severe influenza a(h1n1) in pregnant women higher median levels of creatine phosphokinase-cpk, lactate dehydrogenase-ldh, glutamic oxaloacetic transaminase-got, urea and creatinine, with statistical significance. table 4 presents the variables in the final multiple logistic regression model. having had a previous health visit to a healthcare provider for the influenza episode before hospitalization was a risk factor for death, or 7.93 (95% ci 2.19-28.69). antiviral treatment was a protective severe influenza a(h1n1) in pregnant women factor for death when administered within the first 48 hours after the onset of symptoms, or 0.16 (95% ci 0.05-0.50), and when administered 48 to 72 hours after the onset of symptoms, or 0.09 (95% ci 0.01-0.87). the third trimester of gestation, which was a significant risk factor in univariate analysis, lost significance in the multiple analysis, or 2.13 (95% ci 0.91-5.00), when antiviral treatment was included in the model. the proportion of women who did not receive any antiviral treatment was similar in the three trimesters of gestation (11.1, 11.1 and 12% respectively) and the proportion of those who received the treatment after 72 hours was higher in the 3 rd trimester (14.8%, 25.9% and 32%, respectively for the 1 st , 2 nd and 3 rd trimester of gestation. as shown in table 5 , among the cases, 45.8% of pregnant women had live births, with one twin birth, and 54.1% of the women experienced fetal deaths, of which fetal deaths later (! 23 weeks) represented 65.4% of the total number of deaths. among the controls, 13.5% delivered during hospitalization, with one twin birth. regarding the neonatal outcome in this group, there were 7.7% fetal deaths and 92.3% live births. considering the live births that occurred during hospitalization, in 100% of cases and in 75.0% of controls a cesarean delivery was performed. the distribution of gestational outcomes shows a concentration of miscarriages and premature births among cases compared to controls who delivered during hospitalization: 82.6% and 45.8%, respectively (p = 0.001). among the 144 controls who were discharged before delivery and who completed a home interview, 100% had live births, 62.5% by cesarean delivery and 86.8% at term. regarding the live births, among the cases, there was predominance of gestational age at birth between the 32 nd and 36 th weeks of pregnancy (65.2%), and among the controls who delivered during hospitalization, 54.2% occurred at 37 weeks and over (p = 0.003). among the controls that gave birth after hospital discharge, 86.8% of births were full term (!37 th week). analyzing the weight of live births, there was a higher proportion of low birthweight (<2,500 g) among cases (73.9%) than among controls who gave birth during hospitalization (37.5%), p = 0.011. considering control women who delivered after discharge, low birthweight occurred in only 6.3% of the births. during hospitalization, 8.7% (2 in 23; 28 and 38 days after birth) of the live births of cases and 4.2% (1 in 24; 12 days after birth) of live births of controls evolved to death after giving birth. none of the live births of control women who delivered after discharge evolved to death. the median gestational age, birth weight and apgar score in the 1 st minute were significantly lower among cases than among controls who delivered during hospitalization. among the cases, 73.7% of the newborns were admitted to the intensive care unit (icu), contrasting to only 35.0% of newborns from controls who delivered during the hospitalization. when the weight of the newborns was compared with the gestational age, 27.3% of newborns from cases were classified as small for the gestational age, and 12.5% and 7.5% of those from controls who gave birth during and after hospitalization, respectively, as shown in table 6 . severe influenza a(h1n1) in pregnant women the case control design, including all reported deaths of hospitalized patients who presented with laboratory confirmed influenza a(h1n1)pdm09 with sari in the state of são paulo, and the random selection of four controls for each case, with the collection of hospital data and home interviews, allowed for the expansion of the analysis of risk factors for death. it was possible to evaluate the gestational and neonatal outcomes, including those of pregnant women who delivered after hospital discharge. the main results suggest that an early search for care, the training of physicians for the proper treatment of pregnant women, and early antiviral administration can be protective factors against death. another noteworthy result was the presence of unfavorable neonatal outcomes, with a significant proportion of stillbirths and miscarriages, low birth weights and lower apgar scores among pregnant women who died. after hospital discharge, the patients had a favorable neonatal outcome. pre-eclampsia was present in six percent of women who evolved to death and in less than one percent of those who survived. despite the fact that all women who died had the influenza a (h1n1)pdm09 infection as the main cause of death in their death certificate, we cannot rule out the possibility that pre-eclampsia may have contributed to their unfavourable outcome. age distribution and race were not associated with a risk of death in pregnant women, similar to results in other studies [9, 10, 11] . the third trimester of gestation lost its significance as risk factor for death in the multiple analyses, when antivirus treatment was included in the model. a higher proportion of women in the final period of gestation received the treatment after 72 hours of symptoms than those who were at earlier stages of gestation. a meta-analysis study showed that the third trimester of gestation was a risk factor for death (or 1.22, 95% ci 1.01-1.48) for pandemic influenza, when compared with those in the first or second trimester [12] . although the present study did not confirm the association with the third trimester of gestation, its result indicates that it is important to monitor pregnant women with influenza illness, with special attention during this trimester, for diagnosis and early treatment. the pregnant women who had a previous health visit to a healthcare provider for the influenza episode before hospitalization had a higher risk of death. this increased risk could be an indication of difficulties in accessing hospitalization or lack of perception of the severity of the case by doctors or lack of recognition that, even in cases that are not serious, considering that pregnant women are in a high risk group for severity of the disease, early antiviral therapy should have been introduced. the median time between the first symptoms and hospitalization was twice as high among pregnant women who died. similar results were found when patients in general with influenza during the pandemic were evaluated in são paulo [17] and in mexico [18] . these findings also reinforce the need for pregnant women to have access to health services, particularly hospitalization in serious cases. the training of physicians concerning the proper care for pregnant women and the need to start early treatment are as important as the early search for care. the use of an antiviral medication was a protective factor death when administered within 72 hours of symptom onset. several studies have shown an increased risk of death or worsening disease in pregnant women who started treatment late [10, 11, 19, 20, 21, 22, 23] and in patients in general [17, 18, 24, 25, 26, 27, 28, 29] . regarding the history of previous diseases, no differences in the presence of risk conditions for developing influenza-related complications were found between pregnant women who eventually died and those who recovered. however, surveillance showed a higher proportion of risk conditions among pregnant women who eventually died compared to those who survived [10] . a study in china showed that obesity (bmi ! 30) was a factor associated with mortality in patients with severe disease [9] . this result is in line with the current study. although it was not possible to calculate bmi, a higher rate of obesity among the cases than among the controls was reported. there were a higher proportion of cases with co-infections than controls, consistent with other reported results [17] . the patients who eventually died presented significant alterations in laboratory values when compared to controls. the alterations observed in cpk, ldh, platelets and creatinine were similar to those reported for patients in general [27, 29, 30, 31, 32] . in pregnant women, there was also a decrease in the number of red blood cells. in relation to neonatal outcomes, there were a greater proportion of fetal deaths in patients who died than in controls that delivered either during hospitalization or after discharge. among the live births in the cases, there was a greater proportion of low birth weight, gestational age less than 32 weeks, admitted to icu, lower scores on the apgar scale, when compared to the live births of controls that delivered during or after hospitalization. similar results were reported in studies that evaluated pregnant women infected with influenza a(h1n1) pdm09 compared to women of childbearing age without infection [33] , pregnant women with influenza a(h1n1)pdm09 with severe disease [9, 20] or women who gave birth to live newborns during hospitalization [10, 34] . control women who delivered after discharge from the hospital had favorable outcomes in their offspring, with a median apgar of 9 both in the 1 st and 5 th minutes of life. considering all births in the state of são paulo during 2009, according to the national system of live births-sinasc, 58% were delivered by cesarean, 9% were preterm birth (<37 weeks) and 9% had low birthweight ( 2500 g). in the control women of the study who gave birth after hospitalization, these proportions were also high (62.5%, 13.2% and 7.7%, respectively). this study has limitations. underreporting of sari by health professionals as well as gaps in the sinan may have occurred. the notification of influenza through a new viral subtype associated with sari was initiated during the pandemic; consequently, the sensitivity may have varied with time. the quality of information from medical records can differ between hospitals. the use of standardized hospital and home questionnaires minimized these difficulties during data collection. although all hospital reports were reviewed, we were not able to establish the roles of other factors, such as secondary infections or obstetric factors, which could have contributed to death in the cases. the results presented in this study indicate that early treatment can prevent unfavorable outcomes in pregnant women and in their offspring and reinforce the need for the proper training of doctors for the clinical management of pregnant women and early administration of antiviral treatment. these findings also support interventions in 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experience of 2009 a: h1n1v influenza in pregnant women evaluation of pregnancy as a risk factor in the outcomes of influenza a (h1n1)/ 2009 in women of childbearing age influenza-like illness in hospitalized pregnant and postpartum women during the 2009-2010 h1n1 pandemic hospitalized patients with 2009 pandemic influenza a (h1n1) virus infection in the united states severe hospitalised 2009 pandemic influenza a(h1n1) cases in france hospitalized patients with 2009 h1n1 influenza in the united states critically ill patients with 2009 influenza a(h1n1) in mexico factors associated with death or hospitalization due to pandemic 2009 influenza a(h1n1) infection in california fatalities associated with the 2009 h1n1 influenza a virus pneumonia and respiratory failure from swine-origin influenza a (h1n1) in mexico critically ill patients with 2009 influenza a(h1n1) infection in canada factores asociados a ingreso en unidad de cuidados intensivos en pacientes hospitalizados por influenza pandémica a/h1n1 maternal and neonatal outcomes among pregnant women with 2009 pandemic influenza a(h1n1) illness in florida, 2009-2010: a population-based cohort study the anzic influenza investigators and australasian maternity outcomes surveillance system. critical illness due to 2009 a/h1n1 influenza in pregnant and postpartum women population based cohort study we would like to thank the sao paulo state secretary of health, the santa casa de são paulo medical school for the collaboration in the preparation of the project. this study would not possible without the collaborations of the researchers and field coordinators, the epidemiological surveillance center, the disease control coordination, the adolfo lutz institute, municipal departments of health for data collection. we would also like to thank all those who contributed to this study, especially luiz roberto barradas barata (in memoriam). key: cord-287784-f7usy52w authors: maestre, ana m.; garzón, ana; rodríguez, dolores title: equine torovirus (bev) induces caspase-mediated apoptosis in infected cells date: 2011-06-15 journal: plos one doi: 10.1371/journal.pone.0020972 sha: doc_id: 287784 cord_uid: f7usy52w toroviruses are gastroenteritis causing agents that infect different animal species and humans. to date, very little is known about how toroviruses cause disease. here, we describe for the first time that the prototype member of this genus, the equine torovirus berne virus (bev), induces apoptosis in infected cells at late times postinfection. observation of bev infected cells by electron microscopy revealed that by 24 hours postinfection some cells exhibited morphological characteristics of apoptotic cells. based on this finding, we analyzed several apoptotic markers, and observed protein synthesis inhibition, rrna and dna degradation, nuclear fragmentation, caspase-mediated cleavage of parp and eif4gi, and pkr and eif2α phosphorylation, all these processes taking place after peak virus production. we also determined that both cell death receptor and mitochondrial pathways are involved in the apoptosis process induced by bev. bev-induced apoptosis at late times postinfection, once viral progeny are produced, could facilitate viral dissemination in vivo and contribute to viral pathogenesis. toroviruses are enteric (and probably respiratory) viruses which infect different animal species and humans and cause diarrhea. they are positive-sense, single stranded rna enveloped viruses that, together with coronaviruses, belong to the coronaviridae family of the nidovirales order [1, 2] . there are four torovirus species recognized by the ictv, established according to the animal host that they infect: bovine torovirus (btov), porcine torovirus (ptov), human torovirus (htov) and equine torovirus (etov) (ictv web site: www.ictvonline.org). the latter virus was the first torovirus identified, isolated in 1972 from the faeces of a horse in berne (switzerland), and named berne virus or bev [3] . for a long time bev was the only strain of the genus that could be grown in cell culture, and therefore it is the most thoroughly studied at the molecular level, and is the prototype member of the genus. nonetheless, the propagation of different btov strains in the human hrt-18 cell line has been recently described [4, 5] . the torovirus genome consists of a single rna molecule of about 25-30 kb. the 59 two thirds contain two large and overlapping open reading frames, orf1a and orf1b, that code for the replication machinery. the last third of the genome contains four open reading frames, orfs 2-5, coding for the spike (s), membrane (m), hemagglutinin-esterase (he) and nucleocapsid (n) structural proteins [6] . in bev the he gene is partially deleted when compared with other torovirus strains, though the corresponding mrna is produced [7] . since their identification in 1972, toroviruses have been poorly studied, and many issues regarding torovirus infections remain unexplored. significantly, little is known about the morphological, physiological and biochemical changes that occur in torovirus infected cells. investigating the complex virus-host interactions is important to understand the basis of torovirus-induced disease. there are a few studies about torovirus pathogenesis that were performed with btov, since it is the only torovirus that has been successfully propagated in experimental infections. in those studies, gnotobiotic and colostrum deprived calves, infected orally or intranasally with btov, developed moderate to severe diarrhea. infected animals were sacrificed sequentially during the early stages of infection, and intestinal tissue samples were observed by light and electron microscopy. cytopathological changes associated with btov infection were observed in enterocytes from the lower half of the villi, extending into the crypts throughout the lower small intestine, large intestine and dome epithelial cells [8, 9] . enterocytes showed signs of severe vacuolar degeneration, necrosis and exfoliation. it was suggested that the primary site of infection is in the crypt cells, and that infected cells migrate up to the villous before being shed as the viral cytopathic effect (cpe) develops [10] . on the other hand, the effects of infection in the host with the closely related coronaviruses have been more thoroughly investigated, especially since the emergence of the human coronavirus causing the severe and acute respiratory syndrome (sars-cov). significantly, occurrence of cell death by apoptosis has been observed during infection with several coronaviruses: the mouse hepatitis virus (mhv) [11, 12, 13] , transmissible gastroenteritis virus (tgev) [14, 15, 16] , infectious bronchitis virus (ibv) [17, 18] , canine coronavirus (ccov) [19, 20] , feline infectious peritonitis virus (fipv) [21] , equine coronavirus [22] , and the human coronaviruses oc43 [23] , 229e [24] and sars-cov [25, 26] . the induction of apoptosis represents one of the major components of the host antiviral responses. nonetheless, viruses establish intricate and complex interactions with the host to regulate apoptosis to ensure a successful replication cycle that allows the production and spread of virus progeny to neighbouring cells [27, 28] . in fact, many viruses have been reported to inhibit apoptosis early in infection to facilitate the viral replication and to induce apoptosis at later times postinfection to inhibit the inflammatory response and favour viral dissemination [28, 29, 30] . two cellular proteins which are key effectors of the initial antiviral response triggered by interferon (ifn) and which have been related to the induction of apoptosis, are the protein kinase r (pkr) and the rnase l. both proteins become activated by double-stranded rna (dsrna), and activation of each of these two proteins results in protein synthesis inhibition and apoptosis [31, 32, 33, 34, 35, 36, 37] . upon activation, pkr phosphorylates the alpha subunit of the eukaryotic initiation factor 2 (eif2a) impairing its activity, and then causing inhibition of host-cell translation initiation [38, 39, 40] . on the other hand, rnase l, which is activated by 2959oligoadenylates produced upon dsrna activation of the 2959oligoadenylate synthetase, degrades both viral and host single-stranded rna (ssrna), including ribosomal rna [41, 42] . therefore pkr and rnase l activation effectively inhibits viral and cellular gene expression, and the ultimate consequence of their activation is the induction of apoptotic pathways leading to the removal of the infected cell. caspases are intracellular proteases (cysteine-dependent aspartate-specific proteases) that play an essential role during apoptosis. there are two main apoptosis pathways regulated by caspases: the extrinsic or cell death receptor pathway, and the intrinsic or mitochondrial pathway. the former is initiated mainly by binding of an extracellular ligand to a death receptor, which in turn aggregates and recruits, through adapter proteins, procaspase-8 molecules, leading to their activation into caspase-8. the mitochondrial pathway is activated after an intracellular signal such as dna damage, leading to the release of cytochrome c from mitochondria, which binds to the adaptor molecule apoptotic protease activating factor-1 (apaf-1), and the complex binds to procaspase-9, promoting its autocleavage into caspase-9. both pathways converge at the activation of executioner caspases, namely caspase-3, -6,-7, and -10. caspase-3 is the primary executioner caspase that cleaves most of the cellular substrates examined [43] , leading to the characteristic morphological features of apoptosis, such as nuclear shrinking and membrane blebbing [44] . both cell death receptor and mitochondrial apoptosis signaling pathways can also intersect at the caspase-8-mediated cleavage of bid, a proapoptotic bcl2 family member, which promotes cytochrome c release from the mitochondria, and has been shown to be implicated in apoptosis induced by viruses [45, 46, 47, 48] . here, we report the first biochemical study of the effects caused by a torovirus in the infected cells, and show that typical morphological and biochemical features of apoptosis are induced in bev infected cells. for the elucidation of the underlying mechanisms, we examined several apoptotic markers, and we found that both intrinsic and extrinsic apoptotic pathways are involved, being the second likely activated through bid cleavage by caspase-8. to our knowledge, nothing was known about the torovirus-induced alteration on cellular functions and signaling pathways, which would have a profound impact on cell viability and virus pathogenesis. equine dermal (e. derm) cells (nbl-6, atcc ccl-57), and human fetal lung fibroblasts (mrc-5, attc ccl-171), kindly provided by r. de groot (utrecht university, utrecht, the netherlands) and r. e. randall (university of st. andrews, scotland, uk) respectively, were cultured at 37uc, 5% co 2 and 98% humidity, in dulbecco's modified eagle's medium (dmem; invitrogen, corp) supplemented with 15% and 10% fetal calf serum (fcs) respectively, non-essential aminoacids (1%), gentamicin (50 mg/ml), penicillin (100 u/ml), streptomycin (100 mg/ ml) and fungizone (0.5 mg/ml). equine torovirus bev (strain p138/72) [3] was obtained from r. de groot (utrecht university, utrecht, the netherlands). the virus was grown in e. derm cells and was partially purified by ultracentrifugation over 20% sucrose cushion. the virus was titrated by plaque assay as previously described [49] . a preparation of partially purified virus was placed in an open tube under an uv lamp (philips 15w/g15 t8) inside a laminar flow hood, and maintained exposed to uv light for 30 min. viral inactivation was confirmed by titration of the same viral sample before and after uv irradiation. phase-contrast microscopy e. derm cells were mock-infected and infected with bev [5 plaque-forming units (pfu)/cell] in 12-well plates. at 24 and 48 hours postinfection (hpi) cells were examined under an inverted phase-contrast microscope (leica dmi6000b) with a 106/0.30 objective, and images were acquired with a digital camera (ccd hamamatsu orca r2). monolayers of e. derm cells, mock-infected or infected with bev (5 pfu/cell), were fixed in situ 24 hpi with a mixture of 4% paraformaldehyde (pfa) and 2% glutaraldehyde in 0.1 m phosphate buffer ph 7.4 for 1 h at room temperature. fixed cells were removed from the dishes, washed in phosphate buffer and processed for embedding in the epoxy resin taab-812 (taab laboratories-england uk) by methods previously described [50] . ultrathin sections of embedded samples were stained with saturated uranyl acetate and lead citrate and examined at 80 kv in a jeol jem-1010 (tokyo, japan) electron microscope. pictures were acquired with a bioscan 792 digital camera (gatan, inc.). e. derm and mrc-5 cells were seeded in 24-well plates, and infected with bev (5 pfu/cell). at the indicated times postinfection, cells were subjected to a 30 min pulse, at 37uc with 100 mci/ml of [ 35 s]met-cys (ge healthcare), and dmem without met and cys. cell extracts were lysed in laemmli buffer with 5% b-mercaptoethanol, and proteins were denatured by boiling for 5 min, separated by sds-page in 12% polyacrylamide gels, and visualized after staining of the gel with coomassie blue. labeled proteins were detected by autoradiography of the dried gels. apoptosis quantitation by analysis of the nuclear dna content by flow cytometry the different stages of cell cycle and the percentage of cells with subg 0 -g 1 dna content were analyzed by propidium iodide (pi) staining and flow cytometry [51] . e. derm and mrc-5 cells were seeded in 6-well plates, and infected with bev (5 pfu/cell). in some experiments, after virus adsorption, cells were treated with the caspase inhibitors z-ietd-fmk (caspase-8), z-lehd-fmk (caspase-9) and z-vad-fmk (general inhibitor) (calbiochem), each at a final concentration of 50 mm. at the indicated times, all the cells from each well were collected; the floating cells were taken with the medium and combined with those still adhered to the plastic recovered by trypsinization. cells were washed with icecold phosphate-buffered saline (pbs), and permeabilized with 70% ethanol in pbs at 4uc overnight. later, cells were washed three times with pbs, incubated 45 min at 37uc with rnase-a (0.1 mg/ml) and stained with pi (10 mg/ml). the percentage of cells with hypodiploid dna content was determined by flow cytometry. data were acquired on 10 4 cells per sample in a coulter epics xl-mcl cytometer with argon 488 laser. for nuclei staining of apoptotic cells, e. derm and mrc-5 cells were seeded over coverslips, infected with bev (5 pfu/cell), and fixed with 4% pfa at the indicated times. cells were stained with hoechst 33258 (0.5 mg/ml, sigma), and the images were obtained in a leica dmrd epifluorescence microscope, using the suitable filter (360-400 nm). to analyze rrna degradation, e. derm and mrc-5 cells were seeded in 6-well plates, and infected with bev (5 pfu/cell). at 8, 16, 24 and 48 hpi, medium was removed, cells were washed with pbs and lysed with 350 ml of the rlt buffer from the rneasy mini kit (qiagen), and rna purified following the manufacturer's instructions. finally, rna was eluted with 30 ml of diethyl pyrocarbonate-treated water, and 2 ml of each rna sample was analyzed in 1% agarose gel and stained using ethidium bromide (etbr, 0.5 mg/ml), under rnase free conditions. e. derm and mrc-5 cells grown in 6-well plates were mockinfected or infected with bev at a multiplicity of infection of 5 pfu/cell and collected at 24 or 38 hpi. cells treated with staurosporine (500 nm) for 6 h were included in the assay as a control of cytocrome c redistribution in cells undergoing apoptosis induced through the mitochondrial pathway. at the indicated times cells were collected in pbs and subjected to digitonin permeabilization as previously described [52] . briefly, pelleted cells were resuspended in 100 ml of an isotonic sucrose buffer (250 mm sucrose, 10 mm hepes, 10 mm kcl, 1.5 mm mgcl 2 , 1 mm edta, and 1 mm egta, ph 7.1) containing 0.05% digitonin (sigma), and after 2 min at room temperature disrupted cells were centrifugated at 14.000 rpm 5 min, and the soluble cytosolic fractions were transferred to new tubes containing 25 ml of 56laemmli sample buffer, and the digitonin-insoluble fractions (membrane-bound organellar fractions) were washed once with isotonic buffer and directly disrupted in 125 ml of 16 laemmli sample buffer. mock-infected or bev-infected e. derm and mrc-5 cells were lysed at the indicated times postinfection directly in laemmli buffer with b-mercaptoethanol, or were subjected to subcellular fractionation as described above, and proteins were denatured by boiling for 5 min. protein lysates were fractionated by sds-page and transferred to nitrocellulose membranes (protran, schlei-cher&schuell) in a semidry blotting apparatus (biorad) for 45 min at 200 ma. membranes were blocked for 1 h in pbs containing 5% nonfat dry milk and then probed with different antibodies: rabbit polyclonal anti-parp (1:500, cell signaling), rabbit polyclonal anti-eif4gi, that recognizes the amino (n) and carboxy (c) terminal moieties of the eif4gi protein (1:5000, kindly provided by dr. carrasco from cbmso, madrid, spain), polyclonal anti-phosphorylated (ser51) eif2a (eif2ap) (1:1000, biosource), polyclonal anti-eif2a (1:500; santa cruz biotechnology), rabbit polyclonal anti-phosphorylated pkr (thr451) (1:1000, invitrogen), monoclonal anti-murine pkr (b-10, 1:1000; santa cruz biotechnology), mouse monoclonal anti-b-actin (ab8226, abcam), rabbit polyclonal against bid (1:500, kindly provided by dr. yin from indiana university, indianapolis, eeuu), a mouse monoclonal anti-cytochrome c (1:500; santa cruz biotechnology), two rabbit polyclonal sera against the bev nucleocapsid protein (anti-bev-n, 1:1000) and membrane protein (anti-bev-m, 1:1000) respectively [53] , and a mouse monoclonal anti-s antibody (1:250). proteins were detected using horseradish peroxidase-labeled secondary antibodies (sigma) and an enhanced chemiluminescence western blot detection kit (ecl, ge health care). statistical analyses were performed using student's two tailed t test. means 6 standard deviation for each sample are shown. since little is known about the host cellular responses to torovirus infection, we decided to examine cellular responses to infection with bev virus, as the prototype member of the torovirus group. until very recently, bev was the only torovirus strain that could be grown in cell culture, and e. derm and embryonic mule skin (ems) cells the only cell lines described as being susceptible to infection to date. in e. derm cells infected with bev at a multiplicity of infection of 5 pfu/cell, the first signs of infection could be detected by about 24 hpi, and cpe was seen in some cells (compare panels a and d with b and e in figure 1 ). however, extensive cpe could be observed at 48 hpi ( fig. 1 , panels c and f). on the other hand, examination by electron microscopy of e. derm cells infected for 24 h with bev (5 pfu/cell) showed that some cells presented characteristic signs of apoptosis. figure 2 shows the comparison of the appearance of an uninfected e. derm cell (panel a) with a bev infected cell showing apoptotic characteristics such as a disorganized nucleus, chromatin condensation, membrane blebbing, and cell disassembly in vesicles (panel b) [54] . the number of cells showing this morphology readily increases with the time postinfection (not shown), in agreement with the cpe observed by phase-contrast microscopy. these results suggest that bev causes apoptosis at late times of the infection cycle. the response of mammalian cells to a variety of stimuli and stress-inducing conditions that cause apoptosis, such as viral infections, involves a rapid inhibition of protein synthesis [55, 56, 57] . to analyze whether protein synthesis was affected after bev infection, e. derm cells were infected with bev (5 pfu/cell) and metabolically labeled with [ 35 s]met-cys for 30 min at the indicated times postinfection which ranged between 4 and 32 hpi (fig. 3a) . the proteins were analyzed by sds-page and the gels were stained with coomassie blue and later subjected to autoradiography. a significant general reduction in protein synthesis was observed at 24 and 32 hpi, although a clear decrease was already observed by 16 hpi. the image of the coomassie blue stained gel shows that similar amounts of protein were loaded in all lanes (fig. 3b) . significantly, two bands corresponding to proteins of about 17-and 20-kda of molecular mass could be first appreciated in the autoradiogram at 6 hpi, and the intensity of these bands highly increased by 8 and 10 hpi, but diminished thereafter. these two bands should correspond to bev structural proteins n and m respectively, although their relative mobility in sds-page are slightly higher than previously described [58, 59, 60] . the identity of the 17-and 20-kda proteins was confirmed by analysis of the same samples by western blot with anti-bev-n and anti-bev-m antibodies (fig. 3c ). the patterns of appearance of both proteins were similar to those observed by metabolic labeling (fig. 3a) . interestingly, although the metabolic labeling studies indicated that m and n synthesis decreases at late times of the infection cycle, these proteins were still detected by western blot analysis suggesting that these proteins remain stable at late time points. in addition, in fig. 3a a high molecular weight protein can be observed, the intensity of which increased from 6 to 10 hpi and diminished from 16 hpi onwards (labeled with a solid arrowhead), that could correspond to the 200-kda s protein precursor [61] . moreover, two additional faint bands in the range of 75-to 100-kda (labeled with empty arrowheads) could be observed at 16 hpi, that could correspond to the s cleaved products. by western blot with a monoclonal antibody that recognizes the s protein we observed the precursor at 8 hpi, and both, the precursor and one of the cleaved products from 10 hpi onwards (fig. 3c) . to further investigate the effect of bev infection, we searched for other cell types that could be infected by bev. thus, several cell lines of different origins were subjected to an infection trial with bev, and we found that this virus could also grow in the human mrc-5 cells, though the viral replication cycle is slower in this cell type (garzón et al., unpublished results). therefore we carried out a similar experiment to that described above, and performed a metabolic labeling with [ 35 s]met-cys in mrc-5 cells infected with bev (5 pfu/cell). an overall protein synthesis reduction similar to that observed in e. derm cells was also observed in mrc-5 cells at 24 and 32 hpi (fig. 3d) . again, the intensities of whole protein profiles visualized by coomassie blue staining were similar, indicating that equivalent amounts of proteins were loaded in all lines (fig. 3e) . also, the 17-and 20-kda viral proteins were detected, although they showed a delay in synthesis with respect to e. derm cells, being first detected at 8 hpi. their identity as bev n and m proteins, respectively, was also confirmed by western blot (fig. 3f) . although in the autoradiogram we could not distinguish the high molecular bands corresponding to the s protein precursor and cleaved products among the cellular proteins in these cells (fig. 3d) , by western blot with the anti-s monoclonal antibody we could see the s precursor from 10 hpi, and both precursor and one of the cleaved products from 16 hpi onwards (fig. 3f) . these results show that, in bev infected cells, synthesis of both cellular and viral proteins was drastically inhibited at late times postinfection. eif2a and pkr are phosphorylated in bev infected cells a common mechanism for down-regulating protein synthesis during apoptosis is through the phosphorylation of the translation initiation factor eif2a [62] . pkr is one of the three ser/thr cellular protein kinases known to phosphorylate eif2a in response to viral infection [33] , leading to a block in the translation initiation [31] . activation of pkr occurs by autophosphorylation upon its binding to dsrna, which in the case of virus-infected cells is generated as a result of viral replication, or simply as a byproduct originated during replication of the viral genome. we have observed that there is accumulation of dsrna generated as an intermediate product during viral replication in torovirus infected cells (á vila et al., unpublished results). hence, we sought to investigate whether eif2a becomes phosphorylated in bev infected cells. for that, e. derm and mrc-5 cells were infected with bev (5 pfu/cell) and at the indicated times postinfection cells were collected and subjected to western blot analysis. as shown in figure 4a , a transient eif2a phosphorylation was observed in both cell lines starting at 8 hpi and decreasing by 24 and 32 hpi. therefore, next we wanted to determine if pkr could be responsible for eif2a phosphorylation after bev infection. significantly, in both cell lines pkr phosphorylation was first observed at 6 hpi and increased at 8 hpi (fig. 4a) . in e. derm cells similar phosphorylation dynamics were observed for both eif2a and pkr. western blot analysis of these samples with antibodies against total eif2a and pkr were also performed for sample loading control, however all the antibodies tested against pkr failed to recognize the protein from e. derm cells. these results indicate that the protein synthesis blockade observed after bev infection seems to be related to the phosphorylation of eif2a, which could be caused by pkr. dsrna is also known to activate the cellular 2959-a-dependent rnase l. rna degradation by rnase l is therefore another cellular mechanism implicated in protein synthesis inhibition at a translational level, and an apoptosis inducer [34, 35, 36, 37] . hence, we wanted to determine whether rrna degradation occurs in bev infected cells, and for this we analyzed 18s and 28s rrna pattern. rrna was analyzed in e. derm and mrc-5 cells infected with bev (5 pfu/cell) at the indicated times between 8 and 48 hpi. as shown in figure 4b , a similar rrna degradation pattern was observed in both cell lines, with the 28s rna being degraded at earlier time points than the 18s; degradation of 18s rna is only clearly apparent in both cells lines at 48 hpi.. therefore, these results reinforce the hypothesis that bev causes apoptosis in infected cells, and suggests that the protein synthesis inhibition is also related to rna degradation. caspases, the central executioners of apoptosis, are involved in most of the morphological changes observed in an apoptotic cell. two well-known substrates of the executioner caspases are the poly (adp-ribosylating) protein 1 (parp1), a 116-kda enzyme implicated in the dna-damage repair, and the eif4gi initiation factor, the scaffolding protein of the translation initiation complex. caspase-mediated cleavage of parp1 produces a 89-kda c terminal fragment and a 24-kda n terminal fragment, and renders the protein inactive [63] . the eif4gi is a 220-kda protein that can be cleaved in two different places during apoptosis, giving rise to a 76-kda central fragment and two other fragments of about 50-kda, corresponding to the n and c termini [64, 65] . to determine whether the caspase-cascade system was activated upon bev infection, the potential degradation of both factors was studied. e. derm and mrc-5 cells were infected with bev (5 pfu/cell), and cell extracts were collected at 8, 16, 24, 38 and 48 hpi. mock-infected cells were used as a control. samples were analyzed by sds-page (10% polyacrylamide gel) and subjected to western blot analysis with a commercial anti-parp antibody that recognizes both the 116-kda intact protein and the 89-kda fragment, and with a polyclonal antibody that recognizes both the n and c termini of eif4gi. appearance of the parp 89-kda fragment and a concomitant decrease of the 116-kda band started between 8 and 16 hpi in e. derm cells, and at 38 hpi in mrc-5 cells (fig. 5a, upper panel) . eif4gi presented a similar pattern, with the appearance of the 50-kda band and the concomitant decrease of the 220-kda band at 24 hpi onwards in e. derm cells, and at 38 hpi in mrc-5 cells (fig. 5a, bottom panel) . from these results we concluded that bev-induced apoptosis involves caspase activation. we also sought to determine whether virus replication was a prerequisite for virus-induced apoptosis. for this, e. derm and mrc-5 cells were inoculated with bev (2.5 pfu/cell) or with an equivalent amount of uv-inactivated bev. at 24 hpi cells were harvested and analyzed by western blot with the anti-eif4gi and anti-parp antibodies. we did not observe any cpe on the cells treated with uv-inactivated virus (data not shown), and neither of the two caspase substrates was processed in these cells (fig. 5b) , suggesting that bev apoptosis triggering is dependent upon viral replication. one of the main characteristics of an apoptotic cell is the nuclear morphological alteration, due to dna fragmentation, chromatin condensation, and apoptotic body formation [54] . we had previously observed by electron microscopy that, by 24 hpi, the nuclei of about 10% of bev-infected cells were disorganized, and we wanted to examine the nuclear morphology at later times postinfection. therefore, e. derm cells were infected with bev (5 pfu/cell), or mock-infected, and a kinetic study was performed, fixing cells with pfa at different times between 10 and 48 hpi. nuclei were stained with hoechst and observed by fluorescence microscopy (fig. 6a ). while nuclei from control cells were all evenly stained, abnormal brighter and smaller nuclei were instead observed in some bev infected cells at late times postinfection. at 30 hpi we found a small percentage of apoptotic nuclei (6%), which increases to 18% at 38 hpi. at 48 hpi the percentage of apoptotic nuclei recorded was 12%, but there has to be borne in mind that at this time postinfection many cells were already detached and floating in the culture medium, and therefore, the percentage of apoptotic cells at this time point was underestimated by this approach. therefore, in order to quantify more efficiently the kinetics of appearance of apoptotic nuclei, we analyzed the nuclear dna content by flow cytometry after pi staining. apopotic nuclei show a reduced fluorescence when compared to non-apoptotic nuclei, and appear as a broad subg 0 -g 1 peak [51] . hence, e. derm and mrc-5 cells infected with bev (5 pfu/cell) were harvested (monolayers and medium) at 10, 24, 38 and 48 hpi, stained with pi and analyzed by flow cytometry. fig. 6b shows a representative example of the cell cycle profiles obtained from e. derm and mrc-5 cells where we could observe a gradual increase in the amount of cells present in the subg 0 -g 1 peak with increasing time postinfection, although broader peaks were observed in e. derm cells than in mrc-5 cells. the assay was performed three times, and the results of the three experiments are represented in fig. 6c . as shown in the figure, 11.1763.05% of e. derm cells infected with bev appear in the subg 0 -g 1 peak at 24 hpi, and this percentage progressively increased at 38 and 48 hpi (28.1366.31 and 43.4767.64% of apoptosis respectively). as observed in previous analysis of protein synthesis and cleavage of caspase substrates, in mrc-5 cells the apoptotic process is delayed, with 5.3760.97% of apoptotic cells at 38 hpi, increasing to 9.660.46 at 48 hpi. once we have determined that bev infection causes apoptosis in both e. derm and mrc-5 cells, and knowing that the caspase cascade is involved, we wanted to know which upstream caspase(s), and therefore which apoptotic pathway(s) (death receptor and/or mitochondrial pathway) could be involved. for that, e. derm and mrc-5 cells were infected with bev (5 pfu/cell), and treated with a general caspase inhibitor (z-vad-fmk), a caspase-8 inhibitor (z-ietd-fmk), or a caspase-9 inhibitor (z-lehd-fmk), and the percentage of cells at the subg 0 -g 1 zone was determined at 48 hpi by flow cytometry after pi staining. the assay was performed twice. it was observed that, in both e. derm and mrc-5 cells, the general caspase inhibitor clearly protected cells against bevinduced apoptosis (17.6569.69 and 664.53% of apoptosis respectively in z-vad-fmk treated cells vs 52.4565.87 and 16.462.40 in non-treated cells), while the other two inhibitors produced a partial reduction in the apoptosis levels, being this decrease higher with the caspase-8 inhibitor (35.4067. 35 and 8.7064 .24% of apoptosis respectively) than with the caspase-9 inhibitor (45.5067.21 and 1263.96% of apoptosis respectively) ( fig. 7 a and b) . for both e. derm and mrc-5 cells the difference in the level of apoptosis between non-treated cells and cells treated with the inhibitor of caspase-9 was not statistically significant. nonetheless, by western blot analysis we observed a clear effect of this inhibitor on the cleavage of the caspase substrate eif4gi. for that, untreated and inhibitor-treated e. derm and mrc-5 cells infected with bev were collected at 24 and 38 hpi respectively. the cell extracts were incubated with the antibody against eif4gi, and with anti-bev-n and anti-actin antibodies as infection and load control respectively (fig. 7c) . in untreated, bev-infected cells, an intense 50-kda band corresponding to eif4gi cleaved product was observed (lanes 1), but the protein was almost totally protected from cleavage by the general caspase inhibitor (lanes 2), and partially protected with caspase-8 (lanes 3) and -9 (lanes 4) inhibitors. interestingly, cleavage of parp was not significantly prevented by any of the caspase inhibitors, and it was only slightly inhibited when used at a higher concentration (100 mm) (data not shown). these results, also in agreement with those obtained by observation of infected cells using light field microscopy (not shown), reveal a trend whereby cell damage induced by apoptosis is higher in the untreated cells, followed in first place by cells treated with caspase-9 inhibitor, secondly by cells treated with caspase-8 inhibitor, and then by cells treated with the general caspase inhibitor. as a whole, these results indicate that although both cell death receptor and mitochondrial pathways are implicated in the bev induction of apoptosis, the mitochondrial pathway could be secondarily activated by caspase-8-mediated cleavage of bid. to further ascertain the involvement of the mitochondrial pathway in bev-induced apoptosis we analyzed the cellular distribution of cytochrome c at different times postinfection. for this assay e. derm and mrc-5 mock-infected cells or cells infected with bev (2.5 pfu/cell) for 24 h were fractionated into cytosolic and membrane-bound organellar fractions as previously described [52] , and analyzed by western blot. cells treated with staurosporine were used as positive control of mitochondrial pathway activation. as shown in figure 8 in mock-infected e. derm cells, cytochrome c was exclusively found in the insoluble membranous fraction (lanes 2), while in bev-infected cells cytochrome c release to the cytosol could be observed at 24 hpi (lanes 3), and at a similar rate as in staurosporine-treated cells (lanes 1). in mrc-5 cells cytochrome c was clearly present in the mitochondria containing fraction of the differently treated cells, but it could be only barely observed in the cytosolic fractions of cells infected for 24 hpi, while it was clearly observed in the cytosolic fraction of staurosporine-treated cells. this result is in agreement with all the above results that indicate that in mrc-5 cells the apoptotic process occurs at a lower rate (see fig. 3 , 5a, 6b and 6c). the slight reduction in the percentage of apoptotic cells upon treatment of infected cells with the caspase-9 inhibitor could suggest that the mitochondrial apoptotic pathway may be triggered by caspase-8-mediated cleavage of the pro-apoptotic protein bid. to study the potential involvement of bid in this activation we examined bid proteolytic processing in bev infected cells. for this, the cellular fractions previously obtained were also analyzed with an anti-bid antibody. as shown in figure 8 , cleavage of bid into the 15-kda pro-apoptotic active form (tbid) could be observed in both cell lines infected with bev (lanes 3). on the other hand, in mock-infected cells (lanes 2) and in cells treated with staurosporine (lanes 1) only the intact 22-kda form of bid was detected. these results confirm that the mitochondrial pathway is involved in bev-induced apoptosis, and points to cleavage of bid by caspase-8 as the triggering factor. to analyze the potential effect of apoptosis on bev replication, e. derm and mrc-5 cells were infected with bev (5 pfu/cell) in the absence and presence of z-vad-fmk (50 mm), and supernatants were collected at 8, 16, 24, 32 and 48 hpi. viral titers were determined by plaque assay. the experiment was performed in triplicate. as shown in figure 7d , there were not significant differences between virus yields obtained at any of the time points analyzed in the absence or presence of the general caspase inhibitor in either cell line. similar results were obtained when the experiment was performed infecting both cell lines with bev at a low multiplicity of infection (0.05 pfu/cell) (data not shown). therefore, this result indicates that caspase-mediated apoptosis is not required for virus replication and/or the release of progeny virions. toroviruses have been scarcely studied to date, and hence little is known about both the torovirus effect on the infected cells, and the virus-host interactions established during infection. from studies on pathogenesis caused by btov in experimentally infected calves, it was described that the virus causes a cpe in the epithelial cells of the intestine, that results in severe vacuolar degeneration, necrosis and exfoliation of the enterocytes, inducing villous atrophy, crypt hyperplasia and in some cases fused villi [66] . on the other hand, initial characterization studies about bev infection in e. derm or ems cells infected in vitro showed that cytopathological changes are apparent at about 21 hpi only in about 10% of the cells [67] . we obtained a similar result in e. derm cells infected at a multiplicity of infection of 5 pfu/cell, where at 24 hpi only some cells show characteristic cythopatic titration. graphic representation of the media 6 the standard deviation of three independent experiments. there were no statistically significant differences between the values in the absence and presence of z-vad-fmk for any of the two cell lines and at any of the time points tested (p.0.5; student's t test). doi:10.1371/journal.pone.0020972.g007 features as cell rounding and detachment. however, extensive cpe was observed at 48 hpi, with many cells floating in the medium. to obtain a more detailed image of the cpe caused by bev, infected cells were examined by electron microscopy. we observed that, at 24 hpi, some bev infected cells exhibit morphological changes characteristic of apoptotic cells, such as a condensed nucleus, membrane blebbing, altered mitochondria, and cell disassembly in vesicles. this suggests that bev infection could induce apoptosis, and this fact could be related to the cpe described in btov infected animals. the list of viruses that cause apoptosis in infected cells has expanded significantly in the last two decades, and it includes several coronaviruses [14, 18, 19, 21, 22, 24, 68, 69] . to investigate whether bev actually causes apoptosis we sought to obtain some biochemical evidences. e. derm cells might not be optimal for investigating host-interactions because it is likely that many of the cellular proteins would not be recognized by available antibodies. therefore, we used also mrc-5 cells, that we have identified as susceptible for bev infection. by performing a metabolic labeling in infected e. derm and mrc-5 cells, we observed that there was protein synthesis inhibition, of both cellular and viral proteins, between 16 and 24 hpi in e. derm cells, and between 24 and 30 hpi in mrc-5 cells. there are several factors likely to be involved in the induction of this protein synthesis inhibition, common during the apoptotic onset. the generation of dsrna during genome replication is a usual factor among most rna viruses, including coronaviruses [70] , and in fact, we have observed dsrna accumulation both in e. derm and mrc-5 cells infected with bev (á vila et al., unpublished data). dsrna is, in turn, an activator of the innate immune response against viral infections [71] . dsrna binds and activates two apoptosis triggers that lead to protein synthesis inhibition. on one hand, it activates 29-59-oligoadenylate synthetase, leading to activation of latent rnase l and degradation of ssrnas [34] . cleavage of rrna was observed in both cell lines from 16 hpi, suggesting that rnase l could be activated. cleavage of 28s rrna was described to occur in mhv infected cells, although in that report the authors showed that the 28s cleavage was an early event in mhv infection that was probably independent of apoptosis, and they argued that another rnase of cellular or viral origin other than rnase l was responsible for this cleavage [72] . in addition, it has been proposed that another as yet unidentified rnase is involved in 28s rna specific cleavage during apoptosis [73, 74, 75, 76] . therefore, specific assays will be required to determine whether rrna cleavage observed in bev infected cells is mediated by rnase l and/or other ribonuclease. on the other hand, dsrna also activates endogenous pkr, which leads to eif2a phosphorylation [38, 39, 40] . here we show that both pkr and eif2a become phosphorylated upon bev infection. however, we cannot discard the possibility that any of the two other virus-activated cellular kinases known to phosphorylate eif2a, the pkr-like endoplasmic reticulum kinase (perk) [77] and the general control nonderepressible-2 kinase (gcn2) [78] , might be responsible for bev-induced eif2a phosphorylation. in this regard, it has been described that, in 293/ace2 cells, sars-cov induces apoptosis via pkr and eif2a phosphorylation. however, these two events seemed not to be connected, since inhibition of pkr expression prevented apoptosis but not eif2a phosphorylation. in that case perk was found to be the responsible kinase [70] . therefore, the rna degradation plus eif2a phosphorylation found in bev infected cells could contribute to the protein synthesis inhibition upon bev infection. the unmistakable sign of the apoptosis induction is the activation of a caspase cascade, which results in the proteolytic cleavage of a series of proteins known to exert important cellular functions [45, 79] . here we show that cleavage of parp and eif4gi substrates readily occur in bev-infected cells, both e. derm and mrc-5, in a time-dependent manner. these results also suggest that eif4gi degradation could be another factor contributing to the observed protein synthesis inhibition. in addition, the nuclei morphological changes characteristic of apoptotic cell death and the loss of nuclear dna content with time postinfection determined by pi staining and analysis by flow cytometry, are all consistent with the notion that bev causes caspase-mediated apoptosis. the study of the apoptotic factors and the nuclear dna content at several times postinfection indicates that in e. derm cells the chronology of the processes could be as follows: first pkr and eif2a phosphorylation (starting at 6 hpi), rrna degradation, parp and eif4gi cleavage by caspases and protein synthesis inhibition (between 16 and 24 hpi), and finally, nuclei degradation (between 24 and 38 hpi). the analysis of most of these parameters in bev infected mrc-5 cells indicates that in them the apoptotic process occurs at later times postinfection. this observation is in agreement with the lower infection rate in these cells when compared to e. derm cells, as determined by the lower viral yields (see fig. 7d , untreated cells) and lower viral protein expression (fig. 3) . moreover, this result also indicates that caspase-mediated apoptosis requires active bev replication. in this regard, we could not observe either the characteristic morphological features of apoptosis or the cleavage of parp and eif4gi in e. derm or mrc-5 cells inoculated with uv-inactivated virus. similar results have been described with sars-cov [68] , while in the case of mhv it has been reported that viral replication was not required, and it was suggested that virus fusion triggered after virus binding to the cell receptor was sufficient to initiate the apoptosis signal through fas activation [12] . the analysis of both, dna content and eif4gi cleavage, as well as the de visu examination of cells infected in the presence of caspase inhibitors, showed that both the intrinsic and extrinsic apoptotic pathways are involved. moreover, the observed cytochrome c release to the cytoplasm shows that the mitochondrial pathway is involved. nonetheless, the cell death receptor pathway seems to be predominant in both cell lines since, as determined by the analysis of the nuclear dna content by flow cytometry, caspase-8 inhibitor (extrinsic pathway) causes about 32% and 47% reduction in apoptosis in e. derm and mrc-5 cells respectively, compared to reductions of 13% and 27% respectively observed after treatment with caspase-9 inhibitor (mitochondrial pathway). our results suggest that the mitochondrial pathway can be activated indirectly through cleavage of the pro-apoptotic bcl2 family member bid by caspase-8, as it has been shown to occur in ccov [20] and mhv infected cells [80] . a question arises as to how the extrinsic pathway is activated by bev infection. in this regard, fas-mediated signaling has been implicated in apoptosis in response to infection by different viruses (reviewed in [81] ). the activation of the fas pathway may occur extracellularly through the binding of fas by fas ligand (fasl) or intracellularly through other signaling pathways. several studies have indicated that pkr is involved in activating fas-dependent apoptosis independent of fasl in response to various stress-inducing stimuli, including virus infection [31, 82, 83, 84, 85, 86] . in influenza virus infections, early activation of pkr seems to be important for upregulating fas gene expression and induction of apoptosis [86, 87] . in this regard, we have observed an early induction of fas gene expression at the level of transcription upon bev infection (unpublished results). thus, the possibility that, as in the case of influenza virus and other viruses, fas signaling pathway might be responsible for apoptosis induction in bev infected cells merits further investigation. interestingly, parp cleavage does not seem to be inhibited significantly by any of the caspase inhibitors used. cleavage of parp is considered a biochemical hallmark of apoptosis, however it can be regarded as an ''special'' caspase substrate, since it can be cleaved by both caspases-3 and -7 [79] . therefore, as opposed to most substrates known to be cleaved during apoptosis by caspase-3, such as gelsolin, a-fodrin, lamin, and probably eif4gi, parp can be cleaved in the absence of caspase-3 [43, 88] or in the presence of caspase-3 specific inhibitor [44] . moreover, it has been suggested that caspase-7 is more efficient than caspase-3 for parp cleavage [89] . therefore, it is possible that due to this redundancy, parp cleavage can still proceed in the presence of caspase inhibitors. although it has been reported that cleavage of parp is inhibited in jurkat cells treated with z-vad-fmk (100 mm) [90] , indicating that this inhibitor also blocks caspase-7, cell-type specific differences, such as the abundance of caspases, could explain this discrepancy. specific experiments would be required to ascertain which caspase is cleaving parp in cells infected with bev in the presence of the general caspase inhibitor z-vad-fmk. our results indicate that apoptosis does not affect virus replication and/or release in vitro. we have observed that inhibition of the apoptotic process by treating bev infected cells, either with low or high multiplicity of infection, with a general caspase inhibitor, does not influence the amount of virus released to the medium. significantly, in bev infected cells, maximum viral progeny is already obtained by 24 hpi [67] . therefore, the virus seems to complete its replication cycle before the onset of apoptosis, a common strategy used by several rna viruses, and specifically by some coronaviruses, to overcome the effect of premature cell death [14, 18, 20] . in summary, our study shows that bev infection leads to caspase-dependent apoptotic cell death in e. derm and mrc-5 cells. the apoptotic process induced by bev is triggered at late times postinfection, once the viral progeny is produced. however, in torovirus infected animals, apoptosis could favour the viral infection, likely facilitating the viral dissemination and pathogenesis, as suggested with a great number of viruses, mainly rna viruses that induce apoptosis in the final stages of the replication cycle [27, 28, 91] . the apoptotic death of infected cells causes tissue destruction. in the case of torovirus, the associated gastroenteritis could be related to the apoptosis of the mucosa epithelial cells. in this regard, it has been previously 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fluoromethylketone (z-vad.fmk) inhibits apoptosis by blocking the processing of cpp32 physiological significance of apoptosis in animal virus infection bcl-2 inhibits the caspase-dependent apoptosis induced by sars-cov without affecting virus replication kinetics we thank paco rodríguez, juan ramón rodríguez and randy albrecht for critical readings of the manuscript. we thank various researchers who supplied reagents for the work; their names are listed in the text at appropriate locations. we also want to thank sylvia gutierrez for her expert technical assistance with flow cytometry analyses, and maría teresa rejas for processing the samples for the electron microscopy and for taking the pictures. we wish to thank miriam jiménez for her invaluable technical assistance. conceived and designed the experiments: amm dr. performed the experiments: amm ag dr. analyzed the data: amm dr. wrote the paper: amm dr. key: cord-282202-q2q4vies authors: banerjee, amitava; katsoulis, michail; lai, alvina g.; pasea, laura; treibel, thomas a.; manisty, charlotte; denaxas, spiros; quarta, giovanni; hemingway, harry; cavalcante, joão l.; noursadeghi, mahdad; moon, james c. title: clinical academic research in the time of corona: a simulation study in england and a call for action date: 2020-08-13 journal: plos one doi: 10.1371/journal.pone.0237298 sha: doc_id: 282202 cord_uid: q2q4vies objectives: we aimed to model the impact of coronavirus (covid-19) on the clinical academic response in england, and to provide recommendations for covid-related research. design: a stochastic model to determine clinical academic capacity in england, incorporating the following key factors which affect the ability to conduct research in the covid-19 climate: (i) infection growth rate and population infection rate (from uk covid-19 statistics and who); (ii) strain on the healthcare system (from published model); and (iii) availability of clinical academic staff with appropriate skillsets affected by frontline clinical activity and sickness (from uk statistics). setting: clinical academics in primary and secondary care in england. participants: equivalent of 3200 full-time clinical academics in england. interventions: four policy approaches to covid-19 with differing population infection rates: “italy model” (6%), “mitigation” (10%), “relaxed mitigation” (40%) and “do-nothing” (80%) scenarios. low and high strain on the health system (no clinical academics able to do research at 10% and 5% infection rate, respectively. main outcome measures: number of full-time clinical academics available to conduct clinical research during the pandemic in england. results: in the “italy model”, “mitigation”, “relaxed mitigation” and “do-nothing” scenarios, from 5 march 2020 the duration (days) and peak infection rates (%) are 95(2.4%), 115(2.5%), 240(5.3%) and 240(16.7%) respectively. near complete attrition of academia (87% reduction, <400 clinical academics) occurs 35 days after pandemic start for 11, 34, 62, 76 days respectively—with no clinical academics at all for 37 days in the “do-nothing” scenario. restoration of normal academic workforce (80% of normal capacity) takes 11, 12, 30 and 26 weeks respectively. conclusions: pandemic covid-19 crushes the science needed at system level. national policies mitigate, but the academic community needs to adapt. we highlight six key strategies: radical prioritisation (eg 3–4 research ideas per institution), deep resourcing, non-standard leadership (repurposing of key non-frontline teams), rationalisation (profoundly simple approaches), careful site selection (eg protected sites with large academic backup) and complete suspension of academic competition with collaborative approaches. number of full-time clinical academics available to conduct clinical research during the pandemic in england. in the "italy model", "mitigation", "relaxed mitigation" and "do-nothing" scenarios, from 5 march 2020 the duration (days) and peak infection rates (%) are 95(2.4%), 115(2.5%), 240 (5.3%) and 240(16.7%) respectively. near complete attrition of academia (87% reduction, <400 clinical academics) occurs 35 days after pandemic start for 11, 34, 62, 76 days respectively-with no clinical academics at all for 37 days in the "do-nothing" scenario. restoration of normal academic workforce (80% of normal capacity) takes 11, 12, 30 and 26 weeks respectively. the pandemic sars-cov-2 virus (causing the disease, is unprecedented in its impact on individuals, populations and health systems [1] . since the first cases in wuhan, china in november 2019, every country has been affected [2, 3] but with wide variations in the ability and capacity to respond, with only half estimated to have operational readiness [4] . countries hit later can benefit and learn from acquired knowledge and experience of preceding countries. as part of this response, the research effort is crucial for development, testing and adoption of effective preventative and treatment [5, 6] . a pubmed search (november 2019 to april 2020), using terms "coronavirus" and "covid-19" showed 2206 and 1604 articles respectively, suggesting swift global research mobilisation. however, the publication mix shows the vast majority are reviews, opinions and commentary rather than formal research. many publications on covid-19 are not clinically led, and many are not directly clinically informed. "learning is difficult in the midst of an emergency" [7] , but our ability to deliver timely, high-impact clinical research, relevant to patients and populations, is critical across the academic spectrum [8] , from "bench to bedside to big data", whether basic biology, repurposed and novel therapeutic approaches, vaccines or modelling. obstacles to and strategies for delivering research during a pandemic are poorly characterised. anecdotally, many countries have a baseline shortage of clinical academics in translational science [9] and many leading pathfinder health institutions are within major international transport hubs (london, madrid, new york), which are affected early in the pandemic. lockdowns close university departments and funding bodies, with alternative funding sources (charities, philanthropy) hit by stockmarket falls and competing demand. frontline remoteness impedes communication of urgency to decision makers, themselves usually selected for process delivery rather than dynamic adaptability. critical researchers with relevant virology/immunological/intensive care knowledge are drawn in to local or national clinical responses. other academic staff most likely to redeploy to covid-19 research self-select for immediate response roles [10] with universities prioritising repurposing to frontline care [11] . high disease rates, required self-isolation periods [12, 13] and distractions of remote working degrade the focus needed to create new or repurposed research delivery structures. we therefore wanted to understand the pandemic research process and describe early lessons. our aims were to: (i) model potential impact of the pandemic on clinical academic capacity in england relating to covid-19; and (ii) develop evidence-based recommendations to inform the optimal scientific response to covid-19. based on our previous analysis of covid-19 cases and excess deaths in england [14] , we considered four scenarios of government interventions associated with different levels of population infection rates: 80% ("do-nothing"), 40% ("relaxed mitigation"), 10% ("mitigation") and 6% ("italy model"), since "partial suppression"(1%) and "full suppression"(0.001%) were no longer feasible. the analyses of excess deaths used data in a cohort design with prospective recording and follow-up from the clinical research using linked bespoke studies and electronic health records (caliber) open research platform with validated, reusable definitions of several hundred underlying conditions [15, 16] , linking electronic health records(ehr) from different data sources (via uk unique individual identification data, nhs numbers): primary care (clinical practice research datalink-gold), hospital care (hospital episodes statistics), and death registry (office of national statistics). approval was via the independent scientific advisory committee (16_022r) of the medicines and healthcare products regulatory agency in the uk in accordance with the declaration of helsinki. key variables were population infection rate, background mortality risk based on underlying conditions, and relative risk (rr) of mortality associated with covid-19. we used real-time data until 7 april 2020 for the number of confirmed cases and deaths [17] . we designed and implemented a simple stochastic model to predict number of new cases in the population. since the number of new cases are proportional to the active cases of the previous date (see web appendix), we used official data from 10 april and calculated the ratio new confirmed cases of day n active confirmed cases of day ðnà 1þ from 5 march onwards. we explored four different scenarios of growth of the infection curve, reflecting different government policies (do-nothing, relaxed mitigation, mitigation and the italy model), from april 10 (day 36 in our study which coincides with the date of the analysis) until day 250, see web s1 table in s1 file. we assumed that an individual remains infected for 2 weeks, followed by death or immunity, and that actual cases were~20 times more than confirmed cases, as people with mild or no symptoms are not routinely tested(based on prior estimates of 5-to 100-fold) [18] . further details are specified in the web appendix. we used nhs digital data (december 2019) to quantify number of doctors in england (n = 125,119) [19] . baseline number of clinical academics was estimated as 5% of doctors [20]: 6255 in england. based on uk clinical academic funding [21] , we assumed 50% fte (full time equivalent) overall, equivalent to~3000 100% fte academics, and 25% of doctors off sick and/or socially isolating at any time [22] . there are 1953 intensive care, 7678 emergency medicine, 395 infectious diseases and 2748 respiratory doctors in england [19] . we assumed doctors of any specialty could contribute to the covid-19 academic response, and necessary research skills and training were homogeneously available throughout the medical workforce. clinical academics are not available for research if: (i) they are delivering frontline care due to health system strain, or (ii) they are off sick. we modelled two scenarios with no medical academic capacity at 10% (low strain on the health system) and 5% (high strain on the health system) infection rates respectively. our outcome was the available number of medical academics during the pandemic in england. we assumed that the number of potentially available 100% fte clinical academics in research is 3200, but it is obvious that this number has decreased from the early days of the covid-19 pandemic. we present in detail the assumptions of modelling of available clinical academics in the web appendix. 1. northern italy: gq provided first-hand experience of the pandemic as a physician in bergamo, italy. 2. health care worker (hcw) cohort study: our team recently set up and started recruitment for the "healthcare worker bioresource: immune protection and pathogenesis in sars-cov-2" study (covid-hcw; nct04318314) [23] . 3. nightingale hospital: a new nhs field hospital has been established, providing extra medical and intensive care capacity for provision of care to covid-19 patients with a maximum theoretical capacity of 4000 beds, mainly intensive care [24] . we describe the scenario, staff involved and clinical and research priorities, and constraints. based on our model and our case studies, we have developed pragmatic recommendations for clinical research priorities relating to covid-19. study approval was granted by the independent scientific advisory committee (16_022r) of the medicines and healthcare products regulatory agency in the uk in accordance with the declaration of helsinki. assuming the "low strain on the health system" model (where there is no academic capacity at population infection rate of 10%), fig 2 shows that less than 400 100%fte clinical academics (~13%) will available after april 10 for 11, 34, 62, 76 days for the scenarios of "italy model", "mitigation", "relaxed mitigation" and "do-nothing" respectively. in the "do nothing scenario", no clinical academics are available to do research for 37 days (3/5/2020 to 8/6/2020). the predicted dates to reach 2560 clinical academics (80% normal capacity) are 23/6/2020, 1/7/2020, 3/11/2020 and 10/10/2020 for the scenarios of "italy model", "mitigation", "relaxed mitigation", and "do-nothing", respectively. in the "high strain on the health system" model (where there is no academic capacity at population infection rate of 5%), in the "relaxed mitigation" scenario, no clinical academics can do research for 18 days (13/5/2020 to 30/5/2020) and in the "do-nothing" scenario, from 23/4/2020 to 28/6/2020. the predicted dates to reach 2560 clinical academics (80% normal capacity) is 23/6/2020, 2/7/2020, 7/11/2020 and 11/10/2020 for the scenarios of "italy model", "mitigation", "relaxed mitigation", and "do-nothing" respectively. northern italy. as the first western region to be affected (lombardy, bergamo) there was effectively no warning. almost overnight a huge surge of severely ill patients hit us. it was the beginning of a nightmare. with no approved treatments, we had to re-organize the hospital wards, itu beds, transform simple general into sub-intensive units, commit all doctors from all specialities and research to covid-19 in a matter of hours-days. it was "catastrophe medicine", research was impossible and approaches were empiric based on analogy to other diseases. autopsy was our only science. "mors ubi gaudet succurrere vitae-where the dead are happy to help the living" and we started to appreciate the high rate of thrombotic complications and pulmonary pathology, initiating empiric anticoagulation and corticosteroids. only weeks later with external partnerships were formal randomized trials initiated. the covid-hcw study. italy informed our strategy. the team nucleus was 5 senior lecturers and 1 professor, all of whom had all their clinical work stopped as irrelevant in the pandemic (cardiac mri) and who had a track record of monthly large grant writing and detailed systems knowledge. the hospital had no emergency department, so it was protected with a large institution behind it. "exponential teams" were created to deliver national and local components of research permissions-permissions took 100 documents and~40 staff working at least part-time to deliver in 7 days (covid-consortium.com). following scoping, we rejected all but the most basic of aspirations: to capture (questionnaire, bloods and nasal swab) 400 hcw and track changes over 16 weeks-no clinical trial of an investigational medicinal product (ctimp) and no direct work with covid-19 patients. by day 16 from concept, 400 hcws had been recruited and the study was in follow-up. at this stage, funding, aliquoting, and detailed basic science plans were embarked on [23] . nightingale hospital. the nightingale hospital was the largest field hospital in europe with the largest number of intensive care and step-down facilities for covid-19. it was set up in 14 days from initial concept to first patient admitted nightingale is a learning system, underpinned by research. for patients, staff and wider nhs benefit, the design incorporates a commitment to learning fast and acting fast across all dimensions: clinical, operational, and staff wellbeing. our research approach is: (i) embedded within the quality and learning team, (ii) simple; and (iii) high-quality and high-volume recruitment. the onsite team is backed up by qmul, ucl and uclp, with multidisciplinary expertise, including virology, immunology and therapeutics. from an initial two clinical academic staff (ab and jm), a research governance structure has been set up rapidly and a simple strategy has been established. covid-19 consented studies can be observational or interventional (drugs), in patients or staff. we plan just one initial study in each domain, choosing the simplest possible approaches: patient observational(isaric), patient therapeutic(recovery), staff observational (covid-hcw with expansion to n = 1000) and staff therapy (pre-and/or post-exposure prophylaxis studies-to be confirmed]). the first patient therapeutic trial patient will be recruited on day 8 after first patient admitted. other studies (data, staff surveys) can be conducted at other sites (table 1) or after the initial exponential wave peaks. in addition, there are opportunities for non-consented research, such as epidemiologic and advanced data analytics; e.g. initiatives such as decovid [25] to mobilise data, computer scientists, analysts and analytic infrastructure, including and clinical expertise. there is potential to link effective learning directly to and from clinical questions. after discussion among co-authors, and consensus among a stakeholder group at the nightingale hospital, we produced recommendations for a covid-19 clinical research strategy ( table 2 ). in the first study of clinical academic capacity in the covid-19 era, we show the existential threat to research responses facing the uk and other countries. urgent recognition and mobilisation are required to ensure prioritisation of the most appropriate and clinically imperative science. we have developed recommendations relevant to all health systems. the healthcare and public health emergency caused by covid-19 is not in question, fuelling global discussion, modelling and multidisciplinary research at a pace rarely seen [26, 27] . however, strain on clinical academic workforce and infrastructure in different countries are notable omissions. despite programmes to promote research preparedness in epidemics, covid-19 poses particular challenges [28] to our responses which rest ultimately on research, whether vaccines, drugs, ventilation strategies, risk prediction or machine learning. our experiences are echoed in china, italy and other countries facing the pandemic. a. data analysis using existing systems b. simple delivery of samples 4. resourcing: profound project resourcing to deliver-essentially exponentially more staff than usual (e.g. estimate of up to 50 people per project immediately). a. clinical research: in the community or in large academic health centres b. basic science: using teams without clinical or basic science transferable skills for covid-19 work, and protecting research workers from clinical service duties. 6. dissemination: across uk and internationally to those considering covid-19 clinical research. data sharing and collaboration both nationally and internationally. https://doi.org/10.1371/journal.pone.0237298.t002 there have been quick efforts and advances in fields as diverse as genomics [29] and data science [30] , with rapid-response calls from major funders [5, [31] [32] [33] . however, our data signal a need for a far broader paradigm shift in research design and implementation. at every stage in the traditional research pipeline, there are roadblocks hampering swift reactions necessary to tackle covid-19 within and across countries. even on a "war footing", research processes are unnecessarily time-and resource-consuming, particularly when involving randomized controlled studies. specific hurdles are: (i) staff -doctors and research nurses, but also access to labs; (ii) stuff-consumables difficult to obtain due to challenging supply chains especially if they are competing with clinical service delivery, e.g. personal protective equipment; (iii) siteideally research space near to clinical areas; and systems-approvals in a timely fashion, e.g. research ethics committee, health research authority, local research and development team and standard operating procedures ins relevant institutions. emergencies as far-reaching as the current scenario require total rethinking of research delivery, and aspects that work better when some of the processes are accelerated and the permissions expedited, may well yield long-term benefits outside of covid-19 research. here we have modelled clinical academic time in terms of numbers of staff and time in the pandemic. however, a far deeper examination of the role of clinical academics beyond "hours at the desk" is warranted in times of public health emergency to include the "what" and "how" of their work. for example, certain tasks such as research permissions and data analysis may be diverted away from clinical academics, who may be better placed to act as conduits between the clinical and public health spheres and teams of non-clinical researchers. the needs of the hour are patient-centred, data-driven and time-responsive, and it may be time to usefully change the role and function of the clinical academic. it is worth noting that this is occurring against a backdrop of declining clinical academic numbers [21] . our simulations suggest the pandemic will create health system strain for many critical months. depending on a range of covid-related factors, we show that the clinical academic workforce may be depleted when it is needed most to lead and conduct clinical research, even in a relatively well-resourced context such as the uk, whether by funding, number of universities and staff, infrastructure or policy. therefore, other countries are likely to be worse affected. covid-19 research is least likely to occur where it is most needed, magnifying the well-documented "10-90 research gap", where only 10% of resources for global healthcare research are devoted to low-income settings where 90% of preventable deaths occur [34] . although covid-19 is a unique threat, there are lessons to be learned from prior health research strategies to address structural inequities, such as the global fund for malaria, tb and hiv/aids [29] . without coordinated international responses, including urgent funding and infrastructure, research will be retrospective, patchy and unlikely to have an effect. we provide six clear recommendations for science in the uk and globally in relation to covid-19 (radical prioritisation, leadership, rationalisation, resourcing, careful site selection and dissemination). radical prioritisation is important where field hospitals are being established in rapid timescales in different countries with delivery constraints. high-quality evidence can be obtained, but studies need to be lean with minimal complexity for key operational steps: consenting, randomisation, drug delivery, monitoring, outcomes and follow-up. the number of patients recruited to deliver definitive answers needs to be large, with fast recruitment across multiple sites. furthermore, adaptive trial designs are preferred as new arms (e.g. multi-drug) can be generated swiftly and other arms dropped (e.g. supportive care if one arm has a signal of efficacy) without restarting permissions, via substantial amendments [35, 36] . leadership and rationalisation are the next key steps. balance needs to be struck between clinical researchers in contact with the "frontline" so that research questions are clinically relevant and timely, and having research active leaders who will not be protected from frontline work. rationalisation involves a study selection strategy that is deeply resourced for a limited number (1 or 2) studies per covid cohort. in selecting these studies, a single study of one investigational medical product versus standard of care (supportive care) with 50:50 randomisation is inefficient compared to studies with multiple therapeutic arms. most single agent approaches to covid-19 are likely to have, at most, a modest effect. we used a stochastic model accounting for infection rate, infection growth rate and clinical academic capacity using up-to-date official statistics. there are limitations to our model and its assumptions. our model was simple and was based only on observational patterns of the number of new cases and actual cases from publicly available data [17] . we conducted analyses on 10 april, and on 11 april, some extra~3000 cases were added retrospectively and distributed over the past 10 days-we did not include these data. it did not take into account infectious disease epidemiology parameters, such as the basic reproductive number (r0), and we did not consider differing levels of risk of infection [37] [38] [39] . our model on the availability of clinical academics makes several assumptions (web appendix), including the total number of 100% fte academics as~3200, with a uniform skillset across the workforce. in the first study to model and estimate the impact of covid-19 on the coordinated clinical academic response at system level, we show that all countries face depletion of their clinical academic workforce for several months, which will greatly hamper research in prevention and treatment. the number of studies needs to be rationalised urgently and background problems in clinical academia need to be overcome quickly. to quote sir jeremy farrar, "the only exit from this pandemic is through science" [40] and that requires staffing. novel coronavirus and old lessons-preparing the health system for the pandemic early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study health security capacities in the context of covid-19 outbreak: an analysis of international health regulations annual report data from 182 countries wellcome statements on novel coronavirus (covid-19 covid-19: real-time dissemination of scientific information to fight a public health emergency of international concern thinking globally, acting locally-the u.s. response to covid-19 a call for action to establish a research agenda for building a future health workforce in europe academic factors in medical recruitment: evidence to support improvements in medical recruitment and retention by improving the academic content in medical posts royal college of physicians. covid-19 and its impact on nhs workforce clinical academics freed up to support nhs during covid-19 pandemic protecting healthcare personnel from 2019-ncov infection risks: lessons and suggestions. front med covid-19: protecting health-care workers estimating excess 1-year mortality from covid-19 according to underlying conditions and age in england: a population based cohort using nhs health records in 3.8 million adults uk phenomics platform for developing and validating electronic health record phenotypes: caliber a chronological map of 308 physical and mental health conditions from 4 million individuals in the english national health service. lancet digit health covid-19 coronavirus pandemic country estimates: uk. 5 a fiasco in the making? as the coronavirus pandemic takes hold, we are making decisions without reliable data. statnews nhs digital workforce statistics one in four nhs doctors 'sick or in isolation covid-19: healthcare worker bioresource: immune protection and pathogenesis in sars-cov-2 (covid19-hcw coronavirus: nightingale hospital opens at london's excel centre call for covid-19 rapid response data science taskforce locally informed simulation to predict hospital capacity needs during the covid-19 pandemic projecting demand for critical care beds during covid-19 outbreaks in canada international severe acute respiratory and emerging infection consortium genomic diversity of sars-cov-2 in coronavirus disease 2019 patients modified seir and ai prediction of the epidemics trend of covid-19 in china under public health interventions operational and implementation research within global fund to fight aids, tuberculosis and malaria grants: a situation analysis in six countries. global health announcing the covid-19 therapeutics accelerator coronavirus news, funding and resources for global health researchers the developing world in the randomised evaluation of covid-19 therapy adaptive platform trial for community-acquired pneumonia the effect of control strategies to reduce social mixing on outcomes of the covid-19 epidemic in wuhan, china: a modelling study early dynamics of transmission and control of covid-19: a mathematical modelling study estimates of the severity of coronavirus disease 2019: a model-based analysis opinion: the only exit from this pandemic is through science. we must fund it. globe and mail key: cord-256424-t3dtabi4 authors: bousbia, sabri; papazian, laurent; saux, pierre; forel, jean marie; auffray, jean-pierre; martin, claude; raoult, didier; la scola, bernard title: repertoire of intensive care unit pneumonia microbiota date: 2012-02-28 journal: plos one doi: 10.1371/journal.pone.0032486 sha: doc_id: 256424 cord_uid: t3dtabi4 despite the considerable number of studies reported to date, the causative agents of pneumonia are not completely identified. we comprehensively applied modern and traditional laboratory diagnostic techniques to identify microbiota in patients who were admitted to or developed pneumonia in intensive care units (icus). during a three-year period, we tested the bronchoalveolar lavage (bal) of patients with ventilator-associated pneumonia, community-acquired pneumonia, non-ventilator icu pneumonia and aspiration pneumonia, and compared the results with those from patients without pneumonia (controls). samples were tested by amplification of 16s rdna, 18s rdna genes followed by cloning and sequencing and by pcr to target specific pathogens. we also included culture, amoeba co-culture, detection of antibodies to selected agents and urinary antigen tests. based on molecular testing, we identified a wide repertoire of 160 bacterial species of which 73 have not been previously reported in pneumonia. moreover, we found 37 putative new bacterial phylotypes with a 16s rdna gene divergence ≥98% from known phylotypes. we also identified 24 fungal species of which 6 have not been previously reported in pneumonia and 7 viruses. patients can present up to 16 different microorganisms in a single bal (mean ± sd; 3.77±2.93). some pathogens considered to be typical for icu pneumonia such as pseudomonas aeruginosa and streptococcus species can be detected as commonly in controls as in pneumonia patients which strikingly highlights the existence of a core pulmonary microbiota. differences in the microbiota of different forms of pneumonia were documented. the cause of pneumonia in intensive care units (icus) remains unknown in nearly 30% of cases despite extensive microbiological investigations [1, 2] . microbial communities previously identified, in deep respiratory samples, include bacteria, fungi and viruses for which the role in the observed pathology is not clear. microorganisms frequently identified in respiratory samples from icupneumonia patients included pseudomonas aeruginosa, staphylococci, enterobacteria, candida albicans, influenza virus, herpes simplex virus (hsv) and cytomegalovirus (cmv) [3] [4] [5] [6] [7] . in some investigations, a pathogenic bacterium is isolated, whereas in other cases, the number of colony forming units (cfu) is considered to determine the pathogenic character [8] . recently, the bacterial microbiota of patients with cystic fibrosis and ventilator-associated pneumonia (vap) were studied using 16s rdna gene amplification followed by clone libraries sequencing [9] [10] [11] . our laboratory has contributed to this work and has shown, by different sequencing approaches, that the microbial population of patients with cystic fibrosis was more diverse than expected [10, 11] . here, we use a comparable approach in order to study 185 episodes of icu pneumonia and 25 control cases. these patients were studied using broad-range primer amplification of the 16s rdna gene of bacteria and the intergenic spacer of 18s rdna gene of fungi followed by cloning and sequencing. we also used specific quantitative pcr (qpcr) to target fastidious bacteria and a spectrum of viruses. moreover, we tested samples from our patients by standardized routine culture, amoebal co-culture, blood culture, elisa targeted antibody detection, immunofluorescent assay antigenemia and antigenuria testing as routinely performed in such cases to compare these routine tests with molecular approaches. in preliminary results, we have reported the likely frequency of tropheryma whipplei and the occurrence of vegetable dna in pneumonia patients [12, 13] . in this work, we highlight the different compositions of microbiota in patients with four different types of icu-pneumonia. bacterial microbiota as evaluated by 16s rdna molecular assays were positive for at least one bacterium for 129 out of 185 bronchoalveolar lavage (bal) samples from patients with pneumonia as well as from 13 out of 25 from control individuals (p = 0.07). bacterial clone libraries from amplified 16s rdna genes (nearly 4,000 clones that contained exploitable sequences were included) identified 157 different bacteria at the species level. detailed data about the relative abundance and richness of each species in their corresponding library are summarized in data s1 and s2 in supplementary informations. bacterial clone libraries of patients showed that 44 libraries were characterized by the presence of only one bacterium, 40 libraries were polybacterial but dominated by one bacterium (50% of the clones in the library), whereas 45 libraries were polybacterial without any dominant bacterium (fig. 1) . bacterial clone libraries of controls showed that 2 libraries were characterized by the presence of only one bacterium, 4 libraries were polybacterial but dominated by one bacterium (50% of the clones in the library), whereas 7 libraries were polybacterial but without any dominant bacterium (fig. 1) . patients exhibited up to 15 bacteria in their bal fluids (mean 6 sd; 3.4862.80) (tables 1 and 2 ). overall, patients exhibited 146 different species belonging to 7 different phyla (13 classes, 23 orders, 44 families and 71 genera) of which 73 had not been previously observed in bal from pneumonia, whereas bacterial clone libraries of controls identified 38 species belonging to 4 different phyla (9 classes, 13 orders, 22 families and 27 genera). in patients, aerobic gram-negative bacilli, gram-positive cocci, and anaerobic bacteria from oropharyngeal flora were the most frequent bacteria identified (tables s1,s2,s3,s4, fig. 2) . surprisingly, bacteria that are usually associated with other diseases such as the gram-positive anaerobe atopobium vaginae, or from unexpected animal origins, such as enterococcus canintestini, were alsoc found. moreover, 51 strictly anaerobic bacteria (35%) were found in patients versus 17 anaerobic bacteria (44%) found in controls (p = 0.26). among those bacteria which were identified in controls, 24 bacteria were also identified in patients (fig. s1 , tables s2,s3,s4), including pseudomonas aeruginosa sequences respectively identified in 100% and 86% from 2 different clones libraries from 2 immunocompromised controls. in the second clone library, the 14% of the remaining sequences included achromobacter xylosoxidans, which also is a typical bacterium of nosocomial pneumonia. stenotrophomonas maltophilia sequences were found in 10% of clone library of another immunocompromised control, along with 5 other bacteria. similarly, sequences of streptococcus mitis (7% of the clone library) was identified along with 12 other bacteria in a control with acute respiratory distress syndrome (ards) and a history of aspiration pneumonia (ap) 6 days before bal sampling. additionally, arcobacter cryaerophilus, atopobium parvulum, lachnospiraceae bacterium, prevotella melaninogenica, and prevotella pallens were significantly more frequent in controls than in patients (p = 0.01, 0.01, 0.001, 0.01 and 0.01 respectively) (table s4) . bacterial clone libraries surprisingly showed 37 new phylotypes with 16s rdna sequence similarity lower than 98% to known bacteria available in the genbank database (data s1 and s2). among them, 32 novel bacterial phylotypes were identified in bal from patients, whereas 5 novel phylotypes were identified in bal from controls (fig. 3) . novel bacterial phylotypes identified in patients were more diversified, as they belonged to 6 different phyla including bacteroidetes (11 phylotypes), firmicutes (9 phylotypes), proteobacteria (9 phylotypes), actinobacteria (one phylotypes), acidobacteria (one phylotype) and spirochaetes (one phylotype). novel species identified in controls belong to bacteroidetes (2 phylotypes), firmicutes (2 phylotypes) and actinobacteria (one phylotype). prevotellaceae phylotypes represent 24% of all novel phylotypes identified and they were exhibited in patients with pneumonia as well as in control subjects (fig. 3) . results obtained using routine bal and blood culture are available text s1. fungal microbiota as evaluated by the intergenic spacer of 18s rdna at least one fungus was found in 31 bal patient samples and in 6 from controls (p = 0.37). positive patients exhibit up to 5 fungi in their bal fluids (mean 6 sd; 1.4060.83) (tables 1 and 2) . detailed data about the relative abundance and richness of each species in their corresponding library are also summarized in summary files (data s1 and s2) in supplementary informations. fungal microbiota obtained from patients showed the presence of 22 different species belonging to 2 phyla (8 orders, 11 families and 12 genera) among which 6 phylotypes had not been previously identified in bal fluids from pneumonia. clone libraries from controls, identified 5 fungi belonging to one phylum (2 orders, 4 families and 3 genera) among which 3 fungi were also identified in patients. candida species were the most common fungal species identified (tables s1,s2,s3 and s5). environmental fungi, which usually colonize water, food debris and humid building surfaces, were more notably identified in our study than in previous pneumonia studies. furthermore, tree fungi belonging to basidiomycota phylum, sporidiobolales sp., cryptococcus victoriae and hyphoderma praetermissum, were found for the first time in pneumonia bal samples in the present study, while candida utilis and periconia macrospinosa were identified only in controls. additionally, candida utilis was significantly more frequent in controls than in patients (p = 0.01). results of fungi obtained using a routine bal culture are available in text s1. four pneumonia patients were found to be positive for fastidious bacteria chlamydia pscitacii (1 case), mycobacterium sp. (2 cases) and mycoplasma pneumoniae (2 cases) by qpcr. in addition, qpcr enabled the detection of 7 different viruses. quantitative data of microorganisms identified by qpcr (loads or cycle threshold) are also provided and summarized in supplementary information (data s1 and s2). our study showed that at least one virus was identified in 74 bal samples from patients, and 11 from controls (p = 0.95). hsv and cmv were the most commonly identified viruses. while the prevalence of these two viruses in patients was not significantly different from that of controls (table s6) , cmv was more frequently identified in pneumonia patients than in controls. hsv and cmv coinfection was found in bal samples from 5 vap patients, 2 community-acquired pneumonia (cap) patients and 2 non-ventilator icu pneumonia (nv icu-p) patients and one control subject. coinfection with cmv and respiratory syncytial virus type a was detected in a bal from one nv-icu-p patient, and both hsv and vzv were identified in a bal from a cap patient. rhinovirus was identified in a control with ards, urinary infection and sinusitis. parainfluenza virus-1 was detected in 3 vap patients and an immunocompetent control with a pulmonary contusion. results obtained using routine serology and antigenemia for viruses and fastidious pathogens are available in text s1 overall, bacterial difference between patients and controls showed that bacteria belonging to bacilli and gammaproteobacteria were dominant in patients, whereas anaerobic bacteria related to bacteroidia (represented essentially by prevotella species) and clostridia were dominant in controls ( fig. 4) (p,0.01). mollicutes, which are represented by the mycoplasma genus, were only detected in patients with cap and vap (fig. 4) . as for fungal species, members of saccharomycetes were ubiquitously identified in all cohorts. eurotiomycetes, which are represented by aspergillus, penicillium and cladophialophora genera, were dominant in the cap cohort (fig. 5) . tremellomycetes, represented by the cryptococcus genus, was only identified in the nv-icu-p cohort, whereas figure 3 . a phylogenetic tree inferred from 16s rdna sequences of novel bacterial phylotypes. these novel phylotypes exhibited sequence similarities of less than 98% to known bacteria available in the genbank database, and they were classified in silico using ''classifier'' program. phylotypes are reported according to their genus or by the last possible classification determined by the program. when possible, phylotypes with the same classification are clustered together. the frequency of phylotypes in each cohort is shown on the right.. bacteroidetes are shown in purple, firmicutes in red, proteobacteria in blue, actinobacteria in yellow, acidobacteria in orange and spirochaetes in green. cap, community-acquired pneumonia; vap, ventilator-associated pneumonia; nv icu-p, non-ventilator icu pneumonia; ap, aspiration pneumonia; and cs, control subjects. doi:10.1371/journal.pone.0032486.g003 agaricomycetes and an unclassified ascomycota (melanized limestone ascomycetes) were only identified in the vap cohort. in addition, sordariomycetes, which is represented by the periconia genus, was only identified in controls. at the specie-level, 58 bacteria, 7 fungi and 5 viruses were common to at least two cohorts, among which pseudomonas aeruginosa, streptococcus mitis, prevotella melaninogenica, peptostreptococcus stomatis, candida albicans and hsv were commonly identified irrespective of cohorts, whereas haemophilus influenzae, staphylococcus aureus, streptococcus genomosp. c4, streptococcus parasanguinis and streptococcus pneumoniae were commonly identified in patients regardless of pneumonia type (fig. s2) . additionally, 24 bacteria, 3 fungi and 3 viruses were common to controls and at least one pneumonia cohort, whereas 34 bacteria, 4 fungi and 2 viruses were common to at least two pneumonia cohorts (fig. s2 ). in contrast, many microorganisms (102 bacteria, 17 fungi and 2 viruses) were restricted to one of cohorts (17 bacteria and 5 fungi only were identified in the cap cohort; 54 bacteria and 6 fungi only were identified in the vap cohort; 8 bacteria, 4 fungi and one virus only were identified in the nv icu-p cohort; 9 bacteria only were identified in the ap cohort; 14 bacteria, 2 fungi and one virus only were identified in controls) (fig. s2 ). microbial profiles of positive pneumonia bal fluids showed that 40 (25%) were characterized by the presence of one microorganism, whereas 116 (75%) were polymicrobial. in controls, 4 (20%) of bal fluids were characterized by the presence of one microorganism, whereas 16 (80%) were polymicrobial (data s1 and s2). available clinical data for patients and controls showed that monobacterial patients were more frequently, but statistically insignificant, subjected to initial antibiotic therapy than polymicrobial ones (p = 0.12; table 3 and table s7 ). in ventilated subjects, monomicrobial patients have a slightly shorter period of mechanical ventilation prior to the pneumonia episode as compared to polymicrobials. monomicrobial controls have a remarkably shorter period of mechanical ventilation before sampling compared to polymicrobials (p = 0.25; table 3 ). the same observation was showed for length of icu stay prior to sampling and for total length of hospital stay. according to these observations, the polymicrobial profiles of controls seem to be partially related to the high duration of icu stay before sampling. however, the icu mortality was higher in monomicrobial patients than in polymicrobial ones (p = 0.02). the icu mortality rate was higher in pneumonia patients for whom bal fluids exhibited only viruses or fungi, or both than in monobacterial or polybacterial patients (p = 0.01; table s7 ). a higher but not statistically significant icu mortality was also observed in pneumonia patients for whom bal fluids exhibited only viruses or fungi, or both than in controls with the same profile (p = 0.07; table s7 ). we next compared the bacterial communities found in our study to those found in lung specimens in five previous studies which were based on 16s rdna amplification [9, 11, [14] [15] [16] . comparative analyses of lung microbiota between these studies showed that 147 different genera were found in all of them. among these genera, 70 genera are widely distributed within the studies including gemella, haemophilus, megasphaera, neisseria, porphyromonas, prevotella, pseudomonas, staphylococcus and streptococcus genera which have commonly been found irrespective of study. in contrast, 77 genera were restrictively identified across the studies (table 4) . however, at the species level (only the studies that determined bacterial species were included [9, 11, 14] ), comparative analyses showed that from 59 bacteria commonly distributed within the studies, escherichia coli, haemophilus influenzae, prevotella oris, pseudomonas aeruginosa, staphylococcus aureus and streptococcus mitis were commonly found in the four studies. in contrast, 291 bacteria were restrictively identified across the studies (fig. s3) . consequently, comparative analysis at the specie-level showed that some bacteria, such as pseudomonas aeruginosa and staphylococci, are commonly found in pulmonary specimens. however, the pattern of distribution of many other species is distinctly heterogeneous and depending on the specific study and disease. variation of lung microbiota, from one individual to another and from one study to another, suggests that the repertoire of microorganisms associated with respiratory infections still remains incompletely understood. previous studies performed on respiratory specimens showed that unexpected bacteria are increasingly identified, as well as studies describing isolated cases of respiratory infection due to an unexpected bacterium that was detected using molecular techniques [9, 11, 14, [17] [18] [19] [20] . this study extends the analyses to bacteria, fungi and viruses in a large population of icu pneumonia using comprehensive molecular testing. our results demonstrate that nearly 50% of the microbial species found had not been previously reported in lung samples from pneumonia. therefore, the composition of icu-pneumonia microbiota is more complex, more extensive and more diverse than originally expected. however, we raise the question on the actual role of these microorganisms in pneumonia. indeed, our study reveals that some pathogens that till now had been considered typical for icu pneumonia, such as pseudomonas aeruginosa and streptococcus species, or viruses, such cmv and hsv, can be detected as commonly in controls as in patients (fig. s1 and s2 ). this result is emphasized by more recent studies by erb-downward et al. and hilty et al. who showed that a community of lung-resident bacteria including pseudomonas and streptococcus genera can be identified in patients with chronic obstructive disease or asthma, as well as in healthy people [15, 16] . our study agrees with the recent literature and highlights the existence of a core pulmonary microbiota, confirming the non-sterility of the lung [15, 16] . more interestingly, we showed that pulmonary microbiota heterogeneity can be observed between patients and controls, among pneumonia cohorts and among patients within the same cohort. high pulmonary microbiota heterogeneity was also observed between our study and other previous works performed on cystic fibrosis or vap [9, 11, 14] (fig. s3) . we found that some bacteria were commonly identified in all studies, whereas many others were only identified in one study, and most of these were unexpected. consequently, lung microbiota can vary greatly between individuals, depending on underlying diseases, habits and geographic origin. additionally, these unexpected microorganisms may explain a lack of response to drug therapies in some pneumonia patients. therefore, the possible extension of empiric treatments to cover a large spectrum of microorganisms, especially for patients who do not respond adequately to initial treatment, is questionable. another interesting observation was that mixed infection was observed in many bal fluids from pneumonia patients. interestingly, recent works report that probable interactions between parasitic species can occur in their host, and these reports also show that infection with a given microorganism may increase or decrease susceptibility to infection by another one or can create a cross-immunity response [21] [22] [23] [24] [25] [26] [27] . such interaction remains to be investigated. moreover, by comparing molecular testing to standard routine methods, this study reveals that many pneumonia-associated pathogens are fastidious or uncultured and highlights a wide discrepancy between culture and molecular microorganism repertoire. our study also shows that the molecular assay remains a more efficient method to detect microorganisms in the pneumonia samples, independently of atmospheric conditions and medium nutrient supplements, which are particularly important for culture, especially for fastidious microorganisms. in addition, microorganism diagnosis was obtained for 156 (84%) episodes of pneumonia by molecular tools compared with 120 (65%) pneumonia episodes for which microorganism diagnosis was successfully done by culture (table s8 ). in particular, molecular tools seem to be far more sensitive than culture for bacterial detection. this observation is based on the high number of microorganisms, especially bacteria which were identified by molecular methods compared with those detected by culture. in fact, standard and special bal cultures identified few, essentially easily-grown and strictly aerobic or facultative anaerobic bacteria (23 species) compared to molecular tools which identified 160 bacterial species (p,0.001) (fig s4 and s5 ). molecular tools enabled the identification of unexpected bacteria which usually colonize vaginal tracts, such as atopobium vaginae and peptoniphilus lacrimalis, or of other bacteria coming from unexpected animal origins, such as chlamydia pscittasi, enterococcus canintestini and streptococcus bovis, or of potentially known to be associated with other diseases, such as tropheryma whipplei, which were not identified by culture. furthermore molecular tools allowed the detection of pathogenic bacteria such as mycobacterium sp. and mycoplasma pneumoneae, for which identification attempts by culture using specific media were failed due to culture biases. moreover, all bacteria that were first associated with pneumonia in the present study were exclusively identified by molecular methods. these findings are coherent to results from previous studies on bacterial communities of respiratory diseases, including pneumonia, which showed that molecular assays are more sensitive than culture [9, 11, 14] . however, although molecular approaches identified more fungal species than culture, fungal diagnoses were positive for 79 (42%) episodes of pneumonia by culture compared with 31 (17%) pneumonia episodes for which fungal diagnosis was successfully obtained using molecular tools. thus, fungal bal culture was more sensitive to detect some cultured fungi, such as candida species, than molecular approaches. another important finding was the high number of novel bacterial species never previously described to date (bacteria with blastn similarity less than 98%). this result is concordant with similar studies of pneumonia and cystic fibrosis subjects [9, 14] and shows that in respiratory infections, more complex bacteria populations can exist, among which novel bacteria had never been previously identified. moreover, this finding was also supported by other studies performed on endodontic infections, demonstrating that many novel bacteria essentially resident in the oropharyngeal and dental plaque flora can be detected in these infections [28, 29] . the oropharyngeal and dental plaque flora is potentially suspected to be a reservoir and, thus, the source of icu pneumonia pathogens, which could suggest that these novel bacteria were inhaled through oropharyngeal tracts [30, 31] . nevertheless, molecular tools alone cannot give positive results in some cases, or they could just identify microorganisms known to be commensal or less pathogenic, where it may be useful to perform other tests, such as serology. this was the situation for 5 pneumonia patients for whom serology provided evidence for influenza a virus infection, whereas qpcr performed on their bal fluids was negative. moreover, by combining culture-based methods, blood culture and serology to molecular approaches we significantly increase the probability to detect microorganisms in the pneumonia episodes. in fact, by using these exhaustive laboratory diagnostic tools, we failed to identify a microbial agent in only 7% of the pneumonia episodes, which is significant when compared to previous studies where the microbial agent was not found in more than 30% of episodes of pneumonia (p,0.001) [32] . however, the clinical significance of these microorganisms and their role in the etiology of pneumonia remain difficult to be cleared as their correlation with the disease causation remains to be studied and confirmed in the future. nevertheless, our findings suggest that it would be highly recommended to develop a rapid molecular test to target, besides typical pathogens, potential pathogens known to be fastidious or uncultured (such as anaerobic ones), and that it would be useful to add it to existing routine standard techniques. in summary, our study reveals that the respiratory microbiota is more complex than expected. a large study was implemented in our laboratory over a threeyear period (january 2007 through december 2009) to perform an exhaustive etiologic diagnosis of pneumonia. the study involved three icus in the public hospitals of marseille, france (one medical icu and two medico-surgical icus). a total of 185 bal fluids, 185 blood samples and 185 urinary samples from 130 icu pneumonia patients were studied. a diagnosis of community-acquired pneumonia, ventilator-associated pneumonia and aspiration pneumonia was defined as previously described [32] [33] [34] . bal and blood sampling were performed as previously described [35] . a cohort of 25 icu patients without pneumonia was studied as controls. pneumonia patients exhibited 32 episodes of community-associated table 4 . comparison of lung microbiota between different studies. harris et al. [9] bittar et al. [11] bahranimougeot et al. [14] erb-downward et al. [15] hilty et al. [16] studied nucleic acid extraction, pcr amplification, cloning and sequencing bacterial and fungal dna extraction from bal samples was performed on a magnapure lc workstation (roche diagnostics, meylan, france), using a magna pure lc dna isolation kit ii (roche diagnostics) as previously described [12] . viral nucleic acids were extracted from 200 ml of bal fluids using an mdx workstation and the qiaamp virus biorobot mdx kit according to the manufacturer's instructions. dna was tested by pcr for bacteria using broad-range primers targeting the 16s rdna gene; pcr was also used to test for universal fungi using broad-range primers targeting intergenic spacer of 18s rdna gene (eurogentec, seraing, belgium) ( table 5 ). pcr product was cloned and approximately 48 clones were screened per library. pcr, cloning and sequencing were performed as previously described [12] . the obtained sequences were assembled and analyzed by chromaspro software and then blasted against those available in the genbank database (www.ncbi.nlm.nih.gov) for species identification. chimeric sequence search was performed with black box chimera check (b2c2) program [36] and by examining the blast profile of each sequence. suspected chimeric sequences were discarded from the study. sequences showing a similarity of .98% were considered to be known species, whereas sequences showing a similarity of ,98% were considered to be novel species. legionella sp., afipia sp.,bradyrhizobium sp., azorhizobium sp., mesorhizobium sp., balneatrix alpaca and pneumocystis carinii were tested by pcr using specific primers followed by sequencing of pcr products ( table 5 ). the sequences have been deposited in the genbank database (accession nu jf893554-jf893750). standard bacteriological bal culture and blood culture as phenotypic identification of isolated bacteria were performed as previously described [11, 37] . a 10 4 cfu cut-off defined a positive bal culture. blood culture were processed as previously described [37] . identification of fungi present in bal or blood samples was performed using a standard culture as previously described [38, 39] . viral culture for cytomegalovirus, herpes simplex virus, parainfluenza viruses (types 1 and 3), respiratory syncytial virus, varicella-zoster virus, influenza viruses (type a and b), and enterovirus was performed using shell-vial culture as previously described [4, 40] . amoeba co-culture were performed in microplates on acanthamoeba polyphaga as previously described [41] . tentative isolations of mycobacterium sp., legionella sp. and mycoplasma pneumoniae were performed by using bactec 9000 mb automate, bcye agar plates and sp4 medium as previously described [42] [43] [44] . results obtained using routine culture are available in table s10 ,s11,s12). mycobacterium sp., m. tuberculosis, m. avium group, bosea sp, parachlamydia sp., coxiella burnetii, chlamedia pneumoniae, chlamedia psittaci, mycoplasma pneumoniae, aspergillus sp., mimivirus, cmv, hsv, parainfluenza viruses 1 and 3, respiratory syncytial virus, rhinovirus, metapneumovirus, varicella-zoster virus, influenza viruses a and b, enterovirus, and coronaviruses oc-43, 229-e and nl-63 were detected using quantitative pcr. quantitative pcr was performed using a lightcyclerh instrument (roche diagnostics, meylan, france) in conjunction with the quantitect probe pcr kit. primers and probes used to identify these microorganisms are reported in table 5. the reaction was performed as previously described [13] . for rna viruses, rna was first reverse transcribed using multiscribe tm reverse transcriptase (applied biosystems, courtaboeuf, france) as previously described [13] . sera from patients were tested by immunofluorescent assay (ifa) for coxiella burnetii, bartonella quintana, bartonella henselae, legionella pneumophila, legionella anisa [40, 45, 46] . viral serologies for adenovirus, cytomegalovirus, herpes simplex, parainfluenza viruses 1 and 3, varicella-zoster virus and, influenza viruses a and b were performed using standard serologic methods (immunofluorescent assay or enzyme linked immunosorbent assay) [4] . hemagglutination inhibition, immunoperoxidase staining and elisa techniques were used in-house to identify aspergillosis. l. pneumophila antigenuria and cmv pp65 antigenemia were tested for as previously reported [4, 47] . results obtained using routine serology and antigenemia for viruses and fastidious pathogens are available in table s13 . bacterial and fungal nucleic acid sequences obtained from broad-range primer pcr were aligned with bioedit program (http://www.mbio.ncsu.edu/bioedit/bioedit.html) and phylogenetic trees were create with mega software version 4.1 using the neighbor-joining method and the kimura-2 parameter [48] . species having sequence similarities ,98% with those available in genbank databases were also blasted and classified in silico using ''classifier'' program in the ribosomal database project (http:// rdp.cme.msu.edu/) [49] . statistical analyses were performed using chi square test, fisher's exact test, students t-test or mantel-haenszel's chi square test when appropriate. p values that were less than or equal to 0.05 were considered significant. the pubmed database (www.ncbi.nlm.nih.gov/pubmed/) and google website (http://www.google.fr/) were used to search whether species identified in our study had been previously reported in cases of pneumonia for articles published between 1977 and march 2010, with the combined search term ''species name'' and ''pneumonia'', ''lung'' or ''infection.'' additional articles were identified by hand-searching the references of selected papers. additional search terms included ''microbiology'', ''diagnosis'', ''16s'' and ''molecular detection'' were used. only publications in english were considered. papers in languages other than english were considered only when their abstracts in english were available. figure s1 schematic representation of microorganisms commonly identified in pneumonia and control cohorts, and those only detected in one cohort. fungi are shown in rectangles, viruses in octagons, and bacteria in circles. the name of each microorganism is indicated. (tif) figure s2 schematic representation of microorganisms that were commonly identified between each pneumonia form and controls, and those which were detected in only one cohort. fungi are shown in rectangles, viruses in octagons, and bacteria in circles. actinobacteria are shown in red, bacteroidetes in yellow, chlamydiae in orange, firmicutes in green, fusobacteria in purple, proteobacteria in blue and tenericutes in sky blue. cap, community-acquired pneumonia; vap, ventilatorassociated pneumonia; nv icu-p, non-ventilator icu pneumonia; ap, aspiration pneumonia; and cs, control subjects. (tif) figure s3 comparison of the bacterial communities found in our study with those found in lung specimens in three previous studies. novel phylotypes are not shown. actinobacteria are shown in red, bacteroidetes in yellow, chlamydiae in orange, firmicutes in green, fusobacteria in purple, proteobacteria in blue and tenericutes in sky blue. the name of the first author of each study and the name of each bacterium are indicated. the comparative analysis was conducted using cytoscape software. vap, ventilator-associated pneumonia; cf, cystic fibrosis. (tif) figure s4 molecular methods compared to standard routine culture for bacteria identification. text s1 bal culture, blood culture and serology results. data s1 detailed data about the relative abundance and richness of each species in their corresponding library. data s2 schematic data about the relative abundance and richness of each species in the corresponding library. table s1 species only detected in bal from pneumonia patients by molecular assays. (docx) nosocomial pneumonia in the intensive care unit acquired during mechanical ventilation or not etiology and diagnosis of pneumonia requiring icu admission guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia active cytomegalovirus infection is common in mechanically ventilated medical intensive care unit patients cytomegalovirus. an unexpected cause of ventilator-associated pneumonia viral infections in the icu herpes simplex virus: a marker of severity in bacterial ventilator-associated pneumonia bronchoscopic bal in the diagnosis of ventilatorassociated pneumonia molecular identification of bacteria in bronchoalveolar lavage fluid from children with cystic fibrosis microbial diversity in the sputum of a cystic fibrosis patient studied with 16s rdna pyrosequencing molecular detection of multiple emerging pathogens in sputa from cystic fibrosis patients tropheryma whipplei in patients with pneumonia detection 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society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults amoebaresisting bacteria and ventilator-associated pneumonia aspiration pneumonitis and aspiration pneumonia clinical significance of a positive serology for mimivirus in patients presenting a suspicion of ventilator-associated pneumonia black box chimera check (b2c2): a windows-based software for batch depletion of chimeras from bacterial 16s rrna gene datasets direct identification of bacteria in positive blood culture bottles by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry evaluation of nested and real-time pcr assays in the diagnosis of candidaemia quantification of leishmania infantum dna by a real-time pcr assay with high sensitivity ameba-associated microorganisms and diagnosis of nosocomial pneumonia isolation of new fastidious alpha proteobacteria and afipia felis from hospital water supplies by direct plating and amoebal co-culture procedures cost-effectiveness of blood agar for isolation of mycobacteria diagnosis of legionnaires' disease. an update of laboratory methods with new emphasis on isolation by culture mycoplasma and ureaplasma. 1004-1020 q fever serology: cutoff determination for microimmunofluorescence value of microimmunofluorescence for diagnosis and follow-up of bartonella endocarditis detection of legionella pneumonophila antigen in urine by enzyme-linked immunospecific assay mega4: molecular evolutionary genetics analysis (mega) software version 4.0 naive bayesian classifier for rapid assignment of rrna sequences into the new bacterial taxonomy key: cord-282194-0sjmf1yn authors: cherak, stephana j.; rosgen, brianna k.; amarbayan, mungunzul; plotnikoff, kara; wollny, krista; stelfox, henry t.; fiest, kirsten m. title: impact of social media interventions and tools among informal caregivers of critically ill patients after patient admission to the intensive care unit: a scoping review date: 2020-09-11 journal: plos one doi: 10.1371/journal.pone.0238803 sha: doc_id: 282194 cord_uid: 0sjmf1yn background: the use of social media in healthcare continues to evolve. the purpose of this scoping review was to summarize existing research on the impact of social media interventions and tools among informal caregivers of critically ill patients after patient admission to the intensive care unit (icu). methods: this review followed established scoping review methods, including an extensive a priori-defined search strategy implemented in the medline, embase, psycinfo, cinahl, and the cochrane central register of controlled trials databases to july 10, 2020. primary research studies reporting on the use of social media by informal caregivers for critically ill patients were included. results: we identified 400 unique citations and thirty-one studies met the inclusion criteria. nine were interventional trials–four randomized controlled trials (rcts)–and a majority (n = 14) were conducted (i.e., data collected) between 2013 to 2015. communication platforms (e.g., text messaging, web camera) were the most commonly used social media tool (n = 17), followed by social networking sites (e.g., facebook, instagram) (n = 6), and content communities (e.g., youtube, slideshare) (n = 5). nine studies’ primary objective was caregiver satisfaction, followed by self-care (n = 6), and health literacy (n = 5). nearly every study reported an outcome on usage feasibility (e.g., user attitudes, preferences, demographics) (n = 30), and twenty-three studies reported an outcome related to patient and caregiver satisfaction. among the studies that assessed statistical significance (n = 18), 12 reported statistically significant positive effects of social media use. overall, 16 of the 31 studies reported positive conclusions (e.g., increased knowledge, satisfaction, involvement) regarding the use of social media among informal caregivers for critically ill patients. conclusions: social media has potential benefits for caregivers of the critically ill. more robust and clinically relevant studies are required to identify effective social media strategies used among caregivers for the critically ill. introduction social media is defined as "websites and applications that enable users to create and share content or to participate in social networking" [1] . social media tools are platforms and communities, such as facebook or skype, that facilitate quick communication and enable interaction among several users at any given time [2] . social media participation in older age groups is steadily increasing [3] , contributing to over 3.2 billion active users worldwide [4] . in considering the various user-generated content and social networking platforms, the role of social media conveys different meanings between users and non-users, age groups (e.g., millennials), and demographic populations. since technological change is associated with linguistic and cultural changes, the role of social media is constantly in flux [5] . the use of social media in healthcare for increasing speed of communication, distributing accurate information, and promoting knowledge of support, treatments and self-care options is becoming more widespread [6, 7] . patient-and family-centered healthcare, which acknowledges that patients and their informal caregivers are central figures in decision-making and delivery of care [8] , recognizes that patients and caregivers exist within an online social structure and network of relationships [9] . social media tools, such as real-time communication platforms, educational material, and self-management guides, are now more commonly incorporated in the decision-making process to aid caregivers with making informed decisions regarding their loved one's care [10] . critically ill patients are often unable to communicate their care preferences (e.g., due to mechanical ventilation, coma, etc.) including those that are in line with their individual values and goals [11] . in these situations, critically ill patients rely on their informal caregivers to learn about their diagnosis and treatment options, and to make important decisions on their behalf [12] -these situations can be stressful and distressing for an informal caregiver [13] . family-centered interventions may improve caregiver's comprehension, satisfaction, and long-term psychological outcomes during and after a family member's critical illness [13, 14] . social media tools as family-centered interventions might allow for personalization, presentation, and participation of informal caregivers in their loved one's care, engaging them in the decision-making process and promoting better patient and informal caregiver outcomes [2, 15] . despite their potential value, it is unclear whether social media tools can be meaningfully and systematically deployed in critical care medicine [16] . we therefore asked the question: what is the extent, range, and nature of research evidence on the impact of social media interventions and tools among informal caregivers of critically ill patients? this scoping review was conducted and reported as per the arksey-o'malley 5-stage scoping review method [17] . the approach for this review followed the scoping review methods manual by the joanna briggs institute [18] . the preferred reporting items for systematic reviews and meta-analysis protocols (prisma-p) guideline was used to develop the protocol [19] (s1 table) . we adhered to the prisma-scr extension for scoping reviews [20] to report findings. inclusion criteria were as follows: (1) primary quantitative or qualitative research; (2) reporting on social media use with at least one informal caregiver as an end-user; (3) conducted with informal caregivers of critically ill patients of any age group; and (4) in any language or publication year. studies were excluded if they were not primary research (e.g., reviews or editorials), did not report on caregiver use of social media, or were not conducted in a critical care population. for the purposes of this review, we defined: (1) a caregiver as any informal (i.e., non-clinical) person who regularly provides support to the patient and is in some way directly implicated in the patient's care or directly affected by the patient's health problem (e.g., family, friend); (2) social media as any form of electronic communication that allow users to share information and other content and create online communities; and (3) critically ill patients as any persons who are currently admitted to an intensive care unit (icu) or had previously been admitted to an icu. studies were excluded if only abstracts were available. comprehensive literature searches were conducted in medline, embase, psycinfo, cinahl, and the cochrane central register of controlled trials. the search strategies for each database were developed with a medical librarian (dll) and were revised after reviewing preliminary search results. the search strategies combined synonyms and subject headings from three concepts: 1) caregivers; 2) critical care; and 3) social media. a search of the cochrane database of systematic reviews was undertaken to identify review articles related to the research question and their reference lists were screened to identify potential studies missed in the search. all databases were searched from inception to july 10, 2020. reference lists of included papers were reviewed to identify potential studies missed in the search. no language or date limits were applied. the complete medline search strategy is shown in s2 table. after a subset of the team (sc, ma) achieved 100% agreement on a pilot-test of 50 random studies, all titles and abstracts were reviewed independently in duplicate by two reviewers (sc, ma). any study selected by either reviewer at this stage progressed to the next stage. the fulltext of all articles was reviewed independently in duplicate by two reviewers (sc, ma); articles selected by both reviewers at this stage were included in the final review. disagreements were resolved by discussion or the involvement of a third reviewer (br) when necessary. references were managed in endnote x9 (clarivate analytics, philadelphia, pa, usa). two reviewers (sc, kp) abstracted data independently and in duplicate for each included study using a data collection sheet developed and piloted by the review team. discrepancies were resolved through discussion with a third reviewer (ma). information on document characteristics (e.g., year of publication, geographic location), study characteristics (e.g., setting), caregiver group (e.g., spouses, parents, family caregivers), social media tool used (e.g., communication platform, content community, social networking site, blog or microblog), objectives and outcome measures of social media use, statistical significance, and authors' conclusions were collected. studies that examined social media as one component of a complex intervention were noted as such. findings were synthesized descriptively to map different areas of the literature as outlined in the research question. using a social media framework described in previous research [6] , we categorized social media tools into five categories: collaborative projects (e.g., endnote, slack), blogs or microblogs (e.g., wordpress, twitter), content communities (e.g., youtube, slide-share), social networking sites (e.g., facebook, instagram), and real-time communication platforms (e.g., text messaging, web camera, facetime) (s3 table) . study objectives and outcomes were classified according to an adaptation from those outlined in coulter and ellins [21] proposed framework for strategies to inform, educate and involve patients (s4 table) . the main objective from each study was categorized into one of five categories: to improve health literacy, clinical decision making, self-care, patient safety or other. outcomes reported in each study were classified as patient and caregiver knowledge, patient and caregiver experience, use of services and cost, health behaviors and health status, and usage feasibility. studies that reported statistically significant outcomes determined by p<0.05 related to the main objective of the study were classified as "statistically significant." studies that reported outcomes that were not statistically significant were classified as "not statistically significant," and if a study did not assess significance through statistical equations that study was classified as "not assessed." descriptive statistics were calculated using stata ic 15 (statacorp. stata statistical software: release 15. college station, tx: statacorp llc). we screened 400 unique abstracts and reviewed 72 full-text articles; 41 full-text articles were excluded, the most common reasons being that the study did not report original research (n = 15/41) or that the study did not report on social media use (n = 12/41) (fig 1) . hand searching resulted in the inclusion of seven additional studies. there was 85% agreement on title and abstract screening and 89% agreement on full-text screening. the 31 included studies were published between 2000 and 2020 and primarily conducted in north america (n = 20, 65%) or europe (n = 9, 29%), and with neonatal or pediatric critical care populations (n = 23, 74%) ( table 1 ). fig 2a depicts the different icu types from the included studies. the median start date was 2015 (range: 1997-2016) and the median duration was 19 months (range: 3-95 months). many studies (n = 9, 33%) were interventional studies [22, 23, 27, 29, 30, 32, 33, 39, 48] of which most were conducted in neonatal icus (6/9). we included six qualitative studies and most (4/6) were conducted with neonatal or pediatric critical care populations. caregivers were most commonly parents (n = 19, 61%) [30, 31, 33, 35-38, 41-44, 47-49, 51 , 52] and unspecified family caregivers more broadly-which could include parents, but the term was more broadly defined (n = 7, 23%) [23, 24, 26, 27, 32, 39, 50] . one study was specific to mothers [40] and one study was specific to fathers [45] . few studies reported additional perspectives from members of the clinical care team (e.g., nurses, primary care physicians) (n = 3, 10%) [29, 34, 50] or critical care patients (n = 3, 10%) [22, 28, 49] . more than half of the studies examined real-time communication platforms (e.g., face-time, skype) (n = 17, 55%) [23, 24, 28, 30-40, 47, 51, 52] , which accounted for many of the studies conducted with adult populations (3/7, 43%) and most of the studies conducted with neonatal or pediatric populations (14/22, 64%). included studies were categorized by the type of social media tool used (s3 table) . fig 2b depicts the different specific social media tools from the included studies. real-time communication platforms, that allowed user communication with messages, voice, and/or video, were the most common social media tool used (n = 15, 56%), followed by social networking sites (n = 6, 19%) and content communities (n = 5, 16%). few studies (n = 2, 7%) assessed the use of blog or microblogs and only two studies examined social media use in general. overall, most social media tools included functions that operated like communication platforms, such that they provided the option for users to post and share experiences. many studies (n = 8, 30%) included a social media tool as part of a complex intervention, and most of these studies (n = 6/8) used mobile phones to facilitate the social media component. all of these studies (n = 6/6) reported that the ubiquitous nature and technical capacity of mobile phones were strong motivating factors. several of these studies (n = 5/6) addressed potential misuse of information and privacy concerns over text messaging by an established mobile phone dedicated to the study, and provided recommendations to the clinical care team (i.e., nurses, physicians) for text messaging with informal caregivers. the most common intended use of social media was for caregiver satisfaction (n = 9, 29%). most studies that examined caregiver satisfaction used communication platforms (n = 8/9). social networking sites were often used to improve self-care (n = 2/6, 30%), and content communities were mainly intended to improve patient safety (n = 2/4, 50%). there were few studies that addressed clinical decision making (n = 4, 13%) and half (n = 2/4) used content communities. five studies (16%) did not fit the framework, and were classified as "other"; three of these studies reported the prevalence of social networking use (n = 1) or of internet use more broadly (n = 2), and two compared mothers and fathers use of information and communication technology (n = 1) or frequency and length of webcam viewing (n = 1). usage feasibility and patient and caregiver experience outcomes were most commonly reported (n = 30 and n = 23, respectively) ( table 2 ). patient and caregiver knowledge outcomes were reported in 16 studies (52%), and use of services and cost outcomes, and health behaviors and health status outcomes were reported in eight studies each. among outcomes related to usage feasibility (n = 30), measures of usage and demographics were most common (n = 22, 73%) and were often accompanied by measures of users' attitudes and preferences (n = 20, 67%). measures of patient or caregiver satisfaction or of clinician-patient/caregiver communication were most commonly reported for outcomes related to patient and caregiver experience (n = 13 and n = 12, respectively). fig 3a provides a summary of outcomes as they relate to the study objectives. there were no defining trends between outcomes with regard to objectives for social media use, but measures related to the use of services and cost, or to health behaviors and health status, were generally least reported among any objective. one study reported outcomes related to potential for unintended consequences or harm from social media tools [50]. 2 reported at least one outcome related to social media use by an informal (i.e., non-clinical) caregiver; adult patient defined as >15 years. 3 reporting prevalence of internet use among critically ill septic patients and caregivers. 4 comparing mothers' and fathers' use of information and communication technology. 5 comparing mothers' and fathers' frequency and length of viewing their hospitalized neonate via webcam. 6 reporting prevalence of social networking site use among parents of preterm infants. 7 determining parents perception and preferences for information sharing in the neonatal intensive care unit. https://doi.org/10.1371/journal.pone.0238803.t001 social media use among caregivers of critically ill patients: a scoping review 4a). studies that collected data during and/or after 2016 reported only positive, negative or indeterminate effects of social media use. majority of studies with a sample size >300 reported a negative effect, and majority of studies with a sample size 100-300 or <100 reported a positive effect (fig 4b) . prospective observational studies commonly reported a neutral effect and the majority of prospective intervention studies reported a positive effect (fig 4c) . among the studies that assessed statistical significance, the majority determined that social media use had a positive effect (fig 4d) . the most common type of study design was interventional (n = 9, 29%)-of which 4 were controlled by randomization (i.e., rcts)-followed by prospective cohort (n = 8, 26%) and qualitative (n = 6, 19%). of the quantitative studies (n = 25, 68%), majority assessed statistical significance (n = 20/25) and majority determined there was a significantly positive effect of social media use (n = 12/20). among the randomized interventions (n = 4), two found a significantly positive effect, one found a significantly negative effect and one did not assess statistical significance. fig 3b provides a summary of authors' conclusions of social media use with regard to study objectives. the majority of studies with the objectives of improving health literacy, self-care, patient safety or caregiver satisfaction, reported a statistically significant positive effect. among the four studies that aimed to improve clinical decision making, one study social media use among caregivers of critically ill patients: a scoping review reported a positive effect but did not assess statistical significance, and three studies reported a negative effect but only two assessed significance. we used scoping review methodology to synthesize the literature on the extent, range, and nature of research evidence on the impact of social media interventions and tools among informal caregivers of critically ill patients. there is a growing body of literature, primarily from neonatal or pediatric populations, suggesting that real-time communication platforms are now social media use among caregivers of critically ill patients: a scoping review commonly used social media tools among informal caregivers of critically ill patients. in contrast, there is very little literature regarding caregiver use of social networking sites, blogs, or content communities. the most common intended use for social media was to improve caregiver satisfaction with the experience and role of an informal caregiver of a critically ill patient. outcomes related to usage feasibility, such as measures of user's attitudes, preferences, and demographics, were nearly always reported. few studies assessed cost-effectiveness of using social media tools with informal caregivers, and outcomes related to health behaviors and health status of either the patient or caregiver were reported infrequently. although most studies concluded that the use of social media among informal caregivers is beneficial and meaningful, the potential for unintended consequences or harm specific to informal caregivers were not adequately explored. the low reliability and high variability of content shared on social media highlights the importance of control from medical personnel to avoid the spread of "fake news" [53] . the emerging utilization of social media tools among informal caregivers for critically ill patients have practical implications for critical care medicine. modern mobile phones are powerful computational devices. the technical capacity of mobile phones to facilitate phone-based health interventions was a motivating factor for several included studies. mobile phones are also omnipresent and nearly always at hand [54] , which makes it possible to increase the number of points of care to virtually any place and time [55] . the combination of the technical capacity, personal nature, and convenient proximity of mobile phones has reduced barriers to adoption and increased acceptance of phonebased health interventions in numerous healthcare settings [56] . the immediacy of access of conclusions on social media use with regard to patient and caregiver focused objectives 1,2,4 . 1 adapted from coulter and ellins, 2007; only the main study objective was recorded from a single study; 3 more than one outcome category could be recorded from a single study; 4 only one overall conclusion was recorded from each study. frequency indicated by color: red, very frequent; yellow, moderately frequent; green, infrequent. n, number of studies. https://doi.org/10.1371/journal.pone.0238803.g003 mobile phones might also be useful to informal caregivers after patient discharge by providing prompt advice and support, which may reduce healthcare costs by preventing hospital or icu readmission. mobile phones in healthcare settings also have disadvantages. with regard to nursing, disruption of workflow, interruption of practice, and improper usage have been reported [57] . for example, in the study conducted by piscotty and colleagues [58] , 67% of nurses checked their mobile phone more than 2 times per shift and 22% checked their mobile phone more than 10 times per shift. further, possibility of misuse of information that may violate patient privacy remains an unresolved problem [59] . nursing organizations have responded with guidelines on professional social media use in the workplace [60] [61] [62] . many included studies addressed potential privacy issues by an established mobile phone dedicated to the study, and recommended to refrain from using patient last names and conditions, to keep communications brief, and to destroy caregiver phone numbers after patient discharge [63] . that mobile phones may be useful to facilitate social media interventions in critical care medicine is a noteworthy finding of this review, but further research is needed on how social media strategies can be implemented into practice without violating privacy or ethical considerations. support and encouragement can contribute to caregiver confidence, which can promote better understanding of a stressful illness-related situation and enable the caregiver to provide better care [64] . many included studies found that caregivers reported a more satisfactory critical care experience and increased knowledge of a patient's condition and long-term treatment options when provided with links to online resources with credible information. in the last decade, several members of the united states critical care societies collaborative have started using social media [65] . the society of critical care medicine is one member, which uses web-based education initiatives to provide accurate and reliable information to educate their members and the public [15] . as well, the world federation of societies of intensive and critical care medicine also recognized that social media plays a large role in achieving more and better involvement with other member societies, and actively uses social media to liaise with important groups, such as young clinicians [66] . considering the differences in how critical care societies use diverse approaches to deliver overlapping educational content can provide a rich opportunity to inform development of future web-based education initiatives, targeted specifically at informal caregivers. real-time communication platforms have been studied and implemented in many healthcare settings [67, 68] . several included studies found that in neonatal icu populations, parents who were communicating with the clinical care team using videoconferencing instruments (e.g., facetime, skype) felt significantly more satisfied with their infants' care when they were unable to be physically present. no study conducted in adult icu populations used a social media tool dedicated entirely to videoconferencing, although most social media tools included functions which operated similar to communication platforms. further, no included study from any icu reported the use of communication platforms to engage non-local family members or young children who may benefit from remote communication with their loved one. since many communication platforms are free to download on most electronic devices and allow for multiple users at once, an important area for future research is the use of communication platforms by entire support groups of both adult and non-adult critical care patients. this type of research is warranted to determine if positive outcomes of communication platforms depend on whether the caregivers' relationship to the patient is parent-child (i.e., parent providing support to children) versus child-parent (i.e., children providing support to parents). it is important to recognize that social media tools are exactly that-tools-rather than a substitute for personal interaction with healthcare providers. recent studies in other healthcare settings have found that patients' value in-person interaction with healthcare providers more than social media communication, and that healthcare providers are regarded as the most important source of information [69] . knowledge on the values and preferences of the clinical care team, however, is lacking, and a common concern of many clinicians is that information shared on social media may not always be accurate. more understanding on physician preferences and social media accuracy is important as physicians often rely on patients' informal caregivers to make decisions regarding the patient's care, which frequently contributes to caregiver psychological morbidity [70] . individualized social media interventions adapted to caregiver preferences may improve caregiver's satisfaction and psychological morbidity [13] . more research on accurate, proper and potential use of social media in critical care medicine is required before implementation into daily practice. our review indicates there is untapped potential for social media interventions and tools to provide personalized support to informal caregivers of the critically ill. we recommend future inquiry on this topic examine mental health interventions using social media to determine the effect of social media mental health interventions on psychological outcomes of informal caregivers of the critically ill. this information is particularly relevant to challenges related to restricted visitation and social isolation associated with the covid-19 pandemic [71] . the large numbers of patients experiencing critical illness and visiting restrictions enacted to prevent the spread of covid-19 complicate participation of informal caregivers in patient care and recovery [72] . these factors are likely to make mental health consequences of critical illness on informal caregivers more prevalent and severe [73, 74] . social media interventions and tools may be an effective mode of mental health support for informal caregivers of critically ill patients. this scoping review has several strengths. we conducted an extensive literature search and screened reference lists of included studies in order to identify the full breadth of available literature on social media use in critical care populations. the search was executed in five bibliographic databases and was not restricted by language or dates. it was intentionally broad to ensure that social media use across all critical care populations were included. we followed rigorous methodology defined by adherence to recommended protocols and reporting criteria for scoping reviews. further, the interdisciplinary team of a critical care physician, a critical care nurse, and a psychiatric epidemiologist, offered complementary expertise and knowledge. in spite of these strengths, there are limitations to note. we did not search the grey literature nor did we search social media itself, and could have missed studies, though our search strategy was comprehensive and full-text hand searching was completed. as well, the lack of a universal definition for social media, since social media is a relatively new concept that is continually transforming, added complexity to the process of study selection. however, our broad inclusion of study design allowed us to produce a comprehensive summary of the state of the literature on social media use by informal caregivers in critical care medicine. ultimately, the relatively rapid evolution of social media means studies on usage will nearly exclusively reflect social media use of the past. though such studies are valuable, it is important to note that the medium of social media is evolving faster than it is being studied. there is a growing evidence base to support the use of social media among informal caregivers of critically ill patients. there is untapped potential for social media tools to provide personalized support to informal caregivers. social media tools might enable informal caregivers to gain the knowledge that they need in order to feel empowered, involved, and satisfied. social media users should exercise caution on applications and networking sites so as not to compromise patient privacy. in sum, social media represents a flexible medium to deliver health information, and the individualized support that caregivers can obtain through using social media may promote an invaluable collaborative relationship when caring for critically ill patients. supporting information s1 social media.: oxford dictionary social media in critical care social media-statistics & facts ranked by numbers of active users (in millions). statista social media usage: 2005-2015. pew research ceter users of the world, unite! the challenges and opportunities of social media neurology and the internet: a review patient and family members' perceptions of family participation in care on acute care wards a new dimension of health care: systematic review of the uses, benefits, and limitations of social media for health communication social media and health care professionals: benefits, risks, and best practices a comparison of the opinions of intensive care unit staff and family members of the treatment intensity received by patients admitted to an intensive care unit: a multicentre survey a randomized trial of a family-support intervention in intensive care units association of surrogate decision-making interventions for critically ill adults with patient, family, and resource use outcomes: a systematic review and meta-analysis translating evidence to patient care through caregivers: a systematic review of caregiver-mediated interventions social media engagement and the critical care medicine community evaluation of mobile apps targeted to parents of infants in the neonatal intensive care unit: systematic app review. jmir mhealth and uhealth scoping the field: services for carers of people with mental health problems the joanna briggs institute best practice information sheet: the effectiveness of pelvic floor muscle exercises on urinary incontinence in women following childbirth preferred reporting items for systematic review and meta-analysis protocols (prisma-p) 2015 statement prisma extension for scoping reviews (prisma-scr): checklist and explanation effectiveness of strategies for informing, educating, and involving patients reducing pressure ulcers in patients with prolonged acute mechanical ventilation: a quasi-experimental study. diminuicao das ulceras por pressao em pacientes com ventilacao mecanica aguda prolongada: um estudo quasi-experimental a pilot study of audiovisual family meetings in the intensive care unit prioritizing information topics for relatives of critically ill patients: cross-sectional survey among intensive care unit relatives and professionals reanet", the internet utilization among surrogates of critically ill patients with sepsis. plos one a qualitative study of factors that influence active family involvement with patient care in the icu: survey of critical care nurses a family information brochure and dedicated website to improve the icu experience for patients' relatives: an italian multicenter before-and-after study. intensive care medicine american journal of critical care: an official publication, american association of critical-care nurses effects of a social media website on primary care givers' awareness of music therapy services in a neonatal intensive care unit. the arts in psychotherapy testing the feasibility of skype and facetime updates with parents in the neonatal intensive care unit. american journal of critical care: an official publication, american association of critical-care nurses smartphones and text messaging are associated with higher parent quality of life scores and enrollment in early intervention after nicu discharge a randomized controlled study about the use of ehealth in the home health care of premature infants the use of short message services (sms) to provide medical updating to parents in the nicu web camera use in the neonatal intensive care unit: impact on nursing workflow diabetes self-management care via cell phone: a systematic review patient adherence and accuracy using electronic diaries during remote patient monitoring in type 1 and type 2 diabetes can the ubiquitous power of mobile phones be used to improve health outcomes in developing countries? global health to tweet or not to tweet? nurses, social media, and patient care impact of healthcare information technology on nursing practice mobile phone messaging reminders for attendance at healthcare appointments social media guidelines for nurses registered nurses' association of ontario available from: categorization of objectives and outcomes master's programs in advanced nursing practice: new strategies to enhance course design for subspecialty training in neonatology and pediatrics guidelines for using electronic and social media: the regulatory perspective the effectiveness of mi smart: a nurse practitioner led technology intervention for multiple chronic conditions in primary care patient safety and quality: an evidence-based handbook for nurses. advances in patient safety lessons learned from web-and social media-based educational initiatives by pulmonary the world federation of societies of intensive and critical care medicine newsletter online, social media and mobile technologies for psychosis treatment: a systematic review on novel user-led interventions methods of using real-time social media technologies for detection and remote monitoring of hiv outcomes social media use in healthcare: a systematic review of effects on patients and on their relationship with healthcare professionals retiring the term futility in value-laden decisions regarding potentially inappropriate medical treatment ethical dilemmas due to the covid-19 pandemic bereavement support on the frontline of covid-19: recommendations for hospital clinicians rehabilitation after critical illness in people with covid-19 infection intensive care management of coronavirus disease 2019 (covid-19): challenges and recommendations we thank dr. diane lorenzetti (university of calgary) for the development of the search strategies. key: cord-277357-lpurk7pe authors: gonzález-gonzález, everardo; trujillo-de santiago, grissel; lara-mayorga, itzel montserrat; martínez-chapa, sergio omar; alvarez, mario moisés title: portable and accurate diagnostics for covid-19: combined use of the minipcr thermocycler and a well-plate reader for sars-cov-2 virus detection date: 2020-08-13 journal: plos one doi: 10.1371/journal.pone.0237418 sha: doc_id: 277357 cord_uid: lpurk7pe the coronavirus disease 2019 (covid-19) pandemic has crudely demonstrated the need for massive and rapid diagnostics. by the first week of july, more than 10,000,000 positive cases of covid-19 have been reported worldwide, although this number could be greatly underestimated. in the case of an epidemic emergency, the first line of response should be based on commercially available and validated resources. here, we demonstrate the use of the minipcr, a commercial compact and portable pcr device recently available on the market, in combination with a commercial well-plate reader as a diagnostic system for detecting genetic material of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the causal agent of covid-19. we used the minipcr to detect and amplify sars-cov-2 dna sequences using the sets of initiators recommended by the world health organization (who) for targeting three different regions that encode for the n protein. prior to amplification, samples were combined with a dna intercalating reagent (i.e., evagreen dye). sample fluorescence after amplification was then read using a commercial 96-well plate reader. this straightforward method allows the detection and amplification of sars-cov-2 nucleic acids in the range of ~625 to 2×10(5) dna copies. the accuracy and simplicity of this diagnostics strategy may provide a cost-efficient and reliable alternative for covid-19 pandemic testing, particularly in underdeveloped regions where rt-qpcr instrument availability may be limited. the portability, ease of use, and reproducibility of the minipcr makes it a reliable alternative for deployment in point-of-care sars-cov-2 detection efforts during pandemics. recent epidemic events (i.e., zika in southeast asia and latin-america in 2016 [1, 2] , ebola in west africa in 2013-2015 [3] , and pandemic influenza a/h1n1/2009 [4] ) have clearly evidenced the urgent need for low-cost, portable, and easy-to-use diagnostic systems that can be effectively deployable to address epidemic episodes [5] [6] [7] [8] . however, these portable diagnostic systems have been mainly viewed as solutions for underprivileged or remote places and/or for catastrophic scenarios. nevertheless, the coronavirus disease 2019 (covid19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [9] has broadsided most well developed and developing countries with only a few (i.e., south korea [10] , china, singapore [11] , and taiwan [12] ) showing an ability to deploy massive efforts for rapid and accurate detection of positive infection cases. the swift and massive testing of thousands of possibly infected subjects has been an important component of the strategy of these countries that has helped to effectively mitigate the spreading of covid-19 among their populations [10, [12] [13] [14] . and yet, most nations are still struggling to implement massive testing [15] [16] [17] . current testing methods have exhibited important limitations in widespread reach, flexibility, cost-effectiveness, and scalability during this pandemic. through the last two pandemic events involving influenza a/h1n1/2009 and covid-19 [18] , the centers for disease control (cdc) and the world health organization (who) recommended the reverse transcription quantitative polymerase chain reaction (rt-qpcr) as the gold standard for official detection of positive cases. without any doubt, nucleic acid amplification, and particularly rt-qpcr, is the most reliable technique for the early and accurate detection of viral diseases [19, 20] . unfortunately, conducting rt-qpcr diagnostics often depends on access to centralized laboratory facilities for testing [21] [22] [23] . to resolve this limitation, several different versions of compact pcr platforms (some of them q-pcr systems) have been described recently in the scientific literature [24] [25] [26] [27] . unfortunately, most of these devices have not yet reached the market. during epidemic emergencies, resourcing of incompletely developed technologies is impractical, and the use of commercially available diagnostic platforms becomes the first and arguably the most cost-efficient line of defense. only recently, several miniaturized pcr machines become commercially available [28, 29] . one of them, the minipcr [30, 31] , reached the international market in 2015. the most recent version of this compact pcr machine has an approximate cost of~$800 usd (www.minipcr. com) as compared to $3000 usd for a conventional pcr thermocycler [28] . several papers have documented the value of the minipcr1 system as a portable and robust diagnostic tool [32] [33] [34] [35] [36] . we recently published a comparison of the performance of the minipcr and a commercial thermal cycler for the identification of artificial zika and ebola genetic sequences. our experiments using a wide variety of primers sets and template concentrations revealed no differences in performance between either thermal cycler type [37] . the commercial availability, low price (as compared to conventional thermocyclers), portability, and user friendliness of the minipcr makes it an attractive and tangible solution that effectively brings pcr analysis to the poc. in the present study, we demonstrate the convenience of using the minipcr for the detection and amplification of synthetic samples of sars-cov-2 [18] , the causal viral agent of the current covid-19 pandemic. we ran several sets of amplification experiments in a minipcr from amplyus (ma, usa). the unit has dimensions of 20 × 5 × 15 cm, weighs 0.7 kg, and requires 120v (ac) and 3.5 a to operate. the minipcr can run 8-16 amplifications in parallel (depending on the model employed). a commercial power supply (powerpac from bio-rad, ca, usa) was used to operate the electrophoresis unit used to run the agarose gels to reveal the amplification products obtained by the minipcr thermocycler. a bio-rad chemidoc xrs imaging system was used for endpoint pcr detection. alternatively, the minipcr unit has its own bluegel electrophoresis unit (fig 1a and 1b) , a compact electrophoresis unit (23 × 10 × 7 cm) that weighs 350 g, that is powered by a built-in power supply (ac 100-240 v, 50-60 hz). we also used a synergy ht microplate reader (biotek instruments, vt, usa) to detect the fluorescence induced by an intercalating reagent in positive samples from the pcr reactions. we used a plasmid containing the complete n gene from 2019-ncov, sars, and mers as positive controls at a concentration of 200,000 copies/μl (integrated dna technologies, ia, usa). samples containing different concentrations of synthetic nucleic acids of sars-cov-2 were prepared by successive dilutions from stocks containing 200,000 copies ml -1 ng/l of viral nucleic acids. we used a plasmid containing the gp gene from ebola virus (ebov) as a negative control. the production of this ebov genetic material has been documented previously by our group [37]. we used redtaq ready mix from sigma-aldrich (usa), and followed the recommended protocol: 10 μl readymix, 0.5 μm of forward primer, 0.5 μm of reverse primer,1μl of dna template (~625 to 2x105 dna copies), 1μl of evagreen dye, and nuclease free water to final volume of reaction 20 μl. primers used. three different sets of primers were used to target three different regions of the sars-cov-2 n gene sequence. these primer sets are identical to those recommended by the center of disease control (cdc) for the standard diagnostics of covid-19 (i.e., n1, n2, and n3 assays) using quantitative real time pcr. sequences of all these primers and their corresponding amplicons are presented in tables 1 and 2 . for all pcr experiments, we used the same three-stage protocol (see fig 1d) consisting of a denaturation stage at 94˚c for 5 min, followed by 25 cycles of 94˚c for 20s, 60˚c for 30s, and 72˚c for 20s, and then a final stage at 72˚c for 5 min, for a total duration of 60 minutes in the minipcr1 thermocycler. we analyzed 10 μl of each pcr product using 2% agarose electrophoresis in tris-acetic acid-edta (tae) buffer (sigma-aldrich, mo, usa). gels were dyed with gelgreen (biotium, ca, usa) using a 1:10,000 dilution and a current of 110 v supplied by a bio-rad powerpac hc power supply (bio-rad, ca, usa) for 40 min. we used the quick-load purple 2-log dna ladder (neb, ma, usa) as a molecular weight marker. we analyzed the gels by uv transillumination using a bio-rad chemidoc xrs imaging system. in some of our experiments, we also used the bluegel unit, a portable electrophoresis unit sold by minipcr from amplyus (ma, usa). in these experiments, we analyzed 10 μl of pcr product using 2% agarose electrophoresis tris-borate-edta buffer (tbe). gels were dyed with gel-green (ca, usa) using a 1:10,000 dilution, and a current of 48 v was supplied. photo-documentation was done using a smartphone camera. as a third method of detection and to read the amplification product, we evaluated the amplification products by detecting the fluorescence emitted by a dna intercalating agent, the evagreen1 dye, in the synergy ht microplate reader (biotek instruments, vt, usa). briefly, 20 μl of the pcr reaction mix was placed in distinct wells of a 96-well plate, after completion of the pcr program. each well was made to a final volume of 150 μl by adding 130 μl of nuclease free water and the samples were well mixed by pipetting. combined use of the minipcr thermocycler and a well-plate reader for sars-cov-2 virus detection these experiments were run in triplicate. the following conditions were used in the microplate reader: excitation of 485/20, emission of 528/20, gain of 75. fluorescence readings were made from the above at room temperature. determination of mean values and standard deviations were conducted using excel tools. all experiments were run by triplicate. regression analysis was conducted in excel. time is the most limiting factor in epidemic emergencies. therefore, the integration of welldeveloped and commercially available technologies [31,37,38] becomes an obvious, expedient, and cost-effective first line of defense in the context of covid-19 pandemics. here, we demonstrate that the combined use of a commercial and portable pcr unit (the minipcr) and a 96-well plate reader is potentially adequate for the fast deployment of diagnostic efforts. we show the combined ability of both units to amplify and identify different synthetic genetic sequences of sars-cov-2 (see materials and methods). we conducted a series of experiments to assess the sensitivity of the pcr reactions conducted in the minipcr thermocycler using the three sets of primers recommended by cdc to diagnose infection by sars-cov-2. table 1 shows the sets of primers used to target genetic sequences that code for the expression of the sars-cov-2 n protein. table 2 shows the sequence of the dna products (amplicons) generated by successful targeting of these regions with the n1, n2, and n3 primer pairs. fig 2a-2c show the pcr products of the amplification reactions conducted using three different primer pairs. in all cases, different concentrations of sars-cov-2 genetic material, in the range of 2.0 × 10 5 to 625 dna copies, were used as reaction templates. if we put this range in the proper clinical context, the actual viral load of covid-19 in nasal swabs from patients has been estimated to fall within the range of 10 5 to 10 6 viral copies per ml [18] . the amplification proceeds with sufficient quality to allow proper visualization of the amplification products in electrophoresis gels, even at low nucleic acid concentrations. fig 2a-2c shows agarose gels combined use of the minipcr thermocycler and a well-plate reader for sars-cov-2 virus detection containing the amplification products of each one of three experiments, where the three different sets of primers (namely n1, n2, and n3) were used to amplify the same range of concentrations of template. the minipcr1 was able to generate a visible band of amplification products for all three primer sets and across the whole range of synthetic viral loads. in general, the products of amplification in final point pcr are primarily detected on agarose gels using conventional electrophoresis techniques conducted with conventional lab equipment. the minipcr1 system is commercialized with its own electrophoretic unit ("bluegel1"; fig 1b and 1c) . the bluegel1 has several important advantages and represents a valid and portable solution for detecting pcr amplification products. nevertheless, running an experiment aimed at visualizing amplification products, as with any standard gel electrophoresis procedure requires time. a good separation of bands typically involves a processing time of 35 to 60 minutes from the loading of the amplification product to the final documentation through photography. as an alternative, we show here that the amount of amplification product can be quantitatively evaluated using a commercial 96-well plate reader. to do this, we used an intercalating agent during amplification in the minipcr apparatus. fig 2d-2f shows the fluorescence readings associated with the analysis of the different dilutions of synthetic sars-cov-2 samples previously revealed by gel electrophoresis (s1 fig, s2 fig and s3 fig) . we ran triplicate reactions for each dilution and for each primer data set. the fluorescence readings were capable of clearly discriminating between positive and negative samples across the whole range of dilutions tested (from 2 × 10 5 to 625 copies). this observation holds true for each of the three primer sets tested. note that the use of a plate reader, instead of a conventional gel electrophoresis unit, presupposes a significant savings in time. up to 96 pcr reactions can be read in a matter of 5 to 10 minutes. this implies that an array of 12 minipcr units and a plate reader could equal the throughput of a traditional rt-qpcr platform, but at one third of the capital cost. in addition, during emergencies and particularly in developing countries, attaining or buying regular thermal cyclers and plate readers is much easier than purchasing or accessing rt-qpcr systems. in addition, our results suggest that fluorescence readings using a plate reader exhibit high reproducibility and robustness. overall, we obtained small standard deviations (in the range of 6 to 40 arbitrary fluorescence units [a.f.u.]) and a small average variance coefficient (2.6%) in fluorescence readings across the whole range of values of viral copies tested. we observed similar variability indicators in experiments using different primer pairs. for instance, we observed variance coefficients of 2.31%, 2.15%, and 3.34% when using primer sets n1, n2, and n3, respectively. if we considered only fluorescence readings from positive samples, we observed variance coefficients of 2.23%, 2.34%, and 1.31% when using primer sets n1, n2, and n3, respectively. fig 2g consolidates the fluorescence readings obtained from minipcr amplifications using synthetic sars-cov-2 samples and the primer sets n1 (blue bars), n2 (red bars), and n3 (yellow bars). overall, this data set is consistent. these results suggest that any of the primer sets tested (n1, n2, or n3) may be used to amplify sars-cov-2 genetic material in the minipcr. however, for the experimental conditions tested (i.e., the nature and concentration of the intercalating agent, the concentrations of primers, and the concentration of enzyme, among combined use of the minipcr thermocycler and a well-plate reader for sars-cov-2 virus detection others), we observe differences in the performance of each primer pair. for example, primer sets n1 and n3 appear to promote amplifications in which the observed fluorescence is proportional to the initial concentration of dna template (i.e., the viral load). by contrast, primer pair n2 appears to generate amplification product with high fluorescence emissions even at low values of the initial final copy numbers. note that all fluorescence readings for positive samples shown in fig 2e exhibit a fluorescence reading between 1300 and 1400 a.f.u. furthermore, measuring the fluorescence with the plate reader may add a quantitative element to the analysis of positive covid-19 samples. in principle, samples with higher viral loads will exhibit higher fluorescence if processed through the same pcr program (i.e., exposed to the same number of cycles). for example, for amplifications using primer set n3, we observe a linear relationship between the natural logarithm of the number of viral copies and the natural logarithm of fluorescence signal for the range of 625 to 40,000 viral copies: where f o is the fluorescence reading exhibited by a blank (i.e., a negative sample prepared and processed in the same way than the positive samples) and α = 8.897 (as determined by fitting of the data presented in fig 3a) . for instance, we believe we can adjust the concentration of intercalating reagent to assure linearity of the fluorescence signal with respect to the viral load for experiments with different primer sets. this simple strategy will result in a fully quantitative, reliable, and easily implemented quantitative version of a straightforward final-point pcr protocol. using the primers and methods described here, we were able to consistently detect the presence of sars-cov-2 synthetic dna using a minipcr and a simple plate reader. in the current context of the covid-19 pandemics, the importance of communicating this result does not reside in its novelty but in its practicality. in our experiments, we have used the three sets of primers designed and recommended by the cdc to identify the presence of sars-cov-2, the causal agent of covid-19. these primer pairs, aimed at identifying three different regions encoding for the n protein of sars-cov-2, have been widely validated and used for diagnostic purposes in actual covid-19 patients, here we simply translated widely tested protocols combined use of the minipcr thermocycler and a well-plate reader for sars-cov-2 virus detection from the framework of an rt-qpcr apparatus (the gold standard platform recommended for analyzing and confirming positive cases) to execution in a miniaturized and already commercial poc thermal cycler. while the cost of a commercial rt-qpcr apparatus falls in the range of $10,000 to $40,000 usd, the commercial value of the minipcr is under $800 usd. this difference is significant, especially when considering the need for rational investment of resources during an epidemic crisis. while the quantitative capabilities of testing in a rt-qpcr platform are undisputable, the capacity of many countries for rapid, effective, and massive establishment of diagnostic centers based on rt-qpcr is questionable. the current pandemic scenarios experienced in the usa, italy, france, and spain, among others, have crudely demonstrated that centralized labs are not an ideal solution during emergencies. portable diagnostic systems may provide the required flexibility and speed of response that rt-qpcr platforms cannot deliver. to further illustrate the deterministic and quantitative dependence between the concentration of amplification product and the fluorescence signal, as measured in a plate reader, we simulated some real-time amplification experiments. to that end, we conducted amplification reactions using initial amounts of 4 × 10 4 copies of synthetic sars-cov-2 in the minipcr cycler. we added the intercalating agent, evagreen dye, to the reaction mix at the initial time and extracted samples after 1, 5, 10, 15, 20, 25 , and 30 pcr cycles. the fluorescence from these samples was then measured in a plate reader. we observed a linear increase in fluorescence as more pcr cycles were performed ( fig 3b) ; this highlights the quantitative nature of the intercalating reaction. our results suggest that using a commercial plate reader to determine the extent of advance of pcr amplifications is a practical, reliable, reproducible, and robust alternative to the use of gel electrophoresis. moreover, fluorescence reading of pcr products may lead to precise quantification of viral loads. the current covid-19 pandemic has crudely demonstrated that our available methods of detection have severe limitations in terms of cost-efficiency, scalability, and amenability for rapid implementation. developing and well-developed countries have experienced severe difficulties in intensifying diagnostics, a required condition to stop the pandemic advance in densely populated cities. since time is the most limiting factor in emergencies, the integration of well-developed and commercially available technologies becomes an obvious, expedient, and cost-effective first line of defense during epidemic events. our research extends the validation of the minipcr technology to the as-yet-unexplored topic of detection of covid-19. furthermore, we suggest the combined use of the minipcr and a conventional well-plate reader as a reliable strategy that can expand the testing capabilities of rt-qpcr. we used the set of primers developed by the cdc and recommended by the who for conducting the standard pcr diagnostics of covid-19. these primers target three different regions of the viral nucleic acids encoding for the n protein. in our experiments, we corroborate that the minipcr apparatus is capable of amplifying small amounts of sars-cov-2 synthetic nucleic acids. we were able to detect and amplify 64 copies of genes encoding for the n protein of sars-cov-2. in the context of the covid-19 pandemics, the use of the minipcr thermocycler may be a valuable tool to intensify diagnostics by providing relevant advantages of higher portability, lower capital cost, and easier operation than can be achieved with other rt-qpcr platforms. we found the minipcr1 to be simple and intuitive to use; these are important attributes that would facilitate the widespread adoption of any diagnostic technology. moreover, the combined use of the minipcr thermocycler and a 96-well plate reader enables the possibility of obtaining immediate readings of the amplification products, thereby providing faster (and potentially quantitative) diagnostic results in shorter times than when gel electrophoresis techniques are used. therefore, the integration of these two already commercially available devices-a minipcr thermocycler and a 96-well plate reader-has great potential for use during epidemic emergencies. 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perspectives. acs sensors handheld real-time pcr device the genepoc platform, a rational solution for extreme point-of-care testing pcr heads into the field multi-center evaluation of cepheid xpert® xpress sars-cov-2 point-of-care test during the sars-cov-2 pandemic performance of minipcr tm mini8, a portable thermal cycler validation of use of the minipcr thermocycler for ebola and zika virus detection. ansumana r, editor a simple, economical protocol for dna extraction and amplification where there is no lab real-time dna barcoding in a rainforest using nanopore sequencing: opportunities for rapid biodiversity assessments and local capacity building. gigascience successful amplification of dna aboard the international space station gene expression studies using a miniaturized thermal cycler system on board the international space station key: cord-266799-t7lqlv41 authors: rotejanaprasert, chawarat; lawpoolsri, saranath; pan-ngum, wirichada; maude, richard j. title: preliminary estimation of temporal and spatiotemporal dynamic measures of covid-19 transmission in thailand date: 2020-09-24 journal: plos one doi: 10.1371/journal.pone.0239645 sha: doc_id: 266799 cord_uid: t7lqlv41 background: as a new emerging infectious disease pandemic, there is an urgent need to understand the dynamics of covid-19 in each country to inform planning of emergency measures to contain its spread. it is essential that appropriate disease control activities are planned and implemented in a timely manner. thailand was one of the first countries outside china to be affected with subsequent importation and domestic spread in most provinces in the country. method: a key ingredient to guide planning and implementation of public health measures is a metric of transmissibility which represents the infectiousness of a disease. ongoing policies can utilize this information to plan appropriately with updated estimates of disease transmissibility. therefore we present descriptive analyses and preliminary statistical estimation of reproduction numbers over time and space to facilitate disease control activities in thailand. results: the estimated basic reproduction number for covid-19 during the study ranged from 2.23–5.90, with a mean of 3.75. we also tracked disease dynamics over time using temporal and spatiotemporal reproduction numbers. the results suggest that the outbreak was under control since the middle of april. after the boxing stadium and entertainment venues, the numbers of new cases had increased and spread across the country. discussion: although various scenarios about assumptions were explored in this study, the real situation was difficult to determine given the limited data. more thorough mathematical modelling would be helpful to improve the estimation of transmissibility metrics for emergency preparedness as more epidemiological and clinical information about this new infection becomes available. however, the results can be used to guide interventions directly and to help parameterize models to predict the impact of these interventions. introduction two critical pieces of information when a new emerging infectious disease epidemic occurs are the mechanism of disease transmission and how infectious it is. a new emerging and reemerging infectious disease can occur in one place and have the potential to widely spread, where everyone may be susceptible to the disease. likewise, in thailand, covid-19 caused by the new corona virus is now a major public health concern declared as a national health emergency. in this situation policy makers have to make decisions in the presence of enormous uncertainty and it is crucial to have informed and effective decision making, particularly during the epidemic when the real situation can be very dynamic. during such an outbreak, a large volume of data can be gathered from different sources. these data must be properly transformed into useful and timely information and visualized to effectively assist in the decision-making process. the information should include basic epidemiological descriptions of disease transmission, in terms of person, place, and time. in addition, updated estimates of disease transmissibility should be provided for ongoing planning of appropriate control measures. for transmitted infections transmitted from human to human such as covid-19, the spread intensity depends on factors such as the number of infected and susceptible individuals, human contact patterns, and population demographics. various transmissibility measures can be applied depending on the available data type and can be calculated using different methods. a metric of transmissibility which can be quickly computed is the growth rate, which is estimated from a simple model where disease incidence is exponentially increasing [1] . another important concept in disease dynamic is the reproduction number, commonly noted as r. this quantity measures the expected number of subsequent infections caused by each primary case over the course of their infectious period. the basic reproduction number (r 0 ), probably one of the most widely used of reproduction numbers is defined in a large population where all individuals are susceptible to infection without any control measures. note that a case here means in a generic sense to represent any infection, even if too mild to meet the clinical case definition [2] . an extensive range of reproduction numbers estimation methods have been proposed (see examples [3, 4] ). although these quantities are helpful to understand the transmissibility of an infectious disease, the estimation methods via transmission models often present difficulties in fitting due to context-specific conditions which usually is unsatisfied [5] [6] [7] . however, during the covid-19 epidemic, disease transmission has been changing rapidly in conjunction with dramatic societal and environmental changes. therefore, the reproduction number should be updated continually according to these changes. it has been proposed that the temporal variation of an epidemic can be estimated by the temporal (effective) reproduction number over time, r t [8, 9] . in the who report of thailand covid-19 situation of 6 february 2020, less than 30 laboratory-confirmed covid-19 cases were reported by thai health authorities. most were visitors to thailand from china, including wuhan city in hubei province. furthermore, there were 595 suspected cases in thailand under investigation for covid-19, while others had been treated for symptoms and discharged. among the first thai confirmed covid-19 cases there were two taxi drivers who possibly had been in contact with infected passengers from china [10] . since the new coronavirus outbreak began in january 2020 in bangkok, most newly reported cases were related to transmission clusters including those who had returned from abroad or had occupational exposure to large numbers of people related to businesses in spas, hotels, restaurants, and mall shops. most cases were male and 20-49 years of age. this was likely because so many cases were linked to boxing stadiums, entertainment venues and to attendance at religious events [11] . covid-19 was successfully contained in bangkok for the first two months. however, this was followed by cluster outbreaks in sport and entertainment events, and appearance of the disease in most provinces across the country. since the contact patterns among individuals differ due to differences in the local environment (e.g. population density, weather), human behavior (e.g. social distancing, working from home, travel), as well as use of personal protection (e.g. facemasks, handwashing), and levels of preexisting immunity, disease transmissibility will vary across locations. the spatial variation of transmissibility between locations should be incorporated to provide more detailed information for policy makers in order to monitor areas at risk. this could potentially be helpful for prioritizing healthcare and public health resources during this outbreak. appropriate disease control policies must be planned and implemented in a timely manner. a key ingredient used to guide this public health preparation is the transmissibility metric. this study thus aims to estimate and compare disease dynamic measures in several dimensions that can be augmented with epidemiological summary statistics to monitor the covid-19 situation for each location and time at different stages of the epidemic. this work can be a complement to current control activities to provide a more complete evaluation of the disease transmission situation in thailand. as the new coronavirus pandemic continues with changing risk both locally and globally, we hope this work can be a useful addition to the methodological armory to help ongoing planning efforts. the data in this study were from confirmed covid-19 cases in 77 provinces of thailand from january 12 th 2020 through june 30 th 2020 provided in the daily reports of the department of disease control, thai ministry of public health (moph). suspected cases of covid-19 infection were identified in hospitals and confirmed at designated laboratories by virus polymerase chain reaction of nose and throat swabs. we collected demographic data and place of diagnosis from the official website developed by the digital government development agency (https://data.go.th/dataset/covid-19-daily). as all the data used were publicly available, ethical approval was not required. statistical estimation of reproduction numbers. the first important step to investigate disease transmission is data exploration which combines visualization with calculation of summary epidemiological statistics. epidemiological measures for infectious diseases include incidence rate, standardized ratios, e.g. standardized incidence ratio or mortality rate (smr), and cumulative cases. summary epidemiological statistics are useful information to guide control activities, however these measures are exploratory descriptions that only partially represent disease intensity. alternatively, the transmissibility of a disease can be referred to the rate where incidence arises in an at-risk population and potentially leads to an outbreak [9, [12] [13] [14] . besides an intrinsic characteristic of an emerging infection, transmissibility can also offer the quantification of disease spread in a given epidemic setting and is impacted by various variables including application of personal protection, contact patterns, and pre-existing immunity status. a range of transmissibility metrics can be adopted suitable for different types of available data and can be approximated using several approaches. a transmissibility metric which can be quickly calculated is the growth rate. this quantity can be calculated from a basic model where transmission is exponentially increasing and generally defined as the slope of a linear model on logged incidence [1] . during the early stage of an epidemic curve caused by emerging diseases, the exponential growth (eg) rate, denoted by r, can be related to the initial reproduction rate and can be described as the change in number of new cases per time unit [15] . usually calculation of the basic reproduction number requires careful considerations of fundamental conditions to produce precise interpretation. however, in our situation policy makers have to make decisions with the race of disease transmission in the presence of enormous uncertainty. so we aim to make preliminary estimates of the reproduction number with uncertainty intervals under various assumptions for assisting policy makers in this urgent time. nonetheless, we believe that ongoing investigation and modelling activities should be carried on to assess the effect of public health policies to keep providing updated information to the research community. as incidence data are non-negative integers, a poisson likelihood is perhaps suitable to estimate r 0 , rather than linear gaussian model of the log scaled of incidence [16] . the exponential growth curve can be used to estimate the reproduction rate [15] and then the basic reproduction number can be estimated as r 0 ¼ 1 mðà rþ where m is the moment generating function of the serial interval time distribution [15, 17] and r is the exponential growth rate during the early stage of an outbreak. alternatively, the reproduction number can also be calculated using the maximum likelihood estimator (mle). this method relies on the assumption that the number of secondary cases caused by a primary case follows a poisson distribution parameterized by r 0 in which the likelihood is computed on a period of exponential growth curve [17, 18] . given the set of observation of incident cases, {y t }, over consecutive time units, t = 1,. . .,t, and a generation time distribution over w l , r 0 can be approximated by optimizing the log-likeliwhere μ t is the mean incidence at time t and equal to r 0 x l l y tà l w l , and l is the maximum time for serial interval. temporal and spatiotemporal transmissibility measures. even though the basic reproduction number may be useful for understanding the behavior of an emerging disease and designing various intervention strategies, the classic threshold theoretically assumes that the outbreak first occurs in a population with full susceptibility, and hence this quantity is essentially a mathematically defined number and may be less useful in a real disease control situation. it is practically important to assess time-dependent variations in the covid transmission potential. the pattern of outbreak time series can be partly explained by estimating the effective reproduction number, r t , defined as the expected number of secondary cases per primary case at calendar time t > 0 (for examples see [19] [20] [21] ). the r t can represents temporal variation due to the changes in susceptible individuals as intrinsic factors and the operation of control measures which can be seen as extrinsic factors. it suggests that the outbreak is in decline and may be considered as being under control during time t when r t < 1, and, vice versa, if r t > 1. several methods to estimate the effective reproduction number exist for emerging diseases [19] [20] [21] . here we adopted on a published method [8, 9, 14] that can be calculated with a stochastic process following the renewal equation in which the series of expected cumulative incidence arise from poisson r t x l l y tà l w l � � . from this, a data distribution given a set of model parameters can be calculated, as well as the posterior distribution of r t given collected observations of incidence and knowledge of the serial interval, {w l } [9] . we wanted to provide information that could be used to help design effective control strategies for the current covid-19 situation in thailand after the disease has spread to different provinces across the country much of which was from cluster outbreaks originating from several super spreader events. the spatial variation of disease transmission between locations should be incorporated to provide a more detailed picture for policy makers in order to assess areas at risk and can be potentially helpful for prioritizing healthcare and allocation of public health resources during this outbreak. the spatiotemporal reproductive number, r st , for spatial unit s at time t can be defined as [22] where μ st is the mean incidence in province s on day t. to investigate local behavior of disease transmission, we adopted the exceedance probability to identify unusual elevations and detect clusters of covid-19 incidence. the probability can be estimated by estimating frequency of the measured risk exceeding a threshold and has been used to evaluate how unusual the risk is in an area (see examples [23] [24] [25] [26] ). two cutoff thresholds of r st were chosen as one to represent the null situation (threshold = 1) if the infection was under control and three, a situation where the disease was highly transmitted (threshold = 3) and immediate control policies need to be strictly implemented. details of transmissibility metrics and parameterization are provided in the supplementary document. analyses were performed using r (rstudio version 1.2.5001) and winbugs [27] software. while we are still learning about this new coronavirus, more epidemiological and clinical information is required now in order to specifically design appropriate control strategies. the transmissibility described by the reproduction number is one of the key ingredients. to approximate the disease dynamics at this early phase we adopted statistical methods to estimate disease transmission measures under various assumptions. both the exponential growth (eg) rate and maximum likelihood estimation (mle) were used for estimation of r 0 for the covid-19 situation in thailand with eleven different studies with parameterized serial intervals in a recent review of available evidence [28] . the estimates were selected from studies with the number of sample pairs larger than twenty. four of the studies had a pre-print status and seven were published research articles. most of the estimates were calculated using case information from asian countries, particularly china. some of the calculation also applied data from combinations of countries including italy, germany and the usa. table 1 shows the estimation of r 0 for the covid-19 situation in thailand with different approximation methods and distributional assumptions of serial interval times. for the eg and mle methods we also need to supply the time period over which incidence exponentially grow and different time periods can yield very different estimates. the time period from the primary incidence to the date of maximum number of cases could be one option. nonetheless, because of uncertainty of variation in the early phases of the outbreak, a better choice might be to apply goodness-of-fit criteria to decide the best time period. since the poisson distribution was assumed as the data likelihood, the deviance r-squared metric might be suitable to assess over possible time periods. the largest value of the statistics was considered to be the most appropriate period selected for the analysis. another parameter needed to be defined is the distribution of the serial interval. the empirical distribution could be used from raw data however only the point estimates of mean and standard deviation were reposted in studies and eligible serial-interval distributions are those with positive values. a range of positive-valued distributions were applied to estimate covid-19 serial interval and the gamma distribution was commonly used to estimate serial interval times [28] . on the other hand log-normal modeling also outperformed in a study (both with and without right truncation) [30] . thus, in this study we assumed the serial intervals to follow log-normal and gamma distributions with corresponding parameters for calculation in each scenario. as result, the estimated basic reproduction numbers over our collected data using reasonably exponential curve ranged from 2.23-5.90. the overall means under log-normal and gamma distributions were 3.76 (95% ci: 2.23-5.90) and 3.74 (95% ci: 2.49-5.51) data respectively. besides the basic reproduction number which merely embodies the disease transmissibility in the whole susceptible population, we also investigated temporal disease variations over time using the effective reproduction number. fig 4 shows the number of new cases for the whole country (top), bangkok (middle) and outside bangkok (bottom) with an estimated r t (dash) over february-may 2020. the r t values were approximated by averaging over the serial intervals reported in [30, 36] which were near to the estimated overall mean. plots in fig 4 suggest that the outbreak was under control since the middle of april with r t less than one. after the boxing stadium and entertainment venues, the numbers of new cases had increased until mid-march. then the number of new cases outside bangkok sharply increased about one week thereafter with a large jump in r t while cases in bangkok started to flatten and then decrease thereafter. the number of new cases continued fluctuating likely due to imported cases returning from overseas. this could be partly related to testing capacity and infection residuals. however the thai government has implemented travel restrictions including permission to enter or transit thailand since may. all travelers will be subject to a 14-day state quarantine at a designated facility. so the fluctuation due to traveling might not contribute to local transmission since then. the countrywide spread is also reflected in the incidence and r st maps in fig 2. many provinces had few or no cases on march 16 th . we started to see more cases on march 20 th , increasing further on march 21 st with high r st in several provinces. to evaluate geographical cluster detection, exceedance probabilities were used as elevated risk diagnostics to investigate geographic patterns of possible spatial anomalies of the infection. in the exceedance maps ( fig 5) , we can see only a few scattered provinces with high exceedance probabilities both for thresholds of one (top) and three (bottom) on march 16 th . however, on march 21 st there were increased risks indicated by high exceedance probabilities in the south and west of the country, and some appearing disease clusters spreading over the central region and along the thai-cambodia border. this contrasts with the isolated hot spots also found in northern areas. this method thus helps to identify provinces with more transmission tan others requiring more immediate attention. when a new emerging infectious disease epidemic occurs, a critical challenge for emergency preparation is that the situation can be very dynamic. policy makers need to make decisions based on high level of uncertainty. the novel coronavirus infection has been the primary public health concern in thailand with declaration of a national health emergency. the number of covid-19 cases has rapidly increased in the country during march 2020. most cases were initially contained in bangkok, but this was followed by spread to and transmission in other provinces across the country. transmission dynamics of covid-19 in thailand however remain uncertainly quantified. thus in this study we present descriptive analyses and quantification of transmission dynamic measures both overall and in space-time dimensions to inform the ongoing control activities of covid-19 outbreak in thailand. quantifying transmission dynamics of new emerging diseases is a necessary initial step in understanding the epidemic and pandemic potential. we applied statistical methods to available data on cases of novel coronavirus in thailand to estimate how transmission had varied during february and march, 2020. preliminary estimation of reproduction numbers was presented with different assumptions to provide a range of possible estimates. based on available information, we found that the estimate of r 0 in thailand probably varied around 3.75 (95% ci ranging 2.23-5.90) averaged over parameter and distributional assumptions. a number of studies up to early february were reviewed in liu et al. [38] in which they found that the expected mean r 0 for the infection ranged from 1.4 to 6.49, with a mean of 3.28, a median of 2.79 and interquartile range of 1.16. another recent systematic review [39] showed the estimated basic reproduction number in china was 2.72 (95% ci: 2.08-3.57) while the estimated outside of china was 4.56 (95% ci: 2.28-9.12). note that we applied all cases in thailand available on the website which could include imported cases. however, the sequential cases used to calculate the basic reproduction number during the initial outbreak in bangkok might be more related to local super-spreader sources such as sport stadiums, entertainment venues and religious events. our estimates may also be characterized by a number of incidents in which most initial cases locally infected other people. following initial spread, much of the ongoing transmission was rapidly contained through public health measures including quarantine of infected individuals. established wide spread transmission from those cases would have caused the transmissibility measures to be considerably higher. method estimated r 0 (95% ci) the estimated fluctuations in r t were driven by the rise and fall in the number of cases, both in thailand and internationally, as well as prevalence of infection among passengers on evacuation/repatriation flights, other passenger flights having been severely curtailed. such variations are similar to other studies [40] where causes could include changes in pattern in the population at risk, or specific large spreading events that changed the average transmission estimate. some evidence of a reduction in r t was found in the days after the introduction of restrictions in the city of bangkok, which might have reflected disease control efforts or growing awareness of the infection during that period. there have been no local cases since the end of may and the fluctuation since june was due the imported cases who were subject to a 14-day state quarantine at a designated facility. in this study we applied only publicly available data due to limitations on confidentiality and the uncertainty in our estimates for r t perhaps was a result from a lack of data availability to inform changes in transmission during the study period. it is also possible that the contact pattern related to infection spread varied over time due to the case variation during the period reflected by effective control strategies implemented against the disease spread. moreover, the data could include reporting delays which also could limit the performance of r t in real-time surveillance. however, we have planned to develop a method to correcting for reporting delays which could be beneficial to disease control activities. the spatiotemporal distribution of cases was supposed to be associated with travel of people who attended entertainment events and thai boxing in bangkok in early march. those people perhaps went home unknowingly carrying the coronavirus. a rise in cases later in the same month may be caused by the implementation of a robust set of public health measures to control the disease situation. this can be seen as the incidence had risen in bangkok a week after those events followed by the increase in other provinces (fig 4) . the spread might also be related to the government's lock-down policy in bangkok. during the same period of time the bangkok city hall also imposed the closure of shopping malls, schools, and other venues considered high-risk areas. the workers in those business returned their hometowns and might lead to waves spreading the infection across the country captured in the spatiotemporal transmission dynamics in figs 1 and 5. the prime minister hence forced a state of emergency across the country, set to be effective until at least the entire month of april 2020. since then the number of new cases appeared to decline, yet the strict disease control policies have remained in action. this study aimed to estimate early transmission measures in different dimensions to inform disease control activities. although several scenarios about assumptions were explored in this study, the real situation was challenging to investigate given the limited data. our estimation and analysis relied on the information quality of the serial-time interval of the disease, which was highly uncertain especially with thai data during the study period. in this work, we then employed various generation time scenarios from other studies, some of which also borrowed that information from other similar emerging coronaviruses such as sars as approximations to that of covid-19. the improved determination of those parameters is needed and requires further knowledge of the disease transmission chains with a sufficient number of patient subjects and longitudinal times for follow-up [41] . this is unlikely to be achieved shortly especially for local information. more thorough mathematical modelling would be helpful to improve the estimation of transmissibility metrics for emergency preparedness as more epidemiological and clinical information about this new infection becomes available. more information is urgently needed as a new emerging infectious disease epidemic evolves presenting a critical challenge for emergency preparation. we present descriptive analyses and preliminary statistical estimation of transmission metrics in over space and time. this new infection has happened in china since the middle of december in 2019, and spread globally with the first case found in thailand in january 2020. possible next steps include proposing the most effective control policies to lessen transmission in the country. the working modelling assumptions need to be refined as more is learned about the epidemiological characteristics and outbreak dynamics. our initial inferences have been made on publicly available data; there perhaps soon be too more information of this new pathogen, and other sources of data should be incorporated. we believe that ongoing surveillance and modelling efforts should continue to assess the effect of public health measures. the transmissibility estimation should keep going to determine if the transmissibility might vary in different assumptions and regions including socioeconomic and climatic contexts. new outbreak clusters of this infection have happened across different countries. as this coronavirus pandemic continues to develop and the risk changes on both local and global scales, hopefully our work can provide an addition to the whole picture of this new infection for research communities and policy-makers. a statistical algorithm for the early detection of outbreaks of infectious disease beyond r0: heterogeneity in secondary infections and probabilistic epidemic forecasting the estimation of the basic reproduction number for infectious diseases. statistical methods in medical research the failure of r(0) perspectives on the basic 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handbook of spatial epidemiology winbugs-a bayesian modelling framework: concepts, structure, and extensibility a rapid review of available evidence on the serial interval and generation time of covid-19. medrxiv epidemiology and transmission of covid-19 in 391 cases and 1286 of their close contacts in shenzhen, china: a retrospective cohort study. the lancet infectious diseases serial interval of novel coronavirus (covid-19) infections serial interval of covid-19 among publicly reported confirmed cases. emerging infectious diseases estimating the serial interval of the novel coronavirus disease (covid-19): a statistical analysis using the public data in hong kong from covid-19 serial interval estimates based on confirmed cases in public reports from 86 chinese cities estimating the generation interval for coronavirus disease (covid-19) based on symptom onset data epidemiological parameters of coronavirus disease 2019: a pooled analysis of publicly reported individual data of 1155 cases from seven countries transmission interval estimates suggest pre-symptomatic spread of covid-19 estimating clinical severity of covid-19 from the transmission dynamics in wuhan, china the reproductive number of covid-19 is higher compared to sars coronavirus reproductive number of covid-19: a systematic review and metaanalysis based on global level evidence early dynamics of transmission and control of covid-19: a mathematical modelling study. the lancet infectious diseases preliminary estimation of the basic reproduction number of novel coronavirus (2019-ncov) in china, from 2019 to 2020: a data-driven analysis in the early phase of the outbreak we would like to thank patiwat sa-angchai for assistance with the epidemiological data. key: cord-293794-vudufao5 authors: cuthbertson, leah; oo, stephen w. c.; cox, michael j.; khoo, siew-kim; cox, des w.; chidlow, glenys; franks, kimberley; prastanti, franciska; borland, meredith l.; gern, james e.; smith, david w.; bizzintino, joelene a.; laing, ingrid a.; le souëf, peter n.; moffatt, miriam f.; cookson, william o. c. title: viral respiratory infections and the oropharyngeal bacterial microbiota in acutely wheezing children date: 2019-10-17 journal: plos one doi: 10.1371/journal.pone.0223990 sha: doc_id: 293794 cord_uid: vudufao5 acute viral wheeze in children is a major cause of hospitalisation and a major risk factor for the development of asthma. however, the role of the respiratory tract microbiome in the development of acute wheeze is unclear. to investigate whether severe wheezing episodes in children are associated with bacterial dysbiosis in the respiratory tract, oropharyngeal swabs were collected from 109 children with acute wheezing attending the only tertiary paediatric hospital in perth, australia. the bacterial community from these samples was explored using next generation sequencing and compared to samples from 75 non-wheezing controls. no significant difference in bacterial diversity was observed between samples from those with wheeze and healthy controls. within the wheezing group, attendance at kindergarten or preschool was however, associated with increased bacterial diversity. rhinovirus (rv) infection did not have a significant effect on bacterial community composition. a significant difference in bacterial richness was observed between children with rv-a and rv-c infection, however this is likely due to the differences in age group between the patient cohorts. the bacterial community within the oropharynx was found to be diverse and heterogeneous. age and attendance at day care or kindergarten were important factors in driving bacterial diversity. however, wheeze and viral infection were not found to significantly relate to the bacterial community. bacterial airway microbiome is highly variable in early life and its role in wheeze remains less clear than viral influences. introduction respiratory infections are a significant cause of morbidity in young children, with over half of hospitalisations in children under 5 years of age being related to respiratory disease [1] . evidence suggests that environmental and infectious exposures in early life are a major risk factor for the development of disease [2, 3] , including asthma [4] [5] [6] . acute wheeze is the most common reason for children to present to hospital with between 80-90% of these cases being attributed to viral infection [7, 8] . while a number of respiratory viruses have been implicated in wheeze the most common are respiratory syncytial virus (rsv) and rhinovirus (rv) [9] . in infants aged less than one year rsv infections predominate, but after this rv is also viewed as an important risk factor for acute wheeze [10] . the influence of the respiratory tract bacterial community in the context of wheeze is still being established. recent studies in this area have implicated organisms such as moraxella catarrhalis, haemophilus influenzae and staphylococcus aureus in increased rates of wheeze [11, 12] . it has been suggested this may be due to immune modulation by these organisms, in infants resulting in increased risk of asthma development in later life [13] . using 16s rrna gene sequencing of respiratory samples from children presenting to hospital with acute wheezing, this study aimed to examine whether the bacterial community in the airways of children with acute respiratory wheeze was altered compared with that of nonwheezing children. changes in the bacterial community were also explored to determine if acute rv infection or species had a significant effect on the airway microbiota. children between 0-16 years were recruited as part of the mavric (mechanisms of acute viral respiratory infection in children) study on presentation to princess margaret hospital (pmh) for children, perth, western australia between january 2004 and january 2014. cases and controls were recruited through all seasons, and a questionnaire was administered to all subjects to determine symptoms of any current illness, including coryzal symptoms, and risk factors, including birth history, postnatal and in utero cigarette smoke exposure, daycare attendance, atopy and allergy history, diagnoses of acute illness (children were excluded if they had any chronic illness other than asthma), recent antibiotic use, systemic steroids. recurrence data was collected on cases included in the study from birth. hospital presentation records were used to determine frequency of respiratory presentations both prior to and following presentation. five patterns were determined described as "few", "persistent", "multiple a", "multiple b", and "atypical". (see supplemental methods for further definitions). cases recruited had acute wheezing illness with no other co-morbid conditions besides asthma, eczema, or atopy. controls with no pre-existing chronic disease including chronic respiratory illness were recruited from four sources: siblings and relatives of cases, pmh patients (presenting with minor injury/fractured limbs), volunteers from the local community or day care facilities. a proportion of controls had symptoms of mild acute respiratory infection but no wheeze. blood, oropharyngeal (op) swabs and nasal samples (wash or blow) were collected from each participant. several cases were followed up within 9 months of recruitment and viral, op swab and blood samples were repeated at this time. this study was approved by the pmh for children, perth, western australia human ethics committee (reference: 1761ep). at least one parent or guardian provided informed written consent for children, prior to participation in the study. skin prick tests were performed to 11 allergens (cow's milk, whole egg, cat pelt, dog dander, rye grass, mixed grasses, d. pteronyssinus, d. farinae, cockroach and aspergillus fumigatus, alternaria tenuis), a wheal size �3mm or self-reported allergic reaction to an allergen was used determined atopy. blood cell counts were determined by pathwest laboratory medicine wa (pathwest) at pmh hospital pathology lab. cathelicidin (ll-37) was measured in plasma using a commercially available elisa kit (hycult biotech). a nasal blow or wash was collected at recruitment and placed immediately on ice prior to storage at -80˚c. nasal specimens were typed for rv species using methods previously described [14] using modified primers [15] . briefly, a semi-nested rt-pcr with primers that amplify the 260-bp variable region of the 5' untranslated region of the rv genome was completed. rvpositive samples were sequenced and assigned an rv strain type and species following sequence alignment with sequences of the 101 classic serotypes and the 53 newly assigned genotypes, using clustalx software (university college dublin, dublin, ireland) [16] . a second aliquot from each nasal specimen was tested for rsv, influenza a and b, parainfluenza 1-4, rsv, and human metapneumovirus (hmpv) at pathwest using routine diagnostic methods including pcr [17] , direct or indirect fluorescent antibody testing, or immunofluorescence after cell culture as previously described [18] . whenever possible, samples were also tested for enterovirus, coronavirus, and bocavirus. sterile dry rayon swabs (copan) were used to sample the soft palate of the oropharynx. specific care was made not to contaminate samples with any other part of the mouth. any swab that made contact with the tongue or cheek were disposed of and a new swab was performed, for full details see supplementary materials. samples were immediately put on ice and stored at -80˚c. dna was extracted from samples utilizing an mpbio fastdna spin kit for soil as per manufacturers' instructions and frozen at -80˚c. samples were transported on dry ice to imperial college london, for bacterial analysis. total bacterial burden was measured using a sybr green quantitative pcr assay using the primers 520f, 5'-aytgggydtaaagng and 820r, 5'-tacnvgggtatctaatcc, targeting the v4 region of the 16s rrna gene as described in cuthbertson et al 2017 [19] . all reactions were performed in triplicate and included standards and non-template controls on the viia 7 real-time pcr system (life technologies, paisley, uk) using sybr fast qpcr master mix (kapa biosystems, wilmington, ma, usa). standards were generated from near full length cloned 16s rrna gene of vibrio natregens. plasmids quantified using quantit picogreen dsdna assay kit (promega, madison, usa), samples were then serially diluted 10 fold to form standards ranging from 1 x 10 8 -1 x 10 4 . community analysis was carried out using 16s rrna gene sequencing. custom dual barcoded fusion primers were used to target the previously quantified region of the 16s rrna gene as previously described [19] . each sequencing run contained a pcr negative control and a mock community, consisting of 34 16s rrna gene clones of known bacterial species in equal proportions. sequencing was carried out using the illumina miseq platform using the illumina v2 2x250bp cycle kit. sequences were submitted to the european nucleotide database, project number prjeb32061. downstream sequencing analysis was carried out using quantitative insights in microbial ecology (qiime) version 1.9.0 due to the dual barcoded indexs used in this study. all sequences were trimmed to 200bp and joined with a minimum of 150bp overlap, a maximum of 10% mismatch was stipulated. sequences were then demultiplexed and any phix reads were removed, prior to otu picking using open reference uclust otu picking [20] , clustering at 97% similarity using the silva reference database (www.arb-silva.de), reads with less that 60% id were discarded and 10% of sequences that failed to id were included for de novo clustering. representative sequences were picked from the most abundant read in the cluster. pynast [21] was used to align representative sequences before running the nearest alignment space termination (nast) algorithm [22] . chimeraslayer (http://microbiomeutil.sourceforge. net/) was used to identify and remove any chimeric sequences. the ribosomal database project (rdp) naive bayesian classifier was used to apply taxonomic identification using silva 115 nr database. finally, an otu biom table was created for further downstream analysis. exact sequence variants (esvs) may in some circumstances improve identification of microbial taxa, but genomic sequencing of the airway microbiota is at an early stage. in order to avoid over-splitting of taxa and false inflation of diversity, we have taken the conservative approach of using otus in our analyses rather than amplicon sequence variants. all further analysis was carried out using r version 3.3.2 [23] . pre-processing and primary analysis was carried out in phyloseq [24] . contamination was removed using decontam [25] . a minimum threshold of 2,000 reads was applied to all, samples with less than 2,000 reads were removed from further analysis. all remaining samples were then rarefied to the minimum number of reads present in the data subset. non-parametric wilcoxon sign ranked tests were used to test significant differences between means. pearson correlations were used to test the relationship between continuous clinical variables and diversity measures. adonis permutational anova was used to investigate changes in community composition while random forest analysis was used to identify otu's associated with disease. after removal of sequencing controls and those with less than 2000 reads, a total of 201 samples were taken forward for analysis, see table 1 (further information in s1 table) . this included 109 samples from children with acute wheeze (fig 1) and paired stable follow-up samples from 17 of these children. control samples from 75 children without symptoms of wheeze were collected, this cohort included children from day-care and siblings of those with acute wheeze (fig 1) . table 1 . demographic table of acute cases and healthy controls. data indicates counts for cases (acute wheeze diagnosis) and healthy controls. n indicates the total number of subjects that information was obtained for each group. count data is indicted as a count (% positive of n). continuous data is recorded at median of n (mixmax). differences in clinical variables were calculated using wilcoxon sign rank test, differences in ethnic groups, sampling season an rv type were calculated using chisquared. samples from 109 children with acute wheeze and 75 control children were compared (table 1 and fig 1) . no significant difference in alpha diversity was observed between the 2 groups (richness, p = 0.363; shannon-weiner, p = 0.98; inverse simpsons, p = 0.654). adonis permutational anova revealed a significant difference in bray-curtis dissimilarity (r 2 = 0.016, p = 0.003), fig 2a. however, the variation explained was only 1%, suggesting a relationship driven by a small number of samples (see s1 fig) . a random forest model was used to predict wheeze based on otus in the bacterial community. the predictive power of the model for cases and controls was found to be poor (out of box (oob) error rate = 34.24%, p = 0.37), therefore the model results were considered unreliable and was not used in further investigation. however, indicator species analysis revealed a significant association of a single veillonella otu with the acute group. deseq2 analysis indicated the same veillonella otu associated with cases while several haemophilus otus were found to be associated with controls (s2 fig) clinical variables were investigated to see if they were able to better explain bacterial community variation within this population. wilcoxon rank sum test was used to investigate 33 binary variables across all samples (s2 table) . after bonferroni correction for multiple testing, only the diagnosis of bronchiolitis was found to show a significant difference in bacterial richness (p = 0.006), shannon-weiner (p = 0.017) and inverse simpsons (p = 0.062). significant differences in shannon-weiner and inverse simpsons were observed in those that required oxygen and those that had ever attended kindergarten. it was noted, however, that none of the controls were positive for bronchiolitis or required oxygen. therefore, this relationship was further tested in the acute wheeze population. kruskal-wallis with dunns test was used to investigate categorical variables, however no significant results were observed. we did not find any differences in the microbiota that were attributable to recorded breastfeeding. no data was available to explore the effect of delivery method in this study. pearson's correlations were used to investigate relationships between 20 continuous variables and alpha diversity measures (s3 table) . only bacterial biomass was found to have a weak significant correlation with bacterial richness. significant changes in blood counts were not found to hold up to multiple testing and expected differences in the antimicrobial peptide cathlecidin were not found. adonis permutational anova with 99,999 iterations was used to investigate changes in community composition with 58 variables. only bacterial biomass, bronchiolitis diagnosis and regular attendance at day care were found to be significant (s4 table) . bronchiolitis diagnosis was found to show significant reduction in alpha diversity. due to the control samples coming from a slightly older population true age matched case controls were unable to be compared (wheeze, median = 0.195 (min = 0.08 -max = 1.87), control, 1.38 (0.6-1.91)). however, when the microbiota in children with bronchiolitis were compared to those closest in age there was a significant reduction in alpha diversity (richness; p = 0.01, shannon-weiner, p = 0.003, inverse simpsons, p = 0.005) that was not associated with change in bacterial biomass (p = 0.874). changes in community composition using bray-curtis dissimilarity were found to explain 9.8% of the variation (p = 0.012), s3 fig. to explore variation within the group suffering from acute wheeze, this group was examined as a subset from the whole data set and clinical variables were explored (fig 1) . in the acute wheeze group, significant decrease in all alpha diversity measures (differences in bacterial diversity between subjects) were seen with bronchiolitis. fourteen patients with doctor diagnosed bronchiolitis were included in the study, all of these patients were under the age of 2 years. of those diagnosed, all patients also had wheeze. nine of the patients were positive for rsv at the time of sampling, 5 of these were also positive for rv (fig 1) . a significant increase was observed with attendance at kindergarten (s5 table) . attendance at preschool showed a significant increase in bacterial richness (number of otus) only. only patient age was found to show significant positive correlation with bacterial richness (s6 table) . in the children with acute wheeze, bacterial biomass was found to explain 8.9% of the variation in beta diversity (p < 0.001). bronchiolitis (r 2 = 0.042, p < 0.001) and attendance at kindergarten (r 2 = 0.052, p < 0.001) were also found to be significant in the acute wheeze dataset (s7 table) . within the 109 children with acute wheeze, 71 were below the age of 5. in the acute wheeze group, the under 5s bacterial biomass remained a significant driver of bacterial community composition (r 2 = 0.06, p < 0.001). however, no other variables were significant in the younger cohort. kruskal-wallis was used to investigate wheeze recurrence patterns for the five patterns of wheeze observed in wheeze cases (s4 fig). no significant difference in alpha bacterial diversity was observed within or between any of the above recurrence groups. within the participants suffering from acute wheeze, 71 patients had rv infection. rv strains from two patients were unable to be typed. twenty-six patients were infected with rv-a, 3 with rv-b and 40 with rv-c. the sample numbers only allowed comparison of the bacterial community from patients with rv-a with those with rv-c. no significant difference in alpha diversity was observed. in the under 5s group, 15 patients had rv-a, 1 had rv-b and 27 had rv-c. a significant difference in richness (w = 123.5, p-value = 0.039) was observed between those with rv-a and rv-c, however no differences in diversity were observed. no significant difference in community composition was observed using adonis (r 2 = 0.039, p = 0.328). random forest modelling was unable to identify otus associated with viral species (p = 0.504). stable follow-up samples were collected from 17 individuals after an acute wheezing episode. no significant difference in bacterial biomass (v = 44, p-value = 0.132) or alpha diversity (richness, p = 0.289, shannon, p = 0.145, simpson, p = 0.109) was found when comparing paired samples with wilcoxon signed rank test. between and within sample beta diversity was compared (fig 2b) . significant differences between paired samples were observed with 5.8% of the variation explained by time of sampling (p = 0.013), however 57.6% of the variation was explained by individual (p = 0.013). in this cohort of children, we found substantial diversity and heterogeneity in op microbiota composition regardless of wheeze status. there were no significant microbiome differences between acute wheeze cases and non-wheezing controls on alpha diversity measures. beta diversity was examined using a bray-curtis dissimilarity adonis model and while significant difference (p = 0.003) was found, only 1.6% of the variance is explained by differences between cases and controls. further interrogation with bray-curtis dissimilarity hierarchical clustering (s1 fig) reveals that the correlation is likely driven by a small number of samples and no pattern was observed between those that had wheeze and those that did not (fig 2) . overall, despite cases having an acute wheezing illness serious enough to cause presentation to a children's hospital emergency department, there were no clear differences in the bacterial community between cases and controls. in the small number of cases with matched acute and convalescent samples taken up to 9 months later, we were similarly unable to find differences in alpha diversity between the cases and follow-up samples. significant differences in beta diversity were observed between cases and follow-up samples, however the variation explained was 5.8% compared to within subject changes which explained 57.6% of the variation. the heterogeneous nature of the microbial community between patients makes investigation into microbial changes a challenge and supports the need for large-scale longitudinal investigation into community composition in health and disease. random forest analysis has been used in a number of recent studies to investigate predictive otus in microbiome analysis [26, 27] . in this study, random forest was used to attempt to identify otu predictors for wheeze. we found that the variation in the microbial community observed between these groups was high and this led to low confidence in the predictive power of the model. random forest analysis of the bacterial community found bacterial otus to be poor predictors of wheeze. this analysis was also performed to determine if any otus were significantly associated with rv infection or between rv species. neither model was found to have strong predictive power and estimated accuracy was low. diagnosis with bronchiolitis showed a significant difference in bacterial richness compared with the entire cohort, however this comprised only a small subset of patients (n = 13). when those with a diagnosis of bronchiolitis were compared to other cases that wheezed or age matched control subjects, significant differences in alpha and beta diversity were observed however this may have been driven by subjects with bronchiolitis being significantly younger than other groups of subjects. bacterial diversity was found to be significantly reduced in these subjects with many patients dominated with streptococcus sp. further investigation into the streptococcal species, which is unable to be elucidated from 16s rrna sequencing, would be important in further investigations. longitudinal studies in young children to determine if low bacterial diversity in the respiratory tract is a risk factor for or the result of bronchiolitis would be important for future investigations. in contrast to our findings, there are a number of studies that show significant differences in microbiome in relation to viral infections compared to healthy subjects [11, [28] [29] [30] . these studies show haemophilus, streptococcus, moraxella associated with acute respiratory tract infections and staphylococcus, alloiococcus and corynebacterium in healthy samples. a number of factors account for the differences we find; firstly, they analyse nasopharyngeal (np) samples compared to the op samples analysed in our study. a number of studies have shown that these areas show significantly different bacterial communities, and that op swabs are a better proxy for the lower respiratory tract microbiome. there are also age differences between other cohorts and ours. lastly analytical methods to look at the microbiome differ between studies. analytical methods for microbiota are slowly being standardised, and much of the analytic techniques are borrowed from ecological analysis to try and accurately portray biodiversity and differences. in order to find differences, earlier studies have defaulted to simplistic groupings based on dominant organisms. however, we have observed that many samples are diverse and without a singular dominant organism. for example, man et al, comparing those with lower respiratory tract infection and healthy controls, found no difference in alpha diversity, and using beta diversity measures found, similar to us, very small but significant differences between the groups (r 2 = 0.0031, p = <0.0001) [28] . to find significant differences in species, previous studies grouped samples based on perceived dominance and not necessarily by a dominance measure or beta diversity based on hierarchical clustering. the greater variation in age within our population compared to other studies may have influenced our ability to find significant differences. bacterial biomass was found to explain much of the variation in beta diversity within the acute population. bacterial biomass was also found to show a weak positive correlation with age. age was found to be a key variable in the analysis of this dataset and this relationship has been demonstrated in previous studies [31] . age is interrelated with developmental changes in blood cell counts and immune function [32] . with these considerations, we considered changes in the microbiome while controlling for age. a strength of our study is that we extracted blood from patients to examine differences in blood counts and antimicrobial peptides. however, variation in age in the study resulted in small and non-significant trends in blood cell counts. cathelicidin was also examined as a candidate innate immunity marker as it has both anti-viral and anti-bacterial activity and lower levels have been shown to be associated with worsened severity in bronchiolitis [33] . however, we were unable to find a significant change in the bacterial community with changes in cathelicidin. seasonal influences in the same study location (perth, australia) have shown to have limited effects on microbiome in comprehensive longitudinal studies at least in infants [29] . risk factors that are associated with hospitalisation as a result of respiratory infections in early life include maternal smoking during pregnancy, season of birth, delivery mode and gestational age [34] . when considered in this cohort of mostly hospitalised wheezing children (96%), these factors were not found to be significantly associated with bacterial or viral infection suggesting more work is required to fully understand any potential relationship. at least one other study exists comparing op microbiome samples between healthy adult patients and severe asthmatics showing no significant diversity differences [35] . an abundance of moraxella is found in prior studies in np. hilty et al demonstrate that moraxella (proteobacteria) exists in oropharynx and may account for differences [36] . op samples were specifically chosen as they showed greater representation to lower airway samples at least in stable patients [36, 37] . whether acute op swabs reflect lower airway samples in acute wheeze or acute viral episodes is still unclear but, interestingly, man et al did show that intensive care patients had reasonable similarity between np samples and lower respiratory tracheal samples (bray curtis similarity p = 0.61), but did find key differences, specifically that staphylococcus, corynebacterium, and dolosigranulum sp. were almost exclusively present in np samples and absent from endotracheal aspirates [28] . the bacterial community in the paediatric population is diverse and heterogeneous. the wide range of clinical factors tested did not fully explain the wide variation in the bacterial community in these subjects. age had a significant influence on both the bacterial community and blood cell counts in this study and the wide age range within the study population helped explain some of the variation observed. the simplest interpretation of our results is that acute wheezing illnesses are driven by viral infections and that these infections have little influence on the bacterial community during the acute phase of the illness. however, the heterogeneous nature of the subjects made it difficult to test for significant associations between clinical variables and the oropharyngeal bacterial community. prospective longitudinal investigation of children pre, during and post viral infection may help identify if the bacterial community is either protective or a risk factor for viral infection and respiratory wheeze. supporting information s1 trends in infectious disease hospitalizations in us children childhood asthma after bacterial colonization of the airway in neonates exposure to environmental microorganisms and childhood asthma risk and protective factors for childhood asthma: what is the evidence? wheezing and bronchial hyper-responsiveness in early childhood as predictors of newly diagnosed asthma in early adulthood: a longitudinal birthcohort study impact of parental asthma, prenatal maternal asthma control, and vitamin d status on risk of asthma and 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asthmatic airways topographical continuity of bacterial populations in the healthy human respiratory tract key: cord-273343-als886fe authors: mcclenahan, shasta d.; scherba, gail; borst, luke; fredrickson, richard l.; krause, philip r.; uhlenhaut, christine title: discovery of a bovine enterovirus in alpaca date: 2013-08-12 journal: plos one doi: 10.1371/journal.pone.0068777 sha: doc_id: 273343 cord_uid: als886fe a cytopathic virus was isolated using madin-darby bovine kidney (mdbk) cells from lung tissue of alpaca that died of a severe respiratory infection. to identify the virus, the infected cell culture supernatant was enriched for virus particles and a generic, pcr-based method was used to amplify potential viral sequences. genomic sequence data of the alpaca isolate was obtained and compared with sequences of known viruses. the new alpaca virus sequence was most similar to recently designated enterovirus species f, previously bovine enterovirus (bevs), viruses that are globally prevalent in cattle, although they appear not to cause significant disease. because bovine enteroviruses have not been previously reported in u.s. alpaca, we suspect that this type of infection is fairly rare, and in this case appeared not to spread beyond the original outbreak. the capsid sequence of the detected virus had greatest homology to enterovirus f type 1 (indicating that the virus should be considered a member of serotype 1), but the virus had greater homology in 2a protease sequence to type 3, suggesting that it may have been a recombinant. identifying pathogens that infect a new host species for the first time can be challenging. as the disease in a new host species may be quite different from that in the original or natural host, the pathogen may not be suspected based on the clinical presentation, delaying diagnosis. although this virus replicated in mdbk cells, existing standard culture and molecular methods could not identify it. in this case, a highly sensitive generic pcr-based pathogen-detection method was used to identify this pathogen. alpaca (vicugna pacos, also known as lama guanicoe pacos) are domesticated members of the new world camelid species (lamini), which also include guanaco (lama guanicoe), vicuna (vicugna vicugna), and llama (lama glama). the natural habitat for alpaca is at high altitude (3500-5000 m) in south america (peru, ecuador, bolivia, and chile) where they are kept as livestock in herds and their fiber is used much like wool. approximately 300,000 animals [1] are in the u.s. compared to other livestock, e.g., about 96 million cattle [2] , their number is still relatively small. previously reported viral infections in domestic alpaca include adenovirus, equine viral arteritis virus, rabies, bluetongue virus, foot-and-mouth disease virus, bovine respiratory syncytial virus, influenza a virus, rotavirus, orf virus, bovine papillomavirus, vesicular stomatitis virus, coronavirus, bovine parainfluenza-3 virus, west nile virus, equine herpesvirus-1 [1, 3, 4] and bovine viral diarrhea virus [5] [6] [7] [8] [9] [10] [11] [12] [13] . bovine enteroviruses (bev) have not previously been reported to infect alpaca. the bovine enterovirus species previously contained two types, bev-a and bev-b [14, 15] although a new classification structure was ratified recently, redesignating these as species enterovirus e (ev-e) and enterovirus f (ev-f), respectively [14, 16] . each of the new bev species includes multiple serotypes, with ev-e comprising four described serotypes (previously a1-4, renamed e1-e4), and ev-f containing six reported serotypes (previously b1-6, renamed f1-f6). recently developed approaches to virus detection have the potential to further expand understanding of viral disease in animals, including alpaca. many of these approaches are based on non-specific pcr amplification used in conjunction with standard or high-throughput sequencing to identify pcr products. we utilized such a method [17] [18] [19] to investigate an outbreak of a respiratory infection in alpaca, identifying a bovine enterovirus (ev-f), named enterovirus f, strain il/alpaca, after other techniques had failed to detect any pathogen. four out of 32 alpaca in an illinois herd ranging in age from 1.5 to 14 years of age died from an acute respiratory infection (with some evidence of systemic spread in two of the animals) of unknown etiology or origin. the other animals in the herd remained clinically healthy. necropsy revealed grossly moderate acute diffuse interstitial pneumonia in all four animals and acute renal cortical infarcts in two of the alpaca. microscopically, marked pulmonary congestion and edema were noted in all lungs, as well as moderate erosive gastritis, acute renal infarcts, mild esophageal erosion and ulceration with suppurative esophagitis in two of the alpaca. quantitative rt-pcr for bovine viral diarrhea virus 1 and 2 failed to detect viral genomes. a cytopathic virus was isolated on subpassage from pulmonary tissue of one affected animal using mdbk cells. cytopathic effect (cpe) was not observed in inoculated bovine turbinate, rabbit kidney or uninoculated cells, and therefore these isolation attempts were not pursued. fitc-conjugated fluorescent antibodies against several bovine viruses (adenovirus types 1 and 5; bluetongue; bovine viral diarrhea virus; coronavirus; herpesvirus types 1, 2, and 5; parainfluenza virus 3; parvovirus; reovirus, rotavirus and respiratory syncytial virus) failed to detect a virus in the infected cell cultures. negative staining electron microscopy (em) of frozen and thawed infected mdbk cell culture revealed the presence of numerous, uniformly shaped, non-enveloped virus particles approximately 25 to 30 nm in diameter ( figure 1 ). in order to identify the cytopathic virus isolated from the alpaca, a generic, degenerate oligonucleotide primer (dop) pcr-based virus detection assay [17] [18] [19] was utilized. infected and uninfected cell culture supernatants were enriched for viral capsids by nuclease digestion and ultracentrifugation. extracted nucleic acids were subjected to reverse-transcription, amplified by dop-pcr, and separated by agarose gel electrophoresis ( figure 2 ). the gel electrophoresis pattern of these amplified nucleic acids differed between infected and uninfected mdbk cells. ten bands were excised each from the infected cell lane and from the uninfected cell lane, cloned, and sequenced. sequencing of nucleic acid from the infected cell lane revealed 47 distinct products, encompassing regions with homology to approximately 46% of the ev-f genome, with sequences showing greatest homology to serotypes 1 and 3 ( figure 3 ). one sequence was of cellular origin due to residual mbdk cell dna. thirty-six distinct sequences obtained from the dop-pcr amplicons of the negative control mdbk cells were consistent with amplification of mdbk dna and with amplification of residual dna in dop-pcr reagents that we have observed previously, with no enterovirus-like sequences observed. the remainder of the complete viral genome was identified by specific pcrs and race, based on primers designed from the already-obtained sequence and from bev sequences in genbank ( table 1 ). the complete 7433 bp genome for this virus, named enterovirus f, strain il/alpaca, has been deposited in the genbank database under accession kc748420. based on recently changed nomenclature [14] [15] [16] , the genome of the novel virus was most closely related to ev-f (previously bev type b) serotypes 1 and 3, with homology to ev-f complete genome sequences ranging from 75-83%. homology with ev-e sequences was 67-68% at the genome level. since the capsid is used for typing picornaviruses, the virus identified in this study has to be considered as type 1. in order to analyze the virus for potential recombination and to describe it more accurately, we performed more detailed phylogenetic analyses on several proteins of the novel virus, deduced from the translated nucleic acid sequences. analysis of the full polyprotein and the individual capsid, 2a protease, 3c protease, and polymerase proteins of the alpaca-infecting virus relative to sequences of other representative enteroviruses from bovine ev-e (bev-a serotypes 1-4) and ev-f (bev-b serotypes 1-4), and sequences from three unclassified ev-f viruses [16] , two from bovine sources (ay724744 and ay724745) [20] , and one from a capped langur (jx538037) [21] , possum, porcine (pev), and human (hev) hosts. these analyses revealed the alpaca virus to be most closely related to ev-f (figures 4 to 8) . based on analysis of the full polyprotein, the alpaca-sourced virus clusters most closely with the ev-f, with homologies exceeding 85%, highest with serotype 1 viruses (figure 4 ), and is more distantly related to the ev-f serotypes 2 and 3, followed by the ev-e species. the more diverse capsid protein (comprising the external surface of the virus) sequence of the alpaca-sourced virus was also most closely related to ev-f, serotype 1 ( figure 5 ) sharing 81% and 97% identity at the nucleotide and amino acid levels respectively. the amino acid homology with serotype 2 viruses was 86-87%, and 79% with serotype 3, and 78% with a serotype 4 possum isolate. as compared with serotype 1, the capsid sequence also was less similar to the partial capsid sequences of the unclassified ev-f viruses from bovine (ay424745) and capped langur species (jx538037), each with 87% amino acid identity, although the incomplete nature of these sequences makes it impossible to be certain of the degree of relatedness. because the capsid gene is used for serotyping picornaviruses, this virus is thus considered a type 1. however, based on 2a protease (which cleaves the viral polypeptide into its individual components) sequences, the alpaca-sourced virus groups most closely with serotype 3 with 95% homology, followed by serotype 1 with 89% homology (figure 6 ), indicating that the virus had attributes of type 3 and thus could have been a recombinant between types 1 and 3. the less diverse 3c protease (which the virus also uses to cleave the polypeptide into its individual components) of the enteroviruses groups the alpaca-sourced virus most closely with ev-f, serotypes 1 and 3 ( figure 7) , with 97% amino acid identity. the gene for the polymerase enzyme (which the virus uses to transcribe its rna after infection of a cell) is also highly conserved among the enteroviruses and cannot be used to clearly delineate a serotype for the alpaca-sourced virus, which still clusters most closely with the ev-f species (figure 8 ), with amino acid identity greater than 97% for serotypes 1-3 and 94% for serotype 4. we also compared the 59 untranslated region (utr) of the alpaca virus with the bovine enteroviruses and found the greatest homology with ev-f strains, the highest with serotypes 1-3 and unclassified bovine sequence ay24744 at 87-90% homology. the alpaca virus homology with the ev-e 59 utr was 75-78%. several attempts to perform enterovirus-specific pcrs, using primers developed for the alpaca-source enterovirus and published ev-e and ev-f primers, were made on rna extracted from paraffin-embedded lung tissues from the two of the alpaca (data not shown). in addition we also performed dop-pcr on rna extracted from these embedded tissues. some non-specific pcr bands were evident, but sequencing of these pcr products revealed no enterovirus sequences. pcrs for the housekeeping genes b-actin and glyceraldehyde 3-phosphate dehydrogenase (gapdh) were negative for some of the tissue samples, indicating that rna quality was low in these fixed tissues. in this report, we describe an enterovirus that was isolated on subpassage from pulmonary tissue of an alpaca that died with evidence of respiratory and systemic infection. using a universal virus detection assay, we identified a significant portion of the genome of this picornavirus with a single pcr. this finding is consistent with the em data that visualized non-enveloped viral particles of approximately 25-30 nm in diameter. this is the first report of a bev isolation from alpaca. all four of the diseased animals had similar clinical symptomatology and had similar pulmonary histology on autopsy. because ev-f was the only potential pathogen isolated from any of these animals, the alpaca-adapted ev is a potential cause of this syndrome, although these experiments clearly did not fulfill koch's postulates and limitations in sensitivity of the other tests that were performed do not exclude the potential for other causes. attempts to identify ev-f by pcr of paraffin-embedded pulmonary tissue samples obtained from these animals failed. this could be due to low copy number of ev-f rna in the sections of the paraffin enteroviruses comprise one of the nine genera of picornaviruses; all of which include members that infect vertebrates. picornaviridae members are small, non-enveloped viruses with a single-stranded rna genome of positive polarity. members of the enterovirus genus include human pathogenic poliovirus, coxsackieviruses, enteroviruses, and echoviruses. other mammalian enteroviruses, including those infecting bovine, simian and porcine species, also have been described [22] . the only picornavirus previously reported to infect alpaca is the foot-and-mouth disease virus (fmdv), which belongs to the aphthovirus genus. however, fmdv does not usually cause severe disease in alpaca [12, 23] . ev-e and ev-f are globally prevalent infections in cattle, and while virus can be shed in high titers in the feces [24] , such infections are usually subclinical and their ability to cause disease in any animal is unclear. earlier studies described enteroviruses isolated from calves suffering from respiratory disease [25] [26] [27] . however, in these studies, respiratory disease could not be reproduced using viral isolates from the infected calves. subsequent studies in cattle have not been reported. we hoped to be able to identify sequences that could account for the alpaca infection. while there are insufficient data to determine whether or not the virus adapted to alpaca, the frequent housing of alpaca with cattle without other such reports suggests that these infections are unusual. the alpaca-sourced virus has the interesting characteristic of possessing sequences that are most similar to serotype 1 (including the capsid region that is used to determine picornavirus serotype), but in at least one gene is closest to serotype 3, suggesting that this virus could have arisen by recombination of other ev-f serotypes. it is thus possible that recombination of viruses from two ev-f serotypes led to this unusual infection. the isolate has approximately 80-85% homology in its protein sequence to previously described ev-f strains, which is similar to the degree of homology shared among protein sequences from previously sequenced ev-f strains isolated from cattle, which ranges from 79-99% for ev-f strains, and 50 to 95% when ev-e strains are also considered [15] . the sequence of the alpaca-infecting virus isolate is divergent enough from previously reported strains that it does not provide clear evidence for the basis of its pathogenicity. while it is possible that this virus was transmitted directly from cattle to alpaca, it also is possible that there were one or more intermediate hosts. besides cattle, ev-f has been reported as an infection of possum and of capped langur [28, 29] . we suspect that the absence of previously reported bovine enterovirus infections in u.s. alpaca is related to the relative isolation of alpaca herds, making it less likely that an alpaca-adapted virus would be further transmitted among alpaca. introducing new species (as livestock or as pets) to a habitat potentially increases the risk of an indigenous pathogen causing infections in new species. while most pathogens do not cross the species barrier due to adaptive constraints, those that succeed often cause more severe disease in the new host. notable human examples of this phenomenon are yellow fever virus, hiv and more recently nipah virus [30] , hendra virus [31] and sars virus [32] . animal examples include the devastating infections of canine distemper virus in raccoons and african lions [33] [34] [35] [36] [37] [38] . u.s. alpaca are outside their native south american habitat and are exposed to viruses endemic to the u.s., especially those from u.s. domestic farm animals to which u.s. alpaca herds often have close proximity. thus, recently described alpaca infections include bovine viral diarrhea virus, equine herpesvirus 1, and bluetongue virus. newly introduced animals also can potentially carry pathogens that are relatively benign to them, but not to the indigenous fauna. the risk obviously increases if newly introduced and indigenous livestock are kept in close proximity and if their pathogens are able to remain stable in the environment or persist in the host species. since picornaviruses are non-enveloped viruses, they often are very stable under environmental conditions, increasing the opportunity for infection of different hosts over a prolonged period of time. while the enterovirus infection described in this report was temporally associated with illness in three other alpaca in the affected herd that may have represented limited spread of the virus, the sparse distribution of alpaca, together with the severe and rapid course of disease likely prevented further dissemination of the virus, as evidenced by the absence of other reports of similar illnesses in the herd or other alpaca in the region. however, even though it appears that this outbreak was controlled, bovine enteroviruses should be added to the list of viruses that can infect alpaca, and that could potentially be associated with severe respiratory and systemic infections in alpaca. furthermore, considering the relative stability of enteroviruses, the ubiquity of cattle and likely frequent co-location of domestic cattle with alpaca, it is quite plausible that similar outbreaks may occur in the future. therefore, this alpaca virus infection serves to remind us that viral species are constantly evolving and that the opportunity to infect new hosts may hasten that process. samples were obtained from animals within the affected commercial herd that had been submitted after death for a diagnostic necropsy at the university of illinois veterinary diagnostic laboratory. grossly affected tissues were harvested at necropsy for routine histopathological examination using 10% neutral buffered formalin-fixed, paraffin-embedded, hematoxylin and eosin-stained sections. based on the consistent gross necropsy and microscopic findings of acute diffuse interstitial pneumonia in all four alpaca, lung tissue was used for virus isolation. the animals used in this study met the definition of ''farm animals'', which are not covered by the u.s. animal welfare act (9 cfr 1). thus, iacuc or ethics committee approval was not required for these studies. the owner of the animals provided permission for these studies. negative staining electron microscopy mdbk cells were grown to 90% confluency in 25 cm 2 flasks (midwest scientific) containing 10 ml of growth media then inoculated with the 0.5 ml of the filtered virus isolate. after 90% cpe development, cells were harvested by freeze and thawing one time. the cell culture fluid was then clarified by centrifugation at 9306 g for 20 min at 4uc. four ml of the clarified supernatant containing 1.25% (final) neutral buffered formalin was subjected to ultracentrifugation (76 k6 g for 1 hour at 4uc) to pellet the virus particles. the pellet subsequently was suspended in 100 ml of supernatant, stirred with a wooden stick and vortexed until thoroughly mixed. the sample was then placed as a drop on parafilm and a formvar plastic and carbon-coated copper grid was placed on top of the specimen droplet for 15 minutes. excess sample was then removed with filter paper and the grid placed on 2% ammonium molybdate for 2 minutes. the grid was then dried by removing the excess fluid with filter paper, placed into a grid box and covered with drierite crystals for 10 minutes. the reactions were carried out with 10 ml of template. cycling conditions consisted of initial denaturation for 5 min at 95uc; followed by 5 cycles of 1 min at 94uc, 5 min at 25uc, slow ramping at 0.1uc/sec to 30uc, 4 min at 30uc, slow ramping at 0.1uc/sec to 37uc, 3 min at 37uc, slow ramping at 0.1uc/sec to 42uc, 2 min at 42uc, slow ramping at 0.1uc/sec to 55uc, 55uc for 1 min, 72uc for 2 min; 35 cycles as follows: 94uc for 20 sec, 55uc for 1 min, 72uc for 1 min with the addition of 1 second per cycle to the extension step; final extension at 72uc for 10 min. the dop-pcr products were analyzed and purified by agarose gel electrophoresis. distinct bands were excised, purified (genelute, sigma, st. louis, mo), and ligated into the pcr4-topoh vector (invitrogen). the ligation products were used to transform competent one shoth top10 bacteria (invitrogen) according to the manufacturer's instructions. colony pcr was performed; these pcr products were purified (qiaquick pcr purification, qiagen) and sequenced using m13 primers. sequencing was performed using a 31306l genetic analyzer (applied biosystems, foster city, ca). sequences from the obtained clones were compared with the non-redundant (nr) database in genbank using tblastx (ncbi, bethesda, md). virus genome sequencing. the sequence data obtained from dop-pcr products as well as data for ev-e and ev-f genomes available in genbank (nc_001859.1, ay508697.1, ay508696.1, d00214.1, af123433.1, af123432.1) were used to develop multiple primer pairs (table 1) to amplify and sequence the full genome of the novel virus. pcr conditions were: initial denaturation 2 min 95uc, 40 cycles of 94uc for 30 sec, 54uc for 1 min, 72uc for 1 min, concluding with a final extension step of 72uc for 1 min. pcr products were cloned and sequenced as described above. the 39 end of the viral genome was sequenced using rapid amplification of cdna ends (race) with a commercially available kit (smart race cdna amplification kit, clontech, mountain view, ca) according to the manufacturer's instructions. viral rna was extracted from infected cell cultures and cdna was synthesized using mmlv reverse transcriptase and an oligo dt primer provided with the commercial kit. the cdna was then pcr-amplified with a gene specific primer (gsp) and the universal primer provided in the kit. the gsp (sm-11) was designed based on the cloned alpaca virus cdna sequence and the ev-e and ev-f sequences in the genbank database and was approximately 650-bp upstream of the 39 poly a tail. the resulting pcr products were gel purified cloned into a ta cloning vector. the 39 viral ends were sequenced directly from the purified pcr products and from the cloned cdna to verify the correct sequence. lung tissues from alpaca and horses involved in the initial outbreak were formalin-fixed and paraffin embedded (ffpe) for future analysis and histopathology. following the identification of enteroviruses sequences from mdbk cell cultures by dop-pcr, we attempted to test these ffpe tissues by specific pcr assays for enterovirus sequences. five sections of 10 mm thickness of each tissue were removed from each block with a microtome and rna was extracted with the rneasy ffpe kit according to the manufacturer's instructions (qiagen). rna was reverse tran-scribed into cdna according to the manufacturer's instructions (first strand synthesis kit, invitrogen). several specific pcrs for enterovirus sequences were performed using primers designed for this alpaca sourced enterovirus (sm 9-32, table 1 ) ranging from 100-500-bp and published bovine enterovirus primers [24, 39] (beld, bev, n-bev; table 1 ). pcrs for two bovine housekeeping genes, b-actin and glyceraldehyde 3phosphate dehydrogenase (gapdh) [40, 41] , were also performed to verify the quality of the rna (table 1) . additionally dop-pcr was performed on the cdna as described above. pcr products were cloned and sequenced to verify the identity of the nucleic acid amplified. the deduced amino acid sequences from the alpaca virus polyprotein containing the capsid, polymerase, and protease genes were aligned with other homologous enteroviruses available in the genbank database. the sequences were aligned with clustal w and neighbor-joining phylogenetic trees were constructed and viewed using treeview [42] and phylip software [43] with bootstrap confidence values determined by 1000 replications. genbank accession numbers used in the analyses were: enterovirus f, strain il/alpaca kc748420, bev-a, now ev-e (serotypes 1-4) and bev-b, now ev-f (serotypes 1-4) af123433, d00214, af123432, dq092770, dq092795, ay508696, ay508697, dq092794, nc001859, ay462106, dq092786, dq092787, jq690748, eu886967, hq917060, nc07767, jq690741, ay724745, and jx538037; hev a fm955278, hev b af029859, hev c u05876, and hev d d00820; polio virus mahoney strain n002058; and porcine enterovirus b nc_004441. not all of the viral sequences used for analyses contained the complete polyprotein sequences, and therefore some do not appear in all of the panels of figures 4 to 8. the capsid sequences for the capped langur (jx538037) and unclassified ay24745 sequences are partial. ev-f serotypes 5 and 6 [16] do not have nucleic acid sequences available and do not appear in these analyses. viral diseases of new world camelids census of agriculture chronic weight loss in an immunodeficient adult llama camelid immunoglobulins and their importance for the newborn-a review evaluation of bovine viral diarrhea virus in new world camelids bovine viral diarrhea virus in new world camelids isolation of bovine viral diarrhea virus from an alpaca bvdv in british alpacas genotyping and phylogenetic analysis of bovine viral diarrhea virus isolates from bvdv infected alpacas in north america prevalence of bovine viral diarrhea virus infections in alpacas in the united states persistent infection with bovine viral diarrhea virus in an alpaca update on llama medicine. viral diseases disseminated bovine viral diarrhea virus in a persistently infected alpaca (vicugna pacos) cria virus taxonomy: classification and nomenclature of viruses: ninth report of the international committee on taxonomy of viruses molecular-based reclassification of the bovine enteroviruses the picornavirus pages available: www.picornaviridae use of a novel virus detection assay to identify coronavirus hku1 in the lungs of a hematopoietic stem cell transplant recipient with fatal pneumonia universal virus detection by degenerate-oligonucleotide primed polymerase chain reaction of purified viral nucleic acids use of a universal virus detection assay to identify human metapneumovirus in a hematopoietic stem cell transplant recipient with pneumonia of unknown origin bovine enterovirus 2: complete genomic sequence and molecular modelling of a reference strain and a wild-type isolate from endemically infected us cattle characterizing the picornavirus landscape among synanthropic nonhuman primates in bangladesh enteroviruses: polioviruses, coxsackieviruses, echoviruses, and newer enteroviruses foot-and-mouth disease in camelids: a review survey of bovine enterovirus in biological and environmental samples by a highly sensitive real-time reverse transcription-pcr respiratory viruses of cattle biologic characteristics of certain bovine enteric viruses polioencephalomyelitis of pigs-the identification of viruses related to the teschen and t80 groups in the united states naturally acquired picornavirus infections in primates at the dhaka zoo characterisation of two enteroviruses isolated from australian brushtail possums (trichosurus vulpecula) in new zealand henipaviruses in their natural animal hosts isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus a review of studies on animal reservoirs of the sars coronavirus genetically distant american canine distemper virus lineages have recently caused epizootics with somewhat different characteristics in raccoons living around a large suburban zoo in the usa epizootic canine distemper virus infection among wild mammals canine distemper virus-like infection in a captive african lioness a canine distemper virus epidemic in serengeti lions (panthera leo) the canine distemper epidemic in serengeti: are lions victims of a new highly virulent canine distemper virus strain, or is pathogen circulation stochasticity to blame? climate extremes promote fatal co-infections during canine distemper epidemics in african lions highly sensitive assay for detection of enterovirus in clinical specimens by reverse transcription-pcr with an armored rna internal control performance of a foot-and-mouth disease virus reverse transcription-polymerase chain reaction with amplification controls between three real-time instruments evaluation of realtime pcr endogenous control genes for analysis of gene expression in bovine endometrium treeview: an application to display phylogenetic trees on personal computers an alternating least squares approach to inferring phylogenies from pairwise distances we acknowledge the contributions of debbie cassout (university of illinois), who performed the virus isolation and direct fluorescent antibody work, and lou ann miller (university of illinois), who performed the negative staining em work. key: cord-289093-si8btsab authors: beard, philippa m.; griffiths, samantha j.; gonzalez, orland; haga, ismar r.; pechenick jowers, tali; reynolds, danielle k.; wildenhain, jan; tekotte, hille; auer, manfred; tyers, mike; ghazal, peter; zimmer, ralf; haas, jürgen title: a loss of function analysis of host factors influencing vaccinia virus replication by rna interference date: 2014-06-05 journal: plos one doi: 10.1371/journal.pone.0098431 sha: doc_id: 289093 cord_uid: si8btsab vaccinia virus (vacv) is a large, cytoplasmic, double-stranded dna virus that requires complex interactions with host proteins in order to replicate. to explore these interactions a functional high throughput small interfering rna (sirna) screen targeting 6719 druggable cellular genes was undertaken to identify host factors (hf) influencing the replication and spread of an egfp-tagged vacv. the experimental design incorporated a low multiplicity of infection, thereby enhancing detection of cellular proteins involved in cell-to-cell spread of vacv. the screen revealed 153 proand 149 anti-viral hfs that strongly influenced vacv replication. these hfs were investigated further by comparisons with transcriptional profiling data sets and hfs identified in rnai screens of other viruses. in addition, functional and pathway analysis of the entire screen was carried out to highlight cellular mechanisms involved in vacv replication. this revealed, as anticipated, that many pro-viral hfs are involved in translation of mrna and, unexpectedly, suggested that a range of proteins involved in cellular transcriptional processes and several dna repair pathways possess anti-viral activity. multiple components of the ampk complex were found to act as pro-viral hfs, while several septins, a group of highly conserved gtp binding proteins with a role in sequestering intracellular bacteria, were identified as strong anti-viral vacv hfs. this screen has identified novel and previously unexplored roles for cellular factors in poxvirus replication. this advancement in our understanding of the vacv life cycle provides a reliable knowledge base for the improvement of poxvirus-based vaccine vectors and development of anti-viral theraputics. vaccinia virus (vacv) is a large double-stranded dna virus with a complex cytoplasmic life cycle. it is the prototypical member of the orthopoxviridae genus of the poxviridae family which includes variola virus (the causative agent of smallpox), monkeypox virus and ectromelia virus. vacv was used as a vaccine in the successful global eradication of smallpox in the 20 th century and closely related attenuated strains such as modified vaccinia virus ankara (mva) are now some of the most frequently used recombinant vaccine vectors against a variety of human and animal diseases including hiv, malaria and tuberculosis [1] . understanding the vacv life cycle is therefore important since it provides the base for the development of efficient and safe novel vaccines. vacv, like all other viruses, harnesses the cell to enable its replication. it turns off or subverts multiple crucial anti-viral pathways including cytokine production, toll-like receptor pathways, nf-kb activation and the dsrna pkr response [2] [3] [4] [5] [6] [7] [8] . in addition vacv suppresses both intrinsic and extrinsic proapoptotic pathways [9] and activates numerous anti-apoptotic, pro-survival pathways including the pi3k/akt pathway [10, 11] , the mek/erk pathway [12, 13] , the p38 mapk pathway [14] and the mapk/jnk pathway [14, 15] . modulation of so many different signalling pathways prevents viral-induced premature cell death and contributes to the ability of poxviruses to replicate in a wide range of cell types. to investigate this complex pathogen-host relationship further, a rnai screen of druggable host targets was carried out to analyse the effect of cellular protein depletion on vacv replication, using a multi-cycle vacv infection assay that monitors all stages of virus replication including virus spread. the screen identified a range of previously identified hfs, but also novel hfs and pathways influencing vacv infection that may facilitate the development of broadly effective anti-viral strategies and the optimisation of poxviral-based vaccine vectors. a schematic diagram of the workflow used in the rnai screen is shown in figure 1 . sirna smartpools (4 sirnas per gene, dharmacon) were diluted to 0.3 mm and dispensed in 10 ml volumes using a rapidplate384 liquid handler (qiagen) into eight black 384-well plates (corning). these were stored at 280uc until needed (maximum 48 h). on the day of transfection, plates were thawed and 10 ml transfection reagent (dharmafect 1, dharmacon) diluted in hank's buffered saline solution (hbss, thermo-fisher) was added to each well containing sirna using a multidrop 384 (thermofisher), to give a final transfection reagent concentration of 0.1%. plates were incubated for 20 min at room temperature to allow formation of transfection complexes. during complex formation, low-passage (p20-22) hela cells (ecacc) from approximately 50% confluent flasks were washed in pbs and trypsinised in trypsin-edta (lonza) before diluting in phenol red-free, antibiotic-free transfection medium (dmem/f-12 1:1 with 5% fcs, 15 mm hepes and l-glu; gibco). 3610 3 cells in a volume of 40 ml were added to each well using the multidrop 384. plates were incubated for 48 h at 37uc in a humidified incubator with 5% co 2 before infection. to infect, media was removed from plates by inversion, and 15 ml media (dmem +4.5 g/l dglucose, l-glu and pyruvate with 2.5% fcs and penicillinstreptomycin) or 15 ml media containing vacv strain wr with egfp tagged a5 protein [16] diluted to moi 0.05, was added using the multidrop 384. plates were incubated at 37uc for 1 h before 50 ml of media was added to each well, the plates inverted to remove the media and virus, and a final volume of 50 ml of media added to the plates before they were returned to the incubator. after 48 h the plates were inverted to remove the media and 50 ml of 10% buffered formal saline added to fix the cells. fluorescence levels were measured using a polarstar optima plate reader (bmg labtech). data from eight replicates was used for analysis. background intensity correction was carried out by subtracting the median value of uninfected wells and the data was normalised using the robust z score method [17] , and corrected for the number of cells in each well. the correction for the number of cells in each well was carried out by estimating the linear correlation coefficient between the level of fluorescence (phenotype score) and the number of cells (toxicity score) using least squares optimization. this coefficient was used to linearly adjust the phenotype scores. one replicate of the screen was imaged by a high content screening system. the buffered formal saline was removed from the cells by inverting the plates, and cells were washed in 50 ml of room temperature pbs before permeabilising for 15 min at room temperature in 30 ml of 0.1% tritonx-100 diluted in pbs. plates were inverted and 50 ml of a 1:50 dilution of alexafluor-647 phalloidin (invitrogen molecular probes) diluted in pbs + 1% bsa was added and incubated for 45 min in the dark. the phalloidin was removed by inversion and 50 ml of dapi (1 mg/ml) diluted in pbs was added and left on. cells were analysed by automated microscopy using an opera high content screening system (perkin elmer) and acapella high content imaging and analysis software. to identify sirna smartpools which exerted significantly toxic effects the number of cells in each well was counted and converted to a z-score. a z-score is equivalent to the number of standard deviations away from the mean. sirna treatments that reduced the cell number by two or more standard deviations below the population mean (z-score of 22 or less) were removed from further analysis. a z-score of 22 was equivalent to 250 cells, compared to a population mean of 455. selected sirna smartpools were diluted to 0.3 mm in 1x sirna buffer and dispensed in triplicate in 96-well plates (corning). to this, 10 ml dharmafect 1 diluted in dmem to give a final concentration of 0.15% was added using the multidrop 384. following a 20 min incubation to enable complex formation, 0.4610 4 hela cells in 80 ml transfection media were added and plates were transferred to a 37uc humidified incubator with 5% co 2 . after 48 h, medium was removed and cells rinsed in pbs before lysing in 100 ml trizol (invitrogen). triplicate wells were combined, and rna extracted by purelink (tm) rna mini kit (life technologies). mrna levels were determined by either taqman qpcr with gene-specific primers and probes from the universal probe library (roche), or by sybr green qpcr, using the appropriate one-step rt-qpcr kits (thermofisher). expression levels were normalised to the housekeeping cellular gene hypoxanthine phosphoribosyltransferase 1 (hprt) and calibrated to mock-transfected cells. qpcr was carried out in duplicate for each sample, and normalised expression levels averaged. the phenotype observed in the primary screen was confirmed for a subset of candidate genes with deconvoluted sirna smartpools. the four individual sirnas targeting different regions of each gene were diluted to 0.3 mm in 1x sirna buffer and dispensed to 96-well plates in triplicate. transfection and, 48 h later, infection with vacv-a5egfp was carried out as described above. at 0 and 48 h post infection fluorescence was measured using a synergy ht plate reader (biotek). the experiment was carried out three times to produce a dataset of three biological replicates each containing three technical replicates. the data were analysed using mixed models [18] fitting gene, time-point gene*time interaction and first time-point value as fixed effects. values observed at the first time-point were fitted as a 'baseline covariate' in order to increase the sensitivity of the analysis. the repeated experiments were fitted as random effects, causing the variation in results between the repeated experiments to be taken into account when testing statistical significance. differences between the amount of fluorescence present in wells treated with each sirna and wells treated with a non-specific sirna (targeting the hsv-1 gene vp16) were tested within the mixed models using t-tests. groups of genes were tested on separate plates and each of the groups were analysed using a separate mixed model. a phenotype was considered confirmed if two or more of the four sirnas resulted in a p-value of 0.05 or less. six wells of a 96 well plate were transfected with a sirna smartpool and, 48 h later, infected with vacv-a5egfp as described above. at 24, 36 and 48 h post infection cells were scraped into the overlying media, collected and then frozen and thawed three times and sonicated for 30 seconds (misonix sonicator 3000). the resultant lysate was titrated on bs-c-1 cell monolayers and virus titre quantified as plaque forming units (pfu) per ml [19] . enrichment analysis was performed with respect to pathwayand go-based gene sets defined in msigdb [20] , as well as with respect to gene sets derived from protein complexes curated in the corum [21] and pin [22] databases. specifically, the genes were sorted based on the screening data, and then the propensity of gene sets towards pro-or anti-viral activities were sought out using rank-sum tests with multiple testing [23] . the three gene expression data sets used in the analyses were data set a (gse11238, a microarray of vacv infected hela cells (http://www.be-md.ncbi.nlm.nih.gov/projects/geo/query/acc. cgi?acc = gse11238)), data set b (gse24125, a microarray of macrophages, monocytes and fibroblasts [24] ), and data set c (sra017695, a rna-seq based analysis of gene expression in vacv infected hela cells [25] ). for comparison of the rnai hit list with the two hela cell based cellular expression data sets, the differential expression-based rank for each gene (g) was obtained in each condition (i.e. a timepoint in a data set) and then a ''summerisation q-value'' q g was calculated which reflects how unexpectedly high the ranks are (using order statistics; in particular the rank of the third 4-quantile). therefore q g quantifies how unexpectedly often g is among the most strongly regulated genes (up or down). to identify hfs that influence vacv replication we used a druggable genome small interfering rna (sirna) library (dharmacon) in a high-throughput screen. this library targets genes that are considered potential candidates for therapeutics ( figure 1a) . briefly, smartpool sirnas (a mix of 4 sirnas per gene) targeting 6 719 genes were distributed into 384-well plates and reverse-transfected into hela cells. cells were infected 48 h post-transfection at a low multiplicity of infection (moi 0.05) with the vacv strain vacv-a5egfp. after 48 h (thus allowing multiple complete virus replication cycles), egfp fluorescence was quantified as a measure of infection and compared to controls in order to determine the effect of individual gene depletion on vacv replication. two positive sirna controls known to downregulate vacv-a5egfp growth (targeting prk-ab1 and egfp), two negative controls (mock transfection and rscf sirna which is not processed by the risc machinery) and two nonspecific sirnas (targeting vp16 or vp11/12 from herpes simplex virus type 1) were included in duplicate in each plate (figure 1b) . to confirm that the measurement of virus-expressed fluorescence was a reliable marker of viral replication, fluorescence was correlated to virus-titre, as determined by standard plaque assay, over a range of time-points post-infection after treatment with control or inhibitory sirnas (figure 1c) . this resulted in a pearson product moment correlation coefficient of 0.86, confirming that fluorescence was a reliable determinant of virus replication. the entire druggable screen was repeated four times in duplicate to generate a robust primary data set of eight replicates. pairwise agreement comparing the levels of fluorescence across the eight replicates revealed good reproducibility (median spearman's coefficient 0.55). one replicate of the vacvinfected cells was analysed by automated microscopy using an opera high content screening system and acapella high content imaging and analysis software to quantify the number of cells present in each well. a total of 403 sirna pools (6% of the total) were associated with a significant reduction in cell number (figure 1a) . these were removed from further analysis and are listed in table s1 in file s1. the fluorescence data from the remaining wells in the primary screen was normalised platewise using the robust z-score method [17] . a summary value was calculated for each gene by taking the mean across the replicates, and these values were converted to zscores which were corrected for the number of cells in the well to produce the level of fluorescence per cell for each sirna. a negative z-score indicated a reduction in vacv replication and a positive z-score indicated an increase in vacv replication (figure 1d ). the two positive controls (sirna targeting prkab1 and egfp) produced strongly negative z-scores as expected. the median level of fluorescence (z-score of 0) was very close to the level of fluorescence seen in wells transfected with the non-specific sirna (negative control), indicating that roughly half of the sirna pools caused an increase in fluorescence and half caused a decrease. a ''hit'' was defined as a sirna pool which generated a z-score of $2 or #-2. using these criteria, a hitlist of 302 hfs (4.5% of the total) was generated, consisting of 153 pro-viral hfs which inhibited replication upon depletion and 149 anti-viral hfs which increased replication upon depletion ( table s2 in file s1). to confirm the effect of the sirna smartpools on mrna levels, a subset of smartpools were transfected into hela cells and after 48 h, total rna extracted and subjected to a quantitative rt-pcr to determine the level of mrna of the targeted gene. 62 genes out of 80 tested (78%) had their transcript level reduced by 50% or more, indicating that the majority of the smartpools functioned as expected ( table s3 in file s1). to confirm the effect of gene depletion on vacv replication a subset of hfs, chosen on the basis of their potential for further investigation, were tested using the four individual, deconvoluted sirnas of each smartpool. the level of viral fluorescence was compared to that seen with non-specific sirna and a statistically significant reduction or increase in fluorescence (p,0.05) induced by at least 2 individual sirnas from the original smartpool was required for confirmation of the hit. overall 38 (53%) out of 72 candidate genes tested by this method were successfully confirmed ( table s4 in file s1). this level of validation is commensurate with similar rnai screens of viral replication which have reported confirmation of between 38% and 83% of primary screen hits [26] [27] [28] [29] [30] [31] [32] [33] . within our deconvoluted dataset the validation level of putative pro-viral hfs was notably higher than that for the anti-viral hfs. only 30% of the anti-viral hits (7/24) were successfully validated in comparison to 69% of the pro-viral hits (24/35) and 54% of the sirna pools with no effect in the primary screen (7/13). one potential reason for the lower validation rate of the anti-viral hits might be that the dynamic range of the virus replication assay is such that inhibitory effects are more easily demonstrated, as under normal replication conditions virtually all cells in the well became infected by 48 h post infection (data not shown) suggesting the system reached near saturation. to examine this further, the effect of selected sirnas on traditional viral growth curves was tested and correlated with their perturbation of fluorescence in the primary screen. hela cells were transfected with each one of 6 sirna smartpools (upregulatory map3k14, control vp16, and downregulatory trip, ppap2a, vps52 and cct7) and infected with vacv-a5egfp at an moi of 0.05 after 48 h. analysis of virus titre at 12 h intervals by plaque assay found the endpoint titres correlated very closely with the z-score obtained from the primary rnai screen (figure 2) , further validating the robustness of the primary screen. notably, however, whilst the inhibitory sirnas decreased the maximum virus titre by between 13-(cct7) and 5-fold (vps52) the sirna treatment directed against a candidate antiviral hf (map3k14) only increased peak virus titre by 3-fold. thus the response of the system is more suited to detecting inhibitory perturbation, with relatively reduced sensitivity for detection of individual anti-viral hfs. the list of 302 individual hfs was analysed for genes already known to influence vacv replication. numerous examples were found including clathrin and proteins involved in golgi vesicular trafficking, both of which are required for the production of enveloped vacv forms [34, 35] , as well as multiple components of the ampk complex which has been shown to aid vacv entry [36] . in addition the signalling pathway regulating protein traf2 was identified in the screen as a pro-viral hit; further work demonstrated that it promoted rapid vacv entry [37] . the identification of known host factors for vacv and our follow-up identification of the role of traf2 in vacv replication supports the reliability and significance of this rnai screen dataset. overall, eight replicates of a genome-wide rnai screen of multiple vacv replication cycles identified 302 cellular genes, consisting of 153 hfs that positively support vacv replication and 149 hf with anti-viral effects. to prioritise investigations of the 302 potential vacv hfs, the candidate genes were compared to the hit lists of other viral rnai screens, including two recently published vacv screens [32, 38] . the methodology in the previously published vacv screens varied considerably; mercer et al [32] measured the growth of a thymidine-kinase-deficient vacv (strain western reserve) after only 8 h of infection, thereby identifying cellular proteins involved in the initial stages of virus replication but excluding analysis of viral spread. they reported 188 pro-viral hf but no anti-viral hfs. a second screen by sivan et al [38] used the vacv strain ihd-j (which has a point mutation that accelerates the release of progeny virions from the cell surface) to identify genes which influenced viral replication after 18 h of infection, thus measuring the entire replication cycle with emphasis on viral spread. they reported 576 pro-viral and 530 anti-viral hfs. the overlap between the hit lists reported by the three vacv rnai studies (mercer et al, sivan et al, and this study) is depicted in figure 3a and b and the hfs common to two studies are listed in table s5 in file s1. the number of overlapping hits between two of the screens ranged from 3 to 13 and no hfs were common to all three vacv studies. a small number of common hits between sirna screens of the same virus is a frequent finding [39, 40] and, given the variation in methodology between the three vacv screens (including viral strain, infection time, and data analysis), is not surprising. however, comparison of the enriched functions and pathways identified in each of the three vacv screens revealed marked similarities (discussed below), demonstrating the power of comparative screening approaches to identify significant cellular pathways involved in virus replication. genome-scale sirna screens have been carried out for many viruses other than vacv, including hiv-1 [41] [42] [43] , west nile virus (wnv) [44] , hepatitis c virus (hcv) [30, 45] , vesicular stomatitis virus (vsv) [29] , borna disease virus [46] , enteroviruses [27] , dengue virus [28] , herpes simplex virus 1 (hsv-1) [33] and influenza a virus [26, 31, 47] . host factors common to two or more of these screens could represent broadly acting cellular proteins with a generalised effect on viral replication. a [26, 31, 47] and three published hiv rnai screens with a total of 826 hits [41] [42] [43] . doi:10.1371/journal.pone.0098431.g003 comparison of the vacv hfs identified in this screen with those identified in other viral screens found a small overlap with wnv, vsv, borna disease virus and dengue virus, whilst 21 vacv hfs were shared with hsv-1, 17 with influenza a virus and 13 with hiv-1 (figure 3c) . a list of overlapping genes can be found in table s6 in file s1. amongst the factors in common, the nucleocytoplasmic transport factor nup98 was identified as a proviral hit in the vacv screen reported here as well as hiv, hsv-1 and influenza a virus screens [26, 31, 33, 42] . it is located at both the cytoplasmic and the nuclear faces of the central channel of the nuclear pore complex (npc) [48] , is involved in rev-dependent rna export during hiv infection [49] , and has been shown to play both proand anti-viral functions during influenza a virus infection [31, 50, 51] . nup98 was a somewhat unexpected proviral hit in our screen since poxvirus replication and assembly occur in the cytoplasm. however the two vacv rnai screens published recently also identified a number of nuclear pore proteins as proviral, with one screen demonstrating that knockdown of nup62 strongly inhibited viral morphogenesis [38] . the number of nuclear pore proteins now identified as pro-viral hfs strongly suggests poxviruses require functional nuclear membrane transport for efficient replication. another hf that affects both vacv and influenza a virus replication is map2k3 (also known as mkk3 and mek3), which activates the p38 mapk signalling pathway and is involved in low ph-dependent entry of influenza virus and vsv [31, 47] . vacv also has a low ph-dependent entry mechanism [52] which may be similarly reliant on map2k3. alternatively, it may be required to activate the p38 mapk pathway to promote cell survival post infection [14] . in contrast, ifitms (interferon inducible transmembrane proteins) have been identified in functional genomic screens as mediating resistance to influenza a virus, dengue virus and west nile virus infection in vitro and in vivo [26, 53] as well as marburg and ebola viruses, sars-coronavirus [54] and hiv [55] . these proteins prevent entry of viruses at the plasma membrane, endosomes and lysosomes [56] , however none had an effect on vacv replication in our screen, suggesting vacv is resistant to the repressive effect of this protein family (figure 1d ). to determine whether the expression of hfs identified by rnai is modulated by vacv, the rnai hit list was compared to previously published transcriptional profiling data sets performed on cells infected with vacv. three available expression profiling data sets (designated a, b and c, see materials and methods for sources) which used different cells, virus isolates and time points were compiled and compared. correlation was mainly limited to intra-data set measurements (i.e. different time points or cell lines within a data set) with some examples of agreement between data sets a and c, both of which used hela cells. to test whether there was a general propensity of hfs to be differentially expressed in virus-infected cells the distribution of the 302 rnai hits was compared to that of the whole rnai screen in these three transcriptional profiling data sets. no significant tendency of proviral hfs to be up-regulated or anti-viral hfs to be downregulated was detected (data not shown). subsequently the expression of individual hfs was examined using the transcriptional profiling data of the two hela-based studies (a and c). this revealed specific examples of hfs which are differentially expressed in virus-infected cells (figure 4) . four pro-viral hfs (runx1, eif3c, hbegf, and adm) were significantly upregulated in vacv-infected hela cells (q,0.05), suggesting that vacv might promote expression of these proteins to assist viral replication and spread. runx1 is a subunit of the transcription factor cbf which regulates critical processes in both myeloid and lymphatic haematopoiesis. chromosomal translocations and mutations of runx1 are among the most frequent genomic abnormalities in different types of leukaemia [57] . furthermore, a genome-wide association study found a genetic polymorphism in runx1 to be associated with the serological response to vacv vaccination (dryvax vaccine, wyeth laboratories) in african-americans. this suggests that the runx1 polymorphism may influence replication and viral gene expression of the live-attenuated vaccine in vivo which causes differences in the strength of the adaptive immune response [58] , a hypothesis supported by our identification of runx1 as a pro-viral vacv hf. in addition to upregulated pro-viral hfs, opposite examples were also identified, with two anti-viral hfs (angptl4, rbak) significantly upregulated and one pro-viral hf (sap18) downregulated in vacv-infected cells. this lack of correlation between functional hfs and gene expression at the transcriptional level serves to underscore the complexity of virus-host interactions and highlights the need for further follow-up studies. to assess further the role of candidate hfs and associated functions and pathways in vacv replication, an overrepresentation analysis of the complete vacv rnai data set was performed with respect to pathway(figure 5a ) and go(figure 5b ) based gene sets as defined in the msigdb database, as well as from curated protein complexes defined in the corum [21] and pin [22] databases (figure 5c) . translation was the most strongly enriched theme in all these analyses, particularly the eif3 complex. poxviruses are known to utilise the host translation machinery for production of viral proteins therefore the enrichment of translation as a pro-viral theme is a validation of the screening method. the cellular translation machinery has been highlighted in other viral rnai screens as essential for vsv [29] and hepatitis c virus [45] and hsv-1 [33] . more interestingly, transcriptional initiation and general rna polymerase ii transcription factor activity were identified in the functional analysis of the rnai screen as significantly overrepresented anti-viral go-based gene sets (figure 5b) . sevin et al [38] also reported that interference with dna-dependent rna polymerase ii pathways enhanced vacv spread. our work identified individual anti-viral hfs involved in transcription such as maz (an inflammatory responsive transcription factor also known as saf1), and the transcription factor e2f2. inspection of individual confocal images taken at 48 h pi of cells depleted of these factors showed notably brighter accumulations of egfplabelled virus, in line with the positive z-scores from the overall primary screen (figure 6a ). orthopoxvirus infection results in a rapid shut down of cellular transcription, with a marked reduction in the amount of host mrna present as early as 2 h post infection [25, 59, 60] . this effect is believed to result from cessation of host mrna synthesis and degradation of cellular mrna transcripts. the viral proteins involved in this shut-off of host cell transcription have not been identified, although d9 and d10 have been implicated [61, 62] . in contrast to viral translation which is dependent on host proteins, vacv encodes its own transcription enzymes so is largely unaffected by a general repression of cellular transcription [63] . therefore vacv-induced downregulation of host transcription prevents the host cell from transcribing antiviral, pro-inflammatory gene programmes, such as the nf-kb cascade, while having a minimal negative effect on viral [21] and pin [22] databases. all significantly overrepresented gene sets (-log 10 (q-value).0.1) are shown. each row shows the ranks of genes from a particular gene set that were present in the rnai screen. each tick mark denotes the place of a particular gene from that gene set, placed at the appropriate position in the distribution. genes were sorted from left to right from most pro-viral to most anti-viral. the red and blue colours of the ticks are used for visual contrast. a green diamond is used to denote the median rank of the genes in the set. doi:10.1371/journal.pone.0098431.g005 transcription. whilst vacv has developed mechanisms to shut off host transcription, these data show that virus replication is improved when transcription is impaired prior to infection. thus, despite the efforts of the virus to shut off host transcription, some anti-viral effect of this pathway persists in vacv infected cells. two members of the septin protein family (septin 1 and msf/ septin 9) were identified in the rnai screen as anti-viral hfs (figure 6b) . msf/septin 9 co-purifies from cells with three other septin proteins (nedd5/septin 2, cdc10/septin 7 and septin 11), suggesting they form a functional complex [64] . these were therefore grouped in the pathway analysis, resulting in a significant over representation (q = 0.1) (figure 5c) . consistent with this, depletion of nedd5/septin 2, septin 7 and septin 11 all increased virus replication although not to the stringent cut-off used here to define a 'hit' (figure 6b) . deconvoluted sirnas targeting septin 11 mrna confirmed the enhancement of virus replication, although results with the other family members were more variable ( table s4 in file s1). septin 11 has also been identified as a proviral hit in a recently reported vacv sirna screen [38] . septins are conserved gtp-binding proteins which act as dynamic scaffolds for recruitment of other proteins. they are involved in actin and microtubule function, cytokinesis, cell movement and vesicle trafficking [65] . interestingly, they can be recruited together with autophagic proteins to ''cage'' shigella flexneri in the cytosol of infected cells, restricting bacterial dissemination [66] . the cage assembly is linked with actin polymerisation activity of s. flexneri, suggesting that a similar mechanism may be employed by the host cell to ''cage'' vacv virions (which activate actin polymerisation both early and late in the replication cycle [67, 68] ) and thus invoke an anti-viral effect. two groups of genes involved in dna replication and repair were highlighted in the pathway analysis as having anti-viral properties (figure 5c ). the pcna-mutsa-mutla-dna complex (pcna, msh2, pms2, and mlh1) and the brca1-associated genome surveillance complex (rfc4, brca1, blm, rfc1, msh2 and mlh1) both promoted virus replication when individual group members were downregulated (figure 6c ). these pathways are both involved in dna damage signalling and repair [69, 70] , a cellular process which is targeted by numerous viruses [71] . specifically, dna damage signalling pathways act as a host defence mechanism in poxvirus infection, detecting and responding to foreign poxviral dna and inducing intrinsic apoptosis [72] . the identification of the pcna and brca1 gene sets as strongly anti-poxviral hfs in the rnai screen suggests they are a part of this, or a similar, defence mechanism. the amp-activated kinase complex (ampk) is a key regulator of energy metabolism. it is activated by a reduction in atp which prompts phosphorylation of many target proteins, resulting in the activation of catabolic pathways and inhibition of anabolic pathways [73] . it has also been linked to regulation of the actin cytoskeleton [74] . ampk is a heterotrimer comprising a catalytic a subunit and regulatory b and c subunits. in mammals each subunit has several isoforms (prkaa1, prkaa2, prkab1, prkab2, prkag1, prkag2, and prkag3) [73] . the druggable rnai screen reported here screened all seven genes and identified three (prkaa2, prkab1, and prkab2) as promoters of vacv replication whose depletion led to fewer and dimmer accumulations of cytoplasmic egfp (figure 6d ). this result is in broad agreement with a recently published rnai screen of 440 cellular kinases and phosphatases in a nonpermissive drosophila cell model of vacv infection [36] , which identified seven hits including three ampk subunits. this study performed a loss of function analysis of hfs involved in vacv infection using rnai. previously identified host pathways and protein complexes which aid vacv replication, such as translation and the ampk complex proteins, were highlighted in the rnai screen. in addition, however, a range of novel host pathways and proteins were identified that influenced vacv infection, such as the dna damage and repair pathways, the septin family of proteins, map2k3 and nup98. as many of the genes targeted in this rnai screen have a known drug inhibitor, this work yields a list of hfs that can potentially be targeted by novel therapeutics. file s1 supporting tables. table s1 , list of 403 cytotoxic sirnas which caused significant cell death. mva and nyvac as vaccines against emergent infectious diseases and cancer two vaccinia virus proteins structurally related to the interleukin-1 receptor and the immunoglobulin superfamily a soluble receptor for interleukin-1 beta encoded by vaccinia virus: a novel mechanism of virus modulation of the host response to infection encoding of a homolog of the ifngamma receptor by myxoma virus a46r and a52r from vaccinia virus are antagonists of host il-1 and toll-like receptor signaling the e3l gene of vaccinia virus encodes an inhibitor of the interferon-induced, double-stranded rna-dependent protein kinase inhibition of ikappab kinase by vaccinia virus virulence factor b14 the vaccinia virus k1l gene product inhibits host nf-kappab activation by preventing ikappabalpha degradation near death experiences: poxvirus regulation of apoptotic death activation of the pi3k/akt pathway early during vaccinia and cowpox virus infections is required for both host survival and viral replication role of cell signaling in poxvirus-mediated foreign gene expression in mammalian cells a mitogenic signal triggered at an early stage of vaccinia virus infection: implication of mek/erk and protein kinase a in virus multiplication differential role played by the mek/erk/egr-1 pathway in orthopoxviruses vaccinia and cowpox biology vaccinia virus protein a52r activates p38 mitogen-activated protein kinase and potentiates lipopolysaccharide-induced interleukin-10 vaccinia virus b1r kinase interacts with jip1 and modulates c-jun-dependent signaling vaccinia virus cores are transported on microtubules statistical methods for analysis of high-throughput rna interference screens applied mixed models in medicine the vaccinia virus kelch-like protein c2l affects calcium-independent adhesion to the extracellular matrix and inflammation in a murine intradermal model gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles corum: the comprehensive resource of mammalian protein complexes-2009 pindb: a database of nuclear protein complexes from human and yeast statistical significance for genomewide studies stunned silence: gene expression programs in human cells infected with monkeypox or vaccinia virus simultaneous highresolution analysis of vaccinia virus and host cell transcriptomes by deep rna sequencing the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus comparative rnai screening reveals host factors involved in enterovirus infection of polarized endothelial monolayers discovery of insect and human dengue virus host factors rnai screening reveals requirement for host cell secretory pathway in infection by diverse families of negative-strand rna viruses a genome-wide genetic screen for host factors required for hepatitis c virus propagation genomewide rnai screen identifies human host factors crucial for influenza virus replication rnai screening reveals proteasome-and cullin3-dependent stages in vaccinia virus infection a systematic analysis of host factors reveals a med23-interferon-lambda regulatory axis against herpes simplex virus type 1 replication clathrin potentiates vaccinia-induced actin polymerization to facilitate viral spread evidence against an essential role of copii-mediated cargo transport to the endoplasmic reticulum-golgi intermediate compartment in the formation of the primary membrane of vaccinia virus a kinome rnai screen identified ampk as promoting poxvirus entry through the control of actin dynamics facilitates vaccinia virus replication by promoting rapid virus entry human genome-wide rnai screen reveals a role for nuclear pore proteins in poxvirus morphogenesis host cell factors in hiv replication: meta-analysis of genome-wide studies the use of rnai-based screens to identify host proteins involved in viral replication identification of host proteins required for hiv infection through a functional genomic screen global analysis of host-pathogen interactions that regulate early-stage hiv-1 replication genome-scale rnai screen for host factors required for hiv replication rna interference screen for human genes associated with west nile virus infection a functional genomic screen identifies cellular cofactors of hepatitis c virus replication identification of host factors involved in borna disease virus cell entry through a small interfering rna functional genetic screen human host factors required for influenza virus replication nucleoporin nup98: a gatekeeper in the eukaryotic kingdoms functional analysis of the interaction of the human immunodeficiency virus type 1 rev nuclear export signal with its cofactors influenza virus targets the mrna export machinery and the nuclear pore complex drosophila rnai screen identifies host genes important for influenza virus replication vaccinia virus entry into cells via a low-ph-dependent endosomal pathway ifitm3 restricts the morbidity and mortality associated with influenza distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus the ifitm proteins inhibit hiv-1 infection ifitm proteins-cellular inhibitors of viral entry runx1 translocations and fusion genes in malignant hemopathies genome-wide association study of antibody response to smallpox vaccine sequence complexity and relative abundance of vaccinia virus mrna's synthesized in vivo and in vitro microarray analysis of a549 cells infected with rabbitpox virus (rpv): a comparison of wildtype rpv and rpv deleted for the host range gene, spi-1 down regulation of gene expression by the vaccinia virus d10 protein characterization of a vaccinia virus mutant with a deletion of the d10r gene encoding a putative negative regulator of gene expression vaccinia virus transcription biochemical and cell biological analyses of a mammalian septin complex, sept7/9b/11 the evolution, complex structures and function of septin proteins entrapment of intracytosolic bacteria by septin cage-like structures actin-based motility of vaccinia virus repulsion of superinfecting virions: a mechanism for rapid virus spread mechanisms and functions of dna mismatch repair basc, a super complex of brca1-associated proteins involved in the recognition and repair of aberrant dna structures using or abusing: viruses and the cellular dna damage response cytosolic dna triggers mitochondrial apoptosis via dna damage signaling proteins independently of aim2 and rna polymerase iii amp-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism energy-dependent regulation of cell structure by amp-activated protein kinase the authors thank professor geoffrey smith (cambridge) for the gift of the egfp tagged vacv strain, dr helen brown (the roslin institute) for statistical expertise, and professor paul digard (the roslin institute) for discussion. key: cord-270681-6ayciihs authors: bálint, ádám; farsang, attila; zádori, zoltán; belák, sándor title: comparative in vivo analysis of recombinant type ii feline coronaviruses with truncated and completed orf3 region date: 2014-02-20 journal: plos one doi: 10.1371/journal.pone.0088758 sha: doc_id: 270681 cord_uid: 6ayciihs our previous in vitro comparative study on a feline coronavirus (fcov) pair, differing only in the intactness of their orf3abc regions, showed that the truncated orf3abc plays an important role in the efficient macrophage/monocyte tropism of type ii feline infectious peritonitis virus (fipv). in the present study, we describe a challenge experiment with the same recombinant fcovs in order to gain data on the in vivo characteristics on these viruses. while parent virus fipv df-2 developed feline infectious peritonitis in all the infected cats, its recombinant virus pbfipv-df-2, differing only in seven nucleotides, proved to be surprisingly low virulent, although caused an acute febrile episode similarly to the original fipv df-2. pbfipv-df-2 infection induced significantly lower virus neutralization titers than its parent virus, and lacked the second phase of viremia and development of fatal course of the disease. the recombinant pbfipv-df-2-r3i with completed orf3abc gained biological properties that differentiate between the feline enteric coronavirus (fecv) and fipv biotypes such as intensive replication in the gut, absence of viremia and weak or no serological response. using reverse genetic approaches our study is the first experimental proof that orf3abc is indeed responsible for the restriction of fecv replication to the intestine in vivo. feline coronaviruses (fcovs), members of the alphacoronavirus genus within the coronaviridae family, are major pathogens of felidae with worldwide distribution [1] . fcov occurs in two pathotypes; feline enteric coronavirus (fecv) primarily replicates in the lower portion of intestinal tract, spreads by fecal-oral route, and its clinical appearance is characterized by mild or unapparent enteritis [2, 3] . in contrast, feline infectious peritonitis virus (fipv) efficiently replicates in macrophages/monocytes, and can lead to feline infectious peritonitis (fip), a highly lethal systemic granulomatous disease, [4] [5] [6] [7] [8] . fipvs arise most likely from fecv in the infected cat via genetic changes [9] . characteristic changes can be detected in the spike (s) gene [10, 11] , in the orf7ab [9, 12, 13] and the orf3abc [9, [14] [15] [16] regions. fecvs have three open reading frames (orfs) in the orf3abc region [6] that code proteins conserved both in length and sequence in different isolates. on the contrary, the majority of fipvs contain genetic alterations (non-synonymous mutations, deletions and termination codons) mostly in orf3c but not rarely in orf3a and orf3b [9, 14] . the first in vitro comparison of a recombinant fcov pair differing only in the intactness of their orf3abc revealed that completion of the truncated orf3abc reduces virus replication rate by 2log 10 titer in feline peripheral blood monocytes [17] supporting the long time suspected but never experimentally proved theory that completion of this region alters the in vivo characteristics and pathogenesis of fcov [8] . in the present study using the parent fipv df-2 strain and its recombinant derivates we aimed to collect in vivo data how the completed orf3abc alters virulence, virus shedding, viremia, viral load of organs and humoral immune response against type ii fcov. the data of our experiments show that completion of orf3abc vested the highly virulent fipv df-2 with properties that are characteristic to fecv. felis catus whole fetus 4 (fcwf-4) cells originally purchased from the american type culture collection were used for virus propagation, titration and virus neutralization tests. the cell line was maintained as monolayer culture in dulbecco's modified eagle medium (sigma-aldrich, saint louis, mo, usa) supplemented with 10% fetal bovine serum (fbs), 0.3 mg/ml glutamine, 100 u/ml penicillin, 0.1 mg/ml streptomycin, 0.25 mg/ml amphotericin b, 1 mm sodium pyruvate, and 1% non-essential amino acids (sigma-aldrich). the fipv df-2 strain was kindly provided by berndt klingeborn (sva, uppsala, sweden). fipv df-2 is a regular tissue culture adapted strain that has been well described in the literature, and also used by many other investigators under this name or as fipv-79-1146 or fipv-nor15 [16] . generation of the recombinant pbfipv-df-2 and pbfipv-df-2-r3i was described elsewhere [17] . briefly, pbfipv-df-2 is a virus that originated as a molecular clone of fipv df-2 and then was successfully transfected into cat cells, where it was replicated for several generations before use in this study. pbfipv-df-2-r3i is a derivate of pbfipv-df-2 that was re-engineered to contain the intact orf3abc region of canine coronavirus, and was also transfected into cat cells and cultivated for several generations before being used in this study. the complete genome of pbfipv-df-2 was reverse transcribed using the high fidelity superscript iii first-strand synthesis system (invitrogen, carlsbad, ca, usa) and gene-specific primers. long pcr fragments overlapping the whole genome were amplified with phusion hot start high-fidelity dna polymerase (finnzymes, espoo, finland) and sequenced using the ion proton system (life technologies, carlsbad, ca, usa). sequences were aligned and analyzed with the seqman ngen software (lasergene, madison, wi, usa). specific-pathogen-free iqhsdcpb kittens (isoquimen sl, barcelona, spain) were used in the challenge experiments. kittens arrived at the facility at the age of 8-12 weeks. they were acclimated and used in the studies at the age of 14-18 weeks. the animals were kept in separate groups in a closed facility. their fcov negative status was checked with pcr, elisa and virus neutralization tests. kittens were inoculated oronasally with 10 3 50% tissue culture infective doses (tcid 50 ) of the parent virus fipv df-2 (n = 4) and the recombinant viruses pfipv-df-2 (n = 4) and pfipv-fd-2-r3i (n = 4), respectively. kittens were clinically examined on a daily basis for 42 days. cats were scored for several clinical signs as described earlier [18] . briefly, scoring was based on depression (inactivity for three consecutive days, 1 point), anorexia (not eating for three consecutive days, 1 point), and neurological disorders (swaggering, 1 point) on a daily basis, while fever (40.1uc, 1 point), jaundice (yellow plasma, 1 point), weight loss (loss of 2.5% of body weight per week, 1 point), and lymphopenia (lymphocyte count of ,0.5610 9 /liter) was scored on weekly basis. kittens showing signs of terminal fip were euthanized in order to avoid unnecessary suffering, while healthy animals were exterminated at day 42 postinfection (p.i.), followed by full postmortem examination. all animal experiments were approved and supervised by the ethical and animal welfare committee of national food chain safety office (permission no: 2866/2011). the total number of animals was carefully determined by considering two main principles: i.) the number of animals should be ensured proper amount of samples for statistical analysis and ii.) the 3r rules (replacement, reduction and refinement) must be implemented. to determine virus shedding in feces, fecal swabs were collected at days 0, 7, 14, 21, 28, 35 and 42 p.i., and placed in 500 ml of phosphate buffered saline (pbs). after vortexing and 30 min incubation, the swabs were removed, and the extract was centrifuged at 1000 x g for 10 min to remove cell debris. the supernatant was used for subsequent pcr. viral rna was purified using the qiaamp viral rna mini kit (qiagen, hilden germany). all rna was stored at -80uc until used. to measure the copy numbers of the genome and replicative forms of covs, two taqman-based quantitative real-time pcr (qrt-pcr) assays targeting the 59 end of the fipv df-2 genome and the n gene subgenomic (sg) mrna were applied, respectively [17] . each rna sample was analyzed in duplicates in two different runs. differences in original template rna levels were normalized by using housekeeping gene b-actin pcr [19] . means of the four normalized data per sample were used for further analysis. rna was extracted from whole edta anticoagulated blood taken at days 0, 7, 14, 21, 28, 35 and 42 p.i. using the qiaamp rna blood mini kit (qiagen) according to the manufacturer's instructions, and was subjected to genomic and subgenomic qrt-pcrs. to detect virus load in different organs (liver, spleen, kidney, lung, tonsil, mesenteric lymph nodes, brain and ileum,) approximately 0.5 g pieces of organs diluted in sterile phosphate-buffered saline (pbs) were homogenized with tissue lyser (qiagen, hilden, germany) to obtain 50% w/v suspension and then were centrifuged at 1000 x g for 10 min to remove cell debris. rna was extracted from the supernatant using the qiaamp viral rna mini kit (qiagen), and was subjected to subsequent genomic and subgenomic qrt-pcrs. serum samples were taken using vacuetteh tube (greiner bio-one, germany) at days 0, 7, 14, 21, 28, 35 and 42 p.i. for antibody elisa tests, the fcov eia kit, (bv european veterinary laboratory, the netherlands) was used according to the recommendations of the manufacturer. for virus neutralization (vn) assay, two-fold dilutions of heatinactivated serum from kittens (50 ml) were incubated for 1 hour at 37uc with equal aliquots of fipv df-2 (50 ml of 10 3.5 tcid 50 / ml). the viruses were then added to fcwf-4 cells showing 70% confluency in a 96-well plate, and incubated for 48 h, until the development of cytopathic effect. neutralizing activity was determined by end-point dilution [20] . to determine the statistically significant difference between the vn titers generated after inoculation with the three fcovs, the unpaired two-tailed student t test with equal variances was applied. the p value under 0.05 was considered as a statistically significant difference. cats inoculated with the parent virus fipv-df-2 showed rapid development of fip at day 10-16 p.i. the animals exhibited depression and anorexia, in most cases with fever, jaundice, weight loss and lymphopenia, and they had to be euthanized between days 21-25 (table 1) . pathological examinations proved the characteristic lesions of effusive fip with multiple dispersed pyogranulomas in the abdominal organs such as livers, spleens and kidneys. surprisingly, cats challenged with pbfipv-df-2, a recombinant fcov containing truncated orf3abc like the parent virus, showed only clinical signs of the acute phase of the disease, including transient fever from day 3 to 8, anorexia and slight lymphopenia ( table 1 ). all cats fully recovered and survived until termination of the experiment (day 42 p.i.). macroscopically no lesions were observed in these animals. cats inoculated with pbfipv-df-2-r3i, the recombinant fcov containing complemented orf3abc, showed neither any clinical signs typical of fip nor diarrhea (table 1) . all cats survived, and showed no macroscopic lesions except for slight enlargement of mesenteric lymph nodes in two animals. in order to elucidate the unexpected low-virulent phenotype of pbfipv-df-2, the full-length genomic sequence of the virion was determined using next generation sequencing, and data revealed that besides the 1-nucleotid (nt) change at position 24429 (g/a) that resulted in an amino acid (aa) change in the fusion domain of s protein at position 1332 (v/i) and the 1-nt silent mutation at position 26064 (t/c) in the m gene found also in the infectious clone [17] , additional mutations are present in the viral genome. shedding of fipv df-2 and pbfipv-df-2 was detected from day 3 p.i. to euthanasia of the pip diseased animals at very low and variable amounts of an average value close to the detection limit of the genomic qrt-pcr (1.9610 1 fcov rna copies per ml fecal extract) ( fig. 1) with undetectable virus replication using the subgenomic qrt-pcr assay (data not shown). virus shedding decreased to undetectable levels from day 21 p.i. in the pbfipv inoculated animals. the pbfipv-df-2-r3i infected cats began to shed the virus from day 3 p.i., virus shedding peaked at day 7 p.i. with 8.3610 5 fcov rna copies per ml fecal extract, remained high until day 14 p.i., then began to decrease until reaching 1.2610 2 fcov rna copies per ml fecal extract at day 35 p.i., and remained at this level until the end of the experiment (fig. 1 ). a classic biphasic viremia was observed in fipv df-2 infected cats. fcov rna was detected from day 3 p.i., reached a first peak of 4.8610 3 fcov rna copies per ml blood by day 7 p.i., then decreased quickly after the emergence of neutralizing antibodies. a second wave of viremia was detected from day 14 p.i. until death peaking at 5.8610 4 fcov rna copies per ml blood (fig. 2) . the pbfipv-df-2 infected cats developed only the first phase of viremia, fcov rna was detected in blood from day 3 p.i., reached a peak of 1610 3 fcov rna copies per ml blood by dy 7 p.i., and decreased to undetectable level at day 21 (fig. 2) . in cats inoculated with pbfipv-df-2-r3i, complete absence of viremia was observed, the presence of fcov genomic rna in table 1 . total clinical scores of cats challenged oronasally with the parent virus fipv df-2 (n = 4) and recombinant fcovs pbfipv-df-2 (n = 4) and pbfipv-df-2-r3i (n = 4). in vivo analysis of fipv df-2 recombinants plos one | www.plosone.org blood was not detected until the termination of the experiment (fig. 2) . cats infected with fipv df-2 showed high viral load (4.6610 4 -1.2610 7 genomic rna copies per g tissue) in the examined organs with intensive virus replication but very limited rna copy numbers (1.1610 2 genomic rna copies per g tissue) were obtained from the gut (fig. 3) . in cats infected with pbfipv-df-2, no detectable fcov rna copies were found in the examined organs. the pbfipv-df-2-r3i challenged animals tested highly positive (3.6610 4 genomic rna copies per g tissue) for fcov rna in the ileum. in addition, significantly lower level of positivity was observed in the mesenteric lymph node of two cats (3610 2 genomic rna copies per g tissue) (fig. 3) . no other organs contained genomic rna. the subgenomic qrt-pcr showed replication only in the gut. high copy number (.10 3 ) genomic qrt-pcr results were confirmed with subgenomic qrt-pcr assay not only from organs but all fecal and blood samples (data not shown). according to the pre-experimental data, no fcov antibodies were detected at day 0 p.i. in any cat sera using elisa and vn. in the fipv df-2 inoculated animals, neutralizing antibodies appeared by day 10 p.i., and reached high titers (2.4610 3 ) at the time of euthanasia (fig. 4) . the pbfipv-df-2 challenged cats developed neutralizing antibodies from day 10 p.i. that elevated to lower levels (6.4610 2 ) by day 35 p.i. than in the fipv df-2 inoculated animals (fig. 4) . the difference between vn titers generated after fipv df-2 and pbfipv-df-2 was statistically significant (p = 0.037). the pbfipv-df-2-r3i infected cats showed variable results. as elisa and vn assays showed, two animals did not seroconvert (data not shown). two animals seroconverted by day 14 p.i., and their vn titers remained at low levels (9.6610 1 ) compared with those of the pbfipv-df-2 infected cats (fig. 4) . the difference between vn titers generated after pbfipv-df-2 and pbfipv-df-2-r3i was statistically significant (p = 0.013). the distinctive factor of the different pathogenesis of the two fcov biotypes is the increased macrophage tropism of fipv [10, 21] , while fecv is tropic for the mature intestinal epithelium [22, 23] . although alterations of several different genes of fcov are suspected in the background of phenotypic characteristics, the most widely accepted theory suggests the possible role of the truncated orf3abc in the altered tropism and consequent pathogenesis of the two biotypes [9, 14, 16, 18, 24] . however, an identical fcov pair differing only in the intactness of orf3abc has not been tested yet in vivo due to the lack of a cell culture for propagation of type i fecv and a ''true'' type ii fecv isolate [8] . our previous in vitro experiments added further evidence to the involvement of truncated orf3abc to the increased macrophage tropism of type ii fcov [17] . in the present study, characterizing the parent fipv df-2 and the recombinant fcov pair in in vivo experiments, we were able to distinguish significant differences in their biological properties. development of typical clinical signs and post mortem lesions of classical fip were observed in cats infected with the parent virus fipv df-2 similarly as it was reported earlier [5, 18, 25] . unexpectedly, kittens inoculated with pbfipv-df-2 showed only the acute phase of the disease with similar tropism as its wild-type parent fipv df-2. sequencing of the pbfipv-df-2 infectious clone [17] and the recovered virus pbfipv-df-2 that was passaged in fcwf three times revealed point mutations originated in two waves during cloning (24429 (g/a) and 26064 (t/c)) and passaging (3098 (a/g), 5241 (g/a),7632 (c/t), 27817 (c/g) and 28492 (g/c)). one of the two aa substitutions affecting nsp3 was found in papain-like protease 2 responsible for proteolytic processing of nsp1 and nsp2 [26] , proteins with functions of host gene expression inhibition [27] and cellular signaling disruption [28] . one hydrophobic amino acid change (v to i) was identified close to the second heptad repeat in the interhelical region of s2 fusion domain of s protein responsible for virus-mediated membrane fusion. similar conservative (m to l) aa substitution is suspected to be responsible for attenuation of of type i fcov close to the first heptad repeat in this protein [11] . a nucleotide substitution found in the genome of pbfipv-df-2 affected the trs of orf7 gene. mutations in trs of transmissible gastroenteritis virus (tgev) were shown not to alter virus replication in vitro [29] . accordingly, in our in vitro experiments, no difference was found between the replication dynamics of the parent strain and the recombinant virus [17] , and orf7 mrna transcription was found equal after both fipv df-2 and pbfipv-df-2 infection. the 7b protein is considered as one of the virulence factors of fipv [13, 18, 30, 31] . however, several fipvs have lost virulence upon tissue culture passage do not have 7b mutations. a large number of fipv and fecv genomes in genbank also confirm that mutations in 7b are not associated with the fip mutation. considering that the exact biological function of this protein is unknown, and no single aa mutation in 7b was reported which alter virulence of fcov, the role of the aa substitution (k/n) found in the 7b protein of pbfipv-df-2 is most likely has no effect on the virulence of this cloned virus. the difference in virulence could be the consequence of any or combination of the point mutations in the genome of the recombinant fcov obtained from the infectious clone [17] and during the three passages on fcwf after transfection and virus recovery. similar problems with the attenuation of a virulent type i fcov after bacterial cloning have been also reported by others [32] . a transient fever is often seen during the first few days after infection with virulent fipvs, probably due to early host/virus interactions, but actual disease signs of fip only occur when antibodies start to appear. the aforementioned mutations could lead to less effective replication of pbfipv-df-2 in macrophages/ monocytes in vivo, or this mutated fcov was not reacting in the same manner with antibodies as its wild type counterpart. the low and inconsistent level of fecal shedding following inoculation with the parent fipv df-2 strain and the recombinant pfipv-df-2 containing truncated orf3 is similar to that of previous observations [14, 24] . a classic biphasic viremia detected in fipv df-2 infected cats was similar to earlier experiments using the genetically closest fipv strain 79-1146 [5] . as it was suspected from clinical signs, viremia was different in pbfipv-df-2 challenged animals. the infection kinetics of the two viruses was similar in the first days of infection, with the first replication peak at day 7 p.i. (fig. 2) , confirming our previous in vitro data obtained from feline blood monocytes [17] , although the titer of pbfipv-df-2 was almost one log lower at the day of peak, and decreased rapidly. seroconversion also started at the same time in the fipv df-2 and pbfipv-df-2 inoculated animals but remained at lower level in the latter case until the end of the experiment. these data indicate self-limiting replication of pbfipv-df-2 and complete clearance by the immune system that was further confirmed by the absence of the genomic rna in the organs (fig. 3) . the differences between the biological properties of the two viruses with truncated orf3abc are substantial but by far less pronounced than it can be observed between the orf3abc deleted and orf3abc completed fcovs. pbfipv-df-2-r3i genome was invariably absent in the blood monocytes. as a possible consequence of the absence of viremia, viral load of organs was not detected, the presence of pbfipv-df-2-r3i was found only in the mesenteric lymph node of two animals that showed weak seroconversion. these data indicate rather carrier role of macrophages/monocytes of fcov with completed orf3abc from the sites of the intensive fcov replication. the absence of replication in blood monocytes of pbfipv-df-2-r3i inoculated cats coincide with previous data collected after fecv infection studies [3, 22, 23, 33] , which clearly demonstrated the limited replication of the fecvs in mononuclear cells [5, 34] . the weak or missing seroconversion of pbfipv-df-2-r3i challenged cats is an obvious explanation of the low or absent systemic replication of the virus, and it is comparable with previous experimental type i fecv infections that showed low and often variable antibody titers in serum or plasma [22, 23, 35, 36] , while a proportion of cats remained seronegative despite intensive fecal virus shedding [22] . indeed, intensive fecal shedding and virus replication was detected during the whole period of the experiment and from the ileum of the sacrificed cats challenged with pbfipv-df-2-r3i carrying completed orf3abc, in contrast to the other two investigated viruses. our results are in accordance with the classic theory that mutations (deletions and nonsense mutations) altering the number and size of proteins translated from orf3abc contribute to the altered tissue tropism of fipv. this theory was reinforced by genetic investigations [24] that revealed that nondeleted orf3abc of fipvs accumulates four times more unique non-synonymous amino acid mutations in the orf3abc than the fecvs, possibly modifying the biological function of these proteins. data of pbfipv-df-2-r3i shedding in the present study are similar with those of acute fecv and enteric ccov infection. recent experiments [32] with recombinant type ii fipv showed that orf3c containing stop codon can be restored to code fulllength 3c protein by point mutation during replication in internal organs and the gut. to investigate the mutability of this region we sequenced the 3c region of pbfipv-df-2-r3i from fecal and intestinal samples and we found no genetic alterations in this region (data not shown). in type i fecv challenge studies [23, 35, 37, 38] fcovs with intact orf3abc shed at significantly higher titers compared with fipvs, allowing horizontal spread to contact animals. the type ii fcovs used in our challenge studies are the result of double recombination between type i fcov and type ii ccov [39] . in this respect, results of the present study are also in accordance with ccov shedding pattern gained from canine experimental infection [40] . summarizing the results of our challenge experiment, and comparing with previous experimental and clinical data, we conclude that the orf3abc truncated recombinant virus showed attenuated phenotype with low virulence, transient viremia and complete clearance of the virus. therefore, we cannot draw clear conclusion on the role of truncation of orf3abc in the development of fipv pathogenesis, although the biological properties of pbfipv-df-2 were closer to attenuated fipv than to those of fecv. however, completion of orf3abc vested pbfipv-df-2-r3i with biological properties that differentiate between the fecv and fipv biotypes, such as intensive replication in the gut, absence of viremia and weak or no serological response. the observation of numerous natural and experimental fcov infection in the past decades led to the rise of the idea that completed orf3abc is indispensable for intestinal replication of the virus. however, this theory has never been confirmed due to the lack of an identical virus pair differing only in their orf3abc regions. using such a virus pair our study is the first experimental proof which confirms the decisive role of orf3abc in the intestinal replication of fcov. feline infectious peritonitis: a worldwide serosurvey an enteric coronavirus infection of cats and its relationship to feline infectious peritonitis persistence and 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rt-pcr assays for the detection and quantitation of canine coronavirus type i and type ii rna in faecal samplesof dogs conceived and designed the experiments: ab af sb. performed the experiments: ab af zz. analyzed the data: ab af zz. contributed reagents/materials/analysis tools: af sb. wrote the paper: ab zz. key: cord-294568-12eyo13f authors: fernandes-matano, larissa; monroy-muñoz, irma eloísa; angeles-martínez, javier; sarquiz-martinez, brenda; palomec-nava, iliana donají; pardavé-alejandre, hector daniel; santos coy-arechavaleta, andrea; santacruz-tinoco, clara esperanza; gonzález-ibarra, joaquín; gonzález-bonilla, cesar raúl; muñoz-medina, josé esteban title: prevalence of non-influenza respiratory viruses in acute respiratory infection cases in mexico date: 2017-05-03 journal: plos one doi: 10.1371/journal.pone.0176298 sha: doc_id: 294568 cord_uid: 12eyo13f background: acute respiratory infections are the leading cause of morbidity and mortality worldwide. although a viral aetiological agent is estimated to be involved in up to 80% of cases, the majority of these agents have never been specifically identified. since 2009, diagnostic and surveillance efforts for influenza virus have been applied worldwide. however, insufficient epidemiological information is available for the many other respiratory viruses that can cause acute respiratory infections. methods: this study evaluated the presence of 14 non-influenza respiratory viruses in 872 pharyngeal exudate samples using rt-qpcr. all samples met the operational definition of a probable case of an influenza-like illness or severe acute respiratory infection and had a previous negative result for influenza by rt-qpcr. results: the presence of at least one non-influenza virus was observed in 312 samples (35.8%). the most frequent viruses were rhinovirus (rv; 33.0%), human respiratory syncytial virus (hrsv; 30.8%) and human metapneumovirus (hmpv; 10.6%). a total of 56 cases of co-infection (17.9%) caused by 2, 3, or 4 viruses were identified. approximately 62.5% of all positive cases were in children under 9 years of age. conclusion: in this study, we identified 13 non-influenza respiratory viruses that could occur in any season of the year. this study provides evidence for the prevalence and seasonality of a wide range of respiratory viruses that circulate in mexico and constitute a risk for the population. additionally, our data suggest that including these tests more widely in the diagnostic algorithm for influenza may reduce the use of unnecessary antibiotics, reduce the hospitalisation time, and enrich national epidemiological data with respect to the infections caused by these viruses. introduction acute respiratory infections (aris) represent the leading cause of morbidity and mortality worldwide [1] [2] , are the most common cause of outpatient care in adult patients [3] , and are responsible for 70% of hospitalisations due to respiratory diseases in child populations aged 1-4 years and up to 90% in infants under 1 year of age [4] . aris are a group of diseases with normally less than 15 days of evolution that are caused by different microorganisms. a viral aetiological agent is estimated to be present in up to 80% of cases [5] [6] [7] . these infections can occur in the upper and lower respiratory tract. upper aris may include one or more of the following conditions: rhinopharyngitis, pharyngoamygdalitis, sinusitis, and acute otitis media [8] [9] . lower aris include epiglottitis, laryngitis, laryngotracheobronchitis (croup), bronchitis, bronchiolitis, and pneumonia [9] . these infections are easily transmitted via coughing or sneezing. contagion occurs through the inhalation of aerosols and microdroplets that contain the causative agent. another important form of contagion is through direct contact of hands with objects contaminated with respiratory secretions from infected individuals, which can be self-inoculated into the nasal and buccal mucosae and/or into the ocular cavity [10] . a large amount of information is available concerning the timing and distribution of influenza viruses in the population following the reappearance of avian influenza a subtype h5n1 in 2003 and 2004 and the influenza a subtype h1n1 pandemic in 2009 [11] . influenza viruses are one of the main causative agents of aris worldwide; however, many other respiratory viruses for which insufficient epidemiological information is available can also cause aris. studies performed at the international level have frequently identified human respiratory syncytial virus (hrsv), human parainfluenza virus (hpiv), influenza virus (flu), human mastadenovirus (hmdv), rhinovirus (rv), and enterovirus (ev) and less frequently identified human metapneumovirus (hmpv), primate bocaparvovirus (pbpv), and human coronavirus (hcov) [12] . these viruses can serve as the causative agents for aris that occur outside of influenza season, when the rate of positivity can drop below 10%. although a large percentage of aris are caused by viral infections, the causative viruses have not been specifically identified in the majority of cases due to difficulties such as the high number of possible aetiological agents, similar symptoms among aris caused by different aetiological agents, the emergence of new viruses or new variants of previously described viruses, and the high cost of the detection tests [4] . thus, little information is available concerning the prevalence and seasonality of these viruses, mainly in undeveloped countries, where the possibilities of carrying out this type of study on a regular basis is unusual. the investigation of the causative agents of acute respiratory infections is important in order to lead the development of vaccines to target the most prevalent viruses and to reduce the unnecessary prescription of antibiotics and of antiviral oseltamivir phosphate. in addition, analysis like this can help to identify the age groups more susceptible to infection by each virus, in order to take the necessary actions for prevention and treatment. therefore, our aim was to determine the viral aetiology of aris in samples from patients who presented respiratory symptomatology but were negative for influenza by rt-qpcr testing. to evaluate the presence of noninfluenza respiratory viruses circulating in mexican population in the different seasons of the year, we analized every pharyngeal exudate sample received by the laboratorio central de epidemiología (lce) (central epidemiology laboratory) between the epidemiological week 40 of 2014 and the week 39 of 2015, according to the following criteria. all samples had a previous negative result for influenza by rt-qpcr and met one of the following operational case definitions: influenza-like illness (ili): a person of any age who presents a fever greater than or equal to 38˚c, a cough, and cephalalgia accompanied by one or more of the following symptoms: rhinorrhoea, coryza, arthralgias, myalgias, prostration, odynophagia, thoracic pain, abdominal pain, or nasal congestion. in patients under five years of age, irritability is considered a cardinal sign in place of cephalalgia. in patients older than 65 years, fever is not required as a cardinal symptom. severe acute respiratory infection (sari): a person of any age who presents difficulty breathing accompanied by a fever greater than or equal to 38˚c and a cough with one or more of the following symptoms: poor general condition, thoracic pain, polypnoea or acute respiratory distress syndrome (ards) or any death associated with ili or sari. a total of 872 samples were selected. (s1 table) total nucleic acid extraction the superscript™ iii platinum 1 one-step rt-qpcr system kit (invitrogen, carlsbad, califórnia, eua. catalog: 12574035) was used in a 7500 fast real-time pcr system (applied biosystems 1 , foster city, califórnia, eua) to amplify viral genetic material. primers and probes were used for each of the following viruses: hmpv, hrsv, hpiv 1-4, betacoronavirus 1 (βcov1), human coronavirus (hku1, 229e, nl63) (hcov), hmdv, rv, ev, and pbpv. the human rnase p (rp) gene was used as an internal control ( table 1 ). the viruses were evaluated in uniplex reactions with the following reaction mixture: 12.5 μl of 2x reaction mix, 0.5 μl of each primer and probe, 1 μl of enzyme, 5.5 μl of rnase-free water, and 5 μl of total dna or rna lyophilizates (amplirun1 vircell; granada-spain) were used as the positive controls for all evaluated viruses. all samples that presented amplification for any of the viral markers plus the internal control were considered positive results. all samples without amplification of the viral markers but with amplification of the internal control were considered negative results. samples that did not present amplification of the internal control were considered inadequate. descriptive statistics were used to analyse the prevalence of the viruses included in the study, with the percentages given with a 95% confidence interval. the chi-square test of homogeneity and independence and fisher's exact test were used to compare categorical variables (p-values <0.05 were considered significant). anova and student's t-test were used for hypothesis testing of the quantitative variables. the analyses were performed with the spss statistics version 24.0 software, and the graphics were generated with the statgraphics 1 centurion xvi.ii and microsoft 1 excel 1 2010 software. the information of the biological specimens used in the present study is not traceable to the patient data identity. all the samples were used in an anonymous way. the study was approved by the ethics committee and the research committee of the national committee of scientific research of the instituto mexicano del seguro social with the registration number r-2016-785-041. of the 872 samples analysed, 451 were from males (51.7%) and 421 were from females (48.3%). the average age was 41 years, with minimum and maximum extremes of 0 and 101 years, respectively. the population was divided into 4 age groups based on the age ranges included on the instituto mexicano del seguro social (imss; mexican social security institute) health cards. according to this stratification, the samples were distributed as follows: 265 samples corresponded to children 0-9 years of age (30.4%), 28 to young people 10-19 years of age (3.2%), 263 to adults 20-59 years of age (30.2%), and 316 to adults 60 years and older (36.2%). (table 2) . unintentionallya, the analysed samples were collected from patients from three geographical areas of the country as follows: the north zone (baja california, baja california sur, 25.9% of the samples (fig 1) . additionally, these data were analysed based on the age groups (s2 table and s1 file) . of the total samples analysed, 312 were positive for at least 1 virus (35.8%), with 47.1% from female patients (n = 147) and 52.9% from male patients (n = 165); there was no significant difference between the groups (p>0.05). of the total positive samples, rv had the highest number of detections (33.0%), followed by hrsv (30.8%), hmpv (10.6%), hmdv (9.6%), hpiv3 (8.7%), βcov1 (8.7%), ev (5.8%), and pbpv (4.5%). the other viruses presented percentages below 4%, although together they accounted for 10.3% of all positive cases. only hpiv2 was not detected (table 3 ) the 3 viruses most often identified in cases of co-infection were rv in 48.3% of the cases, followed by hrsv (35.7%) and hmdv (32.1%). interestingly, in the case of pbpv, despite it being the causal agent of only 19.6% of co-infections, these co-infections represented 78.6% of the pbpv cases detected. the behaviour of hmdv was similar, with 60% of all cases in which this virus was present involving a combination with other viruses. both of these cases represent a significant association (p<0.05) with co-infection (table 4) . the most commonly observed viral combination was hmdv + rv (16.1%), followed by hpiv3 + rv and hrsv + βcov1 (both 7.1%). although it occurred only 2 times, the co-infection with 4 viruses involved the same agents in both cases (hrsv + hmdv + βcov1 + pbpv). a comparison was performed between the days of hospitalization required by the patients with negative samples and those with samples positive for a single virus or in whom 2 or more viruses were detected. the analysis of this clinical data showed that the hospitalization was significantly higher in the negative samples than in the positive ones for a virus or with coinfections: 7.5, 5.1 and 4.5 days on average, respectively (p <0.05). we observed the same tendency with the average number of comorbidities and symptoms: 0.65 in the negative samples versus 0.35 in positive samples (p <0.05) and 7.7 in the negative samples against 7.4 in positive samples (p > 0.05), respectively. nevertheless, the number of clinical symptoms and comorbidities were greater in the aris positive for a single virus than in the patients with co-infections (p <0.05) ( table 5 ). the highest number of positive cases occurred in the group aged 0 to 9 years (62.5%) and the second highest in the group aged 60 years or older (20.5%), followed by the 20-to 59-year-old group (14.4%) and finally the 10-to 19-year-old age group (2.6%), which represented a significant difference (p<0.05). consistently, the 0-to 9-year-old age group comprised 91.1% of the co-infections. interestingly this group contained 92.9% of all pbpv infections, 86.7% of the hmdv infections, and 86.5% of the hrsv cases (fig 3) . when conducting the analysis of the overall behaviour of the viruses in the period covered by the study, we observed that the months with high and low positivity differed significantly table 4 . viruses participation in co-infections. (p<0.05). fig 4b-4e shows the monthly behaviour of each virus. the month of november presented the highest percentage of cases during the year ( fig 4a) . when the analysis was performed by season, we observed differences between the four seasons of the year (p<0.05). generally, summer had a significantly lower proportion of positive samples (24.5%; p<0.05), whereas fall had the highest proportion (44.9%; p<0.05). however, the individual trends of the viruses were not the same. for instance, spring (march 21 to june 20) accounted for the greatest proportions of rv, hmdv, hpiv4, and hpiv1 cases (29.5%, 38.5%, 46.8%, and 48.0%, respectively). in contrast, most hrsv (48.2%) and ev (72.6%) cases were detected in the fall (september 21 to december 20), and winter (december 21 to march 20) presented the highest prevalence of hmpv (47.3%) and βcov1 (60.5%). the other viruses (hcov 229e, hcov hku1, hpiv 3, pbpv, and hcov nl63) were detected throughout the year, with no seasonal trends observed (p>0.05). approximately 27 million ari cases occur annually in mexico [14] . these cases can be caused by a large variety of aetiological agents. however, the main purpose of epidemiological surveillance in the country is to detect the antigenic variations of influenza that are presented each season, which determine the changes in the vaccine composition. in 2013, the instituto de diagnóstico y referencia epidemiológicos (indre; institute of epidemiological diagnosis and reference) implemented a differential diagnosis of influenza that included 14 other respiratory viruses [15] . due to this, at this moment mexico does not have enough epidemiological information about the great diversity of viruses causing acute respiratory infection. this lack of information, makes physicians search only for influenza, it is therefore the most requested confirmatory test in the laboratory during the whole year, even when we are out of influenza season (season comprises from october to may), leading to useless negative influenza results in most of the cases, without identifying the real causal agent of ari in mexico. this problem have direct implications for each patient, as the clinician does not know the causal agent, the patient does not receive the suitable treatment. it also has implications that affect the whole society, because the ignorance of the circulation patterns and incidence of other respiratory viruses limit preventive actions by health institutions. according to data from the secretary of health, approximately 80% of the samples from patients with aris that are received for confirmation of influenza virus infection outside of flu season are negative for the different strains of this virus and remain without a defined aetiology. therefore, the objective of this study was to determine the viral aetiology of these infections and to analyse the behaviour of non-influenza respiratory viruses in the mexican population. beginning and ending with the 2015 influenza season, 872 samples collected over one year were evaluated to determine the presence of hmpv, hrsv, hpiv 1-4, βcov1, hcov, hmdv, rv, ev, and pbpv. these 14 respiratory viruses share symptoms with influenza but are rarely suspected or can be confused with influenza. in contrast to other studies in other countries investigating this issue, this study included samples corresponding to all age groups and regions of the country. therefore, the study population best represents the demographic distribution of aris in the country. in contrast to most of the prevalence studies of aris, in which all age groups are not normally analysed and data is limited to a single region of the country, in this study, nor the age of the patient, nor the region of the country from which the sample came were included as inclusion or exclusion criteria. therefore, the population analysed in this study represents better the demographic distribution of aris in our country. this type of study helps provide relevant data for the development of treatment and prevention strategies because the currently available antiviral agents and vaccines are primarily directed at influenza infection. however, the proportion of aris caused by different influenza viral agents is not negligible, with the detection range of non-influenza respiratory viruses spanning from 16.5 to 72.7% in studies conducted worldwide [4;16-20] , depending on factors such as the study design, study population, detection technique used, and period covered by the study. in our case, the prevalence of the analysed viruses was 35.8%, which was within the cited range. the most prevalent viruses were rv, hrsv, and hmpv, and the only virus that was not detected was hpiv2. this result was consistent with other studies in which type 2 parainfluenza virus had the lowest detection frequency [21-24]. however, there is evidence that its prevalence increases when hpiv-3 and hpiv-1 are reduced [25] [26] . consistent with our results, rv was identified as the most common virus in ari cases in several studies [17;20;22;27] in which the presence of influenza and other respiratory viruses from throat swabs is determined by molecular techniques of own design or using commercial panels. equal data were observed in a study of mexican children [28] . this virus is the major aetiological agent of the common cold [29], however, it may also be involved in serious aris [30] [31] [32] , which are primarily observed in patients with underlying comorbidities, such as asthma [33] [34] [35] or other chronic pulmonary diseases [36] , indicating a certain degree of opportunism. similar to rv, hrsv was identified in high proportions in other studies [4;37] , conducted in poland in which 380 individuals were analysed without age selection, and in mexico in which 383 children up to 5 years of age were studied. this trend is also observed in studies where a higher prevalence of influenza was observed [19;21;23;38] , and hrsv has been associated with the pathogenesis of asthma. more importantly, hrsv is the leading cause of child mortality caused by viruses [39] . the third most prevalent virus was hmpv. in mexico, the first study in which the presence of this virus was determined was published in 2005 [40] . subsequently, other mexican studies demonstrated its importance in the child population [16;41-42] . studies such as that of diaz et al. [16] demonstrated that hmpv was mostly associated with severe acute respiratory infections instead of mild and moderate infections. the importance of the differential diagnosis of other respiratory viruses in samples with negative influenza results becomes apparent when we observe the prevalence of the three main viruses identified in this study as well as their associations with severe cases and deaths, especially in the child population. co-infections represented 17.9% of the positive samples. this percentage ranged between 6.9 and 18.6% in other studies that also included various age groups [20;43-44] . however, much higher percentages have been found (above 30%) in study populations composed of children under 5 years of age [16;22-23;45] . our results confirmed that viral co-infection was common, especially in the child population because 91.1% of all co-infections occurred in the 0 to 9 year age group. this result could be attributed to slower viral elimination due to a still-developing immune system [46] . the virus most involved in co-infections was pbpv, as in 78.6% of the cases in which it was identified, it was found together with another virus. studies have reported identification percentages of this virus in conjunction with other agents ranging from 47.4 up to 90% [47] [48] . however, its prevalence in asymptomatic patients is also high (44% or 43%) [49] , which calls into question whether pbpv by itself is capable of generating disease [50] or if it only participates as a facilitator for another agent to infect the host. at present, cellular and animal models are still being developed for this virus [51] , therefore, the evidence needed to confirm this hypothesis does not yet exist. from a clinical perspective, whether the presence of a co-infection results in a more serious case or is a poor prognostic factor is a matter of controversy within the scientific community. although some studies have reported cases with these characteristics [52] [53] [54] , reports by other authors have suggested that co-infections are not synonymous with clinical differences or greater severity [55] [56] . in the study of martínez-roig et al. [57] , an inverse relationship was found between the number of viruses detected and the hospitalization time as well as the need for oxygen therapy. a similar relationship was observed in the study of canducci et al. [58] , where there was a greater hypoxia and lengthier hospitalization time for infections caused purely by hrsv compared to coinfections. in our study, the aris caused by a single virus also presented lengthier hospitalization times than aris caused by 2 or more viruses, although the differences were not statistically significant. notably, similar to the estimation of prevalence, differences in the reported ranges of and discrepancies in the severity associated with viral co-infections can be attributed to the detection techniques employed, the population, the time period of the study, and the viruses studied. because of the short period of time comprised by this study and the limited number of samples, it is not possible to state, but it can be suggested certain seasonal behaviors of some studied viruses. generally, the peak of respiratory infections occurs in the period between november and april in countries of the northern hemisphere [59] [60] . the influenza season is well known in mexico and worldwide; however, the seasons of other respiratory viruses are not well known. according to our results, the highest prevalence of these viruses in mexico appears to occur from october to march (autumn and winter), which coincides with the influenza season. detection was significantly higher in november of 2014; during this month, there was a marked decrease in the national mean temperature from 23.3 to 18.3˚c. according to cui et al. [61] , mean temperature is the key climatic parameter associated with the prevalence of many respiratory viruses because some viruses survive and/or replicate better at low temperatures [61] . on the other hand, viegas et al [62] proposed that mucus release by cilia was reduced when the temperature of the respiratory tract was lowered; consequently, the local innate immune response (neutrophil and natural killer (nk) cell migration) was also reduced, thereby promoting viral infection. climatic factors may also indirectly favour the transmission efficiency because low temperatures induce a change in social behaviour that favours interior overcrowding and increases the likelihood of close contact and transmission of infections [63] . for this reason, it is a common suggestion to avoid going to school or work when you are undergoing an infectious process. our results suggest that some viruses have a more marked seasonal trend than other viruses. in winter, the prevalences of hmpv, hpiv3, and bcov1 were higher than in the other seasons of the year. the circulation of hmpv in particular predominates in the colder seasons because a marked reduction in its prevalence is observed in the spring until it disappears completely in the summer. furthermore, in a study conducted in mexico city, in which 525 children between 1 and 15 years of age were analysed and in other study performed in china with 607 hospitalized patients was reported that the highest prevalence of hmpv occurs in the winter and that its seasonality overlaps with that of other viruses [19;43] . similar to hmpv, some authors agree that hrsv is a virus that circulates preferentially in colder seasons [19;43;64] . this study demonstrates that hrsv can cause aris in all seasons of the year but is consistent with other studies that report that its prevalence is highest during the fall and winter. conversely, rv seemed to be the virus best suited to the climatic conditions of the country because there were no significant differences in its prevalence in the different seasons. according to the results of a study conducted in china [61] , the optimal circulation temperature range of this virus is 15 to 25˚c, which is similar to the range of the mean mexican temperature and may justify its high prevalence in all months of the year. however, despite all of the information reported to date, factors affecting the circulation of different viruses remain unclear. annual research is needed to establish the seasonality of these viruses with more accuracy and precision. although influenza is one of the main causative agents of respiratory infections worldwide, it is of vital importance to determine the prevalence and timing of other causal agents. in this study, we identified 13 non-influenza respiratory viruses that occurred in any season of the year. this study provides evidence for the prevalence and seasonality of a wide range of respiratory viruses circulating in mexico that constitute a risk for the population. additionally, our data suggest that including these tests more widely in the diagnostic algorithm for influenza could reduce the use of unnecessary antibiotics, reduce the hospitalisation time, and enrich national epidemiological data regarding infections caused by these viruses. supporting information s1 human metapneumovirus in adults human metapneumovirus: review of an important respiratory pathogen reforzamiento pulmonar: su relación con la infección respiratoria aguda y la prescripción inadecuada de antibioticos non-influenza viruses in acute respiratory infections among young children. high prevalence of hmpv during the h1n1v.2009 pandemic in poland summary health statistics for u.s. children: national health interview survey viruses and bacteria in the etiology of the common cold bronchiolitis-associated hospitalizations among us children infecciones respiratorias agudas en menores de 5 años intervención educativa sibre infecciones respiratorias agudas 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children with viral respiratory infections? human bocavirus in chile: clinical characteristics and epidemiological profile in children with acute respiratory tract infections does respiratory virus coinfection increases the clinical severity of acute respiratory infection among children infected with respiratory syncytial virus? multipathogen infections in hospitalized children with acute respiratory infections human respiratory syncytial virus in children with lower respiratory tract infections or influenza-like illness and its co-infection characteristics with viruses and atypical bacteria in hangzhou viral coinfection in childhood respiratory tract infections two-year prospective study of single infections and co-infections by respiratory syncytial virus and viruses identified recently in infants with acute respiratory disease health protection agency. respiratory pathogen circulation public health agency of canada. the respiratory virus detection surveillance viral aetiology of acute respiratory infections among children and associated meteorological factors in southern china respiratory viruses seasonality in children under five years of age in seasonality and the dynamics of infectious diseases seasonal patterns of viral and bacterial infections among children hospitalized with community-acquired pneumonia in a tropical region we thank the san angel laboratory for their support for covering the costs of the publication of this manuscript. key: cord-291756-ejh1r8h4 authors: pérez-fuentes, maría del carmen; molero jurado, maría del mar; martos martínez, áfrica; gázquez linares, jose jesús title: threat of covid-19 and emotional state during quarantine: positive and negative affect as mediators in a cross-sectional study of the spanish population date: 2020-06-25 journal: plos one doi: 10.1371/journal.pone.0235305 sha: doc_id: 291756 cord_uid: ejh1r8h4 aims: the objective of this study was therefore to analyze the effect of exceptionally stressful situations, such as the current health risk, on the cognitive and emotive state of the individual, that is, perceived threat and emotional state on affect and mood. method: this was a cross-sectional study with snowball sampling. the sample came to 1014 spanish adults (67.2% women and 32.8% men). the perception of threat from covid-19 questionnaire, the affective balance scale and the mood evaluation scale were used. results: the results showed that the perception of threat from covid-19 was related positively to negative affect and emotional signs, that is, sadness-depression, anxiety and anger-hostility. there was a direct positive effect of perceived threat from covid-19 on sadness-depression, anxiety and anger-hostility moods, while anxiety and anger-hostility had a direct positive effect on perception of threat from the virus. thus, there was a circular relationship, in which perceived threat influenced the presence of negative mood, and negative mood, in turn, linked to emotions of irritation and agitation from a present situation, promoted the feeling of threat. conclusions: a negative affective balance increases both one’s perception of threat from covid-19 and negative mood. thus, knowing the emotional and cognitive effects on the population would enable measures to be put into service to facilitate their effective coping. introduction generalized transmission of the novel coronavirus called sars-cov-2 or covid-19 detected in china in december 2019 has placed international public healthcare in check [1, 2] . within the spanish borders, this has generated a scenario in which the response capacity of the healthcare system depends on the service of healthcare professionals and the disposition of the population to maintain the requirements of isolation, hygiene and social distancing to reduce exposure to contagion [3] . beyond the danger to health, the presence of this state of emergency and constant worry increase stress factors, with a consequent increase in anxiety, and even development of emotional disorders [4, 5] . the perception of threat to one's health is based on perceived susceptibility (that is, belief of vulnerability to and possibility of contracting the disease) and in perceived severity (that is, beliefs related to changes that having the disease would cause in all areas of life) [6] . a state of extended hypervigilance, feelings of life-threatening danger and strong sensitivity to the appearance or recurrence of the disease are characteristic of those who show high perception of threat [7] . thus, beyond physical health, people at risk and patients diagnosed with covid-19 may experience fear from the consequences of infection, such as death or severe physical disability [8] . such emotional disturbance combines with boredom, loneliness and anger that could appear in quarantine [9] . concern for one's health, especially among those who live where the outbreak is worst, is combined with the home confinement decreed by the state of emergency [10] . although there are significant gaps in treatment and primary prophylactic measures, such as vaccines, in this epidemic [11] , the need for those at risk to be isolated is conclusive and requires a high level of coordination and citizen responsibility [12] . this confinement can generate negative consequences to physical and psychological wellbeing, such as anxiety and insomnia, promoted by alteration of physiological and circadian rhythms. however, of much greater concern is the psychological impact [13] . stressful factors such as prolonged confinement, fear of infection, frustration, boredom, inadequate information, lack of contact with other persons outside of those with whom one lives, lack of personal space in the home and financial loss increase worry and individual perception of threat [14] , especially when there is no way to cope constructively with the adversity [15] . in this situation of great stress and uncertainty, it is normal to develop a stronger perception of threat, characterized by assignment of negative meaning to originally neutral stimuli [16] . the perception of threat generates emotional and mood alterations and vice versa, such that the state of anxiety and changes in humor can also lead to heightened perception of threat [17] . in this case, sudden changes and unexpected situations, such as the population is now confronting with covid-19, are perceived as physical or mental threats, challenging one's ability to control the situation. any situation creating an emotional impact dominating one's usual coping strategies can generate anxiety and apprehension in individuals who normally would be mentally balanced and healthy [18] . with regard to emotional control and stress management, individual variables such as emotional intelligence, self-efficacy and optimism have been shown to be determinant [19, 20, 21, 22] . emotion regulation processes based on conscious emotional control facilitate positive affective experiences, contrary to automatic and preconscious, which are related to negative affect [23] . referring specifically to the impact of confinement because of covid-19, moreira de medeiros et al., [8] suggest that quarantine can generate feelings of boredom, loneliness and anger. along this line, studies done in asian populations, where isolation measures began much earlier than in spain, show how individuals during the epidemic experienced a moderate or severe psychological impact and usually showed depressive symptoms and higher anxiety and stress [13] . in view of the above, the objective of this study was to analyze the effect of exceptionally stressful situations, such as the current health risk, on the cognitive and emotional state of the individual, that is on the perception of threat and emotional state, both on effects and mood. the starting hypothesis was that perception of threat in the exceptional state of health emergency caused by covid-19, affects one's emotional situation (model 1), and this, in turn, affects perception of risk (model 2), in which positive and negative affect balances act as mediators in these relationships (fig 1) . the original sample of general spanish population was n = 1043. based on answers to control questions (cq), those cases in which random or incongruent answers were detected were discarded (-29), leaving a final sample of 1014, all of them residents of spain in 19 autonomous regions, but 37.9% were from andalusia and 27.5% from madrid. the mean participant age was 40.87 (sd = 12.42), in a range of 18 to 76. the sample was made up of 67.2% (n = 681) women and 32.8% men, with a mean age of 39.88 (sd = 12.35) and 42.92 years (sd = 12.33), respectively. marital status was 60.1% (n = 609) married or with a stable partner, 30.9% (n = 313) single, 8.1% (n = 82) divorced or separated, and the remaining 1% (n = 10) widowed. 35 .9% (n = 364) of the participants stated they had minors in their care. with respect to education, 78.7% (n = 798) had a higher education, followed by 16% (n = 162) with secondary education, 5% (n = 51) with primary school and 0.3% had no formal education. the whole sample, at the time data were collected, were ordered confined to their homes by the state of emergency decreed by the government of spain in response to the current an ad hoc questionnaire was prepared for collecting sociodemographic characteristics. items included sex, marital status, age, education and if there was anyone with coronavirus close to them. the brief illness perception questionnaire (bip-q) [24] , in the brief [25] spanish version validated for covid-19 [26] was used for this study. this instrument consists of five items answered on a 10-point likert-type scale which finds a single dimension, perception of threat from the disease, for which reliability was ω = 0.68; glb = 0.72. emotional state during the past week was evaluated with the affective balance scale (abs) [27] ; adapted to a spanish population [28] . this instrument consists of 18 items in which the subjects must say whether they have experienced the states indicated in the past week rated on a likert-type scale with three answer choices on the frequency (1 = "little or never", 2 = "sometimes", 3 = "a lot or usually"). the scale directly measures both positive and negative affect. the reliability indices found were ω = 0.82, glb = 0.83 and ω = 0.79, glb = 0.82, respectively. their emotional condition at the time of evaluation was analyzed with the mood evaluation scale (evea) [29] . this instrument evaluates transitory moods, classifying them in four subscales (anxiety, sadness-depression, anger-hostility and joy). it has 16 items rated on a scale of 0 (none) to 10 (a lot) indicating how much the subject identifies with the different moods enumerated. reliability was ω = 0.88, glb = 0.89 for anxiety; ω = 0.88, glb = 0.90 for sadnessdepression; ω = 0.96, glb = 0.97 for anger hostility; and ω = 0.85, glb = 0.88 for joy. this cross-sectional study was carried out in a sample found by snowball sampling, by publicizing it on social networks and by texting during the first week of confinement of the spanish population from march 18 to 23, 2020. a cawi (computer aided web interviewing) survey was used for data collection, including, in addition to the three validated questionnaires, a series of questions for collecting sociodemographic data (age, sex, marital status, education) and others on their current situation (minor children in their care, or positive coronavirus cases in their closest environment). participation was voluntary, and before answering the questionnaire, participants were given information on the study and its purpose on the first page, where they also had to check a box indicating their informed consent before they could start taking the survey. they were also asked to answer sincerely, guaranteeing the anonymity of their answers. for control of random answers, a series of control questions were included throughout the questionnaire. the study was approved by the university of almeria bioethics committee (ref. favorably reported on march 24, 2020). first, pearson's pairwise correlation coefficients were determined. then the descriptive statistics for the variables of the study were presented. participants were grouped according to the affective balance index (abi), on which scores below 0 show a "negative affective balance" and above 0 a "positive affective balance". a third group, which we called the "neutral group", was made up of those whose score was equal to 0. a t-test for independent samples was performed to find out whether there were any differences between the groups (negative and positive affective balance) in perceived threat from the disease. furthermore, the bayesian alternative, which enables estimation of evidence in favor of the hypothesis using the bayes factor, was also calculated. jasp statistical software ver. 0.11.1 [30] was used for estimating the bayesian t-test. the cauchy prior width was 0.707 as predetermined by the software [31] . starting out from the results of the correlation analyses, several mediation models were proposed. specifically, two mediation analyses were done with predictors, mediators and multiple result variables. model estimation was performed applying statistical corrections, including the item on the close presence of a covid-19-positive case as a confusion variable. jasp ver. 0.11.1 [30] based on the lavaan software [32] was used for this. to prove whether there was an indirect effect, bootstrapping was applied, calculating the confidence intervals with the biascorrected percentile method as suggested by biesanz, falk & savalei [33] . to examine the reliability of the instruments used for data collection, mcdonald's omega [34] coefficient was estimated, following the proposal and indications of ventura-león and caycho [35] . the greatest lower bound (glb) was also calculated. as shown in table 1 , perceived threat from the disease correlated positively with negative moods such as sadness-depression, anxiety and anger-hostility, and on the contrary, was negatively correlated with the joy subscale. in relationships with affect, perceived threat from the disease correlated negatively with positive affect and positively with negative affect. moreover, classification of the abi resulted in three groups: negative affective balance, positive affective balance and the group we called "neutral", because they scored an abi = 0. this group (n = 66) was discarded for the comparison of means, using only the differences between the negative (n = 461) and positive affective balance (n = 487). table 2 shows the statistically significant differences between the two groups, where the negative affective balance group had a higher mean score in perceived threat from the disease than the positive affective balance group (fig 2a) . the cohen's d of over 0.8 indicates a large effect size. the bayes factor (bf) was also computed to test the weight of available evidence in favor of the alternative hypothesis (h 1 ) on the existence of differences between groups against the null hypothesis (h 0 ) that there is no significant difference between groups. the data found on the differences between groups support the evidence in favor of the alternative hypothesis (bf 10 = 2.133x10 34 ), which revealed extreme evidence in favor of h 1 . the inference graphs corresponding to the bayesian statistic are shown in fig 2b, 2c and 2d. as a starting point, in line with the hypothetical circular model presented above (fig 1) and to formulate the mediation models, we asked ourselves two questions: 1) how does perceived threat from covid-19 affect mood? (model 1), and 2) how does mood affect perceived threat from covid-19? (model 2). and in both cases, are positive and negative affect mediators in these relationships? as observed in table 3 , the perception of threat had a direct positive effect on moods that could be qualified as "negative" (sadness-depression, anxiety, anger-hostility). indirect effects observed were that positive affect mediated in the relationship of perceived threat and sadness-depression and joy. negative affect exerted a mediating function in the relationship between perceived threat and the four moods, positive for sadness-depression, anxiety, anger-hostility, and negative for joy. the proportion of variance explained for each of the predictor variables in mediation model 1 is the following: 51% (r 2 = 0.510) for sadness-depression, 54.7% (r 2 = 0.547) for anxiety, 29.3% (r 2 = 0.293) for anger-hostility, and 31.9% (r 2 = 0.319) for joy. for the mediators we found 5.2% (r 2 = 0.052) for positive affect and 25% (r 2 = 0.250) for negative affect. in table 4 , anxiety and anger-hostility show a positive direct effect on perceived threat from covid-19. as indirect effects observed, experiencing negative effect exerted a mediating role in the relationship between perceived threat and sadness-depression, anxiety and joy, negative in the last mood. there was no mediation by positive affect in any relationship. finally, in model 2, the proportion of explained variance of the predictor variable (perceived threat) is 39.4% (r 2 = 0.394). for each of the mediators, we found 32.6% (r 2 = 0.326) for positive affect and 55.4% (r 2 = 0.554) for negative. this study analyzed the cognitive and emotional (affective and mood) states of individuals in preventive quarantine. the results confirmed our starting hypothesis. first, they found that perceived threat from covid-19 was related positively to negative affect and emotional states, that is sadness-depression, anxiety and anger-hostility, while the relationship shown with positive affect and feeling of joy was negative. according to the literature, confinement can cause severe consequences for psychological wellbeing [13] , as negative feelings usually appear [8] . it is also an uncertain situation, when it is unclear where events will lead to, which causes high stress, and an increased perception of threat [16] . strong perception of threat for one's health can, in turn, cause an altered affective state and feelings of irritation, anxiety, despondency or sadness [9, 18] . similarly, differences were found in perceived threat from the pandemic according to the affective balance during the previous week of confinement. the analyses found two opposite groups, one framed by positive affect and the other by negative. the latter showed a higher mean score in perceived susceptibility to disease, which coincides with previous literature [5] . finally, a circular relationship, as also observed by other authors [17] , was found, in which perceived threat influenced the presence of negative mood, and negative mood, in turn, was linked to irritation and nervousness in the current situation promoted by the individual's feeling of threat. thus, perceived vulnerability to contagion increased the individual's perception of threat [7, 16] , which promoted negative mood [8, 13] , while emotions of hostility and distress, partly generated by the perception of threat from covid-19 itself and sudden confinement [14] , affected the cognitive state, increasing apprehension [18] . in both cases, the negative affective state mediated in this relationship. this study had some limitations which should be mentioned. first, variables such as age or sex, which have been shown to be determining in the evolution of the virus, were not taken into account. both sociodemographic variables could be closely related to perceived threat from covid-19. since most of the sample were women and the mean age has not been associated with high mortality, the model's generalization to the overall population would have to be tested. neither were covid-19 symptoms or diagnosis in the individual or in close friends and family, or perceived health condition considered, which could have affected the perception of threat from the disease. it should also be mentioned that in spite of the large number of studies on the variables dealt with here in highly stressful situations, there are no previous studies analyzing them together with mediation models, which in turn, decreases the possibility of comparing our results. in addition, it should be mentioned that the data were taken at the beginning of the quarantine, specifically, during the second week of confinement. thus, the results of this study should be taken with caution, since the emotions shown and perception of risk during this initial period may have changed over time as the confinement decree was extended and the virus spread. the current covid-19 health emergency has completely changed the daily life of the spanish population. both the confinement scenario and the spread of the virus, as well as associated consequences could cause alteration of people's cognitive and emotional state through perceived threat from the virus and development of negative affective balance and feelings. therefore, knowledge of the variables associated with the development of these alterations is fundamental to prevention and coping with confinement in similar populations and in the context following the pandemic, where recovery of psychological wellbeing will become a primary goal. the results of this study show how perceived threat is a risk variable for development of negative mood and vice versa, operating as a mediator in this circular relationship of negative affective balance, which increases both effects. as the most effective healthcare measure for reducing the incidence of the coronavirus pandemic is quarantine, and globalization and travel of the population facilitate the probability of similar situations occurring again, knowledge of the emotional and cognitive effects on the population could enable measures that facilitate their more effective coping to be put to use. the results of this study will be put into practice with the implementation of a psychoeducational program for working on the emotional state (emocovid). thus, in view of the affective and emotional alterations found during the quarantine and their relationship to perceived threat, it is intended to design a program to facilitate emotional management with daily activities related to knowledge, connection and management of emotions that could appear during quarantine. supporting information s1 checklist. strobe statement-checklist of items that should be included in reports of cross-sectional studies. (docx) s1 data. (sav) feasibility of controlling covid-19 outbreaks by isolation of cases and contact world health organization declares global emergency: a review of the 2019 novel coronavirus (covid-19) infection prevention and control in the management of patients with covid-19 cigularov kp worry as a mediator between psychosocial stressors and emotional sequelae: moderation by contrast avoidance communicating about infectious disease threats: insights from public health information officers factors influencing seasonal influenza vaccination behaviour among elderly people: a systematic review can hospitalization be hazardous to your health? a nosocomial based stress model for hospitalization the psychiatric impact of the novel coronavirus outbreak mental health care measures in response to the 2019 novel coronavirus outbreak in korea de 14 de marzo, por el que se declara el estado de alarma para la gestió n de la situació n de crisis sanitaria ocasionada por el covid-19 duration of quarantine in hospitalized patients with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection: a question needing an answer the 2019 novel coronavirus outbreak-a global threat immediate psychological responses and associated factors during the initial stage of the 2019 coronavirus disease (covid-19) epidemic among the general population in china the psychological impact of quarantine and how to reduce it: rapid review of the evidence resilience as a protective factor of chronic stress in teacher mechanisms of attentional biases towards threat in anxiety disorders: an integrative review attention bias towards negative emotional information and its relationship with daily worry in the context of acute stress: an eye-tracking study stress, health and social behavior burnout risk and protection factors in certified nursing aides burnout in health professionals according to their self-esteem, social support and empathy profile analysis of burnout predictors in nursing: risk and protective psychological factors optimism and academic burnout in university students cognitive processes of emotional regulation, burnout and work engagement the brief illness perception questionnaire psychometric properties of the questionnaire on threat perception of chronic illnesses in pediatric patients questionnaire on perception of threat from covid-19 on the independence of positive and negative affect the «affective balance scale». psychometric properties of an instrument to measure positive and negative affect in the spanish population [la «escala de balance afectivo». propiedades psicométricas de un instrumento para la medida del afecto positivo y negativo en población española an instrument to evaluate the efficacy of mood induction procedures: the "mood assessment scale" (evea) [un instrumento para evaluar la eficacia de los procedimientos de inducción de estados de á nimo: la "escala de valoració n del estado de á nimo bayesfactor 0.9.12-2. comprehensive r archive network lavaan: an r package for structural equation modeling and more. version 0.5-12 (beta) assessing mediational models: testing and interval estimation for indirect effects test theory: a unified approach the omega coefficient: an alternative method for estimating reliability the present study was undertaken in collaboration with excma. diputación provincial de almería. key: cord-291104-6chpmgry authors: leung, danny t. m.; lim, pak-leong; cheung, tak-hong; wong, raymond r. y.; yim, so-fan; ng, margaret h. l.; tam, frankie c. h.; chung, tony k. h.; wong, yick-fu title: osteopontin fragments with intact thrombin-sensitive site circulate in cervical cancer patients date: 2016-08-05 journal: plos one doi: 10.1371/journal.pone.0160412 sha: doc_id: 291104 cord_uid: 6chpmgry we investigated whether circulating osteopontin (opn) could be used as a biomarker for cervical cancer. we employed a monoclonal antibody (mab 659) specific for the unique and intact thrombin-sensitive site in opn using an inhibition elisa. we found significantly higher levels of opn in 33 cervical cancer patients in both the plasma (mean +/sd, 612 +/106 ng/ml) and serum (424 +/121 ng/ml) compared to healthy subjects [409 +/56 ng/ml, from 31 plasma samples (p < 0.0001), and 314 +/98 ng/ml, from 32 serum samples (p = 0.0002), respectively]. similar results were obtained when the plasma from a bigger group (147 individuals) of cervical cancer patients (560 +/211 ng/ml) were compared with the same plasma samples of the healthy individuals (p = 0.0014). more significantly, the opn level was highest in stage iii-iv disease (614 +/210 ng/ml, from 52 individuals; p = 0.0001) and least and non-discriminatory in stage i (473 +/110 ng/ml, from 40 individuals; p = 0.5318). no such discrimination was found when a mab of a different specificity (mab 446) was used in a similar inhibition elisa to compare the two groups in the first study; a commercial capture elisa also failed. the possibility that the target epitope recognized by the antibody probe in these assays was absent from the circulating opn due to protein truncation was supported by gel fractionation of the opn found in patients’ plasma: 60–64 kda fragments were found instead of the presumably full-length opn (68 kda) seen in healthy people. how these fragments are generated and what possible role they play in cancer biology remain interesting questions. osteopontin (opn), known previously as spp1 (secreted phosphoprotein 1) or eta-1 (early t-lymphocyte activation protein 1), was initially identified as an extracellular matrix protein produced by osteoclasts [1] . it is now considered to be a pleiotropic, pro-inflammatory purchased from r & d systems (minneapolis, mn) (cat. no.: 1433-op) and from sigma (st. louis, md) (cat. no.: o4264). thrombin and glutathione-coupled sepharose 4b beads were obtained from amersham biosciences (piscataway, nj). protein g-coupled sepharose 4b beads, protease inhibitor cocktail, pmsf, tmb, pma, fitc-conjugated goat anti-mouse ig antibodies, and n-hydroxy-sucinimidobiotin were obtained from sigma. bio-gels of various retention limit [p10 (20 kda), p30 (40 kda), p60 (60 kda), p100 (100 kda)] were obtained from bio-rad laboratories (berkeley, ca). human opn assay kit was obtained from immuno-biological laboratories (ibl) co. ltd., gunma, japan. pre-operative paired plasma and serum samples were obtained with consent from a first cohort of 33 patients diagnosed with cervical cancer [27 patients with squamous cell carcinoma, 6 patients with adenocarcinoma; ages ranging from 27-82 years (mean +/-sd = 52.3 +/-12.4 years)], and from a second cohort of 147 cervical cancer patients [40 patients with disease stage i, ages ranging from 39-80 years (mean +/-sd = 57.7 +/-12.2 years); 55 patients with disease stage ii, ages ranging from 31-90 years (mean +/-sd = 62.5 +/-15.5 years); 52 patients with disease stage iii-iv, ages ranging from 35-92 years (mean +/-sd = 64.5 +/-15.0 years)], from the department of obstetrics and gynecology, prince of wales hospital, hong kong. as controls, 31 plasma samples from healthy women (ages ranging from 19-92 years (mean +/-sd = 55.1 +/-21.6 years) and 32 serum samples (ages ranging from 14-86 years (mean +/-sd = 42.0 +/-16.9 years) sent for routine laboratory test were used. all samples were stored at -80°c before use. all the data were analyzed anonymously. this study was approved by the the joint chinese university of hong kong-new territories east cluster clinical research ethics committee. bacterially derived recombinant human opn of n-terminal and c-terminal half were generated as gst fusion proteins, opn-o-gst and opn-p-gst, respectively, in escherichia coli bl21 using the pgex-2t expression vector (amersham biosciences) as previously described [33] . cdna obtained from hep-2 cells was pcr-amplified to generate dna fragments using the following forward (f) primers and reverse (r) primers: opn-o [(aa 1-175), f, 5'-cgtgg atccatgagaattgcagtgatttgc-3'(containing a bamh1 site, underlined); r, 5'-c gatgaattccgcgaaacttcttagattttga-3' (containing an ecor1 site, underlined)], and opn-p [(aa 169-314), f, 5'-cgtggatcctcaaaatctaagaagtttcgc-3' (containing a bamh1 site, underlined); r, 5'-aattcccggggattaattgacctcagaa gatgc-3' (containing a smai site, underlined)] and subcloned into the pgex-2t vector. the fusion proteins derived from the above constructs were induced with isopropyl-β-d-thiogalactopyranoside, purified by affinity chromatography, and examined on sds-page by commassie blue staining and wb. insect-cell derived recombinant human opn were generated as v5 peptide-tagged (v5) fusion proteins (opn-o-v5 or opn-p-v5) in sf9 using the baculodirect baculovirus expression system according to manufacturer's instruction (invitrogen). cdna obtained from hep-2 cells was pcr-amplified to generate dna fragments using the following forward (f) primers (containing a kozak sequence, underlined) and reverse (r) primers: opn-o (f, 5'-ccaccat gggaatgagaattgcagtgatttgc-3'; r, 5'-gcgaaacttcttagattttga-3'), and opn-p (f, 5'-ccaccatgggatcaaaatctaagaagtttcgc-3'; r, 5'-attgac ctcagaagatgcact-3') and subcloned into the entry vector pcr8/gw/topo (invitrogen). purified plasmid with insert of right orientation was recombined with the baculodirect linear dna by incubation with lr clonase overnight at room temperature. sf9 cells growing at log-phase were transfected with the recombined dna mixture by cellfectin (invitrogen) and selected with 100 mm ganciclovir. low titer of virus preparation (p1 viral stock) bearing the opn-o or opn-p gene constructs were harvested from the spent culture supernatant at day 14-21 post-transfection under ganciclovir selection, where less than 10% of sf9 cells was infected as revealed by anti-v5 antibody staining in ifa. high titer virus preparation (p2 viral stock) was harvested from sf9 cells inoculated with an aliquot of p1 viral stock for 5-7 days under ganciclovir selection where 50-80% of cells were infected. fusion proteins were obtained from lysates of sf9 cells incubated with p2 viral stock without selection (72 hr at 27°c) where almost 100% of cells were infected at the time of harvest as revealed by anti-v5 antibody staining. cytosolic extracts were prepared by incubating the infected sf9 cells with lysis buffer (1% np-40, 150 mm nacl, 10 mm tris, ph 7.5, 1x protease inhibitor, 1 mm pmsf) at 4°c for 30 min, followed by centrifugation (14,000 rpm, 10 min, 4°c) the culture supernatant of hl60 (5 x 10 6 /ml) cells with or without treatment with pma (ng/ml) for 72 hr was harvested and concentrated 1:10 the original volume by centrifugation at 13,000 rpm for 20 min at 4°c (centricon ym-10, 10 kda cutoff; millipore, billerica, ma). samples were stored at -80°c before analysis. balb/c mice were hyperimmunized with purified bacterially-derived opn-o-gst or opn-p-gst protein, and spleen cells obtained from these animals were fused with ns0 myeloma cells as described [34] . hybridomas obtained were screened for reactivity against insect cell-derived recombinant proteins (opn-o-v5 or opn-p-v5) by direct-binding elisa; positive hybridomas were further characterized by ifa and wb. monoclonal antibodies from selected clones (mab 659, mab 446, mab 492) were obtained from the spent-culture supernatant or ascites fluid and purified by ammonia sulfate precipitation. in some cases, the antibodies were protein-g selected and biotinylated using n-hydroxy-sucinimidobiotin. opn was detected by determining the ability of the test sample (human serum or plasma, or hl60-derived culture supernatant) to block the binding of the opn-specific indicator antibody (mab 659, mab 446 or mab 492) to the insolubilized antigen [bacterially-derived opn-o-gst (for mabs 659 and 446) or opn-p-gst (for mab 492); ns0-derived fulllength opn]. the concentration of indicator antibody used was determined previously by titration of the antibody against the respective antigen, based on 70% od max . thus, wells of 96-well elisa plates (immunlon ii; dynatech, chantilly, va) were coated with the different antigens (1 μg/ml) at 4°c using bicarbonate buffer (ph 9.6). fifty μl of diluted sample (for plasma and serum, dilution ratio = 1:16.6) were mixed with 50 μl of diluted indicator antibody, and incubated with the immobilized antigen for 16 hr at 4°c. after washing, bound indicator antibody was detected using hrp-conjugated goat anti-mouse ig (1:1000) (1.5 hr at 37°c) and tmb, and the results read at od 450 nm in an elisa reader. a calibration curve was constructed for each experiment using 8 serial dilutions of full-length opn (5 to 625 ng/ml). this was employed to determine whether mab 659 and mab 446 shared a common binding site in opn. thus, 50 μl of serially diluted mab 659 (competitor) were mixed with 50 μl of biotin-coupled mab 446 of a pre-determined concentration and incubated with immobilized opn-o-gst at 4°c overnight. cold (unbiotinylated) mab 446 was used as positive control. following washing, bound indicator antibody was detected by incubation with 100 μl hrpconjugated streptavidin (1:1000) for 1.5 hr at 37°c, and the reaction developed using tmb; the results were read at od 450 nm. binding specificity was further confirmed by a reverse assay, using mab 446 as competitor, and biotin-coupled mab 659 as indicator. cytospin preparations of sf9 cells expressing opn-o-or opn-p-v5 protein were methanolfixed, blocked with 2.5% bsa and stained with the reagent mab for 30 min at room temperature. following incubation with fitc-conjugated goat anti-mouse ig antibodies (1:40) for 30 min at room temperature, the cells were examined by uv microscopy [35] . anti-v5 antibody (invitrogen) was used as a positive control. recombinant proteins or sf9 cell lysates were treated in reducing sample buffer at 95°c for 5 min, resolved on 10-12.5% sds-page gel, and then transferred to pvdf membranes. the membranes were blocked with 2.5% bsa and later incubated with the detecting mab (mab 659 or mab 446) at 4°c overnight; following this, the membrane was incubated with hrp-conjugated goat anti-mouse ig antibodies for 2 hr at room temperature. membranes were then thoroughly washed and used for film development by ecl chemoluminescence (amersham biosciences). full-length opn (25 ng) was incubated with graded doses of thrombin (5, 2.5, 1.25, 0.63, 0.31, and 0.16 u/ml) in tris-buffer, ph 8.0 at 37°c for 1 hr. after heating with reducing sample buffer at 95°c for 5 min, the samples were resolved on 10-12.5% sds-page gel, transferred to pvdf membranes, and examined by wb. in the mab-protection study, opn (25 ng) was preincubated with graded doses of mab 659 in tris-buffer, ph 8.0, at 4°c overnight, followed by incubation with 5 u/ml thrombin at 37°c for 1 hr. samples were then treated with reducing sample buffer at 95°c for 5 min, resolved on 10-12.5% sds-page gel, transferred to pvdf membranes, and examined by wb. mini-columns (1 ml bed-size) were prepared using bio-gel (bio-rad laboratories, berkeley, ca) of different retention limits: p10 (20 kda), p30 (40 kda), p60 (60 kda), p100 (100 kda). these were thoroughly equilibrated with pbs (100 μl) by repeated (5x) loading and subsequent centrifugation (200 x g, 2 min). each column was first calibrated using blue dextran and dnplysine. in the experiment, 100 μl human plasma was loaded to each column and centrifuged (200 x g, 2 min). the eluate (100 μl) was collected and the column was loaded with pbs (100 μl) again, centrifuged and the eluate collected. the procedure of pbs-loading was repeated, so that, for each sample, 10 such eluates (fractions) were obtained. the opn concentration in each fraction was determined using the mab 659 inhibition elisa. as a control, bovine serum albumin (2 mg/ml) was also run through each column using the same procedure, but the protein content in the fractions was determined using the bicinchoninic acid (bca) protein assay kit (pierce, rockford). microdissection of the tumor tissue, rna extraction from the processed tissue, as well as reverse transcription and real-time pcr of the extracted rna, were all performed as described [24] . comparison of difference between two groups was performed using the unpaired t-test. correlation between groups of data was performed using regression analysis, and a p-value of <0.05 was considered statistically significant. variable data were expressed by scatterplot analysis (graphpad prism 3, graphpad software, inc., san diego, ca). recombinant opn proteins were produced in e. coli from dna cloned from hep-2 cells. thus, two gst-linked opn fragments were obtained, one representing roughly the n-terminal half (opn-o) and the other, the c-terminal half (opn-p) of the protein (fig 1a and 1b) . corresponding proteins of these fragments (opn-o-v5, opn-p-v5) were also expressed in insect cells ( fig 1c) . ten hybridomas were produced from mice immunized against opn-o or opn-p. of the clones that bound well to bacterial opn-o, mab 659 and mab 446 are igg antibodies which recognized linear epitopes (elisa + , ifa + , and wb + ), while mab 163 is also an igg but it recognized a conformational epitope (elisa + , ifa + , wb -). all the opn-p-specific antibodies (mab 138, mab 403, mab 454, mab 492) are igms which bound only to linear epitopes. mab 659, mab 446 and mab 492 bound specifically in an elisa to the recombinant opn antigen derived from both bacteria and insect cells (fig 2a and 2b) ; the binding was comparable to that for the full-length opn obtained commercially ( fig 2b) . mab 659 and mab 446 bound to different sites in opn-o since they did not cross-inhibit each other ( fig 2c) . with all three mabs, specificity of binding was also demonstrated by wb analysis ( fig 2d) and immunofluorescence-staining of insect cells transfected with the respective opn genes ( fig 2e) . we discovered, by chance, that mab 659 recognized the evolutionary-conserved and unique thrombin-sensitive site (arg 168 -ser 169 ) in opn (see fig 1a) . first, it is known that digestion of opn by thrombin produces two fragments of roughly equal size. accordingly, we incubated intact opn (obtained commercially) with increasing amounts of thrombin and monitored the result using mab 659 in an elisa to detect how much intact opn remained: increasing degradation was observed with increasing enzyme (fig 3a) . in contrast, this effect was not observable when probed with mab 446 (fig 3a) . secondly, using mab 659 as probe, full-length opn could be observed as a 72 kda band in western blots; this disappeared completely when the protein was pre-treated with 5 u/ml thrombin (fig 3b, upper panel) , while smaller amounts of thrombin had little or no effect. when mab 446 was used to probe the same blot where 5 u/ ml thrombin had been used, there was also significant reduction in the amount of full-length opn but for reasons unclear, this did not disappeared completely; more importantly, an additional antigen of 35 kda appeared (fig 3b, lower panel) . thirdly, mab 659 was able to protect opn from thrombin digestion in a dose-dependent fashion (fig 3c) . concentration of detecting antibody used was pre-determined from the titration curves of these mabs (data not shown), chosen at 70% maximal binding (fig 2b) . full-length opn obtained commercially or produced from mouse ns0 cells as histidine 6 -tagged proteins, was used as the standard. assay sensitivity was determined as the concentration of full-length opn (ng/ml) required to inhibit the maximal antibody binding by 50%. the sensitivities obtained for the different assays are shown in fig 4a. of particular note is the excellent detection by mab 659 (25 ng/ml) when opn-o-gst was used as substrate, compared to the poor detection by mab 446 (200 ng/ml); both antibodies detected the commercially-obtained full-length opn very poorly (200 ng/ml). detection by mab 492 was extremely poor with all opn substrates. inhibition elisas were developed using either mab 659 or mab 446 as the reagent antibody and bacterially-produced opn-o-gst as the substrate. both assays efficiently and specifically detected the appropriate opn produced from insect cells or mouse ns0 cells (fig 4b) . interestingly, however, mab 659 was 2.7 fold more sensitive than mab 446 in detecting the opn produced by hl60 cells (fig 4c) . we next used the mab 659 opn-o-gst inhibition elisa to detect opn from the plasma of 33 cervical cancer patients [26 squamous cell carcinoma, 5 adenocarcinoma, 2 cervical intraepithelial neoplasia (cin) (grade iii)]. we found highly significant (p < 0.0001) levels of opn (mean +/-sd, 612 +/-106 ng/ml) in these 33 patients compared to 31 healthy subjects (409 +/-56 ng/ml) (fig 5a) . based on sample size, this discrimination is highly significant (minimum no. of patients and control required for 99% confidence at 0.99 power is 30 and 12, respectively). the assay sensitivity was 82%, and the specificity, 100%. similar results were found when the serum of these individuals was examined: the cancer patients (424 +/-121 ng/ ml) had significantly (p = 0.0002) higher levels of opn than healthy subjects (314 +/-98 ng/ ml) (fig 5b) , the assay sensitivity and specificity being 18% and 97%, respectively. there is, in fact, good correlation (r 2 = 0.71, p < 0.0001) between the plasma and serum opn levels in the cervical cancer patients (fig 5c) , even though the serum levels were significantly lower than the corresponding plasma levels. however, based on sample size, the discrimination between patients and control is not robust (minimum no. of patients and control required for 85% confidence and 0.8 power is 42 and 28, respectively). for comparison, when mab 446 was used in the inhibition based on plasma, there was no discrimination between the cancer and healthy groups (fig 5d) . this was also the case using a correlation between the small number of patients found positive by this kit or the mab 446 inhibition elisa and the corresponding patients in the mab 659 inhibition elisa (fig 5d and 5e) . we extended the mab 659 inhibition elisa study using more cervical cancer patients and plasma only, this time identifying the stage of the disease as well in order to make a more precise correlation between opn level and disease. thus, again, there was excellent discrimination (p = 0.0014) between the new cohort of 147 patients (560 +/-211 ng/ml) and the 29 healthy subjects (mean 454 +/-142 ng/ml) (fig 5f) . more interestingly, if the patients are subdivided according to the stage of the disease (fig 5f) , discrimination was seen in stage ii (571 +/-249 ng/ml; p = 0.0072) and, more pronouncedly, in stage iii-iv (614 +/-210 ng/ml; p = 0.0001), but not stage i (473 +/-110 ng/ml; p = 0.5318). tumor tissues were available from 20 patients (4 in stage i, 6 in stage ii, 10 in stage i) in this cohort which were used to determine the opn gene expression. as shown in fig 5g, there was good concordance (r 2 = 0.4120, p = 0.0023) between the plasma opn results and the opn gene expression results, thus validating the immunological detection. the finding that the mab 446 inhibition elisa and the commercial opn kit could not detect elevated opn levels in the cancer patients suggested the possibility that the opn present could be fragmented i.e. the target sites for the antibodies used in these assays could be missing, whereas, by virtue of the design of the mab 659-based assay, the thrombin-sensitive site must be present. thus, we fractionated the plasma of cancer patients by gel chromatography using a small but long, thin column made of bio-gel of different pore sizes (p10, p30, p60, p100). serial 0.1 ml fractions were collected and assayed for opn activity using the mab 659 inhibition elisa. each column was pre-calibrated with blue dextran, dnp-lysine and bsa [a protein of similar molecular size (66.4 kda) to intact opn (around 70 kda)]. as shown in fig 6a, bsa was totally excluded in p10 (exclusion limit, 20 kda) and p30 (40 kda); in both cases, the bulk (60%) of the protein appeared in fraction 2 and the rest in fraction 1. in the p60 (60 kda) and p100 (100 kda) elution, however, bsa was not excluded but instead appeared mainly (58%) in fraction 3 and in smaller amounts in fraction 4, but none thereafter. when the plasma pooled from four cervical cancer patients (selected on the basis of high opn activity) was similarly fractionated, only in p10 was opn totally excluded, with the bulk of the activity (82%) appearing in fraction 2 ( fig 6a) . significantly, in p30, the main opn activity (67%) shifted to fraction 3. in the p60 and p100 elution, however, very little activity was recovered from this fraction (fraction 3); instead, there was a spread of activity from fraction 3 to fraction 8, peaking at fraction 4 (p100) or fraction 5 (p60). collectively, the findings suggest that, in the cancer patients, opn exists not as an intact protein but rather as fragments of between 40 kda and 60 kda in size. interestingly, when the plasma pooled from 4 healthy individuals was fractionated as above, the overall fractionation profiles obtained (fig 6a) were slightly different from those of the cancer patients. thus, total exclusion of opn activity was observed with both p10 and p30, and the spread of activity in the included fractions of p60 and p100 was more limited (confined to fractions 3-5). collectively, this suggests a protein size very similar to that of bsa (60-100 kda). we performed western blotting of the p60 fractions obtained from both patients and control subjects (fig 6b) using mab 659 as probe. the results are consistent with those of the elisa detection. significantly, in the patients, no activity was detected from fractions 2 and 3, but fraction 4 revealed an antigen of about 64 kda in size that was present in small amounts. fraction 5 contained a marginally-smaller antigen (62 kda) in significantly greater abundance, while the antigen found in modest amounts in fraction 6 was in turn marginally smaller in size (60 kda). fraction 7 had no activity. in contrast, by western blotting, in healthy subjects, the antigenic activity appeared earlier, in fraction 3 (close to the void volume of the column), which forms the bulk of the activity from the sample, while the molecular size of this antigen (roughly 68 kda) is bigger than those of the fragments found in patients. a smaller antigen of about 60 kda was found in smaller quantities from the subsequent fraction (fraction 4) while no activity was observed thereafter. we sought to answer an important question in cancer diagnosis: is opn present at diagnostic levels in the blood of cervical cancer patients? we found convincing evidence proving this. however, this was only true because we had used a very unique mab in a highly-sensitive but not-so-common detection system. this mouse antibody (mab 659) is highly specific for the unique thrombin-sensitive site in opn, and opn is efficiently detected from a patient by its ability to block the binding between this antibody and the target antigen. thus, although healthy people were found to have nominal amounts of opn in their blood in the assay, cancer patients showed significantly higher levels depending on several factors. this discrimination was found in two separate cohorts of cervical cancer patients. plasma samples gave better results than serum in this assay, the reason being the fact that, as the blood clots to form serum, thrombin is activated which can digest the opn in the sample. presumably the conditions were not met for complete degradation of the protein. validity of the plasma mab 659 inhibition elisa results is shown not only by the good correlation between these results and those of the corresponding serum samples, but also between these and the opn gene expression results. further validation is seen when the patients are re-grouped according to the stage of the disease: the opn levels found by the elisa increased with the severity of the disease. thus, the most severe of these, stage iii-iv, showed the highest level of antigen, while at the other extreme, the least severe, stage i, could not be distinguished from healthy individuals. indeed, the ability to detect stage i patients possibly poses the greatest challenge to all of immunological assays. the most important revelation of the study which sets it apart from past publications is the finding that opn circulates in our patients not as an intact protein but rather, as fragments of about 60-64 kda in size. these opn fragments, which by inference must bear the intact thrombin-sensitive site, are probably truncated somewhere at the n-terminal end of the protein based on the observation that two immunoassays used in parallel with the mab 659 inhibition elisa had failed to detect similar opn increases in the cancer patients: (1) an inhibition elisa which uses mab 446 as the detecting reagent known to bind to the n-terminal half of opn (actual location unknown). (2) a commercially-available capture elisa (human opn detection kit, ibl) which employed polyclonal antibodies directed at two sites in opn separated by 138 amino-acids, one of which ( 17 ipvkqadsgsseekq 31 ) is situated at the n-terminal end of the protein (fig 1) . since this capture assay detects ns0-derived full-length opn as efficiently as the mab 659 inhibition elisa, a likely reason for its failure is the absence of the n-terminal site in the patient's opn. the fact that this commercial kit was successfully used with other types of cancer previously [26] suggests that opn might be cleaved differently (by different enzymes) in different types of cancer or in different individuals. there are many opn detection kits in the market but they all invariably use capture (sandwich) elisa. they can be different, however, in the pair of antibodies utilized not only in terms of fine specificity but also whether these are polyclonal or monoclonal in nature; most polyclonal antibodies target a very small peptide-segment of the protein. differences in the antibody pairs used can affect the type of opn fragment detected and hence the vast discordances among test kits for the same set of plasma samples [32] . the inhibition elisa we developed is the first of its kind for opn detection. a distinct advantage of this format is the fact that the target site needs only be present in fragments or peptides as small as the site itself. it is instructive that at the start of our study, we had actually experimented with pairs of mabs (e.g. biotinylated-mab 659 and -mab 446) in capture eli-sas to detect plasma opn from patients, but none had succeeded. in general, inhibition assays are not common, but one which has proven efficacious in detecting typhoid fever is based on a rapid dual-particle system ('tubex') to detect a single specificity of antibody [36] . the opn fragments were indirectly identified from pooled patient plasma by gel filtration. although the results are preliminary and need to be verified more robustly in terms of sample size and methodology, they nevertheless merit some discussion. to compensate for the imprecision of the fractionation, the bio-gel columns were carefully calibrated using known markers and the results were based on a comparison of the plasma samples between the cancer and healthy groups run under identical conditions. against this background, a notable difference in the p30 elution profiles between the two groups was indeed found: whereas in healthy people the bulk of the opn antigenic activity appeared in the void fraction (fraction 2), in the cancer group, this appeared later (fraction 3)-suggesting an antigen smaller in size i.e. cleaved. support for this is seen when the p60 or p100 elution profiles between these groups are compared. thus, in the case of p60, whereas in healthy people the main antigenic activity was found in fraction 3, this appeared much later in fraction 5 in the cancer group, and there was a greater spread of activity. western blot analysis performed on the p60 fractions using the same antibody probe (mab 659) confirmed the elisa findings. thus, the main antigenic activity was found in fractions 3 and 5 from the healthy and cancer groups, respectively. more instructively, the antigen found in patients was significantly smaller (about 62 kda) than that observed in healthy people (about 68 kda). it seems likely the latter is the intact, full-length opn even though it is slightly smaller than the intact opn (72 kda) produced by hl60 cells used early in our study-this difference could be due to post-translational modification (see later). thus, the 62 kda antigen found in patients is not intact but a major fragment. interestingly, this fragment was also observed in healthy people as a minor component. in the patients too, other fragments (64 kda in fraction 4 and 60 kda in fraction 6) were also found in smaller quantities. it is possible that these three antigens are actually the same fragment but have varying degrees of posttranslational modification. such modifications can increase the molecular weight of the protein considerably. this is highly possible with opn because although the protein is only roughly half as long (amino-acid) as bsa, it nevertheless has a very similar molecular weight. indeed, opn is known to be extensively phosphorylated because of which it is highly active biologically [37, 38] . there are indeed many potential sites in the protein both for phosphorylation (about 36 sites) and for glycosylation (5 for o-glycosylation, 2 for n-glycosylation) [39] [40] [41] . in addition, the protein also undergoes sulfation and transglutamination. a possible candidate for the antigen found in the patients is the major fragment cleaved from whole opn by caspase-8. this enzyme cleaves opn at two sites, asp135 and asp 157 (fig 1) , yielding two major peptides of 180 and 160 amino-acid long, respectively, but both could yield molecular weights greater than 40 kda [15] depending on the extent of post-translational modification. while these glycan or phosphate appendages mattered importantly to the linearized antigen in western blotting by way of increasing the molecular weight, they do not seem to affect the molecular size of the free-form antigen in solution. thus, in the p30 gel chromatography, the opn antigen appeared more like a globular protein with a molecular weight of less than 30-40 kda (i.e. a peptide with fewer than 270 amino-acids) than a 60 kda antigen as deduced by western blotting. the possibility that this antigen could be retarded in its passage through the gel due to interaction between the appended phosphate or carbohydrate groups and the bio-gel resin is belittled by the observation that the opn antigen from healthy people seemed to be unaffected. the surprising finding is the absence of intact opn in our patients. the argument that this could be present but is bound at the thrombin-sensitive site by a co-factor such as syndecan-4 [42] or factor h [43] and becomes masked as such, is ruled unlikely by the western blot results. this is because any factor bound to opn would have dissociated from it under the denaturing conditions used. the question arises whether the opn fragments found in our patients play a role in the cancer biology of the patient. on the one hand, this seems unlikely because the same fragment found in patients was also present in healthy people. indeed, the greater abundance of this fragment in patients may simply reflect the general heightened activity of tumor cells-not only more opn is produced, but also very quickly it becomes cleaved by the caspases and other enzymes in the tissue with the end result that vast amounts of opn fragments are generated and released to the circulation. it is not clear, however, how intact opn produced by other (non-cancerous) tissues becomes fragmented in our patients. on the other hand, there are numerous reports which described the potent biological activities of opn fragments. this includes the opn fragments generated by caspase-8, which bear the rgd domain (fig 1) that was recently shown to promote tumor growth and metastasis [44] . another opn fragment which could remotely fit as the candidate antigen in our patients is the 32 kda c-terminal peptide generated by mmp-3, mmp-7 or mmp-9 (see fig 1) [45] ; this, again, could be bigger in molecular weight if it becomes glycosylated or phosphorylated. the tumorigenic potential of this fragment is not known but recently, takafuji et al. [17] found a small opn fragment (residues 167-210) generated by mmp-9 which seemed able to induce tumor cell invasion via cd44 receptors in hepatocellular carcinoma [46] . thus, the opn fragments which circulate in our cancer patients and which bear both the rgd domain and an intact thrombin-sensitive site, may be more important than just a diagnostic (or prognostic) biomarker-they could very well determine the biology of the cancer. cloning and sequence analysis of rat bone sialoprotein (osteopontin) cdna reveals an arg-gly-asp cell-binding sequence osteopontin: role in immune regulation and stress responses small integrin-binding ligand n-linked glycoproteins (siblings): multifunctional proteins in cancer autocrine activation of an osteopontin-cd44-rac pathway enhances invasion and transformation by h-rasv12 osteopontin: regulation in tumor metastasis osteopontin as a means to cope with environmental insults: regulation of inflammation, tissue remodeling, and cell survival the influence of the proinflammatory cytokine, osteopontin, on autoimmune demyelinating disease osteopontin deficiency protects joints against destruction in anti-type ii collagen antibody-induced arthritis in mice the bridge between dendritic cells and asthma systemic endocrine instigation of indolent tumor growth requires osteopontin osteopontin is a marker for cancer aggressiveness and patient survival lentiviral-mediated mirna against osteopontin suppresses tumor growth and metastasis of human hepatocellular carcinoma osteopontin expression is essential for interferon-alpha production by plasmacytoid dendritic cells intracellular cleavage of osteopontin by caspase-8 modulates hypoxia/reoxygenation cell death through p53 an osteopontin splice variant induces anchorage independence in human breast cancer cells an osteopontin fragment is essential for tumor cell invasion in hepatocellular carcinoma osteopontin-c is a selective marker of breast cancer role of the integrin-binding protein osteopontin in lymphatic metastasis of breast cancer osteopontin overexpression in breast cancer: knowledge gained and possible implications for clinical management osteopontin expression in lung cancer correlation of osteopontin protein expression and pathological stage across a wide variety of tumor histologies osteopontin expression in ovarian carcinomas and tumors of low malignant potential (lmp) genome-wide gene expression profiling of cervical cancer in hong kong women by oligonucleotide microarray osteopontin expression correlates with invasiveness in cervical cancer clinical significance of osteopontin expression in cervical cancer clinical implications of osteopontin in metastatic lesions of uterine cervical cancers plasma osteopontin as a biomarker of prostate cancer aggression: relationship to risk category and treatment response post-operative plasma osteopontin predicts distant metastasis in human colorectal cancer identification of osteopontin as a prognostic plasma marker for head and neck squamous cell carcinomas prognostic significance of plasma osteopontin in patients with locoregionally advanced head and neck squamous cell carcinoma treated on trog 02.02 phase iii trial new dual monoclonal elisa for measuring plasma osteopontin as a biomarker associated with survival in prostate cancer: clinical validation and comparison of multiple elisas antibody response of patients with severe acute respiratory syndrome (sars) targets the viral nucleocapsid molecular mimicry: anti-dna antibodies may arise inadvertently as a response to antibodies generated to microorganisms extremely low exposure of a community to severe acute respiratory syndrome coronavirus: false seropositivity due to use of bacterially derived antigens combined rapid (tubex) test for typhoid-paratyphoid a fever based on strong anti-o12 response: design and critical assessment of sensitivity regulation of vascular calcification: roles of phosphate and osteopontin control of osteopontin signaling and function by posttranslational phosphorylation and protein folding post-translationally modified residues of native human osteopontin are located in clusters: identification of 36 phosphorylation and five o-glycosylation sites and their biological implications cell type-specific post-translational modifications of mouse osteopontin are associated with different adhesive properties post-translational modification and proteolytic processing of urinary osteopontin syndecan-4 protects against osteopontin-mediated acute hepatic injury by masking functional domains of osteopontin factor h binding to bone sialoprotein and osteopontin enables tumor cell evasion of complement-mediated attack the rgd domain of human osteopontin promotes tumor growth and metastasis through activation of survival pathways osteopontin, a novel substrate for matrix metalloproteinase-3 (stromelysin-1) and matrix metalloproteinase-7 (matrilysin) activation of the osteopontin/matrix metalloproteinase-9 pathway correlates with prostate cancer progression we thank mr. man-hin leung for technical assistance. this work was supported in part by a direct grant from the chinese university of hong kong to yfw. designed assays/experiments: pll dtml. key: cord-295339-nzc47itk authors: baker, marissa g.; peckham, trevor k.; seixas, noah s. title: estimating the burden of united states workers exposed to infection or disease: a key factor in containing risk of covid-19 infection date: 2020-04-28 journal: plos one doi: 10.1371/journal.pone.0232452 sha: doc_id: 295339 cord_uid: nzc47itk introduction: with the global spread of covid-19, there is a compelling public health interest in quantifying who is at increased risk of contracting disease. occupational characteristics, such as interfacing with the public and being in close quarters with other workers, not only put workers at high risk for disease, but also make them a nexus of disease transmission to the community. this can further be exacerbated through presenteeism, the term used to describe the act of coming to work despite being symptomatic for disease. quantifying the number of workers who are frequently exposed to infection and disease in the workplace, and understanding which occupational groups they represent, can help to prompt public health risk response and management for covid-19 in the workplace, and subsequent infectious disease outbreaks. methods: to estimate the number of united states workers frequently exposed to infection and disease in the workplace, national employment data (by standard occupational classification) maintained by the bureau of labor statistics (bls) was merged with a bls o*net survey measure reporting how frequently workers in each occupation are exposed to infection or disease at work. this allowed us to estimate the number of united states workers, across all occupations, exposed to disease or infection at work more than once a month. results: based on our analyses, approximately 10% (14.4 m) of united states workers are employed in occupations where exposure to disease or infection occurs at least once per week. approximately 18.4% (26.7 m) of all united states workers are employed in occupations where exposure to disease or infection occurs at least once per month. while the majority of exposed workers are employed in healthcare sectors, other occupational sectors also have high proportions of exposed workers. these include protective service occupations (e.g. police officers, correctional officers, firefighters), office and administrative support occupations (e.g. couriers and messengers, patient service representatives), education occupations (e.g. preschool and daycare teachers), community and social services occupations (community health workers, social workers, counselors), and even construction and extraction occupations (e.g. plumbers, septic tank installers, elevator repair). conclusions: the large number of persons employed in occupations with frequent exposure to infection and disease underscore the importance of all workplaces developing risk response plans for covid-19. given the proportion of the united states workforce exposed to disease or infection at work, this analysis also serves as an important reminder that the workplace is a key locus for public health interventions, which could protect both workers and the communities they serve. to estimate the number of united states workers frequently exposed to infection and disease in the workplace, national employment data (by standard occupational classification) maintained by the bureau of labor statistics (bls) was merged with a bls o*net survey measure reporting how frequently workers in each occupation are exposed to infection or disease at work. this allowed us to estimate the number of united states workers, across all occupations, exposed to disease or infection at work more than once a month. based on our analyses, approximately 10% (14.4 m) of united states workers are employed in occupations where exposure to disease or infection occurs at least once per week. approximately 18.4% (26.7 m) of all united states workers are employed in occupations where exposure to disease or infection occurs at least once per month. while the majority of exposed workers are employed in healthcare sectors, other occupational sectors also have high proportions of exposed workers. these include protective service occupations (e.g. police officers, correctional officers, firefighters), office and administrative support as covid-19 spreads globally, there is public health importance in characterizing the role of the workplace in disease transmission, given the variety of work tasks that could promote the spread of infectious disease (e.g., interfacing with customers, patients, and co-workers; preparing food), and the role of the workplace in spreading previous epidemics or pandemics [1, 2] . it is known that those working in healthcare settings face increased exposure to agents causing infectious diseases such as sars-cov-2, but may also have better infectious disease protection plans and policies than other occupational settings, potentially limiting the transmission of disease to community members [3] . while important, these measures may be inadequate for the effective prevention of infection for such high risk occupations, especially when they are working with inadequate ppe stockpiles, and the hospitals are overwhelmed due to heavy patient loads [4] . nearly 4% of confirmed covid-19 cases in wuhan, china (as of february 11, 2020) were in healthcare workers, indicating the workplace is a potential location of transmission even among workers who are trained to protect themselves from biological hazards [5] . however, other occupational groups which may have more sporadic exposure to infectious or disease-causing agents may not have the same level of planning, or even think that an infection disease control plan is warranted for their workplace. of the first 25 covid-19 cases confirmed in singapore, 17 had probable relation to occupational exposure, including workers in retail stores and casinos, domestic workers, a tour guide, taxi and private hire car drivers, security guards, and workers at the same construction site, further exemplifying the role of the workplace in transmitting disease [6] . understanding the burden of occupational exposure to infection and disease, including how many workers are potentially exposed and what occupations they work in, allows for upstream prevention measures, both at the workplace (e.g. developing appropriate infectious disease response plans, integrating infectious disease trainings into other workplace trainings, developing workplace policies that can support a workforce potentially exposed to sars-cov-2) and regulatory levels (e.g. increased access to paid sick leave, hazard pay for those exposed during a pandemic, etc.). these workplace and regulatory policies will be valuable in helping reduce the transmission of infectious disease from and within the workplace, and their importance may be realized with burden estimates previously, state-level employment data were utilized to estimate the number of workers exposed to a host of occupational exposures in united states federal region x (washington, oregon, idaho, alaska), spanning chemical, physical, ergonomic, and psychosocial hazards [7] . here, utilizing the same data analysis methods as previously detailed in doubleday et al., the number of workers across the united states exposed to disease or infection at work more than once a month is estimated. despite some of the inherent limitations in using these existing data sources, we believe this analysis is valuable for informing risk assessments and prompting protective actions that occupational sectors and regulatory agencies can take during infectious disease outbreaks, such as covid-19. two sources of data were utilized for this analysis, and are detailed below. united states employment data was obtained from the bureau of labor statistics (bls) occupational employment statistics database [8] . the most current employment data at the time of analysis was from may 2018, and is organized by 2010 standard occupational classification codes (2010 soc). soc codes are hierarchical, ranging from two-digits (major group code) to six digits (detailed occupation code), with the six-digit codes being the most detailed [9] . to estimate exposure to disease and infection in the workplace, we used data within the o � net database. o � net is a job characterization tool, generated from survey data, with rich information on tasks performed, skills needed, and job characteristics for different occupations, in order to inform job seekers or researchers [10]. as nearly 600 six-digit soc occupations are updated each year, the entire o � net database is completely refreshed every few years [11] . between 2001 and 2011, nearly 160,000 employees from 125,000 workplaces had responded to o � net questionnaires. o � net uses a deliberate survey sampling scheme, to ensure representation of workers from across the united states, across organizations of different size, and from both government and private workers. for small socs where it may be hard to find respondents, and to complement data from job incumbents, o-net also relies on occupational analysts and occupational experts to answer questionnaires. o � net does not collect data from military occupations; thus, soc codes beginning with 55 "military specific occupations" are not included in o � net data. similarly, employment numbers for "military specific occupations" is not reported in the bls occupational employment statistics database. no other soc codes are excluded from the o � net database, but two soc codes were not included in the measure utilized for this analysis, which were for the occupations of "rock splitters, quarry" and "timing device assemblers and adjusters" employing 4,870 and 780 persons in the united states, respectively [12] . to characterize frequency of workplace exposure to infectious disease, we used the following o � net question: "how often does your current job require you be exposed to diseases or infections?" respondents, who take the survey online or on paper, could select from the following frequencies of exposure: never; once a year or more but not every month; once a month or more but not every week; once a week or more but not every day; every day [13] . respondents are given little context when completing the survey, with interpretation of the question up to the respondent. within o � net, these data are converted to a 0-100 score, representing weighted-average frequency of the metric for each soc code. for this analysis, occupations were retained that had a score of 50-100, representing exposure to disease/infection more than once a month. soc codes were merged with the national employment data to calculate the total number of workers employed in the occupations with exposure to disease/ infection at more than once a month. all data analysis was conducted using the statistical software package r version 3.6.3. as of may 2018, there were a total of 144.7 million persons employed in the united states in employer-employee arrangements counted by bls. of these 144.7 million workers, an estimated 18.4% (26, 669, 810) were employed in occupations where exposure to disease or infection occurs more than once a month. as of may 2018, 10% (14,425,070) of the united states workforce was employed in occupations where exposure to disease or infection occurs at least once a week. table 1 summarizes the number and proportion of workers exposed more than once a week and more than once a month by major occupational sectors (two-digit soc). both healthcare practitioner and technical occupations, and healthcare support occupations have more than 90% of workers exposed more than once a month, and more than 75% of workers exposed more than once a week. other notable major occupation groups with high proportion of exposure are protective service occupations (52% exposed more than once a month, including police officers, firefighters, transportation security screeners), personal care and service occupations (52% exposed more than once a month, including childcare workers, nannies, personal care aides), and community and social services occupations (32.4% exposed more than once a month, including probation officers, community health workers, and social and human health assistants). the 16% of office and administrative support occupations with exposure to disease or infection more than once a month are patient representatives, couriers and messengers, and medical secretaries. the nearly 4% of workers exposed in business and financial operation table 1 . number and percent of workers exposed to infection or disease more than one time per month, and more than one time per week, by major (2-digit) standard occupational classification code (soc). exposure to infection/disease in the workplace occupations are compliance specialists, which includes environmental compliance specialists and coroners. the full o � net dataset, ranking the frequency of exposure for each soc is publicly accessible online [14] , as is employment and wage data [8] . as these databases are periodically updated, they should be referenced for information on frequency of exposure for a specific occupation. during an infectious disease outbreak, the workplace can play an important role in both spreading the disease [15, 16] and helping to stop the spread of disease through workplace practices and policies [1, 17] . understanding the wide range of occupations that could be exposed to infection or disease due to work activities is important for planning risk management and communication to workers, in addition to prioritizing workplace response plans. this analysis estimates that the number of workers who face frequent exposure to an infection or disease at work; estimates of the number of workers who fall ill due to such exposures are not possible in this analysis. however, a primary goal of public health, especially in the face of a global pandemic, is to prevent the spread of disease. therefore, understanding how many workers are frequently exposed, and what occupations they represent, is an important first step in being able to prompt and enact risk reduction strategies prior to disease transmission occurring, and illness manifesting. thus, the results reported here have important public health implications. several limitations must be emphasized. exposure to disease or infection in the workplace, and resultant transmission into the community, is dependent on many factors which were not able to be investigated in this analysis. this includes number of contacts that worker has with the public, workplace emphasis on and access to handwashing, number of interactions with bodily fluids, existing hygiene and cleaning practices in the workplace, availability of appropriate personal protective equipment (ppe) etc. while certainly this could vary between occupations, many of these factors would also vary within occupations, and none of these data were captured with the o � net data. presenteeism, reporting to work despite being symptomatic for disease, is common in the workplace, and is another contributor to the transmission of infectious disease, and potentially to the spread of epidemics or pandemics [2, 18] . one analysis examined the role of workplace transmission in the 2009 h1n1 pandemic, estimating that about 8 million employees in the united states worked while infected, and that these workers may have caused the infection of as many as 7 million of their co-workers [19] . access to paid leave, which could ameliorate the financial burden of staying home while sick, varies substantially by occupation, industry, employer, location, and worker sociodemographic profile (e.g., race/ethnicity) [20, 21] . workers without access to paid leave have higher rates of presenteeism, and are less likely to receive preventative health services such as getting flu shots [22] . occupational sector also influences rates of presenteeism, with studies from various countries showing higher rates of presenteeism among workers in healthcare, public service, and educational sectors, as these essential services often do not have substitute workers available [23] [24] [25] . indeed, a recent systematic review identified occupation type as one of the strongest predictors of presenteeism [2] . as many of these sectors are already exposed to disease due to work activities, it is important that disease response plans for these sectors include not only control methods to reduce exposures at work, but also contingency plans to ensure sick workers do not come back to work with disease. this could be accomplished through cross-training, providing extra paid sick leave during this time, ensuring flexible working conditions, and ensuring substitute workers are identified to fill in if essential workers fall ill. importantly, o � net data are also subject to misclassification and undercounting. o � net data were generated from self-reported subjective questionnaires and therefore are subject to bias and misclassification. respondents may not realize they are exposed to infection or disease at work unless they are in a workplace where these hazards are communicated to them and protective equipment is provided (e.g., healthcare sectors) leading to potential differential misclassification across occupational groups. workers could also be reporting expose to disease or infection that occurs while commuting to work (particularly by public transportation), leading to additional misclassification. additionally, information from the o � net database is applied at the occupation-level, and therefore does not account for within-job exposure variation [26] . many workers are not included in the o � net and bls data sources, including independent contractors (which includes "gig economy" workers), domestic workers, self-employed, undocumented, and continent workers. these workers may be uniquely susceptible to exposure at work due to limited ability to take time off if they or a family member is ill [27] . in sweden and norway, higher rates of presenteeism (coming to work when sick) were found among low-income and immigrant workers [28] . this further emphasizes the importance of continuing to develop occupational surveillance systems that capture exposures and outcomes experienced by these undercounted groups, as well as ensure worker protections extend to protect these undercounted workers. in conclusion, our analysis shows that a large proportion of the united states workforce, across a variety of occupational sectors, are exposed to disease or infection at work more than once a month. these are workers that public health should consider especially at risk for covid-19, due to frequent exposure to disease and infectious agents. however, it should be noted that there are many other workers that could also be exposed to sars-cov-2, or encourage the spread of covid-19, such as workers who are not given access to flexible working, workers who do not feel they can take sick time if they or a family member is sick, workers who do not have access to paid sick leave, or workers that perform essential services and do not have access to substitute workers. work presented here underscores the importance of all workplaces developing sector-specific response plans to keep employees safe, halt the transmission of disease in the workplace, and ensure sick workers do not have to come to work. it also serves as a reminder that the workplace is an important locus for public health interventions, as many workers are frequently exposed to disease and infection at work, and their exposures can increase disease incidence both in worker and community groups. influenza in workplaces: transmission, workers' adherence to sick leave advice and european sick leave recommendations a systematic review of infectious illness presenteeism: prevalence, reasons, and risk factors hospital infectious disease emergency preparedness: a 2007 survey of infection control professionals are powered air purifying respirators a solution for protecting healthcare workers from emerging aerosol transmissible disease? ann work expo heal characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention occupational risks for covid-19 infection estimating the population prevalence of traditional and novel occupational exposures in federal region x soc user guide o*net occupational summary update o*net data collection program work context-exposed to disease or infections impact of a hygiene intervention on virus spread in an office building a large rubella outbreak with spread from the workplace to the community policies to reduce influenza in the workplace: impact assessments using an agent-based model sickness presenteeism today, sickness absenteeism tomorrow? a prospective study on sickness presenteeism and future sickness absenteeism sick at work: infected employees in the workplace during the h1n1 pandemic. inst women's policy res paid sick days access varies by race/ethnicity, sexual orientation, and job characteristics. inst women's policy res effects of social, economic, and labor policies on occupational health disparities paid sick leave and preventive health care service use among u.s. working adults job stress and presenteeism among chinese healthcareworkers: the mediating effects of affective commitment sickness presenteeism at work: prevalence, costs and management presenteeism exposures and outcomes amongst hospital doctors and nurses: a systematic review use of o*net as a job exposure matrix: a literature review at work but ill: psychosocial work environment and well-being determinants of presenteeism propensity sickness presenteeism in norway and sweden the authors gratefully acknowledge annie doubleday for developing the r code that supported this analysis. key: cord-273594-vmbhok1u authors: sichelstiel, anke; yadava, koshika; trompette, aurélien; salami, olawale; iwakura, yoichiro; nicod, laurent p.; marsland, benjamin j. title: targeting il-1β and il-17a driven inflammation during influenza-induced exacerbations of chronic lung inflammation date: 2014-06-11 journal: plos one doi: 10.1371/journal.pone.0098440 sha: doc_id: 273594 cord_uid: vmbhok1u for patients with chronic lung diseases, such as chronic obstructive pulmonary disease (copd), exacerbations are life-threatening events causing acute respiratory distress that can even lead to hospitalization and death. although a great deal of effort has been put into research of exacerbations and potential treatment options, the exact underlying mechanisms are yet to be deciphered and no therapy that effectively targets the excessive inflammation is available. in this study, we report that interleukin-1β (il-1β) and interleukin-17a (il-17a) are key mediators of neutrophilic inflammation in influenza-induced exacerbations of chronic lung inflammation. using a mouse model of disease, our data shows a role for il-1β in mediating lung dysfunction, and in driving neutrophilic inflammation during the whole phase of viral infection. we further report a role for il-17a as a mediator of il-1β induced neutrophilia at early time points during influenza-induced exacerbations. blocking of il-17a or il-1 resulted in a significant abrogation of neutrophil recruitment to the airways in the initial phase of infection or at the peak of viral replication, respectively. therefore, il-17a and il-1β are potential targets for therapeutic treatment of viral exacerbations of chronic lung inflammation chronic obstructive pulmonary disease (copd) is currently ranked the 4 th leading cause of death worldwide by the world health organization (who), and its incidence is increasing. the main risk factor of copd is exposure to tobacco smoke which triggers a cascade of inflammatory pathways leading to disease induction in susceptible people. major hallmarks of the disease pathology are the development of emphysema and chronic bronchitis that lead to a progressive and irreversible airflow limitation resulting in a continuous decline of lung function [1] . copd severity has been associated with acute periods of disease worsening [2, 3] , so-called exacerbations, a key factor in copd morbidity and mortality [4, 5] . by causing acute respiratory distress, they impact on the quality of patient's health [6] and are responsible for most hospital stays related to the disease [4] . exacerbations are primarily caused by respiratory viral or bacterial infections. amongst those, viral-induced exacerbations account for approximately half of the cases [7, 8] and are associated with more severe acute episodes and prolonged recovery time [9] [10] [11] . the most common viral pathogen in exacerbated patients is rhinovirus, followed by influenza virus, rsv and coronavirus [7, 8, 10, 12] . due to targeted vaccination of high risk groups, influenza infections occur less frequently in copd patients of westernized countries [11] . however, they continue to be the predominant cause of viral exacerbations in hong kong [13] and singapore [14] . copd exacerbations have been linked to excessive inflammatory responses, including enhanced recruitment of inflammatory cells [15] and upregulation of a variety of proinflammatory mediators [16, 17] . nevertheless, the underlying mechanisms and the most effective therapeutic strategies are still poorly understood and first-line therapy still predominantly relies on corticosteroids and bronchodilators [18] , which are limited in their efficacy [17, 19] . thus, the study of cellular and molecular mechanisms leading to exacerbations is key for the identification of urgently required therapeutic targets. one of the proinflammatory cytokines that has been associated with copd is il-1b, a major player in initiation and persistence of inflammation. in animal models mimicking features of copd, il-1 has been shown to be key to the induction of emphysema and inflammation [20] [21] [22] [23] [24] [25] [26] [27] . furthermore, its expression is significantly enhanced in copd patients during acute episodes of exacerbations [17, 20, 28, 29] . unraveling the role of il-1b in viral exacerbations might therefore not only result in an overall better understanding of mechanisms of exacerbations, but also indicate whether it qualifies as a valid therapeutic target. a promising candidate for therapeutic inhibition of il-1b signaling is one of its inhibitors, the interleukin-1 receptor antagonist (il-1ra) anakinra (kineret, amgen), which has been used effectively in treatment of rheumatoid arthritis. in order to investigate the role of il-1b during copd exacerbations we utilized a model of lps and elastase induced chronic lung inflammation, followed by infection with influenza in wild type or il-1b deficient mice. we found that il-1b was a key driver of pulmonary inflammation, primarily concerning recruitment of neutrophils and lung dysfunction. il-1b driven neutrophilia was mediated by il-17a in the initial phase of viral infection, but became independent of il-17a during the peak phase of viral replication. treatment with the il-1ra, anakinra, proved efficient in reducing neutrophilic inflammation at the peak of viral replication while blocking of il-17a abrogated neutrophilia in the early phase of viral infection. taken together our data indicate that blockade of il-1b and il-17a could be valid therapeutic approaches for treatment of virus-induced copd exacerbations. all animal experiments were performed according to institutional guidelines and swiss federal and cantonal laws on animal protection. animal experiments were approved by the following ethical committee: service de la consommation et des affaires vétérinaires, affaires vétérinaires, canton de vaud, switzerland (permit numbers 2283 and 2216). c57bl/6 or balb/c mice were between 8-12 weeks of age and were purchased from charles river laboratories. il-1b deficient mice on c57bl/6 background were received from prof. iwakura [30] , tokyo university of science, japan, and bred in house. mice were exposed intranasally to a mixture of 7 mg lps from e. coli o26:b6 (sigma-aldrich) and 1.2 u porcine pancreatic elastase (epc) in a total volume of 100 ml, and treated once per week for four consecutive weeks ( figure 1a ). control mice were treated with pbs. two weeks after the last lps/elastase challenge, mice were infected with 250 pfu influenza a virus, strain pr8 (a/puerto rico8/34, h1n1, viropur). the virus was administered intranasally in a total volume of 50 ml pbs; control mice received only pbs. for all intranasal administrations c57bl/ 6 mice were anesthetized by intraperitoneal injection of 54.17 mg/kg ketamin (ketasol-100, graeub) and 1.28 mg/kg xylazin (xylasol, graeub) and balb/c mice with 78 mg/kg ketamin and 1.93 mg/kg xylazin. lungs were inflated with 1 ml 10% formalin, embedded into paraffin and stained with hematoxylin and eosin. stained slides were analyzed by light microscopy. pulmonary emphysema was quantified using image j software by measuring the mean linear intercept for airspace enlargement and destruction index for alveolar wall destruction. 10 fields of view at 20x magnification per section of lung were used for quantification as described previously [31, 32] . airway cells were recovered by bronchoalveolar lavage and either analyzed by flow cytometry as described below or spun onto slides for differential cell counts. slides were stained with diff-quik (dade) and counts were performed according to standard criteria. total lung resistance was measured using the whole body restrained plethysmograph system flexivent from scireq. mice were anesthetized by intramuscular injection of 100 mg/kg ketamine (ketasol-100, graeub) and intraperitoneal injection of 50 mg/kg pentobarbital (esconarkon, streuli pharma). subsequently, mice were tracheotomized and mechanically ventilated at a rate of 450 breaths/min and a tidal volume of 10 ml/kg bodyweight. single cell suspensions from the whole lung including airways and trachea were obtained by digestion with 2 mg/ml collagenase iv (invitrogen) and 50 u/ml dnasei (roche). neutrophils and monocytes in lung and bronchoalveolar lavage fluid were distinguished by staining with cd11c apc-cy7, cd11b percp-cy5.5, ly-6g biotin, ly-6c pacific blue, streptavidin pe-cy7. neutrophils were defined as cd11c 2 cd11b + ly-6c + ly-6g + and inflammatory monocytes as cd11c 2 cd11b + ly-6c + ly-6g low2intermediate as precised in detail in figure s1a . to analyze il-17a production, cells from lung digests were stimulated with 10 27 m pma, 1 mg/ml ionomycin and 2610 26 m monensin for 4 h at 37uc (indicated chemicals were purchased from sigma-aldrich). subsequently, cell subsets were distinguished by surface staining for cd4 percp-cy5.5, cd8b fitc, cd tcr biotin, cd3 pacific blue, streptavidin pe-cy7 ( figure s1b ) and il-17a production was characterized by intracellularly staining with il-17a alexa700 after fixation with bd lysis buffer (bd biosciences). all antibodies were purchased from biolegend. stained cells were acquired on a bd facs canto or bd facs lsrii and analyzed by using flowjo software (tree star). for neutralization of il-17a, mice were treated with 250 mg of anti-il-17a (clone 17f3) or the corresponding isotype control antibody (clone mpoc-21) from bioxcell. the clone 17f3 uniquely reacts with the il-17a and no other il-17 isoform [33] . antibodies were administered intraperitoneally one day before viral infection and two days post infection. to block il-1b signaling mice received 200 mg of the interleukin-1 receptor antagonist (il-1ra) anakinra (kineret, amgen) twice daily while control mice received only pbs. anakinra was kindly provided by prof. alexander so (centre hospitalier universitaire vaudois, lausanne, switzerland) and mme ghislaine aubel (centre hospitalier universitaire vaudois, lausanne, switzerland). total rna was isolated from lung and trachea with tri reagent (molecular research). all rna samples were checked for purity using a nanodrop 1000 spectrophotometer (thermo scientific) and met standard quality criteria. rna was subsequently transcribed into cdna by the iscript cdna synthesis kit (bio-rad) and quantitative real-time pcr was performed according to the manufacturer's instructions utilizing the ssoadvanced sybr green supermix from bio-rad. expression was determined by comparative delta-threshold cycle method using gapdh as a comparator. the following primers were used: to determine cytokine levels whole lung and trachea were collected and stored in protease inhibitor solution (roche) at 2 20uc until use. tissue homogenate was prepared using a tissuelyser (qiagen). il-1b protein was determined using the mouse il-1b elisa kit ready-set-go! from ebioscience by following the manufacturer's instructions. il-6 and tnfa were measured in a sandwich elisa using 2.5 mg/ml anti-mouse il-6 or tnfa (biolegend) for coating. bound protein was detected by 1 mg/ml biotin labeled anti-il-6 or anti-tnfa antibody (biolegend) and subsequent incubation with alkaline phosphatase conjugated streptavidin (biolegend). plates were developed using the substrate p-nitrophenyl phosphate (sigma-aldrich) and od was measured using an elisa reader (biotek). statistical significant differences were assessed using the student's t test (two tailed, unpaired). p-values below 0.05 were considered significant and were depicted with p#0.05 (*), p# 0.005 (**), p#0.0005 (***). standard error of the mean was applied. copd is a heterogeneous disease in humans but core features of its pathology can be reproduced in mice by repetitive exposure to lipopolysaccharide (lps) and elastase [34] . lps is a bacterial endotoxin present in tobacco smoke [35, 36] , the predominant risk factor of copd. it has been shown to cause inflammation, and particularly when co-administered with elastase, chronic emphysema-like changes develop in mouse lungs [34] . as such, we exposed mice once a week for 4 consecutive weeks to a mixture of 7 mg lps and 1.2 u porcine pancreatic elastase via the intranasal route, as depicted in figure 1a . by this we induced strong emphysema ( figure 1b ,c) and sustained pulmonary inflammation ( figure 1b ,d) in balb/c and c57bl/6 mice. the development of emphysema was scored by increases in the mean linear intercept and the destructive index ( figure 1c ) from lung histology. in addition, we observed a strong airway inflammation, mainly driven by neutrophils and lymphocytes ( figure 1d ). both, emphysema and inflammation, remained above baseline levels for at least 2 months (data not shown). thus, the response induced by repeated challenges with lps/elastase resembled the pathology of copd. as the disease severity was more pronounced in the balb/c strain ( figure 1b-d) , all experiments performed in wild type mice were conducted in balb/c mice. to study viral-induced exacerbations, mice were infected with influenza virus 2 weeks after the last lps/elastase challenge, when the acute inflammation caused by lps/elastase exposure had subsided ( figure 2a ). the peak of viral replication was reached 5 days after the infection ( figure 2b) and was followed by a rapid decline in viral titers ( figure 2b ) until complete viral clearance at day 9 post infection (data not shown). the efficiency of the influenza infection was strikingly reduced in mice pretreated with lps/elastase compared to naïve mice, as revealed by significantly lower viral titers at the peak of viral infection, day 5 and day 7 post infection ( figure s2 ) which was also reflected by reduced amounts of viable virus in the lung on day 5 (data not shown). this is likely to be due to the development of a polyclonal antibody response observed upon lps/elastase treatment ( [37, 38] and data not shown). thus, the response to the influenza infection was not comparable between lps/elastase pretreated and naive mice, and was therefore not included in the remainder of the study. invasive measurements of pulmonary resistance in lps/elastase pretreated mice revealed a viral-induced acute exacerbation of airway dysfunction ( figure 2c ). pulmonary resistance can be influenced by a variety of factors, of which the severity of inflammation is likely to play one of the key roles during exacerbations. in line with this we detected a strong inflammatory response upon infection with influenza virus associated with an augmented absolute number of cells infiltrating into the airways and the lung ( figure 2d ). the cells were primarily neutrophils ( figure 2e , figure s3a ) and inflammatory monocytes ( figure 2f , figure s4a ). the peak of neutrophilic inflammation was reached at day 5 post infection and neutrophil numbers declined afterwards ( figure 2e , figure s3a ), directly correlating with the kinetics of viral replication ( figure 2b ). in line with this we observed increased expression of the proinflammatory cytokines il-6 and tnfa peaking at day 3 or 5 post infection, respectively, and subsiding at day 7 after the infection ( figure 2g ). emphysematous damage upon lps/elastase treatment also resulted in an increase in the lung compliance. however the acute inflammatory changes induced by influenza infection had no impact on the increase in lung compliance (data not shown). acute pulmonary dysfunction, neutrophilic inflammation and enhanced levels of proinflammatory cytokines such as il-6 and tnfa have all been observed during exacerbations of copd patients, indicating that the viral-induced inflammation in our mouse model is in line with that seen in humans. il-1 has previously been shown to be a driving factor in the development of emphysema and inflammation in animal models of copd [20] [21] [22] [23] [24] [25] [26] [27] . as we observed an increase of il-1b protein in the lungs of lps/elastase treated mice upon influenza infection ( figure 3a ), we hypothesized that it also promotes innate immune responses and influences pulmonary function during exacerbations. consequently, we exposed il-1b deficient and c57bl/ 6 wild type mice to lps/elastase and infected them with influenza virus as described above. the c57bl/6 strain showed similar kinetics of neutrophil and inflammatory monocyte recruitment upon viral exacerbation as observed for the balb/c mice ( figure 3d , figure s3b , s4b). of note, viral replication was not altered in il-1b deficient mice ( figure 3b ), demonstrating that any effects seen in the absence of il-1b were not due to differences in the infection rate. c57bl/ 6 wild type mice exhibited a smaller change in pulmonary resistance in response to viral infection ( figure 3c ) in comparison to balb/c mice ( figure 2c) , with a slight increase at day 1 post infection ( figure 3c ). nevertheless, pulmonary resistance in mice lacking il-1b was significantly reduced already after lps/elastase exposure alone ( figure 3c ), thus supporting the described role for il-1b in the development of chronic lung disease [20] [21] [22] [23] [24] [25] [26] [27] . furthermore, pulmonary resistance in il-1b-deficient mice was also completely unaffected by the viral challenge ( figure 3c ). we did not detect any impact of il-1b on inflammatory monocytes ( figure s4b ); however, we found a decreased frequency and number of neutrophils in non-infected il-1b deficient mice upon exposure to lps/elastase ( figure 3d , figure s3c) . similarly, the recruitment of neutrophils to the airways and lung following viral infection was also significantly abrogated in the absence of il-1b ( figure 3d , figure s3c ). we observed significantly lower frequencies and absolute numbers of neutrophils including during the peak of neutrophil infiltration and viral replication at day 5 post infection ( figure 3d , figure s3c ). nevertheless, the control of the virus was unaffected as displayed by unaltered viral titers ( figure 3b ). considering its strong impact particularly upon neutrophils, we sought to address the mechanisms through which il-1b mediated this neutrophilic inflammation. expression of the main neutrophil chemoattractants cxcl1, cxcl2, and cxcl5 were induced upon influenza infection, but were unaffected by il-1b ( figure s5 ). in addition we did not detect any influence of il-1b or the viral infection on the expression of g-csf ( figure s5 ). given there is substantial redundancy in neutrophil chemoattractants we thus next looked upstream at the proinflammatory cytokines il-17a, il-6, and tnfa, which can all stimulate neutrophilic inflamma-tion by inducing chemotactic, growth or survival factors [16, 39, 40] . as expression of il-6 and tnfa were induced in our mouse model upon influenza infection ( figure 2g ), we hypothesized that il-1b drove the observed inflammation by altering their production. the peak expression of il-6 and tnfa upon influenza infection was reached faster in c57bl/6 wild type mice at day 1 post infection ( figure 2g ) as compared to day 3 in balb/c mice ( figure 3e) , and thus coincided with the earlier response in pulmonary resistance observed in the c57bl/6 strain ( figure 3c ). il-6 protein levels followed similar kinetics in both mouse strains ( figure s6a,b) , while tnfa protein production was below the sensitivity limit of our assay. however, lack of il-1b did not impair the expression and production of il-6 ( figure 3e , figure s6b ) and in fact increased the expression of tnfa ( figure 3e ). we therefore focused on il-17a, a proinflammatory cytokine that has been shown to be elevated in copd patients [41, 42] and whose induction partially depends on il-1b [43] [44] [45] . we found significantly reduced expression levels of il-17a in lung homogenate in the absence of il-1b, in both non-infected as well as influenza-infected lps/elastase exposed mice ( figure 3f ). il-17a production was significantly reduced in the predominant cellular sources of il-17a, such as the cd4 + t cells and the cd t cells in il-1b deficient mice ( figure 3g ). further sources of il-17a such as cd3 + cd4 2 cd8 2 cdtcr 2 cells that might comprise nkt cells, and cd3 2 cells also showed reduced levels of il-17a in the absence of il-1b ( figure s7a ). however, even in the wild type animals these cells represented a very minor population of cells relative to the il-17a-producing t cells ( figure s7a ). taken together our data showed that in addition to contributing to lung dysfunction, il-1b played a key role in driving neutrophilic inflammation during influenza-induced exacerbations, an effect that was tightly linked to il-17a expression. to assess whether il-17a was indeed a mediator of il-1b driven neutrophilia we neutralized il-17a during influenza infection of lps/elastase treated mice. neutralization assays were performed in balb/c mice, which exhibited a similar induction of il-17a as c57bl/6 mice ( figure s7b ). mice received either an il-17a neutralizing antibody or an isotype control antibody one day before and two days after the viral infection ( figure 4a ). il-17a neutralization did not impact on the control of viral replication, as viral burden was comparable to the isotype control treated animals ( figure 4b ). we found that influenza-induced neutrophil recruitment to the airways and lung was indeed entirely attenuated 24 h after the infection in absence of il-17a ( figure 4c , figure s3d ). however, neutrophils infiltrated into the lung and airways during the later stages of infection (day 3 and 5 respectively) to finally reach the same frequencies as in mice treated with the isotype control ( figure 4c, figure s3d ); thus indicating that il-17a was only required for the initial but not for the later recruitment of neutrophils. hence, il-1b driven neutrophilia during influenza infection of lps/elastase exposed mice was mediated by il-17a in the early phase of infection, but became independent of il-17a. our data showed that a constitutive lack of il-1b substantially impaired neutrophil infiltration into the airways and lung during influenza-induced exacerbations of chronic lung inflammation. thus, we sought to assess whether it is sufficient to block il-1b signaling only during the course of infection, an important determinant regarding a potential therapeutic intervention. accordingly, recombinant il-1ra (anakinra) or pbs was administered twice daily, starting two days prior to the viral infection ( figure 5a ). mice receiving anakinra displayed an impaired early control of viral infection leading to a higher viral burden at day 3 post infection, although viral titers rapidly declined afterwards to levels similar to non-treated mice at day 7 post infection ( figure 5b) , and virus was completely cleared at day 9 post infection (data not shown). treatment with anakinra was efficient in reducing neutrophil frequencies and numbers in the airways at day 5 post infection, the peak of neutrophilic infiltration and viral replication ( figure 5c , figure s3e ). we did not observe an effect of anakinra on the recruitment of inflammatory monocytes ( figure s4c ). similarly, we did not detect significant changes in lung function upon the treatment with anakinra (data not shown). in conclusion our data demonstrated that il-1b influenced neutrophilic inflammation during influenza-induced exacerbation of chronic lung inflammation in mice throughout the entire phase of viral replication. neutrophil recruitment was mediated by il-17a in the first 24 h following viral challenge and could efficiently be blocked in the early phase of infection by antibodies neutralizing il-17a. during the peak of inflammation and viral replication, il-1b driven neutrophilia was independent of il-17a, but could be significantly reduced by treatment with the il-1ra anakinra ( figure 6 ). in addition to a constant disease burden, copd patients suffer from episodes of acute symptom worsening causing a rapid decline in respiratory function that can necessitate hospitalization and even lead to death [46] . indeed, a meta-analysis study estimated a case-fatality rate of 15.6% following hospitalization due to an exacerbation [47] . as there is a clear need to understand the mechanisms driving exacerbations and responsiveness to therapy, we examined viral-induced exacerbations in mice. in our model (figure 2a ), mice developed a strong inflammatory response characterized by a neutrophilic infiltrate into the airways and lung ( figure 2e ), enhanced expression of proinflammatory cytokines such as tnfa and il-6 ( figure 2g ), and impairment in lung function ( figure 2c ). decline in lung function and neutrophil accumulation are characteristic of exacerbations in humans [48] [49] [50] , and elevated levels of tnfa and il-6 have similarly been measured in the sputum of patients undergoing an exacerbation [17, 51] . thus, our mouse model reflects key pathological characteristics of copd exacerbations in humans. given the significant differences in susceptibility between the pbs treated and lps/elastase treated mice ( figure s2 and data not shown) which might be due to the development of a polyclonal b-cell response upon lps/elastase administration, we believe that conclusions can only be drawn from the comparison between stable disease versus episodes of exacerbation. notably, this is also supported by findings in cigarette smoke (cs) models of pulmonary inflammation where cs exposed mice showed a considerably altered response to influenza infection [37] as well as the accelerated clearance of haemophilus influenza [38] in comparison to control mice. in this study we specifically focused on the role of the proinflammatory cytokine il-1b. il-1 signaling has been shown to be essential for the recruitment of neutrophils in other mouse models mimicking the pathology of stable copd, such as exposure to cigarette smoke [20, 21, 23, 26] or to elastase alone [22] . building upon these studies we found that during influenzainduced exacerbations, pulmonary accumulation of neutrophils was also driven by il-1b ( figure 3d , figure s3c ). our data is in line with results from a study of botelho et al. [20] who investigated il-1 in a mouse model of acute cigarette smoke exposure. they found that interleukin-1 receptor (il-1r) deficiency led to a reduction of neutrophils following infection with influenza and that this effect was independent of il-1a. one could therefore speculate that il-1b may play a critical role in neutrophil recruitment in their model as well. il-17a, a cytokine that plays a central role in amplifying inflammatory cascades by inducing a variety of chemokines and cytokines, has also been reported to contribute to the development of emphysema [52, 53] and to the immunopathology following influenza infections [54] . in line with this, we found that il-17a mediated early inflammation in our model of influenza-induced exacerbations of chronic lung disease ( figure 4c ). our data showed that il-1b driven neutrophil recruitment during the first 24 h of infection was mediated by ll-17a, while it became independent of il-17a at later time points during the exacerbation. we found that in the absence of il-1b the expression of il-17a was completely abrogated upon lps/elastase exposure as well as during exacerbations ( figure 3f ), thus demonstrating that il-1b is required for the induction of il-17a. il-17a expression was induced by lps/elastase exposure alone, and levels were maintained throughout the viral-induced exacerbation ( figure 3f,g) ; however, while il-1b levels increased during the later phase of infection ( figure 3a) , il-17a expression surprisingly remained unaltered and even decreased at the peak of viral replication ( figure 3f ). nevertheless, we could block the recruitment of neutrophils at early time points following the influenza infection by neutralizing il-17a ( figure 4c ). during the peak of viral replication, and thus a more severe state of inflammation, il17a neutralization could not prevent neutrophil influx. this might be due to an induction of cytokines during the progression of the infection, which could overcome the effect of il-17a. therefore, treatment with anti-il-17a seems to be favorable in the early phases of exacerbations while blocking il-1b might be more advantageous during the ongoing infection. whether these findings translate into a clinical setting remains to be investigated. keeping in mind that neutrophil recruitment is elevated in the vast majority of cases of copd exacerbations regardless of their etiology [7] , and furthermore that the increase of neutrophils in the sputum correlates with exacerbation severity [7] , attenuating neutrophilia could be beneficial in patients undergoing a viralinduced exacerbation. current treatment relies mainly on corticosteroids and bronchodilators that have been shown to reduce the frequency of exacerbations [55] [56] [57] , but have no positive effect on an ongoing episode of exacerbation. indeed, no reduction in the inflammatory response including neutrophil influx in mice and cytokine expression in humans could be achieved by treatment with steroids during copd exacerbations [17, 19] . in contrast, corticosteroids have been shown to actually support neutrophil survival [58, 59] . moreover, treatment with corticosteroids, efficient in reducing il-1b levels in stable copd, did not affect the amount of il-1b protein in exhaled breath condensate during exacerbations [17] . as blocking of either il-17a ( figure 4c ) or il-1b ( figure 3d , figure s3c ) efficiently reduced neutrophilic inflammation during viral exacerbation, these two molecules could be considered potential targets for therapeutic intervention. given the redundancies in these two inflammatory pathways, a combination therapy of anti-il-17a and anti-il-1b may prove more beneficial in improving lung function. within the context of targeting il-17a or il-1b in the clinic, one has to keep in mind that altering proinflammatory immune responses harbors the risk of interfering with the control of acute infection and of susceptibility to opportunistic infections. to shorten the duration of intervention and thereby reducing the risk of prolonged or secondary infections, we treated mice directly before and during the viral infection with anti-il-17a ( figure 4a ) or il-1ra ( figure 5a ). this was sufficient to abrogate neutrophil recruitment at the indicated time points. furthermore, neutralizing il-17a did not promote elevated viral replication ( figure 4b ). treatment with il-1ra, and thereby blocking both il-1a and il-1b, led to increased viral titers in the initial phase of infection ( figure 5b ), whereas the absence of il-1b alone did not affect viral replication ( figure 3b ). it is therefore likely that in our model the initial control of the virus might be mediated rather by il-1a than by il-1b. this is in line with data from botelho et al. [20] , who found similarly elevated viral titers upon neutralization of il1-a as in the complete absence of il-1r. our data thus suggest that targeting specifically il-1b, and not its receptor, would be favorable in a therapeutic application. however, as treatment with anakinra did not interfere with final viral clearance it still could be considered as a therapeutic approach with the additional advantage of already being used in the clinic for other indications. taken together our data demonstrated that blocking of il-17a or il-1b signaling during influenza-induced exacerbations diminished neutrophilic infiltration at distinct phases of infection. whether those mechanisms apply also to other respiratory viral infections remains to be elucidated, however is plausible given the common early inflammatory pathways induced by respiratory viral infections. overall, blockade of il-17a and il-1b could be valuable therapeutic options for future treatment of viral induced exacerbations of 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cytokines in induced sputum and severity of chronic obstructive pulmonary disease il-17ra is required for ccl2 expression, macrophage recruitment, and emphysema in response to cigarette smoke cigarette smoke induction of osteopontin (spp1) mediates t(h)17 inflammation in human and experimental emphysema critical role of il-17ra in immunopathology of influenza infection maintenance therapy with budesonide and formoterol in chronic obstructive pulmonary disease efficacy and tolerability of budesonide/formoterol in one hydrofluoroalkane pressurized metered-dose inhaler in patients with chronic obstructive pulmonary disease: results from a 1-year randomized controlled clinical trial efficacy and safety of budesonide/formoterol in the management of chronic obstructive pulmonary disease medicine. neutrophils find smoke attractive beta2-agonists potentiate corticosteroid-induced neutrophil survival key: cord-281161-u896icp9 authors: wang, jing; tricoche, nancy; du, lanying; hunter, meredith; zhan, bin; goud, gaddam; didier, elizabeth s.; liu, jing; lu, lu; marx, preston a.; jiang, shibo; lustigman, sara title: the adjuvanticity of an o. volvulus-derived rov-asp-1 protein in mice using sequential vaccinations and in non-human primates date: 2012-05-17 journal: plos one doi: 10.1371/journal.pone.0037019 sha: doc_id: 281161 cord_uid: u896icp9 adjuvants potentiate antigen-specific protective immune responses and can be key elements promoting vaccine effectiveness. we previously reported that the onchocerca volvulus recombinant protein rov-asp-1 can induce activation and maturation of naïve human dcs and therefore could be used as an innate adjuvant to promote balanced th1 and th2 responses to bystander vaccine antigens in mice. with a few vaccine antigens, it also promoted a th1-biased response based on pronounced induction of th1-associated igg2a and igg2b antibody responses and the upregulated production of th1 cytokines, including il-2, ifn-γ, tnf-α and il-6. however, because it is a protein, the rov-asp-1 adjuvant may also induce anti-self-antibodies. therefore, it was important to verify that the host responses to self will not affect the adjuvanticity of rov-asp-1 when it is used in subsequent vaccinations with the same or different vaccine antigens. in this study, we have established rov-asp-1's adjuvanticity in mice during the course of two sequential vaccinations using two vaccine model systems: the receptor-binding domain (rbd) of sars-cov spike protein and a commercial influenza virus hemagglutinin (ha) vaccine comprised of three virus strains. moreover, the adjuvanticity of rov-asp-1 was retained with an efficacy similar to that obtained when it was used for a first vaccination, even though a high level of anti-rov-asp-1 antibodies was present in the sera of mice before the administration of the second vaccine. to further demonstrate its utility as an adjuvant for human use, we also immunized non-human primates (nhps) with rbd plus rov-asp-1 and showed that rov-asp-1 could induce high titres of functional and protective anti-rbd antibody responses in nhps. notably, the rov-asp-1 adjuvant did not induce high titer antibodies against self in nhps. thus, the present study provided a sound scientific foundation for future strategies in the development of this novel protein adjuvant. the use of an adjuvant is a key element in promoting vaccine effectiveness because it can stimulate the immune system and accelerate, prolong, or enhance antigen-specific immune responses, even when used in combination with weak vaccine antigens [1] . adjuvants have been used in vaccines since the early 20th century following more than 100 years of research. in the u.s., alum remains the sole fda-approved adjuvant for general use of vaccines [2] . however, few adjuvants have been licensed for use around the world [3] . no new adjuvants have been approved in the united states since the 1930s, and only recently has the european heads of medicines agencies licensed the mf59, as03 and as04 adjuvants for use with the fluadh, fendrix tm and cervarix tm defined vaccines, respectively [4] . cervarix tm which uses as04, a combination of aluminum hydroxide and monopho-sphoryl lipid a (mpl), in its formulation was also licensed by fda on october 16, 2009 , to prevent cervical cancer caused by human papillomavirus types 16 and 18. adjuvants are important in guiding the type of adaptive response that is induced after vaccination and that is most effective against incoming infections. the development of novel adjuvants that stimulate discrete subsets of immune cells, in particular, cytotoxic t-lymphocytes (ctl), is required to unleash the full potential of new vaccines and immunotherapy strategies [5] . although tremendous progress has been made in the development of many vaccine platforms, including dna-based vaccines, recombinant subunit vaccines, viruses and conjugates, the absence of safe and effective adjuvants impedes the clinical development of such new generations of vaccines. interest in developing new adjuvants has increased significantly over the past decade, as highlighted by the following issues [6, 7] : 1) the inability of traditional approaches to develop successful vaccines against ''difficult'' organisms such as hiv and hcv; 2) the emergence of epidemics or outbreaks of new infectious diseases with high mortality, especially those causing serious threats to public health and socioeconomic stability worldwide (e.g., sars, ebola, west nile, dengue, pandemic flu and nvcjd); 3) the re-emergence of ''old'' infections like tuberculosis; 4) the continuing spread of antibiotic-resistant bacteria; and 5) the increased threat of bioterrorism. therefore, the molecular design of potently adjuvanted vaccines that would enhance antigen uptake in vivo and potentially also simplify their adjuvant requirements would be highly desirable [8] . there are many reports showing that helminth-derived molecules have potent regulatory or stimulatory effects on the immune system of their mammalian hosts (reviewed in [9] , [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] ). some of these molecules were shown to contain pathogen associated molecular pattern that bind to endocytic-pattern recognition receptors on antigen presenting cells (apcs). three helminth products have also been reported to act as adjuvants in experimental vaccine models. proteins secreted by adult nippostrongylus brasiliensis (nes) induced strong th2 responses in mice immunized with hen egg lysozyme [22] . nes actively matured dendritic cells (dc) and selectively up-regulated cd86 and ox40l, together with il-6 production, while blocking il-12p70 responsiveness in a manner consistent with th2 generation in vivo [23] . similarly, lacto-n-fucopentaose iii (lnfpiii), a carbohydrate found on the surface of the eggs of a human parasite, schistosoma mansoni, acted as a th2 adjuvant for human serum albumin when injected intranasally, subcutaneously or intraperitonealy into mice [24] . it functions as an innate th2 promoter via its action on murine dcs. its ability to drive dc2 maturation was shown to be dependent on signaling via toll-like receptor 4 (tlr4) [25] . finally, when co-administered with an inactivated antiinfluenza vaccine in both young and aged mice, a 19 aa synthetic peptide (gk-1) from taenia crassiceps cysticerci has induced increased levels of anti-influenza antibodies in aged mice, both before and after infection, reduced the local inflammation that accompanied influenza vaccination itself, and favored virus clearance after infection in both young and aged mice [26] . a recombinant onchocerca volvulus activation-associated protein-1, rov-asp-1, has been shown to be a novel protein adjuvant that can increase specific immune responses in mice, both humoral and cellular responses, against recombinant protein or peptide-based figure 1 . anti-rbd antibody responses in vaccinated mice. rbd-specific responses in mice immunized with recombinant sars-cov rbd in the presence of the rov-asp-1, alum or cpg. titer of rbd-specific igg and igg subtypes was detected by elisa using sera from mice before (preimmune) and 10 days after each vaccination. * indicates significant difference (p,0.05) among multiple comparisons; in particular between mice that were immunized in the presence of rov-asp-1 or control adjuvants vs. no adjuvant. doi:10.1371/journal.pone.0037019.g001 antigens when formulated in aqueous mixtures [14, 27, 28, 29] . we have previously suggested that these effects are probably attained through the cellular activation of apcs such as dendritic cells via tlr-2 and tlr-4 [27] . the rov-asp-1 adjuvant is able to induce balanced th2 and th1-associated igg1 and igg2a antibodies to proteins, polypeptides and small peptides. with a few antigens such as rgp41, rbd and hbsag, it also promoted a putative th1biased response based on pronounced induction of th1-associated igg2a and igg2b antibody responses and/or a significantly upregulated production of th1 cytokines, including il-2, ifn-c, tnf-a, and il-6 [14, 27, 29, 30] . its ability to augment th1associated antibody responses was further demonstrated in studies using three commercial inactivated vaccines against hemorrhagic fever with renal syndrome, flu and rabies [29] . moreover, in a novel recombinant configuration, rov-asp-1 fused to 3 copies of the highly conserved extracellular domain of the h5n1 influenza m2 protein sequence was able to induce high levels of m2especific igg, igg1, igg2a providing strong cross-protection from a lethal challenge with 3ld 50 or 10 ld 50 of h5n1 viruses of different clades (clade 1: vn/1194, or clade 2.3.4: sz/406h [28] . in this study, we further demonstrated the adjuvanticity of rov-asp-1 in sequential vaccines in mice and also confirmed its ability to be a potent innate adjuvant in nhps. the rov-asp-1 adjuvant enhances humoral and cellular responses after immunization with rrbd of sars-cov in mice to evaluate the adjuvant activity of rov-asp-1, mice were immunized with sars-cov rrbd in the presence or absence of rov-asp-1 or with alum or cpg for comparison. we previously reported that rov-asp-1 could effectively induce a mixed, but th1-skewed immune response against rs and rrbd in immunized mice [27] . as shown in figure 1 , the titers of rbd-specific igg, igg1, igg2a and igg2b antibodies increase after the first or second boost immunization in the sera of mice immunized with rrbd plus rov-asp-1, which were all significantly higher than in mice immunized with rrbd alone. the igg and igg2a responses in mice vaccinated with rrbd in the presence of alum were significantly higher only after the second boost; only the igg1 response was significantly higher after the first boost. the anti-rrbd igg response was significantly higher in the presence of cpg only after the second boost when compared to mice vaccinated only with rrbd (1:102,400 vs. 1:11,314) . notably, the rov-asp-1 augmented igg antibody response to rrbd was almost 4 times higher than the alum vaccination group (64,000 vs. 16,000) and 5.7 times higher than the cpg vaccination group (64,000 vs. 11,200) already after the first boost. further comparison of the rov-asp-1 and the alum induced rrbd antibody responses after the second boost revealed similar levels of igg1 (1,884,544 vs. 1,722,156); two-fold higher level of igg2a (935,763 vs. 512,000) as well as ten times higher level of igg2b (430,538 vs. 45,255) endpoint titers in the rov-asp-1 vaccine group. comparison of the rov-asp-1 and the cpg induced rrbd antibody responses after the second boost revealed a 27 fold increase in igg1 (1,884,544 vs. 68,000), 14.6 fold increase in igg2a (935,763 vs. 64,000), and 215 fold increase in igg2b (430,538 vs. 2,000) endpoint titers. each adjuvant/rrbd model performed differently depending on the adjuvant, e.g., a skewed th2 response with alum (igg1/igg2a = 3.3), but a mixed th1/ th2 response with cpg (igg2a/igg1 = 0.94) and rov-asp-1(igg2a/igg1 = 2), with a predominance of th1-associated antibodies (when both igg2a and igg2b are taken into account) with rov-asp-1. these results further supported our previous studies showing that rov-asp-1 can induce a more balanced antibody response with some bias towards a skewed th1associated antibody response than other adjuvants used with the same bystander antigen such as alum or cpg (this study) or the mlp plus tdm adjuvant [14, 27, 29] . to further evaluate whether the induced igg antibodies could neutralize infection of sars-cov in vitro (fig. 2) , we tested the antisera from the 10-day post-second boost immunization and found that mice immunized with rrbd in the presence of rov-asp-1 contained a very high titer of neutralizing antibodies against infection by sars-cov pseudovirus (nt 50 = 1:76,592), which was not significantly different than in neutralizing antibodies found in sera from mice immunized with rrbd plus the alum adjuvant (nt 50 = 1:64,666). the subclass of immunoglobulin induced after immunization is an indirect measure of the relative contribution of th1-type cytokines vs.th2-type cytokines. in this study, the data from the cba analyses showed that the levels of th1-and th2-type cytokine secretion were significantly higher in mice immunized with rrbd+rov-asp-1 or alum than in those who were immunized with rrbd alone ( table 1 ). the rov-asp-1 induced the production of type i proinflammatory cytokines (il-2, ifn-c, tnf-a, il-17a and il-6) to the same extent as alum, as well as the th2/regulatory cytokines il-6 and il-10. there was no significant recall induction of the th2 il-4 or il-5 cytokines by rrbd (data not shown). notably, the responses to rrbd when formulated with cpg are in comparison more ifn-c and il-17a dominant with diminished il-2, tnf-a, il-10 and il-6 responses. we found that the variation between individual mice was very low [31, 32] , and therefore we are confident that the results obtained using the pooled spleens are a good representation of what would have been the outcome if we had used individual mice. distinct from the previous studies are the responses to two rbd-specific peptides; n50 (cd8 + t cell epitope) and n60 (cd4 + t cell epitope) (table 1) . interestingly, the cytokine levels in the culture supernatants of the murine splenocytes when stimulated with either n50 or n60 were similar to those produced by stimulation with the full length rrbd in mice vaccinated with rrbd+rov-asp-1, thus further confirming the ability of rov-asp-1 to elicit rbd-specific cd8 + and cd4 + cellular responses in the tested vaccine formulation. naïve balb/c mice or mice ten weeks after immunization with rrbd in the presence of rov-asp-1 were immunized with has of influenza viruses in the absence or presence of yeast expressed rov-asp-1 (100 mg/mice). the mice that were previously immunized with rrbd in the presence of rov-asp-1 had endpoint total anti-rov-asp-1 antibody titers of 1:256,000-1:512,000 at the time of the priming with the second vaccine. as shown in table 2 , similar igg1 and igg2a humoral immune responses against the influenza viruses were induced in the mice vaccinated previously with rrbd plus rov-asp-1 adjuvant and those administered with pbs only. moreover, the igg2b was higher in the group that got the sequential vaccine (162,000 vs. 54,000). the anti-ha igg1 and igg2b antibody responses were much higher in mice that were immunized with the has vaccine in the presence of rov-asp-1 adjuvant than those immunized in the absence of rov-asp-1 adjuvant (igg1: 1,458,000 vs. 607,000 or 729,000; igg2b: 243,000 vs. 162,000 or 54,000). all levels of igg isotype responses in mice when the flu vaccine was formulated with rov-asp-1 were statistically higher than when mice were immunized with no adjuvant (p.0.05; fig. 3 ). there was no significant difference in the igg1 and igg2a responses if the flu vaccine was given to naïve mice or to mice that were previously immunized with another vaccine; rrbd of sars-cov+rov-asp-1. the adjuvanticity of rov-asp-1 was evaluated in a rhesus macaque immunization model using rrbd as the target antigen of table 2 . titers of anti-ha antibody response in rrbd+ rov-asp-1 vaccinated mice and naïve mice after vaccination with an influenza vaccine or an influenza vaccine in combination with rov-asp-1. the sars-cov vaccine. as shown in table 3 , all of the nhps vaccinated with rrbd protein plus 50 mg (n = 2), 100 mg rov-asp-1 (n = 2) or 500 mg cpg (n = 1) as the adjuvant developed rbdspecific igg antibody response with increasing antibody level after each boost. rbd-specific antibodies were not detected in the preimmune sera of the vaccinated rhesus macaques or the rhesus macaques injected with rbd+pbs control (n = 1). immunization with rrbd plus an optimized quantity of the cpg (500 mg) as the adjuvant as used in other vaccine studies [33, 34] was the most effective for the induction of rbd-specific antibodies, with endpoint igg titer of 102,400 after third boost. although this was a limited pilot experiment using only two concentrations of the e. coli expressed rov-asp-1 adjuvant for the formulation (50 mg, 100 mg), which was two or four times the amount used in our mouse model experiments (25 mg), we clearly show that immunization with 50 or 100 mg of the rov-asp-1 adjuvant exhibited a dose-dependent efficacy in the induction of rbd-specific antibodies in the two macaques per group; endpoint igg titers of 3,200 (50 mg ) or 6,400 (100 mg). notably, the anti-rov-asp-1 antibody response titers to the adjuvant itself were lower in the nhps than we might have expected based on the data in mice; the range in all the 4 immunized macaque monkeys was: 1:100-1:800 after first boost, 1:800-1:1,600 after second boost and 1:200-1;1600 after the third boost. although the anti-rbd endpoint tiers were much higher in the nhp that was immunized with cpg, the differences in the nt 50 titers were less prominent. sera from all rhesus macaques vaccinated with the rrbd+adjuvant formulation effectively neutralized the infection of sars pseudovirus in 293t cells expressing the receptor ace2 (ace2/293t) with nt 50 values of 1:3,500-1:6,392. as shown in figure 4 , sera from macaques immunized with rrbd protein plus 100 mg rov-asp-1 induced a slightly higher titer of neutralizing antibody than the 50 mg rov-asp-1 group; 4,167/5,376 vs. 3,724/3,509. the macaques that were immunized with cpg had a higher nt 50 titer at 1:6,392. in an in vitro study, as described above, we showed that mice immunized with rrbd+rov-asp-1 had a strong cytokine production ex vivo when stimulated with rrbd protein or rbd-specific peptides n50 and n60. notably, only a significant tnf-a response was obtained in nhps that were immunized with rrbd in the presence of rov-asp-1 or cpg ex vivo when pbmcs were stimulated with 5 mg/ml of rrbd ( table 4 ). the cytokine response was similar regardless of the amount of rov-asp-1 used as the adjuvant. all other secreted cytokines, such as ifn-c, il-2, il-4, il-5 and il-6 were negligible. vaccines remain the most effective means of preventing or eradicating infectious diseases, and there are ongoing efforts to apply active immunization approaches to prevent and treat table 3 . rbd-specific igg responses in nhps immunized with rrbd in the presence of rov-asp-1 or cpg adjuvant. table 4 . tnf-a response in immunized nhps. autoimmune diseases and cancer [35, 36] . adjuvants potentiate antigen-specific immune responses and can be a key element of vaccine effectiveness [1, 37, 38] . adjuvants can be broadly separated into two classes based on their principal mechanism of action: vaccine delivery systems and immunostimulation. vaccine delivery systems are generally particulate and function mainly to target associated antigens into apcs, e.g., emulsions, microparticles, iscoms, and liposomes [6, 39] . immunostimulatory adjuvants contain residues that are recognized by receptors on apcs, such as tlrs, which play an important role in the innate recognition of pathogens by dcs, and thus directly activate innate immune responses. these adjuvants are now regarded as the most effective means by which an adjuvant-antigen complex can target apcs [40] . adjuvants targeting multiple innate immune receptors may prove to be the most effective adjuvants, as they may induce different arms of the immune responses in the host. a number of microbial products, including bacterial lps, peptidoglycan, dsrna, muramyl peptides, cpg, flagellin and microbial proteins, were shown to act as vaccine adjuvants [41] [42] [43] [44] [45] . some of these immunomodulators could skew acquired immune responses towards a th1-type immune response. adjuvants can also be classified according to their capacity to stimulate either innate or adaptive immunity based on significant differences in their cellular receptors and mechanisms of action [46] . as previously noted, alum is the only adjuvant licensed in the u.s. for general use in humans. yet, alum is not effective in stimulating th1 and/or cytotoxic t cell responses to a number of pathogens and is therefore limited in its applications, in particular for new-generation vaccines [47] . some of the adjuvants being developed in clinical testing include mpl, the saponin derivative qs-21, cpg, flagellin, and combinations of some of these adjuvants [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] . one of the major concerns regarding the use of immunostimulatory adjuvants in humans is the possible increased risk of autoimmune diseases due to targeting pattern recognition receptors by such adjuvants. however, the recombinant ov-asp-1 adjuvant we studied corresponds to a secreted filarial protein, which is presented as an antigen in the o. volvulus exposed or infected individuals in africa. there is no evidence to show that this secreted filarial protein could induce autoimmune disease in the infected patients, thus excluding such a concern. polarized th1-type immunity can be achieved by the addition of complete freund's adjuvant and cpg dna to an antigen [52] [53] [54] . on the other hand, th2 antibody responses can be induced by the alum or incomplete freund's adjuvant, as indicated by increased igg1 relative to igg2a [53] [54] [55] . however, in situations where both th1 and th2 responses are required for protection, the choice of one regimen over another might be counter effective. this has led to additional research for alternative adjuvants or adjuvant combinations that promote balanced mixed th1/th2 responses. the present study clearly demonstrated that rov-asp-1 could effectively induce mixed rbd-or ha-specific th1/th2 antibody associated responses, when used as an adjuvant with recombinant subunit vaccine or as an addition to a commercial flu vaccine (fluvirin; using 20% of the dose recommended for human use). since the rov-asp-1 adjuvant is a protein, it is potentially processed and presented to the immune system and subsequently induces antibodies against self. therefore, concerns might be raised whether preexisting anti-rov-asp-1 antibodies may suppress its adjuvanticity when it is used in subsequent vaccine formulations. previously we demonstrated that antibody response to the adjuvant itself did not hindered the development of ova, rs or rrbd antigen-specific antibody responses after each boost; in all cases the responses in the presence of rov-asp-1 were more elevated than those in the presence of mpl+tmp or alum adjuvants. our present results further confirmed that antibodies induced against the adjuvant had no impact on its ability to induce immune responses against bystander antigens when used as an adjuvant in a sequential vaccine when two vaccine model systems were used: the rbd of sars-cov spike protein and a commercial influenza virus ha vaccine comprised of three virus strains. even though a high level of anti-rov-asp-1 antibodies (1:256,000-1:512,000) was present in the sera of mice before the administration of the second vaccine, the adjuvanticity of rov-asp-1 was retained with efficacy similar to that obtained when it was used as an adjuvant in a first vaccine immunization of naïve mice (table 2 and figure 3) ; no difference in the igg titers was observed between the two vaccine groups, mice pre-immunized with rrbd+rov-asp-1 or pbs. thus, we confirmed that the preexisting anti-rov-asp-1 antibodies induced by a previously administrated rov-asp-1-based vaccine do not have a significant effect on the adjuvanticity of rov-asp-1 when formulated in a subsequent vaccine. notably, immunization of nhps with rov-asp-1 did not induce high titers of anti-self-antibodies, for reasons that are not yet clear. future studies will be needed to further validate that even these reasonably low antibody responses to the adjuvant have no impact on subsequent use of this adjuvant with other vaccines also in nhps. the pilot immunogenicity studies in the nhps have provided us with extremely valuable ''proof of principal'' information in an outbred primate model. firstly, no adverse reactions were observed at the site of the immunizations, indicating the safety of rov-asp-1 as an adjuvant. secondly, using two concentration of the rov-asp-1 adjuvant, 50 or 100 mg, and rrbd as the vaccine antigen, we were able to induce after three immunizations high titers of neutralizing antibodies (1:3,500-1:6,392) that much exceed what is needed for protection against sars-cov infection in vivo (.1:500) [56] . moreover, our studies have shown that rov-asp-1 was able to support the induction of functional antibodies against a pathogen in nhps, thus clearing the way for its future development for human vaccines as well. thus, this pilot nhp study is a definitive step for demonstrating relevance of rov-asp-1 for human vaccine formulations, even though more studies will be needed to find the optimized amount of the adjuvant in vaccine formulations, which will specify the putative starting adjuvant dose for future clinical trials of rov-asp-1-based vaccines. interestingly, we did not see augmented recall rbd specific cellular responses except tnf-á. therefore, future studies will be needed to validate the adjuvanticity of rov-asp-1 in primates from the point of its ability to augment the desired types of cellular responses are associated with protective immunity against possibly other pathogens, duration of immunity and the potential to establish t cell memory. in summary, the present studies have advanced our confidence that the rov-asp-1 protein adjuvant can be further developed for human use, particularly when strong functional balanced antibody responses are needed against the pathogens. importantly, the rov-asp-1 that was used to immunize mice previously immunized with rbd+rov-asp-1 or the naïve mice with the ha vaccine was expressed in the yeast. having a functional yeast expressed adjuvant will support future development of a scalable process for the downstream manufacture of rov-asp-1, including the development of a series of critical biochemical and biophysical assays for in-process, release and stability testing, which will result in a high-yield reproducible production process and a stable product that is highly potent and stimulates the desired antibody and cellular responses to co-administered vaccine antigens in nhps for further analyses and for future clinical trials in humans. animal protocols were approved by the institutional animal care and use committee at the new york blood center (mice, protocols #255 and #194) and the tulane national primate research center (nhps, protocol #p0052). the tulane national primate research center tnprc is a usda-inspected and association for assessment and accreditation of laboratory animal care international (aaalac)-approved facility, and has an animal welfare assurance on file with the office of laboratory animal welfare. all animal studies were carried out in strict accordance with the recommendations of the american veterinary medical association (avma) guidelines and the approved protocols. the animals were handled delicately. any treatment was done with extreme care and professionalism to avoid any unnecessary discomfort for the animals. forty-two female balb/c mice aged 4-6 weeks and six purpose-bred adult male indian-origin rhesus macaques aged 6-10 years were used in this study. animal housing and environmental conditions met all applicable standards. blood was collected retro-orbitally for mice and from the femoral vein using the sarstedt s-monovette system for nhps. the animals were anesthetized using ketamine (0.1 ml/kg im), which ameliorate any suffering of the animals during the blood draw or immunization. the nhp protocol also included a full cbc and chem20 profile, which were taken at each blood drawing, with results falling within normal levels. their body weight was frequently monitored and physical examinations were performed regularly by the attending veterinarian. the rov-asp-1 protein was expressed as a histidine-tagged protein in escherichia coli and purified as previously described [27] . the purified rov-asp-1 was tested negative in a limulus amoebocyte lysate assay. a quantitative lps testing by cambrex bioscience showed that purified rov-asp-1 contained ,0.25 endotoxin units per milligram of the protein (25 pg endotoxin/ mg), and we considered it as an lps-free stock. in addition, we expressed rov-asp-1 in yeast. yeast codon optimized ov-asp-1 cdna sequence (af020586) without the region encoding the n-terminal signal peptide was cloned into the pichia expression vector ppiczaa (invitrogen) using the ecori and xbai restriction sites. the recombinant plasmid was linearized with saci digestion and transformed into pichia pastoris x33 strain as described previously [57] . the positive transformants were screened on zeocin-resistant ypd plates, and the highest expression clone was selected by scale up culturing. the expression of rov-asp-1 with 66his and c-myc tag at c-terminus was induced with 0.5% methanol and scaled up in 10 liters fermentation as described previously [58] . the rov-asp-1 was purified from the fermentation culture with sp sepharose fast flow exchange chromatography as described previously [58] . briefly, the fermentation supernatant was filtered through a 0.22 mm membrane and the ph was adjusted to 4.8 by adding glacial acetic acid. the positively charged rov-asp-1 was captured onto cation sp sepharose ff column and eluted with 50 mm sodium acetate, ph 4.8 containing 200 mm nacl. the eluted rov-asp-1 pool was then purified and buffer exchanged using gel filtration chromatography (sephadex g-25 fine) into pbs, ph 7.2 [28, 57] . the product was tested for its adjuvanticity in mice using ova as the model antigen as previously described [27] and it was established that 100 mg yeast-expressed rov-asp-1 per mouse was as effective as 25 mg per mice of the e. coli expressed rov-asp-1; both eliciting after two immunizations end point anti-ova igg titers of 1:25,600 (data not shown). thus, the e. coli or the yeast expressed rov-asp-1 at their optimal quantities can be used intermittently with assurance. mice were vaccinated subcutaneously with rrbd protein purified from culture supernatant of transfected human embryonic kidney cell-line 293t (hek293t) (atcc, va) according to the previously described protocol [31, 59, 60] using 20 mg/mouse of the protein mixed in aqueous solution with the e. coli-expressed optimized quantity of rov-asp-1 (25 mg/mouse) in 200 ml. as adjuvant controls we immunized mice with rrbd mixed with imject alum (40 mg/ml aluminum hydroxide+40 mg/ml magnesium hydroxide, thermo scientific) diluted 1:1 with the vaccine antigen in a total volume of 200 ml per mouse or with rrbd mixed with 50 mg cpg-odn1826 (invivogen, san diego ca) in a total volume of 200 ml per mouse. mice immunized with rrbd in pbs were used as the negative control. the mice were boosted twice 3 weeks apart with 10 mg/mouse of rrbd protein in pbs, rov-asp-1, imject alum or cpg. all mice were bled retro-orbitally under anaesthesia prior to immunizations and 10 days post injections. sera were stored at 280uc. to test the adjuvanticity of rov-asp-1 in a sequential vaccine model, we immunized intramuscularly naïve mice or mice that were previously immunized with the rrbd+rov-asp-1 with a commercial flu vaccine (100 ml/mouse) containing 3 mg (fluvirin, novartis) . the immunization was done in the presence or absence of the yeastexpressed optimized quantity of rov-asp-1 (100 mg/mouse). the mice were boosted once with the same dose of vaccines three weeks later. all mice were bled prior to immunization and 7 days post immunization as described above and sera were stored at 280uc. rhesus macaques were vaccinated subcutaneously with rrbd protein (50 mg) with either 50 mg (n = 2) or 100 mg (n = 2) of e. coliexpressed rov-asp-1, cpg-c-iss-odn c274 (500 mg; n = 1) (donated by dynavax technologies corporation, berkeley, ca) or pbs (n = 1). they were boosted with the same dose at 1-, 2-and 6month intervals. the macaques were bled before immunization and 7 days post-immunization. sera were collected for serological testing, and pbmcs were purified for in vitro assays. a baseline bleed was also provided. elisa was used to detect specific antibody responses induced in the vaccinated mouse or nhp. briefly, 96-well micro titer plates (costar) were coated with rrbd (1 mg/ml), rov-asp-1 (1 mg/ml) or has (2.5 mg/ml) at 4uc overnight. the plates were blocked with 2% non-fat milk in pbs-tween (0.05%) for 2 h at 37uc. after washing the plates six times with pbs-t, serial diluted sera in binding buffer (1% non-fat milk in pbs-t) were added into each well in duplicate and incubated for 1 h at 37uc. bound antibodies were detected with hrp-conjugated goat anti-mouse igg, igg1, igg2a or igg2b (molecular probes-invitrogen) (1:2000 dilution) or mouse anti-monkey igg-hrp (clone sb108a, southern biotech) (diluted 1:1000) in binding buffer. after incubation for 1 h at 37uc, plates were washed and tmb substrate (kpl) was added, and the reaction was stopped by the addition of 2 n h 2 so 4 . absorbance at 450 nm was measured using spectramax 190 (molecular devices, sunnyvale, ca). end point titers were defined as the highest dilutions giving an a 450 measurement of 0.1. this cutoff value represents the value of mean optical density (od) plus 2 standard deviations (sd) of 10 normal mouse serum samples tested at 1:100 dilutions or the 6 pre-bleeds from the normal nhp serum samples also tested at 1:100 dilutions. the neutralization assay against sars pseudovirus infection was performed as previously described [61] . in brief, plasmid dna encoding sars-cov spike protein and a plasmid dna encoding env-defective, luciferase-expressing hiv-1 genome (pnl4-3.luc.re) were co-transfected into hek293t cells (attc, ga). culture supernatant containing sars pseudovirus was collected at 72 h and used for single-cycle infection in vitro. the sars pseudovirus was incubated in the presence or absence of serially diluted antisera from vaccinated mice or nhps for 1 h at 37uc. subsequently, the antisera-virus mixtures were added to 293t cells expressing the sars receptor angiotensin-converting enzyme 2 (293t/ace2) in 96-well plates, and the infection was allowed to proceed for 48 h, followed by lysing the infected cells using cell lysis buffer included in the luciferase kit (promega). aliquots of cell lysates were transferred to 96-well costar flatbottom luminometer plates (corning costar), followed by addition of luciferase substrate (promega). relative light units were determined immediately in the ultra 384 luminometer (tecan). the neutralization of sars pseudovirus is presented as 50% neutralizing antibody titer (nt 50 ) [60] . th1/th2/th17 cytokine assay was used to estimate the corresponding cytokine production ex vivo from splenocytes of the vaccinated mice. briefly, splenocytes were harvested from the immunized mice and resuspended in rpmi 1640 medium (invitrogen, carlsbad, ca) supplemented with 10% fbs (hyclone laboratories, inc.), 2 mm l-glutamine, 10 mm hepes, and antibiotic-antimycotic (invitrogen, carlsbad, ca). the cells were plated at 4610 5 cells into 96-well u-bottom culture plates for in vitro stimulation with 5.0 mg of rrbd, or with the sars-cov rbd-specific n50 (cd8 + t cell epitope) or n60 (cd4 + t cell epitope) peptide [31] ; a concentration that was pre-determined to be optimal. cells were stimulated with or without pma (10 ng/ml) plus ionomycin (1 mg/ml) as the positive and negative controls, respectively. the plates were incubated at 37uc for 72 h, and the secreted cytokines were quantified from the culture supernatants using the mouse th1/th2/th17 bd cytometric bead array kit (bd biosciences) according to the manufacture's protocols. theoretical limit of detection data is il-2 = 0.1 pg/ml; il-4 = 0.03 pg/ml; il-6 = 1.4 pg/ml; il-10 = 16.8 pg/ml; tnfa = 0.9 pg/ml, inf-c = 0.5 pg/ml; and il-17a = 0.8 pg/ml. detection of the th1/th2 cytokine production in the vaccinated nhps was done using similar protocol as above with some modifications. pbmcs were isolated following a ficoll-hypaque density gradient (sigma). single-cell suspensions were then stimulated at 4610 5 cells with 5 mg of rrbd, n50 or n60 peptides, or pma (10 ng/ml) plus ionomycin (1 mg/ml) for positive control and culture media alone for negative control. the cells were stimulated for 5 days, and cytokines were quantified using the non-human primate th1/th2 bd cytometric bead array kit (bd biosciences) according to the manufacture's protocols. results are expressed as mean 6 sem. the data were analyzed using graphpad version 5.01 for windows (graphpad software). unpaired two-tailed student's t test or mann-whitney test was used to compare means between different groups. one-way anova with bonferroni post-test was considered appropriate for multiple comparisons. p value less than 0.05 was considered significant. alum's adjuvant action: grease is the word alum interaction with dendritic cell membrane lipids is essential for its adjuvanticity immunological mechanisms of vaccination vaccine adjuvants: putting innate immunity to work adjuvants for the future recent advances in vaccine adjuvants rationally-designed vaccine adjuvants: separating efficacy from toxicity enhancing oral vaccine potency by targeting intestinal m cells diversity and dialogue in immunity to helminths immune regulation by helminth parasites: cellular and molecular mechanisms t helper type-2 cytokine responses: potential therapeutic targets helminth antigens modulate tlr-initiated dendritic cell activation tlr11 activation of dendritic cells by a protozoan profilin-like protein rov-asp-1, a recombinant secreted protein of the helminth onchocerca volvulus, is a potent adjuvant for inducing antibodies to ovalbumin, hiv-1 polypeptide and sars-cov peptide antigens immune evasion genes from filarial nematodes helminth parasites-masters of regulation a novel therapeutic approach targeting articular inflammation using the filarial nematode-derived phosphorylcholine-containing glycoprotein es-62 es-62, a filarial nematode-derived immunomodulator with anti-inflammatory potential modulation of a heterologous immune response by the products of ascaris suum taenia crassiceps carbohydrates stimulate il-6 expression in naive murine macrophages via toll-like receptors (tlrs) a novel host-parasite lipid cross-talk. schistosomal lyso-phosphatidylserine activates toll-like receptor 2 and affects immune polarization proteins secreted by the parasitic nematode nippostrongylus brasiliensis act as adjuvants for th2 responses selective maturation of dendritic cells by nippostrongylus brasiliensis-secreted proteins drives th2 immune responses lacto-n-fucopentaose iii found on schistosoma mansoni egg antigens functions as adjuvant for proteins by inducing th2-type response maturation of dendritic cell 2 phenotype by a helminth glycan uses a toll-like receptor 4-dependent mechanism a novel synthetic adjuvant effectively enhances the immunogenicity of the influenza vaccine recombinant ov-asp-1, a th1-biased protein adjuvant derived from the helminth onchocerca volvulus, can directly bind and activate antigen-presenting cells induction of protection against divergent h5n1 influenza viruses using a recombinant fusion protein linking influenza m2e to onchocerca volvulus activation associated protein-1 (asp-1) adjuvant evaluation of recombinant onchocerca volvulus activation associated protein-1 (asp-1) as a potent th1-biased adjuvant with a panel of protein or peptide-based antigens and commercial inactivated vaccines a novel, helminth-derived immunostimulant enhances human recall responses to hepatitis c virus and tetanus toxoid and is dependent on cd56+ cells for its action priming with raav encoding rbd of sars-cov s protein and boosting with rbd-specific peptides for t cell epitopes elevated humoral and cellular immune responses against sars-cov infection intranasal vaccination of recombinant adeno-associated virus encoding receptor-binding domain of severe acute respiratory syndrome coronavirus (sars-cov) spike protein induces strong mucosal immune responses and provides long-term protection against sars-cov infection local and systemic effects of intranodally injected cpg-c immunostimulatoryoligodeoxyribonucleotides in macaques cpg-c iss-odn activation of blood-derived b cells from healthy and chronic immunodeficiency virus-infected macaques anticytokine vaccination in autoimmune diseases cancer vaccines. any future? trends in vaccine adjuvants recent developments in cancer vaccines adjuvants-a classification and review of their modes of action dendritic cell subtypes as primary targets of 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receptorbinding domain of sars-cov spike protein expressed in mammalian, insect and e. coli cells elicits potent neutralizing antibody and protective immunity protocol for recombinant rbd-based sars vaccines: protein preparation, animal vaccination and neutralization detection a 219-mer cho-expressing receptor-binding domain of sars-cov s protein induces potent immune responses and protective immunity we would like to thank dynavax technologies corporation for donating the cpg-c iss-odn c274 used in the nhp experiment. key: cord-011496-r8e19t0c authors: de rooij, doret; rebel, rebekka; raab, jörg; hadjichristodoulou, christos; belfroid, evelien; timen, aura title: development of a competency profile for professionals involved in infectious disease preparedness and response in the air transport public health sector date: 2020-05-21 journal: plos one doi: 10.1371/journal.pone.0233360 sha: doc_id: 11496 cord_uid: r8e19t0c background: recent infectious disease outbreaks highlight the importance of competent professionals with expertise on public health preparedness and response at airports. the availability of a competency profile for this workforce supports efficient education and training. although competency profiles for infectious disease control professionals are available, none addresses the complex airport environment. therefore, the main aim of this study is to develop a competency profile for professionals involved in infectious disease preparedness and response at airports in order to stimulate and direct further education and training. methods: we developed the competency profile through the following steps: 1) extraction of competencies from relevant literature, 2) assessment of the profile in a national rand modified delphi study with an interdisciplinary expert group (n = 9) and 3) assessment of the profile in an international rand modified delphi study with an airport infectious disease management panel of ten european countries (n = 10). results: we systematically studied two competency profiles on infectious disease control and three air transport guidelines on event management, and extracted 61 relevant competencies for airports. the two rand modified delphi procedures further refined the profile, mainly by specifying a competency’s target group, the organizational level it should be present on, and the exact actions that should be mastered. the final profile, consisting of 59 competencies, covers the whole process from infectious disease preparedness, through the response phase and the recovery at airports. conclusion: we designed a profile to support training and exercising the multidisciplinary group of professionals in infectious disease management in the airport setting, and which is ready for use in practice. the many adaptations and adjustments that were needed to develop this profile out of existing profiles and air transport guidelines suggest that other setting-specific profiles in infectious disease control are desirable. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 to the best of our knowledge, there is currently no profile that describes the competencies that professionals involved in infectious disease management at airports need. therefore, this study aimed to develop such a profile that describes the competencies that a multidisciplinary group need-in addition to the basic knowledge and skills required for their profession-when called upon to prepare and respond to infectious diseases in the airport environment. we performed the systematic rand modified delphi consensus procedure [31] to develop a competency profile for professionals involved in infectious disease preparedness and response at airports. competencies were extracted from existing competency profiles and air transport guidelines on event management and consecutively presented to a national and an international panel of professionals. both panels followed a methodologically identical path (fig 1) that consisted of two rounds of data collection. first, panels assessed the relevance of competencies regarding their expertise during a digital questionnaire. next, consensus meetings were held to gather consensus on undecided competencies. the refined final competency profile based on the first national round formed the concept competency profile for the international validation. this method is commonly used for the development of competency profiles [32] , using the collective subjective judgment of professionals [33] . step 2 and 3 were performed twice in this study: first with a national expert panel and after with an international expert panel. the study population of the national panel consisted of professionals involved in infectious disease preparedness and response at schiphol airport. this airport is the only ihr designated airport in the netherlands, one of europe's largest airports and a major hub in international air traffic [34] . the study population of the international panel consisted of professionals involved at a local or national level in infectious disease preparedness and response at major european airports. the research team aimed for two panels consisting out of 8-15 participants [31] , resulting in a national panel of 9 and international panel of 10 participants. in this way, we tried to assure both enough reflection of different opinions and a setting for a safe and fruitful discussion. the panels were recruited using purposive sampling according to inclusion criteria until a representative panel was composed or time was limited for further inclusions. the research team composed an invitation letter by e-mail explaining the background, aim, and method of the study for both panels, through which professionals could confirm their participation. for the national panel, we consulted an experienced nurse from the public health service at schiphol airport to discuss our sample. we invited experienced professionals for the national round from the following disciplines: international medical advice, airport medical services, public health service, air traffic control, flow management of aircrafts and passengers, airport fire officer, continuity & crisis management, and the disaster medicine organization. international medical advice, airport medical services, and the public health service collaboratively ensure that the infectious disease policy is feasible and effective at the airport. the public health service is responsible for the public health response. at their request, international medical advice facilitates medical items on board, such as personal protective equipment, and debrief crew and ground staff of an affected aircraft. in the situation that medical care is required, the ambulance will assist passengers upon arrival. professionals from air traffic control receive warnings from the pilot in command of a possibly affected aircraft and are responsible for informing the flow management of aircrafts and passengers which adjusts logistical processes at the airport. the airport fire officer ensures coordination among various emergency services during the management of an infectious disease case. the continuity & crisis management is responsible for advising on risk, crisis, and physical safety management. disaster management organization provides training in the field of infectious disease control at the airport. the international panel was recruited by selecting professionals who participated in a european, face-to-face 3-day training course on infectious disease control at designated airports. this course was part of the european union (eu) joint action healthy gateways [35] and took place on 18-20 september 2019 in belgrade, serbia. we used these professionals' selfdeclared competence on local and national level before the training to approach a variety of professionals. on the local level, a functional distinction was made between competence in preparedness planning, decision making, and implementation. we invited professionals in all competence areas and tried to include one participant per country, with countries geographically divided over europe. step 1 -literature search and extraction of competencies. we performed a literature search to identify competency profiles and air transport guidelines on event management. the search was executed in the 4th week of march 2019 using pubmed and google scholar. for competency profiles, search terms were 'infectious diseases' or 'public health', and 'competency' and their synonyms. for the airport guidelines, search terms were 'infectious diseases' or 'emergency preparedness' or 'public health event', together with 'airport' or 'air travel' or 'points of entry'. in pubmed, the search terms were limited to the title or abstract. in google scholar, we searched several combinations of search terms and screened the results until 20 hits in a row were irrelevant. the search terms, the search strategy, the screening, and application of the selection criteria are shown in s1 file. regarding the competency profiles, the criteria for title/abstract screening were whether studies aimed at presenting competencies, and were aimed at infectious disease preparedness or control. inclusion criteria for the full-text screening of the competency profiles were the explicit presentation of a competency set in the study, competencies aimed at graduate level or professionals in practice, applicable to infectious disease management. further criteria to select the landmark studies contained a full array of competencies, being a peer-reviewed study, and being relevant for the airport setting. regarding the airport guidelines, the criteria for title/abstract screening were documents describing infectious disease management in the air travel setting with a disease-generic approach. the full-text screening inclusion criteria were documents aimed for the airport or air travel setting, prescribing infectious disease management in terms of specific preparedness and response tasks, with a disease a-specific approach. further selection was made based on the target group at airport level, whether unique topics were prescribed compared with other included studies, and presenting a full array of the preparedness and response process. the competency profiles and air transport guidelines were matched to develop a concept competency profile, using the following steps. first, we extracted main tasks for infectious disease preparedness and control from the guidelines that cover infectious disease management at airports during preparedness, response, and recovery. then, the competency profiles and air transport guidelines were independently reviewed by two researchers (ddr & rr) to extract competencies or textual fragments that could be reformulated into a competency, and which were relevant for a task. results were compared and dissimilarities were discussed until consensus was reached. subsequently, a data reduction round took place by applying three specifying criteria. first, we further narrowed the scope to the airport environment itself instead of competencies required regionally or countrywide. in addition, we restricted competencies on hygiene to the commanding level and excluded all operational cleaning. lastly, competencies or textual fragments regarding general team competencies were excluded in the profile because the team resource management (trm) skill set is already available and includes these [36] . the trm skill set, developed for aviation safety, covers skills such as interpersonal communication, leadership and decision making, and is complementary to our competency profile. finally, we combined overlapping or congruent competencies and fragments. the included textual fragments were reformulated into competencies according to bloom's taxonomy of education objectives [37] . data reduction was performed by rr & ddr collaboratively, all doubts and several versions of the draft profile were discussed with other researchers (eb and at). next to specifying competencies to a task, we also specified them to a role to further enhance the logical structure and the usability of the profile. for this purpose, we adapted the canmeds roles [38] , which are widely used in medical education, into the following roles required in infectious disease management: (1) health expert, (2) organizer (including policy development), (3) scholar. roles with respect to the communicator, professional and collaborator are required in general, and are, therefore, considered general tasks. during several discussion meetings with a third researcher (at), competencies were divided over tasks and roles and overlapping competencies were removed. this first step resulted in a concept competency profile. step 2 -the national panel: digital questionnaire & consensus meeting. the next step contained the assessment of the draft competency profile in the national panel. a digital questionnaire was built in the web-based program formdesk [39] . it was successfully pilot-tested on comprehensibility and usability by an experienced medical trainer working at the national institute for public health and the environment, and a physician specialized in infectious diseases from the maastricht aachen airport. the digital questionnaire started with information regarding the aim and process of the study and key definitions. also, participants received supportive documents on formulating competencies, according to bloom's taxonomy of educational objectives [37] . in addition, a privacy statement was included. the first questions were related to demographic characteristics, such as their gender, profession, the number of years in their current profession, and their experience with infectious disease preparedness and response at the airport on a 5-point likert scale (1 = very inexperienced, 5 = very experienced). we requested e-mail addresses for planning the consensus meeting. subsequently, the relevance of competencies in the concept competency profile was graded by the participants using the following question: 'to what extent do you consider this competency as a relevant element for infectious disease preparedness and response at airports'? scoring was done using a 9-point likert scale (1 = totally irrelevant, 9 = totally relevant). the introduction, privacy statement, and demographic questions were in dutch, but the questions regarding the relevance of competencies, as well as the competencies were in english. all questions on relevance were mandatory. participants could comment on individual competencies as well as suggest new ones per category. after grading each competency, participants were asked if the competency profile reflected their knowledge, skill, and attitude regarding infectious disease preparedness and response at the airport. data were collected in the three weeks following 9 may 2019. participants who completed the digital questionnaire were invited to the consensus meeting. participants received a personal feedback report in advance of the meeting. this report showed the group ratings of each competency, together with the participants' individual ratings. participants could therefore identify major dissimilarities within ratings beforehand. the purpose of the meeting was to reach consensus by discussing these dissimilarities face to face. the moderator asked all participants to keep an incident in mind while considering the relevance of each competency. first, uncertain competencies were discussed, then competencies proposed by participants themselves, and lastly the suggestions for the reformulations of relevant competencies. if the participants could not reach consensus, a voting round was held to either accept, textually amend or reject the competency based on the majority opinion. after discussing all competencies, participants were asked whether the refined final competency profile reflected their knowledge, skills and attitude. finally, the usability level of this profile was discussed, including any further suggestions for improvement. the national meeting was in dutch because this was the native language of all participants. the meeting took place on 21 june 2019 at the training center of schiphol airport. an experienced moderator (at) led the discussion. step 3 -international validation: digital questionnaire & consensus meeting. the competency profile from step 2 functioned as the starting point for international validation. the competencies were already stated in english, but we had to translate the introduction, statements, and demographic questions into english. we asked the respondents in the introduction to keep a recent incident or outbreak in mind during the completion of the questionnaire and asked them to specify which event this was. we added numbers to the tasks to manage expectations on the size of the competency list during completion, and added a suggestion for additional competencies on data protection and privacy. except from these changes, this international study was methodologically identical to the national study. the participants were invited on 12 august 2019 and were reminded twice by e-mail after two and four weeks. the data collection through the questionnaire ended on 13 september 2019. the consensus meeting took place on 19 september 2019 in belgrade, serbia, and was moderated by an experienced moderator. data from formdesk was transferred to an excel file and checked on irregularities. first, a descriptive analysis of demographic characteristics was performed. secondly, the median rating and the amount of dispersion of ratings between participants were calculated for each competency. if the competency had a median within 7-9 range and �70% of the participants scored it in the top tertile (score 7,8 or 9), then the competency was marked as 'accepted'. if the competency had a median of <7 and <70% scored in the top tertile, then the competency was marked as 'not accepted' and excluded. if the median score laid between 7-9 and <70% of the participants scored in the top tertile, then the competency was marked as 'uncertain'. in the national study, competencies with scores spread over all tertiles, despite any median, were also classified as 'uncertain'. table 1 shows the classification of the competencies by the participants' median score and level of agreement. as the digital questionnaire contained openended questions and formulated competencies by the participants, responses were grouped and coded accordingly. to support the reliability, two researchers (ddr & rr) performed the data analysis independently and compared their results. the consensus meetings were audio-taped and in outline transcribed. a distinction was made between individual opinions and contributions to the group consensus. in accordance with the general data protection regulation, no names were used in the transcript. each participant received his or her own code, making it visible which recordings belong to the same professional. the codes of each participant were kept confidential and were only accessible to two researchers (rr and ddr). two researchers (rr and ddr) performed the data coding and analysis of the consensus meeting independently. the researchers discussed dissimilarities until consensus was reached. after the analysis of the international consensus round, an official translator reviewed the competency profile on use of language. the study protocol (lci-413) was reviewed by the clinical expertise centre of the national institute for public health and the environment. based on this review, they determined that the research plan did not fall under the scope of the dutch law on medical research involving humans. all necessary precautions were taken to protect the anonymity and confidentially of the participants; in the invitation letter, participants were informed about their voluntary participation and informed that they were free to decline at any time. furthermore, the participants were informed that their responses were processed anonymously and only used for research purposes. the literature search for competency profiles resulted in 23 unique studies included in titleand abstract screening, and 7 studies included in the full-text screening, of which two had the highest applicability and were therefore selected. these profiles were aspher's european list of core competences for the public health professional [28] and public health emergency preparedness-core competencies for the eu member states [40] . the competency profile for infectious disease management at airports the literature search for airport guidelines resulted in 29 unique documents. nine were included during the full text screening. three guidelines had the highest applicability and were therefore selected, being the handbook for the management of public health events in air transport [29] , coordinated public health surveillance between points of entry and national health surveillance systems [41] and the guide for public health emergency contingency planning at designated points of entry [42] . during data extraction, fifteen tasks and 255 competencies were selected from the source documents. after data reduction, around 110 competencies and exactly eleven tasks were left. aggregating double competencies resulted in 34 competencies which originated from existing competency profiles and 27 from reformulated textual fragments from the air transport guidelines. as a result, the concept competency profile consisted of 61 competencies divided over eleven tasks ( thirty professionals were approached for the national study by e-mail. ten were approached personally; professionals from the afo and ams were approached as a group (n = 20). professionals from phs, ima, ams, aas, and dmo agreed to participate, professionals from atc, afo, and fma could not participate in the study due to time constraints. however, one participant from aas had previously worked at the afo and voluntarily stated as a note in the questionnaire to keep this experience with afo in mind while filling in the questionnaire. nine out of ten included participants that completed the questionnaire had extensive experience in infectious disease preparedness and response at schiphol airport. the input from the dmo participant was not included in the analyses since she pointed out in an e-mail to the researchers that she is solely involved in the organization of training courses and therefore unable to grade the competencies in terms of relevance. sixteen professionals from thirteen countries were approached for the international study, of which ten from ten countries (austria, cyprus, germany, italy, ireland, malta, slovenia, spain, switzerland, and poland) accepted to participate and completed the questionnaire. nine professionals (from austria, cyprus, germany, italy, ireland, slovenia, spain, switzerland, and poland) attended the consensus meeting. their self-assessed competence areas were equally represented among professionals with most representing more than one area: six from the ten professionals were involved in preparedness planning, six in decision making and six in implementation of measures at the airport level. on the national level, five of ten participants were involved with preparedness planning. table 2 shows the demographic characteristics of the included participants. the first questionnaire round resulted in 40 "accepted" competencies, 3 "irrelevant" competencies, and 18 competencies that were marked as "uncertain" (fig 3) . participants provided three main reasons for their low grading of relevance: (1) not relevant to all professionals; (2) concerning individual risks and not aimed at public health; (3) out of scope, primarily focused on other areas (e.g., laboratories). the inconsistency in relevance scores of uncertain competencies reflected different professions. the participants proposed 23 additional competencies. three participants confirmed, five partly, and one denied that the competency profile reflected their competencies in infectious disease preparedness and response at airports. no irregularities were found within the data analysis. five of nine participants attended the consensus meeting. the participants from dmo, ams, and one from aas could not attend the meeting due to time constraints. the"uncertain" competencies were discussed of which three were excluded, ten were included after textual amendment, and five were included without textual amendment. textual amendments included adjusting the target group described within the competency or adapting a verb from an executive to an advisory focus. of the 23 suggestions for additional competencies, three were included. these included competencies entailed the understanding of the logistical structure and functioning of airports, implementing contact tracing, and contacting professionals with extensive epidemiological knowledge for outbreak investigation. ten competencies that were already included were reformulated. the group reached consensus for all competencies, no voting rounds were initiated. the reflection after the consensus meeting resulted in several the competency profile for infectious disease management at airports strengths and difficulties of the profile. during the national study, participants stated that they had enjoyed discussing the profile and this had improved their understanding of roles and responsibilities of different professions involved. several participants involved in design and organization of training and exercises would use the profile either to derive training goals or as a background document for participants. suggestions for improvements were to design a better manageable length or more practical format of the profile, either by splitting it for different disciplines, or connecting competencies to specific tasks. one participant indicated that the content of the competencies had become more evident after the meeting ( table 3 ). the questionnaire round with the international panel resulted in 51 "accepted" competencies, seven that were marked as "uncertain", and one "irrelevant" competency. the excluded competency entailed balancing costs and results during ph response. the inconsistency in relevance scores of uncertain competencies reflected different professions. the participants proposed twelve additional competencies. seven participants confirmed, and three partly confirmed that the competency profile reflected their competencies in infectious disease disaster medicine organization (dmo) 1 0 airport level-ph decision making 6 5 airport level-ph preparedness planning 6 6 airport level-ph measure implementation 6 5 national level-ph preparedness planning 5 5 the competency profile for infectious disease management at airports preparedness and response at airports. no irregularities were found within the data analysis. six of the ten participants stated to have imagined a case during completion of the questionnaire, of which all referred to evd or viral hemorrhagic fevers in general. other named diseases were seasonal influenza, tuberculosis and measles. nine out of ten participants attended the consensus meeting. during discussion of the first competency, the group initiated a voting procedure which was used during the majority of competencies. the"uncertain" competencies were discussed, of which three competencies for surveillance and risk assessment were excluded because they were not considered as tasks at the local level. three competencies were included without textual amendments, and one after further specification of the target group. the only excluded competency in the questionnaire round-on taking the costs in consideration during ph response-was decided yet to be the competency profile for infectious disease management at airports included. of the twelve suggestions for additional competencies, four were added to other existing competencies, five were declined, and three were included. these included competencies entailed knowledge of specific terminology in use, data management, and network management of key partners to assure rapid response and recovery. analysis of the discussions during and after the consensus meeting indicated that people had sometimes doubted the relevancy of certain competencies due to difference between their own functional level and the scope of the profile (airport level). also, the differences between several countries in the division of tasks and responsibilities became clear. participants would use this profile as a source for training goals at the airport or as a background document during training ( table 3) . to enhance the profile in any kind, they suggested to use the profile during a future training or exercise or to discuss it with their entire team at a local airport. the final competency profile (table 4 ) consisted of 59 competencies that were categorized into eleven tasks: communication (c = 3), collaboration (c = 4), professionalism (c = 2), training (c = 3), contingency planning (c = 9), surveillance (c = 5), risk assessment (c = 6), outbreak investigation (c = 8), management of ill / exposed travelers (c = 6), public health measures (c = 9), and evaluation and recovery (c = 4). the majority of competencies entailed the roles of the health expert (c = 26) or organizer (c = 20). four competencies involved the scholar who is needed during outbreak investigation and implementation of health measures. in this study, we developed and validated a competency profile for professionals involved in infectious disease preparedness and response at airports. to the best of our knowledge, this is the first systematically developed competency profile describing the competencies for the air travel environment. the multidisciplinary consensus procedure ensures coverage of all major aspects of preparedness, response and recovery; the international consensus procedure with experts from various countries provides content and face validity to the competency profile and increases its potential for international application. a setting-specific profile is required because of the specific requirements according to international regulations [9] , and the numerous actors which are involved from a range of sectors "i would use it as a source of competencies that you like to address in a training or exercise or anything like that. so, if you are running a table top or live exercise at the airport, these are the aspirational competencies that you would like, not only for the public health staff but for the whole response." "i really like it actually that you make this profile especially for the point of entry. i have never seen anything like this before but it is really nice. i consider it to be a good reflection of someone being responsible for working directly and being involved in public health measures at the airport." pha = public health authority at the airport; ima = international medical advice; md = medical doctor; f = female; m = male. https://doi.org/10.1371/journal.pone.0233360.t003 the competency profile for infectious disease management at airports table 4 . competency profile for professionals involved in infectious disease preparedness and response at airports. communicator • understand and implement the basic principles of risk communication to airport and airline staff, travelers, the public and media. • establish trust with airport and airline staff, travelers, the public and media by using rapid communication channels and ongoing two-way communication. • understand the terminology used by different levels and organizations at the airport. professional • minimize the discomfort or distress associated with public health measures experienced by crewmembers, ground staff, and passengers. • apply relevant laws to data collection, storage, management, dissemination and use of information. • understand the importance of multidisciplinary collaboration during acute outbreak management. • be an effective team member, adopting the role necessary to contribute constructively to the accomplishment of tasks by the group. • participate in the implementation of established plans which ensure the continuity of operations. • create and manage a network of key partners in rapid response and recovery. health expert • provide training and exercises on communication within, and between, involved airport organizations and include healthcare providers in this training. • identify training needs, and plan and organize courses. • periodically practice and test the ability to make decisions in unpredictable circumstances. health expert • be familiar with job-related standards and recommended practices concerning infectious disease control of national and international aviation organizations (iata, icao and capsca). • periodically assess whether the implementation of strategies, standard operating procedures (sops) and action plans requires any changes. • before the response operation, identify which triggers will require key decisions to be made during the outbreak response (keeping in mind that triggers may need modification to fit specific situations). • before the response operation, plan for the storage and stockpiling of medical and non-medical countermeasures. • understand the logistical structure of the airport and the international context of airports and their functioning. • identify key partners and develop a common understanding of roles, resources, planning assumptions, risks or vulnerabilities, and information needs. • support core-capacity-building at the airport and understand the importance of it. • develop, test and evaluate a public health emergency contingency plan (phecp) on a periodical basis. • provide healthcare workers with clinical guidelines for emerging infections from abroad, especially those that may be carried by travelers and the severely contagious. health expert • recognize a potentially infectious disease by key symptoms and signs of events among travelers. • understand the relevance of early detection of public health threats. • understand the components of surveillance systems and how these work. • understand the roles and responsibilities of local, national and international organizations involved in infectious disease control. • be familiar with laws on the surveillance and reporting of infectious diseases at national, european union level and globally (international health regulations). (continued ) health expert • understand risk analysis frameworks, with the elements of risk assessment, risk management and risk communication. • determine when a risk assessment should be carried out, and appropriate measures taken. • perform a risk assessment and continuously review the risk assessment as further information becomes available. • interpret the diagnostic and epidemiological significance of laboratory tests reports. • collect and integrate the facts of an event, based on information from multiple sources, including the traveler, the aircraft operator, ground-based medical services for aircraft in flight (when available) or the agent responsible for the baggage or cargo. • know when case reports or clusters require further investigation, and how to initiate such investigations. health expert • conduct outbreak investigations to identify pathogens and other agents, characterize affected population groups, and sources of exposure. • use reliable systems for disseminating case definitions to standardize both the diagnosis and the reporting of case numbers (e.g. confirmed, suspected, probable, or possible, cases). • systematically generate required information about the number of travelers such as those targeted for screening, screened, referred to secondary screening, and identified as confirmed cases. • implement contact-tracing based on a careful, case-by-case, risk assessment basis, taking into account factors such as feasibility, the severity of the disease and its potential for epidemic spread, the infectivity of index patients, and the duration of the trip. • identify who is responsible at national level for receiving the information on the investigation from the local or intermediate level health authority. • maintain up-to-date and job-specific knowledge about characteristics of infectious diseases such as the reservoir, potential sources, modes of transmission, risk groups, and duration. • be able to contact professionals who have the biological, clinical, and epidemiological knowledge necessary to characterize (potentially novel) pathogens and other agents responsible for an outbreak disease. • use evidence-based methods to identify and recommend control and preventive measures to control an outbreak. health expert • provide ground-based medical support (gbms) regarding infectious disease events, including medical recommendations to manage the discovery of a suspected communicable disease during flights, to support decisions regarding medical treatment and use of on-board medications or equipment. • assess the health status and travel history of travelers arriving from, or going to, an affected region, or who have been exposed to a potential public health risk during air travel. • provide disembarking travelers with information regarding the precautions to take in the event of illness, information sources for any updates on the event and the public health authority (pha) contact information where subsequent enquiries can be made. • provide advice concerning the appropriate parking stand for an incoming affected aircraft and the order of disembarkation of passengers. • provide advice to ensure port health staff respond efficiently so as reduce the time that travelers spend on a board-affected aircraft, and identify space requirements for interviews and health assessments of arriving travelers. • provide advice on a traveler's possible transfer to a medical facility by ambulance and facilitate the rapid transport of suspected cases of an infectious disease. other than public health [43] . as such, airports function as coherent subsystems within the broader public health system. the aspher's european list of core competences, which we used as a main source, acknowledges the challenge to apply their general public health competency profile to such systems [28] . however, the use of competencies can be pivotal in targeted and effective training for professionals, and the evaluation of their competence, as is proven by many studies [18, 36, [44] [45] [46] . to support the development of well-functioning infectious disease systems at airports, we made general formulations more specific and added setting-specific knowledge, skills and attitudes to end up with this airport setting-specific competency profile. we, therefore, demonstrate in this study, that it is possible to design a competency profile for airports across countries. presumably, other points of entry, such as ground-crossings or ports, would also benefit from a setting-specific profile. these have a regulated and sustainable role in prevention of cross-border spreading of diseases and face comparable challenges to airports. examples are fisheries [47] , drilling platforms [43] , and many other mass gatherings [48] or other places imaginable where an international transfer of people and goods take place, and where infectious disease diseases might be introduced. during the development of the profile, we learned more about the possibilities and challenges of competencies. both during the extraction of competencies from the literature, as well as during the consensus meeting we experienced that the concise and theoretical formulations of the competencies sometimes thwart their usability. it happened to us, as well as to the participants, that competencies had to be read and reread out loud before the full meaning of a competency was fully understood. the usability of the competency profile can be enhanced in several ways. for example, the use of entrustable professional activities (epa), as used in medical education, may bridge a potential gap between the theory of competencies and practice. an epa tells whether a professional can be entrusted to carry out all critical activities of a major task [19] . they integrate the demonstration of competence with the respective supervision level and hereby form a usable tool during education or training activities. because our profile covers competencies required by the team of professionals involved in infectious disease management, participants experienced that competencies could only be translated into practice in close collaboration with different disciplines replicating findings of previous studies [49] . another possibility to increase the usability of this profile is to merge disciplinary competencies into functional roles at an airport. in line with recommendations from • equip relevant airport and airline staff with information regarding the public health event so that they can protect themselves and safeguard healthy travelers as required. • organize the use of public health measures underpinned by scientific evidence and expert public health opinions, so as to avoid any contradictory or unnecessary restrictions of individuals. health expert • clearly define goals and objectives of the evaluation of training, exercises or real response. • develop a formal evaluation of the response, including recommendations for prevention and mitigation for future incidents, and share the evaluation with all stakeholders when the public health event is under control or has concluded. this study's participants, the profile could be minimized to a specific group of people, for example, first responders. a third option, is to use the profile and apply it in practice. discussing the profile enhanced usability according to our participants. in addition, we lowered the barrier of translating the theoretically formulated competencies into practice by inviting our participants to think of a possible event. during the international questionnaire, people noted which event they had used during completion. remarkable is that all the events listed comprised a viral hemorrhagic fever. while the chance for an event or suspicion of a viral hemorrhagic fever is still low, it is an interesting finding that its high impact, possibly due to the evd pandemic of 2014-16 and the recent endemic state in the democratic republic of the congo, affects the thoughts of preparations in europe. remarkable findings in the profile are the low number of competencies for recovery and evaluation after an event. in the airport guidelines that we used as a source, evaluation and recovery are scarcely named and hardly elaborated on [29, 41, 42] . however, the use of after action reviews are a required action according to the ihr, and major benefits of either training or real response are made here [50, 51] . it is therefore worrisome that this phase of the event is hardly elaborated upon in current landmark guidelines and competency profiles, and, consequently, is scored as a rather small topic in this study's competency profile. we cannot determine, however, whether the small number of competencies resembles the real need or resembles a lack of awareness. we suggest to critically review the competencies required for recovery and the after action review after a major event. it may seem remarkable that many competencies in our profile are knowledge or skills in comparison to attitudes. this could be the result of several aspects. first, we build upon former general profiles and focused on competencies specific for the airport setting. attitudes such as leadership, flexibility, team work or reflection are mostly general, i.e. not airport-specific. in addition, these are already widely covered in the team resource management skill set, which is additional to our profile and focusses on attitude [36] . however, another possibility is that knowledge and skills are more concrete while attitudes remain harder to distinguish by our participants. the use of this profile in practice should indicate whether attitudes are sufficiently covered. we consider it as a unique opportunity to thoroughly and extensively develop a competency profile by performing two consecutive rand modified delphi studies, one at schiphol airport, one of europe's largest airports, and one internationally with experts from ten european countries. as in every study, we faced several challenges that we tried to cope with. first, composing a draft profile demanded a pragmatic step of combining and formulating competencies of different profiles and guidelines. diminishing the large amount of overlapping competencies with slight differences in word choice, specificity level and combinations of activities was done as systematically as possible based on thoroughly produced profiles in the international literature to present an assessable profile to the participants. another challenge was to compose expert panels with optimal representation of all involved disciplines. in the national study, we could not include all professionals involved in infectious disease management at schiphol airport. however, we assume to have caught the major perspectives, since professionals that are involved with most tasks were represented. in addition, all included participants were highly experienced in outbreak management and collaborated with the missing experts in daily practice. during the international study, we had to select participants based on their self-assessed competence regarding the subject and could not prevent a mixed group regarding experience in real practice. we therefore consider it a strength that the international participants all participated in the european training course, leading to an equally educated group in the consensus meeting. a third challenge is to design a profile that is internationally usable while it turned out that designation of tasks between national, regional or airport levels differs among countries. however, we explicitly demanded participants to look further than the specific situation at their airport, leading to a profile that forms a thorough base for training in european countries. future steps would be to test the usability and implementation of this profile in real practice by means of trainings, exercises and evaluations. it is our aim that this profile is applied at airports worldwide to facilitate a competent workforce, by integrating it in training or exercising schedules as training material or background reference for organizers. standardized and extensive use of this profile could help to standardize terminology among professionals, and contribute to better communication and coordination. subsequent development of tools such as an epa profile on a european level, or the implementation of competencies into discipline specific profiles at specific airports are possibilities. finally, we hope that this competency profile can be used as a basis to develop specific competency profiles for points of entry other than airports, or other settings important in cross-border spreading of disease. in this study, we developed an interprofessional competency profile for professionals involved in infectious disease preparedness and response at airports by means of landmark literature and expertise from professionals in eleven european countries. this profile could be 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global health security. globalization and health the authors are thankful to all participants for sharing their knowledge and practical experience. we thank saskia van egmond for her help in selecting the best representable panel, and for her advice and enthusiasm during the project. we thank eline westland-hogenbirk for the configuration of fig 2; jeannette de boer for her reflections, including on the future applications and usability of our profile; and corien swaan for her contributions before and during the international consensus meeting; and our colleagues from the eu joint action healthy gateways for the opportunity to perform our study during the training-of-trainers in belgrade. conceptualization: doret de rooij, evelien belfroid, aura timen. key: cord-255351-vp19ydce authors: lanata, claudio f.; fischer-walker, christa l.; olascoaga, ana c.; torres, carla x.; aryee, martin j.; black, robert e. title: global causes of diarrheal disease mortality in children <5 years of age: a systematic review date: 2013-09-04 journal: plos one doi: 10.1371/journal.pone.0072788 sha: doc_id: 255351 cord_uid: vp19ydce estimation of pathogen-specific causes of child diarrhea deaths is needed to guide vaccine development and other prevention strategies. we did a systematic review of articles published between 1990 and 2011 reporting at least one of 13 pathogens in children <5 years of age hospitalized with diarrhea. we included 2011 rotavirus data from the rotavirus surveillance network coordinated by who. we excluded studies conducted during diarrhea outbreaks that did not discriminate between inpatient and outpatient cases, reporting nosocomial infections, those conducted in special populations, not done with adequate methods, and rotavirus studies in countries where the rotavirus vaccine was used. age-adjusted median proportions for each pathogen were calculated and applied to 712 000 deaths due to diarrhea in children under 5 years for 2011, assuming that those observed among children hospitalized for diarrhea represent those causing child diarrhea deaths. 163 articles and who studies done in 31 countries were selected representing 286 inpatient studies. studies seeking only one pathogen found higher proportions for some pathogens than studies seeking multiple pathogens (e.g. 39% rotavirus in 180 single-pathogen studies vs. 20% in 24 studies with 5–13 pathogens, p<0·0001). the percentage of episodes for which no pathogen could be identified was estimated to be 34%; the total of all age-adjusted percentages for pathogens and no-pathogen cases was 138%. adjusting all proportions, including unknowns, to add to 100%, we estimated that rotavirus caused 197 000 [uncertainty range (ur) 110 000–295 000], enteropathogenic e. coli 79 000 (ur 31 000–146 000), calicivirus 71 000 (ur 39 000–113 000), and enterotoxigenic e. coli 42 000 (ur 20 000–76 000) deaths. rotavirus, calicivirus, enteropathogenic and enterotoxigenic e. coli cause more than half of all diarrheal deaths in children <5 years in the world. despite global success in the reduction of all cause and diarrheaspecific mortality in the past 30 years, diarrhea remains the second leading cause of death due to infections among children under five years of age worldwide [1, 2] . it is estimated that diarrhea accounted for 9?9% of the 6?9 million deaths among children under 5 in 2011 [2, 3] . several organisms have been implicated as important causes of these deaths [4, 5] , yet there has not been a review using standardized methods to determine the importance of all of the common pathogens. the child health epidemiology reference group (cherg) has estimated the causes of child deaths from major causes since 2001. we have undertaken this review to develop estimates of pathogen-specific diarrhea mortality among children under 5 years of age. we present the results of a systematic literature review of studies of diarrhea etiology in hospitalized children and use these results to estimate the global burden of diarrhea mortality by pathogen for children under 5 years of age for 2011. we searched medline, lilacs, and medscape for studies published between 1990 and 2011. we used the terms ''diarrhea'' (or ''diarrhoea''), ''gastroenteritis'', ''rotavirus'', ''e.coli'' (or ''escherichia coli''), ''salmonella'' (not ''typhi''), ''shigella'', ''campylobacter'', ''giardia lamblia'', ''vibrio'', ''cryptosporidium'', ''entamoeba'', ''norovirus'', ''calicivirus'', ''norwalk agent'', using ''and children'' as a search restriction. an example of one of the search instructions in medline-pubmed is: ''diarrhea'' [mesh] january 1, 1990 -december 31, 2011 . we also included data from the who rotavirus surveillance network for 2011 provided to us by who only from countries that had not introduced rotavirus vaccine as of december 2011 and had data covering the 12-month period. these studies used a standard protocol across the network [6] . we included studies that sought at least one of the above listed pathogens and conducted 12 or more months of surveillance among children less than 5 years of age hospitalized with diarrhea. studies must have included all diarrhea patients at the selected study site or a systematic sampling of cases for the duration of the study. we did not require a minimal number of children evaluated to be included. laboratory tests were performed on rectal swabs or stools samples. we excluded studies conducted during reported diarrhea outbreaks, those that did not discriminate between inpatient and outpatient cases, those that included patients with nosocomial infections, and those conduced in special populations, such as hiv-positive patients. we also excluded studies that did not describe adequate surveillance methods or standard laboratory methods, according to the following criteria: a) salmonella and shigella isolation in salmonella/shigella agar, xylose-lysine-deoxycholate agar, hektoen enteric agar, and selenite enrichment for salmonella [7] ; b) campylobacter isolation by use of transport media with antibiotics (skirrow's supplement or similar) and inoculation into 5% sheep blood with antibiotics (butzlers supplement or similar), cultivated at 42uc in micro-aerobic atmosphere [7] ; c) vibrio cholerae isolation by alkaline peptone water enrichment and subculture at 8 hrs into thiosulfate-citratebile salts -sucrose agar (tcbs) [7] ; d) e. coli isolation from macconkey agar and identification of etec by dna probes or polymerase chain reaction (pcr) for heat-labile (lt) or heatstable (st) toxins, cell cultures (y1, cho cells), ileal loop or mouse models [7] ; e) epec isolation by the use of hep 2 cell cultures or the presence of the plasmid for adherence (bfp) and the intimin gene (eae) identify in dna probes or by pcr [7] ; f) rotavirus, calicivirus (or norovirus), astrovirus and enteric adenovirus identification with the use of enzyme-linked immunoassays (elisa), electronic microscopy, or pcr [7] ; g) giardia lamblia identification by direct microscopic examination, or zinc-sulfate concentration from direct stools or by elisa [7] ; h) cryptosporidium spp. identification by elisa, or the modified ziehl-neelsen stain for microscopy [7] ; i) entamoeba histolytica identification by direct microscopic examination [7] . we did not include studies in areas or countries where the rotavirus vaccine was used but included data from the placebo arm of rotavirus vaccine trials. articles published in languages other than english, spanish, portuguese, italian, german and french were not included. the following enteropathogens were considered: rotavirus, enteropathogenic escherichia coli (epec), enterotoxigenic escherichia coli (etec), salmonella spp. (excluding salmonella typhi), shigella spp., campylobacter spp., vibrio cholerae o1 and o139, giardia lamblia, cryptosporidium spp., entamoeba hystolitica, human caliciviruses (genogroup i and ii norovirus and sapovirus) or astrovirus, coronavirus, and enteric adenovirus. we extracted data for all children less than five years of age for each pathogen. data from more than one hospital in a country were treated as separate studies if the presentation of data permitted. papers that published different etiological data from the same study site were grouped into one study. if co-infections were reported, they were not treated separately so each pathogen was counted as present if isolated alone or in combination. three reviewers (co, cxt, and cfl) did the primary extraction and all selected papers were reviewed by cfl and cfw independently. disagreements were resolved by cfl and/or reb. we calculated overall median proportions of positive diarrheal stool samples for each pathogen for children 0-59 months of age using the overall proportion for all children included in the study; 39 studies enrolled children from a narrower age range so we calculated for these studies an age-adjusted proportion for the 0-59 months of age group by calculating a conversion factor for age group x as the median of 0-59 prevalence over age group x prevalence (median (prev 0-59 /prev x )) using studies that reported both 0-59 and the age group x for a given pathogen. to use this method we required at least 3 studies, where each study reported both 0-59 months and age group x. in situations where less than 3 studies were available we employed an alternative method where the conversion factor for age group x was taken as the ratio of the median prev 0-59 to median prev x (median (prev 0-59 )/median (prev x )). for this approach we required that 3 or more studies contribute to each of the two medians, but dropped the method 1 requirement that individual studies report both age groups. if neither of these sets of conditions were met, we borrowed the conversion factor for the age group x from a similar age group within the same pathogen (for instance, used the conversion factor calculated for studies including infants 0-11 months of age for studies that included infants 0-5 months of age) or from a similar pathogen (conversion factor for age group x for a study on epec borrowed from studies on etec). the 0-59 months prevalence proportion for each pathogen was estimated using the median individual study 0-59 months pathogen prevalence. we stratified studies by the number of pathogens sought and calculated the unadjusted and age-adjusted medians, as described above, separately for single pathogen studies and for studies that sought 5 to 13 pathogens. for estimating the proportion of diarrheal stools due to unknown pathogens, we included 12 studies that sought 8 or more pathogens. for the numbers of diarrheal deaths attributable to each pathogen, we assume that the distribution of pathogens observed among children hospitalized for diarrhea represents the pathogen prevalence among child diarrhea deaths. we applied the ageadjusted median proportion for each pathogen and for unknowns to the overall number of diarrhea deaths of 712 000 estimated for the world in 2011 [3] , adjusting all proportions equally to be constrained to add to 100%. we explored alternative estimates using all studies selected or only those that sought 5 to 13 pathogens, constraining or not all proportions to add to 100%. the uncertainty around each estimate was calculated using bootstrap confidence intervals [8] . 'pseudo-data sets' were created by sampling studies with replacement from the real dataset. each of the 1000 pseudo-datasets was used in the estimation procedure described above to generate a corresponding 1 000 prevalence proportions. the 2?5 th and 97?5 th percentile of these proportions gave the 95% confidence interval (ci). to estimate the uncertainty of the number of deaths for each pathogen, we paired each of the 1 000 pseudo-datasets with random draws from the under 5 total mortality envelope, the proportion of total deaths attributable to diarrhea [2, 3] , and the proportion of diarrhea deaths due to unknown pathogens. the under 5 year global total mortality envelope estimate and standard deviation were calculated by sampling and combining 100 000 random draws from each of the 194 countries in the world [2, 9] . for each country, a normal mean and standard deviation was estimated from the point estimate and associated confidence interval. from 22 643 citations identified in the electronic search, 1 003 articles were selected for further evaluation (fig. 1) ; 840 articles were excluded because they had one or more of the exclusion criteria (about 35% because they were not longitudinal studies or inappropriate laboratory methods were used, 31% because no data was given for children ,5 years of age, 23% for studies that lasted less than 12 months of duration, and the rest because data were reported after rotavirus vaccine introduction, duplicate publications or reporting results on a pathogen not included in our list). a total 163 articles and 31 who rotavirus surveillance network sites were selected representing 286 inpatient studies with data for at least one pathogen [list of the 163 references can be found at www.cherg.org]. the geographical localization of the study sites is shown in figure 2 . the median and age-adjusted median proportions (with 95% ci) of isolation of each enteropathogen in hospitalized diarrhea cases are shown in table 1 . rotavirus, epec, calicivirus, and etec were the most frequently identified organisms. the sum of these age-adjusted median proportions, including unknowns was 138%, indicating a problem with many articles reporting mixed infections as separate causes. different isolation rates were observed in studies in which only one, versus at least 5 enteropathogens were sought (table 2) . rotavirus was more frequently isolated in 180 single-pathogen inpatient studies in comparison with 24 multiple-pathogen studies (39% vs. 20%, respectively, p,0?0001). the same trend was observed between single-and multiple-pathogen studies for most pathogens, but mainly for giardia lamblia (16% vs. 3%, p,0?001), shigella (24% vs. 7%, p,0?001) and v. cholerae (10% vs. 0.2%, p,0?001). very few studies sought a substantial number of pathogens. from the 286 inpatient studies, only 12 (4%) sought 8 or more pathogens (1 study with 13, 2 studies with 10, 5 studies with 9, and 4 studies with 8 pathogens). in these studies, 33?7% of cases had no pathogen identified. adjusting all proportions, including unknowns, to add to 100%, we estimated that rotavirus caused 197 000 (uncertainty range ur 110 000-295 000), enteropathogenic e. coli 79 000 (ur 31 000-146 000), calicivirus 71 000 (ur 39 000-113 000), and enterotoxigenic e. coli 42 000 (ur 20 000-76 000) deaths. these four pathogens were associated with 55% of all diarrhea deaths (table 3) . these estimates varied substantially depending on the methods used. if the proportions were not made to add to 100%, rotavirus would be said to cause 272 000 deaths or if only studies that sought .4 pathogens were selected and the proportions were adjusted to 100% rotavirus would be said to cause 126 000 deaths (table 4 ). when classifying studies by who region, most studies were done in the western pacific region (78 studies) and less in the eastern mediterranean region (19 studies) ( table 5 ). rotavirus was more frequently isolated in the western pacific region (33%) and less in the american region (23%). other comparisons were limited by few or no studies in some regions (table 5 ). in this review, we showed that more than half of the severe diarrhea episodes, most likely to result in death among children under the age of 5 years in 2011, could be attributed to rotavirus, epec, calicivirus, and etec. our estimates have been adjusted for age in studies that did not cover all children ,5 years old, and campylobacter spp to add to 100%, including a fraction of episodes with unknown etiology. such adjustments have not been done in previously published estimates for single diarrhea etiologies [4, 5, [10] [11] [12] . we identified a potential selection bias among studies that focus on a single pathogen. for example, the median proportion of diarrheal episodes with rotavirus identified varied from 39% in single-pathogen studies to 20% in studies that sought more than 4 pathogens. it is possible that studies looking for a particular pathogen are more likely to be conducted in a study site with a high prevalence of that pathogen and/or a low prevalence of other pathogens. an urban hospital that treats children of higher socioeconomic status and living in more hygienic conditions than children in rural areas may find a higher proportion of cases with rotavirus. a study of cholera done in a hospital in an endemic area may not be representative of national or regional populations. because of the low number of studies that sought multiple pathogens, we have not restricted our analysis to only those studies, in an attempt to include as much global data as possible, but it should be recognized that the inclusion of single-etiology studies may result in a biased higher estimate for some pathogens. by including 13 pathogens in this review we are able to address the problem of mixed infections, an important factor ignored in previously published single-pathogen estimates of deaths. no methodology has been developed to identify the true cause of an episode when more than one pathogen is identified in the stool. our adjustment of all percentages to fit 100% is done to correct for this problem, assuming that each pathogen is equally likely to cause the illness. this is probably not correct because some organisms are carried in the feces for a relatively long time after infection-causing illness, like norovirus [13] , or may not cause illness, especially in older children who have acquired immunity that protects against disease, but not carriage of the organism, like some protozoa [14] . this method of including all equally in the constraint to 100% of diarrhea deaths may result in an underestimate of the importance of some pathogens, such as rotavirus in young children, and overestimate the importance of others, such as giardia. we do not have data on the presence of these pathogens in the stools of asymptomatic children in the studies selected in this review so we cannot determine the attributable fraction related to each pathogen as done in other studies [15] . however, controlling for pathogens found in non-ill children does not necessarily eliminate the problem because some pathogens with long excretion periods after illness, like norovirus, may be wrongly classified as not causing diarrhea. carefully conducted longitudinal studies are needed to separate long-term excretors after illness from asymptomatic infections, to reveal the true pathogenic role of these different organisms in developing countries. we estimated that the number of diarrhea episodes for which no pathogen can be identified is 34%, which is based on studies that sought at least 8 pathogens, not necessarily all 13 and thus may be an overestimate. these ''unknowns'' could be due either to the same pathogens not detected because insensitive methods were used to identify them (either the method itself or to using a rectal swab instead of a stool sample) [16] , to the use of antibiotics prior to obtaining the stool sample, to other yet undiscovered infections, or to non-infectious causes of diarrhea. the proportion of samples with unknown causes was based on a selected group of 12 studies that searched for 8 or more pathogens. these studies do not represent the world as the rest of the studies did. the recently conducted studies called the global enterics multicenter study (gems) in 7 countries in africa and asia were designed to fill this gap [15, 17, 18] . however, they studied cases with moderate and severe diarrhea seen in health services (hospitals, emergency rooms and community clinics), not separating those being hospitalized from milder outpatient cases, therefore, those studies would not meet our inclusion criteria. given that we cannot distinguish among the reasons no pathogen was found during the episode, our estimates may represent an under-estimate, at least for some causes. we could not include some pathogens known to cause diarrhea in our review, such as organisms that cause food-borne outbreaks (i.e. clostridium perfringens [19] , or staphylococcus aureus producing enterotoxins [20] ), because there are very little data on their importance in developing countries. a recent review of rotavirus studies estimated that rotavirus caused 453 000 deaths in children ,5 in 2008 [4] . if we would apply the median proportion of 38% rotavirus isolation found in the 242 inpatient studies that sought it in our review, without any adjustment, to the 1 236 million u5 diarrheal deaths in 2008, we would estimate 472 000 rotavirus deaths in 2008. in 2011 it is estimated that diarrhea deaths have been reduced to 712 000 [3] . our estimate of 197 000 deaths due to rotavirus, using our improved methods, still represents an important global public health problem, with 23 children dying due to this condition every hour. this estimate does not account for any recent reduction in rotavirus-specific proportionate mortality due to the introduction of rotavirus vaccine, as seen in some latin america countries [21] , but these countries account for a very small fraction of global diarrhea mortality. wide scale use of the rotavirus vaccine in high mortality countries will allow a more precise estimate of the true proportion of diarrhea deaths caused by rotavirus. our estimate of 28 000 deaths for shigella is much lower than a previous estimate of 667 695 deaths due to shigellosis in children under 5 years in the world in 1995 published by kotloff et al [5] . this initial estimate was not based on a systematic review of the literature; rather, it used a single study in latin america to estimate the proportion of shigella cases that were hospitalized and a bangladeshi study to estimate the case-fatality rate of children hospitalized with shigellosis to estimate the global burden due to this organism. using the same methodology of kotloff et al but with an updated review of the literature and current case fatality rates observed in bangladesh, bardhan p et al [22] estimated that only 14 000 children younger than 5 years of age died due to shigellosis in asia in 2005. our estimates are compatible with this asian estimate. the total number of deaths due to calicivirus of 71 000 deaths has indicated to be the third most common cause of death due to diarrhea in children under 5 years of age. few studies differentiated between gi and gii norovirus and other types of human caliciviruses, but in those few that did, most of calicivirus isolated in children with severe diarrhea have been due to norovirus gii [23, 24] . patel et al [25] estimated 218 000 deaths due to norovirus among children under 5, but this was calculated using very different methods and assumptions: they used an attributable fraction due to norovirus when data on asymptomatic children was available, and applied their mean isolation rate of 12.1% from inpatient studies (not much different from our median isolation rate of 13.8%) to 1.8 million deaths due to diarrhea in the world; they did not adjust for mixed infections or unknowns. the 79 000 deaths estimated to be caused by epec represent different sub-types of this type of pathogenic e. coli, a group that requires further epidemiological studies in different parts of the world to further characterize them since some sub-types are isolated with the same frequency in diarrhea and control children [26] , new ''typical'' and ''atypical'' epec strains have been identified [27] , and in some regions have been identified to cause more persistent than acute diarrhea [28] . these estimates have several limitations. the studies included in this review were conducted in selected sites and in some cases in populations with increased risk of diarrheal diseases. thus, they may not be representative of the countries where they were conducted, nor of the world. for several regions, such as russia and the former soviet states or sub-saharan africa we have limited or no data ( fig. 2 , table 4 ). the gap of information from africa, for pathogens other than rotavirus, is most acute because of the number of diarrhea deaths in this region is very high [1] [2] [3] . no study has been conducted to identify pathogens in children who died due to diarrheal diseases, so we assume that children in need of hospitalization are the best proxy of diarrhea deaths in low to middle income countries, but this may not be true for some pathogens. another limitation is the combination of laboratory methods with different sensitivities to identify a pathogen: from the culture-based identification of salmonella or shigella to the highly sensitive real-time pcr method for norovirus. this may have affected the relative importance of one vs another pathogen in our estimates. we excluded studies on nosocomial infections, on displaced populations and on diarrhea outbreaks, which may have caused us to under-represent deaths due to some pathogens like v. cholerae. we included in our estimates a total of 13 pathogens (4 viruses, 6 bacteria and 3 parasites) that have been incriminated as causes of severe diarrheal diseases. some viruses, like adenovirus, and parasites, like g. lamblia, have not been completely documented as a cause of severe diarrhea in developing countries [14, 29, 30] . the subject of causality of diarrheal diseases is still not completely understood in settings where children are heavily exposed to many pathogens early in life. young infants may be protected by breast milk and trans-placental maternal immunity and very low doses of ingested pathogens early in life may result in subclinical infections and development of immunity. this immunity may not preclude, however, the excretion of these pathogens in the child's feces. practically all studies done in children who were studied when they were healthy as well as when they developed an acute diarrheal episode have found the same pathogens, although usually with lower frequency, in healthy states. thus, the assumption that any pathogen identified in a child with diarrhea is the cause of the episode is naive and additional methods are needed to determine the pathogenicity of microbes. with a better understanding of the pathogenicity of key organisms our estimates could be further adjusted. also, some studies suggest that children ill with a pathogen, as with epec, may excrete higher amounts in the stool, as compared with asymptomatic infections [31] , so future studies may consider quantifying the amount of each pathogen in the stool to help identifying those ill with it. finally, the review period covering studies published between 1990 and 2011 (studies were conducted with a median mid-study period of 2005, only 24 (8%) studies were done prior to 1990). we have not identified a significant change of the proportions assigned to each pathogen over time, so this does not seem to affect our estimates, as shown in fig. 3 for rotavirus. the global burden of disease study recently published cause of death estimates for 187 countries in 2010 [32] . for children ,5 years of age, gbd estimated a total of 666 000 deaths due to diarrheal diseases in 2010 while cherg estimated 712 000 deaths for 2011. gbd also estimated deaths due to 9 etiologies and produced estimates for 0-6, 7-27, and 28-364 days and 1-4 years of age. cherg estimates for 2011 in children ,5 years of age are slightly higher than gbd estimates for rotavirus (198 000 vs 173 000 rotavirus deaths, respectively), similar for epec and etec deaths (79 000 vs 73 000, and 43 000 vs 39 000, respectively), and lower for cholera, salmonella, shigella, campylobacter, entamoeba histolytica, and cryptosporidium spp. (table 6 ). gbd did not estimate deaths due to norovirus, which was the third leading cause of death in our review. gbd used rates reported in diarrhea studies published between 1975 and 2010 done in outpatients, casecontrol, and community-based studies as a reference category to adjust the proportions seen in inpatient studies. cherg only used data from inpatient studies published between 1990 and 2011. both gbd and cherg used modeling to obtain the total number of diarrheal deaths for children ,5, but unlike gbd, cherg has not used models for etiology-specific causes of deaths for each age group and for each country to produce its global estimate. age specific data and modeling may produce spurious results, more so if there are no data. for example, very few studies have been done describing causes of diarrhea in neonates in developing countries, but gbd has estimated deaths caused by each of the 9 pathogens in neonates 0-6 and 7-27 days of age (table 6 ). gbd only produced estimates for 9 etiologies of diarrhea and by subtracting the total of these estimates from the total of diarrheal deaths; they estimated the proportion of other causes of diarrheal deaths. cherg estimated the proportion due to unknowns from studies that searched for 9-13 pathogens, which we feel realistically addresses the fact that a causative agent is not identified in every illness. this also explains why we estimated a higher number of deaths in this category (176 000) than gbd for ''other causes'' which should include unknowns (109 000). gbd and cherg recognized the problem of mixed infections, but the methods used to adjust for it was different: gbd only used proportions for each etiology from inpatient studies that searched for 2-8 etiologies and used that information to produce weights to adjust their estimates in the models. we choose to constrain all proportions, including unknowns, to 100% to correct for mixed infections, which we feel it is more appropriate until better data and analytical tools are available. we have done an extensive search of the literature to include the 286 inpatient studies used in our estimates. gbd has not published the studies included, their search strategy, or modeling methods. until these are published we will not be able to completely compare these estimates. this is the first systematic review attempting to estimate the cause of deaths for these 13 enteric pathogens. rotavirus, calicivirus, enteropathogenic and enterotoxigenic e. coli cause more than half of all diarrheal deaths in children ,5 in the world. we have identified a potential selection bias in studies searching for only one enteropathogen, and the problem when mixed infections (more than one enteropathogen is identified in a stool sample taken from a child with severe diarrhea) are not taken into consideration when estimating causes of diarrheal deaths, factors that has affected previous published estimates. future studies should be done in hospital services dealing with all types of severe diarrhea, searching for all known enteropathogens, removing the effect of asymptomatic excretes, and establishing a mechanism to attribute to one enteropathogen the cause of a diarrheal episode in cases of mixed infections. checklist s1 prisma checklist (doc) estimating diarrhea mortality among young children in low and middle income countries global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since global burden of childhood pneumonia and diarrhea estimate of worldwide rotavirus-associated mortality in children younger than 5 years before the introduction of universal rotavirus vaccination programmes: a systematic review and meta-analysis global burden of shigella infections: implications for vaccine development and implementation of control strategies rotavirus surveillance worldwide -2009 clinical microbiology procedures handbook, 3 rd edition an introduction to the bootstrap global illness and deaths caused by rotavirus disease in children rotavirus and severe childhood diarrhea global mortality associated with rotavirus disease among children in 2004 environmental transmission of norovirus gastroenteritis lack of an adverse effect of giardia intestinalis infection on the health of peruvian children the global enteric multicenter study (gems) of diarrheal disease in infants and young children in developing countries: epidemiologic and clinical methods of the case/control study comparison of direct electron microscopy, immune electron microscopy, and rotavirus enzyme-linked immunosorbent assay for detection of gastroenteritis viruses in children diagnostic microbiologic methods in the gems-1 case/control study burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the global enteric multicenter study, gems): a prospective, case-control study novel insights into the epidemiology of clostridium perfringens type a food poisoning staphylococcal enterotoxins reduction in morbidity and mortality from childhood diarrhoeal disease after species a rotavirus vaccine introduction in latin america -a review decrease in shigellosisrelated deaths without shigella spp.-specific interventions diagnosis of viral gastroenteritis by simultaneous detection of adenovirus group f, astrovirus, rotavirus group a, norovirus genogroups i and ii, and sapovirus in two internally controlled multiplex real-time pcr assays norovirus highly prevalent cause of endemic acute diarrhea in children in the peruvian amazon systematic literature review of role of noroviruses in sporadic gastroenteritis age-related susceptibility to infection with diarrheagenic escherichia coli among infants from periurban characterisation of atypical enteropathogenic e. coli strains of clinical origin pathogens associated with persistent diarrhoea in children in low and middle income countries: systematic review illness and reservoirs associated with giardia lamblia infection in rural egypt: the case against treatment in developing world environments of high endemicity a systematic review and meta-analysis of the association between giardia lamblia and endemic pediatric diarrhea in developing countries quantitative real-time polymerase chain reaction for enteropathogenic escherichia coli: a tool for investigation of asymptomatic versus symptomatic infections global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the global burden of disease study cynthia boschi-pinto of who and theresa diaz of unicef provided coordination of the involvement in cherg of their respective institutions. carolyn weidemann served as coordinator of the grant in support of cherg from the bill and melinda gates foundation. cherg provided advice on methods and interpretation of results. we thank walter mendoza for his initial literature review, cynthia boschi-pinto and laura lamberti for their support in searching for articles, and edda franco for editorial assistance. we thank the countries who provided data through the who-coordinated global rotavirus surveillance network of participating ministries of health, sentinel hospital sites, and the rotavirus laboratory network. we also thank john sanders and theresa j. ochoa for providing useful comments on early drafts of the manuscript. preliminary results of this study has been presented at cherg and food borne epidemiology reference key: cord-279421-rxocrgfu authors: zhang, dan; feng, zhishan; zhao, mengchuan; wang, hao; wang, le; yang, shuo; li, guixia; lu, li; ma, xuejun title: clinical evaluation of a single-tube multiple rt-pcr assay for the detection of 13 common virus types/subtypes associated with acute respiratory infection date: 2016-04-04 journal: plos one doi: 10.1371/journal.pone.0152702 sha: doc_id: 279421 cord_uid: rxocrgfu respiratory viruses are among the most important causes of human morbidity and mortality worldwide, especially for infants and young children. in the past years, a few commercial multiplex rt-pcr assays have been used to detect respiratory viruses in spite of the high cost. in the present study, an improved single-tube multiplex reverse transcription pcr assay for simultaneous detection of 13 respiratory viruses was evaluated and compared with a previously reported two-tube assay as the reference method using clinical nasopharyngeal aspirates samples. of 310 prospectively tested respiratory specimens selected from children hospitalized with acute respiratory illness, 226 (72.90%, 226/310) and 214 (69.03%, 214/310) positive for one or more viruses were identified by the single-tube and the two-tube assays, respectively, with combined test results showing good concordance (kappa value = 0.874). individually, the single-tube assay for adenovirus (adv), human metapneumovirus (hmpv), human rhinovirus (hrv), parainfluenza virus type 1 (piv1), parainfluenza virus type 3 (piv3) and parainfluenza virus type 4 (piv4) showed the significantly superior sensitivities to those of the two-tube assay. no false positives were found. in conclusion, our results demonstrates the one-tube assay revealed significant improvements over the two-tube assay in terms of the better sensitivity, more accurate quality control, less nonspecific amplification, more cost-effective and shorter turn-around time and will be a valuable tool for routine surveillance of respiratory virus infection in china. respiratory viruses are among the most common causes of human morbidity and mortality worldwide, especially for infants and young children [1, 2] . the rapid identification is important for both therapeutic and infection control purposes. diagnoses of viral respiratory tract infection have been performed generally by non-molecular approaches such as direct immunofluorescence and viral culture. they are time-consuming, labor-intensive, and often lack sensitivity or specificity. in the last few years, multiplex rt-pcr assays have been developed to detect respiratory viruses, and many of them have been commercialized, such as xtag rvp, rvp fast (luminex molecular diagnostics, toronto, canada), resplex ii (qiagen, mississauga, canada), filmarray 1 respiratory panel (idaho technology inc., salt lake city, ut, usa), anyplex™ ii rv16 and seeplex rv assays (seegene, seoul, korea), advansure™ real-time rt-pcr (lg life science, seoul, korea) [3] [4] [5] [6] [7] . however, many of these assays are costly, or require specialized laboratory equipment [8, 9] . in our previous study, the two-tube multiplex reverse transcription pcr assay (two-tube assay) to detect sixteen respiratory viruses based on the amplicon size differences using automated electrophoresis system is described. the overall detection rate of the two-tube assay for each virus was comparable to those of the luminex xtag rvp fast assay and seeplex rv15 ace assay, demonstrating the high sensitivity and specificity of the two-tube assay in the analysis of clinical samples [10] . however, two-tube assay has a few drawbacks, such as the size difference of some amplicons are not big enough leading to the difficulty in judging the results by untrained staff, the hands-on two-tube assay is till cumbersome and the internal control is not steadily detected, which limits its wide use in the routine screening in provincial and local center for diseases control and prevention (cdc) in china. in the present study, we adopted the two-tube assay as a reference, and have been progressively optimized and substantially improved the performance of simultaneous detection of thirteen respiratory viruses types/subtypes, the most frequently detected viral agents of respiratory tract infections documented by beijing monitoring network for pneumonia between 2012-2014 (unpublished data), in a single-tube assay while maintaining excellent sensitivity and specificity. the targeted 13 respiratory viruses types/subtypes including influenza a virus (flua), influenza b virus (flub), seasonal influenza a virus subtypes h1n1 2009 pandemic (09h1n1), seasonal influenza a virus subtypes h3n2 (sh3n2), parainfluenza virus type 1 (piv1), human rhinovirus (hrv), adenovirus (adv), parainfluenza virus type 2 (piv2), parainfluenza virus type 3 (piv3), parainfluenza virus type 4(piv4), respiratory syncytial virus a (rsva), respiratory syncytial virus b (rsvb) and human metapneumovirus (hmpv). the aim of this study is to provide a high throughput screening method for routine surveillance of respiratory virus infection in provincial and local centers for disease control and prevention, china. all aspects of this study were performed in accordance with national ethics regulations and approved by the institutional review boards of the center for disease control and prevention of hebei. children's parents were apprised of the study's purpose and of their right to keep information confidential. written informed consent was obtained from parents or caregivers. 2015. of those 110 (35.4%) were female and 200 (64.6%) were male. ages were ranged from 1 months to 11 years old, and 279 (90% were under three years old. trained staff collected nasopharyngeal aspirates (npa) by adding 3.5ml of transport medium and stored at −80°c. total rna/dna was extracted from195μl of clinical sample with addition of 5μl of bacteriophage ms2 as an internal control (10 5 copies) prior to the extraction using the qiaamp viral rna mini kit (qiagen, hilden, germany). the extracts were eluted into 50μl of dnaseand rnase-free water and stored at −80°c. a total 14 pairs of chimeric primers were added to a single-tube to detect 13 respiratory viruses, and one pair of internal control primer (ms2 f, ms2 r) and one universal primer (tag) was also added to the tube. the bacteriophage ms2 coat protein derived virus-like particles (vlps) was used as an internal control to monitor the extraction and detection process of each specimens [11, 12] . the primers sequences, the target genes, the amplicon sizes, and primer working concentrations are listed in table 1 . two-tube assay was performed as described [10] . briefly, one-step rt-pcr kit (qiagen, hilden, germany) was used for the amplification. a total of 25μl pcr mixture containing 2μl of extracted rna and varied primer concentrations was subjected to the following conditions: 50°c for 30min, 95°c for 15min, followed by 10 cycles of 95°c for 30s, 55°c for 30s, and 72°c 30 s; 10 cycles of 95°c for 30s, 65°c for 30s, 72°c for 30s; 25 cycles of 95°c for 30s, 48°c for 30s, and 72°c for 30s, and a final incubation at 72°c for 3min, and the products were analyzed on the qiaxcel automatic electrophoresis using qiaxcel dna high-resolution kit. single-tube assay was performed according to the protocols as described above in two-tube assay with a slight modification of the primer sequences and primer concentration (table 1 ). the x 2 -test and fisher's exact test were performed to analyze the detection agreement between the single-tube assay and the two-tube assay and resolved discordant results. all the comparative detections were double-blind tests performed by trained staff in our laboratory. all of the 310 clinical specimens were simultaneously tested by the single-tube assay and the two-tube assay. the clinical samples are considered as positive when both the targeted pathogen and ms2 are present. the samples are considered to be negative if samples are negative for pathogen but positive for ms2. if both the target and ms2 are absent, the samples are classified as invalid. no invalid sample was found in the optimal single-tube assay. a total of 226 (72.90%, 226/310) and 214 (69.03%, 214/310) samples were detected by the single-tube assay and the two-tube assay, respectively. viral coinfections were found in 57 specimens (18.38%, 57/310) by the single-tube assay, while in 48 specimens (15.48%, 48/310) by two-tube assay. in our study, piv3 and hrv were identified to be the viruses most commonly involved in multiple agent infection, while piv2 and 09h1 were not detected by either of the two methods. all failed results and discordant results were resolved with repeated tests and direct sequencing using the same primers listed in the table 1 . as shown in table 2 , there were 23 specimens with discordant results between the two-tube assay and the single-tube assay, all of them were confirmed by sequencing as true positives, a total of 24 additional viruses were detected only by the single-tube assay, including 5adv, 4hmpv, 3hrv, 2piv1, 9piv3 and 1piv4. the two-tube assay does not include the detection of piv4. the sensitivity, specificity, negative prediction value (npv), positive prediction value (ppv), the accordance rate, and the kappa value of each virus for the single-tube assay compared to two-tube assay are shown in table 3 . early detection of respiratory virus infections allows clinicians to initiate immediate therapeutic interventions that can reduce complications, antibiotic use, and unnecessary laboratory testing. in this study, we compared the performance of a single-tube multiplex reverse transcription pcr assay with the reference method, a two-tube assay, which has been confirmed to be comparable to the commercial luminex x tag rvp fast assay and seeplex rv15 ace detection kit in our previous study. the single-tube assay is also based on the qiaxcel capillary electrophoresis system, which is accessible in most of provincial centers for disease control and prevention in china. of the 310 specimens from hospitalized children, a high prevalence of infection and co-infection with the common respiratory viral pathogens were revealed. hrv was found to be most frequently, followed by piv3. the major discrepancies between the reference two-tube assay and the proposed single-tube assay were the detection rates of hrv, adv, hmpv, piv4, piv1 and piv3. the 24 viruses detected only by the singletube assay (5adv, 4hmpv, 3hrv, and 2piv1, 9piv3, 1piv4) were confirmed by sequencing as true positives. therefore, the single-tube assay is more sensitive than the two-tube assay for the detection of hrv, hmpv, adv, piv, piv3 and piv4. the overall detection rate of the single-tube assay for each virus was comparable to that of the two-tube assay (kappa>0.75) demonstrating the high sensitivity and specificity of the single-tube assay in the analysis of clinical samples. compared with the two-tube assay, the single-tube assay revealed better sensitivity, more accurate quality control, less nonspecific amplification, more cost-effective and shorter turnaround time. this improvement might be attributed to several aspects as follows. first, we have replaced human genome rnase p gene in the two-tube assay with bacteriophage ms2 as an internal control. the utility of ms2-based armored rna as an assay internal control has been documented in many clinical applications [13, 14] . in this study, the internal control ms2 was added to each specimen prior to extraction as an exogenous control to monitor the whole process from nucleic acid extraction to rt-pcr. second, we inserted a few t nucleotides when appropriate between the specific and universal sequences in the primer design to ensure each amplicon can be easily distinguished by the qiaxcel machine. third, homologous tail sequences was added to the 5' end of each chimeric primer [15] . the homo-tag assisted nondimer (hand) system introduced in this study was to prevent dimer formation and allow more, specific, sensitive, and stable detection of the viruses [16] . fourth, for detection of multiple viral targets, simultaneous presence of multiple targets in one specimen may have led to competitive inhibition of amplification of less abundant targets and may explain some loss of assay sensitivity. in this study, we avoided this problem by optimizing temperature switch pcr parameters and further fine-tuning the concentration of primers. in addition, single-tube assay is more convenient and rapidly to apply to clinical specimens, saving the cost and turn-around time. in our study, the most frequently detected pathogen was hrv, while in other study, the rsv was the most commonly detected viral pathogen [17, 18] . this difference might be caused by the following two aspects. first, the detection of rsv increased during the winter, whereas hrv was detected year-round [19, 20] . in this study, the samples collection time is not in winter (may to october). second, hrv is a well-recognized cause of common colds and associated with upper respiratory tract complications. in this study, the patients were hospitalized for both the upper respiratory tract and lower respiratory tract infections. the two-tube assay does not include piv4 while single-tube assay does. for piv4 and piv2, only one piv4 specimen was detected as positive and no piv2 were detected by the single-tube assay with acute respiratory illnesses, which is consistent with previous reports that piv2 and piv4 were not identified as frequently as piv1 and piv 3 [21] [22] [23] . the different geographic location might also lead to the different seasonal distributions of pivs. all of the flua positives detected by the single-tube assay were subsequently sequenced to be h3n2 infection. 09h1n1 were not detected by either of two methods because h3n2 is the predominant seasonal flua circulating in 2015 in china. though piv2 and 09h1n1 were not found in the samples, we detected a few stock clinical samples previously identified as 09 h1n1 or piv2 using both single-tube and two-tube assays, and the results demonstrated both assays worked very well (data not shown). moreover, no false positives were found by the single-tube assay, further suggesting the high specificity of the single-tube assay. however, the limitations of our study are that only the main causes of respiratory infection were investigated. other respiratory tract infection related viruses, such as coronaviruses 229e, hku1, nl63, and oc43, human bocavirus (hbov), enteroviruses (ev) [17, 24, 25] , are not included in this study. besides, the association of clinical outcome with the etiology information, particularly the role of co-infections in the clinical course and severity is not addressed. given the high prevalence (18.38%, 57/310) and diversity of viral co-infections in our study, further research is also needed. in summary, the improved single-tube assay in this study using automatic capillary electrophoresis is a rapid, cost-effective and reliable method with high sensitivity and specificity for the simultaneous detection of thirteen respiratory virus infections, and will be a valuable tool for routine surveillance of respiratory virus infection in china. global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and metaanalysis mortality associated with influenza and respiratory syncytial virus in the united states comparison of the anyplex(tm) ii rv16 and seeplex((r)) rv12 ace assays for the detection of respiratory viruses. diagnostic microbiology and infectious disease comparison of the advansure real-time rt-pcr and seeplex((r)) rv12 ace assay for the detection of respiratory viruses evaluation of the advansure real-time rt-pcr compared with culture and seeplex rv15 for simultaneous detection of respiratory viruses. diagnostic microbiology and infectious disease comparative evaluation of the seegene seeplex rv15 and real-time pcr for respiratory virus detection comparison of filmarray respiratory panel and laboratory-developed real-time reverse transcription-polymerase chain reaction assays for respiratory virus detection. diagnostic microbiology and infectious disease clinical and economical impact of multiplex respiratory virus assays. diagnostic microbiology and infectious disease comparison of fast-track diagnostics respiratory pathogens multiplex real-time rt-pcr assay with in-house singleplex assays for comprehensive detection of human respiratory viruses a two-tube multiplex reverse transcription pcr assay for simultaneous detection of sixteen human respiratory virus types/subtypes. biomed research international armored long rna controls or standards for branched dna assay for detection of human immunodeficiency virus type 1 construction of armored rna containing longsize chimeric rna by increasing the number and affinity of the pac site in exogenous rna and sequence coding coat protein of the ms2 bacteriophage the use of armored rna as a multi-purpose internal control for rt-pcr armored rna technology for production of ribonuclease-resistant viral rna controls and standards multicolor combinatorial probe coding for real-time pcr the elimination of primerdimer accumulation in pcr adenovirus infection in children with acute lower respiratory tract infections in beijing, china community-acquired pneumonia requiring hospitalization among u.s. children. the new england journal of medicine viral aetiology of acute respiratory infections among children and associated meteorological factors in southern china human rhinovirus infections in hospitalized children: clinical, epidemiological and virological features epidemiology and clinical presentation of the four human parainfluenza virus types parainfluenza virus types 1, 2, and 3 in pediatric patients with acute respiratory infections in beijing during human parainfluenza virus infections in infants and young children with acute respiratory infections in beijing zhonghua er ke za zhi chinese journal of pediatrics viral etiologies of hospitalized acute lower respiratory infection patients in china three years surveillance of viral etiology of acute lower respiratory tract infection in children from zhonghua er ke za zhi chinese journal of pediatrics key: cord-260728-4w23kwzu authors: timmermans, ans; melendrez, melanie c.; se, youry; chuang, ilin; samon, nou; uthaimongkol, nichapat; klungthong, chonticha; manasatienkij, wudtichai; thaisomboonsuk, butsaya; tyner, stuart d.; rith, sareth; horm, viseth srey; jarman, richard g.; bethell, delia; chanarat, nitima; pavlin, julie; wongstitwilairoong, tippa; saingam, piyaporn; el, but sam; fukuda, mark m.; touch, sok; sovann, ly; fernandez, stefan; buchy, philippe; chanthap, lon; saunders, david title: human sentinel surveillance of influenza and other respiratory viral pathogens in border areas of western cambodia date: 2016-03-30 journal: plos one doi: 10.1371/journal.pone.0152529 sha: doc_id: 260728 cord_uid: 4w23kwzu little is known about circulation of influenza and other respiratory viruses in remote populations along the thai-cambodia border in western cambodia. we screened 586 outpatients (median age 5, range 1–77) presenting with influenza-like-illness (ili) at 4 sentinel sites in western cambodia between may 2010 and december 2012. real-time reverse transcriptase (rrt) pcr for influenza was performed on combined nasal and throat specimens followed by viral culture, antigenic analysis, antiviral susceptibility testing and full genome sequencing for phylogenetic analysis. ili-specimens negative for influenza were cultured, followed by rrt-pcr for enterovirus and rhinovirus (ev/rv) and ev71. influenza was found in 168 cases (29%) and occurred almost exclusively in the rainy season from june to november. isolated influenza strains had close antigenic and phylogenetic relationships, matching vaccine and circulating strains found elsewhere in cambodia. influenza vaccination coverage was low (<20%). western cambodian h1n1(2009) isolate genomes were more closely related to 10 earlier cambodia isolates (94.4% genome conservation) than to 13 thai isolates (75.9% genome conservation), despite sharing the majority of the amino acid changes with the thai references. most genes showed signatures of purifying selection. viral culture detected only adenovirus (5.7%) and parainfluenza virus (3.8%), while non-polio enteroviruses (10.3%) were detected among 164 culture-negative samples including coxsackievirus a4, a6, a8, a9, a12, b3, b4 and echovirus e6 and e9 using nested rt-pcr methods. a single specimen of ev71 was found. despite proximity to thailand, influenza epidemiology of these western cambodian isolates followed patterns observed elsewhere in cambodia, continuing to support current vaccine and treatment recommendations from the cambodian national influenza center. amino acid mutations at non-epitope sites, particularly hemagglutinin genes, require further investigation in light of an increasingly important role of permissive mutations in influenza virus evolution. further research about the burden of adenovirus and non-polio enteroviruses as etiologic agents in acute respiratory infections in cambodia is also needed. acute respiratory infection (ari) is the leading cause of morbidity and mortality in cambodia [1] . previous studies have attributed the etiology of acute viral respiratory infections in cambodia to rhinovirus, respiratory syncytial virus (rsv), parainfluenza virus (piv), influenza virus a and b, human metapneumovirus (hmpv), bocavirus, adenovirus, enterovirus, and coronavirus [2] . influenza has been described to be one of the most important causes for hospitalization of children with an ari [3] . it was suggested that some southeast asian countries, where influenza a infection is present year round, play an important role in the global spread of influenza that could trigger annual epidemics in temperate regions [4, 5] . until the establishment of the national influenza center (nic) at institut pasteur du cambodge (ipc) and a national influenza-like-illness (ili)-sentinel surveillance system in 2006, little was known about influenza circulation in cambodia. since then, the sentinel surveillance system has, together with event-based surveillance, demonstrated evidence of seasonal, pandemic and avian influenza. previously published data revealed that thailand and cambodia, which are positioned geographically in the northern hemisphere, demonstrate differing influenza epidemiology with regard to seasonality, with thailand exhibiting a northern hemisphere transmission pattern while transmission in cambodia typically occurs from june to december, similar to the southern hemisphere [6] . the rainy season in western cambodia is june to november and the dry season is december to may. the more recent data suggests that influenza seasonality is more similar in thailand and cambodia than previously thought. [5] . depending on the type of genes and the specific residues, phenotypic effects of random or neutral mutation of the virus can be positive, purifying or neutral [7] . influenza virus efficiently escapes from host antibodies through an accumulation of mutations/single amino acid changes (antigenic drift) at the antigenic sites (epitopes) in surface glycoproteins of the hemagglutinin (ha) gene, and to a lesser extent, neuraminidase (na) genes [8] . the antigenic sites are five somewhat overlapping regions (designated a to e for the h3 strains [9] [10] [11] and ca1, ca2, cb, sa, and sb for the h1 strains [12] ). antigenic mapping by smith et al. [13] showed that 11 antigenic clusters of viruses emerged during the 35-year period following the introduction of the a/h3n2 virus in humans in 1968 and that major jumps (or "cluster transitions") occur between antigenically distinct clusters of viral sequences roughly every 3 years (antigenic shift) [13] . until 2009, the cambodian national sentinel surveillance system only covered 8 out of 24 provinces based on a network of urban sentinel-sites at referral hospitals. no data existed about circulating influenza strains and other respiratory pathogens in western-cambodia along the thai-cambodia border, which is a poor and rural area with large volume of crossborder traffic. between 2010 and 2012, the armed forces research institute of medical sciences (afrims) established four additional influenza sentinel surveillance sites in four border provinces in western cambodia, in collaboration with the cambodian communicable disease control (ccdc) department and the ipc. this study describes the circulation of influenza and non-influenza respiratory viruses and the genetic diversity of influenza viruses in western cambodia along the thai-cambodia border which has provided information for treatment, prevention, and control strategies for these populations. the ili and event-based surveillance systems are public health activities organized by the ministry of health in cambodia and as such have a standing authorization from the national ethics committee. samples were all anonymized for the purpose of this study. the study was approved by scientific review committee at afrims in thailand, the institutional review boards at the walter reed army institute of research (wrair) in the united states and the national ethics committee for health research (nehcr) in cambodia. written informed consent was obtained from volunteers or parents or legal guardians of children enrolled in the study. between may 2010 and december 2012, we collected specimens and surveillance data for influenza and other viral respiratory pathogens from a subset of outpatients presenting with influenza-like-illness (ili) at four sentinel sites-located in five health centers and hospitals in battambang, oddar meanchey, pailin and banteay meanchey provinces in cambodia (fig 1) . fig 1 was created using arcview gis version 3.1 (http://arcview.software.informer.com [14, 15] ) sentinel health centers and hospitals were selected based on sufficient overall patient volume and patients with upper respiratory infections (uri), geographical representativeness and ease of specimen transport to the study laboratory. sites were established sequentially: battambang in 2010 (2 sites), oddar meanchey and pailin in 2011, and banteay meanchey in 2012. due to the cross-sectional design of our study in a region for which no previous baseline respiratory surveillance existed, no minimal sample size was calculated. nevertheless, and in accordance with national and world health organization influenza surveillance guidelines [16], we aimed to collect a cross-section of data distributed over time and space. for every site -and pending on local epidemiology and health facility workload, 5 to 10 ili specimens were collected each week (monday to friday) for 52 weeks per year and largely from the first two subjects meeting the predefined inclusion criteria. demographic, clinical data and respiratory specimens were collected from patients who met the case definition for ili defined as persons of 1 year of age or older, arriving at the health center or hospital within 5 days of fever onset with a fever (axillary 38.1°c or oral 38.6°c) and cough or sore throat in the absence of other diagnoses. patients younger than 1 year, who could not provide consent or those with nasal lesions or epistaxis were excluded. demographic data included gender, occupation, presenting signs and symptoms, self-reported influenza vaccination history, medication use and travel within the last 7-days. parents reported for children, which in our study are defined as all study volunteers younger than 18 years old. descriptive statistics of demographic and clinical data including frequencies and cross-tabulations were entered in a study database by double data-entry using microsoft excel 2013 and analyzed using ibm spss statistics 21.0 (spss, ibm inc.) up to three respiratory swabs per volunteer were collected by trained staff with standard procedures. one nasal swab was tested on-site with quickvue influenza a+b (quidel, san diego, calif.) using monoclonal antibodies specific for influenza a and b virus. a combined nasal and throat specimen was placed in a universal transport medium (utm, copan diagnostics, corona, ca, usa), stored between 2-8°c or in liquid nitrogen and shipped to the afrims-cnm (cambodia national malaria center) laboratory at the battambang referral hospital for testing for influenza virus. all ili-specimens were typed and subtyped for influenza a and b at the afrims-cnm laboratory in battambang. influenza-positive specimens were forwarded to the virology unit at institut pasteur du cambodge (ipc) in phnom penh and the who collaborating center (whocc) for reference and research on influenza in melbourne, australia. nucleotide sequence and phylogenetic analysis of influenza viruses was conducted at the virology branch at the walter reed army institute of research (wrair), silver spring, maryland, u.s. influenza-negative specimens were sent to the department of virology at afrims in bangkok, thailand for viral culture and further characterization of other respiratory viruses. rna was extracted from an aliquot of 140 μl of each combined nasal and throat specimen using qiagen viral rna mini kits (qiagen, hilden, germany). the influenza genome was amplified and detected using a standardized the real-time reverse transcription (rrt)-pcr assay on a smartcycler1 ii platform (cepheid, sunnyvale, ca), to test for influenza a and/or b types. samples positive for influenza a and/or b were further tested to confirm influenza a and/or b positivity and to determine influenza a subtypes. the hemagglutinin-gene (ha) specific primers and probes were used for subtyping of human influenza a subtypes h1, h3, h5 and pandemic h1 (ph1, previously known as swh1). nucleoprotein-gene (np) specific primers and probe for universal swine influenza was also used to confirm influenza a subtype ph1 detection in some samples. the 25 μl reaction mixture consisted of 5 μl of rna eluate and 20 μl of the master mixture which included 0.5 μl enzyme mixture (superscript iii rt/platinum1 taq dna polymerase-life technologies), 0.5 μl rnaseout™ recombinant ribonuclease inhibitor (invitrogen™, carlsbad, ca), 12.5μl 2x reaction mix, 0.5μl of both primers and 0.5μl of probe, and 5μl rnase-free water (see s1 table for primer sequences). thermocycling reaction conditions for influenza typing were as follows: initial reverse transcription at 50°c for 30 min, followed by denaturation and pcr activation by taq inhibitor activator at 95°c for 2 min and 45 cycles of pcr amplification (<95°c for 15 sec then 64°c for 30 sec). for influenza a subtyping: reverse transcription at 50°c for 30 min, pcr activation by taq inhibitor activator at 95°c for 2 min and 45 cycles of pcr amplification (<95°c for 15 sec then 62°c for 30 sec). after influenza typing by rrt-pcr, two 500 μl aliquots of every influenza-positive specimen was transferred into cryogenic vials for transport in liquid nitrogen to ipc in phnom penh. specimens testing positive for influenza by rrt-pcr were inoculated onto madin-darby canine kidney (mdck) cells for isolation of influenza strains at the virology unit of ipc. the influenza strains were analyzed by a hemagglutination inhibition test using reference antigens and anti-sera provided by the whocc for reference and research on influenza in melbourne, australia. the na-star1 kit (life technologies, carlsbad, ca, usa), a chemiluminescent neuraminidase (na) inhibition assay which utilizes a 1,2-dioxetane derivative of sialic acid as substrates, was used for na inhibitor susceptibility testing on a subset of isolates, randomly selected across specimen collection sites and dates. the concentration of drug required to inhibit 50% of the na activity (ic 50 ) was calculated using the non-linear curve-fitting function in the graphpad prism 4 package (graphpad software, inc., la jolla, ca, usa). the average ic 50 (nm) (± standard deviation) of two independent determinations was calculated for each virus. outliers of more than 2 standard deviations from the overall mean were retested twice. a subset of isolates were forwarded to whocc in melbourne for confirmation of strain analysis by ha inhibition test and na inhibitor susceptibility using an na enzyme inhibition assay with a fluorescent substrate munana [2'-(4-methylumbelliferyl)-α-d-n-acetylneuraminic acid sodium salt hydrate]. the typical range of ic 50 was calculated as the mean ic 50 ± 3 standard deviations using a panel of well-characterized reference strains kindly provided by the who collaborative center for reference and research on influenza, melbourne, australia. isolates with ic 50 values within or close to the typical ic 50 range were considered to be sensitive isolates. ic 50 values outside of the typical range and between 50 and 200 nm were isolates with mildly reduced sensitivity and ic 50 values well outside the typical range and greater than 200 nm were considered as isolates with highly reduced sensitivity. viral rna segments, extracted from mdck supernatant were sequenced at ipc in phnom penh from 15 samples collected in 2011 and from 10 samples collected in 2012 for both pandemic influenza (ph1n1) and influenza h3n2 (h3n2), representing our four study sites with varying success (s2 table) . cleaned nucleotide sequence data was sent in fasta-format to the bioinformatics section at the virology branch of the wrair in silver spring, maryland, usa. sequences for ph1n1 and h3n2 sequence datasets respectively were combined with references available from the influenza viral resource at genbank [17] and the gisaid epiflu database [18] representing global diversity of ph1n1 from 2009-2013. sequences were aligned using muscle version 3.8.31 [19] implemented in seaview version 4.4.2 [20] and manually inspected for accuracy. best models were determined from alignments using jmodeltest2 version 2.1.4 [21] . maximum likelihood (ml) phylogenetic trees were generated using phyml version 3.0 as implemented in seaview version 4.4.2 [22] . see table 1 for a detailed description of datasets and models used in analysis. ml phylogenies were annotated using mega version 5 and figtree version 1.4.0 [23] . all influenza sequences used in analysis have been deposited into genbank under accessions ku299790-ku299957. alignments were analyzed for diversity, percent nucleotide similarity, antigenic variation and selection using mega version 5 [24] and the hyphy package [25] implemented through the datamonkey webserver [26] [27] [28] . annotation was conducted using genbank's annotation tool in the influenza viral resource [17] and a literature review. all specimens tested negative by influenza pcr were cultured for isolation of respiratory virus and detection by cytopathic effect (cpe) and/or with either immunofluorescence assay (ifa), or direct fluorescence assay (dfa). a subset of 164 culture-negative specimens (collected between may 2010 and april 2012), where we found a higher proportion (5.6%) of non-polio enteroviruses in children less than 5 years old as compared with previous studies (1%) in cambodia [2] , were tested for enterovirus and rhinovirus by two separate nested rt-pcr methods adapted from coiras et al., 2004 and singh et al., 2002 [29,30] , one for simultaneous detection of pan-enteroviruses and rhinoviruses, and the other specific for enterovirus 71 (ev71). pcrproducts positive for enterovirus or rhinovirus were sequenced for nucleotide analysis (fig 2; s3 table) . cell culture of influenza-negative specimens enterovirus ev71 detection included single-step first-round rt-pcr and semi-nested pcr (primers are in s3 table) . the ev71 type specific primers were modified from the previous study by singh et al. 2002 [30] . detection of pan-enteroviruses and rhinoviruses (pan-ev/rv nested rt-pcr) is similar to the procedure for ev71 including 2 rounds of pcr as reported before [29] . the primers for simultaneous detection of enteroviruses and rhinoviruses were designed in the polyprotein gene, between 5 0 non-coding region (5 0 ncr) and vp4/vp2 regions that was previously described by coiras et al. 2004 [29] . ev/rv-2n was modified from primer 2-ev/rv [29] for using in nested pcr reaction. single-step first-round rt-pcr reaction was the same as described above except that 0.25 pmol of each forward and reverse primer (1-ev/rv and 2-ev/rv) was used (s3 table) . the rt step was performed at 42°c for 60 min, followed by 35 cycles of thermocycling 94°c for 30 seconds, 50°c for 1 min, and 72°c for 1 min. the reaction was further incubated at 72°c for 10 min. five micro-liter of 1:20 diluted first round pcr products were added to the nested pcr reaction with the same reagents as in the first-round but without dtt and amv-rt and the primers were 0.25 pmol of each forward and reverse primer, 3-ev/rv and ev/rv-2n. the reaction was incubated at 95°c for 5 min followed by 35 cycles of thermocycling 94°c for 30 seconds, 52°c for 1 min, and 72°c for 1 min. the pcr products (9 μl each) were subjected to electrophoresis in agarose gels, with 100-bp dna ladder serving as a molecular marker. a negative control of rna-free water and a positive control of cdna template from the ev71 reference strain (atcc 1 no. vr-1432 tm ) were included in each experiment. the pcr products from the positive samples were sequenced on both strands with the pcr primers (s3 table) . the specific bands from the first or second rounds pcr were purified using qiaquick gel extraction kit (qiagen, germany) before sending for direct sequencing. sequencing service was performed by ait biotech (singapore). the sequences from both strands were combined for analysis and edited with sequencher (gene code corp., usa). homology searches were through nucleotide blast program [31] along with the percentage of sequence identity of the two given sequences. 586 ili-patients (median age 5 year, range 1-77) were enrolled from five sites from may 2010 to december 2012; among these, 168 (29%) tested positive for influenza by rrt-pcr (table 2) . most frequent symptoms reported were fever (100%, inclusion criterion), cough (100%,), runny nose (90%), congestion (77%), sore throat (75%,) and headache (76%). although gender distribution was similar in ili-patients, more females were positive for enterovirus (71%) or rhinovirus (64%). patients with influenza were slightly older (median age 7 years old) and presented more often with a sore throat (78%). compared with other ili-cases, patients testing positive for enterovirus reported body pain (20% vs. 44%) and headache (50% vs. 77%) less frequently, although chills (50% vs. 32%), vomiting (35% vs. 16%) and abdominal pain (29% vs. 19%) were more common. dyspnea was uncommon, yet most often seen in patients positive for adenovirus (14%). diarrhea was observed in ili-patients (6%-12%) except those infected with enterovirus (0/17). influenza vaccination coverage in 2010 and 2011 approximated 20% in response to the influenza a/ph1n1(2009) pandemic yet decreased to zero in 2012 by subjects' reports. five (table 3 and figs 2 and 3a). vaccination coverage during the entire study period was 11% (63/583). the probability of developing influenza among ili-patients who had been vaccinated (20/63) and who had not (148/520) was similar and not significantly different (0.32 vs. 0.28, p = 0.28). all ic 50 -values of influenza a and b isolates tested from 2011 were within the susceptible range for both oseltamivir and zanamivir (table 3) . overall, at least 1 respiratory virus was detected in 258 out of 586 (44%) ili-specimens collected between may 2010 and december 2012 (fig 2) ; of which most were influenza (168, 29% of ili-cases), followed by rhinovirus (33, 6%), adenovirus (24, 4%), non-polio enterovirus (17, 3%) and parainfluenza virus (piv) (16, 3%). all of the 418 ili specimens that tested negative for influenza a or b by rrt-pcr were sent for viral culture. forty (10%) were culture positive; 24 adenovirus, 11 piv1, 2 piv2, 3 piv3 were isolated (figs 2 and 3b ). no rsv, hmpv or coronaviruses were identified. pan-ev/rv and ev71 nested rt-pcr (s4 table) detected 17 samples positive for enterovirus, which included coxsackievirus a (n = 10, 1 a4, 3 a6, 1 a8, 1 a9, 4 a12), echovirus (n = 5, 2 e6, 3 e9) and coxsackievirus b (n = 2, b3, b4). a single specimen was found to be negative during the first round of rt-pcr but positive by second round pan-ev/rv and ev71 nested rt-pcr. the 662 base pairs pcr product fragment obtained from pan-ev/rv nested rt-pcr was sequenced using the rv/ev inner primers. the 528 bases obtained from sequencing were found to be 99% identical to the enterovirus a71/homo sapiens/vnm/208/2011 strain (accession: kj686294). when the 227 bp pcr product obtained from ev71 nested rt-pcr was sequenced using the ev71 inner primers, 131 bases was obtained. this sequence was found to be 99% identical to enterovirus a71/cambodia: banteay meanchey 2012 strains (accessions: kp308459, kp308453, kp308450, kp308448, kp308430, kp308427, kp308410, kp308406). out of the 33 specimens positive with rhinoviruses, only 18 could be serotyped. of these 18, most were rhinovirus a (n = 11), followed by rhinovirus b (n = 4) and rhinovirus c (n = 3) (fig 4b) . amplicons were sequenced and sequences were analyzed using genbank's blast tool to identify the virus species. pandemic influenza a (ph1n1). all sequences were highly related with no more than 1.67% divergence between the strains detected in the study and respective seed viruses included in the vaccine composition (a/california/7/2009, a/brisbane/10/2010, and a/christchurch/ 16/2010; s5 table, fig 5) for any of the segments analyzed. when compared to the a/california/7/2009 vaccine strain, all cambodian viruses on average had 11 amino acid changes within the ha segment. amino acid changes found in the ha gene can be found in table 4 . no evidence for amino acid substitutions leading to changes in glycosylation was seen in the ha dataset; however, several changes among sequenced samples occurred in antigenic sites, polymorphic sites or had a recorded effect in in vitro analyses per the literature (s6 table) . for the na gene, several amino acid substitutions resulted in addition/loss of glycosylation sites, or table) . for the mp gene, all samples contained the s31n mutation that may confer amantadine resistance as determined by the genbank influenza viral resource annotation tool [32] . no features or changes of interest were seen within the np or ns segment. the ha and na segments contained the most amino acid substitutions when compared to the reference (a/california/7/2009) as well as to the yearly defined groups (2011 and 2012). the mp and np segments showed the fewest substitutions (table 4 ). there were no samples within the ha amino acid analysis that had unique substitutions relative to the global sequences. however, the na gene had unique amino acid substitutions not found with the global references; the function of these substitutions is unknown (s7 table) . selection pressure was determined using the dn/ds statistic. values of dn/ds > 1 are indicative of positive or adaptive selection, values <1 are indicative of purifying or negative selection and a value = 1 indicates neutral or no selection ongoing in the dataset. all samples were under purifying selection for all segments with exception to the ns segment of the 2011 cambodian samples, which had a dn/ds value of 1.0 suggesting neutral selection (s8 table) . for ph1n1 there was no evidence of geographic clustering of sequences within sampling site (colored branches in fig 5) ; nor was there evidence of reassortment among the sequences sampled. sequences (references and samples) from cambodia 2011 distributed throughout the trees; however, the majority of 2011 sequences, including those corresponding to strains detected in this study appeared within a 2011 specific cambodian clade. however, this clade was only evident in ha and na phylogenies and did not appear in np, mp or ns phylogenies (s1-s5 figs). influenza a h3n2 (h3n2). all sequences were highly related with no more than 1.77% divergence between the samples and the furthest related respective vaccine (s9 table, fig 6) for any of the segment. the majority of segments were more closely related to the a/victoria/361/ (1) table) . there were several amino acid changes among the sequenced samples occurring in antigenic sites, polymorphic sites or which resulted in a change of glycosylation (table 5 ). for the na gene, several amino acid substitutions resulted in addition/loss of glycosylation sites, or were found in polymorphic or antigenic/catalytic sites (epitopes) ( table 5 ). for the mp gene, it was noted that all samples contained the s31n mutation that may confer adamantane resistance as determined by the genbank influenza viral resource annotation tool [32] . no features or changes of interest were seen within the ns segment for any sample. the ha and na segments contained the most amino acid substitutions when compared to the reference (a/victoria/361/2011) as well as to the yearly defined groups (2011 and 2012). the mp and ns segments showed the fewest substitutions (s10 table) . there were several amino acid substitution within the ha amino acid analysis that were unique relative to the global sequences (and vaccine references); two of which occurred in antigenic regions (table 5 ) and five additional changes of unknown effect or location significance (s11 table) . one amino acid substitution in the na gene was found to be unique in antigenic site a (table 5 ) and 7 unique substitutions of unknown effect or location significance were also found (s12 table) . all 'unique' aa substitutions were not found with the global sequences or vaccine references. all samples were under purifying selection for all segments with the exception to the ns segment. when samples were separated based on the respective years of collection, the ns gene showed evidence of neutral selection (dn/ds~1). however, when all samples were combined to increase sample size, it showed overall evidence of purifying selection (dn/ds < 1; s10 table) . overall, for h3n2, there was no evidence of geographic clustering of sequences within the same sampling site except samples from oddar meanchey in which all but one sample clustered into the ohio 2012 vaccine cluster (green branches in fig 6) . this clustering was visual only, not statistically significant due to small sample size, though the cluster was well supported (cluster node value 81, value obtained using associated likelihood ratio test (alrt); analogous to bootstrap analysis). there was no evidence of reassortment among the samples analyzed and all samples were consistently grouping with either the a/ohio/02/2012 or a/victoria/361/2011 vaccines. there was one exception; sample a/cambodia/v1221301/2011 consistently fell outside of both the ohio 2012 and victoria 2011 vaccine clusters (fig 7) for all segments analyzed. this is to the best of our knowledge the first influenza surveillance study in remote border areas in western cambodia. over the time course of 31 months, spanning 3 influenza seasons including the last few months of pandemic a/ph1n1(2009) influenza circulation, seasonal influenza virus was the most commonly detected respiratory virus in this predominantly pediatric population of subjects who presented with ili (fig 3) . despite detection of human highly pathogenic avian influenza (hpai) h5n1 virus in western cambodia since early 2011 [36] [37] [38] , h5n1, typically detected in south-central cambodia, was not found at our sentinel sites. influenza subtypes varied by year with a/ph1n1(2009) being predominate in 2010, influenza b in 2011, and influenza a/h3n2 in 2012. we did not expect to detect any h5n1 in our laboratory surveillance system as most suspected h5n1 cases would have been screened out by the health centers based on reported exposure to dead or diseased poultry. the specimen of one patient with reported h5n1 exposure in 2011 was investigated in our laboratory and tested negative for h5n1. table 5 . changes in glycosylation, antigenic and polymorphic sites linked to amino acid (aa) substitutions and sub-antigenic/catalytic sites (epitopes) where aa substitutions were found for hemagglutinin (ha) and neuraminidase (na) segments of h3n2. [39] . our data revealed that the influenza b did not match well with strains included in vaccine composition of trivalent vaccines; therefore, if vaccination is to be implemented, the quadrivalent influenza vaccine that contains the two influenza b lineages, rather than the trivalent vaccine, may be more suitable. aside from a limited quantity of oseltamivir, stockpiled by the government for management of severe disease by pandemic or avian influenza [40] , use of antivirals for influenza is very low in cambodia [40] . as a result, all strains we detected were susceptible to neuraminidase inhibitors (nis) such as oseltamivir and zanamivir, which can be partially explained by the absence of these medications in cambodia. due to widespread adamantane resistance, we did not conduct susceptibility testing on this class of antiviral drugs; however, we did find the s31n mutation conferring adamantane resistance in all of our influenza isolates tested. phylogenetically, all our samples clustered together within each of the two subtypes (a/ ph1n1 or a/h3n2), which is not unusual given the location and specimen collection dates compared with the reference sequences used. our western cambodian ph1n1(2009) isolates were more closely related, based on full genome analysis, to 10 earlier isolates from cambodia (94.4% genome conservation) than to the 13 thai isolates (75.9% genome conservation) or the california 2009-vaccine reference. however, it was also noted that amino acid changes were shared with the thai references suggesting a possibility of mixing between thai and cambodian influenza as expected. isolates from battambang, being western cambodia's major transportation hub, showed the highest diversity of amino acid changes. analysis of sequence data from our influenza patients revealed a random or neutral mutation of the influenza genomes as expected. koel et al. [41] discovered that periodic major antigenic change in influenza a/h3n2 virus was caused mainly by single amino acid substitutions, which occurred at only seven out of 131 possible amino acid positions in ha at antigenic sites immediately adjacent to the receptor binding site (rbs). these substitutions were located in antigenic sites a (position 145) and b (positions 155, 156, 158, 159, 189, and 193) , with none in sites c, d, or e. we did not see these changes within antigenic sites a or b in our dataset. the ha and na genes in our h1 and h3 isolates showed signatures of purifying selection meaning that the virus keeps these genes conserved by removing random mutations as they occur. these single amino acid mutations were found at non-epitope sites of ha [41] that have not been associated with major antigenic changes on their own. however, it is possible that some of these mutations may constitute permissive or compensatory mutations that would be important in enabling co-mutations that could affect viral fitness, and as such, have an incentive to remain fixed under purifying selection. permissive mutations have been described in the spread of oseltamivir resistant influenza a/h1n1 virus that carry the h274y mutation and are increasingly being recognized as a major force in evolution [42, 43] . glycosylation (addition of oligosaccharide chain to the surface protein) is another common form of protein modification. alteration of glycosylation sites can affect folding and conformation changes in the surface glycoprotein, hereby impacting virus survival and transmissibility. in addition, glycosylation can affect interaction with receptors and cause a virus to be more [or less] recognizable by the innate host immune system and antibodies [44] . none of the changes in glycosylation in our isolates have been reported in the literature as having an effect on viral structure or function; but more data may be needed. mapping sites of mutation and glycosylation on epitopes provides a better understanding of antigenic drift and is important for improving vaccine strategies [45] . overall, and despite adequate storage and transportation of our specimens, culture yield was low in our dataset (~10%) with isolation of only adenovirus and parainfluenza. this may be due to an exclusion of children younger than 1 year, the outpatient setting with less severe disease and possibly a later presentation to the health facility (which was possible up to 5 days after fever onset) resulting in a lower viremia. previous work among hospitalized patients has shown presence of human coronavirus, human bocavirus, hmpv and rsv in cambodia [2] but these were not detected by our culture techniques. therefore, the number of non-influenza viruses in our population is possibly underestimated. additional testing is ongoing at afrims in order to elucidate true disease burden of non-influenza viruses on the population. in comparison with previous work in cambodia [46] , we found a relatively higher proportion (9.3% versus 1.3%) of ili-patients testing positive for adenovirus over 2 dry seasons in 2011 and 2012 which may be explained by our younger study population who may be disproportionately affected by this virus. piv-3 (0.9%), which in previous dry seasons was described as the most common parainfluenza-virus in cambodia [2] , was the least common type during our surveillance period, whereas piv-1 (5.6%) was most common, particularly during november 2011 to march 2012. this is not consistent with previous reports for cambodia [46] . the lack of presence of piv-3 in our study population may be due to an outbreak of piv-1 during the surveillance time frame and the fact that we did not include children under 1 year old, an age when 40% of piv-3 is usually detected [47] . by employing a highly sensitive and specific nested rt-pcr assay for enteroviruses and rhinoviruses, we found a higher proportion (5.6%) of non-polio enteroviruses in children less than 5 years old compared with previous studies (1%) in cambodia [2] which could be partially explained by the predominantly outpatient population in our study sample. this is likely an underestimation of the true prevalence in this population since we excluded children less than one year of age, an age group more likely infected by enteroviruses at rates exceeding those of older children and adults by several fold [48] [49] [50] . the most common enteroviruses found among children under 5 years old in our population were coxsackieviruses (7.3%) and echoviruses (3%). although we did not find coxsackievirus a16, the most common etiologic agent for hand-foot-mouth-disease (hfmd), we found other coxsackieviruses that can cause hfmd such as coxsackievirus a6 (n = 3) and a12 (n = 4). coxsackievirus a6 is known to cause either an atypical rash resembling eczema herpeticum or chickenpox (united states 2011-2012 and in europe since 2009) or nail loss one to two months after onset of symptoms (finland 2009, taiwan 2010, japan 2011). coxsackievirus a12 was one of the enteroviruses implicated in hfmd outbreaks in china in 2008 and 2009 [51] . while these patients reported symptoms of fever (38.1-39°c), runny nose, chills, cough, congestion and abdominal pain, they did not report symptoms specific to hfmd such as herpangina and skin rash. we did not detect in our study ev71 virus genotype c4, which was the principal etiologic agent causing the 2012 hfmd outbreak across southeast-asia that manifested as a life-threatening neuro-respiratory syndrome in 78 children under 3 years old from 14 provinces in cambodia [52] . although we did not have ev71 test results from specimens collected during the peak of the outbreak, our data suggests that ev71 virus was not present yet in the border areas of western cambodia before april 2012, the time that the first cases were found elsewhere in cambodia. regarding testing for non-influenza viruses, the use of the less sensitive viral culture as the first-line test supplemented by ifa on only a subset of influenza-negative ili-specimens underestimates overall extent of transmission as well as co-infections. exclusion of children under 1 year of age may also contribute to underestimation. our data was derived from a passive surveillance system with convenience sampling. while we attempted to select surveillance sites representative of the nearby geographic locations, we had no accurate figures of the catchment populations or denominators at our respective sentinel sites. our staggered method of adding sentinel sites suggests that not all sentinel sites enrolled patients evenly over 3 influenza seasons. similarly, with a small sample size and limited number of sampling locations, no inferences could be made with regard to temporal or spatial flow of influenza strains in our phylogenetic analysis. now with a more established system in place, our data may provide a useful baseline for future molecular evolution studies of influenza in cambodia and in the region. despite proximity to thailand, influenza activity, seasonality, antigenicity and anti-viral susceptibility in western cambodian isolates followed patterns observed elsewhere in cambodia rather than thailand. this supports earlier recommendations from the cambodian nic to use the northern hemisphere influenza vaccine on a southern hemisphere vaccination schedule. additionally, use of the quadrivalent versus the trivalent vaccine should improve coverage of influenza b strains circulating in cambodia. neuraminidase inhibitors may still be used for treatment and chemoprophylaxis for seasonal influenza given little evidence for resistance. the amino acid mutations at non-epitope sites, in particular of ha, require further investigation in light of the increasingly important role of permissive mutations in the evolution of influenza virus. further research to clarify the burden of adenovirus and non-polio enteroviruses as etiologic agents in acute respiratory infections in cambodia is needed. table. samples collected for genetic analysis in this study including virus subtype, sample origin, specimen type, sampling date and segments successfully sequenced. (docx) s3 table. primers for rt-pcr and nucleotide sequencing for ev/rv and ev71: the enterovirus 71 type specific primers were modified from the previous study by [53] . the modifications were made by following the alignments of vp1 sequences of different ev71 strains collected during 2002-2011 from thailand, taiwan, philippines, vietnam, and china available in genbank including the sequences with the accession no. jn191177-9, fj969151, fj969163, jq 621835, jq621841, am490141-63, jq315092, and jx203305. the primers for simultaneous detection of enteroviruses and rhinoviruses were designed in the polyprotein gene, between 5 0 non-coding region (5 0 ncr) and vp4/vp2 regions that was previously described by coiras et al. 2004 [29] . ev/rv-2n was modified from primer 2-ev/rv [29] for using in nested pcr reaction. (docx) s4 table. total number of viruses identified by pan-enteroviruses and rhinoviruses (ev/ rv) pcr and nucleotide sequencing † (docx) s5 table. ph1n1 average percent nucleotide sequence identity between vaccine strains and cambodia isolates gene segments. (docx) s6 table. changes in glycosylation, antigenic and polymorphic sites linked to aa substitutions and sub-antigenic/catalytic sites (epitopes) where amino acid (aa) substitutions were found for for the ha and na segments of ph1n1. aa substitution nomenclature is as follows; reference amino acid (a/california/7/2009), amino acid site, sample amino acid. amino acids are numbered from the start codon of the segment (atg:methionine). (docx) s7 table. unique ph1n1 amino acid (aa) changes of unknown function in specific samples for the na gene as compared to a/california/7/2009. aa substitution nomenclature is as follows; reference amino acid (a/california/7/2009), amino acid site, sample amino acid. amino acids are numbered from the start codon of the segment (atg:methionine). (docx) s8 table. ph1n1 sample amino acid substitution summary and selection analysis by group per segment analyzed. (docx) s9 table. h3n2 average percent nucleotide sequence identity between vaccine strains and cambodia isolates gene segments. (docx) s10 table. h3n2 sample amino acid substitution summary and selection analysis by group per segment analyzed. (docx) s11 table. unique h3n2 amino acid changes of unknown function to specific to samples for the ha gene as compared to a/victoria/361/2011. aa substitution nomenclature is as follows; reference amino acid (a/victoria/361/2011), amino acid site, sample amino acid. amino acids are numbered from the start codon of the segment (atg:methionine). (docx) s12 table. unique h3n2 amino acid changes of unknown function to specific to samples for the na gene as compared to a/victoria/361/2011. aa substitution nomenclature is as follows; reference amino acid (a/victoria/361/2011), amino acid site, sample amino acid. amino acids are numbered from the start codon of the segment (atg:methionine). 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avian influenza. cambodian ministry of health avian influenza-situation in cambodia-update influenza vaccination guidelines and vaccine sales in southeast asia responding to pandemic influenza in cambodia and lao pdr: challenges in moving from strategy to operation substitutions near the receptor binding site determine major antigenic change during influenza virus evolution estimating the fitness advantage conferred by permissive neuraminidase mutations in recent oseltamivir-resistant a(h1n1)pdm09 influenza viruses permissive secondary mutations enable the evolution of influenza oseltamivir resistance virus glycosylation: role in virulence and immune interactions predicting the evolution of human influenza a use of a multiplex pcr/rt-pcr approach to assess the viral causes of influenza-like illnesses in cambodia during three consecutive dry seasons parainfluenza viruses a continuing surveillance of enterovirus infections in healthy children in six united states cities. i. viruses isolated during 1960 and 1961 coxsackievirus b5 infection and aseptic meningitis in neonates and children an epidemic of echovirus 18 meningitis aedes hensilli as a potential vector of chikungunya and zika viruses complete sequence analyses of enterovirus 71 strains from fatal and non-fatal cases of the hand, foot and mouth disease outbreak in we gratefully thank all the volunteers for their study participation and study site and provincial health department staff in battambang, oddar meanchey, banteay meanchey and pailin for collecting the data and specimens. we would like to acknowledge the thai-cambodian afrims field team for their dedication and commitment: mr key: cord-256837-100ir651 authors: smith, steven b.; dampier, william; tozeren, aydin; brown, james r.; magid-slav, michal title: identification of common biological pathways and drug targets across multiple respiratory viruses based on human host gene expression analysis date: 2012-03-14 journal: plos one doi: 10.1371/journal.pone.0033174 sha: doc_id: 256837 cord_uid: 100ir651 background: pandemic and seasonal respiratory viruses are a major global health concern. given the genetic diversity of respiratory viruses and the emergence of drug resistant strains, the targeted disruption of human host-virus interactions is a potential therapeutic strategy for treating multi-viral infections. the availability of large-scale genomic datasets focused on host-pathogen interactions can be used to discover novel drug targets as well as potential opportunities for drug repositioning. methods/results: in this study, we performed a large-scale analysis of microarray datasets involving host response to infections by influenza a virus, respiratory syncytial virus, rhinovirus, sars-coronavirus, metapneumonia virus, coxsackievirus and cytomegalovirus. common genes and pathways were found through a rigorous, iterative analysis pipeline where relevant host mrna expression datasets were identified, analyzed for quality and gene differential expression, then mapped to pathways for enrichment analysis. possible repurposed drugs targets were found through database and literature searches. a total of 67 common biological pathways were identified among the seven different respiratory viruses analyzed, representing fifteen laboratories, nine different cell types, and seven different array platforms. a large overlap in the general immune response was observed among the top twenty of these 67 pathways, adding validation to our analysis strategy. of the top five pathways, we found 53 differentially expressed genes affected by at least five of the seven viruses. we suggest five new therapeutic indications for existing small molecules or biological agents targeting proteins encoded by the genes f3, il1b, tnf, casp1 and mmp9. pathway enrichment analysis also identified a potential novel host response, the parkin-ubiquitin proteasomal system (parkin-ups) pathway, which is known to be involved in the progression of neurodegenerative parkinson's disease. conclusions: our study suggests that multiple and diverse respiratory viruses invoke several common host response pathways. further analysis of these pathways suggests potential opportunities for therapeutic intervention. respiratory viruses account for seasonal colds, bronchiolitis, acute otitis, sinusitis, croup, community-acquired pneumonia, and exacerbation of both chronic obstructive pulmonary disease and asthma [1] . the prevalence of pandemic orthomyxoviridae influenza a virus (flu) from april 2009 to 2010 was estimated to be approximately 60 million cases, 270,000 hospitalizations, and 12,000 deaths [2] . paramyxoviridae respiratory syncytial virus (rsv) infection results in nearly two million children requiring medical care with about 57,000 children younger than five years hospitalized annually [3] . in one survey, rsv was the most prevalent pathogen in children under five years with an acute respiratory infection, followed by adenoviridae adenovirus (adeno), and picornaviridae human rhinovirus (hrv) [4] . while initially effective, pathogen gene targeted treatments exert evolutionary selection on the infectious species often leading to the emergence of drug resistant strains. as a result, there are increasing clinical reports of resistance against many drugs that directly act on viral proteins or their dna [5, 6] . in particular, resistance to different classes of antiviral drugs is becoming more clinically prevalent in respiratory virus infections as seen with rsv and flu treated with the antiviral drugs palivizumab [7] , and oseltamivir [8] , respectively. pathogens elucidate two broad types of biochemical responses in the host. first is the activation of the host immune system. while the immune response is critical in combating pathogen infections, its over-activation often exacerbates tissue damage initiated by viral invasion [9, 10] . the second response is the up-regulation of host genes, such as protein biosynthetic pathways, that are crucial for sustaining pathogen invasion, replication and evasion [11] . interestingly, genetically distinct respiratory viruses often modulate common host proteins and biological pathways during infection [1] . for example, many respiratory viruses trigger similar general airway inflammatory responses such as the expression of cytokines interleukin-6 (hugo gene name il6), interleukin-8 (il8) and interleukin-11 (il11), and granulocyte macrophage-colony stimulating factor (csf2). these inflammatory responses in turn initiate iga production, b cell differentiation and t cell stimulation [12] [13] [14] [15] [16] . as a consequence, diagnosis for specific viral infections is difficult since diverse respiratory viruses cause similar, often indistinguishable patient symptoms [1] . however, because distinct respiratory viruses converge on similar immune responses, opportunities also exist for targeting host proteins and pathways which will potentially affect multiple viral pathogens [17] . moreover, human targets might be less susceptible to the evolution of drug resistance due to constraints on the virus to find alternative host pathways for its proliferation. individuals may experience a co-infection or sequential infections of multiple viruses or bacteria which can complicate both disease diagnosis and drug prescription decisions. furthermore, patients infected by multiple pathogens may have further complications due to drug-drug interactions, cumulative drug toxicities and immune system suppression, as observed during hiv and mycobacterium tuberculosis co-infections [18, 19] . indeed, a study in children under five years showed pervasive clinical occurrences of co-infections involving combinations of rsv, hrv, paramyxoviridae parainfluenza virus, flu, coronaviridae sars-coronavirus (coron), paramyxoviridae metapneumonia virus (mpneu), parvoviridae human bocavirus and adeno [4] . therefore, in addition to minimizing drug resistance, there is a need for new therapeutic approaches to safely and effectively treat co-infections by multiple viral and/or bacterial pathogens, particularly where strain-specific diagnostics or treatments are unavailable. the development of new antiviral therapeutics requires a greater understanding of the global host response when challenged by different types of viruses. such knowledge may lead to the identification of novel human genome targets that are shared across multiple viral infections as well as opportunities for repositioning existing drugs for the treatment of infectious diseases [20] . several recent studies have generated multiple mrna microarray gene expression datasets derived from experiments involving the infection of human cell-lines or animal models with one or more of the major respiratory viruses [21] [22] [23] . through a systematic analysis of these respiratory virus-human host gene expression datasets, we determined common sets of genes and pathways involved in host responses to viral infections. among the most significant pathways, we identified several potential new opportunities for repurposing existing drugs for the treatment of respiratory viral infections. we performed a large-scale analysis of published mrna microarray datasets from studies involving a wide range of respiratory viruses in human host infection models. we focused on human mrna array datasets in order to avoid complications inherent in cross-species comparisons. in order to ensure consistency in experimental conditions and reduce biases due to noisy or poor quality datasets, we instituted an iterative process of database querying, data filtering, and common pathway analysis across all published human mrna datasets for twelve relevant respiratory viruses. these viruses initially included the double stranded dna viruses herpesviridae human cytomegalovirus (cmv) and adeno; the positive sense single stranded rna viruses coron, picornaviridae coxsackievirus (cox), hrv, picornaviridae echovirus (echo), and picornaviridae enterovirus (entero); and the negative sense single stranded rna viruses flu, mpenu, rsv, bunyaviridae hantavirus (hant) and sin nombre virus (snv). this list was later narrowed to include only the subset listed in table 1 based on filtering processes outlined in the materials and methods and shown in figure 1 . a total of seven different respiratory viruses were analyzed, represented by fifteen unique gene expression omnibus (geo) datasets (indicated by geo series or gse accession numbers), nine different human cell types, and seven different array platforms for a total of 28 unique comparisons. note that one dataset (gse17156) contained two different viruses (flu and rsv) that were analyzed. after querying the geo database and prescreening for obvious non-candidate datasets such as those not associated with human array platforms, there were at least 23 datasets associated with at least one of the twelve respiratory viruses. however, after considering all conditions for geo dataset candidacy, at least four of these datasets were excluded. in one case, an adeno dataset (gse1291 [pmid unpublished]) had less than three samples per treatment group, as did a cox (gse712 [pmid unpublished]) and a cmv (gse19345 [24] ) dataset. as another example, a cmv dataset (gse675 [25] ) lacked a healthy/control treatment group. additionally, at least four datasets had some comparison groups that did not fit the filters for inclusion. for instance, an hrv (gse13396) dataset's original study design was to observe differences in hrv infectivity between asthmatic and non-asthmatic patients. the asthmatic comparison group data were eliminated from the analysis because of potential difficulties in distinguishing between host inflammatory responses due to viral infections from those associated with chronic asthma. similarly, a combined flu, hrv and rsv dataset (gse17156) contained two main patient groups. one group was classified as developing symptoms after exposure to a single virus under study, while the other group did not develop any symptoms after exposure. only the group that developed symptoms for each of the three viruses was considered for further analysis and the asymptomatic group was omitted. in total, 19 geo datasets, representing 42 unique comparisons (different time points and/or virus strains) were analyzed for quality because they met the four requirements for dataset candidacy. no single dataset exhibited overall poor quality control (qc), and therefore, all 19 datasets representing 42 comparison groups were analyzed for differential expression. however, qc analysis across all candidate datasets revealed two outliers in gse17156 (samples gsm429252 and gsm429279), two in gse11348 (samples gsm286647 and gsm286733), and one outlier each in dataset gse24132 [26] (sample gsm594166), gse1739 (sample gsm30367), and gse19580 (sample gsm487986) for a total of seven samples removed from five different datasets. an illustration of the kernel density and principle component analysis (pca) plots generated during the qc analysis is shown in figure 2 for gse17156's rsv treatment (median of 141 hours post infection) and rsv control (baseline) groups. additional qc analysis results including median of absolute deviation (mad) score plots and pair-wise correlation maps are shown in figure s1 . initially, all samples except gsm429279 showed acceptable kernel density (figure 2a) , pca (figure 2c ), mad score ( figure s1a ) and pair-wise correlation ( figure s1c ) plots. the sample gsm429279 was removed because: a) it did not conform to the kernel density of the other samples; b) it fell outside of the hotelling t2 alpha threshold of 0.05 (represented by the superimposed elliptical on the pca plot), and; c) it was an outlier in both the mad score and pair-wise correlation plots. a second qc round was performed, which resulted in a further non-conforming sample, gsm429252, being discarded. subsequent qc analysis generated acceptable results in kernel density (figure 2b ), pca (figure 2d ), mad score ( figure s1b ), and pair-wise correlation ( figure s1d ), thus this dataset passed our criteria for inclusion in the analysis. all datasets exhibiting acceptable quality were analyzed for probe differential expression. an example volcano plot is shown in figure s2 for rsv treatment group at peak symptoms versus control group (data originating from gse17156). cutoff levels of 1.5-fold increase or decrease in probe expression levels, respectively, and p-values ,0.05 were used throughout (represented by red lines in figure s2 ). all comparison groups had at least some differentially expressed probes, although the number varied greatly indicating potential falsely discovered probes (for example, a comparison group within gse18816 had 111 differentially expressed probes while a comparison group within gse11408 had 2533 differentially expressed probes). however, the conservative pathway enrichment approach we employed tends to attenuate falsely discovered genes. there were three comparison groups that did not meet the least square mean (lsm) threshold requirement and were excluded from the differentially expressed probe list: two of the for each comparison group, the differentially expressed probes were mapped to their corresponding genes, and then a p-value was assigned for each pathway map using the software genego (accessed june 2011). next, the comparison group's significant pathway lists were combined to find the union of all significant pathways (that is, the combined pathway list where all treatment groups have at least one significant pathway). a total of 459 out of the approximately 650 pathway maps available in metabase were determined to be significant. comparison groups having ,5% significant pathways of the total significant pathways (that is, comparison groups containing less than 23 significant pathways) lead to the exclusion of eleven comparison groups from the union list. excluded groups were: hrv at 8 hours (eliminating one comparison group from gse11348), hrv at 72 hours (eliminating one comparison group from gse17156), both strains of flu at 1 hour and 3 hours each and another strain at 6 hours (eliminating three comparison groups from gse18816), rsv at 24 hours (eliminating all comparison groups from gse24132), cmv at 24 and 72 hours (eliminating all comparison groups from gse24434 [27] ), and flu at 8 hours (eliminating all comparison groups from gse24533 [28] ). at the end of the final step in our filtering process, a total of 15 datasets, or 28 comparison groups remained (tables 1, s1 and s2). there were 67 enriched pathways in which all seven respiratory viruses were represented by at least one comparison group (table s3 ). the list is ranked first by the viral frequency, followed by the sum of the normalized viral expression (nve) for each pathway. also shown are the differentially expressed as well as the total number of network objects across all 28 comparisons. the top 20 enriched pathways are listed in table 2 along with the percentage and names of the differentially expressed genes with a viral frequency of at least five in each pathway. of these, the top five pathways were chosen for further analysis and mapping. these pathways are epidermal growth factor receptor (egfr) signaling, cd40 signaling, interferon-gamma (ifng) signaling, histamine receptor h1 (hrh1) signaling, and interleukin-17 (il17) signaling (figures s5. s6, s7, s8, s9; table s4 ). additionally, the parkin-ubiquitin proteasomal system (parkin-ups) pathway was chosen for further analysis because it has not been previously associated with the innate immunity and might be an interesting new mechanism of host response to respiratory viral infection ( figure 3 ). the nves for differentially expressed genes with frequencies of at least six viruses are shown in table 3 along with their associated pathways. the list is ranked by the greatest viral frequency, and then by number of pathways in which the gene is differentially expressed. the nve values for all genes, along with associated pathways, ranked by the greatest viral frequency, followed by the number of pathways in which the gene is differentially expressed are in table s5 . we ensured that the nve was not bias toward any particular comparison group, and indeed no single dataset contributed to the overall nve for any single virus (table s2) . hierarchical clustering on the quantile normalized fold change values for all genes having expression values in at least 26 out of 28 comparisons (at least 90% comparisons) and significant in at least seven comparisons ( figure s3 ) as well as for genes with nve of at least six viruses ( figure s4 ) did not reveal any dominant clustering by gse or virus type. the most consistently up-regulated genes (up-regulated in at least six viruses and down-regulated no more than one virus) are: nuclear factor of kappa light polypeptide gene enhancer in b-cells inhibitor alpha (nfkbia), tumor necrosis factor alpha-induced protein 3 (tnfaip3), chemokine c-c motif ligand 2 (ccl2), interferon regulatory factor 1 (irf1), prostaglandin-endoperoxide synthase 2 (ptgs2), chemokine c-c motif ligand 20 (ccl20), dual specificity phosphatase 1 (dusp1), eukaryotic translation initiation factor 2-alpha kinase 2 (ei-f2ak2), tnf receptor superfamily member 6 (fas), suppressor of cytokine signaling 1 (socs1), tnf receptor-associated factor 1 (traf1), and ubiquitin-conjugating enzyme e2l 6 (ube2l6). there were no consistently down-regulated mrnas (downregulated in at least six viruses and up-regulated in no more than one virus). we sought drug repurposing candidate targets from the top five enriched pathways and the parkin-ups pathway by searching the drugbank database, version 3.0 (http://www.drugbank.ca/ accessed august 2011) [29] [30] [31] , for drugs targeting any of the 67 differentially expressed genes with a viral frequency of at least five (table s6 ). of these, thirteen genes, or almost 20% of the original 67 genes, were associated with at least one approved small molecule or protein therapy. there genes were: prostaglandinendoperoxide synthase 2 (ptgs2), tnf, matrix metallopeptidase 9 (mmp9), jun proto-oncogene (jun), interleukin 1 beta (il1b), ccl2, cd86, coagulation factor iii (f3), phosphoinositide-3kinase regulatory subunit 1 (pik3r1), intercellular adhesion molecule 1 (icam1), nuclear factor of kappa light polypeptide gene enhancer in b-cells 2 (nfkb2), caspase 1 (casp1), and tubulin beta 3 (tubb3). a selection of these genes, along with other characteristics to evaluate their potential as drug targets such as involvement in immune response [29] [30] [31] , jackson laboratory knock-in/knock-out mouse (jax) phenotype [32] , approved or marketed small molecule drug or protein therapy, and current indications for that drug, are listed in table 4 . note that the current indication may not be for the gene target listed. mimosine (gene target: ccl2) and glucosamine (gene targets: nfkb2 and mmp9) did not have a current indication, while the interactions of natalizumab (gene target: icam1) and gallium nitrate (gene target: ilb1) with their gene targets were unclear. additionally, therapies associated with ptgs2 are cyclooxygenase (cox-2) inhibitors which have known side-effect issues thus were not explored further. therefore, nfkb2, icam1 and ptgs2 were excluded from table 4 , leaving ten genes for potential drug repurposing. the potential cases for drug repurposing are discussed more in-depth for four targets; f3, il1b, tnf and casp1. our study used a systematic process to minimize potential technical noise that could have arisen from our comparative analysis of fifteen unique datasets from nine different cell types, and seven different array platforms. these measures included candidate dataset filtering followed by qc, differential gene expression and pathway enrichment analyses. a total of 14 out of 42, about one third of the total comparisons, were removed as a result of this filtering process, which is indicative of our conservative analysis approach. we had previously used largescale and merged-sam analyses in integrating large-scale microarray datasets involving cancer tissues from multiple laboratories [33, 34] . however, the small sample size datasets used in the present study required a more rigorous methodology to identify data outliers. to our knowledge the qc analysis performed with each geo dataset is unique to this study. although no dataset was completely disregarded after qc analysis, some samples were clear outliers, thus potentially skewing the data. kauffmann and huber have demonstrated improvements in signal-to-noise ratios after performing post normalization qc analysis to remove array outliers within an experiment [35] . those authors used ma-plot and boxplots of the log-ratios to determine outliers instead of mad scores, pca and pair-wise correlations employed in this study. fundamentally, the concept of data improvement after outlier removal applies regardless of the qc analysis approach. signaling 26 jun, myc, nfkbia, stat1, fos, jak2, hbegf, dusp1, dusp4, ptk2, gsk3b, mmp9, nfkb2, pik3r1, prkca, sos2, tgfa cd40 signaling 31 il8, jun, nfkbia, tnfaip3, ccl2, fas, il6, irf1, jak2, ptgs2, traf1, ccnd2, cd86, icam1, lyn, map2k3, map3k14, mapk14, nfkb2, pik3r1, tp53, traf5 ifng signaling 24 myc, stat1, cdkn1a, eif2ak2, irf1, jak2, socs1, stat2, camk2g, cebpb, icam1, mapk14, pik3r1, prkca, ptpn11 hrh1 signaling 25 il8, jun, nfkbia, fos, il6, tnf, csf2, f3, gnaq, gnb4, gng12, icam1 despite the diverse nature of the microarray data analyzed here, we found a large overlap between comparison groups in significant pathways, especially the immune system. of the top twenty enriched pathways, eighteen are associated with immune response ( table 2) . for example, egfr signaling is known to be activated during infection by respiratory viruses flu [36] and entero [37, 38] . cd40 signaling is associated with coron [39] , rsv [40] , and the general immune response [41] . interferon gamma (ifng) signaling is initiated by flu [42] and rsv [43] , while interleukin 1 signaling is stimulated by flu [42] . as components of the general immune response, interferon and interleukin pathways are activated by infectious agents such as hepatitis c virus (hcv), hiv and tuberculosis as well as chronic diseases like crohn's disease, diabetes, and metastatic melanoma [44, 45] . the overall relationships between the transitory host immunity response launched by pathogenic infections versus that seen in chronic autoimmune and neurodegenerative diseases are complex and an intense area of investigation [46] . in addition, there are considerations about subtle shifts in gene function roles in different cell tissue types amongst the various diseases. thus, we are cautious about any linkages between pathways involved in infections and those of chronic diseases as implied by our analysis without further validation studies. parkin (park2) is an e3-ubiqutin ligase associated with the progression of the neurodegenerative disorder parkinson's disease. [47] . as a central hub protein in the parkin-ups pathway, park2 ubiquinates proteins encoded by septin 5 (sept5) [48] , tubulin alpha and beta [49] , and the glycosylated form of synuclein, alpha (snca) [50] for degradation by the 26s proteasome. park2 also ubiquinates synuclein, alpha interacting protein (sncaip) for regulation of snca [51] , interacts with stip1 homology and u-box containing protein 1 e3 ubiquitin protein ligase (stub1) to enhance ubiquitination of g proteincoupled receptor 37 (gpr37), [52] (which associates with f-box and wd repeat domain containing 7 (fbxw7)), and cullin 1 (cul1) to ubiquitinate cyclin e [53] . park2 is deactivated by protolytic cleavage by casp1 and caspase 8 (casp 8) [54] and can be activated by either heat shock protein 70kd (hspa4) or stub1 [52] . the parkin-ups pathway is not commonly associated with general immune response to viral infection. however, other ubiquitylation proteins, such as isg15, are known to play roles in host defense [55, 56] . associations between influenza infection and neuroinflammation in early onset autosomal recessive parkinson's disease have been recently suggested [57] [58] [59] . at least one factor in the progression of parkinson's disease is the formation of neuotoxic lewy bodies due to increases in snca. increases in snca are believed to be the result of loss-of-function mutations in park2 which cause disruptions in the protein's localization and solubility [60] [61] [62] . polymorphisms in the gene park2 have also been associated with susceptibility to infectious diseases such as leprosy, typhoid fever and paratyphoid fever, although the exact mechanism is still unclear [63, 64] . jang et al. observed activation of snca in mouse nervous tissue long after pathogenic h5n1 flu infection where the increased levels of snca mirror those found in parkinson's disease [57] . similarly, recent findings from rohn and catlin indicate flu as a potential causative factor for parkinson's disease [58] . indeed, links between flu and other neurodegenerative diseases have been suggested, and these include seizures, transverse myelitis, expressive aphasia, syncope, encephalitis, neuromyelitis optica, and central nervous system disease in general [65] [66] [67] . park2 itself has a low signal at the mrna level which might be due to its significant regulation by post-translation processes [52, 54] . further studies are needed to determine the mechanism our analysis suggests several potential repurposing opportunities for launched drugs against host-viral targets (table 4 ). this assumption is based on the occurrence of genes that are differentially expressed in infection models for at least five of the seven respiratory viruses, have involvement in a number of relevant pathways related to host immune response, and encode for known drug targets. the drugs associated with this gene list do not have current indications as anti-viral therapies, although pranlukast and clenbuterol are prescribed for relief of lung disorders such as bronchospasm after allergic reactions and asthma bronchoconstriction during asthma attacks, respectively. also, minocycline, sometimes called minocin, is a broad-spectrum tetracycline antibiotic as well as a caspase 1 (casp1) inhibitor while chloroquine is a well-known anti-malaria drug [29] [30] [31] . in fact, eight of the ten drug repurposing gene targets are involved in activation of the innate immune response, while the remaining two have some evidence of virus modulation. potential drug repurposing opportunities for f3, il1b, tnf, and mmp9, as well as the parkin-ups pathway gene product casp1, are discussed below. coagulation factor iii (f3). f3 normally binds to the native cofactor vii or viia to induce the blood coagulation cascade. treatment with recombinant coagulation factor viia promotes blood coagulation in hemophiliacs [29] [30] [31] . esmon et al. [68] suggest that coagulation could be used therapeutically to modulate inflammation responses and vice versa, but also caution about the danger of increased incidence of thrombosis. the consistent upregulation of f3 across five viruses suggests that the immunecoagulation axis is already initiated and supplemental f3 activation may cause thrombosis complications. further study is needed to develop therapeutics that could balance between innate immune response triggered by coagulation factor viia therapy and stabilization of the antithrombotic state. interleukin 1 beta (il1b). il1b is a cytokine involved in inflammatory response, cell proliferation, differentiation, and apoptosis. il1b is specifically cleaved into its active form by the protease casp1 after which it activates the nlrp3 inflammasome [29] [30] [31] 69] . indeed, il1b is consistently up regulated across cmv, flu, hrv, mpenu and rsv which likely correlates with inflammasome activation. however, overexpression of il1b causes multiple inflammatory disorders [69] . antagonists or neutralizers of il1b, such as canakinumab, could potentially reduce inflammation damage associated with viral infection. tumor necrosis factor (tnf). tnf has a wide range of biological functions including modulation of immune response to pathogen assault. mouse tnf knock-out phenotypes include abnormal immune system physiology, increased susceptibility to viral infection, and both increased and decreased susceptibility to bacterial infection [29] [30] [31] . in our study, tnf is mostly up regulated in infections by cmv, coron, cox, and flu but directionally ambiguous for mpneu and not expressed under rsv. while total disruption of tnf function would be deleterious to the host, there are instances where partial tnf inhibition provides a clinical benefit in patients with viral complications [70, 71] . pranlukast is a cysteinyl leukotriene receptor-1 antagonist that reduces bronchospasm caused by an allergic reaction, usually with asthmatic individuals. this drug inhibits tnf-alpha by blocking macrophage cysteinyl leukotriene 1 (cysltc4, d4) receptors [72] or suppression of nf-kappa b activation [73] . pranlukast has been recently shown to be beneficial not only in cases of respiratory syncytial virus postbronchiolitis, but also in a wide variety of other diseases with strong inflammatory complications such as cystic fibrosis, cancer, atherosclerosis, eosinophils cystitis, otitis media, capsular contracture, and eosinophilic gastrointestinal disorders [71] . amrinone is a type 3 pyridine phosphodiesterase inhibitor used in the treatment of congestive heart failure and is an inhibitor of tnf [74] . phosphodiesterase inhibitors have been shown to alter immune response [75] [76] [77] [78] and, in one case, specifically through tnf [79] . amrinone is known to modulate pro-and antiinflammatory factors in endotoxin-stimulated cells [80] . type 4 phosphodiesterase inhibitors have been used to treat rsv-induced airway hyper-responsiveness and lung eosinophilia [81] . therefore, indirect evidence suggests that armirone may be beneficial in respiratory viral infection situations by inhibiting tnf via type 4 phosphodiesterase, although this has yet to be seen in clinical studies. matrix metallopeptidase 9 (mmp9). mmp9 encodes a matrix metallopeptidase that degrades type iv and v collagens, and is implicated in arthritis and metastasis [29] [30] [31] . we can only speculate on the role mmp9 plays in infection. our analysis finds the gene to be up-regulated for three viruses while down-regulated for two different viruses. in other studies, mmp9 has been observed to be up-regulated after exposure to double stranded rna and is important to airway injury [82] , specifically by rsv [83] . mmp9 expression is induced by il1b [84] which, as mentioned above, is an activator of the nlrp3 inflammasome [85] . mmp9 inhibitors such as marimastat, minocycline or captopril, could be beneficial assuming that the protein is coopted by the infecting virus for tissue remodeling. blocking mmp9 may also reduce inflammatory damage by down-regulating the inflammasome. caspase 1 (casp1). in the case of the parkin-ups pathway, inhibiting tubulin-beta formation may reduce viral proliferation given that flu utilize acetylated tubulin for protein trafficking [86] and increases in neuronal class iii tubb occur after cox infection [87] . a casp1 inhibitor such as minocycline could be used to increase park2 ubiquitinase activity, in turn decreasing the tuba or tubb availability. as mentioned above, casp1 is a component of the nlrp3 inflammasome, activating the precursor to il1b [69] . therefore, a casp1 inhibitor would have an antagonist relationship with il1b, hence the inflammasome. further, casp1 inhibitors would be agonists for park2, thereby reducing accumulation of snca. in this regard, casp1 inhibitors may not only prevent unnecessary nlrp3 inflammasome activation via ilb1, but may also reduce accumulation of neurotoxic lewy bodies through activation of park2. however, caspases are not specific to the parkin-ups pathway and inhibition in this regard may result in toxicity or other complications [88] . additionally, mouse jax phenotypes for casp1 show both increased and decreased susceptibility to bacterial infection, as well as decreased inflammatory response. while casp1 inhibition may prove beneficial in terms of increasing inflammatory responses, it is ambiguous in terms of benefit for bacterial infections. in our analysis, the expression of casp1 and tubb3 is also somewhat variable across virus types. therefore, more study is needed specifically on the role of caspase and tubulin in host response to respiratory virus infection. modulation of any human host pathway for the treatment of viral infections has potential drawbacks with respect to toxicity and other side-effects. for example, although interferon is widely used to help combat viral pathogens, the treatment is known to cause an array of side-effects related to toxicity including confusion, lethargy, impaired mental status, numbness, tingling, fevers, chills, headaches, anorexia and sepsis [89, 90] . another caveat is that some proteins are beneficial if up-regulated during initial viral infection but have detrimental effects if over-activated for prolonged periods. thus determining the desired mechanism and direction of therapeutic intervention requires careful study. although targeting host-pathogen interactions is a challenging therapeutic approach, there are considerable upside benefits with respect to overcoming pathogen-mediated drug resistance and the capability of treating multiple, co-infecting pathogens. our study suggests several potential human-host proteins that could be targets of future therapeutics as well as some possible drug candidates for further investigations of repurposing against respiratory virus infections. the national center for biotechnology information's geo database (http://www.ncbi.nlm.nih.gov/geo/ (accessed between january and july 2011) was searched for human mrna datasets for twelve respiratory viruses. (figure 1 ) reduced the number of viruses with suitable datasets to seven species (table 1) . all analyzed geo datasets contain at least one ''treatment group'' and ''control group''. ''treatment'' was the experimental variable under study, usually a virus type, strain, or time point. ''group'' was a collection of individual ''samples'', or replicates, each of which originates from their own microarray chip. ''comparison group'' was the treatment group compared to a control group. a particular dataset may have more than one comparison group. all criteria for dataset inclusion in the final analysis were chosen prior to the analysis. dataset candidacy filtering consisted of four criteria: 1) the dataset must contain at least 3 samples per treatment or control group because a sample size any less would mean a loss in statistical power for subsequent analysis; 2) the microarray platform must be supported by either affymetrix, agilent or illumina due to probe mapping abilities of the software used in subsequent analysis; 3) each gene expression profile had to be derived from human cells and probed using a human-based genome microarray platform and not other species; and 4) the dataset must contain at least one wild-type infection treatment group (i.e., unmodified virus strain or infectivity mechanism) and at least one healthy control group (i.e., no genetic or media modifications such as gene knock outs or inhibitors, respectively). prior to quality control (qc) analysis, we pre-screened and preprocessed each dataset. normalized raw data and the study design table were imported from the geo databases (the data was assumed to be normalized by robust multi-array average, but in some cases the published study used an alternative normalization method). where appropriate, the intensity values were log 2 transformation. various experimental parameters such as time point, virus strain and number of replicates were extracted from the study design tables. samples irrelevant to the main study design were marked for segregation or exclusion from our downstream analysis, but not excluded from quality assessment. these were classified as ''failing to meet treatment specification'' at the candidate filtering step. studies that had a large number of missing intensity values (over 10%) were annotated and flagged. the qc analysis assessed each sample in the dataset for kernel density, pca, mad, and pair-wise pearson correlation such that: 1) the kernel density was normally distributed; 2) after pca values were within the hotelling t2 alpha level threshold of 0.05 [91] [92] [93] ; 3) mad score scores were in the range of +3 to 23 with no outliers [94] ; and 4) inner-treatment group pair wise correlations for samples derived from a single cell were $0.97 or $0.90 if taken from individual donors [94] . figures were created using array studio software, version 4.1. (omicsoft corporation, research triangle park, nc, usa [95] ). during subsequent analysis, each comparison group was treated separately, regardless of dataset origination, in order to gain a wider, less bias view of representative genes and pathways. once a comparison group passed the qc analysis filters, lsm values were calculated for each probe using array studio in order to reduce the number of false positives due to low probe intensity values. probes within each of the filtered datasets were tested for biological and statistical relevance using the array studio implementation of fold change and statistical models, respectively. specifically, to determine a probe's fold change expression when compared to control, the geometric mean of each probe's log 2 transformed intensity value within a treatment was generated, and then normalized to the corresponding control group's geometric mean. the treatment versus control data were fitted to a general linear model, and associated p-values for each probe were calculated using a modified t-test [96] . thus, to be considered differentially expressed, each probe within a comparison group must have a p-value ,0.05 after general linear model test and a fold change in either direction of 1.5. to visualize a comparison group's significance and fold change, volcano plots were generated using array studio of a probe's 2log(p-value) versus its transformed fold change (fc) value according to the following piece-wise function: the differentially expressed probes were mapped to their corresponding genes using metacore/metabase (genego), a software/database package that creates biological pathways and networks from gene lists (database accessed june 2011) [97, 98] . if more than one probe mapped to a gene, the probe with the highest magnitude fold change value was used for that gene. thus, the mapped differentially expressed probe list became the differentially expressed gene list for each comparison group. the differentially expressed gene lists from each comparison group were analyzed for enriched pathways using genego. a pvalue for each of the 658 pathway maps in the metabase were generated for each comparison group using a hypergeometric test [99] . in order for a pathway to be considered enriched, each comparison group must contain pathways that have a p-value ,0.01 and occur in .5% of the total studies. the enriched pathway list was ranked by its viral frequency, which is defined by the number of viruses represented by at least one comparison group, and then by the sum of normalized viral expression or nve for each enriched pathway. the nve for each pathway was calculated using the number of comparisons containing significant pathways within a virus type relative to the number of comparisons within that virus type. for example, if one out of four flu comparisons for pathway a were significant, the nve for flu would be 1/4. ranking the pathways in this fashion resulted in a clearer determination of pathways shared across multiple viruses, irrespective of time, strain type, or number of comparison groups. after examining the ranked pathway list described above, the top five significant pathways and an additional pathway representing a unique mechanism were further analyzed. with each map, the proteins were labeled according to the number of viruses in which the transcript was differentially expressed thus yielding the viral frequency for that protein. in cases where a protein complex was made up of subunits, the greatest magnitude fold change value for any subunit was chosen to represent the entire complex. genego was used for the visualization of this pathway map. similar to the pathway nve, the nve for each gene within these six chosen pathways was calculated using the number of comparisons containing either up or down regulated genes for each protein within a virus type relative to the number of comparisons within that virus type. for example, if two out of three rsv comparisons for gene x were up-regulated, gene x's nve for rsv would be 2/3. we performed complete linkage and correlation distance hierarchical clustering using arraystudio on quantile normalized fold change values to determine the separation qualities of the analyzed data [100] . clustering was performed on genes that had expression values for at least 90% of the total number of comparisons. we used the matlab function 'knnimpute' to impute missing fold change values using k-nearest neighbors estimation (matlab version 7.11 (r2010b), mathworks, cambridge ma, 2010) [101, 102] . approved or marketed small molecule and protein therapeutics for each of the differentially expressed proteins modulated by 5 or more respiratory viruses were obtained from the drugbank database, version 3.0 (http://www.drugbank.ca/ accessed august 2011) [29] [30] [31] . we only considered those drugs that were launched products with experimental and clinical evidence of direct interaction with gene product in question. the therapy's interaction with the target and approved indication were identified using a combination of drugbank, the drug manufacturer's information page, and the national center for biotechnology information's pubchem (http://pubchem.ncbi.nlm.nih.gov/ accessed september 2011) [103] and gene (http://www.ncbi.nlm. nih.gov/gene/ accessed september 2011) databases. supplemental evidence of mechanism of action was obtained from immune or infection-related jackson laboratory knock-in/knock-out mouse (jax) phenotype (http://www.jax.org/ accessed september 2011) [32] . (table 3 ). the horizontal axis contains each of the 28 different comparisons labeled by virus, gse and time point. the vertical axis shows the clustering of 27 genes from the top five and parkin-ups pathways that have an nve of at least 6 and have an expression value in at least 26 comparisons. for genes present in more than one of the five pathways, the number of participating pathways is indicated by the count of ''*'' before the gene name. color scheme is as described for figure s3 . (tif) figure s5 epidermal growth factor receptor signaling pathway with viral frequency. viral frequencies superimposed for each of most frequently differentially expressed proteins, where red circles are differential expression of genes by 7 viruses, orange circles are differential expression of genes by at least 6 viruses, and blue circles are differential expression of genes by 5 viruses. see metacore website at http://www.genego.com/pdf/ mc_legend.pdf for figure legend and table s4 for pathway map gene products' corresponding hugo gene names. (tif) figure s6 cd40 signaling pathway with viral frequency. viral frequencies superimposed for each of most frequently differentially expressed proteins, where red circles are differential expression of genes by 7 viruses, orange circles are differential expression of genes by at least 6 viruses, and blue circles are differential expression of genes by 5 viruses. see metacore website at http://www.genego.com/pdf/mc_legend.pdf for figure legend and table s4 for pathway map gene products' corresponding hugo gene names. (tif) figure s7 interferon-gamma signaling pathway with viral frequency. viral frequencies superimposed for each of most frequently differentially expressed proteins, where red circles are differential expression of genes by 7 viruses, orange circles are differential expression of genes by at least 6 viruses, and blue circles are differential expression of genes by 5 viruses. see metacore website at http://www.genego.com/pdf/mc_legend. pdf for figure legend and table s4 for pathway map gene products' corresponding hugo gene names. (tif) figure s8 histamine receptor h1 signaling pathway with viral frequency. viral frequencies superimposed for each of most frequently differentially expressed proteins, where red circles are differential expression of genes by 7 viruses, orange circles are differential expression of genes by at least 6 viruses, and blue circles are differential expression of genes by 5 viruses. see metacore website at http://www.genego.com/pdf/mc_legend. pdf for figure legend and table s4 for pathway map gene products' corresponding hugo gene names. (tif) figure s9 interleukin-17 signaling pathway with viral frequency. viral frequencies superimposed for each of most frequently differentially expressed proteins, where red circles are differential expression of genes by 7 viruses, orange circles are differential expression of genes by at least 6 viruses, and blue circles are differential expression of genes by 5 viruses. see metacore website at http://www.genego.com/pdf/mc_legend. pdf for figure legend and table s4 for pathway map gene products' corresponding hugo gene names. 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title: alphacoronaviruses in new world bats: prevalence, persistence, phylogeny, and potential for interaction with humans date: 2011-05-12 journal: plos one doi: 10.1371/journal.pone.0019156 sha: doc_id: 277306 cord_uid: r8jki3x4 bats are reservoirs for many different coronaviruses (covs) as well as many other important zoonotic viruses. we sampled feces and/or anal swabs of 1,044 insectivorous bats of 2 families and 17 species from 21 different locations within colorado from 2007 to 2009. we detected alphacoronavirus rna in bats of 4 species: big brown bats (eptesicus fuscus), 10% prevalence; long-legged bats (myotis volans), 8% prevalence; little brown bats (myotis lucifugus), 3% prevalence; and western long-eared bats (myotis evotis), 2% prevalence. overall, juvenile bats were twice as likely to be positive for cov rna as adult bats. at two of the rural sampling sites, cov rnas were detected in big brown and long-legged bats during the three sequential summers of this study. cov rna was detected in big brown bats in all five of the urban maternity roosts sampled throughout each of the periods tested. individually tagged big brown bats that were positive for cov rna and later sampled again all became cov rna negative. nucleotide sequences in the rdrp gene fell into 3 main clusters, all distinct from those of old world bats. similar nucleotide sequences were found in amplicons from gene 1b and the spike gene in both a big-brown and a long-legged bat, indicating that a cov may be capable of infecting bats of different genera. these data suggest that ongoing evolution of covs in bats creates the possibility of a continued threat for emergence into hosts of other species. alphacoronavirus rna was detected at a high prevalence in big brown bats in roosts in close proximity to human habitations (10%) and known to have direct contact with people (19%), suggesting that significant potential opportunities exist for cross-species transmission of these viruses. further cov surveillance studies in bats throughout the americas are warranted. bats play important roles in maintaining and transmitting zoonotic viruses [1, 2, 3] . more than 99 different viruses have been detected in and/or isolated from bats of diverse species [2] (and c. calisher, personal communication). rabies virus and other lyssaviruses infect bats of many species, and old world fruit bats (family pteropodidae) are reservoirs for both hendra and nipah viruses [4, 5, 6] . two newly discovered human reoviruses, melaka virus and kampar virus, associated with influenza-like illnesses in humans, may be transmitted from small flying foxes (fruit bats; pteropus hypomelanus) based on the close phylogenetic relationships of these viruses to pulau virus, a bat reovirus [7, 8] . egyptian fruit bats (rousetttus aegyptiacus) are known reservoirs of marburg and certain ebolaviruses [9, 10] . in humans, domestic animals, and birds, coronaviruses are common respiratory and enteric pathogens, and several covs cause systemic disease. among the 5 known human coronaviruses, hcov-229e and hcov-nl63 are alphacoronaviruses (formerly called group 1 covs), hcov-oc43 and hcov-hku1 are betacoronaviruses (formerly group 2a), and the severe acute respiratory syndrome-related coronavirus (sars-cov) and sarslike covs are also betacoronaviruses (formerly group 2b). the sars pandemic of 2002-03 was caused by sars-cov, a zoonotic coronavirus recently emerged from horseshoe bats (suborder microchiroptera, family rhinolophidae, genus rhinolophus) from different locations in southeastern china [11, 12] . extensive worldwide surveillance of bats showed that bats carry an enormous diversity of covs [13, 14, 15, 16, 17, 18, 19, 20, 21, 22] . phylogenetic analysis of complete genome sequences of coronaviruses from bats, humans, birds, and other vertebrates suggests that bats may be the reservoir hosts from which all coronavirus lineages originated [23, 24] . the potential for emergence of zoonotic viruses into the human population depends on the prevalence of the virus in its host species, host range mutations within viral quasispecies, and the degree to which the reservoir host interacts with humans. in 2006, we reported the first detection of alphacoronavirus rna in feces of north american bats sampled in the rocky mountain region of colorado [17] . here we describe a much larger and more comprehensive study of coronavirus prevalence, epizootiology, geographic distribution, and persistence, as well as preliminary phylogenetic analysis of cov genome sequences in bats in colorado. capture, marking, and sampling of bats followed guidelines of the american society of mammalogists [25] and animal protocols were approved by the institutional animal care and use committee of the u.s. geological survey, fort collins science center ('standard operating procedure 01-01 for the capture, handling, marking, tagging, biopsy sampling, and collection of bats') and colorado state university (csu iacuc number 03-096a). bats were captured under authority of a scientific collecting license (permit numbers: 07tr738a3, 08tr2010, and 09tr2010) issued by the colorado division of wildlife. insectivorous bats of the families vespertilionidae (16 species) and molossidae (1 species) were sampled at 16 rural sites (sites #1-16, fig. 1 ) in the rocky mountain region during the summer of 2007. bats were identified to species based on external morphological characteristics as described in regional faunal manuals [26, 27] adopting revised taxonomy for myotis occultus [28] and parastrellus hesperus [29] . to determine whether covs persist in bat populations over the course of several years, additional bat fecal samples were collected during the summers of 2008 and 2009 at two rural sites in north central and southeastern colorado. in addition, big brown bats (eptesicus fuscus) were sampled at 5 different sites (sites #17-21) within a single urban municipality in northern colorado (fort collins) during the summers of 2007 and 2008. these sites were chosen because they were in close proximity to humans [30] . site #17 was in a vintage farmhouse that is currently being used as a family visitation center; site #18 was a natural creek surrounded by suburban neighborhoods; site #19 was in the recreation center of a church; site #20 was within an education building, and site #21 was within a picnic pavilion at a public park. several of these sites had been previously used in rabies ecology studies, and some bats had been tagged with passive integrated transponders (pit tags) for host demographic analysis [31, 32] . this allowed for repeated capture and sampling of known individual bats. all bats were either captured in mist nets during the night as they drank or foraged near open water, or were caught in mist nets or harp traps as they emerged from roosts. whenever possible, the species, sex, reproductive status, age (adult or juvenile), date, and location of capture were recorded for each bat sampled. bats were sampled as previously described [17] , typically within 5-10 min of capture, and then released. anal/rectal swabs or fecal pellets were taken using sterile calcium alginate swabs and stored in rnalater (ambion, austin, tx) and/or m4 viral transport medium (vtm, remel; lenexa, ks). all samples were stored at 270uc prior to analysis. based on sample type and medium results were pooled for analysis of prevalence surveys. in a post hoc analysis we identified differences in the efficacy of different sampling methods (text s1) such that the data represent minimal estimates of the prevalence of cov infection in bats. bat carcasses submitted to the colorado department of public health and environment (cdphe) that were negative for rabies viruses were sent to our laboratory for detection of cov rna. these bats had been submitted from counties throughout colorado for rabies testing to rule out the need for post-exposure rabies prophylaxis of humans who had had close contact with these animals [30, 33] . intestines were removed from the bats and stored at 270uc prior to analysis. extracted rna was eluted in 60 ml of rnase-free water and stored at 280uc. before rt-pcr, 50 microliters of rna was treated with zymo onestep pcr inhibitor removal kit (zymo research, orange, ca) following the manufacturer's instructions. cdna was generated by superscript iii reverse transcriptase (invitrogen, carlsbad, ca) with random hexamers in a 20 ml reaction using 11 ml of rna as a template according to the manufacturer's instructions. all samples were analyzed in duplicate. reverse-transcription products were stored at 220uc. all cdna samples collected from bats at rural sites or during 2007 were screened for cov rna by pcr with a pair of pan-cov consensus primers [13] that amplify a highly conserved region (400 nucleotide amplicon) of the coronavirus rnadependent rna polymerase (rdrp) gene as previously described [17] except that we used 2.0 mmol/l of primers and 1 ml of cdna or pcr product (for hemi-nested reactions). to increase the sensitivity of rna detection, based on our previously published bat cov sequences [17] and new data from this study, we designed specific primers within the amplicons of alphacoronaviruses from bats of several species in the genus myotis and big brown bats (table s1 ). all of the specimens collected from longlegged and big brown bats were also tested with these primers. to obtain longer nucleotide sequences, rt-pcr was performed using consensus degenerate primers from several regions within the rdrp gene in a superscript iii one-step rt-pcr system with platinum taq high fidelity kit (invitrogen, san diego, ca, usa). similarly, we designed consensus primers that targeted a highly conserved region of the s2 region of the alphacoronavirus spike gene, and made primers from an exact s2 sequence obtained from a big brown bat (table s1) . to minimize the possibility of contamination, all rt and pcr reactions were prepared in an enclosed acrylic nucleic acid workstation equipped with a uv light (clone zone, usa scientific, ocala, fl) in a room separate from the main laboratory. water controls without template included in every rt and pcr experiment gave no false-positive results. amplicons were analyzed by agarose gel electrophoresis and sequenced on an abi 3730 dna sequencer (applied biosystems technologies, carlsbad, ca) at the university of colorado school of medicine cancer center dna sequencing and analysis core. samples were scored as positive if cov rna was detected on two pcr runs. statistical significance was determined using fisher's exact test. phylogenetic analyses were conducted using mega version 4, and phylogenetic trees were constructed using the neighbor-joining method [34] . the nucleotide sequences from this study were deposited in genbank under accession numbers hq336973-hq336976 and jf414933-jf414936. table 1) . at site #4, a high-elevation meadow in a mountainous area of north-central colorado, 76 long-legged bats were sampled during three consecutive summers (2007) (2008) (2009) . although the sampled bats were not individually marked, the consistent capture of large numbers of females soon after sunset at the site indicated that most of the sampled bats likely came from a nearby maternity roost. female bats often show year-to-year fidelity to maternity roosts [35] . the percentage of long-legged bats that tested positive for cov rna at site #4 varied by year from 6% to 31% (table 2) . at site #5, an arid grassland bisected by canyons in southeastern colorado [36] , 56 bats of eight different species were sampled during two consecutive summers (2008 and 2009). only big brown bats at site #5 were positive for cov rna. although the number of big brown bats sampled at site #5 was small (4 in 2008 and 14 in 2009), the prevalence of cov rna in these bats during these two summers was high (29% to 100%) ( table 2) . in the five different urban locations (sites #17-21), 465 samples were collected from big brown bats during the summers of 2007 all of the urban bat sampling sites were part of a previous study of the ecology of rabies in big brown bats that emphasized host demography [31, 32] , and 113 (24%) of the 465 bats from these sites sampled for this study had been previously individually tagged. sixteen (14%) of these tagged bats were captured and sampled more than once (14 captured twice, and 2 captured three times). five (31%) of the 16 repeatedly sampled tagged bats captured in 2008 were positive for cov rna, but no cov rna could be detected in subsequent samples ( table 3 ). four of the 5 bats became negative for cov rna within 6 weeks after they tested positive for cov rna. (the fifth bat was not recaptured after turning positive). thus, in this small group of serially sampled bats, individual bats were not continually shedding detectable amounts of cov rna, so did not appear to be persistently infected. the age and sex distributions of the 999 (94%) bats sampled for which these data were available and the subset of big brown bats in the urban maternity roosts sampled are shown in table 4 . juvenile bats were two times more likely to be positive for cov rna than adults bats (13% vs. 6%, p = 0.008). in the urban maternity roosts, as expected, the majority of the big brown bats sampled were adult females, but juvenile bats (10 of 52 tested, 19%) were also more than twice as likely to be positive for cov rna than adult bats (36 of 413 tested, 9%, p = 0.03). from the samples positive for cov rna, we obtained nucleotide sequences of amplicons ranging in length from 93-356 nt from the rdrp region of gene 1b. these formed three clusters (.90% nt identity within each cluster). the first cluster (a) included cov rnas of big brown bats from sites #5 and #17-21, the one big brown bat from site #4, and two long-legged bats from site #4 that were collected in 2007 and 2009. the sequence of the a cluster (representative bat: rm-bt-cov 453/2007 ef) was 96% identical to the same region from a big brown bat (rm-bt-cov 65) reported in our previous study [17] . the second cluster (b) (representative bat: rm-bt-cov 09-07/2009 mv) was found in 2 long-legged bats (one sampled in 2008 and one in 2009) and one western long-eared bat sampled at site #4. these sequences had .97% identity in this region to cov rna obtained from several occult bats (m. occultus; rm-bt-cov 6 and 11) reported previously [17] . the third cluster (c) of cov amplicons (representative bat: rm-bt-cov 429/2007 mv) were from other long-legged bats sampled at site #4. these sequences were 96% identical to that from an occult bat (rm-bt-cov 3) reported previously (table 5 and figure s1 ). cluster a had ,65% identity with clusters b and c, whereas clusters b and c had 83% identity to one another. an 1100 nt sequence encoding the s2 domain of the spike glycoprotein was obtained from a big brown bat collected at site #4 in 2007 (rocky mountain bat-cov 453/2007 ef). we compared this sequence to s2 sequences of other known coronaviruses (table 5 and figure 2 ) and found that this genome was distantly related to other known alphacoronaviruses in group 1a, with ,67% nucleotide identity to covs. we also obtained a 700 nucleotide sequence in the same region of s2 from the long-legged bat (rm-bat-cov 433/2007 mv) that had a similar sequence to this big brown bat in the rdrp gene (both in rdrp cluster a). these s2 amplicons had .98% nt sequence identity. the closest bat coronavirus spike sequence to rm-bt-cov 453/2007 found in genbank, was bt-cov a701, from an old world species, rickett's big-footed bat (myotis ricketti) sampled in southeast china in 2005 [14] (65% nucleotide identity, 65% amino acid identity). an approximately 4000 nt sequence in 2 segments of the rdrp gene was obtained from one of the little brown (rm-bt-cov-15/ 2006/ml) and one of the big brown bats (rm-bt-cov-61/2007/ ef) that were submitted to the cdphe. these nt sequences were only 62% identical, indicating that they represented two unique viruses in bats of these two species. these sequences were distantly related (,75% nt identity) to other known alphacoronaviruses, with ,75% nt identity to covs in this group, including all currently available old world bat covs (table 5 and figure 3 ). this is the first multiyear surveillance project of covs in wild bats in north america. cov rna was detected in approximately 7% of all bats sampled (likely an underestimate of prevalence, text s1), comparable to the prevalence of cov rna detected in various species of bats reported in other parts of the world (ranging from 2-55%) [14, 18, 19, 21, 22, 37, 38, 39] . in our study no cov rna was detected in bats in 13 of the 17 species we sampled (also likely biased negatively). failure to detect covs in bats of these species could be related to the smaller numbers sampled. however, a relatively high prevalence of cov rna was detected in bats of 2 species collected at several different sites: 12% for big brown bats and 8% for long-legged bats, and at lower prevalence, 3% in little brown bats and 2% in western long-eared bats. in marked contrast to the enormous diversity of cov genomes found in old world bats [14, 24, 40] , in this and several other cov surveillance studies of new world bats [17, 18, 22] , all covs detected were alphacoronaviruses. our data indicate that nucleotide sequences of alphacoronaviruses harbored by colorado bats are distinct from those found in old world bats. two recent studies of the bat guano virome using next generation sequence technology also only detected alphacoronaviruses in the new world bats of the species tested, as well as a diverse array of other types of viruses [41, 42] . thus, so far there appears to be much more limited cov diversity in new world bats of the species tested than in old world bats. betacoronaviruses have only been detected in old world bat species belonging to the families pteropodidae (rousettus spp) and rhinolophidae (rhinolophus spp.) which belong to the chiropteran suborder yinpterochiroptera. based on available evidence, betacoronaviruses could be restricted to hosts in the suborder yinpterochiroptera (families pteropodidae, rhinolophidae, megadermatidae, craseonycteridae, rhinopomatidae). no bat families of the suborder yinpterochiroptera occur in the new world. [43] . the finding of only alphacoronaviruses in our study may be because bats of these species are resistant to other covs and/or bats from different parts of the new world have yet to be tested for cov infection, as we sampled bats from only a subset of the hundreds of species that reside in the new world. these observations also support the hypothesis that coronaviruses may have co-evolved with their bat hosts, as no species of bat is found both in the new world and old world [44] . to date, however, only a small subset of new world species of bats has been tested for coronavirus infection. as 75% of living genera of all bats worldwide are found in the new worlds tropics alone, further cov surveillance in bats of additional species from different regions in the western hemisphere may reveal hitherto undetected varieties of coronaviruses. the seasonal epidemiology and persistence of new world cov infections in individual bats and within bat populations has not been elucidated. the most comprehensive epidemiological investigation of covs to date in old world bat populations showed that the prevalence of sars-rh-batcovs in rhinolophid bats over a four-year period at collection sites in hong kong sar and china peaked in the spring and varied from year to year. we found similar results in new world bats. at site #4 long-legged bats had an alphacoronavirus rna prevalence of 31% in 2007, 19% in 2009, but only 6% in 2008. in all five of the urban maternity roosts sampled, covs persisted in bat roosts throughout the course of the non-hibernating part of the year (spring/summer) and persisted from year to year. we also found that the prevalence of cov infection in these bat roosts tended to peak in late spring/ early summer. the prevalence of infection with human covs also shows significant annual variations [45] , possibly depending on environmental conditions and/or fluctuating cov antibody levels in the population. possible seasonal variation in cov infection rates may explain why in our initial 2006 study we found a high prevalence (50%) of alphacoronavirus rna in occult bats [17] , but in 2007 we did not detect any positive individuals (22 tested in the same region). the majority of the bats sampled in our study were adult females because they were primarily captured from maternity roosts. the highest prevalence of infection was noted in juvenile bats. in germany, cov infection was also found to be associated with young age and was more common in female bats from maternity roosts compared to female bats found at foraging or swarming sites [19] . these findings support the hypothesis that younger bats may be more susceptible to cov infection and may serve to propagate and maintain these viruses within bat colonies. no overt clinical manifestations of disease were observed in any of the captured bats, including those that were infected with covs. in the small subset of bats that were tagged and recaptured, no individual bat remained persistently positive for cov rna after 6 weeks. similar findings were made in rhinolophid bats in asia that harbor sars-like-bat-covs [37] and in fruit bats experimentally infected with bat covs which showed no signs of illness [39] . these data suggest that although covs persist within bat populations, individual bats may experience only self-limited infections with covs without apparent illness. phylogenetic studies of cov genomes in old world bats in asia and europe have suggested that some bat covs may infect bats of only one species or several closely related species. in asia and germany, different species of bats roosting in the same cave were found to host different covs, whereas bats of the same species in different locations harbored similar covs [14, 19] . in europe, strict associations were found between bat cov deduced amino acid sequences in an 816 bp fragment of the rdrp gene and their specific bat hosts [40] . in africa, covs found in one species of bat were not detected in bats of different species co-roosting in the same cave [38] . similarly, our study showed that new world bats of the same species in geographically distinct locations and over the course of several years harbor similar covs. in contrast to these findings, in kenya some covs appear to be able to infect old world bats of several different species [21] . our preliminary nucleotide sequence data also suggests that we found very closely related cov nucleotide sequences in new world bats from three different species of myotis (m. volans, m. evotis, and m. occultus). furthermore, in site #4, we found similar nucleotide sequences in the spike and replicase genes in cov rnas from both a big-brown bat and a long-legged bat, suggesting that at least some new world bat covs may be able to infect bats of different genera. these findings are notable, as recent phylogenetic studies of rabies viruses in bats suggest that host species barriers play a key role in cross species transmission of viruses [46] . to assess the potential for zoonotic transmission of bat covs, we focused part of this present work on north american bats that have the closest contact with humans and sampled roosts where big brown bats had histories of contact or potential for contact with people [30] . big brown bats are common inhabitants of buildings in cities and towns in colorado and across the united states, and are the primary species encountered by humans in terms of potential exposure to disease agents [30, 33, 47] these bats had a high prevalence of cov infection, ranging from 0-67% (overall 10%) depending on the site and time of year. big brown bats submitted to the cdphe for rabies testing because of known direct contact with humans also had a very high prevalence (19%) of cov infection. because bats which have known or potential contact with humans have such a high prevalence of cov infection, opportunities exist for potential transmission of these viruses to humans. following the sars epidemic, intensive surveillance detected a great diversity of covs throughout the animal kingdom. covs can undergo a high frequency of rna recombination, both in vitro and in vivo, which may play an important role in their evolution and virulence [48] . old world bat covs of several different genotypes were found to co-exist in a single bat [49] . thus recombination between different bat covs could potentially occur in vivo, giving rise to new cov genomes. two strains of hcov-hku1 have recombined to yield a novel hcov-hku1 genotype [16] , and recombination between different strains of sars-cov-like viruses in bats may have given rise to civet sars-cov [37] . the great diversity of covs, their high frequency of rna recombination, their ability to persist in bat populations, and the finding that some covs can apparently infect bats of divergent genera, suggest that ongoing evolution of covs in bats may pose a continuing threat for emergence of novel covs into new hosts. table s1 primers and rt-pcr programs. a. consensus primers targeted a highly conserved region of the s2 region of the spike gene and from an exact sequence obtained from one of the big brown bats. pcr was performed under the following conditions: one ml of cdna was amplified in a 50-ml reaction containing, 0.2 mmol/l deoxynucleoside triphosphates, 1 u of phusiontaq high-fidelity dna polymerase (finnzymes, espoo, finland), and 2.0 mmol/l primers by the following pcr program: 30 sec at 98uc; 40 cycles for 10 sec at 98uc, 15 sec at 50-52uc (depending on the primer set), and 15 sec at 72uc; and then 10 min at 72uc. b. primers used for detection of cov sequence in bat samples. one microliter of cdna was amplified in a 50-ml reaction containing 1.5 mmol/l mgcl 2 , 0.2 mmol/l deoxynucleoside triphosphates, 2.5 u of hotstartaq (qiagen), and 2.0 mmol/l primers using the following pcr program: 15 min at 95uc; 45 cycles for 1 min at 95uc, 1 min at 48uc for my-f and my-r and 50uc for ef-f and ef-r, and 1 min at 72uc; and 10 min at 72uc. c. to obtain additional sequences for phylogenetic analysis, for two of the cdphe intestinal samples, rt-pcr was performed using consensus degenerate primers from several areas within the rdrp gene in a superscript iii one-step rt-pcr system with platinum taq high fidelity kit (invitrogen, san diego, ca, usa). primers and protocols were kindly provided by suxiang tong, phd and ying tao, phd of the centers for disease control and prevention, atlanta, georgia, usa. text s1 influence of different sampling and analysis techniques on cov rna detection. (doc) emerging viruses: coming in on a wrinkled wing and a prayer bats: important reservoir hosts of emerging viruses bats as a continuing source of emerging infections in humans a novel morbillivirus pneumonia of horses and its transmission to humans infection of humans and horses by a newly described morbillivirus fatal encephalitis due to nipah virus among pig-farmers in malaysia a previously unknown reovirus of bat origin is associated with an acute respiratory disease in humans identification and characterization of a new orthoreovirus from patients with acute respiratory infections large serological survey showing cocirculation of ebola and marburg viruses in gabonese bat populations, and a high seroprevalence of both viruses in rousettus aegyptiacus isolation of genetically diverse marburg viruses from egyptian fruit bats severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats bats are natural reservoirs of sars-like coronaviruses identification of a novel coronavirus in bats prevalence and genetic diversity of coronaviruses in bats from china coronaviruses in bent-winged bats (miniopterus spp comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features detection of group 1 coronaviruses in bats in north america detection of polyoma and corona viruses in bats of canada detection and prevalence patterns of group i coronaviruses in bats coronavirus antibodies in african bat species detection of novel sars-like and other coronaviruses in bats from kenya detection and phylogenetic analysis of group 1 coronaviruses in south american bats evolutionary insights into the ecology of coronaviruses coronavirus diversity, phylogeny and interspecies jumping guidelines of the american society of mammalogists for the use of wild mammals in research distribution and ecology of bats of colorado. denver: university of colorado museum systematics of myotis occultus (chiroptera: vespertilionidae) inferred from sequences of two mitochnodrial genes generic status of the american pipistrelles (vespertilionidae) with description of a new genus ellison ec (in review) bat ecology and public health surveillance for rabies in an urbanizing region of colorado adult survival and population growth rate in colorado big brown bats (eptesicus fuscus) recruitment in a colorado population of big brown bats: breeding probabilities, litter size, and first-year survival risk for rabies transmission from encounters with bats mega4: molecular evolutionary genetics analysis (mega) software version 4.0 ecology of bats dual captures of colorado rodents: implications for transmission of hantaviruses ecoepidemiology and complete genome comparison of different strains of severe acute respiratory syndrome-related rhinolophus bat coronavirus in china reveal bats as a reservoir for acute, self-limiting infection that allows recombination events distant relatives of severe acute respiratory syndrome coronavirus and close relatives of human coronavirus 229e in bats bat coronaviruses and experimental infection of bats, the philippines genomic characterization of sars-related coronavirus in european bats and classification of coronaviruses based on partial rna-dependent rna polymerase gene sequences metagenomic analysis of the virome of three north american bat species: viral diversity between different bat species that share a common habitat bat guano virome: predominance of dietary viruses from insects and plants plus novel mammalian viruses a molecular phylogeny for bats illuminates biogeography and the fossil record a phylogenetic supertree of the bats (mammalia: chiroptera) host phylogeny constrains cross-species emergence and establishment of rabies virus in bats trends in national surveillance for rabies among bats in the united states in vivo rna-rna recombination of coronavirus in mouse brain co-existence of different genotypes in the same bat and serological characterization of rousettus bat coronavirus hku9 belonging to a novel betacoronavirus subgroup the authors are grateful to suxiang tong, phd and ying tao, phd of the centers for disease control and prevention, atlanta, georgia, usa, for sharing their primers and protocols for long range bat cov rt-pcr in advance of publication. field assistance in capturing bats was provided by j. bleak, r. choi, l. ellison, a. englert, a. gann, l. gayton, d. neubaum, m. neubaum, b. smart, e. snider, e. tuttle, and e. valdez. we thank c. willis and w. iko for helpful comments on an earlier draft of this manuscript. any use of trade product, or firm names is for descriptive purposes only and does not imply endorsement by the u.s. government. key: cord-283604-fqc9jp0l authors: chen, meng; zhu, zhen; huang, fang; liu, donglei; zhang, tiegang; ying, deng; wu, jiang; xu, wenbo title: adenoviruses associated with acute respiratory diseases reported in beijing from 2011 to 2013 date: 2015-03-27 journal: plos one doi: 10.1371/journal.pone.0121375 sha: doc_id: 283604 cord_uid: fqc9jp0l background: adenovirus is one of the most common causes of viral acute respiratory infections. to identify the types of human adenoviruses (hadvs) causing respiratory illness in beijing, a sentinel surveillance project on the viral aetiology of acute respiratory infection was initiated in 2011. principal findings: through the surveillance project, 4617 cases of respiratory infections were identified during 2011-2013. throat swabs (pharynx and tonsil secretions) were collected from all the patients, and 15 different respiratory viruses were screened by multiplex one-step pcr method. 45 were identified as adenovirus-positive from sporadic and outbreak cases of respiratory infection by a multiplex one-step rt-pcr method, and a total of 21 adenovirus isolates were obtained. five hadv types among three species, including hadv-3 (species hadv-b), hadv-4 (species hadv-e), hadv-7 (species hadv-b), hadv-55 (species hadv-b), and an undefined hadv type (species hadv-c) were identified. the comparison results of the penton base, hexon, and fiber gene sequences of the beijing hadv-3, hadv-4, hadv-7, and hadv-55 strains in this study and those from the genbank database indicated significant spatial and temporal conservation and stability of sequences within the genome; however, the phylogenetic relationship indicated that both strain bj04 and strain bj09 isolated in 2012 and 2013, respectively, may have recombined between hadv-1 genome and hadv-2 genome within species hadv-c, indicating intraspecies recombination. conclusions: this study confirmed that at least 5 hadv types including hadv-3, hadv-4, hadv-7, hadv-55 and an undefined hadv type were co-circulating and were the causative agents of respiratory tract infections in recent years in beijing. hadv-3, hadv-4, hadv-7, and hadv-55 showed the apparent stability of the genomes, while intraspecies recombination was identified in strain bj04 and bj09. the recombinants carrying penton base gene of hadv-1 as well as hexon and fiber genes of hadv-2 might be a novel type of hadv worthy of further study. human adenoviruses (hadvs) belong to the genus mastadenovirus within the family adenoviridae [1] . adenoviruses are non-enveloped, icosahedral, double-stranded dna viruses with genomes of 26-45 kb [1] . the viral capsid is composed of two types of capsomeres: the hexon and the penton (which consists of the penton base and the fiber). antigens at the surface of the virion are mainly type-specific [2, 3] . hexons are involved in neutralization, and fibers in neutralization and haemagglutination-inhibition. a recombinant that has a unique combination of these three regions (penton base; hexon loops; fiber knob) derived from previously recognized genotypes will be assign a new genotype (http://hadvwg.gmu.edu). traditionally, the only basis for recognizing a new type of hadv is by serology, and on the basis of their biological properties, hadvs have been classified into 7 species (human mastadenovirus a to g, hadv-a to hadv-g), including 52 human hadv types, which are formally recognized by the international committee on taxonomy of viruses (ictv) [4, 5] . in addition, novel hadv genotypes (hadv-53 to hadv-68) were recently identified based on their bioinformatics and genomic analysis of the complete viral genome sequences (http://hadvwg.gmu. edu). novel hadv strains may arise from mutations or recombination among the different types of hadvs [6] . hadv can cause a variety of clinical diseases such as acute respiratory disease [7] , gastroenteritis [8] , and keratoconjunctivitis [9] , which vary depending on the cell tropism of the viruses. among the hadv-associated respiratory diseases, viruses in species hadv-b (hadv3, 7, 11, 14, 16, 21, 50, 55) , species hadv-c (hadv-1, 2, 5, 6), and species hadv-e (hadv-4) [10] [11] [12] [13] [14] are recognized as the main pathogens responsible for the respiratory tract infection. as the capital city of china, beijing covers an area of 16,800 km 2 with a large population of more than 19.72 million (chinese statistics bureau, 2011). in order to elucidate the spectrum of the viral aetiology of acute respiratory infections and provide basic data to guide local disease prevention and control measures, a sentinel surveillance project on the viral aetiology of acute respiratory infections was initiated and sponsored by the beijing municipal health bureau in 2011. adenovirus is one of the most common causes of viral acute respiratory infections. in this study, our primary aim was to identify the types of hadv causing respiratory illness in beijing since 2011, to avoid the overuse of antibiotics and to improve the level of diagnosis and treatment of respiratory viral disease especially hadv associated disease in hospitals, and to provide scientific basis for prevention and control of hadv causing respiratory illness. this study did not involve human experimentation; the only human material used in this study was throat swab specimens collected from cases with respiratory tract infection during the implementation of the surveillance project on viral aetiology of acute respiratory infection. this study was approved by the second session of the ethics review committee of the national institute for viral disease control and prevention in china cdc. written informed consent for the use of the clinical samples has been obtained from all patients involved in this study. pharynx and tonsil secretions of the patients were wiped with disinfection long cotton swabs with gently action, and after samples collection, all samples were transported under a cold chain and preserved at −80°c for further identification. a multiplex one-step reverse transcription-polymerase chain reaction (pcr) was performed to screen for 15 different respiratory viruses (respiratory syncytial virus a and b, influenza virus a and b, parainfluenza virus 1-4, human adenovirus, human enterovirus, human rhinovirus, human metapneumovirus, human bocavirus, and human coronavirus nl63-229e and oc43-hku1) simultaneously by using a commercial kit (seeplex rv 15 ace detection kit; seegene, inc., seoul, korea) [15] . adenovirus-positive specimens were cultured and further analysed. virus isolation for the adenovirus-positive specimens was performed by using hep-2 cell lines (from american type culture collection, atcc number ccl-23) following the standard protocol [13] . cells inoculated with clinical samples were incubated at 37°c for 7 days. if no cytopathic effect was observed, the culture was used to inoculate fresh cells for up to 2 additional passages; the cultures with adenovirus-like cytopathic effects were passaged again to confirm the presence of the virus. determination of the nucleotide sequences of penton base, hexon, and fiber gene the viral dna was extracted from infected cells by using a qiaamp dna mini kit (qiagen, valencia, ca, usa) according to the manufacturer's instructions. for the typing of hadv, the penton base, hexon, and fiber gene sequences were obtained for all hadv strains; pcr was performed with the platinum pcr supermix (invitrogen) following the manufacturer instructions. the primer pairs designed to amplify and sequence the penton base, hexon, and fiber gene sequences are listed in table 1 . the amplification products were analysed with capillary gel electrophoresis by using the qiaxcel dna high resolution kit (qiagen, the netherlands). after the pcr products were purified with a qia gel extraction kit (qiagen, valencia, ca, usa), the amplicons were bi-directionally sequenced using the sanger sequencing method (bigdye terminator, version 3.1, cycle sequencing kit; life technologies, grand island, ny, usa) and an abi prism 3130 genetic analyser (applied biosystems, foster city, ca, usa). sequencher software (version 5.0; gene codes corporation, usa) was used to edit and assemble the raw sequence data. the blastn program (national center for biotechnology information, bethesda, md, usa) was used to identify the homologous nucleotide sequences in the genbank database. phylogenetic trees were generated by using the neighbour-joining method and the kimura-2-parameters model implemented in the mega program (version 5.03) [16] . the reliability of the tree at each branch node was estimated by 1000 bootstrap replicates. bootstrap values greater than 80% were considered statistically significant for grouping. table 2) . all the 21 patients who got hadvs infections presented fever in between 38.2 to 40.0 degrees, seven (33.3%) of 21 patients had radiographic evidence of pneumonia, one patient (4.8%) had bronchitis, and others 13 patients (61.9%) had only upper respiratory tract infection symptoms such as cough and runny nose ( table 2) . among them, hadv-55 infections (2 cases) and hadv-7 infections (4 of 6 cases) seems led to patients of severe symptoms (pneumonia), while hadv-3 and hadv-4 infection caused minor symptoms (symptoms of upper respiratory tract infection or bronchitis), with only one hadv-3 infection causing pneumonia. it is worth noting that the two patients infected with the undefined hadv type appeared to have only mild symptoms such as fever and cough, and both patients affected by this recombinant virus are infants (below 1 years old), while patients infected with other hadvs are all teenagers or adults. in order to further identify possible recombination events, phylogenetic analyses based on the penton base, hexon, and fiber gene sequences were used to analyse the relationship of bj04 and bj09 strains in this study as well as with hadv-c strains in the genbank database. (fig. 2) . in the penton base sequences, strain bj04 and bj09 were close to the corresponding sequences of the hadv-1 strains in the genbank database, and exhibited less similarity with the hadv-2 prototype strain recorded in the genbank database (fig. 2a) . and from the nucleotide sequence alignment based on the penton base gene (fig. 2b) , strains bj04 and bj09 are a little more like hadv-1 sequences, due to hadv-2 sequences having a 9-nt deletion from nt 1099 to nt 1107. in the hexon gene and fiber gene, the sequences of strain bj04 and strain bj09 clustered with that of hadv-2 ( fig. 2c and 2d) . these findings indicated that strain bj04 and bj09 may be recombinants having penton base gene of hadv-1, and hexon gene and fiber gene of hadv-2. in addition, recombination was not found among any of the other strains of hadv-1 and hadv-2 available in the genbank database. phylogenetic analysis of other hadv-3, hadv-4, hadv-7, and hadv-55 beijing strains showed relative genome stability (figs. 3, 4) . beijing hadv strains (representing 4 types) clustered with the sequences from the different provinces of the mainland of china and other hadvs within each corresponding type, and exhibited the highest similarity between those sequences. this indicated that these 4 hadv types' genomes (hadv-3, hadv-4, hadv-7, and hadv-55) are stable. this study documents the hadv types associated with respiratory infection in beijing during 2011-2013 through the surveillance project, and viruses in species hadv-b (hadv-3, 7, 55), hadv-c (undefined hadv type), and hadv-e (hadv-4) were identified. these results are similar to those of a previous study performed between 2005 and 2010 in beijing [17] , and confirmed that at least 5 hadv types were co-circulating and were the causative agents of respiratory tract infections in recent years in beijing. in addition to beijing, these hadvs were also most frequently detected from acute respiratory tract disease cases in other cities of china such as guangzhou in central south china [18] and lanzhou city in northeast china [19] , which indicated that these hadv types are widely distributed in china. this study is also consistent with reports from argentina [20] , usa [21] , egypt [22] , and korea [23] . in this study, a multiplex one-step rt-pcr was performed to screen for 15 different respiratory viruses using a commercial kit (seeplex rv 15 ace detection kit), and hadv is one of the target viruses. the reason for the positivity rate of multiplex one-step rt-pcr (45 adenovirus positive) being higher than that of the viral isolation (21 positive), may be mainly due that this method is more sensitive than viral isolation method for hadvs, and an other possible reason may be that the specimen collection is not timely, etc. the comparison results of the penton base, hexon, and fiber gene sequences between the beijing hadv-3, hadv-4, hadv-7, and hadv-55 strains in this study and the strains from the genbank database indicated significant conservation and stability of the sequences within the genome across time and space. this genome stability of hadv-3, 4, and 7 was also reported in earlier studies, showing that hadv-3 has displayed a relatively stable genome for more than 50 years [24] ; the genomes of the hadv-4 and hadv-7 strains are also remarkably conserved, albeit only extending for at least 20 years [25] . this characteristic has also been found for other hadvs such as hadv-5, whose genome was found to be stable even beyond the 45 years of its circulation in the population [26] . limited mutations and infrequent recombination may contribute to the long-term success of hadv-3, 4, and 7 vaccines. hadv-55 infection has gained attention in the last decade. genomics and bioinformatics data indicate that hadv-55 (earlier name hadv-11a) is an emergent respiratory pathogen due to recombination between hadv-11 and hadv-14 [13, 14] . as a newly identified acute respiratory disease pathogen, hadv-55-associated outbreaks were reported to have occurred in military camps of singapore and turkey in 2005 [27] , and in a senior high school in the shaanxi province of china in 2006 [13] . since 2008, this pathogen has been isolated from cases of respiratory infection and community-acquired pneumonia among adults in beijing, hebei, shandong, chongqing, and gansu provinces of china [28] [29] [30] ; it was also isolated among military trainees in hebei province of china in 2012 [30] . this may raise a serious public health concern, because hadv-55 has the potential to spread and cause severe epidemics in china, and it may become a major etiological agent causing pneumonia among the chinese population. in this study, two patients who were affected with hadv-55 also appeared to have relatively severe symptoms (clinical diagnosis was pneumonia), highlighting the significance of hadv-55 as being an increasing cause of respiratory illness especially pneumonia. therefore, efforts should be focused on a hadv-55 vaccine because of its stable genome. in addition, the genome stability of these hadvs is also a desired property in the application of these viruses as gene delivery vectors. in contrast to the apparent stability of the genomes of hadvs in general, they are also known to undergo recombination. recombination is a well-known feature in hadv genetics and is one of the most important factors driving the evolution of hadvs [31] . in this study, the phylogenetic analysis indicated that strain bj04 and strain bj09 may have recombined between hadv-1 genome (penton base gene) and hadv-2 genome (hexon and fiber genes) within species hadv-c, which are intraspecies recombination events. intraspecies recombinations have already been identified in many emergent hadv pathogens, which were subsequently identified as novel type hadvs, such as hadv-53 to 68 [13, 31, 32] . in this study, strains bj04 and bj09 were identified as intraspecies recombinants, and this recombination pattern (p1h2f2) has not yet been found in elsewhere, which indicated that it may also be a novel hadv type. although the two patients affected with this undefined type hadv appeared to have only mild symptoms (clinical diagnosis was upper respiratory tract infection), the pathogenicity remains unclear and the virus has the potential to cause serious symptoms because of its infection of children below 1-year old. further studies on the whole genomic sequence and virulence determination including a comparison of the growth kinetics and cytopathology of the recombinant virus and the two parental strains (hadv-1 and hadv-2) will be required to elucidate the characteristics of this novel hadv type. genetic content and evolution of adenoviruses phylogenetic analysis of the main neutralization and hemagglutination determinants of all human adenovirus prototypes as a basis for molecular classification and taxonomy molecular evolution of human species d adenoviruses virus taxonomy. seventh report of the 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human adenovirus type 3 provide evidence for relative genome stability across time and geographic space genomic and bioinformatics analyses of hadv-4vac and hadv-7vac, two human adenovirus (hadv) strains that constituted original prophylaxis against hadv-related acute respiratory disease, a reemerging epidemic disease computational analysis of adenovirus serotype 5 (hadv-c5) from an hadv coinfection shows genome stability after 45 years of circulation outbreak of febrile respiratory illness associated with adenovirus 11a infection in a singapore military training camp emergence of community-acquired adenovirus type 55 as a cause of community-onset pneumonia severe community-acquired pneumonia caused by adenovirus type 11 in immunocompetent adults in beijing epidemiology of human adenovirus and molecular characterization of human adenovirus 55 in china evidence of molecular evolution driven by recombination events influencing tropism in a novel human adenovirus that causes epidemic keratoconjunctivitis computational analysis identifies human adenovirus type 55 as a re-emergent acute respiratory disease pathogen we thank all the laboratory staffs and the epidemiologists in beijing center for disease control and prevention and team members in the projects. key: cord-291176-evb6yt0r authors: giorgi rossi, paolo; marino, massimiliano; formisano, debora; venturelli, francesco; vicentini, massimo; grilli, roberto title: characteristics and outcomes of a cohort of covid-19 patients in the province of reggio emilia, italy date: 2020-08-27 journal: plos one doi: 10.1371/journal.pone.0238281 sha: doc_id: 291176 cord_uid: evb6yt0r this is a population-based prospective cohort study on archive data describing the ageand sex-specific prevalence of covid-19 and its prognostic factors. all 2653 symptomatic patients tested positive for sars-cov-2 from february 27 to april 2, 2020 in the reggio emilia province, italy, were included. covid-19 cumulative incidence, hospitalization and death rates, and adjusted hazard ratios (hr) with 95% confidence interval (95% ci) were calculated according to sociodemographic and clinical characteristics. females had higher prevalence of infection than males below age 50 (2.61 vs. 1.84 ‰), but lower in older ages (16.49 vs. 20.86 ‰ over age 80). case fatality rate reached 20.7% in cases with more than 4 weeks follow up. after adjusting for age and comorbidities, men had a higher risk of hospitalization (hr 1.4 95% ci 1.2 to 1.6) and of death (hr 1.6, 95% ci 1.2 to 2.1). patients over age 80 compared to age < 50 had hr 7.1 (95% ci 5.4 to 9.3) and hr 27.8 (95% ci 12.5 to 61.7) for hospitalization and death, respectively. immigrants had a higher risk of hospitalization (hr 1.3, 95% ci 0.99 to 1.81) than italians and a similar risk of death. risk of hospitalization and of death were higher in patients with heart failure, arrhythmia, dementia, coronary heart disease, diabetes, and hypertension, while copd increased the risk of hospitalization (hr 1.9, 95% ci 1.4 to 2.5) but not of death (hr 1.1, 95% ci 0.7 to 1.7). previous use of ace inhibitors had no effect on risk of death (hr 0.97, 95% ci 0.69 to 1.34). identified susceptible populations and fragile patients should be considered when setting priorities in public health planning and clinical decision making. the novel sars-cov-2 (covid19) pandemic in early 2020 has been threatening the entire word [1, 2] . the virus has shown a high reproduction number and to spread rapidly [3, 4] . italy has been one of the first countries facing the epidemic outside of china and surely up to the end of march 2020, it was the most affected western country [5, 6] . the spectrum of disease of covid-19 is wide, ranging from no symptoms at all to severe mixed interstitial-alveolar pneumonia often requiring admission in an intensive care unit and ventilation. fatality rates are high, ranging from 2% to 12%, depending on the country, on reporting systems and definitions, and on length of follow up since disease onset [7] . further, hospitalization rates change according to different approaches to care and to varying availability of hospital beds; the latter also depends on the place and the phase of the epidemic [7] . since we are facing a new disease, very few studies can provide information on the factors explaining the variability observed in the fatality rate and on how to predict whether the disease will be severe or not. therefore, it is hard to define the prognosis both for individuals and for groups of patients. age and sex seem to be the only confirmed and well described prognostic factors, with a higher case fatality rate in older subjects and in males [8, 9] . pre-existing chronic conditions have been generically reported as poor prognosis determinants, but the strength of the association between each specific comorbidity and outcomes has not yet been fully explored [10, 11] . indeed, gaining a better understanding of the role of the main prognostic factors and quantifying the strength of their association with the rate of occurrence of a critical event is essential to identifying patients at high risk of worsening clinical conditions and to assessing the actual needs of different patient groups. in this report, based on the cohort of all residents in the province of reggio emilia who were sars-cov-2-positive at nasal and pharyngeal swab and with symptoms (covid-19 cases) since the inception of the epidemic, we describe patient characteristics and explore their role as putative prognostic factors in predicting the occurrence of hospital admission or death. this is a population-based prospective cohort study on archive data. the province of reggio emilia, located in northern italy, has a population of 532,000. hospital, outpatient, primary, and preventive care to all the resident population is provided by the local health authority, the local public organizational entity of the national health service. the first case of sars-cov-2 disease (covid-19) in the province was diagnosed on february 27, 2020. up to april 8, there were 3264 confirmed cases in the province; the epidemic was still spreading, but at a lower rate, and cumulative incidence reached about 6 per 1000. all schools were closed throughout the province on february 22, and some restrictions were placed on social activities. on march 8, strict control measures limiting people's mobility and a partial lockdown was put in place; on march 11, the lockdown was extended, and only essential work activities were allowed. the cohort of covid-19 patients includes all symptomatic patients who tested positive with pcr between february 27 and april 2, 2020. during the evolution of the epidemic, criteria for testing changed; at an earlier stage (until march 3), all suspected cases with flu-like symptoms, fever, cough, dyspnea, and those who had had a contact with a case or had been in one of the red zones (where the initial cluster occurred) were tested. in this phase, according to the above-mentioned criteria, asymptomatic close contacts of a positive case were also tested. in the subsequent phase, all those with symptoms suggestive of covid-19 were tested, regardless of whether not they had had any contact with a positive case, while asymptomatic contacts were no longer tested at all. access to testing for symptomatic patients was possible through emergency room presentation or through prescription by the public health services or general practitioner, usually by phone. swabs were performed either at the individual's home or in dedicated clinics. since the criteria for testing asymptomatic contacts changed over time, they are excluded from the present cohort. in the province of reggio emilia, data on patients found positive to sars-cov-2 are registered in a special database with a dedicated software made available for the management of each individual case in order to allow epidemiological interviews, contact tracing and surveillance of symptoms through daily phone calls. this dataset registers the date of symptom onset and, for patients in home quarantine, the evolution of symptoms over time hospitalization and death. this sars-cov-2 database was linked with the routinely available administrative databases of the local health authority, which include data for each resident in the province, in addition to demographic information, hospital discharge abstract data, coded according to the international classification of diseases-9-cm (icd-9-cm) of diagnosis and procedure, and admission and discharge dates, vital status at discharge, and outpatient pharmacy data at the individual prescription level. data were anonymized, and record linkage procedures were performed according to the unique identification number which is assigned to each resident. analysis of previous hospitalizations (up to preceding 10 years), as registered in the local administrative databases, made it possible to identify each individual patients' comorbidities; data on drugs prescribed were also used to identify patients with diabetes and chronic obstructive pulmonary disease (copd). (s1 file). the outcomes were hospitalization and death. time to event variable started from symptom inception. events occurring until april 3, 2020, were included. we considered the following patient characteristics: age, sex, place of birth (italy or abroad), time span (in days) from symptom onset to diagnosis/ hospitalization, and comorbidities, whose prognostic role was explored both singly (chronic obstructive pulmonary disease, arrhythmia, diabetes, coronary heart disease, heart failure, vascular diseases, obesity) and by computing the charlson comorbidity index, which provides an overall measure of an individual patient's complexity [12] . in particular, we categorized the index in four classes, ranging from 0 (no presence of relevant comorbidity) to � 3 (indicating the highest level of complexity, in terms of number and/or severity of comorbidities). given the current concerns on their possible impact on the clinical evolution of the disease [11] , we also evaluated exposure to ace inhibitors, a class of drugs targeting molecules involved in covid-19 infection process, and their possible substitute therapy, at1-antagonists. case cumulative incidence and case fatality rates (cfr) in the source population of residents in the province were estimated both overall and by sex and by age. descriptive analyses of patients included in the cohort and rates of hospitalization and death according to the presence of each putative prognostic factor are reported. age-and sex-adjusted hazard ratios (hr) with 95% confidence intervals (95% ci) for each putative prognostic factor were estimated for hospitalization and death through multivariate proportional hazard models on time from symptom onset to event. in particular, a first multivariate model was fitted separately for hospitalization and death, including age, sex, charlson index, and place of birth as covariates. then, in order to estimate the actual association between different types of comorbidities with the events of interest, a second model was used that included with the already mentioned covariates the individual comorbidities instead of the charlson index. in all the multivariate models we included time from symptom onset to diagnosis (assumed to be a proxy of severity of the disease, as worse-off patients seek medical assistance quicker) and calendar week of diagnosis, both because a variation in patient characteristics over time was observed ( table 2 ) and because healthcare services experienced different degrees of difficulty in the clinical and organizational management of patients over the weeks due to the different stages of development of the epidemic. lastly, in order to assess the influence of individual comorbidities on the rate of occurrence of the outcomes of interest, multivariate proportional hazard models were used for each comorbidity, which was included as covariate in the model along with age and sex. multivariate analyses exclude all the patients for whom relevant information was not available. however, excluded cases always represented less than 25% of the whole cohort. we do not report formal test of hypothesis and p-values with predefined threshold. statistical analysis was performed with stata 13.0 statistical package. the study was approved by the area vasta emilia nord ethic committee on 07/04/2020 n2 020/0045199. in accordance with the italian privacy law, no patient or parental consent is required for large retrospective population-based studies approved by the competent ethics committee if data are published only in aggregated form. during the study period, 4551 symptomatic individuals were tested for sars-cov-2 infection. the cohort includes 2653 covid-19 patients, representing all the resident symptomatic patients found positive at rt-pcr from february 27 to april 2, 2020 (table 1) . overall positivity to test was 58%; it was lower in younger patients (49% in males and 44% in females) than in older patients (66% in males and 64% in females) ( table 1) . positivity decreased with the progression of the epidemic, from 52.7% (106/201) in the first period to 31.8% (375/1179) in the last ( table 2 ). the mean age was 63.2 and the median time from symptom onset to diagnosis was 4 days, ranging from 0 to 61 days. males and females were equally represented in the cohort. age and sex distribution of cases changed during the epidemic ( table 2 ). age and sex distribution of the covid-19 cohort are in relation to the whole population of residents in the province in order to draw estimates at the population level of disease prevalence and rates of the events of interest. as shown, females were more represented at younger ages (� 50 years) and at very old age (� 80 years), where women are also much more represented in the general population, while males were more represented between ages 60 and 79. age-specific risks of disease were higher in males than in females, except for below age 51. age-specific risks of hospitalization and death were higher in males than in females by a factor of 2 or more. after a median follow up of 14 days, 1075 (40%) and 217 (8.2%) covid-19 cases experienced hospitalization or death, respectively. the rates of both these events were higher in males than in females (50% vs 31% for hospital admission, and 11% vs 6% for death). for patients followed up for at least four weeks, hospital admission reached 61.3% and death 20.8% (table 2) . the prevalence of individual characteristics are outlined in table 3 , along with the crude rate of hospital admissions and death for each patient group. the frequency of both outcome measures was related to sex, age, and overall patient complexity as defined by the charlson index. comorbidities were more common in males (72% charlson index = 0) than in females (76% charlson index = 0). as for single comorbidities (the most prevalent being hypertension, cancer, and diabetes), all were associated with high (i.e. above 50%) rates of hospitalization and death (except obesity, above 15%). results of the multivariate analysis are reported in table 4 and confirm the association between sex, age, and charlson index with both the outcome measures. immigration status (as represented by place of birth) was found to be associated with hospitalization, with patients born abroad having a 40% higher risk. longer time span from symptom onset to diagnosis had a lower risk of hospitalization and death, thus confirming that a shorter length of that interval indicates worse clinical condition. although not statistically significant, hrs for calendar periods of diagnosis suggest a trend towards better outcomes for patients diagnosed in the second part of the study period (i.e. after the third week) compared to those diagnosed in the early phase of the first three weeks of the epidemic. as shown in table 5 , copd, chronic kidney disease, and heart failure had the strongest association with the risk of hospitalization, adjusting for age and sex. as for the use of at-1 inhibitors and ace inhibitors, exposure to these drugs appeared to be associated with a modest increase in hospitalization risk which, for ace inhibitors, was not compatible with a random fluctuation. however, this association disappeared when limiting the analysis to the subgroup of patients with coronary heart disease, hypertension, or heart failure. the highest risk of death was seen in patients with cardiovascular comorbidities (heart failure, arrhythmia, coronary heart disease), followed by dementia and diabetes. use of at-1 inhibitors or ace inhibitors was not associated with the risk of death. below age 50, females had a higher risk of covid-19 than did males, but in all other age groups the risk was higher in males. hospitalization reached 60% and case fatality rate 20% in patients with at least four weeks of follow up. we confirm better prognosis for women, a strong effect of age (stronger in males than in females), and worse prognosis for immigrants and for patients with heart failure, arrhythmia, dementia, coronary heart disease, diabetes or hypertension but not for patients with copd. table 3 . hospitalization and death rates. characteristics of covid-19 cases, hospitalizations, and deaths for each included putative prognostic factor. hospitalized deaths n % of exposure in the population n % (out of those exposed) n % (out of those exposed) the main limitation of this study is that we do not have any information on treatments administered in hospital or prescribed at home. further analyses, requiring ad hoc data collection, must be conducted to study how therapies interacted with the natural history of the disease and with prognostic factors. another limitation of this study is that it is based only on routinely collected hospitalization data to define comorbidities. this source of information clearly underestimates the prevalence of comorbidities that rarely lead to hospitalization, such as obesity, dyslipidaemia, hypertension, or mild copd. collecting a long history of hospitalization (as we did, up to 10 years) and integrating it with the use of drugs specific to some chronic conditions (i.e. diabetes, copd) has been suggested as an effective measure to reduce misclassification and minimize underestimation of the prevalence. on the other hand, using information registered before the onset of the covid-19 epidemic is the only way to obtain unbiased information on a populationbased cohort including non-hospitalized patients. in fact, the probability of registering comorbidities during anamnesis increases with disease severity; this difference in accuracy of exposure ascertainment introduces a bias toward overestimating the impact of comorbidity on prognosis. this bias may be the cause of the high heterogeneity observed in systematic reviews for comorbidities [13, 14] . we adopted the case definition used by who and the italian ministry of health in which only cases positive to rt-pcr sars-cov-2 test are considered covid-19-confirmed cased. unfortunately, referral to sars-cov-2 testing was not standardized and strongly depended on the availability of human and technical resources to collect swabs and perform tests, but also on the awareness of symptoms and on the accessibility of clinics for testing. during the study period, access to testing for paucisymptomatic patients was strongly limited by the lack of human resources to collect swabs at the patient's home, and covid-19-dedicated clinics outside of the emergency rooms were set up only in the last week of the study period. the absence of uniform criteria for test referral and the context-dependent availability of testing limit the comparability of results between different studies [15] [16] [17] . including only hospitalized patients does not increase comparability, since the availability of hospital resource also changed during the epidemic and from country to country. further, it introduces a collider bias, as our results suggest, since some comorbidities influence both the probability of death and of being hospitalized. thus, restricting the population to only hospitalized people may hide the effect of a such comorbidities [18] . while in this study we focused on the risk of hospitalization and death in a cohort of covid-19 patients diagnosed during the epidemic in northern italy, it also provided us with the opportunity to describe the pattern of distribution of the disease in the whole population. we table 5 . hospitalization and risk of death by comorbidities. effect of each comorbidity on hazard of hospitalization and death. all hazard ratios are adjusted for age and sex. models for hospitalizations include 2143 patients and 782 outcomes; models on deaths include 2362 patients and 201 deaths. observed different age-specific risks for females and males resulting in an overall equal proportion of cases. this observation is consistent with previous studies including all symptomatic cases [8, 19, 20] except for a report on the early phases of the epidemic in lombardy [6] . indeed, females had a higher risk among people below age 50, while males had higher risk in older ages. the cause of this difference is unknown, but both biological reasons, including hormonal factors in women in reproductive age, and different access to testing should be investigated. indeed, we observed a higher probability of being tested below age 50 years in women than in men. surprisingly, we noted a different sex ratio among cases in different phases of the epidemic, with a higher proportion of males at the beginning yet the opposite in the later period under study. this phenomenon, which is unexpected and difficult to explain, could also justify the difference between our study and the report from lombardy, which was conducted in a much earlier phase of the epidemic. consistently with previous findings [9, 10, 19, [21] [22] [23] [24] , while the risk of disease is approximately similar, the clinical condition seems to be more severe in males than in females. we confirm the increased risk with age, which remains extremely high even when adjusting for all others characteristic [9, 10, 19, [21] [22] [23] [24] . the effect of age is stronger for hospitalization and particularly for death than it is for infection and for males rather than females (table 1) . hospitalization and case fatality rates were extremely high in this population-based cohort, reaching 60% and 20%, respectively, in those patients with at least four weeks of follow up. even if most studies are reporting a case fatality rate of between 1% and 10% [8, 10, 25] , cohort studies with sufficient follow up showed similar results [26, 27] . the high fatality rate in our study and in similar studies assessing the prognosis of cases diagnosed during the peak of the epidemic was also due to the limited access to the sars-cov-2 test, resulting in the identification of severe cases only. a previously never-reported finding is the higher hospitalization rate of foreign-born residents than of italians. we previously reported a similar prevalence of positivity and similar probability of testing between the two groups [28] . this finding is surprising because immigrants, particularly when their arrival in the host country is relatively recent (as is the case in italy), are usually healthier than native populations and they usually show lower hospitalization and mortality rates [29, 30] . nevertheless, we could adjust for comorbidities, thus reducing the possible confounding due to the healthy migrant effect. given that excess risk is appreciable only for hospitalization and not for mortality, it is possible that this is due to the difficulty in effective home quarantine for these patients. finally, considering that most of the countries of origin have a high prevalence of tuberculosis and bcg is thus recommended, our data do not support the hypothesis that the previously observed non-specific protective effect of bcg on other viral infections [31] is also protective against sars-cov-2 infection. we also found an interesting trend towards a reduced rate of hospitalization and death over the weeks of the epidemic, taking into account patients' age, sex, comorbidities, and length of follow up. while not explained by differences in patient characteristics, the positive trend observed for the two outcome measures considered could, to some extent at least, represent the effect of health professionals and health services rapidly developing the experience required to better cope with the challenges of the clinical and organizational management of a new disease after the first couple of weeks. nevertheless, as mentioned above, over the 5 weeks representing the time span of this study we saw an increase in diagnoses among females in the last two weeks of the period under study that was not compatible with a random fluctuation, while in the first three weeks we observed more males. this suggests that some underlying characteristics of the case mix may change during the epidemic as the result of changes in the epidemiology of the disease or of changes in the resources available for testing people with less severe symptoms. interestingly, in terms of the comorbidities examined, we found an increased risk of hospitalization for copd but a very small effect on death. this is not consistent with what was reported in a previous study with small numbers [26] . we confirm an important role of several comorbidities, particularly for heart diseases. in general, comorbidities had a stronger association with mortality than with hospitalization, with the only exception being chronic kidney disease. the strongest effects were for heart failure, arrhythmia, dementia, coronary heart disease, diabetes, and hypertension, all with � 50% excess hazard. these data are consistent with recent systematic reviews on the role of cardiovascular diseases and diabetes [13, 14] . lastly, we did not find evidence of any effect of the use of at-1 antagonists and ace inhibitors on hospitalization and death, a reassuring finding that will hopefully be confirmed by others. while an association emerged between ace inhibitors and hospitalization, it was likely due to residual confounding as it was not confirmed when the comparison between users vs non-users of this drug was performed only among the subgroup of patients with cardiovascular comorbidity. surprisingly, we found small or no effect for vascular diseases. this is a quite heterogeneous group of diseases and it is possible that we are missing some important prognostic factor due to this grouping, but numbers did not allow for any further distinction. as assessment of obesity and dyslipidaemia through hospital discharge records is challenging, and in our case resulted in an underestimation of the exposure compared to the known prevalence in the general population, the hr that we obtained should be considered carefully [32, 33] . the mechanisms underlying these associations are mostly unknown. a deeper understanding of the causal chain from infection, disease onset, and immune response to outcomes could lead to an explanation of how these prognostic factors act. nevertheless, quantifying the strength of association between pre-existing conditions and covid-19 outcomes is important to understand the disease. covid-19 map-johns hopkins coronavirus resource center an interactive web-based dashboard to track covid-19 in real time monitoring transmissibility and mortality of covid-19 in europe the reproductive number of covid-19 is higher compared to sars coronavirus covid-19 in europe: the italian lesson the early phase of the covid-19 outbreak in lombardy covid-19) pandemic: increased transmission in the eu/eea and the uk-seventh update the novel coronavirus pneumonia emergency response epidemiology team. vital surveillances: the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) -china, 2020. china: 2020 characteristics of covid-19 patients dying in italy report based on available data on clinical characteristics of coronavirus disease 2019 in china are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? a new method of classifying prognostic comorbidity in longitudinal studies: development and validation diabetes and covid-19: a systematic review on the current evidences epidemiological, comorbidity factors with severity and prognosis of covid-19: a systematic review and meta-analysis survival of hospitalized covid-19 patients in northern italy: a population-based cohort study by the ita-covid19 network the many estimates of the covid-19 case fatality rate case fatality rate in patients with covid-19 infection and its relationship with length of follow up sars-cov-2 positive hospitalized cancer patients during the italian outbreak: the cohort study in reggio emilia task force covid-19 del dipartimento malattie infettive e servizio di informatica. epidemia covid-19 aggiornamento nazionale 6 aprile 2020 who. report of the who-china joint mission on coronavirus disease 2019 (covid-19) clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study clinical characteristics of 140 patients infected with sars-cov-2 in wuhan, china baseline characteristics and outcomes of 1591 patients infected with sars-cov-2 admitted to icus of the lombardy region case-fatality rate and characteristics of patients dying in relation to covid-19 in italy epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study coronavirus disease 2019 in elderly patients: characteristics and prognostic factors based on 4-week follow-up clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study prevalence of sars-cov-2 (covid-19) in italians and in immigrants in northern italy differences in mortality by immigrant status in italy. results of the italian network of longitudinal metropolitan studies the health status of the immigrant population in italy: evidence from multipurpose surveys of the italian national institute of differential covid-19-attributable mortality and bcg vaccine use in countries la sorveglianza passi. i dati per l'italia: sovrappeso e obesità la sorveglianza passi. i dati per l'italia: rischio cardiovascolare the authors have declared that no competing interests exist. key: cord-267189-xq70rn1j authors: wang, xinyu; li, chunqiu; guo, donghua; wang, xinyu; wei, shan; geng, yufei; wang, enyu; wang, zhihui; zhao, xiwen; su, mingjun; liu, qiujin; zhang, siyao; feng, li; sun, dongbo title: co-circulation of canine coronavirus i and iia/b with high prevalence and genetic diversity in heilongjiang province, northeast china date: 2016-01-15 journal: plos one doi: 10.1371/journal.pone.0146975 sha: doc_id: 267189 cord_uid: xq70rn1j to trace the evolution of canine coronavirus (ccov), 201 stool samples from diarrheic dogs in northeast china were subjected to reverse transcription-polymerase chain reactions (rt-pcrs) targeting the partial m and s genes of ccov, followed by an epidemiological analysis. m gene rt-pcrs showed that 28.36% (57/201) of the samples were positive for ccov; of the 57 positive samples, ccov-i and ccov-ii accounted for 15.79% (9/57) and 84.21% (48/57), respectively. a sequence comparison of the partial m gene revealed nucleotide homologies of 88.4%–100% among the 57 ccov strains, and 88.7%–96.2% identity between the 57 ccov strains and the chinese reference strain hf3. the ccov-i and ccov-ii strains exhibited genetic diversity when compared with reference strains from china and other countries. the 57 ccov strains exhibited high co-infection rates with canine kobuvirus (cakv) (33.33%) and canine parvovirus-2 (cpv-2) (31.58%). the ccov prevalence in diarrheic dogs differed significantly with immunization status, regions, seasons, and ages. moreover, 28 s genes were amplified from the 57 ccov-positive samples, including 26 ccov-iia strains, one ccov-iib strain, and one ccov-i strain. a sequence comparison of the partial s gene revealed 86.3%–100% nucleotide identity among the 26 ccov-iia strains, and 89.6%–92.2% identity between the 26 ccov-iia strains and the chinese reference strain v1. the 26 ccov-iia strains showed genetic diversity when compared with reference strains from china and other countries. our data provide evidence that ccov-i, ccov-iia, and ccov-iib strains co-circulate in the diarrhoetic dogs in northeast china, high co-infection rates with cakv and cpv-2 were observed, and the ccov-ii strains exhibited high prevalence and genetic diversity. canine coronavirus (ccov) was first recognized as an enteric pathogen of dogs in 1971 [1] . ccov is a common infection in young dogs, particularly those housed in large groups [2] [3] [4] [5] . ccov is an enveloped, single-stranded, positive-sense rna virus, and it belongs to the family coronaviridae, subfamily coronavirinae, genus alphacoronavirus, species alphacoronavirus-1 [6] . ccov consists of two distinct genotypes, ccov-i and ccov-ii; the ccov-ii viruses are further divided into two subtypes ccov-iia and ccov-iib [3, 7] . the s protein of ccov is a glycoprotein peplomer on the viral surface, and it plays an important role in the induction of neutralizing antibodies, specific receptor binding, and cell membrane fusion. accumulating reports attributed that the increase in the severity of ccov infections in dogs and the emergence of ccov variants to potential recombination events within the s gene, which occur when a host is co-infected with different ccov types [8] [9] [10] . therefore, ccov has received much attention as an emerging cause of infectious disease in dogs [4, [11] [12] [13] [14] [15] [16] . ccov infection is a leading causes of diarrhea in dog population in china. wang et al. (2006) reported that ccov-ii infections were very common in domestic dog, fox, and raccoon-dog populations in china [17] . ma et al. (2008) reported the molecular characterization of the 9.36-kb 3 0 region of the ccov 1-71 strain [18] . gao et al. (2009) reported the isolation and identification of a ccov strain from giant pandas in china [19] . however, information about the epidemiology of ccovs in china is not available in the past five years. in the current study, we conducted a molecular epidemiologic investigation of ccov in heilongjiang province, northeast china. moreover, the genetic evolution and co-infection of the identified ccov strains were analyzed. our aim was to provide insights into the epidemiology and genetic diversity of the ccov strains circulating in northeast china. the animal experiment, sampling, was approved by the animal care and use committee of the harbin veterinary research institute, chinese academy of agricultural sciences, china. the sampling and data publication also were approved by animal's owners. the field study did not involve endangered or protected species. no specific permissions were required for locations of samples because the samples were collected from public areas or non-protection areas. a total of 201 fecal samples were collected in the form of rectal swabs of dogs with diarrhea from animal hospitals in the harbin, daqing, and mudanjiang districts of heilongjiang province in northeast china from may 2014 to april 2015, using 3.5-ml commercial virus sampling tubes (yocon biological technology co. ltd., beijing, china). for all samples, animal age, animal breed, animal gender, collection date, and vaccination were recorded, respectively. of the 201 samples, 141 were collected in harbin, 20 were collected in daqing, and 40 were collected in mudanjiang. all rectal swab samples were stored at −80°c, and they were also used for etiological investigations in our other studies [20, 21] . after 1 ml of fecal samples was centrifuged at 1,500 × g for 10 min at 4°c, the supernatant of each sample was transferred to a 1.5-ml eppendorf tube. viral rna was extracted from each sample using the tianamp virus rna kit (tiangen biotech co., ltd., beijing, china) according to the manufacturer's instructions. the extracted rna were stored at -80°c. molecular detection of ccov was conducted using reverse transcription-polymerase chain reaction (rt-pcr) targeting a 409-bp fragment of the m gene of ccov that was described by pratelli et al. (1999) [22] . the s gene fragments used for ccov genotyping/subtyping, including a 346-bp fragment of ccov-i, a 694-bp fragment of ccov-iia, and a 370-bp fragment of ccov-iib, were amplified using the primers el1f/el1r, s5f/s6r, and cepol-1/tgsp-2, respectively [23, 24] . briefly, first-strand cdna was synthesized using random primers (six nucleotides) using moloney murine leukemia virus (rnase h-) reverse transcriptase (novoprotein scientific inc., shanghai, china) according to the manufacturer's instructions. in this study, the emeraldamp pcr master mix (2× premix) (takara biotechnology co., ltd., dalian, china), and the applied biosystems geneamp pcr system 9700 thermal cycler (thermo fisher scientific, waltham, ma, usa) were used for pcr amplifications of all target genes. other pcr amplification conditions were performed according to the protocols described by [23] and erles and brownlie (2009) [24] . the purified pcr products were directly subjected to sanger sequencing, and each sample was sequenced three times. sequence analysis was performed using the editseq program in the lasergene dnas-tar™ version 5.06 software (dnastar inc., madison, wi, usa). all nucleotide sequences generated in our study were submitted to genbank under accession numbers kt192642-kt192698 for the m gene of the 57 ccov strains, kt222969-kt222994 for the s gene of the 26 ccov-iia strains, kt222995 for the s gene of the ccov-i strain, and kt222996 for the s gene of the ccov-iib strain. for the phylogenetic analysis, partial sequences of the m and s genes of ccov strains, including ccov-i, ccov-iia, and ccov-iib strains, were retrieved from genbank. to construct the phylogenetic trees, a multiple alignment of all target sequences was performed using clustalx program version 1.83. furthermore, phylogenetic trees of all target sequences were generated from the clustalx-generated alignments by mega6.06 software using the neighbor-joining method [25] . neighbor-joining phylogenetic trees were built with the p-distance model, 1000 bootstrap replicates, and, otherwise, the default parameters in mega 6. the 57 ccov-positive samples were screened for cpv-2, cakv, canine astrovirus (caastv), canine norovirus (cnov), canine bocavirus (cbov), and group a-rotavirus (crv-a) by either pcr or rt-pcr, followed by sequencing, according to previously described protocols [26] [27] [28] [29] [30] . all nucleotide sequences generated in our study were submitted to genbank, and the accession numbers of the target genes of co-infected viruses are shown in s1 table. results the characteristics of the 57 ccov-positive dogs, the genotyping of 57 ccov strains, and the amino acid substitutions of m protein of 57 ccov strains are shown in s1 table, and a further analysis of the 57 ccov-positive samples is shown in table 1 . nucleotide sequences of the partial m gene of the 57 ccov strains identified in our study were shown in supporting information (s1 fig). of the 201 samples, 57 samples (28.36%) were positive for ccov following rt-pcr amplification of the partial m gene, and the ccov-positive rates of the harbin, daqing, and mudanjiang districts were 26.95%, 50%, and 22.5%, respectively (table 1 ). of the 57 ccov-positive samples, ccov-i and ccov-ii accounted for 15.79% (9/57) and 84.21% (48/ 57), respectively; immunized animals and non-immunized animals accounted for 43.86% and 36.84%, respectively; 56.14% (32/57) were collected from october to december, and 66.66% (38/57) were collected from dogs aged from 2 to 4 months; the total co-infection rate of ccov with any viruses reached 56.14%, and cakv and cpv-2 accounted for 33.33% (19/57) and 31.58% (18/57), respectively (table 1) . of the 57 ccov strains, only 28 s gene sequences were successfully amplified, of which 26 belonged to ccov-iia, one to ccov-iib, and one to ccov-i (s1 table) . nucleotide sequences of the partial s gene of ccov strains identified in our study were shown in supporting information (s2 fig). the sequence comparison of the partial m gene revealed nucleotide homologies of 88.4%-100% and amino acids homologies of 93%-100% among the 57 ccov strains, while nucleotide and amino acid homologies of 88.7%-96.2% and 92.2%-97.4%, respectively, were observed between the 57 ccov strains and the chinese reference strain hf3. of the 57 ccov strains, the nine ccov-i strains exhibited 94.8%-100% nucleotide identities and 94.8%-100% amino acid identities, and the 48 ccov-ii strains showed 96.8%-100% nucleotide identities and 98.3%-100% amino acid identities (table 1) . a total of 12 amino acid substitutions were found in the partial m protein (s1 table) ; the four substitutions, thr178val, met198ile, his206asn, and gln228lys, occurred in all identified ccov-ii strains (48/48), and two substitutions, ile198met and asn206his, occurred in all identified ccov-i strains (9/9) ( table 1 ). the sequence comparison of the partial s gene revealed nucleotide homologies of 86.3%-100% and amino acids homologies of 87.6%-100% among the the 26 ccov-iia strains, and nucleotide and amino acid homologies of 89.6%-92.2% and 91.1%-97.5%, respectively, between the 26 ccov-iia strains and the chinese reference strain v1 (table 1) . a phylogenetic analysis using the nucleotide sequences of the m gene revealed that the nine ccov-i strains formed two clusters (c1 and c2), while the 48 ccov-ii strains formed six clusters (c1-c6). the 48 ccov-ii strains only exhibited a close relationship to one chinese reference strain, nj17, and differed genetically from most of the reference strains from china and other countries (fig 1) . a phylogenetic analysis using partial s gene sequences demonstrated that the 26 ccov-iia strains were closely related to three reference strains, 5281 (japan), tn-449 (usa), and 1086-iia (brazil), and differed genetically from reference strains from china and other countries (fig 2a) . the ccov-i strain hrb-a4 showed a close relationship to the reference strain from italy (fig 2b) , while the ccov-iib strain hrb-bb9 was closely related to reference strains from european countries (fig 2c) . in our study, 57 of 201 samples (28.36%) were positive for ccov, and the ccov-positive rate differed among the three districts of heilongjiang province (26.95% for harbin, 50% for daqing, and 22.5% for mudanjiang). the ccov-positive rate in feces has been reported to be 43.75% [17] , the republic of korea [31] , japan [32] , albania [33] , brazil [11] , italy, belgium, the netherlands, germany, the united kingdom, spain, and france, respectively [34] . these data demonstrated that ccov infections showed clear differences among the geographical regions. the total ccov-positive rate reported here was lower than that in a previous report in 2006 in china [17] . in our study, ccov-ii, accounting for 84.21% of the 57 ccov strains, was the predominant ccov type in northeast china, which is line with most reports [11, 17, 34] . mixed infections of canine enteric viruses frequently occur in diarrheic dogs. in our study, the total co-infection rate of ccov with one or more cakv, cpv-2, and cbov strains was 56.14%; single co-infections with cakv, cpv-2, and cbov accounted for 33.33%, 31.58%, and 5.26%, respectively. co-infections between ccov and cpv-2 have been documented in dog populations in western europe, japan, and albania [32] [33] [34] . however, the high co-infection rate of ccov with cakv has not been reported in china and other countries. cakv had been reported to be associated with severe enteritis in a litter of puppies [13] . it is speculated that the high prevalence of co-infections of ccov with cakv and cpv-2 may be associated with the occurrence of viral diarrhea in dogs in northeast china. further studies should be conducted to understand the effect of the high co-infection rate with cakv on the severity of clinical symptoms of ccov infections. in our study, ccov-positive rate showed clear differences among seasons and ages. the high prevalence of ccov in the feces of diarrheic dogs aged 2-4 months was partially validated in other studies [11, 32, 33] . the high positive rate (56.14%) of ccov was found between october and december, which may be associated with seasonal variations in northeast china. at present, most dogs in northeast china are vaccinated for cpv-2, canine distemper virus, canine parainfluenza virus, canine adenovirus type 1, and canine adenovirus type 2. in our study, of the 57 ccov-positive samples, the immunized and non-immunized animals accounted for 43.86% and 36.84%, respectively. this result demonstrated that vaccination for other canine viruses did not effect ccov infections in the dog population in heilongjiang province, northeast china. given the high prevalence and co-infection rates of ccov, although it is controversial whether ccov vaccines provide adequate immunity [35, 36] , the immunization for ccov is recommended in the dog population in northeast china in the future. fragments of the m and s genes have been used to genotype ccov [4, 11, 24, 37] . in our study, the ccov-i and ccov-ii genotypes of the 57 ccov strains were successfully identified demonstrated that ccov-i, ccov-iia, and ccov-iib co-circulated in northeast china, and that ccov-iia strains predominated in the dog population. the current study is the first to reveal that ccov-i, ccov-iia, and ccov-iib strains co-circulate in northeast china, and that there are high co-infection rate with cakv and cpv-2. cco-v-ii strains are predominant, and they exhibit genetic diversity. resulting data increase our understanding of the evolution of ccov strains circulating in northeast china, and they provide valuable epidemiological information for further studies of ccov. however, further studies are necessary to clarify the effect of co-infections and amino acid substitutions on the severity of the clinical symptoms of ccov infections. supporting information recovery and characterization of a coronavirus from 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surveillance for pantropic canine coronavirus canine kobuviruses in diarrhoeic dogs in italy characterization of pantropic canine coronavirus from brazil emergence of pathogenic coronaviruses in cats by homologous recombination between feline and canine coronaviruses full-length genome analysis of canine coronavirus type i detection of canine coronaviruses genotype i and ii in raised canidae animals in china molecular characterization of the 9.36 kb c-terminal region of canine coronavirus 1-71 strain isolation and identification of a canine coronavirus strain from giant pandas (ailuropoda melanoleuca) prevalence and phylogenetic analysis of canine kobuviruses in diarrhoetic dogs in northeast china co-circulation of the rare cpv-2c with unique gln370arg substitution, new cpv-2b with unique thr440ala substitution, and new cpv-2a with high prevalence and variation in heilongjiang province, northeast china development of a nested pcr assay for the detection of canine coronavirus two genotypes of canine coronavirus simultaneously detected in fecal samples of dogs with diarrhea sequence analysis of divergent canine coronavirus strains present in a uk dog population mega6: molecular evolutionary genetics analysis version 6.0 antigenic analysis of canine parvovirus strains isolated in italy rotavirus a genotype p[4]g2: genetic diversity and reassortment events among strains circulating in brazil between 2005 and identification and characterization of bocaviruses in cats and dogs reveals a novel feline bocavirus and a novel genetic group of canine bocavirus molecular epidemiology of canine norovirus in dogs from portugal phylogenetic analysis of astrovirus and kobuvirus in korean dogs m gene analysis of canine coronavirus strains detected in korea detection and genotyping of canine coronavirus rna in diarrheic dogs in japan detection and genetic characterization of canine parvovirus and canine coronavirus strains circulating in district of tirana in albania western european epidemiological survey for parvovirus and coronavirus infections in dogs efficacy of an inactivated canine coronavirus vaccine in pups safety and efficacy of a modified-live canine coronavirus vaccine in dogs identification of coronaviruses in dogs that segregate separately from the canine coronavirus genotype genotype-specific fluorogenic rt-pcr assays for the detection and quantitation of canine coronavirus type i and type ii rna in faecal samples of dogs canine coronavirus highly pathogenic for dogs canine coronavirus: not only an enteric pathogen isolation, tissue distribution and molecular characterization of two recombinant canine coronavirus strains key: cord-290539-8ak2tths authors: cagno, valeria; tintori, cristina; civra, andrea; cavalli, roberta; tiberi, marika; botta, lorenzo; brai, annalaura; poli, giulio; tapparel, caroline; lembo, david; botta, maurizio title: novel broad spectrum virucidal molecules against enveloped viruses date: 2018-12-07 journal: plos one doi: 10.1371/journal.pone.0208333 sha: doc_id: 290539 cord_uid: 8ak2tths viral infections are an important cause of death worldwide. unfortunately, there is still a lack of antiviral drugs or vaccines for a large number of viruses, and this represents a remarkable challenge particularly for emerging and re-emerging viruses. for this reason, the identification of broad spectrum antiviral compounds provides a valuable opportunity for developing efficient antiviral therapies. here we report on a class of rhodanine and thiobarbituric derivatives displaying a broad spectrum antiviral activity against seven different enveloped viruses including an hsv-2 acyclovir resistant strain with favorable selectivity indexes. due to their selective action on enveloped viruses and to their lipid oxidation ability, we hypothesize a mechanism on the viral envelope that affects the fluidity of the lipid bilayer, thus compromising the efficiency of virus-cell fusion and preventing viral entry. viral infections are one of the ten leading causes of death worldwide [1] . nowadays, although effective antiviral strategies have been successfully developed for some important pathogens such as hiv and hcv, antiviral drugs or vaccines are still missing for the majority of viruses. as an example, no effective antiviral strategies are yet available for viruses causing chronic infections such as hbv [2] , as well as for tropical viruses like dengue virus that is causing 390 million infections per year [3] . a particular interest is addressed to the recent epidemics of ebola in africa and zika (zikv) in south america, for which, despite the huge effort to find antivirals, the research community was not able to execute in time an efficient antiviral plan. the majority of emerging or re-emerging viruses are zoonoses, and it has been demonstrated that the passage among different species is easier for enveloped viruses in comparison a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 with non-enveloped viruses [4, 5] . it is estimated that there are approximately 500,000 unknown mammalian viruses in animal reservoirs [6] . in this scenario, the possibility to find antiviral molecules directed against viral envelopes is of particular interest in order to identify broad spectrum antiviral drugs [7] . in our previous papers we reported some rhodanine and aminothiazolone derivatives endowed with nanomolar activities against hiv-1 infected cells [8] [9] [10] . in a recent work, we showed that our compounds were also active against hsv-1/2, while they were completely ineffective against hpv, a nonenveloped virus [11] . these results suggested that the mode of action of our molecules could involve the viral envelope. compounds acting on viral envelopes have been previously described: rafi compounds inhibit a broad range of enveloped viruses by inserting into viral envelopes and altering the fusion kinetics in the hemifusion stalk due to their inverted cone structure [12] . liposomes extracting cholesterol from viral envelopes have been reported to inhibit hiv, hcv and hbv [13] . virolytic antiviral peptides derived from mastoparan were shown to inhibit different enveloped viruses acting on the envelope and causing its detachment from the viral core [14] . the selectivity of these strategies is based on the fact that viral envelopes are static and characterized by an absence of repair mechanisms, in contrast with the biogenic membranes of the cells that are endowed with plenty of tools to repair membrane damage or alteration [15] . furthermore, since envelope lipids are derived from host cells, it is more complicated to the virus to develop resistance to these compounds if compared to those targeting the classical viral components; for this reason they are extremely promising targets also for viruses with a high mutation rate. therefore, we investigated the activity on hsv-2 of a new series of compounds. after having selected the most potent derivative of the series, we verified its broad spectrum activity against an hsv-2 acyclovir-resistant strain and other six important enveloped pathogens such as zikv, influenza a virus (iav) and respiratory syncytial virus (rsv), amongst the others, and we confirmed its complete inactivity against three non-enveloped viruses. finally, we investigated its mechanism of action, identifying a lipid oxidizing activity and the impairment of viral entry of hsv-2. the compounds were synthesized using the one pot two step approach previously generated by us for the synthesis of rhodanine derivatives. according to fig 1, aldehydes 4-6 were obtained through suzuki reaction between commercially available iodides 1 or 2 and 5-formyl-2-furanylboronic acid 3. basic hydrolysis of ester 5 provided the corresponding acid 6. nucleophilic substitution between trithiocarbonate and the opportune primary amine led to the rhodanine intermediates 8a-e, which were converted into the final compounds 9a-e by knoevenagel condensation with aldehyde 4 or 6. thiobarbituric derivatives were synthesized as shown in fig 2. reaction between benzoyl chloride 10, ammonium thiocyanate and the opportune amine led to intermediates 11a and 11b; consequent hydrazinolisis and coupling reaction with diethylmalonate 13 in basic conditions furnished monoalkylated thiobarbituric derivatives 14a and 14b. finally knoevenagel condensation with aldehyde 6 in refluxing acidic etoh led to compounds 15a and 15b. having proved that rhodanine derivatives belonging to this series of compounds are potential microbicide active against hiv-1 and hsv-1/2 [11] , we further investigated the inhibitory activity against hsv-2 of the novel derivatives bearing both rhodanine and thiobarbituric scaffolds. the compounds were tested with a plaquing efficiency assay, preincubating with the virus for 1 h at 37˚c and subsequently adding the mixtures on cells. all the compounds exhibited potent inhibitory activities at non-cytotoxic doses, as demonstrated by the high selectivity indexes (fig 3) . compound 9d, which showed the most promising antiviral activity, was subjected to further investigations. the toxicity of compound 9d was evaluated at different time points on vero cells, and also after 96h exposure the cc 50 was evaluated to be 2.09 μm confirming the favorable selectivity index (s1 fig) . due to the lipophilic nature of the compound and the results obtained in previous works, in which derivatives of the same series proved to be effective against hiv and hsv [11] , we investigated the spectrum of antiviral activity of 9d against different enveloped viruses (i.e. hsv-1, hcmv, rsv, zikv, iav, vsv) and non-enveloped viruses (i.e. ad5, hpv and hrov). as reported in table 1 , the compound showed a potent antiviral activity against all enveloped viruses tested at not cytotoxic doses, while it was found to be inactive against nonenveloped viruses, suggesting a possible action on the viral envelope. interestingly, compound 9d retained its antiviral activity also against hsv-2 acyclovir resistant strain, suggesting a different mechanism of action. we performed assays aimed at better understanding the mechanism of action. in the pretreatment assay compound 9d was added on cells before the virus in order to determine if the antiviral activity was due to an interaction with the cells. moreover, an assay was conducted adding virus and compound on cells without the preincubation performed in previous assays. the results showed in fig 4 demonstrate that the antiviral activity of 9d is not due to an interaction with cells, since the compound was not active in pre-treatment assays. on the contrary, we observed a significant reduction of activity in the during-infection assay (ec 50 = 0.147 μm), if compared to the pre-incubation assay (ec 50 = 6.55 nm) suggesting that the loss of activity could be related to a competition between the viral envelope and the cell membrane for the insertion and activity of the compound. we then investigated whether compound 9d was able to inhibit multiple cycles of infection, in viral yield reduction assays, when added post infection. as shown in fig 5, in this condition the compound retained a good antiviral activity, suggesting possible therapeutic uses. to further elucidate the mechanism of action we performed a virucidal assay in which 9d was incubated with the virus at 10 μm 5μm or 1μm concentration for different times (fig 6a) or for 1h with serial dilutions of compound ( fig 6b) ; subsequently, the mixture was titrated on cells and the viral titer was evaluated at dilutions at which the compound concentration was known not to be active in plaquing efficiency assays. in these conditions it was possible to observe that the viral infectivity in presence of the compound was decreased at 15' post treatment and completely abrogated from 30' on when the virus was exposed to 10 μm of compound, and at lower doses ( fig 6a) . while after 1h of exposure, the ec50 was 37.3 nm ( fig 6b) . the irreversibility of the mechanism was also tested with an assay in which the compound was incubated with the virus for 1h and subsequently the mixture has been diluted in drug free medium for additional 1, 2, 3 or 4 hours before the addition on cells (s2 fig) . also in this condition the infectivity was not regained further verifying the permanent virucidal activity. since the observed irreversible effect can be exerted at different stages of viral infection, we investigated more in detail if compound 9d produced a physical disruption of the virus, thus preventing its attachment to the cells or a successive irreversible modification. we performed entry and binding assays, evaluating the amount of bound virus through immunostaining or qpcr. the results shown in fig 7a, 7b and 7c, demonstrate that the compound is not altering viral binding capability, differently from heparin, a known attachment inhibitor [16] . on the contrary, when entry assays were performed, it was possible to observe a reduction in the amount of virus associated to the cells ( fig 7d) . with this assay, the signal from the virus could be related to virus in the cytoplasm or blocked in the endosomes, since the treatment with acidic glycine is affecting only the virus at the surface of the cell. for this reason we performed also an in immunofluorescence (if) in which it was possible to visualize that there is virus signal in discrete dots, possibly endosomes, while in the control the viral signal is diffused in the cytoplasm (fig 7e) , supporting the hypothesis that part of the virus could be blocked in endosomes due to an impaired fusion. due to the chemical structure of compound 9d and its affinity for lipids, we envisioned that its mechanism of action might involve the peroxidation of viral phospholipids, which would lead to altered fusion properties of enveloped viruses. to assess this hypothesis, we carried out an in vitro lipoperoxidation assay based on the oxidation of linoleic acid as model lipid substrate, incorporated in a dppc liposomes. the lipid peroxidation was evaluated by monitoring the production of the lipid degradation end-product malondialdehyde (mda). after the exposure to compound 9d, the linoleic acid underwent a remarkable lipid peroxidation in the liposome system (fig 8) . the results of the tba assay suggest the peroxidant activity of the compound. the peroxidation capability of 9d was further confirmed by the reduction of linoleic acid peroxidation extent in a control sample in which in the preparation of liposomes, an antioxidant such as (±)-α-tocopherol, was added. interestingly the addition of 0.05% w/w (±)-α in this work we demonstrated that the series of compounds herein reported, and in particular compound 9d, are endowed with a broad-spectrum activity against a panel of seven different enveloped viruses including a hsv-2 acyclovir resistant strain, due to an irreversible mechanism that impairs viral entry into the host cell. the selective activity on enveloped viruses and the lipid peroxidation capability of the compound suggest a mechanism of action on the viral envelope that is affecting viral and cell membrane fusion. these results open interesting possible applications for these molecules as antivirals or as components of microbicidal preparations for topical use. this is of particular interest if we consider that all the recent epidemics of emerging and re-emerging viruses are caused by enveloped viruses such as ebola virus, lassa virus, zika virus, sars and mers coronavirus and avian flu [3] , without considering the threatening infections of hendra and nipah viruses and the emergence of novel mutant strains. finally, the activity of the reported compounds against hiv [6] , hsv-1, hsv-2 and zikv, which can be all transmitted through sexual activity, suggest a possible use of this class of compounds as vaginal microbicide components. general information. all commercially available chemicals were used as purchased. anhydrous reactions were run under a positive pressure of dry n 2 . thin-layer chromatography (tlc) was carried out using merck tlc plates: silica gel 60 f254. chromatographic purifications were performed on columns packed with merck 60 silica gel, 23-400 mesh, for the flash technique. 1 h and 13 c nmr spectra were recorded at 400 mhz on a bruker avance dpx400 spectrometer. melting points were measured using a gallenkamp melting point apparatus and are uncorrected. microwave irradiation experiments were conducted using a cem discover synthesis unit (cem corp., matthews, nc, usa). the instrument consists of a continuous focused microwave power delivery system with operator-selectable power output from 0 to 300 w. the temperature of the contents of the vessel was monitored with a calibrated ir temperature control mounted under the reaction vessel. all experiments were performed using a stirring option, whereby the contents of the vessel were stirred by a rotating magnetic plate located below the floor of the microwave cavity and a teflon-coated magnetic stir bar in the vessel. general procedure for the synthesis of aldehydes 4 and 5. methyl-4-iodosalycilate 1 or 4-chloro-3-iodobenzotrifluoride 2 (1.00 mmol) and 5-formyl-2-furan boronic acid 3 were dissolved in 10 ml of dmf and 15 ml of etoh. the reaction mixture was stirred for 10 min under n 2 , then pd(pph 3 ) 2 cl 2 (0.10 mmol) was added and finally na 2 co 3 2m (6.00 mmol). the reaction mixture (light-orange) was stirred under n 2 at room temperature. after 1h the reaction went to completion and was quenched with h 2 o and 2n hcl; then etoac was added and the mixture was stirred until the two layers became clear. the aqueous layer was extracted three times with etoac, then the organic phase was washed several times with h 2 o and brine, dried over na 2 so 4 , filtered and evaporated under reduced pressure. 4-(5-formylfuran-2-yl)-2-hydroxybenzoic acid (6). compound 5 was dissolved in 25 ml of ch 3 oh, then a solution of naoh 1m (5.00 mmol) was added dropwise, after the reaction mixture was heated at reflux. the reaction mixture was stirred overnight until completion (tlc). organic solvent was removed under reduced pressure, then some water was added and the aqueous layer was extracted three times with et 2 o; the aqueous layer was then acidified to ph 1 with hcl 6n and a precipitated appeared. (6) 13 ( 13 ( general procedure for the synthesis of compounds 11a and 11b. benzoylchloride 10 (4.31 mmol, 1.00 eq) was dissolved in 3 ml of acetone. to this, nh 4 scn (5,17 mmol, 1.2 eq) was added in one portion and the mixture irradiated at 60˚c for 15 min at the microwave. after this time, the opportune amine (1.00 eq) respectively benzylamine for 8a, and phenethylamine for 8b, was added, and the mixture was irradiated for further 15 min at the microwave. the resulting suspension was filtered, and the precipitate washed with h 2 o and ch 3 oh, to furnish pure 8a and 8b as white solids. general procedure for the synthesis of compounds 12a and 12b. the opportune thioureido derivative (11a or 11b) (1.61 mmol) was dissolved in 10 ml of hydrazine monohydrate. the solution was stirred at rt for 3 h. then 5 ml of fuming hydrochloric acid were added and the mixture was extracted with ch 2 cl 2 (3 x 15 ml). the organic phase was washed with na 2 co 3 (aq. sol.) (2 x 50 ml), and dried over na 2 so 4 . the solvent was removed at reduced pressure and the corresponding residue purified by flash chromatography (acoet/ch 2 cl 2 1:1). general procedure for the synthesis of compounds 14a and 14b. sodium metal (3,61 mmol, 3 eq) was dissolved in anhydrous etoh (10 ml), then the opportune thiourea compound (12a or 12b) (1.20 mmol) and diethylmalonate 13 (2.41 mmol) were added subsequently, and the mixture was refluxed under argon atmosphere. the solvent was removed at reduced pressure and the corresponding residue was solubilized in water. the mixture was acidified at ph 2 with hcl 1n and filtered. the residue was then purified by flash chromatography (ch 2 cl 2 /ch 3 oh 8:2). 1-benzyl-2-thioxodihydropyrimidine-4,6(1h,5h)-dione (14a). yield 80% red solid. general procedure for the synthesis of compounds 15a and 15b. compound 6 (0,22 mmol, 1 eq) and compound 13a or 13b (0.22 mmol, 1 eq) were suspended in etoh (6 ml); to this, 3 drops of hcl (conc.) were added and the mixture was stirred at 70˚c overnight. after this time 3 ml of hcl were added and the resulting precipitate was filtered at reduced pressure to give a solid that was washed with h 2 o, ch 3 oh and hexane. (z)-4(5-((1-benzyl-4,6179.42, 171.68, 162.05, 161.72, 160.30, 159.71, 158.73, 151.34, 138.28, 136.86, 134.45, 131.61, 130.83, 128.57, 127.31, 116.43, 114.46, 113.68, 113.38, 113. clinical isolates of hsv-1 and hsv-2 were kindly provided by prof. m. pistello, university of pisa, italy. hsv-1 and hsv-2 strains were propagated and titrated by plaque assay on vero cells. a hsv-2 strain with phenotypic resistance to acyclovir was generated by serial passage in the presence of increasing concentrations of acyclovir and tested for acyclovir resistance with dose-response inhibition assay with an ec50 of 319 μm, as previously described [17] . hcmv strain towne was kindly provided by prof. w. brune, heinrich pette institut, hamburg, germany; it was propagated and titrated by plaque assay on helf cells. rsv strain a2 (atcc vr-1540) was propagated in hep-2 and titrated by the indirect immunoperoxidase staining procedure using an rsv monoclonal antibody (ab35958; abcam, cambridge, united kingdom), as described previously [15] . human rotavirus strain wa (atcc vr-2018) was activated with 5 mg/ml porcine pancreatic trypsin type ix (sigma, st. louis, mo.) for 30 min at 37˚c and propagated in ma104 cells using mem containing 0.5 mg trypsin per ml, as described previously [18] . vsv (atcc vr-1238) was propagated on vero cells and titrated by plaque assay. zika virus (prvabc59) was kindly provided by marco alves and was propagated and titrated by plaque assay on vero cells. iav h1n1 isolated from a clinical specimen was propagated and titrated on mdck. adenovirus 5 encoding gfp (gfp-ad5), with a e1/e3 deletion, was purchased from vector biolabs (philadelphia, pa, usa). hpv-16 pseudovirions were produced with 293tt as previously described [19] and their concentration was assessed on hela cells. virus stocks were maintained at -80˚c. cell viability was measured using the mts [3-(4,5-dimethylthiazol-2-yl)-5-(3 carboxy methoxy phenyl)-2-(4-sulfophenyl)-2h-tetrazolium] assay. cell cultures were seeded in 96-well plates and were incubated with different concentrations of compounds in duplicate under the same experimental conditions described for the antiviral assays, (i.e. if the incubation time of the compound on cells was carried for 2 hours and then the evaluation of the antiviral activity was done 72h later, the same timing was used for the cytotoxicity assays, in order to exclude any toxicity effect in the antiviral evaluation). cell viability was determined using the celltiter 96 proliferation assay kit (promega, madison, wi, usa) according to the manufacturer's instructions. absorbances were measured using a microplate reader (model 680, biorad) at 490 nm. the effect on cell viability at different concentrations of the compound was expressed as a percentage, by comparing absorbances of treated cells with those of cells incubated with culture medium and equal volumes of vehicle. the 50% cytotoxic concentrations (cc 50 ) and 95% confidence intervals (cis) were determined using prism software (graph-pad software, san diego, ca). for compound 9d a continuous incubation of 1, 2, 3 and 4 days on vero cells was carried in order to evaluate the toxicity after long exposure the results are shown in s1 fig and fig 3. the antiviral effect on hsv and vsv and zikv infection was evaluated by plaquing efficiency assay. vero cells were pre-plated 24 h in advance in 24-well plates at a density of 10 5 cells. increasing concentrations of compounds were mixed with hsv-2 (moi 0.001 pfu/cell) or hsv-2 acyclovir resistant (moi 0.001) or hsv-1 (moi 0.0005) or vsv (moi 0.005) or zikv (moi 0.005) and incubated for 1 hour at 37˚c. the mixtures were subsequently added to the cells, which were then incubated at 37˚c for 2 h. the virus inoculum was then removed and the cells washed and overlaid with a medium containing 1.2% methylcellulose (sigma). after further incubation at 37˚c for 24 h (hsv-2 and vsv) or 48 h (hsv-1) or 72h (zikv), cells were fixed and stained with 0.1% crystal violet in 20% ethanol and viral plaques counted. the effective concentration producing 50% reduction in plaque formation (ec 50 ) was determined using prism software by comparing drug-treated with wells treated with medium and solvent. the selectivity index (si) was calculated by dividing the cc 50 by the ec 50 value. hcmv inhibition assays. helf cells were pre-plated in a 96-well plate. the following day increasing concentrations of compounds were mixed with hcmv (moi 0.005) and incubated for 1 h at 37˚c. the mixtures were subsequently added to the cells, which were then incubated at 37˚c for 3 h; monolayers were then washed and overlaid with 1.2% methylcellulose medium supplemented with 3% fcs and 1mm sodium pyruvate. after five days incubation, cells were observed under an inverted zeiss lsm510 fluorescence microscope (zeiss, oberkochen, germany) and the percentages of infection were calculated by comparing gfp positive cells in treated and untreated wells. hep-2 cells were pre-plated in a 96-well plate. the following day increasing concentrations of compounds were mixed with rsv (moi 0.005) and incubated for 1 h at 37˚c. the mixtures were subsequently added to the cells, which were then incubated at 37˚c for 3 h; monolayers were then washed and overlaid with 1.2% methylcellulose medium. 72 h later cells were fixed and subjected to specific immunostaining using an rsv monoclonal antibody (ab35958; abcam, cambridge, united kingdom) in order to visualize syncytia. percentages of infection were calculated by comparing numbers of syncytia in treated and untreated wells. hrov inhibition assays. ma104 cell were plated in 96-well trays. the following day virus infectivity was activated by adding 5 μg porcine trypsin (sigma)/ml for 30 min at 37˚c. increasing concentrations of compounds were mixed with hrov (moi 0.02) and incubated for 1 h at 37˚c. the mixtures were subsequently added to the cells, which were then incubated at 37˚c for 1 h; monolayers were then washed and overlaid with medium. after 16 h, cells were fixed with cold acetone-methanol and viral titers were determined by indirect immunostaining using the monoclonal antibody mab-0036 (specific for human 41 kda inner capsid protein-vp6 -of rotavirus) purchased from covalab (villeurbanne, france) and the ultratech hrp streptavidin-biotin detection system (beckman colter). hpv-16 and ad5 inhibition assays. hela cells were plated in 96-well plates. the following day, increasing concentrations of compounds were mixed with hpv-16 (approximately 1 ng/ml l1) or ad5 (moi 0.02) and incubated for 1 h at 37˚c. the mixtures were subsequently added to the cells, which were then incubated at 37˚c for 72 h. the gfp-expressing infected cells were observed under an inverted zeiss lsm510 fluorescence microscope (zeiss, oberkochen, germany) and the percentages of infection were calculated by comparing gfp positive cells in treated and untreated wells. iav h1n1 inhibition assays. mdck cells were pre-plated in 96-well plates. the following day, increasing concentrations of compounds were mixed with iav (moi 0.05) and incubated for 1 h at 37˚c. the mixtures were subsequently added to the cells for 1 h at 37˚c, after a washout cells were overlaid with medium for 16 h at 37˚c. cells were then fixed and subjected to specific immunostaining using a flu a monoclonal antibody (merck 5001) in order to visualize infected cells. percentages of infection were calculated by comparing numbers of infected cells in treated and untreated wells. vero cells were subjected to different assays: pretreatment: cells were pretreated for 2 h at 37˚c with increasing concentrations of compounds, the inocula were then removed, cells washed and hsv-2 was added on cells for 2 h at 37˚c. subsequently the same protocol described above was followed. during infection: cells were subjected to the same experiment described above without the pre-incubation between compounds and virus. post treatment: cells were infected with hsv-2 (moi 0.01) for 2 h at 37˚c, the viral inoculum was removed and cultures were exposed to different compound concentrations and incubated until control cultures displayed extensive cytopathology. supernatants and cells were harvested and cell-free virus infectivity titers were determined in duplicate by plaque assay in vero cell monolayers. percent inhibition was determined by comparing the titer measured in the presence of the compounds to that measured in untreated wells. binding: cells were plated in 96 well plates. the following day 10 μm of compound and hsv-2 (moi 10) were incubated for 1 h at 37˚c and subsequently added on cells for 2 h at 4˚c. cells were then fixed with 4% paraformaldehyde, air dried, and blocked with 5% bovine serum albumin (bsa) in phosphate-buffered saline (pbs)-tween. bound virus was detected using the polyclonal hsv-2 antibody (dako, denmark) (diluted 1:250) incubated for 1 h at room temperature, washed three times with pbs-tween, and incubated for 1 h at 37˚c with anti-rabbit conjugated to horseradish peroxidase (1:500). at the end of incubation, plates were washed three times with pbs-tween, abts [2,2-azinobis(3-ethyl benz thiazoline sulfonic acid)] substrate was added for 30 min (thermo scientific, rockford, il) and the absorbance at 405 nm was read. entry: cells were plated in 96 well plates. the following day 10 μm of compound and hsv-2 (moi 10) were incubated for 1 h at 37˚c and subsequently added on cells for 1 h at 4˚c. cells were then washed and shifted at 37˚c for 1.5 h or 4 h, after they were subjected to a wash with acidic glycine to inactivate unentered virus. cells were then fixed with 4% paraformaldehyde, air dried and permeabilized with pbs-triton (0.5%) and then the same protocol described for binding was followed. virucidal assay: approximately 10 5 pfu of hsv2 plus 10 μm or different concentration of compound were added to mem and mixed in a total volume of 100 μl. the virus-compound mixtures were incubated for different times at 37˚c then diluted serially to the non-inhibitory concentration of test compound on cells or in tubes for additional 1, 2, 3 or 4 hours; the residual viral infectivity was determined by viral plaque assay. cells were plated on coverslips and the following day the binding or entry protocol described above was applied. cells were than fixed with 4% paraformaldehyde, air dried and for entry protocol permeabilized with pbs-triton. blocking was performed with pbs-bsa 1% for 1 h at room temperature and subsequently hsv-2 polyclonal antibody (1:250) was added on cells, after 1 h at 37˚c the inoculum was removed and coverslips were washed with pbs-tween for 3 times. rhodamine conjugated anti-rabbit (santa cruz) was then added on cells for 1 h at 37˚c following 3 washes coverslips were mounted on microscope glasses and observed with zeiss lsm510 fluorescence microscope (zeiss, oberkochen, germany) and images were acquired. qpcr cells subjected to binding assays were then lysed and subjected to total dna/rna extraction and subsequently subjected to qpcr using as primers 5'-ccgtcagcaccttcatcga -3' and 5'-cgctggacctccgtgtagtc -3' and as probe 5'-fam ccacgagatcaagga cagcggcc-tamra for 40 cycles at 94˚c for 15" followed by 60˚c for 60". the results were normalized according to values of rnasep and the % of bound virus were measured comparing the δct of treated wells to wells treated with equal volume of solvent. to evaluate the oxidation capability of 9d an in vitro lipoperoxidation test was carried out. in lipid peroxidation, free radicals attack double bonds of polyunsaturated fatty acids forming lipid hydroperoxides, which undergo homolytic scission to form a variety of cytotoxic compounds, such as aldehydes. this oxidative stress plays an important role in damaging membrane lipids. according to previous literature, the potential oxidative effect of 9d was evaluated toward the oxidation of linoleic acid, a carboxylic acid with two double bonds as a model lipid substrate. the lipoperoxidation of linoleic acid, incorporated in dipalmitoylphosphatidylcholine (dppc) liposomes was determined using the tba assay [20, 21] . dppc liposomes were prepared with the thin film evaporation method and used to mimic a phospholipid bilayer, and to dissolve linoleic acid. tba assay, commonly used as an index of lipid peroxidation, is based on the reactivity of malondialdehyde (mda), a colourless end-product of degradation, with 2-thiobarbituric acid (tba) to produce a pink adduct (tba-mda-tba) that absorbs at 535 nm. mda is indeed one of the final products of polyunsaturated fatty acids peroxidation in the cells and is generally considered an indicator of lipid peroxidation. mda was detected spectrophotometrically according to the method described by bay et al. with the following modifications [22] . for the tba assay,9d compound was dissolved in dmso at the concentration of 10 μm. then, 100 μl of the 9d dmso solution were incubated at 37˚c for 30 minutes with a 0.5% linoleic acid containing liposome aqueous dispersion. blank samples were prepared incubating the liposome aqueous dispersion either with saline solution (0.9% nacl aqueous solution) or dmso in the absence of 9d. after incubation, a volume of the blank and 9d samples (0.2 ml) were withdrawn and introduced in a glass tube closed with a screw cap and added with 0.1 ml of water, 0.2 ml of 4% w/w sds, 1.5 ml of 1.0% w/w phosphoric acid and 1.0 ml of 0.6% w/w tba. the mixture was stirred and heated in water bath at 95-100˚c for 45 min to favor the formation of the complex. after cooling in an ice bath, 4.0 ml of 1-butanol were added to each tube and the tba-mda-tba complex was extracted upon stirring and centrifugation. the organic supernatant was evaluated by spectrophotometry. the calibration curve of tba-mda-tba complex was obtained using a mda precursor 1,1,3,3-tetraethoxypropane. mda can be obtained by acid hydrolysis from 1,1,3,3-tetraethoxypropane in an equimolecular reaction. for this purpose, standard solutions of 1,1,3,3-tetraethoxypropane in sds (4% w/w) within the concentration range 5-250 μm were prepared. the solutions were subjected to tba assay and analyzed at spectrophotometer. the final concentration of mda derived from the reaction of linoleic acid, calculated exploiting the calibration curve, was expressed as micromoles of mda per mg of lipid substrate. to further confirm the oxidant activity of 9d the peroxidation experiment was also carried out using a control sample containing 0.05% w/w of (±)-α-tocopherol, molecule with antioxidant properties, added during the preparation of liposomes. mda formation was monitored in the same conditions above reported. the results are mean and sd of 3 independent experiments. results are presented as the mean values from 2 to 6 independent experiments, in case of 2 independent experiments results are expressed ± square root of the sum of squares, in other cases as ± sd. the ec 50 values for inhibition curves were calculated by regression analysis using the software graphpad prism (graphpad software, san diego, california, u.s.a.) by fitting a variable slope-sigmoidal dose-response curve. for binding and entry assays the significance was analyzed with a one-way anova followed by a bonferroni. results are mean and sd. n = 3. (docx) chronic hepatitis b infection: a review dengue and severe dengue new pharmacological strategies to fight enveloped viruses reservoirs and vectors of emerging viruses a strategy to estimate unknown viral diversity in mammals human ddx3 protein is a valuable target to develop broad spectrum antiviral agents a versatile and practical synthesis toward the development of novel hiv-1 integrase inhibitors 2-aminothiazolones as anti-hiv agents that act as gp120-cd4 inhibitors antimicrob one drug for two targets: biological evaluation of antiretroviral agents endowed with antiproliferative activity rhodanine derivatives as potent anti-hiv and anti-hsv microbicide antivirals acting on viral envelopes via biophysical mechanisms of action polyunsaturated liposomes are antiviral against hepatitis b and c viruses and hiv by decreasing cholesterol levels in infected cells membrane repair: mechanisms and highly sulfated k5 escherichia coli polysaccharide derivatives inhibit respiratory syncytial virus infectivity in cell lines and human tracheal-bronchial histocultures in vitro anti-herpes simplex virus activity of crude extract of the roots of nauclea latifolia smith (rubiaceae) identification of equine lactadherin-derived peptides that inhibit rotavirus infection via integrin receptor competition the agmatine-containing poly (amidoamine) polymer agma1 binds cell surface heparan sulfates and prevents attachment of mucosal human papillomaviruses influence of hydroxypropyl-b-cyclodextrin on the photostability and antiradical activity of trolox lipid peroxidative stress and antioxidative enzymes in brains of milk-supplemented rats conceptualization: valeria cagno, maurizio botta. key: cord-281815-zvs5qe8x authors: subramanian, shoba; hardt, markus; choe, youngchool; niles, richard k.; johansen, eric b.; legac, jennifer; gut, jiri; kerr, iain d.; craik, charles s.; rosenthal, philip j. title: hemoglobin cleavage site-specificity of the plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 date: 2009-04-09 journal: plos one doi: 10.1371/journal.pone.0005156 sha: doc_id: 281815 cord_uid: zvs5qe8x the plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 degrade host hemoglobin to provide free amino acids for parasite protein synthesis. hemoglobin hydrolysis has been described as an ordered process initiated by aspartic proteases, but cysteine protease inhibitors completely block the process, suggesting that cysteine proteases can also initiate hemoglobin hydrolysis. to characterize the specific roles of falcipains, we used three approaches. first, using random p(1) – p(4) amino acid substrate libraries, falcipain-2 and falcipain-3 demonstrated strong preference for cleavage sites with leu at the p(2) position. second, with overlapping peptides spanning α and β globin and proteolysis-dependent (18)o labeling, hydrolysis was seen at many cleavage sites. third, with intact hemoglobin, numerous cleavage products were identified. our results suggest that hemoglobin hydrolysis by malaria parasites is not a highly ordered process, but rather proceeds with rapid cleavage by falcipains at multiple sites. however, falcipain-2 and falcipain-3 show strong specificity for p(2) leu in small peptide substrates, in agreement with the specificity in optimized small molecule inhibitors that was identified previously. these results are consistent with a principal role of falcipain-2 and falcipain-3 in the hydrolysis of hemoglobin by p. falciparum and with the possibility of developing small molecule inhibitors with optimized specificity as antimalarial agents. malaria, especially that caused by plasmodium falciparum, is responsible for hundreds of millions of episodes of disease, and probably at least one million deaths each year [1, 2] . among impediments to effective control of this disease is increasing resistance to most available drugs [3] . new artemisinin-based combination therapies offer excellent efficacy, but resistance to most artemisinin partner drugs has already been seen [4] , and resistance to artemisinins may be emerging [5] . the pipeline for development of other classes of effective antimalarials, especially compounds that act against novel biochemical targets, is limited. to facilitate development of such compounds, it is important to characterize the biochemical features and biological roles of new drug targets. falcipain-2 and falcipain-3 are papain-family cysteine proteases of p. falciparum that are currently the subjects of in-depth drug discovery efforts [6] . these proteases are expressed sequentially across the life cycle of erythrocytic parasites, with expression of falcipain-2 beginning in early trophozoites followed by expression of falcipain-3 in late trophozoites and schizonts [7] . the proteases are hemoglobinases that reside in the food vacuole of intraerythrocytic parasites, where they degrade hemoglobin, in concert with other proteases [8] , to provide the parasite with amino acids for growth and development [9] . treatment of erythrocytic parasites with cysteine protease inhibitors or disruption of the falcipain-2 gene results in accumulation of undegraded hemoglobin in the food vacuole, confirming a role for this enzyme in hemoglobin hydrolysis [6, 10] . disruption of the falcipain-3 gene could not be achieved, but the gene was readily replaced with a tagged functional copy, indicating that falcipain-3 is essential for survival of intraerythrocytic parasites [11] . these results support studies of falcipain inhibitors as new antimalarial agents. the hydrolysis of hemoglobin by malaria parasites is a massive proteolytic enterprise, which appears to be responsible for the degradation of most erythrocytic hemoglobin over the course of a 48 hour developmental cycle [8, 12] . it has been described as an ordered pathway, with initial cleavage by aspartic proteases followed by action of other proteases, eventually leading to free amino acids [12] . however, falcipain inhibitors completely block hemoglobin hydrolysis by erythrocytic parasites [13] , and falcipain-2 and falcipain-3 hydrolyze native hemoglobin under the biochemical conditions of the food vacuole [14] , suggesting that falcipains also initiate hemoglobin hydrolysis in p. falciparum. to better characterize the roles of falcipain-2 and falcipain-3, we evaluated activities of these proteases against a library of small peptide substrates, a series of larger peptides spanning the sequences of a and b globin, and intact human hemoglobin. falcipain-2 and falcipain-3 demonstrated a marked preference for cleavage of small peptide substrates with p 2 leu, but the enzymes showed less specificity against larger peptides and intact hemoglobin, with hydrolysis at multiple sites. these results suggest that hemoglobin hydrolysis by p. falciparum is not a highly ordered process, but rather that falcipain-2 and falcipain-3 rapidly cleave hemoglobin at multiple sites to facilitate rapid hydrolysis of this substrate. expression, solubilization and refolding of falcipain-2 and falcipain-3 were performed as described elsewhere, with slight modifications [15, 16, 17] . in brief, urea solubilized inclusion bodies from bacteria over-expressing the mature domains of either falcipain-2 or falcipain-3 fused to a 66his tag were purified by ultra-filtration and bound to nickel-nitrilotriacetic acid (ni-nta) columns. after several washes with buffer containing 8 m urea and 10-60 mm imidazole, bound protein was eluted sequentially with 500 mm and 1 m imidazole in the presence of urea. refolding of falcipain-2 was achieved by diluting the eluates 100fold in 100 mm tris-hcl, 30% glycerol, 250 mm arginine, 1 mm edta, 1 mm reduced-glutathione (gsh), 1 mm oxidizedglutathione (gssg), ph 9.2 and incubating at 4uc overnight. refolding of falcipain-3 was achieved by diluting the eluates 100fold in 100 mm tris-hcl, 15% sucrose, 250 mm arginine, 1 mm edta, 1 mm gsh, 1 mm gssg, ph 9.2 and incubating at 4uc overnight. the refolded proteins were concentrated 50-fold, run over a q-sepharose column, and eluted with a gradient of nacl starting at 500 mm. eluates were examined by sds-page and for activity against the synthetic substrate, z-leu-arg-amc (benzyloxycarbonyl-leu-arg-7-amino-4-methyl-coumarin). fractions with highest enzyme activity were pooled and concentrated using a 10 kda cut-off ultra-filtration column (amicon). enzymes were stored in 50% glycerol at 280uc. to determine the p 1 -p 4 specificity of falcipain-2 and falcipain-3, complete diverse positional scanning substrate libraries were used [18] . the libraries were composed of peptide-conjugated acc (7-amino-4-carbamoylmethylcoumarin) fluorophore substrates and contained 160,000 different p 1 -p 4 peptide sequences. in the p 1 -, p 2 -, p 3 -, and p 4 -libraries, the p 1 -4 positions were spatially addressed with 20 amino acids (cysteine was replaced with norleucine); whereas the remaining three positions were randomized with equimolar mixtures of 20 amino acids (cysteine was replaced with norleucine). the cleavage of the substrates by a protease released free acc, resulting in a shift of the excitation and emission maxima from 325 and 400 nm to 380 and 460 nm, respectively. falcipain-2 (50 nm) and falcipain-3 (300 nm) were assayed in triplicate at 25uc in 100 mm sodium acetate, 100 mm nacl, 1 mm dtt, 1 mm edta, 0.01% brij-35, 1% v/v dmso, ph 5.5. aliquots of 25 nmol (in 1 ml) from each of 20 sub-libraries of the p 1 -, p 2 -, p 3 -, and p 4 -libraries were added to the wells of a 96-well microfluor-1 u-bottom plate (dynex technologies). the final concentration of each compound was 31.25 nm in 100 ml final reaction volume. the assays were initiated by addition of preactivated enzyme and monitored fluorometrically for 30 min with a spectramax gemini fluorescence spectrometer (molecular devices) with excitation at 380 nm, emission at 460 nm, and cutoff at 435 nm. hydrolysis of globin peptides in the presence of h 2 18 o synthetic peptides of 12 amino acid length scanning the entire sequences of a and b globin, with 4 amino acid overlaps between peptides, were custom-ordered from sigma-genosys. each peptide (250 mm) was incubated in duplicate with 65 nm falcipain-2 or falcipain-3 in 25 mm sodium acetate, ph 5.5, 2 mm dtt, and 50% h 2 18 o at 37uc. reactions were started by adding enzyme after spotting the 0 minute time point. aliquots were spotted after 0, 5, 10, 15, 20, 40, 60, 75, 90, 120, and 180 minutes, and overnight directly onto stainless steel maldi-target plates (applied biosystems) and mixed with an equal volume of matrix (10 mg/ml sinapinic acid in 50% acetonitrile, 0.1% trifluoroacetic acid) which stopped the proteolytic reactions. maldi-tof ms/ ms analysis was performed on a 4800 proteomics analyzer mass spectrometer (applied biosystems) in positive ion mode equipped with tof/tof optics and a 200 hz ndyag laser. for collisioninduced dissociation, the collision cell was floated at 1 kv, the resolution of the precursor ion selection was set to 150 fwhm, and air was used as the collision gas at 5610 27 torr. in the presence of h 2 18 o, protease-peptide interactions can result in the incorporation of up to two 18 o-atoms in carboxylate peptide products [19, 20] . the second incorporation, which depends on the ability of the enzyme to rebind the cleaved peptide product and catalyze the carbonyl oxygen exchange reaction, was rarely observed. therefore, in the presence of 50% h 2 18 o, peptides that were proteolytically generated displayed an 18 o-isotope signature of 1:1 (unlabeled: single labeled) at their newly-formed c-termini. the extent of 18 o incorporation for each peptide was determined by linear regression analysis of the raw spectra using least squares fitting with a model consisting of a linear combination of four components, the theoretical average in isotope clusters positioned at m/z, (m+2)/z and (m+4)/z, and a constant term. linear regression computed four multipliers, the amplitude of the unlabeled, singly-labeled, and doubly-labeled peaks, and the baseline. it was thereby possible to measure the unlabeled, singly labeled, and doubly labeled contributions for each peptide identified in the mass spectrum. we used the following criteria to positively identify new cleavage products: a) a new c-terminus had to be formed to allow 18 o-incorporation; b) the single 18 o-incorporation had to account for 50% of the entire isotopic envelope (610% experimental error); c) peptide needed to be observed in replicate analyses and in consecutive time points (except for the overnight time point); and d) labeled peptide should not be detected at the zero time point, with the following exceptions: e) peptides that started off with .60% unlabeled fraction, with subsequent decrease in unlabeled peptide were included; f) peptides with initial single label, followed by double label were included; g) peptides with single label in a minority of time points slightly .60% were included; h) if the single label significantly increased with time, peptides with slight labeling at 0 min were included; and i) as expected, proteolytic products that were formed while the c-termini stayed intact did not incorporate 18 o. hence, these newly generated peptides with intact c-termini: 1) should display less than 20% labeling (accounting for experimental error); 2) these peptides should be seen in at least two time points and in replicate analyses (unless it is observed only in the overnight time point); and 3) these unlabeled peptides should not be seen at not at the zero time point. for analysis of hydrolysis of intact hemoglobin, reaction conditions and enzyme concentration were as described for experiments with 12-mer peptides, except that the substrate was 2.5 mm human hemoglobin (sigma) and were incubated for 180 minutes or overnight. at the end of the incubation, peptides were separated by ultra-filtration (12,0006g for 40 min; sartorius vivaspin columns, nominal molecular weight limit 3,000 or 5,000 daltons). ultra-filtrates containing the cleavage products were analyzed by lc ms/ms on a qstar xl mass spectrometer (applied biosystems/sciex) equipped with a nano 2dlc system (eksigent) and a nanospray ii source (applied biosystems/sciex). external calibration was performed in ms/ms mode using fragment ions of glu-fibrinopeptide as references. an lc packings pepmap c18 trap column (300 mm i.d., 5 mm length, 300 å pore size, 5 mm particle size) and a column (75 mm i.d., 15 cm length) self-packed with jupiter proteo c12 end-capped material (90 å pore size, 4 mm particle size) were used for desalting and reversed phase peptide separation, respectively. a 20 minute linear gradient from 2% b to 50% b was run at a 250 nl/min flow rate, utilizing solvents a: (2% acetonitrile/0.1% formic acid) and b (80% acetonitrile/0.08% formic acid). precursor ion selection employed an automated routine that consisted of a one survey ms scan (1 s, m/z 400-1700) and two ms/ms scans (1 s, m/z 60-1500); nitrogen served as a collision gas and collision energy was automatically adjusted depending on the size and charge state of the precursor ion. mass spectrometry data were analyzed using a laboratory information system that utilized mascot distiller software (matrix science) for spectral processing and peak detection. peptides were identified by using the mascot algorithm (matrix science; version 2.1) to search against human proteins in the swissprot database via nonspecific digestion (i.e. ''no enzyme'' search). identities of peptides were confirmed by manual interpretation of the ms/ms data. falcipain-2 and falcipain-3 were expressed in bacteria, purified, and refolded to active proteases. these proteases have previously been shown to readily hydrolyze hemoglobin [15, 17] , and also to cleave peptide substrates after leu-arg-, val-leu-arg-, and val-leu-lys[17] . these substrates were tested based on the known specificities of other papain family cysteine proteases [21] . however, more detailed characterization of the substrate specificity of these proteases has not been reported. to better characterize falcipain activities and determine whether a clear pathway of hemoglobin degradation by falcipains is present, activities were studied by three assays, evaluating action against a tetrapeptide library, 12-mer peptides representing the sequences of a and b globin, and intact hemoglobin. all assays were performed under conditions matching, to the best of our knowledge, those of the p. falciparum food vacuole, the site of hemoglobin hydrolysis by the falcipains, notably at ph 5.5. to study the amino acid preferences of falcipain-2 and falcipain-3 at the p 1 to p 4 positions, we studied hydrolysis of positional scanning tetrapeptide libraries (fig. 1) . in each library, each tetrapeptide had a fixed amino acid at one position (p 1 , p 2 , p 3 , or p 4 ) and randomized amino acids at the other positions [18] . p 1 preferences for the two enzymes were similar. both falcipain-2 and falcipain-3 showed a p 1 preference for arg, with activity also high against substrates with p 1 lys, gln, thr, and met. considering the p 2 position, both falcipain-2 and falcipain-3 had a strong preference for leu and a slight preference for val. amino acid preferences were less pronounced at the p 3 and p 4 positions, although specificities for the two enzymes were similar. at p 3 , positively charged amino acids, hydrophobic amino acids, and pro were preferred. p 4 his and asn were preferred by both enzymes, with particularly low activity against peptides with hydrophobic amino acids at p 4 . we next evaluated cleavage of 12-mer peptides spanning the sequences of a and b globin, with 4 amino acid overlaps such that each potential cleavage site was considered with at least 3 p or p9 amino acids present. to best distinguish falcipain cleavage products from minor fragments contaminating synthetic peptides, the proteases and peptide substrates were incubated with 50% h 2 18 o. with proteolytic cleavage, the c-terminal ends of the newly generated peptides, but not false products, bore 18 o either by enzyme catalysis or isotope exchange [19, 20] . peptides were incubated with falcipain-2 or falcipain-3 for varied intervals, and reaction products were assessed by maldi-tof ms/ms analysis. we used stringent criteria to distinguish newly generated peptides from false positive and ambiguous products, based on ratios of labeled to unlabeled peptides and frequencies of appearance at several time points. multiple cleavage sites were identified for both falcipains (fig. 2a-d, top arrows) . considering the sequences most preferred in our positional scanning library analysis, cleavages by falcipain-2 were seen at 25 of 36 positions with p 2 leu and by falcipain-3 at 22 of 36 such positions. analysis of the step-wise hydrolysis of peptides over time yielded a complex picture for both a and b globin and either falcipain-2 or falcipain-3 (fig. 3) . identification of the most common amino acids at cleavage sites was, of course, influenced by the prevalence of different residues in hemoglobin. at the p 2 position, leu was most common, but cleavages of peptides with p 2 val and other amino acids were also well represented (fig. 4) . a more thorough analysis of cleavage for each amino acid at each p 1-4 position is in supplementary material (table s1; figure s1 ). these experiments also showed that the most preferred p 1 9 amino acids were ala and leu. however, this preference was modest, with cleavages demonstrated at sites with multiple different prime-side amino acids. at the p 2 9 position ala was slightly preferred. there was no particular amino acid preference at the p 3 9 or p 4 9 position. for our third analysis, we assessed hydrolysis of full-length hemoglobin by falcipain-2 and falcipain-3. a simple step-wise pathway for hemoglobin degradation was not seen. rather, we identified over a hundred peptides of molecular weight 9 kda or less after 5 minute incubations of human hemoglobin with falcipain-2 or falcipain-3 (data not shown). for simplicity, we therefore concentrated on identification of hemoglobin cleavage fragments after an extended incubation with falcipain-2 or falcipain-3. reactions were performed after incubation of hemoglobin with 50% h 2 18 o, and the identities of hemoglobin cleavage fragments were characterized by lc-ms/ms. data were filtered based on 18 o incorporation, as described above for analysis of peptide hydrolysis. we then mapped cleavage sites on (fig. 2a-d, bottom arrows) . cleavage sites were identified in both the loops (blue color) and helices (red color) of human globin, with no particular preference for buried or exposed cleavage sites on the hemoglobin tertiary structure (fig. 2e) . of note, the data obtained for falcipain-2 were nearly identical to those for falcipain-3 for hydrolysis of hemoglobin and were very similar to those for hydrolysis of globin peptides by both proteases. falcipain-2 and falcipain-3 are critical hemoglobinases of p. falciparum. these enzymes have been extensively studied, with characterization of kinetic properties [15, 17] , confirmation of key functions with inhibitor [22, 23] and gene disruption studies [10, 11] , and identification of unique motifs that are required for protein folding [24, 25] , interaction with hemoglobin [26] , enzyme structure [27, 28, 29] , and targeting to the parasite food vacuole [30, 31] . however, characterization of a specific pathway for hemoglobin hydrolysis by falcipain-2 and falcipain-3 has been difficult. we now present a detailed analysis of hydrolysis of short peptide substrates, 12-mer peptides encompassing a and b globin, and intact human hemoglobin by falcipain-2 and falcipain-3. we show that the proteases have distinct specificity for short peptide substrates with leu at the p 2 position, but that against larger substrates specificity is less marked. indeed, the proteases rapidly cleaved globin peptides and intact hemoglobin at multiple different sequences. these results suggest that falcipain-2 and falcipain-3 do not cleave host hemoglobin via an ordered process, but rather that the proteases efficiently cleave the substrate by rapid hydrolysis at many different sites on the protein. we evaluated hydrolysis of small peptide combinatorial libraries by falcipains, a standard technique. other proteases that have been evaluated in this manner include caspases [32] , the serine protease granzyme b [32, 33, 34] , a coronavirus cysteine protease [35] , a tick asparaginyl endopeptidase [36] , and human kalikreins [37] . although this methodology is most useful for highly selective enzymes, valuable information can also be extracted from substrate profiles with enzymes that are quite promiscuous, as in the case of the falcipains and other papain family cysteine proteases [21] . of note, we observed marked selectivity in p 2 preference for falcipains using small peptide substrates. hydrophobic amino acids at p 2 were strongly preferered by falcipain-2 and falcipain-3, as seen with many other papain-family proteases [21] . however, the specific preference for a single amino acid, leu, at p 2 , was more striking than the case for any of the 8 other papain-family proteases recently studied in the same manner [18] . of the 8 other proteases, the next most specific enzyme was papain, with about 5-fold greater activity for peptides with val at p 2 than any other peptides. other papain-family proteases not included in the prior survey appear to show a stronger preference for p 2 leu, notably the human proteases cathepsin k and cathepsin s [21] . as discussed in more detail below, we found that the striking preference for p 2 leu in small substrates did not predict specificity only for cleavage sites with p 2 leu in larger peptide or protein substrates. nonetheless, this specificity has been seen also in small molecule inhibitors of falcipain-2 and falcipain-3; inhibitors with either phe or leu at p 2 were effective, but typically activity improved ,10-fold for fluoromethyl ketones, vinyl sulfones, and aldehydes with p 2 leu [23,38,39]. thus, although of limited utility in discriminating activity against native protein substrates, the elaboration of enzyme specificity using tetrapeptide scanning libraries provided accurate prediction of optimal small molecule inhibitors. appreciation of the fine specificity of inhibition of falcipains is contributing to drug discovery efforts directed against these proteases [6] . our experimental conditions for globin peptides and native hemoglobin were slightly modified from a previous study [14] , so that they were suited for 18 o incorporation and subsequent mass spectrometry. these conditions were designed to match those in the intact food vacuole, including modest reducing conditions (1 mm dtt) and ph of 5.5. prior studies have estimated the ph of the food vacuole at 4.5-5.8 [40, 41, 42, 43] and shown the ph optima of falcipain-2 and falcipain-3 to be ,4.5-6.0 [15, 17] . the enzyme:substrate ratio was slightly higher than that used previously [14] ; these conditions gave us optimum results with substrate degradation and 18 o incorporation, which is also an enzyme-dependent process. ordered pathways for the hydrolysis of hemoglobin have been demonstrated in studies with isolated aspartic proteases from two parasites that hydrolyze this substrate, schistosome worms that degrade hemoglobin extracellularly [44] and malaria parasites that degrade hemoglobin in an intracellular acidic organelle [12] . however, prior attempts to map out pathways of hemoglobin hydrolysis for cysteine proteases of schistosomes or malaria parasites have been unsuccessful due to an inability to identify the ordered generation of cleavage fragments (shenai br, sajid m, and rosenthal pj, unpublished observations). a key goal of the present study was to more rigorously test the hypothesis that falcipain-2 and falcipain-3 indeed hydrolyze hemoglobin through an ordered pathway of cleavage events and, in any event, to identify globin peptide and hemoglobin cleavage fragments after incubation with falcipains. despite rigorous filtering to identify only bona fide cleavage fragments, we found that falcipain-2 and falcipain-3 cleaved hemoglobin simultaneously (within our limits of analysis) at many sites. thus, the enzymes appear to degrade hemoglobin not via an ordered hydrolytic pathway, but through rapid hydrolysis of multiple sites. this result is consistent with known features of other papain-family cysteine proteases, which also have quite broad specificity against protein substrates [21] . indeed, the prototype enzyme papain is used in a number of industrial applications that take advantage of its broad substrate specificity. lysosomal cysteine proteases similarly demonstrate broad specificity, and appear to be responsible for extensive proteolytic activity in this organelle [21] . in malaria parasites, where the food vacuole appears to serve as the homolog of the lysosome, and where hemoglobin appears to be the primary proteolytic substrate [12] , it seems likely that falcipain-2 and falcipain-3 are responsible for the bulk of cleavages that reduce hemoglobin from a complex tetramer to single amino acids. additional proteases, notably plasmepsin i-iv [12, 45] , dipeptidyl aminopeptidase i [46, 47] and falcilysin [48, 49] , also appear to contribute to the efficient hydrolysis of hemoglobin in the food vacuole, and the presence of multiple proteases of multiple classes suggests a degree of redundancy in this process [9] . despite the presence of multiple proteases in the food vacuole, it is noteworthy that treatment of cultured parasites with specific inhibitors of cysteine proteases fully blocked the hydrolysis of hemoglobin, highlighting the key role for the falcipains in this process [22] . knockout of plasmepsins i-iv, the 4 known food vacuole plasmepsins, was not lethal to p. falciparum (although parasites with knockout of all four food vacuole plasmepsins demonstrated abnormal development) [50, 51] . in contrast, knockout of either falcipain-3 [11] or dipeptidyl aminopeptidase i [46] appears to be lethal, arguing for essential functions for these enzymes, although it is not known if the essential functions are hydrolysis of hemoglobin. of note, it has recently also been shown that the activation of food vacuole plasmepsins is primarily mediated by falcipains, offering further evidence of a primary role for falcipains in mediating the hydrolysis of hemoglobin by erythrocytic p. falciparum parasites [52] . results showing potent hydrolysis of hemoglobin by both falcipain-2 and falcipain-3 [14 and this report] and the localization of these falcipains in the food vacuole [7, 30] argue that a key function of these proteases is hemoglobin hydrolysis. however, it remains possible that they also perform other key functions, including the processing of plasmepsins (as discussed above) and possibly other food vacuole proteases [52] and the hydrolysis of erythrocyte cytoskeletal proteins to facilitate erythrocyte rupture [53, 54, 55] . as falcipain-2 and falcipain-3 are not biochemically identical and as they are expressed sequentially during the parasite life cycle, they may have non-overlapping biological roles, explaining in part the redundancy of papain-family proteases in p. falciparum. of interest, falcipain-2 and falcipain-3 contain a short cterminal insertion that is unique among studied papain-family proteases and mediates interaction between the proteases and hemoglobin independent of the enzyme active site [29] . removal of the insertion from falcipain-2 rendered a protease that remained active against peptide substrates, but could not hydrolyze hemoglobin [26] . this feature of the falcipains likely facilitates particularly efficient hydrolysis of hemoglobin and may contribute to the ability of the proteases to cleave hemoglobin at multiple sites. however, it is difficult to predict interaction between the protease and substrate based on known structures [28, 29] , as once initial cleavages of hemoglobin take place, the structure of hemoglobin will be altered, and multiple peptide bonds will presumably be accessible to cleavage by the falcipains. a prior study identified hemoglobin cleavage fragments after incubation of this protein with crude p. falciparum food vacuole extracts [56] . this analysis identified a series of hemoglobin cleavage fragments, with cleavage sites at mean intervals of 8.4 amino acids. comparison of identified cleavage sites from the prior analysis of food vacuole extracts with our analysis of cleavage by recombinant falcipains identified many shared cleavage sites, but also cleavages identified only after incubation with food vacuole extract or only after incubation with recombinant falcipains. specifically, only 15 of 37 cleavages identified in the prior study after incubation with food vacuole extracts were seen after incubation with falcipain-2 or falcipain-3. therefore, some hemoglobin cleavages appeared to be generated uniquely by proteases other than the falcipains. however, considering both our globin peptides and intact hemoglobin results, we observed over 100 different cleavage sites within hemoglobin, with 90 of these not seen after incubation with food vacuole extract. some falcipain activity may have been missed in studies of vacuole extracts due to chosen assay conditions, the presence of enzyme inhibitors in extracts, or loss of enzymes during sample preparation. in any figure 4 . cleavage site preferences for falcipain-2 and falcipain-3. preferences for amino acids (single letter code) at p 1 -p 4 and p 1 9-p 4 9 are shown schematically. at each position, the height of each amino acid corresponds to its frequency at cleavage sites based on analysis of a and b globin. weblogo (weblogo.berkeley.edu), a free software for plotting consensus sequences, was used to plot p 4-p 4 9 preferences. doi:10.1371/journal.pone.0005156.g004 event, our new results suggest that, although a diverse set of proteases is likely required for optimal hydrolysis of hemoglobin, the sequential expression of falcipain-2 in early trophozoites and then falcipain-3 in late trophozoites and schizonts [7] provides potent hemoglobinolytic activity for erythrocytic p. falciparum parasites. our study has important limitations. first, the precise analysis of cleavage of globin peptides or hemoglobin by falcipains proved to be technically very challenging. analysis of products after incubations of globin peptides with the proteases identified many products, suggesting the presence of contaminating fragments in commercial peptides. we therefore used 18 o labeling and assessments for reproducibility over time to identify genuine cleavage fragments. however, this approach necessarily involved the establishment of arbitrary filtering criteria, and may have inappropriately excluded some cleavage sites and/or included false cleavage sites. second, analysis of cleavage of intact hemoglobin was cost-and labor-intensive, limiting our ability to evaluate a broad range of hydrolytic time points. thus, our ability to identify any single hemoglobin cleavage site for falcipain-2 or falcipain-3 from our studies is somewhat limited, although cleavages seen consistently with both falcipains at multiple time points and with both peptide and intact hemoglobin substrates are very likely to represent true biological cleavage sites. third, our study used ultrafiltration to isolate peptide cleavage fragments generated from intact hemoglobin. this procedure might lead to selective loss of certain peptides. to investigate that possibility, we previously analyzed tryptic digests of proteins prior to and after ultrafiltration, and we did not observe significant differences between the samples (hardt, m., unpublished observation). nonetheless, we cannot exclude the possibility that certain peptides might have been lost due to this separation technology, but, based on our overall sequence coverage of hemoglobin, this potential loss appeared to be minimal. in any event, our overall result is clear: falcipain-2 and falcipain-3 are capable of rapid hydrolysis of hemoglobin at multiple cleavage sites. it is of interest that two common polymorphisms leading to alterations in the sequence of hemoglobin, hbs and hbc, offer protection against severe malaria [57, 58, 59, 60, 61] , and many other polymorphisms in hemoglobin may offer protection against malaria. it is possible that this protection is mediated, in part, by altered susceptibility of mutant hemoglobins to hydrolysis by parasite proteases, including falcipains. this will be an interesting area for future study. in summary, we have characterized the proteolytic specificity of two key cysteine protease hemoglobinases of the malaria parasite p. falciparum. both falcipain-2 and falcipain-3, which are expressed sequentially, contribute importantly to the necessary hydrolysis of hemoglobin by intraerythrocytic parasites. indeed, our results show that the falcipains rapidly hydrolyze hemoglobin at multiple sites, allowing efficient degradation of this substrate, and suggesting that the falcipains are the key proteases responsible for the massive hydrolysis of hemoglobin that occurs over the course of the intraerythrocytic life cycle of p. falciparum. figure s1 graphical representation of amino acid preferences at p1-p4. p1-p4 amino acid preferences were plotted as a percentage of total cleavages, alongside percentage occurrence of each amino acid in the primary sequences of a and b globin. author contributions the global distribution of clinical episodes of plasmodium falciparum malaria the ears of the hippopotamus: manifestations, determinants, and estimates 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cord-294592-zwvr57a0 authors: mukherjee, moumita; goswami, srikanta title: global cataloguing of variations in untranslated regions of viral genome and prediction of key host rna binding protein-microrna interactions modulating genome stability in sars-cov-2 date: 2020-08-11 journal: plos one doi: 10.1371/journal.pone.0237559 sha: doc_id: 294592 cord_uid: zwvr57a0 background: the world is going through the critical phase of covid-19 pandemic, caused by human coronavirus, sars-cov-2. worldwide concerted effort to identify viral genomic changes across different sub-types has identified several strong changes in the coding region. however, there have not been many studies focusing on the variations in the 5’ and 3’ untranslated regions and their consequences. considering the possible importance of these regions in host mediated regulation of viral rna genome, we wanted to explore the phenomenon. methods: to have an idea of the global changes in 5’ and 3’-utr sequences, we downloaded 8595 complete and high-coverage sars-cov-2 genome sequence information from human host in fasta format from global initiative on sharing all influenza data (gisaid) from 15 different geographical regions. next, we aligned them using clustal omega software and investigated the utr variants. we also looked at the putative host rna binding protein (rbp) and microrna binding sites in these regions by ‘rbpmap’ and ‘rna22 v2’ respectively. expression status of selected rbps and micrornas were checked in lungs tissue. results: we identified 28 unique variants in sars-cov-2 utr region based on a minimum variant percentage cut-off of 0.5. along with 241c>t change the important 5’-utr change identified was 187a>g, while 29734g>c, 29742g>a/t and 29774c>t were the most familiar variants of 3’utr among most of the continents. furthermore, we found that despite the variations in the utr regions, binding of host rbp to them remains mostly unaltered, which further influenced the functioning of specific mirnas. conclusion: our results, shows for the first time in sars-cov-2 infection, a possible cross-talk between host rbps-mirnas and viral utr variants, which ultimately could explain the mechanism of escaping host rna decay machinery by the virus. the knowledge might be helpful in developing anti-viral compounds in future. outbreak of novel coronavirus sars-cov-2 has been proclaimed as a pandemic by the world health organization (who). from the first report of infection in wuhan, china on december 31, 2019, the virus has infected 7.21m people worldwide till 9th june, 2020. sars-cov-2 is an enveloped, positive-sense, single stranded rna virus of genus beta-coronavirus (β-covs) with the entire genome size of approximately 30kb. this viral genome is composed of about 14 open reading frames (orf) which encodes both structural and non-structural proteins having a role in their transmission, survival and pathogenesis [1] . the main structural proteins translated from sub-genomic mrnas include spike glycoprotein (s), envelope protein (e), membrane glycoprotein (m) and nucleocapsid protein (n) along with 16 non-structural proteins (nsp [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] . the genomic rna element of sars-cov-2 also includes 5'-untranslated region (5 0 -utr) of 265bp length with a methylated cap and a 3' polyadenylated utr of length 229 bp, according to the reference sequence from wuhan. low fidelity of the rna polymerase makes the rna viruses prone to high frequency mutation and the mutation determines the virus evolution [2] . systemic mutation analysis of the viral genome revealed that the virus had mutated several times in spatio-temporal variation and has evolved into numerous strains [3] . this diversity of rna strains might be correlated to severity and mortality seen in covid-19 (corona virus disease-2019). a recent phylogenetic network analysis on 160 complete sars-cov-2 genome reported that the virus appeared to evolve in three distinct clusters a, b and c, with a being the ancestral type. type a and c seemed to be more prevalent in americas and europe, whereas type b was dominant in eastern asia [4] . during viral replication, sub-genomic mrnas are synthesized by a process of discontinuous transcription with a common 5 0 -untranslated leader sequence [5'-utr] and a 3'-noncoding co-termini [3'utr] , identical to the viral genome [5] . hence, highly structured 3' utr of positive-strand rna viruses is indispensable control element in replication, transcription, and translation of rna viruses along with the 5' utr [6] . extent of structural and functional conservation in the 5'-terminal of genomic rna of different species of genus coronavirus has been found to a distinctive magnitude [7] . these terminal untranslated regions are thus substantial site for rna-rna interaction and binding of viral and host cellular proteins for rna replication and translation [8] . molecular evolution in the untranslated region i.e. the variation in the utr region leads the virus to evolve to a great extent. there are many studies which have considered the mutation in the coding region with respect to geographical location. here, we have enumerated the variants in the 5'-and 3'-utr regions of sars-cov-2 genome. we have studied a total of 8595 viral sequence samples worldwide for this purpose and found that there are some rare variants of utr that are distributed over a global spectrum, while some variants are specifically present in a population at a comparatively higher frequency. this drove us to make a systematic catalogue of the utr variants across six continents of the world that could have a role in emergence of different strains of sars-cov-2. we have also looked at the possible regulation of viral genomic rna through binding of host rna binding proteins (rbps) and mir-nas in specific sequences of the viral utrs. there are experimentally validated evidences of human rbps binding to the regulated signals within the untranslated region of sars-cov rna in order to control the viral rna synthesis and turnover. polypyrimidine tract-binding protein (ptb) is found to bind to the leader sequence and hnrnpa1, hnrnpq is bound to the 3'utr of beta-coronavirus mhv. similarly, a host protein, madp1 (zinc finger cchctype and rna binding motif 1) interacts with the 5 0 -utr of sars-cov, influencing the rna synthesis machinery [9] . likewise, there are reports of mirna binding to the viral genome, both in the coding and non-coding regions [10] . as the mirna mediated decay of mrna is observed mainly through the 3'utr of the gene in mammalian system, we are also interested to explore the host mirna-mediated regulation of the viral genome through the 3'-utr of sars-cov-2 via mrna decay and translational repression. interaction and/ or competition between rbp and mirna-risc is also well studied in mammals [11] and investigation of this interplay of host rbp and mirna in the untranslated region of virus may delineate the role of utrs in sars-cov-2 virulence and survival and how variation in the utr can have an impact in the overall regulation. we have downloaded complete and high-coverage sars-cov-2 genome sequence data in fasta format from global initiative on sharing all influenza data (gisaid), which is a public repository of all influenza virus sequence. our data involved only the human-host specific viral sequence covering six continents of the world: asia, europe, oceania, north america, south america and africa. from asia, we have taken the viral sequence data from china (n = 197), india (n = 205), japan (n = 123), thailand (n = 121) and taiwan (n = 102). from europe, the data is taken from italy (n = 109), spain (n = 671), england (n = 2345), russia (n = 130) and germany (n = 202). viral sequence data is taken only from australia (n = 522), on behalf of oceania. representative countries from north america include usa (n = 3286) and canada (n = 147). there are no further divisions for south america (n = 281) and africa (n = 150). initially the plan was to retrieve viral sequences collected in a span of one month, just after an interval of two month from the date of first report in the corresponding place, in order to catch the diversity in the viral sequence in that region. but, unfortunately, due to less number of sequence deposition in some countries, we had to deviate from the plan and retrieve all the available sequences till may28, 2020 for them ( table 1) . we have downloaded the sequences on 28th may, 2020. the reference sequence from wuhan was taken as our reference from ncbi (nc_045512.2). all the country specific sequences were aligned with the reference sequence separately using multiple sequence alignment tool clustal omega (embl-ebi). as we were interested only in the 5'-and 3'-untranslated region (utr) and these positions are located at the two extreme ends of the sequences, there is a chance of lower coverage and higher error-rate. to resolve this ambiguity, we have put a high filtering cut-off of at least 70% coverage in a particular location of that area. all the alignment files have been shared in 'figshare' and can be accessed using the link: https://figshare.com/articles/supplementary_data_alignment_files/12649109. for prediction of human rbps bound to the utr of a viral genome, we have used the webtool 'rbpmap' that enabled prediction of rbp binding on genome sequences from a huge list of experimentally validated motifs of rbpmap database. weighted-rank algorithm was used for mapping the motifs [12] . we have put the reference and mutated 5'-and 3'-utr sequences separately as query sequence with the in-built human rbp motifs of the database. from the output, we have selected only the predicted rbps with high z-score and p-value. putative human mirna binding sites on viral utr was assessed using the 'rna22 v2 micro-rna target detection' tool [13] . as a target sequence input, we provided the reference and mutated 3'-utr sequences of virus genome. for input mirna sequences, we have obtained a list of all annotated human mirnas with their sequences in fasta format from mirbase database [14] . for all other criteria to be used for the prediction was given as per the default settings and additionally homology with seed sequence and its complementary target sequence were manually examined in the output file of interaction. expression of specific genes was obtained from 'the protein atlas' respective to normal lung tissue. rna-seq data from lung tissue generated by the genotype-tissue expression (gtex) project was reported as mean ptpm (protein-coding transcripts per million), corresponding to mean values of the different individual samples from each tissue. hpa rna-seq data from lung tissue was reported as mean ptpm (protein-coding transcripts per million), corresponding to mean values of the different individual samples from each tissue. tissue data for rna expression also obtained through cap analysis of gene expression (cage) generated by the fantom5 project, reported as scaled transcripts per million. gepia [15] web-tool was used to obtain comparative expression of specific genes in lung adenocarcinoma (luad) and normal lungs. sequence alignment of over 8500 virus samples collected from over 15 geographical locations with the sars-cov-2 reference genome (nc_045512.2) has yielded a total of 74 variants in the 5' utr and 83 variants in the 3'utr. among them, 28 unique variants have been summarized in the s1 table based on a minimum variant percentage of 0.5. but for the variants that are present in at least four populations, no such variant percentage cut off was taken. the most common high frequency variant is the 241c>t, which lies in the leader sequence of the sars-cov-2 viral genome. china, being the ancestral domicile of the virus has a very low percentage of this variant (fig 1) . europe has the maximum frequency of this variant (fig 2) . other than china, other countries in asia also have a lower percentage of this variant (fig 1) . thus, we can a see a clear effect of regional variation on the frequency of this variant. another study reported that this variant has co-evolved with three coding region mutations (3037c > t, 14408c > t, and 23403a > g) of nsp3, rna primase and spike glycoprotein. they have also related this mutation with the increased transmissibility of the virus [16] . another common variation in the 5'utr is 187a>g, that is present in seven of our populations being most prevalent in italy and canada. 29742g>a/t and 29774c>t are the most familiar variants of 3'utr among all the continents. 29742 variant has two alternative alleles with g>a being more prevalent in india, usa and africa, whereas g>t variant is more dominant in thailand, taiwan, germany, australia and canada. china and england has both the variants of almost similar percentage. hence, this variant seems not be associated with any specific region. another important variant of 3'utr is 29734g>c, which is mostly seen in europe and australia, but italy has a quite high percentage of this variant than others (fig 2) . most of the countries in this study seem to have a moderate to significant variation in the 3'utr of the viral genome. furthermore, we have explored the relationship of these variants with the secondary structure of sars-cov-2 viral rna. 5'utr variant 187a>g fall within the sl5a stem-loop and 241c>t variant was within sl5b stem-loop. the above mentioned three 3'utr variants were also within the bulge portion of the stem-loop of viral rna secondary structure. wild type nucleotides of all these variants had high shape-reactivity value which suggested that these nucleotides were less likely to form base-pair and hence had an important role in affecting the secondary structure [17, 18] . all the 15 populations and sub-populations used in this study have a definite pattern of utr variants of their own (figs 1; 2) . even countries in a single continent differ in their utr variation. some variations are merely specific to a particular location with a quite considerable percentage. the overall sequence of 5'utr of sars-cov-2 in italy is exceptionally variable, whereas the 3'utr is substantially stable with a single variant in the position 29701 (g>t). but england, also being a part of europe, has a notable number of variations in the 3'utr having a relatively stable 5'-end. from our analysis, it is evident that russia had no such frequent variation in the utr, except the 241c>t variant which was present in 100% of the population. india had two striking variations in the 3'utr, 29827a>t, 29830g>t, which was found nowhere in this global mutational landscape (s1 table) . all the sequences under this study with these two variants are from indian state of maharashtra, which has the highest number of sars-cov-2 infected people within india. most interestingly, both of these variants occur simultaneously in majority of the cases. similarly, south america also had five unique mutations in the positions 29783g>t, 29786g>c, 29834t>c, 29838c>t and 29839a>g of 3'utr. rna binding proteins belong to a class of proteins which bind to target rna molecules through characteristic binding motifs and perform a variety of functions. in fact, irrespective of the type of rnas, whether is coding or non-coding, specific rbps get associated with rnas right after their birth and subsequently control the processing, stability, transport, translation, regulatory function and turnover. as mentioned earlier, exogeneous viral rna will also encounter host rna decay machinery when released into the cell and there are ample evidences that they attempt to escape the process in order to maintain their successful propagation within the host. one way to take care of the situation is to recruit host rna stabilization factors into the regulatory region, i.e. 5' and 3'-utrs [19] . table 2 lists the predicted host rbps interacting with the 5'-utr of virus sars-cov-2 as obtained using 'rbpmap'. in the next step, the logical exploration was to find out whether the most prevalent variations identified in the 5'-utr of the viral rna genome could alter binding of any of these rbps. the 'a' to 'g' change at position 187 coincided with the binding site of cug-bp (also known as celf1) (fig 3a) , implicating that the variation might have some effect on cug-bp binding. in order to test that, we used the mutated sequence as 'input' and found that the single nucleotide change didn't have any effect on binding of cug-bp to its target sequence. in other words, no matter whether the virus has 'a' or 'g' at position 187 of its 5'-utr, cug-bp binds and probably performs its function. then, we focused on the most important change in the 5'-utr, the position at 241. looking at the distribution of this variation and the reports related to its association with virulence [16] , we had a belief that it might be giving some selective advantage to the virus. however, we didn't find any rbp binding site overlapping with this region. we changed the nucleotide at position 241 from 'c' to 't' (for corresponding 'u' in rna) and used the mutated sequence in rbpmap. to our surprise, we found a tardbp binding site created upon this change ( fig 3b) . tardbp (also called tdp-43) is a well characterized rbp which binds to specific 'ug' (and 'tg') rich sequences of rna (and dna) and reported to facilitate translation when bound to 5'-utr [20] . from protein data bank (pdb) we retrieved the co-crystal structure of tardbp with single strand dna (fig 3c & 3d) which indicated strong binding of the protein to that particular nucleic acid stretch. hence, our finding showed strong binding of tardbp to viral 5'-utr having 'u' at position 241. this could be implicated in facilitating translation of viral proteins resulting in its effective propagation within human host. following the same principle and considering the fact that 3'-utr is the most important site for regulation of rna stability and turnover, we also wanted to find out what are the rbps interacting with this region. the 3'-utr reference sequence of sars-cov-2 was fed into rbpmap and we obtained a list of 3'-utr binding rbps selected on the basis of p-value and z-score ( table 2) . as with 5'-utr, here also we focused on the major variations in 3'-utr to find out if specific nucleotide change could interfere with the binding of specific rbps. we found that 3'-utr variations at positions 29742 and 29774 could affect interaction of srsf5 and hnrnpa1 respectively. however, the regulation at 3'-utr is not that simple. another important factor which needs be taken into account is whether there are host mirna binding sites present within the viral 3'-utr region and how the combinatorial interaction of host rbps and mirnas could dictate the viral rna stability. in order to identify whether there are any host mirna binding site present in viral 3'-utr and if so, what they are, we used the viral 3'-utr sequence as 'input' in the mirna prediction software 'rna22 v2'. the algorithm predicts several of such mirnas and we selected them based on folding energy for heteroduplex formation and p-value ( table 3) . as the most important factor for the functional mirna-target rna interaction is to have the perfect homology between the seed sequence of mirna (2 nd to 8 th nucleotide from 5' end) and the corresponding target rna sequence, we also explored the fact in the identified pairs. furthermore, whether the changes caused by the common variations interfere with the seed sequence https://doi.org/10.1371/journal.pone.0237559.g002 binding was also investigated. the interesting part in this scenario is that the reference sequence at a particular position causes a mismatch in the seed resulting in inefficient binding of a mirna, but the variant base at that position creates a perfect binding site. however, considering the complexity of the region, all these probabilities could not be assessed only in terms of mirnas and hence we set out to probe into the interaction between the host rbp-mirnas and viral sequence variants in 3'-utr region in a comprehensive manner. we investigated the interaction in a stepwise manner. the first situation explored was the case where host mirna mir-34b-5p targets viral rna and no mutation has been identified in the 'seed'-corresponding region. this means that all the viral sub-types will be vulnerable to degradation by this mirna, provided it is expressed in host lungs tissue. careful scanning of the binding site and also looking at the rbp binding information we identified a rbms3 target sequence actually overlapping with the mir-34b-5p seed corresponding region (fig 4a) . this clearly indicated a prevalent competition between rbms3 and mir-34b-5p-risc complex for their respective binding site, with one of them simply presenting a steric hindrance to the other one by blocking the access. this is quite common scenario in regulation of mammalian mrna stability where the final verdict whether the mrna is degraded or stabilized largely depends on the spatio-temporal expression or the availability of both the molecules (rbp and mirna). second scenario was the variation in position 29774. in this case, we identified that change from 'c' to 'a' disrupted the binding of mir-9-5p to its corresponding target sequence at the 'seed' region (fig 4b) . this is clearly an advantage to the virus as the variation would help it to get rid of the mirna mediated degradation. as this overlapping region also had a binding site for rbp hnrnpa1, we wanted to find out what happens to hnrnpa1 binding when there is a change from 'c' to 'a'. interestingly enough, we discovered that hnrnpa1 binding is also not affected, implicating an ensured protection of viral rna by rbp hnrnpa1, no matter whether mir-9-5p is functional or not. again, this situation is also quite common in mammalian mrna regulation where we see some amount of degeneracy among the rbp target sequences, when change of a single nucleotide is often tolerated. the virus uses this phenomenon to its benefit where in spite of utr sequence variation, protection from same rbp is ensured. in case of the third incidence, we looked at position 29742, where the 'g' to 'a' change creates a binding site for mir-3664-5p (fig 4c) , which means that the virus having the variant nucleotide will be prone to degradation by this mirna. we had already commented about the possible binding of srsf5 in this region and found that the variation under investigation had no impact on srsf5 binding. again, it indicated that despite creation of mir-3664-5p binding site, the viral rna is safe until srsf5 is available to bind and compete with mir-3664-5p-risc complex. however, as evident from our 3'-utr analysis, a significant proportion of viral sequence had 'g' to 't' change at position 29742. while srsf5 binding site was still retained, the mir-3664-5p site creation did not take place due to this change. the fourth scenario was found to be very different from what have been mentioned so far. we identified that change of 'g' to 'c' at position 29734 creates a binding site for mir-4701-3p, but no rbp with a overlapping target sequence could be identified (fig 4d) . this means that there is probably no host factor to protect viral rna from mir-4701-3p mediated the phenomenon of viral rna utilizing host stabilizing factors and escaping rna decay machinery is well-established. but, nothing is known so far for sars-cov-2. as mentioned before, the actual incident what is taking place after viral infection largely depends on the expression status of the interacting rbps and mirnas in particular host tissue. since, lungs is the primary site of infection for sars-cov-2, we wanted to find out what is the nature of expression of these molecules in normal lungs tissue. in the first approach we tested their expression in 'the protein atlas' portal (fig 5a and 5b ) and in the second approach we used the tcga dataset for lung adenocarcinoma (luad) from gepia and compared the expression of the selected rbps in adjacent normal lung tissue (fig 5c and 5d ). to our excitement, we found a decent to huge expression of all the rbps in normal lung tissues, clearly indicating their availability to protect viral rna from host decay machinery. as the viral infection will follow a huge inflammatory condition in the lungs and by the time we know that in severe cases this prolonged inflammation leads to pulmonary fibrosis, we also wanted to check whether there are supporting evidences relating the expression of these rbps and mirnas to inflammation or more specific to pulmonary inflammation. as shown in table 4 , almost all the rbps under investigation were either reported to have induced by inflammation or responsible for fibrosis in lungs or other organ. expression of some of them was also reported to be modulated by viral infection. this finding supports our hypothesis and indicates that from the stages of early viral infection to severe advanced conditions sars-cov-2 could utilize the host stabilization factors for its own benefit. following the same tune, we looked at the mirnas also in greater details and several interesting observations were noted. apart from mir-3664-5p, other three mirnas were detectable in lungs tissue and even were closely associated with pulmonary inflammation. in case of mir-3664-5p also, its involvement in breast cancer tumorigenesis and associated inflammation was quite supportive. most of these supporting studies mentioned here for both rbps and mirnas are actually functional interrogations involving rigorous experimental procedures. hence, we are quite confident that that our predictions got validated by experimental reports asking the similar type of questions in a similar set up. it has been more than six months we are experiencing one of the biggest pandemics of the world. investigation of the viral subtypes across the globe has identified several variants in the coding region of the viral rna possibly resulting in key structural or functional changes in spike protein, nucleocapsid proteins or the rna dependent rna polymerase of the virus. as compared to the coding region variants, there have not been so many changes identified in the 5' or 3' untranslated regions of the viral genome. this made us interested about these regions and first we wanted to carry out a systematic exploration of the prevalent changes present in the region using more than 8500 viral sequences isolated from all the continents and explore their significance with respect to host rna decay machinery as well. in spite of some specific changes concentrated in some specific regions of the world, overall pattern of the 5' and 3'-utr variants pinpoint on few predominant ones. the most important of it was at position 241, where we see a clear change of distribution of reference to variant nucleotide from china to south-east asia to europe and americas. this change has also been implicated to higher mortality and/ or infectivity of the virus [16] . for the first time, we report the likelihood of tardbp binding to this region. tardbp has been known to promote translation and rna stability and it has also been shown to play important role in viral infection [42, 43] . therefore, promotion of translation of sars-cov-2 viral proteins by tardbp fits well with possible selection of 't' base over time and its correlation to infectivity. the other rna binding proteins identified in the study to be interacting with viral 5' and 3'-utr also has reported evidences of functioning as rna stabilizing factor or facilitator of translation. cug-bp has been shown to interact with eif2a [44] and promote translation in selected targets [45] . rbms3 is a very well-characterized rna stabilizing factor and known to increase half-life of target mrnas as well as increase protein expression in many instances [29] . similarly, hnrnpa1 is also a rbp known for its role in promoting mrna stability after binding to 3'-utr specific target sites and as mentioned before, this rbp has been experimentally identified to bind to sars-cov 3'-utr [46, 47] . srsf5, although primarily involved in splicing, has been attributed to functions like maintaining mrna stability and translation [48, 49] . the most significant aspect of this study was to identify mirna binding site in the viral 3'-utr and deciphering the cross-talk between rbps and mirnas along with the nucleotide variations at specific sites. our results elaborated five distinct types of such interactions, as summarized in fig 6. we have seen cug-bp binding site is retained and tardbp binding site is created upon change in viral nucleotide sequences at target positions of 5'-utr, providing a continued stability and translational advantage to the virus (shown in arm:a). on the other hand, there exists multiple possibilities in case of 3'-utr, where we first see direct competition over access to target sites between rbms3 and mir-34b-5p (arm:b). lung inflammation causes induction of mir-34b-5p [37] which could be a step to attenuate viral infection. however, the sustained expression of rbms3 in lungs tissue probably acts to prevent mir-34b-5p action after binding to the viral rna at overlapping site. the effect of variation in viral sequence could either disrupt or create mirna binding site, keeping the rbp binding site intact. this result in non-functional mirna site in one case (arm:c) and competition between rbp and mirna in the other (arm: d). expression of both hnrnpa1 and srsf5 is high in lungs, further supporting their possible protective effect on viral genome. the incidence described in arm: e is different as it creates a mirna binding site without an rbp protection. we have seen that expression of mir-4701-3p is less in lungs, provoking the thought that despite having its site created, there might not be enough mirna to act on the viral rna at this site. thereby, we have characterized sars-cov-2 5' and 3'-utr sequences for existing variations in these regions prevailing at major countries of infection spread over all the continents. we further identified interactions between host rbps like cug-bp, tardbp, rbms3, hnrnpa1 and srsf5 with host mirnas mir-34b-5p, mir-9-5p, mir-3664-5p and mir-4701-3p and showed how the interactions changed along with sequence variations at specific positions of untranslated regions of viral genome. our findings elaborate complex relationship between host rna stabilization/ decay factors and sars-cov-2 rna genome highlighting how the virus could manipulate host machinery. the knowledge could also be used to develop antiviral compounds following further experimental studies. supporting information s1 table. list of variations in 5' and 3'-utr of sars-cov-2 across six continents of the world. (xlsx) severe acute respiratory syndrome coronavirus 2 (sars-cov-2): an overview of viral structure and host response. diabetes & metabolic syndrome specific mutations in sars-cov2 rna dependent rna polymerase and helicase alter protein structure, dynamics and thus function: effect on viral rna replication phylogenetic clustering of the indian sars-cov-2 genomes reveals the presence of distinct clades of viral haplotypes among states phylogenetic network analysis of sars-cov-2 genomes coronavirus subgenomic minus-strand rnas and the potential for mrna replicons a phylogenetically conserved hairpin-type 3' untranslated region pseudoknot functions in coronavirus rna replication structural and functional conservation of cis-acting rna elements in coronavirus 5'-terminal genome regions binding of the 5'-untranslated region of coronavirus rna to zinc finger cchc-type and rna-binding motif 1 enhances viral replication and transcription mutations in sars-cov-2 viral rna identified in eastern india: possible implications for the ongoing outbreak in india and impact on viral structure and host susceptibility competing interactions of rna-binding proteins, micro-rnas, and their targets control neuronal development and function rbpmap: a web server for mapping binding sites of rna-binding proteins a pattern-based method for the identification of microrna binding sites and their corresponding heteroduplexes from microrna sequences to function gepia: a web server for cancer and normal gene expression profiling and interactive analyses genotyping coronavirus sars-cov-2: methods and implications genome-wide mapping of therapeutically-relevant sars-cov-2 rna structures de novo 3d models of sars-cov-2 rna elements and small-molecule-binding rnas to guide drug discovery viruses: overturning rna turnover the crystal structure of tdp-43 rrm1-dna complex reveals the specific recognition for ug-and tg-rich nucleic acids cugbp1 promotes cell proliferation and suppresses apoptosis via down-regulating c/ebpalpha in human non-small cell lung cancers mir-574-5p as rna decoy for cugbp1 stimulates human lung tumor growth by mpges-1 induction overexpression of cugbp1 is associated with the progression of nonsmall cell lung cancer cug-binding protein 1 regulates hsc activation and liver fibrogenesis exercise, skeletal muscle and inflammation: are-binding proteins as key regulators in inflammatory and adaptive networks inflammation induces tdp-43 mislocalization and aggregation cytoplasmic translocation, aggregation, and cleavage of tdp-43 by enteroviral proteases modulate viral pathogenesis. cell death and differentiation rbms3 is a tumor suppressor gene that acts as a favorable prognostic marker in lung squamous cell carcinoma rna-binding protein rbms3 is expressed in activated hepatic stellate cells and liver fibrosis and increases expression of transcription factor prx1 knockdown of hnrnpa1 inhibits lung adenocarcinoma cell proliferation through cell cycle arrest at g0/g1 phase prognostic value of heterogeneous ribonucleoprotein a1 expression and inflammatory indicators for patients with surgically resected hepatocellular carcinoma: perspectives from a high occurrence area of hepatocellular carcinoma in china viral modulation of cellular rna alternative splicing: a new key player in virus-host interactions? wiley interdisciplinary reviews rna global profiling of the cellular alternative rna splicing landscape during virus-host interactions trpv4-dependent induction of a novel mammalian cold-inducible protein srsf5 as well as cirp and rbm3. scientific reports srsf5: a novel marker for small-cell lung cancer and pleural metastatic cancer mutually exclusive acetylation and ubiquitylation of the splicing factor srsf5 control tumor growth mir-34b-5p inhibition attenuates lung inflammation and apoptosis in an lps-induced acute lung injury mouse model by targeting progranulin mir-34b-5p knockdown attenuates bleomycin-induced pulmonary fibrosis by targeting tissue inhibitor of metalloproteinase 3 (timp3) mir-9-5p promotes cell growth and metastasis in non-small cell lung cancer through the repression of tgfbr2 mir36645p suppresses the proliferation and metastasis of gastric cancer by attenuating the nfkappab signaling pathway through targeting mtdh. international journal of oncology analysis of differential expression profile of mirna in peripheral blood of patients with lung cancer tdp-43 enhances translation of specific mrnas linked to neurodegenerative disease unconventional rna-binding proteins step into the virus-host battlefront rna cug-binding protein 1 increases translation of 20-kda isoform of ccaat/enhancer-binding protein beta by interacting with the alpha and beta subunits of eukaryotic initiation translation factor 2. the journal of biological chemistry the importance of celf control: molecular and biological roles of the cug-bp, elav-like family of rna-binding proteins viral and cellular proteins involved in coronavirus replication. current topics in microbiology and immunology heterogeneous nuclear ribonucleoprotein a1 regulates rna synthesis of a cytoplasmic virus proteasome-mediated proteolysis of srsf5 splicing factor intriguingly co-occurs with srsf5 mrna upregulation during late erythroid differentiation regulation of mcl-1 by srsf1 and srsf5 in cancer cells we acknowledge the support and advice of prof. saumitra das, director, nibmg and time to time suggestions from dr. bornali bhattacharjee and dr. bhaswati pandit, nibmg. conceptualization: srikanta goswami. cug-bp upregulated in lung cancer/ inflammation [21] cugbp1 stimulates human lung tumor growth [22] overexpression of cugbp1 is associated with the progression of non-small cell lung cancer [23] cug-binding protein 1 regulates hsc activation and liver fibrogenesis [24] exercise, skeletal muscle and inflammation: are-binding proteins as key regulators in inflammatory and adaptive networks [25] 2. tardbp upregulated in inflammation [26] facilitation of viral pathogenesis [27] 3. rbms3 rbms3 is a novel tumour suppressor in lungs squamous cell carcinoma, and its downregulation facilitates development and progression of lscc.[28]rbms3 is upregulated by chronic inflammation and fibrosis [29] 4. hnrnpa1 knockdown of hnrnpa1 inhibits lung adenocarcinoma cell proliferation through cell cycle arrest at g0/g1 phase [30] upregulated in inflammation [31] sars-cov rna synthesis and turnover regulation [9] 5. srsf5 expression upregulated by viral infection [32] upregulated by host-viral interaction [33] upregulated by intracellular stress [34] upregulated in lung inflammation/ cancer [35, 36] mirnas 1. mir-34b-5p upregulated in mouse model of lungs inflammation and fibrosis. [37] upregulated in inflammation [38] 2. mie-9-5p high expression level in normal lungs tissue. further overexpressed in nsclc/ lung inflammation [39] 3. mir-3664-5pno report of detectable expression in normal lungs tissue or in pathogenic condition involved in inflammation/ tumorigenesis associated with breast cancer [40] 4. mir-4701-3pupregulated in luad/ lung inflammation and very less amount of expression in normal lungs tissue [41] https://doi.org/10.1371/journal.pone.0237559.t004 key: cord-289873-6hivjqof authors: lu, rui; huang, tianhui; hu, haiqing; liu, xiao-ping title: patients with mild and general covid-19 should be negative for at least 3 consecutive nucleic acid tests before discharged date: 2020-10-02 journal: plos one doi: 10.1371/journal.pone.0240081 sha: doc_id: 289873 cord_uid: 6hivjqof given the global spread of coronavirus disease (covid-19), strict discharge standard is of great significance for the prevention and control of the epidemic, thus, the purpose of this study is to formulate more strict and scientific discharge standards. a total of 845 patients with mild and general covid-19 who were considered to be discharged from hospital were included in this study. the median time from the onset of covid-19 to the occurrence of two consecutive negative nucleic acid tests of these patients was 21 days. 223 of the 845 patients were tested again after two consecutive negative nucleic acid tests and 17.49% of the patients were positive. moreover, 82.51% (184 of 223) of these patients experienced negative results from three consecutive nucleic acid tests, the median time from the onset of covid-19 to the occurrence of three consecutive negative nucleic acid tests was 23 days (range: 3–56 days), and 38 of which were further tested after three consecutive negative nucleic acid tests, while about 5.26% (2 of 38) patients showed positive nucleic acid test results. thus, we suggested that the patient should be negative for at least 3 consecutive nucleic acid tests before discharge, and the test time should be no earlier than the 23rd day since the onset of the disease. since december 2019, a new coronavirus disease (covid-19) epidemic has occurred in wuhan, hubei province, china. with the spread of the epidemic, covid-19 has appeared in various provincial administrative units and special administrative regions in china. in addition, covid-19 has occurred in many countries around the world, leading to the world health organization (who) made the assessment that covid-19 could be characterized as a pandemic [1] . through a series of preventive control and medical treatment measures across the country, the rising trend of the epidemic in china has been effectively contained. as of 24:00 on april 8, 2020, there were 81,865 confirmed diagnoses in china, and a total of 77,370 patients were discharged from the hospital. the number of new cases per day has fallen below 100 [2] . according to the latest covid-19 clinical diagnosis and treatment guideline [3] , patients who meet the following four criteria can be discharged: (1) the body temperature has returned to normal for more than 3 days; (2) the respiratory symptoms have improved significantly; (3) the pulmonary imaging examination shows that the acute exudative lesions have improved significantly, (4) two consecutive negative nucleic acid test for respiratory specimens such as sputum and nasopharyngeal swabs (sampling interval at least 24 hours). however, based on our clinical observations and experience, some covid-19 patients were tested again for covid-19 nucleic acid when they met the above four discharge criteria, and the results still had a certain positive rate. the clinical observations of this research team are reported as follows. this retrospective study investigated mild and moderate covid-19 patients admitted to the wuchang cabin hospital (wuhan, china) from february 6, 2020 to march 9, 2020. wuchang cabin hospital is a hospital temporarily reconstructed from hongshan gymnasium to solve the "one bed is difficult to find" wuhan crisis. it opened the earliest and shut down latest in wuhan. patients met the following inclusion criteria were included in this study: (1) patients with mild and general covid-19 [3] ; (2) patients with complete self-care ability; (3) patients diagnosed with covid-19 through nucleic acid test. meanwhile, patients who met the following exclusion criteria were excluded: (1) patients with severe diseases of severe respiratory system, cardiovascular and cerebrovascular systems; (2) patients with mental illness or cognitive impairment; (3) patients with a positive influenza virus test; (4) patients who need to be transferred due to worsening condition during hospitalization. all patients received unified antiviral (arbidol, oseltamivir, ribavirin), antibiotic (moxifloxacin), traditional chinese medicine (lianhuaqingwen capsule) and immunomodulatory (interferon, thymopolypeptides) treatment. after the patient met the first three of the above discharge criteria, we began to perform continuous sars-cov-2 nucleic acid testing on the patient using rt-pcr. nucleic acid kits were provided by wuhan covid-19 control command. the interval from the onset of covid-19 to two consecutive negative results of nucleic acid test was recorded, as well as the probabilities of recurrence of a positive nucleic acid test after two and three consecutive negatives. the data of onset of symptoms, admission date, nucleic acid testing date, and discharge date were collected from electronic medical records. the flow chart depicting the composition of patients at different stages was presented in fig 1. description analysis was conducted to reveal the demographic characteristics (gender and age) using the r package "table1" (https://cran.rstudio.com/web/packages/table1/). chi-square test and t test were used to determine the differences gender and age distributions between different groups, respectively. this study was approved by the institutional review board of the wuhan third hospital and the need for informed consent was waived. a total of 845 patients were included in this study, including 369 (43.7%) males and 476 (56.3%) females. the median age of the 845 covid-19 patients was 49 years (range: 9-82 years). the dates of onset of symptoms of these patients were ranging from december 31, 2019 to february 24, 2020. the admission dates of these patients were ranging from february 6, 2020 to february 26, 2020. the dates of nucleic tests were ranging from january 15, 2020 to march 9, 2020. the discharge dates were ranging from february 13, 2020 to march 10, 2020. the median time from the onset of covid-19 to the occurrence of two consecutive negative nucleic acid tests in 845 patients with covid-19 was 21 days (range: 1-56 days). the median interval between the two nucleic acid tests was 2 days (range: 1-33 days). in the total 845 covid-19 patients, 223 patients were tested again after two consecutive negative nucleic acid tests, the characteristics of the 233 patients were summarized in table 1 . the median interval between the two consecutive nucleic acid tests and the further nucleic acid test was 3 days (range: 1-14 days). the positive probability of retesting after two consecutive negative nucleic acid tests was 17.49%. next, we tried to investigate the positive probability of retesting after three consecutive negative nucleic acid tests. a total of 184 patients experienced negative results from three consecutive nucleic acid tests, the median time from the onset of covid-19 to the occurrence of three consecutive negative nucleic acid tests was 23 days (range: 3-56 days), and 38 of which were further tested after three consecutive negative nucleic acid tests, while about 5.26% (2 of 38) patients showed positive nucleic acid test results. as mentioned above, the outbreak of covid-19 is showing a continuous improvement in china. nearly a thousand patients are discharged from hospital every day, but in some countries outside china, there is an outbreak trend. at the same time, china also faces the risk of overseas covid-19 import. as we know, covid-19 is a highly contagious infectious disease, thus, strictly controlling the discharge criteria of patients is of great significance to the prevention of community rebound and spread of the disease. there is a shortage of nucleic acid testing reagents in some countries. therefore, scientific and reasonable nucleic acid testing time and intervals are of great significance for patients diagnosed with covid-19 to receive timely treatment, early isolation and control of the source of infection, to which avoid further spread of the epidemic. this study showed that even if the patient meets the discharge standard of body temperature, respiratory symptoms, imaging findings, and after two consecutive negative nucleic acid tests, 17.49% of patients were positive for nucleic acid tests (that is, the coronavirus had not been completely eliminated in the patient), consistent with previous reports [4] . thus, it could not exclude the possibility that covid-19 is a disease similar to the hepatitis b whose virus cannot be completely cleared in the body, which was worth our vigilance [5] . meanwhile, if covid-19 patients who met other discharge criteria were tested for 3 consecutive nucleic acids (median test time was 23 days), the positive rate of nucleic acid test after three nucleic acid tests might be 5.26%. therefore, we suggest that 3 consecutive nucleic acid tests should be given to patients when they are considered to be discharged. the test should be performed no earlier than the 23rd day after the onset of symptoms and the clinical performance has improved significantly after formal treatment. this study is a retrospective study, so it is inevitable that there are certain defects. first of all, there were a number of patients in this study who did not receive further nucleic acid tests after 2 and 3 consecutive negative nucleic acid tests. this may be due to the insufficient understanding of the phenomenon of positive nucleic acid test after 2 consecutive negative nucleic acid tests in the early stage of the epidemic. the lack of patient data in this section directly reduced our sample size to assess the probability of the nucleic acid test returning positive after two consecutive negative nucleic acid tests. in this regard, we will further improve the follow-up of patients, and their outcomes have been clarified. in summary, we recommend that when the patient is scheduled to be discharged from hospital, the patient should be negative for at least 3 consecutive nucleic acid tests, and the test time should be no earlier than the 23rd day since the onset of the disease. data curation: tianhui huang. formal analysis: xiao-ping liu. writing -review & editing: haiqing hu, xiao-ping liu. director-general's opening remarks at the media briefing on covid-19 update on the outbreak of new coronavirus pneumonia as of 24:00 on april 8 national health commission of the people's republic of china, national administration of traditional chinese medicine. coronavirus disease 2019 (covid-19) diagnosis and treatment guideline (trial version 7) positive rt-pcr test results in patients recovered from covid-19 hepatitis b virus infection key: cord-278087-0nicp0eq authors: garcía-garcía, maría luz; calvo, cristina; rey, cristina; díaz, beatriz; molinero, maria del mar; pozo, francisco; casas, inmaculada title: human metapnuemovirus infections in hospitalized children and comparison with other respiratory viruses. 2005-2014 prospective study date: 2017-03-16 journal: plos one doi: 10.1371/journal.pone.0173504 sha: doc_id: 278087 cord_uid: 0nicp0eq background: human metapneumovirus (hmpv) has an important etiological role in acute lower respiratory infections in children under five years. our objectives were to estimate the relative contribution of hmpv to hospitalization in children with acute respiratory infection, to define the clinical and epidemiological features of hmpv single and multiple infections, and to compare hmpv infections with respiratory syncytial virus (hrsv), rhinovirus (hrv), adenovirus and human bocavirus infections in the same population. methods and findings: a prospective study performed on all children less than 14 years of age with a respiratory tract disease admitted to a secondary hospital between september 2005june 2014. clinical characteristics of patients were analyzed. nasopharyngeal aspirate was taken at admission for viral study with polymerase chain reaction for 16 respiratory viruses. a total of 3,906 children were included. at least one respiratory virus was detected in 75.2% of them. the most common identified virus was hrsv, followed by hrv. hmpv was detected in 214 cases (5.5%); 133 (62%) were single infections and the remaining were detected in coinfection with other viruses. 90.7% cases were detected between february and may. children’s mean age was 13.83 ± 18 months. fever was frequent (69%), and bronchiolitis (27%), and recurrent wheezing (63%) were the main clinical diagnosis. hypoxia was present in 65% of the patients and 47% of them had an infiltrate in x-ray. only 6 (2.8%) children were admitted to the intensive care unit. only the duration of the hospitalization was different, being longer in the coinfections group (p <0.05). there were many differences in seasonality and clinical characteristics between hmpv and other respiratory viruses being more similar to hrsv. conclusions: hmpv infections accounted for 5.5% of total viral infections in hospitalized children. the clinical characteristics were similar to hrsv infections, but seasonality and clinical data were different from other viral infections. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 human metapneumovirus (hmpv), described in the netherlands in 2001 [1] is an rna virus belonging to the pneumoviridae family, genus metapneumovirus [2] . two main genetic lineages a and b have been identified to date. the phylogenetic studies showed a high similarity to the respiratory syncytial virus (hrsv), with which it shares morphological and disease spectrum similarities [3] . upper and lower respiratory tract infections from common colds to pneumonia have been attributed to hmpv, with bronchiolitis being one of the main clinical signs of primary infection in hospitalized patients [4] . a recent meta-analysis has provided evidence that hmpv has an important etiological role in acute lower respiratory infections in children less than five years [5] . our objectives were to estimate the relative contribution of hmpv to hospitalization in children with acute respiratory tract infection in spain and to define the clinical and epidemiological features of hmpv single and multiple infections. also we compared hmpv infections with hrsv infections and with other common respiratory viruses over an extended period. the study was approved by the medical ethics committee of the instituto de salud carlos iii. informed written consent was obtained from parents or legal guardians. the study population comprised all children between the first month of life and 14 years of age with a respiratory tract disease admitted to the secondary public hospital severo ochoa (leganés, madrid), between september 2005 and june 2014 which corresponded to nine consecutive seasons. all patients were evaluated by an attending physician. clinical characteristics of patients were analyzed. during the hospital stay, and as part of the study, a physician filled out a study questionnaire with the clinical data. upper respiratory tract infection (urti) was diagnosed in patients with rhinorrhea and/or cough and no signs of wheezing, dyspnea, crackles or bronchodilator use, with or without fever. acute expiratory wheezing was considered to be bronchiolitis when it occurred for the first time in children aged less than 2 years following the mcconnockie classical criteria [6] . all other episodes of acute expiratory wheezing were considered to be recurrent wheezing [7] . asthma was diagnosed by the national asthma education and prevention program guidelines [8] . laryngotracheobronchitis was associated with inspiratory dyspnea and wheezing. laryngitis was related to inspiratory dyspnea without wheezing. cases with both focal infiltrates and consolidation in chest x-rays were, in the absence of wheezing, classified as pneumonia. clinical specimens consisted of a nasopharyngeal aspirate (npa) taken from each patient at admission. all clinical specimens were sent for virological investigation to the respiratory virus and influenza unit at the national microbiology center (isciii, madrid, spain), npas were processed within 24 hours after collection. upon receipt, three aliquots were prepared and stored at -70˚c. rna and dna from 200 μl-aliquots of npa were extracted by using the qiaamp mini elute virus spin kit in an automated extractor (qiacube, qiagen, valencia, ca). from 2005 to 2010, three conventional multiplex rt-nested-pcr assays were performed to detect a total of sixteen respiratory viruses [9, 10, 11] . from 2011 to 2014, detection of hmpv and the other respiratory virus were performed by real time multiplex rt-pcr assays, not published yet, but based on the same equivalent conventional methods (9, 10, 11) . values were expressed as percentages for discrete variables, or as mean and standard deviation for continuous variables. clinical characteristics of patients with single infections associated to hmpv were compared with those associated with coinfections of hmpv with other respiratory viruses. single hmpv infections were also compared with single infections by hrsv, human rhinovirus (hrv), adenovirus (hadv) and human bocavirus (hbov). clinical characteristics and laboratory variables were compared using the student t-test, the mann-whitney u test, the chi-2 test, and fisher's exact test. a two-sided value of p < 0.05 was considered statistically significant. results were adjusted for age. all analyses were performed using the statistical package for the social sciences (spss), version 21.0. a total of 3906 children under 14 years of age, admitted to the severo ochoa hospital with acute respiratory tract infections were included. the mean age was 21.68 ± 33.8 months. at least one respiratory virus was detected in 75.2% of them. the most common identified virus was hrsv, followed by hrv, and hadv (table 1) . hmpv was detected in 214 cases (5.5%). out of 214 children with an infection associated to hmpv; 133 (62%) were single infections, and the remaining 81 were detected in coinfection with other respiratory viruses (38%). the most frequent coinfections were with adenovirus and rhinovirus (fig 1) . monthly distribution of hmpv infections is shown in fig 2, being 90.7% between february and may. clinical data of hmpv infections are shown in table 2 . children's mean age was 13.83 ± 18 months, 58% (125/214) were males, and 15.5% were preterm infants. fever was frequent (69%; 148/214), and bronchiolitis (27%; 47/214), and recurrent wheezing (63%; 108/214) were the main clinical diagnosis. hypoxia was present in 65% (140/214) of the patients; with a mean duration of 2.9 ± 2 days and 47% (82/214) of them had an infiltrate in x-ray. the length of the stay was 4.4 ± 2.3 days. only 6 (2.8%) children were admitted to the intensive care unit (icu). infants less than 6 months of age have less proportion and duration of fever, but longer duration of hypoxia and stay. diagnosis of bronchiolitis was more frequent in this group (table 3) . single infections were compared with coinfections of hmpv with other viruses (table 2) , and only duration of the hospitalization was different, being longer in the coinfections group (p <0.05). single hmpv infections (n = 133) were selected and compared with single infections of the most frequent viruses detected in the same period; hrsv (n = 766), rhinovirus (n = 651), adenovirus (n = 355) and hbov (n = 84). hmpv vs hrsv. clinical data of single infections associated to hrsv and the comparison with hmpv single infections are shown in table 4 . hrsv was detected in younger infants (p = 0.002), who had hypoxia (p = 0.04) and bronchiolitis diagnosed more frequently than hmpv group (p < 0.001). recurrent wheezing (p< 0.001) and antibiotic treatment (p = 0.05) were more frequent in hmpv group. hmpv vs hrv. clinical data of single hrv infections and the comparison with hmpv single ones are shown in table 5 . children with hmpv infections are younger than hrv group (p< 0.001), and had more frequent fever (p<0.001) and hypoxia (p = 0.02); longer duration of hypoxia (p = 0.001) and hospitalization (p<0.001). a higher level of c-reactive protein was found in hrv group (p = 0.009). clinical data of single hadv infections and the comparison with hmpv infections are shown in table 6 . again, the hmpv group was a younger than the hadv group (p <0.001), had more frequent hypoxia (p = 0.07) and longer duration of fever (p< 0.001). prematurity was also more frequent (p = 0.06). hadv group was diagnosed with pneumonia and laryngitis more frequently and with recurrent wheezing or asthma (p< 0.001) less commonly. a higher value of leucocytes (p = 0.001) and c-reactive protein (p = 0.59) were found in hadv infected children. hmpv vs hbov. clinical data of single hbov infections and the comparison with hmpv group are shown in table 7 . children with hmpv infections were younger (p < 0.001) and bronchiolitis was more frequent (p = 0.002), with longer hospitalization (almost significant; p = 0.08). pneumonia was more common in the hbov group as well as the antibiotic treatment (p = 0.013). leucocytes (p = 0.02) and c-reactive protein (p = 0.04) in blood were higher in hbov infected children. monthly distribution of hmpv was significantly different (p< 0.001) from all other analyzed respiratory viruses. comparison of monthly circulation is shown in fig 2. in relation to the percentage of annual infections, it was variable with a minimum of 2.3% of cases detected in 2006 and a maximum of 19.9% in 2009. according to this large and both long series, i.e. nine consecutive epidemic seasons, of respiratory infections in hospitalized children, hmpv had an important role in infants and was associated with 5.5% of admissions. up to 38% was detected in coinfection with other viruses, and had a typical seasonal distribution being mainly in spring. recurrent wheezing was the most common clinical diagnosis, usually associated with fever and hypoxia. however, infants less than 6 months had less fever, and were usually diagnosed of bronchiolitis. clinical and epidemiological data were significantly different between single hmpv infections and other respiratory viral infections. the burden of hmpv infections in hospitalized children has been confirmed in other countries around the world [12] . in jordanian children [13] of less than 2 years, hmpv was associated with 8.6% of cases, slightly higher than ours, probably because only young children were included. in germany [14] , 11.9% of hospitalized children with bronchitis, pneumonia or pharyngitis were positive for hmpv, but the study was not performed throughout the year, but only during the flu season (weeks 41 to 18). in argentina [15] , the proportion of hmpv infections in hospitalized children ranged from 18% in those less than 6 months of age and 5% in children under 5 years, which is very similar to our data. when children up to 15 year old were included in the u.s by hahn et al [16] , hmpv infections were 3% of respiratory infections in hospitalized children. the proportion of coinfections with other respiratory viruses of up to 38% in our hospital, was higher than in three states of the u.s. [17] where they found 21% of multiple infections, but they did not test as many respiratory viruses as we did (only hrsv, influenza, and parainfluenza virus). nevertheless, in jordan, schuster et al [13] found up to 53% of coinfections, and rhinovirus and adenovirus were frequently detected in coinfections, as in our country, but they also encountered a substantial number of coinfections with hrsv, being clearly higher than in spain. this has also been described in other studies, such as semple [18] in the united kingdom, where coinfections between hmpv and hrsv were frequent and severe. however, in california [19] , as in our study, only 1% of patients had coinfections between hmpv and hrsv. probably, the seasonal circulation of hmpv is different among countries or geographical areas and allows that mixed infections were more or less frequent. hence, in jordan, hmpv was detected in winter and spring, and they were partly coincident with the circulation of hrsv. however, in spain, circulation of both viruses was significantly different making coinfections unlikely. february, march, and april were the peak months of hmpv circulation in our country. clinical data associated with hospitalization due to hmpv was similar to other large studies. in a prospective, population-based surveillance study in the united states [17] , with more than 600 cases, pneumonia (50%), bronchiolitis (22%) and asthma (14%) were the most common diagnosis. in our series, 47% of children had an infiltrate in x-ray, although most of them were diagnosed with recurrent wheezing based on our diagnostic criteria. up to 53% of children in the us study, and 65% of our patients needed oxygen during the admission. in jordanian children [13] , clinical diagnoses were similar to ours, with bronchopneumonia and bronchiolitis being more frequent. prematurity is a well-known risk factor for hospitalization and severity in respiratory viral infections and has also been described in hmpv infections [16, 20] . we also found an important proportion of infants with a history of prematurity (15%) in our series. no other significant proportion of underlying conditions were identified in our hospital, probably because it is a secondary center and the majority of attending children were previously healthy. in our series, clinical differences have been found between hmpv infections and other respiratory viruses infections. the comparison with hrsv is relatively frequent in the literature. as in our results, clinical data of both virus infections are very similar. the global burden of hmpv infections is less than hrsv. in guatemala [21] , hmpv was less prevalent than hrsv (3% vs 41%) in hospitalized children, and hmpv infections were detected in older infants and with less severity. in egypt [22] the proportion was 4% and 46%, clinical differences wer not found. in our study, the proportion was 5.5% for hmpv and 30.8% for hrsv. the older age of infants with hmpv is consistent in different studies [19, 22, 23] and also in our series. hmpv patients had less frequent hypoxia than hrsv ones, the duration of the hospitalization was shorter and the diagnosis of recurrent wheezing was more frequent. in addition, pneumonia was more common in the hmpv group. nevertheless, as far as we know, no other authors have specifically compared hmpv infections with other viruses. we compared single hmpv infections with single hrv, single hadv, and single hbov cases. the hmpv children were the youngest amongst all of them. hypoxia and duration of the hospitalization are usually more frequent in hmpv group than in other respiratory viral infections, probably in relation to the lower age of the infants. pneumonia, a higher rate of leukocytes and c-reactive protein were more frequent in hadv or hbov. recurrent wheezing was more common in hmpv patients. seasonality was also different because of the characteristic and singular circulation of hmpv with the highest incidence being in march and april. in summary, hmpv infections accounted for 5.5% of total viral infections in hospitalized children. the clinical characteristics were similar to hrsv infections, but children were younger, and recurrent wheezing was more common. seasonality and clinical data are different from other viral infections such as hrv, hadv or hbov that affect older children with a higher proportion of pneumonia. hmpv infections have a significant burden of disease, and the development of vaccines could prevent a substantial number of hospitalizations. a newly discovered human pneumovirus isolated from young children with respiratory tract disease taxonomy of the order mononegavirales: update 2016 analysis of the genomic sequence of a human metapneumovirus presence of the new human metapneumovirus in french children with bronchiolitis aetiological role of common respiratory viruses in acute lower respiratory infections in children under five years: a systematic review and meta-analysis what's in the name? role of emerging respiratory virus in severe acute episodes of wheezing in children expert panel report: guidelines for the diagnosis and management of asthma update on selected topics-2002 simultaneous detection of influenza a, b, and c viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-pcr assay simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays pérez-breña p.two rt-pcr based assays to detect human metapneumovirus in nasoharyngeal aspirates prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients human metapneumovirus infection in jordanian children. epidemiology and risk factors for severe disease human metapneumovirus: insights from a ten-year molecular and epidemiological analysis in germany incidence of viral respiratory infections in a prospective cohort of outpatient and hospitalized children aged 5 years and its associated cost in human metapneumovirus infections are associated with severe morbidity in hospitalized children of all ages burden of human metapneumovirus infection in young children dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis prevalence and characteristics of human metapneumovirus infection among hospitalized children at high risk for severe lower respiratory tract infection human metapneumovirus infection is associated with severe respiratory disease in preschool children with comparative epidemiology of human metapneumovirus and respiratory syncytial virus-associated hospitalizations in guatemala. influenza other respir viruses respiratory syncytial virus and human metapneumovirus in severe lower respiratory tract infections in children under two comparison of risk factors for human metapneumovirus and respiratory syncytial virus disease severity in young children key: cord-301811-ykpiorgo authors: tanaka, takuma; yamaguchi, takayuki; sakamoto, yohei title: estimation of the percentages of undiagnosed patients of the novel coronavirus (sars-cov-2) infection in hokkaido, japan by using birth-death process with recursive full tracing date: 2020-10-28 journal: plos one doi: 10.1371/journal.pone.0241170 sha: doc_id: 301811 cord_uid: ykpiorgo estimating the percentages of undiagnosed and asymptomatic patients is essential for controlling the outbreak of sars-cov-2, and for assessing any strategy for controlling the disease. in this paper, we propose a novel analysis based on the birth-death process with recursive full tracing. we estimated the numbers of undiagnosed symptomatic patients and the lower bound of the number of total infected individuals per diagnosed patient before and after the declaration of the state of emergency in hokkaido, japan. the median of the estimated number of undiagnosed symptomatic patients per diagnosed patient decreased from 1.7 to 0.77 after the declaration, and the median of the estimated lower bound of the number of total infected individuals per diagnosed patient decreased from 4.2 to 2.4. we will discuss the limitations and possible expansions of the model. the novel coronavirus (sars-cov-2) spread to the most populated areas of the world in the first few months of 2020. in japan, the first case was reported on january 16th, 2020; on march 31st, the number of cases increased to 2122 [1] . in hokkaido, the largest prefecture of japan, the first case was reported on january 28th, 2020 (fig 1) . the hokkaido government declared a state of emergency on february 28th and lifted it on march 19th. the state of emergency was not legally binding, and the government asked the residents to stay home on the weekends. until the state of emergency was lifted, a total of 157 cases were reported. out of 157, two died, and eight have recently traveled abroad. although the number of diagnosed cases is declining towards the end of march, the situation remains uncertain. to effectively control the spread of the infection, we need to know several parameter values characterizing the infection, such as the basic reproduction number, r 0 , the percentage of asymptomatic patients, and the fatality rate. one of the factors that complicate the decision making in disease control is the uncertainty in the percentage of asymptomatic patients. several lines of evidence indicate that the virus can be transmitted by asymptomatic patients [2, 3] , who may have facilitated the spread of sars-cov-2. to make the disease control strategy successful and less harmful to the society, the transmissibility of asymptomatic patients and the percentage of asymptomatic patients should be measured. another complicating factor is the uncertainty in the percentages of diagnosed and undiagnosed patients. most individuals infected with sars-cov-2 suffer from mild cold-like symptoms and recover without any medical intervention. they remain undiagnosed, disturbing how we monitor the number of total infected individuals. thus, estimating the percentages of asymptomatic and undiagnosed patients is a challenging problem to be answered in epidemiology. it would be useful if these values could be estimated by the information available in the early phase of the outbreak. contact tracing is considered to be one of the effective measures, and health officials have conducted contact tracing of infected patients to prevent the spread of the virus infection for outbreaks of new or reemerging infections [4] [5] [6] [7] [8] [9] . if an infected patient is found, health officials try to find other infected people among those who have come into contact with the infected patient. if any other infected patients are found, officials will repeat tracing from the newly found patients; and if not, tracing is stopped. thus, a "cluster" of infected patients connected by the route of infection is constructed through the process of contact tracing. simultaneously, the dates of onset and diagnosis are obtained for each diagnosed patient. as a result, contact tracing can provide detailed qualitative and quantitative information about diagnosed patients from the infectious disease transmission network of diagnosed and undiagnosed patients. analyzing this network with an appropriate model may enable us to estimate the parameter values of the infection. one promising model for contact tracing is a stochastic model based on the birth-death process, which is a formulation of branching processes [10] [11] [12] [13] [14] [15] [16] [17] , because the number of cases in the early phase stochastically fluctuates and the widely used deterministic sir models are inapplicable. in birth-death processes, a sequence of infectious events generates a tree whose nodes are infected patients and edges are infection routes (fig 2) . when a patient recovers ( fig 2, dotted circle) , the corresponding node and its edges are removed from the tree, which is split into two. the infectious disease transmission network is composed of trees. a connected component of the network is referred to as "cluster" in this paper. contact tracing corresponds to finding a node in the tree and removing nodes connected to the first node found (fig 2, gray filled circle). there are various types of contact tracing based on how to choose nodes to be removed, including backward, forward, or full tracing, and recursive or one-step tracing [17] . we consider only recursive full tracing, that is, all nodes directly and indirectly connected to the first found node are removed (fig 2, dashed rounded rectangle). we propose an analysis based on the birth-death process with recursive full tracing that takes advantage of information obtained by contact tracing to estimate epidemiological parameter values with a small set of data. we focus on the contact tracing of infected individuals in hokkaido. the present analysis uses the distributions of the cluster size and patients' time from onset to diagnosis, which are released by the health officials, to estimate the model parameters. our approach directly models the stochastic dynamics, which is an inherent property of the early phase of the outbreak. this paper is organized as follows. in the methods section, we summarize the sars-cov-2 infection in hokkaido and divide it into those before and after the declaration of the state of emergency. we classify patients into symptomatic and asymptomatic, and diagnosed and undiagnosed. we explain what corresponds to diagnosed and undiagnosed patients in the present model. we describe the formulation of the model and the details of the simulations. in the results section, we estimate the parameter values and the number of asymptomatic and undiagnosed patients. in the discussion section, we relate our results with previous studies and discuss the limitations and possible expansions of the model. this paper reports the analysis of the sars-cov-2 infection in hokkaido, japan [18] . in hokkaido, all cases had not traveled abroad recently except for three cases, which include a tourist from wuhan, china. we concentrate our analysis on the cases whose onset was prior to the lifting of the state of emergency. we excluded the cases with an unknown onset and asymptomatic patients from the analysis. if the date of the diagnosis of a case was not reported, it was estimation of the percentages of undiagnosed patients of sars-cov-2 infection in hokkaido, japan assumed to coincide with the date of the announcement. s1 table summarizes the case reports released by the novel coronavirus response headquarters of the hokkaido government until april 2nd, 2020. we represent the patients with nodes and their contacts with edges in a network. if two patients were in close contact with each other, the corresponding nodes are connected by an edge. the network consists of distinct connected components, which we refer to as clusters. sporadic patients are regarded as size-1 clusters. table) . all cases were divided into datasets 1 and 2 according to the cluster they belong to. if the earliest onset of the cases in a cluster was prior to the declaration of the state of emergency, this cluster was included in dataset 1; if the earliest onset was between the declaration and the lifting thereof, it was included in dataset 2. datasets 1 and 2 contain 78 and 30 clusters, respectively. because the declaration of the state of emergency might have changed the behavior of residents and health officials in hokkaido, we compared the data before the declaration, dataset 1, and the data between the declaration and the lifting thereof, dataset 2. table 1 summarizes the datasets. the patients included in datasets 1 and 2 were all diagnosed and mostly symptomatic. however, not all of the individuals infected with sars-cov-2 were diagnosed and symptomatic; they can be classified into diagnosed symptomatic, diagnosed asymptomatic, undiagnosed symptomatic, and undiagnosed asymptomatic groups. the diagnosed symptomatic group consists of those who developed symptoms and were diagnosed, or those who were found in contact tracing. all individuals belonging to this group were covered by our datasets. although the diagnosed asymptomatic group was also included in the datasets, we ignored this group because this group included only two individuals. the undiagnosed symptomatic group is comprised of individuals who were infected and developed symptoms, but recovered or died without being diagnosed. this group is not directly observable, and thus its percentage is one of the parameters we tried to estimate with the model, which takes this group into account. the undiagnosed asymptomatic group is not directly observable either. it has been suggested that a percentage of sars-cov-2 carriers do not develop symptoms but can infect others [2, 19] . we did not explicitly incorporate the asymptomatic group into the model but estimated its percentage. the birth-death processes have been used to model infectious diseases and population dynamics [10, 11, 16, 17] . the birth-death process is a continuous-time markov process in which the state variable increases and decreases by one. the increase (birth) and decrease (death) of the state variable occur at the rates which depend on the state variable. in the case of infectious diseases, the state variable is the number of infected individuals, and the birth and death rates are the infection and recovery rates positively correlated with the state variable. the birth-death process can be regarded as a discretized sis model if the birth and death rates are appropriately set. however, some modifications are needed to model the infection dynamics sars-cov-2 in hokkaido. since only a fraction of infected individuals are diagnosed and found in the contact tracing, we do not know the exact number of infected individuals. we have to estimate the number of undiagnosed individuals by using the information obtained in the contact tracing. the process of diagnosis and contact tracing must be explicitly included in the model to take advantage of information on the contact between infected individuals. we modeled the contact tracing of sars-cov-2 with a variant of the continuous-time birth-death process, referred to as the birth-death process with recursive full tracing [17] . we used an extended model [10, 13] to take into account the effect of the stochastic diagnosis of individuals and the elimination of the cluster from the population. the model consists of a network whose nodes and edges are continually generated and removed (fig 2) . the lifetime of a node is a random variable drawn from the exponential distribution with the scale parameter 1/γ. during its lifetime, a node gives birth to nodes according to a poisson point process with the stationary rate β 0 and is connected to these offspring nodes (fig 2, green circle) . when the lifetime of a node ends, the node and its edges are removed from the network (fig 2, dotted circle) . for the infection dynamics of sars-cov-2, the nodes and their birth and death can be regarded as symptomatic patients and their onset of symptoms or infection and recovery from the disease. in the limit of an infinite number of nodes, the dynamics of the number of nodes of birth-death processes are approximated by the sir model. asymptomatic infected individuals are not included in this model, and the incubation period is ignored. the difference between the model by müller and colleagues [10] and the present model is as follows. first, in the former, contact tracing can not necessarily find all infected individuals in the cluster of a diagnosed patient whereas, in the latter, contact tracing finds all individuals in the cluster. second, the influx of infected individuals is incorporated only in the present model. the birth-death process with recursive full tracing incorporates the diagnosis and quarantine of patients in addition to the features of continuous-time birth-death processes. the time from infection to diagnosis of a node is a random variable drawn from the exponential distribution with the scale parameter 1/κ. if the diagnosis occurs earlier than the recovery, the node is removed from the network at the diagnosis, representing the quarantine of the diagnosed patient (fig 2, gray filled circle) . at the same time, the nodes in the connected component containing the diagnosed node are also removed from the network, which corresponds to the contact tracing of the infected individuals (fig 2, gray open circles) . infections that are to be caused by these removed nodes later than the removal are abolished because the diagnosed individuals are quarantined. the connected component of the diagnosed individuals corresponds to the clusters in the datasets (fig 2, dashed rounded rectangle) . the nodes in the connected component are counted among diagnosed symptomatic groups. the recovery of a node disconnects its neighboring nodes. for example, if the green node in fig 2 is diagnosed, the cluster of size 2 but not of size 4 is reported. a node is not diagnosed if it has been already removed. if a node recovers before it is diagnosed, this node is counted among undiagnosed symptomatic groups. the simulation of the model is implemented as follows. nodes without edges are generated in the network according to a poisson point process with the stationary rate λ = 10 −5 (fig 2, blue circle). the value of λ is inconsequential if it is small enough to allow for observing the cluster size and time from onset to diagnosis distribution at the steady state. on the generation of node i at time t 0 i , its time of recovery t r i and time of diagnosis t d i are assigned as where the recovery rate γ and the diagnosis rate κ are positive constants. during t 0 i � t � min ðt r i ; t d i þ, node i generates new nodes and connects to them according to a poission point process with the stationary rate β 0 > 0. at t ¼ t r i , if node i is present in the network, node i and its edges are removed from the network. at t ¼ t d i , if node i is present in the network, the nodes in the same connected component containing node i and their edges are removed from the network. let us note that β 0 is the rate of infection giving rise to symptomatic patients, not the rate of infection giving rise to symptomatic and asymptomatic patients. this is because all nodes in the model are capable of being diagnosed, which is not the case with asymptomatic individuals. hence, β, the rate of infection giving rise to any type of patient is greater than β 0 . for κ = 0, the probability that a node directly infects n nodes, that is, the probability that a symptomatic patient directly infects n symptomatic patients, follows whose expected value is β 0 /γ. similarly, the basic reproduction number r 0 is given by β/γ. thus, β must be greater than γ because the number of reported cases is steadily increasing, that is, throughout the simulations reported in this paper, γ was fixed to 1/14 [20] [21] [22] . we performed the approximate bayesian computation of the posterior distribution of κ and β 0 given the average cluster size and the average time from onset to diagnosis. we drew β 0 from u(0.001, 0.2) and κ from u(0.001, 0.12) and accepted the parameter sets with which the average cluster size was identical to 126/78 for dataset 1 and 43/30 for dataset 2, and the average time from onset to diagnosis lied within ±1days of 9.3 for dataset 1 and 6.6 for dataset 2. the cluster size is defined as the number of nodes of the cluster. the average time from onset to diagnosis of clusters is defined as the average of t d i à t 0 i where i runs over the nodes that are diagnosed first in the cluster. we chose these two summary statistics, the average cluster size and the average time from onset to diagnosis of clusters, to fit the model parameters because of the following reasons. first, these can be obtained without using sophisticated techniques. second, these allow the precise determination of κ and β 0 (see the results section). although the number of diagnosed individuals in a given period may be used as a summary statistic, since the number of diagnosed individuals depends on the influx of infected individuals, i.e., the rate of generation of nodes without edge, the parameter value of λ as well as κ and β 0 must be estimated in this case. in contrast, because the average cluster size and the average time from onset to diagnosis of clusters do not depend on λ if λ is small enough, κ and β 0 can be more precisely estimated by using these two summary statistics. third, although the parameter value of the birth-death process can be estimated by the time course of the number of infected individuals, the hokkaido government has not reported the date of recovery of cases, and the time course of the number of infected individuals is unavailable. these two summary statistics can be calculated without information on the recovery of infected individuals. fourth, although the higher order moments of the cluster size distribution and the time from onset to diagnosis of clusters can be used, they provide almost identical information to their average. hence, we used the averages because an average is generally more robust than higher order moments. likewise, other network statistics were not used. in each run of the simulation, we removed c 0 + c clusters of diagnosed symptomatic patients, of which the first c 0 = 100 clusters were discarded to eliminate the dependence of the results on the initial condition and used the following c = 78 (dataset 1) and c = 30 (dataset 2) clusters as the simulated clusters of patients in hokkaido. this procedure is justified by the fact that the average cluster size of birth-death processes converges to its steady-state value on a timescale of 1/β and 1/γ [11] . this fact also suggests that the properties of clusters in the early phase of the spreading of sars-cov-2 can be described by the steady-state of the model. the ratio of undiagnosed symptomatic patients to diagnosed symptomatic patients can be estimated by the number of nodes that recover without being diagnosed in a period divided by the number of diagnosed nodes that recover in the same period. we used the period between the removal of the c 0 -th cluster and the removal of the c 0 + c-th cluster to calculate the ratio. this period is referred to as the target period in the following. the ratio of the number of symptomatic and asymptomatic patients to the number of symptomatic patients is β/β 0 . because β > γ, which follows from eq 4, and β > β 0 , the lower bound of the number of all infected individuals that recover in the target period is estimated by the number of diagnosed and undiagnosed symptomatic patients that recover in the period multiplied by max(γ, β 0 )/β 0 . the estimates presented in this paper are rounded to two significant digits. s1 code is the code that was used to analyse the datasets and generate the figures. we performed simulations with randomly generated 100000 parameter sets and accepted the parameter sets that replicated the average cluster size and the average time from onset to diagnosis of clusters. before applying the parameter estimation to datasets 1 and 2, we tested whether the present model can successfully estimate the parameter values of an artificial data. the artificial data was generated by a simulation run of the model with β 0 = 0.1 and κ = 0.05. in this simulation, the average cluster size was 2.1, and the average time from onset to diagnosis of clusters was 5.6. the orange crosses, blue triangles, and filled circles in fig to examine the number of undiagnosed symptomatic patients, we calculated the number of nodes that recovered without being diagnosed in the target period. fig 5a and 5c show the number of undiagnosed symptomatic patients per diagnosed symptomatic patient for dataset 1 and 2, respectively. the 95% c.i. were [0.95, 3.8] (median 1.7) for dataset 1 and [0.35, 1.9] (median 0.77) for dataset 2. the lower bound of the number of all infected individuals is estimated by the number of diagnosed and undiagnosed symptomatic patients multiplied by max (γ, β 0 )/β 0 (fig 5b and 5d) . the 95% c.i. of the lower bound of the total number of infected individuals per diagnosed patient were [2.3, 8.2] (median 4.2) for dataset 1 and [1.5, 6.3] (median 2.4) for dataset 2, the former of which is consistent with a previous estimate, 1/0.14 [23] . these estimates suggest that around half of infected individuals remain asymptomatic. this is consistent with a previous report on a cruise ship, in which 334 out of 712 infected individuals remained asymptomatic [24] . the 95% c.i. of the lower bound of the total numbers of infected individuals who recovered before the declaration of the state of emergency and those who recovered between the declaration and lifting were [290, 1000] (median 530) and [64, 270] (median 110), respectively. to examine the sensitivity of the estimates on the value of γ, we performed the simulations with γ = 1/10 and γ = 1/20. the medians of the number of undiagnosed symptomatic patients per diagnosed symptomatic patient for dataset 1 and 2 were 5.8 and 1.5 for γ = 1/10 and 0.79 and 0.45 for γ = 1/20, respectively. the medians of the lower bound of the total number of infected individuals per diagnosed patient for dataset 1 and 2 were 8.9 and 3.4 for γ = 1/10 and 2.9 and 2.0 for γ = 1/20, respectively. the medians of the lower bound of the total numbers of infected individuals who recovered before the declaration of the state of emergency and those who recovered between the declaration and lifting were 1100 and 150 for γ = 1/10 and 370 and 88 for γ = 1/20, respectively. hence, a greater γ, i.e., a shorter mean infective period increases the estimated number of undiagnosed patients, and a less γ, i.e., a longer mean infective period decreases the estimated number of undiagnosed patients. in this paper, we have formulated a model to describe the spreading of infection and the quarantine of infected individuals, and estimated the number of undiagnosed symptomatic and asymptomatic covid-19 patients in hokkaido. the estimated percentages of undiagnosed symptomatic and asymptomatic patients coincided with previous studies [23, 25] . the estimation of the percentages of undiagnosed patients of sars-cov-2 infection in hokkaido, japan estimated lower bound of the total number of patients that recovered before the declaration of a state of emergency was also consistent with a previous study, which estimated that the cumulative incidence in hokkaido was 2297 cases on february 27th [26] . one of the previous studies approximated the time evolution of the number of infected individuals with differential equations [23] , while another estimated the number of asymptomatic patients by using rt-pcr (reverse transcription polymerase chain reaction) test results of evacuees from wuhan, china on chartered flights [25] . the present analysis focuses on the stochastic dynamics of a discrete number of infected individuals. thus, the size distribution of clusters, which is a piece of information available in the early phase of the pandemic but difficult to use in differential-equation-based models, can be utilized by the model. although the methods of the previous reports and ours are completely different, quantitative agreement between them suggests the effectiveness of these approaches. there are several reasons we have chosen the cases in hokkaido as the subject of this paper. hokkaido is an island isolated from the other regions of japan. in other words, we can assume that a relatively small percentage of the population commutes between hokkaido and other parts of the world. this makes hokkaido an ideal subject of the investigation. until march 20th, one day after the lifting of the state of emergency, 1549 out of 1707 individuals tested with rt-pcr turned out to be negative for sars-cov-2 [27] , indicating that extensive contact tracing was performed. on the other hand, among the 158 cases diagnosed until march 19th in hokkaido, only two were asymptomatic. this suggests that in most contact tracing in hokkaido, rt-pcr tests for sars-cov-2 were conducted only on symptomatic patients because of restricted resources. we excluded the diagnosed asymptomatic patients from the analysis because they comprise less than 2% of the reported cases. if the individuals who came into contact with diagnosed patients had been so intensively tested that a much larger number of asymptomatic patients had been diagnosed, the analysis would have needed an extended model including asymptomatic diagnosed patients. the criterion of inclusion and exclusion of asymptomatic individuals in rt-pcr tests could have changed over time, but this is beyond the scope of the present study. the claim by the local government that test capability was strengthened after the declaration of the state of emergency [28] is supported by a larger value of κ in dataset 2 than in dataset 1. the hokkaido government started reporting the number of rt-pcr tests on march 3rd, four days after the declaration. until march 3rd, 79 out of 604 were positive (13%), and, from march 4th to march 20th, 79 out of 1103 were positive (7.1%). although the number of rt-pcr tests before march 3rd is not available, this is consistent with the strengthening of the test capability. compared to κ, β 0 did not exhibit a great change before and after the declaration. this seems counterintuitive because the declaration should have changed the behavior of residents and lowered β 0 . there are several possible explanations for this puzzle. each cluster is classified into datasets 1 and 2 depending on the earliest onset of the cases in the cluster. because an incubation period precedes the onset of symptoms, some of the clusters in dataset 2 might reflect the spread of sars-cov-2 before the declaration, blurring the difference between datasets 1 and 2. a much larger dataset may reveal the change of β 0 . another explanation is that, because the state of emergency was not legally binding, the behavioral modification of the residents was not sufficient to reduce β 0 ; in contrast, the declaration urged the health officials to strengthen the test capability. discerning these possibilities needs further study. although the hokkaido datasets were an ideal subject for the present model, the model is not necessarily suitable for other datasets. kyushu and shikoku, both of which are islands in japan, contain several prefectures and, consequently, several local governments. because the cases reported by these local governments must be merged, the analysis of the spread of sars-cov-2 in these islands is much more difficult than that in hokkaido. if a nation-wide dataset was available, it might be an ideal subject for the present model because of the recent travel restrictions. however, the present model cannot be applied to the dataset that exhibits the superspreader phenomenon because the number of individuals directly infected by an individual follows a geometric distribution (eq 3). while the cluster size in hokkaido was moderate as shown in fig 3, there were large clusters in other prefectures in japan, for example, the cluster of a club with live music in osaka [29] . moreover, our model cannot utilize information on asymptomatic diagnosed patients. one of the features of the present model is its simplicity. the model has only three essential parameters. the simplicity of the model allowed us to estimate the number of asymptomatic and undiagnosed patients without using a large number of parameter values estimated by previous studies. in the early phase of the spreading of infectious diseases, this simple model can enable the estimation of the asymptomatic and undiagnosed patients despite limited data. our approach can estimate the number of patients without using costly and time-consuming techniques such as rt-pcr. also, its simplicity might allow for an analytical solution. the model is an extension of the birth-death processes, which has been studied intensively. the birth-death processes with contact tracing is analytically tractable [10, 11, 16, 17] . because the model is simple enough, the analytical solution of the cluster size distribution and the expected time from onset to diagnosis of clusters might be obtained. in future work, the analytical solution would enable us to efficiently estimate the parameter values. the simplicity of the present model allows expansions in several ways. first, we assumed that β 0 is a fixed value in this paper. this is justified by fig 3, which shows that the largest number of individuals infected by an individual, that is, the largest degree of nodes, is eight, which is rather small. however, β 0 may be heterogeneous. heterogeneity in β 0 , which has been suggested for the coronavirus genus [30, 31] , may explain the superspreader phenomenon. also, the spread of sars-cov-2 might be more accurately modeled with the contact process on scale-free networks [32, 33] . the value of β 0 might depend on the severity of the patient [23] . second, the recovery rate γ may depend on the severity of the patient. heterogeneity in γ can affect the estimated number of total infected individuals. third, the incubation period, which is ignored in the present paper, might affect the size and structure of clusters [34] . infectiousness in the incubation period should be included in the model [34] . fourth, the stage of symptoms should be introduced into the model. fig 6 suggests that the time from onset to diagnosis of clusters obeys a unimodal distribution with a peak at around 10 days, although the peak must be at 0 in the present model. assuming that a mildly infected state stochastically develops into a severely infected state would explain this time course. fifth, recursive full tracing might be unrealistic because some of the symptomatic patients can be missed in contact tracing. introducing stochasticity into contact tracing can enable a more precise modeling of clusters. these extensions would be useful in monitoring and controlling the spread of sars-cov-2. supporting information s1 table. the presumed asymptomatic carrier transmission of covid-19 asymptomatic and human-to-human transmission of sars-cov-2 in a 2-family cluster epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong. the lancet transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions role of contact tracing in containing the 2014 ebola outbreak: a review. african health sciences contact tracing and disease control the impact of contact tracing in clustered populations epidemic models of contact tracing: systematic review of transmission studies of severe acute respiratory syndrome and middle east respiratory syndrome contact tracing in stochastic and deterministic epidemic models family trees of continuous-time birth-and-death processes the effectiveness of contact tracing in emerging epidemics estimating the tracing probability from contact history at the onset of an epidemic threshold behaviour of emerging epidemics featuring contact tracing stochastic epidemic models featuring contact tracing with delays the effect of delay on contact tracing exact and approximate formulas for contact tracing on random trees situation of covid-19 in hokkaido prefecture estimating the asymptomatic proportion of coronavirus disease 2019 (covid-19) cases on board the diamond princess cruise ship virological assessment of hospitalized patients with covid-2019 updated rapid risk assessment from ecdc on the novel coronavirus disease 2019 (covid-19) pandemic: increased transmission in the eu/eea and the uk effectiveness of control strategies for coronavirus disease 2019: a seir dynamic modeling study substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov2) press release on march 15th estimation of the asymptomatic ratio of novel coronavirus infections (covid-19) estimation of the actual incidence of coronavirus disease (covid-19) in emergent hotspots: the example of hokkaido press release on hokkaido set to lift coronavirus state of emergency why do some covid-19 patients infect many others, whereas most don't spread the virus at all. science. accessed 11 superspreading and the effect of individual variation on disease emergence the role of superspreading in middle east respiratory syndrome coronavirus (mers-cov) transmission epidemic spreading in scale-free networks epidemic outbreaks in complex heterogeneous networks transmission of 2019-ncov infection from an asymptomatic contact in germany software: takuma tanaka. visualization: takuma tanaka. writing -review & editing: takuma tanaka, takayuki yamaguchi, yohei sakamoto. key: cord-260843-c97kctjz authors: dai, lei; hu, wei wei; xia, lu; xia, mi; yang, qian title: transmissible gastroenteritis virus infection enhances sglt1 and glut2 expression to increase glucose uptake date: 2016-11-16 journal: plos one doi: 10.1371/journal.pone.0165585 sha: doc_id: 260843 cord_uid: c97kctjz transmissible gastroenteritis virus (tgev) is a coronavirus that causes villus atrophy, followed by crypt hyperplasia, reduces the activities of intestinal digestive enzymes, and disrupts the absorption of intestinal nutrients. in vivo, tgev primarily targets and infects intestinal epithelial cells, which play an important role in glucose absorption via the apical and basolateral transporters na(+)-dependent glucose transporter 1 (sglt1) and facilitative glucose transporter 2 (glut2), respectively. in this study, we therefore sought to evaluate the effects of tgev infection on glucose uptake and sglt1 and glut2 expression. our data demonstrate that infection with tgev resulted in increased glucose uptake and augmented expression of egfr, sglt1 and glut2. moreover, inhibition studies showed that egfr modulated glucose uptake in control and tgev infected cells. finally, high glucose absorption was subsequently found to promote tgev replication. transmissible gastroenteritis (tge) is a highly contagious infectious disease of pigs that is characterized by vomiting, diarrhea, and dehydration. notably, the mortality rate of this disease in seronegative suckling piglets can reach up to 100% [1] . tge is caused by the tge virus (tgev), which infects the gastrointestinal tract, causing villus atrophy and crypt hyperplasia, and disrupting intestinal nutrition absorption [2, 3] . glucose is among the nutrients absorbed in the intestinal epithelium, and glucose uptake in epithelial cells depends on two types of glucose transporters, the apically expressed na + -dependent glucose transporter 1 (sglt1) and basolaterally expressed facilitative glucose transporter 2 (glut2). specifically, sglt1 mediates the na+/glucose cotransport function of the kidney and intestine as a secondary active transporter, while glut2 serves as a facilitated diffusion system for transport through lipid bilayers [4] [5] [6] . the epidermal growth factor receptor (egfr) was previously reported to transiently increase glucose transport [7, 8] . moreover, a recently study suggested that egfr may act as another receptor for tgev, in addition to porcine aminopeptidase (papn) [9] . egfr-dependent regulation of glucose uptake has been observed in tumor cells, and egfr has been shown to prevent autophagic cell death by maintaining intracellular glucose levels through interaction with and stabilization of sglt1 [10] . however, the involvement of egfr in virus-induced effects on glucose uptake has yet to be evaluated. therefore, in the study, we aimed to examine the in vitro effects of tgev infection on glucose uptake and the expression of sglt1 and glut2 in porcine intestinal columnar epithelial (ipec-j2) cells, which have been shown to offer a practical model for studying tgev infection [11, 12] . cell lines ipec-j2 cells, which are porcine intestinal columnar epithelial cells that were originally isolated from the middle jejunum of neonatal piglets, were purchased from dsmz (braunschweig, germany), while hek293t cells were purchased from the american type culture collection (atcc; manassas, va, usa). ipec-j2 and hek293t cells were maintained in roswell park memorial institute medium (rpmi) and dulbecco's modified eagle's medium (dmem) with high glucose, respectively, supplemented with hepes, 10% fetal bovine serum (fbs; gibco, grand island, ny, usa), and 1% penicillin-streptomycin (invitrogen, carlsbad, ca, usa) at 37˚c in a 5% co 2 incubator (thermo fisher scientific, waltham, ma, usa). tgev strain shxb was isolated in shanghai, china. the complete genome sequence for this virus is available at the genbank database (id number: kp202848.1) [13] . for experimental assays, cells were incubated with tgev at a multiplicity of infection (moi) of 2 for 1 h at 4˚c in serum-free medium and washed with phosphate-buffered saline (pbs; ph 7.2) at 4˚c three times to remove unbound virus. cells were then cultured in medium containing 2% serum. total rna was extracted from ipec-j2 cells infected with tgev using trizol reagent (invitrogen), according to the manufacturer's instructions. cdna was generated by reverse transcription using hiscript qrt supermix for qpcr (vazyme biotech, nanjing, china), according to the manufacturer's instructions. tgev release was assessed by measuring the levels of viral nucleoprotein (n) gene expression via quantitative rt-pcr using a takara sybr green qpcr kit (takara, shiga, japan). the primer sequences were as follows: n-f (sense), 5'-caattcccgtggtc ggaaga-3', n-r (antisense), 5'-tttacgttggcccttcacca-3'. pcr products were purified using a gel extraction kit (omega bio-tek, inc., norcross, ga, usa) and cloned into the pjet1.2 vector (thermo fisher). plasmids were serially diluted and used as standards for quantitative analysis. the initial copy number of the tgev n gene was calculated using the following formula: x0 = -k log ct + b, where x0 is the initial copy number and k, ct, and b refer to the slope rate, cycle threshold, and constant, respectively. quantitative real-time pcr was performed with an abi prism 7500 detection system (applied biosystems, foster city, ca, usa). at the indicated time points post-infection, cells were washed with pbs and lysed in radioimmunoprecipitation assay (ripa) buffer (thermo scientific) containing a phosphatase inhibitor and protease inhibitor (thermo scientific), according to the manufacturer's instructions. the protein concentrations of the resulting lysates were determined using a pierce bca protein assay kit based on the bicinchoninic acid spectrophotometric method (thermo scientific). after centrifugation at 13,000 × g for 15 min, proteins in the supernatant (15-50 μg protein) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) on 10-12% gradient gels, and transferred to polyvinylidene fluoride (pvdf) membranes (merck millipore, darmstadt, germany). membranes were blocked for 2 h in tris-buffered saline (tbs) containing 5% nonfat dry milk, and probed with the indicated primary antibodies at 4˚c overnight, according to the manufacturer's instructions. the following antibodies were used in this study: anti-p-egfr antibody (d7a5; cell signaling technology, danvers, ma, usa), anti-egfr (c4267; cell signaling technology, danvers, ma, usa), anti-sglt1 (ab14686; abcam, cambridge, uk), anti-glut2 (sc-7580; santa cruz biotechnology, dallas, tx, usa), anti-βtubulin (e12-043; enogene biotech, new york, ny, usa). membranes were then exposed to species-specific horseradish peroxidase (hrp)-conjugated secondary antibodies (dilution: 1:5,000), and proteins were detected by enhanced chemiluminescence (ecl; thermo scientific) and autoradiography. the resulting bands were quantified using quantity one 1-d analysis software (170-9600; bio-rad, hercules, ca, usa). the density of each band was measured and normalized to that of β-tubulin expression. all data were expressed as means ± standard deviations (sd) of the results of three independent experiments. plvx-shrna is a human immunodeficiency virus type 1 (hiv-1)-based lentiviral expression vector designed to express a small hairpin rna (shrna) for rnai studies (clontech laboratories, inc., mountain view, ca, usa). the best silencing efficiencies were observed with clones nm_214007 (porcine egfr) and nm-001012297.1 (porcine sglt1). the shrna for egfr, three shrnas for sglt1, and overexpression plasmid for egfr were designated as shegfr, shsglt1-1, shsglt1-2, shsglt1-3, and plvx-egfr, respectively. hek293t cells were transfected with 1 μg of specific expression plasmid per 10 6 cells using the x-tremegene hp dna transfection reagent (roche, basel, switzerland), according to the manufacturer's instructions, diluted in opti-mem (invitrogen) in a t-25 cell culture flasks. lentiviral particles (moi = 2) were subsequently added to the transfected ipec-j2 cells and gently mixed. after comparing amino acid sequences of egfr, sglt1 and glut2 from ncbi (table 1) , we respectively selected ag1478, phlorizin as the inhibitor of egfr and sglt1. glucose uptake experiments 2-[ 3 h]deoxyglucose (2-dg) uptake experiments were carried out according to the method described by henriksen et al., but with modifications [14] . briefly, the culture medium was removed by aspiration, and cells were gently washed twice with uptake buffer (140 mm nacl, 2 mm kcl, 1 mm kh 2 po 4 , 10 mm mgcl 2 , 1 mm cacl 2 , 5 mm glucose, 5 mm l-alanine, 5 nm indomethacin, and 10 mm hepes-tris; ph 7.4). after washing, cells were incubated in uptake buffer containing 2-dg at 37˚c for 30 min. at the end of the incubation period, cells were washed three times with ice-cold uptake buffer and dissolved in 1 ml of 0.1% sds. the level of intracellular 2-dg uptake was determined by measuring the radioactivity of a 900-μl aliquot of each sample using a liquid scintillation counter (ls 6500; beckman instruments, fullerton, ca, usa). the remainder of each sample was then used to determine protein expression levels of glucose transporters. the radioactivity counts of each sample were normalized with respect to the protein and corrected for time uptake per milligram of protein. all uptake measurements were carried out in triplicate. glucose uptake was calculated as follows: glucose uptake (nm/10 6 cells) = (glucose concentration of the control group-glucose concentration of the experimental group)/cell number. cfse stocks (10 mm in dmso; invitrogen, merelbeke, belgium) were stored at -20˚c prior to use, thawed, and diluted in phosphate-buffered saline (pbs) to the desired working concentrations. ipec-j2 cells were resuspended in pbs (0.1% bsa) at 2 × 10 6 cells/ml and incubated with cfse (final concentration: 1 μm) for 7 min at 37˚c. cells were washed and resuspended in culture medium for 15 min to stabilize cfse staining, and cfse-labeled cells were analyzed by flow cytometry. the viability of raw264.7 cells was determined using a cell counting kit-8 assay kit (beyotime biotechnology, beijing, china), as previously reported. briefly, raw264.7 cells were plated at a density of 1 × 10 4 cells/well in 96-well plates in 100 ml roswell park memorial institute 1640 medium and incubated for 24 h. twenty microliters of cell counting kit-8 reagent was then added to each microwell, and plates were incubated for 2 h at 37˚c. the absorbance of the colored solution was measured using a microplate reader (bio-rad laboratories) at a test wavelength of 450 nm and a reference wavelength of 630 nm. all results are presented as means ± sd of the results of three independent experiments. significant differences between control and experimental groups were analyzed using student's ttest. p-values < 0.05 were considered statistically significant. as shown as in fig 1a, tgev infection triggered a significant increase in glucose uptake in ipec-j2 cells at 48 and 60 h post-infection (hpi). in addition, during late-stage infection, tgev-infected cells were able to continue transporting glucose, even when the concentration of glucose in the cell culture medium was low. these data indicate that tgev infection promotes glucose uptake in ipec-j2 cells. while fig 1b-1f show the mrna and protein levels of sglt1 and glut2 at various time points. notably, while there were no marked changes in the mrna expression of the sglt1 and glut2 genes after tgev infection, there were significant increases in the protein expression of both sglt1 and glut2 at 48 and 60 hpi. to exclude the possibility that cell viability and proliferation may affect glucose uptake, cfse-labeled mock-infected (control), tgevinfected (tgev), and carbonyl cyanide m-chlorophenyl hydrazone (cccp)-treated (cccp) cells were subjected to flow cytometric analysis. cccp is known to reduce cell viability and proliferation, and was therefore utilized as a positive control [15] . as shown in fig 1g-1i , previous studies have shown that the expression of egfr is closely related to glucose transport, especially phosphorylation region of egfr. egfr kinase activity regulates the peak glucose uptake and total egfr may likely be maintaining basal glucose uptake [7, 8] . therefore, we first examined whether egfr expression was stimulated by tgev infection. western blot analysis showed that tgev infection not only increased total egfr expression (fig 2a) but also revealed that more phosphorylated egfr was present in tgev infected relative to control cells (fig 2b) . taken together, these results indicated that tgev infection increased the total and phosphorylation protein expression of egfr at 48 hpi. together, these results indicate that tgev infection promoted the total and phosphorylation protein expression of egfr at 48 hpi. to explore whether egfr contributes to the observed tgev infection increase in glucose uptake, we explored changes in glucose uptake and the expression of glucose transport molecules by inhibiting or overexpressing egfr in mock-infected cells. we choose ag1478 as egfr inhibition because it can inhibit the egfr tyrosine kinase activity and total egfr [16, 17] . as shown in fig 3a-3c , treatment with the egfr inhibitor ag1478 suppressed glucose uptake and markedly decreased egfr and p-egfr protein expression, indicating that ag1478 regulated glucose uptake in mocked-infected cells. furthermore, cells treated with ag1478 exhibited markedly lower levels of sglt1 protein expression than the control cells; in contrast, there was no effect on glut2 protein expression. thus, we concluded that ag1478 modulated glucose uptake in mock-infected cells via downregulation of egfr, which resulted in reduced sglt1 expression. as shown in fig 3d-3f , we sought to then strengthen these findings via modulation of egfr expression. transfection with shegfr resulted in significant downregulation of egfr β-tubulin was also analyzed. doi:10.1371/journal.pone.0165585.g002 and p-egfr protein expression and decreased glucose uptake compared with the cells transfected with the control shrna. conversely, plvx-egfr transfection resulted in significantly increased egfr and p-egfr protein expression and glucose uptake. these data demonstrate that modulation of egfr and p-egfr expression affects glucose uptake in the absence of tgev infection. furthermore, transfection with shegfr and plvx-egfr resulted in decreased and increased sglt1 protein expression, respectively; in contrast, glut2 protein expression was unaffected by either treatment. together, these results indicate that egfr and p-egfr regulates glucose uptake in mock-infected ipec-j2 cells by modulation of sglt1 protein expression. because sglt1 is involved in mediating the functions of egfr in hek-293t cells [18] , we explored the relationship between egfr and sglt1 expression in ipec-j2 cells. as shown in fig 3h and 3j-3l , ipec-j2 cells transfected with three sglt1-specific shrnas for 48 h exhibited reduced sglt1 expression and reduced egfr protein expression. furthermore, we choose phlorizin as sglt1 inhibitor because phlorizin's principal pharmacological action is to block intestinal glucose absorption through inhibition of the sodium-glucose symporters located inmucosa of the small intestine [19, 20] . ipec-j2 cells treated with phlorizin for 48 h showed downregulation of sglt1 expression without inducing cell death, as demonstrated by cck8 assay analyses (data not shown). similarly, western blot analysis showed that ipec-j2 cells treated with phlorizin exhibited lower egfr expression than dmso-treated cells. in conclusion, inhibition of sglt1 by shrna or phlorizin treatment disrupted egfr expression. notably, these treatments also resulted in decreased glucose uptake (fig 3g and 3j) . consistent with these findings, ipec-j2 cells transfected with shegfr or treated with ag1478 also exhibited lower sglt1 expression (fig 3b and 3e) . these data indicate that there is an association between egfr and sglt1 expression in ipec-j2 cells. to further investigate the role of tgev infection-enhanced egfr in the regulation of glucose uptake, we examined the effects of tgev infection after treatment or transfection with ag1478, shegfr, or plvx-egfr on glucose uptake. as shown in fig 4a and 4d , transfection with shegfr and treatment with ag1478 significantly inhibited glucose uptake, whereas transfection with plvx-egfr enhanced glucose uptake. therefore, we concluded that egfr mediates glucose uptake in tgev-infected ipec-j2 cells. moreover, as shown in fig 4b, 4c , 4e and 4f, after tgev infection, western blot analysis showed that transfection with shegfr and treatment with ag1478 resulted in lower protein expression of sglt1 and glut2 than that observed in the control group, whereas transfection with plvx-egfr resulted in increased sglt1 and glut2 protein expression. together, these results indicate that egfr influences glucose uptake in tgev-infected cells by promoting both sglt1 and glut2 expression. previous studies have suggested that the intracellular glucose concentration is closely linked with viral infections, particularly for double-stranded dna viruses [21] [22] [23] . consistent with this conclusion, we found that tgev enhanced glucose uptake in ipec-j2 cells. because glucose is the main energy source for cellular metabolism, tgev replication in ipec-j2 cells requires large amounts of glucose/energy. thus, we explored whether high glucose uptake in tgev-infected cells affected tgev replication. tgev contains a 27.6-31.3-kb singlestranded, positive-sense rna genome; the virion rna functions as an mrna and is infectious. the n protein, in particular, is a critical component of the replication-transcription complex [24] [25] [26] . thus, we treated tgev-infected cells with medium containing 0.1, 1, 10, or 25 mm glucose and harvested the virus at 48 hpi. as shown in fig 5a, glucose absorption concentrations were 0.1, 1, 1.8, and 5.2 mm for medium containing 0.1, 1, 10, and 25 mm glucose, respectively. furthermore, western blot analysis showed that tgev n protein was more abundant when tgev-infected cells absorbed more glucose, particularly in medium containing 10 or 25 mm glucose (fig 5b) . consistent with this finding, there was a concurrent increase in the copy number of the tgev n gene as glucose absorption levels increased (fig 5c) . as described above, in tgev-infected ipec-j2 cells, higher glucose concentrations promoted tgev replication. to exclude the possibility that cell viability and proliferation affect tgev replication, flow cytometric analysis of cfse-labeled cells was performed in the presence of 0.1, 1, 10, and 25 mm glucose. as shown in fig 5d-5f , high glucose concentrations did not significantly affect proliferation and viability. as recently reported, viral infection may result in increased or decreased glucose uptake. for example, the hepatitis b virus x protein regulates hepatic glucose homeostasis via nitric oxide synthase [27, 28] . additionally, human cytomegalovirus (hcmv) enhances glucose uptake during the first 20 h after infection, even when a high level of glucose is constantly present in the medium [22] . hepatitis c virus (hcv) replication suppresses cellular glucose uptake through downregulation of cell surface glucose transporters [29] , while rotavirus infection impairs rabbit intestinal brush-border membrane na + -solute cotransport activities [30] . however, the effects of coronaviruses (including tgev) on glucose uptake have not been reported. we found that tgev infection increased glucose uptake of ipec-j2 cells and the protein expression of sglt1, glut2 after 48 hpi (fig 1) , while tgev infection in ipec-j2 cells reached a peak at 48 hpi [31] , we just provided foresight to explore the interaction between tgev infection and glucose uptake, which is still worth exploring. most of the glucose absorbed in the intestine is transformed to glutamine [32] , which was reported to promote the replication of many viruses, such as porcine circovirus (pcv) and hcmv, in vitro [33, 34] . because viral replication requires a lot of energy, we examined the effects of glucose absorption on tgev propagation. our results demonstrate that increased glucose absorption enhanced tgev replication, suggesting that glucose may be transformed into glutamine in infected cells. in this study, increases in sglt1 and glut2 protein expression directly stimulated glucose uptake in vitro. although the role of other transporters cannot be ruled out, as sglt1 and glut2 dominate intestinal glucose transport, they may play important effect on glucose uptake in tgev infection. however, no significant changes were observed in sglt1 or glut2 mrna expression levels, indicating that tgev infection primarily affects the protein expression of sglt1 and glut2. these findings were consistent with previous reports demonstrating that tgev infection influences protein translation. additionally, sglt1 and glut2 have been shown to mediate intestinal glucose uptake transmissible gastroenteritis virus promotes increased glucose uptake through de novo synthesis of mrna and protein [35] . within the last decade, the mechanisms of passive or diffusive components of intestinal glucose uptake have come under debate [36] . the current paradigm describing intestinal uptake is that glucose enters the absorptive cell through sglt1 in the brush-border membrane and exits into the blood through glut2, a member of the facilitative glucose transporter family, located in the basolateral membrane. we have now provided evidence for an alternative mechanism for the passive component of absorption, i.e., rapid trafficking of glut2 to the brush-border membrane was found to be controlled by the sglt1-dependent activation of a protein kinase c (pkc)-dependent pathway and also by mitogen-activated protein kinase (mapk) intracellular signaling pathways [37] [38] [39] . glut2 exhibits a >10-fold-higher glucose transport capacity than sglt1 and provides a major route of glucose absorption with rapid absorptive capacity [40, 41] . glut2 plays an important role in glucose absorption across the brush border membrane in normal jejunum [42] . although our experiments did not directly demonstrate the rapid trafficking of glut2, we found that tgev infection significantly increased glucose uptake at 48 h, indicating accelerated glucose absorption. thus, tgev infection might promote glucose trafficking through glut2. moreover, because the tgev increased the protein expression of p-egfr ( fig 2b) and tgev spike protein is capable of binding to egfr, thereby activating the downstream pkc-dependent and mapk intracellular signaling pathways [9] , the trafficking of glut2 to the brush-border membrane could be stimulated, allowing rapid transport of glucose. previous studies have shown that glucose transport plays an important role in viral invasion. glut1-mediated glucose transport regulates hiv infection [43] and is a receptor for human t-cell leukemia virus (htlv). additionally, perturbations in glucose metabolism resulting from interactions between htlv envelope proteins and glut1 are likely to contribute to htlv-associated disorders [44] ; thus, analysis of the relationship between viral infection and glucose uptake is critical. notably, in mock-infected cells, egfr mediate sglt1 protein expression. indeed, previous literatures reported that egfr maintains intracellular glucose levels through interaction with and stabilization of sglt1 [10] [18] . importantly, we found that egfr modulated sglt1 expression during infection, but it cannot be concluded unfortunately that the cellular effect of tgev infection on sglt1 is mediated via egfr. because the drug treatment and knock down/overexpression against sglt1 had similar effects with egfr (figs 3 and 4) . in other words, it could also be direct effect on sglt1, which in results in similar effect on egfr. in fact, egfr and sglt1 are co-expressed in many other type cells [45, 46] . moreover, previous studies have shown that egfr is a target receptor for viruses and bacteria, including tgev and escherichia coli [9, 47] . thus, because egfr also interacts with sglt1, this protein might comprise another ubiquitous target receptor. egfr has been reported to increased glucose uptake which is critical for the cell [10] , we found the egfr and p-egfr both promote glucose uptake in ipec-j2 cells during tgev infection. previous literatures reported that egfr kinase activity regulates the peak glucose uptake and total egfr may likely be maintaining basal glucose uptake [7, 8] . in our study, the ipec-j2 cells have been in a rich glucose medium during tgev infection, we also did not explore the respective role of egfr and p-egfr in glucose uptake. the mechanism through which tgev causes diarrhea has not been elucidated. on the other hand, glucose uptake has been shown to cause diarrhea [48, 49] . for example, enteropathogenic e. coli rapidly inactivates sglt1 through multiple mechanisms. indeed, the finding that one mechanism occurs more rapidly than microvilli effacement provides a plausible explanation for the rapid onset of severe watery diarrhea, given the crucial role of sglt1 in the daily uptake of large amounts of fluids from the normal intestine. in contrast, our data indicate that tgev infection resulted in increased sglt1 expression. however, tgev infection did lead to rapid glucose uptake, which in turn supplied glucose to the virus and promoted long-term infection by tgev in the intestine. 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absorption regulation of glut5, glut2 and intestinal brushborder fructose absorption by the extracellular signal-regulated kinase, p38 mitogen-activated kinase and phosphatidylinositol 3-kinase intracellular signalling pathways: implications for adaptation to diabetes stimulation of fructose transport across the intestinal brush-border membrane by pma is mediated by glut2 and dynamically regulated by protein kinase c the diffusive component of intestinal glucose absorption is mediated by the glucose-induced recruitment of glut2 to the brush-border membrane the slc2 family of facilitated hexose and polyol transporters apical glut2: a major pathway of intestinal sugar absorption the facilitated component of intestinal glucose absorption glut1-mediated glucose transport regulates hiv infection the ubiquitous glucose transporter glut-1 is a receptor for htlv localization of the na+-d-glucose cotransporter sglt1 in the blood-brain barrier a protein kinase involved in the regulation of inflammatory cytokine biosynthesis retention of native-like oligomerization states in transmembrane segment peptides: application to the escherichia coli aspartate receptor potent diarrheagenic mechanism mediated by the cooperative action of three enteropathogenic escherichia coli-injected effector proteins. proceedings of the national academy of sciences of the united states of america diarrhea caused by carbohydrate malabsorption key: cord-267307-kyh0xsrp authors: kasting, monica l.; head, katharine j.; hartsock, jane a.; sturm, lynne; zimet, gregory d. title: public perceptions of the effectiveness of recommended non-pharmaceutical intervention behaviors to mitigate the spread of sars-cov-2 date: 2020-11-04 journal: plos one doi: 10.1371/journal.pone.0241662 sha: doc_id: 267307 cord_uid: kyh0xsrp background: the covid-19 pandemic is an unprecedented public health threat, both in scope and response. with no vaccine available, the public is advised to practice non-pharmaceutical interventions (npi) including social distancing, mask-wearing, and washing hands. however, little is known about public perceptions of the effectiveness of these measures, and high perceived effectiveness is likely to be critical in order to achieve widespread adoption of npi. methods: in may 2020, we conducted a cross-sectional survey among u.s. adults (n = 3,474). the primary outcome was a six-item measure assessing perceived effectiveness of recommended behaviors to prevent sars-cov-2 infection from 1 (not at all effective) to 5 (extremely effective). the sample was divided into “higher” and “lower” perceived effectiveness groups. covariates included demographics, healthcare characteristics, and health beliefs. variables that were significant at p<0.01 in bivariate analyses were entered into a multivariable logistic regression and a best-fit model was created using a cutoff of p<0.01 to stay in the model. results: mean age was 45.5 years and most participants were non-hispanic white (63%) and female (52.4%). the high perceived effectiveness group was slightly larger than the low perceived effectiveness group (52.7% vs. 47.3%). almost all health belief variables were significant in the best-fit regression model. covid-19-related worry (aor = 1.82; 95% ci = 1.64–2.02), and perceived threat to physical health (aor = 1.32; 95% ci = 1.20–1.45) were positively associated with perceived effectiveness while perceived severity of covid-19 (0.84; 95% ci = 0.73–0.96) and perceived likelihood of infection (0.85; 95% ci = 0.77–0.94) switched directions in the adjusted model and were negatively associated with perceived effectiveness. conclusions: this research indicates people generally believe npi are effective, but there was variability based on health beliefs and there are mixed rates of engagement in these behaviors. public health efforts should focus on increasing perceived severity and threat of sars-cov-2-related disease, while promoting npi as effective in reducing threat. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 in late december 2019, chinese authorities first notified the world health organization (who) of a cluster of cases of pneumonia in the chinese wuhan hubei province (1) . these pneumonia cases were determined to be caused by a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (sars-cov-2), which is now known to cause the new coronavirus disease (covid-19) [1, 2] . within the first few months of the 2020 calendar year, the virus spread rapidly around the world and, particularly, in the u.s. on march 11, 2020, who officially declared covid-19 a pandemic [1] . by mid-july 2020, the u.s. had over 3 million cases of covid-19 and almost 140,000 deaths [3] . at the time of this research (may 2020), there were no pharmaceutical options for prevention or cure. therefore, to curb the spread of the virus, communities were asked to enact non-pharmaceutical interventions (npi) to prevent infections until a vaccine is developed and protection through herd immunity can be achieved. because sars-cov-2 is thought to be spread mainly through person-to-person transmission, implementation of personal protective practices have been recommended to slow viral spread [4] . specifically, the u.s. centers for disease control and prevention (cdc) suggests several mitigation strategies including (but not limited to): 1. frequent handwashing, 2. avoiding close contact with others (social distancing), 3. wearing masks, 4. covering coughs and sneezes, 5. cleaning and disinfecting surfaces, and 6. being alert for covid-19 symptoms [5] . there has been inconsistent adoption of mitigation strategies in the u.s., despite the fact that covid-19 cases are increasing in large parts of the country [3] . this problem has been exacerbated by inconsistent and, at times, contradictory messaging, particularly at the federal level [6] . specifically, in the early stages of the pandemic, guidance from public health organizations was that wearing masks, in particular, was not necessary if the person was not sick [7] . however, at that point in time, there were personal protective equipment shortages in hospitals and health officials were also attempting to prioritize mask supplies for health professionals [7] . furthermore, the who did not issue a recommendation for the general public to wear masks until june, 2020 [8] . prior to that, they had issued a statement in april 2020 stating that mask use was not supported by the evidence [9] . more recently, there is increasing evidence of the effectiveness of mask-wearing to prevent sars-cov-2 spread [10] and masks are now recommended for use in public by the cdc and mandated in 20 states across the u.s. [5, 11] . nevertheless, some public figures and elected officials have refused to be photographed wearing masks while out in public [12] , despite evidence of the effectiveness of mask-wearing for decreasing the spread of sars-cov-2 across populations [13] . implementing effective mitigation strategies has been further complicated by officials publicly down-playing the risk posed by the virus [14] and stating that it will resolve on its own [15] , while privately acknowledging the severity of the disease [16] . recent polling has indicated only one-third of americans always wear a mask when they are in public [17] . and while a large proportion of the population (84%) reported in late march, 2020 that they practiced social distancing, this proportion decreased by 10% by the end of april, despite a rise in cases and deaths [3, 18] . additional research has found, in general, u.s. residents had lower public approval of npi measures such as those promoted by the cdc, compared to their counterparts in areas of asia and central and south america [19] . furthermore, a systematic review found individuals were ambivalent about adopting personal distancing behavior due to concerns about social stigma [20] . previous research has shown distrust of the government hinders cooperation with public health recommendations during a public health crisis, such as the current pandemic [21] . furthermore, the rapidly emerging new information and naturally evolving science in the face of a novel virus may lead the public to distrust information from public health organizations, if it seems to change frequently [22] . given some of the information related to these npis has changed as the scientific information has evolved, mistrust of information could decrease the perceived effectiveness of mitigation strategies recommended by the cdc, and may therefore lead to decreased engagement in practices that slow the spread of the virus. moreover, previous studies using simulated infectious disease outbreaks found that an individual's intention to engage in social distancing behaviors increased if the person believed their behaviors would reduce the threat of the disease [23] . theoretical models like the health belief model (hbm) and extended parallel process model suggest that cues to action, perceptions of severity and susceptibility of the threat, and perceived response efficacy (e.g., belief that the recommended behavior will mitigate a threat) are important predictors of behavior uptake [24, 25] . however, little is known about how the public perceives npis within the context of the current pandemic. therefore, this study aimed to: 1) understand perceived effectiveness of cdc recommended npis to mitigate the spread of sars-cov-2 and 2) examine factors associated with perceived effectiveness to identify potential targets for future intervention efforts. an online qualtrics survey was conducted the week of may 4 th -may 11 th , 2020. eligible participants were recruited via e-mail invitation by dynata, a survey research company that maintains panels of volunteer survey respondents who receive monetary incentives for participation. dynata employs recruitment quotas based on u.s census data to ensure the sample is balanced by age, gender, race/ethnicity, and geographic location. eligibility criteria included being 18 years or older and able to read english. participants on the dynata survey panel are compensated in points based on study length. the study was given exempt status by the indiana university institutional review board. while this study examined perceptions of sars-cov-2 infection and spread, pretesting of the survey instruments determined the term "covid-19" was more appropriate for lay audiences, because sars-cov-2 is used in lay communication less frequently. therefore, we used the term "covid-19" exclusively in the survey items, even if we were talking about the virus itself. perceptions of non-pharmaceutical interventions to flatten the curve of covid-19 disease. a six-item measure was used to assess participants' perceptions of the effectiveness of npis to prevent sars-cov-2 infection and spread. each item involved a behavior the participant themselves could practice and was measured on a five-point likert scale from 1 (not at all effective) to 5 (extremely effective). the items, exactly as they appeared in the survey, can be found in s1 appendix. three of the six items measured the perceived effectiveness of preventing yourself from catching covid-19 and included: 1) practicing social distancing by leaving at least six feet between you and other people (this does not include people you live with), 2) frequently washing your hands for 20 seconds with warm water and soap, and 3) avoiding touching your face. three of the six items measured the perceived effectiveness of preventing yourself from spreading covid-19 to others and included: 1) wearing a mask anytime you leave the house to go out in public, 2) practicing social distancing by leaving at least six feet between you and other people (this does not include people you live with), and 3) covering your mouth when you cough. the six items created a reliable perceived effectiveness scale with a cronbach's alpha of 0.91. we conducted an exploratory factor analysis, which extracted a single factor that accounted for 69% of the variance of the data, indicating these six items reflect a single construct. we then created a mean effectiveness score so that each person had a score on a scale of 1-5. for purposes of analyses, we divided the sample using a median split, creating two groups: those who had lower perceived effectiveness of the npi measures (score range: 1-3.99; 47.3% of the sample) and those who had higher perceived effectiveness (score range: 4.00-5.00; 52.7% of the sample). covariates fell into three categories: demographic characteristics, healthcare characteristics, and health belief variables. healthcare characteristics included having a health condition that would make covid-19 more severe, believing they have had covid-19, ever having received a sars-cov-2 test (and the result of the test, among those who said "yes"), and personally knowing anyone who had covid-19 disease. health belief variables included: altruism. participants were asked a modified version of a previously validated 18-item altruism scale [26] . this original scale consisted of 18 questions assessing frequency of engagement in various altruistic activities (e.g. helping a stranger push their car out of the snow or mud) on a likert-type scale from 1 = never to 5 = very often. we conducted a principal components exploratory factor analysis, which extracted two factors. we labeled the first factor, which consisted of five items (cronbach's α = 0.83), high commitment altruism (i.e., behaviors that require a relatively high level of personal involvement; e.g., "i have offered my seat to a stranger who was standing"). we labeled the second factor, which consisted of four items (cronbach's α = 0.81), low commitment altruism (i.e., behaviors that require a relatively low level of personal involvement; e.g., "i have given money to charity"). we calculated a mean score for each of the altruism subscales. perceived severity of covid-19 disease was measured using a four-item scale adapted from a measure of perceived ebola severity [27] . items assessed participants' perceptions of the severity of covid-19 disease (e.g. "i am afraid that i may die if i contract covid-19") on a scale of 1 (strongly disagree) to 5 (strongly agree) so that higher scores indicate higher perceived severity. the four items (cronbach's α = 0.71) were summed and averaged to derive a single perceived severity score. the perceived severity questions were only asked of the participants who indicated they did not believe they had previously had covid-19 disease. covid-19-related worry was assessed with a three-item scale modified from the literature [28, 29] . items assessed participants' worry related to getting covid-19 (e.g. "the possibility of getting infected in the future with covid-19 concerns me"). participants responded to each item on a 5-point likert scale from 1 = strongly disagree to 5 = strongly agree. the three items (cronbach's α = 0.82) were summed and averaged into a single scale with higher numbers indicating higher covid-19-related worry. as with the perceived severity variable, the covid-19-related worry questions were also only asked of the participants who indicated they did not believe they had previously had covid-19 disease. perceived personal threat of covid-19 disease was analyzed with two separate items: perceived likelihood of infection and perceived threat to physical health. perceived likelihood of infection was measured by asking participants "how likely do you believe it is that you will get infected with covid-19?" participants responded on a 5-point likert-type scale where 1 = not at all to 5 = extremely. perceived threat to physical health was measured by asking participants "if you got infected with covid-19, how threatening would it be to your physical health?" participants responded on a 5-point likert-type scale where 1 = not at all to 5 = extremely. perceived community threat was assessed with a single item, "do you think covid-19 infection is a major problem in your community?" with a binary yes/no response option. first, we described the study sample using n (%) or means and standard deviations. we then compared the "lower perceived effectiveness" and "higher perceived effectiveness" groups using chi-square or t-tests, as appropriate. any variable that was significant at p<0.01 in bivariate comparisons was included in an adjusted logistic regression model with the binary lower/ higher perceived effectiveness of covid-19 prevention measures as the outcome. we then used a backward selection process to create a reduced model with p<0.01 needed to stay in the model. because participants who believed they previously had covid-19 were not asked perceived severity and perceived susceptibility questions, those participants were excluded from the logistic regression analyses so we could understand perceived effectiveness of covid-19 prevention measures, while accounting for perceived severity and susceptibility, among those not previously thought to be infected. a total of 16,706 invitations were sent out for the survey, 4,042 people opened the survey, 351 indicated they did not wish to participate, and 42 indicated they were younger than 18 years. a total of 3,586 completed the survey and 3,474 answered all of the questions regarding the effectiveness of the recommended prevention measures and were included in the statistical analyses comparing the groups. the mean age of the sample was 45.5 (sd = 16.9); the sample was 47.3% male, 52.4% female, and 0.3% other. the majority of participants identified as non-hispanic white (62.8%), with other groups represented, including non-hispanic black/african american (15.3%), and hispanic (14.0%). participants were fairly evenly split by political ideology with 1,069 (31.9%) indicating their political views were liberal, 1,280 (38.2%) were moderate, and 1,005 (30.0%) were conservative. approximately one-tenth of participants (n = 360; 10.1%) indicated they believed they had previously had covid-19 disease. more than onethird personally knew someone who had covid-19, i.e., the person either had a positive sars-cov-2 test (n = 881; 24.9%) or they had symptoms, but were unable to get tested (n = 354; 10.0%). on average, perceived worry (m = 3.46; sd = 1.08) was higher than perceived severity of disease (m = 3.02; sd = 0.87) (p<0.0001) and most people thought covid-19 was a major problem in their community (n = 2037; 57.8%). for additional sample description, see table 1 . overall, perceived effectiveness of the six cdc recommended items was high. the lowest individual mean of perceived effectiveness value was for wearing a mask to prevent spreading the virus to others (m = 3.68; sd = 1.2) and the highest individual mean was for covering your mouth when you cough to prevent spreading the virus to others (m = 3.99; sd = 1.09). see fig 1 for a graphical representation of the mean scores for all six items. there were differences between the lower and higher perceived effectiveness groups on every healthcare and health belief variable ( , and lower scores on both altruism scales. a larger percentage of the higher perceived effectiveness group reported they did not believe they had been infected with covid-19 (75.7% vs. 70.8%; p = 0.004). while this was significant, as noted above, it was not included in the regression model due to the fact that only those who answered "no" were asked questions regarding severity and worry. however, whether they had received a test to check for covid-19 was asked of everyone and included in the regression model. a larger percentage of the lower perceived effectiveness group had ever received a test to check for covid-19 (14% vs 9% in the higher perceived effectiveness group; p<0.0001) but, of the ones that were tested, they had a lower positivity rate (22.6% vs. 36.2%; p<0.0001). there were some demographic characteristics that were not significantly different between groups, including geographic region (p = 0.052), race/ethnicity (p = 0.450), and whether there were children living in the home (p = 0.129). these variables, along with relationship status (p = 0.024) were not significant at p<0.01 and were not included in the logistic regression models. of the variables that were significant at p<0.01, we conducted further bivariate analyses to determine the odds of being in the higher perceived effectiveness group. these results can be found in table 2 for full regression results, see table 2 . in adjusted analyses, the following variables were not significant at p<0.01 and were removed from the model: having a health condition that would make covid-19 more severe (p = 0.870), high commitment altruism (p = 0.767), having been tested for covid-19 (p = 0.260), education (p = 0.190), income (p = 0.097), being employed in the healthcare field (p = 0.069), believing covid-19 is a problem in their community (p = 0.018), and employment status (p = 0.022). in the reduced model, the only demographic variables that remained were age, sex, and political views. age was positively associated with perceived effectiveness and as age increased, pre-existing condition that would make covid-19 more severe n/a this is among the first studies to examine the public's perception of the effectiveness of npis to prevent the spread of sars-cov-2. despite recent public health messaging attempting to encourage these behaviors [5] , there are media reports that only 65% of people wear masks in public stores and only 44% reported that most people in their communities are wearing masks [30] . furthermore, there have been reports that wearing a mask (or not wearing a mask) has now become a political statement [31] . it is important to understand the public's perceptions of the effectiveness of npis in preventing the spread of sars-cov-2. given the overall high rates of perceived effectiveness of the six behaviors we assessed, as well as a recent cdc study that found widespread support for most npi [32] , it appears the issue is not that people have low perceived response efficacy of npi to mitigate the spread of sars-cov-2. rather, due to the strong effect of perceived worry on the rest of the variables in the model, the issue may be to emphasize the threat of the disease to the person's physical health to highlight it is important to engage in these effective preventive health behaviors. given the strong association of perceived worry and the inverse association of perceived severity and likelihood of infection when accounting for worry, it appears there may be a strong influence of fatalistic beliefs for some individuals when it comes to preventing covid-19 disease. people may believe that infection is inevitable and, once worry is taken into account, increases in perceived severity and likelihood of infection are actually associated with a decrease in perceived effectiveness. this has sometimes been seen in other severe diseases. for example, recent research has shown there are people who have fatalistic beliefs in addition, it may be that working to increase engagement in these npis will need to involve a shift in public opinion and culture, particularly in the u.s. recent reports have shown significantly less opposition to wearing masks in certain countries where mask-wearing was already common, including japan, china, and south korea [35] . in contrast, in the u.s., the culture is highly individualistic and resistance to wearing masks appears to be related to claims that individual liberty is valued over communal well-being [36, 37] . indeed, one report demonstrated men, in particular, were resistant to wearing masks due to the perception that it made them appear weak [38] . our research did show men had lower perceptions of the effectiveness of these covid-19 prevention measures compared to women. furthermore, people with moderate and conservative political ideologies also had lower perception of npi effectiveness. groups with lower perceived effectiveness may be particularly important potential targets for interventions aimed at increasing adoption of npis to reduce the spread of sars-cov-2. another variable that was associated with perceived effectiveness was low commitment altruism, which addressed behaviors like donating money or clothing to a charity. it may be that this scale tapped into a general sense of social responsibility, which would explain why higher scores predicted greater perceived effectiveness of npi. without effective messaging resulting in a shift in public opinion and greater adoption of npis, we may not be able to effectively mitigate the spread of the virus until a vaccine is developed and widely adopted. the results should be interpreted in light of some limitations. first, these data are cross-sectional and causal relationships cannot be determined. related, quantitative data only provide a limited look at this topic, so future work should consider the use of qualitative methods as well. second, the data were collected at the beginning of may, 2020. while this is a rapid turnaround time between data collection and publication under normal circumstances, these are not normal circumstances. it is possible public perceptions of these behaviors have changed in the weeks since data were collected, especially given the recent moves by local governments to reopen public spaces and a resurgence of cases [39]. third, data are self-report and are subject to recall and social desirability biases. however, given the anonymous nature of the survey, we believe social desirability bias has been appropriately reduced. the covid-19 pandemic is unprecedented in our lifetimes. with a novel virus, there is no population immunity, limited effective treatments, and no prophylactic vaccinations. while there have been strides made in the last several months regarding treatment, the safest possibility for each individual and for the public as a whole involves slowing the spread through nonpharmaceutical interventions. this study found people generally perceived as highly effective the wearing of masks, social distancing, washing hands, and covering mouths when coughing or sneezing. however, perceptions of effectiveness varied significantly on the basis of demographics, covid-19-related experiences, and health beliefs. health communication should therefore focus on the importance of npi in protecting oneself as well as social contacts and work to depoliticize perceptions around wearing masks and social distancing. clinical features of patients infected with 2019 novel coronavirus in wuhan covid-19): how to protect 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ahead. the guardian trump says he knew coronavirus was 'deadly' and worse than the flu while intentionally misleading americans who's wearing a mask? women, democrats and city dwellers. the new york times americans social distancing decreases as coronavirus pandemic persists public response to the 2009 influenza a h1n1 pandemic: a polling study in five countries. the lancet infectious diseases public perceptions of non pharmaceutical interventions for reducing transmission of respiratory infection: systematic review and synthesis of qualitative studies trust during the early stages of the 2009 h1n1 pandemic public engagement as a means of restoring public trust in science-hitting the notes, but missing the music? protection motivation theory and social distancing behaviour in response to a simulated infectious disease epidemic putting the fear back into fear appeals: the extended parallel process model chapter 5 the health belief model the altruistic personality and the self-report altruism scale the dynamics of travel avoidance: the case of ebola in the u.s. tour manag perspect attitudes about human immunodeficiency virus immunization: the influence of health beliefs and vaccine characteristics. sexually transmitted diseases behavior and health beliefs as predictors of hiv testing among women: a prospective study of observed hiv testing most americans say they regularly wore a mask in stores in the past month; fewer see others doing it. pew research center face masks make a political statement in era of coronavirus public attitudes, behaviors, and beliefs related to covid-19, stay-at-home orders, nonessential business closures, and public health guidance-united states key: cord-296635-8r3tm966 authors: breed, andrew c.; breed, martin f.; meers, joanne; field, hume e. title: evidence of endemic hendra virus infection in flying-foxes (pteropus conspicillatus)—implications for disease risk management date: 2011-12-14 journal: plos one doi: 10.1371/journal.pone.0028816 sha: doc_id: 296635 cord_uid: 8r3tm966 this study investigated the seroepidemiology of hendra virus in a spectacled flying-fox (pteropus conspicillatus) population in northern australia, near the location of an equine and associated human hendra virus infection in late 2004. the pattern of infection in the population was investigated using a serial cross-sectional serological study over a 25-month period, with blood sampled from 521 individuals over six sampling sessions. antibody titres to the virus were determined by virus neutralisation test. in contrast to the expected episodic infection pattern, we observed that seroprevalence gradually increased over the two years suggesting infection was endemic in the population over the study period. our results suggested age, pregnancy and lactation were significant risk factors for a detectable neutralizing antibody response. antibody titres were significantly higher in females than males, with the highest titres occurring in pregnant animals. temporal variation in antibody titres suggests that herd immunity to the virus may wax and wane on a seasonal basis. these findings support an endemic infection pattern of henipaviruses in bat populations suggesting their infection dynamics may differ significantly from the acute, self limiting episodic pattern observed with related viruses (e.g. measles virus, phocine distemper virus, rinderpest virus) hence requiring a much smaller critical host population size to sustain the virus. these findings help inform predictive modelling of henipavirus infection in bat populations, and indicate that the life cycle of the reservoir species should be taken into account when developing risk management strategies for henipaviruses. hendra virus (hev) and nipah virus (niv) are paramyxoviruses of the genus henipavirus with pteropid bats (i.e. flying-foxes; pteropus sp., family pteropodidae) being the primary wildlife reservoir [1] . evidence of henipavirus infection has been found across the range of pteropid bats from eastern australia, southeast asia, bangladesh, india and madagascar [2] . henipavirus infection has also been found to be present in eidolon helvum, a species of fruit bat (family pteropodidae) that occurs throughout sub-saharan africa [3, 4] . the potential to cross species boundaries from bats to domestic animals and humans causing fatal infection appears to be a consistent feature of henipaviruses wherever they have caused disease (australia and asia). given that over two billion people live in the area where pteropus or eidolon bats are present, even sporadic spillover to humans may result in a significant number of human infections. henipaviruses have the potential to infect a wide range of mammalian species, and hendra virus has spread from flying-foxes to horses in australia on at least 20 reported separate occasions (five involving horse-human transmission), most recently in 2011 [5, 6, 7] . seven humans have become infected with hev via contact with infected horses, resulting in four fatalities [5, 8, 9] . in peninsular malaysia and singapore during 1998 and 1999, nipah virus infected pigs and humans resulting in the death of over 100 humans and the culling of over one million pigs [10] . since that time, there have also been at least 10 outbreaks of niv disease in humans in bangladesh and india, with the resultant death of over 140 people. there is also clear evidence of humanto-human transmission of niv [11, 12] . in spite of the major health concerns, the knowledge of the epidemiology and ecology of these viruses is limited [1, 13, 14] . how the viruses are maintained in bat populations is not fully understood, nor is how the viruses avoid extinction as their host species become immune. in addition, whether these viruses are predominantly horizontally or vertically transmitted is also uncertain [12, 15, 16] , with the apparent viral latency and recrudescence in some human hev and niv infections suggesting that henipavirus infection dynamics may differ significantly from the closely related morbilliviruses [17] . a previous study by plowright et al. [14] on the infection dynamics of hev in the little red flying-fox, pteropus scapulatus, in the northern territory of australia suggested that viral transmission may be predominantly horizontal, with pregnancy and lactation suggested as risk factors for infection. however, plowright et al. [14] sampled multiple colonies over time, leaving the possibility that sampling was not confined to a single population. here, we focus on the transmission of hev in a single colony of flying-foxes over a 25-month period that was approximately 10 km from the location of a spillover of infection to a horse and human in october 2004 [5, 8] . we sampled the spectacled flying-fox, pteropus conspicillatus, a species which is restricted in distribution to the wet tropics bioregion of north queensland [18] . we present data on the infection dynamics of hev within this flying-fox colony over the 25-month period. we investigated the association of antibody response to the lifecycle stage of the host and the hypothesis that hev is maintained by episodic infection with periodic virus outbreaks taking place. a total of 521 pteropus conspicillatus were sampled over the six sampling sessions with an overall seroprevalence to hendra virus of 56% (95% c.i. 51-60). the logistic regression model that included age, sampling session, sex, reproductive status and weight best fitted variance in seroprevalence, thus these variables were analysed in more depth (daic c = 0.00; v i = 0.57; all other models daic c .2 and v i ,0.2; appendix 1 in supplementary information). models that included two-and/or three-way interactions had daicc.5, thus were not investigated further. weight and forearm length predictor variables were highly correlated (r 2 = 0.72), but the model that included forearm length and not weight had limited support (table s1) . temporal variation. seroprevalence steadily increased over the six sampling sessions from 44.7% in january 2005 to 69.4% in february 2007 ( figure 1 ; table s2 ). variation with sex and reproductive status. seroprevalence of female bats did not differ significantly from male bats (female: 58.7%; male: 53.7%; log binomial regression p = 0.25; table 1 ). however, pregnant females had a significantly higher seroprevalence than both males and all other female bats (pregnant females: 70.3%; all other bats: 54.6%), and were 1.3 times more likely to have antibodies to hev than the rest of the bats sampled (95% ci: 1.03-1.61; log binomial regression p,0.05). bats sampled in early lactation had a significantly higher seroprevalence than male and all other female bats (early lactation: 75.0%; all other bats: 54.9%). hence, those in early lactation were 1.4 times more likely to have hev antibodies than the others sampled (95% ci: 1.05-1.78; log binomial regression p,0.05). when seroprevalence was compared among the reproductive categories of adult females, pregnant bats and those in early lactation had a significantly higher seroprevalence than nonreproductive adult females (pregnant: 26 variation with age. seroprevalence was highest in bats of the adult category (60.3%), followed by juveniles (58.3%), while sub-adults had significantly lower seroprevalence (39.8%) than both adults (logistic regression p,0.001) and juveniles (logistic regression p,0.05; figure 2 ; table s3 ). variation with bodyweight and forearm length. seroprevalence did not show statistically significant variation with bodyweight and forearm length although a non-significant linear trend was observed between seroprevalence and bodyweight (table s4 ). hendra virus antibody titre levels were determined by serial dilution of the sera in the virus neutralisation test. significant differences in titre levels were found according to sampling session (kruskal-wallis test p,0.0001), bodyweight (kruskal-wallis test p,0.0001), pregnancy status (wilcoxon test p,0.001) and sex (wilcoxon test p,0.01; table s5 ). age, forearm length and lactation status were not significant risk factors for antibody titre level (kruskal-wallis and wilcoxon tests p.0.05). antibody levels varied greatly across the sampling sessions with animals sampled in january 2005 having a median titre of 15 (mean rank 25.7) followed by consistent increases to a peak median titre of 80 (mean rank 205.6) in september 2006, followed by a drop to 30 (mean rank 58.8) in february 2007, the final sampling session. individuals of greatest bodyweight (.850 g) had the lowest antibody titres with a median of 20 (mean rank of 113.3). females had significantly higher antibody titres than males (median titres 40 and 20; mean ranks 161.2 and 134.2, respectively) with the highest titres observed for pregnant females (median titre 80; mean rank 197.2). those in any stage of lactation did not show a significantly higher antibody titre than the rest of the bats sampled. previous studies have suggested that henipaviruses are maintained in flying-fox populations through episodic infection in a metapopulation structure, and do not persist endemically within a single population [14, 19] (see figure 1 , panel c). our findings do not support this hypothesis, but support an alternative pattern of endemic infection in the population. this endemic infection dynamic is consistent with a study on viral excretion of niv in pteropus lylei in thailand, where seasonal excretion of virus was observed to occur from the same small colonies each year [12] . our findings on hev antibody titre levels show a peak in september 2006 (median titre = 80; mean rank = 205.6) when all adult female bats sampled were at a late stage of pregnancy. this is plausibly consistent with a ''boosted'' immune response subsequent to the previous sampling session (march 2006: median titre = 40; mean rank = 73.96). the following sampling session showed a decrease in titre level (february 2007: median titre = 30; mean rank = 58.8), but an increase in seroprevalence from 62.1% in september 2006 to 69.4%. this finding is consistent with a period of increased viral transmission during late pregnancy that had resolved by the time the majority of females were lactating, as evidenced by the consequent increased seroprevalence. indeed pourrut et al., [20] have suggested that altered immune function in late pregnancy may cause a transient surge in viral replication of filoviruses in african fruit bats. our finding that late pregnancy and lactation were risk factors for hev seropositivity are concordant with results presented by plowright et al. [14] on p. scapulatus. furthermore, the reproductive cycle in other bat species has been linked to seropositivity and viral activity of filoviruses, coronaviruses, lyssaviruses and astroviruses [20, 21, 22] . our study identified the highest seroprevalence in the first few weeks of lactation, indicated by a seroprevalence of 75.0% in females carrying pups ( figure 2 ). our study also supports the conclusions of field (unpublished data) and plowright et al. [14] that maternal transfer of hev antibodies to juveniles likely occurs, evidenced by the higher seroprevalence of juveniles (58.3%) than sub-adults (39.9%), and a correspondingly higher seroprevalence of adults (60.3%). these findings are consistent with horizontal transmission of the virus, however the observed seroprevalence pattern does not preclude the occurrence of vertical transmission. vertical transmission may contribute to viral persistence in bat populations, and there is evidence that vertical transmission of hev occurs from experimental infection studies of flying-foxes and guinea pigs [15] and from natural infection in wild flying-foxes (field unpublished data) [16] . numerous viruses can be transmitted both horizontally and vertically (e.g. transplacentally), including human polyoma virus, bovine viral diarrhoea virus, feline leukaemia virus and parvoviruses (porcine, canine and feline) [17] . for females to be classified as adult in our study they must have shown signs of prior lactation (i.e. enlarged nipples; figure 2), and hence a previous pregnancy and lactation. our finding that hev seroprevalence in early lactation was significantly higher than adult females that were not pregnant or lactating (early lactation; 75.5%; not pregnant or lactating: 48.7%; p-value = 0.047) is evidence for a decline in hev seroprevalence in females in the life stage following lactation. such a decline suggests that detectable immunity to hev is not long lived in p. conspicillatus, and the pattern seen may reflect seasonal variation in response to repeated exposure. this variation is contrary to the assumption that hev induces long-lived detectable immunity in p. conspicillatus and p. poliocephalus (e.g. [14] ), and suggests that the transmission dynamics of henipaviruses may be different to those of the closely related morbilliviruses. indeed, the mechanism of survival of henipaviruses at the population level appears more likely to be one of endemic infection, perhaps similar to that found in bovine viral diarrhoea virus, classical swine fever or some herpes viruses utilising persistent infection, or vertical transmission, as found in arenaviruses or retroviruses [17] . these patterns of infection require much smaller critical host population sizes, in contrast to viruses that demonstrate an acute self-limiting episodic infection pattern determined by: a build-up of susceptibles, introduction of virus, and environmental conditions that promote spread (e.g. measles, newcastle disease virus or canine distemper virus; [17] ; see figure 1, panel b) . a previous serial cross-sectional study by plowright et al. [14] over a 16-month period on little red flying-foxes, p. scapulatus, sought to determine the factors that drive hev spillover. their study suggested that age, sex, body size, pregnancy, lactation, season and mating behaviour were possible risk factors for hev infection, and that horizontal transmission was the major mode of transmission between individuals. they also reported a rapid seroprevalence decline between two successive sampling sessions. however, given their sampling at multiple locations, the expansive geographic distribution and highly nomadic nature of p. scapulatus [23] , it is plausible that they were not sampling the same population over time. in contrast, the species in our study, p. conspicillatus, is not a nomadic species and has a restricted distribution to the wet tropics of northeast queensland [18] . furthermore, our study was conducted within 10 km of a location where an hev outbreak occurred in october 2004 [5] , and we collected samples from a single colony of p. conspicillatus on six separate occasions over a 25-month period. consequently, we are confident that we followed the infection dynamics of a single population of p. conspicillatus over the study period. nonetheless, interpretation of results from studies in wild animal populations should be made with care. capture of bats by mist-netting provides a statistically non-random sample of the population, and the practicalities of sampling from a roost site of many thousands of individuals also precludes following individuals over time. to counter these issues, we sought to investigate potential bias and confounding effects where possible. future studies on henipavirus infection dynamics in wild bats may benefit from: permanent marking of individuals to identify possible repeated capture and sampling of some animals; improved diagnostic capabilities to increase the probability of detection of viral shedding; and improved telemetry methods to enhance the understanding of movement of individuals between roost locations. our findings do not support the episodic infection hypothesis for hev persistence in our study population. rather we suggest that endemic infection of p. conspicillatus occurs, perhaps with periodic pulses of viral transmission associated with late pregnancy and early lactation. the consistent increase in seroprevalence over the duration of our study, together with increasing titres over the first five sessions followed by a drop in titre in the last session, also suggest the presence of inter-annual factors may be affecting viral transmission. an increase in viral transmission associated with pregnancy in flying-foxes is plausibly concordant with the temporal pattern of some hev incidents in horses in australia [5] , and of niv outbreaks in humans in bangladesh and india [12] . this pattern suggests that the risk of henipavirus transmission from flying-foxes to domestic animals and/or humans is higher during the gestation period of flying-foxes. thus, it is plausible that spillover risk may be uniformly spatially distributed wherever pregnant flying-foxes are present. the observed spatial clustering of henipavirus incidents may be confounded by: surveillance intensity (passive surveillance is the only method used, with heightened awareness of disease likely in areas where previous incidents have occurred); variation in flying-fox population density (there is evidence of increasing movement of flying-foxes in eastern australia into urban and rural areas [24] ); variation in horse density and husbandry practices; and as yet unidentified predisposing ecological or environmental factors (e.g. climate). a scenario of persistent henipavirus infection with viral latency and recrudescence in flying-foxes has been proposed by field (unpublished data) and sohayati et al., [25] . viral latency and recrudescence has also been shown to occur in a human hev case [26] , and at least 12 human niv cases [27] . this infection dynamic could lead to the endemic infection pattern seen in our study. however, it is plausible that different social structures of host populations (e.g. panmixia, metapopulation, seasonal aggregation) may favour different mechanisms of maintaining infection at the population level (e.g. predominantly horizontal transmission, predominantly vertical transmission, seasonal explosive outbreaks, repeated viral excretion by persistently infected individuals). consequently, population structures and mechanisms of maintenance of infection may reflect the biology of the host species and level of ecological disruption, rather than only the biology of the virus. as such, it is likely that different host populations may have varying levels of risk of infection spillover to domestic animals and/or humans. an improved understanding of the infection dynamics of henipaviruses in bat populations should facilitate the development of effective risk management strategies for disease spillover from bats to domestic animals and/or humans. here we show that hev infection in a population of pteropus conspicillatus is likely to be endemic rather than episodic, as previously proposed for hev in flying-foxes. we also present evidence for seasonal viral activity suggesting that immunity to the virus may wax and wane on a seasonal basis. these findings should inform disease risk management and approaches for modelling henipavirus infection in bat populations. if the ongoing threat that these viruses pose to public health is to be mitigated, further work is required to clarify the principal mode(s) of transmission of henipaviruses in bats, and to comprehensively determine how these viruses persist in their reservoir hosts. all study animals were captured and sampled at the gordonvale roost site (145u46.749e, 17u4.869s ). this site is in a 4.5 hectare mixed woodland and forest remnant, surrounded by sugarcane plantations and suburban housing. it has been permanently occupied by pteropus conspicillatus for at least ten years, usually containing 40,000 to 60,000 individuals, constituting approximately 20% of the australian population of this threatened species [18, 28] . this is the closest known flying-fox colony to the property where the hev spillover event occurred in october 2004 [5] . the australian population of p. conspicillatus is geographically isolated from other populations of this species in northern papua new guinea and the moluccan islands of indonesia [29] . flying-foxes were caught in mist nets, anaesthetised with isoflurane (isoflurane, laser animal health pty limited) and oxygen via an anaesthetic machine using the protocol of johnson et al. [30] for sampling. data collected were sex, bodyweight, forearm length, approximate age and reproductive status according to descriptions detailed in table s6 . animals were marked with coloured acrylic lacquer on their hind claws to prevent resampling within the same session, and were then released at the site of capture after recovery from anaesthesia. age classification of sampled bats was performed as described by hall and richards ([23] ; figure 3 ; table s6 ). bats being carried by their mother were classified as juvenile (estimated age 0 to 3 months old; figure 3 , a). free flying bats that lacked signs of sexual maturity (e.g. small or non-descended testes in males; lack of enlarged nipples in females) were classified as sub-adults (estimated age 3 months to 2 years; figure 3 , b, c). bats that showed signs of sexual maturity (e.g. large and descended testes in males; visibly enlarged nipples indicating a previous pregnancy and suckling of young in females) but did not show signs of severe wear on all molar teeth were classified as adults (estimated age 2 to 8 years; figure 3 , d, e, f). bats that showed signs of severe molar wear on all molar teeth, including at least two molars worn to the level of the gingiva, were classified as aged (estimated age 8 years and older; figure 3 , g). female bats were further classified according to their reproductive status (table s6 ). if a foetus could be palpated while anaesthetised bats were classified as pregnant (this is likely to represent females in the last trimester of pregnancy; [31] ). bats from which milk could be expressed from their teats and were captured carrying a young were classified as early lactating. bats from which milk could be expressed from their teats, but were not carrying young, were classified as late lactating (p. conspicillatus carry their young continuously for the first month after birth, after which time the young are left in a crèche while the females forage for the remainder of lactation [20] ). bats that were not classified into any of the previous three categories, which would include females in earlymid pregnancy, were classified as non-reproductive. blood samples were collected by venepuncture of the propatagial vein with a 23 or 25 gauge needle and 1 ml or 3 ml syringe depending on the animal size. blood was allowed to clot in 2 ml tubes for 24 hours before centrifugation and separation of serum and storage at 4uc. antibody titres to hendra virus were determined by virus neutralisation test (vnt) according to [32] at the australian animal health laboratory (geelong, victoria); the world organisation for animal health (oie) reference laboratory for hendra and nipah virus diseases. the tests were carried out under biosafety level 4 conditions as hendra virus is a dangerous human pathogen with a high case fatality rate and for which there is no vaccine or effective treatment [33] . a serum sample was considered positive if it neutralised 100 tcid 50 of hev at a dilution of 1:10 or greater in the vnt, since bat sera at lower dilutions have produced a high rate of toxic or non-specific reactions on neutralisation tests. to investigate the association of potential risk factors with serostatus, data were analysed by multivariate logistic regression. we used akaike's information criterion corrected for small sample sizes (aic c ) for model selection [34] . models were ranked according to the difference between the best-fitting model and the aicc value of model i (daicc). the strength of evidence for alternative models was measured by aicc weights (v i ). for the potential risk factors identified by multivariate logistic regression to be important in explaining variation in serostatus, we performed log binomial regression analyses to further analyse their associations with serostatus. the relative risk of being seropositive was determined for these predictor variables. due to the smaller sample sizes, fisher's exact test was used to investigate the hypothesised effect of reproductive status on serostatus in adult females, where serostatus of non-reproductive adult females was compared with those categorised as either pregnant, early lactation or late lactation. as titre levels were not assumed to conform to an a priori distribution, two measures appropriate for comparing titre data among groups where serial dilution of sera produce logarithmic dilutions were used. these measures were the median titres with an interquartile range, to indicate statistical dispersion, and the mean rank titres, which indicates the mean rank value for the titres of animals within a particular category when all animals are ranked according to titre level. subsequently, non-parametric models were fitted to the data. for risk factors with two levels, a simple wilcoxon test was performed. for risk factors with three or more levels, a kruskal-wallis test was performed. all animal work followed the guidelines of the american society of mammalogists guidelines [31] table s1 model selection results for the effects of risk factors and the seroprevalence. the model selection statistics are: number of parameters in model (k), akaike's information criterion corrected for small sample sizes (aic c ), difference between model i and the model with the smallest aic c (daic c ), aic c weights (v i ) and evidence ratios (v i /v j ). only models with daic c ,5 are shown. (doc) figure 3 . features for classification of age categories of pteropus conspicillatus used in this study. age classification features (highlighted by a red circle in b, c, d, e) for both sexes. key features include: juvenile bats (a) carried by their mother (estimated age 0 to 3 months old); sub-adult bats (b, c) were free flying that lacked signs of sexual maturity, including the lack of enlarged nipples for females (b) or small or non-descended testes for males (c; estimated age 3 months to 2 years); adults bats (d, e, f) showed signs of sexual maturity, including visibly enlarged nipples indicating a previous pregnancy and suckling of young in females (d) or large and descended testes in males (e), and but did not show signs of severe wear on all molar teeth (f; estimated age 2 to 8 years); aged bats (g) showed signs of severe molar wear on all molar teeth, including at least two molars worn to the level of the gingiva (estimated age 8 years and older). doi:10.1371/journal.pone.0028816.g003 the natural history of hendra and nipah viruses nipah virus: impact, origins, and causes of emergence evidence of henipavirus infection in west african fruit bats henipavirus rna in african bats epidemiological perspectives on hendra virus infection in horses and flying foxes hendra virus outbreak with novel clinical features queensland government, department of primary industries and fisheries hendra virus infection in a veterinarian human hendra virus encephalitis associated with equine outbreak nipah virus: a recently emergent deadly paramyxovirus person-to-person transmission of nipah virus in a bangladeshi community a longitudinal study of the prevalence of nipah virus in pteropus lylei bats in thailand: evidence for seasonal preference in disease transmission. vector borne zoonotic diseases the ecology of hendra virus and australian bat lyssavirus reproduction and nutritional stress are risk factors for hendra virus infection in little red 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novel paramyxovirus transmitted from horses relapsed and late-onset nipah encephalitis dietary variation in spectacled flying foxes (pteropus conspicillatus) of the australian wet tropics field anaesthesia of three australian species of flying fox some aspects of female reproduction in the greyheaded flying-fox, pteropus poliocephalus (megachiroptera: pteropodidae) laboratory diagnosis of nipah and hendra virus infections hendra and nipah virus diseases. manual of diagnostic tests and vaccines for terrestrial animals model selection and multimodel inference for assistance with fieldwork and provision of photographs we thank les hall, carol de jong, tim kerlin, jonathan lee, lana little, jenny maclean, maria marklund, billie roberts, jack shield, craig smith, justin westrum and the australian quarantine and inspection service. for assistance with diagnostic test result interpretation we thank kim halpin and greer meehan. key: cord-288440-w7g2agaf authors: jia, rui; ding, shilei; pan, qinghua; liu, shan-lu; qiao, wentao; liang, chen title: the c-terminal sequence of ifitm1 regulates its anti-hiv-1 activity date: 2015-03-04 journal: plos one doi: 10.1371/journal.pone.0118794 sha: doc_id: 288440 cord_uid: w7g2agaf the interferon-inducible transmembrane (ifitm) proteins inhibit a wide range of viruses. we previously reported the inhibition of human immunodeficiency virus type 1 (hiv-1) strain bh10 by human ifitm1, 2 and 3. it is unknown whether other hiv-1 strains are similarly inhibited by ifitms and whether there exists viral countermeasure to overcome ifitm inhibition. we report here that the hiv-1 nl4-3 strain (hiv-1(nl4-3)) is not restricted by ifitm1 and its viral envelope glycoprotein is partly responsible for this insensitivity. however, hiv-1(nl4-3) is profoundly inhibited by an ifitm1 mutant, known as δ(117–125), which is deleted of 9 amino acids at the c-terminus. in contrast to the wild type ifitm1, which does not affect hiv-1 entry, the δ(117–125) mutant diminishes hiv-1(nl4-3) entry by 3-fold. this inhibition correlates with the predominant localization of δ(117–125) to the plasma membrane where hiv-1 entry occurs. in spite of strong conservation of ifitm1 among most species, mouse ifitm1 is 19 amino acids shorter at its c-terminus as compared to human ifitm1 and, like the human ifitm1 mutant δ(117–125), mouse ifitm1 also inhibits hiv-1 entry. this is the first report illustrating the role of viral envelope protein in overcoming ifitm1 restriction. the results also demonstrate the importance of the c-terminal region of ifitm1 in modulating the antiviral function through controlling protein subcellular localization. interferon inhibits virus infection by inducing the expression of hundreds of host genes, known as interferon-stimulated genes (isgs), some of which encode antiviral effectors [1] . among these antiviral proteins are a small protein family called interferon-inducible transmembrane (ifitm) proteins. ifitms have an average length of 130 amino acids, contain two predicted transmembrane domains and a conserved intracellular domain [2, 3] . although ifitm1 was shown to modestly inhibit vesicular stomatitis virus (vsv) and hepatitis c virus (hcv) more than a decade ago [4, 5] , the antiviral function of ifitm1, 2 and 3 was established only when their potent restriction of influenza a virus, west nile virus and dengue virus was reported in a genome-wide functional screen [6] . subsequent studies revealed that many viruses are susceptible to ifitm restriction. these include flaviviruses (west nile virus, dengue virus, and yellow fever virus), filoviruses (marburg virus and ebola virus), sars coronavirus, reovirus, rift valley fever virus, human immunodeficiency virus type 1 (hiv-1), jaagsiekte sheep retrovirus (jsrv), and others [6, 7, 8, 9, 10, 11, 12, 13] (reviewed in [14, 15] ). the in vivo importance of ifitm proteins in antiviral defense is supported by their protection of mice from influenza a virus infection [16, 17, 18] . in support of this, single nucleotide polymorphisms in ifitm3 gene have been shown to associate with pathogenesis severity of influenza a virus infection in humans [16, 19] . humans have five ifitm genes, including ifitm1, ifitm2, ifitm3, ifitm5 and ifitm10. they are clustered within a 26.5kb region on chromosome 11 except for ifitm10 that is located 1.4 mb downstream [2, 20, 21] . ifitm5 has a calcium-binding domain at its c-terminal region and is involved in bone mineralization and maturation [22] . ifitm5 has thus also been named bone restricted ifitm-like protein (bril). ifitm1, ifitm2 and ifitm3 are expressed in a variety of tissues. their expression is stimulated by type i and type ii interferon due to the presence of the interferon stimulation response element (isre) in their promoters [2, 23] . in addition to their roles in antiviral defense, ifitm1, ifitm2 and ifitm3 have also been reported to participate in antiproliferative signaling, cell adhesion, and oncogenesis (reviewed in [21] ). for instance, expression of these three ifitm members is significantly upregulated in colorectal tumor cells concurrent with the activation of the wnt/beta-catenin signaling pathway [24] . the function of ifitm10 is unknown except that it is the most conserved among all ifitm proteins across many eukaryotic species [20, 25] . ifitm1, 2 and 3 proteins differ in their abilities to inhibit different viruses. for example, ifitm3 inhibits influenza a virus more potently than inhibits marburg and ebola viruses; in contrast, these latter two viruses are strongly inhibited by ifitm1 [11] . further, ifitm2 and ifitm3, but not ifitm1, inhibit rift valley fever virus [7] . this different restriction profile is likely a result of the sequence divergence between ifitm1, 2 and 3. ifitm2 and 3 share higher sequence homology between each other than with ifitm1 [21] . in this study, we report that human ifitm1 differentially affects the replication of two closely related hiv-1 strains bh10 and nl4-3 and that the viral envelope glycoprotein is responsible for this differential sensitivity to ifitm1 restriction. interestingly, deleting the c-terminal sequence of ifitm1 allows the mutant to potently inhibit both bh10 and nl4-3 viruses, which highlights the important role of this c-terminal domain in regulating the antiviral function of ifitm1. human ifitm1 inhibits the replication of hiv-1 bh10 but not hiv-1 we have previously shown that ifitm1, 2 and 3 inhibit the replication hiv-1 strain bh10 in supt1 cells [10] . when we tested another hiv-1 strain called nl4-3, ifitm1 did not exhibit any inhibitory effect (fig. 1a) , which was in contrast to the marked suppression of hiv-1 nl4-3 replication by ifitm2 and 3 (s1 fig.) . this observation was further confirmed by the resistance of an hiv-1 nl4-3 -derived virus called nleny1-ires to ifitm1 (fig. 1a) . human ifitm1 has a relatively long c-terminal region than ifitm2 and 3. we previously showed that ifitm1 mutants lacking this c-terminal sequence still strongly inhibit hiv-1 bh10 (fig. 1b and 1c ) [10] . given the resistance of hiv-1 nl4-3 to the wild type human ifitm1, we expected that hiv-1 nl4-3 would also be refractory to the c-terminus truncated ifitm1. surprisingly, ifitm1 mutants δ(117-125), δ(112-125) and δ(108-125) all strongly inhibited hiv-1 nl4-3 replication ( fig. 1b and 1c) . these data suggest that the c-terminal sequence of ifitm1 negatively modulates the anti-hiv-1 function of ifitm1. deleting the c-terminal sequence of ifitm1 leads to inhibition of hiv-1 entry we have previously shown that, in contrast to ifitm2 and 3, human ifitm1 does not affect the entry of hiv-1 bh10 [10] . similarly, no inhibitory effect of ifitm1 on hiv-1 nl4-3 entry was observed ( fig. 2a) . interestingly, the c-terminal truncated ifitm1 mutants, δ(117-125), δ(112-125) and δ(108-125), diminished hiv-1 nl4-3 entry by 2 to 3-fold ( fig. 2a and 2b) . this inhibition appears to be specific to hiv-1, since neither the wt ifitm1 nor its c-terminal truncations affected entry that was mediated by the g protein of vesicular stomatitis virus (vsv) ( fig. 2a and 2b) . in order to understand how the c-terminal sequence of ifitm1 might regulate its ability to impair hiv-1 entry, we examined the cellular localizations of ifitm1 and its c-terminal deletion mutants by immunofluorescence and confocal microscopy. the wild type ifitm1 exhibited a predominant intracellular distribution and showed co-localization with the early endosome marker rab5 (fig. 3a) . removing the last 9 or 14 amino acids from c-terminal sequence re-localized the majority of ifitm1 to the cell periphery (fig. 3a) , indicating that these c-terminus truncated ifitm1 mutants are mostly positioned at the plasma membrane where hiv-1 entry occurs. it was also noteworthy that the δ(18-125) deletion mutant exhibited an intracellular localization similar to the wild type ifitm1 (fig. 3a ), yet δ(18-125) markedly diminished hiv-1 entry (fig. 2 ). this suggests that the δ(18-125) mutant may employ a distinct mechanism to deter hiv-1 entry. we next asked how the c-terminal sequence of ifitm1 modulates protein subcellular distribution. no apparent endocytic sorting signals are present within the sequence 117-qii-qekrgy-125 of ifitm1, which was deleted in δ(117-125). one possibility is that this sequence may serve as a retention signal to sequester ifitm1 in the endoplasmic reticulum (er) or golgi [26] . this hypothesis is refuted by the results showing that blocking endocytosis with the dynamin inhibitor dynasore caused relocation of ifitm1 to the plasma membrane, as opposed to a ifitm1-kdel variant that had the er retention signal kdel inserted at the cterminus and exhibited er localization even in the presence of dynasore (fig. 3b) . these data suggest that ifitm1 is an endocytic protein and that removal of its c-terminal sequence causes its relocation to the plasma membrane where it interferes with hiv-1 entry. mouse ifitm1 diminishes hiv-1 entry ifitm1 orthologs from different species are highly conserved, except for mouse and rat ifitm1 that have shortened c-termini (fig. 4a ). these two ifitm1 proteins are 19 and 16 amino acids shorter at their c-termini compared to the human ifitm1. this prompted us to test whether they would act like c-terminal truncations of human ifitm1 and inhibit hiv-1 deletion mutations are indicated. both the wild type and mutated ifitm1 have a flag tag attached to the n-terminus. levels of the wild type and mutated ifitm1 in stably transduced supt1 cells were examined by western blotting. an amount of 500 μg/ml doxycycline was used to induce ifitm1 expression. levels of tubulin were probed as internal controls. (c) replication of nl4-3 and bh10 in supt1 cells stably expressing ifitm1 mutants δ(117-125), δ(112-125) or δ(108-125). virus production was determined by measuring levels of viral rt activity in culture supernatants. a representative result of three independent infections is shown. fig. 4b show that mouse ifitm1 suppressed hiv-1 replication in supt1 cells. further, mouse ifitm1 markedly decreased the entry of hiv-1 but not the entry that was mediated by vsv g protein ( fig. 4c and 4d ). these data further highlight the importance of the c-terminal sequence of ifitm1 in modulating its antiviral function and also suggest the greater antiviral potential of mouse ifitm1. we next performed mutagenesis to identify which viral protein(s) accounts for the resistance of hiv-1 nl4-3 to human ifitm1. to this end, we generated chimeric viruses by exchanging dna fragments between hiv-1 bh10 and hiv-1 nl4-3 and tested which viral dna fragment(s) bears the genetic information that determines the resistance of hiv-1 nl4-3 to ifitm1 (fig. 5a) . the results showed that exchange of the 2.7kb dna fragment, located between the sali and bamh1 restriction sites (called sb fragment), allowed bh(sb) resistant to ifitm1 and, reciprocally, nl(sb) sensitive to ifitm1 inhibition (fig. 5b ). this sb dna fragment encodes four viral proteins, tat, rev, vpu and env. to further determine which viral protein plays the countering role, we exchanged between hiv-1 bh10 and hiv-1 nl4-3 only the env sequence. the results of infection experiments showed that hiv-1 nl4-3 env enabled hiv-1 bh10 to replicate in ifitm1-expressing cells, whereas inserting the env of hiv-1 bh10 into hiv-1 nl4-3 dramatically reduced virus replication in the presence of ifitm1 (fig. 5c) , supporting the role of nl4-3 env in overcoming ifitm1 inhibition. no significant effect in this regard was observed for the small dna fragment that is located between the sali site and the beginning of env (fig. 5c ). hiv-1 nl4-3 is more efficient than hiv-1 bh10 in cell-to-cell transmission one mechanism behind the ifitm1 inhibition of hiv-1 bh10 is the reduction in gag/p24 levels in the infected cells as well as the diminution in virus production ( fig. 6a and 6b ) [10] . similar defects were also observed for hiv-1 nl4-3 infection of ifitm1-expressing supt1 cells ( fig. 6c and 6d ), which indicates that hiv-1 nl4-3 escapes ifitm1 in the spread infection without the need to restore this decrease in gag/p24 expression. we recently reported that hiv-1 bh10 became resistant to ifitm1 restriction in the spread infection through acquiring mutations in viral env and vpu proteins that together enhance the virus transmission between cells [27] . in order to test whether the same mechanism supports the resistance of hiv-1 nl4-3 to ifitm1, we compared the cell-to-cell transmission efficiency of hiv-1 bh10 and hiv-1 nl4-3 . indeed, hiv-1 nl4-3 was 3-fold more efficient than hiv-1 bh10 in transmitting between supt1 cells (fig. 7a ). this phenotype was readily reversed when the env sequence was exchanged between hiv-1 bh10 and hiv-1 nl4-3 , either by exchanging the sb fragment or the env sequence alone (fig. 7a) . similar observations were made for virus transmission from control supt1 cells to supt1 cells expressing ifitm1 (fig. 7b) . collectively, these data suggest that the env protein of hiv-1 nl4-3 mediates greater cell-to-cell transmission, a property that may allow hiv-1 to escape ifitm1 inhibition under the virus spread infection condition. transfected with plasmid dna expressing the wild type ifitm1 or its variant ifitm1-kdel that has the er retention signal attached to the c-terminus. cells were either treated with dynasore (160 μm) or dmso as control. ifitm1 and ifitm1-kdel were detected by immunostaining with anti-flag antibody. the endogenous calreticulin was stained with anti-calreticulin antibody. nuclei were stained with dapi. images shown represent the major subcellular distribution of each protein. this study was based on the observation that two closely related hiv-1 subtype b strains bh10 and nl4-3 were differentially inhibited by human ifitm1 in virus spread infection. this finding allowed us to identify the viral component(s) in nl4-3 that gave rise to ifitm1 resistance. results of mutagenesis and virus replication studies revealed an essential role of viral env protein in hiv-1 nl4-3 evasion from ifitm1. exchanging the env sequences between bh10 and nl4-3 reversed the susceptibility of the parental strains to ifitm1 inhibition. this role of hiv-1 env protein in determining viral sensitivity to ifitm1 corroborates our recent report showing that hiv-1 bh10 was able to develop resistance to ifitm1 through mutating env and vpu [27] . there are precedents illuminating the function of viral env in overcoming host restriction. one example is the hiv-2 env that antagonizes tetherin [28] . in addition, hiv-1 env has been shown to regulate the establishment of latent infection in resting cd4+ t cells [29] , likely through modulating the functionality of certain cellular pathways [30] . in the case of ifitm1, it remains to determine whether hiv-1 env serves as the viral target of ifitm1 or env acts as a viral antagonist to counter ifitm1. although ifitm1 does not affect the entry of either hiv-1 bh10 or hiv-1 nl4-3 , production of both viruses is diminished in the one-round infection assay, which alludes to inhibition of a post-entry step of hiv-1 infection by ifitm1. to our surprise, in spite of being inhibited to similar degrees in the one-round infection assay, only hiv-1 bh10 , but not hiv-1 nl4-3 , is crippled by ifitm1 in the long-term virus replication assay (fig. 1) . these seemingly conflicting observations could be reconciled by the major difference between the one-round infection and the long-term infection, i.e. the latter infection mode involves virus cell-to-cell transmission that actually dominates hiv-1 transmission over infection by free virus particles [31] . it is thus possible that the superiority of hiv-1 nl4-3 over hiv-1 bh10 in replicating in ifitm1expression supt1 cells, despite that both viruses are equally impaired by ifitm1 in the oneround infection, might be a result of the greater ability of hiv-1 nl4-3 to transmit between cells. indeed, we found that hiv-1 nl4-3 was 3-fold more efficient in transmission from cell to cell than hiv-1 bh10 . hiv-1 nl4-3 has therefore utilized its strong cell-to-cell transmission to compensate for the deficiency in virus production caused by ifitm1. the efficiency of cell-to-cell transmission is determined by viral env protein. inserting the env sequence of hiv-1 bh10 into hiv-1 nl4-3 diminishes cell-to-cell transmission of the parental virus. reciprocally, hiv-1 nl4-3 env protein increases cell-to-cell transmission of hiv-1 bh10 by 3-fold. concurrent with the change in cell-to-cell transmission, this exchange in env sequences also switches the sensitivity of hiv-1 bh10 and hiv-1 nl4-3 to human ifitm1. one possible scenario is that hiv-1 env protein may modulate virus sensitivity to human ifitm1 restriction by virtue of its ability to mediate and regulate virus cell-to-cell transmission. the env sequences of hiv-1 bh10 and hiv-1 nl4-3 differ at 23 amino acid positions. changing each of these 23 amino acids in hiv-1 bh10 env to the counterpart in hiv-1 nl4-3 did not increase the resistance of hiv-1 bh10 to human ifitm1 (data not shown). it can be conceived that two or more of these 23 amino acids in env are required to generate resistance to ifitm1. human ifitm1 does not affect the entry of either hiv-1 bh10 or hiv-1 nl4-3 (fig. 2) [10]. interestingly, ifitm1 gains the ability to inhibit hiv-1 entry when its c-terminal sequence is truncated (fig. 2) . in agreement with this observation, c-terminal truncation enhances the ability of ifitm1 to promote the infection of coronavirus oc43 [32] . this gained function appears to be virus specific, since the truncated ifitm1 does not affect virus entry mediated by vsv g protein (fig. 2) . one possible explanation of this differential inhibition is that hiv-1 and vsv adopt different pathways for entry. hiv-1 entry is ph-independent and occurs at the plasma membrane, whereas vsv enters cells in the endosomes of low ph [33, 34] . in support of this scenario, removal of the last 9 amino acids from the c-terminus of human ifitm1 relocates this protein from intracellular sites to cell periphery (fig. 3) . this cellular locationdependent inhibition phenomenon was previously reported for ifitm3. deleting the endocytic sorting signal 20-yeml-23 in ifitm3 led to redistribution of ifitm3 to the plasma membrane and the consequent loss of inhibition of influenza a virus entry that takes place at late endosomes [35, 36, 37, 38] . in addition to the regulation by their own sequences, subcellular distribution of ifitms can also be modulated by other factors. for example, compared to its intracellular localization in 293t cells as shown in this study, the exogenous ifitm1 has been observed at the plasma membrane in a549 cells [39] . in addition to the cell type-dependent effect, other factors including expression levels and treatment with interferon may also affect where ifitm proteins are localized in cells. it is unclear how the c-terminal sequence modulates the subcellular distribution of ifitm1, especially in light of the recent report showing that ifitm1 c-terminal domain is extracellular [39] . one mechanism by which extracellular/luminal protein domain regulates protein subcellular localization is the er-luminal retention signal [26] . our data rule out the presence of erluminal retention signal within the c-terminal region of ifitm1, because inhibiting endocytosis with the dynamin inhibitor dynasore redistributes ifitm1 to the plasma membrane, but the same treatment fails to do so to an ifitm1 variant with the exogenous er retention signal inserted at the c-terminus (fig. 3b) . alternatively, deleting the c-terminal sequence may change the membrane topology of ifitm1 and, as a result, disturb its subcellular localization. it remains to be determined if other hiv-1 strains are also differentially inhibited by human ifitm1. we suspect that the chance of finding human ifitm1-sensitive hiv-1 strains, either lab-adapted or naturally-existing or circulating, would be low if ifitm1 exerts selection pressure in vivo on hiv-1 replication. since both hiv-1 bh10 and hiv-1 nl4-3 are strains that have been propagated in cultured human t cell lines, they have adapted to the in vitro culture conditions and as a result, they could have lost the original viral sequences that confer resistance to ifitm1, which may have happened to hiv-1 bh10 . we envision that since hiv-1 has never encountered mouse ifitm1, most, if not all, of the hiv-1 strains would be sensitive to the mouse ifitm1 inhibition. work is ongoing to examine the effect of ifitm proteins from different species on other strains of hiv and different lentiviruses. the hiv-1 proviral dna clone hiv-1 nl4-3 was obtained from the nih aids research and reference reagent program. the nleny1-ires dna is a derivative of hiv-1 nl4-3 [40] . the nleny1-ires-es dna is similar to nleny1-ires except for insertion of stop codon at the beginning of env gene and is therefore env-negative [40] . rab5-gfp was a gift from stephen ferguson and michel tremblay. the tet-ifitm1 dna construct has the cdna of human ifitm1 gene inserted into the pretrox-tight-pur vector. the ifitm1 mutants δ(117-125), δ(112-125) and δ(108-125), which have different lengths of the c-terminal sequence (illustrated in fig. 1) , have been previously reported [10] . the er retention signal kdel was added to the c-terminus of ifitm1 by a pcr-based method. the cdna clone of mouse ifitm1 was purchased from atcc, amplified by pcr, and cloned into pretrox-tight-pur to generate tet-miftm1. like all human ifitm1 constructs, a flag tag was added to the n-terminus of mifitm1 to facilitate detection by western blotting. all these dna clones were verified by sequencing. the anti-flag antibody was purchased from sigma, anti-tubulin antibody from santa cruz biotechnology, fitc-conjugated anti-hiv-1 p24 antibody from beckman, anticalreticulin antibody from abcam, and bmqc from invitrogen. the hek293 cells and supt1 cells were originally obtained from atcc. supt1 is a human cd4+ t cell line. they are cultured in rpmi1640 medium supplemented with 10% fetal bovine serum (fbs), 2 mm l-glutamine, 100 u of penicillin/ml and 100 μg of streptomycin/ml. in order to stably transduce supt1 cells, we first prepared retrovirus stocks by transfecting the gp-2 packaging cells (clontech) with the pretrox-tight-pur vector together with the pcmv-vsv-g plasmid (expressing the g protein of vesicular stomatitis virus (vsv)). the viruses were then used to infect supt1 cells together with another retrovirus carrying the rtta activator gene (clontech). the infected supt1 cells were cultured in the rpmi1640 medium supplemented with 10% tetracycline-free fbs (clontech), 2 μg of puromycin/ml and 1 mg g418/ml to select for stably transduced cells. hiv-1 stocks were produced by transfecting human embryonic kidney 293t (hek293t) cells with hiv-1 proviral dna clone using lipofectamine2000 (invitrogen). the culture supernatants were clarified by centrifugation at 3,000 rpm in a beckman bench-top centrifuge at 4°c for 30 min. the viruses were aliquoted, used immediately or stored at -80°c for future use. amount of viruses was determined by measuring viral p24(ca) in elisa. supt1 cells stably transduced with the tet-ifitm retroviral vectors were treated with doxycycline (0.5 mg/ml) before exposure to hiv-1 equivalent to 20 ng viral p24(ca) per 2x10 6 cells. virus growth was monitored by measuring viral reverse transcriptase activity in the supernatants at different time intervals. the experiments were performed as described in [41] . briefly, two virus stocks were first produced by transfecting the hek293t cells using either hiv-1 nl4-3 proviral dna or the nleny1-ires-es dna (env-) and the pcmv-vsv-g dna together with pcmv-blam-vpr. the virus stocks were named hiv-1 nl4-3 /blam-vpr that has hiv-1 envelope protein, and nleny1/vsv-g/blam-vpr that has the vsv-g protein as the envelope, respectively. the amount of viruses in each stock was determined by measuring viral p24(ca) in elisa. the supt1 cell lines were first treated with doxycyline (0.5 mg/ml) for 16 hours, then infected with either hiv-1nl4-3/blam-vpr (an amount equivalent to 100 ng viral p24(ca)) or nleny1/ vsv-g/blam-vpr (an amount equivalent to 50 ng viral p24(ca)) by spinnoculation at 1,800 x g for 45 min at room temperature. following a 2-hour incubation at 37°c, cells were washed twice with co 2 -independent medium, then incubated in the loading solution at room temperature for one hour. the loading solution contained 2 μl of ccf2/am (1 nm) and 8 μl of 0.1% acetic acid (containing 100 mg/ml pluronic-f127surfactant) in 1 ml of co 2 -independent medium. after washing with development media (containing 10 μl of probenecid (250 mm) and 100 μl tetracycline-free fbs in 1 ml co 2 -independent medium, invitrogen), cells were kept in dark at room temperature for 16 hours before being fixed in 1% paraformaldehyde and analyzed by flow cytometry. first, supt1 cells were infected with hiv-1 particles bearing vsv-g protein to enhance infection efficiency. forty hours post infection, the infected cells (designated as donor cells) were washed with complete medium, then mixed with equal number of un-infected supt1 cells (designated as target cells), which had been labeled with a cell tracker bmqc (invitrogen). after 8 hours, the cell mixtures were fixed with 1% paraformaldehyde and permeabilized with 0.1% triton x-100. after staining with fitc-conjugated anti-hiv-1 p24 antibody, the p24positive donor and target cells were scored by flow cytometry. hek293 cells were seeded onto poly-lysine-coated coverslips and transfected with the indicated ifitm1 dna constructs. cells were fixed in 4% paraformaldehyde in phosphate buffered saline for 10 min at room temperature, permeabilized with 0.1% triton x-100 for 10 min, and incubated with blocking buffer containing 3% bsa and 6% skim milk. cells were then stained with indicated primary antibodies (1:100 dilution) for 2 hours at room temperature, followed by staining with fitc or tritc conjugated secondary antibodies (1:100 dilution). the nuclei were stained by dapi. dynasore treatment was performed by incubating cells with 160 μm dynasore for 1 hour at 37°c prior to fixation. images were acquired with leica tcs sp5 laser scanning confocal microscope using 100x objectives with sequential-acquisition setting. all the images were obtained using the laser power and digital gain below saturation at a resolution of 1024 x 1024 pixels (8 bit). imagej software was used for post-acquisition measurement of the fluorescent intensity. supporting information s1 fig. effect of ifitm2 and 3 on the replication of hiv-1 nl4-3 . supt1 cells were treated with doxcycyline to induce the expression ifitm2 and 3 before being exposed to hiv-1 nl4-3 . virus production was determined by measuring viral reverse transcriptase activity in culture supernatants at various time intervals. (eps) conceived and designed the experiments: cl wq. performed the experiments: rj sd qp. analyzed the data: rj sd sll cl. contributed reagents/materials/analysis tools: sll cl. wrote the paper: sll wq cl. interferon-stimulated genes: a complex web of host defenses molecular analysis of a human interferon-inducible gene family small isgs coming forward partial inhibition of vesicular stomatitis virus by the interferon-induced human 9-27 protein interleukin-1 inhibits hepatitis c virus subgenomic rna replication by activation of extracellular regulated kinase pathway the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus ifitm-2 and ifitm-3 but not ifitm-1 restrict rift valley fever virus interferon-induced cell membrane proteins, ifitm3 and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms identification of five interferon-induced cellular proteins that inhibit west nile virus and dengue virus infections the ifitm proteins inhibit hiv-1 infection distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus interferon inducible transmembrane protein 3 (ifitm3) restricts reovirus cell entry ifitm proteins restrict viral membrane hemifusion the broad-spectrum antiviral functions of ifit and ifitm proteins ifitms restrict the replication of multiple pathogenic viruses ifitm3 restricts the morbidity and mortality associated with influenza ifitm3 limits the severity of acute influenza in mice enhanced survival of lung tissue-resident memory cd8(+) t cells during infection with influenza virus due to selective expression of ifitm3 interferon-induced transmembrane protein-3 genetic variant rs12252-c is associated with severe influenza in chinese individuals the dispanins: a novel gene family of ancient origin that contains 14 human members the small interferon-induced transmembrane genes and proteins bril: a novel bone-specific modulator of mineralization interferome: the database of interferon regulated genes identification of the ifitm family as a new molecular marker in human colorectal tumors evolution of vertebrate interferon inducible transmembrane proteins retention of a type ii surface membrane protein in the endoplasmic reticulum by the lys-asp-glu-leu sequence hiv-1 mutates to evade ifitm1 restriction antagonism to and intracellular sequestration of human tetherin by the human immunodeficiency virus type 2 envelope glycoprotein the hiv envelope but not vsv glycoprotein is capable of mediating hiv latent infection of resting cd4 t cells hiv envelope-cxcr4 signaling activates cofilin to overcome cortical actin restriction in resting cd4 t cells avoiding the void: cell-to-cell spread of human viruses interferon induction of ifitm proteins promotes infection by human coronavirus oc43 hiv entry and envelope glycoprotein-mediated fusion acid-dependent viral entry the n-terminal region of ifitm3 modulates its antiviral activity by regulating ifitm3 cellular localization identification of an endocytic signal essential for the antiviral action of ifitm3 phosphorylation of the antiviral protein interferoninducible transmembrane protein 3 (ifitm3) dually regulates its endocytosis and ubiquitination the cd225 domain of ifitm3 is required for both ifitm protein association and inhibition of influenza a virus and dengue virus replication a membrane topology model for human interferon inducible transmembrane protein 1 dynamics of hiv-1 recombination in its natural target cells a sensitive and specific enzyme-based assay detecting hiv-1 virion fusion in primary t lymphocytes key: cord-296487-m4xba78g authors: macintyre, chandini raina; costantino, valentina; kunasekaran, mohana priya title: health system capacity in sydney, australia in the event of a biological attack with smallpox date: 2019-06-14 journal: plos one doi: 10.1371/journal.pone.0217704 sha: doc_id: 296487 cord_uid: m4xba78g planning for a re-emergent epidemic of smallpox requires surge capacity of space, resources and personnel within health systems. there are many uncertainties in such a scenario, including likelihood and size of an attack, speed of response and health system capacity. we used a model for smallpox transmission to determine requirements for hospital beds, contact tracing and health workers (hcws) in sydney, australia, during a modelled epidemic of smallpox. sensitivity analysis was done on attack size, speed of response and proportion of case isolation and contact tracing. we estimated 100638 clinical hcws and 14595 public hospital beds in sydney. rapid response, case isolation and contact tracing are influential on epidemic size, with case isolation more influential than contact tracing. with 95% of cases isolated, outbreak control can be achieved within 100 days even with only 50% of contacts traced. however, if case isolation and contact tracing both fall to 50%, epidemic control is lost. with a smaller initial attack and a response commencing 20 days after the attack, health system impacts are modest. the requirement for hospital beds will vary from up to 4% to 100% of all available beds in best and worst case scenarios. if the response is delayed, or if the attack infects 10000 people, all available beds will be exceeded within 40 days, with corresponding surge requirements for clinical health care workers (hcws). we estimated there are 330 public health workers in sydney with up to 940,350 contacts to be traced. at least 3 million respirators will be needed for the first 100 days. to ensure adequate health system capacity, rapid response, high rates of case isolation, excellent contact tracing and vaccination, and protection of hcws should be a priority. surge capacity must be planned. failures in any of these could cause health system failure, with inadequate beds, quarantine spaces, personnel, ppe and inability to manage other acute health conditions. smallpox is a category a bioterrorism agent, despite being declared eradicated in 1980 [1] . the virus is retained in high security biosafety level 4 laboratories in the united states and russia [2] . the variola genome is fully sequenced and could be synthesized in a laboratory [3] . this a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 to determine the capacity of the health system in sydney, a city of 5.3 million people in australia, during an epidemic of smallpox. specifically, we aimed to determine hospital bedcapacity for isolation, public health workforce capacity for contact tracing and health care worker (hcw) personal protective equipment (ppe) requirements under different attack scenarios. we also aimed to test a worst case scenario among the range of possible attack scenarios and identify modifiable factors which would prevent a worst case scenario. we constructed a modified seir model for smallpox transmission based on a model published in our previous study [7] . model parameters and their estimation have been previously described [7] . we assumed that the virus has not been genetically modified and that there is minimal residual immunity in the population from previous vaccination, as described in our previous study [7] . we assumed an initial attack size of 100, 1000 or 10,000 infected. case isolation was assumed to reduce transmission to zero [2] . given antivirals would be commenced after diagnosis and isolation, we assumed this effect would only apply in the healthcare setting and would not add to interruption of transmission above the effect of isolation alone, with the main transmission risk being in the community for undiagnosed or early cases prior to hospitalisation. we assumed that antivirals would therefore have no effect on community transmission, acknowledging that they would likely reduce morbidity and mortality for treated cases. we estimated number of hospital beds needed to control the epidemic, ppe requirements for clinical hcws and public health workers required for contact tracing, under different scenarios. we constructed a modified model for smallpox transmission. population residual immunity and contact mixing are based on assumptions used in our previous study [7] . using ordinary differential equations as described in s1 file, the population moves through the disease compartmental epidemiological states of being susceptible, exposed, infected and recovered (seir) from smallpox. once infected, people move into the next state following disease duration rates. euler's approximation was used to estimate age-specific force of infection, assuming contact would be similar to observed patterns in the uk [17] [18] [19] . different infectivity levels were based on the reproduction number for hemorrhagic, flat, ordinary and modified smallpox as previously described [7] . the force of infection was multiplied by a parameter (α1, α2, α3, α4) to account for different population susceptibility levels. model parameters and their estimation are described in our previous study [7] . the model runs for 300 days. the model assumptions are shown in table 1 . to test the preparedness of the health system we assumed the base case response would be case isolation and treatment, contact tracing and ring vaccination. as the study was examining health system factors, ring vaccination was kept constant in the model at 90% with assumed adequate vaccine supply and trained vaccinators, and 95-98% vaccine efficacy for uninfected people and 50-53% for latent infected [2, [20] [21] [22] as described in s1 file. hospital bed requirements. we estimate the number of hospital beds in sydney using data published for 2015-2016 in nsw [18] . in nsw for 2015-2016 there were 21152 beds available from public hospitals (2.78 per 1000 population) and 8184 beds available from private hospitals (1.07 per 1000 population). this was resized for the sydney population. the number of hospital beds needed for case isolation was then modelled under different scenarios based on variation of response time (t), the percentage of infected cases isolated each day and how many contacts were traced. we tested if the number of available hospital beds in sydney will be enough to isolate up to 95% of all new infected cases every day, under different scenarios. clinical health workforce and ppe requirements. the clinical health workforce was estimated by the number of hcws in sydney for 2016/2017, including aboriginal and tsi health practitioners, chinese medicine practitioners, dental practitioners, medical practitioners, radiation therapists, nurses and midwives, occupational therapists, pharmacists and ambulance services workers [23] . the total estimated health workforce number was 147997 for nsw, with a total population of 7.7 million in 2016 [24] . we applied the same percentage adjusted for the sydney population from the same year, 5.25 million [25] . hcw distribution by age average number of contacts per case 11 [19] proportion of contacts traced around an infected case 90%, sensitivity analysis with 70% and 50% [2] [proportion of cases that get isolated once infected and symptomatic 95%, sensitivity analysis on with 70% and 50% [2] group was estimated using national and global health worker data [26, 27] . we estimated, based on epidemic size and duration, the amount of respiratory ppe (n95 respirators) required for sydney clinical hcws assuming two respirators per shift per hcw. this is based on recommendations that disposable respirators should not be re-used, and the fact that a standard shift for a hcw would include at least one break, after which a new respirator would need to be used. public health workforce for epidemic control. there are no published data to estimate the public health workforce, comprising trained public health officers working in health departments and capable of conducting contact tracing and outbreak investigation, as public health workers are not registered health practitioners. the only uniform qualification in public health is a master of public health (mph) and similar degrees. whilst there are a large number of mph graduates in australia, the number working in government public health roles would be a minority. it should also be noted that a mph does not equip people with the skills for field response to an epidemic. there are approximately 300 alumni of the national field epidemiology training program (fetp). in addition, there is a medical specialisation in public health medicine for a relatively small number of medical doctors, with an estimated 244 full time equivalent public health physicians nationwide in 2017 [17] . based on discussions with national experts we estimated there are approximately 1000 skilled public health officers in australia, although the actual number may be lower. the public health workforce was calculated using estimates of mph graduates currently working in government, fetp graduates or current fetp trainees and public health physicians. an optimistic assumption of 1500 public health officers nationally was used to estimate the number working in sydney. contact tracing requirements. in the base case, we assumed 90% of contacts would be traced and 95% infectious people would be isolated. we used age specific contacts rates, with an average of 11 contacts per case based on european social mixing data [19] . we estimated the number of public health workers required to conduct contact tracing under different scenarios. given contact tracing may require complex communications and travel over large geographic distances, we assumed one public health officer could trace 10 contacts per day. australian guidelines for management of smallpox state that isolation is needed for nonimmune category a (high risk) contacts, in individual rooms with supervision by vaccinated staff [28] . we conservatively assumed at least 50% of contacts traced would be category a, and would require supervised quarantine. data from tuberculosis studies [29] as well as estimated social contact matrices suggest one person [19] , on average, has 9-11 contacts at reasonable risk of infection. the closest of these contacts would include household contacts (about 3-4 people), plus 1-2 others in work or friendship circles. this would be about half of the 9-11 contacts. the number of contacts needed to be traced and managed was estimated based on attack size, time to response (t) and the percentage of infected cases isolated each day. a contact tracing day was defined as one day entirely spent tracing contacts per public health worker. sensitivity analysis. a sensitivity analysis was conducted on attack size, the proportion of infectious cases isolated and contacts traced, as well as time to commencing the response. to illustrate the difference in epidemic size between a single index case of smallpox imported from overseas, compared to a primary attack which results in 100 or 1000 simultaneous firstgeneration cases, we modelled the epidemic resulting from 1, 100 or 1000 initial first generation cases. the size of an attack is unknown, and would depend on the technical sophistication of aerosol dispersion of variola. to account for this uncertainty, we explored the influence of attack scenarios of 100, 1000 and 10000 initial infected as a wide range of possible attack sizes, to determine the impact of attack size on epidemic control. delays in diagnosis and time to obtaining laboratory confirmation could vary the time of onset of the response. we therefore varied the time of the response commencing between t = 15, 20 and t = 30 days following virus release. given an average incubation period of 12 days for smallpox [2] , this corresponds to day 3, 8 and 18 after the onset of symptoms of the index case. we estimated 14595 public hospital beds and 5618 private hospital beds in sydney. we estimated there are 100638 clinical hcws in sydney, the majority (65%) aged 30-49 years old, 51% nurses and 18% doctors [27] . we estimated a public health workforce of 1500 nationally, with approximately 330 public health workers in sydney. fig 1 shows the relative epidemic size of a deliberate release scenario with 100 or 1000 initial infected compared to a single importation of smallpox from an epidemic overseas, to illustrate the potential scale of the required public health response. the higher the initial number infected, the more rapid and severe the epidemic. without intervention, the death rate will reach an incidence (number of new infected people per day) rate of 8 deaths per 1000 population per day. the overall reproductive number was estimated to be 4.6. fig 2 shows the influence on infections and deaths by varying time to response and case isolation rates. both timing and isolation rates of infected cases are highly influential in outbreak control. with 100 infected initially, when isolation decreases, the deaths increase from a maximum of 2, 4 and 9 per day in the best scenario with 95% isolation (for t = 15,20 and 30 respectively), to 3.5, 5.5 and 12 per day with only 50% of cases isolated (fig 2) . fig 3 shows the influence of varying time of starting the intervention and varying percentage of contacts traced on the incidence of infections and deaths with case isolation constant at 95%. with a high proportion of cases isolated, outbreak control can be achieved within 100 days even with only 50% of contacts traced. fig 4 shows the effect of varying both case isolation and contact tracing rates, with the intervention commencing at 20 days-case isolation is more influential, with epidemic control severely impacted when isolation and contact tracing falls to 50%. in table 2 we show the impact of the epidemic (total cases and contacts needed to be traced) by varying case isolation rates. the total number of cases range from 528 to 285,400; and contacts that need to be traced between 1277 and 940350 in the best-and worst-case scenarios respectively. we estimated 14595 public hospital beds and 5618 private hospital beds in sydney. the modelled maximum number of people isolated at the same time is about 2.3 and 5.5 times the initial number of infected if we start intervention respectively at t = 20 and at t = 30 and peaks 28 days after the response commences. therefore, if the initial number of infected is 100 or 1000, the available beds will not be completely exhausted, but treatment capacity for other illnesses may be impacted. in the case of 100 initial infected, the maximum beds usage will reach 1.6% and 3.8% of sydney public available beds, if the response starts at time t = 20 and t = 30 days from the virus release respectively. if the initial number of infected is 1000, the maximum beds usage will reach 16.1% and 37.9% of sydney public available beds 28 days after the response commences, if the response starts at time t = 20 and t = 30 days from the virus release respectively. however, in the case of 10000 initial infected, the available hospital beds will be all used in the first few days of the response. fig 5 shows the hospital bed usage in the worst-case scenario of 10000 initially infected, with varying start times of the response. maximum number of beds needed at the same time and day shown in the square windows. with 10000 initial infected if we start the intervention at t = 20 from virus release, 1703 beds will be used the first day, 3356 the second day. at 7 and 13 days after commencing the intervention (at day 27 and 33) more than 50% and 80% of the total beds in sydney hospitals will be needed. at day 39 post-attack, 19 days after starting the intervention, 100% of all public and private beds will be used. if the intervention is delayed to day t = 30, almost 50% of the available beds will be used in the first 2 days, 80% at day 5 of response will be used and at day 6 of the response (t = 36 after the attack) all available public and private beds will be used. table 3 https://doi.org/10.1371/journal.pone.0217704.g005 results showed for 95% of new infected isolated. 50% 80% 100% maximum number of beds used in the same day (% of the total) 10000 initial infected 19 23 shows the time to occupancy of all available hospital beds at levels of 20% or greater. whilst an attack of 100 initial infections does not reach 20% of beds under any scenario, in the worstcase scenario (response commencing at day 30) 100% of beds will be used by day 36. the number of hcws required and the ppe they need will be proportionate to the number of cases requiring treatment (table 2 ). in scenarios described above where cases (beds) exceed 100% of available beds, staffing requirements will increase 100%, unless reduced staff/patient ratios are implemented. estimating a minimum of 2 disposable respirators a day per hcws for 150 days, over 30 million respirators will need to be stockpiled for all 100638 clinical hcws in sydney. this number can be used to estimate requirements based on the estimated percentage of the clinical workforce needed for the epidemic, which will be proportionate to the number of cases requiring treatment ( table 2 ). if 10% of clinical hcws are involved in care of smallpox patients, over 3 million respirators will be needed. if the epidemic is not controlled within 300 days this number will be doubled. the public health staff (phs) required to conduct contact tracing will depend on how many contacts one person can trace per day and the number of available phs, estimated to be 330 in sydney. if one phs can trace 10 contacts a day, then in the best-case scenario of 1277 contacts, over 127 contact tracing days are required, based on the number of contacts in table 2 above. in the worst-case scenario, 940,350 contacts and 94,035 contact tracing day are required. in the worst-case scenario, 330 phs would work over 285 days each doing contact tracing. if half of contacts are high-risk, quarantine spaces will be required for 638 to 470,175 contacts. in the case of a smallpox release in sydney, a high-income, well-resourced city of over 5.3 million people, health system impacts may be substantial under some scenarios as shown in our model. we showed if smallpox arises overseas and is imported as a single case into australia by travel, control will be far easier than under an attack scenario. we showed that influential factors on epidemic impact are the size of the initial attack, time to commencing the response, case isolation rates and contact tracing for ring vaccination. whilst both are influential, case isolation is more influential than contact tracing. these public health interventions depend on physical and human resources, including clinical and public health workforce. whilst the size of an attack may not be within our control, other the influential factors are modifiable and potentially within our control. if the initial attack size is 100-1000 and the response is rapid, an outbreak of smallpox can be controlled with case isolation, contact tracing and vaccination. however, if the response is delayed to 30 days or longer (which equates to about 2 weeks after the first symptoms occur), or if the attack infects 10000 people, epidemic control will be much more challenging, and the health systems impacts will be substantial. in the worst-case scenario, available hospital beds will be exceeded in less than 40 days. the requirement for hospital beds for isolation of cases will vary from up to 4% to 100% of all available beds depending on the size of initial release and speed of response. even in the mid-range scenario of 1000 initial cases, up to 40% of all available hospital beds will be required for smallpox control. this does not account for the facilities required for quarantine of contacts, which must additionally be planned for, and in the worst-case scenario would require over 400,000 high risk contacts to be quarantined. quarantine and isolation capacity are critical to epidemic control. planning for surge bed capacity using available guidelines should be undertaken [30] , and back up plans such as the use of community halls, school buildings, hotels or other large buildings should be made to ensure that that other viable isolation sites are pre-designated as smallpox treatment centres and available. during the 2009 pandemic of influenza, which was reportedly not as severe as expected, studies reported a tripling of patient presentations to hospital [31] . plans for managing hospital bed capacity in the event of a large initial attack should also be made, including designation of specific treatment facilities, cancellation of elective surgery and decanting of patients with non-urgent other conditions into private hospitals or other facilities. the capacity for hospital beds for non-smallpox patients who require urgent hospitalisation must also be considered, and in some scenarios, the care of patients with urgent non-infectious conditions such myocardial infarction or stroke, may be compromised by lack of hospital capacity and staffing shortages. rapid response time is critical and becomes even more critical when the initial infected number is higher. responding more than 20 days from the virus release (which means commencing the public health response within 7 days of symptom onset, given an average 12 day incubation period) will result in a more severe outbreak. whether it is feasible to commence response within the best-case scenario of 15 days post-release (or 3 days after symptom onset) is unknown, but unlikely. a rapid response depends on very early detection and diagnosis, as well as prompt commencement of case finding, isolation, contact tracing and vaccination. practically, the target for reducing the time to response is in early diagnosis, which depends on awareness first, followed by diagnostic test. delays may occur if the diagnosis is missed in the index case. recent examples of serious emerging infectious diseases where the diagnosis was missed include ebola in nigeria and the us, both of which occurred during the height of the west african epidemic when media reports were at a peak and awareness should have been high [32, 33] . the largest epidemic of mers coronavirus outside the arabian peninsula occurred in south korea following a missed diagnosis and failure of triage in a patient with a relevant travel history and a respiratory clinical syndrome [34] . the last european epidemic of smallpox in 1972 also involved a missed diagnosis, when a traveller to the middle east returned to yugoslavia, which had been free of smallpox for 30 years. the patient had haemorrhagic smallpox, which was misdiagnosed as a severe adverse reaction to an antibiotic, and smallpox was not suspected until second generation cases began occurring, resulting in an outbreak of 175 cases [35] . excellent surveillance systems and triage protocols for early detection of low probability, high impact outbreaks such as smallpox is recommended. improving diagnosis requires triage protocols and rapid diagnostics, the latter being useful only if the diagnosis is suspected clinically in the first instance. other avoidable delays in response could include having pre-vaccinated first responder teams, pre-designated isolation and quarantine facilities, and rapid human resources and surge capacity scale up plans [36] . we have showed that epidemic control is highly sensitive to case isolation rates, which need to be maintained at high levels. identifying and isolating less than half the new infected cases and tracing only half of all contacts will result in a blow-out of the epidemic. space and human resource requirements for case finding, isolation, contact tracing, vaccination and quarantine are therefore essential for preparedness planning. physical space requirements extend beyond isolation of smallpox cases, to quarantine of contacts. in the worst-case scenario, almost one million contacts need to be traced and there will be a lack of physical space for quarantine of high-risk contacts. plans for home quarantine and surveillance of contacts should also be undertaken and will require adequately trained personnel. the speed and effectiveness of contact tracing is also critical to the success of ring vaccination and will require an adequately trained critical mass of public health workers and epidemiologists, separate from the clinical workforce. in australia, as a federation, this will rely on state and territory capacity, and cross-border mobilisation of jurisdictional capacity in the event of a smallpox epidemic, and estimation of the current and required capacity for such an event. the fact that public health personnel are not registered as health practitioners or documented in any other centralised way, makes it more challenging to rapidly mobilise suitably qualified and experienced personnel for a large-scale epidemic response. this is a policy consideration that could be addressed as part of pandemic and health emergency planning, which may strengthen response. contact tracing may need to rely on community volunteers, as the available public health workforce will be inadequate in a large epidemic. staff surge requirements would track parallel to bed requirements and would be over 100% in some scenarios. the need for a clinical health workforce to treat smallpox will be high, with case numbers in the 1000s to 100,000s in many of the scenarios modelled, and just over 100,000 clinical hcws in sydney. limiting the number of hcws working in designated smallpox facilities is a sensible strategy. a possible approach to such a scenario would be a reduction of staff to patient ratios, as well as using trainee hcws. protection of these clinicians is key, with vaccination being the mainstay. up to 100,000 doses of vaccine will need to be reserved for clinical hcws and plans in place to commence vaccination. ppe will not be an alternative to vaccination, but an additional protective measure for hcws. today, work health and safety requirements would dictate that paprs or disposable respirators with a hood and coveralls be available to clinicians treating smallpox cases. perceived lack of protection during a serious emerging infection outbreak may result in refusal to work or industrial action by hcws [37] . hcw may be well protected by ppe, but there is large uncertainty around effectiveness of ppe. studies of other viruses transmitted by the respiratory route suggests good effectiveness of respirators against smallpox [38] . it should be noted that surgical masks are unlikely to offer protection to hcws based on available data [38] . stockpiling may provide a short duration of supplies. the modelled epidemic may run for 150-300 days or more, depending on the scenario. a very large quantity of respirators may need to be stockpiled, depending on the percentage of hcw involved in direct care of smallpox patients. given the likely duration of an epidemic, plans should put in place for rapid procurement of ppe supplies beyond the stockpiled capacity. strategies to minimise the number of hcw treating each case of smallpox, including using designated smallpox hospitals, will reduce the quantity of ppe required. early identification of the epidemic, high rates of case isolation, excellent contact tracing and vaccination, and protection of hcws are the key influential components of epidemic control. failure in any of these could severely compromise the capacity of the health system. australia has a detailed plan for smallpox response, [39] and we have outlined key influential parameters for disease control which can add further guidance on mitigating severe outcomes in both the planning and response stages. excellent surveillance systems and triage protocols for early detection of low probability, high impact outbreaks such as smallpox can make a difference, given the criticality of timing of the response and better prospects of epidemic control in the early stages. planning for the health system should consider rapid surge capacity for beds, strategies to create and staff make-shift designated smallpox treatment facilities, and protection of hcws at all levels of care. requirements for contact tracing are substantial and may require mobilisation of community volunteers and additional space for quarantine and surveillance of high-risk contacts. designated surge smallpox facilities and plans for management of other urgent health conditions should be considered. we have outlined several modifiable factors which, with good planning, can ensure adequate health system capacity in the event of a smallpox epidemic. general fact sheets on specific bioterrorism agents smallpox and its eradication: the pathogenesis, immunology, and pathology of smallpox and vaccinia the de novo synthesis of horsepox virus: implications for biosecurity and recommendations for preventing the reemergence of smallpox. health security how canadian researchers reconstituted an extinct poxvirus for $100,000 using mailorder dna biopreparedness in the age of genetically engineered pathogens and open access science: an urgent need for a paradigm shift development of a risk-priority score for category a bioterrorism agents as an aid for public health policy influence of population immunosuppression and past vaccination on smallpox reemergence who (world health organizazation). smallpox. geneva, switzerland: world health organization smallpox vaccines: past, present, and future public health and health reform in australia respiratory protection for healthcare workers treating ebola virus disease (evd): are facemasks sufficient to meet occupational health and safety obligations? planning for smallpox outbreaks modelling disease outbreaks in realistic urban social networks containing bioterrorist smallpox modeling the effect of herd immunity and contagiousness in mitigating a smallpox outbreak planned and unplannedfutures for the public health physician workforce in australia. a labour market analysis for the australasian faculty of public health medicine: australasian faculty of public health medicine:sydney hospital resources 2015-16: australian hospital statistics canberra: australian institute of health and welfare social contacts and mixing patterns relevant to the spread of infectious diseases transmission potential of smallpox in contemporary populations effectiveness of postexposure vaccination for the prevention of smallpox: results of a delphi analysis smallpox in europe ambulance service of nsw. workforce statistics staff turnover counting health workers:definitions, data, methods and global results. geneva: world health organization annual report 2016/2017: melbourne. australian health practitioner regulation agency smallpox response plan and guidelines (version 3.0). archive-use only for research purposes preventability of incident cases of tuberculosis in recently exposed contacts. the international journal of tuberculosis and lung disease a survey of emergency department 2009 pandemic influenza a (h1n1) surge preparedness office of the chief health officer. public health workforce surge guidelines transmission dynamics and control of ebola virus disease outbreak in nigeria ebola us patient zero: lessons on misdiagnosis and effective use of electronic health records hospital outbreaks of middle east respiratory syndrome smallpox as actual biothreat: lessons learned from its outbreak in ex-yugoslavia in 1972. annali dell'istituto superiore di sanita exercise mataika: white paper on response to a smallpox bioterrorism release in the pacific uncertainty, risk analysis and change for ebola personal protective equipment guidelines the efficacy of medical masks and respirators against respiratory infection in healthcare workers. influenza and other respiratory viruses smallpox cdna national guidelines for public health units. communicable diseases network australia key: cord-294023-knaxr7t0 authors: murri, rita; segala, francesco vladimiro; del vecchio, pierluigi; cingolani, antonella; taddei, eleonora; micheli, giulia; fantoni, massimo title: social media as a tool for scientific updating at the time of covid pandemic: results from a national survey in italy date: 2020-09-03 journal: plos one doi: 10.1371/journal.pone.0238414 sha: doc_id: 294023 cord_uid: knaxr7t0 in the face of the rapid evolution of the covid-19 pandemic, healthcare professionals on the frontline are in urgent need of frequent updates in the accomplishment of their practice. hence, clinicians started to search for prompt, valid information on sources that are parallel to academic journals. aim of this work is to investigate the extent of this phenomenon. we administered an anonymous online cross-sectional survey to 645 italian clinicians. target of the survey were all medical figures potentially involved in the management of covid-19 cases. 369 questionnaires were returned. 19.5% (n = 72) of respondents were younger than 30 years-old; 49,3% (n = 182) worked in infectious diseases, internal medicine or respiratory medicine departments, 11.5% (n = 42) in intensive care unit and 7.4% (n = 27) were general practitioner. 70% (n = 261) of respondents reported that their use of social media to seek medical information increased during the pandemic. 39.3% (n = 145) consistently consulted facebook groups and 53.1% (n = 196) whatsapp chats. 47% (n = 174) of respondents reported that information shared on social media had a consistent impact on their daily practice. in the present study, we found no difference in social media usage between age groups or medical specialties. given the urgent need for scientific update during the present pandemic, these findings may help understanding how clinicians access new evidences and implement them in their daily practice. on march the 30th, dr m. r. attended the morning meeting of the recently born "columbus covid ii hospital". during the summit, a message was read from a whatsapp chat held by clinicians working in northern italy, where the pandemic was displaying striking proportions. in the communication, it was said that a worrying number of cases of pulmonary thromboembolism was being reported. this was a new and previously unknown observation. after the meeting, dr m. r. came back to the ward where mr a, a previously healthy man in his fifties, currently recovering after a mild pneumonia due to sars-cov-2, was about to be discharged. he complained of a mild, yet worsening dyspnea and a lumbar pain. he had normal body temperature, blood pressure and was hemodynamically stable. arterial blood gas analysis showed po2 87 mmhg, pco2 34 mmhg and sato2 96%. keeping the recent meeting in mind, we decided to request a chest ct scan, that revealed massive pulmonary thromboembolism. low-molecular-weight heparin (lmwh) at therapeutic dose was started, the patient improved within a few days and was then safely discharged. to what extent the recent informal communication accelerated the diagnosis is difficult to define. the first confirmed case of sars-cov-2 infection in italy was identified in rome on january 31th, 2020. since then, the coronavirus disease 2019 (covid-19) pandemic has spread around the world, catching many countries unprepared to face its enormous burden [1]. at the time this article was written, italy was among the countries with the highest number of covid-19 cases in the world, counting more than 162.000 total confirmed cases, with approximately 600 deaths and 3.000 new diagnosis per day. full commercial and movement restrictions had been in place on all national territory for more than three weeks [2] . national guidelines recommended an antiviral therapy based on protease inhibitors and chloroquine [3] for nearly all hospitalized patients. available literature focused almost exclusively on the respiratory tract manifestations, but clinical practice and not peerreviewed evidences suggested that clinical features of the disease could be more varied and umpredictable [4, 5] . by the end of july, the total number of confirmed cases and deaths raised to 246.000 and 35.000. clinicians on the frontline thus felt compelled to search for rapidly available, yet accountable information and started to share, in turn, any relevant finding coming from their daily practice or from preliminary data analysis. aim of this study is to investigate to what extent physicians sought information on social media and other not conventional sources. in order to investigate to what extent clinicians are seeking and using information coming from social media, or other sources that are parallel to the scientific literature, we designed an anonymous, voluntary questionnaire on surveymonkey. the questionnaire was built as a nationwide, cross-sectional survey, targeting all medical figures potentially involved in the management of covid-19 cases. the online form included 17 questions about basic demographic characteristics, personal involvement in the sars-cov2 pandemic, frequency of social media utilization and the perceived impact of social media in the respondent's practice. a total of 645 italian clinicians received the form. data were collected from the 5 th to the 14 th of april 2020 and analyzed from the 15 th to the 19 th of the same month. categorical variables were reported as proportions and compared using chi-square test. binary logistic regression was used to determine the relationship between demographical variables and self-reported impact of social media on clinical practice. results were adjusted for demographical variables in the multivariable analysis. an a priori p-value <0.05 was considered to be significant. all statistics were conducted using ibm spss statistics version 25 (ibm corporation, armonk, ny). the present study has been approved by the fondazione policlinico universitario a. gemelli ethics committee. consent was obtained by written statement. three hundred sixty-nine questionnaires were returned. twenty percent of respondents (n = 72) were younger than 30 years-old and 10% (n = 37) were more than 60 years-old; 21.9% (n = 81) of the respondents worked in an infectious diseases department before the pandemic, 27.4% (n = 101) in internal medicine or respiratory medicine, 11.5% (n = 42) in intensive care unit and 7.4% (n = 27) were general practitioner. two-hundred and twelve respondents (57.5%) answered from central italy (including lazio, our region), 112 (30.4%) from northern italy and 39 (10.6%) from southern italy. fifty-two percent of respondents (n = 191) were visiting patients with covid-19 at least once per week, and 46.6% (n = 172) visited confirmed covid-19 cases every day. data about how our colleagues sought information to obtain guidance for covid-19 medical practice are presented in table 1 . almost 80% of respondents (n = 285) reported seeking information in peer-reviewed papers, yet an equal rate (78.4%; n = 288) recurred to personal communications from colleagues working in other centers at least twice per week, 39.3% (n = 145) consistently consulted facebook groups and more than the half (53.1%; n = 196) reported to use whatsapp chats for the same purpose at least once per week. respondents characteristics are summarized in table 1 . seventy percent (n = 261) of respondents reported that their use of social media to find medical information increased during the current pandemic (fig 1) . in terms of covid-19 medical practice, information coming from social media were considered "enough" or "much" or "very much" useful by 82.9% (n = 306) of the sample. to the question "during the last week, do you think that information shared on social media had an impact on your clinical practice for patients with covid?" 28.7% (n = 106) answered "enough" and 47.1% (n = 174) "much" or "very much". in 2016, during the zika epidemic, a protocol for data sharing during public health emergencies was issued by the world health organization [6] . currently, several academic journals are trying to meet the instances of the medical community by hosting open-access covid sections while speeding up their peer-reviewing process. special web pages have also been created to accelerate data sharing on this disease, such as the nejm coronavirus page [7] , the lancet covid-19 resource centre [8] , the bmj's coronavirus (covid) hub however, although providing great opportunities, social networks create the ideal framework for misinformation to spread [12, 13] . this is particularly true in time of pandemics, when a substantial increase in demand pressure, along with poor supervision of online contents can easily lead to misinformation dissemination [14] . alarmingly, this phenomenon has the potential to undermine trust in health institutions and programmes, especially when governments rely almost solely on empirical evidence for policy-making [15] . in fact, studies conducted during the covid-19 pandemic showed that the rate of tweets with false or unverifiable contents may be as high as 24% [16] . similar results were described for other pandemics, such as for 2009 h1n1 [17] and 2014 ebola outbreaks. interestingly, data from the ebola crisis showed that statements that were political in nature were particularly at risk to spread misinformation [18] . however, to our knowledge, few studies have explored the role of information dissemination through social media on clinicians and other healthcare professionals. our survey shows that, at the time of covid pandemic, many clinicians react to their urgent need for updates by seeking information through unconventional sources instead of academic journals publications. data obtained from colleagues working on different centers, facebook groups and informal whatsapp chats seem to be highly valued and trusted. these findings may reflect the need of a more flexible, user-friendly way to seek for medical information and updates, while the current epidemic is boosting the usage of social media to access to the complex, rapidly evolving amount of evidence that is increasingly emerging from all around the world. interestingly, 150 responders (40.7%) reported to actively share medical information via social media "often" or "everyday". this, on one hand, is coherent with the purpose of social media themselves but, on the other hand, it entails a broader shift in how professionals conceive the access to medical information, technological advances and scientific knowledge. we believe that it is important to acknowledge this phenomenon, as well as the risk of spreading misinformation, fear or research exceptionalism, with potentially dangerous consequences for public health [5, 12, 13, 18] . we strongly suggest that, during a pandemic, academic journals implement dedicated sections for rapid communications in the form of forum sections, rapid responses or comments, and reserve peer reviewing for key points as needed [19, 20] , in accordance to the cited who protocol for data sharing during public health emergencies [6] . facilitated focus groups on social media could be another way to encourage discussion, even though, to our knowledge, no protocols are currently available. our study has several limitations. first, our sample was not uniformly distributed to all medical figures involved in covid-19 epidemic, being intensive care doctors, primary care physicians likely underrepresented. also, few doctors from southern italy responded to the questionnaire. second, the results have to be interpreted with caution due to the small sample size. finally, our findings about the impact of social media on clinical practice are based upon the personal perspective of the respondents. in conclusion, rapidly sharing information could have an invaluable impact during a pandemic such as that caused by sars-cov-2. methods to promote a safe open and rapid dissemination of relevant findings, as long as new technologies capable to identify relevant information [21] could provide a substantial benefit during the ongoing and future public-health crises. decreto del presidente del consiglio dei ministri-22 linee guida sulla gestione terapeutica e di supporto per i pazienti con infezione da coronavirus covid-19 -versione 2 clinical features of patients infected with 2019 novel coronavirus in wuhan, china case-fatality rate and characteristics of patients dying in relation to covid-19 in italy data sharing in public health emergencies: a call to researchers the bmj's coronavirus (covid-19) hub social media and health care professionals: benefits, risks, and best practices. pharmacy and therapeutics systematic literature review on the spread of healthrelated misinformation on social media covid-19 related misinformation on social media: a qualitative study from iran the lancet infectious diseases coronavirus goes viral: quantifying the covid-19 misinformation epidemic on twitter pandemics in the age of twitter: content analysis of tweets during the 2009 h1n1 outbreak misinformation and the us ebola communication crisis: analyzing the veracity and content of social media messages related to a fear-inducing infectious disease outbreak open peer-review platform for covid-19 preprints sharing research data and findings relevant to the novel coronavirus (covid-19) outbreak. wellcome press release humanitarian health computing using artificial intelligence and social media: a narrative literature review key: cord-294372-pec1886j authors: greene, dina n.; jackson, michael l.; hillyard, david r.; delgado, julio c.; schmidt, robert l. title: decreasing median age of covid-19 cases in the united states—changing epidemiology or changing surveillance? date: 2020-10-15 journal: plos one doi: 10.1371/journal.pone.0240783 sha: doc_id: 294372 cord_uid: pec1886j background: understanding and monitoring the demographics of sars-cov-2 infection can inform strategies for prevention. surveillance monitoring has suggested that the age distribution of people infected with sars-cov-2 has changed since the pandemic began, but no formal analysis has been performed. methods: retrospective review of sars-cov-2 molecular testing results from a national reference laboratory was performed. result distributions by age and positivity were compared between early period (march-april 2020) and late periods (june-july 2020) of the covid-19 pandemic. additionally, a sub-analysis compared changing age distributions between inpatients and outpatients. results: there were 277,601 test results of which 19320 (7.0%) were positive. the median age of infected people declined over time (p < 0.0005). in march-april, the median age of positive people was 40.8 years (interquartile range (iqr): 29.0–54.1). in june-july, the median age of positive people was 35.8 years (iqr: 24.0–50.2). the positivity rate of patients under 50 increased from 6.0 to 10.6 percent and the positivity rate for those over 50 decreased from 6.3 to 5.0 percent between the early and late periods. the trend was only observed for outpatient populations. conclusions: we confirm that there is a trend toward decreasing age among persons with laboratory-confirmed sars-cov-2 infection, but that these trends seem to be specific to the outpatient population. overall, this suggests that observed age-related trends are driven by changes in testing patterns rather than true changes in the epidemiology of sars-cov-2 infection. this calls for caution in interpretation of routine surveillance data until testing patterns stabilize. introduction since its first detection in china in december 2019, the sars-cov-2 virus has emerged to cause a global pandemic. to date, over 31 million confirmed sars-cov-2 infections have been detected globally with 965,000 deaths due to covid-19 [1] . understanding of the demographics of persons infected with sars-cov-2 is essential for informing the public health response to the covid-19 pandemic. age is a major factor in determining the risk of severe illness outcomes [2, 3] , so data on the age distribution of infected persons can help guide expectations about demands on hospital resources. knowledge of which age groups are highly infected is also important for designing effective interventions [4] . in the united states, surveillance data suggest that mean age of infected patients is decreasing compared to the early stages of the covid-19 pandemic. in washington state, for example, 35% of detected cases in march were aged 60 years or older, compared to 12% in july [5] . however, testing practices for sars-cov-2 have changed dramatically over the course of the covid-19 epidemic in the united states. the average daily number of sars-cov-2 tests has increased from approximately 35,000 in march to 676,000 in july [6] . thus, it is unclear whether changes in the age distribution of detected cases represent changes in the epidemiology of covid-19, changes in testing practices, or a combination of the two. we used sars-cov-2 testing data from a national reference laboratory to characterize the age distribution of detected cases between march and july of 2020. the study was covered under exemption umbrella deidentified data (utah irb 00082990). we reviewed of all sars-cov-2 test results performed at arup laboratories from march 10, 2020 to july 8, 2020. arup laboratories (salt lake city, utah) is a national reference laboratory that provides clinical testing for over 1000 hospitals across the united states. arup has offered sars-cov-2 testing since march 2020. patients who received positive test results were presumed to been infected. we refer to this group as the positive population which is an estimate of the infected population. all testing was performed on combination of three high throughput, automated molecular assays: hologic (75%), roche (18%) and thermofisher (7%). these assays have similar limits of detection and are among the most sensitive assays developed to date. we divided the results into two periods: 1) march 10 th -april 30 th (early period), 2) june 1 st -july 8 th (late period). we compared the age distribution of positive cases in the early and late periods by calculating the medians and interquartile ranges (iqr). to explore differential changes in testing for severely ill patients vs. patients with milder disease, we also stratified combined, 277,601 test results were reported in the early and late periods. of these, 19,320 (7.0%) were positive. approximately half (48%, n = 134,253) of the results were from utah. samples were obtained from 40 additional states. in addition to utah, 22 states had over 1000 results each, which accounted for 49% of the total results (table 1 ). there was no significant difference between the percentage of positive cases in the utah (6.94%) and non-utah (6.97%) cohorts (p = 0.81). the median age of positive patients in the non-utah cohort (42.1 years iqr: 27.1-58.4) was greater than the utah cohort (35.8 years; iqr: 24.2-48.0) (w 2 1 ¼ 254, p<0.0005). we were able to determine inpatient status for 6973 of 9327 (75%) positive patients in the utah cohort, of whom, 6865 (98.5%) were outpatients and 108 (1.5%) were inpatients. the median ages of positive outpatients and inpatients were 35.4 years (iqr: 23.8-47.8) and 53.6 years (iqr: 43.1-67.4), respectively (p < 0.001). the overall median age for positive cases was 38.4 years (iqr: 25.7-53.3). the median age for all positive cases decreased by 5.0 years between the early and late periods from a median age of 40.8 years (iqr: 29.0-54.1) to 35.8 years (iqr: 24.0-50.2) late period (june-july) (p < 0.001) (fig 1, table 2 ). the pattern was similar when comparing utah to all other states combined (fig 1, table 2 ). the distribution of positive cases contains several outliers with advanced age (fig 1) . within utah, the overall median age for all positive results decreased from 38.5 to 34.3 years. when the utah cohort was stratified based on inpatient and outpatient medical care, the median age of inpatients increased by 5.8 years between the early and late period ( table 2 , fig 2) . in contrast, the median age of outpatients decreased by 3.9 years. the positivity rate increased over time for almost all age groups; however, the increase was greatest among younger people (table 3) . for example, among those younger than 18 years, the positivity rate increased from 3.3% to 10.0% between the early and late periods. similarly, the positivity rate increased from 6.1% to 11.5% and from 6.2% to 10.1% for people in the 18-29 and 30-39 age groups. in contrast, the infection rate decreased from 5.5% to 4.4% for people within 60-69 age group and decreased from 6.1% to 3.6% for those 70 years old and older. overall, the positivity rate of patients under 50 years old increased from 6.0% to 10.6% and the percent of positive results for those over 50 years old decreased from 6.3% to 5.0%. (patients aged 50.00-50.99 years were included in the over 50 age group). surveillance data in the united states have shown a trend toward decreasing age among persons with laboratory-confirmed sars-cov-2 infection. this study found a similar pattern among patients tested by a national reference laboratory, with the median age among patients testing positive being five years lower in june and early july compared to march and april. this pattern holds true for patients in utah (which has not yet experienced a major covid-19 epidemic) as well as for patients outside of utah (which include states with early major epidemics, recent major epidemics, and no major epidemics to date) [7, 8] . if the median age of sars-cov-2 infection were truly declining over time, we would expect to see decreases in the median age of covid-19 patients in both inpatients and outpatients. instead, we found that the median age of covid-19 patients showed opposite patterns over time between inpatients and outpatients in utah: the median age of inpatients increased while the median age of outpatients decreased. this finding suggests that the overall decreasing age of persons with detected sars-cov-2 infection is driven by changes in sars-cov-2 testing rather than a true change in the epidemiology of covid-19. given the striking increase in risk of severe covd-19 with increasing age, our findings suggest that the increasing availability of sars-cov-2 tests has increased testing of low-acuity patients or asymptomatic persons in ambulatory care settings, who tend to be younger than the more severely ill patients. a key limitation this study is that, although arup is a national reference laboratory, the majority of specimens (and the only specimens with data on inpatient vs. outpatient providers) were available from utah. although the aggregated non-utah specimens show similar agerelated trends to the utah specimens, results from this study may not generalize to any specific non-utah state. understanding how sars-cov-2 infection varies across the age spectrum is key for developing responses to the covid-19 epidemic. our findings suggest that age-related differences in infection from the early epidemic until now are driven by changes in testing patterns rather than true changes in the epidemiology of sars-cov-2 infection. this calls for caution in interpretation of routine surveillance data until testing patterns are stabilized with regard to illness acuity. an interactive web-based dashboard to track covid-19 in real time incidence, clinical outcomes, and transmission dynamics of severe coronavirus disease 2019 in california and washington: prospective cohort study features of 20 133 uk patients in hospital with covid-19 using the isaric who clinical characterisation protocol: prospective observational cohort study covid-19 worldwide: we need precise data by age group and sex urgently novel coronavirus outbreak (covid-19 the covid tracking project: the atlantic monthly presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new york city area characteristics of hospitalized adults with covid-19 in an integrated health care system in california conceptualization: dina n. greene. key: cord-297835-ukrz8tlv authors: leith, douglas j.; farrell, stephen title: measurement-based evaluation of google/apple exposure notification api for proximity detection in a light-rail tram date: 2020-09-30 journal: plos one doi: 10.1371/journal.pone.0239943 sha: doc_id: 297835 cord_uid: ukrz8tlv we report on the results of a covid-19 contact tracing app measurement study carried out on a standard design of european commuter tram. our measurements indicate that in the tram there is little correlation between bluetooth received signal strength and distance between handsets. we applied the detection rules used by the italian, swiss and german apps to our measurement data and also characterised the impact on performance of changes in the parameters used in these detection rules. we find that the swiss and german detection rules trigger no exposure notifications on our data, while the italian detection rule generates a true positive rate of 50% and a false positive rate of 50%. our analysis indicates that the performance of such detection rules is similar to that of triggering notifications by randomly selecting from the participants in our experiments, regardless of proximity. there is currently a great deal of interest in the use of mobile apps to facilitate covid-19 contact tracing, see e.g. [1] [2] [3] . the basic idea of a contact tracing app is that if two people carrying mobile handsets installed with the app spend significant time in close proximity to one another (e.g. spending 15 minutes within 2 metres) then the apps on their handsets will both record this contact event. contact tracing apps based on the google/apple exposure notification (gaen) api [4] are currently being rolled out across europe, with apps already deployed in italy, switzerland and germany. these apps use bluetooth received signal strength to estimate proximity and will likely be used as an adjunct to existing manual contact tracing and test systems. existing manual systems can usually readily identify the people with whom an infected person share accommodation and with work colleagues with whom the infected person is in regular contact. more difficult is to identify people travelling on public transport with whom an infected person has been in contact, since the identities of these people are usually not known to the infected person and are generally not otherwise recorded. public transport is therefore potentially an important use case where effective contact tracing apps may be of significant assistance in infection control. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 we report on the results of a covid-19 contact tracing app measurement study carried out on a commuter tram. the tram is of a standard design widely used in europe. measurements were collected between 108 pairs of handset locations and are publicly available [5] . in summary, our measurements indicate that in the tram there is little correlation between received signal strength and distance between handsets. similar ranges of signal strength are observed both between handsets which are less than 2m apart and handsets which are greater than 2m apart (including when handsets are up to 5m apart). this is likely due to reflections from the metal walls, floor and ceiling within the tram, metal being known to be a strong reflector of radio signals [6, 7] , and is coherent with the behaviour observed on a commuter bus [8] . we applied the detection rules used by the italian, swiss and german contact tracing apps to our measurement data and also characterised the impact on performance of changes in the parameters used in these detection rules. we find that the swiss and german detection rules trigger no exposure notifications, despite around half of the pairs of handsets in our data being less than 2m apart. the italian detection rule has a true positive rate (i.e. correct detections of handsets less than 2m apart) of around 50%. however, it also has a false positive rate of around 50% i.e. it incorrectly triggers exposure notifications for around 50% of the handsets which are greater than 2m apart. this performance is similar to that of triggering notifications by randomly selecting from the participants in our experiments, regardless of proximity. we observe that changing the people holding a pair of handsets, with the location of the handsets otherwise remaining unchanged, can cause variations of ±10db in the attenuation level reported by the gaen api. this is pertinent because this level of "noise" is large enough to potrentially have a substantial impact on proximity detection. the experimental protocol was reviewed and approved by the ethics committee of the school of computer science and statistics, trinity college dublin. the ethics application reference number is 20200503. oral consent was obtained from participants. our experimental measurements were collected on a standard light-rail tram carriage used to carry commuters in dublin, ireland, see fig 1(a) . we recruited seven participants and gave each of them google pixel 2 handsets. we asked them to sit in the relative positions shown in fig 1(b) . this positioning aims to mimic passengers respecting the relaxed social distancing rules likely during easing of lockdown and with the distances between participants including a range of values < 2m and a range of values > 2m, see fig 2. each experiment is 15 minutes duration giving around 3 scans by the gaen api when scans are made every 4 mins (per measurements reported in [9] ). a wifi hotspot was set up on the tram and the participants were asked to hold the handset in their hand and use it for normal commuter activities such as browsing the internet. after the first experiment was carried out participants were then asked to switch seats (they chose seats themselves) and a second 15 minute experiment run. after the second experiment participants were again asked to change seats for the third 15 minute experiment and, in addition, two participants were asked to place their handsets in their left trouser pocket (in an orientation of their choice). each handset had the gaen api and a modified version of the google exemplar exposure notification app [10] installed, and was registered to a gmail user included on the google gaen whitelist so as to allow use of the gaen api by the exposure notification app. each handset also had a gaenadvertiser app developed by the authors installed. this app implements the transmitter side of the gaen api and allowed us to control the tek used and also to start/stop the broadcasting of bluetooth le beacons. at the start of each 15 minute experiment participants were asked to configure the gae-nadvertiser app with a new tek and then to instruct the app to start broadcasting gaen beacons. at the end of the experiment the gaenadvertiser stopped broadcasting beacons. in this way a unique tek is associated with each handset in each experiment, and these can be used to query gaen api to obtain separate exposure information reports for each handset in each experiment. following all three experiments the handsets were collected, the teks used by each handset extracted and the gaen api on each then queried for exposure information relating to the teks of the other handsets. in total, therefore, from these experiments we collected gaen api reports on bluetooth le beacon transmissions between 108 pairs of handset locations. this measurement data is publicly available [5] . to provide baseline data on the radio propagation environment we also used the standard android bluetooth le scanner api to collect measurements of rssi as the distance was varied between two google pixel 2 handsets placed at a height of approximately 0.5m (about the same height as the tram seating) in the centre aisle of the tram carriage. we used google pixel 2 handsets running gaen api version 202512001 as reported in the settings-covid 19 notifications handset display, which includes a major update by google issued on 13th june 2020. we used a version of the google exemplar exposure notification app modified to allow us to query the gaen api over usb using a python script (the source code for the modified app is available on github [10] ). in addition we also wrote our own gaenadvertiser app that implements the bluetooth le transmitter side of the gaen api [4] . gaenadvertiser allows us to control the tek, and in particular reset it to a new value at the start of each experiment. in effect, resetting the tek makes the handset appear as a new device from the point of view of the gaen api, and so this allows us to easily collect clean data (the gaen api otherwise only resets the tek on a handset once per day). we carried out extensive tests running gaenadvertiser and the gaen api on the same device to confirm that under a wide range of conditions the responses of the gaen api on a second receiver handset were the same for beacons from gaenadvertiser and the gaen api, see [9] for further details. subsequent to our measurement study google has now published the code for the transmitter side implementation and details of the receiver side attenuation calculation [11, 12] . these also confirm that the gaenadvertiser implementation is essentially identical to the google transmitter-side implementation. gaenadvertiser is open source and can be obtained by contacting the authors (we have not made it publicly available, however, since it can be used to facilitate a known replay attack against the gaen api [13] ). the basic idea of a contact tracing app is that if two people carrying mobile handsets installed with the app spend significant time in close proximity to one another (e.g. spending 15 minutes within 2 metres) then the apps on their handsets will both record this contact event. if, subsequently, one of these people is diagnosed with covid-19 then the contact events logged on that person's handset in the recent past, e.g. over the last two weeks, are used to identify people who have been in close contact with the infected person. these people might then be made aware of the contact and advised to self-isolate or take other appropriate precautions. for this approach to be effective it is, of course, necessary that the app can accurately detect contact events. the gaen api uses bluetooth le wireless technology as the means for detecting contact events. bluetooth le devices can be configured to transmit beacons at regular intervals. to distinguish between beacons sent by different handsets each handset running gaen generates a random temporary exposure key (tek) once a day. this tek is then used to generate a sequence of rolling proximity identifiers (rpis), approximately one for each 10 minute interval during the day (so around 144 rpis are generated). the gaen system running on a handset transmits beacons roughly every 250ms. each beacon contains the current rpi value. approximately every 10 minutes the beacons are updated to transmit the next rpi value. by constantly changing the content of beacons in this way the privacy of the system is improved. in addition to the rpi each beacon also carries encrypted metadata containing the wireless transmit power level used. although beacons are emitted roughly every 250ms, on the receiving side, devices only scan for beacons roughly every 4 minutes [9] . the basic idea is that the signal strength with which a beacon is received provides a rough measure of the distance between transmitter and receiver. namely, when the received signal strength is sufficiently high then this may indicate a contact event and, conversely, when the received signal strength is sufficiently low then this may indicate that the handsets are not in close proximity. this is based on the fact that in general the radio signal gets weaker as it travels further since the transmit power is spread over a greater area. however, many complex effects can be superimposed upon this basic behaviour. in particular, obstacles lying on the path between the transmitter and receiver (furniture, walls etc) can absorb and/or reflect the radio signal and cause it to be received with higher or lower signal strength. a person's body also absorbs radio signals in the 2.4 ghz band used by bluetooth le and so the received signal strength can be substantially reduced if their body lies on the path between the transmitter and receiver. the relative orientation of two handsets can strongly affect the received signal strength owing to way antennae are packaged within the handset body. in indoor environments walls, floors and ceilings can reflect radio signals even when they are not on the direct path between transmitter and receiver, and so increase or decrease the received signal strength. see, for example, [14] for measurements illustrating such effects in real environments. the gaen system presents an interface to health authority apps this interface allows these apps to submit a request that includes an exposure configuration data structure to the gaen system [4] . the exposure configuration data structure allows specification of the tek to be queried, the start time and duration of the interval of interest (specified in 10 minute intervals since 1st jan 1970) and a low and high attenuation threshold (specified in db). the gaen system responds with one or more exposure information data structures that report an exposure duration (field durationminutes) and an array with three atttenuation duration values, giving the duration (in minutes) that the attenuation level is below the low threshold, the duration the attenuation level is between the low and high thresholds and the duration above the high threshold. it is also possible to query for an exposure summary response, but we did not make use of this since the relevant information that this contains can be derived from the exposure information reports. for each tek and time interval we made repeated queries to the gaen api holding the low threshold constant at 48db and varying the high threshold from 49db to 100db (in 1db steps up to 80db, then in 5db steps since noise tends to be higher at higher attenuation levels). by differencing this sequence of reports we can infer the attenuation duration at each individual attenuation level from 48db through to 100db. at the time of our meaurement study the gaen documentation did not precisely state how the attenuation level is calculated, nor did it give details as to how the attenuation duration is calculated. the analysis in [9] , indicated the attenuation level is calculated as p tx − p rx , where p tx is the transmit power level sent in the beacon metadata and p rx is given by a filtered rssi for google pixel 2 handsets (and others) the rssi is recorded only from beacons transmitted on one of the three radio channels used by bluetooth le for transmitting beacons, see [9] . measurements plus a calibration offset. google has subsequently published documentation [12] that confirms this. for the google pixel 2 handsets and gaen api version 202512001 used in our experiments p tx is -31db and the calibration offset is -6db. google supplied us with the calibration and offset values used for all handset models in gaen version 202512001 and we have posted these in our online study archive [5] . note that we observed that the noise floor (the rssi below which beacons can no longer be reliably decoded) is around -100db in a pixel 2, giving a maximum measureable attenuation of around 75db i.e. above this attenuation level beacons are generally not decoded successfully and so no rssi values are reported by bluetooth scans. fig 3(a) plots the attenuation measured between two handsets placed at seat height in the aisle of the tram as the distance between them is varied. these measurements were taken using the standard android bluetooth le scanner api (rather than the gaen api). this scanner api reports an rssi value for each received beacon. following [9] updated to reflect gaen calibration changes pushed by google on 13th june 2020, for the google pixel 2 handsets used in our experiments we map from rssi to attenuation level using the formula -31-(rssi-6) db. it can be seen that the attenuation initially increases as the distance is increased from 0.5m to 1.5m, as might be expected. but thereafter the attenuation level stays roughly constant with increasing distance out to 2.5m. there is then a sharp rise in the attenuation at 3m. this corresponds to the end of a group of seats and the start of a flexible joint between two carriages. as the distance is increased further it can be seen that the attenuation starts to fall. the attenuation is around 52db at 1.5m and around 60db at 4m. the baseline measurements in fig 3 indicate that the radio attenuation within the tram does not simply increase with the distance between handsets. this is similar to the behaviour observed in previous gaen measurements taken on a bus [8] , and is of course pertinent to the use of attenuation level as a proxy for distance. although further measurements are needed to confirm this, it seems likely that this effect is due to reflections from the tram walls, ceiling and floor, all of which are made of metal and highly reflective at the bluetooth radio frequency. the full attenuation duration data reported by gaen api is given in the appendix and is publicly available online [5] . in this section we analyse two aspects of this data: (i) the relationship, if any, between attenuation level and distance between handsets and (ii) the magnitude of the variations in the attenuation level induced by differences in the way participants hold their handsets. fig 4 plots the mean attenuation level vs the distance between participants in the three tests. the mean is calculated by weighting each attenuation level by the duration at that level reported by the gaen api and then summing over all attenuation levels. it can be seen that there is no clear trend in the mean attenuation level as the distance changes, with similar ranges of attenuation levels observed at all distances, except perhaps for distances below 1m where the attenuation level is more tightly clustered. the gaen api records the duration at each attenuation, and so effectively the full distribution of attenuation levels rather than just the mean. fig 5 plots the sum-duration that the measured attenuation level is below 55db, 63db, 68db and 73db. for each pair of handsets these values are the rescaled empirical cdf of the attenuation level evaluated at the specified values. recall that a typical definition of a proximity event is spending 15 minutes or more at a distance of 2m or less apart. we have therefore indicated the 2m distance with a vertical line in fig 5, and attenuation durations greater than 15 minutes by the shaded areas. for reliable detection of proximity events what one might like is that for an appropriate choice of threshold value the attenuation levels lie within the shaded area when the distance is less than 2m and outside the shaded area when the distance is greater than 2m. unfortunately we do not see such behaviour in between each of the three experiments the participants switch seats. the seat positions themselves remain the same, only the person sitting in the seat changes, allowing us to see the impact of differences in the way that each participant uses their handset. for beacons transmitted from each seat position fig 6 shows the mean attenuation level observed at the other seat positions (see the appendix for the full attenuation duration data). the attenuation level observed in test 1 is plotted vs the attenuation level observed in test 2. it can be seen that the points are clustered around the 45˚line, but variations of ±10db between the two tests are common. since the seating locations and environment within the tram are the same between experiments, participants have similar build and height and use the same model of handset, these variations can be attributed to differences in the way each particpant holds their handset and/or changes between tests in the way the same particpant holds their handset. such substantial variations in attenuation level are obviously pertinent to the use of attenuation level for proximity detection. the gaen api is intended for use by health authority covid-19 contact tracing apps [4] . when a person is found to be infected with covid-19 the teks from their handset are uploaded to a central server. the health authority app on another person's handset can then download these teks, and use them to compare against the set of beacons received by the handset. if there is a match, the attenuation duration values reported by the gaen api can then be used to estimate the risk of infection and trigger an exposure notification is this risk is sufficiently high. a typical requirement is for a person to have spent at least 15 minutes within 2m of the infected person in order to trigger an exposure notification. the mapping from gaen attenuation durations to exposure notification is therefore largely based on use of attenuation level as a proxy for proximity between handsets. switzerland deployed a covid-19 contact tracing app based on the gaen api on 26 may 2020 [15] . the documentation for this app states that it queries the gaen api with low and high attenuation thresholds of t1 = 50db and t2 = 55db and then bases exposure notifications on the quantity es = b1 + 0.5b2, where b1 is the attenuation duration below 50db reported by the gaen api and b2 is the attenuation duration between 50db and t2 [16] . an exposure notification is triggered when es is greater than 15 mins, see table 1 . germany deployed a covid-19 contact tracing app based on the gaen api on 15 june 2020 [17] . the app is open source. by inspecting the documentation and code, and querying the server api to obtain the app configuration settings this means that the app configuration table 1 . summary of detection rules studied. b1 is the the attenuation duration below threshold t1, b2 is the attenuation duration between t1 and t2, b3 is the attenuation duration below t2. can be dynamically updated. we downloaded the detection settings from the server on 21 june 2020 and they are included in the study data repository [5] , we determined that the german app follows an approach similar to the swiss app for triggering an exposure notification, but uses values t1 = 55db and t2 = 63db and an exposure duration on 10 minutes. we applied the swiss and german exposure notification rules to the tram dataset. fig 7( a) plots the true and false positive rates for t1 = 50db and as t2 is varied from 55db upwards and the es threshold varied from 10 minutes to 15 mins. the mean rates are shown with one standard deviation indicated by the error bars. the mean and standard deviation are obtained by a standard bootstrapping approach the dataset was resampled with replacement n = 1000 times, the exposure notification percentage calculated for each sample and then the mean and standard deviation of these n estimates calculated. we selected n by calculating the mean and standard deviation vs n and selecting a value large enough that these were convergent. fig 7(b) plots the true and false positive rates when t1 = 55db. it can be seen from fig 7(a) that selecting t1 = 50db and t2 = 55db (the values used in the swiss app) yields no positive detections, despite approximately 50% of the handset pairs in the tram dataset being within a 2m distance of one another. increasing t2 to 62db and above yields a small increase in detection rate, with true and false detection rates roughly equal (we comment further on the implications of this below). it can be seen from fig 7(b) that selecting t1 = 55db and t2 = 63db (the values used in the german app) there are are no detections when the threshold for es is 15 minutes but when a threshold of 10 minutes is used, as in the app, then the true and false positive detection rates both rise to 9%. increasing t2 does not increase these detection rates. italy deployed a covid-19 contact tracing app based on the gaen api on 1 june 2020 [18, 19] . the app is open source. by inspecting the documentation and code, and querying the server api to obtain the app configuration settings we downloaded the detection settings from the italian app server on 21 june 2020 and they are included in the study data repository [5] , we determined that the app follows a different approach to the swiss and german apps, triggering an exposure notification whenever the attenuation duration is above threshold t2 = 73db i.e. without the weighting of 0.5 used in the swiss and german exposure notification rules. we applied this exposure notification rule to the tram dataset. fig 8(a) plots the true and false positive rates as threshold t2 is varied from 55db upwards and the threshold for es is varied from 10 minutes to 15 mins. for t2 = 73db the true and false positive detection rates are both around 50% when the threshold for es is 15 minutes, rising to 80% when the threshold for es is reduced to 10 minutes. as noted in section 2.5, with the calibration values used in the gaen api the maximum observable attenuation level with google pixel 2 handsets is around 75db (above this level beacons are generally no longer successfully received). selecting t2 = 73db therefore means that almost the full range of possible attenuation levels will trigger an exposure notfication. high detection rates are therefore unsurprising, but the detection has little discrimination and essentially would trigger exposure notifications for all participants in our tests regardless of proximity. fig 8(a) also shows the true and false positive detection rates for other choices of threshold t2. while the detection rates are generally substantially higher than with the swiss and german detection rules, it can be seen that the false positive rate increases almost exactly in line with the true positive rate. this can be seen more clearly when this data is replotted in roc format, see fig 8(b) . it can be seen that the true vs false positive curve lies close to the 45˚line (to avoid clutter we do not plot the error bars from fig 8(a) on fig 8(b) but the small deviation from the 45˚line is not statistically significant.). that is, the detection performance is poor, and comparable to simply selecting from participants at random when making exposure notifications. a limitation of this study is that it is confined to handsets using the android operating system. the gaen api is also implemented on apple ios devices, but apple have severely limited the ability of testers to make measurements (each handset is limited to querying the gaen api a maximum of 15 times a day, and apple has no whitelisting process to relax this constraint. our measurement approach uses 34 queries to extract fine-grained attenuation data per pair of handset locations. we equipped participants with the same model of handset in order to remove this as a source of variability in the data and instead focus on variability caused by the radio environment and the way that people hold their handsets. google and apple are currently undertaking a measurement campaign to select calibration values within the gaen api with the aim of compensating for differences between handset models. we therefore expect that our measurements should also be applicable to a range of handsets, although this remains to be confirmed. with regard to calibration, we note that bluetooth received signal strength is affected by several factors including (i) differences between different models/makes of handset, (ii) fluctuations in the relative orientation of handsets (even small changes can have a large impact), (iii) absorption by human bodies (especially when phone is in a pocket), bags etc, (iv) radio wave reflection from walls, floors, furniture. see [14] for measurements highlighing the potential for significant impact of these factors. calibration may mitigate (i), although this remains unclear at present and variations between handsets might be expected to degrade performance compared to our measurements, but not (ii)-(iv). in both the tram measurements reported here and previous measurements in a commuter bus [8] only a weak correlation between received signal strength and distance between handsets is observed. a direct comparison of detection accuracy in these two datasets is unfortunately not possible since in the bus measurements all pairs of handsets were within 2m of one another and so only the rate of false negatives can be evaluated. we report on the results of a covid-19 contact tracing measurement study carried out on a commuter tram in dublin, ireland. our measurements indicate that in the tram there is little correlation between received signal strength and distance between handsets. we applied the detection rules used by the italian, swiss and german apps to our measurement data and also characterised the impact on performance of changes in the parameters used in these detection rules. we find that the swiss and german detection rules trigger no exposure notifications on our data, while the italian detection rule generates a true positive rate of 50% and a false positive rate of 50%. our analysis indicates that the performance of such detection rules is similar to that of triggering notifications by randomly selecting from the participants in our experiments, regardless of proximity. bin, with the rest of the time roughly evenly split between 62-64db, 66-68db, 70-72db. the weighted average attenuation is 70db. plot the exposure information between each pair of handsets reported by the gaen api for each of the three experiments. to assist with interpreting the plots the reports in each plot are ordered by increasing distance between the pairs of participants (see fig 1(a) ). no data is shown when no beacons were received between a pair of handsets, e.g. between particpants 2 and 3 in fig 9(b) and 9(c). it can be seen that occasionally there is an increasing trend in attenuation, for example see figs 9(c) and 11(c), but this is infrequent. occasionally there is a decreasing trend in attenuation, for example see figs 9(e) and 11(d). overall, however no consistent trend is evident in the change in attenuation level with increasing distance. in fig 11 participants 1 and 3 place their handsets in their left trouser pocket rather than their hand. intuitively, one might expect this change to increase the attenuation level since the particpants body is now more likely to affect transmission and reception of radio signals. however, comparing fig 11(a) and 11(c) with figs 9 and 10 it can be seen that this change does not cause any consistent change in the observed attenuation level. for example, comparing figs 11 (a) and 10(a) the attenuation level between participants 1 and 5 decreases from test 2 to test 3, while the attenuation level between participants 1 and 3 increases. quantifying sars-cov-2 transmission suggests epidemic control with digital contact tracing eu urges vigilance to avoid coronavirus second wave apple and google partner on covid-19 contact tracing technology exposure notifications: android api documentation dublin luas tram gaen attenuation durations dataset experimental study of propagation characteristics for wireless communications in high-speed train cars experimental characterisation and modelling of intra-car communications inside highspeed trains. iet microwaves, antennas and propagation measurement-based evaluation of google/apple exposure notification api for proximity detection in a commuter bus verifying the google/apple covid exposure notification api modified exposure notification app exposure notifications: source code snippets exposure notifications: exposure notifications ble attenuations a coronavirus contact tracing app replay attack with estimated amplification factors coronavirus contact tracing: evaluating the potential of using bluetooth received signal strength for proximity detection coronavirus: first google/apple-based contact-tracing app launched dp-3t exposure score calculation summary accessed 14 open source project; accessed 23 immuni app web site; accessed 23 immuni apple store version history the authors would like to extend their thanks to the irish health & safety executive (hse) for arranging with google for us to have whitelisted access to the gaen api, and to transdev dublin light rail for kindly providing access to one of their trams. we emphasise that any views expressed in this report are the authors own, and may not be shared by the hse, transdev or trinity college dublin. we present the full attenuation duration data using a coloured heatmap. we split the range of attenuation values into 2db bins, i.e. 70-72db, 72-74db and so on, up to 80db when 5db bins are thereafter used since the data is noisier at these low signal levels. within each bin the colour indicates the percentage of the total duration reported by the gaen api that was spent in that bin, e.g bright green indicates that more than 90% of the time was spent in that bin. the mapping from colours to percentages is shown on the righthand side of the plot. bins with no entries (i.e. with duration zero) are left blank. where appropriate we also include a solid line in plots that indicates the average attenuation level at each transmit power level (the average is calculated by weighting each attenuation level by the duration at that level and then summing over all attenuation levels).for example, in fig 9(a) the left-hand heatmap shows the attenuation durations measured between participants 1 and 4. the attenuation spends around 60% of its time in the 74-76db conceptualization: douglas j. leith, stephen farrell. key: cord-295559-yc8q62z8 authors: qian, zhaohui; dominguez, samuel r.; holmes, kathryn v. title: role of the spike glycoprotein of human middle east respiratory syndrome coronavirus (mers-cov) in virus entry and syncytia formation date: 2013-10-03 journal: plos one doi: 10.1371/journal.pone.0076469 sha: doc_id: 295559 cord_uid: yc8q62z8 little is known about the biology of the emerging human group c betacoronavirus, middle east respiratory syndrome coronavirus (mers-cov). because coronavirus spike glycoproteins (s) mediate virus entry, affect viral host range, and elicit neutralizing antibodies, analyzing the functions of mers-cov s protein is a high research priority. mers-cov s on lentivirus pseudovirions mediated entry into a variety of cell types including embryo cells from new world eptesicus fuscus bats. surprisingly, a polyclonal antibody to the s protein of mhv, a group a murine betacoronavirus, cross-reacted in immunoblots with the s2 domain of group c mers-cov spike protein. mers pseudovirions released from 293t cells contained only uncleaved s, and pseudovirus entry was blocked by lysosomotropic reagents nh(4)cl and bafilomycin and inhibitors of cathepsin l. however, when mers pseudovirions with uncleaved s protein were adsorbed at 4°c to vero e6 cells, brief trypsin treatment at neutral ph triggered virus entry at the plasma membrane and syncytia formation. when 293t cells producing mers pseudotypes co-expressed serine proteases tmprss-2 or -4, large syncytia formed at neutral ph, and the pseudovirions produced were non-infectious and deficient in s protein. these experiments show that if s protein on mers pseudovirions is uncleaved, then viruses enter by endocytosis in a cathepsin l-dependent manner, but if mers-cov s is cleaved, either during virus maturation by serine proteases or on pseudovirions by trypsin in extracellular fluids, then viruses enter at the plasma membrane at neutral ph and cause massive syncytia formation even in cells that express little or no mers-cov receptor. thus, whether mers-cov enters cells within endosomes or at the plasma membrane depends upon the host cell type and tissue, and is determined by the location of host proteases that cleave the viral spike glycoprotein and activate membrane fusion. coronaviruses cause respiratory, enteric, renal and/or neurological disease in humans, many other mammals and birds. in 2002-03 a previously unknown coronavirus emerged from a wild animal reservoir to cause the sars pandemic, with about 8,000 human cases and more than 770 deaths [1, 2] . previously, cross-species transmission from coronaviruses of bat and bovine origin had allowed human respiratory coronaviruses oc43, nl63 and 229e to become established in the human population worldwide [3] [4] [5] [6] [7] [8] . in the arabian peninsula in 2012, another novel human cov, now called middle east respiratory syndrome coronavirus (mers-cov), was isolated in vero e6 cells from sputum from a fatal case of severe respiratory disease with kidney failure. since then, mers-cov rna has been detected by rt-pcr in over 70 patients with severe to moderate respiratory disease, 39 of whom have died [9, 10] . genome sequence analysis showed that mers-cov is a novel betacoronavirus in genogroup c, closely related to two prototype group c betacoronaviruses of asian bats, btcov-hku4 from a tylonycteris pachypus bat and btcov-hku5 from a pipistrellus abramus bat [11] , and to partial sequences of a group c betacoronavirus from a pipistrellus pipistrellus bat in the netherlands [12] . recently group c betacoronaviruses were also detected in a nyctinomops laticaudatus bat in mexico [13] , and nycyteris cf. gambiensis bats in ghana [14] . mers-cov, like sars-cov, is probably a zoonotic betacoronavirus that has spilled over into humans, directly or indirectly, from one of the species of bats that harbor group c betacoronaviruses or from other unknown animal reservoirs [13, 15, 16] . the ~200 kda spike glycoprotein (s) of coronaviruses is an important determinant of virus virulence, tissue tropism and host range. trimers of s form the characteristic large spikes on the coronavirus envelope that bind to receptors, mediate membrane fusion, virus entry and syncytia formation, and elicit virus neutralizing antibodies. coronavirus s proteins are class i viral fusion proteins like the hiv envelope (env), influenza hemagglutinin (ha) and paramyxovirus fusion (f) glycoproteins [17] , which typically require protease cleavage between the s1 and s2 domains ( figure 1a ) to permit conformational changes in s2, activated by receptor binding and/or low ph, that mediate membrane fusion leading to virus entry and syncytia formation [3, 17, 18] . in different cell types and tissues, coronavirus s proteins may be cleaved by a variety of host proteases including furin, trypsin, human airway trypsin-like protease (hat), transmembrane protease serine protease-2 (tmprss-2), tmprss-4, or cathepsins [18] [19] [20] [21] [22] . functional analysis of mers-cov s glycoprotein is needed to identify susceptible cell types and host species that affect viral tissue tropism and host range, and to determine how various host proteases promote mers-cov virus entry and syncytia formation. identification of the receptor or receptors is an important first step in understanding the host range and tissue tropism of coronaviruses. four receptor proteins for spike proteins of different coronaviruses are now known: murine carcinoembryonic antigen cell adhesion molecule 1a (mceacam1a) for mouse hepatitis virus (mhv) [23] , a betacoronavirus in group a; aminopeptidase n (apn) for human coronavirus 229e (hcov-229e) and several other alphacoronaviruses [24, 25] ; and angiotensin-converting enzyme 2 (ace2) for both sars-cov, a betacoronavirus in group b and hcov-nl63, an alphacoronavirus [26, 27] . raj and co-workers [28] recently demonstrated that mers-cov uses dipeptidyl peptidase 4 (dpp4) as a receptor. in contrast, s proteins of several group a betacoronaviruses including bovine coronavirus and hcov-oc43 use sialic acid moieties as receptors [29, 58] . we have used lentivirus pseudotypes with mers-cov spike glycoprotein to identify cells susceptible to infection with mers-cov and to study the role of mers s protein in virus entry and syncytia formation. expression of coronavirus s proteins on 293t cell membranes for incorporation into lentivirus pseudovirions can be enhanced by using codon-optimized spike cdna and deleting an er/golgi retention motif and an endosomal recycling motif from the cytoplasmic tail of s [30] [31] [32] . codonoptimized cdna encoding s of mers-cov (derived from genbank: afs88936) [15] , with the 16 c-terminal amino acids replaced by a linker, ggggs, and a flag tag (here called mers-cov sδ16) ( figure 1a ) was expressed on 293t cell figure 1b, lane 2) . no protease cleavage products of the ~200kda s protein were detected in transfected 293t cells or pseudovirions ( figure 1b) . in marked contrast, the mers lentivirus pseudovirions used to identify cells susceptible to entry of mers-cov in the poehlmann laboratory [33] , contained a high proportion of cleaved mers-cov s protein at about 100 kda. this important difference in the mers pseudovirions is likely due to differences between our 293t cells and those used in the poehlmann laboratory. surprisingly, when these mers pseudovirions and cell lysates were blotted with polyclonal goat antibody ao4 to spikes purified from detergent-disrupted virions of mhv-a59, a betacoronavirus in group a, the mers s protein bands were detected ( figure 1b ). immunoblotting of soluble, truncated mers s proteins with c-terminal flag tags showed that the ao4 antibody did not recognize the s1 domain of mers s ( figure 1c ), so the cross-reactivity between these proteins from betacoronavirus groups a and c must lie within the s2 domain. vero e6 and llcmk2 monkey kidney cell lines are susceptible to infection with mers-cov virus and to sars-cov [10, 34] , and also susceptible to sars pseudovirions and to mers pseudovirions with uncleaved s protein ( figure 2a ). cell entry was quantitated by expression of the luciferase reporter gene in pseudovirus-transduced cells. compared to control pseudovirions with no spike protein, mers pseudovirions showed a 100 to 1,000 fold increase in luciferase activity in vero e6 and llcmk2 cells (figure 2a and b), and sars pseudovirions showed a 1,000 increase in luciferase activity in vero e6 cells. because the uncleaved mers-cov s protein mediated virus entry into vero e6 and llcmk2 cells, transduction by mers pseudovirions was used to identify additional cell lines that express functional receptors for mers-cov [10, 34] . mers pseudovirions detected strong mers-cov receptor activity on the calu3 line of human airway epithelial cells (figure 2a and b) , and weaker receptor activity on the a549 line of human alveolar basal epithelial cells ( figure 2a ) as also shown by mers-cov infection [35] . interestingly, the eff embryo cell line from eptesicus fuscus bats was susceptible to mers pseudovirions, increasing luciferase activity by nearly 100-fold compared to the no spike control, but the tb1lu lung cell line from tadarida brasiliensis bats, murine fibroblasts and hela cells were not susceptible to mers pseudovirions ( figure 2c ). expression of human ace2 in 293t cells did not significantly increase susceptibility to mers pseudovirions ( figure 3a ), although as expected hace2 greatly increased susceptibility of 293t cells to sars pseudovirions ( figure 3a ). figure 3b and c show that neither human ceacam1, or four related human ceacam proteins or human apn functions as a receptor for mers-cov spike protein. these experiments confirm the observation that mers-cov does not use the receptor proteins known for other coronaviruses [33] or related human membrane proteins. instead dpp4 is the principal receptor protein for mers-cov [28] . mers pseudovirions induced a small but consistent 5 to 10-fold increase in luciferase activity in 293t human embryo kidney cells compared to the no spike control virus ( figure 2c ), suggesting that our 293t cells expressed either a low level of dpp4, or an alternative but less efficient receptor, such as cd209l or lsectin for sars-cov [36, 37] . to determine whether entry of mers pseudovirions with uncleaved s protein required endocytosis and acidification in endosomes, the effects of ammonium chloride and bafilomycin a, lysosomotropic agents that inhibit the acidification of endosomes, were studied. in vero e6 cells, 20mm nh 4 cl inhibited entry of sars pseudovirions by about 99.9% compared to entry of sars pseudovirions without inhibitor, and nh 4 cl also inhibited entry of mers pseudovirions by about 99.6% ( figure 3a ). bafilomycin a specifically inhibits the vacuolar-type h+-atpase that is required for acidification of lysosomes. figure 3a shows that bafilomycin a inhibited entry of sars pseudovirions into vero e6 cells by 99.8% as previously reported [21, 38] , and also inhibited entry of mers pseudovirions by more than 99.9% compared to mers pseudovirions without inhibitor. in llcmk2 cells, although bafilomycin a inhibited 99.7% of mers-cov s mediated entry, nh 4 cl reduced mers-cov s-mediated entry only 6-fold (data not shown), suggesting that the inhibition of endosomal acidification by nh 4 cl may be cell type dependent. these experiments show that mers pseudovirions with uncleaved s protein can enter monkey kidney cells only by endocytosis. cathepsins are a diverse group of acid-activated cysteine proteases located within endosomes and lysosomes. cathepsin activity is essential for infection by several viruses that enter by the endosomal route, including reovirus [39] , sars-cov [22] , and ebolavirus [40] . e64d, an inhibitor of the cysteine protease activities of cathepsins b, h, and l and calpain, reduced transduction of vero e6 cells by sars pseudovirions by 80% as previously reported ( figure 3b ) [41] . since cell entry mediated by vsv-g glycoprotein does not require protease activation [17] , e64d treatment of vero e6 ( figure 3b ) and llcmk2 cells (data not shown) did not inhibit entry of vsv pseudovirions. however, e64d decreased entry into vero e6 cells of mers pseudovirions with uncleaved s by 96.7% ( figure 3b ) and llcmk2 cells by 99.2% (data not shown). thus, cleavage of mers-cov s protein by one of the cathepsins or calpain was required for triggering s-mediated membrane fusion and virus entry at low ph in endosomes. as previously reported [41] , in vero e6 cells 10 µm of cathepsin l inhibitor iii, a specific and irreversible inhibitor of cathepsin l, significantly inhibited entry mediated by sars s protein, but did not inhibit vsv-g-mediated entry ( figure 3b ). cathepsin l inhibitor iii reduced entry into vero e6 cells of mers pseudovirions with uncleaved s protein by 97% relative to entry without inhibitor ( figure 3b ), and similar results were seen in llcmk2 cells (data not shown). thus, mers-cov s protein on pseudovirions must be cleaved in endosomes by the acidactivated cysteine protease activity of cathepsin l to trigger receptor-dependent entry into vero e6 and llcmk2 cells. trypsin cleavage of mers-cov s on pseudovirions adsorbed to receptors on the cell surface triggers virus entry at the plasma membrane at neutral ph sars-cov can enter susceptible cells at the plasma membrane, instead of by endocytosis, if virions adsorbed at 4°c to ace2 on the cell membrane are treated with trypsin, then warmed to 37°c in the presence of an inhibitor of endosomal acidification [21] . trypsin treatment at either 4°c or 37°c cleaved the s protein of mers pseudovirions and generated a ~65kda subunit in the s2 domain of the protein recognized by antibody to mhv-a59 s protein ( figure s1 ). mers pseudovirions with uncleaved s protein were adsorbed at 4°c to cell surface receptors on vero e6 cells in the presence of 20mm nh 4 cl, and then the cells with bound virions were briefly treated with trypsin at ph 7.4 at room temperature to cleave the ~200 kda s protein and activate its membrane fusing activity. figures 3a and 4a show that nh 4 cl strongly inhibited infection of vero e6 cells by mers pseudovirions with uncleaved s. however, trypsin treatment of the mers pseudovirions bound at neutral ph and 4°c to the vero e6 cell membrane triggered both virus entry at the plasma membrane and formation of small syncytia by 40 hours post inoculation ( figure 4a and b). thus, receptor binding together with protease cleavage and activation of s at neutral ph was sufficient to trigger entry of mers pseudovirions and syncytia formation. in this experiment membrane fusion did not depend upon synthesis of s protein, but syncytia formation was mediated by the cleaved s protein on pseudovirions adsorbed to virus receptor on the cell membrane. although acidic ph is required to activate the cathepsin l activity that allows mers pseudovirions to enter at endosomes, low ph is not required for the conformational changes in trypsin-cleaved mers-cov s protein that mediate entry at the plasma membrane. 293t cells expressing uncleaved mers-cov s protein or control cells stably transfected with the empty pcdna3.1 vector were overlaid on monolayers of vero e6 cells in the presence or absence of tpck trypsin ( figure 4c ). no syncytia formation was induced by 293t cells with empty vector or 293t cells expressing mers-cov sδ16 without trypsin ( figure 4c ), but addition of tpck trypsin to the medium triggered formation of massive syncytia in the vero e6 cells co-cultured for 20 hr with mers-cov s-expressing 293t cells ( figure 4c , arrows). large syncytia were also formed after even a brief 20 minute trypsin pre-treatment at ph 7.4 and 4°c of 293t cells expressing mers-cov s protein, followed by incubation with a 5-fold excess of soybean trypsin inhibitor before layering the cells over confluent monolayers of vero e6 cells and incubating at 37°c for 20 hours ( figure 4c, lower central panel, arrows) . thus, trypsin cleavage at neutral ph of mers-cov s protein on 293t cells triggered syncytia formation when s was bound to receptors on susceptible vero e6 cells. type ii transmembrane serine proteases, including tmprss-2 and tmprss-4, which like trypsin are expressed in the respiratory tract, play important roles in triggering entry of influenza a virus, human metapneumovirus and sars betacoronavirus in group b [19, 20, [42] [43] [44] [45] . we therefore transfected 293t cells with plasmids encoding tmprss-2 or -4, mers-cov sδ16 protein, pspax2 and plenti-gfp-luc and investigated whether s proteins on pseudovirions produced in these cells were cleaved and whether they could infect vero e6 cells in the presence of nh 4 cl. surprisingly, the pseudovirionproducing 293t cells expressing either tmprss-2 or -4, formed large syncytia by 40 hrs after transfection ( figure 5a ), but the mers pseudovirions produced by these cells could not transduce vero e6 cells in the presence or absence of nh 4 cl ( figure 5b ). in contrast, without tmprss-2 or -4, the 293t cells expressing uncleaved mers-cov sδ16 did not form syncytia, and pseudovirions that they produced efficiently infected vero e6 cells, but virus entry was inhibited by nh 4 cl ( figure 5b ). immunoblots with antibody ao4 to mhv s or anti-flag (data not shown) revealed that the mers pseudovirions produced in 293t cells expressing tmprss-2 or -4 contained little or no immunoreactive s protein or fragments of s. although the novel group c betacoronavirus mers-cov is highly virulent in humans and can infect cells from several different species, including humans, monkeys, pigs, and some species of bats [10, 16, 34] , little is known about the biology of this virus. because the spike glycoprotein is essential for coronavirus entry, elucidating the functions of the mers-cov spike can provide valuable insight into the pathogenesis of mers-cov, and suggest potential therapeutic interventions. here we used lentivirus pseudovirions with mers-cov spike protein to study s-mediated cell entry at biosafety level 2. we found that mers pseudovirions, like infectious mers-cov virions [10, 34] , readily infected the vero e6 and llcmk2 lines of monkey kidney cells, several human respiratory epithelial cell lines, and embryo cells from eptesicus fuscus bats. others have also recently demonstrated that human respiratory tract cells, and also primary human bronchus and alveolar cells are susceptible to mers-cov in accord with the severe respiratory disease in mers patients [10, 33, 35, 46] . muller et al. [34] recently reported that mers-cov can infect cells from four genera of old world bats, rousettus, rhinolophus, pipistrellus, and myotis, and one new world genus, carollia. we found that mers pseudovirions could also infect cells from one new world bat, e. fuscus, but not from another, t. brasiliensis. the ability of mers-cov to infect cells from multiple mammalian species directly and without adaptation [47] , including a diverse array of both old world and new world bats, suggests that the receptor for mers-cov, dpp4 [28] , is broadly conserved among many species, an important property of many emerging viruses [47, 48] . e. fuscus figure 5a were inoculated onto vero e6 cells in the presence (striped bars) or absence (white bars) of 20mm nh 4 cl to inhibit acidification of endosomes. doi: 10.1371/journal.pone.0076469.g005 bats, commonly known as big brown bats, are the bats most commonly encountered by humans in north america, and they are a reservoir for an alphacoronavirus [49, 50] . it will be important to learn whether these new world bats are susceptible to mers-cov or related group c betacoronaviruses. the recent detection in n. laticaudatus bats in mexico of a group c betacoronavirus with 96% similarity to mers-cov [13] , coupled with the diverse array of alphacoronaviruses previously discovered in north american bats [49] [50] [51] [52] , justify increased surveillance to identify additional species of new world bats that may also harbor group c betacoronaviruses like mers-cov or other coronaviruses with the potential to cause severe disease in humans. mers-cov is a betacoronavirus in group c, and we were surprised that its s protein was recognized in immunoblots by a polyclonal antibody to the spike protein of mhv-a59, a group a betacoronavirus. the cross-reactive epitope(s) was mapped to the s2 domain, which is more highly conserved than the s1 domain of betacoronaviruses. chan et al. [33, 53] found that mers-cov s protein was recognized in immunofluorescence and in vitro neutralization assays by sera of some convalescent sars patients, and suggested, based on bioinformatics, that epitope(s) in s2 could account for the observed serological cross-reactivity. these observations that the s protein of mers-cov, a group c betacoronavirus, contains crossreacting epitope(s) with s proteins of both some group b (sars-cov) and group a (mhv) betacoronaviruses, indicate that serological studies may not accurately distinguish between different phylogenetic groups of betacoronaviruses. identification and characterization of the cross-reacting epitope(s) is an important research priority to show whether there is a common epitope in s that could be used as an immunogen to vaccinate against all betacoronaviruses. enveloped viruses infect cells by fusion of the viral envelope with host cell membranes, a process mediated by a series of conformational changes in the viral fusion protein that are regulated by receptor binding, protease activation, and/or ph [17] . the classes of viral fusion proteins are determined based on their structures and conformational changes during membrane fusion. most class i viral fusion proteins require proteolytic cleavage upstream of the hydrophobic fusion peptide in the viral spike protein to enable these conformational changes to occur, as well as subsequent steps that trigger membrane fusion including either binding to receptor like hiv gp120, low ph in endosomes like influenza ha, or both like avian sarcoma leukosis virus (aslv) [9, 17, [54] [55] [56] . coronaviruses in different phylogenetic groups differ in the sequence of steps leading to virus entry [57, 58] . the s protein on virions of group a betacoronavirus mhv-a59 requires protease activation--either by furin during virus maturation [18] , by trypsin or other serine proteases in extracellular fluids before either receptor binding, or by cathepsin in endosomes at acidic ph-to trigger the conformational changes that lead to membrane fusion and virus entry [59] . in contrast, virions of group b betacoronavirus sars-cov that contain uncleaved s [22] , first bind to the viral receptor protein, ace2, and are endocytosed, and then s is cleaved within endosomal vesicles by acid-dependent cathepsin l enabling the conformational changes in s that lead to virus entry [21, 22, 60] . here we analyzed the steps needed to trigger conformational changes in mers-cov s and their roles in virus entry and syncytia formation. in our laboratory, mers pseudovirions released by 293t cells contained only uncleaved s protein, and, as for most coronaviruses, cleavage between s1 and s2 was necessary to enable its membrane fusing activity. the mers pseudovirions bound to receptors on susceptible cells and were endocytized, and within the endosomes cleavage of s by the acid-dependent cysteine protease cathepsin l mediated virus entry. gierer et al [33] reached similar conclusions using mers pseudovirions that, unlike ours, contained more cleaved than uncleaved s protein, although both labs had made the pseudovirions in 293t cells. gierer et al. [33] showed that batches of 293t cells differ markedly in expression of the mers-cov receptor and susceptibility to transduction by mers pseudovirions. in our laboratory, the 293t cells showed minimal susceptibility to transduction with mers-cov pseudovirions with uncleaved s protein. in addition to entry by endocytosis, we showed that, like sars-cov [21, 22] , mers pseudovirions could enter susceptible vero e6 cells at the plasma membrane if virions were first bound to cell surface receptors at 4°c at neutral ph in the presence of nh 4 cl to inhibit acidification of endosomes, and also treated briefly at room temperature with trypsin to cleave the viral s protein. upon warming to 37°c at neutral ph, the mers pseudovirions fused with the plasma membrane and transduced the cells. thus, mers s protein does not require acidification to mediate virus entry, and the acidification required for endosomal entry [33] was required to activate the protease activity of cathepsin. although treatment of coronavirus virions or pseudovirions with proteases can activate virus entry, it may also make them lose infectivity if the cleaved s1/s2 heterodimer dissociates before receptor binding. we found that mers pseudovirions released from cells expressing tmprss-2 contained reduced amounts of s protein and had lost the ability to transduce susceptible cells. we postulate that the mers pseudovirions that contain large amounts of cleaved s protein, detected by a c-terminal tag [33, 53] , could not enter cells at the plasma membrane because s1 may have dissociated from the cleaved spikes on virions. development of antibodies specific for the s1 domain of mers s protein are needed to test this hypothesis. coronavirus s proteins expressed on cell membranes can trigger receptor-dependent syncytia formation if the membranebound s protein is cleaved within the infected cells by furin or other proteases. mers-cov infection of calu-3 and caco-2 cell lines induced syncytia formation [35] . our 293t cells were only minimally susceptible to entry of mers-pseudovirions, and did not form syncytia when producing mers pseudovirions with uncleaved s. however, when 293t cells expressing mers-cov sδ16 protein were co-cultured in the presence of trypsin with vero e6 cells that express the mers-cov receptor, enormous syncytia formed. these observations suggest that in tissues such as the lung, where trypsin, tmprss-2 or -4 and hat and other serine proteases are available, mers-cov virus infection might spread directly from cell to cell by s-mediated, receptor-dependent syncytia formation, potentially escaping from virus-neutralizing antibodies, as do other syncytia-forming viruses such as respiratory syncytial virus, parainfluenza viruses, and measles. it will be important to learn whether syncytia are formed in lungs or other tissues of mers-cov patients or animal models of mers-cov. some coronavirus s proteins can also trigger receptorindependent syncytia formation [61, 62] . when s proteins expressed on the plasma membrane are cleaved, the s1 domain can detach from the spike, exposing the hydrophobic fusion peptide of the membrane-anchored s2 domain that can directly induce fusion with any nearby cell membranes or lipid bilayers even if they lack receptors. this "receptor-independent spread (ris)" allows an infected cell to fuse with adjacent noninfected, receptor-negative cells that can, in turn, produce virus and fuse with additional receptor-negative cells. ris activity depends on the stability of s1/s2 interactions, and low stability of s1/s2 heterodimers correlates with rapid spread of infection through tissues that express little receptor protein [63, 64] . we found that transmembrane serine proteases tmprss-2 and -4 could activate the syncytia forming activity of mers-cov sδ16 protein expressed in 293t cells as these proteases do for class 1 fusion proteins of other respiratory viruses including influenza, sars-cov and human metapneumovirus [19, 20, [42] [43] [44] [45] . we were surprised that 293t cells, which express very little mers-cov receptor, were so extensively fused, and we hypothesize that this syncytia formation may be due to ris. due to its high case fatality rate, therapeutic interventions for mers-cov are urgently needed. pooled purified human immunoglobulin containing neutralizing antibody has been used to treat a variety of infectious diseases. not surprisingly, since mers-cov is an emerging pathogen, we found that human immunoglobulin from the usa could not neutralize the infectivity of mers pseudovirions (data not shown). however, sera from patients infected with either sars-cov or mers-cov contain antibodies that can neutralize mers-cov [33, 53] . most neutralizing antibodies would likely target the receptorbinding s1 domain of mers-cov s, which is less conserved than the s2 domain and can mutate, likely generating antibody escape mutants [65] . here we showed that a polyclonal antibody to the s protein of mhv, a group a betacoronavirus, cross reacts with the s2 domain of mers s protein in immunoblots. as we and others have proposed for sars-covs [66, 67] , the more highly conserved s2 domain, and especially its c-terminal heptad repeat (hrc) region, can be important targets for blocking conformational changes in s, inhibiting syncytia formation and virus entry, and also eliciting neutralizing antibodies. if mers-cov virions made in the lung have uncleaved s protein and must therefore enter cells through endocytosis, then inhibitory hrc peptides, or neutralizing antibodies to hrc might not penetrate into the endosomes to prevent virus entry. however, mers-cov virions in the lung likely have cleaved s protein due to lung proteases, so that virus entry at the plasma membrane might be inhibited by hrc-targeted peptides or antibodies. in summary, we demonstrated that, similar to sars-cov, cleavage of mers-cov-s protein by trypsin, tmprss2 or -4 or cathepsin l is required to activate the membrane fusion activity of s, leading to virus entry and syncytia formation, and that the location of the protease determines whether virus enters via endocytosis or by fusion at the plasma membrane. mers-cov s-mediated binding and entry mechanisms and protease triggering of conformational changes required for mers-cov-s virus entry and syncytia formation present potential targets for development of drugs or vaccines against this newly emerging and lethal group c human betacoronavirus. codon-optimized cdna encoding the spike glycoprotein of mers-cov [15] was synthesized with the c-terminal 16 amino acids replaced with a ggggs linker and a flag tag (genscript, piscataway, nj), and for eukaryotic expression was cloned into pcdna3.1(+) (invitrogen) between the bamhi and noti sites. to make constructs for expression of truncated soluble mers s aa1-351, s aa1-384, and s aa1-748 proteins, pcr reactions were performed using the same forward primer aatgaaaagcttcaccatgattcactccgtgttcctc pairing with the following reverse primers, for s aa1-351: tagttttctagaacttccgcctccaccataa ctacagtggagctggct; for s aa1-384: tagttttctagaacttccgcctccac cgtcgcactccacgccttctgcc; or for s aa1-748 tagttttctagaacttc cgcctccacctggggtcagtgtgctgggggt, and cloned into p3xflag-cmv 14 (sigma, st louis, mo) between hindiii and xbai sites for expression. the vsv-g plasmid and lentiviral packaging plasmid, pspax2, were obtained from addgene (cambridge, ma). the lentiviral reporter plasmid, plenti-gfp-luc, which expresses green fluorescent protein (gfp) and luciferase, was kindly provided by fang li, duke university [68] the vero e6 line of african green monkey kidney cells, the 293t line of human embryonic kidney cells transformed with sv40 large t antigen, the calu 3 line of human airway epithelial cells, the a549 line of human alveolar epithelial cells, and the tb1lu lung cell line from t. brasiliensis bats were obtained from atcc (manassas, va). hela cells stably expressing recombinant human ceacam proteins and the control hela cell line containing the empty vector were kindly provided by scott gray-owen, university of toronto [69] . the bat eff cells were prepared by macerating mid-gestation fetuses of eptesicus fuscus bats, briefly trypsinizing the cells, and plating them for expansion. cells were passaged twice, frozen, and kindly provided by richard bowen, colorado state university. isolation of the eff cells was conducted under approval 03-096a from the colorado state university iacuc. murine nih3t3 cells stably expressing recombinant human aminopeptidase n (hapn) or control cells with empty vector were previously described [70] . these cell lines were maintained in dulbecco's mem with 10% fetal bovine serum (fbs) and 2% penicillin, streptomycin, and fugizone (psf) (life technologies inc, grand island, ny). the llc-mk2 line of rhesus monkey kidney cells from atcc ccl-7 was maintained in opti-mem1 (life technologies inc, grand island, ny) with 10% fbs and 2% psf. pseudotyped lentiviruses were produced as described previously [71] with minor modifications. briefly, plasmids that encode viral spike glycoproteins mers-cov s∆16, sars s∆19, or vsv-g were co-transfected into 293t cells with pspax2 and plenti-gfp-luc using polyetherimide (pei) (polyscience inc, warrington, pa). forty to 60 hr later the supernatant media containing pseudovirions were centrifuged at 800g for 5min to remove debris, and passed through a 0.45µm filter. to quantitate entry of pseudovirions into different cell types, 250µl of pseudovirions with 8 µg/ml of polybrene (sigma) was inoculated onto cells in 24-well plates, incubated overnight at 37°c, and cells were fed with fresh medium. at 40hr post inoculation (pi) cells were lysed at room temperature with 120µl of medium with an equal volume of steady-glo (promega, madison, wi). transduction efficiency was monitored by quantitation of luciferase activity. living cells transduced by pseudovirions were detected by gfp expression. for all experiments triplicate samples were analyzed, data are representative of two or more experiments, and the standard error is shown. pseudovirions with mers-cov sδ16, sars sδ19, or vsvg glycoprotein or control pseudovirions with no spike were centrifuged through a 20% sucrose cushion at 30,000 rpm at 4°c for 3 h in a beckman sw41 rotor [71] . spike proteins in the virions were separated on 4-15% sds page, blotted to nitrocellulose and detected with mouse anti-flag antibody m2 (sigma, st louis, mo) for mers-cov sδ16, or polyclonal goat antibody ao4 to purified spikes from detergent-disrupted mhv-59 virions [72] , followed by horseradish peroxidase (hrp)-conjugated antibody to mouse or goat igg, and visualized with chemiluminescent reagent plus (perkinelmer, boston, ma). vero e6 cells or llcmk2 cells were incubated for 1hr at 37°c with medium alone or medium containing either 20mm nh 4 cl or 200nm bafilomycin a to inhibit acidification of endosomes, and then spin-inoculated with pseudovirions and no spike controls in the presence of either 20mm nh 4 cl or 200nm bafilomycin a for 90 min at 1,200g at 4°c. at 40 hr pi cells were lysed and luciferase activity was quantitated as a measure of virus entry. in figure 3 , the luciferase activities of vero e6 cells transduced with vsv-, sars-, and mers-covspseudovirions without endocytosis inhibitor were 1x10 6 , 1.5-2x10 6 , and 5-6x10 5 , respectively. monolayers of vero e6 or llcmk2 cells were incubated with 20mm nh 4 cl in medium containing 10% fbs for 1hr at 37°c, then shifted for 15min to 4°c with 40mm nh 4 cl. pseudovirions were adsorbed to cells at 4°c by spin-inoculation for 90 min at 1,200g. the virus inocula were removed and replaced with prewarmed, serum-free dmem with or without 20mm nh 4 cl. after 15min at 37°c, the media were replaced with serum-free dmem with or without 15 µg/ml of tpck-trypsin at room temperature to activate the membrane fusing activity of the s protein on virions adsorbed to the plasma membrane. trypsin activity was then inhibited by incubation for 15 min on ice with 75µg/ml of soybean trypsin inhibitor (worthington biochemical corporation, lakewood, nj) in dmem with 10% fbs, and cells were incubated overnight at 37°c, fed with fresh medium, and incubated at 37°c. at 40 hr pi, virus entry was quantitated by luciferase activity and cells were photographed to detect viral cytopathic effects. monolayers of vero e6 cells or llcmk2 cells were preincubated for 2 hrs at 37°c with either 30µm of e64d, that inhibits cathepsin b, h, l and calpain, or 10µm of specific cathepsin l inhibitor iii (millipore, billerica, ma). pseudovirions and no spike controls with or without cathepsin inhibitors were spin-inoculated onto the cells at 4°c, then incubated at 37°c for 5 hours with or without the endocytosis inhibitors. cells were then incubated at 37°c without inhibitors, and at 40 hr pi, luciferase activity in lysed cells was determined. 293t cells in 6-well plates were transfected using pei with 3µg of either empty vector or mers-cov s∆16 plasmid. for brief trypsin pre-treatment, cells were lifted with 1mm edta in pbs and washed, then incubated on ice with 20 µg/ml of tpck trypsin or control medium for 20 min. typsin activity was then inhibited by incubation with a five-fold excess of soybean trypsin inhibitor for 15min. the trypsin-pretreated 293t cells and control cells were then layered over monolayers of vero e6 cells and incubated for 20 hr. syncytia formation was detected by phase contrast microscopy or by imaging fixed cells stained with crystal violet. figure s1 . detection of trypsin-cleaved mers-cov s protein on pseudovirions by antibody to mhv s protein. pseudovirions containing either mers-cov s protein or no spike (control) were incubated with 20 µg/ml of trypsin either at 4°c or 37°c for 20 min. after digestion, glycoproteins on pseudovirions were analyzed by immunoblot using ao4 antibody to the s protein of murine betacoronavirus mhv-a59 that cross-reacts with mers-cov s protein. lanes 1 to 3: no spike control; lanes 4 to 6: mers pseudovirions; lanes 1 and 4: no trypsin; lanes 2 and 5: trypsin at 4°c; lane 3 and 5: trypsin at 37°c. the band at ~90kda was seen in pseudovirions without spike, indicating that it is not due to mers-cov s protein. uncleaved mers-cov s protein, 200kda, on pseudovirions is shown in lane 4, and trypsin treatment cleaved all of the s protein, and generated a subunit of ~65kda that was recognized by ao4 antibody (lanes 5 and 6). 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for prediction of peptide retention time in reversed-phase high-performance liquid chromatography: hydrophilicity/hydrophobicity of side-chains at the n-and c-termini of peptides are dramatically affected by the end-groups and location apoptotic caspases regulate induction of ipscs from human fibroblasts cd66 carcinoembryonic antigens mediate interactions between opaexpressing neisseria gonorrhoeae and human polymorphonuclear phagocytes human myeloid plasma membrane glycoprotein cd13 (gp150) is identical to aminopeptidase n complementation of a binding-defective retrovirus by a host cell receptor mutant isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid we thank scott d. gray-owen (university of toronto) for providing hela cells stably expressing human ceacam proteins, linda shapiro (university of connecticut) for mouse cells producing human apn, richard bowen (colorado state university) for the bat cell lines, stefan poehlmann (hannover medical school and german primate center) for pca7-tmprss2 and pcmv-tmprss4 plasmids, and fang li (duke university) for plenti-gfp-luc. conceived and designed the experiments: zq srd kvh. performed the experiments: zq. analyzed the data: zq srd kvh. contributed reagents/materials/analysis tools: zq srd kvh. wrote the manuscript: zq srd kvh. key: cord-278123-mq56em3z authors: hasan, mohammad rubayet; mirza, faheem; al-hail, hamad; sundararaju, sathyavathi; xaba, thabisile; iqbal, muhammad; alhussain, hashim; yassine, hadi mohamad; perez-lopez, andres; tang, patrick title: detection of sars-cov-2 rna by direct rt-qpcr on nasopharyngeal specimens without extraction of viral rna date: 2020-07-24 journal: plos one doi: 10.1371/journal.pone.0236564 sha: doc_id: 278123 cord_uid: mq56em3z to circumvent the limited availability of rna extraction reagents, we aimed to develop a protocol for direct rt-qpcr to detect sars-cov-2 in nasopharyngeal swabs without rna extraction. nasopharyngeal specimens positive for sars-cov-2 and other coronaviruses collected in universal viral transport (uvt) medium were pre-processed by several commercial and laboratory-developed methods and tested by rt-qpcr assays without rna extraction using different rt-qpcr master mixes. the results were compared to that of standard approach that involves rna extraction. incubation of specimens at 65°c for 10 minutes along with the use of taqpath(™) 1-step rt-qpcr master mix provides higher analytical sensitivity for detection of sars-cov-2 rna than many other conditions tested. the optimized direct rt-qpcr approach demonstrated a limit of detection of 6.6x10(3) copy/ml and high reproducibility (co-efficient of variation = 1.2%). in 132 nasopharyngeal specimens submitted for sars-cov-2 testing, the sensitivity, specificity and accuracy of our optimized approach were 95%, 99% and 98.5%, respectively, with reference to the standard approach. also, the rt-qpcr c(t) values obtained by the two methods were positively correlated (pearson correlation coefficient r = 0.6971, p = 0.0013). the rate of pcr inhibition by the direct approach was 8% compared to 9% by the standard approach. our simple approach to detect sars-cov-2 rna by direct rt-qpcr may help laboratories continue testing for the virus despite reagent shortages or expand their testing capacity in resource limited settings. the ongoing pandemic of coronavirus disease (covid-19) caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (sars-cov-2) posed an unprecedented public health threat to the entire world. in the absence of an effective vaccine or specific treatment against the virus, early detection and contact tracing, physical distancing measures and plos one plos one | https://doi.org/10.1371/journal.pone.0236564 july 24, 2020 1 / 9 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 quarantine of cases are considered the cornerstones to curb the community transmission of sars-cov-2 [1] [2] [3] . since the virus was identified and its genome sequenced in early january 2020, detection of viral rna in respiratory specimens by real-time reverse transcription pcr (rt-qpcr) remains the main approach to manage the outbreak by allowing early detection of cases and targeted measures to prevent transmission of the virus [4] . the massive demand for sars-cov-2 rt-qpcr has brought about a global shortage and supply chain irregularities of rna extraction kits that are crucial for rt-qpcr testing [5] [6] [7] . detection of viral pathogens directly from clinical specimens without rna extraction has been described earlier in cases such as detection of norovirus from fecal specimens [8] , human papilloma virus from crude cell extracts [9] , and zika virus from blood or serum samples [10] . in this study, we tested a number of commercial and laboratory-developed, specimen pre-treatment procedures to optimize the performance of direct rt-qpcr for sars-cov-2 avoiding the rna extraction step. this method was validated against a standard approach that included extraction of viral rna on a commercial automated extraction platform. standard approach for detection of sars-cov-2 rna from nasopharyngeal specimens in our laboratory involves extraction of total nucleic acids from specimens in an ivd-labeled, automated extraction platform followed by rt-qpcr, based on one of the assays (table 1) suggested by world health organization (who) [11] . the performance standards of the assay were established in our laboratory according to college of american pathologists (cap) standards, and at time of writing this paper, the assay was used to test more than 2000 respiratory specimens to screen patients for potential infection with sars-cov-2. therefore, the assay was used as the reference method for all other alternative approaches assessed in this study. all specimen preparations, pre-treatments and pcr setup were performed in a class ii biosafety cabinet in a biosafety level 2 (bsl2) facility. clinical specimens: nasopharyngeal flocked swab (npfs) specimens collected in universal viral transport (uvt) medium (becton, dickinson and company) (n = 180) submitted for testing sars-cov-2 or other respiratory viruses at sidra medicine, doha, qatar were used in this study. to maintain patient anonymity, each sample was coded, and all patient identifiers were removed to ensure that personnel involved in this study were unaware of any patient information. ethics approval was not sought because the study involves laboratory validation of test methods and the secondary use of anonymous pathological specimens that falls under the category 'exempted' by sidra medicine institutional review board. for spiking experiments, npfs specimens (n = 6) were collected from laboratory volunteers after obtaining npfs specimens were either subjected to viral rna extraction by standard method using the nuclisens easymag automated extraction system (biomerieux), or diluted 4-fold with nfw followed by incubation at 65˚c for 5 minutes. all samples were tested for sars-cov-2 rna by standard rt-qpcr using different master mixes in duplicate and mean c t values were compared. written informed consents. no personal data were collected, and specimens were labeled with random numbers so that test results cannot be linked to an individual. nucleic acid extraction: nucleic acids from 0.2 ml of npfs specimens were extracted on a nuclisens 1 easymag platform (biomérieux, france) according to the methods described by the manufacturers. pre-treatment of specimens: unless otherwise stated, specimens were diluted using nuclease free water. heat treatment of specimens at different temperature was performed in a ther-momixer (eppendorf). specimens processed with arcis coronavirus rna extraction research kit (arcis biotechnology) were performed according to manufacturer's instructions. briefly, 90 μl specimen was mixed with 6 μl reagent 1 rtu and either left unheated or heat lysed at 60˚c for 5 minutes. 40 μl of lysate was then mixed with 2 μl of reagent 2a and was used directly for rt-qpcr. for takara primedirect™ probe rt-qpcr mix (takara bio), 0.2 ml specimens were heated at 99˚c for 10 mins and then centrifuged at 4000rpm for 5 min. the supernatants were collected and 7μl of supernatant was directly used in rt-qpcr reaction mixture. rt-qpcr: primers and probes for detection of sars-cov-2, hcov-hku1, rnasep and ms2 bacteriophage are listed in s1 table [4, [12] [13] [14] . for detection of hcov-hku1 using quantifast pathogen rt-pcr + ic kit (qiagen), five μl of extracted or pre-treated samples were mixed with 20 μl of a master mix containing 5 μl of quantifast pathogen rt-pcr + ic master mix, 0.25 μl of quantifast rt mix and 0.5 μl of 50 x rox dye solution and primers and probes to final concentrations shown in s1 table. thermal cycling was performed in a abi7500 fast instrument (thermofisher scientific) with 1 cycle of reverse transcription at 50˚c for 20 min followed by 1 cycle of pcr activation at 95˚c for 5 min, followed by 40 amplification cycles each consisting of 95˚c-15s and 60˚c-60s. for detection of hcov-hku1 using taqpath™ 1-step rt-qpcr kit (thermofisher scientific), five μl of extracted or pre-treated samples were mixed with 5 μl of a master mix containing 5 μl of taqpath™ 1-step rt-qpcr master mix and primers and probes to final concentrations shown in s1 table. thermal cycling was performed in a abi7500 fast instrument (thermofisher scientific) with 1 cycle of reverse transcription at 50˚c for 15 min followed by 1 cycle of polymerase activation at 95˚c for 2 min, followed by 40 amplification cycles each consisting of 95˚c-15s and 60˚c-60s. for detection of sars-cov-2 with standard method, five μl of nucleic acid extracts from nuclisens 1 easymag system were mixed with 7.5 μl of a master mix containing 2.5 μl of quantifast pathogen rt-pcr + ic master mix, 0.125 μl of quantifast rt mix and 0.25 μl of 50 x rox dye solution and primers and probes to final concentrations shown in s1 table. rna extraction and pcr inhibition were monitored by an internal control pcr assay or an rnasep assay, using the primers and probes shown in s1 table. specimens were spiked with a tittered preparation of ms2 bacteriophage to serve as a template for internal control assay. for direct pcr on specimens, pre-treated specimens were assessed in the same way except that extracted ms2 bacteriophage rna was mixed with pcr master mix to serve as an inhibition control. a synthetic rna (idt) based on e-sarbeco assay [4] amplicon sequence was used as a positive control. as negative control, 0.2 ml neonatal calf serum (ncs) (thermofisher scientific) was extracted along with specimens and used with each pcr run. sars-cov-2 rt-qpcr with takara primedirect™ probe rt-qpcr mix was performed as described by the manufacturer. briefly, 7 μl of processed samples were mixed with 18 μl of master mix containing 12.5 μl of primedirect probe rt-qpcr mix and primers and probes to final concentrations shown in table 1 . thermal cycling was performed in a abi7500 fast instrument (thermofisher scientific) with 1 cycle of denaturation at 96˚c for 10 sec, 1 cycle of reverse transcription at 60˚c for 5 min followed by 45 amplification cycles each consisting of 95˚c-5s and 60˚c-30s. sars-cov-2 rt-qpcr with taqpath™ 1-step rt-qpcr kit with the optimized approach (specimens heat treated at 65˚c for 10 min) was performed as above except that 8 μl of specimen was used per 20 μl reaction. a total of 132 nps that were previously tested by the standard approach were also tested by the optimized, direct approach and the results were compared. an additional 30 nps was tested in the same way except that rnasep was detected as an internal control or specimen control instead of ms2 bacteriophage. in addition, an external quality assessment (eqa) panel (8 specimens) from quality control for molecular diagnostics (qcmd) was tested simultaneously with the standard and the direct approach, as well as with a qiastat-dx respiratory 2019-ncov panel (qiagen) and the results were compared with expected results. limit of detection (lod) study: a sars-cov-2 positive nasopharyngeal specimen (c t = 23.3) was titered by the optimized approach using synthetic rna, and serially diluted (up to 5x10 -7 -fold) using a negative specimen. all diluted specimens were pre-treated by heating at 65˚c for 10 min and then assessed by sars-cov-2 rt-qpcr in replicates of 8. the c t values obtained were used to calculate limit of detection (lod) and intra-assay reproducibility of direct rt-qpcr. statistical analysis: sensitivity, defined as the number of true positive results divided by the sum of true positive and false negative results; specificity, defined as the number of true negative results divided by the sum of true negative and false positive results; and accuracy (concordance), defined as the sum of true positive and true negative results divided by the total number of test samples, were calculated and expressed as percentages. ninety-five percent confidence intervals (ci) for sensitivity, specificity and accuracy were calculated by the clopper-pearson interval or exact method using an online, diagnostic test evaluation calculator (medcalc, 2018) . correlation between the rt-qpcr ct values between standard approach and optimized direct approach was determined by pearson's coefficient calculator. the limit of detection with 95% endpoint (c 95 ) was determined by probit regression analysis [15] . using a human coronavirus hku1 (hcov-hku1) positive specimen as a surrogate for sars-cov-2, we first assessed whether specimens can be used directly for rt-qpcr after 2-10 fold dilution with nuclease free water (nfw), simple heat treatment (100˚c for 5 min) and centrifugation to remove any insoluble material that may be present in the specimen. the pre-treated specimens were then assessed in parallel with extracted specimens by a previously described, laboratory developed rt-qpcr for hcov-hku1 (s1 table) . a significant loss of sensitivity was observed with a rt-qpcr δc t ranging from 10-14 (s2 table) . to determine whether any components of uvt medium (becton, dickinson and company) were inhibitory to rt-qpcr, we collected nasopharyngeal flocked swabs (npfs) from laboratory volunteers in nfw along with swabs in utm. we then spiked a sars-cov-2 positive specimen to all specimens and assessed them by sars-cov-2 rt-qpcr. however, no significant improvement in sensitivity was observed (s3 table) . similar results were observed when two commercial test kits were used for direct rt-qpcr: arcis coronavirus rna extraction research kit comes with lysis reagents that can be used directly in rt-qpcr; and takara primedirect™ probe rt-qpcr kit provides a master mix that is compatible with heat-treated specimen extracts. however, in our evaluation both test kits failed to demonstrate an acceptable level of sensitivity (s4 and s5 tables). our attempts to further optimize the pre-treatment conditions showed modest improvement with a non-ionic detergent, tween-20, and further improvement with a heating step at 65˚c for 10 min without centrifugation (δc t = 5.2) (s6 table) . we then tested this low heat approach with more sars-cov-2 positive specimens and using 3 different rt-qpcr master mixes. interestingly, we found that with taqpath™ 1-step rt-qpcr master mix, 4/4 positive samples were correctly detected with a δc t range 0.8-3.8 (table 1) . on the other hand, two other master mixes gave higher δc t and 1/4 positive results were missed by both. based on these results, the optimal pre-treatment and reaction conditions for the direct approach were: i) transfer and dilute (4-fold) 10 μl of npfs specimen in nfw; ii) incubate at 65˚c for 10 min; and iii) test 8 μl of heat lysed specimen in a 20 μl reaction using taqpath™ 1-step rt-qpcr master mix. the analytical sensitivity of the direct rt-qpcr assay using specimens prepared in this manner was determined by serially diluting a specimen positive for sars-cov-2 with a negative specimen as a diluent. the positive specimen was titered based on the c t value obtained by the direct approach using a standard curve prepared with sars-cov-2 synthetic rna. the c t values were linear (r 2 = >0.99) over the range of 10 3 copy/ml to 10 8 copy/ml, the highest concentration used in this analysis (fig 1a and 1b) . the 95% limit of detection (lod; c 95 ) of the assay with the direct approach was 6.6x10 3 copy/ml. the c t variation between rt-qpcr replicates across different concentration of analytes were <1 with an average coefficient of variation (cv%) of 1.2%. a total of 132 npfs specimens that were previously tested with standard approach including viral rna extraction were re-tested with the new direct approach. the direct approach detected all except one positive case with c t >38. on the other hand, the direct approach detected (c t >37) sars-cov-2 in one specimen that was negative by standard approach. overall agreement of results between two approaches was >98% (kappa = 0.939; 95% ci = 0.854 to 1.000). the sensitivity and specificity of the new approach compared to the reference method were 95% and 99%, respectively ( table 2 ). the rt-qpcr c t values for all specimens that were positive by both methods (n = 18) were positively correlated with a pearson coefficient (r) of 0.6971 (p<0.01) (fig 2) . the rate of pcr inhibition among the specimens that gave negative rt-qpcr results by the direct approach was 8% compared to 9% by the standard approach. the direct approach accurately detected sars-cov-2 rna in all except one specimen in an external quality assessment (eqa) panel provided by quality control for molecular diagnostics (qcmd) (s7 table) . the specimen that gave discrepant result was reported as 'borderline' by qcmd, and sars-cov-2 rna was also undetectable in this finally, we also tested our direct rt-qpcr approach using rnasep as an internal control or a specimen quality control within a duplex rt-qpcr with sars-cov-2 and compared the results with that of the standard method involving rna extraction (s8 table) . in 30 npfs specimens, rnasep was detected in all specimens, but with an average loss of c t (δc t ) by 4.2 ±1.7 as compared to an average loss of sars-cov-2 c t by 2±1. 3 . surprisingly, rnasep was not detected in two samples by the standard method that were positive for rnasep by the direct method. this could potentially be due to interfering substances concentrated by the sars-cov-2 detection by rt-qpcr without rna extraction extraction process. in this set, one sample that was positive for sars-cov-2 by standard method with high c t (36.3) was missed by the direct method. success in rt-qpcr testing depends on multiple factors. rna extraction is preferable to the use of direct specimens because the extraction process concentrates and purifies the rna targets and excludes pcr inhibitory substances. the use of pre-treated or untreated specimens directly in rt-qpcr is challenging because of the presence of inhibitors and rna loss due to heating and/or rnases. after many attempts with various pre-treatment agents and conditions, we have determined an optimal pre-treatment protocol complemented with specific rt-qpcr reagents, that generates results equivalent to standard methods that involve rna extraction. minimizing rna loss through the low heat approach, appropriate dilution of inhibitory substances and the higher sensitivity of taqpath™ 1-step rt-qpcr master mix may have played a combinatory role in achieving equivalency of the direct rt-qpcr compared to a standard approach requiring viral rna extraction. by the direct approach, the greater loss of rna-sep c t compared to sars-cov-2 c t may be due to the fact that heating of specimens at 65˚c is more efficient for lysing viruses than nasopharyngeal epithelial cells and that a significant fraction of viruses remains extracellularly. indeed, we have observed that sars-cov-2 is distributed approximately 50:50 between the supernatant and pellet after centrifugation of npfs specimens (data not shown). in summary, our new approach demonstrated high sensitivity and specificity in the detection of sars-cov-2 rna, and the rate of rt-qpcr inhibition was similar to that of a standard approach. by skipping the rna extraction step, the new approach will also significantly reduce the cost and improve the turn-around time of the assay. delayed and inadequate laboratory testing can significantly hamper efforts to control the pandemic. our results will help many labs all over the world who are struggling with a shortage of reagents to continue testing for sars-cov-2. also, because of a significant reduction in cost, the optimized direct approach we described, will be useful to resource-limited countries to expand their capacity for rt-qpcr testing. supporting information s1 table. primers and probes used in this study. (docx) s2 table. direct rt-qpcr on a nasopharyngeal specimen positive for human coronavirus hku1 after heat treatment at 100˚c for 5 minutes. npfs specimens were either i) subjected to viral rna extraction by standard method using a nuclisens easymag automated extraction system (biomerieux), or ii) serially diluted with nuclease free water (nfw) followed by incubation at 100˚c for 5 minutes, or iii) freeze thawed once prior to heat treatment. all heattreated samples were centrifuged at 13,000 rpm for 5 minutes at 4˚c and supernatants were collected. all samples were tested for sars-cov-2 rna by standard rt-qpcr in duplicate and mean c t values were compared. (docx) s3 table. direct rt-qpcr on simulated, dry npfs using a spiked specimen positive for sars-cov-2. specimens were collected from laboratory members who volunteered to provide specimens. npfs specimens were either i) collected in vtm or ii) collected in sterile empty tubes as dry swabs and later resuspended in 1 ml of nfw. five μl of a patient specimen positive for sars-cov-2 rna (c t = 22) were spiked into 0.2 ml of specimens collected in vtm of nfw. vtm specimens were extracted by standard method using a nuclisens easy-mag automated extraction system (biomerieux). specimens collected in nfw were incubated at 100˚c for 5 minutes and centrifuged at 13,000 rpm for 5 minutes at 4˚c and supernatants were collected. all samples were tested for sars-cov-2 rna by standard rt-qpcr using quantifast pathogen rt-pcr + ic master mix in duplicate and mean c t values were compared. (docx) s4 table. direct rt-qpcr on sars-cov-2 positive and negative npfs specimens processed using arcis pathogen kit. npfs specimens were processed according to manufacturer's protocol (arcis biotechnology ltd.). all samples were tested for sars-cov-2 rna by standard rt-qpcr in duplicate and mean c t values were compared. (docx) s5 table. direct rt-qpcr on sars-cov-2 positive and negative npfs specimens processed using takara primedirect probe rt-qpcr mix. npfs specimens were processed according to manufacturer's protocol (primedirect™ probe rt-qpcr mix, takara bio inc.). all samples were tested for sars-cov-2 rna by standard rt-qpcr in duplicate and mean c t values were compared. (docx) s6 table. direct rt-qpcr on sars-cov-2 positive and negative npfs specimens with or without heating at 65˚c for 5 minutes or after lysis using a non-ionic detergent. npfs specimens were either i) subjected to viral rna extraction by standard method using a nucli-sens easymag automated extraction system (biomerieux), or ii) diluted 4-fold with nuclease free water (nfw) iii) diluted 4-fold with nuclease free water (nfw) followed by incubation at 65˚c for 5 minutes or iv) diluted 4-fold with tween-20 to final concentration of 0.2% followed by incubation at room temperature for 10 min. all samples were tested for sars-cov-2 rna by standard rt-qpcr using quantifast pathogen rt-pcr + ic master mix. (docx) s7 table. direct rt-qpcr on qcmd eqa specimens. qcmd eqa specimens were simultaneously tested by standard and direct approach and by qiastat-dx respiratory 2019-ncov panel (qiagen). (docx) s8 a novel coronavirus from patients with pneumonia in china world health organization declares global emergency: a review of the 2019 novel coronavirus (covid-19) a hundred days into the coronavirus disease (covid-19) pandemic, eurosurveillance detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr critical supply shortages-the need for ventilators and personal protective equipment during the covid-19 pandemic fda chief warns of supply 'pressure' on reagents for coronavirus tests laboratory testing of sars-cov, mers-cov, and sars-cov-2 (2019-ncov): current status, challenges, and countermeasures coronavirus disease (covid-19) pandemic detection of noroviruses in fecal specimens by direct rt-pcr without rna purification hpv direct flow chip: a new human papillomavirus genotyping method based on direct pcr from crude-cell extracts development of a direct reverse-transcription quantitative pcr (dirrt-qpcr) assay for clinical zika diagnosis a novel real-time pcr assay panel for detection of common respiratory pathogens in a convenient, strip-tube array format detection of pathogenic leptospira spp. through taqman polymerase chain reaction targeting the lipl32 gene use of bacteriophage ms2 as an internal control in viral reverse transcription-pcr assays validation of laboratory-developed molecular assays for infectious diseases we are grateful for the efforts of all technologists in the molecular infectious disease laboratory at sidra medicine. key: cord-289892-yh1lioyz authors: bai, bingke; hu, qinxue; hu, hui; zhou, peng; shi, zhengli; meng, jin; lu, baojing; huang, yi; mao, panyong; wang, hanzhong title: virus-like particles of sars-like coronavirus formed by membrane proteins from different origins demonstrate stimulating activity in human dendritic cells date: 2008-07-16 journal: plos one doi: 10.1371/journal.pone.0002685 sha: doc_id: 289892 cord_uid: yh1lioyz the pathogenesis of sars coronavirus (cov) remains poorly understood. in the current study, two recombinant baculovirus were generated to express the spike (s) protein of sars-like coronavirus (sl-cov) isolated from bats (vacbs) and the envelope (e) and membrane (m) proteins of sars-cov, respectively. co-infection of insect cells with these two recombinant baculoviruses led to self-assembly of virus-like particles (bvlps) as demonstrated by electron microscopy. incorporation of s protein of vacbs (bs) into vlps was confirmed by western blot and immunogold labeling. such bvlps up-regulated the level of cd40, cd80, cd86, cd83, and enhanced the secretion of il-6, il-10 and tnf-α in immature dendritic cells (dcs). immune responses were compared in immature dcs inoculated with bvlps or with vlps formed by s, e and m proteins of human sars-cov. bvlps showed a stronger ability to stimulate dcs in terms of cytokine induction as evidenced by 2 to 6 fold higher production of il-6 and tnf-α. further study indicated that ifn-γ+ and il-4+ populations in cd4+ t cells increased upon co-cultivation with dcs pre-exposed with bvlps or sars-cov vlps. the observed difference in dc-stimulating activity between bvlps and sars cov vlps was very likely due to the s protein. in agreement, sl-cov s dna vaccine evoked a more vigorous antibody response and a stronger t cell response than sars-cov s dna in mice. our data have demonstrated for the first time that sl-cov vlps formed by membrane proteins of different origins, one from sl-cov isolated from bats (bs) and the other two from human sars-cov (e and m), activated immature dcs and enhanced the expression of co-stimulatory molecules and the secretion of cytokines. finding in this study may provide important information for vaccine development as well as for understanding the pathogenesis of sars-like cov. severe acute respiratory syndrome (sars) is an emerging infectious disease caused by a novel coronavirus (cov) variant, sars cov [1] [2] [3] [4] [5] . during the 2002-2003 epidemic, sars cov was highly transmissible in humans. as of 31 december 2003, 8,096 cases had been identified worldwide and 774 people had died with a mortality rate of about 9.6% (world health organization statistics (http://www.who.int/csr/sars/country/ table 2004_04_21/en/). although the sars epidemic was successfully contained by july 2003, the pathogenesis of sars cov remains poorly understood. the viral envelope of sars cov contains at least three structural membrane proteins, spike (s), membrane (m), and small membrane or envelope (e) proteins [6, 7] . the cellular receptor of sars cov and the receptor-binding domain (rbd) on s protein have been identified, indicating that rbd in the s1 region of s protein plays a critical role in neutralizing antibody induction, angiotensin-converting enzyme 2 (ace-2) binding and viral entry [8] [9] [10] . in the absence of s protein, expression of m and e proteins does not induce a detectable serum sars-covneutralizing antibody response [11] . m and e proteins are the only requirements for the assembly of viral particles; the s protein is dispensable but is incorporated when present [12] [13] [14] [15] [16] . the isolation of sars cov from himalayan palm civets (paguma larvata) and raccoon dogs (nyctereutes procyonoides) in wild live markets in china suggests that the virus may be originally from animal reservoirs [17] . recently, two independent groups reported the identification of sars-like coronavirus (sl-cov) in different chinese horseshoe bat species, providing strong evidence that bats could serve as natural reservoirs of sars cov [18] [19] [20] . the s coding sequence of sl-cov isolated from bats displays low level of similarity to those isolated from human (76%) or civets (78%) [18] [19] [20] , while the e and m proteins of sl-cov have high sequence similarity (96% to 100%) to those from human or civets [19, 20] . it is worthy to note that the nucleotide sequence identity among these coronaviruses from different sources was greatly increased when the s region was excluded [19, 20] . the host range of coronaviruses was reported to correlate with the degree of s protein variations [21] , suggesting that the variations in s protein are responsible for the interspecies transmission and the adaptation to new hosts. virus-like particles (vlps) represent a specific class of subunit vaccine that mimics the structure of authentic virus particles. vlps present viral antigens in a more authentic conformation than other subunit vaccines and are readily recognized by the immune system [22] . many studies have documented that vlps are capable of inducing b-cell-mediated immune responses and are highly effective in stimulating cd4 t cell proliferation and cytotoxic t lymphocyte (ctl) responses [23] [24] [25] . the stimulating activities of ebola, marburg and hiv vlps in dendritic cells (dcs) were previous investigated [26] [27] [28] , indicating that, unlike native virus, vlps are effective stimulators of dcs and can enhance innate and adaptive immune responses. our previous study described that vlps formed by e, m and s proteins of sars cov elicited strong sars cov-specific humoral and cellular immune responses in mice [29] . however, whether sl-cov vlps can elicit cytokines secretion or/and activate human dcs remains to be determined. in this study, we have investigated whether the s protein of sl-cov isolated from bats can incorporate into vlps formed by the e and m proteins of human sars cov. in addition, because in vitro infection model of bat sl-cov has not so far been established, we intended to use vlps as an alternative to study the immune responses induced in dcs. therefore, we compared the phenotypic and functional changes of immature dcs inoculated with bvlps or with sars cov vlps. the s-specific immune activation was further confirmed in mice using s dna vaccines. findings described in this report may have significance for understanding the evolution and the pathogenesis of sars cov. the bs gene was amplified by rt-pcr and cloned into pfastbac dual vector. the recombinant baculovirus vacbs was generated following transfection in sf21 cells. the recombinant baculovirus vacme, expressing the e and m proteins of sars cov wh20 strain (genbank accession number ay772062), was previously described [29] . vlps formed by the s, e and m proteins of sars cov were successfully constructed in our previous study using a baculovirus system [30] . the main variation between sl-cov and sars cov of human or civet locates in the s protein [18] [19] [20] . to study whether bs protein can be incorporated into vlps formed by the e and m proteins of sars-cov, we co-infected sf21 cells with the recombinant baculoviruses vacbs and vacme. after 72 h of coinfection, the cells were collected and thin-sections were prepared and examined. as shown in figure 1a , numerous viral particles, ,100 nm in diameter, were mainly observed in the cytoplasm of infected cells. western blot and immunogold labeling were subsequently performed to detect the incorporation of bs in vlps. as shown in figure 1b , the presence of bs, m and e proteins was detected in bvlps by using an anti-sars cov antibody. there were no corresponding bands in the negative control of preimmune rabit antiserum. the genomic differences between sl-cov and sars cov largely locate in the s gene, particularly at the 59 end of s region (equivalent to s1 coding region). therefore, it is not surprising that serum raised against human sars cov recognized bat sl-cov s protein. the s2 coding region of s protein is highly conserved (,96%) [5, 10, 21] . using a specific anti-bs2 (bat s2 region 667-1242 aa) antibody ( figure 1c ), the gold particles were detected around the bvlps in immunogold labeling analysis, further confirming the results of western blot. in addition, the anti-bs2 antibody also recognized the human sars cov vlps but not vlps formed by e and m proteins in the absence of s protein ( figure 1c ). taken together, our data indicate that the bs protein of sl-cov was successfully incorporated into vlps formed by the e and m proteins of human sars cov. immature dcs were incubated with 10 mg/ml bvlps for 16 h. the expression of costimulatory molecules (cd40, cd86 and cd80) and the maturation marker cd83 were analyzed. dcs treated with pbs or ac were used as negative controls. as shown in fig. 2 , similar to those on lps-treated dcs, all of the four markers were up-regulated to much higher levels than those on pbs or ac-treated dcs (fig. 2) . collectively, our results indicate that bvlps can lead to the activation and maturation of dcs. in contrast, the heated bvlps induced much lower levels of cd83, cd40, cd86 and cd80 expression on dcs, suggesting that the structural proteins incorporated into vlps are essential for dc activation and maturation. dc maturation not only leads to up-regulation of adhesion and co-stimulatory molecules, but also results in secretion of proinflammatory cytokines that can activate innate and adaptive immune responses [31] . to assess the effects of bvlps on dcs, immature dcs were treated under various conditions and supernatants were collected for cytokine (il-6, il-10 and tnf-alpha) quantification. il-6 and tnf-a are proinflammatory cytokines while il-10 is an anti-inflammatory cytokine. as shown in fig. 3 , incubating dcs with bvlps significantly enhanced the secretion levels of these three cytokines and were all similar to those in lps-treated dcs. the boiled-bvlps lost the ability to induce cytokine production. the secretions of il-6, il-10 and tnf-a were 5-10 folds lower in heated bvlps-treated dcs than those in bvlps-treated dcs, although the secretion levels were still 5-15 folds higher than those in pbs or ac treated groups. this enhancement is likely due to nonspecific stimulation by remaining denatured proteins in heated bvlps. moreover, cytokine secretion profiles were similar in dcs inoculated with bvlps with or without polymyxin b pretreatment, excluding the possibility of lps contamination. in contrast, lpstreated dcs significantly lost the cytokine-secretion ability with polymyxin b pretreatment. combining the flow cytometry results in fig. 2 , it is reasonable to draw a conclusion that the structure of bvlps, not lps contamination, contributed to cytokine production in bvlps-treated dcs. we previously constructed sars cov vlps and investigated the humoral and cellular immune responses induced by sars cov vlps in mice [29] . whether sars cov vlps can activate dcs remains to be addressed. in the current study, 10 mg/ml of either sars cov vlps or bvlps was incubated with immature dcs, and the expression of surface makers and the secretion of cytokines were subsequently analyzed. similar to lps treatment, both sars cov vlps and bvlps enhanced the expression of cd40, cd86, cd80 and cd83 (fig. 4) . however, bvlps showed a stronger ability to induce the secretion of il-6 and tnf-a, with 2 and 4 fold higher, respectively, while the secretion of il 10 was similar (fig. 5) . because the only difference between sars cov vlps and bvlps lies in the s protein, the difference in il-6 and tnf-a induction is highly likely due to the variation between the two s proteins. to understand the types of t cells stimulated by vlps-exposed dcs, ifn-c and il-2 intracellular staining was measured by flow cytometry. as show in fig. 6 , ifn-c+ population in cd4+ t cells stimulated by either bvlps-or sars cov vlps-treated dcs were much higher than il-4+ popoulation. in pbs-or ac-treated groups, the percentage between ifn-c+ and il-4+ populations in cd4+ t cells did not have significant difference. thus, the bvlps or sars cov vlps treated dcs could polarize th cells toward a th1 phenotype. the balance between th1 and th2 responses to an infectious agent can influence both pathogen growth and immunopathology. th1-dominant cellular immunity may be a crucial factor for long-term protection against virus infection. the observed difference in dc-stimulating activity between sars cov vlps and bvlps was very likely due to the s protein. to further confirm our conclusion, we cloned s and bs gene into pcdna3.1 to construct the recombinant plasmids pcdna-s and pcdna-bs, respectively. these two plasmids were then immunized in mice intramuscularly every two weeks. ten days after the final immunization, igg1 and igg2a were measured to determine the humoral immune response profiles by elisa. as shown in although the level of igg1 was similar, the level of igg2a in the pcdna-bs vaccinated group was appreciably higher than that in pcdna-s immunized group. elispot assay was also used to assess the magnitudes of sspecific ifn-c (th1) and il-4 (th2) t-cell responses after the mice were vaccinated with pcdna-s or pcdna-bs. compared with pcdna-s immunization, antigen-specific ifn-c-secreting cell number was 2-fold higher and il-4-secreting cell number was 3 times higher in mice immunized with pcdna-bs (p,0.05) (fig. 8) . moreover, in all groups, ifn-c was induced to a much higher level than that of il-4. combing with the higher igg2a elevated by bs immunization, it is reasonable to draw a conclusion that bs protein could induce a stronger th1 bias immune response than s protein. the spike (s) protein of sars cov is a key protein involved in viral entry and therefore the main target for vaccine design [32] . in the absence of s protein, expression of other structure proteins, such as m and e proteins, does not induce a detectable neutralizing antibody response [33] . antibodies against sars cov s protein inhibit pulmonary viral replication and protect against sars cov challenge [34] . variations in the s proteins can dramatically affect the virulence and host tropism of the virus [35, 36] . for instance, sl-covs isolated from bats display greater genetic variations than sars covs from humans or civets [19] . here, we intended to investigate whether the s protein of sl-cov isolated from bats can incorporate into viral like particles (vlps) formed by the e and m proteins of human sars cov and whether such vlps can activate dcs. using baculovirus expression system, we constructed bvlps incorporated with the e and m proteins of human sars cov and the s protein of sl-cov of bats. electron microscopy (em) detection confirmed the formation of vlps, which were very similar to the morphology of the native virus. western blot indicated that bs protein was incorporated into bvlps. because the major difference of s proteins between sars cov and sl-cov locates in the s1 region (,64%) and the s2 region is more conserved (92% to 96%) [19, 20] , successful incorporation of the sl-cov s protein into vlps formed by the e and m proteins of sars cov suggests that such bvlps likely assembled through s2 region other than s1 region. these genome-free bvlps formed by structure proteins from different species provide us a physical means to investigate their immunogenicity in immune cells. sars cov mainly targets lung tissues and the clinical syndrome of sars indicates that the host immune system is greatly damaged [37] [38] [39] . dendritic cells (dcs) are antigenpresenting cells that play key role in innate and adaptive immunity [31, [40] [41] [42] . exposure of dcs to infectious sars cov leads to the phenotypic and functional maturation of dcs, including costimulatory molecule expression, t cell-stimulation and cytokine production. [43] . in addition, uv-inactivated sars cov also activates immature dcs [44] . in this study, we have demonstrated that bvlps, morphologically and antigenically similar to the native virus, exhibited stimulating ability in dcs and induced dc maturation by enhancing the expression of cell-surface costimulatory molecules including those essential for optimal activation of t cells: cd40, cd86, cd80 and cd83. phenotypic maturation of dcs exposed to bvlps was similar to those observed in dcs incubated with lipopolysaccharide (lps), a well-known stimulator of dcs maturation. in contrast, heat-denatured bvlps lost the ability to enhance the expression of cell surface molecules and the secretion of cytokine, suggesting that the specific structure of viral proteins might significantly contribute to the immune modulating activity. taken together, our data indicate that the baculovirus derived-bvlps can interact with human dcs to induce dc maturation and activation. our previous study found that vlps formed by the s, e, and m proteins of human sars cov elicited strong sars cov-specific humoral and cellular immune responses in mice and conferred protective immunity against the infection of s protein -pseudotyped murine leukemia viruses (mlv) [29] . here, we showed that both bvlps and sars cov vlps activated dcs and induced dc maturation by enhancing the expression of costimulatory molecules (cd40, cd86 and cd80) and the maturation marker cd83. a study in sars cov-infected patients indicated that the figure 6 . evaluation of cd4 t cell types stimulated by bvlps-exposed dcs. immature dcs (1610 4 ) were exposed to bvlps (10 mg/ml), ac or pbs for 24 h and then co-cultured with cd4+t cells (1610 5 ) levels of il-6 and tnf-a were markedly increased in the acute stage and returned to normal after given adequate immunosuppressive treatment [45] . corticosteroid has been used for the treatment of sars on the basis that proinflammatory cytokines is responsible for the immunopathology in the lungs [46] . of interest, in the current study, bvlps induced much higher levels of il-6 and tnf-a than did sars cov vlps. it seemed that bvlps vs dcs served as a better model to simulate the acutephase response between the virus and the host. thus, this model may be applicable in the development of vaccines and drugs against the virus. tnf-a and il-6 are important proinflammatory cytokines mediating apoptosis, immunity, and inflammation, and may play important role in antiviral responses and inflammation [31] . a previous study by wang et al indicated that the s protein of sars cov played a key role in the production of proinflammatory cytokine at a stage of virus-host cell interaction. s protein could activate nf-kb signaling pathway and thus upregulate the secretion of il-6 and tnf-a. here we demonstrate that bvlps are stronger immune modulators in terms of dc activation and maturation. since the only difference between sars cov vlps and bvlps was the s protein incorporated, the enhanced immune modulating ability of bvlps is very likely contributed by the s protein. different types of dcs may play different roles in the onset of a th1 or th2 immune response. the balance between th1 and th2 cells is important and may determine the outcome of an immune response toward infection and allergy. we therefore studied the influence of bvlps-stimulated dcs on t cell polarization by intracellular cytokine staining. compared to mock-treated dcs, bvlps-exposed dcs increased the population of ifn-c or il-4 positive cd4 t cells. the ifn-c secretion level was much higher than that of il-4, indicating a bias of th1 immune response. because matured dcs can enhance native t cells differentiation, these results further confirm that bvlps can induce dcs activation and maturation. bvlps had stronger ability to stimulate dcs by inducing the production of il-6 and tnf-a in our study. to address whether such difference was s protein specific, we constructed two recombinant plasmids pcdna-s and pcdna-bs, and detected their immune responses in mice. the higher level of igg2a and the higher number of s protein-specific ifn-c or il-4-secreting cells in mice immunized with pcdna-bs indicated that the s protein of bat sl cov was more immunogenic. our in vivo data provide evidence that the difference of immune modulating ability between bvlps and sars-cov vlps was highly likely contributed by the s protein. taken together, we have successfully constructed bvlps formed by membrane proteins of different origins, one from sl-cov isolated from bats (bs) and the other two (e and m) from human sars cov. the formed bvlps demonstrated stimulating activity in immature dcs. in the absence of an in vitro cell culture model for sl-cov, our study has together demonstrated that bvlps vs dcs can be used as a model to study the interaction between sl-cov and host cells. further study by using this model may provide important information for vaccine development and for understanding the pathogenesis and the evolution of sl-cov. dna coding the s protein (bs) of bat sl-cov was amplified from rp3 strain (genbank accession number dq071615) by reverse transcription-polymerase chain reaction (rt-pcr) using the following primers: 59-tttggatccccaccatgaaaattttaatt-ctt and 59-ggg gaattcttatgtgtagtgtaattttac. bs gene was cloned into pfastbac dual vector [47] under the control of a strong viral promoter, polyhedron promoter. gfp gene was cloned under the control of p10 promoter to generate pfbs. recombinant baculoviruses were generated using autographa californica multiple nucleopolyhedrovirus (acmnpv) genome. in brief, vacbs was generated by transfecting the bs plasmids into insect cells sf21 using lipofectin (invitrogen). recombinant baculovirus vacme was generated as previously described [30] . in addition, s and bs gene were subcloned into pcdna3.1(+) (invitrogen, carlsbad, calif.) to construct the recombinant plasmids pcdna-s and pcdna-bs, respectively. the accuracy of the constructs was confirmed by restriction digestion and sequencing. dna plasmids were purified using qiagen megaprep columns (qiagen) and dissolved in endotoxin-free pbs to a final concentration of 2 mg/ml and stored at 220uc. insect cells were co-infected with vacbs and vacme at a multiplicity of infection (moi) of 5. at 4 days post-infection, cells and medium were harvested and centrifuged at 90,100 g for 4 hours. bvlps in the pellets were resuspended in pbs and further purified on a discontinuous 30-50% (w/v) sucrose gradient. a visible band between 30% and 40% sucrose layers was collected, concentrated by centrifugation and then resuspended in pbs at a total protein concentration of 2 mg/ml (measured in eppendorf biophotometer, germany). the purity of the vlps was then checked by sds-page. the presence of bs proteins in the purified preparations was confirmed by western blot using rabbit-derived anti-sars-cov antibody, kindly provided by prof. lin-fa wang in australian animal health laboratory (b.t. eaton and l.-f. wang, unpublished results). immunogold labeling was detected using antiserum against bs2 (667-1242 aa in s2 region of bat s protein). to generate bs2 antiserum, the cdna of 667-1242 aa in s2 region was cloned into pmal-c2x expression vector (neb). bs2 protein was purified by using amylose resins (neb) and then was used to immunize rabbit. for em study of bvlps budding from insect cells, sf21 cells were co-infected with vacbs and vacme at a moi of 5. at 72 h post-infection, the cells were collected and fixed with 2% glutaraldehyde and then with 1% osmium tetroxide. thin-section samples were prepared and examined under a hitachi-h7500 transmission electron microscope. for immunogold labeling, bvlps were loaded onto a collodion-coated em grid for 5 min. after removal of the excess sample solution, antibody against bs2 protein was added onto the grid and incubated for 1 h at room temperature. grids were washed three times and then incubated with 15 nm gold conjugated anti-rabbit igg for 1 h. the samples were stained with 2% phosphotungstic acid (pta) for 1 min, then drained, and examined under the em. human peripheral blood mononuclear cells (pbmc) were isolated from healthy donors by isopaque-ficoll (lymphoprep; nycomed, oslo, norway) following the manufacturer's instructions. immature dcs were generated from pbmc as previously described [48] with minor modifications. briefly, pbmc in rpmi 1640 (gibco laboratories, grand island, ny) were cultured in sixwell plates (2610 6 cells/ml) for 2 hours. adherent monocytes were washed with rpmi 1640 and then cultured for 6 days in complete medium containing 10% fetal calf serum (life technologies) and supplemented with rhgm-csf (1000 u/ml) and rhil-4 (500 u/ ml) (both from peprotech). half of the medium was replaced every two days. the resulting differentiated dcs were .97% cd1a positive and ,1% cd14 positive. all participants gave written informed consent. this study was conducted in accordance with the declaration of hubei and approved by health department of hubei province and wuhan blood center of hubei province, china. dcs (2610 6 cells) were incubated with either 10 mg/ml of bvlps or 10 mg/ml of lipopolysaccharide (lps, sigma, st. louis, mo). meanwhile, immature dcs were treated with pbs, ac (culture supernatant of sf21 cells transfected with baculovirus expression vector pfastbac dual with gfp under the control of p10 promoter) or heat-denatured bvlps (100uc for 10 min). alternatively, an equivalent amount of bvlps or lps was incubated with 10 mg/ml polymyxin b (a compound that binds to lps and inactivates lps's biological functions [49] ) at room temperature for 1 h before added to dcs. after different treatments of dcs at 37uc for 16 h, supernatants were removed and frozen at 270uc until cytokine detection. dcs were stained for specific cell-surface markers and analyzed by flow cytometry. culture supernatants were collected for cytokine (tnf-a, il-6 and il-10) quantification using enzyme-linked immunosorbent assay (elisa) according to the manufacturer's instructions (u-cytech, sensitivity: 2 pg/ml). dc surface markers were analyzed by flow cytometry. briefly, cells were washed in cold pbs and cell surface staining was carried out using the following antibodies: fluorescein isothiocyanate (fitc)-conjugated anti-cd40, fitc-conjugated anti-cd80, phycoerythrin (pe)-conjugated anti-cd83, pe-conjugated anti-cd86 (all from pharmingen). after incubation with antibodies for 30 min at 4uc, cells were washed and analyzed using flow cytometer (beckman, epics altra, usa). data were analyzed using exopo analysis software. a minimum of 10,000 events were collected and analyzed for each sample. isotype-matched controls (pe-conjugated mouse-anti-human-igg1, fitc-conjugated mouse-anti-human-igg1, both from bd pharmingen) were included in each staining. evaluation of t cell types stimulated by bvlps-exposed dcs cd4+t cells were prepared from pbmc by positive selection using anti-cd4 antibody (bd pharmingen) and the purity was consistently above 95%. immature dcs (1610 4 ) were exposed to bvlps (10 mg/ml), ac or pbs for 24 h and washed thoroughly. treated dcs were then co-cultured with cd4+t cells (1610 5 ) in 96-well plates (costar) in triplicate for another 72 h. for intracellular detection of il-4 and ifn-c, dcs/t cell mixtures were further stimulated with 10 ng/ml pma (phorbol 12myristate 13-acetate) and 0.5 mmol/l ionomycin for 6 h. after stimulation, mononsesin (1.5 ml/ml) was added to each well and plates were incubated at 37uc for 3 h to block reaction. the cells were then washed, fixed and permeabilized. intracellular ifn-c and il-2 (both pe-conjugated) staining and flow cytometry analysis (beckman, epics altra, usa) were performed in succession. data were analyzed using exopo analysis software. a minimum of 10,000 events were collected and analyzed for each sample. isotype-matched controls (pe-conjugated mouse-antihuman-igg1, bd pharmingen) were included in each staining. analysis of the humoral immune response 6-8 wk-old female balb/c mice were immunized intramuscularly with purified plasmid pcdna-s or pcdna-bs 3 times at a 2-wk interval. s-specific igg and the subclasses (igg1 and igg2a) in serum were assessed by elisa. recombinant s2 protein expressed in e. coli was purified and used as the detection antigen. horseradish peroxidase (hrp)-conjugated goat anti-mouse igg, igg1 or igg2a (sigma) was used as the secondary antibody. optical density (od) was read at 490 nm (a 490 ). cellular immune responses to sars-cov were assessed by ifn-c and il-4 elispot assays using mouse splenocytes. assays were performed according to the instruction manual (u-cytech, netherlands). absorbance was read using an elispot reader (hitech instruments) and spot-forming cells (sfc) per 10 6 splenocytes were calculated. the medium background response in the absence of antigen was consistently ,10 sfc per 10 6 splenocytes. all data 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and downregulated by tumor necrosis factor alpha inhibition of the mitogenic response to lipopolysaccharide (lps) in mouse spleen cells by polymyxin b we thank prof. lin-fa wang in australian animal health laboratory for kindly providing the rabbit-derived anti-sars-cov antibody. key: cord-274268-0ucqp3uz authors: chorus, caspar; sandorf, erlend dancke; mouter, niek title: diabolical dilemmas of covid-19: an empirical study into dutch society’s trade-offs between health impacts and other effects of the lockdown date: 2020-09-16 journal: plos one doi: 10.1371/journal.pone.0238683 sha: doc_id: 274268 cord_uid: 0ucqp3uz we report and interpret preferences of a sample of the dutch adult population for different strategies to end the so-called ‘intelligent lockdown’ which their government had put in place in response to the covid-19 pandemic. using a discrete choice experiment, we invited participants to make a series of choices between policy scenarios aimed at relaxing the lockdown, which were specified not in terms of their nature (e.g. whether or not to allow schools to re-open) but in terms of their effects along seven dimensions. these included health-related effects, but also impacts on the economy, education, and personal income. from the observed choices, we were able to infer the implicit trade-offs made by the dutch between these policy effects. for example, we find that the average citizen, in order to avoid one fatality directly or indirectly related to covid-19, is willing to accept a lasting lag in the educational performance of 18 children, or a lasting (>3 years) and substantial (>15%) reduction in net income of 77 households. we explore heterogeneity across individuals in terms of these trade-offs by means of latent class analysis. our results suggest that most citizens are willing to trade-off health-related and other effects of the lockdown, implying a consequentialist ethical perspective. somewhat surprisingly, we find that the elderly, known to be at relatively high risk of being affected by the virus, are relatively reluctant to sacrifice economic pain and educational disadvantages for the younger generation, to avoid fatalities. we also identify a so-called taboo trade-off aversion amongst a substantial share of our sample, being an aversion to accept morally problematic policies that simultaneously imply higher fatality numbers and lower taxes. we explain various ways in which our results can be of value to policy makers in the context of the covid-19 and future pandemics. the outbreak of covid-19 in the netherlands, as in many other countries, was followed by an unprecedented package of measures, summarized by the dutch government under the name "intelligent lockdown". as of mid-march, schools closed, as did bars and restaurants and countless other service providers (the so-called 'contact professions' such as barbers). working from home became the norm, large scale events such as professional football matches were banned, and a variety of other commandments and urgent stipulations were issued [1] . in this first, acute phase of the crisis, there was a sense of unanimity and focus in dutch society: the goals (i.e., delaying and limiting the spread of covid-19, protecting the vulnerable, preventing the collapse of the healthcare system) justified the means (i.e., a lockdown that in effect crippled large parts of society). in this acute and early phase of the crisis, deontological ethics were dominant in the public debate: most agreed that everything had to be done to keep the healthcare sector afloat and to keep the loss of human lives at an absolute minimum. about a month and a half after the lockdown was put in place, pressure on the healthcare system started to gradually decrease, accompanied by a downward trend in the number of covid-19 related fatalities. this signaled a gradual transition into the chronic phase of the crisis, with public attention shifting to the question of how the country should deal with the lurking threat of a virus that can always re-emerge until a vaccine or medicine is found, while at the same time keeping society functioning at a reasonable level. in the public debate it is becoming clear that this chronic phase also entails a different-consequentialist-ethical perspective in which all possible effects of government policy are taken into account. in addition to health related effects, this includes, for example, the impacts of the lockdown on the economy at large and one's personal income, as well as possible educational disadvantages due to distance learning. during this transition to the chronic phase of the crisis, the call for further opening up society has been getting louder and louder. the dutch government-like many other ones-as a result found itself in a position where diabolic dilemmas had to be faced; these were the words uttered by prime minister mark rutte, during an april 21 press conference watched by almost half of the dutch population [2] . the morning after this press conference, our survey including a choice experiment went live. our aim was to explore the preferences of the dutch population in terms of the weights they assigned to various effects of policies aimed at relaxing the lockdown. specifically, we wanted to know if the dutch were willing to trade health effects (such as avoiding fatalities) against other effects (e.g. on the economy), and if so, what would be their willingness to sacrifice economy-and education-related suffering for a reduction in fatalities and in pressure on the national healthcare system be. their choices in the experiment would also allow us to learn to what extent the transition from a deontological ethics perspective (requesting a full focus on saving human lives) was indeed gradually being replaced by a consequentialist one which puts weight on all foreseeable consequences of government policy. this paper reports the outcomes of this choice experiment. to be sure, the main contribution of this paper is not a methodological one: the use of choice experiments to study citizens' preferences and trade-offs involving fatalities and injuries has a long tradition in health economics [3] , traffic safety analysis [4] and environmental and climate change economics [5] . more related to the topic of our study, choice experiments have been deployed for measuring preferences for a covid-19 contact tracing app in the netherlands [6, 7] , in the united kingdom [8] and in the united states (e.g. [9] ); as well as to measure preferences for attributes of a covid-19 vaccine among australian citizens [10] . in addition, related survey techniques have been used to study beliefs about the effectiveness of covid-19 policies [11] , public sentiment toward covid-19 policies [12] and willingness to be vaccinated against covid-19 [13] . what makes our study unique is that it is the first one, to the best of our knowledge, that applies this tool to survey and investigate a society's willingness to make the highly salient and morally troubling trade-offs associated with policies aimed at relaxing a lockdown that was imposed in the wake of the covid-19 crisis. while our study is confined to the dutch society, and we acknowledge that countries differ widely in terms of their culture, the actions taken by their governments and preferences towards covid-19 policies [11, 12] , we nonetheless observe that in many other countries, debates are raging that are similar to the one being held in the netherlands; take for example the heated exchange in the united states of america (e.g., [14] ) between governor cuomo of new york who emphasizes that avoiding fatalities takes priority and that one cannot weigh a human live against the economic impact of the lockdown, versus president trump who is keen to re-open the economy and professes that the cure cannot be worse than the disease. given the similarities between various countries in terms of the societal tension between health-and economy-related effects of lockdowns, we expect our results to hold lessons well beyond the dutch context. the remainder of this paper is organized as follows: section 2 presents the method of discrete choice experiments and the econometric methodology used to analyze the obtained choice data. section 3 elaborates the data collection effort, followed by section 4 where we present and interpret our results. section 5 concludes by discussing the main conclusions and policy recommendations that can be drawn from our study. the method of using discrete choice experiments (dces) to elicit latent preferences and tradeoffs of citizens regarding the effects of government policies has a long pedigree in domains as diverse as transport (e.g. [15] ), environment and climate adaptation [16, 17] , immigration [18] and health care [19] [20] [21] . also when it comes to morally challenging trade-offs involving human lives, dces have been widely used in these contexts (e.g. [22, 23] ). the core idea behind using dces is that choices made by respondents between policy scenarios specified in terms of their outcomes on various dimensions, can be used to identify the weights that respondents assign to each of these dimensions. these weights may then be used to: i) learn about the relative importance attached by society to various policy-impact dimensions, ii) predict levels of support for and opposition against specific policies, and iii) convert various policy dimensions into monetary terms in order to allow for a cost-benefit assessment. in this paper, the emphasis is on the first of these three potential uses of dces. compared to other approaches to identify citizens' preferences and trade-offs, such as directly asking respondents to assign a weight to various policy-impact dimensions, the dce methodology has important advantages. for example, it is well known that people find it very difficult to make explicit their preferences and decision making processes [24] , especially in the context of morally sensitive topics such as the one we study [25] . that is, people have great difficulty in reliably answering questions such as "how many cases of lasting mental health effects are you willing to tolerate, to avoid one covid-19 related fatality?". the dce approach circumvents such difficulties, by asking respondents to choose between policy scenarios specified in terms of these and other impacts; based on choices made, the implied relative weights attached to different dimensions can then be inferred by the analyst. furthermore, in contrast to standard opinion polls which typically ask questions that are too generic to be of much policy relevance ("should lockdown policies be focused more on reducing health effects or economic effects?"), the dce approach presents very specific policy scenarios (e.g. in terms of numerically expressed policy effects), allowing for the assessment of particular policies based on the estimated weights. it is important to note however, that despite its advantages and the resulting widespread use of dces, there is a continuing debate about their reliability [26, 27] . important insights from this debate can be put as follows: i) if available, the analysis of choices observed in real life (e.g. during referenda) is to be preferred over the analysis of choices made in hypothetical conditions such as a dce; ii) if a dce is used, care must be exercised to ensure 'consequentiality', i.e. respondents must feel that their choices will have consequences in real life [28] ; iii) the choice situations presented in the dce must be realistic and must align with experiences and considerations held by respondents (see e.g. [29, 30] ). a recent study in the context of immigration policies in switzerland, which compared the outcomes of a dce with those obtained by an actual referendum, shows that a properly designed dce is likely to generate reliable insights into the weights assigned by the population to various policy dimensions, also in morally salient contexts [31] . translating these generic lessons to our specific dce, we are confident that the policy scenarios presented to respondents were well aligned with the current public debate in the netherlands; in fact, the dimensions covered in our study were widely discussed in the media during the weeks preceding the data collection. furthermore, we made a substantial effort to ensure consequentiality, by (truthfully) informing respondents that the outcomes of this study would be shared with high-ranking policy makers at relevant ministries and the netherlands institute of public health (rivm). qualitative statements provided by respondents after having completed our survey (not reported here) strengthen our belief that most took the experiment very seriously. in the absence of an actual referendum on the topic, we believe that given these precautions our dce provides a useful and reliable alternative to collect choice data. in our dce, we vary policy scenarios along seven dimensions, covering those impacts of the lockdown that received the most attention in the public debate during the weeks preceding the data collection effort. table 1 shows the different policy dimensions ('attributes') and the ranges of their scores ('attribute levels'). note that these attributes and their levels were selected in an iterative process of pilot testing and discussions with colleagues at other dutch universities as well as analysts at relevant ministries and the netherlands institute of public health (rivm). for instance, we decided in consultation with analysts from the dutch government to select the three health dimensions ('increase in the number of deaths caused by the coronavirus', 'increase in the number of lasting physical injuries caused by the coronavirus', 'increase in the number of lasting mental injuries caused by the coronavirus') instead of using the concept of quality adjusted life years (qaly) which is popular in health economics studies [32, 33] . the reason being that the public debate in the netherlands focused on these three dimensions and not on qalys. moreover, in a draft version of the dce we made a distinction between increases in the number of deaths in different age groups (younger than 50 years, 50-75 years, older than 75 years); however, policy makers and analysts from the rivm argued that this was not a relevant variable for the trade-offs they faced in their decision-making. hence, we decided not to distinguish between different age-groups in our final experiment. more generally speaking, we constructed the attribute levels in three stages. firstly, we analyzed studies which provided projections of the impacts of the corona crisis on the seven policy impact dimensions. for instance, we analyzed rough estimates on the increase in the number of deaths [34] , projections regarding the increase in the number of people with lasting physical injuries caused by postponed operations [35] , data on the increase in domestic violence caused by the corona crisis in the united kingdom [36] , data regarding domestic violence in the netherlands prior to the corona crisis [37] , data on the number of children with educational disadvantages prior to the crisis [38] and projections about bankruptcies, unemployment and income loss [39] [40] [41] . secondly, we discussed the realism of these figures with epidemiologists and policy makers. the epidemiologists warned us to lower our predictions on the number of deaths and gave suggestions for determining the levels for 'number of people with lasting physical injuries'. moreover, we asked policy makers from the ministry of finance to provide a prediction of the one-off corona tax per household in 2023. thirdly, we tested in a pilot survey whether the levels that we constructed were salient and relevant in the eyes of participants. based on the results of the pilot survey we decided to increase the difference between the levels of 'increase in number of deaths'. we chose to execute a so-called unlabeled dce, which did not specify policies in terms of their nature (e.g. reopening schools, or sport clubs) but rather focused on the impact of policies on a range of dimensions. the advantage of such an unlabeled approach is that it allows policy makers to use our results for the assessment of (combinations of policies), including those that are currently not on the table but might be considered in later phases of the crisis. the experiment was designed using statistical techniques which ensure that every choice task contains a maximum amount of information on the weights assigned by respondents to different policy impacts [42] . more specifically, respondents were asked to choose between two policy packages described by seven attributes or policy impacts. the attributes and their levels (note that each attribute had three possible levels) were combined into 18 choice tasks using a d-efficient design with priors obtained from a pilot study [43] . the 18 choice tasks were blocked into two blocks of 9 choice tasks and respondents were randomly allocated to a block when entering the survey. we show a sample choice task and the text shown to respondents in fig 1. to see the relation between the exit strategies presented in the choice task exhibited in fig 1 with the attribute levels presented in table 1 , note that the left-hand side strategy in the choice task ("exit strategy 1") consists of level 2 of attribute deaths, level 3 of attribute injuries, level 3 of attribute mental injuries, level 2 of attribute educational disadvantages, level 0 of attribute income losses, level 0 of attribute corona tax, and level 0 of attribute work pressure in health sector. our econometric approach for analyzing the choices collected in the discrete choice experiment involves three model types. first, we estimate a classical linear in parameters binary logit model, where each attribute is assigned a corresponding parameter (its weight) in a process of maximum likelihood estimation; see ben-akiva and lerman [44] for details of this model and how it is estimated. second, we estimate a latent class model where we identify different classes in the population with people assigned to the same class have the same weight-parameter, i.e., have the same preferences [45, 46] . third, acknowledging the moral salience of the choice context, we estimate a so-called taboo trade-off aversion model [47] . this model estimates a penalty parameter for each policy scenario that involves higher numbers of fatalities and lower taxes. such a policy may be considered taboo by some respondents, as it in effect assigns a monetary value to a human life. the taboo trade-off aversion effect has a rich tradition in moral psychology [48] ), and in the context of a dce analysis it can be captured by means of an interaction effect, which indicates a potential dislike for a combination of particular attribute values beyond the separate direct effects of those attributes. following chorus et al. [47] we implement the taboo trade-off by constructing a dummy variable which equals 1 if a policy scenario presented in a choice task featured lower taxes and more fatalities than the other scenario that was presented in the same choice task. the accompanying parameter estimate represents the level of taboo trade-off aversion (we estimate such a parameter for each latent class, to explore heterogeneity in terms of taboo trade-off aversion). to introduce notation, let respondent n's utility from choosing alternative i in choice situation s be described by the linear-in-the-parameters random utility function in eq 1. here, β is a row-vector of parameters to be estimated, column vector x nis contains the levels of the attributes of the alternative and ε nis represents an extreme value type i distributed error term. under these standard assumptions, the probability that respondent n chooses alternative i in choice situation s can be expressed by the multinomial logit model [49] . the multinomial logit model (mnl) is the workhorse in discrete choice analysis, but is limited by its inability to describe unobserved preference heterogeneity and its inability to accommodate for the fact that each respondent made a series of choices (panel effect). a latent class model allows for capturing such unobserved preference heterogeneity and panel effects. let π qn be the probability that respondent n's preferences can be described by the q th vector (class). here, α q is a class specific constant, γ q a vector of parameters to be estimated, and z n a vector of respondent specific variables. we set the qth constant and parameter vector to zero for identification. every class q has a unique vector of attribute weights β q which describes the behavioral preferences of respondents assigned to the specific class. this enables us to take the panel structure of the data into account by allowing parameter weights to vary across classes while at the same time ensuring that they are constant across choices made by the same individual. now, we can express the probability that respondent n chooses alternative i in a particular choice task s as: likewise, for example, we can express the choice probability that respondent n chooses alternative i in every choice task s as: given our linear treatment of attributes, society's willingness-to-sacrifice a particular attribute (e.g. a certain number of households facing an enduring and substantial reduction in net income) in order to avoid one fatality which is directly or indirectly related to covid-19 can be calculated straightforwardly: it is given by the ratio between the fatality parameter and the parameter associated with the other policy effect (e.g. the parameter associated with the number of households facing income reductions). in the context of the latent class models, the sample level willingness to sacrifice is the weighted sum of the willingness to sacrifice per latent class, where the weights are the unconditional class probabilities. the data were gathered on 22 april, the day following a widely watched press conference by prime minister mark rutte (21 april) during which he emphasized that most of the lockdown-policies and regulations would remain in place until further notice, while some others were slightly relaxed. in hindsight, the days surrounding this press conference can be considered the height of the public debate in the netherlands about how to weigh the health-related effects of the lockdown with other societal impacts such as economic standstill, educational problems and mental health crises, among others. note that early may, after our data were collected, another press conference was held in which the government-quite unexpectedly in the eyes of many-announced a series of lockdown relaxations to take place in the months of may through september (also in this, the netherlands was not alone, as various other countries were contemplating and deciding for relaxations of lockdowns in this same time period). respondents were sampled from the online kantar public panel, with a view to be representative with respect to the dutch population in terms of age, gender and education level. kantar public approached members of their panel by e-mail to take part in our online survey. sampled respondents were informed about the purpose of the study, the methods employed and the specific policy impacts that were part of the choice experiment. our data collection effort was approved by the ethics board of the delft university of technology. a total of 1260 respondents of the panel were invited to participate. of these, 1121 respondents started the survey and we received 1,009 completed and usable surveys (implying a drop-out rate of 10%). for our analysis, we excluded two respondents who stated 'other' gender because parameters associated with this socio-demographic variable could not be identified empirically due to insufficient variation in the data. all models are run with these respondents excluded for comparability. table 2 compares the sociodemographics of our sample with those of the population, and finds a close correspondence in terms of gender and age, but an over-representation of highly educated respondents. in the next section, we will explore what this means in terms of the general applicability of our findings. table 3 shows the estimation results of our final model specifications. the binary logit model is our point of departure; note that the outcomes of this base model have been published in a dutch-language economics journal [50] . also note that in order to ensure that all obtained parameters were within the same order of magnitude (which helps guaranteeing parameter stability in latent class models), we rescaled our attributes; see table 3 for the resulting units. signs are as expected: people dislike increases in: the number of fatalities related (directly or indirectly) to covid-19; the number of people with lasting physical and mental injuries; the number of children left with an educational disadvantage; the number of households with persistent income loss; personal income taxes; and work pressure in the health care sector. all parameters are highly significant. the implied average (in the dutch society) willingness to sacrifice in order to avoid one fatality directly or indirectly related to covid-19 equals (rounded to the nearest integer): • 10 cases of lasting physical injuries; • 15 cases of lasting mental health problems; • 18 children with lasting educational disadvantage; • 77 households with long term decline in net income. in terms of the relative importance of working pressure in the health care system, we find that a one-step increase in the working pressure corresponds (in terms of disutility to dutch society) to 3,636 additional fatalities, underscoring the dominant role this variable has been playing in the public debate. in terms of the willingness to accept a higher (one-off) tax to help avoid fatalities, we find that the average dutch citizen weighs an additional 10,000 fatalities as heavily as a one-off tax increase per household of 2,912 euro. this implies that dutch society as a whole (which consists of approximately eight million households) is willing to sacrifice a total additional one-off tax burden of approximately 2.32 million euros. this 'value of life' estimate allows us to indirectly validate our dce: the dutch road authority [51] employs a value of life metric of 2.61 million euro in the context of road safety analysis, which is in line with our results. note that the average life lost in road accidents is likely to be that of a younger person than the average life lost due to covid-19 directly or indirectly, e.g. due to postponed treatment-this may partially explain that our estimate is lower than the official number used by dutch authorities. a meta-study done by the oecd [52] reports a median (across studies) value of life of 2.8 million euros, which also is of the same order of magnitude as our result. as a final indirect validation of our estimates, it is worth pointing out that the factor ten which we obtain between the weight of a fatality versus the weight of a lasting physical injury is about the same as the factor used for dutch road safety analysis [51] . before moving to the more sophisticated latent class model, we briefly illustrate how the basic binary logit model can be used to forecast support for policy scenarios. take the choice between the two policy scenarios depicted in fig 1. based on the parameter estimates (and applying eqs 1 and 2 given in the previous section), the model predicts that 54% of dutch society would prefer exit strategy 1, while the remaining 46% would prefer exit strategy 2. if the government would be able to reduce the impact of strategy 1 on mental health problems (e.g. by means of aggressively increasing funding in mental health care programs), in effect reducing the number of effected individuals from 200 thousand to 20 thousand (i.e., the same number as in strategy 2), the model predicts that support would rise to 70%. of course, such a mental health program would come with a cost. if the government decides to increase the oneoff corona tax in strategy 1 from 1,000 euro to 1,250 euro (which would generate approximately 2 billion euro in taxes) to achieve these mental health benefits, our model predicts that that would still imply a 69% preference level for strategy 1 over strategy 2. the latent class (lc) model with three classes fits the data significantly better than the binary logit model, even when adjusting for additional parameters; this is to be expected, given that opinions expressed in the public debate on this topic vary widely. the latent class model with four classes did fit the data even better, but we are of the opinion that reasonable class sizes and interpretability are important factors to consider in model selection when the purpose is to generate behavioral insights for policy analysis. we tested a wide range of alternative models and specifications; these are available from the corresponding author upon request. it should be noted, however, that the improvement in fit cannot be entirely attributed to the model describing unobserved heterogeneity, since the lc model also takes the panel structure of the data into account whereas the binary logit model does not. we see that all parameters in all classes are of the expected sign and significant at the 1% level, except for the increase in the number of deaths in class 1, which is insignificant, and income loss (number of households facing a loss in income) in class 1, which is only significant at the 5% level. looking at the weights obtained per class, the classes can be interpreted as follows: class 1, in which older people are over-represented, is not sensitive to changes in the number of fatalities, but it does care about the other policy-impacts and in particular puts a very high negative weight on tax increases. this finding is interesting and somewhat surprising in light of the fact that individuals in this class are known to be much more likely to die when contracting the virus, compared to younger people. this class contains about 20% of the sample. class 2, in which higher educated people are over-represented, is highly sensitive to each policy-impact, except for the tax increase (to which they are equally sensitive as the average respondent). this class contains about 29% of our sample. class 3 (containing about 51% of our sample) is as sensitive as the average respondent to fatality numbers and working pressure in the healthcare sector, while being considerably less sensitive to the other policy effects. in terms of ethical perspectives, class 2 can be described as a typical consequentialist class, weighing every single impact of the exit strategies. class 1 weighs most impacts, but not the one which moral psychologists call the sacred attribute (human life), while class 3 can be considered to combine the consequentialist and deontological perspectives as it weighs all effects, but puts less (compared to the average respondent) weight on effects other than the increase in fatalities and the working pressure in the healthcare system. in table 4 , we show the sample level willingness-to-sacrifice estimates for each sub-group (segment) of respondents implied by our class probability functions, as well as the associated 95% confidence intervals. standard errors were calculated using the delta method [53] . in terms of willingness to accept an increase in the number of people with lasting physical injuries to avoid one fatality directly or indirectly related to covid-19, we find several differences between subgroups of respondents. for example, highly educated women between 66 and 74 years old are only willing to accept an increase of 10 people with lasting physical injuries for each avoided fatality, whereas men between 18 and 25 with low education are willing to accept an increase of more than 16 people with lasting physical injuries for each avoided fatality. looking at the other policy dimensions, we see the same general trend: older and more highly educated people in general, and women in particular, are willing to sacrifice (per fatality avoided) fewer people with mental injuries, fewer children at an educational disadvantage and fewer households with an income loss, compared to younger people with lower education, and younger men in particular. in many cases the differences in willingness to sacrifice between segments of the population are large and significant judging by the non-overlapping confidence intervals. returning to the fact that our sample, while representative in terms of gender and age, is somewhat skewed towards highly educated people (see table 2 ), these segment-specific estimation results imply that our aggregate level estimates for society's weighing of various policy impacts of exit strategies is likely to somewhat underestimate the weight attached to fatalities and to somewhat overestimate the weight attached to other policy impacts such as physical and mental injuries, educational advantage, and income loss. more generally, these results suggest that segmentation along socio-demographic lines is needed to obtain a nuanced view of society's preferences related to covid-19 policies. with a view to exploring potential causes behind differences in weights attached to fatalities and other policy impacts, we estimated a series of two-class latent class models, where class membership was determined by respondents' perceived risk that they or a relative would contract covid-19, would become severely ill if having contracted the virus, would be hospitalized if contracting the virus, or would die if contracting the virus. none of these variables, which were all measured using five-point likert-scales, turned out to have a significant effect on class membership (and hence, on respondents' weighing of the policy impacts). we also tested a model where class membership was a function of whether or not one or more of a respondent' relatives had contracted the virus; 58 respondents indicated that this was the case. (note: only two respondents indicated that they had contracted the virus themselves, this number being too low to use in our analyses) using this variable as a predictor of class membership, table 5 shows that respondents whose relative(s) had been infected by the virus were significantly less likely to belong to class 1 which does not put a significant weight on fatalities. this is in line with intuition. our results for taboo trade-off aversion (see table 6 for the full estimation results) can be summarized as follows: in a three class model where we allowed all parameters, including the taboo trade off aversion parameter, to vary between classes, we find two classes with a significant and negative taboo-penalty associated with policies that involve (simultaneously) a lower tax and a higher number of fatalities than the alternative policy. for one of these classes, containing 21% of our sample, the taboo penalty is large (-1.44) whereas in the other class, containing 54% of our sample, the taboo penalty is of moderate size (-0.42). a third class (containing a minority of 25% of our sample), surprisingly features a large and positive taboo parameter (2.35) , implying that individuals in this class actually favor the combination of lower taxes and higher fatality rates. some caution is in place though, when interpreting these taboo aversion-related outcomes: correlations between the taboo penalty-parameters on the one hand, and the tax-and fatalityrelated parameters on the other hand, were quite high (>0.80). this indicates, that the model struggles to disentangle the direct effects of the tax-and fatality-variables on the one hand, and their joint indirect effect through the taboo-dummy variable on the other hand. this is not surprising, given that the experiment was not designed specifically to pick up these two separate but subtly related effects in an econometrically efficient way. we performed additional analysis to verify our results, including a three class model where the taboo parameter was allowed to vary across classes while the attribute weights were constrained to be the same across classes, as well as a three class model where the taboo parameter was allowed to vary across classes while the attribute weights were fixed to their binary logit sample-level estimates. estimation results for these models (which can be obtained from the corresponding author) showed that, as expected, they had a much lower model fit than the original taboo trade off aversion model which allowed the taboo parameter and the attribute weights to vary across classes, but they avoid high correlations between parameters. these models identified a taboo aversion for only about 10% of the sample. this indicates that more research is needed to draw definite conclusions about the role of taboo trade off aversion in the context of lockdown relaxation policies. this paper presented the results of an empirical study into dutch society's preferences of covid-19 related government policies, specifically in terms of the weights attached to various impact-dimensions of such policies. at the aggregate level-i.e., combining choices made by all respondents-as well as for particular segments of the population, we obtain estimates for society's willingness to accept or sacrifice fatalities in order to avoid physical, mental, educational, and economic impacts of lock-downs. the fact that the implied 'value of life' estimate obtained on our sample is close to the value that is used by the dutch government in other contexts, lends credibility to our results. what is perhaps the most striking finding of our study, is the large heterogeneity within the population in terms of the weights attached to various policy impacts: first, while some groups appear to weigh all impacts in line with what would be prescribed by consequentialist ethics, other groups appear to put much weight on some impacts while ignoring other impacts. second, while a majority dislikes policies that imply a taboo trade-off between lives and taxes, a sizeable minority in fact favors such policies. third, also within each group, there appears to be a substantial variation in terms of the weights attached to various policy impacts. some of this heterogeneity can be traced back to whether one has a relative that has contracted the virus (this leads to a higher weight on avoiding fatalities), but classical sociodemographic differences (gender, age, education level) appear to play an important role as well. this high level of preference heterogeneity should come as no surprise to those who have been following the heated debates about covid-19 policies in various countries including the netherlands; this result is also in line with the high levels of heterogeneity found in several other covid-19 choice experiments and surveys (see papers cited in the introduction). in the face of the covid-19 crisis, policy makers worldwide need to make choices with far-reaching consequences, based on a wide variety of considerations and limited by a high degree of uncertainty. the results of our research are no more than a small piece of this immense puzzle, and are subject to a number of limitations: first, while great care was exercised to develop a choice experiment aimed at obtaining trustworthy responses, it must be acknowledged that the choices made by respondents were made between hypothetical policies. as such, care must be exercised when interpreting and applying our estimation results. second, although our sample was representative in terms of gender and age, and despite having a high response rate among members of a representative panel, we did obtain an over-representation of highly educated people. combining this notion with our estimation results, this implies that our aggregate level results are slightly overestimating society's willingness to accept a fatality in order to reduce negative impact on educational disadvantage, economic impacts and other policy impacts. third, our data were collected at a specific point in time in a specific geographical and cultural context, which in itself limits the generic applicability of our results. notwithstanding these limitations, we believe that our results can be useful in four ways in developing policies with regard to a possible (further) relaxation of the intelligent lockdown in the netherlands as well as in other countries. first, upon inspection of our estimation results, including the latent class models, we find that most of our respondents appear to have an eye for both the direct health-related effects of relaxation policies and their indirect impacts on mental health, the economy, education and their personal financial situation. although health impacts are of great importance to our respondents, most seem to employ a consequentialist ethical perspective by balancing various dimensions of policies. this suggests that an open view of policy makers, taking into account the broad range of effects of policies, would be appreciated by citizens. nonetheless, our result that about three quarters of our sample have a moderate to high aversion against making 'taboo tradeoffs', suggest that governments need to be careful in their decision-making and in how policies and their effects are communicated to the public. secondly, our results enable policy makers to determine whether the net valuation in society is positive or negative for particular combinations of policy effects. take, for example, the situation in which a certain policy measure leads to an expected reduction in deaths, but also to an increase in the number of people who will suffer from lasting mental health problems such as depression. our results indicate that, if the number of people who experience such problems as a result of the policy is fewer than about fifteen per avoided death, the policy is assessed positively by the average dutch person. however, if the number is higher, the net valuation is negative. thirdly, our results enable policy makers to identify levels of support and opposition among dutch citizens for different policy packages. for example, the binary logit model, fed by the estimated parameters, can be used to determine the percentage of dutch people who support a certain policy package over another one, as was illustrated in section 4. for this, the effects of the policy package must of course be within the scope of those presented in table 1 . and fourthly, our model can be used to determine whether the average adult citizen prefers a proposed policy package over a particular reference case. this reference case can be "a continuation of current policy" or "no restrictive measures". for this, it is important that the reference case can be specified in terms of the impacts on (a selection of) the policy impacts included in our experiment. in terms of avenues for further research, we believe that assessing the spatial and temporal transferability of our results is important, before conclusions for other countries, and for future situations, can be derived. regarding geography, given the considerable differences across countries in terms of culture, covid-19 policies and the effects of the virus on society, we expect that weights for various policy effects will differ across countries. moreover, some effects included in our experiment might be less relevant in other countries, while factors excluded in our study may be highly relevant in different geographical contexts. regarding timing, we expect that as the situation changes in terms of the threat of the virus and the visibility of e.g. economic effects, societal preferences and trade-offs will change, too. it would be interesting to test for this, by repeating our experiment in due time. nonetheless, we feel that having these timely estimates available helps (local) policy makers in the short term. a final suggested avenue for further research relates to the hypothetical nature of our experiment: if, as seems likely, one or more countries will in due time ask their citizens to vote for particular covid-19 policies in a referendum style voting process, such 'real' data could be used to make a comparison with data obtained by means of hypothetical choice experiments such as ours; see hainmueller et al. [31] for an example of such a comparison. in general, we hope that future study efforts aimed at measuring citizens' tradeoffs and preferences concerning the policy impacts of lockdowns in different countries (triggered by covid-19 or another pandemic) can use our study as a stepping stone. supporting information s1 appendix. description of the policy impacts. 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conditional logit analysis of qualitative choice behavior nederlanders maken een brede afweging bij afbouw coronamaatregelen mortality risk valuation in environment, health and transport policies calculating errors for measures derived from choice modelling estimates we received valuable input from the following individuals during the development and implementation of the experiment: marion collewet, shannon spruit, anatol itten, ardine de wit, key: cord-284275-bqo203pf authors: lu, roujian; yu, xiaoyan; wang, wen; duan, xijie; zhang, linglin; zhou, weimin; xu, jin; xu, lingjie; hu, qin; lu, jianxin; ruan, li; wang, zhong; tan, wenjie title: characterization of human coronavirus etiology in chinese adults with acute upper respiratory tract infection by real-time rt-pcr assays date: 2012-06-15 journal: plos one doi: 10.1371/journal.pone.0038638 sha: doc_id: 284275 cord_uid: bqo203pf background: in addition to sars associated coronaviruses, 4 non-sars related human coronaviruses (hcovs) are recognized as common respiratory pathogens. the etiology and clinical impact of hcovs in chinese adults with acute upper respiratory tract infection (urti) needs to be characterized systematically by molecular detection with excellent sensitivity. methodology/principal findings: in this study, we detected 4 non-sars related hcov species by real-time rt-pcr in 981 nasopharyngeal swabs collected from march 2009 to february 2011. all specimens were also tested for the presence of other common respiratory viruses and newly identified viruses, human metapneumovirus (hmpv) and human bocavirus (hbov). 157 of the 981 (16.0%) nasopharyngeal swabs were positive for hcovs. the species detected were 229e (96 cases, 9.8%), oc43 (42 cases, 4.3%), hku1 (16 cases, 1.6%) and nl63 (11 cases, 1.1%). hcov-229e was circulated in 21 of the 24 months of surveillance. the detection rates for both oc43 and nl63 were showed significantly year-to-year variation between 2009/10 and 2010/11, respectively (p<0.001 and p = 0.003), and there was a higher detection frequency of hku1 in patients aged over 60 years (p = 0.03). 48 of 157(30.57%) hcov positive patients were co-infected. undifferentiated human rhinoviruses and influenza (flu) a were the most common viruses detected (more than 35%) in hcov co-infections. respiratory syncytial virus (rsv), human parainfluenza virus (piv) and hbov were detected in very low rate (less than 1%) among adult patients with urti. conclusions/significance: all 4 non-sars-associated hcovs were more frequently detected by real-time rt-pcr assay in adults with urti in beijing and hcov-229e led to the most prevalent infection. our study also suggested that all non-sars-associated hcovs contribute significantly to urti in adult patients in china. human coronaviruses (hcovs) are enveloped viruses with a single-strand rna genome [1] . 5 species are known to infect humans, 229e and oc43 first were identified in the 1960s, nl63 and hku1 identified in 2004 and 2005, respectively [1] [2] [3] , and sars-cov was identified during the severe acute respiratory syndrome epidemic in 2003 [4] [5] . hcovs are associated with respiratory syndromes ranging from mild upper to severe lower respiratory tract infections including pneumonia and bronchiolitis [1, [6] [7] [8] [9] . specifically, hcovs can elicit a more serious respiratory disease in children, the elderly and people with underlying disease [1, 6, [10] [11] . hcovs circulate worldwide, although their frequencies differ from country to country depending on seasonality and detection annum [1, [12] [13] [14] [15] [16] . most studies have focused on children or adults with more serious respiratory disease including lower respiratory tract infections [1, [17] [18] [19] [20] [21] [22] . upper respiratory tract infections (urti) or ''the common cold'' are a significant health burden, especially among children. the major viral agents of urti in children include rhinoviruses, hcovs (-oc43 and -229e), rsv,piv, adenovirus(adv) and influenza(flu). newly identified viruses (hmpv, hcov-nl63,hcov-hku1 and hbov) are also included. although hcovs are recognized as one of the most frequent causes of urti or common colds in adults [7] , epidemiological data and clinical profiles are limited on hcovs infection in adults with urti continuously for several years with sensitive molecular methods [1, 13, 20, [23] [24] [25] , especially for hcov-nl63 and hcov-hku1 in china after 2009 h1n1 pandemic. ren et al reported the prevalence of hcovs was 1% in adults with arti in beijing from 2005 to 2009 by rt-pcr assays [24] , which was significantly lower than in previous literatures indicated above 2.1% [1, 13, 20, 23] . in the present study, 981 [19, 23, 26] , other 11 respiratory viruses infection among all specimens were also detected by sensitive molecular assays [25] [26] [27] [28] . hcovs infection and their clinical and epidemiological characteristics were analyzed. all aspects of the study were performed in accordance with the national ethics regulations and approved by the institutional review boards of the centre for disease control and prevention of china, as well as the ethics committee of peking union medical college hospital. participants were received "written informed consent" on the study's purpose and of their right to keep information confidential. written consent was obtained from all participants or their guardians. nasopharyngeal swabs were collected from adults presenting with urti to the peking union medical college hospital, beijing, china, between march 2009 and february 2011. patients provided informed consent for specimen collection and testing. all patients over 14 years of age were selected according to a set of criteria that included respiratory symptoms, a body temperature above 37.5uc, and a normal or low leukocyte count(#10 10 /l), but not lrti identified by pulmonary abnormalities on radiography and clinical descriptions(without bronchitis, bronchiolitis and pneumonia). demographic data and clinical findings at the time of diagnosis were recorded on a standard form. all swabs were collected into viral transport medium and transported for immediate testing to the national institute for viral disease control and prevention (nivdc), china center for disease control and prevention. viral rna was extracted from 200 ml of sample using qiaamp minelute virus spin kits (qiagen, germany). cdna was synthesized with amv reverse transcriptase and random hexamer primers (promega, usa) as described previously [19, 23, 25] . real-time rt-pcr amplification was performed using taq-manh one-step rt-pcr master mix reagents kit (applied biosystems, usa) [19, 23, 26] . a real-time reverse transcription (rt)-pcr technique was used to screen for each of the 4 non-sars hcovs (table 1) . pcr was performed under the following conditions: 48uc for 30 min, 95uc for 15 min, followed by 40 cycles at 95uc for 15s, 68uc for 1min.the lower limit of detection of each hcov real-time rt-pcr assay was 50-100 copies/ 25ul.the assay sensitivity, specificity and coefficients of variability(cvs) were evaluated and validated as previously described [19, 26] . specimens were also screened for influenza virus types a and b, parainfluenza virus types 1 to 3, respiratory syncytial virus (rsv), picornaviruses (including enteroviruses and rhinoviruses) and adenoviruses using three multiple-nested pcr assays [25] [26] ( table 1) , for human metapneumovirus (hmpv) and human bocavirus (hbov) using pcr and nested-pcr assays [26] [27] [28] ( table 1) . multiple-nested pcr and nested pcr programs were run as follows: 1 st round: 94uc for 2 min; 94uc for 30 s, 55/50uc statistical analysis was performed using the predictive analysis software (pasw) statistics 18 package with x 2 -test and fisher's exact test. a total of 981 nasopharyngeal swabs were collected. the male: female ratio was 438:543 (1:1.24) and the median age was 29 years (age range 14 years to 91 years). hcovs were detected by realtime rt-pcr in 157 (16.0%) of the 981 specimens: 229e in 96 (9.8%), oc43 in 42 (4.3%), hku1 in 16 (1.6%) and nl63 in 11(1.1%). the detection rates of hcovs are summarized in table 2 . differences in the detection rates of oc43 and nl63 during 2009/10 and 2010/11 were statistically significant (p,0.001 and p = 0.003, respectively). other 11 respiratory viruses infection among all specimens were also detected by sensitive molecular assays (table 1 ). during the same period, flu a virus was detected in 87(8.9%) patients, flu b virus was detected in 18(1.8%) patients, piv-1 was detected in 5(0.5%) patients, piv-2 was detected in 1(0.1%) patient, adv was detected in 20(2.0%) patients, rsv was 1(0.1%) patient, rhinoviruses was detected in 71(7.2%) patients, enterovirus was detected in 7(0.7%) patients, hmpv was detected in 26(2.7%), and hbov was detected in 2(0.2%) patients( table 2 ). the seasonal distribution of hcovs is shown in fig.1 . hcov-229e was the most common species, appearing in most months and without an obvious seasonal distribution. peaks in 229e detections occurred in may 2009, december 2009 and august 2010, corresponding with spring, winter and summer, respectively, in beijing. hcov-oc43 detections peaked in april 2009. this virus continued to circulate for the next 10 months but only sporadic infections were detected in the second half of the study. in 2009, when most coronavirus infections occurred, 229e and oc43 were detected in every evaluable month, with the exception of 229e in september. a peak of nl63 activity occurred in march and april 2009 with little or no subsequent activity. hcov-hku1 activity was sporadic throughout, with no obvious seasonal distribution. hcovs were detected across all age groups, although nl63 cases were absent in the 30-39 years age group (fig.2) . a significantly higher detection frequency of hku1 occurred in patients aged over 60 years (p = 0.03), but there were no significant differences in age group distribution for 229e, oc43 and nl63. approximately equal numbers of males and females were infected with oc43, hku1 and nl63; 229e infected significantly more female than males (62.5% versus 37.5%, p = 0.009). the clinical characteristics are summarized in table 3 . all hcov positive patients presented with urti. fever, sore throat and headache were the most common symptoms at presentation. gastrointestinal tract associated symptoms were rare. overall there were no statistically significant differences in the clinical symptoms between speciesspecific hcovs except for rhinorrhea, which was observed less in 229e-infected patients than in the other cases (p = 0.025). high rates of co-infection with non-hcov respiratory viruses were observed (table 4 ). of the 157 hcov positive patients, 48 (30.57%) were co-infected, 38 of them with one other virus and 10 with 2 other viruses. most hcov-associated co-infections involved human rhinoviruses (all picornaviruses positive samples were confirmed to be rhinoviruses by dna sequencing) and flu a virus (more than 30%). compared to single hcov infections, there was no evidence to indicate that patients with co-infections had more serious illnesses (results not shown). hcovs circulate worldwide and the prevalence of the different species varies by year and geographical location [1] . recent reports show that hcovs have been detected in hong kong [29] , italy [13] , france [22, 30] , usa [18, 21, 31] , uk [23] , netherlands [19] , finland [32] , and china [24] [25] in respiratory and/or stool samples obtained from young children [18, [21] [22] 31] and adults [20, [23] [24] 33] . in our retrospective study, a 16.0% detection rate for hcovs infection was found in adults with urti; 229e was the most common infection (9.8% of all cases), followed by oc43 (4.3%), hku1 (1.6%) and nl63 (1.1%). the detection rates for 229e and oc43 are consistent with other studies performed worldwide (0.1-26% &0.5-10.1%) [1, 11, 22, 26, [29] [30] [31] [32] [33] [34] [35] [36] [37] , but higher than in a recent study undertaken in beijing [24] , perhaps due in our study to the exclusion of patients with lower respiratory tract infections via clinical descriptions and radiography, the use of a screening criteria leukocyte count of #10 10 /l which is likely to have excluded most patients with bacterial infection, and the use of real-time pcr with excellent sensitivity [17, 19, 23] . the studies were also undertaken in different years, suggesting that the presence or absence of circulating hcov species during these times may have influenced these differed outcomes [1, 17, 24] . in addition, flu a detection rate was 15.6% among urti adult patients between 2009/2-2010/3 with the occurrence of pandemic h1n1 virus 2009, which was signicantly higher than that (0.7%) of patients between 2010/2-2011/3 (table 2 ). whether the circulating patterns of hcovs might be affected by flua pandemic, warrants further studies. hcovs exhibit variable circulation approximately every 2-3 years [1, 17, 24, [34] [35] , and this was confirmed by our observation that detection rates for both oc43 and nl63 were statistically significant between 2009/10 and 2010/11. nevertheless, 229e strains circulated throughout the 2 years of study, often in high numbers and without discernible seasonality, in contrast to that reported in the uk [23] . a previous study in beijing reported higher detection rates for hcovs in individuals over 65 years of age [24] . in contrast, in this study hcovs were detected essentially in all adult age groups. of note, however, hku1 infection predominated in patients aged over 60 years, suggesting that adults in this category may be at higher risk for infection with this species. urti occurs commonly in both children and adults and is a major cause of mild morbidity which have a high cost to society. urti among adults are responsible for missed work and unnecessary medical care (antibiotic prescriptions), also occasionally associated serious sequelae. the clinical diagnosis of all hcovs positive patients we studied was urti. the main clinical signs at presentation were fever, cough, sore throat and headache, in accord with several previous studies [1, 7, 22] . some cases also showed gastrointestinal symptoms except in hcov-hku1 infection patients [1, 32] . rhinorrhea was observed in significantly fewer patients infected with 229e. many previous reports have indicated that hcovs have high rates of co-infection with other respiratory viruses [1, 19, 23, 26] , including rhinoviruses, influenza, hmpv, adv and rsv. in this study, co-infections were associated with each of the non-sars hcovs at rates similar to previous studies [1, 18, 23, 26] . human rhinoviruses and influenza a viruses were the most common respiratory viruses detected in hcovs-positive patients, possibly reflect the level of their circulation at the time, and which were consistent with rhinoviruses being the most common respiratory viruses for common cold [1, 36] . in contrast with virus infection among children with urti [1, 7, 23, 36] , rsv, piv and hbov were detected in very low rate(less than 1%) among adults with urti in this study. it was consistent with the previous reports [26, 37] . comparison of clinical symptoms experienced by patients with and without coinfections indicated that co-infection with hcovs did not affect illness severity. in summary, we found that the four non-sars hcovs are regularly associated with urti in adult patients in china, although distinct seasonal patterns of circulation are not obvious. our observation that elderly adults may be are at high risk of hku1 infection requires further study. our results suggest that testing algorithms that include sensitive hcovs detection contribute to the likelihood of obtaining a laboratory diagnosis when respiratory virus infection is suspected [26, [37] [38] . to the best of our knowledge, this is the first comprehensive analysis of the potential impact of four non-sars hcovs (hcov-oc43, hcov-229e, hcov-nl63 and hcov-hku1) as causes of urtis in adults admitted to hospital in china by real time rt-pcr assays. to identify the role of hcovs infection in urti of adults, more specimens include control subjects with clear clinical and epidemiological profiles warrant be investigated. update on rhinovirus and coronavirus infections identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome a previously undescribed coronavirus associated with respiratory disease in humans viruses and bacteria in the etiology of the common cold a cluster of cases of severe acute respiratory syndrome in hong kong croup is associated with the novel coronavirus nl63 respiratory coronavirus infections in children younger than two years of age coronavirus 229e-related pneumonia in immunocompromised patients coronavirus hku1 infection in the united states human respiratory coronavirus hku1 versus other coronavirus infections in italian hospitalised patients detection of human coronavirus nl63, human metapneumovirus and respiratory syncytial virus in children with respiratory tract infections in southwest sweden evidence of human coronavirus hku1 and human bocavirus in australian children detection of human coronavirus-nl63 in children in japan human coronaviruses infections in rural tailand: a comprehensive study using real-time reverse-transcription polymerase chain reaction assays detection of four human coronaviruses in respiratory infections in children: a one-year study in colorado impact of human coronavirus infections in otherwise healthy children who attended an emergency department human coronavirus and acute respiratory illness in older adults with chronic obstructive pulmonary disease coronavirus infection and hospitalizations for acute respiratory illness in young children an outbreak of coronavirus oc43 respiratory infection in normandy, france epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex real-time pcr method prevalence of human coronaviruses in adults with acute respiratory tract infections in beijing laboratory diagnosis and surveillance of human respiratory viruses by pcr in etiology and clinical characterization of respiratory virus infections in adult patients attending an emergency department in beijing molecular assays for detection of human metapneumovirus epidemiological profil and clinical associations of human bocavirus and other human parvoviruses coronavirus hku1 and other coronavirus infections in hong kong human (nonsevere acute respiratory syndrome) coronavirus infections in hospitalized children in france clinical disease in children associated with newly described coronavirus subtypes detection of human coronaviruses in children with acute gastroenteritis a prospective hospital-based study of the clinical impact of non-severe acute respiratory syndrome (non-sars)-related human coronavirus infection seroepidemiology of group i human coronaviruses in children acute respiratory infection and influenza-like illness viral etiologies in brazilian adults the common cold molecular diagnosis of respiratory virus infections use of sensitive, broad-spectrum molecular assays and human airway epithelium cultures for detection of respiratory pathogens the authors thank chris birch and julian druce of the victorian infectious diseases reference laboratory, north melbourne, australia, for their assistance when developing multiplex rt-pcr for other common respiratory viruses and preparation of the manuscript. we also thank the staff of the peking union medical college hospital for providing samples. key: cord-293072-giakcaki authors: xu, wan-xiang; wang, jian; tang, hai-ping; chen, ling-han; lian, wen-bo; zhan, jian-min; gupta, satish k.; ji, chao-neng; gu, shao-hua; xie, yi title: a simpler and more cost-effective peptide biosynthetic method using the truncated gst as carrier for epitope mapping date: 2017-10-12 journal: plos one doi: 10.1371/journal.pone.0186097 sha: doc_id: 293072 cord_uid: giakcaki there is a need to develop better methods for epitope mapping and/or identification of antibody-recognizing motifs. here, we describe improved biosynthetic peptide (bsp) method using a newly developed plasmid pxxgst-3 as vector, which has a viral e7 gene in the cloning sites of pxxgst-1. it is crucial to employ pxxgst-3 instead of pxxgst-1, since it makes use of the bsp method simpler and easier to perform, and more cost-effective for epitope mapping. these merits are embodied in two aspects: i) convenient recovery of double enzyme-digested product due to the existence of 315 bp inserted between bamh i and sal i sites, and thus greatly reducing the production of self-ligation clones, and ii) no longer requiring control protein when screening recombinant (r-) clones expressing 8/18mer peptides by running polyacrylamide gel electrophoresis. the protocol involves the following core steps: (i) design of plus and minus strands of dna fragments encoding overlapping 8/18mer peptides; (ii) chemical synthesis of the designed dna fragments; (iii) development of r-clones using pxxgst-3 vector expressing each 8/18mer peptide fused with truncated gst188 protein; (iv) screening r-clones by running the cell pellets from each induced clone on sds-page gel followed by sequencing of inserted dna fragments for each verified r-clone; and (v) western blotting with either monoclonal antibodies or polyclonal antibodies. this improved gst188-bsp method provides a powerful alternative tool for epitope mapping. for rational vaccine design or development of diagnostics, it is imperative to map all functionally relevant, specific and/or conserved linear (continuous) b cell epitopes (bce) of target proteins. several methods have been described to define linear bces, which can be broadly classified into four categories: i) chemically synthetic peptide (csp) and its chip method [1] [2] [3] [4] [5] ii) biosynthetic peptide (bsp) by gene fragment expression, phage display random-peptide or antigen-fragment libraries and expression-pcr technique, etc [6] [7] [8] [9] [10] [11] ; iii) epitope prediction based on computational tools relating to several physical or chemical parameters of a given protein; and iv) sequencing of proteolytic fragments applying enzymatic and chemical reagents. however, performance of these methods, including their numerous improved or derivative methods, is far from ideal because of the various limitations. in particular, for classical csp method commonly used, it is not easy to identify antibody-recognizing bce minimal motifs even when using monoclonal antibodies (mabs) in most cases, or impossible to reveal igg-epitome on a target antigen using polyclonal antibodies (pabs) that is the basis of finding all desired functional bces. recently, many research groups tried to use bsp method similar to csp method for antibody identification or bce mapping of antigenic segments of interest, on which several genes encoding core streptavidin (stv), glutathione s-transferase (gst), and β-galactosidase (gal) were used as carriers of bsp [12] [13] [14] [15] [16] [17] . these explorations, including our construction of minimum short fragment encoding 4 amino acids (aa) fused with stv gene [12] and finding of weak antigenic range (~21 to 30 kda) in bacterial proteins (fig 1a and 1b) , resulted finally in the development of original bsp method capable of arbitrarily expressing overlapping 8/ 18mer peptides. it employed a truncated gst gene encoding 188 residues (gst188) as carrier in pxxgst-1 ( fig 1c) [18] , and the subsequent improvement of applying the enhanced chemiluminescence (ecl) reagents in western blotting for bce mapping [19] . although the method has been used to reveal igg-epitomes of three major proteins from human papillomavirus type 58 (hpv58) and in other related studies [18] [19] [20] [21] [22] [23] [24] [25] , it still had potential faults that needed to be overcome for convenience of users, which were: i) it is unable to purify the double enzyme-digested pxxgst-1 product with 4190 base pairs (bp), because it is only 6 bp shorter than the 4196 bp product generated by inadequate digestion leading to lower efficiency of constructing recombinant (r-) clones; and ii) setting gst188 control was required to screen the r-clone of expressing gst196 (gst188 + 8mer peptide) protein due to the~1 kda of migration rate difference between both proteins, otherwise it is inconvenient to distinguish the~0.5 kda difference between gst196 and gst192 (gst188 + 4 aa) proteins expressed by r-and self-ligation clones. it also has same trouble for determining self-ligation clone by running sds-15% polyacrylamide gel electrophoresis (page), due to the~0.5 kda difference between the gst192 and control gst188 proteins that were expressed by pxxgst-1 and pxxgst-2 ( fig 1d) . it is likely that these clones expressing gst192 protein could be mistakenly regarded as r-clones and subsequently followed-up for sequencing leading to wasteful expenditure. in our newly improved method (named gst188-bsp), these shortcomings have been well addressed through using pxxgst-3 ( fig 1e) , which can make it more convenient and efficient for cloning and screening r-clones of overlapping 8/18mer peptides, because of the strategy to insert a longer dna fragment between both target cloning sites of pxxgst-1. obviously, the 315 bp in length of selected e7 gene is sufficient to distinguish two different enzyme-cut products of 4190 bp and 4511 bp, and the~11.5 kda of e7 protein composed of 105 aa can make the gst188-e7 fusion protein out of the 21 to 30 kda weak antigenic area, and thus no longer require any protein control for screening r-clones of 6/18mer peptide due to non-existence of any nonspecific band like gst192 protein within the area of cell proteins from a self-ligation clone of pxxgst-3. the gst188-bsp method presented here not only keeps characteristics and virtues of original bsp approach but also is simpler and easier to perform, and more costeffective. here, we describe the method and its standardized protocol for general laboratory application. our data suggest that the gst188-bsp method reported herein is an excellent alternative tool for bce/epitome mapping and identification of mab-recognizing minimal motif. the preparation of rabbit antisera to r-huzp4c 227-463 segment was conducted in accordance with the guidelines for the care and use of laboratory animals, and was approved by the ethics committee of shanghai institute of planned parenthood research (sippr). the procedures were in accordance with guidelines established by the ethics committee of sippr. ethical approval for the unpublished research project no. 2012bai31b07 was granted by the ethical committee of the sippr (2012-08). three male new zealand white rabbits purchased from sippr-bk lab animal co., ltd. (shanghai, china) were caged at controlled temperature of 25˚c. all the performed experiments were in full compliance with standard laboratory animal care protocols approved by the institutional animal care committee of sippr. these rabbits were immunized intramuscularly with 1 mg of purified e. coli-expressed recombinant human zona pellucida glycoprotein-4 corresponding to amino acid residues from 227 to 463 (r-huzp4c 227-463 ) emulsified in 0.5 ml of complete freund's adjuvant and 0.5 ml of pbs at multiple sites on rabbit's back. three booster immunizations of 0.5 mg r-huzp4c 227-463 protein in incomplete freund's adjuvant were administered at 3-week intervals. sera were collected from each rabbit seven days after each vaccination and stored at -70˚c. the thermo-inducible plasmids of pxxgst-1 and pxxgst-2 were constructed as described earlier [18] . the plasmid pxxgst-3 was constructed by our group, which had a hpv18-e7 gene (genbank no: x05015) inserted between bamh i and sal i sites in pxxgst-1, but failed to express gst188-e7 fusion protein in the various prokaryotic expression systems used by us. the rabbit antisera raised against r-huzp3b 177-348 [26] and r-huzp4c 227-463 segments were prepared previously, the mab c1p5 [27] (american research products inc., usa), and goat anti-rabbit/mouse igg conjugated to horseradish peroxidase (hrp) (proteintech group, usa) were purchased from shanghai sangon co., china. all gene fragments encoding short peptides of interest were synthesized by the sbs genetech co., ltd, shanghai. dna sequencing of inserts in each r-clone was performed by shanghai generay biotechnology co., ltd, china. the molecular cloning of synthesized dna fragments was done as described previously [28] , which has been briefly described in the 'protocol' as described later in this section. step one: designing series of overlapping short peptides. design all 16/18mer peptides with an overlap of 8 aa residues covering the full-length of the target protein or a longer segment sequence for the first round of antigenic peptide mapping, as well as design a set of 8mer peptides with overlapping 7 aa residues and covering each reactive 16/18mer peptide sequence for the second round of precise bce motif identification. note: it is recommended to design overlapping 16mer peptides, as the cost for synthesizing oligonucleotide of less than 60 nt in length is lower than that of oligonucleotide of more than 60 nt. also, when using a mab or cross-reactive mab to map bce, it is desirable to express two or several longer segments of the target protein so as to determine its shorter antibody reactive segment followed by designing overlapping 8/16mer peptides. doing so can save the cost, time and workload in following steps 4-7. step two: designing oligonucleotides of plus and minus strands. design all plus and minus strands of dna fragments encoding each designed 8/18mer peptides of the target protein, having cohesive ends for bamh i and sal i restriction enzymes at their 5'and 3' ends, so that they could be directly used to insert into the bamh i and sal i sites downstream of the gst188 gene in plasmid pxxgst-3 after their annealing in pairs. also, a taa termination codon is designed between each 8/18mer peptide and sal i site, so as to avoid possible false blotted bands of 8/18mer peptides for bce and minimal motif mapping, which will be caused by four gsvd residues encoded by 12 bp present in cloning site region of pxxgst-3 ( fig 1e) . in short, the designed plus and minus strands of encoding fragment should include sequences of 5'-gatcc and taag-3', as well as 5'-tcgactta and g-3' at their both ends (fig 2a and 2b ). as mentioned in step one, it is just close to 60 nt that is oligonucleotide corresponding to 57 nt encoding 16mer peptide together with 9 nt at their both ends. note: it is emphasized that design of the target gene fragments at this step is very convenient, that is, the consideration of the optimal codon usage for expression of each short peptide fused with gst188 in e. coli is not essential as more than 1000 of the constructed 8/18mer peptide fusion proteins have been successfully expressed by using their dna fragments from known prokaryotic and eukaryotic genes [12] [13] [14] [19] [20] [21] [22] [23] [24] [25] . however, it should be ascertained that whether there is another potential bamh i or sal i site within the oligonucleotide sequence, if any, it needs to be modified because it will disturb annealing of the paired oligonucleotides and its subsequent cloning. step three: chemical synthesis of the designed dna fragments. once the plus and minus stands of encoding gene fragments have been designed, they can be synthesized and page-purified by the sbs genetech co., ltd, shanghai. all page-purified dna fragments should be stored at -20˚c prior to use. step four: conduct annealing reactions of page-purified dna fragments in pairs. 1. to make 100 μm stock solution of each synthesized dna fragment by adding appropriate volume of double distilled h 2 o (ddh 2 o) into tubes based on the report provided by the biotechnical service company, respectively. 2. take respectively 5 μl of the stock solution from two tubes in pairs into a 1.5 ml of new tube, and add 90 μl of ddh 2 o to make 5 μm final concentration of each dna fragment. 3. heat tubes up to 94˚c in an electric heating block, anneal for 5 min, and then allowed them to cool gradually to room temperature (rt). step five: development of r-clones for expressing each 8/18mer peptide. 1. perform the ligation reactions using above each annealed dna fragment and constructed vector of pxxgst-3 digested with bamh i and sal i enzymes, and then purified using qiaquick gel extraction kit after running 0.7% agarose gel electrophoresis at 50 v for 90 min. the ligation reaction was carried out at 16˚c overnight using the following 15 μl of reaction mixture in 0.5 ml tube (table 1) , in which all components were mixed by flicking and spin briefly (refrigerated mikro 120 centrifuge). 2. employ the above ligation mixture in each tube to transform the e. coli bl21 (de3) competent cells according to the standard procedures [28] . grow overnight each clone on solid luria broth (lb)-ampicillin (amp) plates at 37˚c, respectively. it should be observed that there are about 10 to 20 clones on the lb plates next morning. note: if there was no growth of clones on a lb-amp plate after repeating twice, it suggests that they were incorrect or wrongly paired dna fragments and need to be resynthesized. 3. grow overnight each clone from lb-amp plates in 3 ml of liquid lb-amp medium at 30˚c, subsequently dilute 1:50 in fresh lb-amp medium and were grown at 30˚c at 220 rpm to an od 600 of 0.6-0.7. the clones were induced at 42˚c for 4-5 h. harvest each cell pellets by centrifugation at 5000 x g for 5 min at 4˚c. cell pellets obtained from 3 ml culture of expressed 8/18mer peptide fusion proteins were boiled in 400 μl of 1x sample loading buffer (50 mm tris-hcl, ph 6.8, 2% sds, 0.1% bromophenol blue, 10% glycerol and 100 mm β-mercaptoethanol) for 5 min. peptide biosynthetic method for epitope/epitome mapping step six: screening of r-clones by running sds-page. take~3 μl of each cell suspension from prepared individual thermo-induced bacterial cultures to run sds-15% polyacrylamide gel for screening r-clones, and cell proteins resolved by conducting electrophoreses at 100 v for 2 h. the gels were stained with coomassie brilliant blue g250 for analyzing the bands of fusion proteins. note: as shown in fig 3, all r-clones can be easily determined by observing difference between gst188 fusion proteins expressed in lanes 3-6 and control gst188 protein expressed by pxxgst-2 in lane 2. approximately 1 kda difference can be observed even when expressing overlapping 8mer peptide fusion proteins as compared to gst188 protein. however, as shown in lane 7, there was no specific 8/18mer-peptide fusion protein band appeared as compared to lanes 3-6 when self-ligation of pxxgst-3 happened due to inadequate enzyme digestion, suggesting that it no longer needs any additional control (lane 2 or 7) on the gels for the 8/18mer peptides expressed specifically in r-clones when using pxxgst-3 vector in the improved gst188-bsp method. step seven: sequencing of insert for each determined r-clone. conduct sequencing of all synthesized dna fragments inserted into plasmid pxxgst-3 from r-clones verified by above sds-page analysis through the shanghai biosune biotechnology co., ltd, china. it is recommended to use the primer 5'-ggccatcatacgttatatag-3'. note: for ensuring that all amino acid sequences of expressed short peptides are accurate, it is important to check the correctness of each synthesized dna fragments by sequencing after determining r-clones and before conducting bce and minimal motif mapping (fig 2c) , because this step can avoid possible error in bce mapping due to possible bp missense mutation or tubes numbering errors happened when synthesizing a large number of dna fragments. therefore, elimination of deleterious errors becomes necessary in many cases. of course, for saving cost and time if needed, it might be carried out after finishing two rounds of bce and fine motif mapping to check those dna fragments encoding reactive 8/18mer peptides. step eight: determination of bce recognized by mabs/pabs in western blot. proteins on sds-page gel were electrotransferred onto 0.2 μm nitrocellulose membrane, on which complete transfer was ensured by staining membrane with 0.1% ponceau s. non-specific antibody-binding sites on the membrane were blocked with blocking phosphate-buffered saline (pbs) containing 0.05% tween 20 and 1% skim milk powder or preimmune rabbit serum sample overnight at 4˚c, and probed with antisera raised against r-huzp4c that was pre-adsorbed with host cell lysates or mab c1p5 (1:1000 or 1:5000 dilution in pbs) for 2 h at rt. following washing four times, specific bands on the membrane were visualized by using goat anti-rabbit igg or goat anti-mouse igg conjugated with hrp at 1:10000 dilution. the blot was developed by using the ecl plus western blotting detection reagents. peptide biosynthetic method for epitope/epitome mapping note: to produce clear blotted bands in western blotting, it is suggested that before use, the antiserum against r-protein may be absorbed on host cell lysates as described earlier [28] , which facilitate in removal of cross-reactive antibodies to e. coli proteins. in order to know how many bces are there among three neighboring reactive 18mer-peptides of p32-34 (fig 4a and 4b) , which were blotted in first round of antigenic peptide mapping in our study on epitome decoding of huzp4 protein, we first expressed a set of 8mer-peptides (p110-120) covering the full-length sequence of reactive p32 according to procedures of steps 1 to 8 in the protocol, and then they were subjected to sds-page, and transferred onto 0.2 μm nitrocellulose membrane. the western blotting was performed by standard method with 1:2000-diluted rabbit antisera to r-huzp4c. specific antigen-antibody reactions on the membrane were visualized by applying goat anti-rabbit igg conjugated to hrp and ecl plus western blotting detection kit with higher sensitivity according to the manufacturer's instructions. as shown in fig 4c and 4d , a precise bce motif (pltlelq) was defined in reactive p32 according to the common sequence highlighted in yellow color within reactive p115-116 peptides. the mapped motif was in good agreement with the pltlel motif of huzp4 that was recognized by rabbit anti-native porcine zp igg in another previous study [18] , which only had a residue q at its c-terminus more than that of the latter, suggesting that rabbit immune system could recognize the common antigenic site shared among homologous proteins of pig and human, no matter whether they were against native protein or not. obviously, it is impossible to identify a bce among these reactive peptides (p32-34) using rabbit antisera to r-huzp4c, if not applying the gst188-bsp method. by the way, other two bce motifs in reactive p33 and p34 have also been identified using a set of overlapping 8mer peptide of p34 in completed study on epitome decoding of huzp4 glycoprotein, which one (dknygsy 324-330 ) was present in the 10 aa overlapping region of p33-p34 and another (yygvgdyp 330-337 ) at the c-terminus of p34 (fig 5) . there is a need to identify the fine bce motif recognized by a non-conformational mab, and the present gst188-bsp method is a powerful and economic technique for such purpose. to save cost and workload, express two or more bigger segments of a large protein at first so as to determine a reactive segment, and then map bce motif using 18/8mer peptides for a specific mab. for a known cross-reactive mab; however, another strategy may be adopted to find one or several potential segments of 100% conservation or with a residue difference among homologous proteins through sequence alignment. the following is a practical example for the latter case. the fine bce e6-2 motif (ygd/xtl) of hpv58-e6 was found to be highly conserved among most known high risk-hpvs (hr-hpvs) including hpv16 and hpv18 in our previous study [20] , and the commercially available mab c1p5 of hpv18-e6 can cross-react with hpv16-e6 protein [27] . therefore, in order to know whether the mab also could recognize the e6-2 epitope, the western blotting tests were performed using our previously expressed p35 octapeptide containing ygdtl motif and r-e6 protein of hpv58. the mab did not react with p35, but recognized r-protein of hpv58-e6. thus, another highly conserved site (el/ yrhy) with only a residue difference among them was found via sequence alignment of e6 proteins from hpv16, 18 and 58 (fig 6a) . based on bce minimum motif of three aa [19, 20] , thus, a panel of dna inserts encoding 3mer to 5mer peptides (the addition of three aaa residues at their n-terminus is only for the convenience of constructing r-clones) were designed, synthesized, annealed, and cloned into bamh i and sal i sites within pxxgst-3, resulting in five r-vectors. after constructing and expressing each r-clone, cell pellets from induced r-clones were used to perform western blotting with mab c1p5. finally, the bce fine motif (elrhy) of hpv18-e6 and a cross-reactive pentapeptide (eyrhy) among several homologous proteins were characterized, which the consensus pentamer sequence el/yrhy was based on blotted results of p5 and p6 (fig 6b and 6c ). meanwhile, it was yet found from the mapping result that besides hpv16-e6, the cross-reactive mab c1p5 can recognize hpv58-e6 as well as oncogenic hpv33-and 52-e6 proteins that were according to subsequent eyrhy alignment of homologous e6 proteins (fig 6a) , suggesting the importance of identifying fine bce motif for a cross-reactive nonconformational mab. in shanghai, people's republic of china, the cost of oligonucleotide synthesis is currently about usd 0.17/nt (rmb yuan 1.20/nt) for oligonucleotides of length less than 60 nt (57 nt for encoding 16mer peptide that included 9 nt of cohesive sequence at their both ends), and usd 0.29 (rmb yuan 2.00) per base for length more than 60 nt (63 nt for encoding 18mer peptide). enzymes (dna ligase, bamh i and sal i), molecular weight markers and other reagents until got a sequence-confirmed r-clone, which is far below present market price (usd~15.7 to 19.40 or rmb yuan~110.00 to 135.00 per residue in shanghai sangon co., china and the biomatik co., usa) of 6/18mer csp with >98% purity used in bce mapping. the cost-effectiveness of the gst188-bsp method is obvious when conducting bce fine motif or epitome mapping to a macromolecular protein. most importantly, it can obtain more definitive and trustworthy results of bce mapping by applying such bsps than csps [20] , and these r-plasmids expressing 8/18mer bsps can also be stored at -20˚c for future possible use. most of the methods pertaining to mapping of bces have employed csp strategy [29] , but it failed to reveal epitome of a target antigen using this strategy due to the limitation of being unable to use pabs to map a bce fine motif and/or more within several contiguous reactive peptides as shown in fig 4 that resulted in a limited number of mapped bces [30] [31] [32] . for example, not including a bce present in the c-terminus transmembrane-like domain, only three bces on mature huzp3 were identified by rabbit pabs in elisa assay when using a panel of overlapping 8mer csps covering huzp3 sequence [30] , whereas it was shown by our group that there are at least seven bces present in the protein by western blotting with rabbit pabs and sets of 8mer bsps [19] . for non-conformational mabs, there are several reports on using sets of overlapping 8/ 10mer csps or 12mer peptide library to identify bce minimal motifs [33] [34] [35] . due to limited availability of 8/10mer peptides synthesized on polypropylene pins and the high cost to purchase csps of high-purity, this method has limited applications. further, most of mapped 16mer antigenic peptides were inadvertently referred as "epitopes" [14] [15] [16] [17] , even being 37mer peptide [36] , although it is well recognized that smaller number of the aa residues are needed for binding to the antibody [37] . it is likely that within a mapped 18/20mer peptide there may be two and more bces [19, 20, [38] [39] [40] . moreover, the mab-recognizing fine motif has not yet been stipulated as a needful criterion or parameter for manufacturing a mab(s) as drug, although all know it is one of the major features to distinguish one mab from another, and could broaden application range of a mab. for instance, the commercially available mab c1p5 raised against hpv18-e6 protein can be used to identify hpv33/52/58-e6 as well, besides known hpv16-e6 according to the mapping results in fig 6. in early explorations of bsp, the stv118 (expression plasmid ptsa18), gst245 (pgex-6p) and gal590 (pwr590) proteins were used as carriers to express 4/16mer peptides for bce mapping or antibody identification [12] [13] [14] [15] [16] [17] . these endeavors suggested the feasibility to develop bsp approach in addition to csp strategy. however, these efforts failed to show the merits of bsp approach leading to a standard protocol suitable for general laboratory application. major drawbacks of applied bsps in above studies were: i) expressed 16mer peptide fusion proteins need to be purified before western blotting even when using chicken sera to sars-cov and sars convalescent sera [14] [15] [16] , with this operation step it obviously increases the research cost and work load, and takes much longer time; ii) it is inconvenient to select each r-clone of 16mer bsps before sequencing of insert, which needs to conduct the double enzyme restriction to identify insert, and iii) the position of expressed short peptides fused with gst245 or stv118 was not in the widest weak antigenic range of bacterial proteins, and thus was unsuitable for bce mapping with pabs, etc. the present gst188-bsp method as described has fully embodied practical approach. the outstanding advantages of gst188-bsp method are as follows: i) using the truncated gst188 as carrier, it makes the expressed 8/18mer peptides fusion proteins in the weak antigenic area of bacterial proteins, and thus permits directly using them to map each bce fine motif and epitome of target protein; ii) employing pxxgst-3 enables users to extract double enzymecut pxxgst-3 by running agarose gel electrophoresis, and thus greatly reduce the self-ligation of pxxgst-3 via removing products produced by inadequate digestion that may happened sometimes; iii) screening r-clones on the sds-page gels no longer needs any control, because as shown in lane 7 of fig 3, there are no band similar to gst192 protein in the 21 to 30 kda area of cell proteins, which will interfere with screening r-clones of 8mer peptide in the 21~30 kda area for self-ligation of pxxgst-3; iv) employing thermo-inducible expression system instead of isopropyl-β-d -thiogalactoside (iptg) induction reduces cost; v) it is less expensive than csp method due to cheaper cost of oligonuleotide synthesis than peptides; vi) it is clearer and more credible for the blotted bands in western blotting due to use of the ecl luminescence reagents than others such as 3,3'-diaminobenzidine (dab) or alkaline phosphatase (ap) kit; vii) it is adaptable for most laboratories due to its relative simplicity. in conclusion, based on the improved gst188-bsp method using the pxxgst-3 vector, we have constructed more than one hundred of r-clones to express 8/18mer and minimum 3mer peptides (via adding three alanine residues to its n-terminus, fig 6c) fusion proteins in our ongoing studies of epitome mapping. the epitopes recognized by pabs to r-huzp4c and mab cip5 against hpv18-e6 have been mapped. hence, we believe that the gst188-bsp method offers a much simpler option which is more cost-effective, reliable and adaptable for general laboratories. it will facilitate studies on epitope/epitome mapping of numerous target proteins and bce motif identification of non-conformational mabs. use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid general method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids strategies for epitope analysis using peptide synthesis epitope mapping using synthetic biotin-labeled peptides applications of peptide arrays prepared by the spot-technology efficient mapping of protein antigenic determinants vaccination with a synthetic zona pellucida peptide produces long-term contraception in female mice mapping of viral epitopes with prokaryotic expression products universal promoter for gene expression without cloning: expression-pcr expression polymerase chain reaction for the in vitro synthesis and epitope mapping of autoantigen. application to the human thyrotropin receptor high-resolution mapping of gens3 and b11f7 epitopes on myelin-associated glycoprotein by expression pcr expression and purification of three fusion proteins containing a single b-cell epitope (β5, β9 or β8) of human chorionic gonadotropin beta subunit immunogenic comparison for two different recombinant chimeric peptides (cp12 and cp22) containing one or two copies of three linear b cell epitopes from β-hcg subunit identification of two antigenic epitopes on sars-cov spike protein expression and antigenic epitopes mapping of receptior binding domain on the spike protein of severe acute respiratory syndrome coronavirus identification and antigenic epitope mapping of immunodominant region amino residues 510 to 672 on the spike protein of the severe acute respiratory syndrome coronavirus a peptide of foot-and-mouth disease virus serotype asia1 generating a neutralizing antibody response, and an immunostimulatory peptide minimal motif mapping of a known epitope on human zona pellucida protein-4 using a peptide biosynthesis strategy mapping of minimal motifs of b-cell epitopes on human zona pellucida glycopotein-3 epitomics: igg-epitome decoding of e6, e7 and l1 proteins from oncogenic human papillomavirus type 58 mapping of epitopes relevant for induction of acrosome reaction on human zona pellucida glycoprotein-4 using monoclonal antibodies fine epitope mapping of the central immunodominant region of nucleoprotein from crimean-congo hemorrhagic fever virus (cchfv) fine mapping and conservation analysis of linear b-cell epitopes of peste des petits ruminants virus nucleoprotein antisera preparation and epitope mapping of a recombinant protein comprising three peptide fragments of the cystic fibrosis transmembrane conductance regulator vaccination with an epitope peptide of izumo1 to induce contraception in female mice immunogenicity of recombinant human zona pellucida-3 peptides expressed in e. coli and efficacy of their antisera to inhibit in vitro human sperm-egg binding identification of human papillomavirus type 18 e6 polypeptide in cells derived from human cervical carcinomas molecular cloning: a laboratory manual surface conformational and linear epitopes on hpv-16 and hpv-18 l1 virus-like particles as defined by monoclonal antibodies evaluation of the contraceptive potential of recombinant human zp3 and human zp3 peptides in a primate model: their safety and efficacy mapping of dominant b-cell epitopes of a human zona pellucida protein (zp1) mapping of b cell epitopes on the zona pellucida 2 protein of a marsupial, the brushtail possum (trichosurus vulpecula) identification of epitopes of monoclonal antibodies to porcine zona pellucida 3β glycoprotein, a homologue of the mouse/human sperm receptor identification of b-epitopes in the human papillomavirus 18 e7 open reading frame protein human papillomavirus type 18 e6 and e7 antibodies in human sera: increased anti-e7 prevalence in cervical cancer patients epitope mapping of antibodies using a cell array-based polypeptide library epitopes on protein antigens: misconceptions and realities papillomavirus-like particle (vlp) vaccine displaying hpv16 l2 epitopes induces cross-neutralizing antibodies to hpv11 a pan-hpv vaccine based on bacteriophage pp7 vlps displaying broadly cross-neutralizing epitopes from the hpv minor capsid protein, l2 identification of broad-genotype hpv l2 neutralization site for pan-hpv vaccine development by a cross-neutralizing antibody writing -original draft: wan-xiang xu, satish k. gupta. wan-xiang xu, satish k. gupta. key: cord-294768-bs6thjw2 authors: alonso-fernández, alberto; toledo-pons, nuria; cosío, borja g.; millán, aina; calvo, néstor; ramón, luisa; de mendoza, sara hermoso; morell-garcía, daniel; bauça-rossello, josep miquel; núñez, belén; pons, jaume; palmer, juan a.; martín, luisa; peñaranda, maría; pou, joan a.; sauleda, jaume; sala-llinas, ernest title: prevalence of pulmonary embolism in patients with covid-19 pneumonia and high d-dimer values: a prospective study date: 2020-08-25 journal: plos one doi: 10.1371/journal.pone.0238216 sha: doc_id: 294768 cord_uid: bs6thjw2 introduction: coronavirus disease 2019 (covid-19) pneumonia is associated to systemic hyper-inflammation and abnormal coagulation profile. d-dimer elevation is particularly frequent, and values higher than 1μg/ml have been associated with disease severity and in-hospital mortality. previous retrospective studies found a high pulmonary embolism (pe) prevalence, however, it should be highlighted that diagnoses were only completed when pe was clinically suspected. material and methods: single-center prospective cohort study. between april 6(th) and april 17(th) 2020, consecutive confirmed cases of covid-19 pneumonia with d-dimer >1 μg/ml underwent computed tomography pulmonary angiography (ctpa) to investigate the presence and magnitude of pe. demographic and laboratory data, comorbidities, ctpa scores, administered treatments, and, clinical outcomes were analysed and compared between patients with and without pe. results: thirty consecutive patients (11 women) were included. pe was diagnosed in 15 patients (50%). in patients with pe, emboli were located mainly in segmental arteries (86%) and bilaterally (60%). patients with pe were significantly older (median age 67.0 (iqr 63.0–73.0) vs. 57.0 (iqr 48.0–69.0) years, p = .048) and did not differ in sex or risk factors for thromboembolic disease from the non-pe group. d-dimer, platelet count, and, c reactive protein values were significantly higher among pe patients. d-dimer values correlated with the radiologic magnitude of pe (p<0.001). conclusions: patients with covid-19 pneumonia and d-dimer values higher than 1 μg/ml presented a high prevalence of pe, regardless of clinical suspicion. we consider that these findings could contribute to improve the prognosis of patients with covid-19 pneumonia, by initiating anticoagulant therapy when a pe is found. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the world health organization (who) has declared coronavirus disease 2019 (covid19) as a pandemic and a major public health emergency [1] . the clinical spectrum of the disease caused by the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is wideranging, from asymptomatic infection to acute respiratory distress syndrome (ards) with high mortality [2] . risk factors for severe disease and death have been reported in retrospective cohorts [3] [4] [5] [6] . among them, older age, a higher sequential organ failure assessment (sofa) score, and a d-dimer higher than 1 μg/ml at admission were risk factors for death [3] . patients with severe covid-19 may also exhibit features of systemic hyper-inflammation or cytokine storm [7] . besides, coagulation disorders are frequently encountered among covid-19 patients, especially among those with severe disease [3, 5] . this has been further confirmed in larger studies showing that nearly 50% of patients with laboratory confirmed covid-19 infection had elevated d-dimer and fibrin degradation products, being the elevation more pronounced among severe cases [2, 8] . in addition, in a multicenter retrospective cohort study from china, increased d-dimer levels (>1μg/ml) were significantly associated with in-hospital death in the multivariable analysis (p = 0.003) [3] . on the other hand, it is worth noting that in a retrospective study, including 449 severe covid-19 patients in wuhan, low molecular weight heparin administration among patients with markedly elevated d-dimer was significantly associated with better 28-day overall survival [9] . first series of autopsies in covid-19 patients showed thrombosis in small pulmonary vessels [10] , and first clinical cases with pe in covid-19 patients has already been published [11] . interestingly, klok et al. [12] found, in a retrospective study in 184 intensive care unit (icu) patients with severe covid-19 pneumonia, a high prevalence of thrombotic complications and, by far, pulmonary embolism (pe) was the most frequent. similarly, some other retrospective studies found a high frequency of pe in covid-19 patients [12] [13] [14] [15] [16] . however, the real prevalence was not known, since computed tomography pulmonary angiography (ctpa) was only performed when thrombotic complications were clinically suspected. overall, all the above seems to indicate that d-dimer elevation may be common in patients with severe form of covid-19 infection. it is reasonable to conceive that covid-19 infected patients could be at high risk for venous thromboembolic events. although the published data are very limited, it seems rational to suggest that d-dimer evaluation could offer useful information for the search of pe in severe covid-19 infected patients. accordingly, we aimed to evaluate prospectively the prevalence of pe in patients admitted to hospital with covid-19 pneumonia and d-dimer >1μg/ml. as a secondary objective we evaluated clinical, radiological and biological variables that potentially could be related with this event. we performed a single-center prospective cohort study (hospital universitario son espases, palma de mallorca, spain) from april 6 to april 17, 2020. we selected consecutive adult patients with confirmed covid-19 pneumonia admitted to the hospital and with at least one d-dimer value higher than 1 μg/ml during hospitalization. the diagnosis of covid-19 pneumonia was done according to the case definition established by who interim guidance. 17 patients were excluded if they fulfilled at least one of the following exclusion criteria: (1) unwillingness or inability to participate in the study; (2) previous allergic reactions to iodinated contrast media; (3) previous anticoagulant treatment in the three months prior to admission; (4) ctpa performed before d-dimer rising above 1 μg/ml and/or (5) any other concurrent severe medical condition that would, in the investigator's judgment, contraindicate patient participation in the study. the clinical suspicion of pe was not and exclusion criterion. all patients were followed up until hospital discharge, death or until april 17th 2020, whichever came first. the strobe standards for reporting observational studies were followed. epidemiological, demographic, clinical and laboratory examinations were collected in all subjects by the time of admission. the recorded data included time from symptoms onset to hospital admission, time from symptoms onset to cpta, medical treatment during hospitalization, vte prophylaxis, respiratory support, clinical outcomes (acute respiratory failure, arrhythmia, intensive care unit admission or death) and permanent and temporary pe risk factors. temporary pe risk factors were considered if they were present within the 3 months before the admission. laboratory data included complete blood count (cell-dyn sapphire platform, abbott diagnostics), coagulation (including d-dimer), kidney and, liver function tests, collected at admission, on days 1, 3 and then at the discretion of the attending physician onwards. in addition, we analysed in each patient, baseline, peak and prior to ctpa values of the following biomarkers: d-dimer (acl top 700, instrumentation laboratory), c-reactive protein (crp), lactate dehydrogenase (ldh), erythrocyte sedimentation rate (esr), ferritin, lymphocytes and, neutrophil-to-lymphocyte ratio (nlr). the d-dimer-to-ferritin, d-dimer-to-ldh and d-dimerto-crp ratios were measured at baseline moment. besides, we measured high sensitive troponin i, interleukin-6 (il-6, by elisa, r&d systems), n-terminal pro hormone b-type natriuretic peptide (nt pro-bnp) (test 1 thl module, ali fax; architect platform, abbott diagnostics) and, fibrinogen values. the severity of pneumonia was defined according to the curb-65 score [17] . laboratory confirmation of sars-cov-2 infection was defined as a positive result of realtime reverse transcriptase-polymerase chain reaction (rt-pcr) assay of nasal and pharyngeal swabs [18] . the ctpa was requested per protocol only if the d-dimer determination was higher than 1ug/ml, regardless of symptoms. diagnosis of pe was performed by an expert radiologist based on direct visualization of the endoluminal thrombus in the pulmonary arteries. in order to provide a quantitative assessment of the magnitude of the embolism, the pulmonary artery obstruction index (paoi) was calculated according to the formula proposed by qanadli et al. [19] . the institutional ethic committee of the balearic islands approved the study (ib 4197/20 pi), and all subjects gave their written informed consent. only, those patients with a severe and critical clinical condition gave verbal consent with at least two witnessed. descriptive statistics included frequency analysis (percentages) for categorical variables and medians and interquartile ranges (iqrs) for continuous variables. comparisons were determined by mann-whitney u or kruskall-wallis test for continuous variables as appropriate and by the use of the χ2 test or fisher exact test for categorical variables. rho spearman correlation was used in order to assess significant relationship between severity pe markers (paoi) and inflammatory biomarkers. receiver-operating characteristic (roc) curve analysis was performed to identify thresholds, and the sensitivity and specificity of different cut-off points of the inflammatory markers for the discrimination of pe. differences are considered statistically significant at 2-tailed p<0.05. the statistical software used to analyse the data was spss v.26 (ibm). one hundred and twenty-four covid-19 confirmed patients were hospitalized during the study period (6 to 17 april). sixty-three of those patients (50.8%) presented a d-dimer > 1 μg/ ml at some time-point during the admission. thirty-one patients presented at least one exclusion criteria. the ctpa was performed on 32 patients, but 2 of them were excluded for technical reasons (fig 1) . finally, 30 patients were included in the analysis. the anthropometric and clinical characteristics are described in table 1 . the median age was 64.5 (iqr 55.8-71.3) years and 63.3% were males. the median time from symptoms onset to hospital admission was 8 (iqr 4.8-14.0) days. at the time of admission the median fractional inspired oxygen (fio2)/partial pressure of arterial blood oxygen (pao2) ratio was 264 (iqr 211-343) and the median curb-65 score was 1 (iqr 1-2). eleven patients (37.9%) were admitted in the intensive care unit (icu) at certain time-point of their clinical evolution. the prevalence of pe was 50% (15 patients). one patient presented one strong risk factor for pe (previous medical history of deep vein thrombosis (dvt)). but, even after excluding this patient from the analysis, the prevalence of pe in the remaining population was 48.3%, being in this case, a population without any strong risk factor for pe. hospitalization were not significantly different between cases with pe and subjects without pe (table 1) . nine patients with at least one minor vte risk factor were found to have pe, and seven patients with at least one minor risk factor did not show evidence of pe (p>0.05). only one patient presented one major risk factor (5%, p>0.05) as mentioned above. the proportion of patients with pe diagnosed during the first days (when laboratory data was obtained by protocol) was higher (66.7%) than the proportion of pe patients diagnosed during follow-up (when laboratory data was obtained at the discretion of the clinician) (42.9%). all pe patients started anticoagulation after being diagnosed of pe. fourteen out of 15 non-pe patients and 12 pe patients received thromboprophylaxis with enoxaparin (40 mg per day) by the time of admission. the remaining 3 pe subjects were diagnosed of pe at the time of admission, and they were started on anticoagulant therapy from the first day. pulmonary embolism in covid-19 pneumonia with high d-dimer values: a prospective study table 1 chest pain, n (%) 2 (6.7%) 1 (6.7%) 1 (6.7%) 1.000 the median and iqr of the covid-19 pneumonia ct severity score was 10 (5. [8] [9] [10] [11] [12] [13] . this score did not present differences between the two subpopulations (pe 10 (iqr 5.0-15.0); non-pe 10 (iqr 7.0-13.0); p = 0.75). regarding the pe radiological characteristics, the vascular allocation of emboli showed a predominantly peripheral and bilateral (60%) distribution, affecting mainly segmental and subsegmental arteries (53% and 7%, respectively). the overall paoi was 15% (iqr 8-25%). the baseline laboratory findings and inflammatory and pe biomarkers are shown in table 2 and s1 table. patients with covid-19 pneumonia showed lymphopenia (1. 2) vs 0.9 (0.2-4.5) mg/dl) were found in pe patients compared with non-pe patients (p value for all comparisons<0.05). on the other hand, it is worth noting that, despite the results were not statistically significant, there were also differences in the d-dimer peak values and d-dimer values prior to ctpa, being this biomarker higher in the pe group (3.4 (iqr 2.6-11.2) vs 2.3 (iqr 1.6-7.3) μg/ml, p = 0.07 and 2.6 (iqr 1.8-7.1) vs 1.6 (0.6-3.5) μg/ml, p = 0.07, respectively). moreover, the baseline d-dimer-to-ferritin, d-dimer-to-ldh and d-dimerto-crp ratios were significantly higher in pe patients compared with non-pe subjects (s1 fig) . the roc analysis carried out on the different peak inflammatory and pe biomarkers revealed that the highest area under the curve (auc) was reached by peak d-dimer, with an auc of 0.68 (95% ci 0.51-0.89; p = 0.065). different cut-off points of peak d-dimer were tested in order to analyze the sensitivity and specificity reached in each one. d-dimer value >2.5 μg/ml was predictive of pe with a sensitivity of 80% and a specificity of 51%. s2 table summarizes sensitivity, specificity, and positive and negative predictive values of different cutoff points of peak d-dimer (>1, >1.5, >2, and, >2.5 μg/ml). bivariate analysis showed significant correlations between the paoi and peak (rho = 0.592, p = 0.020), and prior to ctpa (rho = 0.795, p<0.0001) d-dimer values, crp prior to ctpa levels (rho = 0.518, p = 0.048), and baseline (rho = 0.574, p = 0.025), and, peak lymphocytes count (rho = 0.605, p = 0.017) (figs 2 and 3 ). pulmonary embolism in covid-19 pneumonia with high d-dimer values: a prospective study in this study, we have shown that patients with covid-19 pneumonia and d-dimer levels higher than 1 μg/ml had a remarkably high prevalence of pe. in addition, we found that patients with pe were older and had higher inflammatory (platelet counts, crp) and table 2 . initial inflammatory profile and pulmonary embolism biomarkers of patients admitted because of covid-29 pneumonia with and without pulmonary embolism. pulmonary embolism in covid-19 pneumonia with high d-dimer values: a prospective study procoagulant (d-dimer) markers that correlated with the extend of the thromboembolic episode compared to those patients without pe. we included patients with d-dimer values higher than 1 μg/ml, which have been associated with disease severity and in-hospital mortality in patients with covid-19 infection. this is a relatively common condition, ranging from 40-70% in the largest retrospective studies [3, 5] . pe prevalence in our patients was particularly high, since the incidence of pe in acutely ill hospitalized medical patients on prophylaxis with enoxaparin is very low (0-0.4%) [20] [21] [22] . our data do not allow us to distinguish whether pe started before or during admission. only one pulmonary embolism in covid-19 pneumonia with high d-dimer values: a prospective study of our patients, allocated in the non-pe group, was not on enoxaparin prophylaxis during hospitalization. we also found a trend to longer time from covid-19 infection symptoms onset to hospital admission among those patients with pe. moreover, pe patients were older than those ones without pe, but we did not find significant differences in the number of pe risk factors between both groups. it could be speculated that up-regulation of procoagulant activity in covid-19 infection may additively or synergistically increase the risk of pe, although this hypothesis needs to be proven in further studies. one might think, that the proportion of patients with pe could be higher at the time when blood tests (d-dimer) were performed at clinician discretion, compared to the first days when laboratory tests were performed according to protocol, resulting in a bias of selection. however, the proportion of patients with pe diagnosed during the first days was higher (66.7%) than the proportion of pe patients diagnosed during follow-up (42.9%). furthermore, the time from onset of symptoms to ctpa was not different in the pe group vs. the non-pe group (p = 0.802). the coexistence of pneumonia and pe has been known for years, and it is still today a diagnostic challenge [23] . data from the international cohort riete showed that patients with respiratory infections had higher risk of pe than patients with other types of infections [24] . some other studies showed that up to 90% of patients admitted to the hospital for pneumonia had high procoagulant markers, with d-dimer being one of the most common [25] . d-dimer is a very useful biomarker for excluding pe in the general population when clinical probability is low. however, it is usually not useful to diagnose the presence of pe since other inflammatory conditions may increase its value. moreover, this biomarker is not suitable to rule out or confirm pe in patients with pneumonia in whom d-dimer levels are also increased [26] . this same circumstance seems to occur in covid-19 disease. the mechanisms by which covid-19 could increase the risk of pe are unknown. pe is the result of virchow's classic risk triad, namely vascular endothelial impairment, stasis of blood flow, and/or increased coagulability. covid-19 could hypothetically affect all the 3 mechanistic pathways [27] . sars-cov-2 appears to use the angiotensin-converting enzyme receptor 2 to enter into lung cells. these proteins are also expressed in endothelial cells, and therefore this type of cells could be a target for the virus. furthermore, hypoxia, which is present in a significant proportion of seriously ill patients with covid-19, may lead to thrombosis by increasing blood viscosity and by increasing systemic inflammatory response [28] . lastly, patients with severe covid-19 pneumonia can trigger a state of sepsis that can induce the release of inflammatory cytokines such as il-6, il-8, tnf-α, among others that can promote the activation of a hypercoagulability disorder [28] . some patients even have a more prominent inflammatory response, which is associated to high d-dimer levels [2] [3] [4] [5] [6] [7] [8] . we found higher d-dimer levels, baseline d-dimer-to-ferritin, d-dimer-to-ldh and d-dimer-to-crp ratios in patients with pe compared to those without pe. this secondary analysis could represent that despite the clear link between inflammation and subsequent activation of coagulation in severe covid-19, patients with higher d-dimer values in relation to their inflammatory response could be at a higher risk of pe. nonetheless, further studies with larger sample size are needed to explore this hypothesis at a deeper level. besides, we also found a significantly higher platelet count in the group of covid-19 patients with pe compared with non-pe subjects, although median values were within normal limits. increase of both platelets and acute phase reactants has been detected due to the severe inflammatory state of these patients [5, 8, 29] . interestingly, yin et al. [30] found higher mortality rates, and platelet count in consecutive patients with covid-19 severe pneumonia compared with non-covid pneumonia patients. nevertheless, an alternative hypothesis is that the virus induce a direct alveolar inflammation which triggers hemostasis activation causing direct vascular thrombosis in the lungs [31] . recent data showing that consecutive covid-19 pneumonia patients and d-dimer >1 μg/ml did not have higher frequency of dvt to that reported in general internal medicine subjects could support this latter theory [32, 33] . there are only few studies that have evaluated pe in patients with covid-19 infection. a retrospective study evaluated 106 patients who underwent a ctpa during hospitalization and they confirmed pe in 32 subjects. furthermore, they found higher d-dimer levels in pe patients compared with those patients without pe [13] . similarly, klok et al. [12] found a cumulative incidence of arterial or venous thrombosis of 31% in patients with covid-19 pneumonia who had been admitted to icu. pe was diagnosed in 25 patients and it was the most common thrombotic disorder. unfortunately, no d-dimer values were reported. one more retrospective study detected venous thromboembolism (vte) in 35 of 74 (11 with pe) icu patients despite being on thrombosis prophylaxis [16] . interestingly, mean d-dimer values were associated with higher vte risk. a recent analysis from a french group showed that the rate of thromboembolic complications in 150 covid-19 patients with ards was much higher (11.7%) than what observed in a historical control group of non-covid-19 ards patients (2.1%) despite anticoagulation [15] . although these preliminary data suggested a high prevalence of pe in severe patients with covid-19 pneumonia, true prevalence was not known, since diagnostic tests were only performed if thrombotic complications were clinically suspected. on contrary, we diagnosed underlying pe in 50% of patients with covid-19 pneumonia and d-dimer levels higher than 1 μg/ml, regardless of clinical suspicion. pe is a challenging diagnosis that may be ignored because of non-specific clinical presentation. however, early diagnosis is essential, since well-timed treatment is highly effective and significantly influences clinical outcomes [34] . previous studies in non covid-19 patients have shown that paoi was correlated to pao2, right ventricle dysfunction, and to systolic blood pressure in pe [35] . moreover, it has been reported that patients with higher paoi, had an increased short-term risk of death [36] . it should acknowledge that 60% of our patients had principally segmental and subsegmental vessels and its clinical relevance it is unknown, but we found significant correlations between the paoi and d-dimer values, therefore, those patients with higher d-dimer values were found to have more clot burden in pulmonary arteries (fig 2) . as mentioned above, some retrospective studies found that a high d-dimer level was a mortality risk factor, and that anticoagulation treatment in severe covid-19 could improve the survival rate [3, 9] . besides, a recent autopsy study included 12 consecutive patients who died of covid-19. interestly, dvt was detected in 7 of them, and pe was the direct cause of death in 4 patients (one third), which was not detected while they were still alive [10] . taken together, all these preliminary available data could support the hypothesis that severe covid-19 infection, as a procoagulant disease, may have an increased risk of pe, which could represent one of the reasons for the increased morbidity and mortality. however, to clarify the influence of d-dimer levels, pe, and paoi as mortality risk factors in patients with covid-19 pneumonia, more prospective and rigorous investigations are still needed. the present study has several strengths. firstly, this is the first study to demonstrate prospectively the role of pe in the pathogenesis of covid-19 pneumonia in patients without a clear suspicion; and secondly, all the patients were exhaustively selected and characterized from the clinical, imaging and inflammatory point of view. yet, as in any study, there are some potential limitations that deserve comment. firstly, the sample size was small. however, the magnitude of the signal and its clinical implication precluded us to extend the observation period. secondly, although most patients had neither signs nor symptoms of dvt, lower limb venous statuses were not routinely and objectively investigated, therefore, asymptomatic events could not be ruled out. finally, the design of the present study does not permit to determine if pe was a direct consequence of covid-19 infection, systemic inflammation or hypoxia. moreover, it was not possible to assess whether pe happened previous or during hospitalization. in summary, our results demonstrate a pe prevalence of 50% in patients with covid-19 pneumonia and d-dimer values higher than 1 μg/ml. we consider that these findings could have clinical relevance in the management of patients with covid-19, since many of these patients would benefit from starting anticoagulant therapy, which could have a beneficial impact on the prognosis. these data should encourage the scientific community to perform further and larger studies to clarify the impact of an early pe diagnosis, and a prompt treatment initiation on the prognosis of patients with covid-19 pneumonia. supporting information s1 table. baseline laboratory data. values represent median (iqr). abbreviations: alt, alanine aminotransferase; pt, prothrombin time; pao2, partial pressure of arterial blood oxygen; fio2, fractional inspired oxygen; paco2, partial pressure of arterial blood carbon dioxide. world health organization. coronavirus disease (covid-19) outbreak clinical characteristics of coronavirus disease 2019 in china clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study. the lancet coronaviruses and the cardiovascular system: acute and long-term implications abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia clinical features of 85 fatal cases of covid-19 from wuhan: a retrospective observational study. american journal of respiratory and critical care medicine the role of cytokines including interleukin-6 in covid-19 induced pneumonia and macrophage activation syndrome-like disease prominent changes in blood coagulation of patients with sars-cov-2 infection. clinical chemistry and laboratory medicine anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy autopsy findings and venous thromboembolism in patients with covid-19. annals of internal medicine arterial and venous thromboembolic disease in a patient with covid-19: 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tratamiento de los pacientes con tromboembolia pulmonar randomized comparison of enoxaparin with unfractionated heparin for the prevention of venous thromboembolism in medical patients with heart failure or severe respiratory disease a comparison of enoxaparin with placebo for the prevention of venous thromboembolism in acutely ill medical patients risk factors for pulmonary embolism in patients preliminarily diagnosed with community-acquired pneumonia: a prospective cohort study infection as cause of immobility and occurrence of venous thromboembolism: analysis of 1635 medical cases from the riete registry prevalence and significance of coagulation abnormalities in community-acquired pneumonia diagnostic value of d dimer in pulmonary embolism and pneumonia. respiration; international review of thoracic diseases coagulation and sepsis. thrombosis research cardiovascular implications of fatal outcomes of patients with coronavirus disease 2019 (covid-19) hypercoagulability of covid-19 patients in intensive care unit. a report of thromboelastography findings and other parameters of hemostasis difference of coagulation features between severe pneumonia induced by sars-cov2 and non-sars-cov2 microvascular covid-19 lung vessels obstructive thromboinflammatory syndrome (microclots): an atypical acute respiratory distress syndrome working hypothesis. critical care and resuscitation: journal of the australasian academy of critical care medicine sars-2 coronavirus-associated hemostatic lung abnormality in covid-19: is it pulmonary thrombosis or pulmonary embolism? seminars in thrombosis and hemostasis incidence of asymptomatic deep vein thrombosis in patients with covid-19 pneumonia and elevated d-dimer levels esc guidelines for the diagnosis and management of acute pulmonary embolism developed in collaboration with the european respiratory society (ers) are clinical parameters and biomarkers predictive of severity of acute pulmonary emboli on ctpa right ventricular dysfunction and pulmonary obstruction index at helical ct: prediction of clinical outcome during 3-month follow-up in patients with acute pulmonary embolism we thank all the patients and their families for their willingness to participate in the study. we are also grateful to the many front-line health-care staff for their dedication in the face of this outbreak, despite the potential threat to their own lives and the lives of their families. key: cord-257217-f9sdt7ax authors: nunes, marta c.; kuschner, zachary; rabede, zelda; madimabe, richard; van niekerk, nadia; moloi, jackie; kuwanda, locadiah; rossen, john w.; klugman, keith p.; adrian, peter v.; madhi, shabir a. title: clinical epidemiology of bocavirus, rhinovirus, two polyomaviruses and four coronaviruses in hiv-infected and hiv-uninfected south african children date: 2014-02-03 journal: plos one doi: 10.1371/journal.pone.0086448 sha: doc_id: 257217 cord_uid: f9sdt7ax background: advances in molecular diagnostics have implicated newly-discovered respiratory viruses in the pathogenesis of pneumonia. we aimed to determine the prevalence and clinical characteristics of human bocavirus (hbov), human rhinovirus (hrv), polyomavirus-wu (wupyv) and –ki (kipyv) and human coronaviruses (cov)-oc43, -nl63, -hku1 and -229e among children hospitalized with lower respiratory tract infections (lrti). methods: multiplex real-time reverse-transcriptase polymerase chain reaction was undertaken on archived nasopharyngeal aspirates from hiv-infected and –uninfected children (<2 years age) hospitalized for lrti, who had been previously investigated for respiratory syncytial virus, human metapneumovirus, parainfluenza i–iii, adenovirus and influenza a/b. results: at least one of these viruses were identified in 274 (53.0%) of 517 and in 509 (54.0%) of 943 lrti-episodes in hiv-infected and -uninfected children, respectively. human rhinovirus was the most prevalent in hiv-infected (31.7%) and –uninfected children (32.0%), followed by cov-oc43 (12.2%) and hbov (9.5%) in hiv-infected; and by hbov (13.3%) and wupyv (11.9%) in hiv-uninfected children. polyomavirus-ki (8.9% vs. 4.8%; p = 0.002) and cov-oc43 (12.2% vs. 3.6%; p<0.001) were more prevalent in hiv-infected than –uninfected children. combined with previously-tested viruses, respiratory viruses were identified in 60.9% of hiv-infected and 78.3% of hiv-uninfected children. the newly tested viruses were detected at high frequency in association with other respiratory viruses, including previously-investigated viruses (22.8% in hiv-infected and 28.5% in hiv–uninfected children). conclusions: we established that combined with previously-investigated viruses, at least one respiratory virus was identified in the majority of hiv-infected and hiv-uninfected children hospitalized for lrti. the high frequency of viral co-infections illustrates the complexities in attributing causality to specific viruses in the aetiology of lrti and may indicate a synergetic role of viral co-infections in the pathogenesis of childhood lrti. pneumonia is a leading cause of mortality in children under 5 years age worldwide, including in hiv-infected children [1] [2] [3] . the aetiology of childhood pneumonia may include infection with bacteria and/or respiratory viruses. although respiratory viruses are more frequently identified than bacteria in children with pneumonia, this may be biased by lack of availability of sensitive and specific tests for diagnosing bacterial causes of pneumonia [4] . furthermore, respiratory viral infections may heighten the susceptibility to developing a super-imposed bacterial infection resulting in severe pneumonia [5, 6] . traditionally, respiratory viruses that have been associated with lower respiratory tract infections (lrti) include respiratory syncytial virus (rsv), parainfluenza viruses i-iii (piv i-iii), influenza viruses a/b and adenovirus. two human coronaviruses (cov), oc43 (cov-oc43) and 229e (cov-229e) were initially identified as causes of upper respiratory tract infections (urti) in the 1960s using classical culture methods [7, 8] . more recently, advances in molecular diagnostics have resulted in the discovery of other respiratory viruses which have also been associated with lrti. included among these are human metapneumovirus (hmpv) [9] , human bocavirus (hbov) [10] , human coronavirus nl63 (cov-nl63) [11] and hku1 (cov-hku1) [12] and wu and ki polyomaviruses (wupyv, kipyv) [13] [14] [15] . also, human rhinovirus (hrv), which was previously mainly associated with mild urti, has increasingly been implicated in having a role in the pathogenesis of lrti and asthma exacerbations [16, 17] . due to impaired humoral and cell-mediated immunity, hiv infection in children has been described as a risk factor for severe illness and mortality caused by respiratory-viral associated lrti, such as rsv, hmpv and influenza virus [18, 19] . there are, however, limited data on the role of other respiratory viruses, including the more recently-discovered viruses which occur as single or co-infecting pathogens in hiv-infected children hospitalized with lrti, and of these studies, most have small sample sizes [20] [21] [22] . the aim of this study was to identify the prevalence of hbov, hrv, wupyv, kipyv, cov-oc43, cov-nl63, cov-hku1 and cov-229e among hiv-infected and -uninfected children who were hospitalized for lrti using real-time reverse transcriptase-polymerase chain reaction (rt-pcr). the study-cohort had been previously investigated for rsv, influenza a/b, piv i-iii and adenovirus by immunofluorescence assay and hmpv by nested-pcr as described [5, 23] . the main 9-valent pneumococcal conjugated vaccine (pcv9) efficacy trial and subsequent retrospective analysis of study participants were approved by the human research ethics committee (medical) of the university of the witwatersrand. the main study did not have a clinical trials number since it was undertaken prior to registration of clinical trials having been made mandatory. signed informed consent was obtained from the parent/legal guardians of all the study participants as part of the main trial. the ethics committee did not require additional consent for this study. this was a retrospective study of children who participated in a phase iii trial in south africa which investigated the efficacy of a pcv9 as described [5, 24] . briefly, 39836 children recruited from march 1998 to october 2000 were randomized (1:1) to receive 3 doses of either pcv9 or placebo. vaccination occurred at a mean of 6.661.2 (6 standard deviation), 11.262.5, and 15.963.9 weeks of age [24] . hospital-based surveillance for all-cause hospitalization was undertaken at chris hani-baragwanath hospital, the only public hospital in the study community. hospitalized children had their signs and symptoms recorded and underwent hiv testing according to the study protocol [24] . nasopharyngeal aspirates (npa) were obtained for respiratory viral studies from children hospitalized with lrti [5] , and archived from january 2000 onward. in the present study only npa collected from 1 st february 2000 to 31 st january 2002 from children less than 2 years old were analysed. if a child had recurrent lrti hospitalizations, only npa collected at least 28 days apart were included in the analysis. blood cultures were performed with the use of an automated blood-culture system (bact-alert, organon teknika). total nucleic acids were extracted from archived npa using a nuclisens easymag platform (biomerieux), and eluted in a final volume of 60 ml of elution buffer [25] . rna was reverse transcribed with high capacity cdna reverse transcriptase (invitrogen, life technologies) and primed with oligo-dt primers (invitrogen, life technologies). real-time pcr was done in an abi 7500 rt-pcr system (applied biosystems, life technologies), reactions were performed in 20 ml using taqman universal pcr master mix (applied biosystems, life technologies) and the primers and probes listed in table s1 . five duplex rt-pcr reactions, targeting the 8 respiratory viruses, were developed. internal controls (the human genes: ribonucleoprotein and glyceraldehyde-3-phosphate dehydrogenase; or the spiked viruses: lambda and newcastle disease virus) were included to check the efficiency of the extraction step and to detect the presence of pcr inhibitors. positive controls were included in each experiment. the clinical definitions used in this study are the same as previously described [5] . briefly, lrti was defined as any episode with clinical diagnosis of pneumonia or bronchiolitis done by a study physician. children with lrti were categorized as having clinical pneumonia if they had evidence of alveolar consolidation on chest x-ray (cxr-ac) or if they fulfilled the clinical diagnosis of lrti without wheeze on chest auscultation but had rales and/ or bronchial breathing. children were categorized as having bronchiolitis in the presence of wheezing on chest auscultation and in the absence of documented cxr-ac or bronchial breathing on chest wall auscultation. a clinical diagnosis of who severe/very severe pneumonia was made if the child had a cough ,14 days in duration and lower chest wall in-drawing and/or any of the following signs and symptoms of severe pneumonia: feeding difficulties, convulsions, central cyanosis, or encephalopathy. the previously developed respiratory index of severity in children (risc) score was used to compare disease severity between single and multiple viral infections [26] . demographic, clinical and laboratory characteristics at admission were compared between hiv-infected and -uninfected children, chi-square or fisher's exact tests were used to compare the distribution of categorical variables and mean or median of continuous variables were compared by two-tailed student t-test or mann-whitney test, respectively. viral prevalence was compared between hiv groups and clinical and laboratory characteristics were compared between episodes with single and multiple viral detection using multivariate logistic regression models adjusted for study intervention arm (i.e. whether received pcv9 or placebo), year of sampling, detection of a virus previously-tested and age. the results are presented as adjusted odds ratios (aor) with 95% confidence intervals (95% ci) and p-values. a p-value, 0.05 was considered significant. analyses were performed using stata version 11.0 (statacorp, texas, usa). during the follow-up period included in this analysis, there were a total of 2147 hospitalizations for lrti, including 2094 (97.5%) in which npa had been collected. of the initial collected samples 69.7% were available for rt-pcr analysis. the same proportion of samples were available for rt-pcr from pcv9-recipients (69.2%) and placebo-recipients (70.2%; p = 0.621), from hivinfected (78.2% vs. 73.1%; p = 0.130) and hiv-uninfected children (65.5% vs. 68.6%; p = 0.218); children from whom npa were available for rt-pcr testing compared to those in whom samples were unavailable were older (median age: 10 vs. 8 months; p,0.001), were 1.3-fold less likely to have tested positive for one of the previously-tested respiratory viruses (33.3% vs. 42.3%; p,0.001), had a higher prevalence of cyanosis (11.4% vs. 8.1%; p = 0.025), higher evidence of cxr-ac (26.6% vs. 22.1%; p = 0.041), higher c-reactive protein (crp) levels (median: 15 vs. 12 mg/l; p = 0.003) and higher procalcitonin (pct) concentration (median: 0.26 vs. 0.15 ng/ml; p = 0.006); table s2 . there were no other demographic, clinical or laboratory differences observed between the lrti-episodes with npa available versus unavailable for rt-pcr. a total of 517 npa samples from hiv-infected children were analysed by rt-pcr, including 45.0% from pcv9-recipients and 55.0% from placebo-recipients. among hiv-uninfected children, 943 specimens were available for rt-pcr analysis, including 49.5% from pcv9-recipients and 50.5% from placeborecipients. on admission hiv-infected children compared to hiv-uninfected were younger (median age: 9 vs. 11 months; p, 0.001), more frequently presented with cyanosis (23.8% vs. 4.6%; p,0.001), had lower mean oxygen saturation (89.8% vs. 92.2%; p,0.001), had a higher median respiratory rate (54 vs. 48 breaths per minute; p,0.001), were more likely to present as pneumonia (88.8% vs. 49.6%; p,0.001) than bronchiolitis, had higher crp (18 vs. 14 mg/l; p = 0.007) and pct levels (0.47 vs. 0.17 ng/ml; p,0.001), had a longer hospital stay (4 vs. 1 median days; p, 0.001), had a higher case-fatality rate (17.8% vs. 0.95%; p,0.001) and more frequently had bacteraemia (7.8% vs. 2.7; p,0.001); table 2 . of the 1460 specimens analysed, all but 13 (1 from hivinfected and 12 from hiv-uninfected children) were previously tested for hmpv by nested-pcr [23] and 1458 were tested by an immunofluorescence assay for rsv, which if found negative, were further tested for influenza a/b, piv i-iii and adenovirus as described [5] . among hiv-infected children, the rt-pcr viral panel was positive in 274 (53.0%) lrti-episodes for at least one of the newly-tested viruses; table 3 . the prevalence of any of the newlytested respiratory viruses was similar between pcv9-and placeborecipients in hiv-infected children, except for wupyv (11.2% vs. 6.3%; p = 0.047, respectively) and hbov (12.5% vs. 7.0%; p = 0.034, respectively); table s3 . in hiv-infected children hrv was the most frequently detected virus (31.7%) followed by cov-oc43 (12.2%), hbov (9.5%), kipyv (8.9%), wupyv (8.5), cov-nl63 (1.7%) and cov-hku1 (1.4%); table 3 . the newlytested viruses were frequently identified as co-infecting viruses among hiv-infected children, including 49.4% of lrti-episodes associated with hrv; table 4 . the most common viral coinfections with hrv included kipyv (14.6%), wupyv (11.6%), cov-oc43 and hbov (11.0%, each); table 4 . among the 486 children on whom blood culture was done, bacteria were isolated on 38 (7.8%) occasions, 20 (52.6%) of which were associated with concomitant detection of one of the newly-tested viruses and 22 (57.9%) of any of the viruses. in hiv-uninfected children at least one newly-tested virus was detected in 509 (54.0%) lrti-episodes, with hrv also being the most common (32.0%), followed by hbov (13.3%), wupyv (11.9%), kipyv (4.8%), cov-oc43 (3.6%), cov-nl63 (2.6%), cov-hku1 (1.6%) and cov-229e (0.42%); table 3 . comparing hiv-uninfected pcv9 and placebo recipients, differences in the prevalence of newly-tested viruses were evident for kipyv (2.1% vs. 7.4%; p,0.001), cov-hku1 (0.21% vs. 2.9%; p = 0.001), cov-oc43 (2.1% vs. 5.0%; p = 0.017) and hrv (36.2% vs. 27.9%; p = 0.007); table s3 . of the 302 lrti-episodes in which hrv was identified, 51.3% had at least one other virus detected, including 13.6% with rsv, 12.3% with wupyv or hbov and 2.0% (n = 6) with both wupyv and hbov; table 5 . the prevalence of bacteraemia in hiv-uninfected children among those with blood culture results was 2.7% (n = 24/881) of which 12 (50.0%) occurred in the presence of infection with one of the newly-tested viruses and 15 (62.5%) in presence of any of the studied viruses. by multivariate analysis, adjusting for pcv-vaccination status, period of collection and age, single infections with a newly-tested virus were more frequent in hiv-infected (30.2%) than hivuninfected children (25.5%) (adjusted odds ratio [aor] 1.3; p = 0.033); table 3 . also, hiv-infected compared to hivuninfected children had a higher prevalence of kipyv (aor table 1 . number of specimens analysed in the current study and total specimens collected. when compared to lrti-episodes associated with the identification of only a single virus the detection of multiple viruses in hiv-infected children was significantly associated with a higher frequency of bronchial breathing (aor 2.11; p = 0.015) and in hiv-uninfected children with a higher prevalence of cyanosis (aor 2.50; p = 0.008) and wheezing (aor 1.55; p = 0.006); table 6 . no differences were detected in the severity of lrti-episodes with single and multiple viral infections using the risc score previously developed. in both hiv-infected and -uninfected children with bacteria isolated from blood, there were no significant differences in the clinical and laboratory characteristics between children in whom viruses were detected and children without any virus detected. the same was observed restricting the analysis to streptococcus pneumoniae isolation (data not shown). to our knowledge, this study provides the most in-depth analysis of the prevalence of hrv and some of the newlydiscovered respiratory viruses in hiv-infected children. our study identified hrv to be the most frequently detected virus both in hiv-infected and -uninfected children followed by cov-oc43 in hiv-infected and hbov in hiv-uninfected. most cases of hospitalizations associated with single infections overall were noted for hrv and rsv, the rate of co-infections was high in children infected with the other newly-discovered viruses. : previously-tested by immunofluorescence assay. 4 : previously-tested by nested pcr. 5 : including viruses previously-tested by immunofluorescence assay (rsv, influenza a, piv i-iii and adenovirus) and nested-pcr (hmpv). 6 : all multiple infections included at least one newly-tested virus except in 2 hiv-uninfected children. viral co-infections with at least one newly-tested virus in hivinfected children 118 (22.8%) and in hiv-uninfected children 269 (28.5%). 7 : not adjusted for detection of viruses previously-tested. 8 : : previously-tested by immunofluorescence assay. 5 : previously-tested previously by nested-pcr. 6 : including viruses previously-tested by immunofluorescence assay (influenza a, rsv, piv, adenovirus) and nested-pcr (hmpv). 7 : 487 patients had specimens available for culture. bacteria isolated included: hbov with escherichia coli (n = 2) and salmonella sp (n = 1); wupyv with streptococcus pneumoniae (n = 2) and escherichia coli (n = 1); kipyv with streptococcus pneumoniae (n = 3), escherichia coli (n = 3) and haemophilus parainfluenzae (n = 1); cov-nl63 with streptococcus pneumoniae (n = 2); cov-oc43 with escherichia coli (n = 3), salmonella sp (n = 1), streptococcus viridans (n = 1) and other streptococcus (n = 1); hrv with streptococcus pneumoniae (n = 6), escherichia coli (n = 3), streptococcus viridans (n = 1) and citrobacter freundii (n = 1); hmpv with salmonella sp (n = 2); piv with streptococcus viridans (n = 1); influenza a with streptococcus pneumoniae (n = 1) and escherichia coli (n = 1); adenovirus with pseudomonas aeruginosa (n = 1 table 5 . respiratory viruses co-infections in hiv-uninfected children hospitalized with lower respiratory tract infections. : previously-tested by immunofluorescence assay. 5 : previously-tested previously by nested-pcr. 6 : including previously-tested by immunofluorescence assay (influenza a, rsv, piv, adenovirus) and nested-pcr (hmpv). very few viral aetiology studies have been conducted in africa: in a mozambican study of virus-associated acute respiratory infections (ari) in infants with an estimated 3-5% hiv prevalence, the most frequently detected viruses were hrv (26%), influenza (15%) and adenovirus (14%) [27] . a recent study from south africa on children with ari requiring medical attention, 61% (n = 383) being hiv-infected or hiv-seropositive, also reported that hrv was the most frequently detected virus (33%) followed by rsv (30%), piv (8%) and hbov (6%) [21] . in the south african study hospitalized children with respiratory disease had higher hrv detection rates compared to healthy children (36% vs. 19%; p = 0.047) which may indicate a causality effect of hrv in disease [21] . in kenya in older children (5-17 years old), despite hrv being the most commonly detected virus (38%) in patients presenting with ari, its detection rate was similar among asymptomatic controls [28] . indeed two other studies in kenya found that with the exception of rsv, viral detection in the nasopharynx did not have a significant association with pneumonia in hospitalized children under 5 years [29] or under 12 years, [30] compared to children both without symptoms of urti or with respiratory symptoms but not meeting any criteria for pneumonia. it is uncertain as to what the impact of hiv infection on the duration of shedding of these viruses might be. an analysis of serial respiratory samples from otherwise healthy children detected shedding of cov-nl63 for up 21 days [31] , hrv up to 41 days and of hbov up to 44 days [32] . detection of cov-hku1 and cov-229e in respiratory specimens of transplanted children were also reported for at least 38 days and 11 weeks, respectively, possibly suggesting that immunocompromised children may have a prolonged duration of shedding of these viruses [33, 34] . such prolonged shedding in immunocompromised individuals such as hiv-infected children, may result in a greater frequency of coincidental identification of these viruses when investigated for respiratory illness, affect the seasonal occurrence of the viruses as well as potentially present a threat to greater nosocomial transmission of these viruses because of higher incidence of hospitalization of hiv-infected children. the casual relationship between viral dna detection, development of disease and mixed infections is very complex. in the case of hbov serological studies have shown that the presence of viral dna in respiratory samples was not proof of an acute primary infection and that prolonged viral shedding could be an explanation for viral detection at high rates in asymptomatic controls [35] . enrolment of healthy non-hospitalized children and quantification of viral loads may help to elucidate the problem of co-infections and is currently being under-taken in a multi-centre study on aetiology of pneumonia in children [36] . contradictory findings on whether infection with multiple viruses contribute to disease severity in hospitalized children have been reported, with some studies reporting an increased severity among children with viral co-infections [21, 37] and others finding decrease severity associated with co-infections [38] . in some studies no differences were observed [39] . we found that multiple viral infections were associated with bronchial breathing in hiv-infected children and with cyanosis and wheezing in hiv-uninfected children. bacterial co-infections can also increase the severity of viral diseases [40] . furthermore, in children with invasive pneumococcal disease, superimposed viral co-infections are common and may lead to higher mortality rates [41] . of the participants in our study from whom bacterial cultures performed, no differences in clinical and laboratory characteristics were observed between children with or without viral infections. our analysis, however, included pcv9 vaccinees who probably present with less severe lrti cases since it was shown before that pcv9 vaccination was associated with a decrease in lrti viral-associated hospitalizations [5, 23] . we detected a high prevalence of bacterial co-infections with the newly-tested viruses especially in hiv-infected children (8%), what may have contributed to the higher c-reactive protein and procalcitonin levels, the longer hospital stay and the higher mortality in hiv-infected children compared to hiv-uninfected. our high co-infection detection rates are similar to other molecular detection studies of paediatric respiratory samples, with coronaviruses (40-75%) [42, 43] , hbov (up to 78%) [13, 44, 45] and polyomaviruses (68-79%) [46] [47] [48] . our results may, however, have under-estimated the true prevalence of viral co-infections since the previously-studied viruses were tested by immunofluorescence [5] or conventional pcr [23] . although the sensitivity of the conventional methods is reported to be high, comparative studies have shown that rt-pcr is considerably more sensitive for detection of respiratory viruses [21, 49] . moreover the specimens tested by immunofluorescence were initially screened for rsv and only if negative were further tested for influenza a/ b, piv i-iii and adenovirus [5] . limitations of our study include that this was a post-hoc analysis and only 70% of specimens collected during the study period were available for further testing [24] . compared with the specimens available for rt-pcr the specimens unavailable were collected from younger children and were more likely to have tested positive for a previously-studied virus. this selection bias may have also contributed to an under-estimation of the number of co-infections. along the same line rsv-associated lrti are more common among younger children [21, 39] , for whom we had less available samples increasing our uncertainty on the prevalence of rsv coinfections with the newly-tested viruses. an equal proportion of pcv9-and placebo-recipients hospitalized with lrti were investigated in this study and our analyses were adjusted for the initial intervention arm. further detailed analyses on the effect of pcv9 on the incidence of hospitalization associated with the individual newly-tested viruses will be reported on in future. although the development of new diagnostic techniques allows more detailed investigation and has led to the discovery of a number of new putative respiratory pathogens, the role for some of these potential pathogens in respiratory illness remains speculative in the absence of fulfilling koch's postulates for causality. in the present study we demonstrate that at least one respiratory virus was identified in the majority of hiv-infected and hiv-uninfected children hospitalized for lrti. the difficulty in attributing disease causality to specific viruses due to the high frequency of viral co-infection suggests a possible synergy among different pathogens during childhood lrti. table s1 primer and probe sequences for real-time reverse transcriptase-polymerase chain reaction viral detection used in the study. 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nl63 associated with lower respiratory tract symptoms in early life epidemiology of multiple respiratory viruses in childcare attendees human respiratory coronavirus hku1 versus other coronavirus infections in italian hospitalised patients detection of four human coronaviruses in respiratory infections in children: a one-year study in colorado clinical assessment and improved diagnosis of bocavirus-induced wheezing in children rationale and expectations of the pneumonia etiology research for child health (perch) study the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children the association of newly identified respiratory viruses with lower respiratory tract infections in korean children prevention (2012) severe coinfection with seasonal influenza a (h3n2) virus and staphylococcus aureus-maryland viral coinfections in children with invasive pneumococcal disease clinical disease in children associated with newly described coronavirus subtypes coronavirus causes lower respiratory tract infections less frequently than rsv in hospitalized norwegian children human bocavirus commonly involved in multiple viral airway infections frequent and prolonged shedding of bocavirus in young children attending daycare presence of the newly discovered human polyomaviruses ki and wu in australian patients with acute respiratory tract infection wu polyomavirus in children with acute lower respiratory tract infections clinical and epidemiologic characterization of wu polyomavirus infection advances in the laboratory diagnosis of viral respiratory disease the authors thank the essential contribution of the members of the vaccine trialist group [24] for their involvement in the original study, all the trial participants, all rmpru staff involved in the study and biomérieux south africa for providing reagents. key: cord-267566-gdjl0qmu authors: kweon, oh joo; lim, yong kwan; kim, hye ryoun; kim, min-chul; choi, seong-ho; chung, jin-won; lee, mi-kyung title: antibody kinetics and serologic profiles of sars-cov-2 infection using two serologic assays date: 2020-10-22 journal: plos one doi: 10.1371/journal.pone.0240395 sha: doc_id: 267566 cord_uid: gdjl0qmu background: coronavirus disease 2019 (covid-19) is an emerging threat worldwide. this study aims to assess the serologic profiles and time kinetics of antibodies against severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in patients with covid-19 using two immunoassays. methods: a total of 97 samples serially collected from 17 patients with covid-19 and 137 negative control samples were analyzed for igm and igg against sars-cov-2 using the afias covid-19 ab (boditech med inc., chuncheon, republic of korea) and the edi(™) novel coronavirus covid-19 elisa kit (epitope diagnostics, inc., san diego, ca). results: with both assays, igm and igg rapidly increased after 7 days post symptom onset (pso). igm antibody levels reached a peak at 15–35 d pso and gradually decreased. igg levels gradually increased and remained at similar levels after 22–35 d. the diagnostic sensitivities of igm/igg for ≤14d pso were 21.4%/35.7~57.1% and increased to 41.2~52.9%/88.2~94.1% at >14 d pso with specificities of 98.5%/94.2% for afias covid-19 ab and 100.0%/96.4% for edi(™) novel coronavirus covid-19 elisa kit. among 137 negative controls, 12 samples (8.8%) showed positive or indeterminate results. conclusions: the antibody kinetics against sars-cov-2 are similar to common findings of acute viral infectious diseases. antibody testing is useful for ruling out sars-cov-2 infection after 14 d pso, detecting past infection, and epidemiologic surveys. coronavirus disease 2019 , the lung disease caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), is an emerging threat to individual and public health. on march 11, 2020 , the world health organization (who) declared covid-19 a pandemic [1] . as of june 26, 2020, the number of patients infected with sars-cov-2 has exceeded 9,296,202 globally, and more than 479,133 people have died of covid-19 in 216 countries [1] . timely and accurate diagnosis of covid-19 infection is important for appropriate treatment and limiting further spread of the virus [2] . the current gold standard for diagnosing sars-cov-2 infection is a molecular test using real-time reverse transcription-polymerase chain reaction (rrt-pcr) with respiratory tract specimens [2, 3] . however, the quality of pcr testing is critical in obtaining accurate results. diagnostic accuracy of pcr testing is highly dependent on many factors, including sample types, sample collection methods, sample handling, sample transport and storage, presence of interfering substances, manual errors, and variety of targeted genes for diagnosis and primer designs [2, 4] . obtaining nasopharyngeal or throat swabs is an uncomfortable procedure that can cause coughing and sneezing, which may generate aerosols as a potential hazard for healthcare workers [5] . serologic testing is emerging as an additional diagnostic tool for covid-19 [6] . although the current role of serologic tests remains confined to epidemiologic purposes [7] , accurate assessment of immunologic response may provide several benefits, including diagnosis of patients several days after symptom onset, diagnosis of suspected cases with repeatedly negative results by molecular test, identification of individuals who could serve as donors for plasma immunotherapy, determining the immune status of individuals, and monitoring immune responses to candidate covid-19 vaccine candidates [8] . understanding the kinetics of the immune response and antibody dynamics against sars-cov-2 is the cornerstone for serologic testing. this study reports the time kinetics of igm and igg antibodies in covid-19 and assesses the serologic profiles of patients with covid-19 using two commercially available serologic assays based on fluorescent immunoassay (fia) and enzyme-linked immunosorbent assay (elisa) from a single medical institution in seoul, republic of korea. the protocol was approved by the institutional review board (irb) of chung-ang university hospital (seoul, republic of korea; approval no. 2042-002-412), and informed consent was obtained from all study subjects. from february to june 2020, a total of 17 symptomatic patients with covid-19 who were positive for sars-cov-2 from upper respiratory tract specimens were enrolled in the study from chung-ang university hospital. of these, a total of 97 serial serum samples (median 5 samples per patient, minimum 4, and maximum 9) were collected, and their serological profiles were assessed. twelve (70.6%) patients were male, the median age was 62 yrs (minimum 20, and maximum 91), and all patients were korean except for one polish patient. symptoms used for patient selection included upper/lower respiratory symptoms, fatigue, headache, body ache, fever, chills, diarrhea, nausea, vomiting, loss of taste or smell, and/or chest pain [9] . as a negative control, 137 serum samples were obtained from 137 subjects with negative results on sars-cov-2 molecular tests with respiratory samples. the median age of the negative control group was 61 yrs, and 73 patients (53.3%) were male. among 137 patients, 6 (4.4%) patients had a history of cov other than sars-cov-2, such as human cov (hcov)-229e, hcov-nl63, or hcov-oc43. they were infected with other covs several months ago (median 4 months, minimum 2 months, and maximum 8 months). all serum samples used in this study were remnant samples after analysis for clinical care and were stored at -70˚c until analysis. detection of anti-sars-cov-2 antibody igm/igg was performed with two serologic assays, afias covid-19 ab (boditech med inc., chuncheon, republic of korea), an fia, and edi™ novel coronavirus covid-19 igg/igm elisa kit (epitope diagnostics, inc., san diego, ca). the afias covid-19 ab (hereafter, afias) assay is a sandwich fia for automatic qualitative/semiquantitative (through signal intensity, cut-off index (coi)) determination of igg and igm antibodies separately against sars-cov-2 using recombinant nucleocapsid protein as an antigen [10] . this assay is a point-of-care test and reports results within 20 minutes using whole blood, serum, or plasma. for testing, 100 ul of serum was dispensed into the sample well on the cartridge. after loading the cartridge into the afias-6 system (boditech med inc.), all procedures from loading the buffer into the cartridge to obtaining test results were conducted automatically. briefly, a fluorescence-labeled conjugate in dried detection buffer binds to the antibody in a sample to form antibody-antigen complexes. the complexes migrate onto the nitrocellulose membrane and are captured by immobilized-anti-human igg and anti-human igm on the test strip. the presence of more antibodies in the sample results in formation of more antigen-antibody complexes and leads to stronger intensity fluorescence signal on detector antigen, which is processed to determine the relative concentration of anti-sars-cov-2 antibodies in the sample [10] . results were interpreted according to the coi. the coi was determined by an equation based on the sample-to-positive control signal ratio. coi<0.9 was interpreted as negative for igm/igg, 0.9�coi<1.1 was indeterminate, and coi�1.1 was positive for igm/igg. edi™ novel coronavirus covid-19 igm elisa kit (hereafter, igm elisa) and edi™ novel coronavirus covid-19 igg elisa kit (hereafter, igg elisa) utilize the microplatebased enzyme immunoassay technique for qualitative/semiquantitative antibody detection [11, 12] . elisa with the serum samples was performed according to the manufacturer's instructions. in the assay procedures, "anti-human igm antibody-human covid-19 igm antibody-horseradish peroxidase (hrp) labeled covid-19 nucleoprotein antigen" and "covid-19 recombinant antigen-human anti-covid-19 igg antibody-hrp labeled antihuman igg tracer antibody" are formed in the microplate for igg and igm tests, respectively. these immunocomplexes were incubated with substrate solution, and the optical densities (ods) of each well were measured in a spectrophotometric microplate reader. with the od of negative controls, positive and negative cutoffs of each microplate were calculated according to package inserts. ods between the negative and positive cut-off were interpreted as indeterminate results. additionally, the sample-to-positive control od ratio (s/p ratio) was obtained; s/p ratio higher than 1.0 was interpreted as a positive result. dataset was entered into microsoft excel (microsoft, wa, usa) and analyzed using r version 3.6.1 (http://www.r-project.org/). seropositive rates were calculated according to the time frame, and graphical representation of time kinetic results with smoothing splines was carried out using r version 3.6.1. to determine the diagnostic accuracy of serologic assays for covid-19, diagnostic sensitivity and specificities were calculated subdivided by pso of �14 d and >14 d. to determine the concordance of serologic assays, cohen's κ value and total agreement percentage were calculated using 3-by-3 crosstab analysis. the κ values were interpreted according to the criteria proposed by landis & koch [13] as bad for values of 0.01-0.20, fair for 0.21-0.40, moderate for 0.41-0.60, strong for 0.61-0.80, and almost perfect for 0.81-1.00. fig 1 shows the kinetic results for patients with covid-19 at different pso periods assessed by two serologic assays according to coi and s/p ratio values. the two assays showed similar antibody time kinetics to each other. the smoothing splines of both assays showed that igm and igg antibody titer rapidly increased after 7 d pso. for igm, the smoothing splines of both assays reached a peak at 15-35 d pso and then gradually decreased. for igg, the smoothing splines gradually increased and remained at similar levels at 22-35 d pso. detailed results from two serologic assays are provided in s1 table. seropositive rates of patients with covid-19 according to pso days are listed in table 1 . igm seropositive rates before 7 d pso was 12.5% (1/8) from both assays. both assays showed the highest igm seropositive rate in the period of 15-21 d pso. igg seropositivity of samples obtained before 7 d pso was lower than 12.5%, and it reached 100% at 22-35 d pso. one patient showed negative results on 36-49 d pso from igg elisa, but positivity by afias. the diagnostic accuracy of serologic assays is listed in tables 2 and 3 for the 137 negative controls, 12 (8.8%) samples showed positive or indeterminate results from any of the two serologic assays. the details of their antibody profiles are listed in table 4 . for igm, 2 samples (1.5%) showed positive or indeterminate results by afias, and they were all negative by elisa. for igg, 9 (6.6%) samples showed positive or indeterminate results. among them, 1 sample (0.7%) was positive by both assays. among 6 negative controls with past medical history of cov other than sars-cov-2 infection, there was no seropositivity or cross-reactivity in igm or igg with any assay. the concordance between serologic assays was assessed. for igm, κ value was 0.40 (0.25-0.55, fair agreement) with a total agreement rate of 87.0% (82.2%-90.7%). for igg, the κ value was 0.75 (0.67-0.84, strong agreement) with a total agreement rate of 89.8% (85.4%-93.1%). a beta-coronavirus, sars-cov-2 is the seventh member of the coronaviridae family of viruses and is the causative agent of covid-19 in humans [14] . molecular testing of respiratory tract samples to detect sars-cov-2 rna remains the preferred diagnostic test for patients with covid-19. however, interest in using serologic assays to detect antibodies against sars-cov-2 is also increasing [8] . unlike molecular testing, serologic assays are indirect viral testing, and it is important to understand the kinetics of the immune response against sars-cov-2. currently, serologic assays targeting covid-19 are highly variable in their assay principles or formats, which include elisa, lateral flow immunoassays, chemiluminescent immunoassays (clia), and microsphere immunoassays [2, 8, [15] [16] [17] [18] [19] [20] . they also differ in the sars-cov-2 antigens used, such as recombinant nucleocapsid protein or spike protein [8] . furthermore, there is insufficient performance characteristics data for each assay and lack of commercially accessible reference tests. rigorous verification study with serially collected serum from covid-19 patients is required to ensure analytic performance and clinical accuracy. the observed antibody kinetics against sars-cov-2 were consistent with common findings of acute viral infectious diseases [21, 22] . time kinetics results showed that most signal intensities (coi for afias, s/p ratio for elisa) of igm and igg from both tests were below the cut-off at 1-7 d pso. after that, the signal values of igm and igg from both tests increased rapidly. the coi of igg continued to increase until 22-35 d pso and was maintained at similar levels thereafter. the coi of igm reached a peak at 15-35 d pso and then gradually decreased. the patterns of igg and igm titer kinetics were also similar to those of previous studies of covid-19 [2, 15, 20, [23] [24] [25] . however, the timing of igm seroconversion varies among published studies. among 8 patients at �7 d pso in the present study, only 1 patient (12.5%) showed positive results by both assays. this was discordant to several other studies in which a significant number of patients showed igm positivity in this period. guo [20] , and yang et al. concluded that values were lower than 15% using cyclic enhanced fluorescence assay [25] . those discordances may stem from differences in severity, antibody detecting methodology, and/or definition of the date of first symptom onset. until now, the number of studies investigating time of igm seroconversion is limited. further studies with various assay techniques and large subject numbers are needed to elucidate this discordance. the sensitivities of igm antibody detection were as low as 21.4% (same in both assays) in the samples collected �14 d pso and 41.2%~52.9% in samples >14 d pso. these findings indicated that in patients infected with sars-cov-2, igm seroconversion may not develop or might not be detected until the middle or late stages of infection. in other words, sars-cov-2 infection may be missed based on igm seropositivity; thus, igm tests must not be solely used in covid-19 diagnosis and should be used only as a supportive tool in addition to molecular tests. in addition, although the specificities of igm tests were satisfactory (above 98.5% in this study), igm titers in patients with sars-cov-2 infection showed a significant reduction after 35 d pso, their utility in detecting past infection or epidemiologic studies is limited. taken together, we did not observe any benefit of using igm testing for covid-19. the sensitivity of igg samples from �14 d pso was as low as 35.7%~57.1%, but it sharply increased for >14 d pso to 88.2%~94.1%. this means that almost all patients with covid-19 showed seroconversion after 14 d pso, and igg seronegative subjects in this period are considered less likely to be infected with sars-cov-2. in addition, both assays showed 94.2~96.4% of igg specificities and increased igg titers in covid-19 patients were maintained. thus igg serologic assays can be useful for ruling out sars-cov-2 infection after 14 d pso, detecting past infection, and epidemiologic surveys. nevertheless, caution is always needed for immunocompromised patients or those with mild symptoms after sars-cov-2 infection because their antibody titers may be lower than those of other patients [26, 27] . in the present study, 7.3% (10 of 137) of the negative control group showed igg (10 samples, 7.3%) seropositivity from one or both assays; 5 samples were from afias, 2 samples were from elisa, and 3 samples were from both assays. the possible causes of igg positivity in non-covid-19 patients include non-specific reaction by serum substances or true positivity. the former can be resolved by assessing samples with other serologic assays, using different antigens to detect antibodies, and the latter implies that subjects had past asymptomatic infections that are currently not vigorously investigated. xiang et al. reported that 5% (3/60) of the negative control group showed igg seropositivity [15] , similar to our results. one concern regarding sars-cov-2 serologic assay specificity is potential cross-reactivity with antibodies to common circulating covs, including hcov-229e, hcov-nl63, hcov-oc43, or hku1 [8] . among 6 non-covid 19 patients in this study who had past infection history of other hcovs, no patient showed seropositivity by both assays. however, our study included a small number of patients who had history of other common hcov. further investigation of crossreactivity to common hcov is needed using a large number of study subjects and various assays. in this study, we did not report the ppv and npv. the ppv and npv of a test depend heavily on the prevalence of what that test is intended to detect; however, we do not currently know the exact prevalence of sars-cov-2 antibody positive-individuals in the community, and the prevalence may change based on the duration [28] . a prevalence based on molecular tests may be helpful but may also be inaccurate or underestimated [29] ; this is because all suspected patients are not tested, and asymptomatic infection may also be present. assuming a prevalence of 0.1% for afias, the estimated ppvs of igm/igg for �14 d pso and >14 d pso were 1.4%/1.0% and 2.7%/1.6%, respectively, with estimated npvs of 99.9%/100% and 99.9%/ 100%, respectively. for elisa, the estimated ppvs of igm/igg for �14 d pso and >14 d pso were 100%/1.0% and 100%/2.4%, respectively, with estimated npvs of 99.9%/99.9% and 100%/100%, respectively. nevertheless, these are estimated values, and the exact ppv and npv must be calculated with an appropriate prevalence for each community. afias, the point-of-care fia test, has several advantages compared to other assays. the assay time for antibody analysis is relatively faster (less than 20 min.) than other assays, and the analysis procedure is simpler especially compared to elisa format tests [15, 24] . it also has relatively simple requirements. an automated fluorescent immunoassay system is utilized for analysis. the point-of-care bench-top analyzer is relatively compact compared to other automated assays. for these reasons, afias covid-19 ab could be useful, especially in resource-limited settings. this study has several limitations. first, the relatively small number of patients with covid-19 and their sample sizes could have affected the study outcomes. the small sample size of the negative control group could have also affected the specificities of the assays. second, antibody detection with another reliable validated method was not available for comparison studies. because the analytical performances of immunoassays vary, another serologic assay would be helpful to interpret disagreement results between afias and elisa. finally, because the number of days pso was highly dependent on patient memory, it is likely inaccurate to within a few days. in conclusion, we demonstrated that igm and igg antibodies against sars-cov-2 in patients with covid-19 were mainly detected after 14 d pso, igm levels reached a peak level at 15-35 d pso and gradually decreased, and igg levels reached a peak level and remained at similar levels after 22-35 d pso. testing for antibodies against sars-cov-2, especially igg, has the potential for ruling out sars-cov-2 infection after 14 d pso, detecting past infection, and epidemiologic surveys. supporting information s1 world health organization. coronavirus disease (covid-19) pandemic: world health organization analytical performances of a chemiluminescence immunoassay for sars-cov-2 igm/igg and antibody kinetics. clinical chemistry and laboratory medicine a rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019-ncov) infected pneumonia (standard version). military medical research potential preanalytical and analytical vulnerabilities in the laboratory diagnosis of coronavirus disease 2019 (covid-19). clinical chemistry and laboratory medicine temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study. the lancet infectious diseases assessment of immune response to sars-cov-2 with fully automated maglumi 2019-ncov igg and igm chemiluminescence immunoassays. clinical chemistry and laboratory medicine 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of infection serodiagnostics for severe acute respiratory syndrome-related coronavirus-2: a narrative review. annals of internal medicine detection of sars-cov-2 antibodies using commercial assays and seroconversion patterns in hospitalized patients. the journal of infection antibody response and viraemia during the course of severe acute respiratory syndrome (sars)-associated coronavirus infection innovative and new approaches to laboratory diagnosis of zika and dengue: a meeting report clinical infectious diseases: an official publication of the infectious diseases society of america viral kinetics and antibody responses in patients with covid-19 sars-cov-2 antibody characterization in emergency department, hospitalized and convalescent patients by two semi-quantitative immunoassays clinical and immunological assessment of asymptomatic sars-cov-2 infections inadequate antibody response to rabies vaccine in immunocompromised patient eua authorized serology test performance: u.s. food and drug administration covid-19: us cases are greatly underestimated, seroprevalence studies suggest we thank boditech med inc. for providing the afias covid-19 ab for this study. boditech med inc. provided technical support only and had no role in the study design, data collection, and interpretation. the authors have declared that no competing interests exist. conceptualization: jin-won chung, mi-kyung lee. key: cord-282668-bs634hti authors: niang, mbayame ndiaye; diop, ndeye sokhna; fall, amary; kiori, davy e.; sarr, fatoumata diene; sy, sara; goudiaby, déborah; barry, mamadou aliou; fall, malick; dia, ndongo title: respiratory viruses in patients with influenza-like illness in senegal: focus on human respiratory adenoviruses date: 2017-03-22 journal: plos one doi: 10.1371/journal.pone.0174287 sha: doc_id: 282668 cord_uid: bs634hti background: human adenoviruses (hadvs) are highly contagious pathogens that are associated with a wide spectrum of human illnesses involving the respiratory tract. in the present study, we investigate the epidemiologic and viral molecular features of hadvs circulating in senegal after 4 consecutive years of sentinel surveillance of influenza-like illness cases. methodology and results: from january 2012 to december 2015 swabs were collected from consenting ili outpatients. adenoviral detection is performed by rrt-pcr with the anyplex(™) ii rv16 detection kit (seegene) and molecular characterization was performed using a partial hexon gene sequence. 6381 samples were collected. more than half of patients (51.7%; 3297/6381) were children of ≤ 5 years. 1967 (30.8%) were positive for hadv with 1561 (79.4%) found in co-infection with at least one another respiratory virus. the most common co-detections were with influenza viruses (53.1%; 1045/1967), rhinoviruses (30%; 591/1967), enteroviruses (18.5%; 364/1967) and rsv (13.5%; 266/1967). children under 5 were the most infected group (62.2%; 1224/1967; p <0.05). we noted that hadv was detected throughout the year at a high level with detection peaks of different amplitudes without any clear seasonality. phylogenetic analysis revealed species hadv-c in majority, species hadv-b and one hadv4 genome type. the 9 hadv-b species like strains from senegal grouped with genome types hadv-7, hadv-55 and hadv-11 as shown by a phylogenetic branch with a high bootstrap value of (88%). conclusion: in conclusion, the results of the present study suggest strong year-round hadv activity in senegal, especially in children up to 5 years of age. molecular studies revealed that the dominant species in circulation in patients with ili appears to be hadv-c and hadv-b species. the circulation of though hadv-7 and hadv-55 genome types is of note as these serotypes are recognized causes of more severe and even fatal acute respiratory infections. from january 2012 to december 2015 swabs were collected from consenting ili outpatients. adenoviral detection is performed by rrt-pcr with the anyplex™ ii rv16 detection kit (seegene) and molecular characterization was performed using a partial hexon gene sequence. 6381 samples were collected. more than half of patients (51.7%; 3297/6381) were children of 5 years. 1967 (30.8%) were positive for hadv with 1561 (79.4%) found in co-infection with at least one another respiratory virus. the most common co-detections were with influenza viruses (53.1%; 1045/1967), rhinoviruses (30%; 591/1967), enteroviruses (18.5%; 364/1967) and rsv (13.5%; 266/1967). children under 5 were the most infected group (62.2%; 1224/1967; p <0.05). we noted that hadv was detected throughout the year at a high level with detection peaks of different amplitudes without any clear seasonality. phylogenetic analysis revealed species hadv-c in majority, species hadv-b and one hadv-4 genome type. the 9 hadv-b species like strains from senegal grouped with genome types hadv-7, hadv-55 and hadv-11 as shown by a phylogenetic branch with a high bootstrap value of (88%). in conclusion, the results of the present study suggest strong year-round hadv activity in senegal, especially in children up to 5 years of age. molecular studies revealed that the dominant species in circulation in patients with ili appears to be hadv-c and hadv-b a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 species. the circulation of though hadv-7 and hadv-55 genome types is of note as these serotypes are recognized causes of more severe and even fatal acute respiratory infections. human adenoviruses (hadvs) are highly contagious pathogens that are associated with a wide spectrum of human illnesses involving the respiratory, ocular, gastrointestinal, and genitourinary systems [1] . they belong to the family adenoviridae, genus mastadenovirus with seven species (a-g), including each various types [2] . ubiquitous in the environment, hadvs are non-enveloped, double stranded dna viruses that vary in size from 70 to 100 nm [3] . hadvs are recognized as a common cause of respiratory infection in persons of all ages. the illnesses range from influenza-like fever and discomfort to pneumonia and death [4] . indeed, hadvs infections are usely mild but some groups such as very young children, elderly, immunocompromised persons, or persons with underlying pulmonary or cardiac disease, might be at higher risk degree for severe disease [5, 6, 7, 8] . the most common hadvs species that cause respiratory tract infections in children are b (hadv-b3 and b7) and c (hadv-c1, c2, and c5). serotypes b3, b7, and b21 are the most frequent strains responsible for epidemics of acute febrile respiratory disease [9] . circulating hadvs can vary temporally and geographically with possibility of emergent genomic variants which can be associated with more severe illness [10, 11] . in the present study, we investigate the epidemiologic and viral molecular features of hadvs circulating in senegal after 4 consecutive years of sentinel surveillance of influenzalike illness cases. from january 2012 to december 2015 we collected specimens (nasal-pharyngeal and oral-pharyngeal swabs) and surveillance data for influenza and other viral respiratory pathogens from outpatients presenting with influenza-like-illness (ili) at different sentinel sites in senegal. once collected, swabs are placed in 2-ml cryovials with viral transport medium (universal transport medium; copan diagnostics inc., murrieta, ca), and transported at a controlled temperature of 2˚c-8˚c to the laboratory. an ili patient was defined as a person presenting with sudden onset of fever (>38˚c) or history of sudden onset of fever in the recent past ( 3 days) and either cough or sore throat and/or rhinorrhea in the absence of other diagnosis, according to the cdc case definition. each sample is accompanied by a case report form collecting demographic and clinical data. the questions included information on date of enrollment and symptom onset, sex, age, clinical symptoms, previous treatments, travelling history, vaccination status for influenza, and whether or not the patient was hospitalized. upon arrival at the laboratory, the specimens were processed immediately for virus diagnosis. aliquots of samples were also stored at −80˚c for additional analysis (isolation and/or molecular characterization). the data obtained daily were entered into an epi info database (centers for disease control and prevention, atlanta, ga) and analyzed using epi info. total viral nucleic acid (dna and rna) was extracted from 140 μl of each clinical specimen using the purelink™ viral rna/dna mini kit (invitrogen, carlsbad ca, usa) according to the manufacturer's recommendation. dna/rna are eluted with 60 μl nuclease-free water and stored at −80˚c until use. a two-step multiplex real-time rt-pcr was performed with a bio-rad cfx-96 thermocycler (bio-rad laboratories) and the anyplex™ ii rv16 detection kit (seegene) for a simultaneous testing of influenza viruses (flua and flub), human respiratory syncytial virus (rsva and rsvb), human adenoviruses (hadv), human metapneumovirus (hmpv), human coronavirus (229e, nl63, oc43), human parainfluenza virus (piv1, -2, -3 and -4), human rhinovirus (hrv), human enterovirus (hev) and human bocavirus (hbov), as previously described [12] . in consideration with low ct-values, 80 hadv positives samples (20 per year) were selected using a random number generator on ms excel for further molecular characterization using classical pcr and sequencing. viral dna was extracted as previously described and eluted with 50 μl water nuclease-free. dnas were stored at −20˚c until pcr reactions. for hadv molecular characterization the last 300 base pairs (bp) of the hexon gene were amplified with the following specific primers: adeno3 (5'-cctttggcgcatcccattct-3') and adeno4 (5'-tgggcacctatgacaagcgc-3') previously used by garcia et al, [13] . the phusion high-fidelity pcr master mix with hf buffer (new england biolabs, ipswich ma, usa) was used for amplifications. for each sample, pcr was carried out in a total reaction volume of 50 μl consisting of 15 μl h2o rnase free, 2.5 μl of each primer (diluted at 10μm), 25 μl of 2x phusion master mix and 5 μl of dna template. cycling conditions were as follows: denaturation step of 15 min at 95˚c, 40 pcr cycles including 30 s at 95˚c, 60 s at 55˚c, 60 s at 72˚c followed by an extension step of 10 min of 72˚c. five microliters of the pcr product was then mixed with 1 μl of 10x 5prime loading dye and loaded on to a 1% agarose gel along with an appropriated molecular weight markers (100 bp ladder, new england biolabs), and gels were stained with ethidium bromide (0.5 μg/ml) before visualization under uv. for positive samples (380 bp size band), amplicons were cut and purified using the gene-jet gel extraction kit (thermo scientific). purified products are then sent for sequencing to beckman coulter services. sequencing was performed in both directions with the same pcr primers (adeno3 and adeno4) on an abi prism bigdye terminator v3.1 ready reaction cycle sequencing kit (applied biosystems) on a 96-capillary abi prism 3730-xl (applied biosystems). data in fasta format were then sent to the laboratory for analysis. sequences successfully obtained were aligned with representative genbank sequences of previously published genotypes using the bioedit sequence alignment editor [14] . the search for sequence similarities were carried out using the basic local alignment search tool (blastn) from ncbi blast web portal. phylogenetic trees were performed in mega 6 software [15] using the neighbor-joining method, and the statistical significance of the tree topology tested by bootstrapping (1,000 replicates). the evolutionary distances were derived using the tamura-nei method. bootstrap replicates with values !70 are shown on the trees. statistical analysis. regarding hadv infection comparisons between age groups were performed using the fisher's exact test. p value < 0.05 was considered statistically significant and the 0-5 year age group was used as reference group. hadv mono-infections were also compared to hadv co-infections. the r.3.0.1 tool was used to perform the analyses. this study is a component of the 4s network syndromic surveillance [12] . the principles of the 4s network were approved by the ministry of health in its guidelines for influenza surveillance policy, finalized with the support of pasteur institute in dakar and the strengthening influenza sentinel surveillance in africa (sisa) project funded by the who. the protocol and oral consent were determined as routine surveillance activity, and therefore non-research by the senegalese national ethics committee and the steering committee for 4s network, an entity representing moh, ipd, who and clinicians in compliance with all applicable national regulations governing the protection of human subjects. data were collected in an objective of surveillance and are anonymous. the information provided to participants was an informal description of the study. respiratory specimens were collected, only after informed consent was granted, verbally, to local health care workers by the patients or parents in the case of minors. oral consent was documented in the patient form with two questions about received information and about oral consent. patients could refuse to participate, no specimen will be taken. for the surveillance activities, written consent is judged not necessary by the senegalese national ethics committee, which has also previously approved the work of the national influenza center. collections of non-sensitive data or an observation from normal care in which participants remain anonymous do not require ethics committee review. the patients included in this study were of all ages and consulted the sentinel sites due to influenza-like symptoms; the patients, or parents in the case of minors, accept the tests for respiratory viruses largely because they are free and safe. of 6381 specimens tested, 1967 (30.8%) were positive for hadv (table 2) . detection rates over the study period are almost similar in the first 3 years (2012, 2013 and 2014) while in 2015 there is a marked decrease in adenoviral infections. the mean age of infected patients was 8 years 7 months and median age was 3 years. regarding the viral detection per age group, most of hadv infected cases (62.2%; 1224/1967) were under 5 years patients, a statistically significant finding (p <0.05). however, the detection rates in the other groups including the elderly (above 50 years old) remain high. no significantly gender distribution of adenoviral infection was observed. the comparison of symptoms prevalence between ili patients with adenoviral infection and patients without adenoviral infection showed that cases of myalgia (p = 0.0014), cough (p = 0.0028), diarrhea (p < 0.001), rhinitis (p < 0.001) and headache (p = 0.01) are significantly higher in patients infected by adenoviruses ( table 3 ). the fig 1 shows the temporal distribution of hadv positivity rate per month in senegal from 2012 to 2015. we noted that hadv was detected throughout the year at a high level with detection peaks of different amplitude. the highest peak, with 62% of detection rate, was recorded on december 2013. hadv circulation pattern shows no seasonality even if results suggest a higher activity of these viruses during cold periods. it should be pointed out that the cold periods (between december and february) experience some instability in senegal with possibilities of shifting. for phylogenetic analysis, we were able to obtain the partial hexon gene sequence from 54 hadv-positive samples: 8 were from samples in 2012, 13 from 2013, 11 from 2014, 16 from 2015 and 6 from 2016. unfortunately, some samples showed no amplification or poor-quality sequences. the low sensitivity of conventional pcr compared with real time pcr on samples with low viral load, and certainly non-specific amplifications could be the cause of these failures. the nucleotide sequence alignment clustered the majority of senegalese isolates into hadv-c species (44/54). 9 isolates grouped with hadv-b species and the remaining isolate, from 2012, seems close to the hadv-4 genome type belonging to the hadv-e species. in all cases bootstrap values are high (more than 85%). within the hadv-c species, 16 senegalese isolates are grouped with the type hadv-6 (36.4%); 2 isolates with hadv-2 type (4.5%), 4 with hadv-5 type (9%), and 22 isolates formed a subcluster with hadv-1 and 57 types (fig 2) . the 9 hadv-b like species from senegal grouped with genome types hadv-7, hadv-55 and hadv-11 as shown by a phylogenetic branch with a high bootstrap value of (88%). we also noted that this dominance of species c and b is confirmed over the years. in this four-year retrospective study, we characterized hadv isolates derived from an ili surveillance program conducted as collaboration between pasteur institute of dakar and the senegalese ministry of health between 2012 and 2015. it is the first nationally molecular epidemiology investigation of hadvs and even in west africa. our study suggests that hadv are strongly associated with ili syndrome in senegal with an overall detection rate of 30.8% among 6381 patients. this rate seems very higher in comparison with available data. indeed, much lower rates are reported in similar other studies conducted in other countries. a study conducted in kenya [16] on refugees from different countries (somalia, sudan, ethiopia and kenya) yielded a detection rate of 21.7%, in gabon douki et al [17] detected hadv in 16.3% of outpatients with ili. these detection rates are still lower in other geographical regions: south korea with 10.1% or 0.6% [18, 19] , china with 2.7% or 6.3% [20, 21] , philippines with 0.9% [22] , malaysia less than 2% [23], usa with 5.7% or 2.8% reported [24, 25] , canada in the ontario provence with 0.9% [26] , peru with 6.2% [27] , venezuela with 1.6% [28] , england with 6.6% [29] . the analysis of these data tends to confirm a higher prevalence of adenoviruses in the respiratory sphere in african populations. this trend was largely confirmed when we investigated the importance of hadv in children with acute respiratory infections. indeed we observed that proportions in cameroon (27.3%) [30] and senegal (29.2%) (fall et al, on submission) were considerably higher than those found in other geographical areas: nascimento-carvalho et al., [31] in brazil with 3%, moe et al., [32] in norway (1.7%), wansaula et al., [33] in usa (1%) or lu et al., [34] in china. however, these discrepancies in hadv detection rates can be also due to differences in technical approaches, virus burden geographical differences, the number of patients tested, the periods during which samples were collected and even the duration of the study. it should be also noted that adenoviral detection does not necessarily prove disease causation as coincidental upper airway infection, asymptomatic viral carrier state [35] , or prolonged shedding [36] in a previous infection could explain adenoviral detection. regarding the group age, as expected, results showed that most patients with hadv infection were younger than 5 years (62.2%), a statistically significant finding. these results are in concordance with those of other studies which findings concluded that most children are infected by adenovirus at an early age [25, 37, [38] [39] [40] [41] . indeed, it is well established that by 5 years of age, 70% to 80% of children demonstrate antibodies to at least one serotype [42] . additionally more than 80% of diagnosed hadv infections occur in children < 4 years old (due to lack of humoral immunity) [43] . although most cases exhibit low to mild symptoms are and indistinguishable from other viral causes, acute respiratory infections caused by hadv can be severe [44] , or even fatal [7, 45] , and are associated with the highest risk of long term respiratory sequelae [46] . consistent with the report from many other studies, results here showed that 79.4% of hadv infected participants were co-infected with one or more other respiratory tract viruses. the most frequently co-detected viruses were influenza viruses (53.1%), rhinoviruses (30%), enteroviruses (18.5%) and rsv (13.5%). however, we noted no significant differences in clinical characteristics and laboratory findings between patients with single hadv infection and those co-infected. the same observation was reported in studies conducted in diverse geographical contexts [27, 41] . a previous study conducted in chilean children stated that the clinical severity in patients with single hadv infection and those with mixed infections was the same [47] . the overall finding is that the clinical value of such co-infections is not clear and still requires independent investigations in order to assess the association between co-infection and severe illness or symptoms. regarding the four years of surveillance, hadv circulation pattern shows no clear seasonality even if results suggest a higher activity of these viruses during cold periods. this lack of seasonality of hadv infection has been largely reported elsewhere [27, 48, 49] . however, seasonal peaks for hadv infection were noted in summer in some china areas [50] or in spring in northern china [51] , mexico [52] and taiwan [53] . in our study, the last 300 bp region of the hexon gene were used for molecular studies of the different hadv isolates. phylogenetic analysis showed that among the 54 sequenced strains hadv-c species were the most common hadv detected (81.5%) in patients with ili in senegal from 2012 to 2016. despite some divergences, the strains from senegal were close to types 1, 2, 5, 6 and 57. this hadv-c species predominance was reported in malaysia [23], in italy [54] , in many latina america countries [27, 52, 55] in contrast with studies done in the united states of america [56] , united kingdom [37] , korea [57] , in argentina [58] and china [59] , where hadv-b species were the most commonly isolated hadv. hadv-b species were the second most common in senegal with 9 strains, and only one type belonging to hadv-e species was sequenced. hadv-b species from senegal clustered with genome types hadv-7, hadv-7d, hadv-55 and hadv-11 (88% bootstrap value). hadv-7d serotype, firstly identified in 1980 in beijing [59] , is of particular concern as it was often associated with illnesses presenting with more severe and higher levels of morbidity than other respiratory hadv pathogens, and also may result in higher levels of fatalities [60] [61] [62] . the hadv-55 genome type, formerly known as hadv-11a, is a genotype resulting from recombination between hadv-11 and hadv-14 [63] . the serotype has recently reemerged as a highly virulent pathogen, causing severe [64] and sometimes fatal pneumonia among immunocompetent adults, particularly in asia [65] [66] [67] . so the circulation of such hadv genome types in senegal emphasizes the need to reinforce hadv surveillance, especially in hospitalized patients, by including hadv genome detection and genotyping in the documentation of severe respiratory infections. the single hadv-e species strain was typed as hadv-4, the unique human type in this species, which is more commonly associated with high rates of febrile respiratory illness in us military recruits [68] though associated with viral conjunctivitis outbreak in australia [69] for example. we observed some limitations in our study. first, considering the vast number of hadv positive samples, only a small number of hadv were typed. so the sequencing results do not reflect the full spectrum of hadv strains that may be circulating in ili patients in senegal, and even for selected samples it may have a bias toward samples with a high viral load. another limitation concerned the molecular methods used for typing hadvs in this study, a method which targeted a short hexon hypervariable region that has been shown to correlate closely with serotype. this method does not provide genomic detail and might miss recombination events located in other regions of the genome. therefore, full-genome sequencing would be more informative on senegalese strains, especially for hadv-b7 and hadv-b55 types. the results of this study should also be interpreted with caution especially for hadv ili causality (carriage in healthy or asymptomatic individuals). in conclusion, the results of the present study suggest strong year-round hadv activity in senegal, especially in children up to 5 years of age. molecular studies revealed that the dominant species in circulation in patients with ili appears to be hadv-c, hadv-b species. the circulation of though hadv-7d and hadv-55 genome types is of note as these serotypes are recognized causes of more severe and even fatal acute respiratory infections. so in the interest of global public health we strongly suggest molecular surveillance and genotyping of newly detected hadv strains in senegal and even by whole genome sequencing for some especial strains. our study offers also an important perspective on the burden of adenovirus-associated respiratory illness in senegal. such a perspective, especially among children, should include asymptomatic controls, sari cases, information on disease outcome, atypical clinical signs, duration of symptoms, and treatment. data regarding viral load, shedding, and other possible etiologies (e.g., bacterial and other viruses) would also enable a more thorough assessment of the viral effective disease (or symptom) causality. adenovirus infections in immunocompetent and immunocompromised patients family adenoviridae structural studies on adenoviruses worldwide epidemiology of human adenovirus infections adenovirus infections in transplant recipients clinical severity of respiratory adenoviral infection by serotypes in korean children over 17 consecutive 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11a infection in a singapore military training camp epidemiology of human adenovirus and molecular characterization of human adenovirus 55 in china fatal pneumonia cases caused by human adenovirus 55 in immunocompetent adults respiratory diseases among u.s. military personnel: countering emerging threats outbreak of adenovirus type 4 conjunctivitis in south australia this study would not have been possible without the excellent support from all the health-care workers of the 4s network who contributes, every day, to the surveillance network. we convey our special thanks to kathleen victoir from the international network of pasteur institutes for her unwavering support to the 4s network. we acknowledge the senegalese ministry of health for their help in implementing the 4s network. key: cord-272655-qeojdpez authors: remolina, yuly andrea; ulloa, maría mercedes; vargas, hernán; díaz, liliana; gómez, sandra liliana; saavedra, alfredo; sánchez, edgar; cortés, jorge alberto title: viral infection in adults with severe acute respiratory infection in colombia date: 2015-11-17 journal: plos one doi: 10.1371/journal.pone.0143152 sha: doc_id: 272655 cord_uid: qeojdpez objectives: to identify the viral aetiology in adult patients with severe acute respiratory infection (sari) admitted to sentinel surveillance institutions in bogotá in 2012. design: a cross-sectional study was conducted in which microarray molecular techniques for viral identification were used on nasopharyngeal samples of adult patients submitted to the surveillance system, and further descriptions of clinical features and relevant clinical outcomes, such as mortality, need for critical care, use of mechanical ventilation and hospital stay, were obtained. setting: respiratory infections requiring hospital admission in surveillance centres in bogotá, colombia. participants: ninety-one adult patients with acute respiratory infection (55% were female). measurements: viral identification, intensive care unit admission, hospital stay, and mortality. results: viral identification was achieved for 63 patients (69.2%). comorbidity was frequently identified and mainly involved chronic pulmonary disease or pregnancy. influenza, bocavirus and adenovirus were identified in 30.8%, 28.6% and 18.7% of the cases, respectively. admission to the intensive care unit occurred in 42.9% of the cases, while mechanical ventilation was required for 36.3%. the average hospital stay was 9.9 days, and mortality was 15.4%. antibiotics were empirically used in 90.1% of patients. conclusions: the prevalence of viral aetiology of sari in this study was high, with adverse clinical outcomes, intensive care requirements and high mortality. acute respiratory infection is one of the main causes of hospitalization and death worldwide, although identification of the aetiological agent is not achieved in a majority of cases. instead, the infections are treated empirically and often successfully with antimicrobial therapy. nonetheless, the roles of viruses in the aetiology of these infections are becoming clear, especially after the 2009 pandemic of the new influenza a subtype h1n1 [1] . the presence of a virus does not imply either a more benign clinical course or that systemic inflammatory responses or complications will be absent [2] . due to its implications for public health, the efforts in reinforcing and improving the epidemiological surveillance of respiratory infections have increased. under this initiative, countries have developed surveillance systems by following cases of influenza-like illness and severe acute respiratory infections (saris), which are clinically diagnosed among patients with fever, coughing or sore throat, difficulty breathing and the need for hospitalization [3] . the main aims of surveillance have been to provide information on circulating viruses and the susceptibility of influenza to available antivirals and also to promote and define vaccination needs in different populations. the true impact of viral infections in the aetiology of acute respiratory disease requiring hospitalization is unknown [4] . the aims of this study were to identify viral aetiologies in hospitalized adult patients with sari in bogotá in 2012 and to describe the characteristics and clinical outcomes among these patients. this study was performed in colombia's capital city of bogotá, which is located in the andes in south america, near the equator and 2,600 metres above sea level. a total of 7 tertiary care hospitals performing sentinel surveillance of sari during 2012 participated in the study. such hospitals forwarded all respiratory samples from patients with sari to the district health department. sari was defined as any respiratory infection with a possible viral and/or bacterial origin requiring inpatient management and a clinical presentation of fever of less than 14 days after onset and higher than 38°c, shortness of breath, cough, hypoxia and systemic compromise (systemic inflammatory response syndrome or organ failure), depending on symptom severity [5] . the samples were taken via nasopharyngeal aspiration or swab. the samples were sent, together with the required basic epidemiological data collection form, through the epidemiological surveillance system (sistema de vigilancia epidemiológica nacional-sivigila). samples were sent in a viral transport medium to the public health laboratory of the district health department, where they were stored between 4 and 8°c in refrigerators intended for this purpose. need for intensive care, the use of mechanical ventilation and the length of hospital stay. inclusion criteria consisted of patients over 18 years of age who provided a respiratory sample in 2012 at one of the hospitals performing sentinel surveillance. patients with incomplete medical records from the sampling institution were excluded. the following research and ethics committees of each participating institution approved the study: , with the exception of one institution that requested written informed consent to access medical records (fundación cardioinfantil, instituto de cardiología). written consent was obtained from 25 participants or their proxies (in case of death) from that institution (39 patients were not found, did not respond or refused to provide written consent). written informed consent, in the cases in which was requested and obtained, was kept at universidad nacional de colombia. a total of 288 nasopharyngeal aspiration or swab samples were taken during 2012 from adult patients of the 7 hospitals. the sample was randomly selected, with the only inclusion criteria being patients older than 18 years of age with sari reported to the surveillance system during that year. a sample size calculation found that 117 respiratory samples were needed for a prevalence of 25%, with the poorest accepted result of 20%, 90% power and a 95% confidence level. however, because 150 molecular tests could be processed, the sample was increased to 150 patients. subsequently, the medical records were reviewed, and after the approval and authorisation of the research and ethics committee of each institution, the information was gathered from each hospital. a microarray diagnostic assay using clart 1 pneumovir equipment by genomica (madrid, spain) was used to identify the viruses involved [6] . this assay detects and characterises viruses that most frequently cause respiratory symptoms in humans. the following viruses were analysed: respiratory syncytial virus (rsv) a and b, influenza a (h1n1, h3n2, 2009 a/h1n1 pdm), b and c virus, parainfluenza virus subtypes 1, 2, 3, 4a and 4b, metapneumovirus a and b, adenovirus, enterovirus, rhinovirus, coronavirus subtype 229e and bocavirus. this kit amplifies specific fragments of the viral genome via a reverse transcription polymerase chain reaction (pcr) or via a pcr with hybridisation detection using specific capture probes [7] . the following variables were collected from the patients' medical histories according to international definitions: age, gender and comorbidities such as chronic obstructive pulmonary disease (copd), diabetes mellitus and heart failure. the inclusion criteria took into account the systemic inflammatory response syndrome (sirs), which was defined as a heart rate over 90 beats per minute, a breathing rate of over 20 breaths per minute, leukocytosis of over 12,000 cells per millilitre or leukopenia of fewer than 4,000 cells per millilitre and fever; pneumonia among sari patients was diagnosed from radiological findings of consolidation or alveolar infiltrates. the confusion, urea, respiratory rate and blood pressure (curb)-65 scores for pneumonia were applied using the criteria of age greater than or equal to 65 years, impaired consciousness, blood urea nitrogen (bun) over 20 mg/dl, breathing rate above 30 breaths per minute and a systolic arterial pressure under 90 mmhg or a diastolic pressure under 60 mmhg [8] . additionally, severe pneumonia was considered among patients complying with the following major and minor criteria of the american thoracic society/infectious disease society of america (ats/idsa): shock, need for mechanical ventilation, thrombocytopenia (platelets under 100,000), an arterial oxygen partial pressure to fractional inspired oxygen ratio (pao 2 /fio 2 ) ratio, multilobar compromise, impaired consciousness, leukopenia (under 4,000) and bun over 20 mg/dl [9] . shock was also considered when the systolic arterial pressure was below 90 mmhg or when the diastolic arterial pressure was under 60 mmhg. moderate oxygenation impairment was defined by a pao 2 /fio 2 below 220 mmhg and over 160 mmhg, and severe oxygenation impairment was defined by an index less than or equal to 160 mmhg. institutions that identified viral antigens following institutional protocols performed and interpreted these tests in their hospitals. colonisation of the airway was defined as the presence of microorganisms in a gram test or in a culture of the airway. gram tests and sputum cultures or other respiratory samples were performed based on clinical decisions made by the doctor from the institution. a viral co-infection diagnosis was made when 2 or more different viruses were identified in the same sample. the chi-square test and fisher's exact test were used to compare categorical variables, and the comparison of continuous variables was performed with either student's t test or the mann-whitney u test on a case-by-case basis. the variable analysis was performed using stata (ver. 11.0), and p values less than 0.05 were considered significant. odds ratios (or) with corresponding 95% confidence intervals (ci) were used to analyse outcomes. in 2012 in bogotá, 288 samples from respiratory secretions were collected in the sentinel surveillance system from patients over 18 years of age. through randomised sampling, 150 cases of sari were selected from those respiratory samples (fig 1) . the medical histories of 99 patients with sari reported through the surveillance system were examined. these histories were obtained from 7 bogota hospitals in 2012 according to the following distribution: 45 (45.5%) were from the san rafael university hospital clinic, 23 (23%) were from fundación cardioinfantil, 10 (10%) were from suba hospital, 9 (9.1%) were from el tunal hospital, 6 (6%) were from santa clara hospital, 3 (3%) were from san ignacio university hospital, and 3 (3%) were from the occidente de kennedy hospital. of the 99 patients, 8 showed disease progression beyond 14 days and were dismissed from the final analysis (fig 1) . of the 91 patients included in the analysis, 50 (55%) were female; the patients' average age was 50.6 years (range, 18 to 95 years). table 1 shows the most frequent comorbidities identified. the average length of disease progression was 5.1 days, with a minimum duration of hours and a maximum duration of 14 days (table 2) . radiographical abnormalities were observed in 65 patients. the following radiological findings of chest images were described in the medical histories, in order of frequency: 32 patients had interstitial infiltrates (35.1%), 27 had alveolar infiltrates (29.6%), 26 had consolidation (28.6%), 21 had multilobar compromise (23.1%), and 19 had pleural effusion at admission (20.9%). pleural effusion was found in 15.3% of the patients with alveolar infiltrates and 13.2% of the patients with interstitial infiltrates. we among the sari patients, 82 (90.1%) were treated with antibiotics; patients received between 1 and 7 antibiotics, with an average of 2.3 antibiotics per patient. antibiotics were started a median of 1 day after admission (range, 0 to 21 days, 95% ci: 0-7 days). the beta-lactam group was the most frequently used. oseltamivir was used in 72.5% of cases (66 patients), with a minimum duration of 1 day, a maximum duration of 10 days and an average duration of 4.2 days. oseltamivir was started a median of 1 day after admission (range, 0 to 21 days, 95% ci: 0-9 days). at least one virus was identified in 63 patients (table 3) . influenza virus was the most common and was isolated in 28 patients (30.8%), of whom 21 (75%) had influenza a and 7 (25%) had influenza b (table 3) . of the parainfluenza virus subtypes identified, five cases were piv 3, and one case was piv 1. in the group with more than one viral infection ( influenza and bocavirus were identified throughout the year, though cases of influenza were commonly found in may, august and september, and cases of bocavirus were commonly found in june and november. influenza b was identified only in the second semester, while the two cases of influenza a/h2n3 were seen in may. adenovirus was seen only during the rainy season (april to may and august to november); most cases of metapneumovirus, parainfluenza and rsv were seen only in the first semester, specifically in april and may; the two cases of coronavirus were identified in may (fig 2) . patients with and without viral detection did not differ in terms of comorbidity, with the exception of the frequency of diabetes mellitus. diabetes was detected in 4.8% of patients with viral detection and 20.8% of patients without viral detection (fisher's exact test, p = 0.034). of the patients with pneumonia, a virus was identified in 28 patients (73.7%). severe pneumonia was diagnosed in 62.5% of patients without viral detection and 60.1% of patients with viral detection. no significant differences were observed between the groups with and without viral detection with regards to sirs, neutropenia during hospitalization or shock. of the 22 patients with sari and a history of copd, viruses were identified in 16 (72.7%), 5 of whom had viral co-infections. the viruses were distributed as follows: 6 cases of bocavirus, 4 of adenovirus, 3 of influenza a, 3 of parainfluenza, 3 of rsv, 2 of metapneumovirus, 1 of coronavirus and 1 of rhinovirus. of the 91 patients, 13 were active smokers and 9 (69.2%) had a viral infection, with bocavirus being the most frequently isolated (33.3%) virus. with regards to complications, pleural effusion developed in 12.5% of patients without viral identification and 19% of patients with viral identification; closed thoracotomy was required in 4.2% of patients without viral identification and 3.2% of patients with viral identification; inpatient infection was present in 8.3% and 3.2% of patients without and with viral identification, respectively; and underlying disease complications were found in 25% of patients without viral identification and 38.1% of patients with viral identification. these differences were not statistically significant. patients with chronic pneumopathy showed more complications in their underlying pathologies (77.3% vs. 20.3%, or: 13.5; ci 95%: 3.5-52.7). a total of 14 (15.4%) patients died. however, two cases included either an inadequate sample for pcr or no pcr amplification; thus, the outcome analysis was performed for 12 patients. mortality was reported for 4 cases (16.7%) in the group without viral identification and 8 cases (12.7%) in the group with viral identification (p = 0.23, or: 0.72; 95% ci: 0.17-3.68) ( table 4) . mechanical ventilation was required for 33 patients (36.3%); this intervention was invasive in 24 (26.4%) patients, non-invasive in 13 (14.3%) patients and invasive and non-invasive in 4 (3.6%) patients. this outcome occurred among 7 patients (29.2%) without viral identification and 23 patients (36.5%) with viral identification (p = 0.41, or: 1.4; 95% ci: 0.46-4.59). the average length of mechanical ventilation was 7 days, with a minimum of 1 day and a maximum of 20 days. thirty-nine (42.9%) patients were admitted to the intensive care unit (icu); 3 had unsuitable samples or non-amplified pcr results, 9 (37.5%) were from the group without an isolated virus, and 27 (42.9%) were from the group with an isolated virus (p = 0.21, or 1.25; 95% ci: 0.43-3.75). one case with viral identification was readmitted to the unit with an identification of influenza subtype ah1n1. the average length of icu stay was 8.4 days, with a range between 1 and 21 days. the group with an isolated virus had an average icu stay of 3.8 days, with a range between 1 and 21 days, and the group without an isolated virus had an average stay of 2.8 days, with a range between 1 and 13 days (p > 0.05). the average hospital stay in the sari group was 9.9 days, with a range between 1 and 45 days, with average stays of 10.6 and 9.8 days for patients with and without a virus, respectively (p = 0.6). among patients without a virus, the hospital stay varied between 1 and 45 days, while patients with a virus had hospital stays that varied between 1 and 28 days. of the 8 patients from the viral infection group who died, bocavirus was isolated in 5, influenza was isolated in 4 (all cases of influenza a), metapneumovirus was isolated in 3, and rsv was isolated in 1. of the patients with bocavirus isolation, 19.2% died. death occurred in 14.3% of those in whom influenza was identified, 50% of those in whom metapneumovirus was identified and 20% of those in whom rsv was identified. among patients with viral identification who died, 5 (62.5%) had viral infections of 2 or more viruses (p = 1.7, or: 2.4; 95% ci: 0.46-18.89), and fewer had only one virus detected. no statistically significant differences were observed in the outcomes of the remaining patients with mixed viral infection, which does not confirm higher morbidity in patients with more viruses isolated. the diagnosis of acute respiratory infections is common and apparently easy to perform; however, the determination of the infection's causal agent is more complex, as current diagnostic tools are limited and rarely available in primary health care centres or even in hospitals in much of the world [10] . the role of viruses and their prevalence is a matter of debate and discussion, and findings vary worldwide. the results from studies in new zealand, spain and more recently in the united kingdom report prevalence rates of 28% [11] , 18% [12] ,and 44% [13] , respectively. in our study, viruses were identified as the most frequent causal agents of sari requiring hospitalization in 2012, with most cases showing a high rate of viral co-infection, a high degree of morbidity, prolonged hospital stays and frequent needs for icu management and mechanical ventilation. the definition of sari used in this study is of great utility from an epidemiological surveillance perspective, which is known as syndrome surveillance or surveillance of syndromes [14] . nonetheless, the results reported here demonstrate its clinical relevance and potential utility in this field, as it appears to identify potentially severe patients and those with high complication rates; thus, the definition's routine use should be considered during the performance of clinical duties. furthermore, current challenges in the epidemiological surveillance of viral respiratory tract infections include the early and fast identification of aetiological agents, especially at the beginnings of outbreaks, and the optimal and timely management of a large number of samples [14] . our study suggests that molecular technology makes it possible to closely follow circulating viruses in these groups of patients. in contrast to the epidemiological definition used, the curb-65 index of pneumonia was not useful, as only 10 (26%) scored for general hospitalization and none scored for icus using these criteria. this finding contrasts with the high number of our patients who required icu care, which agrees with findings in the literature that applied this scale in cases of viral pneumonia during the influenza a subtype h1n1 pandemic of 2009; these results demonstrate its low value for detecting either severity or the need for icu admission in patients with viral pneumonia [15] . these findings represent a marked difference in severity stratification between influenza-related pneumonia and pneumonia caused by other aetiological agents, indicating the importance of clinical judgement in this scenario [16] . the application of these scales in non-influenza viral pneumonia has yet to be assessed. moreover, a recent multicentre study of adults with radiographically confirmed pneumonia has shown that viruses are now the most commonly identified pathogens. human rhinovirus and influenza virus are more frequently found than streptococcus pneumoniae [17] . together, viruses represent a quarter of patients and more than half of the pathogens identified. the mortality rate was relatively high for patients both with and without communityacquired pneumonia. a study performed in the united states reported a low mortality rate in patients hospitalized for viral infection; nonetheless, bacterial co-infection increased both the morbidity and the mortality [18] . this variable should be considered for our patients. countries with marked seasons report a correlation of influenza with high mortality due to respiratory infections, which is usually related to community-acquired pneumonia [19] . this seasonal pattern of acute respiratory infections, especially viral infections due to influenza, is dependent on temperature, humidity and host factors, such as serum vitamin d levels [20] . colombia is located in the tropics and thus lacks seasons; however, this study confirms that these infections, especially those caused by influenza and rsv infections, increase during the rainy season in countries at these latitudes [21] . this pattern shows the importance of epidemiological surveillance, especially during the seasons when viruses circulate, because it may increase control and prevention strategies such as timely vaccinations. techniques based on identifying nucleic acids, such as those used in this study, can obtain more rapid and precise results for diagnosing viral infections in order to provide appropriate and managed medical care [22] . there is limited information on the diagnostic utility of these new tests. a study by sultankulova et al. compared viral isolation of influenza a with dna [23] . microarray technology showed a higher sensitivity (99.5%) and similar specificity (98.5%) to real-time pcr. however, another study with the same microarray kit used in this study showed a high specificity (100%) and low sensitivity (52%) in the clinical scenario of atypical pneumonia [24] . a study in japan using near patient automated microarray technology showed not only a higher sensitivity and specificity compared to immunochromatographic antigen detection (the gold standard used was virus isolation) but also quicker results for children infected with influenza and rsv [25] . a recent multicentre study in the united states using real-time pcr technology was able to precisely and reproducibly detect all adenovirus infections in a group of children and adults with respiratory infections, which increased the possibility of establishing a clear diagnosis [26] . taken together, use of molecular technology for the diagnosis of viral infections can improve detection and identify the cases in which antibiotic use might be inadequate. the use of antibiotics in acute respiratory infections is indiscriminate and excessive, and according to worldwide literature, is employed in more than half of all respiratory infections [27] . this study determined that 90% of the cases are treated with antibiotics, although most of the infections were viral in origin. additional strategies, such as the measurement of procalcitonin, can identify patients who would not benefit from antibiotic therapy [28] . this study has several limitations. although it was multicentre in design, the information was gathered from only one city in colombia. one important limitation is that it is a retrospective study; thus, it was not possible to control viral and bacterial sampling, and there was limited access to relevant data, such as previous influenza or pneumococcal vaccination history or co-morbidity measurement. the sample size was smaller than expected, although the prevalence of viral infection was higher than expected. together, all of these limitations lead to difficulties in establishing significant comparisons among the groups and in appropriately assessing the impact of co-morbidity and bacterial co-infection in the outcomes, especially in relation to the role of pneumococcus [29] . other limitations include the difficulty of defining the actual roles of certain viruses, such as bocavirus and rhinovirus, in respiratory infection, which are also detected in asymptomatic patients according to descriptions in the literature [4] . the empirical use of antibiotics in most cases, without using additional tools to confirm bacterial infection, is also a limitation. viral pneumonia epidemic viral pneumonia and other emerging pathogens guia opertativa para la vigilancia centinela de eti e irag what is the role of respiratory viruses in community-acquired pneumonia?: what is the best therapy for influenza and other viral causes of community-acquired pneumonia? protocolo de vigilancia en salud publica-infección respiratoria aguda (ira) caracterización de virus causantes de infecciones respiratorioas humanas mediante identificación genomica para diagnostico in vitro respiratory viruses in children admitted to hospital intensive care units: evaluating the clart(r) pneumovir dna array defining community acquired pneumonia severity on presentation to hospital: an international derivation and validation study infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults the current epidemiology and clinical decisions surrounding acute respiratory infections incidence and characteristics of viral community-acquired pneumonia in adults viral community-acquired pneumonia in nonimmunocompromised adults adults hospitalised with acute respiratory illness rarely have detectable bacteria in the absence of copd or pneumonia; viral infection predominates in a large prospective uk sample surveillance for emerging respiratory viruses pandemic influenza (h1n1) 2009 pneumonia: curb-65 score for predicting severity and nasopharyngeal sampling for diagnosis are unreliable severity of influenza a 2009 (h1n1) pneumonia is underestimated by routine prediction rules. results from a prospective, population-based study community-acquired pneumonia requiring hospitalization among u.s. adults bacterial complications of respiratory tract viral illness: a comprehensive evaluation estimates of mortality attributable to influenza and rsv in the united states during 1997-2009 by influenza type or subtype, age, cause of death, and risk status roles of humidity and temperature in shaping influenza seasonality epidemiology and seasonality of respiratory tract virus infections in the tropics utilization of nucleic acid amplification assays for the detection of respiratory viruses comparative evaluation of effectiveness of iavchip dna microarray in influenza a diagnosis microorganisms in respiratory tract of patients diagnosed with atypical pneumonia: results of a research based on the use of reverse transcription polymerase chain reaction (rt-pcr) dna microarray method and enzyme-linked immunosorbent assay the clinical utility of a near patient care rapid microarray-based diagnostic test for influenza and respiratory syncytial virus infections in the pediatric setting multi-center evaluation of the adenovirus r-gene us assay for the detection of adenovirus in respiratory samples antibiotic prescribing in ambulatory care settings for adults with colds, upper respiratory tract infections, and bronchitis procalcitonin to initiate or discontinue antibiotics in acute respiratory tract infections a role for streptococcus pneumoniae in virus-associated pneumonia we acknowledge the following institutions for their participation in this study: suba hospital, hospital de occidente kennedy, santa clara hospital (gerson arias), el tunal hospital (diana marcela bejarano), san ignacio university hospital (sandra valderrama), fundación cardioinfantil (alvaro arango), and hospital san rafael (carlos restrepo). key: cord-254025-j1l0mder authors: de melo, andreia c.; thuler, luiz c. s.; da silva, jesse l.; de albuquerque, lucas z.; pecego, ana c.; rodrigues, luciana de o. r.; da conceição, magda s.; garrido, marianne m.; quintella mendes, gelcio l.; mendes pereira, ana cristina p.; soares, marcelo a.; viola, joão p. b. title: cancer inpatients with covid-19: a report from the brazilian national cancer institute date: 2020-10-26 journal: plos one doi: 10.1371/journal.pone.0241261 sha: doc_id: 254025 cord_uid: j1l0mder objective: this study aimed to describe the demographic and clinical characteristics of cancer inpatients with covid-19 exploring clinical outcomes. methods: a retrospective search in the electronic medical records of cancer inpatients admitted to the brazilian national cancer institute from april 30, 2020 to may 26, 2020 granted identification of 181 patients with covid-19 confirmed by rt-pcr. results: the mean age was 55.3 years (sd ± 21.1). comorbidities were present in 110 (60.8%) cases. the most prevalent solid tumors were breast (40 [22.1%]), gastrointestinal (24 [13.3%]), and gynecological (22 [12.2%]). among hematological malignancies, lymphoma (20 [11%]) and leukemia (10 [5.5%]) predominated. metastatic disease accounted for 90 (49.7%) cases. in total, 63 (34.8%) had recently received cytotoxic chemotherapy. the most common complications were respiratory failure (70 [38.7%]), septic shock (40 [22.1%]) and acute kidney injury (33 [18.2%]). a total of 60 (33.1%) patients died due to covid-19 complications. for solid tumors, the covid-19-specific mortality rate was 37.7% (52 out of 138 patients) and for hematological malignancies, 23.5% (8 out of 34). according to the univariate analysis covid-19-specific mortality was significantly associated with age over 75 years (p = .002), metastatic cancer (p <0.001), two or more sites of metastases (p < .001), the presence of lung (p < .001) or bone metastases (p = .001), non-curative treatment or best supportive care intent (p < .001), higher c-reactive protein levels (p = .002), admission due to covid-19 (p = .009), and antibiotics use (p = .02). after multivariate analysis, cases with admission due to symptoms of covid-19 (p = .027) and with two or more metastatic sites (p < .001) showed a higher risk of covid-19-specific death. conclusion: this is the first brazilian cohort of cancer patients with covid-19. the rates of complications and covid-19-specific death were significantly high. the mean age was 55.3 years (sd ± 21.1). comorbidities were present in 110 (60. 8% [18.2%] ). a total of 60 (33.1%) patients died due to covid-19 complications. for solid tumors, the covid-19-specific mortality rate was 37.7% (52 out of 138 patients) and for hematological malignancies, 23.5% (8 out of 34). according to the univariate analysis covid-19-specific mortality was significantly associated with age over 75 years (p = .002), metastatic cancer (p <0.001), two or more sites of metastases (p < .001), the presence of lung (p < .001) or bone metastases (p = .001), non-curative treatment or a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the novel coronavirus, named severe acute respiratory syndrome coronavirus-2 (sars-cov-2) that causes the coronavirus disease 2019 [1], was first detected in wuhan, the provincial capital of hubei, china, in december 2019. sars-cov-2 has rapidly spread to many other countries worldwide becoming an unprecedented astounding and devastating pandemic in a short period of time. following an exponential upward trend, the increasing number of cases and death toll remain to concern the scientific community around the globe. currently, more than 13.8 million cases are confirmed worldwide, with more than 593,000 deaths [2] . the first case of covid-19 in brazil was detected on february 26, 2020. standing out worldwide for having one of the steepest epidemiological curves, the country has 2.04 million confirmed cases and more than 77,800 deaths so far [3] . as the most populous areas, the states of são paulo and rio de janeiro have predictably concentrated the highest numbers of cases and deaths. thus far, brazil has been considered the new epicenter of the global pandemic [4] . in general, the vast majority of covid-19 patients develop mild symptoms or remain asymptomatic over the course of the disease. an intermediate group of patients have moderate symptoms requiring hospitalization and some noninvasive intervention. another group of patients have a more severe course of the disease with desaturation, dyspnea, septic shock, and/or multiple organ dysfunction leading to life-threatening consequences or death [5] . patients with cancer are more likely to have severe complications and even death when affected by covid-19 [6] [7] [8] , mainly due to the effects of the immunosuppressive anticancer treatments, frequent use of corticosteroids, advanced age, comorbidities and pulmonary involvement (primary tumors or secondary lung metastases). particularly in low-and middleincome countries, covid-19 has brought a heavy burdening to the public health systems and induced new planning and adjustments in the clinical approach to cancer patients [9] . based on a few series previously published around the world, data evaluating the impact of covid-19 outbreak in management and survival of patients with cancer are still very scarce, incomplete, with heterogeneous outcomes and descriptions [10] [11] [12] [13] [14] [15] . brazilian data in this specific field are still unknown due to the lack of publications. the aim of this report was to describe the demographics, clinical characteristics and laboratory abnormalities of cancer inpatients with covid-19 admitted to the hospital ward of the brazilian national cancer institute (inca), exploring factors associated with death. this retrospective cohort was performed through a search on electronic medical records and compiled data of cancer inpatients admitted to inca with laboratory-confirmed sars-cov-2 infection between april 30, 2020 and may 26, 2020. the hospital admission occurred for various medical reasons, including covid-19 symptoms or any other clinical condition (for those cases with onset of symptoms throughout hospitalization or cancer inpatients who had contact to other covid-19 cases). outpatients tested positive for sars-cov-2 infection and patients with only non-invasive cancer (or pre-malignant conditions) were not the object of this study. covid-19 was diagnosed on the basis of the who interim guidance [16] , in which confirmation was defined as a positive result on real-time reverse transcriptase polymerase chain reaction (rt-pcr) assay of nasal-and oropharyngeal swab specimens using the u.s. centers for disease control and prevention (cdc) reagents and protocol [17] . specimens were collected right after the hospital admission from those patients with covid-19 symptoms and immediately after clinical suspicion from those admitted to hospital for diverse reasons unrelated to covid-19. the study was approved by the national commission of ethics in research (conep) and conducted in accordance with the good clinical practice guidelines. written informed consent was waived due to the retrospective design and the emergency feature of the research. only anonymized data were analyzed. the demographic and clinical characteristics, including tumor site, histological subtype, staging, site of metastases, cancer treatment within the last 60 days, the presence of comorbidities, covid-19-related clinical signs and symptoms, and laboratory tests at diagnosis and throughout hospitalization were obtained from the electronic medical records. covid-19-specific clinical treatments were also collected. the variables analyzed in order to feature disease severity were admission to the intensive care unit (icu), mechanical ventilation, renal failure, hemodialysis, septic shock, and death. patients transferred out from inca to another hospital were censored on the date of transfer. patients who had not been discharged from hospital were censored in the date of the last follow-up on may 31, 2020. the statistical software package spss, version 21.0 (são paulo, brazil) was used for the analyses performed by accessing the database between june 1st and 6th, 2020. all continuous variables were evaluated by the kolmogorov-smirnov test of normality. categorical variables were shown in percentages or absolute values. the study endpoint was covid-19-related mortality. time of follow-up was calculated from the date of swab collection to hospital discharge, death, or censorship of patients who were transferred or still hospitalized at the end of the study. risk factors for death were assessed using logistic regression. crude and adjusted odds ratios (or) were calculated. variables with a p-value < .20 at the univariate analysis were included in the multivariate model by stepwise forward selection with the entry order based on their level of significance. all p-values < .05 were considered statistically significant. a total of 181 patients had the diagnosis of covid-19 confirmed at inca and were considered eligible for this study. the median follow-up for the general population was 5 days (interquartile range, iqr 2-10.3). the demographic and clinical characteristics are described in detail in table 1 . the mean age was 55.3 years (standard deviation, sd ±21.1) and 92 (50.8%) patients aged 60 or older. female gender was more prevalent (110 [60.8%]) and 40 patients (22.1%) were former or , and 21 (11.6%) patients had three or more comorbidities. long-term use of corticosteroids was seen in 21 (11.6%) cases. ) and leukemia (10 [5.5%]) predominated. a more detailed list of the frequency of cancers by site is detailed in the supporting information section (s1 table) . patients with metastatic disease accounted for almost half of the cases (90 [49.7%]) and 32 (17.7%) patients had lung metastases. more than a third of the cases (63 [34.8%]) had recently undergone cytotoxic chemotherapy within the last 60 days before the covid-19 diagnosis, 32 (17.7%) were in best supportive care and 27 (14.9%), in post-treatment clinical surveillance. only nine patients (5%) were receiving targeted therapy or immunotherapy with checkpoint inhibitors as the current treatment line (table 1) . information regarding the conditions of hospital admission and clinical evolution of patients are summarized in table 2 -1488) , the mean c-reactive protein levels were 19.4 mg/ dl (sd ±15.0) and median d-dimer levels were 2099 ng/ml (iqr 909-5948). under the clinical diagnosis of a severe acute respiratory syndrome, where influenza infection was considered one of the causative hypotheses and before the final rt-pcr result was confirmed as sars-cov-2 infection, oseltamivir was administered to 41 (22.7%) cases. during the course of covid-19, more than half of the patients (98 [54.1%]) received corticosteroids, 148 (81.8%) were treated with antibiotics (including those against bacterial coinfections), and therapeutic anticoagulation was prescribed to 39 (21.5%) patients. eight (4.4%) patients received chloroquine and 36 (19.9%), ivermectin. at the time of analysis, a total of 60 patients (33.1%) had died due to covid-19. for solid tumors, the covid-19-specific mortality rate was 37.7% (52/138) and for hematological malignancies (leukemia, lymphoma and multiple myeloma) was 23.5% (8/34). four out of seven (57.1%) patients with lung cancer died from covid-19, as well 52.5% (21/40) of breast cancer patients (fig 2) . as shown in table 3 , mortality related to covid-19 was significantly associated to older age (p < .001 for patients between 60 to 74 years and p = .002 for patients aged 75 years or older), metastatic cancer (p < .001), two or more sites of metastases (p < .001), the presence of lung (p < .001) or bone metastases (p = .001), non-curative treatment or best supportive care intent (p < .001), higher c-reactive protein levels (p = .002), admission due to covid-19 (p = .009), and antibiotics use (p = .02). isolated or combined comorbidities and elevated d-dimer levels did not demonstrate increased risk of dying from covid-19. different modalities of cancer therapy, including systemic agents (chemotherapy, hormone therapy, targeted therapy, immunotherapy), surgical procedures or radiotherapy within 60 days before covid-19 were not associated with mortality. also, specific therapies during the covid-19 course, such as oseltamivir, therapeutic anticoagulation, corticosteroids, ivermectin and chloroquine did not influence the risk of death (s2 table) . according to the multivariate analysis patients admitted due to covid-19 symptoms (or 2.3, 95% ci-1.1 to 4.9, p = .027) and with two (or 10.0, 95% ci-3.6 to 28.3, p < .001) or more metastatic sites (or 14.8, 95% ci-4.1 to 53.2, p < .001) showed a significantly higher risk of covid-19-specific death. the findings of this cohort highlight, in detail, several significant aspects of the covid-19 course in patients already diagnosed with cancer. although the emergency period for case selection was considerably short, due to the large number of cancer patients admitted to inca with covid-19, 181 patients were successfully included for analysis. women had greater representation, more than half of the patients aged 60 years or older and almost a quarter of the patients had also reported smoking. patients with two or more comorbidities accounted for more than a quarter of the study population as well, in which hypertension and diabetes prevailed. almost half of the patients (83 [45.9%]) were hospitalized due to conditions unrelated to the sars-cov-2 infection which can be explained by patients asymptomatic for covid-19 having been admitted to hospital for other cancer-related clinical complications. an intra-hospital transmission may also be considered, raising an important issue about the remarkable risk of infection to patients admitted for elective procedures. as for the symptoms present at diagnosis, similarly to data of other series [7, 8, 10, 12, 14] , in the current cohort, dyspnea, cough and fever were all highly frequent. the odds of some covid-related complications were quite similar to the findings reported by kuderer et al.(14) in an international prospective series in which more than 928 patients were analyzed. the rate of icu admission of 14% (versus 17.7% in the current study) and the mechanical ventilation requirement rate of 12% (versus 19.3% in the current study) were also alike in proportional terms. zhang et al. [6] also showed paralleled data with respect to other variables such as the demand of supplemental oxygen in 78.6% of cases (versus 71.8% in the current study). early data from non-cancer patients suggested that 14-19% of cases progress with severe complications, such as septic shock, respiratory failure, acute kidney injury and multiple organ failure [5, 15, 18] . in the present study, these rates were much higher, ranging from 18.2 to 38.7%, highlighting the increased likelihood of severe complications in cancer patients. conversely, cerebrovascular and cardiovascular events were less frequent. in total, 69 (38.1%) of 181 patients died. herein, covid-19-related mortality was considered as the endpoint. consequently, nine patients who clearly have recovered from covid-19, and died due to other cancer-related reasons, were excluded from this mortality analysis. therefore, the overall covid-19-related mortality rate reached almost one third of the cases (60 [33.1%]), which was higher than that reported by other series with cancer patients [7, 8, 10, 12, 14] , and far exceeding the mortality reported for non-cancer patients [5] . it is important to point out that some patients had the definition of non-invasive support after or even before the diagnosis of covid-19 due to the severity of their advanced malignancies, which may have overestimated the mortality rate. in addition to this, a noticeable number of the analyzed patients (103 [56.9%]) were under non-curative treatment or best supportive care at the time, reflecting the advanced stage of their respective diseases. unlike in the multicenter studies recently published by mehta et al. [10] and dai et al. [15] , the mortality rate was higher in patients with solid tumors than in patients with hematological malignancies, possibly due to the small number of hematological patients in this cohort. but likewise, lung cancer, breast cancer and gastrointestinal cancer stood for the highest numbers of cases progressing to death. in line with the results published by kuderer et al. [14] , the characteristics associated with clinical fragility, such as being elderly, having advanced stage, a greater number of metastases, pulmonary metastases, non-curative treatment or supportive treatment and symptomatic patients at hospital admission were significantly associated with a higher risk of death. in this same context, the type of anti-cancer treatment received by patients within the previous 60 days did not influence survival outcome. except for the association of c-reactive protein with mortality (or 1.04; p = 0.002), none of the laboratory markers were likely to predict a higher risk of mortality. other laboratory exams, including inflammatory markers such as lactate, ferritin, fibrinogen, and lactate dehydrogenase were not regularly collected in this cohort, preventing related analyses. a prospective study in order to evaluate the immune response markers in our cohort of cancer patients with covid-19 is being currently conducted. as in the study performed by kuderer et al. [14] , none of the specific therapies prescribed such as antiviral oseltamivir (used for the initial suspicion of influenza infection), therapeutic anticoagulation, ivermectin or chloroquine influenced the risk of death in the current cohort. the strong association between the use of antibiotics and the outcome of death can be explained by the fact that these patients showed a more serious condition than covid-19, including coinfections. lastly, finishing on a positive note, some strengths of the current study can also be recognized. the brazilian national cancer institute is the most important national reference center for the treatment of cancer patients through the brazilian public health system (sus), with a high admission charge, enabling a quick inclusion of patients for this study. this was one of the largest series ever undertaken to explore the impacts of sars-cov-2 infection specifically in cancer patients. throughout the analysis, many variables were presented, allowing us to explore the possibility of their association with risk of death. ultimately, this is the first set of brazilian data in this field, ever. some important limitations are also worth mentioning in this study. as a single-center cohort in a country of continental proportions, such as brazil, a selection bias may well exist, hindering an external validity. the missing data rate for some variables was considerably high due to the retrospective design of the study. there was no paired sample with non-cancer patients with covid-19 or cancer patients without covid-19 to provide a better comparison between the outcomes of morbidity and mortality. due to the in-hospital follow-up only, there was no report of long-term morbidity. finally, the general population of the study was very heterogeneous with several types of neoplasia and anti-cancer treatment, making it difficult to design a more reliable portrait by tumor site. like other comorbidities, cancer is suggested to be an important prognostic factor for patients with covid-19, probably due to the greater clinical fragility and the negative impact of immunosuppressive treatments. despite having formulated early institutional emergency measures, since the onset of the pandemic in brazil, to reduce the exposure of this group of patients to sars-cov-2, inca had a considerable burden of infected patients in need of hospitalization. the rates of complications and covid-19-specific death were significantly high. supporting information s1 all membership of inca covid-19 task force is affiliated to brazilian national cancer institute funding acquisition supervision references 1. who director-general's remarks at the media briefing on 2019-ncov. world health organization covid-19 situation reports. world health organization brazilian ministry of health covid-19: brazil has 438,238 cases; death toll up to 26,754. agência brasil clinical characteristics of coronavirus disease 2019 in china clinical characteristics of covid-19-infected cancer patients: a retrospective case study in three hospitals within wuhan covid-19 mortality in patients with cancer on chemotherapy or other anticancer treatments: a prospective cohort study. the lancet clinical characteristics, outcomes, and risk factors for mortality in patients with cancer and covid-19 in hubei, china: a multicentre, retrospective, cohort study does covid-2019 have an impact on the purchase intention of commercial long-term care insurance among the elderly in china? healthcare case fatality rate of cancer patients with covid-19 in a new york hospital system covid-19 outcomes in patients with hematologic disease clinical characteristics and risk factors associated with covid-19 disease severity in patients with cancer in wuhan, china: a multicentre, retrospective, cohort study covid-19 in breast cancer patients: a cohort at the institut curie hospitals in the paris area clinical impact of covid-19 on patients with cancer (ccc19): a cohort study. the lancet patients with cancer appear more vulnerable to sars-cov-2: a multi-center study during the covid-19 outbreak clinical management of severe acute respiratory infection when covid-19 is suspected. world health organization cdc 2019-novel coronavirus (2019-ncov) real-time rt-pcr diagnostic panel characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention key: cord-272878-6f0q661e authors: schnepf, nathalie; resche-rigon, matthieu; chaillon, antoine; scemla, anne; gras, guillaume; semoun, oren; taboulet, pierre; molina, jean-michel; simon, françois; goudeau, alain; legoff, jérôme title: high burden of non-influenza viruses in influenza-like illness in the early weeks of h1n1v epidemic in france date: 2011-08-17 journal: plos one doi: 10.1371/journal.pone.0023514 sha: doc_id: 272878 cord_uid: 6f0q661e background: influenza-like illness (ili) may be caused by a variety of pathogens. clinical observations are of little help to recognise myxovirus infection and implement appropriate prevention measures. the limited use of molecular tools underestimates the role of other common pathogens. objectives: during the early weeks of the 2009–2010 flu pandemic, a clinical and virological survey was conducted in adult and paediatric patients with ili referred to two french university hospitals in paris and tours. aims were to investigate the different pathogens involved in ili and describe the associated symptoms. methods: h1n1v pandemic influenza diagnosis was performed with real time rt-pcr assay. other viral aetiologies were investigated by the molecular multiplex assay respifinder19®. clinical data were collected prospectively by physicians using a standard questionnaire. results: from week 35 to 44, endonasal swabs were collected in 413 patients. overall, 68 samples (16.5%) were positive for h1n1v. in 13 of them, other respiratory pathogens were also detected. among h1n1v negative samples, 213 (61.9%) were positive for various respiratory agents, 190 in single infections and 23 in mixed infections. the most prevalent viruses in h1n1v negative single infections were rhinovirus (62.6%), followed by parainfluenza viruses (24.2%) and adenovirus (5.3%). 70.6% of h1n1v cases were identified in patients under 40 years and none after 65 years. there was no difference between clinical symptoms observed in patients infected with h1n1v or with other pathogens. conclusion: our results highlight the high frequency of non-influenza viruses involved in ili during the pre-epidemic period of a flu alert and the lack of specific clinical signs associated with influenza infections. rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management. in order to monitor the spread of influenza and alert health handlers, several epidemiological tools have been developed. in france, a network of 1300 general practitioners, ''réseau sentinelles'', working throughout the country, provides real-time clinical data used to evaluate regional and national influenza spreading [1, 2] . the criteria used by this network to define clinical influenza-like illness (ili) are the occurrence of a sudden fever above 39uc with myalgia and respiratory signs. in general no formal viral diagnosis is carried out. the groupes régionaux d'observation de la grippe (grog) is a second french network that surveys the emergence and the spread of the influenza viruses [3, 4] . this network is based on clinical surveillance of acute respiratory infections and laboratory analysis of nasal specimens collected from adults and children by volunteer general practitioners and pediatricians. according to the sentinel network's criteria, french health authorities proclaimed that flu epidemic level was reached during the second week of september 2009 (week 37) [5, 6] . on the contrary, data provided by the grog showed only sporadic h1n1v activity until the last week of october (week 44) [6, 7] . thus, it became rapidly obvious that a variety of viruses were circulating in the community and that an overestimation of myxovirus infection was at stake [8, 9, 10, 11] . as a better knowledge of the epidemic status was a key feature for national healthcare organization, hospital preparedness, patient management and disease control, unambiguous viral diagnosis appeared critical. in france, data on viral aetiologies associated with ili were at best sporadic and correlations with clinical symptoms were often lacking. extensive molecular assays to screening for respiratory viruses were not available countrywide for routine diagnosis. therefore the epidemiological pattern of respiratory pathogens with overlapping seasonality was poorly known. the aim of the present study was to investigate respiratory pathogens involved in ili during the early weeks of the 2009-2010 h1n1v diffusion in france (weeks 35 through 44) and describe the associated symptoms in paediatric and adult populations. this study was a non-interventional study with no addition to usual proceedures. biological material and clinical data were obtained only for standard viral diagnostic following physicians' prescriptions (no specific sampling, no modification of the sampling protocol, no supplementary question in the national standardized questionnaire). data analyses were carried out using an anonymized database. according to the french health public law (csp art l 1121-1.1), such protocol does not require approval of an ethics committee and is exempted from informed consent application. in the two academic hospitals, saint-louis hospital (sls) in paris and tours hospital (trs), influenza-like illness (ili) was defined as a patient suffering from at least one general symptom (fever above 38uc, asthenia, myalgia, shivers or headache) and one respiratory symptom (cough, dyspnoea, rhinitis or pharyngitis), in agreement with the guidelines from the french institut de veille sanitaire (invs), a governmental institution responsible for surveillance and alert in all domains of public health [12] . criteria for severe clinical presentation were temperature below 35uc or above 39uc despite antipyretic, cardiac frequency above 120/min, respiratory frequency above 30/min, respiratory distress, systolic arterial pressure below 90 mmhg or altered consciousness. predisposing factors of critical illness were children younger than one year old, pregnant women, diabetes, chronic pre-existing disease (such as respiratory, cardiovascular, neurologic, renal, hepatic or hematologic diseases) and immunosuppression (associated with hiv infection, organ or hematopoietic stem cells transplantation, receipt of chemotherapy or corticosteroids) [13, 14] . a cluster of suspected influenza infections was defined as at least three possible cases in a week in a closed community (household, school,…) [15] . in the two institutions, the prescription of h1n1v molecular testing was recommended for patients with ili and with either a severe clinical presentation, an underlying risk factor of complications or a condition which was not improving under antiviral treatment. investigation of grouped suspected cases was also recommended. from week 35 (last week of august) to 44 (last week of october), 413 endonasal swabs were collected in 3 ml of universal transport medium (copan diagnostics inc, murrieta, ca) from adults and children seen in emergency rooms for suspected ili (table 1 ) and sent to sls and trs laboratories for h1n1v detection. the two microbiology laboratories participated in the reference laboratories network for the detection of pandemic influenza h1n1v. clinical data were collected at the time of medical attention and reported by clinicians on a national standardized questionnaire provided by invs [1, 12] . this questionnaire included the presence or absence of the main general and respiratory symptoms associated with ili (fever, asthenia, myalgia, shivers, headache, cough, rhinitis, pharyngitis, sudden onset) [12] . total nucleic acid was extracted from 400 ml of universal transport medium using the easymag system (biomérieux, marcy l'etoile, france) in sls or the ez1 advanced xl (qiagen, courtaboeuf, france) in trs, according to the manufacturers' instructions (elution volume: 100 ml in sls or 90 ml in trs). before extraction, 5 ml of an internal amplification control (iac) which contained an encephalomyocarditis virus (emc) rna transcript was added into the sample. pandemic h1n1v infection was diagnosed by real-time reverse transcription-pcr (rt-pcr) assay on a 7500 real time pcr system (applied biosystems, foster city, ca) according to the protocol of the centers for disease control (cdc) [16] . other respiratory infections were investigated by a multiplex molecular assay based on the multiplex ligation-dependent probe-amplification (mlpa) technology (respifinder19h, pathofinder, maastricht, the netherlands) that allows the detection and differentiation of 14 respiratory viruses, including influenza virus a (infa), influenza virus b (infb), rhinovirus (rhv), parainfluenza viruses 1 to 4 (piv-1 to piv-4), human metapneumovirus (hmpv), adenovirus (adv), respiratory syncytial virus a (rsva), respiratory syncytial virus b (rsvb) and human coronaviruses 229e, oc43 and nl63 (cor-229e, cor-oc43, cor-nl63) [17] . the test allows also the detection of h5n1 influenza a virus and of four bacteria: chlamydophila pneumoniae (cp), mycoplasma pneumoniae (mp), legionella pneumophila (lp) and bordetella pertussis (bp). the amplified mlpa products were analyzed on an abi 3100 genetic analyzer (applied biosystems, foster city, ca). fragment sizing analysis was performed with the genemarker software (softgenetics, llc, state college, pa). further testing for h1n1v was carried out with simplexa tm influenza a h1n1 (2009) (focus diagnostics, cypress, california) when the cdc real time rt-pcr assay was negative for h1n1 and the respifinder19h assay was positive for influenza a. if this latter assay was negative, h3n2 typing was performed as previously described [18] . data from our study are summarized as frequencies and percentages for categorical variables. quantitative variables are presented as medians, 25th and 75th percentiles. to compare those variables according to the viral infection status, fisher tests by using cdc reference assay, h1n1v was detected in 66 samples out of 413 (16.6%), more frequently in sls (38 samples) than in trs (28 samples) (p,10 24 ). overall, weekly percentage of h1n1v positive endonasal swabs remained under 10% until week 41 and increase significantly after (p trend ,0.0001) ( figure 1 ). rate of h1n1v detection reached 30% in sls at week 42 and in trs at week 44. overall, this rate was in agreement with results provided by the grog network, showing an earlier start of h1n1v epidemic in paris area [7, 19] . all 413 nucleic acid extracts were analyzed using the respifinder19h assay ( figure 2 ). sixty six patients tested h1n1v positive with cdc real time rt-pcr assay were confirmed with the multiplex assay. thirteen were also co-infected by one or two other respiratory pathogens (multiple infections) ( figure 2 ). three of the 347 h1n1v negative samples could not be studied with the multiplex assay because they contained rt-pcr inhibitors (no amplification of the internal control). two hundred and fifteen (62.5%) of the remaining 344 h1n1v negative samples were found positive for at least one respiratory pathogen ( figure 2 ). two hundred and twelve were positive for non influenza pathogens (189 single infections and 23 mixed infections with two, three or four viruses) and three additional single infections by influenza a were identified in sls, including two by pandemic h1n1v and one by seasonal h3n2, as determined after molecular typing (data not shown). overall, 68 patients (16.5%) were then positive for h1n1v, one for h3n2 and 212 for non influenza pathogens. there were 245 single infections (55 with h1n1v and 190 with other respiratory pathogens) and 36 mixed infections (13 with h1n1v and 23 without h1n1v) ( figure 2 ). among h1n1v negative single infections, the most prevalent viruses were rhinovirus (62.6%, 119 patients), followed by parainfluenza viruses 1 to 4 (24.2%, 46 patients), adenovirus (5.3%, 10 patients), human coronavirus 229e, oc43 and nl63 (3.2%, 6 patients) and respiratory syncytial virus a and b (2.6%, 5 patients) (figure 2 ). in addition, respifinder19h assay identified three patients with bacterial infection, two with mycoplasma pneumoniae (one 25 years old female in sls and one 39 years old female in trs) and one with bordetella pertussis (one 60 years old male in sls). no single infection by influenza b, hmpv, chlamydophila pneumoniae or legionella pneumophila was identified ( figure 2 to analyze if viral co-infections occurred more frequently for some viruses, we carried out a two by two comparisons, that showed a higher proportion of co-infection only for adv (p = 0.05). non-influenza respiratory viruses presented a different epidemic profile compared to h1n1v. overall, in both hospitals, weekly rate of non-h1n1v respiratory viruses whether alone or involved in co-infection increased between week 37 and 39 (from 51.4% to 81.3%) and then consistently decreased ( figure 3 ). rhv infections that represented nearly half of non-h1n1v viral infections (141 out of 213, 66.2%) were a significant contributing factor. in both hospitals, emergence of h1n1v cases was associated with a rapid decline of rhv rate of infection from 50-60% down to less than 20% with a one to two weeks gap between sls and trs. data on age ( in both institutions, 85.5% (106/124) children younger than 15 years of age were infected by at least one respiratory pathogen ( table 2 ). h1n1v infected patients were not significantly younger than h1n1v non infected patients (27 years old vs. 25 years old, p = 0.80) (figure 4) . however, 70.6% (48/68) of h1n1v cases were identified in patients under 40 years old (22 in sls and 26 in trs) and no case was observed in patients older than 65 years ( table 2) . piv infection occurred in very young patients (median (figure 4) . consequently, piv and adv were more frequently detected in the younger population of trs versus sls (p,10 24 and p,10 23 respectively). in contrast, although individuals with rhv infection were slightly younger than individuals without (median age = 24 vs. 29 for patients without rhv, p = 0.05) (figure 4) , influenza-like illness associated with rhv was more frequent in sls than in trs (p = 0.012). finally, patients with viral multiple infection were significantly younger than those with single infection (median, idr: 4, 2-18.5 vs. 25, 6-43) and rates of mixed infection at the time of medical attention, 383 (92.7%) standardized clinical questionnaires were collected out of 413 patients. four of them could not be exploited because they were too incomplete. a review of the 379 workable questionnaires showed that 90.8% (344/379) of the patients included in this study fulfilled the criteria of ili as defined above, and 52.5% had either a severe clinical presentation or an underlying risk factor of complications (45.9%, 174/379), or were in a suspected cluster of grouped cases (6.6%, 25/379). overall, most patients have fever (93.9%) and cough (86.1%) ( table 3) . other classical clinical signs associated with ili such as asthenia, myalgia, shivers, headache, rhinitis or pharyngitis were less frequent. a sudden onset was also described in 59.2% of cases. only 32.5% of the patients had a temperature above 39uc; the age of these patients ranged from zero to 86 years, with a median age of 32 years and a mean age of 34 years (data not shown). in h1n1v infected patients (including single and multiple infections), the main symptoms were also fever (98.2%) and cough (89.5%) ( we then compared clinical characteristics between patients positive for h1n1v, patients positive for other respiratory pathogens and negative for h1n1v and patients without any detection of respiratory pathogens (as detected with respifin-der19h) ( table 3 ). there was no difference between the three groups except for fever, cough, pharyngitis. however for these latter symptoms, the comparison between patients positive for h1n1v and those positive for other respiratory pathogens or between patients positive for h1n1v and those without any detection of respiratory pathogens, showed no difference except for pharyngitis, which was less frequent in patients positive for h1n1v than in patients positive for other respiratory pathogens ( table 3) . as rhv was the most frequent aetiology in ili, we also compared clinical symptoms observed in patients with a single infection by rhv or by h1n1v (data not shown). there was no difference except that rhinitis and pharyngitis were significantly more frequent in rhv infection (62.7% vs. 34.1% [p = 0.006] and 39.0% vs. 10.0% [p = 0.001], respectively). viral multiple infection (including samples with h1n1v) was not associated with a different clinical presentation. fever and cough were observed in over 90% of the patients (90.6% and 90.3%, respectively), but only 33.3% of these patients had a temperature above 39uc, which was not different from patients with single viral infection (28.6%). our results highlight the high frequency of non-influenza viruses involved in acute respiratory infections during the epidemic period of a flu alert as defined by the réseau sentinelles according to ili definition (a sudden fever above 39uc accompanied by myalgia and respiratory signs). these data extent previous observations in europe reporting high prevalence of rhv infections before seasonal influenza [4, 20] or in 2009, before h1n1v pandemic influenza [1, 8, 9, 11, 21] . we confirm that rhv represent the most frequent aetiology of acute respiratory table 2 . age of patients with respiratory samples positive for h1n1v, positive for other respiratory pathogens or negative. infections both in adult and paediatric populations and may represent more than 50% of cases. we show that other viral infections than influenza and rhv may represent up to 30% of aetiologies. we observed differences between the two hospitals, with a higher frequency of parainfluenza and adv infections in tours in contrast with a higher frequency of rhv in paris, likely explained by the higher proportion of paediatric samples collected in tours. however, despite the distance between the two institutions (about 250 km) and differences between the two populations, both presented similar patterns of high frequency of non-influenza viruses in acute respiratory infections before the flu epidemic wave and a decline when influenza reached epidemic levels. in the two cities, high frequencies of rhv were seen at the same level with a likely different evolution speed, with sudden increase and decrease in sls and more progressive variation in trs. in both institutions, there was a decrease in the proportion and number of rhv diagnoses roughly in parallel with the increase of influenza diagnoses. indeed, h1n1v exceeds 20% of positive detection's rate only when rhv dropped under 40%. these data are thus consistent with negative interaction of the two epidemics at the population level. it was previously hypothesised that rhv epidemic could interfere with the spread of pandemic influenza [20, 21, 22] . few in vitro data support this hypothesis. it has been reported that interferon and other cytokines production by rhv infected cells induced a refractory state to virus infection these data include the three patients whose respiratory samples could not be studied with the multiplex assay because of rt-pcr inhibitors. of neighbouring cells [23] . further work is needed to confirm in vitro and in vivo such negative interactions and if viral interference are really translated to a population level. analysis of rhinovirus and influenza epidemics in previous years should also help to determine if similar interferences were observed with seasonal influenza and to elaborate modelling and prediction of the spread of influenza according to respiratory viruses' circulation. systematic extensive screening of respiratory viruses at a national level should be implemented for this purpose. very few rsv infections were observed in contrast to usual epidemiology which was characterized the last four past years by a start of epidemics in weeks 44-45 [1] . it has been confirmed by other laboratories and the french invs that the 2009-10 rsv epidemic was delayed and had a lower impact compared with the previous winter season [1, 24] . delayed and reduced rsv spread may be due to viral interference between rsv and influenza. another possible explanation is better prevention behaviour about respiratory infections as recommended by a national campaign including recommendations for hands washing after sneezing and the use of mask [1] . influenza infections were mainly detected in patient under 40 years old and no case was found in patients older than 65. these results corroborate previous data suggesting that past seasonal h1n1 infections or vaccination may give partial crossed protection [10, 13, 25] . we have previously shown that the neutralizing titers against pandemic h1n1v virus correlate significantly with neutralizing titers against a seasonal h1n1 virus, and that the h1n1v pandemic influenza virus neutralizing titer was significantly higher in subjects who had recently been inoculated by a seasonal trivalent influenza vaccine [26] . viral co-infections were predominantly seen in paediatric patients, as previously described [4, 27, 28, 29] , both in influenza and non-influenza cases at a similar rate. no evidence of more pronounced respiratory impact was seen in these patients. our results showed the lack of specific clinical signs associated with proven h1n1v infections. clinical characteristics did not differ between influenza infections or other viral infections. in particular, the proportion of patients with fever above 39uc was not higher in h1n1v positive patients. in addition, the patients without any evidence of respiratory viral infections did not have different symptoms. these patients may have been infected with other virus not included in the multiplex assay (human bocavirus, coronavirus hku1) [9, 10, 11] or were seen too late at the time of viral shedding was cleared [30] . however, to determine how specific the symptoms are for influenza would require to assess also the distribution of respiratory pathogens (h1n1v and other respiratory viruses) and related symptoms in patients presented at the emergency departments in sls and trs with respiratory syndromes, but not tested for h1n1v. in addition, despite some underlying conditions that were associated with complications not previously observed in seasonal influenza, most illnesses caused by the h1n1v virus were acute and self-limited [13, 31] . the higher proportion of non influenza viruses reported in ili in 2009 was thus most likely a consequence of more frequent visits to a doctor for respiratory tract infections than usually observed for fear of the flu pandemic. the general lack of difference in symptoms in the particular context of h1n1v pandemic has therefore to be considered with caution and does not rule out that more significant differences may arise in future influenza epidemics with other influenza viruses. our data confirm that it may be virtually impossible to recognize symptoms heralding h1n1v infections and virological data should be helpful along with clinical reports to monitor influenza epidemic [10] . molecular multiplex detection has recently emerged as a potent diagnostic tool to determine acute respiratory infections' aetiologies [11, 32, 33] . these data show that sensitive molecular multiplex detection of respiratory viruses is feasible and efficient for the detection of virus involved in acute respiratory infections and provides insights into their epidemic profile. our results confirm the performance of respifinder19h assay to detecting respiratory viruses in the general population as recently shown in transplant patients with ili [34] . respifinder19h confirmed all h1n1 infections detected by the cdc reference assay and was able to identify two additional h1n1 cases suggesting a high sensitivity of this multiplex assay to detect influenza a infections. in conclusion, our results highlight that successive and mixed outbreaks of respiratory viral infections may affect influenza epidemiology and can lead to misinterpret the early development of a flu epidemic. rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management. impact of the 2009 influenza a(h1n1) pandemic wave on the pattern of hibernal respiratory virus epidemics virtual surveillance of communicable diseases: a 20-year experience in france a new influenza surveillance system in france: the ile-de-france ''grog''. 1. principles and methodology surveillance of community-acquired viral infections due to respiratory viruses in rhone-alpes (france) during winter 1994 to 1995 bulletins hebdomadaires surveillance de la grippe en france. bulletins hebdomadaires a variety of respiratory viruses found in symptomatic travellers returning from countries with ongoing spread of the new influenza a(h1n1)v virus strain frequency of detection of upper respiratory tract viruses in patients tested for pandemic h1n1/09 viral infection novel virus influenza a (h1n1sw) in south-eastern france rapid detection of respiratory tract viral infections and coinfections in patients with influenza-like illnesses by use of reverse transcription-pcr dna microarray systems surveillance de la grippe en france clinical aspects of pandemic 2009 influenza a (h1n1) virus infection severe hospitalised 2009 pandemic influenza a(h1n1) cases in france modified surveillance of influenza a(h1n1)v virus infections in france cdc protocol of real-time rt-pcr for influenza a (h1n1) respifinder: a new multiparameter test to differentially identify fifteen respiratory viruses application of a fluorogenic pcr assay for typing and subtyping of influenza viruses in respiratory samples bulletins épidémiologiques de la grippe a (h1n1) rhinoviruses, a(h1n1)v, rvs: the race for hivernal pandemics rhinoviruses delayed the circulation of the pandemic influenza a (h1n1) 2009 virus in france does viral interference affect spread of influenza? the interferon response circuit: induction and suppression by pathogenic viruses situation épidémiologique de la bronchiolite portrait of a year-old pandemic detection of extensive cross-neutralization between pandemic and seasonal a/ h1n1 influenza viruses using a pseudotype neutralization assay multiplex real-time pcr for detection of respiratory tract infections frequent detection of viral coinfection in children hospitalized with acute respiratory tract infection using a real-time polymerase chain reaction detection of multiple respiratory pathogens during primary respiratory infection: nasal swab versus nasopharyngeal aspirate using real-time polymerase chain reaction prospective evaluation of a novel multiplex real-time pcr assay for detection of fifteen respiratory pathogens-duration of symptoms significantly affects detection rate improving the clinical diagnosis of influenza-a comparative analysis of new influenza a (h1n1) cases molecular diagnosis of respiratory viruses detection of respiratory viruses by molecular methods comprehensive diagnostics for respiratory virus infections after transplantation or after potential exposure to swine flu a/h1n1: what else is out there? we gratefully acknowledge the contribution of the members of the two virology laboratories and sandrine picco for their excellent technical assistance in the detection of h1n1v pandemic virus and other respiratory viruses, and catherine scieux for her help in epidemiological data analysis. key: cord-263978-jk82bk1a authors: karaivanov, alexander title: a social network model of covid-19 date: 2020-10-29 journal: plos one doi: 10.1371/journal.pone.0240878 sha: doc_id: 263978 cord_uid: jk82bk1a i construct a dynamic social-network model of the covid-19 epidemic which embeds the sir epidemiological model onto a graph of person-to-person interactions. the standard sir framework assumes uniform mixing of infectious persons in the population. this abstracts from important elements of realism and locality: (i) people are more likely to interact with members of their social networks and (ii) health and economic policies can affect differentially the rate of viral transmission via a person’s social network vs. the population as a whole. the proposed network-augmented (nsir) model allows the evaluation, via simulations, of (i) health and economic policies and outcomes for all or subset of the population: lockdown/distancing, herd immunity, testing, contact tracing; (ii) behavioral responses and/or imposing or lifting policies at specific times or conditional on observed states. i find that viral transmission over a network-connected population can proceed slower and reach lower peak than transmission via uniform mixing. network connections introduce uncertainty and path dependence in the epidemic dynamics, with a significant role for bridge links and superspreaders. testing and contact tracing are more effective in the network model. if lifted early, distancing policies mostly shift the infection peak into the future, with associated economic costs. delayed or intermittent interventions or endogenous behavioral responses generate a multi-peaked infection curve, a form of ‘curve flattening’, but may have costlier economic consequences by prolonging the epidemic duration. i construct and compute a dynamic social network-based model of the covid-19 epidemic and use it to evaluate a range of simulated health and economic policies-herd immunity, distancing, lockdown, testing, quarantine, and contact tracing. endogenous behavioral responses are also analyzed. the analytical framework superimposes the classic sir model of infectious diseases ( [1] [2] [3] [4] among many others) onto a social-network graph of interactions. previous work on infection spread via networks is mostly theoretical and includes [5] [6] [7] [8] [9] [10] . an economic module can be overlaid onto the disease dynamics, similar to [11] or [3] . additionally, since the network model tracks individual nodes over time, heterogeneity (e.g., in savings, employment status; ability to pay rent or bills) can be incorporated. sir (or seir) markov models characterize the spread of an epidemic over time in a population of agents who pass through the states of 'susceptible', ('exposed'), 'infectious' and 'resolved' (recovered or dead). biological and socioeconomic parameters determine the duration and transition probabilities between the states. health and economic policies such as physical distancing, testing and quarantine also influence the spread of the disease by affecting the rate and number of contacts between agents. the standard sir framework assumes random (uniform) mixing of infectious persons with the rest of the population. while helpful for simplifying the dynamics and computing outcomes, this population-level random matching assumption abstracts from important elements of realism and locality: (i) people are more likely to interact with members of their social network, broadly defined (e.g., family, work, or distance based); (ii) health and economic policies targeting disease mitigation, as well as individual behavioral responses, can affect the rate of viral transmission via a person's network of contacts vs. the population as a whole differently. for example, [12] use facebook data and show that areas with stronger social ties to two early covid-19 "hotspots" in the u.s. and italy had more confirmed covid-19 cases; (iii) social contact heterogeneity can induce path-dependence and role for 'superspreaders' or 'clusters' in the epidemic dynamics, see [13, 14] . incorporating local, social-network based transmission in the sir epidemiological model can yield quantitatively different outcomes and policy implications compared to the standard framework with uniform mixing. using simulations i show that, for the same biological parameters, the standard sir model can overstate the reproduction rate and infection peak of the epidemic. relative to the sir model, the network structure and degree heterogeneity introduces uncertainty and unpredictability in the epidemic dynamics and duration as well as in policy outcomes, since the infection can spread in a non-uniform, state-dependent way. the observed broad range of covid-19 infection rates across countries, the presence of clusters and superspreaders and the prolonged plateau of new daily infections in some countries despite long lockdown periods may be related to the social network structure and the underlying number and frequency of contacts. an advantage of the network-augmented model, relative to the standard sir model, is that the network model (hereafter nsir) allows tracking (including via contact tracing) and distinguishing infections occurring through social contacts vs. at the population-level (unknown origin or community infections). the nsir approach also allows modeling and analyzing richer behavioral responses, e.g., based on the disease state of an agent's social contacts or deaths among one's contacts, in addition to responses based on aggregate states. the main challenge to the network approach is the choice or calibration of the social network of contacts which is a key model input. the social-network augmented nsir model allows the researcher to specify and vary, via model and policy parameters, the relative rate of viral transmission within agents' social network vs. the population and thus nests the standard sir model as a special case. since, unlike sir, the nsir model is simulated at the agent level it incorporates agent heterogeneity, via the agent's position in the network by construction, but also extendable in other economically relevant dimensions. the nsir model is solved via a stochastic monte carlo approach using the gillespie algorithm ( [15, 16] ), a numerical method for generating statistically correct trajectories (possible solutions) of a stochastic system. the proposed network-augmented model of covid-19 is used to assess a broad set of simulated health and economic policies and behaviors, applying to all or subset of the population and including but not limited to: (i) physical distancing-by varying the network structure (a reduction in the nodes' degree / social contacts) and/or by varying the network-level vs. population-level mixing parameter. (ii) testing and quarantine with or without contact tracing-by keeping track of and varying each agent's network of allowed contacts in the simulation. (iii) policy timing and duration-imposing or lifting health or economic policies at specific times or conditional on observed epidemic aggregates; both contiguous and intermittent policy interventions are considered. (iv) endogenous behavioral responses by the agents (e.g., self-quarantine, avoiding contacts) based on observed infections or deaths among the agent's contacts or in the population at large. i am not an epidemiologist and all analysis and conclusions in this paper should be interpreted with the appropriate caveats. in addition, at the time of writing there is still a lot of uncertainty about the covid-19 epidemiological parameter values and the policy outcomes are sensitive to that (robustness is explored). my objective is therefore primarily descriptiveto explore via simulations the implications, interactions and joint effects of epidemiological dynamics, social networks and policy or behavioral counterfactuals on health and economic outcomes. my main findings are summarized as follows: 1. viral transmission over a network-connected population can proceed slower and reach a lower peak than transmission via uniform/random contacts as assumed by standard sir models. this is consistent with the findings of [17] using new york social interactions data. the resulting longer epidemic duration could imply larger overall economic costs, e.g., if accompanied by longer lockdown periods. in the nsir model with network-based viral transmission: 2. lockdown, quarantine and physical distancing policies which reduce the agents' contacts are on average more effective in slowing down the viral transmission compared to in the sir model with uniform mixing. even partial lockdown or distancing can break or significantly reduce the transmission in the nsir model by removing and isolating key network links, paths and nodes while these policies are less effective with uniform mixing. largescale and persistent testing and contact tracing are required to lower and flatten the infection rate curve. a low testing rate or a one-off mass testing campaign are not likely to be effective because of the relatively short serial interval of covid-19. 3. if lifted early, lockdown or distancing policies mostly shift the infection peak into the future, with associated economic costs. simulations show that one-, two-and four-month distancing policies starting from 0.5% infected share initially steadily reduce the number of active cases but could fail to contain the epidemic since a large number of susceptible nonimmune agents remains at large. mass vaccination, herd immunity (at the cost of many deaths), or a combination of mass-scale and persistent testing, contact tracing and enforced (self-)isolation appear the only reliable ways to stop the epidemic from reigniting if lockdown policies are lifted early. it may still take long to contain the covid-19 epidemic when a vaccine is available. unlike the virus, a vaccine does not replicate and spread on its own. hence, a vaccine is only effective if introduced on a sufficiently large and/or optimally chosen subset of the population. for example, [18] show that a non-uniform (proportional to node degree) distribution of antidote in a network can control an epidemic while uniform antidote distribution cannot. 4. the epidemic dynamics are sensitive to policy timing and duration. the social-contacts network structure and infection time path (which nodes are infected when) also affects the spread of the epidemic unlike in the sir model. delayed lockdown or distancing policies or endogenous behavioral responses generate a multi-peaked infection rate over time, a form of 'curve flattening', but may have costlier economic consequences by prolonging the epidemic duration. 5. intermittent ("on", "off", "on" again) lockdown or distancing policies and behaviors are demonstrated to be effective in flattening the infection curve. intermittent policies can be politically easier to implement and enforce but may entail larger overall economic or healthcare costs. 6. behavioral responses, through reducing the number or rate of social contacts based on observed infections, on aggregate or in one's own network, can be a powerful and economically less costly alternative to mandated lockdowns but could induce a cyclical pattern of tightening and relaxation over a prolonged period. consider a large population of n persons modeled as the nodes of a social network/graph g. the graph edges capture (regular) social interactions which are possible vectors of infection transmission. the assumed baseline network structure is an input of the model, however, health policies (e.g., lockdowns, quarantine, etc.), can be interpreted as (temporarily) changing the social network by eliminating edges (contacts). in addition to network-level contact, persons/nodes can also interact with any other node (connected or not) with rate/probability p 2 [0, 1]. the limiting case p = 1 thus approximates the random mixing assumption in the standard sir model. each node i = 1, . . ., n has an individual state x it at time t. the basic model states are five: s for susceptible to the disease; e for exposed (infected but not yet infectious); i for infectious; r for recovered and f for dead. additional states for 'tested positive' (known infected) or 'in lockdown' will be introduced in the policy simulations. the nsir model is initialized by randomly assigning #i 2 (0, n) nodes to the infectious state, that is setting x i0 = i, i 2 i 0 and the rest of the nodes to the susceptible state, x j0 = s for j 2 s 0 , where from now on x t denotes the set of nodes/agents with state x it = x at time t. conditional on current state x it , the next state x it 0 for node i is determined as follows. the probability for any state transition not specified below, e.g., s to i or e to r is set to zero. (a) susceptible agents where a t denotes the number of active agents at t (for example, all living agents, a t = n − f t ) and where c g (i), with dimensionality (node degree) c g (i), denotes the set of contacts / edges of node i in the social graph g. are the probabilities that the contact is infectious, in the population or in one's social network, respectively. the first term (multiplied by p) in (1) captures the rate of infection from contact with an infectious person in the population at large (e.g., public transit, shopping, etc.) this term corresponds to the uniform mixing (random meeting) transmission vector in the standard sir model. the second term in (1) (multiplied by 1 − p) captures viral transmission that occurs because of an existing infection(s) among i's social contacts in g, the set c g (i). in section 3 i show how (1) can be modified to include testing and quarantine/isolation. (b) exposed agents the transition from the exposed to the infectious state happens at rate σ set to match the disease's incubation period. (c) infectious agents the expected recovery rate is γ. the fatality rate conditional on being infected is μ. (d) recovered agents and deaths death (state f) and recovery (state r) are absorbing states. possible transition from state r back to the susceptible state s is ruled out in the simulations but is very easy to incorporate via an additional parameter. base population birth or death rates can be also modeled but i abstract from this here. there are two main groups of parameters in the model. the parameters β (infectiousness), σ (incubation period), γ (survivability) and μ (mortality) are assumed biologically fixed in the baseline simulations. it is computationally feasible to allow state-based mortality rate, μ(i t ) (for example, because of exceeding hospital capacity) as in [3] . in contrast, the parameter p and the social network structure g on which agents interact are interpreted as socioeconomic variables affected by policy or behavioral responses. in section 3 i introduce additional policy parameters and graphs to model testing, contact tracing, lockdowns, distancing and quarantine. the model is initialized by choosing the initial number of infectious nodes (i 0 ), with the rest of the n nodes set in susceptible s state. a baseline network graph, g of size n is also chosen (see section 4.1 for details and section 6 for alternative specifications and robustness). model time evolves stochastically from t to t 0 = t + τ, by having the time index t increased by the amount τ computed from the state-transition probabilities in (1), (3) and (2) using gillespie's algorithm, see [16] . one unit of time equals one day. only the state of a single randomly selected node is modified at each time increment τ. all other nodes retain their previous states. the matlab codes used in this paper (available at the author's website) draw on and significantly extend publicly shared python code by ryan mcgee, see https://github.com/ ryansmcgee. formally, for each t, the gillespie algorithm executes the following steps: (i) draw two random scalars r 1 and r 2 from the uniform distribution on (0, 1) (ii) compute the total probability of any node changing to a new state using (1), (3) and (2), call it p (iii) use r 1 to draw the time interval τ until the next state change event as t ¼ 1 p ln 1 r 1 � � , using that the state change time interval is exponentially distributed with mean 1 p . call t 0 = t + τ. (iv) use the draw r 2 to select one of all positive-probability state transitions, x lt to x lt 0 with x lt 0 6 ¼ x lt implied by (1) , (3) and (2), with corresponding transitioning node l 2 {1,..n}. the chance of selecting a specific transition is proportional to its probability. (v) perform the state transition from step (iii) by updating node l's state and keeping all other nodes' states the same as at t (vi) forward model time to t 0 = t + τ and go back to step (i) model time is forwarded by larger intervals when transition events are relatively rare (e.g., few initial infections or low values of β, γ, σ and μ) and by small intervals when transition events are frequent (many nodes with high total transition probability around the same t). the total rates of susceptible, exposed, infectious, recovered and dead agents are calculated at any model time t by adding up over i the individual states x it . for example, the total number of infectious persons is i t = ∑ i 1 xit = i. in sum, the nsir model allows keeping track of and simulating: (i) each node's individual disease state (s, e, i, r or f) over time (ii) the evolution of aggregates over time, including total infections, total recoveries, total deaths, etc. (iii) daily changes in the aggregates (over model time intervals with length δt = 1) (iv) state transitions over time and over the social network g by using g's adjacency matrix; for example, this allows tracking the states of nodes with large vs. small number of contacts (edges in g) and comparing and tracing the spread of the disease via social-contacts vectors vs. at the population level (random mixing). in epidemiology the basic reproduction number, r 0 is the expected number of cases that the first infected person generates, when all other agents are susceptible but not yet infected. in the standard s(e)ir model without social network component, where r = γ + μ is the removal rate and s t � s t a t is the fraction of susceptible agents at time t out of all active agents a t . early on, or with few deaths, a t ' n yielding an effective reproduction number evaluating at s 0 ' n gives the familiar sir r 0 value, r sir 0 ¼ b r . if b r > 1 the epidemic grows (if unchecked) as long as there is a sufficiently large fraction of susceptibles, in contrast, if r sir 0 < 1 the epidemic would die out on its own. i define the effective reproduction number r t in the nsir model analogously. define corresponding to the agent i's probability of infection from one of her social contacts in graph g at time t. using (1), in the (no-intervention) nsir model we have, the number of infected agents (exposed plus infectious) would grow if the expression in the brackets is positive. hence, for s t ' s t n , define the nsir model effective reproduction number at p = 1 this expression equals r sir t but in general, including at t = 0, the nsir model reproduction number r nsir t differs from r sir t and depends on the graph g. for example, [9] emphasize the importance of the ratio between the second and first moment of the degree distribution for the infection growth rate. i next compare the reproduction numbers for the sir model (p = 1) and the network-only transmission nsir model (p = 0) for given values of i t and s t . using (6), for p = 0 we have where is the average chance of infection across all susceptible nodes i 2 s t at time t, given the set i t of infectious agents i with x it = i. comparing (5) and (7), observe that intuitively, the standard sir model assumes a uniform chance of infection for each susceptible agent which is proportional to the population infection rate i t n . in contrast, in the networkaugmented nsir model an individual's chance of infection is heterogeneous and is a function of the social network g. the average time-t infection probability in the network, � s t ðgþ determines the reproduction number r nsir t . for example, consider the first infection, i 0 = 1 of some agent i 0 , at which r sir 0 ' b r . in contrast, the value of the nsir effective reproduction number r nsir 0 would depend on � s 0 ðgþ, which is a function of the graph g and of which node was initially infected (path dependence). using (5) and (7), it is clear that the growth rate of the disease would differ in general in the sir (p = 1) vs. nsir model (p = 0) and the counts i t and s t would generally differ in calendar time t. thus, to proceed with the comparison, i compare the sir vs. nsir reproduction numbers for the same cumulative infection count m for several example graphs g. to avoid potential confusion with calendar time, for the rest of this section i will use i(m) and s(m) to denote the number of infectious and susceptible agents at the time of the m-th infection. result 1: for the same infection count, the sir model effective reproduction number equals that of the nsir model on a complete graph. proof sketch: suppose g is a complete graph (each node is connected to all other nodes) and n is large. the first infection, m = 1 yields n − 1 susceptible agents with average chance of infection ' iðmþ n -the sir and nsir reproduction numbers are equal. if a formerly infectious node recovers or dies in the process, then i(m) is reduced in both sir and nsir. example 1. regular graph. suppose g is a connected regular graph in which each node has degree k 2 [2, n). after the first infection, k susceptible agents have infection probability consider now the second infection, of some node j 1 which by construction is one of the contacts of the first infected node j 0 . hence only k − 1 susceptible agents could be infected by j 0 and j 1 each. if a node h is connected to both j 0 and j 1 we can think of splitting the total probability σ h as 1/2 coming from each. thus, p i2sð2þ s ið2þ ¼ ið2þðk à 1þ 1 k and so � s 1 ðgþ ' ið2þ n kà 1 k which is strictly less than ið2þ n and so r nsir < r sir . a similar argument applies for further infections. as a result, for the same infection count m, the average chance of infection in a regular-graph nsir model is lower than the population infection rate iðmþ n in the sir model. example 2. ring graph. suppose g is a connected ring graph, such that each node i = 1,..n is only connected to two nodes, i − 1 and i + 1 (where node index 0 maps to n and n + 1 maps to 1). for the first infection � sð1þ ' ið1þ n and r nsir 0 ¼ r sir 0 , as in example 1. by construction, any subsequent infectious node must be a contact of a previously infectious node, thus at any time the set of infectious and recovered/dead nodes, i is contiguous (consists of nodes that are neighbors on an arc j, j + 1, ..j + l). hence, at each next infection count step, m = 2, 3, . . . there are only at most 2 susceptible nodes in a ring graph (the outside neighbors of the set iþ which have positive probability of infection σ i = 1/2. for example, if the set i consists of nodes 2,3,4,5 positioned in order on the ring graph, the end-nodes are 2 and 5. there would be 1 or 0 susceptible nodes that can be infected if an end-node of i has already recovered/died. for the rest of the susceptible nodes σ i = 0, since in a ring graph they are not connected to any nodes in i. therefore, � sðmþ � 1 sðmþ which is (much) smaller than iðmþ n for m small. thus, using (8) we obtain r nsir < r sir . if, as time progresses, both end-nodes of the set i become recovered/dead before a new node is infected (this can occur with positive probability) then the epidemic dies out in the network model but not necessarily in the sir model (if interior nodes in i remain infectious). suppose g is a star graph in which a single node j is connected to all n − 1 other nodes and there are no other edges. if the first infected node is j then � sð1þ ' 1 > if the first infected note is instead one of the 'rays', then � sð1þ ' 1 n but since the second infected node is necessarily j, we obtain again � sð2þ ' 1 > ið2þ n . these examples show that the graph structure and the network node path followed by the infection over time (the subgraph of infected nodes) are key determinants of the effective reproduction number and hence infection growth (see also fig 11 in section 4.5) . degree heterogeneity combined with high-degree nodes infected early on could raise the nsir reproduction number above the sir value (see also [9] ), while graphs in which the degree distribution is relatively homogeneous are likely to have lower reproduction rates than in the sir model. degree heterogeneity could also be critical in determining policy outcomes, e.g., the infection reaching a superspreader can accelerate or re-ignite the epidemic-see table 3 and no interventions). fig 1 is just an example, for this section only (the main simulation results use the graphs described in section 4.1). the regular graph in fig 1 when d is even (d = 20 or 50) is constructed by setting all nodes on a circle and then each node i is connected to the d/2 nodes immediately before (i − 1, . . .i − d/2) and immediately after it (i + 1, . . ., i + d/2). the ring graph in example 2 corresponds to the case d = 2. thus, for d even, each node is only connected to other nodes in its locality, that is, an infectious node can only infect susceptible nodes near it (up to distance d/2). in contrast, when d is odd(d = 21 or 51 in fig 1) each node of g is connected to the (d − 1)/ 2 nodes immediately before and after it (analogously to the d even case) but, in addition, to node i + n/2, that is, the node "across" from i on the graph circle. this means that each infectious node now has a positive probability of spreading the virus to a new, "far" area of the graph g-a "bridge" link. fig 1 shows that a minor difference in the graph degree (20 vs. 21 or 50 vs. 51) can have a significant effect on the infection and death rates. specifically, when bridge links are present in the social contacts graph g (the odd-degree cases d = 21 and 51) the share of active infections and total deaths can be 2 or 3 times larger than in the 'local contacts only' cases (even-degree, d = 20 and 50). in contrast, there is almost no difference between the simulations using d = 18 vs. 20 or 48 vs. 50 (not displayed on the figure) . the conclusion is that interventions that aim at restricting the epidemic on a local level and eliminate bridge contacts (e.g., air travel) can be effective in suppressing the epidemic. on fig 2 i explore further the role of the network structure for viral transmission dynamics and the infection rate over time. the figure computes the infection curve for a series of graphs, starting with a regular (ring-style) graph in which each node is only connected to nearby nodes (the dotted line) and comparing it to three watts-strogatz graphs with the same median degree 12 and number of nodes (n = 10, 000) but with different values for the parameter b that governs the probability of re-wiring an edge to a new node. larger values of b correspond to more re-wiring. i.e., adding more bridging contacts with non-local nodes. the results show that an increased number of cross-links added to the contacts graph can raise the infection peak, total infections, and cumulative deaths significantly (e.g., the peak infection rate is 0.8% in the regular graph, b = 0 vs. 7.8% in the watts-strogatz graph with b = 0.5), consistent with the theoretical discussion above. the nsir model can be used or extended to incorporate a wide variety of health and socioeconomic policies and scenarios related to mitigating or failing to control the spread of the disease. 1. herd immunity-simulating the nsir model without any policy intervention or behavioral response. testing is modeled by introducing an additional state p ("tested positive"). assume for simplicity that only infectious (state i) agents can test positive. the transition probabilities in (3) are modified to: where θ is the fraction of currently infectious agents tested per unit of time. agents with x it 6 ¼ i are assumed to always test negative (allowing the possibility of positive test for state e is simple). keeping track of "tested negative" agents can be easily incorporated too (e.g., to keep track of testing costs or testing coverage over time). the transition probabilities for the agents who have tested positive (state p) are: where the recovery and fatality parameters γ p and μ p can be the same of different than γ and μ in (3) and (9). the agents who test positive, x it = p are assumed to be isolated or in (self-)quarantine and not mixing with others in the population; that is, (1). however, the p agents could still infect contacts in their immediate social network q, defined as a sub-graph of g with the same nodes but fewer edges per node (see more details below). that is, the second term in (1) is modified to the network aspect of the nsir model is well-suited to study contact tracing, that is, following up, identifying and isolating the contacts of agents who have tested positive. contact tracing is modeled by adding a parameter ϕ and a new term in (9) , interpreted as the additional probability of identifying an agent i as infectious (and moving i to state p) for each of i's contacts j who have tested positive. 4. distancing and quarantine-physical (social) distancing can be incorporated in two complementary ways, both of which are explored in the simulations in section 4. the first way of modeling distancing is by decreasing the value of the parameter p. this corresponds to setting a lower rate of global (population level) interactions and higher rate of local (networklevel) interactions in (1) . a second way of modeling distancing is by varying the network structure, that is, replacing the baseline social network g with another network d which is a subgraph of g with fewer edges connected to each node (lower degree). quarantine, an extreme form of distancing is modeled by setting p = 0 and assuming a very small number or zero social contacts for each quarantined node (their narrow social graph q). 5. lockdown-assume that fraction λ 2 (0, 1) of all agents are locked down and only the remaining fraction 1 − λ of agents interact, similar to [3] . this is done by introducing an indicator variable ('locked down', l or 'not locked down', ¬l) for each node i and modifying (1) as follows: the first term in (11) assumes that the probability of contact with a random person remains unchanged for the agents not in lockdown (e.g., interact with others at work). an alternative would be to assume reduced frequency of contacts, for example, pbð1 à lþ ð1à lþi t ð1à lþnà f t . locked down agents, the second line in (11) , are assumed to be exposed only to their narrow social network q, a sub-graph of g (e.g., close family) with the same number of nodes but fewer edges per node. the nsir model allows incorporating a rich set of endogenous behavioral responses to the epidemic. the agents can decide to reduce the number or rate of their contacts, based on observable information or individual cost-benefit calculations (see also [19, 20] or [21] in nonnetwork models). specifically, suppose p < 1 and define the following social-contact graphs: e 0 = g and e k � e k−1 for k = 1, . . .m, where � x denotes a sub-graph of x with the same nodes but fewer edges/contacts per node. for example, if m = 2 we can think of e 0 = g as the "normal times" social network; e 1 � g as a "reduced contacts" network (e.g., work and necessity shopping); and e 2 � e 1 as a "close family" network. in the simulations in section 4.3 each agent is assumed to switch to a more restricted (lower-degree) network, based on the observed infection rate in the population (aggregatelevel information) or, alternatively, based on positive case(s) in their own social network c g (i) (individual-level information). each policy or behavioral scenario 1 through 6 can be imposed or lifted in the simulations at a pre-specified model time t 2 (0, t max ) or conditional on reaching a specific aggregate state value (e.g., number of positive tests or deaths per day, total positive cases, etc.). i investigate a range of scenarios in the following sections. table 1 reports the baseline parameter values used in the model simulations. the baseline expected removal rate r is set to 0.2 which corresponds to a 5-day average period of infectiousness ( [22, 23] ), following a 5.2-day average exposed stage duration (the parameter σ). i also explore a longer infectiousness period, r = 0.1 in the robustness checks in section 6. the baseline infection fatality rate (ifr) is set to 0.37% using streeck et al.'s german randomized study, [24] . an 0.66% estimated ifr with wuhan data, (e.g., [25] ) and 1% ifr are also explored in the robustness section 6.2. the ifr value is important for the death total but, since μ is small and death is an absorbing state, it otherwise changes very little the infection rate dynamics (see fig 13 in section 6.2). the value of the covid-19 infectiousness β is calibrated to fit the observed approximately three-day early doubling time of the disease (e.g., [26] ) and/or a basic reproduction number r 0 of 2.5. the calibrated parameters are actual as of early may 2020. versions of most figures using alternative parameter values, corresponding to slower infection growth and higher ifr (r = 1/16, μ = .0066r and β = .156) are available on request. in the simulations below i set the recovery and mortality rates for positive agents (in state p) to be the same as the baseline values, γ p = γ and μ p = μ. a key ingredient of the nsir model is the social contacts graph g. i use as baseline a modified (pruned) version of a barabasi-albert (b-a) graph, constructed starting from a barabasi-albert graph with 9-edge preferentially attached nodes and then randomly removing a fraction of edges to generate node degrees lower than 9. the resulting social contacts graph g has median degree 10 and mean degree 12.6. the reason for choosing this baseline graph is that neither the standard scale-free b-a graph nor the standard small-world watts-strogatz (w-s) graph match well certain network properties documented in actual covid-19 or other epidemic transmission networks (see [14] , [13] , [27] [28] [29] ), namely broad degree heterogeneity and long/heavy right tail (superspreaders). standard b-a graphs match well the breadth and long right tail of the degree distribution (allow for superspreaders) but truncate the minimum node degree to a value close to the median, essentially ruling out nodes with few contacts. watts-strogatz graphs capture well short paths and local clustering realistic in many social networks but feature a relatively homogeneous degree distribution (all nodes have similar degree) and lack a long right tail, that is, they exhibit insufficient heterogeneity and broadness in the number of contacts and lack of superspreaders. the modified b-a graph g used in this paper matches both the broad heterogeneity of the degree distribution, including nodes with zero or low degree, and a long/heavy right tail-features also emphasized in the theoretical analysis in section 2.3. robustness simulations with w-s graphs are reported in fig 2 and section 6 showing that the main patterns and results remain robust. fig b in the s1 appendix compares the degree distributions of the baseline graph g with that of a standard albert-barabasi graph (the input graph used in the edge removal procedure described in section 4.1) and a standard watts-strogatz graph with mean degree 12. fig c in s1 appendix depicts the degree distribution of the baseline graph g and the closed-contacts graph q constructed in the same way as g but with larger number of removed edges. i report simulation results from different policy and behavioral scenarios in the nsir model. all graphs in this section show sample simulation paths (one possible time path of the dynamic system), however, the same pseudo-random number sequences are used so the graphs are comparable across the scenarios. summary table 3 in section 6.1 and tables 1 and 2 in s1 appendix in report average values from 100 simulations each, using the same parameters but 100 different pseudo-random number sequences (these sequences are held constant across the different parameter/policy specifications for comparability). the black lines on fig 3 assume epidemic dynamics absent any intervention and/or behavioral responses (herd immunity). the blue lines use a testing rate θ = 0.05, that is, 5% of the currently infectious agents are assumed to be detected per day. i.e., a 25% average total chance of positive test for an infectious agent. this hypothetical testing rate is much higher compared to current daily testing rates in the world so these results should be interpreted as a "mass testing" counterfactual. the simulations assume persistent testing at rate θ, not a one-off testing campaign. a one-off campaign would only detect fraction θ of the currently infectious agents and thus is much less effective. the agents who test positive (enter state p) are assumed to be quarantined and interact only on a close-contacts social network q (see section 3). the red lines on table 3 ), however, these reductions are larger in the nsir, p = 0 setting. tables 1 and 2 in s1 appendix further quantify these results by reporting averages over 100 simulations. the larger policy impact in the network setting is especially pronounced for contact tracing ( table 2 in s1 appendix)-the decrease in total infections or deaths in the nsir setting can be double that in the sir setting, relative to the respective no-tracing baseline. intuitively, testing and contact tracing in the network setting (p = 0) can isolate high-degree infectious nodes (superspreaders) early and thus reduces the infection rate by a larger amount -this effect is absent in the uniform-mixing sir, p = 1 model setting, as previously discussed in section 2.3. tables 1 and 2 in s1 appendix also show that a 0.1% testing rate has very small effect on the infection aggregates, except a 3.9% reduction in deaths in the p = 0 setting. to make a serious dent in overall infections and deaths, very intensive testing and quarantine is required (θ = 10%), with the downside of a significantly prolonged (+41%) epidemic duration. table 2 in s1 appendix further shows that, holding the testing rate constant, increasing the intensity of contact tracing yields additional large reductions in total infections, deaths and the infection peak with this effect being stronger in the nsir, p = 0 model. in this section i simulate several physical distancing policies in the nsir model with network-level transmission, p = 0. the duration and timing of the policy is represented by the shaded area on the graphs. during the distancing period it is assumed that all agents' interactions occur on the truncated social network q defined as a sub-graph of the original social network g whereby each node's degree is randomly scaled down (an agent's contacts are reduced by 10 times on average). to explore different policy lengths and timings all simulations are initialized with 0.5% infectious agents; the timelines on figs 2 and 3 and table 3 are relative to that moment. shorter or less strict policies can be effective at lower initial infection rates. fig 4 ( panels a to f) exhibits six different example scenarios which vary the assumed distancing policy duration ('short'-30 days; 'medium-long' -60 days; 'long'-120 days) and the policy timing ('early', at t = 0; or 'delayed', at t = 30). at the calibrated parameters, distancing policies of short and medium-long duration fail to contain the epidemic in the simulated outcomes. in panel c, even a 4-month long distancing policy imposed at the 0.5% infection rate mark may only delay the epidemic (this happens in 20% of the simulation runs with different random seeds; in the remaining runs policy c contains the epidemic with 1.6% total infection rate and 0.01% death rate). scenario c also illustrates how the network path dependency in the nsir setting relative to the sir model (which node infects when) matters (see fig e in the s1 appendix). in example scenario f the epidemic is successfully suppressed by imposing a sufficiently long (120 days) distancing policy with delay. the simulation results in fig 4 show that delaying the introduction of a distancing policy may be beneficial in some cases-the left-side panels with the right-side panels. intuitively, an appropriately-timed delayed policy can create a two-peaked infection curve (as opposed of a single high peak), which is a form of "curve flattening". however, such delays may possibly overwhelm a country's health system capacity (not modeled here) or result in larger economic costs, an issue explored further in sections 5 and 6. in fig 5 ( panels g through l) i evaluate intermittent distancing policies, that is, policies consisting of two separate periods of physical distancing (contacts on social network q), with "back to normal" (contacts on social network g) time in between. the notation (x)-y-(x) in the panel captions means x days of distancing, followed by y days of policy relaxation, followed by x days of distancing again. current events as of may 2020 suggest that such intermittent policies may be easier to implement or enforce politically in many countries. there are two main takeaways from the hypothetical policy evaluations in fig 5 ( see also table 3 in section 6). first, two shorter distancing periods spaced farther apart (as in panels i or j of fig 5) could be more effective in flattening the infection peak compared to a single longer distancing period imposed early on (panels b and c of fig 4) or compared to two early distancing periods close to each other (panel g of fig 5) . on average, scenario i results in 7% (6.6%) less total infections and 4% (2.5%) lower annualized economic cost than scenario b (scenario g). second, the policy timing matters a lot-for example, longer distancing period early on, or a second period of distancing that is too late, are less effective in flattening the infection peak (compare panels k and l with panels h and j in fig 5) . fig 6 i simulate and compare the effectiveness of a lockdown policy with fraction of locked down agents λ equal to zero (no lockdown), 30%, 70% and 90% for the pure sir model (p = 1) and the network-only nsir model (p = 0). the lockdown intervention is defined as in section 3 and is assumed indefinitely long (there is no testing or contact tracing). the simulations are initialized with 0.5% initial infection rate. the main difference between the lockdown and the distancing policies explored in the previous sub-section is that lockdowns affect both the population-level transmission and the network-level transmission, by reducing the contact rate for fraction λ of the population, see (11) . in contrast, in the sir model with only population-level uniform mixing (p = 1, the left-side panels of fig 6) , the effectiveness of lockdowns is limited since both the numerator and denominator in the infection probability term b ð1à lþi t ð1à lþnà f t in (11) are reduced nearly proportionately for low death counts f t and hence the reproduction number among the agents not in lockdown remains high. expression (11) assumes that the individual contact rate for agents not in lockdown remains the same as without lockdown; the lockdown effectiveness would be higher if the contact rate is also reduced, e.g. as in [3] . the simulation shows that even a 90% (indefinitely long) lockdown only reduces the infection rate and peak but does not eliminate the epidemic. in contrast, in the nsir model with network-level transmission only (p = 0, the rightside panels of fig 6) , a mild λ = 30% lockdown flattens the infection curve significantly by taking out many potential contacts and vectors of transmission while a moderate 70% (indefinitely long) lockdown contains the epidemic. while these are simulated examples, the robust implication is that the global vs. network-level mixing degree (the parameter p) plays a key role in lockdown efficiency. in fig 7 i further investigate the effectiveness of a 70% lockdown with different finite durations in the network-only nsir model, p = 0, staring from a 0.5% initial infection rate. without testing (the left-side panels), lockdowns with duration shorter than 120 days mostly delay the infection peak but do not contain the epidemic. summary table 3 in section 6 further quantifies that a 30-day lockdown only reduces total infections by 0.5% and the infection peak by 5% on average. a longer 90-day lockdown in contrast reduces total infections by 49%, the infection peak by 52% and total deaths by 48% on average, relative to the no-intervention benchmark. these averages are, however, composed by two types of outcomes-the 90-day lockdown either fully contains the epidemic or only delays the peak and makes a small dent in infections and deaths (see fig 7 for the latter case) . these results do depend on the assumed initial infection rate (0.5% in fig 7) -the minimum required lockdown period is shorter if started at a lower infection rate. next, fig 8 explores several illustrative example simulations of lockdowns followed by relaxation. a short or inefficiently timed (panels a, b, c, e) and/or lax (c, d) lockdown may result in (i) a slow and prolonged decline in infections, with asymmetry in the growth rates of infection ramp-up vs. decrease (e.g., as observed in italy or spain) and (ii) a second, larger and/or longer epidemic wave (panels b, e). in contrast, in panel f a strict well-timed lockdown reduces the infection rate significantly below the peak, although in this simulation the epidemic carries on at lower intensity for a long time. lockdown exit-role of testing and contact tracing. i next perform simulations to investigate the complementarity between lockdown policies and follow-up testing and contact tracing. specifically, fig 9 considers a 30 -day lockdown for 70% of the population in the nsir model with p = 0. i simulate alternative lockdown exit scenarios, varying the testing and/or contact tracing rate. all agents who test positive are assumed to be quarantined or (self-)isolating and interact on the reduced degree close-contacts graph q with average node degree 1 defined in section 4.1. the values for the testing rate θ and the contact tracing rate ϕ used in the simulations on fig 9 are hypotheticals, corresponding to continuous mass testing and contact tracing. the results show that opening up social and economic interactions after a relatively short lockdown without testing or with little testing in place can soon result in a new, higher infection peak and larger total number of deaths, because of the large remaining fraction of susceptible persons. second, testing and contact tracing are complementary-mass testing combined with intensive contact tracing can significantly mitigate the epidemic while mass testing alone may be insufficient to prevent a new infection wave. the simulations suggest that, for the calibrated parameters, very high rates of testing and tracing are needed to prevent a new peak after a short 30-day lockdown. prolonging the lockdown period (assuming that moving it forward in time is not possible) is likely to be more effective in reducing infections and deaths although it carries larger economic (and possibly political) costs. in the appendix (fig d in s1 appendix) i perform the same set of simulations for the sir model with global transmission only, p = 1. comparing fig 9 with fig d in s1 appendix reveals that testing and contact tracing are less effective with population-level mixing compared to in the network-contacts model, for the reasons discussed in section 4.2.1. the nsir model allows incorporating behavioral responses by the agents, based either on individual-level information (from their own social contacts) or aggregate-level information. in fig 10 and table 3 i analyze five simulated scenarios of behavioral responses in the networkonly nsir model, p = 0. behavioral response scenarios a, b and c assume testing rate θ = 0.05 and model a (e.g., fear-driven) reduction in an agent's number of contacts if the agent learns that one of his social contacts has tested positive, that is, if x jt = p for some agent j 2 c g (i). this behavioral response works as self-triggered contact tracing. formally, a susceptible agent i for whom 9j 2 c g (i) with x jt = p at some time t, switches to a lower-degree social networkq with contacts cqðiþ whereq is a sub-network of g with lower degree per each node. the median degree of networkq is set to 5 in simulation a and to 1 in simulation b. scenarios a and b assume a permanent switch, to assess the upper bound of the effect. compared to the baseline setting, fig 10 (a single simulation path) and table 3 (average over 100 simulation paths, see section 6.1) show that these behavioral responses reduce the infection rate, peak and death toll by significant amounts. the total number of infected is reduced on average by 25% in scenario a and 48% in scenario b; the infection peak is reduced by 45% and 60% respectively, and the total death count is reduced by 17% and 41% respectively (see table 3 ). behavioral response simulation c (see fig 10 and table 3 ) assumes that the switch to the restricted-contacts networkq is temporary. specifically, a susceptible individual i with x it = s switches to graphq only for the times t for which s/he has a social contact who has tested positive and still in state p, i.e., 9j 2 c g (i) with x jt = p while i uses the baseline social network g otherwise. such adaptive behavior still lowers the infection peak but the reduction in the overall infection and death rates is smaller compared to that in simulations a and b-15% reduction in total infections and 10% reduction in total deaths (see table 3 ). in behavioral response scenarios d and e (fig 10, middle and bottom panel and table 3 ) i assume that agents react to new positive cases in the population. specifically, the susceptible agents observe the infection aggregates and choose to reduce their contacts by switching from graph g to graphq if there is a large increase δ in new active cases p t (δ is set to 100 per 10,000), over the preceding 20 days. the agents revert back to contact graph g if there are less new cases than the threshold δ over the preceding 20 days. simulation d uses a 2% testing rate θ while simulation e uses 5% testing rate. fig 10 shows that these behavioral responses result in multiple but low infection peaks, corresponding to alternating periods of endogenous distancing and relaxation. compared to the scenarios with 2% or 5% testing only and no endogenous behavioral response, the behavioral response scenarios d and e reduce total infections by on average 16% and 25%, the infection peak by 34% and 63%, and total deaths by 7% and 22%, respectively (see table 3 ). as explained earlier, the effective reproduction number in the nsir model depends on the network structure and the social contacts of the currently infectious nodes. to illustrate this point further i explicitly look at the role of superspreaders, that is, nodes with a large number of edges. specifically, i take the baseline no-intervention p = 0 simulation from fig 3 and compute the percent increase in the effective reproduction number r nsir t , as defined in (7), registered immediately after a 'superspreader' node becomes infectious (see row 3 in table 2 ). i define as superspreaders the ten nodes with largest degree in the baseline graph g. from these ten nodes, seven become infected in the simulation, listed in table 2 . the immediate change (increase) in r t because of a superspreader node turning infectious (row 3 in table 2 ) is compared to the average preceding r t change (computed as the average change in r t over the preceding 10 model time-steps / state transitions) in row 4. these results show that superspreaders can lead to significant 'jumps' in r t in the nsir model. in contrast, in the sir model r t changes continuously no matter which node becomes infections since only the total number of currently infectious nodes i t matters for the effective reproduction number, see (5) . fig a in the s1 appendix illustrates further the importance of superspreaders and node degree heterogeneity in the nsir model, compared to the sir model with population-level transmission. fig a in s1 appendix compares the infection curves resulting from a single initial infectious person who is either a superspreader (node 34 with degree 200) or an average spreader (node 21 with degree 10). i do this for p = 1 (random matching, sir setting), p = 0.5 (mixed nsir) and p = 0 (network-only nsir). in the sir setting the identity of the initial spreader (or any later one) has no effect on the infection dynamics by construction-only the total number of infectious i t matters. in contrast, in settings with network-transmission (p < 1) an early superspreader results in much earlier and higher infection peak. in the p = 0 setting the initial superspreader node generates large number of secondary cases very quickly, who in turn infect others, leading to 758 infected nodes (7.6% of the population) at t = 60 as opposed to only 24 infected nodes at t = 60 in the simulation with average initial spreader. these examples highlight the necessity for quickly identifying superspreaders or for restricting the situations in which superspreader scenarios are common (e.g., mass gatherings, bars, cruise ships, etc.), as also documented in the medical literature (e.g., [13] or [14] ). i compute and compare the effective reproduction number r t across different model scenarios. in general, for any scenario, with corresponding endogenous s t , i t and e t , we can compute (agents who tested positive, if any, are included in i t ): the top panel of fig 11 plots the reproduction number of the sir model (p = 1) vs. the nsir model with network transmission only (p = 0) in the absence of any interventions or behavioral responses. the lines in the top panel are plotted against cumulative infection count, as analyzed in examples 1-3 above. the infection count is not equally spaced in calendar time since there are many new infections when the disease is peaking than in the its early or late stages. the figure shows that for the chosen modified barabasi-albert graph g the nsir model has lower reproduction rate r t than the sir model, with the gap being the largest in the early stages (see appendix b for more formal discussion on comparing the reproduction numbers in the sir vs. nsir models). the middle and bottom panels in fig 11 plot the reproduction number r nsir t over actual calendar time (days) for several of the simulation scenarios considered in the previous sections. compared to the sir model (p = 1) baseline (the thick dashed line on fig 11) , network-level transmission (p < 1) 'flattens' the reproduction number-for the assumed social network g, the value of r nsir t is initially below that of the sir model but it is higher later on (after approximately 60 days on the figure) and may stay around 1 for a prolonged time if the infection rate is slowed down by testing and contact tracing (see the bottom panel). the impact of distancing policies in bringing r nsir t below 1 is fast and strong, however, the figure also shows that, when the policy is lifted, the reproduction number may quickly rise above 1 again. as a simple illustration of the economic costs analysis of the covid-19 epidemic using the nsir model, i follow [11] to define and compute an index of economic activity based on the number and relative productivity of active vs. quarantined or sick agents in the economy. clearly this measure is very rough and excludes indirect (e.g., additional costs from deaths and hospitalization or psychic costs), long-term (job loss, inability to pay debts, destruction of employment attachment), sectoral (e.g., hospitality vs. it), or general equilibrium effects associated with the (duration of) epidemic or lockdown policies. define the following simple index of economic activity over time, y t which keeps track and varies with the numbers of active and healthy agents vs. locked-down / quarantined agents vs. sick or dead agents. where λ 2 [0 1] is the fraction of agents in lockdown, ρ 2 (0, 1) is the factor with which the productivity of locked down agents is reduced, and α is the fraction of infectious agents (i t or p t ) who are asymptomatic (assumed as productive as healthy agents). the rest, 1 − α of sick agents are assumed to have zero productivity. all "tested positive" agents are assumed in quarantine (productivity ρ). in the simulation results reported in table 3 and fig 12 i assume ρ = 0.5 as in [11] and α = 0.18 as estimated in [30] . the lockdown rate λ is a policy variable. a value of 1 for the index y t is interpreted as "normal times", that is, all agents being healthy and fully productive. in these simulations it is assumed that during the distancing period all agents' productivities are reduced by the factor ρ d = 0.7. these results are just illustrative and assume large economic costs from broad lockdown or distancing (self-isolation). table 3 in section 6 reports the economic loss measured by the index y t across the multiple scenarios considered. specifically, column "gdp loss" reports the average losses, compared to the baseline y t = 1 and annualized to account for the different durations of the epidemic. column "max gdp fall" in table 3 displays the largest decrease in the economic index y t over the epidemic duration. as expected, because of the mandated reduction in production, the distancing or lockdown scenarios entail the largest average (up to 9% annualized decrease in y t ) and maximum economic losses (up to 35% decrease), with the losses increasing in the intervention duration. the second largest economic costs are observed in the no-intervention scenarios (7-9% maximum decrease in y t ), because they result in a large share of infected agents who are assumed less productive. in contrast, the lowest economic losses result in the testing / contact tracing and behavioral response simulation scenarios, where the combination of no mandated lockdown and low infection shares mitigates economic costs. clearly, these results should be interpreted only as illustrative of the productivity losses and the trade-off of between lockdown/distancing vs. infections/deaths since only direct reductions in productivity or output are considered and multiple other factors are omitted: additional cost of deaths, healthcare costs, job losses or (except in the behavioral response scenarios) reductions in economic activity due to fear (e.g., restaurants, travel), etc. table 3 summarizes the simulation results from the previous sections (see there for the corresponding discussion). each table row reports averages over 100 simulations with the same model parameters and policy setting but different pseudo-random number generator seeds that are held constant across the rows (scenarios). there is still a lot of uncertainty and variation in the covid-19 epidemiological parameter estimates and other model ingredients in the current early state of the literature. the baseline parameters i have used in this paper are believed to be current as of early may 2020, however, depending on different data sources and clinical studies, different authors use different values for the removal and mortality rates tied in the model to the parameters r and μ (see section 2), e.g., larger mortality rate or slower removal rate. note that the observed removal rate may be 'contaminated' by policy effects (e.g., if health authorities isolate symptomatic individuals) so using data from policy-treated time periods and locations to estimate the epidemiological parameters should be treated with caution. in fig 13 ( top panel) i explore the implications of using alternative epidemiological parameters relative to the baseline calibration in section 4.1. in simulation 'slower removal a' i keep the initial doubling time the same, so β − r = 0.3 but assume lower removal rate r = 0.1, corresponding to a longer, 10-day on average, infectious period instead of 5 days (this raises the sir r 0 to 4). this creates a higher infection peak and shifts the active infected curve i t forward in time, since there is slower exit from state i. alternatively, in specification 'slower removal b', i assume r = 0.1 but keep r 0 = 2.5 as in the baseline (i.e., use β = 0.25). the result is a higher infection peak but the infection rate curve moves back in time as the epidemic spreads slower due to the lower infectiousness rate. a higher mortality rate, μ = 0.0066r or μ = 0.01r, corresponding to ifr of 0.66% or 1%, has a very minor effect on the infection curve. it does, however, impact total deaths f tmax (not reported on the figure) since they are a fraction μ of cumulative infections, f tmax ' μ(n − r tmax ). finally, i keep r = 0.2 as in the baseline but explore raising the sir r 0 to 5 (i.e., β = 1)-this results in a much earlier and higher infection peak. the middle panel of fig 13 displays simulations exploring lower initial infection rates i 0 = 0.1% or i 0 = 0.01%. the result of these alternative initial conditions is largely just a time shift in the infection curve, suggesting that in empirical work it is important to carefully calibrate the initial condition to match the infection peak. finally, in the bottom panel of fig 13 i perform simulations with alternative specification of the social contacts graph g-by assuming a higher density of contacts (median node degree equal to 13, instead of 10 in the baseline); lower density of contacts (median node degree 8); or a watts-strogatz graph g with mean degree 12. the specification using a denser graph moves the infection peak slightly forward and upward in time, while the opposite is true for the specification with less dense graph. otherwise i find that the shape of the infection curve i t is not very sensitive to these alternative assumptions about the contacts graph g. a necessary step for future empirical work is to calibrate the network g using actual data, e.g., as in [17] . i analyze the combination and interaction of a compartmental epidemiological model and a network model of social contacts (an nsir model). i explore, via calibration and simulations, how network-based transmission and the network structure affect the epidemic dynamics as well as the outcomes and effectiveness of a broad range of policy interventions and behavioral responses, compared to the standard sir model with population-level uniform mixing. i find that viral transmission over a network-connected population can proceed slower and reach lower peak compared to transmission via uniform/random mixing. network-based viral transmission introduces uncertainty and path dependence in the epidemic dynamics, with important role for bridge links and superspreaders. testing, quarantine and contact tracing tend to be more effective in the network model, as these policy interventions can quickly isolate infectious nodes with a large number of contacts. similarly, interventions that can break major transmission vectors across local sub-populations, such as restrictions on non-local travel or bans on mass gatherings are also very effective. other implications of the nsir model remain in line with those in the standard sir models. if lifted early, distancing policies mostly shift the infection peak into the future, while intermittent interventions or endogenous behavioral responses can generate a flattened, multi-peaked infection curve but may have costlier economic consequences by prolonging the epidemic duration. the main advantage of the network approach, compared to standard aggregate sir-type models is that the nsir model captures heterogeneity and locality of social contacts as possible vectors of transmissions. this allows a micro-level, agent-based modeling of health and economic policy outcomes and individual behavioral responses. in addition, the social contact heterogeneity induces path-dependence and role for superspreaders or clusters in the epidemic dynamics (see [13] or [14] for empirical evidence). the main challenge to the network approach is that, in addition to the standard epidemiological parameters governing disease incubation, infectiousness, recovery and mortality, the specification and identification of the social contacts graph, initial conditions and node path followed by the epidemic require additional attention in future empirical work. adding further detail, including agent-level, on the economics side of the model can also yield important insights. formal analysis: alexander karaivanov. a contribution to the mathematical theory of epidemics what will be the economic impact of covid-19 in the us? rough estimates of disease scenarios a simple planning problem for covid-19 lockdown. working paper phase-adjusted estimation of the number of coronavirus disease 2019 cases in wuhan epidemic spreading in scale-free 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equilibrium social distancing. working paper, cambridge u estimating the impact of covid-19 control measures using a bayesian model of physical distancing. pre-print estimating and simulating a sird model of covid-19 for many countries, states, and cities. working paper infection fatality rate of sars-cov-2 infection in a german community with a super-spreading event estimates of the severity of coronavirus disease 2019: a model-based analysis internal and external effects of social distancing in a pandemic. working paper the effect of anti-covid-19 policies on the evolution of the disease: a complex network analysis of the successful case of greece the french connection: the first large population-based contact survey in france relevant for the spread of infectious diseases networks and epidemic models estimating the asymptomatic proportion of coronavirus disease 2019 (covid-19) cases on board the diamond princess cruise ship key: cord-301180-ndiwmnv0 authors: lin, min-hsuan; sivakumaran, haran; apolloni, ann; wei, ting; jans, david a.; harrich, david title: nullbasic, a potent anti-hiv tat mutant, induces crm1-dependent disruption of hiv rev trafficking date: 2012-12-10 journal: plos one doi: 10.1371/journal.pone.0051466 sha: doc_id: 301180 cord_uid: ndiwmnv0 nullbasic, a mutant of the hiv-1 tat protein, has anti-hiv-1 activity through mechanisms that include inhibition of rev function and redistribution of the hiv-1 rev protein from the nucleolus to the nucleoplasm and cytoplasm. here we investigate the mechanism of this effect for the first time, establishing that redistribution of rev by nullbasic is not due to direct interaction between the two proteins. rather, nullbasic affects subcellular localization of cellular proteins that regulate rev trafficking. in particular, nullbasic induced redistribution of exportin 1 (crm1), nucleophosmin (b23) and nucleolin (c23) from the nucleolus to the nucleus when rev was coexpressed, but never in its absence. inhibition of the rev:crm1 interaction by leptomycin b or a non-interacting revm10 mutant completely blocked redistribution of rev by nullbasic. finally, nullbasic did not inhibit importin βor transportin 1-mediated nuclear import, suggesting that cytoplasmic accumulation of rev was due to increased export by crm1. overall, our data support the conclusion that crm1-dependent subcellular redistribution of rev from the nucleolus by nullbasic is not through general perturbation of either nuclear import or export. rather, nullbasic appears to interact with and disrupt specific components of a rev trafficking complex required for its nucleocytoplasmic shuttling and, in particular, its nucleolar accumulation. both the human immunodeficiency virus type-1 (hiv-1) tat and rev proteins are encoded by two exons arranged in alternative reading frames on fully spliced viral mrna [1] . tat and rev are similar in size; tat is typically 101 amino acids long and rev is typically 116 amino acids long, and both have rna binding domains composed of arginine and, in the case of tat, lysine residues which bind to different hiv-1 rna stem loop structures. tat binds to an rna structure in the 59 untranslated region (utr) of all viral transcripts called the trans-activation response element (tar), while rev binds to an intronic region retained by incompletely spliced transcripts called the rev response element (rre). the rna binding domains of both proteins also function as a nuclear/nucleolar localization signal (nls/nols), although recent evidence implies that tat may passively enter the nucleus by diffusing through nuclear pores [2] . both proteins are localized primarily in the nucleus; tat is observed throughout the nucleoplasm with nucleolar accumulation, whereas the nucleocytoplasmic shuttling rev concentrates in the nucleolus in addition to localizing to the nucleoplasm and, to a lesser extent, to the cytoplasm. trafficking of rev in cells has been studied extensively ( fig. 1 ) [3, 4] . in the nucleolus, rev promotes the nuclear export of various hiv-1 mrnas by directly binding to singly-spliced and unspliced viral transcripts via the rre contained therein (fig. 1, step 1 ). exportin 1 (also called crm1 and xpo1) binds to rev through a nuclear export signal (nes; hiv-1 nl4-3 rev amino acids 73 to 84, lqlpplerltld) [5, 6, 7] , which leads to colocalization of rev and crm1 in the nucleolus and subsequent export of the rev:mrna complex from the nucleus to the cytoplasm (fig. 1 , step 2). many other cellular proteins can contribute to rev nuclear export, including hrip/rab, eif5a, ddx3, ddx1, rna helicase a, and pimt that act through rev, and matrin 3 and sam68 that bind to viral mrna [3, 8, 9, 10, 11, 12] . the rev:mrna complex disassembles in the cytoplasm (fig. 1, step 3) allowing rev to recycle back to the nucleus using the transportin 1 or importin b nuclear import pathways (fig. 1, step 4) [3] . once rev enters the nucleus, nucleophosmin (b23) facilitates transport of rev to the nucleolus (fig. 1 , step 5) [13] . b23 is reported to be necessary for the nucleolar localization of both rev and tat through interaction with their respective basic domains [13, 14, 15, 16, 17] . we recently described a mutant of the two-exon hiv-1 tat protein, termed nullbasic, that exhibits antiviral properties by inhibiting multiple steps of the hiv-1 replication cycle [18] . nullbasic was created by replacing the entire arginine-rich rna binding domain of wild type tat with glycine and alanine residues. like similarly mutated one-exon tat mutants, nullbasic exhibits transdominant negative effects on tat-dependent hiv-1 gene expression [18] . however, unlike previously reported mutants [19, 20, 21] , nullbasic also effectively suppresses the steady state levels of unspliced and singly-spliced viral mrna, an activity caused by the inhibition of hiv-1 rev activity [18] . the inhibition by nullbasic was attributed, in part, to a subcellular redistribution of rev from the nucleolus to the nucleoplasm and cytoplasm, but precisely how nullbasic mediates this effect is unknown. in this study, we investigated the effect of nullbasic on the nucleocytoplasmic transport machinery responsible for rev trafficking. not surprisingly, rev recruited and colocalized with several cellular proteins in the nucleolus, including crm1, b23 and nucleolin (c23). however, coexpression of nullbasic with rev resulted in the unexpected redistribution of crm1 and other nucleolar proteins from the nucleolus to the nucleoplasm in a revdependent manner, whereas nullbasic did not affect the distributions of these nucleolar proteins when expressed alone. our experiments support the possibility that nullbasic interferes with an unknown component of the rev nucleocytoplasmic transport complex required for the nucleolar organization and function of rev. hek293t and hela cells were cultured in rpmi 1640 medium supplemented with 10% (v/v) newborn bovine serum (invitrogen) and 1% (v/v) penicillin-streptomycin. all cells were typically incubated at 37uc in a humidified 5% co 2 atmosphere. when 50% -80% confluent, cells were transfected with desired plasmids using fugene 6 transfection reagent (roche applied science) according to the manufacturer's instructions. at 24 h post-transfection, cells were harvested for further analysis. in certain experiments where rev nuclear export was blocked, leptomycin b (sigma-aldrich) was added to growth medium at a final concentration of 20 nm and incubated for 2 h before cell fixation. the two-exon tat (101 amino acids) expression plasmid with flag epitope (tat-flag) was a gift from monsef benkirane, institute de génétique humaine, france. gfp 2 -cnls and gfp 2 -m9core plasmids were gifts from ralph kehlenbach, georgaugust-university of göttingen, germany [22] . the gfp 2 expression plasmid was obtained from addgene, plasmid #20738. the construction of the nullbasic-flag and myc-rev plasmids have been previously described [18] . the pgch infectious molecular clone is a hiv proviral plasmid which expresses authentic hiv-1 rna using the cmv immediate early promoter. the plasmid consists of a pgem7z (-) (promega) backbone containing the cmv promoter fused so that the transcription initiation site uses the correct hiv-1+1 position. with respect to the hiv-1 transcription start site, hiv-1 sequences +1 to +933 from the hiv-1 pnl4-3 proviral plasmid (obtained from the adis research and reference reagent program, plasmid #114) were inserted into the pgem7z (-) via mlui and sphi restriction enzymes sites and +934 to +9251 from the hiv-1 pnl4-3 were inserted into the pgem7z (-) via sphi and xbai restriction enzymes sites. the hiv-egfp expression plasmid was engineered by inserting an egfp sequence from pcdna3.1-egfp into the hiv-1 nef gene in pgch proviral expression vector through bamhi and xbai restriction enzyme sites. the expression vector encoding nullbasic-mcherry was generated by polymerase chain reaction (pcr) amplification of mcherry from pmcherry-lacrep (obtained from addgene, plasmid #18985), which was then inserted into the nullbasic-flag plasmid via bsrgi and xhoi restriction enzyme sites. the eluates were then separated by sodium dodecylsulfatepolyacrylamide gel electrophoresis (sds-page) and electroblotted onto a polyvinylidene fluoride (pvdf) membrane (pall) using a semi-dry transfer system (bio-rad laboratories). tat-flag and nullbasic-flag proteins were detected with a rabbit anti-flag polyclonal antibody (cell signalling technology). myc-rev was detected with a rabbit anti-myc polyclonal antibody (cell signalling technology). endogenous cdk9 was detected with a rabbit anti-cdk9 monoclonal antibody (cell signalling technology). primary antibodies were detected with houseradish peroxidase (hrp)-conjugated goat anti-rabbit antibody (invitrogen). hela cells were grown on glass coverslips and transfected with appropriate expression vectors. after 24 h, the cells were fixed in 3% (w/v) paraformaldehyde at room temperature for 10 min and quenched with 50 mm nh 4 cl for 5 min. cells were then permeabilized with 0.1% (v/v) triton x-100 for 15 min and blocked in 10% (v/v) normal goat serum (millipore) for 15 min. cellular endogenous proteins were detected with rabbit anti-crm1 polyclonal (santa cruz biotechnology), rabbit antifibrillarin monoclonal (cell signalling technology), mouse antinucleophosmin monoclonal (invitrogen) and mouse anti-nucleolin monoclonal (santa cruz biotechnology) antibodies, and myc-rev was probed with either mouse anti-myc monoclonal antibody (millipore) or rabbit anti-myc polyclonal antibody (cell signalling technology). rev expressed from proviral hiv-1 was probed with a mouse anti-hiv-1 rev monoclonal antibody (santa cruz biotechnology). primary antibodies were detected with fitcconjugated goat anti-mouse or anti-rabbit antibodies (invitrogen) and alexa fluor 647-conjugated goat anti-rabbit (invitrogen), cy5-conjugated goat anti-mouse antibodies (invitrogen) or cy3congugated goat anti-rabbit antibodies (invitrogen) (to detect the rabbit anti-flag polyclonal antibody). nuclei were stained with 1 mm 49,6-diamidino-2-phenylindole (dapi, invitrogen). finally, coverslips were mounted onto slides with prolong gold antifade reagent (invitrogen). fluorescent images were captured using a leica tcs sp2 confocal scanning microscope (leica microsystems) and deltavision deconvolution microscope (applied precision) with 636 objective lenses and standard lasers and filters for fitc, mcherry, cy5, cy3 and dapi fluorescence. we previously observed that nullbasic induces considerable redistribution of rev within the cell, from the nucleolus to the nucleoplasm and cytoplasm, leading to significant down regulation of steady state levels of unspliced or singly-spliced viral mrna [18] . to investigate whether nullbasic is able to alter rev's subcellular localization in the context of hiv-1, we visualized the subcellular distribution of rev derived from hiv-1 provirus in the presence or absence of nullbasic. to overcome transcriptional inhibition caused by nullbasic [18] , we utilized a cmv-driven hiv-1 proviral plasmid, pgch, which expresses viral factors independent of tat. the plasmid also has the egfp gene inserted into the nef reading frame (here called hiv-egfp) to enable visualization of hiv expression in cells. hiv-egfp was transfected into hela cells with or without flag-tagged nullbasic fused to the mcherry fluorescent reporter protein (nullbasic-mcherry). we noted strong nuclear accumulation of nullbasic-mcherry compared to a previous study [18] , whereas one exon tat proteins with basic domain mutation were also shown to predominantly localized in the cytoplasm [20, 23, 24] . further comparison of nullbasic localization in hela cells showed that nullbasic fused to flag (fig. s1 , rows 3 and 5) or mcherry ( fig. 2a , row 2) had very similar patterns of distribution in the nucleus and cytoplasm. the localization of rev was determined by indirect immunofluorescence microscopy using an anti-rev antibody 24 h post-transfection. as expected, myc epitope-tagged rev (myc-rev) expressed alone was prominent in the nucleolus and the nucleoplasm but to a lesser extent ( fig. 2a, row 1) . when rev was expressed by hiv-egfp, it was distributed throughout the nucleus (fig. 2b, row 1 ). this nuclear distribution of rev is consistent with its role in nucleocytoplasmic trafficking of viral mrna [3, 25] . coexpression of hiv-egfp and nullbasic-mcherry resulted in the distinct redistribution of rev to the nucleoplasm and cytoplasm (fig. 2b, row2) , comparable to the subcellular redistribution of myc-rev by nullbasic-mcherry ( fig. 2a, row2) . although nullbasic was shown to inhibit revmediated viral mrna transport leading to limited expression of viral proteins from unspliced or singly spliced viral mrna [18] , these data indicate that nullbasic-induced redistribution of rev is independent of other hiv-1 factors under these experimental conditions. others have shown that wild type tat does not interact with rev within cells [26, 27, 28] . to formally exclude the possibility that nullbasic may interact with rev, co-immunoprecipitation reactions were performed using hek293t cells transfected with plasmids encoding flag epitope-tagged nullbasic (nullbasic-flag) and myc-rev. immunoprecipitation of wild type tat-flag with anti-flag beads did not co-immunoprecipitate myc-rev ( fig. 2c ) but did, as expected, co-immunoprecipitate endogenous cdk9, a member of the ptef-b protein complex that interacts with the tat activation domain [29] . similarly, nullbasic-flag did not show evidence of co-immunoprecipitation of myc-rev but did with cdk9 (fig. 2c ). the latter interaction is most likely similar to the interaction between wild type tat and cdk9, through the activation domain preserved in nullbasic-flag. these experiments imply that a nullbasic:rev interaction is not likely to be responsible for the observed mislocalization of rev. in order to investigate the effects of nullbasic on rev nucleocytoplasmic transport, we performed the following analyses on myc-rev expressed by our myc-rev expression plasmid instead of hiv-expressed rev, since the subcellular localization of myc-rev and proviral rev is similarly altered by nullbasic. crm1 can directly bind to the nes sequence in rev to facilitate its export from the nucleus [3] . we therefore investigated the effect of nullbasic-mcherry on the association between myc-rev and endogenous crm1 in transfected hela cells. in control cells transfected with the parental pcdna3.1 plasmid, crm1 was observed widely throughout the nucleus and along the nuclear membrane (fig. 3, row 1) . as previously observed [28, 30, 31] , myc-rev expression induced crm1 relocalization and prominent colocalization of rev and crm1 in nucleoli (fig. 3, row 2) . this colocalization could be reversed by treating cells with leptomycin b (lmb), a specific crm1 inhibitor that directly binds to and inactivates crm1 [32] , thereby preventing its interaction with rev (fig. 3, row 3) . expression of nullbasic alone had no apparent effect on crm1 distribution (fig. 3, row 4) . colocalization between rev and crm1 could also be partially disrupted by the expression of nullbasic-mcherry, in the presence of which myc-rev, but not crm1, redistributed to the cytoplasm (fig. 3, row 5) . importantly, treatment of myc-rev and nullbasic-mcherry expressing cells with lmb resulted in the complete restoration of myc-rev nucleolar accumulation (fig. 3, row 6) , suggesting that nullbasic requires functional crm1 to mislocalize rev most likely by an indirect interaction. to test this hypothesis further, we utilized the rev mutant revm10 which contains a non-functional nes and hence is unable to be recognized by crm1 [33] . as reported for revm10 [17, 34] , myc-revm10 (fig. 4, row 1 ) was unable to interact and colocalize with crm1 in the nucleolus. interestingly, coexpression of nullbasic-mcherry had no disruptive effect on the nucleolar localization of myc-revm10 since, unlike wild type myc-rev, no redistribution to the cytoplasm was observed (fig. 4, row 2) . we thus conclude that an intact rev:crm1 interaction is required for nullbasic to relocalize rev to the cytoplasm. b23 is a nucleolar chaperone and shuttling protein that, in hela cells, is observed in both the nucleolus and nucleoplasm [35] , being associated with nucleolar ribonucleoprotein structures and involved in ribosome biogenesis. b23 is reported to assist rev and tat transport into the nucleolus through interactions with their respective basic domains [13, 14, 15, 16, 17, 36] . mutant tat proteins lacking a basic domain, like nullbasic, have been reported not to interact with b23 in vitro [14] . as previously observed, myc-rev colocalized with b23 in nucleolar structures (fig. 5, row 1) . while expression of nullbasic-mcherry alone had no apparent effect on b23 nuclear distribution (fig. 5, row 2) , coexpression of myc-rev with nullbasic-mcherry resulted in the strong redistribution of both myc-rev and b23 throughout the nucleus, and the observation of myc-rev in the cytoplasm (fig. 5, row 3) . since b23 is known to strongly bind to rev [13] , it is likely that the redistribution of b23 by nullbasic is rev-dependent. the data thus far suggest that nullbasic can alter the localizations of nucleolar proteins in a rev-dependent manner. to determine if nullbasic can generally disrupt nucleolar composition, we monitored the localization of c23 and fibrillarin, two nucleolar proteins that have not been reported to interact with rev. both c23 and fibrillarin colocalized with myc-rev within the nucleoli of transfected cells (fig. 6 , rows 1 and 4, respectively). while expression of nullbasic-mcherry alone had no apparent effect on the nucleolar localization of either protein (fig. 6 , rows 2 and 5), coexpression of nullbasic-mcherry with myc-rev induced relocalization of c23 but not fibrillarin from the nucleolus to the nucleoplasm, in a manner concomitant with the redistribution of myc-rev (fig. 6, rows 3 and 6 , respectively). we therefore conclude that nullbasic does not cause a generalized mislocalization of nucleolar proteins, but effects the relocalization of only certain, specific proteins (including crm1, b23 and c23) in a manner dependent on the presence of rev. five different importin b family members, including importin b, transportin 1, importin 5, importin 7 and importin 9, have been reported to recognize the rev nls and facilitate the transport of rev into the nucleus, with different pathways being used in a cell line-dependent manner [22, 37, 38, 39] . in hela cells, for example, transportin 1 is the dominant rev nuclear import pathway [22, 40] , while the importin b pathway is favored in hek293t cells [40] . to eliminate the possibility that nullbasic-mediated blockade of a nuclear import pathway leads to the observed cytoplasmic accumulation of rev, two nls-containing reporters, gfp 2 -cnls and gfp 2 -m9core, which mimic cargo for nuclear import by importin b and transportin 1 respectively, were expressed individually or along with nullbasic-mcherry in hela cells. as shown in figure 7 , there was no change in the nuclear accumulation of either reporter in the presence of nullbasic-mcherry (fig. 7, rows 2 and 4) compared to in its absence (fig. 7, rows 1 and 3) . nullbasic-mcherry did not affect localization of gfp 2 lacking a nuclear import signal (fig. 7, rows 5 and 6) . similar experiments indicated that coexpression of myc-rev with nullbasic-mcherry likewise had no effect on the nuclear localizations of the respective reporters (data not shown). while we cannot exclude interference of other pathways, we conclude that the accumulation of rev in the cytoplasm induced by nullbasic is not caused by a general inhibition of the importin b and transportin 1 nuclear import pathways. this study aimed to elucidate a possible mechanism by which the two-exon anti-viral acting tat mutant, nullbasic, perturbs rev subcellular localization through the active mislocalization of a set of cellular proteins that control rev trafficking. nucleocytoplasmic shuttling of rev is critical for hiv-1 infectivity since viral protein synthesis, and subsequent virion assembly, require the coordinated transport of incompletely spliced viral mrnas from the nucleus. nullbasic's action in downregulating rev's mrna transport function by mechanisms that include the substantial mislocalization of rev from nucleolus to nucleoplasm and cytoplasm (see fig. 2 ) [18] is therefore of great importance. immunoprecipitation of either nullbasic-flag or tat-flag from lysates of cells coexpressing myc-rev revealed no co-immunoprecipitation of myc-rev (fig. 2c) , supporting the idea that nullbasic does not interact with rev. furthermore, fluorescence resonance energy transfer analyses between a tat-gfp fusion protein and a rev-bfp fusion failed to detect any interaction in living cells [26, 27] , a result which we reason will likewise apply to nullbasic and rev. it is thus reasonable to conclude that nullbasic interferes with rev nucleolar localization by an indirect mechanism. the fact that nullbasic strongly disrupts the nucleolar localization of rev in an indirect manner suggests that nullbasic may interfere with a cellular factor important for rev nucleolar accumulation. the nucleolus is a complex structure where several thousands of different cellular proteins have been identified [41] . among these, crm1 is a rev binding protein that regulates rev trafficking throughout the nucleus. crm1 is a cellular receptor that recognizes and binds to the leucine-rich nes domain of rev, an interaction which facilitates the egress of rev/viral mrna complexes through nuclear pores [3, 4, 42] and is readily visualized in cells as a colocalization in the nucleolus (fig. 3, row 2 ). our evidence strongly indicates that crm1 is a mediator through which nullbasic induces rev redistribution, since disrupting the interaction between crm1 and rev mitigates the effect of nullbasic. this was evident through the use of lmb (fig. 3) , a specific inhibitor of crm1, and the revm10 mutant, whose altered nes is no longer recognized or bound by crm1 (fig. 4) . in both cases, rev retained a nucleolar localization despite high levels of nullbasic expression. as sole expression of nullbasic does not appear to directly affect crm1 localization (fig. 3 , row 1 compared to row 4), the precise mechanism by which crm1 mediates nullbasic-induced redistribution remains to be elucidated. b23, c23 and fibrillarin are among the most abundant proteins in the nucleolus [43] . b23 is a nucleolar shuttling factor that recognizes and binds to the nols of rev [13] , tat [44] and c23 [45] . while primarily located in the nucleolus, b23 can display a nucleocytoplasmic distribution as a consequence of its shuttling function [46] . nullbasic-mediated redistribution of rev in this study was accompanied by the mislocalizations of b23 (fig. 5) and c23 (fig. 6 ), but not fibrillarin (fig. 6) . it is likely that the mislocalization of b23 in the presence of redistributed rev is a consequence of their strong interaction [13] rather than through an interaction between nullbasic and b23 because, while tat has been reported to interact with b23 through its basic domain [44] , the same domain in not present in nullbasic [18] . due to the lack of evidence of a direct interaction between c23 and rev, the mislocalization of c23 may be a result of its interaction with mislocalized b23 [45] . the observed mislocalizations, however, were largely confined to the nucleoplasm even when rev was substantially redistributed into the cytoplasm. it is important to note that nullbasic did not affect the localizations of crm1, b23 and c23 in the absence of rev (figs. 3, 4, 5 and 6 ). that nullbasic did not affect the nucleolar distribution of fibrillarin (fig. 6) , either in the presence or absence of rev, indicates that nullbasic is not inducing a generalized deformation or reconfiguration of cellular nucleoli. further, the retention of the importin b and transportin 1 nuclear import reporters in the nucleus in the presence of nullbasic demonstrates that nullbasic does not effect a general perturbation of these trafficking pathways (fig. 7) . it is evident, therefore, that nullbasic induces the redistribution of crm1, b23 and c23 only in the presence of rev. considering all of the available data, we propose that rev induces the formation of a nuclear or nucleolar complex to reconcile why nullbasic redistributes only certain nucleolar proteins in a rev-dependent manner. this rev containing complex would be enriched for crm1 and colocalized with b23 and c23 in order to facilitate the proper processing and nuclear export of hiv-1 unspliced and singly-spliced transcripts [27, 47, 48] . nullbasic may destabilize the complex that includes rev by binding to an unidentified factor that is important for its formation, resulting in the redistribution of rev and concomitant redistributions of b23 and c23 throughout the nucleoplasm. the liberated rev:crm1 complexes would freely traffic out of the nucleus, resulting in the observed redistribution of rev into the cytoplasm, and which would be counteracted by lmb treatment or the revm10 mutation. similar nucleolar hijacking and modification have been described for other viruses. the replication of certain dna and rna viruses (such as adenovirus, herpes simplex virus, hepatitis b virus, hiv-1, hepatitis c virus, japanese encephalitis virus, hendra virus, nipah virus, sars coronavirus and dengue virus) require the trafficking of viral proteins through the nucleolus [42] . in some instances, for example during herpes simplex virus and adenovirus infections, this results in the dramatic mislocalization of cells were immunostained with anti-myc (green) and either anti-c23 (magenta, upper panels) or anti-fibrillarin (magenta, lower panels) antibodies before the subcellular localizations of myc-rev, endogenous c23 or endogenous fibrillarin, and nb-mcherry (red) were analyzed by fluorescence microscopy. nuclei were stained with dapi. the overlay panels show the merge of the myc-rev panel with either the c23 or fibrillarin panels (rows 1, 3, 4 and 6) and the nb-mcherry panel with either the c23 or fibrillarin panels (rows 2 and 4). all figures are representative of five fields each from four independent experiments. the white bar in last panel is equal to 10 mm. doi:10.1371/journal.pone.0051466.g006 c23 and b23 [42, 49] . given such evidence from other viruses, it is entirely consistent that rev may similarly manipulate nucleoli factors in hiv-1 infected cells. identification of nullbasic interacting factors that are required for rev nucleolar accumulation may provide insights into the design of potent yet specific antiretroviral inhibitors. overall, our data provide evidence that nullbasic appears to interact with and disrupt specific components of a rev trafficking complex important for rev's nucleocytoplasmic trafficking and nucleolar accumulation, resulting in crm1-mediated rev redistribution. mutant hiv-1 proteins such as nullbasic thus not only provide insights into the functions of their wild type counterparts, but may also reveal novel drug targets in previously unrecognized host/ pathogen interactions. crm1-dependent subcellular redistribution of rev from the nucleolus by nullbasic is not through general perturbation of either nuclear import or export. rather, our data suggest that nullbasic appears to interact with and disrupt specific components of a rev trafficking complex required for rev nucleocytoplasmic shuttling and, in particular, nucleolar accumulation. figure 7 . nullbasic does not affect the nuclear import of importin b and transportin 1 cargoes. hela cells were transfected to express the importin b-dependent nuclear import reporter gfp 2 -cnls, or the transportin 1-dependent nuclear import reporter gfp 2 -m9core, either alone (rows 1 and 3, respectively) or with nullbasic (nb)-mcherry (rows 2 and 4, respectively). the subcellular localizations of the reporter proteins (green) and nb-mcherry (red) were analyzed in fixed cells by fluorescence microscopy. nuclei were stained with dapi. the overlay panels show the merge of the gfp reporter and dapi panels. plasmid expressing gfp 2 was also transfected into cells either alone or with nb-mcherry as a vector control (rows 5 and 6, respectively). figures are representative of five fields each from four independent experiments. the white bar in last panel is equal to 10 mm. doi:10.1371/journal.pone.0051466.g007 crm1-dependent inhibition of rev trafficking hiv-1 replication in vivo study of hiv-1 tat arginine-rich motif unveils its transport properties cellular proteins and hiv-1 rev function virus-host interactions: role of hiv 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transdominant tat protein localized to both the nucleus and cytoplasm immunodeficiency virus rev trans-activator modulates the expression of the viral regulatory genes automatic and quantitative measurement of protein-protein colocalization in live cells in vivo hiv-1 rev multimerization in the nucleolus and cytoplasm identified by fluorescence resonance energy transfer kinetic and molecular analysis of nuclear export factor crm1 association with its cargo in vivo interactions between tat and tar and human immunodeficiency virus replication are facilitated by human cyclin t1 but not cyclins t2a or t2b a synthetic hiv-1 rev inhibitor interfering with the crm1-mediated nuclear export nucleoporins nup98 and nup214 participate in nuclear export of human immunodeficiency virus type 1 rev leptomycin b inactivates crm1/exportin 1 by covalent modification at a cysteine residue in the central conserved region functional dissection of the hiv-1 rev trans-activator-derivation of a trans-dominant repressor of rev function inhibition of human immunodeficiency virus rev and human t-cell leukemia virus rex function, but not mason-pfizer monkey virus constitutive transport element activity, by a mutant human nucleoporin targeted to crm1 npm1/b23: a multifunctional chaperone in ribosome biogenesis and chromatin remodeling intracellular trafficking and interactions of the hiv-1 tat protein the arginine-rich domains present in human immunodeficiency virus type 1 tat and rev function as direct importin betadependent nuclear localization signals interactions between hiv rev and nuclear import and export factors: the rev nuclear localisation signal mediates specific binding to human importin-beta multiple importins function as nuclear transport receptors for the rev protein of human immunodeficiency virus type 1 intermolecular masking of the hiv-1 rev nls by the cellular protein hic: novel insights into the regulation of rev nuclear import nopdb: nucleolar proteome database-2008 update involvement of the nucleolus in replication of human viruses involvement of the plant nucleolus in virus and viroid infections: parallels with animal pathosystems protein b23 is an important human factor for the nucleolar localization of the human immunodeficiency virus protein tat c23 interacts with b23, a putative nucleolar-localization-signal-binding protein major nucleolar proteins shuttle between nucleus and cytoplasm the roles of nucleolar structure and function in the subcellular location of the hiv-1 rev protein recruitment of the crm1 nuclear export factor is sufficient to induce cytoplasmic expression of incompletely spliced human immunodeficiency virus mrnas adenovirus protein v induces redistribution of nucleolin and b23 from nucleolus to cytoplasm we thank dongsheng li and kylie warren for their advice and comments. key: cord-306278-c4q4la5c authors: esposito, susanna; zampiero, alberto; bianchini, sonia; mori, alessandro; scala, alessia; tagliabue, claudia; sciarrabba, calogero sathya; fossali, emilio; piralla, antonio; principi, nicola title: epidemiology and clinical characteristics of respiratory infections due to adenovirus in children living in milan, italy, during 2013 and 2014 date: 2016-04-05 journal: plos one doi: 10.1371/journal.pone.0152375 sha: doc_id: 306278 cord_uid: c4q4la5c to evaluate the predominant human adenovirus (hadv) species and types associated with pediatric respiratory infections, nasopharyngeal swabs were collected from otherwise healthy children attending an emergency room in milan, italy, due to a respiratory tract infection from january 1 to february 28 of two subsequent years, 2013 and 2014. the hadvs were detected using a respiratory virus panel fast assay (xtag rvp fast v2) and with a hadv-specific real-time polymerase chain reaction; their nucleotides were sequenced, and they were tested for positive selection. among 307 nasopharyngeal samples, 61 (19.9%) tested positive for hadv. hadv was the only virus detected in 31/61 (50.8%) cases, whereas it was found in association with one other virus in 25 (41.0%) cases and with two or more viruses in 5 (8.2%) cases. human enterovirus/human rhinovirus and respiratory syncytial virus were the most common co-infecting viral agents and were found in 12 (19.7%) and 7 (11.5%) samples, respectively. overall, the hadv strain sequences analyzed were highly conserved. in comparison to hadv-negative children, those infected with hadv had a reduced frequency of lower respiratory tract involvement (36.1% vs 55.2%; p = 0.007), wheezing (0.0% vs 12.5%; p = 0.004), and hospitalization (27.9% vs 56.1%; p<0.001). antibiotic therapy and white blood cell counts were more frequently prescribed (91.9% vs 57.1%; p = 0.04) and higher (17,244 ± 7,737 vs 9,565 ± 3,211 cells/μl; p = 0.04), respectively, in children infected by hadv-c than among those infected by hadv-b. on the contrary, those infected by hadv-b had more frequently lower respiratory tract involvement (57.1% vs 29.7%) but difference did not reach statistical significant (p = 0.21). children with high viral load were absent from child care attendance for a longer period of time (14.5 ± 7.5 vs 5.5 ± 3.2 days; p = 0.002) and had higher c reactive protein levels (41.3 ± 78.5 vs 5.4 ± 9.6 μg/dl; p = 0.03). this study has shown that hadv infections are diagnosed more commonly than usually thought and that hadvs are stable infectious agents that do not frequently cause severe diseases. a trend toward more complex disease in cases due to hadv species c and in those with higher viral load was demonstrated. however, further studies are needed to clarify factors contributing to disease severity to understand how to develop adequate preventive and therapeutic measures. frequently cause severe diseases. a trend toward more complex disease in cases due to hadv species c and in those with higher viral load was demonstrated. however, further studies are needed to clarify factors contributing to disease severity to understand how to develop adequate preventive and therapeutic measures. human adenoviruses (hadvs) are a group of at least 68 non-enveloped viruses containing double-stranded linear dna [1] . they belong to the family adenoviridae, genus mastadenovirus and are categorized into seven species (a-g) according to their biophysical, biochemical, and genetic characteristics. moreover, in each of these species, several types have been identified [1] . species identity strongly correlates with antigenicity, epidemiologic characteristics, clinical manifestations of hadv infection, and in vitro response to some antiviral drugs [2, 3] . from a clinical point of view, species d (hadv-d8, hadv-d19, and hadv-d37) is usually associated with the development of conjunctivitis; species f (hadv-f40 and hadv-f41) is usually associated with gastroenteritis; and species b (hadv-b3 and hadv-b7), c (hadv-c1, hadv-c2, and hadv-c5), and e (hadv-e4) are usually associated with respiratory diseases [2] . recombination between members of the same species and between members of different species has been frequently described [4] . as a result, certain new types may acquire different pathogenicity and have strong potential for widespread and epidemic outbreaks. consequently, surveillance of hadv circulation with an early evaluation of the relationships between clinical manifestations and molecular characteristics of new infecting strains may be important for the development of adequate diagnostic, prophylactic, and therapeutic measures against hadv infection. hadvs play an important role in the determination of respiratory infections, particularly in children. hadvs are responsible for a number of lower respiratory tract diseases in children, including community-acquired pneumonia (cap). wo et al. reported that among 3,089 nasopharyngeal aspirates collected in children with cap in china, 186 (6.0%) tested positive for hadv [5] . although hadvs are associated with mild to moderate disease in most cases, lifethreatening disease can occur in some patients, particularly if they are immunocompromised, [2] . overall, little data on hadv circulation have been collected in europe and no recent data regarding the epidemiology, molecular characterization, and clinical features of respiratory hadv infections in children have been collected in italy. the main aim of this study was to evaluate the predominant hadv species and types associated with pediatric respiratory infections in milan, italy, during two consecutive winter seasons. clinical features related to hadv types and genetic characteristics were also studied. to evaluate the circulation of the different hadv types and the possible relationship between viral load, viral genetic characteristics, and the severity of infection, nasopharyngeal swabs were collected from otherwise healthy children consecutively attending the emergency room of the fondazione irccs ca' granda ospedale maggiore policlinico, university of milan, italy, due to a respiratory tract infection. the study was carried out during the period from january 1 to february 28 in two subsequent years, 2013 and 2014, and was approved by the ethics committee of the fondazione irccs ca' granda ospedale maggiore policlinico, milan, italy. written informed consent from a parent or legal guardian was required, and children 8 years of age were asked to give their written assent. patient demographic characteristics and medical histories were systematically recorded before the visit to the emergency room using standardized written questionnaires. the study patients were classified into disease groups (i.e., acute otitis media, rhinosinusitis, pharyngitis, croup, infectious wheezing, acute bronchitis, pneumonia) on the basis of signs and/or symptoms using well-established criteria and were subdivided into two subgroups: upper respiratory tract infections (urtis) and lower respiratory tract infections (lrtis) [6] . nasopharyngeal secretions were collected from all of the children immediately after admission to the emergency room using a paranasal flocked swab (1 swab per child), which was stored in a tube containing 1 ml of universal transport medium (kit cat. no. 360c, copan italia, brescia, italy). viral nucleic acids were extracted from the swab by means of a nuclisens easymag automated extraction system (biomeriéux, craponne, france), and the extract was tested for respiratory viruses using the respiratory virus panel (xtag rvp fast v2) (luminex molecular diagnostics, inc., toronto, canada) fast assay in accordance with the manufacturer's instructions (luminex molecular diagnostics inc.) this assay simultaneously detects the following viruses: influenza a virus (flua subtypes h1 or h3); influenza b virus (flub); respiratory syncytial virus (rsv); parainfluenzaviruses (hpiv) 1-4; adenoviruses (hadv); human metapneumovirus (hmpv); coronaviruses (hcov) -229e, -nl63, -oc43, and -hku1; enterovirus/rhinovirus (hev/hrv) and human bocavirus (hbov). moreover, considering the risk of false negative results reported for the rvp fast v2 assay [7] , the negative samples were also tested with an alternative hadvspecific real time-pcr [8] . positive results were also quantified with a hadv-specific real-time polymerase chain reaction (pcr), as previously described [9] . viral nucleic acid extracts were tested using a specific hadv plasmid by a single-plex realtime pcr using taqman universal master mix ii (applied biosystems, foster city, california, usa). amplification and detection of viral dna was performed with a 7900ht real-time pcr system instrument (applied biosystems). the real-time hadv-specific primer sequences were as follows: 5'-gccacggtggggtttctaaactt-3', adenoquant 1 (aq1) and 5'-gcccc agtggtcttacatgcacatc-3', adenoquant 2 (aq2). the sequence of the probe was 5'-tgcaccagacccgggctcaggtactccga-3' (adenoprobe) labeled with fam on the 5'end as a fluorescent dye and labeled with tamra on the 3'-end as a fluorescence quencher dye. cycling conditions were as follows: 50°c for 2 min, 95°c for 8 min and 50 cycles of 95°c for 15 sec and 59°c for 1 min. the plasmid amplified target fragment was verified by sequencing [7] . plasmid dna concentrations were detected using an nd-1000 spectrophotometer (nanodrop products, wilmington, de). real-time fluorescence quantitative pcr was carried out in a total reaction volume of 20 μl consisting of 10 μl of taqman universal master mix (applied biosystems), 0.8 μl (0.4 mm) of each primer, 0.6 μl (0.3 mm) of the probe, 5 μl of template, and 2.8 μl of double-distilled water. the real-time pcr thermal cycling reaction and quantitative measurement were performed in a stepone real-time pcr instrument (applied biosystems) using the following conditions: one cycle at 50°c for 2 min, one cycle at 95°c for 10 min, 45 cycles at 95°c for 15 s, and one cycle at 60°c for 1 min [9] . each run included plasmid and negative controls. standard precautions were taken throughout the pcr process to avoid cross-contamination. negative controls and serial dilutions of the plasmid positive control were included in every pcr assay. finally, quantitative results were reported as dna copies/ml of respiratory samples. hadv typing was performed by sequencing the hypervariable region (1-6) of loop 1 of the hexon protein using a protocol proposed by lu and erdman [10] . pcr products ranging in size from 764 to 896 bp (first pcr) and 688 to 821 bp (nested pcr). first-round amplification was carried out using primers for adhexf1 (nt 19135-19160 geneamp pcr system 9700 (applied biosystems) with the following settings: 94°c for 2 min denaturation followed by 35 cycles of 94°c for 1 min, 45°c for 1 min, and 72°c for 2 min, with a final extension of 72°c for 5 min. for the nested reaction, 0.5 μl of the first pcr product was amplified as above. amplified products were separated on 1% agarose gels and purified with the qiaquick pcr purification kit (qiagen, chatsworth, california, usa). sequencing was performed in both directions using adhexf2/adhexr2 primers and the abi prism bigdyetm terminator cycle sequencing ready reaction kit ver. 3.1 on an abi 3100 dna sequencer (applied biosystems). sequencher 3.1.1 software (gene codes, ann arbor, minnesota, usa) was used for sequence assembly and editing. all sequences were aligned using clustalx 2.1 and bioedit (version 7.1.3.0) software (ibis biosciences, carlsbad, california, usa). phylogenetic trees were generated using the maximum likelihood method with molecular evolutionary genetics analyses (mega) software, version 5.05 [11] , and adenovirus prototype strains. bootstrap probabilities for 1,000 iterations were calculated to evaluate confidence estimates. the graphs were made using graphpad prism version 5.01 for windows (graphpad software, san diego, california, usa). all the hadv sequences originated from this study were submitted to genbank (accession numbers kt963953-kt964000). descriptive statistics of the responses were generated. continuous variables were presented as the mean values and standard deviations (sds), and categorical variables were presented as numbers and percentages. for categorical data, comparisons between groups were performed using a contingency table analysis with a χ 2 or fisher's exact test when appropriate. for ordered categorical data, a cochran-armitage test for trends was used to compare the groups. continuous data were analyzed using a two-sided student's t-test after ensuring the data were normally distributed (based on the shapiro-wilk statistic) or using a two-sided wilcoxon's rank-sum test if the data were non-normal. all analyses were two tailed, and p-values of 0.05 or less were considered to be statistically significant. all analyses were conducted using sas version 9.2 (cary, nc, usa). during the two study periods, a total of 307 nasopharyngeal samples were collected in the emergency room. of these, 61 (19.9%) tested positive for hadv. the luminex xtag rvp fast v2 assay identified 30 cases, all confirmed by real-time pcr. this method revealed 31 positive cases that tested negative with the rvp fast v2 assay. among the hadv infected children, 14.8% were <1 year old, whereas 42.6% and 42.6% were 1-2 and 3 years old, respectively ( table 1) . the prevalence of hadv detection was similar in the two studied periods: 28 (45.9%) and 33 (54.1%) positive samples were collected in 2013 and 2014, respectively. hadv was the only virus detected in 31/61 (50.8%) cases, whereas it was found in association with one other virus in 25 (41.0%) cases and with two or more viruses in 5 (8.2%) cases. hev/hrv and rsv were the most common co-infecting viral agents and were found in 12 (19.7%) and 7 (11.5%) samples, respectively. molecular typing assignments were based on the identity of the closest matching sequences after both blast and phylogenetic analysis. the 61 hadvs belonged to species b in 7 cases (11.5%; all hadv-b3), species c in 37 cases (60.7%; 10 hadv-c1, 25 hadv-c2, and 2 hadv-c5), species d in 1 case (1.7%; hadv-d26), species e in 2 cases (3.4%; hadv-e4), and species f in 1 case (1.6%; hadv-f41) (fig 1) . it was not possible to identify the species and type of 13 (18.6%) samples due to inadequate sample volume. no peculiar clustering was observed among hadv strains. hadvs circulating in 2013 were closely related to strains identified in 2014. however, among hadv-c2 sequences, two distinctive branches were observed with 15 and 10 italian strains (fig 1) . similarly, in the branch of the tree corresponding to the hadv-c1 strains, two strains appeared to cluster separately from the other 8 strains. using 10 6 dna copies/ml as a cut-off, the viral load was classified as low in 37 (62.7%) and as high in 22 (37.3%) cases (2 hadv-b, 14 hadv-c, and 7 of 11 without species identification). for hadv-b, viral load varied from 1.4 x 10 2 to 3.9 x 10 8 copies/ml, whereas for hadv-c it was between 3.4 x 10 3 and 3.0 x 10 9 copies/ml. no significant difference in viral load was observed between each hadv species the sequence identity matrix of the hadv partial hexon gene for groups with at least 7 sequences (hadv-c2, -c1 and -b3) showed a minimum to maximum identity range of 97.6-100.0% for hadv-c2, 98.7-100% for hadv-c1, and 99.4-100% for hadv-b3. overall, the hadv strain sequences analyzed were highly conserved and only few amino acid changes were observed. in detail, among the hadv-c2 sequences, 15/25 strains (60.0%) had an insertion of one glutamic acid (e) in position 151 and the m305l change. in two of serotype was not available for 13 positive subjects, and viral load was not available for 2 positive subjects. viral load was categorized in two groups and was considered "low" for values <6 log(copies/ml) and "high" for values 6 log(copies/ml). no statistically significant result emerged for the relationship between adenovirus types and age or between viral load and age. doi:10.1371/journal.pone.0152375.t001 these strains, the additional change s195t was identified. among the hadv-c1 sequences, only one amino acid change (a190t) was evidenced in 2/10 strains (20.0%). finally, in 5/7 strains (71.4%) belonging to the hadv-b3 group, two changes, g205v and t254i, were observed. in table 2 , demographic, clinical, and laboratory characteristics of children infected by hadv alone or co-infected with hadv and one or more other respiratory viruses are compared with those of children with respiratory infection due to other agents. a preliminary analysis revealed that no statistically significant difference between cases infected by hadv alone or co-infected with other viruses, in particular rsv or rhinovirus could be evidenced, all the children with hadv infection were considered together. as shown, in comparison to hadv-negative children, those infected with hadv were younger (4.3 ± 3.3 vs 3.2 ± 2.5 years; p = 0.01) and had high-grade fever more frequently (56.4% vs 72.4%; p = 0.03). moreover, children infected with hadv had lower respiratory tract involvement less frequently (55.2% vs 36.1%; p = 0.007) and never suffered from wheezing unlike children with disease due to other etiologic agents. crp, c reactive protein; sd, standard deviation; spo 2 , peripheral oxygen saturation."38.0°c or more any time during the illness (before or at enrolment, or during follow-up);°39.0°c or more any time during the illness (before or at enrollment or during follow-up). a data were extracted from datasets of different studies that collected different information; therefore, the denominators vary across characteristics. however, when not indicated the reported number refers to the whole enrolled sample. children infected with other agents wheezed in 12.5% of the cases (p = 0.004) and were hospitalized more frequently (56.1% vs 27.9%; p<0.001). no other significant differences between groups were observed. in table 3 , comparisons based on demographic, clinical, and laboratory variables between subjects with hadv b and c species are shown. two significant differences were found between the groups: antibiotic therapy was more frequently prescribed (91.9% vs 57.1%; p = 0.04) and white blood cell count was higher (17,244 ± 7,737 vs 9,565 ± 3,211; p = 0.04) in children infected by hadv-c. table 4 shows data regarding characteristics of children with hadv infection according to viral load. children with high viral load were younger, had high-grade fever more frequently, were more frequently hospitalized, were absent from the community for a longer period of time, and had a higher c reactive protein (crp) level. however, differences were statistically significant only for absence from the community (14.5 ± 7.5 vs 5.5 ± 3.2 days; p = 0.002) and crp level (41.3 ± 78.5 vs 5.4 ± 9.6 μg/dl; p = 0.03). several previous epidemiological studies have shown that hadvs are considered the cause of respiratory infections in otherwise healthy children in approximately 4-10% of the cases [12] [13] [14] [15] . in this study, similarly to what has been found in asia by other authors [16] , a prevalence of approximately 20% was found, suggesting that the relevance of this infectious agent in the determination of respiratory problems could be higher than previously thought. the methods used to identify hadvs might partially explain this finding. in the past, most of the epidemiological studies of viral respiratory infections were carried out using methods that could underestimate viral presence in respiratory secretions, such as viral culture, antigen detection by immunofluorescence, and visualization by electron microscopy [17] . to overcome this problem, molecular methods were suggested. multiplex assays, including the rvp fast v2 assay, were developed to obtain the simultaneous identification of all the most common respiratory viruses and are now commonly used in routine practice. as previously reported [8] and confirmed by this study, the rvp fast v2 assay has poor sensitivity for hadv and can lead to undervaluation of the real importance of these viruses in the determination of respiratory infections. the addition of a specific real-time pcr can solve this issue. moreover, the higher than expected prevalence of hadv infection evidenced by this study could be strictly related to the period during which it was carried out. samples were collected in two winter months of two consecutive years. although hadvs circulate during the whole year, peak periods are in winter and early spring [1] . consequently, it is possible that the study was carried out during epidemics leading to the higher prevalence values reported here. in this study, the most commonly identified species were b and c, types 3, 2, and 1. this is not surprising because, despite possible temporal and regional changes in predominant type [18] , these types are more commonly reported as the cause of respiratory infection worldwide. hadv-b3 has been identified in successive outbreaks of severe acute respiratory illnesses in korea [19, 20] , brazil [21] , and taiwan [22, 23] , where this virus was the predominant type for respiratory hadv infection from 1981 to 2002. moreover, together with other hadv types, it has been the causative agent in epidemic outbreaks of respiratory diseases in europe, america, and oceania [24] [25] [26] . finally, hadv-b3 is known to be a causative agent of a characteristic syndrome of acute pharyngo-conjunctival fever in older children and adults, especially in summer camps and swimming pools [27] . types c1 and c2 have been more frequently reported as the cause of endemic or sporadic cases [28] , although there have been reports of epidemics [19] . in italy, a survey carried out approximately 10 years ago found that hadv-c1 and -c2 were the most common hadvs isolated in patients with infection due to these agents [28] . the same was shown by this study, showing that epidemics of infection due to the same hadv in a given geographic area can be prolonged compared with outbreaks of rsv, parainfluenza virus, and influenza viruses, which are well defined and have duration limited to some months [29, 30] . the severity of hadv infection varies according to age, socioeconomic status, environmental status, and above all, the immunological characteristics of the patient. detection of hadv in severely immunocompromised children has been implicated as a risk factor for poor outcome [31] . however, severe cases have been frequently described in otherwise healthy children [5, 19, 20] . in this study, most of the children with hadv infection had a mild infection and, globally, the severity of respiratory infection of children in whom hadv alone or in association with other respiratory viruses was identified was lower than that due to other infectious agents considered together. both the prevalence of lrtis and hospitalization rate were significantly lower in hadv-infected children than in children not infected by hadv. the long-term circulation of hadvs with similar genetic characteristics could partially explain the generally poor clinical relevance of infections due to the strains identified in this study. in italy, the most common hadvs identified in children during the periods of this study were of the same species and of the same types of those identified several years before. moreover, despite sequencing analyses that were focused on one of the more variable genes (hexon) [32] , no significant variation in hadv genetic characteristics was seen and no recombination between viruses was found. hadv-c2 strains were the only strains to have two slightly different clusters, suggesting the circulation of two different hadv-c2 strains with indistinct pathogenetic roles. as a result, it is possible that many of the children had previously had contact with these viruses and developed sufficiently high immunity to limit the clinical expression of subsequent infections. however, the number of non-hadv infection children included in this study is significantly higher than that of hadv infected patients and this difference could have led comparison to wrong results. a longer period and expanded surveillance may help to construct the complete picture on the hadv circulation, other infections and related clinical features. prevalence of lrti was higher in children infected by type b3, although the difference compared with type c was not statistically significant and the total number of patients with this type of infection is too small to draw definitive conclusions. potentially increased virulence of type b3 in comparison to other hadvs is not surprising because this virus has already been associated with a number of severe lrtis [5, 19, 20] and to the development of acute meningo-encephalopathy [33] . in this study, higher viral load was more common in children with some markers of more severe disease, such as higher fever, higher hospitalization rate, higher crp values, and delayed return to normal activities, independent of the infecting hadv type. however, clinical differences between patients with high or low viral load were not always significant, and it is not possible to state that hadv load can be a marker for severity of infection. by contrast, hadv load was found to be significantly higher in patients developing severe hadv infection after transplantation, especially in pediatric stem cell transplant recipients [31] . consequently, the measurement of hadv load is considered a possible method for an early diagnosis of disseminated hadv disease and for the initiation and monitoring of antiviral therapy in these subjects [34] . further studies are needed to evaluate whether high viral load could indicate which subjects might have to receive antiviral therapy to avoid negative evolution of the infection even if they are not immunocompromised. in conclusion, this study has shown that when adequately investigated, hadv infections are diagnosed more commonly than usually thought. moreover, these data seem to indicate that hadvs are stable infectious agents that do not frequently incur genetic variations and, for this reason, do not cause frequently severe diseases. it seems that there are some differences in the severity of disease outcome between types and according to viral load, with hadv type c and high viral load apparently associated with a more severe disease. however, further studies are needed to identify the potential pathogenetic role of the different species and types of hadv and the importance of viral load in the severity of infection. clarification of these unsolved problems may be useful for deciding how to develop adequate preventive and therapeutic measures for immunocompromised and otherwise healthy children who suffer from hadv infection. family adenoviridae adenovirus infections in immunocompetent and immunocompromised patients differential susceptibility of adenovirus clinical isolates to 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single-tube multiplex reverse transcription-pcr: feasibility study rapid detection of respiratory viruses by centrifugation enhanced cultures from children with acute lower respiratory tract infections viral etiology of acute respiratory infections among children in porto alegre, rs, brazil high-incidence of human adenoviral co-infections in taiwan respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology lower respiratory tract infections due to adenovirus in hospitalized korean children: epidemiology, clinical features, and prognosis genome type analysis of adenovirus types 3 and 7 isolated during successive outbreaks of lower respiratory tract infections in children antigenic and genomic characterization of adenovirus associated to respiratory infections in children living in northeast brazil respiratory adenoviral infections in children: a study of hospitalized cases in southern taiwan in 2001-2002 a two-decade survey of respiratory adenovirus in taiwan: the reemergence of adenovirus types 7 and 4 adenovirus type 3 pneumonia causing lung damage in childhood an outbreak of adenovirus type 3 disease at a private recreation center swimming pool on the aetiology of whooping cough molecular characterization of human adenoviruses isolated in italy epidemiology of acute lower respiratory disease in children adenovirus type 7; 1971-74 epidemic outbreaks of adenovirus 7 with special reference to the pathogenicity of adenovirus genome type 7b adenoviruses in immunocompromised hosts comparative sequence analysis of the hexon gene in the entire spectrum of human adenovirus serotypes: phylogenetic, taxonomic, and clinical implications adenovirus infection associated with central nervous system dysfunction in children adenoviral load diagnostics by quantitative polymerase chain reaction: techniques and application key: cord-303187-ny4qr2a2 authors: belo, vinícius silva; struchiner, claudio josé; werneck, guilherme loureiro; teixeira neto, rafael gonçalves; tonelli, gabriel barbosa; de carvalho júnior, clóvis gomes; ribeiro, renata aparecida nascimento; da silva, eduardo sérgio title: abundance, survival, recruitment and effectiveness of sterilization of free-roaming dogs: a capture and recapture study in brazil date: 2017-11-01 journal: plos one doi: 10.1371/journal.pone.0187233 sha: doc_id: 303187 cord_uid: ny4qr2a2 the existence of free-roaming dogs raises important issues in animal welfare and in public health. a proper understanding of these animals’ ecology is useful as a necessary input to plan strategies to control these populations. the present study addresses the population dynamics and the effectiveness of the sterilization of unrestricted dogs using capture and recapture procedures suitable for open animal populations. every two months, over a period of 14 months, we captured, tagged, released and recaptured dogs in two regions in a city in the southeast region of brazil. in one of these regions the animals were also sterilized. both regions had similar social, environmental and demographic features. we estimated the presence of 148 females and 227 males during the period of study. the average dog:man ratio was 1 dog for each 42 and 51 human beings, in the areas without and with sterilization, respectively. the animal population size increased in both regions, due mainly to the abandonment of domestic dogs. mortality rate decreased throughout the study period. survival probabilities did not differ between genders, but males entered the population in higher numbers. there were no differences in abundance, survival and recruitment between the regions, indicating that sterilization did not affect the population dynamics. our findings indicate that the observed animal dynamics were influenced by density-independent factors, and that sterilization might not be a viable and effective strategy in regions where availability of resources is low and animal abandonment rates are high. furthermore, the high demographic turnover rates observed render the canine free-roaming population younger, thus more susceptible to diseases, especially to rabies and leishmaniasis. we conclude by stressing the importance of implementing educational programs to promote responsible animal ownership and effective strategies against abandonment practices. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the relationship between dogs (canis familiaris) and men goes back to the beginning of civilization, about 13,000 years ago [1, 2] . it is generally accepted that dogs were domesticated from the wolf (canis lupus pallipes or c. lupus variabilis) in a process of symbiosis that evolved through selective breeding [3] . indeed, dogs are termed as 'domestic' or 'domesticated' animals due to its association with humans and to the role that humans exercised in the emergence of this lineage [4] . since domestication, this relationship became even more intense and dogs are ubiquitous in the cultural context of every society, constituting the most abundant carnivore animal on the planet [5] . dogs have been associated with their owners' welfare and well-being [6, 7] and have started to play different functions [2] due to their malleable personalities, docile behavior and utility as guardians and hunters [8] . dogs that are not under immediate human supervision and have unrestricted access to public property are named "free-roaming" or free-ranging [1] . these terms encompass both owned dogs (family and some neighborhood dogs) and ownerless dogs (stray or feral) [3] . the existence of these dogs that can circulate freely in the streets can be harmful to both the animals and to human beings [1] . the abandonment and breeding of dogs in unrestricted environments have been attributed to behavioral, religious, cultural, ecological and socioeconomic factors, constituting important issues in public health and animal welfare [9, 10] . unrestricted dogs, in general, have their psychological and physical health compromised, are more likely to acquire infectious diseases and have a lower life expectancy compared to pet dogs [11] [12] [13] . their presence can be detrimental to humans since they are associated with the occurrence of biting incidents, transmission of diseases, damage to wild animal populations, accidents and pollution [14] [15] [16] [17] [18] [19] . different strategies are used to control the population of unrestricted dogs [20] . elimination by killing is not considered effective, since the number of removed animals is compensated for the increased entry and survival of the remaining ones. in addition, this method is the subject of much criticism based on ethical issues [20] [21] [22] . as a result, actions towards promoting responsible animal ownership, the strengthening of legislation against abandonment, and surgical control have been established in different countries [20, 23] . annually, thousands of unrestricted dogs are sterilized in veterinary clinics and in campaigns run by governments and non-governmental organizations. nevertheless, the effectiveness of this measure, in the long term, has been poorly evaluated [24, 25] a proper evaluation of actions that aim at controlling the free-roaming canine population requires non-biased estimates of parameters driving the dynamics of the target population [26, 27] . even though several studies have yielded estimates of the population size of free-ranging dogs, most of them used inadequate analytical methods and were susceptible to biases, which casts doubt on the validity of these estimates, as evidenced in a recent systematic review [28] . additionally, there is no published data of capture and recapture procedures that consider the canine populations as open, that is, subjected to deaths, births and migrations [29] . despite the perceived need and usefulness of such parameter estimates and recommendations for the most appropriate approaches applicable under such study designs [30] , survival and recruitment estimates of free-ranging dogs had not been obtained using methods of capture and recapture. in this study, we present estimates of abundance, survival and recruitment rates, and the probabilities of capture of two free-roaming dog populations by means of analytical models for open populations, so far unexplored in previous studies. these dogs were followed for 14 months in a city located in the southeast region of brazil. we report temporal variations of the estimates during the study period regarding gender and the effectiveness of surgical sterilization. been approved by cepea-commission of ethics in research involving animals of the federal university of são joão del rey (protocol no. 24/2010). prior to the implementation of the activities described in the next section, we performed a pilot study to define the areas of study and to identify potential problems requiring further attention. the pilot study lasted for four days, each day spent in a different neighborhood of the city. each one of the four neighborhoods belonged to four different eligible areas defined with municipal health authorities based on previous information on the occurrence of freeroaming dogs and on the feasibility of carrying out the research. those with similar features and with the highest raw number of captured and released animals were selected. data acquired in this stage were not used in the analyses. we conducted seven capture and recapture procedures in a period of one year and two months, one every two months. dogs found wandering in the streets during the capture period were included in the study, provided an owner with a dog leash did not accompany them. adapted vehicles drove around all the streets of the study areas screening for free-roaming animals. the work team consisted of one driver, two municipal health agents, one veterinarian and two individuals responsible for collecting and recording data. in area a, activities took place in the first week of the collecting months, while in area b, they took place in the second week of the same month. screenings always followed the same route, so that they covered all the streets of each region at least once. the same team collected the data in both areas. captured dogs of areas a and b were taken to the health surveillance reference center of the city (crevisa) in adapted vehicles of public health service. in crevisa they underwent clinical tests conducted by veterinarians and were screened for canine leishmaniasis (canl). the diagnosis of canl was made in the parasitology laboratory of universidade federal de são joão del rei through the techniques of "enzyme-linked immunosorbent assay" and "indirect fluorescent antibody test". seropositive dogs were euthanized according to the recommendations of the brazilian ministry of health [31] . dogs that tested negative were microchipped for identification if recaptured. these animals were also de-wormed and received a vaccine against rabies and another against distemper, leptospirosis, hepatitis, parainfluenza, parvovirus, coronavirus and adenovirus. dogs captured in the area b (intervention) were sterilized (s1 appendix). healthy animals were returned to the same place where they had been captured, after screening for canl and total rehabilitation of the surgical procedure (area b dogs) (s1 appendix). recaptured animals were re-examined. animals screened and found negative for canl were released again. animals that tested positive were euthanized. all dogs, even those not captured, were photographed for posterior identification in the same sample interval and in recaptures. for identification, distinctive characteristics of the dogs were sought in their craniolateral and/or dorso-caudal portions. for analytical purposes, dogs not physically captured but photographed were considered captured. this information was added to the database serving as input for the estimation of the population dynamic parameters. we released informative materials to the local population with the purpose of increasing their awareness regarding responsible animal ownership and visceral leishmaniasis (s2 appendix). we entered the individual history of capture and recapture of each animal into a microsoft excel (2013) database formatted as "encounter history" for captured animals tagged alive [30] . euthanized animals carried a negative sign, indicating the occurrence of death during the capture procedure. there were no deaths attributed to other factors. general procedure. the jolly-seber model with popan parametrization served as the starting structure for model fitting [34] . we estimated the following three parameters using his approach: φ i (survival): probability of a marked or unmarked animal surviving (and not migrating) between the captures i and i+1. p i : (capture probability): the probability of finding or seeing a marked or unmarked animal in a given capture i, given the animal is alive and in the area of capture. b i : (probability of entrance): considering the existence of a "super-population", comprised of all animals that would ever be born to the population, this parameter constitutes the probability of an animal of this hypothetical "super-population" entering the population between the occasions i and i+1. the parameters above allow for the estimation of recruitment (b: number of animals that enter the population between two capture procedures) and population size (n). we used mark, version 6.2 for fitting the statistical models. goodness of fit of highly parameterized models. we evaluated the goodness of fit (gof) of the model with the largest number of parameters prior to fitting models that were more parsimonious [35] . this step was necessary to check the premises of the jolly-seber approach. we checked model's gof based on tests 2 and 3 of the release suite of mark software, the gof statistics obtained via the bootstrap, as well as the "median c-hat" statistics. among the procedures, we adopted the one that indicated the highest variance inflation factor (c-hat). we first considered the model with the variables "sex" (male or female), "area" (a or b), "time" (sampling period), and their respective interaction terms. a c-hat value of 2.52 suggested sparsity in different periods of capture. we changed our model search strategy accordingly and partitioned the previous model into two models: a model with "sex", "time" and interactions and a second model with "area", "time" and interactions. c-hat estimated in these cases was 1.17 and 1.25, respectively for each model, and there were few indications of sparse data. modeling procedures. the gof analysis reported above prompted us to investigate factors associated with survival estimates, probability of capture and probability of entry separately for the variables "sex" and "area". in both cases, we built models considering timedependent or time-independent parameters and the presence of interactions between the variables "sex" and "time" or "area" and "time". we also fitted additive models containing parameters expressed as a function of two or more factors, in this case, area and time or sex and time, without the presence of interactions. in total, we fitted 50 models in both groups (s3 appendix). all models supported temporal variations for the "probability of entry" (b i ). model selection followed the usual approach by searching for the most parsimonious structure that retained the best balance between explained variability and precision of estimates. we ranked all models based on akaike's information criterion corrected for finite sample sizes (aicc). this statistic provides a summary balance between the goodness of fit to the data of each model and the number of necessary parameters. "data cloning" was used to identify the correct number of estimated parameters [36] . the presence of overdispersion in the data indicated the need to further correct the aicc statistics by the c-hat values to obtain the quasi-aicc statistics (qaicc). lower values of these latter statistics point to models that were more parsimonious [37] . after ranking all models based on qaicc, we evaluated the force of evidence in favor of each model (aic weight-"w"). this statistic can be interpreted as the conditional probability of a given model being the best among the set analyzed. thus, higher values of "w" indicate higher force of evidence in favor of the model. models with values of "w" lower than 0.01 were disregarded. we further evaluated the importance of each variable in a context of a set of models by adding up the weight (w) of each model containing a given variable [37] . we repeated this procedure for all predictors considered. variables with higher weights are considered more important than those with lower weights in explaining the variance observed in the data. parameter estimation. estimates for the parameters survival probability, probability of capture, probability of entry in the population, abundance and recruitment rate relied on the technique known as "model averaging" [38] . under this approach, we calculated the weighted average of parameter estimates from all models fitted to data using as weights the relative support (w) of the respective model. therefore, this technique accounts for both sources of variance: the specific conditional variation present in each one of the models and the nonconditional variation present in the model selection process. in this way, parameter estimates express more faithfully the sources of uncertainty associated with the estimation process. in time-dependent models under popan not all parameters are identifiable [30] . this is the case of the probability of capture in the first and in the last captures (p 1 and p k ), the probability of entry between the first and the second captures (b 1 ) and between the penultimate and the last captures (b k-1 ), and the survival probability between the penultimate and the last captures (φ k-1 ). thus, only the remaining parameters whose estimation was possible are described here. the effectiveness of sterilization was analyzed by comparing the evolution of abundance and of the other parameters estimated in the areas: a (control) and b (intervention). we estimated dog:human ratio by the ratio of population size and the dog mean abundance in each of the areas. during the study period, 171 dogs were identified individually in region a (control) and 157 in region b (intervention). the proportion of males in areas a and b was 56% (96 dogs) and 62% (98 dogs) respectively. one hundred and thirty-three animals (77 males and 56 females) were captured in more than one effort of captures. one hundred and thirty-eight dogs (88%) were sterilized. twenty-four were euthanized for testing positive to canl. sixty-six different individual histories of captures were registered and 38 of them included animals not captured in the first effort. all recaptures and visualizations took place in the same area where the dogs were initially detected. most free-roaming dogs were neighborhood dogs, i.e. several human residents in the area provided the needed resources to them [3] . models including the variable "gender". we fitted 50 models containing the variable gender to the data (s3 appendix). five had w-statistics greater than 1% and are shown in table 1 , along with statistics qaicc, δqaicc (difference, in module, between qaicc values of the best model and the analyzed model). the relative support for each model is also expressed as the ratio of its w-statistics to the largest value of this statistic among the five models considered. the model in which survival, probability of capture and probability of entry varied with time, but not between male and female dogs, was considered the most parsimonious (w = 73.24%). the weight for this model was 6.48 times higher than the model in which survival varied additively with gender; and 8.37 times higher in relation to the model in which the probability of capture varied between genders. other models had weights lower than 5% and low support, when compared to the most parsimonious model. the sum of each variable's weight (w) considering all models (table 1 ) are presented in s5 appendix. time-dependent parameters displayed higher weights. survival and capture probabilities varied between genders but these variables' "w" conferred weak support to this statement. for entrance probability, there was no evidence for the existence of group variation. models with the variable "area". models containing the variable "area" behaved similarly to the previous set containing the variable "gender". table 2 presents the six models in this group with w-statistics greater than 1%. results for the remaining models are described in s3 appendix. the model containing time-dependent parameters, but constant between areas control (a) and intervention (b), was the most parsimonious in this group. its weight, however, was lower when compared to the models containing the variable "gender" (table 1 ). it had 3.11 times more support from the data than the model in which there was variation in survival between areas, and 3.99 more support than the model in which the probability of capture also varied between areas. differences were significantly higher when comparing the most parsimonious model to the remaining models since the latter models received even lower support from the data. we observed stronger weights associated with time-dependent variables (s5 appendix). however, the observed weights were lower than those associated to the variables in the set of models containing the variable "gender". we also observed stronger weights in variables describing differences in survival and probabilities of capture between areas. the remaining variables were associated with lower weights. in particular, the probabilities of entry did not vary between control and intervention areas. models with the variable "gender". we estimated a population abundance of 148 females and 227 males in the target population in the entire study period. table 3 depicts gender-specific parameter estimates and their respective confidence intervals (ci). they result from model-specific estimates weighted by the relative support (w) of the respective model. taken together, these results show that gender-specific differences regarding the estimated parameters were not relevant. in contrast, time-dependent differences were significant. survival probabilities increased steadily, going from 0.75 in the interval between the first and second captures, to 0.99 between the fifth and sixth. on the other hand, probability of entry in the population was close to zero between the fifth and the sixth captures, and varied between 0.12 and 0.15 in other intervals. probability of capture reached the highest value in the second capture (0.68), and decreased subsequently until the fifth capture when it reached its lowest value (0.39). estimates of abundance highlight the majority of males in its composition. additionally, there was a higher entry of male dogs in all intervals in which the number could be estimated. population increased in size during the study. we estimated the presence of approximately 59 females and population dynamics and effectiveness of sterilization of free-roaming dogs 92 males in the second capture, 71 and 69 females and 104 and 105 males, respectively, in the fifth and sixth captures. models with the variable "area". we estimated the presence of 199 dogs in area a (control) and 177 in area b (intervention) throughout the study period. estimates of additional parameters stratified by control and intervention areas are presented in table 4 . they reflect the weighting mechanism by the relative support of all models analyzed as explained in the methods section. analogously to the models containing gender, differences between the estimates of each area were small, even though they were slightly higher than those seen between genders. owing to the fact that stronger weights were attributed to models that did not show a difference between strata in either set of models, estimates of survival, capture probabilities and entry probabilities for the areas were similar to those described for gender. on the other hand, the recruitment was similar in both areas, contrasting with our findings comparing males and females. population size increased in both areas. abundances seen in the second capture were smaller, 82 animals in area a and 70 in area b, contrasting with abundances observed in the fifth capture, 96 dogs in area a and 83 in area b. dog:human ratio in area a was one dog to 42 human beings. in area b, this ratio was one dog to 51 humans. we estimated critical parameters (survival, recruitment and abundance) that describe the population dynamics of free-roaming dogs based on a capture and recapture study design and on models suitable for open populations. our study demonstrated the increase in population size in both areas, the predominance and greater recruitment of males, the temporal variability in recruitment and in survival probabilities, the lack of effect of sterilization on population dynamics, the influence of abandon and of density-independent factors and a high demographic turnover. such information on the dynamics of free-ranging dogs are useful for informing control interventions of unrestricted dog populations and against canine visceral leishmaniasis and rabies, both neglected tropical diseases endemic to various countries. the dog:man ratio observed in our study was smaller than that observed in counts performed in urban regions of nigeria (1 dog to 25 men) [39] and that among rural dog populations in india (1 dog to 35 men) analyzed by mean of beck's method [40] . it was larger, however, than the counts obtained by hossain et al. [41] in a rural area of bangladesh. demographic, socioeconomic, environmental and cultural factors able to explain differences in abundances between and within regions have been underexplored in the literature [28] . abundance of free-roaming dogs in general is lower in rural than urban areas [42, 43] . regions under poorer socioeconomic conditions and higher population densities tend to have a larger concentration of dogs [44] . in the present study, abundance possibly reflects the intermediary socioeconomic condition, the urban environment and low population density of the study areas as well as the different methodology applied. for most animal species, survival is the demographic parameter with highest impact on population size [45] . few studies, however, aimed at estimating the survival of free-ranging dogs in urban environment. reece et al. [46] used data from a sterilization program to estimate the survival of castrated females in jaipur, india. annual survival of females aged over one year old was 0.70 and of females in their first year of life was 0.25. the assumptions leading to these estimates were implausible and might have biased the results. pal [47] conducted four annual capture efforts, in bengal, india, and estimated the canine mortality from the number of dogs observed in the captures after the first one. annual survival for adult dogs was 0.91, and for dogs in their first year of life, 0.18. this study did not report capture probabilities and included in estimation only dogs found dead. this approach possibly contributed to an overestimation of the survival probability. survival probability reported by beck [1] , in a study conducted in baltimore, canada, with dogs of all age groups, was 0.70. this author relied only on existing information regarding the number of dead dogs, also possibly leading to an underestimation of mortality and consequently to an overestimation of survival probability. although limited, estimates obtained in the literature suggest that survival is lower in young free-roaming dogs [46, 47] , a pattern already seen in different animal species [48, 49] . once the proper identification of the dogs' ages was outside the scope of our project, estimates in the present study refer to the general survival probability of the population and not age-specific probabilities. annual survival in our study was higher than that estimated for dogs aged less than one year [46, 47] , and lower than the survival probability estimated for adult dogs [46, 47] and for beck's study population [1] . the low survival probability identified in the population results from the different sources of mortality experienced by free-roaming dogs in the study setting. residents often reported roadkill and poisoning episodes during the study period. the high prevalence of canl-seropositive dogs, especially in the first months of the study, is another relevant factor leading to the removal of many animals by euthanasia. additionally, government actions towards street dogs were restricted to rabies vaccination. the lack of additional prophylactic measures or treatment may have contributed to the increased susceptibility of dogs to infections and other conditions. females have lower survival rates [50, 51] in a large number of animal species due primarily to the effects of reproduction. given the predominance of males in different studies, it is hypothesized that this pattern also happens in the canine populations [52] . we observed no difference in survival probabilities between genders, although a higher abundance and recruitment of males occurred. most pet owners prefer male dogs since they do not get pregnant and are better guard dogs [28, 41] . therefore, the higher survival of male puppies of owned but free-ranging dogs or of pet dogs subsequently abandoned by their owners could probably explain the predominance of males in the free-roaming dog population. to our knowledge, we report for the first-time the temporal evolution of the survival probability of free-roaming dogs. annual point estimates of survival probability found in the literature do not bear a longitudinal structure. our results show that survival of unrestricted dogs displays variations, even in short temporal scales. among the models fitted to the data, those in which survival did not vary with time had significantly lower weights, indicating that a constant value is not appropriate to representing the entire period. estimates of survival probabilities in other mammal species also show a temporal dependence, especially in young individuals [53] [54] [55] . long-term studies are required to uncover the intrinsic and extrinsic determinants driving these temporal dependencies. this would be useful for understanding the population dynamics of free-ranging dogs and improving the validity and precision of predictive modelling procedures. such studies are difficult to perform, and thus are rare in the literature [56] . despite being a short-term study, survival, recruitment and population size displayed an increasing tendency. this pattern suggests that density-independent factors could be responsible for driving the variations observed in survival probabilities of dogs in both areas. density-dependent mechanisms are the subject of several studies focusing on different animal species [57] [58] [59] [60] . in epidemiological and ecological modeling, one assumes that survival and recruitment rates in free-ranging dogs are driven by the availability of resources in the environment, a density-dependent mechanism [61] . however, as pointed out by de little et al. [56] , extrinsic factors not regulated by density may determine fluctuations in population size when those populations have not yet reached their carrying capacity or when environmental conditions are favorable. according to morters [61] , human beings are the major agents responsible for providing care and adequate food for dogs. as a result, human related factors such as living together with free-ranging dogs, the low dog-human ratio and the availability of residents' resources to maintain these animals, may explain why the increase in density had no influence upon mortality and recruitment. reducing the availability of shelters and food is an ethically questionable measure for population control of free-roaming dogs. however, this alternative has been presented in a recent study [62] . it is not possible to affirm whether population growth, attributed to the large number of animals entering the population, would keep the reported increasing trend constant if the study had a longer duration. maximum survival and lack of recruitments between the fifth and the sixth captures suggest potential instabilities. in the presence of increasing abundance, density-dependent factors could start to play a stronger role in regulating the population [63, 64] and in the behavior of residents regarding their support to dogs. there is considerable uncertainty in assessing the role played by vital rates and intrinsic and extrinsic factors in driving the population size of free-ranging dogs and other mammals [54, 61, 65, 66] . estimates of recruitment obtained from capture and recapture models do not allow us to disentangle the sources of entry attributable to births and immigration. we observed no females with their brood along the study period. we might infer that breeding females were located in less visible areas or put to adoption by the city public service and returned to the streets after the lactation period, even though such registries were rare. in the study of morters et al. [61] , as well as in the present study, recruitment was driven, predominantly, by the arrival of adult animals. the recruitment contingent may comprise dogs born in the region and not identified as puppies, dogs from other regions that migrated to the study region or were relocated by residents who raised them unrestrictedly, previously restricted dogs that changed status to being freely raised, or dogs abandoned nearby that later joined the population. the study areas are geographically isolated from their neighboring regions and are located next to a highway where dogs were frequently abandoned. therefore, the latter mechanism seems more plausible to account for the increase in population size rather than the spontaneous immigration of dogs. although there are heterogeneities [67] , free-ranging dogs are territorial animals that, in general, do not move across long distances, unless forced by unfavorable environmental conditions [1] . the low mobility of dogs in a favorable environment is supported by our data, since there were no animal movements between the areas a and b. the replacement of a great number of dogs that died or emigrated by dogs that are born or immigrate, as observed in our study populations, drives the population structure and gives rise to health problems that result from these structures. a population with a high turnover may be more susceptible to diseases [24] . a high population turnover is the major obstacle for the success of control strategies against rabies in developing countries [68] . vaccination strategies under such population dynamics must occur in short intervals and achieve high coverage in order to maintain proper levels of immunization. on the other hand, the replacement of euthanized dogs by susceptible animals and new individuals entering the reservoir compartment are the main causes of the low effectiveness of the euthanasia of seropositive dogs, a control strategy adopted in brazil against leishmaniasis [69] . in addition, the population also becomes younger and more likely to acquire other infections under the high turnover regime [70] . the field of mammal ecology identifies two main reproductive strategies driving population size, each focusing on specific stages of the life cycle. the so-called "slow breeding" animals experience late maturation and their reproductive strategy depends on the survival of juveniles and young adults. on the other hand, "fast breeding" mammals complete their reproductive cycle within their first year of life and place emphasis on fertility as their survival strategy as a species [71, 72] . control of the population size of "fast breeding" animals, such as dogs [73] , is more effectiveness when relying on measures that restrict entry of new individuals into the population as opposed to subjecting animals to euthanasia, a practice that reduces adults' survival. the fast versus slow breeding rationale, the sensitive ethical issues, and the low effectiveness of euthanizing animals observed in regions where this practice has been applied [20, 61, 74] prompted us to only consider sterilization, and not culling, as an alternative control strategy in our study. its use as a population control measure against hydatid disease in developing countries, however, has been recommended [19] . it is worth noting that in the present study sterilization did not affect the canine population dynamics. after one year and two months, we observed no difference in survival, entrance or recruitment probability between the control region and the intervention area where 88% of the dogs were sterilized. the impact of sterilization takes place slowly as suggested by modeling exercises. it might take up to five years for the first impact of sterilization to become apparent and up to 30 years of uninterrupted efforts to reach its maximum impact [75] . reece and chawla [24] evaluated a program that surgically sterilized 19,129 neighborhood dogs in jaipur, india, for eight years and showed that the population declined by only 28 per cent. on the other hand, frank and carlisle-frank [76] observed only a small impact of a sterilization program on the number of dogs joining a shelter in the united states. amaku et al. [77] , based on results from a mathematical model developed specifically for stray dogs, concluded that sterilization becomes inefficient in the presence of high abandonment rates, even after prolonged periods of use. natoli et al. [78] reached the same conclusion after studying for 10 years the impact of a castration and devolution program on a non-restricted cat population. continuous negligent practices of animal ownership, including abandonment, had a negative impact on the sterilization strategy rendering it ineffective and countering the effect of 8,000 surgical interventions undertaken in that study. finally, in a study conducted in brazil, dias et al. [79] concluded that it is counter-productive to invest in sporadic sterilization campaigns of owned dogs, the currently strategy adopted in most of brazilian municipalities. the small impact in controlling the population size, especially in areas with high abandoning rates as in our case, the need to reach high coverage rates without interruptions, the absence of behavioural benefits for castrated dogs [80] , high costs and null impact on a shortterm perspective, minimize the relevance of sterilization of free-ranging dogs in managing the population and controlling diseases. in this context, it becomes apparent that public health services and non-governmental organizations must develop and prioritize more effective strategies against abandonment practices. in countries where free-ranging dogs are considered a humanitarian or a public health issue the implementation of educational programs addressing responsible animal owning at different levels, the registration of dogs and their owners and the improvement of legislation aimed at those who wish to have a pet becomes imperative [81] . probability of capture is a useful parameter in the identification of essential population features [82] . it is known to vary in space, time and among individuals [83] . although we observed no significant differences in this parameter between genders and areas, it varied over time even in the presence of standardized procedures. such fluctuations may be attributable to social organization features not yet investigated in the population, or to environmental and climatic factors. dias et al. [84] showed that weather exerts an influence on dogs' activity, and consequently influences the probability of finding a dog in a given capture effort. in our study, even with the vehicles driving around in all the streets of the target regions, there was a large number of animals present but not visualized in all captures. our observations indicate that individual counts based on a census do not adequately estimate the abundance of unrestricted dogs and that the majority of the estimates available in the literature show important biases. different studies aimed at estimating the abundance of free-roaming dogs did not model or even consider the existence of differences in the probabilities of dog detection [28] . as pointed out before [28] , counting techniques should be carried out only in short periods of time and when no other alternative becomes available considering logistics, geography and culture of the study region. values of capture probabilities obtained in the present study are similar to those estimated by kalati in a population of urban free-ranging dogs in kathmandu [85] and may be used as correction factors for the previously published estimates of abundance. in addition to the limitations already mentioned regarding the observation and sampling techniques applied in capture and recapture studies, issues related to the choice of appropriate analytical methodology deserve mentioning. environmental and individual variables, relevant to helping understand the population dynamics [30] were not included in our models. the logistics of fieldwork turned out to be complex and difficult, requiring the participation of at least six individuals in each capture effort and extensive fieldwork journeys. direct contact with animals was unavoidable since assessing the effectiveness of sterilization was one of the study objectives. studies assessing the population dynamics of dogs, however, could rely on only photographic methods, these being less complex and onerous [86, 87] . our choice of modeling procedures for open populations allowed for the estimation of survival and recruitment probabilities of unrestricted dogs. in addition, we tested the data for the statistical assumptions required by each model. model selection followed the aic technique, which compares favorably with the classic statistical and hypothesis testing [37, [88] [89] [90] . lastly, our parameter estimates and confidence intervals express more faithfully the sources of uncertainty present in the whole estimation process, due to the use of the "model averaging" technique. the analytical procedures adopted here addressed methodological limitations of previous publications and propose a new starting point for future studies. in our view, longer periods of observation, larger sample sizes, and the choice of more variable study settings including different social, cultural and geographic characteristics are important topics that need the attention of researchers in the field of unrestricted dogs' ecology. the agenda of the investigation of factors influencing the canine population dynamics must consider the variables addressed in the present study, and further consider the stratification of these population parameters by age groups, as well as by intrinsic animal features and environmental conditions not yet investigated. our estimates of population size in the studied regions in general were small compared to previous estimates in the literature. survival probability was small and probability of animal entry in the population was high during the 14 months period of follow-up. high turnover, attributed mostly to the abandonment of pet dogs, has important implications to the population composition and the control of zoonosis. estimates of survival, recruitment and capture probabilities varied over time. survival and recruitment showed an increasing tendency. mortality patterns did not differ between genders. the probability of entry in the population was higher among males. the observed population dynamics seem to be driven by density-independent factors. sterilization, in turn, had no influence upon the parameters analyzed. our observations are useful for a better understanding of the population dynamics of free-roaming dogs and may aid in the planning, designing and evaluation of population control actions. in this context, it becomes imperative that public health services and nongovernmental organizations develop educational training programs addressing responsible animal ownership and better strategies against abandonment practices. parameter estimates may also be used as input to new predictive mathematical models. even though our study generated important answers and new hypotheses, the scarcity of existent knowledge and the misuse of the proper methodology raise numerous relevant questions yet to be elucidated about the population dynamics of free-roaming dogs. the ecology of stray dogs: a study of free-ranging urban animals a 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the wellington region of new zealand urban rabies transmission dynamics and economics of rabies control in dogs and humans in an african city estratégias adicionais no controle populacional de cães de rua %20caes%20de%20rua.pdf?sequence=1 control of rabies in jaipur, india, by the sterilisation and vaccination of neighbourhood dogs impact of publicly sponsored neutering programs on animal population dynamics at animal shelters: the new hampshire and austin experiences free-roaming dog control among oie countries which are members approaches to canine health surveillance population estimation methods for free-ranging dogs: a systematic review métodos para estimativas de parâmetros populacionais por captura, marcação e recaptura analysis and management of animal populations secretaria de vigilância em saúde. departamento de vigilância epidemiológica infecção por leishmania em uma população de cães: uma investigação epidemiológica relacionada ao controle da leishmaniose visceral secretaria de vigilância em saúde. departamento de vigilância epidemiológica. normas técnicas de profilaxia da raiva humana. 1st ed. brasília: editora ministério da saúde a general methodology for the analysis of capture-recapture experiments in open populations u-care: utilities for performing goodness of fit tests and manipulating capture-recapture data estimability and likelihood inference for generalized linear mixed models using data cloning model selection and multi-model inference: a practical information-theoretic approach information-theoretic model selection and model averaging for closed-population capture-recapture studies studies on dog population and its implication for rabies control assessing demographic and epidemiologic parameters of rural dog populations in india during mass vaccination campaigns a survey of the dog population in rural bangladesh free-roaming dog population estimation and status of the dog population management and rabies control program in dhaka city demography of domestic dogs in rural and urban areas of the coquimbo region of chile and implications for disease transmission spacing and social organization: urban stray dogs revisited is survivorship a better fitness surrogate than fecundity? fecundity and longevity of roaming dogs in jaipur population ecology of free-ranging urban dogs in west bengal age, sex, density, winter weather, and population crashes in soay sheep sex-and age-dependent effects of population density on life history traits of red deer cervus elaphus in a temperate forest the evolution of parental care effects of maternal care on the lifetime reproductive success of females in a neotropical harvestman a seroepidemiologic survey of canine visceral leishmaniosis among apparently healthy dogs in croatia persistent instability and population regulation in soay sheep temporal changes in key factors and key age groups influencing the population dynamics of female red deer assessing the impact of climate variation on survival in vertebrate populations complex interplay between intrinsic and extrinsic drivers of long-term survival trends in southern elephant seals loss of density-dependence and incomplete control by dominant breeders in a territorial species with density outbreaks stochasticity and determinism: how density-independent and density-dependent processes affect population variability bayesian inference on the effect of density dependence and weather on a guanaco population from chile density-dependent spacing behaviour and activity budget in pregnant, domestic goats (capra hircus) the demography of freeroaming dog populations and applications to disease and population control defining priorities for dog population management through mathematical modeling population regulation in mammals: an evolutionary perspective population growth rate and its determinants: an overview population dynamics of large herbivores: variable recruitment with constant adult survival population dynamics of owned, free-roaming dogs: implications for rabies control domestic dog roaming patterns in remote northern australian indigenous communities and implications for disease modelling transmission dynamics and prospects for the elimination of canine rabies control of visceral leishmaniasis in latin america-a systematic review dog culling and replacement in an area endemic for visceral leishmaniasis in brazil life histories and elasticity patterns: perturbation analysis for species with minimal demographic data carnivora population dynamics are as slow and as fast as those of other mammals: implications for their conservation estudo do programa de esterilização das populações canina e felina no município de são paulo efecto del sacrificio de perros vagabundos en el control de la rabia canina an interactive model of human and companion animal dynamics: the ecology and economics of dog overpopulation and the human costs of addressing the problem analysis of programs to reduce overpopulation of companion animals: do adoption and low-cost spay/neuter programs merely cause substitution of sources? dynamics and control of stray dog populations management of feral domestic cats in the urban environment of rome (italy) dog and cat management through sterilization: implications for population dynamics and veterinary public policies effects of surgical and chemical sterilization on the behavior of free-roaming male dogs in stray dog and cat laws and enforcement in czech republic and in italy is heterogeneity of catchability in capture-recapture studies a mere sampling artifact or a biologically relevant feature of the population? revisiting the effect of capture heterogeneity on survival estimates in capture-mark-recapture studies: does it matter? size and spatial distribution of stray dog population in the university of são paulo campus, brazil street dog population survey, kathmandu: final report to wsp spot the match-wildlife photo-identification using information theory mark-recapture and mark-resight methods for estimating abundance with remote cameras: a carnivore case study choosing among generalized linear models applied to medical data null hypothesis testing: problems, prevalence, and an alternative model selection in ecology and evolution the authors are grateful for the invaluable support provided by members of staff of the municipal health service and by veterinarians who participated in this study. key: cord-305303-82n96ukr authors: shapira, assaf; shapira, shiran; gal-tanamy, meital; zemel, romy; tur-kaspa, ran; benhar, itai title: removal of hepatitis c virus-infected cells by a zymogenized bacterial toxin date: 2012-02-16 journal: plos one doi: 10.1371/journal.pone.0032320 sha: doc_id: 305303 cord_uid: 82n96ukr hepatitis c virus (hcv) infection is a major cause of chronic liver disease and has become a global health threat. no hcv vaccine is currently available and treatment with antiviral therapy is associated with adverse side effects. moreover, there is no preventive therapy for recurrent hepatitis c post liver transplantation. the ns3 serine protease is necessary for hcv replication and represents a prime target for developing anti hcv therapies. recently we described a therapeutic approach for eradication of hcv infected cells that is based on protein delivery of two ns3 protease-activatable recombinant toxins we named “zymoxins”. these toxins were inactivated by fusion to rationally designed inhibitory peptides via ns3-cleavable linkers. once delivered to cells where ns3 protease is present, the inhibitory peptide is removed resulting in re-activation of cytotoxic activity. the zymoxins we described suffered from two limitations: they required high levels of protease for activation and had basal activities in the un-activated form that resulted in a narrow potential therapeutic window. here, we present a solution that overcame the major limitations of the “first generation zymoxins” by converting mazf ribonuclease, the toxic component of the e. coli chromosomal mazef toxin-antitoxin system, into an ns3-activated zymoxin that is introduced to cells by means of gene delivery. we constructed an expression cassette that encodes for a single polypeptide that incorporates both the toxin and a fragment of its potent natural antidote, maze, linked via an ns3-cleavable linker. while covalently paired to its inhibitor, the ribonuclease is well tolerated when expressed in naïve, healthy cells. in contrast, activating proteolysis that is induced by even low levels of ns3, results in an eradication of ns3 expressing model cells and hcv infected cells. zymoxins may thus become a valuable tool in eradicating cells infected by intracellular pathogens that express intracellular proteases. hepatitis c virus (hcv) is a small, enveloped rna virus belonging to the hepacivirus genus of the flaviviridae family. hcv has been recognized as a major cause of chronic liver disease and affects approximately 200 million people worldwide at the present time. persistent infection is associated with the development of chronic hepatitis, cirrhosis and hepatocellular carcinoma. a protective vaccine for hcv is not yet available and even the most recent combination of antiviral therapy is often poorly tolerated [1] . the hcv genome encodes one large open reading frame that is translated as a polyprotein which is proteolytically processed to yield the viral structural and nonstructural (ns) proteins. the non-structural proteins include the p7 ion channel, the ns2-3 protease, the ns3 serine protease/rna helicase and its co-factor ns4a, the ns4b and ns5a proteins and the ns5b rna-dependent rna polymerase (rdrp) [2, 3] . two virally encoded proteases participate in polyprotein processing, the ns2-3 autoprotease (which cleaves in cis at the ns2-3 junction) and the ns3-4a serine protease (which cleaves at four downstream ns protein junctions). ns3 is an extensively studied hcv protein that possesses multiple enzymatic activities that are essential for hcv replication, making ns3-4a the most attractive target for anti hcv drug development [4] . the n-terminus of ns3, in complex with its endoplasmic reticulum (er) membrane-anchored cofactor ns4a, primarily functions as a serine protease, which cleaves the viral polyprotein precursor downstream to ns3. the remaining 2/3 of the protein has a helicase and ntpase activities, both of which are essential for hcv replication [5] . zymogens are inactive enzyme precursors that are converted to their active form following a biochemical modification, such as proteolytic processing. among the known and important groups of enzymes that are proteolytically activated are secreted digestive enzymes like pepsin and trypsin [6, 7] , the cysteine aspartic acid proteases (caspases) which play an essential role at various stages of the apoptotic process [8] ; and blood coagulating factors [9] . recently we described a proof of concept for potential new antiviral agents that can specifically eradicate virally infected cells (thus limiting virus production and spread). these agents, which we named ''zymoxins'' (for ''zymogenized toxins''), were composed of a fusion between the binding and translocation domains of pseudomonas exotoxin a and ns3-activable modified catalytic domains of the bacterial or the plant toxins diphtheria toxin (dta) or ricin toxin (rta), respectively. upon binding and translocation into cells cytoplasm by virtue of the corresponding pseudomonas exotoxin a domains, activation of these toxins is mediated by hcv-ns3 protease cleavage that separates between the toxic domains and a fused, rationally designed inhibitory peptide. these zymoxins showed a higher level of cytotoxicity when applied to ns3 expressing cells or to hcv infected cells, demonstrating a potential therapeutic window. still, the zymoxins we described had two limitations: first, they required high levels of ns3 protease for efficient activation, and second, they were not totally inactive in the zymogenized state, and their basal cytotoxic activity without proteolytic activation resulted in quite narrow therapeutic windows [10] . apparently, the zymoxins were only partially inhibited by the rationally designed inhibitory peptides. toxic proteins that are completely inhibited by a natural inhibitor can be found in bacterial ''toxin-antitoxin'' systems. natural bacterial plasmids ensure their survival within the bacterial host by replicating inside their host cell and using mechanisms that function to segregate them prior to cell division [11] . in addition, several low copy number plasmids use a unique system called ''addiction module'' or ''toxin-antitoxin (ta) system'' which functions to prevent the proliferation of plasmidfree cells by executing the so called ''post-segregational killing effect''. the system consists of a pair of genes encoding for a stable toxin and an unstable antitoxin organized in a bicistronic operon that is transcriptionally autoregulated either by the toxin-antitoxin complex itself or by the antitoxin alone. when coexpressed in plasmid harboring cells, the antitoxin component interferes with the lethal action of the toxin. if a cell loses the plasmid, the cellular concentration of the labile antitoxin (that is degraded faster than the more stable toxin) is rapidly diminished, enabling the toxin to exert its action which eventually results in cell death (reviewed by [12, 13, 14, 15, 16] ). toxin-antitoxin systems have also been found integrated to the chromosome of various bacteria species where their function has been the subject of considerable speculation [14, 17, 18, 19, 20, 21] . one of the most studied ''genomic'' ta modules was found on the chromosome of e. coli as a negatively autoregulated bicistronic operon. the system, which is activated by several stress conditions, was denoted mazef after its two active protein components: the long lived mazf toxin and the labile maze antitoxin. mazf induced toxicity is executed by blocking denovo protein synthesis through its endoribonuclease activity that catalyze the cleavage of single-stranded mrnas at aca sequences [22] . when coexpressed with mazf, the maze antitoxin complexed with the toxin and a catalytically inactive heterohexamer is formed in which a maze dimer is sandwiched between two mazf dimers (mazf 2 -maze 2 -mazf 2 ) (reviewed by [21, 23] ). the crystal structure of the maze-mazf complex indicates that the interactions between the toxin and the antitoxin are primarily mediated by the acidic c terminus of maze which wraps around the mazf homodimer crossing the edge of the dimer interface [24] . later on, li et al had discovered that a short acidic peptide corresponding to maze c-terminal 23 amino-acids, which they denoted ''mazep'', binds strongly to the homodimer of mazf which possesses two identical active sites. interestingly, it was found that one inhibitory peptide, occupying a single active site on the mazf homodimer, affects the conformation of both sites that consequently become catalytically inactive. this unique mechanism also explains the inhibitory activity of maze toward mazf at a 1:2 molar ratio [25] . the discovery of the mazf mechanism of action was soon followed by demonstrations that it is also toxic to eukaryotic cells, causing bak-dependent programmed cell death in mammalian cells, suggesting it may be used as a tool for gene therapy against diseases such as cancer and aids [26, 27] . here we describe the design of a different zymoxin than those described in [10] , based on ns3-activable mazf ribonuclease that is delivered as a transgene by an adenoviral vector. the delivered transgene encodes for a fusion between mazf, and a potent inhibitory peptide derived from its natural antidote maze, through an ns3 cleavable linker. we show that the self-inhibited, zymogenized toxin is well tolerated when expressed in naïve cells. in contrast, in ns3 expressing or in hcv infected cells, ns3mediated cleavage separates between the toxin and its inhibitor which results in inhibition of protein synthesis followed by death of the cells. finally, we demonstrate that treatment with the mazf based zymoxin has a ''curing effect'' when applied to mixed culture of healthy and hcv infected cells, leading to specific eradication of the infected cell population. for the construction of ns3-activated mazf based zymoxin, the mazf coding sequence was fused through its c terminus to the hcv p10-p10' ns3 cleavage sequence derived from the 2a genotype (strain jfh1) ns5a/b junction. a short inhibitory peptide corresponding to maze c-terminal 35 amino-acids (which encompass the 23 amino-acids inhibitory peptide (mazep) that has been described by li et al. [25] ) was fused, preceded by a short flexible linker, to the c terminus of the mazf-ns3 cleavage site sequence. a flexible linker, followed by the c-terminal er membrane anchor of the tyrosine phosphatase ptp1b [28] , was than fused to the c terminus of the inhibitory peptide and the whole construct was fused through its n terminus to the monomeric red fluorescence protein mcherry [29] (see figure 1 ). the rationale behind the design of this construct, which was denoted ''mcherry-ns3 activated mazf'', was that the coupling between the ribonuclease and its antidote may enable high level of expression of the non-toxic fusion on the er membrane of uninfected mammalian cells without causing any deleterious effect. in contrast, in hcv infected cells, the fusion protein is expected to colocalize with the er membrane-bound viral ns3 protease (in infected cells, ns3 is localized to the cytosolic side of the er membrane and membranes of er-like modified compartments [30, 31, 32, 33, 34] ). as a result, the ns3cleavable linker between the toxin and its inhibitory peptide is expected to be cleaved. the toxic ribonuclease, no longer covalently tethered to its er membrane-anchored inhibitor, is now free to diffuse into the cytoplasm (which lacks the antidote) and exert its cytotoxic activity. finally, fusion to the fluorescent protein mcherry makes the whole construct trackable and facilitates the determination of its expression level and intracellular localization by fluorescence microscopy. as a control, an uncleavable construct (denoted ''mcherry-uncleavable mazf'') was constructed in which the ns3 cleavage sequence was replaced by a mutated 14 amino acids cleavage sequence (p10-p4') from hcv genotype 1a ns5a/b junction in which the p3 valine was substituted by alanine, the p2 cysteine by glycine, the p1 cysteine by glycine and the p4' tyrosine by alanine. a schematic representation of the ns3-activated mazf-based zymoxin (''mcherry-ns3 activated mazf'') and the hypothetical mechanism of its cleavage by ns3 protease on the cytoplasmic side of the er membrane are shown in figure 1 . the aminoacid sequence of the mazf based zymoxins can be found in text s1. to verify that expression of ''mcherry-ns3 activated mazf'' is tolerated by cells that do not express the ns3 protease, a colony formation assay was carried out. in this assay, the toxicity of an expressed transgene can be comparatively and qualitatively assessed by testing its effect on the competency of transfected cells to evolve into colonies under selection. hek293 t-rex cells where transfected with plasmids encoding either mcherry-ns3 activated mazf, mcherry (only the fluorescent protein) or egfp-mazf (where mazf is not fused to its inhibitory peptide). 48 hours (h) later, expression of the encoded transgenes was confirmed by fluorescence microscopy (data not shown) and transfected cells were seeded in 3 fold dilutions and were treated with g418 (to which all three plasmids confer resistance). after 10 days of selection, surviving colonies were stained. as shown in figure 2 , similar numbers of surviving colonies were observed when the cells were transfected with the plasmids encoding mcherry-ns3 activated mazf or the red fluorescent protein alone, suggesting that expression of ns3-activable ribonuclease in naïve hek293 t-rex cells (that do not express ns3) cause minimal toxicity, if any. as expected, growth was severely inhibited when cells were transfected with the egfp-fused active (uninhibited) toxin. the er membrane-targeted zymoxin colocalizes with ns3 protease in vivo previously we described a hek293 cell line which inducibly expresses (by addition of tetracycline) a fusion between egfp and the coding sequence of the full length ns3 (including the helicase domain) followed by ns4a from hcv 1a genotype [10] . these cells, denoted ''tet-inducible full ns3-4a expressing cells'', were stably transfected with a plasmid encoding the ns3-activated zymoxin ''mcherry-ns3 activated mazf'' or with the uncleavable control (''mcherry-uncleavable mazf'') encoding plasmid. following selection, stable clones that constitutively express high level of the cleavable construct (denoted ''tet-ns3/activated mazf cells'') or the uncleavable control (denoted ''tet-ns3/uncleavable mazf cells'') were isolated. in order to characterize the intracellular distribution of the mcherry fused zymoxins, tet-ns3/activated or uncleavable mazf cells were subjected to immunofluorescence microscopy analysis. as shown in figure 3 (a and b), both zymoxins have a similar cellular distribution, colocalizing with the er marker calnexin at the juxtanuclear region of the er. importantly, upon induced expression of ns3-a4 in tet-ns3/uncleavable mazf cells, a colocalization of the two fluorescent fusion proteins could be observed ( figure 3 , c and d).these observations confirm that indeed both the protease and the modified ribonuclease are tethered to a common cellular compartment, presumably the cytoplasmic side of the er membrane (see scheme in figure 1 ). since expression of active mazf was found to inhibit de-novo protein synthesis in mammalian cells [27] , we hypothesized that such an effect may be observed also in cells in which mazf based zymoxin is proteolytically activated. in order to validate this assumption, tet-ns3/activated mazf and tet-ns3/uncleavable mazf cells were supplemented with tetracycline for 24 or 48 h, or left untreated. levels of de-novo protein synthesis were than determined by [ 3 h]-leucine incorporation assay. as shown in figure 4 (upper panel), a complete shutoff in protein synthesis was observed as soon as 24 h post ns3 induction in cells that express the cleavable construct, indicating proteolytic activation of the zymoxin. as expected, protein synthesis was not impaired following ns3 induction in cells that express the uncleavable toxin. figure 1 . schematic representation of the construct ''mcherry-ns3 activated mazf'' and hypothetical mechanism of activation by ns3 protease. the ns3-activated mazf zymoxin was constructed by fusing 5 elements in the following order (from the n terminus): monomeric red fluorescence protein mcherry, e. coli mazf ribonuclease, hcv p10-p10' ns3 cleavage sequence derived from 2a genotype (strain jfh1) ns5a/b junction, a short inhibitory peptide corresponding to maze c-terminal 35 amino-acids (which encompass the 23 amino-acids inhibitory peptide (mazep) that has been described by li et al. [25] ) and the c-terminal er membrane anchor of the tyrosine phosphatase ptp1b [28] . after being anchored to the er membrane, the ns3 cleavage site that is located between the ribonuclease and the inhibitory peptide in the ''mcherry-ns3 activated mazf'' construct (which is active as a dimer but for convenience is illustrated here in its monomeric form) is cleaved by the hcv-ns3 protease which is also localized to the cytoplasmic side of the er membrane. the toxic ribonuclease, no longer covalently tethered to its er-anchored inhibitor, is now free to diffuse to the cytoplasm (which lacks the antidote) and exert its destructive activity. doi:10.1371/journal.pone.0032320.g001 in support of these findings, an immunoblot assay revealed a near complete cleavage of the zymoxin following tetracycline-induced expression of ns3 protease in ns3-activated mazf expressing cells ( figure 4 , lower panel). presumably, this proteolytic activation of the ribonuclease toxin resulted in a cessation of cellular protein synthesis soon after induction, what explains the detection of relatively low levels of ns3 protease in these cells. as expected, no zymoxin cleavage could be detected in cells expressing the uncleavable form of the toxin. a faint band, corresponding to a cleaved zymoxin, can also be detected in uninduced cells that express the ns3 cleavable construct. this cleavage can be attributed to the ''leakiness'' of the tet inducible system, allowing a very low figure 2 . colony formation assay for the assessment of ''mcherry-ns3 activated mazf'' cytotoxicity toward naïve cells. a day before transfection, 7.5610 5 hek293 t-rex cells where seeded per well in 6 wells plate and subsequently transfected with 2 mg of plasmids encoding either mcherry-ns3 activated mazf, mcherry (only the fluorescent protein) or egfp-mazf (where mazf is not fused to its inhibitory peptide). 48 hours later, transfection efficiency was assessed by fluorescence microscopy and was determined as equal between the plasmids. transfected cells were than trypsinized, counted and seeded in 3 fold dilutions (starting from 150,000 cells/well) in 6 well plates and were incubated for 10 days in the presence of 1 mg/ml of g418 (to which all three plasmids confer resistance). surviving colonies were fixed and stained with giemsa (upper panel). number of surviving colonies from wells that were seeded with 5556 cells was determined by manual counting. each bar represents the mean 6 standard deviation (sd) of a set of data from two wells (lower panel). doi:10.1371/journal.pone.0032320.g002 zymoxin-mediated removal of hcv infected cells plos one | www.plosone.org basal transcription of the protease in the absence of externally added tetracycline. such cleavage could not be detected in ''naïve'' cells following transfection with ns3-cleavage substrate encoding transgene ( [10] , data not shown). apparently, this very low, basal proteolytic activity is well tolerated by ns3 activated zymoxin expressing cells that shows no indication of de-novo protein synthesis inhibition in comparison to cells that express the uncleavable toxin (see cpm values in the upper panel of figure 4 ). to evaluate the potential of the ns3-cleavable mazf based zymoxin in eradication of ns3 expressing cells, three 96 well plates were seeded with tet-inducible full ns3-4a, tet-ns3/activated mazf or tet-ns3/uncleavable mazf cells and were supplemented with 3 fold serial dilutions of tetracycline starting with 1000 ng/ ml. after 72 h, the relative fraction of viable cells was determined using an enzymatic mtt assay. as shown in figure 5 (lower panel), the expression level of ns3 can be roughly tuned by modulation of the final tetracycline concentration in the growth figure 4 . inhibition of de-novo protein synthesis by ns3-activated mazf based zymoxin in ns3-expressing cells. 1610 5 tet-ns3/ activated mazf or tet-ns3/uncleavable mazf cells were seeded per well in 24-wells plate. 24 or 48 h later, cells were supplemented with tetracycline to a final concentration of 1000 ng/ml, or left untreated (48 h tet, 24 h tet and no tet, respectively). 72 h after seeding, levels of de-novo protein synthesis were determined by [ 3 h]-leucine incorporation assay, as described in ''materials and methods''. results are expressed as percent of the value obtained for cells which were not induced to express the ns3 protease (no tet). each bar represents the mean 6 sd of a set of data determined in triplicates. numbers above each bar represent mean counts per minute (cpm) values for 7 micrograms total protein samples (upper panel). 30 micrograms of total protein from lysates of the described cells were analyzed by immunoblotting with mouse anti-mcherry (for detection of the zymoxin), mouse anti-gfp (for the detection of egfp-ns3) and mouse anti actin antibodies (loading control) followed by hrp-conjugated secondary antibodies and ecl development. solid arrow: full length zymoxin. hollow arrow: n' terminal portion of ns3-cleaved zymoxin (lower panel). doi:10.1371/journal.pone.0032320.g004 media, with around 10 ng/ml as an intermediate concentration for induction of low ns3 expression level. indeed, strong cytotoxicity was clearly evident when tet-ns3/activated mazf cells where treated with tetracycline concentrations of down to 4 ng/ml. no cytotoxic effect was detected when the controls tet-ns3/uncleavable mazf or tet-inducible full ns3-4a (protease only) expressing cells were similarly treated ( figure 5 , upper panel). these findings demonstrate the deleterious effect of the mazf based zymoxin specifically toward ns3 protease expressing cells, as well as its competence to be activated by very low cellular levels of protease. in order to obtain a visual insight at the cell level, tet-ns3/activated mazf and tet-ns3/uncleavable mazf cells were supplemented with tetracycline to a final concentration of 10 ng/ml or 1000 ng/ml (for low and high induction levels of ns3 expression, respectively), or left untreated. 36 h later, nuclei were stained and cells were examined under a fluorescence microscope. the results show that both lower and higher induction levels of ns3 protease caused growth inhibition and rounding of cells that constitutively expresses the cleavable mazf. furthermore, both green and red fluorescence were faint in these cells, probably as a result of the destructive ribonuclease activity of the cleaved toxin toward the ns3 protease and its own mrna. as expected, none of the above observations was evident when these cells were not supplemented with tetracycline or when ns3 expression was induced to high level in cells that constitutively express the uncleavable toxin ( figure 6 ). adenovirus-mediated delivery of mcherry-ns3 activated mazf encoding cassette specifically eradicates ns3 expressing hepatocytes to achieve efficient dna delivery into mammalian cells, the expression cassette encoding mcherry-ns3 activated mazf was cloned into a ''first generation'' de1/de3 human type 5 adenoviral vector plasmid dna by homologous recombination in bacteria [35] . virus particles were propagated in hek293 packaging cells as described in the ''materials and methods'' section. in addition, a control adenoviral vector was constructed for the delivery of a similar cassette that encodes for the . after 24 h, cells were supplemented with 3 fold dilutions of tetracycline starting with concentration of 1000 ng/ml, or left untreated. 72 hours later, the fraction of viable cells (relatively to the untreated controls) was determined using an enzymatic mtt assay. each bar represents the mean 6sd of a set of data from six wells. lower panel: 30 ng of total protein from lysates of tet-ns3/uncleavable mazf cells that were supplemented with 3 fold dilutions of tetracycline for 48 h were analyzed by immunoblotting with mouse anti-gfp (for the detection of egfp-ns3) and mouse anti-actin antibodies (loading control) followed by hrp-conjugated secondary antibodies and ecl development. doi:10.1371/journal.pone.0032320.g005 uncleavable version of the construct (mcherry-uncleavable mazf). red-fluorescent comet-like adenovirus-producing foci were apparent upon infection of packaging cells with both recombinant viruses (encoding cleavable or uncleavable constructs) (see figure s1 ). the production yields for both viruses were ,3610 8 plaque forming units (pfu)/ml, after two ''cycles'' of virus amplification (see text s2). in order to evaluate the ability of adenovirus-mediated delivery of ns3 activated mazf encoding cassette to eradicate ns3 expressing hepatocytes, wild-type huh7.5 hepatoma cells and our previously described egfp-full ns3-4a expressing huh7.5 cells [10] were infected with the ns3-activated or uncleavable mazf encoding viruses. since our previous unpublished observations and studies of others have indicated that high level of transgene expression and adenoviral infection, per se, may adversely affect cell viability and growth [36, 37, 38, 39] ; the cells were infected with a series of multiplicity of infection (moi) ratios in order to find an optimal moi that leads to eradication of ns3-expressing cells while maintaining minimal toxicity to naïve cells. as shown in figure 7a (left panel), infection with the ns3activated zymoxin resulted in a considerable cytotoxic effect against ns3-expressing hepatocytes, leading to their almost complete eradication at moi's$12. however, infection at these ratios also adversely affected the growth of wild-type hepatocytes. in contrast, infection at moi of ,3 decreased the viability of the ns3-expressing huh7.5 cells to about 35% of the untreated control without affecting the viability of the wildtype hepatocytes (see also microscopic examination in figure 7b ). therefore, infection at this moi was applied during the next experiments. as expected, no substantial enhancement in cytotoxicity against ns3 expressing huh7.5 cells (relatively to wild-type huh7.5 cells) was observed, at any moi, upon infection with the uncleavable mazf encoding viruses ( figure 7a , right panel). adenovirus-mediated delivery of ns3-activated mazf encoding cassette specifically eradicates hcv infected hepatocytes to test the competence of the mazf based zymoxin to specifically eradicate hcv infected hepatocytes, we utilized the infectious chimeric virus hj3-5 [40] . this is one of the currently used models to study hepatitis c virus in which recombinant infectious hcv particles are produced in cell culture (hcvcc) (for review, see [41, 42] ). as the main purpose of zymoxins based treatment is the specific eradication of hcv infected cells from a background of healthy cells, ''mixed culture'' experiments were carried out. in these experiments, huh7.5 hepatoma cells were infected with the hcv 1a/2a chimeric virus hj3-5 (encoding the ns3 protease of genotype 2a strain jfh1 [40] ). when infection reached ,50% (about 50% of the cultured cells showed expression of the hcv-core protein, as detected by immuno-staining and fluorescence microscopy), the mixed culture and a culture of uninfected cells were treated with ns3 activated mazf or uncleavable-mazf encoding adenoviruses at moi of ,3. control cells remained untreated. 72 h post adenoviral infection; viability assay and microscopic examination that included immunostaining for hcv-core protein were performed. as shown, treating the mixed culture with ns3-activated mazf zymoxin-encoding adenovirus reduced the viable cells population to about 65% relatively to untreated control; while viability of the uninfected cells (''hcv negative'') was barely affected by this treatment (figure 8, upper panel) . microscopic examination of the treated mixed culture revealed two cell populations that differ in their appearance. while one population is characterized by a ''typical'' huh7.5 cell morphology (hollow arrows, figure 8, lower panel) , the other is composed of partially detached cells with round, condensed or distorted shape (filled arrows) that are hypothesized to be zymoxin-intoxicated hcv infected cells. in order to validate this assumption, the fraction of hcv infected cells from the general population was evaluated by immunofluorescence analysis using anti-hcv core protein specific antibodies. indeed, treatment with the ns3activated zymoxin showed a ''curing effect'' upon the partially infected culture, considerably reducing the fraction of the hcv infected cells from the general population ( figure 9 ). as expected, no significant effect upon the hcv infected cell population was observed following treatment with the uncleavable zymoxin. fight against viral infections is considered as one of the most challenging areas in modern medicine. while vaccination is considered to be the most efficient method for fighting viral infections; for some viral pathogens which cause world-wide health problem like human immunodeficiency virus (hiv) and hepatitis c virus (hcv), no efficient vaccine has yet been developed. over the last years, efforts have been focused on the discovery and development of anti-viral agents that target crucial steps in the viral replication cycle which includes viral entry, rna translation and post-translational processing, reverse transcription, genome integration, viral assembly and release [43, 44, 45] . an essential step in the replication cycle of many viruses is the processing of a polyprotein precursor by a viral-encoded protease. a partial list of human diseases associated viruses encoding protease(s) in their genome include flaviviruses such as hepatitis c virus (hcv), west nile virus (wnv), dengue fever virus (dfv) and yellow fever virus (yfv); retroviruses such as hiv-1; picornaviruses such as coxsackievirus, poliovirus and hepatitis a virus; nidoviruses such as coronaviruses (cov), including the severe acute respiratory syndrome (sars) causative sars-cov and herpesviruses such as varicella-zoster virus (vzv) and epstein-bar virus (ebv) [46, 47] . therefore, a large part of antiviral drug discovery is focused on inhibiting viral proteases [46] . our group too has published several studies where hcv replication was inhibited by intracellular expression of antibodies and peptide aptamers [48, 49, 50] . the opposite ''side of the coin'' -taking advantage of the activity of a viral protease per-se as an antiviral approach has been rather neglected. recently we described a study that was based on the concept of ''sitoxins'' which are anti-viral agents that are designed to eradicate viral-infected cells by taking advantage of a specific viral activity instead of inhibiting it [51, 52] . specifically, a sitoxin is comprised of an effector domain (e.g. a toxin); a domain bearing an intracellular signaling moiety (e.g. a degradation or an intracellular localization signal); and a domain located between the effector domain and the domain bearing the intracellular signaling moiety which specifies a cleavage site for a predetermined protease (e.g. a viral encoded protease). following the introduction of the sitoxin into the target cell (that expresses the specific protease), cleavage by the predetermined protease separates the toxic effector domain of the sitoxin from the intracellular signaling moiety, resulting either in a longer-lived (and therefore more toxic) effector domain or in an effector domain that moves from a cellular compartment where the domain is nontoxic to a cellular compartment where the domain is able to exert its effect [51] . we developed and successfully evaluated two rationally designed viral-protease-activated chimeric toxins which we named ''zymoxins'' for ''zymogenized toxins'' [10] . in contrast to sitoxins, in which the inhibitory activity is mediated by intracellular components which recognize a cleavable signaling polypeptide fused to a constitutively active toxin; the concept of zymoxins is based on the idea of reengineering a toxic enzyme into an inactive zymogen which is specifically activated by a predetermined protease. this general approach was demonstrated by other elegant studies in which several enzymes, including bovine rnase a, vip2 and the maize ribosome inactivating protein (maize-rip) were converted into protease activated forms [53, 54, 55, 56, 57] . our previous work focused on the design of protein-delivered toxins that were converted into protease-activated forms by means of fusion to specific, rationally designed inhibitory peptides through hcv-ns3 protease cleavable linker. when tested in-vitro and on ns3-overexpressing or hcv infected cells, a clear ns3 protease cleavage-dependent enhancement in activity/cytotoxicity was observed. however, these constructs had two major drawbacks, as mentioned in the introduction: the first is the incomplete inhibition of the toxic enzymatic activity that is conferred by the rationally designed fused peptide. the second relates to the necessity of relatively high level of expressed viral protease for achieving adequate zymoxin activation inside the cells [10] . in the current study, we turned to evaluate a similar strategy for eradicating hcv-infected cells, namely, by converting a constitutively active enzyme into an ns3 activated zymogen. as a toxic moiety, we chose mazf, an endoribonuclease that together with its polypeptidic antidote, maze, constitute one of the most studied ''genomic'' toxin-antitoxin systems in e. coli. in order to convert mazf into a zymoxin, a fusion polypeptide was constructed in which an inhibitory peptide derived from the maze antitoxin was fused to the c terminus of the mazf toxin via an ns3-cleavable linker. regarding the issue of incomplete inhibition of zymoxin's activity in uncleaved form, it should be mentioned that in contrast to our previously described constructs, in which a ''rationally designed'' peptide is appended to provide the inhibition of the toxin's activity; in the mazf based-zymoxin, a ''natural'' inhibitory polypeptide was chosen for that purpose. antidote polypeptides in toxin-antitoxin systems were ''evolutionary shaped'' to strongly inhibit the destructive activity of their toxic counterparts. thus, a very efficient inhibition may be achieved when using them as inhibitory peptides in the construction of zymoxins. indeed, our results show that the fused maze-derived inhibitory peptide diminishes the toxin's activity to such an extent that enables its non-lethal overexpression in naïve cells. in order to improve the responsiveness of the zymoxin to the presence of low levels of cellular-expressed viral protease, an er membrane ''anchoring peptide'' was fused to the c terminus of the construct, subsequently to the maze derived inhibitory peptide. by using that design, a colocalization between the er-bound ns3 protease and its zymoxin substrate might be achieved, resulting in an improved cleavage efficiency. indeed, such a colocalization could be observed, as shown above (figure 3) . moreover, activation of the zymoxin was evident also upon expression of very low cellular levels of the viral protease ( figures 5 and 6) . it should also be mentioned that while being extremely sensitive to minute amounts of expressed ns3 protease, zymoxin expressing cells can still tolerate some degree of activating proteolysis (as shown above for ns3-activated mazf expressing cells that show a very low, basal ns3 proteolytic activity without tetracycline induction (figure 4) ). this property may, in fact, contribute to the specificity and general safety of zymoxin-based therapeutics, even though ''spontaneous'', unspecific proteolytic activity toward ns3 substrates could not be detected in naïve cells during our experiments (data not shown). as discussed, tethering the zymoxin to the er membrane through its inhibitory peptide may also enhance its cleavage-dependent toxicity. this might be achieved by enabling spatial separation between the activated toxin and its antidote following ns3-cleavage and diffusion of the active mazf to the inhibitory peptide-free cytoplasm. supporting this assumption, we found that although cleaved, a cytoplasmic version of the ns3-activated mazf based zymoxin is barely toxic to ns3 protease-expressing cells (data not shown). in contrast to our previous study, in which the toxins were delivered into mammalian cells as purified recombinant proteins; in the current research an adenoviral vector gene-delivery system was used. widely applied in gene therapy, recombinant viral vaccines and basic science studies, this system enables efficient delivery and high level of transgene expression in a wide variety of human cells. using this system, we have demonstrated specific eradication of full ns3-4a expressing huh7.5 cells, sparing ''naïve'' cells which do not express the protease. furthermore, specific eradication of hcv infected cells has also been achieved, demonstrating a prominent ''curing effect'' when tested on mixed cultures of healthy and hcv infected hepatocytes. the use of viral gene delivery for treating hcv infection by eradication of infected cells has been proved successful in previous study on mice with chimeric human livers. in that study, hsu et al designed a modified proapoptotic bid molecule in which its endogenous cleavage sites were replaced by ns3 recognition site. as bid requires proteolytic processing in order to activate its apoptotic function, infection with adenoviral vector that delivers a transgene encoding the engineered bid molecule was demonstrated to induce activation of apoptosis in cells expressing the hcv ns3 protease. moreover, a significant reduction in hcv titer in the serum of the hcv infected mice was detected following injection of the engineered adenoviral vector into the jugular vein [58] . although such an approach had proved successful; it should be taken into account that a delivered transgene which encodes for a modified form of a protein that is naturally expressed in the target cell has the potential to negatively influence normal cellular processes in which its endogenous counterpart plays a role [59, 60, 61, 62, 63] . for example, when overexpressed in noninfected cells, an ns3-cleavable apoptotic molecule, such as that described above, may act as a ''dominant negative'' by competing with the natural, endogenous protein for interactions with activators, regulators or substrates. delivery of a transgene encoding for a foreign protein which has no endogenous counterpart in target cells, such as mazf, may reduce that risk. furthermore, it should be noted that mazf possess a ribonuclease activity that is also capable of processing multiple potential cleavage sites in the hcv genome. although not evaluated in this study, a direct attack on the parasite's genetic material may represent an additional mode of anti-viral activity that works in parallel with host-cell protein synthesis shutoff. of note, the applicability of the described zymoxin may be extended by replacing the protease cleavage site that separates between the toxic moiety and the inhibitory peptide. this could facilitate eradication of cells that are infected with other protease expressing viruses other than hcv (a partial list of protease expressing viruses is given at the beginning of the discussion) or different strains of hcv that differ in their ns3 protease cleavage specificity. in addition, one may replace the c terminal er anchoring peptide with sequence that tethers the construct to a different intracellular location in which the viral protease resides. in that way, a colocalization between the zymoxin and the predetermined viral protease may be achieved. in conclusion, the presented anti-viral agent was designed following our previously described ''zymoxins'' concept in which a constitutively active toxin is converted into a ''zymogenized'', viral-protease activated from. the mazf-based zymoxin, that is introduced to target cells by means of adenovirus mediated gene delivery, shows very low toxicity to naïve cells and enhanced responsiveness to low viral-protease expression level, when compared to our previously presented constructs. as evident from our results, the mazf based zymoxin eradicates ns3-expressing model cells and hcv infected cells with remarkable efficiency and specificity, providing further proof to the concept of zymoxins and a potential new means of fighting viral diseases. obviously, further optimizations and pharmacological assessments in animal models may be required in order to determine the safety and efficiency of zymoxins treatment in the context of the whole organism. the following escherichia coli (e. coli) strains were used: xl-1 blue and dh5a (stratagene, usa) for plasmid propagation and bj5183 (stratagene, usa) for the generation of recombinant adenovirus plasmid dna. recombinant dna techniques were carried out according to standard protocols or as recommended by the suppliers. nucleotide sequences were determined using the prism 3100 genetic analyzer (applied biosystems, usa) according to the supplier's recommendations. the eukaryotic cmv promoterbased gfp-fusion expression vector pegfp c2, which was used for expression of mcherry, mazf and mazf-based zymoxins, was from clontech (usa). the adeasy plasmid system (pshuttle and padeasy-1) [35] , that was used for generation of recombinant human type 5 adenoviral vectors for gene delivery of the zymoxins expression cassettes, was a generous gift from prof. nadir arber, integrated cancer prevention center, tel aviv sourasky medical center, israel. all plasmid and dna fragment purifications were carried out with high-speed plasmid mini kit and gel/pcr dna fragments extraction kit (geneaid biotech ltd., taiwan) unless mentioned otherwise. t4 dna ligase and restriction enzymes were purchased from new england biolabs (usa). dna ligations were carried out at 16uc overnight. oligonucleotides. all the oligonucleotides that were used in this study were purchased from hylabs, israel. oligonucleotides that were used in this study are listed in table s1 . construction of the vector encoding for ''mcherry-ns3 activated mazf''. a polymerase chain reaction (pcr) was carried out using a single colony of escherichia coli strain xl-1 as a template, the forward primer: 40-clvmazf and the reverse primers: 41-clvmazf, 42-clvmazf, 43-clvmazf, 44-clvmazf, 45-clvmazf, 46clvmazf and 47-clvmazf. the pcr product, encoding for a fusion polypeptide composed of (from the n terminus) mazf, hcv p10-p10' ns3 cleavage sequence derived from 2a genotype (strain jfh1) ns5a/b junction, a short flexible linker, a short inhibitory peptide corresponding to maze c-terminal 35 amino-acids (which encompass the 23 amino-acids inhibitory peptide (mazep) that has been described by li et al. [25] ), a flexible linker and the cterminal er membrane anchor of the tyrosine phosphatase ptp1b [28] was digested with xhoi and ecori and was cloned between the corresponding sites in the plasmid pegfp-c2, generating plasmid ''pegfp-ns3 activated mazf''. next, the sequence of the red fluorescent protein mcherry [29] was amplified by pcr from an expression cassette (kindly provided by prof. adi avni, department of molecular biology and ecology of plants, tel-aviv university, israel) using the forward primer: 48clvmazf and the reverse primer: 49-clvmazf. the pcr product was digested with nhei and xhoi and was cloned between the corresponding sites of the plasmid ''pegfp-ns3 activated mazf'' (replacing the egfp coding sequence), generating plasmid: ''pmcherry-ns3 activated mazf''. the amino-acid sequence of the encoded cleavable zymoxin can be found in text s1. construction of the vector encoding for ''mcherryuncleavable mazf''. a pcr was carried out using dna of plasmid ''pmcherry-ns3 activated mazf'' as a template, the forward primer: 40-clvmazf and the reverse primers: 50-unclmazf and 51-unclmazf. the pcr product was digested with ecorv and nrui, and the digestion product of 233 bp was cloned between the corresponding sites of the same plasmid that has been used as a template, generating the plasmid: ''pmcherry-uncleavable mazf''. this plasmid encodes for an uncleavable construct in which the ns3 cleavage sequence was replaced by a mutated 14 amino acids cleavage sequence (p10-p4') from hcv genotype 1a ns5a/b junction in which p3 valine was substituted by alanine, p2 cysteine by glycine, p1 cysteine by glycine and p4' tyrosine by alanine. the amino-acid sequence of the encoded uncleavable zymoxin can be found in text s1. construction of the vector encoding for ''egfp-mazf''. this is a mutated variant of an ''intermediate'' vector used in the construction process of the ''mcherry-ns3 activated mazf'' encoding vector. in this variant, a nonsense mutation was inserted instead of the tyrosine in the smsy sequence of the ns3 recognition site, generating the plasmid ''pegfp-mazf'' that encodes for a truncated egfp-mazf fusion protein that lacks the maze derived inhibitory peptide and the er anchor. construction of the vector encoding for mcherry. the sequence of the red fluorescent protein mcherry [29] was amplified by pcr from an expression cassette (see construction of the vector encoding for ''mcherry-ns3 activated mazf'') using the forward primer: 48-clvmazf and the reverse primer: 49clvmazf. the pcr product was digested with nhei and bglii and was cloned between the corresponding sites of the plasmid pegfp c2 (replacing the egfp coding sequence), generating the plasmid ''pmcherry''. construction and propagation of recombinant adenoviral vectors. construction and propagation of recombinant human type 5 adenoviral vectors for gene delivery of the mcherry-ns3 activated mazf and mcherry-uncleavable mazf expression cassettes was carried out using the adeasy system essentially as described in [35, 64] . a more detailed description of the procedure is provided in text s2. human embryonic kidney cells hek293, stably expressing the tetracycline repressor protein (t-rex hek293 cell line, invitrogen, usa), and human hepatoma cells huh7.5 [65] were used throughout this study. cell lines were maintained in dulbecco's modified eagle medium (dmem) supplemented with 10% fetal calf serum (fcs), 2 mm l-glutamine, 100 u/ml penicillin and 100 mg/ml streptomycin (biological industries, israel) in a humidified 5% co 2 incubator at 37uc. the calcium-phosphate transfection method was applied for introducing 2 mg of the plasmid ''pmcherry-ns3 activated mazf'' or the plasmid ''pmcherry-uncleavable mazf'' into t-rex hek293 cells inducibly expressing egfp-full ns3-4a [10] seeded 1.5610 6 cells per 60 mm plate 24 h before transfection. stable transfectants, inducibly expressing egfp-full ns3-4a and constitutively expressing mcherry-ns3 activated mazf (denoted ''tet-ns3/activated mazf cells'') or mcherry-uncleavable mazf (denoted ''tet-ns3/uncleavable mazf cells'') were selected in a medium containing 1 mg/ml of g418 (a.g. scientific, usa). cell clones that express high level of the cleavable construct or the uncleavable control were identified by fluorescence microscopy and isolated. for protein extraction, 48 h post-transfection the cells were washed with pbs, scraped and lysed in a buffer containing 150 mm nacl, 5 mm edta, 0.5% np-40, 10 mm tris(hcl) ph 7.5, and protease inhibitors cocktail (sigma, israel). following 30 minutes of incubation on ice, lysates were cleared by centrifugation at 20,000 g for 10 minutes, at 4uc. for immunoblotting, protein samples were electrophoresed on 12% sds/polyacrylamide gel, transferred to nitrocellulose and detected using mouse monoclonal anti-mcherry antibody (clontech, usa), mouse monoclonal anti-gfp antibody (santa-cruz, usa) or mouse monoclonal anti-actin antibody (abcam, usa), followed by horseradish peroxidase (hrp)-conjugated goat anti-mouse antibodies (jackson immunoresearch laboratories, usa) and enhanced chemiluminescence (ecl) detection using supersignal west pico chemiluminescent substrate (thermo scientific/pierce, usa). virus assays were carried out with an inter-genotypic chimeric hepatitis c virus (hcv) produced by replacing the core-ns2 segment of the jfh-1 virus genome with the comparable segment of the genotype 1a h77 virus. this chimeric virus, hj3-5 (kindly provided by prof. stanley lemon, university of texas at galveston), contains two compensatory mutations that promote its growth in cell culture as described previously [40] . hcv rnas were transcribed in vitro and electroporated into cells essentially as described previously [66, 67] . in brief, 10 mg of in vitro-synthesized hcv rna was mixed with 5610 6 huh7.5 cells in a 2-mm cuvette and pulsed twice at 1.4 kv and 25 mf. cells were seeded into 12well plates or 25-cm 2 flasks, and passaged at 3-to 4-day intervals posttransfection by trypsinization and reseeding with a 1:3 to 1:4 split into fresh culture vessels. when infectivity reached ,50%, as was monitored by immunofluorescent staining with anti hcv core protein mouse monoclonal antibody (affinity bioreagents, usa) and cy2-conjugated goat anti-mouse igg (jackson immunoresearch laboratories, usa) (see ''visualizing hcv infected cells (infectivity assay)'' below), the mixed culture (of uninfected and hcv infected cells in 1:1 ratio) was taken for cytotoxicity assays. confocal fluorescence microscopy analysis of t-rex hek293 cells inducibly expressing egfp-full ns3-4a and constitutively expressing mcherry-ns3 activated or uncleavable mazf. 1610 5 tet-ns3/activated mazf or tet-ns3/uncleavable mazf cells were seeded on poly-l-lysine coated cover-slips in a 24 well-plate. 12 h later, the cells were supplemented with 1 mg/ml of tetracycline, or left untreated. 24 h later, the cells were fixed with 4% formaldehyde in pbs. uninduced cells were permeabilized with triton x-100 (0.1% in pbs) for 5 minutes, blocked with 90% fetal calf serum/10% pbs at room temperature for 25 minutes, incubated with 1:200 diluted rabbit-polyclonal anti-calnexin antibody (sigma, usa) as primary antibody for 1 h and followed by 1:200 diluted cy2conjugated anti-rabbit igg (jackson immunoresearch laboratories, usa) secondary antibody for 30 minutes. nuclei of induced and uninduced cells were then stained by hoechst 33258 for 1 h at room temperature. slides were washed with pbs, mounted in mowiol 4-88 solution (calbiochem, usa) (immunostained uninduced cells) or immuglo mounting medium (immco diagnostics, usa) (induced tet-ns3/uncleavable mazf cells) and examined using a zeiss lsm 510 meta laser scanning confocal microscope. visualizing t-rex hek293 cells inducibly expressing egfp-full ns3-4a (supplemented with different tetracycline concentrations) and constitutively expressing mcherry-ns3 activated mazf or mcherry-uncleavable mazf. 1610 5 tet-ns3/activated mazf or tet-ns3/uncleavable mazf cells were seeded on poly-l-lysine coated cover-slips in a 24 well-plate. 12 h later, cells were supplemented with 10 ng/ml or 1000 ng/ml of tetracycline, or left untreated. 36 h later, cells were fixed with 4% formaldehyde in pbs. following nuclear staining by hoechst 33258 for 1 h at room temperature, slides were washed with pbs, mounted in immuglo mounting medium and examined using a fluorescence microscope. visualizing hek293 cells infected with recombinant adenovirus encoding for mcherry-ns3 activated mazf or mcherry-uncleavable mazf. 3610 5 hek293 cells were seeded per well in 6 wells plate. when the culture reached 90% confluence, the growth medium was replaced by fresh medium containing 10 fold dilutions of recombinant adenoviruses encoding for mcherry-ns3 activated mazf or mcherry-uncleavable mazf, starting from 2.5610 6 pfu per well. after 36 h, cells were fixed with 4% formaldehyde in pbs and examined using a fluorescence microscope. visualizing hcv infected cells (infectivity assay). huh7.5 cells infected with hcv hj3-5 chimeric virus were seeded into 8well chamber slides (nalge nunc, usa). after 24 h, cells were fixed and permeabilized with 1:1 acetone/methanol mixture and stained with 1:300 diluted mouse monoclonal antibody c7-50 (affinity bioreagents, usa) specific for the hcv core protein followed by staining with 1:100 diluted cy2-conjugated goat anti-mouse igg (jackson immunoresearch laboratories, usa). nuclei were then stained with dapi (sigma, israel) and slides were washed with pbs, mounted (southernbiotech, usa) and examined using a fluorescence microscope. visualizing zymoxin-treated mixed culture of uninfected and hcv infected cells. see ''cytotoxicity assay of recombinant adenoviruses encoding for mcherry-ns3 activated mazf or mcherry-uncleavable mazf on a mixed culture of uninfected and hcv-infected huh7.5 cells'' below. colony formation assay 7.5610 5 hek293 t-rex cells were seeded per well in 6 wells plate. 24 h later, cells were transfected with 2 mg of the plasmids ''pmcherry-ns3 activated mazf'', ''pmcherry'' or ''pegfp-mazf'' encoding for mcherry-ns3 activated mazf, mcherry (only the fluorescent protein) or egfp-mazf (where mazf is not fused to its inhibitory peptide), respectively. transfection was carried out using fugene 6 reagent (roche, germany) according to the manufacturer instructions. after 48 h, transfection efficiency was assessed by fluorescence microscopy and was determined as equal between the plasmids. transfected cells were then trypsinized, counted and seeded in 3 fold dilutions (starting from 150,000 cells/well) in 6 well plates and were incubated for 10 days in the presence of 1 mg/ml of g418 (to which all the three plasmids confer resistance). surviving colonies were fixed with 4% formaldehyde in pbs and stained with giemsa (sigma, usa). number of surviving colonies from wells that were seeded with 5556 cells was determined by manual counting. the cell-killing activities of ns3-activated mazf and uncleavable mazf zymoxins were measured by a thiazolyl blue tetrazoliam bromide (mtt) assay as described below: cytotoxicity assay of intracellularly expressed mcherry-ns3 activated mazf or mcherry-uncleavable mazf in t-rex hek293 cells inducibly expressing egfp-full ns3-4a. tet-inducible full ns3-4a, tet-ns3/activated mazf or tet-ns3/uncleavable mazf cells were seeded in 96 well plates (2610 4 cells per well). after 24 h, cells were supplemented with 3 fold dilutions of tetracycline starting with concentration of 1000 ng/ml, or left untreated. 72 h later, the media was replaced by fresh media (100 ml per well) containing 1 mg/ml mtt (thiazolyl blue tetrazoliam bromide (sigma, israel) dissolved in pbs) reagent and the cells were incubated for further 30 minutes. mtt-formazan crystals were dissolved by the addition of extraction solution (20% sds, 50% n, n-dimethyl formamide (dmf), ph 4.7) (100 ml per well) and incubation for 16 h at 37uc. absorbance at 570 nm was recorded on an automated microtiter plate reader. the results were expressed as percentage of living cells relatively to the untreated controls. cytotoxicity assay of recombinant adenoviruses encoding for mcherry-ns3 activated mazf or mcherry-uncleavable mazf on full ns3-4a expressing huh7.5 cells. 1610 4 wildtype or egfp-full ns3-4a expressing huh7.5 cells [10] were seeded per well in 96 well plates. after 24 h, growth media were replaced by fresh media containing recombinant adenoviruses encoding for mcherry-ns3 activated mazf or mcherry-uncleavable mazf at indicated multiplicity of infection (moi) ratios. control cells remained untreated. four days post infection, the media was replaced by fresh media (100 ml per well) containing 1 mg/ml mtt (except in representative wells in which cells were fixed and microscopically examined) and the cells were incubated for further 60 minutes. the next steps were identical to theses described above. cytotoxicity assay of recombinant adenoviruses encoding for mcherry-ns3 activated mazf or mcherry-uncleavable mazf on a mixed culture of uninfected and hcv-infected huh7.5 cells. uninfected huh7.5 cells and mixed culture of hcv infected and uninfected cells at 1:1 ratio (50% infected culture) were seeded in 96-well plates (1610 4 cells/well). after 24 h, cells were treated with recombinant adenoviruses (at moi of ,3) encoding for mcherry-ns3 activated mazf or mcherry-uncleavable mazf zymoxins. control cells remained untreated. 72 h later, the media was replaced by fresh media (100 ml per well) containing 1 mg/ml mtt (except in representative wells in which cells were fixed and microscopically examined) and the cells were incubated for further 60 minutes. the next steps were identical to these described above. for hcv-infection immunofluorescence analysis, 3610 4 cells from the hcv infected and uninfected mixed culture were seeded per well into 8-well chamber slides (nalge nunc, usa). 24 h later, cells were treated with recombinant adenoviruses (moi of ,3) encoding for the mcherry-ns3 activated mazf or mcherryuncleavable mazf zymoxins. control cells were remained untreated. 72 h post treatment, cells were fixed and permeabilized with 1:1 acetone/methanol mixture and stained with 1:300 diluted mouse monoclonal antibody c7-50 (affinity bioreagents, usa) specific for the hcv core protein followed by staining with 1:100 diluted fitc-conjugated goat anti-mouse igg (jackson immu-noresearch laboratories, usa). nuclei were then stained with dapi and slides were mounted (southernbiotech, usa) and examined using a fluorescence microscope. for each treatment, evaluation of the fraction of the hcv-infected cells from the general cell population was performed by dividing the number of the green, hcv-core positive cells by the general number of cells (dapi stained) from five representative microscopic fields. [ 3 h]-leucine incorporation assay 1610 5 tet-ns3/activated mazf or tet-ns3/uncleavable mazf cells were seeded per well in 24-wells plate. 24 or 48 h later, cells were supplemented with tetracycline to a final concentration of 1000 ng/ml, or left untreated. 72 h after seeding, cells were supplemented with [ 3 h]-leucine (perkin elmer, usa) to a final concentration of 1 mci/ml and returned to incubation. after 6 h, cells were scraped, washed with pbs and lysed by four freeze/thaw cycles. 7 mg total protein form the lysate of each treatment were then add to a solution containing pbs and a final concentration of 150 mg bovine serum albumin (bsa) in a total volume of 75 ml. the solution was then mixed with an identical volume of ice-cold 10% trichloro acetic acid (tca). mixtures were then incubated on ice for 30 minutes and centrifuged for 10 minutes at 20,000 g, 4uc, after which the pellet was washed with ice-cold 5% tca, followed by washing with ice-cold 80% ethanol. the pellet was then dissolved in 300 ml of 0.1 m naoh, transferred to a scintillation tube and neutralized with 200 ml 1 m hcl. 4 ml of scintillation liquid was added and radioactivity was counted by a beta-counter device. figure s1 fluorescence microscopy analysis of adenovirus producing foci. 3610 5 hek293 cells were seeded per well in 6 wells plate. when reached 90% confluence, cells were infected with 10 fold dilutions of recombinant adenoviruses encoding for mcherry-ns3 activated mazf or mcherry-uncleavable mazf, starting from 2.5610 6 pfu per well. after 36 h, cells were fixed and examined under a fluorescence microscope. red fluorescent adenovirus-producing foci from wells infected with 2.5610 3 pfu are shown. the bar represents 200 mm. table s1 oligonucleotides that have been used in this study. text s1 amino-acid sequences of mazf based zymoxins. text s2 construction and propagation of recombinant adenoviral vectors. stanley lemon (university of texas at galveston, usa) for the hj3-5 hcvcc clone. we thank prof. nadir arber (integrated cancer prevention center, tel aviv sourasky medical center, israel) for the adeasy plasmid system used for generation of recombinant human type 5 adenoviral vectors. treatment failure and resistance with direct-acting antiviral drugs against hepatitis c virus replication of hepatitis c virus hepatitis c viral life cycle advances in the development of new therapeutic agents targeting the ns3-4a serine protease or the ns5b rnadependent rna polymerase of the hepatitis c virus the ns3/4a proteinase of the hepatitis c virus: unravelling structure and function of an unusual enzyme and a prime target for antiviral therapy role of proteolytic enzymes in biological regulation (a review) mechanism of activation of the gastric aspartic proteinases: pepsinogen, progastricsin and prochymosin structure and zymogen activation of caspases a brief historical review of the waterfall/cascade of blood coagulation engineered toxins ''zymoxins'' are activated by the hcv ns3 protease by removal of an inhibitory protein domain bacterial mitotic machineries bacterial death by dna gyrase poisoning addiction modules and programmed cell death and antideath in bacterial cultures prokaryotic toxin-antitoxin stress response loci toxins-antitoxins: plasmid maintenance, programmed cell death, and cell cycle arrest programmed cell death in bacterial populations hypothetical functions of toxin-antitoxin systems occurrence of mazef-like antitoxin/toxin systems in bacteria toxin-antitoxin loci are highly abundant in freeliving but lost from host-associated prokaryotes bacterial toxin-antitoxin systems: more than selfish entities? mazef: a chromosomal toxinantitoxin module that triggers programmed cell death in bacteria mrna interferases, sequence-specific endoribonucleases from the toxin-antitoxin systems bacterial programmed cell death and multicellular behavior in bacteria crystal structure of the maze/mazf complex: molecular bases of antidote-toxin recognition characterization of dual substrate binding sites in the homodimeric structure of escherichia coli mrna interferase mazf the discovery of mrna interferases: implication in bacterial physiology and application to biotechnology nbk/ bik antagonizes mcl-1 and bcl-xl and activates bak-mediated apoptosis in response to protein synthesis inhibition characterization of the c-terminal er membrane anchor of ptp1b improved monomeric red, orange and yellow fluorescent proteins derived from discosoma sp. red fluorescent protein subcellular localization, stability, and trans-cleavage competence of the hepatitis c virus ns3-ns4a complex expressed in tetracycline-regulated cell lines membrane association of hepatitis c virus nonstructural proteins and identification of the membrane alteration that harbors the viral replication complex structural determinants for membrane association and dynamic organization of the hepatitis c virus ns3-4a complex expression of hepatitis c virus proteins induces distinct membrane alterations including a candidate viral replication complex a dynamic view of hepatitis c virus replication complexes a simplified system for generating recombinant adenoviruses adenoviral vector cytotoxicity depends in part on the transgene encoded recombinant, replication-defective adenovirus gene transfer vectors induce cell cycle dysregulation and inappropriate expression of cyclin proteins effect of adenoviral vector infection on cell proliferation in cultured primary human airway epithelial cells recombinant e1-deleted adenovirus vector induces apoptosis in two lung cancer cell lines compensatory mutations in e1, p7, ns2, and ns3 enhance yields of cell culture-infectious intergenotypic chimeric hepatitis c virus hepatitis c virus molecular clones and their replication capacity in vivo and in cell culture studying hepatitis c virus: making the best of a bad virus review article: investigational agents for chronic hepatitis c novel targets for hiv therapy perspectives on antiviral drug development antiviral drug discovery targeting to viral proteases protease inhibitors as antiviral agents inhibition of protease-inhibitor-resistant hepatitis c virus replicons and infectious virus by intracellular intrabodies inhibition of hepatitis c virus rna replicons by peptide aptamers hcv ns3 serine protease-neutralizing single-chain antibodies isolated by a novel genetic screen the n-end rule toxins that are activated by hiv type-1 protease through removal of a signal for degradation by the nend-rule pathway a ribonuclease zymogen activated by the ns3 protease of the hepatitis c virus creation of a zymogen design and characterization of an hiv-specific ribonuclease zymogen from enzyme to zymogen: engineering vip2, an adp-ribosyltransferase from bacillus cereus, for conditional toxicity a switch-on mechanism to activate maize ribosome-inactivating protein for targeting hivinfected cells modified apoptotic molecule (bid) reduces hepatitis c virus infection in mice with chimeric human livers mutant p53 exerts a dominant negative effect by preventing wild-type p53 from binding to the promoter of its target genes caspase-8 prevents sustained activation of nf-kappab in monocytes undergoing macrophagic differentiation cytochrome c and datp-dependent formation of apaf-1/caspase-9 complex initiates an apoptotic protease cascade a c-jun dominant negative mutant protects sympathetic neurons against programmed cell death structure and activation mechanism of the drosophila initiator caspase dronc a protocol for rapid generation of recombinant adenoviruses using the adeasy system highly permissive cell lines for subgenomic and genomic hepatitis c virus rna replication adaptive mutations producing efficient replication of genotype 1a hepatitis c virus rna in normal huh7 cells production of infectious genotype 1a hepatitis c virus (hutchinson strain) in cultured human hepatoma cells we thank prof. matti sä llberg (karolinska institute, sweden) for the plasmid carrying the ns3-4a gene of the 1a genotype. we thank prof. key: cord-263276-keyu60in authors: zhou, weimin; lin, feng; teng, lingfang; li, hua; hou, jianyi; tong, rui; zheng, changhua; lou, yongliang; tan, wenjie title: prevalence of herpes and respiratory viruses in induced sputum among hospitalized children with non typical bacterial community-acquired pneumonia date: 2013-11-18 journal: plos one doi: 10.1371/journal.pone.0079477 sha: doc_id: 263276 cord_uid: keyu60in objective: few comprehensive studies have searched for viruses in infants and young children with community-acquired pneumonia (cap) in china. the aim of this study was to investigate the roles of human herpes viruses (hhvs) and other respiratory viruses in cap not caused by typical bacterial infection and to determine their prevalence and clinical significance. methods: induced sputum (is) samples were collected from 354 hospitalised patients (infants, n = 205; children, n = 149) with respiratory illness (cap or non-cap) admitted to wenling hospital of china. we tested for hhvs and respiratory viruses using pcr-based assays. the epidemiological profiles were also analysed. results: high rate of virus detection (more than 98%) and co-infection (more than 80%) were found among is samples from 354 hospitalised infants and children with respiratory illness in this study. of 273 cap samples tested, cmv (91.6%), hhv-6 (50.9%), rsv (37.4%), ebv (35.5%), hbov (28.2%), hhv-7 (18.3%) and rhinovirus (17.2%) were the most commonly detected viruses. of 81 noncap samples tested, cmv (63%), rsv (49.4%), hhv-6 (42%), ebv (24.7%), hhv-7 (13.6%) and hbov (8.6%) were the dominant viruses detected. the prevalence of several viral agents (rhinovirus, bocavirus, adenovirus and cmv) among is samples of cap were significantly higher than that of non-cap control group. we also found the prevalence of rsv coinfection with hhvs was also higher among cap group than that of non-cap control. conclusions: with sensitive molecular detection techniques and is samples, high rates of viral identification were achieved in infants and young children with respiratory illness in a rural area of china. the clinical significance of rhinovirus, bocavirus, adenovirus and hhv (especially cmv) infections should receive greater attention in future treatment and prevention studies of cap in infants and children. lower respiratory tract infections (primarily pneumonia) are the leading cause of death worldwide in infants and children [1, 2] . there are approximately 150 million cases of childhood community-acquired pneumonia (cap) each year [1, 3] . cap is a major cause of morbidity and mortality among children in developing countries, which is 10-50 times more common than in developed countries [1, 3] . bacteria as the principal cause of cap in children has been widely investigated [3] [4] [5] . in more than 50% of cases, however, there is still a considerable deficit in the aetiologic diagnosis resulting in unnecessary or inappropriate antibiotic prescription [2, 6] . it is clear that the involvement of viruses in cap have been underestimated due to a lack of understanding of the viral etiology in a clinical setting [7] [8] [9] [10] [11] [12] . in addition, the appropriate sample from infants and young children is critical for the aetiologic diagnosis of cap. lung itself is rarely sampled directly, and sputum, representing lower-airway secretions, can rarely be obtained from children [11, 14, 15] . among children, cap may be caused by a wide variety of microbes, including ''typical'' bacteria (e.g., streptococcus pneumonia) and atypical bacteria, mycobacterium tuberculosis and fungi. viral infections are also involved with 80% of episodes of cap in children under 2 years old and over 40% of older children [6] [7] [8] [9] [10] [11] . studies of cap have traditionally focused little on viral causes [2] . so for, very few studies have included an extensive and appropriate evaluation of the role of viruses in the aetiology of cap in developing countries, including china. in recent years, the introduction of better-quality diagnostic tests has markedly improved the ability to detect multiple viral pathogens [11] [12] [13] , shifting attention to the important role of viruses as a cause of cap [6] [7] [8] [9] [10] [11] . according to previous studies, up to two-thirds of childhood pneumonia cases are associated with a viral infection [3] [4] [5] [6] [7] [8] [9] [10] [11] . respiratory syncytial virus (rsv), influenza virus (ifv), rhinovirus (rv), human metapneumovirus (hmpv) and parainfluenza viruses (pivs) are the most common viruses associated with pneumonia [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] . in addition, the roles of cytomegalovirus (cmv), other herpes viruses (hhvs), recently identified human coronaviruses (hcov-nl63 and -hku1) and human bocavirus (hbov) as causes of cap in infants and children remain controversial [3, [19] [20] [21] [22] [23] [24] [25] . so far, pathogenic profiles of hhv and its role in cap among infants and young children from rural areas have not been well characterized. the present study was undertaken to describe the profiles of hhvs and other respiratory viruses associated with hospital-based cap and non-cap among infants and young children in a rural area of china using comprehensive and sensitive molecular diagnostic techniques. all aspects of this study were performed in accordance with national ethics regulations and approved by the institutional review boards of the centre for disease control and prevention of china and the ethics committee of wenzhou medical college. the participants received written information regarding the purpose of the study and of their right to confidentiality. individual written informed consent was obtained from the parents or guardians of all participants. wenling is located in a rural area on the southeast coast of china with a sub tropical monsoon climate. it has a population of approximately 1,000,000. according to world health organisation clinical criteria [3, 8, 11] , cap was defined as the presence of pneumonic infiltrates (alveolar or parenchymal) on chest radiography with simultaneous signs and/or symptoms of acute infection in which the reading of x-ray films by specialist were blinded to the clinical results. all cap patients were also selected according to a set of necessary criteria based on respiratory symptoms (i.e., dyspnea or respiratory distress, cough, tachypnea) or evidence of parenchymal infiltrates on chest radiography. a total of 354 highquality induced sputum (is) samples (,25 squamous epithelial cells and .25 leukocytes per low-power field) were obtained from 948 hospitalised infants and young children with respiratory illness in wenling hospital from september of 2007 to april of 2008. two hundred and seventy-three samples were preselected from hospitalised children patients who diagnosed as non typical bacterial cap within 48 hrs of admission, while 81 samples from hospitalized children patients were set as a control group, whom were clinical diagnosis as non-cap patients based on chest x-ray and other respiratory signs (asthma, chronic bronchitis or cystic fibrosis) at admission. typical bacterial cap based on microbiologic tests, treatment algorithms and an elevated leukocyte count ($10 10 /l) were excluded. in addition, all patients were selected as immunocompetent at baseline and negative for hiv-1 and tb test. all the immunosuppressed or typical bacterial cap patients were excluded. the children with presumed nosocomical cap and lower-quality induced sputum (is) samples were also excluded. sputum production was induced by the inhalation of a 5.0% hypertonic saline solution; the sputum was sampled during the 1 st week after hospital admission by aspiration through the nostrils. our sputum collection method was described in detail elsewhere [14] [15] [16] . nucleic acid was extracted from 200 ml of the virus transport medium (vtm) using a qiaamp minelute virus spin kit (qiagen, germany) according to the manufacturer's instructions. polymerase chain reaction (pcr) or multiplex pcr was performed as described previously [26] [27] [28] [29] [30] for hhvs, including hsv-1 and -2, varicella zoster virus (vzv), cmv, epstein barr virus (ebv), hhv-6 and -7. adenoviruses (advs), ifv types a and b, piv types 1-3, rsv, picornaviruses (pic, including enteroviruses and rhinoviruses) using multiple rt-pcr assays [26, 27] (table s1 ); and for human coronavirus (hcov)-oc43, -229e, -nl63 and -hku1, human metapneumovirus (hmpv) using rt-pcr or and hbov using nested-pcr assays [27] [28] [29] (table s1). a positive (virus stock or dna) and negative control (vtm only) in each set pcr assay was included to survey the possibility of laboratory contamination. all the methods were reported previously and validation in our lab [26] [27] [28] [29] [30] . the amplicons of positive for pic were gel-purified for dna sequencing using a qiaquick gel extraction (qiagen, germany), according to the manufacturer's instructions. dna sequencing was performed with specific primers using an abi prism bigdye terminator cycle sequencing reaction kit (version 3.1) on an abi prism 3130 dna sequencer (applied biosystems, foster city, ca), following the manufacturer's instructions. enteroviruses (ev) or rhinoviruses were identified based on sequence alignment of amplicons. eligibility and classification of the clinical syndromes of pneumonia were determined from the original record of each item on the medical history and examination in the database. the frequency distribution of viral pathogens between cap and non-cap were analysed by the x 2 test and fisher exact test. all statistical analyses were performed with the statistical package for the social sciences (spss, version17, spss inc., chicago, il). statistical significance was assessed by tukey's test and p-values ,0.05 were considered to be statistically significant. all is samples were collected from hospitalised patients with severe cap (n = 273) and non-cap (n = 81) in wenling area between september 2007 and april 2008. the age and sex distributions are shown in in addition, we found that the prevalence of several viral agents (rhinovirus, hbov, adv and cmv) among is samples of cap were significantly higher than that of non-cap control group (p,0.05), while the prevalence of inf b (5,6.2%) among is samples of non-cap were significantly higher than that of cap group (p = 0.001). a total of 271 cap cases were positive for hhvs, accounting for about 99.3% of the hospitalised infants and young children with cap included in this study. 73 of 81 non-cap cases, however, were also identified as positive (90.1%) for hhvs. among other 15 respiratory viruses, rsv was the most dominant for both cap and non-cap groups, which was present significantly less frequent than hhvs. to further study the epidemiological profiles of virus infections, the distribution of viruses by age and season in this study were characterised (figure 1 and 2) . no significant difference was found for flua, piconavirus (enterovirus/rhinovirus), piv and hmpv among various age groups of cap cases ( figure 1a) . however, the infection rate of hbov and adv showed a peak among cap patients aged 6 months to 3 years (p,0.05). in contrast, rsv detection peaked in the infant group (0-12 months) of cap and decreased significantly with advancing age. in addition, no significant differences for hsv and cmv were observed among various age groups of cap cases ( figure 1b) . however, the rate of infection with ebv, hhv-6 and hhv-7 increased with age among cap cases (p,0.05). the infection rate was more than 73.7% for cmv, ebv, hhv-6 and hhv-7 among children older than 3 years. the distribution of viruses by age among the control group with non-cap was also investigated ( figure 1c and 1d) . no significant differences for rsv, ebv, hhv-6 and cmv were shown among various age groups of non-cap cases. however, the rate of infection with hhv-7 increased with age, which is similar to that of cap group. interestingly, co-infections were found in 241 (88.28%) of the cap cases and 65 (80.25%) of the non-cap cases (table 1) . single virus infection were detected in 31 is samples of cap cases (25 for cmv only, 5 for hhv-6 only and 1 for hbov only). in this study of co-infection, 77 patients were found to be infected with 2 viruses, 76 with 3 viruses, 48 with 4 viruses, 36 with 5 viruses, 3 with 6 viruses and 1 with 7 viruses. in addition, hhvs were the most detected co-infection agent with other respiratory viruses. among 102 rsv infections of cap cases, hhv (102 cases, 100%) and hbov (23 cases, 22.54%) were the most common concomitantly detected viruses, which were significantly higher than that of coinfection among non-cap group. the clinical manifestation of cap patients included cough, fever ($38uc), asthma and sputum. a few cases also showed signs of diarrhoea, rhinorrhoea, dyspnea and rale (data not shown). since the high virus detection rate (more than 98%) and coinfection rate (more than 80%) were among is samples from both cap and non-cap groups in this study (table 1) , it was difficult to associate the clinical symptoms of patients with cap with individual virus infection. however, the prevalence of several viral agents (rhinovirus, hbov, adv and cmv) among is samples of cap were drastically higher that of non-cap control group (p,0.05). in addition, the prevalence of rsv coinfection with hhvs (102/102, 100%) and hbov (23/102,22.54%) among cap group was significantly higher than that among non-cap controls (p,0.05).these data indicated that several viral agents (such as hbov and cmv) may contribute to the occurrence of cap. cap is more common and severe in the developing areas than developed areas [1] [2] [3] , and is a major cause of death among infants and children in rural areas [3] . previous investigations of paediatric cap in us and europe emphasised the importance of infections with common respiratory viruses (rsv, inf, piv and adv) [3, 4, 6] . the roles of hhvs and more recently identified viruses (hbov, hcov-nl63 and hcov-hku1) as causes of cap remain controversial [2, [5] [6] [7] 12, 14] . in this study, the viral prevalence in sputum specimens of childhood with non typical bacterial cap was investigated using sensitive molecular diagnostic methods for hhvs and 15 respiratory viruses, and viruses were detected in 99.6% of the children. this is not surprising considering that the samples included were highly selected for the discovery of viral etiology and addition hhvs detection in this study. to our knowledge, this is the first comprehensive study of the prevalence of hhvs in sputum samples among infants and young children with cap [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] . a few reports have described the detection of dna from several hhvs in respiratory samples, with most of them focusing on immunosuppressed individuals or adults with cap [2, [19] [20] [21] [22] 31, 32] . in this study, we screened for dna of hhvs in is samples among infants and young children with cap using a sensitive multiple pcr assay; of the hhvs considered, only vzv was not detected. the highest positive rate was found for cmv infection (91.6%). cmv infection, which is usually congenital, showed no significant difference among various age groups in this study. these data are consistent with those of previous reports [2, 22, 31] . in addition, it was reported here that cmv and hhv-6 were the only detected viral agent among 25 cap cases and 5 cap cases, respectively. furthermore, the prevalence of cmv among is samples of cap were significantly higher than that of non-cap control group. the prevalence of rsv coinfection with hhvs among cap group was drastically higher than that among non-cap control (p,0.05). these data suggest an association between infection with hhvs (especially cmv) and cap in infants and children. few comprehensive studies have searched for viruses in is samples among infants and children with cap in rural areas [8, 10, 11] . in this study, 15 common and recently identified viruses associated with acute respiratory infection were screened using molecular methods. our results are consistent with previous studies conducted in china or other areas [8, 10, 11, 33] , which showed that the most-detected agent was rsv, followed by hbov, rv, hmpv, adv and piv3 in is samples from infants and children with cap [3, 11, [14] [15] [16] . rsv and hbov were also the dominant viruses detected in is samples from non-cap group. moreover, our data show a higher hbov detection rate (28.2%) compared with previous reports [23, 24] . the prevalence of hbov among is samples of cap were significantly higher that of non-cap control group. similar trends were also observed in the prevalence of rhinovirus and adv among is samples of cap when compared with non-cap control group. unlike previous data [34] , the prevalence of hcovs among is samples was significantly lower in present study. these differences might primarily due to the specimens [11, 15, 16] -is vs. a nasopharyngeal aspirate or nasopharyngeal wash. in the meantime, the impact of other factors, such as area and the duration of the study period on infection, could not be ruled out. one interesting finding of present study was that hhv coinfections were found in 70% of the cap cases, compared with rates between 15 and 45% in previous etiological studies of childhood cap [7, [35] [36] [37] . the clinical consequences of mixed infections have not been fully understood yet. evidence suggests that mixed viral infections can lead to more severe condition than individual viral infections [2, 7, [35] [36] [37] [38] . in this study, it was unable to determine whether the hhvs were reactivated from a latent reservoir after another respiratory virus infection, or if an immunosuppressed condition caused by hhv infection increases the potential risk of other respiratory virus infections. consequently, it is difficult to estimate the true association between the clinical manifestations and virus infection. at the same time, we understand that the detection of viruses in an is sample by pcr does not necessarily mean that they are the causative agents of the concomitant cap. the is samples included in this study may only represent part of hospitalized infants and children with cap in this hospital. to evaluate the real pathogenic role played by virus in hospitalized children with cap in this study, non-cap hospitalized children with chronic respiratory illness were set as control group. however, this study still has two major limitations. one limitation of this study is that viral detection from non-hospitalised children (due to limitation of ethics) and patients with bacterial cap, which would have provided a control group for this study, were not included. therefore, some viral agents detected in this study may represent asymptomatic persistence, prolonged shedding, or other situations. it is also not possible to evaluate the exact importance of each virus responsible for cap in most cases. nevertheless, it is believed that overall this study highlights the importance of hhvs (mainly cmv) and respiratory viruses in children with cap. another limitation of this study is that no samples were collected during summer, which could lead to the missing of some viral agents such as parainfluenza viruses and some enteroviruses. in summary, our study on the prevalence of hhvs and other respiratory viruses in infants and young children with cap identified a detectable virus in more than 99.6% of case participants, in which cmv, hhv-6, ebv, rsv and hbov were clearly predominant (.25%) and contributed significantly to the spectrum of cap in a rural area of china. although the hhvs were the most commonly identified pathogens in this study, which were not previously thought of as typical causes of cap in immunocopetent individuals [3] [4] [5] [6] [7] [8] 20, [38] [39] [40] [41] , further studies are required to determine the relationship of the presence of hhvs and severity of disease, thus the clinical significance of hhv infections or co-infections should receive greater attention in future treatment and prevention studies of cap in infants and children. who. global burden of disease in 2002: data sources, methods and results community-acquired pneumonia in children etiology of community-acquired pneumonia in 254 hospitalized children etiology of community-acquired pneumonia in hospitalized children based on who clinical guidelines access to a polymerase chain reaction assay method targeting 13 respiratory viruses can reduce antibiotics: a randomised, controlled trial epidemiology and clinical characteristics of community-acquired pneumonia in hospitalized children 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(herpes simplex virus, varicella-zoster virus, epstein-barr virus, cytomegalovirus, human herpes virus 6a/b, and human herpes virus 7) by multiplex pcr assay etiology and clinical characterization of respiratory virus infections in adult patients attending an emergency department in beijing molecular assays for detection of human metapneumovirus epidemiological profile and clinical associations of human bocavirus and other human parvoviruses characterization of human coronavirus etiology in chinese adults with acute upper respiratory tract infection by real-time rt-pcr assays the role of viruses in the aetiology of community-acquired pneumonia in adults lower respiratory tract viral infections in hospitalized adult patients molecular monitoring of causative viruses in child acute respiratory infection in endemoepidemic situations in shanghai effects of coronavirus infections in children mixed respiratory virus infections multipathogen infections in hospitalized children with acute respiratory infections viruses in community-acquired pneumonia in children aged less than 3 years old: high rate of viral coinfection severe respiratory syncytial virus pneumonia associated with primary epstein-barr virus infection cytomegalovirus and herpes simplex virus effect on the prognosis of mechanically ventilated patients suspected to have ventilator-associated pneumonia cytomegalovirus pneumonia in immunocompetent host: case report and literature review cmv in critically ill patients: pathogen or bystander we thank the medical and technical staffs from the wenling hospital for their assistance and support. we also thank all the participants involve in this study for providing samples. key: cord-289305-mfjyjjer authors: lee, min hye; lee, gyeoung ah; lee, seong hyeon; park, yeon-hwan title: a systematic review on the causes of the transmission and control measures of outbreaks in long-term care facilities: back to basics of infection control date: 2020-03-10 journal: plos one doi: 10.1371/journal.pone.0229911 sha: doc_id: 289305 cord_uid: mfjyjjer background: the unique characteristics of long-term care facilities (ltcfs) including host factors and living conditions contribute to the spread of contagious pathogens. control measures are essential to interrupt the transmission and to manage outbreaks effectively. aim: the aim of this systematic review was to verify the causes and problems contributing to transmission and to identify control measures during outbreaks in ltcfs. methods: four electronic databases were searched for articles published from 2007 to 2018. articles written in english reporting outbreaks in ltcfs were included. the quality of the studies was assessed using the risk-of-bias assessment tool for nonrandomized studies. findings: a total of 37 studies were included in the qualitative synthesis. the most commonly reported single pathogen was influenza virus, followed by group a streptococcus (gas). of the studies that identified the cause, about half of them noted outbreaks transmitted via person-to-person. suboptimal infection control practice including inadequate decontamination and poor hand hygiene was the most frequently raised issue propagating transmission. especially, lapses in specific care procedures were linked with outbreaks of gas and hepatitis b and c viruses. about 60% of the included studies reported affected cases among staff, but only a few studies implemented work restriction during outbreaks. conclusions: this review indicates that the violation of basic infection control practice could be a major role in introducing and facilitating the spread of contagious diseases in ltcfs. it shows the need to promote compliance with basic practices of infection control to prevent outbreaks in ltcfs. four electronic databases were searched for articles published from 2007 to 2018. articles written in english reporting outbreaks in ltcfs were included. the quality of the studies was assessed using the risk-of-bias assessment tool for nonrandomized studies. a total of 37 studies were included in the qualitative synthesis. the most commonly reported single pathogen was influenza virus, followed by group a streptococcus (gas). of the studies that identified the cause, about half of them noted outbreaks transmitted via person-toperson. suboptimal infection control practice including inadequate decontamination and poor hand hygiene was the most frequently raised issue propagating transmission. especially, lapses in specific care procedures were linked with outbreaks of gas and hepatitis b and c viruses. about 60% of the included studies reported affected cases among staff, but only a few studies implemented work restriction during outbreaks. outbreak of an infectious disease is defined as the occurrence of a disease above the expected level [1] . over the past several years, many countries have experienced serious economic and health consequences due to outbreaks of infectious diseases such as the middle east respiratory syndrome in 2015 and severe acute respiratory syndrome in 2003. long-term care facilities (ltcfs) are facing a great need for preparation for infection outbreaks because of an increase in the number of residents with global aging. ltcfs are exposed to the risk of outbreaks owing to several factors. first, older residents in ltcfs are susceptible to infectious diseases because of aging and health conditions [2] and are known to be dependent with regard to activities of daily living. thus, among residents, self-hygiene is observed to be poor. loss of independence in residents creates unique and frequent contact opportunities between healthcare workers (hcws) and residents [3] . second, hcws in ltcfs tend to be poorly informed about infection prevention and control (ipc), and compliance with ipc is generally low [2, 4] . third, the environment in ltcfs offers challenges for ipc, like the sharing of rooms, group living, and difficulty with the isolation of infected persons [5, 6] . finally, ltcfs have limited resources and capacities for diagnosis of infection [7] . this leads to a delay in the detection of hidden carriers and infection. all these factors contribute to the onset and spread of outbreaks in ltcfs. outbreaks in ltcfs threaten the life and health of both residents and hcws, and thus, eliminating the risk of outbreaks is a matter of concern in such facilities. however, ltcfs vary in their individual capacities to respond to outbreaks [8] . the keys to outbreak control are as follows: identification of the transmission causes and minimization of the spread through early initiation of control measures. therefore, it is essential to understand the causes of transmission and the applied measures to control outbreaks in ltcfs. there are several gaps and limitations in previous studies to comprehensively understand outbreaks in ltcfs. first, in many previous researches that addressed outbreaks, the focus was on pathogens, burdens, and adverse outcomes such as mortality [9, 10] . based on the perceived importance of control measures, one of the purposes of this review was to explore and analyze in detail the control measures reported in studies. secondly, pharmaceutical measures may have some limitations on effectiveness against newly emerging infectious diseases or resistant strains [3] and some pathogens may not have pharmaceutical interventions to be considered during outbreaks. non-pharmaceutical interventions (npis) such as hand hygiene and precautions should be utilized to prevent the transmission of outbreak pathogens, regardless of the evolution of infectious diseases. however, little attention has been paid to npis in studies concerning outbreaks in ltcfs [3] . this review focused on npis and ascertains the control measures based on the guideline for prevention and control of influenza outbreaks in ltcfs of the world health organization (who) [11] . finally, to our knowledge, there is no published systematic review addressing overall outbreaks in ltcfs in the past 5 years. after pilot data extraction, two reviewers independently extracted data such as information on participants, pathogens, case definitions, number of cases and non-affected persons, overall attack rate, causes and problems that led to transmission, and control measures. control measures. general control measures considered were the formation of outbreak control team, active surveillance, standard precautions, transmission-based precautions, training and education, employee work restriction, environmental control, containment measures, and prophylaxis based on the who guideline [11] . the quality of studies was assessed using risk of bias assessment tool for nonrandomized study (robans) by two reviewers [13] . robans is an evaluation tool for the risk of bias of non-randomized studies, with moderate reliability and acceptable validity and compatible with domains of the cochrane risk-of-bias tool [13] . six domains were evaluated including the selection of participants, confounding variables, exposure measurement, blinding for outcome assessment, incomplete outcome, and selective outcome reporting. according to the instruction for evaluation [13] , the risk of bias for each domain was determined as low risk, unclear risk and high risk. studies with full-text including case-control analysis, cohort study was involved in the quality assessment. studies that simply described the results of investigation without comparative analysis were not able to evaluate the domains of the tool. thus, the quality evaluation was not conducted with this type of study. any difference was discussed between the two reviewers, and, if necessary, an agreement was reached with the corresponding author. cinahl ti (infection or infections or outbreak � or transmission) and ti ("nursing home � " or "skilled nursing � " or "long-term care") and ti (control � or outcome � or factor � ) not ti (surgery or cancer or "neoplasm" or "intensive care unit" or child or children or "operative") the result of quality assessment was displayed using review manager (revman) version 5.3 software (the cochrane collaboration, oxford, uk). a total of 2,789 studies were retrieved from 4 databases and hand searched. the duplicate records were removed (n = 1180), and the eligibility criteria were applied for the selection process. after reviewing the full text, 76 articles were excluded for the following reasons: irrelevant for the research topic (n = 38) and population (n = 4), unavailable full-text (n = 32), review article (n = 1), and duplicated report (n = 1). finally, 37 articles were included in this review (fig 1) . characteristics of the eligible studies are presented in table 2 . over half of the included studies (n = 22) were published since 2013. the majority of the included studies were reported in the united states (n = 15) and europe (n = 13) followed by asia (n = 5). the quality of 20 studies was assessed and the results are summarized in fig 2. one study was at high risk for five criteria [14] . six studies were at low risk for all criteria [15] [16] [17] [18] [19] [20] . problems related to recall bias and standardization of self-reported measurement created a high risk of bias for the measurement of the exposure domain in seven studies [14, [21] [22] [23] [24] [25] [26] . lack of consideration for confounders led to a high risk of bias in four studies [24, [27] [28] [29] . the problem related to missing data resulted in a high risk of bias for the incomplete outcome domain in five studies [14, 26, 28, 30, 31] . characteristics of the outbreaks are presented in tables 2-4 . fifteen studies reported outbreaks caused by bacteria [15-17, 20, 22, 24, 27, 32-39] and 22 studies were outbreaks by viruses [14, 18, 19, 21, 23, 25, 26, [28] [29] [30] [31] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] . the largest number of a single pathogen was influenza viruses [40] [41] [42] [43] [44] [45] , followed by group a streptococcus (gas) [17, 20, 24, 37, 38] . the most affected site was respiratory tract (n = 12) [32, 35, 36, 39-45, 48, 50] , followed by gastrointestinal (gi) tract (n = 10) [14, 16, 23, 26, 27, 31, 33, 46, 47, 49] . other sites including skin and soft tissue and eyes were affected. the majority of the eligible studies reported one outbreak involving one facility (n = 31), while the study by nguyen and middaugh [49] described a gastroenteritis outbreak that was transmitted to eight facilities. three studies analyzed the data of multiple outbreaks of viral gastroenteritis and influenza-like illness that occurred in multiple facilities for a certain period of time [14, 46, 48] . the outbreaks in 23 studies affected both the residents and hcws [14, 20, 22, 23, 25, 26, 29, 31, 33, [35] [36] [37] [38] [39] [40] [41] [43] [44] [45] [46] [47] [48] [49] , and the outbreaks of 14 studies affected only the residents [15-19, 21, 24, 27, 28, 30, 32, 34, 42, 50] . in total, 37 studies reported 1,332 outbreaks (affecting 1,122 residents and 385 staff members) in 1,182 facilities. there were three prolonged gas outbreaks of multiple consecutive clusters for over 6 months. the overall attack rates ranged widely from 0.84% to 73.17% in 29 studies. among the 29 studies, the median of the overall attack rate was 15.73%: 8.27% for bacterial outbreaks and 19.25% for viral outbreaks (table 3 ). in 8 studies, it was not possible to calculate the rate due to lack of information. the highest attack rate of 73.17% was reported in an outbreak of respiratory syncytial virus (rsv) and human metapneumovirus (hmpv) [50] , followed by clostridium difficile (51.97%) [27] and viral gastroenteritis caused by norovirus and rotavirus (48.6%) [26] . influenza-like illness had the median overall attack rate of 24.50%. the median attack rate among staff was highest for the acute gastroenteritis outbreaks. the duration of outbreaks ranged from less than one month to over 6 months. outbreaks in 13 studies lasted for over 6 months: 3 studies by hepatitis b virus, 3 by gas, 2 by tuberculosis (tb), 2 by multi-drug resistant organisms (mdros), 1 by viral gastroenteritis, 1 by c. difficile, and 1 by hepatitis c virus [15, 17, 19-22, 27, 28, 30, 35, 37, 39, 46] . outbreaks in long-term care facilities: sr if only one study of the outbreaks was reported, the attack rate of the study was displayed. if the number of reports was 2, only the range of attack rate was displayed. outbreaks in long-term care facilities: sr causes and critical problems contributing to transmission. causes of transmission in the eligible studies were reported as person-to-person transmission, contaminated water and food, and problems in practice (tables 2 and 4 ). the following studies (n = 12) did not report or could not identify the cause of the outbreaks: 5 studies on influenza viruses, 2 on non-typeable haemophilus influenzae, 1 on hepatitis c virus, 1 on c. difficile, 1 on adenovirus, 1 on norovirus, and 1 on rsv and hmpv [14, 27, 29, 30, 32, 36, 40, 42, 44, 45, 48, 50] . the most commonly reported route of transmission was person-to-person [17, 22-24, 26, 31, 33, 35, 38, 41, 43, 46, 47, 49] . of these studies, the human source of transmission was identified as the hcws in six outbreaks [22, 24, 26, 38, 41, 49] and the residents in two outbreaks [17, 33] . the skin of a hcw was a reservoir for methicillin-resistant staphylococcus aureus (mrsa) outbreaks, leading to cross-infection, in the study by maltezou et al. [22] . the large gastroenteritis outbreak affecting 394 people in 8 facilities was attributed to staff who worked at multiple facilities [49] . one study showed that three outbreaks of gas recurred for three years because of continued person-to-person transmission from colonized residents [17] . in the study by šubelj and učakar [23] , person-to-person transmission resulted from delayed implementation of control measures. contaminated water and food were sources of infection in five studies [16, 25, 26, 33, 46] . hepatitis e outbreak was caused by contaminated tap water after heavy rain [25] , while the consumption of tap water was the suspected cause of one viral gastroenteritis outbreak [26] . foodborne causes such as contaminated cake or meals were noted in three studies regarding clostridium perfringens, salmonella enteritidis, and gastroenteritis [16, 33, 46] . most of the reviewed studies pointed out several issues in practice that might have facilitated the occurrence and spread of outbreaks. the most frequently observed problem was suboptimal hand hygiene, followed by personal protective equipment (ppe), and cleaning and disinfection. investigation for the gas outbreak in the study by nanduri et al. [37] revealed that hand hygiene compliance among employees was 14-25%. additionally, poor hand hygiene became a more critical factor that facilitated the transmission of acute gastroenteritis, particularly in ltcfs having close living conditions with frequent close contact between the staff and dependent residents [31] . issues related to ppe had been addressed including inappropriate use of glove and improper storage of ppe [20, 24, 30, 31, 34, 37] . there were reports indicating the potential to cross-contamination by not-changing gloves between residents or by storage of ppe in the room of the index case [31, 34] . breaches in disinfection and cleaning of the environment and equipment were associated with many outbreaks, most of them were gas [20, 37, 38] or hepatitis b and c outbreaks [28, 30] . three reports of gas outbreaks found lapses in wound care practice such as inconsistent cleaning and disinfection [20, 37, 38] . the outbreaks of mdros and hepatitis b reported device related issues including sharing of a device and inappropriate use of reusable devices [15, 18, 21, 34] . hepatitis b and c outbreaks commonly reported that lapses during podiatry care and point-of-care testing procedures (blood glucose test and international normalized ratio monitoring) caused the transmission of bloodborne pathogens among residents [18, 19, 21, 28, 30] . the lapses included the sharing of contaminated equipment, improper disinfection, and poor hand hygiene adherence. some studies noted failure of environmental infection control [32, 35, 39, 43] . two of those studies were tb epidemics, and the investigation revealed that the case residents were exposed to insufficient room ventilation. an influenza outbreak in the summer was facilitated by a heating preventive measure that placed all the residents in one limited area [43] . the response to outbreaks also could influence the progress of outbreaks. nine reports underlined early notification of outbreaks to public health authorities and implementation of control measures within 3 days of onset of the first case, which affected the attack rates and duration of the outbreaks [23, 29, 35, 41, 44, 46, [48] [49] [50] . some studies on influenza outbreaks discussed issues related to vaccines. of the three influenza outbreaks in a well-vaccinated population, two studies pointed out that a mismatch between the circulating strains and the vaccine strains affected this population [41, 45] , and the other study noted an insufficient vaccine effectiveness [42] . especially, the study by burette et al. [41] identified that in addition to the mismatch, several defects including a vaccination rate of 0% among staff and untimely vaccination among residents led to the outbreaks. in addition, they raised the issue of the knowledge and proficiency of general practitioners in influenza diagnosis, treatment, and prevention. moreover, three studies suspecting transmission from staff to residents placed emphasis on work restriction of the ill staff [38, 40, 49] . there were other problems including poor personal hygiene of staff members [22] , lack of communication between institutions [17] , and understaffing [24] . several studies demonstrated host factors associated with the outbreaks in case-control analysis, that were identified as: age [29] , sex [16] , cognitive impairment [29] , nutritional status [27] , comorbidities [23, 24] , use of an indwelling device [15, 17] , and dependence level [31] . although not the result of the case-control analysis, the study by spires et al. [50] reported that all cases of rsv and hmpv were dependent dementia patients, implicating that dependence was an important factor. control measures. strategies to control outbreaks were reported in 30 of the 37 reviewed papers, as summarized in table 5 . all 30 studies reported that one or more npis were applied to control the outbreaks. from a rigorous perspective, only one study on a multidrug-resistant pseudomonas aeruginosa (mrpa) outbreak implemented all the measures recommended to control an outbreak by pathogens [15] . work restriction of ill workers was less frequently reported compared to other measures. only five studies reported the creation of outbreak control teams for effective management of the outbreaks [15, 17, 23, 27, 43] . most facilities notified public health authorities or institutions about the outbreaks and received advice and assistance to manage the outbreaks. all four studies applying limitation or cessation of group activities were recently published since 2014 [31, 42, 44, 50] . gastroenteritis outbreaks (n = 5). three studies on gastrointestinal infection, in which adherence to hand hygiene among hcws was crucial to prevent its spread, reported control measures including stringent hand hygiene practice and reinforcement of standard precautions [23, 27, 31] . only two studies implemented barrier precautions by use of ppe [23, 31] . all five studies that reported control measures used various types of social distancing measures including isolation, restriction of new admission and visitors, or cessation of group activities [23, 26, 27, 31, 49] . active surveillance by symptom reporting for early detection of new cases was reported in two studies [27, 49] . although four studies reported that staff members were affected by the outbreaks, only one study implemented work exclusion for ill employees and showed the lowest attack rate among staff [23] . four studies reported intensive cleaning and disinfection of the environment [23, 26, 27, 31] . the implementation of more stringent procedures for cleaning and disinfection with diluted bleach was reported for outbreaks of c. difficile [27] . the study by luque et al. [26] on viral gastroenteritis reported a relatively small number of interventions, showing a high attack rate of 48.63%. on the other hand, the study by šubelj and učakar [23] with the largest number of control measures among the five studies had a lower attack rate of 11.21% compared to the other outbreaks. influenza virus outbreaks (n = 6). five of the six reports implemented prophylactic oseltamivir for the residents and/or hcws [40] [41] [42] [43] [44] . both droplet precaution and active surveillance were reported in 3 of the six studies. five of the studies on influenza outbreaks reported a total of 172 cases among staff, but only three of the studies implemented the measure of work restriction [40, 42, 44] . the study by burette et al. [41] reported the lowest number of control measures including prophylaxis and isolation and had the highest attack rate of 42.05% among the five reports on influenza a. tuberculosis outbreaks (n = 2). following the detection of the index case, two reports on tuberculosis outbreaks conducted case finding among residents and staff by contact tracing [35, 39] . responding to the outbreaks, measures for the cases included isolation and transfer to a hospital in one study [39] , but the other study only restricted new admissions [35] . neither of them mentioned airborne precautions taken such as n95 respirators. investigations in both reports found that the air exchange rates of the rooms were inadequate. the study by lai et al. [39] corrected the failure of the environmental infection control by increasing the ventilation rates in the building. both outbreaks involved cases among workers, but there was no description about the work status of the affected staff after the occurrence. mdros outbreaks (n = 3). three outbreaks of mdros were caused by mrsa, mrpa, or klebsiella pneumoniae carbapenemase-producing klebsiella pneumonia (kpc-kp) [15, 22, 34] . the mrsa outbreak study applied mupirocin eradication for the residents and staff [22] . all three outbreaks used transmission-based precautions and quarantine measures to prevent the spread of mdros. in addition, they all provided re-education for the staff to improve infection control practice. furthermore, two of the reports on mrpa and kpc-kp intensified the cleaning of the environment to interrupt the contamination of the environment. the study by kanayama et al. [15] demonstrated that the sharing of a device such as a suction device was linked with the mrpa cases; thus, the control measures included stopping the sharing of devices. the unexpected occurrence of the kpc-kp cases led to contact surveillance for additional exposure cases [34] . the investigation for the kpc-kp outbreak revealed poor hand hygiene compliance among staff; thus, interventions including frequent audit and feedback were implemented. gas outbreaks (n = 5). three of the five gas outbreaks provided antibiotic prophylaxis to the residents and staff [17, 24, 37] . all five studies conducted surveillance culture for active case finding. none of the five outbreaks reported droplet precautions, but the study by thigpen et al. [24] mentioned an enhanced respiratory hygiene practice. although three of the gas outbreaks lasted for a long period due to an unsolved person-to-person transmission [17, 20, 37] , none of the studies implemented social distancing measures. two studies improved the availability of hand dispensers to address the suboptimal hand hygiene practice that was revealed during their observation [17, 24] . none of the studies on the three outbreaks involving sick employees reported encouragement of work exclusion for ill staff [20, 37, 38] , but some studies reported that there were voluntary sick leaves of employees before the recognition of the outbreaks. hepatitis virus outbreaks (n = 5). prophylaxis of hepatitis b vaccine and immuno-globulin were implemented for hepatitis b virus outbreaks in two studies [21, 28] . there is not much generally recommended npis for the hepatitis b and c outbreaks; thus, the studies on these outbreaks reported fewer npis than those on the other outbreaks. three of the studies on the hepatitis virus outbreaks tried to find additional cases by serologic screening [21, 25, 30] . all the studies on the hepatitis b and c virus outbreaks employed the principle of single-use device or individual equipment to break the chain of infection [18, 21, 28, 30] . improvement of the care room was done in two studies that found lapses in the environment of the procedure room [28, 30] . interventions for drinking water standards and toilets were reported in the hepatitis e virus outbreak caused by contaminated water [25] . heamophilus influenzae outbreaks (n = 2). one of the two h. influenzae outbreaks reported droplet precaution during the outbreak [32] , and the other study restricted new admissions to prevent additional transmission [36] . other outbreaks (n = 2). the study on the rsv and hmpv outbreak reported various measures including active surveillance, isolation, contact precaution, antiviral prophylaxis for residents and work restriction for ill staff to control respiratory pathogen transmission [50] . in the epidemic keratoconjunctivitis outbreak, control measures involved universal precaution with enhanced hand hygiene, isolation and restriction of visitors, and work restriction for affected workers [29] . we updated the understanding of outbreaks in ltcfs with more recently published reports. this review also explored and summarized critical issues facilitating the spread of the outbreaks and the control measures, which have not been addressed in detail in the previous review [9] . lessons learned from the results of this review would enable better prevention and control of outbreaks in ltcfs in the future. implications and suggestions for achieving the best response to epidemics by ltcfs, and for future research concerning outbreaks, have been described in this review. the most common outbreaks in ltcfs in this review were respiratory infections followed by gi infections, showing consistency with the findings of a previous study on nursing homes [51] . interestingly, there is a difference in the outbreak reports for mdros compared to the previous review [9] . this review identified three reports including mrsa, mrpa, and kpc-kp, suggesting the increase of multidrug-resistant organisms, given that the prior review found only two reports of mrsa [9] . as the prevalence of mdros is increasing in ltcfs [2] , they become a particular concern in these facilities. with drug resistance on the rise, mdro related outbreaks may occur in ltcfs with growing frequency. it shows that staff and managers of ltcfs need to be aware of the significance of this trend and to prepare a plan. influenza viruses and gas accounted for a large number of single pathogens. this is similar to the results of the previous review showing that the largest number of aetiologic agents affecting outbreaks was influenza viruses in ltcfs from 1966 to 2008 [9] . first, in this review, five of the six influenza virus outbreaks occurred by the influenza a virus and the other by influenza b virus. influenza-like illness included in the studies showed a median attack rate of 24.50%, similar to that of seasonal influenza, usually 20-30% [40] . vaccine-related issues have been raised in influenza outbreaks that have occurred in ltcfs of highly immunized residents. this finding suggests some implications to prevent influenza outbreaks in ltcfs. regarding vaccination coverage among staff, the study by thomas [52] found that influenza episodes were reduced if an employee was vaccinated, and the centers for disease control and prevention (cdc) recommend that all healthcare worker get vaccinated annually [53] . therefore, influenza vaccination among healthcare personnel should be considered to mitigate the risk of influenza outbreaks in ltcfs. additionally, because vaccination does not provide complete protection, active daily surveillance for influenza-like illness is still recommended for all persons in ltcfs during influenza season [53] , which is evident by the outbreaks occurring in highly immunized ltcf population. secondly, the gas infection rate among older adults in ltcfs is from 3 to 8-fold higher than that of community-dwelling older adults, due to risk factors such as grouped living conditions, and underlying diseases [54, 55] . five of the studies on the gas outbreaks in this review reported a median attack rate of 2.43% in ltcfs, which is within the range of 1-30% reported in the previous study [56] . three of them were long-lasting outbreaks with multiple clusters for more than 6 months, which suggested that an accurate identification of how pathogens spread was a fundamental step in outbreak control. this review also explored critical issues on practices that propagated the occurrence and spread of outbreaks. consequently, failure to adhere to basic infection control practice, including hand hygiene, disinfection, and cleaning, was found to be a practical issue of great importance on the transmission of the outbreaks in ltcfs. some reports even mentioned that this issue ultimately caused their outbreaks [19, 21, 28] . most studies showed that this problem contributed to their outbreaks by causing cross-contamination between hands, environments, and equipment. first, the hands of hcws may be the sources of the outbreaks. frequent close contacts between residents and hcws in ltcfs increase the risk of widespread outbreaks. incorrect hand hygiene among hcws can result in hands remaining contaminated, and this may lead to the transfer of organisms to the environment and to other residents [57] . like previous studies that already confirmed poor compliance with hand hygiene among hcws [4] , one of the included studies reported that hand hygiene compliance was less than 30% [37] . semmelweis demonstrated the role of hand hygiene in preventing infections transmitted by person-to-person [58] . hand hygiene has a significant effect on reducing gi and respiratory infections [59] . the who recommends that hand hygiene should be performed at the following 5 key moments: before and after touching a patient, before clean/aseptic procedures, after body fluid exposure risk, and after touching a patient's surroundings [57] . promotion of hand hygiene compliance through multimodal strategies has been proven to reduce healthcare-associated infection [60] . multifaceted interventions such as who-5 strategies (including system change, training and education, monitoring and feedback, reminder and communication, and culture of safety) are generally effective in increasing and sustaining hand hygiene compliance at various settings [61] [62] [63] [64] [65] . the same evidence has been reported from studies on ltcfs, suggesting improved hand hygiene reduces the infection rate or respiratory outbreaks [66, 67] . secondly, lapses in cleaning and disinfection could make equipment and the environment become a reservoir for transferring pathogens [68] . most of the studies regarding this issue were on outbreaks of gas, gastroenteritis, and hepatitis b and c virus, and they found a failure to adhere to proper disinfection and cleaning principles. first of all, the outbreaks of gas and hepatitis b and c were linked with breaches in specific procedures. gas outbreaks were usually relevant to wound care and hepatitis b and c to point-of-care testing. with the aforementioned hand hygiene, disinfection and cleaning were basic infection control practices that are included in standard precautions. the standard precautions consist of hand hygiene, environmental cleaning, reprocessing and disinfection of care equipment, waste and linen management, the prevention of needle stick injuries, and the use of personal protective equipment (ppe), if necessary [11] . the practice of standard precautions is the imperative basic approach for ipc that was applied to all residents assuming they had the potential for pathogen transmission [11] . standard precautions are necessary practice, especially in ltcfs where the systems for diagnostic tests are poor and active surveillance is not generally done. tailored ongoing education with multimodal strategies for hcws would ensure that basic infection control principles and standard precautions are integrated into daily practice such as point-ofcare testing [69] . as a result, a reduction in threats of outbreaks can be guaranteed, as well as the safety and health of persons residing or working in ltcfs. meanwhile, the studies on the outbreaks of gastroenteritis reported that there were lapses in decontaminating environment. environmental contamination may have a critical role in the spread of these outbreaks. importantly, norovirus and c. difficile that are capable of surviving in the environment for long periods of time require more consideration in environmental disinfection [70, 71] . for norovirus, the cdc has recommended more frequent cleaning and disinfection of rooms and high-touch surfaces with a hypo-chlorite (1000-5000 ppm) or other proper disinfectant [72] . the most effective control method for c. difficile was reported as disinfection and cleaning of rooms and high-touch surfaces with a chlorine-based solution (5000 ppm) [70, 71] . this systematic review identified control measures taken during the outbreaks, especially non-pharmaceutical interventions. the results showed that the actual application of control measures may be far from what is recommended and implied that there are several challenges to overcome in outbreak management at ltcfs. first, acute care facilities like hospitals can successfully manage outbreaks through collaborative efforts with multiple experts [73] . however, most of the ltcfs in this review requested advice from public health authorities and organizations for unexpected outbreaks instead of organizing a multidisciplinary team. this may imply that ltcfs do not have sufficient capacity and expertise to individually plan, implement, and evaluate the management of outbreaks. forming a local support network between acute hospitals and ltcfs at a regional level would be a potential way to close the gaps and to enhance outbreak control practices in ltcfs without adequate capacity [74] . furthermore, training infection control professionals in facilities could facilitate early detection of outbreaks and timely interventions. secondly, this review found that many ltcf employees were affected by the outbreaks, which is consistent with the finding of the previous review [9] . however, it also revealed that work restriction for ill staff was not implemented well during the outbreaks in the ltcfs, which was not reviewed in the prior review [9] . gastroenteritis outbreaks in this review had a higher median attack rate among staff than the other outbreaks, but only one study among them reported the application of work exclusion. moreover, there were some reports that implied transmission attributable to sick employees. these results pointed out the role of presenteeism in ltcfs. presenteeism among sick employees may have a role in either introducing pathogens or facilitating the transmission of outbreaks. the cdc recommends work restrictions for health care workers infected with or exposed to diseases such as diarrheal diseases, gas, tuberculosis, and viral respiratory infection [75] . however, it may be challenging for ltcfs with fewer available resources to implement the exclusion of ill staff during outbreaks, given the fact that one study reported difficulty from understaffing as a result of work restriction [50] . the study by widera et al. [76] suggested that daily screening of all staff members for symptoms during outbreaks on every shift may mitigate the impact of presenteeism. considering that presenteeism is associated with various factors such as job security and lack of paid sick leave [77] , further discussion is needed for plans addressing this issue in ltcfs. lastly, additional challenges in managing the outbreaks in ltcfs were reported. they included understaffing, insufficient supply of products such as ppe, lack of expertise, and limited application of isolation [41, 46, 47, 50] . many long-term care facilities have difficulty in applying isolation of infected persons due to the limited availability of isolation rooms [5] and concern about the adverse effects of isolation and additional precautions may affect the compliance with related practices [78] . for the same reason, some studies in this review used minimal types of isolation like enteric isolation. if single rooms are not available, facilities should consider applying the cohort measure or bed curtains as another method of isolation. in regard to this challenge, a study by dumyati et al. [73] suggested a shift towards enhanced standard precautions or risk-based application of transmission-based precautions to uphold the quality of life of residents by hcws. future research should identify the rationale for the safety and effectiveness of this strategy or other options. additionally, one qualitative study found that misunderstanding of the key concepts and recommendations of ipc contributed to under-utilization of transmission-based precautions [78] . thus, emphasis should be on training and education of hcws on transmission-based precautions. a majority of the reviewed studies assessed infection control practice as part of the investigation to identify the problem areas of the outbreaks. most studies attempted on-site direct observation of infection control practices and product availability. some studies of retrospective design used survey and interview among employees. however, it is difficult to find a study that investigated the compliance rate of control measures during the outbreaks. only a few studies described gaps in the actual application after recommendations for control measures were made. even several studies overlooked reporting control measures, especially npis [3] . although this does not necessarily mean that they did not apply measures, for the purpose of this review some studies were excluded from the analysis of control measures. the outbreak reports and intervention studies of nosocomial infection (orion) statement was developed and recommended, to improve the quality of outbreak reporting [79] . according to the statement, control measures should be included in the paper. future studies should consider following the orion statement for reporting of outbreaks [79] , which would facilitate the formation of a body of evidence for outbreak management in ltcfs. this study has several limitations. first, we only included studies written in english. it is possible that our review missed articles of interest written in other languages. second, we conducted only a qualitative review due to the variability of the outbreak reports. third, the quality assessment was conducted on studies of certain design including case-control and cohort study due to applicability of the quality assessment tool. fourth, the results of this review have limited generalizability due to publication bias, given that either successfully controlled outbreaks or outbreaks with higher attack rates or fatality rates tend to be published. this update for understanding outbreaks in ltcfs by reviewing recent studies indicates that staff members and residents are still at risk for contagious disease outbreaks including influenza, gastroenteritis, and gas infection. as for the problem aspects, rather than by new or unexpected issues, violation of basic infection control practices was found to facilitate the occurrence and onward transmission of pathogens. the results of this review suggest that ltcfs need to inspect basic infection control practice and to implement them thoroughly in daily care as priorities. efforts should be directed to promoting consistent and optimal adherence to the basic practice of infection control among hcws at all times in ltcfs. when an outbreak occurs, non-pharmaceutical control measures should be utilized to interrupt transmission. however, work restriction was infrequently taken compared to other measures. given the fact that over half of the included studies reported at least one employee ill and their possible role in the spread of pathogens, it is necessary that symptomatic staff members temporarily preclude themselves from working. in addition to work restriction, though, ltcfs with poor resources have faced various challenges in outbreak management. further discussion and studies are needed to identify the way addressing these challenges. world health organization. disease outbreaks shea/apic guideline: infection prevention and control in the long-term care facility influenza outbreak control 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and meta-analyses: the prisma statement testing a tool for assessing the risk of bias for nonrandomized studies showed moderate reliability and promising validity use of alcohol-based hand sanitizers as a risk factor for norovirus outbreaks in long-term care facilities in northern new england: december successful control of an outbreak of ges-5 extended-spectrum β-lactamase-producing pseudomonas aeruginosa in a longterm care facility in japan a mild outbreak of gastroenteritis in long-term care facility residents due to clostridium perfringens investigation of a prolonged group a streptococcal outbreak among residents of a skilled nursing facility hepatitis b outbreak in a nursing home associated with reusable lancet devices for blood glucose monitoring, northern germany acute hepatitis b outbreaks in 2 skilled nursing facilities and possible sources of transmission: north carolina the role of wound care in 2 group a streptococcal outbreaks in a chicago skilled nursing facility risk 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analysis of an epidemic outbreak of keratoconjunctivitis hepatitis c virus transmission in a skilled nursing facility the role of dependency in a norovirus outbreak in a nursing home an outbreak of infections caused by non-typeable haemophilus influenzae in an extended care facility protracted outbreak of s. enteritidis pt 21c in a large hamburg nursing home an outbreak of colistinresistant klebsiella pneumoniae carbapenemase-producing klebsiella pneumoniae in the netherlands tuberculosis outbreak in a long-term care facility outbreak of a beta-lactam resistant non-typeable haemophilus influenzae sequence type 14 associated with severe clinical outcomes prolonged and large outbreak of invasive group a streptococcus disease within a nursing home: repeated intrafacility transmission of a single strain a cluster of group a streptococcal infections in a skilled nursing facility-the potential role of healthcare worker presenteeism a pulmonary tuberculosis outbreak in a long-term care 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guidelines for transparent reporting of outbreak reports and intervention studies of nosocomial infection key: cord-305071-4ck8nd24 authors: calvo, cristina; garcía-garcía, maría luz; sanchez-dehesa, rosa; román, cristina; tabares, ana; pozo, francisco; casas, inmaculada title: eight year prospective study of adenoviruses infections in hospitalized children. comparison with other respiratory viruses date: 2015-07-06 journal: plos one doi: 10.1371/journal.pone.0132162 sha: doc_id: 305071 cord_uid: 4ck8nd24 human adenovirus (hadv) cause upper and lower respiratory tract infections. however, there are few large prospective studies focused on hadvs acute infections requiring hospitalization. from 2005 to 2013 a prospective study was conducted on children admitted with acute respiratory infections. specimens of nasopharyngeal aspirate were taken for virological study by pcr and clinical data was recorded. hadv specimens were genotyped. frequency and clinical course of hadv infections were compared with rsv, rhinovirus (rv), human bocavirus (hbov) and influenza in the same population. hadv was detected in 403 cases of 2371 confirmed viral infections (17.2%) , of which 154 were single virus infections (38%). we genotyped 154 hadvs. the most frequent genotypes were hadv-3 (24%), hadv-6 (21%), and hadv-5 (20%). a total of 262 children had fever (64.9%); 194 suffered hypoxia (48%), and 147 presented infiltrate in chest x-rays (36.4%). the most frequent diagnoses were recurrent wheezing or asthma (51.7%), bronchiolitis (18.3 %), and pneumonia (11.9%), and 46 (11.4%) episodes required prolonged hospitalization (>7 days) due to the severity. adenovirus single infections were compared with single infections of 598 rsv, 494 rv, 83 influenza and 78 hbov. significant clinical differences were found between hadv, rsv and rv infections. human adenovirus (hadv), a double-stranded dna virus, causes a wide range of clinical syndromes and is a well-recognized agent of upper and lower respiratory infections in children [1, 2] . less frequently adenoviruses can cause gastrointestinal, ophthalmologic, genitourinary and neurological infections. hadvs are classified into seven species, a to g [3] . different serotypes may be implicated in different clinical syndromes. serotypes 1,2,3,5 and 7 have been described to be associated with pharyngitis. pharyngoconjuntival fever is usually caused by serotypes 2,3,4 and 7, and pneumonia has been related to 3,7 and 21 [2, 4] . less information exists about other lower respiratory syndromes such as bronchiolitis, recurrent wheezing or asthma [5] . adenovirus infections can occur sporadically or in outbreaks and are frequent throughout the year. the severity of hadv infections varies from mild upper respiratory cases to those that require hospitalization, intensive care admission and occasionally fatal cases, mainly in immunocompromised children [6] . hadv 7 and 14 have been associated with fatal pneumonia [7] . although the literature on adenoviral infections in children is increasing, there are few prospective, long term studies, designed specifically to evaluate the role of hadv in acute respiratory infections requiring hospitalization. this work is part of a prospective study performed in all hospitalized children with respiratory diseases in the pediatrics department of the severo ochoa hospital in madrid (spain). we have designed a specific sub-study with the objective of describing the clinical impact of the adenovirus' infections and comparing clinical and epidemiological features with other respiratory viruses in the same population. the study was approved by the medical ethics committee of the instituto de salud carlos iii. informed written consent was obtained from parents or legal guardians. the study population comprised all children < 14 years of age with a respiratory tract disease admitted to the secondary public hospital severo ochoa (leganés, madrid), between september 2005 and august 2013. all patients were evaluated by an attending physician and clinical characteristics of patients were analyzed. during the hospital stay, and as part of the study, a physician filled out a study-questionnaire with the clinical data. upper respiratory tract infection (urti) was diagnosed in patients with: rhinorrhea and/or cough and no signs of wheezing, dyspnea, crackles or bronchodilator use, with or without fever. asthma was diagnosed on the basis of the national asthma education and prevention program guidelines [8] . all other episodes of acute expiratory wheezing were considered to be recurrent wheezing. acute expiratory wheezing was considered to be bronchiolitis when it occurred for the first time in children aged under 2 years. laryngotracheobronchitis was associated with inspiratory dyspnea and wheezing. laryngitis was associated with inspiratory dyspnea without wheezing. cases with both focal infiltrates and consolidation in chest x-rays were, in the absence of wheezing, classified as pneumonia. specimens consisted of nasopharyngeal aspirates (npa) taken from each patient at admission (monday to friday). each specimen (one for each patient) was sent for virological investigation to the respiratory virus and influenza unit at the national microbiology center (isciii, madrid, spain). npas were processed within 24 hours after collection. upon receipt, three aliquots were prepared and stored at -80°c. both, the reception and the npa sample processing areas were separate from those defined as working areas. detected by using previously described primer sets only to amplify influenza viruses in a multiplex pcr assay [9] . a second multiplex pcr was used to detect parainfluenza viruses 1 to 4, human coronaviruses 229e and oc43, enteroviruses and rhinoviruses (rv) [10] . presence of respiratory syncytial virus (rsv) a and b types, human metapneumovirus (hmpv), human bocavirus (hbov) and adenoviruses were investigated by a third multiplex rt-nested pcr-brq method [11] . with several modifications, genotyping of detected adenoviruses were performed by amplifying and analyzing a partial hexon genomic region as described previously [12] . briefly, 5 μl of the nucleic acid extraction was added to 45 μl of reaction mixture containing 60 mm tris-hcl (ph 8.5), 15 mm (nh4)2so4, 0.2 mm each of dntps (ge healthcare, uk), 60 pmol of each primer (genadv1s 5'gtigayytgcaigacagraayaciga3' and genadv1r 5'tttiagickig traaiswccaicc3') and 1.25u amplitaq dna polymerase (applied biosystems, branchburg, new jersey usa). temperature and time profiles were: 95°c for 4 min and 40 cycles, 95°c for 30 sec, 50°c for 2 min, 72°c for 30 sec. for nested reactions, the same reagents, temperature and time profiles were used as in first reaction as well as 60 pmol of primers (hadv2s + 5'agitayttywgiatgtggaa3' and panadv1r (5'tgrtcrttggtitcrttickiag cat3'). amplification products (consensus 768nt) were visualized by agarose gel electrophoresis and sequenced in both directions using an automated abi prism 377 sequencer. values were expressed as percentages for discrete variables, or as mean and standard deviation (sd) for continuous variables. clinical characteristics of patients with infections associated to adenovirus were compared with those associated with single infection by rsv, rv, hbov and influenza. clinical characteristics and laboratory variables were compared using the student t test, the mann-whitney u test, the χ 2 test, and fisher's exact test. a two-sided value of p < 0.05 was considered statistically significant. results were adjusted for age. all analyses were performed using the statistical package for the social sciences (spss), version 21.0. the study population consisted of 3092 cases of hospitalization for respiratory causes in children < 14 years of age. a total of 2371 cases (76.7%) had a positive respiratory viral identification; 70.2% were single infections. finally, 403 cases had adenovirus detection (17% of the respiratory viral cases). of the positive adenovirus infections, 154 were single virus infections (38%) and 249 children had dual or multiple viral infections (121 with rv and 60 with rsv were the most frequent associations). the total of 403 cases corresponded to 387 children; 13 children had two episodes, separated between six weeks and one year, and one girl had 3 episodes (in different years). one child had two episodes, two weeks apart, both in coinfection with rv and cov. since normal excretion of adenovirus in nasopharyngeal aspirate lasts 3-10 days, all were analyzed, although in the latter case it could be not guaranteed that these were two different infections. adenovirus infections peaked in november-december although they were present throughout the year except for august. the proportion of infections was higher in 2006, 2008, 2009, 2010 and 2011 than in the other studied years (fig 1) . of the total number of cases, 235 were males (58.2%), 262 had fever (64.9%), 194 suffered hypoxia (48%) for 2.5 (sd 2.3) days, and 147 presented infiltrate in chest x-rays (36.4%). mean higher temperature was 38.8 (sd 0.8)°c, and the duration of the fever was 3.6 (sd 3.5) days. mean c-reactive protein, in those cases in which it was determined, was 49 (sd 57) mg/ l and leucocytes count 15330 (sd 8274)/ mm 3 . antibiotic therapy was prescribed in 129 cases (31.9%). only 25 of the children had been born preterm (6.2%). the mean age of the group was 20 (sd 17) months, and the length of the stay at the hospital was 4 (sd 2.5) days. diagnosis in order of frequency was recurrent wheezing or asthma (51.7%), bronchiolitis (18.3%), pneumonia (11.9%) and fever syndrome (4%). two patients were admitted to the intensive care unit suffering from pneumonia with pleural effusion and had negative blood cultures. on the other hand, 4 patients were found positive for streptococcus pneumoniae (3) and enterococcus (1) in blood culture as well as presenting pneumonia (2), bacteremia (1) and bronchiolitis (1). these 4 cases had leukocytosis higher than 22,000 cells/mm 3 and creactive protein above 100 mg/l. adenovirus single infection was detected in 154 cases. clinical characteristics of these single cases were compared with coinfections due to adenovirus and other viruses (table 1) . children with single infections were older (p<0.001), with more prolonged fever (p = 0.047), higher values of leukocytosis (p = 0.04), more frequent pneumonia (p = 0.0015) and these received antibiotics more frequently (p<0.001). between 2005 and 2008 a total of 154 adenoviruses (154 episodes) were genotyped, corresponding to 80% of adenoviruses detected in this period (fig 2) . the most frequent genotypes identified were hadv-3 (24%) and hadv-6 (21%) followed by hadv-5 (20%) and hadv-2 (19%). genotype hadv-7 was only detected in one patient. genotype distribution was different between cases with single or multiple viral detection. genotype hadv-3 was more frequent among patients with single infection, and was present in 37.3% of them (p = 0.003). we compared clinical data among the four more prevalent genotypes (hadv 2,3,5,6) and we found that patients infected with genotype hadv-3 had higher c-reactive protein levels than those infected with genotype hadv-5 (47.8 ± 37) vs 16.9(sd 15), p = 0.05) and longer duration of the fever than those with genotype hadv-6 (4.7 ± 3 vs 2.9 ±1.5, p = 0.06). genotype hadv-3 was the most frequent in patients with pneumonia (12.5% of cases), but other genotypes also found in these processes were hadv-1, hadv-2, hadv-5 and hadv-6. genotype hadv-6 was the most frequent in patients with bronchiolitis (14.9% of cases), recurrent wheezing and asthma (20% of cases). of the13 patients with repeated hadv infections, 12 of them could be genotyped. all were different genotypes except a case, with separate episodes six weeks apart, who presented with genotype hadv-5 on both occasions. severe cases of adenovirus infection. we analyzed the group of patients hospitalized for more than 7 days (mean stay in total group was 4 ± 2.5 days). we found 46 (11.4%) cases requiring such prolonged hospitalization. mean stay was 9.4 ± 2.5 days. this group of patients also had a longer duration of fever; 4.8 ± 3.1 day (p = 0.07), but the main severity marker was hypoxia, present in 71% of these patients, vs. 49% in the other patients (p = 0.017) and for 6.08± 3.3 days (p<0.001). infiltrate in the chest x-ray was present in 41% (no differences with the total group). other clinical data were similar between this group and the total of the number of cases. out of those 46 patients, 19 were genotyped, and no statistically significant predominant genotype was found (although type 2 was detected in 6 patients). clinical differences between adenovirus and other respiratory viruses. adenovirus single infections (154 episodes) were compared with single infections of 598 rsv, 494 rv, 83 influenza and 78 hbov that were detected in the same period (table 2 ). other less prevalent viruses were not included in this analysis. clinical data for infections caused by hadv were similar to infections associated to hbov and influenza. patients with influenza have fever more frequently (p = 0.028) and have a lower leukocytes count in blood (p<0.001), than children infected by hadv. comparison hadv/rv. nevertheless, infections caused by rsv and rv were significantly different to those associated with hadv. rsv patients were younger than hadv ones (mean age of 9 months vs 22, p<0.001); diagnosis of bronchiolitis was more frequent (p<0.001), the patients needed oxygen more frequently (p<0.001), had less and shorter fever (p< 0.001); and they needed less antibiotic treatment (p<0.001), but their hospital stay was slightly longer (p = 0.029). leukocytes and acute phase reactants in blood tests were significantly higher in hadv infections. patients with rv infections were also different from those with hadv. although age and diagnosis were similar, the rv group had less and shorter fever (p<0.001), fewer abnormal xrays (p = 0.002), less antibiotic treatment (p<0.001), and lower duration of the oxygen therapy (p<0.001) and hospital stay (p = 0.017). regarding the seasonal distribution of the virus, we found significant differences between hadv monthly circulations and each of the other viruses studied (fig 3) . hadv predominated in spring with another peak in december, while rsv circulates in november, december and january (p = 0.001), similar to hbov circulation (p = 0.001). higher influenza incidence takes place in january and february (p = 0.001). rv infections occur throughout the year, with a higher incidence in september and october (p = 0.001) adenovirus respiratory tract infections are an important cause of hospitalization in children. we report one of the longest prospective studies with the largest number of patients published to date. hadv was associated with 17% of viral respiratory cases in our series, over 8 consecutive years. children affected were usually under 2 years old, and clinical data associated were often episodes of recurrent wheezing, with fever but mainly with hypoxia. hadv frequently (11% of cases) caused lengthy hospitalizations (more than 7 days) 21% of the single infections were diagnosed with pneumonia. genotypes hadv-2, hadv-3, hadv-5 and hadv-6 were most frequently identified in our patients. previously reported prevalence of adenovirus in acute respiratory tract infections in children ranges between 6-18% of the patients depending on the geography and the study population. in china, jin et al, found hadv in 6.3% of the infections in hospitalized and outpatient children [13] . in argentina [14] , the proportion increases to 14.3% in hospitalized children, very close to brazilian hospitalized children [15] (15.8%). our proportion is slightly higher (17%), possibly due to the prospective nature of our study, conducted in all hospitalized children, and not only in selected cases. we have also included coinfections in our analysis. single adenovirus infections were 38% of the study cases. in any case, the burden of the adenovirus infections in hospitalized children is considerable. although clinical data of children with infections due to hadv can be considered similar to other viral infections, there are some specific findings. the age of the children is mostly under 5 years but mean age is around 2 years (20 months in our patients). high fever of more than 38.5°c is frequent and both recurrent wheezing and asthma crisis are the most common diagnosis. hypoxia and fever are often the cause of prolonged hospitalizations. leukocyte count and c-reactive protein are usually higher than in other viral infections, this being a confounding factor with a bacterial infection. as a result, children are often treated with antibiotics. these data are consistent with the literature [13, 15, 16] . in our series these clinical characteristics were more evident in single infections than in coinfections, but we have not found other groups that compare single and multiple infections. our rate of coinfection (62%) was the same as found by jin [13] . the severity of adenovirus infections in immunocompetent children is well known and has been characteristically associated to with pneumonia caused by genotypes 3 and especially 7 [2, 7, 16] . in our patients, 11% had a hospital stay of more than 7 days. hypoxia and not pneumonia (present in 21% of all single infected children) was the cause of such prolonged hospitalization. we have not found a higher proportion of pneumonia (10.8%) in these patients, and we failed to identify any predominant genotype either in these cases or in children with pneumonia. genotype 7 appears to be more prevalent in other countries than in our series, and especially in outbreaks, and we have not identified any outbreak in our patients. genotypes 2,3,5 and 6 were more frequently found in our children, and in our series genotype 3 was not associated with more severity as previously reported by other groups [16] . again, regional differences could be involved. we have been able to genotype 154 of the total identified hadv and although it is a significant number, since there are many genotypes, the groups are not numerous enough to detect significant differences among genotypes. finally, we have performed a comparison between hadv single infections, and the most important circulating virus in the same populations and period. as far as we know, such a complete comparison has not been performed previously, although partial comparisons can be found. hadv infections are quite similar to bocavirus and influenza. children with influenza infections have high fever more frequently than hadv infections. leukocytes in blood are higher in the hadv group. our data are similar to the comparison performed by chan et al in the usa, in children with pandemic influenza a with respiratory illness [17] . to our knowledge, this is the first comparison between human bocavirus and hadv, except for a short series previously published by our group [18] . although these three viruses are clinically quite similar, their seasonality is different. hadv is more frequent in spring months, with another peak in december. the highest influenza incidence is in january and february in characteristically annual epidemics after the rsv epidemic, whereas the highest incidence of bocavirus is in november and december. on the other hand, infections caused by hadv are different from infections caused by rv and rsv. some groups have performed comparisons between hadv and rsv. in brasil, ferone et al [15] found in hospitalized infants, that children with hadv infections are older than those with rsv, need antibiotic treatment more frequently and have fewer leukocytes and less c-reactive protein in their blood. jin et al [13] , in china, describe similar findings, that children with rsv are younger than patients with hadv, and have lower respiratory tract infections such as bronchiolitis and bronchitis more frequently. we did not find any specific comparisons between rv and hadv infections. in our series, patients with rv had less fever (grade and frequency), less pneumonia and x-ray infiltrate, less antibiotic treatment and a shorter process in general (stay, hypoxia and fever duration). hadv infections are found in an important proportion of the hospitalized children with respiratory illnesses (17% in our series), and circulate mainly in spring and december. they are associated with characteristic clinical data, such as higher and more prolonged fever, recurrent wheezing or pneumonias and elevated acute phase reactants, and frequently require antibiotic treatment. there is a fairly significant proportion (11.4%) of hadv cases that are severe enough to require prolonged hospitalization of more than 7 days, mainly caused by hypoxia. in our country, the different genotypes are not related to specific diagnoses, although hadv 2,3,5 and 6 are the most frequent in our patients. although clinical characteristics are similar to influenza and bocavirus infections, seasonality and epidemics could lead us to consider one or another virus. on the other hand, rv and rsv infections are clinically and epidemiologically different from hadv infections in children. clinical presentation and characteristics of pharyngeal adenovirus infections clinical features of radiologically confirmed pneumonia due to adenovirus in children the role of adenovirus in respiratory tract infections molecular epidemiology and clinical presentation of human adenovirus infections in kansas city children adenoviruses in immunocompromised hosts acute lower respiratory tract infections by adenovirus in children: histopathologic findings in 18 fatal cases expert panel report: guidelines for the diagnosis and management of asthma update on selected topics-2002 simultaneous detection of influenza a, b, and c viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-pcr assay simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays detection of new respiratory viruses in hospitalized infants with bronchiolitis: a three-year prospective study molecular identification of adenoviruses in clinical samples by analyzing a partial hexon genomic region prevalence of adenovirus in children with acute respiratory tract infection in lanzhou adenovirus type 7 associated with severe and fatal acute lower respiratory infections in argentine children clinical and epidemiological aspects related to the detection of adenovirus or respiratory syncytial virus in infants hospitalized for acute lower respiratory tract infection adenovirus serotype 3 and 7 infection with acute respiratory failure in children in taiwan distinguishing characteristics between 2009-2010 pandemic influenza a (h1n1) in other viruses in patients hospitalized with respiratory illness clinical characteristics of human bocavirus infections compared with other respiratory viruses in spanish children key: cord-285749-0ejhd9nw authors: hoffmann, markus; krüger, nadine; zmora, pawel; wrensch, florian; herrler, georg; pöhlmann, stefan title: the hemagglutinin of bat-associated influenza viruses is activated by tmprss2 for ph-dependent entry into bat but not human cells date: 2016-03-30 journal: plos one doi: 10.1371/journal.pone.0152134 sha: doc_id: 285749 cord_uid: 0ejhd9nw new world bats have recently been discovered to harbor influenza a virus (fluav)-related viruses, termed bat-associated influenza a-like viruses (batfluav). the internal proteins of batfluav are functional in mammalian cells. in contrast, no biological functionality could be demonstrated for the surface proteins, hemagglutinin (ha)-like (hal) and neuraminidase (na)-like (nal), and these proteins need to be replaced by their human counterparts to allow spread of batfluav in human cells. here, we employed rhabdoviral vectors to study the role of hal and nal in viral entry. vectors pseudotyped with batfluav-hal and -nal were able to enter bat cells but not cells from other mammalian species. host cell entry was mediated by hal and was dependent on prior proteolytic activation of hal and endosomal low ph. in contrast, sialic acids were dispensable for hal-driven entry. finally, the type ii transmembrane serine protease tmprss2 was able to activate hal for cell entry indicating that batfluav can utilize human proteases for hal activation. collectively, these results identify viral and cellular factors governing host cell entry driven by batfluav surface proteins. they suggest that the absence of a functional receptor precludes entry of batfluav into human cells while other prerequisites for entry, hal activation and protonation, are met in target cells of human origin. influenza a viruses (fluav) are enveloped, negative stranded rna viruses that pose a major threat to public health [1] . the ability of fluav to constantly adapt to immune pressure allows these viruses to continuously circulate in the human population, resulting in annual influenza epidemics (seasonal influenza [2, 3] ). infants, children and the elderly are at particular risk of developing severe disease upon infection with seasonal fluav and it has been estimated that world-wide 250,000 to 500,000 people die each year of seasonal influenza [1] . were detached by either resuspension in fresh culture medium (hek-293t cells) or by the use of trypsin/edta (paa laboratories). shuttle vectors harboring codon-optimized (for expression in human cells) open reading frames coding for the published amino acid (aa) sequences of the hal of the two batfluav, a/little yellow-shouldered bat/guatemala/153/2009 (h17/n10) (genbank: cy103876.1, hal17) and a/flat-faced bat/peru/033/2010 (h18/n11) (genbank: cy125945.1, hal18), were purchased from a commercial service (eurofins mwg operon) and cloned into the pcg1 expression vector, that was kindly provided by r. cattaneo, via bamhi and xbai restriction sites. the pcaggs-based expression plasmid for nal of batfluav a/little yellowshouldered bat/guatemala/153/2009 (h17/n10) (genbank: cy103878.1, nal10) was provided by m. schwemmle. hal equipped with a c-terminal flag epitope (dykddddk, hal17-flag and hal18-flag) were constructed by pcr and controlled for sequence integrity by automated sequence analysis. in addition, we used pcaggs-based expression plasmids for the ha and na of a/wsn/33 (h1n1) and a/singapore/1/57 (h2n2) (genbank: ay209895.1) [32, 33] . furthermore, we employed pcaggs-based expression plasmids for the ha of a/south carolina/1/18 (h1n1) (genbank: af117241.1) and the na of a/brevig mission/1/1918 (h1n1) (genbank: af250356.2) that were generated from previously used constructs [32] . the expression plasmid for the glycoprotein (g) of vesicular stomatitis virus (vsv, indiana strain, vsv-g; genbank: aj318514.1) was generated by inserting the vsv-g orf into the pcg1 expression vector and has been used in previous studies [28, 31, 34, 35] . furthermore, expression plasmids for nipah virus fusion protein (f, genbank: af212302) and glycoprotein (g, synthetic, genbank: af212302; derived from niv/my/hu/1999/cdc) were used [36] . for experiments analyzing proteolytic activation of hal17 and hal18 by human type-ii transmembrane serine proteases (ttsps), we employed expression plasmids for tmprss2, tmprss11e (desc-1) and tmprss13 (mspl), which have been described previously [32, 33] . we employed a replication-deficient vsv vector for pseudotyping that contains two separate open reading frames, coding for enhanced green fluorescent protein (egfp) and firefly luciferase (fluc), instead of the genetic information for vsv-g, vsv ã δg-fluc [28, 31, 35] . propagation of vsv ã δg-fluc was performed in a previously described vsv-g-expressing, transgenic cell line [37] . generation of vsv pseudotypes (vsvpp) was performed as follows: hek-293t cells were transfected by calcium-phosphate precipitation with expression plasmids encoding viral surface proteins, vsv-g (positive control) , niv-f/g, fluav-ha and/or na and bat-fluav-hal and/or nal, or empty plasmid (pcaggs) as negative control. in order to investigate the potential of human ttsps to proteolytically activate batfluav-hal for host cell entry, we additionally cotransfected the cells with expression plasmids for tmprss2, desc-1 or mspl. at 16 h post transfection, the cells were inoculated with vsv ã δg-fluc at a multiplicity of infection of 3 for 1 h at 37°c and 5% co 2 . subsequently, the cells were washed and incubated with an anti-vsv serum to neutralize residual input virus. finally, the cells received fresh culture medium and were further incubated for 16-20 h, before the vsvpp-containing supernatants were collected, clarified from cell debris by centrifugation and aliquoted. aliquots were stored at 4°c for a maximum of 7 days. for proteolytic activation of ha/hal by trypsin, pseudotypes were incubated with bovine trypsin (sigma-aldrich; final concentration: 50 μg/ml) for 20 min at 37°c. subsequently, trypsin was inactivated by addition of soybean trypsin inhibitor (sigma-aldrich; final concentration: 50 μg/ml). to investigate the roles of sialic acids and endosomal acidification in batfluav entry, we used recombinantly-produced, bacterial sialidase (clostridium welchii, sigma-aldrich) and ammonium chloride (sigma-aldrich). for treatment, the cell culture supernatant was removed and the cells were washed with phosphate buffered saline (pbs) before culture medium containing water (negative control), ammonium chloride (50 mm) or different concentrations of bacterial sialidase (1.5, 15 or 150 mu) was added. after 2 h of incubation at 37°c and 5% co 2 , the supernatant was removed, the cells washed and then inoculated with pseudotypes, as described below. transduction of cell lines with rhabdoviral pseudotypes and quantification of fluc activity all transduction experiments were performed in 96-well plates using quadruplicate samples. at 24 h post seeding, the cell culture medium was removed and the cells were washed with pbs. the cells were either directly inoculated with vsvpp or treated as specified above and then inoculated. vsvpp inoculation was performed for 1 h at 37°c and 5% co 2 . afterwards, the inoculum was removed and the cells were again washed and incubated with fresh culture medium for 16-18 h at 37°c and 5% co 2 . for the quantification of the fluc activity as an indicator of transduction efficiency, the cell culture supernatant was removed and the cells were washed with pbs. next, 50 μl of 1x luciferase cell culture lysis reagent (promega) in pbs was added to each well and incubated for 30 min at room temperature, before the cell lysate was transferred to a white, opaque-walled 96-well plate (thermo scientific). the measurement of the fluc activity was carried out in a microplate reader, plate chameleon (hidex), using the microwin2000 software (version 4.44, mikrotek laborsysteme gmbh) and fluc substrates from the luciferase assay system (promega) or beetle-juice (pjk) kits. transduction efficiency, represented by fluc activity, was either displayed in counts per second (cps) or as normalized values. to assess expression of hal proteins, we transfected bhk-21 cells that were grown on coverslips with expression plasmids for hal17-flag or hal18-flag using the lipofecta-mine2000 reagent (thermofisher scientific) according to manufacturers' protocol. cells transfected with an empty expression vector served as negative control. at 24 h post transfection, cells were fixed with 4% paraformaldehyde/pbs, permeabilized by incubation with 0.2 m triton x-100/pbs (10 min at room temperature) and subsequently incubated with anti-flag (mouse, 1:1,000, sigma-aldrich) and cy3-labeled anti-mouse (1:750, sigma-aldrich) antibodies. after each antibody incubation, the cells were washed three times with pbs and finally incubated with dapi (roth, 5 min/37°c) to stain the nuclei before the coverslips were mounted on glass slides using mowiol (applichem) supplemented with anti-bleaching reagent (dabco, roth). representative pictures were taken at a 10x magnification using a nikon eclipse ti fluorescence microscope and the nis elements ar software (nikon). batfluav-hal cleavage was investigated by cotransfection of hek-293t cells, which were grown in 6-well plates, with expression plasmids for batfluav-hal and different ttsps (tmprss2, desc-1 or mspl) or by incubation of hal-expressing cells with trypsin (1, 5, 10, 50 μg/ml; 20 min/37°c) directly before cell lysates were produced. cells cotransfected with empty plasmid and not subjected to trypsin treatment served as negative control. the 1918-ha served as positive control, since cleavage by ttsps and trypsin has been previously shown [33] . at 24 h post transfection, the cells were washed with pbs, resuspended in 100 μl 2x sds-containing lysis buffer (50 mm tris [ph 6.8], 10% glycerol, 2% sds, 5% β-mercaptoethanol, 0.1% bromophenol blue, 1 mm edta) and boiled for 20 min at 96°c. to assess incorporation of hal into vsvpp, 1 ml of the respective pseudotypes was loaded onto a 20% sucrose cushion in pbs and centrifuged at 17,000x g for 2 h at 4°c. after discarding the supernatant, the pelleted pseudotypes were mixed with 30 μl 2x sds-containing lysis buffer and boiled for 20 min at 96°c. finally, all samples were subjected to immunoblot analysis. for this, anti-flag (mouse, 1:1,000, sigma-aldrich), anti-fluav (goat, 1:1,000, millipore), anti-vsv-m (mouse, 1:1,000, kerafast), anti-vsv-g (i1, mouse hybridoma supernatant from crl-2700, atcc, 1:200) and anti-ß-actin (mouse, 1:1,000, sigma-aldrich) served as primary antibodies. peroxidase-coupled anti-mouse (1:10,000, dianova) and anti-goat (1:5,000, dianova) antibodies served as secondary antibodies. signal detection was carried out in a chemocam imager together with the chemostar professional software (both intas) using a self-made chemiluminescence substrate (recipe available upon request). in order to assess statistical significance, two-tailed student's t-tests were performed. to test whether hal17 and hal18 are comparably expressed and incorporated into rhabdoviral pseudotypes, both proteins were equipped with a c-terminal flag epitope (hal17-f-lag, hal18-flag), since no batfluav-hal-specific antibody was available. upon transfection of bhk-21 cells similar numbers of hal-expressing cells were detected by fluorescence microscopy ( fig 1a) and the intensity of the fluorescence signals emitted by the cells was comparable, indicating robust expression of both batfluav-hal proteins. in order to assess hal incorporation into rhabdoviral pseudotypes, we pelleted pseudotype preparations through a sucrose cushion and subjected the samples to sds-page and immunoblotting. using antibodies specific for the flag epitope, vsv-g and vsv matrix protein (vsv-m), we found that vsv-g, as expected, as well as both hal17 and hal18 proteins were incorporated into particles, with incorporation of hal17 being more efficient than that of hal18. (fig 1b) . thus, both hal17 and hal18 were robustly expressed and incorporated into vsvpp, allowing their functional characterization. it has been previously reported that hal and nal of batfluav are not compatible with viral spread in the cell lines tested so far [18, 19] , suggesting that these proteins mediate entry into a restricted set of target cells or are even inactive. in order to gain insights into the functional activity of batfluav-hal and nal, we employed rhabdoviral vectors pseudotyped with these proteins. we inoculated cell lines from different host species, including those standardly used for fluav research, as well as cell lines derived from different bat species (table 1) , as they are the natural reservoir for batfluav. we chose bat cell lines known to be susceptible to infection by viruses of different families [28, 29, 31] and previously used to functionally characterize surface glycoproteins of bat-borne viruses [28, 36, 38] . it is well established that fluav-ha depends on proteolytic cleavage by host cell proteases to transit into an active form [12] and it has been previously reported that batfluav can be activated by exogenous trypsin [39] [40] [41] . therefore, we assessed whether trypsin treatment of the pseudotypes impacts transduction efficiency. as controls, we included the surface glycoprotein(s) of well-characterized fluav strains, a/wsn/33 (h1n1) (wsn-ha, wsn-na), a/south carolina/1/18 we found that none of the human, simian and canine cell lines was susceptible to entry driven by batfluav surface proteins (fig 2a) . in contrast, pseudotypes bearing vsv-g, niv-f/g or wsn-ha/na could readily enter these cells, whereas pseudotypes that harbored 1918-or h2n2-ha/na required activation by exogenous trypsin for efficient transduction (fig 2a) . these results are in agreement with expectations, since activation of wsn-ha is known to be independent of trypsin [42] [43] [44] , although viral infectivity can be enhanced by trypsin treatment. when we focused on bat-derived cell lines, vsv-g, niv-f/g and wsn-ha/na again permitted pseudotype entry without prior trypsin treatment, while pseudotypes harboring 1918-ha/na or h2n2-ha/na were only able to transduce some of the bat cell lines after incubation with trypsin ( fig 2b) . cpkd cells remained refractory to entry mediated by 1918-ha/na or h2n2-ha/na but also showed the lowest susceptibility to all other tested pseudotypes. notably, three bat cell lines (eidni/41, hypni/1.1 and eponi/22.1) were susceptible to entry of pseudotypes bearing hal and nal of batfluav (fig 2b) , demonstrating that surface glycoproteins of batfluav can mediate cellular entry. entry into the three bat cell lines was robust (1-3 log units above the threshold) and required prior treatment of pseudotypes with trypsin, which presumably resulted in the proteolytic activation of hal. furthermore, pseudotypes bearing hal17/nal10 or hal18/nal10 were both able to enter hypni/ 1.1 and eponi/22.1 cells while eidni/41 cells could only be transduced by pseudotypes bearing hal18/nal10 (fig 2b) , suggesting that batfluav of the hl17nl10 and hl18nl11 subtype might exhibit subtle differences in entry efficiency or cell tropism. finally, hal-proteins with a c-terminal flag tag facilitated host cell entry, although with somewhat reduced efficiency as compared to their untagged counterparts, indicating that the proteins used to study hal expression and virion incorporation (fig 1) were functional (data not shown). taken together, we showed that batfluav surface proteins can mediate entry into certain bat cell lines. for further studies on the entry process, we focused on eponi/22.1 cells since they showed the highest susceptibility to entry driven by batfluav surface proteins. the na proteins of human fluav facilitate release of progeny particles from infected cells by removing sialic acids from the cell surface. to study the impact of the batfluav-nal on transduction efficiency, we produced pseudotypes bearing batfluav-hal, -nal or both proteins. for comparison, we generated pseudotypes harboring wsn-ha, wsn-na or both proteins. these pseudotypes were then used for inoculation of mdck (inoculated with pseudotypes bearing wsn proteins) and eponi/22.1 (inoculated with pseudotypes bearing wsn or batfluav proteins) cells. pseudotypes harboring only wsn-na were not able to transduce target cells (fig 3) while pseudotypes bearing either wsn-ha alone or in combination with wsn-na transduced both mdck and eponi/22.1, as expected. transduction efficiency was 500-1,500-fold higher when wsn-na was expressed in cells used for pseudotype production, in keeping with the findings that the presence of na is required for efficient release of ha-bearing vectors and infectious fluav [32, 45, 46] . pseudotypes harboring nal were not infectious while pseudotypes bearing hal robustly transduced eponi/22.1 cells, indicating that batfluav-hal, like wsn-ha, is sufficient to mediate host cell entry. however, unlike wsn-na, the expression of nal in pseudotype producer cells did not increase transduction efficiency of hal-harboring pseudotypes (fig 3) , suggesting that nal is not required for release and/or infectivity of hal containing particles, at least in the experimental system chosen. batfluav-hal does not use sialic acids for host cell entry fluav employ alpha-2,3-(avian viruses) and alpha-2,6-linked (human viruses) sialic acids as receptors for host cell entry [47] [48] [49] [50] [51] . in order to assess the potential role of sialic acids in haldriven entry, we pre-treated the cells with escalating doses of bacterial neuraminidase before transduction. neuraminidase treatment of eponi/22.1 cells reduced transduction mediated by the fluav-ha proteins, as expected (fig 4) . in contrast, sialidase treatment had no effect on pseudotype entry mediated by batfluav-hal, niv-f/g or vsv-g. moreover, pre-treatment of eponi/22.1 cells at the highest dose (150 mu) even enhanced transduction driven by hal and vsv-g (fig 4) . these results indicate that hal does not use sialic acids for host cell entry and suggest that removal of sialic acids might even increase batfluav infectivity, potentially by increasing accessibility of a cellular receptor. endosomal low ph triggers fluav-ha for membrane fusion. therefore, we investigated whether increasing the endosomal ph in eponi/22.1 cells by ammonium chloride treatment impacts hal-driven entry. as expected, ammonium chloride treatment led to a decrease in transduction efficiency mediated by pseudotypes bearing the ha-proteins of fluav of the h1n1 and h2n2 subtype and vsv-g (fig 5) . in contrast, pseudotype entry orchestrated by niv-f and -g was unaffected, again in keeping with published data [52, 53] . finally, haldriven entry was markedly reduced by ammonium chloride, demonstrating that the membrane fusion activity of batfluav-hal is triggered by acidification (fig 5) . fluav-ha is synthesized as an inactive precursor and requires activation by host cell proteases to be responsive to low ph, the trigger for ha-driven membrane fusion [12] [13] [14] . members of the ttsp family activate fluav-ha in cell culture [33, [54] [55] [56] [57] [58] and tmprss2 was previously shown to be essential for fluav-ha activation and viral spread in mice [59] . therefore, we asked whether ttsps able to activate fluav-ha can also activate batfluav-hal. for this, we first investigated batfluav-hal cleavage by tmprss2, desc-1 and mspl, and compared it to cleavage by trypsin. cleavage of the 1918-ha served as positive control. we found that 1918-ha was efficiently processed by all proteases tested, as expected. moreover, we could show that coexpression of tmprss2, desc-1 and mspl, and trypsin treatment resulted in cleavage of the hal precursor (hal 0 ) determined by the appearance of bands corresponding to the hal 2 subunit (fig 6a) . while hal18 was comparably cleaved by all tested ttsps, hal17 cleavage by tmprss2 was more pronounced than proteolysis by desc-1 and mspl (fig 6a) . moreover, hal18 was generally more sensitive to cleavage by ttsps than hal17 (fig 6a) . in order to assess whether batfluav-hal cleavage by ttsps also leads to hal activation for host cell entry, we produced pseudotypes harboring batfluav-hal (hal17 or hal18) in the presence of tmprss2, desc-1 and mspl. as a control, pseudotypes bearing 1918-ha and -na were included in this experiment. the pseudotypes were treated with trypsin to activate ha/hal or were mock-treated before addition to eponi/22.1 cells. pseudotypes bearing 1918-ha and -na and produced in the presence of tmprss2, desc-1 and mspl or treated with trypsin robustly transduced target cells ( fig 6b) . in contrast, infectivity of fluav-ha pseudotypes produced in the absence of ttsps or not treated with trypsin was in the background range (fig 6b) . similarly, batfluav-halbearing pseudotypes were activated by trypsin or ttsps, including tmprss2 (fig 6b) . however, differences in the activation of hal17 and hal18 were observed and correlated with the efficiency of hal protein cleavage, as determined above (fig 6a) . thus, expression of tmprss2 but not desc-1 and mspl conferred robust infectivity to hal17-bearing pseudotypes while all proteases were able to efficiently activate hal18. moreover, transfection of escalating amounts of tmprss2-encoding plasmids increased infectivity of hal17-bearing pseudotypes in a concentration-dependent manner. in contrast, transfection of even the lowest amount of tmprss2 plasmid was sufficient to confer maximal infectivity of hal18bearing pseudotypes, confirming that the efficiency of tmprss2-mediated activation of hal is subtype specific (fig 6c) . in sum, proteolytic activation of batfluav-hal is critical for hal-driven cell entry and proteases able to activate ha can also activate hal. host cell entry by bat-associated influenza viruses the identification of two new fluav, subtypes h17n10 (hl17nl10) and h18n11 (hl18nl11), in new-world bats [16, 17] suggests that bats could serve as a natural reservoir of fluav [16, 17] . unraveling the zoonotic potential of batfluav is of great importance since fluav are major human pathogens, responsible for influenza epidemics and pandemics. while the batfluav replication machinery appears to be functional in different mammalian (including human) cells [16, [18] [19] [20] [21] 60] , reassortment with fluav and flubv is unlikely [18, 19] . regarding batfluav tropism of the viral surface proteins, hal and nal, only human proteases that activate fluav-ha for cell entry also activate batfluav-hal. (a) hek-293t cells were transfected with plasmids encoding ha or hal proteins and either trypsin treated or cotransfected with plasmids encoding type ii transmembrane serine proteases. transfection of empty vector served as negative control. cleavage of ha/hal proteins was analyzed by sds-page and western blotting, employing antibodies against fluav-ha (α-fluav) and the flag epitope (α-flag). detection of ß-actin served as loading control. signals corresponding to uncleaved precursor proteins are marked by black circles, while products of proteolytic cleavage are indicated by white circles. the results were confirmed in a separate experiment. to assess proteolytic activation of ha/hal proteins, vesicular stomatitis virus-based pseudotypes (vsvpp) were produced in cells transfected to express the indicated type ii transmembrane serine proteases (b) or different amounts of tmprss2 (c). pseudotypes were either directly used for transduction of eponi/22.1 cells (black bars) or previously treated with trypsin (white bars). at 24 h post inoculation, transduction efficiency was measured by quantification of the activity of vsvpp-encoded luciferase in cell lysates. for normalization, transduction by ha-or hal-bearing pseudotypes that were produced in the absence of type ii transmembrane serine protease expression (empty vector) and not treated with trypsin was set as 1. the result of a single representative experiment carried out with quadruplicate samples is presented. similar results were obtained in three independent experiments carried out with separate pseudotype preparations. error bars indicate standard deviations. a two-tailed, unpaired student's t-test was used to test statistical significance (* = p < 0.05). doi:10.1371/journal.pone.0152134.g006 host cell entry by bat-associated influenza viruses limited information is available, which indicate that batfluav do not engage with canonical fluav receptors [17, [22] [23] [24] . however, until very recently no proof of functional activity of batfluav surface proteins was available [17, [22] [23] [24] [25] [26] [27] . here, we employed a vector system to analyze batfluav-hal and -nal. we show that hal mediates entry into certain bat cell lines and that entry does not depend on the presence of sialic acids on the cell surface. moreover, we demonstrate that nal is not required for production of infectious hal-bearing particles, at least under the conditions examined. finally, our studies revealed that trypsin and ttsps activate hal for host cell entry. we used a vsv-based vector system to study cellular entry of hal and nal-bearing particles. vsv pseudotypes allow convenient analysis of entry driven by diverse glycoproteins [28, 31, 61] , although one should keep in mind that due to differences in particle shape and efficiency of glycoprotein incorporation pseudotypes might not adequately mirror all aspects of cellular entry of authentic viruses [62, 63] . we found that cell lines frequently used for fluav propagation were not susceptible to transduction by hal and nal bearing particles, which is in agreement with the finding that replacement of batfluav-hal and -nal by their fluav counterparts is required for spread of batfluav in the cell lines studied so far [18, 19] . in contrast, inoculation of bat cell lines originating from five different species of micro-and megachiropteran bats revealed that three cell lines, eidni/41, hypni/1.1, eponi/22.1, were susceptible to entry mediated by batfluav surface proteins. eponi/22.1 cells showed the highest susceptibility and were thus used for further studies, while eidni/41 cells were found to be robustly susceptible only to transduction by pseudotypes harboring the hal18. collectively, these findings suggest that batfluav surface proteins can mediate entry into certain bat cells and that entry efficiency might differ between batfluav subtypes. our finding that certain bat cell lines are susceptible to pseudotypes harboring hal of batfluav are in line with observations very recently documented by maruyama et al. who found that out of a diverse panel of bat cell lines tested, cells from miniopterus fuliginosus, miniopterus schreibersii and pteropus giganteus were susceptible to ph-dependent, hal-driven entry [64] . a cell line derived from eidolon helvum spleen cells was found to be non-susceptible in contrast to our findings with a kidney cell line established from the same species, suggesting that receptor expression might differ between organs. somewhat more surprising, maruyama and colleagues also observed hal-driven entry into mdck cells [64] , which was not detected in the present study, and these discrepant results might be attributed to use of mdck cells from different sources or to differences in the method used to quantify pseudotype entry. finally, it is noteworthy that cell lines from bats inhabiting different geographical locations (africa, asia, europe) were found to be susceptible to hal-driven entry, suggesting that entry is not a bottleneck for spread of batfluav between bat species. the finding that batfluav surface proteins can facilitate entry into bat-derived target cells allowed us to investigate which viral and cellular components contribute to the entry process. we first focused on nal. the expression of this protein, unlike expression of na, in pseudotype-producing cells did not increase the titers of vectors harboring the corresponding bat-fluav-hal protein. however, this finding does not exclude that nal, like the na of fluav, acts as a receptor-destroying enzyme. thus, hek-293t cells used for pseudotype preparation were not susceptible to hal-driven entry, most likely because they do not express the appropriate receptor. it thus remains to be analyzed whether batfluav-nal increases release of hal-bearing vectors and authentic batfluav from susceptible bat cell lines. these endeavors might be challenging since transfection of bat cell lines by calcium-phosphate precipitation and liposome-based reagents was inefficient (not shown). the fluav-ha is sufficient to mediate viral binding and entry into target cells and our findings indicate that the same applies to batfluav-hal. in contrast to fluav-ha, however, hal does not depend on the presence of sialic acids for entry. thus, treatment of eponi/22.1 cells with sialidase did not decrease hal-mediated pseudotype entry. these findings are in keeping with the work by maruyama et al. [64] and with structural data indicating that hal does possess a distorted putative sialic acid binding site [17, 24] . contrarily, high amounts of sialidase increased entry efficiency, potentially by increasing access to the elusive receptor. in addition, removal of sialic acids might increase electrostatic interactions of batfluav-hal with cell surface factors, since sialic acids are negatively charged. although hal-driven entry was independent of sialic acids, it did require endosomal acidification (in accordance with maruyama et al. [64] ), which is known to trigger the membrane fusion activity of ha. most likely, protonation also triggers bat-fluav-hal for membrane fusion. however, it cannot be disregarded that the inhibitory effect of ammonium chloride was due to blockade of ph-dependent endosomal cysteine proteases, which activate the surface proteins of several coronaviruses and ebolaviruses [65] [66] [67] [68] . cleavage-activation of fluav-ha by host cell proteases is essential for fluav infectivity. several ttsps can cleave and activate ha in cell culture and recent studies demonstrated that tmprss2 is essential for fluav spread in mice [32, 33, 58, 59] . moreover, polymorphisms in tmprss2 were shown to impact severity of influenza in humans [69] . treatment of bat-fluav-hal-expressing cells with trypsin led to proteolytic cleavage of hal and exposure of hal-bearing pseudotypes to trypsin was required for efficient transduction of target cells, indicating that proteolytic processing is also required for hal function. moreover, coexpression of batfluav-hal with tmprss2, desc-1 or mspl resulted in proteolytic cleavage of hal and rendered the particles infectious in the absence of trypsin treatment, suggesting that bat-fluav-hal can utilize human proteases for their activation. finally, titration of the amounts of tmprss2 had differential effects on the proteolytic cleavage of hal17 and hal18 and on infectivity of pseudotypes bearing these proteins, hinting towards subtle differences in the efficiency of tmprss2 use by these subtypes. whether bat tmprss2 is also able to cleave and activate batfluav-hal remains to be investigated. collectively, our results are most compatible with a scenario in which human cells allow for batfluav-hal activation and triggering but fail to express a receptor, which can be employed by hal for host cell entry. these results, jointly with the documented observation that the batfluav replication machinery is functional in human cells [16, 18, 19] suggest that hal adaptation to a human receptor might be the major hurdle batfluav need to overcome to spread in humans. it will thus be highly interesting to identify the nature of this receptor and its interface with batfluav-hal. of note, during the preparation of this manuscript, maruyama and colleagues published a manuscript reporting batfluav-hal-driven entry into bat cell lines different from those used in the present study (maruyama et al., 2015, doi: 10 .1016/j.virol.2015.11.002.). both studies show that hal-driven entry requires prior proteolytic hal-activation by trypsin and endosomal acidification but is independent of sialic acids. the present work extends these findings by demonstrating that hal can utilize the human enzyme tmprss2 for its activation. seasonal) fact sheet no. 211: world health organisation global epidemiology of influenza: past and present. annual review of medicine the origins of new pandemic viruses: the acquisition of new host ranges by canine parvovirus and influenza 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endosomal compartment activation of the nipah virus fusion protein in mdck cells is mediated by cathepsin b within the endosome-recycling compartment novel type ii transmembrane serine proteases, mspl and tmprss13, proteolytically activate membrane fusion activity of the hemagglutinin of highly pathogenic avian influenza viruses and induce their multicycle replication cleavage activation of the human-adapted influenza virus subtypes by matriptase reveals both subtype and strain specificities hat, and tmprss2 activate the hemagglutinin of h9n2 influenza a viruses influenza virus activating host proteases: identification, localization and inhibitors as potential therapeutics proteolytic activation of influenza viruses by serine proteases tmprss2 and hat from human airway epithelium tmprss2 is essential for influenza h1n1 virus pathogenesis in mice the n-terminal domain of pa from batderived influenza-like virus h17n10 has endonuclease activity severe fever with thrombocytopenia virus glycoproteins are targeted by neutralizing antibodies and can use dc-sign as a receptor for ph-dependent entry into human and animal cell lines lentiviruses inefficiently incorporate human parainfluenza type 3 envelope proteins pseudotyping viral vectors with emerging virus envelope proteins characterization of the glycoproteins of bat-derived influenza viruses endosomal proteolysis of the ebola virus glycoprotein is necessary for infection inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry protease-mediated entry via the endosome of human coronavirus 229e endosomal proteolysis by cathepsins is necessary for murine coronavirus mouse hepatitis virus type 2 spike-mediated entry identification of tmprss2 as a susceptibility gene for severe 2009 pandemic a(h1n1) influenza and a(h7n9) influenza. the journal of infectious diseases we would like to thank c. drosten, m. a. müller and m. schwemmle for cell lines and expression plasmids. furthermore, we thank e. berger and i. nehlmeier for excellent technical support. key: cord-292237-45hi4iz2 authors: calvo-pinilla, eva; rodríguez-calvo, teresa; anguita, juan; sevilla, noemí; ortego, javier title: establishment of a bluetongue virus infection model in mice that are deficient in the alpha/beta interferon receptor date: 2009-04-09 journal: plos one doi: 10.1371/journal.pone.0005171 sha: doc_id: 292237 cord_uid: 45hi4iz2 bluetongue (bt) is a noncontagious, insect-transmitted disease of ruminants caused by the bluetongue virus (btv). a laboratory animal model would greatly facilitate the studies of pathogenesis, immune response and vaccination against btv. herein, we show that adult mice deficient in type i ifn receptor (ifnar((−/−))) are highly susceptible to btv-4 and btv-8 infection when the virus is administered intravenously. disease was characterized by ocular discharges and apathy, starting at 48 hours post-infection and quickly leading to animal death within 60 hours of inoculation. infectious virus was recovered from the spleen, lung, thymus, and lymph nodes indicating a systemic infection. in addition, a lymphoid depletion in spleen, and severe pneumonia were observed in the infected mice. furthermore, ifnar((−/−)) adult mice immunized with a btv-4 inactivated vaccine showed the induction of neutralizing antibodies against btv-4 and complete protection against challenge with a lethal dose of this virus. the data indicate that this mouse model may facilitate the study of btv pathogenesis, and the development of new effective vaccines for btv. bluetongue (bt) is an infectious noncontagious viral disease of ruminants caused by the bluetongue virus (btv). the virus, consisting of 24 different serotypes, is transmitted to its vertebrate host by a few species of biting midges of the culicoides genus (diptera: ceratopogonidae) [1] . bt is a reportable disease of considerable socioeconomic concern and of major importance in the international trade of animals and animal products. from 1998 through 2005, at least 6 btv strains belonging to 5 serotypes (btv-1, btv-2, btv-4, btv-9, and btv-16) were continously present in the mediterranean basin [2, 3, 4] . since august 2006, btv-8 has caused severe epizootic outbreakes in northern europe [5] . the emergence of bt in parts of europe never before affected was attributed mainly to climate change and linked to the northern expansion of the major old world vector culicoides imicola. additionally, there is also evidence for the involvement of other novel indigenous european vector species of culicoides (c. obsoletus and c. pulicaris ) [6] . btv has a genome composed of ten linear segments of doublestranded rna (dsrna) and is classified as the type species of the genus orbivirus within the family reoviridae [7] . the btv genome encodes 7 structural (vp1 through vp7) and 4 nonstructural proteins (ns1 through ns3/ns3a). the outer capsid is composed of two major structural proteins, vp2 and vp5 (segments 2 and 6, respectively), involved in cell attachment and virus entry. vp2 is known to contain the major neutralization determinant of btv while vp5 influences virus neutralization through its conformational interaction with vp2 [8] . the outer capsid covers the inner capsid that is composed of two major structural proteins vp3 and vp7 (encoded by segments 3 and 7, respectively) and three distinct minor proteins (vp1, vp4, and vp6 corresponding to segments 1, 4, and 9 respectively) [9] , in addition to the viral genome. four other nonstructural proteins, produced during the viral cycle (ns1, ns2, and ns3/ns3a) (segments 6, 8, and 10 respectively), are more conserved among serotypes [10] . studies involving the natural hosts of btv are limited by the complexity of the system, the scarce knowledge of their immune system, and the need to have an animal facility with biosafety level 3. to circumvent some of these problems, an adequate system would be the use of adult mice because of the knowledge of its genetics and its manageability. btv infects newborn mice [11, 12, 13] , but an adult mouse model will be necessary to allow studies of acquired immune responses and vaccination against btv. bluetongue virus is a potent interferon alpha (ifn-a) inducer [14, 15, 16] . in addition, a temporal relationship between viremia and ifn-a activity has been observed in sheep infected with btv, where ifn peak concentrations induced approximately a 90% decrease in virus titer [17] . ifn-a plays an essential role in the antiviral innate immune response. virus-derived dsrnas are detected by toll-like receptors on type i ifn-producing cells (mostly notably on plasmacytoid dendritic cells). secreted ifns bind to the type i ifn receptor (ifnar) on the surface of neighbouring cells, activating the janus kinase (jak)/signal transducer and activator of transcription (stat) signalling pathway. this in turn induces the transcriptional activation of target genes, the products of which render the cells resistant to virus replication through various mechanisms, including degradation of viral mrnas, inhibition of viral translation, and inhibition of cell growth [18, 19, 20, 21] . blocking ifn-a/b activity in mice leads to a dramatically increased sensitivity to many viruses. genetically targeted (knockout) mice lacking the b subunit of the ifna/b receptor (ifnar (2/2) mice) are unable to establish an antiviral state and, as a consequence, are highly susceptible to many viral infections, despite the presence of an otherwise intact immune system [20, 22] . recently, ida-hosonuma et al [23] found that the deletion of the ifnar gene in the ifnar (2/2) mice resulted in the disruption of ifn-a/b-induced signaling, which is an important determinant of the tissue tropism and pathogenicity of poliovirus. similarly, it has been reported that ifn-a/b plays an important role in the pathogenicity and tissue tropism of some viruses. the lack of an ifn system allows the virus to replicate more efficiently and ifnar (2/2) mice have been used as a laboratory animal model to study the immune response and pathogenicity of coronaviruses, vaccinia virus ankara strain (mva), measles virus, rift valley fever virus and west nile virus [24, 25, 26, 27] . all these data, and the presence of an otherwise intact immune system in these mice [20, 22] suggest that ifnar (2/2) mice could be a good animal model to study btv infections and to evaluate vaccine strategies against this virus. here, we report the establishment of a new laboratory animal model suitable for the evaluation of vaccination strategies against btv. adult ifnar (2/2) mice support the in vivo growth of btv-4 after intravenous inoculation. btv-4 replicated in spleen, lung, thymus, and lymph nodes (popliteal, inguinal, mediastinal, and mesenteric) of ifnar (2/2) mice reproducing the tropism observed during calf and sheep infections. in addition, if-nar (2/2) mice vaccinated with a btv-4 inactivated vaccine show complete protection against a lethal dose of btv-4 in order to develop an adult murine model for btv infection in which mice showed disease symptoms, we tested the susceptibility of c57bl/6 and ifnar (2/2) mice to btv infection. since blood is the natural route of btv infection in ruminants, adult c57bl/6 and ifnar (2/2) mice (males, 8 weeks old) were infected intravenously (i.v.) with 10 6 pfus of btv-4. under these conditions, c57bl/6 mice did not show any disease symptom or death following viral infection. by contrast, ifnar (2/2) mice were susceptible to btv-4 infection (fig. 1a) , showing disease symptoms characterized by ocular discharges and apathy starting at 48 h.p.i. disease progression led to animal death within 60 h.p.i. the ld 50 value was obtained by i.v. inoculation with 10-fold dilutions of btv-4, resulting in a ld 50 value of 10 2.6 pfu (fig. 1b) . at low infectious doses (10 2 pfu or less) mice survived up to 21 days, at which point the experiment was terminated (data not shown). to determine virus dissemination, viral titers in blood samples after i.v. inoculation with 10 4 pfus of btv-4 were analyzed. according to the previous data, no viremia was detected in c57bl/6 mice (data not shown). in contrast, viremia was observed in ifnar (2/2) mice at day 2 post-infection ( fig. 2a) , with peak titers of 5610 4 pfu/ml at day 4 post-infection (p.i.), before animal death. ifnar (2/2) mice inoculated with 10 2 pfu did not show any viremia but titers up to 3610 4 pfu/ml were observed at 3 and 4 days p.i. in mice inoculated with 10 3 pfus. viral spread was determined in tissue samples. the first tissue to be reached by the virus was the spleen (fig. 2b) where infectious virus was detected as early as 24 h.p.i. (5610 3 pfu/gr), with viral titers increasing thereafter until death (reaching titers of 2610 6 pfu/gr). by 48 h.p.i. significant titers of btv-4 were detected in spleen, lung, thymus, and popliteal and mesenteric lymph nodes. at 72 h.p.i., titers up to 10 6 pfu/gr of btv-4 were recovered from the spleen, lung, thymus, and lymph nodes (popliteal, inguinal, mediastinal and mesenteric). no infectious virus was detected in liver, brain, heart, tongue, skin, and testicles at any time points examined, even when the tissues were analyzed by rt-pcr (data not shown). interestingly, the virus was not detected in the blood until 48 h.p.i. these results suggest that the virus leaks into the blood stream after replicating in the spleen. to determine whether ifnar (2/2) mice were susceptible to btv of different serotypes, ifnar (2/2) mice were inoculated with ten-fold serial dilutions of btv-8. mice infected with serotype 8 showed similar symptoms to those shown after infection with serotype 4. however, btv-8 showed higher virulence than btv-4 with only 10 pfus of btv-8 being enough to kill 100% of the mice at day 7 p.i. (fig. 3a) , while btv-4 was not lethal at the same infective dose (fig. 1b) . the viremia was determined after i.v. inoculation with 10-fold dilutions of btv-8 ( fig. 3b ). btv-8 was first detected in blood of mice inoculated with 10 pfus at day 4 post-infection, and the titer increased up to 2610 4 pfu/ml at day 5 p.i. titers up to 8610 3 pfu/ml were observed at 3 and 4 days p.i. in mice infected with 10 3 and 10 2 pfus, respectively. in the three dilutions of virus analyzed, viral titers increased thereafter until animal death. although btv-8 showed higher virulence than btv-4 in ifnar (2/2) mice, btv-8 and btv-4 titers in blood were equivalent, indicating that the higher virulence of serotype 8 is not due to a higher level of viral replication. these data suggest that ifnar (2/2) mice could be an adequate small animal model to study differences in virulence among btv serotypes. in order to study the pathological effects of the infection in the organs where btv replicates, histological analysis were performed on material of several organs extracted from btv infected and uninfected ifnar (2/2) mice at 48 h.p.i. gross pathological alterations were characterized by widespread oedema, haemorrhages especially in spleen and lungs, and enlarged spleen and lymph nodes. histological examination of lungs of btv infected mice showed hyperemia and increased septum size with infiltration of lymphocytes, inactivated macrophages and a few neutrophils (fig. 4) . the peribronchial and perivascular connective tissue contained a few infiltrating round cells (fig. 4) . these histopathological findings are consistent with bronchointerstitial pneumonia. there was also a moderate oedema in the alveolar cavity with presence of abundant alveolar macrophages and a few detached epithelial cells (descamative alveolitis). the infected spleen showed a marked lymphoid depletion with infiltration of neutrophils in the white pulp (fig. 4 ). this lymphoid depletion was also observed in the thymus as well as the loss of thymic architecture with the medulla and the cortex becoming not distinguishable (data not shown). the results suggest that btv-4 produces similar tissue lesions in ifnar (2/2) mice than in the natural host. immunized ifnar (2/2) mice are completely protected against a lethal btv-4 challenge all the previous data indicated that btv-4 caused a lethal infection in adult ifnar (2/2) mice. to provide further proof that btv-4 was the causative agent of the disease and death in these animals and that ifnar (2/2) mice are a good animal model for btv vaccination studies, vaccination protection experiments were performed. adult ifnar (2/2) mice were immunized with a zulvac-btv-4 inactivated btv-4 preparation. the vaccine was administered by two consecutive subcutaneous injections of the equivalent to 3610 5 tcid 50 of btv-4 at 3 weeks intervals. after the second immunization, vp2 antibody titers were determined by elisa. all immunized animals, but not the control animals, developed an antibody response (fig. 5a) indicating a successful immunization. in addition, the immunization induced neutralizing antibodies against btv-4 in ifnar (2/ 2) mice (vnt 1.5360.32) as detected by virus neutralization tests. three weeks after the second immunization, immunized and control ifnar (2/2) mice were challenged intravenously with 10 3 pfus of btv-4. while all nonimmunized animals died, 100% of the immunized animals were protected against a lethal challenge (fig. 5b) . infectious viral titers were analyzed in the blood of immunized and nonimmunized ifnar (2/2) mice by plaque assay after intravenous infection with btv-4 (fig. 5c ). no infectious viruses were detected in immunized mice; however, we observed titers up to 3610 4 pfu/ml in nonimmunized animals at day 5 post-challenge. in addition, we analyzed by rt-qpcr the presence of btv genomes in the blood of inmmunized and nonimmunized ifnar (2/2) mice challenged with btv-4. the results are summarized in table 1 . btv genomes were readily detected in nonimmunized mice at days three and four after btv infection (ct: 27-29) and increased (ct: 23-26) thereafter until the death of the animal. in contrast, the rt-qpcr reaction yield no positive results for the majority of the immunized mice (n = 5) at all days post-challenge analyzed (ct$38). viral genomes were detected in one of the vaccinated mice (ct: 30) at days four and five post-challenge and in two of them at day 5 (ct: 32). however, in these three immunized mice the ct was higher than in the nonimmunized mice and the presence of btv-genomes reverted to negative at day 7 post-challenge. overall, these data indicate that protective immunity was achieved after vaccination. these results confirm that btv-4 was the causative agent of disease and death of adult ifnar (2/2) mice. for years, different groups have tried to establish a laboratory animal model to facilitate the studies of pathogenesis, immune response and vaccination against btv. natural hosts, although excellent tools for studies of pathogenesis and vaccination, are expensive and required specialized laboratories. here, we characterize a new animal model based on adult ifnar (2/2) mice that support the in vivo growth of btv-4 and btv-8 after intravenous inoculation. a tremendous advantage of this mouse model is the availability of a wide variety of reagents that can be used to study many aspects of the immune response to the virus. in addition, we propose this animal model as an adequate system for testing btv vaccines, an important issue because the cost of testing new vaccines in target species is a major obstacle for laboratories and industries. the new mouse model for the study of btv infection has unique features that open the possibility to study in the same host-virus system susceptibility, virulence, immunobiology of infection, and vaccine efficacy, in ways that are not approachable with the natural host. btv infects newborn mice [11, 12, 13, 28] , but an adult animal model will be necessary to allow studies of acquired immune responses and vaccination against btv. different approaches have been followed to avoid this problem. the protective effects of baculovirus expressed btv-10 minor and non-structural proteins were assessed by measuring virus titers in the ovaries of mice challenged with homologous recombinant vaccinia virus (rvv). protection mediated by ctls specific for the btv minor or nonstructural proteins could not be evaluated by challenging mice with btv-10, as the virus does not cause disease in adult mice [29] . the infection of fetal mice with btv leads to severe cerebral malformation. infection of 2-week-old mice resulted in very limited multiplication without sequelae, and infection of 4-weekold mice did not show clinical disease and no viral multiplication was detected [30] . thus, the use of highly immature mice results in pathologies that do not resemble those found in the natural host. another aspect to consider is the route of inoculation that may determine the outcome of infection. in this work, we have used the intravenous inoculation to resemble the natural infection route by mosquito biting. thus, mice infected with btv i.v. develop clinical symptoms, as it happens in natural hosts. this makes the mouse model even more atractive to be used as a tool for studying immune response to btv and vaccination strategies. up-regulation of type i ifn is one of the earliest cellular responses upon contact with infectious agents. the rapid induction of type i ifn reflects the crucial role that this cytokine plays in the inhibition of viral spread before the generation of a specific immune response. given that viruses must at least partially circumvent the ifn response, it is not surprising that an inability to do this can restrict host range. there are many examples where this is known. for example, neither measles virus nor polio virus will replicate efficiently in mouse models unless the ifn system has been compromised [24, 31] . btv is a potent type i interferon inducer in mice and cattle [14, 16, 32] . our results show that btv is pathogenic for ifnar (2/2) mice mainly due to the lack of the ifn-i receptor, allowing the virus to circumvent the ifn-i response in the host. in addition, ifnar (2/2) mice serve as a good animal model for btv, reproducing many aspects of its natural host infection. first, ifnar (2/2) mice infected with btv showed viremia and disease symptoms, in contrast with c57bl/6 mice, which did not show them, even when these animals were infected at the highest viral dose tested (10 6 pfus/mouse). the appearance of viremia after inoculation in infected ifnar (2/2) mice was dependent on the viral dose, althought the highest titers observed in blood were not dose-dependent. second, the differential virulence of serotypes 4 and 8 were maintained in this animal model. some btv serotypes such as serotype 8, which recently caused infection in northern europe, exhibit enhanced virulence in cattle [5] , in contrast to btv-4 that usually exhibits subclinical infections in this species. in infected ifnar (2/2) mice, btv-8 killed 100% of the animals with a dose as low as 10 pfus per mouse. in contrast 10 3 pfus of btv-4 were needed to kill 100% of the mice. in addition, the virus titers found in the blood of infected mice were similar at the same virus doses for the animals infected with btv-8 than with btv-4, indicating that btv-8 exhibited a higher virulence than btv-4 in the ifnar (2/2) model, as is observed in cattle, one of the btv natural hosts. third, btv dissemination in ifnar (2/2) mice reproduced btv infection in cattle and sheep. high titers of btv are present in the lungs, precapsular and mesenteric lymph nodes, thymus, and spleen of infected calves [33, 34] . in ifnar (2/2) mice, the virus was detected in spleen before was isolated from other organs, including blood. release of btv from spleen was followed by an increased in viremia and dissemination to other organs (lymph nodes, lung and thymus), followed by its dissemination to lymph nodes, lung, and thymus after intravenous infection. this has been suggested also for calves [34] indicating a similar btv spreading pattern in ifnar (2/2) mice. histological analyses of btv infected mice at 48 h.p.i. showed many similarities with lessions described in natural hosts. the permeability disorders of the vascular system described for ruminants [35] is reflected in the petecheias observed in spleen in btv infected mice. the enlargement of lymph nodes reflects an ongoing immune response. however, the observed lymphoid depletion suggests that other cells are migrating to these organs or that a general aedema results in organ enlargement. the lungs are especially susceptible to permeability disorders of the vasculature induced by btv, and this is consistent in btv infected mice as well as in ruminants. in general, pathology in natural hosts has not been profoundly studied. the described pathology similarities between ifnar (2/2) mice and ruminants after infection with btv indicate that our mouse model may be a good tool to study new findings in btv pathology. the cost of testing new vaccines in target species is a major obstacle for laboratories and industries. for this reason, the intracerebral inoculation of newborn mice with btv vaccines has been used as an animal model to evaluate the level of attenuation of live attenuated btv vaccines [28] . our results show that adult ifnar (2/2) mice serve as a good animal model to test btv vaccines. even though the lack of type i interferon signals may have an effect in the development of acquired immune responses, our results and previous studies [20, 22, 24, 25, 26, 27] show that the ifnar (2/2) infection model is useful for the definition of effective vaccine candidates against btv. indeed, in our study all immunized animals developed an antibody response specific of btv-4 with the production of neutralizing antibodies against the same serotype, indicating a successful immunization. the protection mediated by inactivated whole btv vaccine in ifnar (2/2) mice infected with a lethal dose of btv-4 was complete, and 100% of the animals did not show any symptoms associated with infection or died. viremia was not detected in the blood of immunized animals after challenge with btv-4 when analyzed by plaque assay. the presence of btv genomes in the blood of immunized mice after btv-4 challenge analyzed by rt-qpcr, a more sensible method than the plaque assay, showed the presence of viral genomes in the blood of some of the immunized mice but in all the cases, the cts were higher than in the nonimmunized mice and those that were rt-qpcr positive reverted to negative at day seven post-challenge. similar rtqpcr results have been observed in cattle and sheep vaccinated with zulvac-btv-4 inactivated btv-4 preparation [36] these results strongly support the involvement of btv in the pathological processes described in this work and, moreover, suggest that the mouse model is adequate to evaluate vaccine candidates. future work will determine whether the ability to protect mice with other vaccine formulations mimics the capacity to protect the natural host. in summary, we have characterized a novel small animal model for btv infection based on ifnar (2/2) adult mice that reproduces many aspects of its natural host infection. this animal model may facilitate the understanding of the mechanisms of btv pathogenicity in its natural host and a faster advance in the development of new btv vaccines. mice were infected intravenously with different doses of virus. mice were examined for clinical symptoms daily. ld50 values were calculated by the method of reed and muench (1938) [37] , after inoculation of mice with 10-fold serial dilutions of virus (from 10 6 to 10 1 pfu/mouse). whole blood was collected in edta from all animals at regular intervals after inoculation. at varying times postinfection, several mice were sacrificed by perfusion with phosphatebuffered saline (pbs), and several organs (spleen, lung, thymus, liver, brain, heart, tongue, skin, and testicles), and lymph nodes (popliteal, inguinal, mediastinal, and mesenteric) were harvested. tissues were homogenized in pbs using a tissue lyser homogenizer (quiagen). the viruses were released from whole blood and homogenized tissues by three freeze/thaw cycles. the amount of infectious virus was measured by plaque assay on vero cells. groups of 6 ifnar (2/2) mice were immunized by two consecutive subcutaneous injections of either zulvac-btv-4 (1.5610 6 tcid 50 btv-4) inactivated btv-4 preparation (fort dodge veterinaria, s.a.) or phosphate-buffered saline (pbs) (controls), administered 3 weeks apart. mice were intravenously inoculated with 10 3 pfus of btv-4 (lethal dose) 3 weeks after the last immunization. mice were bled before each immunization and virus challenge. sera were tested for btv-4 neutralizing antibodies by virus neutralization test (vnt). samples from different tissues and organs were taken and fixed in 10% buffered formalin (ph 7.2) for histopathological studies. after fixation, samples were dehydrated through a graded series of alcohol to xylol and embedded in paraffin wax. sections of 4-imthick were cut and stained with hematoxylin and eosin (h & e) for histopathological analyses. maxisorp plates (nunc, usa) were coated with vp2 baculovirus expressed protein (164 ng per well) and incubated overnight at 4uc. plates were saturated with blocking buffer (pbs-0.05% tween 20 and 5% skimmed milk). the animal sera, diluted in blocking buffer were added and incubated for 1 hour at 37uc. after three washes in pbs-0.05% tween 20, plates were incubated for 1 hour at 37uc with an anti-mouse-hrp secondary antibody (biorad, usa) at a 1/2,000 dilution in blocking buffer. finally, after three washes in pbs-0.05% tween 20, the reaction was developed with 50 ml of substrate solution 3,39, 5,59-tetramethylbencidine liquidsupersensitive (tmb) (sigma) and stopped by adding 50 ml of 3n h 2 so 4 . results were expressed as optical densities (ods) measured at 450 nm. the vnt was used to determine neutralizing antibody titers against btv-4. for plaque reduction assays, 2 fold dilutions of sera were mixed with 100 pfu of btv-4, incubated for 1 hour at 37uc and then plated onto monolayers of vero cells. after 1 hour, agar overlays were added and the plates were incubated for 5 days. the titer was determined as the highest dilution that reduced the number of plaques by 50%. whole blood was collected in edta from all animals at regular intervals after inoculation and btv challenge. total rna was extracted from blood with tri reagent solution (ambion), according to the method recommended by the manufacturer. the real-time rt-qpcr specific for btv segment 5 was performed as described by toussaint et al. 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a simple method of estimating fifty percent endpoints bluetongue virus detection by two real-time rt-qpcrs targeting two different genomic segments we thank fort dodge veterinaria sa for facilitating the btv inactivated vaccine zulvac-btv-4. key: cord-303845-y6ws3u6x authors: delisle, sylvain; south, brett; anthony, jill a.; kalp, ericka; gundlapallli, adi; curriero, frank c.; glass, greg e.; samore, matthew; perl, trish m. title: combining free text and structured electronic medical record entries to detect acute respiratory infections date: 2010-10-14 journal: plos one doi: 10.1371/journal.pone.0013377 sha: doc_id: 303845 cord_uid: y6ws3u6x background: the electronic medical record (emr) contains a rich source of information that could be harnessed for epidemic surveillance. we asked if structured emr data could be coupled with computerized processing of free-text clinical entries to enhance detection of acute respiratory infections (ari). methodology: a manual review of emr records related to 15,377 outpatient visits uncovered 280 reference cases of ari. we used logistic regression with backward elimination to determine which among candidate structured emr parameters (diagnostic codes, vital signs and orders for tests, imaging and medications) contributed to the detection of those reference cases. we also developed a computerized free-text search to identify clinical notes documenting at least two non-negated ari symptoms. we then used heuristics to build case-detection algorithms that best combined the retained structured emr parameters with the results of the text analysis. principal findings: an adjusted grouping of diagnostic codes identified reference ari patients with a sensitivity of 79%, a specificity of 96% and a positive predictive value (ppv) of 32%. of the 21 additional structured clinical parameters considered, two contributed significantly to ari detection: new prescriptions for cough remedies and elevations in body temperature to at least 38°c. together with the diagnostic codes, these parameters increased detection sensitivity to 87%, but specificity and ppv declined to 95% and 25%, respectively. adding text analysis increased sensitivity to 99%, but ppv dropped further to 14%. algorithms that required satisfying both a query of structured emr parameters as well as text analysis disclosed ppvs of 52–68% and retained sensitivities of 69–73%. conclusion: structured emr parameters and free-text analyses can be combined into algorithms that can detect ari cases with new levels of sensitivity or precision. these results highlight potential paths by which repurposed emr information could facilitate the discovery of epidemics before they cause mass casualties. as the outbreak of severe acute respiratory syndrome (sars) [1] and the continued threat of novel influenza strains that could produce pandemics illustrate [2, 3] , human populations remain vulnerable to epidemics of acute respiratory infections (ari). these outbreaks must be discovered as soon as possible if control measures are to be implemented in time to curb morbidity, mortality and socioeconomic disruptions [4] . the increasing availability of electronic health data offers the opportunity to enhance public health surveillance for infectious diseases, a process that has historically relied on slow and incomplete paper-based reporting [5] . early electronic surveillance efforts have collected codified diagnoses, chief complaints, school absenteeism, over-the-counter medication sales and other independent data streams to detect the early stages of infectious illnesses, when mild signs and symptoms may not alarm clinicians [6, 7, 8, 9, 10, 11] . while the utility of these pre-diagnostic, so-called ''syndromic'' surveillance systems (sss) has thus far been less than persuasive, there is now increasing evidence that the electronic transfer of diagnostic test results can lead to more complete and timely reporting of notifiable diseases to the public health authorities [12, 13, 14] . these results encourage the continued search for new approaches aimed at improving the timeliness and usefulness of surveillance alerts. by assembling a rich array of clinical data around individual patients, a surveillance system rooted in the electronic medical record (emr) could provide early and nuanced illness detection compared to current-generation sss. notwithstanding the challenges of protecting patient privacy, an emr-based surveillance system could also support the efficient flow of information necessary to promptly investigate alerts and manage evolving epidemics [15, 16, 17] . to gain insight into the potential of the emr as a pre-diagnostic surveillance tool, we asked how information automatically retrieved from a comprehensive emr could be arranged to detect patients with symptoms suggestive of ari. more specifically, we hypothesized that structured and freetext clinical entries into the veterans administration's (va) comprehensive emr could be automatically extracted, transformed and combined to improve ari detection compared to the sole use of icd-9 diagnostic codes, a common basic avenue to electronic disease detection. the maryland (n = 5,127) and the utah (n = 10,250) study samples were representative of their respective outpatient populations and included mostly male veterans. they differed in racial composition: the utah population was more than 90% caucasian, whereas almost half of the maryland veterans were african american (table 1) . our review of the 15,377 sampled records identified a total of 280 ari cases. ari incidence during the 6-month study period was lower at the ut site (1.3%) than at the md site (2.8%). on average, ari cases were younger than the study population at both sites, with patients over the age of 70 representing 46% of the study sample but only 21% of ari cases ( table 1 ). the distribution of ari cases across outpatient or telephone care areas is shown in table 2 . two-thirds of ari cases were seen in the emergency room or in the same-day/walk-in primary care areas, where they made up ,8% of the visits. comparatively, ari cases amounted to less than 1% of visits to routine care areas. cough was the most commonly documented symptom of ari (88% of cases), followed by fever/chills/night sweats (58%) and sore throat (45%). eight (8) percent of ari patients were febrile at the time of the encounter. table 3 displays the frequency with which these and other less common symptoms and signs occurred, either alone or in selected combinations. note that gastrointestinal symptoms were common complaints, even though they were not part of the ari case definition. six of the emr parameters considered were either not found or present in only one ari patient, and did not contribute to ari detection at either site. these included orders of diagnostic tests for influenza or for other respiratory tract pathogens, prescriptions for antivirals, antidiarrheals or antiemetics, and orders for sinus xrays. the overall frequency at which other emr parameters occurred at both sites is shown in table 4 . providers wrote prescriptions aimed at relieving cough or cold symptoms in 65% of ari patients, and blood tests or imaging in 27%. rates at which cbc, chest imaging and blood cultures were obtained were low (table 4 ) but increased with the number of abnormal vital signs ( fig. 1) . cdas that did not include icd-9 codes achieved a peak sensitivity of 80% and specificity of 69%, and were not retained (data not shown). table 5 reports the composition and performance of illustrative cdas that used icd-9 codes either alone (cdas 1-3) or a combination with other structured emr parameters (cda 4-9). icd-9-only cdas used by the biosense (cda 1) or the essence (cda 2) sss during our study period could be improved by adding three ari-compatible icd-9 codes (acute maxillary sinusitis (461.0), acute sinusitis nos (461.9), and bronchitis nos (490)) to the essence code set. the resulting icd-9 code set, labeled ''va'' (cda 3), was used as a starting the text-only cda was as sensitive as a cda that best combined icd-9 codes with other structured clinical parameters ( table 6 , compare cda 10 to cda 8). the ''va icd-9 codes'', ''cough remedies'' and ''text'' cda components could be coupled through an or operand to bring sensitivities up from 88% (cda 10) to 97-99% (cdas 11 and 12). while this maximized the estimated area under the roc curve, gains in sensitivity were accompanied by losses in specificity (from 93 to 89%, cda 10 to cda 12). because of the low incidence of ari in this broad outpatient population (an average of 1.8% during our 6-month study period), these losses in specificity halved the positive predictive value (ppv) that had originally been obtained with cda that used icd-9 codes alone ( table 6 , compare cda 3 to cda 12). we could design cdas with high ppv (47-60%) using only structured emr parameters, for example by requiring that both the ''icd-9 codes'' and the ''temperature $38uc'' parameters be satisfied (cda 13). however, the resulting cdas exhibited low sensitivities (e.g. 6%, cda 13, and 38%, cda 14) . cdas that had to satisfy both a query of structured emr parameters as well as the text analysis exhibited similarly high ppvs (52-54%), but retained much higher sensitivities (69%, cda 15, and 73%, cda 16). the centers of disease control and prevention (cdc) defines an influenza-like illness (ili) as an acute febrile illness (temperature of $37.8uc) accompanied by a cough and/or a sore throat. thus defined, patients with ili represent a subset of our broad ari population. a text-only cda achieved a higher sensitivity for ili than its icd-9-only counterpart ( table 7 , 92%, cda 18 vs. 79%, cda 17) . despite specificities of 91-96%, both detection approaches yielded ppvs in the 2-3% range. as could be expected, restricting cases retrieved through icd-9 codes or text analysis to those patients who also have a temperature of at least 37.8uc increased the ppv more than 10-fold (cdas 19 and 20) without decreasing sensitivity. selecting febrile patients who also satisfied the [icd-9-or-text] clause (cda 21) identified all ili cases and yet maintained a ppv of 34%. alternatively, requiring patients to simultaneously satisfy the icd-9 codes, text and temperature parameters maximized the ppv (68%) while keeping sensitivity above 70% (cda 22). in this work, we combined selected structured emr entries with a computerized analysis of free-text documentation to detect reference patients with acute respiratory infections with sensitivities up to 99%, or ppv up to 68%. the results support our working hypothesis that judicious use of information contained in the emr can improve early disease detection compared to the sole use of diagnostic codes, and raise the possibility that harnessing the emr as a data source may improve the overall performance of automated outbreak detection systems. case definitions used for influenza surveillance vary widely [18] , reflecting both the variability of influenza's clinical presentation and differing views on what distinguishes an influenza-like illness from other ari [19, 20] . absent a standard, we crafted a case definition that would identify a broad population of patients with ari. most (78%) of our ari patients had normal vital signs and thus probably suffered from mild, uncomplicated ari [21] . nevertheless, twothird of them felt ill enough to visit urgent or same-day care areas. thus, the majority of patients who satisfied our case definition were seeking health services despite a mostly mild illness. these patients lie at the crosshair of syndromic surveillance. we used a manual abstraction of respiratory symptoms from the emr as a reference standard to uncover cases of ari. this casefinding method may have underestimated the absolute incidence of ari encounters, as busy providers may not document symptoms that are impertinent to a focused outpatient visit. nevertheless, the abstraction, which was done on a large random sample of records, relied on information committed to a real-world emr and thus represents a realistic benchmark against which to compare the performance of ari detection algorithms relative to each other. the reference review allowed an evaluation of a stalwart component of sss, icd-9 diagnostic codes. our data support the suggestion that an early step toward system enhancement is to scrutinize the icd-9 codes used to categorize cases [22] : additions of only a few codes improved both detection sensitivity and specificity. while our observed level of performance may not be duplicated in health systems where diagnostic codes are assigned by non-providers and/or have reimbursement repercussions, our findings reinforce results obtained in adult and pediatric emergency room populations [23, 24] . note that the icd-9 code parameter was retained statistically in all of the most successful ari and ili detectors, suggesting that diagnostic codes are not made redundant with the availability of a broad array of structured emr parameters. our results therefore suggest that icd-9 codes represent a valuable, perhaps indispensible component of automated ari detection algorithms, and bolster their widespread use. some of the non-icd-9 parameters considered in this study had previously emerged as potentially useful for ari surveillance. in particular, sales of selected over-the-counter or prescription medications [25, 26, 27, 28, 29, 30, 31, 32] and requests for diagnostic tests for influenza or for other respiratory pathogens [12, 33] have been shown to correlate with the seasonality of influenza and respiratory syncytial virus infections. our study revisits similar parameters, adds new ones not yet formally investigated, but evaluates them in a different way i.e. for the accuracy with which they can detect reference ari cases not already found by alternative parameters. this evaluation was made possible because emr-derived parameters were not utilized as independent data streams. rather, emr parameters were related to each other at the single-patient level. amongst a broad field of candidate structured emr entries, we found two that could complement icd-9 codes to detect patients with ari: new prescriptions directed at the symptom of cough and an elevation in measured body temperature recorded in an emr field dedicated to vital signs. with 88% of ari patients experiencing a new onset of cough, it is not surprising that prescriptions for cough remedies uncovered new ari cases. while these results lend further support to the use of medications-related data elements for biosurveillance [29, 30, 31] , the practical difficulty is to devise database queries that, over time, will continue to reflect the concept of ''cough remedies''. cough suppressants belong to a variety of drug classes (opioids, non-opioid antitussives, decongestants, antihistamines) that are often variably combined with each other and with other agents to relieve flu-like symptoms. efforts to standardize competing drug terminologies and to develop methods to automatically update medication groupings [27, 28] must be encouraged if symptom-relief medications are to be routinely incorporated into surveillance systems. though significant statistically, the added contribution of the non-icd-9 structured emr parameters to ari detection was admittedly modest. we attribute this low marginal utility at least in part to the mildness of the disease in the majority of our ari patients. to wit, providers did not order any blood tests or chest imaging in 73% of these encounters. our efforts at devising cdas that target the subset of ari patients with ili hint at how fruitful incorporating structured non-icd-9 emr parameters may become as disease severity increases. for example, a parsimonious ili cda (va icd-9 codes and temperature .37.8uc, cda 19) increased ppv by an order of magnitude with little losses in sensitivity compared to its icd-9 codes-only counterpart. while we acknowledge that an icd-9 code set optimized for ili could narrow this performance gap, our observations that more diagnostic tests are ordered with increasing vital signs abnormalities ( fig. 1 ) raise the prospect that as disease severity increases, so will the value of the structured emr data. in contrast to the potential utility of the ''cough remedies'' and the ''temperature'' parameters, specific tests and therapies targeting viral ari were not utilized, and thus did not contribute detection value. perhaps the simplest explanation for why providers didn't order tests for viral respiratory pathogens or anti-influenza drugs is that neither were felt to offer a meaningful therapeutic impact in non-hospitalized patients during our study period. because these practice patterns could change quickly with the emergence or fear of a more severe illness, and for the important situational and logistical insight they may provide during an epidemic, we would certainly retain disease-specific diagnostic and therapeutic emr entries in ari cdas meant for an operational surveillance system. for surveillance purposes, text analyses have been applied to limited data sources within the emr, including ''chief complaint'' fields [34, 35, 36, 37, 38] , laboratory or imaging reports [34, 35, 38, 39, 40] or dictated hospital discharge summaries [41, 42] . we are aware of only one published study where ari surveillance was performed by applying text analysis to the full outpatient notes typed in by the providers themselves [43] . this study illustrated the feasibility of using the full narrative text for surveillance in the ambulatory setting, and showed a temporal correlation between total daily ili counts obtained using text analysis, queries of structured emr data, categories of chief complaints or influenza isolates. our work extends this knowledge in two significant ways. first, our reference review allowed us to provide insight into the performance of free-text mining per se. despite ongoing challenges with non-standard grammar and abbreviations, spelling mistakes, lack of punctuation, copy-and-pasting, personal templates and checklists, our simple approach to free-text analysis discovered ari cases with a sensitivity (88%) and a specificity (93%, cda 10) not dissimilar to what could be reached with structured parameters (e.g. cda 8) . second, the relational nature of emr data allowed us to observe the extent to which free-text and structured data sources could complement each other for case detection. on the one hand, we found that combining the results of the two mining approaches through an ''or'' logical operand yielded near-perfect sensitivity, albeit at the cost of a much reduced ppv. these results support the common-sense belief that, at least with a disease characterized by relatively mild symptoms, significant information resides exclusively in the provider's note. on the other hand, we found that combining the two approaches through an ''and'' logical operand could increase ppvs well above 50% while retaining sensitivities in the 70% range (cdas 15, 16, 22) . these results suggest that cross-validating information obtained from structured data with that obtained from clinical narrative can reduce false-positive findings from either data sources and improve the noise environment through which a surveillance system must operate to uncover disease clusters. whether or not the achievable gains in signal-to-noise ratio will outweigh the losses in detection sensitivity to improve the overall ability of the surveillance system to detect disease outbreaks will have to be determined empirically. several factors, some of which have already been mentioned, may limit the generalizability of our results: 1) factors related to the performance of our study at the va health care system: a) the veterans study population is mostly male and excludes the pediatric population, a key target for ari surveillance [44] ; b) veterans health care utilization may differ from that observed in uninsured or privately insured individuals; c) clinical practices, documentation and coding habits by va practitioners may differ from those observed in solo or group practices or in health systems subject to different financial or quality-control incentives; 2) factors related to our study period: optimal cdas could differ outside the respiratory infection season, or during periods of heightened apprehension for an influenza epidemic; 3) factors related to our iterative cda development process, which may have over adapted cdas to va's particular emr implementation and to our sample dataset in particular, this despite our efforts to maintain a separation between development and validation data subsets; 4) factors related to our text mining approach: a) we did not employ a spell checker prior to applying the negex algorithm. instead, we added common misspellings of negation and search terms (e.g. ''deneis'' instead of ''denies'', ''cocuphing'' instead of ''coughing'') identified during the chart review process. thus, unencountered misspellings are left out of our final umls concept table and negation list; b) our adaptation of the negex algorithm is specific to the types of emr note from only two va medical centers. further adaptations could therefore be needed for text documents extracted from the vista systems at other va facilities. our results confirms that it is feasible to harness the emr for biosurveillance purposes and that judicious parameter selection coupled with free-text analyses can yield case-detection tools with performance characteristics not previously available through diagnostic codes only. much more work remains to be done both to replicate our results under different health delivery environments and to determine which case-detection strategy shortens outbreak detection delay and/or minimizes the burden that false alarms impose on public health professionals. this study was approved by the institutional review boards (irbs) of the participating va health systems and those of the university of maryland, the university of utah and the johns hopkins university. the risks were limited to information confidentiality involving data generated during the course of routine patient care. the study did not interfere with such care and thus did not adversely affects the rights and welfare of the participants. the irbs granted a waiver of consent on that basis and because, given the large number of participants, the work would not have been practicable otherwise. the data collected are specified in the ''description of procedures'' section below. we randomly sampled outpatient clinical encounters from october 1, 2003 through march 31, 2004 at va maryland (vamhcs) and at va salt lake city (vaslchcs) health care systems. while we aimed at sampling as broad an outpatient visit population as possible, it was nevertheless not useful to include specialized areas of care that neither diagnose nor treat ari, such as psychiatry or ophthalmology. we therefore limited our sampling to outpatient care areas where at least two encounters were assigned icd-9 codes related to ari (see below) during the study period. individual patients were sampled only once. reference record review. a manual review of emr entries constituted the reference standard for ari case detection. the unit of analysis was the calendar day of an index outpatient encounter. a trained abstractor reviewed all emr entries during the calendar day of each index encounter for documentation indicative of ari. predefined ari symptoms and signs were recorded individually on an abstraction instrument (ms access, microsoft corp., redmond wa). ari was defined as follows: [1) positive influenza culture/antigen; or 2) any two of the following, of no more than 7 days duration: a) cough; b) fever or chills or night sweats; c) pleuritic chest pain; d) myalgia; e) sore throat; f) headache] and [3) illness not attributable to a noninfectious etiology]. all uncovered ari cases and a 10% random sub-sample of negative records were re-reviewed by a physician, who validated the ari-defining elements, and who could also access any part of the emr for documentation that would indicate that a non-infectious disease could best explain the current illness. a panel of specialists in pulmonary and infectious diseases arbitrated cases where there was disagreement. sample size was adjusted so that we could reach ,140 reference ari cases at each study site, a goal estimated by the need to have sufficient power to perform a regression analysis that included our planned candidate explanatory variables (epiinfo, version 2.2, centers for disease control and prevention). development of ari case-detection algorithms (cda) that use structured emr parameters. practicing clinicians from the research team systematically reviewed and adjucated structured or semi-structured emr parameters that could potentially identify patients with ari and that would be available within 24 hours of an index encounter. the parameters chosen were: a) icd-9 diagnostic code(s) included in the existing ''respiratory'' groupings from sss of national scope (either the centers for disease control and prevention (cdc) biosense [45] or the department of defense's essence [46] systems) or the icd-9 code for fever (780.6). note that icd-9 codes are assigned by va health care providers when they complete their clinical note for an outpatient visit in emr, and are thus rapidly available for surveillance; b) vital sign abnormalities (temperature $38uc, respiratory rate $22 breath per minute, heart rate $100 beats per minute); c) orders for tests (complete blood count (cbc) with or without differential cell count, influenza antigen or culture, diagnostic tests for respiratory organisms other than influenza (respiratory syncytial virus, adenovirus, legionella), streptococcal throat screen, sputum culture or gram stain, gram stain for other respiratory specimens, blood cultures); d) requests for diagnostic imaging (chest x-ray, chest computerized tomography, any respiratory sinus imaging); and e) new prescriptions (none similar in the last 90 days), selected from the va national formulary and grouped into the following parameters by expert consensus: cough remedies (from va national formulary (vanf) drug classes re-200, -301, -302, -502, -503, -507, -508, -513, -516, or codeine), ''other cold remedies'' (from vanf cn-900, ms-102, nt-100, -200, -400, -900, re-99, -501); antiemetics (from vanf ga-700), antidiarrheals (from vanf ga-400), influenza-targeting antivirals (neuraminidase inhibitors or adamantanes), and alternative groupings of antibacterials (i: 33 antibiotics that could be prescribed for ari (from vanf am-051, -052, -053, -101, -102, -103, -200, -250, -300, -650 and -900); ii: 11 antibiotics commonly prescribed for ari (clarithromycin, erythromycin, azithromycin, clindamycin, amoxicillin/clavulanate, amoxicillin, penicillin v, gatifloxacin, levofloxacin, cefaclor, tetracycline and doxycycline); iii: the three antibiotics most commonly associated with an encounter ascribed an icd-9 code from the essence ''respiratory'' grouping at both sites (amoxicillin/clavulanate, azithromycin and clarithromycin). all of the data elements required to construct the above emr parameters in the study population were transferred from the veterans integrated service technology architecture (vista) hierarchical database to a structured query language (sql) relational database using the mumps data extractor software (strategic reporting systems inc., peabody, ma). data elements were included if they were entered in the emr within the calendar day of the index encounters. subsequent data transformations and database queries were implemented using sql server 2000 (microsoft corp., redmond, wa). ari cdas were developed independently for each study site. for a given site, the clinician-selected emr parameters were reduced to include only those that contributed significantly to detection of reference ari cases using backward elimination logistic regression with 95% confidence intervals [47] . supplemental statistics in those analyses included akaike's information criterion, wald's chi square test, likelihood ratio test statistic, and drop-in-deviance. explanatory parameters were explored for multicollinearity with variance inflation factors and spearman correlations. the performance of retained cdas was summarized with standard statistical descriptors (sensitivity, specificity, positive and negative predictive value, and an estimate of the area under the receiver-operating characteristic (roc) curve [48] ). bootstrap analysis with 95% confidence intervals was conducted to test the reliability of the most successful ari cdas [49] . for a given cda, the dataset was divided by placing a random sample of 80% of the data in a first subset, and the remaining 20% in a second subset. the previous analyses for the selected case detectors were performed on the 80% subset. the statistical descriptions for the case detectors in each dataset were compared and the results were found to be similar to those for the entire dataset. the case detectors were then used to predict the ari cases in the 20% subset. data for each case detector was resampled 100 times to ensure consistent results. all statistical analyses and roc estimates were performed using splus (version 6.1, insightful corp., seattle, wa). development of ari cdas that use unstructured clinical text. of the many free-text data sources within the emr, we focused on the notes typed in by providers to document outpatient visits. our goal was first to apply simple methods using character string matching coupled with negation detection to extract information documenting ari symptoms. if a clinical note related to an index visit contained non-negated strings related to two or more symptoms from our ari case definition, then the index visit was labeled as ''positive'' for the presence of ari. to develop a list of search strings, we began by mapping ari symptoms from our case definition to the unified medical language system (umls) using the national library of medicine umls metathesaurush search tool [50] . the umls incorporates many common source vocabularies (including icd-9, mesh, and snomed) and maps concepts to a standard vocabulary represented by concept unique identifier (cui) codes [41, 50, 51, 52, 53, 54] . we examined all of the umls-supplied lexical variants and semantic types related to ari [50] to build the final list of strings. this list included 186 synonyms, term variants, and common misspellings (table s1) . we searched for the text strings identified above in the full text of all emr clinical notes completed on the calendar day and related to each index encounter (76,500 notes related to 15,377 encounters). to determine if concepts identified by the text strings were affirmed or negated, for example whether a patient was ''coughing'' or denied ''coughing'', we used the publicly available negex version 2 algorithm [55] . we sought to improve the performance of the native algorithm by iteratively reviewing 10-15% of notes associated with false negative and false positive cases, then correcting problems with negation detection. reusable text templates and checklists, commonly imported into va note documents, drove most of the modifications to the negex algorithm, which included: 1) adding pre-processing steps to remove extraneous white spaces, carriage returns and line feeds; 2) identifying special characters commonly associated with imported templates (e.g. series of ----------------, ********** to indicate section breaks) or indicating embedded data-mining software objects used to automatically retrieve vital signs or medication lists; 3) identifying headings of templates that often contained a list of non-negated ari-related strings (e.g. ''cough assessment'', ''clinician instructions'', ''problems to report to your doctor'', ''needed for'', ''observe for''); 4) modifying the term list used by negex for possible negation status (e.g. adding the term ''absent''); 5) using regular expressions to identify non-textual patterns where the concept identified was either affirmed or negated by special characters or abbreviations (e.g. neg, pos, a novel coronavirus and sars update on avian influenza a (h5n1) virus infection in humans emergence of a novel swine-origin influenza a (h1n1) virus in humans strategies for containing an emerging influenza pandemic in southeast asia outbreak investigations-a perspective what is syndromic surveillance? essence ii and the framework for evaluating syndromic surveillance systems the rods open source project: removing a barrier to syndromic surveillance generating a reliable reference standard set for syndromic case 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databases: a feasibility study indexfinder: a method of extracting key concepts from clinical texts for indexing evaluating the umls as a source of lexical knowledge for medical language processing a simple algorithm for identifying negated findings and diseases in discharge summaries the authors would like to thank shawn loftus and robert sawyer, md for technical assistance with the institutional databases, shobha phansalkar for help with automated text analyses, guillermo giangreco, md for help with the validation of the record review, and kathleen speck for coordinating our scientific and administrative activities. key: cord-295352-b8kztgt8 authors: maksimowski, nicholas; williams, vanessa r.; scholey, james w. title: kidney ace2 expression: implications for chronic kidney disease date: 2020-10-30 journal: plos one doi: 10.1371/journal.pone.0241534 sha: doc_id: 295352 cord_uid: b8kztgt8 angiotensin-converting enzyme 2 (ace2) has been implicated in the pathogenesis of chronic kidney disease (ckd) and is a membrane receptor for severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the virus responsible for coronavirus disease (covid-19), whereas transmembrane protease, serine 2 (tmprss2) is involved in viral attachment. together, tissue expression of ace2 and tmprss2 may determine infection. sex, age, body mass index (bmi), and ckd are clinical risk factors for covid-19 severity, but the relationships between kidney ace2 and tmprss2 expression and these clinical variables are unknown. accordingly, we obtained renal tubulointerstitial and glomerular microarray expression data and clinical variables from healthy living donors (hld) and patients with ckd from the european renal cdna bank. ace2 expression was similar in the tubulointerstitium of the two groups, but greater in females than males in hld (p = 0.005) and ckd (p < 0.0001). ace2 expression was lower in glomeruli of ckd patients compared to hld (p = 0.0002) and lower in males than females. tmprss2 expression was similar in the tubulointerstitium but lower in glomeruli of ckd patients compared to hld (p < 0.0001). there was a strong relationship between ace2 and tmprss2 expression in the glomerulus (r = 0.51, p < 0.0001). in ckd, there was a relationship between tubulointerstitial ace2 expression and estimated glomerular filtration rate (r = 0.36, p < 0.0001) and age (r = -0.17, p = 0.03), but no relationship with bmi. there were no relationships between tmprss2 expression and clinical variables. genes involved in inflammation (ccl2, il6, and tnf) and fibrosis (col1a1, tgfb1, and fn1) were inversely correlated with ace2 expression. in summary, kidney expression of ace2 and tmprss2 differs in hld and ckd. ace2 is related to sex and egfr. ace2 is also associated with expression of genes implicated in inflammation and fibrosis. angiotensin-converting enzyme 2 (ace2) modifies angiotensin peptide metabolism and affects the progression of chronic kidney disease (ckd) [1] . in the kidney, ace2 is analyses were performed using graphpad prism 7 (graphpad software, san diego, ca). data are presented as the mean ± sem, unless otherwise stated. tubulointerstitial and glomerular median-centered log2 mrna expression of ace2 or tmprss2 from renal biopsy samples were compared in ckd and hld, as well as in male and female subgroups. ace2 or tmprss2 expression levels were correlated against estimated glomerular filtration rate (egfr), age, and bmi. ace2 expression was also correlated against expression of monocyte chemoattractant protein 1 (mcp-1 or ccl2), interleukin 6 (il6), tumor necrosis factor alpha (tnf), collagen, type i, alpha 1 (col1a1), transforming growth factor beta 1 (tgfb1), fibronectin (fn1), and tmprss2 in the ckd population. for comparisons between two groups, two-tailed p values were determined by χ 2 tests for categorical variables and unpaired student's t test for continuous variables. for comparisons between more than two groups, p values were determined by one-way analysis of variance. pearson's correlation coefficient (r) with two-tailed p values were calculated. linear regression was used to generate the line of best fit with 95% confidence intervals. there were 31 subjects in the hld tubulointerstitial cohort (table 1) including 10 females and 12 males. the sex was not specified for 9 subjects. the average age was 47.3 ± 2.4 years with a mean egfr mdrd of 104.3 ± 6.5 ml/min/1.73 m 2 . the ckd tubulointerstitial cohort (table 1) included 170 subjects with a range of primary kidney diseases ( table 2 ) and included 75 females and 95 males. the average age was 46.8 ± 1.4 years with a mean egfr mdrd of 66.4 ± 2.9 ml/min/1.73 m 2 . there were 21 subjects in the hld glomerular cohort (table 1 ) including 9 females and 12 males, and the average age was 47.2 ± 2.6 years with a mean egfr mdrd of 105.4 ± 6.7 ml/min/ 1.73 m 2 . the ckd glomerular cohort (table 1) included 178 subjects with a range of primary we compared ace2 mrna expression in both kidney compartments in the two cohorts and examined ace2 mrna expression based on sex. tubulointerstitial ace2 mrna expression was similar between the two groups, with more variability in ckd ( fig 1a) . ace2 mrna expression in the tubulointerstitium was greater in females compared to males in ckd (p < 0.0001; fig 1c) and in hld (p = 0.005; fig 1e) . in the glomerular compartment, ace2 mrna expression was lower in ckd compared to hld (p = 0.0002; fig 1b) , due primarily to a difference between males and females in the ckd cohort (p = 0.04; fig 1d) that was not observed in hld ( fig 1f) . mean values for ace2 mrna expression in both the tubulointerstitial and glomerular compartments were similar in all of the disease categories represented in the ckd cohort (s1 fig) . there was a relationship between ace2 mrna expression and egfr in the tubulointerstitium (r = 0.36, p < 0.0001; fig 2a) and the glomerulus (r = 0.16, p = 0.03; fig 2b) . ace2 mrna expression was related to age in the tubulointerstitium (r = -0.17, p = 0.03; fig 2c) but not glomeruli ( fig 2d) . ace2 mrna expression was not related to bmi in either compartment ( fig 2e and 2f ). ccl2 mrna expression was related to ace2 mrna levels in the tubulointerstitium (r = -0.39, p < 0.0001; fig 3a) and glomeruli (r = -0.18, p = 0.02; fig 3b) . il6 mrna expression was also related to ace2 mrna levels in the tubulointerstitium (r = -0.20, p = 0.008; fig 3c) and glomeruli (r = -0.17, p = 0.02; fig 3d) . tnf mrna levels were also related to ace2 in the tubulointerstitial (r = -0.22, p = 0.005; fig 3e) and glomerular (r = -0.25, p = 0.0007; fig 3f) compartments. there was a relationship between col1a1 mrna expression and ace2 mrna levels in the tubulointerstitium (r = -0.35, p < 0.0001; fig 4a) and glomeruli (r = -0.27, p = 0.0003; fig 4b) . tgfb1 mrna expression was also related to ace2 in the tubulointerstitium (r = -0.30, p < 0.0001; fig 4c) and glomeruli (r = -0.24, p = 0.001; fig 4d) . fn1 mrna expression was related to ace2 in both the tubulointerstitial (r = -0.38, p < 0.0001; fig 4e) and glomerular (r = -0.35, p < 0.0001; fig 4f) compartments. we compared tmprss2 mrna expression between hld and patients with ckd in both the glomerular and tubulointerstitial compartments. we also examined tmprss2 mrna expression on the basis of sex. tubulointerstitial tmprss2 mrna expression was similar between the hld and ckd groups ( fig 5a) . in both hld and patients with ckd, tmprss2 mrna expression in the tubulointerstitium was similar in females and males (fig 5c and 5e ). in the glomerular compartment, tmprss2 mrna expression was lower in ckd compared to hld (p < 0.0001; fig 5b) , although there were no sex differences in either group (fig 5d and 5f ). there were no relationships between tmprss2 mrna expression and egfr in the tubulointerstitium ( fig 6a) or glomerulus (fig 6b) . in a similar manner, tmprss2 mrna expression was not related to either age ( fig 6c and 6d ) or bmi (fig 6e and 6f ). there was no correlation between tmprss2 and ace2 expression in the tubulointerstitial compartment ( fig 7a) , but there was a strong positive correlation between ace2 mrna expression and tmprss2 mrna expression in the glomerulus (r = 0.51, p < 0.0001; fig 7b) . we compared the expression of ace2 in the hld to the subgroup of ckd subjects with dn. the dn tubulointerstitial cohort included 17 subjects with 5 females and 12 males ( table 3 ). the average age was 58.3 ± 2.6 years with a mean egfr mdrd of 44.3 ± 6.0 ml/min/1.73 m 2 . the dn glomerular cohort included 12 subjects (table 3 ). there were 4 females and 8 males, and the average age was 54.8 ± 3.2 years with a mean egfr mdrd of 52.9.6 ± 8.8 ml/min/1.73 m 2 . diabetic subjects had lower egfr values compared to the hld (p < 0.0001). we compared ace2 mrna expression in both kidney compartments in subjects with dn and hld. in subjects with dn, we also explored whether ace2 mrna expression differed between females and males. in the tubulointerstitium, ace2 mrna expression was similar between the two groups ( fig 8a) . glomerular ace2 mrna expression was lower in dn fig 8b) . ace2 expression in either kidney compartment was similar between males and females with dn ( fig 8c and 8d ). in both the tubulointerstitial ( fig 9a) and glomerular ( fig 9b) compartments, there were weak positive associations between ace2 expression and egfr, although neither reached statistical significance, likely based, in part, on the number of subjects available for analysis (n = 17 for the tubulointerstitium and n = 12 for the glomerulus). opposing trends were observed in the tubulointerstitial and glomerular compartments regarding the relationships between ace2 expression and age (fig 9c and 9d ) and ace2 expression and bmi ( fig 9e and 9f ), but only the relationship between ace2 expression and age in the glomerulus reached statistical significance (r = 0.59, p < 0.05; fig 9d) . ccl2 mrna expression was inversely correlated to ace2 mrna levels in the tubulointerstitium (r = -0.50, p = 0.04; fig 10a) but not in glomeruli (fig 10b) of patients with dn. tnf mrna expression was also related to ace2 mrna levels in the tubulointerstitium (r = -0.52, p = 0.03; fig 10c) but not in glomeruli ( fig 10d) . there were negative correlations between ace2 expression and col1a1 expression in both the tubulointerstitial ( fig 10e) and glomerular ( fig 10f) compartments, although neither reached statistical significance. interestingly, opposing trends were seen in the relationships between ace2 expression and tgfb1 mrna expression in the tubulointerstitial ( fig 10g) and glomerular (fig 10h) compartments. the relationship between ace2 and tgfb1 mrna expression in the tubulointerstitium reached statistical significance (r = -0.55, p = 0.02; fig 10g) . ace2, found mainly in the brush border of the proximal tubule and the glomerular podocyte, is a determinant of experimental kidney injury [1] [2] [3] 33] . there are few studies of ace2 expression in ckd in humans [26, 28, 29], and interestingly, studies have shown that ace2 is a receptor for sars-cov and sars-cov-2 [12, 34] . accordingly, we accessed publicly available data from the ercb (nephroseq.org) to study the effect of ckd on ace2 expression and to examine the relationships between clinical variables and ace2 expression. given the role of the membrane-bound protease, tmprss2, along with ace2, in sars-cov-2 infection [12, 13] , we also investigated tmprss2 expression in the glomerular and tubulointerstitial compartments of the kidney. in addition, loss of ace2 accelerates the progression of experimental kidney disease [35-37]; therefore, we also studied the relationships between kidney ace2 expression and genes implicated in inflammation and fibrosis in ckd. our first major observation was that mean levels of ace2 expression were similar in the kidney tubulointerstitium in ckd and hld but lower in glomeruli in ckd. there was considerable variability in ckd, likely due, in part, to the variety of kidney pathologies in the ckd cohort. we then examined the impact of sex on ace2 expression. ace2 expression was lower in males than in females in both kidney compartments. the ace2 gene is located on the x chromosome [38] , but x inactivation should limit any effect on ace2 expression. male sex is a risk factor for the progression of ckd [39] . although generally attributed to the effect of sex hormones on kidney cells, differences in ace2 expression may also contribute, at least in part, to this differential risk. our second set of observations was related to the associations between ace2 expression and clinical variables. we first examined the relationship between ace2 expression and egfr in the ckd cohort. lower values of egfr were associated with lower levels of ace2 expression in the kidney. this relationship was more marked in the tubulointerstitium than in glomeruli. we next examined the relationship between age and ace2 expression in the kidney and focused on the ckd cohort for this analysis. there was a very modest negative correlation between age and ace2 expression in the tubulointerstitium and no relationship in glomeruli. this finding contrasts with reports that showed an age-dependent increase in upper and lower respiratory tract ace2 gene expression [40, 41] . finally, we did not see any relationship between bmi and ace2 expression in the kidney in the ckd cohort; although we cannot rule out a threshold effect for bmi [42, 43] , especially in a healthy population. taken together, these findings suggest that the impact of egfr and age on ace2 expression is more important in the tubulointerstitial compartment than in the glomerular compartment. we found that tubulointerstitial tmprss2 mrna expression was similar in the kidneys of hld and subjects with ckd, but it was lower in the glomerular compartment of ckd subjects than in hld. there was no impact of sex, egfr, age, or bmi on expression in either compartment. there was a strong relationship between ace2 mrna expression and tmprss2 mrna expression in glomeruli. batlle and coworkers reported single-cell rna expression of ace2 and tmprss2 in the kidney [33]. they found that ace2 was predominantly expressed in proximal tubule cells. li et al. have reported that ace2 is also expressed in the glomerulus [44] . in contrast, batlle and coworkers found that tmprss2 expression was greatest in the distal convoluted tubule and type a intercalated cells of the collecting duct, with modest expression in other cell types, including the podocyte. this cell-specific pattern of expression may account for our observation that there was no correlation between ace2 and tmprss2 expression in the tubulointerstitium. it has been postulated that co-expression of ace2 and tmprss2 underlies the susceptibility of other tissues to infection [14] . the colocalization and co-expression of ace2 and tmprss2 in the glomerulus and their strong correlation in this compartment may underlie the recent clinical observation of an emerging sars-cov-2-associated nephropathy (covan) which is characterized by a collapsing form of glomerulosclerosis not unlike hivassociated nephropathy [45] . notwithstanding these observations, it is important to note that we did not study ace2 expression in subjects that were infected with sars-cov-2. we next examined the relationships between ace2 expression and genes implicated in inflammation and fibrosis. the rationale was that ace2 reduces inflammation and fibrosis by limiting ang ii and promoting ang-(1-7). in kidneys of mice with ischemia-reperfusion injury or unilateral ureteral obstruction, the loss of ace2 exacerbated infiltration of neutrophils, f4/80 + macrophages, and cd3 + t cells, and increased mrna levels of cytokines and chemokines, including il6, tnf, and mcp-1 to reduce expression of il6 and tnf in macrophages [46] . our third major observation was that there were significant relationships between ace2 mrna expression and mrna expression of ccl2, il6, and tnf in both the tubulointerstitium and glomeruli of the ckd cohort. studies have also shown that ace2 limits kidney fibrosis and ace2 deficiency was associated with higher expression of col1a1, tgfb1, and fn1 in mice [36, 37] . lower levels of ace2 mrna expression were related to increased mrna expression of col1a1, tgfb1, and fn1. taken together with experimental studies of ace2 null mice [37, [47] [48] [49] and studies of recombinant ace2 treatment [50] [51] [52] , these findings suggest that low ace2 expression may contribute to the progression of ckd, by enhancing early inflammation and contributing to longterm fibrosis. finally, we performed a subgroup analysis of the subjects with dn. we saw a trend towards decreased ace2 expression in the tubulointerstitium and a statistically significant decrease in glomerular ace2 mrna in dn compared to hld. these findings confirm our original observations in dn [26] . in our previous study, we used laser-capture microdissection to examine ace2 expression in both the glomerular and tubulointerstitial compartments. diabetes was associated with decreased ace2 expression in both kidney compartments [26] . more recently, gilbert and coworkers reported increases in kidney ace2 mrna expression in patients with dn [53] , a finding that may reflect the use of formalin-fixed paraffin-embedded biopsy cores that were not microdissected. we then extended our studies of the dn subset to look at the relationships between ace2 mrna expression and egfr, age, and bmi. there was a positive association between glomerular ace2 expression and age in dn. interestingly, age appears to be a determinant of reninangiotensin system (ras) activation in the kidney [54] . the relationships between ace2 expression and the other clinical variables were not statistically significant, likely due, in part, to the fewer number of subjects that we were able to analyze. however, decreased ace2 expression in the tubulointerstitium was associated with increased ccl2, tnf, and tgfb1 mrna expression. tissue depletion of ace2 may promote inflammation and fibrosis in dn. our study has several limitations. first, although we were able to associate higher kidney ace2 expression with lower expression of inflammation and fibrosis genes, it is important to note that our findings do not imply causation. second, a wide range of underlying kidney pathologies was included in the ckd cohort, limiting our ability to look at disease-specific relationships. there were relatively few specimens (31 tubulointerstitial and 21 glomerular samples) in the hld cohort, and this limited the analysis of relationships between ace2 expression and age and bmi. important clinical data regarding ethnicity and race, and treatment with aceis and/or arbs were not available, so we could not address the impact of these variables on kidney ace2 expression in our study. finally, our study focused exclusively on ace2 and tmprss2 mrna expression and did not address protein expression or enzyme activity. in conclusion, kidney ace2 and tmprss2 mrna expression differs in hld and ckd. ace2 expression is greater in females than in males in the tubulointerstitium and glomeruli, whereas there is no impact of sex on tmprss2 expression. there is a relationship between ace2 expression and egfr in ckd. ace2 and tmprss2 expression levels are strongly correlated in the glomerulus but not in the tubulointerstitium. finally, lower ace2 expression is associated with higher expression of genes implicated in inflammation and fibrosis in both kidney compartments, suggesting that loss of ace2 may promote inflammation and fibrosis in the tubulointerstitium and glomerulus. angiotensin-converting enzyme 2 and renal disease tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis renal ace2 expression in human kidney disease 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function and raas activation over the natural history of type 1 diabetes key: cord-302189-3xab3yxc authors: tillmann, ramona liza; simon, arne; müller, andreas; schildgen, oliver title: sensitive commercial nasba assay for the detection of respiratory syncytial virus in clinical specimen date: 2007-12-26 journal: plos one doi: 10.1371/journal.pone.0001357 sha: doc_id: 302189 cord_uid: 3xab3yxc the aim of the study was to evaluate the usability of three diagnostic procedures for the detection of respiratory syncytial virus in clinical samples. therefore, the fda cleared ce marked now® rsv elisa, the nuclisens® easyq rsv a+b nasba, and a literature based inhouse rt-pcr protocol were compared for their relative sensitivities. thereby, nasba turned out to be the most sensitive method with a total number of 80 rsv positive samples out of a cohort of 251 nasopharyngeal washings from patients suffering from clinical symptoms, followed by the inhouse rt-pcr (62/251) and elisa (52/251). thus, nasba may serve as a rapid and highly sensitive alternative for rsv diagnostics. despite an increasing number of newly detected respiratory pathogen the human respiratory syncytial virus (rsv) remains the single most prevalent etiologic agent in pediatric viral respiratory tract infection [1, 2, 3] . rsv is responsible for the majority of episodes of acute wheezing triggered by infection [4] , bronchiolitis [5] and pneumonia [6] predominantly during the first 24 months of life. an estimated percentage of about 1-2% of all rsv-infected children require hospital care. the rsv-related hospitalization rate and the risk of severe complications are increased in prematurely born infants with chronic lung disease (cld) [7] and in children with hemodynamically relevant congenital heart disease (chd) [8, 9] , other forms of chronic lung disease or severe neuromuscular impairment [2] . forster and coworkers estimated (95% confidence interval) a total of 26,524 (23,812-29,432) rsv-related hospitalizations per year in children under 3 years of age in germany (i.e. 38% of all pediatric hospitalizations for viral lower respiratory tract infection) [10] . the same group calculated j2.772 as median total costs per hospitalised rsv-infection [11, 12, 13] . others recently calculated even higher costs [14] . specific therapeutic agents with proven efficacy against rsv are still not available [1, 5] . meticulous hand hygiene after patient contact together with other barrier precautions and rapid laboratory diagnostic are considered to be of utmost importance for the prevention of nosocomial transmission [16, 17] . rapid laboratory detection of rsv is mainly performed by elisa [17, 18] or by the use of nucleic acid amplification and detection methods. the latter methodology includes a high number of rt-pcr protocols, but for reasons of quality assurance in quality management systems the need for standardized nucleic acid amplification procedures with quality marks like the ce mark increases more and more. recently, the nuclisens easyq rsv a/b (biomerieux, nürtingen, germany), a ce-labeled nucleic acid sequence based amplification (nasba) based kit for the rapid detection of rsv, became available. in search for options to optimize the rapid laboratory diagnostics of rsv we have compared this nasba method with a published rt-pcr protocol and a rapid elisa, the latter both used in our routine procedures for the detection of rsv. the patient cohort consisted of a total number of 251 pediatric patients hospitalized with respiratory tract infection. only one clinical sample per patient was included in the study, resulting in a total number of 251 nasopharyngeal aspirates. these aspirates were used freshly for all subsequent procedures and were not frozen before usage. all specimens were previously tested negative by pcr or rt-pcr as previously described [16, 2] for any of the following viruses: human bocavirus, human metapneumovirus, influenzaviruses a and b, and human coronaviruses nl63, hku1, sars, oc43, and 229e. additional tests to detect rhinoviruses, adenoviruses, parainfluenzaviruses, or bacteria, were not performed in our laboratory. the main focus of the present study was to evaluate the sensitivity and specificity of the nasba method and the rapid elisa compared to rt-pcr. native samples were tested by the fda cleared ce marked nowh rsv elisa (inverness medical, cologne, germany). nowh rsv elisa tests were carried out strictly following the manufacturer's protocol and considered positive according to the manufacturer's guidelines. for nsaba and rt-pcr rna was automatically extracted by the nuclisensh easymag tm (bio-merieux, nürtingen, germany) using the manufacturer's extraction protocol for nasopharyngeal specimen, using 100 ml of specimen preincubated for 30 min at 37uc with 10 ml dnase and 12 ml dnase buffer (promega, germany). subsequent nasba reactions were carried out using the nuclisensh easyq (biomerieux, nürtingen, germany) system strictly following the manufacturer's guidelines. rna used for rt-pcr was extracted as described above. rt-pcr was performed essentially as previously described by mentel and coworkers [19] . briefly, the results are summarized in table 1. from the 251 specimen 52 (20.7%) were tested positive for rsv by nowh rsv elisa (inverness medical, cologne, germany), 62 (24.7%) were tested positive for rsv by rt-pcr, and 80 (31.9%) were tested positive for rsv by nuclisensh easyq nasba (biomerieux, nürtingen, germany). thus, as the highest sensitivity was observed for the ce marked nuclisensh easyq, this relative sensitivity was set to 100%. thereby it was assumed that with nuclisensh easyq according to the ce marked guarantee a sensitivity and specificity of this test of 99% as earlier described [20] . in relation to the positive test results obtained with the nuclisensh easyq nasba, the relative sensitivity of the rt-pcr was 77,5% compared to 65% obtained with the nowh rsv elisa. a total number of 43 (17.1%) samples were tested positive for rsv by all methods, 16 samples (6.37%) were tested positive for rsv by both rt-pcr and elisa but not by elisa, 4 samples (1.59%) were tested rsv positive by rt-pcr and elisa but not by nasba, 1 sample (0.4%) was tested rsv positive only by rt-pcr, 4 samples (1.59%) were tested rsv positive only by elisa, and 17 samples (6.77%) were tested rsv positive only by nasba. the overall accordance of all techniques was 53.75%, the accord-ance of rt-pcr and nasba was 76.25%, the accordance of nasba and elisa was 60%, and the accordance of rt-pcr and elisa was 78.69%. the negative predictive values for were 97.1% for nasba, 83.4% for elisa, and 87.8% rt-pcr, the positive predictive values were 94.1% for nasba, 92.9% for elisa, and 100% rt-pcr (table 2). the results showed that from the three tested methods for molecular diagnosis of rsv the nuclisensh easyq nasba (biomerieux, nürtingen, germany) detected the most rsv positive samples in a cohort of 251 nasopharyngeal samples of pediatric patients hospitalized with respiratory disease. taking into account data published in the manufacturer's manuals and on their respective websites, it can be assumed that the specificity of both nuclisensh easyq nasba and the nowh rsv elisa is very high. furthermore, as demonstrated last year by manji and coworkers, the nuclisensh easyq nasba assay specifity has a positive sample value of $1.100 with an acceptable ic value of $1.100 [21] . thereby, the absolute assay specifity turned out to be $95%. for the nowh rsv elisa cruz et al. [22] determined that the sensitivity was 81% and specificity 93.2%. moreover, with our in house rt-pcr we have not yet any false positive as all detections were confirmed by sequencing (simon, schildgen et al., unpublished data) . it is commonly known that antibody based methods elisa like for detection of rsv in clinical samples is less sensitive than nucleic acid amplification techniques [23] . however, the rapid results are of high importance for clinicians in order to initiate therapy and/ or isolation of the patients in order to avoid nosocomial outbreaks. in this earlier study which solely compared rapid elisa methods, the nowh rsv elisa was found to be the most sensitive at least for the cohort of pediatric patients [17] with hands on time of about 10 to 20 min. however, taken into account the higher relative sensitivity and the acceptable predictive values accompanied by short hands on time and final results in nearly of 90 min, the nuclisensh easyq nasba may serve alternative method as it is both a fast but also a highly sensitive method. it thus should be taken into account whenever rapid and sensitive rsv diagnostics are required, such as in clinical setting involving high risk patients for which nosocomial outbreaks may be a fatal event. systematic review of the biology and medical management of respiratory syncytial virus infection dsm rsv ped study group. hospitalized children with respiratory syncytial virus infection and neuromuscular impairment face an increased risk of a complicated course human metapneumovirus infections cause similar symptoms and clinical severity as respiratory syncytial virus infections viral etiology of acute respiratory tract infections in children presenting to hospital: role of polymerase chain reaction and demonstration of multiple infections bronchiolitis: assessment and evidence-based management community-acquired pneumonia in children selected populations at increased risk from respiratory syncytial virus infection respiratory syncytial virus infections in the pediatric intensive care unit: clinical characteristics and risk factors for adverse outcomes palivizumab prophylaxis reduces hospitalization due to respiratory syncytial virus in young children with hemodynamically significant congenital heart disease prospective population-based study of viral lower respiratory tract infections in children under 3 years of age (the pri.de study) the use of palivizumab monoclonal antibody to control an outbreak of respiratory syncytial virus infection in a special care baby unit economic impact of community-acquired and nosocomial lower respiratory tract infections in young children in germany defining the timing of respiratory syncytial virus (rsv) outbreaks: an epidemiological study anticipated costs of hospitalization for respiratory syncytial virus infection in young children at risk nosocomial respiratory syncytial virus infection: impact of prospective surveillance and targeted infection control rsv outbreak in a paediatric intensive care unit evaluation of the binax now, bd directigen, and bd directigen ez assays for detection of respiratory syncytial virus prospective evaluation of a dot-blot enzyme immunoassay (directigen rsv) for the antigenic detection of respiratory syncytial virus from nasopharyngeal aspirates of paediatric patients real-time pcr to improve the diagnosis of respiratory syncytial virus infection enhanced clinical utility of the nuclisens easyq rsv a+b assay for rapid detection of respiratory syncytial virus in clinical samples nucleic acid sequence based amplification (nasba) and molecular beacon detection for the real-time detection of respiratory syncytial virus (rsv) in pediatric respiratory specimens performance of a rapid assay (binax now) for detection of respiratory syncytial virus at a children's hospital over a 3-year period comparison of the bd directigen flu a+b kit and the abbott testpack rsv with a multiplex rt-pcr elisa for rapid detection of influenza viruses and respiratory syncytial virus key: cord-305547-e66o5j85 authors: bénet, thomas; sylla, mariam; messaoudi, mélina; sánchez picot, valentina; telles, jean-noël; diakite, abdoul-aziz; komurian-pradel, florence; endtz, hubert; diallo, souleymane; paranhos-baccalà, gláucia; vanhems, philippe title: etiology and factors associated with pneumonia in children under 5 years of age in mali: a prospective case-control study date: 2015-12-22 journal: plos one doi: 10.1371/journal.pone.0145447 sha: doc_id: 305547 cord_uid: e66o5j85 background: there are very limited data on children with pneumonia in mali. the objective was to assess the etiology and factors associated with community-acquired pneumonia in hospitalized children <5 years of age in mali. methods: a prospective hospital-based case-control study was implemented in the pediatric department of gabriel touré university hospital at bamako, mali, between july 2011-december 2012. cases were children with radiologically-confirmed pneumonia; controls were hospitalized children without respiratory features, matched for age and period. respiratory specimens, were collected to identify 19 viruses and 5 bacteria. whole blood was collected from cases only. factors associated with pneumonia were assessed by multivariate logistic regression. results: overall, 118 cases and 98 controls were analyzed; 44.1% were female, median age was 11 months. among pneumonia cases, 30.5% were hypoxemic at admission, mortality was 4.2%. pneumonia cases differed from the controls regarding clinical signs and symptoms but not in terms of past medical history. multivariate analysis of nasal swab findings disclosed that s. pneumoniae (adjusted odds ratio [aor] = 3.4, 95% confidence interval [95% ci]: 1.6–7.0), human metapneumovirus (aor = 17.2, 95% ci: 2.0–151.4), respiratory syncytial virus [rsv] (aor = 7.4, 95% ci: 2.3–23.3), and influenza a virus (aor = 10.7, 95% ci: 1.0–112.2) were associated with pneumonia, independently of patient age, gender, period, and other pathogens. distribution of s. pneumoniae and rsv differed by season with higher rates of s. pneumoniae in january-june and of rsv in july-september. pneumococcal serotypes 1 and 5 were more frequent in pneumonia cases than in the controls (p = 0.009, and p = 0.04, respectively). conclusions: in this non-pcv population from mali, pneumonia in children was mainly attributed to s. pneumoniae, rsv, human metapneumovirus, and influenza a virus. increased pneumococcal conjugate vaccine coverage in children could significantly reduce the burden of pneumonia in sub-saharan african countries. pneumonia is the leading cause of child mortality from infectious diseases, accounting for an estimated 1 million deaths annually, and mainly affecting children in developing countries [1, 2] . mortality attributed to pneumonia has decreased since 2000, but remains a major public health concern [3, 4] . the main known causative pathogens reported are streptococcus pneumoniae, haemophilus influenzae type b, and respiratory syncytial virus (rsv) [5] . however, their distribution varies by season and location. data on the etiology and epidemiology of pneumonia in children in developing countries are still insufficient, particularly in sub-saharan africa [6] . mali is one of the poorest countries in the world, with a under-five year mortality rate of 123 per 1,000 live births in 2013 according to the un inter-agency group for child mortality estimation. it has been estimated that among its 14.9 million inhabitants, each year more than 900,000 pneumonia episodes occur in children under 5 years of age, leading to almost 8,000 deaths annually [7] . a previous descriptive study reported that pneumonia was the most frequent cause of admission, representing 18% of total hospital admissions [8] . however, detailed information was not available on clinical presentation and on the etiology of suspected pneumonia cases [8] . moreover, no control group was included. the presence of a control group of children without pneumonia would allow better interpretation of microbiological findings, particularly with nasal sampling [9] . in march 2011, mali included pneumococcal vaccination (pcv13) in a routine immunization program; but vaccine coverage is still low [10] . the study objective was to assess the etiology and factors associated with communityacquired pneumonia in hospitalized children in mali. the gabriel touré university hospital is a 447-bed tertiary-care general hospital located in bamako. it is a primary care hospital for people living in bamako and a national reference centre for other patients. various medical and surgical specialities, including pediatrics, are located in the hospital. the pediatrics department, with a capacity of 150 beds, includes a general pediatrics unit and a neonatal/emergency unit. it receives sick children for primary care and severe cases referred from other healthcare settings. on average, 50,000 consultations and 10,000 hospital admissions occur in the pediatrics department annually. acute respiratory infections represent 34% of admissions in children, and 15% of child hospitalizations. pneumonia cases were hospitalized children who fulfilled the following criteria: -cough and/or dyspnea, and -tachypnea, as characterized by the world health organization (who) in children between 2 and 12 months of age: breathing rate !50 cycles per minute; in children between 12 and 59 months of age: breathing rate !40 cycles per minute) [13] , and -absence of wheezing at auscultation, and -first symptoms appearing within the last 14 days, and -radiological confirmation of pneumonia as per who guidelines [14] . the exclusion criterions for cases were presence of wheezing at auscultation, or minors whose parents or legal guardian declined to sign the informed consent statement. controls were patients hospitalized for surgery or in a routine outpatient practice environment, aged between 2 and 59 months, without any symptoms suggestive of respiratory illness; suspicion of infection of other site was not an exclusion criteria. cases and controls were matched for age (±1 year) and calendar date of hospital admission (±1 month) to take seasonality into account. thus, 61% of patients were recruited during the rainy season (may to october) while 39% were recruited during the dry season (november to april). samples were collected in the first 48 hours of patient hospitalization. nasal swabs were taken from all pneumonia cases and controls. to document antibiotic usage history, urine was sampled from cases for broad spectrum antibiotic detection. whole blood and pleural effusions (in applicable cases) were collected from cases only. whole blood was distributed in ethylenediaminetetraacetic acid (edta) vials for preservation and in dry tubes for serum specimens. edta-whole blood samples allowed complete blood count, blood culture and real-time multiplex polymerase chain reaction (pcr) assay for the identification of staphylococcus aureus, streptococcus pneumoniae and haemophilus influenzae type b. c reactive protein (crp) and procalcitonin (pct) were measured quantitatively in serum. respiratory specimens from nasal swabs and pleural effusions were characterized by ftd respiratory pathogens 21 plus (fast-track diagnostics, luxembourg) based on rt-pcr which included a panel of 19 demographic characteristics, underlying diseases, medical history, clinical examination at enrollment, radiological findings, vaccinations, and outcome were recorded prospectively for each patient on a standardized form. continuous variables were described as median and interquartile range (iqr), categorical variables as number and percentage. pneumonia cases we compared to the controls by the mann-whitney u test for continuous covariates, and by the chi 2 test for categorical variables. univariate and multivariate logistic regression analyses were performed to assess factors associated with pneumonia. multivariate analyses were adjusted for gender, age, period per quarter, and other pathogens significantly associated with increased risk of pneumonia. concordance between serotypes detected in nasopharyngeal samples and blood was tested by kendall rank correlation. crude population attributable fraction (paf) was calculated after univariate logistic regression analysis to quantify the contribution of the each microorganism to pneumonia occurrence. conceptuality, paf permits the estimation of the proportional reduction in pneumonia occurrence that would occur if the pathogen was absent (alternative ideal scenario). the formula was the following: with : pr c ¼ prevalence of microorganim detection in controls; or ¼ crude odds ratio: no patient was excluded because of missing data. p<0.05 was considered significant; all tests were bilateral, and statistical analysis was conducted with stata version 13.0 (stata corp.). with at least 100 cases and 100 controls, analyses had 80% power to detect odds ratios (or)!3 with !20% prevalence of control exposure, whatever the prevalence of case exposure. the study protocol, informed consent statement, clinical research form, any amendments and all other study documents were submitted to and approved by the institutional ethics committee (comité national d'ethique pour la santé et les sciences de la vie). written informed consent was obtained from all participants. among the 119 enrolled cases, 118 conformed with the inclusion criteria and were all sampled. one patient without radiological confirmation was excluded from the analysis. among the 100 included controls, 2 were excluded because of missing data. overall, 216 patient, accounting for 118 (54.6%) pneumonia cases and 98 (45.4%) controls, were analyzed. among them, 93 (44.1%) were female. the male/female gender ratio was 0.79, and median age was 11 months (iqr: 5-55 months). among the 118 patients with pneumonia, 16 (13.6%) were referred from another health center; median length of hospital stay was 7 days (iqr: 6-10 days) for a total of 909 days of hospitalization ( table 1 ). the median time period between first symptoms and hospitalization was 6 days (iqr: 4-7 days). four (3.9%) pneumonia cases presented a ventricular septal defect. none had previous tuberculosis or underlying lung disease. among cases with pulmonary crackles, 36 (35.3%) were unilateral and 66 (64.7%) were bilateral. other signs or medical history of pneumonia cases included sickle-cell anaemia (n = 1), dehydration (n = 1), epilepsy (n = 1), goiter (n = 1), edema (n = 1), pallor (n = 1), and trisomy (n = 3). among the 47 patients who took antibiotics before hospitalization, 17 (14.4%) received amoxicillin, 8 (5.6%) ceftriaxone, 4 (2.8%) amoxicillin/clavulanic acid, and 3 (2.1%) cotrimoxazole. among the 63 cases tested for antibiotic detection testing in urine, 22 (65.1%) were positive; in this population, sensitivity of declared antibiotic use compared with urine detection was low (sensitivity = 51.2%, 95% confidence interval: 35.1-67.1%). median crp level at admission was 21 mg/l (iqr: 6-63, mean: 56.6 mg/l), median pct level was 4.6 ng/ml (iqr: 0.4-26.4, mean: 31.5 ng/ml). median crp and pct levels were higher in hypoxemic compared with non-hypoxemic pneumonia cases (p = 0.03 and p = 0.007, respectively). median white blood cell count was 15,750 ã 10 9 cells/l (iqr: 10,000-25,700, mean: 20,600 ã 10 9 cells/l), and median neutrophil percentage was 52.5% (iqr: 31-68, mean: 50.3%). x-rays showed generalized, dense, homogeneous opacification in 24 (20.3%) patients, interstitial syndrome in 24 (20.5%), and alveolar infiltrate in 65 (55.1%), without significant differences according to the pattern of infection (viral, bacterial or mixed). no patient had abscess or pneumothorax, but 4 (3.4%) had pleural effusion. during hospital stay, pneumonia cases received the following antibiotics: amoxicillin (n = 111, 94.1%), ceftriaxone (n = 3, 2.6%), amoxicillin/clavulanic acid (n = 3, 2.5%), ciprofloxacin (n = 1, 0.8%), and vancomycin (n = 2, 1.6%). fifty-one (43.2%) received oxygen for a median length of 2 days (iqr: 1-3 days), and 3 (2.5%) had blood transfusions. three patients with pleural effusions were tested for microbial detection. five patients died (lethality: 4.2%); death was directly related to pneumonia in 4 patients ( table 2 ). table 1 compares the clinical signs and symptoms at admission between cases and controls, underlying conditions and biological findings. pneumonia cases differed from controls regarding clinical signs and symptoms as well as vital signs at admission, but not in terms of demographic factors or past medical history. pneumonia cases were more frequently hypoxemic (defined as oxygen saturation<90%) at admission than the controls (30.5% vs. 0%, p<0.001). pcv coverage was zero in both groups. at least 1 microorganism was detected on nasal swabs in 96.6% of cases and 82.3% of controls (crude or = 6.4, 95% confidence interval [95% ci]: 2.1-19.7, p<0.001). overall, 78.8% of cases and 54.2% of controls were co-infected or co-colonized (crude or = 3.3, 95% ci: 1.8-6.0, p<0.001). co-detection on nasal swab of s. pneumoniae and rsv was more frequent in cases than in controls (respectively, 15 between cases and controls (respectively, 5.9% [n = 7] vs. 3.1% [n = 3], p = 0.32). a doseresponse relationship was apparent between the number of microorganisms found in nasal swabs and the risk of being a case (fig 1) . distribution of s. pneumoniae and rsv differed by season with higher rates of s. pneumoniae in january-june and of rsv in july-september (fig 2) . univariate analysis revealed that s. pneumoniae, human metapneumovirus, rsv, and influenza a virus detection in nasal swabs were significantly associated with pneumonia in mali (table 3) . multivariate analysis reinforced linkage of these pathogens with pneumonia, independently of patient age, gender, period per quarter and the presence of other pathogens significantly coupled with increased risk of pneumonia (table 3) . paf was the highest for s. pneumoniae (paf = 46%, 95% ci: 30-59%). contribution of human metapneumovirus, rsv, and influenza a were lower, with pafs of 9% (95% ci: 7-11%), 21% (95% ci: 16-25%) and, 8% (95% ci: 6-10%), respectively. fig 3 reports the distribution of pneumococcal serotypes detected in nasal swabs from cases and controls. the most prevalent serotype in pneumonia cases and controls was serotype 6a/b (18.6% vs. 11.2%, p = 0.13). serotypes 1 and 5 were more frequent in pneumonia cases than in the controls: 6.8% vs. 0%, p = 0.009, and 4.2% vs. 0%, p = 0.04, respectively. in pneumonia cases, s. pneumoniae was positive in 16 (13.6%) patients, s. aureus in 6 (5.1%) patients and h. influenza in 5 (4.2%) patients by pcr blood sample detection. most patients with s. pneumoniae detection by pcr had also s. pneumoniae nasal carriage (93.5%, 15/16), while only 17.6% (15/85) of patients with s. pneumoniae nasal carriage had positive detection by pcr (p = 0.04). concordance of serotype 1 detected in nasal swabs and blood in pneumonia cases was high (κ = 0.70, p<0.001). coronavirus 63 was identified from pleural effusion in 1 patient. microbiological findings, including s. pneumoniae serotypes distribution, from pcr nasal swab or blood sample were not different in pneumonia cases according to the result of urine antibiotic testing (data not shown). blood culture was positive in 36 (30.5%) pneumonia cases; most microorganisms were probably related to contamination of samples. the following bacteria were detected: coagulase-negative staphyloccoci (n = 26), salmonella spp. (n = 3), gram-positive bacilli (n = 2), acinetobacter baumannii (n = 1), aerococcus viridans (n = 1), enterococcus faecium (n = 1), granulicatella elegans (n = 1), and staphylococcus aureus (n = 1). the primary objective of this prospective case-control study was to assess the etiology and factors associated with community-acquired pneumonia in hospitalized children in mali. in this non-pcv population, we observed that s. pneumoniae, human metapneumovirus, rsv, and influenza a were the main microbial agents associated with pneumonia among children in mali, independently of patient age, gender, period, and other pathogens. h. influenzae was not associated with pneumonia, but the vaccination rate against this bacteria, at least for the first dose, was above 85% in both cases and controls. in addition, while most patients with s. pneumoniae by pcr in blood had also nasal carriage, frequently with the same serotype, positive s. pneumoniae test from nasal sample was not highly predictive of s. pneumoniae detection in blood, meaning that the bacteria was frequently not the cause of pneumonia. few studies have assessed the etiology of pneumonia in sub-saharan africa. howie et al. recently investigated pneumonia etiology in children in gambia, observing that s. pneumoniae was the leading cause with a high rate of microbial agent co-detection [15] . conversely, they found that viral pneumonia was not predominant whereas we observed that 3 viruses, namely human metapneumovirus, rsv, and influenza a virus, were associated with pneumonia in mali. major differences between the 2 studies were sample type (lung aspirates vs. nasal swabs in our investigation) and the lack of a comparative group in the study by howie et al. which did not permit comparison of microbial prevalence in pneumonia patients and healthy subjects [15] . in a western kenya case-control study, s. pneumoniae, rsv, and influenza a virus were the predominant causes of pneumonia in children [16] . we noted almost similar results in mali. thus, s. pneumoniae and rsv could still be considered as the primary causes of pneumonia in several sub-sharan african countries. all patients who died had severe pneumonia, according to who criteria, with significant dyspnea, very marked chest indrawing and severe hypoxia. two patients had confirmed pneumococcal pneumonia. two patients had acute malnutrition linked with pneumonia. in 2 cases, death occurred within 24 hours of admission. despite active treatment with antibiotics and oxygen, the management of severe respiratory distress is often difficult in this context because the hospital does not have assisted ventilatory support. mortality was higher than in a recent birth cohort in south africa [17] , but was similar to what was reported previously in other developing countries [18, 19] . some invasive serotypes were detected selectively in pneumonia cases but not in the controls (i.e., serotypes 1 and 5 were associated with the risk of pneumonia). a previous study of serotypes involved in invasive pneumococcal disease in mali found that serotype 5 was the most prevalent (54%) [20] . it was also linked with pneumonia in our series. in other sub-saharan african countries, serotype 1 was described as the most prevalent serotype of invasive pneumococcal disease [21] . it was the second serotype significantly associated with pneumonia in children in mali. thus, the introduction of pcv in routine immunization programs in mali would substantially reduce pneumonia caused by s. pneumoniae because most serotypes eliciting pneumonia would be covered by the vaccine [22] . in addition, pneumococcal pneumonia seasonality was similar to that observed previously in burkina faso, a neighbouring country, in 2007-2011 with higher incidence during the dry season between january and may [23] . interestingly, among the 4 microbial agents associated with pneumonia, 3 were viruses: human metapneumovirus, rsv, and influenza a virus. self et al. recently observed, in a pneumonia cases-control study implemented in hospitals of utah, that detection respiratory syncytial virus, human metapneumovirus and influenza from nasopharyngeal or oropharyngeal sample of patients with pneumonia probably indicates an etiologic role [24] . we observed similar results in a different context. despite the dearth of vaccination against pneumococcus in our population, viral pneumonia is a major segment of the pneumonia burden. treatment of these infections is often problematic because the empirical use of antibiotics or antivirals is not consensual [25] . vaccine development, particularly against rsv, is warranted to prevent pneumonia in children. moreover, the impact of influenza vaccine policies in developing countries should be evaluated because this virus often causes pneumonia [26] . rhinovirus was detected in almost 25% of pneumonia cases of our series, but the detection rate was not different between cases and controls. however, this observation does formally excludes that rhinovirus could play a role in the etiology of pneumonia. jain et al. recently observed that rhinovirus was detected in 27% of u.s. children with community-pneumonia requiring hospitalization [27]; this large study had however no control group, it was also not possible to estimate the prevalence of respiratory detection in children without pneumonia. growing evidence suggests the role of rhinovirus in the etiology of viral pneumonia [28] or related to the risk of secondary bacterial pneumonia [29] . other viruses, such as bocavirus or adenovirus were also equally prevalent in cases and controls. again, this finding does not exclude completely their implication in pneumonia etiology in children from mali. however these viruses were poorly described as causative agents of pneumonia in children from other sub-saharan african countries. several microorganisms have been detected in most pneumonia cases but also in the controls. the clinical significance of microbial detection by pcr is problematic at the individual level because most detected pathogens are not causes of pneumonia. however, number of pathogens is predictive of the risk of pneumonia, suggesting that simple quantification of species detected would permit the evaluation of pneumonia risk. at the population level, nasal samples are interesting because the prevalence of carriage in the controls is considered. the main strengths of our study were its prospective design with the inclusion of controls that permitted us to assess pneumonia etiologies while taking into account the prevalence of pathogen detection in non-infected patients. ours is the first case-control investigation of pneumonia etiology in children from mali (s1 strobe checklist). the results should serve to better manage pneumonia not only in these children but also in those from neighboring countries. data quality was enhanced by centralized microbiological analysis in the emerging pathogens laboratory (lyon, france). in addition, this analysis should serve to better focus multicentric pneumonia studies that will permit us to assess the global etiologies of pneumonia and to compare etiologic agents between countries and continents. some limitations should be underlined. first, microbiological diagnosis of pneumonia is difficult due to the lack of single reliable test. then, at the individual level, it is difficult to establish if a positive nasal swab denotes etiology or nasopharyngeal colonization; particularly for bacterial agents such as s. pneumoniae because of the high asymptomatic carriage. however, at the population level, addition of a control group permits to evaluate and control for the prevalence of carriage in asymptomatic children. in a pneumonia etiology study, it would be interesting to correlate the threshold cycle (ct) values obtained from rt-pcr with lower airway specimens to establish more precisely the relationship between positive nasal swab and etiology of pneumonia, according to the microorganism [30] . second, because the study was implemented in one hospital in mali, external validity might be limited. however, gabriel touré university hospital is a reference center in the country, and then source population is not limited to the city of bamako. third, study power was confined to detecting some linkages (i.e. serotypes associated with the risk of pneumonia). community-acquired pneumonia was mainly attributable to s. pneumoniae, human metapneumovirus, rsv or influenza a among children in mali. increased pneumococcal conjugate vaccine coverage in children would significantly reduce the burden of pneumonia in this country. the addition of a control group to assess etiologies of pneumonia in children is critical to properly interpret the microbiological results of diagnostic testing with high sensitivity. supporting information s1 strobe checklist. (doc) s1 global burden of childhood pneumonia and diarrhoea global health observatory data repository global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since global, regional, and national causes of child mortality in 2008: a systematic analysis community-acquired pneumonia in children epidemiology and etiology of childhood pneumonia in 2010: estimates of incidence, severe morbidity, mortality, underlying risk factors and causative pathogens for 192 countries the causes of hospital admission and death among children in bamako, mali identification and selection of cases and controls in the pneumonia etiology research for child health project the impact of introducing new vaccines on the health system: case studies from six low-and middleincome countries enhancing research capacities in infectious diseases: the gabriel network, a joint approach to major local health issues in developing countries multicenter case-control study protocol of pneumonia etiology in children: global approach to biological research, infectious diseases and epidemics in low-income countries (gabriel network) case management of childhood pneumonia in developing countries standardized interpretation of paediatric chest radiographs for the diagnosis of pneumonia in epidemiological studies etiology of severe childhood pneumonia in the gambia, west africa, determined by conventional and molecular microbiological analyses of lung and pleural aspirate samples viral and bacterial causes of severe acute respiratory illness among children aged less than 5 years in a high malaria prevalence area of western kenya incidence and severity of childhood pneumonia in the first year of life in a south african birth cohort: the drakenstein child health study variations in mortality in children admitted with pneumonia to kenyan hospitals risk factors for mortality in community acquired pneumonia among children aged 1-59 months admitted in a referral hospital invasive pneumococcal infections among hospitalized children in bamako, mali molecular typing of the pneumococcus and its application in epidemiology in sub-saharan africa licensure of a 13-valent pneumococcal conjugate vaccine (pcv13) and recommendations for use among children-advisory committee on immunization practices (acip) streptococcus pneumoniae invasive infections in burkina faso respiratory viral detection in children and adults: comparing asymptomatic controls and patients with community-acquired pneumonia strategic priorities for respiratory syncytial virus (rsv) vaccine development global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis. the lancet community-acquired pneumonia requiring hospitalization among u.s. children rhinovirus associated with severe lower respiratory tract infections in children viral and bacterial interactions in the upper respiratory tract respiratory virus detection in nasopharyngeal aspirate versus bronchoalveolar lavage is dependent on virus type in children with chronic respiratory symptoms this protocol was developed on behalf of members of the global approach to biological research, infectious diseases and epidemics in low-income countries (gabriel) network: http://gabriel.globe-network.org. key: cord-296550-wkmnfph3 authors: hossain, mohammad anwar; jahid, md. iqbal kabir; hossain, k. m amran; walton, lori maria; uddin, zakir; haque, md. obaidul; kabir, md. feroz; arafat, s. m. yasir; sakel, mohamed; faruqui, rafey; hossain, zahid title: knowledge, attitudes, and fear of covid-19 during the rapid rise period in bangladesh date: 2020-09-24 journal: plos one doi: 10.1371/journal.pone.0239646 sha: doc_id: 296550 cord_uid: wkmnfph3 the study aims to determine the level of knowledge, attitude, and practice (kap) related to covid-19 preventive health habits and perception of fear towards covid-19 in subjects living in bangladesh. design: prospective, cross-sectional survey of (n = 2157) male and female subjects, 13–88 years of age, living in bangladesh. methods: ethical approval and trial registration were obtained before the commencement of the study. subjects who volunteered to participate and signed the informed consent were enrolled in the study and completed the structured questionnaire on kap and fear of covid-19 scale (fcv-19s). results: twenty-eight percent (28.69%) of subjects reported one or more covid-19 symptoms, and 21.4% of subjects reported one or more co-morbidities. knowledge scores were slightly higher in males (8.75± 1.58) than females (8.66± 1.70). knowledge was significantly correlated with age (p < .005), an education level (p < .001), attitude (p < .001), and urban location (p < .001). knowledge scores showed an inverse correlation with fear scores (p < .001). eighty-three percent (83.7%) of subjects with covid-19 symptoms reported wearing a mask in public, and 75.4% of subjects reported staying away from crowded places. subjects with one or more symptoms reported higher fear compared to subjects without (18.73± 4.6; 18.45± 5.1). conclusion: bangladeshis reported a high prevalence of self-isolation, positive preventive health behaviors related to covid-19, and moderate to high fear levels. higher knowledge and practice were found in males, higher education levels, older age, and urban location. fear of covid-19 was more prevalent in female and elderly subjects. a positive attitude was reported for the majority of subjects, reflecting the belief that covid-19 was controllable and containable. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 bangladesh is among the top 20 countries in terms of confirmed cases of covid-19, with a positive case rate of 19.09% -22.91% as of june 1, 2020 [1] . however, questions remain regarding the actual number of cases and the scarcity of testing facilities [2] . there are also concerns about bangladesh's ability to mount an effective response to the covid-19 pandemic [3] . one newspaper also states [4] that bangladesh is a developing economy and is mainly dependent on remittances, ready-made garments, and small trades. the country is mid-phase in a few financial megaprojects. natural calamities and covid-19 pose challenges for the bangladeshi government and its residents at home and abroad [5] . due to economic concerns, bangladesh did not impose a countrywide lockdown. instead, the authorities sub-sectioned the country into red, yellow, and green zones based on the level of community contamination [6] . additionally, the government website for coronavirus briefing measures is being used to improve the situation, raising individual awareness by improving individual knowledge, attitudes, and practices, which has helped alleviate unnecessary fears and social stigmas [7] . battling the covid-19 pandemic is a lengthy process and requires the combined efforts of individuals and the government; adequate testing, isolation, and supportive treatment provision are the best ways to overcome the pandemic [8] . there is ongoing research to develop a vaccine. nevertheless, measures to raise the general population's knowledge and implementation of recommended health practices are some of the best approaches to combating covid-19 [9] . the world health organization (who) [10] stated that only 15% of cases were projected to have severe symptoms, and one-third of the severe cases required critical care; the main priority of the who is to mobilize resources to improve community healthcare practices. there is an emphasis on developing a community's receptiveness to staying at home. moreover, the who raised concerns regarding mental health needs [11] . mental health needs related to covid-19 are emerging regardless of age, occupation, and education and are related to isolation, financial uncertainty, quarantine effects, excessive time spent online, gaming, physical inactivity, insomnia, anxiety, depression, and fear of covid-19 [12] . the study also suggests that extreme fear and anxiety led individuals in china to have more physical and psychological signs, even with mild to no symptoms reported. bangladesh reported a few cases of suicide due to extreme fear of covid-19, with some cases showing negative outcomes after the administration of the real-time polymerase chain reaction (rt-pcr) test postmortem [13] . bangladesh responded relatively early in march 2020, with no cases for nearly a week. the subsequent arrival of travelers from italy who defied quarantine regulations could have been the source of the virus [14] . besides, religious gatherings and the lack of travel restrictions are considered the primary reasons for the sharp upward projections in covid-19 cases [15] . a population-based study was required to determine general knowledge about the disease and what practices were being taken by bangladeshi individuals to combat covid-19. fear is thought to be one of the main contributors to mass anxiety and depression. fear has been shown to predict inadequate health overall, insomnia, and the suppression of immunity. other influencing factors of anxiety and depression include occupation, knowledge, attitudes, and practice of health-related habits, as well as other environmental indicators [12] . determining knowledge, attitudes, practices (kap), and fear will provide a glimpse of how bangladesh is responding to the pandemic in this state of rising cases. this will further help to evaluate their overall preparedness. the covid-19 crisis is assumed to be a long-term process, and the only way to battle the pandemic is to know the right information and practice the recommended health advisories. it is also necessary to examine the relationship among demographic variables with kap and fear to explore the in-depth understanding of factors contributing to the preparedness for and response towards covid-19. the study objectives were to determine the level of knowledge, attitudes, and practices related to covid-19 preventive health habits and the underlying fear of covid-19 in the bangladeshi population and how they are affected by socio-demographic factors. this study was a prospective cross-sectional survey conducted online through a structured questionnaire from april 4 to may 2, 2020. both male and female bangladeshi subjects, and aged 13 to 90 years, were able to respond to the questionnaire and were eligible for the study. subjects with an intellectual disability or an inability to communicate were excluded from the survey. a structured questionnaire has been designed by the authors to fulfill the objectives of the study. the questionnaire included socio-demographic variables (table 1) , questions on kap, and fear. questions related to kap adapted from the survey questions used in a study conducted during a period of rapid case increases in china [16] . the kap section of the questionnaire related to a total of 12 score knowledge questions on covid-19, categorical answers to attitudes towards the control of the pandemic, and practices of wearing masks and avoiding public gatherings. the co-morbidities and symptoms of covid-19 were obtained from who resources and asked to the respondents' whether present or not [17] . the fear of covid-19 scale (fcv 19s) was used and reported to be valid and reliable in measuring fear attributed to coronavirus disease [18] . the questionnaire complied with the forward and back-translation into bangla by a bilingual british researcher and sent to two renounced bilingual epidemiologists in bangladesh to examine the difference and suitability of the questionnaire. also, a pilot study was conducted before the commencement of the research. ethical permission was obtained from the institutional review board (bpa iprr/irb/29/03/ 2020/021) of the institute of physiotherapy, rehabilitation, and research (iprr). participation was voluntary, consent was obtained, and confidentiality of the information was assured. the trial registration was obtained prospectively from a primary trial registry of the who (ctri/ 2020/04/024413). from april to may 2020, the questionnaire was disseminated online, and through email and social media, targeting students, professionals, and public groups on facebook. recipients of the questionnaire were encouraged to complete it themselves and to send it to family members and neighbors for completion. a video tutorial was also provided to ensure an appropriate response. for illiterate family members, another member assisted them in responding to the questionnaire. the survey was requested to be sent back after completion. a total of 3500 questionnaires were sent, and 2200 questionnaires were returned. the data auditor found 2157 responses that could be included in the study and analyzed. the respondents were from all areas of bangladesh and may represent the whole population. descriptive statistics were employed for correct answers to knowledge, and diverse attitudes and practices were presented. knowledge, fear scores, attitudes, and practice variables of respondents were presented and compared with independent sample t-tests or chi-square tests to determine associations (tables 1 and 3 ) between continuous data (knowledge and fear score) and categorical or nominal data (demographic variables) [19] . binary logistic regression analysis using dichotomous demographic variables as dependent variables and knowledge and fear scores as covariates ( table 2 ) was performed to measure the relationship between categorical dependent variables and continuous variables. the chi-square test was employed to determine the relationship between attitude and practice with demographic variables, and knowledge and fear score (table 3 ). data analysis was completed using the ibm statistical package for the social sciences (spss) version 20.0. the alpha level of significance was set at p < .05. among 2157 respondents, 1166 (54.1%) were male and 991 (45.9%) were female. the mean population age was 33.48±14.65 years. the participants' ages ranged from 13 years to 88 years, and the majority of the respondents were aged 21-30 years (33.1%). respondents were categorized as adolescents (10-20 years), youth (21-40 years), adults (41-60 years), and elderly (above 60 years). there was a larger response among those with higher secondary education (32.3%) and graduates (27.6%). a total of 11.2% of respondents were either undergoing primary education or reported low levels of literacy. respondents were from all divisions of bangladesh; the highest response was from dhaka (23%), and the lowest was from sylhet (4.5%). the majority of the respondents were non-public servants (93%), 4% were healthcare professionals, 13.4% worked or did business in a crowded place, 43.1% were students, and 7.7% of the respondents reported that a relative, colleagues or a neighbor had been diagnosed with covid-19. other sociodemographic profiles are described in table 1 . multiple response analyses found that 28.69% of the respondents (n = 619) reported one or more symptoms related to covid-19 in the last 14 days, but none reported completing a covid-19 test during the response. the most prevalent symptoms were dry cough 19.5% (n = 121), cough with sputum (14.2%), sore throat (10%), fever of more than 100˚f (4.7%), anosmia or taste loss (4.5%), and shortness of breath (3.7%); 4 patients were diagnosed with pneumonia, and 3 patients were hospitalized for pneumonia. multiple response analyses also found 463 respondents (21.4%) who reported one or more co-morbidities, including diabetes (7.1%), chronic obstructive pulmonary disease (copd) (1.9%), and heart disease or hypertension (2.8%). nine subjects reported a chronic neurological disability, including stroke; 20 subjects reported chronic kidney disease (ckd), and 4.6% reported chronic smoking habits. in the population knowledge score, the mean was 8.71 out of 12, and the standard deviation was 1.64. knowledge regarding covid was similar in both males (8.75± 1.58) and females (8.66± 1.70). there was a significant relationship found between knowledge scores and age (p < .005), an education level (p < .001), and geographical distribution (p < .001). no significant differences in knowledge scores were found in the following comparisons: between public servants (8.93± 1.52) and others (8.69± 1.65); between healthcare professionals (8.99± 1.05) and others (8.70± 1.65); working in a crowd (8.80± 1.46) or working alone (8.70± 1.67); or in people who reported covid-19-positive relatives, friends or colleagues (8.80± 2.02) vs. those with associates without covid-19 (8.70± 1.60). significant differences were found between subjects with symptoms of covid-19 (9.04± 1.20) and subjects without covid-19 symptoms (8.58± 1.78) (p < .001). additionally, a significant difference (p < .001) was found in knowledge scores between subjects with co-morbidities (9.03± 1.26) and subjects without comorbidities (8.62± 1.72). the detailed associations are available in table 1 . binary logistic regression analysis showed a significant correlation between knowledge scores and gender (p < .005). logistic regression associations were found between knowledge and education levels, with the lowest knowledge scores found in primary education compared to all other education groups (p < .001). dhaka "urban dwellers" reported significantly higher knowledge of covid-19 symptoms and precautions than did subjects from rural areas of bangladesh (p < .001). knowledge and education levels were directly associated, with bachelor of science (bsc) degree holders reporting higher knowledge of covid-19 symptoms and precautions than any other education group (p < .005). public servants reported higher knowledge than did other non-public servant groups (p < .05), and students reported higher knowledge of covid-19 symptoms and precautions than did other non-student groups (p < .05). subjects without symptoms showed a significant inverse relationship with knowledge than did those with symptoms (p < .001) ( table 2 ). attitudes were measured concerning "beliefs" regarding whether bangladesh can overcome the challenge of covid-19 or "positive synergy" towards disease control. females reported a higher belief that covid-19 could be controlled (p < .05). similarly, graduates or more qualified respondents were confident that covid-19 can be controlled (p < .001) and agreed that bangladesh was capable of overcoming the challenge (p < .001). the majority of subjects who identified as public servants in bangladesh also reported belief that the disease was controllable (p < .05); however, they did not believe covid-19 would be overcome easily (p < .001). the subjects with a higher knowledge score of covid-19 and a higher score on the covid-19 fear scale also showed higher scores in "belief" that the virus was controllable (p < .005) and that eradication of the virus nationwide would be achieved (p < .001). subjects with covid-19 symptoms and co-morbidities reported a higher prevalence of the "belief" that the virus was both controllable and containable (p < .001). details are presented in table 3 . additionally, table 2 shows that the subjects with a higher knowledge score of covid-19 and a higher score on the covid-19 fear scale also showed higher scores on the "belief" that the virus was controllable (p < .001) and that eradication of the virus nationwide would be achieved (p < .001). practices were measured by the report of the subject's attendance in crowded areas and reports of wearing a mask. the majority of female subjects in the study followed the practice of staying home (37.5%) and wearing a mask (36.8%) to prevent the spread of covid-19 (p < .001). similarly, 27.9% of qualified personnel who were graduates and above reported staying home and avoiding crowded spaces (p < .005). the majority of the population from dhaka followed the health advisory by staying home (55.7%) (p < .001) and reported wearing a mask (65.3%) (p < .005). no significant relationship was found between knowledge score and practice, but a highly significant association was found between fear scores and adhering to the health advisory and between fear scores and reporting mask-wearing (p < .001). the majority of subjects with covid-19 symptoms reported wearing a mask (p < .05) but also reported going to a crowded place. the majority of subjects with co-morbidities also reported staying at home but did not report wearing a mask (p < .005) ( table 3) . table 2 explores a significant association (p < .001) between knowledge scores and wearing masks. the population means the fear score was 18.53 out of 35, with a standard deviation of 5.013. fear scores were strongly associated with gender, education and geography (p < .001), with females reporting a higher score (19.07± 5.04) and respondents aged 61-70 years, 71-80 years and 80-90 years reporting a higher score of fear of contracting covid-19 (19.00± 4.89; 19.44± 4.96; 20.00± 4.35). dhaka urban dwellers also reported a higher fear status than did rural dwellers (19.15± 5.02) (p < .001). the demographic relationship of fear scores is listed in table 1 . binary logistic regression found gender differences in fear scores (p < .001). other regressions are described in table 2 . an indirect, strong, but significant relationship (p < .001) was found between the fear scores and practices of recommended health advisory habits of subjects (table 3 ). there were significant differences (p < .005) in fear scores between subjects with symptoms and those without symptoms (18.73± 4.6; 18.45± 5.1) ( table 1) . inverse relationships were found among persons with positive covid-19 symptoms and fear scores (p < .005). table 2 shows a significant association (p < .001) between fear scores and wearing masks. the study intended to explore the knowledge, attitudes, and practices of recommended health advice for the prevention of covid-19 and to explore the impact of fear towards contracting covid-19 on people living in bangladesh. there is little to no research published on this important topic for bangladesh to date. the study covered every geographical area of bangladesh according to administrative distribution and provided a glimpse of the time frame. questions of knowledge, attitudes, and practices have been used following a chinese study [16] , which was relevant in terms of geographical distribution, as both bangladesh and china are on the asian continent. additionally, the time was relevant, the questionnaire was prepared for a rapid rise in cases, and bangladesh was the process of experiencing a rapid rise in cases from april to may 2020 as per who case definition [17] . the questionnaire development and translation completed in a structured and standard process. the fear scale was a valid questionnaire [18] and until the start of data collection, the bangla language validation had not been published. therefore, all the questionnaires have been translated with the who guidelines for translating questionnaires [20] . among the respondents, there were more males (54.1%) than females (45.9%). that dhaka had the majority of sars-cov-2 cases, and it is considered to have the most challenges regarding the level of practice of healthcare advisory precautions [22] . the baseline characteristics of subjects in comparative studies varied across the world. studies in india, china, and egypt had more responses from females, and the usa had more responses from males [16, [22] [23] [24] [25] indian, chinese, and egyptian studies had similar responses by age group and education, while the usa study reported a mean age higher than that in our study. our study found a satisfactory level of knowledge by gender, geography, occupation, and education (table 1 ) and relatively higher fear scores than those observed in similar studies across the world. one study in china showed similar scores of fear by age concerning knowledge and occupation, while another study completed in india reported that 80% of people in need of mental health care for covid-19 experienced fear, anxiety, and depression [16, 25] . twenty-nine percent (28.69%) of the respondents (n = 619) reported one or more symptoms related to covid-19 in the last 14 days, including cough 19.5% (n = 121), cough with sputum (14.2%), sore throat, (10%), fever (4.7%), anosmia or taste loss (4.5%), and shortness of breath (3.7%). the symptoms were related to covid-19, as per the cdc [26] . the who south-east asia region reported the test positivity in bangladesh to be 20%, with positive tests being reported only for a person with one or more covid-19-related symptoms [2] . eleven percent (11%) of subjects reported co-morbidities, including subjective disabilities. besides, 4.4% of the respondents were over 60 years of age. the who south-east asia region country profile and the iedcr covid-19 update states that the number of deaths is higher among elderly persons, males, and those with pre-existing co-morbidities in bangladesh [21, 27] . overall, we found a low number of elderly patients with symptoms, low reported levels of comorbidities, and a slightly higher rate of infection among males than among females. the reason behind the higher rate of infection among males is their greater exposure outside. at that time, bangladesh was in a state of "movement restriction", and no nationwide "lockdown" had been imposed [6, 15] , so more male cases were expected. the symptoms were matched with the who statements [17] of symptoms until then, and many cases were found to be relevant, but they were not tested. many reports have been published on unwillingness, droughts, and fear regarding covid-19 testing in bangladesh [3, 4] . knowledge regarding covid-19 by the subject was satisfactory and similar across age, gender, and occupation. there were a few variations in perspectives by occupation. young, graduates and urban dwellers had more knowledge than did older adults, those with lower education, and those living in rural areas. several similar articles in the preprint found that more than half of the respondents reported "good knowledge" of covid-19, with age and education showing a significant linear association with knowledge [27] [28] [29] . this study is similar to one study in china that found a significant relationship between knowledge and age and knowledge and educational level, with males reporting higher levels of knowledge than females regarding covid-19 symptoms, precautions, and health advisory practices [16] . however, in our study, subjects living in bangladesh reported similar knowledge for both males and females regarding covid-19 symptoms, precautions, and health advisory practices. this finding may be attributed to a similar degree of access to information through print and electronic media and internet access in bangladeshi populations, as the country's digital gateway is currently being prioritized. overall, a high prevalence of "positive attitudes" among the subjects regarding disease control was reported. female subjects and subjects with higher levels of education were more likely to believe that covid-19 can be controlled, but they doubted the ability of bangladesh to contain it. subjects with "good knowledge" or "high scores for fear" were more likely to believe that covid-19 can be controlled and that a collective effort can contain the spread of the disease. similar studies in bangladesh, india, and china all found similar results regarding the relationship between knowledge and fear of covid-19 regarding "practices", [16, 25, 30] , and our study reported similarities to previous studies across the world [23, 24] . our study found that 37.5% of women reported "staying home", and 36.8% reported wearing masks in public places. the majority of the population outside of dhaka, i.e., those living in the more rural regions, reported staying home (55.7%) and wearing a mask (65.3%). however, no statistical relationship was found between knowledge scores and practices. this is similar to results reported in studies in both india and china. however, our study did find subjects with high fear scores and who were also more likely to follow good preventive practices, as recommended by the health advisory. the fear score was significantly associated with female gender, higher education, and urban dwelling. senior citizens aged 61-70 years, 71-80 years, and 80-90 years reported the highest fear scores among individuals in all categories. one study suggests that fear comes from a longer duration of isolation, greater movement restriction, and greater reactivity to news and rumors from social media [31] . women, senior citizens, and young adults had limited movement, were isolated from quarantining, and were attached to media. a study reports that fear and stress can lead to insomnia and psychological illness [25] . fear is an important component in both positive and negative ways, as illustrated in the positivity explained earlier; hence, there have been cases of suicide due to the fear of covid-19 in bangladesh [13] . however, although important as an indicator, this was not evaluated in this study and may be considered a limitation of the study. our study faced challenges regarding structured questionnaires, reporting, and resources. limitations included the response rate (62.85%) and the completion of the questionnaire by none covid-19-positive individuals. we recommend that future studies include information on the long-term observation of corvid-19-positive cases or cases with symptoms with respect to movement, function, physical signs, mental health, and quality-of-life issues. in a resource-challenged country such as bangladesh, individual knowledge, positive attitudes, and practices of suggested precautionary and preventive health advisories are crucial to controlling the vicious community transmission of covid-19. the study found that knowledge levels were adequate in the majority of the population and were directly and significantly related to higher education levels, younger age, and female gender. there were positive attitudes among respondents regarding the control of the disease and the overcoming of challenges related to covid-19 in bangladesh. the majority of the population had high fear scores, with significantly higher scores found in women and elderly adults. surprisingly, those with higher fear scores had good practices of staying at home and wearing masks. future studies on explanatory issues related to activity, function, social issues, and quality of life might add more insight into the bio-psychological impact of covid-19 in the most densely populated country in the world. covid-19: bangladesh records highest 42 deaths in a day, cases cross 65,000 mark official covid-19 numbers disguise undercounting bangladesh cannot afford to close its garment factories [internet]. the economist. 2020 [cited 3rd covid-19 infections are rising fast in bangladesh, india and pakistan fighting cyclones and coronavirus: how we evacuated millions during a pandemic covid-19 tracker. bangladesh computer council (bcc) coronavirus disease 2019 (covid-19) information bangladesh.corona.gov.bd epidemiology, causes, clinical manifestation and diagnosis, prevention and control of coronavirus disease (covid-19) during the early outbreak period: a scoping review preventing a covid-19 pandemic covid-19 strategy update-14 mental health and psychosocial support aspects of the covid-19 response pandemic fear" and covid-19: mental health burden and strategies first covid-19 suicide case in bangladesh due to fear of covid-19 and xenophobia: possible suicide prevention strategies covid-19 and bangladesh: challenges and how to address them. frontiers in public health fears grow over bangladesh's covid-19 response knowledge, attitudes, and practices towards covid-19 among chinese residents during the rapid rise period of the covid-19 outbreak: a quick online cross-sectional survey the fear of covid-19 scale: development and initial validation the chi-square test of independence who | process of translation and adaptation of instruments covid-19: bangladesh records highest 45 deaths, 3,171 cases in a day knowledge, perceptions, and attitude of egyptians towards the novel coronavirus disease (covid-19) knowledge and behaviors toward covid-19 among us residents during the early days of the pandemic: cross-sectional online questionnaire. jmir public health and surveillance study of knowledge, attitude, anxiety & perceived mental healthcare need in indian population during covid-19 pandemic centers for disease control and prevention attitude, and practice regarding covid-19 outbreak in bangladeshi people: an online-based cross-sectional study attitude and practices (kap) towards covid-19 and assessment of risks of infection by sars-cov-2 among the bangladeshi population: an online cross sectional survey knowledge and attitude towards covid-19 in bangladesh: population-level estimation and a comparison of data obtained by phone and online survey methods the covid-19 outbreak: crucial role the psychiatrists can play authors acknowledge rubayet shafin and ahnaf al mukit, research assistants for their contribution in data collection and input, also students of bangladesh health professions institute helped in collecting data. the authors are also grateful to professor dr. md. forhad hossain for his support in statistical analysis. key: cord-286613-cmtsu73g authors: lee, sung woo; yu, mi-yeon; lee, hajeong; ahn, shin young; kim, sejoong; chin, ho jun; na, ki young title: risk factors for acute kidney injury and in-hospital mortality in patients receiving extracorporeal membrane oxygenation date: 2015-10-15 journal: plos one doi: 10.1371/journal.pone.0140674 sha: doc_id: 286613 cord_uid: cmtsu73g background and objectives: although acute kidney injury (aki) is the most frequent complication in patients receiving extracorporeal membrane oxygenation (ecmo), few studies have been conducted on the risk factors of aki. we performed this study to identify the risk factors of aki associated with in-hospital mortality. methods: data from 322 adult patients receiving ecmo were analyzed. aki and its stages were defined according to kidney disease improving global outcomes (kdigo) classifications. variables within 24 h before ecmo insertion were collected and analyzed for the associations with aki and in-hospital mortality. results: stage 3 aki was associated with in-hospital mortality, with a hazard ratio (hr) (95% ci) of 2.690 (1.472–4.915) compared to non-aki (p = 0.001). the simplified acute physiology score 2 (saps2) and serum sodium level were also associated with in-hospital mortality, with hrs of 1.02 (1.004–1.035) per 1 score increase (p = 0.01) and 1.042 (1.014–1.070) per 1 mmol/l increase (p = 0.003). the initial pump speed of ecmo was significantly related to in-hospital mortality with a hr of 1.333 (1.020–1.742) per 1,000 rpm increase (p = 0.04). the pump speed was also associated with aki (p = 0.02) and stage 3 aki (p = 0.03) with ors (95% ci) of 2.018 (1.129–3.609) and 1.576 (1.058–2.348), respectively. we also found that the red cell distribution width (rdw) above 14.1% was significantly related to stage 3 aki. conclusion: the initial pump speed of ecmo was a significant risk factor of in-hospital mortality and aki in patients receiving ecmo. the rdw was a risk factor of stage 3 aki. the initial pump speed of ecmo was a significant risk factor of in-hospital mortality and aki in patients receiving ecmo. the rdw was a risk factor of stage 3 aki. receiving continuous renal replacement therapy when ecmo were initiated (n = 66), if they initiated continuous renal replacement therapy on the date of ecmo insertion (n = 77). therefore, 322 patients were ultimately analyzed in the present study. the physiologic and laboratory data within 24 h before ecmo initiation were collected retrospectively through a review of the electronic medical records. the clinical parameters that were recorded included the following: age, sex, causes of admission; causes of ecmo support, mode of ecmo, whether to perform cardiopulmonary resuscitation within 24 h, use of an intra-aortic balloon pump (iabp), ecmo settings, duration of ecmo, urine output, and ventilator settings. initial blood findings, including blood urea nitrogen (bun), total bilirubin, albumin, white blood cells, hemoglobin level, platelet number, red cell distribution width (rdw), sodium, potassium, chloride, and c-reactive protein (crp) were measured. for the severity index, we used the simplified acute physiology score 2 (saps2) [15] . to calculate the saps2, the worst values during the first 24 h before ecmo initiation were used. aki and the stage of its severity were defined according to the guidelines proposed by kdigo [16] . aki was defined in a case with either an increase in serum creatinine by 26.5 μmol/l or 1.5 times the baseline within 48 h. the changes in serum creatinine according to the aki stages were as follows: stage 1, an increase of more than or equal to 26.5 μmol/l or an increase to more than or equal to 1.5-to 2-fold of the baseline; stage 2, an increase to more than 2-to 3-fold of the baseline; stage 3, an increase to more than 3-fold of the baseline or more than or equal to 353.6 μmol/l with an acute increase of at least 44.2 μmol/ l or on renal replacement therapy. the maximum aki stage reached during ecmo support was used to define the incidence of aki [17] . in-hospital mortality was determined whether a death certificate had been issued or not at 90 d after ecmo insertion. the applied ecmo console was composed of a centrifugal pump and membrane oxygenator. the products utilized included capiox ebs (terumo corporation, tokyo, japan) and quadrox pls (maquet, hirrlingen, germany). the values were expressed as the mean ± standard deviation in continuous variables and n (%) in categorical variables. for the severely skewed variables, such as follow-up duration, the median (interquartile range, iqr) was used. the difference was analyzed by an independent ttest in continuous variables and chi-square test in categorical variables. for the estimated survival, the kaplan-meier method was employed, and the statistical significance was calculated using the log-rank test. for multivariate analysis, logistic regression analysis for aki and coxproportional hazard analysis for in-hospital mortality were carried out. the variables in the multivariate analysis were chosen by p <0.05 in the univariate analysis. calibration was done using the hosmer-lemeshow goodness-of-fit test to compare the numbers of predicted and observed in-hospital mortality and aki. discrimination was analyzed using auroc. the best threshold was calculated by obtaining the best youden index (sensitivity + specificity-1). we consider p <0.05 to be statistically significant. all of the analyses were performed using the spss statistics software (version 22, ibm, usa). the mean age of the study participants was 60.3 ± 15.3 years and 213 (66.1%) of the participants were male. the reasons for admission were cardiovascular disease (203, 63.0%), lung disease (49, 15.2%), malignancy (35, 10 .9%) and others (35, 10 .9%). one hundred and thirty seven (42.5%) patients had received cardiopulmonary resuscitation within 24 h prior to ecmo initiation. after the median (iqr) 2 (0-10) days of admission, the patients received ecmo insertion because of cardiotomy (31, 9. 6%), non-operative cardiovascular causes (185, 57.5%), adult respiratory distress syndrome (ards) (43, 13.4%), non-ards lung causes (44, 13.7%) and other causes (19, 5.9%) . two hundred and thirty (71.4%) and 92 (28.6%) patients received va and vv ecmo support, respectively. one hundred and six (32.9%) patients were undergoing iabp on the date of ecmo insertion. the median (iqr) duration from ecmo initiation to death or discharge was 21 days. the incidence of aki comprising all kdigo grades was 82.3%. in-hospital mortality was 51.6%. the median (iqr) durations for aki and in-hospital mortality were 2 (1-7) days and 9 (4-23) days, respectively. we explored the factors associated with in-hospital mortality. aki developed less frequently in the survivor group than in the non-survivor group. moreover, stage 3 aki developed significantly less in the survivors than in the non-survivors. saps2 and the serum sodium level were significantly lower in the survivors than in the non-survivors. ventilator settings, such as positive end expiratory pressure and peak inspiratory pressure before ecmo insertion, did not affect the survival rate. the ecmo pump speed was significantly lower in the survivors than in the non-survivors. age, causes of admission, causes of ecmo support, mode of ecmo, use of iabp, length of stay before ecmo insertion, duration of ecmo support, initial urine output, bun, creatinine, rdw and crp were associated with in-hospital mortality ( table 1) . we performed a multivariate cox-proportional hazard regression analysis to adjust confounding effects among the selected variables. compared to the non-aki group, the stage 3 aki group significantly increased the risk of in-hospital mortality whereas the stage 1 and 2 aki groups did not (table 2 ). in the kaplan-meier survival curves according to the stages of aki, the estimated mean (95% ci) survival in the non-aki group and the stage 1, 2, and 3 groups were 65.7 (55.2-76.2) days, 54.0 (45.8-62.3) days, 53.8 (38.7-69.0) days and 33.6 (27.9-39.4) days, respectively (p < 0.001 by log-rank test). in the post-hoc analysis, the stage 3 aki group, but not the stage 1 (p = 0.14) or 2 (p = 0.43) aki groups, showed a significant difference in survival compared with the non-aki group (fig 1) . with every increment in saps2, serum sodium level, and ecmo pump speed (1 score in saps2, 1 mmol/l in serum sodium level, and 1,000 rpm in ecmo pump speed), the risks of in-hospital mortality were increased, with hrs (95% ci, p-value) of 1.02 (1.004-1.035, 0.01), 1.042 (1.014-1.070, 0.003) and 1.333 (1.020-1.742, 0.04), respectively ( table 2) . we performed a calibration and discrimination analysis of saps2, serum sodium level, and ecmo pump speed to predict in-hospital mortality. all three variables were well-calibrated. the auroc analysis showed the discriminative power of these variables. the cut-off values of saps2, serum sodium level, and ecmo pump speed for in-hospital mortality were a score of 69.5, 147.6 mmol/l, and 2.19 x 10 3 rpm, respectively (table 3) . we compared clinical characteristics according to the mode of ecmo. the length of the hospital stay before ecmo insertion was shorter in patients with va mode than in those with vv mode. the level of crp was lower in the va mode group than in the vv mode group. nonetheless, saps2 was not different between the two groups. the initial ecmo settings were also comparable between the two groups. according to the linear regression analysis, there was no correlation between saps2 and ecmo speed either in vv mode (r 2 = 0.003, p = 0.59) or va mode (r 2 = 0.001, p = 0.709). the mortality within 2 weeks after ecmo insertion was significantly higher in patients with va mode than in those with vv mode (p = 0.03), whereas the overall in-hospital mortality was significantly lower in the va mode group than that in the vv mode group (p = 0.02). compared to the patients with the vv mode, those with the va mode had shorter stays in the intensive care unit and hospital; however, there was no difference in the occurrence of aki between the two groups (table 4 ). because aki, especially stage 3 aki, showed a significant association with in-hospital mortality, we attempted to detect the risk factors associated with aki and stage 3 aki. we compared the characteristics between the patients with and without aki. the initial ecmo pump (table 5) . these variables were also significant risk factors for developing stage 3 aki (table 6) . there was an additional risk factor in stage 3 aki. the rdw was significantly lower in those without stage 3 aki than in those with stage 3 aki. in the multivariate logistic regression analysis, the rdw was still statistically significant, with an or (95% ci, p-value) of 1.308 (1.053-1.625, 0.02) for every 1% increase (table 6 ). in the calibration and discrimination analysis, stage 3 aki was well-calibrated and discriminated by a cut-off value of 14.1% for rdw (table 3) . we compared patient characteristics according to the rdw status. patients with an rdw above 14.1% showed significantly higher level of crp than did those with an rdw below 14.1%. moreover, patients with an rdw above 14.1% showed considerably lower hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration than did those with an rdw below 14.1% (table 7) . in this work, we investigated the risk factors of aki and in-hospital mortality in patients receiving ecmo support. here, we found that the initial pump speed of ecmo was associated with in-hospital mortality and aki. the elevated rdw could be suggested as the risk factor for severe aki in these patients. this was the first study to identify the risk factors of aki in adult patients receiving ecmo support. because aki is the most common complication and a major risk factor of mortality, defining the risk factors for aki in these patients is extremely important [9] [10] [11] [12] [13] [14] . this study is the largest ecmo assessment ever reported. moreover, the association of pump speed with aki and mortality is a novel finding. we showed that aki, especially stage 3 aki, was a significant risk factor for in-hospital mortality in patients receiving ecmo support. saps2 and serum sodium level were also important risk factors of in-hospital mortality. along with these well-known and expected findings [11] [12] [13] [18] [19] [20] , we found that the initial pump speed of ecmo was significantly related to in-hospital mortality, with a 33% increased risk for every 1,000 rpm increase. the initial pump speed of ecmo was also a risk factor for both aki and stage 3 aki. on the other hand, the blood flow rate of ecmo was not associated with in-hospital mortality or aki. why a high pump speed, but not a high blood flow rate of ecmo, increases the risk of in-hospital mortality and aki is not clear at this time. however, the ecmo pump can induce hemolysis, leukocyte and platelet destruction, and complement activation [21, 22] . blood flow through the ecmo circuit is driven by centrifugal pump. a rotating impeller in centrifugal pumps spins, which creates a constrained vortex that suctions blood into the pump and propels it out toward the membrane oxygenator [23] . hemolysis has been reported to be associated with aki [24] . in addition, lou et al. found that the pump speed was a risk factor for hemolysis and that hemolysis was associated with adverse outcomes in pediatric patients receiving ecmo [25] . although we did not evaluate the degree of hemolysis in our patients, we postulate that hemolysis caused by high revolutions of the ecmo pump might result in aki and in-hospital mortality. to provide stable cardiac output in the va mode and adequate oxygenation in the vv mode, adequate blood flow should be maintained. therefore, clinicians raise the ecmo pump speed as much as possible to maintain adequate blood flow. the blood flow rate that was applied to 90% of our patients was less than 4.1 l/min. a high blood flow extracorporeal circuit that pumped up to 7 l/min [26] did not apply to our patients; however, 43.8% (141/321) of our patients were treated with a pump speed higher than the cut-off value of 2.19 x 10 3 rpm. for these reasons, we speculate that pump speed, but not a blood flow, is a predictor of death in this study. we compared the clinical characteristics of patients from the va and vv ecmo modes. patients with the vv mode had higher levels of crp, showed higher mortality, and had longer stays in the hospital compared with those with the va mode; however, the mortality within 2 weeks after ecmo insertion was higher in patients with the va mode. we speculated that the patients with the va mode deteriorated rapidly but recovered soon if they were not severe enough for death. in contrast, patients with the vv mode seemed to show slower but poorer outcomes than those with the va mode. the different disease process of the patients treated with the va and vv ecmo modes [27] might be related to these findings. future prospective studies will be needed to investigate whether ecmo mode determines outcomes. in this study, the higher the rdw was, the more frequently stage 3 aki occurred. to the best of our knowledge, this is the first study to suggest a potential role of the rdw in aki. recently, the use of the rdw as a simple and inexpensive biomarker to predict mortality in chronic heart failure [18, 28] , liver disease [29] , and critical illness [30] has increased. moreover, the rdw has been reported to be associated with many pathological conditions such as colon cancer, inflammatory bowel disease, celiac disease, rheumatoid arthritis, alzheimer's disease, and contrast-induced nephropathy [31, 32] . although the exact mechanism of this relationship is not clear, inflammation is a proposed underlying factor [33, 34] . this proposed factor can also be supported by our data, which indicate that the elevated rdw was associated with high crp levels in the patients. in this study, patients with an rdw greater than 14.1% showed lower rbc indices than did those with an rdw less than 14.1%. because anemia is a risk factor for aki [35] , the low rbc indices found in the elevated rdw group might contribute to increase the odds of stage 3 aki occurring. the current study suffered from several limitations. first, this study is a retrospective cohort study; however, the variables before ecmo insertion were well retrieved with a less than 10% missing rate. moreover, this is the largest study to explore the association of aki and mortality in patients receiving ecmo support [11] [12] [13] . a low level of missing data and a large number of patients could partially compensate for the weakness of the study design. second, we classified the patients into their kdigo stage based only on their serum creatinine concentration. urine volume is a sensitive marker for the early detection of aki in patients on ecmo. decreased urine volume during ecmo treatment and/or on the day of ecmo removal can be attributed to decreased cardiac output following decannulation, and can be correlated with acute cardiorenal syndrome type 1 [27, 36, 37] . third, we could not provide direct evidence that hemolysis due to a high pump speed resulted in aki in this study. we should have measured plasma-free hemoglobin, which is an indicator of hemolysis. furthermore, we did not obtain information on the cannulation site and mean venous pressure in the ecmo circuit. finally, this study was composed of data from two centers, which could limit the generalizability. in conclusion, aki is a significant risk factor for in-hospital mortality in patients receiving ecmo support. the initial pump speed of ecmo is associated with in-hospital mortality and strongly related to aki, especially stage 3 aki. therefore, once adequate blood flow is maintained, clinicians must be careful not to further increase the ecmo pump speed. because the elevated rdw was also strongly related to stage 3 aki, special attention should be paid to patients with abnormal rdw values to prevent aki. extracorporeal membrane oxygenation in severe acute respiratory failure. a randomized prospective study low-frequency positivepressure ventilation with extracorporeal co2 removal in severe acute respiratory 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extracorporeal membrane oxygenation: the impact of acute kidney injury on mortality predictors of mortality in patients successfully weaned from extracorporeal membrane oxygenation key: cord-295293-ickp2n47 authors: latsuzbaia, ardashel; herold, malte; bertemes, jean-paul; mossong, joël title: evolving social contact patterns during the covid-19 crisis in luxembourg date: 2020-08-06 journal: plos one doi: 10.1371/journal.pone.0237128 sha: doc_id: 295293 cord_uid: ickp2n47 we conducted an internet survey using survey monkey over six weeks to evaluate the impact of the government interventions on social contact patterns in luxembourg. participants were recruited via the science.lu website on march 25, april 2, april 16, may 1 during lockdown, and june 12 and june 25 after the lockdown to provide an estimate of their number of contacts within the previous 24 hours. during the lockdown, a total of 5,644 survey participants with a mean age of 44.2 years reported 18,118 contacts (mean = 3.2, iqr 1–4). the average number of contacts per day increased by 24% from 2.9 to 3.6 over the lockdown period. the average number of contacts decreased with age: 4.2 (iqr 2–5) for participants below 25 years and 1.7 (iqr 1–2) for participants above 64 years. residents of portuguese nationality reported a higher number of contacts (mean = 4.3, iqr 2–5) than luxembourgish (mean = 3.5, iqr 2–4) or other foreign residents, respectively. after lockdown, 1,119 participants reported 7,974 contacts with 7.1 (iqr 3–9) contacts per day on average, of which 61.7% (4,917/7,974) occurred without a facemask (mean = 4.9, iqr 2–6). while the number of social contacts was substantially lower during the lockdown by more than 80% compared to the pre-pandemic period, we observed a more recent 121% increase during the post lockdown period showing an increased potential for covid-19 spread. monitoring social contacts is an important indicator to estimate the possible impact of government interventions on social contacts and the covid-19 spread in the coming months. covid-19 has become a global public health emergency affecting more than 200 countries and territories resulting in more than 10 million reported cases by june 30 and over 500,000 deaths [1] . as of june 30, luxembourg reported 4,299 cases and 110 deaths (fig 1) [2] . following the closure of schools, sports facilities, non-food shops, bars and restaurants on march 16, the luxembourg government declared a state of emergency on march 18 implementing strict social distancing measures and instructing the local population to stay at home except for essential work and to avoid all unnecessary social interactions. although social gatherings were prohibited, people were free to go outside while maintaining a physical distance of two a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 meters or more. five surgical masks were distributed to every resident on april 17-24 followed by the first easing of lockdown phase on april 20 with reopening of construction sites and recycling centres. the second phase started with reopening of final grades of secondary schools on may 4 and secondary schools one week later with reduced class size. wearing a facemask became mandatory in a public area if a two-metre distance could not be maintained. fifty additional surgical masks were distributed to every resident during the second easing of lockdown phase and gatherings of up to six people (plus household members) indoors and 20 people outdoors were authorised. the third phase was initiated on 25th of may reopening elementary schools with reduced class size, restaurants (maximum of 10 persons per table) and cafes (only table service) including mandatory face masks for staff and guests when not sitting at the table. covid-19 spreads via the respiratory route to close contacts and social contact patterns are therefore a key factor shaping the spread of covid-19 and other infectious agents in a population [3, 4] . contact surveys are an important methodological approach to assess social mixing as well the impact of control measures such as quarantine, travel restrictions or social distance measures, or lockdown in general [4] [5] [6] . previous work from the uk suggests that lockdown measures may have decreased the reproduction number from 2.6 to 0.62 [6] . similarly, researchers from china have shown a significant decrease of the reproduction number below one following physical restriction measures [7] . social contact patterns differ across european countries. according to the polymed study luxembourg resident reported 17.5 social contacts per day before the pandemic, similar to numbers reported for italy (19.8). belgian, british and german residents, for example, reported 11.8, 11.7 and 8.0 social contacts per day, respectively [4] . additionally, luxembourg has a unique demographic structure with a foreign population of nearly 50%, hence this population requires specific communication strategies targeting local and foreign communities. we repeatedly conducted an internet survey to follow up the impact of the local government interventions on social contact patterns in luxembourg shortly after the lockdown was implemented due to the rapid local spread of the covid-19. in addition, our study provides insights on social contact patterns by age group and nationality, which can be important for identifying groups less compliant to imposed restrictions. recruitment of participants occurred via sharing of a survey link on the social media platforms facebook and twitter to followers and readers of the science.lu website following the publication of a general interest article on covid-19 [8] . individuals were requested to fill in an online questionnaire to self-report their daily number of contacts. the first survey was for the first wave, the survey collected age category, number of individuals living in the household other than the respondent and number of contacts within the last 24 hours excluding members of the household. from april 2, we expanded the questionnaire by recording nationality and the location where most contacts occurred (s1 file). the post lockdown survey included additional question to identify the number of contacts without wearing a facemask (s2 file). two more categories were added to identify the place of contact with a multiplechoice answer. a social contact was defined as a face-to-face conversation with more than three words at a distance of less than two meters. the total number of contacts was estimated by adding the reported number of contacts outside the household to the number of individuals living in the household. similarly, contacts without a facemask were calculated by adding the number of contacts without a facemask to individuals living in the household (assuming participants from the same household do not wear a mask at home). the survey was purposely designed to have only a small number of questions (duration less than a minute) with available translations in three languages (german, french and english) to ensure high participation and completion. ethical approval for this study was waived by the luxembourg ministry of health and the national ethics committee for research. study participants were informed on how collected data was processed and utilized. the mean number of social contacts per person was calculated and stratified by age category, nationality, household size, location of most contacts and sampling week. the number of contacts in the questionnaire "10 or more" was counted as 10 contacts and "6-9" was averaged to 7.5. similarly, in the post lockdown follow up survey, the number of contacts in the questionnaire "25 or more" was counted as 25 contacts. to estimate the number of contacts related to the place of occurrence, the total number of contacts was divided by the total number of places where contacts occurred assuming that participants would have a similar number of contacts in indicated places of contact. we compared the mean number of daily participants to number of contacts from a large contact survey conducted in luxembourg before the pandemic between may 2005 and september 2006 [4] . the total number of social contacts was adjusted representative to the population age structure (s1 table) . the age adjusted average number of reported contacts was calculated by multiplying the average number of contacts in each age group by the actual proportion of that age group from national population data [9] . a poisson regression was performed to evaluate factors influencing the number of contacts. variables significantly associated with the number of social contacts in univariate regression were selected for the multivariable model. the language variable was excluded from the model due to significant correlation with nationality (r = 0.45, p<0.05). the statistical analysis was performed in stata 14 (college station, texas usa). the effective basic reproduction number estimates and graph were downloaded from the epiforecasts platform (https://epiforecasts.io/) [10, 11] . between march 25 and may 1, a total of 5,644 (mean age 44.2 years) respondents participated in the online survey, of which 4.9% were under 25 years of age and 5.0% of participants were over 64 years of age (table 1) . of 3,683 respondents reporting nationality, 60.3% (2,221/3,683) were luxembourgish and 39.7% (1462/3,683) were foreign residents ( table 1) . the total number of reported contacts was 18,118, while the average number of daily contacts was 3.2 (95% ci 3.1-3.3, iqr 1-4). after adjusting for age structure, the average number of daily contacts was 3.1. the average number of contacts reported by luxembourg residents in a study before the pandemic was 17.5 [4] , suggesting that contacts during lockdown had decreased by 81.7%. we observed a consistent decline across all age groups and household sizes. the mean number of reported contacts was significantly higher (p<0.001) in young participants: 4.2 (95% ci 3.9-4.8, iqr 3-5) reported by participants below 25 years compared to 1.7 (95% ci 1.6-1.9, iqr 1-2) for participants above 64 years ( table 1) . residents of portuguese nationality reported a significantly higher (p<0.001) number of contacts (mean = 4.3 [95% ci 3.9-4.8, iqr 2-5]) than luxembourgish residents (mean = 3.5 [95% ci 3.3-3.6, iqr 2-4] or other foreign residents ( table 1 ). the mean number of contacts was significantly higher (p<0.001) for the survey when conducted in french language (mean = 3.6 [95%ci 3.5-3.7, iqr 2-5]) compared to german (mean = 3.2 [95% ci 3.1-3.3, iqr 1-4]) and english (mean = 2.8 [95%ci 2.6-2.9, iqr 1-4]) language. we observed a significant variation of the average number contacts depending on the place where most contacts occurred. the highest number of contacts was reported for most contacts at work (mean = 6.2 [95% ci 5.9-6.5, iqr 3-9]), while the lowest number of contacts was reported for most contacts during leisure (mean = 3.2 [95%ci 3.0-3.5, ) and at the supermarket (mean = 2.9 [95%ci 2.7-3.0, iqr 1-4]) ( table 1 ). the average number of contacts reported at work increased by 6.7% from 5.9 (95% ci 5.2-6.6, iqr 3-8) to 6.3 (95% ci 5.9-6.8, iqr 4-9), while average number of contacts during leisure activities increased by 34.6% from 2.6 (95% ci 2.2-3.1, iqr 1-4) to 3.5 (95% ci 3.5-4.0, iqr 2-5) ( table 1 and fig 2) . the average number of contacts per day significantly increased (p<0.001) by 24.1% over the lockdown period from 2.9 (95%ci 2.8-3.0, iqr 1-4) to 3.6 (95%ci 3.49-3.79, iqr 2-5) ( table 1 and fig 3) . in the post lockdown period, 1,119 participants filled in the survey (mean age = 46.3) reporting 7,974 contacts. the average number of daily contacts significantly increased from 3.2 during the lockdown to 7.1 after lockdown (95% ci 6.8-7.5, iqr 3-9) (fig 3) (p<0.05) . after adjusting for age structure, the average number of daily contacts was 6.7. the increase was consistent across all categories (table 2, figs 4 and 5). of the total number of contacts, 61.7% (4,917/ 7,974) reported a contact without a facemask (mean = 4.9, iqr 2-6). univariate poisson regression analysis showed that age above 55 years, foreign nationality (other than portuguese), as well as english survey language were associated with a lower number of contacts (table 3) . survey sampling week, portuguese nationality and french survey language were associated with higher number of contacts. in multivariable regression, age, foreign nationality (other than belgian) and calendar date remained significant predictors of the number of social contacts (table 3 ). as shown in s1 fig, the effective reproduction number in luxembourg dropped below one shortly after lockdown (s1 fig) [10, 11] and remained below one during the full lockdown period. the effective reproduction number increased to levels close to unity from the beginning of june onwards, although large confidence intervals were observed due to very low number of new daily cases. our study suggests that the strict physical distancing measures implemented in luxembourg had a substantial and immediate impact on social mixing patterns resulting in a large reduction of the average number of contacts per day. during the early lockdown period, survey participants reported 3.2 contacts on average, which is 82% lower than during the non-pandemic period [4] . this decline was consistently observed across all age groups and household sizes. our study findings are similar to those from shanghai, wuhan and the uk showing 88%, 86% (2, 6) and 73% reduction in the average number of daily contacts, respectively [6, 7] . in these studies, the reduction of contacts was estimated to have resulted in a significant decrease of the basic reproduction number r 0 below one [5] . similar to these estimates, our results explain the rapid decline in sars-cov-19 transmission, also resulting in the rapid decline of covid-19 cases observed since the beginning of the lockdown in luxembourg. although in luxembourg the incidence of infections has been dropping to single figures by early may, further relaxing of physical distance restrictions poses significant risk for transmission. relaxing restrictions too early could lead to an earlier second wave leading to further tightening of restrictions [12, 13] . between march 25 and may 1, the average number of contacts increased by 19% associated with an increasing number of contacts at work and during leisure activities. this increase occurred after the easing of lockdown phase 1 on april 20, when construction sites and recycling centers were reopened. in june, during the post lockdown period the number of average contacts increased to 7.1, nevertheless it remains 59% lower than during the pre-pandemic period [4] . this increase in social contacts most likely resulted in the increase of the reproduction number followed by growing covid-19 incidence that had been observed by the end of june. in addition, more than half of the contacts in the post lockdown period occurred without wearing a facemask increasing the transmission risk [14] . our results suggest that older individuals are more compliant with restriction measures compared to younger persons, which is expected since the risk of hospitalization and death from covid-19 increases with age [5] . similarly, results of a large survey study conducted by del fava et al. showed that participants older than 65 years have a decreased number of contacts in belgium, france, germany, italy, netherlands, spain and united kingdom [15] . residents of portuguese nationality had more daily contacts compared to other residents, which could be work related, as 27% of portuguese participants recorded contacts at work during lockdown. in luxembourg, portuguese residents represent the largest foreign community accounting for 15% of the total population and appear to be at increased risk for transmission [9] . direct communication with these foreign communities could help to ensure compliance with physical distancing measures. one limitation of our study is that we potentially overestimated the effect of lockdown due to selection bias. compliant individuals following strict restrictions might be more active on social media and thus more likely to come across the survey. secondly, we have to take into account that our study sample was not representative of the general population in terms of age structure and nationality: participants below 24 and above 65 years of age were underrepresented. similarly, participants of luxembourgish, french, belgian and german nationality were overrepresented, portuguese residents were underrepresented. nevertheless, the adjusted mean number of contacts during lockdown was similar to the non-adjusted number of contacts. another limitation of our study is that the pre-pandemic survey conducted in 2007 was conducted using a paper diary approach and our online approach might lead to lower ascertainment of contact numbers. furthermore, the online survey does not account for multiple responses by a single respondent. we did not collect any further data on contacts (e.g. age), thus were unable to construct age matrix and estimate exact reduction of basic reproduction number. on the other hand, 6,766 respondents filled in the survey representing over 1% of total population in luxembourg. in conclusion, our stud shows that physical distance measures resulted in significant reduction in social contacts and therefore decreased the spread of covid-19 in luxembourg. monitoring social contacts patterns using online surveys provides valuable early evidence of the effects of both lockdown and easying of lockdown measures on transmission and could be easily adapted to be used in different countries and regions in the world. situation update worldwide, as of the contribution of social behaviour to the transmission of influenza a in a human population social contacts and mixing 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modelling the differential impact of physical distancing strategies on social contacts relevant for the spread of covid-19 data curation: ardashel latsuzbaia, jean-paul bertemes. key: cord-281364-syg0wo77 authors: caì, yíngyún; yú, shuǐqìng; postnikova, elena n.; mazur, steven; bernbaum, john g.; burk, robin; zhāng, téngfēi; radoshitzky, sheli r.; müller, marcel a.; jordan, ingo; bollinger, laura; hensley, lisa e.; jahrling, peter b.; kuhn, jens h. title: cd26/dpp4 cell-surface expression in bat cells correlates with bat cell susceptibility to middle east respiratory syndrome coronavirus (mers-cov) infection and evolution of persistent infection date: 2014-11-19 journal: plos one doi: 10.1371/journal.pone.0112060 sha: doc_id: 281364 cord_uid: syg0wo77 middle east respiratory syndrome coronavirus (mers-cov) is a recently isolated betacoronavirus identified as the etiologic agent of a frequently fatal disease in western asia, middle east respiratory syndrome. attempts to identify the natural reservoirs of mers-cov have focused in part on dromedaries. bats are also suspected to be reservoirs based on frequent detection of other betacoronaviruses in these mammals. for this study, ten distinct cell lines derived from bats of divergent species were exposed to mers-cov. plaque assays, immunofluorescence assays, and transmission electron microscopy confirmed that six bat cell lines can be productively infected. we found that the susceptibility or resistance of these bat cell lines directly correlates with the presence or absence of cell surface-expressed cd26/dpp4, the functional human receptor for mers-cov. human anti-cd26/dpp4 antibodies inhibited infection of susceptible bat cells in a dose-dependent manner. overexpression of human cd26/dpp4 receptor conferred mers-cov susceptibility to resistant bat cell lines. finally, sequential passage of mers-cov in permissive bat cells established persistent infection with concomitant downregulation of cd26/dpp4 surface expression. together, these results imply that bats indeed could be among the mers-cov host spectrum, and that cellular restriction of mers-cov is determined by cd26/dpp4 expression rather than by downstream restriction factors. in 2012, a novel human coronavirus causing frequently fatal disease emerged in western asia [1] and was named ''middle east respiratory syndrome coronavirus (mers-cov)'' [2] . as of june 11, 2014, mers-cov caused 699 laboratory-confirmed human infections in 21 countries, including 209 deaths (proportion of fatal cases <29.9%) [3] . increasing evidence points to dromedaries (camelus dromedarius) as an intermediate reservoir contributing to the emergence of middle east respiratory syndrome (mers) in humans. several seroepidemiology studies have found mers-cov-neutralizing antibodies in dromedaries from egypt, jordan, oman, and saudi arabia [4] [5] [6] [7] [8] [9] . more recently, coronaviral genomes detected in nasal swabs obtained from dromedaries proved to be identical to genomes of human mers-cov isolates [4, 5, 9] . one such genome was detected in a patient who had been caring for a sick dromedary and directly from that animal [10] . in addition, mers-cov was directly isolated from a dromedary in qatar [11] . the source of dromedary mers-cov infection remains to be elucidated, but it is not unlikely that they serve only as intermediary hosts [12] . bats have been proposed as additional mers-cov hosts. this hypothesis is based on the fact that several betacoronaviruses related to mers-cov (e.g., severe acute respiratory syndrome-like coronaviruses, tylonycteris bat coronavirus hku4, pipistrellus bat coronavirus hku5) are known to infect bats in africa, europe, and asia [1, [13] [14] [15] [16] . in addition, mers-cov genome fragments encoding parts of the rnadependent rna polymerase were detected in one egyptian tomb bat (taphozous perforates) living close to a mers-cov-infected patient [14] . finally, a novel coronavirus (neocov) closely related to mers-cov was discovered in cape serotines (neoromicia capensis) in south africa [12] . therefore, bats could possibly maintain mers-cov in nature and may occasionally infect dromedaries and thereby may infect humans similar to the batshorse-human or bats-pig-human transmission cycle observed for henipaviruses [17, 18] . recently, cd26, also known as dipeptidyl peptidase 4 (dpp4) was identified as the human mers-cov cell entry receptor [19] and also as a receptor for tylonycteris bat coronavirus hku4 [20, 21] . cd26/dpp4 receptor is conserved among different mammals (e.g., bats, dromedaries, humans), and the possibly broad species tropism of mers-cov may partly be the result of this conservation [19, 22] . to further evaluate the hypothesis that bats may be implicated in transmission of the virus, we inoculated ten cell lines from phylogenetically diverse bats living in geographically distinct areas with mers-cov. six bat cell lines were productively infected. the susceptibility or resistance of the ten cell lines to mers-cov infection directly correlated with the absence or presence of naturally expressed cd26/dpp4 on the cells surface. anti-human cd26/dpp4 antibodies reduced mers-cov yield in susceptible bat cell cultures in a dose-dependent manner. similar to other studies using mers-cov-resistant (non-bat) cell lines transfected with cd26/dpp4 [19] , expression of human cd26/dpp4 in resistant bat cells rendered these cell lines susceptible to infection. finally, we demonstrate that persistent mers-cov infections can be established in permissive bat cell lines after sequential virus passage, leading to downregulation of natural cd26/dpp4 cellsurface expression. together, our data indicate that the host cell tropism of mers-cov may largely depend on the expression of suitable cd26/dpp4 orthologs, and that bats cannot be excluded as mers-cov reservoirs at this point in time. huh-7 (a kind gift from hideki ebihara, rocky mountain laboratory, hamilton, mt), vero e6 (atcc, #crl-1568, manassas, va), and vero (atcc, #ccl-81) cells were grown in dulbecco's modified eagle's medium (dmem) supplemented with 10% heat-inactivated fetal bovine serum (fbs, sigma-aldrich, st. louis, mo). the bat cell lines used in this study are described in table 1 . r05t, r06e, and hypni/1.1 cell lines were grown in dmem/f-12 (lonza, walkersville, md) supplemented with 10% fbs. all others bat cell lines were maintained in dmem supplemented with 10% fbs. all cells were incubated at 37uc in a humidified 5% co 2 atmosphere. bat cell lines were seeded in collagen-coated 24-well plates (becton dickinson labware, bedford, ma) at 2610 5 cells/well. one day later, media were removed, and cells were washed once with dmem without fbs (0% dmem). cells were then exposed to mers-cov/emc or mers-cov/jor at an moi of 1. after 1 h of incubation at 37uc, viral inocula were removed and cells were washed once with 0% dmem and then supplemented with dmem containing 2% fbs (2% dmem). at 1, 3, or 5 days postexposure, supernatants were harvested and cleared of cellular debris by centrifugation. plaque assay mers-cov particle yields were quantified by plaque assay [23] . briefly, confluent monolayers of vero cells in 6-well plates were exposed to serial dilutions of mers-cov, incubated at 37uc for 1 h under gentle rocking every 15 min, followed by removal of inocula and addition of a 0.8% tragacanth overlay (sigma-aldrich, st. louis, mo). infected cells were then incubated at 37uc for 72 h. the tragacanth overlay was removed, and the cells were stained with 2% crystal violet (sigma-aldrich) in 10% neutral buffered formalin (nbf, fisher scientific, kalamazoo, mi). plaques were enumerated manually. roni/7.1 or huh-7 cells were incubated with different concentrations (0, 1.25, 2.5, 5, 10, 20 mg/ml) of goat anti-human cd26/dpp4 antibody (r&d systems, minneapolis, mn) or control goat igg antibody at 37uc for 1 h. antibody-treated cells were exposed to mers-cov/emc at an moi of 1 at 37uc for 1 h in the presence of antibodies. virus-antibody inocula were then removed, cells were washed in 0% dmem, fresh dmem (2% fbs) was added, supernatants were harvested 24 h postexposure, and viral yields were determined by plaque assay. at the same time, plates were fixed with 10% nbf and then stained with rabbit polyclonal anti-mers-cov spike protein antibody (sino biological, beijing, china) followed by secondary alexa fluor 488conjugated goat anti-rabbit igg antibody (life technologies, carlsbad, ca). hoechst 33342 dye was used to stain nuclei. the percentage of infected cells was measured and analyzed using the operetta high content imaging system (perkinelmer waltham, ma) and analysis software (harmony 3.1). bat cells were washed with phosphate-buffered saline (pbs) and then dissociated with cell dissociation buffer (life technologies). cells were spun down, washed, and resuspended in 4% paraformaldehyde for fixation. cells were stained with goat antihuman cd26/dpp4 antibody followed by alex fluor 488conjugated rabbit anti-goat igg antibody. as a control, the cells were stained with the same concentration of isotype control goat igg antibody followed by the same secondary antibody. samples were collected using an lsr fortessa flow cytometer (bd biosciences, san jose, ca). flowjo software version 9.7.5 (treestar, ashland, or) was used to analyze the data. confluent bat cells were inoculated with mers-cov/emc at an moi of 1 for 1 h at 37uc. after viral inocula were removed, cells were washed once with 0% dmem and then supplemented with 2% dmem. media were removed 24 h later, and electron microscopy grade fixative, 2.5% glutaraldehyde (e.m. sciences, warrington, pa) in millonig's sodium phosphate buffer (tousimis research, rockville, md), was added directly to the dishes. after 10 min, bat cells were scraped off the dishes with a cell scraper, collected into 15-ml tubes, and immediately centrifuged at 5006g for 20 min. to complete fixation, cells were kept in fixative for 24 h at 4uc and were post-fixed in 1% osmium tetroxide (electron microscopy sciences, hatfield, pa). post-fixed cells were stained en bloc with 2% uranyl acetate, dehydrated in a series of graded ethanols, and infiltrated and embedded in spurr plastic resin (electron microscopy sciences). a leica em uc7 ultramicrotome (leica microsystems, buffalo grove, il) was used to section the embedded blocks into ultra-thin sections (60-80 nm). these sections were collected, mounted on 200-mesh copper grids (electron microscopy sciences), and contrasted with reynold's lead citrate. a fei g2 tecnai transmission electron microscope (fei, hillsboro, or), operating at 80 kv, was used to examine and image the grids. fluor 488-conjugated chicken anti-rabbit igg antibody (life technologies). images were acquired using the operetta high content imaging system. 2 flasks were infected with mers-cov/emc or mers-cov/jor at an moi of 1. after 7 days, supernatants were harvested for virus yield analysis by plaque assay, and the cells were subcultured at a 1:10 dilution in new flasks. subsequently, the infected cells were passaged at a 1:10 dilution weekly for a total of nine passages. from each passage, supernatants were harvested, and virus yields were determined by plaque assay. eidni/41.3 cells (non-infected or persistently infected with mers-cov, day 63) were washed with pbs and lysed in cell lysis buffer (cell signaling, danvers, ma) according to the manufacturer's instruction. equivalent amounts of total cellular lysates were resolved in 4% to 12% bis-tris gradient gels (life technologies) and then dry-transferred to polyvinylidene difluoride (pvdf) membranes (life technologies) by using the iblot gel transfer system (life technologies). after blocking in 5% nonfat milk powder in pbs with 0.1% tween (sigma-aldrich), membranes were incubated overnight with goat anti-human cd26/dpp4 antibody (1:500) or anti b-actin antibody (1:500, abcam, cambridge, ma), followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies (sigma-aldrich). signals were detected by supersignal west femto chemiluminescent substrate (thermo fisher scientific, rockford, il), and images were acquired using a syngene g: box chemiluminescene imaging system (syngene, frederick, md). six of ten tested bat cell lines are susceptible to mers-cov infection figure 1a, 1b) . these results were also confirmed by immunofluorescence assay (ifa) in cell lines inoculated with mers-cov/emc ( figure 1c ) or mers-cov/jor ( figure 1d ). infected cells were detected by mers-cov spike protein ifa. representative images were picked from those taken on day 1 or day 3 post-exposure, as cytopathic viral effects diminished immunofluorescence in some cell lines (e.g., eidni/41.3, roni/7.1, roni/7.2 cells, figure 1c ). images of bat cells (day 1 post-exposure) inoculated with mers-cov/emc taken by a transmission electron microscope (tem, figure 1e to evaluate whether cd26/dpp4 expression is related to susceptibility of bat cells to mers-cov infection, surface expression of cd26/dpp4 was analyzed in ten bat cell lines by flow cytometry using a polyclonal anti-human cd26/dpp4 antibody. none of the four mers-cov-resistant cell lines tested in this study (pesu-b5l, r05t, r06e, and tb1lu) were recognized by anti-human cd26/dpp4 antibody in this assay, whereas all susceptible bat cells (eidni/41.3, eponi/22.1, hyplu/45.1, hypni/1.1, roni/7.1, and roni/7.2) tested positive for cd26/dpp4 expression (figure 2 ). to confirm the role of cd26/dpp4 in mers-cov bat cell entry, roni/7.1 cells or control human huh-7 cells were incubated with increasing concentrations of a monoclonal antihuman cd26/dpp4 antibody and subsequently exposed to mers-cov/emc. cd26/dpp4 antibody treatment reduced mers-cov particle production in both roni/7.1 cells and huh-7 cells, as evidenced by a dose-dependent reduction in viral yield in plaque assays ( figure 3a ). immunofluorescent images of infected cells stained against mers-cov spike protein confirm these results ( figure 3b ). thus, cd26/dpp4 plays a crucial role in mers-cov cell entry into bat cells. figure 4d ). in vitro studies revealed that mers-cov can infect cell lines derived from nonhuman primates, civets, rabbits, goats, cows, sheep, chickens, and pigs, but not cell lines derived from cats, dogs, hamsters, or mice [22, 24, 25] . in this study, we explored the potential of bats to be a reservoir for mers-cov infection [13] [14] [15] northern africa. in these geographic areas, domestic and wild dromedaries can be found and natural human mers-cov infections are recorded (table 1) . cd26/dpp4 is the cellular receptor for mers-cov [19] . this evolutionary conserved dimeric ectopeptidase is differentially expressed in various tissues but may not be the sole determinant for susceptibility at the organism level [25] . importantly, cellular susceptibility to mers-cov infection not only depends on the expression of cd26/dpp4, but also on its sequence. for instance, five amino-acid variations in the mers-cov-binding domain of hamster, ferret, and mouse cd26/dpp4 compared to human cd26/dpp4 have been linked to the resistance of hamster, ferret, and mouse cell lines to mers-cov infection [22] . our study confirms the role of cd26/dpp4 as receptor for two divergent mers-cov isolates and correlates its presence or absence on the surface of bat cells directly with bat cell susceptibility or resistance to productive mers-cov infection (figures 2 and 3 ). bat cells that tested negative for cd26/dpp4 expression by flow cytometry using the polyclonal anti-human cd26/dpp4 antibody may possibly express a cd26/dpp4 ortholog that is not recognized by this antibody. although the cells were derived from bats of different geographic origins, they could be rendered permissive by ectopic expression of human cd26/dpp4 ( figure 4a-c) . overexpression of human cd26/dpp4 in already mers-cov-susceptible bat cell lines led to an increase in virus yield in some bat cell lines and to a decrease in virus yield in others ( figure 4d ). we hypothesize that this variation may be due to differences in the number of bat cd26/dpp4 molecules on the cell-surface of each bat cell. for instance, overexpression of human cd26/dpp4 in a bat cell line with naturally high bat cd26/ dpp4 surface expression may not have an effect on virion entry efficiency since all virions already find enough binding partners in the untransfected cell. vice versa, if a bat cell line expresses little cd26/dpp4, finding a binding partner would present a bottleneck for mers-cov virions, and overexpression of human cd26/dpp4 might overcome this bottleneck and thereby increase virus yield. second, we hypothesize that overexpression of human cd26/dpp4 may interfere with transport and/or functionality of certain bat cd26/dpp4 orthologs due to heterodimerization and consequent structural changes. our results indicate that, at least on the cellular level, presence or absence of functional cd26/dpp4 with a suitable mers-cov-binding domain is a major determinant for mers-cov cellular tropism in bats. in addition to potential differences in the mers-cov-binding domain of distinct bat cd26/dpp4 orthologs, presence or absence of yet-to-be-identified co-receptors and bat species-specific cellular factors acting downstream of virion adsorption and fusion may further influence to what extent a productive infection can be established. however, these considerations alone are not sufficient to pinpoint bats as epidemiologically relevant mers-cov hosts, as the immune system at the level of the organism may interfere with infection prior to cell entry or lead to rapid viral clearance. one characteristic of natural virus host reservoirs is that hosts frequently are persistently infected with virus in the absence of clinical signs. continuous transmission of viruses from such a reservoir to other animals, including humans, is a hallmark of zoonoses and explains repeated introduction of viruses into susceptible animal populations [26] . a number of zoonotic viruses are known to persistently and subclinically infect rodents (e.g., arenaviruses, hantaviruses) or bats (henipaviruses), which then directly or indirectly infect humans [17, [27] [28] [29] [30] [31] . among the betacoronaviruses, the group of coronaviruses including mers-cov, murine coronavirus and severe acute respiratory syndrome (sars)-related coronaviruses that are related to mers-cov, are known to cause persistent infection in their mammalian hosts [16, 32] . during persistent sars-cov infection in vero e6 cells and also during acute infection in laboratory mice, the sars-cov entry receptor, angiotensin converting enzyme 2 (ace2), is downregulated [33, 34] . ace2 downregulation contributes to the severity of lung pathology of sars [34] . in figure 5 ). the observation that cultures continued to release significant virion amounts on day 63 post-inoculation, but also exhibited low to absent cd26/dpp4 cell-surface expression, is consistent with receptor downregulation as one mechanism for persistent infection with mers-cov in our experiments. virus carryover between cell/virus passages probably did not influence these results as excreted viruses should be unable to re-infect cells in the absence of cd26/dpp4 receptor. the mechanism of cd26/dpp4 downregulation upon persistent mers-cov infection requires further study. western blot-based examination of lysates of persistently infected cells at day 63 indicate that cd26/dpp4 is either not expressed anymore or is very efficiently degraded ( figure 5g ). the reason for the observed differences in establishing persistent infection in these cell lines among the two different mers-cov isolates is unclear, but may have epizootiological significance and needs to be examined in future studies. our results are consistent with the suggested role of bats in mers-cov transmission. if cd26/dpp4-positive cells of bats become infected with mers-cov and if subsequently the receptor surface expression is downregulated as we observe in our experiments, a persistent infection could be established. such animals may provide a reservoir that continuously sheds infectious virus, possibly transmitting the virus to other mammals, such as dromedaries. further evaluation is needed to determine whether downregulation of cd26/dpp4 is a general hallmark of persistent mers-cov infection in animal models of mers and whether the receptor influences pathogenesis in a manner similar to that seen with ace2 downregulation in persistent sars-cov infection. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group middle east respiratory syndrome coronavirus (mers-cov) summary and literature update -as of 11 middle east respiratory 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room temperature, in the plaque assay of fish and other animal viruses differential cell line susceptibility to the emerging novel human betacoronavirus 2c emc/2012: implications for disease pathogenesis and clinical manifestation the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters how to find natural reservoir hosts from endemic prevalence in a multi-host population: a case study of influenza in waterfowl persistence of lymphocytic choriomeningitis virus in immune animals and its relation to immunity density thresholds for mopeia virus invasion and persistence in its host mastomys natalensis emergence and persistence of hantaviruses hantavirus reservoirs: current status with an emphasis on data from brazil recrudescent infection supports hendra virus persistence in australian flying-fox populations persistent infection promotes cross-species transmissibility of mouse hepatitis virus jnk and pi3k/ akt signaling pathways are required for establishing persistent sars-cov infection in vero e6 cells a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury type i interferon reaction to viral infection in interferon-competent, immortalized cell lines from the african fruit bat eidolon helvum comparative analysis of ebola virus glycoprotein interactions with human and bat cells evidence supporting a zoonotic origin of human coronavirus strain nl63 cell lines from the egyptian fruit bat are permissive for modified vaccinia ankara differential sensitivity of bat cells to infection by enveloped rna viruses: coronaviruses, paramyxoviruses, filoviruses, and influenza viruses we would like to thank eric donaldson (fda) for providing pesu-b5l cells. we are grateful to our colleague, jiro wada (irf-frederick), for assisting us with the preparation of figures. the content of this publication does not necessarily reflect the views or policies of the us department of the army, the us department of defense, the us department of health and human services, or of the institutions and companies affiliated with the authors. key: cord-309194-jtouafgd authors: lu, xiao; zhang, mao; qian, anyu; tang, luping; xu, shanxiang title: lung ultrasound score in establishing the timing of intubation in covid-19 interstitial pneumonia: a preliminary retrospective observational study date: 2020-09-03 journal: plos one doi: 10.1371/journal.pone.0238679 sha: doc_id: 309194 cord_uid: jtouafgd purpose: to investigate the role of lung ultrasound score (lus) in assessing intubation timing for patients with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) pneumonia. materials and methods: seventy-two patients with critical coronavirus disease 2019 (covid-19) were admitted to a makeshift intensive care unit (icu). all patients underwent bedside lung ultrasonography one to two times per day. the patients were either intubated, treated with noninvasive ventilation (niv), or given high-flow nasal cannula (hfnc) after a discussion with the multidisciplinary group after their conditions worsened. bedside lung ultrasound was performed daily after intubation, and patients received mechanical ventilation. lung ultrasound was performed on days 1, 2, 3, 5, and 7 after patients were admitted to the icu; if the patient was intubated, lus determination was performed before intubation within 24 h (t1) and on days 1, 2, 5, and 7 after intubation (t2, t3, t4, and t5, respectively).the goal of this study was to evaluate the severity of lung aeration loss in intubated and non-intubated patients with sars-cov-2 pneumonia by ultrasound at different time points within one week. results: a total of 16 patients were included in this study, including nine who were intubated and mechanically ventilated and seven patients without intubation. the number of elderly individuals in the intubated group was higher than in the non-intubated group (p < 0.05). in addition, there were more male than female patients in both groups. patient characteristics (bmi, sofa, and pao(2)/fio(2) value) were similar between the two groups (p > 0.05). the 28-day mortality rate of intubated patients was higher than that of non-intubated patients; six patients in the intubated group and two patients in the non-intubated group died. nine intubated patients showed changes in lus within seven days (n = 9). the mean lus within 24 h before intubation was 12.8 ± 1.3. lus was significantly higher on t1 than on t5 (p <0.05), and did not significantly differ from t1 to t4. comparing lus between intubated and non-intubated patients on t1 showed that the lus of intubated patients was significantly higher than that of non-intubated patients (p <0.05). between the two patient groups, oxygenation index was 140.1 ± 7.7 vs. 137.8 ± 5.9 on t1, and the respiratory rate of the two groups was 26 ± 5 vs. 28 ± 4 breaths/min. neither oxygenation index nor rr significantly differed between the two groups. conclusion: lus may be an effective tool for assessing intubation timing in critically ill patients with covid-19 interstitial pneumonia. a total of 16 patients were included in this study, including nine who were intubated and mechanically ventilated and seven patients without intubation. the number of elderly individuals in the intubated group was higher than in the non-intubated group (p < 0.05). in addition, there were more male than female patients in both groups. patient characteristics (bmi, sofa, and pao 2 /fio 2 value) were similar between the two groups (p > 0.05). the 28-day mortality rate of intubated patients was higher than that of non-intubated patients; six patients in the intubated group and two patients in the non-intubated group died. nine intubated patients showed changes in lus within seven days (n = 9). the mean lus within 24 the coronavirus disease 2019 (covid-19) outbreak began in december 2019 and rapidly spread to different areas of mainland china. on march 14, 2020, who declared covid-19 a pandemic. the most common and severe complication in patients with covid-19 is acute hypoxemic respiratory failure or acute respiratory distress syndrome (ards), which requires oxygen and ventilation therapies [1] . some critically ill patients require intubation and invasive ventilation [2] . a number of patients with covid-19 present with "silent" or "happy hypoxia," in which the body's oxygen levels are well below 90%, yet patients are still able to breathe normally. for these patients, there is no shortness of breath, fast or shallow breathing, and likely no signs, symptoms, or sense that something may be wrong. few studies have addressed the timing of intubation for patients with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) pneumonia due to the high mortality of patients treated with invasive ventilation. the histopathology of initial covid-19 pneumonia is characterized by alveolar damage, which includes alveolar edema, while the inflammatory component is patchy and mild [3] . the analysis of the available ct data from patients with covid-19 pneumonia [4] shows largely bilateral lesions, which are patchy and confluent, with a ground glass or mixed consolidative and ground glass pattern. to date, current literature from china and italy doses not recommend the use of lung ultrasounds to diagnose covid-19. however, lung ultrasounds are known to potentially assist in monitoring lung conditions [5] . it has also been proven that lung ultrasound findings can be suggestive of a wide range of conditions, including pulmonary edema, pleural effusion, and pneumothorax [6] . lung ultrasounds have also proven capable of detecting lung lesions before the development of hypoxemia in ards patients [7] . ultrasounds can accurately quantify the loss of pulmonary aeration before, after, and during the weaning trial by calculating the lung ultrasound score (lus) [8] . one prospective two-center study involving 100 patients has demonstrated that the intensity of lung aeration loss occurring during the weaning trial is predictive of post-extubation respiratory distress within 48 h of extubation. the study found that lus � 14 could identify patients at high risk of developing post-extubation respiratory distress [9, 10] . other studies recently found that 80 patients weaned from mechanical ventilation showed a 30% reduction in respiratory distress during post-extubation [11, 12] . however, these studies examined diseases similar to covid-19, but their findings have not yet been proven in covid-19. this study was designed to retrospectively analyze changes in lus for 16 critically ill patients with covid-19. our study examined covid-19 patients with interstitial pneumonia, using the clinical application of lus to assess intubation timing. a retrospective study was conducted in one makeshift icu in wuhan. from february 14, 2020 to march 6, 2020, covid-19 patients were admitted in the icu of the cancer center of union hospital, tongji medical college, huazhong university of science and technology. a total of 72 patients with critical covid-19 who were admitted to this icu within one month were included in this study. patients with severe covid-19 met any of the following criteria: 1. respiratory distress, rr � 30 breaths/min; 2. pulse oxygen saturation (spo 2 ) � 93% on room air in the resting state; 3. arterial partial pressure of oxygen (pao 2 )/oxygen concentration (fio 2 ) � 300 mmhg (1 mmhg = 0.133 kpa); and 4. > 50% lesion progression within 24 and 48 h on pulmonary imaging. routine bedside lung ultrasound (one to two times each day) was performed on patients who were treated via non-rebreather mask, non-invasive ventilator, or hfnc after admission to the icu. the patients were treated according to the guidance for corona virus disease 2019 of china (editions 6, 7) for antiviral and proprietary chinese medicine. for patients with acute hypoxemic respiratory failure due to covid-19, deciding whether to proceed with intubation and invasive ventilation can be challenging. in our icu, if the patient's condition became more severe during the treatment, the multidisciplinary discussion group (mdt) discussed whether to intubate that patient. after the patients received mechanical ventilation treatment, lung ultrasound assessment was performed daily. retrospective analysis of lus of patients at different time points was performed for one week. the patient's gender, age, body mass index (bmi index), sequential organ failure assessment (sofa score), and 28-day mortality rate were recorded; and the evolution of respiratory parameters between the two groups on time point t1 (lung ultrasound performed before intubation within 24 h) were also recorded. this study was approved by the national health commission of china and ethics commission of second affiliated hospital, zhejiang university school of medicine (ky-2020-186). written informed consent was waived by the ethics commission of the designated hospital for emerging infectious diseases. a mindray m9 echography (mindray co, shenzhen, china) and a 2 to 4 mhz round-tipped or convex probe was used for the examination. for each patient, 12 areas in the right and left lung were examined, and as delineated by a parasternal line, anterior axillary line, posterior axillary line, and paravertebral line, the anterosuperior, anteroinferior, laterosuperior, lateroinferior, posterosuperior, and posteroinferior lung regions were identified. scoring of each area was performed according to the most severe lung ultrasound detected in the corresponding intercostal spaces: 0) normal aeration: presence of lung sliding with horizontal a lines or fewer than two isolated vertical b lines; 1) moderate loss of lung aeration: either multiple welldefined and spaced b1 lines, issued from the pleural line or from small juxtapleural consolidations and corresponding to interstitial edema or coalescent b1 lines or issued from the pleural line or from small juxtapleural consolidations, present in a limited portion of the intercostal space and corresponding to localized alveolar edema; 2) severe loss of lung aeration: multiple coalescent vertical b2 lines issued either from the pleural line or from juxta-pleural consolidations, detected in the whole area of one or several intercostal spaces and corresponding to diffuse alveolar edema; 3) lung consolidation: the presence of a tissue pattern containing either hyperechoic punctiform images, which are representative of static air bronchograms, or hyperechoic tubular images, which are representative of dynamic air bronchograms, corresponding to the complete loss of aeration. lus was calculated as the sum of the points ranging from 0-36. lung ultrasound was performed on days 1, 2, 3, 5, and 7 after patients were admitted to the icu; these time points are called t1, t2, t3, t4, and t5, respectively. if the patient was intubated, the lus scoring was performed before intubation within 24 h (t1) and on days 1, 2, 5, and 7 after intubation (t2, t3, t4, and t5, respectively). statistical analyses were performed using stata v12 (stata corp, college station, tx, usa). all statistical tests were performed at an α risk of 5% (excluding interim analysis). continuous variables are presented as the mean and standard deviation, subject to the normality of distribution (shapiro-wilk). in case of non-normality, these are presented as the median, quartiles, and extreme values. qualitative variables are expressed as numbers and associated percentages. graphic representations are associated with the analysis. comparisons between groups were conducted systematically: (1) without adjustment, and (2) adjusted by regression model based on factors whose distribution could be unbalanced between the groups despite randomization. during the study period, 72 patients presented to the icu in wuhan with covid-19 within one month; 43 patients were confirmed to be severe cases. twenty-seven patients were excluded for the following reasons: five patients were intubated before transferring to our icu, four patients died within 72 h, and 18 patients were not assessed by transthoracic lung ultrasound, and thus had no data. a total of 16 patients were included this study, of whom nine were intubated within 72 h after admission into the icu, and seven patients were not intubated and treated with a non-rebreather mask, non-invasive ventilator (niv), or high-flow nasal cannula oxygen therapy (hfnc). the demographics, clinical features, and outcomes of the sample are presented in table 1 . the mean participant age was 58.0 (47.0-68.0) years old, with a higher proportion of older patients in the intubated group than in the non-intubated group (p < 0.05). there were more male patients than female patients in both groups. patient characteristics (bmi, sofa, and pao 2 /fio 2 value) were similar between the two groups (p > 0.05). the 28-day mortality rate of intubated patients was higher than that of non-intubated patients, and six patients in the intubated group and two patients in the non-intubated group died. table 2 shows the evolution of respiratory parameters between the two groups on t1; no differences in respiratory rate, pulse rate, ph, paco 2 , pao 2 /fio 2, and spo 2 were found between the two groups. . the average lus was 10.7 ± 1.4 on t1 and 10.3 ± 2.9 on t5, and the lowest lus was 10.7 ± 1.1 on t3. there was no significant difference in lus among time points. fig 2 shows the changes in lus of intubated patients (n = 9) at different time points. the lus within 24 h before intubation was 12.8 ± 1.3. lus was significantly higher at t5 than t1 (p < 0.05), but the scores did not significantly differ from t1 to t4. fig 3 shows that the lus of intubated patients, 12.8 ± 1.3, was significantly higher than the lus of the non-intubated patients at t1, 10.7 ± 1.4 (p < 0.05). fig 4 shows a comparison of the oxygenation index of the two groups of patients: 140.1 ± 7.7 vs. 137.8 ± 5.9 on t1. there was no significant differences in oxygenation index between the two groups. the high contagiousness of sarscov-2 and the risk of transporting unstable patients with hypoxemia and hemodynamic failure make chest ct a limited option for patients with suspected or established covid-19. lung ultrasonography generates results that are similar to those of chest ct and superior to standard chest radiography when evaluating pneumonia and/or adult respiratory distress syndrome (ards), with the added advantages of ease of use at point of care, repeatability, absence of radiation exposure, and low cost [13] . the most important clinical manifestations of respiratory failure in patients with covid-19 are hypoxemia and increased work of breathing. attention should be paid to the different effects of different oxygen therapy concentrations to avoid prolonged high-concentration oxygen therapy [14] . during the treatment process, deciding when to switch from non-invasive ventilation or high-flow oxygen therapy to invasive mechanical ventilation and how to dynamically assess lung condition changes can be challenging. for critically ill patients with covid-19, the difficulty of accurately assessing lung conditions is greatly increased due to the inability of doctors to use stethoscopes in protective clothing, as well as due to the use of lung ct to repeatedly evaluate patients' lung conditions in special circumstances. several studies on non-covid-19 patients have demonstrated that lung ultrasound is accurate for assessing positive end-expiratory pressure, prone position-induced lung recruitment, lung reaeration following antimicrobial therapy in ventilator-associated and community-acquired pneumonia, and lung reaeration data are presented as the number of patients, mean ± sd, or median and interquartile interval (25 to 75%). https://doi.org/10.1371/journal.pone.0238679.t001 table 2 . evolution of respiratory parameters between the two groups on t1. associated with the resolution of various forms of pulmonary edema [8] [9] [10] . soummer et al. first proposed and successfully applied this method to assess the changes in lung ventilation for patients after offline extubation, and they confirmed that patients with lus > 14 had a significant increase in intubation rate after offline extubation [15] . an experienced sonographer can perform this examination within five min, and brief training and about 25 supervised exams seem to be sufficient to achieve a basic ability to perform the task [16] . a prior study showed lus's ability to influence clinical decisions in up to 50% of icu patients [17] . in this study, the lus of the intubated group on t1 (within 24 h before intubation) was higher than that of the non-intubated group. however, oxygenation index and respiratory rate, the conventional indicators to decide whether to intubate critical care patients, did not significantly differ between the two groups at the same time point. the obvious difference indicates that the lus may be a more accurate indicator of ideal intubation timing than the oxygenation index and respiratory rate. lus could dynamically assess the ventilation status of the two patient groups during treatment, and provided earlier prediction of pulmonary ventilation status and disease deterioration. an lus = 12 may be a warning for intubation or exacerbations in critically ill covid-19 patients. clinicians need to closely observe the patient's condition and consider the possibility of tracheal intubation according to the patient's situation. a unique syndrome of hypoxic covid-19 patients has been described (labelled "the happy hypoxic") who are mentally alert and lack significant respiratory distress despite hypoxia that would usually prompt treatment, sometimes with profoundly low oxygen saturations [18] . according to our experience in treating patients with severe covid-19 in wuhan, most patients are more tolerant to hypoxia, and patients with a low oxygenation index often have no obvious symptoms of chest tightness, shortness of breath, or mild symptoms, which are inconsistent with clinical indicators. there is no evidence that choosing early intubation instead of niv or hfnc improves outcome: early reports suggest intubated patients remain on ventilators for extended periods and mortality rates appear high. a retrospective cohort study of 191 patients in wuhan showed that mortality rate was significantly increased after intubation; 57% of patients died after mechanical ventilation treatment, and patients often had complications such as septic shock, ventilator-associated pneumonia (vap), and deep vein thrombosis after intubation [19] . intubated patients are thought to pose a lower risk of viral dispersion to staff. however, both the process of intubating and extubating are high risk to staff, and there are reports of accidental ventilator circuit disconnection when intubated. therefore, judging the timing of intubation and mechanical ventilation treatment is a concern for doctors in the emergency department and icu, but no recent study has found the best clinical indicators for evaluation. lung ultrasound is an ideal inspection method in addition to the gold standard ct examination, and may be one of the most effective tools for judging intubation time. according to our experience, the accuracy of bedside ultrasound in evaluating severe and critical covid-19 patients is significantly better than its accuracy in evaluating mild patients. the reason may be that the exudation of mild covid-19 patients is often not obvious, and the lesions are deep and/or pleural, and not accumulated. lung ultrasound's usefulness is limited, especially in cases of early or mild covid-19. according to previous studies, lus is a semiquantitative assessment of lung ventilation by ultrasound. an lus of 14 (scored out of 36) may thus be an indicator for intubation in patients with respiratory failure. in this study, the lus before intubation was 12.8 ± 1.3, which is lower than the score suggested by the previous study (14 points) . the reason for this may be that the primary ct manifestation of the patients in the group was pulmonary exudation without obvious consolidation or atelectasis, which accounts for 3 points in the lus. because the behavior of ards caused by covid-19 differs from that caused by other diseases, the patient's lus for covid-19 shows mainly a large number of b lines in both lungs, and is lower than normal ards patients, which show consolidation in some parts of the lung. most patients with mechanical ventilation have secondary bacterial infections or poor sputum drainage, which may cause some consolidation of the lungs and atelectasis, leading to a significant increase in lus after intubation [20] [21] [22] . this study indicates that lus may be an effective tool for assessing the timing of intubation in patients with sars-cov-2 pneumonia/ards. the intubation of critically ill patients with covid-19 is mostly due to respiratory failure, but there is also a small number of patients who undergo intubation due to secondary acute heart failure or airway obstruction [23] . thus, an ultrasound may be employed to evaluate cardiac function and other conditions as part of a comprehensive covid-19 assessment. as this study is a retrospective study, the number of patients included in this investigation is relatively small, and further large-scale prospective studies are needed to confirm our findings. supporting information s1 data. (xlsx) the novel coronavirus originating in wuhan, china: challenges for global health governance severe acute respiratory syndrome-related coronavirus: the species and its viruses-a statement of the coronavirus study group short term outcome and risk factors for adverse clinical outcomes in adults with severe acute respiratory syndrome (sars) early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia essentials for radiologists on covid-19: an update-radiology scientific expert panel lung ultrasound: a useful tool in the assessment of the dyspnoeic patient in the emergency department. fact or fiction? lung ultrasound for daily monitoring and management of ards patients ultrasound assessment of antibiotic-induced pulmonary reaeration in ventilator-associated pneumonia bedside ultrasound assessment of positive end-expiratory pressure-induced lung recruitment lung ultrasound in acute respiratory distress syndrome and acute lung injury clinical review: bedside lung ultrasound in critical care practice comparative diagnostic performances of auscultation, chest radiography, and lung ultrasonography in acute respiratory distress syndrome findings of lung ultrasonography of novel corona virus pneumonia during the 2019-2020 epidemic analysis of clinical features of 29 patients with 2019 novel coronavirus pneumonia. zhonghua jie he hu xi za zhi ultrasound assessment of lung aeration loss during a successful weaning trial predicts postextubation distress* training for lung ultrasound score measurement in critically ill patients impact of lung ultrasound on clinical decision making in critically ill patients why covid-19 silent hypoxemia is baffling to physicians clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study our italian experience using lung ultrasound for identification, grading and serial follow-up of severity of lung involvement for management of patients with covid-19 epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study noninvasive positive-pressure ventilation for postextubation respiratory distress: a randomized controlled trial pathological findings of covid-19 associated with acute respiratory distress syndrome we thank letpub (www.letpub.com) for their linguistic assistance during the preparation of this manuscript. key: cord-293393-kbndie8e authors: braesch-andersen, sten; beckman, lena; paulie, staffan; kumagai-braesch, makiko title: apod mediates binding of hdl to ldl and to growing t24 carcinoma date: 2014-12-16 journal: plos one doi: 10.1371/journal.pone.0115180 sha: doc_id: 293393 cord_uid: kbndie8e apolipoprotein (apo) d is an important protein produced in many parts of the body. it is necessary for the development and repair of the brain and protection from oxidative stress. the purpose of this study was to investigate the extent to which apod interacts with lipoproteins in human plasma. by using detergent-free elisa, we show that immobilized monoclonal antibodies against apod very efficiently bind to low density lipoprotein (ldl) from plasma; this binding is as equally efficient as binding to an anti-apob monoclonal antibody. adding detergent to the plasma inhibited the binding, suggesting that the binding is dependent on the presence of intact lipoprotein particles. reversing the system by using immobilized anti-apob revealed that the affinity of apod for ldl is rather low, suggesting that multiple bindings are needed for a durable connection. biosensor experiments using purified lipoproteins also showed that purified apod and high density lipoprotein 3 (hdl3), a lipoprotein fraction rich in apod, were both able to bind ldl very efficiently, indicating that the hdl3-ldl interaction may be a physiological consequence of the affinity of apod for ldl. furthermore, we found that apod increases the binding of hdl to actively growing t24 bladder carcinoma cells but not to quiescent, contact-inhibited, confluent t24 cells. this result is especially intriguing given that the t24 supernatant only contained detectable levels of apod after growth inhibition, raising the possibility that alternating the expression of apod and a putative apod-receptor could give direction to the flow of lipids. in the current paper, we conclude that apod mediates binding of hdl to ldl and to growing t24 carcinomas, thereby highlighting the importance of apod in lipid metabolism. introduction upon growth arrest (quiescence) in cell lines [25, 26, 27, 28, 29] . addition of purified apod to vascular smooth muscle cells inhibits pdgf-bb-induced proliferation [29] and induces apoptosis in colorectal carcinoma cells [30] . an important function of hdl particles is to deliver lipids to ldl and vldl particles, and it is reasonable to assume that this interaction is facilitated/regulated in some way. miller et al. [31] showed that hdl and ldl mutually inhibited each other when binding to fibroblasts. their results indicate either that ldl and hdl compete for the same receptors on the fibroblasts or that the interaction between hdl and ldl in solution interfered with the binding to the fibroblasts. in the present study we have investigated the potential role of apod in the interaction between lipoproteins and between lipoproteins and cells. our results show that apod may provide an important link in the interaction between hdl and ldl particles and between hdl particles and cells, thus, apod may serve as an important regulator of lipid trafficking. monoclonal antibodies (mab) to apod were generated by immunizing mice with human recombinant apod. antibody specificity was confirmed by immunoprecipitation followed by mass spectrometry, and three mabs (d544, d201 and d263) were selected and used in the study. other mabs used were the ldl20 and ldl17 mabs against human apob, the e981 mab against apoe, the h219 and h464 mabs against apoh, the j29 and j84 mabs against apoj and two control mabs, the 7-b6-1 mab against human ifn-c and the il4-i mab against il-4 (all from mabtech, nacka strand, sweden). a positive control antibody against human thioredoxin reductase (trxr1), which was used for immunostaining the t24 cells, was a kind gift from anders rosén, university of linköping. recombinant apod was obtained from biovendor (modrice, czech republic). to increase the solubility, this recombinant apod was modified at the following positions: trp99-his, cys116-ser, ile118-ser. other modifications include leu23-pro, pro133-val and asn134-ala. for more information, see the biovendor homepage. purified apoa1, ldl, vldl, hdl, hdl2 and hdl3 were obtained from the academy bio-medical company inc. (houston, tx, usa). native apod was purified from plasma by affinity chromatography. in short, 6.5 ml edta/ aprotinin plasma was centrifuged first at 300 g for 10 minutes to remove cells, followed by centrifugation at 14000 g for 3 minutes to remove platelets and particles. irrelevant mouse antibodies were added to the plasma to a final concentration of 90 mg/ml in order to block hama-activity (human anti-mouse antibodies). the plasma was then diluted 4x with pbs tween20 for a final concentration of 0.025% tween20 and passed at a flow rate of 0.2 ml/min over a 5 ml affinity-column coupled with the anti-apod mab d544. the column was washed with pbs, and apod was eluted with 0.7% hac and immediately flashfrozen (s1 figure) . the bladder carcinoma cell line t24 (atcc, manassas, usa) was cultured in either dmem or rpmi 1640 medium (gibco, brl, life technology ltd. paisley, scotland) with 10% fbs (hyclone, thermo scientific). finger blood was obtained from consenting healthy volunteers (approved by regionala etikprövningsnämnden stockholm, 2006/227-31/1) by piercing the top of the finger with a lancet and recovering 20 ml blood with an adjustable pipette and a sterile tip. the blood was immediately transferred to eppendorf tubes containing 480 ml dilution buffer (pbs, 0.5 mm edta, 0.2% bsa and 20 mg/ml irrelevant mouse igg), and cells were removed by spinning for 3 minutes at 800 g. finally, the supernatant was transferred to a new tube and centrifuged at 14000 g for 3 minutes to remove platelets. for the purpose of this publication, we arbitrarily calculated the volumes of the finger blood plasma based on the observation that the finger blood samples were approximately half blood cells and half plasma. except when stated differently in the figure legends the finger blood plasma came from the same donor, although repetitions of the finger blood experiments were partly conducted using different donors. elisa assays to measure apod, apoa1 and apob (mabtech) were performed according to the manufacturer's instructions. dual-specific elisa, capable of detecting direct or indirect interactions, was performed using antibodies with different target specificities for capture and detection. to not disturb the integrity of the complexes, the incubation and washing buffers did not contain detergent. in short, the coating antibodies were diluted to 2 mg/ml in pbs with 0.02% azide, except for mab ldl20, which was used at 1 mg/ml, and 100 ml per well were added to 96-well elisa plates (costar, corning, ny, usa). after a two hour incubation at room temperature, the plates were blocked with 0.1% bsa in pbs azide (incubation buffer) for a further two hours at room temperature or in the refrigerator overnight. the plates were then washed in an automatic elisa washer (elx50, biotek instruments, winooski, vt, usa), using pbs as the wash buffer. purified apolipoproteins or finger blood plasma was diluted in the incubation buffer, and 100 ml/well were added to the elisa plates for a one hour incubation. after washing with pbs, the biotinylated antibodies were diluted in the incubation buffer to 1 mg/ml, except for ldl20-biotin, which was used at 0.5 mg/ml, and antibodies were added to the plates. following a one hour incubation, plates were again washed, streptavidin-alp (mabtech), diluted in the incubation buffer, was added, and the plates were incubated for 1 hour. after further washing, the plates were developed by adding the substrate pnpp (para-nitrophenylphosphate, s0942, sigma-aldrich, st. louis, mo, usa) in a 30 mm, ph 9.0, tris-hcl buffer supplemented with 2 mm mgcl 2 . the readings were performed at 405 nm in a thermo max microplate reader (molecular devices, ca. usa). t24 bladder carcinoma cells were seeded onto 48-well plates (costar) at varying concentrations to generate both confluent and non-confluent cell layers. to obtain approximately 50% confluent cultures, cells were seeded 24 hours before the experiment, whereas confluent cells were seeded 5 days before to obtain achieve full contact (confluency) and a cobblestone-like pattern. the cells were washed three times with serum-free rpmi buffered with 20 mm hepes. next, 200 ml of 1 mg/ml hdl were added to each well, with or without 100 ng/ml of apod or apoa1, in an incubation buffer consisting of rpmi with hepes and 0.1% bsa. after a 1 hour incubation at room temperature, the cells were washed three times with 500 ml of rpmi and 200 ml of pbs with 0.1% bsa and 0.05% tween20 was added. after 10 minutes, the supernatants were transferred to a lowbinding 96-well v-shaped polypropylene plate (no. 651201, greiner bio one, germany) and centrifuged at 1100 g for 5 minutes to remove non-solubilized material. finally, 100 ml from each well were collected, and the concentration of apoa1 was determined by elisa. the binding between apod and lipoprotein particles was assessed using a blitz biosensor (fortebio, pall life science, menlo park, ca, usa). this biosensor measures mass by how it changes optical layer thickness and thus the interference patterns of reflected light. for more details, see the pall life science homepage. in brief, biotinylated mab d544 (anti-apod) and control mabs e981 (anti-apoe) and 7-b6-1 (anti-ifnc isotype control for d544) were added to streptavidincoated biosensor surfaces, followed by rinsing and addition of purified apod and/ or lipoprotein particles. pbs with 0.1% bsa and 0.02% sodium azide was used as a buffer, and both the rinsing and loading steps were performed using a volume of 250 ml. antibodies were loaded at a concentration of 5 mg/ml, apod at 2 mg/ml and hdl2, hdl3 and ldl at 100 mg/ml. the loading time was 3 minutes for antibodies and 5 min for lipoproteins, and the rinsing time was 30 seconds. t24 cells were grown in dmem supplemented with 10% fbs in 25 cm 2 cell culture flasks (costar). supernatants from the confluent cultures, corresponding to approximately 2.5 million cells per flask, and non-confluent cultures, corresponding to 1-1.5 million per flask (70-80% confluent), were collected 24 hours after changing the media. for each condition, the supernatants from three flasks were probed four times and tested for apod content via apod elisas (mabtech), with a detection limit of approximately 0.1 ng/ml. the same cells were also harvested and used for flow cytometry (see below). for intracellular staining, t24 cells were removed from 25 cm 2 tissue culture flasks by washing once with 5 ml pbs containing 1 mm edta, followed by a 10 minute incubation at 37˚c with 3 ml of the same buffer. detached cells were transferred to a 15 ml centrifuge tube and resuspended with a pipette to obtain a single cell suspension. the cells were washed once in pbs and, after sedimentation at 250 g for 10 minutes, the cell pellet was re-suspended in 100 ml pbs and 3 ml of a freshly prepared buffer of pbs with 0.5% tritonx100 and 2% paraformaldehyde. after 10 minutes fixation at room temperature, the cells were resuspended by adding 9 ml pbs and then pelleted again by centrifugation at 1000 g for 5 minutes. the supernatant was removed, and the remaining reactive groups were inactivated by addition of 1% glycine in pbs, azide, and 0.1% bsa. after another 20 minutes, the cells were pelleted again at 1000 g for 5 minutes and then re-suspended in 1 ml of pbs, azide, 0.1% bsa. cell staining was performed in v-shaped 96-well plates (type 651201, greiner bio one, frickenhausen, germany) using 250k cells/well, and primary antibodies were added to a final concentration of 2 mg/ml. after a 30 minute incubation at room temperature, the cells were washed twice with pbs before adding a secondary fitc-conjugated rabbit f(ab)2 anti-mouse igg antibody (f0313, dako, glostrup, denmark) diluted 1/100 in pbs, azide, 0.1% bsa. after incubation for 30 min, the cells were again washed, followed by analysis in in a guava easycyte flow cytometer (millipore, billerica, ma, usa). cells were seeded onto glass microscope slides placed in petri dishes to maintain humidity. after incubation for 2 days (non-confluent) or 9 days (confluent) in a 37˚c incubator, the cells were washed in pbs and fixed for 20 min in pbs containing 2.7% paraformaldehyde and 0.3% tritonx100. the cells were then washed with pbs and blocked with pbs containing 0.1% bsa and 1% glycine for 30 min, followed by washing and incubation with primary antibodies at room temperature. in addition to the anti-apod mab d544, a negative isotype-control mab (il4-1) and a positive control mab (trxr1) were used (all at 5 mg/ml). after 1 h, the cells were washed and incubated with a secondary rabbit anti-mouse-igg fitc-conjugated f(ab)2 (dako) antibody (1:100) for another hour. after washing, cell nuclei were stained for 5 min with dapi (1 mg/ml) (pierce) and, after a final wash, were covered with a cover glass. cells were analyzed and photographed using a leica dmrb fluorescent microscope equipped with a nikon coolpix 4500 camera (shutter speed: fitc, 2 sec; dapi, 1/30 sec). results are presented as means with standard deviation. statistical analysis was performed with the graphpad prism 6 program using the mann-whitney-u-test. differences were considered statistically significant if p-values were less than 0.05. apod in plasma is found primarily in hdl particles and to a lesser extent in ldl and vldl particles. to investigate the presence of particles containing both apod and apob, we performed a dual-specific elisa using the anti-apod mab d554 as a capture antibody and a biotinylated anti-apob mab, ldl20, for detection. to avoid disturbing the integrity of the lipoprotein particles, the elisa was performed in the absence of detergents. using this approach, we could detect not only lipoprotein particles carrying both apod and apob but also potential complexes between hdl and ldl or vldl particles, as depicted in fig. 1 . the dual-specific apod/b elisa analysis of plasma from finger blood yielded a surprisingly strong signal that was higher than that seen when immobilized apod was probed with the anti-apod antibody, d263-biotin ( fig. 2a) . similar results were obtained when performing the dual-specific elisa with two other anti-apod capture antibodies, d201 and d263 (data not shown). similarly, the detection mab ldl20-biotin in fig. 2a could be replaced by a different anti-apob antibody, ldl17-biotin, with comparable results (s2 figure) . furthermore, diluting the plasma yielded very similar titration curves from the dual-specific elisa as the same plasma from an apob-specific elisa (fig. 2b) . as apod has been reported to be scarce in ldl and vldl particles, much of this signal is likely attributable to the recognition of complexes between apod-containing hdl and apob-containing ldl (or vldl) particles. using the same type of dual-specific elisa with antibodies against apoe (fig. 2c) , apoh (fig. 2d ) or apoj (fig. 2e ) for capture resulted in weak or absent signals. to demonstrate the presence of these putative lipoprotein complexes in the general population, finger blood from ten healthy volunteers was tested using the apod/b elisa, and as seen in fig. 3a , all samples displayed elisa values comparable to those seen in fig. 2 . for reference, the apob and apod levels in the same samples were measured separately ( fig. 3b and 3c ). to determine the importance of having intact lipoprotein particles, we repeated the dual-specific apod/b elisa in the presence of detergent, using anti-apod-d544 as the capture mab and anti-apob-ldl20 as the detecting mab. as seen in fig. 4a , only small amounts of apob bound to d544 when detergent was present, indicating that intact lipoprotein particles are needed for the apod-ldl interaction. more surprisingly, when we reversed the system, using ldl20 as the capture antibody and biotinylated anti-apod d544 as the detecting antibody, no complexes could be detected, independent of the presence of detergent (fig. 4b ). to further investigate the potential interaction between apod-containing particles and ldl and the role of apod in this interaction, we used both purified apod and isolated hdl preparations in combination with purified ldl in the same dualspecific elisa. apod was purified from plasma by affinity chromatography in the presence of detergent, and a single band corresponding to apod was observed on sds-page (s1 figure) . we also used hdl2 and hdl3 particles, which differ both in size (hdl2 is significantly bigger) and apod content (hdl3 contains more apod). the apod and apob content in the different preparations was determined by elisa, confirming the differences in apod content (table i) . hdl2 contained some apob, which often happens when hdl2 is contaminated with small dense ldl. in fig. 5 , purified ldl alone and ldl co-incubated with hdl2, hdl3 or purified apod were analyzed via the dual-specific elisa. as expected, ldl itself gave a weak to moderate positive signal due to the concomitant presence of apod and apob in some of these particles. this signal was roughly doubled by the addition of either hdl2 or hdl3, while addition of purified native apod led to a 6-fold binding enhancement. interestingly, recombinant apod, in which 5 amino acids had been removed to reduce the hydrophobicity, did not promote ldl binding. purified vldl also binds to anti-apod in a detergent-sensitive manner and the binding is also enhanced by the addition of extra apod (fig. 6) . however, the binding gives lower values and these values are not raised as much by addition of purified apod. fig. 2 . anti-apod antibodies capture apob in a detergent-free dual specific elisa. the following capture antibodies functional under detergent-free conditions were used: a) anti-apod (d544), b) anti-apob (ldl17), c) anti-apoe (e981), d) anti-apoh (h219), e) anti-apob (ldl20) and f) anti-apoj (j29). human edta finger blood plasma, prepared as described in the methods, was diluted with pbs containing 0.1% bsa. detecting antibodies in a-d were ldl20-biotin (anti-apob), d263-biotin (anti-apod), h464-biotin (anti-apoh), 7b6-biotin (anti-ifnc) and e981 (anti-apoe), and in e and f were d201-biotin (anti-apod), d263-biotin (anti-apod), ldl17-biotin (anti-apob) and j84-biotin (anti-apod). note that e981 was also used for capture in c). the means ¡ sd of four replicates are shown. results within each panel are from the same donor. experiments a-d were repeated three times using the same donor, and experiments e and f were repeated four times using four different donors. doi:10.1371/journal.pone.0115180.g002 to measure binding in a more direct manner, we also used biosensor technology. we were able to not only register binding but also obtain information about the relative association affinities of the interactions. in agreement with the previous elisa results, the anti-apod mab d544 bound purified ldl due to the presence fig. 3 . the interaction between apod and apob is a general mechanism. finger blood from ten donors was assayed for a) apod/apob interaction in a detergent free dual-specific elisa using d544 for capture and biotinylated ldl20 for detection b) elisa for apob levels and c) elisa for apod levels. note that finger blood plasma, in figures b and c, was arbitrary calculated as half the finger blood volume, with the remaining volume assumed to be cells. of the ten donors, no. 1-5 were women, and no. 6-10 were men, all between 30-62 years old. means ¡ sd of four replicates are shown. apod mediates binding of hdl to ldl of apod in some of the ldl particles. (fig. 7a) . however, ldl binding was considerably faster and stronger when the d544 mab was first loaded with purified apod. the mab d263 yielded similar results (s3 figure) . as expected, the antibody also bound hdl3 and hdl2 to an extent reflecting the amount of apod in the respective particle. similar to the enhanced ldl binding observed in the presence apod, preloading the d544 mab with hdl3, and to some extent with hdl2, also increased the initial speed of ldl binding, though this effect was less strong compared to using purified apod (fig. 7b ). as apod is expressed by many cell types, we also investigated the interaction between apod and lipoprotein particles in a cellular setting. for this we used the fig. 4 . binding between apob and apod is disturbed by detergents and is dependent on the immobilized antigen. finger blood plasma was treated with or without detergent before being added to elisa plates. a) anti-apod (d544) was used as the coating antibody, allowing for multiple apod bonds to each ldl particle, as detected using anti-apob (ldl20-biotin). b) ldl20 was used as the coating antibody, and biotinylated d544 was used as the detection antibody. the means ¡ sd of four replicates are shown. experiments were repeated three times with same donor. doi:10.1371/journal.pone.0115180.g004 urinary bladder carcinoma cell line t24, which produces apod and displays contact inhibition when cultured in vitro, which has previously been shown to affect apod production [25, 26, 27, 28, 29] . in agreement with this latter finding, apod production was high in confluent, growth-arrested t24 cell cultures (2,5 million cells in 9 ml), but was not detectable in non-confluent proliferating anti-apod (d544) was used as the capture antibody and anti-apob (ldl20-biotin) was used as the detection antibody in a detergent free dualspecific elisa. ldl (333 ng/ml) was added either alone or in combination with apoa1 (100 ng/ml), hdl2 (1000 ng/ml), hdl3 (1000 ng/ml), apod (native) (100 ng/ml) or recombinant apod (100 ng/ml). as a the control, ldl was omitted, but the same amounts of apoa1, hdl2, hdl3, apod or recombinant apod were added. the control without ldl is shown in grey bars. relative absorbance was calculated by dividing the absorbance value by the absorbance of ldl alone. the experiment was repeated 6 times, each time using four replicates, and mean of the 6 experiments ¡ sd is shown. ** p,0.01 compared with ldl alone; p-values were calculated using the mann-whitney test with graphpad prism 6. the effect of adding extra apod (100 ng/ml) to vldl (1000 ng/ml) was analyzed using a dual-specific elisa with or without detergent present. the capture antibody was anti-apod (d544), and the detection antibody was anti-apob (ldl20-biotin). the means ¡ sd of four replicates are shown. experiments were repeated three times. * p,0.05; p-values were calculated using the mann-whitney test with graphpad prism 6. cultures (1,5 million cells in 7 ml) (fig. 8a) . however, despite the absence of apod in the supernatant of proliferating cells, apod was abundant intracellularly, as shown by flow cytometry (fig. 8b ) and by immunocytochemistry (fig. 9) . to measure potential interactions between cells and lipoprotein particles, cells were incubated with hdl in the presence or absence of apod. after removing unbound material by washing, the bound particles were solubilized, and the apoa1 (hdl) content in the soluble fraction was analyzed by elisa. as fig. 7 . biosensor monitoring of apod-mediated binding of ldl. in the blitz biosensor analysis, the signal corresponds to the mass collected on the biosensor surface. here we used streptavidin-coated sensor surfaces that were loaded with biotinylated antibody (10 mg/ml for 120 seconds) followed by apod (2 mg/ml for 300 seconds) or buffer-control (300 seconds). the surfaces were subsequently loaded with the ldl, hdl2 or hdl3 lipoprotein particles (100 mg/ml for 300 seconds). between loading reagents, the sensors were rinsed for 30 seconds. the buffer used for all steps and dilutions was pbs, azide, 0.1% bsa. a) the green, black, grey and red lines represent experiments preloaded with anti-apod d544. the blue line indicates the control preloaded with isotype control antibody 7b6 (anti-ifnc). the green and blue lines indicate that apod was preloaded, while the black, grey, and red lines indicate the buffer controls. the panel shows the final step in which the lipoprotein particles bind to the antibody loaded with or without apod. the green, black and blue lines show binding of ldl, the grey line shows binding of hdl3 and the red line shows binding of hdl2. b) the binding step of the biotinylated antibody was omitted. green, black, red and grey lines indicate experiments that used d544-biotin, and the blue line indicates that e981-biotin (anti-apoe) was used. subsequent reactions show the addition of hdl2 in red, hdl3 in grey, apod in green and blue, or buffer in black to the preloaded antibodies. in the last step, ldl was loaded in all the conditions. doi:10.1371/journal.pone.0115180.g007 demonstrated in fig. 10 , significant amounts of hdl were bound to both the confluent and non-confluent cultures. however, addition of purified apod to the non-confluent, proliferating cells significantly promoted the binding of hdl, whereas the same addition to confluent cells only had a marginal positive effect. similar experiments with ldl showed that apod did not help ldl bind to the cells (data not shown). fig. 8 . apod production in t24 cells was assayed by elisa for culture supernatant and by facs for intracellular staining. a) t24 cells were grown in dmem with 10% fbs in 25 cm 2 flasks. three flasks each of confluent cells (2.5 million in 9 ml) and non-confluent cells (1.5 million in 7 ml) were sampled 4 times. apod content was assayed by apod elisa; detection limit 50.1 ng/ml. b) d544 (anti apod) was used to stain confluent (black) and non-confluent (grey) t24 carcinoma cells. the isotype control is depicted with a dotted black line for confluent t24 cells and with a dashed grey line for non-confluent t24 cells. experiments were repeated three times. doi:10.1371/journal.pone.0115180.g008 we have here shown that apod, immobilized by specific monoclonal antibodies on a solid surface, mediates the binding of ldl to the surface. we used a dualspecific elisa in which antibodies to apod bound intact apob-containing lipoproteins equally efficient as antibodies specific for apob. this property was unique for apod and was not seen with other exchangeable apolipoproteins such apoe, apoh and apoj, all of which are present in hdl and ldl/vldl lipoprotein particles [32, 33, 34] . adding detergent inhibited the apod-mediated binding of apob, demonstrating that changes in the ldl structure affect the formation of the complex. surprisingly, ldl/vldl particles immobilized by antibodies to apob could only marginally capture apod, suggesting that the affinity of a single apod is not sufficient to withstand the repeated washings and incubations of the elisa protocol. therefore, several apod molecules will likely be needed to achieve sufficient avidity to immobilize ldl to a surface via apod-binding. this moderate affinity between apod and ldl is likely suitable for the rapid encounters between fig. 9 . non-confluent t24 cells stain positive for apod. cells were seeded on objective glasses in petri dishes. after either 2 days (non-confluent) or 9 days (confluent), the cells were washed and incubated with a negative isotype-control mab (anti-il4), a positive control anti-thioredoxin reductase mab (anti-trxred), or anti-apod d544 mab. bound monoclonal antibodies were stained with polyclonal anti-mouse fitcconjugated fab fragments. nuclei were visualized with dapi. cells were photographed with a leica dmrb fluorescent microscope (shutter speed fitc 2 sec, and dapi 1/30sek). staining was repeated three times. hdl-containing apod and ldl particles in the plasma, as a high affinity interaction could yield more stable complexes and aggregation. notably, adding purified apod helped d544 to bind more ldl, indicating that the apod contact with ldl is a very rapid event. hydrophilized recombinant apod, on the other hand, was not capable of binding ldl, probably because some of the hydrophobic amino acids required for binding have been removed in the recombinant apod. these results emphasize the need to use native apod for functional studies of this lipocalin. biosensor analysis is a nice complement to elisa as it measures binding in a direct way. mass binding to the sensor-tip results indiscriminately in a stronger signal, and the speed of binding can be monitored and is proportional to the fig. 10 . apod facilitates binding of hdl to growing t24 cells. t24 cells grown in 24-well plates were washed with protein-free rpmi two times, and 200 ml/well rpmi supplemented with 0.1%bsa was added with or without hdl (total hdl, 200 ng/well) and with or without apod (20 ng/well) or apoa1 (20 ng/well). after incubation for 1 hour at 21˚c, the plate was washed three times with protein-free rpmi, and bound apoa1 was released using detergent and assayed by apoa1 elisa. in a) the cells were non-confluent and in b) the cells were confluent. means ¡ sd of four experiments are shown. ** p,0.01, ****p,0.0001; p-values were calculated using two-way anova with holm-sidal multiple comparison test, graphpad prism 6. doi:10.1371/journal.pone.0115180.g010 affinity/avidity of the reaction. we demonstrate that although ldl contains some apod and will bind to anti-apod (d544), the binding was considerably faster if the d544 antibodies were preloaded with apod. in addition, hdl3 bound both more quickly and with more mass to d544-coated surfaces than did hdl2. as mentioned above, hdl3 has a higher density than hdl2 but is also considerably smaller and carries more apod. preloading the surface with purified apod is the most efficient way to tie down ldl, but hdl3 also yields a high initial speed of ldl binding compared to d544 alone or loaded with hdl2. one explanation for the difference between the quick binding of ldl to apod-or hdl3-coated surfaces and the slower linear binding of ldl to d544 alone is that, in the latter case, apod needs to reposition to allow multiple bonds between the surface and ldl. in animal experiments, apod was shown to be important in protecting against oxidative stress [6, 7, 24] . interestingly, hdl3 has been reported to be more important than hdl2 for protection against ldl-copper-catalyzed oxidation [35, 36] . the biosensor results obtained here show that hdl3 bound ldl more avidly, supplying a potential explanation for the protection of ldl by hdl3. t24 carcinoma cells display contact inhibition if the cells are washed with fresh medium after reaching confluency. washing removes growth factors and ensures that the cells are kept below the threshold needed to overcome contact inhibition [37] . interestingly, purified apod may aid growing t24 cells in binding hdl, but not contact-inhibited, growth-arrested cells, indicating a possible growthregulated receptor for apod. additionally, only supernatants from growthinhibited t24 cells contained detectable amounts of apod. these results agree with several reports showing that apod production increases upon growth arrest . however, intracellular staining with d544 revealed that non-confluent t24 cells contained substantial amounts of apod, albeit less than confluent cells. the reason for these unexpected results may be that non-confluent cells produce, but do not export, apod. another possibility is that non-confluent cells secrete apod but then internalize it efficiently via receptors not expressed by quiescent cells. one might speculate that apod plays a role in the lipid efflux to and from t24 cells. a putative mechanism is that apod helps to supply lipids to growing cells, presumably via an apod-recognizing receptor. once contact inhibition is induced, the cells downregulate this receptor and secrete surplus lipids via apod-containing lipoproteins. however, verifying such a mechanism would require extensive studies to find an apod binding receptor. large amounts of apod accumulate at sites of nerve injury [8] . human genetic studies and studies in apod knockout mice have revealed that apod is important not only for brain and nerve functions but also for controlling inflammation and macrophage homeostasis and to maintain a normal lipid profile [7, 20] . here we propose that apod acts a mediator/cofactor in the interaction of hdl particles with ldl particle and with cells. apod would presumably anchor hdl to ldl or cells with an affinity/avidity that result in a suitable on-off rate for lipid trafficking. this mechanism could partly explain several of the physiological roles of apod. for example, apod has been reported to help regulate lcat activity [21, 22, 23] . if apod is an important mediator of lipid trafficking, one might speculate that the intense up-regulation of apod at the site of nerve injury is necessary to attract and increase the local concentration of hdl particles for lipid transport between glial cells or macrophages and nerve cells and schwann cells. apod is an enigmatic protein, but the results of the current study reveal an important function of apod as a mediator of the interaction between hdl particles to ldl particles and to growing cells. however, further studies are needed to fully elucidate the physiological function of apod. several important questions remain, such as determining whether other cofactors are necessary for these lipoprotein interactions and how the lipid hormone-carrying role of apod affects these interactions. will the lipid load of this lipocalin change its function? identifying and characterizing the potential cellular receptor for apod and how it is regulated during cell growth will also be necessary. supporting information s1 figure. affinity purified apod. the mabtech apod standard was purified by affinity purification from edta aprotinin plasma in the presence of a non-ionic detergent. approximately 0.5 mg was analyzed by sds-page in the presence of a reducing agent and stained with simplyblue, invitrogen. doi:10.1371/journal.pone.0115180.s001 (tif) s2 figure. ldl/vldl captured by anti-apod can be detected by either ldl20biotin or ldl17-biotin. d544 was used to capture lipoproteins from human edta finger blood plasma, prepared as described in the methods. biotinylated detection antibodies were ldl20 (anti-apob), and ldl17 (anti-apob). d263biotin was used as a positive control. means ¡ sd of three replicates are shown. doi:10.1371/journal.pone.0115180.s002 (tif) s3 figure. biosensor monitoring of apod-mediated binding of ldl. in the blitz biosensor analysis, the signal corresponds to the mass collected on the biosensor surface. here we used streptavidin-coated sensor surfaces that were loaded with either d263-biotin or the isotype control 7b6-biotin (10 mg/ml for 120 seconds). sensors were rinsed (30 seconds) and loaded with apod (2 mg/ml) or buffer-control (120 seconds), and then rinsed again and loaded with 100 mg/ml ldl (120 seconds). doi:10.1371/journal.pone.0115180.s003 (tif) author contributions preparative isotachophoresis of human plasma high density lipoproteins hdl2 and hdl3 apolipoprotein d apolipoprotein d in lipid metabolism and its functional implication in atherosclerosis and aging structural insight into the dual ligand specificity and mode of high density lipoprotein association of apolipoprotein d apolipoprotein d is the major protein component in cyst fluid from women with human breast gross cystic disease human apod, an apolipoprotein up-regulated in neurodegenerative diseases, extends lifespan and increases stress resistance in drosophila apolipoprotein d is involved in the mechanisms regulating protection from oxidative stress accumulation of apolipoproteins in the regenerating and remyelinating mammalian peripheral nerve. identification of apolipoprotein d, apolipoprotein a-iv, apolipoprotein e, and apolipoprotein a-i apod, a glia-derived apolipoprotein, is required for peripheral nerve functional integrity and a timely response to injury regeneration-associated high level expression of apolipoprotein d mrna in endoneurial fibroblasts of peripheral nerve apolipoprotein d: an overview of its role in aging and age-related diseases apolipoprotein d takes center stage in the stress response of the aging and degenerative brain identification of apolipoprotein d as a cardioprotective gene using a mouse model of lethal atherosclerotic coronary artery disease neuroprotective effect of apolipoprotein d against human coronavirus oc43-induced encephalitis in mice apolipoproteins in the brain: implications for neurological and psychiatric disorders genetic variation in apolipoprotein d affects the risk of alzheimer disease in african-americans genetic variation in apolipoprotein d and alzheimer's disease association between polymorphisms in the apolipoprotein d gene and sporadic alzheimer's disease a role of apolipoprotein d in triglyceride metabolism genetic deficiency of apolipoprotein d in the mouse is associated with nonfasting hypertriglyceridemia and hyperinsulinemia separation of free and apolipoprotein d-associated human plasma lecithin: cholesterol acyltransferase activation of lecithin-cholesterol acyltransferase by apolipoprotein d: comparison of proteoliposomes containing apolipoprotein d, a-i or c-i a cholesteryl ester transfer complex in human plasma selective reduction of hydroperoxyeicosatetraenoic acids to their hydroxy derivatives by apolipoprotein d: implications for lipid antioxidant activity and alzheimer's disease apolipoprotein d transcription occurs specifically in nonproliferating quiescent and senescent fibroblast cultures inverse relationships between cell proliferation and basal or androgen-stimulated apolipoprotein d secretion in lncap human prostate cancer cells growth inhibition of human breast cancer cells by 1,25-dihydroxyvitamin d3 is accompanied by induction of apolipoprotein d expression modulation of apolipoprotein d and apolipoprotein e mrna expression by growth arrest and identification of key elements in the promoter apolipoprotein d inhibits platelet-derived growth factor-bb-induced vascular smooth muscle cell proliferated by preventing translocation of phosphorylated extracellular signal regulated kinase 1/2 to the nucleus expression and potential role of apolipoprotein d on the death-survival balance of human colorectal cancer cells under oxidative stress conditions interaction between high density and low density lipoproteins uptake and degradation by cultured human fibroblasts proteomic analysis of electronegative low-density lipoprotein a 70-kda apolipoprotein designated apoj is a marker for subclasses of human plasma high density lipoproteins genetic variation in the apolipoprotein h (beta2-glycoprotein i) gene affects plasma apolipoprotein h concentrations hdl3 exerts more powerful antioxidative, protective effects against copper-catalyzed ldl oxidation than hdl2 hdl, lipid peroxidation, and atherosclerosis apod mediates binding of hdl to ldl matrix stiffening sensitizes epithelial cells to egf and enables the loss of contact inhibition of proliferation key: cord-296399-vvbjulm9 authors: brinkmann, constantin; hoffmann, markus; lübke, anastasia; nehlmeier, inga; krämer-kühl, annika; winkler, michael; pöhlmann, stefan title: the glycoprotein of vesicular stomatitis virus promotes release of virus-like particles from tetherin-positive cells date: 2017-12-07 journal: plos one doi: 10.1371/journal.pone.0189073 sha: doc_id: 296399 cord_uid: vvbjulm9 vesicular stomatitis virus (vsv) release from infected cells is inhibited by the interferon (ifn)-inducible antiviral host cell factor tetherin (bst-2, cd317). however, several viruses encode tetherin antagonists and it is at present unknown whether residual vsv spread in tetherin-positive cells is also promoted by a virus-encoded tetherin antagonist. here, we show that the viral glycoprotein (vsv-g) antagonizes tetherin in transfected cells, although with reduced efficiency as compared to the hiv-1 vpu protein. tetherin antagonism did not involve alteration of tetherin expression and was partially dependent on a gxxxg motif in the transmembrane domain of vsv-g. however, mutation of the gxxxg motif did not modulate tetherin sensitivity of infectious vsv. these results identify vsv-g as a tetherin antagonist in transfected cells but fail to provide evidence for a contribution of tetherin antagonism to viral spread. vesicular stomatitis virus (vsv) is a negative-stranded rna virus within the rhabdoviridae family, and vsv new jersey and indiana are major vsv serotypes. vsv is transmitted from insects to ungulates (mainly cattle, horses and pigs), in which it can cause mucosal lesions [1] [2] [3] . in addition, the virus can be transmitted to humans and such infections usually induce influenza-like symptoms [3] . vsv replicates fast, is highly immunogenic and is frequently used to model infection by negative-stranded rna viruses. moreover, vsv is used as a tool for diverse scientific endeavors [4] . for instance, vsv has oncolytic properties [5] and is developed for cancer therapy [6] . moreover, vsv variants in which the open reading frame for the viral glycoprotein (vsv-g) has been replaced by that of the ebola virus (ebov) glycoprotein (gp) are currently tested as vaccines against ebov infection [7] [8] [9] . the interferon (ifn) system is an integral component of innate immunity and constitutes the first line of defense against viral infection. sensors of the ifn system, including toll-like receptors and retinoic acid inducible gene i-like receptors, can detect pathogen-associated plos one | https://doi.org/10.1371/journal.pone.0189073 december 7, 2017 1 / 19 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 molecular patterns (pamps), which triggers signals that commandeer the cells to express ifn [10, 11] . binding of ifn to uninfected cells in turn triggers further signaling events that induce the expression of ifn-stimulated genes (isg), many of which exert antiviral activity [12, 13] . vsv spread can be blocked by ifn in cell culture, although the viral matrix protein vsv-m interferes with ifn signaling [14] [15] [16] . the isg-encoded proteins that are responsible for ifninduced blockade of vsv infection are not fully known, although ifitm3 and tetherin were shown to block vsv infection in transfected cells [17, 18] . the ifn-induced antiviral host cell protein tetherin (cd317, bst-2) blocks release of diverse enveloped viruses from infected cells [19, 20] . the particular membrane topology of tetherin is key to its antiviral activity: tetherin harbors an n-terminal transmembrane domain and a c-terminal gpi-anchor which allows the protein to simultaneously insert into viral and cellular membranes, thereby forming a physical tether between virus and host cell [21] . several viruses encode tetherin antagonists which allow viral spread in tetherin-positive cells [22] . the prototypic tetherin antagonist, the hiv-1 protein vpu, and most other viral tetherin antagonists block tetherin by reducing its expression at the plasma membrane [23] [24] [25] , which is used by these viruses as platform for budding of progeny particles. in contrast, the ebov-gp, another tetherin antagonist, interferes with tetherin's antiviral activity without modulating tetherin expression or cellular localization [26] [27] [28] [29] and the mechanism underlying tetherin antagonism by ebov-gp is largely unclear. two studies reported that vsv is inhibited by tetherin. weidner and colleagues showed that directed expression of tetherin resulted in a profound decrease in vsv release from infected cells [18] . liberatore and coworkers dissected cell-cell spread of vsv from viral dissemination to distal cells via free particles and found that only the latter process was markedly inhibited by tetherin [17] . however, it is at present unknown whether vsv encodes a tetherin antagonist, which is responsible for residual viral spread in tetherin-positive cells. here, we show that vsv-g counteracts tetherin in transfected cells. however, no evidence for a contribution of tetherin-antagonism to spread of authentic vsv in tetherin-positive cells was obtained. human embryonal kidney-293t, vero (african green monkey, kidney) and hela (human, cervix carcinoma) cells were maintained in dulbecco's modified eagle medium (dmem) supplemented with 10% fetal bovine serum (fbs, biochrome, berlin) and penicillin/streptomycin (pan-biotech, aidenach; final concentration penicillin 100 units/ml, streptomycin 0.1 µg/ml). bhk-21 cells (baby hamster kidney) were cultivated in dmem supplemented with 5% fbs (biochrome) and penicillin/streptomycin. cells were cultured at 37˚c in humidified atmosphere containing 5% co 2 . for seeding and subcultivation, cells were washed with phosphatebuffered saline (pbs) and detached by incubation in a trypsin/edta solution (pan-biotech, aidenach; hela, vero and bhk-21 cells) or by directly resuspending them in dmem (293t). cell numbers were determined under a light microscope using a neubauer chamber. transfection of vero, bhk-21 and hela cells was performed using lipofectamine2000 (thermofisher scientific, dreieich) according to manufacturer's protocol, while 293t cells were transfected using the calcium phosphate method. selection and maintenance of vero cells stably transfected with the retroviral pqcxip vector was achieved using puromycin (cayman chemical, ann arbor). 293t cells were obtained from dsmz (acc-635, leibniz institute dsmz-german collection of microorganisms and cell cultures). vero and bhk-21 cells were obtained from collaborators. hela cells were obtained through the nih aids reagent program, division of aids, niaid, nih and their identity was confirmed by str dna typing employing a published protocol [30, 31] . plasmids encoding vpu, ebov-gp and hiv-1 p55-gag were previously described [27, 32] . expression plasmids for the coding sequences of vsv nucleoprotein (vsv-n), phosphoprotein (vsv-p), matrix protein (vsv-m), glycoprotein (vsv-g) and rna-dependent rnapolymerase (vsv-l) were generated by amplifying the respective open reading frame (orf) from a plasmid-encoded vsv anti-genome (indiana strain, kindly provided by g. zimmer) and inserting them via standard cloning procedures into plasmids pcaggs (vsv-n, vsv-p, vsv-m (mutant ncp, harboring four amino acid substitutions associated with reduced cytotoxicity [33] ) and vsv-l) or pcg1 (vsv-g). this was achieved by overlap-extension pcr technique using primers that introduce the desired nucleotide exchanges. generation of vsv-g mutants a133r and lxxxl was achieved by the same strategy using the expression plasmid for wt vsv-g as a template. the expression plasmid for human tetherin was generated by amplifying the orf from a previously described plasmid [34] and inserting it into plasmid pcaggs using kpni and xhoi restriction sites. similarly, porcine tetherin was pcr amplified and cloned via ecori and nhei restriction sites. for detection of human tetherin expression, the sequence for a c-myc antigenic tag was added to the 5' end of human tetherin using pcr. in order to generate transgenic cells stably expressing human tetherin, the genetic information for human tetherin was cloned into the retroviral vector pqcxip-mcs, a modified form of pqcxip (clontech, palo alto), in which the multiple cloning site was modified to contain sites for noti-bamhi-agei-hpai-mlui-xhoi-nrui-ecori. to generate recombinant vsv that expresses egfp (enhanced green fluorescent protein), we constructed a plasmidencoded vsv anti-genome (genbank: j02428.1) with an additional transcription unit for egfp between vsv-g and vsv-l orfs. first, unique mlui and nhei restriction sites were introduced upstream and downstream of the orf for vsv-g, respectively. next, a cassette consisting of the coding sequences of vsv-g and egfp, separated by a minimal intergenic region [35] was cloned and inserted into the parental vsv genome making use of mlui and nhei restriction sites, thereby replacing the genetic information of vsv-g and thus generating vsv ã (the asterisk stands for egfp). for convenient exchange of vsv-g or egfp orfs by other transcription units, unique asci and noti restriction sites were added upstream and downstream of the vsv-g and egfp orfs, respectively. in order to generate vsv ã mutant vsv-g (lxxxl), the respective orf was amplified from the corresponding vsv-g (lxxxl) expression plasmids with primers adding 5' mlui and 3' asci restriction sites and inserted into vsv ã , thereby replacing the parental orf coding for wt vsv-g. the integrity of all pcramplified sequences was confirmed by automated sequencing. release of virus-like particles (vlps) and its inhibition by tetherin has been examined as described [27, 32] . in brief, 293t cells were seeded in 6-well plates and cotransfected with plasmids encoding hiv-1 p55-gag, tetherin and a potential tetherin antagonist or empty plasmid, using the calcium phosphate method. at 16 h post transfection, the transfection medium was replaced by fresh culture medium. for blockade of vsv-g-dependent tetherin antagonism, anti-vsv-g hybridoma supernatant (i1, concentrated mouse hybridoma supernatant from crl-2700, atcc) was added to the culture medium at a final dilution of 1:1,000. at 48 h post transfection the supernatants were collected and cells were lysed in 200 μl of 2x sds-containing lysis buffer (30 mm tris [ph 6.8], 10% glycerol, 2% sds, 5% β-mercaptoethanol, 0.1% bromophenol blue, 1mm edta). the lysates were incubated at 95˚c for 30 min. the supernatants were cleared by centrifugation and vlps were pelleted from cleared supernatants by centrifugation through a 20% sucrose cushion. the concentrated vlps were lysed in 50 μl 2 x sds loading buffer and incubated at 95˚c for 30 min. subsequently, cell lysates and supernatants were analyzed for the presence of gag employing western blot. for immunoblotting, the proteins were separated via sds-polyacrylamid (paa) gel electrophoresis using a 12.5% paa gel and transferred onto a nitrocellulose membrane (ge healthcare life sciences, solingen, 0.2 μm). the membranes were blocked in 5% milk powder in pbs supplemented with 0.1% tween 20 and gag-protein was detected using 1:100 diluted supernatants of hybridoma cells secreting a mouse anti-gag antibody (183-h12-5c). expression of vsv-g wt and mutants was detected using the aforementioned anti-vsv-g hybridoma supernatant at a dilution of 1:100 while vsv-m was detected using mouse monoclonal antibody 23h12 (kerafast, boston). tetherin expression was detected employing anti c-myc-hybridoma supernatants (c-mycl-9e10 (ecacc 85102202)). expression of vsv proteins, vpu and ebov-gp was detected using previously described rabbit sera [36] [37] [38] . expression of ß-actin was detected using mouse anti-ß-actin antibody (sigma-aldrich, munich) at a dilution of 1:1,000. bound antibodies were detected using hrp-coupled anti-mouse and anti-rabbit secondary antibodies (dianova, hamburg) at a final concentration of 0.1 μg/ml. binding of secondary antibodies was detected using a self-made ecl solution (0.1 m tris-hcl ph 8.6, 250 μg/ml luminol (sigma-aldrich, münchen), 1 mg/ml para-hydroxycomaric acid (sigma-aldrich, münchen); 0.3% h 2 o 2 ) and signals were visualized using the chemocam imaging system and the chemostar-professional software (intas). for quantification of the signal intensity, the program imagej (fiji distribution) [39] was used. gag signals detected in the supernatants were normalized against the respective signals obtained in cell lysates. for the rescue of vsv ã harboring wt or mutant vsv-g, we employed a strategy developed by others [40] with slight modifications. first, bhk-21 cells were grown in 12-well dishes until they reached~70% confluency. at this point, the cells were infected with recombinant modified vaccinia virus ankara expressing t7 polymerase ( [41] , vmva-t7, kindly provided by g. sutter) at a multiplicity of infection (moi) of 3. at 1 h post infection, the inoculum was removed and the cells were transfected with expression plasmids for vsv-n, -p and -l (see above) as well as the respective, plasmid-encoded vsv ã anti-genome (the genome is preceded by a t7 promotor sequence and followed by a hepatitis delta virus ribozyme and a t7 terminator sequence for cytoplasmic production of negative-sensed viral genomes that are template for transcription and genome replication) using lipofectamine2000 (thermofisher scientific, dreiech) as transfection reagent. the following dna concentrations were used (per well): 0.4 μg vsv-n, 0.35 μg vsv-p, 0.25 μg vsv-l and 1.0 μg plasmid-encoded vsv ã or vsv ã (lxxxl) genome. transfection was carried out in optimem medium (thermofisher scientific, dreiech) for 6 h, before the transfection medium was replaced by standard culture medium. at 12 h post transfection, the culture medium was replaced by medium containing 100 μg/ml rifampicin and 40 μg/ml cytosine β-d-arabinofuranoside (both from sigma-aldrich, münchen) to limit rmva-t7 replication. after an additional 24 h, the culture medium was collected, clarified from cellular debris by centrifugation (4,700 rpm, 10 min, 4˚c) and twice filtered through a membrane filter with a pore size of 0.2 μm to eliminate rmva-t7 from the preparation. next, fresh bhk-21 cells grown in t-75 flasks were inoculated with a 1:100 dilution of the passage 0 (p0) to amplify vsv ã for virus stock production (p1). quantification of titers from vsv ã stocks and cell culture supernatant was carried out on confluently grown bhk-21 cells seeded in 96-well plates. after removal of the cell culture supernatant, cells were inoculated with 10-fold serial dilutions of virus (diluted in serum-free medium). at 1 h post infection, the inoculum was removed and cells were overlaid with culture medium containing 2% methylcellulose (sigma-aldrich, münchen). after an incubation of 16-18 h, egfp-positive cells were counted under the fluorescence microscope to determine viral titers (displayed as focus forming units per ml, ffu/ml). in addition, vsv ã titers were also determined for hela cells as this cell line is less susceptible to vsv infection (~100x) and therefore titers determined on bhk-21 cells are not useful to calculate the optimal infectious dose for hela cells. to verify the authenticity of wt and mutant vsv-g in vsv ã , viral rna was isolated from p1 stocks and reverse-transcribed into cdna using the superscript iii first-strand synthesis system (thermofisher scientific, dreiech) according to the manufacturer's protocol (for random hexamers). next, a~1,800 bp fragment was amplified with primers binding in the intergenic region upstream of vsv-g (forward) and the 5' end of the egfp orf (reverse) using phusion polymerase (thermofisher scientific, dreiech), separated by agarose gel electrophoresis and extracted from the gel by commercial kits (macherey & nagel, düren), before being subjected to automated sequence analysis (seqlab, göttingen). to assess the effect of directed tetherin expression on the spread of vsv ã , 293t cells were transfected with increasing amounts of expression plasmid encoding for human tetherin. to avoid unspecific effects due to differences in the total dna amounts of the tetherin vector, empty expression plasmid was used for equilibration. cells transfected only with empty expression vector served as controls. at 16 h post transfection, the transfection medium was replaced by culture medium and the cells were further incubated for 24 h before they were infected. additionally, vero cells stably expressing human tetherin (vero-tetherin) or stably containing empty vector were used. in order to analyze the impact of sirna-mediated knockdown of endogenous tetherin expression on vsv spread, hela cells were transfected with sirna specific to human tetherin or scrambled sirna (control) (both santa cruz, dallas; 10 pm final concentration) using lipofectamine2000 and optimem medium (both from ther-mofisher scientific, dreieich). at 6 h post transfection, the transfection medium was replaced by culture medium and the cells were further incubated for 24 h before they were infected. for infection, vsv ã stocks were diluted with serum-free medium to obtain the desired moi and inoculated onto target cells for 1 h. afterwards, the inoculum was removed and cells were washed with pbs before receiving fresh culture medium. next, cells were further incubated and supernatants were collected for quantification of vsv ã titers at different time points. to analyze vsv-g-driven host cell entry, vsv vectors were pseudotyped with either vsv-g wt or mutants a133r and lxxxl. for this, 293t cells were transfected with the respective expression plasmids or empty plasmid as negative control. at 24 h post transfection, cells were inoculated with vsv-g trans-complemented vsv ã δg that lacks the genetic information for vsv-g but codes for egfp and firefly luciferase from two independent transcription units [42] (kindly provided by g. zimmer) at an moi of 3. at 1 h post infection, the inoculum was removed and the cells were washed with pbs. next, medium containing a neutralizing antibody directed against vsv-g (i1, produced from crl-2700 hybridoma cells, atcc) was added to the cells and left for 1 h in order to neutralize residual input virus that had not entered the cells so far. subsequently, the cells were again washed and further incubated with fresh culture medium for 16-18 h. then, the supernatant was collected, clarified from cellular debris by centrifugation (4,700 rpm, 10 min, 4˚c) and used for transduction experiments. for this, 293t cells were inoculated with identical volumes of the respective vsv pseudotypes and firefly luciferase activity in cell lysates was quantified at 20 h post inoculation using a plate luminometer (hidex) and commercial substrates (pjk and promega) as described elsewhere [43] . for analysis of the surface expression of tetherin, 293t cells were cotransfected with tetherin plasmid and plasmid encoding viral antagonist or empty plasmid as negative control. at 48 h post transfection, the cells were washed and harvested in pbs. expression of tetherin at the cell surface was detected by employing a tetherin-specific mouse monoclonal antibody (biolegend) at a dilution of 1:50 and an alexa 647-conjugated anti-mouse secondary antibody at a dilution of 1:100 (thermofisher scientific, dreieich). subsequently, cells were fixed with 2% paraformaldehyde and staining was analyzed employing a lsr ii flow cytometer (bd biosciences, heidelberg) and the facs diva software (bd biosiences, heidelberg). the data were further analyzed using the fcs express 4 flow research software (de novo software). to ensure that directed tetherin expression did not lead to unwanted cytotoxic side-effects at the concentrations used, we analyzed cell viability employing the celltitre-glo kit (promega, mannheim). for this, 293t cells grown in 12-well plates were transfected with increasing amounts of tetherin expression plasmid or empty vector, which was also used to equilibrate total dna amounts. at 16 h post transfection, the transfection medium was replaced by fresh culture medium and the cells were further incubated for 24 h. next, the cell culture supernatants were removed and 200 μl of the celltitre-glo reagent were added. after an incubation period of 10 min at room temperature, 100 μl of the lysates were transferred into a white, opaque-walled 96-well plate, before luminescence was recorded using a plate luminometer (plate chameleon v, hidex, turku). all samples were analyzed in triplicates. for normalization, viability of cells transfected only with empty expression vector was set as 100% and relative viability of tetherin-transfected cells was calculated. if not explicitly stated in the figure legends, unpaired and paired student t-tests were performed to analyze data pairs originating from individual experiments or mean data of multiple experiments, respectively. one-way anova with bonferroni post-test analysis was carried out for combined comparison of multiple groups ( ã , p 0.05; ãã , p 0.005; ããã , p 0.001). in order to analyze tetherin antagonism by vsv proteins, we employed a previously reported virus-like particle (vlp) release assay, which measures release of hiv-gag-based vlps from transfected 293t cells and its blockade by tetherin [27, 32] . release of vlps was markedly diminished upon coexpression of tetherin and tetherin's antiviral activity was blocked by the well-established tetherin antagonists vpu and ebov-gp (fig 1a and 1b) , as expected. in addition, vsv-g but not vsv-l, m (mutant m(ncp), [33] ) and p interfered with tetherin's antiviral activity, although less efficiently than vpu and ebov-gp (fig 1a and 1b) . finally, none of the viral proteins tested modulated vlp release from tetherin-negative control cells, indicating that the effects observed in tetherin-positive cells were specific. thus, vsv-g can antagonize tetherin in transfected cells, although with reduced efficiency as compared to ebov-gp and vpu. the studies described above were conducted with human tetherin. since vsv can infect livestock, we next analyzed if vsv-g also antagonizes pig-derived tetherin. we found that vpu failed to antagonize porcine tetherin (fig 1c) , in keeping with the well-established notion that the anti-tetherin activity of vpu is highly species specific [44] [45] [46] . in contrast, both ebov-gp and vsv-g rescued vlp release from blockade by porcine tetherin (fig 1c) , indicating that vsv-g might be able to promote viral spread in infected pigs by antagonizing tetherin. evidence that tetherin antagonism by vsv-g can be blocked by a gprotein specific antibody and is not due to vsv-g overexpression we next sought to investigate whether tetherin counteraction by vsv-g can be inhibited by antibodies. this question was triggered by our previous observation that antibodies directed against ebov-gp can interfere with gp-mediated tetherin counteraction [32] . to this end, we added supernatant of hybridoma crl-2700 (which secretes the vsv neutralizing antibody i1) to cells expressing vsv-g and releasing vlps in the presence and absence of tetherin. the hybridoma supernatant did not modulate vlp release from tetherin-negative control cells coexpressing vsv-g but abrogated vlp-release from cells coexpressing tetherin and vsv-g (fig 1d and 1e) . we cannot exclude that the antibody triggered vsv-g internalization and a control antibody remains to be tested. however, the results available at present suggest that antibodies directed against vsv-g might not only block viral entry into target cells but may also interfere with tetherin antagonism. moreover, our findings indicate that the ectodomain of vsv-g plays an important role in tetherin counteraction. we next investigated whether the vsv-g expression levels attained in transfected cells exceeded those found in infected cells, which would suggest that tetherin counteraction by vsv-g could have been due to overexpression. for this, vsv-g expression levels in cells transfected to express vsv-g and cells infected with a recombinant vsv encoding gfp (vsv ã ) were compared. cells were harvested at the same time points at which vlp release and (figs 1a-1e and 5e and 5f) and vsv release (fig 3a-3c and fig 6a-6d ) from tetherin-positive cells were examined within functional assays. the immunoblot revealed that less g-protein was expressed in transfected as compared to infected cells (fig 1f) , indicating that tetherin antagonism was not due to overexpression. in this context, it should be noted that the lack of correlation between infectious dose and g-protein expression levels resulted from the fast replication kinetics of vsv, which led to 100% infected cells at the time of analysis. moreover, the reduced arrow heads indicate bands exhibiting the molecular weight expected for unglycosylated (white), partially (grey) and fully (black) n-glycosylated tetherin. (c) quantification of four experiments performed as described for panel (b). the intensities measured for all tetherin signals with a molecular weight between 17 and 30 kda were added to yield total tetherin expression. tetherin expression in the absence of antagonist (mock) was set to 100%. error bars indicate sem. one-way anova with bonferroni post-test analyses were performed for panels a and c to test for statistically significant differences between samples with and without (mock) antagonist (*, p 0.05). signals for actin in vsv-versus mock-infected cells can be attributed to the well-established cytotoxic effects associated with vsv infection [33, 47, 48] . collectively, the results discussed tetherin antagonism by viral proteins is usually due to removal of tetherin from the site of viral budding, the plasma membrane [20, 22] . in order to investigate whether vsv-g also interferes with tetherin expression at the plasma membrane, we performed flow cytometry with cells transfected to coexpress tetherin and viral antagonists. coexpression of vpu but not ebov-gp reduced tetherin surface levels (fig 2a) , as expected from previous reports [27, 28] . in contrast, coexpression of vsv-g did not modulate surface levels of tetherin (fig 2a) . in agreement with these findings, neither vsv-g nor ebov-gp coexpression reduced tetherin levels in cell lysates while a marked decrease in tetherin levels was observed upon coexpression of vpu (fig 2b and 2c) . thus, vsv-g, like ebov-gp, employs a mechanism different from that of vpu for tetherin counteraction. upon coexpression of vsv-g and ebov-gp, a band with the size expected for unglycosylated tetherin accumulated (fig 2b) , which is known to exert reduced antiviral activity as compared to the fully glycosylated form [21] . therefore, it is conceivable that vsv-g and ebov-gp inhibit tetherin by interfering with its n-glycosylation, similarly as reported for sars-coronavirus orf7a protein [49] . weidner and colleagues reported that vsv spread can be efficiently blocked by directed expression of tetherin [18] , and a subsequent study demonstrated that tetherin mainly inhibits cell-free but not cell-associated vsv spread [17] . we sought to confirm these findings and to establish a cellular system which allows investigating the potential contribution of vsv-gmediated tetherin counteraction to viral spread. for this, we transfected 293t cells with rising amounts of tetherin plasmid, infected the cells with vsv and quantified the number of infectious units present in culture supernatants at 8 h post infection. we observed a modest but dose-dependent reduction of viral titers upon transfection of increasing amounts of tetherinplasmid (fig 3a) in the absence of appreciable cytotoxic effects (s1 fig). moreover, sirnamediated knock-down of endogenous tetherin expression in hela cells reduced the amount of cell surface associated tetherin (fig 3b) and increased viral titers roughly 10-fold as compared to cells transfected with scrambled sirna or mock transfected cells (fig 3c) . collectively, these findings confirm that vsv is inhibited by tetherin and indicate that knock-down of tetherin expression in hela cells affords the opportunity to examine whether vsv-g-mediated tetherin antagonism is operative in the context of infected cells. program is presented. expression of vsv-g wt was set to 100%. (c) incorporation of vsv-g wt and mutant lxxxl into vsv pseudotypes was investigated by western blot analysis using an anti-vsv-g antibody (concentrated supernatants from hybridoma cl-2700). to ensure that similar amounts of pseudotypes were analyzed, levels of particle-associated m proteins were determined using an anti-vsv-m antibody. pseudotypes harboring no glycoprotein (mock) served as negative control. the results of a single immunoblot are shown from which irrelevant lanes were cut out. similar results were obtained in two separate experiments. (d) 293t cells were transduced with equal volumes of the vsv pseudotypes described in panel (c). at 24 h post transduction, luciferase activity in cell lysates was measured. transduction driven by vsv-g wt was set as 100%. the average of three independent experiments is shown. error bars indicate sem. (e) 293t cells were cotransfected with plasmids encoding gag, the indicated glycoproteins and tetherin. expression of gag in supernatants and cell lysates was determined by western blot. detection of β-actin expression served as loading control. (f) the average of three independent experiments conducted as described for panel (e) and quantified via the imagej program is presented. error bars indicate standard error of the mean (sem). release of gag from cells coexpressing the highest amount of vsv-g and tetherin was set to 100%. for all graphs (b, d, f), paired two-tailed students' t-tests were performed to assess whether differences between vsv-g wt and lxxxl mutant were of statistical significance. https://doi.org/10.1371/journal.pone.0189073.g005 a gxxxg motif in the transmembrane domain of vsv-g contributes to tetherin antagonism in transfected cells but is dispensable for vsv spread in tetherin-positive cells having established that vsv-g can antagonize tetherin upon directed expression and that vsv spread is reduced but not abrogated by tetherin, we finally sought to determine whether vsv-g-mediated tetherin antagonism contributes to viral spread. for this endeavor, we needed to identify a mutation in vsv-g that selectively interferes with tetherin antagonism. to this end, we tested a vsv-g mutant in which a gxxxg motif in the transmembrane domain was changed to lxxxl (fig 4) . this mutation was chosen, since our unpublished results indicate that a gxxxa motif in the transmembrane domain of ebov-gp contributes to tetherin antagonism. in addition, we analyzed vsv-g mutant a133r. this mutation is known to interfere with viral entry by rendering vsv-g defective for membrane fusion [50] . both mutation a133r and mutation of the gxxxg motif (mutant lxxxl) were compatible with robust g-protein expression (fig 5a and 5b ) and particle incorporation (fig 5c) . moreover, mutation of the gxxxg motif (mutant lxxxl) did not interfere with host cell entry of vsv-g pseudotypes (fig 5d) . in contrast, mutation a133r markedly reduced entry efficiency (fig 5d) , as expected [50] . finally, mutant a133r was able to counteract tetherin while mutant lxxxl failed to efficiently antagonize tetherin (fig 5e and 5f ). thus, mutation of the gxxxg motif afforded an opportunity to investigate whether g-protein-mediated tetherin antagonism contributes to viral spread. for this, the lxxxl mutation was introduced into the vsv genome, employing a reverse genetics system, and infectious vsv was rescued. infection of sirna-transfected hela cells with vsv wt and mutant lxxxl at equal moi revealed that knock-down of tetherin expression increased vsv wt release, as expected. however, a comparable rescue of viral release was observed for the lxxxl mutant (fig 6a and 6b) . moreover, spread of vsv wt and mutant lxxxl was comparably reduced by directed expression of tetherin in vero cells (fig 6c and 6d) , suggesting that tetherin antagonism by vsv-g might not appreciably contribute to viral spread in tetherin-positive cells. tetherin and other effector proteins of the ifn system are responsible for the establishment of an antiviral state in ifn exposed cells [12, 13] . many viruses evolved countermeasures against these antiviral effectors by either multi-functionalizing their structural proteins or by acquiring non-structural proteins with antagonistic activity. whether vsv, which is sensitive to blockade by tetherin [17, 18] , encodes an antagonist that allows residual viral spread in tetherin-positive cells is unknown. here, we show that the surface protein of vsv, vsv-g, can antagonize tetherin, at least upon directed expression. tetherin counteraction by vsv-g did not involve modulation of tetherin expression and was partially dependent on a gxxxg motif in the vsv-g transmembrane domain. however, mutation of the gxxxg motif in the context of infectious vsv did not affect viral spread in tetherin-positive cells. thus, vsv-g is a novel tetherin antagonist but the potential contribution of its antagonistic activity to viral spread in tetherin-positive cells requires further investigation. previous studies reported that directed expression of tetherin in nih 3t3 cells and 293 cells markedly diminished viral release [17, 18] . however, tetherin expression failed to reduce viral release to background levels in both studies, although tetherin was expressed at high levels, which leaves the possibility that vsv encodes a modestly active tetherin antagonist. in keeping with such a scenario, we found that vsv-g antagonizes human and pig tetherin but does so less efficiently than vpu and ebov-gp. tetherin antagonism could only be demonstrated in the context of directed vsv-g expression in the present study. nevertheless, tetherin counteraction was not due to g-protein overexpression, indicating that vsv-g could block tetherin in infected cells, as discussed below. several viral tetherin antagonists inhibit tetherin by interfering with its expression or appropriate cellular localization. for instance, vpu can interfere with the anterograde transport of tetherin [51, 52] and directs tetherin towards lysosomal degradation [23] [24] [25] , which ultimately results in reduced tetherin levels at the cell surface. in contrast, vsv-g did not modulate total tetherin expression or tetherin levels at the cell surface. these findings are similar to those previously reported for ebov-gp [27, 28] , which antagonizes tetherin by a so far unknown mechanism. moreover, vsv-g, like ebov-gp, was active against diverse tetherins, as demonstrated by the counteraction of porcine tetherin, which shares only 48% sequence identity with human tetherin. finally, our finding that antibody i1 directed against the vsv-g ectodomain interferes with tetherin antagonism recapitulates findings previously made for ebov-gp [32] and suggests that both vsv-g and ebov-gp depend on their ectodomains for tetherin counteraction. how vsv-g (and ebov-gp) counteracts tetherin remains unknown and at present a role of direct interactions (potentially disrupted by anti-ectodomain antibodies) cannot be discounted. our finding that coexpression of vsv-g resulted in increased generation of unglycosylated tetherin, which is known to display little antiviral activity [21] , suggests that vsv-g might block tetherin, at least in part, by interfering with its posttranslational modification. a similar inhibitory strategy has previously been reported for the sars-coronavirus orf7a protein [49] but the underlying mechanism is incompletely understood. in order to get initial insights into the contribution of vsv-g-mediated tetherin antagonism to vsv release from tetherin-positive cells, we examined previously characterized mutations in the ectodomain (a133r) and the transmembrane domain (g473l and g477l, mutant lxxxl) of vsv-g for their effect on tetherin antagonism. a133r markedly reduced vsv-gdriven entry, as expected [50] , but did not affect tetherin antagonism. in contrast, mutation of the gxxxg motif in the transmembrane domain had no impact on entry but reduced tetherin antagonism by roughly 60%, as discussed below. the finding that the gxxxg motif was dispensable for vsv-g-driven virus-cell fusion was unexpected, since the same motif has been reported to be required for cell-cell fusion induced by exposure of vsv-g expressing cells to low ph [53] . in this context, it is important to state that the integrity of the lxxxl mutations after amplification of the rescued viruses in bhk-21 cells (passage 1 stocks) has been confirmed by sequencing. therefore, the gxxxg motif seems to be important for vsv-g-driven cell-cell but not virus-cell fusion and the underlying reasons remain to be determined. the finding that the gxxxg motif is required for tetherin antagonism is in keeping with our unpublished observation that a gxxxa motif in the ebov-gp transmembrane domain, which has been reported to be important for ebov-gp-mediated cellular detachment [54] , is required for full tetherin antagonism by ebov-gp. although mutation of the gxxxg motif in vsv-g reduced tetherin antagonism in transfected cells, it had no impact on viral spread in hela cells, which express endogenous tetherin [19] , and vero cells, which were engineered to express tetherin. these observations could indicate that vsv-g-mediated tetherin antagonism is not operative in vsv infected cells. in fact, one could speculate that in infected cells the interaction between vsv-g and other viral proteins, which is required for assembly of progeny particles [55] , compromises the ability of vsv-g to counteract tetherin. alternatively, it is conceivable that the modest reduction of tetherin antagonism observed in transfected cells upon mutation of the gxxxg motif precluded detection of anti-tetherin effects in infected cells. the identification of a mutation in vsv-g that selectively and efficiently reduces tetherin antagonism is required to address this question. collectively, our results and published data indicate that vsv-g, ebov-gp [26] and possibly other viral glycoproteins can interfere with tetherin's antiviral activity via a mechanism that is relatively independent of the tetherin sequence. glycoprotein-mediated interference with tetherin's n-glycosylation status might contribute to this effect. moreover, our results indicate that findings made for tetherin-antagonism in transfected cells might not always adequately reflect the situation in infected cells. finally, our findings suggest the possibility that vsv might acquire robust tetherin antagonism upon infection of humans. supporting information s1 fig. directed tetherin expression is not associated with major cytotoxic effects. 293t cells were transfected with increasing amounts of expression vector for human tetherin. empty vector was used for equilibration of total dna amounts. at 48 h post transfection, intracellular atp levels were quantified. the average of three independent experiments performed with triplicate samples is shown, error bars indicate standard error of the mean (sem). a paired two-tailed student's t-test was used to examine whether differences in cell viability between cells transfected with empty vector (0 μg tetherin plasmid) and tetherin-expressing cells were of statistical significance (ns, not significant; ã , p 0.05). (pdf) vesicular stomatitis transmission of vesicular stomatitis virus from infected to noninfected black flies co-feeding on nonviremic deer mice emergence and re-emergence of vesicular stomatitis in the united states vesicular stomatitis virus: re-inventing the bullet oncolytic activity of vesicular stomatitis virus is effective against tumors exhibiting aberrant p53, ras, or myc function and involves the induction of apoptosis oncolytic vesicular stomatitis virus as a viro-immunotherapy: defeating cancer with a "hammer" and "anvil assessing the safety and immunogenicity of recombinant vesicular stomatitis virus ebola vaccine in healthy adults: a randomized clinical trial six-month safety data of recombinant vesicular stomatitis virus-zaire ebola virus envelope glycoprotein vaccine in a phase 3 double-blind, placebo-controlled randomized study in healthy adults safety and immunogenicity of the rvsvg-zebov-gp ebola virus vaccine candidate in healthy adults: a phase 1b randomised, multicentre, double-blind, placebo-controlled, dose-response study prrs are watching you: localization of innate sensing and signaling regulators pattern recognition receptors and the innate immune response to viral infection interferon-stimulated genes: a complex web of host defenses a diverse range of gene products are effectors of the type i interferon antiviral response the vesicular stomatitis virus matrix protein inhibits transcription from the human beta interferon promoter factors affecting the sensitivity of different viruses to interferon rhabdovirus evasion of the interferon system tetherin inhibits cell-free virus dissemination and retards murine leukemia virus pathogenesis interferon-induced cell membrane proteins, ifitm3 and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms tetherin inhibits retrovirus release and is antagonized by hiv-1 vpu the antiviral activities of tetherin tetherin inhibits hiv-1 release by directly tethering virions to cells counteraction of the multifunctional restriction factor tetherin differential effects of human immunodeficiency virus type 1 vpu on the stability of bst-2/tetherin vpu directs the degradation of the human immunodeficiency virus restriction factor bst-2/tetherin via a {beta}trcp-dependent mechanism vpu antagonizes bst-2-mediated restriction of hiv-1 release via beta-trcp and endo-lysosomal trafficking tetherin-mediated restriction of filovirus budding is antagonized by the ebola glycoprotein the ebola virus glycoprotein and hiv-1 vpu employ different strategies to counteract the antiviral factor tetherin ebola virus glycoprotein counteracts bst-2/tetherin restriction in a sequence-independent manner that does not require tetherin surface removal anti-tetherin activities of hiv-1 vpu and ebola virus glycoprotein do not involve removal of tetherin from lipid rafts recommendation of short tandem repeat profiling for authenticating human cell lines, stem cells, and tissues short tandem repeat typing technologies used in human identity testing the tetherin antagonism of the ebola virus glycoprotein requires an intact receptor-binding domain and can be blocked by gp1-specific antibodies fusionactive glycoprotein g mediates the cytotoxicity of vesicular stomatitis virus m mutants lacking host shutoff activity tetherin-driven adaptation of vpu and nef function and the evolution of pandemic and nonpandemic hiv-1 strains the minimal conserved transcription stop-start signal promotes stable expression of a foreign gene in vesicular stomatitis virus use of influenza c virus glycoprotein hef for generation of vesicular stomatitis virus pseudotypes human immunodeficiency virus type 1 vpu protein is an oligomeric type i integral membrane protein modulation of virion incorporation of ebolavirus glycoprotein: effects on attachment, cellular entry and neutralization fiji: an opensource platform for biological-image analysis recombinant vesicular stomatitis viruses from dna non-replicating vaccinia vector efficiently expresses bacteriophage t7 rna polymerase a vesicular stomatitis virus replicon-based bioassay for the rapid and sensitive determination of multi-species type i interferon the glycoproteins of all filovirus species use the same host factors for entry into bat and human cells but entry efficiency is species dependent hiv-1 antagonism of cd317 is species specific and involves vpu-mediated proteasomal degradation of the restriction factor species-specific activity of siv nef and hiv-1 vpu in overcoming restriction by tetherin/bst2 species-specific activity of hiv-1 vpu and positive selection of tetherin transmembrane domain variants the cell-rounding activity of the vesicular stomatitis virus matrix protein is due to the induction of cell death vesicular stomatitis virus matrix protein inhibits host cell gene expression by targeting the nucleoporin nup98 severe acute respiratory syndrome coronavirus orf7a inhibits bone marrow stromal antigen 2 virion tethering through a novel mechanism of glycosylation interference molecular architecture of the bipartite fusion loops of vesicular stomatitis virus glycoprotein g, a class iii viral fusion protein hiv-1 vpu antagonizes bst-2 by interfering mainly with the trafficking of newly synthesized bst-2 to the cell surface hiv-1 vpu blocks recycling and biosynthetic transport of the intrinsic immunity factor cd317/tetherin to overcome the virion release restriction the transmembrane domain in viral fusion: essential role for a conserved glycine residue in vesicular stomatitis virus g protein inhibition of ebola virus glycoprotein-mediated cytotoxicity by targeting its transmembrane domain and cholesterol subunit interactions of vesicular stomatitis virus envelope glycoprotein stabilized by binding to viral matrix protein the following reagents were obtained through the nih aids reagent program, division of aids, niaid, nih: anti-hiv-1 nl4-3 vpu polyclonal antiserum from dr. klaus strebel, hela cells from dr. richard axel. polyclonal antiserum against vsv was kindly provided by dr. georg herrler. key: cord-286708-igu984oc authors: chua, kaw bing; voon, kenny; crameri, gary; tan, hui siu; rosli, juliana; mceachern, jennifer a.; suluraju, sivagami; yu, meng; wang, lin-fa title: identification and characterization of a new orthoreovirus from patients with acute respiratory infections date: 2008-11-25 journal: plos one doi: 10.1371/journal.pone.0003803 sha: doc_id: 286708 cord_uid: igu984oc first discovered in the early 1950s, reoviruses (respiratory enteric orphan viruses) were not associated with any known disease, and hence named orphan viruses. recently, our group reported the isolation of the melaka virus from a patient with acute respiratory disease and provided data suggesting that this new orthoreovirus is capable of human-to-human transmission and is probably of bat origin. here we report yet another melaka-like reovirus (named kampar virus) isolated from the throat swab of a 54 year old male patient in kampar, perak, malaysia who was suffering from high fever, acute respiratory disease and vomiting at the time of virus isolation. serological studies indicated that kampar virus was transmitted from the index case to at least one other individual and caused respiratory disease in the contact case. sequence analysis of the four small class genome segments indicated that kampar and melaka viruses are closely related. this was confirmed by virus neutralization assay, showing an effective two-way cross neutralization, i.e., the serum against one virus was able to neutralize the other. although the exact origin of kampar virus is unknown, epidemiological tracing revealed that the house of the index case is surrounded by fruit trees frequently visited by fruit bats. there is a high probability that kampar virus originated from bats and was transmitted to humans via bat droppings or contaminated fruits. the discovery of kampar virus highlights the increasing trend of emergence of bat zoonotic viruses and the need to expand our understanding of bats as a source of many unknown viruses. reoviruses (respiratory enteric orphan viruses), members of the family reoviridae, are a large and diverse group of non-enveloped viruses with segmented dsrna genomes, which are taxonomically classified into ten genera [1, 2] . members of the genus orthoreovirus contain 10 genome segments and have been isolated from a broad range of mammalian, avian and reptilian hosts. although orthoreoviruses have been identified as the causative agents of diseases in animals, infections in humans are generally benign with very rare cases of mild upper respiratory tract illness or enteritis in infants or children [3] . orthoreoviruses are divided into two subgroups, fusogenic and nonfusogenic, based on the ability of the virus to induce cell-cell fusion and syncytium formation [4] . a fusogenic orthoreovirus, the melaka virus (melv), was isolated from a human patient suffering acute upper respiratory disease [5] . melv was shown to be capable of human-to-human transmission and has close sequence relatedness to two bat-borne orthoreoviruses, the nelson bay virus (nbv) isolated from fruit bats in australia and the pulau virus (pulv) isolated from fruit bats in malaysia [6, 7] . epidemiological tracing suggested that melv originated from bats and was transmitted directly to the index case, followed by subsequent transmission to other members of the same family [5] . bats have been shown to be the reservoir hosts of many recently emergent zoonotic viruses, including hendra virus, nipah virus, menangle virus, and potentially sars and ebola viruses [8] [9] [10] [11] [12] [13] . nbv was the first reovirus of bat origin, which was isolated in 1968 from the heart blood of a flying fox (pteropus poliocephalus) in new south wales, australia [6, 14] . nbv was also the first mammalian reovirus to display fusogenic properties [6] , a characteristic previously only known for avian reoviruses (arvs). in 1999, during a search for nipah virus in pteropid bats on tioman island, pulv was isolated from pteropus hypomelanus [7, 15] . here, we report the discovery and characterization of kampar virus (kamv), the fourth member in the nbv species group and its isolation from a human patient with fever and acute respiratory illness. although there is no direct evidence to suggest that kamv originated from bats, the close relationship of kamv with other members of the nbv group and preliminary epidemiological data suggest that kamv is most likely a batborne orthoreovirus. to mitigate the potential health as well as socio-economic impact of emerging diseases, in particular, the potential emergence of pandemic influenza, the ministry of health malaysia undertook nation-wide influenza-like illness surveillance for early detection, identification and control of these emerging diseases. in august 2006, as part of the surveillance process, a patient with acute influenza-like illness was investigated in kampar, a town situated in the north-western part of peninsular malaysia, about 36 kilometres south of ipoh, the capital city of the state perak. case 1 (index case). subject 1 (s1), a 54-year old chinese man, was well until 19 august 2006 when he developed sudden onset of high fever with chills and rigor. this was associated with cough, sore-throat and headache. there was no associated dyspnoea, tachypnoea, haemoptysis or chest pain on coughing. besides the severe frontal throbbing headache, he had generalized body aches, myalgia and severe malaise. on the following day, he developed nausea, vomiting and diarrhoea. the vomitus consisted of food taken and was not bile-stained. the stool was described as watery without excessive mucous and it was non-malenic. the gastrointestinal symptom was associated with abdominal pain and loss of appetite. his illness was not relieved with self-medication of antipyretics. on review, there was no associated giddiness, blurring of vision, photophobia, skin bleeding or arthritis. he sought medical treatment at the government health clinic in kampar on 21 august 2006. at the outpatient clinic, he was noted to be febrile (an axillary temperature of 40.1 degree celsius), illlooking with a generalized body erythema that blanched on pressure and was more prominent over the face and upper trunk. he had mild conjunctivitis but there was no jaundice. his tonsils were enlarged and injected but there was no white exudate noted over the tonsils. he was not in any respiratory distress and his lungs were clear with good air entry on auscultation. other systemic examination was essentially normal and there was no significant lymphadenopathy noted. a provisional diagnosis of influenza-like illness was made at which he was given a higher dose of anti-pyretic. his illness was noted to resolve on 23 august 2006 although he still appeared weak and lethargic. venous blood samples were taken from the patient for full blood count analysis, and the results are shown in table 1 . these results indicated that his white blood cell and platelet counts were within normal limits although there was a relative lymphopenia in the blood sample taken at first examination. his throat swab was taken on august 21 for virus isolation as described below. case 2 (contact case). subject 2 (s2) is a 28 year old female chinese medical officer who attended to the index patient in the government health clinic in kampar on 21 august 2006. on 25 august, she developed nasal obstruction with mild runny nose. this was associated with a mild sore-throat and hoarseness of voice. there was no associated cough or breathing difficulty. apart from the subjective feeling of mild lethargy and general unwellness, she did not experience fever, headache or myalgia. her upper respiratory symptoms resolved within three days. case 3 (suspected contact case). subject 3 (s3), a 46 year old female, is the wife of the index patient. she regularly stays and takes care of the family home. due to the nature of their work in other parts of the country, her husband and their 17-year old son only come back intermittently to stay in the same house. she was unable to re-call having a similar illness as her husband previously, although she did remember experiencing incidents of mild headache and fever in late august to early september, which resolved with self-medication. since she did not seek medical help, there was no record of medical examination at the time of suspected infection. on august 21, during the examination by the local doctor, a throat swab was taken from the index case patient (s1) and sent in viral transport medium (vtm) to the national public health laboratory for virus isolation. after 3 days of culturing, a syncytial cytopathic effect (cpe) was noted in mdck and vero cells, but not in hep-2 cells. after 2 passages in mdck cells, the virus was able to replicate and cause syncytial cpe in all types of mammalian cell-lines available in the laboratory inclusive of c6/36 (atcc crl-1660) cell-line which is of mosquito cell in origin (data not shown). the virus was named kampar virus (kamv) after the location of the index case. due to the similar cpe morphology ( figure 1 ) and cell line susceptibility patterns between kamv and the recently discovered melaka virus (melv), which also causes acute respiratory diseases in humans [5] , immunofluorescent antibody testing was conducted to examine cross reactivity. as shown in figure 2 , the strong cross reactivity between kamv antigen and human anti-melv sera, and vice versa, suggested these viruses are closely related. this was later confirmed by further serological characterization and molecular analyses (see below). the small township of kampar is bordered by forested limestone hills on the eastern side and a scattering of abandoned old tin mining ponds on the other side. s1 is a part-time carpenter and lives with his wife and their youngest son (17 years old) in an old wooden house with metal zinc roof and plain cement floor. his house is situated on reclaimed old tin mining land which is about 80 metres from the central market of kampar town. many tall trees, comprising of mango trees, coconut palms and natural wild fruiting trees (in particular, the ''sea-almond'' or locally named ketapang trees), are found surrounding his house and between neighbouring houses. a tall fruiting mango tree is planted close to the house which is of less than three feet from the side wall of the house and directly adjacent to the window of the living room. at night, fruit bats were noted to use this mango tree to feed on the ''sea-almond'' fruits which they obtained from the nearby ketapang trees. partially eaten fruits and leftover stones of the fruits abandoned by the fruit bats were noted on the ground just outside the window of the living room or on the roof of the house. only occasionally, the fruit bats were noted to feed on the ripened mangoes. during night time, s1 and his wife often rested in the living room while watching television. there was no recollection of fruit bats flying into his house or roosting inside the house. there was also no record of dead fruit bats being found in the house compound. it is not clear at the present time whether kamv is carried by a specific fruit bat species or by multiple bat species circulating in the region. a preliminary survey of bat sera collected in peninsular malaysia from our previous studies indicated a low prevalence of kamv-specific antibodies in at least two different bat species, pteropus vampyrus (1/55) and pteropus hypomelaunus (2/77). considering that the closely related pulv virus was isolated from pteropus hypomelaunus [7] and nbv from pteropus poliocephalus [6] , it seems likely that these orthoreoviruses may have a broad host range among different bats. it should be noted that smaller fruit bats, such as eonycteris spelaean and cynopterus brachyotis, are more commonly found in the areas where kamv and melv were discovered. further field study is required to elucidate the bat species mainly responsible for the spill over of these viruses into human population in malaysia. the family keeps a couple of chickens in the backyard and a few pet birds in the patio. a week prior to the onset of s1's illness, his wife bought 6 chickens from a neighbour and one of them died on day 2 of illness. on follow-up investigation, venous blood samples were taken from s1, his wife (s3) and the doctor (s2) who examined him on august 21. all sera were tested for the presence of antibodies against kamv by immunofluorescent antibody testing using kamv-infected mdck cells. as indicated by the results in table 2 , anti-kamv antibodies were present in all three subjects, with the index case patient showing the highest level of igg antibodies. to exclude the possibility that kamv was just a passenger virus which might be commonly present in the human population in this region and was not the cause of the observed sickness in s1 and s2, we have since conducted serological surveillance of 31 human serum samples collected in the same region for an unrelated study in early 2008. none of these sera had any specific igm antibodies against kamv and only one serum sample contained low level (1:80) of kamv-reactive igg antibodies. to further establish the antigenic relationship between kamv and three other known orthoreoviruses in the nbv species group, a four way cross-neutralization was conducted as described in the materials and methods with the results presented in table 3 . it is clear that all of the four viruses share significant antigenic relatedness as evident from the cross-neutralization activities of each serum. the failure of any of the four sera to neutralize the control virus, mammalian reovirus 3 (mrv3), and of the mrv3 serum to neutralize any of the four viruses confirmed the specificity of the assay. while the kamv human serum neutralized the homologous virus better than heterologous viruses, such a difference was not observed for the melv human serum, which seemed to be equally effective in neutralizing all four viruses. the close genetic relationship between kamv and melv was confirmed by the comparison of genome segment mobility or electropherotypes using sds-page. the two viruses have similar but not identical rna genome segment profiles (figure 3) . the genetic relatedness of kamv and three other viruses in the nbv species group was further corroborated by the following molecular characteristics: (i) the deduced protein products encoded by the four small (s) genome segments of these viruses are very similar in size and share significant levels of sequence identity (table 4 ); (ii) one s-class genome segment of orthoreoviruses may be polycistronic, and its coding arrangement is used as a useful molecular marker for differentiation of different species groups (duncan et al., 2004) . the s1 segment of kamv has an identical coding arrangement to that of the three other viruses (data not shown); (iii) orthoreoviruses have highly conserved genome terminal sequences at the 59 end of the positive sense rna which can be used as a genetic marker for virus classification [3] . for kamv, the consensus sequence is 59 gcuuww (w = u or a), which is identical to that of melv, pulv and nbv (figure 4 ), but different from other orthoreoviruses. the evolutionary relationship between kamv and other known orthoreoviruses was investigated by conducting phylogenetic analyses for all the proteins encoded by the s-class genome segments. representative phylogenetic trees based on deduced amino acid sequences of the major outer capsid protein and the major inner capsid protein are shown in figure 5a and 5b, respectively. it is evident that kamv is a close member of the nbv species. similar trees were obtained based on other proteins (data not shown). respiratory tract infections remain the main infectious disease of humans and account for a large proportion of public health spending worldwide. despite recent success in discovery of novel respiratory pathogens during the last decade [16] [17] [18] , there is still a substantial proportion of rtis remaining undiagnosed. isolation of melv from a patient suffering acute respiratory disease marked a new beginning of linking orthoreoviruses to acute human rtis [5] . this was especially significant in that epidemiology studies indicated that melv originated from bats. the last decade has experienced a surge in the discovery of emerging viruses of bat origin, several of which have had significant impact on human and animal heath, tourism and trade [8, [19] [20] [21] . although the exact reason is not fully known, it seems that bats are an ideal reservoir for a number of zoonotic viruses covering many different virus families. in this context, the isolation of kamv confirms the observed trend of zoonotic virus emergence out of bat populations in the pacific region. although there is no direct evidence to prove the origin of kamv, the close genetic relationship of kamv with melv, pulv and nbv and the location of the house of the index case in close proximity to fruit trees frequently visited by fruit bats indicated a high probability that kamv is also a bat-borne virus. subsequent serological surveillance confirmed low levels of antibodies to kamv circulating in two bat species commonly found in peninsula malaysia. retrospective serological studies indicated that the index has transmitted the virus to at least one other person, his doctor, presumably via contact droplet nuclei or aerosol. it is more difficult to assess how his wife was infected by kamv although serological results suggested she could have been infected earlier in the same manner as her husband. the risk of virus spillover from bats as a result of increasing encroachment by animals and humans into bat habitats is enhanced by the wide distribution of a large number of bat species and the seemingly great genetic diversity of newly emergent bat-borne viruses. this is true for henipaviruses [22] [23] [24] [25] , coronaviruses [26, 27] , and now orthoreoviruses. henipaviruses were initially discovered in pteropid bats, but a subsequent seroprevalence survey indicated that henipaviruses or henipa-like viruses are also circulating among non-pteropid bats [28] [29] [30] . since the discovery of sars-like coronaviruses in horseshoe bats, a large number of new coronaviruses have been detected in many different bat species [26, 27, [31] [32] [33] [34] [35] . it is important to conduct similar serological and virological surveillance studies for bat orthoreoviruses to better understand their geographic distribution, their host range and their genetic diversity. such information will be essential for an effective risk assessment of future spill over events and potential large scale outbreaks. during the preparation of this manuscript, the partial s segment sequences of a new orthoreovirus were made available in the genbank by a group in hong kong (genbank accession numbers eu165526 and eu170365-7). this virus, tentatively named reovirus strain hk23629/07, was isolated from a patient suffering an acute respiratory infection. phylogenetic analysis of the available sequences confirmed that the hk strain is very closely related to other members of the nbv group. a closer look at the phylogenetic tree based on the most variable protein, cell attachment protein sigma c (figure 6 ), indicated that the malaysian bat isolate (pulv) is more closely related to the three human isolates (melv, kamv and hk) than to the australian bat isolate (nbv). this suggests that geographic location, rather than the host of isolation, is a more important determinant for genetic relatedness. although the exact reservoir species of kamv is yet to be determined, preliminary serological studies suggest that kamv is likely able to infect multiple fruit bat species in malaysia. the isolation of three related viruses from human patients within such a short period and the close relation between bat and human isolates would suggest that spill over by this group of orthoreoviruses is not an uncommon event and systematic surveillance among rti patients is needed to provide a more accurate assessment of this newly discovered viral infection among human population in different countries in this region and beyond. in conclusion, the discovery and characterization of kamv corroborate our previous work on melv and demonstrate the increasing risk posed by unknown bat viruses which are capable of infecting and causing disease in humans. this further highlights the urgent need to systematically survey bat-borne viruses in the international community so as to enable us to conduct more effective risk assessment, to provide forecast for potential future outbreaks and to devise better prevention and control strategies. a throat swab was taken from the patient at the time of his first clinical examination and transported in viral transport medium (vtm) to the national public health laboratory for virus isolation. the sample was treated with antibiotics (c. penicillin 100,000 i.u./ml and streptomycin 100 mg/ml) for an hour before being inoculated in duplicate (100 ml and 200 ml, respectively) into freshly confluent monolayers of mdck (atcc, ccl-34), vero (atcc, ccl-81) and hep-2 (atcc, ccl-23) cells cultured in a 24-well tissue culture plate. the plate was incubated at 37uc in 5% co 2 and examined daily for the presence of cpe in cultured cells. supernatant from cultures with visible syncytial cytopathic effect (cpe) after 3 days was taken for further analysis by serial passage in different cell lines available in the laboratory. the investigation conducted in this study was approved by the ethics committee of the malaysian national public health laboratory. all patients (subjects) in this manuscript have given written informed consent (as outlined in the plos consent form) to publication of their case details. no identification of the subjects is to be revealed in any publication. immunofluorescence antibody testing (ifat) and virus neutralization assay were conducted as previously described [5] . briefly, for ifat a freshly confluent monolayer of mdck was infected with kamv and at full cpe, the infected cells were harvested, washed four times and suspended in sterile pbs at a cell concentration of approximately 3000 cells per millilitre. an aliquot of the infected cell suspension was carefully spotted onto each well of teflon coated slides, followed by air-drying over a warm plate and subsequent fixation in cold acetone for 10 min. serial 2-fold dilutions of serum samples were then added to detect specific reactivity. for detection of igm, igg was removed by absorption with protein a prior to serum dilution. bound antibodies were detected using fluorescein conjugated rabbit anti-human igm or igg (dako, usa). specific reactivity/labelling were read under a uv fluorescence microscope (olympus bx50, japan). for vnt, serial 2-fold dilutions of control and test sera were prepared in duplicate starting at 1:10. an equal volume of virus working stock containing 150 tcid 50 was added to the diluted sera and incubated for 30 min. the pre-incubated virus/ serum mix was added to confluent cell monolayers and incubated for 1 h. the inoculum was removed, monolayers washed three times with pbs and cell media replaced. ability of sera to neutralize virus was determined by scoring the extent of cpe observed in duplicate wells three days later. virion from culture supernatant was harvested and concentrated by centrifugation and viral rna was extracted and purified using the qiamp viral rna kit (qiagen, germany). for each virus, an aliquot of 15 mg rna was run on a 10% sdspolyacrylamide/bis gel under denaturing and reducing conditions at 150 v for 4 hrs at room temperature. the gel was washed with distilled water, stained with ethidium bromide before the photo was taken. extraction and purification of dsrna and synthesis of randomly primed cdna were carried out as previously described [5, 7, 36] . the majority of sequence information was obtained by pcr with primers designed from conserved regions of melv, pulv and nbv (primer sequences will be supplied upon request). to obtain the genome segment terminal sequences, a two-step pcr strategy was used. first, the single primer amplification technique or spat [36] was used to generate primary pcr product followed by a semi-nested pcr using the combination of kamv genome segment-specific primers and the adaptor-specific primer used in spat. pcr products were sequenced directly without cloning. all regions of the genome segments were sequenced at least three times. phylogenetic analysis was conducted using the mega4 software package [37] . phylogenetic trees were constructed using the neighbour-joining algorithm with bootstrap values determined by 1,000 replicates. complete genome sequences of the four s segments were deposited in genbank under accession numbers eu448334 to eu4488337 for s1, s2, s3 and s4 segments, respectively. family reoviridae orthoreoviruses and their replication genus orthoreovirus extensive sequence divergence and phylogenetic relationships between the fusogenic and nonfusogenic orthoreoviruses: a species proposal a previously unknown reovirus of bat origin is associated with an acute respiratory disease in humans structure and cytopathic effects of nelson bay virus pulau virus; a new member of the nelson bay orthoreovirus species isolated from fruit bats in malaysia bats: important reservoir hosts of emerging viruses a novel morbillivirus pneumonia of horses and its transmission to humans nipah virus: a recently emergent deadly paramyxovirus an apparently new virus (family paramyxoviridae) infectious for pigs, humans, and fruit bats bats are natural reservoirs of sars-like coronaviruses fruit bats as reservoirs of ebola virus nelson bay virus. a novel reovirus a novel approach for collecting samples from fruit bats for isolation of infectious agents severe acute respiratory syndrome characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia identification of a new human coronavirus anthropogenic deforestation, el nino and the emergence of nipah virus in malaysia zoonotic viruses of wildlife: hither from yon host range and emerging and reemerging pathogens molecular biology of hendra and nipah viruses bat nipah virus genetic characterization of nipah virus nipah virus in lyle's flying foxes prevalence and genetic diversity of coronaviruses in bats from china molecular diversity of coronaviruses in bats antibodies to nipah-like virus in bats (pteropus lylei) the natural history of hendra and nipah viruses nipah virus infection in bats (order chiroptera) in peninsular malaysia covdb: a comprehensive database for comparative analysis of coronavirus genes and genomes evolutionary relationships between bat coronaviruses and their hosts detection and prevalence patterns of group i coronaviruses in bats coronavirus antibodies in african bat species detection of group 1 coronaviruses in bats in north america strategies for the sequence determination of viral dsrna genomes mega4: molecular evolutionary genetics analysis (mega) software version 4.0 we thank g. marsh and m. tachedjian for critical reading of the manuscript, k. selleck for technical assistance, and t. pye and e. hansson for help with dna sequencing. key: cord-291360-z19ri377 authors: lan, fan-yun; filler, robert; mathew, soni; buley, jane; iliaki, eirini; bruno-murtha, lou ann; osgood, rebecca; christophi, costas a.; fernandez-montero, alejandro; kales, stefanos n. title: covid-19 symptoms predictive of healthcare workers’ sars-cov-2 pcr results date: 2020-06-26 journal: plos one doi: 10.1371/journal.pone.0235460 sha: doc_id: 291360 cord_uid: z19ri377 background: coronavirus 2019 disease (covid-19) is caused by the virus sars-cov-2, transmissible both person-to-person and from contaminated surfaces. early covid-19 detection among healthcare workers (hcws) is crucial for protecting patients and the healthcare workforce. because of limited testing capacity, symptom-based screening may prioritize testing and increase diagnostic accuracy. methods and findings: we performed a retrospective study of hcws undergoing both covid-19 telephonic symptom screening and nasopharyngeal sars-cov-2 assays during the period, march 9—april 15, 2020. hcws with negative assays but progressive symptoms were re-tested for sars-cov-2. among 592 hcws tested, 83 (14%) had an initial positive sars-cov-2 assay. fifty-nine of 61 hcws (97%) who were asymptomatic or reported only sore throat/nasal congestion had negative sars-cov-2 assays (p = 0.006). hcws reporting three or more symptoms had an increased multivariate-adjusted odds of having positive assays, 1.95 (95% ci: 1.10–3.64), which increased to 2.61 (95% ci: 1.50–4.45) for six or more symptoms. the multivariate-adjusted odds of a positive assay were also increased for hcws reporting fever and a measured temperature ≥ 37.5°c (3.49 (95% ci: 1.95–6.21)), and those with myalgias (1.83 (95% ci: 1.04–3.23)). anosmia/ageusia (i.e. loss of smell/loss of taste) was reported less frequently (16%) than other symptoms by hcws with positive assays, but was associated with more than a seven-fold multivariate-adjusted odds of a positive test: or = 7.21 (95% ci: 2.95–17.67). of 509 hcws with initial negative sars-cov-2 assays, nine had symptom progression and positive re-tests, yielding an estimated negative predictive value of 98.2% (95% ci: 96.8–99.0%) for the exclusion of clinically relevant covid-19. conclusions: symptom and temperature reports are useful screening tools for predicting sars-cov-2 assay results in hcws. anosmia/ageusia, fever, and myalgia were the strongest independent predictors of positive assays. the absence of symptoms or symptoms limited to nasal congestion/sore throat were associated with negative assays. we performed a retrospective study of hcws undergoing both covid-19 telephonic symptom screening and nasopharyngeal sars-cov-2 assays during the period, march 9-april 15, 2020. hcws with negative assays but progressive symptoms were re-tested for sars-cov-2. among 592 hcws tested, 83 (14%) had an initial positive sars-cov-2 assay. fiftynine of 61 hcws (97%) who were asymptomatic or reported only sore throat/nasal congestion had negative sars-cov-2 assays (p = 0.006). hcws reporting three or more symptoms had an increased multivariate-adjusted odds of having positive assays, 1.95 (95% ci: 1.10-3.64), which increased to 2.61 (95% ci: 1.50-4.45) for six or more symptoms. the multivariate-adjusted odds of a positive assay were also increased for hcws reporting fever and a measured temperature � 37.5˚c (3.49 (95% ci: 1.95-6.21)), and those with myalgias (1.83 (95% ci: 1.04-3.23)). anosmia/ageusia (i.e. loss of smell/loss of taste) was reported less frequently (16%) than other symptoms by hcws with positive assays, but was associated with more than a seven-fold multivariate-adjusted odds of a positive test: or = 7.21 (95% ci: 2.95-17.67). of 509 hcws with initial negative sars-cov-2 assays, nine had coronavirus 2019 disease (covid-19) has become pandemic since being first reported in china [1] . covid-19 can present along a clinical spectrum from asymptomatic, mild symptoms (e.g. cold-like) [2] [3] [4] , influenza-like (e.g. fever, malaise and myalgia) to severe lower respiratory disease with dyspnea and pneumonia [3] [4] [5] . evidence supports person-to-person transmission, including from asymptomatic patients [6] [7] [8] [9] . severe acute respiratory syndrome coronavirus-2's (sars-cov-2) environmental persistence suggests transmission may also result from hand contact with contaminated surfaces [10] followed by facial selfcontamination. healthcare workers (hcws) potentially experience greater risks for emerging infectious diseases [11] [12] [13] due to occupational exposure to sick patients and virus-contaminated surfaces [14] . contagious hcws may infect patients, co-workers and family members. moreover, the removal of ill hcws from duty can threaten essential healthcare staffing during an epidemic [15] . therefore, infection prevention and quick, accurate diagnosis of potential covid-19 in hcws are crucial to maintaining hospital operations [16] . testing hcws with early/mild symptoms has been approved and prioritized [17, 18] . because they are more likely to be tested, characterization of hcws' presentations and viral assays provide valuable clinical and epidemiologic perspectives on covid-19, to compare with hospitalized patients generally representing more severe cases [19] . there is no reference diagnostic test for covid-19. currently available tools are symptom reports and a reverse transcriptase polymerase chain reaction (rt-pcr) assay that detects sars-cov-2 rna from naso-/oropharyngeal specimens [20] , but whose test characteristics are not established at this time [21] . because of limited testing capacities, and potential latency of up to 14 days in illness onset from exposure [22] , hcw testing is largely restricted to persons reporting compatible symptoms [17, 18] . nonetheless, many hcws and other first responders desire wider testing because of potential exposures, often without symptoms. therefore, we investigated the presenting symptoms most predictive of positive/negative sars-cov-2 rt-pcr results among hcws. since march 9, 2020, the occupational health service of a massachusetts community healthcare system has implemented a staff "hotline" system to maintain a viable/healthy workforce and operational continuity during the pandemic. accordingly, the service was re-configured to perform telephonic triage of hcws for covid-19-related concerns; electronically document related clinical information; manage and communicate pandemic-related testing results; and oversee hcws' safe return to work. reasons for contacting the covid-19 hotline have included: a) travel; b) potential contact with a covid-19-positive/suspect person; and/or c) possible viral symptoms. a standard triage form (s1 file) was completed by occupational nursing and medical personnel based on contemporaneous telephonic interviews with each hcw. the form contains demographics, administrative information, potential exposure history, and eleven potential viral symptoms: fever (subjective, as well as highest measured temperature), cough (new or worse), shortness of breath (new or worse), myalgia, malaise, sore throat, nasal symptoms (including runny nose, sneezing, congestion, and sinus symptoms), gastrointestinal symptoms (including nausea, vomiting, and diarrhea), rash, anosmia/ageusia (i.e. loss of smell/loss of taste), and headache. headache and anosmia/ageusia were added to a revised form after several hcw reports. in accordance with expert guidelines [17, 18] , symptomatic hcws were referred for sars--cov-2 testing (see below). employees tested elsewhere forwarded their rt-pcr results to the occupational health service and were triaged via a telephonic visit. in this retrospective cohort study, we included all adult employees/personnel or contractors of the hospital who had undergone both covid-19-related triage and a sars-cov-2 pcr assay between march 9 and april 15, 2020. sars-cov-2 assays were performed at designated sites, where trained healthcare system staff collected nasopharyngeal swabs and transferred each specimen to a 3ml vial with viral transport media (vtm), universal transport media (utm) or saline. after collection, specimens were refrigerated at 2-8˚c, unless transported immediately, and refrigerated at 2-8˚c during transport. hcws samples were sent to one of three laboratories (the massachusetts department of public health (madph), a commercial lab, or a tertiary hospital partner), whichever offered the fastest turnaround-time on the day of testing. all three laboratories used real-time, reverse-transcriptase-polymerase-chain-reaction (rt-pcr) diagnostic panels for the qualitative detection of nucleic acid from the 2019 sars-cov-2 (madph, cdc 2019-novel rt-pcr; commercial laboratory, roche cobas sars-cov-2; and hospital partner, abbott real time sars-cov-2). the limit of detection at the laboratories testing the majority of our samples was 100 copies of viral rna/ml. positive assay results represented detection of sars--cov2 rna, while for negative results, the virus was not detected. in case of an invalid or indeterminate result, a repeat sample is requested. for operational needs, the occupational health service maintains data from the triage encounters, pcr results and clinical/work status on a secure "live" spreadsheet. the presence/absence of the eleven possible symptoms, travel, and exposure history in the spreadsheet entries were verified from triage forms by an occupational medicine physician for every hcw undergoing a rt-pcr assay. for body temperature, we recorded the highest value before testing as selfreported by each hcw. sars-cov-2 pcr assay results for hcws tested outside of the healthcare system were verified and entered in the database. because there is no reference diagnostic test, we considered false negative sars-cov-2 assays as occurring when the initial test was negative, but symptoms had progressed/persisted at telephonic follow-up and a repeat assay was subsequently sars-cov-2 positive. the institutional review board of the healthcare system reviewed the study protocol, determined it to be exempt and waived informed consent based on the use of existing, hipaa-deidentified data. prior to statistical analyses, all data were deidentified and transferred to a spreadsheet without linkages to the "live" database. continuous characteristics were presented as means and standard deviations and compared between groups with the parametric t-test. categorical variables were presented as counts and percentages and compared between groups using the chi-square test of independence with the yates' continuity correction or the fisher's exact test, as appropriate. no imputations were made for missing data. principal component analysis (pca) was utilized to identify dominant symptoms (those contributing more than 5% variability to the identified principal component explaining most data variability). logistic regression models were then fit to evaluate symptom association with the probability of having a positive sars-cov-2 assay, after age-, sex-and other symptom adjustment. regression results are provided as odds ratios (ors) and corresponding 95% confidence intervals (cis). the hosmer-lemeshow goodness of fit test with 15 groups was also performed to check the model fit. additionally, the c-statistic was calculated to assess each model's predictive accuracy. the clinical covid-19 attack rate during the study period was calculated as: (the number of initial positive sars-cov-2 assays + the number of false negatives) divided by the system's estimated total hcw population (n = 4600). covid-19 complication rates were calculated as hcws who required an emergency room visit or hospitalization before the end of the study period (april 15, 2020) and/or had intubation or death before the end of follow-up (april 20, 2020); divided by the number of total covid-19-diagnosed hcws. statistical analyses were conducted using the r software (version 3.6.3). all tests were twosided and a p-value < 0.05 was considered statistically significant. during the study period, we identified 592 unique hcws who underwent triage and sars--cov-2 rt-pcr. eighty-three (14.0%) had positive sars-cov-2 rt-pcr results on the initial assay. the cohort's presentation at triage is summarized in table 1 . the average age was 43.6 ± 12.9 years old, with no significant difference between hcws testing positive or negative (p = 0.84). the proportion of men with positive tests (27.7%) was non-significantly higher than among those with negative tests (20.0%) (p = 0.15). sars-cov-2 positive hcws self-reported several symptoms more frequently than those with negative assays: fever (55% vs. 27%), myalgia (57% vs. 35%), headache (41% vs. 28%), and anosmia/ageusia (16% vs. 3%) (all p<0.05). nasal symptoms (runny, sneezing, congestion, sinus) were more frequently associated with negative assays (52% vs. 35%) (p = 0.006) ( table 1, fig 1) . among hcws reporting fever, 87% (40/46) of those testing sars-cov-2 positive and 89% (121/136) of those testing negative had measured their body temperature. the mean peak temperature reported was higher in hcws with a positive assay (38.0 ± 0.7˚c) compared to those with negative assays (37.6 ± 0.7˚c) (p = 0.006). dichotomizing the peak temperature (� or <37.5˚c), the measured temperature exceeded the threshold in 85% of hcws with positive rt-pcr, compared to 56% of those with negative assays (p = 0.002). assay results for asymptomatic or mildly symptomatic hcws are shown in table 2 . none of the hcws with only sore throat and/or nasal symptoms had a positive sars-cov-2 assay (0%), while all 34 (100%) had a negative pcr (p = 0.009). when we combined asymptomatic and mildly symptomatic hcws, 59/61 (97%) had negative initial assays (p = 0.006). total symptoms at triage ranged from zero to ten, with only 40 (7%) hcws reporting seven or more symptoms. table 3 shows the counts and percentages of hcws with positive and negative assays, and age-and sex-adjusted odds ratios for increasing numbers of total reported symptoms. hcws workers reporting three or more symptoms had an increased likelihood of a positive sars-cov-2 assay (or = 1.95 (95% ci: 1.10-3.64). the odds ratio of a positive rt-pcr generally increased with additional symptoms and reached 2.61 (95% ci: 1.50-4.45) for six or more symptoms. further multivariate logistic regression analyses of pca-determined dominant symptoms are shown in table 4 . hcws reporting fever had an age-and sex-adjusted odds ratio of 3.34 (95% ci: 2.07-5.41) of having a positive sars-cov-2 assay, which remained significant after additional adjustment for other symptoms (or = 2.88, 95% ci: 1.66-5.01). when measured body temperature �37.5˚c was considered together with reported fever, the odds of a positive sars-cov-2 assay increased to 4.47 (95% ci: 2.66-7.48) in the age-and sex-adjusted model and to 3.49 (95% ci: 1.95-6.21) in the all-symptom-adjusted model. the odds ratios remained similar when the body temperature threshold changed to 38˚c (or = 4.45, 95% ci: 2. 30-8.44 in the age-and sex-adjusted model; or = 2.85, 95% ci: 1.36-5.86 in the full model). hcws reporting anosmia/ageusia had an increased age-and sex-adjusted odds ratio of 6.50 (95% ci: 2.89-14.51), and multivariate odds ratio of 7.21 (95% ci: 2.95-17.67) of having a positive sars-cov-2 assay. myalgia was also associated with increased odds ratios of having a positive sars-cov-2 assay in both models: or = 2.41 (95% ci: 1.50-3.89) and or = 1.83 (95% ci: 1.04-3.23), respectively. headache was a significant predictor in the age-and sexadjusted model: or = 1.84 (95% ci: 1.13-2.96), but not in the all-symptom-adjusted model. nasal congestive symptoms were a significant negative predictor in both multivariate models: or = 0.51 (95% ci: 0.31-0.82) and or = 0.40 (95% ci: 0.23-0.68), respectively. the symptoms shaded in salmon-red are the symptoms more frequently seen with positive tests, and the symptoms shaded in blue-green are more frequently seen with negative tests. gi symptom denotes a gastrointestinal symptom (nausea/ vomiting/ diarrhea). nasal symptom includes runny nose, sneezing, congestion, and sinus symptoms. no/mild symptom denotes no symptom or only sore throat and/or nasal symptoms. the asterisks above the bars denote different statistically significance levels when comparing hcws with positive assays and hcws with negative assays ( � : p<0.05, �� : p<0.01, ��� : p<0.001). https://doi.org/10.1371/journal.pone.0235460.g001 the hosmer-lemeshow goodness of fit test for all models demonstrated no evidence of poor fit. therefore, we also present c statistics (i.e. area under the curve) for each symptom in table 4 . after age-and sex-adjustment, fever had the best discrimination between hcws with positive and negative sars-cov-2 assays (c-statistic = 0.663). nine hcws with symptom progression after an initial negative sars-cov-2 assay are described in s1 table. upon re-testing, all nine had positive rt-pcr for sars-cov-2. these cases represent 1.8% of originally negative hcws (9/509), yielding an estimated negative predictive value of 98.2% (95% ci: 96.8-99.0%) for excluding clinically relevant covid-19 disease. the cumulative attack rate for clinically symptomatic covid-19 during the study period (march 9-april 15, 2020) was 92 incident cases among an estimated employee cohort of 4600 or 2.0% (95% ci: 1.6-2.5%). a total of nine among the 92 incident hcw covid-19 cases (9.8%) (all nine diagnosed with the initial pcr assay) experienced a complication during the study period: four required an emergency room visit, five needed hospitalization, two of the hospitalized required intubation, and one of those died (1.1%). this original investigation describes initial symptoms and their associations with sars-cov-2 rt-pcr assays among all 592 hcws in our system tested between march 9 and april 15, 2020. overall, we tested 13% of our workforce, which is a testing rate 6.7-fold greater than that of the massachusetts population during the same period [23] . we found that a total of 16% (including 14% with initial positive assays and 2% with initial false negative assays) of these hcws were diagnosed with clinical covid-19, yielding a cumulative attack rate of 2% for our workforce. the absence of symptoms or those limited to the throat/nose (excluding anosmia/ageusia) were significantly associated with having negative sars-cov-2 assays. in contrast, hcws reporting three or more symptoms had 2-fold greater age-and sex-adjusted odds of having positive assays. these odds generally increased for each additional symptom reported, reaching 2.6-fold for six or more symptoms. our results are consistent with previous reports suggesting that rt-pcr results correlate with viral shedding, covid-19 symptom onset and clinical severity [24, 25] . in multivariate adjustment models, hcws with anosmia/ageusia had higher than seven-fold odds of having positive rt-pcr; those with fever and a measured temperature � 37.5˚c had almost 3.5-fold odds of having positive rt-pcr; and those with myalgia had almost 2-fold odds of having positive assays, while nasal symptoms were associated with a 60% reduced risk of a positive assay. studies support varying clinical manifestations among covid-19 patients [2] [3] [4] [5] . an investigation of critically ill cases found half presenting with temperature � 38˚c during their icu stay, while more than 80% had cough and/or shortness of breath, accompanied by tachypnea [19] . in contrast, a study of subclinical covid-19 patients showed 40% of cases initially presented fever and non-specific symptoms, such as cough and sore throat, but none exhibited dyspnea [4] . regarding hcws, it is useful to compare our results with two recent hcw series, which reported average ages (42-43 years-old) and female predominance (73-77%) similar to ours. in the large us national series, 78% of hcws with covid-19 had cough, 68% had fever, 66% reported myalgia, and 41% presented shortness of breath [26] . in the second smaller series from king county, wa (usa), the most common initial symptoms were cough (50%), fever (42%) and myalgias (35%) [27] . in general agreement, our hcw covid-19 cases commonly reported fever (55%), myalgia (57%) and cough (71%), but only 17% reported dyspnea. however, only our investigation collected data on those testing negative as well, demonstrating that cough is a very non-specific symptom that was also reported by 60% of those testing negative. on the other hand, fever (particularly if measured) and myalgia were independent predictors of positive sars-cov-2 assays, as were anosmia/ageusia in hcws symptomatic for longer periods. regarding the latter findings, they are in accordance with a cross-sectional study of the general population, which reported adjusted odds ratios for covid-19 of 6.6 and 3.1 for anosmia/ageusia and fever, respectively [28] , similar to our findings of fully-adjusted odds of 7.2 and 3.5 for the same symptoms. in our study, anosmia/ageusia was reported less frequently (16%) as compared to 59% in the cross-sectional study, most likely because our symptoms reports were captured early in the course of illness. our results are also comparable to a dutch study examining the point prevalence of positive sars-cov-2 rt-pcrs among hcws with mild respiratory symptoms, during the early outbreak in the netherlands, finding 4% of hcws across nine hospitals positive, ranging from 0-10% for each individual hospital [14] . we found an overall positive initial test rate of 14% during a five-week period, which overlapped early spread with a later surge in community transmission and hospital covid-19 traffic. we have observed that the proportion of positive tests increased along with epidemic's progression in our region. false negative results of sars-cov-2 rt-pcr have been reported at rates from 10-30% [21, 29] . we did find nine hcws who initially tested negative, developed progressive symptoms and had subsequent positive rt-pcr. none of these likely false-negatives were among the hcws with the most severe cases requiring emergency/hospital care. further study of symptomatic patients, their household contacts and of random population samples with both rt-pcr and serologies [30] are needed to better elucidate the true prevalence of asymptomatic carriers as well as the frequency of false negative rt-pcrs. nonetheless, our estimated negative predictive value of 98% for excluding clinically relevant covid-19 disease is very reassuring regarding the test's performance when done properly. our findings have potential implications for hcw covid-19 surveillance. first, hcws with no symptoms or mild symptoms limited to sore throat/nasal congestion and a measured body temperature lower than 37.5˚c, have a low probability of a positive rt-pcr and covid-19. thus, many hcws with no symptoms or symptoms more compatible with allergy or a common cold could refrain from testing and continue to self-monitor symptoms and body temperature. in contrast, early systemic symptoms, especially fever and myalgia are predictive of possible clinical covid-19, while anosmia/ageusia are fairly specific later findings. our results support expert guidelines for temperature monitoring [18] , which should not be limited to healthcare settings, and that a measured temperature � 37.5˚c may be predictive of a positive sars-cov-2 rt-pcr result. our study does have some limitations. first, the hcws tested were not a random or convenience sample, but rather all hcws who self-reported covid-19-related concerns. thus, our results do not necessarily reflect total cumulative incidence of covid-19 among hcws in our healthcare system. however, our triage and testing process were conducted in accordance with expert guidelines [3, 17, 18] , and therefore, our results can inform other healthcare systems employing comparable protocols. second, because there is no reference diagnostic test for covid-19, the test performance characteristics of the rt-pcr are not established [21] . potentially corroborating serology tests were not available during the study period, but may be helpful in the future to identify additional infections that occurred with mild or no symptoms [30] . our study also has several strengths. first, hcws generally reported their symptoms at triage before their sars-cov-2 pcr test results were available, eliminating recall bias. second, all symptoms reports were validated by occupational medicine physicians, strengthening their accuracy. third, all hcws had complete symptom and testing data. moreover, unlike other hcw studies, we used identical methods to collect data on persons testing negative and were able to compare these two groups. in addition, all hcws tested had telephonic follow-up visits, allowing us to identify and re-test hcws with potential false negative results. finally, our present study included all eligible employees in the healthcare system. the study population comprised not only healthcare professionals but also staff/contractors such as maintenance or it (information technology) persons. therefore, our results may be generalized to other working populations during the pandemic. among hcws, systemic symptoms/signs (fever, body temperature � 37.5˚c, myalgia, and headache) and anosmia/ageusia were predictive of positive sars-cov-2 assays, with fever, anosmia/ageusia, and myalgia being the strongest independent predictor. in contrast, no symptoms or isolated sore throat/nasal congestion were associated with negative sars-cov-2 assays. supporting information s1 file. the healthcare system's occupational health covid-19/influenza like illness triage form. (pdf) s1 table. presentation of hcws with initial negative assays but repeat positive assays (i.e. presumed initial false negative assays). (docx) covid-19 and community mitigation strategies in a pandemic first mildly ill, non-hospitalized case of coronavirus disease 2019 (covid-19) without viral transmission in the united states-maricopa county covid-19 in south korea-challenges of subclinical manifestations imaging and clinical features of patients with 2019 novel coronavirus sars-cov-2: a systematic review and meta-analysis presumed asymptomatic carrier transmission of covid-19 transmission of 2019-ncov infection from an asymptomatic contact in germany first known person-toperson transmission of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in the usa clinical characteristics of 24 asymptomatic infections with covid-19 screened among close contacts in nanjing aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 work-related covid-19 transmission in six asian countries/areas: a follow-up study infection rates and risk factors for infection among health workers during ebola and marburg virus outbreaks: a systematic review effectiveness of masks and respirators against respiratory infections in healthcare workers: a systematic review and meta-analysis rapid assessment of regional sars-cov-2 community transmission through a convenience sample of healthcare workers, the netherlands ensuring and sustaining a pandemic workforce covid-19 and healthcare workers: emerging patterns in pamplona, asia and boston massachusetts department of public health guidance for risk assessment and public health management of healthcare personnel with potential exposure in a healthcare setting to patients with coronavirus disease (covid-19); c2020 covid-19 in critically ill patients in the seattle region-case series detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr incorporating test characteristics into sars-cov-2 testing policy-sense and sensitivity the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application massachusetts department of public health covid-19) cases in ma: as of april 15 temporal dynamics in viral shedding and transmissibility of covid-19 viral dynamics in mild and severe cases of covid-19 characteristics of health care personnel with covid-19-united states symptom screening at illness onset of health care personnel with sars-cov-2 infection in king county, washington loss of smell and taste in combination with other symptoms is a strong predictor of covid-19 infection covid-19 testing: the threat of false-negative results developing antibody tests for sars-cov-2 key: cord-277409-q5wx313k authors: resende, lucilene aparecida; aguiar-soares, rodrigo dian de oliveira; gama-ker, henrique; roatt, bruno mendes; de mendonça, ludmila zanandreis; alves, marina luiza rodrigues; da silveira-lemos, denise; corrêa-oliveira, rodrigo; martins-filho, olindo assis; araújo, márcio sobreira silva; fujiwara, ricardo toshio; gontijo, nelder figueiredo; reis, alexandre barbosa; giunchetti, rodolfo cordeiro title: impact of lbsapsal vaccine in canine immunological and parasitological features before and after leishmania chagasi-challenge date: 2016-08-24 journal: plos one doi: 10.1371/journal.pone.0161169 sha: doc_id: 277409 cord_uid: q5wx313k dogs represent the most important domestic reservoir of l. chagasi (syn. l. infantum). a vaccine against canine visceral leishmaniasis (cvl) would be an important tool for decreasing the anxiety related to possible l. chagasi infection and for controlling human visceral leishmaniasis (vl). because the sand fly salivary proteins are potent immunogens obligatorily co-deposited during transmission of leishmania parasites, their inclusion in an anti-leishmania vaccine has been investigated in past decades. we investigated the immunogenicity of the “lbsapsal” vaccine (l. braziliensis antigens, saponin as adjuvant, and lutzomyia longipalpis salivary gland extract) in dogs at baseline (t(0)), during the post-vaccination protocol (t(3rd)) and after early (t(90)) and late (t(885)) times following l. chagasi-challenge. our major data indicated that immunization with “lbsapsal” is able to induce biomarkers characterized by enhanced amounts of type i (tumor necrosis factor [tnf]-α, interleukin [il]-12, interferon [ifn]-γ) cytokines and reduction in type ii cytokines (il-4 and tgf-β), even after experimental challenge. the establishment of a prominent pro-inflammatory immune response after “lbsapsal” immunization supported the increased levels of nitric oxide production, favoring a reduction in spleen parasitism (78.9%) and indicating long-lasting protection against l. chagasi infection. in conclusion, these results confirmed the hypothesis that the “lbsapsal” vaccination is a potential tool to control the leishmania chagasi infection. leishmania infantum (syn. l. chagasi) is an intracellular protozoon that cause a severe systemic disease named visceral leishmaniasis (vl) [1] . it is widely distributed in the mediterranean basin, middle east, and south america. vl has an annual incidence of approximately 500,000 cases [2] . brazil declared a total of 50,060 clinical vl cases between 1990 and 2006, and this number accounts for 90% of all reported vl cases in the americas; however, it is subject to substantial under-reporting [3] . different geographical regions of the globe where visceral leishmaniasis is endemic, present the dogs as main reservoir for the l. chagasi that play a relevant role in transmission of the parasite [4, 5] . dogs are also excellent models for the study of vl because the natural history of the disease in dogs and in humans is similar [6] , especially regarding parasite-host interaction, immune response, and the development of new vaccines [7] . the natural history of canine vl (cvl) has been well-described, particularly in regard to the parasite load in different tissues and the immunopathological changes according to progression of clinical forms [8, 9, 10, 11, 12, 13, 14, 15, 16, 17] . a vaccine against cvl would be an important tool in the control of vl and would decrease the anxiety associated with l. chagasi infection in humans [18] . the induction of long-lasting cell-mediated immune response triggering high levels of protection against cvl is considered as prerequisite of an ideal vaccine for controlling the l. chagasi transmission. different vaccine candidates against cvl have been reported showing the ability to induce immunoprotective mechanisms [19, 20, 21, 22, 23, 24, 25, 26, 27] . in this sense, it has been shown that leishmune 1 (fucose manose ligand plus saponin as adjuvant; zoetis, são paulo, brazil) presented 95% protection in a phase iii study [28] . leishmune 1 provided protection associated with the ability to trigger early and persistent activation of neutrophils and monocytes, in addition to activation of t cd4 + and t cd8 + lymphocytes displaying high levels of ifn-γ in the cd4 + t-cell subset [29, 30] . additionally, the leish-tec 1 vaccine (hertape calier, juatuba, brazil; a2 antigen plus saponin as adjuvant) has also been shown to induce increased levels of ifn-γ in four out of seven dogs with l. chagasi-infected bone marrow [21] . more recently, a vaccine composed of excreted/secreted antigens of l. infantum promastigotes, referred as liesap has been shown to induce increased levels of ifnγ and nitric oxide (no), thus supporting its leishmanicidal effect [31] . the efficacy of the lie-sap vaccine was reported as 92% in a double-blind randomized study [19] . liesap is commercially available as canileish 1 (virbac, carros, france) and is able to induce a th1 profile and reduce the parasitic load of infected macrophages co-cultured with lymphocytes from immunized dogs [32] . the lbsap (l. braziliensis crude antigens plus saponin as adjuvant) and "lbsapsal" (l. braziliensis crude antigens plus saponin as adjuvant and lutzomyia longipalpis saliva) vaccines against cvl has also shown to induce a strong immune-mediated response. the lbsap and "lbsapsal" presented higher levels of circulating t-cell subsets (cd4 + , cd8 + ) and b lymphocytes (cd21 + ), as well as leishmania-specific cd8 + and cd4 + t cells [20, 22, 25, 27] . furthermore, lbsap-vaccinated dogs presented high ifn-γ and low interleukin (il)-10 and transforming growth factor (tgf)-β1 expression in the spleen, resulting in a significant reduction of parasite load in this tissue [25] . additionally, lbsap has been shown to induce a prominent pro-inflammatory immune response characterized by increased levels of both il-12 and ifn-γ and decreased levels of tgf-β by peripheral blood mononuclear cells (pbmcs), which were associated with parasite control in dogs [26] . the incorporation of salivary proteins of sand flies have been widely used in experimental challenge studies, and the results suggest that this could be a good strategy to protect against leishmania infection [22, 33, 34, 35, 36, 37, 38, 39, 40, 41] . previous studies of dogs using the "lbsapsal" vaccine displayed higher counts of circulating and leishmania-specific cd8 + t cells in addition to high nitric oxide (no) production [22] and reduction of splenic parasite load [27] . considering the inclusion of saliva from sand flies as a promising compound in vaccines against leishmania infection, we describe additional biomarkers induced by the "lbsap-sal" vaccine in dogs. we considered the previous vaccine protocol (t 0 ), post-vaccine protocol (t 3rd ), and early (t 90 ) and late (t 885 ) periods of the l. chagasi-challenge. all research involving dogs was carried out according to the regulations of the brazilian society of science in animal research, adopted by the federal university of ouro preto ethics committee in animal experimentation, that approved the technical procedures using dogs under protocol number 2010/71. all dogs included in this study, were born and bred in the kennel facility at universidade federal de ouro preto in ouro preto, minas gerais, brazil. this kennel facility was established with male and female mongrel dogs, with negative serological and molecular/parasitological diagnosis for leishmania infection, donated by the local zoonosis control center from belo horizonte, minas gerais, brazil. the filial generations, comprising of offsprings resulting from the cross between the original mongrel dogs pairs were used further maintain the mongrel dog colony. in the present study, twenty mongrel dogs (10 males and 10 females), with seven months of age and negative results for indirect fluorescence immunoassay to anti-leishmania antibodies, were selected and received an anti-helmintic treatment and were submitted to polyvaccination protocols, including rabies (tecpar, curitiba-pr, brazil), leptospira, parvovirus, canine distemper, type ii adenovirus, parainfluenza and coronavirus (vanguard 1 htlp 5/ cv-l; pfizer animal health, new york, ny, usa). prior the experimental onset, the animals were maintained in quarantine in kennel runs (4m length x 2m width x 3m height) completely covered with stainless steel wire mesh to prevent the entry of sand flies. the kennel facilities were sprayed with pyrethroid insecticide, every three months, as a measure to control insect access. each run was installed with an infrared lamp heating (250 watts) to ensure the thermal comfort of animals during the night and on cold days. all the dogs were housed without environmental enrichment (e.g. toys, exercise regimes, etc). the dogs were monitored twice a day, as routine inspection, during which the responsible veterinary was in charge of stimulating and playing with them to aiming to avoid behavior problems and minimize their suffering or distress throughout the study. all the experimental procedures described in this study, including this aspect of animal care was approved by federal university of ouro preto ethics committee in animal experimentation (protocol number 2010/71). the animals were maintained with water and food ad libitum throughout the experiment time. the euthanasia of all dogs was performed under the supervision of a veterinarian physician, using barbiturate anesthetic (thiopental sodium, 35mg/kg, iv) followed by intravenous injection of saturated solution of potassium chloride. four groups of dogs involved in experiments were treated as follows: (i) "control" (c) group (n = 5; subcutaneous injections of 1ml of sterile 0.9% saline); (ii) "sal" group (n = 5; subcutaneous injections of salivary lutzomyia longipalpis gland extract (sge; obtained as previously described[22] ) in 1ml of sterile 0.9% saline); (iii) "lbsal" group (n = 5; subcutaneous injections of 600μg of l. braziliensis promastigote antigen and sge in 1ml of sterile 0.9% saline); and (iv) "lbsapsal" group (n = 5; subcutaneous injections of 600μg of l. braziliensis promastigote antigen plus 1mg of saponin and sge in 1ml of sterile 0.9% saline). all animals received three injections in the right flank at 28days intervals. the l. chagasi-experimental challenge in all dogs was performed after 100 days of vaccination protocol, using intradermal 10 7 promastigotes during the stationary phase of cultivation. the challenge was performed in the inner side of the left ear including five lutzomyia longipalpis salivary gland acini. all the analyzed dogs were euthanized 885 days after l. chagasi-experimental challenge and the spleens were collected to evaluate parasite loads. the rationale for choosing such a long endpoint (885 days after challenge) was based on the chronic course of the experimental canine visceral leishmaniasis after intradermal l. chagasi-challenge as previously reported [27] . the vaccine was obtained as previously described [22] . briefly, l. braziliensis (mhom/br/75/ m2903) promastigotes were obtained by in vitro culture in neal, novy, nicolle/liver infusion triptose media [20] , and the parasite was fully disrupted by ultrasound treatment (40w, 1min, 4°c), aliquoted and stored at −80°c. protein concentration was determined according to the method of lowry [42] . the sge used in this study as the antigenic component of the vaccine was obtained from lutzomyia longipalpis females that were not fed, aged 4 days, and dissected in slightly hypotonic unbuffered saline 0.8%, as described in [22] . after collection, the glands were disrupted in a sonicator for 10 seconds and centrifuged at 10,000g for 2min. the supernatant was collected and stored in a freezer at -80°c until use. jugular vein was used for collecting 20ml of peripheral blood in sterile heparinized syringes. we analyze distinct times: baseline before vaccination (t 0 ), 15 days after third immunization dose (t 3rd ) as well as early (90 days-t 90 ) and late (885 days-t 885 ) after experimental l. chagasi-challenge. the pbmcs were isolated as previously described [20] . briefly, the whole blood samples were added over 10ml of ficoll-hypaque (histopaque 1 1077; sigma, usa), centrifuged at 450g for 40 min at room temperature, and the pbmcs were washed twice with rpmi 1640 (450g for 10 min at room temperature). the pbmcs were resuspended in rpmi 1640 at 10 7 cells/ml. the pbmcs cultures were performed in 48-well flat-bottom tissue culture plates (costar, cambridge, ma, usa). the in vitro assays were performed using 50μl of pbmcs (5.0×10 5 cells/well) with 100μl of vaccine l. braziliensis-soluble antigen (vsa; 25μg/ml) or 100μl of soluble l. chagasi antigen (slca; 25 μg/ml). the control cultures (cc; unstimulated) were analyzed using 100μl of rpmi in place of the antigenic stimulus. incubation was performed in a humidified 5% co 2 atmosphere at 37°c for 5 days, after which time the supernatants were collected and stored in a freezer at −80°c for detection of cytokine and no. the quantification of cytokines was carried out by enzyme-linked immunosorbent assay (elisa) according to the manufacturer's instructions (r&d systems, minneapolis, mn, usa), as previously described [26] . minimum cytokine sensitivity detection levels were 62pg/ml (il-12), 63pg/ml (tnf-α and ifn-γ), 78pg/ml (il-4 and il-10) and 31pg/ml (tgf-β). the analysis of il-12 (anti-canine il-12/il-23 p40, catalog number dy1969), ifn-γ (anti-canine ifn-γ, catalog number dy781b), tumor necrosis factor (tnf)-α (anti-canine tnf-α/tnfsf1a immunoassay; catalog number dy1507) and il-10 (anti-canine il-10, catalog number dy735) cytokines were performed using duoset elisa. quantikine 1 kit (mouse/rat/porcine/canine tgf-β1 immunoassay, catalog number mb100b) (r&d systems, minneapolis, mn, usa) was used to measure tgf-β levels. the analysis of il-4 levels employed the capture antibody (monoclonal anti-canine il-4 antibody-catalog number mab7541); the standard curve was obtained using recombinant canine il-4 (catalog number 754cl); and streptavidin (dy998; r&d systems/usa), biotinylated anti-canine il-4 antibody (catalog number: baf754) and substrate solution (1:1 mixture of h 2 o 2 and tetramethylbenzidine; product code 50-76-4) were used. all experiments were performed according to the instructions of r&d systems using 96-well plates (corning incorporated, costar 1 , washington, dc, usa). the microplate automatic reader (el800; biotek, winosski, vt, usa) was employed at a wavelength of 450nm. the measurement of no levels in supernatants of pbmcs cultures was performed by indirect method that quantify the nitrite concentration by griess reaction [43, 44] . briefly, 100μl of griess reagent (1% sulfanylamide, 0.1% naphthylethylene-diamide-dihydrochloride, and 2.5% phosphoric acid; all from sigma, usa) was mixed with 100μl aliquot of cell-free culture supernatant. the microplate reader (biotek, el800) was used to analyze the absorbance at 540nm, after 10 min of incubation at room temperature in the dark. the final nitrite concentration was determined based on a standard curve interpolation constructed by using sodium nitrite solutions in the range of 0-100μm. the interference of nitrites already present in the culture medium was discounted; data were calculated by taking into account the blank, as control reaction, assayed by using the pbmcs cultures medium. the data were expressed as nitrite concentration (μm). the spleen specimens (5mm) were collected during necropsy and stored at -80°c until use for dna extraction. the wizard™ genomic dna purification kit (promega, madison, wi, usa) was used to extract total genomic dna in 20mg of spleen following manufacturer's recommendations. primers that amplified a 90-bp fragment of a single copy of the dna polymerase gene of l. chagasi were used to analyze the spleen parasite burden by quantitative polymerase chain reaction (qpcr). for the q pcr analysis 200nm forward and reverse primers, 5μl of template dna and 16syber green reaction master mix (applied biosystems, grand island, ny, usa) were used in a final volume of 25μl. as previously described [45] , we used the targets the dna polymerase gene l. chagasi (genbank accession: af009147) and the pair of primers (forward: 5' tgt cgc ttg cag acc aga tg 3'; reverse: 5' gca tcg cag gtg tga gca c 3'). the pcr reactions employed an initial denaturation at 95°c (10 min), 40 denaturation cycles at 95°c (15 seconds), in addition to annealing and extension at 60°c (1min.). the pgemh t plasmids (promega) was used for constructing stardart curves, for each run, containing inserts of interest [46] . the gapdh gene (115-bp fragment genbank accession number ab038240) was used to analyze the integrity of the samples. the primers 5'ttccacggcacagtcaag 3' (forward) and 5' actcagcaccagcatcac 3' (reverse) were used for gapdh gene amplification. the spleen parasite load was performed in duplicate and calculated by interpolation from the standard curve included in the same experimental batch. the data was expressed as number of l. chagasi organisms/20ng of total dna. the prism 5.0 software package (prism software, irvine, ca, usa) was used to evaluate data distribution normality by kolmogorov-smirnoff test and for further statistical analyses. oneway analysis of variance (anova) followed by tukey's multiple comparison were used for comparisons of cytokine profiles, no levels and parasite load amongst the experimental groups. student's t-test was used for intra-group comparisons between the control cultures (cc) and antigen-stimulated cultures (vsa or slca-stimuli in vitro). pearson correlation analysis was further applied to evaluate the relationship between spleen parasite burden and no profiles. additionally, the cytokine networks were assembled using cytoscape software version 2.8.2 (institute of systems biology, seattle, usa), for each experimental group ("control", "sal", "lbsal", and lbsapsal) in all times analyzed (t 0 , t 3rd , t 90 , and t 885 ), based on the correlations indices obtained by pearson correlation analysis. the network constructed using distinct edges between nodes to identify negative or positive correlations, referred as moderate (0.37<r>0.67) or strong (r>0.68). in all cases, the significance were considered at p<0.05. "lbsapsal" immunization induced increased levels of ifn-γ before and after experimental l. chagasi-challenge aiming to verify whether the immunization protocols were able to induce the production of pro-inflammatory cytokines, we evaluated the profiles of tnf-α, ifn-γ, and il-12 in cc or upon vsa or slca-stimuli in vitro. no significant differences were observed amongst the experimental groups at baseline (t 0 ) ( fig 1a, 1b and 1c, upper panel) . data analysis performed at (t 3rd ) post-vaccination did not show any differences in the tnf-α levels amongst the experimental groups, regardless the culture conditions. analysis of il-12 demonstrated that the "lbsapsal" group displayed increased levels (p<0.05) upon vsastimulation as compared with the same cultures in the "control", "sal", and "lbsal" groups ( fig 1b, middle panel) . moreover, the "lbsal" group showed reduced il-12 levels (p<0.05) upon slca-stimulation as compared with the "control" group ( fig 1b, middle panel) . interestingly, the analysis of ifn-γ showed that "lbsapsal" group presented increased levels (p<0.05) in cc as compared to the "control" group ( fig 1c, middle panel) . additionally, "lbsapsal" group displayed increased ifn-γ levels (p<0.05) upon vsa and slca-stimuli in comparison with "control", "sal", and "lbsal" groups ( fig 1c, middle panel) . analysis of the cytokine profile early after l. chagasi-challenge (t 90) demonstrated that "lbsapsal" group showed a significant increase of tnf-α levels (p<0.05) upon vsa-stimulation as compared to "sal" and "lbsal" groups ( fig 1a, middle panel) . no differences in the il-12 levels were observed amongst the experimental groups, regardless the culture conditions. analysis of ifn-γ, "lbsapsal" group displayed increased levels (p<0.05) upon vsa-stimulation as compared to "control" and "lbsal" groups at t 90 (fig 1c, middle panel) . additionally, "lbsapsal" group showed a significant increase of ifn-γ (p<0.05) upon slca-stimulation as compared with the "control" group ( fig 1c, middle panel) . data analysis late after l. chagasi-challenge (t 885 ) demonstrated that the "lbsal" group displayed increased tnf-α levels (p<0.05) upon slca-stimulation as compared with the "control" group ( fig 1a, bottom panel) . in addition, the "lbsapsal" group displayed higher levels of tnf-α (p<0.05) upon vsa-stimulation as compared with "lbsal" group ( fig 1a, bottom panel) . no differences in the il-12 levels were observed amongst the experimental groups, regardless the culture conditions. interestingly, the analysis of ifn-γ revealed that "lbsapsal" and late (885 days-t 885 ) after experimental l. chagasi-challenge. the groups are represented as follows: c ("control"; white bars); "sal" (lutzomyia longipalpis salivary glands; light gray bars); "lbsal" (antigen of l. braziliensis plus lutzomyia longipalpis salivary glands; dark gray bars); and "lbsapsal" (l. braziliensis antigen plus saponin and lutzomyia longipalpis salivary glands; black bars). the x-axis displays the different experimental groups ("control", "sal", "lbsal", and "lbsapsal") according to the in vitro stimuli (control culture [cc], vsa or slca). the y-axis represents the cytokine levels (pg/ml) for tnf-α (a), il-12 (b) and ifn-γ (c). data are presented as mean values ± standard deviations. the connecting lines represent significant difference (p <0.05) between the cc, vsa or slca-stimulated cultures. the symbols c, sal and lbsal indicate significant differences in comparison to the "control", "sal" and "lbsal" groups, respectively. group showed higher levels (p<0.05) upon vsa-stimulation as compared with the "control", "sal" and "lbsal" groups ( fig 1c, bottom panel) . "lbsapsal" induced lower levels of il-4 and tgf-β but while unvaccinated dogs presented higher amounts of il-10 after l. chagasichallenge aiming to evaluate whether the immunization protocols would induce regulatory/anti-inflammatory cytokines, we further quantified the levels of il-10, il-4, and tgf-β upon vsa or slca-stimuli in vitro. no significant differences were observed amongst the experimental groups at baseline (t 0 ) (fig 2b, upper panel) . the results observed at the post-vaccination period (t 3rd ) demonstrated that the "lbsal" group showed a significant reduction in the il-10 levels (p<0.05) upon vsa-stimulation as compared to the "sal" group ( fig 2b, middle panel) . no differences in il-4 and tgf-β levels were observed amongst the experimental groups, regardless the culture conditions. data mining performed early after l. chagasi-challenge (t 90 ) revealed no differences in the il-10 production amongst the experimental groups, regardless the culture conditions. however, "lbsapsal" group showed a significant reduction of il-4 levels (p<0.05) upon slca-stimulation as compared to the "sal" group (fig 2a, middle panel) . interestingly, "lbsapsal" group also presented a significant reduction of tgf-β levels (p<0.05) upon slca-stimulation as compared the "control" group (fig 3, middle panel) . analysis carried out late after l. chagasi-challenge (t 885 ) did not differences in il-10 levels amongst the experimental groups, regardless the culture conditions. the results showed that the "lbsal" group presented a significant reduction in the il-4 levels (p<0.05) upon vsa-stimulation as compared with the "sal" group ( fig 2b, bottom panel) . interestingly, "lbsapsal" group displayed a significant reduction of tgf-β levels (p<0.05) upon slca-stimulation as compared with the "control" group (fig 3, bottom panel) . the hallmark of the cytokine network in the "lbsapsal" group is a balanced immune response regarding positive correlation between proinflammatory cytokines (il-12, ifn-γ, tnf-α) and regulatory cytokines (il-10) aiming to identify the overall balance of the evaluated cytokines, we have applied machinelearning approaches to analyze the cytokine network for all immunization protocols generated upon slca and vsa-stimuli in vitro (s1 fig and fig 4) . the cytokine balance was calculated as the ifn-γ/il-4 and ifn-γ/il-10 ratio for all experimental groups and culture conditions and data provide in the s1 fig. the results re-enforce that ability of "lbsapsal" vaccine to induce a long lasting ifn-production over the synthesis of both il-4 and il-10, regardless the antigen-stimuli (s1 fig). data mining by system biology tools were used to generate biomarker networks for each experimental group using both vsa and slca-stimuli. the results demonstrated that upon vsa-stimulation, the "control" group presented negative correlations between il-4 with tnfα and il-10 (vsa stimulus). positive correlation was also observed between tnf-α and ifn-γ. upon slca-stimulation, positive correlation was observed between il-12 and ifn-γ along with a negative correlation between tnf-α and il-4 (fig 4, upper left panel) . the "sal" group presented, upon vsa-stimulation, a range of positive correlations, including tnf-α with il-12, ifn-γ and il-10 along il-12 with il-10. in addition, negative correlation was observed between ifn-γ and il-4. analysis upon slca-stimulation demonstrated data were analyzed at baseline before vaccination (t 0 ), 15 days after third immunization dose (t 3rd ) as well as early (90 days-t 90 ) and late (885 days-t 885 ) after experimental l. chagasi-challenge.the groups are represented as follows: c ("control"; white bars); "sal" (lutzomyia longipalpis salivary glands; light gray bars); "lbsal" (antigen of l. braziliensis plus lutzomyia longipalpis salivary glands; dark gray bars); and "lbsapsal" (l. braziliensis antigen plus saponin and lutzomyia longipalpis salivary glands; black bars). the x-axis displays the different experimental groups ("control", "sal", "lbsal", and "lbsapsal") according to the in vitro stimuli (control culture [cc], vsa or slca). the y-axis represents the cytokine levels (pg/ml) for il-4 (a) and il-10 (b). data are presented as mean values ± standard deviations. the connecting lines represent significant difference (p <0.05) between the cc, vsa or slca-stimulated cultures. the symbol sal indicates significant differences in comparison to the "sal" group. positive correlations amongst the pro-inflammatory cytokines (tnf-α, ifn-γ and il-12) along positive correlation of them with il-10 (fig 4, upper right panel) . analysis of the "lbsal" group upon vsa-stimulation demonstrated a single negative correlation between tnf-α and il-4 (fig 4, bottom left panel) . "lbsapsal" (l. braziliensis antigen plus saponin and lutzomyia longipalpis salivary glands; black bars).the xaxis displays the different experimental groups ("control" and "lbsapsal") according to the in vitro stimuli (control culture [cc] and slca). the y-axis represents the tgf-β levels (pg/ml). data are presented as mean values ± standard deviations. the connecting lines represent significant difference (p <0.05) between the cc or slca-stimulated cultures. the symbol c indicates significant differences in comparison to the "control" group. data analysis demonstrated that upon vsa-stimulation, the "lbsapsal" group showed strong positive correlation among tnf-α and ifn-γ along with positive correlation between tnf-α and il-10. negative correlations were also observed amongst tnf-α and ifn-γ with il-4 (fig 4, bottom right panel) . moreover, upon slca-stimulation, positive correlation was observed between il-12 with ifn-γ and il-10 (fig 4, bottom right panel) . a sustained prominent reduction in spleen parasite load was observed in "lbsapsal" group later on after l. chagasi-challenge the parasitological analysis performed in spleen samples later on (t 885 ) following l. chagasichallenge and reported as number of l. chagasi organisms/20ng of total dna in spleen samples and presented in table 1 . data analysis demonstrated that all vaccination protocols were able the groups are represented as follows: "control"; "sal" (salivary glands of lutzomyia longipalpis); "lbsal" (antigen of l. braziliensis plus lutzomyia longipalpis salivary glands) and "lbsapsal" (antigen of l. braziliensis plus saponin and lutzomyia longipalpis salivary glands). the letter "a" indicate significant difference in relation to the sal, lbsal and lbsapsal groups. reduction (%) in parasite load was calculated as the proportion of number of amastigotes organisms/20ng of total dna observed in "sal", "lbsal" and "lbsapsal" groups in relation to "control" group. no clinical signs and mortality were observed throughout the experimental design. doi:10.1371/journal.pone.0161169.t001 to induce a reduction in the splenic parasite load as compared to the "control group". indeed, "sal" and "lbsal" groups yielded 74.6% and 66.5% of reduction in the spleen parasite load (1.56 and 2.06 amastigotes/20ng of total dna, respectively) as compared to the "control" group (6.14 amastigotes/20ng of total dna). "lbsapsal" groups showed the lowest parasite load (1.30 amastigotes/20ng of total dna), leading to 78.9% of reduction rate in relation to the "control" group (table 1) . moreover, no clinical signs and mortality were observed throughout the experimental design (table 1) . "lbsapsal" group showed after l. chagasi-challenge enhanced no production with negative association with the spleen parasite load the levels of nitric oxide produced by pbmcs were evaluated upon vsa or slca-stimuli in vitro early (t 90 ) and late (t 885 ) after l. chagasi-challenge. data analysis carried early (t 90 ) after l. chagasi-challenge, demonstrated that the "lbsap-sal" group presented upon control culture condition enhanced no levels (p<0.05) as compared to the "sal" group ( fig 5, upper left panel) . additionally, "sal", and "lbsapsal" groups showed increase in the no levels (p<0.05) upon vsa-stimulation as compared to the "control" and "lbsap" groups ( fig 5, upper left panel) . the analysis performed late (t 885 ) after l. chagasi-challenge showed the "lbsapsal" group displayed higher no levels (p<0.05) upon slca-stimulation as compared to the "control" group ( fig 5, upper right panel) . additional analysis revealed that the no production at t 885 displayed a negative correlation with the spleen parasite load selectively upon slca-stimulation (fig 5, bottom right panel) . interestingly, it was observed that in the "lbsapsal" group, four out of five animals (80%) showed simultaneous high no production and low spleen parasite load, compatible with a protection profile (fig 5, bottom right panel) . in contrast, in the "control" group, only one out of five (20%) showed high no production but all animals presented high spleen parasitism (fig 5, bottom right panel) . the control of leishmania chagasi (syn. leishmania infantum) infection in dogs is essential to stop the current spread of zoonotic visceral leishmaniasis. therefore, a vaccine against vl would be an important tool for controlling cvl and would dramatically decrease anxiety regarding l. chagasi infection in humans [18, 47] . in this sense, the establishment of biomarkers of immunogenicity is considered critical in the rational approach for analyzing candidate vaccines against cvl, and it contributes to identifying the pattern of immune response in dogs and the search for vaccine candidates against cvl [48, 49] . for this reason, in this work, the immunogenicity and protective effects of the "lbsapsal" vaccination in dogs were investigated using levels of no and cytokines and evaluations related to the spleen parasite load. furthermore, the addition of sand fly saliva extract to vector-based vaccines can enhance the ability of the host to control or block leishmania infection [38, 33] . the major results observed after "lbsapsal" immunization revealed a reduction in il-4 levels during the early (t 90 ) post-challenge period. importantly, previous studies have identified that the presence of il-4 in splenocytes from dogs naturally infected with l. chagasi would be an important biomarker during ongoing cvl [12, 50] . however, in some reports, the role of il-4 was related to the resistance profile or susceptibility profile of cvl [50, 51] ; the higher levels of il-4 are considered the hallmark of dogs naturally infected with l. chagasi [52] and sustained reduction of il-4 has been also reported by other immunogen candidates. in fact, reduction in the levels of il-4 after vaccine immunization against cvl has been considered a biomarker for protection against leishmania infection [53] . in the present study, the evaluation of il-10 demonstrated a possible association with events related to the susceptibility to infection by l. chagasi; the "control" group has presented increased amounts during the late (t 885 ) post-challenge period. in fact, this cytokine has been fig 5. impact of distinct immunization protocols on the no production elicited early and late after l. chagasi-challenge. no levels (μm) were determined in supernatants from pbmcs cultures maintained upon vaccine-soluble antigen (vsa) or soluble leishmania chagasi antigen (slca) stimuli in vitro. data were analyzed early (90 days-t 90 ) and late (885 days-t 885 ) after experimental l. chagasi-challenge. the groups are represented as follows: c ("control"; white bars); "sal" (lutzomyia longipalpis salivary glands; light gray bars); "lbsal" (antigen of l. braziliensis plus lutzomyia longipalpis salivary glands; dark gray bars); and "lbsapsal" (l. braziliensis antigen plus saponin and lutzomyia longipalpis salivary glands; black bars). top panels: the x-axis displays the different experimental groups ("control", "sal", "lbsal" and "lbsapsal") according to the in vitro stimuli (control culture [cc], vsa or slca). the y-axis represents the nitrite levels [μm] . data are presented as mean values ± standard deviations. the connecting lines represent significant difference (p <0.05) between the cc, vsa or slca-stimulated cultures. the symbols c, sal and lbsal indicate significant differences in comparison to the "control", "sal" or "lbsal" groups, respectively. bottom panels: correlation between no levels and spleen parasite load (# amastigotes/20ng of total dna) at t 885 considering cc (bottom left panel) or the presence of a stimulus (vsa: bottom middle panel; or slca: bottom right panel) in all groups. the groups are distinguishable by colors as follows: as follows: "c" (white circles); "sal" (ligh gray circles); "lbsal" (dark gray circles) and "lbsapsal" (black circles). the quadrants represented in the bottom panels delimit the low and high no producers (y-axis) and the low and high spleen parasite load (x axis). associated with severity of vl [54] and cvl [12, 16, 50, 52, 55, 56] . importantly, the "lbsapsal" group did not have increased il-10 production, even after the late l. chagasi-challenge period. additionally, analysis of tgf-β revealed reduced production in the "lbsapsal"-immunized group during the early and late post-challenge periods. interestingly, the presence of tgf-β has been associated with inducing immunosuppression characteristics during the course of vl [57, 58] . moreover, the presence of tgf-β in vitro has a protective effect for amastigotes in macrophages, favoring the maintenance of parasitism [59] . in addition, alves et al. [50] reported high levels of tgf-β associated with increased parasite load in lymph nodes from symptomatic dogs and concluded that tgf-β is associated with morbidity in cvl. in this context, it is possible to hypothesize that lower levels of tgf-β in the "lbsapsal" group would indicate the establishment of immunoprotective mechanisms, whereas higher amounts in the "control" group would be associated with the susceptibility pattern after l. chagasi-challenge. analysis of pro-inflammatory cytokines has been considered a prerequisite for composing immunogenicity analyses before and after experimental challenge with l. chagasi in clinical trials of anti-cvl vaccines [48, 26] . the analysis of tnf-α before vaccine immunization (t 0 ) demonstrated increased levels in slca-stimulated cultures in the "control" and "lbsapsal" groups and demonstrated a tendency for enhanced amounts in the "sal" and "lbsal" groups. similar results were observed for il-12 and ifn-γ at t 0 . these results seem to indicate that slca stimulation would induce increased levels of these cytokines, pointing to an inherent feature of the antigenic stimulus. for this reason, we evaluated all groups and analyzed the same stimulus in each group to identify a cytokine profile regarding type i immune response. this strategy demonstrated enhanced levels of il-12 and ifn-γ post-vaccination and sustained production of both tnf-α and ifn-γ even after early and late post-challenge periods in the "lbsapsal" group. some studies have described tnf-α as being associated with a resistance profile in cvl [52, 60, 61, 62, 63] or in dogs vaccinated against cvl [53] , and has been associated with susceptibility when associated with high levels of il-4 and il-10 [64] . results obtained by strauss-ayali et al. [65] showed that after stimulation with exogenous il-12, pbmcs from l. infantum−infected dogs were able to reverse an apparent state of anergy, resulting in increased production of ifn-γ. moreover, menezes-souza et al. [62] found that low levels of il-12 concomitant with high levels of il-10 and tgf-β represent a favorable condition for the persistence and replication of parasites in cvl. it has been described that ifn-γ is linked to a resistance profile in different experimental models for vl [50, 61, 66, 67, 68] and in dogs vaccinated against cvl [21, 26, 29, 30, 53] . our data regarding the cytokine network indicated a balanced immune response in the "lbsapsal" group. in this sense, we describe positive (tnf-α versus ifn-γ and il-10; il-12 versus ifn-γ and il-10) and negative (il-4 versus tnf-α or ifn-γ) correlations demonstrating a prominent pro-inflammatory immune response in the "lbsapsal" group. in addition to a non-significant increase in il-4 or il-10 levels in the "lbsapsal" group, a balanced immune response was demonstrated by taking into account the positive correlations between il-10 and type i cytokines (ifn-γ, il-12, tnf-α). these data should be related to the regulation of the prominent pro-inflammatory immune response induced by the "lbsapsal" vaccination and should aim to control potential tissue damage by type i cytokines. the parasitological evaluation revealed in "lbsapsal" group a remarkable reduction in spleen parasite load (78.9%) after experimental l. chagasi-challenge, in concordance with a prominent pro-inflammatory immune response induced by this vaccine. in fact, we have been described a parasite load reduction of 69% in the lbsapsal group [27] , indicating the capacity of this vaccine to control parasite replication even long after challenge (885 days). sand fly saliva displays an important role in the first steps of leishmania infection, due to the vast repertoire of pharmacologically active molecules that surround host's hemostatic system [69] . the re-exposure to the salivary components seems to display an immunogenic activity, eliciting antibody production and cell mediated immunity by the host that could be block or limit the leishmania infection [70] . some studies have been shown that l. longipalpis salivary proteins induce an immune response associated with protection in dogs [22, 27] . furthermore, in a hamster model, salivary proteins of a sand fly protects against the fatal outcome of visceral leishmaniasis [40] . similarly, we observed 74.6% parasite load reduction in "sal" group, showing that salivary components have a high potential to limit infection in dogs. the experimental challenge in vaccine studies against canine visceral leishmaniasis is considered as crucial to analyze the protection performance. distinct studies have been published aiming to determine the experimental l. chagasi-challenge plus sand fly saliva using intradermal route in dogs would be more similar to natural infection than intravenous challenge [71, 72] . however, using intradermal challenge, the dogs would be asymptomatic during all the study, besides to present lower parasitism [71] . since the "lbsapsal" vaccine presents saliva as antigenic compound, the ideal experimental challenge to test the protection should ideally be performed by intradermal route, as analyzed in our study. importantly, we observed increased no levels in the "lbsapsal" group during the early and late post-challenge periods. interestingly, four out of five dogs immunized with "lbsapsal" presented higher no amounts and low spleen parasite burden during the late post-challenge period, indicating long-term immunogenicity and resulting in reduction of parasitism. in fact, we have previously demonstrated that "lbsapsal" induced resistance biomarkers specifically related to expansion of circulating cd4 + and cd8 + t-cells and leishmania-specific subsets and lower levels of parasitism [22, 27] . panaro et al. [73] also observed an increase in no production and anti-leishmanial activity of macrophages, as well as increased levels of ifn-γ in pbmcs supernatants, in dogs immunized with a vaccine comprising crude antigens of l. infantum. taken together, our major data indicate that immunization with "lbsapsal" is able to induce a protection profile characterized by enhanced amounts of type i (tnf-α, il-12, ifnγ) cytokines and reduction in type ii cytokines (il-4 and tgf-β), even after experimental challenge. the establishment of a polarized type i immune response after "lbsapsal" immunization supported increased levels of no production, favoring a reduction in parasitism and indicating long-lasting protection against l. chagasi infection. these results encourage further studies that can provide important information for a better understanding of the effectiveness of the "lbsapsal" vaccine and strategies for addressing leishmania antigens in combination with sand fly proteins such as those present in the saliva in the vector. supporting information s1 fig. impact of distinct immunization protocols on the pro-inflammatory/regulatory cytokine balance. the balance of inflammatory cytokine ifn-γ and regulatory/anti-inflammatory (il-4 and il-10) were analyzed in the supernatant of pbmcs maintained upon vaccinesoluble antigen (vsa) or soluble leishmania chagasi antigen (slca) stimuli in vitro. data were analyzed early (90 days-t 90 ) and late (885 days-t 885 ) after experimental l. chagasi-challenge. the groups are represented as follows: c ("control"; white bars); "sal" (lutzomyia longipalpis salivary glands; light gray bars); "lbsal" (antigen of l. braziliensis plus lutzomyia longipalpis salivary glands; dark gray bars); and "lbsapsal" (l. braziliensis antigen plus saponin and lutzomyia longipalpis salivary glands; black bars).the x-axis displays the different experimental groups ("control", "sal", "lbsal" and "lbsapsal") according to the in vitro stimuli (control culture [cc], vsa or slca). the y-axis represents the cytokine ratio (ifn-γ/il4 and ifn-γ/il-10). data are presented as mean values ± standard deviations. the connecting lines represent significant difference (p <0.05) amongst the cc, vsa or slca-stimulated cultures. the symbols c and sal indicate significant differences in comparison to the "control" or "sal" groups, respectively. 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hengartner, nick; meadors, grant; ke, ruian title: change in global transmission rates of covid-19 through may 6 2020 date: 2020-08-06 journal: plos one doi: 10.1371/journal.pone.0236776 sha: doc_id: 275395 cord_uid: w2u7fq1g we analyzed covid-19 data through may 6th, 2020 using a partially observed markov process. our method uses a hybrid deterministic and stochastic formalism that allows for time variable transmission rates and detection probabilities. the model was fit using iterated particle filtering to case count and death count time series from 55 countries. we found evidence for a shrinking epidemic in 30 of the 55 examined countries. of those 30 countries, 27 have significant evidence for subcritical transmission rates, although the decline in new cases is relatively slow compared to the initial growth rates. generally, the transmission rates in europe were lower than in the americas and asia. this suggests that global scale social distancing efforts to slow the spread of covid-19 are effective although they need to be strengthened in many regions and maintained in others to avoid further resurgence of covid-19. the slow decline also suggests alternative strategies to control the virus are needed before social distancing efforts are partially relaxed. since its initial outbreak in wuhan, china in late 2019 and early 2020 [1] , covid-19 caused repeated rapid outbreaks across the global from february through april 2020. the extremely rapid spread of covid-19 in china [2] does not appear to be an anomaly: the disease has shown a short doubling time (2.4-3.6 days) outside of china as well [3] . as of may 21, 2020, the virus caused 5,034,458 reported infections, and 328,730 deaths globally [4] . in response, most affected countries/regions have implemented strong social distancing efforts, such as school closures, working-from-home, shelter-in-place orders. as a result, the spread of covid-19 slowed down substantially in some countries [5] , leading to a flattening of the epidemic curve. as social distancing induces high costs to both society and individuals, plans to relax social distancing are discussed. however, changes in both the transmission rates and detection probabilities over time coupled with stochasticity due to reporting delays makes differentiating between truly subcritical dynamics and simply reduced transmission difficult. in this report, we developed a deterministic-stochastic hybrid model and fitted the model to case incidence and death incidence time series data from 55 countries. following the a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 approach suggested by king et al. [6] , we use a (partially) stochastic model and base our estimates on incidence rather than cumulative incidence data. using both case count and death count of data allowed us to disentangle changes in surveillance intensity from changes in transmission [3] . we found evidence for large decreases in the country-level transmission rates, in several of the worst-affected countries. importantly, using data up to may 6, 2020, we computed 99% confidence intervals to test whether or not the data were consistent with subcritical dynamics (i.e. the reproductive number r was below 1 on may 6, 2020). most countries showed large decreases in transmission rates over time, and more than half of studied countries have transmission rates below the epidemic threshold. on the other hand, many countries still appear to be showing rapid exponential growth. given its highly contagious nature, covid-19 can spread rapidly when strong social distancing measures are lifted, even partially [3] . alternative strategies that can effective control the virus are needed when social distancing measures are relaxed. case count and death data were downloaded from the johns hopkins github repository (https://github.com/cssegisanddata/covid-19) through may 6, 2020. data included aggregate counts of reported cases and deaths at the country level and contained no identifying information. any country in the data that had more than 2000 cumulative cases and 100 deaths by may 6, 2020 was included. to minimize the effect of repatriated cases we started each time series on the first day when the cumulative number of cases exceeded 100. all data processing and model fitting were otherwise done on the incidence scale. to address obvious bulk-reporting issues in the data (e.g. sudden zeros in the data followed by very large numbers), we smoothed the data using tukey's 3-median method. because several countries had days with a single death surrounded by days with no deaths, which the smoothing method would set to zero, days with a single death were not replaced with smoothed values. the original data and the smoothed data used for estimation are shown in s1 fig in s1 file. we model the spread of covid-19 as a partially observed markov process with real-valued states s (susceptible), e (exposed), i (infected), and r (removed) to describe the latent population dynamics, and integer-valued states c 0 (to be counted), y 1 (counted cases), d 0:3 (dying), and y 2 (counted deaths) to model sampling into the data. we use multiple states to model the counted deaths to produce an erlang distribution with mean 21 days and standard deviation 11 days in the time to death based on previous estimates of the time to death [7] . the model and all of its parameters (table 1 ) have time units of days. the latent population model is governed by the following ordinary differential equations, where χ(t) is the time-variable transmission rate and n = s + e + i + r is assumed to be fixed over the run of the model. λ ei is the rate at which exposed persons become infectious and λ ir is the rate at which cases recover (i.e. are either no longer infectious or die). at every time interval, we sample persons moving from the e to i states into a stochastic arm of the model that is used to calculate the likelihood of the data. to relate the latent population model to data, we randomly sample individuals from the unobserved population into a stochastic process that models the random movement from infection to being either counted as an observed case or counted as an observed death. the number of persons sampled into the observation arm of the model over a time interval dt is given by and ω are the probabilities that an infected person will be counted or die respectively, ρ c (t) and ω c are the probabilities of being not sampled or not dying respectively, and g(u) is a stochastic function that maps from real-value u to integer g(u); it takes value buc with probability u mod 1 and value due with probability 1 − (u mod 1). in plain english, the model tracks the random fate of each newly infected person as they move from the exposed to infected state with respect to eventually being observed as a case and/or a death in data. at each time step of length δt, the change in state space is given by, where f t |c 0 is a random variate from binðc 0 ; 1 à expðà l y 1 dtþþ, and h it is a random variate from bin d ià 1 ; 1 à exp à 4 21 dt à � à � . x i indicates the i th element of multinomial random variate defined above. the rate λ y1 determines the rate at which persons who will be counted are counted (i.e. lower values of λ y1 mean a longer delay in cases showing up in the data). the values of y 1:2 are set to zero at the beginning of every day such that they accumulate the simulated number of cases and deaths that occur each day. both the transmission rate and detection model probability are allowed to vary with time in the following way: where t f is the final time in the given dataset. in plain english, the transmission rate is constant up to some time, t ð1þ w , where it linearly increases or decreases to the value χ f by time t ð2þ w . the model is constrained such that t ð2þ w < t f à 20, that is, the transmission rate must be constant for at least 20 days before the end of the data collection period. this constraint is in place to avoid overfitting the final transmission rate. the detection rate likewise has a linear increase or decrease beginning at time t ρ ; however, the increase or decrease continues to a fixed point that is 20 days before the final datum. variation in the detection rate (i.e. the probability that an infected case will be counted in a fixed interval) over the course of the epidemic can strongly bias estimates of the population growth rate (derivation for the exponential growth case in s1 file); not allowing the detection probability to change over time could lead to discordance in the case count and death count time series. we assume that the data are negative binomial-distributed, conditional on the simulated number of cases and deaths that occur in a given time interval, i.e. the number of cases in the i th observation period has density negbin(y 1 , � 1 ) and the number of deaths in the i th observation period has density negbin(y 2 , � 2 ). the negative binomial is parameterized such that the first argument is the expectation and the second is an inverse overdispersion parameter that controls the variance of the data about the expectation; as � i becomes large, the data model approaches a poisson with parameter y i . both � 1 and � 2 were estimated from the data. parameter estimation had two distinct steps: model selection and computation of confidence intervals. in the model selection phase, the model was fit to the data using an iterated particle-filtering method, implemented in the pomp r library [8] . to optimize the likelihood of the data, we used 1500 particles in 125 iterations for each country. to determine whether or not 1500 particles was sufficient to minimize the variance in the estimated the mean likelihood at a given parameter value we ran 20 independent particles filters on a single data set and found that the average deviation from the mean was less than 0.1 log units suggesting that we would be able to detect differences in the log likelihood greater than 0.1 log units. the reported likelihoods were measured using 4500 particles at the optimized maximum likelihood estimates (mles). for all fits, the initial state at time zero is computed by assuming there were i(0) infected persons, 21 days before the first reported death (by definition time one). the number of initial number of susceptibilities was assumed to be the predicted 2020 population size of the given country [9] . the model is then simulated forward for 21 days, assuming exponential growth with transmission rate χ 0 , which is taken at the initial state of the model at time zero. because the data were highly variable in the complexity of the patterns they showed, we considered three nested models of increasing complexity for each country. the first model (model 1) assumed simple exponential growth with a constant sampling probability, i.e. ρ 0 = ρ f and t ð1þ w ¼ t ð1þ w ¼ 0. this amounts to a period of exponential growth with transmission rate χ 0 in the pre-data period and then a constant transmission rate χ f over the observation period. the second model (model 2) allowed the transmission rate to vary but kept the detection probability constant, i.e. ρ 0 = ρ f . the third model (model 3) allowed both the transmission rate and detection rate to vary, i.e. all parameters estimated. we determined the best model for each country by a sequence of likelihood ratio tests first comparing model 1 and model 2 and then model 2 to model 3. because we are using an optimization method, we do not have access to samples from the likelihood surface directly. therefore, to obtain estimates of the parametric uncertainly in the final transmission rate, χ f , we computed 99% confidence intervals using the profile likelihood method. for each country computed the profile likelihood by optimizing the model along a fixed grid with points every 0.1 units centered on the mle of χ f value from the previous fit. points were added to the grid until the measured likelihood on either side of the mle was greater than 9 log units lower than the measured maximum likelihood. we then fit a local polynomial regression (loess in r) to those points and found the predicted maximum likelihood parameter value [10] and the 99% ci by locating the points on either side of the mle that were 3.84 log units below the maximum likelihood. we fixed several parameter values based on published work and our scientific judgement. the probability that a case would eventually die, ω = 1%, is based on estimates of the case fatality ratio for both asymptomatic and symptomatic patients (95% ci 0.5-4%) [11] . the latency rate, l ei ¼ 1 3 , was initial set based on our general sense of what was consistent with the pre-print literature available at the time, however, that value is has proven to be consistent with later reports [12] . the recovery rate, l ir ¼ 1 10 , was similarly set to be consistent with the available literature when the model was being developed. likewise, longitudinal studies have shown that our assumption of an average infectious period of 10 days is reasonably consistent with the clinical data [13, 14] . although the clinical data paints a picture of the natural history of infection that is far more complex than our model can capture, the formulation of our model is consistent with the available data. our primary outcome of interest is the growth rate, r, at the end of observation period, which is derived from the transmission rate estimated from the data. using the equation in [15] we can express the growth rate in terms of the model parameters we found that for most countries, the fraction of the population that was still susceptible at the end of the observation period was greater than 95%, therefore we omitted the term prðsþ ¼ s between countries. from the same paper we also have the equation showing that r 0 = 1 when r = 0. we also compare predicted deaths due to covid-19 in each country through july 5th 2020 to the average number of deaths in a period of the same length (fig 3) . predicted deaths were computed by simulating the number of daily deaths from the first observation though july 5th 2020 for each country and taking the mean value. confidence intervals were computed as the relevant quantiles of the sum over 1000 simulations. pre-covid-19 deaths were based on allcause death data were downloaded from the who mortality database (https://www.who.int/ healthinfo/mortality_data/en/) for each country for which it was available. using data from the most recent year, we computed the death rate and multiplied the death rate by the length of the interval from the first observation to july 5th 2020 for each country. we fit our model to data collected from 55 countries. model fits are shown in fig 1, and parameter values are given in table 1 . the model can capture the data well, with a few exceptions. the model was not able to find a robust fit to the data from bangladesh; in general, the upside down 'v' shape to the deaths could not be captured well in such a short time series. algeria had a similarly odd pattern in the deaths time series that the model could not capture in detail, although the overall trend in deaths was recovered. in previous versions of this paper, the model had a hard time fitting data from both italy and spain. however, given the longer time series and modifications to the model form, we now find that both italy and spain are well-captured by the model. overall, the model slightly overestimates the number of deaths around the time where deaths begin to decrease. however, this was generally corrected if the time series was long enough. including temporal heterogeneity in the time from infection to detection of a covid-19 death would likely correct this; however, it is not clear that this is advisable, as death counts are likely under-reported. all countries except japan and saudi arabia were found to have lower transmission rates on may 6, 2020 than at the beginning of the observation period, suggesting a global decline in the transmission of covid-19 though may 6, 2020. however, the initial transmission rate should be interpreted cautiously, as we allowed a wide range of infected persons to exist 21 days before the first observation. that is, the initial transmission rate parameter is rather a convolution of the unknown number of infected persons and initial growth rate consistent with the data. we found significant evidence for subcritical dynamics in 27 countries (3 countries had subcritical point estimates but their cis contained the epidemic threshold). fig 2 shows the point estimates and cis of the final transmission rate on may 6, 2020 for all countries, stratified by continent [16] . european countries had the highest probability of being subcritical (21 of 25) with asia (7 of 15) and the americas (2 of 11) having fewer subcritical countries. none of the countries in africa that met the inclusion criteria had subcritical dynamics. generally, countries that were found to have both variable transmission rates and variable detection probabilities (model 3 in table 1) show a pattern of level or increasing deaths coupled with a level or slightly declining incidence in number of reported cases. this pattern illustrates how viewing the case count data alone can be potentially misleading as declines in reported cases can be confounded by variation in the probability of detection (e.g. comparing canada to denmark). some countries were found to have detection probabilities lower than 1% on may 6 2020; however, these values should not be over-interpreted as the simple linear model for changing detection probabilities imposes strong assumptions that are focused on capturing the general trend. fig 3 shows the predicted number of deaths projected out to july 5, 2020 assuming that all parameter values are constant over the period may 6 through july 5 2020. the average duration of the country-level epidemic in european countries is longer than in asia, leading to a higher level of death, despite asian countries having on-average higher growth rates. however, in the americas, the predicted deaths are higher with 8 of 11 countries having total predicted deaths greater than 0.1% of the total population by july 5, 2020. the model predicts 1,320,170 total covid-19 deaths in all 55 countries on july 5, 2020; of those deaths, 21% are predicted to occur in the us. the deaths due to covid-19 in europe are lower than the average number of reported deaths in a period of the same length for all countries in the data set that also had all-cause death counts from previous years. however, in the americas, the covid-19 death counts are approaching the all-cause death levels in several countries, suggesting that covid-19 deaths are approaching a doubling of all-cause deaths in those countries. our model found evidence for reductions in transmission rates of covid-19 in 53 of 55 examined countries. encouragingly, of those countries, we found statistical evidence that the size of the epidemic is decreasing in 27 countries, i.e. the effective reproductive number is less than 1, using data up to may 6, 2020. this suggests that, despite the highly heterogeneous populations represented by these countries, the growth of covid-19 outbreak can be reverted. although our model cannot attribute exact causes to the global decline in transmissions rates, most countries implemented sustained, population-level social distancing efforts over a period of weeks to months. these efforts are highly likely to play a major role in reducing the transmission of covid-19 [5] . we estimated that in countries with decreasing transmission, the rate of decrease is in general less than 0.1/day (average -0.04/day). based on data from 8 european countries, the us, and china, we previously estimated that in the absence of intervention efforts, the epidemic can grow at rates between 0.19-0.29/day [2] . this means that the outbreak can grow rapidly and quickly wipe gains made though public heath efforts made if social distancing measures are completely relaxed. for example, if the rate of decrease under strong public health interventions is 0.1/day and the growth rate in the absence of public heath interventions is 0.2/ day, then, the number of cases averted in two weeks of intervention will be regained in only one week. social distancing measures have their own social costs. our results suggests alternative strategies to control the virus are needed in place when social distancing efforts are relaxed. due to the uncertainties in the impact of each specific control measures, changes to policies should be made slowly because the signal of changing transmission can take weeks to fully propagate into current data streams as a result of the long lag between infection to case confirmation (as we estimated to be on average approximately 2 weeks). our goal in this paper was to develop a model that could be applied very broadly to multiple countries and we have made assumptions that facilitate that goal. however, our model makes key assumptions that should be considered when thinking about where these results fit into the vast collection of covid-19 modeling papers. for example, we slightly privilege death counts over case counts in linking the population model to the data by assuming that the distribution of the time to death is known and that the probability of being a detected death is fixed over time. likewise, we assume that at the country level, the change in transmission rates can be modeled by a simple linear function, which we believe is reasonable as interventions implemented at the local level are likely to lead to a smooth change when aggregated up to the population level. our model produces reasonable fits to the global data, but out approach does not allow us to have unique models for each country that could almost certainly capture country-level trends with greater accuracy. our model also makes strong simplifying assumptions about the natural history of infection, about which we are continuously learning. for example, our model assumes that contagiousness is constant over the infection, which is possibly variable over time [12] . we also assume that diagnosis does not affect transmission from infected persons. however, given that we are inferring broad, population-average parameters and allowing those parameters to change over time to reflect broad changes in the transmission dynamics, we believe that our results are are reasonable portrayals of reality despite using a simple model of the natural history of infection. overall, our results suggest that covid-19 is controllable in diverse settings using a full range of strong and comprehensive non-pharmaceutical measures, and that future deaths from the disease are avoidable. supporting information s1 file. (pdf) early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia early release -high contagiousness and rapid spread of severe acute respiratory syndrome coronavirus 2 -emerging infectious diseases journal -cdc fast spread of covid-19 in europe and the us and its implications: even modest public health goals require comprehensive intervention an interactive web-based dashboard to track covid-19 in real time. the lancet infectious diseases 2020-03-30-covid19-report-13.pdf avoidable errors in the modelling of outbreaks of emerging pathogens, with special reference to ebola high contagiousness and rapid spread of severe acute respiratory syndrome coronavirus 2. emerging infectious diseases pomp: statistical inference for partially observed markov processes maximum smoothed likelihood estimation severity of 2019-novel coronavirus (ncov) temporal dynamics in viral shedding and transmissibility of covid-19 virological assessment of hospitalized patients with covid-2019 clinical and virologic characteristics of the first 12 patients with coronavirus disease 2019 (covid-19) in the united states estimating epidemic exponential growth rate and basic reproduction number countrycode: an r package to convert country names and country codes conceptualization: ethan obie romero-severson, nick hengartner, ruian ke. key: cord-296691-cg463fbn authors: wang, ren; xu, sheng; jiang, yumei; jiang, jingwei; li, xiaodan; liang, lijian; he, jia; peng, feng; xia, bing title: de novo sequence assembly and characterization of lycoris aurea transcriptome using gs flx titanium platform of 454 pyrosequencing date: 2013-04-09 journal: plos one doi: 10.1371/journal.pone.0060449 sha: doc_id: 296691 cord_uid: cg463fbn background: lycoris aurea, also called golden magic lily, is an ornamentally and medicinally important species of the amaryllidaceae family. to date, the sequencing of its whole genome is unavailable as a non-model organism. transcriptomic information is also scarce for this species. in this study, we performed de novo transcriptome sequencing to produce the first comprehensive expressed sequence tag (est) dataset for l. aurea using high-throughput sequencing technology. methodology and principal findings: total rna was isolated from leaves with sodium nitroprusside (snp), salicylic acid (sa), or methyl jasmonate (meja) treatment, stems, and flowers at the bud, blooming, and wilting stages. equal quantities of rna from each tissue and stage were pooled to construct a cdna library. using 454 pyrosequencing technology, a total of 937,990 high quality reads (308.63 mb) with an average read length of 329 bp were generated. clustering and assembly of these reads produced a non-redundant set of 141,111 unique sequences, comprising 24,604 contigs and 116,507 singletons. all of the unique sequences were involved in the biological process, cellular component and molecular function categories by go analysis. potential genes and their functions were predicted by kegg pathway mapping and cog analysis. based on our sequence analysis and published literatures, many putative genes involved in amaryllidaceae alkaloids synthesis, including pal, tydc omt, nmt, p450, and other potentially important candidate genes, were identified for the first time in this lycoris. furthermore, 6,386 ssrs and 18,107 high-confidence snps were identified in this est dataset. conclusions: the transcriptome provides an invaluable new data for a functional genomics resource and future biological research in l. aurea. the molecular markers identified in this study will provide a material basis for future genetic linkage and quantitative trait loci analyses, and will provide useful information for functional genomic research in future. the genus lycoris is an important group of amaryllidaceae composed of approximately 20 species of flowering plants native to the moist warm temperate woodlands of eastern and southern asia, of which 15 (10 endemic) are distributed in china. most of the lycoris species are commonly cultivated in china, korea, japan and vietnam as bulbous plants [1, 2] . in comparison with other well-known bulb flowers, such as narcissi and lilies, lycoris has its own characteristics and merits. it comes into flower at a time when few other bulbous plants are active. the flowers are characterized by their pastel and plentiful colors as well as by beautiful and varied shapes [1] . so the lycoris species are all very popular with considerable acceptance as ornamental plant [3] and most of them have been successfully cultivated. in the past several decades, some of the lycoris species, cultivars, and hybrids such as lycoris radiata and lycoris aurea have been used worldwide. meanwhile, the demand for lycoris as a commercial horticultural product has been increasing steadily, so the breeding of varieties with new flower forms and/or colors has become desirable for lycoris. moreover, lycoris species are all of medical values. the bulbs of lycoris have been used in traditional chinese medicine (tcm) for a long time and some amaryllidaceae-type alkaloids isolated from these plants have been reported to exhibit immunostimulatory, anti-tumor, anti-viral and anti-malarial activities [4] [5] [6] [7] . for example, lycorine, a pyrrolophenanthridine alkaloid, has been demonstrated to suppress cell growth of the human leukemia cell line hl-60 [8] as well as the multiple myeloma cell line km3 [9] by arresting the cell cycle, subsequently inducing apoptosis of tumor cells. more recently, lycorine causes a rapid turnover of protein levels of myeloid cell leukemia-1 (mcl-1), which may play an important survival role in a variety of tumor cells including leukemia were reported [10] . so lycorine might be a good candidate therapeutic agent against leukemia. additionally, it has also been reported that lycorine was an active component in the alkaloid portion and a good candidate for the development of new antiviral medicine in the treatment of severe acute respiratory syndrome (sars) [11] . galanthamine, another major amaryllidaceae alkaloid, has also been widely used in medicine as a strong reversible inhibitor of cholinesterase to increase acetylcholine sensitivity [12] . it is a specific remedy for myasthenia gravis and poliomyelitis sequela and has also been used in the therapy of glaucoma [13] and alzheimer's disease [13] [14] [15] . hence, galanthamine also has important medicinal value and broad application prospect [16] . at the same time, because of their several biological activities and their potential diversity in pharmacology, amaryllidaceae alkaloids have also attracted great interest of synthetic organic chemists [17] [18] [19] [20] [21] [22] [23] [24] [25] . it is well known that the generation of large-scale expressed sequenced tags (ests) is a very useful approach to describe the gene expression profile and sequence of mrna from a specific organism and stage (especially in non-model species). ests represent a valuable sequence resource for research and breeding, as they provide comprehensive information regarding the transcriptome [26] . they have played significant roles in functional genomics research for discovery of novel genes together with identifying different protein groups (e.g. proteins with signal peptides) other than the whole genome [27] [28] [29] , developing ssrs and snps markers [30] [31] [32] [33] [34] , allowing large-scale expression analysis [35] , improving genome annotation [36] , and elucidating phylogenetic relationships [37] . next-generation sequencing (ngs) technologies such as the illumina solexa, roche 454, and abi solid platforms have greatly decreased the cost and time required for receiving genomic and transcriptomic data [38] . by generating sufficiently long sequence reads, roche 454 pyrosequencing using genome sequencing (gs) flx technology makes it possible to compensate for the lack of a reference genome during de novo sequence assembly with the concurrent improvements of de novo assembly software [39] . meanwhile, it is particularly useful as a shotgun method for generating est data and a powerful method for whole genome transcriptome analysis and gene discovery with pyrosequencing of uncloned cdnas [40] . so far, a large number of plants [26, 34, [40] [41] [42] including arabidopsis [43] , artemisia annua [44] , cucumber [45] , medicago [46] , maize [47] , barley [48] and jatropha curcas (barbados nut) [49] have been performed for transcriptome analyses by roche 454 pyrosequencing. also, many est libraries of a wide range of plant species have been constructed for genes involved in plant growth and differentiation [46, 50] , biochemical pathways [47, 48] , secondary metabolism [51, 52] as well as responses to environmental stresses and pathogen attack [53] . the goal of this study was to characterize the transcriptome of l. aurea in lycoris species using high-throughput roche 454 pyrosequencing. as one of the amaryllidaceae plants, l. aurea is an indigenous and popular ornamental herb in china [54] . it is wellknown not only for the high economic value in horticulture but also for the alkaloids it produces, among which galanthamine and lycorine are the major ingredients [55, 56] . in recent years, studies have reported that l. aurea is a good material for extraction of galantamine and other alkaloids [55, 57] . however, little research has been performed to address the amaryllidaceae alkaloids biosynthesis-related genes (especially for galanthamine biosynthesis). additionally, to date, there are only less than 9,000 ests available for lycoris. and limited by the availability of genomic information, studies of lycoris have mainly focused on karyotypes analysis [1,3,58,59], morphology [60] , medicine [11, [13] [14] [15] , and molecular aspect [2, [61] [62] [63] [64] . hence, determination of the genetic pathways and specific genes involved in amaryllidaceae alkaloids biosynthesis and some other aspects of lycoris could be beneficial for humans and enrich our knowledge and understanding of functional genomics and biological research. transcriptome sequencing might provide such a useful tool. after preparing a cdna library by pooling total rna from various organs and tissues, including leaves with sodium nitroprusside (snp), salicylic acid (sa), or methyl jasmonate (meja) treatment, stems and flowers at the bud, blooming, and wilting stages, we sequenced ests from this library. the transcriptome sequences were then annotated by blasting against public databases. subsequently, the annotated sequences were clustered into putative functional categories using the gene ontology (go) framework and grouped into pathways using the kyoto encyclopedia of genes and genomes (kegg). this transcriptome dataset represents the first exploration of l. aurea and provides an invaluable new resource for functional genomics and biological research in l. aurea. the results described herein provide a material basis for future genetic linkage and quantitative trait loci (qtl) analysis and may serve to guide further gene express and functional genomic research in future. in order to achieve l. aurea transcriptome, total rna was extracted from a variety of adult organs and tissues, including the leaves, stem, and flowers. it has been reported that in some plants of amaryllidaceae family, the improved production of galanthamine was examined in meja-treated tissues and 1-aminocyclopropane-1-carboxylic acid (acc)-treated somatic embryos respectively [65, 66] . and in our previous study, we also found that the content of galanthamine in lycoris chinensis and lycoris radiata seedlings would have been affected after treating with sodium nitroprusside (snp), salicylic acid (sa), or meja [67, 68] . for the purpose of improving mrna abundance of genes related to amaryllidaceae alkaloids biosynthesis, the leaves were treated with those abiotic elicitors for rna extraction. quality of the rna as determined by agarose gel electrophoresis and od 260 /od 280 ratio (2.0 6 0.10) was found to be suitable for cdna synthesis. after that, equal quantities of rna from different samples were mixed together and normalized cdna was synthesised. it has been reported that normalization of the cdna greatly reduces the frequency of abundant transcripts, and increases the rate recovery of unique transcripts [69] . after subjecting to quality control experiment, the normalized cdna was used to construct a cdna library. then the library was sequenced by a roche 454 gs flx. one-plate 454 pyrosequencing reaction of the normalized cdna was done using gs flx titanium platform. the reads produced by the roche 454 gs flx were used for clustering and de novo assembly. after eliminating primer and adapter sequences and filtering out the low-quality reads, a total of 937,990 highquality transcriptomic raw sequence reads with a total size of 308,633,593 bp were obtained. size distribution of these reads is shown in figure 1a . length of these reads ranged between 150 and 854 bases with an average length of 329 bp per read ( figure 1a ). clustering and assembly of these raw reads was done using gs de novo assembler [70, 71] . this assembler can assemble the data under genomic or cdna option. after clustering and assembly, a non-redundant set of 141,111 expressed sequence tags (ests), comprising 24,604 contigs and 116,507 singletons, respectively (table 1) were obtained. most of these contigs (95.04%) were distributed in the 200,1,400 bp region ( figure 1b ). and most of these singletons fell between 161 and 500 bp in length ( figure 1c ). so far, the number of ests that are available from lycoris is less than 9,000. recently, by sequencing clones from three non-normalized cdna libraries, 32,521 est sequences were obtained and most of them were used for floral transcription factors prediction from lycoris longituba [64] . therefore, this transcriptome dataset provides a useful resource for future analyses of genes related to amaryllidaceae alkaloids synthesis. to the best of our knowledge, this is the first comprehensive study of the transcriptome of l. aurea. the gc content (ratio of guanine and cytosine) of all unique sequences of l. aurea was determined. the content of gc was 40.42% and 39.58% in contigs and singletons, respectively, giving rise to an overall gc content of 40.03%, indicating a low gc content in the cdna of l. aurea. the l. aurea contigs were further assembled into 16,828 isogroups (all the splice variants of individual transcripts). more contigs than isogroups were found because some contigs (called isotigs, 24,463) are attributed to the same isogroups due to alternative splicing. a large number of alternative splicing could improve the utilization rate of the encoding genes. alternative splicing is an important mechanism for regulating gene expression in eukaryotic cells, and it contributes to protein diversity. similarity search for all the unique sequences was done against genbank non-redundant protein sequences database (nr) using blastx. a total of 66,197 (46.91%) l. aurea unigenes including 18,397 contigs and 47,800 singletons were significantly matched to known genes in the public databases (with an e-value of 10 26 ) (table s1 ), representing putative functional identifications for almost half of the assembled sequences. previous studies have shown that approximately 87% of arabidopsis 454-derived ests could be aligned to predicted genes [43] , while 72% could be similarly identified in cucumber [45] and 54.9% in bamboo [50] . as such, our results succeeded in assigning putative identification to a significant proportion of the discovered l. aurea transcripts given the lack of genomic information for this species. amongst the unique sequences derived from contigs and singletons, coding sequences with homology to 'nadh dehydrogenase', 'cytochrome c oxidase', 'atp synthase', 'splicing factor', 'cytochrome p450', 'ubiquitin-protein ligase', and 'zinc finger protein' were the most abundant. although our research mainly focused on finding putative genes related to amaryllidaceae alkaloids synthesis, other putative functional transcripts identified here could provide a foundation for future investigations of the roles of stress response, reproduction and defense reaction. the transcriptomic findings could also be the best source for deciphering the putative functions of novel genes, but further studies would need to be conducted to understand their molecular functions. go provides a structured and controlled vocabulary for describing gene products in three categories: molecular function, biological process and cellular component [72] . we added go terms using blast2go [73] , which is based on the automated annotation of each unigene using blast results against the genbank non redundant protein database (nr) from ncbi. according to the database, a total of 36,188 unigenes could be assigned to one or more ontologies based on their similarity to sequences with previously known functions, including 43,970 sequences assigned to the molecular function category, 72,628 to the biological process category and 79,853 to the cellular component category. the assigned sequences were divided into 58 functional terms (table s2 ). because several of the sequences were assigned to more than one go term, the total number of go terms obtained in our dataset was bigger than the total number of the unique sequences. in total, 196, 451 go terms were retrieved, 22.38%, 40.65% and 36.97% in the molecular function, in the we used the go annotations to assign each unigene to a set of go slims of the three categories, which are a list of go terms providing a broad overview of the ontology content. a summary with the number and percentage of unigenes annotated in each go slim term is shown (figure 2 ). go annotations for the unigenes showed fairly consistent sampling of functional classes. in the molecular function category, 'binding', 'catalytic activity', 'transporter activity' and 'structural molecule activity' comprised the largest proportion, accounting for 93.35% of the total. whilst the cellular component category showed that many unique sequences were to likely possess 'cell' (29.88%), 'cell part' (29.88%) and 'organelle' (21.38%) functions. moreover, 'metabolic processes' (27.75%) and 'cellular process' (27.29%) were among the most highly represented groups under biological functions category. this might be indicating the analyzed tissues were undergoing rapid growth and extensive metabolic activities. genes involved in other important biological processes such as biological regulation (6.59%), regulation of biological process (6.27%) and response to stimulus (5.83%) were also identified ( figure 2 ). in summary, these terms account for a large fraction of the overall assignments in l. aurea transcriptomic dataset. understandably, genes encoding these functions may be more conserved across different species and are thus easier to annotate in the database. assignments of cog were used to predict and classify possible functions of the unique sequences. based on sequence homology, 2,142 unique sequences had a cog functional classification. these sequences were classified into 23 cog categories (figure 3 ). 'translation, ribosomal structure and biogenesis' represented the most common category (426, 19.89%), followed by 'posttranslational modification, protein turnover, chaperones' (362, 16.90%) and 'general function prediction only' (254, 11.86%). 'cell motility' (1, 0.05%), 'defense mechanisms' (2, 0.09%) and 'cell wall/membrane/envelope biogenesis' (7, 0.93%) were the smallest cog categories. besides go analysis, kegg [74] pathway mapping based on enzyme commission numbers for assignments was also carried out for the assembled sequences, which is an alternative approach to categorize genes functions with the emphasis on biochemical pathways. ortholog assignment and mapping of the contigs and singletons to the biological pathways were performed using kegg automatic annotation server (kaas). according to the kegg results, 21,274 l. aurea unigenes comprising 7,097 contigs and 14,177 singletons were mapped onto a total of 295 predicted metabolic pathways, representing compound biosynthesis, degradation, utilization and metabolism (table s3) . it also assigned ec numbers for 3,222 contigs and singletons, and they were mapped to respective pathways. transcripts identified as related to the following global map or cellular processes were the most abundant: metabolic pathways (6,048 unigenes), biosynthesis of secondary metabolites (2,606), ribosome (1,444), microbial metabolism in diverse environments (1,305) and protein processing in endoplasmic reticulum (793). the largest category was metabolism (13,923) which included carbohydrate metabolism (3,541), energy metabolism (2,289), amino acid metabolism (2,044), lipid metabolism (1,647), nucleotide metabolism (875), metabolism of cofactors and vitamins (659), biosynthesis of other secondary metabolites (625) and other subcategories (figure 4 ). in the secondary metabolism category, the most represented subcategories were phenylpropanoid biosynthesis (226), terpenoid backbone biosynthesis (161), tropane, piperidine and pyridine alkaloid biosynthesis (112), metabolism of xenobiotics by cytochrome p450 (102), carotenoid biosynthesis (99), limonene and pinene degradation (96), flavonoid biosynthesis (84), stilbenoid, diarylheptanoid and gingerol biosynthesis (76) , and chloroalkane and chloroalkene degradation (69) was also classified. in addition to metabolism pathways, genetic information processing genes (6,850) were highly represented categories. transcription, sorting and degradation, replication and repair, folding, and translation were included in these categories. kegg pathway analysis and cog analysis are helpful for predicting potential genes and their functions at a whole transcriptome level. the predicted metabolic pathways, together with the cog analysis, are useful for further investigations of gene function in future studies. the transcriptome of l. aurea was primarily examined to identify a wide range of candidate genes that might be functionally associated with amaryllidaceae alkaloids biosynthesis. since the isolation of the first alkaloid, lycorine, from narcissus pseudonarcissus in 1877, substantial progress has been made in examining the amaryllidaceae plants, although they still remain a relatively untapped phytochemical source. at present, over 100 alkaloids have been isolated from different amaryllidaceae plants [75] , although their structures vary considerably, these alkaloids are considered to be biogenetically related. mainly, the large numbers of structurally diverse amaryllidaceae alkaloids are classified into 9 skeleton types, for which the representative alkaloids are: norbelladine, lycorine, homolycorine, crinine, haemanthamine, arciclasine, tazettine, montanine and galanthamine. most of the biosynthetic research done on amaryllidaceae alkaloids was carried out in 1960s and 1970s. since then, studies have been reported that the biosynthesis of amaryllidaceae alkaloids belongs to different ring type subgroups [75] [76] [77] [78] . and the noteworthy study could be the biosynthesis of galanthamine and related alkaloids [76] . for example, it has been considered that l-phenylalanine (l-phe) and l-tyrosine (l-tyr) would be the precursors of amaryllidaceae alkaloids biosynthesis. although lphe and l-tyr are closely related in chemical structure, they are not interchangeable in plants. the presence of the enzyme phenylalanine ammonia-lyase (pal) has been demonstrated in amaryllidaceae plants [68, 79] and the elimination of ammonia mediated by this enzyme is known to occur in an antiperiplanar manner to give trans-cinnamic acid, with loss of the b-pro-s hydrogen [80] . besides, it has been proposed that amaryllidaceae alkaloids could be regarded as derivatives of the key intermediate 49-o-methylnorbelladine [77] . there are three different groups of amaryllidaceae alkaloids that are biosynthesized by three modes of intramolecular oxidative phenol coupling (para-para9, ortho-para9 and para-para9) [75, 76, 78] . moreover, plant cytochromes p450 (p450s), as one of the biggest gene superfamilies in plant genomes, might also be involved in the amaryllidaceae alkaloids biosynthesis. it has been well-known that p450s catalyze a wide variety of monooxygenation/hydroxylation reactions in primary and secondary metabolism. they participate in a variety of biochemical pathways to produce primary and secondary metabolites such as phenylpropanoids, alkaloids, terpenoids, lipids, cyanogenic glycosides, and glucosinolates, as well as plant hormones [81] [82] [83] [84] . for example, in some kinds of plants, several p450s in the cyp80 and cyp719 families, known to catalyze reactions (such as c-o and c-c phenol-coupling reaction) atypical for p450s, function in benzylisoquinoline alkaloids (bias) biosynthesis [85] [86] [87] [88] . although little is known about the relationship between p450s and amaryllidaceae alkaloids biosynthesis, it could also be postulated that p450s might catalyze the stereospecific reactions in some steps of amaryllidaceae alkaloids biosynthesis pathways. additionally, omethyltransferase (omt) acts as an important enzyme could also have participated in the galanthamine biosynthesis [76] . according to our sequence analysis and published literatures, many genes might be involved in amaryllidaceae alkaloids synthesis, including phenylalanine ammonia-lyase (pal), tyrosine decarboxylase (tydc), omt, p450s, n-methyltransferase (nmt), and other potential candidates were identified (table 2) . for example, 26 unique sequences were identified as pal1, pal2, and pal3 with similarities ranging from 62%,100%, respectively. 91 unique sequences were identified as omt with similarities ranging from 51%,100%, respectively. additionally, only 6 unique sequences were identified as tydc with similarities ranging from 55%,88%, respectively. to the best of our knowledge, these putative tydc genes are first reported in l. aurea. ssrs, or microsatellites, are neutral molecular markers that widely distribute in a genome. they consist of repeated core sequences of 2,6 base pairs in length. among the various molecular markers, ssrs have been proven to be an efficient tool for performing qtl analysis, constructing genetic linkage and evaluating the level of genetic variation in a species because of the high diversity, abundance, neutrality and co-dominance of microsatellite dna [31] [32] [33] . in total, 9,740 ssrs were obtained from the transcriptomic dataset. of these, the most frequent repeat motifs were trinucleotides, which accounted for 68.37% of all ssrs, followed by di-nucleotide repeats (19.83%), tetranucleotides (6.98%), pentanucleotides (2.77%), and hexanucleotides (2.05%) ( figure 5 ). based on the distribution of ssr motifs, (ga/ag) n , (ct/tc) n and (ca/ac) n were the three predominant types among the dinucleotide repeats motifs, with frequencies of 31.12%, 27.76% and 15.12%, respectively. in the 20 types of tri-nucleotide repeats, ctt (19.39%) was the most common motif, followed by aag (13.47%), gat (8.50%) and atc (7.94%). to date, only a few microsatellites have been available for l. aurea from ncbi. thus, the development of ssrs for this species is highly desirable. snps were identified from alignments of multiple sequences used for contig assembly. by excluding those that had mutation frequency of bases lower than 1%, we obtained a total of 55,800 snps, of which 5,160 were putative indels (in), 32,440 were putative transitions (ts) and 18,220 were putative transversions (tv), giving a mean in: ts: tv ratio of 1:6.29:3.53 across the transcriptome of l. aurea ( figure 6 ). the ag/ga, ct/ tc and at/ta snp types were the most common. in contrast, gc/cg types were the smallest snp types because of the differences in the base structure and the number of hydrogen bonds between different bases. multiple sequence alignment also identified a total of 5,160 indels across the transcriptome. it should be treated with caution because of technical problems associated with roche 454 gs flx pyrosequencing [42] . in this study, de novo transcriptome sequencing for l. aurea using the 454 gs flx was performed for the first time. a total of 937,990 high-quality transcriptomic reads were obtained, giving rise to an average of 329 bp per read. a significant number of putative metabolic pathways and functions associated with the unique sequences were identified. moreover, a large number of snps and ssrs were predicted and can be used for subsequent marker development, genetic linkage and qtl analysis. many candidate genes that are potentially involved in amaryllidaceae alkaloids synthesis were identified for the first time and are worthy of further investigation. our study provides the largest number of ests to date and lays the initial groundwork for indepth, functional transcriptomic profiling of l. aurea. l. aurea used in this study were collected from institute of botany, jiangsu province & chinese academy of sciences, nanjing, china. in order to achieve l. aurea transcriptome, samples were collected from a variety of adult organs and tissues, including the stem, flowers, and leaves. the stem and flowers collected for the rna extraction were at their bud, blooming, and wilting stages respectively. for the leaves collection, the seedlings grown in illuminating incubator (25 6 1uc ; 14/10 h photoperiod) were treated with 500 mm sodium nitroprusside (snp), 250 mm salicylic acid (sa), or 100 mm methyl jasmonate (meja) for 1, 6, 12, 24, and 48 h. at above indicated time point of treatment, the samples were harvested. all of the samples were immediately frozen in liquid nitrogen and stored at -80uc until use. total rna was extracted from these materials using trizol reagent (invitrogen, usa) according to the manufacturer's instructions. the quality of total rna was determined using a nanodrop spectrophotometer (thermo, usa) and rna samples with a 260 of 280 ratio from 1.9 to 2.1were selected for the next analysis. after that, equal quantities of total rna from each sample (,0.35 mg total rna) were mixed together and delivered it to shanghai majorbio bio-pharm biotechnology co., ltd. (shanghai, china) for the construction of the cdna library. the cdna library was constructed using the creator tm smart tm cdna library construction kit (clontech laboratories inc., mountain view, ca, usa) and following the manufacturer's protocol step-by-step. with agarose gel electrophoresis and extraction of dna from gels, dna bands (500,800 bp) were purified, blunt ended followed by ligation with adapters and finally immobilized on beads. the quality control of a double dna library was performed using high sensitivity chip (agilent technologies). the concentration and the proper ligation of the adapters were examined by using tbs 380 fluorometer. after the examination, one-plate, whole-run sequencing was performed on roche 454 gs flx titanium chemistry (roche diagnostics, indianapolis, in, usa) by shanghai majorbio bio-pharm biotechnology co., ltd. following the manufacturer's protocol. the initial assembly comprised 937,990 reads. for each sequence, low-quality bases and the sequencing adapter were trimmed using lucy (http://lucy.sourceforge.net/) and seq-clean (http://compbio.dfci.harvard.edu). the remained sequencing reads were assembled using the newbler software package (a de novo sequence assembly software) with the ''extend low depth overlaps'' parameter. all of the ests from the roche 454 were used to run the final assembly of l. aurea. blastx searches [89] of the genbank nr database hosted by ncbi (http://www.ncbi.nlm.nih.gov/) were performed on all unique sequences to identify the putative mrna functions. additionally, go terms (http://www.geneontology.org) were extracted from the best hits obtained from the blastx against the nr database using blast2go. these results were then sorted by go categories using in-house perl scripts. blastx was also used to align unique sequences to the swiss-prot database (http://web. expasy.org/docs/swiss-prot_guideline.html), kyoto encyclopedia of genes and genomes (kegg) and clusters of orthologous groups (cog) (http://www.ncbi.nlm.nih.gov/cog/) (with the e-value of 10 26 ) to predict possible functional classifications and molecular pathways [90, 91] . the unique sequences were screened for microsatellites using software mreps (http://bioinfo.lifl.fr/mreps/) with default parameters. perfect di-, tri-, tetra-, penta-, and hexa-nucleotide motifs were detected, and all ssr types required a minimum of 6 repeats. potential snps were extracted using varscan (http://varscan. sourceforge.net) with the default parameter only when both alleles were detected from 454 reads. since no reference sequences were available, snps were identified as superimposed nucleotide peaks where two or more reads contained polymorphisms at the variant allele. the roche 454 reads of l. aurea were submitted to ncbi sequence read archive under the accession number of srp018374. synopsis of the genus lycoris (amaryllidaceae) phylogenetic relationships and possible hybrid origin of lycoris species (amaryllidaceae) revealed by its sequences karyotypes of six populations of lycoris radiata and discovery of the tetraploid amaryllidaceae and sceletium alkaloids amaryllidaceae and sceletium alkaloids amaryllidaceae and sceletium alkaloids ethanol extract of lycoris radiata induces cell death in b16f10 melanoma via p38-mediated ap-1 activation effects of lycorine on hl-60 cells via arresting cell cycle and inducing apoptosis apoptosis induced by lycorine in km3 cells is associated with the g0/g1 cell cycle arrest lycorine induces apoptosis and down-regulation of mcl-1 in human leukemia cells identification of natural compounds with antiviral activities against sars-associated coronavirus physicochemical methods for the analysis of galanthamine (review) the pharmacology of galanthamine and its analogues pharmacological evaluation of novel alzheimer's disease therapeutics: acetylcholinesterase inhibitors related to galanthamine plants used in chinese and indian traditional medicine for improvement of memory and cognitive function galantamine for alzheimer's disease phenol oxidation and biosynthesis. part v. the synthesis of galanthamine the total synthesis of (6)-lycoramine. part i the total synthesis of (6)-lycoramine. part ii total synthesis of dl-lycoramine general methods for alkaloid synthesis. total synthesis of racemic lycoramine total synthesis of racemic lycoramine a short stereospecific synthesis of (dl)-lycoramine. control of relative stereochemistry by dipole effects oxidative intramolecular phenolic coupling reaction induced by a hypervalent iodine (iii) reagent: leading to galanthamine-type amaryllidaceae alkaloids an efficient total synthesis of (6)-lycoramine transcriptome characterization and high throughput ssrs and snps discovery in cucurbita pepo (cucurbitaceae) generation and analysis of expressed sequence tags from the tender shoots cdna library of tea plant (camellia sinensis) analysis of expressed sequence tags in apomictic guineagrass (panicum maximum) the molecular ecologist's guide to expressed sequence tags the first set of est resource for gene discovery and marker development in pigeonpea (cajanus cajan l.) exploiting est databases for the development and characterization of gene-derived ssr-markers in barley (hordeum vulgare l.) development of est-ssrs in cucumis sativus from sequence database analysis of expressed sequence tags from grapevine flower and fruit and development of simple sequence repeat markers de novo assembly of chickpea transcriptome using short reads for gene discovery and marker identification melogen: an est database for melon functional genomics a populus est resource for plant functional genomics generation and analysis of expressed sequence tags from six developing xylem libraries in pinus radiata d. don sequencing technologies-the next generation evaluating characteristics of de novo assembly software on 454 transcriptome data: a simulation approach transcriptome analysis of sarracenia, an insectivorous plant transcriptomic signatures of ash (fraxinus spp.) phloem transcriptome sequencing in an ecologically important tree species: assembly, annotation, and marker discovery sampling the arabidopsis transcriptome with massively parallel pyrosequencing global characterization of artemisia annua glandular trichome transcriptome using 454 pyrosequencing transcriptome sequencing and comparative analysis of cucumber flowers with different sex types sequencing medicago truncatula expressed sequenced tags using 454 life sciences technology deep sampling of the palomero maize transcriptome by a high throughput strategy of pyrosequencing sequencing put to the test using the complex genome of barley de novo assembly and transcriptome analysis of five major tissues of jatropha curcas l. using gs flx titanium platform of 454 pyrosequencing de novo sequencing and characterization of the floral transcriptome of dendrocalamus latiflorus (poaceae: bambusoideae) rapid transcriptome characterization for a nonmodel organism using 454 pyrosequencing deep sequencing of the camellia sinensis transcriptome revealed candidate genes for major metabolic pathways of tea-specific compounds analysis of the pythium ultimum transcriptome using sanger and pyrosequencing approaches growth and photosynthetic responses of three lycoris species to levels of irradiance alkaloids from the bulbs of lycoris aurea extracts of lycoris aurea induce apoptosis in murine sarcoma s180 cells photosynthetic characteristics of lycoris aurea and monthly dynamics of alkaloid contents in its bulbs a new chromosome number and karyotype in l. radiata variation and evolution in the karyotype of lycoris, amaryllidaceae. iv. intraspecific variation in the karyotype of l. radiata (l'herit.) herb. and the origin of this triploid species natural variation in petal color in lycoris longituba revealed by anthocyanin components polymorphic microsatellite loci for the genetic analysis of lycoris radiata (amaryllidaceae) and cross-amplification in other congeneric species genome differentiation in lycoris species (amaryllidaceae) identified by genomic in situ hybridization genetic variations in the chloroplast genome and phylogenetic clustering of lycoris species analysis of floral transcription factors from lycoris longituba improved production of galanthamine and related alkaloids by methyl jasmonate in narcissus confusus shoot-clumps effects of ethylene on somatic embryogenesis and galanthamine content in leucojum aestivum l. cultures effect of abiotic and biotic elicitors on growth and alkaloid accumulation of lycoris chinensis seedlings molecular cloning and characterization of a phenylalanine ammonia-lyase gene (lrpal) from lycoris radiata gene discovery from jatropha curcas by sequencing of ests from normalized and full-length enriched cdna library from developing seeds genome sequencing in microfabricated high-density picolitre reactors comparing de novo assemblers for 454 transcriptome data gene ontology: tool for the unification of biology. the gene ontology consortium blast2go: a comprehensive suite for functional analysis in plant genomics kegg: kyoto encyclopedia of genes and genomes chemistry, biology, and medicinal potential of narciclasine and its congeners biosynthesis of the amaryllidaceae alkaloid galanthamine phenol oxidation and biosynthesis. part vi. the biogenesis of amaryllidaceae alkaloids the biosynthesis of plant alkaloids and nitrogenous microbial metabolites biogenesis of the amaryllidaceae alkaloids. ii. studies with whole plants, floral primordia and cell free extracts studies of enzyme-mediated reactions. part i. syntheses of deuterium-or tritium-labelled (3s)-and (3r)-phenylalanines: stereochemical course of the elimination catalysed by l-phenylalanine ammonia-lyase molecular-genetic analysis of plant cytochrome p450-dependent monooxygenases comparison of cytochrome p450 genes from six plant genomes functional genomics of p450s diversification of p450 genes during land plant evolution molecular cloning and characterization of cyp719, a methylenedioxy bridgeforming enzyme that belongs to a novel p450 family, from cultured coptis japonica cells molecular cloning and characterization of cyp80g2, a cytochrome p450 that catalyzes an intramolecular c-c phenol coupling of (s)-reticuline in magnoflorine biosynthesis, from cultured coptis japonica cells molecular cloning and heterologous expression of a cdna encoding berbamunine synthase, a c-o phenol-coupling cytochrome p450 from the higher plant berberis stolonifera cyp719b1 is salutaridine synthase, the c-c phenol-coupling enzyme of morphine biosynthesis in opium poppy gapped blast and psi-blast: a new generation of protein database search programs from genomics to chemical genomics: new developments in kegg kegg for linking genomes to life and the environment we thank dr. zhengzhi zhang (south dakota state university, south dakota, united states of america) for his kindly help in writing this manuscript. we thank yan cheng (shanghai majorbio bio-pharm biotechnology co., ltd.) for her kindly help in sequencing and bioinformatics analysis. key: cord-270683-982eqtog authors: pavel, shaikh terkis islam; yetiskin, hazel; aydin, gunsu; holyavkin, can; uygut, muhammet ali; dursun, zehra bestepe; celik, i̇lhami; cevik, ceren; ozdarendeli, aykut title: isolation and characterization of severe acute respiratory syndrome coronavirus 2 in turkey date: 2020-09-16 journal: plos one doi: 10.1371/journal.pone.0238614 sha: doc_id: 270683 cord_uid: 982eqtog coronavirus disease 2019 (covid-19) caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and associated with severe respiratory illness emerged in wuhan, china, in late 2019. the virus has been able to spread promptly across all continents in the world. the current pandemic has posed a great threat to public health concern and safety. currently, there are no specific treatments or licensed vaccines available for covid-19. we isolated sars-cov-2 from the nasopharyngeal sample of a patient in turkey with confirmed covid-19. we determined that the vero e6 and ma-104 cell lines are suitable for supporting sars-cov-2 that supports viral replication, development of cytopathic effect (cpe) and subsequent cell death. phylogenetic analyses of the whole genome sequences showed that the hcov-19/turkey/eragem-001/2020 strain clustered with the strains primarily from australia, canada, england, iran and kuwait and that the cases in the nearby clusters were reported to have travel history to iran and to share the common unique nucleotide substitutions. coronaviruses (covs) are members of the family coronaviridae, which consists of a group of enveloped, positive-sense, single-stranded rna viruses [1] . transcription of coronaviruses requires a polymerase template switch, characterized as a discontinuous process unique among rna viruses [2] [3] [4] . based on the difference in protein sequences, covs are classified into four genera, alpha-cov, beta-cov, gamma-cov, and delta-cov2 [1, 2, 5] . there are hundreds of coronaviruses are circulating broadly among mammals and birds that cause respiratory, enteric, hepatic, and neurologic diseases [1, 6, 7] . until recently, six coronavirus species have been known to cause disease in humans. the 229e, oc43, nl63 and hku1 viruses are prevalent and cause mild illness, such as the common cold [1, 8] . however, the other two viruses have been considered highly pathogenic in humans, and cause the diseases sars (severe acute respiratory syndrome), which resulted from an outbreak in 2002 and disappeared by 2004, and mers (middle east respiratory syndrome), which emerged in 2012 and continues to circulate in the middle east [9] [10] [11] [12] [13] . at the end of 2019, severe pneumonia cases of unknown etiology were reported in wuhan, a city in the hubei province of china [14] [15] . sequencing analysis revealed that this unidentified pneumonia was considered to be caused by a novel coronavirus [14, 16] . the world health organization (who) termed the disease as coronavirus disease-2019 (covid-19) on february 11, 2020 [17] . on the same day, the international committee on taxonomy of viruses (ictv) named this novel coronavirus as severe acute respiratory syndrome coronavirus 2 (sars-cov-2). sars-cov-2 has become the seventh coronavirus that known to infect humans. even though the first cases had a contact history with the huanan seafood market the studies have clearly showed that sars-cov-2 can be transmitted by person-to-person and frequently cause asymptomatic infections. with transmission of the virus possible before the onset of clinical signs, the covid-19 outbreak has quickly expanded to worldwide [18] [19] [20] . it was declared a pandemic by the who on march 11, 2020. as of august 18, 2020, a total of 21,756,357 confirmed cases of covid-19 and 771,635 deaths have been reported in more than 200 countries and territories. (https://www.who.int/emergencies/diseases/novelcoronavirus-2019/situation-reports/). the first case of covid-19 in turkey was confirmed on march 112020. as of august 18, 2020, there have been 251,805 cases and 6,016 deaths (the ministry of health, turkey). in this study, we report the isolation of the hcov-19/turkey/eragem-001/2020 strain from a patient in turkey with confirmed covid-19. the whole genomic sequence and replication characteristics of the hcov-19/turkey/eragem-001/2020 strain are described. this is the first known report of the isolation and characterization of sars-cov-2 from a human clinical sample in turkey. the successful isolation and characterization of the virus will be essential for continued investigations of sars-cov-2 pathogenicity and will provide valuable information for vaccine design and drug target. this study protocol was approved by the kayseri training and research hospital ethics committee (2020-3-/23), which allowed sampling for diagnostic and surveillance purposes. a written informed consent was obtained from the patient for being included in the study. all cell lines used in this study purchased from atcc cell culture company. african monkey green kidney cells (vero e6, atcc crl-1586), rhesus monkey kidney cells (ma-104, atcc crl-2378), human adrenal carcinoma cells (sw-13, atcc ccl-105) and human cervical adenocarcinoma cells (hela, atcc ccl-2) were cultured in dulbecco's modified eagle's medium (dmem) (sigma, germany) supplemented with 10% heat-inactivated fetal bovine serum (fbs) (gibco, usa), 100 mm l-glutamine, 100 u/ml penicillin, 100 μg/ml streptomycin (biological industries, usa). all cell lines tested were found to be free of mycoplasma using the ez-pcr mycoplasma detection kit (biological industries, usa). a patient was admitted to the kayseri city training and research hospital on march 17, 2020 due to respiratory symptoms. the patient's nasopharyngeal sample was obtained by using a utm™ kit containing 1 ml of viral transport media (copan diagnostics, usa) on day 4 of his illness. the diluted sample was inoculated onto monolayers of vero e6 cells and gently agitated at 37˚c for 1 h. consequently, dmem with 2% fbs was added and the infected cells were monitored for the appearance of cytopathic effect (cpe). all handling of the virus was conducted in a biosafety level 3 enhanced facility (bsl-3). viral rna was isolated from 140 μl of the infected culture supernatant using the qiaamp viral rna mini kit (qiagen, germany) according to the manufacturer's recommendations. the viral rna was reverse transcribed using the moloney murine leukemia virus reverse transcriptase (m-mlv rt) (thermo scientific, usa) using random hexamers according to the manufacturer's recommendations. the reaction mixtures were incubated for 60 min at 42˚c, and the reaction was stopped by heating the mixture at 95˚c for 5 min and chilling it on ice. the primers used in pcr reactions were designed according to the sequences published by the centers for disease control and prevention (cdc) [21] . the pcr was conducted in a 50 μl reaction mixture containing 3 μl of cdna template, 10 mm tris-hcl, 50 mm kcl, 1.5 mm mgcl 2 , 2019-ncov_n1 forward primer (5 0 -gaccccaaaatcagcgaaat-3 0 ), 2019-ncov_n3 reverse primer (5 0 -tgtagcacg att gcagcattg -3 0 ), 1 u of taq polymerase (thermo scientific, usa), and 1.25 mm dntps. the cycling conditions were 94˚c for 3 min, followed by 35 cycles of 94˚c for 45 sec, 55˚c for 45 sec, and 72˚c for 1 min with a final extension at 72˚c for 10 min. the pcr products were visualized by ethidium bromide staining after 1% agarose gel electrophoresis. the pcr reactions were also set up with two different combinations of the primers under the same conditions as described above. the primers used in the pcr reactions were 2019-ncov_n1 forward primer (5 0 -gaccccaaaatcagc gaaat-3 0 ), 2019-ncov_n2 reverse primer (5'-gcgcgacattccgaagaa-3') and 2019-ncov_n3 forward primer (5'-gggagccttgaatacaccaaaa-3'), and 2019-ncov n2 reverse primer (5'-gcg cgacattccgaagaa-3'), respectively. twenty-four-well plates were seeded with vero e6 cells and incubated at 37˚c with 5% co 2 . the monolayer was inoculated with 10-fold serially dilutions of the virus. after incubation for 1 h at 37˚c with shaking, the monolayer was overlaid with 0.5 ml overlay medium containing 0.3% low melting agarose (sigma, germany). after incubation at 37˚c for 3 days, the cells were fixed with 10% formalin (v/v) for 90 min at room temperature. the agarose overlay was discarded, and the plaques were visualized by staining the monolayer with 1% crystal violet (w/v) in 20% ethanol (v/v). we cultured to sars-cov-2 passage 1 (1:100 dilution) in vero e6 cells to make virus the passage 2 virus stock. the sars-cov-2 virus lysate was then harvested at 48 h post-infection and the supernatants were collected, clarified, and stored at -80˚c. to determine the titer of the passage 2 virus a focus forming assay (ffa) was performed as described previously [22] . briefly, vero e6 cells were seeded on 96 well-plates and incubated at 37˚c with 5% co 2 for overnight. the cell monolayers were inoculated with 10-fold serial dilutions virus at 2nd passage. the diluted samples were added in triplicate to confluent vero cell monolayers. after absorption for 1 h at 37˚c, the supernatants were removed and the cells were washed with pbs. the cell monolayers were overlaid with virus medium containing 1% cmc (carboxymethyl cellulose) then incubated at 37˚c with 5% co 2 for 72 h. after fixation with 10% neutral buffered formaldehyde at room temperature for 20 min, the cells were permeabilized with 0.1% triton x-100 in pbs for 20 min with gentle rocking and blocked with 5% skim milk in pbs. the wells were then incubated with a human antibody to sars-cov-2 nucleocapsid protein (1:2500) (genscript; hc2003) for 1h in tbst (100 mm tris-hcl ph 8.0, 1.5 m nacl, 1% tween 20) at 37˚c and washed 3 times with tbst. the cells were incubated for 1 h with goat anti-human igg conjugated to fluorescein isothiocyanate (fitch) (southern biotech, usa) and diluted 1:1000 in tbst and then washed three times with tbst and once with distilled water. the antibody-labeled cells were detected and analyzed by immunofluorescence microscopy (leica, uk). the fluorescent foci in each well were counted, and the virus titers were calculated and expressed as fluorescent focus units (ffu) per ml as described previously [22] . to obtain the virus passage 3 virus stock, the vero e6 cells were infected with the virus passage 2 virus at an moi of 0.01, and the viral lysate were was harvested at 48 h post-infection and the supernatants were collected. subsequently, the virus passage 4 virus was generated in vero e-6 cells infected with virus passage 3 virus at an moi of 0.01. cell lysates were harvested in laemmli sodium dodecyl sulfate-polyacrylamide (sds) gel electrophoresis sample buffer containing 2% sds and 5% β-mercaptoethanol. the lysates were boiled and loaded onto a polyacrylamide gel. the samples were separated on 12% resolving and 5% stacking sds-page gels in a mini electrophoresis unit (bio-rad, usa) at 100 v for 1 h. the proteins were transferred onto a nitrocellulose membrane (millipore, usa) under wet conditions using a trans-blot apparatus (bio-rad, usa). after blocking with 5% skimmed milk, the membrane was incubated either with a rabbit polyclonal to sars-cov-2 spike glycoprotein (1/1000) (abcam; ab272504) or a human antibody to the sars-cov-2 nucleocapsid protein (1:2500) (genscript; hc2003) followed by a goat anti-rabbit horseradish peroxidase (hrp)-conjugated antibody (1:2000 dilution, invitrogen; usa) and a goat anti-human horseradish peroxidase (hrp)-conjugated antibody (1:2000 dilution, invitrogen; usa), respectively. b-actin was used as a loading control in western blot. the membrane was reacted with the ecl substrate solution (pierce ecl, usa). the membrane was exposed to an autoradiograph film (kodak x-omat, sigma germany), and was developed using a kodak developer (x-omat 1000a, sigma germany). vero e-6 and ma-104 cells cultured in 24-well-plates were infected with an moi at an 0.1 (passage 4 virus). the cultures were harvested by scraping cell monolayers from at different time points (6, 12, 18, 24, 48 and 72 h) and stored at -80˚c. the vero e-6 cells were then inoculated with 10-fold serial dilutions of the samples in triplicate per dilution. the viral inoculum was removed. the cell monolayers were overlaid with virus medium containing 1% cmc. the cells were fixed with 10% neutral buffered formaldehyde after infection of 72 h at room temperature for 20 min, and permeabilized with triton x-100. the wells were then incubated with a human antibodt to the sars-cov-2 nucleocapsid protein (1:2500) (genscript; hc2003) for 1h in tbst at 37˚c and washed 3 times with tbst. the cells were incubated for 1 h with goat anti-human igg conjugated to fluorescein isothiocyanate (fitch) (southern biotech, usa) and diluted 1:1000 in tbst and then washed three times with tbst and once with distilled water. antibody-labeled cells were detected and analyzed by immunofluorescence microscopy (leica, uk). the fluorescent foci in each well were counted, and the virus titers were calculated and expressed as fluorescent focus units per ml. for whole genome sequencing of hcov-19/turkey/eragem-001/2020, vero e6 cells infected with the virus were used for rna extraction. the rna was extracted by using the qiaamp viral rna mini kit (qiagen, germany). the viral rna was reverse transcribed by m-mlv rt using random hexamers according to the manufacturer's recommendations. the 26 dna amplicons the from full genome amplification [23] were quantified using the quant-it dsdna hs assay kit (invitrogen, usa) and pooled in equal concentrations. the libraries were prepared using pooled amplicons with nextera dna flex library prep kit (illumina, san diego, ca) and sequenced on an illumina nextseq 500 (illumina, usa) platform with a 2x150 cycle kit (gen era diagnostics inc., turkey). the quality of the raw data was evaluated by fastqc v.0.11.5 (babraham bioinformatics) and low-quality bases, primers and remnant adapters were trimmed using trimmomatic v.0.32 [24] . the reads were aligned to the previously assembled sequence of the sars-cov-2 genome (genbank accession: mn908947.3) using the burrows-wheeler aligner v.0.7.1 with the mem algorithm [25] . the variants were called by using genome analysis toolkit (gatk) v.3.8.0 with the haplotypecaller algorithm [26] and were manually inspected in genomebrowse v2.1.2 (goldenhelix). the variants that had low quality and a low variant fraction (%<60) were filtered. the filtered variants and reference sars-cov-2 genome were used to generate the consensus sequence using bcftools v1.9 [27] . for multiple sequence alignment, complete (>29,000bp) and high-coverage genomes (n = 3970) were used from the gisaid database. the gisaid strain genomes including the genome of our strain were aligned using the mafft v7.450 tool [28] . phylogenetic analysis of the alignment was performed using the iq-tree v. 1.6.12 with a general time-reversible (gtr) model [29] . the whole genome sequence was submitted to genbank (id:mt327745.1) and gisaid (id: epi_isl_424366) and the raw data deposited on sra (samn15062833). graphpad prism 7 software (graphpad, usa) was used to perform all statistical analysis and graphics. mann-whitney u test was used to find significant differences between viral passages. the significance level was set as a p value of less than 0.05 where � p<0.05. after 24 h of incubation, very little but visible cpe was detected in virus passage 1 virus ( fig 1b) . the time for the onset of cpe was typically 48 h post-infection ( fig 1c) and major cpe was observed within 72 h post-infection ( fig 1d) . the cells showed some morphological changes such as cell rounding, detachment/floating and degeneration whereas no such changes were observed in the uninfected cells (fig 1a, 1b, 1c and 1d) . as we observed cpe in the infected monolayers, rt-pcr was used as a confirmatory assay. the primer set from cdc [21] targeting the nucleocapsid protein gene (np) of sars-cov-2 was used for the pcr reactions. a pcr product size of 469 bp was amplified with the 2019-ncov_n1 forward and 2019-ncov_n3 reverse primers (fig 1e, lane 2) . we amplified two pcr products, with sizes of 945 bp and 549 bp, with the 2019-ncov_n1 forward, and 2019-ncov_n2 reverse primers and with the 2019-ncov_n3 forward and 2019-ncov_n2 reverse primers, as in shown fig 1e lane 3 and fig 1e lane 4 , respectively. we also performed to plaque assay to purify of sars-cov-2 for subsequent use in further experiments. representative sars-cov-2 plaques in the vero e6 cell monolayers infected with sars-cov-2 are shown in fig 1f and 1g . taken together, these results suggest that a sars-cov-2 strain named hcov-19/turkey/eragem-001/2020 was successfully isolated from the nasopharyngeal sample of a patient in turkey with confirmed covid-19. we cultured to sars-cov-2 passage 1 in vero e6 cells to make the virus passage 2 virus stock. subsequently, the passage 2 virus stock was passaged two more times in vero e6 cells. the virus stocks were quantified by using ffa (fig 2) . we determined that the titer of the passage 2 virus was 2.8x10 4 ffu/ml, while the titers of the passage 3 and passage 4 viruses were 4.3x10 5 ffu/ml and 4.9x10 6 ffu/ml, respectively (fig 2) . these results indicated that propagation of the hcov-19/turkey/eragem-001/2020 strain in vero e6 cells led to an increasing in the viral titers in each passage. in addition to vero e6 cells, we examined the susceptibility of ma-104, sw-13 and hela cell lines to infection by sars-cov-2. all cell lines were infected with an moi of 0.1 (virus passage 4 virus) and monitored for cpe until 72 h post-infection. only the ma-104 cell line developed cpe. in abnormal areas, small clusters of rounded cells, cell detachment and degeneration were observed (fig 3) . similar to vero e6 cells infected with sars-cov-2, cpe formation in the ma-infected cells began at 24 h post-infection ( fig 3b) and increased at 48 h post-infection (fig 3c) . the complete cpe was observed within 72 h post-infection (fig 3d) . to confirm the results of the susceptibility of the ma-104, sw-13 and hela cell lines to infection by sars-cov-2, all cell lines were infected with an moi of 0.1 (passage 4 virus) and incubated at 48 h post-infection. an immunofluorescence assay (ifa) confirmed that the vero isolation of sars-cov-2 in turkey e6 and ma-104 cell lines supported the replication of sars-cov-2. in contrast, sars-cov-2 did not replicate in sw-13 and hela cells. (fig 4) . to expand these observations, we examined the expression of the sars-cov-2 proteins. all cell lines were infected with an moi of 0.5 (passage 4 virus). cell lysates from infected cell lines were harvested at 24 h post-infection and were probed either with the rabbit polyclonal antibody to sars-cov-2 spike glycoprotein or with a human antibody to the sars-cov-2 nucleocapsid protein. sars-cov-2 spike protein (s) expression was detected in vero e6 and ma-104 cell lines that supported sars-cov-2 replication (fig 5a) . the vero e6 and ma-104 cell lines also showed a sars-cov-2 nucleoprotein (np) band, as shown in fig 5b. consistent with the ifa results (fig 4) , viral antigen expression was not detectable in the nonsusceptible sw-13 and hela cell lines (fig 5) . overall, these results showed that the vero e6 and ma 104 cell lines can be efficiently infected by sars-cov-2. to assess the replication kinetics, vero e6 and ma-104 cells were infected with at an moi of 0.1, and the supernatants were harvested at different time points (6, 12, 18, 24, 48 and 72 h post-infection). the vero e6 and ma-104 cell monolayers were then inoculated with 10-fold serial dilutions of the samples. viral titers of the samples were determined by ffa (fig 6a) . the growth kinetics study showed that sars-cov-2 replicated rapidly and efficiently and could be detected within 6 h post-infection in vero e6 and ma-104 cells (fig 6a and 6b ). the western blot assay was performed to examine the production of viral proteins using a rabbit polyclonal antibody to the sars-cov-2 spike glycoprotein (s) (1/1000) (abcam; ab272504) and a human antibody to the sars-cov-2 nucleocapsid protein (np) (1:2500) (genscript; hc2003). the membrane was reacted with the ecl substrate solution (pierce ecl, usa). the membrane was exposed to an autoradiograph film (kodak x-omat, sigma germany), and was developed using a kodak developer (x-omat 1000a, sigma germany). the arrows indicate that the bands at approximately 180 kda ( fig 5a) and 48 kda (fig 5b) sars-cov-2 replicated in vero e6 and ma-104 cells with similar kinetics and achieved similar peak titers of 6.1xlog 6 ffu/ml and 1.6xlog 6 ffu/ml at 48 h of post-infection, respectively ( fig 6a) . however, the viral titers decreased after 48 h infection. at 72 h post-infection, the titers from the samples vero e6 and ma-104 infected with sars-cov-2 were 5.4xlog10 5 ffu/ml and 2xlog10 5 ffu/ml, respectively (fig 6a) . the sequencing produced approximately 4.9 m paired reads (150 bp x2), of which 95.2% of the reads were mapped to the reference genome. after alignment, 99.6% of the genome was covered with a 26200x sequencing depth on average. the whole-genome sequencing of hcov-19/turkey/eragem-001/2020 revealed that the strain had 6 variants, compared to the mn908947.3 reference genome. the detected 2 non-synonymous, 3 synonymous, and 1 utr variants are listed in table 1 . the variants were found to correspond to the genomic positions 1397 and 11083 (orf1ab gene), 23876 (s gene), 26688 (np gene), 29563 (orf 10 gene) and 29742 (3' utr) . the mutations at 1397 and 23876 are non-synonymous, leading to a change from valine to isoleucine in genes orf1ab and s (table 1) . the phylogenetic analysis showed that hcov-19/turkey/eragem-001/2020 was located outside of the main clades (s, g, and v) and clustered with sars-cov-2 isolates from australia, canada, england and kuwait (fig 7) . this geographically dispersed cluster is known to be branched in the early period of the epidemic and genetically clustered very closely together. our strain shared three distinct mutations (g1397a, t28688c, and g29742t) with all members of this cluster, and those mutations were not observed in other known clusters. according to published case reports and gisaid metadata entries, at least 9 sars-cov-2 samples in the nearby clusters had a recent travel history to iran [30] . those samples in nearby clades include several cases from pakistan, kuwait, canada, and norway (fig 7) . the genome sequences of all those cases with a history of travel to iran share three nucleotide substitutions (g1397a, t28688c, and g29742t) in the sars-cov-2 genome, which were also found in the hcov-19/ turkey/eragem-001/2020 strain. the gisaid had only one full genome sequence of sars-cov-2 from iran, which also included the two key mutations (g1397a and g29742t). in addition, analysis of the np partial gene sequences of the iranian samples in gisaid showed that all of the iranian partial sequences (n = 15) also contain the t28688c, which is another key single nucleotide polymorphism (snp) of this clade. our strain also contained g23876a, a non-synonymous mutation in the s gene, which was not observed in any other full genome sequences in gisaid. the mutation leads to a val to ile change at the 772nd position, which is located at one of the inner coil motifs of the s protein. another mutation in the orf10 gene (c29563t) is also quite rare and found only in two cases in australian samples. this study generated the second whole genome sequence of a sars-cov-2 strains in turkey. the first sequence was generated in march 2020 and deposited in gisaid (epi_isl_417413). the first sequence was not included in the phylogenetic analysis because of its high number of unique mutations (0.44%). however, the sequence comparison showed that both sequences carry key iranian cluster mutations including g1397a, t28688c, and g29742t. the g11083t, g23876a, and c29563t nucleotide changes in our strain were not found in the first sequenced strain in turkey. as of april 2020, a total of 17 sars-cov-2 sequences have been submitted to gisaid samples from turkey. two of those sequences (turkey/hgsm/5516/2020 and turkey/hgsm/8010/2020) were located in the same cluster and share cluster-specific variants (g1397a, t28688c, g29742t). the rest of the submitted strains clustered with several other european clades. sars-cov-2 is an emerging coronavirus that is highly infectious and efficiently transmitted through droplets and close contact. the virus has been able to spread promptly to several countries throughout the world [31] [32] [33] [34] [35] . the sars-cov-2 pandemic is likely to have serious effects on not only people's health but also societies, health system worldwide and the global isolation of sars-cov-2 in turkey economy [36] [37] [38] . in the current outbreak situation, it is crucial to isolate of the causative virus for vaccine strain production, initial characterization of antiviral candidates and evaluation of diagnostic tools. sars-cov-2 was first isolated by using human airway epithelial cells on january 7, 2020 [14, 39] . following to the first isolation of sars-cov-2 in china, several groups have also isolated sars-cov-2 by using the vero cell line [40] [41] [42] [43] . in this study, we isolated the sars-cov-2 from the nasopharyngeal sample of a patient in turkey with confirmed covid-19. isolation of sars-cov-2 was successfully achieved in vero e6 cells in the absence of trypsin. sars-cov-2 caused morphological changes such as rounding, detachment/floating and degeneration (fig 1b, 1c and 1d ). it is essential to define different target cells for sars-cov-2 for further studies on virushost interactions. chu and colleagues identified different cell lines in which both sars-cov and sars-cov-2 replicated efficiently, but the cytopathic effects were only seen in the nonhuman primate kidney cell lines veroe6 and frhk4 [40] . recently, a study showed that the vero e6 and vero ccl81 cell lines were infected with sars-cov-2 and exhibited to sars--cov-2 specific cpe. these authors found that huh7.0 and 293t cells showed only modest viral replication but no cpe was observed, suggesting that both vero cell types support amplification and replication of sars-cov-2 but that vero e6 cells are more suitable for amplification and quantification [42] . in this study, we assessed the susceptibility of vero e6, ma-104, sw-13 and hela cell lines to infection by sars-cov-2. we determined that the vero e6 and ma-104 cell lines were permissive for sars-cov-2 infection. initial cpe formation in vero e6 and ma-104 cell lines infected with sars-cov-2 was observed as early as at 24 h postinfection (figs 1b and 3b, respectively) . immunoblotting analysis also confirmed that only vero e6 and ma-104 cell lines infected with sars-cov-2 showed the expression of the virus specific proteins expression (fig 5) . in addition to vero e6 cells, the ma104 cell line pertaining to its suitability for sars-cov-2 proliferation and facilitate further study of sars-cov-2. our results are in agreement with the previous reports showing that sars-cov replicated efficiently and caused cpe in vero and ma-104 cell lines [44] [45] [46] [47] . in order to evaluate the viral growth kinetics of sars-cov-2, the vero e6 and ma-104 cell lines were infected with at an moi of 0.1. viral replication was assessed at different time points (6, 12, 18, 24, 48 and 72 h post-infection). we used ffa for all virus titration experiments in this study, since ffa is independent of cell death. sars-cov-2 replicated with a similar kinetics in vero e6 and ma-104 cells, but the ma-104 cells supported replication of sars-cov-2 slightly less well than the vero e6 cells (fig 6a) . viral replication could be detected at 12 h post-infection, and continued to increase gradually, peaking at 48 h post-infection (fig 6a and 6b ). the decline in virus titer at 72 h post-infection which might be due to death of the infected cells or to the cell lysis ( fig 6a) . our data are in agreement with that of harcourt et.al., in which sars-cov-2 replicated rapidly in vero cells after an initial eclipse phase and increased gradually, peaking at 48 h post-infection [42] . lastly, we have compared the whole genome of hcov-19/turkey/eragem-001/2020 with a dataset of 3970 available sars-cov-2 complete genomes from different countries retrieved from gisaid. the hcov-19/turkey/eragem-001/2020 strain was closely clustered with other strains primarily from australia, canada, england, iran, and kuwait. the reason for the high fraction of australian cases in this cluster was possibly due to the high number of submitted sequences, as australia was the third country that had the most sequences submitted to gisaid by april 2020. the cases in the nearby clusters were reported to have a travel history to iran and shared the common unique nucleotide substitutions, g1397a, t28688c, and g29742t, which were also found in our strain and partially in several iranian cases [30] . the common key mutations and similar travel histories of the closely clustered cases may indicate possible links between our case and the iranian epidemic. interestingly, hcov-19/turkey/ eragem-001/2020 has the g23876a mutation on the s gene, which is not found in any other full genome sequences in gisaid, including the australian cluster. the lack of some variants (g11083t, g23876a, and c29563t) in the previously sequenced turkey strains may also show multiple introductions of strains in the early stages of the epidemic. in this regard, the potential limitation of this finding is the limited number of sequences available for analysis. further studies with more sequences are needed to determine the distribution of this variant in turkey and tracing the origin of this strain. we have describe the successful isolation of sars-cov-2 from a patient in turkey with confirmed covid-19. we determined that the vero e6 and ma-104 cell lines might be a good choice of cell culture model for sars-cov-2 that supports viral replication kinetics, development of cpe and subsequent cell death. we also showed that hcov-19/turkey/eragem-001/2020 was closely clustered with other strains primarily from australia, canada, england, iran, and kuwait and that the cases in the nearby clusters were reported to have a travel history to iran and to share the common unique nucleotide substitutions. (docx) fields virology. philadelphia continuous and discontinuous rna synthesis in coronaviruses downstream sequences influence the choice between a naturally occurring noncanonical and closely positioned upstream canonical heptameric fusion motif during bovine coronavirus subgenomic mrna synthesis brian bovine coronavirus 5'-proximal genomic acceptor hotspot for discontinuous transcription is 65 nucleotides wide zoonotic origins of human coronaviruses coronavirus pathogenesis emerging coronaviruses: genome structure, replication, and pathogenesis epidemiology, genetic recombination, and pathogenesis of coronaviruses mers coronaviruses in dromedary camels origin and evolution of pathogenic coronaviruses the sars wake-up call sars and mers: recent insights into emerging coronaviruses middle east respiratory syndrome: emergence of a pathogenic human coronavirus a novel coronavirus from patients with pneumonia in china return of the coronavirus: 2019-ncov gisaid database. 2020 coronavirus the new pneumonia "covid-19 a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study centers for disease control and prevention home page application of the pseudo-plaque assay for detection and titration of crimean-congo hemorrhagic fever virus a new coronavirus associated with human respiratory disease in china trimmomatic: a flexible trimmer for illumina sequence data fast and accurate short read alignment with burrows-wheeler transform a framework for variation discovery and genotyping using next-generation dna sequencing data bcftools/roh: a hidden markov model approach for detecting autozygosity from next-generation sequencing data mafft multiple sequence alignment software version 7: improvements in performance and usability iq-tree: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies an emergent clade of sars-cov-2 linked to returned travellers from iran an interactive web-based dashboard to track covid-19 in real time characteristics and outcomes of 21 critically ill patients with covid-19 in washington state host and infectivity prediction of wuhan 2019 novel coronavirus using deep learning algorithm first case of 2019 novel coronavirus in the united states the novel zoonotic covid-19 pandemic: an expected global health concern economic impacts of wuhan 2019-ncov on china and the world the socio-economic implications of the coronavirus and covid-19 pandemic: a review multidisciplinary research priorities for the covid-19 pandemic: a call for action for mental health science. lancet psychiatry who. novel coronavirus(2019-ncov) situation report-1 virus isolation from the first patient with sars-cov-2 in korea serological and molecular findings during sars-cov-2 infection: the first case study in finland severe acute respiratory syndrome coronavirus 2 from patient with 2019 novel coronavirus disease, united states. emerg infect dis inhibition of sars-cov-2 infections in engineered human tissues using clinical-grade soluble human ace2. cell comparative tropism, replication kinetics, and cell damage profiling of sars-cov-2 and sars-cov and implications for clinical manifestations, transmissibility, and laboratory studies of covid-19: an observational study sars-associated coronavirus replication in cell lines severe acute respiratory syndrome coronavirus envelope protein regulates cell stress response and apoptosis exogenous ace2 expression allows refractory cell lines to support severe acute respiratory syndrome coronavirus replication our gratitude is expressed towards the staff at the infectious diseases ward at the kayseri city training and research hospital for providing us with the fresh material for this study. conceptualization: aykut ozdarendeli. key: cord-286404-eggkqq3b authors: strayer, david r.; young, diane; mitchell, william m. title: effect of disease duration in a randomized phase iii trial of rintatolimod, an immune modulator for myalgic encephalomyelitis/chronic fatigue syndrome date: 2020-10-29 journal: plos one doi: 10.1371/journal.pone.0240403 sha: doc_id: 286404 cord_uid: eggkqq3b background: rintatolimod is a selective tlr3 agonist, which has demonstrated clinical activity for me/cfs in phase ii and phase iii double-blind, placebo-controlled, randomized, multi-site clinical trials. methods and findings: a hypothesis-based post-hoc analysis of the intent to treat (itt) population diagnosed with me/cfs from 12 independent clinical sites of a phase iii trial was performed to evaluate the effect of rintatolimod therapy based on disease duration. the clinical activity of rintatolimod was evaluated by exercise treadmill tolerance (ett) using a modified bruce protocol. the itt population (n = 208) was divided into two subsets of symptom duration. patients with symptom duration of 2–8 years were identified as the target subset (n = 75); the remainder (<2 year plus >8 year) were identified as the non-target subset (n = 133). placebo-adjusted percentage improvements in exercise duration and the vertical rise for the target subset (n = 75) were more than twice that of the itt population. the non-target subset (n = 133) failed to show any clinically significant ett response to rintatolimod when compared to placebo. within the target subset, 51.2% of rintatolimod-treated patients improved their exercise duration by ≥25% (p = 0.003) despite reduced statistical power from division of the original itt population into two subsets. conclusion/significance: analysis of ett from a phase iii trial has identified within the itt population, a subset of me/cfs patients with ≥2 fold increased exercise response to rintatolimod. substantial improvement in physical performance was seen for the majority (51.2%) of these severely debilitated patients who improved exercise duration by ≥25%. this magnitude of exercise improvement was associated with clinically significant enhancements in quality of life. the data indicate that me/cfs patients have a relatively short disease duration window (<8 years) to expect a significant response to rintatolimod under the dosing conditions utilized in this phase iii clinical trial. these results may have direct relevance to the cognitive impairment and fatigue being experienced by patients clinically recovered from covid-19 and free of detectable sars-cov-2. trial registration: clinicaltrials.gov: nct00215800. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 myalgic encephalomyelitis/chronic fatigue syndrome (me/cfs) is a debilitating disorder characterized by an incapacitating fatigue that is not improved by bed rest and is associated with a diverse combination of variable signs and symptoms [1] [2] [3] . a significant majority of patients are female [4] [5] [6] ; this gender predisposition establishes me/cfs as an important women's health issue. the etiologic/pathogenic basis for me/cfs is unknown, although the evidence indicates that it is multifactorial with a variety of microbial, hormonal, and immunological abnormalities linked to its pathogenesis and dependent upon genetic signatures [7] . although controversial, the best available data suggests that patients with me/cfs have a life span reduced by about 18 years due to an apparent early incidence of cancer, heart disease, and suicide [8, 9] . in the amp-516 phase iii clinical trial, patients with severe me/cfs demonstrated significant improvement in the primary endpoint, exercise treadmill tolerance (ett), compared to placebo controls following the twice weekly for 40 weeks systemic administration [12] of the selective tlr3 dsrna agonist, rintatolimod (ampligen 1 ) [10, 11]. a hypothesis based posthoc analysis of ett response in a subset of study patients was performed. this subset (n = 75), selected primarily on baseline me/cfs symptom duration, has revealed �2 fold higher placebo-adjusted rintatolimod improvements compared to the itt population (n = 208). the amp-516 study was a prospective, double-blind, placebo-controlled, randomized, phase iii trial with equal parallel cohorts; it was conducted in accordance with the ethical principles of the delaration of helsinki at 12 sites in the united states to evaluate the safety and efficacy of rintatolimod in me/cfs [12] with patient informed consent and irb approval (irb #1: essex institutional review board, inc., 121 main street, lebanon, new jersey 08833, irb #2: the umdnj-rwjms irb, 97 paterson street, new brunswick, nj 08903, irb #3: dean institutional review board, 2711 allen boulevard, middleton, wi 53562). this pivotal trial enrolled patients meeting both the original holmes cdc 1988 diagnostic criteria [1] and the revised fukuda 1994 cdc case definition [2] . although post-exertional malaise (pem) was not an absolute requirement of the cdc case definitions, the cdc case definitions had pem as a symptom of the disease. in 2015, the institute of medicine (iom) recommended that pem be required for a diagnosis of me/cfs and pem is now widely accepted as a key symptom for me/cfs. a karnofsky performance score (kps) of 40-60 indicating severe debilitation [12] was an inclusion criterion for the clinical trial. the design of the study, including endpoints, was reviewed by the fda prior to its initiation. many of the me/cfs patients were unable to physically perform the standard bruce exercise protocol (s1 table, panel a) commonly used for the evaluation of cardiac function [13] . to insure patient safety, a modified bruce me/cfs protocol (s1 table, panel b) was used, with reduced energy requirements similar to protocols (s1 table, panel c) designed for the debilitated and elderly [14] . the primary endpoint was established as a change in ett from baseline to week 40 of the study [12] . in order to decrease the likelihood of spontaneous remissions, the amp-516 protocol required a diagnosis of me/cfs for �1 year, which was extended to �2 years for performing the post-hoc analysis. to test the hypothesis that patients with a shorter duration of symptoms (illness) would respond better than patients with a longer duration of the disease, the total amp-516 itt population (n = 208) was divided into 2 subsets. because the median duration of symptoms for the entire population was 8.5 years, we selected 8 years as the maximum duration since we wanted the subset with the shorter duration of illness to represent less than 50% of the total population. accordingly, we stratified the subsets based on a 2-8 year duration of symptoms vs <2 and >8 years. in the post-hoc data analysis patients having onset of symptoms between 2 and 8 years, pem lasting more than 24 hours and the ability to walk on a moving treadmill for longer than one minute but less than 16 minutes (target subset, n = 75) were compared with the remainder of the itt population (non-target subset, n = 133). the data analyses employed sas (version 9.2) statistical software (cary, nc). analysis of the raw ett data showed consistency with normality and equality of the variances between treatment groups. therefore, no transformation was performed (skewness was -0.16 for all patients), -0.18 for rintatolimod, and -0.19 for placebo). all statistical analyses were two-sided. exercise treadmill duration and vertical rise were analyzed using the two-sided student's t-test. both the pooled test for equal variances and the satterthwaite test for unequal variances were calculated. if the equality of variance test indicated that there was a significant difference between the two variances, the result from the satterthwaite test was reported. if there was no significant difference, the result from the pooled test was reported. comparison of the proportion of patients who improved ett by at least 25% was analyzed by the chi-squared test. multivariable regression models were used to analyze the possible confounding factors of age, sudden onset, pem, and gender on ett response. study participants were required to undergo ett testing using the modified bruce protocol, which incorporated progressive increases in the treadmill inclination/grade from 0% to 21% in seven separate increments of 3% (s1 table, panel b). the vertical rise component of the ett testing protocol was calculated for each of the inclination stages completed. the last stage attempted, which was only partially completed in most cases, was also included in the calculation based on the percentage of completion. the increase in vertical rise from baseline to week 40 was calculated for each patient and was expressed as vertical feet ascended (vertical rise). the target subset of patients (n = 75) primarily identified as having a 2-8 year duration of symptoms had twice the placebo-adjusted percent increase in ett response of the entire itt population (n = 208). the remainder, identified as the non-target subset (n = 133), failed to show any clinically significant ett response to rintatolimod when compared to placebo. the baseline demographics of the target and non-target amp-516 subsets are shown in table 1 . mean age, gender, sudden onset and kps were well matched between the subsets and the original itt population. however as expected, a significant difference was observed between the two subsets with regard to the duration of me/cfs baseline symptoms (p<0.001). the target subset had a mean duration of me/cfs symptoms of 5.0±1.6 years for the rintatolimod cohort and of 4.9±1.9 years for the placebo cohort; in contradistinction, the non-target subset had mean durations of 12.5±4.8 and 12.0±6.2 years, respectively, for the rintatolimod and the placebo cohorts. the me/cfs patients enrolled into this trial had severe debilitation, with a median kps of 50 (s2 table) , indicating a requirement for considerable daily assistance to care for their daily activities (s2 table) . table 2 illustrates the significant difference between the target and non-target subsets in treadmill endurance. the difference between the rintatolimod and placebo cohorts in the target subset was 122.5 seconds, compared to 30.1 seconds in the non-target subset and 67.5 seconds in the total itt population. the difference in ett duration was statistically significant for the itt population and the target subset. table 2 also reveals the percent increase in intra-group mean exercise duration from baseline to week 40. the placebo-adjusted mean increase seen in the target subset (δ = 23.6%) is twice that seen for the itt population as a whole (δ = 11.8%). the placebo-adjusted mean increase seen in the non-target subset (δ = 4.7%) was not statistically significant. despite effect of me/cfs duration in response to rintatolimod fewer patients and reduced statistical power compared to the itt population (n = 208), the placebo-adjusted mean increase in ett (δ = 23.6) within the target subset (n = 75) was statistically significant. a value of a �25% increase in ett improvement was used as a high-bar element of our target subset analysis. this was based on a request from the fda (division of antiviral drug products) to establish a percent change in the clinical protocol that was above the variability of the intra-patient exercise tolerance at baseline. the percentage of ett responders (i.e., exhibiting �25% improvement in exercise duration) seen in the rintatolimod cohort (39.0%) vs. placebo (23.1%) was statistically significant for the itt population (p = 0.013). the majority of the patients receiving rintatolimod in the target subset (51.2%) were ett responders vs placebo (17.6%) (p = 0.003). in fig 1, the placebo-adjusted difference in percent responders (shown as the δ value below the p-value in the figure) for the target subset is twice that seen for the overall itt population (δ = 33.6% vs δ = 15.9%). there was only a 4.8% difference seen in fig 1 between the rintatolimod and placebo cohorts for the non-target subset, which was not significant (p = 0.54). s1 and s2 figs show each individual patient's disease duration at baseline, along with their corresponding percent improvement in ett from baseline for the target and non-target subsets, respectively, visually showing results in fig 1. multivariable regression models were used to analyze the possible confounding factors of age, sudden onset, pem, and gender on ett response. age, sudden onset, and pem were found not to be confounding factors, while gender was a confounding factor with male patients responding better than females. fortunately, both of the two subsets, the target (2-8 year) and non-target (<2 and >8 year) subsets, as well as the entire itt population, were each well-balanced with no significant gender differences found when comparing their rintatolimod vs. placebo cohorts. within the 2-8 year subset, the response rates for improving ett duration by �25% for patients receiving rintatolimod were 44.8% (13/29) for females (p = 0.035) and 67.7% (8/12) for males (p = 0.057) compared to placebo. the percent of patients with �25% ett improvement was 2.4 fold (females) and 4.7 fold (males) greater for the rintatolimod cohort compared to placebo. there was no significant difference in ett response between the rintatolimod and placebo arms for either the female or male cohorts in the non-target (<2 and >8 year) subset. the ett protocol included progressive increases in treadmill grade from 0% to 21% in seven increments of 3% (s1 table, panel b). the vertical rise in feet was calculated for each patient. table 3 documents the mean change in vertical rise from baseline to week 40 for the itt population and for each subset. the improvement in vertical rise in the itt population was significantly greater (p = 0.033) for the rintatolimod cohort (56.9 feet) when compared to the placebo cohort (22.5 feet). the increase in vertical rise seen for the target subset was 85.2 feet for the rintatolimod cohort vs. 23.3 feet for the placebo cohort. the difference in vertical rise was 61.9 feet and was at the threshold for statistical significance (p = 0.050). nonetheless, the difference in percent increase in intra-group means between the rintatolimod and placebo cohorts seen for the target subset of 70.3% was over twice that seen for the itt population of 28.6%. the increases in rise for the rintatolimod and placebo cohorts in the non-target subset were 37.3 and 22.2 feet, respectively, however, this difference of 15.1 feet between the rintatolimod and placebo cohorts was not statistically significant (p = 0.401). this post-hoc analysis of the successful amp-516 double-blind, placebo-controlled, randomized, phase iii trial has identified a subgroup of patients defined primarily by the length of me/cfs symptoms (2-8 years) with an increased likelihood of a clinically beneficial response to rintatolimod. patients enrolled in the phase iii clinical trial amp-516 met both the original cdc 1988 [1] diagnostic criteria and the revised 1994 cdc case definition [2] . an international consortium proposed in 2011 that myalgic encephalomyelitis was a preferable term for the syndrome complex [3] . in 2015, the institute of medicine (iom) recommended that pem be required for a diagnosis of the disease [15] ; we have incorporated pem as a requirement for the target subset, although only one patient was excluded from the target subset because of a lack of pem. the percentage of patients with pem was well-balanced with no significant effect of me/cfs duration in response to rintatolimod differences seen between the rintatolimod and placebo cohorts within the itt population and within both of the subsets (target and non-target). the cdc 1988 case definition [1] defines "description of the main symptom complex as initially developing over a few hours to a few days" as minor symptom criteria number 11, which is commonly known as "sudden onset". in amp-516, 63.0% of the itt population reported sudden onset of their illness. the proportion of rintatolimod patients improving ett by �25% was statistically similar for those with sudden onset (38.7%) and for those with slow onset (39.5%). the symptom common to all cases of me/cfs is fatigue. for severe me/cfs, the fatigue is debilitating. cardiopulmonary exercise tolerance testing (ett) is an objective measure of efficacy for physical fatigue and is accepted as a regulatory standard for approval of drugs ameliorating exertional fatigue. an improvement of �6.5% was based on prior increases in exercise tolerance recognized by the fda for drugs ameliorating fatigue in non-me/cfs indications. the amp-516 phase iii protocol pre-specified a �6.5% improvement in intra-group mean exercise tolerance as demonstrating efficacy of rintatolimod in me/cfs. a total of five drugs have been approved by the fda based, in part, on improvement in exercise tolerance for indications that include congestive heart failure [16, 17] , chronic angina [18, 19] , and pulmonary hypertension (6 minute walk) [20, 21] . four of the five approved agents provided approximately 6.5% improvement in placebo-adjusted exercise tolerance (fig 2) . rintatolimod elicited an 11.8% improvement for the itt study population for this exercise parameter and was similar in improvement to tracleer (approved for pulmonary hypertension). the target subset within the itt population (with symptoms between 2-8 years) had an increased response to rintatolimod with 23.6% improvement, which is over twice the clinical improvement/quality for the entire rintatolimod treated itt population, 39% of the patients improved ett by �25%; those patients were able to ascend the equivalent of 174.6 more vertical feet at week 40 as compared to baseline. this increase in~175 vertical feet was, on average, accomplished over approximately 6-8 additional minutes on the treadmill at inclinations between 12-21%. the patients who did not improve ett by at least 25% had a mean decrease in vertical rise of 18.3 feet compared to baseline. similar results were seen for the target subset, with 51.2% of these patients improving ett by �25% (181.1 vertical feet). even patients in the non-target subset with �25% ett improvement in the rintatolimod arm were able to ascend about 167 additional vertical feet, similar to the itt population. rintatolimod treatment significantly increased the number of these responders in the itt population (p = 0.013) and in the target subset (p = 0.003) when compared to placebo (fig 1) , demonstrating a substantial reduction in the profound and universal fatigue of severe me/cfs and a corresponding significant improvement in quality of life. although fatigue is a universal symptom which can be quantified by ett, a number of symptoms and signs including cognitive impairment, sleep disturbance, dysautonomia, neuroendocrine abnormalities and pem [22, 23] are observed with variable penetrance in me/cfs. the rintatolimod subset of the itt population exhibiting a �25% improvement in exercise duration also demonstrated a correspondingly significant improvement in two quality of life secondary clinical endpoints, kps and vitality score within the short form-36 (sf-36), when compared to the <25% cohort [24] . the patient self-evaluated vitality score increased from 9.49 at baseline to 24.10 at week 40, a 14.6 point increase (p = 0.008), and almost three times effect of me/cfs duration in response to rintatolimod the minimum clinically important difference (mcid) of 5 points [24] . the vitality score is considered to be one of the best sf-36 subscales for measuring the reduction in function seen in patients with me/cfs [25] . a similar statistically significant improvement was seen with the physician evaluated kps. the median kps increased from 50 to 60 (p = 0.005). a 10-point increase in kps was pre-specified as a clinically significant improvement in the amp-516 protocol reviewed by the fda prior to authorizing the study to proceed. a kps of 50 requires considerable assistance from a caregiver to complete common daily activities (i.e., bathing, dressing, grooming, food preparation, eating, etc.); at a kps of 60, a patient requires occasional assistance (once or twice weekly) for these same daily activities (s2 table) . importantly, the 10 point increase in kps and the 14.6 point increase in vitality score are both clinically relevant, representing objective and decisive improvements in quality of life [24, 26] . over 90,000 doses of rintatolimod have been administered by intravenous infusion across all clinical trials and have been generally well-tolerated. safety data from the two pivotal clinical trials (phase 2 and phase 3) in severely debilitated me/cfs patients with kps �60 (amp-502 and amp-516) show that the number of serious adverse events (saes) associated with rintatolimod is no different than placebo-related saes [7] . the most frequent adverse event is a limited flu-like syndrome (consisting of headache, chills, fever, flushing, and myalgia) that occurs in approximately 44-45% of rintatolimod patients vs. 30-33% of placebo patients. the toll-like receptors (tlrs) act as a first line of defense against microbial pathogens by the induction of innate immunity; they further provide the initial cellular orchestration for the induction of adaptive immune responses to provide specific humoral and cellular immunity mediated in part by inflammatory cytokines [27] . tlrs are abundant in dendritic cells, central to the host adaptive immune response system [28, 29] . all of the tlrs employ an inflammatory myd88-dependent signaling pathway with the exception of tlr3 that utilizes the myd88-independent trif pathway [30] . two other dsrna inducers of gene expression that initiate innate immune responses are the cytosolic helicases, mda5 and rig-1, which activate the inflammatory cytokine-inducing mitochondrial antiviral signaling protein (mavs) [31] . the introduction of a non-paired uridine into the polycytidine chain of rintatolimod as poly i:poly c 12 u creates a mismatched region of thermodynamic instability in the dsrna, restricting the activity of rintatolimod to that of a tlr3 agonist [10, 11] . the importance of this unique property of rintatolimod as a selective tlr3 agonist is a reduction of the systemic inflammatory cytokines [32] that have limited the clinical utility of other tlr3-activating ligands, such as poly i:poly c and viral dsrna, that also activate mda5 and rig-1 [33] . rintatolimod (poly i:poly c 12 u) induction of innate and adaptive immunity is restricted to tlr3 [10, 11, 33] . this restriction of rintatolimod to tlr3 is responsible for the absence of systemic cytokine induction in primates, including humans [32] . of significance to the aberrant immune responses observed in me/cfs [34, 35] is a recent seminal observation in cancer that rintatolimod increases the ratio of teff/treg cells in the tumor microenvironment, in contrast to non-restricted dsrna tlr3 agonists [33] . this provides a relative increase in the killer tcell balance between immune rejection and tolerance. other tlr-activating agonists that employ the systemic inflammatory cytokine-inducing, myd88-dependent or mavs signaling pathways, inextricably engender greater levels of toxicity when compared to rintatolimod. the potential role of chronic stress mediated by post-infection damaged-associated molecular patterns (damps) in genotypes at-risk and its association with chronic hyper-activation of nfκb has been recently reviewed by morris et al. [36] . the selective activation of tlr3 by rintatolimod through the myd88-independent/nfκb minimizing trif pathway is believed to be responsible for its generally well-tolerated clinical safety profile. the presence of damps in me/cfs has been reported by multiple investigators and epigenetic mechanisms in immune response genes play an important role in the development of damps in individual patients post-infection [36] . early in the course of me/cfs there are definite patterns of plasma cytokine activation that are not seen in subjects with longer disease durations [34] . indeed, a higher correlation of these cytokine signatures was found for illness duration than for illness severity. with short illness duration (�3 years) levels of il-1a, il-8, il-12p40, il-17a, and tnfα were significantly higher (p<0.03) when compared to subjects with a longer duration of illness. russell et al. (2016) [37] also studied cytokine expression as a biomarker in me/cfs as a function of disease duration. levels of il-1a were high in adolescent subjects with recent onset of me/cfs and progressively decreased with increased illness duration. similarly, high levels of il-8 in early me/cfs dropped in subjects with the illness for more than 2 years. in contrast, in subjects 18 years and older, low levels of il-6 were found in early me/cfs, while the opposite was seen after more than 2 years of the disease. these results suggest that the immunopathology of me/ cfs is not static, but changes over time. the mechanism for the ett improvement seen in the rintatolimod cohort compared to placebo as a function of baseline disease duration is not known. time-dependent epigenetic-based gene regulation including cytokine expression provides an attractive potential mechanism. genomic polymorphisms [38] affecting expression or function of me/cfs associated genes [39] [40] [41] [42] in a time-dependent manner may be influenced by time-dependent epigenetic mechanisms of dna methylation, micrornas, and transposable elements. dna methylation. cytosine methylation at cpg dinucleotide genomic sites regulates gene expression without disrupting the nucleotide sequence. differential dna methylation in promoter regions has been reported in greater numbers of me/cfs patients versus matched controls. these methylation patterns occurring on gene coding regions as well as promoters and regulatory elements suggest a dysregulation of the immune system in me/cfs [43] . micrornas(mirna). non-coding dna provides the origin of mirnas which serve as 22-nucleotide "sculptors" of gene regulation. although mirnas principally inactivate target mrna, most commonly by binding to the rna 3'utr with resultant target mrna degradation, 5'utr or promoters are infrequent targets. extracellular mirnas act in cell-to-cell communication as chemical messengers [44] . the differential expression reported in several studies in me/cfs patients is possibly limited by the use of differing me/cfs case definitions [45] . the reported differential levels of mirna based on gender in me/cfs [46] may explain our observed gender differences in the magnitude of ett responses to rintatolimod. transposable elements (te). non-coding dna provides the source of repetitive te, which independently replicates and translocates to new locations within the genome. the vast majority are retrotransposons, which replicate by reverse-transcription into dna and are inserted into other loci within the cell genome. the most common repetitive elements are two families denoting long interspersed repetitive element (line) and short interspersed repetitive element (sine). next generation sequencing (ngs) frequently masks sine sequences in wide genome sequencing requiring directed reporting of these repetitive elements. ltr elements are a third type of retrotransposition resembling the integrated form of proviruses. although abundant in number (8% of the human genome), most are defective, restricted to vertical transmission, and are non-infectious, although there are some exceptions. human endogenous retroviruses (herv) transcription has been reported for organ-specific (multiple sclerosis) and systemic (lupus erythematosus) autoimmune mediated diseases. herv expression in human pbmc has been associated with immune responses [47] . external factors, such as infections and stress, have been shown to have an ability to activate te-associated epigenetic control mechanisms. me/cfs is associated with several stressors, including altered cytokine profiles and infections [45] . it is possible that aberrant te activation could also be playing a pathogenic role in me/cfs. rintatolimod is the only drug to have completed successful advanced placebo-controlled clinical trials (phase ii/iii) for me/cfs and is approved for severe me/cfs in argentina. to the best of our knowledge, there are no other drugs with a nda for me/cfs under review at the fda or other international regulatory bodies. rituximab, a monoclonal antibody which binds to cd20 expressed on b-cells, was originally reported in open-label trials [48, 49] and in a small double-blind placebo trial as being active in me/cfs [50] . however, a 152-patient, double-blind, placebo-controlled clinical trial in norway concluded that b-cell depletion using several infusions of rituximab over 12 months was not associated with clinical improvement in patients with me/cfs [51, 52] . significantly, and in contrast to rintatolimod, rituximab was associated with increased drug related serious adverse events compared to placebo. there is increasing creditable anecdotal evidence that patients recovering from covid-19 can develop a me/cfs-like illness [53, 54] . individuals with this condition have been referred to as covid-19 "long-haulers". this post-viral syndrome can be incapacitating with brain fog, fatigue, and difficulty concentrating and is reported to last for many weeks following covid-19 clinical recovery and clearance of sars-cov-2. whether rintatolimod would have any beneficial activity if administered early to patients with this condition needs to be investigated. the subset of patients reported herein represents a population with onset of symptoms between 2-8 years prior to the initiation of therapy for severe me/cfs. the majority of these patients (51.2%) receiving rintatolimod substantially improved physical performance and quality of life. moreover, there is a time-sensitive window for expected rintatolimod efficacy under the conditions employed in the clinical trial. whether non-targets could benefit from a treatment duration longer than 40 weeks, or from rintatolimod combined with other drugs, or whether clinically recovered, sars-cov-2 negative covid-19 debilitated patients with "long hauler" post-viral syndrome would benefit from rintatolimod are topics for further investigation. chronic fatigue syndrome: a working case definition the chronic fatigue syndrome: a comprehensive approach to its definition and study. international chronic fatigue syndrome study group myalgic encephalomyelitis/ chronic fatigue syndrome: clinical working case definition, diagnostic and treatment protocols american myalgic encephalomyelitis and chronic fatigue syndrome society how many people have me/cfs office on women's health efficacy of rintatolimod in the treatment of chronic fatigue syndrome/ myalgic encephalomyelitis (cfs/me) a double-blind, placebo-controlled, randomized, clinical trial of the tlr-3 agonist rintatolimod in severe cases of chronic fatigue syndrome maximal oxygen intake and nomographic assessment of functional aerobic impairment in cardiovascular disease exercise assessment of arthritic and elderly individuals beyond myalgic encephalomyelitis/chronic fatigue syndrome: an iom report on redefining an illness effects of fosinopril on exercise tolerance and clinical deterioration in patients 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agents dendritic cells and the control of immunity myd88-dependent and myd88-independent pathways in synergy, priming, and tolerance between tlr agonists pattern recognition receptors and the innate immune response to viral infection discordant biological and toxicological species responses to tlr3 activation helicase-driven activation of nfκb-cox2 pathway mediates the immunosuppressive component of dsrna-driven inflammation in the human tumor microenvironment distinct plasma immune signatures in me/cfs are present early in the course of illness cytokine signature associated with disease severity in chronic fatigue syndrome patients myalgic encephalomyelitis or chronic fatigue syndrome: how could the illness develop? illness progression in chronic fatigue syndrome: a shifting immune baseline use of single-nucleotide polymorphisms (snps) to distinguish gene expression subtypes of chronic fatigue syndrome/myalgic encephalomyelitis (cfs/me) seven genomic subtypes of chronic fatigue syndrome/myalgic encephalomyelitis: a detailed analysis of gene networks and clinical phenotypes identification of marker genes for differential diagnosis of chronic fatigue syndrome a gene signature for post-infectious chronic fatigue syndrome light kc gene expression alterations at baseline and following moderate exercise in patients with chronic fatigue syndrome and fibromyalgia syndrome identification of myalgic encephalomyelitis/chronic fatigue syndrome-associated dna methylation patterns overview of microrna biogenesis, mechanisms of actions, and circulation epigenetic components of myalgic encephalomyelitis/chronic fatigue syndrome uncover potential transposable element activation unravelling myalgic encephalomyelitis/chronic fatigue syndrome (me/cfs): gender-specific changes in the microrna expression profiling in me/cfs ltr-retrotransposon transcriptome modulation in response to endotoxin-induced stress in pbmcs clinical impact of b-cell depletion with the anti-cd20 antibody rituximab in chronic fatigue syndrome: a preliminary case series b-lymphocyte depletion in myalgic encephalopathy/chronic fatigue syndrome. an open-label phase ii study with rituximab maintenance treatment benefit from b-lymphocyte depletion using the anti-cd20 antibody rituximab in chronic fatigue syndrome. a double-blind and placebo-controlled study myalgic encephalomyelitis/chronic fatigue syndrome: trial fails to confirm earlier observations of rituximab's effectiveness b-lymphocyte depletion in patients with myalgic encephalopathy/chronic fatigue syndrome. a randomized, double-blind, placebo-controlled trial we thank angus shieh, richard chiacchierini, and frank canale for data analyses and biostatistical considerations. w. neal burnette, phd, and charles stratton, md, provided helpful critiques for which we are grateful. key: cord-293946-4bquxdqa authors: huong, nguyen quynh; nga, nguyen thi thanh; long, nguyen van; luu, bach duc; latinne, alice; pruvot, mathieu; phuong, nguyen thanh; quang, le tin vinh; hung, vo van; lan, nguyen thi; hoa, nguyen thi; minh, phan quang; diep, nguyen thi; tung, nguyen; ky, van dang; roberton, scott i.; thuy, hoang bich; long, nguyen van; gilbert, martin; wicker, leanne; mazet, jonna a. k.; johnson, christine kreuder; goldstein, tracey; tremeau-bravard, alex; ontiveros, victoria; joly, damien o.; walzer, chris; fine, amanda e.; olson, sarah h. title: coronavirus testing indicates transmission risk increases along wildlife supply chains for human consumption in viet nam, 2013-2014 date: 2020-08-10 journal: plos one doi: 10.1371/journal.pone.0237129 sha: doc_id: 293946 cord_uid: 4bquxdqa outbreaks of emerging coronaviruses in the past two decades and the current pandemic of a novel coronavirus (sars-cov-2) that emerged in china highlight the importance of this viral family as a zoonotic public health threat. to gain a better understanding of coronavirus presence and diversity in wildlife at wildlife-human interfaces in three southern provinces in viet nam 2013–2014, we used consensus polymerase chain reactions to detect coronavirus sequences. in comparison to previous studies, we observed high proportions of positive samples among field rats (34.0%, 239/702) destined for human consumption and insectivorous bats in guano farms (74.8%, 234/313) adjacent to human dwellings. most notably among field rats, the odds of coronavirus rna detection significantly increased along the supply chain from field rats sold by traders (reference group; 20.7% positivity, 39/188) by a factor of 2.2 for field rats sold in large markets (32.0%, 116/363) and 10.0 for field rats sold and served in restaurants (55.6%, 84/151). coronaviruses were also detected in rodents on the majority of wildlife farms sampled (60.7%, 17/28). these coronaviruses were found in the malayan porcupines (6.0%, 20/331) and bamboo rats (6.3%, 6/96) that are raised on wildlife farms for human consumption as food. we identified six known coronaviruses in bats and rodents, clustered in three coronaviridae genera, including the alpha-, beta-, and gammacoronaviruses. our analysis also suggested either mixing of animal excreta in the environment or interspecies transmission of coronaviruses, as both bat and avian coronaviruses were detected in rodent feces on wildlife farms. the mixing of multiple coronaviruses, and their apparent amplification along the wildlife supply chain into restaurants, suggests maximal risk for end consumers and likely underpins the mechanisms of zoonotic spillover to people. human-wildlife contact with a bat or an intermediate host species in china likely triggered a coronavirus spillover event that may have involved wildlife markets and led to the pandemic spread of sars-cov-2 [1, 2] . the pandemic risk of commercial trade in live wildlife was first recognized during the 2002-2003 severe acute respiratory syndrome (sars) outbreak due to sars-cov [3] . this virus spread to countries in asia, europe, and the americas with 8,096 people infected and 774 deaths, costing the global economy about $us 40 billion in response and control measures [4, 5] . unfortunately, the impact of covid-19, the disease caused by sars-cov-2, has reached nearly every country and greatly surpassed those numbers by many orders of magnitude [6] . while bats are thought to be the ancestral hosts for all groups of coronaviruses [7] , for both sars-cov and sars-cov-2 wildlife trade supply chains are suspected to have contributed the additional conditions necessary for the emergence, spillover, and amplification of these viruses in humans [8, 9] . in viet nam, between 2013 to 2014, we conducted coronavirus surveillance to understand the presence and diversity of coronaviruses in wildlife at sites identified as high-risk interfaces for viral spillover from wildlife to humans [10] . we sampled at three sub-interfaces along the live field rat trade (rattus sp. and bandicota sp.) including field rats sold by rat traders, by vendors in large markets, and rats butchered and sold in restaurants as prepared dishes. we also sampled rodents raised on wildlife farms to assess risk from different wildlife supply chains destined for human consumption. we sampled bat guano, primarily on bat guano farms to assess the potential occupational risk of this practice given that bat guano farm artificial roost structures are often erected near human dwellings. in the early 2000s, the vietnamese field rat trade was estimated to process 3,300-3,600 tons of live rats annually for consumption, a market valued at us$2 million [11] . although rats are still commonly traded in wet markets and sold live for food consumption along the mekong delta in southern viet nam, no recent published data on the scale and scope of the trade is available [12] . this human-wildlife interface involves the capture of wild free-ranging field rats, subsequent trade, and consumption along a supply chain involving the entire mekong delta region, particularly cambodia and viet nam [13] . driving this trade are consumers in viet nam and cambodia, some of whom report eating rats at least once per week because of their good flavor, low cost, and perception of rats as 'healthy, nutritious, natural, or disease free' [13] . rat parts (heads, tails, and internal organs discarded at slaughter) are also often fed to domestic livestock or herptiles raised in captivity including frogs, snakes, and crocodiles [12] . over the past three decades, commercial wildlife farming has developed in many countries in southeast asia, including viet nam. although there are historic references to the occurrence of wildlife farms in viet nam dating back to the late 1800s, the rapid expansion in terms of farm numbers, species diversity, and scale of operations has occurred in recent decades in response to growing domestic and international demand for wildlife [14] . a 2014 survey across 12 provinces in southern viet nam identified 6,006 registered wildlife farms of which 4,099 had active operations. the surveyed farms were stocked with approximately one million wild animals including, rodents, primates, civets, wild boar, oriental rat-snakes, deer, crocodiles, and softshell turtles. ninety-five percent of the farms held 1-2 species of wildlife, and 70% of the farms also raised domestic animals on the same premises [15] . a key component of the wildlife farm industry in viet nam is the raising of wild species for meat for human consumption [15] . these farms sell to urban wild meat restaurants serving increasingly affluent populations throughout the country and also supply international markets with wild meat [16] . commercial wildlife farming in viet nam is part of the expanded international trade of wildlife that has been hypothesized to contribute to the cause of global epidemics, such as sars [17] and now covid-19. emerging evidence suggests zoonotic virus spillover risk is a concern at bat-human interfaces in asia. guano harvested from a cave in thailand were positive for a group c betacoronavirus, which includes mers-cov, and 2.7% of 218 people living in close proximity to bats known to carry viruses related to sars-cov tested positive for sars-related antibodies in china [18, 19] . the traditional practice of guano farming in parts of cambodia and viet nam involves the construction of artificial bat roosts in gardens or backyard farms, under which domestic animals and crops are raised, and children often play [20, 21] . cambodian development programs promoted the practice in 2004 to enhance soil fertility, reduce reliance on chemical fertilizers, generate income ($us 0.50/kg), control insect pests, and protect the lesser asiatic yellow bats (scotophilus kuhlii) that were being hunted [20] [21] [22] . no personal protection measures are taken when harvesting the guano, which is reported to improve the growth rate in five economically important plant species [23] . in this study we investigated the presence and diversity of coronavirus sequences in the field rat trade distribution chain, wildlife farms specializing in raising rodents for human consumption, and bat guano "farms" and roosts near human dwellings to better understand the natural hosts of coronaviruses and the risk for these interfaces to facilitate spillover into humans. sampling was performed at multiple sites representing several high-risk interfaces for contacts among people, rodents, and bats. rodent sampling focused on the live field rat trade supply chain and wildlife farms specializing in raising rodents [malayan porcupines (hystrix brachyura) and bamboo rats (rhizomys sp.)] for meat. along the field rat supply chain, we targeted eight sites involved in the private sale and butchering of rats for consumption, defined as 'traders' for the purpose of this study in dong thap and soc trang provinces, 14 large market sites where rats were butchered and sold in dong thap and soc trang provinces (>20 vendors), and two restaurant sites in soc trang province where live rats were kept on the premises and butchered and served as food (fig 1) . the 28 rodent farm sites targeted in dong nai province produced malayan porcupines and bamboo rats for human consumption (fig 2) . other species observed or raised at the wildlife farm sites included dogs, cattle, pigs, chickens, ducks, pigeons, geese, common pheasant, monitor lizards, wild boar, fish, python, crocodiles, deer, civets, non-human primates as pets or part of private collections, free-flying wild birds, and free-ranging peri-domestic rats. bat sampling occurred at bat guano "farms" and a natural bat roost located at a religious site. bat guano farms consisted of artificial roosts constructed with a concrete base and pillars topped with fronds of coconut palm or asian palmyra palm (borassus flabellifer) (fig 3) . seventeen bat guano farms were sampled in the two provinces of dong thap and soc trang. the natural bat roost was located at a religious site in soc trang province known as the "bat pagoda", where pteropus sp. have historically roosted in trees protected from hunting, and light and noise pollution [24] . all study sampling occurred from january 2013 to march 2014 at 41 sites in the wet (south viet nam: may 1 st -november 30 th ) and 30 in the dry (south viet nam: december 1 st -april 30 th ) seasons. given the distances between sites, 69 sites were sampled once except the bat pagoda natural roost in soc trang province, which was visited three times and sampled in both seasons. samples from animals were humanely collected using standard and previously published protocols and no animals were purchased for this study (s1 table) [25]. samples from rats at all three sub-interfaces in the field rat trade were collected from individual carcasses after the rats were slaughtered by a trader, market vendor, or restaurant kitchen staff as part of the rat meat preparation process during normal sales to customers. oral swabs were collected from the severed heads of all the rats, with at least one additional tissue sample collected from the discarded internal organs of each individual. the small intestine was the additional tissue most frequently collected with brain, kidney, lung, rectal swab, and urine also collected from some individuals. rats were usually butchered at a common site for each observed time period that was only cleaned intermittently following the trader's, vendor's, or restaurant's regular practices. feces, urine, and swabs of the pen floors (environmental samples), were collected non-invasively (without handling the animals) from rodents on wildlife farms. samples were classified as 'fecal sample' or 'urine sample' when voided feces or urine was collected from an animal housed individually in its own cage, and as 'environmental sample' when collected as a swab from cages housing multiple individuals. fecal samples and a small number of urine samples excreted by bats in guano farms and the natural roost site were collected on clean plastic cover sheets within 1-2 hours after placement under bat roosts, and thus each sample may represent one or multiple bats. oral and rectal swabs were also collected from live-captured bats during one sampling visit at the natural pagoda roost site. all animals were identified in the field to the lowest taxonomic level possible based on morphological characteristics, and species was identified in a subset of animals through genetic barcoding [15] . due to difficulty of morphologic identification in the field, unless barcoded, rodents (rattus argentiventer, r. tanezumi, r. norvegicus, r. exulans, r. losea, and bandicota indica; [12, 26] ) were categorized as "field rats". bats were classified as "microchiroptera" following the traditional taxonomic classification (new classification of two new suborders yangochiroptera and yinpterochiroptera, was only published near the end of the study, so for consistency we used the historical classification [27] ). all samples were collected in cryotubes containing rnalater (rna stabilization reagent, qiagen), and stored in liquid nitrogen in the field before being transported to the laboratory for storage at -80˚c. samples were tested by the regional animal health office no. 6 (raho6) laboratory in ho chi minh city. the study and sampling activities for specified dates and locations were approved by the department of animal health of the ministry of agriculture and rural development and animal sampling protocols were reviewed by the institutional animal care and use committee at the university of california at davis (protocol number 16048). rna was extracted (rna miniprep kit, sigma-aldrich) and cdna transcribed (superscript iii first strand cdna synthesis system, invitrogen). coronavirus rna was detected using two broadly reactive consensus nested-pcr assays targeting the rna dependent rna polymerase (rdrp) gene [28, 29] . the positive control was a synthetic plasmid containing the primerbinding sites for both assays. distilled water was used as a negative control and included in each test batch. pcr products were visualized using 1.5% agarose gels, and bands of the correct size were excised, cloned, and sequenced by sanger dideoxy sequencing using the same primers as for amplification. for sequence analysis and classification operating taxonomic units were defined with a cut off of 90% identity, i.e. virus sequences that shared less than 90% identity to a known sequence were labelled sequentially as predict_cov-1, -2, -3, etc. and groups sharing � 90% identity to a sequence already in genbank were given the same name as the matching sequence [7] . a phylogenetic tree was constructed for sequences amplified using the watanabe protocol, as this pcr protocol yielded longer sequences and more positive results than the quan protocol. several representative sequences for each viral species found in our study were included for analysis and are available in genbank (s3 table) . alignments were performed using muscle, and trees were constructed using maximum likelihood and the tamura 3-parameter model in mega7 [30] . the best-fit model of dna substitution was selected in mega7 using bic scores (bayesian information criterion) and maximum likelihood values (lnl). bootstrap values were calculated after 1,000 replicates. in addition, a median-joining network was constructed using network 5.0.0.3 [31] to explore phylogenetic relationships among bat coronavirus 512/2005 sequences at the intraspecies level, as haplotype networks may better represent the relationships among viral sequences with low sequence diversity compared with phylogenetic trees [32] . visualization of sampling locations in provinces in viet nam, along with the distribution by species and interface was constructed with the ggplot2 and sf packages [33] . the source of the viet nam provincial map is geoboundaries v. 3.0.0 (https://www.geoboundaries.org; [34] ) and open development mekong (https://vietnam.opendevelopmentmekong.net). all analyses were done using r version 3.5.0 or higher (r development core team, vienna, austria). data (s1 data) and code (s1 r code) are available in the supplementary materials. the effect of risk factors (season, sub-interface type) was examined and limited to interfaces for which the distribution of samples across factors could support the analysis. these included season for pteropus bat samples collected in the bat pagoda natural roost and the effect of season and sub-interface for samples collected in the rodent trade in southern viet nam. given the low sample size, the effect of season for pteropus bats samples positive for coronaviruses was assessed using a fisher exact test. the effect of season (dry, wet, with dry season as reference category) and sub-interface type (trader, large markets, restaurants, with trader as reference category) in traded rodent samples positive for coronaviruses was assessed with a mixed effect multivariable logistic regression, with sites as random effect (i.e. grouping variable) using the lme4 r package [35] . a p-value of less than 0.05 was considered statistically significant. the 95% binomial confidence intervals for proportions were calculated using binom.test in r. the comparison of the proportion of coronavirus positives in different sample types was performed on positive individuals sampled in the live field rat trade with multiple sample types collected per individual. we then calculated the proportion of individuals positive for each sample type, as a proxy for the probability of detection by each sample type. a total of 2,164 samples collected between january 2013 and march 2014 from rodents and bats were tested for coronaviruses (table 1, s1 table) . assuming that non-invasive samples from bats and farmed rodents represented unique distinct individuals, these samples came from 1,506 individuals, including 1,131 rodents (702 field rats and 429 wildlife farm rodents) and 375 bats from 70 sites sampled in dong thap, soc trang, and dong nai provinces in the southern region near the mekong river delta (fig 4) . out of 70 sites, coronavirus positives were detected at 58 including 100% (24/24) of live rat trade sites, 60.7% (17/28) of rodent wildlife farm sites, 94.1% (16/17) of bat guano farm sites, and at the one natural pteropid bat roost. wildlife farms were only sampled in dong nai province and the live rat trade and bat interfaces were sampled in dong thap and soc trang provinces (fig 4) . coronaviruses were detected in the field rat trade (a mix of rattus and bandicota genera) at all sites in dong thap (n = 16) and soc trang (n = 8) provinces, with 34.6% (95% ci 29. table) . it should be noted, however, that since sites were only visited during one season, both independent variables were defined at the site level and confounding effects with other site-level characteristics cannot be excluded. among the positive field rats with more than one sample tested (n = 220), the proportion positive by sample type was 79.9% (95% ci 73.9-84.9%, 175/219) in oral swabs, 52.9% (95% ci a field rat here refers to a mix of rattus sp. and bandicota sp. b this environmental sample collected from a porcupine cage on a porcupine farm was barcoded as rattus sp., suggesting this species was free-ranging at the site (fig 2) . 50 .0% in feces (1/2), 100% in spleen (1/1), and 0% in urine/urogenital swabs (0/1). at the rodent wildlife farm interface, 6.0% (95% ci 3.8-9.3%, 20/331) of hystrix brachyura and 6.3% (95% ci 2.6-13.6%, 6/96) of rhizomys sp. were positive. the overall proportion of positives was 6.3% (95% ci 4.3-9.1%, 27/429) ( table 1 and fig 4) . there was no difference among species or season and proportion positive in rodent farms, and low sample size and unequal sampling limited analysis. the proportion of coronavirus positives at the two bat interfaces differed by an order of magnitude as 74.8% (95% ci 69.5-79.4%) of the non-invasive samples collected from microchiroptera bats at bat guano farms were positive, and 6.7% (95% ci 2.2-17.0%) of the pteropus predict_cov-17 and predict_cov-35 were first reported by anthony et al. [17] . we found predict_cov-17 in pteropus bats and in microchiroptera ( table 1 ). the predict_ cov-17 sequences from pteropus detected in this study clustered closely with predict_ cov-17 sequences from pteropus giganteus bats in nepal and pteropus lylei bats in thailand [36] (fig 6, s3 table) . predict_cov-35 was found in microchiroptera in bat guano farms the analysis included 17 sequences from this study (red from bat hosts, blue from rodent hosts), six sequences (in gray) from a previous study in viet nam [26] , and 26 reference sequences (in black) available in the genbank database (s3 table) . the tree was rooted by a strain of night-heron coronavirus hku19 (genbank accession no. nc_016994). https://doi.org/10.1371/journal.pone.0237129.g006 and in a pteropid bat (table 1) . predict_cov-35 sequences from viet nam clustered with other predict_cov-35 sequences found previously in samples from hunted scotophilus kuhlii bats in cambodia (s3 table; dr. lucy keatts personal communication), and with sequences found in bats from an earlier study in the mekong delta region in viet nam (fig 6) . bat coronavirus 512/2005 was detected in microchiroptera bat guano; and in h. brachyura (feces and environmental samples), r. pruinosus (feces barcoded), and r. argentiventer (barcoded environmental sample) in wildlife farms (table 1 and s1 table) . in microchiroptera, bat coronavirus 512/2005 was frequently found in co-infection with predict_cov-35 (table 1 , s1 table) . network analysis showed the relationships among the bat coronavirus 512/2005 sequences from the three provinces in south viet nam (fig 7) . we observed two main clusters and a shallow geographic structure of genetic diversity, perhaps illustrative of sampling effort but also of localized transmission and circulation of bat coronavirus 512/2005 strains in these provinces. one cluster was exclusively detected in microchiroptera and mostly restricted to dong thap province and another cluster included sequences shared among all hosts and distributed in the three provinces (fig 7) . parts of the network showed a star-like topology (fig 7) , typical of populations in expansion that have recently increased size. there were two sequence types that were shared among microchiroptera and farmed rodents. the remaining 11 sequence types isolated from rodents on wildlife farms were not identical to those isolated from bats and were characterized by several nucleotide differences (fig 7) . murine coronavirus and longquan aa coronavirus were detected in 209 and 56 field rat samples, respectively, and 26 were coinfected with both (table 1) . two sequences of ibv were detected in rodent feces collected on two wildlife farms, one in a bamboo rat and another in a malayan porcupine. the rodent farm interface where bat and avian coronaviruses were detected in feces were not full containment facilities and possibly had bats and birds flying and roosting overhead (fig 2) . the ibv positives were detected in fecal samples from wildlife farms that had chickens, pigs, and dogs on site. significant findings of this study are the high proportion of coronavirus positive wildlife (bats and rodents) and the increasing proportion of positives found along the rat trade supply chain from sub-interfaces close to the capture site (rat traders) to restaurants. the transit of multiple rat species through the supply chain, and admixing with other species and taxa at sub-interfaces along the supply chain, offers opportunities for inter-and intra-species viral exchange and recombination. capture and transport of wildlife combined with overcrowding and close confinement of live animals in cages results in increased animal contact, likely leading to stress. while methodologically similar to rodent surveys in zhejiang province, china (2%), dong thap province, viet nam (4.4%), and globally (0.32%), our overall proportion of coronavirus positives was much higher among field rats (34.5%) and somewhat higher among farmed rodents (6.3%) [7, 26, 37] . stress, dehydration, and poor nutrition reduce animal condition and alter immune function and likely contribute to both increased shedding of viruses by infected animals, and increased susceptibility to infection of animals in the wildlife trade chain for human consumption [38] . the amplification of coronavirus along the supply chain may be seasonal as field rats were significantly more positive in the wet season. rattus argentiventer generally reproduce yearround in viet nam, but are particularly abundant in the wet season (may through october) following the rice harvest when an abundance of food supports the population increase [39] . if these seasonal population increases affect density dependent contact, there could be increased coronavirus prevalence and shedding in wild field rats during certain times of the year, which could then be further amplified along the trade. our survey was not a comprehensive multi-year evaluation of the field rat supply chain and was restricted to two provinces with this human-wildlife interface. these limitations mean we are not able to make inferences about larger spatial patterns or the inter-annual variability of coronavirus prevalence in wildlife populations found in this interface, which spans into neighboring cambodia. field rat carcasses were sampled immediately after they were slaughtered by traders, market vendors, or restaurant kitchen staff to optimize viral detection. some viral cross contamination of carcasses during the butchering process may have increased the proportion of coronaviruses detected in individual animals. the degree to which cross-contamination may have elevated the proportion of coronaviruses detected in individual animals is unknown, however, this proportion accurately reflects the risk of human exposure from handling and consumption of field rats at sub-interfaces along this wild meat food chain. from a mechanistic perspective as animals progress along the wildlife supply chain, opportunity for human contact increases, including close direct contact with traders, butchers, cooks, and consumers [40] . the combination of increased coronavirus prevalence in traded wildlife and greater opportunity for human-wildlife contact as well as intra-and inter-species contact in trade systems is likely to increase the risk of zoonotic transmission of coronaviruses in wildlife markets, restaurants, and other trade interfaces. we detected avian and bat coronaviruses in rodents raised on wildlife farms, including malayan porcupines and bamboo rats, but we did not detect rodent-associated coronaviruses. the only previously published coronavirus testing of malayan porcupine samples carried out in china were negative [41] . it is unclear if the malayan porcupine samples from animals screened in this study were infected with the avian or bat viruses or if environmental contamination or mixing occurred with avian and bat guano. chickens were present at the two sites where the ibv-positive rodents were detected, and bats fly and potentially roost overhead at most farms. 'artificial market' studies of influenza a viruses have found cage-stacking of species on top of other species and shared water sources facilitate viral transmission [42, 43] . nevertheless, viral sharing between species and environmental contamination or mixing (i.e. bat/ bird guano landing on rat feces) are two equally likely explanations for the presence of bat and avian coronaviruses detected in rodent fecal and environmental samples. the field rats were co-infected with the longquan aa coronavirus and the murine coronaviruses, both of which are from the lineage a (embecovirus) betacoronavirus genus. co-infections with multiple coronaviruses deserve particular attention as this co-occurrence may facilitate viral recombination leading to the emergence of new viruses [44, 45] . at the very least, we conclude that rodents in the field rat and farmed rodent supply chains are being exposed to coronaviruses from rodents, bats, and birds and perhaps creating opportunities for coronavirus recombination events, which may lead to viruses that could spill over into humans [46, 47] . our findings indicate a high risk of consumer exposure even if crosscontamination due to shared butchering materials influenced the proportion of positive individuals. repeated and more direct individual sampling of these species at these interfaces would be advantageous to determine if viral sharing was occurring versus environmental contamination of samples. the high proportion of positive bat feces at bat guano farms indicates the potential risk of bat guano farmers, their families, and their animals being exposed to bat coronaviruses. the overall proportion of positives (74.8%) was higher than previous studies using similar testing methods targeting bats in viet nam (22%), thailand (7.6%), lao pdr (6.5%), and cambodia (4.85%) [26, 48, 49] . in this region of viet nam, artificial roosts are typically erected in backyard family owned plots that incorporate a mosaic of duck, goat, or pig production and crops such as guava tress or other fruit trees and large scale kitchen gardens. bats have been shown to be an important evolutionary hosts of coronaviruses, including those infecting humans [7, [50] [51] [52] [53] . both predict_cov-17 and predict_cov-35 have been detected previously in the pteropus and microchiroptera bats in viet nam, cambodia, and nepal, which confirms that coronaviruses are capable of infecting distantly related hosts [7] . the finding of the same virus in different bat species raises the question of whether they coroost and/or share viruses through contact during other activities. utilizing shared resources such as water or feeding on and around crops and fruit could lead to contact and facilitate a host jump. the presence of the same virus in bat species in multiple neighboring countries supports the suggestion by others that virus distribution coincides with their bat host distribution [7, 54, 55] . while there has been no testing of the pathogenicity of these bat coronaviruses in humans or animals, they are found at close contact bat-human interfaces and further characterization is needed to understand their host range and potential for spillover. any general persecution of bats because of zoonotic viruses they may carry can actually increase the number of susceptible bats and increase transmission risk to people [56] , and would interfere with the important ecosystem services that bats provide, such as controlling insect pests of rice fields [57] , plant pollination, and seed dispersal. beyond the viral findings, this work represented an important opportunity for capacity development in field, laboratory, and scientific disciplines, as well as opportunities for social engagement and education of high-risk communities on zoonotic disease threats. the consensus pcr approach for viral detection provides a cost-effective tool to detect emerging viruses in low-resource settings. our work adds to the growing body of research demonstrating the utility of this approach to detect both known and novel viruses and co-infections in a variety of taxa, sample types, and interfaces. in viet nam, the direct result is an enhanced one health surveillance capacity to detect important emerging or unknown viruses in humans, wildlife, and livestock. in the communities with which we partnered, strong engagement enabled teams to sample a wide diversity of wild animals at high-risk interfaces. importantly, we have returned to these same communities to share the viral findings and to educate participants with an outreach program on how to live safely with bats [58] . large percentages of coronaviruses were detected in bats and rodents at sites where people have close contact and interact with wildlife including sub-interfaces along wildlife trade chains, wildlife farms, and artificial bat roosts where bat guano is collected for use as fertilizer. the high proportion of coronavirus positive samples at these human-wildlife interfaces highlights the potential for human exposure to wildlife origin coronaviruses. the observed viral amplification along the wildlife trade supply chain for human consumption, illustrated by the field rat trade in this study, likely resulted from the admixing of different species or sub-populations, and the close confinement of stressed live animals. this highlights the potential for coronavirus (and other virus) shedding and amplification along other wildlife supply chains (e.g., civets, pangolins) where similarly large numbers of animals are collected from a wide range of locations, transported, and confined. the detections of rodent, bat, and avian coronaviruses confirm concerns about productions systems and supply chains that increase contact between wildlife and domestic species. livestock and people living in close contact with rodents, bats, and birds shedding coronaviruses provides opportunities for intra-and interspecies transmission and potential recombination of coronaviruses. human behavior is facilitating the spillover of viruses, such as coronavirus, from animals to people. the wildlife trade supply chain from the field to restaurant and end consumer provides multiple opportunities for such spillover events to occur [1] . since the sars outbreak, broad scientific consensus exists that long term, structural changes, and wildlife trade and market closures will be required to prevent future epidemics. to minimize the public health risks of viral disease emergence from the consumption of wildlife and to safeguard livestock-based production systems, we recommend precautionary measures that restrict the killing, commercial breeding, transport, buying, selling, storage, processing and consuming of wild animals. the time has come for the global community to collectively assume responsibility through targeted wildlife trade reform. the world must also increase vigilance through building and improving detection capacity; actively conducting surveillance to detect and characterize coronaviruses in humans, wildlife, and livestock; and to inform human behaviors in order to reduce zoonotic viral transmission to humans. the more opportunities we provide for humans to come into direct contact with a multitude of wildlife species, the higher the likelihood of another spillover event. the costs of inaction are astronomically high and we must ensure that future food production and security is sustainable, just, and supports global health. supporting information s1 table. summary of all testing results by genus, interface, sub-interface, sample types, sites, percentage of samples testing positive, and viral species. (pdf) s2 table. multivariate mixed effect logistic regression showing the association between season and sub-interface with coronavirus positives in field rats. (pdf) s3 table. genbank accession numbers for coronavirus sequences detected in this study and for reference sequences. (pdf) s1 data. (txt) s1 r code. code used to conduct the analysis described. 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resurgence in kitaka mine bat population after extermination attempts the wrinkle-lipped free-tailed bat (chaerephon plicatus buchannan, 1800) feeds mainly on brown planthoppers in rice fields of central thailand living safely with bats. usaid we are thankful to the government of viet nam, the wildlife conservation society health team for conducting field sampling, partnering laboratories for running diagnostic tests, and many other agencies for collaborations on this project. specifically, we would like to acknowl key: cord-295781-b831y105 authors: vanleuven, james t.; ridenhour, benjamin j.; gonzalez, andres j.; miller, craig r.; miura, tanya a. title: lung epithelial cells have virus-specific and shared gene expression responses to infection by diverse respiratory viruses date: 2017-06-02 journal: plos one doi: 10.1371/journal.pone.0178408 sha: doc_id: 295781 cord_uid: b831y105 the severity of respiratory viral infections is partially determined by the cellular response mounted by infected lung epithelial cells. disease prevention and treatment is dependent on our understanding of the shared and unique responses elicited by diverse viruses, yet few studies compare host responses to viruses from different families while controlling other experimental parameters. murine models are commonly used to study the pathogenesis of respiratory viral infections, and in vitro studies using murine cells provide mechanistic insight into the pathogenesis observed in vivo. we used microarray analysis to compare changes in gene expression of murine lung epithelial cells infected individually by three respiratory viruses causing mild (rhinovirus, rv1b), moderate (coronavirus, mhv-1), and severe (influenza a virus, pr8) disease in mice. rv1b infection caused numerous gene expression changes, but the differential effect peaked at 12 hours post-infection. pr8 altered an intermediate number of genes whose expression continued to change through 24 hours. mhv-1 had comparatively few effects on host gene expression. the viruses elicited highly overlapping responses in antiviral genes, though mhv-1 induced a lower type i interferon response than the other two viruses. signature genes were identified for each virus and included host defense genes for pr8, tissue remodeling genes for rv1b, and transcription factors for mhv-1. our comparative approach identified universal and specific transcriptional signatures of virus infection that can be used to distinguish shared and virus-specific mechanisms of pathogenesis in the respiratory tract. viruses from several different families, including picornaviridae, orthomyxoviridae, paramyxoviridae, coronaviridae, and adenoviridae, infect and cause diseases in the respiratory tract. these diseases range from mild infections of the upper respiratory tract, to severe diseases when viruses infect the lungs. respiratory viruses commonly target epithelial cells of the a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 airways and lungs. these epithelial cells are responsible for detecting viral pathogens and initiating antiviral responses at the level of infected cells and the immune system, and therefore their response to infection has an important role in determining disease outcomes. knowledge of the shared and virus-specific responses of respiratory epithelial cells to infection by diverse viruses is fundamental to understanding viral pathogenesis and developing therapies to treat severe respiratory infections. murine models of respiratory viral infections have been widely used to identify the mechanisms that determine disease severity in the respiratory tract. while these models are invaluable for evaluating pathology and host responses to infection, parallel in vitro studies can be used to identify gene expression and signaling pathway changes that occur in infected cells to mediate pathogenesis. in this study, we compare the gene expression response of murine lung epithelial cells to infection by three respiratory viruses used in murine models: rhinovirus (serotype rv1b) in the family picornaviridae, mouse hepatitis virus (mhv strain 1) in the family coronaviridae, and influenza a virus (strain pr8) in the family orthomyxoviridae. viruses from different families and with different replication strategies were chosen to identify which responses to infection in respiratory epithelial cells are shared and which are virus-specific. in the following paragraphs, we describe some of the key features of these three viruses in murine models. minor group rhinoviruses, including rv1b, use low-density lipoprotein receptor to enter either human or murine cells. because rv1b can replicate in mouse cells, it has been used to study immune responses and mechanisms of asthma exacerbation in infected mice [1] [2] [3] [4] [5] . intranasal inoculation of mice with a high dose of rv1b results in viral replication and an early inflammatory response in the respiratory tract without clinical signs of disease [1, [3] [4] [5] . rv1b antigens have been detected in cells of the epithelia and lamina propria of the upper respiratory tract of infected mice [2, 4] . mhv-1 is a strain of mouse hepatitis virus that preferentially replicates and causes disease in the respiratory tract of specific mouse strains and thus serves as a model for respiratory coronaviral infections [6, 7] . mhv-1 causes severe disease in a/j-strain mice and milder pathology in other mouse strains [6, 8] . mouse strain-dependent disease severity corresponds to inflammatory responses, fibrin deposition, and reduced type i interferon (ifn) production [6] . further, type i ifn-mediated signaling is required for protection from severe disease during mhv-1 infection of resistant mice [8] . mhv-1 has been detected in pulmonary macrophages of infected mice, but has not been reported to infect respiratory epithelial cells in vivo [6] . although primary murine alveolar epithelial cells are susceptible to mhv-1 infection in vitro, their role during in vivo infection is not clear [9] . mice have been used for decades to study the pathogenesis of influenza. one of the most commonly used strains of influenza a virus, pr8, has been serially passaged in mice to produce a model for pulmonary infection. pr8 infection results in a range of disease severities that is mouse strain-dependent [10] . although susceptible mice mount a type i ifn response to pr8 infection, lethal infection is associated with spread of virus to the alveoli and an excessive inflammatory response [10] [11] [12] [13] . pr8 replicates in bronchiolar and alveolar epithelial cells of the lower respiratory tract in vivo and in primary murine respiratory epithelial cells in vitro [9, 12, 14, 15] . in this study, we used a murine lung epithelial cell line (la4) to compare the gene expression response to these three unrelated viruses. la4 cells were derived from neoplastic lung epithelia from strain a (a/he) mice and have some properties of alveolar type ii cells, although they do not maintain a highly differentiated type ii phenotype [16] . strain a (a/j) mice are highly susceptible to respiratory viral infections, including mhv-1 and influenza a viruses [6, 10] . other studies have demonstrated that la4 cells are susceptible to infection by pr8 and rv1b [15, 17] . in this study, we show that la4 cells are also susceptible to infection by mhv-1 (hereafter referred to as mhv). the gene expression response of la4 cells to infection by mhv, pr8, and rv1b (hereafter referred to as rv) differed in timing and magnitude of the changes. while we expected to see highly divergent transcription responses to these three viruses, they induced expression of a surprisingly large overlapping set of shared genes, including genes involved in antiviral responses. however, and more in line with our expectation, each virus also altered expression of unique genes, which highlight their different replication strategies and mechanisms of pathogenesis in murine hosts. virus stocks were generated by 24 h of growth from a low dose inoculum in the cell lines described below. supernatant medium from infected cells was centrifuged to remove cell debris, aliquoted, flash frozen and stored at -80˚c. pr8 (a/puerto rico/8/1934 (h1n1)), obtained from bei resources (nr-3169), was grown and titrated by plaque assay in mdck cells from atcc (ccl-34) [18] . mhv, obtained from atcc (vr-261), was grown and titrated by plaque assay in 17cl.1 cells [19] (provided by dr. kathryn holmes, university of colorado denver school of medicine). rv, obtained from atcc (vr-1645), was grown and titrated by tissue culture infectious dose 50% (tcid 50 ) assay in hela (atcc: ccl-2) cells [17] . la4, a murine lung epithelial cell line from atcc (ccl-196), was cultured in ham's f12k medium (mediatech) with 10% fbs (atlanta biologicals) and 1x antibiotic/antimycotic (gibco). our experimental approach was to inoculate la4 cells with the three viruses at times t = 0 h and t = 12 h and harvest rna for microarray analysis at t = 24 h. controls were mock-inoculated at both time points. preliminary experiments were done to establish the growth kinetics of each virus and determine a multiplicity of infection (moi) that resulted in comparable numbers of cells positive for viral antigen at 24 h post-infection (s1 fig) . based on this, la4 cells were inoculated with 3 tcid 50 /cell rv, 1 pfu/cell pr8, or 3 pfu/cell mhv. triplicate wells of la4 cells in 6-well plates were inoculated with each virus diluted in serum-free medium or were mock-inoculated with serum-free medium for 1 h at 37˚c. viral inocula were removed and the cells were rinsed twice with serum-free medium. the cells were incubated in ham's f12k medium with 2% fbs until the 24 h time point at which time rna was isolated from cell cultures using an rnaeasy plus kit (qiagen) and transcript levels were measured by microarray (nimblegen mus musculus mm9 expression array). for the 24 h samples, the media were removed and replaced with fresh media 12 h after inoculation, which is the same time that 12 h samples were inoculated. raw and processed data are available from ncbi gene expression omnibus under accession number gse89190. nimblescan v2.5 software (niblegen, madison, wi) was used to extract raw intensity data for each probe on each array. intensity data were read into the r statistical computing environment and checked for quality [20] . data were prepared for processing using the pdinfobuilder package and then normalized using the robust multichip average (rma) function in the oligo package [21] . statistical tests for differences in expression between treatments were conducted on the normalized expression data using a linear mixed-effect model followed by linear contrasts corrected for multiple comparisons. more specifically, expression was modeled as a function of treatment, while probes for a particular gene were treated as random effects. the models used the nlme::lme function in r. the data contained seven treatments: three viruses tested at two time points (12 h and 24 h) each plus the mock treatment (rv 12 , rv 24 , mhv 12 , mhv 24 , pr8 12 , pr8 24 , and mock). the following nine post hoc, two-sided contrasts were then performed on the fitted models using the multcomp::glht function in r: each virus-time combination vs. mock (rv 12 vs. mock, rv 24 vs. mock, mhv 12 vs. mock, mhv 24 vs. mock, pr8 12 vs. mock, pr8 24 vs. mock) and each pairwise combination of viruses at the 24 h time point (rv 24 vs. mhv 24 , rv 24 vs. pr8 24 mhv 24 vs. pr8 24 ). these 9 tests had their p-values adjusted by the multcomp::summary.glht function according to their joint distribution. any factors detected to be significant at the family (gene) level were then subsequently corrected using the benjamini-hochberg algorithm [22] with a false discovery rate set at 1%. genes associated with type i ifn responses were identified among the sets of genes with differential expression for each virus compared to mock at 12 h and 24 h using the interferome v.2.01 database (http://interferome.its.monash.edu.au; [23] ). this database was queried using the search criteria: type i ifn (all), in vitro, mus musculus, 2.0 fold change (up or down). interferome genes were manually sorted into functional categories: antiviral, ifn signaling, viral detection, immune response, mhc class i, inhibitory, apoptosis, ubiquitination, miscellaneous, and unknown. the significance of each virus having genes in the specific categories was tested using a chi-squared test. gene expression responses to rv1b were compared between our data from mouse cells and published data using human cells [24] using the mgi vertebrate homology database provided by the jackson laboratory [25] as well as the annotate package in r. mhv, pr8, and rv alter cellular gene expression by different magnitudes and with different timing our goal was to evaluate the gene expression response of murine respiratory epithelial cells to infection by three unrelated respiratory viruses studied in murine models. a preliminary study was undertaken to identify a murine cell line that was susceptible to infection by the three viruses. la4 cells were chosen because mhv, rv, and pr8 established productive infections in this line, as shown by increased viral titers released from infected cultures over a 24 h infection (s1 fig). additionally, comparable numbers of viral antigen-positive cells were observed for the three viruses 24 h after inoculation of la4 cells (s1 fig) . to compare how unrelated respiratory viruses (mhv, pr8, and rv) alter gene expression of murine epithelial cells, we inoculated la4 cells with each of the three viruses and evaluated cellular gene expression by microarray analysis after 12 and 24 h of infection compared to mock-inoculated controls. fig 1 shows the log 2 -fold change in expression level of genes that were differentially expressed in virus-infected, compared to mock-inoculated, cells. by plotting the changes in gene expression at 12 vs. 24 hours, we observed differences in magnitude and timing of gene expression changes mediated by the three viruses. the genes with significantly different expression in mhv-infected cells had low fold change values (fig 1a) . at 24 h, when gene expression changes were the highest, genes that were up-regulated by mhv infection had log 2 -fold change values of less than five. in contrast, pr8 and rv induced expression of many genes by greater than five log 2 -fold at 24 h, and genes were spread consistently across the full range of values. by 24 h, the genes most strongly up-regulated by pr8 and rv induced changes of 7-9.5 log 2 -fold and 6-7.5 log 2 -fold compared to mock, respectively. this same pattern was observed with the down-regulated genes (fig 1) . in addition to the differences in fold change values, the three viruses also differed in the timing of gene expression changes. mhv altered expression of relatively few host genes, most of which were only significantly different from mock at 24 h ( fig 1a) . while both pr8 and rv induced expression of large subsets of host genes, they did so with different timing. pr8-induced changes in gene expression occurred at a constant rate: the expression level of most genes at 24 h was approximately twice the expression value at 12 h ( fig 1b) . in contrast, rv infection altered expression of a large number of genes by 12 h and the expression levels were maintained at approximately the same levels at the 24 h time point (fig 1c) . taken together, we observed differences in magnitude and timing of gene expression changes mediated by the three viruses. the limited response to mhv infection is in agreement with studies of other coronaviruses, such as mhv-a59 [26] and sars-cov [27, 28] . in addition to inducing minor transcriptional up-regulation of host genes, mhv-a59 shuts down host gene expression by enhancing mrna degradation [26] . a related coronavirus, sars-cov, also induces degradation of host mrnas [29] . the low numbers of host mrnas that were altered in response to mhv infection in our study could be due to one or both of these mechanisms. while rhinoviruses are known to down-regulate host gene expression by inhibiting transcription [30], upon rv infection we saw robust, early increases in host mrna expression ( fig 1c) . this is in agreement with other transcriptome studies of major and minor serogroup rhinoviruses in human respiratory epithelial cells and experimental infections of humans [24, [31] [32] [33] [34] . the plateau in gene expression changes in rv-infected cells at 24 h may be due to transcriptional inhibition later in infection, or the death of infected cells. pr8 infection induced a strong transcriptional response in la4 cells, which has also been seen with multiple strains of influenza a viruses in primary human and mouse airway or lung epithelial cells [35] [36] [37] [38] . interestingly, the differences in kinetics of host cell gene expression did not correspond with differences in the kinetics of viral replication in this cell line (s1 fig). despite inducing 1) . these data also demonstrate that the limited response of cellular gene expression to mhv infection was not due to limited infection of la4 cells (s1 fig). host genes have shared and unique responses to rv, pr8, and mhv infection we identified which genes were altered by each virus at 24 h compared to mock and the degree of overlap among the differentially expressed genes. at 24 h rv infection resulted in up-regulation of the largest number of genes, followed by pr8 then mhv (fig 2a) ; a similar pattern was seen with down-regulated genes (fig 2b) . while one might worry that some of the small number of significant genes that were altered by mhv could be false positives, the majority of these genes (65% of up-regulated and 86% of down-regulated genes) were also significantly altered by at least one other virus suggesting that most of these genes are true positives. for both up-and down-regulated gene sets, rv had the largest proportion of unique genes, while the majority of genes affected by pr8 and mhv were shared by at least one other virus. s1 table contains the list of genes whose expression was significantly up-regulated by all three viruses compared to mock-inoculated cells. these genes may reflect a global response of epithelial cells to viral infection. several of the genes with the highest fold change values are involved in antiviral defense at the level of infected cells (eg., mx1, bst2, oas2, gbp10) or recruitment of immune cells (eg. , cxcl10, cxcl11, cxcl1) . these genes are up-regulated by type i ifns, suggesting that induction of a type i ifn response is shared by these viruses. in contrast to the shared up-regulated genes, genes that were significantly down-regulated by all three viruses have diverse functions (s2 table) . some examples of genes that were down-regulated by all three viruses included genes that encode transmembrane proteins (tmem 119, 231, 19 , 50a, and 14c), extracellular matrix proteins (spon2, ogn, aspn), and apoptotic signaling proteins (sdpr, bmf, bnip3l). as a measure of validation, the expression levels of five genes (tnf, cxcl10, bst2, icam1, and oas1a) were also quantified by rt-qpcr at 24 h post-infection (s2 fig). a strong correlation was observed between rt-qpcr and microarray measurements of gene expression (slope = 1.02, r 2 = 0.87). identification of signature genes that were uniquely altered by each virus comparing the number of genes altered by each virus provides insight into shared and unique cellular responses elicited by the viruses, but it does not provide information on the relative magnitudes of gene expression changes between viruses. to compare gene expression changes between viruses, we plotted the log 2 -fold change of each gene at 24 h for mhv vs. rv vs. pr8 (fig 3a) . we only included genes that were differentially expressed in at least one viral infection compared to mock. like fig 1, this 3d plot illustrates that pr8 and rv not only caused a larger number of genes to be up-regulated compared to mhv, but they also induced higher fold change values (fig 3a) . for each of the three viruses, we defined a signature gene as a gene that is both differentially regulated at 24 h compared to the mock treatment and has an effect size significantly larger than the other two viruses (i.e. fold change on the x axis is significantly different from y-axis, z-axis, and mock). these genes are colored in fig 3a and appear along the diagonal in fig 3b. as expected, rv had the largest number of signature genes, followed by pr8, then mhv ( fig 3b) . interestingly, the genes with the highest fold change values compared to mock were not signature genes, but were up-regulated by both pr8 and rv infection. a pairwise analysis was performed to identify the number of genes with altered expression compared with mock in two viruses compared with the third. this analysis, shown in fig 3b, reveals that rv and pr8 had the most similarities in both up-and down-regulated genes (fig 3b, purple blocks) . the pattern of up-regulated gene expression changes during mhv infection was more similar to pr8 (24 genes) than rv (6 genes). this may reflect the fact that pr8 and mhv cause severe pathogenesis in mice, whereas rv-infected mice do not exhibit clinical signs of disease. among the six genes co-upregulated by mhv and pr8 was tnf-α, a key proinflammatory cytokine (not shown). several host defense genes were identified as signature genes uniquely up-regulated by pr8 infection (s3 table) . these genes included cytokines and chemokines (cxcl9, ccl5, il12b, ccl8), ifn response genes (ifitm6, ifi27l2a, ifna2, ifit2, ifitm5, ifna11) , and genes involved in processing mhc class i antigens (psmb10, tap2, h2-q2, h2-k1, psmd9, psme2, psme1) . the significant up-regulation of host defense genes in response to pr8 in the la4 cell line corresponds with the expression profile of murine type ii alveolar epithelial cells in response to pr8 infection in mice [39] . pr8 infection in highly susceptible mouse strains results in dramatic up-regulation of inflammation-associated genes when compared to resistant mouse strains [11] . many studies in murine models of influenza a virus infection have demonstrated a relationship between an excessive inflammatory response and disease severity [10] [11] [12] 15] . furthermore, infection of tlr3-/-mice with influenza a virus results in a reduced inflammatory response and increased survival [40] . our data support the role of alveolar epithelial cells in generating this excessive inflammatory response in vivo. several genes that were uniquely down-regulated by pr8 are involved in cellular metabolic pathways (cdo1, aldh1a7, acad11, hsd17b4) or intracellular transport (myl6b, ift88, anxa8). although rv induced expression of several genes involved in host defense, these were largely shared by pr8 so were not identified as signature genes. the signature genes up-regulated by rv included kallikrein-1 and ten kallikrein-1-related peptidases and additional proteins involved in tissue remodeling (s4 table) . although tissue remodeling is not likely to be relevant in murine models of rhinovirus infection alone, due to the limited damage, it may be an important factor in murine models of rhinovirus-induced allergic asthma [1, 2, 41] . rhinovirus infections are a significant cause of asthma exacerbations, which correspond with inflammatory responses in the airways. kallikreins generate kinins and contribute to many disease processes, including inflammation. kinins are induced by rhinovirus infections and kallikrein-1 is up-regulated by rhinovirus infection in humans, especially those with asthma [42, 43] . upregulation of these genes in mouse cells upon rv infection would provide a tractable animal model in which to study the roles of kallikreins in rhinovirus-induced asthma exacerbations. mucins, which contribute to mucus hypersecretion, are up-regulated by rhinovirus infection of airway epithelial cells in vitro and in mice [1, 44] . muc2 was the only mucin gene up-regulated by rv in our study, and was unique to rv infection (s4 table) . mhv infection resulted in regulation of a small set of signature genes (fig 3b, s5 table) . signature genes that were uniquely up-regulated by mhv infection included multiple transcription factors from the double homeobox (duxf3, dux, dux4) and zinc finger and scan domain (zscan4d, zscan4c, zscan4-ps1, 2 and 3) families. despite up-regulating expression of transcription factors, mhv infection had a minor impact on the host cell transcriptome. this may be due to enhanced degradation of mrnas as discussed above, which has been shown to occur during other coronaviral infections [26, 29] . therefore, la4 cells may be up-regulating transcription in response to mhv infection through expression of various transcription factors while mhv causes degradation of these transcripts, which would reflect the small number of up-regulated transcripts in mhv infected samples. in contrast to mhv-a59, mhv-1 infection did not cause down-regulation of a substantial number of host genes. differences could be due to virus strain, host cell type, and timing differences between the studies. in contrast to the robust up-regulation of genes involved in innate immunity and inflammatory responses by pr8 and rv, the limited response of infected epithelial cells to mhv infection may reflect the ability of mhv to replicate (s1 fig) without being detected by the host cell. coronaviruses, including mhv, delay induction of antiviral responses. multiple mechanisms have been proposed to account for this, including replicating within double membrane vesicles, ribose 2'-omethylation of viral mrna, and endonuclease activity within the rna polymerase complex [45] [46] [47] [48] . this would allow mhv to replicate to higher levels before triggering antiviral defenses, which might promote pathogenesis in the murine respiratory tract [6] . alternatively, the reduced response to mhv by epithelial cells may reflect the different cellular tropism of mhv. in contrast to pr8 and rv, which are known to infect epithelial cells in the murine respiratory tract, mhv-1 has only been reported in alveolar macrophages [2, 6, 12] . type i ifn-related genes had increased expression in la4 cells infected by pr8, rv, and mhv as described above, several of the genes with up-regulated expression in response to all three viruses, as well as those that were unique to pr8, are induced by type i ifns. to specifically evaluate how ifn response genes were altered by the three viruses, genes that were significantly up-regulated by each virus at the 24 h time point were used to query the interferome v2.01 database (see materials and methods). a venn diagram was generated to visualize the degree of overlap in ifn-related genes whose expression was induced by at least one of the three viruses (fig 4) . pr8 induced expression of the greatest number of ifn-related genes, a majority of which were shared by at least one other virus. rv up-regulated slightly fewer ifninduced genes compared to pr8 and mhv infection resulted in up-regulation of the fewest ifn-induced genes. it was somewhat surprising that pr8 induced a higher type i ifn response than rv, given that rv induced expression of nearly twice as many genes than pr8 (fig 2) . however, some of these genes contribute to inflammatory responses, which could explain the excessive inflammatory response to pr8 infection vs. the self-limited inflammation during rv infection in mice [10] [11] [12] 15] . there was strong overlap between the ifn-induced genes up-regulated by each virus. the timing of ifn-related gene expression followed the same trend as was seen for all significant genes in fig 1 (data not shown) . most of the ifn-related genes up-regulated by mhv were only increased at 24 h. pr8 induced expression of 110 ifn-related genes at 12 h and these genes were a subset of the 179 genes up-regulated at 24 h. in contrast, rv infection induced expression of more ifn-related genes at 12 h (148 genes) than at 24 h (123 genes). relative to up-regulation, few ifn-related genes were down-regulated at the 24 h time point (mhv = 5, pr8 = 10, rv = 26). type i ifns induce expression of genes with different functions during an antiviral response. to determine whether there were specific patterns in expression of ifn-induced genes that correspond with function, the ifn-induced genes that had significantly increased expression by any of the three viruses were separated into functional groups. heatmaps that demonstrate differences in fold change (color scale) and significant differences (outlined boxes) in expression compared to mock-inoculated controls were generated (figs 4 and s3) . as shown in the venn diagram, this analysis also demonstrates that pr8 infection resulted in up-regulation of the most genes involved in type i ifn responses, followed by rv then mhv. the fold change values induced by pr8 infection were also generally higher than the other two viruses. however, there was not a significant association between virus identity and functional group. for most of the functional groups, mhv up-regulated expression of a smaller subset of the same genes as pr8 and rv, with the exception of the mhc class i pathway (fig 4) . mhv significantly up-regulated expression of only one gene involved in the mhc class i pathway (blmh), which was not significantly up-regulated by the other two viruses. this observation suggests that cytotoxic t cell responses may differ in mhv infections compared to pr8 and rv. t cell responses have complex roles in mhv-1 infections, as they contribute to protection in resistant mouse strains but mediate pathology in susceptible strains [49] . however, mice with the cd8+ t cell repertoire of a resistant strain in the background of a susceptible strain remain susceptible to severe mhv-1 infection [50] . the failure of mhv-1 to activate processing and presentation of mhc class i antigens could explain the inability of a broadly reactive cd8+ t cell response to protect these mice. the interferome analysis focuses on ifn-induced gene expression, but not expression of the type i ifns that induce these responses. multiple type i ifns exist, including ifn-β and 14 subtypes of ifn-α, all of which signal through the type i ifn α/β receptor [51] . type i ifns can induce autocrine and paracrine signaling; thus the ifn-induced genes we detected could be from both infected and uninfected cells in the cultures. to determine if differential expression of type i ifns explains the differences in ifn-induced gene expression upon infection by pr8, rv, and mhv, we analyzed the expression of type i ifn and receptor genes for each virus compared to mock (fig 5) . probes for ifn-β1 and ten subtypes of ifn-α were present on the arrays. in agreement with expression of ifn-induced genes, pr8 induced expression of the largest set of type i ifns, followed by rv. both viruses induced expression of ifnb and ifna4, which encode type i ifns known to be expressed early during antiviral responses [52, 53] . five subtypes of ifna were up-regulated by both pr8 and rv, while three ifna subtypes were uniquely up-regulated by pr8 and only ifnab was uniquely up-regulated by rv. only pr8 induced expression of ifnar2, which encodes the high affinity chain of the type i ifn α/β receptor [54] . this may allow for enhanced positive-feedback signaling and account for the larger number of ifn-induced genes up-regulated by pr8 infection. differential signaling through mda-5 and rig-i pathways may contribute to the differences in type i ifn responses by rv and pr8. rhinoviruses and influenza a viruses are known to induce type i ifn responses through recognition by mda-5 and rig-i, respectively [55, 56] . furthermore, both viruses are recognized by tlr3 in infected epithelial cells [55, 56] . however, tlr3 predominantly induces expression of pro-inflammatory genes, rather than type i ifn-dependent genes, during influenza a virus infection [55] . zaritsky et al. have demonstrated that the type i ifn response to sendai virus differs when cells are infected by different doses [57] . they further showed that these differences were mediated by differential signaling through the ifn α/β receptor, with robust signaling in uninfected cells. this supports our findings that pr8 induces expression of ifnar2 and additional type i ifn genes that are not up-regulated by rv (fig 5) . none of the type i ifns or receptors had significantly altered expression upon mhv infection (fig 5) , despite up-regulation of a modest number of ifn-stimulated genes (fig 4) . this could be due to ifn-independent expression of these genes, or induction by a type i ifn that was not represented on the microarray. coronaviruses are notorious for being able to replicate within cells without triggering type i ifn responses, or delaying ifn induction until late in the replication cycle [36, [58] [59] [60] . other studies have shown that the ifn response to mhv-1 is a critical determinant of susceptibility. severe disease in a/j mice compared to c57bl/6 mice correlates with lower type i ifns detected in the lungs of a/j mice upon mhv-1 infection [6, 61] . similarly, the expression of various type i ifns in response to mhv-1 infection in vitro is cell line-dependent [61] . because the cell line we used, la4, was derived from the lungs of a/ he mice, we would expect it to have a similar response as a/j mice. thus the lack of type i ifns induced by mhv-1 in la4 cells in vitro corresponds with pathogenesis observed in a/j mice in vivo. the finding that la4 cells mount a stronger response to pr8 than rv or mhv infection may be due to differences in the viral recognition and signaling pathways used to detect these different viruses and amplification of the type i ifn response as discussed above. alternatively, it could be due to the timing of our analysis. rv-infected cells have started dying by the 24 h time point (not shown), therefore expression of host genes may be declining by that time point. in contrast, coronaviruses are known to delay cellular responses to infection [62] , so the 24 h time point may be too early to evaluate the innate response to mhv infection. alternatively, the cells may detect mhv and up-regulate transcription of ifn response genes, but mrna degradation would mask this process. by quantifying mrna transcripts at two time points after viral infection, our study cannot distinguish between these possibilities. one limitation of our study is the analysis of three viruses that do not share a natural host. mhv is a natural pathogen of mice and pr8 is a highly mouse-adapted strain of influenza a virus. however, rv1b is a human rhinovirus whose receptor is conserved between mice and humans. rv1b is increasingly being used in mouse studies [1] [2] [3] [4] 63] . despite the difference in host, we found similar changes to gene expression in murine cells as studies with rv1b in human cells [24] . of the 24,204 and 12,438 genes represented on our mouse microarray and the human microarray chip used by chen et al., respectively, 10,847 genes are shared. using the same 2-fold increase in expression cut-off and restricting our list only to homologous human genes studied by chen et al., we found that 196 mouse genes were up-regulated by rv1b infection. comparing this list of 196 genes to the 48 up-regulated human genes identified by chen et al., we found that 20 genes (s6 table) were up-regulated by rv1b infection in both human and mouse cells. a chi-squared test confirmed the significance of this shared number of up-regulated genes (χ 2 = 431.7, d.f. = 1, p<0.001). interestingly, all 20 of the shared genes we identified are involved in type i ifn responses. while far from identical, the similarity of the responses in the two cell types suggests conserved activation of type i ifn responses by these different hosts and supports the validity of a murine model for studying rhinovirus infections in humans. alveolar epithelial cells have a key role in alerting the immune system to infection by respiratory viruses and shaping immune responses [39, 64, 65] . as viruses from several different families all target respiratory epithelial cells, it is important to understand the similarities and differences in how these cells respond to a diverse set of viruses. a significant number of genes were up-or down-regulated in response to infection by three distinct viruses from different families. genes that were associated with a shared response to the three viruses included those involved in defense against viruses and particularly genes induced by type i ifns. however, there were differences in the timing, numbers of genes altered, and expression levels of these genes. this may reflect differences in viral replication cycles and signaling pathways that are activated by infection, which may reflect differences in pathogenesis of these viruses in murine models. la4 cells were inoculated with virus using the same moi's as for the microarray study and rna was extracted at 24 h post-infection using trizol (ambion). rna was converted to cdna using random hexamers and super-script vilo (invitrogen). five genes with differential expression by microarray analysis were validated by qpcr analysis using sybr green (powerup, applied biosystems) and the primer pairs listed in the figure on a stepone plus instrument (applied biosystems). ct values from triplicate qpcr reactions were averaged and normalized to β-actin before calculating the fold change values of virus-infected samples vs. mock. linear regression was used to compare fold change values between qpcr and microarray with removal of outliers ( ã ) with cook's distance > 0.5. (pdf) genes with significantly up-regulated expression compared to mock at 24 h (see fig 2) were used to query the interferome v.2.01 database. interferon-regulated genes were divided into functional groups and heat maps were generated using log 2 -fold change values for each virus at 24 h compared to mockinoculated controls. heat maps of additional functional groups can be found in fig 4. gene names are indicated to the right of each row and statistically significant values are outlined in black. (pdf) s1 table. genes whose expression was significantly up-regulated by all three viruses compared to mock-inoculated cells. these genes were from the center of the venn diagram in fig 2a. (xlsx) s2 table. genes whose expression was significantly down-regulated by all three viruses compared to mock-inoculated cells. these genes were from the center of the venn diagram in fig 2b. (xlsx) s3 table. signature genes for pr8. these genes were significantly different in pr8 infection compared to mock, rv, and mhv. (xlsx) s4 table. signature genes for rv. these genes were significantly different in rv infection compared to mock, pr8, and mhv. (xlsx) s5 table. signature genes for mhv. these genes were significantly different in mhv infection compared to mock, rv, and pr8. (xlsx) s6 table. genes up-regulated by rv in both murine and human cells. these genes were identified to be up-regulated in our study by rv1b in murine cells and also were found by chen et al. (23) to be up-regulated by rv1b in human cells. 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journal the interferons and their receptors-distribution and regulation cutting edge: influenza a virus activates tlr3-dependent inflammatory and rig-i-dependent antiviral responses in human lung epithelial cells role of double-stranded rna pattern recognition receptors in rhinovirus-induced airway epithelial cell responses virus multiplicity of infection affects type i interferon subtype induction profiles and interferon-stimulated genes murine coronavirus cell type dependent interaction with the type i interferon response group 2 coronaviruses prevent immediate early interferon induction by protection of viral rna from host cell recognition mouse hepatitis virus does not induce beta interferon synthesis and does not inhibit its induction by double-stranded rna distinct signature type i interferon responses are determined by the infecting virus and the target cell dynamic innate immune responses of human bronchial epithelial cells to severe acute respiratory syndrome-associated coronavirus infection toll-like receptor 2-expressing macrophages are required and sufficient for rhinovirus-induced airway inflammation host-pathogen interactions during coronavirus infection of primary alveolar epithelial cells virus-infected alveolar epithelial cells direct neutrophil chemotaxis and inhibit their apoptosis the authors are grateful to dr. kathryn holmes, university of colorado at denver school of medicine, dr. elizabeth fortunato, university of idaho, and dr. julian leibowitz, texas a&m university for cells and antibodies that were used in this study. the following reagents were key: cord-310678-33c3mp1o authors: morgantini, luca a.; naha, ushasi; wang, heng; francavilla, simone; acar, ömer; flores, jose m.; crivellaro, simone; moreira, daniel; abern, michael; eklund, martin; vigneswaran, hari t.; weine, stevan m. title: factors contributing to healthcare professional burnout during the covid-19 pandemic: a rapid turnaround global survey date: 2020-09-03 journal: plos one doi: 10.1371/journal.pone.0238217 sha: doc_id: 310678 cord_uid: 33c3mp1o background: healthcare professionals (hcps) on the front lines against covid-19 may face increased workload and stress. understanding hcps’ risk for burnout is critical to supporting hcps and maintaining the quality of healthcare during the pandemic. methods: to assess exposure, perceptions, workload, and possible burnout of hcps during the covid-19 pandemic we conducted a cross-sectional survey. the main outcomes and measures were hcps’ self-assessment of burnout, indicated by a single item measure of emotional exhaustion, and other experiences and attitudes associated with working during the covid-19 pandemic. findings: a total of 2,707 hcps from 60 countries participated in this study. fifty-one percent of hcps reported burnout. burnout was associated with work impacting household activities (rr = 1·57, 95% ci = 1·39–1·78, p<0·001), feeling pushed beyond training (rr = 1·32, 95% ci = 1·20–1·47, p<0·001), exposure to covid-19 patients (rr = 1·18, 95% ci = 1·05–1·32, p = 0·005), and making life prioritizing decisions (rr = 1·16, 95% ci = 1·02–1·31, p = 0·03). adequate personal protective equipment (ppe) was protective against burnout (rr = 0·88, 95% ci = 0·79–0·97, p = 0·01). burnout was higher in high-income countries (hics) compared to lowand middle-income countries (lmics) (rr = 1·18; 95% ci = 1·02–1·36, p = 0·018). interpretation: burnout is present at higher than previously reported rates among hcps working during the covid-19 pandemic and is related to high workload, job stress, and time pressure, and limited organizational support. current and future burnout among hcps could be mitigated by actions from healthcare institutions and other governmental and non-governmental stakeholders aimed at potentially modifiable factors, including providing additional training, organizational support, and support for family, ppe, and mental health resources. to assess exposure, perceptions, workload, and possible burnout of hcps during the covid-19 pandemic we conducted a cross-sectional survey. the main outcomes and measures were hcps' self-assessment of burnout, indicated by a single item measure of emotional exhaustion, and other experiences and attitudes associated with working during the covid-19 pandemic. a total of 2,707 hcps from 60 countries participated in this study. fifty-one percent of hcps reported burnout. burnout was associated with work impacting household activities (rr = 1�57, 95% ci = 1�39-1�78, p<0�001), feeling pushed beyond training (rr = 1�32, 95% ci = 1�20-1�47, p<0�001), exposure to covid-19 patients (rr = 1�18, 95% ci = 1�05-1�32, p = 0�005), and making life prioritizing decisions (rr = 1�16, 95% ci = 1�02-1�31, p = 0�03). adequate personal protective equipment (ppe) was protective against burnout (rr = 0�88, 95% ci = 0�79-0�97, p = 0�01). burnout was higher in high-income countries (hics) compared to low-and middle-income countries (lmics) (rr = 1�18; 95% ci = 1�02-1�36, p = 0�018). plos one | https://doi.org/10.1371/journal.pone.0238217 september 3, 2020 1 / 11 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 more than 200 countries worldwide are impacted by the spread of the novel coronavirus (sars-cov-2), the pathogen responsible for the coronavirus disease 2019 . their healthcare systems are frantically maximizing efforts to deploy resources in order to mitigate spread and reduce morbidity and mortality from covid-19. large numbers of healthcare professionals (hcps) on the frontlines face high adversity, workloads, and stress, making them vulnerable to burnout [1, 2] . burnout, defined by emotional exhaustion, depersonalization, and personal accomplishment, is known to detract from optimal working capacities, and has been previously shown to be similarly prevalent among hcps in hics (high-income countries) and lmics (low-to-middle-income countries) [3] [4] [5] [6] . burnout has been found to be driven by high job stress, high time pressure and workload, and poor organizational support. these factors are common between hics and lmics despite their differences in healthcare and socioeconomic structures [3] . researchers have begun exploring the impact of the covid-19 pandemic on hcps' mental health. barello et al. assessed 376 italian hcps who interacted with covid-19 infected patients for their reported burnout, psychosomatic symptoms and self-perceived general health, finding in their study population high emotional burnout, physical symptoms, and work-related pressure [7] . the society of critical care medicine surveyed 9492 intensive care unit clinicians in the u.s. and found that median self-reported stress, measured on a scale from 0 to 10, increased from 3 to 8 during the pandemic [8] . the principal stressors included concern for lack of personal protective equipment (ppe), and work impacting household activities and interactions [8] . shanafelt et al. identified the necessity for hcps to care for patients that required clinical skills beyond their training as an additional stressor, among others [9] . the pandemic has not affected all hcps in the same manner, as there have been demonstrated differences based on occupation and patient population. lai et al. demonstrated how hcps in wuhan, especially nurses and frontline workers, were experiencing the highest psychological burden in late january 2020 [1] . zerbini et al. identified how german nurses working in covid-19 wards reported worse burnout scores compared to their colleagues in regular wards, while physicians reported similar scores independently from their covid-19 workload [10] . in contrast, wu et al. reported in their study of 190 hcps in wuhan how individuals working in their usual ward reported a higher frequency of burnout and fear of being infected, when compared to their colleagues working with covid-19 patients [11] . differences in the perception of the pandemic, the local spread of the pandemic at the time of study, support structures, or definition of burnout that may explain these diametrically opposed results [12] . because each study focused on hcps working in a particular region or country, it is impossible to draw conclusions about the impact on hcps globally. the objective of this study was to understand the impact of covid-19 on hcps around the world working during the pandemic. this was the first intercontinental survey examining the perceptions of hcps during the covid-19 pandemic without restriction on geographic location or covid-19 exposure. given that the pandemic has not affected all nations in the same time frame, gathering opinions from hcps worldwide within a single time range offers a unique snapshot of how the pandemic affects hcps at that moment. our aim was to describe current contributing factors associated with hcps burnout during the pandemic and to provide data that will drive future research on mitigating burnout. the university of illinois at chicago (uic) institutional review board (irb) determined on april 1 st , 2020 that this study, with assigned protocol number 2020-0388, met the criteria for exemption as defined in the u.s. department of health and human services regulations for the protection of human subjects [45 cfr 46. 104(d)]. before initiating the survey, respondents were informed that their responses would be shared with the scientific community. survey responses were recorded and stored without participant identifiers using the redcap electronic data capture software hosted by uic servers [13, 14] . redcap (research electronic data capture) is a secure, web-based software platform designed to support data capture for research studies, providing 1) an intuitive interface for validated data capture; 2) audit trails for tracking data manipulation and export procedures; 3) automated export procedures for seamless data downloads to common statistical packages; and 4) procedures for data integration and interoperability with external sources. inclusion criteria was restricted to hcps. platforms including facebook, whatsapp, and twitter, as well as e-mail, were used for global recruitment and dissemination from april 6 to april 16, 2020. potential study participants were approached via irb-approved messages containing a link to the survey shared on the aforementioned social media. study participants were also asked to share the link with their colleagues via personal networks. demographic data collected from the survey participants was limited to the country of provenience and occupation. the survey contained 40 questions covering three major domains of hcps experience (exposure, perception, and workload) that were validated by experts in infectious diseases, public health, occupational medicine, psychology, and clinical psychiatry. elements of these domains were previously proposed as contributing toward hcp anxiety during the covid-19 pandemic [15] . the main outcome, hcps-perceived burnout in its core domain of emotional exhaustion, was assessed by a single item on a 7-point likert scale (1: strongly disagree to 7: strongly agree) using the statement, "i am burned out from my work [16] . only the core domain of emotional exhaustion was assessed as previous research has demonstrated that the depersonalization and personal accomplishment domains represented a western concept not generalizable across different cultures [12] . the questionnaire was developed with a pilot group of 10 hcps and 40 questions were included based on expert opinion (s1 questionnaire) that were then translated into 18 languages by professional translators. the country of the respondents was categorized as highincome or low-and middle-income as defined by the world bank classification system in order to reduce confounding factors such as differences in the number of covid-19 cases (fig 1) , healthcare system, and socioeconomic structure [3, 17] . covid-19 deaths and cases per 1 million population were obtained from a widely used web-based dashboard [18] . a descriptive assessment was performed for each variable surveyed for all data, country by country, and according to the income level (high vs. low-middle). covariates collected as ordinal variables were transformed into binary (s1 table) . for burnout, scores � 5 were considered burned out [16] . quasi-poisson regression analysis was performed using the binary burnout outcome to compare factors associated with low and average burnout against high emotional exhaustion burnout [19] . relative risk (rr) was reported with nominal 95% confidence intervals and 2-sided p values. only the participants who responded completely to the variables of interest were included in regression analyses. a total of 2,707 responses were received from hcps in 60 countries. fig 1 demonstrates the study period in context of the covid-19 pandemic (s2 table) [17, 18] . table 1 summarizes participant characteristics and responses (additional responses in s3 table) . half (51�4%) of the respondents from 33 countries reported emotional exhaustion burnout related to their work during the covid-19 pandemic. the u.s. had the highest reported burnout among all countries at a rate of 62�8%. across all countries (fig 2) , in the multivariable regression analysis, reported burnout was associated with work impacting household activities (rr = 1�57, 95% ci = 1�39-1�78, p<0�001), feeling pushed beyond training (rr = 1�32, 95% ci = 1�20-1�47, p<0�001), exposure to covid-19 patients (rr = 1�18, 95% ci = 1�05-1�32, p = 0�005), and making life prioritizing decisions due to supply shortages (rr = 1�16, 95% ci = 1�02-1�31, p = 0�03). adequate ppe was protective against reported burnout (rr = 0�88, 95% ci = 0�79-0�97, p = 0�01). the answers of the individuals that were not included in the regression analyses due to missing data did not significantly differ from those who did completely respond. country-level analysis revealed lower reported burnout in italy (rr = 0�72, 95% ci = 0�61-0�84, p<0�001) and sweden (rr = 0�43, 95% ci = 0�30-0�59, p<0�001) compared to the u.s. predictors of burnout differed between lmics and hics (s1 fig). among the 314 respondents from lmics, reported burnout was associated with work impacting household activities (rr = 2�31, 95% ci = 1�61-3�43, p<0�001) and adequate ppe (rr = 0�68, 95% ci = 0�52-0�90, p = 0�007). in the 1334 respondents from hics, reported burnout was associated with feeling pushed beyond training (rr = 1�41, 95% ci = 1�06-1�88, p = 0�02), difficulty obtaining covid-19 testing (rr = 1�43, 95% ci = 1�04-1�94, p = 0�03), work impacting quality of life (rr = 1�67, 95% ci = 1�12-2�59, p = 0�02), work impacting household activities (rr = 1�75, 95% ci = 1�16-2�75, p = 0�01), and mental health support (rr = 0�72, 95% ci = 0�54-0�96, p = 0�03). among respondents, half of hcps from 33 countries reported burnout. previously reported rates of hcp burnout have ranged from 43% to 48% [3] . burnout for hcps working during the covid-19 pandemic was associated with factors that typically increase the likelihood of hcp burnout [1, 3] . these included feeling pushed beyond training (high workload), making life-or-death prioritizing decisions (high job stress), work impacting the ability to perform household activities (high time pressure), and lack of adequate ppe (limited organizational support). burnout among hcps could be reduced by actions from healthcare institutions and other governmental and non-governmental stakeholders aimed at potentially modifiable factors. these could include providing additional training and mental health resources, strengthening organizational support for hcps' physical and emotional needs, supporting family-related issues (e.g. helping with childcare, transportation, temporary housing, wages), and acquiring ppe. a systematic review showed that both individual-and organizational-level strategies are effective in meaningfully reducing burnout. some of the most commonly utilized methods focused on mindfulness, stress management and small group discussion [20] . future studies should examine if and how the implementation of such strategies can reduce burnout among hcps during the pandemic. recent studies regarding hcps' mental health in response to covid-19 from china, as well as prior studies of other pandemics, have demonstrated that hcps may experience depression, anxiety, and posttraumatic stress disorder. shanafelt et al. highlighted common sources of anxiety from listening sessions with hcps that align with our findings, such as access to adequate ppe, unknowingly bringing the infection home, and lack of access to up-todate information and communication [9] . some hcps who worked extensively during the sars pandemic in beijing later demonstrated posttraumatic stress symptoms (ptss), and many hcps in the areas hardest-hit by covid-19 in china have already started exhibiting similar complaints [21, 22] . to prevent adverse psychological outcomes, mental health support for hcps is critical [2, 23] . key interventions include access to psychosocial support including web-based resources, emotional support hotlines, psychological first aid, and self-care strategies. burnout can impact not only mental health but also can correlate with physical ailments. a systematic review found that burnout was a predictor for conditions including musculoskeletal pain, prolonged fatigue, headaches, gastrointestinal and respiratory issues [24] . some factors included in our survey, such as increased workload hours, inadequate ppe or lack of updated guidelines, contributed to higher rates of infection among hcps at the beginning of the outbreak in late january [25] . burnout was higher in those countries where the covid-19 pandemic was surging at the time of data collection (e.g. the u.s.) compared with those where it was declining (e.g. italy) or had not reached the peak (e.g. turkey). the lower reported burnout among hcps in lmics may reflect resilience due to more experience working in conditions with high adversity and limited availability of supplies [26] . additionally, the greater reported burnout by hcps in hics could be attributed to their greater covid-19 burden. addressing burnout in all countries is crucial, but our findings indicate that different strategies should be tailored to the phase of pandemic and the sociocultural and healthcare organizational contexts. despite this study's major strengths, including the breadth of responses from across the globe, there are multiple limitations including a non-validated questionnaire, not providing the definition of burnout to participants before the initiation of study, a single item indicator for burnout, minimal demographic data collection, and sampling method using social media. by utilizing recruitment and dissemination strategies dependent on social media, there is a potential selection bias resulting in overrepresentation of hcps more active on social media forums. the lack of extensive demographic collection, designed to increase participation, limits the ability to assess the representativeness of the study sample. future studies should consider expanding beyond the single item to explain the complexity of burnout in hcps as this study only represents an indication of reported emotional exhaustion in the study participants [16] . causality between the covid-19 pandemic and hcps' reported burnout cannot be determined due to the nature of this observational study, and additional studies are needed to ascertain causality. furthermore, drawing comparisons between countries is limited by the differences in cultures, languages, and healthcare systems. the definition and perception of burnout varies across countries, although the domain of emotional exhaustion, as investigated in this study, remains consistent and validated across all translated languages [12] . while hcps wage a war against covid-19, institutions must support these individuals as they face enormous stress that can negatively impact their emotional and physical well-being. our study is the first worldwide survey of hcps during the covid-19 pandemic and demonstrates the presence of reported burnout among respondents at a rate higher than previously reported. reported burnout was significantly associated with, among others, limited access to ppe as well as making life-or-death decisions due to medical supply shortages. furthermore, reported burnout was associated with different factors in hics and lmics. current and future burnout among hcps could be mitigated by actions from healthcare institutions and other governmental and non-governmental stakeholders aimed at potentially modifiable factors, including providing additional training, organizational support, support for hcps' families, ppe, and mental health resources. factors associated with mental health outcomes among health care workers exposed to coronavirus disease mental health care 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during the covid-19 epidemic in wuhan burnout: a short socio-cultural history research electronic data capture (redcap)-a metadata-driven methodology and workflow process for providing translational research the red-cap consortium: building an international community of software partners healthcare workers' attitudes towards working during pandemic influenza: a multi method study single item measures of emotional exhaustion and depersonalization are useful for assessing burnout in medical professionals how does the world bank classify countries?-world bank data help desk. datahelpdesk.worldbank. org an interactive web-based dashboard to track covid-19 in real time [published online ahead of print quasi-likelihood estimation for relative risk regression models interventions to prevent and reduce physician burnout: a systematic review and meta-analysis. the lancent the psychological impact of the sars epidemic on hospital employees in china: exposure, risk perception, and altruistic acceptance of risk prevalence and predictors of ptss during covid-19 outbreak in china hardest-hit areas: gender differences matter impact on mental health and perceptions of psychological care among medical and nursing staff in wuhan during the 2019 novel coronavirus disease outbreak: a cross-sectional study physical, psychological and occupational consequences of job burnout: a systematic review of prospective studies reasons for healthcare workers becoming infected with novel coronavirus disease 2019 (covid-19) in china resilience of primary healthcare professionals working in challenging environments: a focus group study we acknowledge the support received from uic center for clinical and translational sciences' community engagement and collaboration core, dr. craig niederberger and dr. ervin kocjancic. the authors acknowledge the research open access publishing (roaap) fund of the university of illinois at chicago for financial support towards the open access publishing fee for this article. key: cord-268429-drejl99k authors: almberg, emily s.; mech, l. david; smith, douglas w.; sheldon, jennifer w.; crabtree, robert l. title: a serological survey of infectious disease in yellowstone national park’s canid community date: 2009-09-16 journal: plos one doi: 10.1371/journal.pone.0007042 sha: doc_id: 268429 cord_uid: drejl99k background: gray wolves (canis lupus) were reintroduced into yellowstone national park (ynp) after a >70 year absence, and as part of recovery efforts, the population has been closely monitored. in 1999 and 2005, pup survival was significantly reduced, suggestive of disease outbreaks. methodology/principal findings: we analyzed sympatric wolf, coyote (canis latrans), and red fox (vulpes vulpes) serologic data from ynp, spanning 1991–2007, to identify long-term patterns of pathogen exposure, identify associated risk factors, and examine evidence for disease-induced mortality among wolves for which there were survival data. we found high, constant exposure to canine parvovirus (wolf seroprevalence: 100%; coyote: 94%), canine adenovirus-1 (wolf pups [0.5–0.9 yr]: 91%, adults [≥1 yr]: 96%; coyote juveniles [0.5–1.5 yrs]: 18%, adults [≥1.6 yrs]: 83%), and canine herpesvirus (wolf: 87%; coyote juveniles: 23%, young adults [1.6–4.9 yrs]: 51%, old adults [≥5 yrs]: 87%) suggesting that these pathogens were enzootic within ynp wolves and coyotes. an average of 50% of wolves exhibited exposure to the protozoan parasite, neospora caninum, although individuals’ odds of exposure tended to increase with age and was temporally variable. wolf, coyote, and fox exposure to canine distemper virus (cdv) was temporally variable, with evidence for distinct multi-host outbreaks in 1999 and 2005, and perhaps a smaller, isolated outbreak among wolves in the interior of ynp in 2002. the years of high wolf-pup mortality in 1999 and 2005 in the northern region of the park were correlated with peaks in cdv seroprevalence, suggesting that cdv contributed to the observed mortality. conclusions/significance: of the pathogens we examined, none appear to jeopardize the long-term population of canids in ynp. however, cdv appears capable of causing short-term population declines. additional information on how and where cdv is maintained and the frequency with which future epizootics might be expected might be useful for future management of the northern rocky mountain wolf population. several high-mortality disease outbreaks among carnivore populations have demonstrated the potential for pathogen-induced population declines [1] [2] [3] [4] [5] [6] and have emphasized the role of infectious disease in carnivore conservation [7, 8] . these outbreaks have underscored both the need for better baseline data on disease prevalence, and a better understanding of the ecology of disease in wild populations [7, 9] . yellowstone national park (ynp) is home to one of the largest, protected, intact suites of carnivores in the contiguous united states. gray wolves (canis lupus) were reintroduced into the yellowstone ecosystem after a . 70 year absence, and as part of recovery efforts, the population is closely monitored [10, 11] . in 1999 and 2005, pup survival was significantly reduced, suggestive of a disease outbreak [12] . thus we sought to use long-term serological data to identify temporal, spatial, and demographic patterns of pathogen exposure among wolves, coyotes (canis latrans), and foxes (vulpes vulpes) in ynp. we screened for exposure to canine parvovirus (cpv), canine adenovirus type-1 (cav-1), canine distemper virus (cdv), and canine herpesvirus (chv), all of which can inflict morbidity and mortality in canids (table s1 ) [13] [14] [15] [16] [17] [18] [19] . in wolves, we also screened for exposure to neospora caninum, a protozoan parasite whose life cycle includes canids, the definitive hosts where sexual reproduction takes place, and ungulates, the intermediate hosts [20, 21] . n. caninum is transmitted between canids and ungulates when a canid consumes infected ungulate tissue. n. caninum reproduces in the canid's intestines, and oocysts are shed in feces and then consumed by ungulates through contaminated forage and water. n. caninum infection can cause high rates of abortion in cattle, and thus is a pathogen of special interest to the local ranching community. we assessed whether each of the pathogens of interest were enzootic or epizootic in the ynp canid community, and whether pathogen exposure varied by region of the park in relation to canid density. we investigated if behavioral differences between resident and transient coyotes, the latter potentially interacting with many more individuals across many different packs, and thus potentially at greater risk for pathogen exposure, contributed to differences in exposure risk. host age was used primarily to examine temporal patterns of exposure, but it was also evaluated as a risk factor for recent or current infection with chv and n. caninum. survival data were not available for coyotes or foxes. however, we did examine the relationship between pathogen exposure and wolf-pup survival. furthermore, we used comparisons of exposure patterns among the canids to assess the likelihood of single versus multi-host pathogen transmission within ynp. all wolves, coyotes, and foxes used in this study were handled in strict accordance with recommendations from the american society of mammalogists [22, 23] , and all animal work was approved by a national park service veterinarian, a ynp review committee, and by the ynp superintendent. ynp encompasses 8,991-km 2 of protected land in northwestern wyoming and adjacent parts of montana and idaho in the western united states (44u339 n, 110u309 w). ynp is surrounded by the greater yellowstone ecosystem (gye), a 60,000-km 2 area that includes yellowstone and grand teton national parks, national forests, wildlife refuges, and a mosaic of state and private lands. ynp is mountainous (elevation range: 1,500 to 3,800 m), and its steep gradients in elevation, soil, and climate contribute to varied land cover, including riparian vegetation, shrubland, grassland, alpine meadows, and mixed coniferous forests. we divided the park into two units, the northern range (nr) and the interior, based on ecological and physiographical differences [24] . the 1,000-km 2 area of the nr within ynp is characterized by lower elevations (1,500-2,200 m), serves as prime wintering habitat for the park's ungulates [25] , and supports a higher density of wolves than the interior (20-99 wolves/1000 km 2 versus 2-11 wolves/1000 km 2 [26] ; minimum population count for entire ynp ranged between 118 and 172 wolves between 2000 and 2007 [27] ). the interior of the park (7,991 km 2 ) is higher in elevation (.2,500 m), receives higher annual snowfall, and generally supports lower densities of wolves and ungulates, with the exception of a large migratory herd of bison (bison bison). ynp has an intact suite of terrestrial carnivores, including gray wolves, grizzly (ursus arctos) and black bears (ursus americanus), cougars, coyotes, red foxes, badgers (taxidea taxus), river otters (lontra canadensis), american martens (martes americana), short (mustela erminea) and long-tailed weasels (mustela frenata), striped skunks (mephitis mephitis), and wolverines (gulo gulo) [28] . although extremely rare inside ynp, raccoons (procyon lotor) are present in the surrounding gye. wolves. since wolf reintroduction to ynp, the national park service has captured and radio-collared an annual average of 26 wolves (range spanning all known packs in the park (mean packs sampled per year = 8, range = 4-12). collaring efforts have generally targeted breeders and ,50% of each year's young, with an emphasis on maintaining contact with each pack. we darted wolves from a helicopter during november-march and anesthetized them using a 10 mg/kg dose of telazolh (tiletamine & zolazepam). we fitted them with radio-collars (telonics, inc. mesa, az), drew 6-8 ml of blood from the saphenous vein, and categorized the animals as pups (,12 months) or adults, with precise ages estimated from tooth wear [29] . we stored whole blood and serum (serum collected by centrifuging whole blood for 15 minutes after 30 minutes of rest) at 280 c until analysis. following capture, each wolf was identified as belonging to a particular pack. coyotes and foxes. staff from the yellowstone ecological research center (bozeman, mt, usa) captured coyotes on the nr of ynp during three, multi-year sampling intervals (1991-1992, 1996-1999, and 2003-2005) . foxes were also captured on the nr of ynp, but trapping efforts were less intense and less frequent (1993, 1996, 2003, and 2005) . coyotes and foxes were captured from three regions (lamar valley, blacktail plateau, and gardiner river basin) spanning east to west on the nr inside ynp from september through october. juvenile and adult coyotes and foxes were captured using padded, offset, centerswivel, foot-hold traps (victor soft-catch, woodstream corp., lititz, pa, usa) baited with species-specific urine lures. sex, weight, condition, dentition, and body measurement data were collected for each animal. individuals were classified as juveniles (0.5-1.5 yrs), young adults (1.6-4.9 yrs), or old adults ($5 yrs) based on tooth wear [30] . technicians drew blood and isolated serum as described for wolves and radio-collared (advanced telemetry systems, isanti, mn, usa; telonics, mesa, az, usa) the animals. monitoring of radio-collared coyotes permitted classifying individuals as residents (i.e., member of a territorial pack) or transients (i.e., solitary individuals, typically inhabiting an area overlapping one or more pack territories). however, we did not have detailed information on individual coyotes' pack membership or territory location. thus, exposure data from resident and transient individuals captured in the same region were assumed to be non-independent. 1991-1992, 1996-1999, and 2003-2005) , and foxes (n = 9 samples [3 females, 3 males, 3 unk.] during 1993, 1996, 2003, and 2005) were screened for antibodies to cpv, cav-1, cdv, chv, and n. caninum (wolf samples only due to insufficient quantities of coyote and fox sera) by the new york state animal health diagnostic center (ithaca, ny, usa). serum neutralization tests [31] were used to detect cav-1 (positive titer: $8), chv (positive titer: $8), and cdv (positive titer: .12) antibodies (titer cutoff selected so as to minimize false positives; data not shown) [32] . a hemagglutination inhibition test was used to detect cpv antibodies (positive titer: $20) [33] , and an indirect fluorescent antibody test was used to detect n. caninum antibodies (positive titer: $50) [34, 35] . data from wolf and coyote pups were used only for animals $5 months old to avoid the influence of maternal antibodies [36] [37] [38] . repeat samples from the same individual were excluded from the statistical analysis unless they seroconverted or tested negative for two consecutive sampling periods for a given pathogen. we identified wolf dens by tracking radio-collared adult females throughout april. dens were monitored and pups counted weekly in may and june. pup counts in the remote interior were primarily conducted from airplanes. aerial monitoring of nr dens was often supplemented with ground counts using spotting scopes. we estimated pups born per pack based on high counts observed between may-june. we also estimated pup survival per pack between may and december by calculating the proportion of pups in a pack still alive at the end of december based on weekly (at minimum) aerial and ground counts. survival data were not available for coyote-pups and fox-kits. to accommodate the available datasets and the biological differences between both the canid hosts and the pathogens, our analyses involved several different approaches outlined below. age effects. the viral pathogens cpv, cdv, and cav-1 generally produce long-lasting immunity in their hosts [14] [15] [16] [17] [18] , so we assumed that once a wolf, coyote, or fox was exposed to one of these pathogens they remained seropositive for life (although mech and goyal [unpublished] have found exceptions to this for cpv among wolves). under this assumption, the serological status of pups, as compared to adults, offers the most precise information about whether a pathogen is circulating in a given year or region. therefore, we examined wolf-pup and coyote-juvenile data separately from wolf adult ($1 yr) and coyote adult ($1.6 yrs) data in the analysis of cpv, cdv, and cav-1 serological data. by contrast, chv, a herpesvirus, produces life-long infections characterized by periods of latency where the virus is present but does not provoke a strong immune response [39] . a negative chv test result most likely reflects an uninfected individual, although a latent infection cannot be ruled out, whereas a positive result suggests exposure, a more recent infection, or recrudescence [19, 40] . canids acquire n. caninum infections by consuming ungulate tissue infected with the asexual stage of the parasite [41] , and a positive n. caninum test suggests an active or recent infection with the parasite [35] . because neither chv nor n. caninum induce consistent, long-term immunity, and because positive results suggest a recent or active infection, we evaluated age class (juvenile (wolf: 0.5-1.9 yrs; coyote: 0.5-1.5 yrs), young adult (wolf: 2-4.9 yrs; coyote: 1.6-4.9 yrs), and old adult (wolf & coyote: $5 yrs)) for both wolves and coyotes as a risk factor for recent infection in our analyses of these two pathogens. temporal, spatial, and demographic patterns of pathogen exposure. positive and negative test results were analyzed using a logistic, generalized, linear, mixed model with random ''pack,'' or in the case of coyotes, ''region'' effects [42, 43] . these random effects were considered important because they allowed for the nonindependence of individuals sampled from the same pack or trapping region. we developed sets of a priori candidate models including factors such as year, spatial location (wolves only; nr versus interior), resident versus transient status (coyotes only), and age class (chv and n. caninum analyses only), thought to potentially influence the probability of pathogen exposure ( table 1, table 2 ). year effects were evaluated to test the evidence for temporal variation in exposure, location effects to determine whether nr wolves, living at higher densities, exhibited a higher risk of exposure compared to interior wolves, and a year*location interaction to allow for the possibility that pathogens circulate at different times between the nr and interior regions of the park. among coyotes, we asked whether behavioral differences between residents and transients might contribute to differences in their risk of infection. finally, as described above, we evaluated age class as a risk factor for recent infection with chv or n. caninum. sets of candidate models for wolves and coyotes were evaluated for each pathogen using model-selection procedures based on akaike's information criterion, corrected for small samples (aic c ) [44]. all candidate models within ,2 aic c units from the bestsupported model (lowest aic c value) were considered to have reasonable support, given the data and set of models [44] . relative support for each model was evaluated based on its akaike weight, [1995] [1996] [1997] [1998] [1999] [2000] [2001] [2002] [2003] [2004] [2005] [2006] [2007] and location effects on wolf-pup survival were evaluated using a logistic, generalized, linear, mixed model with random pack effects and aic c modelselection procedures. not all monitored dens were visible from the air or ground, so we did not always have pup counts to match the serological results from a particular pack to directly test the relationship between seroprevalence and survival. therefore, while the strength of inference was reduced, we used regression analyses to examine the relationship between annual wolf-pup survival and annual wolf-pup seroprevalence (n = 11 years of estimates), broken down by location (i.e., nr and interior). temporal, spatial, and demographic patterns of exposure wolf cpv seroprevalence was 100% across all years, locations, pups, and adults ( table 3 ; thus none of the wolf models were relevant for table 4 or s2). the best-supported models ( by contrast, wolf and coyote exposure to cdv varied annually. the best-supported models for cdv exposure suggested constant, low pup exposure and a year effect among adults. there was also marginal support for a model with year and location effects among both wolf pups (d aic c = 2.04, weight = 0.24) and adults (d aic c = 2.11, weight = 0.26) which exhibited a much better fit, particularly to the pup data. among adult coyotes, the bestsupported models included year and resident effects. while the best-supported model for juvenile coyote seroprevalence suggested constant, near-zero exposure, the model exhibited poor fit to 2 years of the data (i.e., 1999 and 2005; figure 1 ). the probability of cdv exposure among wolf pups was highest in 1999, 2002, and 2005, a pattern less clearly mirrored in the adult data (no year effect was significant) (figure 1 , table s3 ). between these three outbreak years, there was evidence for a small amount of seroconversion among pups (20-33% in 2000, 2001, 2003 , and 2004; figure 1 ). in addition, both nr pups and adults had as much as a 36% and 14% positive difference, respectively, in their probability of exposure compared to their interior counterparts (pups: or = 4.25, 95% ci: 0.97, 18.54; adults: or = 1.72; 95% ci: 0. 36, 8.25) . both juvenile and adult coyote seroprevalence mirrored the temporal patterns among nr wolf pups; cdv seroprevalence was 100% in 1999 and 2005 among both age groups and 0% otherwise among juveniles (year effects were not significant; figure 1 ). furthermore, adult resident coyotes had as much as an 18% positive difference in the probability of cdv exposure compared to adult transients (or = 2.05, 95% ci: 0.41, 10.18), although this difference was not statistically significant. the best-supported model for wolf exposure to chv included a covariate for age class; however, wolf chv seroprevalence was uniformly high (87%) and estimated probabilities of exposure were 1.0 for all three age classes (95% cis, juveniles: 0.97-1.0; young adults: 0-1.0; old adults: 0-1.0). among coyotes, the two competing models with nearly equal aic c weights suggested support for age class and resident status covariates in the risk of chv exposure. the probability of chv exposure among coyotes significantly increased with age class; juveniles had the lowest probability of exposure (pr[e] = 0.20, 95% ci: 0.09, 0.38; seroprevalence = 23%), followed by young adults (pr[e] = 0.81, 95% ci: 0.69, 0.89; seroprevalence = 51%), and old adults (pr[e] = 0.96, 95% ci: 0.83, 0.99; seroprevalence = 87%). although not statistically significant, resident coyotes had as much as an 11% positive difference in their probability of chv exposure compared to transients (or = 1.58, ci: 0.59, 4.18). the four best-supported models for n. caninum exposure among wolves suggested that age class, year, and location were important covariates (table 4 ). wolves' probability of exposure increased with age; old adults had the greatest probability of exposure to n. caninum there were too few fox samples to look for patterns of exposure, but we did find evidence for fox exposure to cpv, cav-1, cdv, and chv (table 5) . wolf-pup survival and correlations with pathogen exposure between 1995 and 2007, we annually monitored an average of 9 (sd = 4, range = 2-15) wolf dens, or an average of 84% (sd = 14%) of reproducing packs. although the best-supported model for annual wolf-pup survival included only year and location covariates, there was also model support for a year*location interaction (table 4 ). pup survival was significantly lower on the nr than in the interior (or = 0.25, 95% ci: 0.12, 0.49) (figure 3) . although there was no significant year effect common to both nr and interior wolves, the probability of survival was significantly lower among nr pups in 2005 (pr[survival] = 0.13, 95% ci: 0.04, 0.33) (survival = 13%) when compared to most years, and lower than average, but not significantly so, in 1999 (pr[survival] = 0.37, ci: 0.12, 0.71) (survival = 37%) (see figure 3 for a comparison of 95% cis). although the 95% cis on the nr survival estimates from 2005 overlap with those of 1995 and 1996, the latter's confidence intervals are almost certainly too large. survival estimates in these two years were derived from censuses of the small, closely monitored, reintroduced population, and thus were both accurate and precise. annual wolf-pup cdv seroprevalence coincided with significant variation in annual pup survival on the nr (r 2 = 0.69, t = 24.51, df = 10, p = 0.001), although this was not the case in the interior (r 2 = 0.02, t = 0.48, df = 10, p = 0.65). none of the other viral pathogens (cpv, cav-1, and chv) exhibited significant temporal variation capable of explaining temporal patterns of pup survival. annual wolf-pup survival was independent of annual pup exposure to n. caninum (nr: r 2 = 0.18, t = 21.39, df = 10, p = 0.20; interior: r 2 = 0.09, t = 20.91, df = 10, p = 0.38). the discussion that follows must be qualified by the fact that overall, our sample sizes were quite small. small samples reduced our accuracy and precision in estimating exposure rates as well as our power to detect significant differences in exposure between groups, hence limiting the strength of our inferences based on our data. this is particularly apparent in our analysis of cdv exposure, where our supported models included many estimable parameters. however, in some cases, small samples were unavoidable. for example, in 1999 and 2005, pup survival was so poor that only 13 and 8 pups, respectively, were known to be alive on the nr, making it very difficult to capture pups in those years. our conclusions must further be qualified by the fact that our serological assays were not specifically validated or optimized for wolves, coyotes, or foxes. without knowing the sensitivity and specificity of our tests for these species, we do not know the degree to which our positive and negative test results reflect true exposure status. we cannot rule out, for example, false positive results caused by non-specific antibody binding or exposure to closely related or cross-reacting viruses. however, there is good biological reason to believe that wolf and coyote immune systems would behave very similarly to those of closely related domestic dogs, for which the tests have been optimized. previous serological work with foxes (including cdv assay validation via vaccination trials) suggests our titer cutoffs were appropriate for this species as well [32] . furthermore, the fact that multiple species exhibited similar patterns of exposure suggests that we did detect 'real' signals of disease exposure. our findings suggest cpv, cav-1, and chv are enzootic, and that cdv is epizootic, within yellowstone's canid community. among wolves, n. caninum appears enzootic, although it does exhibit some temporal variation, possibly reflecting complex dynamics between the parasite, its intermediate hosts (domestic and wild ungulates), and definitive hosts (wild canids and domestic dogs). resident status of coyotes was the one variable that consistently emerged as a possible risk factor, regardless of age group or pathogen. contrary to our original hypothesis, resident coyotes tended to have a greater probability of pathogen exposure than their transient counterparts. we had hypothesized that transient coyotes, whose home-ranges overlap multiple resident packs' territories, might contact a greater variety of individuals and be at greater risk for pathogen exposure. however, it is possible that transients actually make fewer contacts with other coyotes compared to residents, whose frequent interactions among packmates may provide the best opportunity for pathogen transmission. an alternative explanation, at least among adult coyotes ($1.6 yrs), is that the sampled residents tended to be slightly older (10% of transient adult coyotes were old adults [i.e., the other 90% were young adults] compared to the 28% of resident adult coyotes that were old adults), and that perhaps age, which should be positively correlated with exposure risk, was a confounder. from our study alone, it is not clear whether social status among coyotes has a true effect on the pathogen-exposure risk as hypothesized for other social-mammal systems [45, 46] . following the emergence of cpv in the late 1970s, studies throughout north america have reported high seroprevalences for table s3 for number of packs sampled and 95% cis). where points overlap, the top number refers to nr, the bottom to interior. doi:10.1371/journal.pone.0007042.g001 cpv among wild canids [47] [48] [49] . nearly all wolves and coyotes that we tested in ynp were positive for cpv exposure by 0.5-0.75 yrs of age. this high seroprevalence suggests low levels of diseaseinduced host mortality [50, 51] and high rates of transmission, perhaps aided by the stability of cpv in the environment [52] . we did not detect evidence for cpv-induced wolf-pup mortality, contrary to reports of suspected or confirmed cpv-induced mortality in the 1980s and early 1990s [53] [54] [55] , including among coyote pups in ynp [47] . furthermore, cpv seroprevalence offered no explanation for pup-survival patterns because there was no annual variation in exposure to cpv among wolves or coyotes. however, it is possible that cpv either causes a constant, low level of mortality or periodic mortality when combined with other factors such as nutritional stress or co-infection with other pathogens, both scenarios of which our current methods would fail to detect. nearly all wolves also exhibited exposure to cav-1, but there was no evidence for or against disease-induced mortality. cav-1 seroprevalence has generally been high in other canid surveys [47, [56] [57] [58] , suggesting that transmission among wild canids is high. juvenile coyotes had much lower seroprevalences to cav-1 than did wolf pups, but this may have been due, in part, to the slightly younger age at which coyotes were sampled. none of the studies that screened for chv antibodies among wild canids found evidence for exposure ( [59] (canis lupus); [56] (canis latrans); [60] (chrysocyon brachyurus)). by contrast, chv seroprevalence was high among ynp wolves, but somewhat lower and age-dependent in coyotes. canine herpesvirus is primarily spread though direct contact, so wolves' higher seroprevalence may be attributed to higher contact rates or a greater variety of contacts compared to coyotes or foxes. similarly, relatively high intra-pack contact rates may help explain the trend towards a slightly higher risk of pathogen exposure among resident coyotes compared to their transient counterparts. furthermore, increasing risk of exposure with age, as observed among wolves and coyotes, is common for enzootic diseases. neospora caninum. n. caninum exposure among wolves suggests that a sylvatic cycle of this protozoan parasite exists in ynp. domestic livestock, except horses, are prohibited in ynp. as hoofed-stock-to-canid transmission occurs through ingestion of infected tissue, livestock is likely not the source of canid exposure to n. caninum in ynp. while there is no information on n. caninum [20, 64] , infected wolves probably shed them as well. although we had insufficient quantities of coyote and fox sera to screen for n. caninum, we suspect that exposure levels in at least coyotes would be similar to that of ynp wolves. we did not sample wild canids outside of ynp, and thus future research could employ a combination of serologic and genetic tools to look at the relationships between n. caninum in wild and domestic canids and ungulates in regions where n. caninum is of concern to local livestock producers. however, at this time, there is no evidence to suggest that n. caninum has been or will be significantly impacting either domestic or wild ungulates or canids in the gye. the dynamics of highly immunizing, fast acting, epidemic-type pathogens such as cdv are challenging to decipher within the usual 3-5-year time frame of most wildlife studies. in these situations, reports of average seroprevalence, without regard to year or age of the sampled animal, can be misleading and of limited value for comparisons across different study sites and populations. in many of the serosurveys among coyotes [47, [65] [66] [67] and wolves [55, 68] , dynamic temporal patterns may have been masked by examining cdv seroprevalence averaged across years or age classes at spatial scales likely too small for cdv to be enzootic [69] . the supported cdv seroprevalence models suggested that (1) coyotes experienced cdv outbreaks in 1999 and 2005, (2) all wolves experienced cdv outbreaks in 1999, 2002, and 2005 (although 2000 and 2006 adult wolf seroprevalence was also high, these were likely individuals that were exposed in 1999 and 2005 and were thus positive upon capture the following year), and (3) nr wolves experienced a greater probability of cdv exposure than interior wolves. this last finding was consistent our hypothesis that high wolf densities on the nr may result in higher inter-pack contact rates and thus higher levels of pathogen exposure compared to the less-dense interior. although we do not have interior density estimates for the other canids, it is quite possible that coyote and fox densities are also higher on the nr than in the interior, and thus higher canid densities in general may contribute to higher rates of wolf exposure observed on the nr. however, as the seroprevalence data suggested, these aforementioned generalities obscured some potentially important differences in spatial and temporal cdv dynamics. for example, none of the interior wolf pups handled in 1999 and 2005 had been exposed to cdv in contrast to the high levels of exposure found among the limited samples of interior adults and nr adults and pups. these inconsistencies may be the result of small samples or differences in case-fatality rates across sampling locations. if all infected pups in the interior died due to disease, those available for sampling would all be negative. it is also possible that the timing and point of disease introduction into ynp could account for these differences. cdv is generally thought to move quickly through populations as it is highly contagious, infected individuals shed virus for a relatively short time (mean duration of infectiousness = 14 days, maximum 90 days), and the virus rapidly degrades in the environment (within hours at $20uc, and within several weeks at 0-4uc) [15, 16] . thus, if cdv had entered from the south before pup birth or weaning, it could have swept through the interior adults, sparing the young interior pups protected by maternal antibodies but arriving on the nr when pups would be most vulnerable. furthermore, if there was in fact a 2002 outbreak, it seems to have been confined to the interior wolves; none of the nr pups and only a few of the nr adults were exposed that year. however, there may be reason to suspect false positives in this particular case. in 2002, the two positive interior pups had antibody titers just over the positive titer cutoff value (positive antibody titer: $16), in contrast to marked increases in the titers observed among nr pups in 1999 and 2005 ( figure s1 ). adult titers in the interior and nr were not particularly high in 2002, either. in the absence of larger samples and more conclusive evidence (e.g. virus isolation or identification via pcr), we cannot be sure that cdv actually swept through the interior of the park in 2002. the wolf-pup data suggested low rates of seroconversion between the discrete outbreak years of 1999, 2002, and 2005. once a wild or domestic canid is infected with cdv, the animal either recovers rapidly (mean time from infection to recovery [including latency and infectiousness] = 21 days, maximum 120 days) with life-long immunity or dies [15, 16] . thus, cdv requires a large population of susceptibles to persist, a population likely larger than ynp's canid community [69] . the low seroconversion between epizootics, if representative of true positives, suggests reexposure from some wild or domestic host outside ynp or mistaken assumptions about the disease. for example, although no evidence exists for carrier states, loss of immunity, or imperfect protection against novel strains of cdv among canids, loss of cdv immunity has been documented in raccoons (procyon lotor) [70] . if any of these factors pertained to canids, that could help explain the apparent persistence of cdv in ynp. perhaps more likely, as there are multiple competent hosts for cdv within the gye (e.g. short and long-tailed weasels, american martens, striped skunks, and raccoons), multi-host transmission might allow localized cdv persistence within the gye. although a thorough analysis of factors influencing wolf-pup survival would evaluate multiple hypotheses such as population density and food availability, the strong negative correlation between nr cdv seroprevalence and nr wolf-pup survival supports the hypothesis that cdv may have contributed to high nr pup mortality in 1999 and 2005. although $8 young wolfpup carcasses were located in 2005, all were too degraded for cdv isolation. we found several pup mandibles (n = 4) and handled two live pups during the winter of 2005-2006 displaying the distinctive tooth-enamel hypoplasia diagnostic of cdv [12, 71, 72] . furthermore, several coyote dens appeared to experience high pup loss in 2005, with pups displaying neurologic symptoms consistent with late-stage cdv infection (e. almberg, personal observation) [16] . more recent data suggests that cdv swept through the park again in 2008, and in addition to observing the same patterns of high cdv seroprevalence and very low wolfpup survival, we recovered cdv ribonucleic acid via pcr from 3 dead wolves, all of which had been born after 2005 and thus presumably had no acquired immunity against cdv (almberg, unpublished data). despite the negative correlation between cdv exposure and pup survival, however, the ultimate causes of death could have been due to synergistic effects of cdv and another pathogen (e.g. cpv, cav-1, canine coronavirus, or protozoan or helminth infections), such as with cdv and babesia in serengeti's lions [73] . population impacts of pup mortality were short term, for the wolf population rebounded in both years following the 1999 and 2005 lows [27] . we found no relationship between interior cdv seroprevalence and interior wolf-pup survival. aside from the hypothesis that the timing of cdv introduction into the interior either happened to be too early (e.g. 1999 and 2005) or too late (e.g. in 2002) to cause significant pup mortality, other plausible explana-tions for this lack of relationship include 1) that there was no cdv outbreak in 2002, and thus insufficient variation in exposure to detect a relationship with survival, and 2) that we failed to detect pup mortality due to bias in our sampling methods. the interior packs' dens were remote and only visible from the airplane, and thus, on average, we made our first pup observations and obtained our first high counts of pups over a month later than those made on the nr (first pup observations, nr: m date = 5/24, sd = 21 days, interior: m date = 6/26, sd = 27 days; first high pup count, nr: m date = 6/19, sd = 34 days, interior: m date = 7/22, sd = 37 days). because much microparasite-induced (e.g., viruses and bacteria) pup mortality takes place following weaning (i.e., at 10-12 weeks of age) in late june through early july, it is quite possible that we failed to detect most interior pup mortality, yielding artificially high survival estimates. the results of two previous studies on pathogen exposure in ynp carnivores further support the patterns of cdv exposure that we observed in wolves and coyotes. gese et al. [47] suggested that ynp coyotes experienced a cdv outbreak between 1989 and 1991, which fits with the ,50% seroprevalence we detected in adult coyotes sampled during 1991. also, cougars in ynp appeared to experience isolated outbreaks of cdv in 1991 and 1999 [74] , lending support to the pattern of discrete, multi-host, cdv epizootics. our own extremely limited fox data at least did not contradict the pattern of discrete cdv outbreaks; the single positive animal sampled in 1996 was $5 years old and thus could have been exposed as a kit during the 1989/90 outbreak, and the only other two positive animals were sampled in 2005. furthermore, mustelids are highly susceptible to cdv, and the badger population on the nr appeared to have crashed in 2005 (e. almberg, personal observation). however, there are no data on cdv exposure or survival patterns among mustelids in ynp. these correlations among multiple hosts suggest regular cdv spillover but might also suggest multi-host transmission contributing to cdv persistence in the larger region. domestic animals cannot be ruled out as a reservoir for cdv. however, reported cdv cases in montana's domestic animals are uncommon, with 18 possible cases recorded between 1994 and 2008 (montana veterinary diagnostic lab, bozeman, mt, usa, unpublished data). furthermore, while the percentage of local domestic animals vaccinated for cdv is unknown, it is probably safe to assume that the unvaccinated population of dogs and cats is too small to serve as a cdv reservoir [69] . ynp and the gye are not closed biological systems. on an annual basis, an unknown number of visitors from around the u.s. bring their pets to ynp and the gye. there is currently no proof of dog health or immunization required for entry into the national parks. visiting domestic animals certainly constitute a plausible route for new or emerging pathogens (particularly those that are vector-borne or indirectly transmitted) to enter into local, wild canid populations. furthermore, ynp is a small fraction of the overall gye, and pathogen dynamics within ynp may be in part a product of much larger-scale dynamics driven by inter-connected canid and carnivore populations in the rocky mountains. in summary, the constant high canid exposure to cpv, cav-1, and chv in ynp suggest that these pathogens are established in the wolf and coyote populations and that they are unlikely to be causing acute mortality in their hosts [50, 51] . although n. caninum is unlikely to impact canid health, wolf exposure indicates a sylvatic cycle in the park, which may or may not be related to the parasite's dynamics among regional livestock. canine distemper appears to cycle through ynp's carnivores in periodic epizootics, and may have contributed to low wolf-pup survival in 1999 and 2005 on the nr. although cdv does not appear to jeopardize the long-term population survival of ynp wolves, it can cause short-term population decreases. additional information on how and where cdv is maintained and the frequency with which future epizootics might be expected would be useful for regional managers working on canids in the northern rocky mountains. (k = number of estimable parameters, increasing differences from the best model (h) indicate decreasing model adequacy, and akaike weights (w) express model support relative to all other models in the set. additive effects are expressed with a plus sign, and interactions between factors are connected with an asterisk.) found at: doi:10.1371/journal.pone.0007042.s002 (0.14 mb doc) table s3 wolf and coyote canine distemper seroprevalence and associated 95% score confidence intervals. sample sizes and the number of packs (for wolves) or regions (for coyotes) sampled are noted. found at: doi:10.1371/journal.pone.0007042.s003 (0.14 mb doc) figure s1 mean wolf antibody titers to canine distemper virus in yellowstone national park, 1997 park, -2007 . mean log 2 (antibody titers) are displayed with corresponding 95% confidence intervals for northern range (nr) and interior pups (a) and adults (b). found at: doi:10.1371/journal.pone.0007042.s004 (7.71 mb doc) disease and endangered species: the 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community a serologic assessment of exposure to viral pathogens and leptospira in an urban raccoon (procyon lotor) population inhabiting a large zoological park the effect of canine distemper virus on the ameloblastic layer of the developing tooth multiple dental abnormalities following canine distemper infection climate extremes promote fatal co-infections during canine distemper epidemics in african lions factors associated with pathogen seroprevalence and infection in rocky mountain cougars the authors would like to thank staff from the national park service and the yellowstone ecological research center for their assistance with data collection, analysis, and interpretation. we thank d. andersen, r. key: cord-273175-bao8xxe2 authors: tran, viet-thi; ravaud, philippe title: covid-19–related perceptions, context and attitudes of adults with chronic conditions: results from a cross-sectional survey nested in the compare e-cohort date: 2020-08-06 journal: plos one doi: 10.1371/journal.pone.0237296 sha: doc_id: 273175 cord_uid: bao8xxe2 background: to avoid a surge of demand on the healthcare system due to the covid-19 pandemic, we must reduce transmission to individuals with chronic conditions who are at risk of severe illness with covid-19. we aimed at understanding the perceptions, context and attitudes of individuals with chronic conditions during the covid-19 pandemic to clarify their potential risk of infection. methods: a cross-sectional survey was nested in compare, an e-cohort of adults with chronic conditions, in france. it assessed participants’ perception of their risk of severe illness with covid-19; their context (i.e., work, household, contacts with external people); and their attitudes in situations involving frequent or occasional contacts with symptomatic or asymptomatic people. data were collected from march 23 to april 2, 2020, during the lockdown in france. analyses were weighted to represent the demographic characteristics of french patients with chronic conditions. the subgroup of participants at high risk according to the recommendations of the french high council for public health was examined. results: among the 7169 recruited participants, 63% patients felt at risk because of severe illness. about one quarter (23.7%) were at risk of infection because they worked outside home, had a household member working outside home or had regular visits from external contacts. less than 20% participants refused contact with symptomatic people and <20% used masks when in contact with asymptomatic people. among patients considered at high risk according to the recommendations of the french high council for public health, 20% did not feel at risk, which led to incautious attitudes. conclusion: individuals with chronic conditions have distorted perceptions of their risk of severe illness with covid-19. in addition, they are exposed to covid-19 due to their context or attitudes. the novel coronavirus disease 2019 (covid-19) pandemic threatens to saturate healthcare systems all around the world [1] . on april 7 th of july 2020, 6,416,828 cases were confirmed in 213 countries, with 382,867 deaths [2] . in france, 152,444 cases were confirmed, with 29,065 deaths [3] . severe acute respiratory distress develops in about 16% to 26% of patients hospitalized with covid-19, thus requiring oxygen supplementation and/or intensive care [4] . as the number of cases grows worldwide, in order to avoid a surge of demand on the healthcare system and shortages of equipment such as ventilators needed to care for critically ill patients [5] [6] [7] , many countries have imposed quarantine and recommended physical distancing to reduce transmission to people likely to have a severe illness (i.e., older patients and those with chronic comorbidities). those individuals with chronic comorbidities should also, in return, avoid contacts and/or use appropriate measures to prevent potential infection. yet, in france and around the world, specific advice for individuals with chronic conditions and their households is scarce with most information is intended for the general public. for example, information from the european centre for disease prevention and control refer to only "people with chronic diseases" without specifying specific groups of individuals. this was confirmed by a recent study showing that adults with comorbid conditions lacked critical knowledge about covid-19 [8] . in this study, we aimed to understand the perceptions, context and attitudes toward covid-19 of individuals with chronic conditions in order to clarify their potential risk of infection. this study was a cross-sectional survey nested in compare, a nationwide e-cohort of patients with chronic conditions in france [9] . participants were adults with chronic conditions recruited from the community of patients for research (compare, http://compare.aphp.fr), a nationwide e-cohort of patients with chronic conditions in france. participants of compare are adults (>18 years old) who reported having at least one chronic condition (defined as a condition requiring healthcare for at least 6 months) and who joined the project to donate time to accelerate research of their conditions by answering regular patient-reported outcomes and experience measures online [9] . all participants provide electronic informed consent before participating in the ecohort. compare was approved by the comité de protection des personnes ile de france 1 (irb: 0008367). all methods were performed in accordance with the relevant guidelines and regulations. data from this study were collected between march 23 and april 2, 2020 at the peak of the french epidemic. during that time, 27 475 new cases of covid-19 were confirmed, with a total of 56 261 cases on april 2, 2020. this time period includes the maximum number of daily cases in france (april 1, 2020) [10] . since march 17, france had been under lockdown (movement restrictions and closure of non-essential businesses), and people with chronic conditions were encouraged to stay at home [11] . during this time, knowledge of covid-19 was still limited and information for the public was imprecise. for example, information available on the website of the french ministry of health referred to "people at risk", mixing older people and patients with chronic conditions [12] . of note, at the time of the study, benefits of using face masks were debated in france and in europe. participants' demographic and clinical data were collected as part of their participation in the compare e-cohort. all variables are updated yearly. conditions and medications are selfreported by patients by using the international classification of primary care-version 2 [13] and the thesorimed database of medications (french database of medications developed by the national health insurance) [14] . in addition, participants answered a dedicated survey designed by vtt and pr by use of the literature and their own expertise [8] . it was then face-validated by two other researchers (ip and cr) with expertise in questionnaire development before dissemination. the questionnaire was not tested with patients; however, the first respondents provided comments in a dedicated open-ended question at the end of the questionnaire, which led to minor reformulations. final survey questions are available in s1 and s2 data. this survey covered 3 topics. • for perception of risk of severe illness with covid-19, we asked participants whether they felt at high risk of severe illness with covid-19 with the question: "do you feel at increased risk of severe illness with covid-19 as compared to people of the same age as you but without chronic disease?" (yes/no). • for their context. participants described their activity (e.g., whether they continued working outside of the home); their household (i.e., whether any member of their household worked outside of the home and were in contact with the public); and their recent physical visits to healthcare professionals. • for their attitudes to prevent infection, participants were presented four theoretical situations involving different types of contacts: frequent (e.g., family member frequently visiting, child care, etc.) or occasional (e.g., during shopping) and discerning whether these contacts showed symptoms or not. in each situation, participants reported whether they would refuse contact, enact physical distancing or wear personal protective equipment (mask, gloves, etc.). results of the survey were described globally and for the subgroup of patients considered at high risk of a severe illness according to the french high council for public health (box 1). these patients were those with a severe cardiac or vascular disease (high blood pressure with complications, history of stroke or ischemic heart disease, cardiac surgery, heart failure), insulin-dependent diabetes, chronic lung disease or lung disease likely to be exacerbated by a viral infection, chronic kidney disease under dialysis, cancer under treatment, immunodeficiency (due to a drug [cancer chemotherapy, immunosuppressive medications, biotherapy and/or corticosteroids], an uncontrolled hiv infection, transplantation, or cancer), liver cirrhosis, or severe obesity (body mass index [bmi] >40 kg/m 2 ), or pregnant in the third trimester [15] . to operationalize these criteria with the data available in compare, one physician (vtt) matched the conditions and treatments reported by patients in compare with the list of high-risk conditions and treatments presented above. cancer chemotherapy immunosuppressive medications, biotherapy and corticosteroids were those classified as such in manufacturers' prescribing information, by using the vidal dictionary (https://www.vidal.fr/classifications/vidal/). descriptive statistics (mean with sd and frequency with percentage) were calculated for all patient characteristics and survey responses. associations between participant characteristics and responses to the survey items were then examined in bivariate analyses by chi-square or t test, as appropriate. in addition, we fitted two logistic regressions aimed at exploring the association between participants' characteristics and 1) their perception of their risk for severe infection and 2) their attitudes to prevent infection with occasional contacts with asymptomatic people. variables included in the model were sex, age (as a continuous variable), household with > 1 person (including the patient), low educational level, smoking status (current smoker vs. others), treatment considered at risk according to the french high council for public health, bmi �40 kg/m 2 , high blood pressure, diabetes (under insulin treatment or not), history of stroke or cardiac ischemic disease, heart failure (any new york heart association stage), asthma, chronic obstructive pulmonary disease, thyroid disease, chronic kidney failure (under dialysis or not), cancer (under treatment or not) and osteoarthritis. analyses were performed on complete cases only. p < 0.05 was considered statistically significant. no corrections for multiple testing were performed. • third trimester of pregnancy analyses involved using a weighted dataset obtained by calibration on margins with weights for age categories (<24, 25-34, 35-44, 45-54, 55-64, 65-74, >75 years), sex and educational level (low, middle school or equivalent, high school or equivalent, associate's degree, higher education). weights were derived from national census data describing the french population reporting chronic conditions [16, 17] . analyses involved use of r v3.6.1 (http://www.r-project.org, the r foundation for statistical computing, vienna, austria). between march 23 and april 2, 2020, we invited 18,651 patients from compare to complete our survey and 7169 (38.4%) answered (s1 fig). participants were mostly female (5616 [78.3%]) with mean (sd) age 46.1 (14.7) years. in the non-weighted data, diseases most frequently reported were high blood pressure (11.6%), diabetes (7.1%), asthma (6.2%) and cancer (5.2%); 3684 (51.4%) participants reported �2 chronic conditions. differences between respondents and non-respondents are shown in s1 table. in the weighted sample, 39.4% were at high risk for a severe illness according to the french recommendations: 33.0% because of their conditions, 8.8% because of their treatments, 1.9% with bmi > 40 kg/m 2 , and 0.5% in their third trimester of pregnancy. patients' characteristics before and after weighting are presented in table 1 . in the weighted sample, 63% of participants felt at risk of severe illness with covid-19, of whom 51% (32% of the whole sample) reported a high-risk situation according to the french high council for public health. conversely, 37% participants did not feel at risk of severe covid-19, of whom 20% (7.4% of the whole sample) reported a high-risk situation according to the french high council for public health (fig 1) . patients' characteristics associated with a perceived risk of severe covid-19 identified in the logistic regressions are presented in table 2 . in total, 7041 (98%) participants answered the survey section regarding their risk of infection due to their context. risk of infection involved working outside of the home (8.8% of participants, of whom 29% were care professionals), visits to health facilities for a consultation or test (54.7% of participants) or to a pharmacy (82% of participants); their household (74.9% of participants lived with at least on other person, of whom 18% worked outside of the home and 13% were children < 15 years old), or regular contacts with people outside of their home (e.g., family, friends, housekeeping, child care, etc.) (5% of participants). in all, 23.7% were exposed to some risks because of their work, their household members working outside of the home, or regular visits from external contacts. among patients at high risk of a severe illness according to the french high council for public health, 5% continued working, 15% had a household member working outside of the home and 7% reported regular contacts with people outside of their home. in all, 21.1% were exposed to some risks because of their work, their household members working outside of the home, or regular visits from external contacts. in total, 6940 (97%) participants answered the survey section regarding their attitudes to prevent infections. independent of the type of contact, participants reported that they would enact physical distancing under all situations presented to them. about one quarter of patients would refuse any contact with symptomatic people (17.8% and 23.4% for occasional and covid-19-related perceptions, context and attitudes of adults with chronic conditions frequent contacts, respectively). concerning the use of personal protective equipment, use of masks ranged from 19% (for occasional contacts with asymptomatic people) to 65% (for frequent contacts with symptomatic people). similarly, use of gloves ranged from 19% (for occasional contacts with asymptomatic people) to 50% (for frequent contacts with symptomatic people) (fig 2) . we found similar results in the subgroup of patients at high risk of a severe illness according to the french high council for public health. only 18.2% and 23.2% patients would refuse contact with symptomatic people for occasional and frequent contacts, respectively. concerning the use of personal protective equipment, use of masks ranged from 30% (for occasional contacts with asymptomatic people) to 63% (for frequent contacts with symptomatic people). similarly, use of gloves ranged from 21% (for occasional contacts with asymptomatic people) to 44% (for frequent contacts with symptomatic people). patients' characteristics associated with a perceived risk of severe illness with covid-19 identified on logistic regression are presented in table 3 . the only variable found associated with use of face masks with asymptomatic people (or refusal to see these people) was patients' perception of high risk of severe infection by covid-19 (odds ratio 1.93, 95% confidence interval 1.53-2.43). table 2 . association between participant characteristics and their perceived risk of severe covid-19. results of logistic regression analysis of complete cases, accounting for weights obtained after calibration on margins for sex, age categories and educational level by using data from a national census describing the french population self-reporting at least one chronic condition. odds ratio (95% confidence interval) we involved 7169 individuals with chronic conditions in a nationwide survey nested in an existing cohort and described their perception of risk of severe covid-19 and their potential risk of infection due to context and attitudes. first, our study highlighted that patients with chronic conditions have distorted perceptions of their risk of severe covid-19. among patients with criteria for high risk of severe covid-19 by the french high council for public health (40% of our sample), about 20% did not feel at risk and could therefore adopt incautious attitudes. this figure may even be conservative in light of recent works suggesting that all patients with hypertension, diabetes, cardiovascular disease, or chronic lung disease are at risk, not just those with complicated diseases [18] [19] [20] . data from the he chinese center for disease control and prevention showed increased case fatality rate among patients with preexisting comorbid conditions-10.5% for cardiovascular disease, 7.3% for diabetes, 6.3% for chronic respiratory disease, 6.0% for hypertension, and 5.6% for cancer [20] . especially, our findings highlight that patients with a bmi � 40 kg/m 2 or who smoked did not feel at risk nor took extra precautions when in contact with other people despite these two factors being associated with risk of severe complications and mortality from covid-19 [21, 22] . these results are of importance because of the confluence of two elements. first, preventing infection for people at risk of severe disease is difficult. in our study, 21.2% of patients at high risk of a severe illness according to french recommendations were in frequent contact with "the outside world" during the quarantine because of their work, their household members working outside of the home, or regular visits from external contacts. second, feeling at risk seems to be the major factor for using face masks with asymptomatic people. at the time of the table 3 . association between participant characteristics and the use of face masks for occasional contacts with asymptomatic people (or the refusal to see these people). results of logistic regression analysis of complete cases, accounting for weights obtained after calibration on margins for sex, age categories and educational level by using data from a national census describing the french population self-reporting at least one chronic condition. odds ratio (95% confidence interval) covid-19-related perceptions, context and attitudes of adults with chronic conditions study, it was still unknown that 40% to 80% of transmission events could occur from people who are presymptomatic or asymptomatic [23] . therefore, specific communication clearly identifying patients at risk for severe illness by covid-19 is mandatory. communication should also target the household of these patients because the rate of secondary transmission among household contacts of patients with sars-cov-2 infection was estimated at 30% [24] . our results are important given the cumulative amount of evidence showing that patients with chronic conditions, about 20 million individuals in france, are at increased risk of severe covid and death. in a small case series conducted at the beginning of the epidemic in china, among 102 patients hospitalized for covid-19, those with comorbidities (especially hypertension, diabetes, cardiovascular and respiratory diseases) were more likely to be hospitalized in intensive care units [25, 26] . similar findings were observed in europe. in a large case series of 4000 patients hospitalized in icus in italy, the highest risk of death was for patients with chronic obstructive pulmonary disease (adjusted hr [ahr] 1.68, 95% ci 1. 28-2.19 ) and type 2 diabetes (ahr 1.18, 95% ci 1.01-1.39) [27] . reasons underlying these findings are still unclear, with hypotheses related to meta-inflammation or use of angiotensin-converting enzyme inhibitors (aceis)/angiotensin receptor blockers (arbs) in these populations, despite recent controversial findings about this latter point [28, 29] . our study complements the literature on the awareness and attitudes of patients with chronic conditions related to covid-19. to date, most works have focused on the general public [30, 31] . knowledge and attitudes of patients with chronic conditions is unknown, apart from a study of 600 patients with chronic conditions in the united states that showed gaps in awareness and knowledge of covid-19 among patients with chronic conditions [8] . our findings confirm these results and provide details on individuals' risks associated with their context and their attitudes to prevent infection. this study has several limitations. first, all data were self-reported, with risk of desirability bias regarding their attitudes. second, individuals at high risk of severe illness with covid19 are not yet well known; recommendations from the french high council for public health are mostly based on case reports in china and precaution measures [15] . third, the response rate was relatively low (38%) owing to the short duration of data collection (10 days) and the sole use of e-mails for the invitation and reminders. yet, such response rate is consistent with the literature of online surveys for the general public [32, 33] . non-respondents were younger, less multimorbid and had fewer conditions considered at high risk according to recommendations than respondents. despite statistical weighting, results should be generalized with caution. in conclusion, we found that individuals with chronic conditions may have distorted perceptions of their risk of severe illness with covid-19. targeted communication may increase the use of personal protective equipment and prevent infection, which is fundamental because 20% of these individuals are exposed to infection because of their work, their household or regular visits from external contacts, despite quarantine. table. demographic characteristics of respondents and non-respondents to the survey (raw data). (docx) preventing a covid-19 pandemic world health organization. novel coronavirus (covid-19) situation 2020 clinical infectious diseases: an official publication of the infectious diseases society of america critical supply shortages-the need for ventilators and personal protective equipment during the covid-19 pandemic. the new england journal of medicine the effect of control strategies to reduce social mixing on outcomes of the covid-19 epidemic in wuhan, china: a modelling study. the lancet public factors associated with hospitalization and critical illness among 4,103 patients with covid-19 disease awareness, attitudes, and actions related to covid-19 among adults with chronic conditions at the onset of the u.s. outbreak: a cross-sectional survey. annals of internal medicine collaborative open platform e-cohorts for research acceleration in trials and epidemiology (cooperate) european centre for disease prevention and control. data on the geographic distribution of covid-19 cases worldwide: european centre for disease prevention and control emmanuel macron annonce une série de mesures 2020 page du ministère de la santé international classification of primary care avis provisoire: patients à risque de formes sé vères de covid-19 et priorisation du recours aux tests de diagnostic virologique institut national de la statistique et des etudes économiques. la macro sas calmar: institut national de la statistique et des etudes é conomiques direction de la recherche dé, de l'évaluation et des statistiques. l'é tat de santé de la population en france-rapport 2017. paris: ministère des solidarité s et de la santé covid-19: risk factors for severe disease and death clinical research ed). 2020; 368:m1091. epub 2020/03/29. pmid: 32217556 www.icmje.org/coi_disclosure.pdf and declare: support from the tongji hospital for pilot scheme project and the chinese national thirteenth five years project in science and technology, national commission of health, people's republic of china, for the submitted work; no financial relationships with any organisation that might have an interest in the submitted work in the previous three years characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention severity and mortality associated with copd and smoking in patients with covid-19: a rapid systematic review and meta-analysis www.icmje.org/coi_disclosure.pdf and declare: support from the kenneth c griffin charitable fund for submitted work; no financial relationships with any organizations that might have an interest in the submitted work in the previous three years substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov2) household transmission of sars-cov-2 clinical features and short-term outcomes of 18 patients with corona virus disease 2019 in intensive care unit clinical infectious diseases: an official publication of the infectious diseases society of america risk factors associated with mortality among patients with covid-19 in intensive care units in lombardy, italy obesity, and metabolic inflammation create the perfect storm for covid-19 dr torp-pedersen reported receiving grants from bayer and novo nordisk. dr køber reported receiving speaker honoraria from novartis us public concerns about the covid-19 pandemic from results of a survey given via social media adoption of personal protective measures by ordinary citizens during the covid-19 outbreak in japan response rate and completeness of questionnaires: a randomized study of internet versus paper-and-pencil versions challenges using online surveys in a danish population of people with type 2 diabetes the authors thank isabelle pane and carolina riveros for their help in the survey development and isabelle pane for data management. key: cord-265138-i5m3ax7g authors: wang, xi-ling; yang, lin; chan, king-pan; chiu, susan s.; chan, kwok-hung; peiris, j. s. malik; wong, chit-ming title: model selection in time series studies of influenza-associated mortality date: 2012-06-20 journal: plos one doi: 10.1371/journal.pone.0039423 sha: doc_id: 265138 cord_uid: i5m3ax7g background: poisson regression modeling has been widely used to estimate influenza-associated disease burden, as it has the advantage of adjusting for multiple seasonal confounders. however, few studies have discussed how to judge the adequacy of confounding adjustment. this study aims to compare the performance of commonly adopted model selection criteria in terms of providing a reliable and valid estimate for the health impact of influenza. methods: we assessed four model selection criteria: quasi akaike information criterion (qaic), quasi bayesian information criterion (qbic), partial autocorrelation functions of residuals (pacf), and generalized cross-validation (gcv), by separately applying them to select the poisson model best fitted to the mortality datasets that were simulated under the different assumptions of seasonal confounding. the performance of these criteria was evaluated by the bias and root-mean-square error (rmse) of estimates from the pre-determined coefficients of influenza proxy variable. these four criteria were subsequently applied to an empirical hospitalization dataset to confirm the findings of simulation study. results: gcv consistently provided smaller biases and rmses for the influenza coefficient estimates than qaic, qbic and pacf, under the different simulation scenarios. sensitivity analysis of different pre-determined influenza coefficients, study periods and lag weeks showed that gcv consistently outperformed the other criteria. similar results were found in applying these selection criteria to estimate influenza-associated hospitalization. conclusions: gcv criterion is recommended for selection of poisson models to estimate influenza-associated mortality and morbidity burden with proper adjustment for confounding. these findings shall help standardize the poisson modeling approach for influenza disease burden studies. numerous studies have demonstrated that influenza causes substantial burden on mortality and morbidity [1] [2] [3] . reliable estimates for disease burden associated with influenza in the community are essential for public health policy-making. however the case numbers of influenza infections derived from medical records grossly underestimated the true burden [4] . during 2001 to 2009 there were only 138 deaths registered in hong kong with underlying cause of influenza infection [5] . underreporting of influenza cases was due to the fact that influenza infections usually caused relatively mild symptoms and many infected people did not seek medical care from hospital or clinic. among outpatients and inpatients with influenza-like illness, few were tested for influenza to get confirmed diagnoses. even for those with laboratory confirmed infections, influenza was rarely recorded as underlying cause of death on their death certificates. several statistical models have been used to quantify the disease burden attributable to influenza [6] . among these models, poisson regression models have become increasingly popular in recent years [7] [8] [9] . unlike most of the other models, the poisson model does not require clear seasonality of influenza to define influenza epidemic and nonepidemic periods. therefore, it is particularly suitable for tropical and subtropical regions where influenza seasonality is less clear and influenza viruses could be circulating throughout the whole year. another advantage of the poisson model lies in its ability to adjust for multiple seasonal confounders simultaneously. there are two types of confounders that are often considered in poisson models: measured confounders, such as meteorological factors, circulation of other respiratory pathogens and air pollution [10] ; and unmeasured confounders, such as seasonal change in host susceptibility and health seeking behavior [11] . however, overadjustment of confounders may result in underestimation of true effects, as some variations caused by influenza were allocated to confounders. likewise, inadequate adjustment could lead to residual confounding that causes spurious association between influenza proxy variable (such as proportions of specimens testing positive for influenza) and health outcome of mortality or hospitalization. therefore, proper adjustment of confounders is critical for obtaining reliable estimates of influenza-associated disease burden. previous studies using poisson models adjusted for unmeasured confounders by incorporating sinusoidal pairs [12] [13] [14] [15] or a smoothing function of time trend into the poisson model [8, 16, 17] . however, few studies has properly discussed on how to determine the adequacy of adjustment for seasonal confounders in the model. here we conducted a simulation study with the aim to compare the performance of several commonly adopted model selection criteria, in terms of selecting the best-fit poisson model with adequate adjustment for confounders. four model selection criteria were considered in this study: quasi akaike information criterion (qaic), quasi bayesian information criterion (qbic), partial autocorrelation functions of residuals (pacf), and generalized cross-validation (gcv). we obtained weekly all-cause mortality data from 1998 to 2008 from the census & statistics department, and daily meteorological data of temperature and relative humidity from the hong kong observatory. daily concentrations of air pollutants nitrogen dioxide (no 2 ), sulfur dioxide (so 2 ), ozone (o 3 ) and particulate matters with diameter less than 10 mm (pm 10 ) were obtained from the environmental protection department. weekly numbers of specimens positive for influenza and respiratory syncytial virus (rsv) as well as total numbers of tested specimens were obtained from the microbiology laboratory of queen mary hospital. influenza virology data of this single laboratory have been demonstrated representative of the virus activity in the entirety of hong kong [16] . we performed a simulation study by generating mortality data from a poisson model with adjustment for over-dispersion [18] . this model is similar to the models used in our previous studies on influenza-associated mortality and morbidity [17] , in which an influenza proxy variable is added to assess influenza effects. to derive a proper estimate for influenza-associated mortality or morbidity, it is important to adjust for confounding to separate the effect of influenza from those of other seasonal factors. cocirculation of rsv, together with two meteorological factors of temperature and humidity, are adjusted for as confounders in this study given their association with both health outcomes and influenza [19, 20] . weekly concentrations of four major ambient air pollutants are also included as confounders based on recent findings on the association between influenza virus and ambient air pollutants [21] . unmeasured confounding is adjusted for by including the long-term and seasonal trends of outcome variables. a typical model was as follows: y t *quasipoisson(m t ,wm t ), log (m t )~b 0 zbflu t zb 1 rsv t zs(t, df~11|k) zs(temp t , df~3) zs(hum t , df~3)zb 2 no 2t zb 3 so 2t zb 4 o 3t zb 5 pm 10t zb 6 sars t : y t denotes the weekly number of all-cause deaths which was assumed to follow a poisson distribution with an over-dispersion parameter w [22] . flu t and rsv t denote the proxy variables for influenza and rsv, which are weekly proportions of specimens testing positive for influenza or rsv. three natural cubic spline smoothing functions of s(t, df = 116k), s(temp t ) and s(hum t ) are added to adjust for time (t = 1,2,…,574), weekly mean temperature (temp t ) and relative humidity (hum t ), where the degrees of freedom (df) of time k ranges from 1 to 10 per year. we used a natural cubic spline with fixed degrees of freedom so that the locations of knots were evenly distributed [23] . no 2t , so 2t , o 3t and pm 10t denote the weekly mean concentrations of four air pollutants, respectively. to adjust for the increased mortality during the outbreak of severe acute respiratory syndrome (sars) in 2003, we added into the model a dummy variable sars for the sars period of week 1-30 of year 2003. before simulation, we first estimated the coefficients of model (1) by fitting it to the all-cause mortality data during 1998 to 2008 in hong kong. the degrees of freedom were fixed to three for weekly temperature and humidity based on our previous experience [17] and to one per year for long term and seasonal trends. mean mortality for weekm m t was then predicted from this fitted model with the b coefficient for influenza variable flu t fixed to 0.33 (i.e. mortality increasing 3.3% when the influenza positive proportion increases 10%). the over-dispersion parameter wwas also derived from this model. because there was no statistical package available for data simulation based on the over-dispersed poisson distribution, we simulated 500 mortality datasets by assuming that mortality followed a negative binomial distribution, i.e. y t *negbin(m t ,h) when h~m t w{1 [24] . given the uncertainty in degrees of freedom for unmeasured seasonal confounders, we repeated the above simulation process with the degrees of freedom of s(t, df) changing from 1 to 2,3,…, 10 per year. hence, we got a total of 5000 weekly mortality datasets and 500 for each fixed degrees of freedom for t. we then applied model (1) with degrees of freedom varying from 1-10 per year for s(t,df) to each set of 500 simulated data), and selected the best-fit model with the minimal value for each of the following model selection criteria: 1) quasi-akaike information criterion (qaic) [25] : 2) quasi-bayesian information criterion (qbic) [26] : where n is the number of observations. 3) residual autocorrelation: the sum of the absolute value of the partial autocorrelation function (pacf) of the residuals up to 5 lag weeks. 4) generalized cross validation (gcv) [26] : where tr(r) is the trace of weighted additive-fit operator corresponding to the last iteration of the local-scoring procedure; y t is the observed number of death at week t;m m t is the predicted number of death at week t; d(y t ;m m t ) is the deviance of y t fromm m t ; n is the number of observations. we calculated the bias as the average difference between the estimated coefficients of the influenza variable from the best-fit model and the true coefficient of 0.33. standard error and rootmean-square error (rmse) were defined as the standard deviation and square root of the mean square error of estimated coefficients, respectively. in this study we took rsme as the primary measure to compare the performance of different model selection criteria, as it could evaluate both accuracy and variation of the estimates [25] . the criterion that obtained the minimal rmse under the different assumptions of confounding was considered as the best criterion in selecting the model with adequate adjustment for confounders. to investigate the robustness of our results, we conducted a sensitivity analysis by changing the pre-determined coefficient of influenza variable from 0.33 to 0.1 and 0.5, which were the lower and upper boundaries of influenza effects based on our previous experience. because a short study period might offer less reliable estimates with large standard error, we did another sensitivity analysis with the data of 2003 to 2008 or those of 2006 to 2008. given that influenza effects on mortality might lag several weeks behind the increase of influenza activity [15] , we separately added the influenza proxy variables up to 3 weeks before (lag 1-3 weeks) into the models to assess any lag effects. all the analyses were conducted using r software (version 2.13.0) [27] . we applied the four model selection criteria to an empirical dataset of weekly hospitalization numbers of pediatric patients younger than 18 years. these patients were admitted into two major public hospitals on hong kong island from october 2003 through september 2008, with acute respiratory disease (ard) listed in the first five discharge diagnoses. these data were retrieved from the computerized database of the hong kong hospital authority, according to the international classification of disease 9 th revision (icd9) codes of 4602466 or 4802487. five age groups were considered: 021, 122, 225, 5210, 10218 years. this poisson model was similar to model (1), except that two proxies for adenovirus (adeno t ) three types of parainfluenza viruses (p1 t , p2 t , p3 t ) were added as confounders, because these data were only available after 2003. influenza-associated hospitalization rates were defined as the difference between the observed and expected hospitalization under the assumption of no circulating influenza viruses. these rates were separately estimated from the best-fit models chosen by each criterion, and the bias and rmse were calculated by comparing with the observed admission rates of a pediatric cohort of influenza hospitalization cases. as previously described [17] , this cohort was composed of all the pediatric patients who were recruited from the same two hospitals and diagnosed with influenza infection by immunofluorescence tests and viral culture. ethics approval for collecting specimens from pediatric patients was obtained from the ethics committee of li ka shing faculty of medicine, the university of hong kong (ec1880-02). figure 1 shows the weekly number of deaths simulated under the scenario of low and high seasonal confounding (df = 1 and df = 10 per year for the seasonal trend smoothing functions). the simulated data fluctuated within the range of 400 to 1100 with a steadily increasing annual trend. as expected, the data simulated under df = 10 was rougher and closer to the true mortality than those simulated under df = 1 (figure 1) . overall, the simulated allcause mortality data were generally comparable with the true mortality data. most models overestimated influenza effects with a few exceptions observed for the models selected by the minimal gcv ( figure 2) . estimates tended to have larger biases as the seasonal confounding of the simulated data increased. overall the models selected by pacf, qaic and qbic had the larger biases (ranging from 0.0022 to 0.2909) than did those selected by gcv (ranging from 0.0008 to 0.007) (figure 2 ). standard error of influenza coefficients was comparable between these four criteria, ranging from 0.0012 to 0.0045. rmse was similar between the models selected by pacf, qaic or qbic when the df of smoothing functions for time were less than 5 per year, but dramatically increased when the df increased to 5 or more per year ( figure 2 ). the rmse of gcv criterion remained lower than those of the other three criteria (figure 2 ). biases and rmse did not markedly change when the predetermined coefficient for influenza proxy variable of weekly positive proportions changed from 0.33 to 0.1 and 0.5 ( figure s1 ). among the four criteria, gcv still provided the smallest bias and rmse under the different simulation scenario. sensitivity analysis of a shorter study period of 2003-2008 or 2006-2008 showed slightly higher rmse than those from the whole study period, but gcv provided smaller biases and rmse compared to the other three criteria ( figure s2 ). in the models with the lag effects of up to 3 weeks, gcv still provided the smallest biases and rmse for influenza coefficients ( figure s3 ). figure 3 shows the percentage difference between the estimated excess ard hospitalization rates and directly observed admission rates of influenza cases in the pediatric cohort from 2003 to 2008. for the age groups of 021and 10218 years, the best-fit models selected by gcv provided the estimates closer to the observed rates than did those selected by qaic, qbic and pacf ( figure s4 ). estimates from the four criteria were comparable for the 122 and 225 age groups. all the poisson models respectively selected by the four criteria slightly overestimated the true rates for all the age groups, except that the pacf and gcv criteria provided the estimates smaller than the observed rates in the 5210 age group. among the four criteria, gcv had the smallest biases and rmse, whereas qaic and qbic had the largest (table 1 ). as underreporting of influenza cases is common in clinical practice, the poisson modeling approach has been widely accepted in estimating disease burden of influenza [28] . two recent studies in canada and hong kong have demonstrated the estimates of influenza-associated hospitalization derived from poisson regression models reasonably matched the numbers of patients with laboratory confirmed influenza infections [17, 29] . however, it is extremely difficult to obtain the gold standard data on influenza associated deaths to assess the validity of the statistical models, because patients with influenza infection could have died from secondary bacterial infections and exacerbation of their preexisting conditions [30, 31] . therefore, their presenting problems may not be directly linked to influenza. moreover, given the potential lag time between severe complications and primary influenza infection, influenza virus might have become undetectable in these patients in the time of admission. hence recorded influenza deaths could still seriously underestimate the true numbers of deaths due to influenza even if laboratory tests for influenza are intensively conducted. in this study we performed a simulation study to assess the performance of poisson regression models. the small biases and rmse of most estimates may give further evidence to support the validity and reliability of poisson models. in this study, we adopted a semi-parametric model with smoothing functions to adjust for potential confounders, whereas most of the other studies just used linear terms of confounding variables in their poisson models [13, 15] . this semi-parametric model is preferred over the traditional parametric model in that it does not require any pre-determined relationships between the independent and dependent variables and thereby allows us to assess both linear and nonlinear relationships. although we chose natural spline smoothing functions in this study, there are also other smoothing functions available. however, previous studies have found that having a sufficient number of degrees of freedom is more important than the type of smoothing functions for adequate adjustment of confounding in the semi-parametric model [32] . therefore, in this simulation study, we mainly focused on the determination of degrees of freedom by an appropriate model selection criterion. among the four criteria under study, gcv consistently provided the smallest biases and rmse under the different assumptions of seasonal confounders, particularly when this confounder was assumed to have a high seasonal variation. our findings were robust to the various assumptions of influenza coefficients in simulation and also to the length of study period. increase of rmse was observed when the study period was shorten, which is not surprising as using less data points would increase the variation of estimates. all the four model selection criteria were developed under the different schemes. pacf measures the autocorrelation of the residuals, whereas both qaic and qbic evaluate the relative goodness of fit of a statistical model by quantifying the relative lost of information when a given model is used to describe the reality. therefore the latter two reflect the tradeoff between accuracy and simplicity, but qaic penalizes the number of model parameters to a lesser extent than qbic does. unlike other criteria, gcv assesses the model validity by cross-validation, i.e. randomly sampling data as training and test datasets to compare the accuracy and variation of prediction. our findings that gcv outperforms the other criteria in poisson models are also in line with previous studies on air pollution [25] . we chose the best-fit model based on the adequacy of confounding adjustment in terms of providing reliable estimates for influenza effect. although many seasonal factors could confound influenza effects on mortality, we only focused on the confounding of long term and seasonal trends of mortality in the present study, as this factor affected our estimates to a greater extent than any other confounders according to our previous experience. in this study the best-fit model was selected by minimizing each of the selection criteria, but the magnitude of their difference was not assessed. some studies suggested that the models selected under the different criteria might perform equally well if the difference between these criteria were small [33, 34] . however, it is not easy and somewhat arbitrary to define the cutoff points for small difference. burnham and anderson (2002) developed a set of cutoff points for aic to select the models with meaningfully different estimates [33] . similar thresholds for the bic value were also introduced by kass and raftery [34] . however, so far there are no commonly accepted cutoff points for those selection criteria used in our study. therefore, we did not take into consideration of the difference magnitude between these values, in order to achieve the simplicity and efficiency in the model selection procedure. there are several limitations in our study. first we assessed the parameter uncertainty in poisson regression models based on the data of subtropical city hong kong, where influenza seasonality is less clear than that in the temperate regions [16] . given the well defined winter peaks of influenza in temperate regions, it can be expected that the data from these regions probably required less complex adjustment for seasonal confounders. nevertheless, the framework developed in our study can still be applied to a wide range of data. second, we did not separately estimate the effects of different influenza virus subtypes, although previous studies have demonstrated their difference in excess mortality and mutation frequency [35, 36] . unfortunately, the virus subtype data during the study period are not available to us. future studies are needed to assess the performance of these model selection criteria in assessing the disease burden associated with each subtype. by applying poisson regression models to an empirical dataset of influenza hospitalization, we demonstrate that our findings can be generalized to other health outcomes. the best-fit models were validated by comparing the estimates of age-specific excess hospitalization rates with the observed rates in a pediatric cohort undergoing intensive laboratory tests for influenza infections. consistent with the findings of our mortality simulated study, gcv criterion outperformed qaic, qbic and pacf with smaller biases and rmse. given the enormous cost in money and manpower by such a prospective cohort study, statistical modeling is relatively easier to conduct and able to provide reliable estimates for influenza associated disease burden. in conclusion, our results suggested that the gcv criteria should be recommended for selection of the best-fit model in the future disease burden studies using poisson models. standardization of this modeling procedure shall increase the reliability of estimates and facilitate the comparison across countries or regions. abbreviations: qaic, quasi-akaike information criterion; qbic, quasi-bayesian information criterion; pacf, partial autocorrelation function; gcv, generalized cross validation; rmse, rootmean-square error; (tif) figure s4 weekly numbers of observed and fitted hospitalization by age group. the fitted hospitalization data were derived from the best-fit models selected by the generalized cross validation (gcv) criterion. (tif) the burden of influenza in east and south-east asia: a review of the english language literature the underrecognized burden of influenza in young children the annual impact of seasonal influenza in the us: measuring disease burden and costs estimating influenza-associated deaths in the united states department of health, the government of the hong kong special administrative region (2012) death statistics -by sex and age, cause of death in tabulation list of the international classification of diseases a practical guide for designing and conducting influenza disease burden studies influenza-attributable mortality in australians aged more than 50 years: a comparison of different modelling approaches circulating influenza virus, climatic factors, and acute myocardial infarction: a time series study in england and wales and hong kong excess mortality monitoring in england and wales during the influenza a(h1n1) 2009 pandemic air pollutants and health outcomes: assessment of confounding by influenza confounding by season in ecologic studies of seasonal exposures and outcomes: examples from estimates of mortality due to influenza estimates of us influenza-associated deaths made using four different methods influenza-associated hospitalization in a subtropical city mortality associated with influenza and respiratory syncytial virus in the united states influenza-associated mortality in hong kong seasonal effects of influenza on mortality in a subtropical city validation of statistical models for estimating hospitalization associated with influenza and other respiratory viruses regression models for count data in r winter viruses: influenza-and respiratory syncytial virus-related morbidity in chronic lung disease influenza and the rates of hospitalization for respiratory disease among infants and young children part 4. interaction between air pollution and respiratory viruses: time-series study of daily mortality and hospital admissions in hong kong generalized linear models generalized additive models overdispersed poisson regression models for studies of air pollution and human health model choice in time series studies of air pollution and mortality generalized additive models r: a language and environment for statistical computing. r foundation for statistical computing time series methods for obtaining excess mortality attributable to influenza epidemics the need for validation of statistical methods for estimating respiratory virus-attributable hospitalization influenza virus induces bacterial and nonbacterial otitis media invasive bacterial infections in relation to influenza outbreaks advances in statistical methods for the health sciences model selection and multimodel inference: a practical information-theoretic approach bayes factors the genomic and epidemiological dynamics of human influenza a virus influenza associated mortality in the subtropics and tropics: results from three asian cities we thank the census and statistics department of hong kong for providing mortality data. the hong kong hospital authority for providing hospitalization data. the hong kong observatory for providing meteorological data, the environmental protection department for providing air pollution concentration data. we also thank ms yk chau, dr tq thach and dr hk lai for their comments. key: cord-299509-7xjdryoq authors: scholte, florine e. m.; tas, ali; martina, byron e. e.; cordioli, paolo; narayanan, krishna; makino, shinji; snijder, eric j.; van hemert, martijn j. title: characterization of synthetic chikungunya viruses based on the consensus sequence of recent e1-226v isolates date: 2013-08-01 journal: plos one doi: 10.1371/journal.pone.0071047 sha: doc_id: 299509 cord_uid: 7xjdryoq chikungunya virus (chikv) is a mosquito-borne alphavirus that re-emerged in 2004 and has caused massive outbreaks in recent years. the lack of a licensed vaccine or treatment options emphasize the need to obtain more insight into the viral life cycle and chikv-host interactions. infectious cdna clones are important tools for such studies, and for mechanism of action studies on antiviral compounds. existing chikv cdna clones are based on a single genome from an individual clinical isolate, which is expected to have evolved specific characteristics in response to the host environment, and possibly also during subsequent cell culture passaging. to obtain a virus expected to have the general characteristics of the recent e1-226v chikv isolates, we have constructed a new chikv full-length cdna clone, chikv ls3, based on the consensus sequence of their aligned genomes. here we report the characterization of this synthetic virus and a green fluorescent protein-expressing variant (chikv ls3-gfp). their characteristics were compared to those of natural strain ita07-ra1, which was isolated during the 2007 outbreak in italy. in cell culture the synthetic viruses displayed phenotypes comparable to the natural isolate, and in a mouse model they caused lethal infections that were indistinguishable from infections with a natural strain. compared to ita07-ra1 and clinical isolate nl10/152, the synthetic viruses displayed similar sensitivities to several antiviral compounds. 3-deaza-adenosine was identified as a new inhibitor of chikv replication. cyclosporin a had no effect on chikv replication, suggesting that cyclophilins -opposite to what was found for other +rna virusesdo not play an essential role in chikv replication. the characterization of the consensus sequence-based synthetic viruses and their comparison to natural isolates demonstrated that chikv ls3 and ls3-gfp are suitable and representative tools to study chikv-host interactions, screen for antiviral compounds and unravel their mode of action. chikungunya virus (chikv) re-emerged in 2004 and has caused unprecedented outbreaks in asia and africa since 2005. the estimated number of cases exceeds 2 million and over a thousand infected travelers have returned to europe and the usa since 2006 [1, 2] . chikv generally causes a fever that resolves within several days, a maculopapular rash, and a characteristic arthralgia that can be extremely painful and may persist for months. during the recent outbreaks also more severe clinical manifestations have been reported occasionally, such as neurological complications and even deaths, usually in the elderly, patients with underlying conditions, and newborns [3, 4] . a licensed vaccine or specific antiviral therapy are currently not available. chikv is an alphavirus with an 11.7 kb positive-stranded rna genome that contains two open reading frames (orfs). the 59 orf encodes the nonstructural polyproteins p123 and p1234. the latter results from translational read-through of an opal termination codon that is present at the end of the nonstructural protein (nsp) 3 coding sequence of most chikv isolates. assuming that chikv follows the typical alphavirus life cycle, proteolytic processing of the nonstructural polyproteins by the protease domain in nsp2 will ultimately lead to the release of nsp1, nsp2, nsp3, and nsp4. these nsps and their precursors possess a variety of functions and the enzymatic activities, including protease, helicase, methyltransferase, and rna-dependent rna polymerase (rdrp) activity that drive chikv replication [5] . in addition to replication of its genomic rna, chikv also transcribes a subgenomic (sg) rna encoding a precursor polyprotein that is processed by viral and cellular proteases into the structural proteins c, e3, e2, 6k and e1. chikv nsps willpresumably together with host factors -assemble into replication and transcription complexes (rtcs) that associate with membrane structures derived from the plasma membrane and/or endosomes, as observed for other alphaviruses [5] [6] [7] . the chikv strains that emerged during the 2005-2006 outbreaks had acquired a mutation (a226v) in the e1 envelope glycoprotein, which facilitated transmission of the virus via a new vector, the asian tiger mosquito aedes albopictus, and consequently dramatically increased the epidemic potential of chikv [8, 9] . later studies suggested that recent indian and indian ocean epidemics have emerged separately as the result of at least three independent events, and that convergent evolution of east-central-south african lineage strains in different geographical regions ultimately led to the emergence of strains with the a226v substitution in e1 [10] [11] [12] [13] . more recently, other amino acid positions and epistatic interactions were also shown to play an important role in the emergence of these new chikv variants, which are now even replacing endemic strains that have been circulating in asia for decades [14] . aedes albopictus also thrives in more temperate climates and its geographical distribution has rapidly expanded. over the past decades, parts of southern europe and large areas of the usa have been invaded by this mosquito, providing imported cases of chikv with a competent mosquito vector, thus paving the road for outbreaks in nonendemic-areas such as the usa and europe. indeed, autochthonous infections have been reported from italy in 2007 and france in 2010 [15, 16] . the recent and ongoing chikv outbreaks are characterized by their rapid geographical spread, high numbers of infected people and high morbidity, emphasizing the need to gain more insight into the replicative cycle of this important human pathogen. infectious cdna clones of viruses have become invaluable tools that allow reverse genetics studies to elucidate the contribution of specific amino acids or rna structures to viraemia, virulence, antigenicity, replication kinetics, interactions with host factors, adaptation to new vectors, and many other aspects of the viral life cycle. the use of cdna clones is also instrumental in mechanism of action studies to pinpoint the viral target of antiviral compounds by selecting for and genotyping compound-resistant viruses, followed by reverse engineering of the identified mutations to assess their individual phenotypic contribution to resistance. finally, the generation of cdna clones of reporter viruses, like those expressing green fluorescent protein (gfp), greatly facilitates high throughput screening, e.g. for antiviral compounds or host factors that affect replication. several chikv cdna clones have been constructed in the past, which -except for the west african lineage strain 37997 strain that was isolated from a mosquito -were all based on clinical isolates from infected humans [17] [18] [19] [20] [21] [22] [23] . each natural isolate is expected to have evolved its own specific characteristics in terms of sequence, virulence and virus-host interactions as a result of specific selective pressures within the infected host (tissue) and possibly also during subsequent passaging in cell culture. intrahost evolution and quasispecies diversity is expected to be substantial, especially compared to the relatively low level of interhost variation when the consensus sequences of chikv genomes isolated from different hosts are aligned. the low level of interhost variation is a typical trait of arboviruses, due to evolutionary constraints imposed by the alternating replication in vertebrate and arthropod hosts. a recent study on the distantly related ross river virus indeed reported a high level of intrahost diversity [24] . the existing chikv molecular clones can be considered to represent a single individual genome (or fragments of several individual genomes) out of the whole spectrum of viruses present in the chikv quasispecies population that has been shaped by intrahost evolution and probably a complex set of environmental factors. in contrast, most deposited chikv genome sequences represent the consensus (or master sequence) of a viral quasispecies population. to obtain a virus that -in terms of virulence, sensitivity to antiviral compounds, and chikv-host interactions -is expected to have the general characteristics of the e1-226v chikv strains that were circulating during the 2005-2009 outbreaks, we have constructed a completely synthetic chikv cdna clone based on the consensus sequence of the aligned genomes of these recent isolates. this new infectious clone, chikv ls3 (leiden synthetic 3), and a variant that expresses the egfp reporter gene under control of a duplicated subgenomic promoter (chikv ls3-gfp), were created by custom dna synthesis. the properties and replicative cycle of the new synthetic viruses were characterized in detail, and comparison with a field isolate (ita07-ra1) from the 2007 chikv outbreak in italy demonstrated that they have similar characteristics. the sensitivity of ls3 to a number of antiviral compounds was compared to those of ita07-ra1 and clinical isolate nl10/152. all compounds tested had a similar antiviral activity against ls3 and the natural isolates. these experiments also identified 3-deaza-adenosine as a novel inhibitor of chikv replication. this study describes a detailed characterization of the chikv replication cycle at the molecular level and demonstrates that a new synthetic infectious clonederived virus is a useful and representative tool to gain more insight into the replicative cycle of chikv, its interactions with the host, and the mode of action of antiviral compounds, which should aid in the development of antiviral strategies against this important human pathogen. vero e6, ae. albopictus c6/36 [25] and 293/ace2 cells [26] were maintained in dulbecco's modified eagle's medium (dmem; lonza), supplemented with 8% fetal calf serum (fcs; paa), 2 mm l-glutamine, 100 iu/ml of penicillin and 100 mg/ml of streptomycin. 293/ace2 cells were grown in the presence of 12 mg/ml blasticidin (paa) and c6/36 medium was supplemented with non-essential amino acids (lonza). bhk-21 cells were cultured in glasgow's modified eagles medium (gibco) supplemented with 7.5% fcs, 10 mm hepes ph 7.4, 8% tryptose phosphate broth (gibco), and antibiotics. the mammalian cell lines were grown at 37uc and c6/36 cells at 30uc in 5% co 2 . chikv strain ita07-ra1 (genbank accession number eu244823) was isolated from ae. albopictus during the 2007 outbreak in ravenna, italy, and was passaged twice on bhk-21 cells. chikv nl10/152 (genbank kc862329) was isolated at the erasmus medical center in rotterdam from the serum of an infected traveler that returned from indonesia and was passaged twice on vero cells. working stocks of chikv were routinely produced in vero e6 cells at 37uc, typically yielding titers of ,10 7 pfu/ml. infections were performed in eagle's minimal essential medium (emem; lonza) with 25 mm hepes (lonza) supplemented with 2% fcs, l-glutamine, and antibiotics. after 1 h, the inoculum was replaced with fresh culture medium. all procedures with live chikv were performed in a biosafety level 3 facility at the leiden university medical center. a cdna clone of the synthetic chikv strain ls3-gfp, which contains a duplicated subgenomic promoter and expresses the egfp reporter gene, was designed in silico as described in the results section. three dna fragments together forming a cdna copy of chikv ls3-gfp were chemically synthesized (geneart, germany). using standard cloning techniques, these fragments were assembled and cloned into the asci-noti sites of vector puc19an, a puc19-derived plasmid in which the original polylinker was replaced by one with asci-ncoi-ecorv-xhoi-noti sites. the resulting plasmid (pchikv-ls3-gfp) contains the genomic cdna of chikv ls3-gfp directly downstream of a phi2.5 promoter and followed by a unique spei linearization site for dna linearization prior to in vitro transcription. the 'wild type' synthetic pchikv-ls3 construct was made by deleting the 920 bp egfp-containing saci fragment from pchikv-ls3-gfp (fig. 1) . a third variant with a duplicated subgenomic promoter and a multiple cloning site behind subgenomic promoter 1 (pchikv-ls3-mcs), which allows the introduction of e.g. a reporter gene, was generated by removing the 737 bp asisi-paci fragment from pchikv-ls3-gfp. the constructs were verified by sequencing. in vitro transcription and rna transfection rna was transcribed from plasmids with the phi2.5 promoter [27] using the ampliscribe t7 high yield transcription kit (epicenter), the m 7 gpppa rna cap structure analog (neb) and 0.7 mg of template dna that had been linearized with spei. after a 3-h reaction at 37uc, template dna was digested with dnasei and rna was purified by precipitation with 7.5 m licl (ambion). the concentration of in vitro transcribed rna was determined with a nanodrop spectrophotometer (thermo scientific) and its integrity was checked by agarose gel electrophoresis. bhk-21 cells (2610 6 ) were electroporated with 1 mg of rna using program t-20 of the amaxa nucleofector and kit t (lonza) according to the manufacturer's instructions. electroporated cells were plated in 6well clusters and incubated at 37uc in the same medium used for chikv infection experiments. chikv rna was isolated from virions using the qiaamp viral rna mini kit. four overlapping amplicons were generated by a two-step reverse transcriptase (rt) pcr. in the first step cdna was synthesized using revertaid h minus reverse transcriptase (fermentas) and primers at-39 (gactgca-gatgcccgccatt), at-41 (cgctcggtccagg-caactct), at-43 (cgtggtgtttgccaacaggc), or at-52 (cgccgttttttttttttttttttttttttt). in the second step 4 pcr products were generated using combinations of primers at-38 (atggctgcgtgagacacacg) and at-39, at-40 (tgcacccaagtgtaccacaa) and at-41, at-42 (caggagagtgcatccatggc) and at-43, or at-44 (gaatgcgcgcagatacccgt) and at-52. the resulting rt-pcr products were purified and directly sequenced (50 ng template) using the bigdye terminator cycle sequencing kit v1.1 (applied biosystems) and a 3130 genetic analyzer automatic sequencer (applied biosystems). pcr conditions and primer sequences are available upon request. viral titers were determined by plaque assay on vero e6 cells. six-well clusters containing confluent monolayers of vero e6 cells were incubated with 0.5-ml volumes of 10-fold serial dilutions of chikv-containing samples. after a 1-h incubation at 37uc, the inoculum was replaced with 2 ml of dmem containing 1.2% avicel rc-581 (fmc biopolymer), 2% fcs, 25 mm hepes, and antibiotics. after a 66-h incubation at 36uc, monolayers were fixed with 3.7% formaldehyde in pbs and plaques were visualized by crystal violet staining. for infectious center assays 10-fold serial dilutions of electroporated cells were added to 6-well clusters already containing a monolayer of 1610 6 bhk-21 cells per well. after a 1-h incubation at 37uc, a dmem/avicel overlay was applied and cells were incubated at 37uc for 48 h. plaques were visualized as described above. total rna was isolated from 7610 5 cells by lysis in 0.5 ml of 20 mm tris-hcl (ph 7.4), 100 mm licl, 2 mm edta, 5 mm dtt, 5% (w/v) lithium dodecyl sulfate, and 100 mg/ml proteinase k. after acid phenol (ambion) extraction, rna was precipitated with isopropanol, washed with 75% ethanol, and dissolved in 1 mm sodium citrate (ph 6.4). samples containing rna from 4.7610 4 cells were mixed with 3 volumes of 67% formamide, 23% formaldehyde, 6.7% glycerol, 13 mm mops (ph 7.2), 6.7 mm naac, 2.7 mm edta, 0.07% sds, and 0.03% bromophenol blue. after denaturation for 15 min at 75uc, rna was separated in 1.5% denaturing formaldehyde-agarose gels using the mops buffer system as described [28] . rna molecules were detected by direct hybridization of the dried gel with 32 p-labeled oligonucleotides essentially as described previously [29] . positive-stranded genomic and subgenomic chikv rnas were visualized with probe chikv-hyb4 (59-tgtgggttcggagaatcgtg-gaagagtt-39) that is complementary to the 39 end of the genome. negative-stranded rna was detected with probe chikv-hyb2 (59-aacccatcatggatcctgtgtacgtg-ga-39) that is complementary to the 39 end of the minus strand. 18s ribosomal rna (loading control) was detected with the oligonucleotide probe 59-atgcccccggccgtccctct-39. probes (10 pmol) were labeled with 10 mci [c-32 p]atp (perki-nelmer) in a 1h reaction using 10 u of t4 polynucleotide kinase (invitrogen) in 10 ml of the supplied forward reaction buffer (invitrogen). prehybridization (1 h) and hybridization (overnight) were done at 55uc in 56 sspe (0.9 m nacl, 50 mm nah 2 po 4 , 5 mm edta, ph 7.4), 56 denhardt's solution, 0.05% sds, and 0.1 mg/ml homomix i. hybridized gels were washed twice in 56 sspe with 0.05% sds before they were exposed to storage phosphor screens. after scanning with a typhoon-9410 scanner (ge healthcare), quantification of rna levels was done with quantity one v4.5.1 (biorad) and corrections for loading variations were made based on the quantity of 18s ribosomal rna in the same lane. the results of two or three independent experiments were quantified (one representative experiment is shown in figures). total protein samples were prepared by lysing 7610 5 cells in 0.5 ml of 46 laemmli sample buffer (100 mm tris-hcl, ph 6.8, 40% glycerol, 8% sds, 40 mm dtt, 0,04 mg/ml bromophenol blue). proteins were separated by sds-page in 12% polyacrylamide gels and were transferred to hybond-lfp membranes (ge healthcare) by semi-dry blotting. after blocking with 1% casein (sigma) in pbs with 0.1% tween-20 (pbst), membranes were incubated overnight with rabbit antisera against chikv nsp1 (raised using the peptide eveprqvtpndhan), nsp4 (raised using the peptide assrsnfeklrgpv) or e2 [30] in pbst with 0.5% casein. mouse monoclonal antibodies against b-actin (sigma), or the transferrin receptor (zymed) were used for detection of loading controls. biotin-conjugated swine-a-rabbit (dako) or goat-a-mouse (dako), and cy3-conjugated mousea-biotin (jackson) were used for fluorescent detection of the primary antibodies with a typhoon-9410 scanner (ge healthcare). at various time points post infection approximately 2610 5 chikv-infected or mock-infected 293/ace2 cells in 12-well clusters were incubated with 40 mci of 3 h-uridine in medium and incorporation was allowed to proceed for 60 minutes at 37uc. total rna was isolated and analyzed in a denaturing agarose gel as described above. for fluorographic detection of 3 h-labeled rna, the gel was soaked in methanol for 1 hour (one change) and then incubated with 3% 2,5-diphenyloxazole in methanol for at least 3 hours. after incubation in milliq for 30 minutes, the gel was dried and a fuji rx film was placed on top. films were developed after a 1-4 day exposure at 280uc and scanned with a biorad gs-800 densitometer. to check equal sample loading, the gel was hybridized with a 32 p-labeled 18s ribosomal rna-specific probe as described above. in addition, incorporation of 3 h-uridine into rna was quantified by analyzing 2-ml samples of isolated total rna with a liquid scintillation counter (beckman ls 6500 ic). in control samples, cellular transcription was inhibited by adding actinomycin d (sigma) to a final concentration of 5 mg/ml. at various time points post infection approximately 2610 5 chikv-infected or mock-infected 293/ace2 cells in 12-well clusters were starved in dmem lacking l-methionine and lcysteine (invitrogen) for 30 min., and subsequently incubated with 44 mci easytag express 35 s protein labeling mix (perkinelmer) for 30 min. total protein samples were analyzed by sds-page as described above. gels were stained with coomassie to check equal sample loading and 35 s-labeled proteins were detected by drying the gels and exposing them to a storage phosphor screen, which was scanned 1-2 days later with a typhoon-9410 scanner (ge healthcare). chikv-or mock-infected vero e6 cells grown on coverslips were fixed with 3% paraformaldehyde in pbs. after quenching with 10 mm glycine in pbs, cells were permeabilized with 0.1% triton in pbs for 10 min. and coverslips were incubated with primary antibodies diluted in pbs with 5% fcs for 1 h. doublestranded rna was detected with a 1:200 dilution of mouse monoclonal antibody j2 (english & scientific consulting). chikv e2 was visualized with a 1:8000 dilution of a polyclonal rabbit antiserum [30] . detection of primary antibodies was done with donkey-a-mouse-cy3, goat-a-rabbit-cy3 or goat-a-rabbit-alexa488 (1:500; jackson). nuclei were stained with hoechst 33342. the coverslips were mounted with prolong (invitrogen) and analyzed using an axioskop2 mot plus fluorescence microscope with axiocam hrc camera and axiovision software (zeiss). mouse monoclonal antibodies raised against chikv particles of strain ita07-ra1 (izsler, brescia, italy) were heatinactivated for 30 min. at 56uc. two-fold serial dilutions of the neutralizing monoclonal antibody 1h7 and non-neutralizing control antibody 3h9 [31] were incubated with an equal volume of medium containing 100 pfu of chikv. these mixtures were incubated for 60 min. at 37uc and transferred to 96-well clusters containing 2610 4 vero e6 cells per well. after incubation at 37uc for 2 days, the wells were fixed with 3.7% formaldehyde and cpe was detected by staining with crystal violet. chloroquine, 6-aza-uridine and ribavirin were dissolved in pbs. cyclosporin a and 3-deaza-adenosine were dissolved in dmso. mycophenolic acid was dissolved in ethanol. all compounds were obtained from sigma. for cpe reduction assays, 96-well clusters with ,1610 4 vero e6 cells/well were incubated with 50 pfu of virus per well, corresponding to a multiplicity of infection (moi) of 0.005, and 2-fold serial dilutions of the compound in medium. wells without cells, uninfected cells, infected untreated cells and infected cells treated with solvent alone were included as controls. four days post-infection cell viability was assessed using the celltiter 96h aqueous non-radioactive cell proliferation assay (promega). cpe reduction experiments with ribavirin were done with bhk-21 cells in a similar way, except that viability was assessed 2 days post infection. for egfp reporter gene assays, ,1610 4 vero e6 cells/well in black 96-well plates were infected with chikv ls3-gfp at an moi of 0.05. after a 42-h incubation in medium containing the compound, the cells were fixed with 3% paraformaldehyde in pbs. egfp expression was quantified using a berthold mithras lb 940 plate reader, with excitation and emission wavelengths of 485 and 535 nm, respectively. the fluorescence in wells containing mock-infected cells was used to correct for background signal. ic 50 and cc 50 values were calculated with graphpad prism 5 using the nonlinear regression model. all animal experiments described in this paper were carried out in the bsl3 facilities of the erasmus medical center in accordance with the dutch guidelines for animal experimentation and were approved by the institute's independent animal ethics committee. twelve-day old c57bl/6 mice were injected intraperitoneally with 100 tcid 50 of chikv s27, chikv ls3 or chikv ls3-gfp. after the challenge the mice were monitored daily for signs of illness or death. the infection was considered lethal when the animals reached humane end-points and needed to be euthanized. viral rna was extracted from brain samples using the automated magnapure method (total nucleic acid isolation kit, roche diagnostics, the netherlands) according to the manufacturer's instructions, and quantified using a one-step rt-pcr taqman protocol (ez-kit, applied biosystems) and an abi prism 7500 detection instrument. the primers and probe used for chikv rna quantification were essentially as described [32] except that probe fam-ccaatgtcttcagcctgga-caccttt-tamra was used. dilutions of virus suspensions of known titer were included to make a calibration curve, which was used to express results as tcid 50 equivalents per gram of brain tissue. all animal experiments described in this paper were carried out in the bsl3 facilities of the erasmus medical center in accordance with the dutch guidelines for animal experimentation. a dutch government-approved and independent animal experimentation ethical review committee (stichting dec consult) approved the animal studies (permit nr. emc2838/122-12-29). the genbank accession numbers for the full-length cdna clones pchikv-ls3, pchikv-ls3-gfp and pchikv-ls3-mcs are jx911334, jx911335, and jx911336 respectively. the genbank accession numbers for the genomic rna sequences of chikv ls3, ls3-gfp, lcs3-mcs and nl10/152 are kc149888, kc149887, kc149889, and kc862329, respectively. the complete genomes of the 13 chikv strains carrying the e1-a226v mutation ( table 1 ) that were available in genbank at the time of in silico design (november 2009) were aligned using mafft [33] and the resulting consensus sequence formed the basis for the synthetic full-length cdna clones. a 40 nucleotide polya tail was added to the 39 end of the consensus sequence and an a7435g point mutation was introduced to create a translationally silent saci restriction site required for cloning. the virus encoded by the resulting sequence was termed chikv ls3 (genbank accession kc149888). variants containing a duplicated subgenomic promoter and a multiple cloning site (chikv ls3-mcs; genbank kc149889) or an egfp reporter gene (chikv ls3-gfp; genbank kc149887) were also designed. the egfp reporter gene was placed under control of the native subgenomic promoter and upstream of a second subgenomic promoter that drives expression of the viral structural polyprotein, as this configuration was previously reported to result in a more stable reporter gene expression [18] . the chikv cdnas were placed downstream of a phi2.5 t7 promoter, and a unique spei site for linearization prior to in vitro transcription directly followed the polya tail. the phi2.5 promoter was used because the 59 ends of capped transcripts generated from this promoter with t7 polymerase and the m 7 gpppa cap analog are identical to the 59 end of the genomes of naturally occurring chikv strains. in contrast, capped rnas generated by in vitro transcription from the frequently used sp6 promoter will contain m 7 gpppg at their 59 terminus, i.e. will contain an additional 59terminal g residue. however, existing chikv cdna clones that contain the sp6 promoter also efficiently yield infectious virus and it is assumed that the additional 59-terminal g residue is removed during subsequent rounds of replication. in line with this, in vitro transcribed rna from pchikv-ls2, a variant of plasmid pchikv-ls3 in which the phi2.5 promoter was replaced with the sp6 promoter also yielded infectious virus. plasmid pchikv-ls3-gfp, the infectious clone encoding the egfp-expressing reporter virus, was created by assembling the chemically synthesized dna fragments as described in the materials and methods section. plasmid pchikv-ls3, the infectious clone encoding the synthetic 'wild type' strain chikv ls3, and plasmid pchikv-ls3-mcs were generated from pchikv-ls3-gfp by deleting specific restriction fragments, as described in materials and methods (fig. 1) . in the original alignment, strains drde-07 (genbank u372006) and d570/06 (genbank ef012359) shared the highest sequence similarity with ls3, with 3 amino acid differences (table 2) . however, a blast search performed in march 2013, three years after the design of chikv ls3, and alignment of the retrieved complete chikv genomes revealed that strains ind-06-ap3 (genbank ef027134), ind-gj53 (genbank fj000065), and chik31 (genbank eu564335) share the highest degree of nucleotide sequence identity with chikv ls3 (.99.9%), with only 5-7 nucleotide differences respectively (table s1). interestingly, these indian strains were not included in the original alignment on which the ls3 sequence was based, as they do not contain the e1-a226v mutation (table 1) . however, nsp1234 of ls3 is identical to that of ind-06-ap3. at the amino acid level, chikv ls3 differs at 4 positions from lr2006_opy1 and at 3 positions from ita07-ra1 (table 2 ). to determine whether infectious virus could be generated from the synthetic chikv clones, in vitro transcribed rna was electroporated into bhk-21 cells. strong egfp fluorescence was readily detected 16 h after transfection of chikv ls3-gfp rna. for chikv ls3 and ls3-gfp rna specific infectivities of approximately 10 5 pfu/mg of rna were found in infectious center assays, which is similar to what has been found for other chikv cdna clones [17, 18] . virus titers in cell culture supernatants 16 h after electroporation, were generally in the range of 10 5 -10 6 pfu/ml. this is lower than the peak viral titers that are obtained during infection experiments, but can be explained by the early time point of harvesting and the fact that not all cells were transfected. as expected, electroporation of bhk-21 cells with uncapped chikv rna did not result in the release of infectious virus. to assess the stability of egfp reporter expression, chikv ls3-gfp was serially passaged (moi 0.5) in both 293/ace2 and vero e6 cells. virus harvested during each passage was used to infect vero e6 cells at an moi of 0.2 and immunofluorescence microscopy revealed that at passage 10, over 95% of the e2positive foci still displayed robust egfp expression. sequencing of cdna obtained by rt-pcr amplification of rna extracted from extracellular virions revealed that, after 3 passages on vero e6 cells, the consensus genome sequence of chikv ls3-gfp was identical to the original in silico designed sequence. these results demonstrated that the synthetic viruses are viable, genetically stable, and able to retain stable expression of the reporter gene. since we aim to use chikv in sirna screens and proteomics studies to identify host factors involved in replication, various human cell lines were evaluated for their ability to support chikv replication. chikv ls3-gfp was able to productively infect hela, mrc-5, huh7, 293, and 293/ace2 cells (data not shown). infection of hela and huh7 cells was not very efficient and these cells were therefore not used for any further experiments. 293/ace2 cells were selected for this study, as they supported high levels of chikv ls3-gfp replication, could be efficiently transfected with sirnas, and -unlike regular 293 cellsadhered well to tissue culture plastics. 293/ace2 cells stably express angiotensin-converting enzyme 2 (ace2), the receptor for sars-coronavirus. ace2 expression is not required for chikv infection, but these cells were chosen because of the aforementioned advantages and the fact that they have been previously used in our lab in sirna screens for host factors that affect corona-and arterivirus replication ( [34] ; de wilde et al. submitted; wannee et al., in preparation). using these cells in similar sirna screens with chikv and other alphaviruses would allow direct comparison of data sets with those obtained for corona-and arteriviruses, which could lead to the identification of common (broad spectrum) pro-and antiviral host factors. to determine whether the synthetic viruses behave like natural isolates, their growth kinetics in vero e6, 293/ace2, and c6/36 cells were compared to those of ita07-ra1, which was isolated during the 2007 chikv outbreak in italy ( fig. 2a-c) . the growth curves of chikv ls3 on all three cell lines were found not to differ significantly from those of ita07-ra1, with virus titers reaching a maximum 14-18 h post infection (p.i.). peak virus titers on mosquito cells were approximately 1-log higher than those on mammalian cells. chikv ls3-gfp replicated slightly slower than the other viruses in all three tested cell lines, which is not unusual for recombinant reporter viruses. egfp expression could be detected as early as 6 h p.i. and peaked around 22 h p.i. the plaque morphology of the synthetic viruses was similar to that of ita07-ra1 (fig. 2d) . chikv ls3 induced a cytopathic effect (cpe) indistinguishable from the natural isolate. on vero e6 cells early signs of cpe started to appear around 12 h p.i. and cpe was complete by 24 h p.i. (fig. 2e) . to study chikv-induced transcriptional host shut-off, the incorporation of 3 h-uridine into cellular and viral rna was analyzed by metabolic labeling of infected 293/ace2 cells at various time points post infection (moi of 5). a strong reduction in the incorporation of 3 h-uridine into rna was observed at 10-12 h p.i. in cells infected with chikv ls3 or ita07, as determined by liquid scintillation counting of total rna samples table 2 . comparison of the amino acid sequences of chikv ls3 and various natural isolates. the chikv ls3 amino acid sequences were aligned with those of several highly similar natural isolates, and clinical isolate nl10/152. only amino acid differences are indicated and identity is represented by dots. the numbering is based on the sequence of ls3, which is also equal to that of lr2006_opy1. it is important to note that -like all other chikv strains in this ( fig. 3a) . inhibition of cellular transcription with actinomycin d for 30 min. prior to metabolic labeling at 12 h p.i. revealed the contribution of viral rna synthesis to the total signal. fluorographic detection of 3 h-labeled rna analyzed in denaturing gels also showed a decrease in cellular transcription during the course of the infection, while the synthesis of chikv rna became clearly detectable by 6 h p.i (fig. 3b) . transcriptional shut-off occurred around 10-12 h p.i. and was induced by chikv ls3 and ita07 with similar kinetics, although ls3 seemed to act slightly faster. to examine chikv-induced translational shut-off, the synthesis of 35 s-labeled viral and cellular proteins during the course of chikv ls3 infection was analyzed by metabolic labeling of infected 293/ace2 cells with 35 s-met and 35 s-cys (fig. 3c) . a clear shut-off of host translation was observed 8-9 h p.i. beyond 9 h p.i. the bulk of newly produced protein appears to be of viral origin, likely c, e1, e2 and their precursors (indicated with * in fig. 3c ). chikv ita07 and ls3 induced translational host shut-off in a similar manner (only results obtained with ls3 are shown in fig. 3c ). both chikv ita07-ra1 and the synthetic viruses established non-cytopathic persistent infections in c6/36 mosquito cells. all characterization experiments have been performed in both 293/ ace2 and vero e6 cells, with similar results. for simplicity only the results for 293/ace2 cells are shown, except for immunofluorescence experiments, which were done with vero e6 cells as they had a more suitable morphology. the replication cycle of the synthetic viruses and ita07-ra1 was characterized in more detail to assess whether the synthetic viruses behaved like their natural counterpart. the kinetics of rna synthesis was analyzed by isolating total rna from 293/ ace2 cells infected with chikv ls3, ls3-gfp, or ita07-ra1 at various time points post infection. negative-and positivestranded rnas were detected by hybridization with 32 p-labeled oligonucleotide probes (fig. 4a ). both negative-and positivestrand rnas were readily detected in cells infected with the various strains starting at 6 h p.i. the negative-strand rna was less abundant than the positive strand, it was easily detected relatively early in infection ( fig. 4a top panel, fig. 4b) , and appeared to decrease at later time points as has also been observed for other alphaviruses. this apparent decrease is probably not only due to degradation of minus strands, but at least partly due to a hampered detection caused by the large excess of positive-strand rna present at late time points. this excess of positive-strand rna competes with the radioactively labeled minus-strand specific probe. in support of this, we observed that mixing rna isolated from chikv-infected cells at 6 h p.i. with in vitro transcribed positive-strand rna reduced the amount of negative strand that could be detected (data not shown). furthermore, when samples taken at 6 and 14 h p.i. were treated with singlestrand-specific rnase a/t1 before hybridization, the negativestrand levels at the late time point were approximately 70% of that at 6 h p.i, instead of the approximately 50% in untreated samples (data not shown). using a positive-strand-specific probe, both the 49s genomic and 26s sgrna could be detected, and both rnas accumulated until 12 h p.i (fig. 4a middle panel, fig. 4c ). the ratio of genomic to sgrna varied between 1:3.5 and 1:5.5 during the course of infection, similar to the ratios reported for semliki forest virus and sinv [35] . the kinetics of rna synthesis and rna accumulation levels were similar in chikv ls3-and ita07-ra1-infected cells. in cells infected with chikv ls3-gfp, the additional subgenomic rna encoding the egfp reporter gave rise to an extra band above the 26s rna band, and its expression level was calculated to be approximately half of that of the 26s rna. the individual levels of the two sgrnas expressed by chikv ls3-gfp were lower than those of ita07 or ls3, but their combined abundance was comparable to that of the viruses expressing a single sgrna (fig. 4c ). to monitor viral protein expression, 293/ace2 cells were infected with chikv ls3, ls3-gfp, or ita07-ra1 and total protein was isolated at various time points post infection. these samples were analyzed by western blotting with antisera against the nonstructural proteins nsp1 and nsp4, and the structural protein e2. expression of nsp1, e2, and the e3e2 precursor could be detected as early as 6 h p.i. and the proteins accumulated over time, reaching a plateau around 12 h p.i. (fig. 5) . the rdrp nsp4 could not be detected in infected cells using a chikv nsp4specific antiserum capable of detecting the purified bacterially expressed protein. this was probably due to the low affinity of the antibody, the low expression level and relative instability of nsp4 in infected cells, as was also observed for other alphaviruses [36] . in addition, a quantitative proteomics study on chikv-infected cells also suggested that at 10 h p.i. the amount of nsp4 was at least 200-fold lower than that of nsp1 (treffers, tas, de ru, van veelen, snijder and van hemert, manuscript in preparation). indirect immunofluorescence analysis of vero e6 cells infected with chikv ls3, ls3-gfp, or ita07-ra1 at various time points showed that the localization and expression kinetics of e2 and dsrna were similar for the natural isolate and the synthetic viruses (fig. 6) . double-stranded rna, which is assumed to be generated during replication of chikv in infected cells [37] , could be detected as early as 4 h p.i. and remained clearly visible throughout the infection. the dsrna localized to foci throughout the cytoplasm. the e2 protein could be detected from 6 h p.i. onwards with maximum expression reached by 12 h p.i. the e2 protein mainly localized to the plasma membrane of infected cells. egfp produced by the reporter virus was visible from 6 h p.i. onwards, reaching a maximum level around 12 h p.i. (fig. 6c ). chikv ls3 and ita07-ra1 were compared in a neutralization assay using the neutralizing monoclonal antibody 1h7 that was raised in mice against chikv ita07-ra1 virions, and appears to recognize a linear epitope in e2 [31] . the nonneutralizing mab 3h9 was used as a control. both the natural isolate and chikv ls3 were neutralized with similar characteristics by 1h7, while their infectivity was not affected by 3h9 (fig. 7) . newborn mice are highly susceptible to chikv infection and they develop symptoms as lethargy, dragging of hind limbs, flaccid paralysis, and reduced weight gain [38] . 12-day old mice were injected intraperitoneally with 100 tcid 50 of chikv ls3, ls3-gfp or prototype strain s27 as a control. the animals were euthanized when their humane end points were reached 3 or 4 days post infection and viral rna levels in brain tissue were analyzed (fig. 8) . both synthetic viruses behaved like the natural isolate in vivo, causing lethal infections with similar kinetics (fig. 8a ). in addition, the viral titers in the brains of chikv s27-infected mice were similar to those of mice infected with the synthetic viruses (fig. 8b ). to evaluate their suitability for analyzing the potency and mechanism of action of antiviral compounds, the sensitivity of chikv ls3 and ls3-gfp to a number of such compounds was determined and compared to ita07-ra1. cyclosporin a, which through its effect on cellular cyclophilins inhibits the replication of a variety of viruses, had no specific effect on chikv replication, not even at a (cytotoxic) dose of 32 mm (data not shown). the compounds 3-deaza-adenosine, 6-aza-uridine, chloroquine, and mycophenolic acid were tested in cpe reduction assays with vero e6 cells infected at an moi of 0.005 and analyzed 4 days p.i. they were all found to inhibit chikv replication with ic 50 s in the low micromolar range and with minimal cytotoxicity (fig. 9a-d) . no substantial differences were observed between the ic 50 values calculated for ita07-ra1, ls3 and ls3-gfp. the four compounds also clearly reduced egfp reporter gene expression in vero e6 cells infected with chikv ls3-gfp (fig. 9f) . slightly lower ic 50 values were obtained for 6-aza-uridine and chloroquine, and a significantly higher ic 50 was observed for 3-deazaadenosine in this assay, compared to the cpe-based assay. this might be due to the mode of action of 3-deaza-adenosine and/or due to differences in experimental set-up compared to the cpebased assay (moi 0.05 vs. 0.005; measurement 42 h p.i. vs. 4 d p.i). ribavirin is a known inhibitor of chikv replication, but in our cpe reduction assay with vero e6 cells it was not very effective in inhibiting replication of the various strains, as ic 50 values of over 400 mm were obtained (fig. 9e, gray lines) . this is likely due to the inefficient conversion of ribavirin to its active phosphorylated form in vero e6 cells [39] . therefore, we have also analyzed the effect of ribavirin in a 2-day cpe reduction assays with bhk-21 cells, which are able to metabolize ribavirin [40, 41] and found ic 50 s of 15-21 mm for the various strains. clinical isolate nl10/152 was also included in the assays and appeared to be somewhat more sensitive to the antiviral compounds than ls3 and ita07-ra1. however, the slower replication kinetics of this strain made it impossible to directly compare nl10/152 and ls3 in the same cpe reduction assays, despite the fact that virus yields and cytopathic effect of nl10/152 and ls3 were comparable (data not shown). the massive chikv outbreaks that have been occurring in asia and the indian ocean region since 2005 are associated with the emergence of strains with the a226v substitution in the e1 glycoprotein, which allowed their transmission by a novel mosquito vector, aedes albopictus [8] [9] [10] [11] [12] [13] . these east-central-south african lineage-derived strains even appear to be replacing the the relative abundance of rna was calculated as before, except that data were normalized to the value measured for ls3 sgrna at 12 h p.i (100%). genomic rna levels are indicated with black lines, sgrna levels with gray lines. the total level of both sgrnas expressed by ls3-gfp is indicated with the gray dotted line. doi:10.1371/journal.pone.0071047.g004 asian lineage chikv strains that have been endemic in the region for decades. since the 1980s, the geographic distribution of aedes albopictus has dramatically expanded and now also includes large parts of the usa and several european countries. this creates concern for locally transmitted outbreaks in europe and the usa, which could be initiated by viraemic travelers arriving from countries where chikv is endemic, like india and indonesia. locally transmitted chikv infections have indeed already been reported from italy in 2007 and france in 2010 [15, 16] and recent studies suggest that also the usa is at risk for locally transmitted chikv outbreaks [42, 43] . besides its large medical and societal impact in endemic countries, the increased risk of chikv outbreaks in europe and the usa underlines the importance of studying the replication of this important human pathogen and its interactions with the host to develop safe and effective vaccines and antiviral therapy. infectious cdna clones have proven to be important tools to study many aspects of the viral life cycle, and molecular clones of a variety of natural isolates have been instrumental in several recent chikv studies [17] [18] [19] [20] [21] [22] [23] . the existing chikv molecular clones can be considered to be derived from a single genome (or fragments of single genomes) out of the whole spectrum of viruses present in a chikv quasispecies population. in contrast, most of the complete chikv genome sequences that have been deposited in genbank represent the consensus (or master sequence) of a viral quasispecies population. the diversity (and evolution) of a chikv quasispecies population has probably been shaped by the characteristics of the individual host and the specific tissue source (serum) from which it was isolated. for ross river virus it was observed that the level of intrahost genetic variation in patient serum samples, was considerably larger than that observed at the epidemiological scale, which can be explained by the purifying selection for replication in both arthropod and vertebrate hosts [24] . advances in sequencing techniques now allow a more detailed view on quasispecies diversity and intrahost evolution, and also for chikv a recent study has provided more insight into quasispecies dynamics and the effect of purifying selection by host alternation [44] . a link was observed between increased fitness as a result of alternating passaging and reduced quasispecies complexity, which restricted adaptability to novel selective pressures like antiviral treatment or antibody-mediated neutralization [44] . individual chikv isolates or molecular clone derived viruses could have their specific properties in terms of replication kinetics, vector specificity, dissemination within the host, virulence, virus-host interactions or sensitivity to antiviral compounds. we were interested in studying the general characteristics of the life cycle and virus-host interactions of the e1-226v chikv strains that were circulating during the 2005-2009 outbreaks. therefore, we have constructed a fully synthetic cdna clone, chikv ls3, based on the consensus sequence of the aligned genomes of these recent e1-226v isolates, rather than on a single genome from a clinical isolate. in addition, a variant that expresses the egfp reporter gene under control of a (duplicated) subgenomic promoter was created (chikv ls3-gfp). the current possibilities of gene synthesis allowed the design of these clones in silico, with sequences tailored to our requirements, e.g. already containing a reporter gene under control of a duplicated subgenomic promoter and including (translationally silent) mutations to create restriction sites that facilitate cloning and reverse genetics studies. alignment of all 148 complete chikv genomes that were in genbank by june 2013 yielded a consensus sequence that differed only at 3 nucleotide positions from the sequence of ls3 that was designed 3 years earlier. these were position 7,435 at which we introduced a translationally silent restriction site (g7435a), a synonymous urc substitution at position 3,397, and position 10,670, which is a c in 68% of all deposited genomes (strains with e1-226a), while the remaining (e1-226v) strains have a u at this position. an interesting observation was that 6% of the sequenced chikv strains, including the prototype strains s27 and ross, contain an arginine codon instead of the opal stop codon that is present between the nsp3-and nsp4-coding regions of most chikv isolates. the presence or absence of this opal codon might be influenced by the passage history of the isolate as has been observed for other alphaviruses [45] . this might also explain why the sequence of the original clinical isolate of lr2006-opy1 (genbank dq443544.2) contains the opal termination codon near the end of the nsp3 coding region, while the infectious clone of this strain (genbank eu224268.1) contains an arginine codon at this position. to assess whether the synthetic viruses are representative models, their characteristics were compared to those of the natural strain ita07-ra1. like the natural isolate, the synthetic viruses caused cytopathic infections in vero e6 and 293/ace2 cells (fig. 2e) , whereas non-cytopathic persistent infections were observed in the mosquito cell line c6/36. in vertebrate cells all strains caused a shut-off of cellular translation around 8-9 h p.i. and a strong inhibition of cellular transcription by 10-12 h p.i. the accumulation of negative-and positive-strand viral rna, the kinetics of non-structural and structural viral protein expression, as well as the growth kinetics and plaque morphology of the synthetic viruses were indistinguishable from those of chikv ita07-ra1 ( fig. 2-6 ). in addition, the synthetic viruses caused lethal infections in 12-day old mice, with virus spreading to the brain, as observed for natural isolates (fig. 8) . although this demonstrates that the synthetic viruses replicate in vivo, this mouse model does not allow comparison of strains for more subtle differences in virulence and pathogenesis. the genomic stability of chikv ls3-gfp was assessed and after 3 passages its (consensus) sequence was found to be identical to the original in silico designed sequence. the expression of the egfp reporter gene was stable for at least 10 passages, making the synthetic viruses suitable tools for highthroughput screens for antiviral compounds, (reverse genetics) studies into their mechanism of action, and systematic functional genomics screens for host factors affecting chikv replication. to evaluate whether chikv ls3 and ls3-gfp are suitable to analyze the potency and mechanism of action of antiviral compounds, their sensitivity to a number of such compounds was determined and compared to ita07-ra1. the lysosomotropic agent chloroquine and nucleoside analog 6-aza-uridine inhibited the replication of the synthetic viruses and natural isolates with ic 50 s that were in the same range and comparable to values previously reported by others [46] [47] [48] [49] [50] [51] . the inhibitory effect of chloroquine on the replication of many viruses including alphaviruses has been known for decades. for chikv it is a useful reference compound in cell-based studies, but a small scale clinical trial on the island of la reunion suggested it is not effective in the treatment of chikv infections in patients [46] . the nucleoside analog 6-aza-uridine has previously been reported to inhibit the replication of a variety of viruses, including chikv [47, 51] . the compound could interfere with cellular utp metabolism and may be incorporated into chikv rna, leading to chain termination and/or increased error frequency, ultimately resulting in 'error catastrophe'. mycophenolic acid is a noncompetitive inhibitor of inosine monophosphate dehydrogenase (impdh), causing a depletion of the intracellular guanosine pool. it is a known inhibitor of various viruses, including chikv [47, 52] . ribavirin is a synthetic nucleoside analog with broad spectrum antiviral effect due to potential effects on the cellular impdh enzyme, viral rna synthesis and capping [39] . however, not all cell lines are able to perform the necessary conversion of this compound to its active phosphorylated form, explaining the contradictory reports on the antiviral activity of this compound [40, 41, 53] . in our hands, ribavirin inhibited chikv replication in bhk-21 cells with an ic 50 of around 18 mm, while it was hardly effective in vero e6 cells, with ic 50 values of over 400 m. the ic 50 that we obtained with bhk-21 cells is in the same range as those previously reported for the antiviral effect of ribavirin on chikv replication [47, 51] . cyclosporin a, which through its effect on the cellular cyclophilins, inhibits the replication of a variety of viruses (for recent review see [54] ), had no effect on chikv replication. we identified 3-deaza-adenosine as a novel inhibitor of chikv replication with an ic 50 of approximately 6 mm and a cc 50 .50 mm. this compound has previously been identified as inhibitor of a broad spectrum of viruses, although many other +rna viruses appeared to be rather insensitive or not affected at all (reviewed in [55] ). the antiviral activity of 3-deazaadenosine was attributed to its inhibitory effect on the cellular enzyme s-adenosylhomocysteine hydrolase, leading to an accumulation of s-adenosylhomocysteine, which inhibits s-adenosylmethionine-dependent methylation reactions [55] . in this manner the enzyme plays a key role in s-adenosylmethionine-dependent methylation reactions and inhibition of viral methylation reactions (e.g. of viral rna) apparently can be achieved at compound concentrations that do not notably interfere with cellular methylation reactions. our observation warrants a more detailed analysis of the mode of action of 3-deaza-adenosine and analogs, also to evaluate their potential for use in antiviral therapy to treat chikv infections. overall, no large differences were observed between the ic 50 values calculated for ita07-ra1, ls3 and ls3-gfp, indicating that the synthetic viruses are suitable for use in antiviral screens. for most compounds, a faster and simpler assay with chikv ls3-gfp reporter virus showed a good dosedependent response that correlated well with results obtained in the cpe-based assay. clinical isolate nl10/152 exhibited slightly slower replication kinetics and appeared to be more sensitive to antiviral compounds than ita07 and the synthetic viruses. differences in the sensitivity to antiviral compounds among clinical isolates is not an uncommon phenomenon. nl10/152 differs at 7 amino acid positions from ls3 and it will be interesting to determine the contribution of these mutations, in particular the r88s substitution in nsp4, to the slower replication kinetics (and higher sensitivity to antivirals). taken together the detailed characterization of the chikv replication cycle at the molecular level demonstrated that our new synthetic consensus-based viruses behave like natural isolates and are suitable tools to study various aspects of the chikv life cycle, which should ultimately provide a basis for the development of antiviral therapy. table s1 comparison of chikv ls3 with the genome sequences of various closely related natural isolates. only differences between ls3 and each of the other strains are summarized. dots indicate that the nucleotide at that position is identical to that at the corresponding position in the sequence of ls3. genomes were aligned with mafft and analyzed in jalview. numbering is based on the sequence of lr2006_opy1 (and is equal to ls3 numbering). the nucleotide at position 10670 (indicated in gray) determines whether the strain has the a226v mutation in the e1 protein. strains with a t at this position have the a226v mutation. differences not included in the comparison are the 35 nt, 5 nt and 23 nucleotides that are missing from the 3'utr of the sequences of drde-07, d570/06 and ita07-ra1, respectively. the missing first 19 nt, missing last 13 nt and the insertion of an a after position 11564 in the sequence of ind-06-ap3 were also not included in this comparison. 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virus in the united states host alternation of chikungunya virus increases fitness while restricting population diversity and adaptability to novel selective pressures genetic and fitness changes accompanying adaptation of an arbovirus to vertebrate and invertebrate cells on chikungunya acute infection and chloroquine treatment inhibitors of alphavirus entry and replication identified with a stable chikungunya replicon cell line and virus-based assays characterization of reemerging chikungunya virus chikungunya disease and chloroquine treatment assessment of in vitro prophylactic and therapeutic efficacy of chloroquine against chikungunya virus in vero cells in vitro inhibition of chikungunya and semliki forest viruses replication by antiviral compounds: synergistic effect of interferon-alpha and ribavirin combination cellular impdh enzyme activity is a potential target for the inhibition of chikungunya virus replication and virus induced apoptosis in cultured mammalian cells cell type mediated resistance of vesicular stomatitis virus and sendai virus to ribavirin cyclophilin involvement in the replication of hepatitis c virus and other viruses carbocyclic adenosine analogues as s-adenosylhomocysteine hydrolase inhibitors and antiviral agents: recent advances we are grateful to dr. gorben pijlman (wageningen university, the netherlands) for his generous gift of chikv e2 antiserum and to adriaan de wilde and emmely treffers for helpful discussions and sharing their unpublished data. key: cord-263684-3535k3op authors: tang, julian w.; nicolle, andre; pantelic, jovan; klettner, christian a.; su, ruikun; kalliomaki, petri; saarinen, pekka; koskela, hannu; reijula, kari; mustakallio, panu; cheong, david k. w.; sekhar, chandra; tham, kwok wai title: different types of door-opening motions as contributing factors to containment failures in hospital isolation rooms date: 2013-06-24 journal: plos one doi: 10.1371/journal.pone.0066663 sha: doc_id: 263684 cord_uid: 3535k3op hospital isolation rooms are vital for the containment (when under negative pressure) of patients with, or the protection (when under positive pressure) of patients, from airborne infectious agents. such facilities were essential for the management of highly contagious patients during the 2003 severe acute respiratory syndrome (sars) outbreaks and the more recent 2009 a/h1n1 influenza pandemic. many different types of door designs are used in the construction of such isolation rooms, which may be related to the space available and affordability. using colored food dye as a tracer, the qualitative effects of door-opening motions on the dissemination of potentially contaminated air into and out of a single isolation room were visualized and filmed using reynolds-number-equivalent, small-scale, water-tank models fitted with programmable door-opening and moving human figure motions. careful scaling considerations involved in the design and construction of these water-tank models enabled these results to be accurately extrapolated to the full-scale situation. four simple types of door design were tested: variable speed single and double, sliding and hinged doors, in combination with the moving human figure. the resulting video footage was edited, synchronized and presented in a series of split-screen formats. from these experiments, it is clear that double-hinged doors pose the greatest risk of leakage into or out of the room, followed by (in order of decreasing risk) single-hinged, double-sliding and single-sliding doors. the relative effect of the moving human figure on spreading any potential contamination was greatest with the sliding doors, as the bulk airflows induced were large relative to those resulting from these door-opening motions. however, with the hinged doors, the airflows induced by these door-opening motions were significantly greater. further experiments involving a simulated ventilated environment are required, but from these findings alone, it appears that sliding-doors are far more effective for hospital isolation room containment. isolation rooms to contain infectious patients or to protect vulnerable (e.g. immunocompromised) patients from infection are an important facility to protect patients and staff against the risk of infection by airborne pathogens [1, 2] . recommendations for their use features in many guidelines related to the control of airborne pathogens, especially for tuberculosis [3] [4] [5] [6] . in the aftermath of the severe acute respiratory syndrome outbreaks of 2003, the demand for such rooms increased, dramatically [7] [8] [9] [10] . many of these were eventually utilized in the management of patients infected with the 2009 pandemic influenza a/h1n1 virus [11] [12] [13] [14] . although there have been many studies evaluating the performance of such rooms with regard to the maintenance of the pressure differential across the doors when closed [15] [16] [17] [18] , there have been relatively fewer studies assessing how dooropening motions and healthcare worker passage through the door can affect the performance of such rooms [19] [20] [21] [22] . yet, at least one analytical case report has demonstrated that containment failure may result from simply opening isolation room doors [23] . this study is part of a longer-term project that aims to demonstrate the effects of door-opening motions using a variety of doors, with and without the passage of a human figure, on the movement of potentially contaminated air into and out of an isolation room, using both a small-scale, reynolds-number-equivalent model in water, and a full-scale model in air. in this study, baseline measurements were made using colored food dye visualization in still water (i.e. to simulate still air) for each of the moving figure-door systems, with no simulated ventilation system imposed. it was decided to conduct the experiments in water, at reynolds number equivalent lengths and velocities, such that the results obtained in water could be directly extrapolated to the full-scale situation in air [20] . water was chosen because it was easier to visualize flows, qualitatively, using coloured dye, or, more quantitatively, using neutrally buoyant, suspended, reflective particles for particle image velocimetry (piv). two simplified, one-tenth scale (1:10) models were designed and constructed, as shown in figures 1 and 2 (cys engineering & trading, singapore). all the dimensions of the one-tenth scale models were taken from full-scale models that were being constructed at the same time by collaborators at the finnish institute of occupational health (fioh) and their collaborators oy halton group (finland). one of the larger aims of this study involving the parallel construction of these two models was to test and validate more fundamental scaling assumptions about flow dynamics, using flow visualization methods as well as computational fluid dynamical (cfd) modeling. one tank was designed to accommodate single-and doublesliding doors, the other single-and double-hinged doors, as the door-opening mechanisms were significantly different for these two types (i.e. sliding versus hinged) of doors. each of the tanks had interchangeable door modules so that the single-and doubleversion of the doors could be swapped as required for the experiments. otherwise the tanks were of virtually identical dimensions. the same scale model of a human male figure was used in each tank to maintain similarity. the figure moved on a sliding track that was built into the floor of each tank, and which could be programmed to move at realistic human walking speeds. this track was regularly greased to ensure a smooth sliding action without juddering. the movement controller chip (arduino mega 1280, atmega1280 (silicon core), arduino, chiasso, switzerland) for each of the tanks was manually programmed to allow the figure and doors to move at speeds similar to those observed in real isolation facilities. once each of the figure-door movement systems in each tank was reset, the edges around the door module (including the door and surrounding wall partition) were carefully sealed, manually, with vaselineh petroleum jelly (unilever, london, uk) to prevent any leakage of the food dye across the door partition prior to the running of the experiment. once the seal was in place, the tank was slowly filled with water from the bottom up via a supply pipe entering the floor of the tank. this facilitated the removal of air bubbles from the tank, as it filled, which would otherwise obstruct clear views of the tank from the top that were required for filming. the tank was also placed on a custom-built table which allows a tilt of up to 45u when the tank was full, to allow any air bubbles to escape through small drainage air-holes that were drilled into the roof along its edge. any remaining bubbles were manually suctioned using a 30 ml or 50 ml syringe and small-bore catheter that was passed through these air-holes, as required. once the bubbles had been removed and the tank leveled again, 50 mls of blue food dye (true blue, star brand, kuala lumpur, malaysia) was injected into one side of the tank, as required by the experimental protocol, i.e. into the side containing the human figure (the 'from dye' protocol), or into the side without the figure (the 'to dye' protocol). the syringe catheter was used to mix the food dye carefully within the chamber to ensure a relatively uniform dye distribution. the tank was back-lit using a lighting rig that consisted of a bank of 18 spotlights (240 v, 120 w, par 38 spot 12u, philips, amsterdam, holland) arranged in a 3-row by 6-column grid, whose beams were diffused by a series of cloth diffusers and greaseproof paper that was taped to the back of the water-tank model. three cameras were used to obtain video images: a nikon d7000 with a 28-105 mm nikkor af lens in manual mode (nikon inc., melville, ny), at 24 frames per second (fps), high definition (hd, color, 192061280 pixels) from the front; a nikon 3100 with a 18-55 mm nikkor af-s lens in manual mode, at 24 frames per second (fps), high definition (hd, color, 192061280 pixels) from the top; a photron sa1.1 camera (dynamic analysis system, pte ltd, singapore), at 500 fps (black and white) with a 28-85 mm nikkor (manual) lens, also from the front -to allow extreme slow motion playback. once the lighting was in place and switched on, and the cameras were in position and recording, the controller was activated and the figure and doors moved according to a pre-set program: the figure would accelerate almost instantly to its designated scaled-down walking speed, slide towards the door. it would then stop just before the door to allow it to open, before passing through the doorway to enter the room. the doors would then close behind it, completing the programmed movement cycle. note that the figure's movements were not quite the same for the sliding and hinged doors. a larger clearance in front of the figure was required for the hinged (i.e. when opening towards the figure) than the sliding doors so as to avoid any collision between it and the door. several movement combinations were tried with the figure and the various types of doors, including scenarios where the doors opened and closed alone, with no figure movement, as these defined a baseline airflow behavior for the action of these doors in the absence of any human movement. after each experimental run, the tank was drained of water (now coloured with food dye) and cleaned. if necessary, a different door module was inserted and the edges sealed with vaselineh, before the tank was refilled and the cameras repositioned as required, for the next run. for this baseline series of experiments, all of them were performed in a still water environment, with no pressure differential simulated across the doorway. the programmed velocities of the door-opening and human figure movements were based on the principles of reynolds (re) number equivalence, i.e. that motion in the 1:10 scale model in water should be equivalent to the same motion at full-size in air, i.e. re air = u air l air /n air = re water = u water l water /n water where u, l and nare the velocity, representative lengths and kinematic viscosities in the two media, respectively. if the length scale in the water-tank model is one-tenth of that in air (i.e. l air = 10lwater ), and the kinematic viscosities of water at 20uc and air at 25uc (the approximate operating temperatures in this tropical climate) are 1.004610 26 m 2 /s and 15.6610 26 m 2 /s, respectively, then the ratio of the velocities u air /u water = 1.56/1.004 = 1.55. for the angular velocities of the hinged-doors in air and water (? air and ? water , respectively?, their equivalent linear velocities must also scale as u air /u water = l air ? air /l water ? water = 1.55, so that the ratio of the angular velocities are related as ? air = 0.155? water , or ? air / ? water = where l is the width of the door. due to the qualitative nature of the food dye tracer used for visualization, the descriptions of its movements are best demonstrated as a series of photographs and videos (available as videos s1, s2, s3, and s4), with some relative comparisons presented in the results below. multiple repeats of each experimental scenario produced very similar results at this qualitative level, which were sufficient to show the relative differences between the performance of the different door and human motion combinations. velocities of door-opening and figure movement in each scenario were defined and programmed into the controller chip to be within a realistic parameter range when scaled up to their full-scale motions in an air medium. realistic walking speeds equivalent to , 1-1.2 m/s in air were chosen for these model parameters, though obviously walking speeds may vary considerably between individuals. only one sliding door opening-speed was examined (table 1 , figures 3 and 4 , videos s1-s2), as other settings showed relatively little difference. for the hinged-doors, both slow (table 1 , videos s3-s4) and fast (table 2, figures 5 and 6) angular velocities were investigated. this was done because the effect of these door-opening motions on the movements of the food dye were much more dramatic and it was of interest to capture these flows at these two speeds, with the fast parameters being approximately twice those of the slow parameters. brief descriptions of each door-opening scenario are also included in tables 1 and 2 . from a qualitative visual inspection of these images, it can be clearly seen that the single-doors produce less disturbance than the double-doors, and sliding doors produce far less air exchange than hinged-doors, i.e. single-sliding,double-sliding,single-hinged,double-hinged, when the doors are graded in terms of the potential for their door-opening motion to induce bulk air flow movement across the doorways. for both the single-and double-sliding doors, the movement of the human figure through the door caused a significant additional amount of food dye to be exchanged (both into and out of the room) because the motion of the sliding doors themselves caused very little disturbance to the food dye. in contrast, the relative effect of the manikin movement on the food dye was much smaller when moving through the single-and double-hinged doors because the opening motion of these hinged doors caused significantly more movement of the food dye across the doorway. there have been relatively few formal, published studies on the effects of door-opening motions on the integrity of containment in hospital isolation rooms, and even fewer where the effects of a healthcare worker moving through the doorway has also been considered. this study aims to fill some of these gaps in our knowledge. overall, the results of this qualitative visualization study are not surprising and relatively intuitive, however being able to visualize these relative differences may emphasize the advantages and disadvantages of these different door designs in a more emphatic manner for consideration by hospital managers and administrators, infection control and hospital building design teams. for general infection control purposes, it is clear that the sliding doors (single or double) offer some obvious advantages over the more conventional hinged-door design in the amount of air exchanged across isolation room doorways each time they are opened. yet, the majority of hospital isolation rooms still use the more traditional hinged-door design. this may possibly be due to the space requirements and the practicalities of higher installation (sliding doors may be more expensive to make and install) and maintenance costs (there are more moving parts in a slidingcompared to a hinged-door). in addition, where air-tight, 'nonleaky' containment facilities are required, it is much easier to ensure an airtight seal around a hinged-door than a sliding-door. an early study used tracer gas (sf 6 ) and measured how its concentration changed when a technician exited the isolation room into an adjoining anteroom and then the outside corridor. other variables included the time intervals of sampling from the anteroom and the outside corridor, the room size (31.3-49.3 m 3 ) and the ventilation rate (15-21 air changes/hour). the results showed that the concentration of the tracer gas decreased dramatically, as measured by a 'dilution factor' that was defined by the authors, ranging from 122-211 (5 minutes after gas release) between the isolation room and the anteroom, and 1260-3670 (10 minutes after gas release) between the isolation room and the outside corridor [19] . however, the types of doors used for the isolation and anterooms (i.e. hinged or sliding) were not described, though it was probably a hinged door design. it can be seen from the results presented here that the type of door may dramatically affect the results as hinged door motions can rapidly accelerate any mixing and therefore have an impact on the apparent 'dilution factor' that is measured. tang et al. [23] described a clinical situation where a severe case of adult chickenpox (i.e. primary varicella zoster virus, vzv, infection) managed in a negative pressure isolation room (with no anteroom) caused an infection of a vzv-susceptible nurse whose only contact with the patient was when he stood outside the room for at least 2 minutes, several times a day, handing supplies to the vzv-immune nurse inside the room that was directly caring for the patient. the access to the isolation room was a standard single hinged-door that opened into the room. the non-immune nurse developed chickenpox 10 days later. transmission of the same virus between the patient and the nurse was confirmed by viral sequencing of skin lesion samples taken from both individuals. the negative pressure difference across the doorway was measured to be only 3 pa and it was postulated that this was readily reversed each time the door was opened to receive the supplies. this may have been a fairly typical pressure differential found in many hospital isolation units at this time, and it is clear from this incident that this was probably insufficient to maintain the containment during a door-opening motion event. additional qualitative flow visualization studies were performed using food dye in a smallscale, water-tank model under the principle of reynolds number equivalence, to exam the action of the hinged-door opening motion on the airflow across the doorway. these additional experiments confirmed that the hinged-door opening motion into the isolation room was likely to have caused a transient reversal of the negative pressure, allowing airborne virus to leak out and be inhaled and infect the non-immune nurse standing outside. however, the water-tank model developed for this study (with a hand-operated hinged-door) was relatively simple and crude, and this study extends this model and tests double and sliding-door options, also. later, tang et al. [1] , reviewed various other factors potentially involved in the aerosol transmission of infection, and also provide a simple estimate of the effect of a human body moving through the doorway, suggesting that an adult of 1.7 m height, 0.3 m width and 0.15 m depth, giving an approximate cross-sectional area of 0.51 m 2 , weighing about 76.5 kg (assuming a body density equivalent to water), walking at 1 m/s through the doorway produces an air volume flux of 255 l/s, with an attached wake of 76-230 l/s. eames et al. [20] further refined these estimates, using a more complex, reynolds number-equivalent, small-scale water-tank model, suggesting that the contribution from a human body wake may be as much as that produced the motion of a single hinged-door, and as much as 10% of the total room volume for a typical isolation room volume of 31 m 3 , which is similar to those described earlier by rydock and eian [19] . this investigation takes some aspects these earlier studies further in a qualitative manner, i.e. by investigating the impact of the moving human figure on different door designs, moving at different speeds, in a 1:10 motorized scale water-tank model of a single isolation room. johnson et al. [21] simulated the effect of a moving healthcare worker using a life-sized manikin passing through a doorway made of curtains, using airborne, fluorescent beads produced by a nebulizer within the room as tracer particles. air samplers captured escaping fluorescent beads onto filters as the manikin was moved through the curtains, both of which were moved by fine wires. fluorescence microscopy of filters obtained from samplers placed inside and outside the room allowed a 'particle containment efficiency to be calculated, which indicated that the manikin movement through the doorway did induce the escape of particle tracers to the outside of the room. this phenomenon is also confirmed in this study -the movement of the human figure through the doorway does induce some backflow/backwash of food dye out of the room as the figure enters. a follow-up study from the same team [22] using similar a methodology used a real hospital isolation facility with a human volunteer to simulate the actions of a healthcare worker. they specifically stated that hinged-doors were used between the various room compartments (i.e. between the isolation room and anteroom, and anteroom and outside corridor). in this more real-life situation, they also reported that the presence of a healthcare worker moving into and out of the isolation room increased the escape of airborne particles, but also that increasing degrees of negative pressure decreased the amount of particles escaping from the isolation room. however, in this and the previously described studies, none of the teams compared and contrasted single-and double-hinged doors, and sliding doors were not investigated or discussed. this study has shown the different effects of single-and double-hinged and sliding doors on the figures 3 and 4 , and in videos s1, s2, s3, and s4). airflow movement generated by door-opening motions alone, as well as in combination any manikin movement through the door to simulate the movement of a healthcare provider. there are several limitations with the small-scale, water-tank model used here: no ventilation airflow has been simulated as this is a baseline study, and the airflow motion indicated by the colored food dye is only qualitative. further experiments are required to investigate the effects of various ventilation modes, though the scaling issues for thermal buoyancy and pressure effects may make this difficult in a small-scale, water-tank model like the one used here. computational fluid dynamical (cfd) modeling of the airflows across doorways has been performed recently [24] , but this is difficult to compare directly to these experimental results as they also take into account pressure and thermal differences across the doorway, which are both equal on either side of the doorway in the baseline experiments presented here. however, one interesting finding from the cfd modeling, the phenomenon of the back-flow of potentially contaminated air when a hinged-door is opened, has been observed in our qualitative experimental findings here, and demonstrated in the case report by tang et al. [23] . however, the conclusions from the images obtained (and in the accompanying online videos) clearly demonstrate that sliding doors induce much less airflow across the doorway than hingeddoors; single-doors cause less disturbance than double-doors (assuming that the single-doors are smaller than the double-doors, which is not always the case in some facilities); and that the movement of a single healthcare worker through the doorway in either direction induces additional airflow movement, thereby increasing the amount cross-contamination across the doorway. the case report by tang et al. [23] suggested that door-opening motions will almost certainly reverse any low level pressure differentials (i.e. ,5 pa) across doorways, and these experiments provide a visual representation of how and why this may occur, with several combinations of door opening moving human figure parameters. perhaps the most important implication from this study is that whatever door design is used, there is likely to be some leakage across the doorway to a lesser or greater degree as a human figure moves through the door at a reasonable walking speed -which strongly supports the requirement for anterooms. in the small-scale water-tank models used in these experiments, the compartment outside the isolation room, into or from which the series of 4 snapshots with each dooropening, manikin movement scenario were taken with respect to the following events, rather than at specific times: food dye movement due to door-opening motions alone then with any initial manikin movement -manikin interaction and any entrainment food dye -final food dye movements once the manikin had come to rest at its destination position. all movement parameters are shown in table 1 for these single-sliding door scenarios. note that with the sliding doors, the scenarios where the manikin enters or leaves the isolation room are effectively symmetrical (unlike with the hinged-door scenarios). a. manikin moving into/out of the isolation room (seen from outside/inside, respectively), v = 0.79 in water the manikin enters, can be considered as an anteroom or a corridor area. the opening of the sliding-or hinged-doors induces a variable amount of leakage (as indicated by the movement of the food dye) from the 'dirty' area across the doorway to the 'clean' figure 4 . double-sliding door snapshots (side and top views; left-to-right, top-to-bottom). the series of 4 snapshots with each dooropening, manikin movement scenario were taken with respect to the following events, rather than at specific times: food dye movement due to door-opening motions alone then with any initial manikin movement -manikin interaction and any entrainment food dye -final food dye movements once the manikin had come to rest at its destination position. all movement parameters are shown in table 1 for these double-sliding door scenarios. note that with the sliding doors, the scenarios where the manikin enters or leaves the isolation room are effectively symmetrical (unlike with the hinged-door scenarios). a. manikin moving into/out of the isolation room (seen from outside/inside, respectively), v = 0.79 in water figure 1 for the meaning of these v1 and v2 parameters. these additional parameter settings were used with relatively similar qualitative outcomes (as shown in figures 5 and 6 ). doi:10.1371/journal.pone.0066663.t002 area -much more so for the hinged than the sliding doors. in addition, with the moving manikin, when entering or leaving an isolation room there is clearly either a backwash or an entrained drag wake flow into this outer area, arising from the passage of a person into or out of the room, respectively. both of these sources of contamination argue for the use of an anteroom area adjacent to the isolation room, in which the air should be completely exchanged (and filtered) before allowing an exit into the corridor. however, it is acknowledged that other more economical and space limitation factors may also influence the availability of anterooms with isolation units in individual hospitals or healthcare facilities. it is difficult to make a direct comparison with contact transmission as to which route is more clinically significant for acquiring hospital-related infections. one recent observational study examined the number of viable bacteria found on hospital door handles of different designs in certain high traffic areas in a tertiary referral hospital in the uk. the authors found that the door handle's location, design and mode of use were all factors that affected their degree of contamination, with the traditional lever-style handles being the most highly contaminated [25] . however, in most cases when sliding doors are installed, they are often controlled by automated detection systems that detect the approach of people and open automatically, without any touching of the door surface being required. where a contact plate is required for opening such sliding doors (particularly those leading into dedicated isolation units), these are often 'foot' plates at floor level that are operated by a person's foot pressure, or no-touch sensor plates at higher positions that just require the hand to be passed over them, without any contact with them at all. in short, the way that sliding doors are installed often preclude the need to touch these door surfaces at all, which is another advantage of these style of doors, particularly in areas where the isolation of potentially infectious (or especially vulnerable) patients is of prime clinical importance. this study is part of an international collaboration between a small-scale modeling facility (singapore) and a full-scale modeling facility (finland). further results from the large-scale modeling figure 5 . single-hinged door snapshots (sideviews only; left-to-right). the series of 4 snapshots with each door-opening, manikin movement scenario were taken with respect to the following events, rather than at specific times: food dye movement due to door-opening motions alone then with any initial manikin movement -manikin interaction and any entrainment food dye -final food dye movements once the manikin had come to rest at its destination position. all movement parameters are shown in table 2 experiments are currently in preparation for publication. in addition, this experimental data will be used to validate the cfd modeling of these airflow patterns across the doorway under different ventilation modes, which is also being performed in both the singapore and finnish facilities as part of this international project. video s1 single sliding-door. the door was programmed to open in various combinations involving the moving manikin (simulating a healthcare worker) entering/leaving the isolation room. details of the movement parameters used for the door and manikin motions are included within the video. all experiments were performed in still water i.e. no simulated ventilation was present in these baseline experiments. . double-hinged door snapshots (sideviews only; left-to-right). the series of 4 snapshots with each door-opening, manikin movement scenario were taken with respect to the following events, rather than at specific times: food dye movement due to door-opening motions alone then with any initial manikin movement -manikin interaction and any entrainment food dye -final food dye movements once the manikin had come to rest at its destination position. all movement parameters are shown in table 2 factors involved in the aerosol transmission of infection and control of ventilation in healthcare premises airborne transmission of disease in hospitals practical and affordable measures for the protection of health care workers from tuberculosis in low-income countries guidelines for environmental infection control in health-care facilities. recommendations of cdc and the healthcare infection control practices advisory committee (hicpac) guidelines for preventing the transmission of mycobacterium tuberculosis in health-care settings control and prevention of healthcare-associated tuberculosis: the role of respiratory isolation and personal respiratory protection infection control practices for sars in lao people's democratic republic, taiwan, and thailand: experience from mobile sars containment teams rapid creation of a temporary isolation ward for patients with severe acute respiratory syndrome in taiwan infection control for sars in a tertiary paediatric centre in hong kong epidemiologic study and containment of a nosocomial outbreak of severe acute respiratory syndrome in a medical center isolation rooms for highly infectious diseases: an inventory of capabilities in european countries healthcare infection control special interest group of the australian society for infectious diseases asid (hicsig) position statement: infection control guidelines for patients with influenza-like illnesses, including pandemic (h1n1) influenza 2009, in australian health care facilities optimal control for pandemic influenza: the role of limited antiviral treatment and isolation medical response planning for pandemic flu an evaluation of hospital special-ventilationroom pressures a simple method for tracer containment testing of hospital isolation rooms a performance assessment of airborne infection isolation rooms development of an empirical model to aid in designing airborne infection isolation rooms containment testing of isolation rooms movement of airborne contaminants in a hospital isolation room containment effectiveness of expedient patient isolation units the effect of pressure differential and care provider movement on airborne infectious isolation room containment effectiveness door-opening motion can potentially lead to a transient breakdown in negative-pressure isolation conditions: the importance of vorticity and buoyancy airflows accumulation and transport of microbial-size particles in a pressure protected model burn unit: cfd simulations and experimental evidence hospital door handle design and their contamination with bacteria: a real life observational study. are we pulling against closed doors? key: cord-260647-7bjhobg7 authors: coudray-meunier, coralie; fraisse, audrey; martin-latil, sandra; delannoy, sabine; fach, patrick; perelle, sylvie title: a novel high-throughput method for molecular detection of human pathogenic viruses using a nanofluidic real-time pcr system date: 2016-01-29 journal: plos one doi: 10.1371/journal.pone.0147832 sha: doc_id: 260647 cord_uid: 7bjhobg7 human enteric viruses are recognized as the main causes of foodand waterborne diseases worldwide. sensitive and quantitative detection of human enteric viruses is typically achieved through quantitative rt-pcr (rt-qpcr). a nanofluidic real-time pcr system was used to develop novel high-throughput methods for qualitative molecular detection (rt-qpcr array) and quantification of human pathogenic viruses by digital rt-pcr (rt-dpcr). the performance of high-throughput pcr methods was investigated for detecting 19 human pathogenic viruses and two main process controls used in food virology. the conventional real-time pcr system was compared to the rt-dpcr and rt-qpcr array. based on the number of genome copies calculated by spectrophotometry, sensitivity was found to be slightly better with rt-qpcr than with rt-dpcr for 14 viruses by a factor range of from 0.3 to 1.6 log(10). conversely, sensitivity was better with rt-dpcr than with rt-qpcr for seven viruses by a factor range of from 0.10 to 1.40 log(10). interestingly, the number of genome copies determined by rt-dpcr was always from 1 to 2 log(10) lower than the expected copy number calculated by rt-qpcr standard curve. the sensitivity of the rt-qpcr and rt-qpcr array assays was found to be similar for two viruses, and better with rt-qpcr than with rt-qpcr array for eighteen viruses by a factor range of from 0.7 to 3.0 log(10). conversely, sensitivity was only 0.30 log(10) better with the rt-qpcr array than with conventional rt-qpcr assays for norovirus giv detection. finally, the rt-qpcr array and rt-dpcr assays were successfully used together to screen clinical samples and quantify pathogenic viruses. additionally, this method made it possible to identify co-infection in clinical samples. in conclusion, given the rapidity and potential for large numbers of viral targets, this nanofluidic rt-qpcr assay should have a major impact on human pathogenic virus surveillance and outbreak investigations and is likely to be of benefit to public health. human enteric viruses constitute a serious public health concern, since they are capable of causing a variety of acute illnesses, including the most commonly reported acute gastrointestinal illness. they are mainly transmitted via the fecal-oral route either by person-to-person contact or by ingestion of contaminated water and food, particularly shellfish, soft fruits and vegetables. enteric viruses are shed in enormous quantities in feces (10 9 to 10 10 /g) and have an infectious dose on the order of tens to hundreds of virions. enteric viruses are host-specific and are not capable of replicating in the environment, but they survive for long periods of time on food or food contact surfaces or in water (ground, surface, and drinking water) [1] . these characteristics enable enteric viruses to play a significant role in food-and waterborne outbreaks. aside from noroviruses, which have been recognized as the largest cause of outbreaks, the viruses most often implicated in outbreaks include hepatitis viruses (hepatitis a virus and hepatitis e virus), rotavirus, adenovirus (40, 41) , astrovirus, enterovirus [2, 3, 4, 5, 6, 7] . additional viruses of lesser epidemiologic importance include human bocavirus, cosavirus, parvovirus, sapovirus, tick-borne encephalitis virus (tbev), aichi virus, and coronavirus [8, 9, 10, 11] . tools for rapid detection of viral pathogens are important for analyzing clinical, environmental and food samples. detection of these enteric viruses based on their infectivity is complicated by the absence of a reliable cell culture method and the low levels of contamination of food and environmental samples [12, 13] . to date, real time rt-pcr has been one of the most promising detection methods due to its sensitivity, specificity, and speed. recently, the iso/ts 15216-1 and 15216-2 standards covering real time rt-pcr for both quantitative determination and qualitative detection of nov and hav in foodstuffs were published [14, 15, 16] . the aim of this study was to develop real time rt-pcr assays for detection of a total of 19 human enteric viruses (including 3 genogroupes of norovirus and 4 coronaviruses) and two control process viruses (mengovirus and murine norovirus) generally used for monitoring the recovery of viral foodstuff extraction methods. limits of detection of the viral genomes were determined with the conventional rt-qpcr system and with the fluidigm's biomark system by using the qualitative nanofluidic real-time rt-pcr array and the quantitative digital rt-pcr array. the advantages of these new detection techniques were determined by detecting and quantifying pathogenic viruses in clinical samples. hav strain hm175/18f, clone b (vr-1402), was obtained from the american type culture collection (atcc). this clone replicates rapidly and has cytopathic effects in cell culture [17] . hav stock was produced by propagation in foetal rhesus monkey kidney (frhk-4) cells (atcc, crl-1688) [18] and titrated by plaque assay [19] . the titer of viral production was established in hav rna genomic copies with an rt-qpcr standard curve obtained with the ten-fold diluted in vitro rna transcripts. based on this approach, hav stocks had titres of 9.33 x 10 8 genome copies / ml. dr. h. virgin from washington university in the usa provided anses's fougères laboratory in france with mnv-1 (cw1 strain) which was then propagated in mouse leukemic monocyte macrophage (raw 264.7, atcc tib-71) cell line [20] . mnv-1 stock was produced as previously described [21] . the extracted rna was confirmed with rt-qpcr and quantified by measuring absorbance at 260 / 280 nm with a nanodrop nd-1000. based on this approach, the production stock of mnv-1 had titres of approximately 1.36 x 10 12 genome copies / ml. mengovirus (strain mc 0 ) was obtained from clarified supernatant provided by dr. albert bosch from the "enteric virus group" of the university of barcelona. mengovirus stock was produced by propagation in hela cells (atcc, ccl-2™) [22] . the extracted rna was confirmed with rt-qpcr and quantified by measuring absorbance at 260 / 280 nm with a nanodrop nd-1000. based on this approach, the production stock of mnv-1 had titres of approximately 6.68 x 10 11 genome copies / ml. rotavirus strain wa (human rotavirus) was obtained from the pasteur institute (paris, france) and was propagated in ma-104 rhesus monkey epithelial cell line (atcc crl-2378) [23] . the extracted rna was confirmed with rt-qpcr and quantified by measuring absorbance at 260 / 280 nm with a nanodrop nd-1000. based on this approach, the production stock of wa had titres of approximately 3.21 x 10 11 genome copies / ml. four viral strains were obtained from infected cell culture supernatants: two enterovirus strains (human b6 coxsackievirus (schmitt strain: atcc 1 vr-1037as/mk™) and human echovirus 19 (burke strain)), one adenovirus 40 strain (atcc vr-931) and one astrovirus gi strain. the study was conducted in accordance with the ethics principles of the declaration of helsinki. hepatitis a virus infection is a notifiable disease in france. the current system of mandatory reporting was approved by the commission nationale de l'informatique et des libertés (deliberation n°02-082, november 19 2002) . patients receive oral and written information on the finality of the notification and on the modalities of information recording. this information is available on line on the web site of the institut de veille sanitaire (ivs) at http://www. invs.sante.fr/content/download/6498/42945/version/2/file/fiche_info_patient.pdf for hav samples and on the web site of the nrc at www.cnr-ve.org for enteric virus samples. all clinical and biological parameters are treated anonymously. the virological surveillance of strain diversity is performed on stored samples obtained for hepatitis a diagnosis (no need for any additional blood draw). diagnostic laboratories are asked to contribute to hav and enteric virus strains surveillance by sending samples to the national reference centre (nrc). the study was not specifically approved by an ethics committee. human samples were collected before the study and they are anonymously collected and analyzed. the following human stool samples were provided by the national reference center (nrc) for enteric viruses (dijon, france): adenovirus 41 (stool n°e5669), astrovirus gi (e4883), norovirus gi (e5486; e5569; e8050), norovirus gii (e6929; e6618; e7859; e7022; e6992), rotavirus (rv g12p8 = e7622; rv g1p8 = e8097), aichi virus (e6841) and norovirus gii.13 + norovirus giv. the following human stool samples were provided by the national reference center (nrc) for hav/hev (villejuif, france): hav gia (stools no. 780627147; 1181216151), hav gib (1280210015; 1280514230), hev g3c (1280511146), hev g3f (1280418084; 1280530128) and hev g4 (1280615097; 1280522166). the faecal samples were suspended in 1x phosphate buffered saline (pbs), ph 7, to obtain a final 10% suspension (w/v), vortexed and centrifuged at 3000 g for 30 min at 4°c. aliquots of 100 μl supernatant were kept frozen at -80°c for later use. the extracted genomic rna/dna of adenovirus, astrovirus and rotavirus were confirmed with rt-qpcr and quantified by measuring absorbance at 260 / 280 nm with a nanodrop nd-1000. the extracted genomic rna of norovirus gi, gii and giv, sapovirus, aichi virus, hav and hev were confirmed and quantified with rt-qpcr by using in vitro rna standard curves (see "dna and rna standards"). sequences from reference strains were inserted into recombinant plasmids ( table 1 ). the hev, hav, nov gi and nov gii cdna were cloned in pgem-t easy vector (promega, charbonnières-les-bains, france) and propagated in e. coli one shot 1 top10f' (life technologies, saint aubin, france). high-quality dna plasmids containing hav or nov regions were purified using the qiagen plasmid midi kit (qiagen, courtaboeuf, france) according to the manufacturer's protocol. then, nov gi plasmid was digested with ncoi (life technologies), and hev dna, hav dna and nov gii dna plasmids were digested with spei (life technologies) and transcripts were obtained by using a megascript 1 kit (life technologies) according to the manufacturer's protocol. synthesized rna were treated with turbo™ dnase (life technologies) according to the manufacturer's protocol in order to remove the dna template following transcription, and purified by using the megaclear™ kit (life technologies). the synthesized dna and rna were confirmed with (rt)-qpcr and quantified by measuring absorbance at 260 / 280 nm with a nanodrop nd-1000 (thermoscientific, courtaboeuf, france) and the free software available on the "http://endmemo.com/bio/dnacopynum.php" website. aliquots of 10μl containing 10 9 genome copies / μl were kept frozen at -20°c for later use and used as standards. sapovirus, tbev, norovirus giv, aichi virus, 229e, hku1, cosavirus, oc43 and nl63 cdna were cloned into the pbluescriptiisk+ vector by genecust (dudelange, luxembourg). all recombinant plasmids were purified by genecust and used to produce rna transcripts. sapovirus, tbev, norovirus giv, 229e, cosavirus and nl63 dna plasmids (0.5 μg) were digested with ecorv (life technologies) and aichi virus, hku1 and oc43 dna plasmids were digested with spei (life technologies). digested plasmids were transcribed by using the megascript 1 kit (life technologies) according to the manufacturer's protocol. synthesized rna was treated with turbo™ dnase (life technologies) according to the manufacturer's protocol in order to remove the dna template following transcription, and purified by using the megaclear kit (life technologies) according to manufacturer's instructions. the synthesized rna was confirmed with rt-qpcr and quantified by measuring absorbance at 260 / 280 nm with a nanodrop nd-100 (thermoscientific, france) and the free software available on the "http://endmemo.com/bio/ dnacopynum.php" website. rna stocks were diluted to contain 10 9 copies / μl. aliquots of 10 μl were kept frozen at -20°c for later use as standards. bocavirus and parvovirus cdna were cloned into the pbluescriptiisk+ vector by genecust (dudelange, luxembourg). both recombinant plasmids were purified by genecust. dna plasmids (0.5 μg) were digested with spei (life technologies) to be linearized. the synthesized dna was confirmed with qpcr and quantified by measuring absorbance at 260 / 280 nm with a nanodrop nd-100 (thermoscientific, france) and the free software available on the "http://endmemo.com/bio/dnacopynum.php" website. dna stocks were diluted to contain 10 9 copies / μl. aliquots of 10 μl were kept frozen at -20°c for later use as standards. the primers and probes used to detect all the viruses of this study are described in table 2 . those used to detect nov gi, nov gii, hav and mengovirus are described in iso/ ts 15216-1 / 15216-2 (2013). all the primers and probes were purchased from life technologies or eurofins mwg operon (les ulis, france). one-step rt-qpcr amplifications were performed on a cfx96™ real time pcr detection system from bio-rad (marnes-la-coquette, france). reactions were performed in a 15 μl reaction mixture containing 1x of rna ultrasense™ master mix and 0.63 μl of rna ultrasense™ enzyme mix, which are components of the rna ultrasense™ one-step quantitative rt-pcr system (life technologies), 2 u rnase inhibitor (life technologies), 5 μg of bovine serum albumin (life technologies), 500 nm of forward primer, 900 nm of reverse primer, 250 nm of probe, and 5 μl of rna extract. a negative control containing all the reagents except the rna template was included with each set of reaction mixtures. the one-step rt-qpcr program involved 60 min reverse transcription of rna at 55°c, followed by a 15 min denaturation step at 95°c, 45 cycles of 15 s at 95°c, 1 min at 60°c and 1 min at 65°c. fluorescence was recorded by the apparatus at the end of the elongation steps (1 minute at 65°c) for each amplification cycle. all samples were characterised by a corresponding ct value. negative samples gave no ct value. a standard curve for each target was generated with reactions were performed in a 10 μl reaction mixture containing 1x of rna ultrasense™ master mix, 1x of rox reference dye and 0.44 μl of rna ultrasense™ enzyme mix, which are components of the rna ultrasense™ one-step quantitative rt-pcr system (life technologies), 1x of 20x ge sample loading reagent (fluidigm), 2 u rnase inhibitor (life technologies), 500 nm of forward primer, 900 nm of reverse primer, 250 nm of probe, and 5.8 μl of rna extract. a negative control containing all the reagents except the rna template was included with each set of reaction mixtures. 6 μl out of ten reaction mix was charged onto the chip with the ifc controller mx, but 0.65 μl were effectively partitioned into the 770 chambers of one panel, including 0.38 μl of rna extract. the one-step rt-dpcr program involved 60 min reverse transcription of rna at 55°c, followed by a 15 min denaturation step at 95°c, and lastly 45 cycles of 15 s at 95°c, 1 min at 60°c and 1 min at 65°c. fluorescence was recorded by the apparatus at the end of the elongation steps (1 minute at 65°c) for each amplification cycle. the digital pcr analysis software (fluidigm) was used to count the number of positive chambers out of the total number of chambers per panel. the poisson distribution was used to estimate the average number of template copies per chamber in a panel [24, 25] . all samples were characterised by a corresponding absolute quantity. no positive chambers were observed in negative samples. the 48.48 dynamic arrays were automatically loaded using an integrated fluidic circuit (ifc) controller (fluidigm corporation), and real-time reactions were performed and analyzed using a biomark real-time pcr system and analysis software (fluidigm corporation), respectively. as a quality control, negative control samples were included on every array for each viral genome. rt reactions were performed in a 25 μl reaction mixture containing 1x of first-strand buffer, 10mm of dtt and 1 μl of superscript 1 iii rt enzyme, which are components of superscript 1 iii reverse transcriptase (life technologies), 2 u rnase inhibitor (life technologies), 2μm of random hexamer (life technologies), 200 μm of dntp (life technologies), and 10 μl of nucleic acids. a negative control containing all the reagents except the rna template was included with each set of reaction mixtures. the rt program involved 5 min at 25°c, followed by 60 min at 55°c, and lastly 15 min at 70°c. aliquots were kept frozen at -80°c for later use. preamplification reactions were performed in a 10 μl reaction mixture containing 1x of supermix, a reagent of perfecta preamp supermix (quanta), 0.2μl of 0.2x primer pool (1x = 500nm forward and 900nm reverse), and 5 μl of cdna. a negative control containing all the reagents except the cdna template was included with each set of reaction mixtures. the preamplification program involved 10 min at 95°c, followed by 14 cycles of 15 s at 95°c and 4 min at 6°c. immediately after the pre-amplification pcr, products were diluted (1:4) and stored at -80°c prior to use in qpcr. for the qpcr array, 48 x 6 μl reaction mixture containing 1x of rna ultrasense™ master mix, 1x of rox reference dye and 0.27 μl of rna ultrasense™ enzyme mix, which are components of the rna ultrasense™ one-step quantitative rt-pcr system (life technologies), 1x of 20x ge sample loading reagent (fluidigm) and 2.7 μl of dna extract were charged on the right part of the "48.48 dynamic array ifc" plate (biomark). negative controls containing all the reagents except the dna template were included with each set of reaction mixtures. in addition, 48 x 5 μl of a mix of 500 nm of forward primer, 900 nm of reverse primer and 250 nm of probe were deposited on the left part of the plate. nine nl of reaction volume mix were charged onto each of the 2304 chambers on the chip with the ifc controller mx. the qpcr program involved a 15 min denaturation step at 95°c followed by 45 cycles of 15 s at 95°c, 1 min at 60°c and 1 min at 65°c. fluorescence was recorded by the apparatus at the end of the elongation steps (1 minute at 65°c) for each amplification cycle. negative samples gave no ct value. the sensitivity of conventional qpcr assays targeting 21 viral genomes was compared to the quantitative digital rt-pcr array and to the qualitative nanofluidic real-time pcr array performed on fluidigm's biomark system. digital rt-pcr's potential for sensitive and accurate quantification was assessed on 10-fold dilution series of 21 viral genomes ( table 3 ). the sensitivity was slightly better with rt-qpcr than with rt-dpcr for ten viruses by a factor ranging from 0.3 to 0.9 log 10 and for four viruses by a factor ranging from 1.3 to 1.6 log 10 . conversely, sensitivity was better with rt-dpcr than with rt-qpcr for seven viruses by a factor ranging from 0.1 to 1.4 log 10 . the expected numbers of genome copies calculated via the standard curve by rt-qpcr were close to the direct measurement of the target concentrations by rt-dpcr only by testing dna from plasmids. by testing rna transcripts, the numbers of genome copies as determined by direct rt-dpcr measurement of the target concentrations were 0.9 to 2.1 log 10 lower than table 3 . comparison of rt-qpcr, rt-dpcr and rt-pcr array assays. characteristics of standard curves based on the rt-qpcr assays and limit of detection (lod) of viral targets by rt-qpcr, by rt-dpcr and rt-pcr array assays. the differences between relative quantification (by rt-qpcr) and absolute quantification (by rt-dpcr) were indicated. nanofluidic pcr system for enteric virus detection the expected copy numbers calculated via the standard curve by rt-qpcr. similarly, by testing genomes from viruses in stools and rna from virus production in cells, the limit of detection (lod) as determined by rt-dpcr was respectively 1.5 to 3.4 log 10 and 1.6 to 2.1 log 10 lower than the expected copy numbers calculated via the standard curve by rt-qpcr. the potential of the rt-pcr array for sensitive detection was assessed on a dilution series of 21 viral genomes ( table 3 ). the limits of detection obtained with rt-qpcr array assays ranged from 1 to 10 3 genome copies / μl of rna / dna extracts for 11 viruses and from 10 4 to 10 5 genome copies / μl of rna extracts for the others. rt-qpcr array assays commonly showed a slightly lower sensitivity than conventional rt-qpcr. the sensitivity of both rt-qpcr and rt-qpcr array assays was found to be similar for two viruses (enterovirus, adenovirus 41), and was slightly better with the rt-qpcr than with the rt-qpcr array for 18 viruses by a factor ranging from 0.7 to 3.0 log 10 . conversely, sensitivity was only 0.3 log 10 higher with the rt-qpcr array than with conventional rt-qpcr assays for norovirus giv detection. the nanofluid-based (rt)-pcr assays developed were applied to characterize 25 samples (4 culture supernatants and 21 clinical samples previously characterized by nrc) for detection of hepatitis (hav, hev) and enteric virus genomes. first, the samples were tested on the rt-pcr array to perform a qualitative screening of the 19 viral genomes. then the viral-positive samples were specifically quantified by rt-dpcr and by conventional rt-qpcr. results are shown on table 4 . rt-qpcr array assays detected the previously determined viruses in 100% of the samples. furthermore, positivity for more than one virus was found in two clinical samples. a stool previously identified as positive for hav ib was found positive for hav and aichi virus and a stool identified as positive for aichi virus was found positive for aichi virus, adenovirus and astrovirus. the stool previously identified as co-infected by nov gii.13 and nov giv was confirmed positive for both viruses. following the viral screening of 25 samples, the 29 detected viral genomes were successfully quantified by both rt-qpcr and rt-dpcr. the number of genome copies determined for 28 viruses was lower by rt-dpcr with a difference of quantification comprised between 0 and 1 log 10 for 7 out of the 29 samples (24%), between 1 and 2 log 10 for 17 out of the 29 samples (59%) and higher than 2 log 10 for 4 out of the 29 samples (14%). so the numbers of genome copies calculated by absolute quantification (rt-dpcr) were lower than the expected numbers of genome copies calculated by using standard curve of rt-qpcr except in the sample coinfected with nov gii and nov giv. in the latter sample, the nov gii quantification was 0.4 log 10 higher by rt-dpcr than by the rt-qpcr assays (1 out of the 29 samples, i.e. 3%). enteric viruses are able to persist for long periods in the environment and can be transmitted with a low infectious dose by human contact, water, food and fomites [26] . they pose a significant public health concern. they are associated with gastroenteritis in humans, but also with hepatitis and other diseases including respiratory infections, conjunctivitis, aseptic meningitis, encephalitis, myocarditis, and paralysis which have high mortality rates, particularly in immunocompromised individuals [27] . commonly studied groups of enteric viruses include noroviruses and hepatitis viruses, but new tools for detecting the full range of pathogenic viruses are table 4 . screening and viral quantification in clinical samples (stools and viral supernatants from cell culture) by rt-qpcr and novel nanofuidic approaches (rt-qpcr and rt-dpcr). samples were firstly screened by rt-pcr array and then quantified by rt-dpcr. absolute viral quantification (by rt-dpcr) was compared to relative quantification (by rt-qpcr). needed for their surveillance in the environment, food samples and for outbreak investigations [28] . microfluidic digital pcr (rt-dpcr) is an accurate endpoint-sensitive absolute quantification approach that makes it possible to determine the number of target copies without a standard curve. digital pcr ((rt)-dpcr) was compared to real-time (rt)-pcr for quantifying 19 human enteric viruses and two control process viruses. for detecting viral rna and cdna, rt-dpcr assays were often found to be comparable in terms of sensitivity to rt-qpcr. the number of rna genome copies determined by digital rt-pcr was often lower than the number of copies expected using spectrophotometry. one potential cause of discrepancy between relative and absolute quantification could be errors introduced by spectrophotometric determination of the nucleic acid concentration, leading to an overestimation of the copy genome number [29, 30] . this could explain why samples from viral stocks and stools potentially containing cellular genomes (non-target rna) and degraded (non-amplified) targets were particularly affected by quantification discrepancies. both quantification methods were close when dna targets were tested. one other potential cause of discrepancy might be the rt step, which is not 100% effective, so that all the rna may not be transcribed into cdna and therefore is not quantified by the digital pcr. digital rt-pcr may provide more accurate measurements than rt-qpcr, as it is not dependent on amplification efficiency. moreover, the advantage of this novel technology is that it is more tolerant to inhibitory substances and may reduce the difficulty of quantifying viruses when inhibitors linked to the matrix-type components analysed in food or environmental virology are present [31, 32, 33] . recent innovations in pcr miniaturization made it possible to conduct high-throughput qpcr in which the reactional volumes are reduced to a nanolitre, leading to a decrease in the cost per assay per sample. recently, a microfluidic quantitative pcr (mfqpcr) system was developed to simultaneously quantify 11 major human viral pathogens and two process controls (murine norovirus, mengovirus). this system included a specific target amplification (sta) reaction to increase the amount of target genes prior to mfqpcr [34] . in this study, the rt-qpcr array assays were developed and enabled simultaneous detection of 48 samples with 22 targeted virus assays. the preamplification step was also necessary because low amounts of target molecules had to be detected in very small volumes of reaction (9nl). thus, rt-qpcr array assays involve three separate steps (rt, preamplification and pcr). nineteen enteric viruses and two control process viruses (mnv and mengovirus) were targeted. the sensitivity of the rt-qpcr array assays was lower (by 0.8 to 3.8 log 10 ) than the limits of detection obtained with conventional rt-qpcr and rt-dpcr. however, all the clinical samples tested with the rt-qpcr array assays were identified and matched the nrc results. moreover, two stools contained more than one viral genome, and these results completed the nrc analysis. this assay is therefore useful for rapid sample screening. in conclusion, a combination of rt-qpcr array and rt-dpcr assays could be applied to screen contaminated samples and quantify pathogenic viruses in case of outbreaks investigation and surveillance. the choice of techniques should take into account the aim of analysis, the number of targets involved and the analytical costs. to date, the rt-qpcr array includes enteric viruses frequently reported as the causes of foodborne outbreaks and some additional viruses of lesser epidemiologic importance. in future, this technology could be updated by extending the range of viral targets to gain information during epidemiological studies. for this purpose, biomark real-time pcr system (fluidigm) can be also used for high-throughput microfluidic real-time pcr amplification with 96.96 dynamic arrays (fluidigm) leading to an increase of detected targets. concerning rt-dpcr assays, it could be helpful for standardizing the quantification of enteric viruses in samples and 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detection in food: current and future prospects international standardization of a method for detection of human pathogenic viruses in molluscan shellfish iso/ts 15216-1. microbiology of food and animal feed-horizontal method for determination of hepatitis a virus and norovirus in food using real-time rt-pcr-part 1: method for quantification. international organization for standardization microbiology of food and animal feed-horizontal method for determination of hepatitis a virus and norovirus in food using real-time rt-pcr-part 2: method for qualitative detection. international organization for standardization antigenic and genetic variation in cytopathic hepatitis a virus variants arising during persistent infection: evidence for genetic recombination development of a plaque assay for a cytopathic, rapidly replicating isolate of hepatitis a virus intra-laboratory validation of a concentration method adapted for the enumeration of infectious f-specific rna coliphage, enterovirus, and 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using droplet digital pcr evaluation of digital pcr for absolute rna quantification a comparative study of digital rt-pcr and rt-qpcr for quantification of hepatitis a virus and norovirus in lettuce and water samples one-step rt-droplet digital pcr: a breakthrough in the quantification of waterborne rna viruses quantitative analysis of food and feed samples with droplet digital pcr microfluidic quantitative pcr for simultaneous quantification of multiple viruses in environmental water samples duplex rt-qpcr for the detection of hepatitis e virus in water, using a process control increased sensitivity for various rotavirus genotypes in stool specimens by amending three mismatched nucleotides in the forward primer of a real-time rt-pcr assay increased detection of rotavirus using a real time reverse transcription-polymerase chain reaction (rt-pcr) assay in stool specimens from children with diarrhea evaluation of removal of noroviruses during wastewater treatment, using real-time reverse transcription-pcr: different behaviors of genogroups i and ii etiological role of viruses in outbreaks of acute gastroenteritis in the netherlands from 1994 through real-time rt-pcr for norovirus screening in shellfish broadly reactive and highly sensitive assay for norwalk-like viruses based on real-time quantitative reverse transcription-pcr use of taqman real-time reverse transcription-pcr for rapid detection, quantification, and typing of norovirus sapovirus detection by quantitative realtime rt-pcr in clinical stool specimens aichi virus shedding in high concentrations in patients with acute diarrhea diagnosis of viral gastroenteritis by simultaneous detection of adenovirus group f, astrovirus, rotavirus group a, norovirus genogroups i and ii, and sapovirus in two internally controlled multiplex real-time pcr assays epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex realtime pcr method design and validation of consensus-degenerate hybrid oligonucleotide primers for broad and sensitive detection of corona-and toroviruses development of a quantitative real-time rt-pcr assay with internal control for the laboratory detection of tick borne encephalitis virus (tbev) rna detection of five rash-associated viruses using multiplex real-time pcr during a sensitive and quantitative single-tube real-time reverse transcriptase-pcr for detection of enteroviral rna quantification of enterovirus rna in sludge samples using single tube real-time rt-pcr cosavirus infection in persons with and without gastroenteritis development of a real-time pcr assay for detecting and quantifying human bocavirus 2 comparison of two extraction methods for the detection of hepatitis a virus in semi-dried tomatoes and murine norovirus as a process control by duplex rt-qpcr risk assessment in shellfish-borne outbreaks of hepatitis a we thank am roque (nrc for hepatitis a virus, france) and p pothier (nrc for enteric viruses, france) for providing contaminated stools. this work is part of the thesis by coralie coudray-meunier, a phd student who received financial support from anses. conceived and designed the experiments: ccm af sml sd pf sp. performed the experiments: ccm af sd. analyzed the data: ccm af sml sd pf sp. contributed reagents/materials/analysis tools: ccm af sml sd pf sp. wrote the paper: ccm af sml sd pf sp. key: cord-283976-jgae7r2q authors: armstrong, melissa j.; gamez, noheli; alliance, slande; majid, tabassum; taylor, angela; kurasz, andrea m.; patel, bhavana; smith, glenn title: research priorities of caregivers and individuals with dementia with lewy bodies: an interview study date: 2020-10-07 journal: plos one doi: 10.1371/journal.pone.0239279 sha: doc_id: 283976 cord_uid: jgae7r2q background: funding bodies are placing increased emphasis on patient and public involvement in research, but the research priorities of individuals and caregivers living with dementia with lewy bodies (dlb) are unknown. method: investigators conducted telephone interviews with individuals living with dlb and caregivers. participants were recruited from a lewy body dementia association research center of excellence. interviews employed a semi-structured questionnaire querying research needs in different categories and then asking participants to select their top priorities. investigators used a qualitative descriptive approach to analyze transcripts and identify themes. results: twenty individuals with dlb and 25 caregivers participated. seventeen from each group participated as part of a patient-caregiver dyad. twenty-three of the caregivers were spouses, two were daughters. individuals with dlb and caregivers identified research needs relating to focusing on awareness, determining the cause of dlb, improving diagnosis, and investigating what to expect/disease stages. participants also highlighted dlb symptoms needing additional research, therapies to prevent, cure, or slow the progression of dlb, and research targeting daily function and quality of life, caregiving, and improving education. conclusions: these findings support the research priorities defined in the national institutes of health dementia care summits in addition to adrd priority-setting summits. research is needed across all domains of dlb. funding should be informed by the priorities of all relevant stakeholders and support research investigating causes, natural history, biomarkers, and treatment in addition to research targeting themes regarding living with disease (e.g. independence, quality of life, caregiving, and education). funding bodies including the national institutes of health (nih) are placing increased emphasis on patient and public involvement in research. patients can have a role throughout the research process, from study design to dissemination [1, 2] . before studies are even initiated, patients, caregivers, and the public have a role in identifying research priorities [2] . this is critical given a mismatch between the research priorities of patients, caregivers, clinicians, and researchers [3] . the advisory council on alzheimer's research, care and services recommends that all funders "should establish the engagement of the ad/adrd [alzheimer disease/ alzheimerdisease related dementia] community as a standard practice in. . . participating in setting national research priorities for ad/adrd. . ." [4] . the nih has adopted multiple strategies to increase the role of patient and public involvement in dementia priority setting. adrd national research priority summits include sessions led by nongovernmental organizations to increase non-expert stakeholder input and public-private partnerships [5] . the 2017 national research summit on care, services, and supports for persons with dementia and their caregivers included "persons living with dementia" and family caregiver stakeholder groups as two of the six stakeholder groups informing summit planning and recommendations [6] . one aim of the planned 2020 national research summit on care, services, and supports for persons with dementia and their caregivers was to develop recommendations for research priorities to inform federal agencies and other research funders, but the in-person meeting was cancelled due to the global covid-19 pandemic. the summit will now occur via a series of virtual meetings in summer 2020. involvement of patient representatives alongside experts in priority setting reflects a "participation" engagement strategy. through participation, representatives have an active and equal voice in the process. participation strategies usually rely on small numbers of representatives, potentially missing other perspectives. consultation strategies (e.g. surveys, interviews, focus groups) collect a variety of views from larger groups of people, but fail to give patient representatives an active voice in the process. employing participation and consultation approaches as complementary may be the optimal approach to incorporate the views of a diverse group while also giving patient representatives an equal and active voice in decision making [7] . stakeholders representing individuals with lewy body dementia participated in nih ad/ adrd priority setting [5] and the 2017 care summit [6] . however, no prior research investigates the research priorities of individuals and caregivers living with dementia with lewy bodies (dlb). dlb is a subset of lewy body dementia, the 2 nd most common neurodegenerative dementia in the united states [8] . dlb is a dementia with clinical and pathological overlap with parkinson disease (pd) and both fall in the pathological category of lewy body diseases. however, dlb has important differences from both pd and ad/other dementias which could meaningfully impact research priorities. for example, symptoms such as cognitive fluctuations, hallucinations, and dream enactment behavior distinguish individuals with dlb from ad [9] . individuals with clinically-diagnosed dlb survive a median of 3-4 years after presentation [10] [11] [12] , shorter than individuals with pd [13, 14] or ad dementia [11, 15] . caregiver burden in dlb may be similar to pd dementia [16] , but it is higher in dlb compared to ad dementia [17] [18] [19] . quality of life is also worse in dlb compared to ad dementia [20, 21] . given these and other important differences between dlb and other dementias and parkinsonisms, prior publications reporting on research priorities in pd [22, 23] and dementia in general [24] [25] [26] [27] may not capture the priorities of individuals with dlb. we thus aimed to identify the research priorities of individuals with dlb and caregivers, particularly to guide research at our center. the study used telephone interviews to investigate the research priorities of individuals living with dlb and caregivers of individuals living with dlb. investigators used a qualitative descriptive approach [28] to analyze interview transcripts. a qualitative descriptive approach involves reporting and summarizing straightforward accounts of participants' views without an intent to generate or test theory. a qualitative descriptive approach was employed given the aim of identifying research priorities, rather than investigating theories underpinning those priorities. consolidated criteria for reporting qualitative research guided study reporting (s1 checklist) [29] . individuals with dlb and caregivers of individuals with dlb were recruited from the uf health norman fixel institute for neurological diseases, a lewy body dementia association research center of excellence. inclusion criteria were: (1) patient or caregiver of a patient followed at the fixel institute, (2) personal diagnosis of dlb [9] or caregiver for an individual diagnosed with dlb, (3) if a person with dlb, clinician-judged mild-moderate severity such that the individual could understand the study and provide personal opinions, and (4) willingness to participate in a telephone interview. individuals and caregivers were not required to participate as a dyad. participants were recruited when presenting for routine clinical visits or through a consent-to-contact research database at the institute. the university of florida institutional review board provided approval (irb201500996). the study used a waiver of documentation of informed consent. the pi (mja), a dlb specialist, drafted the semi-structured interview guide (s1 file) and revised it based on suggestions from a neuropsychologist (gs), a dementia specialist (not further involved in the project), phd specializing in qualitative research (tm), and former caregiver of an individual with dlb (at). the interview included questions regarding clinical care priorities, which will be analyzed separately. research questions asked about participant priorities for dlb research in general and then specifically queried participant priorities regarding research on dlb symptoms, daily challenges, caregiving/family life, and diagnosis. finally, the interview guide asked the participant to identify how they would divide $1000 to spend on dlb research and to prioritize what research was most important to them. a research assistant with qualitative research experience conducted the telephone interviews (sa). the research assistant had no relationship with participants prior to the study. interviews occurred via telephone to allow individuals to participate from home. participants selected the interview times. the preferred approach was to interview individuals with dlb and their caregivers separately (if both participated), but the interviewer accommodated requests for the caregiver to be present during the patient interview. any caregiver opinions offered during a patient interview were coded as belonging to the caregiver, not the individual with dlb. audio recording started after allowing for participant questions and verbal consent at the beginning of the call. a professional service transcribed the interviews verbatim, so member checking was not performed. investigators used tables in microsoft word 1 and excel 2016 1 to organize data and a qualitative descriptive approach to identify and organize themes [28] . broad topics/categories were defined by interview questions, but themes were identified from interview transcripts. the pi and two research assistants independently analyzed interview transcripts to create a codebook and then reached consensus regarding emerging themes (open coding). the research assistants analyzed remaining transcripts using a constant comparative technique, revising themes and subthemes with the pi if needed (axial coding) (s2 file). coders assessed saturation during analysis. co-investigators gave feedback after the initial coding. participants were numbered in the analysis such that participants who enrolled in the study as a dyad shared participant numbers (with "p" indicating patient participants and "cg" indicating caregiver participants). interviews occurred between 1/22/2018 and 5/6/2019. twenty individuals with dlb and 25 caregivers participated (table 1) . seventeen from each group participated as part of a patientwell, maybe the breaker tripped. maybe the bulb burned out. maybe the wire broke." you know, there's a lotta maybes. well, you've got to find the root cause. . . you know, you find the cause or the problems, and you can treat it or correct it. (cg21, daughter) in addition to desiring to know causes in general, multiple participants wanted further research regarding whether other health problems, prior injuries (e.g. head trauma), exposures (e.g. from mining), a history of shift work, or a traumatic event could contribute to developing dlb. multiple individuals with dlb and almost half of caregivers discussed the importance of research to improve early diagnosis ( table 2 ). while these respondents discussed the need for earlier diagnosis, particularly in circumstances where a diagnosis was delayed, one individual (cg22, husband) multiple individuals with dlb and caregivers described needing more research to help individuals with dlb, caregivers, and healthcare professionals recognize the signs of dlb (table 2 ). caregivers wanted research to focus on improving the accuracy of diagnosis, including distinguishing dlb from pd and dementia generally, particularly given widespread confusion regarding lewy body terminology. several individuals with dlb and caregivers desired more research into tests and biomarkers to assist with dlb diagnosis ( participants highlighted the need for more research for many of the core and supportive features of dlb (table 3 ) [9] . many discussed that research should investigate the symptoms and also medications to help: medicine-trying to find the right combination. anything you can give him to tamp down his symptoms-anything you can give to make it so it's easier for him to exercise, it's easier for him to live his daily life, is very important. participants also desired research on non-pharmacologic strategies to address dlb symptoms, including therapy, exercise, meaningful activities, "natural" therapies, changes in diet and nutrition, and unconventional approaches such as hyperbaric chambers. multiple individuals with dlb and caregivers discussed the need for pharmacologic or nonpharmacologic disease-modifying therapies that will prevent, cure, or slow dlb: how it could possibly be prevented or mitigated or slowed down in its decline. (p4) individuals with dlb and caregivers offered many topics pertaining to daily life that they felt were deserving of more research, including how to maintain socialization, maintain the ability to drive, identify emergencies, keep individuals with dlb safe, communicate well between couples (patient-caregiver), find support (e.g. good healthcare providers, external programs), handle non-dlb healthcare issues, make medical decisions, and strategize longterm planning. research aiming to improve quality of life was another common theme, particularly from caregivers: several individuals with dlb and most caregivers identified the need for additional research addressing different realms of caregiving, including caregiver burden and support, depression and how to handle impatience and frustration, balance external work and caregiving, identify alternative care options and ways to keep the individual with dlb at home, and identify resources to pay for caregiving needs. almost all caregivers and half of participants with dlb described a need for improved education surrounding dlb and how research could improve educational efforts. while these opinions were provided in response to questions about research, it was sometimes unclear whether participants were expressing a need for more education clinically or a specific desire for research on this topic. participants described a need for education in general and also specifically for physicians, medical trainees, therapists, patients, and caregivers/families. the most commonly described needs were education for non-specialist healthcare professionals and for caregivers. i think there should be probably some more, uh, i guess they have to have yearly training or whatever, you know. i think that should be something that's sort of at the forefront, because so many doctors don't even know what you're talkin' about when you tell 'em your disease. and, you know, all of them, a lot of 'em. education for regular doctors would be really good. (cg19, wife) [it] would be helpful for everybody to have a caregiver course, to help take care of the person, but also to learn how to take care of themselves. most of the research topics queried in the interviews or mentioned by interviewees were identified by at least some participants as their top priorities for dlb research (table 4 ). symptoms described as priorities for research included cognition, hallucinations, movement, mobility, sleep, and mood. a few participants mentioned that obtaining the patient and caregiver voices is the most important thing to them. (cg3, wife). the study guide queried a variety of research categories and individuals with dlb and caregivers identified topics important for research in all of them-focusing on awareness, determining the cause of dlb, improving diagnosis, investigating what to expect and disease stages, dlb symptoms needing additional research, therapies to prevent, cure, or slow the progression of dlb, targeting daily function and quality of life, caregiving, and improving education. furthermore, when participants were prompted to assign virtual money to research topics and identify their highest research priorities, at least one participant endorsed each of these areas. this highlights that research is needed across the dlb spectrum-basic science, translational, biomarker, natural history, drug development/therapeutic, quality improvement, and outcomes. published lewy body dementia research priorities were developed primarily by experts and focus on disease mechanisms and processes (including animal model development), diagnostic criteria, terminology, risk factors/prodromal stages, longitudinal cohorts, pathological staging, imaging, biomarkers, genetics, new treatment targets, and preventative/disease-modifying and symptomatic treatments [5, 30] . the individuals with dlb and caregivers in this study supported and prioritized research on many of these topics. however, many topics proposed in this study were outside the national research priorities, possibly because the adrd summit aimed to inform prevention and effective treatment of ad/adrds [5] . individuals with dlb and caregivers described needing symptomatic treatments but also more research on topics pertaining to how families should handle dlb symptoms in daily life, improving daily life in general, targeting quality of life, caregiving research, understanding disease progression, and improving education regarding dlb across populations (public, patients, caregivers, healthcare professionals). recommendations form the 2017 national research summit on care, services, and supports for persons with dementia and their caregivers address some of the themes from current study participants-the need for research to understand different dementia trajectories, determinants of behavioral and psychological symptoms, caregiving, decision-making, and support and financial considerations, as well as research investigating pharmacologic and nonpharmacologic treatment strategies and studying complex, multicomponent programs accommodating care needs [31] . the care summit also identified the need to understand what outcomes are important to persons living with dementia and to engage persons living with dementia and caregivers as part of research teams [31] . nearly half of the final research recommendations expressed ideas contributed by the persons living with dementia summit working group [6] . this emphasizes the need for funders (federal and otherwise) to consider research priorities from both dementia summits (adrd and caring) when developing funding announcements and selecting projects to support. in this study, individuals with dlb and caregivers described several topics as needing research which professionals might put in the category of clinical care. for example, the need for more education across populations was a common theme. many participants mentioned needs relating to daily life (decision making, independence including driving, socialization), practical caregiving considerations, when and how to obtain and use extra support, and finances. these practical research priorities are consistent with previously published research priorities of individuals with dementia and their caregivers. the top six overall research priorities identified through modified delphi consensus involving participants from dementia advocacy organizations were (1) cure and treatment, (2) caregiving, (3) education and training, (4) quality of life, (5) complementary therapies, and (6) care settings [25] . similarly, dementia priority setting through the james lind alliance identified 10 research priorities, including maintaining independence, optimal care, pharmacologic and non-pharmacologic care, and caregiver support [24] . a more recent dementia priority setting process in canada identified 10 research priorities including investigating stigma, supporting emotional well-being, early treatment, health system changes, caregiver support, connection to education and support, necessary dementia-related skills and knowledge for healthcare professionals, dementiafriendly communities, best dementia practices, and non-pharmacologic treatments [27] . research priority setting for people with pd identified needed research on physical functioning, symptoms, coping, stress, socialization, relationships, support, autonomy, and good care and communication alongside prioritizing research addressing pd causes, diagnosis, subtypes and medication for both motor and non-motor symptoms [22, 23] . differences in research priority-setting studies likely reflect the populations participating (e.g. country of origin; combination of patients, caregivers, advocates, healthcare professionals), the types of dementia represented, and the focus of the organizers or project leadership. the current study is also distinct from prior formal priority-setting work, as the goal of the current project was to investigate the views of individuals with dlb and caregivers at a single center, and not to use survey or formal consensus-building processes to build a top-ten list. the current results could serve as background for such a project specific to dlb. regardless of methodology, it is clear from the current study and prior work that individuals with dementia and caregivers of individuals with dementia, including dlb, prioritize research targeting issues relating to living with disease alongside efforts to better understand the cause of dlb and identify a cure. indeed, research into the topics discussed by participants is sorely needed. it is estimated that that 1 in 3 cases of dlb may be missed [32] and initial misdiagnosis is common [32, 33] . it takes over a year for half of individuals with dlb to receive a diagnosis [33] . family members of individuals who died with dlb described both lack of knowledge of what to expect and negative experiences relating to lacking healthcare professional education and knowledge [12, 34, 35] . while î±-synuclein deposition has been the presumed pathogenic cause of dlb for years, recent re-analysis questions this assumption [36] . there are no biomarkers for dlb and no treatments for dlb approved by the u.s. food and drug administration. caregiver burden in dlb is high [17] [18] [19] and quality of life is negatively affected by dlb [20, 21] . this study is the first to evaluate the research priorities of individuals living with dlb and caregivers of individuals with dlb. this study can inform survey development to identify research priorities of a larger group of individuals living with dlb, serve as the background for formal research priority-setting in dlb, and help guide research planning. the study recruited individuals with dlb and caregivers from a single united states-based center of excellence, affects generalizability, though the identified topics were consistent with publications enrolling stakeholders with other dementias and pd. it is plausible that a different cohort could have identified different or additional themes. for example, because enrolled individuals with dlb had to have mild-moderate dementia (such that they could participate), they and the caregivers who enrolled with them as part of dyads may not have considered end of life research questions, which could potentially be a priority for families dealing with advanced dlb. furthermore, interview questions were open-ended within categories and additional prompting regarding research topics could have resulted in different responses. the study specifically sought the opinions of individuals with dlb to give them an active voice and role, but several of the participants struggled to understand and answer the questions or perseverated on certain answers, limiting the information that could be gained from those interviews. most participants were of white non-hispanic backgrounds and had high educational attainment, potentially affecting generalizability. similarly, most participating caregivers were wives and it is plausible that other caregiving roles (e.g. husbands, children caring for parents) would have different priorities. individuals with dlb and caregivers of individuals with dlb identified areas needing more research related to dlb awareness, causation, diagnosis, prognosis and dlb stages, core and supportive symptoms, disease-modifying and symptomatic treatments, issues relating to daily function and quality of life, caregiving, and education across populations. these findings support the research priorities defined in the nih care summits in addition to the nih adrd summits. further research is needed across all domains of dlb. research funding should be informed by the priorities of all relevant stakeholders and support research investigating causes, natural history, biomarkers, and treatment in addition to research targeting themes regarding living with this disease (e.g. independence, quality of life, caregiving, and education). continuous patient engagement in comparative effectiveness research patient involvement in clinical research: why, when, and how patients', clinicians' and the research communities' priorities for treatment research: there is an important mismatch. research involvement and engagement department of health & human services alzheimer's disease-related dementias summit 2016: national research priorities contributions of persons living with dementia to scientific research meetings. results from the national research summit on care, services, and supports for persons 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revisiting protein aggregation as pathogenic in sporadic parkinson and alzheimer diseases key: cord-292777-oztmw8jo authors: wong, michelle; barqasho, babilonia; öhrmalm, lars; tolfvenstam, thomas; nowak, piotr title: microbial translocation contribute to febrile episodes in adults with chemotherapy-induced neutropenia date: 2013-07-16 journal: plos one doi: 10.1371/journal.pone.0068056 sha: doc_id: 292777 cord_uid: oztmw8jo in this study we sought to determine the contribution of microbial translocation to febrile episodes with no attributable microbiological cause (fever of unknown origin, fuo) in an adult febrile neutropaenic cohort. endotoxin concentrations were measured with the chromogenic limulus amoebocyte assay and used as a direct measure of bacterial products whilst soluble cd14 (scd14), measured with elisa was selected as an indicator of the early host response to endotoxins. endotoxin concentrations in this cohort were generally elevated but did not differ with the presentation of fever. further stratification of the febrile episodes based on the microbiological findings revealed significantly (p = 0.0077) elevated endotoxin concentrations in fuo episodes compared with episodes with documented bacterial and viral findings. scd14 concentrations were however, elevated in febrile episodes (p = 0.0066) and no association was observed between scd14 concentration and microbiological findings. however, fuo episodes and episodes with gram-negative bacteraemia were associated with higher median scd14 concentrations than episodes with gram-positive bacteraemia (p = 0.030). in conclusion, our findings suggest that in the absence of microbiological findings, microbial translocation could contribute to febrile episodes in an adult neutropaenic cohort. we further observed an association between prophylactic antibiotic use and increased plasma endotoxin concentrations (p = 0.0212). neutropenia is a common complication in patients undergoing chemotherapy for malignancies and is one of the most important risk factors for infections. infections account for substantial morbidity and mortality and fever is often the only indication of infection in this patient group. [1] empirically, prompt administration of broad-spectrum antimicrobials has been one of the important contributions to increased event free survival and overall survival among these patients. [2] today, bacteraemia is identified in 23%-33% [3, 4, 5, 6, 7] of all patients, and studies of the viral contribution to the aetiological panorama are still limited but estimated to be 30% [8] . this leaves, approximately a third of febrile episodes unexplained despite extensive microbiological investigations and these are thus termed fever of unknown origin (fuo). [9, 10] . cytotoxic agents administered as treatment for the underlying malignancies often cause intestinal mucosal damage and subsequent inflammation-mucosistis. this, process disrupts the balance between the normal bowel bacterial flora and host immune system, creating a milieu where microbial products could translocate into systemic circulation. endotoxins, more specifically, lipopolysaccharides (lps) are found commonly on the outer membrane of gram negative bacteria. their presence in the peripheral circulatory system could thus signify potential bacterial invasion, requiring rapid host response. potent pyrogens, endotoxins activate the inflammatory host defence via binding to soluble sc14 (scd14) which, in turn initiates downstream cytokines important for the clearance of bacterial infections. elevated endotoxins had been reported in studies with immunocompromised cohorts, for example, hiv-1 [11] and patients with haematological malignancies and is used as a measure of microbial translocation. [12, 13, 14, 15] . since the manifestation of febrile state is dependent upon the pro-inflammatory response towards endotoxins, scd14, a receptor for lps could be a suitable immediate early response biomarker for fevers associated with microbial translocation. in this study we thus sought to determine the contribution of microbial translocation to fuo episodes in an adult febrile neutropaenic cohort using endotoxin and scd14 concentrations in the plasma as markers for microbial translocation. during a 26 month period (january 2008 to february 2010), adult patients with haematological disorders at the karolinska university hospital, stockholm were, after informed consent, included in a cross sectional study where the inclusion criterion was chemotherapy-induced neutropenia (absolute neutrophil count #500/mm 3 ). patients that developed fever (auricular temperature .38.0uc twice within an hour or $38.5uc at one occasion) were sampled within 72 hours from fever onset, whereas patients without fever were sampled upon routine medical appointments during the neutropenia episode. whole blood was collected in edta-tubes and plasma was prepared by centrifugation before storage at 280uc until use. additional blood and nasal pharyngeal aspirates (npa) samples were collected for microbiological testing. medical records were retrospectively acquired for all included patients. the study was approved by the regional ethical review board in stockholm, permit numbers 2007/1213-31/4 and 2008/1300-32. all patients were screened with the same diagnostic panel. [16, 17, 18, 19, 20, 21, 22] and ohrmalm et al. [23] . lps was detected in patient plasma by the endpoint chromogenic limulus amebocyte lysate (lal) method using the qcl-1000 kit (lonza group ltd., switzerland) with modifications in the visualisation of the cleaved substrate. [24] all reagents and consumables were endotoxin -free (lonza). briefly, plasma was diluted 1:10 with 10 mm mgcl 2 solution and heat inactivated at 80uc for 12 minutes. the products were then cooled at room temperature for 15 minutes and 50 ml of the sample was added to empty wells of an endotoxin free flat bottomed 96-well plate (lonza). the background absorbance was measured at 540 nm (labsystems multiskan mcc 340, deltasoft multiskan iii 2.26) and the plate was placed in a 37uc block heater to start the microplate assay (qcl-1000). thereafter, 50 ml of pre-warmed (37uc) lal lysate (lonza) was added to each well and incubated for 10 min followed by 100 ml of pre warmed 37uc chromogenic substrate solution (lonza) and incubated for a further 6 minutes. the reaction was stopped by addition of 50 ml of sodium nitrate (0.3 mg/ml) in 0.48 n hcl to each well. subsequently, 50 ml of ammonium sulphamate (2.25 mg/ml) dissolved in water was added to each well, followed by 50 ml of n.e.d.a [n-(1-naphthyl) ethylene-diamine dihydrochloride, 0.525 mg/ml], producing a brilliant magenta-colored derivative. the color change was measured at 540 nm within 15 minutes. endotoxin standards (1 eu/ml to 0.0625 eu/ml) prepared in endotoxin free water were analysed alongside the samples as were, lps spiked samples as controls for assay inhibition. to calculate the lps levels, the final absorbance reading was subtracted from the background reading and derived from a linear regression of the absorbance readings of the known endotoxin standard concentrations. plasma concentrations of scd14 were measured with a commercially available, quantikine h human scd14 immunoassay kit (r&d systems, inc., minneapolis, usa). absolute neutrophil and leucocyte counts were obtained retrospectively from medical records of routine blood analyses. in addition, the absolute count of lymphocytes, cd4+ t cells, cd8+ t cells, cd 19+b cells, cd56+nk cells, and monocytes was determined in 59 episodes where the material was available, by flow cytometry using the bd multitest antibodies and trucount tubes (bd biosciences, nj, usa). statistical analyses were made with the prism suite (graphpad inc., ca, usa). fisher's exact test and mann-whitney test were used for investigating the patients' general characteristics. mann-whitney test was further used for analyses involving continuous data for two groups and kruskal-wallis test was used for all other analyses where comparison of continuous data for 3 groups or more were required. correlations were calculated by using spearman's rank test. a p-value ,0.05 was considered statistically significant. a total of 103 febrile neutropaenic episodes and 42 afebrile neutropaenic episodes were included in the study ( table 1 ). the majority of patients were treated for acute leukaemias and none were diagnosed with invasive fungal infections. microbiological findings were assumed, for the purpose of this study, to be the primary cause of fever and episodes with no microbiological findings are classified as 'fever with unknown origin' (fuo). in this neutropaenic cohort, endotoxin concentrations were not different between the febrile and afebrile episodes (figure 1a interestingly, use of prophylactic antibiotics (ciprofloxacin and combination therapy with trimetoprim and sulfamethoxazole) was correlated with higher (p = 0.0212) endotoxin levels at a median of 110.9 pg/ml (iqr = 93.2 pg/ml-139.1 pg/ml) compared to a median of 101.4 pg/ml (iqr = 90.4 pg/ml-110.9 pg/ml) in episodes where prophylactic antibiotics were not administered ( figure 2) . no association was seen between endotoxin concentration and neither other forms of antibiotics (administered before sampling), the underlying disease nor the general treatment regime. a positive association was observed between scd14 concentrations and manifestation of febrile status (p = 0.0066) with scd14 concentrations higher in febrile episodes (3.03610 6 pg/ml [iqr = 2.17610 6 pg/ml -3.89610 6 pg/ml]) compared with afebrile episodes (2.51610 6 pg/ml [iqr = 1.86610 6 pg/ml-3.08610 6 pg/ml]) (figure 3a) . stratification of the febrile episodes into those with bacterial findings, viral findings or fuo showed no significant correlation (p = 0.0740) (figure 3b ). upon further classification of episodes with bacterial findings into gram-positive bacteraemia and gram-negative bacteraemia, a difference (p = 0.030) was observed (figure 3c ). scd14 concentration in fuo episodes (3.20610 6 pg/ml [iqr = 2.29610 6 pg/ml-3.80610 6 pg/ml]) were similar to those with gram-negative bacteraemia (3.34610 6 pg/ml [iqr = 2.52610 6 pg/ml-4.01610 6 pg/ml]) whilst gram-positive bacteria findings were observed with a lower median scd14 concentration of 2.55610 6 pg/ml (iqr = 1.87610 6 pg/ml-3.07610 6 pg/ml). no correlation was observed between viral findings and scd14 concentrations. association between scd14 concentration and underlying disease was not observed. the endotoxin and scd14 concentrations were not intercorrelated in this cohort. similar results were obtained when analyses were limited only to febrile episodes. in this cohort of iatrogenic neutropaenic adults, we found elevated plasma levels of endotoxin and scd14 in febrile neutropaenic episodes where no other microbiological findings have been documented, implicating microbial translocation as a potential contributor towards their febrile status. we further observed a correlation between the use of prophylactic antibiotics and increased plasma endotoxin concentrations. previous studies attempting to determine relation of microbial translocation to febrile episodes in a similar patient cohort had presented mixed results. this stem primarily from differences in endotoxin detection methods [13, 15] , degree of immunosuppression in patients [12] and small sample sizes. it should also be noted that these studies limited their work to only systemic concentrations of endotoxins [12, 13, 14] or positive faecal bacterial cultures as indicators of microbial translocation. [15] in only one of these studies were pro-inflammatory cytokines measured as well [12] . however, the patients were less immunosuppressed compared to the current study, leading to difficulties for adequate comparison. to this end, effort had been placed to amass a larger study cohort with strict adherence to the inclusion criteria and, endotoxin concentrations measured with established and commonly used commercial assays. adding to the field, we have, in our study, in addition to measuring plasma endotoxin concentrations, also measured the host receptor for lps, scd14 in an attempt to determine the contribution of microbial translocation to fever as a clinical outcome. with this adult iatrogenic neutropaenic cohort, no apparent correlation between fever and endotoxin concentrations was observed. the median endotoxin concentrations were however, elevated in the whole cohort compared to healthy controls in earlier reports. [11] the corresponding association of scd14 concentrations to febrile status then suggests that a host response mediated through scd14 binding of lps has been initiated, leading to the development of fever as a symptom, implicating afebrile patients potentially as inadequate responders. admittedly, microbiological findings should be considered as the most likely grounds of fevers and further stratification was performed to tease apart this confounder. endotoxin concentrations in fuo episodes were significantly elevated compared to episodes with bacteria findings. while one might expect that presence of bacteria findings in blood should correlate with elevated endotoxin concentrations, it is pertinent to bear in mind that the detection target of the endotoxin assay is the immunoreactive lipid a portion of lps. [25] the lower endotoxin concentrations observed in episodes with bacterial findings, as well as the similarities in endotoxin concentrations between episodes with bacterial and viral findings, could reflect a situation of availability. an anchor of the lps molecule to the bacterial outer membrane, exposure occurs only if they are shed or, upon disruption of the bacterial cell wall. thus, in line with previous studies [26] , it should not be surprising that bacteria findings do not necessarily correlate with increase in measurable endotoxins. the lack of accuracy and precision undoubtedly limits the use of the lal assay in routine clinical practice but it has been the most widely used standardised method for the measure of endotoxin concentration in recent years. the tendency of hydrophobic regions of lipid a to aggregate or binding to the lps binding protein, could also potentially limit detection. a corresponding trend was however, not observed with scd14 concentrations; there were no differences when episodes were stratified based on microbiological findings or lack thereof, with respect to the fuo group. scd14 concentrations in episodes with gram negative bacteria findings were higher than those with gram positive findings, indicating differential responses to bacteria sub-groups as expected. further, scd14 concentrations observed in our study were similar to levels found in plasma of hiv-1 infected patients where microbial translocation had been documented. [11, 27, 28] . unlike endotoxins, scd14 concentrations in fuo episodes were not higher than in episodes with gram negative bacteria. scd14 performs dual roles; in low levels transfers lps to mcd14 and tlr4 to initiate pro-inflammatory response however at high systemic concentrations, scd14 act as an attenuator to cell responses by shuttling lps to lipoproteins. [29] with the elevated endotoxins observed in this cohort, our findings contend that scd14 could be acting in an inhibitory way to limit excessive bystander damage from prolonged systemic pro-inflammatory responses. thus, despite higher endotoxin concentrations in the fuo group, there is no corresponding increase in scd14. the inherent immunosuppressed state of this cohort could also be implicated in the saturation of scd14 response observed. monocytes and to a lesser extent, neutrophils have been suggested to be the main sources of scd14, either by shedding of mcd14 or secreted directly. antineoplastic treatment for the underlying disease leaves these patients with reduced monocytes and neutrophils hence a limit on scd14 production is not entirely surprising. a dampened scd14 secretion could also explain the lack of correlation between endotoxin and scd14 concentration in this study. overall, our findings show evidence of microbial translocation, particularly in the fuo episodes through elevated endotoxin and scd14 concentrations. another interesting finding in this study was that the use of prophylactic antibiotics was associated with higher plasma endotoxin concentrations. liberal use of prophylactic antibiotics is commonplace in iatrogenic neutropaenic patients as a pre-emptive measure to bacterial infections in this highly susceptible patient group. yet, the extensive use of oral prophylactic antibiotics might predispose a patient to increased risk of microbial translocation with the subsequent presentation of fever and delays in chemotherapy cycles for their underlying haematological malignancy. one could speculate that the use of prophylactic antibiotics had led to excessive remnant bacterial products or altered the bowel microflora, promoting the overgrowth of resistant variants [30, 31] . correlation of endotoxin concentrations and duration of prophylactic antibiotic use would have been ideal but we were unfortunately not able to accurately determine this parameter in our study. collectively, our findings suggest that in these patients, translocation of bacterial products, rather than whole bacteria, from the intestines has occurred. correlation of our findings with enterocyte function and integrity could provide more insight into the possible mechanisms of translocation and should be considered for future studies. arguably, bacterial findings based solely on positive bacterial cultures could lack sensitivity and be an underestimation of episodes with blood-borne bacterial findings. broad spectrum pcr targeting conserved bacterial genes like the 16 s rdna could improve bacterial diagnostics once the inherent difficulties with cross-contamination and false positivity have been resolved. [32] regardless, pcr diagnostics is a viable option to circumvent this limitation and should be considered for future studies. in this study, we have chosen to follow conventional clinical bacteriological routines in order to obtain the current clinical picture. given the transient nature of circulating endotoxins and scd14, care had been taken to ensure samples were obtained within a 72 hr window within which, no sampling bias was observed. additional information with regards to mucositis grading was unfortunately unavailable in this study and should be considered for inclusion in future studies as a measure of mucosal damage. further, it would have been desirable to perform longitudinal studies in order to determine if there were correlations between endotoxin concentrations and progression to severe infection or, even septic conditions. unfortunately, this was not possible with the current study but should be considered in other studies with the same patient cohort. in this cross-sectional study, our findings suggest that in the absence of microbiological findings, microbial translocation could contribute to febrile episodes in an adult neutropaenic cohort. this not only adds to the repertoire of potential causes of fever, it also indicates the urgency for suitable biomarkers to distinguish between drug and tumour related fevers (dtrf) and fevers of infectious causes. further, an association between prophylactic antibiotic use and increased plasma endotoxin concentrations was observed. unusual presentations of infection in neutropenic patients fever in immunocompromised patients molecular diagnosis of sepsis in neutropenic patients with haematological malignancies bacteremia and fungemia in patients with neoplastic disease infections in patients with febrile neutropenia: epidemiology, microbiology, and risk stratification the infectious diseases society of america 2002 guidelines for the use of antimicrobial agents in patients with cancer and neutropenia: salient features and comments bacteraemia in febrile neutropenic cancer patients viral findings in adult hematological patients with neutropenia fever of unknown origin-reexamined and redefined fever of unknown origin in adults: 40 years on microbial translocation is a cause of systemic immune activation in chronic hiv infection endotoxaemia and inflammatory mediators in febrile patients with haematological disease endotoxaemia as a cause of fever in immunosuppressed patients endotoxaemia, fever and clinical status in immunosuppressed patients: a preliminary study bacterial translocation and gram-negative bacteremia in patients with hematological malignancies multiplex real-time pcr for detection of respiratory tract infections submitted) multiplex real-time pcr for simultaneous detection of cytomegalovirus, epstein-barr virus and adenovirus in combination with bk polyomavirus or parvovirus b19 real-time reverse transcription-pcr assay for comprehensive detection of human rhinoviruses rapid and sensitive routine detection of all members of the genus enterovirus in different clinical specimens by real-time pcr quantitative rt real time pcr and indirect immunofluorescence for the detection of human parainfluenza virus 1, 2, 3 development and implementation of a molecular diagnostic platform for daily rapid detection of 15 respiratory viruses who (2009) cdc protocol of realtime rtpcr for influenza a (h1n1). accessed 15 submitted) virus association to fever in adult neutropenic patients with hematological disorders: a cross sectional study diazo-coupling option with pyrochrome chromogenic lal. lal update, associates of cape cod incorporated 16 bacterial lipopolysaccharides: relationship of structure and conformation to endotoxic activity, serological specificity and biological function does gram-negative bacteraemia occur without endotoxaemia? a meta-analysis using hierarchical summary roc curves microbial translocation, the innate cytokine response, and hiv-1 disease progression in africa plasma levels of soluble cd14 independently predict mortality in hiv infection plasma cd14 decreases monocyte responses to lps by transferring cell-bound lps to plasma lipoproteins long-term impacts of antibiotic exposure on the human intestinal microbiota metronidazole is still the drug of choice for treatment of anaerobic infections use of broad range16s rdna pcr in clinical microbiology we are extremely grateful to all study participants and thank the study nurses and doctors at the haematology centre as well as the staff at the department of accident and emergency, karolinska university hospital, for their dedication to the study. assistance from the staff at the department of clinical microbiology at the karolinska university hospital is much appreciated. key: cord-289510-spmknns5 authors: curado, evaldo m. f.; curado, marco r. title: a discrete-time-evolution model to forecast progress of covid-19 outbreak date: 2020-10-29 journal: plos one doi: 10.1371/journal.pone.0241472 sha: doc_id: 289510 cord_uid: spmknns5 here we present a discrete-time-evolution model with one day interval to forecast the propagation of covid-19. the proposed model can be easily implemented with daily updated data sets of the pandemic publicly available by distinct online sources. it has only two adjustable parameters and it predicts the evolution of the total number of infected people in a country for the next 14 days if parameters do not change during the analyzed period. the model incorporates the main aspects of the disease such as the fact that there are asymptomatic and symptomatic phases (both capable of propagating the virus), and that these phases take almost two weeks before the infected person status evolves to the next (asymptomatic becomes symptomatic or symptomatic becomes either recovered or dead). a striking advantage of the model for its implementation by the health sector is that it gives directly the number of total infected people in each day (in thousands, tens of thousands or hundred of thousands). here, the model is tested with data from brazil, uk and south korea, presenting low error rates on the prediction of the evolution of the disease in all analyzed countries. we hope this model may be a useful tool to estimate the propagation of the disease. a newly identified coronavirus capable of infecting humans was found in patients hospitalized in wuhan, china [1] (and, possibly, in europe [2] ) in december 2019. named severe acute respiratory syndrome coronavirus-2, or sars-cov-2, and causing the respiratory syndrome known as covid-19, it joins a group coronaviruses which originated from animals and resulted in human diseases, including severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers). however, while these diseases share to some extent similar clinical symptoms, including fever, dry cough and dyspnoea [3] , the covid-19 outbreak quickly spread worldwide and became a global public health crisis. in 30 january 2020 who declared covid-19 a public health emergency of international concern (pheic) [4] , and soon thereafter, on 11 march 2020 it was declared a global pandemic. notably, while on 30 january 2020 there were 8.324 confirmed cases worldwide, on 28 may 2020 this number increased to almost 6 million [5] . economically, it is expected that the covid-19 pandemic a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 results in a recession at least as severe as the 2009 crisis [6] . as such, to reduce the social and economic burden caused by the pandemic, the development of reliable models to forecast its propagation is a promising strategy to assist public policies aiming at controlling further dissemination of the disease. previously published models of propagation of diseases include sir [7] , seir, sis and others [8] [9] [10] [11] . these are powerful models, as they are relatively simple and perform well on distinct infectious diseases. however, these models are in general based on differential equations and their implementation with available data are rather challenging, what makes them difficult to be widely understood and implemented. notably, for covid-19 the accessible data set is extensive and updated in a daily basis, including number of confirmed cases, deaths, and recovered people per country. this increases the relevance of simple models that can be easily implemented and fed with daily updated data from the pandemic, facilitating tracking of its propagation and assessment of the efficacy of public policies aiming to control it in a daily basis. moreover, the recent history of infectious diseases have demonstrated that emergence of outbreaks are relatively frequent, to a point that such events are being included in scientific [12, 13] and political [14] discussions on preparedness for future outbreaks. therefore, as current (in case of and future, yet unknown, infectious diseases will continue to impact us, the development of simple but efficient models to track pandemics evolution based on daily updated data may be valuable not only for covid-19 but also for potential future outbreaks to come. the data set of the covid-19 pandemic can be found online by distinct sources, including governmental, e.g., the european centre for disease prevention and control (ecdc) [15] , and non-governmental, private sources, e.g. the johns hopkins university [5] (jhu, from usa), and the website worldometer [16] . these sources commonly present small differences in their data sets, but as common characteristics all present daily updated data and, among other quantities, the total number of confirmed cases of infected people and the total number of deaths per country. here, we present a model that can easily incorporate these available data sets and is based on discrete-time equations to forecast the number of confirmed cases by covid-19 in any given country for the next 14 days. among its strengths, the presented model: (i) presents low relative error rates, as tested in data from brazil, south korea and uk; (ii) provides easily interpretable results, specifically the predicted number of infected people in the next 14 days; (iii) presents results that are directly comparable across countries; and (iv) incorporates the average time related to the disease incubation period (asymptomatic phase) and the average time related to the symptomatic phase, both parameters adjustable according to the pandemics' characteristics. the main points about the covid-19 pandemic taken into account in our model are the following: • in a population, we can distinguish five types (classes or phases) of people. they are: a) people immune to the virus. b) people susceptible (s) to the virus but not infected. c) people infected but still in the asymptomatic phase (a). they can contaminate other people but present no symptoms. this phase takes roughly two weeks before clinical symptoms become apparent. d) people already presenting the clinical symptoms of the infection (i). this phase takes roughly two weeks before either recovery or death. e) people recovered (r) from the disease or dead. • the disease follows the sequence: s ! a ! i ! r. the time interval, t, going from a ! i commonly vary from few days to two weeks (according to the centers for disease control and prevention, usa [17] ) and we will take, for simplicity, t = 14 days. the time interval going from i ! r can also vary from 10 to more than 20 days and again, for simplicity, we will take the same value t = 14 days. clearly, this is a crude simplification since these time intervals can significantly vary per individual. for example, in patients where lungs are severely affected the recovery period is expected to take considerably longer than for patients presenting symptoms of a mild flu only. therefore, putting an average value t = 14 for both phases a and i is a simplification that can only be supported by fitting with the real data set: if the fitting produces a reasonably accurate forecast, then this average value can be kept. otherwise, the value for t may be fine-tuned by more sophisticated generalizations of the model. nevertheless, in all cases we have studied, the average error rate of tested predicted values were lower than 5%. • immune people are not included in the model as they are assumed to not be affected by the virus, nor transmit it. furthermore, it is assumed that a recovered person does not become susceptible again, i.e., after phase i the person is assumed to not be vulnerable to be reinfected by sars-cov-2. we propose that the daily evolution of covid-19 can be modelled by the following discretetime equations: aðt þ 1þ ¼ aðtþ þ a sðtþ aðtþ þ b sðtþ iðtþ à aðt þ 1 à tþ þ aðt à tþ iðt þ 1þ ¼ iðtþ þ aðt þ 1 à tþ à aðt à tþ à iðt þ 1 à tþ þ iðt à tþ ð3þ where t means the day, t means the duration of asymptomatic (incubation time) and symptomatic infected phases, both herein assumed to be 14 days. the term as(t)a(t) indicates that a number of the susceptible people becomes asymptomatic if in contact with asymptomatic people; the same with the term bs(t)i(t) where susceptible people becomes asymptomatic if in contact with symptomatic infected people, from now on simply called infected people. the parameters a and b are constants, measuring the intensity of contagion coming from asymptomatic or infected people and having, in principle, different values. finally, it is important to note that the sum s(t) + a(t) + i(t) + r(t) is a constant value and does not depend on time. in fact, it accounts for a fixed part of the studied population, in the order of tens of millions or eventually more for countries with large populations. as such, for these countries, the model may be described in a simplified manner if we consider that s is in practical terms a constant (see next section). the parameter t is a key component of the model as it indicates the influence of one class into another. for example, the difference of the number of infected people from time t to time t + 1, i(t + 1) − i(t), see eq (14) , is consequence of two factors. the first one is the change in the number of asymptomatic people from time t − t to t + 1 − t, because after a time t these asymptomatic people become infected people. the second one is that we have to discount the number of people leaving the class of infected ones from t − t to t + 1 − t, because they have more than 14 days since the symptoms of the disease manifested, i.e., i(t + 1 − t) − i(t − t). hence, those become part of the class of recovered (or dead) people, r. for simplicity, we have included in the same parameter, r, the healed and dead people. if we want to study these two cases in more detail, we can replace the eq (4) by two equations, with the new variables h(t) (healed) and d(t) (dead) given by: where r(t) = h(t) + d(t) and 0 < μ < 1 is a parameter that indicates the percentage of infected people dead by covid-19 after t days in a given country. importantly, this parameter may vary between countries but for most countries this percentage is accessible by publicly available sources [18] . with these equations, the total number of healed and dead people can be individually predicted by the model. here we focus on parameters a(t) and i(t), but in future works these two other variables will be analyzed. the model presented in the last section can still be simplified for countries with large population. first, as remarked before, it should be noted that the number of susceptible people can be a large number for countries with millions of inhabitants. the epidemic starts with a very small number of infected people, that increases and can reach (as currently seen) hundreds of thousands of people. for countries with large population, e.g., several tens (or even more than a hundred) million habitants, we can further simplify the model described in eqs (1)(4) assuming that the number of susceptible people is practically constant in time, s(t) ' s, where s is a constant, so there is no time evolution for s. this can be assumed for countries with large populations as the difference between infected (asymptomatic a + symptomatic i, which theoretically may reach up to few hundred thousand people in a given country) and susceptible people s (depending on the country reaching over a hundred million people) is still so huge that the decrease in the sample of susceptible people s may be negligible. as such, calling 1 + as � α and bs � β, where α and β are positive constants, the equations for covid-19 for largepopulation countries can be written as: where a, i and r are counted by hundreds, thousands or hundred of thousands. hence, here the sum a + i + r is not a constant anymore, but an increasing function on time. the susceptible people are, in these cases, considered as a 'thermostat', practically not changing in time. notice that here the total number of inhabitants of a country is not an important parameter, as it is in other disease models. this simplified model can be used while the number of a + i + r is lower than a small percentage of the total number of susceptible people of that country. conservatively, we recommend to use it while this sum is lower than 5%. however, this threshold of 5% is not strict and may be modified to some extent, but if the number of a + i + r is larger than 10% of susceptible people, the accuracy of the simplified version of the model may be affected. in these cases, the complete model represents a better alternative to forecast progress of covid-19. an analysis of these equations shows that eqs (15) and (16) are the key equations of the model, with the eq (9) being a supportive equation. this system of equations has only two parameters, α and β, that can be found phenomenologically for each country and roughly measures the capacity of contagion of asymptomatic and symptomatic people. this model could have more parameters, but it seems that all other parameters can be condensed in these two, with the only requirement of having positive values. the data set provided by the above-mentioned sources are updated in a daily basis and have at least two useful information for the proposed model: the number of deaths and the total number of confirmed cases (total symptomatic infected) people, i tot (t) per country and globally. the main variables in our model are the number of infected (i.e., asymptomatic a(t) + symptomatic i(t)) people at time t, i.e. people that at time t can infect susceptible people. there are many definitions of symptomatic people in these sources, and they are not completely equivalent. for our model, we will define the class of symptomatic people by means of the total number of confirmed cases by country, as the definition of confirmed cases is the same on all sources. with our hypothesis we can simply define: that is, the number of infected people at time t, defined as i(t), is the total number of infected people at time t minus the total number of infected people at time t − t. in turn, the total number of infected people at time t − t represents people who either recovered or died after the infection, since their symptoms manifested in more than t days. hence, by defining t 0 as the first day when someone is identified with the symptoms of the infection in a given country, i(t 0 ) as the "first infected people", i.e., the number of people infected at day t 0 , and t = t n as the last day when data is collected, we can construct the corresponding data set of the variable i(t) from t = t 0 until t = t n . here, we are assuming that i tot (t 0 − τ), where τ is a positive integer, is equal to zero since we are considering t 0 as the day of the first infected people (with symptoms). this ensues that i(t) = 0 for 1 � t < t 0 . in fact, in our model, we always have t 0 = t + 1 = 15. our day one for a specific country is always t 0 − t, the day of the first asymptomatic people. now, we can construct, from the recent built i(t) time series, the time series of the other variable, a(t). for that we use eq (16) rewritten as, with this equation we can construct the time series of a(t) from t = t 0 − t = 1 until t = t n − t. as t 0 − t = 1, i.e., the first day when people got contaminated (but still asymptomatic), we can define the initial condition in order to solve eq (11), after t days these people will lead to the first infected people, at time t = t 0 = t + 1. so, from the real data set of total infected people we can construct the time series of i(t) from t = 1, � � �, t n and, from eqs (11) and (12), the time series of a(t) from t = 1, � � �, t n − t. in the next step we use eqs (15) and (16) to generate the theoretical time series for a(t), from t = t n − t + 1, and for i(t) from t n + 1. for that we need the values of α and β. we need the series of a(t) and i(t) to forecast the next t days for the total number of infected people starting on day t n . one way to try to get good values for the parameters α and β is to make the prediction using eqs (15) and (16) not from t n but, say, from t n − 5 until t n , that is, we consider the real data until the day t n − 5 for i(t) and the real data until t n − t − 5 for a(t). this way it is possible to check the theoretical values of a(t), from t n − t − 5 until t n − t, i(t) and i tot (t) from t n − 5 until t n with the real ones, obtained from the real data set until t n . adjusting α and β to best fit these, say, five values, the forecast of the values of a(t), i(t) and i tot (t) from t n until t n + t + 1 ensue. in fact, the prediction for a(t) and i(t) has no upper bound for the chosen values of α and β, only the prediction for i tot (t) is limited to the next t days due to eq (10) if we want to keep using real data. other ways to obtain good values for the parameters α and β can be conceived, but we propose this simple way as it worked well in the tested cases. the values of α and β can change over time following changes on social behaviour, e.g. due to measures of confinement by local governments or increasing consciousness of the population, which in turn affect the interactions of susceptible people with asymptomatic and symptomatic infected people. therefore, it is recommendable to test the adequacy of the parameters α and β along the progression of the disease. in the next section we will see examples of the application of the model in some countries. if we regard the graphs of total infected people for several countries, we can notice three standard patterns. the first one shows a curve increasing faster than a linear one. these are the cases of brazil, russia, india, among others. the second pattern shows an almost linear increasing curve, e.g., uk and us. finally, the third one presents a strong increasing in the beginning and then the curve tends to saturate, e.g., south korea, germany and china. we will discuss examples of each one of these three patterns. in all cases we are considering the data until may 28th. then, we use the days from may 23th or 24th until may 28th to adjust good values for α and β. these values can then be used for prediction from may 28th until june 11th. the first infected person in brazil was identified on february 26th, defined here as day t 0 . using t = 14, the first infected person according to our model was identified on february 12th (i.e., 14 days before), defined here as day t = 1. the time series of the total infected people from the day 01, february 12th, until may 28th (107 days in total), can be obtained from the sources [5, 15, 16] . we are using herein, in this case, the data set from [5]. based on this data set we can construct the time series of i br (t) from t = 1, � � �, t n = 107 and a br (t) from t = 1, � � �, t n − 14 = 93 (fig 1) . here, we can implement our proposed model to predict 14 days after day 107. with the intention to test the method and find good values for the parameters α and β, we considered our fictitious t f n ¼ 102-th day, so it is possible to compare the total number of infected people as provided by the original source (jhu) with the predicted data generated by the model. for this comparison we will focus on days 103 to 107 and we consider only the real data of i br (t) up to day 102 and data of a br (t) up to day 88. adjusting the predicted data generated by the method with the real data values, we found that for α = 1.01 and β = 0.17 the relative error from days 103 to 107 is lower than 2%. the relative error in this period of five days is less than 2%. these values can then be applied to estimate the total number of infected people t = 14 days following day 107 (fig 2) . the empty triangles (red) are the real data obtained from [5] until day 121, june 11th. the full circles (red) are the numbers predicted by the model starting from day 108 and based on the real data until day 107. the maximal relative error, defined as errorðtþ ¼ ði tot ðtþ à i tot model ðtþþ=ði tot ðtþþ registered on the last day of the forecast, was 5.27%. i tot (t) is the total registered number of infected people in a specific country and i tot model ðtþ is the total number of infected people given by the numerical simulation of the model, at day t. the inset shows a shorter scale, from day 100 until day 121. it should be noted that in the case of brazil the values for parameters α and β are practically constant from april to early may, but from may to june it is possible to notice a slight decrease on these values. this does not happen with the other countries here presented, where the values of the parameters decrease faster with time. accordingly, the parameters α and β should be updated for each change on the monotony of the curve of infected people, since these changes may reflect new social behaviours affecting the contagion parameters α and β, with consequences on the time evolution of the disease. clearly, these changes are unpredictable because they depend on public health polices and population awareness. here, we consider the real data of the total number of infected people by covid-19 in uk. the first people identified in uk presenting symptoms of covid-19 were registered on january 31th, which we define here as day t 0 . using t = 14, the date of contagion for those people was on january 17th, defined here as day t = 1. the data set of the total infected people from the day 01, january 17th, until may 28th (133 days in total), can be obtained from the sites [5, 15, 16] . here we are using the data set from the johns hopkins university [5] . from this data set we can build the time series of symptomatic infected people fi uk ðtþg 133 t¼1 , and asymptomatic infected people fa uk ðtþg 119 t¼1 (fig 3) . in uk, the curves for symptomatic infected people and asymptomatic people suggest that the peak of the contagion ended roughly 100 days after the first registered case of infection. besides that, some oscillations can be found following the peak and prior to the pronounced decline in contagion. this is in contrast with the curve profiles from brazil, as there they are monotonically increasing over time. here, in order to capture the oscillatory profile from uk into the model and estimate good values for parameters α and β, it is better to consider a small interval of time for the test, with all data points belonging to the slope of the new tendency. therefore, to test the method in uk, we will consider our fictitious t n = 129-th day, four days less the true t n = 133. as a consequence, it will be possible to test the days from 129 until 133, generated by the method (for this test we are considering only the real data of i uk (t) up to the day 129 and of a uk (t) up to day 115). the agreement among the real data and the points predicted by the method from day 129 until day 133 is quite good for α = 0.94 and β = 0.001. using these values for the parameters we can predict the evolution of the total number of infected people in uk from day 133 until day 147 (june 11th). fig 4 shows the real data, with empty triangles (blue), and the numbers predicted by the method, with full circles (red), for α = 0.94 and β = 0.001. the inset shows a window from day 128 until day 147. the agreement is quite good for 14 days and the maximum error in these period is always smaller than 4%. the first infected person with symptoms in south korea was identified on january 20th, our day t 0 . in our model this means that the first asymptomatic person was infected on january 6th, here defined as day 1. the data set from january 06 (day 1) until may 28th (day 144), the last day where data was considered for forecast, was obtained from the ecdc site [15] (i.e. demonstrating the implementation of the model based on data set from a distinct source). based on the data of total infected people, we can build the time series for the (symptomatic) infected people, fi kor ðtþg 144 1 , see eq (10) . from the time series of {i kor (t)}, and using eqs (11) and (12), we can build the time series for the asymptomatic people, fa kor ðtþg in the present study we build and present a discrete-time model to study the time evolution of the covid-19 pandemic based mainly on the numbers of asymptomatic and symptomatic infected people. the model has only two parameters, α and β, what facilitates its implementation by health sector personnel and interested people in general. it is possible to use the daily updated data set of total confirmed cases of infected people to choose values for these parameters and predict how this number of infected people changes in the next 14 days if the parameters are kept constant. the model gives the effective numbers of total infected people, in tens or hundreds of thousands, that can be directly compared with the data set available from distinct sources, including johns hopkins university, ecdc, worldometer and others. also, the model incorporates the average time-delay asymptomatic people take to become symptomatic (incubation period) and the average time-delay the symptomatic infected people take to recover or, eventually, die. here, the model has been implemented in three countries (brazil, uk and south korea), and the predicted values generated by the model from may 29th until june 11th, based on the real data set until day may 28th, agrees very well with the real numbers of total infected people during this period. the maximum daily error for these three cases, each one with a period of 14 days, is around 5%. as such, we have found that the model is able to provide a reliable estimate of the status of the disease at least two weeks ahead of analysis time. remarkably, this is achieved with a rather simple algorithm, based only on fitting two parameters and on publicly available data [5, 15, 16] . altogether, we believe these characteristics facilitate its implementation by public sector, and may be a useful tool to track disease propagation and efficacy of public policies aiming to control it. it may be argued that the model is over-simplistic and do not account for complexities from the pandemic evolution. for instance, it has been reported that the coronavirus can still be detected in throat swabs from recovered patients up to 13 days after they present no symptoms of the disease (as evaluated by clinical assessments), indicating that even people considered to be recovered from covid-19 may still potentially infect others [19] . furthermore, it has been speculated the possibility of reinfection for patients who recovered from covid-19 (in this regard, preliminary data from primates support the hypothesis of immunization and protection against reinfection [20] ). in fact, we are aware that the model does not cover extensively the possible complexities related to disease propagation, but it focuses on the main course of propagation instead. as the model indicates a small error rate in its prediction, we considered it to fulfil its purpose adequately, exchanging complexity for simplicity in its comprehension and implementation while maintaining satisfactory predictive power (accuracy). these, in our understanding, are core principles for its assimilation by public health sector. moreover, the assessment of some complexities related to the pandemic may be heavily influenced by regional factors, what makes them challenging to reliably model, interpret or compare results between countries. for instance, some patients may develop from asymptomatic phase a to recovered phase r without undergoing the symptomatic phase i, a situation not included in the model. here, a challenge for modelling those cases is that not only the number of covid-19 testing varies dramatically between countries but also the infectious status of the tested population may vary significantly between countries. for example, it is expected that countries testing the population more extensively are detecting more asymptomatic subjects (including those in the above-mentioned scenario), while countries with less testing are prioritizing diagnosing symptomatic patients, what risks biasing the model towards countries with more data available. still, having discussed the challenges related to reliably assessing these cases, one could evaluate their effects in our model by adding a new parameter γ in eqs (2) and (3), and modifying them into the equations: aðt þ 1þ ¼ aðtþ þ a sðtþ aðtþ þ b sðtþ iðtþ à g ½aðt þ 1 à tþ à aðt à tþ� iðt þ 1þ ¼ iðtþ þ g ½aðt þ 1 à tþ à aðt à tþ� à iðt þ 1 à tþ þ iðt à tþ ; implying the changes in eqs (5) and (6): iðt þ 1þ ¼ iðtþ þ g ½aðt þ 1 à tþ à aðt à tþ� à iðt þ 1 à tþ þ iðt à tþ ; ð16þ where 0 < γ < 1. this means that not all asymptomatic people between days t − t and t + 1 − t will become infected people t days after. it should be emphasized, though, that to explicitly include these cases into the model comes at the cost of (a) increasing the complexity of the model by introducing another parameter, γ, (b) risking accuracy, as this information is challenging to reliably assess in any country (unless massive testing is implemented in the population in a narrow time window), and (c) potentially impacting the comparability across countries, as the assessment of this population may be largely different between countries, as discussed above. in the simplified model (i.e., without parameter γ), we assume that this parameter is integrated in the parameter α, decreasing its value, and consequently reducing the number of infected people without increasing the number of parameters. considering the good predictive power presented by the simplified model, we believe that its simplistic approach based on the main course of the pandemic has the advantages of being easier to implement, and providing more standardized, interpretable and comparable results across countries. we would like to remark that the prediction for the total number of infected people is restricted to 14 days because of the definition given in eq (10), where we want to use the real data for the total number of infected people. if we relax this requirement to use real data, we can get points for the whole future, keeping the parameters constant. certainly, this is not expected and this prediction for a long time is likely not reliable. it only gives an estimate of how will the disease propagate in the future if a given country does not change the value of the parameters. in fact, in practically all tested cases the parameters α and β decreased on time, likely reflecting measures against the propagation of the disease. for this reason, predictions provided by the model here presented are expected to decrease its accuracy for longer periods. the people from phase r incorporates those who recovered as well as those who died following the infection. thus, it is also possible to estimate separately the number of recovered and the number of dead people in two weeks, by simply applying in the r estimate the percentage of recovered and dead people from the studied country. as this is straightforward, we did not analyze this aspect in this study, but it is planned to be done in future works. a last word about the model: it is noteworthy, the model may also be used in other similar diseases-having both asymptomatic and symptomatic phases and both being contagious-as long as these other diseases have a daily (or some periodic) update of the data set. clearly, the parameter t has to be adapted to the life cycle of the studied pathogen and it also is plausible to require two distinct t 0 's, one for the asymptomatic and other for the symptomatic phase. this, however, needs to be fitted to the specific characteristics of the studied disease. finally, a more extensive work is being prepared, with the analysis of many other countries and a study of the parameter space. we hope the present model can contribute to global efforts made to understand and control the covid-19 pandemic. clinical characteristics of coronavirus disease 2019 in china sars-cov-2 was already spreading in france in late clinical features of patients infected with 2019 novel coronavirus in wuhan world health organization and others covid 19 public health emergency of international concern (pheic). global research and innovation forum: towards a research roadmap shared responsibility, global solidarity: responding to the socio-economic impacts of covid-19 a contribution to the mathematical theory of epidemics mathematical models in population biology and epidemiology modeling infectious diseases in humans and animals a model based study on the dynamics of covid-19: prediction and control predicting covid-19 peaks around the world the science behind preparing and responding to pandemic influenza: the lessons and limits of science polio infrastructure strengthened disease outbreak preparedness and response in the who african region what recent history has taught us about responding to emerging infectious disease threats european centre for disease prevention and control (ecdc) worldometer: covid-19 coronavirus pandemic centers for disease control and prevention (cdc): coronavirus disease 2019 (covid-2019). available from positive rt-pcr test results in patients recovered from covid-19 will we see protection or reinfection in covid-19? we would like to acknowledge the fruitful discussions with ignacio bediaga and constantino tsallis from centro brasileiro de pesquisas físicas, rio de janeiro, brazil. key: cord-272445-0xauff51 authors: naaber, paul; hunt, kaidi; pesukova, jaana; haljasmägi, liis; rumm, pauliina; peterson, pärt; hololejenko, jelena; eero, irina; jõgi, piia; toompere, karolin; sepp, epp title: evaluation of sars-cov-2 igg antibody response in pcr positive patients: comparison of nine tests in relation to clinical data date: 2020-10-27 journal: plos one doi: 10.1371/journal.pone.0237548 sha: doc_id: 272445 cord_uid: 0xauff51 sars-cov-2 antibody tests are available in various formats, detecting different viral target proteins and antibody subclasses. the specificity and sensitivity of sars-cov-2 antibody tests are known to vary and very few studies have addressed the performance of these tests in covid-19 patient groups at different time points. we here compared the sensitivity and specificity of seven commercial (snibe, epitope, euroimmun, roche, abbott, diasorin, biosensor) and two in-house lips assays (lips n and lips s-rbd) igg/total ab tests in serum samples from 97 covid-19 patients and 100 controls, and correlated the results with the patients’ clinical data and the time-point the test was performed. we found a remarkable variation in the sensitivity of antibody tests with the following performance: lips n (91.8%), epitope (85.6%), abbott and in-house lips s-rbd (both 84.5%), roche (83.5%), euroimmun (82.5%), diasorin (81.4%), snibe (70.1%), and biosensor (64.9%). the overall agreement between the tests was between 71–95%, whereas the specificity of all tests was within 98–100%. the correlation with patients’ clinical symptoms score ranged from strongest in lips n (ρ = 0.41; p<0.001) to nonsignificant in lips s-rbd. furthermore, the time of testing since symptom onset had an impact on the sensitivity of some tests. our study highlights the importance to consider clinical symptoms, time of testing, and using more than one viral antigen in sars-cov-2 antibody testing. our results suggest that some antibody tests are more sensitive for the detection of antibodies in early stage and asymptomatic patients, which may explain the contradictory results of previous studies and should be taken into consideration in clinical practice and epidemiological studies. introduction currently, more than 300 tests are available for sars-cov-2 antibody detection [1] . these tests are produced in several formats and they detect different types of antibodies including igg, igm or iga subtypes or total immunoglobulin. in addition, the target proteins used to detect antibodies vary between the tests. commercial tests are usually designed to detect antibodies against sars-cov-2 nucleocapsid (n), spike1 (s1), spike2 (s2), or receptor-binding domain of the spike (s-rbd) protein, or their combinations, though not all commercial providers specify the viral proteins used. given the large variability in antibody tests, discrepancies between test results are expected. concordantly, at the moment no agreement exists upon which viral protein should be used as a gold standard in serodiagnosis of covid-19 patients. although the producers have usually reported high sensitivity and specificity for their tests, variable clinical sensitivity has been reported by independent studies [2] [3] [4] [5] . however, minimal data is available on their sensitivity and specificity for their differences in target proteins. the majority of clinical studies and validations of commercial tests have been performed in patient groups with severe disease and thus reported sensitivity data may not be the same for covid-19 patients with mild symptoms. only a few studies have investigated the antibody responses in pauci-symptomatic or asymptomatic persons [6, 7] . several studies have shown stronger antibody response in patients with severe disease as compared with mildly symptomatic ones. also higher rate of absence of seroconversion in asymptomatic patients has been described. however, other studies have failed to find any correlation between clinical course and immune response [3, 6] . since the majority of covid-19 cases are asymptomatic, the performance of the tests in this group is important to evaluate the reliability of antibody tests in seroepidemiological studies and clinical diagnostics [8] . it is known that the sensitivity of the antibody test depends on sampling time. different studies have reported variable time of appearance of antiviral igg antibodies but in most publications the median seroconversion time has been between 6 and 14 days from symptoms onset. although several studies have shown high igg at least for seven weeks, rapid decline of igg in convalescence phase has been reported in asymptomatic covid-19 patients [2, 3, 6] . it seems, that the optimal time for igg detection (with the highest sensitivity rate) may also depend on clinical course of covid-19 and is not clearly defined yet. there is variation in antibody tests design (different target viral proteins has been used) on the one hand, and the conflicting results of clinical studies (tests sensitivities, antibody response dependence on clinical cause, optimal testing time) on the other hand. thus, there is lacking information on how different sars-cov-2 antibody tests perform in subgroups of covid-19 patients and at variable time points. our study aimed to compare the performance characteristics of seven commercial and two in-house igg/total ab tests, which analyze the reactivity to several target proteins, and to correlate the results with the patients' clinical data (with different symptoms score and age), and time from disease onset. the study has been approved by research ethics committee of the university of tartu on april 23, 2020 (nr 311/t-1). patients signed informed consent before recrutement into the study. serum samples from 97 persons with covid-19 were collected between april 28 and may 07 2020 from kuressaare hospital located on the island of saaremaa in estonia. persons who had been diagnosed covid-19 by positive sars-cov-2 rt-pcr regardless of clinical symptoms were invited to participate. these persons included hospitalized and ambulatory patients as well as healthy contacts of confirmed covid-19 cases selected randomly. the time from symptoms onset or positive pcr to serum collection had to be at least one week. included persons signed informed consent agreeing with sampling and usage of clinical data. medical personnel in kuressaare hospital performed blood sampling (10ml from each patient), recorded time of clinical onset and patients' symptoms related to covid-19 using modified who questionnaire. samples and patients' data were sent to synlab estonia central laboratory in coded manner. patients were scored based on a number of different symptoms present during covid-19 episode. on that basis we classified patients as asymptomatic (no symptoms before or after positive sars-cov-2 rt-pcr test), patients with 1-6 symptoms and patients with �7 different symptoms. serum was separated and aliquoted before storage. all aliquots were stored at-30˚c and analyzed within one month applying one freezing/thawing cycle before testing. for testing the specificity of applied tests, 100 anonymous serum samples collected before covid-19 pandemic and stored in synlab estonia were used. these samples were taken from healthy persons for various health control laboratory tests and have not been screened for virus related antibodies. five laboratory tests for igg (snibe, euroimmun, abbott, epitope, diasorin), one laboratory total ab (roche) test, one rapid igg test (sd biosensor) and 2 in-house igg tests (lips n and lips s-rdb) were compared. different protein markers have been used in these tests (s1, s2, s-rbd, n or their combinations). detailed information about the tests is summarized in s1 table. commercial tests were performed and interpreted according to manufacturer instruction, in-house lips as described previously [9] . the following analysis was made: correlation by spearman's test, comparison of qualitative data (positivity rate) by fisher exact test, groups' comparison by kruskal-wallis test, pairwise comparison by conover-iman test with holm-bonferron correction using past 4.03 and stata 14.2 software. according to medical history and patients' anamnesis, 20 patients (20.6%) had no symptoms before or after the positive pcr test. in 43 patients (44%), one to six different symptoms has been recorded, and 34 patients (35%) had seven or more symptoms related to covid-19 episode. detailed clinical data is shown in table 1 . we found no false-positive results in testing 100 pre-covid-19 sera by roche, abbott and biosensor tests. other tests gave one to two false positive results. additionally, euroimmun test gave 2% (2/100) and epitope 4% (4/100) borderline results with pre-covid-19 sera. the specificity and sensitivity of applied tests is shown in table 2 . in total, 15% (15/100) of control samples gave false positive results by any test. in most cases, only one test was positive per sample but 2% (2/100) of samples showed positive/borderline results by two (epitope and euroimmun) or three (snibe, epitope, lips s-rbd) tests. the distribution of quantitative values of controls is presented in s1 fig. out of 97 covid-19 patients' samples, 53 (55%) were positive and two (2%) negative by all nine tests used. results varied by tests in the case of 42 (43%) samples. sensitivity by different tests is shown in table 2 . the highest positivity rate was found in in-house lips n test (91.8% cases) and lowest in biosensor rapid test (64.9%). the distribution of quantitative test values of covid-19 cases in comparison with control samples is presented in s1 fig. the agreement between the tests (using qualitative interpretations) ranged between 95% (euroimmun and diasorin) and 71% (lips n and biosensor). the correlation between quantitative results was significant in all test combinations (p<0.001) except between in-house lips n and lips s-rbd that gave non-significant results. the strongest correlation was found between euroimmun and diasorin tests (ρ = 0.95). the agreement between qualitative results and the correlation between quantitative values is shown in table 3 comparing patients' symptom scores and the tests' quantitative values, we found a significant positive correlation in all cases except with lips s-rbd. the strongest correlation was table 4 . we found a significant correlation between ttt and the quantitative results of the test in case of roche (ρ = 0.38; p<0.001), euroimmun (ρ = 0.21; p = 0.04) and lips n (ρ = 0.28; p = 0.009) test. we also found significant differences between ttt groups in quantitative test data as well as in positivity rate in case of some but not all tests (table 5 ). no correlations between test results and patients' age or sex were found. this is the first study evaluating the performance characteristics of several commercial and inhouse sars-cov-2 antibody tests in different patient groups and different time to test points. we found that different tests gave diverse antibody results if applied to heterogeneous covid-19 patient group. in less than 60% of covid-19 cases, all tests gave identical positive or negative result. considering that all covid-19 patients (confirmed by positive sars-cov sars-cov-2 igg response 2 pcr test) should develop igg antibodies, the sensitivity of tests varied from 65 to 92%, which is much less than reported by the manufacturers [1] . in agreement, similar low sensitivity rates have been reported in a recent meta-analysis on antibody tests [2] . a combination of two tests with different protein markers may increase sensitivity. for example according to our results re-testing abbott igg (n protein) negatives with index value 0.3-1.39 by diasorin igg (s1 and s2 proteins), as practiced in synlab estonia, increases sensitivity by ca 7%. although this combination did not affect the specificity in our control group, comparisons in larger study groups should be done. however, even if several different tests are combined, some confirmed covid-19 cases remained negative for antibodies. correlation and agreements between the tests studied here varied highly in our covid-19 group. the best correlation was found between epitope and diasorin, which could be explained by detecting the antibodies to the same viral antigen (spike protein). one of the weakest agreements in positive/negative results and absence of correlation in quantitative test values was found when comparing igg antibodies to nucleocapsid and to rbd of spike protein detected by lips tests. it is thus plausible that the patients develop diverse igg antibody reactivities to sars-cov-2 proteins and their epitopes. while analyzing the results from different tests in patient groups with different symptoms scores we found that patients with more different symptoms usually had a higher positivity rate and higher levels of antibodies. this is in accordance with some previous studies [3] . however, this relationship was not uniform in all tests. for example, lips detected significant differences in anti-n igg levels between asymptomatic, pauci-symptomatic and polysymptomatic covid-19 patients. at the same time anti-rbd igg variation within the patients groups was higher and no significant differences between groups were found. according to our data production of igg antibodies against some virus proteins are more symptom-dependent than others. thus, according to our study, patients with more symptoms develop a higher immune response against virus proteins than asymptomatic ones. this makes the diagnosing of previous covid-19 patients by antibody test more difficult in asymptomatic and pauci-symptomatic patients since the sensitivity of several tests is much lower in this subgroup than in highly symptomatic group. since the majority of covid-19 patients are asymptomatic or have only a few mild symptoms the sensitivity of antibody tests to detect disease in the general population could be lower [8] . this may affect the reliability of antibody-based epidemiological studies. however, for some tests (such as abbott), the absence of clinical severity seems not to affect positivity rate so much than for others (such as biosensor rapid test or snibe) where the positivity rate in asymptomatic covid-19 cases was about two times lower than in polysymptomatic ones. more studies are needed to confirm the finding that some antibody tests (that use specific antigens) are more suitable to diagnose asymptomatic covid-19 cases than others. we also found test-dependent differences while comparing antibody detection among different ttt groups. for example, anti-n igg was detected by abbott in high level already at 7-14 days ttt group and no differences were found with later ttt groups. in some other test (such as euroimmun) lower detection rate and antibody levels were associated with shorter ttt. the reason could be in an analytical sensitivity of the test and also in the usage of different viral proteins-antibodies to some virus proteins may appear earlier and at increased levels than others. our study has several limitations. firstly, although the onset of disease could be dated relatively precisely in symptomatic cases, in asymptomatic cases the disease detection depends on random pcr screening of risk groups. thus, the first positive sars-cov-2 pcr is not equivalent to disease onset and one should be careful drawing conclusions based of ttt in such heterogeneous group of patients that includes symptomatic and asymptomatic patients. secondly, we only analyzed igg and total antibody values. the main reasons were the absence of igm tests from several manufacturers (abbott, diasorin) in the time of testing and questionable reliability of available ones. snibe and epitope igm tests gave significantly lower positive results than igg tests and added no additional positive cases (all igm positive cases were also igg positive). euroimmun iga had a high positivity rate (ca 90% in covid-19 patients) but also a high proportion of nonspecific reactions (21% of positive and borderline cases) in pre covid-19 control group. thus, until reliable commercial igm and iga tests are available, we can't evaluate the role of these antibodies in infection and applicability of these tests in clinical practice or surveillance studies. in conclusion, our study gave new theoretical insight to covid-19 diagnostic testing and has practical implications. we confirmed that sars-cov-2 antibody response depends on clinical symptoms and time of testing, but we also found that this relation is dependent on test type and viral antigens used in the tests. this means that not all antibody tests work uniformly well in symptomatic and asymptomatic cases and in different time periods from disease onset. this explains some contradictory results of previous studies and should be taken into consideration in clinical practice and epidemiological studies. supporting information s1 table. detailed information about applied sars-cov-2 antibody tests. sars-cov-2 diagnostic pipeline diagnostic accuracy of serological tests for covid-19: systematic review and meta-analysis evidence summary of the immune response following infection with sars-cov-2 or other human coronaviruses evaluation of nine commercial sars-cov-2 immunoassays test performance evaluation of sars-cov-2 serological assays clinical and immunological assessment of asymptomatic sars-cov-2 infections sars-cov-2 serological analysis of covid-19 hospitalized patients, pauci-symptomatic individuals and blood donors the incidence of novel coronavirus sars-cov-2 among asymptomatic patients: a systematic review lips method for the detection of sars-cov-2 antibodies to spike and nucleocapsid proteins we thank keith orchard for language corrections. conceptualization: paul naaber, pärt peterson, piia jõgi. key: cord-267605-efb10j3u authors: zheng, li-zhen; liu, zhong; lei, ming; peng, jiang; he, yi-xin; xie, xin-hui; man, chi-wai; huang, le; wang, xin-luan; fong, daniel tik-pui; xiao, de-ming; wang, da-ping; chen, yang; feng, jian q.; liu, ying; zhang, ge; qin, ling title: steroid-associated hip joint collapse in bipedal emus date: 2013-10-21 journal: plos one doi: 10.1371/journal.pone.0076797 sha: doc_id: 267605 cord_uid: efb10j3u in this study we established a bipedal animal model of steroid-associated hip joint collapse in emus for testing potential treatment protocols to be developed for prevention of steroid-associated joint collapse in preclinical settings. five adult male emus were treated with a steroid-associated osteonecrosis (saon) induction protocol using combination of pulsed lipopolysaccharide (lps) and methylprednisolone (mps). additional three emus were used as normal control. post-induction, emu gait was observed, magnetic resonance imaging (mri) was performed, and blood was collected for routine examination, including testing blood coagulation and lipid metabolism. emus were sacrificed at week 24 post-induction, bilateral femora were collected for micro-computed tomography (micro-ct) and histological analysis. asymmetric limping gait and abnormal mri signals were found in steroid-treated emus. saon was found in all emus with a joint collapse incidence of 70%. the percentage of neutrophils (neut %) and parameters on lipid metabolism significantly increased after induction. micro-ct revealed structure deterioration of subchondral trabecular bone. histomorphometry showed larger fat cell fraction and size, thinning of subchondral plate and cartilage layer, smaller osteoblast perimeter percentage and less blood vessels distributed at collapsed region in saon group as compared with the normal controls. scanning electron microscope (sem) showed poor mineral matrix and more osteo-lacunae outline in the collapsed region in saon group. the combination of pulsed lps and mps developed in the current study was safe and effective to induce saon and deterioration of subchondral bone in bipedal emus with subsequent femoral head collapse, a typical clinical feature observed in patients under pulsed steroid treatment. in conclusion, bipedal emus could be used as an effective preclinical experimental model to evaluate potential treatment protocols to be developed for prevention of on-induced hip joint collapse in patients. steroid-associated osteonecrosis (saon) is a common orthopaedic problem although steroids are initially prescribed for many non-orthopedic medical conditions, such as systemic lupus erythematosus (sle), organ transplantation, asthma, rheumatologic arthritis (ra), and severe acute respiratory syndrome (sars) [1] [2] [3] [4] [5] . part of saon patients even evolved to hip joint collapse with subsequent total joint replacement [6, 7] , and its long-term durability however still remained a big challenge [8] . how to prevent accumulation of saon lesions is the first-line strategy for avoiding joint collapse. therefore, establishment of appropriated saon animal models that mimic clinical etiology and even evolves to joint collapse is desirable prior to translating prevention and treatment experimental protocols into clinical validation and applications. up to now there is lack of ideal animal models to exam the treatment efficiency or therapeutic strategy for saon-associated joint collapse. the limitation of the existing animal models, such as rabbit [9] [10] [11] [12] [13] , rat [14] [15] [16] [17] , mouse [18, 19] , pig [20, 21] and chicken [22] is that they fail to progress to the end-stage of saon, i.e. structural collapse of the weight-bearing joints. with bipedality, high activity level and large enough bodyweight similar to that of human beings, on model to be developed in emu femoral head could provide a unique opportunity to progress to human-like femoral head collapse [23, 24] . focal cryogenic (liquid nitrogen) insults [23, 25] and alternative cooling and heating insults [26] have also been tested to induce on in emus with femoral head collapse. however, these models are not etiology-and or pathophysiology-orientated for saon research. accordingly, the aim of the current study was to establish a saon model in bipedal emus, with potentials to bone structural deterioration with subsequent femoral head collapse, a condition seen in saon patients attributed to similar biomechanics or loading ratio imposed onto the hip joint [23, 24] . such a model would be essential for testing strategies to be developed for potential clinical applications for prevention and treatment of steroid-associated joint collapse. of all available animal models, rabbits were intensively used for establishing on model where either lipopolysaccharide (lps) [27] or methylprednisolone (mps) [12, 28, 29] or their combination (lps+mps) [11, 13] were tested. all of them showed effectiveness in on induction, yet with varying degrees of on lesions and mortality of animals. based on our established saon rabbit model with a high incidence of on and low or no mortality that was induced by a combination of lps and mps [11, 30] , we hypothesized that such a combination of pulsed lps and mps injections might also be able to induce saon in bipedal emus with subsequent hip joint collapse. the research ethics committee of shenzhen second peoples' hospital reviewed and approved the experimental protocols [licence no. 2009-001] (appendix s1). both the guide for the care and use of laboratory animal (1996) [31] and the arrive (animals in research: reporting in vivo experiments) guidelines [32] were followed. eight 24 months old young adult male emus were used for this study. they were kept in shenzhen emu institute and received food and water ad libitum. five emus assigned to the saon group were treated with a combination of lps and mps. three emus were used as controls without receiving either lps or mps. the emus were euthanized by intravenous injection of overdose of pentobarbital via jugular vein at 24 weeks post injection. the details of this combined protocol were described as follows: each emu was intravenously injected with lipopolysaccharide (escherichia coli o111:b4; sigma-aldrich, st. louis, mo, usa) twice via jugular vein with 8 mg/kg body weight at an interval of 4 days from day 0. thereafter, three injections of methylprednisolone (pharmacia & upjohn, peapack, nj, usa) with 10 mg/kg body weight were given intramuscularly at gluteus muscle at an interval of 2 days. in addition, each emu was intramuscularly injected at gluteus muscle with 40 mg omeprazole sodium and orally with 250 mg amoxicillin dispersible per day for 7 days immediately after induction to prevent potential stomach ulcers and systemic infection ( figure s1 ). mri was performed with a 1.0 t mr unit (magnetom harmony; siemens, erlangen, germany) at baseline, week 2 and then at monthly basis on saon induced emus for in vivo examination on bilateral proximal femora until 12 weeks post induction. for facilitating in vivo bioimaging examination, a specific posture fixture was designed to obtain a highly reproducible image during mri scanning ( figure s2) . a phased-array body coil was used for mri scanning. coronal turbo spin-echo fatsaturated t2-weighted images (4000 ms repetition time, 96 ms echo time) were obtained with a slice thickness of 3 mm and interslice gap of 0.3 mm from a field of view of 300 mm 6 300 mm with a matrix of 3206320 pixels. blood was sampled at baseline, week 2, 4 and 8 post induction for routine blood examination and serum was prepared for examination of both coagulation and lipid metabolism. the serum parameters related to lipid metabolism, including total cholesterol (tc), total glycerin (tg), low-density lipoprotein (ldl) and high-density lipoprotein (hdl), and the serum parameters related to coagulation, including prothrombin time (pt), prothrombin time and international normalized ratio (pt/ inr), fibrinogen (fbg) and thrombin time (tt), were tested using standard clinical laboratory protocols for blood chemistry in shenzhen second peoples' hospital. gait of emus with saon was observed regularly after induction by recording abnormal gait pattern using a video camera. normal gait of the control emus was also recorded for comparison (video s1 and s2). emus walked or ran on a 20-meter walkway, and the sagittal plane motion was videotaped at 300 hz by a highspeed video camera (casio ex-f1, japan). the videos were analyzed by a motion analysis system (kwon3d xp, korea) to obtain the included ankle joint angle during a gait stride cycle with respect to the time presented in term of percentage stride. gait pattern was defined abnormal when there was a deviation of the included ankle joint kinematics (more than 20 degrees at any time) or the proportion of the stance and swing time (more than 10% deviation from control case). bilateral proximal femora from both control group and saon group were sampled and fixed in 10% buffered neutral formalin solution for 10 days, and then soaked in 70% ethyl alcohol for measurement of trabecular morphology within and around on lesions. in brief, the proximal femur was scanned using a highresolution peripheral ct (hr-pqct) (xtreme ct, scanco medical, brüttisellen, switzerland) at a voltage of 70 kv and a current of 114 ua, with entire scan length of 40 mm in a spatial resolution of 40 mm used for animal experimental studies [33, 34] . after the initial scanning, 2-dimentiaonal (2-d) images were realigned in the z-axis along the direction of femoral neck for further evaluation. for separating the signals of the mineralized tissue from the background signal, noise was removed using a lowpass gaussian filter (sigma = 2.5, support = 2) and mineralized tissue was then defined at a threshold of 85. 3-d structures of entire femoral heads were then reconstructed. the collapse was identified when fracture and/or clear deformation appeared in femoral head. a 10 mm 610 mm 68 mm region in the centre of femoral head was defined as the region of interest (roi) for analysis and comparison. roi was defined within subchondral region centered in the collapsed region or the corresponding region of the non-collapsed femoral heads, where the largest thickness of this roi was 1/3 diameter of the femoral head. bone mineral density (bmd), bone tissue volume fraction (bv/tv), trabecular number (tb. n), trabecular thickness (tb. th) and trabecular separation (tb. sp) in the roi were measured separately by the workstation with the built-in hr-pqct software. after micro-ct scanning, femoral heads were sawed in half longitudinally along the coronal plane. halves were decalcified and embedded in paraffin; and halves were embedded in methyl methacrylate (mma) without decalcification. 1. decalcified sections. for paraffin embedded samples, sections were sliced along the coronal plane for hematoxylin-eosin (h&e) and fast green and safranin o staining, respectively. a microscope imaging system (leica q500mc; leica microsystems, wetzlar, germany) was used to digitalize the histological sections for histomorphometric evaluation. 1) examination of on: the entire area of each h&e stained section was examined for the presence of on with the established criteria, i.e. diffuse presence of empty lacunae or pyknotic nuclei of osteocytes in the trabeculae, accompanied by surrounding necrotic bone marrow [13, 28] . the femoral head with at least one on lesion was considered as on+, while that with no on lesion was considered as on[11, 35] . 2) quantification of fat cells in bone marrow: fat cells were quantified for both average of fat cell size and fat cell area fraction. the total bone marrow in subchondral bone was 200 fold magnified and captured. the fat cells were manually traced and then quantified. the fat cell size was interpreted by fetet's diameter, i.e. the longest distance between any two points along a region of interest (roi) boundary [11] . the fat cell area fraction was defined as total marrow fat cell area normalized by total marrow tissue area [35] . 3) histomorphometry of the subchondral plate: the thickness of subchondral bone plate of the femoral head was measured on the 506 images. at least 10 views per section were randomly selected for measuring. the thickness was examined by measuring point-to-point distance from the top of the calcified cartilage to the deep surface of the subchondral bone plate [36] . 4) examination of articular cartilage: the average thickness of cartilage were examined by the total area of cartilage divided by the length of cartilage band measured from the manually traced region on the entire frontal sections for evaluation under 2006 magnification. the thickness of cartilage at the collapsed region or the corresponding region of the non-collapsed femoral head was also examined. the proteoglycans content was quantified by measuring the thickness of safranin o stained articular cartilage. 5) calculation of osteoblasts: the osteoblast perimeter percentage (%ob.pm) was calculated as the ratio of the total perimeter of the trabecular surface covered by osteoblasts to the whole perimeter of the trabecular surface [37] . 6) blood vessel quantification: the number of blood vessels within the collapsed region or the corresponding region of the non-collapsed femoral head was quantified on h&e sections [38] . 2. undecalcified sections. the mma embedded femoral heads were sectioned along the sawed plane using a diamond saw (isomet, buehler). the cut surface was polished on a soft cloth rotating wheel [39] . the surfaces were acid-etched with 37% phosphoric acid for 2-10 seconds, followed by 5% sodium hypochlorite for 20 minutes. the samples were then sputtercoated with gold and palladium, as described previously [40, 41] and examined for bone matrix and features of osteocytes in the scanning electron microscope (sem) (jsm-6300, jeol, japan). statistical power was set .0.8 and the type i error probability was set ,0.05 for calculating sample size (n = 5) using ps (power and sample size calculations version 3.0) for establishing saon model based on our previous rabbit model with a saon incidence of 93% [11] . the incidence of collapse was defined as the number of collapsed hips divided by total number of hips in each group. the incidence of saon was defined as the number of saon emus divided by total number of emus in each group, and analyzed with fisher's exact test. the serum parameters were expressed as mean 6 sd, and analyzed by one way analysis of variance (anova) with a post hoc bonferroni's multiple comparison test to compare the differences between every time point and baseline. micro-ct and histomorphometry data were expressed as mean 6 sd, and analyzed with mann whitney test to compare the differences between the control group and saon group. spss 10.0 was used. the significance for comparison was set at p,0.05. in t2 weighed mri images, intense signals of edema in the proximal femur were found at week 2 post-induction when compared to the baseline. the edema signals in proximal femur decreased in t2 weighed mri image at week 12 post induction ( figure 1 ). no collapse was found from mri images in the first 12 weeks after induction. the results of routine blood examination showed abnormal increase in percentage of neutrophils (neut %) at 2 weeks post induction (figure 2a ). for the time course changes in serum parameters related to lipid metabolism, tc was significantly increased at each time point post-induction, and ratio between ldl and hdl as well as ldl was also significantly increased at each time point post-injection (p,0.05 for all) while tg did not show significant increase post-induction ( figure 2b ). however, as compared with baseline, no significant difference was found for the time course changes in serum parameters related to coagulation post-induction ( figure 2c ). all emus were observed to be less active but with normal gait in the next day after the first lps injection; while an asymmetric limping gait pattern was firstly observed at week 12 post induction during loading and unloading gait cycle ( figure 3 ). in the early post-induction phase the asymmetric limping gait was only observable when the emus were running. with time the asymmetric limping gait was observed when emus were walking; closing to week 24 post induction some of the emus were observed no more active and kept sitting most of the time. no emus died during the experiment period over 24 weeks. all five emus in saon group developed edema, an early stage sign of on at bilateral hip based on mri observations as described above, in part also shown by gross morphology after sample harvesting ( figure 4a and b) , and confirmed histologically as described below. the incidence of hip joint collapse was 70% (7 out of 10 hips from 5 emus) found in the saon group, including 3 emus with bilateral collapse, 1 emu with unilateral collapse, and 1 without hip joint collapse ( figure 4a-f) . there was no collapse of the hip found in control group. micro-ct analysis was performed for the trabecular bone microarchitecture in the subchondral region ( figure 4e and f) and in the center of the femoral head ( figure 4g and h) , respectively. within the subchondral region, the bmd, bv/tv, tb. n and tb. th in the saon group were found significantly lower than those in the control group (p,0.05 for all), while the tb. sp in the saon group was significantly higher than that in the control group (p,0.05) ( table 1) . however, no statistically significant difference was found for all micro-ct indices of trabecular bone in the centre of the femoral head between the control and saon group (table 1) . gross coronal view of h&e stained femoral head in the control group showed well-arranged trabecular bone supporting the subchondral plate and the articular cartilage ( figure 5a1 ); while the collapsed femoral head in saon group was lack of vertical arranged trabecular bone to support the subchondral plate and the articular cartilage, and bone fracture shown at collapsed site ( figure 5a2 ). though 70% of 10 hips from 5 emus in the saon group developed hip collapse, 100% of hips from all emus in the saon group developed on at bilateral hips as confirmed histologically while as expected no on lesion was found in the control emus (p,0.05) ( figure 5b , c, d, e). osteonecrosis was distributed in whole femoral head, including subchondral bone ( figure 5b2, e) , middle of the femoral head ( figure 5c2 ) and femoral neck ( figure 5d ), with numerous empty lacunae in the trabeculae and marrow tissue degenerated. compared with the control group, the marrow fat cell size (fetet's diameter of fat cell) and fat cell area fraction in the saon group were increased significantly with decreased number of mononuclear cells (p,0.05 for both, figure 5b and figure 6a , b). the thickness of subchondral plate of femoral head of saon emus was decreased significantly (p,0.05, figure 6c ). the articular cartilage of saon emus also showed pathological alteration with significantly thinner thickness (p,0.05, figure 5f , g and figure 6d ). the proteoglycans content as interpreted by maximum thickness of safranin o staining was decreased significantly in saon group (p,0.05, figure 6e ), with osteonecrosis located at the collapsed region ( figure 5e ). the osteoblasts perimeter percentage (% ob. pm) of the saon group was significantly lower than those in the control group (p,0.05, figure 6f ). the blood vessels at subchondral region of saon group were surrounded with enlarged and compacted arranged fat cells, with some blood cells effused out of the vessel (figure 5b ). at the collapsed region or the corresponding region of the non-collapsed femoral head, the number of blood vessels of saon group was significantly less than that of the normal group (p,0.05, figure 6g ). sem images showed that in the collapsed region there was no osteo-like structure; instead, there were more osteo-lacunae outline, with more removal of bone mineral or less matrix; and that in the normal control, there was osteon-like structure and there were few osteo-lacunae outline with much solid bone matrix (figure 7 ). using a combined pulsed lps and mps induction protocol previously established for saon quadrupedal rabbits [11, 13] , the present study established a saon model in bipedal emus characterized with subchondral bone deterioration and hip joint collapse, an experimental model mimicking human on often developed at hip joint with femoral head collapse. as bioimaging evidence, we used mri to evaluate in vivo alteration of mri signals in the first 12 weeks post induction as mri could diagnose early-stage on, even presymptomatically [42] . abnormal signals were firstly detected at week 2 postinduction, showing large scales of edema at the proximal femora. this phenomena is similar to clinical on where the bone marrow edema was found from mri earlier than either formation of the necrotic lesion or the collapse of the necrotic fragment [43] . however, mri of emu hips merely shows bone marrow edema without typical band pattern shown in initial mri signaling of on patients [44, 45] . this could be explained by the differences in anatomy and physiology of human (mammals) and emu (aves), such as that the bone marrow of emu hip mainly presented in subchondral bone in a shell-shaped region and there was hollow structure in emu's bone marrow cavity at both femoral neck and head. irrespective such differences, there were common structural features, i.e. on lesion and hip joint collapse found in saon emus and this would provide a platform, i.e. a large bipedal animal model for testing biomaterials developed for bone defect repair, including for that after surgical core-decompression in hip joints, a condition indicated for patients with early stage of on. for functional evidence, asymmetric limping gait was observed at week 12 post-induction, suggesting that emus might suffer from joint pain caused by the on lesion formation and structural damage confirm histologically after harvesting the samples for detailed analysis. this symptom was also common in patients at early stage of on [46] . the asymmetric limping gait shown in bipedal emus was similar to the low limb dysfunction in saon patients with subchondral lesions [47] . for biochemical evidence, the current study suggested hyperlipidemia occurred after saon induction, which was evidenced by a significant increase in tc at all examined time point postinduction. in addition, abnormal higher lipid transportation to the peripheral tissues also occurred after induction, as evidenced by a significant increase in ldl/hdl, which was consistent with the results found in both saon rabbit model [9, 11] and on patients [48] . however, there was no significant difference in coagulationrelated parameters after induction as compared with baseline, while hypercoagulable and hypofibrinolysis state were reported in saon rabbit model [9, 11] and sars on patients [49] . this inconsistency suggested that the pathogenesis of saon in emu might be explained by the dominant disorder in lipid metabolism. histological and histomorphometric analysis was performed in three rois of the femoral head, including the articular cartilage, subchondral bone region and the central part of the femoral head. under higher magnification of the subchondral bone region where edema was shown in mri images, remarkable necrotic changes were observed in emu after induction, including 1) avascular zone at load bearing region, referring to the blood vessels quantification within the collapsed region; 2) extravascular adipogenesis (fat cell enlargement); 3) apoptosis of osteocytes in regions next to the marrow region packed with fat cells; and 4) thinning of subchondral bone, poor quality of bone matrix and more osteolacunae that weakens the mechanical integrity of the femoral head, which could also be weakened by the necrosis and thinning of articular cartilage. these were essential indices of later joint collapse, typically seen in saon patients [50, 51] that were substantiated in our histomorphometric analysis. on the contrary, our micro-ct evaluation for the central region of the femoral head, where no bone structural collapse was demonstrated, implied that the pulsed lps-mps induction protocol did not result in general osteoporosis as no difference in boney structural and bmd was found between saon emus and normal controls in regions away from the subchondral bone. this finding was also similar to a bone densitometry study in sars patients who underwent pulsed steroid treatment in hong kong [52] . no obvious osteoporosis found in saon patients with joint collapse or animals were mainly explained by short-term effects of pulsed corticosteroid administration that resulted in local damage of vasculature in the subchondral bone region and impairment of bone marrow mononuclear cells, which triggered up-regulation of osteoclastic activities and inadequate bone formation known as destructive repair [1, 6, 10, 34, 53, 54 ]. yet, long-term steroid administration was known to be able to induce secondary osteoporosis in patients [55] or animals [14] . in spite of the presence of difference in hip anatomy, the similarity in biomechanics of the hip joint with regard to proportion of joint loading between human and bipedal emu was well characterized by iowa biomechanics research group [56, 57] . the contact stress of femoral head was reported with a maximum hip contact force of approximately 5.5 times of body weight in emus, directed axially along its femoral neck, which was moderately larger than that of humans [56] and the contact stress magnitude and the sites of habitual loading on the femoral head was also comparable between emu hip and the human hip [57] . that the joint biomechanics plays a crucial role for no-induced hip joint collapse was also well supported by findings obtained from bipedal and quadrupedal animals. in fact, the on-induction protocol with a combination of lps and mps successfully induced saon in quadruped animals, yet without resulting in subchondral collapse [11] , although inadequate repair was also demonstrated in quadruped saon animals [35] . besides emu study, another report showed that the chicken was also bipedal animal suitable for building up saon animal model [22] , yet their induction protocol did not result in hip joint collapse. in order to mimic on and hip joint collapse, we tested large bipedal emus not because of its similarity in hip joint biomechanics to that of human but mainly that emu hip was sizeable to perform core decompression and testing porous scaffold biomaterials developed for potential clinical applications [58] [59] [60] . the saon induction protocol tested in the present study successfully induced hip joint collapse in large bipedal emus. the occurrence of hip joint collapse in emus was apparent more (7 out of 10 hips from 5 emus) than clinical data, such as 32.7% saon incidence reported by li zr and co-workers in their welldocumented clinical study in sars patients who were treated with a high dose of corticosteroid for life-saving [61] . as saon incidence is often dose-dependent and the purpose of the current preclinical study is to establish a saon bipedal large animal model with high incidence of on and hip joint collapse, we tested much higher dose of mps as compared with clinical recommended dose for sars patients [61] . differ to human situation where only mps was used, we used both lps and mps pulsed treatment, where lps served the purpose to mimic clinical conditions, i.e. disease-related tissue inflammation [11, 27] , in addition to higher mechanical loading imposed to the hip joint in emus where emu hip joint share 65-70% of the body weight as compared with around 60% of body weight in human [56] . in present emu study, we selected two lps injections with 4 days interval that showed safe and effective to induce saon. neut % increased 2 weeks post induction and decreased gradually to baseline level from 2 weeks to 8 weeks post induction which indicated inflammation reaction induced by lps. no typical clinical shwartzman reaction was observed in emus as we did not systemically study the non-skeletal tissue thrombosis or reticuloendothelial blockage for comparison with that in saon patients. it would be of interest to study in details the differences in physical or immune responses between human and birds in saon in future. in our previous study, we established saon in rabbit with 16lps (10 mg/kg) +36mps (20mg/kg) [11] . as emu was estimated with similar body surface area to that of human, we calculated the current experimental dose based on the conventional human-rabbit dose conversion. however, the dose for the initial experiment for emu was 6.67 mg/kg. as we found that emus could tolerate larger dosage from our pilot study, we increased mps dose to 10 mg/kg for the current study at a time interval of 2 days for avoiding potential side-effect of mps injection. specific design for studying dosing effects (amount and frequency of lps and mps treatment) would also be of interests for further studies. the limitation of the current experimental study is that we are not able to delineate if the cause of joint collapse is attributed to saon and/or cartilage and subchondral bone thinning as we did not observe typical destructive repair by uncoupling of osteoclasts and osteoblasts as well as extensive local fibrosis found within necrotic regions reported in both quadrupedal rabbit model [35] or patients [62, 63] . future studies shall be designed to monitor such pathological changes at various time points after oninduction. larger sample size would also be appreciated although our findings did reach statistical significance where we estimated sample size using ps (power and sample size calculations version 3.0) for establishing our current emu saon model based on our previous rabbit model with a saon incidence of 93% [11] . in conclusion, this was the first experimental study to confirm that a combined injection protocol of pulsed lps and mps was able to induce on and deterioration of subchondral bone microarchitecture in bipedal emus, with subsequent femoral head collapse. the establishment of this bipedal emu model 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the proximal femur in corticosteroid-induced osteonecrosis osteoporosis management in patients with rheumatoid arthritis: evidence for improvement hip joint contact force in the emu (dromaius novaehollandiae) during normal level walking contact stress distributions on the femoral head of the emu (dromaius novaehollandiae) comparative study of osteogenic potential of a composite scaffold incorporating either endogenous bone morphogenetic protein-2 or exogenous phytomolecule icaritin: an in vitro efficacy study exogenous phytoestrogenic molecule icaritin incorporated into a porous scaffold for enhancing bone defect repair plga/tcp composite scaffold incorporating bioactive phytomolecule icaritin for enhancement of bone defect repair in rabbits magnetic resonance imaging and histology of repair in femoral head osteonecrosis osteonecrosis of the jaws in patients treated with bisphosphonates -histomorphologic analysis in comparison with infected osteoradionecrosis the authors would like to thank medical staff from shenzhen second people's hospital, including dr. j xia for mri scanning and dr. jy xiong for providing anesthesia materials for animal experiments and dr. xh pan from the first peoples' hospital (shenzhen, china) for providing technical support. key: cord-284016-zb6cv8ik authors: li, wei; liu, yan; mukhtar, muhammad mahmood; gong, rui; pan, ying; rasool, sahibzada t.; gao, yecheng; kang, lei; hao, qian; peng, guiqing; chen, yanni; chen, xin; wu, jianguo; zhu, ying title: activation of interleukin-32 pro-inflammatory pathway in response to influenza a virus infection date: 2008-04-16 journal: plos one doi: 10.1371/journal.pone.0001985 sha: doc_id: 284016 cord_uid: zb6cv8ik background: interleukin (il)-32 is a recently described pro-inflammatory cytokine that has been reported to be induced by bacteria treatment in culture cells. little is known about il-32 production by exogenous pathogens infection in human individuals. methods and findings: in this study, we found that il-32 level was increased by 58.2% in the serum samples from a cohort of 108 patients infected by influenza a virus comparing to that of 115 healthy individuals. another pro-inflammatory factor cyclooxygenase (cox)-2-associated prostaglandin e2 was also upregulated by 2.7-fold. expression of il-32 in influenza a virus infected a549 human lung epithelial cells was blocked by either selective cox-2 inhibitor ns398 or aspirin, a known anti-inflammatory drug, indicating il-32 was induced through cox-2 in the inflammatory cascade. interestingly, we found that cox-2-associate pge(2) production activated by influenza virus infection was significantly suppressed by over-expression of il-32 but increased by il-32-specific sirna, suggesting there was a feedback mechanism between il-32 and cox-2. conclusions: il-32 is induced by influenza a virus infection via cox-2 in the inflammatory cascade. our results provide that il-32 is a potential target for anti-inflammatory medicine screening. influenza a virus (iv) is a highly contagious single-stranded rna virus that infects both the upper and lower respiratory tracts of humans. the host innate immune toll-like receptor 3 (tlr3) was shown previously in cells of myeloid origin to recognize the viral replicative, intermediate double-stranded rna (dsrna). thus, dsrna is critical for the outcome of the infection and appears to be an active component of viral infection that stimulates antiviral activities. it accumulates during the replication of many viruses [1, 2] , including influenza virus. prominent sources of dsrna include viral rnas containing double-stranded secondary structure and dsrna formed during viral replication [2] . furthermore, tlr3 contributes directly to the immune response of respiratory epithelial cells to influenza a virus and dsrna [3, 4] . therefore dsrna treatment was always used to mimic the viral infection in cell lines [5, 6, 7] . in macrophages, dsrna and viral infection stimulate the expression of pro-inflammatory cytokines such as il-1alpha, il-1beta, tumor necrosis factor, and il-6 [1, 8, 9, 10] . cyclooxygenase (cox) is the rate-limiting enzyme in the biosynthetic pathway of prostaglandins and thromboxanes. prostaglandins play an important role in many biological processes. altered prostanoid production is associated with a variety of illnesses, including acute and chronic inflammation, cardiovascular disease and colon cancer [11] . two isoforms of cox were described: cox-1 and cox-2. cox-1 is constitutively expressed in almost all tissues [12] ; cox-2 is the inducible form of the enzyme, which is expressed in response to inflammatory stimuli and growth factors and is involved in mediating pain and inflammatory processes [13, 14] . in our previous studies, we identified two viral proteins, the nucleoprotein and spike protein of sars-cov, which were involved in up-regulating cox-2 [15, 16] . prostaglandin e 2 (pge 2 ) is synthesized from pgh 2 in a variety of cells, which itself is synthesized from arachidonic acid by the enzyme prostaglandin synthetase cox-2. its levels can be measured to access the cox-2 activity as described in reference [5, 6, 17, 18] . interleukin-32 (il-32), previously called natural killer cell transcript 4, has been recognized as a pro-inflammatory cytokine recently. it is mainly expressed in natural killer cells, t cells, epithelial cells and blood monocytes. il-32 can induce the pro-inflammatory cytokines tnf-a and il-1b in murine peritoneal macrophages as well as in phorbol ester-differentiated human thp-1 cells [19] . it plays an important role in innate and adaptive immune responses, synergism between il-32 and other well-characterized players in innate immunity has recently been documented [20] . furthermore, il-32 contributes to the synovitis during rheumatoid arthritis [21] . it is stimulated by mycobacterium tuberculosis [22] and it synergizes with nod1 and nod2 ligands to stimulate il-1b and il-6 release in a caspase-1-dependent manner [23] . recently proteinase 3 has been identified as a specific il-32a binding protein which cleaves the cytokine to enhance its activity [24] . a number of investigations demonstrated that viral infections stimulate cox-2 expression, followed by pge 2 accumulation [6, 15, 16, 25, 26] . however, the role of influenza a virus infection in the regulation of the newly described pro-inflammatory factor il-32 expression is still unclear. furthermore, since cox-2 and il-32 are obligatory mediators of inflammation, the question arises as to whether there is an interaction between the two proteins or they act as independent effectors of host inflammatory response to viral infection. because cox-2 and il-32 gene expression are associated with inflammatory processes, the aim of this study is to investigate the role of influenza a virus infection in the regulation of il-32 expression and to determine the molecular mechanisms responsible. our results showed that influenza a virus infection or poly(ic) treatment activates cox-2 and il-32 expression by a heretofore unrecognized mechanism, in which influenza a virus stimulates il-32 expression through cox-2, and il-32 feedback inhibits cox-2 expression. the study examined 108 consecutive adults with influenza a virus infection (59 male, 49 female, aged 39.2613.5 yr) seropositive for influenza a antigen and 115 healthy adults (62 male, 53 female, aged 37.6611.3 yr) seronegative for influenza a antigen. all adults were seronegative for markers of hepatitis b virus (hbv), hepatitis c virus (hcv), hepatitis delta virus, and hiv. all of the investigated serum samples were obtained with the help from hubei provincial center for disease control and prevention (hubei cdc). informed consent was obtained from each of the patients. the collection of blood samples for research was approved by the institutional review board of the college of life sciences, wuhan university in accordance with guidelines for the protection of human subjects. the influenza virus strain a/chicken/hubei/327/2004 (h5n1) used in this study was provided by china center for type culture collection (cctcc). stock virus was propagated in 10-day-old embryonated chicken eggs for 36 to 48 h at 37uc. the allantoic fluid was then harvested, and aliquots were stored at -80uc before being used. the final concentration of h5n1 virus infection used in this study was 1 multiplicity of infection (moi). pcdna3.1-cox-2 was a gift from dr. kenneth k. wu (university of texas-houston medical school, houston, texas, usa). luciferase reporter vector (pgl3) containing a cox-2 promoter region (2891/+9) was constructed previously [27] . il-32 promoter (2746/+25) was amplified from human genomic dna by pcr, using the following primers: 59-gttacgcgt-cctatttcaatatgactggt-39 (sense), 59-gttaagct-tagggaaagtccagactcggg -39 (antisense); and then inserted into pgl3-basic vector to generate il-32 promoter and luciferase gene fusion plasmid (pil-32-luc). an il-32 construct was created by rt-pcr amplification of the open reading frame from a549 human lung epithelial cells. to create il-32-encoding vector, the il-32 beta gene was amplified using the following primers: 59-catgaattccatgtgcttcccgaagg -39 (sense), 59-ctactcgaggtatcttcattttgaggattg -39 (antisense); in which ecori and xhoi sites were introduced, respectively. the pcr product was cloned into ecori and xhoi sites of pcmv-tag2a (stratagene) to generate plasmid flag2a-il-32, in which il-32 beta protein was tagged by flag. all of the ten genes of influenza a virus h5n1 were obtained by rt-pcr from h5n1 infected a549 cells and constructed into pcmv-flag2a vector to generate iv-gene expressing plasmids ; il-32 sirna plasmid was constructed by ligating the corresponding pairs of oligonucleotide to psilencer tm 2.1-u6 neo (ambion, inc., augstin, tx, usa) based on the sequences described previously [28] . all constructs was confirmed by dna sequencing. monoclonal mouse antibody against human cox-2 was purchased from cayman chemical company (ann arbor, mi, usa). polyclonal goat antibody against human il-32 was purchased from r&d systems, inc. usa. polyclonal goat antibody specific for human b-actin (sc-1616) were purchased from santa cruz biotechnology, inc (santa cruz, ca, usa). n-(2-cyclohexylosy-4-nitrophenyl)-methanesulphonamide (ns398) (promega, madison, wi) was dissolved in dmso and used as indicated concentrations according to reference [29] . aspirin cell culture hek 293t cells were cultured in dmem (gibcobrl, usa), human lung epithelial cells a549 were cultured in f12k media (gibcobrl, usa), respectively. all media were supplemented with 10% fetal calf serum, 100 u/ml penicillin and 100 mg/ml streptomycin sulfate and all cell cultures were maintained at 37uc in a 5% co 2 incubator. cells were plated at density of 4.0610 5 cells per 24-well plate or 6-well plate and grown to confluence reaching about 80% at the time of transfection. the plasmids which express cox-2, il-32, the pgl3-promoter plasmids, prl-tk (promega) were cotransfected into the cells by using lipofectamine 2000 reagent (invitrogen). if necessary, poly(ic), ifn-c, ns398, and aspirin were added into the culture media after transfection. 24 h posttransfection, cells were serum-starved for another 24 h before being harvested. luciferase activities were measured 48 h after transfection according to the manufacturer's instructions (promega) and renilla luciferase activities were determined as internal control for transfection efficiency as previously described [15] . assays were performed in triplicate and expressed as means6s.d. relative to vector or mock control as 100 (rlu) or luciferase activity (luc). total rna, isolated from a549 cells using trizol reagent (invitrogen, carlsbad, ca, usa), was treated with dnase i and reverse-transcribed with mlv reverse transcriptase (promega) and random primers (takara). pcr was performed in 25 ml reactions with the detection primer pairs described as follows: il-32 sense: 59-catgaattccatgtgcttcccgaagg-39; il-32 antisense: 59-ctactcgaggtatcttcattttgaggattg -39. detection primers for cox-2, and b-actin were described previously [15] . b-actin was amplified by pcr for normalization in all reactions. the primers for il-32 were designed for detecting all known isoforms of human il-32. pcr products were analyzed by electrophoresis on 1% agarose gel containing ethidium bromide. protein extracts of cultured cells were prepared by suspending cells in lysis buffer (0.01% edta, 0.1% triton x-100, 10% proteinase inhibitors cocktail), sonication, and centrifugation. concentrations of proteins in supernatant were quantified using protein assay kit (bio-rad). western blot analysis was performed using cox-2 and il-32 antibodies and sample loading was normalized by using b-actin antibody. immunoblots were visualized with the ecl detection system (pierce, rockford, il, usa). because increased pge 2 is the metabolite of cox-2 enzyme catalysis in epithelial cells, cox-2-derived pge 2 levels in the culture medium was assayed by the biotrak prostaglandin e2 enzyme immunoassay system (r & d systems) according to the manufacturer's protocol. elisa for il-32 measurement elisa assays were developed using il-32 antibody and a polypeptide, h-keeltpqkcsepqssk-oh, (gl biochem ltd. shanghai china) was synthesized as an antigen for making an il-32 protein standard curve. elisas were prepared by coating the bottom of a 96-well plate with diluted serum samples or culture media. 50 ml of 2 mg/ml capture antibody was incubated for one hour then washed three times with tbst, followed by hrp-labeled secondary antibody incubation for 15 minutes, and finally washed six times with tbst. elisas were developed with tmb (sigma) substrate and the absorbance at double-wavelength 450 nm/ 630 nm was measured. il-32 concentrations were calculated from the standard curve. all experiments were reproducible and were carried out in triplicate or quadruplicate. each set of experiments was repeated at least three times with similar results, and a representative one is shown. the results are presented at the means6s.d. student's t test for paired samples was used to determine statistical significance. differences were considered statistically significant at a value of p#0.05. the serum levels of pge 2 and il-32 were significantly higher in the cohort of patients with influenza a virus infection investigated in comparison with healthy control individuals (mean6sem for pge 2 , 808.7652.9 vs. 299.5645.4 pg/ml; for il-32, 183.8643.6 vs. 116.2629.3 pg/ml, respectively, p,0.01 mann whitney-u) as described in table 1 . it has been reported that influenza virus can activate the expression of cox-2 in cell culture systems [26] and that dsrna can induce the production of cox-2, followed by pge 2 release [5] . initially, we investigated whether influenza a virus infection and dsrna plays a role, if any, in the regulation of il-32 expression. a549 cells were infected with influenza a virus or treated with poly(ic)+ifn-c, results showed that a549 cells were susceptible to influenza virus infection and apparent cytopathic effect was observed after viral infection. the culture supernatants and cell lysates were harvested at different time points after infection or induction as follows: 0 h, 6 h, 12 h, 24 h, 48 h. pge 2 accumulation (fig. 1a) and il-32 production (fig. 1b) in culture supernatants were measured as described in materials and methods section. cox-2, il-32 mrna (fig. 1c) and protein (fig. 1d ) expression levels in lysated cells were examined by semiquantitative rt-pcr and western blot analyses, respectively. these results suggested that both influenza a virus infection and dsrna treatment could activate the expression of cox-2 and il-32 in a549 cells in a time-dependent manner. to identify the viral components which play important roles in iv-stimulated pro-inflammatory factors cox-2 and il-32 expression, we screened all ten proteins of influenza virus: ha, na, np, ns1, ns2, m1, m2, pa, pb1, pb2 and poly(ic) (to mimic viral replicative intermediate dsrna) by luciferase assays. results showed that poly(ic), poly(ic)+ifn-c, and ns1 are the most important factors in the induction of either cox-2 ( fig. 2a) or il-32 (fig. 2b) promoter activities, which means dsrna and ns1 are the key viral components involved in iv-triggered cox-2 and il-32 expression during viral infection. in this study, we focused on the function of dsrna in inflammatory response to iv infection. to define the role of cox-2 in the regulation of influenza a virus induced pro-inflammatory factor il-32, poly(ic)+ifn-c treatment was used to mimic the influenza a virus infection in following experiments as reported previously [30] . first, the effects of cox-2 on the activation of il-32 promoter were determined. a549 cells were cotransfected with the reporter plasmid pil-32-luc and pcdna3.1, pcdna3.1-cox-2 plus different concentration of ns398 as mentioned in fig. 3a , pcdna3.1 was used as empty vector control. results from luciferase activity assay showed that the level of il-32 promoter activity was increased by cox-2 over-expression and it was blocked by cox-2 inhibitor ns398 in a dose-dependent manner. secondly, to determine the effects of cox-2 on the activation of il-32 mrna level and protein expression, a549 cells were transfected with different amounts of pcdna3.1-cox-2. results from rt-pcr using il-32-specific, or b-actin-specific primers showed that the levels of il-32 mrna were increased as the amount of pcdna3.1-cox-2 increased, but the levels of b-actin mrna remained relatively constant (fig. 3b) . furthermore, il-32 production in culture supernatants was stimulated by cox-2 over-expression in a dose-dependent manner (fig. 3c) . thirdly, results from rt-pcr analyses showed that the level of il-32 mrna activated by poly(ic)+ifn-c was suppressed by cox-2-specific inhibitor ns398 in a dose dependent manner (fig. 3d) . furthermore, il-32 accumulation in culture supernatants stimulated by poly(ic)+ifn-c were suppressed by 80 mm ns398 (fig. 3e) . taken together, these data suggest that cox-2 is an upstream regulatory factor of dsrna-triggered il-32 production. il-32 feedback inhibits dsrna-induced cox-2 expression. the effect of il-32 on the regulation of cox-2 promoter was determined. a549 cells were cotransfected with the reporter plasmid pcox-2-luc and pcmv-flag2a, flag2a-il-32. results from luciferase activity assay showed that the level of cox-2 promoter activity was decreased by il-32 over-expression (fig. 4a) . to determine the effects of il-32 on the regulation of cox-2 mrna expression, pge 2 production, a549 cells were transfected with different amounts of flag2a-il-32 and treated with poly(ic)+ifn-c as the inducer. results from rt-pcr using cox-2-specific, or b-actin-specific primers showed that the levels of cox-2 mrna was decreased as the amount of flag2a-il-32 increased, but the levels of b-actin mrna remained relatively constant (fig. 4b) . furthermore, pge 2 production in culture supernatants was suppressed by il-32 over-expression in a dosedependent manner (fig. 4c) . to confirm the above results, il-32-specific sirna was cotransfected along with reporter plasmid pcox-2-luc into a549 cells and treated with poly(ic)+ifn-c. results from luciferase activity assay showed that the level of cox-2 promoter activity was increased by knocking down il-32 (fig. 4d) . furthermore, pge 2 production in culture supernatants stimulated with poly(ic)+ifn-c was enhanced by knocking down il-32 (fig. 4e) . taken together, these data suggest that il-32 plays a very important role in the inflammatory response following an influenza a/cox-2/il-32 dependent positive regulatory order, while a negative feedback to cox-2 biosynthesis was also first observed. to confirm the cox-2 and il-32 regulatory loop, a549 cells were infected by influenza a virus and treated with or without selective cox-2 inhibitor ns398, or non-selective cox inhibitor aspirin, a widely used anti-inflammatory drug. results showed that iv-triggered pge 2 release (fig. 5a ) and il-32 (fig. 5b ) production in the culture supernatants were inhibited by either ns398 or aspirin, which indicated that il-32 is an important proinflammatory factor downstream of cox-2 during influenza a virus infection. influenza a-induced pge 2 production was increased by knocking down il-32 the effect of il-32 on the regulation of iv infection induced pge 2 production was also determined. we found that pge 2 release in a549 cell culture supernatants stimulated by iv infection was increased (fig. 5c ) and il-32 production decreased (fig. 5d ) by knocking down il-32. taken together, these data demonstrate that a novel inflammatory pathway in response to influenza a virus infection and dsrna treatment was identified as described in fig. 6 . our results demonstrate that significant increase of serum cox-2-derived pge 2 and il-32 levels were observed in influenza a infected patients compared with healthy individuals. viral infection resulted in 2.7-fold increase in pge 2 synthesis and 58.2% increase in il-32 production. both influenza a virus infection and poly(ic)+ifn-c treatment in a549 human lung epithelial cells were able to induce cox-2/ il-32 mrna and protein expression as well as pge 2 and il-32 accumulation in the cell culture supernatants. il-32 was induced by influenza virus infection through cox-2-dependent mechanism. although pro-inflammatory factor cox-2 has been identified as an obligatory mediator in the airway inflammation during influenza virus infection [26] , virtually little is currently known regarding the regulation of a newly identified proinfammatory factor il-32 in influenza a virus infection or the mechanism whereby influenza a virus upregulates il-32 expression. the present study provides considerable new information relevant to these issues. we identified first a cox-2/il-32 regulatory loop in which cox-2 upregulated il-32 expression and il-32 feedback inhibited cox-2 expression in influenza a virus infected a549 lung epithelial cells. at present, a possible ''cross-talk'' between two important pro-inflammatory factors nitric oxide synthase (nos) and cox has been extensively examined in different cell lines, tissues and under different pathophysiological conditions with conflicting results [31] . but the relationship between cox-2 and il-32 has not been evaluated. comparing the iv infection induced il-32 level in cell culture with that in human individuals, a significant difference between them was observed, over 10-fold increase in cell culture versus 58.2% in human individuals. it is likely the pro-inflammatory factor is not able to be increased sharply under physiological conditions due to complex inflammation network in which proinflammatory factors regulate each other. two viral components, the ns1 and viral replicative intermediate dsrna were identified to be involved in iv infection triggered cox-2 and il-32 expression in a549 cells. because tlr3 contributes directly to the inflammatory response of respiratory epithelial cells to both influenza a virus and dsrna, we focused on the function of dsrna in the regulation of cox-2 and il-32 expression in this study. role of viral ns1 protein in the pro-inflammatory process need further investigation. cyclooxygenase metabolizes arachidonic acid to prostaglandins (pgs) and thromboxane [32] . cox-2 has been elucidated to be crucial to the inflammatory response [33] and tumorigenesis [34, 35] , so nonsteroidal anti-inflammatory drugs (nsaids) selective for its inhibition are used clinically to treat inflammatory arthropathies but always cause many side effects such as gastrointestinal (gi) mucosa damage and cardiovascular adverse events [36, 37, 38] . aspirin is one of the classic nsaids, and its anti-inflammatory activity is associated with cyclooxygenase inhibition directly or indirectly [39] . nsaid-associated gi mucosal injury is an important clinical problem and the increased knowledge of physiological roles of cox-2 enzyme in a variety of tissues, including stomach and kidney, together with the withdrawal from the clinic trials of rofecoxib and valdecoxib because of cardiovascular toxicity, have challenged the benefits of selective cox-2 inhibition [38] . our study identified a novel cox-2 downstream pro-inflammatory factor il-32 which could be a potential target for screening anti-inflammatory drugs with less adverse effects. in summary, these studies provide new insights into a novel model as described in fig. 6 in which influenza a virus or poly(ic)+ifn-c triggers pro-inflammatory factors cox-2 and il-32 expression, cox-2-derived pge 2 production, and subsequent host inflammatory responses. in this model, we further demonstrate that cox-2 is located up-stream of il-32 in the positive regulatory pathway. the cross-talk between the two genes is described as follows: cox-2 upregulates il-32 production, and conversely, il-32 attenuates cox-2 activity. double-stranded rna-induced inducible nitric-oxide synthase expression and interleukin-1 release by murine macrophages requires nf-kappab activation when two strands are better than one: the mediators and modulators of the cellular responses to double-stranded rna involvement of toll-like receptor 3 in the immune response of lung epithelial cells to doublestranded rna and influenza a virus diesel exhaust enhances virus-and poly(i:c)-induced toll-like receptor 3 expression and signaling in respiratory epithelial cells role of mapk in the regulation of double-stranded rna-and encephalomyocarditis virus-induced cyclooxygenase-2 expression by macrophages regulation of cyclooxygenase-2 expression by macrophages in response to double-stranded rna and viral infection intratracheal doublestranded rna plus interferon-gamma: a model for analysis of the acute phase response to respiratory viral infections activation of the p38 mitogen-activated protein kinase pathway by epstein-barr virus-encoded latent membrane protein 1 coregulates interleukin-6 and interleukin-8 production potential role of pkr in double-stranded rna-induced macrophage activation transient exposure to hiv-1 tat protein results in cytokine production in macrophages and astrocytes. a hit and run phenomenon cyclooxygenase-2-10 years later arachidonic acid oxygenation by cox-1 and cox-2. mechanisms of catalysis and inhibition up-regulation of p300 binding and p50 acetylation in tumor necrosis factor-alpha-induced cyclooxygenase-2 promoter activation an essential role of the nuclear factor of activated t cells in the regulation of the expression of the cyclooxygenase-2 gene in human t lymphocytes spike protein of sars-cov stimulates cyclooxygenase-2 expression via both calcium-dependent and calcium-independent protein kinase c pathways nucleocapsid protein of sars-cov activates the expression of cyclooxygenase-2 by binding directly to regulatory elements for nuclear factor-kappa b and ccaat/enhancer binding protein contrasting effects of cyclooxygenase-1 (cox-1) and cox-2 deficiency on the host response to influenza a viral infection cox-2 expression and cell cycle progression in human fibroblasts interleukin-32: a cytokine and inducer of tnfalpha il-32: an emerging player in the immune response network against tuberculosis il-32, a proinflammatory cytokine in rheumatoid arthritis mycobacterium tuberculosis induces interleukin-32 production through a caspase-1/il-18/interferon-gamma-dependent mechanism il-32 synergizes with nucleotide oligomerization domain (nod) 1 and nod2 ligands for il-1beta and il-6 production through a caspase 1-dependent mechanism proteinase 3 is an il-32 binding protein the hepatitis b virus x protein promotes tumor cell invasion by inducing membrane-type matrix metalloproteinase-1 and cyclooxygenase-2 expression role of mitogen-activated protein kinases in influenza virus induction of prostaglandin e2 from arachidonic acid in bronchial epithelial cells dynamic regulation of cyclooxygenase-2 promoter activity by isoforms of ccaat/enhancer-binding proteins interactions between il-32 and tumor necrosis factor alpha contribute to the exacerbation of immune-inflammatory diseases mechanism of selective inhibition of the inducible isoform of prostaglandin g/h synthase cutting edge: influenza a virus activates tlr3-dependent inflammatory and rig-idependent antiviral responses in human lung epithelial cells effects of nitric oxide and nitric oxide-derived species on prostaglandin endoperoxide synthase and prostaglandin biosynthesis discovery of prostaglandins, thromboxane and leukotrienes inducible isoforms of cyclooxygenase and nitric-oxide synthase in inflammation transcription of cyclooxygenase-2 is enhanced in transformed mammary epithelial cells prostaglandin h synthase 2 is expressed abnormally in human colon cancer: evidence for a transcriptional effect cardiovascular effects of the selective cyclooxygenase-2 inhibitors nsaid-associated adverse effects and acid control aids to prevent them: a review of current treatment options gastrointestinal safety of novel nonsteroidal antiinflammatory drugs: selective cox-2 inhibitors and beyond aspirin and other cyclooxygenase inhibitors: new therapeutic insights we thank hubei provincial center for disease control and prevention (hubei cdc) for providing serum samples with seronegative for influenza a antigen in this study, and thank dr. kenneth k. wu for kindly providing plasmid pcdna3.1-cox-2. key: cord-263464-fdosch11 authors: nuvey, francis sena; edu-quansah, elijah paa; kuma, george khumalo; eleeza, john; kenu, ernest; sackey, samuel; ameme, donne; abakar, mahamat fayiz; kreppel, katharina; ngandolo, richard bongo; afari, edwin; bonfoh, bassirou title: evaluation of the sentinel surveillance system for influenza-like illnesses in the greater accra region, ghana, 2018 date: 2019-03-14 journal: plos one doi: 10.1371/journal.pone.0213627 sha: doc_id: 263464 cord_uid: fdosch11 background: influenza-like illness (ili) is a medical diagnosis of possible influenza or another respiratory illness with a common set of symptoms. the deaths of four schoolchildren, during a pandemic influenza outbreak in december 2017 in ghana, raised doubts about the ili surveillance system’s performance. we evaluated the ili surveillance system in the greater accra region, ghana, to assess the system’s attributes and its performance on set objectives. methods: cdc guidelines were used to evaluate the data of the ili surveillance system between 2013 and 2017. we interviewed the surveillance personnel on the system’s description and operation. additionally, routinely entered ili data from the national influenza center provided by the six sentinel sites in accra was extracted. we sampled and reviewed 120 ili case-investigation forms from these sites. surveillance activities were examined on system’s performance indicators, each being scored on a scale of 1 to 3 (poorest to best performance). results: all population and age groups were under ili surveillance over the period evaluated. overall, 2948 suspected case-patients, including 392 (13.3%) children under-five were reported, with 219 being positive for influenza virus (predictive value positive = 7.4%). the predominant influenza subtype was h3n2, recorded in 90 (41.1%) of positive case-patients. the system only met two out of its four objectives. none of the six sentinel sites consistently met their annual 260 suspected case-detection quota. samples reached the laboratory on average 48 hours after collection and results were disseminated within 7 days. of 120 case-investigation forms sampled, 91 (76.3%) were completely filled in. conclusions: the ili surveillance system in the greater accra region is only partially meeting its objectives. while it is found to be sensitive, representative and timely, the data quality was sub-optimal. we recommend the determination of thresholds for alert and outbreak detection and ensuring that sentinel sites meet their weekly case-detection targets. introduction influenza-like illnesses (ili), often also called acute respiratory infection or flu-like syndrome, are acute viral infections of the respiratory tract with similar signs and symptoms to influenza. ili is a syndrome and affected persons may become infectious before, during or after the onset of symptoms. the pathogen can be transmitted both directly (by droplets) and indirectly through contact with contaminated fomites. children, the elderly and pregnant women, as well as persons with chronic illnesses or immunosuppression are at the highest risk for morbidity and mortality from ilis [1, 2] . according to the world health organization (who), most influenzas in the global circulation are of zoonotic origin. sub-types implicated in epidemics include h1n1, h5n1, h7n9, h7n7 and h3n2 [3] . influenza has a global annual attack rate of 5-10% in adults and 20-30% in children, causing between 3-5 million cases of severe illness and about 500,000 deaths yearly [4] . pandemics of influenza have had high fatality rates in the past and robust surveillance systems are key to global efforts to prevent similar outbreaks [5] . due to the epidemic-prone nature of influenza pathogens and their high propensity for mutations, the world health organization (who) recommends strict adherence to infection control and prevention measures including increased handwashing during peak flu seasons [2] . in ghana, laboratory-based surveillance for respiratory tract infections (rtis) only tests for influenza in suspected cases. rtis remain a major cause of morbidity and mortality in ghana ranking second among the top 10 diseases seen at outpatient departments (opds) in healthcare facilities across the country, in 2016 [6] . respiratory diseases may occur because of an invasion of a susceptible host by microbes including bacteria and viruses. three main types of influenza viruses exist, namely; influenza a, b, and c but epidemics are often linked to the influenza a strain [7] . influenza surveillance data between 2012 and 2014 in ghana indicated 1041 positive influenza cases out of a total 8601 respiratory samples tested, with 6 different subtypes [influenza a (h3, h1n1, h1, h5) and influenza b (victoria, yamagata)] identified [8] . ghana began influenza surveillance in 2007 to obtain data on strains in circulation. the national influenza center (nic) was formally recognized by the who in 2010 after the 2009 pandemic influenza [9] . the nic is a member of the who global influenza surveillance and response system (gisrs) and is located in the department of virology of the noguchi memorial institute of medical research (nmimr). in collaboration with the ghana health service (ghs) and the ministry of defense (mod), it currently operates sentinel surveillance for influenza in 27 sites across all regions in ghana with support from the u.s. naval medical research unit no. 3 (namru-3), centers for disease control and prevention (cdc) and who [8] . ili surveillance is conducted all year round across the sentinel sites. the ili surveillance system aims to detect early unusual events indicating a change in circulating influenza sub-types, identify and monitor vulnerable groups for influenza, determine influenza thresholds and detect antiviral resistance. in december 2017, an outbreak of influenza in a school in the ashanti region of ghana was followed by the death of four children (case fatality rate = 5.2%) [10] . this raised concerns about the effectiveness of the influenza surveillance system, particularly that in the greater accra region of ghana, which recorded no alerts over the past five years. we evaluated the effectiveness of the ili sentinel surveillance system to determine if its objectives are being met and to assess its attributes and usefulness. the findings of this study are important in order to give useful recommendations to improve the current system. we described the attributes and effectiveness of the ili sentinel surveillance system in the greater accra region (gar) of ghana using a descriptive cross-sectional survey. we extracted and evaluated routinely recorded ili data between january 2013 and december 2017. the gar is one of ten regional demarcations in ghana with a population of about 5 million people [11] . as the most densely populated region in ghana, it is mainly an urban settlement and lies in the southeastern part of the country along the coast of the gulf of guinea. it is the administrative capital of ghana with over 500 public and private health care facilities. six of these facilities in the region conduct ili surveillance including four ghs facilities, one military and one quasi-government facility (see fig 1) . they fall under the three main levels of healthcare delivery in ghana; primary, secondary and tertiary. the manhean health center provides primary health services, while secondary healthcare providers involved in ili surveillance are tema polyclinic, achimota hospital and university of ghana hospital, legon. lastly, the greater accra regional hospital (garh) and 37 military hospital provide tertiary care. data on ili from all six sentinel facilities in gar was extracted and abstracted from the nic database into microsoft office excel format. additionally, we selected three sentinel facilities, based on the different levels of care they provided, and obtained permission for site visits. these sentinel sites; garh, achimota hospital and manhean health center, were visited for at least one week each and their records were collected and reviewed. the researchers interviewed all personnel directly involved in ili surveillance and also partook in surveillance activities while observing practices. the staff members were interviewed using a structured questionnaire and guided interviews on their knowledge on ili surveillance activities. forty case investigation forms were randomly sampled from each of the three facilities, using microsoft excel. the unique serial numbers on case investigation forms for each sentinel site were used to retrieve the randomized forms for examination. in addition, we collected and reviewed ili registers at the sites. the evaluation was conducted using the cdcs guidelines for evaluating public health surveillance systems [12] . nine attributes: simplicity, flexibility, data quality, acceptability, sensitivity, predictive value positive, representativeness, timeliness and stability, usefulness and the utility of the system to achieve its objectives were evaluated. the indicators for each attribute or characteristic measured were scored one point each. based on the evaluator's assessment, an indicator is scored zero (0) if the key finding from evaluation do not support the indicator assessed in relation to the attribute. a score of one (1) is given to each indicator that supports the attribute assessed. the assessment scores were then summed and divided by the total number of indicators used in evaluating each attribute. attributes with relative scores of more than twothirds of the total score, were considered as major strengths of the system and scored 3 overall. those with less than one-third were major weaknesses (overall score = 1). scores between one-third and two-thirds were relative strengths (overall score = 2). scores were on a scale of 1 to 3: poorest to best performance, respectively. the evaluation was done within the framework of integrated disease surveillance and response matrix implemented by the ghana health service and therefore did not have to receive formal review by ethical review committees. the field epidemiology and laboratory training programme and national influenza center in ghana approved the study. permission was sought and obtained from the public health directorate of ghana health service and authorities in the sentinel facilities before commencement of the evaluation. all respondents provided informed, written consent and were assured of confidentiality. the surveillance system was found to be utilizing the syndromic approach by screening suspected cases and thereafter conducting further laboratory confirmatory tests on collected nasopharyngeal or oropharyngeal specimen. a reverse transcriptase-polymerase chain reaction (rrt-pcr) is used to confirm real influenza cases from among those suspected using the influenza case definition and determine the influenza virus sub-type. we found that data on patients meeting the ili case definition (s2 table) from the sentinel sites are collected together with nasopharyngeal or oropharyngeal specimen. specimen are stored in sample bottles with virus transport media (vtm) in a cold chain system and submitted to nmimr for laboratory confirmation. each site is required to suspect five cases weekly using the case definition. thus, the expected annual total for each site is 260 suspected cases. firstly, clinicians at the opds suspect cases and trained personnel (i.e. nurses, laboratory scientists and surveillance officers) collect the specimen for storage in designated refrigerators at the disease control units. case detection is, in most cases, directly done by ili surveillance team members at the opds. socio-demographic, epidemiologic and clinical data are collected on each case using case investigation forms. the nic supplies the sentinel sites with the tools including case investigation forms, vtm, and specimen bottles, for the system's operation. they contact the sentinel sites via phone calls and visit the sentinel sites in the gar at least twice every week to pick up specimen. the nic reports results from the polymerase chain reaction laboratory test to each sentinel site, the national surveillance department (nsd), and who via e-mail. data is entered into the flu-net system, a who (gisrs) global database specifically designed for influenza surveillance, weekly. sentinel sites receive printed laboratory results from the nic averagely every 7 days. at the facility level, health information units (hiu) enter the data into the district health information management system 2 (dhims 2). ili data entry into dhims 2 started in 2017. the ghs, nic and other stakeholders periodically hold review meetings to discuss influenza activities and publish the data generated by the system in reports and journals. the information and data flow in the ili surveillance system is further illustrated in fig 2. of the 2948 suspected cases tested, 219 (7.4%) cases were tested positive for influenza viruses. s1 table) . utility of the ili sentinel surveillance system in attaining set objectives. the ili surveillance system has four objectives: early detect events that show change in severity or patterns of influenza infection or emergence of new strains, determine influenza thresholds, identify and monitor at risk groups and detect antiviral resistance in circulating strains. the system met two out of its four objectives over the period evaluated. it was able to detect and characterize influenza viruses in circulation, as from influenza a and b lineages, and established the groups of persons most at risk of influenza infection. it however did not set thresholds, which is the minimum number of suspected cases above which the system is alerted, and was not performing antiviral resistance testing. surveillance system attributes. usefulness: data generated by the system informed the choice of vaccines used in controlling influenza outbreaks in ghana. in addition, who and cdc use the system's information to monitor influenza activity globally, through sharing of confirmed influenza samples. tables 1 and 2 show the key findings on the indicators and scores for all the qualitative and quantitative attributes evaluated respectively. sensitivity: the ili system in gar has been able to confirm influenza cases in each of the years evaluated (s1 table) . evidence of aggregate data from ghana can also be found in the who flunet system, available free at http://apps.who.int/flumart/default?reportno=1. predictive value positive (pvp): the predictive power of the ili case definition is tested using rrt-pcr. overall pvp for the period was 7.4% (range 4.7%-14.8%). see s3 table for yearly pvps over the evaluated period. simplicity: surveillance personnel interviewed identified case definition to be simple. only opds partake in ili surveillance and follow-up of confirmed cases done within 5 days. however, specialized training is required for specimen collection and laboratory confirmation, data collected on each suspected case is comprehensive and laboratory testing is not done at sentinel sites. flexibility: modification of the ili system in 2016, enabled detection of severe acute respiratory infections (sari) with no difficulty. furthermore, addition of other common respiratory symptoms to case definition at the sites did not disrupt the system. data quality: key surveillance information was completely provided in 76% (91/120) of case investigation forms sampled and data extracts were comparable at sentinel facilities and the nic. in spite of this, only two sites: garh and achimota hospital, had 2017 influenza data table 1 entered in the dhims-2 platform and laboratory results were not entered in ili registers at the three sites. acceptability: on average, five out of the six sentinel sites detect influenza cases each year (83%) and about 80% of case investigation forms sampled were completely filled out by ili personnel. all sentinel staff interviewed reported satisfaction with feedback from nic and test results from collected specimen are on average tested within 2 days. most of the sites (5/6) however, do not meet the case detection quota for each year with some recording as low as three suspected cases per year only (see fig 3) . representativeness: age and sex distribution of total cases detected reflect the general distribution of ghana's population. the median age of cases was 30 years (range: 3 weeks to 90 years). females constituted 60% (1775/2948) of cases. timeliness: it takes on average 10 days between symptom onset and detection at facilities. the majority of specimens are tested within 48 hours after collection and results are disseminated within 7 days. this conforms with the who set standard timelines for influenza surveillance. stability: data flow in the system conforms to set standards; testing is routinely done within 48 hours after case detection and results are released within 7 days to the sites on average. the system is sustainable with donors mainly funding its operation. there was not a time influenza surveillance in the greater accra region, ghana during the period evaluated when no site recorded cases, but all sites detected cases only for 60% of the period. influenza causes considerable morbidity and about 500,000 deaths per year globally. the disease is highly infectious with high pandemic potential. therefore, worldwide surveillance systems to record influenza-like illnesses (ili) in real time and detect possible influenza outbreaks are essential to prevent and control epidemics. ghana's ili surveillance system since its inception in 2007, aims to early detect changes in circulating influenza, identify vulnerable groups to influenza infection, determine influenza thresholds and detect antiviral resistance to influenza viruses. the late detection of a pandemic influenza outbreak in ghana raised doubts about the ili surveillance system's performance. our study provides evidence, that the ili sentinel surveillance system in the greater accra region (gar), ghana, is only partially meeting its objectives because it did not have thresholds for alerting the health system and does not perform antiviral resistance testing. it is sensitive and timely in detecting influenza cases but of low predictive value positive (pvp). it is representative of the population under surveillance and flexible to modifications. the system is fairly stable and acceptable to key stakeholders; the quality of data is relatively high. predominant influenza subtype in circulation is influenza a (h3n2) virus. the sentinel sites consistently failed to meet their case detection quotas annually over the period evaluated. despite these apparent weaknesses in the ili system, the good performance of the laboratory component is commendable and key to detection of novel influenza viruses for prompt response. even though the who's standards for influenza surveillance alluded to the possibility of resource limitation hindering achievement of all influenza surveillance system objectives, it advocates for influenza surveillance systems capable of collecting the minimum amount of data needed for decision making [5] . for the past two decades of influenza surveillance in ghana, the system proved its utility by its ability to detect and classify circulating strains as well as providing key information for public health action. similar findings were made in the south african [13] and madagascar [14] influenza surveillance systems. even though the pvp is low, it generally conforms to other syndromic surveillance systems with broad case definitions but specific for respiratory diseases in this case, aimed at maximizing influenza case detection [15] . nevertheless, the unmet objectives, lack of thresholds and antiviral resistance testing, of the system require attention. the lack of antiviral resistance testing to detect the emergence of treatment resistant strains and the absence of thresholds preventing the issuing of alerts, are a major drawback. this situation is not specific to the gar alone but is found in all sites in ghana. in addition, the mainly conservative management of clinical signs and symptoms, of confirmed influenza cases in the health system, without the use of recommended antiviral agents, may partly explain the absence of antiviral susceptibility testing. these shortfalls may, however, be caused by a lack of capacity or resources (antiviral drugs and laboratory reagents) at the sentinel facilities to determine thresholds for influenza alerts and test for antiviral resistance, as was observed in evaluations done in other settings [16, 17] . threshold establishment for disease surveillance is paramount to be able to alert the health system early when outbreaks occur for prompt public health action [1] . a study to test the moving epidemic method in determining thresholds for ili and sari surveillance systems in europe, was able to detect epidemic periods with few or no false alarms in different countries [18] . other methods employed in cambodia [19] and australia [20] had similar findings. the nic must take the lead using the who manual [21] , to choose an epidemic threshold determination method and train focal persons in the facilities to use it, to ensure this key information is available. even though implementing an integrated (human and animal) surveillance could be costeffective and improve case detection and response [22] , discussions with ili surveillance personnel at sentinel sites revealed the absence of a link between influenza surveillance in humans and animals. human infection with zoonotic influenza strains is possible and may cause mild to severe form of the disease. there have been recorded episodes of pandemics in humans over a century due to cross-species transmission of zoonotic respiratory viruses including influenza viruses, notably spanish flu (1918), severe acute respiratory syndrome (2003) and swine flu (2009), resulting in high morbidity and mortality globally [23, 24] . thus, the importance of a one-health approach in the surveillance and response with control of pandemics in an increasingly globalized world is evident. research findings in africa, europe and the americas, have shown incremental gains derived when interventions are integrated between human and animal health systems [25] . the nic and veterinary service department in ghana should collaborate to formalize protocols for engaging each other to integrate influenza surveillance systems in humans and animals, using a one health approach, as this can provide additional key information on possible cross-species transmission in the country and enhance savings. the usage of one laboratory to test for both human and animal pathogens in canada was shown to cost about 30% less than the combined original operational costs of testing individually in both laboratories [26] . in ghana, the timeliness of the system in case detection, laboratory confirmation and result dissemination is commendable. this strength may be as result of the weekly visits nic makes to sentinel sites to collect specimen, supply virus transport media in specimen bottles and transfer results. the nic should further take advantage of these visits and collaborative meetings to ensure each site meets weekly case detection quotas. focal persons at the various sites must take the lead. in spite of the usefulness and fair performance of ili surveillance in gar on indicators evaluated, it is only partially meeting its set objectives. it is sensitive in detecting circulating influenza types, representative of the population under surveillance and timely. however, sentinel sites do not consistently meet annual case detection quotas. there is the need to address shortfalls in the system's objectives as well as improving case detection at sentinel facilities. this would ensure that the successes chocked are not undermined, thereby preventing increasing morbidity and mortality related to influenza infections. failure of the system to address these shortfalls would also affect ghana's contribution to the who global influenza surveillance and response system. considering the zoonotic character of most influenza viruses, it is important that a one health approach is adopted with influenza surveillance in ghana. we propose strict adherence to case detection targets by individual sentinel sites and determination of alert thresholds for the system to allow for a more effective monitoring of influenza activity in the region for prompt public health actions. supporting information s1 table. ili case definitions used for screening and enrolment by the ili sentinel surveillance technical guidelines for integrated disease surveillance and response in the african region world health organization. community case management during an influenza outbreak: participant's handbook influenza at the human-animal interface; sumary and assessment influenza (seasonal) fact sheet [internet]. who. world health organization world health organization. global epidemiological surveillance standards for influenza virological surveillance of influenza-like illness among children in ghana ghana health service/ministry of health. interim report: surveillance response to influenza type a (h1n1) outbreak in kumasi academy, asokore -mampong municipality population projections by districts updated guidelines for evaluating public health surveillance systems: recommendations from the guidelines working group evaluation of two influenza surveillance systems in south africa evaluation of the influenza sentinel surveillance system in madagascar overview of syndromic surveillance: what is syndromic surveillance? morb mortal wkly rep strategy to enhance influenza surveillance worldwide a summary of influenza surveillance systems in australia influenza surveillance in europe: establishing epidemic thresholds by the moving epidemic method establishing seasonal and alert influenza thresholds in cambodia using the who method: implications for effective utilization of influenza surveillance in the tropics and subtropics exploring a proposed who method to determine thresholds for seasonal influenza surveillance world health organization. who interim global epidemiological surveillance standards for influenza creating a framework towards integrated health syndromic surveillance and response in africa global burden of influenza: contributions from resource limited and low-income settings sars, the first pandemic of the 21st century one health: the theory and practice of integrated health approaches. croydon: cab international the world bank. people, pathogens and our planet: towards a one health approach for controlling zoonotic diseases we acknowledge support received from the national influenza center and noguchi memorial institute of medical research, and afrique one alliance. in addition, we would like to thank the physicians, nurses, surveillance officers, laboratory scientists and health information officers who contribute to the influenza-like illnesses sentinel surveillance system in the greater accra region of ghana, as well as the staff and cohort 11 residents of the ghana field epidemiology and laboratory program. key: cord-304993-t4rua95e authors: jung, kwonil; wang, qiuhong; kim, yunjeong; scheuer, kelly; zhang, zhenwen; shen, quan; chang, kyeong-ok; saif, linda j. title: the effects of simvastatin or interferon-α on infectivity of human norovirus using a gnotobiotic pig model for the study of antivirals date: 2012-07-23 journal: plos one doi: 10.1371/journal.pone.0041619 sha: doc_id: 304993 cord_uid: t4rua95e the lack of an animal model for human norovirus (hunov) has hindered the development of therapeutic strategies. this study demonstrated that a commonly used cholesterol-lowering statin medication, simvastatin, which increases hunov replication in an in vitro replicon system, also enhances hunov infectivity in the gnotobiotic (gn) pig model. in contrast, oral treatment with interferon (ifn)-α reduces hunov infectivity. young piglets, all with a or h1 histo-blood group antigens on enterocytes, were treated orally with 8 mg/kg/day of simvastatin; 5 days later, the pigs were inoculated orally with a gii.4 hunov (hs194/2009/us strain) and then treated with simvastatin for 5 more days. simvastatin induced significantly earlier onset and longer duration of hunov fecal shedding in treated pigs, frequently with higher fecal viral titers. simvastatin impaired poly (i:c)-induced ifn-α expression in macrophages or dendritic cells, possibly due to lowered toll-like receptor (tlr) 3 expression; however, the mechanisms were not related to interferon regulatory factor 3 or nuclear factor kappa b signaling pathway. thus, the enhanced, earlier infectivity of hunov in simvastatin-treated pigs coincided with the inhibitory effect of simvastatin on innate immunity. in contrast to the increased hunov shedding that simvastatin induced, viral shedding during the treatment period was reduced or curtailed in the hunov-inoculated pigs pre-treated/treated with human ifn-α. our findings are the first to indicate that ifn-α has potential as antiviral therapy against hunov. based on these intriguing and novel findings using the gn pig model, we confirmed that hunov infectivity is altered by treatment with simvastatin or ifn-α. collectively, these findings indicate that gn pigs are a useful model to test immunomodulators or efficacy of antivirals against hunov. human norovirus (hunov), a single-stranded, positive sense rna virus, is a member of the caliciviridae family. this virus is the leading pathogen causing food-or water-borne gastroenteritis [1] . hunovs are estimated to cause 54 million cases of illness annually in the us, and they account for approximately 58% of the foodborne illnesses caused by 31 different bacteria, parasites and viruses [2] . clinical and pathological features of hunov infections include: i) a short incubation period (10-51 hr) prior to onset of clinical signs such as vomiting and diarrhea, although asymptomatic infections occur frequently [3] ; ii) acute and self-limiting infection, but often with prolonged fecal virus shedding [1] ; and iii) lymphocytic, atrophic enteritis [4] . hunovs are classified into 3 distinct genogroups (gi, gii, and giv), which are further subdivided into 26 or more different hunov genotypes [5, 6, 7] . in the last 15 years, the gii. 4 hunovs have been responsible for the majority of hunov outbreaks, possibly due to several viral and host factors that have been reviewed recently [8] : i) the broader binding of host receptor to gii.4 hunovs, ii) incomplete herd immunity against gii.4 or its variants, and iii) higher mutation rate of their polymerases. in the viral capsid protein, the p2 domain is the most protruding and variable region. it is believed to recognize host cellular receptors and to contribute to establishment of viral infection. histo-blood group antigens (hbgas) are considered as cellular receptors or coreceptors that determine host susceptibility to certain hunovs. individuals of blood type a or o (h) of secretors were more susceptible to gi.1/norwalk/1968/us virus infection than individuals who were non-secretors or secretors of blood type b [9, 10] . however, an emerging hunov, gii.12 strain did not bind to hbgas when tested in vitro [11] . these observations imply a host factor affecting infection by certain genogroups or genotypes of hunov. hunov infection is generally self-limiting, but it can induce severe illness and fatal disease in immunocompromised patients, specifically, organ recipients receiving long-term chemotherapy or hematopoietic stem cell transplantation [12, 13] . the young and elderly are also at risk, due to their high exposure rates to hunov infection in community settings (child care centers, nursing homes, hospitals, etc). notably, the common use of statin medications, which lowers serum cholesterol levels and prevents cardiovascular disease, is a significant risk factor for exacerbating hunov disease severity and increasing the related fatality rates [14] . these conditions require effective therapeutic strategies against hunov infection. however, the lack of small animal model for hunovs has hindered development and testing of hunov antivirals or vaccines [15] . gnotobiotic (gn) pigs are susceptible to oral infection by a gii.4 hunov strain (hs66/2001/us) [16, 17] and an emerging gii. 12 hunov strain (hs206/2010/us) [11] . most hunov-infected gn pigs shed virus in feces. the incubation period (12-48 hrs) for gii.4 hunov in gn pigs was similar to that (19-41 hrs) observed in humans experimentally infected with the gii.2 snow mountain virus [16, 18] . the longer fecal hunov shedding in gn pigs infected with the gii.12 hs206 strain was similar to that observed in human cases [11] . also, like humans possessing secretor phenotype, gn pigs express a or h1 hbga on enterocytes. different hbga phenotypes (a or h) were shown to influence susceptibility of gn pigs to hunov infection [19] . in a norwalk virus (gi.1) replicon-harboring cell system, the viral rna and protein levels were increased after treatment with cholesterol lowering drugs, such as simvastatin, that act as 3hydroxy-3-methylglutaryl-coenzyme a (hmg-coa) reductase inhibitors [20] . after statin treatment, reduced cellular cholesterol levels are followed by high expression of low-density lipoprotein receptor (ldlr) gene that compensates for the lower cellular cholesterol levels. these results indicated that cholesterol pathways may be associated with enhanced replication of hunov in vitro, although the related mechanisms are unclear. statins are also involved in a variety of immune responses and are immunosuppressive. they inhibit major histocompatability complex (mhc) class ii expression on antigen presenting cells in mice, promote the generation of foxp3+ t regulatory cells in mice, and impair lipopolysaccharide-induced toll-like receptor (tlr) 4-mediated inflammatory responses in human embryonic kidney-293 cells (hek-293) [21, 22, 23] . the aim of our study was to determine if statins also enhance hunov infectivity in vivo in our gn pig model of hunov infection [16] . the gn pigs were treated with high-doses of simvastatin and then inoculated with gii.4 hunov (hs194/2009/us strain). we further investigated if the enhanced, earlier infectivity of hunov seen in simvastatin-treated pigs correlated with inhibitory effects of simvastatin on innate immunity. finally, we investigated the effect of an innate immunity mediator, ifn-a on hunov infectivity in the gn pig model. simvastatin treatment lowered the serum cholesterol level in gn pigs and resulted in increased early ldlr gene expression in a porcine enterocyte cell line (ipec-j2) serum cholesterol levels were monitored to measure the pharmacological activity of simvastatin in gn pigs. simvastatintreated pigs had significantly decreased serum cholesterol levels (81.567.5 to 90.467.4 mg/dl) at 8 to 14 days after treatment began, which were 1.6 to 2.4 times lower than the levels in the untreated pigs (147.0611.0 to 202.3625.5 mg/dl) during the same period (fig. 1a) . we further investigated if a reverse relationship existed between the cholesterol level and the ldlr gene expression in a porcine jejunal epithelial cell line, ipec-j2, because intestinal epithelial cells are a cell type critical for initiation of hunov infection [16] . cells were treated with multiple concentrations (0 mm, 1 mm, 20 mm, and 80 mm) of simvastatin, and ldlr gene expression was analyzed by quantitative real-time rt-pcr (qrt-pcr). at 12 hours after treatment with 80 mm simvastatin, ldlr gene expression levels in ipec-j2 cells were significantly increased compared to 0 to 20 mm treated groups (fig. 1b) . at 12 and 24 hours after treatment with 1 to 80 mm simvastatin, the ldlr gene expression levels were significantly increased by 2.4 to 2.7 times compared to untreated groups (fig. 1b) . significantly earlier onset of hunov shedding was observed in simvastatin-treated pigs, which began shedding at mean postinoculation day (pid) 1.960.2, compared to mean pid 4.860.7 in untreated pigs (p,0.01) ( fig. 2a) . significantly longer duration of hunov shedding was also observed in simvastatin-treated pigs, which shed for a mean of 15.161.1 days, compared to a mean of 8.861.7 days in untreated pigs (p,0.01) (fig. 2b ). the mean daily fecal hunov titers were compared statistically between simvastatin-treated and untreated pigs in each trial, due to high variability in mean viral titers among the 3 independent trials. significantly higher viral rna titers were detected in simvastatintreated pigs than untreated pigs in trial 1 (5.4460.16 log 10 genomic equivalents (ge)/ml vs. 4.7960.05 log 10 ge/ml] (p,0.01) and trial 2 (5.2760.10 log 10 ge/ml vs. 4.9760.07 log 10 ge/ml) (p,0.05) (fig. 2c) . however, no such significant difference was observed in trial 3. immunohistochemistry (ihc) results showed hunov antigens in the cytoplasm or on the surface of enterocytes, but not in lamina propria cells (fig. 3) , supporting that fecal virus shedding is a result of hunov replication and infection in the intestine. the ihcpositive cells were in the small intestine, but not the large intestine, in which epithelial cells also expressed similar levels of hbga as in the small intestine (fig. 4a ). however, after hunov inoculation of simvastatin-treated or untreated gn pigs, no pronounced histological changes were evident in the small and large intestines of gn pigs or as a side-effect after oral treatment with high-doses of simvastatin in controls. under our experimental conditions no hunov-infected pigs showed diarrhea, whereas mild diarrhea was noticeably observed in all of the statin-treated pigs up to 5 days after statin treatment. both hbga a+ and h+ type pigs were equally susceptible to gii.4 hunov infection all gn pigs used in this study were positive for either hbga a or h1 (fig. 4a-c) . the hbga antigens were distributed on the surface or in the cytoplasm of epithelial cells lining the intestine, the salivary glands, and pulmonary (bronchial) and renal tubular epithelial cells (fig. 4a-c) . under similar ihc conditions, amounts of a antigens in a + pigs were greater in the intestine and other positive tissues, as compared to those of h1 antigens in h + pigs (fig. 4a) . however, no significant differences in the onset and duration of fecal virus shedding were found between the a + and h + pigs ( fig. 2a and b) . despite the higher expression levels of a antigens, significantly lower viral rna titers (4.9460.05 log 10 ge/ml) were detected in the feces of a + pigs than that (5.2860.09 log 10 ge/ml) in the h + pigs (p,0.05), but no difference was observed between the simvastatin-treated, a + and h + pigs (fig. 2c ). simvastatin impaired tlr3-mediated induction of ifn-a in macrophages or dendritic cells, possibly due to lowered expression of tlr3 after treatment our in vivo data showing enhanced early infectivity of hunov suggested potential subversion of innate immunity related to simvasatin treatment. thus, we further investigated if simvastatin inhibits the capacity of macrophages or dendritic cells (dcs) to produce ifn-a after stimulation with poly (i:c), which triggers tlr3-mediated induction of ifn-a. in general, ifn-a was not detected in culture supernatants of macrophages or dcs treated with either simvastatin only or mock. in porcine pulmonary alveolar macrophages (pams), ifn-a levels (50.0613.4 u/ml) released in simvastatin + poly (i:c)-treated pams were significantly lower than those (173.3648.1 u/ml) of poly (i:c) onlytreated pams at 24 hours after poly (i:c) treatment (p,0.05) (fig. 5a) . peripheral blood mononuclear cell (pbmc)-derived macrophages responded less to treatment with 50 mg/ml of poly (i:c), as compared to pams treated with 25 mg/ml of poly (i:c) ( table 1) , possibly due to lower ratios of harvested adherent macrophages from pbmc. similar to the observations for pams, significantly lower ifn-a levels (68.9612.9 u/ml) were observed in simvastatin-treated, enriched intestinal dcs at 12 hours after poly (i:c) treatment (p,0.05), as compared with those (93.3615.3 u/ml) after poly (i:c) treatment alone ( table 1 ). the mean percentages (6 sem) of tlr3+ cells (3.2260.85%, n = 6) from simvastatin + poly (i:c)-treated intestinal macrophages at 24 hours after poly (i:c) treatment were significantly lower (p,0.01), as compared to that (6.5660.47%, n = 6) from poly (i:c) alone. a representative flow cytometric profile is illustrated in fig. 5b and c. gene expression levels of irf3 and nfkb, mainly involved in poly (i:c)-induced, tlr3-mediated ifn production, were analyzed to investigate possible mechanisms underlying the subversion of innate immunity induced by simvastatin in pams and intestinal dcs. although simvastatin alone did not induce ifn-a production in pams (fig. 5a ), increased expression of irf3 and nfkb genes was found in simvastatin-treated pams ( fig. 6a and b). cotreatment with simvastatin and poly (i:c) synergistically resulted in increased gene expression of irf3 and nfkb. at 24 hours after poly (i:c) treatment, however, gene expression levels of irf3 and nfkb were reduced in poly (i:c) only-treated pams, but not in simvastatin + poly (i:c)-treated pams, as compared to no treatments. swine ifn-a levels were decreased in the poly (i:c) and simvastatin-treated pams or dcs, and hunov infection was enhanced in vivo. therefore, we investigated whether fecal hunov shedding, i.e. hunov replication in the gut of infected gn pigs, was altered by treatment with ifn-a. oral treatment of gn pigs with natural human ifn-a (nhifn-a) [300 international unit (iu)/ kg/day] reduced or curtailed virus shedding in treated animals during the treatment period (pid 1 to 4), compared to untreated animals ( fig. 7a-c) . the treatment significantly delayed the onset of virus shedding by 1.7 day in treated pigs, which began shedding at mean pid 3.060.8, compared to mean pid 1.360.2 in untreated pigs (p,0.05) (fig. 7a ). during the nhifn-a treatment period (pid 1 to 4), a significantly shorter duration of hunov shedding was observed in the nhifn-a-treated pigs, which shed for a mean of 0.860.5 days, compared to a mean of 2.060.3 days in untreated pigs (p,0.05) (fig. 7b ). during the treatment period, a significantly lower qrt-pcr-positive rate of the fecal samples tested (p,0.01) was also observed in the nhifn-a-treated pigs (3/ 16; 18.8%) than in the untreated pigs (12/16; 75%), with significantly lower viral rna titers in the feces (4.8860.11 log 10 ge/ml in the treated pigs vs. 5.0660.14 log 10 ge/ml in the untreated pigs) (p,0.01) (fig. 7c) . however, at pid 5 to 18 after nhifn-a-treatment was discontinued, significantly increased viral shedding titers were noted in the nhifn-a-treated pigs (5.1860.08 log 10 ge/ml), compared to the untreated pigs (4.9260.05 log 10 ge/ml) (p,0.05) (fig. 7c) . at pids 5-18, there were no significant differences in the duration of fecal virus shedding and the qrt-pcr-positive rate of the fecal samples tested between the nhifn-a-treated pigs and untreated pigs (fig. 7b) . further repeated studies in additional pigs are needed to investigate how termination of nhifn treatment results in higher viral shedding titers post ifn-a treatment. no negative control pigs shed detectable viral rna in the feces throughout the experiment. we demonstrated that use of simvastatin enhances hunov infectivity in the gn pig model. thus, its use may also support growth of hunov in cell culture. we also verified that the increased infectivity of hunov may be associated with the inhibitory effect of statins on innate immunity (ifn-a). this observation might explain the exacerbated hunov disease and the related higher mortality described in statin-treated humans [14] . because of the immunosuppressive effects, use of statins have been proposed for immunomodulatory therapy against severe influenza a virus infections in which large amounts of innate (ifna) cytokines are involved [24] . in addition, we showed that oral treatment with nhifn-a can curtail early hunov fecal shedding in the gn pig model. because no hunov vaccines are available, use of effective antivirals such as ifn-a should be tested to control multiple genogroups and genotypes of hunovs, including the gii.4 variants that have emerged each year [25] . our findings that hunov infectivity in gn pigs can be enhanced by simvastatin treatment or reduced by oral treatment with nhifn-a, suggest that gn pigs are a useful model to test efficacy of antivirals against hunov. as a surrogate model for hunovs, murine nov (mnv) infection of mice was useful for investigating the roles of specific immunologic factors such as type i or ii ifns in host defense [15] . however, the different pathogenesis of mnv infection with its systemic spread raises concerns about extrapolation of these findings to the hunov restricted gastrointestinal infection. a chimpanzee model was recently established to evaluate the efficacy , and the daily viral titers (mean) as monitored by qrt-pcr are shown (c). monitoring continued until 3 to 4 weeks after infection and terminated when pcr results were negative (,4.7 log 10 ge/ml) for 3 consecutive days. data from 3 independent animal trials were combined, and the pcr test was performed in duplicate or triplicate. additional analysis was also conducted according to the hbga a or h1 phenotype of each animal. each bar represents the mean 6 sem. *p,0.05; **p,0.01 for simvastatin + hunov vs hunov alone or for a+ pigs vs h+ pigs by the unpaired two-tailed mann-whitney test. animal numbers (n) are indicated at the bottom of each graph. the dotted line indicates the detection limit (4.7 log 10 ge/ml) of the qrt-pcr. doi:10.1371/journal.pone.0041619.g002 of vlp-derived vaccines against infection with gi or gii hunovs [26] . chimpanzees developed serum antibody responses after intravenous injection of gi.1/norwalk virus or intramuscular injection with norwalk vlps and were protected from gi.1/ norwalk virus challenge (but not gii hunov infection). the chimpanzee model, however, is compromised by the lack of availability of chimpanzees, and the finding that oral infection of chimpanzees with hunovs failed to induce gastroenteric disease comparable to human cases [26] , as well as by the intravenous route required for viral challenge. in our study, although hunov infection of gn pigs induced mild enteric disease, gn pigs were susceptible to oral infection by the gii.4 hs194 strain, which reaffirms the results of our previous studies using a closely related gii.4 hunov (hs66 strain) [16, 17] and the emerging gii.12 hunov (hs206 strain) [11] . fecal hunov shedding patterns in gn pigs, with peak viral titers during an early stage of infection are also typical for other acute enteric viral infections in pigs, such as rotavirus and porcine epidemic diarrhea virus [27, 28] . however, how hunov shedding in infected gn pigs is maintained for 2 or 3 weeks after viral inoculation is unclear and requires further investigation. cholesterol biosynthesis and metabolism are mainly mediated by hepatic enzymes, such as hmg-coa reductase [29] . statins act as competitive inhibitors of hmg-coa reductase and reduce production of cholesterol in the liver. as in humans, our study showed that statins lower serum cholesterol levels in gn pigs, possibly due to similar cholesterol pathways between swine and humans as reported previously [30] . when hepatic cholesterol stores are depleted, the liver increases the expression of ldlr which leads to uptake of ldl from plasma to compensate for the lower cellular cholesterol levels. several rna viruses manipulate cholesterol pathways in diverse ways for more efficient viral infection and replication as exemplified for novs in comparion to hepatitis c virus (hcv) and coronavirus [20, 31, 32, 33] . for example, low cellular cholesterol levels (or high cellular ldlr expression) following statin treatment contributed to increased gi.1/norwalk virus replication, as verified in an in vitro replicon system [20] . our study also showed that increased ldlr expression levels in ipec-j2 cells (a porcine jejunal cell line) treated with simvastatin might similarly contribute to enhanced hunov replication in the gastrointestinal tract. our other ongoing in vitro studies also found that hunov rna titers in supernatants or lysate samples of ipec-j2 cells treated with simvastatin were slightly increased in trials using gii.12 hs206 strain compared to those of controls (without simvastatin), but did not differ significantly in cell cultures using the gii.4 hs194 strain. the latter was previously reported [34] . the data indicate a positive but inconsistent effect of simvastatin on hunov replication in vitro, possibly depending on the hunov strains or different environmental conditions for hunov replication in vitro versus in vivo. a paper describing more detailed and comprehensive in vitro cell culture findings is in preparation by takanashi et al. (unpublished data, 2012) . in addition to the cholesterol lowering effects, the inhibitory effects of statins on innate immunity also might influence the immunological and cellular microenvironment for more efficient hunov replication. in our study, simvastatin impaired tlr3-mediated innate immunity and inhibited production of ifn-a induced by poly (i:c) in pams or intestinal dcs. these observations are similar to the results of an in vitro study using hek-293 cells, showing the inhibitory effect of simvastatin on tlr4-mediated immune responses, such as tumor necrosis factor (tnf)-a and interleukin-6 [23] . nevertheless, it is notable that observations for gn pigs and humans infected with hunovs [14] are contrary to the effect of statins that reduced replication of hcv in replicon-harboring cells [31] and a positive correlation between cellular cholesterol levels and entry of coronaviruses [32] and of gv/mnv into host cells [33] . further confirmatory data are needed to define the role of the cholesterol pathway in the pathogenesis of hunov. although simvastatin treatment inhibited ifn-a production, we found that gene expression of irf3 and nfkb in simvastatintreated pams was increased rather than being decreased. because activation of nfkb kinase is a shared property among tlrs, including tlr3 [35] , simvastatin or its cellular byproducts could trigger other tlrs that stimulate nfkb gene expression. notably, at 24 hours after poly (i:c) treatment, gene expression levels of irf3 and nfkb in poly (i:c) only-treated cells were reduced remarkably, as compared with other treatments or those at the earlier time-point. this observation could be explained by a cellular negative feedback effect to mediate production of ifna. it is also notable that a similar result did not occur in simvastatin + poly (i:c)-treated cells, possibly due to reduced ifn-a levels after simvastatin treatment. at least four families of transcription factors are activated by dsrna and relate to tlr3: nfkb, irf-3, c-jun, and activating transcription factor 2 [35] . besides lowered tlr3 expression by macrophages after simvastatin treatment, which was thought to be mainly responsible for reduction of ifn-a production in statin + poly (i:c)-treated cells, other tlr3-or ifn-mediated signaling pathways might be involved in the impaired innate immunity by simvastatin. type i ifns are essential for early viral clearance and development of adaptive immune responses. as a crucial mediator of the innate antiviral immune responses, ifn-a has been an effective antiviral treatment for viral infections, such as hcv and influenza [36, 37] . the signal transducer and activator of transcription-1 (stat-1)-dependent ifn stimulation was essential for controlling murine nov (mnv) infection. although mnv did not cause disease in immunocompetent mice, oral mnv infection caused fatal systemic disease in mice lacking either type i and type ii interferon receptors or stat-1, which is critical for ifn signaling [15, 38] . several studies have suggested that repeated oral treatment with nhifn-a may be effective in treating acute viral gastroenteritis related to coronavirus and rotavirus in domestic pigs [39, 40] . similarly, our study showed that oral administration of nhifn-a inhibits infection by or replication of hunov, as fecal hunov shedding is curtailed in the gn pig model. the nhifn-a is formulated to be stable at low ph of the stomach. degradation of nhifn-a by a variety of intestinal enzymes appears to be slow enough to allow nhifn-a to reach some ifn-a receptors of cells in mucosal lymphoid tissues of the oral cavity and intestine [41] . the table 1 . therapeutic effectiveness of nhifn-a might be related to its immunostimulatory effects. orally delivered nhifn-a promoted systemic innate immunity by increasing expression levels of innate immunity-related genes, such as ifn-stimulated genes (isgs) and tnf-a, and phagocytic capacity of phagocytes [42, 43] . further studies with larger numbers of animals are needed to determine the most effective dose and regimen of nhifn-a to prevent or treat hunov infections, and to elucidate the immunological and molecular mechanisms related to the antiviral effects of ifn-a. based on the effectiveness of a combination of nhifn-a pre-and post-treatment, the nhifn-a treatments need to be tested therapeutically in future studies using the gn pig model or in five or six day-old piglets were treated orally with 300 iu of nhifn-a once a day from pid -1 to pid 4. on day 2 after nhifn-a treatment, they were inoculated orally with gii.4 hs194 hunov, and subsequently treated with nhifn-a (300 iu) for 5 more days. after nhifn-a treatment or hunov inoculation, clinical signs and fecal virus shedding were monitored daily until shedding terminated. data from 2 independent animal trials were combined, and the pcr test was performed in duplicate or triplicate. duration of virus shedding in nhifn-a-treated and untreated pigs were analyzed based on nhifn-a treatment period, i.e. during treatment at pids 1-4; post-treatment at pids 5-18; and overall at pids 1-18. each bar represents the mean 6 sem. *p,0.05; **p,0.01 for nhifn-a + hunov vs hunov alone by the unpaired two-tailed mann-whitney test. animal numbers (n) are indicated at the bottom of each graph. the dotted line indicates the detection limit (4.7 log 10 ge/ml) of the qrt-pcr. doi:10.1371/journal.pone.0041619.g007 clinical trials. the mechanisms by which fecal virus shedding recurred and the increased viral rna titers in nhifn-treated gn pigs compared to untreated pigs after nhifn treatment was discontinued need to be investigated. however, we hypothesize that during the period of nhifn treatment, ifn signaling pathways might be regulated by a negative feedback in some ifn producing cells in the intestine. thus, on the termination of treatment such a distinct condition of the ifn system might hinder induction or production of ifn-a in most ifn containing cells or its antiviral activity against hunov. in conclusion, simvastatin treatment increased hunov infectivity in the gn pig model, possibly due to its inhibitory effect on innate immunity as well as its cholesterol lowering effect as reported previously [20] . these findings could partially explain the exacerbated hunov disease in statin-treated humans [14] . testing of nhifn-a as an antiviral for hunov using the gn pig model also revealed that ifn-a has potential as a hunov antiviral therapy. development of hunov antivirals is important because hunovs cause large-scale epidemics with significant mortality in immunocompromised, elderly and young patients. thus, the gn pig model for hunov will allow testing of new treatment modalities for hunov infection and new knowledge on the antiviral mechanisms of innate and adaptive immunity. the ipec-j2 cells were kindly provided by dr. bruce d. schultz (kansas state university) [44] . cells were maintained in dulbecco's modified eagle's medium/nutrient ham's mixture f-12 (invitrogen, carlsbad, ca) with 5% fetal bovine serum (fbs; hyclone laboratories, inc., logan, ut), 1% insulin-transferrinsodium selenite (roche, mannheim, germany), and epidermal growth factor (5 ng/ml) (invitrogen). the gii.4/hs194/2009/ us (hs194) strain (genbank accession number: gu325839) used as viral inoculum in this study was isolated from stool samples of a young child with watery diarrhea [11] . stool samples were screened for other enteric viruses, including gi hunov, rotavirus groups a, b and c, sapovirus, astrovirus, and adenovirus by reverse transcription (rt)-pcr or pcr, respectively, as described previously [11] . near-term pigs were derived by hysterectomy and maintained in sterile isolator units [16] . by ihc using monoclonal antibodies to human a (immucor, norcross, ca) and h1 (covance research products, inc., dedham, ma), we determined the a/h phenotype on fresh bucal cells or formalin-fixed, paraffin-embedded intestinal and salivary glandular tissues of gn pigs. the institutional animal care and use committee (iacuc) of the ohio state university approved all protocols related to the animal experiments in this study. all animals used in this study were also handled in accordance with the guidelines of the iacuc of the ohio state university. piglets were randomly assigned to one of four groups: simvastatin + hunov (n = 11), hunov alone (n = 10), mock (n = 4), and simvastatin alone (n = 4). to lower serum cholesterol levels prior to virus infection, five or seven day-old piglets were first treated orally with 8 mg/day/pig (approximately 1 kg of body weight) of simvastatin (zocor; merck and co, inc., whitehouse station, nj). on day 6 after treatment, they were infected orally with 2.4610 9 or 3610 10 ge of the gii.4 hunov hs194, and then subsequently treated with the half doses of pre-treatment for 5 more days. after simvastatin treatment or hunov inoculation, we monitored clinical signs daily. at an acute (pid 3 to 5) stage of hunov infection, 1 to 4 pigs per group were euthanized for histopahological examination. when virus fecal shedding terminated as determined by qrt-pcr, i.e. at a later (pid 20 to 29) stage of infection, 2 to 8 pigs per group were euthanized. the nhifn-a was kindly provided by dr. joseph cummins (amarillo biosciences, inc., amarillo, tx). piglets were randomly assigned to one of three groups and constituted 2 independent trials: nhifn-a-treated, hunov-infected (n = 4), nhifn-a-untreated, hunov-infected (n = 4), and negative control (n = 4). five or six day-old piglets (approximately 1 kg of body weight) were treated orally with 300 iu of nhifn-a once a day from pid -1 to pid 4. on day 2 after nhifn-a treatment, they were infected orally with 1.3610 10 ge of the gii.4 hs194 hunov, and subsequently treated with nhifn-a (300 iu) for 5 more days. after nhifn-a treatment or hunov inoculation, clinical signs and fecal virus shedding were monitored daily until shedding terminated. total serum cholesterol levels were assessed in simvastatin treatment trials by using an amplex red cholesterol assay kit (invitrogen), as described previously [20] . total cholesterol was extracted in chloroform-methanol-double-distilled water [(4:2:1) (vol/vol/vol)]. the chloroform phase was separated, mixed with a 1:100 volume of polyoxyethylene 9-lauryl ether (sigma-aldrich, st. louis, mo), dried, and resuspended in the assay reaction buffer in the kit. each treatment was duplicated in additional sixwell plates, and cell lysates were prepared for the measurement of protein contents by using a bca protein assay kit (bio-rad, hercules, ca). the concentrations of total cholesterol were normalized with the protein contents. rectal swabs were collected daily from each animal throughout the experiment. the gii hunov fecal shedding titers were determined by the taqman real-time rt-pcr (cog2f/2r primer set and ring2 probe), as described previously [11, 45] . the detection limit of this pcr assay was 10 ge per reaction determined based on the standard curve generated using serially diluted plasmid dna carrying hs194-specific cog2f/2r amplicons. the limit of viral rna detection in the qrt-pcr assay was 4.7 log 10 ge/ml. the recombinant baculovirus carrying the capsid protein (vp1) gene (orf2) of hs194 strain (genbank accession number: gu325839) was generated by using the baculodirect tm baculovirus expression system (invitrogen) according to the manufacturer's instructions. briefly, a gateway entry clone containing the orf2 of hs194 (pentr tm /sd/d-topo-hs194) was generated and used with the baculodirect tm linear dna to perform a lr recombination reaction to generate recombinant baculovirus dna carrying the orf2 of hs194. the insect sf9 cells were transfected by the recombination reaction products and the cells containing the recombinant baculovirus dna were positively selected by ganciclovir. the expression of hs194 capsid proteins in the sf9 cells and culture supernatants were examined by immunoblot using the guinea pig antiserum against hu/nov/ gii.4/hs66/2001/us strain [46] . the recombinant baculoviruses (rbac-hs194) were propagated to prepare virus stocks with high titers for routine vlp expression. the production and purification of hs194 vlps were performed as described previously [46] . the protein concentration was quantified using the bradford method and the vlps were negatively stained with 3% phosphotungstic acid (ph 7.0) and examined by transmission electron microscopy as described previously [11] . hyperimmune serum against hunov gii.4 hs194 vlps was generated using guinea pigs according to an approved iacuc protocol, as previously described [16] . small (duodenum, proximal, middle and distal jejunum, and ileum) and large (cecum and colon) intestinal tissues and other major organs (lung, liver, heart, kidney, spleen, and lymph node) were examined grossly and histologically and tested by ihc for nov antigen detection. tissues from age-matched mock controls were tested for histological comparisons and as a negative control for ihc. the ihc was performed on formalin-fixed, paraffinembedded tissues or fresh frozen tissues using the guinea pig hyperimmune antisera to vlps of the gii.4 hunov hs194, as described previously [47] . the pams (2,4610 6 cells/ml) were collected from the lungs of 5 ten-day-old, uninfected gn pigs using aseptic techniques, as previously described [48, 49] . cells (4,8610 5 cells/well) were seeded onto 12-well cell plates, and the wells were randomly assigned to 4 treatment groups: no treatment, simvastatin alone, simvastatin + poly (i:c), and poly (i:c) alone. pams were first treated with 1 mm simvastatin (sigma), and 24 hours later, treated with either 1 mm simvastatin or poly (i:c) (25 mg/ml) (sigma), or treated with both. the cell culture supernatants were harvested at 4 and 24 hours after poly (i:c) treatment to measure ifn-a levels released from pams. intestinal mononuclear cells or pbmc were isolated from ileum or blood of gn pigs, as previously described [27] . gut or pbmc monocyte-derived macrophage or dc-enriched cell preparations were obtained by plating gut mononuclear cells or pbmc at 2,4610 6 cells/ml in rpmi-1640 supplemented with 8% fbs, 1% gentamicin, 0.1% ampicilin, 20 mm hepes, 2 mm lglutamine, and 1 mm sodium pyruvate for 2-3 days before harvesting adherent cells. non-adherent cells were used for dcenriched cells. macrophage-or dc-enriched cells (4,8610 5 cells/well) were seeded onto 12-well cell plates, and the wells were randomly assigned to 4 treatment groups: no treatment, simvastatin alone, simvastatin + poly (i:c), and poly (i:c) alone. cells were treated with simvastatin in the same manner as for pams. mardin-darby bovine kidney (mdbk) cells were grown in mem with 5% fbs and 1% antibiotic-antimycotic. the cell supernatant samples, serially diluted 1:2 in mem, were added to the confluent cell monolayers seeded in 96-well plates, as previously described [50] . at 24 hrs after incubation at 37uc, media was removed and 100 ml of vesicular stomatitis virus (3610 5 plaque forming unit/ml) was added. at 48 hrs after incubation, 10 ml of alamar blue was added to each well and incubated for 3 hrs at 37uc. fluorescence was measured at 530-560 nm. antiviral ifn-a levels (u/ml) were expressed as the reciprocal of the sample dilution which resulted in a 50% reduction in cytopathic effects. expression levels of porcine ldlr, nfkb, and irf3 mrna were measured in ipec-j2 cells or pams by taqman real-time pcr, as described previously [51, 52, 53] , with slight modifications. the mrna expressions were normalized to the expression levels of porcine b-actin [54] . primers and probes of ldlr and b-actin and probes of nfkb and irf3 were designed by geneious primer design software, as follows (59-39): ldlr f, cgccctccaaaacggtggct; ldlr r, acttcggc-gagcgtgggttg; and ldlr probe, fam-ac-ctgtgtctgccagctccaca-3iabkfq. b-actin f, cccacgccatcctgcgtctg; b-actin r, gtagccccgctccgtcagga; and probe, fam-ggccgggacctgaccgacta-3iabkfq. nfkb probe, fam-accaggctggcagctctcctcaaagcagca-3iabkfq. irf3 probe, fam-ccggtctgccctgaaccg-gaa-3iabkfq. all values are expressed as the means 6 standard error of the means (sem). cholesterol level data among the treatment groups were analyzed by the kruskal-wallis test (nonparametric) using the statistical analysis systems. all gene expression and ifna level data and numbers of tlr3+ cells were analyzed by oneway analysis of variance (anova). virus titers that were undetectable (,4.7 log 10 ge/ml) during the shedding period were assigned as a value of 4.7 log 10 ge/ml for statistical analysis. the mean onset and duration of virus shedding and viral titers between simvastatin + hunov and hunov alone groups, between a + and h + pigs, and between nhifn-a-treated and untreated pigs, were compared by unpaired two-tailed mann-whitney tests. specifically, duration of virus shedding and viral 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transmissible gastroenteritis with natural human interferon alpha: a field study treatment of rotavirus infection in neonate and weanling pigs using natural human interferon alpha systemic effects of interferons after oral administration in animals and humans evidence for the immunostimulatory effects of low-dose orally delivered human ifn-alpha in cattle immunostimulatory effects of natural human interferon-alpha (huifn-alpha) on carps cyprinus carpio l epithelial barrier modulation by a channel forming peptide broadly reactive and highly sensitive assay for norwalk-like viruses based on real-time quantitative reverse transcription-pcr binding of human gii.4 norovirus virus-like particles to carbohydrates of romaine lettuce leaf cell wall materials porcine reproductive and respiratory syndrome virus modifies innate immunity and alters disease outcome in pigs subsequently infected with porcine respiratory coronavirus: implications for respiratory viral co-infections nitric oxide is elicited and inhibits viral replication in pigs infected with porcine respiratory coronavirus but not porcine reproductive and respiratory syndrome virus role of toll-like receptors in activation of porcine alveolar macrophages by porcine reproductive and respiratory syndrome virus application of an objective biological assay of human interferons to clinical specimens and a survey of a normal population classical swine fever virus npro interacts with interferon regulatory factor 3 and induces its proteasomal degradation activation of the transcription factor, nuclear factor kappa-b, during the estrous cycle and early pregnancy in the pig analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method selection of reference genes for gene expression studies in pig tissues using sybr green qpcr we thank dr. joseph cummins who kindly provided the natural human ifn-a and gave helpful suggestions for the ifn-a treatment of our experimental pigs. we also thank dr. j. hanson and r. mccomick for assistance with animal care and t. aubrecht, c. siegismund, a. vlasova, s. takanashi, and k. chatta for assistance with immunological assays. key: cord-276898-ia80cy8j authors: yamanaka, atsushi; iwakiri, akira; yoshikawa, tomoki; sakai, kouji; singh, harpal; himeji, daisuke; kikuchi, ikuo; ueda, akira; yamamoto, seigo; miura, miho; shioyama, yoko; kawano, kimiko; nagaishi, tokiko; saito, minako; minomo, masumi; iwamoto, naoyasu; hidaka, yoshio; sohma, hirotoshi; kobayashi, takeshi; kanai, yuta; kawagishi, takehiro; nagata, noriyo; fukushi, shuetsu; mizutani, tetsuya; tani, hideki; taniguchi, satoshi; fukuma, aiko; shimojima, masayuki; kurane, ichiro; kageyama, tsutomu; odagiri, takato; saijo, masayuki; morikawa, shigeru title: imported case of acute respiratory tract infection associated with a member of species nelson bay orthoreovirus date: 2014-03-25 journal: plos one doi: 10.1371/journal.pone.0092777 sha: doc_id: 276898 cord_uid: ia80cy8j a japanese man suffered from acute respiratory tract infection after returning to japan from bali, indonesia in 2007. miyazaki-bali/2007, a strain of the species of nelson bay orthoreovirus, was isolated from the patient's throat swab using vero cells, in which syncytium formation was observed. this is the sixth report describing a patient with respiratory tract infection caused by an orthoreovirus classified to the species of nelson bay orthoreovirus. given the possibility that all of the patients were infected in malaysia and indonesia, prospective surveillance on orthoreovirus infections should be carried out in southeast asia. furthermore, contact surveillance study suggests that the risk of human-to-human infection of the species of nelson bay orthoreovirus would seem to be low. genus orthoreovirus is one of 15 current genera in family reoviridae comprises nonenveloped virus with segmented double stranded (ds) rna genomes that are taxonomically classified into 15 genera, each containing 10 genome segments. orthoreoviruses have been isolated from a variety of mammalian, avian, reptilian, and piscine hosts. orthoreoviruses are divided into the fusogenic and nonfusogenic subgroups, based on the ability of the virus to induce cell-to-cell fusion and syncytium formation [1] . the majority of orthoreoviruses are fusogenic, including avian, baboon, reptilian and nelson bay orthoreoviruses; while the mammalian and piscine orthoreoviruses (mrv) are nonfusogenic. in humans, mrv infections are common, but are usually asymptomatic or mildly symptomatic [1, 2] . in the last decade, however, several orthoreoviruses, which seem to have originated in fruit bats including the grey-headed flying fox (pteropus poliocephalus) and small flying fox (pteropus hypomelanus), have been identified as causative agents for respiratory tract infection (rti) in malaysia and indonesia [3] [4] [5] [6] . another orthoreovirus, xi river virus, was isolated from fruit bats in the people's republic of china, although the virus was not demonstrated to be an agent for rti in humans [7] . these orthoreoviruses including pulau virus, which was isolated from pteropus hypomelanus urine samples from tioman island, an island locates off the eastern shore of malaysia [8] and xi river virus [7] , were genetically and antigenetically related to nelson bay orthoreovirus, which was isolated from pteropus policephalus heart blood samples from nelson bay, in the hunter region of new south wales, australia [9] . here, we report an imported case of an rti associated with an orthoreovirus, which is classified to the species of nelson bay orthoreovirus, in a patient who returned to japan from bali, indonesia in november 2007. the isolate was named ''miyazaki-bali/2007''. the patient of the present study was a 38-year-old japanese man, who visited bali, indonesia in november 2007. after returning to japan, he presented to a local hospital with high fever, joint pain, sore throat, and cough. a throat swab specimen collected from the patient was mixed with viral transport medium, and transported to the department of microbiology, miyazaki prefectural institute for public health and environment. the throat swab sample was centrifuged for 10 min at 150006g at 4uc, and the supernatant fractions were inoculated into the monolayers of the rd-18s, hep-2, vero, and caco-2 cell lines. cells were cultured in eagle's minimum essential medium supplemented with heat-inactivated 2% fetal bovine serum (mem-2fbs). cell morphologies were observed daily to detect the appearance of cytopathic effect (cpe). virological examination was also performed to assess the presence of other respiratory viruses, including highly pathogenic avian influenza virus a h5n1 and severe acute respiratory syndrome coronavirus. furthermore, presence of viral rna in the supernatant fractions of cell culture medium was evaluated by using the method, a rapid determination system of viral genome detection (rdv), as reported previously [10] . the rdv method, which was developed by our group, is a rapid method for the direct determination of viral rna sequences without using the cdna cloning step. the rdv method uses whole-genome amplification and direct nucleotide sequencing techniques, in which multiple steps; 1) effective destruction of cellular rna and dna for semipurification of viral particles, 2) effective elimination of dna fragments by using a prefiltration column system and elution of small amounts of rna, 3) effective synthesis of first-and secondstrand cdna library, 4) construction and amplification of a cdna library, 5) construction of a second cdna library, and 6) direct sequencing using optimized primers. the method is effective in the identification of unknown emerging viruses [10] [11] [12] [13] [14] [15] . orthoreovirus miyazaki-bali/2007 isolated in this study was used. additionally, avian orthoreovirus [16] , mammalian orthoreovirus 1 [17] , and mammalian orthoreovirus 3 [18] were used for evaluation of migration pattern of miyazaki-bali/2007-dsrna segments in comparison with those of these viruses. the culture supernatant of vero cells, in which cpe appeared, was used for electron microscopic observation. samples were fixed with 4% glutaraldehyde, negatively stained with 2% phosphotungstic acid, and then observed using a jem-1400 transmission electron microscope (jeol ltd, tokyo, japan). viral dsrnas were purified as previously described [19] . rna genomes were purified from each virus solution by using trizol (life technologies). each of the purified rna was mixed with licl at its concentration of 2 m, followed by incubation of the mixture at 4uc overnight. the mixture was then centrifuged at 14,000 rpm for 30 min for separation of ssrna from dsrna. the supernatant fraction containing dsrna was treated with ethanol precipitation. precipitated dsrna was solubilized with water and separated by 10% sds-polyacrylamide gel electrophoresis. genome segments were visualized by ethidium bromide staining. infectious dose of miyazaki-bali/2007 was determined by a plaque assay using vero cell monolayers. briefly, vero cell monolayers were inoculated with each of serially diluted virus solutions and incubated for 1 hr for adsorption. the cell monolayers were washed with phosphate buffered saline solution and the cells were cultured with mem-2fbs supplemented with 0.8% agarose for 3 days. plaque was visualized by staining the cells with neutral red solution. virus solution containing approximately 100 plaque forming units was mixed with heat-inactivated and diluted serum samples for 1 h at 37uc. the mixtures were then inoculated onto a monolayer of vero e6 cells. cells were cultured for 4 days. the neutralization titer was defined as the reciprocal of the highest dilution at which no cpe was observed. when specific cpe appeared in the cells inoculated with serum samples, which were diluted 5 times with pbs, the serum was identified as neutralization activity negative. cdna was synthesized by reverse transcription using random hexamer from dsrna purified by using qiamp viral rna kit (qiagen, germany). partial genes of s1-, s2-, s3-, and s4segments, which codes sigma c protein, major inner capsid protein, nonstructural replication (ns) protein, and major outer capsid protein, respectively, were amplified by rt-pcr. primer sets were designed based on the nucleotide sequences of the conserved regions of the melaka, hk23629/07, kampar, and sikamat orthoreoviruses. the nucleotide sequence of the genomes amplified was determined using the direct sequencing method. the orthoreovirus nucleotide sequence data obtained in this study or from genbank (table 1) were phylogenetically analyzed using mega5.2 (pmid: 21546353). the nucleotide sequences of the open reading frames (orfs) for sigma c protein (s1-segment), major inner capsid protein (s2-segment), sigma ns protein (s3segment), or major outer capsid protein (s4-segment) extracted from genbank (table 1) were aligned using muscle and program built into mega5.2. evolutionary distances between amino acid sequences were estimated using the poisson model and phylogenetic trees were constructed using the neighbor-joining method. the robustness of the trees was tested using 1000 bootstrap replication. the regions used for the phylogenetic analyses are shown in table 1 . the nucleotide sequences of the s1-, s2-, s3-, and s4-segments of miyazaki-bali/2007 are deposited in genbank with the accession numbers of ab521793, ab521794, ab521795, and ab521796, respectively. serum samples were collected from 46 people who may have had contact with the patient (including family members, the patient's hospital caregivers, and workers in local health stations). samples were subjected to neutralization antibody assay as described above. information on symptoms after possible contact with the patient was collected. serum samples collected from the patient diagnosed as having orthoreovirus infection were obtained only for etiological analyses under the informed consent with the written document from him. furthermore, travel history, clinical and laboratory data of the patient with the orthoreovirus infection described in this study were also disclosed under the written informed consent obtained through the easy-to-understand explanation including the human rights for him. human rights of the patient were protected as much as possible. serum samples collected from the contact persons with the index patient were also collected only for detection of the neutralization antibody to the orthoreovirus isolated, miyazaki-bali/2007, under the written informed consent. all the protocols and procedures were approved by the research and ethical committees for use of human subjects of the national institute of infectious diseases, tokyo, japan (no. 452). a 38-year-old japanese man visited bali, indonesia, with his wife on november 8, 2007 . eleven days later, on november 19, he showed symptoms of high fever, joint pain, sore throat, and cough. he returned to miyazaki, japan on november 21, and presented to a local hospital. the patient was hospitalized on the same day. at the time of presentation, the patient's vital signs were stable, he had no skin rashes or purpura and his throat was neither reddish nor swollen. a physical abdominal examination revealed no abnormal findings. a small lymph node in his right neck was palpable with mild tenderness. chest x-ray examination showed no signs of lower respiratory tract infection. laboratory findings were as follows. proteinuria and hematuria were negative. peripheral total blood cell counts revealed that the patient's white blood cell, red blood cell, hemoglobin, and platelet counts were within normal ranges. serum chemistry analyses showed normal values of total bilirubin, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and amylase. no abnormalities were observed in serum electrolyte concentrations. blood urea nitrogen and creatinine levels were normal. at 2.66 mg/dl, the patient's c-reactive protein was elevated in comparison to the normal range of 0.00-0.30 mg/dl. on november 29, eight days after admission, all of the patient's symptoms resolved and he was discharged without any sequelae. cytopathic effect, characterized by syncytium formation, appeared in vero e6 cells one day after inoculation with the throat swab specimens (fig. 1a) . virological examination showed the patient to be seronegative for parainfluenza, mumps, and herpes viruses. next, a rapid determination system of viral genome detection, which was developed in the authors' laboratory, was carried out with supernatants of cpe-positive cultures [10] , revealing the presence of a virus genome sequence that was similar to that of the species of nelson bay orthoreovirus such as hk23629/07 and melaka orthoreovirus [3, 6] . rt-pcr was performed against the isolated virus with primer sets designed specifically for the amplification of the s4 segments of hk23629/07. genetic analysis indicated that the amplified dna was 98% homologous to hk23629/07 (data not shown). comparison of genome segments of the cpe agent by gel electrophoresis revealed an electropherotype that was almost identical to the previously reported electropherotypes of melaka and pulau orthoreoviruseses (fig. 1c) [3] . electron microscopic examination of the supernatant of the isolated virus revealed icosahedral particles resembling those of the family reoviridae, genus orthoreovirus (fig. 1b) [3] . the particles possessed a mean diameter of approximately 75 nm. neutralization antibody titers rose significantly against the isolated virus; from 10 in the acute phase to 320 in the convalescent phase. the cpe agent was identified as a species of the nelson bay orthoreovirus based on the above results and the virus was named ''miyazaki-bali/2007.'' homology of nucleotide and amino acid sequences among the species of nelson bay orthoreovirus is summarized ( table 2 ). s1segment of miyazaki-bali/2007 had the highest homology with being 97% to those of hk46886/09 and hk50842/10, which also seemed to have originated in indonesia. interestingly, s1segment of miyazaki-bali/2007 showed 94% homology to that of kampar virus originated in malaysia, while it did 48-59% homology to those of the other orthoreoviruses in the genus of nelson bay orthoreovirus. s2-segment of miyazaki-bali/2007 had the highest homology with being approximately 92-94% in nucleotide sequences to those of hk23629/07, hk46886/09, and hk50842/10, which also seem to have originated in indonesia [6, 20] , while it showed 83-90% homology to those of kampar, melaka, pulau, sikamat, and nelson bay viruses. when the homology was evaluated using the nucleotide sequences of s3segment, similar results were demonstrated ( table 2 ). the nucleotide sequence was conserved at the rate of more than 80% among the orthoreoviruses in the genus of nelson bay orthoreovirus. the homology of miyazaki-bali/2007 s4-segment was approximately 94% to those of hk23629/03, hk46886/09, and hk50842/10 and less than those to other orthoreoviruses. the homology pattern was almost the same as that observed in s2segment. miyazaki-bali/2007, together with the other species of nelson bay orthoreovirus, formed a cluster that was independent to avian, mammalian, and baboon reoviruses (fig. 2) . miyazaki-bali/2007 was found to be most closely related to hk23629/07, hk46886/ 09, and hk50842/10 based on the phylogenetic analyses with s2and s4-segments. [6, 20] . however, miyazaki-bali/2007 did not form a subcluster with hk23629/07, when analyzed with s1segment sequences ( fig. 2a) . furthermore, miyazaki-bali/2007 formed a subcluster with hk23629/07, but not with hk46886/ 09 and hk50842/10, when analyzed with s3-segment sequences (fig. 2c) , although all the 4 viruses including miyazaki-balihk23629/07, hk46886/09, and hk50842/10 originated from indonesia. serum samples collected from all the persons, who came into contact with the index patient, showed negative activity in the neutralization antibody test, in spite of the fact that one of the patient's family members and three of his caregivers in the hospital had experienced fever and sore throat within one week of the contact. these data suggest that human-to-human transmission of the virus did not easily occur. the first orthoreovirus of bat origin, nelson bay orthoreovirus, was isolated in 1968 from the heart blood of a grey-headed flying fox (pteropus poliocephalus), a species of fruit bat, in new south wales, australia, followed by the isolation of pulau virus, which was also isolated from a fruit bat, specifically a small flying fox (pteropus hypomelanus). pulau virus was found to be serologically and genetically related to nelson bay orthoreovirus [8, 9, 21] . at the time of isolation, it was not yet known whether these bat orthoreoviruses were capable of infecting or causing diseases in humans and/or other animals. an orthoreovirus, melaka virus, was first reported as a causative agent for rti in malaysia [3] . this was followed by several reports describing rti in humans being caused by the viruses with similar characteristics [4] [5] [6] 20] . the characteristics of these sporadic outbreaks are summarized in table 3 . table 2 . homology in nucleotide and amino acid sequences of sigma c protein (s1-segment), major inner capsid protein (s2-segment), sigma ns protein (s3-segment), and major outer capsid protein (s4-segment) between the orthoviruses of nelson bay group as well as avian, baboon and mammalian orthoreoviruses. sigma c (s1-segment) [ this is the sixth report describing a patient infected with an orthoreovirus classified to the species of nelson bay orthoreovirus. three patients were infected in malaysia, and the other 4 patients including the patient in the present study were infected in indonesia and imported to hong kong [6, 20] or miyazaki. it is noteworthy that both the present and 2 of the hong kong cases were infected with their respective viruses in bali. the phylogenetic analyses indicated a close association among these isolates, when analyzed with s2-and s4-segments ( fig. 2b and 2d) . the association pattern based on the s2-and s4-segments' sequences was different from those based on the s1-and s3-segments' sequences (fig. 2) . the cause behind this difference might be the reassortment event among these orthoreoviruses in the endemic regions. further study is needed to confirm this assumption. although the travel information on the patient infected with hk23629/07 was not described in detail [6] , it was described that the patient was infected in april, 2007 [20] , while the patient in the present study was infected in november, 2007. there seemed to be no direct epidemiological link between the two patients. an epidemiological investigation to clarify the epidemiology of the infections associated with the species of nelson bay orthoreovirus among residents in southeastern asia would be valuable. we found no evidence of human-to-human transmission of the virus among the family members and hospital caregivers, who were in contact with the patient. given that only a small number of possible human-to-human transmission episodes were reported in the sporadic outbreaks of melaka, kampar, and sikamat viruses [3] [4] [5] , currently the risk of human-to-human infection of these orthoreoviruses would seem to be low. the antigenicity of miyazaki-bali/2007 virus was not compared with other orthoreoviruses in the genus of nelson bay orthoreoviruses. while neutralization activity was examined during the convalescent phase in the miyazaki-bali/2007 case of the present study, further study is needed to clarify the neutralization activity of the serum sample collected from the patient in the convalescent phase against other species of nelson bay orthoreoviruses. the orthoreoviruses isolated from patients with rti were classified to the species of nelson bay orthoreovirus are more closely associated in the phylogenetic analyses with pulau virus than nelson bay virus (fig. 2) . thus far, there is no direct evidence of bat origin in the reported cases. however, epidemiological studies that were performed in each of the reports, with the exception of the hong kong case, indicated the potential for bat exposure before the onset of the disease [3] [4] [5] . that each of the melaka, kampar, and sikamar virus patients resided in areas, in which fruit bats were present, supports this possibility. in the hong kong case, where the patient was also infected in bali, no detailed information was recorded on the environment, in which the patient resided [6] . one patient, who might be infected in bali, indonesia, in 2009, visited a safari park and entered bat caves during the stay in bali [20] . this suggests the fruit bat as a possible vector in these orthoreovirus infections. however, the infection mode of the species of nelson bay orthoreoviruses in humans is still unknown and should be a subject for prospective studies. miyazaki-bali/2007 orthoreovirus caused respiratory tract infection in mouse model, in which mice were infected with the virus through intranasal inoculation route (data not shown, but presented at the 56th annual meeting of the japanese society for virology, okayama, japan, in october, 2008 with the presentation number ''3c18''). furthermore, the virus was isolated from and detected at the lesions in the lung. it suggests that miyazaki-bali/2007 orthoreovirus caused rti in the patient. bats have recently been identified to be the reservoir hosts of a variety of viruses responsible for severe zoonotic virus infectious disease outbreaks with very high mortality [22] . examples of such infections are encephalitis caused by hendra virus [23, 24] , nipah virus [25] (family paramyxoviridae), menangle virus (another new paramyxovirus) [26, 27] , and australian bat lyssavirus (family rabdoviridae) [22, 28] . marburgvirus (family filoviridae), the etiological agent for viral hemorrhagic fever in the continent of africa, has been isolated from egyptian fruit bats (rousettus aegyptiacus), suggesting that fruit bats are a viral reservoir for marburgvirus [29] . the virus genome of ebolavirus, another genus of family filoviridae and also an etiological agent for hemorrhagic fever, was detected in serum samples collected from three species of fruit bats: hypsignathus monstrosus, epomops franqueti, and myonycteris torquata [30] . this suggests that these species might serve as a reservoir for ebolavirus. in summary, the authors described an imported case of an rti associated with an orthoreovirus, which was classified to the species of nelson bay orthoreovirus, from bali, indonesia, to miyazaki, japan, that occurred in 2007. the virus was closely related to the viruses isolated from patients possibly infected with the orthoreoviruses in indonesia. prospective surveillance of the nelson bay orthoreovirus species group should be performed in southeast asia. orthoreoviruses and their replication piscine reovirus encodes a cytotoxic, non-fusogenic, integral membrane protein and previously unrecognized virion outer-capsid proteins a previously unknown reovirus of bat origin is associated with an acute respiratory disease in humans identification and characterization of a new orthoreovirus from patients with acute respiratory infections investigation of a potential zoonotic transmission of orthoreovirus associated with acute influenza-like illness in an adult patient a novel reovirus isolated from a patient with acute respiratory disease xi river virus, a new bat reovirus isolated in southern china pulau virus; a new member of the nelson bay orthoreovirus species isolated from fruit bats in malaysia structure and cytopathic effects of nelson bay virus rapid genome sequencing of rna viruses an improved procedure for rapid determination of viral rna sequences of avian rna viruses novel reovirus isolation from an ostrich (struthio camelus) in japan bat coronaviruses and experimental infection of bats, the philippines ligationmediated amplification for effective rapid determination of viral rna sequences (rdv) novel virus discovery in field-collected mosquito larvae using an improved system for rapid determination of viral rna sequences (rdv ver4.0) pathogenic characteristics of highly virulent avian reovirus, strain 58-132, isolated from a chicken with tenosynovitis a post-entry step in the mammalian orthoreovirus replication cycle is a determinant of cell tropism a plasmid-based reverse genetics system for animal double-stranded rna viruses the great island subgroup of tick-borne orbiviruses represents a single gene pool virulence potential of fusogenic orthoreoviruses a novel approach for collecting samples from fruit bats for isolation of infectious agents bats: important reservoir hosts of emerging viruses a morbillivirus that caused fatal disease in horses and humans hendra and nipah viruses: different and dangerous nipah virus: a recently emergent deadly paramyxovirus an apparently new virus (family paramyxoviridae) infectious for pigs, humans, and fruit bats probable human infection with a newly described virus in the family paramyxoviridae. the nsw expert group australian bat lyssavirus infection in three fruit bats from north queensland isolation of genetically diverse marburg viruses from egyptian fruit bats fruit bats as reservoirs of ebola virus we thank the following people, who contributed to the active contact surveillance undertaken in this study: minako saito, hisako nakamura, (nichinan health station), daizo nagatomo, chigusa sonoda, noriko yamada, hiroshi terazono (miyazaki city health station), shoichi yamashita, kyoko oura, and yoko nakamura, (health promotion division, miyazaki prefecture government). key: cord-306466-y4yg42p8 authors: nofal, ahmed maged; cacciotti, gabriella; lee, nick title: who complies with covid-19 transmission mitigation behavioral guidelines? date: 2020-10-08 journal: plos one doi: 10.1371/journal.pone.0240396 sha: doc_id: 306466 cord_uid: y4yg42p8 during the past 6 months, the world has lost almost 950,000 lives because of the outbreak of covid-19, with more than 31 million individuals diagnosed with covid-19 worldwide. in response, lockdowns, and various other policies have been implemented. unfortunately, many individuals are violating those policies and governments have been urging people to comply with the behavioral guidelines. in this paper, we argue that personality traits need to be considered to understand and encourage more effective public compliance with covid 19 transmission mitigation behavioral guidelines. using a sample of 8,548 individuals from japan, we show that certain personality traits are related to the tendency to comply with covid-19 transmission mitigation behavioral guidelines. we emphasize the importance of understanding why people respond differently to the same authority’s messages and provide actionable insights for government policy makers and those who implement policies. since the outbreak of covid-19, a number of initiatives have been put in place with the objective of mitigating transmission of the virus, such as imposing lockdowns, closing schools, and encouraging social distancing. medical scientists are racing to find a vaccine. geographers and software developers are developing applications to track people's movements in an attempt to identify and contain the virus in specific regions. health and media officials are promoting social distancing and persuading the population to follow health behavioral guidelines, often involving significant behavioral changes. despite these initiatives, as of mid-2020 the world is still losing thousands of lives every day. in a recent statement, tedros adhanom ghebreyesus, director-general of the world health organization, stated that "although many countries have made some progress, globally, the pandemic is actually speeding up" [1] . therefore, there is a clear need to explore further ways to help mitigate the spread of covid-19. in this regard, "social and behavioral science can provide valuable insights for managing the pandemic and its impact" [2, p. 1] , by enhancing our understanding of the role of human behavior in the spread of the virus, and thus contribute to the public good. while the covid-19 pandemic has resulted in the development and implementation of numerous behavioral guidelines intended to mitigate transmission, evidence suggests that many of these guidelines are not being followed by enough people to make them optimally effective [3] . governments and health authorities are still pleading for individuals to behave responsibly, by complying with these imposed behavioral guidelines and rules [4, 5] . however, laboratory studies show that "persuasive appeals are more effective in influencing behavior when they are tailored to individuals' unique psychological characteristics" [6, p. 12714 ]. in simple terms, what convinces one person to behave in a particular way, might not do so for another. moreover, some people are by nature more likely than others to be rule-breakers [7] . for instance, evidence shows that emotionally unstable [8] , and disagreeable people [9, 10] are more likely to break rules in general. accordingly, while it is important to understand which behaviors influence the speed of spread of covid-19 [11] , it is also important to understand the specific personality characteristics that could plausibly be related to the propensity of the population to comply with covid-19 transmission mitigation behavioral guidelines. to examine this, we investigate whether certain personality traits are related to compliance with transmission mitigation behavioral guidelines. particularly, we examine if major personality traits (i.e. conscientiousness, agreeableness, openness to experience, extraversion, and emotional stability) [12] , yield differences in the tendency of people to comply with 21 implemented covid-19 transmission mitigation behavioral guidelines (see s1 appendix). this paper makes a number of contributions. first, it draws attention to the importance of individuals' personalities when assessing the likelihood that an outbreak of the virus will occur among some people but not others. in this regard, we respond to research questions raised by davidai, day [13] regarding who complies with covid-19 transmission mititgation behavioral guidelines. second, this is not the first crisis the world has faced and will, unfortunately, not be the last. thus, our work provides evidence of how different personality traits can help people face the outbreak of crises such as global pandemics, and emphasizes the importance of implementation measures which take account of individuals' personality traits. in fact, this should help to ensure that we learn important lessons from the crisis, so that the responses of governments and organizations will be more effective when future crises occur [11] . third and most importantly, to our knowledge, the paper provides the first empirical evidence using an exogenous shock (i.e. the covid-19 outbreak) of the importance of personality traits and motivational propensities when monitoring people's tendency to comply with covid-19 transmission mitigation behavioral guidelines. in fact, there has been work exploring the effect of covid-19-related messages on people's actual compliance with covid-19 transmission mitigation behavioral guidelines [14] [15] [16] , research showing gender differences with regards to the compliance of transmission mitigation behavioral guidelines [17] , and studies examining the effect of emotional responses to pro-social messages on the tendency of people to comply with self-isolation restrictions [18] . however, there has been no work examining the influence of personality traits, such as the big five (i.e. conscientiousness, agreeableness, openness to experience, extraversion, and emotional stability) on the tendency of people to comply with covid-19 transmission mitigation behavioral guidelines. connecting literatures of emotional responses to covid-19 messages, and personalized/tailored communication, we believe that this research not only supports prior work in highlighting the importance of pro-social messages, but also the importance of knowing the specific personality traits that may make individuals less likely to comply with covid-19 transmission mitigation behavioral guidelines. hence, the aim of this paper is threefold. first, drawing from research on persuasive mass communication [e.g., 19] , we aim to demonstrate if personality traits, specifically, conscientiousness, agreeableness, openness to experience, extraversion, and emotional stability, relate to the tendency of people to comply with covid-19 transmission mitigation behavioral guidelines. second, we highlight the importance of incorporating behavioral science into governments, and organizations' responses to crises, such as covid-19. third and most importantly, we aim to provide some practical insights, such as occupational targeting and the use of social network platforms, such as facebook, twitter, and linkedin, to minimize the risk of not complying with covid-19 transmission mititgation behavioral guidelines. in psychology, persuasive mass communication refers to the process by which large groups of individuals are encouraged to adopt beliefs, attitudes and behaviors that are consistent with the communicator's viewpoint [6] . persuasive mass communication theories aim, thus, to identify strategies to improve the effectiveness of persuasive mass communication messages [e.g. 20, 21] . one such theory, psychological persuasion, assumes that persuasive communication is most effective "when tailored to people's unique psychological characteristics and motivations" [6, p. 12714 ]. this means that messages that are more congruent with an individual's motivational tendencies are processed more smoothly and assessed more positively than incongruent messages [22] [23] [24] . unfortunately, in some situations where mass communication messages are needed, such as covid-19 transmission mitigation behavioral guidelines, it is often impractical to make the messages either personalized or individually-targeted. in these situations, it becomes even more important to understand how individual differences in motivational tendencies result in differences in the attitudes and behaviors that individuals may adopt in response to the same message [25, 26] . because motivational tendencies are strongly influenced by personality traits [27] , the latter can yield differences in beliefs, attitudes and/or behaviors even when individuals are exposed to the same mass communication message. in this respect, the behavior or the attitude towards the same authority's messages (as in the case of covid-19) can be more/less pronounced among people who possess certain personality traits. therefore, building on this literature, we consider how different personalities respond differently to the same message from authorities, and hence comply with behavioral guidelines contained in those messages. to do so, we use the big five model of personality which relies on the assumption that individual differences are best captured by five global traits [12, 28] : extraversion, emotional stability, openness to experience, agreeableness, and conscientiousness. each personality trait reflects a different motivational system that influences how individuals perceive the context around them, process authority's messages, and in turn decide to adopt (or not) compliance behavior. we explore these differences and build our hypotheses next. extraversion refers to the extent to which individuals are assertive, dominant, energetic, active, talkative, impulsive, and enthusiastic [12] . individuals who score high on extraversion tend to be cheerful, are positively disposed towards other people and large groups, and seek excitement and external stimulation. meanwhile, individuals who score low on extraversion prefer to spend more time alone and can be described as reserved, quiet, and independent [29] . in this sense, messages containing requests to respect self-isolation and social distancing are incongruent with a motivational system that gains rewards and satisfaction from active engagement with the world, social interaction and social recognition [30] . accordingly, such messages are not processed smoothly and effectively which results in a tendency to violate such policy measures. there is a negative association between extraversion and the tendency of people to comply with covid-19 transmission mitigation behavioral guidelines. emotionally unstable individuals are especially sensitive to threats and uncertainty [31, 32] . individuals who score low on emotional stability tend to experience a number of negative emotions such as anxiety, depression, hostility, self-consciousness, and vulnerability [12] . individuals who score high on emotional stability can be described as self-confident, calm, and relaxed [29] . these psychological characteristics suggest that messages containing rules and policies aiming at providing structures and formalized procedures are positively accepted by a motivational system that is normally inhibited by uncertainty and unpredictability [31, 33] . however, enacting self-isolation and keeping social distancing for long periods of time can result in increased stress and negative emotions for emotionally unstable individuals, than for more emotionally stable individuals. this could possibly motivate the former to break rules as an attempt to protect their psychological wellbeing [8] . as such, while individuals who score low on emotional stability can respond to authorities' messages in different ways, we assume that the opportunity of feeling secure by complying with rules and policies will not be perceived as such if it results in increased risk of anxiety and depression. accordingly, such messages are not processed fluently and effectively which leads to a tendency for those lower in emotional stability to violate authorities' rules and behavioral guidelines. hypothesis 2: there is a positive association between emotional stability and the tendency of people to comply with covid-19 transmission mitigation behavioral guidelines. openness to experience is a personality trait that characterizes someone who values creativity, innovation, and intellectual stimulation [34] . individuals who score high on openness to experience can show active imagination, creativity, preference for variety, and intellectual curiosity. individuals who score low on openness to experience can be described as conventional, narrow in interests, and unanalytical. these psychological characteristics suggest that open individuals can perceive the self-isolation and social distancing rules as an opportunity to cultivate their curiosity and imagination. the lockdown can create the conditions (e.g., by increasing time for themselves) to engage in activities that promote absorption, self-examination, and creative solutions [12] . accordingly, they have little interest in escaping a situation that allows to satisfy their needs for intellectual stimulation and cognition [35] . hypothesis 3: there is a positive association between openness to experience and the tendency of people to comply with covid-19 transmission mitigation behavioral guidelines. agreeable individuals value communal goals and interpersonal harmony [36] . people who score high on agreeableness have strong cooperative values and a preference for positive interpersonal relationships [9, 10] . people who score low on agreeableness can be described as manipulative, self-centered, suspicious, and aggressive [12, 28, 29] . because agreeableness may lead one to be particularly empathetic, individuals at the high end of this personality construct tend to show great concern for the welfare of others [36] . accordingly, they are likely to be very motivated to comply with rules and behavioral guidelines that present self-isolation and social distancing as a way to protect themselves and those around them. hypothesis 4: there is a positive association between agreeableness and the tendency of people to comply with covid-19 transmission mitigation behavioral guidelines. conscientious individuals value achievement, order, hard work, and efficiency [37, 38] . individuals high on conscientiousness are described as careful and diligent as opposed to easy going and disorderly [39] . the psychological characteristics of conscientious individuals suggest that violation of rules and behavioral guidelines aiming at controlling social mobility to limit the spread of the virus is not consistent with their tendency and motivation to show selfdiscipline, act dutifully, and achieve efficiency [12, 40] . hypothesis 5: there is a positive association between conscientiousness and the tendency of people to comply with covid-19 transmission mitigation behavioral guidelines. our sample is drawn from japanese citizens aged 20 to 64 years old. the data was collected between the 26 th and 28 th of march 2020. a sample of 11,342 individuals responded to the survey. a quota sampling approach was adopted to match the sample distribution to the japanese population in terms of gender, age, and employment status. after excluding individuals who did not respond to any of the items measuring our variables of interests, missing values, and individuals who responded with "don't know" to any of our variables of interest, we were left with a sample of 8,548 individuals. further information on the study is available at https:// www.openicpsr.org/openicpsr/project/118584/version/v1/view. it is also worth-noting that this research does not require an ethics committee approval because it is a secondary analysis of anonymous data. dependent variable. adoption of transmission mitigation behavioral guidelines. the measure of our outcome variable is measured using policy statements that assess the extent to which individuals adopt the transmission mitigation behavioral guidelines. the data includes 21 behavioral guidelines developed to prevent the spread of the virus, such as respondents' tendency to "always wear a surgical-style mask when going out", "stockpile surgical-style masks", "avoid large gatherings", and "participate in virtual events using online tools" (see s1 appendix for the full scale items). for each policy, respondents selected the extent to which they adopted the policy on a 5-point scale; 1-very true, 2-true, 3-neither, 4-not true, and 5-not at all. we reversed the scale so that a higher score corresponds to a more likelihood of compliance with the transmission mitigation behavioral guidelines. internal consistency analysis of the measure returned a cronbach's alpha value of .89. independent variables. personality traits. we use a short scale, very well-established in personality research, to assess individuals' psychological traits, specifically the ten item personality inventory (tipi) [41, p. 289, 42, 43] . the tipi is comprised of 10 items, each consisting of a pair of traits (see s1 appendix). for consistency of interpretation, we reverse some responses, such that the higher the value, the higher the level of extraversion, openness to experience, agreeableness, conscientiousness, and emotional stability. the cronbach's alpha value of each of the big five ranged from 0.3 to 0.6, which is a limitation that we consider below. control variables. we control for the effects of a number of covariates. specifically, we control for the influences of gender (1-male, 2-female) because research shows that gender is related to various psychological traits [44] . further, as socio-economic conditions could have an effect on both personality traits, and individual behaviors [45] , we control for the dummies of individuals' annual household income, with the first dummy corresponding to having "less than 2,000 k japanese yen", and the highest dummy corresponding to "more than 20,000k jpy" (see s1 appendix for further details). following prior research on the relationship between the big five and education [46] , we also account for the influence of education by controlling for whether respondents are university or college graduates. furthermore, as age captures various factors that could influence individuals' personality traits [47, 48] , we control for age categories as dummies. we also control for marital status (1-married, 2-unmarried) because marital status might confound the effects of the big five on different behaviors [48, 49] . in addition, having a partner might impact certain behavioral guidelines, such as gathering with friends. furthermore, because the sources of information used by individuals may affect the tendency of people to comply with the covid-19 transmission mitigation behavioral guidelines, we control for the sources of information, such as "tv news programs", "information sent by the ministry of health, labor and welfare", and "information sent by local (prefecture) government" (see s1 appendix for further details). all our sample is drawn from japan, and therefore location is automatically controlled for. we use stata mp version 16 for the analyses. table 1 presents the correlations between the variables (see s1 table in s1 appendix for further details). our sample consists entirely of japanese citizens. approximately 54% of the sample are males and 46% are females. in terms of marital status, approximately 38% of the sample are married and 62% are not married. the sample includes various age groups ranging from the age of 20 to the age of 64 years old. table 2 presents the results of a number of ols models examining the influence of personality traits on the compliance with covid-19 transmission mitigation behavioral guidelines. specifically, model 1 examines the effect of personality traits on the compliance with covid-19 transmission mitigation behavioral guidelines without any controls. we find support for all our hypotheses apart from extraversion which supports a significant positive effect on compliance with covid-19 transmission mitigation behavioral guidelines. model 2 adds our control variables, and again we find support for our hypotheses. importantly, when adding controls in model 2, we now find that extraversion negatively influences the tendency of people to comply with covid-19 transmission mitigation behavioral guidelines, meanwhile agreeableness, conscientiousness, and openness to experience positively influence the tendency of people to big five personality traits and covid-19 behavioral guidelines while we are cautious about causality issues given the cross-sectional nature of our data, we emphasize that covid-19 transmission mitigation behavioral guidelines are a result of an exogenous shock (i.e. the sudden outbreak of covid-19). as dunning [50] explains, exogenous shocks constitute natural experiments in which subjects are-as-if-randomly assigned to different levels of a treatment and therefore may allow researchers to uncover causal relationships. therefore, causality issues are reduced. however, despite our inclusion of a number of key controls, omitted variables might still bias the estimates of our model parameters. one way to deal with these potential biases is the use of multilevel modeling [51, 52] . as such, we perform an additional multi-level analysis to overcome the aforementioned issues of omitted variable bias. the rationale for applying a multilevel estimator is the fact that for a global phenomenon as the covid-19 pandemic, government communications have often been aimed at certain specific societal groups, including different age groups [53] , and household characteristics [54] . for instance, older people are asked to be more cautious compared to younger ones [53] . therefore, multilevel models can be used to capture variations at both levels; individuals at the first level, as well as age and household annual income groups at the second level. hence, for robustness reasons, we re-estimate our models using the multilevel estimator we find similar results to the ols analysis; with and without the control variables (see s1 appendix). because our large sample size increases our power to detect statistical significance of even small effects, we also consider the substantive importance of our results in terms of effect size, using the odds ratio. specifically, our findings indicate that the highest odds ratio is for conscientiousness, with people high in conscientiousness 31% more likely to adopt covid-19 transmission mitigation behavioral guidelines compared to people low in conscientiousness, followed by 19% for openness to experience, and 17% for agreeableness (i.e. these percentages are calculated based on the odds ratios computed based on a 1 unit increase/decrease on the scale items used). we also found that people high in extraversion are 7% less likely to adopt covid-19 transmission mitigation behavioral guidelines compared to introverts (see fig 1) . it is worth mentioning that our outcome is assessed using scale items and therefore the linear coefficients estimated might overestimate/underestimate the effect size of our independent variables [56, 57] . therefore, we have calculated the effect sizes based on odds ratios estimated through ordinal logit models (see also fig 1 for linear effect sizes) . in this respect, we emphasize that in the case of an ordinal outcome such as the scale item being used, the interpretation is not read as a contrast between extremes as in the binary outcome case, but as a contrast for each unit change. in other words, on a scale demarcated as 1, 2, 3, 4, 5 as in the case of our compliance measure, an or of 1.1, for instance, entails a 10% increase in the odds of the dependent variable for each unit change in the independent variable (e.g. moving from 1 to 2, or 2 to 3, or 3 to 4, or 4 to 5 on the scale demarcated). for robustness, we re-test our hypotheses after splitting our sample into males and females. we find consistent results across both samples for agreeableness, conscientiousness, and openness to experience. however, extraversion shows a negative influence on the adoption of covid-19 transmission mitigation behavioral guidelines only for the male sample, but not for the female sample. the covid-19 pandemic has resulted in the introduction of an unprecedented number of governmental policy measures intended to contain the health crisis and mitigate its effects on the economy [58] . however, as of mid-2020, most governments are still struggling to contain the outbreak of covid-19. in this paper, we argue that the effectiveness of governmental messaging, in terms of its influence on behavioral change of individual citizens, is dependent on individual differences in personality traits. there are a number of key implications of our findings, which translate into actionable insights for government policy makers and those who implement policies. a first key implication is that governments need to tailor messaging towards different personality traits. at the top of the list in this regard is conscientiousness. in essence, using a sample of 8,549 individuals from japan, we find that conscientiousness increases the likelihood of adopting covid-19 transmission mitigation behavioral guidelines by 31%. in fact, identifying individuals low in conscientiousness can be helpful in knowing the communities where transmission mitigation behavioral guidelines are least likely to be adopted. though we do not find consistent patterns for other personality traits, we find tentative evidence of the role of extraversion, agreeableness, and openness to experience in the adoption covid-19 transmission mitigation behavioral guidelines, with the highest effect size detected for openness to experience, followed by agreeableness, and then extraversion. we emphasize that these effect sizes are not deterministic. personality influences imply probabilistic propensities rather than hard-wired patterns of covid-19 guidelines compliance behavior. thus, our suggestion that the influence of our independent variables on the tendency of people to comply with covid-19 transmission mitigation behavioral guidelines should not be confounded with determinism. that said, when dealing with a societal-level communication task such as that required for pandemic-mitigation guidelines, understanding these probabilistic propensities is highly valuable, even if they are not deterministic per se. our study provides several contributions. to begin with, the value of our findings can be seen in the example of efforts to increase the number of people who comply with covid-19 transmission mitigation behavioral guidelines. this can be accomplished by implementing initiatives that can help trigger some personality traits, including relatively static ones [59, 60] . for instance, since our findings show that conscientiousness considerably increases the likelihood that people adopt covid-19 transmission mitigation behavioral guidelines, governments are encouraged to boost citizens' sense of belonging and obligation to their communities-which has been suggested to develop conscientiousness [61, 62] . moreover, because our findings show that openness to experience, especially for males, positively influences the tendency of people to adopt transmission mitigation behavioral guidelines, promoting online inductive reasoning training can be beneficial for developing openness to experience [63] and therefore the adoption of transmission mitigation behavioral guidelines. in addition, as our results indicate that agreeableness positively influences the adoption of transmission mitigation behavioral guidelines, especially for females, governments are urged to promote training on balancing criticism and empathy to trigger higher levels of agreeableness [64] and hence higher adoption of behavioral guidelines. further, researchers argue that contact tracing is of significant importance to contain the spread of the virus [65] . yet, many countries are still struggling to develop effective ways of tracing. given our findings, we have three suggestions. first, countries can trace groups that are less likely to have developed high levels of conscientiousness, openness to experience, and agreeableness. one quite feasible way of doing this is through sending out short surveys on personality traits using social network platforms, such as facebook, twitter, and linkedin. based on those surveys, governments and organizations can provide different covid-19 messages. another way is through occupational categorizing, for instance, entrepreneurs are known for their high levels of conscientiousness, and openness to experience, unlike full-time employees [27, 49] . hence, tracing certain occupational groups (i.e. entrepreneurs and fulltime employees) might be informative in this respect (indeed, we empirically examined this suggestion and found that fulltime employees are less likely to comply with covid-19 transmission mitigation behavioral guidelines compared to entrepreneurs, part-time employees, and unemployed individuals). as a result, governments can send different messages to different occupational groups and therefore increase the likelihood that those occupational groups comply with the announced covid-19 transmission mitigation behavioral guidelines. second, assessing individuals' personalities could be beneficial to identify people who tend to violate the transmission mitigation behavioral guidelines. for instance, using the tipi which is an "ultra-short scale" [41, p. 289 ], countries can collect information about the people who tend not to adopt the covid-19 transmission mitigation behavioral guidelines. further, recent research shows that digital footprints, such as their facebook likes or tweets, can predict people's personality traits [6] . thus, observing individuals' digital footprints could inform about their personalities, and therefore help us identify the people who are least likely to adopt the transmission mitigation behavioral guidelines. that said, we recognise that the privacy implications of such initiatives would also have to be balanced with their potential effectiveness. in sum, with our study, we respond to the question of davidai, day [13, p. 3] on who complies with covid-19 transmission mitigation behavioral guidelines. based on our findings, we provide one plausible answer, that is: those who are high in conscientiousness, introverts, and agreeable people as well as individuals who are high in openness to experience. we also provide insights for covid-19 media campaigns. we demonstrate the potential importance of tailoring campaigns to people's psychological characteristics-which may help provide an explanation for why the significant resources that have been expended on campaigns to encourage transmission mitigation behaviors have yielded sometimes little effect. this research has some limitations. first, the tipi that is used to measure the big five might be inferior to other scales, given its internal-consistency issues [27] . however, as park, wiernik [66, p. 1] explain, different big-five factor models are recommended based on "specific studies, research questions, and contexts". as the tipi is characterized by having a high level of content validity and is highly urged in situations "when brevity is a priority" [41, p. 289 ], such as the outbreak of the deadly pandemic, we see its relevance to our study. meanwhile, future research is urged to retest our hypotheses using other scales. second, although our findings can be insightful to many countries, our study applies to japan. countries have implemented to a great extent the same covid-19 transmission mitigation behavioral guidelines, yet there have been some differences. even proximate countries showed different reactions to covid-19. for example, sweden has implemented very few behavioral guidelines to face the pandemic unlike its neighboring finland which has implemented stricter quarantine conditions. therefore, further research focusing on other countries is urged. third, the small amount of variance explained by the big five (4%) suggests that the big five personality traits alone do not completely predict compliance with covid-19 transmission mitigation behavioral guidelines. rather, they may play a role in one of the many mechanisms explaining compliance with covid-19 transmission mitigation behavioral guidelines. therefore, it is important for future research to examine different interactive effects of the big five with other factors on compliance with covid-19 transmission mitigation behavioral guidelines [67] . over 100 years ago, science magazine published an article that put down some lessons from the spanish flu pandemic [68] . as bavel, baicker [2] explain, the paper demonstrates that three main factors contribute to the outspread of a pandemic: (1) individuals do not appreciate the risks they undertake, (2) people are resistant to shutting themselves up in strict isolation as a means to contain the outbreak of a pandemic, and (3) people in many 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covid-19 epidemic? the lancet meta-analytic five-factor model personality intercorrelations: eeny, meeny, miney, moe, how, which, why, and where to go big five aspects of personality interact to predict depression the lessons of the pandemic key: cord-278099-ypov9ha3 authors: kumar, surender; subbarao, burra l.; kumari, reenu; hallan, vipin title: molecular characterization of a novel cryptic virus infecting pigeonpea plants date: 2017-08-03 journal: plos one doi: 10.1371/journal.pone.0181829 sha: doc_id: 278099 cord_uid: ypov9ha3 a new member of the genus deltapartitivirus was identified containing three dsrnas with an estimated size of 1.71, 1.49 and 1.43 kb. the dsrnas were extracted from symptomless pigeonpea [cajanus cajan (l.) millspaugh] plants cv. erra kandulu. this new virus with 4.64 kb genome was tentatively named arhar cryptic virus-1 (arcv-1). the genomic rnas were amplified and characterized by sequence independent single primer amplification. the dsrnas shared a highly conserved 16 nt 5’ non-coding region (5’-gataatgatccaagga-3’). the largest dsrna (dsrna-1) was identified as the viral rna dependent rna polymerase (replicase), predicted to encode a putative 55.34 kda protein (p1). the two other smaller dsrnas (dsrna-2 and dsrna-3) predicted to encode for putative capsid proteins of 38.50kda (p2) and 38.51kda (p3), respectively. phylogenetic analysis indicated that arcv-1 formed a clade together with fragaria chiloensis cryptic virus, rosa multiflora cryptic virus and rose cryptic virus-1, indicating that arcv-1 could be a new member of the genus deltapartitivirus. arcv-1 3d(pol) structure revealed several interesting features. the 3d(pol) in its full-length shares structural similarities with members of the family caliciviridaeand family picornaviridae. in addition, fourth dsrna molecule (dsrna-2a), not related to arcv-1 genome, was found in the same plant tissue. the dsrna-2a (1.6 kb) encodes a protein (p4), with a predicted size of 44.5 kda. p4 shares similarity with coat protein genes of several cryptic viruses, in particular the bipartite cryptic viruses including raphanus sativus cryptic virus-3. this is the first report of occurrence of a cryptic virus in pigeonpea plants. pigeonpea [cajanus cajan (l.) millspaugh], having originated in india, is one of the major grain legume (pulse) crops in the indian subcontinent. the crop is known to be susceptible to a few virus and virus-like pathogens, belonging to different genera [1] [2] [3] [4] . while characterizing the dsrna from the sterility mosaic disease (smd) [3] affected pigeonpea plants, we noticed that two sets of symptomless controls plants containing four dsrna molecules while none were noticed in the remaining sets of healthy samples. healthy control plant samples (mg1-h1 and mg1-h2) that contained the dsrna were collected from one of the farmers' fields in chevella area (near hyderabad, telangana state). the pattern and the number of dsrna segments did not match with dsrnas associated with pigeonpea plants infected by ppsmv-p sub isolate-chevella (separate manuscript under preparation). sequence of the four dsrnas showed neither homology nor alignment to any region of ppsmvs. however, these dsrnas share close similarities with individual genomic segments of several plant cryptoviruses. evidence is provided to show that three of the four dsrnas constitute the genome of a new cryptovirus tentatively named "arhar cryptic virus-1" (arcv-1) with a genome size of 4.64 kb. pigeonpea is referred to as "arhar"in hindi. the genomic sequences showed its phylogenetic relationship to members of the genus delatapartitivirus. the fourth dsrna (dsrna-2a) contained cryptic virus coat protein-like sequences with no similarity to that of arcv-1 genome. we discussed different possibilities of its occurrence with arcv-1 genome and its relevance. cryptic viruses usually contain 2 to 3 monocistronic genomic dsrna segments which are encapsulated individually [5, 6] , and referred to as bipartite and tripartite viruses. occurrence of tripartite cryptoviruses is less frequent [7] . plant cryptoviruses, in general are either pollen or seed transmitted, belong to the family partitiviridae which include five genera viz. alphapartitivirus; betapartitivirus; gammapartitivirus; deltapartitivirus and cryspovirus [8] . members of the family partitiviridae are known to infect mainly fungi, plants and protozoans. this is the first report detailing association of a cryptic virus with pigeonpea (cajanus cajan(l.) millspaugh) plants which belongs to the family fabaceae, harboring one third of the reported cryptovirus infections. mixed infections involving different cryptoviruses as well as host specific pathogenic viruses are common [5, 9, 10] , effecting a single plant or more. beet cryptic virus-1 (bcv-1) and beet cryptic virus-2 (bcv-2) were reported infecting the same host [9] , as a mixed infection, similarly in the case of pepper where pepper cryptic virus-1 (pepcv-1) and pepper cryptic virus-2 (pepcv-2) were found as mixed infection [11] . in the present study, arcv-1 genomic segments were characterized by sequence independent single primer amplification (sispa) method. this comprehensive method was found to be precise with a high degree of specificity to investigate the evolution and genetic diversity in dsrna viruses. the four dsrnas eluted from the agarose gel were purified and have been used as templates for rt-pcr amplification employed in sispa to generate fulllength cdnas. it is of interest to examine if arcv-1 rna dependent rna polymerase (rdrp) structurally resembles the known rdrp of the dsrna bacteriophage փ-6, reovirus, or with other viruses like calciviruses and picornaviruses [12] [13] [14] [15] [16] . their rdrp molecules, whose structural details have been described, are larger than rdrp of arcv-1. rdrps of several important human and animal picornaviruses have been extensively studied that correlated functions with structural details, which contributed to the understanding of the mechanics of this enzyme activity leading to viral replication [12] [13] [14] [15] [16] . similar efforts were made in the past decade to study the 3d structural characterization of rdrps of a very few plant viruses and detailed studies remained to be carried out for rdrps of double-stranded plant viruses including partitiviruses. arcv-1 rdrp sequence analysis revealed the presence of several conserved amino acid sequence motifs common in other tripartite cryptoviruses. these motifs have been described to be important for biological functions in several rdrps [17] . we report here the results of elaborated computer-assisted analysis of arcv-1 replicase which revealed the presence of conserved sequence motifs (a to g) present in the fingers and palm subdomains of the polymerase that are shared in most of the rdrps. interestingly, arcv-1 replicase has more structural resemblances with several members of ssrna (+) mono-partite picornaviruses (viral replication by primer-dependent initiation), than the de novo dsrna bacteriophage փ-6 and reovirus polymerases. variations found in arcv-1 motifs' sequence that may be involved in polymerization mechanism and the conserved motifs unique to cryptoviruses have been described. this report illustrates several interesting features of the arcv-1 3d pol and its complete structure was determined. symptomless pigeonpea leaf samples, mg-h1, mg-h2 collected from mg-1and mg-2 fields contain four dsrna species with an estimated size of~1.71,~1.49,~1.43 and~1.6 kb (figs 1a and 2), whereas as symptomless leaf sample (mg-h3) collected from another field (mg-3) were devoid of similar dsrnas. the pure dsrnas yields ranged 700 to 800 ng was obtained from 7g of fresh leaves with consistent dsrna unique profile which is entirely different from the ppsmvs. post sispa, pcr amplification product profile was similar to the dsrnas obtained from the initial dsrna extraction from the symptomless leaves (fig 1) . these amplification products were cloned and characterized. the cloned amplification products corresponding to the four dsrnas were sequenced. blast analyses of the full-length sequences revealed their identity with the orthologs of several cryptoviruses. arcv-1 genomic segments shared distinctive conserved 16 nt stretch (gataatga tccaagga) at the 5'-non-translated region (ntr), a feature commonly observed in multipartite cryptic viruses and constituted as the genome of arcv-1 with a size of 4.64 kb (fig 3) . whereas the dsrna-2a, 5'-terminus sequence (agaatttgccctgtat) did not share the conserved sequences and thus treated as a separate entity. the genome organization of arcv-1 was thus established during the present study (fig 2) , together with dsrna-2a depicting the single open reading frames (orfs) of dsrna-1 (rdrp), dsrna-2 (cp-1) and dsrna-3 (cp-2) with the predicted molecular weights of proteins encoded by the rnas (table 1) . unlike the 5' end, the 3'-region is least conserved in several cryptic viruses. the majority of the members of the family partitiviridae typically contain few pyrimidine bases conserved at the 3'-terminal and in tripartite viruses, conservation is seen mainly in two of the rna segments. arcv-1 rnas encoding capsid proteins are conserved in the last three nt (ttc), like in raphanus sativus cryptic virus-2 (rscv-2), the three arcv-1 rnas end with tc. the majority of the deltapartitiviruses contain rna segments consistently ending with nucleotides tc except in rosa multiflora cryptic virus (rmcv) and cannabis cryptic virus (cancv) (betapartitivirus) interestingly, the 3' termini, each with a poly (a) tract. the dsrna-1 (hg797710) is 1,714 nt long, containing a single open reading frame (orf) between nt 1,606 and nt 179 with 178-and 108-nt-long 5' and 3' ntrs, respectively (fig 2, table 1 ). this orf encodes for a 55.34 kda protein (p1) containing 475 amino acids. p1 protein was identified as viral rdrp and contains conserved short amino acid sequence motifs (a to g) which are essential for viral replication [18] [19] [20] [21] , and known in several picornavirus rdrps. the a-g motifs are conserved in the rdrps of several genera of plant and animal viruses containing monopartite, linear, ssrna (+) genome [17, 18, 22] , segmented, tripartite ssrna (+) viruses [23] , as well as multi-segmented linear ssrna (-) viruses [24] [25] [26] [27] . arcv-1 rdrp similar to the other cryptoviruses contained these signature motifs in the central region of the polypeptide. specific amino acid sequences mostly conserved formed these seven motifs: ssaagygyvglk (motif g; 117-128) kvrnvw//dpl//sfyfigqd(motif f; 181-219), fdwsgfd (motif a; 241-247), gipsgscftniigsitn (motif b; 302-318), thgdd (motif c; 338-342), dksd (motif d; 372-375), and tfl (motif e; 384-386) known to be functionally active in viral replication [17] . computer analysis predicted the active residues in each of the conserved motives (s1 and s2a figs), indicated in bold letters. motif g is less conserved amongst the human and animal viruses belonging to the picornavirus ssrna (+) super family as well as viruses containing dsrna genome. active residues in motif g are comparatively well conserved in the dsrna cryptovirus rdrps (s1 fig). aided by rdrp sequence of փ-6 and reovirus active residues in motif g were identified [12, 13] . conserved proline (in this motif) of most of the picornaviruses is substituted in arcv-1 rdrp by glycine in the c-terminus. ssaagygy-g-k sequence is highly conserved in cryptic viruses. in addition to the above mentioned seven conserved motifs, several conserved sequences in arcv-1 rdrp were present flanking the central region motif c represented by gdd sequence (fig 4d, s2a fig) is almost universal and has been described as the center of catalytic activity of the rdrp in all rdrp classes [18, 28] . the 3d table 1 . genome characteristics of aarcv-1, encoded proteins and associated dsrna-2a. structural analysis revealed that the gdd sequence present in the loop formed by antiparallel β-sheets 6 and 7 is located in the bottom part of the central cavity of the molecule in the palm region ( fig 4b, s2a fig) . several investigators conducted mutational studies of the catalytically important residues of the polymerase of different viruses [29] [30] [31] [32] , and determined that the first aspartate is an essential residue of this highly conserved motif for the enzyme activity and its coordination in the binding of a magnesium ion that would be part of the rntp binding site [12, 33] . it was found that the first asp (d341 in arcv-1) is a strict requirement and any change leads to loss of the enzyme activity. some flexibility was suggested with regard to glycine and the second aspartate and subtle changes in the position of these two residues are tolerated with exceptions [34, 35] . similar observations were made when the conserved aspartate of brome mosaic virus (+ssrna) was replaced in the 2a gene with glutamate (d470e), as the dr4 mutant did not replicate in the barley protoplasts [36] . the polymerase structural cryptic virus from pigeonpea domains and the regulatory mechanism of the enzyme with respective different viruses are well documented in a recent review [17] . possible functions of the residues of the a to g motifs described for identical rdrps was conserved with respect to the arcv-1 3dpol structure and was discussed in structural analysis of arcvtable 1 ) and the 3' terminus contained the sequence "gca cccatattc". the p2 was identified as the capsid protein-1, having 40.6% amino acid sequence identity with corresponding protein of rscv-1-(eu024677) ( table 2 ). the dsrna-3 (hg967638) is 1,433 nt long, containing a single orf between nt 171 and 1,205 with 170-and 227nt long 5' and 3' ntrs, respectively (fig 2, table 1 ) and the 3' terminus contained the sequence "gcaccacgtttc". this orf encodes a 38.51 kda protein (p3) of 344 amino acids, identified as the second capsid protein. the p3 protein has 42.7% sequence identity with coat protein (rna-3) of rscv-1 (eu024677) ( table 2) . the dsrna-2a (hg917912), extracted along with the arcv-1 genome was found in lower concentration in comparison to the genomic dsrnas ( fig 1a) . the rna is 1,608 nt long, contains a single orf that encodes a 44.5 kda protein (p4) of 389 amino acids. the orf starts at nt. 85 and terminates at 1,254 nt ( fig 2b, table 1 ) with 84-and 353-nts as the 5' and unusually long 3'-ntrs, respectively. its 5' terminus has agaatttgccctgtat sequence and did not share sequence identity neither with the corresponding region of arcv-1 genomic segments (fig 3) , nor with the c-terminus (ttc) of the capsid proteins. amino acid sequence identity analysis of p4 revealed similarity with the putative genomic capsid proteins of several cryptic viruses (table 3) , as well as few eukaryotic genes of plant table 3) . interestingly, the p4 protein has high degree of sequence identity with cryptic virus coat proteins of bipartite viruses (table 3 ). efforts were made to confirm the presence of a second rdrp assuming there may be in existence and dsrna-2a belongs to another cryptic virus, but were not successful. in each attempt similar four dsrnas were isolated. it is hard to explain, in absence of a related rdrp gene, and to speculate that the dsrna-2a coat protein like sequence belong to a yet unknown second cryptovirus infecting pigeonpea. apart from this, the four rna showed significant sequence homology with plant genes. interestingly in blastx, rna-1 showed 64% sequence identity with unknown protein of picea sitchensis or sitka spruce (ade76327), an evergreen conifer tree. rna-2 showed 34% sequence identity with unknown protein (xp008459635) of cucumis melo. further, rna-3 showed highest sequence identity with plant genes as 61% with uncharacterized proteins kom46435 and xp017431256 of vigna angularis. similarly, dsrna-2a shared 34% sequence identity with atp-binding cassette (abc) transporter f family member-1 (khf98106) of gossypium arboretum (tree cotton) found conserved in eukaryotes and prokaryotes. these eukaryotic lineages are not the known host plants for cryptic viruses. the peptide sequence of arcv-1 rdrp (p1) was used for developing the 3dpol structure. structural templates were identified from the pdb and the 3d model created using zhang lab server [37, 38] . pymol was used to develop the three dimensional figures. crystal structure of rdrp has been described depicting the "closed right hand" configuration [17, 39] . rdrps belonging to various genera of viruses containing amino acid sequence motifs are most conserved and occur in a typical order [20, 28, 40] , reflecting structural conservation in most cases. besides size, origin, and some structural variations, all classes of rdrp including several known picornaviruses share two main features by (1) having the 3d structure divided into three major subdomains described as palm, thumb and fingers [17, 39, 41, 42] , and (2) the interconnected subdomains that encloses the conserved catalytic region (fig 4, s2 fig) within a largely hollow center referred to as the catalytic center [43] . the 55 kda arcv-1 3d pol is a spherical molecule resolved at 7.9å resolution showed the basic features described above comprising of characteristic 16 α-helices, 7 β-sheets interconnecting with coiled structures ( fig 4a) forming a structure of a cupped right hand. unlike in picornaviruses the partitivirus genome is devoid of 5' terminus genome-linked protein (vpg) or particularly the polymerase with polyadenylated c'-terminal with the exception of cancv (jn196536) and rscv-1rna-3 (dq181927). the n-terminal residues (1-55) in fingers subdomain (fig 4a, blue) of the rdrp molecule extend over to the thumb subdomain binding them together (fig 4a and 4d ) encircling the molecule, ensuing central cavity. this architecture makes the molecule spherical or globular ( fig 4a and 4c ), and not u shape normally seen in dna polymerases and dna-dependent rna polymerases [44] . arcv-1 3d pol has a big cleft opening in to central cavity groove in the front and one at the top left channel referred to as template channel leads to the centre of the cavity supposed to be allowing the access of the rna template and ntp substrates to the central cavity [17, 33, 45, 46] and that central cavity opens out to the side on the right (rear channel) (fig 4e) . the n-terminal portion of the fingers subdomain is mainly composed of random coiled web (residues 1-55) on the top portion molecule sometimes referred as index finger with the n-terminal (met 1) protruding out. n-terminal coiled structure runs across, connecting the fingers and the thumb subdomains, folds back on itself into a loop providing the conserved residues gwars (53-57) seem to be unique to the bi-and tripartite cryptoviruses (s2a fig the fingers subdomain (residues 56-232 and 252-309) consist of eight α-helices (α2 to α7 and α9, α10) and four β-strands (β1-β2 and β4-β5). broad structural variations are seen in fingers subdomain followed by the thumb in general than the palm in the viruses belonging to picornaviruses. whereas these variations in fingers subdomain being less significant in cryptic viruses. the fingers region contains motif f and motif g, the latter being geometrically closer to the catalytic center of the palm subdomain. motif f: a stretch of 42 amino acid residues mostly conserved in cryptoviruses spanning few residues before β-sheet 2 to α-6 and connecting coil containing conserved active residues as motif f. like the n-terminal long loop, a portion of motif f extends from the fingers to interact with the thumb. the size of the loop, however, varies in different rdrps [21] . motif f contains predominantly hydrophobic residues, along with at least six active residues (r183, w186, l203, y214, g217 and d219) supposed to be interacting with the rna in the arcv-1 3d complex (s1 fig) along with the conserved lys181. basic residues lys181 and arg183 of arcv-1 3d rdrp have been identified associated with motif f of other rdrps; fmdv (k172). lys172 residue along with two arginine residues (r168 and r179) predicted to interact with the incoming ntp substrate in fmdv rdrp [33] . motif f of cryptoviruses unlike the other classes of rdrps is well conserved. motif g: the motif consists of residues from amino acid positions 117 to 128 (ssaagy-gyvglk). this motif is highly conserved in cryptoviral rdrps. amino acid residues of motif g form part of a long coiled loop connecting α-helices 4 and 5 in the fingers subdomain ( fig 4b) of the 3d pol. motif g is not well conserved in general and does not exist in human immunodeficiency virus-1 (hiv-1) rt, but observed across several genera of plant, animal and human picornaviruses, as noted from rdrp sequences [47] . glycine and lysine seem to be a strict requirement of motif g as they are conserved (gly121 and lys128 in arcv-1) in all classes of rdrp. the conserved proline residue noticed in some picornavirus [avian infectious bronchitis virus (aibv), severe acute respiratory syndrome-associated coronavirus (sars-cov), rhdv] and dsrna virus [infectious bursal disease virus (ibdv), infectious pancreatic necrosis virus(ipnv), bacteriophage փ-6] rdrps, is not present in the highly conserved motif g of tripartite cryptoviruses (fccv, rmcv, rocv-1, and rscv-2) including arcv-1. proline is substituted by glycine (gly123), suggesting some flexibility at this position. it has been described that the residues of motif g contact the nucleic acid at its 5' template overhang and form part of the channel for the template strand observed in the structures of reovirus and bacteriophage փ-6 rdrps [12, 13] , and the motif also predicted to be involved in positioning of the 5'template strand [21] . the palm subdomain(residues 233-251 and 310-398) consists of three β-sheets (β-3, β-6, β-7) sandwiched between helices α-11 and α-12 and a long loop from α-12 connecting α-13 in the thumb, α-7 being at the junction of palm and thumb ( fig 4a) . the palm domain shows the maximum conservation in the conserved motifs a, b, c, d and e (s1 fig, fig 4b) , and has been shown to play different roles in the catalytic activity of the enzyme, found in most of the known rdrps [20, 28] . palm sub-domain contained the structural architecture to suit to the functions, has remarkable alignment with the known rdrps. arcv-1 rdrp sequence identity with cryptoviruses is high, like the structural alignment. interestingly, with less sequence homology (table 4 ) a close structural alignment was noticed between arcv-1 and sapovirus (sv) and norwalk virus (nv) (+ssrna) rdrps as evident from the panels shown in fig 5e-5h . residues playing key roles in active site interactions, distributed across the motifs a to e, in different classes of rdrps have been identified in arcv-1 dsrna rdrp as well as the rdrps of other cryptoviruses have been discussed as below. motif a consists of conserved residues present between the β-sheet 3 running into α-helix 8. the rdrp contained active residues d242, f246 and d247 are conserved in motif a of several picornaviruses. motifs a and c located in the 3d pol structurally adjacent to each other contain aspartate residues (d341), involved in a two-metal (mg2 + and/or mn2 + ) mechanism of catalysis [48] [49] [50] . motifs a, b, and f are key for nucleotide triphosphate (ntp) binding, motifs a and c are also involved in binding of metal ions [51] . motif b: it consists of conserved residues as a single block (s1 fig) containing ser305, cys308, thr310, ile312 and asn311 (α-helix 11; fig 4) and are predicted to play a major role in the catalytic process conserved in picornaviruses as well (fig 6) . in the n-terminal of the motif, the coil forms a loop commonly referred to as b-loop (ser, gly, ser and cys), connecting to the long α-helix11. motif b provides important orienting interactions with the incoming deoxyribonucleoside rntp [52] , and the b-loop has been described structurally flexible and moves its conformations [53] , the key residue glycine (g306 in arcv-1) was described to act like a "hinge" for the movement. substitutions of this glycine residue in 3dpol basically abolished rna synthesis in vitro [35] . the high sequence and structural conservation of the b-loop among viral rdrps have been reported [17] . rearrangement residues (sgscftniigsi) of arcv-1 motif b which are conserved in cryptoviruses (s1 fig and fig 6) did not affect the structural sequence identity of the motif with picornaviruses. while describing the function of b-loop in fmdv the authors speculated that the rearrangements in the template channel and the b-loop occur in a concerted manner and that these changes collectively serve to regulate rna replication, processivity and fidelity, and the active residues in motif b, each has a definite role to play [17] . motif b takes part in template binding, incoming nucleotide recognition [39] and determining whether rna or dna will be synthesized by selecting rntps and dntps and correct positioning of the sugar in the ribose-binding pocket [54] , asparagine residue thought to be playing a vital role in its functioning. briefly, asn residue in this motif (asn307 in fmdv) has been confirmed to interact with incoming rntp (play a role in differentiating between dntps and rntps and thus determines whether rna or dna is synthesized) during rna elongation in a number of picornavirus elongation complexes [39, 49, [54] [55] [56] . similar positioning of asn residue (n297) is present in the long helix α-h [39], 6.6å away from the active aspartate of the catalytic center. however, the highly conserved asn residue which is critical for enzymatic activity was not evident in motif b of the arcv-1 rdrp (fig 6) . this is a conspicuous difference between the rdrps of picornaviruses and cryptoviruses. the asn residue appears to be substituted by glycine (g314), present at the same position in the α-11 at a distance of 6.9å away from the active aspartate of the catalytic center and is conserved in most of the dsrna cryptovirus rdrps and also exist in phage փ-6 motif b (gly403). crotty et al. [49] demonstrated that an absolutely conserved residue (asn) of motif b within the nucleotide binding pocket of the poliovirus polymerase can be substituted for a different amino acid, yielding replication-competent virus. glycine that replaced the asn in cryptoviruses does not have amide group [49] . employing one of the pv mutants (pv-297g) developed; authors explained that the molecular modeling suggests that a glycine at position 297 leaves sufficiently a large pocket for an additional water molecule [49] . therefore, glycine may substitute for asparagine 297 by allowing a water molecule to become the hydrogen bonding partner for the ntp 2 0 oh. these results attest to the extreme evolutionary flexibility of the viral rdrp, in terms of both structure and cation usage [49] , and could explain, devoid of asn (fig 6) , in motif b of arcv-1 and other cryptoviruses the polymerase is able to function. motif c is comprised of β-sheets-6 and 7 in an anti-parallel manner connected by a loop towards the central cavity and this loop bares the highly conserved gly340-asp341-asp342 observed in most of the rdrps, now observed in plant cryptic virus rdrps. like the b-loop the beta bend gdd motif is structurally similar in all classes of rdrps, described as the center of catalytic activity of the enzyme in coordinating the metal ion. the first aspartate (asp341in arcv-1) is known to facilitate the rntp transfer and by binding to the metal ion at the catalytic site. it has been shown that first n-terminus aspartate is absolutely required for enzyme function [29, 32, 35, 57, 58] . the presence of the second aspartate almost is universal in gdd motif with a couple of exceptions [replaced by asparagine (adn) in ibdv and glutamate cryptic virus from pigeonpea (gde) in case of kf of e. coli dna polymerase i] signifying flexibility at this position. but any substitutions made in the second aspartate were not tolerated as reported in poliovirus rdrp [35, 58] . phage փ-6 and ibdv (dsrna) rdrps contain the catalytic motif as sdd and adn respectively that makes the requirement for the glycine residue of gdd motif also somewhat flexible in vitro. however, substitutions at glycine position in encephalomyocarditis virus (emcv) 3dpol make the rdrp completely inactive in vitro [35] , as in the case of potatovirus x (pvx) rdrp in vivo were not tolerated [57] , suggesting that the requirements for this position may be more strict [59] , with some rdrps to function. aspartate in motif a interacts in catalytic reaction as they, together with the first aspartate of gdd, coordinate the rntp transfer [60] . arcv-1 3dpol contains a couple of highly conserved aspartate "active residues" in motif a; d242 a and d247 a present at a distance of 12.4å to each other (s3 fig) and the d242 a, 7.0å from d341 are similar to those referred above [60] , could be interacting with asp341 by coordinating catalytically essential metals as noticed in many rdrps [23, 39, 61] . many of the active residues of the conserved motifs (s1 and s2 figs) present in the central conserved region of the rdrp were located in the central cavity or close by. this region forms a domain that is partially resistant to protease digestions [59] . motif d has been identified in arcv-1, 3dpol in a long loop between α-helix12 and α-helix13 (fig 4a and 4b) . a positively charged residue lysine373 along with serene is conserved in this motif (dksd) across all cryptoviruses (s1 fig) . lysine seems to be sustained in the sequences of motif d of all rdrps [20] . motif d is inconsistent and not so well conserved in members of the family picornaviridae and the cryptoviruses analyzed in the present study. structural variability was noticed in the reported rdrps as well, but the majority showed motif d in a coil. the n-terminal asp372 in the motif of the 3dpol was conserved in many primerdependent rdrps of calciviruses and picornaviruses, but not in the rest of cryptoviruses and in de novo rdrps (dsrna-phage փ-6, ibdv and reovirus). active residues in motif d have been postulated to play critical roles in catalysis [62] . motif d for long considered "scaffolding" for the palm, keeping the structural integrity [39, 59] . movement of motif d facilitates active site closure and is sensitive to incorrect ntp binding and mismatches at the rna terminus. these studies link the conformation of motif d to the efficiency and fidelity of nucleotide addition in elongation [63] . closing and reopening of the active site happens as a result of association of motif d with motif a, which undergo coordinated conformational changes [64] . motif e was located at the junction of the palm and thumb, and was described as not integral to the conserved core structure [39] , contains characteristic motif xfl where leucine was predicted as active residue. the conserved sequence at amino acids position 384 to 388 (tflsr) was detected in the arcv-1 3dpol at the mentioned location α-helix turn α-helix as part of the long moving loop structure, adjacent to motif d close to the catalytic center (fig 4b and 4c ). there was variation in the amino acid t384 position in the cryptovirus primary sequence (s1 fig) as well as in picornaviruses. the hydrophobic l386 residue of this motif was found in the analysis to be active residue; described essentially for catalytic activity. this motif described interacts with the elements of β-sheets of the palm core structure and the elements in the thumb subdomain in both poliovirus rdrp and rt [39, 65] . substitution in the vicinity of active leucine residue is not tolerated in polio virus and an insertion of a leucine after r376 of pv (r388 in arcv-1) was shown to abolish replication in vivo and in vitro [30] , and a double mutant k375a/r376a of the same virus was found to abolish viral replication in vivo [66] , suggesting that the critical role these amino acids play in rdrp functioning. thumb subdomain (residue 399 to residue 475) constitutes mainly of helical structures. out of four α-helices, α-13 and α-14 helices adjacent to each other runs antiparallel while α-16 helix runs across and ends with protruding gln 475 residue (fig 4a) . the α-13-α-14, and α-15-α-16 are interconnected resulting in larger loops at the crest of the thumb subdomain and bending over forward to tie into the n-terminal region of the finger tips, creating a complete annulus around the catalytic site on the palm (fig 4a) . the small size of the domain contributes to the formation of a large central cleft [45] , which is located at the front of the molecule (fig 4a, 4c and 4d ) and leads to the active site shown in the surface representation of the molecule. arcv-1, 3d pol visually seems to be having similar central cavity architecture and dimensions with fmdv, pv and hrv-16. the c-terminus region is conserved amongst the cryptoviruses studied showing conserved residues in motifs that are not observed in the single stranded picornaviruses or in the dsrna viruses. clrml (402-06) motif as part of α-13, facing the central cavity, the rest ypey (408-11), dag (429-31), and wpd (461-63) motifs are present in the remaining 3 helices. the overall topology of arcv-1 3d pol smaller thumb subdomain structure resembles more with ssrna (+) rdrp analogs of nv, sv, hrv, rhdv, pv and fmdv than the de novo dsrna viruses. the main structural differences between rdrps of arcv-1 and the closely related fccv and rocv-1, rmcv, rscv-2 (deltapartitivirus) are in the thumb region ( fig 5a-5d) . the thumb subdomain of these cryptoviruses contained four α-helices (α-α-α-α) on the contrary fccv thumb had an extra helix making total thumb α-helices to five (β-β-α-α-αα-α). the second difference noticed was the palm and thumb interface region. arcv-1 3d pol, consists of long coiled loops connecting α-12 (palm) and α-13 (thumb) with no β-sheets in this region. while the rdrps of other cryptoviruses consists of two antiparallel β-sheets followed by motif e as was observed in nv and sv (fig 5a-5d ) making the thumb configuration as β-β-α-α-α-α. the thumb c-terminus observed in arcv-1 3dpol was protruded out away from central cavity like other cryptoviruses except in case of rscv-1 (alphapartitivirus) where the c-terminus helix (α-16) extends in to a compact tuft of coil packs against the front face of the molecule and the end pointing towards the central cavity, a similar structure (s3 fig) seen in rice tungro virus [rtsv; ssrna (+)] a plant picornavirus and nv (s3d fig, fig 5d) . the c-terminus of rmcv (eu024675-77) genomic segments has a unique poly "a" tract. flaviviruses, ibdv, bacteriophage փ-6and reo (dsrna) viruses with larger polymerase molecule contain elaborate thumb architecture with 7 to 15 α-helices and 2-4β-sheets. the phage phi6 pol c-terminal extends back ploughing parts of it through the molecule and ends in the middle finger region of the other side [12] . interestingly, substitutions or deletion of specific residues in the thumb subdomain of brome mosaic virus (bmv 2a) and poliovirus 3dpol abolish rna replication [30, 67] . the analysis showed arcv-1 rdrp structural alignment with the viruses belonging to picornaviridae and deltapartitiviridae (fig 5 and s3 fig). the range of identity varied with the ranking viruses which had high identity with the rdrp of arcv-1. different genera of picornaviruses showed high template modeling (tm) score with arcv-1 3d pol; nv (0.898) [68] , sv (0.898) [69] ,rabbit hemorrhagic disease virus (rhdv) (0.895) [70] , all members of the family caliciviridae (fig 5g and 5h ) and with moderate tm score with alignments of viruses members of the family picornaviridae; fmdv (0.834) [33] , emcv (0.830) [71] and poliovirus (pv) (0.823) [39] . these economically important viruses are human and animal pathogens and the structural details are available in pdb archives which doesn't have the information on the rdrps of cryptic viruses to compare. for the first time using computer analysis we are reporting structural details of rdrps of few plant cryptic viruses with high structural similarity to arcv-1 3dpol (fig 5e and 5f ). plant cryptovirus and human picornovirus rdrps that showed structural identity were superimposed on arcv-1 3d rdrp in a cartoon and backbone representations. arcv-1, 3dpol (blue) was superimposed by fccv (green) rdrp (fig 5e) in a cartoon representation showed exact structural alignment. similar high-level alignment was noticed when arcv-1 3dpol (blue), superimposed by fccv (grey) rdrp (fig 5f) , by sv (green) rdrp (pdb id, 2wk4a) ( fig 5g) and by nv (brown) rdrp (pdb id, 2b43d) ( fig 5h) in a backbone representation. every 40 th residue from n-terminal to c-terminal was numbered in a stereo back the bone representation of the rdrps. a high degree of alignment was noticed arcv-1, 3dpol with fccv and sv followed by nv especially in the palm region containing the a to e motifs with conserved residues. structural comparison analysis provided for sv and nv an rmsd value of 2.10å and 2.15å, respectively. our findings validate the analysis predictions and reveal a possible evolutionary linkage between cryptoviruses (dsrna viruses) and the picornaviruses (+ssrna viruses). phylogenetic analysis. blast analysis involving several tripartite cryptoviruses revealed that the arcv-1 genome shared a low level of identity with the orthologs, mentioned in table 2 . besides 5' terminus 16 nt similarity, a high degree of sequence homology between the arcv-1 5'ntr sequences of the three genome segments was noticed while lacking decipherable identity in the 3'proximal un-translated regions. the genomic segments of several partitiviridae members usually have sequence homology in the terminus ends [72, 73] . arcv-1 rdrp (dsrna-1) and dsrna-2a sequences were used in the phylogenetic analysis with corresponding sequences of members of the genera alphapartitivirus, betapartitivirus, gammapartitivirus, deltapatitivirus and cryspovirus (fig 7) mentioned in the s1 table. the analysis revealed that the viruses used in the study assembled in five groups, as recognized previously [8] . arcv-1, dsrna-1 clustered with tripartite rocv-1, fcv and fccv and with members of the genus deltapartitivirus (fig 7a) . phylogeny of dsrna-2a revealed close relationship with members of bipartite deltapartitivirus; pepcv-1, pepcv-2, rscv-3 and cilcv (fig 7b) . the findings further support the hypothesis that dsrna-2a does not belong to the tripartite arcv-1. in this study, the complete sequence of four dsrnas isolated from asymptomatic pigeonpea plants was determined. three dsrna segments formed the genome of a new cryptovirus and named arhar cryptic virus-1 (arcv-1). the fourth dsrna (dsrna-2a; p4) was not related to the arcv-1 genome and has characteristics like a viral coat protein. arcv-1 dsrna-1 dsrna-2 and dsrna-3 have been identified as encoding rdrp, cp-1 and cp-2, respectively (fig 2a) . concentration of dsrnas varied, dsrna-1 (rdrp) was the most predominant transcript followed by the two capsid proteins (fig 1) . the arcv-1 genome segments shared high degree of sequence identity in the 5'ntr including the marker 5'terminus 16 nt conserved sequences. similar conservation in the tripartite viruses (fig 3) was noticed; fccv, rocv-1, rscv-2 [7, 74, 75] , and were clustered together phylogenetically to form the genus deltapartitivirus, a member of the family partitiviridae (fig 7a) . the 3' ntr (50 nt) was rich in gc content and the three dsrnas of arcv-1 terminated with tc (fig 2, s1 fig) . furthermore, these findings were supported by a phylogenetic analysis which revealed that arcv-1 formed a unique phylogenetic cluster with fccv, rmcv and rocv-1 within the genus deltapartitivirus, infecting different genera of the family rosaceae. the status and role of dsrna-2a which encodes 44.5kda protein (p4), which was found in least concentration, is not known. association of an additional dsrna segment (s), discrepancies with cryptic virus infections was not uncommon. in an earlier finding, the association of an extra component in many vicia faba cultivars infected with vicia crypticvirus (vcv) was reported [72, 76] . while characterizing the cryptovirus infection of radish (raphanus sativus-root cv.yidianghong) the authors suggested that more than one partitivirus was co-infecting radish leaves [73, 75] . the second virus in the complex has two smaller segments (rasr4 and rasr5) contained conserved 5' termini sequence with rasr3 (rdrp) deem to be with unknown functions not having sequence identity with the known capsid proteins. in the present study, the four dsrna shared sequence identity with members of deltapartitivirus. the dsrna-2a, (p4) contains sequence that resembles viral coat protein showed an unusually long 3'-terminal 353nt ntr region. the 5'-terminus of this dsrna is different, does not share sequence identity with the 5'-terminus conserved genome segments of arcv-1. however, p4 showed sequence identity with bipartite and tripartite cryptovirus coat proteins with pronounced resemblance to the bipartite cryptoviruses (table 3) . phylogenetic analysis indicated that the p4 was closely related to rscv-3 segment-2; coat protein (fj461350). this indicates that the origin of p4 is related to partitiviridae, but we have no evidence that p4 may interact with arcv-1 in its replicative cycle or it is a derivative of rearranged sequences of the arcv-1 coat proteins-highly unlikely or it could be part of the genome of evolving yet another cryptic virus. members of group 1 and 2 clustered separately but originate from common point however origin of members of group 3 were different. group 4 and 5 originate from common sub-clade, which is different from other groups. arcv-1 rna-1 clusters with tripartite members of the genus deltapartitivirus and is closely related to fccv and rscv-2. *beet cryptic virus-2 was an assigned member of deltapartitivirus but it was noticed to fit in with alphapartitivirus. (b) phylogenetic analysis of dsrna-2a (p4 protein), displays its proximity with bipartite pepcv-2 and rscv-3. the bar represents base substitution per site. sequences of different partitiviruses from ncbi database were used in the analysis (s1 table) . rdrps of hpbv (ab186898) and opbv (jq776552) and cp sequence (hpbv: ab186897 and q776551) were used to provide an out group root in the analysis. https://doi.org/10.1371/journal.pone.0181829.g007 primary sequence analysis of arcv-1 rdrp revealed, besides several conserved sequences amongst the cryptoviruses; at least seven conserved signature motifs in a particular order which were well characterized [20, 28, 59] . these motifs were aligned with the rdrps of picornaviruses ssrna (+) that led us to examine the structural similarity, if any, of arcv-1 3dpol with known rdrps in further detail. however, detailed studies of rdrps encoded by partitiviruses have not been reported. amongst the double-stranded viruses studied; representatives of cystoviridae [12] , reoviridae [13] , and birnaviridae [14, 77] , in terms of the structure, mechanism of initiation of replication and transcription by their active rdrp was explained with the 3d structural analysis. this report illustrates several interesting features of the arcv-1 rdrp and a cryptovirus 3d pol and full-length structure was determined for the first time. it was revealed that the 3d pol shared structural details more with calciviruses, members of the family picornaviridae. computational analysis was used to develop and characterize arcv-1 3d pol structure at 7.9 å resolution. we studied several rdrp analogs in the pdb which showed homologous structural identity with arcv-1 rdrp mostly of animal and human picornaviruses. unlike the genomic rna of picornaviruses, the arcv-1 dsrna-1 that encodes rdrp (55kda) protein does not show 5' cap or vpg or 3' polyadenylation, but a stretch of ntrs present on both the termini with no internal structures. the 3dpol structures of norwalk virus, poliovirus and sapporo virus which were previously determined and the respective sequences used, produced identical 3d structures in our computer analysis, confirming authenticity of arcv-1 structural analysis. the overall topology of the arcv-1 rdrp structure resembles the general description of the three-dimensional structures of the primer dependent initiating single stranded rna (+) rdrps [33, 39, 45, [68] [69] [70] [71] [78] [79] [80] , depicted as a closed right hand (fig 4a) , differentiated as fingers, palm and thumb sub domain [52] . 3d pol structure of arcv-1 and other cryptoviruses analyzed are found to be different from the dsrna viruses; bacteriophage փ-6, reovirus and ibdv [12] [13] [14] 77] . in general, the topology of the cryptoviruses; bcv-2, fccv, rmcv, rocv-1, rscv-2, rscv-3 (deltapartitivirus) and rscv-1 (alphapartitivirus) rdrps, had close identity with arcv-1 with minor differences. the arcv-1 rdrp sharing 72.4% sequence identity with fccv and only 14.8% sequence identity with rscv-1 (fig 4) . despite low amino acid identity (15%), interestingly the arcv-1 rdrp shares more structural identity with the members of the family caliciviridae; nv and sv, (fig 5, s3 fig) . the structural identity of nv at 2.17 å resolution (1sh0, 2b43) and sv at 2.3 å resolution (2ckw) 3d pol with the arcv-1 rdrp, indicate a closer evolutionary link between the very divergent groups of viruses, and the polymerase has no structural alignment with other dsrna, bacteriophage փ-6, reovirus and ibdv which have larger rdrp molecules with complex dense subdomain structures. the analysis of rdrp sequence and structure revealed the occurrence of motifs a to g which play an important role in ntp binding and catalysis [70] , described for calicivirus and picornavirus rdrps has structural similarity ( fig 4b) as described earlier. the similarity was confirmed by the visual observation of their superimposed structures (fig 5e and 5f ). the unique feature noticed in arcv-1 rdrp is an n-terminal portion of the fingers reaches to the thumb bridging the fingers and thumb sub domains [43] , was observed with all cryptoviruses studied. this feature was noticed in ssrna (+) and dsrna rdrps visualized in less cluttered ibdv rdrp. fingers subdomain contains motifs f and g. motif f of cryptoviruses unlike the other classes of rdrps is well conserved and extends from few residues before β-sheet 2 to αhelix 6 and the connecting coils. computer analysis predicted that motif f contains several active residues supposed to be interacting with the rna in the arcv-1 3d complex were detected (fig 5b, green colour, s1 fig) . basic residues lys181 and arg183 of this motif were also conserved in other rdrps; for example, in fmdv motif f, lysine residue along with two arginine residues (k172, r168 and r179) predicted to interact with the incoming ntp substrate [33] . motif g forms part of a long coiled loop connecting α-helices 4 and 5 in the fingers subdomain (fig 4b, red) . motif g is highly conserved in the cryptoviral rdrps. the conserved gly121 and lys128 (in arcv-1) of this motif in all classes of rdrps seem to be a strict requirement of motif g. mutational analyses in pv and fmdv also have demonstrated that some substitutions in residues located far from the active site, in particular at the rdrp n-terminus, have significant effects on catalysis and fidelity [17] . several highly conserved stretches of sequence motifs of arcv-1 in the n-terminus to the central region, have been identified uniquely to cryptic virus rdrps. motifs 1 and 2 are observed in the rdrp sequence away from the palm subdomain could play a role in catalysis (s1 fig). the palm sub domain contains a to e motifs in a series. β-sheet 3 to α-8 consists of residues 241-251 were identified as motif a (fig 4b, blue color) contains conserved asp242 and asp247. like sv and nv, rhdv 3d pol also has a high degree of structural homology with arcv-1 (fig 5 and s3 fig) and the aspartate residues share identical positions in motif a and c (asp a 250 and asp c 354 in rhdv). it has been described that metal ions that are likely to be involved in the nucleotide transfer reaction, interact with aspartate residues at positions 250, 354, and 355 in rhdv [70] . similarly, in arcv-1 3d pol the corresponding two aspartate residues asp a 242 and asp a 247 which are separated by a distance of 12.4å and asp a 242 being closer (7.0 å) to the catalytic aspartate asp c 341, could be performing the same functions. ser, gly, thr and ns/t are identified as highly conserved active residues in motif b (fig 4b, purple), each with specific function in many picornaviruses. asn residue (asn307 in fmdv) of motif b plays a role in differentiating between dntps and rntps and thus determines whether rna or dna is synthesized [39, 55] . later it was determined that the active asp residue of motif a, and asn of motif b (asp240, asn307 in fmdv) play a critical role in rntp selection as evident from the fmdv replication process [33] . asparagine residue is highly conserved in the long helix, about 6.6å away from the active aspartate of the catalytic center in picornaviruses. on the contrary, the asn residue was substituted by glycine (314) present almost at the same position (fig 6) , in the α-helix (α-11 in arcv-1), conserved in most of the dsrna cryptovirus rdrps. in poliovirus mutational studies, asn (297) was substituted by glycine (pv-297g) resulted in replication-competent virus, which probably explains how cryptoviruses are able to multiply with altered functional residue (gly314) involved in rntp selection (s3a fig) [49] . rearrangement (sgscftniigsi) of few residues of the arcv-1 motif b which are conserved in cryptoviruses did not affect the structural similarity of the motif with picornaviruses. the architecture of the palm subdomain of arcv-1 3d pol with three β-sheets (β-3, β-6 and β-7) sandwiched between two α-helices (α-7 and α-12) is highly conserved feature in most of the rdrps as motif c (fig 4b, pale pink) . the anti-parallel β-sheets 6 and 7 connected by a short loop containing gdd (asp 341 and asp 342) sequence described as catalytic center. structural properties and catalytical aspects of palm and thumb (residue 399 to residue 475) sub-domains are discussed in greater detail in the results section. understanding structural details of known viral rdrps and associated mechanism described for the initiation of rna synthesis and chain elongation reveal, that these viruses use different strategies to initiate replication. it has been described that viral replication can be initiated by two principally differing mechanisms; the primer-dependent [42] , and primer independent or de novo initiations [81] [82] [83] . however, influenza virus employs a combination of the two mechanisms with the choice being determined by the type of rna to be synthesized [84] . the de novo synthesis involving the interactions of required components, the initiation essentially a one-nucleotide primer provides the 3'-hydroxyl for the addition of the next nucleotide [42] . the de novo rdrps have specific structural elaborations that function to stabilize the initiation complex. such initiation platforms have been found in the crystal structures of viral rdrps of cystoviridae, reoviridae and birnaviridae [12, 13, 77] . in the second mechanism, the primer-dependent initiation requires the use of either an oligonucleotide or a protein primer to provide the hydroxyl nucleophile. members of the picornaviridae family use exclusively the protein-primed mechanism of initiation [17, 81] . viruses belonging to different groups containing ssrna (+), dsrna as genomes adopt de novo initiation mechanism. for example, members of the genus flavivirus: hepatitis c virus (hcv) [85] , and dengue virus (denv-2) [86] , as well as enterobacteria phage q beta (qb) [87] , all have ssrna (+) genome. the second group to adopt de novo initiation are the dsrna viruses namely, cystoviral bacteriophage փ-6 [12] , infectious bursal disease virus(ibdv) (family birnavirdae), [14, 77] , and reovirus (family reoviridae) [13] . by contrast, very little is known about knowledge of the mechanisms underlying dsrna cryptovirus (partitiviridae) replication as the polymerases of this group have never been studied. arcv-1 has segmented linear dsrna genome, each genomic segment is monocistronic: dsrna-1 that encodes rdrp does not have either 5' termini or 3'-end structures like templatedirected nucleic acid polymerases and does not resemble the vastly intricate 3dpol structure of known dsrna bigger molecules that adopt de novo, to speculate the polymerase preferred mechanism. unlike the de novo rdrps, primer dependent rdrps of the viruses of caliciviridae and picornaviridae have significantly smaller thumb subdomains, wider template tunnels and large central cavity with exposed active sites [33, 45] as observed in arcv-1 3d pol. fmdv rdrp was shown to have the wide enough central cleft to accommodate the bulky priming peptide during the initiation of rna (complexed with rna, divalent cation and protein primer vpg), synthesis [46] . similar structural details of the polymerase mentioned above share a number of unique features by arcv-1 and other cryptoviruses (fig 4a, 4c and 4e ). if the structural architecture reflects the functions of a polymerase as often supposed [17] , the simple thumb organization, arrangement of the motifs (a to g) in a structural order (fig 4b) , large central cavity and the entry and exit channels like in picornaviruses and having remarkable structural conservation with sv, nv and rhdv 3dpol, lead to a strong possibility that arcv-1 rdrp could adopt primer dependent initiation mechanism. this important aspect of research of cryptovirus rdrps is yet to be explained. the remarkable structural similarities between arcv-1 and the other tripartite cryptoviruses (dsrna) and the positive-stranded rna viruses of the picornavirus family, in particular, caliciviral rdrps, offer evidence of probable functional and evolutionary relationships between these two virus groups. several highly conserved stretches of sequence motifs have been identified that seem unique to cryptic virus rdrp; motifs 1-2, observed in the rdrp sequence away from the palm subdomain are inimitable and observed in both bi-and tripartite cryptoviruses (s1 fig) , whose exclusive or overlapping functions are yet to be determined. we hope in the near future, biochemical analysis experiments would reveal the function (s) and their relevance in rna synthesis. substantial data that has been generated on arcv-1 3d pol is being authenticated by xray crystallography studies and biochemical analysis. leaf samples of pigeonpea were collected from fields near the chevella area of hyderabad; (telangana state, india). a popular local pigeonpea cultivar erra kandulu, is being traditionally cultivated in this area which is susceptible to sterility mosaic disease (smd), fungal blight, and wilt diseases. leaves from four healthy looking plants, with no disease symptoms, were collected randomly from three fields mg-1, mg-2 and mg-3 (approximately three km apart). collected leaf samples were designated as mg-h1, mg-h2 and mg-h3 respectively, along with the smd samples. leaf samples were placed in separate ziplock bags placed in ice chest with dry ice and transported to the lab. for sample collection, no specific permissions were required from these locations, as these were collected from a private farm land. also, this study did not involve endangered or protected plant species. leaf samples, mg-h1 mg-2 and mg-h3 were used for dsrna extraction [88] . briefly, 7g leaf tissue were crushed to fine powder in liquid nitrogen and homogenized in 20 ml of 2× ste extraction buffer (0.1 m tris-hcl, ph 7.0; 0.2 m nacl; 2 mm edta), containing 1.5% β-mercaptoethanol, 1.5% (w/v) polyvinylpyrrolidone (pvp), 2% (w/v) sds, and 16 mg of bentonite powder and incubated at 37˚c for 15 min. the clarified contents were extracted with equal volume of tris (ph 8; phenol ph 4.5 ± 0.2) saturated phenol: chloroform: isoamyl-alcohol (25:24:1) mixture followed by vigorously shaking at room temperature for 20 min. the contents were centrifuged at 11,000 rpm (heraeus multifuge 35r+, thermo scientific, usa) for 10 min. one fifth volume of ethanol was added to the supernatant and mixed well, followed by the addition of 1g of cellulose cf-11 (whatman, usa) and the slurry was incubated at room temperature for 30 min with continuous shaking. the mixture was centrifuged at 5,000 rpm for 5 min. and the cellulose pellet with dsrna was resuspended in 40 ml wash buffer (1× ste with 16% ethanol) and washed twice for 5 min with mild agitation. the cellulose slurry was applied to a 20 ml syringe blocked with glass wool and washed with 20 ml of washing buffer. the bound dsrna was eluted stepwise (two times) with 15 ml elution buffer (1× ste without ethanol). the eluted dsrna was further purified by adding 1/5 th volume of ethanol and 0.8 g cf-11 cellulose and the suspension was vigorously shaken for 30 min at room temperature. the suspension was transferred to a new 20 ml syringe and the dsrna was eluted again twice with 2.5 ml elution buffer. the extracted dsrna was precipitated by adding 1/10 th volume of 3 m sodium acetate ph 5.2, and 2.5 volumes of absolute ethanol and incubated overnight at -20˚c. the contents were centrifuged at 14,000 rpm for 20 min at 4˚c and the resulting dsrna pellet was washed with 80% (v/v) ethanol, air dried and resuspended in 500 μl milliq water. mgcl 2 was added to the extracted dsrna to a final concentration of 300 mm and was digested with 500 ng rnase a and 2u dnase i (rnase-free) at 37˚c for 45 min followed by phenol/chloroform extraction and precipitating with ethanol. the precipitates were suspended in 500 μl of milliq water. purified dsrna were separated by electrophoresis on a 1.5% agarose gel in 1x tae buffer and stained with ethidium bromide and documented by gel doc system. the obtained dsrna was resolved on 1.5%agarose gel and the dsrna segments were excised. dsrna molecules that were close to each other were excised together as a mixed population. these dsrnas were further purified by gel elution kit (macherey nagel, germany). 200-300 ng of dsrna was used for self-priming anchor primer ligation in a 60 μl reaction with t4 rna ligase (fermentas, thermo scientific, usa). self-priming oligo (5 0 p-gacctctgagga ttctaaac/isp9/tccagtttagaatcc-oh 3 0 ), having c9 internal spacer region (isp9) was used for ligation at 3 0 end -oh group of dsrna [89] . the ligation buffer [90] was modified to contain 50 mm hepes/naoh buffer ph 8.0, 15 mm mgcl 2 , 0.01% bsa, 0.75 mm atp (fermentas, thermo scientific, usa), 1.5 mm dtt (fermentas, thermo scientific, usa), 8% dmso (sigma, usa), 15% polyethylene glycol 6000 (peg 6000 ) (himedia, mumbai, india) and 10 u t4 rna ligase (fermentas, thermo scientific, usa). ligation was performed at 37˚c for 12-16 hrs. ligated dsrna was purified using gel extraction columns (macherey-nagel, germany) in 15 μl volume. about 50-70 ng of ligated dsrna were denatured in the presence of 1.5% dmso and 2m betaine at 98˚c for 2 min (optimal) and immediately chilled on ice. the denatured dsrna was used for the first strand cdna synthesis by reverse transcription (rt) reaction. the rt reaction mixture consisted of 1x rt buffer, 1 μl dntps (10 mm each), 1 μl (200 units) of rtase (primescript, takarabio, japan) and the final volume was adjusted to 25 μl with nuclease free water. the rt reaction mixture was incubated at 42˚c for 1 hr and then 70˚c for 15 min to deactivate the enzyme. the cdna obtained was amplified by a primer (5 0 -gagggatccagtttagaatcctcagaggtc-3 0 ) complementary to anchor sequence [89] . briefly, pcr reaction contained 25 μl reaction mixture [1.5 μl of first strand cdna, 1.0 μl of 2.5 mm dntps mix, 1x gc melt buffer, 0.5 μl of 10 pmole primer and 0.2 μl (1unit) gc la taq (takarabio, japan)]. amplification cycle was: 94˚c for 1min followed by 35 cycles of denaturation at 94˚c for 30 s, annealing at 62˚c for 30 s and extension at 72˚c for 2.30 min. final extension was carried for 72˚c for 5 min. rt-pcr amplicons were resolved on 1% agarose gel containing ethidium bromide and visualized in gel doc. amplicons corresponding to the dsrna bands were excised from gel, purified using genelute gel extraction kit (sigma-aldrich, usa) and cloned into pgemt-easy vector (promega, madison, wisconsin, usa). recombinant plasmids were purified using genelute plasmid miniprep kit (sigma-aldrich, usa) and the target cdnas were sequenced in an automated dna sequencer (abiprism 1 3130xl genetic analyzer) using abi prism big dye™ terminator v3.1 ready reaction cycle sequencing kit (applied biosystems, usa), with 2x coverage. nucleotide sequences were translated using expasy server (http://web.expasy.org/translate), and the obtained amino acid sequence was used to determine homology by blastp (http:// www.ncbi.nlm.nih.gov/blast) analysis. we studied the three dimensional crystal structure of rdrp using computer aided analysis. the amino acid sequence of arcv-1 (table 1) , rdrp (p1), was used to develop 3d structure with characteristic folding. i-tasser (http://zhanglab.ccmb.med.umich.edu/i-tasser), an online server was used to study the rdrp [37, 38] . this integrated platform generated threedimensional atomic models from multiple threading alignments and iterative structural assembly simulations. data provided on topology similarity (tm) with the proteins structurally close to the target protein (s) in the pdb is helpful contrivance to assess the relationships of the polymerase. the outputs of the i-tasser data of possible predicted models and the molecular graphics are of high quality that contained full-length secondary and tertiary structure predictions. developed structures were subjected to simulations by an open-source viewer, the pymol (https://www.pymol.org) and jmol (http://jmol.sourceforge.net/) an interactive graphics program [91, 92] , for illustrating the three-dimensional (3d) chemical structures of the crystal. these programs were used to alter the scheme of the images to characterize the proteins. using this program, individual and conserved residues in the motifs were identified which facilitated understanding over all nature of the rdrp. structural adjustment was made for this model of arcv-1 3d pol of about 90˚to study from top surface and side ways to visualize the orientation of channels. amino acid sequences of 3dpol structures of norwalk virus (1sh0), poliovirus (4r0e) and sapporo virus (2wk4a) from pdb and rtsv sequence (ncbi np734463) was used in the analysis and to develop 3dpol structures. multiple alignments and sequence identity matrix of rdrp and dsrna capsid protein-like sequences were carried out by clustalw [93] . to deduce the evolutionary relationship, the phylogenetic tree was prepared by mega 6.0 program [94] , by using neighbor-joining method [95] , and evolutionary distances were computed using the maximum composite likelihood method with 1000 bootstrap value [96] . the bar represents base substitution per site. sequence identity matrix and phylogeny were studied by using ncbi genbank database (http://www.ncbi.nlm.nih.gov/genbank). an annotated bibliography of pigeonpea diseases the occurrence of mosaic mottle, a viroid disease of pigeonpea (cajanus cajan) in india deep sequencing of dsrnas recovered from mosaic-diseased pigeonpea reveals the presence of a novel emaravirus, pigeonpea sterility mosaic virus 2 first report of tobacco streak virus infecting pigeonpea (cajanus cajan) in india cryptic plant viruses virus taxonomy: eighth report of the international committee on taxonomy of viruses molecular characterization of a tripartite cryptic virus from rose taxonomic reorganization of family partitiviridae and other recent progress 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general acid for nucleotidyl transfer motif d of viral rnadependent rna polymerases determines efficiency & fidelity of nucleotide addition structural basis for active site closure by the poliovirus rna-dependent rna polymerase crystal structure at 35 å resolution of hiv-1 reverse transcriptase complexed with an inhibitor clustered charged-to-alanine mutagenesis of poliovirus rna-dependent rna polymerase yields multiple temperature-sensitive mutants defective in rna synthesis deletion analysis of brome mosaic virus 2a protein: effects on rna replication &systemic spread crystal structure of norwalk virus polymerase reveals the carboxyl terminus in the active site cleft structural and functional characterization of sapovirus rna-dependent rna polymerase crystal structures of active and inactive conformations of a caliciviral rna-dependent rna polymerase the crystal structure of a cardiovirus rna-dependent rna polymerase reveals an unusual conformation of the polymerase active site 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fast and scriptable molecular graphics in web browsers without java3d clustal w and clustal x version 2.0 mega6: molecular evolutionary genetics analysis version 6.0 the neighbor-joining method: a new method for reconstructing phylogenetic trees confidence limits on phylogenies: an approach using the bootstrap the authors are thankful to the director, csir-institute of himalayan bioresource technology, palampur, himachal pradesh, india for encouragement and providing the necessary facilities to carry out the work. authors are highly thankful to prof. ahmed hadidi, united states department of agriculture, agricultural research service, beltsville, md, usa, (emeritus lead scientist) for critical reading and language editing of the manuscript. sk acknowledges academy of scientific and innovative research (acsir), india for phd registration. csir-ihbt publication number of this manuscript is 3663. key: cord-271660-5sfkhg19 authors: sun, hsin-yun; wang, jann-yuan; chen, yee-chun; hsueh, po-ren; chen, yi-hsuan; chuang, yu-chung; fang, chi-tai; chang, shan-chwen; wang, jung-der title: impact of introducing fluorescent microscopy on hospital tuberculosis control: a before-after study at a high caseload medical center in taiwan date: 2020-04-03 journal: plos one doi: 10.1371/journal.pone.0230067 sha: doc_id: 271660 cord_uid: 5sfkhg19 background: undiagnosed tuberculosis (tb) patients hospitalized because of comorbidities constitute a challenge to tb control in hospitals. we aimed to assess the impact of introducing highly sensitive fluorescent microscopy for examining sputum smear to replace conventional microscopy under a high tb risk setting. methods: we measured the impact of switch to fluorescent microscopy on the smear detection rate of culture-confirmed pulmonary tb, timing of respiratory isolation, and total non-isolated infectious person-days in hospital at a high-caseload medical center (approximately 400 tb cases annually) in taipei. multivariable cox regression was applied to adjust for effects of covariates. the effect attributable to the improved smear detection rate was determined using causal mediation analysis. results: after switch to fluorescence microscopy, median non-isolated infectious duration decreased from 12.5 days to 3 days (p<0.001). compared with conventional microscopy, fluorescence microscopy increased sputum smear detection rate by two-fold (for all patients: from 22.8% to 48.1%, p<0.001; for patients with cavitary lung lesion: from 43% to 82%, p = 0.029) and was associated with a 2-fold higher likelihood of prompt respiratory isolation (odds ratio mediated by the increase in sputum smear detection rate: 1.8, 95% ci 1.3–2.5). total non-isolated infectious patient-days in hospital decreased by 69% (from 4,778 patient-days per year to 1,502 patient-days per year). conclusions: in a high tb caseload setting, highly sensitive rapid diagnostic tools could substantially improve timing of respiratory isolation and reduce the risk of nosocomial tb transmission. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 pulmonary tuberculosis (tb) is an airborne disease [1] . unless promptly isolated, hospitalized patients with active pulmonary tb can transmit to both healthcare workers (hcws) and other patients [2] . hcws could have an incidence rate of active tb 2-to 20-fold higher than that in general population [3] . the discovery of hospital-acquired extensively drug-resistant tb, with an extremely high mortality among hiv-positive patients, further highlights this deadly hazard [4, 5] . rapid isolation of hospitalized patients with pulmonary tb is the pivotal step to prevent nosocomial transmission [6] . however, undiagnosed tb patients hospitalized for treatment of comorbidities constitute a challenge to tb control within hospitals. clinical predictive rules had been proposed to guide the decision to implement respiratory isolation [7] . an expanded isolation policy which pre-emptively isolated all patients with possibility of tb achieved immediate isolation of >95% of patients with tb in low tb risk settings [8] . nevertheless, the same policy would be impractical in a high tb risk setting because there would be too many patients to be pre-emptively isolated [9, 10] . for a laboratory diagnosisbased respiratory isolation policy, an important barrier is the limited sensitivity of conventional sputum smear [11, 12] . compared with conventional microscopy, fluorescence microscopy has a superior sensitivity for detecting tb bacilli [13] . in 2011, world health organization (who) recommends switching conventional to fluorescence microscopy [14] , under the condition that the switch should be carefully planned at country level with training, quality assurance, and monitoring on tb detection rate [14] . who also endorsed highly sensitive tb nucleic acid amplification test (tb-pcr) such as xpert 1 mtb/rif (cepheid, sunnyvale, ca) [15] . point-of-care xpert 1 mtb/rif reduces all-cause 12-month mortality in patients positive for tb symptoms at the time of hiv diagnosis [16] . nevertheless, to our knowledge, the effect of introducing these more sensitive diagnostic tools on reducing risk of nosocomial tb transmission has not been documented. taiwan is a developed country with an incidence of tb at the range of approximately 70 per 100,000 general population in 2001 [17] . in 2003, severe acute respiratory syndrome (sars)related chest radiograph screenings led to the discovery of a large nosocomial tb outbreak at a rehabilitation facility in taipei, involving 65 cases of active tb in hcws [18] . the index case of this outbreak was an elderly patient hospitalized for stroke rehabilitation without suspicion of tb [18] . subsequent investigations found that the problem of delayed isolation of undiagnosed tb patients also existed in other hospitals [19, 20] . in response, taiwan centers for disease control (cdc) issued guidance on nosocomial tb control [21] . to facilitate laboratory diagnosis-based isolation, medical center a started to roll out auramine-rhodamine staining with fluorescence microscopy since 2006 and completed the switch by early 2010s. we aimed to assess whether switching from conventional microscopy to a more sensitive rapid diagnostic tool improves early detection and prompt isolation of hospitalized patients with undiagnosed tb. medical center a, a leading university-affiliated general hospital having the second-highest tb caseload (approximately 400 cases annually) in taiwan, was chosen for this study. the center in taipei had a 2,200-bed capacity and provided both primary and tertiary referral care reimbursed by national health insurance (nhi). the service amount steadily increased from 2001 to 2014. there were 3,454,724 outpatient visits and 91,645 admissions in 2014, nearly 2-fold than that in 2001. medical center a followed the guidance on hospital respiratory isolation policies issued by taiwan cdc [21] . contact investigation had been expanded to all hcws who were exposed to tb patients since 2004. sputum smear auramine-rhodamine staining with fluorescent microscopy started in 2006. the national laboratory personnel training programs for quality assurance of acid-fast staining [21] , mandated by taiwan cdc, helped to ensure the technical proficiency and performance quality of fluorescence microscopy (see details in s1 table) [22] . this before-after retrospective cohort study included all hospitalized patients with cultureconfirmed pulmonary tb in 25 wards/units in 2001 and those in 2014. we compared the duration from admission/arrival to respiratory isolation in 2001 (conventional microscopy with ziehl-neelsen staining, represented the baseline situation before 2003 sars outbreak) with that in 2014 (after full switching to fluorescent microscopy with auramine-rhodamine staining and the quality assurance program). cox regression was used to adjust for effects of covariates. the effect mediated by improved smear detection rate was precisely identity using causal mediation analysis. the study procedure and exemption of informed consent were reviewed and approved by research ethics committee of national taiwan university hospital (taipei, taiwan). for each included tb case, medical and administrative records were reviewed to determine the infectious duration. a computerized data collection form was used to systematically collect the following information from the medical records: demographic data, sputum smear and culture results, presentations, comorbidities, reasons of hospitalization, and other relevant data, with pre-defined criteria. hospitalized patients had typical presentations of pulmonary tb if they had: (a) a prolonged cough for >3 weeks; (b) clinical suspicion of pulmonary tb based on chest radiography, such as cavitary pulmonary lesions, upper lobe diseases, or miliary lesions; or (c) already received a confirmed diagnosis of pulmonary tb by a positive sputum culture of mycobacterium tuberculosis, positive acid-fast stain (afs), or positive tb pcr, before the hospitalization. the hospitalization was considered as tb-related if the chief complaint suggested an infectious etiology or the admission was for inpatient tb treatment. the hospitalization was considered comorbidities-related when the patients was admitted for management of acute complications of non-infectious diseases, such as myocardial infarction, pulmonary edema, malignancy, or acute exacerbation of chronic lung diseases. we retrospectively identified all cases of culture-confirmed pulmonary tb patients in 2001 (january 1 to december 31, 2001 ) and in 2014 (january 1 to december 31, 2014), using a computerized registry of mycobacteriology laboratory. the diagnosis was verified in each case with review of medical records. for each included infectious tb case, the zero time was the date of admission to the hospital or the date of arrival to emergency department (er). the end of follow-up was the date when the patients was sent to a respiratory isolation room (event), the date of discharge (from hospital or er) before respiratory isolation can be implemented (censored), the date of completion 14-day anti-tb treatment (censored), or the date of mortality due to any cause (censored). for patients who had multiple admissions or multiple positive sputum cultures, only the admission with or following the first positive sputum culture (the index culture) was used to calculate the kaplan-meier estimates for time to respiratory isolation. to estimate the total non-isolated infectious patient-days in hospital, each tb case/patient was considered infectious from 3 months prior to the first positive sputum culture unless being put in a respiratory isolation room or had already received a 14-day course of at least two in vitro active anti-tuberculous agents after the last positive sputum culture. for those who had multiple hospitalization or had ever been transferred between wards/units before being diagnosed with pulmonary tb or adequately treated, all hospitalizations or stay in each ward/unit were counted in the calculation of total infectious patient-days. statistical analyses were performed using spss 21.0 (ibm, armonk, new york, usa). cox regression, with backward selection, was used to adjust for covariates. causal mediation analyses were performed using proc causalmed (based on logistic regression for isolation status on day 7) in sas 9.4 (sas institute, cary, north carolina, usa). all analyses were two-sided. p values less than 0.05 was considered statistically significant. in 2001 and 2014, 180 of 403 (45%) and 81 of 301 (27%) patients with culture-confirmed pulmonary tb were hospitalized, respectively ( table 1 ). the median non-isolated infectious duration decreased from 12.5 in 2001 to 3 days in 2014 (p<0.001) ( table 1) . improvement occurred over all subgroups of patients (s2 table) . fig 1 shows kaplan-meier estimates for time to respiratory isolation (discharge of undiagnosed tb is counted as censored rather than as the end of infectiousness) in 2001 versus that in 2014 (median: 46 vs. 19 days, p = 0.028, logrank test). patients with cavitary lung lesions were more quickly isolated, while lack of typical clinical presentation and hospitalized due to comorbidities were associated with delayed respiratory isolation ( switching to auramine-rhodamine staining with fluorescent microscopy doubled the overall positive sputum smear rate from 22.8% (2001) to 48.1% (2014) (p<0.001), particularly in patients with non-cavitary lung lesions (18.8% to 42.9%, p<0.001) ( table 1 ). cox regression analyses shows that a positive sputum smear was associated with an earlier respiratory isolation (ahr 3.2, 95% ci 2.1-4.9, p<0.001) ( table 2 : model 2). causal mediation analyses show that the two-fold higher sputum smear detection rate of fluorescence microscopy doubled the (fig 2) . in patients with positive sputum smear, the use of tb-pcr grew from 9.8% (2001) alertness of physicians, measured by duration from patient arrival to physician's ordering of smear or culture, also improved from 2001 to 2014 (median: 5 vs. 2 days, p<0.001) (s2 table) . cox regression analysis shows that physician alertness was also associated with earlier respiratory isolation (ahr 0.98 for each additional day before physician ordering tb smear/culture, 95% ci 0.96-0.99, p = 0.004) ( in 2001, there were a total of 4,778 infectious patient-days in hospital (582 from smear-positive patients, 4,196 from smear-negative patients). in 2014, the total non-isolated infectious patient-days in hospital decreased by 69%, to 1,502 infectious patient-days (229 from smearpositive patients and 1,273 from smear-negative patients). improvement occurred over all types of wards/units, including er, internal medicine wards, surgical wards, and intensive care units (fig 3, s2 and s3 figs) . our study showed that introducing highly sensitive rapid diagnostic tools decreases the risk of nosocomial tb transmission from hospitalized patients with undiagnosed tb in a high tb risk setting. switching from conventional to fluorescence microscopy doubles the sputum smear detection rate and was associated with a two-fold increase in likelihood of prompt respiratory isolation. a genuine improvement in time-to-respiratory-isolation of hospitalized tb patients should reduce nosocomial tb transmission, especially to hwcs. this is precisely what we observed in medical center a. our previous survey on age/sex-standardised tb incidence ratio of hcws (using general population as reference)---the excess tb risk that are attributable to nosocomial tb transmission---in medical center a showed a drop of this risk, from 3.11 in 2006 to 1.37 in 2012 [23] , and the decrease in time-to-isolation and total non-isolated infectious patient-days was in parallel in the present study. traditional ziehl-neelsen staining and conventional light microscopy have unsatisfactory sensitivity in detecting acid-fast bacilli [24] . a systematic review showed that, compared with conventional method, fluorescence microscopy has higher sensitivity and similar specificity [13] . this present study found that, after switch to fluorescent microscopy, the overall sputum smear detection rate doubled (23% vs. 48%, p<0.001), particularly in patients with non-cavitary lung lesions (18.8% to 42.9%, p<0.001) but also in patients with cavitary lesion (43% to 82%, p = 0.029). furthermore, the superior detection rate of fluorescence microscopy translated to a more timely respiratory isolation. the 20%-40% difference in sensitivity between fluorescence and conventional microscopy from our real life data is much larger than the 10% (on-average) reported in previous studies that compared the diagnostic performance of fluorescent versus conventional microscopy under the optimal conditions [13] . this highlights an often overlooked problem of traditional ziehl-neelsen staining, i.e. the majority of clinical laboratories just did not have the manpower to adequately check 300 high-power fields [13] (which takes around 4 minutes) per sample that are required for conventional microscopy [24] , particularly in a busy, high clinical caseload settings. in contrast, it takes only 30-60 seconds per sample for an adequate check with fluorescent microscopy [24] which would have a decisive advantage in the real world implementation. although tb-pcr has been considered an important rapid diagnostic tool, this study did not show a significant role of tb-pcr in the reduction in time-to-respiratory isolation from 2001 to 2014. there were several probable reasons. first, the low adoption rate (due to cost issues) may not be enough to make an impact on shortening the overall infectious duration. second, because of the lower sensitivity of tb-pcr in smear-negative respiratory specimens, tb-pcr was performed predominantly in specimens with positive afs to distinguish m. tuberculosis from non-tuberculous mycobacteria. the negative result does not exclude the possibility that universal use of automatic tb-pcr, such as xpert 1 mtb/rif, may further shorten the time to respiratory isolation. in keeping with previous observations (s3 table) , the absence of cough and other typical symptoms is a barrier to prompt respiratory isolation of tb patients (s2 table) . another cause of delay is hospitalization due to comorbidities, a problem that was previously neglected or confused with the lack of typical clinical presentations. to distinguish these two different situations, we defined hospitalization due to comorbidities as the reason of admission being for the management of acute complications from non-infectious diseases, while lack of typical presentation was defined as the absence of prolonged cough for more than 3 weeks, clinical suspicion of pulmonary tb based on chest radiography, or already having a confirmed diagnosis. multivariable regression established that hospitalization due to comorbidities was a risk factor independent from lack of typical presentations ( table 2) . the diverse distribution of hospitalized infectious tb patients across 25 medical/surgical sub-specialities wards/units (s2 and s3 figs) further supports that hospitalization of patients with undiagnosed tb for treatment of comorbidities is an unrecognized but important issue requiring to be addressed. the hcws in specialties units often concentrate on the management of acute complications of non-infectious chronic diseases and are not trained or prepared for diagnosing concomitant tb in their patients. the 2003 large nosocomial tb outbreak in taipei [18] involving more than 65 hcws, occurred exactly under such scenario -an elderly patient with acute stroke was hospitalized to a rehabilitation unit, without being suspected to also have active tb. the harm is two-way. first, the patients are at increased risk for morbidities and mortality from delayed diagnosis and treatment of tb. second, the hcws are at increased risk of nosocomial tb outbreak, which could be fatal if the strain is multidrugresistant or extensively drug-resistant [25] . the interplay between tb and chronic diseases further complicates the clinical scenarios. chronic non-communicable diseases increase the risk of tb-the associations have been well established for diabetes mellitus [26] and rheumatoid arthritis treated with anti-tumour necrosis factor (tnf) agents [27] . on the other hand, tb may increases the risk of complications from chronic diseases, e.g. hyperglycaemia in diabetes mellitus and ischemic stroke in people with atherosclerosis [28] . moreover, certain risk factor, such as smoking, increases the risk of both tb and chronic diseases [29] . therefore, hospitalization of undiagnosed tb patients for treatment of comorbidities is more than just a coincidence by chance. in low hiv prevalence countries, tb is a disease of elderly [30] . with population aging, concurrence of tb and comorbidities could be an increasing challenge to clinicians. currently, most hospitals in taiwan still used traditional ziehl-neelsen staining (or kinyoun staining, a similar method) and conventional light microscopy in laboratory diagnosis of tb. an analysis of 2003-2010 taiwan national health insurance database revealed that frequent exposure to hospital environment is a risk factor for contracting tb in taiwan (adjusted odds ratio: 1.77, for those with � 30 outpatient care visit annually) [31] . our findings on the impact of switching to auramine-rhodamine staining with fluorescence microscopy suggest that a nationwide adoption and roll-out might cut the risk of nosocomial tb transmission to both patients and healthcare workers. to address the remaining barriers to respiratory isolation of hospitalized tb patients, "fast (find cases actively by cough surveillance and rapid molecular sputum testing, separate safely, and treat effectively based on rapid drug susceptibility test)" is an option to decrease the time to respiratory isolation [32, 33] although the substantial cost of rapid molecular testing could be a barrier to nhi reimbursement. alternatively, environmental controlsthe second level in the hierarchy of tb control-with adequate ventilation (>6-12 air changes per hour) or upper room ultraviolet germicidal irradiation (to disinfect the air) can be applied to reduce the concentration of infectious droplet nuclei in hospital indoor air, and decrease risk of nosocomial transmission [6, 34] . the present study has several important limitations. first, it was not a randomized controlled trial (which cannot be performed in the context, due to ethical reasons). physician education and expansion of respiratory isolation facilities were simultaneously implemented along with the switching to fluorescence microscopy during the same intervention period. nevertheless, we applied multivariable regression and causal mediation analyses to estimate the effect attributable to switching to fluorescence microscopy. second, the study hospital closely followed the national guidance on nosocomial tb control policies issued by taiwan cdc. therefore, our findings might not be generalizable to hospitals with less proficiency. third, the study hospital is a medical center, and therefore might not represent situations in smaller hospitals. nevertheless, under nhi in taiwan, people can and did directly seek health care in medical centers, without the need of referral from general practitioners. the 3 million annual outpatient visits and more than 90 thousand hospitalizations per year in the study hospital were predominantly from primary care services, rather than tertiary referral care. finally, since taiwan has a low hiv prevalence [35] , only two of tb patients had hiv coinfection in our study. thus, our conclusions might not be generalized to countries with a high hiv prevalence. in conclusion, highly sensitive rapid diagnostic tools could substantially improve timing of respiratory isolation and reduce risk of nosocomial tuberculosis transmission in high tb risk settings. lack of typical presentations and hospitalization due to comorbidities continued to be main reasons of delayed isolation. studies will be required to assess whether routine sputum smear or tb-pcr of all hospitalized patients with cough or abnormal chest radiograph is effective in overcoming these remaining barriers. supporting information s1 table. tuberculosis aerial dissemination of pulmonary tuberculosis. a two-year study of contagion in a tuberculosis ward world health organization. guidelines for prevention of tuberculosis in healthcare facilities in resourcelimited settings. geneva: world health organization risk of tuberculosis infection and disease associated with work in health care settings extensively drug-resistant tuberculosis as a cause of death in patients co-infected with tuberculosis and hiv in a rural area of south africa 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screening for tuberculosis with xpert mtb/rif assay versus fluorescent microscopy among adults newly diagnosed with human immunodeficiency virus in rural malawi: a cluster randomized trial (chepetsa) trends in tuberculosis in taiwan nosocomial transmission of mycobacterium tuberculosis found through screening for severe acute respiratory syndrome-taipei delayed suspicion, treatment and isolation of tuberculosis patients in pulmonology/infectious diseases and non-pulmonology/infectious diseases wards why is in-hospital diagnosis of pulmonary tuberculosis delayed in southern taiwan? guidance on prevention and control of nosocomial tuberculosis transmisison sputum smear microscopy: evaluation of impact of training, microscope distribution, and use of external quality assessment guidelines for resource-poor settings tuberculosis in healthcare workers: a matched cohort study in taiwan fluorescence microscopy for tuberculosis diagnosis extensively drugresistant tuberculosis (xdr-tb) among health care workers in south africa diabetes mellitus increases the risk of active tuberculosis: a systematic review of 13 observational studies safety and effectiveness of tocilizumab in treating patients with rheumatoid arthritis-a three-year study in taiwan tuberculosis and the risk of ischemic stroke: a 3-year follow-up study effects of smoking and solid-fuel use on copd, lung cancer, and tuberculosis in china: a time-based, multiple risk factor, modelling study treatment delay and fatal outcomes of pulmonary tuberculosis in advanced age: a retrospective nationwide cohort study health care visits as a risk factor for tuberculosis in taiwan: a population-based case-control study a refocused, intensified, administrative tuberculosis transmission control strategy fast implementation in bangladesh: high frequency of unsuspected tuberculosis justifies challenges of scale-up guidelines for preventing the transmission of mycobacterium tuberculosis in health-care facilities we thank all staff in the hospital for their commitment to improving patient safety and reducing healthcare-associated infection.supervision: yee-chun chen, chi-tai fang, shan-chwen chang. hsin-yun sun, jann-yuan wang, yee-chun chen, po-ren hsueh, yi-hsuan chen, yu-chung chuang, chi-tai fang, shan-chwen chang, jung-der wang. key: cord-274241-biqbsggu authors: shaw, timothy i.; srivastava, anuj; chou, wen-chi; liu, liang; hawkinson, ann; glenn, travis c.; adams, rick; schountz, tony title: transcriptome sequencing and annotation for the jamaican fruit bat (artibeus jamaicensis) date: 2012-11-15 journal: plos one doi: 10.1371/journal.pone.0048472 sha: doc_id: 274241 cord_uid: biqbsggu the jamaican fruit bat (artibeus jamaicensis) is one of the most common bats in the tropical americas. it is thought to be a potential reservoir host of tacaribe virus, an arenavirus closely related to the south american hemorrhagic fever viruses. we performed transcriptome sequencing and annotation from lung, kidney and spleen tissues using 454 and illumina platforms to develop this species as an animal model. more than 100,000 contigs were assembled, with 25,000 genes that were functionally annotated. of the remaining unannotated contigs, 80% were found within bat genomes or transcriptomes. annotated genes are involved in a broad range of activities ranging from cellular metabolism to genome regulation through ncrnas. reciprocal blast best hits yielded 8,785 sequences that are orthologous to mouse, rat, cattle, horse and human. species tree analysis of sequences from 2,378 loci was used to achieve 95% bootstrap support for the placement of bat as sister to the clade containing horse, dog, and cattle. through substitution rate estimation between bat and human, 32 genes were identified with evidence for positive selection. we also identified 466 immune-related genes, which may be useful for studying tacaribe virus infection of this species. the jamaican fruit bat transcriptome dataset is a resource that should provide additional candidate markers for studying bat evolution and ecology, and tools for analysis of the host response and pathology of disease. bats are an ancient and diverse group [1] and are the second largest taxonomic group of mammals with more than 1,200 identified species among the 5,499 known mammals [2, 3] . bats are the only mammals to have evolved powered flight, which has allowed dispersal across all continents other than antarctica. bats are critical components of ecosystems, serving as major predators of insects, pollinating flowers and dispersing seeds of keystone plant species worldwide. the body sizes of bats range from less than 2 gm with 8 cm wingspans to more than 1 kg with 2 m wingspans. most contemporary species of bats are insect-, nectar-, or fruit-eaters, but about 1% are carnivores, including fish-eating and blood-drinking species. the evolutionary origin of bats remains controversial [4, 5] . in early work, bats were thought to be closely related to rodents and primates [6] . bats are now established within laurasiatheria; however, the placement of bats within laurasiatheria has been difficult to resolve because the major groups diverged from one another within a relatively short period of time [7] . different placements recently hypothesized for bats include: (a) sister to perissodactyla (horse) [8] ; (b) sister to cetartiodacyla (cattle+dolphin) [5] , (c) sister to perissodactyla+cetartiodacyla (horse, cattle, dolphin) [9] , (d) sister to ferungulata (cattle+dolphin, dog+horse) [4, 10] and (e) the pegasoferae hypothesis which places bat with perissodactyla and carnivora (horse+dog) [11] (see [5] for a review). two bat genomes have been sequenced to date [12] , the little brown bat (myotis lucifugus, 76 coverage) and the large flying fox (pteropus vampyrus, 2.66 coverage), but neither has been extensively annotated. these species represent the two major clades within bats: the microbats and megabats. transcriptome sequencing for another megabat species, the australian flying fox (pteropus alecto), has recently been published [13] . thus, a transcriptome for a microbat species is needed. many highly pathogenic viruses are hosted, or suspected to be hosted, by bat reservoirs, including ebolaviruses, marburg virus, hendra virus, nipah virus, rabies virus and coronaviruses [2] . in total, more than 100 viruses have been isolated from, or detected in, bats of dozens of species, yet many of the viruses that cause disease in humans cause little or no disease in the bats. significantly, the great majority of bat species have not been examined for infectious agents and are, thus, likely underappreciated as reservoir hosts. the continued encroachment of humans upon bat habitat and bat migrations caused by climate change may lead to novel infectious diseases among humans and livestock. moreover, some infectious diseases cause significant morbidity and mortality in bats that could have dramatic impacts on population numbers and cascading ecological effects [14] . thus, the study of bats and their infectious agents is an important but neglected aspect of zoonotic and wildlife disease research. jamaican fruit bats (artibeus jamaicensis) are one of the most common bats in the tropical americas, ranging from the caribbean islands, tropical south and central america, mexico and the florida keys [15] . the jamaican fruit bat is a microbat in the family phyllostomidae, which contains 56 genera and 192 species. they are a frugivorous generalist and fig specialist of medium size; about 80 mm in length with a wingspan of 130 mm and mass of about 50 grams. they can readily fly 20 km per night, although they typically maintain a smaller home range as long as food is available, and can live 9 years or more in the wild. females typically produce two offspring per year and provide maternal care for about 50 days, with pups reaching adult body weight by about 80 days. several microbes of interest have been isolated from or detected in jamaican fruit bats, including histoplasma capsulatum, trypanosoma cruzi, eastern equine encephalitis virus, mucambo virus, jurona virus, catu virus, itaporanga virus and tacaiuma virus, suggesting the species may be an important reservoir and vector of infectious diseases [15] [16] [17] [18] . it is unknown what diseases these pathogens may cause in bats. tacaribe virus (tcrv) was isolated from 11 artibeus bats (6 a. lituratus, 5 a. jamaicensis) in the late 1950s in and near port of spain, trinidad [19] . tcrv was the first arenavirus isolated in the americas and during the next decades other arenaviruses with substantial similarity to tcrv were identified that cause the south american hemorrhagic fevers (sahf) [20, 21] . the known reservoir hosts for all other arenaviruses are rodents, making tcrv exceptional for its repeated isolation from artibeus bats. because exhaustive searches for evidence of other potential reservoir hosts of tcrv failed to suggest another reservoir species [19, 22] , it has been suspected that artibeus bats are reservoirs of the virus. however, recent work by us demonstrated that trlv-11573, the only remaining isolate of tcrv, causes a fatal infection resembling the sahf in jamaican fruit bats, one of the species from which tcrv was isolated, or is cleared without disease, suggesting this species is not a suitable reservoir host for tcrv and the virus may be a significant pathogen for bats [23] . because of the equivocal role of artibeus bats as a reservoir host species for tcrv, and because of the similarities with human sahf, the jamaican fruit bat may be a novel model for studying the pathology of the disease. however, as an unusual, non-model organism, very little is known about its physiology, immunology or host response to tcrv. no antibodies are available with known specificity to jamaican fruit bat proteins, which dramatically limits its usefulness. to address some of these deficiencies, we have performed transcriptome sequencing and analysis of spleen, lung, kidney and poly-ic-stimulated primary kidney cells to identify genes of interest for assessing the host response to tcrv infection. more than 240,000 454 reads and 142 million illumina reads were obtained ( table 1 ). the reads were submitted to short read archive (sra) under srr539297 and srr538731. reads from lung, kidney, and poly-ic-stimulated primary kidney cell libraries were pooled for a combined de novo assembly using the 454 gs assembler program, yielding 6,450 contigs. for the illumina spleen sequences, we first corrected reads using the soapdenovo correction tool and further assembled them using soapdenovo, yielding 214,707 contigs. a total of 367,317 snps and 44,679 indels were detected through gigabayes. at least 16 reads covering a site were required to ensure the snp was of high quality. using tgicl, a combined assembly of the 454 and illumina contigs was constructed that contained 102,237 contigs with n10, n50, and n90 of 3,882 bp, 1,004 bp, and 289 bp, respectively. human and mouse genomes were used as references to estimate the distribution of bat contigs within known gene transcripts. human and mouse genomes were chosen for completeness of their annotations. genomic features were divided into 5 groups: 1 kb upstream of 59 utr, 59 utr, cds (coding sequence), 39 utr, 1 kb downstream of 39 utr. we found 58.03% and 23.18% mapped cds region for human and mouse genome respectively ( figure 1a ). because we performed transcriptome sequencing, we expected a majority of the sequences to map to cds and utr regions of the genome. many rna genes were also mapped, including long noncoding rnas and a substantial number of micrornas ( figure 1b ). annotation was concentrated on identifying micrornas because they could be cross validated through their rna secondary structure features. to further obtain a confident set of microrna sequences, a microrna prediction pipeline was used to cross validate the blast mapping of prediction. in the process, 42 confident microrna candidates were found that have been deposited within mirbase [24, 25] . we present the list of predicted micrornas as table s1 . mapping the predicted snps on the genomic features indicates that the vast majority of snps are in the cds region ( figure 1c-1d) . although humans and mice are both outside laurasiatheria the relatively fast rate of molecular evolution of mice is expected to result in more differences between bats and mice than bats and humans [26] [27] [28] [29] . the presence of sequences mapped 1 kb upstream or downstream of the known transcript indicated possible alternative splicing from human and mouse transcripts. blast2go was used to functionally annotate contigs. a total of 20,020 contigs (19.58% overall) had significant matches to known proteins in the ncbi non-redundant protein (nr) database. horse and human were identified as the top two species with best blast hits for bat contigs ( figure 2 ). the blastx annotation process is biased by the completeness of the annotation for each respective genome; therefore, despite the lack of a completely annotated horse genome, a high similarity between bat and horse genomes was apparent. the human genome is well annotated, which explains the high number of blast hits between bat and human. the go annotation divides the functional annotation into three main components: biological process, cellular process, and molecular [30] . a majority of the annotated genes encoding proteins that function within a cell or organelle are involved in metabolic and cellular processes. the primary molecular functions of these genes are catalytic and binding activities ( figures 3a-c) . a total of 466 immune-related genes were annotated by blast2go. these immune genes include toll-like receptors, cytokines, transcription factors, kinases and several chemokine receptors. in addition, categorizer was used to categorize the immune class using the goslim database, resulting in 30 categories representing a broad range of immune activities ( figure 4 ). the immune response and lymphocyte activation genes represented the largest proportion of theses transcripts. there were 82,218 unannotated contigs. a total of 16,869 sequences had open reading frames longer than 300 nt; 5,417 were identified through blastp to the nr database with evalue,1e 23 . for the remaining contigs, 54,892 mapped to the assembled myotis lucifugus genome, and 48,809 mapped to the assembled pteropus vampyrus genome. there were 20,145 contigs that mapped to pteropus alecto, australian flying fruit bat, and 18,359 that overlapped between genomic and transcriptome sequences for all three datasets ( figure 5 ). through this process, we were able to account for 65,828 (80%) unannotated contigs. the completeness of genes mapped to immunological pathways was examined using human and mouse as reference species. based on the ortholog data obtained, all contigs were mapped onto immune system related kegg pathways ( table 2 ) and determined that many genes were missing from these pathways. this could be due to the low expression within bat tissues or due to the overly stringent e-value cutoff of 1e 220 during reciprocal blast annotation that we chose to limit the number of false positives. a kegggraph visual representation of contigs mapped onto the mouse pathway was generated (figures s1, s2, s3, s4, s5, s6, s7, s8, s9, s10, s11, s12, s13, s14, s15, s16, s17, s18). pathways involved in the adaptive immune response, t and b cell signalling pathways, generally had more mapped genes than did those involved in innate response or natural killer (nk) cell-mediated cytotoxicity pathways ( figures 6a and 6b ). the nk cell cytotoxicity pathway appears to have almost half of its genes missing, whereas the b cell receptor pathway appears to have most of its genes present. nucleotide substitution in the coding region can be synonymous or non-synonymous. the ratio between the rate of synonymous (ds) and non-synonymous mutation (dn) can be used to infer the degree of selection operating on the system. we used the human genome as a reference for dn/ds calculations because the human genome is well annotated. reciprocal blast was used to identify human, mouse, and bat orthologs. macse was used to generate codon alignments. the alignments were trimmed for excessive gap codon triplets, and paml was used to calculate dn/ds for each gene. when genes are highly conserved, synonymous mutations (ds) tend to be estimated as 0, resulting in a larger dn/ds ratio, therefore those results were removed from the analysis. after filtering, dn/ds results were obtained for 14,717 genes. the majority of the genes have close to zero dn/ds with clear evidence of purifying selection, a feature common among mammalian genes [31] [32] [33] . for investigation of positive selection, tang et al. [34] have argued that a dn/ds threshold of greater than 1 for positive selection might be overly stringent. because of this, a dn/ds cutoff of 0.7 was chosen to investigate genes that might be experiencing weak purifying selection. a total of 138 genes above the 0.7 threshold were found (table s2) . for genes with evidence of positive selection, 32 exceeded the 1.0 dn/ds threshold (figure 7) . through annotation by david [35, 36] , there were 14 genes involved in transcriptional activation and regulation processes. there were 9 genes associated with cellular signaling. in particular, we found dna-damage-inducible transcript 4 (ddit4) gene with dn/ds 1.4053; this protein is involved in the mtor signaling pathway and it regulates cell growth and promotes neuronal cell death [37, 38] . ectodysplasin a (eda), involved with cytokine:receptor interaction pathways, had a dn/ds value of 1.23. the phylogenetic placement of bats within laurasiatheria is still unresolved. through reciprocal blast, we identified 8,785 putative orthologs across mouse, rat, cattle, horse and human (table s3) . afterward we filtered out alignments with greater than 5% gap, the 2,378 genes remaining were used to construct 500 iteration multilocus bootstrap species tree (see methodology). this resulted in a highly supported species tree placing bat sister to the clade containing cattle, horse, and dog ( figure 8 ). the jamaican fruit bat transcriptome described here is a major new resource for genetic studies of bats. this bat is an important seed dispersing and pollinating species found in most of the tropical americas. it is likely susceptible to infectious diseases, could be a zoonotic reservoir and vector, and may be a suitable model for the pathogenesis of sahf. considering the importance of immunological functions in response to infections, we conducted a transcriptome assessment of genes from spleen, kidney and lungs so that genetic tools and methods can be used to study this species as well as other microbats. genes were identified that mapped to immune response pathways; based on categorize classification of immune classes, we found 40 different immune classes. recently, the transcriptome sequencing for the australian black flying fox was performed [13] . our data contain a greater proportion of lymphocyte related immune classes than does the flying fox's transcriptome dataset. however, our dataset also contained a lesser proportion of cytokine related immune classes than the flying fox's transcriptome dataset. genes involved in adaptive immune response generally had more mapped genes compared to genes involved in innate responses. from figures 6a and 6b , more genes were mapped to the b cell receptor signalling pathway than to the nk cellmediated cytotoxicity pathway. this bias is likely due to the large number of b cells found in the spleen. due to our stringent blast criteria, it is also possible that lowering the e-value threshold could obtain additional genes mapped but at the risk of more false positives. we deposited 42 microrna genes for a. jamaicensis into mirbase, and according to mirbase this gene set is the first deposited bat microrna genes. estimates of substitutions within the orthologous contigs found 32 genes with a dn/ds ratio.1. this ratio provides a guide for indicating potential genes that are under positive selection. many genes were involved in transcriptional activation/regulation processes, suggesting potential differences in the transcriptional regulating architectures of bats and humans. the ddit4 and eda have a dn/ds ratio.1, suggesting these genes are under positive evolutionary selection. ddit4 is involved in regulation of cell death and its positive selection suggests a potential difference in cell death regulation between human and bats; further analysis will need to be performed to verify the functional differences. another potential positively selected gene, eda is associated with ectodermal dysplasia type 1 [39] , a disorder associated with abnormal development of physical structures, including skin, hair, nails, teeth, and sweat glands. we suspect the bat's eda gene could be used as a potential reference for future studies of the disorder. for transcripts that failed to be identified by reciprocal blast searches, we predicted the orf for the unannotated contigs and used blastp against the nr database to identify 5,349 unannotated contigs. for the remaining unannotated contigs, we used genomic data from myotis lucifugus and pteropus vampyrus, as well as transcriptome data from pteropus alecto to identify additional unannotated contigs. existing artibeus contigs that were not present within the nr database, but overlapped among myotis and pteropus genomic and transcriptomic sequences indicated the possibility for bat specific transcripts. we also found contigs that mapped only to the myotis lucifugus genome indicating the possibility for microbat specific contigs. in total, we were able to account for 80% of the unannotated trancripts, and the remaining unannotated transcripts likely include misassembled contigs, contigs not sequenced sufficiently in the other bats to be included in their genome assemblies, as well as a few transcripts specific to artibeus jamaicensis. many additional analyses are warranted to further refine the transcriptome information from artibeus and other bats. phylogenomics is an important tool for resolving the tree of life, and this transcriptome data set provides an opportunity to study the evolutionary history of bats. bats were once thought to be closely related to primates [6] ; however, further work using molecular information placed them within laurasiatheria [40] . our finding of bat as sister to the clade containing horse, dog, and cattle is consistent with the recent study by mccormack et al. [4] and zhou et al [10] . here, we used 2,378 loci from a microbat and species tree analyses to obtain 95% bootstrap support, whereas mccormack et al. use 683 loci to obtain 64%bootstrap support. a recent study by nery et al. [5] obtained a concatenated data from 3,733 loci from megabat with 100% bootstrap support and 1.0 posterior probability placing bat as sister to cattle. our phylogenetic tree is less resolved than nery et al., probably because we did not include the more limited transcriptome data available from dolphin and hedgehog. maximum likelihood analyses are powerful, yet can lead to incorrect conclusions in certain situations, whereas species tree analyses are less powerful but more robust to well-known violations of the models used for maximum likelihood phylogenetic analysis, such as incomplete lineage sorting (see [5] and references therein). additional work is clearly warranted, especially by using additional taxa, testing for convergence and specific violations of gene-tree models, and other sources of conflict among protein-coding genes and other portions of the genome. a principal difficulty for identifying mechanisms of pathogenesis of the sahf, in which the immune response may play a contributory role, is a lack of animal model resources that faithfully recapitulate human disease [41] . although laboratory mice (mus musculus) and rats (rattus norvegicus), which have substantial experimental methodologies and reagents, can be infected with junã­n virus (junv), the etiologic agent of argentine hemorrhagic fever, the pathogenesis is markedly different than human disease. the guinea pig (cavia porcellus) typically exhibits signs of disease that closely resembles human disease; however, there are few immunological or genetic tools for assessing the host response to infection. junv is also a bsl-4 and select agent, thus use of virulent strains is confined to only a few laboratories with highly specialized containment facilities. the pathogenesis of tcrv, a bsl-2 agent, in jamaican fruit bats exhibits many similarities to the sahf in humans, thus the use of transcriptome data could be useful for studying pathogenesis using a variety of new technologies, such as pcr arrays for pathway discovery, and for the development of antibodies to specific artibeus proteins that are important in the pathogenesis of disease. the transcriptome resource provided will facilitate research into artibeus host responses to infectious agents, including mechanisms of pathogenesis of arenavirus disease and will also provide further resource for additional understanding for the bat species evolution and physiological development. all procedures were approved by the unc institutional animal care and use committee (iacuc) and were in compliance with . substitution estimation scatter plot. we calculated the nonsynonymous mutation rate (dn) and synonymous mutation rate (ds) using orthologous genes between bat and human. two lines were drawn representing the two dn/ds cutoffs of 0.7 (green) and 1.0 (blue). doi:10.1371/journal.pone.0048472.g007 figure 8 . unrooted species tree from the orthologous dataset across six boreoeutheria mammals. the species tree was generated from 2378 gene loci. there was 95% bootstrap support for placing bats (chiroptera) sister to perissodactyla, cetartiodactyla, and carnivora. doi:10.1371/journal.pone.0048472.g008 the usa animal welfare act. unc animal care and use committee approval number, 1207c-ra-b-15. five bats from the university of northern colorado jamaican fruit bat colony were used for this work. male and female a. jamaicensis bats were euthanized by respiratory hyperanesthesia followed immediately by thoracotomy. tissues were aseptically removed and flash frozen in liquid nitrogen for subsequent rna extraction. tissues were homogenized in buffer rlt (rneasy kit, qiagen, valencia, ca) containing 2me using a bead beater and silicone beads. the homogenate was passed over a qiashredder column prior to total rna extraction according to manufacturer's instructions. for cell culture, one kidney from one bat was collected in serum-free hbss and minced under aseptic conditions, then trypsinized (trypsin-versene) at room temperature in a sterile 50 ml trypsin flask. cells were washed 36 in 10% fbs-emem, then seeded into a vented t-25 flask. the next day, unattached cells were removed and fresh 10% fbs-emem added. when cells approached confluence they were passaged with trypsin at a split ratio of 1:4. poly-ic was added to 50 mg/ml in two t-75 flasks containing 20 ml each of 10% fbs-emem and incubated for 6 hours, after which rna was extracted according to manufacturer's instructions (qiagen). total rna was extracted using the rneasy minelute cleanup kit (qiagen) and then shipped on dry ice to seqwright (houston, tx) for cdna library construction and sequencing. rna concentrations and quality were assessed by a260/a280 and a260/a230 absorbance values and agarose gel electrophoresis. a260/a280 values were all above 2.0 and a260/a230 were all above 1.9. electrophoresis of the rna samples demonstrated that 28s and 18s rrna were not degraded. libraries for the 454 were prepared from three tissues (kidney, lung, poly-ic-stimulated kidney cells). for 454 library construction, full-length cdna was synthesized with two set of primers for driver and tester cdna [42, 43] . single-stranded cdna was used for hybridization instead of double-stranded cdna. excess amounts of sense-stranded cdna hybridized with antisense-stranded cdna. after hybridization, duplex was removed by hydroxyapatite chromatography. normalized tester cdna was re-amplified with tester specific primer l4n. driver cdna was unable to amplify using l4n. an illumina truseq rna library was made from spleens according to manufacturer's instructions. the libraries were then sequenced according to manufacturer's recommendations: 454 using titanium chemistry and illumina using 26100 nucleotide paired-end sequencing on a hi-seq 2000. the 454 and illumina libraries were assembled individually and also by combining both libraries. bases from the 454 reads were called from the 454 generated sff file using pyrobayes [44] and 454 gs assembler (version 2.5) was used to perform the assembly. soap denovo [45] (version 1.04) was used to assemble reads obtained from spleen (illumina library). only contigs greater than 200 bases were used in the final analysis. prior to performing the combined assembly, duplicates from pre-assembled contigs of lung, kidney and spleen tissues were removed with cd-hit [46] (cd-hit-2009-0427) at default criterion and then combined into longer fragments with tgicl [47] . gigabayes [48] -a short-read snp/indel discovery program was used to detect polymorphisms. snp/indel detection was performed for both libraries separately. to make snp/indel predictions more reliable, we used the criterion that minor allele and major allele (alleles with fewer reads are minor alleles, and alleles with more reads are major alleles) occur at least twice and 8 times for 454 and illumina libraries, respectively. to identify the approximate relative position of conserved mammalian genes, we mapped the bat contigs on to the genome of mouse mm9 and human grch37 (downloaded from the ucsc genome browser) using blat v.34 [49] with a minimum score of 80 used as a filter. coordinates of the protein coding genes were obtained from ensemble (http://uswest.ensembl.org/index.html) xenoref and gtf files. we also normalized the number of blat hits based on the total annotated transcript regions (1000 nt upstream of 59 utr, 59 utr, cds, 39utr, and 1000 nt downstream of 39utr) that were present in the mouse and human. to predict precursor-microrna genes within assembled sequences, we downloaded precursor micrornas for mouse, rat and human from mirbase [24, 25] . we performed a blast search focused on high quality candidates, hits with $95% sequence identity [50] . based upon rnafold [51] secondary structure prediction, we further filtered out sequences that did not possess any hairpin loop structure. previously, it had been demonstrated that micrornas tend to have deterministic folding [52] and, therefore, we used unpaired structural entropy (use) to evaluate the rna secondary structure base pairing distribution (cutoff 0.83 use score). mir-abela, a support vector machine learning program [53] , was used to cross validate the prediction. the final remaining unfiltered sequences are considered as highly confident microrna candidates. orthologous contigs (against human, mouse, dog, cattle and horse) were identified using the reciprocal blast (blastn) approach [54] as it has been found to be superior to sophisticated orthology detection algorithms [55] . a stringent cutoff of 1e 220 was used to separate paralogs from orthologs. cdna sequences from human (homo_sapiens.grch37. 64 the substitution rate is inferred from orthologous genes between bat and mouse. sequences were aligned using macse [56] and an in-house java script was used to trim/remove codon gap triplets from the alignment. substitution rate was estimated using a maximum likelihood method implemented in the codeml program of paml 4.5 [57, 58] . the pairwise maximum likelihood analyses were performed in runmode-2. estimated rates of non-synonymous to synonymous substitutions (dn/ds) were plotted as a scatter plot. blast2go [59] was used to functionally annotate contigs. a combined graph was generated for each go category. to prevent overloading graphs, the sequence filter value was changed to 500 in all 3 categories (biological process, molecular function and cellular component). functional annotation was performed separately for all assembled contigs present in the combined assembly. based on categorizer [60] , we further classified the genes using the go slim database immune classes. the completeness of mapping the bat genes using euarchontoglires as a reference was further examined through kegg. to do this, we first downloaded the xml file of annotated kegg pathways [61, 62] for human and mouse. to identify genes that are functionally important within kegg pathways, kegggraph was used to represent a graph form of the kegg pathway. we further used kegggraph to compute the relative betweenness centrality, which is the algorithmic representation of the involvement of a node within a network. we chose to set a cutoff of grabbing the top 4 nodes within each network, or selecting the top 4 functionally important genes within each pathway [63] . open reading frame was predicted from the assembled contigs through the orfpredictor web server (http://proteomics.ysu.edu/ tools/orfpredictor.html) [64] . a customized java program was used to parse through the prediction to identify sequences longer than 300 nt. to perform additional annotation of the predicted open reading frame we used blastp with an e-value of 1e 23 against the most recent nr database that is available from ncbi during our analysis (august 26 th , 2012). using contigs that were functionally unannotated, we compared the jaimacan fruit bat contigs against three other available bat sequence dataset. myotis lucifugus and pteropus vampyrus genomes were downloaded from the ncbi tracedb ftp server (ftp://ftp.ncbi.nih.gov/pub/tracedb/). the pteropus alecto transcriptome was obtained from dr. a. papenfuss [13] . an evalue threshold of 1e 25 was used to indicate blast hit. we then used an r package venndiagram [65] for displaying the mapped unannotated contigs that overlapped between different bat genome and transcriptomes. to resolve the evolutionary relationship for the artibeus bat species, we filtered the putative bat orthologs between human, mouse, dog, cattle, and horse. insectivores such as hedgehog and dolphin were not used in our analysis due to limited gene annotation in these taxa. to obtain the best multiple sequence alignment for each putative orthologs, we used aqua's pipeline for performing multiple sequence alignment; the pipeline consists of multiple sequence alignment through muscle and mafft which is refined by rascal and assessed by normd [66] [67] [68] [69] . a customized java program was used to filter alignments (obtained through aqua) with greater than 5% gap per sequence. additionally, we filtered for sequences that are at least.1,000 bp long. phyml 3 was used to generate a maximum likelihood gene tree [70, 71] . mraic, a perl script wrapper for phyml, was used to infer the best substitution model for each gene tree based on aic, aicc, bic, and akaike weights [72] . aic was used as the objective function since not much variation was observed across different objective function. njst was used to calculate the unrooted species tree based on our gene trees [73] . a customized rprogram is used for performing a nonparametric bootstrap species tree through resampling nucleotides within loci as well as resampling the loci within the data set as described by seo [74] . fossil evidence and the origin of bats bats: important reservoir hosts of emerging viruses iucn red list version 2011.2: tabel 3a -status category summary by major taxonomic group (animals) ultraconserved elements are novel phylogenomic markers that resolve placental mammal phylogeny when combined with species-tree analysis resolution of the laurasiatherian phylogeny: evidence from genomic data mammalian phylogeny: shaking the tree mammalian phylogenomics comes of age using genomic data to unravel the root of the placental mammal phylogeny confirming the phylogeny of mammals by use of large comparative sequence data sets phylogenomic analysis resolves the interordinal relationships and rapid diversification of the laurasiatherian mammals pegasoferae, an unexpected mammalian clade revealed by tracking ancient retroposon insertions a highresolution map of human evolutionary constraint using 29 mammals the immune gene repertoire of an important viral reservoir, the australian black flying fox emerging diseases in chiroptera: why bats? artibeus jamaicensis identification of a new venezuelan equine encephalitis virus from brazil humoral and cell-mediated immunity to histoplasma capsulatum during experimental infection in neotropical bats (artibeus lituratus) experimental rabies virus infection in artibeus jamaicensis bats with cvs-24 variants tacaribe virus, a new agent isolated from artibeus bats and mosquitoes in trinidad, west indies the phylogeny of new world (tacaribe complex) arenaviruses phylogenetic analysis of the arenaviridae: patterns of virus evolution and evidence for cospeciation between arenaviruses and their rodent hosts serological evidence of infection of tacaribe virus and arboviruses in trinidadian bats tacaribe virus causes fatal infection of an ostensible reservoir host, the jamaican fruit bat mirbase: the microrna sequence database mirbase: integrating microrna annotation and deep-sequencing data fast genes and slow clades: comparative rates of molecular evolution in mammals determinants of rate variation in mammalian dna sequence evolution determination of mitochondrial genetic diversity in mammals correlates of substitution rate variation in mammalian protein-coding sequences the gene ontology categorizer natural selection on protein-coding genes in the human genome a scan for positively selected genes in the genomes of humans and chimpanzees positive selection on the human genome a new method for estimating nonsynonymous substitutions and its applications to detecting positive selection systematic and integrative analysis of large gene lists using david bioinformatics resources bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists rtp801 is elevated in parkinson brain substantia nigral neurons and mediates death in cellular models of parkinson's disease by a mechanism involving mammalian target of rapamycin inactivation rtp801 is a novel retinoic acid-responsive gene associated with myeloid differentiation mutations within a furin consensus sequence block proteolytic release of ectodysplasin-a and cause x-linked hypohidrotic ectodermal dysplasia parallel adaptive radiations in two major clades of placental mammals junin virus. a xxi century update construction of a uniformabundance (normalized) cdna library construction and characterization of a normalized cdna library pyrobayes: an improved base caller for snp discovery in pyrosequences de novo assembly of human genomes with massively parallel short read sequencing cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences tigr gene indices clustering tools (tgicl): a software system for fast clustering of large est datasets a general approach to single-nucleotide polymorphism discovery blat-the blast-like alignment tool a uniform system for microrna annotation fast folding and comparison of rna secondary structures analyzing modular rna structure reveals low global structural entropy in microrna sequence identification of clustered micrornas using an ab initio prediction method gapped blast and psi-blast: a new generation of protein database search programs phylogenetic and functional assessment of orthologs inference projects and methods macse: multiple alignment of coding sequences accounting for frameshifts and stop codons paml 4: phylogenetic analysis by maximum likelihood paml: a program package for phylogenetic analysis by maximum likelihood blast2go: a universal tool for annotation, visualization and analysis in functional genomics research categorizer: a web-based program to batch analyze gene ontology classification categories kegg for integration and interpretation of large-scale molecular data sets kegg: kyoto encyclopedia of genes and genomes kegggraph: a graph approach to kegg pathway in r and bioconductor orfpredictor: predicting proteincoding regions in est-derived sequences venndiagram: a package for the generation of highly-customizable venn and euler diagrams in r muscle: multiple sequence alignment with high accuracy and high throughput mafft: a novel method for rapid multiple sequence alignment based on fast fourier transform aqua: automated quality improvement for multiple sequence alignments rascal: rapid scanning and correction of multiple sequence alignments morephyml: improving the phylogenetic tree space exploration with phyml 3 estimating maximum likelihood phylogenies with phyml model selection and multi-model inference estimating species trees from unrooted gene trees calculating bootstrap probabilities of phylogeny using multilocus sequence data we thank jason shaw for assistance with bat handling and stephanie james for laboratory assistance, and uga's georgia advanced computing resource center and institute of bioinformatics for computational resources and support. we thank anthony papenfuss for providing the assembled transcriptome for p. alecto. contributed reagents/materials/analysis tools: ts tg ll. wrote the paper: tis tg ts. key: cord-306958-8bx8kxxh authors: christensen, sarah r.; pilling, emily b.; eyring, j. b.; dickerson, grace; sloan, chantel d.; magnusson, brianna m. title: political and personal reactions to covid-19 during initial weeks of social distancing in the united states date: 2020-09-24 journal: plos one doi: 10.1371/journal.pone.0239693 sha: doc_id: 306958 cord_uid: 8bx8kxxh objective: to examine perceptions, behaviors, and impacts surrounding covid-19 early in the pandemic response. materials and methods: a cross-sectional survey of 1,030 u.s. adults was administered on march 31st, 2020. this survey examined attitudes toward media, government, and community responses to covid-19 by political ideology and sociodemographic factors. knowledge, anxieties, and impacts of covid-19 were also assessed. results: conservatives were more likely to report that covid-19 was receiving too much media coverage and people were generally overreacting; liberals were more likely to report the government had not done enough in response to the pandemic. females and those with lower income experienced more covid-19 related economic anxieties. those working and with children at home reported higher social, home, and work disruption. social distancing behaviors were more common among liberals and were associated with increases in depressive symptoms. general knowledge about covid-19 was widely exhibited across the sample, however, black and hispanic respondents were less likely to correctly answer questions about the availability of a vaccine and modes of transmission. conclusions: public health experts should consider the political climate in crafting messaging that appeals to the values of those across the political spectrum. research on the covid-19 pandemic should continue to monitor the effects of social distancing on mental health and among vulnerable populations. and adherence to preventative behaviors in the wake of this ongoing global pandemic. in light of differences in vulnerability to covid-19, access to information, sources of information, and timing of national, state, and local responses to covid-19, it is reasonable to assume that individual responses in the midst of the pandemic vary widely. this study examines individual attitudes, behaviors, anxieties, mental health impacts, and knowledge early in the pandemic response, as well as those outcomes by sociodemographic characteristics and political ideology. the purpose of this study was to determine how complex factors within society shaped early perceptions of and responses to covid-19. an anonymous cross-sectional internet survey was administered to 1,030 adults residing in the u.s. on march 31st, 2020. the sample was recruited by qualtrics (provo, ut, usa). quotas for sex, race, and income, derived from u.s. census data, [10] were used to increase demographic representation. implied consent was provided prior to the survey. participants were presented with information about the survey as well as potential risks, benefits and compensation. following this information participants were presented with a statement which read, "the completion of this survey implies your consent to participate. if you choose to participate, please proceed with the questions." compensation was valued at <$5 u.s. the study was approved by the brigham young university institutional review board. questions assessed political ideology, scientific trust, and media consumption, as well as attitudes, anxieties, impacts, and knowledge related to covid-19. respondents also assessed mental health and demographic information. three author-constructed questions assessed attitudes toward the response to covid-19. respondents were asked about pandemic media coverage (too much, right amount, or too little), government action (not enough, responding correctly, or too much), and public response (overreacting, responding correctly, or too much). respondents also answered two true/false questions: if they believed their state would experience a major outbreak of the virus and if they would isolate if they contracted the virus. we used the political polarization in the american public survey, [11] which has been validated as a reliable measure of political partisanship, [12] to examine personal political ideologies through a series of 10 dichotomous statements on political issues. responses were scored (liberal = -1 vs. conservative = +1), summed, and categorized as leans liberal, moderate, and leans conservative. respondents also self-characterized their political views on a 7-point scale (extremely liberal to extremely conservative). two additional items asked about attitudes toward global warming (most scientists think global warming is happening vs. there is a lot of disagreement as to whether global warming is happening) [13] and trust in government to make vaccination decisions (i trust that the government makes the best decisions when it comes to vaccination requirements vs. i do not trust the government to make decisions about what vaccinations are required). [14] a subset of questions from the reuters institute digital news report [15] was used to assess media consumption. respondents indicated their usual sources of media (abc, fox, npr etc.) which were each given a bias score based on ad fontes media's source evaluations. [16] pandemic-related behavior change was assessed by eleven author-constructed items asking respondents to compare their behavior on march 31st with their behavior before the pandemic on a 5-point scale from "much less than usual" to "much more than usual". behaviors included virtual communication, face-to-face contact, visiting restaurants/bars, stores, work, and travel. four items asked respondents to indicate agreement on a 7-point scale that "events related to covid-19 had interrupted" their social life, home life, work or vocational life, and/or hurt their mental health. fourteen author constructed items assessed pandemic-related anxieties using a 7-point agreement scale. four statements related to fear if they, an older family member, a young family member, or a healthy adult family member became ill with covid-19. two statements assessed anxieties related to healthcare equipment and personnel, three statements assessed economic concerns, and four items assessed concerns related to children at home (e.g. routines affected, children without care, etc.). questions regarding work-and child-related anxieties were only asked of those working for pay before the pandemic and those who had children under 18 in the household respectively. the last question assessed concerns that mental health would suffer due to social distancing measures. respondents assessed change in their own mental health from before the covid-19 pandemic on a 7-point scale from much worse to much better. respondents completed the phq-9 (a valid measure of depressive severity) [17] retrospectively for the two-week period preceding social distancing and for the current two-week period. higher phq-9 scores indicated more depressive symptoms. an increase in depressive symptoms is indicated by a positive change score. true/false and multiple-choice items developed by the authors were used to assess respondent knowledge regarding common symptoms of covid-19, recommended preventative measures, viral spread, and comparisons of covid-19 and seasonal influenza. respondents reported age, biological sex, race, ethnicity, marital status, education level, whether they were currently in school, employment status prior to the pandemic, average hours worked currently and prior to the pandemic, household size, household income, whether they received government nutritional program assistance, children at home, state of residence, and flu vaccination history. frequencies, proportions, and means were calculated. chi-square, t, and f tests were used to examine the influence of demographic characteristics, political ideology, and mental health on attitudes, knowledge, anxieties, behavior change, and impact variables. logistic regression was used to assess the relationship between political ideology and attitudes towards media, government, and community responses to covid-19 while controlling for sociodemographic characteristics, political ideology, media bias, global warming agreement, and trust in government vaccination requirements. initial covariate selection included all variables that were significant (p <0.05) in bivariate tests, including: political ideology, bias score for consumed news media, attitudes toward global warming and vaccination, sex, race, poverty level, and education. the final model was achieved by sequentially removing non-significant predictors and assessing the impact on model fit using the bayesian information criterion and akaike information criterion. non-significant predictors were retained if removing them worsened model fit. two attitude questions were dichotomized for logistic regression, due to small cell counts. those who responded there was "too little media coverage" (n = 88) were merged with those who responded "the right amount" of media coverage. similarly, those who responded the government had "done too much" (n = 51) were merged with those who responded the government had "done the right amount" in response to the pandemic. logistic regression models were tested with the grouped categories as outlined above, and with the small categories coded as missing; results were similar. logistic regression was also used to assess the relationship between knowledge about covid-19 while controlling for sociodemographic characteristics and media bias. initial covariate selection included all variables that were significant (p <0.05) in bivariate tests, including: bias score for consumed news media, sex, race, poverty level, and education. the final model was achieved by sequentially removing non-significant predictors and assessing the impact on model fit using the bayesian information criterion and akaike information criterion. non-significant predictors were retained if removing them worsened model fit. all analyses were completed in sas 9.4. the sample included 1,030 u.s. adults from 48 u.s. states and d.c. no respondents resided in vermont or wyoming. the sample was 47.5% male, 49.0% white, 23.4% black, 12.0% hispanic and 12.3% asian. about 29% of the sample had children under 18 living at home, about 23% were living under the federal poverty line, and about 24% had received government benefits in the last six months. sample demographics overall and stratified by political ideology are presented in table 1 . the majority (63.1%) felt that covid-19 was receiving the right amount of media coverage, with 28.4% responding that the pandemic was receiving too much media coverage. conservatives were most likely to feel the pandemic was receiving too much media coverage (50%) compared to moderates (30.1%) and liberals (21.0%; p-value <0.001). table 2 provides the adjusted logistic regression analysis for the attitude questions. compared to conservatives, liberals had three times the odds (aor 3.3; 95%ci: 2.1-5.2) and moderates had twice the odds (aor 2.3; 95%ci: 1.5-3.6) of reporting that the media coverage was the right amount/too much. the majority (55.2%) responded that the u.s. government had not done enough in response to covid-19, while 40% felt the government responded correctly. just 5% felt the government had done too much. liberals (70.8%) were most likely to respond that the government had not done enough in response to covid-19, compared to 19.6% of conservatives. nearly 10% of conservatives reported that the government had done too much in response to the pandemic (p-value <0.001). in the adjusted logistic regression model ( table 2 ) liberals had 5.7 (95%ci: 3.3-9.7) and moderates had 2.5 (95%ci 1.5-4.3) times the odds of responding that the government had not done enough in response to covid-19 compared to conservatives. those who consumed liberal leaning news media and who indicated there was scientific agreement about global warming also had higher odds of feeling the government had not done enough. approximately 19% felt that people were generally overreacting to covid-19 and 40% felt that people were generally under-reacting. conservatives (36.5%) were most likely to feel that people were overreacting while liberals (44.6%) were most likely to feel that people were under-reacting (p-value <0.001). in the multinomial logistic regression model (table 2) , compared to conservatives, liberals had approximately three times the odds of reporting people were responding correctly (aor 2.9; 95%ci 1. 6-5.3) or under-reacting (aor: 3.0; 95%ci: 1.6-5.5). females and those who consumed liberal news had significantly higher odds of feeling people were under-reacting to covid-19. despite variation in opinions regarding the response to covid-19, 80% felt that their state would experience a major outbreak of the disease. a similar percentage of liberals and moderates felt a major outbreak would occur (82.4%, 79.3%), while a smaller percentage of conservatives (67.6%; p-value = <0.001) agreed. regardless of political ideology nearly all respondents (95.8%) reported they would self-isolate in the event that they became ill with covid-19. respondents reported moderate agreement with all four statements evaluating fear related to becoming sick or having a family member become sick with covid-19. respondents agreed most strongly that they would be scared if an elderly family member contracted covid-19 (mean: 5.28; sd: 1.21), followed by a young family member (mean: 5.07; sd: 1.34), regarding events surrounding covid-19, a majority (64.9%) agreed they were afraid they may not be able to purchase supplies, food, and/or medication they needed. similarly 64.4% of those who were working before the pandemic agreed they were afraid they may not be able to financially provide for themselves or their families if asked not to work due to social distancing, and 66.8% agreed that they were afraid they would not be able to provide for themselves or their families if they became sick with covid-19. table 3 shows the distribution of economic anxieties by sociodemographic factors. after adjusting for the other factors in the table, females and those with lower income had higher mean agreement with all three economic anxieties statements as compared to males and those with higher income. in general, respondents reported changing their behaviors consistent with public health guidelines for social distancing. table 4 shows the distribution of changes in behavior. the degree to which people reported their behavior changed differed by political ideology. liberals were more likely to report a change in their behavior in the desired direction compared to conservatives. in answer to a direct question, 33.3% reported that their perceived mental health was worse than before the pandemic, while 15.8% reported their mental health was better. we examined the change in depressive symptoms using the change in phq-9 scores. for behavior changes related to in-person contact with family, close friends, and colleagues, as contact decreased, there was a slight, but statistically significant increase in depressive symptoms (table 4) . a similar pattern was seen for frequenting your usual place of work, restaurants and bars, and stores. there was no statistically significant association between reduction in travel or contact with strangers and depressive symptoms. overall, respondents indicated highest mean agreement that covid-19 had interrupted their social life (mean: 4.44; sd: 1.61). table 5 provides the mean agreement scores and t-test analyses for differences in social life, work life, home life, and mental health interruptions due to covid-19 overall and across child and work status. those with children at home indicated higher agreement that the pandemic had interrupted social, work, and home lives and hurt their mental health compared to those who did not have children at home. those who were working before the pandemic similarly reported higher levels of interruption for social, work, and home life and worse mental health as opposed to those who were not working. the sample was generally knowledgeable about covid-19. the vast majority (93.6%) correctly identified that the world health organization had declared covid-19 a global pandemic. nearly all correctly identified that covid-19 was spread by respiratory droplets from coughs and sneezes (90.0%), and by touching infected surfaces followed by touching your face (91.9%). a smaller, but still large percentage, (77.7%) correctly identified that at the time the survey was distributed (march 31st, 2020) there was no vaccination for covid-19. a bivariate analysis showed that general knowledge largely differed by media bias and sociodemographic characteristics (sex, race, poverty level, and education). however, after adjusting for all parameters using logistic regression, education was no longer significant, sex was only significant on two of the four questions, while race and income were significant on three of the four questions (table 6 ). while media bias was not significant for most questions, removing it from the model worsened model fit. ) of correctly reporting that one can contract covid-19 by touching infected surfaces and then touching one's nose or mouth. compared to those whose news bias score leaned conservative, those whose news bias score was moderate or leaned liberal had 2.2 (95%ci: 1.2-3.9) and 2.2 (95%ci: 1.3-3.8) times the odds of correctly reporting that covid-19 is primarily transferred through respiratory droplets. greater than 90% of respondents correctly identified fever, cough, and shortness of breath as symptoms for covid-19. however, a majority also said that nausea (69.5%), aches (53.0%), and nasal congestion (66.7%) were common symptoms of covid-19. likewise, more than 85% of respondents correctly identified hand washing (94.1%), not touching your face (90.4%), avoiding contact with sick persons (88.0%), avoiding large groups (89.8%) and sanitizing surfaces (88.6%) as recommendations from the centers for disease control and prevention (cdc) to prevent covid-19. a smaller, but still large percentage identified avoiding eating in restaurants and bars (70.6%) as recommended. a little over half (50.4%) correctly identified wearing a facemask in public as not being an official recommendation of the cdc. this recommendation was released on april 3rd, 2020 (four days after the survey was administered). in comparing covid-19 to seasonal influenza, the majority (65.5%) correctly identified covid-19 as having a higher case-fatality rate. however, 22% felt that seasonal influenza and covid-19 had similar risk of death and 12% reported that seasonal influenza was more deadly than covid-19. those who were politically conservative were more likely (p-value <0.001) to say that the seasonal influenza was more deadly than covid-19 (25.7%) compared to moderates (10.3%) and liberals (9.9%). political ideology was the strongest factor associated with attitudes toward the covid-19 response. this finding is consistent with research suggesting that as new politicized issues emerge, ideology is predictive of adopting beliefs which are suggested to be consistent with an ideology. [18] suggestions of beliefs that correspond with ideology may be implied by the deliverer of information (e.g. a conservative or liberal lawmaker) or through language cues in information sources, such as media. political ideology was further associated with behavior change surrounding covid-19. this finding is consistent with the theory of planned behavior [9] . as political ideology was associated with attitudes toward the covid-19 response, it is reasonable to assume that those with attitudes suggesting government or community over-response to the pandemic would be associated with beliefs that recommended behavior changes were unnecessary. the u.s. political climate continues to affect individual, organizational, and governmental responses as the pandemic evolves. however, our results suggest that, even early in the pandemic, political ideology played a large role in the attitudes and behaviors adopted by u.s. adults. the ability of political ideology (and related measurements such as news source bias) to predict an individual's attitudes about and adherence to recommended behaviors in response to a public health crisis raises concerns about the efficacy of existing strategies to manage such crises in this era of extreme politicization. [8] this suggests the necessity of developing politically neutral strategies that facilitate effective communication surrounding public health crises. sociodemographic characteristics were associated with pandemic-related economic anxieties (sex, income, and race), attitudes toward the community response (sex and race), and knowledge (race) about covid-19. many of these discrepancies point to persistent gender, income, and racial inequality in the u.s. these phenomena are particularly well illustrated when analyzing the disproportionate burden of economic anxieties felt by minority races, lower income individuals, and females. higher economic anxiety would be expected among those in lower income brackets, as they have a reduced ability to weather income loss or unexpected expenses. it is also unsurprising that racial minorities in the u.s. are experiencing higher economic anxieties, given the conflation of poverty and race in the u.s. increased economic anxiety in females is consistent with other research. this may be at least partially explained by poorer perceived economic stability relative to males. [19] disparities in care-giving responsibilities may also help explain sex differences in economic anxieties. females generally have more care-giving responsibilities for home, children, and family, as dictated by societal tradition. [20] responsibility for maintaining family schedules and routines during this pandemic would likely add disproportionately to the physical and emotional strain on females in the u.s. while general knowledge about covid-19 was high, most respondents also identified symptoms including nausea, aches, and nasal congestion which were not part of the initial symptom list. this finding may reflect the emerging nature of information about covid-19 or inaccurate information spreading by word-of-mouth rather than official sources. while general knowledge about covid-19 was widely exhibited across most sociodemographic and political characteristics (a promising demonstration of the wide reception of public health messages and recommendations), black and hispanic respondents were generally less likely to respond correctly to knowledge questions, which may be a result of a larger proportion of black and hispanic respondents lacking access to adequate resources or receiving misinformation. this is particularly concerning given racial differences in the rate of severe complications and deaths from covid-19. [21] at the time of writing, 48% of deaths due to covid-19 in chicago had occurred in african americans, despite the fact that their percent of confirmed cases (33%) mirrored their proportion of the chicago population (30%). [21] unfortunately, these racial and economic disparities mirror well-documented disparities for many other respiratory infectious diseases, including severe outcomes from influenza. these disparities, rooted in historic, racially-motivated policies, limit african americans' access to care and information and exacerbate factors that place them at higher risk for pre-existing conditions. after only two weeks of social distancing in most areas of the country, one-third of respondents reported worse mental health than before covid-19. this finding is consistent with research which identifies social isolation as a significant factor in mental health. as social distancing fundamentally requires separation from most sources of community (i.e. work, religious communities, friends, family, etc.), increases in loneliness as the pandemic progresses may be expected. [22] social isolation and loneliness are linked to significant increases in morbidity and mortality, which raises concerns about population well-being in the event of protracted social distancing and supports the need to find means of social connection that are consistent with social distancing recommendations. [23] disruption to social, work, and home life and worsened mental health due to covid-19 were higher for those with children at home and for those who were working for pay before the pandemic. due to school closures, many with children at home are managing new roles as full-time caregivers and managing educational activities, often while maintaining their own employment responsibilities. higher levels of disruption and worsened mental health among the employed likely results from disruption of daily routines, job insecurity, or an absence of valued social interaction. we acknowledge that these results were based off a cross-sectional study regarding an emerging infection. at the time of data collection, information about covid-19 was nascent. knowledge, best practices, attitudes, and impacts have rapidly changed since the collection of these data. therefore, the generalizability of our results are limited to adult populations in the u.s. during the early weeks of the pandemic's influence in the u.s. nevertheless, early stage information regarding this pandemic, may prove useful for future outbreaks of emerging infections. this study was conducted approximately two weeks after implementation of initial social distancing guidelines. as such, it provides the opportunity to examine the early impacts of covid-19 and associated social distancing in the u.s. population. although this provides useful information, it is unlikely to represent the attitudes, anxieties, and behaviors of the population throughout the pandemic. quota sampling for sex, race, and income provided a sample that is statistically similar to the overall population of u.s. adults; however, samples derived from internet panels may differ in unmeasurable ways from the u.s. population. our sample under-represents households with children at home (28.9% of sample vs. 45.0% u.s. households). [10] conclusion these findings underscore the need to develop public health messaging that considers the influence of the political climate. strict fact-based messaging may simply be insufficient to engage the community in desired public health actions, particularly for highly politicized events such as covid-19. public health experts should consider differential messaging that appeals to the values of those across the political spectrum. covid-19): cases and deaths in the factors associated with mental health outcomes among health care workers exposed to coronavirus disease 2019 a nationwide survey of psychological distress among chinese people in the covid-19 epidemic: implications and policy recommendations mental health outcomes among frontline and second-line health care workers during the coronavirus disease 2019 (covid-19) pandemic in italy mental health outcomes of the covid-19 pandemic. rivista di psichiatria immediate psychological responses and associated factors during the initial stage of the 2019 coronavirus disease (covid-19) epidemic among the general population in china effective communication during an influenza pandemic: the value of using a crisis and emergency risk communication framework. health promotion practice crisis leadership and hurricane katrina: the portrayal of authority by the media in natural disasters the theory of planned behavior. organizational behavior and human decision processes families by presence of own children under 18 political polarization in the american public: how increasing ideological uniformity and partisan antipathy affect politics, compromise and everyday life. pew research center political polarization in the american public global warming's six americas vaccine hesitancy survey questions related to sage vaccine hesitancy matrix: examples of survey questions designed to assess determinants of vaccine hesitancy. who the media bias chart version 5 the phq-9: validity of a brief depression severity measure more than ideology: conservative-liberal identity and receptivity to political cues women and economic anxiety. marketplace-edison research poll housework: who did, does or will do it, and how much does it matter? social forces chicago's coronavirus disparity: black chicagoans are dying at nearly six times the rate of white residents, data show an overview of systematic reviews on the public health consequences of social isolation and loneliness loneliness and social isolation as risk factors for mortality: a meta-analytic review we thank william f. christensen for his comments and feedback. key: cord-298679-w0yp4u19 authors: iftimie, simona; lópez-azcona, ana f.; vicente-miralles, manuel; descarrega-reina, ramon; hernández-aguilera, anna; riu, francesc; simó, josep m.; garrido, pedro; joven, jorge; camps, jordi; castro, antoni title: risk factors associated with mortality in hospitalized patients with sars-cov-2 infection. a prospective, longitudinal, unicenter study in reus, spain date: 2020-09-03 journal: plos one doi: 10.1371/journal.pone.0234452 sha: doc_id: 298679 cord_uid: w0yp4u19 spain is one of the countries that has suffered the most from the impact of severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the strain that causes coronavirus disease 2019 (covid-19). however, there is a lack of information on the characteristics of this disease in the spanish population. the objective of this study has been to characterize our patients from an epidemiological point of view and to identify the risk factors associated with mortality in our geographical area. we performed a prospective, longitudinal study on 188 hospitalized cases of sars-cov-2 infection in hospital universitari de sant joan, in reus, spain, admitted between 15(th) march 2020 and 30(th) april 2020. we recorded demographic data, signs and symptoms and comorbidities. we also calculated the charlson and mccabe indices. a total of 43 deaths occurred during the study period. deceased patients were older than the survivors (77.7 ± 13.1 vs. 62.8 ± 18.4 years; p < 0.001). logistic regression analyses showed that fever, pneumonia, acute respiratory distress syndrome, diabetes mellitus and cancer were the variables that showed independent and statistically significant associations with mortality. the charlson index was more efficient than the mccabe index in discriminating between deceased and survivors. this is one of the first studies to describe the factors associated with mortality in patients infected with sars-cov-2 in spain, and one of the few in the mediterranean area. we identified the main factors independently associated with mortality in our population. further studies are needed to complete and confirm our findings. in january 2020, a new type of coronavirus was identified as the causative factor in a series of cases of severe pneumonia in the city of wuhan, province of hubei, in the people's republic of china [1] . the world health organization gave the official name 'covid-19' for this coronavirus disease, as well as the term 'severe acute respiratory syndrome coronavirus 2' (sars-cov-2) for the virus [2] . this virus is currently the cause of a global pandemic, producing hundreds of thousands of hospital admissions and deaths, with enormous effects on the health and life of the population and serious economic consequences for society. on 1 st february, 2020, the first case of a sars-cov-2 positive patient in spain was reported on the island of la gomera [3] and, following that, the first cases diagnosed in the autonomous region of catalonia date from 5 th march [4] . the incubation period for sars-cov-2 ranges from 5 to 6 days on average, with cases being possible from 0 to 14 days [5] . the most common period of transmission of the virus begins 1-2 days before the onset of symptoms, and lasts for up to 5-6 days after the onset of symptoms [6] . the basic reproductive rate r (the average of new cases secondary to a primary case) in our country is, at the time of writing, estimated to be <1; globally, the r number ranges from 0 to 6 depending on various factors, in particular the political and public health measures imposed by the various governments that focus on complete cleaning of public spaces and a decrease in contact between individuals [7] . identifying the epidemiological characteristics of this disease will help appropriate decisions to be made and thus to control the epidemic. certain clinical symptoms of covid19 have been reported previously. the most frequent are: fever, dry cough, asthenia, expectoration, dyspnea, sore throat, headache, myalgia, arthralgia, chills, nausea or vomiting, nasal congestion, diarrhea, hemoptysis and conjunctival congestion (from highest to lowest frequency) [8, 9] . occasionally, symptoms of a different nature appear: neurological, such as altered consciousness or dizziness; cardiological, such as acute myocardial damage or heart failure; or ophthalmological, such as dry eye, blurred vision, foreign body sensation and conjunctival congestion [10] [11] [12] [13] . to date, there is still a lack of information on the characteristics of sars-cov-2 infection outside china. spain is one of the western european countries that has suffered the most from the impact of covid-19 and this pandemic has had a great impact on our public health system. the present study reports the results of an analysis of all cases hospitalized in the hospital universitari de sant joan, which is affiliated to the universitat rovira i virgili, in reus, catalonia, spain. the objective of the present study has been to characterize our patients' epidemiology and to identify the risk factors associated with mortality for this disease in our geographical area. this is a prospective longitudinal study conducted on all hospitalized cases of sars-cov-2 infection in hospital universitari de sant joan, in reus, spain admitted between 15 th march 2020 and 30 th april 2020. this hospital has 392 beds provided for hospitalization and social health care and is part of the hospital network for public use in catalonia. it acts as a general hospital for a population of over 175,000 inhabitants, including primary care centers and residences for the elderly in the area. it is a reference center for the specialities of oncology and radiotherapy for the whole of the tarragona province, which has a population of 550,000 inhabitants. sars-cov-2 infection was confirmed by reverse transcription-polymerase chain reaction (rt-pcr) using swab samples from the upper respiratory tract (nasopharyngeal/oropharyngeal exudate), from the lower respiratory tract (sputum/endotracheal aspirate/ bronchoalveolar lavage/bronchial aspirate) or from the lower digestive tract (rectal smear). tests were carried out with the viasure sars-cov-2 real time pcr detection kit that detects orf1ab and n genes (certest biotec, zaragoza, spain). rna was extracted in a qiacube apparatus with rneasy reagents (qiagen n.v., hilden, germany) according to the manufacturer's instructions, and analyses were carried out in a 7500 fast rt-pcr system (applied biosystems, foster city, ca,usa). we recorded demographic data, comorbidities, and other acute or chronic infections. we also calculated the mccabe score as an index of clinical prognosis [14] and the charlson index (abbreviated version) as a way of categorizing a patient's comorbidity [15] . the only inclusion criterion was to be a hospitalized patient with an analytical diagnosis of sars-cov-2. we excluded hospitalized patients with suspected sars-cov-2 infection but without laboratory confirmation, or patients who did not require hospitalization, with or without laboratory diagnosis of sars-cov-2 infection. thirty-four patients required transfer to the intensive care unit based on the attending specialist's criteria, and taking into account the curb65 scale and the ats/idsa criteria [16, 17] . this study was approved by the data are shown as means and standard deviations or as numbers and percentages. statistical comparisons between two groups were carried out with the student's t test (quantitative variables) or the χ-square test (categorical variables). logistic regression models were fitted to investigate the combined effect of selected variables on mortality. the diagnostic accuracy of the mccabe and charlson indices in predicting mortality was assessed by receiver operating characteristics (roc) analysis [18] . statistical significance was set at p �0.05. all calculations were made using the spss 25.0 statistical package (spss inc., chicago, il, usa). the raw data for this article are shown as supporting information. during the study period, a total of 188 patients were hospitalized for sars-cov-2 infection. the mean age was 66.4 ± 18.4 years (range: 0-102) and a small majority were men (55.8 vs. 44.2%; p < 0.001). one hundred and eighteen patients were admitted to the department of internal medicine, 34 to the intensive care unit, and 36 to the social health unit. thirty-two patients were admitted to hospital due to causes unrelated to the suspicion of covid-19 infection but gave a positive result in the rt-pcr. a total of 43 deaths occurred during the entire study period (fig 1) , so the case fatality rate was 22.9% based on the total number of covid-19 hospitalized patients. deceased patients were significantly older than the survivor patients (77.7 ± 13.1 vs. 62.8 ± 18.4 years; p < 0.001). thirty-seven patients died of respiratory failure, 4 of multi-organ failure and 2 of cardiogenic shock. a total of 125 patients (66.5%) had chronic underlying diseases. some seriously ill patients could not be admitted to the icu due to their pathological history and/or comorbidities associated with their advanced age and who made aggressive treatments inadvisable. the relationships between covid-19 and the demographic and clinical variables are shown in table 1 and fig 2. most of the cases and deaths were of patients between 70 and 89 years old (fig 2a) . the signs and symptoms present in more than 50% of the patients were, in descending order, fever (64.9%), dyspnea (58.0%), pneumonia (57.4%), and cough (51.6%) (fig 2b) . the most relevant comorbidities were cardiovascular diseases (50.5%), type 2 diabetes mellitus (26.0%), and chronic neurological diseases (19.1%) (fig 2c) . we also evaluated whether patients had had any behaviour that might be considered risky in the days prior to admission, and we observed that a high proportion of patients had attended another health center in the previous month or had been in contact with people infected with sars-cov-2 or with respiratory problems over the previous 14 days (fig 2f) . five employees of our institution or the associated residences were hospitalized for covid-19, although not requiring either intensive measures or ventilatory support. most of the patients presented low values on the charlson and mccabe indices and, as expected, higher scores were associated with higher mortality (fig 2d and 2e ). when comparing the diagnostic accuracy of the roc curves of these indices in their ability to discriminate between deceased patients and survivors, we found that charlson index was more efficient, with higher values of the area under the curve (fig 3) . finally, since the different symptoms and comorbidities can be mutually interdependent and present cause-effect relationships between them, we wanted to identify which factors were independently associated with mortality. logistic regression analyses showed that the presence of fever, pneumonia, acute respiratory distress syndrome, type 2 diabetes mellitus and cancer were the only variables that showed an independent and statistically significant association with mortality when they were adjusted for differences in age, gender, smoking status and alcohol intake (tables 2 and 3) . one hundred and thirty-seven patients (72.9%) required one or more than one type of respiratory intervention, including noninvasive (face mask) or invasive (endotracheal tube) mechanical ventilation, high flow oxygen therapy (up to 60 l/min.) or conventional oxygen therapy (table 4 ). no significant differences in mortality were observed in patients requiring globally analyzed respiratory intervention (25.5 vs. 17.6%, p = 0.173), but there was a non-significant trend towards higher mortality in the subgroup of patients receiving high flow oxygen therapy (38.9 vs. 21.8%, p = 0.094). moreover, we did not find any significant difference in mortality in relation to whether patients were treated with anticoagulants or corticosteroids or not (anticoagulants: 24.1 vs. 17.6%, p = 0.399; corticosteroids: 26.0 vs. 21.8%, p = 0.318). we carried out a rt-pcr determination for all the patients admitted to our center, regardless of the diagnosis. thirty-two patients (17.0%) admitted for reasons other than suspected sars-cov-2 infection gave a positive result despite not presenting any symptoms. we believe that this is important since it highlights the need to perform diagnostic tests for this disease in all hospitalized patients, something which has not been given sufficient attention in the scientific literature. most of our patients were over 60 years old and mortality was very high (47.0%) among those over 80 years old. these results are consistent with those published so far, which show that age is one of the most important risk factors for covid-19 [19] [20] [21] [22] . it is accepted that age is a risk factor for respiratory diseases [19, 23, 24] and impairment of immune function associated with age has been identified as a major cause of high mortality due to severe pneumonia [23] . we observed a higher mortality rate in patients treated with high flow oxygen therapy, but the observed differences did not reach statistical significance. the limited size of our sample prevents us from obtaining reliable conclusions in this regard. among the signs and symptoms of the disease, we found that fever, pneumonia, and acute respiratory distress syndrome were the only factors independently associated with mortality when adjusted for age, smoking and alcohol intake. these factors are among those that have been most frequently found in patients with covid-19 in most of the studies conducted in china [19, 25, 26] . we did not observe any independent relationship between cough, chills or gastrointestinal disturbances and mortality, despite being present in a relatively high proportion of subjects, something which differs from what has been published previously [19] . the comorbidities showing a significant relationship with mortality were type 2 diabetes mellitus and cancer. we did not find any independent association with any other chronic metabolic disease, such as cardiovascular disease or others. the univariate analysis showed a high number of patients with these chronic alterations and the logistic regression analysis identified diabetes as the most relevant. indeed, all of these metabolic diseases are closely related. diabetes is a causative factor of hypertension and metabolic syndrome and these, in turn, can cause heart, vascular, liver, neurological and kidney diseases. our study therefore suggests that diabetes might be a triggering factor for these disorders and therefore is related to mortality in patients infected with sars-cov-2. type 2 diabetes mellitus has also been reported to be one of the most important factors related with covid-19 severity in previous investigations conducted in china, israel and italy [25, [27] [28] [29] . indeed, the italian study reported that 2/3 of the patients who died were diabetic [29] . furthermore, diabetes is linked to a higher mortality in other viral infections, such as those caused by influenza a(h1n1), mers-cov and sars-cov viruses [30, 31] . we also found a close relationship between cancer and covid-19 mortality. one aspect that caught our attention is that, despite our hospital being the reference center for oncology in our province, the number of cancer patients infected with covid-19 was relatively low. it might be that the corticosteroids often prescribed for the treatment of these patients offered some protection, as some studies have suggested [32, 33] . however, we did not observe any significant difference in mortality in relation to whether patients were treated with corticosteroids or not. an alternative explanation is that, perhaps, these patients were more careful than the general population during confinement, which unfortunately cannot be proven. having said that, the relationship between cancer and the mortality of our patients was evident. patients with cancer are often immunosuppressed and as a result they are more likely to worsen rapidly if infected by sars-cov-2. wuhan studies report that the incidence of cancer is higher in covid-19 patients than in the general population [34, 35] . however, definitive conclusions on this issue are hampered by the small sample size, the retrospective nature of most studies, the limited follow-up duration, and the heterogeneity of the disease and treatment strategies [36, 37] . the influence of smoking on covid-19 is controversial. an unusually low prevalence of current smoking among infected patients was observed in china [38] and the plausibility of using medicinal nicotine to lower infection and mitigate disease severity has been proposed [39] . however, other studies indicate that smokers might be at higher risk because nicotine can directly impact the putative receptor for the virus (angiotensin-converting enzyme 2) and lead to harmful signaling in lung epithelial cells [40] . in the present study, we have found no firm positive or negative relationship between tobacco use and mortality because only 9 patients (4.8%) were active smokers at the time of the study. that might be explained by their generally advanced age and because many of them were suffering from chronic ailments that had advised them to quit tobacco use. a novel aspect of our study has been to investigate the usefulness of some frequently used clinical scores in the evaluation of infectious diseases. we found that the charlson index, which categorizes comorbidity might be more useful than the mccabe index in predicting death in these patients. we chose these indices because they are easy to apply in all patients, both in the less severe, and in those receiving palliative treatment or admitted to the social health unit. other scores are difficult to apply in less severe patients. for example, the curb 1 scale only applies to patients with pneumonia [41] . the quick sofa index requires measurement of respiratory rate, a data not frequently collected in milder patients [42] , and sofa and saps-ii require arterial blood gas analysis, which is difficult to justify in patients not admitted to the icu [43] . a limitation of the present study is the small sample size. ours is not a big hospital and covers a relatively small geographical area. however, we believe that the results obtained are relevant since they might be representative of many similar centers in western europe and in the mediterranean area, and little information is yet available on this issue. this is one of the first studies to describe the factors related with death in patients infected with sars-cov-2 in spain, and one of the few from the mediterranean basin. our results identify age, fever, pneumonia, acute respiratory distress syndrome, type 2 diabetes mellitus and cancer as independent factors predicting lethality. further studies are needed in similar centers to complete and confirm our findings. a novel coronavirus from patients with pneumonia in china naming the coronavirus disease (covid-19) and the virus that causes it the virus that changed spain: impact of covid-19 on people with hiv chasing the ghost of infection past: identifying thresholds of change during the covid-19 infection in spain early transmission dynamics in wuhan, hina, of novel coronavirus-infected pneumonia how will country-based mitigation measures influence the course of the covid-19 epidemic? estimation of the reproductive number of novel coronavirus (covid-19) and the probable outbreak size on the diamond princess cruise ship: a data-driven analysis clinical presentation of covid-19: a systematic review focusing on upper airway symptoms virology, epidemiology, pathogenesis, and control of covid-19 neurological manifestations in covid-19 caused by sars-cov-2 covid-19 and the cardiovascular system characteristics of ocular findings of patients with coronavirus disease 2019 (covid-19 ocular manifestations of a hospitalised patient with confirmed 2019 novel coronavirus disease gram-negative bacteremia. i. etiology and ecology emotional support and survival after myocardial infarction. a prospective, population-based study of the elderly modified idsa/ats minor criteria for severe community-acquired pneumonia best predicted mortality defining community acquired pneumonia severity on presentation to hospital: an international derivation and validation study receiver-operating characteristic (roc) plots: a fundamental evaluation tool in clinical medicine risk factors for mortality in 244 older adults with covid-19 in wuhan, china: a retrospective study risk factors for predicting mortality in elderly patients with covid-19: a review of clinical data in china clinical features of covid-19 in elderly patients: a comparison with young and middle-aged patients epidemiological characteristics of coronavirus disease 2019 (covid-19) patients in iran: a single center study pneumonia in the elderly: a review of the epidemiology, pathogenesis, microbiology, and clinical features pneumonia in the elderly: understanding the characteristics clinical findings of patients with coronavirus disease 2019 in jiangsu province, china: a retrospective, multi-center study imaging and clinical features of patients with 2019 novel coronavirus sars-cov-2 clinical characterization of 162 covid-19 patients in israel: preliminary report from a large tertiary center covid-19 and diabetes mellitus: may old anti-diabetic agents become the new philosopher's stone? covid-19 and italy: what next? prevalence of diabetes in the 2009 influenza a (h1n1) and the middle east respiratory syndrome coronavirus: a systematic review and meta-analysis plasma glucose levels and diabetes are independent predictors for mortality and morbidity in patients with sars use of corticosteroids in coronavirus disease 2019 pneumonia: a systematic review of the literature effect of dexamethasone in hospitalized patients with covid-19 -preliminary report sars-cov-2 transmission in patients with cancer at a tertiary care hospital in wuhan, china clinical characteristics of covid-19-infected cancer patients: a retrospective case study in three hospitals within wuhan, china risk of covid-19 for patients with cancer covid-19 and cancer: what we know so far systematic review of the prevalence of current smoking among hospitalized covid-19 patients in china: could nicotine be a therapeutic option? beyond smoking cessation: investigating medicinal nicotine to prevent and treat covid-19 is nicotine exposure linked to cardiopulmonary vulnerability to covid-19 in the general population? defining community acquired pneumonia severity on presentation to hospital: an international derivation and validation study the third international consensus definitions for sepsis and septic shock (sepsis-3) a new simplified acute physiology score (saps ii) based on a european/north american multicenter study the authors are indebted to all the staff of the hospital universitari de sant joan, doctors, nurses, assistants, cleaning and security personnel, and all the volunteer students, who with their enormous effort are managing to overcome this dramatic situation. editorial assistance was provided by phil hoddy at the service of linguistic resources of the universitat rovira i virgili. key: cord-291417-p49ukyhx authors: mikulska, malgorzata; nicolini, laura ambra; signori, alessio; di biagio, antonio; sepulcri, chiara; russo, chiara; dettori, silvia; berruti, marco; sormani, maria pia; giacobbe, daniele roberto; vena, antonio; de maria, andrea; dentone, chiara; taramasso, lucia; mirabella, michele; magnasco, laura; mora, sara; delfino, emanuele; toscanini, federica; balletto, elisa; alessandrini, anna ida; baldi, federico; briano, federica; camera, marco; dodi, ferdinando; ferrazin, antonio; labate, laura; mazzarello, giovanni; pincino, rachele; portunato, federica; tutino, stefania; barisione, emanuela; bruzzone, bianca; orsi, andrea; schenone, eva; rosseti, nirmala; sasso, elisabetta; da rin, giorgio; pelosi, paolo; beltramini, sabrina; giacomini, mauro; icardi, giancarlo; gratarola, angelo; bassetti, matteo title: tocilizumab and steroid treatment in patients with covid-19 pneumonia date: 2020-08-20 journal: plos one doi: 10.1371/journal.pone.0237831 sha: doc_id: 291417 cord_uid: p49ukyhx introduction: coronavirus disease 2019 (covid-19) can lead to respiratory failure due to severe immune response. treatment targeting this immune response might be beneficial but there is limited evidence on its efficacy. the aim of this study was to determine if early treatment of patients with covid-19 pneumonia with tocilizumab and/or steroids was associated with better outcome. methods: this observational single-center study included patients with covid-19 pneumonia who were not intubated and received either standard of care (soc, controls) or soc plus early (within 3 days from hospital admission) anti-inflammatory treatment. soc consisted of hydroxychloroquine 400mg bid plus, in those admitted before march 24(th), also darunavir/ritonavir. anti-inflammatory treatment consisted of either tocilizumab (8mg/kg intravenously or 162mg subcutaneously) or methylprednisolone 1 mg/kg for 5 days or both. failure was defined as intubation or death, and the endpoints were failure-free survival (primary endpoint) and overall survival (secondary) at day 30. difference between the groups was estimated as hazard ratio by a propensity score weighted cox regression analysis (hr(ow)). results: overall, 196 adults were included in the analyses. they were mainly male (67.4%), with comorbidities (78.1%) and severe covid-19 pneumonia (83.7%). median age was 67.9 years (range, 30–100) and median pao(2)/fio(2) 200 mmhg (iqr 133–289). among them, 130 received early anti-inflammatory treatment with: tocilizumab (n = 29, 22.3%), methylprednisolone (n = 45, 34.6%), or both (n = 56, 43.1%). the adjusted failure-free survival among tocilizumab/methylprednisolone/soc treated patients vs. soc was 80.8% (95%ci, 72.8–86.7) vs. 64.1% (95%ci, 51.3–74.0), hr(ow) 0.48, 95%ci, 0.23–0.99; p = 0.049. the overall survival among tocilizumab/methylprednisolone/soc patients vs. soc was 85.9% (95%ci, 80.7–92.6) vs. 71.9% (95%ci, 46–73), hr(ow) 0.41, 95%ci: 0.19–0.89, p = 0.025. conclusion: early adjunctive treatment with tocilizumab, methylprednisolone or both may improve outcomes in non-intubated patients with covid-19 pneumonia. coronavirus disease 2019 can lead to respiratory failure due to severe immune response. treatment targeting this immune response might be beneficial but there is limited evidence on its efficacy. the aim of this study was to determine if early treatment of patients with covid-19 pneumonia with tocilizumab and/or steroids was associated with better outcome. this observational single-center study included patients with covid-19 pneumonia who were not intubated and received either standard of care (soc, controls) or soc plus early (within 3 days from hospital admission) anti-inflammatory treatment. soc consisted of hydroxychloroquine 400mg bid plus, in those admitted before march 24 th , also darunavir/ ritonavir. anti-inflammatory treatment consisted of either tocilizumab (8mg/kg intravenously or 162mg subcutaneously) or methylprednisolone 1 mg/kg for 5 days or both. failure was defined as intubation or death, and the endpoints were failure-free survival (primary a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 pandemic coronavirus disease 2019 (covid-19) caused by sars-cov-2 coronavirus has recently emerged [1] . although most of the infected patients will remain asymptomatic or develop mild symptoms, up to 20% may develop severe disease with pneumonia and respiratory failure [2] . oxygen administration is the cornerstone of supportive treatment and is required in approximately 15% of cases, while invasive mechanical ventilation is necessary in up to 5-7% of severe cases [3] [4] [5] . since mortality in patients with invasive ventilation can be very high, halting the progression from moderate to severe respiratory failure should reduce the mortality in covid-19 [6] . at the beginning of covid-19 pandemics, based on the experience with previous studies in viral pneumonia, including sars-cov and mers, the use of steroids was discouraged, mainly due to undocumented benefit and fearing potential increase in viral proliferation and side effects [7, 8] . however, with the increasing knowledge about covid-19, a biphasic model of the disease has been proposed [9, 10] . according to this model, the first phase is caused directly by viral replication, while in the second phase, the symptoms and respiratory failure are due to inflammatory response, and could be treated with agents which reduce inflammation, such as corticosteroids, or inhibitors of pro-inflammatory interleukins and janus kinase (jak) [9] [10] [11] . indeed, some real life experiences in covid-19 patients showed that the use of antiinflammatory treatments might be beneficial [12] . in fact, short-term steroid therapy was associated with lower mortality in 201 patients with acute respiratory distress syndrome (ards) [13] . additionally, following the data on presence of inflammatory cytokine storm in severe covid-19, tocilizumab use has been advocated. this monoclonal antibody, which binds to interleukin 6 (il-6) receptor and blocks the il-6 mediated inflammatory response, is approved for treatment of rheumatologic disorders and cytokine-release syndrome associated with chimeric antigen receptor t-cell (car-t) administration. it was reported to reduce covid-19-associated inflammation, and was approved in china for this indication [12, 14] . based on the first evidences, we formulated the hypothesis of potential benefit of antiinflammatory treatment, and progressively modified our therapeutic approach to covid-19. we started using tocilizumab in patients with respiratory failure, and subsequently, we introduced into our protocol early administration of methylprednisolone treatment, followed in more severe cases by the administration of tocilizumab. we hypothesized that outcomes such as no need for intubation and survival of patients with severe covid-19 pneumonia in whom tocilizumab and/or methylprednisolone were administered in addition to standard of care (soc) could be better than in those who received only soc. in this observational single-center study, adult patients admitted to the san martino university hospital, genova, italy, for covid-19 pneumonia were included as cases if treated with tocilizumab and/or methylprednisolone, not intubated, not treated with remdesivir and not pregnant. the outcomes of patients treated with tocilizumab/methylprednisolone were compared to data from consecutive patients admitted to our hospital for covid-19 pneumonia who received only soc, mainly because they were admitted before the routine use of tocilizumab/ methylprednisolone (control group). all patients provided a verbal informed consent because of isolation precautions for treatment with off label agents according to the local protocol approved by hospital authorities, for data collection and for participation in the study, in accordance with national drug agency communication, ver. 2 april 7 th 2020. the study was carried out in accordance with the principles of the declaration of helsinki and approved by the regional ethic committee (n. cer liguria 114/2020-id 10420). data were collected from hospital information system by a standard based automatic procedure and stored in an online database with pseudo-anonymization features suitable for secondary use of clinical data [15] . controls were identified through this prospectively collected database of hospital-admitted covid-19 patients. patients who had any of the following features at the time of, or after, admission were classified as having severe pneumonia: (1) respiratory distress (�30 breaths per min); or (2) oxygen saturation at rest �93%; or (3) ratio of partial pressure of arterial oxygen to fractional concentration of oxygen inspired air (pao 2 /fio 2 ) �300 mm hg; or (4) severe disease complications (e.g., respiratory failure, requirement of mechanical ventilation, septic shock, or non-respiratory organ failure) [7, 14, 15] . systemic inflammation was defined as presence at baseline of one of the following: fever > 38˚c, c-reactive protein (crp) 10 times above the upper limit of normal (uln) of 5 mg/dl, ferritin 2 times above the uln (400 μg/l), or il-6 10 times above the uln of 3.4 ng/l. the first results obtained at hospital admission, and in any case not later than within day 3 of admission were considered. adverse events possibly or probably related to steroid and tocilizumab treatment, such as neutropenia, anemia, thrombocytopenia, increase in alanine aminotransferase (alt) levels, (unl < 40 u/l), microbiologically documented infections and allergic reactions were evaluated for both treated patients and controls. adverse events were collected up to the last available follow up from starting of tocilizumab and/or methylprednisolone in treatment group and from hospital admission in the control group. the common terminology criteria for adverse event (ctcae) version 5.0 was used. the diagnosis of covid-19 was made with a positive rt-pcr assay performed on nasal swab or broncoalveolar lavage fluid according to world health organization interim guidance [16] . patients received treatment with oral hydroxychloroquine 400mg bid, unless glucose-6-phosphate dehydrogenase deficient. until march 24 th , darunavir/ritonavir 800/100 qd was also administered [17] . thereafter, the protocol was amended and darunavir/ritonavir was withdrawn [18] . short-term antibiotic coverage was prescribed at admission. low-molecularweight heparin prophylaxis was administered unless contraindicated. these treatments were defined as soc. since march 11 th , we started adding tocilizumab to soc in case of severe covid-19 pneumonia and systemic inflammation. tocilizumab was administered intravenously at the dose of 8mg/kg (maximum 800mg), with the possibility of repeating the dose after 24 hours if no response was obtained. due to a temporary shortage of intravenous formulation, the available subcutaneous formulation (162 mg) was administered. following an internal review of risks and benefits of steroid treatment in patients with severe covid-19, since march 16 th methylprednisolone (1mg/kg for 5 days intravenously, then 0.5mg/kg for 5 days) was included in the protocol. tocilizumab was added in case of systemic inflammation or rapid respiratory function deterioration. no sample-size calculations were performed. the primary end point was time to failure, defined as intubation and mechanical ventilation or death, whichever occurred first, within 30 days from the hospital admission. the secondary endpoint was overall survival (os). time was calculated from time of hospitalization for the comparison between tocilizumab/methylprednisolone/soc and soc patients and from the date of starting anti-inflammation treatment for the comparison among treatment groups. the landmark analysis was applied in order to minimize the potential immortal time bias that can arise in non-randomized studies and is related to the fact that patients treated after a longer time from admission must have not experienced the event up to that time, and that patients with a very early event (e.g. death) were more likely assigned to the untreated group. this is a conservative analysis which reduced the risk that the treatment choice was motivated by the patient's disease course. therefore, day 3 from hospital admission was set as a landmark time point: those who died, were intubated or discharged from the hospital before day 3 were excluded, while patients were included in the tocilizumab/methylprednisolone treatment group if the treatment was started within 3 days from hospital admission (see fig 1) . to minimize baseline differences between treated and untreated patients a propensity score-based analysis was performed. propensity score (ps) was derived by a logistic regression model including the following baseline variables: age, gender, presence of comorbidities and week of treatment start, ratio of partial pressure of arterial oxygen to fractional concentration of oxygen inspired air (pao 2 /fio 2 ), non-invasive ventilation (niv), time from symptoms onset to hospital admission, il-6, ferritin, c-reactive protein (crp) and d-dimer serum levels. positivity assumption of ps was checked after the calculation. for each patient, the overlap weight (ow) based on ps was calculated [19] . to assess the balance of covariate distribution between the two groups, cohen's standardized mean differences were calculated between the two groups in the original samples and after weighting. an absolute value of difference < 0.10 was considered an acceptable balance. the ow-weighted cox proportional hazard regression model was used to calculate the adjusted hazard-ratio (hr ow ) of tocilizumab/methylprednisolone/soc vs soc patients. weighted cumulative probability of failure or death was calculated by mean of kaplan-meier (km) survival curves. to define risk factors associated with outcomes and to compare the three treatment groups, adjusted hrs were estimated by a multivariable cox proportional hazard regression model. the same baseline variables used in the calculation of ps were considered for the multivariable analysis. to avoid overfitting, only those characteristics who showed a pvalue � 0.15 at univariable analysis and after inclusion in the multivariable model were considered, with age and gender forced into the model. for a better interpretation and to avoid the influence of outliers on estimation, the il-6, ferritin, crp and d-dimer were logtransformed before the analysis due to the highly skewed distribution. all results were reported as hr with 95% confidence interval (95%ci). a subgroup analysis was performed to assess if the treatment effect of tocilizumab versus methylprednisolone on primary outcome was different between subgroups defined according to categorized baseline variables. an interaction test was used to assess a different treatment effect in subgroups. the sensitivity analysis was pre-planned, and the comparison between tocilizumab/methylprednisolone/soc and soc patients was reassessed excluding from the soc group the patients that received tocilizumab or methylprednisolone after 3 days from hospitalization. the ps and ow were recalculated. p was considered significant if � 0.05. stata (v.16; statacorp.) was used for the computation. overall, 215 patients were evaluated: 135 (62.8%) received tocilizumab/methylprednisolone within 3 days from hospitalization, 20 (12.9%) were treated after 3 days from hospitalization and were included in the soc group, and 60 (38.7%) patients received only soc. after excluding the patients who were discharged or developed a failure event (intubation or death) before the day 3 set for landmark analysis, 196 patients were included (130 tocilizumab/methylprednisolone/soc and 66 soc patients) (fig 1) . patients were mainly male (67.4%), with a median age of 67.9 years (range, 30-100), and most of them (78.1%) had comorbidities ( table 1 ). the median pao 2 /fio 2 was 200 mmhg (interquartile range, iqr 133-289), and 164 (83.7%) had severe pneumonia (pao 2 /fio 2 <300mmhg). the median time from the onset of symptoms to anti-inflammatory treatment in 130 patients was 8 days, iqr: 9-15; range 5-23. in univariable and multivariable analyses, older age, male gender, higher baseline inflammatory markers, especially il-6, and pao 2 /fio 2 < 100 mmhg were identified as risk factors for failure ( table 2) . differences between the two groups were consistently reduced after ow. ow weighted characteristics of two groups of patients are shown in table 3 . after (fig 2) . the cox regression analysis adjusted by ow weighted propensity score estimated a significant effect of therapy in reducing the risk of failure (hr ow = 0.48 95%ci, 0.23-0.99; p = 0.049). within the cohort of 130 treated patients, 45 (34.6%) received methylprednisolone, 29 (22.3%) tocilizumab and 56 (43.1%) combined therapy. patients in combined treatment group were younger and with fewer comorbidities but with comparable inflammatory markers, the frequency of low pao 2 /fio 2 and niv (table 4) . after a median follow-up of 53 days (range 4-70, interquartile range 33-57), 28 failures were observed: in 14/45 (31.1%) patients receiving methylprednisolone, 6/29 (20.7%) tocilizumab (1 subcutaneously and 5 intravenously) and 8/56 (14.3%) in combined treatment group. tocilizumab and steroid treatment in patients with covid-19 pneumonia at 14 days of follow-up from treatment start, the failure-free survival (fig 3) was 80% (95% ci, 65.1-89.1) in methylprednisolone group, 79.3% (95%ci, 59.6-90.1) in tocilizumab group and 87.5% (95 ci, 75.6-93.8) in combined therapy group. no significant differences between the three treatment groups were identified in a multivariable analysis adjusted for the baseline risk factors (p for heterogeneity among treatment groups = 0.45) ( table 5 ). it was not possible to identify subgroups, based on demographic or clinical characteristics, with different benefits of tocilizumab versus methylprednisolone or tocilizumab versus combination therapy. as a sensitivity analysis, we run the comparison between tocilizumab/methylprednisolone/ soc and soc patients excluding the 20 patients that received tocilizumab/methylprednisolone treatment after 3 days from hospital admission (6 methylprednisolone, 8 tocilizumab and 6 both), and experienced 5 (25%) failures. the weighted failure-free survival in the soc group was now 72.5% (95%ci, 58.8-81.5) at 14 days and 67.1% (95%ci, 52.8-77.9) after 30 days. the ow weighted difference between tocilizumab/methylprednisolone/soc and soc patients was amplified (hr ow = 0.39; 95%ci, 0.17-0.89; p = 0.026). among 155 patients who received tocilizumab/methylprednisolone/soc at any time, 106 (68%) developed alt increase (grade 1-2: 98/155, 63% and grade 3: 8/155, 5%): 32.4% of those who received methylprednisolone/soc and 77% of those who received tocilizumab/ soc +/-methylprednisolone. microbiologically documented infections were recorded in 12 crp, c reactive protein; il-6, interleukin 6; niv, non invasive ventilation; pao 2 /fio 2 , ratio of partial pressure of arterial oxygen to fractional concentration of oxygen inspired air. https://doi.org/10.1371/journal.pone.0237831.t004 (7.7%) patients: 10 bloodstream infections, 2 ventilator-associated pneumonias and 2 urinary tract infections. in tocilizumab/methylprednisolone/soc group, grade 4 transient neutropenia and grade 2 maculopapular rash occurred in 1 patient each. among 60 soc patients, grade 1-2 alt increase occurred in 5 (8%) and 1 patient developed urinary tract infection. there were no cases of significant decrease of hemoglobin or platelet levels. in this observational study in non-intubated patients with mainly severe covid-19 pneumonia, the early addition of tocilizumab and/or methylprednisolone to soc resulted in adjusted failure-free survival of 86.5% and 80.8% at day 14 and 30, which was, respectively, 10.7% and 16.7% higher than in soc patients. even though only the minority of patients develop the severe form of covid-19 (14% in the chinese cohort of 72.314 patients), the outcomes in this group are poor, with 24.9% rate of failure (intubation or death) reported in 173 patients with severe disease [2, 20] . the observation that covid-19-associated respiratory failure can be caused by cytokine storm rather than viral progression is the rationale for administering anti-inflammatory treatments, including tocilizumab [9, 10, 21] . the initial studies on tocilizumab in covid-19 reported a clinical benefit in retrospective cohorts of 21 and 15 patients with moderate to critical covid-19 pneumonia, in whom steroids were also administered [12, 22] . however, none of studies reported and evaluated the impact of steroid co-administration, nor included a control group which did not receive tocilizumab. subsequently these treatments were recommended by chinese diagnosis and treatment protocol for novel coronavirus pneumonia (version 6 and 7) and experience from larger cohorts in europe have been published [14, 23, 24] . however, considering the rapid widespread increase in severe cases of covid-19 worldwide, the availability and the cost of anti-il-6 treatment might limit its use. therefore, at the peak of covid-19 epidemics in our city, we implemented the early use of corticosteroids and the use of subcutaneous tocilizumab if intravenous formulation was not readily available. we acknowledge that the benefit of subcutaneous formulation might be lower and slower than in case of intravenous drug, but no standard intravenous dose for covid-19 has been established, as one study used 400 mg and the other the range of doses from 80 mg to 600 mg, irrespective of the patients' weight [12, 22] . the first study that reported the impact of steroid treatment in covid-19, showed that it was administered to 30.8% of patients, mainly in case of more severe disease, and was associated with a significant reduction of the risk of death in patients with ards (hr = 0.38) [13] . while other cohorts reported steroid use in approximately 44% of severely ill patients, they did not analyze their influence on outcome [20, 25] . subsequently, an observational study reported a benefit of early administration of steroid therapy on composite outcome of icu admission, mechanical ventilation or death at 14 days (34.9% vs. 54.3%) and overall survival (86.4% vs. 73.7%) [26] . in addition to these data, we were able to demonstrate that, after minimizing as much as possible the differences between the groups through ow adjustment, the outcome of patients was better in case of early treatment with tocilizumab and/or methylprednisolone. indeed, in soc group the rate of failure at day 14 of 24.2% was very similar to what reported in other cohorts with severe pneumonia (24.9%), while it was reduced to 13.5% in our tocilizumab/methylprednisolone/soc group [20] . our data show that this benefit was also present with month-long follow up (overall hr ow of 0.48), which is important in establishing long term prognosis of these patients. moreover, the benefit of early tocilizumab/methylprednisolone was also noted on overall survival, both at 14 and 30 days (respectively, 92.7% vs. 78.2% and 85.9% vs. 71.9%). compared to the study with steroid use only, the 14-day survival was higher in our cohort, providing background for the hypothesis that combined tocilizumab/steroid treatment might be warranted [26] . finally, preliminary results of the recovery trial reported higher survival rate in patients with severe covid-19 pneumonia treated with dexamethasone, although the detailed results are not available yet. interestingly, our observational study documented that these patients were treated at a median time of 8 days after the onset of symptoms, which is compatible with the timing of cytokine storm, and therefore might be optimal for the effect of anti-inflammatory treatment. consistent with other studies, we identified older age, high il-6 levels and poor respiratory function as independent predictors of failure, with possible impact also of crp and d-dimer levels [27] . possibly due to a limited sample size, we were unable to document which of three treatment groups provided most benefit, and if there were predictors of better response to tocilizumab/methylprednisolone compared to methylprednisolone alone in any subset of patients. however, the rate of failure-free survival was the highest in the combination treatment group. in addition, based on our sensitivity analyses, adding the anti-inflammatory treatment later after hospital admission might still provide some clinical benefit. in fact, including in the soc group patients who received anti-inflammatory treatment later during the infectious course possibly reduced the difference between the study arms, supporting the overall benefit of an early anti-inflammatory treatment. the limitations of this study include the non-randomized design, yet the inclusion of consecutive patients using the same soc but not treated with tocilizumab or methylprednisolone, and adjustment for the outcome-associated variables, allowed to note the improvement in patient outcomes. nonetheless, it is possible that some benefit observed was partially due to general improvements in patient clinical care that occur with time. additionally, this being a single-center experience might limit the applicability to other settings, since our hospital managed to rapidly increase the capacity for hospitalization and ventilation support, potentially improving general patient care. however, the adjustment for the differences between patient groups through propensity score and conservative approach with the use of landmark analysis were directly at minimizing the risk associated with an absence of randomization. finally, we believe that the rate of failure observed in this study of 7.3% at 14 days in those with severe covid-19 treated with soc and tocilizumab/methylprednisolone might help to better define the expected rate of response and calculate the number of patients needed to include in the studies assessing various treatment options. in conclusion, the negative impact of immune response in covid-19 might be mitigated by early administration of anti-inflammatory therapy with tocilizumab, methylprednisolone or both. randomized studies are warranted to establish the best treatment options, their timing and limitations. pao 2 /fio 2 , median (iqr), mmhg clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention baseline characteristics and outcomes of 1591 patients infected with sars-cov-2 admitted to icus of the lombardy region severe acute respiratory infections treatment centre: practical manual to set up and manage a sari treatment centre and sari screening facility in health care facilities. geneva: world health organization clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study. lancet respir med presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new york city area clinical management of severe acute respiratory infection (sari) when covid-19 disease is suspected. interim guidance 13 clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury. the lancet the use of anti-inflammatory drugs in the treatment of people with severe coronavirus disease 2019 (covid-19): the perspectives of clinical immunologists from china covid-19 illness in native and immunosuppressed states: a clinical-therapeutic staging proposal pharmacologic treatments for coronavirus disease 2019 (covid-19): a review effective treatment of severe covid-19 patients with tocilizumab risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease issued by: china national health commission from liguria hiv web to liguria infectious diseases network: how a digital platform improved doctors' work and patients' care laboratory testing for 2019 novel coronavirus (2019-ncov) in suspected human cases balancing evidence and frontline experience in the early phases of the covid-19 pandemic: current position of the italian society of anti-infective therapy (sita) and the italian society of pulmonology (sip) a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 overlap weighting: a propensity score method that mimics attributes of a randomized clinical trial clinical characteristics of coronavirus disease 2019 in china the cytokine release syndrome (crs) of severe covid-19 and interleukin-6 receptor (il-6r) antagonist tocilizumab may be the key to reduce the mortality tocilizumab treatment in covid-19: a single center experience impact of low dose tocilizumab on mortality rate in patients with covid-19 related pneumonia tocilizumab for the treatment of severe covid-19 pneumonia with hyperinflammatory syndrome and acute respiratory failure: a single center study of 100 patients in brescia clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan early short course corticosteroids in hospitalized patients with covid-19 clinical predictors of mortality due to covid-19 based on an analysis of data of 150 patients from wuhan, china. intensive care med we would like to thank all the patients and the hospital staff, with particular mention of mrs enrica lombardi, who helped us to get through these difficult weeks.this study was supported by the efforts of all members of gecovid group. key: cord-305811-987dhnf7 authors: cho, che-pei; lin, szu-chieh; chou, ming-yuan; hsu, hsiu-ting; chang, kung-yao title: regulation of programmed ribosomal frameshifting by co-translational refolding rna hairpins date: 2013-04-29 journal: plos one doi: 10.1371/journal.pone.0062283 sha: doc_id: 305811 cord_uid: 987dhnf7 rna structures are unwound for decoding. in the process, they can pause the elongating ribosome for regulation. an example is the stimulation of -1 programmed ribosomal frameshifting, leading to 3′ direction slippage of the reading-frame during elongation, by specific pseudoknot stimulators downstream of the frameshifting site. by investigating a recently identified regulatory element upstream of the sars coronavirus (sars-cov) −1 frameshifting site, it is shown that a minimal functional element with hairpin forming potential is sufficient to down-regulate−1 frameshifting activity. mutagenesis to disrupt or restore base pairs in the potential hairpin stem reveals that base-pair formation is required for−1 frameshifting attenuation in vitro and in 293t cells. the attenuation efficiency of a hairpin is determined by its stability and proximity to the frameshifting site; however, it is insensitive to e site sequence variation. additionally, using a dual luciferase assay, it can be shown that a hairpin stimulated +1 frameshifting when placed upstream of a +1 shifty site in yeast. the investigations indicate that the hairpin is indeed a cis-acting programmed reading-frame switch modulator. this result provides insight into mechanisms governing−1 frameshifting stimulation and attenuation. since the upstream hairpin is unwound (by a marching ribosome) before the downstream stimulator, this study’s findings suggest a new mode of translational regulation that is mediated by the reformed stem of a ribosomal unwound rna hairpin during elongation. sequence complementarities and three-nucleotide based genetic codes in messenger rna (mrna) imbue interesting features for translation. these include a) an intra-molecular duplex (formed via sequence complementarities) having to be unwound for decoding, and b) one of three potential reading-frames having to be maintained for faithful protein synthesis. the ribosome possesses helicase activity that allows for the unwinding of rna duplexes [1, 2] while reading-frame maintenance is closely coupled with translocation [3] . however, unwinding of specific rna structures can pause or stall the ribosome for further elongation regulation [4] . in particular, specific mrna signals can program a ribosome to switch reading-frames during elongation, with the ribosome slipping backward (toward the 59-direction) or forward (toward the 39-direction) by a single nucleotide. it then continues translation in the new21 or +1 reading-frame. such21 or +1 programmed ribosomal frameshifting (prf) has been characterized in prokaryotes and eukaryotes and is usually related to specific cellular functions [5] . a slippery sequence (xxxyyyz) and optimally placed downstream stimulator structures on mrna are the two in-cis elements required for efficient eukaryotic21 prf [6] . however, the precise21 prf stimulation mechanism remains unclear [7] . most models of21 prf stimulation propose that a specific structural or mechanical feature of the stimulator resists the unwinding activity of ribosomal helicases [2] . this is done either passively by serving as a roadblock to pause ribosomal movement or actively by creating tension/strain to communicate with transfer rna (trna)-mrna linkages to destabilize the p site codon-anticodon helix in the 0-frame (xxy). it also eventually facilitates re-pairing of trna with the21 frame mrna (xxx) [2, 3, 8, 9, 10] . other factors have been suggested in the modulation of frameshifting efficiency [5] . in particular, an optimally placed internal shine-dalgarno (sd) sequence in prokaryotic mrna may serve as a 21 prf stimulator by pairing with the anti-sd element in 16s ribosomal rna (rrna) of the 70s ribosome [11] . in addition, 21 frameshifting efficiency can be affected positively or negatively by flanking sequences upstream of a slippery site [12, 13] . previously, we identified a 170-nucleotide rna element (att), upstream of the21 prf slippery site of sars-cov mrna, capable of down-regulating viral21 prf [14] . recently, att was shown to optimize viral replication and was suggested to act by causing a fraction of the elongating ribosome to fall-off in front of the att [15] . understanding how21 prf attenuation is achieved would not only shed light on how a 21 prf stimulator promotes21 frameshifting and provide insight into the mechanism governing reading-frame control, but also may have potential for antiviral applications because21 prf efficiency is crucial for the replication of several human viral pathogens, including hiv and sars coronavirus (sars-cov) [15, 16] . here, we identify a minimal element in sars-cov att as the major determinant of21 prf attenuation function. additionally, we show that attenuation efficiency is not sensitive to e site sequence variation, suggesting flanking-sequences effect is not the main cause of attenuation. we further demonstrate that this minimal element acted through a hairpin form with attenuation efficiency determined by hairpin stability and spacing to the slippery site. importantly, this potential hairpin also enhanced +1 frameshifting in yeast, indicating that in addition to being a 21 frameshifting attenuator, it can serve as a +1 frameshifting stimulator. together, these results indicate that the upstream rna hairpin functions as a cis-acting rna motif in programmed frameshifting regulation. finally, our findings also indicate basepair reformation involving the terminal sequences of the 39-half of the hairpin stem as being crucial for attenuation. this implies the existence of a refolding hairpin stem in close proximity to the ribosomal e site. to search for a minimal determinant within att, we performed sequential 59sequence deletions of att and compared the relative frameshifting activity of the different deletion variants (fig. 1 ). we found that viral sequences, covering nucleotides 13363 to 13387, possessed substantial21 prf attenuation activity ( fig. 1 ) and the ability to form a stable stem-loop structure ( fig. 2a) . however, the base of the predicted hairpin stem is only 4 nucleotides away from the 59-edge of the slippery site. it is possible that a hairpin stem cannot be formed when the slippery site occupies ribosomal p and a sites. in the past, the refolding pathway of unwound rna structures within ribosome cores has not been comprehensively investigated. interestingly, the crystal structure of an elongation mode of the 70s ribosome indicates that nucleotides within the first codon upstream of the e codon are flexible [17] . the implication being that these nucleotides are accessible for base-pair formation. therefore, we disrupted two potential au base pairs in the lower stem of the predicted hairpin to generate two ac mismatched mutations at the 13363-13520 construct. we found that the resultant 59cc-wt construct lost two-thirds of its attenuation activity compared with an intact hairpin (figs. 2b and 2c). because both 59cc-wt and 13363-13520 constructs share 27 identical nucleotides upstream of their slippery sites, the attenuation activity difference is not likely to be caused by an e-site flanking sequences effect [12, 13] but rather by the disruption of the two potential au base pairs. similar results were observed for gc-sb-wt and 6bpgc hairpins when potential watson-crick base pairs were disrupted (figs. 2b and 2c). together, these results suggest that base-pair formation and the composition of the predicted hairpin stem are crucial for efficient attenuation. next, we swapped six gc base pairs for six corresponding wild-type base pairs in the predicted hairpin stem within a longer sars-cov viral sequence (13318-wt) (fig. 3a) and found that the attenuation activity of the chimera was further enhanced both in vitro and in 293t cell cultures ( fig. 3b and 3c) , indicating that the predicted stem-loop is a major determinant of 21 prf attenuation activity in sars-cov att. we noticed a potential to form four extra base pairs between 59and 39-flanking sequences (gacg and cguu, respectively) of the 6bpgc hairpin stem (and other deletion mutants) due to the existence of a 59 sali cloning site (fig. s1a ). in particular, the formation of two base pairs involving 39-flanking uu invaded the 0 frame e site of the ribosome when the slippery sequence occupied ribosomal p and a sites. this invasion could interfere with the proposed reading-frame maintenance function of the e site [18] . however, mutagenesis analysis indicates that in vitro attenuation activity of the 6bpgc hairpin is not sensitive to basepair formation involving 39-flanking uu sequences (figs. s1a and s1b). on the other hand, disrupting potential base pairs at 39flanking cg impaired attenuation activity by a third (fig. s1b compares uucg-6bpgc with gaaa-6bpgc). especially important is the disruption of two gc base pairs at the bottom of the hairpin stem (6bpgc12ag). such a disruption dramatically reduced attenuation activity. this evidence seems to show that two extra gc base pairs involving 39-flanking cg lead to an extended stem and contribute to attenuation activity. however, only one potential gc base pair exists in the corresponding region of wildtype sars-cov viral rna sequences (fig. 3a) , suggesting that this extra base pair is not essential for attenuation activity by the wild-type sars-cov attenuator hairpin. to see if these observations are valid and applicable to a different stimulator in other biological systems, selected 6bpgc 59-flanking sequence mutants were placed upstream of a distinct 21 prf stimulator, the du177 pseudoknot [19] , and examined for their attenuation activity in 293t cells. the results (fig. s1c ) indicate that the two potential base pairs involving e-site sequences are not the main cause of observed attenuation activity in 293t cell cultures. the identified attenuator hairpin contained a single nucleotide g bulge and a ugcg tetra-loop in the upper part of its stem. we examined the roles of both motifs in attenuation; however, neither insertion of a c nucleotide to convert the g bulge into a gc pairing nor the six nucleotides inserted to interrupt ugcg loop sequences impaired attenuation efficiency significantly (figs. 4a to 4c). by contrast, deletion of 6 nucleotides at the 59-half of the lower stem in the wild-type sars cov 13318-13520 construct abolished attenuation activity (figs. 4a to 4c). thus, the apical ugcg loop and g bulge are not major determinants of attenuation. next, we investigated the role of stability in the potency of attenuator hairpins by designing a simplified rna hairpin with only 6 gc base pairs (6gc-hairpin). we found that it possessed attenuation activity comparable to that of a 6bpgc hairpin (figs. s2a and s2 b). we further modified the composition of basepairings along the hairpin stem to create variants of different attenuation activity (figs. s2a and s2b). a plot of attenuation efficiencies against predicted free energy values for these 6gchairpin variants reveals a positive correlation between both parameters (fig. 4d) , indicating that hairpin stability is crucial for attenuation efficiency. in addition to reducing hairpin stability, base-pairing disruption at the lower stem leads to changes in the spacing between the bottom of the hairpin stem and the slippery site. to address this issue, we created mutants by inserting different numbers of nucleotides between the two extra gc base pairs of the extended 6bpgc hairpin stem and the slippery site (fig. 5a ). when the spacing was increased from 2 to 5 nucleotides, attenuation activity was reduced by about a half. it was reduced further by the insertion of additional nucleotides (figs. 5b and 5c). the spacing dependency of attenuation activity in yeast and a construct containing the du177 pseudoknot stimulator was also examined and confirmed (fig. 5d ). varying the ratio between mrna and ribosomes in in vitro assays so that the amount of ribosomes available for each mrna was different did not affect attenuation activity significantly (figs. 5e and 5f). this result indicates that the lower attenuation activity attributable to a distant hairpin is not likely due to hairpin unwinding by an adjacent marching ribosome. cumulatively, these results establish the following: 1) attenuation activity of a hairpin depends on its spacing to the slippery site; 2) attenuation functions are preserved among distinct eukaryotic systems; and 3) attenuators down-regulate distinct 21 prf stimulators. in contrast to a downstream 21 prf stimulator promoting 21 frameshifting, a potential hairpin attenuator upstream of the slippery site possesses an opposing effect. as e site sequences can affect the potency of downstream stimulators [12, 13] , we ask whether the potency of attenuators can be affected by either proximal e site sequences or downstream stimulators. a weakened m1 attenuator hairpin, derived from a 6bpgc hairpin with a disrupted gc base pair (in the middle of the hairpin stem ( fig. 6a )), and a potent du177 21 prf stimulator were used to address these issues. they were chosen because a potent 6bpgc hairpin attenuated a weaker sars 21 prf stimulator so efficiently that the resultant intensity of radioactivity in in vitro assays gave uncertain results (data not shown). consistent with flanking sequences effects, changing sequences in the 21 frame e site led to variations in 21 frameshifting efficiencies. these were promoted by the same stimulator regardless of the absence or presence of an m1 attenuator. however, calculated 21 prf attenuation efficiencies of the m1 attenuator remained virtually unchanged among different e-site sequence variants (fig. 6b) , indicating that the attenuator down-regulates 21 prf to a similar extent under flanking-sequences effects. by contrast, attenuation efficiencies of the m1 attenuator were different among constructs containing du177 or sars 21 prf stimulator (fig. 6c ), implying discrimination toward distinct stimulators. we considered the possible mechanisms by which an attenuator may interact with downstream stimulator or sequester soluble factors involved in 21 frameshifting stimulation. however, no obvious variation in attenuation activity was observed in 21 prf reporters (with or without the in-cis attenuator hairpin) in the presence of different dosages of in-trans rna attenuator hairpins (fig. s3 ). this suggests that 21 prf attenuation is not mediated by either mechanism. we then investigated the possibility that an attenuator could actively alleviate the proposed strain triggered by ribosomal helicase resistance to a 21 prf stimulator [2, 8] and thus offset 21 frameshifting. should this prove to be true, we predict that an efficient 21 prf attenuator could facilitate frameshifting in the +1 direction under appropriate circumstances. a hepta-nucleotide sequence (cuuaggc), derived from ty1 retrotransposon of yeast saccharomyces cerevisiae, can efficiently induce +1 prf. it is caused by a ribosomal pause at the agg codon due to the low expression levels of decoding trna in yeast. mutating agg to cgg partially impairs +1 frameshifting activity [21] . we reasoned that a partially functional cuucggc sequence represented an ideal platform for evaluating the properties of an upstream 6bpgc hairpin (fig. 7) . indeed, a reporter construct of agg-containing hepta-nucleotides possessed high +1 frameshifting activity (compared with random-sequence negative controls) whereas the +1 frameshifting activity of a reporter with cgg-containing hepta-nucleotides decreased significantly (fig. 7) . in this study, the smaller difference in +1 frameshifting efficiency between agg and cgg constructs compared with that of ty1 retrotransposon may result from a difference in e site sequence identity (cgc versus cac) [22] . importantly, we found that an upstream 6bpgc hairpin with 59flanking sequences designed to prevent direct e site invasion stimulated +1 frameshifting of the cgg-containing shift site (fig. 7) . by contrast, mutants carrying mutations to disrupt base pairs at the hairpin stem (59-wt construct of fig. 7) lost the ability to stimulate +1 frameshifting. therefore, this upstream 6bpgc hairpin may act as a +1 frameshifting stimulator and is indeed a programmed reading-frame switch regulator. in addition to affecting hairpin stability, mutations that change nucleotide composition in a hairpin stem can alter the encoded amino acids. to see if the nature of amino acids encoded by a hairpin is responsible for the observed variation in 21 prf attenuation, mutation data and the amino acids encoded in particular attenuator hairpin variants in this study were further analyzed (table s1 ). comparison of the encoded amino acids between constructs 13363-13520 and 59cc-wt (fig. 2) in mutation sites that disrupts 2 au base pairs reveals a leucine to proline change. by contrast, a similar leucine to proline change from constructs 39gg-wt to gc-sb-wt (fig.2) increased 21 prf attenuation activity. thus, reduced 21 prf attenuation activity in 59cc-wt is not caused by the replacement of encoded leucine with proline in the mutation sites. additionally, amino acid compositions encoded by 6bpgc, r-bulge, and 6gc hairpin ( fig. 4 and fig. s2 ) are changed further (table s1 ) with the maintenance of substantial 21 prf attenuation activity. together, these observations suggest that changes in the encoded amino acid composition caused by nucleotide mutations at the predicted hairpin stems are not a major determinant of 21 prf attenuation. refolding of a ribosomal unwound rna structure within a ribosome has not been fully addressed; however, a previous study indicates that a length of about 30 nucleotides is protected from ribonuclease digestion when mrna is occupied by a prokaryotic ribosome [23] . our observation of a positive correlation between hairpin stability and attenuation efficiency (fig. 4d ) strongly suggests that base-pair reformation of the upstream hairpin stem plays a crucial role in the reduction of 21 prf efficiency. furthermore, mutating two nucleotides (27 nucleotides upstream of the e site) to disrupt watson-crick base pairs in the lower hairpin stem dramatically impairs attenuation activity (fig. 2) , indicating that attenuation is not caused by primary sequencemediated flanking-sequences effects [12, 13] . together, these observations support the idea that a ribosomal unwound hairpin stem can partially reform when the final codon in the 39-half of the lower stem leaves the e site. the proposed ribosomal fall-off hypothesis that has original att impeding ribosome processivity [15] could work here. however, such a mechanism should decrease the observed efficiencies for both +1 prf and 21 prf. additionally, the 6gc hairpin used in this work is not likely to cause a ribosome to fall off because the ribosomal helicase is capable of unwinding a duplex of 27 base pairs [1] . this raises the question of what the other potential mechanisms responsible for programmed reading-frame regulation by a refolding hairpin are. although final proof of the existence of a refolding hairpin stem proximal to the ribosomal e site during 21 prf stimulation awaits direct physical evidence such as ribosome crystallography and mrna : rrna cross-linking analysis, the available 70s ribosome structure mimicking the elongation stage of translation indicates that duplex formation between sd and anti-sd can exist 5 to 6 nucleotides upstream of the p site [17] and overlaps the region where the cis-acting hairpin stem reforms. additionally, cryo-em structures of the 80s ribosome-bound viral internal ribosomal entry site indicate that a folded structure can be accommodated in the space surrounding the mrna exit site [24, 25] . in this context, sd ? anti-sd duplex formation has been shown to change the mrna exit channel pathway and create numerous ribosomal interactions [26] . thus, formation of a refolding cis-acting hairpin stem could create contacts with the 80s ribosome to modulate the e site network and regulate a programmed reading-frame switch. in a non-mutually exclusive model, the +1 frameshifting stimulation and 21 prf attenuation properties of a refolding hairpin can be explained by a pulling force in the 59-direction generated by hairpin stem closure. this explanation is consistent with proposed mechanical tension triggered by a 21 prf stimulator [2] . in agreement with this active role, a refolding gc-rich hairpin has been shown to exert a 59-pulling force on rna-dna hybrids at the active site of rna polymerase [27] . alternatively, the upstream hairpin may serve as a wheel chock that blocks 21 ribosomal movement during late stage of hairpin refolding. thus, a stable hairpin upstream of the slippery site represents a cis-acting rna motif for 21 prf attenuation. this is in contrast to the downstream 21 prf stimulator. these revelations should be of much interest when further studies on the programmed frameshifting mechanism are being planned. by contrast, a much more stable structure, such as the original att in sars cov, could still affect viral 21 prf efficiency by serving as a translational attenuator as previously proposed [15] . however, deletion of six nucleotides to disrupt the minimal upstream hairpin stem in an att-containing construct (construct 13318-13520 d6 in fig. 4b ) restored 21 prf efficiency to that of the att-lacking 13390-13520 construct in vitro (compare lanes 1 and 4 of fig. 4b and fig. 1b) . interestingly, both the in-cis acting hairpin revealed here and the internal sd ? anti-sd interaction in 70s ribosome can stimulate +1 prf as well as attenuate 21 prf when placed in close proximity upstream of the corresponding shift sites [11, 18] . however, moving the cis-acting attenuator hairpin 59 further reduced its 21 frameshifting attenuation activity (fig. 5) , whereas moving the internal sd ? anti-sd duplex further upstream of the slippery site converted the duplex into a 21 prf stimulator [11] . a possible reason is that the 16s rrna component of 70s ribosome is part of the functional duplex, whereas the eukaryotic ribosome does not have an anti-sd sequence. it will be interesting to see if an in-cis acting rna hairpin can replace the functionality of internal sd ? anti-sd interaction in the 70s ribosome. further experiments, such as measuring stimulator unwinding and attenuator hairpin refolding times (by the single-molecule approach) [2] as well as elucidating how translational machinery responds to a refolding hairpin should help reveal the interplay responsible for the intricacies of reading-frame switch adjustment. finally, the search for overlooked cis-acting regulators in the programmed reading-frame switches of genomes using bioinformatics should benefit from the stability and proximity features revealed in this study. because widely distributed rna structures along an open reading-frame [28] are unwound and refolded repeatedly during translation, the involvement of refolding rna hairpins in the regulation of translational elongation may be more common than first thought. the plasmid encoding the gene for orf 1ab junction region of sars-cov, pcrii-sars 12265-13653 was a gift from professor pei-jer chen at national taiwan university. the p2luc recoding reporter [29] suitable for both +1 and 21 frameshifting assays was a kind gift from professor john atkins at the university of utah. plasmids pjd-375 & 378 [30] were obtained from professor jonathan dinman at the university of maryland, while the yeast strain yrp1674 [31] was a gift from professor roy parker at the university of arizona. forward and reverse dna primers, respectively carrying the sali and bamhi restriction sites and appropriately designed annealing sequences, were used for pcr amplification of the desired cdnas encoding sars-cov viral rnas by using pcrii-sars 12265-13653 as the template. the pseudoknot stimulator sequences of du177 [19] with or without the 6bpgc attenuator hairpin were chemically synthesized. the amplified inserts of interests were then cloned into the sali/bamhi sites of p2luc using standard procedures and the resultant recombinant vectors were transformed into dh5a strain of e. coli cells for maintenance and selection by ampicillin. all of the base-pairing disruption and restoration mutants were constructed using a quik-change mutagenesis kit from stratagene according to the manufacturer's instructions. for cloning reporter constructs suitable for in vivo frameshifting assays in yeast, inserts of interest were treated as above and cloned into sali/bamhi restriction sites of the pydlempty reporter (see below). identities of all cloned and mutated genes were confirmed by dna sequence analysis. as the original pjd378 plasmid possesses an inserted hiv recoding signal between bamhi/saci restriction sites, an insert-free vector, derived from pjd378, was created to facilitate subsequent cloning of recoding signals of interests. to this end, the gene fragment corresponding to the hiv recoding signal and ensuing firefly luciferase orf in pjd378 were removed by treatment with restriction enzymes, bamhi and xhoi. an insert-free region corresponding to that in p2luc plasmid was obtained by pcr amplification using a set of forward and reverse primers containing bamhi and xhoi recognition sequences, respectively. the amplified inserts were treated by the same set of restriction enzymes after purification. both fragments were then purified, recovered and ligated to obtain a recombinant insert-free pjdl-empty vector. this pjdl-empty vector has the same set of cloning sites as those in p2luc, making it suitable for insertion with other recoding signals. identities of all cloned and mutated genes were confirmed by dna sequencing analysis. synthetic dna oligonucleotides used in this study were chemically synthesized and purchased from mission bio-tech. synthetic rnas used in this study were transcribed by t7 rna polymerase with designed dna templates using in vitro transcription methods [32] . after being purified by 20% denaturing polyacrylamide gel electrophoresis in the presence of 8 m urea, gels of bands containing rna of desirable sequences were cut out and electro-eluted using a biotrap device (schleicher & schuell). the eluted rnas were then ethanol precipitated and recovered by centrifugation. finally the concentration of a particular dna or rna was determined by uv absorbance at 260 nm. capped reporter mrnas were prepared using the mmes-sage mmachine high-yield capped rna transcription kit (ambion) following the manufacturer's instructions. reticulocyte lysate (progema or ambion) was used to generate shifted and nonshifted protein products. in each assay, a reaction totaling 5 ml of reactants (i.e., 50-250 ng of capped reporter mrna, 2.5 ml of reticulocyte lysate, and 0.2 ml of 10 mci/ml 35 s-labeled methionine (nen)) was incubated at 30uc for 1.5-2 hours. samples were then resolved by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page), and exposed to a phosphorimager screen for quantification on bas-2500 phosphorimager (fujifilm) or typhoon fla7000 phosphorimager (ge) after drying. to facilitate 21 prf activity analysis in vitro, the sars-cov 13222-13520 fragment was originally cloned into sali/bamhi sites of p2luc so that shifted ribosomes would encounter a premature 21 frame stop codon, located 33 nucleotides downstream of the corresponding bamhi site, and produce a shortened 21 frame product during translation [19] . all the other 59deletion mutants derived from the sars 13222-13520 construct in fig. 1 (including the 13363-13520 construct) possess this property. all radioactivity-based 21 prf activity measurement in vitro was performed assuming that the ribosome drop-off effect [29] was minimized for the translation of a shortened 21 frame product. as we present most of our in vitro 21 prf results in terms of relative 21 prf activity, ribosome drop-off effect is removed. experiments were performed in triplicate and reported as one standard deviation from the mean. frameshifting efficiencies were calculated by dividing the counts of the shifted product by the sum of the counts for both shifted and non-shifted products. calibration was conducted for the methionine content in each protein product. we also used relative frameshifting activity to compare attenuation activity among constructs with variations in attenuator composition, spacing to the slippery site, and e site sequence identity in the same gel. attenuation efficiency of an upstream hairpin was defined as the difference in frameshifting efficiency between two constructs with or without an upstream hairpin, divided by the frameshifting efficiency value of the construct without the upstream hairpin. experiments were performed in triplicate and reported as one standard deviation from the mean. human embryonic kidney hek-293t cells were cultured in dulbecco's modified eagle medium (gibco) supplemented with 10% fetal bovine serum. one day before the transfection, 0.5-1610 5 hek-293t cells per well were placed in a 24-well culture plate with 1000 ml growth medium. transfection was conducted by adding the mixture of 0.5 mg plasmid dna and jetpei tm transfection reagent (polyplus) into each well, according to the manufacturer's instructions. luciferase activity measurements for transfected 293t cell lysates were performed using the dual luciferase tm reporter assay (promega) according to the manufacturer's instructions on a chameleon tm multi-label plate reader (hidex). all the experiments were repeated three times with four to six assays for each reaction. frameshifting efficiency was then calculated according to previously described procedures [29] . the firefly/renilla activity ratio generated from the control reporter was divided into that from frameshift reporters carrying frameshifting signals of interest and multiplied by 100 to obtain frameshifting efficiencies (expressed as percentages) for each recoding signal. to measure 21 and +1 prf activity in yeast, yrp1674 cells (mat a his3d1 leu2d met15d ura3d) [31] harboring pydl-based reporter constructs were grown in liquid media composed of a minimal sd base with -ura do supplement (clonetech) to an o.d. 595 nm value of 1.0 on a 1 ml scale. cells were harvested by centrifugation, washed once with 1 ml of ice cold lysis buffer (16 pbs ph 7.4, 1 mm pmsf) and then re-suspended in 0.3 ml of the same buffer. cells suspensions were lysed with glass beads by agitation in a vortex mixer at 4uc for 3 minutes. dual-luciferase activities were determined using 20 ml lysate/sample by a dual-luciferase assay system (promega) and on a chameleon tm multi-label plate reader (hidex). frameshift efficiencies were calculated using the method previously described [29] , except that the pjd375 reporter [30] was used as a control to measure the firefly/renilla activity ratio. all assays were performed in triplicate. figure s1 potential base pairs involving the e site sequences are not essential for attenuation. (a) the 59flanking sequences gacg (typed in green) of 6bpgc hairpin are part of the sali restriction site (underlined) used during cloning, and have the potential to form base pairs with the 39-flanking sequences cguu (also typed in green) of the hairpin to generate four extra base pairs (connected by dashed lines) in the bottom of an attenuator hairpin stem. the 59-flanking nucleotides mutated for disrupting potential base-pairings are listed below the drawing and typed in red with the number of potential base pairs left after disruption shown in parentheses. the 2 terminal gc base pairs disrupted in 6bpgc12ag for comparison are boxed. (b) in vitro -1 prf assays by sds-page analysis of 35 s methionine-labeled translation products for reporter constructs in (a) (left) and the relative frameshifting activity calculated by treating that of construct 13390-13520 as 100% (right). error bars, s.d.; n = 3. (c) relative frameshifting activity calculated from dual-luciferase assay data obtained from 293t cells harboring transiently expressed p2luc reporters. the reporters contain 6bpgc 59flanking sequence mutants with the sars-pk replaced by du177 pseudoknot. the frameshifting efficiency of a reporter construct containing a disrupted 6bpgc hairpin attenuator (6bpgc59wt-du177) was used for comparison and treated as 100%. error bars, s.d.; n = 3. (tif) figure s2 attenuation efficiency and predicted free energy of the 6gc-hairpin variants. (a) the predicted secondary structures and free energy values (in kcal/mol) of all the 6gc-hairpin variants using mfold [20] . free energy prediction was performed using sequences that include the two extended gc base pairs involving spacer (boxed). the base pairs, which changed along the hairpin stem in each mutant, are typed in bold. all the variants share the same cguu 39-flanking sequence to minimize the e site flanking sequence effect. (b) in vitro -1 prf assays by sds-page analysis of 35 s methionine-labeled translation products for constructs containing variants of 6gc-hairpin of (a) above. (tif) figure s3 the -1 prf efficiency of a reporter with or without an in-cis potent attenuator is not affected by titration of an attenuator rna in-trans. (a) schematic drawing of the reporter construct and the wild-type attenuator rna hairpin used for in-trans titration. the sars-pk was used as the stimulator in these -1 prf reporters. (b) in vitro -1 prf assays by sds-page analysis for the 6bpgc hairpin containing reporter in the presence of different amounts of in-trans wt attenuator hairpins (left), and the relative frameshifting activities in comparison with that of the reporter alone (right). the concentrations of the rna hairpin are labeled as indicated. error bars, s.d.; n = 3. (c) in vitro -1 prf assays by sds-page analysis for attenuator-less reporter in the presence of different amounts of in-trans wt attenuator hairpins (left), and relative frameshifting activities in comparison with that of reporter alone (right). error bars, s.d.; n = 3. (tif) table s1 the nucleotide sequences and encoded amino acids of selected upstream -1 prf attenuator hairpin variants. the amino acids encoded by each 0-frame codon are shown below the codons, and the sequences corresponding to the 59-half and 39-half of each hairpin stem are boldly typed with the nucleotides involving particular base pairs disruption in two sets of constructs colored in red or blue. (tiff) mrna helicase activity of the ribosome the ribosome uses two active mechanisms to unwind messenger rna during translation ribosome structure: revesting the connection between translational accuracy and uncovential decoding halting a cellular production line: responses to ribosomal pausing during translation programmed translational frameshifting an rna pseudoknot and an optimal heptameric site are required for highly efficient ribosomal frameshifting on a retroviral messenger rna frameshifting rna pseudoknots: structure and mechanism the 9-å solution: how mrna pseudoknots promote efficient programmed -1 ribosomal frameshifting p-site trna is a crucial initiator of ribosomal frameshifting a mechanical explanation of rna pseudoknot function in programmed ribosomal frameshifting rrna-mrna base pairing stimulates a programmed -1 ribosomal frameshift comparative mutational analysis of cis-acting rna signals for translational frameshifting in hiv-1 and htlv-2 the three transfer rnas occupying the a, p and e sites on the ribosome are involved in viral programmed -1 ribosomal frameshift an atypical rna pseudoknot stimulator and an upstream attenuation signal for -1 ribosomal frameshifting of sars coronavirus achieving a golden mean: mechanisms by which coronaviruses ensure synthesis of the correct stoichiometric ratios of viral proteins importance of ribosomal frameshifting for human immunodeficiency virus type i particle assembly and replication structural aspects of messenger rna reading frame maintenance by the ribosome maintaining the ribosomal reading frame: the influence of the e site during translational regulation of release factor 2 an intermolecular rna triplex provides insight into structural determinants for the pseudoknot stimulator of -1 ribosomal frameshifting mfold web server for nucleic acid folding and hybridization prediction ribosomal frameshifting in the yeast retrotransposon ty: trnas induce slippage on a 7 nucleotide minimal site genetic analysis of the e site during rf2 programmed frameshifting polypeptide chain initiation: nucleotide sequences of the three ribosomal binding sites in bacteriophage r17 rna cryo-em visualization of a viral internal ribosome entry site bound to human ribosome: the ires functions as an rna-based translational factor structure of the ribosome-bound cricket paralysis virus ires rna structural basis for messenger rna movement on the ribosome applied force reveals mechanistic and energetic details of transcription termination genome-wide measurement of rna secondary structure in yeast a dualluciferase reporter system for studying recoding signals an in vivo dual-luciferase assay system for studying translational recoding in the yeast saccharomyces cerevisiae analysis of dom34 and its function in no-go decay synthetic polyamines stimulate in vitro transcription by t7 rna polymerase we thank professor jin-der wen for reading the manuscript. key: cord-267644-guzn0peq authors: livadiotis, george title: statistical analysis of the impact of environmental temperature on the exponential growth rate of cases infected by covid-19 date: 2020-05-29 journal: plos one doi: 10.1371/journal.pone.0233875 sha: doc_id: 267644 cord_uid: guzn0peq we perform a statistical analysis for understanding the effect of the environmental temperature on the exponential growth rate of the cases infected by covid-19 for us and italian regions. in particular, we analyze the datasets of regional infected cases, derive the growth rates for regions characterized by a readable exponential growth phase in their evolution spread curve and plot them against the environmental temperatures averaged within the same regions, derive the relationship between temperature and growth rate, and evaluate its statistical confidence. the results clearly support the first reported statistically significant relationship of negative correlation between the average environmental temperature and exponential growth rates of the infected cases. the critical temperature, which eliminates the exponential growth, and thus the covid-19 spread in us regions, is estimated to be t(c) = 86.1 ± 4.3 f(0). the daily number of new cases infected by covid-19 is currently exponentially growing for most countries affected by the virus. however, this exponential growth rate varies significantly for different regions over the globe. it is urgent and timely to understand the reasons behind this regional variation of the exponential growth rates. little information is known about this matter, while there are indications that the environmental temperature may be a factor; for instance, northern and colder us and italian regions experienced much more incidents than others. typically, the evolution curve of the spread of the coronavirus initiates with a pre-exponential phase, which is characterized by a mild logarithmic growth, followed by the outbreak, that is, the phase of the exponential growth. social-distancing measures against the spread may affect the evolution curve in a way that the exponential growth slows down (decelerated phase) and starts to decline (decline or decay phase [1] ), depending though on the effectiveness and applicability of these measures. however, after the decline of the spread at some place, new infected cases may outbreak in other places, marked with insignificant number of cases until that moment. then, a newly growth phase may appear. for example, fig 1 (left) shows the evolution curve of the spread for the infected cases in mainland china; clearly, we observe the whole growth−decay cycle, as well as, a new re-growth phase. super-strict measures, such as complete shut down and quarantines, can successfully lead to the deceleration of the exponential growth of infected cases [2] . unfortunately, they cannot be successfully applied and followed within vast regions, and especially, for a long and indefinite period of time. inevitably, measures may be loosened during the decay phase − if not earlier, leading to the birth of an equally disastrous re-growth phase. the exponential growth is the most effective phase for the evolution curve of infected cases; and the most important question regarding this evolution is still open [3] : what can influence the exponential growth rate, and thus, "flatten the curve"? measures, strict or not, may affect the evolution of new infected cases, by shifting the spread curve from the exponential to the decelerated growth. it should be noted though that measures do not affect the exponential growth rate itself, but only the period of time that this exponential phase applies. then, what factors do affect the exponential growth rate? the age distribution in the place where the outbreak occurs is unlikely to be a factor; indeed, the number of new cases is known to be positively correlated with age, however, the exponential growth rate (china: 0.169; us: 0.121; italy: 0.090 -decreasing rate) appears to be negatively correlated to the age median of these countries (china: 37.4; us: 38.1; italy: 45.5increasing age); hence, the age is likely irrelevant to the rate variations. in addition, culture in social activities may be a factor; for example, this might be contributing in the observed differences among the exponential rates in the cases of china, italy, and us (fig 1) . however, what is causing the major variation of exponential rates among different regions of the same culture? it is apparent that culture does not constitute the main factor influencing the exponential rate. fig 2 shows the regional variation of infected cases (left) during the exponential growth phase and the average winter temperature (right) in italy. the possible negative correlation, observed between regional number of infected cases and winter temperature in italy, is an indication of the influence of temperature on the exponential growth, but it certainly does not constitute a necessary condition. the reason is that the map plots the total number of the infected cases n t , which is not dependent only on the exponential growth rate λ, but also on the initial number of cases n 0 . it is generally accepted that the initial infected cases in italy were travelled directly from china; since some destinations are more favorable than others, then, the initial number of cases n 0 , as well as the current number of cases n t (which is proportional to n 0 ), should be subject of regional variation. therefore, there is a non-negligible possibility, the observed regional variation of the number of infected cases n t to be caused by the regional distribution of the initial cases n 0 . in such a case, main airport cities would have incredibly high number of infected cases outplaying a possible negative correlation of daily infected cases with regional average temperature t; the latter may be one of the reasons of the high numbers of cases observed in new york city and rome. on the other hand, in their letter to the white house, members of a national academy of sciences committee said that "there is some evidence to suggest that [coronavirus] may transmit less efficiently in environments with higher ambient temperature and humidity; however, given the lack of host immunity globally, this reduction in transmission efficiency may not lead to a significant reduction in disease spread without the concomitant adoption of major public health interventions" [4] . nevertheless, it has to be stressed out that there were no statistical analyses focused on the exponential growth rates of the infected cases in regions with different temperatures. for instance, several authors (e.g., [5, 6] ) found insignificant correlations between temperatures and confirmed cases. however, their analysis was performed on the number of the infected cases n t , which is subject to the randomness of the initial cases n 0 as explained above, and not pre-exponential (pre-exp), exponential (exp) growth, decelerated growth, decay (or decline), and possibly, a re-growth. day t = 1 corresponds to 1/15/2020 for china, 2/20/2020 for italy, 2/27/2020 for us. evolution in china cases follows the whole growth-decay cycle, and a new re-growth phase. italian cases are characterized by a milder exponential rate, entered the phase of decelerated growth on march 12. us suffers with a larger exponential rate, and it is not clear whether has entered the decelerated growth phase. the exponential growth rate for china rose as high as λ = 0.169±0.017, while for italy and us the rates were λ = 0.090±0.004 and 0.121±0.003, respectively (with correlation coefficient > 0.99). https://doi.org/10.1371/journal.pone.0233875.g001 impact of environmental temperature on growth rate of covid-19 cases on the exponential growth rate λ, which is clearly dependent on physical characteristics of the coronavirus, binding protein, and environment. analysis of regional cases can show whether the speculated negative correlation between temperature and number of infected cases is true, meaning a negative correlation between temperature and exponential growth rate. if the environmental temperature plays indeed a substantial role on the virus spread, then, this can provide promising results, such as, the estimation of the critical temperature that may eliminate the number of daily new cases in heavily infected regions. the purpose of this paper is to improve our understanding of the effect of environmental temperature on the spread of covid-19 and its exponential growth rate. in particular, we calculate the exponential growth rates of infected cases for us and italian regions, derive the relationship of these rates with the environmental temperature, evaluate its statistical confidence, and determine the critical temperature that eliminates this rate. a standard model for describing the evolution of the infected cases by viruses can be constructed as follows where is the number of total infected cases evolved from the initial n 0 �n(0) cases, n max is the maximum possible number of infected cass; λ is the exponential growth rate, and becomes clear for x(t) <<1 where i is negligible, leading to: typically, data of infected cases are daily provided and updated. thus we set the readout of n(t) on a daily basis, such as: n t �n(t/[d]). hence, we may write with where x t �n t /n max with t indicating the time on a daily basis, (t = 1d, 2d, . . .). the function of negative feedback i models the factors that flattens the curve, such as, the measures taken against spreading. while these factors are not affecting the exponential growth rate λ, they become more effective as the number of cases increases, getting closer to n max ; exponent b controls the effectiveness of these factors; strict {loose} measures correspond to smaller {larger} values of b. as observed in fig 3(a) , stricter measures, nicely modeled by decreasing b, do not affect the exponential rate λ but they successfully flatten the curve. however, the same can be achieved by downgrading the exponential growth rate, as shown in fig 3(b) . it is apparent, then, how much useful would it be to know the factors that can flatten the curve by decreasing directly the exponential growth rate, instead of applying stricter measures. applied measures could be loosen and shorter! model (1) originates from the logistic map family (e.g., [7] , and references therein; [8] ); other complicate versions, such as, the susceptible-infectious-recovered models (e.g., [9] ) may be expressed by multi-dimensional differential or difference equations (e.g., [10] , and references therein; [11] ), but still, the curve flattening is governed by the same features. the two composites, the exponential growth e and the negative feedback i, are just the main and necessary conditions for reproducing the growth-decay phases of the spread curve. their interplay shows how the spread curve can be flattened as a result of stricter measures, independently of the existent exponential rate. what are the main factors that can affect the exponential growth rate λ of covid-19 spread? the rate λ is expected to have positive correlation with the reproduction number r 0 (e.g., proportional to its logarithm), and negative correlation with the incubation period τ (e.g., inverse proportional) [12] . the number r 0 is a measure of how contagious a disease is; it provides the average number of people in a susceptible population that a single infected person will spread the disease over the course of their infection [9] , and depends on the physical characteristics of coronavirus [13] . the incubation period τ is the time elapsed between exposure to coronavirus and first symptoms; during this period, an infected individual cannot infect others; other characteristic periods and time intervals are the latent period between exposure and infection, and the generation time, mostly concerned with transmission process [14] . characteristic values for covid-19 are τ~5-6 days and r 0~2 -4 [15] . the rate expression can be written as λ/lnr 0 /τ, and involves all the physical characteristics of the mechanisms of infection and the environmental interactions; this can be easily derived, considering difference equations (that is, iterated discrete maps) (e.g., see: [16] [17] [18] ). setting the time to be given in discrete τ-steps (t = 1τ, 2τ,. . .), then, by definition of r 0 (average number of people that a single infected person will spread the disease), we have n t = r 0 n t−τ , that is, we note that the number of the infected cases does not vary for times t taken in-between the integer multiples of τ, but this is not expected in mixtures of populations with random characteristics. indeed, in a mixture of m evolving infected populations with different initial number of cases n 0 (m) and starting times t 0 (m) , m = 1, 2, . . ., m, the total number of infected cases n at a continuous time t is given by which coincides with (3b), but with time t varying on a daily basis, independently of the larger value of τ~5 days [15] . therefore, we set the readout of the total number of infected cases n on a daily basis, such as: ; then, eq (3d) matches eq (2b), where the exponential rate is given by: the main factors that can affect the exponential rate λ are: (a) culture in social activities, and (b) environmental temperature and/or other thermodynamic parameters. intense cultural and social activities have reasonably a positive correlation with r 0 . as previously mentioned, measures against the virus spread do not effectively influence the exponential growth rate; e.g., they do not change the culture in social activities, which are characteristics of the particular population, but they just cease these social activities for some period of time. in terms of modeling, measures appear only in the negative feedback factor i and not in the e factor of model in eq (1), while the culture, together with the environmental temperature, are the two main parameters affecting r 0 directly. potentially, the environmental temperature t can affect all the parameters influencing exponential rate. we approach this dependence by (i) a linear approximation of the phenomenological relationship between exponential rate and temperature, and (ii) the connection of reproduction number with arrhenius behavior (with negative activation energy): (i) the temperature can affect the physical properties of coronavirus, such as, the incubation time τ, as well as, the reproduction number r 0 that depends on these physical properties [13] . a linear approximation absorbs the (weak) temperature dependence of any parameters involved in the exponential rate; then, eq (5) is linearly expanded as: where we set the intercept to be given in normal conditions of atmospheric temperature and pressure (ntp) (that is, t = 20 c 0 , p = 1atm). then, we rewrite the exponential rate as: where λ 0 and p 2 are the intercept and slope of the linear relation (7a). (ii) coronavirus uses their major surface spike protein to bind on a receptor-another protein that acts like a doorway into a human cell [19] . the whole process is a slow chemical reaction, where the mechanism behind can lead to rates negatively correlated with temperature, i.e., increasing rate with decreasing temperature. this is consistent to reaction rate expressed by the arrhenius exponential with negative activation energy exp[|e a |/(k b t)] [20] . then, the effective reproduction number r 0 (t) is expressed as a product combining the reproduction number in the absence of temperature effect, r 0 1 , and the arrhenius exponential rate, namely, then, eq (5) gives we rewrite this expression as: where −λ 0 and p 2 are the intercept and slope of the linear relation (10a), respectively. reactions of negative activation energy are barrier-less, relying on the capture of the molecules in a potential well. increasing {decreasing} the temperature leads to a reduced {gained} probability of the colliding molecules capturing one another. due to the negative activation energy, decreasing the environmental temperature reduces the probability of virus-protein reaction, thus the virus may stay inactive on air or surfaces and eventually die. exponential growth is related to community spread through outdoors activities, while the decelerated growth caused by effective measures is related to indoors activities: exponential growth exists once the disease is still effective and the measures are loosened, allowing people to outdoor social activities; however, exponential growth decelerates followed by the decay phase, once effective measures hold people in small groups indoors. therefore, exponential growth rate must be related to outdoors (rather than indoors) activities, and thus to the environmental temperature. as long as the exponential growth takes place, the environmental temperature has an effective role on the chemical reaction between virus and spike protein. it should be noted that both the models (7b) and (10b) consider that the exponential rate λ, or the reproduction number r 0 , are subject to a component influenced by the culture in social activities (intercept λ 0 ) and a component mostly influenced by the temperature (linear term with slope p 2 ). in this way, the slope may indicate some universal quantity involved, such as, the (negative) activation energy. next, we employ the above two expressions of exponential rate λ and temperature t, eqs (7a and 10a), in order to set the two types of statistical models for fitting (τ, λ) measurements for us and italian regions. we use publicly available datasets of: (1) average environmental temperature of us and italian regions (e.g., see: www.ncdc.noaa.gov/data-access/land-based-station-data/land-baseddatasets/climate-normals; it.climate-data.org; www.weather-atlas.com); (2) time series of the number of daily infected cases of us and italian regions (e.g., see: www.thelancet.com; www. protezionecivile.gov.it). we analyze the datasets of regional infected cases in us and italy, derive the relationship of the exponential growth rate of the number of cases with temperature, and evaluate its statistical confidence. first, we derive the exponential growth rates of the infected cases characterizing each examined region of us and italy; then, we plot these values against the environmental temperatures of each region, and perform the corresponding statistical analysis. we proceed according to the following steps: i. collect the time series of the current infected cases n t for all us and italian regions. ii. for each of the us and italian regions, we plot log (n t ) and log (δn t ) with time t, detect the time intervals of linear relationship corresponding to the phase of exponential growth, fit the data-points within this region, and derive the slope (on linear-log scale), that is, the exponential growth rate λ. the total n t and new cases δn t should be characterized by the same exponential rate, λ, thus the slopes resulted from the linear fits of log (n t ) and log (δn t ) with time are (weighted) averaged (fig 4) . iii. collect environmental temperature data, and calculate the temperature averaged over the whole examined region. the incubation period τ is longer than the time scale of a single day or night, thus the temperature is averaged over the daily and nightly measurements. iv. co-plot all the derived sample values (τ±δτ, λ±δλ), where each pair corresponds to each examined region; then, apply a linear fitting in order to derive the linear relationship between t and λ, as well as, evaluate the statistical confidence of this relationship; repeat the same for all us and italian regions. v. determine the critical temperature t c for which the rate becomes negligible; to eliminate the uncertainties of t c as a fitting parameter, we perform the linear fitting with the statistical model λ = λ 0 (1−τ/t c ) instead of λ = p 1 +p 2 �t. vi. repeat (iv) and (v) with pairs of (t -1 ±δt -1 , λ±δλ); we estimate again t c by performing the linear fitting with the statistical model λ = λ 0 (-1+τ c /t) instead of λ = p 1 +p 2 �t -1 . the hypothesis to be tested is that the exponential growth rate λ varies linearly with temperature; (x is set to be the temperature or its inverse). this is tested by examining the chi-square corresponding to the fitting of the two-parameter linear statistical model λ(x;p 1 ,p 2 ) = p 1 +p 2 x to the given n data points, (the number of data point, n, should not to be confused with the number of cases, n t ). therefore, we minimize the chi-square w 2 ðp 1 ; where the total variance that characterize each data point is now given by σ i (p 2 ) 2 = σ λi 2 +p 2 2 σ xi 2 [21] . the global minimum of the chi-square function χ 2 (p 1 ,p 2 ) gives the optimal parameter values, ðp � 1 ; p � 2 þ, by solving the normal equations @χ 2 (p 1 ,p 2 )/@p 1 = 0 and @χ 2 (p 1 ,p 2 )/@p 2 = 0; the minimum chi-square value is w 2 min ¼ w 2 ðp � 1 ; p � 2 þ. the statistical errors of these values are given by dp a; the statistical confidence of the dependence of the exponential growth rate on the environmental average temperature may be sufficiently high, leading to the acceptance of any of the two statistical models. the goodness of the fitting of each model is evaluated using two types of statistical tests, the "reduced chi-square", the "p-value of the extremes", and their combination (e.g., [24] [25] [26] ), while student's t-test is also used for evaluating the statistical confidence of the derived slopes: • reduced chi-square: the goodness of fitting is estimated by the reduced chi-square value, the meaning of w 2 red is the portion of w 2 min that corresponds to each of the dof, and w 2 red has to be~1 for a good fit. therefore, fitting is characterized as "good" when w 2 red~1 , otherwise there is an overestimation, w 2 red <1, or underestimation, w 2 red >1, of the errors. one order of magnitude less, w 2 red = 0.1, or more, w 2 red = 10, can be set as the accepted limits, i.e., 0.1� w 2 red �10. • p-value of the extremes: the goodness of fitting is evaluated by comparing the estimated minimized chi-square value, w 2 min , and the chi-square distribution, , that is, the distribution of all the possible χ 2 values (parameterized by the dof = m). the likelihood of having a χ 2 value, equal to or larger than the estimated value w 2 min , is given by the complementary cumulative distribution. the probability of taking a result χ 2 , larger than the estimated value w 2 min , defines the p-value that equals the larger the p-value, the better the fitting. according to this method, the probability of taking a result with χ 2 being extremer than the observed value w 2 min , defines the p-value of the extremes; this equals the minimum between the two probabilities, pð0 � w 2 � w 2 min þ and its complementary, pðw 2 min � w 2 < 1þ. fits associated with p-values smaller than the significance level of 0.05 are typically rejected. • combined p-value and chi-square: the p-value of the extremes has very similar behavior with the reduced chi-square [27, 28] , because, (i) the p-value attains the optimal value (p = 0.5) when chi-square does (w 2 red = 1), (ii) larger values of 1-2p corresponds to larger values of jw 2 red à 1j, (iii) both fractions (1−2p)/(1+2p) and j1 à w 2 red j=ð1 þ w 2 red þ range from 0 to 1, reduced to 9/11 when the accepted limits, p = 0.05 or 0.1�w 2 red �10, are reached. then, a combined measure can be defined by the sum of the squares of these fractions, i.e., ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ½ð1 à 2pþ=ð1 þ 2pþ� 2 þ ½ð1 à w 2 red þ=ð1 þ w 2 red þ� 2 q . • student's t-test: this is another test for evaluating the statistical confidence of the slope derived from the linear fitting of the temperature-rate sample points (t i ±δt i , λ i ±δλ i ) and , λ i ±δλ i ). we examine, whether the slope p 2 ±δp 2 has significant difference from the zero slope (null hypothesis: slope is zero), by performing the student's t-test with t m = p 2 /δp 2 , where the corresponding p-value is derived from the integration of t-distribution p t ðt; mþ ¼ for t�t m <1, i.e., p t ðt m ; mþ ¼ r 1 t m p t ðt; mþdt. the student's t-test is not passed for the null hypothesis that the examined slope equals zero, when the corresponding p t -value is smaller than the acceptable confidence limit of 0.05; then, the null hypothesis is rejected, imposing that the slope has statistically significant difference for zero. in addition, we compare the slopes estimated for us with those estimated for italian regions, by deriving t m ¼ jb us à b it j= ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi and then, finding again p t (t m ;m m ); the t-test is passed for the null hypothesis that the examined slopes are equal, when the corresponding p t -value is larger than 0.05; in this case, the null hypothesis is accepted, thus there is no statistically significant difference between the two slopes. the linear fitting of log (n t ) or log (δn t ) with respect to time t within the region of exponential growth phase, resulted to the respective rates (which are given by the fitted slopes); their weighted averages are shown in table 1 for us and in table 2 for italian regions, while plotted against the average regional temperature in figs 5 and 6, respectively. the method of weighted fitting for double uncertainties (x i ±δx i , λ i ±δλ i ), as described by [21] , is used for estimating the fitting parameters λ 0 , t c , together with their statistical, propagation, and total errors. the fits of the linear statistical model with temperature, x i = t i , (left panels in figs 5 and 6) , as well as of the alternative statistical model with inverse temperature, , (right panels in figs 5 and 6) , are both characterized with high statistical confidence, attaining high p-values (>0.05) and reduced chi-squares w 2 red values (close to 1); also, both fits provide similar estimations of t c . the fitting results are shown in table 3 . we also examine whether the sample points (t i ±δt i , λ i ±δλ i ) are subject to statistically significant concentrations or rarefactions, namely, whether possible heterogeneities within the distribution of sample points plays significant role in the fitted relationship. for this, we derive the temperature-rate relationship and its statistical confidence by fitting the homogenized set of sample points, instead of the raw sample points; then, we examine whether the fitting parameters differ from those derived from fitting the raw sample points. we homogenize the sample points by grouping them in temperature bins of δt~1 c 0 (e.g., see: [29] ). we estimate the weighted mean and error of the rates included in each bin. in the case of us regions we also performed a homogenization of rates, by grouping the temperature-binned means in rate bins of δλ~0.01 d -1 . in the case of sample points with inverse temperatures, (t i , λ i ±δλ i ), the procedure is exactly the same. homogenized datasets result in a smooth relationship between the values of binned temperature and rate, as it can be observed in the plots of rate against temperature or inverse temperature (left or right lower panels, respectively), and for both us and italy regions (figs 5 and 6 , respectively). the results are highly supportive of the negative correlation between rate and temperature. the results are shown in table 4 . we observe that the linear relationships of the growth rate with temperature or inverse temperature are characterized by high statistical confidence for the homogenized datasets (p-values much higher than the significant limit of 0.05; w 2 red far from the significant limits of 0.1 and 10). therefore, the arrangement of sample points do not affect significantly the fitting results. in addition, as shown in tables 3 and 4 , the linear fits of sample points (t i ±δt i , λ i ±δλ i ) and , λ i ±δλ i ) do not pass the student's t-test for the null hypothesis that their slopes equals zero, i.e., the corresponding p t -values are smaller than the acceptable confidence limit of 0.05; therefore, the negative correlation of environmental temperature with the exponential rate is statistically significant (accepted with confidence 95%). in order to improve the statistics of the estimated critical temperature, we combine the sample points (t i ±δt i , λ i ±δλ i ) of us and italian regions. first, we perform the student's t-test to compare the slopes from these regions; we find high p t -values (>0.05) for both fits of x = t and x = t -1 , thus, the two populations are likely characterized by the same slope. the respective intercept λ 0 does not pass the same test, i.e., the intercepts corresponding to us and italian regions are likely different; (that is expected, given of the different culture). a universality may characterize the slopes of two countries, either for the fits with x = t or x = t -1 , i.e., p 2 = (@λ/ @t) ntp or p 2 = |e a |/(τk b ), respectively. next, we perform the linear fits of the sample points (t i ±δt i , λ i ±δλ i ) and (t i , λ i ±δλ i ) for the mixed set of us and italian data, once the rates of the italian regions are shifted by δλ; (this is allowed, since it has just be shown that a universality is likely characterized the slopes). the optimal fitting is obtained for that shift δλ, for which the reduced chi-square is 1, the p-value of the extremes is~0.5, and the combined measure~0 (see previous section). fig 7 shows how the combined datasets of temperature-rates from us and italian regions lead to the optimal fitting. (note that the optimization is not performed for the binned datasets, since they are characterized by smaller p-values-see, figs 5 and 6) . the results are shown in table 5 ; we observe that the optimization is reached for two values of the shift δλ; we estimate the weighted average of the results corresponding to the two shifts. the weighted mean is performed separately for the fitting cases of x = t and x = t -1 ; however, the weighted mean of the critical temperature is performed for all four results. table 5 includes the weighted means of slopes for the fits x = t or x = t -1 , with slopes p 2 = −|@λ/@t| ntp and p 2 = |e a |/(τk b ), respectively. the latter can be used for deriving the activation table 3 . fitting parameters of temperature-rate values for us and italian regions. 19b) we derive the reproduction number r 0 , i.e., the two formulae in eq (12b) provide the value of 1 t lnðr ntp 0 þ as 0.0572±0.0098 and 0.0534 ±0.0146, respectively, with weighted mean 0.0560±0.0084; then, we find r ntp 0 ffi 1:34 � 0:10, that is, the reproduction number for t = 20 c 0 . the corresponding number at t = 0 c 0 is r 0 (0c 0 )ffi2.47±0.45, while by substituting the estimated parameters in eq (8), we derive the up-to-date there is no systematic statistical analysis of the effect of the environmental temperature t (and possibly other weather parameters) on the exponential growth rate of the cases infected by covid-19, while a statistically confident relationship between temperature and growth rate (either with positive or negative correlation) was unknown. the presented analysis led to the first statistically confident relationship of negative correlation between the exponential growth rate and the average environmental temperature, derived for us and italian regions. in particular, we analyzed datasets of regional infected cases in us and italy, derived the exponential growth rates for each of these regions and plotted them against environmental temperatures averaged within the same regions, derived the relationship of temperature-growth rate, and evaluated its statistical confidence. the performed statistical analysis involved fitting of linear statistical models with the datasets of environmental temperature (or its inverse) and exponential growth rate. the two linear models developed and used for the statistical analysis are (a) λ(t) . the statistical confidence of fitting was evaluated using the reduced chi-square values, the p-value of extremes, and a testing measure that combines both of these values; also, the student's t-test was used to compare the derived slopes. the sample points of temperature (or inverse temperature) and exponential growth rate were also tested for statistically significant concentrations or rarefactions, that is, for possible heterogeneities within the distribution of sample points that could have significant role in the results. the statistical analysis of the homogenized temperature-rate data points concluded that the negative correlation between temperature and exponential rate is stable, having no statistically significant variability due to concentrations or rarefactions, and it is characterized by a high statistical confidence. we also performed a student's t-test and ensured that the difference between the sample means of us and italian regions is not statistically significant. a universality is likely characterizing the slope of the temperature-rate relationship. this verifies the modeling developed and used by this analysis, where the exponential rate λ, or the reproduction number r 0 , are subject to a component influenced by the culture in social activities (intercept λ 0 ) and a component influenced by the temperature (slope p 2 ). in this way, the slope may indicate to a universal quantity involved, such as, the (negative) activation energy. having shown that the derived slopes for us and italian regions are characterized by no statistically confident difference, we improved the statistics of the estimated fitting parameters by combined the sample points of us and italian regions. from the derived relationship, among others, we were able to estimate the values of the (negative) activation energy e a , as well as the reproduction number r 0 at normal conditions and how this depends on temperature. therefore, the results clearly showed that there is indeed statistically significant negative correlation of temperature on the exponential growth rate of the cases infected by covid-19. fig 9 shows the anti-correlation between the mapped exponential rates and average environmental temperature of the us regions examined by this analysis, which they are characterized by a readable exponential growth phase in their evolution spread curve. the plot shows also the may-june daily, nightly, and 24h-averaged environmental temperatures in san antonio, texas, averaged over the last three years. the daily average temperatures will be clearly above the estimated t c threshold in the second half of may; thus, the plot suggests a possible date for loosening the strict measures in san antonio, that is, may 24. https://doi.org/10.1371/journal.pone.0233875.g010 given the negative correlation of the environmental temperature with the exponential growth rate, it was reasonable to ask for the critical temperature that eliminates the exponential rate, and thus the number of daily new cases in infected regions. this was found to be t c~8 6.1 ± 4.3 f 0 for us regions. it is straightforward to ask when the environmental temperature will climb above this critical value. as an example, fig 10 plots the daily average temperatures in san antonio, texas, shown that it will be clearly above the estimated t c threshold by the end of may. the resulted high statistical confidence of the negative correlation of the environmental temperature on the exponential growth rate of the cases infected by covid-19 is certainly encouraging for loosening super-strict social-distancing measures, at least, during the summery high temperatures. however, we are, by no-means, recommending a return-to-work date based only on this study. but we do think that this should be part of the decision, as well as an inspiration for repeating the same analysis in other heavily infected regions. the steps of these analyses may be followed as: i. identify different outbreaks in regions with the same culture in social activities and different environmental temperature; ii. estimate the exponential growth rates for these regions from the time series of infected cases; iii. plot the derived rates against the environmental temperature averaged for these regions, and repeat the analysis of this study to determine the temperature-rate relationship and its statistical confidence. conceptualization: george livadiotis. formal analysis: george livadiotis. funding acquisition: george livadiotis. investigation: george livadiotis. methodology: george livadiotis. an accurate two-phase approximate solution to an acute viral infection model greece shows how to handle the crisis how to flatten the curve of coronavirus, a mathematician explains rapid expert consultation on sars-cov-2 survival in relation to temperature and humidity and potential for seasonality for the covid-19 pandemic. the national academies press effects of temperature on covid-19 transmission no association of covid-19 transmission with temperature or uv radiation in chinese cities numerical approximation of the percentage of order for one-dimensional maps generalized logistic growth modeling of the covid-19 outbreak in 29 provinces in china and in the rest of the world how covid-19 and other infectious diseases spread: mathematical modeling an introduction to difference equations kappa function as a unifying framework for discrete population modeling vaccinology: an essential guide infection dynamics of coronavirus disease 2019 (covid-19) modeled with the integration of the eyring's rate process theory and free volume concept time variations in the generation time of an infectious disease: implications for sampling to appropriately quantify transmission potential a mathematical model for simulating the phase-based transmissibility of a novel coronavirus general allee effect in two-species population biology nearly exact discretization of single species population models mathematical model for the aggregation of β-amyloid cryo-em structure of the 2019-ncov spike in the prefusion conformation negative activation energies and curved arrhenius plots. 1. theory of reactions over potential wells fitting a straight line with errors on both coordinates approach to general methods for fitting and their sensitivity geometric interpretation of errors in multi-parametrical fitting methods decades-long changes of the interstellar wind through our solar system solar radiation pressure and local interstellar medium flow parameters from ibex low energy hydrogen measurements low energy neutral atoms from the heliosheath chi-p distribution: characterization of the goodness of the fitting using l p norms general fitting methods based on l q norms and their optimization plasma-field coupling at small length scales in solar wind near 1 au key: cord-290446-43h1r4pm authors: vazquez, leonardo; e lima, luis mauricio trambaioli da rocha; almeida, marcius da silva title: comprehensive structural analysis of designed incomplete polypeptide chains of the replicase nonstructural protein 1 from the severe acute respiratory syndrome coronavirus date: 2017-07-27 journal: plos one doi: 10.1371/journal.pone.0182132 sha: doc_id: 290446 cord_uid: 43h1r4pm the cotranslational folding is recognized as a very cooperative process that occurs after the nearly completion of the polypeptide sequence of a domain. here we investigated the challenges faced by polypeptide segments of a non-vectorial β-barrel fold. besides the biological interest behind the sars coronavirus non-structural protein 1 (nsp1, 117 amino acids), this study model has two structural features that motivated its use in this work: 1its recombinant production is dependent on the temperature, with greater solubility when expressed at low temperatures. this is an indication of the cotranslational guidance to the native protein conformation. 2conversely, nsp1 has a six-stranded, mixed parallel/antiparallel β-barrel with intricate long-range interactions, indicating it will need the full-length protein to fold properly. we used non-denaturing purification conditions that allowed the characterization of polypeptide chains of different lengths, mimicking the landscape of the cotranslational fold of a β-barrel, and avoiding the major technical hindrances of working with the nascent polypeptide bound to the ribosome. our results showed partially folded states formed as soon as the amino acids of the second β-strand were present (55 amino acids). these partially folded states are different based on the length of polypeptide chain. the native α-helix (amino acids 24–37) was identified as a transient structure (~20–30% propensity). we identified the presence of regular secondary structure after the fourth native β-strand is present (89 amino acids), in parallel to the collapse to a non-native 3d structure. interestingly the polypeptide sequences of the native strands β2, β3 and β4 have characteristics of α-helices. our comprehensive analyses support the idea that incomplete polypeptide chains, such as the ones of nascent proteins much earlier than the end of the translation, adopt an abundance of specific transient folds, instead of disordered conformations. protein folding is linked to one of the principles of life: most of the functions in a cell are carried out by proteins that have to fold properly. incorrectly, folded proteins have to be directed to proteasomal degradation; otherwise, they can lead to misfolded protein diseases, also known as proteopathias, which can culminate in prion disease, alzheimer's disease, parkinson's disease, amyloidosis, or cancer. the process of translation, consisting of the ribosome molecular machinery with all the accessory molecular elements including mrna, trnas, and chaperones, is the first process that calls attention when one wants to learn about protein folding. ribosomes synthesize polypeptide chains, and during their synthesis, the nascent proteins can acquire its structure or only obtain in the end of the process. the cotranslational fold has been studied essentially in parallel to the description of protein synthesis by the ribosome since the middle of the last century. early observations made it clear that some nascent chains of β-galactosidase still bound to the ribosome are enzymatically active [1] . more direct evidence that a polypeptide still attached to the ribosome, can adopt its native fold came from the use of conformational antibodies raised against β-galactosidase and tailspike protein of phage p22. [2, 3] initial efforts to characterize the folding propensity of nascent polypeptide chains were performed with synthetic fragments of chymotrypsin inhibitor-2 from barley seeds, an alpha/beta protein with 64 amino acids that readily refolds by a two-state mechanism [4, 5] . as measured by circular dichroism and ans binding, the acquisition of secondary and tertiary structure starts simultaneously after completion of 80% of the polypeptide chain. native-like tertiary structure, measured by intrinsic fluorescence of tryptophan, starts after the polypeptide chain is 95% complete. it is worth noting that even though the experimental conditions used in these studies were native-like, the purification process involved denaturing conditions, mainly because of the poor solubility of the polypeptides. of special interest is the fact that the longest construct, with 63 residues, was recovered from bacterial inclusion bodies. important evidence that the process of folding occurs during the synthesis of a protein comes from the lower recovery of the native fold after the refolding of a full-length protein in comparison to a protein that has been synthesized in a vectorial fashion by the ribosome. the presence of chaperones is significant but not mandatory in the cellular milieu, and in fact is not sufficient to increase the in vitro folding recovery to the efficiency level of a polypeptide emerging from the ribosome. a well-studied example is the β-barrel of the green fluorescence protein (gfp), which becomes active after folding from a nascent polypeptide at a much higher efficiency than during in vitro refolding of its full-length polypeptide chain and independently of the cytosolic crowding or cellular chaperones [6] . in fact, the study of co-translational fold has greatly improved after the use of gfp. this protein has a very complex fold composed of a non-vectorial 11 stranded parallel/antiparallel β-barrel, and very importantly, its activity is the emission of fluorescence, which can be assayed in several experimental conditions, including inside the cells, while still bound to the ribosome. with this system, it was possible to identify that nascent gfp with 10 of the 11 β-strands outside the ribosome exit tunnel forms a non-native conformation that is remarkably stable [7] . more recently, the sequential compaction of sub-domains of the first nucleotide-binding domain from the cystic fibrosis transmembrane conductance regulator was detected using fluorescence resonance energy transfer [8] . protein folding coupled to the polypeptide synthesis in the ribosome seems to be more important to some proteins than others. in fact, the speed of translation can dictate the folding competence. sequences of rare codons in the mrna, which slows down the translation at specific lengths of the protein, are concentrated at the end of the coding sequence for a protein domain [9] [10] [11] . curiously, silent mutations can lead to the impairment of protein folding and function [12, 13] . likewise, it has been shown that slowing down the bacterial translation enhances the amount of natively folded heterologous eukaryotic proteins [14] . nevertheless, the amount of data actually showing the presence of folding intermediates is almost inexistent for polypeptides representing the beginning of the translation, which includes either free designed polypeptides or nascent polypeptide chains still bound to the ribosome. important technical hindrances account for this lack of data: first the nascent polypeptide chains have high tendency to aggregate; secondly, working with the large ribosomal complex with nascent polypeptide chains still bound to them causes an enormous signal interference when they are accessed by the current spectroscopic methods; finally, the scarce concentration of the staled ribosomes with the nascent polypeptide chains can be as low as in the range of nanomolar. in this work, we want to address the folding of vectorial polypeptide intermediates designed from a complex 3d structure: the six-stranded mixed parallel/antiparallel β-barrel of nsp1 from the sars coronavirus. this viral protein is expressed as a soluble protein in escherichia coli only at lower temperatures, such as 18˚c, indicating a strong dependence on cotranslational folding events in order to achieve its native fold. we show that designed nascent polypeptide chains of the nsp1 adopt intermediates with hydrophobic clusters and significant 3d compaction. these intermediates appear with 2 of the 6 β-strands and the α-helix, represented by the designed protein nsp1 . additionally, after the presence of 4 β-strands and the αhelix, in the designed protein nsp1(13-100), there is the observation of significant formation of secondary structure. these events could be detected since we designed a recombinant fusion protein that allowed the use of complementary biophysical techniques to identify folding intermediates earlier in the translation of a polypeptide than detected previously. the study of cotranslational folding involves the use of purified ribosome-bound nascent polypeptide chains or free c-terminally truncated proteins to mimic the growth of a polypeptide [15] [16] [17] [18] [19] [20] [21] [22] [23] . these ingenious experimental setups have been developed since the 60´s and they have provided fascinating clues regarding the nature of protein synthesis. however, these systems present three intrinsic experimental drawbacks: 1-the ribosomal-bound nascent chain is a complex of more than 2 mda, precluding studies by many biophysical approaches, including nmr spectroscopic methods, to achieve high-resolution structural data; 2-the free c-terminally truncated chains are very unstable, and are frequently expressed in inclusion bodies. because of that, they need to be denatured in order to allow them to be purified; 3-in both systems, the effective concentration of purified sample is limited (lower micromolar). here we develop an experimental setup that attaches gb1, the soluble and small (56 amino acids) immunoglobulin-binding domain of streptococcal protein g to the c-terminus of the truncated polypeptides, to mimic a growing chain appended to a structured scaffold [24] -sebastian hiller, personal communication. besides the intrinsic solubility and stability characteristics of the gb1 domain, it has a fast and very efficient fold, which avoids interactions with any n-terminal incomplete polypeptide chain during its synthesis by the ribosome [25] . we verified by the comparison of nmr chemical shifts that the gb1 domain does not have significant interactions with the n-terminal constructs (s1 fig) . this analysis relies on the combined chemical shift difference (δδ) to indicate the level of conformational and chemical environment similarity among identical polypeptide sequences. the δδ values were calculated according to the following equation [26, 27] : where: δδ is chemical shift difference, δδ hn are the values of chemical shift of the amidic hydrogens and δδ n are the values of chemical shift of the nitrogen. the combined chemical shift differences were very small (δδ < 0.1 ppm) throughout the polypeptide sequence, but there were significant differences in the chemical environment of the n-terminal residues q2 and y3, as well as residues a20 and v21, which are in a loop very close to the n-terminus of gb1. we infer that the alteration in the chemical shifts of these four residues was an effect of the presence of the spacer placed n-terminally to the gb1 domain. it is worth noting that the attached gb1 domain behaved like the native, well-folded globular domain, with well-defined secondary structures and high 15 n{ 1 h} noes (s2 fig) . additionally, to separate the n-terminal truncated domains of nsp1 constructs from the gb1 c-terminal domain during ribosomal synthesis we included a 20-residue spacer, which encompasses an extended loop of bovine beta-crystallin [28, 29] . this linker is long enough to allow the n-terminal polypeptides to reach the surface of the ribosome during the beginning of the synthesis of the gb1 domain. this spacer provided a reasonably dynamic loop, according to our nmr data, which included narrow chemical shift dispersion (s2a fig as depicted in fig 1a, we designed the nsp1 constructs so as to avoid truncating their secondary-structure elements. constructs included six intermediates and the full-length chain of the nonvectorial nature of the β-barrel fold in nsp1 is evident in these representations. for instance, the intermediate nsp1 includes β-strand 3, which is not paired with β-strand 2 in the native fold, nsp1(13-111) includes β-strand 5, which is not paired with β-strand 4, and finally the full-length nsp1 globular domain includes β-strand 6, which is not paired with β-strand 5. the constructs ranged in size from 11 to 22 kda and included, from the n-to the c-terminus ( fig 1b) : an nsp1 construct; a spacer, designed to occupy the ribosome exit tunnel before the synthesis of the gb1 domain; a gb1 solubility domain; and a histag for purification. these constructs behaved similarly during their purification and provided us with stable samples for at least two weeks, with concentrations as high as 4 mm. secondary structure begins to stabilize in construct nsp1(13-100)-two β-strands before the formation of the β-barrel we evaluated the overall content of secondary-structure elements by circular dichroism (cd) in the range of 200-260 nm (fig 2a) . the fusion construct containing the nsp1 domain as well as the six constructs containing incomplete polypeptide chains presented cd spectra distinct from the gb1 spectrum with an increase of negative ellipticity around 200-230 nm. an further increase in a negative band at 208 nm was noted especially for fusion constructs nsp1 (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) , nsp1 , nsp1(13-66) and nsp1(13-100), which is correlated with the presence of random coil conformation [30] . the full-length nsp1 fusion construct displayed a cd spectrum very similar to the nsp1(13-111) truncated fusion construct, which includes a positive band in the range of 200-205 nm, typical of β-strand conformation in the presence of coil. this characteristic appeared even earlier, in the fusion construct nsp1(13-84), but was somehow obliterated in fusion construct nsp1(13-100). even though the signal of secondary structure is significantly bigger in the intermediate fusion construct nsp1(13-100) than in the shorter fusion constructs, it does not resemble the cd spectrum of full-length nsp1. this most likely reflects an intermediate state of folding distinct from the other fusion constructs. the transition for the formation of significant secondary-structure elements is better demonstrated in fig 2b, since the negative ellipticity at 220 nm is commonly used to characterize the formation of both β-strands and α-helices. it is clear that the formation of secondary structure depends on the presence of strands β1-4 and helix α, represented by construct nsp1 . the substantial emergence of stable secondary structure in this construct is related to the large increase in the amount of native long-range contacts after the presence of strand β4. the long-range contacts in the native structure of nsp1 are illustrated in the s3 fig. the segment coding up to strand β3, represented by construct nsp1(13-84) has 43 long-range contacts, considering the native full-length nsp1 fold. in contrast, the construct nsp1(13-100) has 47 additional long-range contacts compared to construct nsp1 . furthermore, the native pairing among native strands β3 and β4 is maintained by 33 long-range contacts, which is more than three times the amount between β1 and β2 (9 contacts), β2 and β3 (7 contacts), or between each strand and the helix α (1-11 contacts). the further increase of negative ellipticity in fusion construct nsp1 in comparison to nsp1(13-100) also follows the trend observed with the transition between construct nsp1 (13-84) and nsp1(13-100), both for the increase in the 220 nm negative ellipticity and the amount of native long-range contacts. considering the native structure, strand β5 has an extensive network of long-range contacts (36 contacts) with β3, and the construct nsp1(13-111) has 61 additional contacts compared to nsp1(13-100). finally, the full-length protein forms a globular β-barrel fold, by the addition of the remaining 52 long-range contacts with βstrand 6. in denaturing conditions (7 m urea), the fusion constructs and the gb1 construct lost most of their secondary structure signal, reaching an ellipticity level around -12 to -6 deg.cm 2 .dmol -1 ( fig 2c) . we used nmr spectroscopy to characterize the tertiary structure of each construct. the 2d [ 1 h, 15 n]-hsqc spectrum of the full-length fusion construct has a wide dispersion of chemical shifts, which resembles the spectra of the free nsp1 and the free gb1 domains (fig 3) . this data indicates that the nsp1 domain in the fusion construct has the same fold as the free domain and that it does not interact with the gb1 domain to a significant extent. the clear similarity of the resonance chemical shifts of the spacer and gb1 domain among all the fusion constructs, including the ones shown in s1 fig domain. the spectrum showing exclusively the signals of the nsp1 segment in each designed fusion construct was then back-calculated and analyzed for 1 h dispersion and number of peaks (fig 4) . the intermediate constructs have much lower dispersion than the well-folded, full-length domain, showing that the intermediates of nsp1 do not have a well-defined fold ( fig 4a) . it is clear that the spectra of nsp1 (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) , nsp1(13-50) and nsp1(13-66) proteins do not overlap, indicating that these three fusion constructs have different conformations. considering the longer fusion constructs, from nsp1(13-66) up to nsp1(13-111), there is considerable equivalence among the spectra; however, this can be merely a superposition of peaks from different amino acids and because of that cannot be considered fold similarity. nevertheless, if we compare the median and the dispersion of signals of each fusion construct (fig 4b) , we see that the medians of the spectra from fusion construct nsp1(13-66) towards the full-length construct are very similar, around 8 ppm. on the other hand, the median signals of the two shortest fusion constructs, nsp1(13-25) and nsp1 , are very similar to the urea-denatured samples nsp1(13-84), nsp1(13-111) and the full-length nsp1, all around 8.3-8.4 ppm. this indicates that the nsp1 polypeptides from nsp1(13-66) onward adopt different conformations than that of an unfolded structure. it is also noticeable that the dispersion of the fusion construct nsp1(13-111) is greater than the other intermediates, or urea-denatured samples, indicating that the nsp1 polypeptide segment of this sample adopted a more compact 3d fold. the number of missing peaks in the 2d [ 1 h, 15 n] correlation spectra ( fig 4c) is a straightforward and widely used tool to identify the quality of the fold for a sample, especially for relatively small proteins such as the fusion constructs used here (less than 22.2 kda). the greater the number of missing peaks in 2d spectra, the more likely it is that the protein adopts an intermediate fold with intermediate conformational dynamics. it is worth noting that there are several examples in the literature showing that for mostly unfolded and highly dynamic proteins their 2d [ 1 h, 15 n] correlation spectrum will present low dispersion but only a few missing peaks, because of superposition or intermediate dynamics of their 3d structures. this is in fact the case for the fusion construct nsp1 (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) , missing only 3% of its peaks. the number of missing peaks increases substantially to 44% with the length of the polypeptide chain from the fusion construct nsp1 to nsp1 , and then decreases until it reaches approximately 11% in the full-length nsp1 domain. this increased percentage of missing peaks is an indication for the existence of folding states with intermediate conformational dynamics. the full-length nsp1 fusion protein has only a few missing peaks, indicating a well-folded 3d domain, which causes a wide dispersion of chemical shifts and allows straightforward identification of backbone hn signals. a classic approach to identifying 3d structures with intermediate folding uses the fluorescence of bis-ans dye [31, 32] . a blue shift of the emission maximum and an increase of quantum yield of the bis-ans fluorescence spectrum occur with decreasing dielectric constant of its surroundings, such as in the partition from aqueous solvent to protein hydrophobic microdomains. the binding of bis-ans to proteins is dominated by hydrophobic interactions, and as such, its interaction with hydrophobic clusters and stable hydrophobic pockets, which are well-known indicators of intermediate folding structures, protein aggregation, or even specific active sites, such as nucleotide binding sites. as shown in fig 5, there is an increase in the bis-ans fluorescence from fusion constructs nsp1 to nsp1(13-111). significant extrinsic fluorescence is also observed in the fulllength nsp1 sample, but only a residual signal is observed in the samples nsp1 (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) and nsp1 as well as the gb1 domain alone. the most likely cause for the binding of bis-ans to these samples is the formation of hydrophobic clusters in partially collapsed states or in oligomers. the increase of bis-ans binding does not correlate linearly with the accumulated hydrophobicity [33] in each construct (primary sequence shown in fig 5b) , indicating that there are specific conformations forming hydrophobic clusters in the fusion constructs nsp1(13-66)-nsp1(13-111), which enable a very efficient binding of this hydrophobic dye. the fusion constructs present an average hydrophobicity of 0.51 ± 0.03 (accumulated hydrophobicity divided by the number of residues in each construct ± sd), ruling out the possibility of the existence of abnormally hydrophobic polypeptide chains that would have more affinity for bis-ans. it is also worth mentioning that the urea-denatured samples do not bind significantly to bis-ans dye (data not shown). nonetheless, the results from bis-ans fluorescence indicate that after a specific polypeptide length, represented by the nsp1(13-66) fusion construct, there is a critical accumulation of hydrophobic residues that is sufficient to form hydrophobic clusters. finally, there is a large decrease in accessible hydrophobic clusters in the full-length well-folded globular domain of nsp1, which is usually attributed to native hydrophobic collapses [34] . in order to obtain deeper insight into the morphological features of the nsp1 fusion constructs in solution we collected small-angle x-ray scattering (saxs) data [35, 36] , as shown here by the i (q) scattering function. the gb1 domain alone behaved as a globular particle with r g of 2 nm and d max of about 6.8 nm (fig 6) , behaving as a compact structure as suggested by the kratky curve (s4 fig). from the kratky plots we found indications of flexible elongated polypeptide segments in all designed fusion constructs, characterized by an increase of i (q) × q 2 plateauing at a given threshold. these data correlates with the other experimental observations presented here, which indicates the presence of a globular gb1 domain fused to distinct partially folded nsp1 polypeptide segments. the fusion construct nsp1 in particular has a consistent increase of the i (q) × q 2 along the q values (s4 fig), indicating a high content of disordered structure. for the full-length nsp1 fusion construct, a sinusoidal function in the kratky plot is observed, indicative of a compact, globular structure with no major intrinsically disordered components, compatible with the expected structure of two globular domains (gb1 and nsp1). from the pair distance distribution p(r) data (fig 6a) , we calculated the r g and d max for each of the designed fusion proteins, as well as for gb1 domain alone, and compared with the hypothetical parameters for ideal folded and unfolded polypeptides of similar polypeptide chain length [37] (fig 6b) . in these calculations we used the equations calc-rh folded = (0.475 × number of residues 0.29 ) and calc-rh unfolded = (0.221 × number of residues 0.57 ) for folded or unfolded proteins, respectively (blue and red lines in fig 6b) . the r g and d max of gb1, as well as the designed fusion proteins nsp1 (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) , nsp1(13-50) and full-length nsp1, follows the expected dependence of dimensional parameter upon the chain length. a pronounced upper-deviation from the theoretical function is evident for the fusion construct nsp1 (13-84), most likely identifying the expansion of the mean occupied conformational space. the fusion constructs nsp1(13-100), nsp1(13-111) show a reduction in r g and d max compared to the theoretical and to the other fusion constructs, indicating protein condensation upon crossing a given threshold between chain length as of the completion of the segment nsp1(13-84)-nsp1(13-100). we assume that this behavior may be attributed to additional structural stability for the previous strands (β1-β3). moreover, according to the plots of the distance distribution function p(r) (fig 6a) , all the fusion constructs are clearly represented by a small globular domain with a radius of approximately 3 nm, most likely from the gb1 domain, elongated up to approximately 10 nm (or 15 nm in the nsp1(13-84) protein), which we interpret as the contribution of the nsp1 chains, the spacer and the his-tag. the full-length nsp1 fusion construct presents two clearly defined minor radii of approximately 3 nm and 6 nm, which fit with the presence of the gb1 and nsp1 globular domains. with the exception of constructs nsp1(13-25) and nsp1 , the intermediates of nsp1 have consistent indications for the formation of hydrophobic clusters and, after construct nsp1 (13-100), the presence of defined secondary structure. in order to characterize the structure of the intermediates of nsp1 at atomic level we performed sequence-specific resonance assignment, which afforded us a more detailed view on the conformation of selected constructs: nsp1(13-25)-should not have any secondary structure; nsp1(13-50)-contains the polypeptide sequence that codes helix α, a secondary-structure element that is well known to have a significantly higher intrinsic folding propensity; nsp1(13-100) emerge as the smallest sample to show evidence for conformational collapse (fig 6b) , and might represent an important intermediate with two β sheets (see fig 1a) . we were able to assign most of the backbone resonances for these three samples regardless of significant signal overlaps, typical for samples with non-globular 3d structure. the most dissecting the folding of replicase nonstructural protein 1 representative exception is the segment from residues 74-87 in fusion construct nsp1(13-100). this segment corresponds to the first half of the most flexible loop in the native structure, which connects native strands β3 and β4. the signals belonging to this segment were absent in the spectra collected, most likely due to intermediate polypeptide dynamics, in the range of milliseconds. polypeptide chain motions in this range are usually represented by conformational exchange of loops that are flexible but not thermally disordered. this intermediate dynamics causes nmr line broadening due to the sampling of different chemical environments in a ratio close to the difference in chemical shift of each state, a fundamental nmr property observed since the first studies of protein conformation [38] . with the 13 c α , 13 c β and h α chemical shifts we calculated the secondary-structure propensity (ssp) for the three fusion constructs nsp1 (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) , nsp1(13-50), nsp1(13-100) and the globular domain of nsp1. the ssp indicates the propensity ranging from -1 to 1 to adopt backbone conformations typical for extended β-strands or helical structures, respectively, according to the effect of these conformations on the chemical shifts of backbone atoms, [39] [40] [41] as shown in fig 7, with the ssp algorithm it was possible to define very well the secondarystructure elements of the folded globular domain of nsp1 (orange bars in the uppermost graph). moreover, most of the native secondary structures can be predicted solely based on amino-acid composition (blue bars in the fig 7a) , with striking exceptions of β-strand 4 and the first half of β-strand 6. the intermediate nsp1(13-100) presents mainly a propensity to form helical conformations, under non-denaturing buffer. one segment with significant helical propensity is in a region that includes the native helix α, but also encompassing strand β2. this helical propensity is present in the same segment of the nsp1(13-50) intermediate, while the nsp1 (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) construct, which has only the sequence coding for the first β-strand and part of the first loop, has propensities close to zero (data not shown). one quite intriguing behavior of the nsp1(13-100) intermediate is that none of the native β-strands have propensity to be in extended conformation. furthermore, there is the presence of helical propensity for the polypeptide segments that form the native strands β2 and β3, peaking at 0.25, and to a lesser extent strand β4, peaking at 0.15. to the best of our knowledge, this effect has not been previously detected in any intermediate of nascent polypeptide chains. interestingly, the opposite propensity was shown for sperm-whale apomyoglobin, an all-αhelical protein [42] . at relatively short lengths of the apomyoglobin, a predominantly nonnative β-sheet is present, but as chain length increases, α-helical conformation progressively takes over. we also evaluated the polypeptide backbone dynamics by measuring 15 n{ 1 h} noes, which gives information on the motion of the h n moiety for individual residues in a protein [43] . the 2d 15 n{ 1 h}-noe experiment is very useful for characterizing the backbone h n dynamics at pico to nanosecond timescales. noes with intensity of around 0.8, identify rigid polypeptide segments, and are usually found in secondary structure elements and folded core of proteins. residues that undergo fast picosecond motion are identified by negative or noes with decreased intensity (minimum intensity at around -3.5). the 15 n{ 1 h}-noe profile for nsp1(13-100) indicated the existence of flexible, disordered residues (ps-ns time scale) in its n-terminus up to residue v26, residue v35, and a few residues around strand β3 and the end of the loop connecting this strand to strand β4. residues with apparent lower flexibility (noe peaking at~0.6) are clustered in the polypeptide segments that correspond to the native helix α, 3 10 helix and strands β2 and β4. we collected 3d noesy data for the three samples mentioned in this subsection, and noticed that they are very sparsely populated with homonuclear noes within the nsp1 polypeptide segments, showing almost exclusively the expected sequential d αn and d nn (data not shown). it is worth noting that the gb1 globular domain in these fusion constructs exhibited a very reasonable number of noes, compatible with the number observed for the isolated domain (data not shown). the paucity of noes, especially long-range ones, is a strong indication for the lack of stable tertiary structure within the studied nsp1 segments, but does not exclude the existence of partially folded species in these samples, which in fact corroborate with the data presented here. finally, we performed a pairwise comparison of the backbone h n chemical shifts of nsp1 (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) with nsp1(13-50), nsp1(13-50) with nsp1(13-100), and nsp1(13-100) with the full-length globular nsp1. as noted in the fig 7d, the chemical shifts of nsp1(13-100) are quite distinct from those of the full-length globular domain, evidence that the conformations of these constructs are not alike. nonetheless, it is noticeable that the loop connecting strand β1 and helix α, as well as the polypeptide sequence surrounding helix 3 10 , have the highest δδ, suggesting that these are the regions that suffer the greatest conformational change, from nsp1(13-100) to full-length globular nsp1. the smaller δδ in strand β1 and helix α indicate that these two elements are already in a chemical environment more similar to the hydrophobic core of stably folded nsp1. as revealed previously by ssp, the δδ analysis also indicated that the conformation of nsp1 (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) is very similar to that of its corresponding sequence in nsp1 and nsp1(13-100), since the δδ values between nsp1(13-25) and nsp1 and between nsp1(13-50) and nsp1 (13 even though it is intuitive to expect the formation of a β-barrel only after the translation of the last β-strand, there is the possibility of the existence of intermediates on the cotranslational folding of nsp1, including (shown in fig 1) : 1-the presence of the α-helix, first seen in the construct nsp1(13-50); 2-the existence of one β-sheet formed by β-strand 1 and β-strand 2, in constructs nsp1 , nsp1(13-84) and nsp1(13-100); 3-the formation of a second β-sheet with β-strands 3 and 4 in the construct nsp1(13-100); 4-the union of the two intermediate, double-stranded β-sheets by the translation of β-strand 5 in construct nsp1 . however, in spite of nsp1 having an α-helical propensity in a very similar polypeptide segment as the native helix α, our data support a different scenario. first, there is no evidence for the formation of any intrinsic β-sheet intermediate. in its place, at fusion construct nsp1(13-100) there is a significant α-helical propensity for the polypeptide segments that form the native β-strands 2 and 3, and to a lesser extent β4. the detection of non-native folding intermediates with α-helix propensity within segments of native β-strands has been described in the literature for full-length β-lactoglobulin and canine milk lysozyme [44] [45] [46] [47] , and these elements are considered intermediates on the protein-folding pathway. the formation of the native α-helix must be aided by the long-range contacts involving at least strands β1-β5 of nsp1, since there is no stabilization of this secondary structure up to the fusion construct nsp1(13-100), but probably in nsp1(13-111), which has the most similar cd spectra to the full-length fusion construct, at the same time very distinguishable from the other conformation such as β-strands. the secondary structure elements extracted from the 3d solution structure of nsp1 are identified across the top. (b) secondary structure propensity of the nsp1(13-100) designed fusion protein. the white hatched bars identify positions without data. (c) heteronuclear 15 n{ 1 h}-noe values of the nsp1(13-100) designed fusion protein (green bars). positive values represent less dynamic to rigid residues, while close-to-zero and negative values identify highly dynamic residues. the standard errors are indicated. (d) differences in the h n backbone chemical shifts (δδ) of the nsp1(13-100) designed fusion protein compared to the full-length nsp1 protein (grey bars). the average δδ for each secondary structure segment as well as their connecting loops are represented in red. https://doi.org/10.1371/journal.pone.0182132.g007 fusion constructs (fig 2) . in parallel, the abundance of long-range contacts indicates that the α-helix is important for the stabilization of the β-barrel. the fact that this helix has the highest propensity among all the secondary-structure elements of nsp1 that are formed after the intermediate nsp1 corroborates this hypothesis. we envisage testing this by comparing a construct lacking this helix with a synthetic polypeptide encoding the native α-helix. it is clear that the incomplete polypeptides of nsp1 do not adopt stable conformations. this is common sense with regard to the cotranslational fold, including for proteins that have been studied by the use of truncated polypeptides, such as barnase (rnase from bacillus amyloliquefacies), chymotrypsin inhibitor 2 (ci2), staphylococcal nuclease and sperm-whale apomyoglobin [48] [49] [50] [51] . in polypeptide chains nearing completion (>95% of the final length), these authors identified compact structures with long-range interactions, perhaps non-native, but lacking stable secondary structures. it is worth mentioning that the purification of these truncated proteins involved chemical denaturation; we infer that the results were obtained with refolded samples, which is an additional variable in the experimental setup, not present in the natural cellular environment. our experimental results indicate a different scenario, where intermediate polypeptide lengths of nsp1, represented by designed proteins nsp1(13-100) and nsp1 , start to adopt stable secondary structure and then tertiary structure before the completion of the polypeptide chain, observed for the designed protein nsp1(13-111) (fig 8) . nevertheless, we identified the formation of dynamic hydrophobic clusters, which are distinct from the chemically denatured samples. this effect is also observed in the full-length globular domain of nsp1 and reaches a peak with the fusion construct nsp1(13-111) (fig 5) , which acquires tertiary structure according to our nmr data (fig 4) , albeit still with substantial flexibility as detected by saxs (s4 fig). the main core for the folding of nsp1 might be the helix α, which starts to form with the designed fusion construct nsp1 . this helix presents one of the lowest chemical shift deviations (δδ) when one compares different intermediates with the full-length globular nsp1, and it makes long-range contacts with most of the native β-barrel. we summarized the events occurring during the hypothetical folding pathway studied here in fig 9. the shortest intermediates nsp1 (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) and nsp1 are mostly intrinsically disordered, but with residual native α-helix in nsp1 . subsequently, there is the formation of hydrophobic clusters in nsp1 with concomitant formation of residual non-native αhelices, which reaches a maximum in nsp1(13-100). the fusion constructs nsp1(13-100) and nsp1(13-111) presents a visible condensation of 3d structure, as detected by saxs, but only the latter has some stable tertiary structure. a hydrophobic collapse to the native 3d fold might happen only after completion of β-strand 6. the fusion constructs starting from nsp1 , which presents β-strands 1 and 2 and helix α, until nsp1 , resemble molten globules [52] . it is noticeable that the native amphipathic helix α start to be formed within the fusion construct nsp1 and may provide a template to initiate the tertiary structure of nsp1. a second segment with helical propensity in fusion construct nsp1(13-100) does not show amphipathicity (last panel in fig 9b) , which reinforces the argument that this segment is unstable in this conformation, leaving it with more propensity to form strand β4 and the loops flanking it. based on our experimental evidence and theoretical analysis, we suggest that nsp1 (13-100) represents a crucial step in the cotranslational folding of the β-barrel of nsp1. the plasmid pet25b containing the cdna encoding the six nsp1 intermediates truncated at different positions at their c-termini and the full-length globular domain c-terminally fused with a 20-amino-acid-linker, followed by the gb1 domain (residues 354-407 of immunoglobulin g-binding protein g-uniprotkb: p19909) and a his-tag, as well as gb1 fused to his-tag, were synthesized by genescript company. the plasmid containing the full-length globular domain of nsp1 (residues 13-128 of replicase polyprotein 1ab-uniprotkb: p0c6x7; pdb id.: 2gdt), consisting of residues 13 to 128, was prepared previously by our group [53] . the truncated constructs are identified in the table 1 . the plasmids were transformed into the escherichia coli strain bl21 (de3). protein expression was achieved by growing the cells in lb (luria bertani) medium. the cell culture was shaken at 37˚c until an od 600nm of 0.6 was achieved and then induced with 1 mm of isopropyl β-d-1-thiogalactopyranoside (iptg). cells were grown for approximately 3 h at the same temperature and harvested by centrifugation. uniformly 13 c-and 15 n-labeled proteins were expressed by growing the cells in m9 minimal medium containing 15 nh 4 cl (1 g/l) and ( 13 c 6 )-d-glucose (4 g/l) as the sole nitrogen and carbon sources, respectively. procedures for expression were similar to those used for unlabeled proteins. after expression, the cell cultures were centrifuged at 3000 × g for 20 min, at 4˚c; the supernatant was discarded. (fig 2b) or nmr spectroscopy ( fig 4b) and plotted together to highlight these events within the designed fusion polypeptides. signals are normalized to the highest value (full-length nsp1). https://doi.org/10.1371/journal.pone.0182132.g008 dissecting the folding of replicase nonstructural protein 1 plos one | https://doi.org/10.1371/journal.pone.0182132 july 27, 2017 for protein purification, the cells were lysed by sonication in an ice bath, in the presence of buffer a (50 mm hepes ph 7.0, 250 mm nacl, 1 mm dithiothreitol, 3 mm nan 3 ) and protease inhibitors (complete™, edta-free from roche). the debris was removed by centrifugation at 7000 × g for 30 min, at 4˚c; the supernatant was filtered (0.45 μm) and loaded onto a ni 2+ affinity column (histrap hp column; ge healthcare) equilibrated with 90% buffer a and 10% buffer b (500 mm imidazole in buffer a) at 2 ml/min. a linear gradient of 10-100% buffer b was used to elute the target protein at 4 ml/min. the fractions containing recombinant proteins (determined by sds-page) were pooled, concentrated using centrifugal filter devices (vivaspin 20; ge healthcare 1 ) and loaded onto a size-exclusion column (16/60 super-dex™ 75, ge healthcare 1 ) equilibrated with buffer c (sodium phosphate 50 mm, ph 7.0, 250 the interpretation of the 3d structure formation is a combination of experimental evidences obtained by bis-ans fluorescence, saxs and nmr spectroscopy. the native secondary structure topology is shown above the amino-acid sequence of nsp1, which is colored to distinguish each nsp1 segment used to design the incomplete nsp1 polypeptides. the number of additional long-range contacts between each secondary structure element and the rest of the polypeptide was calculated with molmol on the basis of the native nsp1 3d structure. thicker arrows and bold number indicate the transitions to more compact 3d structures as identified by saxs in fusion constructs nsp1(13-100) and nsp1 and the acquisition of native globular fold in the fusion construct full-length nsp1. (b) helical topology calculated with the online server rzlab. dashed lines highlight the interface between hydrophobic and hydrophilic faces of the helices. helix 1 and 2 were experimentally identified by ssp in the nsp1(13-100) construct (fig 7) . https://doi.org/10.1371/journal.pone.0182132.g009 small angle x-ray scattering saxs experiments were performed in the d11-saxs1 beam line [54] at the national laboratory for synchrotron radiation (lnls). saxs data were collected using a two-dimensional detector (pilatus 300k; dectris, usa) at a wavelength of 1.548 å with the sample-detector distance providing a q-range from 0.07 nm -1 to 2.5 nm -1 , where q is the modulus of the scattering vector (calculated according to q = (4π/λ) sinθ, where λ is the wavelength and 2θ is the scattering angle). three successive frames of 300 sec were collected per sample in order to rule out radiation-induced damage. frames behaved similarly, and thus we assumed no detectable sample instability during measurements. all three frames were averaged. the data reduction routine was performed with fit2d [55] , including normalization of the one-dimensional scattered data to the intensity of the transmitted incident beam; correction for detector response, incident beam intensity and sample absorption; and blank subtraction using scattering from buffer collected under the same experimental protocol. the r g and the scattered intensity extrapolated to zero q, i (q) , were inferred from the slope and the intercept of the linear fit of ln[i (q) ] versus q 2 in the q-range q×rg < 1.3 [56] and also computed from the indirect fourier transform program gnom [57] . from these data we inferred the monodispersity of all protein constructs. we also used gnom to compute the distance-distribution function, p (r) , its r g and the maximum dimension, d max . nuclear magnetic resonance spectroscopy nmr spectra were collected at 22˚c with bruker ascend 500 mhz, ascend 700 mhz, and avance 800 mhz spectrometers equipped with z axis gradient 5-mm triple gradient probes and avance 600-mhz, equipped with a z axis gradient 5-mm triple resonance cryogenic probe. 2d [ 1 h, 15 n]-hsqc, 3d hnco, 3d hncacb, 3d cbca(co)nh, 3d hbha(co) nh, and 3d 15 n-edited [ 1 h, 1 h] noesy spectra [58] were used to obtain sequence-specific assignments for the polypeptide backbone of nsp1 (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) , nsp1(13-50) and nsp1(13-100) constructs in buffer c. for the resonance assignments of gb1 solubility domain we followed the j-unio protocol [59] . briefly, we performed automated backbone assignment using 5d apsy-cbcaconh, 4d apsy-hacanh and 5d apsy-hacaconh data sets [60, 61] as input for the software unio-match [62] , which yielded 98% of the expected chemical shifts after interactive validation using the 3d 15 n-edited [ 1 h, 1 h] noesy spectra. the chemical shift assignments for nsp1 globular domain have been published elsewhere [63] . steady-state 15 n{ 1 h} noes [64, 65] were measured on a bruker avance 800-mhz spectrometer, using a saturation period of 3 s and an interscan delay of 5 s. the errors in the primary intensity data were taken from the root-mean-square noise of background regions in the spectra [66] . the secondary structure propensity was calculated using the algorithm ssp [67] . the c α , c β and h α chemical shifts of nsp1 (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) , nsp1(13-50) and nsp1(13-100) constructs, as well as gb1 and nsp1 globular domains, were used as input to calculate the propensity to form αhelix (ssp > 0), or extended conformation such as in β-strands (ssp < 0). the 1 h chemical shifts were referenced to internal sodium-3-(trimethylsilyl) propanesulfonate (dss). using the absolute frequency ratios (0.251449530 and 0.101329118), the 13 c and 15 n chemical shifts were referenced indirectly to dss [68, 69] . the nmr spectra were processed with topspin 3 and analyzed with cara 1.9.1.5 [70] . the nmr chemical shifts of the protein constructs have been deposited in the biomagresbank under bmrb codes 27169, 27176, 27177, and 27178. cd experiments were carried out using a chirascan tm , cd spectrometer (applied photophysics) with a 0.1-cm path length quartz cuvette. cd spectra were recorded using 30 μm protein in buffer c. far-uv spectra were recorded from 190 to 260 nm, averaged over three scans at a speed of 0.5 nm/min, and collected in steps of 0.5 nm. the buffer baselines were automatically subtracted from the respective sample spectra. the raw data were processed using the software proview, provided by the manufacturer. cd data was reported as mean residue molar ellipicity (deg×cm 2 ×dmol -1 ). the compactness of the protein hydrophobic cores was assessed using bis-ans (4,40-dianilino-1,10-binaphthyl-5,50-disulfonic acid) fluorescence spectroscopy. the fluorescence emission spectra of bis-ans were recorded from 400 to 600 nm using an excitation wavelength of 360 nm, after 5 minutes of incubation of the fluorescent die with each protein. the experiments were conducted with 30 μm protein plus 30 μm bis-ans, in buffer c. spectra of bis-ans without protein and protein without bis-ans were used as controls. all of the fluorescence experiments were recorded using a varian cary eclipse 1 fluorimeter (palo alto, ca) at 22˚c, and the data analyzed with graphpad prism 6.01. (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) , nsp1(13-50) and nsp1(13-100) had their nmr chemical shifts assigned and then pairwise compared among each protein, as a probe for 3d structure comparison. (a) combined chemical shift differences of 1 hn and 15 n, between the gb1 domains fused to his-tag or to the designed fusion protein nsp1 (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) . (b) cartoon of the gb1 domain polypeptide backbone fold highlighting in orange the locations of residues that exhibit a combined chemical shift difference greater than one standard deviation above the average (δδ = 0.16 ppm) and in blue for residues that exhibited chemical shift difference greater than the average (δδ = 0.06 ppm). (c) combined chemical shift differences of 1 hn and 15 n, between the gb1 domains in the designed fusion proteins nsp1 (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) and nsp1 , or nsp1(13-50) and nsp1(13-100). the identification of a ribosomal-bound β-glucosi-dase* in vitro and ribosome-bound folding intermediates of p22 tailspike protein detected with monoclonal antibodies immunological activity of ribosome-bound, growing polypeptide chains folding of a nascent polypeptide chain in vitro: cooperative formation of structure in a protein module conformational pathway of the polypeptide chain of chymotrypsin inhibitor-2 growing from its n terminusin vitro. parallels with the protein folding pathway cotranslational folding increases gfp folding yield kinetic analysis of ribosome-bound fluorescent proteins reveals an early, stable, cotranslational folding intermediate translational tuning optimizes nascent protein folding in cells transient ribosomal attenuation coordinates protein synthesis and co-translational folding a pause for thought along the co-translational folding pathway ribosome-mediated translational pause and protein domain organization synonymous codons direct cotranslational folding toward different protein conformations synonymous mutations and ribosome stalling can lead to altered folding pathways and distinct minima slowing bacterial translation speed enhances eukaryotic protein folding efficiency folding of a nascent polypeptide chain in vitro: cooperative formation of structure in a protein module conformational pathway of the polypeptide chain of chymotrypsin inhibitor-2 growing from its n terminusin vitro. parallels with the protein folding pathway a structural ensemble of a ribosome-nascent chain complex during cotranslational protein folding quantitative determination of ribosome nascent chain stability cotranslational protein folding inside the ribosome exit tunnel protein folding on the ribosome studied using nmr spectroscopy the ribosome modulates nascent protein folding transient tertiary structure formation within the ribosome exit port tertiary interactions within the ribosomal exit tunnel design of an expression system for detecting folded protein domains and mapping macromolecular interactions by nmr fast folding of a prototypic polypeptide: the immunoglobulin binding domain of streptococcal protein g mapping the binding site for matrix metalloproteinase on the n-terminal domain of the tissue inhibitor of metalloproteinases-2 by nmr chemical shift perturbation † using chemical shift perturbation to characterise ligand binding crystal structure of truncated human βb1-crystallin x-ray analysis of βb2-crystallin and evolution of oligomeric lens proteins the use of circular dichroism in the investigation of protein structure and function dimer formation from 1-anilino-8-naphthalenesulfonate catalyzed by bovine serum albumin. fluorescent molecule with exceptional binding properties extrinsic fluorescent dyes as tools for protein characterization development of hydrophobicity parameters to analyze proteins which bear postor cotranslational modifications initial hydrophobic collapse in the folding of barstar structural characterization of proteins and complexes using small-angle xray solution scattering hydrodynamic radii of native and denatured proteins measured by pulse field gradient nmr techniques † internal motion in globular proteins the 13c chemical-shift index: a simple method for the identification of protein secondary structure using 13c chemical-shift data the chemical shift index: a fast and simple method for the assignment of protein secondary structure through nmr spectroscopy sensitivity of secondary structure propensities to sequence differences between α-and γ-synuclein: implications for fibrillation chain length dependence of apomyoglobin folding: structural evolution from misfolded sheets to native helices † backbone dynamics of proteins as studied by 15n inverse detected heteronuclear nmr spectroscopy: application to staphylococcal nuclease rapid formation of secondary structure framework in protein folding studied by stopped-flow circular dichroism a non-native α-helix is formed in the -sheet region of the molten globule state of canine milk lysozyme transient non-native helix formation during the folding of b-lactoglobulin non-native alpha-helical intermediate in the refolding of beta-lactoglobulin, a predominantly beta-sheet protein chain length dependence of apomyoglobin folding: structural evolution from misfolded sheets to native helices transient folding intermediates characterized by protein engineering truncated staphylococcal nuclease is compact but disordered exploring the folding funnel of a polypeptide chain by biophysical studies on protein fragments the active form of the steroidogenic acute regulatory protein, star, appears to be a molten globule novel β-barrel fold in the nuclear magnetic resonance structure of the replicase nonstructural protein 1 from the severe acute respiratory syndrome coronavirus the small-angle x-ray scattering beamline of the brazilian synchrotron light laboratory fit2d: an introduction and overview. eur synchrotron radiat facil intern rep esrf97ha02t small angle scattering of x-rays determination of the regularization parameter in indirect-transform methods using perceptual criteria methodological advances in protein nmr. nmr of proteins the j-unio protocol for automated protein structure determination by nmr in solution automated projection spectroscopy (apsy) apsy-nmr with proteins: practical aspects and backbone assignment automated sequence-specific protein nmr assignment using the memetic algorithm match nmr assignment of the sars-cov protein nsp1 protein dynamics measurements by trosy-based nmr experiments practical aspects of the 2d 15n-${$1h$}$-noe experiment backbone dynamics of proteins as studied by nitrogen-15 inverse detected heteronuclear nmr spectroscopy: application to staphylococcal nuclease sensitivity of secondary structure propensities to sequence differences between α-and γ-synuclein: implications for fibrillation 13c and 15n chemical shift referencing in biomolecular nmr recommendations for the presentation of nmr structures of proteins and nucleic acids computer-aided resonance assignment (cara) we thank prof. martha sorenson and prof. jerson l. silva (instituto de bioquímica médica leopoldo de meis, ufrj), for critical reading and proofing of the manuscript. the centro nacional de ressonância magnética nuclear jiri jonas (cnrmn-ufrj) is gratefully acknowledged for providing access to nmr instrumentation. we also thank prof. dr. kurt wüthrich for helpful discussions and advising the phd thesis of the leonardo vazquez at universidade federal do rio de janeiro. key: cord-303490-rixuuytu authors: pazos, michael a.; kraus, thomas a.; muñoz-fontela, césar; moran, thomas m. title: estrogen mediates innate and adaptive immune alterations to influenza infection in pregnant mice date: 2012-07-05 journal: plos one doi: 10.1371/journal.pone.0040502 sha: doc_id: 303490 cord_uid: rixuuytu pregnancy is a leading risk factor for severe complications during an influenza virus infection. women infected during their second and third trimesters are at increased risk for severe cardiopulmonary complications, premature delivery, and death. here, we establish a murine model of aerosolized influenza infection during pregnancy. we find significantly altered innate antiviral responses in pregnant mice, including decreased levels of ifn-β, il-1α, and ifn-γ at early time points of infection. we also find reduced cytotoxic t cell activity and delayed viral clearance. we further demonstrate that pregnancy levels of the estrogen 17-β-estradiol are able to induce key anti-inflammatory phenotypes in immune responses to the virus independently of other hormones or pregnancy-related stressors. we conclude that elevated estrogen levels result in an attenuated anti-viral immune response, and that pregnancy-associated morbidities occur in the context of this anti-inflammatory phenotype. the immunological implications of pregnancy have motivated immunologists for more than half a century [1] . various mechanisms have been proposed to explain how rejection of an antigenically foreign fetus is prevented, from the absence of classical mhc class i molecules [2] , to regulatory t cell expansion [3] and specific suppression of paternally-derived antigens [4, 5] . pregnancy is also associated with reduced pathology and alleviation of symptoms in inflammatory autoimmune diseases such as multiple sclerosis [6] and rheumatoid arthritis [7] . the profound immunological changes in these disease states during pregnancy suggests that there exists a broad impact on immune regulation that is not specific to fetal antigens. empirical evidence suggests that pregnant women fair poorly in response to certain pathogen challenges, including listeria monocytogenes [8] , malaria [9] , sars coronavirus [10, 11] , varicella zoster [12] , rhinovirus [13] and influenza virus [14] , among others. of these, the increased risk of severe influenza infection is of particular public health concern. pregnancy has been an acknowledged risk factor for severe complications from influenza virus infection for nearly a century [15] , and has been observed during every major influenza pandemic including the pandemics of 1918 [15] , 1957 [16] , 1968 [17] , and 2009 [18] [19] [20] . implications of a severe influenza infection during pregnancy are life threatening for mother and child. significantly increased risks of cardiopulmonary events have been reported [14] , and mortality for the mother can be as high as 45% [21] . moreover, consequences to the fetus range from behavioral alterations and low birthweight, to preterm birth and spontaneous abortion [21] [22] [23] [24] . significant effort has been put into determining the safety and efficacy of vaccination during pregnancy [25, 26] , as well as the efficacy of neuraminidase inhibitors [20, 27] , but a deeper understanding of the changes in maternal immunity during pregnancy are needed in order to devise adequate therapeutic strategies. many clinical studies have been performed to empirically define the increased severity of influenza infections during pregnancy [14, 18, 21] . for safety and ethical reasons, these studies are conducted in a setting without viral challenge, and rely on observations made only after the onset of symptoms. although earlier work has established that the increased mortality to influenza infection during pregnancy can be observed in mouse models [28] [29] [30] [31] , recent work has primarily focused on characterization of associated morbidities in lethal infections with little insight into how immunity is impacted early in infection. increased morbidity due to influenza infection is typically associated with the third trimester of pregnancy, and correlates well with the highest levels of circulating estrogen. estrogens have long been known to possess potent immunomodulatory effects in various models of disease [32] [33] [34] , and have been proposed for therapeutic use in some clinical situations [35] . although pregnancy levels of estrogen have been associated with reduced levels of immune-mediated morbidity, severe morbidity is a hallmark of influenza infection during pregnancy. we sought to reconcile these observations and determine what role high levels of estrogen play in influenza infections during pregnancy. we have developed a model of influenza infection in pregnant mice and documented the major changes in the anti-viral response. moreover, using implantable hormone pellets, we have determined that estrogen alone is able to recapitulate many key alterations observed in pregnancy. estrogen treatment leads to early reductions in cytokine production, in particular type-i interferon (ifn), impacting downstream activation of adaptive immunity. additionally, estrogen treatment compromises effective antigen-specific cytotoxicity. ultimately these deficiencies result in significantly delayed viral clearance in pregnant animals, highlighting the presence of an anti-inflammatory mechanism in the context of enhanced morbidity. to determine the response to influenza virus infection during pregnancy, we challenged mice at gestational day 10.5 (e10.5) and non-pregnant controls with aerosolized pr8 virus in a controlled chamber. peak viral titers were reached in both sets of mice by 3 day post-infection (dpi) ( figure 1a) , as described previously [36] . by 7 dpi, non-pregnant mice showed significantly lower pulmonary viral titers, suggesting they were able to clear virus more efficiently than their pregnant counterparts. weight gain resulting from pregnancy outpaced any potential weight loss incurred during infection, although pregnant infected mice did lose weight compared to mock controls. this weight loss was delayed by approximately 2 days when compared to the weight loss of non-pregnant controls, suggesting that immuneassociated morbidities were delayed in pregnant animals ( figure 1b ). significant morbidity was readily evident in measures of gestational health. the number of fetuses per mother were not effected by infection in our model ( figure 1c ), but fetuses from influenza-infected mice failed to achieve the weight of their counterparts from mock-infected mothers. by e18.5 they were nearly 35% below the weight of controls ( figure 1d ). the length of gestation was also significantly impacted by infection with influenza virus. infected pregnant mice undergo labor much earlier than their mock-infected controls ( figure 1e ). all mock infected pregnant mice continued gestation beyond the bounds of our experiments, while infected mice universally delivered by e19. pups delivered by influenza-infected mothers were therefore not only premature, but additionally underweight for their gestational age. these results reinforce the evidence that the viability of the pregnancy is negatively impacted by infection with influenza virus, with a significant burden borne by the fetus, as is seen in clinical situations [23, 37] . as most clinical incidents of influenza infection are non-lethal, we wanted to model a viral infection with a milder pathology. pr8 is extremely virulent, and useful for establishing morbidities, but is 100% lethal in wild-type mice. as a non-lethal infection model, we made use of the reassortant mouse-adapted virus x31 (h3n2). infection of pregnant animals with this virus leads to full recovery, but with a significantly more pronounced delay in viral clearance ( figure 1f ). these data suggest that despite significant morbidity associated with fetal gestation, pregnant mice have an impaired ability to control influenza virus infection in both lethal and nonlethal models. given the well documented effects of sex hormones on immune modulation [38, 39] , as well as the increased attention estradiol has received in modulating disease during pregnancy [35, 40] , we decided to investigate the contribution of pregnancy-level 17-bestradiol independently of other hormonal and immunological modulations. we made use of a biodegradable pellet that was implanted beneath the skin and release 17-b-estradiol (e2). serum levels of 17-b-estradiol were found to be at third trimester levels [38] through the experiment ( figure 2a ). as we did with the pregnant animals, we tracked weight loss in estradiol-treated mice using a virulent pr8-derived virus (figure 2b ). initial weight loss was nearly identical before stabilizing in e2-pelleted mice, while placebo-pelleted mice continued to lose weight throughout the latter part of infection. these observations are compatible with our results in pregnant mice, where we found more significant weight loss in non-pregnant controls. viral growth and clearance in e2-pelleted mice mirrored pregnancy extremely well ( figure 2c ). using x31 virus to analyze viral clearance, we found peak titers were achieved in both groups by 3dpi. virus was cleared in the placebo-pelleted mice by 7dpi, but clearance was delayed in e2-pelleted mice. taken together, these data support the hypothesis that e2 is at least partially responsible for the delayed viral clearance found in pregnant animals. one of the critical early signaling events during an influenza infection is the release of type-i interferon (ifn). we have previously demonstrated that e2 was able to significantly impair type-i interferon signaling in human dendritic cells [41] . in order to extend these observations, we measured transcription of ifnb and interferon-stimulated genes (isgs) by quantitative real-time pcr on rna collected from whole lung during infection with x31. we observed a significant early impairment in this pathway in both pregnant and estrogen-treated mice ( figure 3a , 3b). the deficit was not only in ifnb mrna expression, but also activation of downstream isgs, suggesting a significant, biologically relevant impairment in ifn protein expression. we have previously shown that interferon signaling plays an important role in tissues outside the of lung [42] . antiviral instruction of peripheral leukocytes is important for effective control of viral infection and a feature of a robust immune response. using the spleen as a tissue that is not in direct contact with virus, we detected an impairment of isg upregulation in the periphery ( figure 3c ). this data suggests that cytokine-mediated early immune activation is inefficient in pregnant animals. to eliminate the possibility that estrogen treatment interferes with interferon signaling pathways in addition to interferon induction, we treated splenocytes directly with murine ifnb ( figure 3d ). we found e2 to have no effect on ifn signaling. in order to further dissect the mechanism of altered immune responses in pregnancy, we collected whole lung digests throughout x31 infection for analysis by elisa. at 3dpi, we observed several key early cytokines were under expressed in pregnant mice ( figure 4a ), including il-1a, il-1b, and tnf-a. we observed reduced ifn-c expression early in infection, as well as decreased expression of chemokines such as mcp-1 and kc. it is important to note that at this time point we do not detect any difference in viral loads. we repeated the experiment using our estrogen model and observed a similar modulation in many of the same cytokines ( figure 4b ). ifnc, kc, and tnf-a all were under expressed in the lungs of influenza infected e2 mice compared to placebo controls. these results suggest that both models establish a significant anti-inflammatory environment at critical early time points in infection. given the significant changes observed in early cytokine and chemokine expression, we investigated whether these changes lead to variations in migratory kinetics of early immune mediators. we did not observe any significant differences in the absolute number of cells infiltrating lung tissue during infection with x31 in pregnant ( figure 5a ) or e2-treated mice (data not shown). we found no differences in populations of natural killer cells, neutrophils, or cd11c + mhcii + cells that represent a mixture of activated exudate macrophages, dendritic cells and infiltrated monocyte-derived cells. in order to determine whether emigration from the lung was impaired, we investigated the number of dendritic cells in the draining mediastinal lymph node at key time points of infection and found no significant differences between pregnant mice and their non-pregnant controls ( figure 5b ), or between e2-pelleted mice and their placebo controls (data not shown). we next analyzed whether the function of cells migrating into the mediastinal lymph nodes was impaired. we found strongly reduced levels of cd86 expression on cd11c+ cells at critical early time points after infection in both pregnancy ( figure 5c ) and estrogen-treatment ( figure 5d ), suggesting a deficiency in proper dc maturation. the maturation state of dcs at these time points is a critical junction bridging innate and adaptive immunity. the upregulation of cd86 expression, as well as other markers of activation, is extremely sensitive to ifn signaling [43] [44] [45] . the adaptive immune response is critical for efficient clearance of viral infection. of particular importance is the activity of cytotoxic cd8 t cells [46] . in order to determine whether functional deficiencies in adaptive immunity may account for delayed viral clearance, we made use of an in vivo cytotoxicity assay. in this assay, animals are infected with pn-1, an influenza virus bearing the model antigen siinfekl. cytotoxic t cells are endogenously generated towards this peptide during infection. at 8dpi, two populations of target splenocytes are adoptively transferred. each differentially labeled with a fluorescent dye and incubated with either siinfekl or an irrelevant peptide. specific lysis of the antigen-bearing population is then calculated by comparing the relative proportion of the two target populations 16-18h after adoptive transfer ( figure 6a ). we observed significant reductions in antigen-specific cytotoxicity in both pregnant ( figure 6b ) and estrogen-treated ( figure 6c ) animals. these differences represent delays in the ability of pregnant and e2-treated mice to clear antigen specific targets. we next used intracellular cytokine staining techniques to determine the effect of estrogen on granzyme b production by t cells during x31 infection. granzyme b is a serine protease that serves an important role in cd8 t cell cytotoxicity. despite the fact that more virus is present in e2-pelleted mice at 7dpi, we found significant reductions in granzyme b production by intracellular cytokine staining ( figure 6d ). in order to determine whether cytokine signaling in cd8 t cells was more broadly affected, we also measured ifn-c production, a common measure of t cell activation. ifn-c levels were equivalent in both placebo and estrogen treatment ( figure 6e ). these results suggest that while we observe strongly reduced granzyme b levels and impaired cytotoxicity, lung-migrating cd8 t cells were capable of secreting equivalent levels of ifnc. we next investigated the antigen-specific proliferative response. animals were again infected with the pn-1 virus and adoptively transferred with cfse-labeled ot-i t cells, which are a transgenic cytotoxic t cells specific for the siinfekl antigen. at 4dpi, we analyzed draining lymph nodes for cfse dilution and found fewer cd8 t cells proliferating in estrogen treated mice ( figure 6f , 6g). those cells that did proliferate underwent fewer divisions than their placebo-treated counterparts ( figure 6h ). these results are consistent with our findings of immature dendritic cells in the lymph node at early time points. in addition to delays in proliferation, migration of total and influenza-specific cd8 t cells to the infected lung was delayed in x31 infection ( figure 6i ). these observations correlate well with the observed delays in viral clearance. similarly, at 7dpi, antigen specific cd8 t cells in the draining lymph node continue to be reduced in estrogen-treated animals ( figure 6j ). the total number of cd8 t cells is not significant reduced in the draining lymph node. this likely reflects the large number of t cells present in a lymphoid tissue. in order to distinguish between the effects of estrogen on delayed t cell activation by antigen presenting cells and the effects independent of antigen presentation, we used a single-shot model of e2-treatment. a single 100mg dose of e2 was administered to infected mice 6h prior to adoptive transfer of target cells. this dose was sufficient to partially replicate the defect in cytotoxicity observed in e2 mice ( figure 6k) . therefore, e2 impairs t cell cytotoxicity after dc stimulation has occurred. taken together, these data suggest two mechanisms of cytotoxic t cell deficiency. the first involving delayed proliferation and migration of antigenspecific cytotoxic t cells to the lung. the second involves a functional effect of estrogen treatment that occurs after activation of t cells and can be replicated in a short period of time after treatment. establishing a model of influenza infection during pregnancy in order to determine mechanism and intervention strategies is of primary importance. the 2009 h1n1 pandemic outbreak reestablished the risk of severe morbidity and increased mortality that influenza exposure presents to pregnant women, especially in the second and third trimesters [18] [19] [20] . significantly elevated mortality has been reported in mouse models [28] [29] [30] [31] . using an aerosol model of infection, we were able to detect quantifiable and clinically relevant increases in morbidity independent of lethality. notably, fetal pups were found to be significantly underweight, and were delivered prematurely, consistent with clinical observations [23, 37] . in addition to these measures of morbidity, we also observed evidence of an anti-inflammatory phenotype in pregnant mice. viral clearance was delayed in pregnancy, using both lethal and non-lethal infections. additionally, weight loss in pregnant mice was attenuated and delayed. altered pathology in response to influenza virus infection during pregnancy is likely mediated by multiple factors including changes to sex hormones and increased cardiopulmonary demands [47] . we focused on the impact of pregnancy-levels of estrogen for its well-defined impacts on immune function. estrogen is a particu-larly interesting immune modulator as it has been described as having bimodal effects on immunity [38] . circulating levels of estrogen in female mice have been associated with stimulating immune function in general, including exacerbating influenza virus pathogenesis [48] . this accounts for the relative susceptibility to influenza virus infection in non-pregnant females compared to males. this effect can be abolished by gonadectomy. gonadallyintact females were used in this study in order to directly compare pathogenesis in pregnancy or high-dose estrogen-treatment to healthy wild-type females. high levels of estrogen, on the other hand, have been associated with an anti-inflammatory phenotype, including in the context of influenza a virus infection [48] , and cannot be directly linked to the susceptibility of pregnant females to high morbidity. high-dose estrogen treatment has been used to alleviate symptoms in models of rheumatoid arthritis [34, 49] and multiple sclerosis [32, 33, 50] in response to clinical observations showing a reduction of symptoms in pregnant women with these diseases. excessive immune responses mediate much of the pathology associated with influenza virus [51, 52] . pregnancy-specific morbidities have also been associated with inflammation including premature parturition [53] [54] [55] . anti-inflammatory treatment can prevent loss of pregnancy. blocking tnf-a or administering antiinflammatory cytokines such as il-10 [56] have been shown to reduce morbidity. additionally, the placenta has been shown to employ negative regulatory mechanisms aimed at reducing inflammatory cytokine signaling [57] . given the well described anti-inflammatory phenotype of estrogen, and specifically the high while other groups have reported on increased morbidity to influenza infection during pregnancy, anti-inflammatory mechanisms in the context of infection have not been extensively characterized as they have been in the context of disease [6, 7] and of fetal rejection [3, 5] . because of a focus on morbidity, mouse models of influenza virus infection during pregnancy often rely on a high dose intranasal inoculum of pathogenic virus strains such as the 2009 pandemic h1n1 strain. using these models, morbidities such as elevated mortality and low fetal birth weight have been observed [30, 31] . clinical observations of preterm delivery have not been reported in a mouse model, and was not observed in one study [30] , despite increased cytokines levels. additionally, proinflammatory cytokine phenotypes have been reported in these models, and delays in viral clearance are often not supported, although these reports often focus on events late in infection or reflect divergent viral kinetics. we were concerned that models using highly pathogenic intranasal inoculation may not adequately model clinical pre-sentations of influenza virus infection and obscure relevant phenotypes. aerosolized infection closely models clinical disease [58, 59] and was able to replicate clinical observations of influenza infection during pregnancy. we focused on a early cytokine events within the first 24h of the onset of immunity in our model [36] . these inflammatory events have broad impacts on the outcome of immunity, and serve as the best indicators early inflammation. by making use of a model of infection using pregnancy-levels of 17-b-estradiol, we were able to measure its impact on immunity independently of other pregnancy-associated variables. in addition to demonstrating a similar delay in viral clearance, estrogentreated animals were also protected from the weight loss experienced by placebo controls. it is not clear how estrogen may protect mice from weight loss associated with influenza infection, but a likely mechanism may be decreased cytokineassociated cachexia. in both pregnant and estrogen-treated animals, early cytokine responses were reduced. estrogens have been linked to modulation of cytokine signaling, such as through antagonism of nf-kb pathway [38, 60] . sources often disagree as to the degree and direction of these changes depending on the concentration of estrogen, model organism, and method of stimulation [38, 39, 61] . indeed, we saw a strong trend towards elevated levels of the chemokine kc in the steady state of pregnancy, followed by significant deficit in kc expression upon infection. this complex regulation highlights the elaborate mechanisms involved in estrogen signaling [62] . pregnancy presents additional layers of complexity in cytokine regulation. several hormones circulating in pregnancy are immunologically active, including progesterone and prolactin [39, 63] . we did not observe identical regulation of all cytokines in pregnancy and estrogen-treatment, and some of these other hormones may account for some of this variation. the overlap remains significant, and both models demonstrate defects in early immune responses. over-expression of several regulated cytokines including il-1a, il-1b, tnf-a, and ifn-c have been associated with morbidities in influenza infection [52] , and spontaneous pregnancy loss [56, 64] . the ability of estrogen to suppress cytokine expression has been proposed as protective in influenza virus pathogenesis [48] . indeed, it may be that estrogen plays a protective role during pregnancy by muting early cytokine responses that would otherwise threaten fetal development [22, 54] . enhanced morbidities experienced during pregnancy may rely on the failure to clear virus following an impaired immune response. prolonged infection may result in a late cytokine response triggering premature delivery and significant morbidity. delayed weight loss in pregnant animals supports the hypothesis of a late development inducing pathology. alternatively, prolonged infection may lead to significant lung damage. others have reported that delayed lung repair during pregnancy leads significant cardiopulmonary demands during pregnancy, and may contribute to morbidity [31] . type-i ifns play a critical role in early immune responses to influenza, and impairment of this pathway during pregnancy implicates a significant alteration in the response to virus. transcription of ifn-b is a prototypical marker of early type-i interferon expression, and was found to be significantly reduced in both of our models. we did not find any evidence that estrogen treatment interfered with downstream signaling pathways, suggesting that this effect is mediated by reduced type-i interferon expression. isg levels were reduced not only in the lung, but additionally in the periphery. this is an important observation, as interferon signaling in the periphery is necessary for efficient antiviral immunity [42] . this reduction is well conserved in human models of both e2 treatment and pregnancy [13, 41] . the mechanism of interaction between estrogen and type-i ifn induction pathways is not currently understood, but could lead to significant insights. after the onset of inflammation, pregnant and e2-treated animals were able to control early virus titers as well as wild-type counterparts, despite reduced levels of interferon. influenza virus ns1 is a powerful inhibitor of inflammation and functions to counteract early checks on viral growth [36] , likely minimizing the impact of interferon deficiency at the earliest time points. interferon signaling also plays an important role in the activation and maturation of antigen-bearing dendritic cells [65, 66] , and serves as a bridge between innate and adaptive immunity [44, 45] . pregnancy-levels of estrogen were able to interfere with proper dendritic cell maturation. the reduced levels of the co-stimulatory molecule cd86 is particularly relevant for its role in cd8 t cell activation [67] . the primary goal of dendritic cell maturation is the efficient initiation of adaptive immunity, and in particular initiating proliferation of antigen-specific cd8 t cells [46] . as expected, we observed significant estrogen-associated delays in cd8 t cell proliferation in the lymph node at 4dpi, and significantly reduced numbers of antigen-specific cytotoxic t cells in the lung and lymph node at 7dpi. these numbers eventually recovered by 9dpi, correlating nicely with the kinetics of viral clearance in this model. because the observed phenotype results in delays in cd8 t cell recruitment to the lung, the timing of this observation is critical. observations made late in infection may not reflect this phenotype and may explain why others have failed to note significant differences in the numbers of cytotoxic t cells in similar models [31] . dysregulation of interferon function can be linked to delays in appropriate activation of cytotoxic cd8 t cells, and delays in viral clearance [68] . in addition to experiencing delays in proliferation and migration, these t cells are functionally compromised. cytotoxicity was decreased in an in vivo cytotoxicity assay, and granzyme b levels were strongly reduced in the lungs of infected mice. interestingly, ifn-c expression in cd8 t cells was unchanged, suggesting that these cells that reach the lung were at least partially activated, even while functionally compromised. a single dose of estrogen administered immediately prior to encountering target cells was sufficient to impair cytotoxic function, suggesting that this effect is not entirely mediated by inefficient dendritic cell stimulation. estrogen signaling pathways have been associated with lysosome biogenesis in cytotoxic t cells, and may represent a relevant mechanism [69] . notably, two mechanisms of impaired adaptive immunity can be observed. the first involves delayed proliferation in the draining lymph nodes and can account for delayed migration to the lung. this mechanism is likely the product of weak early interferon signaling events and impaired antigen presentation. those cells that do proliferate and are activated are additionally functionally compromised. this second mechanism is independent of antigen presentation and is at least partially responsible for a deficiencies in cytotoxic function. this mechanism has a rapid onset and can be observed within hours of estrogen treatment. this mechanism likely accounts for the observed decreases in granzyme b production that do not effect ifn-c levels in the same t cells. although pregnancy is a complex immunological state, it is clear that estrogen plays a key role. initially, estrogen reduces early cytokine responses and may function to protect from cytokineassociated morbidities. early recruitment of leukocytes is equivalent and early viral titers are controlled equally well in pregnant and wild type mice. the key deficit appears to be in priming of dendritic cells that migrate from the lungs to lymph nodes to initiate t cell expansion. this disruption in interferon signaling leads to suboptimal cd8 t cell activation and delayed viral clearance. estrogen also appears to impair cd8 t cell cytotoxicity independently of dendritic cell activity. it is not currently clear how these anti-inflammatory effects impact the viability of pregnancy, particularly fetal development and premature delivery, but evidence suggests it may play a protective role. deficits in viral clearance and prolonged exposure to infection may be act as stressors that trigger delayed morbidities despite the anti-inflammatory effects of estrogen. it is also not clear whether prolonged infection is of significant impact on morbidity absent pregnancy, as would be the case in hormone treatment. these are key questions that require further study and the establishment of novel models. there is overwhelming clinical value to understanding the mechanisms and contributions of sex hormones to immune modulation during pregnancy. animal models, such as those described in this report, allow for controlled infection conditions and detailed observation of early events during infection. understanding the mechanisms and immunological outcomes of pregnancy, particularly the role of steroid hormones, may lead to the development of therapeutic options for protection of women, their children, and patients receiving hormone replacement therapy. establishing the tenets of hormone mediated immune modulation may provide testable hypotheses for treatment of autoimmune diseases such as multiple sclerosis. mice c57bl/6 wild type, transgenic ot-i and syngeneic pregnant mice were purchased from jackson laboratories (bar harbor, ma). timed pregnant mice were delivered at e8.5 and allowed two days to rest before commencing the experiment. nonpregnant mice were age matched to timed pregnant mice. wildtype mice were implanted with placebo or estrogen pellets purchased from innovative research of america. estrogen pellets contained 35mg of 17-b-estradiol and were designed for 21-day release. surgery was performed 4-5 days prior to infection. mice were anesthetized with avertin (tribromoethanol, acros organics) and shaved behind the right ear. the pellet was implanted via small incision between the shoulder and ear and secured with a surgical clip. mice were monitored for signs of infection. for experiments using a single dose of estradiol, mice received a single 100mg subcutaneous injection of water soluble 17-b-estradiol (sigma) six hours prior to receiving target cells for in vivo cytotoxicity assay. the animals were housed in specific pathogenfree conditions. influenza virus strains a/pr/8/1934 (h1n1) (pr8), recombinant pr8-oti (h1n1) (pn-1), and a/x-31(h3n2) (x31) were propagated in 10-day-old embryonated eggs. virus titers were determined by tissue culture infectivity assay (tcid50) as previously described [70] . briefly, lungs were extracted and homogenized in pbs-gelatin (0.1%) and frozen in dry ice-ethanol for storage at 280uc. presence of the virus was detected by infecting mdck cells at 1:10 dilutions for 72h in the presence of trypsin. presence of virus was measure by testing for the presence of hemagglutination of chicken red blood cells. the limit of detection for this assay is approximately 8610 2 tcid50 per lung. all mice were infected using an inhalation exposure system a42x (glass-col, usa). virus was diluted in pbs to obtain a solution of 10 7.9 tcid50 in12ml. this solution was placed in a glass nebulizer and aerosolized for a total exposure of 30 min. this leads to 100% infection rate as described previously [36, 71] . we estimate between 10 and 100 infectious particles are passively inhaled during this period [72] . mock infected animals are exposed to aerosolized pbs. lungs or spleen at indicated time points were homogenized in 3ml of trizol reagent (invitrogen). rna was isolated as indicated by the manufacturer, and converted to cdna by rt-pcr using the transcriptor first strand cdna synthesis system (roche). qpcr reactions were performed using a lightcycler 480 ii (roche). all reactions were normalized to three housekeeping genes: a-tubulin, rps-11, and b-actin, as previously described [73] . the following primers were used in this study: b-actin -forward: aggtgacagcattgcttctg, reverse: gctgcctcaacacctcaac; a-tubulin lungs were perfused with cold pbs containing 0.5mm edta and immediately ground in hank's buffered saline solution containing 0.5mm edta and 0.5% fbs using the gentlemacs tissue dissociator (miltenyi biotec). mediastinal lymph nodes were mechanically disrupted. both tissues were then incubated with 0.25mg/ml collagenase (liberase type iii, roche) and 8000u/ml dnase i (invitrogen) for 20 min at 37uc. collagenase was inactivated with sterile hbss containing 2% fbs and the suspension was passed through a 70mm strainer. suspensions were then treated with red blood cell lysis buffer (bd biosciences), and resuspended in hbss containing 10mg/ml fc-receptor block (bd biosciences). total cell counts were then attained by hemocytometer. cells were then stained with antibodies for multiple surface antigens: cd8 (53-6.7), cd3 (17a2), cd11c (hl3), mhcii (m5/ 114.15.2), cd86 (gl-1), nk1.1 (pk136), ly6g (1a8). for intracellular cytokine staining, total lung was digested and a single cell suspension was incubated with brefeldin a-containing golgiplug (bd biosciences) for six hours, according to manufacturer's instructions. following staining of extracellular antigens, intracellular cytokine staining was performed using the cytofix/ cytoperm system (bd biosciences). intracellular antigens granzyme b (gb11), and ifn-c (xmg1.2) were stained following permeabilization. antibodies were purchased from bd bioscience, ebiosciences, and biolegend. kb/siinfekl pentamer was purchased from proimmune. influenza np-specific tetramers were kindly provided by dr. david woodland (trudeau institute). samples were acquired using cytomic fc500 coulter station (beckman coulter) and analyzed using flow jo software (treestar corp). whole lung was homogenized in pbs-gelatin (0.1%). cytokine concentration was measured by bead-based multiplex elisa (millipore) using a luminex 200 (luminex corporation). serum was used for detection of circulating 17-b-estradiol using a competitive estradiol elisa (cayman chemicals). mice were infected as described with pn-1 virus or mock infected with pbs. at 8dpi, 1-2.5610 6 target cells were adoptively transferred. splenocytes from a congenic naive donor mouse were used as target cells, and injected intravenously as a 1:1 ratio of two populations. one population was incubated with 100mg/ml of the h2-k b -restricted ova peptide, siinfekl, and labeled with 2.5mm cfse. the second population was incubated with irrelevant peptide and labeled with 0.25mm cfse. 16-18h posttransfer mice were sacrificed. spleens of recipient mice were disrupted, and suspended in red blood cell lysis buffer. total spleen was analyzed by flow cytometry for the relative proportion of the cfse-labeled cells. specific killing was calculated as previously described [74] . briefly, a normalization value (n) was calculated as (% low cfse/% high cfse) in mock infected animals for each treatment group. percent specific cytotoxicity was then calculated for each infected mouse as follows: (% low cfse x n -% high cfse)/(% low cfse x n) x 100. spleens from ot-i mice were mechanically disrupted and passed through a 70mm strainer. cells were suspended in red blood cell lysis buffer (bd biosciences), washed and suspended in hbss containing 0.5% fbs and 10mg/ml fc-receptor block (bd biosciences). cells were then incubated with a cocktail of biotinlabeled antibodies including: cd19 (1d3), b220 (ra3-6b2), mhcii (m5/114.15.2), gr-1 (rb6-8c5), nk1.1 (pk136), cd11b (m1/70), ter119 (ter119), and cd4 (gk1.5). cell suspensions were then washed and incubated with anti-biotin magnetic beads and passed over a macs magnetic column (miltenyi biotec). flow through was collected and verified by flow cytometry to have a purity of greater than 80%. for the cfse dilution assay, these cells were stained with 2.5mm cfse. 1610 6 -2.5610 6 labeled cd8 t cells were intravenously injected into recipient mice on the day of infection. at 4dpi, mice were sacrificed and lymph nodes were collected for analysis. results are expressed as mean +/2 standard deviation. data sets with multiple groups were analyzed by 1-way anova followed by newman-keuls multiple comparison test. weight loss was evaluated using a two-way anova. single time point analyses were evaluated using unpaired two-tailed student's t test. p values # 0.05 (95% confidence) were considered to be significant. data was analyzed and graphs were prepared using prism 5 software. actively acquired tolerance of foreign cells the non-expression of mhc class ii in trophoblast cells regulatory t cells mediate maternal tolerance to the fetus t cell awareness of paternal alloantigens during pregnancy pregnancy induces a fetal antigen-specific maternal t regulatory cell response that contributes to tolerance rate of pregnancy-related relapse in multiple sclerosis. pregnancy in multiple sclerosis group rheumatoid arthritis and pregnancy the epidemiology of listeriosis in the united states-1986. listeriosis study group the burden of malaria in pregnancy in malaria-endemic 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responses during influenza infection: getting and keeping control the impact of sex, gender and pregnancy on 2009 h1n1 disease elevated 17b-estradiol protects females from influenza a virus pathogenesis by suppressing inflammatory responses suppression of the inflammatory response in experimental arthritis is mediated via estrogen receptor a but not estrogen receptor b suppression of experimental autoimmune encephalomyelitis using estrogen receptor-selective ligands a question of selfpreservation: immunopathology in influenza virus infection the pathology of influenza virus infections animal models of preterm birth cachectin/ tumor necrosis factor-alpha formation in human decidua. potential role of cytokines in infection-induced preterm labor nod1 activation by bacterial ie-dap induces maternal-fetal inflammation and preterm labor spontaneous pregnancy loss mediated by abnormal maternal inflammation in rats is linked to deficient uteroplacental perfusion cytokines, prostaglandins and parturition-a review human influenza resulting from aerosol inhalation time lines of infection and disease in human influenza: a review of volunteer challenge studies crossroads of estrogen receptor and nf-kappab signaling peripheral blood cytokine profiling during pregnancy and post-partum periods estrogen receptors: how do they signal and what are their targets prolactin and autoimmunity cytokines and neonates interferon-beta pretreatment of conventional and plasmacytoid human dendritic cells enhances their activation by influenza virus antiviral-activated dendritic cells: a paracrine-induced response state cd86 has sustained costimulatory effects on cd8 t cells serves as a host antiviral factor that enhances innate and adaptive immune responses to influenza a virus the tumor-associated antigen ebag9 negatively regulates the cytolytic capacity of mouse cd8+ t cells a mouse model for immunization with ex vivo virus-infected dendritic cells influenza virusinduced dendritic cell maturation is associated with the induction of strong t cell immunity to a coadministered, normally nonimmunogenic protein experimental transmission of influenza virus infection in mice. i. the period of transmissibility a novel role for viral-defective interfering particles in enhancing dendritic cell maturation cutting edge: rapid in vivo ctl activity by polyoma virus-specific effector and memory cd8+ t cells we thank dr. david woodland for providing influenza np-specific tetramers, and olga herrera for technical assistance. key: cord-280386-a8qr7nl6 authors: pires, sara m.; fischer-walker, christa l.; lanata, claudio f.; devleesschauwer, brecht; hall, aron j.; kirk, martyn d.; duarte, ana s. r.; black, robert e.; angulo, frederick j. title: aetiology-specific estimates of the global and regional incidence and mortality of diarrhoeal diseases commonly transmitted through food date: 2015-12-03 journal: plos one doi: 10.1371/journal.pone.0142927 sha: doc_id: 280386 cord_uid: a8qr7nl6 background: diarrhoeal diseases are major contributors to the global burden of disease, particularly in children. however, comprehensive estimates of the incidence and mortality due to specific aetiologies of diarrhoeal diseases are not available. the objective of this study is to provide estimates of the global and regional incidence and mortality of diarrhoeal diseases caused by nine pathogens that are commonly transmitted through foods. methods and findings: we abstracted data from systematic reviews and, depending on the overall mortality rates of the country, applied either a national incidence estimate approach or a modified child health epidemiology reference group (cherg) approach to estimate the aetiology-specific incidence and mortality of diarrhoeal diseases, by age and region. the nine diarrhoeal diseases assessed caused an estimated 1.8 billion (95% uncertainty interval [ui] 1.1–3.3 billion) cases and 599,000 (95% ui 472,000–802,000) deaths worldwide in 2010. the largest number of cases were caused by norovirus (677 million; 95% ui 468–1,153 million), enterotoxigenic escherichia coli (etec) (233 million; 95% ui 154–380 million), shigella spp. (188 million; 95% ui 94–379 million) and giardia lamblia (179 million; 95% ui 125–263); the largest number of deaths were caused by norovirus (213,515; 95% ui 171,783–266,561), enteropathogenic e. coli (121,455; 95% ui 103,657–143,348), etec (73,041; 95% ui 55,474–96,984) and shigella (64,993; 95% ui 48,966–92,357). there were marked regional differences in incidence and mortality for these nine diseases. nearly 40% of cases and 43% of deaths caused by these nine diarrhoeal diseases occurred in children under five years of age. conclusions: diarrhoeal diseases caused by these nine pathogens are responsible for a large disease burden, particularly in children. these aetiology-specific burden estimates can inform efforts to reduce diarrhoeal diseases caused by these nine pathogens commonly transmitted through foods. diarrhoeal diseases are a major cause of disease burden worldwide [1] . the global burden of disease study 2010 (gbd 2010) ranked diarrhoeal diseases as the fourth largest disease burden, accounting for 3.6% of the total disease burden globally. diarrhoeal diseases accounted for an even higher proportion (5%) of the total disease burden in children <5 years of age [1] . diarrhoeal diseases have a substantially higher impact in low-income countries and regions with poor water quality, sanitation and food safety. diarrhoeal diseases are caused by a variety of bacteria, viruses, and parasites, many of which are commonly transmitted through food [2] . despite the large disease burden caused by these pathogens, the global contribution of specific aetiological agents of diarrhoeal diseases is largely unknown. for example, recent studies have estimated the worldwide incidence of diarrhoeal diseases in children <5 years of age [3] , and in older children and adults [4] , but did not provide aetiology-specific estimates. lanata et al. [5] and fisher walker et al. [6] provided aetiology-specific estimates, but only for diarrhoeal deaths among children <5 years of age, and diarrhoeal cases in persons 5 years of age, respectively. another large-scale study estimated diarrhoeal aetiologies in children <5 years of age in specific study sites, particularly in sub-saharan africa and south asia [7] . other studies have estimated the incidence of specific foodborne diseases, many of which cause diarrhoea, but each focused on a single developed country [8] [9] [10] [11] [12] [13] [14] . to identify and prioritize targeted interventions to reduce the public health impact of foodborne diseases, public health policy makers and other stakeholders need aetiology-specific regional and global estimates of the incidence and mortality of diarrhoeal diseases caused by pathogens that are commonly transmitted through foods. these estimates, combined with knowledge on the proportion of this burden that is derived from foods, will form the basis for the estimation of the global and regional burden of foodborne diseases. as part of the effort by the world health organization (who) foodborne disease burden epidemiology reference group (ferg; http://www.who.int/foodsafety/areas_work/ foodborne-diseases/ferg/en/) to estimate the disease burden of foodborne diseases, we estimated the global and regional incidence and mortality of diarrhoeal diseases which are commonly transmitted through foods. after reviewing the epidemiology of diseases caused by bacteria, viruses, and protozoa that are commonly transmitted to humans through food, and which are included in ferg, we selected nine pathogens which commonly cause diarrhoea for inclusion in our study: campylobacter global spp., cryptospordium spp., entamoeba histolytica, enterotoxigenic escherichia coli (etec), enteropathogenic e. coli (epec), giardia lamblia, norovirus, salmonella spp., and shigella spp. we did not include shiga-toxin producing e. coli (stec), and vibrio cholerae, diarrhoeal pathogens included in ferg, because the incidence and mortality of diseases caused by these agents have been estimated using different approaches and published elsewhere [15, 16] . although some countries have published national incidence and mortality estimates for diseases caused by most of these nine pathogens, such estimates of foodborne diseases, which are considered to be the highest quality burden estimates for these diseases [17] , are only available from a limited number of countries. we therefore used two approaches to estimate the incidence and mortality of the diseases caused by the nine selected diarrhoeal pathogens, and applied one or the other approach in each country in the six regions. approach 1 was applied to all countries in the european region (euro) and all other countries with overall low mortality as defined by who (http://www.who.int/choice/demography/mortality_strata/en/); approach 2 was applied to all remaining countries. using these two approaches allowed us to utilize the highest quality available data in countries with overall low mortality which have similar sanitary and public health infrastructure and presumed incidence of foodborne diseases. the final results of the two approaches were merged in a last step to determine global estimates and estimates for the 6 regions. the first approach utilized available national incidence and mortality estimates of diseases caused by the nine selected pathogens and was applied to 61 countries in euro (sub-regions a, b, and c), and low-mortality countries in american region (amro; sub-region a) and western pacific region (wpro; sub-region a). we conducted a literature review and consulted with the international collaboration of foodborne disease burden of illness studies (http://www.cdc.gov/ncezid/dfwed/international/enteric-burden-collaboration.html) to identify all published national estimates (with associated uncertainty intervals) of foodborne diseases. such estimates were derived through studies that corrected national surveillance data to account for under-diagnosis and underreporting [8] . in cases where national estimates were expressed as number of cases and deaths, and not as incidence and mortality rates (per 100,000 population), we used united nations population figures for the national study year to create such rates. if a national estimate only included domestically-acquired infections, we used the proportion of infections acquired during international travel in a neighbouring country to derive revised estimates that included infections acquired abroad. we applied the national incidence and mortality estimates, with the associated uncertainty intervals, to each country with such national estimates, and the median incidence and mortality of all studies, with uncertainty intervals associated with those median estimates, to each country in euro, amro sub-region a, and wpro sub-region a that did not have national incidence or mortality estimates. this approach resulted in incidence and mortality estimates (per 100,000 population), with uncertainty intervals, for each of the 61 countries for each of the nine diseases. overall estimates were partitioned to the age groups <5 years of age and 5 years of age on the basis of the age distribution of the population in each region. further details of this approach are available in s1 appendix. regional incidence and mortality of diarrhoea. first, we identified the total number of diarrhoeal cases in the 133 countries for 2010 by combining estimates based on systematic reviews for children <5 years of age and persons 5 years of age [3, 4] . for the estimate of the total number of diarrhoeal deaths, we obtained data on the total number of deaths in the 133 countries in 2010 attributed to diarrhoeal diseases from who (http://www.who.int/gho/en/; accessed 6 june 2014); we used the range of diarrhoeal deaths estimated in the global burden of disease 2010 study (gbd2010) to derive an uncertainty interval for the death envelope. we derived the final number of diarrhoeal cases ("diarrhoeal envelope") and diarrhoeal deaths ("diarrhoeal death envelope") by subtracting published estimates of the number of diarrhoeal cases and deaths caused by stec [15] and v. cholerae [16] . aetiology proportions. our next step was to estimate the proportion of diarrhoeal illnesses and deaths due to the nine pathogens by extracting the aetiological proportions of diarrhoeal cases and deaths for each pathogen by region from systematic reviews of studies reporting stool sample isolation and detection from inpatient, outpatient, and communitybased studies of persons with diarrhoea. we assumed that the distribution of pathogens observed among outpatient and community studies represented the pathogen prevalence among diarrhoeal cases, and that the distribution of pathogens among inpatients hospitalized for severe diarrhoea represented the pathogen prevalence among diarrhoeal deaths for all age groups. due to data scarcity, we also included inpatient studies to estimate the aetiological proportions of diarrhoeal cases in persons 5 years of age (i.e. all available studies). we assumed that g. lamblia infection was unlikely to result in death based upon the data available from the national foodborne disease mortality estimates, and therefore excluded it from mortality estimates. to determine aetiological proportions, we used 3 systematic reviews to identify studies that reported isolation or detection of pathogens from stool specimens or rectal swabs collected from persons with diarrhoea. for norovirus, we used a previously published systematic review including studies published between 1990 and 2012 and then updated through 2014 [17] . the norovirus-specific review was conducted because of the increased recent use of molecular diagnostics globally and as part of a parallel effort to estimate the global burden of norovirus disease [17] . for all other pathogens we included two previously published systematic reviews: 1) the aetiology of diarrhoeal disease studies for children 5 years of age published between 1980 and 2008 [3] and 2) the aetiology among children <5 years of age published between 1990 and 2011 [5] . we updated each of these reviews to include studies published through 2012. s2 appendix details the systematic review methodologies and results of the updated reviews, including search terms and inclusion/exclusion criteria. studies focusing only on norovirus collected in the general reviews were excluded to avoid duplicates with the norovirus-specific review. the general systematic reviews collected data on isolation or detection of the nine pathogens included in our study and on pathogens not commonly transmitted by food (e.g. rotavirus, sapovirus, astrovirus, and coronavirus); the latter were grouped together as "other pathogens". we initially calculated study-specific aetiology-proportions by dividing the number of samples positive for the pathogen by the total number of samples tested in that study (studyproportions); we then estimated regional proportions by calculating the median aetiology-proportion of all study-estimates within each region (regional-proportions). for example, if 10 studies conducted in the western pacific region (wpro) provided data on the number of salmonella isolates among diarrhoeal cases, this region's salmonella-proportion was estimated as the median of the estimated 10 study-specific aetiology proportions. since several of the studies among children <5 years of age used narrower age ranges, in analyses a and b we calculated an age-adjusted proportion for this age group by calculating a conversion factor as the ratio of the median proportion in the age group 0-59 months to the median proportion in age group x ((median (prev0-59)/median (prevx)). we applied this approach when three or more studies for each pathogen contributed to each of the two medians. if this condition was not met, we borrowed the conversion factor for the age group from a similar age group within the same pathogen (for example, used the conversion factor calculated for studies including infants 0-11 months of age for studies that included infants 0-5 months of age). for persons 5 years of age, we have assumed that differences between narrower age categories would be diluted in this very broad population group and have chosen not to age-adjust medians. to estimate the proportion of diarrhoeal stools due to unknown aetiology, we included studies that sought 8 pathogens and reported patients with an unknown cause of disease. if only one study tested for a given pathogen in one region, we applied criteria to identify outliers and prevent potentially non-representative studies from misrepresenting the final regional aetiology-proportions. an outlier was defined as a regional estimate ±5 times the global median. if a regional estimate derived from 2 studies was identified as an outlier, the individual studies' prevalence and sample size were evaluated, and a particular study was excluded if it represented an outlier when compared to remaining studies within the region. outliers were excluded from the results and taken as a missing value. if a regional median aetiological proportion estimate was missing (due to either missing data or exclusion of an outlier), the missing regional estimate was replaced by the global median of the aetiological proportions for that pathogen. all global medians were estimated after the exclusion of potential outliers. statistical analysis. to account for uncertainty in these calculations, we used monte carlo simulation in all steps of the analysis. we applied a bootstrapping analysis to derive 95% uncertainty intervals (ui) around aetiology-proportions. 'pseudo-data sets' were created by sampling each study with replacement from the real dataset. in each of these 1,000 pseudo-datasets, a different random number of positive samples for each study was generated from a binomial distribution defined on the basis of the number of samples and the expected proportion in that study. all pseudo-datasets were used in the estimation procedure described above to generate corresponding 1,000 study-specific aetiology proportions, and regional aetiology proportions. the 2.5th and 97.5th percentile of these proportions gave the 95% ui. data management and analyses were conducted in sas enterprise guide 4.3. we then constrained the aetiological proportions for the nine diarrhoeal pathogens, for other pathogens not commonly transmitted by food, and unknown aetiology to ensure that they did not sum to more than 100% in each region. for this purpose, we first fitted univariate beta(α, β) distributions to the median proportions and simulated quantiles (i.e., the 2.5th and 97.5th percentiles). the distributions' optimized parameters were estimated via one dimensional optimization, minimizing the squared distance between the estimated and fitted quantiles, and ensuring the median of the fitted distribution to be similar to the estimated median. next, 10,000 random deviates were sampled from the fitted beta distributions. if needed, an "unknown aetiology" category was created as 1 minus the sum of simulated proportions across aetiologies, per iteration. finally, the random deviates were normalized iteration-wise by dividing them by the sum of simulated aetiological fractions. once estimates of aetiological proportions for cases and deaths for the nine pathogens were derived, the regional aetiological proportions for each disease were multiplied by the respective estimates for total diarrhoeal illnesses and deaths in those regions, accounting for uncertainty using a stochastic model with 10,000 iterations. we then derived age-stratified incidence and mortality estimates, with associated uncertainty, for each disease for each region using country-level population data for 2010 from the 2012 revision of the united nations world population prospects (esa.un.org/wpp, accessed june 10, 2014); among the 133 countries, all countries in the same region were assumed to have the same incidence and mortality, and associated uncertainty, for each pathogen. in order to aggregate results with estimates derived for low-mortality countries (approach 1), age-specific estimates were then grouped into overall estimates. the stochastic model was implemented in r version 3.1.1 (r core team, 2014). global estimates (combining approach 1 and approach 2). the incidence and mortality estimates, with associated uncertainty, for each disease for each region obtained through approach 1 and approach 2 were combined in a final step to produce final global estimates and estimates for each of the six regions. we identified national incidence and mortality estimates of the nine diarrhoeal diseases for seven countries: australia, canada, france, netherlands, new zealand, united states of america, and the united kingdom [8] [9] [10] [11] [12] [13] [14] . the national estimates from australia and canada included only domestically-acquired infections; revised national estimates were derived using data on international travel-acquired infections from the new zealand and the united states, respectively. estimates of incidence and mortality for each of 7 countries are available in s1 appendix. the systematic reviews conducted to collect data to estimate the proportion of diarrhoeal illnesses and deaths in the 133 other countries yielded 136 outpatient and community studies (fig 1) and 254 inpatient studies (fig 2) among children <5 years of age, and 97 inpatient, outpatient, and community studies (fig 3) among persons 5 years of age. table 1 lists the number of studies for each of the nine pathogens by region. the final number of studies providing data for individual pathogens in different regions varied substantially. the data available for persons 5 years of age were scarcer and the number of studies for each region was low, particularly for the afro and amro regions. table a in s1 file presents the estimates of the regional envelopes of diarrhoeal cases and deaths, and tables b1 to b4 in s1 file present the contribution of each pathogen for these envelopes. we estimated that the nine pathogens included in our study resulted in a total of 4.6 billion cases (95% ui 3.5-6.5) and 1.6 million deaths (95% ui 1.3-1.9) in 2010 worldwide (table 2 ). in the 61 countries in euro and amro and wpro a regions, we estimated that these nine pathogens resulted in 852.2 million cases (95% ui 649.8-1,095.7) and 23,898 deaths (95% ui 14,607-34,212. in the remaining 133 countries, we estimated these pathogens resulted in a total of 3.4 billion cases (95% ui 1.4-8.4 billion) and 1,560,986 deaths in 2010 (95% ui 1,322,987-1,845,688). global and regional estimates of incidence and mortality rates caused by these pathogens are presented in s1 there were marked regional differences in incidence and mortality caused by the nine diseases (table 3 ; figs 4 and 5). among the nine pathogens, norovirus was the most common cause of cases in all regions, particularly among persons 5 years of age (fig 4) . among these nine pathogens, norovirus and g. lamblia were the cause of the highest incidence in children <5 years of age; in afro, amro, emro and wpro campylobacter caused the third highest incidence in afro and wpro, and cryptosporidium spp. made a substantial contribution in emro. the second highest incidence of cases among persons 5 years of age was caused by salmonella spp. in emro, searo and wpro. for deaths caused by the nine pathogens, epec and norovirus were the most frequent causes in children <5 years of age in afro, amro, searo and wpro (fig 5) . etec was also estimated to be an important cause of mortality, particularly in afro and amro, and shigella spp. was the leading cause in emro and third leading in afro. salmonella spp. caused the largest proportion of diarrhoeal deaths in this age group in euro. in persons 5 years of age, norovirus was the leading cause of mortality in all regions. these are the first global and regional estimates of incidence and mortality caused by nine pathogens that cause diarrhoea and are commonly transmitted through foods. of these agents, we showed that norovirus was responsible for the largest number of cases followed by etec, g. lamblia and shigella spp. norovirus caused the most deaths among these nine pathogens, followed by epec and shigella spp. nearly 40% of all cases and 43% of deaths caused by these nine pathogens occurred in children <5 years of age, who represent only 8% of the global population. we also demonstrated regional differences in both the incidence and mortality of diseases caused by these nine pathogens. these differences are important to consider when defining interventions to reduce the burden of diseases commonly transmitted through foods. as an example, campylobacter and salmonella, which are pathogens that have mainly domestic food-producing animals as reservoirs and usually infect humans due to contamination in the food production chain, play a major role in the incidence of foodborne enteric disease in the euro region but are less frequently observed in who-defined high mortality countries. in high mortality countries where poor sanitary conditions and food and water contamination are more important, pathogens such as g. lamblia and etec play more important roles. the predominance and relative ubiquity of norovirus across all regions suggests that targeted interventions, such as vaccines, may be necessary to reduce the burden of this human reservoir pathogen. our study focused only on diarrhoeal pathogens that are commonly transmitted through foods. it is important to recognize that the estimates we present include disease from all modes of transmission, including contaminated food, water, and environments, along with infected persons and animals. our inclusion of only pathogens that are commonly transmitted through food also meant that we did not present estimates for important diarrhoeal agents, such as rotavirus. by including them into other pathogens, we have assured that we did not overestimate the incidence and mortality of remaining pathogens when distributing the diarrhoeal total cases and deaths to the nine diseases of interest. we used two approaches to estimate the relative contribution of different aetiologies for disease because the quality and availability of data varied substantially between countries and regions. national incidence and mortality estimates were available only from seven low mortality countries, but given the high quality of these estimates, we gave priority to these data by using them as the basis for estimating the incidence and mortality of the diseases caused by the nine pathogens for all euro and other low mortality countries (sub-region a in amro and wpro). for all other countries, we adapted the cherg approach for estimating the burden of diarrhoeal diseases in these countries [5] . this approach was facilitated by the availability of estimates of the envelope of diarrhoeal deaths, along with recent advances in diarrhoeal disease diagnosis, such as widespread application of polymerase chain reaction (pcr) for norovirus detection. we have divided countries for the two approaches on the basis of their overall mortality status, as defined by who, which we assume can be used as a proxy for differences in the public health status of the countries. the two approaches differed in terms of methodology, assumptions and type of data used. while approach 1 analysed national incidence and mortality of disease by pathogens commonly transmitted through foods estimated primarily by correcting surveillance data to account for underreporting and under-diagnosis, approach 2 relied on systematic reviews of studies identifying causative agents in patients with diarrhoea. applying approach 2 for all regions was not possible due to the scarcity of community, inpatient and outpatient studies from low mortality countries for most pathogens. because of these differences, we acknowledge that comparisons between regions should be made with care. we relied on studies that estimated the incidence of potentially foodborne diseases in a specific country by correcting public health surveillance data for underreporting and under-diagnosis for approach 1. such studies have their own limitations, such as relying on a variety of data with variable quality and representativeness, multiple modelling approaches and a wide range of assumptions. as an example, most use population surveys on care-seeking behaviours of patients with diarrhoea, which are subject to recall bias that can influence the results [18] . furthermore, in approach 1 we have assumed that the median aetiology-proportion of the seven collected studies (along with its uncertainty interval) represented the relative *not all studies provide data for pathogens included in our analysis. remaining studies were used to estimate the aetiology-proportion of "others". †studies conducted in countries in the euro region, amro sub-region a, or wpro sub-region a were not included in the analyses. doi:10.1371/journal.pone.0142927.t001 contribution of each agent for the diarrhoeal morbidity and mortality estimates, and have taken a simplified approach to estimate uncertainty. approach 2 to estimate the contribution of aetiologies for diarrhoeal cases and deaths used studies that tested for varying numbers of pathogens. studies that focused on a single pathogen may tend to overestimate the importance of that pathogen because they are potentially more likely to be conducted in a study site with a high prevalence of that pathogen [5] or have selective inclusion criteria e.g. acute watery diarrhoea. however, we needed to include them due to estimates exclude the proportion of the incidence envelope attributable to "other pathogens", because these were not considered in approach 1 (applied to 60 countries within euro, amro sub-region a, and wpro sub-region a). doi:10.1371/journal.pone.0142927.g004 the low number of studies focusing on multiple pathogens, particularly in the older age group. due to scarcity of data, some regional aetiological proportion estimates were informed by a single study. to avoid that studies with potentially over or underestimated aetiology-proportion estimates misrepresented the estimate for that region, we have defined criteria to identify outliers, and have excluded these estimates from the results. these overestimations or underestimations can have various causes, such as study locations with a particularly high or low prevalence of that pathogen, a small sample size, or a country not representative of the whole region. excluded outliers were treated as missing values, and the aetiology-global median used to represent the regional estimate. in this approach, we also assumed that the distribution of pathogens among inpatients hospitalised with severe diarrhoea represented the relative importance of these pathogens for diarrhoeal deaths. this meant that we used severity of disease as a proxy for mortality, which may lead to an over-or under-estimation of the number of deaths caused by some pathogens. in the analyses for persons over five years of age, due to sparseness of data we decided to use data from inpatient studies to estimate aetiology-proportion of cases, which may also have led to an erroneous estimation of the contribution of pathogens to diarrhoeal incidence. additionally, this approach relied on stool samples, but some patients that tested positive for a pathogen may have had asymptomatic infections. this is more likely to happen for pathogens that have long excretion periods after illness (e.g. norovirus). carefully conducted longitudinal studies would be needed to distinguish clinical cases from asymptomatic infections [5] . in approach 2, we estimated the proportion of the diarrhoeal cases and deaths that was caused by unknown aetiology. the defined strategy was to base our estimates on studies that had collected data for eight or more pathogens and had reported "aetiology unknown". undiagnosed cases could be caused by truly unrecognized agents, but also could be due to e.g. the use of incorrect or insensitive testing methods, to antimicrobial-therapy prior to stool sampling, or to non-infectious diarrhoeal causes. this strategy could only be adopted for the analyses for children <5 years of age, and still there were few studies available for the calculations. in remaining analyses, unknown was calculated as the difference between 100% and the sum of all remaining aetiologies (including others). in both approaches, we assumed that studies conducted in a given country would be representative of the region or sub-region of this country. when several studies providing data for a given pathogen were available for that region, calculating a median estimate ensured that the influence of extreme values was restricted. on the contrary, when only one or few studies were available, this study's/studies' estimate was assumed to represent all countries within a region. in addition, the six regions' classification was based on the geographic distribution of countries, and countries within each are very diverse. we tried to avoid inappropriate extrapolations by applying distinct approaches to low mortality and medium and high mortality countries, assuming that a country's mortality status may also reflect the nation's public health and food safety situation. in the euro region, we made an extra extrapolation and assumed that all countries were similar to sub-region a countries in terms of foodborne diseases' incidence and mortality. this region includes countries with other mortality status (euro b and euro c), and some of these countries may have a different distribution of aetiologies. due to lack of data, we were not able to explore the extent of these differences. a recent large prospective matched case-control study in children <5 years of age conducted over three years at selected sites in africa and asia (the gems study) estimated the burden of aetiology-specific diarrhoea in these sites, and concluded that rotavirus, cryptosporidium spp., etec and shigella spp. were the most important causes [7] . this study was fundamentally different from ours: among other differences, it calculated attributable cases of diarrhoea to each aetiology, comparing patients (moderate to severe diarrhoeal cases seen in health facilities) and controls (un-matched healthy children in a community sample). the gems study excluded mild diarrhoeal cases, which along with their moderate diarrhoeal cases would constitute our non-fatal incident cases. by including moderate and severe cases instead of only severe hospitalized cases, the gems study does not have a good proxy for fatal cases as we defined it. still, the study's findings are generally consistent with ours; epec, etec, shigella spp. and cryptosporidium spp. were among the most important causes of diarrhoea. one important difference was on the relative contribution of norovirus for diarrhoea incidence and mortality: our results suggest that norovirus is amongst the most important causes of diarrhoeal cases and deaths, whereas the gems study estimated a lower contribution of this pathogen. this discrepancy may indicate that our approach may lead to an overestimation of the proportion of diarrhoea attributable to norovirus or other pathogens that are likely to be carried asymptomatically after the first exposure (like certain types of etec, e.g. lt-etec) because it does not account for the potential detection of norovirus in the stool of healthy individuals [17] . however, the gems study required that control individuals did not have a diarrhoeal episode in the 7 days prior to sampling; because norovirus patients can excrete the virus for 3 to 4 weeks after infection, it is likely that some controls included in this study corresponded to asymptomatic carriers of norovirus [5] . these findings may suggest limitations in ascribing pathogenicity based on case-control studies, particularly in developing country settings in which frequent exposures and a high degree of transmission results in common reinfections [19] . a recent community based prospective cohort study (the "etiology, risk factors, and interactions of enteric infections and malnutrition and the consequences for child health and development project", the mal-ed study), supported our results and estimated that norovirus was the most important cause of diarrhoea in young children [20] . our study provides information on the incidence and mortality of nine pathogens that cause diarrhoeal and are commonly transmitted through food. most of these agents can also infect humans through other sources (e.g. environmental, contact with animals and person-toperson transmission), and the relative contribution of these sources for disease varies between aetiologies and regions [21] . to estimate the number of foodborne aetiology-specific diarrhoeal cases and deaths, our results need to be combined with regional source attribution-proportions and other aetiological agents [22] . these results, as well as the foodborne disease burden of each disease in terms of disability adjusted life years (dalys) are presented elsewhere [23] . our results provide public health policy makers, including risk managers, and other stakeholders with information for advocacy for improved regulation and control of diseases commonly transmitted through foods. we highlight the most important diarrhoeal diseases in different regions and age groups, which will allow policy makers to define and improve control strategies targeted at different pathogens, settings and countries. supporting information s1 appendix. estimating the aetiology-specific incidence and mortality of diarrhoea in 60 countries in the region of the americas (amro) sub-region a, western pacific region (wpro) sub-region a, and european region (euro; sub-regions a, b and c). (docx) s2 appendix. detailed description of the methods of the systematic reviews used to identify studies that provided data to derive aetiology-proportion estimates for all included pathogens except norovirus. (docx) table b1: regional and global etiology-proportions of diarrhea cases in children <5, 2010 (median % and 95% ci). table b2: regional and global etiology-proportions of diarrhea cases in persons >5, 2010 (median % and 95% ci). table b3: regional and global etiology-proportions of diarrhea deaths in children <5, 2010 (median % and 95% ci). table b4: regional and global etiology-proportions of diarrhea deaths in persons >5 total global incidence and mortality rate (per 100,000) of 10 foodborne diarrhoeal pathogens (median and 95% uncertainty intervals) disability-adjusted life years (dalys) for 291 diseases and injuries in 21 regions, 1990-2010: a systematic analysis for the global burden of disease study evolving public health approaches to the global challenge of foodborne infections diarrhoea morbidity and mortality in older children, adolescents, and adults. epidemiology and infection global burden of childhood pneumonia and diarrhoea global causes of diarrhoeal disease mortality in children <5 years of age: a systematic review etiology of diarrhea among older children, adolescents, and adults: a systematic review burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the global enteric multicenter study, gems): a prospective, case-control study foodborne illness acquired in the united states-unspecified agents disease burden of foodborne pathogens in the netherlands longitudinal study of infectious intestinal disease in the uk (iid2 study): incidence in the community and presenting to general practice estimates of the burden of foodborne illness in canada for 30 specified pathogens and unspecified agents, circa foodborne illness, australia, circa 2000 and circa foodborne infections in france risk ranking for foodborne microbial hazards in new zealand: burden of disease estimates global incidence of human shiga toxin-producing escherichia coli infections and deaths: a systematic review and knowledge synthesis. foodborne pathogens and disease the global burden of cholera. bull world health organ the global prevalence of norovirus among cases of gastroenteritis estimating the burden of acute gastroenteritis, foodborne disease, and pathogens commonly transmitted by food: an international review epidemiologic implications of asymptomatic reinfection: a mathematical modeling study of norovirus pathogen-specific c burdens of community diarrhoea in developing countries: a multisite birth cohort study (mal-ed) assessing the applicability of currently available methods for attributing foodborne disease to sources, including food and food commodities who foodborne disease burden epidemiology reference group's (ferg) expert elicitation for estimating the relative contribution of food to the global burden of diseases commonly but not only acquired through the consumption of food who foodborne disease burden epidemiology reference group (ferg) 2010 estimates of the global and regional disease burden of 21 foodborne bacterial, protozoal and viral diseases the world health organization funded this study under the foodborne disease burden epidemiology reference group through contributions from member states and international agencies. we would also like to acknowledge the support from the bill and melinda gates analyzed the data: smp. wrote the paper: smp clfw cfl bd ajh asrd reb mdk fja. key: cord-310438-744r7gc3 authors: chan, ta-chien; hsiao, chuhsing kate; lee, chang-chun; chiang, po-huang; kao, chuan-liang; liu, chung-ming; king, chwan-chuen title: the impact of matching vaccine strains and post-sars public health efforts on reducing influenza-associated mortality among the elderly date: 2010-06-25 journal: plos one doi: 10.1371/journal.pone.0011317 sha: doc_id: 310438 cord_uid: 744r7gc3 public health administrators do not have effective models to predict excess influenza-associated mortality and monitor viral changes associated with it. this study evaluated the effect of matching/mismatching vaccine strains, type/subtype pattern changes in taiwan's influenza viruses, and the impact of post-sars (severe acute respiratory syndrome) public health efforts on excess influenza-associated mortalities among the elderly. a negative binomial model was developed to estimate taiwan's monthly influenza-associated mortality among the elderly. we calculated three winter and annual excess influenza-associated mortalities [pneumonia and influenza (p&i), respiratory and circulatory, and all-cause] from the 1999–2000 through the 2006–2007 influenza seasons. obtaining influenza virus sequences from the months/years in which death from p&i was excessive, we investigated molecular variation in vaccine-mismatched influenza viruses by comparing hemagglutinin 1 (ha1) of the circulating and vaccine strains. we found that the higher the isolation rate of a (h3n2) and vaccine-mismatched influenza viruses, the greater the monthly p&i mortality. however, this significant positive association became negative for higher matching of a (h3n2) and public health efforts with post-sars effect. mean excess p&i mortality for winters was significantly higher before 2003 than after that year [mean ± s.d.: 1.44±1.35 vs. 0.35±1.13, p = 0.04]. further analysis revealed that vaccine-matched circulating influenza a viruses were significantly associated with lower excess p&i mortality during post-sars winters (i.e., 2005–2007) than during pre-sars winters [0.03±0.06 vs. 1.57±1.27, p = 0.01]. stratification of these vaccine-matching and post-sars effect showed substantial trends toward lower elderly excess p&i mortalities in winters with either mismatching vaccines during the post-sars period or matching vaccines during the pre-sars period. importantly, all three excess mortalities were at their highest in may, 2003, when inter-hospital nosocomial infections were peaking. furthermore, vaccine-mismatched h3n2 viruses circulating in the years with high excess p&i mortality exhibited both a lower amino acid identity percentage of ha1 between vaccine and circulating strains and a higher numbers of variations at epitope b. our model can help future decision makers to estimate excess p&i mortality effectively, select and test virus strains for antigenic variation, and evaluate public health strategy effectiveness. increased influenza vaccination coverage for the elderly, one of the highest risk groups for influenza-related deaths [1] , has prevented influenza-related complications and deaths, based on 64 studies from 1964 to 2006 [2] . in taiwan, elderly populations (aged $65 years) have received free influenza vaccination since 1998. vaccine coverage rates have increased from 9.9% in 1998 to 49.1% in 2007 . despite similar expansions in coverage, pneumonia and influenza (p&i) mortality among the elderly have continued to rise in italy [3] and the united states [4, 5] . such findings contribute to the current international debate on the influenza vaccine's effectiveness in preventing elderly influenzaassociated deaths. to examine this issue, we investigated the impact of potential vaccination mismatches with co-circulating viral strains of influenza virus types/subtypes, and public health efforts after the 2003 outbreak of sars on vaccination effectiveness in subtropical regions like taiwan. routine virological surveillance has been crucial for early detection of influenza viral changes [6] . understanding epidemiological pattern changes of influenza in taiwan, located geographically close to several past influenza pandemic epicenters in china and southeast asia, has larger implications for global virological surveillance. taiwan's dominant circulating a(h3n2), a(h1n1), and b wild-type influenza virus strains appeared about one to two years earlier than selected vaccine strains recommended by world health organization (who) for the northern hemisphere, implying that taiwan has the potential to play a key role in early pandemic and epidemic detection and control [7, 8] . the aims of this study were: (1) to evaluate the effectiveness of matching or mismatching influenza vaccine strains on influenzaassociated mortality, (2) to assess whether public health improvements during the post-sars period might have decreased elderly mortality, and (3) to investigate molecular variation among vaccine-mismatched influenza viruses that may be associated with increased excess influenza-associated mortality. data was collected on taiwan's annual and monthly influenzaassociated mortality rates for the elderly population, monthly meteorological conditions (obtained from taiwan's central weather bureau), annual influenza vaccine strains (collected from the who) [9] , dominant types/subtypes of influenza viruses for winter epidemic seasons, and monthly influenza isolation rates [compiled from the centers for disease control in taiwan (taiwan-cdc)] for the 1999-2000 through 2006-2007 influenza seasons. rates of both winter and annual influenza-associated excess mortality among the elderly were calculated. winters periods were designated as december 1 st to february 28 th of the following year. annual periods were marked as beginning on october 1 st and concluding on september 30th of the following year. the elderly population was calculated as the average of two mid-years' elderly population (acquired from the census database of the ministry of the interior). monthly isolation rates of influenza virus types/subtypes for each studied year were obtained from the virological surveillance database of contract laboratories and compiled by taiwan-cdc ( figure s1 ) [10] . wild-type, dominant circulating influenza virus strains were also collected from the contract laboratories, as designated by taiwan-cdc and the literature [8] . comparisons between influenza vaccine strains and taiwan's dominant influenza epidemic strains are summarized in table s1 . to better evaluate influenza's disease burden, we used influenzaassociated mortality rates that were calculated under broad definitions [4] . using international classification of diseases, ninth revision (icd-9) codes and clinical data obtained from taiwan's department of health, mortality was divided into three categories: (1) pneumonia and influenza (p&i, icd-9: 480-487), (2) respiratory and circulatory (r&c, icd-9: 390-519), and (3) all-cause deaths (icd-9: 000-999) with the exclusion of non-natural deaths. we developed a negative binomial regression model and added two variables of vaccine match/mismatch and pre/post-sars effect for multivariate analyses with a modification of a thompson-like model [4] , because of dispersed distributions of the three influenza-associated mortality rates (variance/mean .20). explanatory variables for the above three outcome measures include monthly meteorological parameters (monthly means of temperature and humidity), annual periodic cycle (i.e., sine/cosine function of seasonal periodicity), monthly virus isolation rates for different subtypes/types of influenza viruses [a (h3n2) or a (h1n1) or b], matching status of different vaccine strains for each subtype/type in each of the studied years, post-sars effect, and linear temporal monthly trends. matching status for each subtype/ type of influenza viruses was defined as the consistency between the nomenclature of the vaccine strain and the nomenclature of that season's dominant wild-type strain in taiwan. if no wild-type subtype was isolated for a certain year, the status of the flu vaccine was thus coded as ''matching'' for that subtype/type and year. to assess whether the public health effort after the 2003 unique outbreak of sars in taiwan might also play a role in mortality, the indicator variable ''post-sars effect'' was applied to the period beginning october 2003 (the first month of the 2003 influenza season) till after the conclusion of the outbreak in june 2003. model selection was based on akaike's information criterion (aic) and likelihood ratio test [11] . when modeling p&i mortality, both mean temperature and relative humidity were found to be without statistical significance and were thus excluded from the full model (table 1) . the model was implemented by sas (version 9.1; sas institute inc, cary, nc). the final model for ''influenza-associated deaths'' was devised as follows: winter and annual influenza-associated excess elderly mortalities to evaluate the impact of influenza vaccination and/or post-sars effect on influenza-associated elderly mortalities, we listed both variables in table 2 . excess mortality (95% confidence interval) was calculated for each winter and year. these excess deaths were assessed by calculating the difference between observed data and expected baselines that were derived from the negative binomial model ( figure s2 ). when we modeled vaccinematching status and post-sars effect, we coded data as ''1's'' or ''0's'' depending on the actual data for each year. ''post-sars'' was defined as all months after october 2003. after calculating residual deaths for each month, we replaced negative residuals (e.g. observed values less than the expected value) with zero and summed up excess deaths for each influenza season. winter (or annual) excess mortality rates were calculated based on each winter's (or annual) total excess deaths divided by mid-year mean populations. in other words, we assessed temporal differences between each winter's monthly observed influenza-associated deaths and monthly expected value [obtained from our multivariate modeled deaths (baseline)], and then divided these monthly differences by the mid-year mean population for two years to calculate the actual excess mortality rate in each winter for statistical comparison among the three time periods (prior to sars, during sars, and post-sars) ( table 2, table s2 ). the mean values for each winter's excess mortality before and after sars were evaluated by independent t-test. to better understand why 2001-2002 exhibited the highest winter and annual excess p&i mortality rates, ha1 sequences of the 2002 epidemic influenza virus a (h3n2) fujian strain (a/ fujian/411/2002 (h3n2)), vaccine strains (red squared symbol) from 1999 to 2007, and other taiwanese h3n2 isolates (circular symbol) from 1996 to 2008 were gathered from the national center for biotechnology information (ncbi) for genetic comparison. we then constructed a phylogenetic tree of ha1 amino acids among taiwanese a (h3n2) virus strains using the neighbor-joining method. this tree was bootstrapped 1,000 times with mega 4.0 software [12] . percentages of amino acid sequence identity in the ha1 between dominant wild-type circulating and vaccine strains were calculated for vaccine-mismatched influenza a(h3n2) viruses isolated from months and years (1999-2000, 2003-2004, and 2004-2005) with high annual/monthly influenza-associated excess p&i mortality. the numbers and percentages of amino acid difference at specific epitope locations of ha1 that have been documented in literature [13] were further analyzed for vaccine-mismatched influenza a (h3n2) viruses isolated from these three years. temporal patterns of influenza-associated mortality rates indicate that taiwan's elderly p&i mortalities have been increasing since the beginning of the 2001-2002 influenza season despite significant increases in vaccine coverage ( figure s3a ). as described in figure 1 , the mean p&i mortality rate, 12 [14] . these increasing patterns were consistent throughout the study period among the three oldest age groups (65-74, 75-84, and 85+) ( figure s3b ). in addition to this temporal pattern, seasonal cycles (cosine and sine function in table 1 ) were also found to be significant (p,0.01) for all three influenza-associated mortality rates (except for the cosine function for r&c). furthermore, the corresponding coefficients in table 1 's model demonstrate the considerable reduction in elderly p&i mortality in years with either matching vaccines or post-sars effect. temporal patterns of monthly isolation rates for human influenza viruses in taiwan are displayed in figure s1 . during the study period, a (h3n2) and b were the dominant type/subtypes of influenza viruses. regarding to vaccine matching rate, a (h1n1) was the most frequently matched subtype (87.5%, 7/8) in eight epidemic seasons. a (h3n2) had the second highest (62.5%, 5/8) and b had the worst (50%, 4/8). using multivariate negative binomial regression models, we found that four significant variables -the monthly isolation rate of table 1 . estimated coefficients ( " b), standard errors (se) and p-values (p) of three fitted negative binomial models for influenzaassociated deaths: (1) pneumonia and influenza (p&i), (2) respiratory and circulatory, and (3) all-cause in taiwan, from october 1999 to september 2007, respectively. (1) p&i deaths (2) respiratory and circulatory deaths (3) all-cause deaths influenza a (h3n2), vaccine mismatch with a (h3n2), linear temporal trends, and sine function -were all positively correlated with annual p&i deaths (p,0.05, table 1 ). in contrast, post-sars effect and cosine function were negatively correlated with annual p&i mortality (p,0.05). influenza a (h3n2)'s monthly isolation rate and sine function, as well as vaccine mismatch for influenza b, were positively correlated with r&c deaths (p,0.05). mean temperature and relative humidity were both negatively correlated with r&c deaths (p,0.05). values for observed and estimated deaths were proximate for all three influenza-associated mortality models (as illustrated in figure s2 ). , excess p&i mortality rates (11.0 per 100,000) were 2-5 times higher than rates for the same months in prior years and about 8 times higher than rates for the four months preceding the outbreak (table s2 ). in may 2003, when interhospital nosocomial infection was the most severe, the three influenza-associated excess mortality rates of p&i (22.09 per 100,000), r&c, and all-cause ranked the highest throughout the studied months. after june 2003, excess p&i mortalities in july and august declined dramatically to 0 per 100,000 ( figure s2 ). 3. stratification analysis. totally, four of the eight studied years showed increased winter excess influenza-associated mortalities ("3/100,000) plus one higher excess mortality in march, 2005 (5.28/100,000), and five of them had vaccinemismatches. of the latter five, three occurred in the pre-sars period (1999-2000, 2000-2001, and 2001-2002) and the remaining two happened in the post-sars period (2003-2004 and 2004-2005) . to examine the winter seasons' excess mortality in those years with vaccine-matched versus vaccine-mismatched strains before and after sars, we first reviewed the data on vaccine-matches or not during the pre-sars years only (e.g. without effect of sars). then, we focused on vaccine-mismatches table 2 . annual and winter excess mortality rates of influenza-associated deaths (per 100,000) among the elderly ("65 years). in summary, stratification of these two variables showed substantial trends toward lower excess p&i mortality during: (1) influenza vaccine-matched winters during the pre-sars period (without post-sars effect), and (2) figure s4 ). during the 1999-2000 flu season, a/sydney/05/97(h3n2) and a/ moscow/10/99(h3n2) were the dominant circulating strains affecting taiwan while a/sydney/05/97-like was the vaccine strain and provided inadequate protection against a/moscow/ 10/99. as shown in table s3 , five variations were identified at known epitopes [13] , including two variations (y137s, s142r) at (table s4 ). most notably, we discovered one variation at the undefined epitope (position 3, i3l) previously identified by shih et al. [13] , and three new variations (positions of a138s, i194l, y233h, figure s5 (table s4 ). we also observed that the excess mortality in 2004-2005's winter was much lower than in 1999-2000. this can be supported by higher identity percentage between vaccine and circulating strains, and a lower number of variations at epitope b. this is the first study to analyze the impact of the dominant types/subtypes of influenza viruses, the matching status of influenza vaccine strains, and the 2003 sars outbreak on three influenza-associated mortality rates among the elderly in taiwan. the study is unprecedented in its molecular-level investigation of vaccine-mismatched influenza viruses associated with excess mortality. while the limitations of our data prevent us from drawing definitive conclusions about these potential factors, we did observe five associations that merit discussion. first, higher a (h3n2) subtype isolation rates were associated with increased influenza-associated mortality. second, lower p&i mortality rates were observed when circulating strains of influenza viruses were vaccine-matched. third, increased influenza p&i excess mortality was associated with vaccine-mismatched circulating influenza h3n2 and b viruses with fewer amino acid identities. fourth, influenza disease burden after the 2003 sars outbreak (i.e. with post-sars effect) was significantly lower than before this sars outbreak. lastly, patterns of taiwan's influenza types/subtypes became more diversified after 2001 when mini links with china facilitated open travel exchanges [14] . co-circulation of h3n2 subtype with h1n1 subtype or b type viruses resulted in higher p&i mortality than any subtype/type acting alone. our observations suggest that improvements in public education and public health efforts (as a result of post-sars effects and better vaccine matching) may have contributed to a reduction in p&i mortality. this trend would be further supported if mortality reductions persist in the presence of adequately sustained prevention measures. our study suggests that the future deployment of epidemiological measures such as virological surveillance (obtaining more specimens), timely molecular analysis of viral isolates and their accompanying vaccine strains, and identification of vaccinemismatched strains would support public health efforts to minimize complications and deaths. we recommend that public health resources be allocated to include both pharmaceutical [18] and non-pharmaceutical interventions [19] for minimizing elderly deaths whenever vaccine-mismatched h3n2 viruses are dominant. daily syndromic surveillance data integrated with virological surveillance information and statistical methods for detecting abnormal signals and trends can provide timely information for identifying the occurrence of vaccine-mismatched or novel influenza viruses. these efforts can jump-start prevention at the initial phase of an epidemic, when there is a higher risk of humanto-human transmission (e.g., increased epidemic/pandemic potential of newly emerged influenza viruses). as outbreaks of emerging infectious diseases (eid) and novel influenza viruses continue to increase [20] , we believe our results will help countries have not affected by sars to evaluate the effectiveness of their preventive and/or control measures for reducing influenza disease burdens (including vaccination programs for the current influenza h1n1 pandemic in 2009-2010). in addition to determining the effectiveness of vaccine strains in a given flu season, variant epitopes on the surface of the virus may also result in varying immunological responses. certain epitope variants might be less effective at stimulating the development of b-cell humoral immunity or interfere with the ability of cytotoxic t-lymphocytes to recognize epitopes presented by hla class i proteins on the surface of infected cells [21] . analysis of data from pre-sars winters-before the initiation of public health intervention efforts prompted by sars-may provide more clues as to the impact of vaccine-mismatched influenza viruses on excess mortality. in addition, taiwan's vaccine-mismatched influenza viruses appeared prior to the introduction of who's recommended vaccine strain [22] . therefore, it is explainable why we observed higher excess p&i mortality during pre-sars vaccinemismatches than post-sars. the mismatched a (h3n2) fujian strain which appeared in 2002 and circulated for several months may have steadily increased the population's herd immunity leading up to 2004-2005. lower influenza-associated excess mortality during the winter of 2004-2005, particularly in comparison to 1999-2000, may also be attributed to a higher identity percentage between vaccine strains and circulating strains, a lower number of variations at epitope b, the development of herd immunity, and the post-sars effect. these two vaccinemismatching examples suggest that both quantitative and qualitative variations of amino acids, as well as the locations and epitopes involved, are important considerations when monitoring vaccine-mismatched influenza viruses. antigenic differences thus need to be identified efficiently by serological testing for isolated influenza viruses with high monthly/weekly excess p&i mortality [23] . our results suggest that timely identification of vaccinemismatched circulating influenza viruses and their antigenic variations is crucial for effective evidence-based public health planning and preparedness. from january 2001-october 2001, documented vaccine effectiveness (ve) was 53% for preventing pneumonia deaths (when b was mismatched) and 44% for preventing all-cause deaths among the elderly in taiwan [24] . matched vaccines reached a ve as high as 80% for preventing influenza among healthy adults [25] . this ve declined to 50% when vaccines mismatched with circulating influenza viruses. we can surmise from past data that the ve for mismatched vaccines would be even lower for elderly populations because of their weakened immune responses [26] . the variation of ve across different countries may be attributed to different age distributions, variable influenza vaccination coverage rates [27] , and variation in post-sars effects. these variations may account for the higher overall p&i mortality and excess p&i mortality rates observed in italy and u.s compared to taiwan. the 2003 sars outbreak posed a significant challenge to taiwan's health care system but also had the potentially beneficial effect of educating the public about the need for seeking health care earlier [28] and protective behaviors [29, 30] . public education measures and behavioral changes prompted by the sars outbreak may have contributed to the ensuing decline in excess p&i mortality in taiwan and other sars-affected countries/areas [29, 31] . although our observation periods were not long, the evidence suggests that the sars outbreak not only spurred behavioral change among taiwanese citizens [32] but also prompted government officials to reform the infectious disease surveillance system [33] as well as policies for hospital management [34] and infection control [35] in taiwan these changes, in combination with increasing awareness of infectious diseases among physicians, may have contributed to the sustained post-sars effect that we propose had a significant impact on taiwan's elderly p&i mortality rates. three studies in taiwan [36] , in wuhan city of hubei province in mainland china [30] and in hong kong [31] support the claim that public health efforts were sustained after the sars epidemic. interestingly, influenzaassociated mortalities in taiwan, southern china and hong kong in the post-sars period were all lower than in the pre-sars period (personal communication in the march 15-19, 2010 misms oceania regional influenza meeting and workshop in melbourne, australia). moreover, many public health measures taken in response to sars-including advising sick students to stay at home, teaching coughing etiquette, providing hand-cleaning facilities in front of elevators and at building entrances, closing classes if more than three influenza-like illnesses occur in one class, and vaccinating high-risk populations-all had been applied during the 2009 influenza h1n1 pandemic. the effectiveness of these public health efforts is supported by the lower total number of p&i mortalities observed during the 2009-2010 winter season (ending february 26, 2010) than in previous years [37] . in addition, we found that people had a greater understanding of health protection measures during the 2009-10 pandemic influenza in sars-affected areas/countries such as taiwan and hong kong [38] . this protective effect may have contributed to the lower numbers of p&i deaths in 2009-10 compared to seasonal influenza in 2008-2009 and to the reduction in total laboratory-confirmed pandemic influenza h1n1 deaths in taiwan (42 deaths from july 1, 2009 to may 8, 2010)) and hong kong (80 deaths from july 17, 2009 to april 15, 2010) [39] . this study has five major limitations. first, taiwan's monthly influenza virus isolation rates prior to 1999 were not comprehensive. second, weekly and monthly matching statuses were not available. third, the benefit of vaccination may be underestimated because older elderly populations are at higher risk of developing severe complications and deaths [40] . fourth, the unknown temporality of vaccinations and the presence of many possible uncontrolled confounders (such as variations in age-specific attack rates, prior accumulated immunity, socioeconomic conditions, nutrition factors, public health efforts, and viral characteristics of individual influenza viruses including infectivity, pathogenicity, transmissibility and virulence) could not be fully accounted for in this retrospective ecological study. causal effects cannot be determined with certainty from observational studies comparing these groups (e.g., vaccine-matched versus vaccine-mismatched groups or pre-sars versus post-sars groups in this study). additionally, uncontrolled confounders at the individual level are a major limitation of an ecological study design. fifth, accounting for epitope variations in a (h3n2) viruses will require more amino acid sequencing and serological data of the ha1 over the course of several years. enhanced virological surveillance in asia, where mostly past pandemic influenza viruses have originated [7, 41] , is urgently needed because most new wild-type influenza virus strains have appeared much earlier in taiwan and south-east asia [42] than in who-recommended vaccine strains [8, 22] . furthermore, viral changes and co-circulating subtypes/types have been documented in the later periods of influenza epidemics [43] . antigenic distance between the vaccine and circulating strains can be best measured by serologically testing simultaneously for vaccine strains and 20-30 local influenza isolates obtained from various time intervals of the same year (in which excess influenzaassociated mortality is identified). unfortunately, we did not have enough monthly retrospective samples to incorporate serological results into the model during the study period. the major limitation of this study was a lack of long-term data that prevents us from drawing definitive conclusions. however, we have illustrated possible associations between the observed reduction in p&i mortality and vaccine match on the one hand, and the post-sars effect on the other hand. our model is sufficiently flexible to apply to different scenarios in various countries. for minimizing a country's/global influenza disease burden, further studies will be required to validate the interpretations of our results that we have suggested. international collaboration on an integrated clinical, epidemiological, and virological/serological influenza surveillance system will be necessary to monitor for potential increases in clinical severity as well as viral sequence and antigenic changes in any parts of the world. this study points to a number of possible directions for improving influenza vaccination policy and provides a means for public health officials to monitor for possible occurrences of vaccine-mismatched influenza viruses at the population level. moreover, our study attempts to lay a foundation for a molecular explanation of influenza-associated deaths that integrates macrolevel mortality data with micro-level amino acid sequence identity percentage. we hope that our findings can prompt the discovery of better and more effective mechanisms for selecting strains for future serological testing. in the future, we hope to collect more data domestically and internationally in order to reevaluate and refine our recommendations. public health professionals in sarsaffected countries can also examine the post-sars impact and vaccine-mismatched effect using data sets from their countries. a concerted effort to obtain more evidence can bring the international community closer to devising more effective guidelines for the global control of next pandemic influenza. future research efforts should include: (1) weekly/daily monitoring of influenza viral sequences, antigenic changes of the ha [44] , and excess influenza-associated mortality; (2) an evaluation of vaccine efficacy through measurement of antigenic distances [45, 46] , and b-and t-cell epitopes [47] ; and (3) improvements in influenza vaccines through enhancement of innate immunity [48, 49, 50] . figure s5 the 3d structure of the three newly undefined epitopes of human influenza a (h3n2) viruses during the three vaccine-mismatched influenza seasons in taiwan, 1999 taiwan, -2007 prevention and control of influenza: recommendations of the advisory committee on immunization practices (acip) vaccines for preventing influenza in the elderly influenzarelated mortality in the italian elderly: no decline associated with increasing vaccination coverage mortality associated with influenza and respiratory syncytial virus in the united states impact of influenza vaccination on seasonal mortality in the us elderly population seasonal trends of viral respiratory tract infections in the tropics the global circulation of seasonal influenza a (h3n2) viruses influenza in taiwan: seasonality and vaccine strain match world health organization. who recommendations for influenza vaccine composition molecular characterization of the ha gene of influenza type b viruses simultaneous amino acid substitutions at antigenic sites drive influenza a hemagglutinin evolution cabinet expands mini links with mainland exploration of the emergence of the victoria lineage of influenza b virus increasing appearance of reassortant influenza b virus in taiwan from 2002 to process of standardized antiserum and antigen of human influenza viruses in taiwan mortality from pandemic a/h1n1 2009 influenza in england: public health surveillance study nonpharmaceutical interventions implemented by us cities during the 1918-1919 influenza pandemic global trends in emerging infectious diseases t-cell tolerance for variability in an hla class i-presented influenza a virus epitope laboratorybased surveillance and molecular epidemiology of influenza virus in taiwan mapping the antigenic and genetic evolution of influenza virus impact of influenza vaccination on major cause-specific mortality vaccines for preventing influenza in healthy adults antibody response to influenza vaccination in the elderly: a quantitative review international data base (idb) impact of sars on healthcare utilization by disease categories: implications for delivery of healthcare services factors influencing the wearing of facemasks to prevent the severe acute respiratory syndrome among adult chinese in hong kong severe acute respiratory syndrome epidemic and change of people's health behavior in china handwashing practice and the use of personal protective equipment among medical students after the sars epidemic in hong kong taipei's use of a multi-channel mass risk communication program to rapidly reverse an epidemic of highly communicable disease establishing a nationwide emergency department-based syndromic surveillance system for better public health responses in taiwan the reform of the hospital accreditation system in taiwan the impact of sars on hospital performance influenza express in taiwan -week 8 widespread public misconception in the early phase of the h1n1 influenza epidemic flu pandemic by country complications of viral influenza influenza vaccine strain selection and recent studies on the global migration of seasonal influenza viruses influenza vaccine strain selection and recent studies on the global migration of seasonal influenza viruses vaccination and antigenic drift in influenza influenza virus antigenic variation, host antibody production and new approach to control epidemics on the use of hemagglutinationinhibition for influenza surveillance: surveillance data are predictive of influenza vaccine effectiveness on the use of hemagglutination-inhibition for influenza surveillance: surveillance data are predictive of influenza vaccine effectiveness influenza virus ctl epitopes, remarkably conserved and remarkably variable influenza a viruses with truncated ns1 as modified live virus vaccines: pilot studies of safety and efficacy in horses live attenuated influenza viruses containing ns1 truncations as vaccine candidates against h5n1 highly pathogenic avian influenza influenza b virus ns1-truncated mutants: live-attenuated vaccine approach the authors appreciate the laboratory surveillance efforts of taiwan's virological surveillance contract laboratories and taiwan-cdc. we would also like to express our gratitude to ms. peggy lee, mr. andres su, and mr. james steed for their help in editing this paper in english. we are also grateful to dr. chi-tai fang for his critical viewpoints and the department of health -national taiwan university (ntu) infectious diseases research and education center for their administrative support. key: cord-302200-9gekjgr0 authors: kilich, eliz; dada, sara; francis, mark r.; tazare, john; chico, r. matthew; paterson, pauline; larson, heidi j. title: factors that influence vaccination decision-making among pregnant women: a systematic review and meta-analysis date: 2020-07-09 journal: plos one doi: 10.1371/journal.pone.0234827 sha: doc_id: 302200 cord_uid: 9gekjgr0 background: the most important factor influencing maternal vaccination uptake is healthcare professional (hcp) recommendation. however, where data are available, one-third of pregnant women remain unvaccinated despite receiving a recommendation. therefore, it is essential to understand the significance of other factors and distinguish between vaccines administered routinely and during outbreaks. this is the first systematic review and meta-analysis (prospero: crd 42019118299) to examine the strength of the relationships between identified factors and maternal vaccination uptake. methods: we searched medline, embase classic & embase, psycinfo, cinahl plus, web of science, ibss, lilacs, africawideinfo, imemr, and global health databases for studies reporting factors that influence maternal vaccination. we used random-effects models to calculate pooled odds ratios (or) of being vaccinated by vaccine type. findings: we screened 17,236 articles and identified 120 studies from 30 countries for inclusion. of these, 49 studies were eligible for meta-analysis. the odds of receiving a pertussis or influenza vaccination were ten to twelve-times higher among pregnant women who received a recommendation from hcps. during the 2009 influenza pandemic an hcp recommendation increased the odds of antenatal h1n1 vaccine uptake six times (or 6.76, 95% ci 3.12–14.64, i(2) = 92.00%). believing there was potential for vaccine-induced harm had a negative influence on seasonal (or 0.22, 95% ci 0.11–0.44 i(2) = 84.00%) and pandemic influenza vaccine uptake (or 0.16, 95% ci 0.09–0.29, i(2) = 89.48%), reducing the odds of being vaccinated five-fold. combined with our qualitative analysis the relationship between the belief in substantial disease risk and maternal seasonal and pandemic influenza vaccination uptake was limited. conclusions: the effect of an hcp recommendation during an outbreak, whilst still powerful, may be muted by other factors. this requires further research, particularly when vaccines are novel. public health campaigns which centre on the protectiveness and safety of a maternal vaccine rather than disease threat alone may prove beneficial. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 maternal vaccination aims to reduce maternal and neonatal morbidity and mortality caused by infection [1] . the world health organisation (who) recommends the inactivated influenza, tetanus-toxoid-containing vaccine (ttcv), and combined tetanus, diphtheria, and acellular pertussis (tdap) vaccines for pregnant women in settings where the disease burden is known [2] . historically, maternal tetanus vaccination was limited to areas of significant transmission. in areas where there is ongoing maternal to neonatal transmission of tetanus, two doses of ttcv (preferably tetanus-diphtheria) are recommended in pregnancy in addition to tdap or dtap (for pertussis) and seasonal influenza vaccines. [2] pertussis vaccination was limited to childhood, however the resurgence of pertussis during outbreaks that disproportionately affected younger infants led to national policy changes between 2011 and 2015 in countries such as the united kingdom and the united states, that introduced routine maternal pertussis vaccination. [2, 3] similarly, the widespread influenza immunisation programs during the 2009 h1n1 pandemic resulted in public health bodies particularly in europe, the united states and australia introducing guidance to implement recommendations for routine antenatal seasonal influenza vaccination during the subsequent decade. the united states healthy people 2020 campaign sets a target to achieve influenza vaccination coverage of 80% among pregnant women [4] . suboptimal maternal vaccination coverage (estimated between 0-70%) of seasonal influenza and pertussis vaccines globally represents a missed opportunity to improve maternal and neonatal health [3] [4] [5] [6] . understanding the features that contribute to reduced uptake of vaccines used in outbreaks is also of particular importance given the increased morbidity and mortality seen with infections contracted during the vulnerable period of pregnancy [7] . in the last decade, the world health organisation has declared multiple public health emergencies of international concern for diseases including outbreaks of the ebola virus (west africa, north kivu), zika virus, and the novel coronavirus (wuhan) [8, 9] . ebola and zika are known to cause significant morbidity and mortality if contracted during pregnancy, whereas the effect of the covid-19 is unknown [10] . a vaccination strategy has been developed for ebola, and vaccine research is underway for zika virus and covid-19. the concern of disease risk may be amplified during an outbreak, but concerns about using a novel vaccine may also be enhanced. it is important to identify factors that appear to affect antenatal vaccine uptake during routine use (pertussis and influenza) versus vaccinations recommended during an outbreak setting (h1n1 influenza) to help prepare for future outbreaks. understanding the influence of personal beliefs and experiences on maternal vaccination uptake is key to designing, testing and deploying interventions that are tailored to improve vaccine acceptance and coverage in routine and outbreak settings. researchers have investigated the underlying reasons for low coverage using surveys, focus group discussions, and indepth interviews to explore the perceptions and experiences of pregnant women. previous reviews have established a narrative of evidence that suggests a broad range of factors (vaccine cost, accessibility, maternal knowledge, social influences, context, healthcare professional (hcp) recommendation and the perception of risks and benefits) all contribute to vaccine uptake. consensus within the field and across four prior literature reviews indicate that receiving a recommendation from an hcp for vaccination is the most important factor in maternal decision-making, irrespective of geographic or social context [11] [12] [13] [14] [15] . in general, there is limited data on maternal vaccination uptake and records of hcp recommendations at a national level. however, for the united states of america (usa), which monitors antenatal seasonal influenza and pertussis vaccination coverage, data suggest approximately one-third of women who receive an hcp recommendation for the vaccine will choose to remain unvaccinated [5] . in 2018, the centers for disease control and prevention (cdc) found that 79.3% of pregnant participants received a recommendation or an offer for tdap vaccine, but 45.6% of them chose to remain unvaccinated [5] . for seasonal influenza, fewer women chose to vaccinate when recommended to do so; 81.1% received a recommendation or an offer yet 50.9% of pregnant women surveyed remained unvaccinated [5] . understanding why women remain unvaccinated despite an hcp recommendation is key. we also sought to discriminate factors that influence specific vaccines since seasonal influenza vaccination coverage is lower than other routine vaccines (tdap, tetanus) during pregnancy. prior literature and systematic reviews tend to characterize the factors influencing maternal vaccination decisions as either barriers or facilitators [11] [12] [13] [14] [15] . we sought to quantify the association between beliefs, attitudes and prior behaviours that influence maternal vaccination uptake. we selected the h1n1 influenza vaccine, deployed globally and recommended to pregnant women during the pandemic of 2009, to be included alongside our analysis of other who routinely recommended vaccines, the pertussis and seasonal influenza vaccine. thus, we performed a systematic review and meta-analysis of qualitative and quantitative literature to provide comprehensive evidence on the magnitude of effect that factors influence maternal vaccination decisions globally with the aim to inform policy makers, public health strategists and researchers involved in designing vaccine interventions to increase uptake. we conducted a systematic review of literature, unrestricted by language or location, to identify qualitative and quantitative studies that reported on the cognitive, psychological, and social factors associated with maternal vaccination among pregnant and recently pregnant women (within two years of birth). we searched medline, embase classic and embase, psycinfo, cinahl plus, web of science, ibss, lilacs, africawideinfo, imemr, and global health for studies published by 22 november 2018 (appendix p3-10 in s2 file). additional studies were identified by screening reference lists (ek, sd) of previous reviews and through suggestions by experts in the field. titles and abstracts were independently screened and agreed upon (ek, sd) for potential eligibility. a final arbiter (pp) resolved any conflicts of agreement on inclusion. we excluded pre-clinical research, behavioural intervention studies, and any research that exclusively examined experimental vaccines in pregnancy or sociodemographic variables (appendix p11-15 in s2 file). to be included in the meta-analysis, studies had to report an estimated odds ratio (or (or could be calculated from raw data)) for the association between a specific factor and vaccination status (excluding intention to be vaccinated). research groups from studies in which the data were unclear or had not been reported were contacted for clarification (appendix p51 in s2 file). the data from included studies were extracted (ek, sd) and input into microsoft excel (microsoft corporation, redmond, wa, usa) and a coding template was developed by authors to categorise factors influencing maternal vaccination uptake (expanded methodology appendix p18-19 in s2 file details why established frameworks were not used). coding into broad themes (e.g. accessibility and convenience) using grounded theory was completed independently (ek, sd) with nvivo 12 (qsr international, melbourne, australia) (inter-rater reliability kappa score 0.76). the quantitative studies were independently assessed (ek, mrf) for inclusion in the metaanalysis based on first cycle broad codes to capture data that could be synthesized (appendix p38-40 in s2 file). qualitative data underwent a second round of coding to identify specific patterns within the broad themes (inter-rater reliability kappa score 0.88). a third round of coding (subdividing the first cycle codes) was conducted to ensure that only data that was directly comparable were included in each meta-analysis (appendix p41 in s2 file). twentythree narrow definitions were agreed upon by two authors (ek, mrf) to ensure consistency of the data included (appendix p42-43 in s2 file). these definitions were used to pool studies for specific vaccines (seasonal influenza, pandemic influenza, and pertussis) generating 31 separate meta-analyses (ek, mrf). any discrepancies in data extraction were resolved by both authors. quality appraisal was performed (ek, sd) using checklists for cross-sectional, cohort, and qualitative research studies from the joanna briggs institute quality assessment tools (appendix p21-32 in s2 file). [16] studies were ranked based on a framework developed by authors with an attributed quality score. where authors disagreed on final point allocation, the arbiter (pp) intervened to resolve the disagreement. quality analysis was not used to define inclusion or exclusion. however, pre-specified sensitivity analyses were performed investigating the robustness of results to the inclusion of only high-quality studies (joanna briggs institute scores >10) (appendix: p90, p92 in s2 file). we wished to conduct a sensitivity analysis assessing the robustness of results by gross domestic product of countries included to assess the influence of geographic context. when two or more studies reported ors for a specific factor, random-effects models were used to calculate a summary or [17] with heterogeneity assessed with i 2 . funnel plots were used to examine the potential for publication bias. specific factors reported by only one study are summarised in the appendix (appendix p93 in s2 file). a secondary analysis was performed to assess factors associated with intention to be vaccinated during pregnancy. all meta-analyses were conducted in stata 15 (statacorp, college station, tx, usa) [18, 19] . the prisma checklist (preferred reporting items for systematic review and meta-analysis checklist) (appendix p16-17 in s2 file) [20] was used and the study was registered with prospero (international prospective register of systematic reviews) (crd42019118299). an expanded methodology can be found in the appendix (appendix p19-21 in s2 file). countries (appendix p33-37 in s2 file). the majority of studies were quantitative only (n = 99), then qualitative only (n = 18) with three studies using mixed methods (table 1) . studies were predominantly from the usa (39 studies), australia (22), and canada (9) . seasonal influenza vaccine was the most commonly investigated vaccine, either independently or as part of a study of factors influencing the uptake of multiple vaccines (63% of studies n = 75), followed by vaccines against pertussis (27% n = 32), pandemic influenza (24% n = 29), tetanus (8% n = 9), and antenatal vaccines generally (2% n = 2). we identified eight categories of factors that influence maternal vaccination across both qualitative and quantitative studies: accessibility and convenience (55 studies), personal values and lifestyle (43), awareness of information regarding the specific vaccine or disease of focus (90), social influences on vaccine use (109) , emotions related to vaccination (85), perceptions of vaccine risk (110) , perceptions of vaccine benefit (93), and personal vaccination history (80). from these eight categories, five could be synthesised quantitatively (appendix p40-41 in s2 file). results from all meta-analyses are presented in fig 2. no data for tetanus vaccination were suitable for meta-analysis. a list of the most common barriers or facilitators cited in studies excluded from meta-analysis is available in the appendix (appendix p52-53 in s2 file). from the 21 qualitative studies, we identified 30 sub-categories of factors that appear to influence maternal vaccination decision-making (table 2 ). for our primary analysis we conducted 33 meta-analyses which assessed the relationship between a specific belief or behaviour and maternal vaccination status (338-14,099 participants (average 2,955) included in each meta-analysis)) (appendix p55-89 in s2 file for individual meta-analyses and summary table) (fig 2) . for our secondary analysis we conducted 15 metaanalyses assessing the relationship between a specific belief or behaviour and maternal vaccination intentions, rather than prior behaviour (531-2,215 participants (average 1,344) included in each meta-analysis)) (appendix p91 in s2 file when the number of studies included in the meta-analysis exceeded seven, funnel plots were used to assess the potential for publication bias (appendix p54 in s2 file). although based on a small number of studies, there was incomplete agreement between the primary and secondary analyses (intention to be vaccinated meta-analysis results are provided in appendix p91-92 in s2 file). sensitivity analyses including studies with joanna briggs institute scores >10 were conducted for both vaccination status and intention to be vaccinated outcomes (appendix: p90, p92 in s2 file). whilst results were generally consistent, differences were difficult to interpret due to the low number of higher-quality studies. all qualitative studies reported on the perceived effect of hcp influence on decision-making, and to a lesser extent the influence of other social networks or the internet. often an offer (or lack of an offer) of vaccination during an antenatal visit was a key factor in final behaviour [58] [59] [60] . participants also expressed willingness to receive information from hcps, but were disappointed with a perceived overuse of leaflets to convey information in lieu of direct conversation with an hcp [58, 61, 62] . other studies reported that some pregnant women sought vaccination information through media or the internet, but these avenues were not regarded as the most reliable for accurate information [62] [63] [64] [65] [66] . almost all qualitative studies indicated that being aware of maternal vaccination and/or the respective disease, regardless of information source, was key to receiving the vaccine but rarely sufficient (17 studies in eight of the 17 qualitative studies that examined perceptions of disease severity, participants were unaware of the additional risks of influenza to pregnant women [56, 62, 64, 66-68, 74, 75] . qualitative studies also highlighted differences in participants' perceptions of severity for different diseases. for example, h1n1 or pandemic influenza was perceived as more severe than seasonal influenza [68, 74] . additionally, pertussis was correctly seen primarily to present danger to infants, whereas influenza was viewed as a significant risk to the pregnant woman [60] . whilst the disease risk was used as a contributing factor to final decision other factors were weighed against it. whilst factors such as convenience, personal values, and emotions related to vaccinations during pregnancy were not captured in our meta-analyses, they were highlighted in qualitative analyses. [64, 73] , and pain [52, 61, 77] . one study reported that vaccinated and unvaccinated pregnant women expressed similar fears, but unvaccinated women often described their fears in more detail [70] . the fear of perceived vaccine harms (including the ideas of unknown risks for novel vaccines) were used to explain the rejection of maternal vaccination despite a connected fear of the disease it was aimed to protect against [59, 64-68, 70-73, 75]. despite the challenges of synthesizing an extensive and varied body of research, we have been able to quantify the relative effect size for a large number of specific beliefs and behaviours around maternal vaccination uptake. prior attempts to weight factors influencing maternal vaccination uptake have largely been confined to ranking the most commonly cited barriers or facilitators within studies, listing the latter as predictors. this approach is likely to conflate several individual factors which are important to designing better interventions aimed at increasing maternal vaccination acceptance and uptake. our major finding is that vaccine-specific factors and previous vaccination behaviour have a strong influence on antenatal vaccine uptake. disease-related perceptions have a modest effect on final vaccination uptake. beliefs that vaccine would benefit the mother or cause no harm to the pregnancy were associated with four-to-nine-times greater odds of vaccination-acceptance during pregnancy. prior systematic reviews were unable to characterise the nature or strength of effect of vaccine safety concerns on maternal vaccination decisions. lutz et al. described that 2.9 to 77% of pregnant women had safety concerns for their foetuses [13] . wilson et al. reported that safety concerns were the most frequently cited barriers (64 of 155 studies) but the relationship of this concern to final vaccination uptake was not defined. the underlying vaccination status of pregnant women was unreported in this prior study, limiting the interpretation of this finding [11] . in our study, beliefs that vaccine could cause birth defects or general harm in pregnancy served as strong deterrents to both seasonal and pandemic influenza vaccination (seasonal or 0.22, 95% ci 0.11-0.44; pandemic or 0.11, 95% ci 0.06-0.22). similarly, perceptions of vaccine utility had a strong positive influence on uptake. for the seasonal influenza vaccine, perceiving the vaccine as beneficial in general was an important factor associated with pregnant women's vaccination status (or 7.22, 95% ci 3.49-14.93). for pandemic influenza vaccination, despite the wide confidence intervals, our data suggest that perceptions that vaccine can protect pregnant women (or 8.44 95% ci 2.90-24.61) is strongly associated with vaccine uptake. our study did not find clear evidence that a belief of susceptibility to pandemic or seasonal influenza was associated with increased maternal pandemic influenza vaccination (or 1.11, 95% ci 0.56-2.19) or seasonal influenza (or 1.76, 95% ci 1.26-2.47) vaccine uptake. there was some evidence to support an association between perceptions of the severity of pandemic influenza and pregnant women's vaccination status (or 2.04, 95% ci 0. 98-4.26 ) with the belief that pandemic influenza can result in hospitalisation increasing vaccine uptake threefold (or 2.91, 95% ci 2.02-4.18). we would recommend additional studies to explore the role of disease severity and susceptibility in greater detail to clarify their importance when other factors are present. for seasonal influenza, the data is inconclusive since women who believed that the disease could be harmful to their pregnancy or baby had four-times greater odds of being vaccinated than those who did not (or 3.70, 95% ci 1.37-9.94) yet there was no evidence to suggest belief in the risk of the disease generally (or 1.56, 95% ci 0.88-2.76) or its ability to result in hospitalisation (or 0.57, 95% ci 0.22-1.45) were related to vaccine uptake. this was mirrored by our qualitative research which indicated that the influence of a belief in the severity and susceptibility to a disease does not in isolation determine vaccination decision [56, 58, 59, [63] [64] [65] [66] [67] [68] [69] [70] [71] [73] [74] [75] [76] . this has important implications for public health communication strategies around maternal vaccination since campaigns, particularly during an epidemic or influenza outbreak, have centred around disease threat. based on our findings we caution any communication approach which highlights only the threat of disease when publicising vaccination. we suggest this requires further review of the messaging strategies comparing those with and without explicit details of vaccine safety to the public and/or a discussion of disease threat with attention to language which might inadvertently promote fear [78] . the global communication strategies during the h1n1 2009 pandemic have been widely criticised as lacking an evidence-base and not appropriately targeting specific vulnerable groups [79] . we suggest that future research investigates disease-focused communication strategies versus vaccine-centred communication when discussing maternal vaccination to help prepare for future pandemics. our study was conducted prior to the north kivu ebola vaccine deployment among pregnant women in 2019. this study precedes vaccine candidate deployment for sars-cov-2 with immediate implications for future studies analysing potential acceptance of a maternal vaccine and the associated communication strategy. this is an important area of investigation to analyse the factors that influence maternal uptake when the vaccine is still in an experimental part of outbreak control. whilst the analysis of purely experimental vaccines was outside the remit of this work, we suggest further investigation into assessing the importance of factors identified in this review (including fear-conflict, anxiety and specific safety concerns) and their influence on uptake of vaccine during its developmental phase at the time of an outbreak. our findings also have potential implications for future study design. many studies included in this systematic review and meta-analysis were designed using the framework of the health belief model [24-26, 28, 39, 40, 53, 54, 60, 62-64, 69, 73, 80-88] . in brief, the health belief model describes final vaccination acceptance or rejection based on the interacting beliefs of seriousness and susceptibility to the target disease of the vaccine, benefits of the intervention, and barriers in order to predict health behaviour. our study suggests that the model should be adapted to highlight the importance of the latter two categories in maternal vaccination behaviour predictions. consistent with the extensive body of evidence on this topic, an hcp recommendation for routine vaccinations (seasonal influenza and pertussis vaccination) was a very strong factor influencing maternal vaccine acceptance that is associated with ten-times greater odds of being vaccinated over those who did not receive an hcp recommendation (pertussis or 10.33, 95% ci 5.49-19.43; seasonal influenza or 12.02, 95% ci 6.80-21.44). although based on a small number of studies, our meta-analysis suggests that the influence of an hcp recommendation for pandemic influenza vaccination moderately-to-strongly influences uptake. pandemic vaccine uptake was closely related to prior vaccination behaviour. vaccination (with a different vaccine) during a prior pregnancy (or 9.12 95% ci 1.99-41.76) had a strong influence on pandemic vaccine uptake. interestingly, this did not appear as evident for receiving an antenatal seasonal influenza vaccine in a subsequent pregnancy (or 1.51, 95% ci 0. 71-3.24 ). this suggests a possibility that decision-making for seasonal influenza vaccines made in second and third pregnancies may not be consistent with the decisions in the first pregnancy [89] . whilst studies have often included a sample of second-or third-time mothers, there is less extensive evidence of temporal changes in decision-making factors for maternal vaccines. whilst a general awareness about maternal vaccination did not increase the odds greatly of pandemic vaccine uptake, it appeared key for routine antenatal vaccines. policy awareness was strongly associated with seasonal influenza vaccination uptake. national recommendations from the authoritative health bodies appear to carry weight in maternal vaccination decision-making at a population level [90] . this is important for the rollout of new maternal vaccines, as vaccinations not endorsed by national policy may be less accepted. publicising such policies could improve trust in maternal vaccination programmes and facilitate improved uptake. qualitative findings from focus group discussions and in-depth interviews were generally consistent with the quantitative results: an unambiguous recommendation from an hcp to vaccinate against seasonal influenza or pertussis is key to pregnant women being vaccinated [58-66, 68, 72, 73, 76, 77] . it is difficult to draw conclusions about which specific hcp (e.g. obstetrician, general practitioner, and midwife) or service provider (e.g. community or hospital-based practitioners) is the most influential. similar to the quantitative literature, qualitative studies have shown that a recommendation by an hcp was not always sufficient [26, 34, 36, 41, 45, 46, 62, 63, 67] . reasons for refusal despite hcp recommendation from the qualitative analysis provide insights into the effects of fear, mistrust, and a feeling of accountability [61, 63, 67, 73, 75] . in the face of uncertainty about a vaccine, a guarded state prevails despite concern for disease risk [61, 68, 71, 73, 76] . this was most notably captured by meharry et al. as, ". . .fear if i do (vaccinate), fear if i don't (vaccinate), and do nothing when i fear both" [73] . this analysis, combined with the finding of a very strong relationship between the belief of vaccine harm and reduced uptake indicates that the perceived risk of self-intervening (i.e. taking a vaccine) can be very powerful. this can overshadow the belief in environmental risk (e.g. contracting a severe disease). this suggests that mothers feel accountable for a perceived risk when choosing to vaccinate during pregnancy, which can result in inaction if the disease is also feared. whilst it is essential that pregnant women are informed about the risks of the disease in order to be appropriately consented, the manner in which this is communicated should be evaluated. it appears that, in some cases, fear may be counter-productive. this has been seen in childhood vaccines: if parents already fear the vaccine, making them fear the disease leads to decision conflict, and hesitancy [91]. by including qualitative analysis, we were also able to unravel specific, participant-driven concerns that ranged from possible adverse events such as narcolepsy, infertility, and autism spectrum disorder as well as pregnancy-related concerns such as suspected risks of miscarriage, preterm birth, and birth defects. since the non-pregnancy related health concerns occur rarely in the general population or require long-term follow up over decades, post-marketing surveillance studies are used to measure if there are any vaccine-related effects [92] . however, data on these specific concerns during pregnancy are often unavailable to general practitioners or midwives during counselling. we recommend that hcps are given ready access to clear and concise language on the safety of vaccines during pregnancy. the cdc has launched an extensive response to the relationship between antenatal vaccines and guillain barre syndrome, autism, febrile seizures and sudden infant death syndrome (sids) [93] [94] [95] [96] [97] [98] [99] [100] [101] [102] [103] . however, discussions on narcolepsy are made in reference to childhood rather than prenatal vaccination. additionally, there is limited availability of summarized reports for the public or general practitioners that synthesize the abundance of safety evidence on miscarriage, infertility, and birth defects. health bodies should make this widely generated safety evidence more accessible to the public and to hcps to facilitate uptake where concerns in practice arise [103, 104] . it is reassuring that our meta-analysis reinforces some of the existing evidence surrounding factors that influence maternal vaccine uptake. attempting to quantify an effect size adds a useful summary measure. however, our study has a number of limitations that potentially impact inferences drawn from these data. the evidence-base exploring the factors determining antenatal vaccination decisions is extensive but of mixed quality, and synthesis of the results was complicated by the contextual and methodological heterogeneity between studies. a particular challenge was synthesizing questionnaire data which used variable phrasings and posed different assortments of questions limiting the volume of data that could be synthesised. ninety-seven percent of quantitative studies pooled employed a cross-sectional design. we acknowledge that in some settings, where data are obtained using variable questionnaires, examining a different number of factors, pooling information may obtain inconsistent results. however, in practice, it is unclear how these differences are likely to influence the obtained summary estimates. this has highlighted the need for more standardised procedures for data collection and reporting for individual studies. this is of particular importance during outbreaks when research is delivered in a timely manner. whilst we intended to compare results from the meta-analyses with vaccination status and intention to be vaccinated as outcomes, data were insufficient to draw meaningful comparisons. all the data for vaccine intention is found in the appendix 23 p91 in s2 file. this is important since the majority of data available for pertussis vaccination in pregnancy focused on beliefs associated with vaccination intentions only [81, 88, 105, 106] . whilst previous literature has shown intention to vaccinate can be a proxy for actual vaccination status, this may not always be the case with maternal vaccination and additional research is needed [68, 107] . our findings were largely consistent across countries; however, we recognize that the majority of data come from high-income settings where national vaccine policies exist and, therefore, the generalisability of these findings may be limited. we were unable to conduct a sensitivity analysis to stratify results by gross domestic product of each country as there were too few studies included in each meta-analysis. additionally, data from many studies relied on self-reported vaccination status and did not verify medical records or vaccine registry data which may have introduced a recall bias (reflected in the jbi quality assessment scoring). however, previous research in the field has indicated that this bias is unlikely to be substantial [108, 109] . a number of studies recruited participants using purposive or convenience sampling (table 1) . thus, we applied the joanna briggs institute (jbi) critical appraisal tools (jbi) to assess potential biases among individual studies. based on our results, we then performed a pre-defined sensitivity analysis (of high quality/low quality) presented in the appendices (appendix 22, appendix 24 p90, p92 in s2 file). we were unable to detect an influenced study quality on our pooled analyses. a final limitation is that the exposures in our meta-analyses were dichotomized, whereas in reality beliefs exist on a spectrum. vaccine refusal is undoubtedly multifactorial. however, our study has demonstrated that factors specific to the vaccine, perhaps more so than the disease are highly influential. interventions recommended to improve maternal vaccination uptake 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during the antenatal periods in cavan monaghan general hospital pandemic (h1n1) 2009 influenza vaccine uptake in pregnant women entering the 2010 influenza season in western australia influenza and pertussis vaccination in pregnancy: portrayal in online media articles and perceptions of pregnant women and healthcare professionals. vaccine determinants of influenza and pertussis vaccination uptake in pregnancy: a multicenter questionnaire study of pregnant women and healthcare professionals pregnant women's intention to take up a post-partum pertussis vaccine, and their willingness to take up the vaccine while pregnant: a cross sectional survey acceptability of tetanus toxoid vaccine by pregnant women in two health centres in abidjan (ivory coast) pregnant women's knowledge of influenza and the use and safety of the influenza vaccine during pregnancy survey of attitude toward immunization of h1n1 influenza a vaccine of pregnant women in guangzhou key: cord-292396-8w06oc5i authors: leger, thomas; jacquier, alexis; barral, pierre-antoine; castelli, maxime; finance, julie; lagier, jean-christophe; million, matthieu; parola, philippe; brouqui, philippe; raoult, didier; bartoli, axel; gaubert, jean-yves; habert, paul title: low-dose chest ct for diagnosing and assessing the extent of lung involvement of sars-cov-2 pneumonia using a semi quantitative score date: 2020-11-03 journal: plos one doi: 10.1371/journal.pone.0241407 sha: doc_id: 292396 cord_uid: 8w06oc5i objectives: the purpose is to assess the ability of low-dose ct (ldct) to determine lung involvement in sars-cov-2 pneumonia and to describe a covid19-ldct severity score. materials and methods: patients with sars-cov-2 infection confirmed by rt-pcr were retrospectively analysed. clinical data, the national early warning score (news) and imaging features were recorded. lung features included ground-glass opacities (ggo), areas of consolidation and crazy paving patterns. the covid19-ldct score was calculated by summing the score of each segment from 0 (no involvement) to 10 (severe impairment). univariate analysis was performed to explore predictive factor of high covid19-ldct score. the nonparametric mann-whitney test was used to compare groups and a spearman correlation used with p<0.05 for significance. results: eighty patients with positive rt-pcr were analysed. the mean age was 55 years ± 16, with 42 males (53%). the most frequent symptoms were fever (60/80, 75%) and cough (59/80, 74%), the mean news was 1.7±2.3. all ldct could be analysed and 23/80 (28%) were normal. the major imaging finding was ggos in 56 cases (67%). the covid19-ldct score (mean value = 19±29) was correlated with news (r = 0.48, p<0.0001). no symptoms were risk factor to have pulmonary involvement. univariate analysis shown that dyspnea, high respiratory rate, hypertension and diabetes are associated to a covid19-ldct score superior to 50. conclusions: covid19-ldct score did correlate with news. it was significantly different in the clinical low-risk and high-risk groups. further work is needed to validate the covid19-ldct score against patient prognosis. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the outbreak of severe acute respiratory syndrome coronavirus 2 (sars-cov-2), that began in wuhan, china, spread worldwide, and is now in the ascending phase of the epidemic in europe [1] . sars-cov-2 is responsible for coronavirus disease 2019 (covid-19) pneumonia. respiratory involvement has a wide variety of clinical features, ranging from simple nasal congestion to pulmonary failure [2] . moreover, many patients remain asymptomatic but a vector of the disease, allowing the epidemic to spread easily. the main problem of covid-19 pneumonia is the risk of saturation of our health system due to an uncommonly massive inflow of patients requiring intensive care. patient selection is therefore essential in order for health care practitioners to focus on patients with comorbidities and on severe cases. this selection is currently essentially based on clinical criteria. reverse-transcription polymerase chain reaction (rt-pcr) is currently the standard of reference for diagnosis of covid 19 pneumonia. chest x-ray might be replaced by low-dose computed tomography (ldct) in covid-19 to assess lung involvement. thus, computed tomography (ct) has an important role in the management of patients, both for early screening and diagnosis and for establishing disease severity [3] [4] [5] . covid-19 pneumonia lung lesions are now well characterized: they are typically asymmetric, with ground-glass opacity (ggo) lesions or, less commonly, pulmonary areas of consolidation, they have a peripheral distribution, and they preferentially affect the lower territories [6] . it seems that there is a continuum in the lung lesions visible on ct scans, first with ggo lesions; then reticulations appear with crazy-paving patterns, parenchymatous areas of consolidation and finally, healing [7, 8] . the objective is to evaluate the ability of (ldct) to analyze well-known imaging abnormalities as well as to establish a covid19-ldct score reflecting disease severity and correlate it with clinical risk scores to allow better selection and follow-up of patients. the first 80 patients with a diagnosis of covid-19 pneumonia confirmed by rt-pcr method [9] were included in this single-centre retrospective study conducted from the 13 th to the 20 th of march 2020. the inclusion criteria and patient enrolment included all consecutive patients presenting to the department of infectious disease for 12 consecutive days with a diagnosis of covid-19 confirmed by rt-pcr. all patients underwent ldct. the protocol was approved by the local institutional review board: institut hospitalo-universitaire méditéranée-infection n˚:2020-0012. the exclusion criteria was protocol refusal. for each patient, the following clinical parameters were recorded: age, sex, date of first symptoms, date of chest ct scan, delay between the first symptom and chest ct scan, fever, cough, dyspnea, diarrhea, myalgia, rhinorrhea, abnormalities at lung auscultation, temperature, heart rate, blood pressure, respiratory rate, oxygen saturation, and oxygen needed. medical history parameters were recorded as follows: heart disease, tobacco use, copd, asthma, diabetes, obesity, sleep apnea syndrome, oncologic status and immunosuppression status. the national early warning score (news) was calculated for each patient using respiratory rate, oxygen saturation, supplemental oxygen needed, temperature, systolic blood pressure, heart rate, and avpu score, as described previously [10] . the news determines the degree of illness of a patient and prompts critical care intervention. according to previously published data, patients were divided into the following two groups based on the news in order to compare the radiological data in these populations: low news (news � 4) and high news (news > 4) groups [10] . all departments had to be reorganized in response to the increase in covid-19 pneumonia, and ct scans were dedicated for this activity with a specific patient circuit. technicians and workers in contact with covid19 patients took all preventative means to protect themselves against transmission of the virus following the local institutional recommendations. a video was performed in our department to highlight the work of radiology technicians and available on https://youtu.be/mi-l_zrl__u. all patients underwent ldct on the same system (revolution evo-ge healthcare). all ldct scans were unenhanced in profound and maximal inspiration with the following parameters: detector collimation: 0.625 mm; field of view: 500 mm; matrix: 512x512; pitch: 1.375; gantry speed 0.35s; 120 kv; 45 mas; and reconstructed slice thickness 1.2mm. all imaging data were reconstructed using high resolution and standard algorithms. ldct data were sent directly to a picture archiving and communicating system. monitors were used to view both mediastinal (width, 400 hu; level, 20 hu) and lung (width, 1500 hu; level, -2700 hu) windows. the pre-established top anatomic border was the lower part of the neck. the preestablished anatomic bottom boundary was the estimated location of the adrenal glands below the costophrenic angle. all ldct scans were analysed by a single chest radiologist with more than 25 years of experience in chest imaging (jyg). all abnormalities were described according to the fleischner glossary [11] . the main features encountered during sars-cov-2 have been described elsewhere as ggos, areas of consolidation, crazy paving patterns, and extension of these lesions might evolve dramatically to acute respiratory distress syndrome (ards). the type and distribution of the lesions were analysed [12, 13] . ldct scores were used to quantify the extent of lung abnormalities. the score was obtained by summing the notes attributed to each segment. the extent of the lesions of covid-19 pneumonia was visually classified into 4 types for each segment: lack of lesions and minimal, intermediate and severe involvement. lack of involvement was defined as a normal pattern and was equivalent to 0. minimal involvement was defined as the presence of maximum 10 secondary lobules of any features and was equivalent to 1. intermediate involvement was defined as less than 50% involvement of the segment by any features and was equivalent to 4. severe involvement was defined as more than 50% involvement of the segment by any feature and was equivalent to 10. the score was obtained by summing the score of all segments for the right and left lungs, with the result ranking between 0 corresponding to a normal chest ct and 200 if all segments had involvement of more than 50% of their volume (fig 1) . chest-ct was considered with high impairment if the global score was superior to 50, we compared two groups to assess if clinical data could be predictor of severe involvement of lung parenchyma. virological diagnosis of sars-cov-2 infection was performed using a sample nasopharyngeal swabs with an hydrolysis probe-based real-time reverse transcription-pcr system that targets the envelope (e) protein-encoding gene [9, 14] . quantitative data were expressed as mean, standard deviation and range. qualitative data were expressed raw numbers, proportions and percentages. the differences between the variables were compared by the chi 2 or fischer test for qualitative variables and by u mann-whitney for quantitative variables. the relationship between quantitative parameters was examined with spearman correlation test. binary logistic regression analysis was used to test the relationships between covid-ldct score and clinically relevant variables. a value of p <0.05 indicated a statistically significant difference. statistical analysis was performed on xlstat v19.1 (addinsoft, new york, usa). between the 13 th and the 20 th of march 2020 the first 80 patients with positive rt-pcr results for covid-19 were enrolled in a single center. all data could be recorded for all patients. the mean age of the population was 55 years ± 16, with 42 males and 38 females (47%). for 75 of the patients (94%), the medical consultation was carried out following a symptom; for the others, it followed close contact with an affected case. initially the most frequent symptoms were fever in 60 patients (75%) and cough in 59 patients (74%). myalgia, rhinorrhea, dyspnea and diarrhea were less frequent and were found in 35 (44%), 18 (23%), 11 (14%) and 11 (14%) patients, respectively. lung auscultation abnormalities were found in only 14 patients (18%). five patients needed resuscitation for an ards 5/80 (6%). at the first examination before ldct, the mean temperature was 37.5˚c ± 0.9, heart rate was 83 bpm ± 13, systolic blood pressure was 134 mmhg ± 21, diastolic blood pressure was 76 mmhg ± 14, respiratory rate was 19/min ± 5 and oxygen saturation was 97% ± 2. the mean news was 1.7 ± 2.3 ( table 1) . the delay between the first symptom and the ldct scan was 7 ± 4 days. the dose-length product mean was 41.7 mgy.cm ± 15.5. all patients could have their images analysed 80/80 low-dose chest ct scoring for sars-cov-2 pneumonia (100%). on ldct 23 patients showed no abnormalities (28%), 56 patients demonstrated ggos (67%), 21 patients had areas of consolidation (25%). an exclusive peripheral distribution was found in 33 patients (40%). only 9 patients showed crazy paving patterns (11%) (fig 2) . the mean covid19-ldct score was 19 ± 28 with a mean right lung score of 10 ± 15 and a mean left lung score of 8 ± 15 (table 2) . a significant correlation was found between the low-dose chest ct scoring for sars-cov-2 pneumonia news and the covid19-ldct score (r = 0.48, p<0.001). the correlation was also significant for the right lung and the left lung (fig 3) . no clinical symptoms were significant risk factors to find abnormalities on ldct and especially cough was not a relevant clinical symptom to determine if patients had risk to have lung involvement on ldct. mean pulse oxymetry show a tendency to decrease in patient with pneumonia on ldct than without pneumonia: 97.6% ± 1. (fig 4) . sixty-nine patients were classified as having low news, and 11 patients had high news. the covid-ldct score was 13 ± 19 in the low-news group and was significantly higher in the high-news group (52 ± 52; p = 0.001). there was no significant difference between groups according to their symptoms or blood pressure at the time of admittance. patients with a news > 4 had a significantly higher temperature, heart rate, and respiratory rate and a lower oxygen saturation than patients with a low news (38.3˚c ± 1.0 versus 37.3˚c ± 0.8; p < 0.0001, 95 bpm ± 8 versus 81 bpm ± 12; p<0.0001, 25/min ± 9 versus 18/min ± 4; p < 0.0001, 94% ± 2 versus 98% ± 2; p < 0.0001; respectively). oxygen was needed in 7 of 11 patients (64%) in the high-news group and in 3 of 69 patients (7%) in the low-news group ( table 3 ). the clinical factors associated with high covid19-ldct score in univariable analysis were diabetes (or = 6.9 [1.32-36.2], p = 0.02), the main findings of the presented work are that 1) ldct scans can depict the typical features of sars-cov-2 pneumonia with limited irradiation; and 2) the covid19-ldct score is correlated with the news used routinely to assess disease severity and patient prognosis. https://doi.org/10.1371/journal.pone.0241407.g004 table 3 . low-and high-risk groups. unenhanced chest ct was performed using a low-dose acquisition protocol. all the ct scans performed allowed optimal analysis of parenchyma involvement. recently, recommendations have been made regarding the use of ldct for covid19 that indicate preference for ldct [15] . they reported a mean dose-length product of 14.4 mgy.cm. a patient with covid-19 pneumonia could have 3 to 6 chest ct scans in a short period of time, and a healthy patient could have 1 or 2 chest ct scans to ensure that they did not have covid-19 [16] . irradiation awareness is needed, especially for younger patients [17] . the present study showed that ct image quality using ldct is sufficient for depicting all the typical features of covid-19 pneumopathy. acquisition parameters settings should be adapted to each patient to maintain good contrast-to-noise ratio and signal-to-noise ratio, allowing adequate quality of the lung parenchyma images [18] . debray et al. have already shown in patients with lung transplantation that the use of ldct could be interesting to decrease global irradiation because of repeated imaging for follow-up [19] . however, in patients with covid-19 an enhanced chest ct scan might be required for patients with clinical worsening considering an over-risk of pulmonary embolism caused by inflammatory syndrome and bed rest. as recently described, the clinico-radiological correlation is good, and diffuse lung involvement is frequently observed in severe forms [20, 21] . lesions extent is higher in patients with a need for intensive care units than for ordinary units. in particular, lymph nodes and pleural effusion are highly frequent in severe patients. the present paper is in favor of a correlation between disease extent and clinical severity. there are some patients with a rt-pcr positive result and a normal chest ct 23 of 80 (28%), that is not consistent with the whole studies on ct scan sensitivity. we explained that because patients came from a screening center and some patients are tested because they are contact case with infected patients. the sensitivity of the ct scan depends on the level of pre-test risk of the population to present lung involvement. disappointingly, we did not highlight any symptoms that could predict pulmonary involvement, particularly cough, which is generally one of the main symptoms of manifestations of alveolar involvement. it is in line with a recent publication from yang et al. showing that patients could be covid-19 infected and had few or no clinical symptoms with a normal chest ct and/or negative rt-pcr test [22] . the covid19-ldct score may provide the quantitative measurement of covid-19 injury. patients can be easily classified in various degrees of the disease. this type of ct score was first described in 2004 for severe acute respiratory syndrome (sars). investigators divided each lung into 3 parts (upper, middle and lower) and evaluated the involvement on a scale of 0-4: 0, <25%, 25-50 >50% and >75% involvement: the maximum possible score was 24 [23] . the present approach uses the simplest scale, comprising 3 kinds of impairment: minimal, intermediate, and severe. it focuses on the anatomical distribution of lesions based on segmental analysis. this scoring system is based on the decreased in lung function applayed in thoracic surgery. it has been shown that wedge resection spare parenchyma loss more than segmentectomy and segmentectomy more than lobectomy [24] . we guess that a minimal involvement will have only few consequences on hematosis and more than 50% of segment involvement seems lead to a total shunt effect and we grade it 10. the score over 50/200 had been arbitrary chosen for severe involvement because that mean involvement of 50% of the lung corresponding to a lobectomy. patients with comorbidities such as emphysema or former thoracic surgery have a higher risk of death in case of lobectomy [25] . we considered that a score > 50/200 could lead to death according to the cormobidities and need to classify patients in the severe group. there are a few limitations in the present study. our population was mainly paucisymptomatic patients which did not allow us to have a wide repartition of news and ct scores for correlation, especially for high scores. this could be explained because we enrolled the 80 first patients with covid-19 pneumonia in our centre and the severe forms are not the most frequent. another limit is the low number of patient that does not allowed us to perform a multivariate analysis and further studies are needed to establish this score a clinical prognosis tool. low-dose ct scan could depict the typical features of covid-19 pneumonia. in this population of 80 covid-19 patients detected positive by rt-pcr, there was a significant correlation between the clinical news and the covid19-ldct score, showing the close relationship between signs and the extent of disease in the lung. more work is required to assess whether this score could add clinically relevant information to assess the prognosis of patients with covid-19 pneumonia. supporting information s1 file. irb. (pdf) s1 data. (xlsx) a novel coronavirus emerging in china-key questions for impact assessment clinical features of patients infected with 2019 novel coronavirus in wuhan, china correlation of chest ct and rt-pcr testing in coronavirus disease 2019 (covid-19) in china: a report of 1014 cases a patient with covid-19 presenting a false-negative reverse transcriptase polymerase chain reaction result sensitivity of chest ct for covid-19: comparison to rt-pcr ct imaging features of 2019 novel coronavirus (2019-ncov) clinical and high-resolution ct features of the covid-19 infection: comparison of the initial and follow-up changes temporal changes of ct findings in 90 patients with covid-19 pneumonia: a longitudinal study rapid viral diagnosis and ambulatory management of suspected covid-19 cases presenting at the infectious diseases referral hospital the ability of the national early warning score (news) to discriminate patients at risk of early cardiac arrest, unanticipated intensive care unit admission, and death fleischner society: glossary of terms for thoracic imaging clinical features and chest ct manifestations of coronavirus disease 2019 (covid-19) in a single chest ct manifestations of new coronavirus disease 2019 (covid-19): a pictorial review testing the repatriated for sars-cov2: should laboratory-based quarantine replace traditional quarantine recommendation of low-dose ct in the detection and management of covid-2019 the progression of computed tomographic (ct) images in patients with coronavirus disease (covid-19) pneumonia: the ct progression of covid-19 pneumonia the effect of long-term x-ray exposure on human lymphocyte optimization of acquisition parameters for reduced-dose thoracic ct: a phantom study diagnostic accuracy of low-ma chest ct reconstructed with model based iterative reconstruction in the detection of early pleuropulmonary complications following a lung transplantation relation between chest ct findings and clinical conditions of coronavirus disease (covid-19) pneumonia: a multicenter study the clinical and chest ct features associated with severe and critical covid-19 pneumonia patients with rt-pcr confirmed covid-19 and normal chest ct | radiology severe acute respiratory syndrome: temporal lung changes at thin-section ct in 30 patients lobectomy versus segmentectomy and wedge resection in the treatment of stage i non-small cell lung cancer risk factors and cancer recurrence associated with postoperative complications after thoracoscopic lobectomy for clinical stage i non-small cell lung cancer. thorac cancer we would like to thank all the technicians for their professionalism during this crisis. thanks to health managers to the optimal organization of workflow in the department. conceptualization: alexis jacquier, jean-yves gaubert. key: cord-305900-ht7hb2rc authors: van den brand, judith m. a.; stittelaar, koert j.; van amerongen, geert; reperant, leslie; de waal, leon; osterhaus, albert d. m. e.; kuiken, thijs title: comparison of temporal and spatial dynamics of seasonal h3n2, pandemic h1n1 and highly pathogenic avian influenza h5n1 virus infections in ferrets date: 2012-08-08 journal: plos one doi: 10.1371/journal.pone.0042343 sha: doc_id: 305900 cord_uid: ht7hb2rc humans may be infected by different influenza a viruses—seasonal, pandemic, and zoonotic—which differ in presentation from mild upper respiratory tract disease to severe and sometimes fatal pneumonia with extra-respiratory spread. differences in spatial and temporal dynamics of these infections are poorly understood. therefore, we inoculated ferrets with seasonal h3n2, pandemic h1n1 (ph1n1), and highly pathogenic avian h5n1 influenza virus and performed detailed virological and pathological analyses at time points from 0.5 to 14 days post inoculation (dpi), as well as describing clinical signs and hematological parameters. h3n2 infection was restricted to the nose and peaked at 1 dpi. ph1n1 infection also peaked at 1 dpi, but occurred at similar levels throughout the respiratory tract. h5n1 infection occurred predominantly in the alveoli, where it peaked for a longer period, from 1 to 3 dpi. the associated lesions followed the same spatial distribution as virus infection, but their severity peaked between 1 and 6 days later. neutrophil and monocyte counts in peripheral blood correlated with inflammatory cell influx in the alveoli. of the different parameters used to measure lower respiratory tract disease, relative lung weight and affected lung tissue allowed the best quantitative distinction between the virus groups. there was extra-respiratory spread to more tissues—including the central nervous system—for h5n1 infection than for ph1n1 infection, and to none for h3n2 infection. this study shows that seasonal, pandemic, and zoonotic influenza viruses differ strongly in the spatial and temporal dynamics of infection in the respiratory tract and extra-respiratory tissues of ferrets. humans may be infected with different categories of influenza a virus-seasonal, pandemic, and zoonotic-each with their own epidemiology and pathogenesis. seasonal influenza viruses cause annual epidemics during autumn and winter in temperate regions. they predominantly cause upper respiratory tract disease with rare extension to the lower respiratory tract, resulting in severe and even fatal pneumonia [1] . pandemic influenza viruses, like the pandemic h1n1 (ph1n1) virus in 2009, cause sporadic pandemics with variable mortality. in fatal cases of ph1n1 infection, virus antigen expression occurred throughout the respiratory tract and was associated with both upper and lower respiratory tract disease [2] . zoonotic influenza viruses, such as highly pathogenic avian influenza (hpai) h5n1 virus, are sporadically transmitted from poultry and other animals to humans, but do not transmit efficiently from human to human [3] . human infection with hpai h5n1 virus involves primarily the lower respiratory tract, resulting in diffuse alveolar damage (dad) and a fatality rate of almost 60% in confirmed cases [4] . the pathogenesis of both human and avian influenza virus infections in ferrets resembles that in humans [5] . in part, this is because the distribution of receptors for human and avian influenza viruses in the respiratory tract of ferrets is similar to that in humans [6] . therefore, the ferret is often used in animal models to study the pathogenesis of different influenza virus strains and to evaluate the efficacy of vaccines and antiviral agents against influenza [7, 8, 9, 10] . in these studies, it is critical to collect respiratory tract samples for virological, pathological, and molecular analyses at both the appropriate time point after infection and the appropriate location along the respiratory tract. this is because influenza virus infection is a highly dynamic process, both temporally and spatially. we recently compared the pathogenesis of infections with seasonal human h1n1, ph1n1, and hpai h5n1 virus in ferrets [9] . our results showed that, at 4 days post inoculation (dpi), ph1n1 caused pneumonia intermediate in severity between that caused by seasonal h1n1 and hpai h5n1. this was associated with virus replication throughout the lower respiratory tract for ph1n1, while seasonal h1n1 replicated mainly in the bronchi, and hpai h5n1 replicated mainly in the alveoli. however, the location of virus replication and extent and severity of associated pathological changes were recorded at a single time point. other experiments have used multiple time points (usually 1, 3, 5, and 14 dpi) to study the dynamics of influenza virus infection in the ferret respiratory tract [10, 11, 12, 13, 14, 15, 16] ; however, these experiments often lacked detailed pathological or virological analyses of samples collected along the full length of the respiratory tract on all time points. the goal of our study was to describe and compare the temporal and spatial dynamics of different influenza virus infections and associated pathology in the respiratory tract of the ferret. to this end, we inoculated ferrets with either seasonal human h3n2, ph1n1, or hpai h5n1 virus, and performed detailed virological and pathological analyses at time points from 0.5 to 14 dpi, as well as measuring virus excretion, clinical signs, and hematological parameters. additionally, we compared the results of histopathological analyses with digital microscopical scoring of tissue sections. clinical data and gross pathology. the survival rate was 100% in all groups. clinical signs (table 1) were mild with sneezing from 2 to 14 dpi and nasal discharge from 2 to 3 dpi, as shown by increased licking on the nose. nasal and pharyngeal swabs revealed excretion of virus from 0.5 to 4 dpi with a peak on 1 dpi in the nasal swabs (figure 1a and b). the mean body weight loss was around 10% (figure 1d ). on gross pathology, a few dark red and raised areas were seen in the lungs of some animals and were consistent with mild pulmonary consolidation. estimated areas of lung affected ranged from 0 to 10% (figure 2a ) between 0.5 and 14 dpi. the relative lung weight was comparable to that of non-infected ferrets (figure 2b ). there was a mild splenomegaly from 0.5 to 14 dpi (data not shown). the trachea-bronchial lymph nodes were slightly enlarged between 0.5 and 4 dpi, with a peak on 2 dpi (table 1) . histopathology and virus antigen expression. by histopathology, there was a mild to severe multifocal rhinitis with necrosis of the epithelium and mild multifocal tracheitis. in the lungs, there was a mild multifocal bronchitis and bronchiolitis with intra-epithelial neutrophils, mild peribronchiolar and perivascular cuffing, mild broncho-adenitis, and mild multifocal alveolitis with mild intra-epithelial infiltration of neutrophils with mild epithelial necrosis and mild edema in the alveolar lumina. by immunohistochemistry, influenza virus antigen expression was visible as diffuse to granular red staining, which usually was stronger in the nucleus than in the cytoplasm ( figures 3, 4, 5, 6, 7, 8) . antigen expression in the nose was present from 0.5 to 4 dpi, while no antigen expression was seen in the tracheal, bronchial, bronchiolar, and tracheo-bronchial glandular epithelium at any time point, and little antigen expression in few type ii pneumocytes in the alveoli. changes in the histological lesions and antigen lowest titers for h5n1. (b) virus titers in pharyngeal swabs demonstrate initially comparable virus titers for ph1n1 and h3n2 and lower titers for h5n1, and on 4 dpi comparable titers for ph1n1 and h5n1 and lower titers for h3n2. (c) body temperatures in ferrets inoculated with ph1n1 and h5n1 are higher than for h3n2 and the sham-inoculated group. the large sem for h5n1 is due to the low body temperatures of moribund animals. (d) body weight loss in animals inoculated with ph1n1 and h5n1 was comparable and higher when compared to h3n2. doi:10.1371/journal.pone.0042343.g001 expression over time are described in the supporting information (supporting information s1). semiquantitative histological scoring (table 2) showed that the extent and severity of the alveolar lesions were comparable for all days of sampling with no obvious changes. no expression of virus antigen was seen in extra-respiratory tissues on any days. by digital scoring, there was on average 63 to 73% of air in the pulmonary tissue (table 3) . additionally, the percentage of antigen-expressing tissue and counts of virus antigen expression were absent and negligible, respectively ( table 3) . virology of tissues. comparable with the pattern of antigen expression in the respiratory tissues, high virus titers were seen in the nasal concha from 0.5 to 4 dpi with a peak on 1 dpi (figure 3) . low virus titers were present in the trachea, bronchi, lungs, tracheo-bronchial lymph nodes and tonsil ( figure 3 and table 4 ). all other tissues did not contain replication competent virus. hematology and comparison of leucocytes in blood and alveolar lumina. total leucocyte counts after infection were slightly increased on all days except for 14 dpi compared to those in negative control animals (table 5 ). in blood, there was a slight increase in the number of mononuclear cells on 1 dpi and of neutrophils on 2 dpi, followed by a mild decrease up to 4 dpi after which again a small increase of both was observed ( figure 9 ). in the alveolar lumina, the number of mononuclear cells was only slightly increased on 2 dpi ( figure 8 ). generalized linear model of viral excretion. we used generalized linear models (glm) to define possible predictors of viral excretion, based on viral production and damage scores in upper (nose and trachea) and deeper regions of the respiratory tract (bronchi, bronchioles and alveoli). these models were used to determine significant linear relationships between these scores and viral excretion (as measured by viral titers in nose and throat swabs; see methods). the final glm predicting excretion of h3n2 included only viral production in the upper regions of the respiratory tract as a significant predictor (lr x 2 = 16.8, df = 1, p,0.0001). excretion of h3n2 was a positive linear function of viral production in the upper regions ( table 6 ). as such, viral excretion of h3n2 could be directly estimated based on viral production scores in the nose and trachea, strongly suggesting that these regions were the main sources of excreted virus. clinical data and gross pathology. the survival rate was 100%. clinical signs per group were observed from 2 to 14 dpi ( table 1) . the activity status varied over the different time points with the highest score (status 3) seen in one animal between 5 and 8 dpi. dyspnea was seen only in this animal. inappetence, sneezing and nasal discharge was seen in all animals. virus excretion was shown in nasal and pharyngeal swabs with a peak on 1 dpi and higher values in the nasal swabs. the virus titers for the pharyngeal swabs were comparable to those of the animals inoculated with h3n2 ( figure 1b ). an increase in body temperature was seen with a peak on 1 dpi (figure 1c ). the mean body weight loss was up to 15% on 7 dpi (figure 1d ). by gross pathology, multifocal pulmonary consolidation was seen on 0.5 dpi with grey-red raised, and slightly firmer than normal areas. on 1 dpi, the percentage of affected lung tissue was increased (figure 2a ). on 2 dpi the lesions were dark red and firmer with increased relative lung weight. on 14 dpi the percentage of affected lung tissue decreased again (figure 2a ). the relative lung weight was increased from 1 to 7 dpi and decreased on 14 dpi (figure 2b ). the trachea-bronchial lymph nodes were enlarged from 0.5 to 7 dpi, with a peak on 7 dpi (table 2 ). there was a mild splenomegaly from 0.5 to 14 dpi (data not shown). histopathology and virus antigen expression. by histopathology, there was a mild to severe multifocal rhinitis with necrosis of the epithelium and mild to moderate multifocal tracheitis. in the lungs, there was a mild to severe multifocal bronchitis and bronchiolitis with intra-epithelial and intraluminal neutrophils, mild to severe necrosis of epithelium, mild to severe peribronchiolar and perivascular cuffing, mild to severe bronchoadenitis, and mild to severe multifocal alveolitis with infiltration of neutrophils in alveolar walls and lumina, mild to moderate epithelial necrosis and mild to moderate edema in the alveolar lumina with hypertrophy and hyperplasia of epithelium ( figures 10 and 11 ). the tracheo-bronchial lymph nodes and tonsils demonstrated lymphadenopathy, and in the palatine roof of two animals there was severe inflammation and necrosis of the sero-mucous glands. antigen expression in the nose was present from 0.5 to 7 dpi starting on 0.5 dpi in the respiratory epithelium and on 1 dpi in the olfactory epithelium. in the trachea there was antigen expression from 0.5 to 6 dpi and in the bronchi and bronchioles from 0.5 to 7 dpi with peak values on 1 dpi. bronchial glandular (table 3) . when digital scoring for antigen expression was performed, antigen expression as well as the number of positive counts showed that virus antigen was clearly present on 0.5 dpi with peak scores on 1 dpi, albeit with high variation between the different animals (table 3) . virology. in the respiratory tissues, the changes in virus titers over time showed a comparable pattern to that of viral antigen expression, with high virus titers from 0.5 to 4 dpi ( figure 3 ). in the extra-respiratory tissues, virus was isolated from the olfactory bulb, cerebellum, cerebrum, and heart (table 4 ). hematology and comparison of leucocytes in blood and alveolar lumina. total leucocyte counts showed higher values on all time points when compared to those from negative control animals (table 5 ). on 1 dpi, there was an increase of neutrophils in the blood and in the alveoli. on 2 dpi, there was a severe decrease in the number of neutrophils in the alveoli, but the number of neutrophils in the blood remained high. the number of mononuclear cells in the alveoli increased during the first 3 days while the number of mononuclear cells in the blood decreased. on 14 dpi, the numbers of neutrophils and mononuclear cells were decreased in both the blood and the alveoli. generalized linear model of viral excretion. similarly to h3n2, the final glm predicting excretion of ph1n1 included only viral production in the upper regions of the respiratory tract (nose and trachea) as a significant predictor (lr x 2 = 58.9, df = 1, p,0.0001). excretion of ph1n1 was a positive linear function of viral production in the upper regions (table 6 ). clinical data and gross pathology. on 2.5 dpi, one animal died and one animal was euthanized because of its moribund state. on 3 dpi, another animal died. from 2 to 4 dpi all animals developed severe clinical signs characterized by decreased activity, dyspnea, and inappetance (table 1) . two animals showed nervous signs, characterized by ataxia, drifting to the right, walking into a corner and aggression. virus was excreted in nasal and pharyngeal swabs, with lower virus titers than the ph1n1 and h3n2 groups during the first 3 days for the nose and the first 2 days for the pharynx (figures 1a and b) . in contrast to what was seen in the other virus groups, the nasal swabs showed lower titers than the pharyngeal swabs. this indicated lower replication in the nose, as was also suggested by the low levels of virus antigen expression and low virus titers in the nose ( figure 3 ). the increase in body temperature showed a peak on 1 dpi. at later time points, the average temperature decreased below baseline values due to the progressively moribund state of most of the ferrets (figure 1c ). the body weight loss increased during time and was comparable to that of the ph1n1 group ( figure 1d ). by gross pathology, there was multifocal pulmonary consolidation on 0.5 dpi with dark red, raised, and firmer than normal areas affecting an average of 13% of the lung tissue ( figure 2a ). on 1 dpi, there was fluid in the bronchi and lung parenchyma, and the percentage of affected lung tissue was increased. on 2 dpi, the percentage of affected lung tissue had increased dramatically to almost 60% ( figure 2a ). the relative lung weights were increased on all time points and were much higher than in the other virus groups (figure 2b ). tracheobronchial lymph nodes were enlarged starting on 0.5 dpi, and up to two times the normal size on 4 dpi. there was a mild splenomegaly on all days (data not shown). histopathology and virus antigen expression. by histopathology, there was a mild to moderate multifocal rhinitis with necrosis of the epithelium and mild multifocal tracheitis. in the lungs, there was a moderate multifocal bronchitis and bronchiolitis, with intra-epithelial and intraluminal neutrophils, mild to moderate epithelial necrosis, and mild peribronchiolar and perivascular cuffing; a mild to severe broncho-adenitis; and a severe multifocal to diffuse alveolitis, with severe epithelial necrosis, intra-epithelial and intraluminal infiltration of neutrophils, severe intraluminal edema, and hypertrophy and hyperplasia of epithelial cells (figures 4, 5, 6, 7, 8) . the tracheo-bronchial lymph nodes and tonsils demonstrated lymphadenopathy. in the extra-respiratory tissues there was lymphadenopathy in a sternal lymph node and there was hyperplasia and necrosis in the gut associated lymphoid tissue (galt) of the jejunum and in the spleen of few animals. in the nose there was antigen expression from 1 to 4 dpi with highest values on 2 and 3 dpi. antigen expression in the trachea was present from 0.5 to 3 dpi, predominantly on 1 dpi. the bronchi demonstrated antigen from 2 to 4 dpi, and in the bronchioles from 0.5 to 4 dpi. antigen expression in the alveoli was present in type ii pneumocytes (figure 8 ), and less in type i pneumocytes and alveolar macrophages. it was seen from 0.5 to 4 dpi with large percentages from 1 to 3 dpi. in the tracheobronchial lymph nodes there was antigen expression from 1 to 3 dpi and in the tonsils on 2 and 3 dpi in mononuclear cells. one animal had virus antigen expression in squamous stratified epithelium in the tip of the nose and one animal showed expression in endothelial cells in the pharynx. in the extrarespiratory tissues there was virus antigen expression in mononuclear cells in the galt of the jejunum on 2 and 3 dpi and in the spleen on 4 dpi. no antigen expression was seen in other extrarespiratory tissues. changes in the histological lesions and antigen expression over time are described in the supporting information (supporting information s1). digital scoring demonstrated that the amount of air-containing tissue was lower than in the other virus groups, and decreased over time (table 3) . digital scoring for antigen expression showed positive values on all time points with highest values on 1 dpi, but with high variation ( table 3) . virology of tissues. in the nose, the virus titers were higher than expected based on virus antigen expression in the nose, indicating a different origin of the virus ( figure 3 ). in the lungs, virus titers on 1 dpi were higher than on the other days. this could be due to slower replication on 0.5 dpi and necrosis of virusreplicating cells on 4 dpi. in the extra-respiratory tissues, virus was isolated from the olfactory bulb, cerebrum, cerebellum, heart, spleen, liver, kidney, adrenal gland, pancreas, and jejunum in one or more animals from 0.5 to 4 dpi. the highest virus titers were seen on 2 dpi in olfactory bulb and jejunum, and on 3 and 4 dpi in the spleen (table 4) . hematology and comparison of leucocytes in blood and alveolar lumina. the total leucocyte counts were decreased from 1 to 4 dpi ( table 5 ). the number of neutrophils in the alveolar lumina was low on 0.5 dpi but increased strongly with a peak value on 1 dpi. however, in the blood, after a small peak on 1 dpi, there was a decrease of the number of neutrophils ( figure 9 ). the number of mononuclear cells in the alveolar lumina showed an increase on 4 dpi. this increase was later than in the ph1n1 group, while the number of mononuclear cells in the blood did not increase. the number of lymphocytes in the blood was decreased on all days (table 5 ). generalized linear model of viral excretion. the final glm predicting excretion of the h5n1 included 6 significant predictors (lr x 2 = 14.1, df = 6, p = 0.029): viral production and damage in the deeper regions of the respiratory tract (bronchi, bronchioles and alveoli; p = 0.018 and p = 0.024, respectively); their product (p = 0.025); damage in the upper regions of the respiratory tract (nose and trachea; p = 0.001); its product with viral production in these regions (p = 0.002); and its product with viral production in the deeper regions (p = 0.031). excretion of h5n1 was a negative linear function of each of the three single predictors and a positive linear function of each of the three products. coefficients are given in table 6 . clinical signs and gross pathology. no clinical signs were seen and there was a survival rate of 100%. the body weight loss in the control group increased slightly in time ( figure 1d ). by gross pathology, the lungs had few dark red and slightly raised areas consistent with mild pulmonary consolidation. the area of affected lung tissue varied between 0 and 10% of the lung area (figure 2a) . the relative lung weight remained constant at around 0.6% on all time points (figure 2b ). tracheo-bronchial lymph nodes were slightly enlarged (table 1) and mild splenomegaly was present on all time points (data not shown). histopathology and virus antigen expression. by histopathology, there was mild multifocal rhinitis, tracheitis, bronchitis and bronchiolitis with few neutrophils, mild perivascular and peribronchiolar cuffing, mild broncho-adenitis, and mild focal alveolitis with infiltration of few neutrophils in the alveolar septa. no virus antigen expression was seen in any of the tissues. changes in the histological lesions over time are described in the supporting information (supporting information s1). digital scoring demonstrated that the amount of air-containing tissue was slightly lower on 3 and 4 dpi ( table 3) . virology. all swabs and tissues were negative in virus titration for influenza virus. hematology and comparison of leucocytes in blood and alveolar lumina. no trends were observed in total leucocyte, segmented neutrophil, mononuclear cell and lymphocyte counts in the blood, or in comparison of leucocyte counts between blood and alveolar lumina (figure 9 and table 5 ). our time course experiments show how strongly the spatial and temporal dynamics of infection and associated lesions in the ferret respiratory tract differ between a seasonal human influenza virus (h3n2), a pandemic human influenza virus (ph1n1), and a zoonotic avian influenza virus (h5n1): h3n2 infection was restricted to the nose and peaked at 1 dpi; ph1n1 infection also peaked at 1 dpi, but occurred at similar levels throughout the respiratory tract; and h5n1 infection occurred predominantly in the alveoli, where it peaked for a longer period, from 1 to 3 dpi ( figure 3 ). the associated lesions followed the same spatial distribution as virus infection, but their severity peaked between 1 and 6 days later ( table 2) : at 4 to 7 dpi for h3n2 and ph1n1, after which lesions decreased in severity by 14 dpi; and at 4 dpi for h5n1, when all h5n1-infected ferrets had either died or been euthanized on humane grounds. an important implication of these results is that location and timing of sample collection need to be chosen carefully in any comparative study of infection and pathology by different influenza viruses, otherwise the comparison will not be valid. the main source of excreted virus likely differs between h3n2 and h5n1 infections, based on comparison of virus titers in nasal and pharyngeal swabs ( figure 1 ) with virus antigen expression in respiratory tract tissues ( figure 3 ). for h3n2, the temporal dynamics of nasal swab virus titers, pharyngeal swab virus titers, and virus antigen expression in the nose were comparable. together with the lack of virus antigen expression in the lower respiratory tract, these results suggest that the nose is the main source of excreted h3n2. this is supported by the final glm for h3n2 viral excretion, which included only viral production in the upper regions of the respiratory tract as significant predictor. in contrast, for h5n1, virus titers in both nasal swabs and pharyngeal swabs were already near peak levels at 1 dpi, while virus antigen expression in the nose remained relatively low until 3 dpi. together with the higher levels of virus antigen expression in the trachea, bronchioles, and especially alveoli at 1 to 2 dpi, these results suggest that the lower respiratory tract is the main source of excreted h5n1, at least until 3 dpi. in fact, the final glm for there is high antigen expression with associated lesions characterized by bronchiolitis in bronchiolar epithelium of ferrets inoculated with ph1n1 on 1 dpi that was low on 4 dpi, consistent with severe loss of bronchiolar epithelium, and for h5n1 there was no expression on 1 dpi and moderate antigen expression with associated lesions on 4 dpi. there were no antigen expression and no lesions for h3n2. he and immunoperoxidase counterstained with hematoxylin, 2006. doi:10.1371/journal.pone.0042343.g007 h5n1 viral excretion suggests that viral production and damage in the deeper regions of the respiratory tract (bronchi, bronchioles and alveoli) may have a strong negative impact on viral excretion. this may be due to physical obstruction of the deeper airways due to damage caused by h5n1 virus infection and immune responses. for ph1n1, it is more difficult to determine the main source of excreted virus, because virus antigen expression was high at all levels of the respiratory tract. however, the final glm for ph1n1 viral excretion revealed that only the viral production in the nose and trachea was a significant predictor of viral excretion, suggesting that as for the h3n2 virus, the upper regions of the respiratory tract may be the main sources of excreted virus. the pattern of h3n2 and h5n1 antigen expression in the ferret respiratory tract corresponded only in part with the pattern of attachment for these viruses (for the ph1n1 virus, the pattern of attachment in ferrets has not been determined). attachment of an influenza virus to a host cell is the first step in the virus replication cycle and is considered to be necessary, but not sufficient, for subsequent infection of that cell. previously, we found that h3n2 attached to many tracheal epithelial cells (predominantly ciliated cells), rare or few bronchial and bronchiolar epithelial cells, and a moderate number of alveolar epithelial cells (predominantly type i pneumocytes, which have a low metabolism) in the lower respiratory tract of the ferret [6] . however, in the current study, we found very little h3n2 infection (based on virus antigen expression) in any cell types of the ferret lower respiratory tract ( figure 3 ). this suggests that other factors were necessary for h3n2 to infect these cell types. these factors may be the virus load that may need to be higher for infection with h3n2 and the influence of surfactant that protect against influenza in humans and pigs [17] . we previously found that h5n1 did not attach to tracheal or bronchial epithelial cells, and attached to rare or few bronchiolar epithelial cells and a moderate number of alveolar epithelial cells (predominantly type ii pneumocytes, which have a high metabolism) in the lower respiratory tract of the ferret [6] . in the current study, we found abundant h5n1 infection in alveolar and bronchiolar epithelial cells, which fits with the pattern of virus attachment ( figure 3 ). however, we also found h5n1 infection in tracheal and bronchial epithelial cells, which does not. next to the fact that this could be related to a different h5n1 strain (a/ vietnam/1194/04 [6] , while in the present experiment we used a/indonesia/5/05), the replication of h5n1 in alveolar and bronchiolar epithelial cells was so abundant that tracheal and bronchial epithelial cells became infected despite the relatively weak attachment of h5n1 to the latter cell types. different parameters were used to measure the severity of lower respiratory tract disease in our ferrets. of these parameters, relative lung weight and affected lung tissue allowed the best quantitative distinction between the virus groups: they showed clear differences between virus groups from 2 dpi onwards (table 7) , with relatively little within-group variation (figure 2 ). these parameters have been used with success before for studies on severe acute respiratory syndrome [18] and on influenza [19] . immunohistochemistry score for virus antigen expression in the table 2 . histopathology scores in ferrets inoculated with different influenza viruses. (table 7) , alveolar edema score, and alveolar hemorrhage score (table 2 ) also showed clear differences between the virus groups. however, between-group differences differed according to the dpi, and within-group variation was large. comparing the histopathology scoring for extent and severity of alveolitis and alveolar damage with the digital scoring for aircontaining space in pulmonary tissue showed that the latter demonstrated less statistically significant results, and therefore is less useful for discriminating between viruses (table 7) . comparing the antigen expression scoring in the alveoli by hand with the digital scoring and the virus titers, the p-values are similar and allow distinction between the virus groups ( table 7) . the other parameters did not allow a clear distinction between the three virus groups. besides quantitative differences in severity of lower respiratory tract disease, the three virus infections also showed clear differences in the qualitative character of lower respiratory tract disease, related to the tropism of the virus and the ability of the host to counter infection. this is particularly well illustrated in the alveoli. for the mildest disease, caused by h3n2 infection, there was rare evidence of virus infection in the alveolar epithelium ( figure 3 ). there was a mild acute inflammatory response with small numbers of neutrophils and macrophages, peaking at 2 dpi. damage to the alveolar epithelium was so mild that there was no visible evidence of epithelial cell hypertrophy or hyperplasia to reepithelialize the alveolar walls (table 2) . for the intermediate disease, caused by ph1n1 infection, virus infection already was high at 0.5 dpi and peaked soon afterwards at 1 dpi (figure 3 ). there was a moderate acute inflammatory response characterized by influx of neutrophils, peaking at 1 dpi (figure 9 ). at the same time, there was evidence of damage to the alveolar wall, characterized by necrosis of the epithelium and edema and hemorrhage in the alveolar lumina, which was maximal at 4 to 7 dpi (table 2) . after 1 dpi, the alveolar lesion progressed to a more chronic inflammatory response, with neutrophils being replaced by mononuclear cells, which peaked at 7 dpi (figure 9 ). at the same time, repair of the alveolar epithelial damage was visible as hypertrophy and hyperplasia of type ii pneumocytes, which was maximal at 7 dpi. during this period, the level of virus infection decreased to a low level. by 14 dpi the damage to and inflammation of the alveoli was completely resolved, except for a remnant of mononuclear cells in table 3 . digital histopathology scores in ferrets inoculated with different influenza viruses. table 4 . virus isolation from tissues in ferrets inoculated with different influenza viruses. the alveolar lumina, and virus infection was no longer detectable. the resolution of edema and hemorrhage could be explained on the one hand by the action of alveolar macrophages [20] , and on the other hand by re-epithelialization of the alveolar walls [21] . for the most severe disease, caused by h5n1 infection, the virus infection reached about the same level as that by ph1n1 infection at 1 dpi but remained high until death or euthanasia at 4 dpi, indicating that the host was not able to control virus infection. there was concurrent alveolar damage, which was so severe and extensive that all ferrets either died or had to be euthanized on humane grounds by 4 dpi. this was despite clear attempts at reepithelialization, based on prominent type ii pneumocyte hypertrophy and hyperplasia from 1 dpi onwards (table 2 ). there was a marked acute inflammatory response characterized by neutrophil influx peaking at 1 dpi but much greater than in ph1n1 infection, with a neutrophil count that was nearly twice as high (figure 9 ). this could be explained by a higher induction of pro-inflammatory cytokine production (e.g., il-6 and il-8) and no production due to h5n1 infection [22, 23] . maines et al. [10] demonstrated a relatively high production of il-6 and il-8 in the lower respiratory tract of ferrets infected with h5n1, but not with ph1n1. similarly, humans infected with h5n1 had high levels of il-6 and il-8 in the blood [24] . another major difference with ph1n1 infection was the low influx of mononuclear cells, with a mononuclear cell count of less than half that in ph1n1 infection by 4 dpi, suggesting that the inflammatory response never progressed beyond the acute stage. comparison of the dynamics of neutrophil and mononuclear cell influx in the alveolar lumina with neutrophil and monocyte counts in peripheral blood helps to explain the differences in hematological dynamics among the three virus infections (figure 9 ). for h3n2 infection, the slight and transient increase in the numbers of neutrophils and monocytes in the blood at 1 dpi correlates with the mild influx of these cell types into the alveolar lumina at that time. this corresponds with a low demand for neutrophils and monocytes during h3n2 infection. for ph1n1 infection, the marked increase in first the number of neutrophils and then of monocytes in the blood correlates with the moderate influx of first neutrophils and then mononuclear cells in the alveolar lumina. this corresponds with increased demand for both cell types, both of which can be met adequately by the myelopoietic compartment. neutrophils and monocytes are back to normal levels in the blood by 14 dpi, indicating resolution of the respiratory tract inflammation. for h5n1 infection, the initial increase of neutrophils in the blood at 1 dpi correlates with a massive influx of neutrophils into the alveolar lumina. the steep decline in neutrophils in the blood on subsequent days, despite continued high influx in alveolar lumina, corresponds to a demand that has outstripped the available supply by the myelopoietic compartment. this is corroborated by the presence of immature neutrophils with rod-shaped nuclei in the blood from 1 dpi onwards ( table 5 ). the lack of increase in monocytes in the blood corresponds with the failure of the acute inflammatory response in the lungs to transform into a more chronic inflammatory response. in addition to changes in neutrophil and monocyte counts, there was an overall leucopenia from 2 dpi (table 5) . such a leucopenia has been described previously for h5n1 infection in both humans and ferrets [11, 12, 25, 26] , whereas ferrets infected with ph1n1 showed higher leucocyte counts [12] . together, this comparison between inflammatory cell dynamics in alveoli and blood provides insight into the interpretation of hematological analyses of influenza pneumonia. besides the respiratory tract, there was evidence, by virus isolation, of extra-respiratory spread for h5n1 and ph1n1, but not for h3n2 ( table 4 ). the failure to detect virus antigen in any of these virus-isolation-positive tissues except jejunum may be because virus isolation is more sensitive than immunohistochemistry, or because virus was present but not replicating in these tissues. previously, h5n1 virus antigen expression in extrarespiratory tissue has been found in the central nervous system table 5 . leucocyte concentrations in the peripheral blood of ferrets inoculated with different influenza viruses. and liver of experimentally inoculated ferrets [27, 28, 29] . in our experiment, we additionally found virus antigen in other extrarespiratory tissues: for ph1n1 in sero-mucous glandular epithelial cells in the palatine roof, and for h5n1 in mononuclear cells in tracheo-bronchial lymph nodes, tonsils, sternal lymph node, spleen, and galt of the jejunum, and in squamous stratified epithelium on the tip of the nose. the implication of these sites of replication for the pathogenesis and transmission of these viruses is yet to be elucidated. the extensive h5n1 infection in extra-respiratory tissues known from previous studies [11, 28, 27, 30] likely occurs by spread from the respiratory tract via the blood [24, 31, 32] . however, the expression of h5n1 antigen in olfactory mucosa ( figure 5 ) together with high virus titres in the olfactory bulb (table 4) suggest that h5n1 may reach the central nervous system from the nasal cavity via the olfactory route, as demonstrated recently by schrauwen et al. [33] . this h5n1 infection of the central nervous system resulted in clinical disease, judging by the presentation of neurological signs (table 1 ). in addition to extra-respiratory spread of h5n1, there also was evidence of ph1n1 in extrarespiratory tissues, especially the central nervous system (table 4) , and ph1n1 expression in olfactory mucosa ( figure 5 ). this suggests that ph1n1 also may be neurotropic and enter the central nervous system via the olfactory route. animals were housed and experiments were conducted in strict compliance with european guidelines (eu directive on animal testing 86/609/eec) and dutch legislation (experiments on animals act, 1997). the protocol was approved by the independent animal experimentation ethical review committee of the netherlands vaccine institute (permit number 200900201) and was performed under animal biosafety level 3 conditions. animal welfare was observed on a daily basis, and all animal handling was performed under light anesthesia using a mixture of ketamine and medetomidine to minimize animal suffering. after handling atipamezole was administered to antagonize the effect of medetomidine. three viruses were used: seasonal h3n2 virus (a/netherlands/ 177/2008), isolated from a patient during the 2008 influenza season [34] ; pandemic (h1n1) 2009 virus (ph1n1) (a/netherlands/602/2009), isolated from a specimen of a human patient who had recently visited mexico during the pandemic in 2009 [35] ; and hpai h5n1 virus (a/indonesia/5/2005) as described earlier [35] . the h3n2 virus was chosen as a representative from a recent seasonal h3n2 influenza epidemic. like virtually all recent h3n2 viruses the present virus exhibits the oseltamivirsensitive neuraminidase-associated agglutination of turkey erythrocytes [36] . the ph1n1 virus which had been initially isolated in embryonated chicken eggs was chosen as a representative virus from the 2009 pandemic because it has been used in previous experiments by others [37] and ourselves [19, 35, 38] . the h5n1 figure 10 . histopathologic changes in the alveoli during time in ferrets inoculated with ph1n1. on 0.5 dpi there was mild dad that was characterized by neutrophils in the alveolar walls. on 1 dpi the alveolar walls were thickened with mild necrosis of the lining epithelium and increased number of neutrophils, in the lumina there were more mononuclear cells, little amount of edema fluid mixed with fibrin, hemorrhage and little cellular debris. on 2 dpi there was increased presence of necrosis, edema, hemorrhage and hyperplasia. on 3 dpi there were both new lesions with acute necrosis and old lesions with type ii hyperplasia present, with increased amounts of edema and hemorrhage. on 4 dpi the type ii hyperplasia and hypertrophy was more pronounced with increased amount of edema and high numbers of mononuclear cells. on 7 dpi there was severe epithelial hyperplasia and hypertrophy as well as intraluminal edema and hemorrhage. on 14 dpi the walls were mildly thickened. he, 1006 (first column), 4006 (second column). doi:10.1371/journal.pone.0042343.g010 virus was chosen as a representative of clade 2 (subclade 1.3.2) of hpai h5n1 virus, and has been used in several previous experiments [19, 29] . the different isolates were passaged three times in madin-darby canine kidney (mdck) cells and titrated according to standard methods. subsequently, the viruses were clarified and reached an infectious virus titer of 1610 7.4 median tissue culture infectious dose (tcid 50 ) per ml for h3n2 virus, and 1610 7.8 tcid 50 for both ph1n1 and h5n1 virus [8, 39] .the inoculum for the control group was prepared as follows: mdck cells were grown up to a monolayer of 80 to 90% in two 75 cm 2 flasks (a and b). virus medium (emem supplemented with hepes, sodium bicarbonate, bsa fraction v, l-glutamin, penicillin, streptomycin, trypsin and amphothericin-b) was added and the cells were incubated for 2 days at 37uc. to mimic cellular damage in mdck cells during virus infection when preparing the virus stock we aimed to reach a cytopathic effect (cpe) of 75%. therefore, the cells in bottle b were collected by disrupting the monolayer with 3 mm glass beads (vwr, amsterdam, the netherlands), sonication (3 times 20 seconds in melting ice) and freezing at 280uc, leading to lysis of all cells. the cells in bottle a were not damaged and the final inoculum consisted for 25% of the supernatant of bottle a and for 75% of the supernatant of bottle b. for every virus and the sham inoculated group, 7 groups (5 groups for h5n1) of four ferrets were inoculated under anesthesia with ketamine (4-8 mg/kg; nimatek, eurovet animal health b.v., bladel, the netherlands) and medetomidine hydrochloride (0.1 mg/kg; domitor, orion pharma, espoo, finland) with each of these three viruses with 10 6 tcid 50 in a 3-ml volume intratracheally and in a 0.3-ml volume intranasally evenly divided over the two nostrils. intratracheal and intranasal inoculation was performed to ensure that the inocula would reach both the lower and the upper respiratory tract [19, 29] . after inoculation, the ferrets received atipamezole hydrochloride (0.5 mg/kg antisedan; orion pharma, espoo finland) and were monitored daily for clinical signs until maximally 14 dpi [8] . the animals were predestined to be sacrificed at 12 hours (0.5 day), 1, 2, 3, 4, 7 and 14 dpi (for h5n1 not at 7 and 14 dpi) in order to avoid any bias that could follow from clinical observation, or earlier when they were moribund before the selected time point of euthanasia (h5n1 only). the animals were euthanized by exsanguination after anesthesia with ketamine. at euthanasia, body weight was measured, nose, throat and rectal swabs were collected, blood was taken, and samples were taken from both respiratory and extrarespiratory tissues for virological, pathological, and immunohistochemical analyses. the inoculum used for the sham control group induced comparable body weight loss, affected lung tissue, histological scores, digital scoring, and leucocyte counts as the h3n2 group. as an extra comparison, we therefore used non-inoculated healthy ferrets with the same age and background as the other ferrets as negative control animals to score and compare the above parameters. ferrets were used in this experiment since they resemble disease in humans when infected with influenza a viruses [5, 7] . hundredand-four eleven-month-old purpose-bred ferrets, seronegative for antibodies against circulating influenza viruses h1n1 (a/brisfigure 11 . histopathologic changes in the bronchioles during time in ferrets inoculated with ph1n1. on 0.5 dpi there was infiltration of neutrophils in the bronchiolar epithelium and lumen with mild peribronchiolar cuffing. on 1 dpi the number of neutrophils increased with necrosis of the epithelium, intraluminal cellular debris and peribronchiolar cuffing. on 2 dpi the necrosis was more severe than on 1 dpi. on 3 dpi there was denudation of the basement membrane and little regeneration of the bronchiolar epithelium with hyperplasia, peribronchiolar cuffing and occasionally cellular debris in the lumen. on 4 dpi there was increased epithelial hyperplasia with moderate cuffing. on 7 dpi the epithelium covered the basement membrane of the bronchiole with hypertrophic cells and moderate clinical scores in all groups were assessed every day. activity status was scored as follows: 0, alert and playful; 1, alert and playful only when stimulated; 2, alert but not playful when stimulated; 3, neither alert nor playful when stimulated. for diarrhea, sneezing, nasal and conjunctival discharge, inappetence and dyspnea we scored: 0, not present; 1, present [11] . inappetence was measured by the amount of food that was still present in the cages at the time of feeding. as a control we also assessed the amount of food that was present in the stomach and intestine of the animals on the day of necropsy. dyspnea was characterized by open-mouth breathing with exaggerated abdominal movement. additionally, we used four eleven-month-old purpose-bred ferrets, seronegative for antibodies against circulataleutian disease virus. the four ferrets were euthanized immediately by the same method as described above and necropsied to provide control data of non-inoculated ferrets, and blood was taken blood for hematologic analyses and respiratory tract tissues for histopathological scoring. for every virus four animals per time point were euthanized by exsanguination under ketamine/medetomidine anesthesia at 12 hours (0.5 day), 1, 2, 3, 4, 7 or 14 dpi and were necropsied according to a standard protocol. the trachea was clamped off to prevent the lungs from deflating upon opening the thoracic cavity, allowing visual estimation of the area of affected lung parenchyma. the lungs were weighed and the relative lung weight was calculated by the following formula: (lung weight on day of death/body weight on day of death)*100, and presented as percentage. the following tissues were collected for histological examination: left lung, left nasal concha, nasal septum, larynx, trachea, bronchus, tracheo-bronchial lymph node, left tonsil, heart, liver, spleen, kidney, pancreas, duodenum, jejunum, colon, adrenal gland, left olfactory bulb and left brain (cerebrum and cerebellum). lung samples were taken in a standardized way, not guided by changes as seen in the gross pathology. tissues were stored in 10% neutral-buffered formalin (lungs after careful inflation with formalin), embedded in paraffin, sectioned at 4 mm, and stained with hematoxylin and eosin (he) for examination by light microscopy. semiquantitative assessment of influenza virus-associated inflammation in the lung (four slides with longitudinal section or cross-section of cranial or caudal lobes per animal) was performed on every slide as reported earlier [9]: for the extent of alveolitis and alveolar damage we used: 0, 0%; 1, 1-25%; 2, 25-50%; 3, .50%. for the severity of alveolitis, bronchiolitis, bronchitis, and tracheitis we scored: 0, no inflammatory cells; 1, few inflammatory cells; 2, moderate numbers of inflammatory cells; 3, many inflammatory cells. for the presence of alveolar edema, alveolar hemorrhage, and type ii pneumocyte hyperplasia we scored: 0, no; 1, yes. finally, for the extent of peribronchial, peribronchiolar, and perivascular infiltrates we scored: 0, none; 1, one to two cells thick; 2, three to ten cells thick; 3, more than ten cells thick. slides were examined without knowledge of the treatment allocation of the animals. the cumulative scores for size and severity of inflammation of all slides provided the total score per animal. to assess the number of neutrophils and alveolar macrophages in the alveolar lumina and the number of neutrophils in the alveolar walls, we counted them in 5 arbitrarily chosen 1006 objective fields per he-stained slide, with a total of 20 fields per animal. neutrophils were identified on the basis of their size (approximately 12 to 15 mm in diameter) and the morphology of their nucleus (heterochromatic and segmented, with 3 to 5 lobes joined by thin strands). neutrophils in the alveolar walls were counted when present in small capillaries, but were excluded if present in larger blood vessels. pulmonary alveolar macrophages were identified on the basis of their morphology and location: large, oval to round cells with a distinct cell border, foamy cytoplasm and large, oval to bean-shaped nucleus, located in the alveolar lumen separate from the alveolar wall [40] . for detection of influenza a virus antigen, tissues were stained with a primary antibody against the influenza a nucleoprotein as described previously [34] . in each staining procedure, an isotype control was included as a negative control and a lung section from a cat infected experimentally with h5n1 was used as positive control [41] . semiquantitative assessment of influenza virus antigen expression in the lungs was performed as reported earlier [19] : for the alveoli, twenty-five arbitrarily chosen, 206 objective, fields of lung parenchyma of four lung sections were examined by light microscopy for the presence of influenza virus nucleoprotein, without the knowledge of the identity of the animals. the cumulative scores for each animal were presented as number of positive fields per 100 fields. for the nose, trachea, bronchi and bronchioles, the percentage of positively staining epithelium was estimated on every slide and the average of the four slides was taken to provide the score per animal: 0, 0%; 1, 1-25%; 2, 25-50%; 3, .50%. for computerized scoring of slides we used the cross-section of the left caudal lung of all animals to make a digital scan using the nanozoomer with accompanying software (nanozoomer digital pathology and ndp.scan and ndp.view, hamamatsu, higashiku, hamamatsu city, japan). of every scan, 20 pictures of the alveoli were made in a randomized order with the 206 objective. from every picture, we calculated: the quantity of tissue (visualized in blue staining) and the quantity of red staining consistent with virus antigen expression using zeiss ks 400 version 3.0 image analysing system (carl zeiss vision gmbh, eching, germany). the images were 10246768 pixels with 0.455 mm per pixel. the percentage of air-containing space present in the pulmonary tissue was calculated as 100% minus the total percentage of pulmonary tissue (blue staining) that was present in the pictures. the quantity of virus replication was calculated either as the percentage of antigen expression (red staining) relative to quantity of tissue (blue staining), or as the number of times red staining was detected in a picture. after collection on day 0 and the day of euthanasia, nose, pharyngeal and rectal swabs were collected in virus transport medium (emem containing bovine serum albumin (fraction v), penicillin, streptomycin, amphothericin-b, l-glutamine, sodium bicarbonate and hepes), aliquotted and stored at 270uc. upon necropsy, samples were collected from the following tissues: nasal concha, trachea, bronchus, tracheo-bronchial lymph node, tonsil, heart, liver, spleen, kidney, pancreas, duodenum, jejunum, colon, adrenal gland, olfactory bulb and brain. specifically from the lungs, sections of the cranial, median and caudal lobe of the right lung and of the accessory lobe from each animal were collected and pooled (total average weight of about 0.4-0.5 g/animal); lung samples were taken in a standardized way, not guided by changes as seen in the gross pathology. tissue samples were homogenized with a fastprep-24 (mp biomedicals, eindhoven, the netherlands) in influenza infection medium (emem containing bovine serum albumin (fraction v), penicillin, streptomycin, amphothericin-b, l-glutamine, sodiumbicarbonate, hepes and trypsin) and centrifuged briefly before titration. quadruplicate 10-fold serial dilutions of tissue and swab supernatants were used to determine the virus titers in confluent layers of mdck cells as described previously [39] . on day 0 and the day of euthanasia (0.5, 1, 2, 3, 4, 7 or 14 dpi or when an animal was sacrificed due to a moribund state) blood was collected from the euthanized animals. total leucocyte counts were determined in blood collected in edta vacutainer tubes (vacuette, greiner bio-one gmbh, kremsmünster, austria) using an automated hematology analyser sysmex poch-100i (sysmex europe gmbh, norderstedt, germany). thin blood films were prepared from edta blood and stained with may-grünwald-giemsa (merck, darmstadt, germany). differential cell counts were obtained by counting 100 cells per slide, and the numbers of lymphocytes, mononuclear cells, blastocytes, rodshaped neutrophils, segmented neutrophils, eosinophils, basophils and normoblasts were calculated by multiplying these percentages by the leucocyte counts obtained for the same sample. by using the total leucocyte counts we calculated the absolute number of different leucocytes in 10 9 /l. the non-parametric mann-whitney test was used to compare several parameters of the different virus infection per day for; percentage of affected lung tissue, relative lung weight, extent and severity of alveolitis and alveolar damage, percentage of air containing space in pulmonary tissue, virus titers in lung tissue, antigen expression in the alveoli by immunohistochemistry, percentage of antigen expression in the alveoli by digital scoring, and number of antigen expression counts in the alveoli by digital scoring. the outcomes were considered significant when p,0.05. generalized linear models (glm) were used to determine the most appropriate predictors of viral excretion for each virus, based on viral production and damage in the respiratory tract. these models were used to determine the linear equations of the form y = g(a i x i +b i ) that best fit the data, where y is viral excretion, x i is a predictor, and a i and b i are the predictor and intercept coefficients, respectively. viral excretion was measured as the sum of nasal swab and pharyngeal swab viral titers. predictors initially included in the models were measures of viral production and measures of damage in upper (nose and trachea) and deeper regions of the respiratory tract (bronchi, bronchioles and alveoli), as well as all two-ways interactions. measures of viral production for each region were calculated as the product of the viral titers and immunohistochemistry scores in the respective regions. this was done to take into account the fact that virus isolated in a particular region of the respiratory tract may not be produced locally. measures of damage for each region were calculated as the sum of the severity scores in the respective regions. the severity scores were used because they were similarly assessed in all regions of the respiratory tract. only predictors or interactions of predictors with p,0.05 were retained in the final glm. supporting information s1 detailed description per virus of histopathologic changes and antigen expression by immunohistochemistry. 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is critical for systemic spread along the olfactory and hematogenous routes in ferrets modification of the ferret model for pneumonia from seasonal human influenza a virus infection pathogenesis and transmission of swine-origin 2009 a(h1n1) influenza virus in ferrets neuraminidase receptor binding variants of human influenza a(h3n2) viruses resulting from substitution of aspartic acid 151 in the catalytic site: a role in virus attachment? in vitro and in vivo characterization of new swine-origin h1n1 influenza viruses pandemic h1n1 vaccine requires the use of an adjuvant to protect against challenge in naive ferrets comparison of rna hybridization, hemagglutination assay, titration of infectious virus and immunofluorescence as methods for monitoring influenza virus replication in vitro textbook of veterinary histology. philadelphia: lea & febiger avian h5n1 influenza in cats we thank c. van hagen, r. boom, w. van aert, r. van lavieren, l. leijten, p. van run, e. veldhuis kroeze, d. van riel, d. van de vijver, and t. bestebroer for technical assistance, a. gomersbach and w. vos for biotechnical assistance, f. van der panne for figure preparation, a. nigg for assistance with the digital scoring and m. van leeuwen (uvdl, utrecht) for reading the thin blood slides. key: cord-303331-xolksoy3 authors: pourghasemi, hamid reza; pouyan, soheila; farajzadeh, zakariya; sadhasivam, nitheshnirmal; heidari, bahram; babaei, sedigheh; tiefenbacher, john p. title: assessment of the outbreak risk, mapping and infection behavior of covid-19: application of the autoregressive integrated-moving average (arima) and polynomial models date: 2020-07-28 journal: plos one doi: 10.1371/journal.pone.0236238 sha: doc_id: 303331 cord_uid: xolksoy3 infectious disease outbreaks pose a significant threat to human health worldwide. the outbreak of pandemic coronavirus disease 2019 (covid-19) has caused a global health emergency. thus, identification of regions with high risk for covid-19 outbreak and analyzing the behaviour of the infection is a major priority of the governmental organizations and epidemiologists worldwide. the aims of the present study were to analyze the risk factors of coronavirus outbreak for identifying the areas having high risk of infection and to evaluate the behaviour of infection in fars province, iran. a geographic information system (gis)-based machine learning algorithm (mla), support vector machine (svm), was used for the assessment of the outbreak risk of covid-19 in fars province, iran whereas the daily observations of infected cases were tested in the—polynomial and the autoregressive integrated moving average (arima) models to examine the patterns of virus infestation in the province and in iran. the results of the disease outbreak in iran were compared with the data for iran and the world. sixteen effective factors were selected for spatial modelling of outbreak risk. the validation outcome reveals that svm achieved an auc value of 0.786 (march 20), 0.799 (march 29), and 86.6 (april 10) that displays a good prediction of outbreak risk change detection. the results of the third-degree polynomial and arima models in the province revealed an increasing trend with an evidence of turning, demonstrating extensive quarantines has been effective. the general trends of virus infestation in iran and fars province were similar, although a more volatile growth of the infected cases is expected in the province. the results of this study might assist better programming covid-19 disease prevention and control and gaining sorts of predictive capability would have wide-ranging benefits. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 in december 2019, several pneumonia infected cases were reported in wuhan, china [1, 2] . in january 2020, a novel coronavirus (2019-ncov) that was later formally named covid-19 was approved in wuhan [3] . it was announced that the disease is a severe acute respiratory syndrome coronavirus 2 (sars-cov-2). the virus elevated concerns within china as well as the global community as it was believed to be transmitted from human to human [4] . initially, china witnessed the largest outbreak in hubei and other nearby provinces. the spread in china was controlled soon thereafter through stringent preventive measures, but other parts of the world (europe, the middle east, and the united states) were increasingly affected by the outbreak through transmission by infected travellers from china. a similar outbreak soon followed in other asian countries [5] . its global spread to more than 150 countries led to the declaration in mid-march 2020 that covid-19 was a pandemic [6] . by june 18, 2020, there were nearly 8.60 million cases worldwide, with 455575 deaths attributed to covid-19 [7] . currently, the united states (2263651), brazil (983359) and russia (561091) have the largest number of confirmed cases, whilst the united states (120688), brazil (47869) and uk (42288) have the highest number of casualties, respectively [7, 8] . iran with 197647 recorded cases and 9272 deaths is the most affected country in the middle east (as of june 18, 2020) and infected cases are expected to surge in the coming days [7, 9] . the outbreak of covid-19 has disrupted and depressed the world economy, whereas iran is among the most severely affected by massive economic losses, largely compounded by politically motivated sanctions imposed by other governments [10] . the problem has been exacerbated as no specific medicine is yet realized for covid-19 disease treatment, though there are a few pre-existing drugs that are being tested, so regions are presently concentrating their efforts on maintaining the infection rate in a level that assists in reducing virus spread [11] . this has led to most states imposing lockdowns, encouraging social distancing, and restricting the sizes of gatherings to limit transmission [12] . there is a pressing necessity for scientific communities to aid governments in their efforts to control and prevent transmission of the virus [13] . during previous virus outbreaks stemming from zika, influenza, west nile, dengue, chikungunya, ebola, marburg, and nipah, geographic information systems (giss) have played significant roles in providing significant insight via risk mapping, spatial forecasting, monitoring spatial distributions of supplies, and providing spatial logistics for management [13] . in this current situation, risk mapping is critical and may be used to aid governments' need for tracking and management of the disease as it spread in places with the highest risk. sánchez-vizcaíno et al. [14] used a multi-criteria decision making (mcdm) model to map the risk of rift valley fever in spain. traditional statistical techniques had also been used to detect the risk of an outbreak [14] . reeves et al. [15] employed an ecological niche modelling (enm) technique for mapping the transmission risk of mers-cov; the middle eastern name for the coronavirus known as sars-cov-2. similar techniques have been in the nyakarahuka et al. [16] study to map ebola and marburg viruses risks in uganda. they assessed the importance of environmental covariates using the maximum entropy model. more recently, the use of machine learning algorithms (mlas) for mapping the risk of transmission of viruses has been increasing which is due to the demonstrated superior (and more accurate) predictive abilities of the mla models over traditional methods [17] . jiang et al. [18] employed three mlas-backward propagation neural network (bpnn), gradient boosting machine (gbm), and random forest (rf)-to map the risk of an outbreak of zika virus. tien bui et al. (2019) compared different mlas-artificial neural network (ann) and support vector machine (svm) with ensemble models including adaboost, bagging, and random subspace-for modelling malaria transmission risk. similarly, gbm, rf, and general additive modelling (gam) were used by carvajal et al. [19] to model the patterns of dengue transmission in the philippines. mohammadinia et al. [20] employed geographically weighted regression (gwr), generalized linear model (glm), svm, and ann to develop a forecast map of leptospirosis; gwr and svm produced highly accurate predictions. saba and elsheikh [21] , also used the nonlinear autoregressive ann model to forecast covid-19 outbreak. another statistical-based model that recently has been applied to forecast the behaviour of covid-19 outbreak and death cases is arima in which the forecast process is as a function of time. recently, the significant ability of this model to forecast covid-19 outbreak in egypt [21] and coronavirus related deaths in iran [22] has been reported. benvenuto et al [23] performed arima model on the johns hopkins epidemiological data and they found that the spread of virus tends to be slightly decreasing. however, ahmar and del val [24] combined the α-sutte indicator with arima and developed a model to forecast covid-19 outbreak in spain. their combined model presented more accurate forecast compared to the arima model. the literature shows that very few studies have tried to use gis for analysis of covid-19 outbreak in human communities. kamel boulos and geraghty [25] described the use of online and mobile gis for mapping and tracking covid-19 whilst zhou et al. [13] revealed the challenges of using gis for sars-cov-2 big data sources. to our knowledge, there has been no study with a focus on mapping the outbreak risk of the covid-19 pandemic. the aims of the present study were to analyze the risk factors of coronavirus outbreak and test the svm model for mapping areas with a high risk of human infection with the virus in fars province, iran. in addition, the growth trend of the covid-19 infestation in fars province was analyzed and compared with the growth rate (gr) of iran and several other countries. the outcome of the present study lays a foundation for better planning and understanding the factors that accelerate the virus spread for use in disease control plans in human communities. the methodology of this research can be used for mapping the outbreak risk of covid-19 and for detecting the trend of covid-19 infections in other parts of the world. this study also can aid local authorities in imposing strict social distancing measures in the regions with high outbreak risk. furthermore, this study can be helpful in determining the significant effective factors that influence the covid-19 outbreak risk. the study area is in the southern part of iran with an area of 122608 square kilometres located between 27˚2 0 and 31˚42 0 n and between 50˚42 0 and 55˚36 0 e. fars is the fourth largest province in iran (7.7% of total area) with a population density of 4851274 (based on in 2016 report). fars province is divided into 36 counties, 93 districts, and 112 cities (fig 1) . the multi-phased workflow implemented in this investigation (fig 2) is described comprehensively below. a dataset of active cases of covid-19 in fars was prepared to analyze the relationships between the locations of active cases and the effective factors that may be useful for predicting outbreak risk. the data utilized in this research (s1 file) was collected on april 10, 2020 from iranian's ministry of health and medical education (imhme: http://ird.behdasht.gov.ir/). choosing the appropriate effective factors to predict the risk of pandemic spread is vital as its quality affects the validity of the results [17] . since, there have been no previous studies of risk for covid-19 distribution, the selection of effective factors is a quite challenging task. also, there is no approved universal factors for mapping the outbreak risk of covid-19. ongoing research on the pandemic has revealed that local and community-wide transmission of the virus largely happens in public places where the most people are likely to come into contact with largest number of potential carriers of the infection [26] . wang et al. [27] indicated that meteorological conditions, such as rapidly warming temperatures in 439 cities around the world resulted in a decline of covid-19 cases. accordingly, in this research, we selected sixteen most relevant effective factors for the outbreak risk mapping of covid-19 in fars province of iran, which includes minimum temperature of coldest month (mtcm), maximum temperature of warmest month (mtwm), precipitation in wettest month (pwm), precipitation of driest month (pdm), distance from roads, distance from mosques, distance from hospitals, distance from fuel stations, human footprint, density of cities, distance from bus modelling outbreak risk and infection behavior of coronavirus stations, distance from banks, distance from bakeries, distance from attraction sites, distance from automated teller machines (atms) and density of villages. all the effective factors employed in this research are generated using arcgis 10.7. a few studies have established that variation in temperature would impact the transmission of covid19 [27] . it has also been reported that alteration in temperature would have impacted the sars outbreak, which was caused by the identical type of coronavirus as sars-cov-2 [28] . recently, ma et al. [2] disclosed that surge in temperature and humidity conditions have resulted in the decline of death caused by sars-cov-2. thus, climatic factors such as temperature and precipitation can have an impact on the outbreak of sars-cov-2. the temperature and precipitation data, namely mtwm, mtcm, pdm and pcm of fars province is acquired from world climatic data (https://www.worldclim.org/). in this study, the mtwm of the fars province ranges from 27.7˚c to 41.8˚c (fig 3) whereas mtcm ranges between -15.3˚c and 10.4˚c (fig 3) . the pwm of the study area varies between 28 mm and 86 mm (fig 4) , and also the pdm is presented in fig 3. the proximity to various public places including roads, mosques, hospitals, fuel stations, bus stations, banks, bakeries, attraction sites, and atms where people come in close contact to each other can also be considered as significant factors that influence the distribution of covid-19. the data was acquired from open street map (https://www.openstreetmap.org). the distance from roads ranges from 0 to 45 in the study area (fig 4) whereas the distance from mosques varies between 0 and 0.71 (fig 4) and the distance from fuel stations spans 0 to 0.67 (fig 4) . the distance from bus stations, banks, bakeries, attraction sites, and atms of fars province have the minimum value of 0 and maximum value of 1.31, 0.68, 0.97, 0.79, and 0.78 respectively (figs 4, 5) . since humans are the potential carriers of the covid-19, the use of human footprint (hfp) can aid in understanding the terrestrial biomes on which humans have more influence and access [29] . in this study, hfp of the study area is acquired from the global human footprint dataset. the hfp of fars province ranges from 6 to 78 (fig 5) where the minimum value represents the places having least access by humans, and the maximum value refers to those regions having highest human influence and access. the density of population is also considered to be an important factor for the spread of the disease [30, 31]. gilbert et al. [32] revealed that the number of covid-19 cases was proportional to the population density in africa. accordingly, in this research, density of cities and villages were assessed, and the outcome displays that density of cities in fars province ranges between 0 and 0.60 ( fig 5) while the density of villages varies from 0 to 0.58 (fig 5) . the distance from hospitals ranged from 0 to 1.11 ( fig 5) . the association among the location of covid-19 active cases and effective factors were evaluated using ridge regression in order to assess the significance of individual effective factor in predicting the outbreak risk [17] . to our knowledge, no previous study in epidemic outbreak risk mapping has utilized ridge regression in determining the significance of effective factors. however, the ridge regression algorithm has been utilized for modelling purposes in various fields [33] . it was first given by hoerl and kennard [34] which exploits l 2 norm of regularization for lessening the model complication and controlling overfitting. ridge regression was also developed to avoid the excessive instability and collinearity problem caused by leastsquare estimator [35] . the 'caret' package (https://cran.r-project.org/web/packages/caret/ caret.pdf) of r 3.5.3 was utilized for assessing the variable importance using ridge regression. support vector machine. svm is an extensively exercised mla in diverse fields of research that functions on the principle of statistical learning concept and structural risk minimization given by vapnik [36] , which is utilized for classification as well as regression intricacies [37, 38] . svm has high efficacy in classifying both linearly separable and inseparable data classes [39] . it utilizes an optimal hyperplane to distinguish linearly divisible data, whereas kernel functions are employed for transforming inseparable data into a higher dimensional space so that it can be easily categorized [40] . assume a calibration dataset to be (s m , t m ), where m is 1, 2, 3. . ., x; s m refers to the sixteen independent factors; t m denotes 0 and 1 that resembles risk and non-risk classes and x represents the total amount of calibration data. this algorithm tries to obtain an optimal hyperplane for classifying the aforementioned classes by utilizing the distance between them, which can be formulated as follows [41]: where, kpk denotes the rule of normal hyperplane; a refers to a constant. when lagrangian multiplier (λ m ) and cost function is introduced, the expression can be given as follows [42]: in case of an inseparable dataset, a slack covariate δ m is added into the previs eq (2) that is provided as follows [36] : accordingly eq (3) can be described as follows [36]: moreover, svm contains four kernel functions (linear, polynomial, radial basis function: rbf and sigmoid) for making an optimal margin in case of inseparable dataset [36] . mohammadinia et al. [20] revealed that rbf kernel type produces high prediction accuracy than other kernel types for epidemic outbreak risk mapping. thus, in this study, rbf is used for creating decision boundaries, and the kernel function is expressed as follows [36]: where, k(z a , z b ) refers to kernel function and v represents its parameter. analysis of growth rate for active and death cases of covid-19. in this study, the growth rate (gr) of active and death cases around the world, iran, and fars province were evaluated using the data acquired from who and imhme between february 25, 2020 and june 10, 2020 for active cases and from march 2, 2020 to june 10, 2020 for death cases. validation of outbreak risk map. the cross-checking of the calibrated model using untouched testing data is vital for determining the scientific robustness of the prediction [37] . in this research, we utilized roc-auc curve values for the validation of covid-19 outbreak risk map generated using the svm model. it is a widely utilized validation technique for analyzing the predictive ability of a model [39] . a model is determined to be perfect, very good, good, moderate and poor if the auc values were 1.0-0.9, 0.9-0.8, 0.8-0.7, 0.7-0.6 and 0.6-0.5, respectively [43] . models for infection cases trend. the behavior of the variable infection cases in fars province was captured by a third-degree polynomial or cubic specification while for those of iran the fourth-degree polynomial specifications was found to be more appropriate as follows: where, infection(t) represents the total infected cases in day t and t denotes the days starting from 19th of february for iran and one week later for fars province. a quadratic specification was examined and based on the fitted model, the cubic form (for fars province) and fourthdegree polynomial (for iran) were selected. in the literature, the cubic form of specification has been applied by aik et al. [44] to examine the salmonellosis incidence in singapore. we also used an arma model to compare the process generating the variable for iran and fars province. this model includes two processes: autoregressive (ar) and moving average (ma) process. an arma model of order (p,q) can be written as [45] : where x is the dependent variable and ε is the white noise stochastic error term. in the applied model, x shows the total infected cases and t is the days starting from the first day of happening infection cases. in building a time series model, the data are expected to be stationary [24] . in other words, the model (eq 8) is based on the assumption that the data series are stationary. briefly, a time series process x(t) is stationary if the mean and variance are constant and independent of time and the covariance between x(t) and x(t+s) (x with s period apart) is time-invariant or is dependent only upon the distance between the two time periods considered [46, 47] . thus, if a time series have time-varying mean or a time-varying variance or both will be nonstationary. using nonstationary time series for the forecasting purposes has little practical value. if the applied time series data is not stationary, after differencing it d times an stationary time series was obtained. this series is called integrated of order d. after differencing d times, we may apply the arma (p, q) model which is called arima (p, d, q) [46] . the arima (p,d,q) model is an arma(p,q) that applies d times differencing data. benvenuto et al. [23] applied an arima model to predict the epidemiological trend of covid-2019. also, saba and elsheikhb [21] used this model to forecast the outbreak of covid-19 in egypt. the analysis of variable importance using ridge regression revealed that distance from bus stations, distance from hospitals, and distance from bakeries have the highest significance whereas distance from atms, distance from attraction sites, distance from fuel stations, distance from mosques, distance from road, mtcm, density of cities and density of villages exhibit moderate importance. the effective factors such as distance from banks, mtwm, hfp, pwm and pdm were the least influential factors (fig 6) . the covid-19 outbreak risk map generated using svm displays that risk of sars-cov-2 ranges from -0.25 to 1.22 (march 29) and -0.35 to 1.21 (april 10) where -0.25 and -0.35 represent the lower risk of sars-cov-2 outbreak and 1.22 and 1.21 indicates the regions of fars province which is likely to experience a higher risk of covid-19 outbreak (fig 7a and 7b) . it can be observed from fig 7b (april 10) that shiraz county and its surrounding counties including firouzabad, jahrom, sarvestan, arsanjan, marvdasht, sepidan, abadeh, khorrambid, rostam, larestan and kazeron of fars province has the highest risk of being the epicentre of sars-cov-2 outbreak. apart from which counties like eghlid, and fasa also lie in the high risk zone. the results of gr of active cases in the world, iran, and fars province are presented in table 1 . percentage of death cases in china was related to february 29, whereas for iran and fars province it is related to march 14 and may 4, respectively. table 1 show that age class > 50 years old lie in the highest class of death rate. so, this age class of above 50 years is highly sensitive to covid-19. the roc-auc curve cross-validation technique is utilized in this research for validating the covid-19 outbreak risk map generated by svm. the model achieved an auc value of 0.786 and a standard error of 0.031 indicating a good predictive accuracy when cross-verified using the remaining 30% testing dataset collected on march 20, 2020 (fig 11 and table 2 ). when tested with active case locations on march 29, 2020, the model achieved an increased auc value of 0.799 which proves the stable and good forecast precision of the outbreak risk map (fig 12 and table 3 ). also, change detection on april 10, 2020 show that accuracy of the built models is increased to 86.6% (auc = 0.868) (fig 13 and table 4 ). two tools have been applied to compare the general trend of infection in fars province and iran. the first includes a third-degree (for fars province) and a fourth-degree (for iran) polynomial models that are presented in fig 14. another quantitative model is an arima model presented in table 5 . fig 12 shows the trend of infection cases in iran and fars province, where predicted values extraordinarily keep pace with the actual values. coefficients of determination () values also indicate that estimated models have significant predictive power. the infection cases are increasing over the selected horizon. the first derivative of the estimated model represents the daily infection cases. based on the daily infection model, there is a turning point for both iran and provincial cases. it was found that the turning point for provincial daily infection is 134. in other words, after 134 days the decreasing trend in the daily infection is expected. however, the corresponding value for iran is much higher than the provincial one. there are some evidences showing that a turning point in infection is expected. for instance, it has been reported for sars incidence [48] , hav [49] , ari [50] , and for a (h1n1)v. it is worth noting that a turning point means that after passing the peak, it is expected to show a decreasing trend. in the 107 th day of infection, fars province accounts for around 4.34% of the total iranian cases while its population share is more than 6% (statistical center of iran, 2016). regarding the values obtained for turning points and the infection share, up to the present, the measures taken by the provincial government may be considered more effective than those taken in other provinces as a whole. however, it should be taken into consideration that fars province experienced its first infection cases 7 days after qom and tehran, provinces that are considered as starting point for virus outbreak in iran. this might have given the provincial governmental body and the households to take measures to cope with the widespread outbreak. it is worth noting that the comparison of the specified models is more appropriate to investigate the effectiveness of the measures taken by the corresponding health body rather than using it to predict future values. the arima time series models for infection variables of the fars province and iran are presented in table 5 . these models may show the generating process of the variables in time horizon. it is worth noting that in order to have more comparable models, a 107-day time horizon is selected. this is the period of time that data are available, starting on 19th of february for iran and one week later for fars province. as shown in table 5 , provincial data are generated by an arima (2,1,1) process while arima (2,0,2) was found more appropriate for iran's data. regarding the orders for ar and ma processes, the country model shows more complicated behaviour. in addition, the fars data was applied after differencing since it was not stationary; indicating a more explosive process of an increasing trend for fars province compared to those of iran in the following days. the provincial data indicated more volatility which was captured by variance-related variable garch that was not easily captured in the trends as shown in fig 14. benvenuto et al. [23] also used an arima model and found that covid-2019 spread tends to reveal a slightly decreasing spread. generally speaking, the diagnostic statistics indicate that the estimated models are acceptable since q-statistics reveal that the residuals are not significantly correlated and the jarque berra statistic supports the normality of residuals at conventional significance level. in addition, all ar and ma roots were found to lie inside the unit circle, indicating that arima process is (covariance) stationary and invertible. there is a great necessity for new robust scientific outcomes that could aid in containing and preventing the covid-19 pandemic from spreading. the spatial mapping of covid-19 outbreak risk can aid governments and policy-makers in implementing strict measures in certain regions of a city or a country where the risk of an outbreak is very high. it is, therefore crucial to identify the regions that would have high outbreak risk through predictive modelling with the help of machine learning algorithms (mlas). in recent times, mlas have demonstrated promising results in forecasting the epidemic outbreak risk [17] . in this research, the svm model showing good forecast accuracy was used for mapping the outbreak risk of covid-19. similarly, mohammadinia et al. [20] revealed that gwr and svm had the highest precision in mapping the occurrence of leptospirosis. ding et al. [51] employed three mlas including svm, rf and gbm, for mapping the transmission risk assessment of mosquito-borne diseases and disclosed that all three mlas acquired excellent validation outcome. machado et al. [52] also applied rf, svm and gbm in modelling the porcine epidemic diarrhoea virus and demonstrated 90% specificity values in case of svm. tien bui et al. [17] stated that svm achieved an auc value of 0.968 in mapping the susceptibility to malaria. the ability to classify inseparable data classes is the greatest benefit of the svm model [53] . it is among the most precise and robust mla [54] . svm can be useful and has higher prediction accuracy when it comes to handling a small dataset. however, huang and zhao [55] demonstrated that svm also yields excellent precision in predictive modelling when a large dataset is utilized. the algorithm has a very low probability of overfitting and is not disproportionately impacted by noisy data [53] . behzad et al. [56] revealed that svm had huge capacity in simplification and had enduring forecast accuracy. it should also be noted that the predictive exactness of svm model largely depends on the choice of kernel function [54] . among the four kernel functions of svm, rbf has been proved to generate high accuracy models [54] . svm includes diverse kinds of categorization functions which are responsible for assessing overfitting and simplifying data that needs a minor tuning of model parameters [57] . the significance of each effective factor employed in this research is assessed using ridge regression. since, there is no previous study in covid-19 that outlines the proper effective factors. the outcome of this research can be very helpful for scientists in experimenting the same and additional effective factors for covid-19 outbreak risk mapping. the proximity factors including distance from bus stations, distance from hospitals, distance from bakeries were most influential in forecasting the covid-19 outbreak risk whereas other proximity factors such as distance from atms, distance from attraction sites, distance from fuel stations, distance from mosques and distance from road had the moderate influence which is followed by mtcm, density of cities and density of villages. it should be noted that climatic factors including mtwm, pwm and pdm had the least significance in mapping the outbreak risk. from this, it can be concluded that precipitation factors pwm and pdm are not associated with the transmission of covid-19 in fars province whereas in case of temperature factors mtcm had moderate influence in mapping covid-19 outbreak risk but mtwm exhibited a least significance. this outcome reveals that proximity factors had high influence in the transmission of sars-cov-2. in addition, the study conducted disclosed that increase in temperature will not decline the sars--cov-2 cases, although it has been also revealed that increase in temperature and absolute humidity could decrease the death of patients affected by covid-19 [58] . the polynomial and arima models were applied to examine the behaviour of infection in fars province and iran. the general trend of infection in iran and fars province are similar while more volatility for provincial cases is expected. the methodology and effective factors used in this research can be adapted in studies investigated in other parts of the world for preventing and controlling the outbreak risk of covid-19. mapping of sars-cov-2 outbreak risk can aid decision-makers in drafting effective policies to minimize the spread of the disease. in this research, gis-based svm was used for mapping the covid-19 outbreak risk in fars province of iran. sixteen effective factors including mtcm, mtwm, pwm, pdm, distance from roads, distance from mosques, distance from hospitals, distance from fuel stations, human footprint, density of cities, distance from bus modelling outbreak risk and infection behavior of coronavirus stations, distance from banks, distance from bakeries, distance from attraction sites, distance from automated teller machines (atms) and density of villages were selected along with the locations of active cases of sars-cov-2. the results of ridge regression revealed that distance from bus stations, distance from hospitals, and distance from bakeries had the highest influence in covid-19 outbreak risk mapping whereas the climatic factors had the lowest influence. the generated model using svm had a good predictive accuracy of 0.786 and 0.799 when verified with the locations of active cases during march 20 and march 29, 2020. however, the weakness of the svm model lies in managing a very large dataset and inferring with the model outcome that is due to the black box nature of the model. the gr average for active cases in fars for a period of 107 days was 1.15, whilst it was 1.06 in the country and the world. the iranian government should take restrict preventive measures for controlling the outbreak of sars-cov-2 in shiraz as a tourism destination and the counties having high risk. based on the results of polynomial and an arima model, the infection behavior is not expected to reveal an explosive process, however; the general trend of infection will last for several months especially in iran as a whole. a more slowly trend is expected in fars province, demonstrating extensive home quarantine and travel and movement restrictions were good strategies for disease control in fars province. the main policy implication is that the infection cases, to some extent, may be controlled using more effective measures. although, the estimated models may be used to predict the infection in following days, however; this contribution is less significant than the other implications derived from them. generally speaking, it is expected to encounter a decreasing trend, however; this may be reversed if the ongoing attempts are slowed down, pointing out the need to keep the measures like quarantine or even to try more restricting attempts. as a policy implication, it is worth noting that the applied models clearly show the extent that the measures taken by the central and provincial governments body have been efficient, allowing them to consider more effective measures. this contribution will be more valuable when the dynamic and the complicated nature of the virus is taken into consideration. several extensions may be recommended for further investigation. it is possible to apply the developed models to examine the behaviour of other related variables including recovered cases and critical cases. if more detailed data is provided, the effectiveness of the location-specific measures deserves to be investigated more deeply. supporting information s1 file. (xlsx) early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia effects of temperature variation and humidity on the death of covid-19 in wuhan clinical features of patients infected with 2019 novel coronavirus in wuhan, china. the lancet a novel coronavirus outbreak of global health concern. the lancet world health organization declares global emergency: a review of the 2019 novel coronavirus (covid-19) who characterizes covid-19 as a pandemic who, 2020b. coronavirus disease 2019 covid-19 and italy: what next? the lancet mapping the incidence of the covid-19 hotspot in iran-implications for travellers. travel medicine and infectious disease covid-19 battle during the toughest sanctions against iran chloroquine and hydroxychloroquine in the treatment of covid-19 with or without diabetes: a systematic search and a narrative review with a special reference to india and other developing countries mass gathering events and reducing further global spread of covid-19: a political and public health dilemma. the lancet covid-19: challenges to gis with big data. geography and sustainability identification of suitable areas for the occurrence of rift valley fever outbreaks in spain using a multiple criteria decision framework. veterinary microbiology mers-cov geography and ecology in the middle east: analyses of reported camel exposures and a preliminary risk map ecological niche modeling for filoviruses: a risk map for ebola and marburg virus disease outbreaks in uganda understanding spatial variations of malaria in vietnam using remotely sensed data integrated into gis and machine learning classifiers mapping the transmission risk of zika virus using machine learning models machine learning methods reveal the temporal pattern of dengue incidence using meteorological factors in metropolitan manila prediction mapping of human leptospirosis using forecasting the prevalence of covid-19 outbreak in egypt using nonlinear autoregressive artificial neural networks spatial modelling, risk mapping, change detection, and outbreak trend analysis of coronavirus (covid-19) in iran (days between application of the arima model on the covid-2019 epidemic dataset suttearima: short-term forecasting method, a case: covid-19 and stock market in spain climate variability and salmonellosis in singapore-a time series analysis applied econometric times series the mcgraw−hill has sars infected the property market? evidence from hong kong hepatitis a incidence, seroprevalence, and vaccination decision among msm in amsterdam, the netherlands a computational approach to investigate patterns of acute respiratory illness dynamics in the regions with distinct seasonal climate transitions mapping the spatial distribution of aedes aegypti and aedes albopictus identifying outbreaks of porcine epidemic diarrhea virus through animal movements and spatial neighborhoods testing a new ensemble model based on svm and random forest in forest fire susceptibility assessment and its mapping in serbia's tara national park prioritization of effective factors in the occurrence of land subsidence and its susceptibility mapping using an svm model and their different kernel functions review on landslide susceptibility mapping using support vector machines. catena comparative study of svms and anns in aquifer water level prediction text categorization with support vector machines: learning with many relevant features association between ambient temperature and covid-19 infection in 122 cities from china key: cord-295201-u2dola34 authors: morimoto, konosuke; suzuki, motoi; ishifuji, tomoko; yaegashi, makito; asoh, norichika; hamashige, naohisa; abe, masahiko; aoshima, masahiro; ariyoshi, koya title: the burden and etiology of community-onset pneumonia in the aging japanese population: a multicenter prospective study date: 2015-03-30 journal: plos one doi: 10.1371/journal.pone.0122247 sha: doc_id: 295201 cord_uid: u2dola34 background: the increasing burden of pneumonia in adults is an emerging health issue in the era of global population aging. this study was conducted to elucidate the burden of community-onset pneumonia (cop) and its etiologic fractions in japan, the world’s most aged society. methods: a multicenter prospective surveillance for cop was conducted from september 2011 to january 2013 in japan. all pneumonia patients aged ≥15 years, including those with community-acquired pneumonia (cap) and health care-associated pneumonia (hcap), were enrolled at four community hospitals on four major islands. the cop burden was estimated based on the surveillance data and national statistics. results: a total of 1,772 cop episodes out of 932,080 hospital visits were enrolled during the surveillance. the estimated overall incidence rates of adult cop, hospitalization, and in-hospital death were 16.9 (95% confidence interval, 13.6 to 20.9), 5.3 (4.5 to 6.2), and 0.7 (0.6 to 0.8) per 1,000 person-years (py), respectively. the incidence rates sharply increased with age; the incidence in people aged ≥85 years was 10-fold higher than that in people aged 15-64 years. the estimated annual number of adult cop cases in the entire japanese population was 1,880,000, and 69.4% were aged ≥65 years. aspiration-associated pneumonia (630,000) was the leading etiologic category, followed by streptococcus pneumoniae-associated pneumonia (530,000), haemophilus influenzae-associated pneumonia (420,000), and respiratory virus-associated pneumonia (420,000), including influenza-associated pneumonia (30,000). conclusions: a substantial portion of the cop burden occurs among elderly members of the japanese adult population. in addition to the introduction of effective vaccines for s. pneumoniae and influenza, multidimensional approaches are needed to reduce the pneumonia burden in an aging society. guardians. the requirement for obtaining written consent from all participants was waived by all irbs because of the study's observational nature without any deviation from the current medical practice. hospital doctors verbally described the study objectives and methods to eligible patients and their guardians during their consultations. we also provided the necessary information to patients and their guardians using a standardized questionnaire sheet and a poster presentation at the outpatient department. anonymized data were used for the analysis. according to the national statistics, the total population in japan was 127 million in 2013 [7] . of this population, 25.1% were !65 years of age, and 3.6% were !85 years of age [7] . although no national recommendation for the 23-valent polysaccharide pneumococcal vaccine (ppv23) existed at the time of study, the cost of ppv23 was partially or fully subsidized by the local government. the estimated coverage rate of ppv23 for adults aged !65 years was approximately 25% in 2013 [15] . the study was conducted at four community-based hospitals in four prefectures (hokkaido, chiba, kochi, and nagasaki) in japan from september 2011 through january 2013. one hospital is located on each of the four main islands (ebetsu city hospital in hokkaido, kameda medical center in honshu, chikamori hospital in shikoku, and juzenkai hospital in kyusyu). because of japan's universal health insurance system, 70% of the medical costs for people aged <70 years and 80-90% of the medical costs for people aged !70 years are covered, regardless of whether the individual is treated in the private or public sector [8] . therefore, we assume that the characteristics of the pneumonia patients visiting these hospitals do not significantly differ from those visiting neighboring hospitals. all the outpatients were screened by hospital physicians, and eligible patients were identified using a standardized case definition. patients who fulfilled all the following criteria were enrolled in the study: 1) age !15 years, 2) with symptoms compatible with pneumonia (e.g., fever, cough, sputum, pleuritic chest pain, and dyspnea), and 3) with new pulmonary infiltrates on chest x-ray (cxr) or computed tomography (ct) scan films that were consistent with pneumonia. to ensure that all eligible cases were enrolled, the study investigators screened the hospital database for international classification of diseases, 10th revision (icd-10) codes and reviewed hospital medical records. all the enrolled cases were classified into cap and hcap groups according to the definitions in the ats/idsa guideline [16, 17] . if a patient developed the disease 48 hours after admission, he or she was classified as having hap and was excluded. repeated episodes of pneumonia in the same patient within a 2-week period were regarded as a single episode. demographic and clinical information were collected from patients and medical charts using a standardized data collection form. sputum and blood samples were collected from the participants on admission; sputum was induced with the inhalation of hypertonic saline solution if the patients were unable to cough up sputum. cxrs were taken from all patients within 24 hours of admission, and ct scans were ordered by clinicians based on their judgment. clinical specimens were immediately transported to the laboratory at each hospital. gram staining was performed on each sputum specimen, and the specimen quality was evaluated by trained laboratory technicians according to miller and jones' classification [18] . all sputum samples were examined using semi-quantitative or quantitative culture methods. sputum samples were further tested at the institute of tropical medicine, nagasaki university, using in-house multiplex polymerase chain reaction (pcr) assays to identify bacterial and viral pathogens. three typical bacterial pathogens (s. pneumoniae, h. influenzae, and m. catarrhalis), three atypical bacterial pathogens (mycoplasma pneumoniae, chlamydophila pneumoniae, and legionella pneumophila), and thirteen viral pathogens ( [19, 20] . we also performed urinary antigen tests for s. pneumoniae and l. pneumophila using commercial kits (binax now streptococcus pneumoniae, binax now legionella; alere inc., waltham, ma, usa). all pneumococcal isolates were serotyped using the capsular quelling method. the etiological category of pneumonia was defined according to the microbiological findings of sputum, blood, and urine samples. the causative pathogens were determined by hospital clinicians and study investigators. because no culture, pcr or rapid urine test is perfect, we estimated the prevalences of s. pneumoniae and h. influenzae using two different methods: 1) positivity was defined if either a sputum culture or a urinary antigen test showed a positive result (standard estimation), and 2) positivity was defined if a sputum culture, sputum pcr, or urinary antigen test showed a positive result (maximum estimation). to estimate the etiology-specific incidence of pneumonia, aspiration-associated pneumonia was defined independent of microbiological profiles. cases were classified as aspirationassociated pneumonia when the patients had any of the following known risk factors: episodes of aspiration, the presence of dysphagia, consciousness disturbances, neuromuscular diseases, cerebrovascular diseases, tube feeding, and bedridden status [21] . the prevalence of aspiration-associated pneumonia was calculated separately from those of pathogenspecific category. the age-group specific incidence rates of pneumonia, hospitalization and death in the four prefectures were estimated using the surveillance data and the national statistics. we used the pneumonia-outpatient ratio (por)-based estimation model that was used in our previous study [22] . in this model, the number of age group-specific pneumonia cases was estimated using the age group-specific number of outpatients and the age group-specific por. pors were calculated for hospitals and clinics separately. the model used to estimate the number of pneumonia outpatient visits was as follows: c ijk : the annual number of outpatients reported to the patient survey in age group i, prefecture j, and facility type k (k = 1, hospitals; k = 2, clinics) α ijk : the ratio of confirmed pneumonia patients to the number of total outpatients in age group i, prefecture j, and facility type k p ij : the population in age group i and prefecture j c ijk was obtained from the patient survey conducted by the mhlw in 2011 [23] . the survey was conducted at hospitals and clinics on one designated date set for each hospital on one of three days in october. p ij was obtained from the national demographic survey in 2012 [7] . the proportion of pneumonia cases among outpatients in hospitals (α ij1 ) was calculated from the surveillance data. to estimate the proportion of pneumonia cases among outpatients in clinics (α ij2 ), we obtained the clinic databases from two clinics in nagasaki city. pneumonia diagnosis was confirmed using the case definition identical to that used for the hospital-based surveillance. α was estimated for each age group using curve fitting. age-standardized rates were calculated using the who world standard population [24] . the number of pneumonia cases in the entire japanese population was estimated assuming that the incidence rates (i ij ) were constant across all prefectures. during the study period, a total of 932,080 patients visited the study hospitals; 1,935 of these patients were enrolled in the study. after excluding 163 cases that did not meet the criteria, 1,772 patients were eligible for our analysis (see supplementary material, s1 fig) . ct scans were administered to 1,332 patients (75.2%), including 110 (8.3%) who did not demonstrate any infiltrates on their cxrs. the demographic and clinical characteristics of the enrolled cases are shown in table 1 . fifty-nine percent of all patients were male, and the median age was 77 years (range: 15 to 103). seventy-five percent of the patients were elderly people aged !65 years, and 57% were aged !75 years. the case fatality rate was 8%. seventy-two percent of our cases were hospitalized, and these cases were more likely to be male, older, smokers, and classified as having hcap; more likely to have underlying conditions, aspiration-associated conditions, and severe conditions; more likely to visit the hospital early; and more likely to have a fatal outcome compared with outpatients. among 1,594 sputum samples tested using conventional cultures, causative bacterial pathogens were isolated from 759 (48%) samples; 719 (45%) were monoclonal and 40 (3%) were polyclonal (table 2 ). h. influenzae was the most common bacterial pathogen isolated (10%), followed by s. pneumoniae (9%). h. influenzae was more frequently isolated from outpatient cases, while staphylococcus aureus, klebsiella pneumoniae, and escherichia coli were more frequently isolated from hospitalized cases. among the 718 sputum samples tested using multiplex pcr, 359 (50%) were positive for any bacterial pathogens; 20% were positive for s. pneumoniae, and 18% were positive for h. influenzae. h. influenzae and m. pneumoniae were more frequently detected in samples from outpatient cases than those from hospitalized cases. s. pneumoniae was isolated by blood culture from 2.9% (n = 7/1,039) of cases, and s. pneumoniae urinary antigen was detected in 13% (n = 132/992). taken together, 26.2% of samples were positive for s. pneumoniae either by culture, pcr, or urinary antigen tests. among 142 s. pneumoniae isolates, 100 were serotyped; serotype 3 was the most dominant (n = 22, 22%), followed by serotypes 11a (n = 10, 10%) and 19f (n = 8, 8%). ppv23 covered 67% of all serotypes, and 54% were covered by 13-valent pneumococcal conjugate vaccine (pcv13) (see supplementary material, s2 fig) . multiplex pcr was used to test 1,201 sputum samples for rvs, and 23% of the samples were positive for at least one rv (table 2) . hrv was the leading virus identified, followed by infa and rsv. the positivity rates for rvs were similar between the outpatient cases and the hospitalized cases. among 717 sputum samples tested for viral and bacterial pcrs, 105 (15%) were both positive. among all 1,722 patients, 677 (38.2%) were with aspiration-associated conditions (ie, aspiration-associated pneumonia): 373 (21.1%) had episodes of aspiration, 149 (8.4%) dysphagia, 81 (4.6%) consciousness disturbances, 143 (8.1%) neuromuscular diseases, 373 (21.1%) cerebrovascular diseases, 21 (1.2%) tube feeding, and 148 (8.4%) bedridden status. among 629 sputum samples available from aspiration-associated pneumonia, staphylococcus aureus was the most common bacterial pathogen isolated by culture (10%), followed by s. pneumoniae (7%) and klebsiella pneumoniae (7%) (see supplementary material, s1 table) . s. pneumoniae and h. influenzae were less frequently detected in samples from aspiration-associated pneumonia than those from non-aspiration-associated pneumonia by culture and pcr. the burden of cop the overall annual incidence rate of adult pneumonia in the four prefectures was 16.9 per 1,000 py (95% ci, 13.6 to 20.9 per 1,000 py) in japan, and the asr was 10.2 (7.7 to 13.3; table 3 ). the incidence rate was highest in kochi (22.8 per 1,000 py) and lowest in chiba (14) 33 (14) 65 (14) 0.855 m. pneumoniae 38 (5) 24 (10) 14 (3) <0.001 (13.7 per 1,000 py), while the asr did not differ significantly by prefecture, ranging from 9 to 11.5 per 1,000 py (see supplementary material, s2 table) . the rate was higher in males than in females (15.6 vs 9.3 per 1,000 py in all age groups; 120.6 vs 36.4 per 1,000 py in people aged !85 years). it was lowest in those aged 35-44 years (4.5 per 1,000 py), increased sharply with age, and became highest in the population aged !85 years (79.3 per 1,000 py; 95%ci, 65.7 to 95.5; fig 1) . this age-associated increase in the rate was more apparent in males than in females (the point estimates of the rate ratios comparing males and females in people aged 55-64 years, 65-74 years, 75-84 years, and 85 years were 1.6, 2, 2.4, and 3.2, respectively). this increasing trend was also observed for pneumonia-related hospitalizations and in-hospital death. for the etiology-specific incidence estimates, the etiology-associated incidence rate was calculated according to age group (table 3) . although aspiration-associated pneumonia had the highest incidence, there were substantial numbers of infection-associated pneumonia cases among older people; s. pneumoniae-, h. influenzae-, and rv-associated pneumonia followed. the exception was atypical pneumonia; the incidence of atypical bacteria-associated pneumonia was highest among younger people (aged 15-54 years). in our study population, only 7 out of 1,039 cases had positive blood cultures, indicating that the incidence of bacteremic pneumococcal pneumonia was 12 per 100,000 py (95% ci, 9.8 to 14.5 per 100,000 py). assuming that these proportions of pneumonia etiologies were constant across all prefectures, the estimated annual number of cop in the entire japanese adult population was 1,880,000; of these, 1,300,000 cases (70%) occurred in people aged !65 years (fig 2) . seventy percent were hospitalized cases, and 70% of all pneumonia cases were cap. among cop cases, 74,000 died in hospitals. 630,000 cases were aspiration-associated, and 90% of patients with this condition were aged !65 years. 530,000 cases were s. pneumoniae-associated, 420,000 were h. influenzae-associated, and 420,000 were rv-associated pneumonia. to our knowledge, this study is the first to estimate the burden of pneumonia in the entire japanese adult population. in 2012, the overall incidence of community-onset pneumonia, including cap and hcap, among people aged !15 years was 16.9 per 1,000 py; the incidence sharply increased with age and reached up to 79.3 per 1,000 py among people aged >85 years. the estimated annual number of adult cop patients in japan was 1,880,000, 70% of which were elderly people aged !65 years. aspiration was the leading etiologic category of pneumonia, though a substantial number of cases were still associated with infections, such as s. pneumoniae. our findings clearly indicate that pneumonia is an age-related disease that causes an enormous burden in this aging population. the incidence of pneumonia in japanese elderly people was higher than the incidences observed in large-scale population-based studies in both the united states and european countries. in japan, the incidence of cop among people aged !65 years was 42.3 per 1,000 py; in contrast, cop incidences were 28.4 per 1,000 py in the united states [25] , 14 per 1,000 py in spain [26] , and 8 per 1,000 py in the united kingdom [27] . the high pneumonia incidence in japan may be partially explained by its high proportion of extremely elderly people aged !85 years (14% of the elderly population in 2012 [7] ). however, the trend did not fundamentally change after the incidence was standardized using the who world standard population: the asrs of cop among the elderly in japan, the united states, spain, and the united kingdom were 37.1, 25.3, 13.5, and 7.2 per 1,000 py, respectively. the in-hospital mortality rate for cop in japan (11.5%) was lower than that reported for other countries (12.4% in the united states [25] , 15% in spain [26] , and >25% in the united kingdom [28] ). these findings suggest that patients with mild pneumonia cases that may be overlooked in other countries are visiting clinics and being diagnosed in japan. in fact, 75% of our pneumonia cases were examined with ct scans to diagnose pneumonia. the good access to healthcare facilities resulting from universal health coverage and the wide use of sensitive diagnostic tools may explain this high incidence. however, it does not indicate that the hospitalized cases in japan are milder than those in other countries; the proportion of severe cases (curb65 score !3) in the current study was 30%, compared with 38% in the united kingdom [29] and 10% in spain [30] . the pneumonia incidence and in-hospital morality were higher among males than among females, especially among the older age group, confirming previous works [4, 25, 26] . considering the higher incidence of childhood pneumonia among males, they may be genetically vulnerable to pneumonia; however, there is no evidence to support this hypothesis. according to our estimates, aspiration was the leading cause of pneumonia, and the burden of pneumonia associated with aspiration was higher than that associated with any single pathogen, including s. pneumoniae. the burden was particularly high among the elderly population; 85.8% of aspiration-associated pneumonia cases occurred in patients aged !65 years. the inhospital mortality for aspiration-associated pneumonia (10.9%) was higher than that for other pneumonia categories (6%). aspiration-associated pneumonia has been overlooked in current pneumonia control programs. although previous studies have shown that this condition is common among hospitalized pneumonia patients [2, 31] , its burden has never been evaluated at the population level in the past. aspiration-associated pneumonia is a multi-factorial condition observed in older people. impaired swallowing and an abnormal cough reflex increase the risk of oropharyngeal aspiration; the aspiration of colonized pathogens and gastric acid causes lower respiratory tract infection and/or lung injury [12] . compromised immunity, comorbidity and changes in lung function in this age group underlie this condition and are associated with the high mortality. nursing home residents are at high risk for aspiration, but hcap and aspiration-associated pneumonia are not identical conditions. in fact, in our study, 25.4% of cap and 64.3% of hcap cases were associated with aspiration. effective clinical management and preventive measures targeting aspiration-associated pneumonia remain underdeveloped. ats guidelines recommend using β-lactam/β-lactamase inhibitors for this condition [1, 13] , but the management of recurrent and refractory cases is challenging. for prevention, oral hygiene care and dysphagia rehabilitation have been suggested for reducing the risk of aspiration pneumonia, but with limited supporting evidence [32] . the burden of aspiration-associated pneumonia may further increase as the number of elderly people who require long-term care increases. effective clinical and public health intervention measures are urgently needed. in the current study, s. pneumoniae was the leading single etiological pathogen and was associated with 20-28% of pneumonia, confirming previous reports [4] . recent studies in japan have shown that the positivity of s. pneumoniae among cap cases was 17% [33] to 24% [34] . according to a recent meta-analysis, the proportion of pneumococcal pneumonia among cap cases was 26-28% [9] . the proportion of pneumococcal pneumonia among all pneumonia cases is declining in high-income countries, reflecting the wide use of antibiotics and pneumococcal vaccines [11] . in our study, the positivity of s. pneumoniae by sputum culture was only 9%. considering the low sensitivity of sputum culture, we included urinary antigen test-positive cases for the standard estimation and further included pcr-positive cases for the maximum estimation. the true value must lie between these values (i.e., 20 to 28%). the proportion of bacteremia among pneumococcal pneumonia cases was 6% in our study. a meta-analysis showed that approximately 25% of pneumococcal pneumonia is bacteremic [9] ; our figure was lower than this estimate. however, our results showed that the incidence of bacteremic pneumococcal pneumonia among japanese adults was 12 per 100,000 py, a figure that was comparable with those reported for other countries, such as the united states [35] and australia [36] . the findings suggest that pneumococcal pneumonia, either bacteremic or non-bacteremic, remains the leading target for pneumonia control programs in japan. ppv23 reduces the risk of invasive pneumococcal diseases (ipds) among adults; however, its effectiveness against pneumococcal pneumonia is still controversial, particularly for the elderly [37] . the recently approved pcv13 is expected to prevent almost half of the pneumococcal pneumonia cases in the elderly [38, 39] ; however, the vaccine covers only 13 serotypes of pneumococcus, and its long-term effects remain unknown. in japan, before the introduction of pcv7 for children in 2010, 85% of ipd isolates were ppv23 serotypes, and 62% were pcv13 serotypes [40] . in the current study, 67% of the isolates were ppv23 serotypes, and 54% were pcv13 serotypes. the vaccination policy for pneumococcus has been dramatically changing in japan. pcv7 for children was replaced by pcv13 in late 2013, and ppv23 was also included in the ministry of health, labour and welfare recommended vaccines for elderly people in late 2014. the proportion of vaccine-covered serotypes is known to decline after widespread use of pcv [41] ; thus, these figures will decrease in coming years. the true efficacy of pcv13 for adult pneumonia among the japanese population must be evaluated along with cost-effectiveness analyses before it is introduced into the national immunization program. a substantial proportion of pneumonia was associated with rvs (23% of all pneumonia cases). recent studies suggest that rvs play crucial roles in the development of pneumonia, including severe cases [10, 22, [42] [43] [44] ; however, their biological mechanisms remain largely unknown. rvs such as influenza, rsv, and human metapneumovirus (hmpv) cause outbreaks among the elderly in nursing homes [45, 46] , and these rvs are potential targets for vaccination. currently, only seasonal influenza vaccines are available for adults, but their effects on pneumonia prevention have not yet been established [47] . further investigations are needed to clarify the public health impact of rv-associated pneumonia in aging societies. our findings have important implications for effective pneumonia control programs in the aging society. the burden of pneumonia is higher in older people, and the pneumonia etiology largely varies by age group: the incidences of aspiration-, s. pneumoniae-, h. influenzae-, rv-, and pdr pathogen-associated pneumonia increase with age, while the incidence of atypical bacteria-associated pneumonia decreases. it must be noted that the proportion of pneumonia caused by unknown pathogens is higher among elderly people. this category most likely represents multifactorial conditions. therefore, in coming decades, the pneumonia burden will likely increase, and its etiology will become more diverse. in this situation, the current etiologyspecific approach (i.e., vaccinations for pneumococcus and influenza, guidelines for appropriate antibiotics use) must have only a limited impact. a multidimensional approach integrating vaccination programs, clinical management guidelines, training for health care workers, and education for people must be needed; further studies are warranted. this study is the first to estimate the national burden of cop in japan. although pneumonia is a common disease, its true burden remains unclear, even in high-income countries. a number of studies have reported the incidence of adult pneumonia, but their estimates substantially varied from setting to setting [4, 22, 25-28, 30, 48, 49] . several factors explain this variation. first, the definition of pneumonia differs among studies. some studies have reported incidences of cap that include outpatients and hospitalized patients [25, 30, 49] , while other studies have reported hospitalized cases only [28, 48] . it was not clear whether these studies included hcap cases. additionally, the diagnosis of pneumonia is not standardized in clinical settings; thus, the burden estimates based on existing database are unreliable. second, study designs vary. pneumonia is a common disease, and it is not included in national surveillance. cohort studies may not represent the entire population of a country, while hospital-based studies do not capture all the cases in the community. different designs may produce different estimates in an identical population. third, the health care-seeking pattern affects the incidence estimates. mild cases must be overlooked in countries in which access to health care is limited. in the current study, we enrolled pneumonia cases prospectively, and all were confirmed by study clinicians using the standardized case definition. considering the high reliability of national statistics in japan, our estimates can be reasonably assumed to reflect the true pneumonia burden among japanese adults. our study has limitations. for the incidence estimation, we assumed that the pneumonia-outpatient ratios in the study hospitals were constant across all hospitals in the four prefectures. additionally, to calculate the national burden, we assumed that the incidence of cop in the four prefectures was constant across all prefectures. our hospitals were community-based general hospitals that provided primary, secondary, and tertiary care for residents; thus, the patients visiting these hospitals reflected the general population. in fact, the asrs in the four prefectures were almost identical in our study. furthermore, according to the national patient survey, the proportion of reported acute respiratory infections among all outpatients in the four prefectures was identical to the national average (see supplementary material). we believe that these assumptions are reasonable. in contrast, we did not consider seasonal differences in the pneumonia etiology. our etiology-specific burden estimates must be confirmed using multi-year surveillance data. in this study, aspiration-associated pneumonia was defined based on the presence of known risk factors; thus, we could not distinguish between "aspiration pneumonia" caused by aspiration of colonized oropharyngeal pathogen or "aspiration pneumonitis" caused by aspiration of gastric contents [12] . however, there is no reliable marker to identify aspiration [2] . further studies are needed to better define this pneumonia category. a substantial portion of the cop burden in the japanese adult population occurs in the elderly. aspiration was the leading etiology of pneumonia, followed by s. pneumoniae. in addition to the introduction of vaccines for s. pneumoniae and influenza, multidimensional approaches are urgently needed to reduce the pneumonia burden in this aging society. supporting information s1 database. database of community-onset pneumonia cases. table. microbiological profiles of patients with community-onset pneumonia with and without aspiration-associated conditions. 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pathogens we are grateful to all the laboratory staff at the participating hospitals. we would like to thank rina shiramizu and kyoko uchibori for performing pcr and yumi yamasaki for administrative work. we also wish to thank hidefumi yamamoto and masatoshi ide for sharing their data. adult pneumonia study group-japan (apsg-j) are: masahiko abe 1 , takao wakabayashi 1 , masahiro aoshima 2 , naoto hosokawa 3 , norihiro kaneko 2 , naoko katsurada 2 , kei nakashima 2 , yoshihito otsuka 4 , eiichiro sando 5 , kaori shibui 5 , daisuke suzuki 3 , kenzo tanaka 6 , kentaro tochitani 3 , makito yaegashi 5 , masayuki chikamori 7 , naohisa hamashige 7 , masayuki ishida 7 , hiroshi nakaoka 7 , norichika aso 8 , hiroyuki ito 8 , kei matsuki 8 , yoshiko tsuchihashi 8 , koya ariyoshi 9 , bhim g dhoubhadel 9 , akitsugu furumoto 9 , sugihiro hamaguchi 1,9 , tomoko ishifuji 9 , shungo katoh 1,9 , satoshi kakiuchi 9 , emi kitashoji 9 , takaharu shimazaki 9 , motoi suzuki 9 , masahiro takaki 9 , konosuke morimoto ã9 , kiwao watanabe 9 , lay-myint yoshida 10 key: cord-286837-j2sqs20q authors: koetsier, antonie; van asten, liselotte; dijkstra, frederika; van der hoek, wim; snijders, bianca e.; van den wijngaard, cees c.; boshuizen, hendriek c.; donker, gé a.; de lange, dylan w.; de keizer, nicolette f.; peek, niels title: do intensive care data on respiratory infections reflect influenza epidemics? date: 2013-12-31 journal: plos one doi: 10.1371/journal.pone.0083854 sha: doc_id: 286837 cord_uid: j2sqs20q objectives: severe influenza can lead to intensive care unit (icu) admission. we explored whether icu data reflect influenza like illness (ili) activity in the general population, and whether icu respiratory infections can predict influenza epidemics. methods: we calculated the time lag and correlation between ili incidence (from ili sentinel surveillance, based on general practitioners (gp) consultations) and percentages of icu admissions with a respiratory infection (from the dutch national intensive care registry) over the years 2003–2011. in addition, icu data of the first three years was used to build three regression models to predict the start and end of influenza epidemics in the years thereafter, one to three weeks ahead. the predicted start and end of influenza epidemics were compared with observed start and end of such epidemics according to the incidence of ili. results: peaks in respiratory icu admissions lasted longer than peaks in ili incidence rates. increases in icu admissions occurred on average two days earlier compared to ili. predicting influenza epidemics one, two, or three weeks ahead yielded positive predictive values ranging from 0.52 to 0.78, and sensitivities from 0.34 to 0.51. conclusions: icu data was associated with ili activity, with increases in icu data often occurring earlier and for a longer time period. however, in the netherlands, predicting influenza epidemics in the general population using icu data was imprecise, with low positive predictive values and sensitivities. a limitation of current influenza surveillance systems is that timely information on severe influenza illness requiring hospital admission is not available. influenza surveillance in most countries is based upon sentinel general practitioners (gp) networks and the collected information on influenza like illness (ili) is dependent on the health care seeking behavior of the general population, which can fluctuate with for example media attention. the implementation of a hospital based surveillance system for severe acute respiratory infection (sari) is now promoted by the world health organization (who) and european centre for disease prevention and control (ecdc) as a public health priority worldwide, both for routine surveillance and for preparedness [1, 2] , such as in the case of the middle east respiratory syndrome coronavirus (mers-cov). sari surveillance can focus on admissions for respiratory infections in general hospital wards or in intensive care units (icu) . during the pandemic period, hospitalization for laboratory confirmed influenza a(h1n1)pdm09 infection was notifiable in the netherlands. in the season 2009/2010 as well as in the season 2010/2011, ili incidence as measured by gp sentinel practices, reached the epidemic threshold of 5.1 consultations per 10.000 enlisted patients at a time when already more than 100 patients had been hospitalized, with several icu admissions and deaths from laboratory confirmed influenza (national institute for public health and the environment, unpublished surveillance data). hospital admission for influenza is not notifiable anymore and in the netherlands sari cases are not routinely collected. an alternative source of information could be the dutch national intensive care evaluation registry (nice) [3], wherein diagnostic, and physiologic information from the first 24 hours of adult icu admissions, as well as length of stay and in-hospital mortality of all icu patients are registered. patients are admitted to the icu if they have a high severity of illness, and require constant monitoring of their vital functions, regardless of their expected outcome. respiratory infections, such as pneumonia, are among the most common conditions for which patients are admitted to the icu. in the early onset of an influenza epidemic, patients with multiple comorbidities are more likely to develop more severe influenza related diseases like pneumonia. they possibly get submitted to the icu before there is an epidemic in the general population [4] . therefore, increases in the number of admissions at the icu with a respiratory infection can possibly occur before a detectable increase in ili incidence in the gp sentinel network. in this study we explore whether icu data on respiratory infections reflect ili activity in the general population, and which relevant time lag exists between both data sources. additionally we assessed whether icu data can predict ili defined influenza epidemics (further referred to as influenza epidemics). in the netherlands, patients that consult the gps with symptoms of ili are reported on a weekly basis to the continuous morbidity registration sentinel general practice network (further referred to as sentinel gp registry), covering 0.8% of the dutch population being nationally representative by age, gender, regional distribution, and population density [4] . the sentinel gp data consists of the weekly number of patients presenting with ili at the gp. in 2009, the registry had 42 participating gp practices with 59 gps covering a population of approximately 130,000 patients [4] . ili was defined according to the criteria of pel [5] , by (1) acute onset, with prodromal stadium of three to four days, (2) a temperature increase to at least 38 degrees celsius, and (3) at least one of the following symptoms: cough, nasal congestion, raw throat, frontal headache, retrosternal pain, and body aches. incidence of ili is calculated on a weekly basis, using number of patients registered at the reporting gp practices as the denominator. this is acceptable as almost every person in the netherlands is registered with a gp. in the netherlands, an influenza epidemic is defined as more than 5.1 patients with ili per 10,000 inhabitants per week consulting the gp for at least two consecutive weeks [6] , combined with influenza a virus isolation from laboratory samples. data quality is assured by training of gps for data entry, and a pop-up appearing in the system when an ili diagnosis is entered reminding the gp to register the ili case in the sentinel gp registry. the nice registry was founded in 1996, with initially six participating icus. in 2003 this number had grown to 33 and in 2011 85 icus participated covering approximately 90% of the dutch adult icus. for each admission, among other items, the acute physiology and chronic health evaluation ii (apache ii) [7] reason for icu admission diagnosis is registered and sent to the nice registry on a monthly basis. upon receipt, the data is usually entered into the nice registry database and available for participants within one day. there is currently no distinct variable describing whether an icu patient has influenza like illness or was diagnosed with an influenza virus infection, or receives antiviral drugs. therefore, we defined an icu admission with a possible sari (further referred to as icu admission with respiratory infection) when the following four criteria were met: (1) the patient was admitted to the hospital less than two days before icu admission, (2) there was a medical (non-surgical) reason for admittance, (3) the icu admission was not a readmission to the icu within the hospitalized period, and (4) the apache ii reason for admission was 'respiratory infection. we used the percentage of icu admissions with a respiratory infection (relative to the total number of medical icu admissions), instead of the absolute number of icu admissions to adjust for the growing number of nice registry participants throughout the study period. thus our study dataset included study year, week number, number of patients with ili, population size of reporting gps, number of icu admissions with respiratory infection, and number of medical icu admissions. data quality is assured by regular on site visits of the icus [8] . in this study, we used weekly time series of patients presenting with ili from the sentinel gp registry and icu admissions of respiratory nature from the nice registry. in the nice registry, few icus were participating in the years 2000 until 2002, leading to large variations in the percentage of icu admissions with respiratory infections. therefore, from both registries, we used data from 2003 through 2011. as influenza generally occurs between week 40 and week 20 of the subsequent year [9] , we defined an influenza year (i.e. season) from july 1st until june 30th the next year, thereby having ten influenza years in our dataset. to explore the association between the weekly incidence of ili patients and the percentage of icu admissions with a respiratory infection, we plotted per week the percentage of icu admissions with a respiratory infection and the incidence of ili over the period january 1, 2003, through december 31, 2011. in addition, we performed a generalized estimating equations (gee) [10] additive poisson regression analysis [11] using an autoregressive working correlation matrix over this time period. the dependent variable was weekly incidence of patients with ili, and the independent variables were chronological week number, percentage of icu admissions with respiratory infection in the current week, and one to five weeks before the current week and one to five weeks after the current week, and sine and cosine terms to adjust for seasonality [12] . the sine term was sin(k2pt/t) and the cosine was cos(k2pt/t), where k is a constant with values 1 (yearly seasonality) or 2 (half year seasonality), t is current week number, and t is total number of weeks in the specific influenza year, e.g. 52 weeks (years 2004 and 2009 had 53 weeks, and the cases were added to week 52). we adjusted for autocorrelation in the residuals, where the unit of clustering was influenza year. we calculated the average time lag between the sentinel gp and icu data by computing the weighted average of the time lag in weeks (25, …, 0, …, +5), using the corresponding regression coefficients as weighting factors. the r-squared (r 2 ) value based on the deviance residuals [13] was also calculated. to assess the possibility of using icu data for predicting influenza epidemics, we used a subset of three years of training data to develop three gee models with the same characteristics as the aforementioned model, to predict the incidence of ili patients one to three weeks ahead. in each of these models, independent variables were removed by pseudo stepwise selection [14] , using a fixed scheme for removal. we first considered the time trend (chronological week number) for removal, then seasonal terms, and finally time lagged percentage of icu admissions with respiratory infection. in order for the final models to be useful in surveillance, the following restrictions also applied: (1) the percentages of icu admissions with respiratory infection one to five weeks after the current week were not included as they are unavailable and useless for prospective surveillance, (2) the percentage of icu admissions with respiratory infection in the current week cannot be removed from the model, (3) additional variables of lagged icu admissions should correspond to a range of subsequent weeks (e.g., one and two weeks before the current week, not one and three weeks before the current week), and (4) a seasonal term is always a combination of a sine and cosine function. we used data of the first three influenza years (january 1, 2003 1, , through june 30, 2005 to generate the final model for predicting the incidence of ili one week ahead. to accomplish this, we varied the decay factor l of the full model, giving weeks further back in time exponentially lower weights, from 0.97 to 1.00 with increments of 0.005, resulting in seven candidate models. variable selection for these seven models was performed with pseudo stepwise selection, with the quasi-likelihood information criterion (qic) [15] as performance measure. to determine the optimal value of l, 10-fold cross validation [16] was performed for the seven candidate models using the same three influenza years that they were built with. the model with the best r 2 value was selected as final model. the above steps were repeated for variable selection of the models predicting ili two and three weeks ahead. using the final models based on the 3 training years of data we started predicting the incidence of ili patients week by week starting from the fourth influenza year in our dataset (july 1, 2005) onward. before predicting each successive week, the model parameters were recalculated with an updated dataset that included the data of the previous week to make the model dynamic. we continued updating the model parameters week by week, until all remaining seven influenza years were predicted. from the fourth influenza year onward, the predicted incidence of ili patients was plotted together with the observed incidence of ili patients. we used the same threshold as the ili sentinel surveillance to define an influenza epidemic in the predicted ili numbers (incidence . = 5.1 ili patients per 10,000 inhabitants for at least two consecutive weeks). we compared the predicted epidemic weeks using icu data with observed epidemic weeks based on ili data. accordingly, we calculated the positive predictive value (ppv), and sensitivity of the predicted epidemic weeks on a weekly basis. for comparison, we also predicted the weekly incidence of ili with models that used only seasonal terms and auto-regressive ili variables, but excluded icu data. these models were also created with pseudo stepwise selection. the resulting ppv and sensitivity were also calculated. the statistical analyses were performed using the statistical package r, version 2.15.1 (http://www.r-project.org/; vienna, austria). in the period january 1, 2003, through december 31, 2011 there were a total of 477,422 icu admissions, of which 133,615 (28.0%) had a medical (non-surgical) reason for admittance. of the medical icu admissions, 17,786 (13.3%) were for a respiratory infection ( table 1 ). the incidence of ili was on average 3.2 per 10,000, with a standard deviation of 2.8, and a minimum of 0.1 per 10,000 and a maximum of 17.5 per 10,000. there were nine epidemics, consisting of 72 weeks in total, and an average length of eight weeks. on average, 43 gps supplied data on ili. both the incidence in ili and percentage of icu admissions with a respiratory infection show a similar timing of seasonal peaks (figure 1 ). the amplitude of the yearly peaks in the icu data were relatively lower than ili peaks, and often lasted for a longer time period. increases in the incidence of ili showed a yearly pattern and increased in a smoother pattern compared to icu data. while trends were roughly comparable, they differed in some instances since peaks in icu data occasionally occurred when ili increases were absent in the general population. the association between the different lags of icu admissions and ili incidence is shown in table 2 (assessed using gee additive poisson regression analysis). the r 2 value of the full model was 0.58. of each variable, the contribution to the r 2 of the full model is also shown, which is the r 2 value of the full model minus the r 2 of the model with the corresponding variable omitted. figure 1 also shows the predicted ili incidence (black line) according to the full model. statistically significant time lags were percentage of icu admissions with respiratory infection were one week before, current week, one week after, two weeks after, and four weeks after current ili incidence. the time lags mostly associated with increases in ili incidence one week before and in the current week, with coefficients of 0.12 and 0.11. for example, if the percentage of icu admissions with a respiratory infection in the current week increased by one percent, then the incidence of ili in the general population increased by 0.11 per 10,000 population. according to the contribution to r 2 , also icu admissions one week later was strongly associated with current ili (coefficient of 0.08). there is no linear time trend present, but seasonality exists in the data reflected by a half year sine function (sine term with k = 2) with a p-value of ,0.01 and a large contribution to r 2 . looking at figure 1 , a yearly time trend would be expected but is now partly reflected in the different icu time lagged variables. using the coefficients of the time lags in table 2 , the average of the weighted relative week numbers was 20.24 weeks implying that the increase in percentage of icu admissions with a respiratory infection was on average 1.68 days earlier than the increase in ili incidence. for our second research question, whether icu data can predict ili incidence ahead in time, we generated three gee models predicting the incidence of ili patients one to three weeks ahead using icu data. table 3 shows these three different gee models, and their ppv and sensitivity. predicting two weeks ahead yields the largest sensitivity of 0.51 and predicting one week ahead has the largest ppv of 0.78. for comparison, models using only auto-regressive ili variables and seasonal terms, showed the sensitivity to range between 0.21-0.22 and the ppv between 0.31-0.37. figure 2 shows three figures plotting the predicted incidence of ili patients one to three weeks ahead versus the actually observed ili incidence. the epidemic threshold of 5.1 patients (or more) with ili per 10,000 population is plotted and the weeks in which an influenza epidemic occurred according to the predicted versus the actual data is shown. predicting one week ahead detected three of the six epidemics, of which two were longer and one shorter according to the icubased predictions. one epidemic was predicted earlier, one at the same time and one later. using icu data to predict two weeks ahead resulted in five of the six epidemics detected, one is missed, and one false epidemic is predicted in the autumn of 2010 (which was not present in the ili data). according to the icu data, the predicted epidemics were longer in time except one. besides, most predictions were shifted in time compared to the actual occurrence: two epidemics were predicted earlier and three later. when predicting three weeks ahead all six epidemics were detected, however four were shorter in length, one longer, and one had the same length, again shifted in time: two epidemics were predicted earlier, three later, and one at the same time with icu data. the study showed that the percentage of medical icu admissions for respiratory infection was associated with weekly incidence of ili in the current week, and with one week positive and negative time lag. an increase in the percentage of icu admissions for respiratory infections on average preceded the increase in the incidence of ili (gp data) by 1.68 days, implying that before an epidemic the severely ill influenza cases get admitted to the icu. despite this precedence, our analyses showed that with the current models icu data do not accurately predict influenza epidemics in the general population, but including icu data showed an improvement in sensitivity and ppv compared to only including auto-regressive ili variables and seasonal terms. in our study we built three additive poisson gee regression models with icu data to predict the incidence of ili patients, thereby detecting influenza epidemics and aimed at detecting opportunities for enhancing the current national surveillance method. previous studies also aimed at enhancing their current surveillance of influenza epidemics, using laboratory or hospital data. steiner et al. [17] used an exponentially weighted moving average control chart to enhance and automate influenza epidemic detection. weekly laboratory notifications data of seven years were used instead of the ili data that we studied. the predicted influenza epidemics were compared to retrospective inspection of the same notification data by epidemiologists. the predictions were, just like our study, not the same as their reference data. however in their study there was a maximum of one week difference only, except for one year where there was a difference of eight weeks. a study by closas et al. [18] used a kolmogorov-smirnov test with virologic laboratory data of five years to detect influenza epidemics. the test provides a binary signal indicating epidemic activity and a quantitative measure of its confidence. they sequentially updated the test as new data became available. the results differed one to nine weeks with the retrospective data of the sentinel network, which is comparable to our results. google flu trends also aimed to detect influenza epidemics, but overestimated peak influenza levels [19] whilst our study underestimates peak influenza levels. these methods complement the current surveillance networks, but cannot replace them. a study by van den wijngaard et al. [20] did not aim to predict influenza epidemics, but instead explored whether excesses in influenza severity per season can be detected by combining gp, hospital, laboratory, and mortality data (7 years of data). their finding was that combining these data sources is of added value, allowing for better understanding of increases in severe morbidity and mortality due to influenza infections. also from our data we see that trends in icu related sari differ from the trends of ili in the general population and may thus be of value in offering additional information on severity of influenza seasons which need to be explored further. however, both respiratory icu admissions and ili in the general population are not necessarily caused by influenza alone. microbiological laboratory results would provide better insight but to date, these data are not available at the icu patient level. the major strength of our study is that we had access to two large historical datasets from the nice registry and the sentinel gp registry. this allowed us to retrospectively analyze ten influenza years of data, which, to our knowledge, is a longer time period than in comparable studies. a second strength of our study is that we used gee in our additive poisson model, thereby correcting for correlations between weeks. the last strength of our method is that for each additional week we sequentially updated the coefficients of the covariates in the model used for prediction of ili, adding a decay factor giving historic data less weight, and adjusted our models for seasonal changes. with these adjustments, our models always incorporated the most recent information on icu and ili trends. a limitation of the nice registry data is that there is no distinct variable describing whether a patient has an influenza like illness or whether an patients has been diagnosed with an influenza virus infection. furthermore it only contains adult patients thus representing an older population compared to the ili surveillance which also includes children. we extracted admissions with a medical respiratory infection, admitted to the icu within two days after hospital admission, and excluding readmissions. these admissions represent community-acquired respiratory infections and, therefore, included influenza virus infections. additionally, the data of the sentinel gp registry is weekly updated, whereas the nice registry is updated on a monthly basis. this frequency is developed because outcome data, e.g. mortality, is measured at hospital discharge. for sentinel purposes this delay is too long and more frequent updates are needed. however our results can give incentives to set up an additional registry of near real-time surveillance of sari cases at the icu. our statistical analysis also has some limitations. due to the weekly scope of the ili data, we aggregated the icu data on a weekly basis, losing detail as they are available on a daily basis. with regard to our chosen models to predict ili, in the ideal situation stepwise variable selection is combined with 10-fold cross validation. since automating this process is not possible in gee, we first performed stepwise selection and then 10-fold cross validation on the seven remaining candidate models. additionally, we used three years of data as training set to determine the best models, whereas a longer period would also have been an option but not necessarily better, since we continuously added data to the baseline data. another limitation is that during the 2009-2010 pandemic, the ili peaks were not detected or later. this means that our models were not sensitive to large or unexpected changes. apparently the association between icu admissions and ili in the general population can change greatly from season to season. icu related sari might occur at a very different rate (compared to symptoms in the general population) during a pandemic or unexpected seasons [9] . a probable explanation is that the influenza pandemic caused by the a(h1n1)pmd09 virus targeted another patient population than the previous epidemics with severe illness in younger patients, and fewer elderly with a severe infection. this could explain why increases in icu admissions during the pandemic were later than usual. additionally, the icu data reflects only sari cases. therefore, we do not know if the icu data reflect an influenza epidemic in the general population or possibly very different influenza dynamics in the icu population alone. icu data on respiratory infections was associated with ili incidence, with highest association in the same week and in the week before and the week after. increases in icu data on average occur two days sooner and for a longer time period than increases in ili. icu data thus contains additional information on icu related sari cases during a specific influenza epidemic. predicting influenza epidemics one, two or three weeks ahead in the general population using icu data was imprecise, reflected by the low ppvs and sensitivities. thus, icu data cannot improve the current surveillance method to detect influenza epidemics. due to the association between both data sources, a next step is to investigate the possibility of using icu data in combination with microbiological laboratory results for surveillance of severity of illness, and icu capacity prediction when an (severe) influenza epidemic is present. the performance of the gee models in predicting the start, end and length of an influenza epidemic is expressed by the positive predictive value (ppv), and sensitivity (n = 338 weeks) based on comparing the signals for an epidemic predicted with intensive care unit (icu) data with the reference standard from the observed influenza like illness data. doi:10.1371/journal.pone.0083854.t003 surveillance trends of the 2009 influenza a(h1n1) pandemic in europe responding to new severe diseases-the case for routine hospital surveillance and clinical networks in europe continuous morbidity registration sentinels: netherlands proefonderzoek naar de frequentie en de aetiologie van griepachtige ziekten in de winter 1963-1964 modelling influenza epidemics: can we detect the beginning and predict the intensity and duration? apache ii: a severity of disease classification system defining and improving data quality in medical registries: a literature review, case study, and generic framework comparing pandemic to seasonal influenza mortality: moderate impact overall but high mortality in young children longitudinal data analysis using generalized linear models fitting additive poisson models studying seasonality by using sine and cosine functions in regression analysis r-squared measures for count data regression models with applications to health-care utilization a biometrics invited paper. the analysis and selection of variables in linear regression akaike's information criterion in generalized estimating equations how biased is the apparent error rate of a prediction rule detecting the start of an influenza outbreak using exponentially weighted moving average charts sequential detection of influenza epidemics by the kolmogorov-smirnov test detection of excess influenza severity: associating respiratory hospitalization and mortality data with reports of influenza-like illness by primary care physicians key: cord-294591-793ywpcd authors: hu, xiaoyun; zhang, zhidan; li, na; liu, dexin; zhang, li; he, wei; zhang, wei; li, yuexia; zhu, cheng; zhu, guijun; zhang, lipeng; xu, fang; wang, shouhong; cao, xiangyuan; zhao, huiying; li, qian; zhang, xijing; lin, jiandong; zhao, shuangping; li, chen; du, bin title: self-reported use of personal protective equipment among chinese critical care clinicians during 2009 h1n1 influenza pandemic date: 2012-09-05 journal: plos one doi: 10.1371/journal.pone.0044723 sha: doc_id: 294591 cord_uid: 793ywpcd background: critically ill patients with 2009 h1n1 influenza are often treated in intensive care units (icus), representing significant risk of nosocomial transmission to critical care clinicians and other patients. despite a large body of literature and guidelines recommending infection control practices, numerous barriers have been identified in icus, leading to poor compliance to the use of personal protective equipment (ppe). the use of ppe among critical care clinicians has not been extensively evaluated, especially during the pandemic influenza. this study examined the knowledge, attitudes, and self-reported behaviors, and barriers to compliance with the use of ppe among icu healthcare workers (hcws) during the pandemic influenza. methodology/principal findings: a survey instrument consisting of 36 questions was developed and mailed to all hcws in 21 icus in 17 provinces in china. a total of 733 physicians, nurses, and other professionals were surveyed, and 650 (88.7%) were included in the analysis. fifty-six percent of respondents reported having received training program of pandemic influenza before they cared for h1n1 patients, while 77% reported to have adequate knowledge of self and patient protection. only 18% of respondents were able to correctly identify all components of ppe, and 55% reported high compliance (>80%) with ppe use during patient care. in multivariate analysis, vaccination for 2009 h1n1 influenza, positive attitudes towards ppe use, organizational factors such as availability of ppe in icu, and patient information of influenza precautions, as well as reprimand for noncompliance by the supervisors were associated with high compliance, whereas negative attitudes towards ppe use and violation of ppe use were independent predictors of low compliance. conclusion/significance: knowledge and self-reported compliance to recommended ppe use among chinese critical care clinicians is suboptimal. the perceived barriers should be addressed in order to close the significant gap between perception and knowledge or behavior. on april 29, 2009 , the world health organization (who) announced the outbreak of a novel influenza a (h1n1) 2009 virus to be a public health emergency of international concern [1] , which ultimately led to the declaration of the first phase 6 global influenza pandemic on june 11, 2009 [2] . as of september 6, 2009 , the who had reported more than 277,607 laboratoryconfirmed cases, with at least 3,205 deaths [3] . studies estimated that up to 2.7 million patients would be hospitalized, and about 25% of these patients might experience rapid deterioration, leading to intensive care unit (icu) admission within 1 day after hospitalization, equivalent to an increase in the volume of mechanical ventilation of 23% to 45% over the current use [4, 5] . all these data suggested an excessive workload during the initial period of the pandemic, as perceived by 80% of frontline healthcare workers (hcws) [6] . a simulation study by swaminathan and the colleagues reported that, for a patient with suspected avian or pandemic influenza who was not clinically unwell or hypoxic, the mean number of close contacts was 12.3 (range 6-17; 85% hcws), and mean exposures were 19.3 (range [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] during the first 6 hours in the emergency departments [7] . in comparison, critical care clinicians are likely to encounter even more repeated close contacts, and are at significantly high risk of acquiring such an infectious disease during patient care. evidence does exist suggesting nosocomial transmission within hospital settings. apart from earlier findings that more than 20% of patients who acquired severe acute respiratory syndrome (sars) were hcws [8] , possible healthcare-related 2009 h1n1 influenza transmission was identified in 9 out of 63 exposed hcws [9] . protection of hcws from acquisition of infectious diseases can be achieved by compliance to established infection control guidelines [10] [11] [12] [13] , including rigorous infection control practices, prescriptive instructions for the use of personal protective equipment (ppe), and postexposure antiviral prophylaxis [7] . however, reported compliance to ppe use might be extremely low. in response to a survey conducted by the center for disease control and prevention following the pandemic influenza, among 11 hcws with probable or possible patient-to-hcw transmission, only 3 reported always using either a surgical mask or an n95 respirator [14] . a variety of barriers have been identified to hinder compliance to infection prevention and control guidelines, including knowledge, attitude, belief and behavioral factors [15] . daugherty and colleagues explored the behavior, knowledge, and attitudes of critical care clinicians about recommended precautions for prevention of healthcare-associated influenza infections in an anticipated influenza pandemic [16] . with the same methodology using a modified questionnaire, we previously reported that 82.3% of the icu hcws expressed willingness to work in a pandemic, with professions, knowledge training prior to patient care, and the confidence to know how to protect themselves and the patients independently associated with more likelihood to care for h1n1 patients [17] . however, little is known about their behavior and factors influencing compliance during a real influenza pandemic. as the second part of the above survey, we wish to evaluate the self-reported compliance to the use of ppe during the current influenza pandemic among critical care clinicians in chinese icus, as well as independent predictors of the compliance. this study was approved by the institutional review board (irb) of peking union medical college hospital. all participants were informed about the study. however, the irb waived the need for written informed consent from the participants because the identities of all respondents would be completely anonymous during data collection and analysis, and there would be minimal risk as perceived by the irb for being involved in this study. the design of this study was described in details elsewhere [17] . in brief, this study was conducted in 21 adult icus in 17 provinces in china. all participating icus admitted patients with 2009 h1n1 influenza during the pandemic. a 36-item survey questionnaire was designed based on the study of daugherty and coworkers [16] , to assess the knowledge, attitudes, and behaviors of icu hcws related to the 2009 h1n1 influenza pandemic, which was available as supporting information; see questionnaire s1. on december 25, 2009 , the questionnaire with an instruction was sent by e-mail to the contact persons of individual participating icus, who encouraged as many hcws as possible to participate the study. all questionnaires were collected and sent back by e-mail before january 15, 2010. any hcws not responding after the deadline were regarded as non-respondents. data on the demographic characteristics of respondents, including age, sex, marital status, living status, status of influenza vaccination, and profession, were recorded. the professional status of the respondents was categorized as physicians, and nurses, and others (including respiratory therapists, student nurses, and nurse assistants). for the purpose of this study, we only included physicians and nurses in the final analysis. the respondents were asked to report their experience of caring for h1n1 patients, as well as relevant training. they were also required to report the level of knowledge and the level of confidence in their ability to protect themselves and their patients from exposure to influenza at work. a 5-point likert scale (complete agree, agree, neither agree nor disagree, disagree, and complete disagree) was used to elicit preferred answers. we defined recommended ppe as use of hand hygiene, gloves, gown, mask (including surgical mask and n95 respirator), and goggles [13] . in the final analysis, answers with a higher level of protection than recommended (e.g. use of goggles when no aerosolgenerating procedures were anticipated) were deemed as correct because they represented adequate protection [16] . as a response to the 2009 h1n1 influenza pandemic, all hospitals were required by local healthcare authorities to provide training programs to all hospital staffs during seminars. these training programs were mainly 2 to 3-hour lectures, developed based on the guidelines issued by ministry of health, often involving diagnosis, treatment, and infection control of 2009 h1n1 influenza. there was no posttest to evaluate the extent of information attainment by the attendees. all likert-scale responses were dichotomized into complete agree/agree versus neither agree nor disagree/disagree/disagree/ complete disagree, and expression in proportions. continuous variables were compared with student's t-test or mann-whitney u test. categorical variables were compared with chi-square test or fisher's exact test when appropriate. self-reported compliance to ppe use of .80% was considered as high compliance [16] . correlations were measured using kendall rank correlation coefficient. for determination of independent predictors for high compliance to ppe use during patient care, odds ratio (or) was estimated on the basis of both univariate analysis and multivariate logistic regression analysis. variables including clinicians characteristics, knowledge, attitudes, and behaviors were added into the model using stepwise conditional forward entry, if p,0.1 in univariate analysis. an or of less than 1 was associated with low compliance to ppe use, while an or of greater than 1 was associated with high compliance to ppe use during patient care. in the 21 icus surveyed, 733 eligible participants were identified, and 695 returned completed surveys, for an overall response rate of 94.8%. forty-five respondents were excluded (including 25 other professionals, and 20 with missing data), therefore 650 respondents (including 229 physicians and 421 nurses) were included in the final analysis (table 1) . compared with physicians, more nurses were single, and living with parents or living alone. more than half respondents received vaccination for 2009 h1n1 influenza. five hundred and eighty-six respondents (90.2%) reported that they had received the pandemic training program, although only 364 (56.0%) claimed to complete the pandemic training program before they cared for h1n1 patients. in comparison, about three-fourths of respondents reported to wear goggles and gown during aerosol-generating procedures, and to wear n95 respirator in droplet precaution or close contact, respectively. however, 435 respondents (66.9%) reported to wear goggles and gown during entire treatment and/or nursing care, indicating overprotection. significant correlation was found between self-reported adequate knowledge of pandemic influenza and correct identification of ppe and knowledge of goggles (kendall tau-b 0.184 and 0.131, p,0.001 and p = 0.001, respectively), but not knowledge of hand hygiene or mask (kendall tau-b 20.007 and 20.029, p = 0.851 and 0.467, respectively). about 80% of respondents believed that they knew self-and patient protection during the pandemic (table 2 ). in particular, 87.5% of respondents believed that use of appropriate ppe would confer adequate protection for hcws, while only 68.5% stated that this protection was adequate for vulnerable patients. half of respondents reported that ppe use was inconvenient, while 21.2% believed that ppe use would interfere with patient care, with no difference observed between physicians and nurses. no significant correlation was found between self-reported adequate knowledge of both self-protection and patient protection and correct knowledge of hand hygiene, goggles, or masks. however, selfreported adequate knowledge was significantly correlated with the perception of further improvement of ppe compliance (kendall tau-b 0.143, p,0.001). with regards to organization factors, 63.2% of respondents reported that appropriate ppe was readily available in their icus (table 2 ). more physicians than nurses knew when influenza precautions were initiated in their patients (p = 0.015). by contrast, significantly more nurses than physicians (92.1% vs. 86.4%, p = 0.020) reported being reprimanded by the supervisor for noncompliance. as to behaviors of ppe use, about 21% of respondents reported that their colleagues often forgot to use ppe during patient care, while a similar proportion reported themselves to forget to change ppe between patients. among all respondents, 361 (55.5%) reported high compliance (.80%) to ppe use, with significant inter-institutional variation ranging from 0% (0/5) to 88.1% (37/42 (table 3 ). to our knowledge, this study represents the first effort to examine self-reported knowledge, attitude, behavior and influencing factors of ppe use during the pandemic influenza in chinese icus. among 650 respondents, although up to 77% reported to have adequate knowledge of self-and patient protection, fewer than 20% could correctly identify all components of ppe or exhibited correct knowledge of ppe use during patient care. this suggested significant gaps in the perception and actual knowledge with regards to infection control practices, in particular ppe use, among our critical care clinicians [17] . moreover, about 55% of respondents reported high compliance to the recommended ppe use. vaccination status, positive attitudes towards ppe use, cultural factor (perceived reprimand for noncompliance), and organizational factors (availability of ppe in icu, notice of influenza precautions) were identified as independent predictors of high compliance, while negative attitudes towards ppe use and violation of recommended ppe use were associated with low compliance. ppe referred to a variety of barriers and respirators used alone or in combination to protect mucous membranes, airways, skin, and clothing from contact with infectious agents [12] . the critical importance of compliance to ppe use was not only recognized in a variety of practice guidelines of infection control [9] [10] [11] [12] [13] , but also demonstrated during the outbreak of sars in 2003 [8, 18] . unfortunately, compliance by professionals was often suboptimal [19, 20] , due to knowledge, attitudes, and behavior among professionals, as well as to organizational and other factors [15, 21] . in this survey of chinese critical care clinicians, only 55% of respondents reported high compliance (.80%) to recommended ppe use, consistent with other relevant studies [16, 19] . however, significant gaps between perception and practice were a common finding in icu [22] , indicating overestimation of clinical practice judged by self-reported behavior, especially for infection control measures, such as hand hygiene [23] and ppe use [16, 19] . similar to the study of daugherty and coworkers [16] , we found a similar proportion (74%) of respondents claiming their confidence to improve compliance to ppe use, again suggesting perception of inadequate ppe use among most respondents. our results indicated that a number of factors, including attitudes, behavior, and organization, might significantly influence clinical practice. although behavior could be changed without knowledge or attitude being affected, behavior change (i.e. selfreported high compliance to ppe use) based on improving knowledge and attitude (e.g. ppe use could confer adequate protection for hcws) was probably more sustainable than indirect manipulation of behavior alone [15] . in the meanwhile, it was also self-intuitive that a negative attitude (e.g. perception that ppe use might interfere with patient care) often predicted low compliance. likewise, daugherty and coworkers found that the belief that ppe use was inconvenient was predictive of poorer adherence [16] . the perception that ppe use interfered with patient care was supported by previous studies. despite the fact that critical care clinicians were probably highly compliant with ppe use, patients in contact isolation might suffer from adverse effect of inadequate patient care, including less time spent in patient rooms not explained by severity of illness [24, 25] , less time examining patients [26] , more incomplete records of vital signs and progress notes, and increasingly likelihood of preventable adverse events. moreover, almost half hcws reported difficulty in communicating with patients through enhanced infection precautions during the sars outbreak [27] . organizational factors were commonly acknowledged as barriers that impede and hamper professionals' compliance to ppe. compliance to ppe use was closely related to the professionals' perception about the risks they were exposed to and their susceptibility to these risks. our study showed that, if critical care clinicians were aware of the patients on isolation precautions, they were twice likely to report high compliance to ppe use. similarly, in a survey of physicians working in canadian pediatric emergency departments, almost 90% considered identifying patients with complaints requiring ppe use prior to the physician entering the room as an important factor promoting ppe use [19] . in a study performed during the first wave of 2009 h1n1 influenza, banach and coworkers observed more unprotected exposures in patients who did not present with influenzalike illness [28] . this finding was not unexpected because such patients would not have been identified by the screening protocol, which might result in delays in consideration of influenza as a potential diagnosis when these patients were subsequently evaluated by clinicians, as well as delays in implementation of recommended infection control measures. studies have consistently demonstrated significant association of the availability of ppe in icu and self-reported compliance, as in our study, indicating unavailability as the major reason for noncompliance [16, 19, 21] . however, among the 256 critical care clinicians surveyed by daugherty and coworkers, self-reported high compliance was only 62%, despite the fact that 72% reported that recommended ppe was readily available near patients' rooms [16] . this evidenced the complexity of compliance to ppe, which might go beyond availability, confirming the interference of individual factors, perceptions, and relations in the work environment in decision making towards protection. professional's behavior was an important factor that determined the commitment to, and the style and proficiency of, an organization's health and safety management [29] . a study in 1986 examined the role of organizational factors in 13 hospitals in the united states, and found that severity-adjusted mortality were related more to the interaction and coordination of each hospital's icu staff than the icu administrative structure, amount of specialized treatment used, or the hospital's teaching status [30] . similar to other studies [16] , our study found close association between self-reported compliance and safety culture (i.e. hcw behavior, and perceived reprimand for noncompliance by the supervisors), underscoring the importance of icu safety culture in promoting behavior change, or even patient outcome [29] . perceived barriers of compliance to ppe use as described above should be addressed during development of practice guidelines, in order to prevent transmission of infectious diseases within hospital setting. despite the lack of data validating such concept with regards to 2009 h1n1 influenza in icu, studies did suggest that implementation of protocoled care and/or educational program, by addressing knowledge, attitude, and behavioral barriers, might significantly reduce catheter-related bloodstream infection [31] , and improve mortality in patients with severe sepsis [32] . the major limitation of our study was that it might be subject to social desirability bias (individuals may wish to present themselves or their organization in a favorable way) due to its reliance on self-reporting [33] . in addition, cause-effect relationship could not be determined due to the inherent ''chicken or egg'' caveat of the observational study. nevertheless, these data provided clue of the barriers that existed with regard to the implementation of infection control guidelines in icus and provided useful suggestions for the implementation. only 55% of chinese critical care clinicians reported high compliance to ppe use during pandemic influenza, putting hcws and their patients at risk. both attitudes towards ppe use and perceived organizational norms have been recognized as predictors of compliance, which should be addressed while developing educational program and/or practice guidelines, in order to prevent nosocomial transmission of influenza. questionnaire s1 survey questionnaire. (doc) swine influenza. statement by who director-general, dr margaret chan world now at the start of 2009 influenza pandemic. statement to the press by who director-general dr margaret chan h1n1 flu: international situation update swine origin influenza a (h1n1) virus and icu capacity in the us. are we prepared? hospitalized patients with 2009 h1n1 influenza in the united states impact of the 2009 influenza a(h1n1) pandemic on public health workers in the netherlands personal protective equipment and antiviral drug use during hospitalization for suspected avian or pandemic influenza sars plague: duty to care or medical heroism transmission of 2009 pandemic influenza a (h1n1) virus among healthcare personnel -southern california guideline for infection control in health care personnel healthcare infection control practices advisory committee guideline for hand hygiene in health-care settings. recommendations of the healthcare infection control practices advisory committee and the hicpac/shea/apic/idsa hand hygiene task force guideline for isolation precautions: preventing transmission of infectious agents in healthcare settings pandemic influenza: guidance for infection control in critical care. dh novel influenza a (h1n1) virus infections among health-care personnel -united states why don't physicians follow clinical practice guidelines? a framework for improvement the use of personal protective equipment for control of influenza among critical care clinicians: a survey study knowledge and attitudes of healthcare workers in chinese intensive care units regarding 2009 h1n1 influenza pandemic effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory distress syndrome (sars) use of personal protective equipment in pediatric emergency departments a method for evaluating health care worker's personal protective equipment technique barriers to implementing infection prevention and control guidelines during crises: experience of health care professionals practice and perception -a national survey of therapy habits in sepsis understanding adherence to hand hygiene recommendations: the theory of planned behavior adverse effects of contact isolation contact isolation in surgical patients: a barrier to care? do physicians examine patients in contact isolation less frequently? a brief report the immediate psychological and occupational impact of the 2003 sars outbreak in a teaching hospital factors associated with unprotected exposure to 2009 h1n1 influenza a among healthcare workers during the first wave of the pandemic intensive care unit safety culture and outcomes: a us multicenter study an evaluation of outcome from intensive care in major medical centers an intervention to decrease catheter-related bloodstream infections in the icu improvement in process of care and outcome after a multicenter severe sepsis educational program in spain implementing quality indicators in intensive care units: exploring barriers to and facilitators of behavior change key: cord-290120-fd26t8ja authors: tan, chew yee; opaskornkul, keerati; thanawongnuwech, roongroje; arshad, siti suri; hassan, latiffah; ooi, peck toung title: first molecular detection and complete sequence analysis of porcine circovirus type 3 (pcv3) in peninsular malaysia date: 2020-07-24 journal: plos one doi: 10.1371/journal.pone.0235832 sha: doc_id: 290120 cord_uid: fd26t8ja porcine circovirus type 3 (pcv3) is a newly emerging virus in the swine industry, first reported recently in 2016. pcv3 assembles into a 2000 bp circular genome; slightly larger than pcv1 (1758–1760 bp), pcv2 (1766–1769 bp) and pcv4 (1770 bp). apart from being associated with porcine dermatitis and nephropathy syndrome (pdns), pcv3 has been isolated from pigs with clinical signs of reproductive failures, myocarditis, porcine respiratory disease complex (prdc) and neurologic disease. given that pcv3 is increasingly reported in countries including thailand and u.s. with whom malaysia shares trade and geographical relationship; and that pcv3 is associated with several clinical presentations that affect productivity, there is a need to study the presence and molecular characteristics of pcv3 in malaysian swine farms. twenty-four commercial swine farms, three abattoirs and retail shops in peninsular malaysia were sampled using convenience sampling method. a total of 281 samples from 141 pigs, including 49 lung archive samples were tested for pcv3 by conventional pcr. twenty-eight lung samples from wild boar population in peninsular malaysia were also included. nucleotide sequences were analyzed for maximum likelihood phylogeny relationship and pairwise distances. results revealed that pcv3 is present in peninsular malaysia at a molecular prevalence of 17.02%, with inguinal lymph nodes and lungs showing the highest molecular detection rates of 81.82% and 71.43% respectively. despite wide reports of pcv3 in healthy animals and wild boars, no positive samples were detected in clinically healthy finishers and wild boar population of this study. pcv3 strain a1 and a2 were present in malaysia, and malaysian pcv3 strains were found to be phylogenetically related to spanish, u.s. and mexico strains. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 circoviruses of swine comprise of porcine circovirus type 1 (pcv1), porcine circovirus type 2 (pcv2) porcine circovirus type 3 (pcv3) and most recently reported porcine circovirus type 4 (pcv4). pcv1 was discovered in 1974 as a cell culture contaminate [1] . in contrast, pcv2 is associated with a group of complex multi-factorial diseases classified under the umbrella term of porcine circovirus associated diseases (pcvad) [2] . the novel detection of pcv4 was described in a herd with severe clinical signs of respiratory disease, enteritis and porcine dermatitis and nephropathy syndrome (pdns) [3] . pcv3 is a newly emerging virus in the swine industry, first reported in 2016 in the united states [4] . pcv3 assembles into a 1999-2001 bp circular genome; slightly larger than other known porcine circoviruses which ranged from 1758-1760 bp (pcv1) and 1766-1769 bp (pcv2) [5] [6] [7] [8] . pcv4, very recently reported in hunan province, china, was described to be 1770 bp [3] . albeit their different lengths, genome of all porcine circoviruses encodes for three known proteins: replication-associated (rep) protein, capsid (cap) protein and open reading frame (orf) 3, which function has yet to be determined. international committee on taxonomy of viruses (ictv) defines a distinct circovirus species based on sequence similarity: a novel circovirus must share < 75% nucleotide (nt) identity over its entire genome and < 70% amino acid (aa) identity of its cap protein with other species in the genus [9] . pcv1 and pcv2 shares < 80% similarity of overall nt identity, 86% and 65% similarity of aa identity in their orf1 and orf2 respectively [10] [11] [12] . in a complete genome of pcv3, orf1 and orf2 genes encode for 296 -297aa rep protein and 214aa cap protein respectively [4, 7] . pcv3 shares even lower similarity of only < 50% of overall nt identity, 48% and 26-36% aa identity of orf1 and orf2 respectively with pcv2 [4, 9, 13] . the genome of pcv4 showed 50.3%, 51.5% and 43.2% nt similarities to pcv1, pcv2 and pcv3 respectively. most strikingly, aa identities of cap protein of pcv3 and pcv4 differ by 75.5% [3] . while the conserved nonanucleotide stem loop motif (t/n)a(g/ t)tattac representing the origin of replication can be found in all porcine circoviruses, their motif differ. pcv1 and pcv3 have an identical motif of tagtattac [4, 9, 14] whereas the motif on pcv4 rep gene was shown to be cagtattac [3] . pcv2 has a nonamer sequence unique among circovirus species-aagtattac [14] . pcv3 was associated with a pdns outbreak in north carolina, where there was an increase in sow mortality rate and decrease in conception rate, presented with skin and kidney lesions suggestive of pdns [4] . several other clinical presentations have been associated with pcv3, including reported reproductive failures [15] , neonatal congenital tremor [16] , myocarditis and multi-organ inflammation [13] . role of pcv3 in porcine respiratory disease complex (prdc) has also been discussed [13, 17] . although many common swine pathogens especially pcv2, and others such as porcine reproductive and respiratory syndrome virus (prrsv) and ungulate protoparvovirus 1 (ppv) have been ruled out in these reports, there could still be other co-infections that elude the role of pcv3 in pathogenesis. a successful reproduction of pdns-like clinical disease following experimental inoculation of 4-and 8-week-old specific-pathogen-free piglets with infectious pcv3 dna clone has been demonstrated, thus implying that pcv3 may have a direct role in disease process [18] . considering the presence of pcv3 in increasing number of countries including thailand [17, 19] and u.s. [4, 13] with whom malaysia shares trade and geographical relationship; and that pcv3 is associated with several clinical presentations that affect productivity, molecular prevalence of pcv3 in malaysia and molecular characteristics of malaysian pcv3 strains reported in this study may contribute practical knowledge to the malaysian swine farming industry. commercial swine farms involved in this study were located in perak, selangor, melaka and johor states representing different regions of malaysia. from these 24 farms, 123 pigs were subjected to convenience sampling method. from three abattoirs and retail shops, 18 clinically healthy finishers were sampled by random selection. a grand total of 141 pigs were included in this study. the sampled animals were also categorized by age group (foetuses, piglets, weaners, growers or finishers and sows), health status (clinically healthy or clinically ill), standing sow population of origin farm (< 800 or > 800 heads) and distance between origin farm and neighbouring farms (< 1km, 1 -10km, > 10km). the 281 organ samples collected were comprised of 49 archived lung samples of year 2016-2017, 18 lung samples from clinically healthy finishers and 214 tissues samples of various organs from clinically ill pigs of year 2018-2019. for each animal sampled, at least lung and/or inguinal lymph node was collected. other organs (spleen, tonsil, kidney, heart, mesenteric lymph nodes, liver and brain) were collected on the basis of availability. clinically ill pigs were those showing various clinical signs such as wasting, moribund, dyspnea, neurological signs and sudden death. majority of the sampled pigs were of weaner and grower stage between the age of 4-12 weeks old. detailed breakdown of each sampled farm and pig is provided in s1 and s2 tables. in addition, 28 archived wild boar lung samples of year 2018-2019 were also included in this study. this study was granted approval from the universiti putra malaysia (upm) institutional animal care and use committee (iacuc) under aup code upm/iacuc/aup-r030/2019 and was conducted adhering to the guidelines as stated in the code of practice for care and use of animals for scientific purposes as stipulated by universiti putra malaysia. all samples were collected under the supervision of veterinarians from faculty of veterinary medicine, universiti putra malaysia. dna extraction was performed using dneasy blood & tissue kit extraction kit (qiagen, germany) in accordance to manufacturer's instructions. conventional pcr was performed to amplify the orf2 region of pcv3, by using mytaq™ red mix 2x (bioline, united kingdom) and published primers, kf-5'-ttacttagagaacggacttgtaac g-3' and kr-5'-aa atgagacacagagctatattcag-3' [15] . briefly, 12.5 μl of taq dna polymerase master mix and 1.0 μm each from the primer pair were used in a 25 μl total pcr reaction volume. cycling conditions of the conventional pcr were as described by ku et al. [15] . pcr products were stained using redsafe™ nucleic acid staining solution (intron biotechnology, south korea) and analysed by agarose electrophoresis. expected pcr product was a 649 bp band indicating pcv3 cap gene sequence spanning from nt position 1343-1987. samples that showed a positive band at the 650 bp region as marked by gelpilot 100 bp plus ladder (qiagen, germany) were further sequenced (macrogen, south korea). to test for association between pcv3 molecular detection status and age group, health status, farm standing sow population and distance from neighbouring farms, chi-square tests were performed with statistical significance level set at p < 0.05. fisher's exact tests were run in place of chi-squared tests for variables with expected count of less than five. post hoc tests of cell residuals were ran on statistically significant chi-square and fisher's exact test values, with p-values adjusted with bonferroni correction. animals that tested positive for pcv3 in lung and/or inguinal tissues, with at least one other organ samples (spleen, tonsil, kidney, heart, mesenteric lymph nodes, liver and/or brain) tested for pcv3 were identified. molecular detection rate comparison across tissues from different organs was then similarly evaluated as described above. all statistical tests were performed using ibm spss statistics for windows v23 software programme [20] . upon confirmation of the nt sequences as cap gene of pcv3 by ncbi nucleotide blast 1 [21] , the positive samples were subjected to pcr with another three pairs of primers (fig 1, table 1 ) to generate the complete 2000 bp genome of pcv3. two pairs of published sequencing primers [4] were run under modified cycling conditions, chiefly adjusting annealing temperature and final extension time as detailed in table 1 . primer pair v4f/v4r were designed based on pcv3 strain ky075986 [15] , using primer-blast 1 [22] . all pcr assays were performed using the same reagents, reaction volumes and primer concentration as described in method of pcv3 cap gene detection. pcr protocols used in this study is accessible at http://dx. doi.org/10.17504/protocols.io.bdd9i296 [protocol doi]. sequencing was done to confirm the identity of the pcv3 nt sequences. sequence assembly and multiple sequence alignment were generated using mega v7.0.26 software programme [23] . the resulting sequences were analysed using ncbi nucleotide blast 1 [21] for a final identity confirmation as pcv3 by comparing their similarity with reference pcv3 sequences deposited in the genbank. maximum likelihood (ml) phylogenetic trees were constructed with mega7 programme, using 1000 bootstrap replicates with either general time reversible (gtr) model for species-specific circoviruses comparison or tamura-nei model for porcine circoviruses analyses. pairwise distance analysis with p-distance model was performed using the same software, similarly with 1000 bootstrap replicates. both transitions and transversions nt substitutions were included. after number of base differences per site was computed, percentage nt identities were calculated by subtracting the computed p-distance values from 1.0 and multiplying by 100. the same 45 pcv3 strains included in the phylogenetic methods were further analysed. tajima's d, fu and li's d, and fu and li's f statistical tests of neutrality were performed on nt sequences of pcv3 orf1 and orf2 genes using dnasp v6.12.03 software programme [24] [25] [26] . dna polymorphism data of pairwise nt differences were analysed to measure balancing selection and negative selective processes over the sequences. statistical significance was set at p � 0.05 for all three tests. shannon's entropy h(x) values were calculated with bioedit software v7.2.5 using the shannon entropy formula: à ð p 4 j¼1 p ij log 2 p ij þ where i; j is equal to 1, 2, 3 and 4, corresponding to a, c, g and t nt and pij being the proportion of nt j in site i. entropy plots of aa sequences of the orf1 and orf2 genes were constructed to plot the diversity of aa residues at a given position [27, 28] . range, mean and standard error of mean (sem) were calculated using ibm spss statistics for windows v22 software programme. positive and negative selective pressures acting specifically on each codon of the orf1 and orf2 nt sequences were estimated based on calculated difference between non-synonymous (dn) and synonymous (ds) substitution rates per codon. single-likelihood ancestor counting (slac), fixed-effects likelihood (fel), internal branches fixed-effects likelihood (ifel), fast, unconstrained bayesian approximation (fubar) and mixed effects model of evolution (meme) selection pressure methods were run in the datamonkey web server (http://www.datamonkey.org/) [29] [30] [31] [32] . to infer dn and ds rates, fubar uses bayesian approach; fel, ifel and meme utilize ml approach; while slac incorporates additional counting approaches. fel, ifel, slac and fubar detects both positive and negative selection, but meme aims to detect aa sites evolving under positive selection. comparison between rates of dn and ds substitutions were expressed as dn-ds < 0, = 0 and > 0 (dn / ds < 1, = 1 and > 1 for meme method) and interpreted as indication of negative selection, neutral evolution and positive selection respectively. statistical significance was set at p � 0.05 for fel and ifel methods and p � 0.1 for slac and meme method. fubar method was run with posterior probability of 0.9. the dn-ds and h(x) entropy values for every codon were plotted against their respective aa positions along the orf1 and orf2 genes. out of the 141 pigs, 24 pigs from 10 farms were positive for pcv3 based on pcr detection ( table 2 ), representing a molecular prevalence of 17.02% of pcv3 in peninsular malaysia. pcr results for each 141 animals are provided in s2 table. notably, all 18 samples from clinically healthy pigs and all 28 lung samples from the wild boar population were tested negative for pcv3. statistical significance was observed between pcv3 molecular detection status and age group (p: 0.022), as well as health status (p: 0.043) of the test animals. in the age group set of variables, only the weaners group (p: 0.0007) was shown to be statistically significant at adjusted p < 0.005. no statistical differences in the frequency of animals molecularly positive for pcv3 were observed across farms with different standing sow population (p: 0.180) and distance from neighbouring farms (exact p: 0.512). statistical significance for all chi-square and fisher's exact tests is summarized in table 3 . detailed results of statistical tests are tabulated in s3 table. the molecular detection rate across different organs (lung, inguinal lymph node, mesenteric lymph node, spleen, tonsils, kidney, heart, liver, brain) was tabulated for the positive animals (table 4 ). for an accurate representation of the distribution, positive animals with only one organ sample type tested were excluded from the analysis. the presence of pcv3 genetic material was detected in all nine group of organ samples included in this study. the highest frequency of pcr positive results was observed in inguinal lymph node and lung samples, with detection rates of 81.82% and 71.43% respectively. for other organs, positive pcr detection rate ranged from 12.5% to 54.55%. pcr detection rate was moderately high in five organs, namely tonsil, spleen, kidney, mesenteric lymph node and brain. the lowest detection rates were seen in heart and liver. in terms of statistical significance, it was found that only the inguinal lymph nodes group (p: 0.0002) was statistically significant at adjusted p < 0.003, as shown in table 3 . twelve complete genome sequences and three cap gene sequences were obtained successfully. the local pcv3 sequences showed over 99% similarity with pcv3 sequences recorded in gen-bank. the genome of malaysian pcv3 strains in this study is 2000 bp in length, encoding a 296 aa rep protein and a 214 aa cap protein. ml phylogenetic trees were constructed to analyze malaysian pcv3 strains in relation to known member species of circovirus genus using nt sequences of exemplar isolates listed by ictv [33] . pcv1 and pcv2, as well as pcv3 strains from different countries were also included in the analysis. the phylogenetic relationship among species in circoviridae was inferred using ml method based on gtr model (fig 2) . pcv1, pcv2 and pcv3 were clustered into a same clade with >50% bootstrap value. supported by a lower bootstrap value of 30%, malaysian strains of pcv3 were phylogenetically related to bat associated circovirus 7 (batacv7) and starling circovirus (stcv). the phylogenetic relationship among porcine circoviruses was inferred using ml method based on tamura-nei model (fig 3) . all pcv3 strains analyzed in this study were clustered in a distinct clade with the longest branch length, separated from pcv1 and pcv2. further, percentage nt identities between 42 pcv3 sequences (12 malaysian sequences and 30 genbank reference strains) were compared using pairwise distance method with p-distance model (s4 and s5 tables). all 2000 nt positions were included in the final dataset. overall, all 42 pcv3 sequences in the comparison dataset are closely related to each other, given that only 0.58% (5 / 861) of the p-distance values are � 0.020. the p-distance values range from 0.002 to 0.021, averaging at 0.010. this is equivalent to a shared percentage nt identities of 97.9%-99.8%. among the 12 malaysian sequences, while the p-distance range maintains, the average is slightly higher at 0.012. a similar pairwise distance analysis was run to compute the p-distance values among cap gene sequences of 15 malaysian pcv3 strains and 30 pcv3 genbank reference strains (s6 and s7 tables). the range of p-distance values widens from 0.000 to 0.026, with an increased average of 0.013. this is equivalent to a shared percentage nt identities of 97.36%-100%. compared to the complete genomes, the cap gene sequences are less closely related to each other, given that 8.28% (82 / 990) of the p-distance values are � 0.020. among the 15 malaysian sequences, the p-distance range is slighter smaller at 0.000 to 0.020, and the average is slightly lower at 0.012. based on the phylogenetic analysis of pcv3 cap gene sequences (fig 4) , malaysian pcv3 strains appear to be grouped into two main clusters within one clade. in the first cluster, malaysian pcv3 strains were grouped with pcv3 strains from italy (genbank accession no.: to determine positive and/or negative selections in the orf1 and orf2 genes of pcv3 sequences, neutrality test values, dn-ds values and h(x) entropy values of the two genes were evaluated. results from the statistical tests of neutrality ran on pcv3 orf1 and orf2 gene sequences were summarized in table 5 analysis of the selective pressures revealed minor differences between orf1 and orf2 genes at both global and site levels. global dn-ds value of orf1 and orf2 were 0.015 and 0.091 respectively, as determined by slac method. at site levels, dn-ds values for every codon were determined to identify aa positions under positive or negative selection pressure, as tabulated in table 6 . large majority of the calculated dn-ds values were < 0, indicating negative selection. at the statistical significance thresholds applied in this study, no positive selection was reported. within the orf1 and orf2 gene, 24 / 296 (8.11%) and 26 / 214 (12.15%) aa sites respectively were identified as negatively selected sites. among the four selection pressure methods used, descending detection rates were observed from fubar, fel, slac to ifel method. only 3 sites, codon 203 of the orf1 gene, codon 75 and 85 of the orf2 gene, were identified as negative selection sites across all four methods. [34, 35, 38] . pcv2 is known to have tropism for lymphoid tissues in pigs, with lymphoid depletion and histiocytic replacement of lymphoid tissues being hallmark lesions of pcv2 infection [39, 40] . interstitial pneumonia and bronchiolitis with mononuclear infiltration are also part of pcv2 disease manifestation, which correlate with clinical sign of respiratory distress [41, 42] . since high molecular detection rates of 81.82% and 71.43% respectively were found in inguinal lymph nodes and lungs, with demonstrated statistical significance in molecular detection rate of pcv3 in the inguinal lymph nodes group, further research may be focused on these tissues to study potential tissue tropism and relationship with pcv3 pathogenesis. weaners have been shown to have the highest prevalence of pcv3 [7, 43, 44] across different production stages and statistical results of this study supports this finding. pcv3 has been reported in pigs with various pathological conditions including respiratory, reproductive, neurological and gastrointestinal disorders. statistics calculated in this study also showed high pcv3 molecular detection rate in the clinically ill group. nevertheless, the virus has also been detected in clinically healthy animals [45] [46] [47] . however, in this study, all lungs samples of clinically healthy pigs sourced from abattoir and retail shops were tested negative for pcv3 in spite of their origins in pcv3 positive farms. this 100% negative results may not be accurate, considering the low sample number which constitute only 12.76% (18 / 141 pigs) of the study population, and that sampling healthy animals solely from finishers stage may not be representative for the clinically healthy population. apart from domestic pigs, pcv3 was found to infect wild boar population at rates of 23%-42.66% [37, 48, 49] . notably, all 28 lung samples from wild boar population in peninsular malaysia were tested negative for pcv3. this finding may suggest that spillover infection from wild boar reservoir hosts is not implicated in introduction of pcv3 into malaysian commercial swine population. pcv3 infection susceptibility has been suggested to be associated with the age of wild boar, with juvenile animals showing statistically lower detection rates, unlike reports described in domestic pigs [37, 48] . in our study, 46.42% (13 / 28 animals) were identified as adults of > 12 months old, with another 17.86% (5 / 28 animals) categorized as 6-12 months old subadults, thus eliminating the concern of age group bias. high pcv3 prevalence of 33.15% was reported by franzo et al. [48] , despite sampling mostly apparently healthy wild boars (60 / 62 animals), suggesting that pcv3 may be unlikely to cause overt clinical diseases in wild boars. if such is the case, serum samples might be more suitable for study of pcv3 prevalence in wild boar population, as compared to lung samples used in this study. nevertheless, 57.14% (20 / 35 samples) and 54.29% (19 / 35 samples) detection rates in lung and spleen samples respectively have been reported [37] . possibility of false negative prevalence of pcv3 in wild boar population in this study due to low sample number still need to be considered. pcv3 sequences from different years and countries analysed to date showed nt similarities ranging from 97 to 100% [50] . the complete genome sequences of malaysian pcv3 strains in this study showed similarities of 97.9%-99.8%, concurring with the summarized global findings. in the case of pcv2, orf1 is the most conserved region of circovirus genome spanning the entire genome sequence [51] . in contrast, orf2 has been identified as the most variable [6] and most immunogenic viral protein [52, 53] . the assumption that the pcv3 cap gene is also a variable region like its pcv2 counterpart was supported as the 45 pcv3 cap gene sequences analyzed in this study showed higher p-distance values, when compared to the complete genomes in pairwise distance analysis. this indicates that the cap genes show higher variability than the complete genomes, in terms of number of base differences per site. tajima's d, fu and li's d, and fu and li's f statistical tests of neutrality share a common foundation of utilizing the frequency distribution of mutations. the unanimous negative values of all three neutrality tests signify an excess of low frequency polymorphisms relative to expectation, an effect resulting from either purifying selection or expansion of population size [25, 26] . considering the general negative trend of dn-ds values as shown in figs 5 and 6, we suggest that the orf1 and orf2 genes of pcv3 may be heavily influenced by negative selection pressure. in this study, up to 12.15% (26 / 214) of orf2 codons were predicted to be evolving under negative selection pressure. orf1 has a lower percentage of 8.11% (24 / 296). a recent study predicted up to 17% (37 / 214) of pcv3 orf2 codons were negatively selected and suggested a strong negative selection acting over orf2 gene of pcv3, which will likely cause the gene to be subjected to strong restrictions and thus unable to tolerate high levels of variation on its sequence [54] . in the context of diversity of aa residues at a given position, entropy h(x) values range from 0.0 (single residue present) to 4.322 (all 20 residues equally represented). as the h(x) value increases, it would be more likely to observe different aa residues diversity at same codon position. aa with h(x) � 2.0 are considered variable, while highly conserved aa would be expected to have h(x) of � 1.0 [55] . for pcv3, just as orf2 has higher dn-ds values as compared to orf1, orf2 also have slightly more aa sites with h(x) entropy > 0.0, at 7.94% (17 / 214). this again suggests that orf2 has higher genetic variability, in terms of number of base differences per site. however, all the h(x) values were well below � 1.0, supporting the conjecture of pcv3 orf2 being under strong purifying selection processes [53] . the highest h(x) value of orf1 was seen at aa position 122, with a value of 0.683. the highest h(x) values of orf2 ranged from 0.432-0.663, seen at aa position 24, 27, 77 and 150. the aa substitutions resulting from nt mutations at these five codons were suggested as criteria to classify pcv3 into different strains [7] . apart from being a component of classification criteria, orf2 aa position 24 was the convergence of several studies, proposed as a potential epitope region and determinant of antigen effect [38, [56] [57] [58] . since the cap genes are much more divergent, differences over nt sequences may be resolved by phylogenetic comparison of the cap gene coding sequence, orf2. hence, the genomic variability of malaysian pcv3 strains were compared by having their 645 bp long cap gene sequences phylogenetically analysed. though the malaysian pcv3 strains are rather closely related to each other, as reflected by p-distance values of < 0.021, they were grouped into two distinct clades. this study suggests a homogeneous pcv3 frequency among farms with different size of standing sow population and proximity with neighbouring farms, since no statistically significant differences were found among those tested groups. there was no apparent geographical distribution, strains from central and southern malaysia are present in both clades. however, it was observed that strains from the same farm are closely related, as shown by strains my008 (genbank accession no.: mn725080) and my010 (mn725082); my001 (mk585347), my002 (mk585348), my009 (mn725081), my012 (mn725084) and my013 (mn725085). interestingly, from the same farm, strains my012 and my013 which were obtained on a later time point in 2018/2019 showed further phylogenetic distance from strains my001, my002 and my009 obtained in 2016/2017. in another farm, a phylogenetic distance gap was seen between strain my008 (mn725080) and strain my010 (mn725082) collected five months apart. this may suggest that pcv3 may have the tendency to mutate rapidly, as what was seen with pcv2. high mutation rates of pcv2 were widely reported [59] [60] [61] . over the years, new pcv2 antigenic variants have evolved with the variability of pcv2 attributed to high evolutionary rates of 1.2x10 −3 substitution/site/year, higher than expected for a dna virus and instead resembling rna viruses [62] . in addition, low frequency mutations that allow rapid adaptation of the virus in changing environments had been identified in pcv2 genomes [60] . in the first clade, notably, malaysian strain my006 (mk585352) was singled out in its own cluster with a spanish strain (mf805720), at a high bootstrap value of 52%. the singling out of my006 strain may be attributed to the isolated location and strict biosecurity practice of the origin farm that possibly reduced the likelihood of introducing circulating pcv3 strains from other local farms. given that pcv3 can be found in semen of healthy animals [15] and that potential persistent infection nature of pcv3 has been suggested [37] , the phylogenetic relationship between malaysian and spanish strains might be related to semen and breeder importation. malaysian pcv3 strains also showed phylogenetic relationship with pcv3 strains from italy, thailand, brazil, japan and germany, though with lower bootstrap values. however, of these countries, only thailand and germany have trade activities involving porcine products with malaysia in the past 10 years [63] . in the second clade, malaysian pcv3 strains were closely related to a u.s. strain (kx458235) and a mexico strain (mh192340). since 2001, malaysia imports live breeders from u.s., from 30 to 200 heads annually though not consistently [63] . u.s. is also the main provider of live swine imports for mexico, with a 72% share of the mexican import market [64] . hence, it may be speculated that the phylogenetic relatedness among malaysian, spanish, u.s. and mexico pcv3 strains might be a result of live breeder and semen stock movement. the first and second clades discussed above are evidently grouped together in a larger cluster. this observation is in accordance with the pcv3 strain classification system proposed by (table 7) . similarly, there was no geographical distribution pattern observed, for both malaysian and global strains of pcv3 a1, a2, b1 and b2. the arrangement of the motif was reflected in the phylogenetic clade arrangement, with strain a and b each clustered into separate clade (fig 4) . further clustering into strain 1 and 2 were more evident in the a group, where clade a1 and a2 were clearly branched apart. however, more pcv3 sequences are needed to validate this genotype classification, and further work is required to determine if these genetic differences correlate to specific biological properties of pcv3 [7] . nevertheless, with the rapid emergence of pcv2 genotype 2d and 2e unfurling the speed of worldwide distribution of a new type of pcv [65] , these phylogenetic and epidemiological data may give us a head start with pcv3. in terms of relationship with other species of circovirdae, china strains of pcv3 was reported to share a clade with bat associated circovirus 8 (batacv8) at a high 82% bootstrap value [38] ; while the u.s. strains of pcv3 traced back to a common ancestor bat associated circovirus 2 (batacv2) [4] . malaysian strains of pcv3 showed phylogenetic relatedness to batassociated circovirus 7 (batacv7) and stcv with a 30% bootstrap value. another instance of involvement of bats in porcine diseases would be fruit bats of pteropid species acting as natural reservoir hosts of nipah virus, where a nipah outbreak in the 1990s almost costed the entire malaysian swine industry [66, 67] . as for starlings (sturnus vulgaris), they are native to europe, asia and north africa and have successfully established populations on nearly every continent [68] . starling fecal material has been shown to be one of the transmission sources of transmissible gastroenteritis (tge) coronavirus, with history of tge outbreaks in swine farms attributed to starlings as outbreak vector [69] . although circovirus genus members are known to infect a wide host range [33] , cases of unspecific cross-species transmission had been reported. pcv1 and pcv2 has been detected human stool samples, and avian circovirus-like dna was found in wild chimpanzee feces samples [14] . pcv3 has also been detected in chamois, roe deer [70] , cattle [71] and canine [72] . considering the possibility of cross-species transmission, documented pathogenicity of closely related circovirus species, high mutation and recombination rate of certain ssdna viruses [14] , and history of bats and starlings transmitting porcine pathogens, further investigation into the relationship between pcv3 with batacv and stcv may be warranted. on the other hand, the most recently discovered pcv4, showed closest genomic and phylogenetic relationship with mink circovirus (micv) [3] . the pathogenicity of pcv4 remains unclear and to date, no direct relationship between pcv3 and pcv4 has been reported. pcv3 is present in peninsular malaysia at a molecular prevalence of 17.02%, with inguinal lymph nodes and lungs showing the highest molecular detection rates of 81.82% and 71.43% respectively. among the nine organ types tested, only the molecular detection rate in inguinal lymph nodes was statistically significant. although pcv3 positive samples spanned across all age group from foetuses to finishers and sows, only the weaners group was shown to be statistically significant. despite wide reports of pcv3 in healthy animals and wild boars, no positive samples were detected in the clinically healthy finishers and wild boar population in this study. pcv3 strains included in this study were found to be heavily influenced by negative selection pressure. in both orf1 and orf2, aa positions with the highest h(x) values correspond with the distinct mutation patterns included in the current pcv3 strain classification system. malaysian pcv3 strain a1 and a2 were phylogenetically related to spanish, u.s. and mexico strains. supporting information s1 a very small porcine virus with circular singlestranded dna history of porcine circoviral disease (pcvd) and current western canadian situation novel circovirus species identified in farmed pigs designated as porcine circovirus 4 a novel porcine circovirus distantly related to known circoviruses is associated with porcine dermatitis and nephropathy syndrome and reproductive failure sequence of porcine circovirus dna: affinities 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investigation of porcine circovirus 3 infection in cattle in shandong province, china first molecular detection of porcine circovirus type 3 in dogs in china the research team is thankful to veterinary clinical laboratory and virology laboratory, faculty of veterinary medicine, universiti putra malaysia for assistance in this postgraduate research. resources: peck toung ooi. ooi. writing -review & editing: roongroje thanawongnuwech, siti suri arshad, latiffah hassan, peck toung ooi. key: cord-299953-sasfvcun authors: whitehead, ashley b. r.; butcher, gary d.; walden, heather s.; duque, viviana; cruz, marilyn; hernandez, jorge a. title: burden of exposure to infectious bursal disease virus, infectious bronchitis virus, newcastle disease virus, mycoplasma gallisepticum, and intestinal parasites in introduced broiler chickens on the galapagos date: 2018-09-24 journal: plos one doi: 10.1371/journal.pone.0203658 sha: doc_id: 299953 cord_uid: sasfvcun diseases in introduced broilers can possibly spill over to wild birds on the galapagos. knowledge about the current burden of exposure to pathogens in broilers on the galapagos is very limited. the objective of the study reported here was to measure the burden of exposure to infectious bursal disease virus (ibdv), infectious bronchitis virus (ibv), newcastle disease virus (ndv), mycoplasma gallisepticum (mg), and intestinal parasites in a sample of broiler chickens on 13 farms on santa cruz island and san cristobal island in july 2017. blood serum samples were tested for detection of antibodies to ibdv, ibv, ndv, and mg by using an idexx enzyme-linked immunosorbent assay. in addition, fecal samples and pen bedding environmental samples were processed and analyzed for diagnosis of intestinal parasite eggs under a compound light microscope. the frequency of seropositive broilers to ibdv was 74/130 or 56% (95% ci = 48, 65%), to ibv was 27/130 or 20% (14, 28%), and to ndv was 1/130 or 0.7% (0.1, 4%). all broilers tested negative to mg antibodies. eimeria spp. infection was common in study broilers. finally, we observed interaction between broiler chickens and wild birds (finches) inside broiler pens, as well as the presence of backyard chickens inside property limits of study farms. this study produced evidence that exposure to ibdv, ibv, and intestinal parasites in broilers on santa cruz island and san cristobal island is important. study results are relevant because (i) they provide new baseline data on the burden of exposure to avian pathogens in broiler farms, (ii) justify the need to verify standard operating procedures in hatcheries that supply (non-vaccinated) day-old chicks to the galapagos and (iii) to implement enhanced biosecurity standards on broiler chicken farms to mitigate risk of disease transmission between broilers, backyard poultry, and wild birds on the galapagos. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the rapid expansion of the human population and tourism industry has created a demand for poultry products on the galapagos in the past two decades. for example, the importation of one-day-old broiler chicks into santa cruz island increased by 115% from 143,000 chicks in 2005 to 308,500 chicks in 2016. currently, the number of broilers per farm varies from 500 to 9,000. the average duration of the production cycle is six weeks, when broilers reach a body weight of about 2.7 kg (6 lb). broilers are harvested on the farm by attending personnel and sold chilled or frozen in the local market (e.g., meat shops, grocery stores, restaurants, tourist boats). introduced broilers are susceptible to avian pathogens that can possibly spill over to wild birds on the galapagos. for example, infections with infectious bursal disease (birnavirus) in broiler chickens can potentially spillover to lava gulls and galapagos penguins [1] . infections with infectious bronchitis virus (coronavirus) can be transmitted to galapagos doves [1] . infections with newcastle disease virus (paramyxovirus-1) can cause morbidity and mortality in the flightless cormorant, brown pelican, galapagos penguin, lava gull, galapagos finches, mockingbirds, and galapagos pintail [1, 2] . in addition, mycoplasma gallisepticum infections can cause morbidity and population declines in darwin's finches, mockingbirds, galapagos doves, dark-billed cuckoos, and yellow warblers [1, 2] . the use of vaccines in day-old chicks shipped to the galapagos and after arrival is prohibited. this policy requires high biosecurity standards to mitigate risk of disease transmission between broilers, backyard chickens, and wild birds on the archipelago. knowledge of disease burden in introduced broilers on the galapagos is limited to two studies. during 2001-2003, a study conducted on san cristobal island produced evidence of prior exposure to several pathogens in 72 broilers including: (i) infectious bursal disease virus, (42%); (ii) infectious bronchitis virus (46%); (iii) newcastle disease virus (22%); and (iv) m. gallisepticum (7%) [1] . in 2005, a study on santa cruz island revealed that the burden of previous exposure to these four pathogens in 88 broilers varied from 3% to 73% [2] . in both studies, indirect contact with infected backyard chickens or vaccination (although illegal) were suspected as potential risk factors associated with broilers demonstrating positive antibody titers to investigated pathogens. finally, the study on san cristobal did not test broilers for intestinal parasites [1] , and the study on santa cruz did not find evidence of exposure to intestinal parasites in study broilers [2] . to our knowledge, no other studies have investigated or confirmed the burden of exposure to pathogens in broilers on san cristobal and santa cruz since 2003 and 2005, respectively. in october 2012, ecuador's ministry of the environment established the agencia de regulación y control de la bioseguridad y cuarentena para galápagos (galapagos biosecurity agency) (abg). an important mandate of the abg is to regulate, control and prevent the introduction and dissemination of introduced species that represent a hazard to galapagos native species and their habitat. an issue of concern is diseases in poultry that represent a health hazard to poultry, people, and wild birds on the archipelago. the objective of the study reported here was to produce new baseline data on the burden of exposure to infectious bursal disease virus (ibdv), infectious bronchitis virus (ibv), newcastle disease virus (ndv), m. gallisepticum (mg) and intestinal parasites in a sample of introduced broilers on 13 farms on santa cruz island and san cristobal island, galapagos in july 2017. this information is important to support current abg science-based policymaking efforts aimed at reducing the burden of diseases in broilers that can spill over to wild birds on the galapagos. this study received approval from the university of florida's institute of animal care and use committee. the study was conducted during 10-14 july 2017 as part of a poultry farmers' training workshop on biosecurity, diagnosis and risk management of diseases in broilers on santa cruz island and san cristobal island, galapagos. the workshop was organized by the abg. in july 2017, there were 25 broiler farms on santa cruz (farm bird maximum capacity: 500 to 9000) and seven broiler farms on san cristobal (2500 to 8000). on all farms, the source of broilers was one-day-old chicks supplied by four commercial chicken egg hatcheries in guayaquil, ecuador. based on a current law and regulations for control of introduced species on the galapagos (libro vii del regimen especial: galapagos, capítulo iii, artículo 6 http:// bioseguridadgalapagos.gob.ec/lista-de-productos-2/) the use of vaccines (e.g., against ibdv, ibv, ndv) in one-day-old chicks shipped to galapagos, and after arrival, is prohibited. local authorities do not approve the use of poultry vaccines because poultry are not native island species, and because the use of vaccines in introduced species requires vaccination guidelines supported by a risk assessment that must be approved by local authorities. chicks are transported by commercial air carriers from guayaquil to baltra island (then crossed by a small ferry to santa cruz island and after by land transportation to the farm) or to san cristobal. poultry pens are made of concrete or dirt floors, chicken-wire or wire mesh for fencing, galvanized metal roof, and tarp, roll-up side curtains for ventilation. poultry pen bedding material is made out of wood shavings and is changed after each production cycle. currently, there is no policy that prohibits the use of poultry litter as fertilizer on agricultural fields. the average production cycle is six weeks, when broiler chicken body weight reaches about 2.7 kg. broilers are fed using commercial diets and tap water. farms use two to four feed formulations for different age groups (starter 1 and 2, grower, finisher diets). the first three formulations can include the use of anti-coccidials. broilers are harvested on the farm by attending personnel. harvested broilers are distributed chilled or frozen in the local market (restaurants, meat stores, tourist boats). a non-random, convenience sample of 13 broiler farms was selected based on available funds, interest and willingness of farmers to participate. six of 25 broiler chicken farms on santa cruz island (1300 to 5200 broilers per farm) and all seven broiler chicken farms on san cristobal island (1050 to 6400 broilers per farm) were included. on each study farm, 10 clinically healthy broilers from one pen at harvesting age (six weeks) were selected by the farm manager and included. the sample size justification of 10 broilers per farm was based on input from abg's management office and farm managers, as well as on funding available for the study. on each study farm, all 10 broilers were offered for blood sample collection. in addition, two of the 10 broilers were harvested for a postmortem examination and collection of intestinal (fecal) samples as part of the training workshop. all study farms practiced biosecurity control measures to prevent exposure to contaminated vehicles, equipment, clothing and footwear by visitors. personal protective equipment including disposable plastic footwear, coveralls, and gloves were used by study personnel on each study farm. in addition, several study farm managers required the use of disposable facemasks and hairnets and that disposable footwear and the study truck be sprayed with a disinfectant prior to entering the premises. study farms had two or more poultry pens with broilers of different age groups. in addition, poultry pens were not 100% rodent-or native bird-proof. finally, backyard poultry were present within farm property limits. approximately 2-3 ml of blood were collected from the ulnar vein using new, disposable 3 ml syringes (20-g 1-inch needle) on each study bird. blood samples were poured into new, individual 10-ml vacutainer tubes (rubber stopper was removed from the tube before use to release the vacuum and reduce risk of red blood cell hemolysis). in addition, approximately 5 g of feces were collected from the intestinal tract of study birds. finally, one environmental sample (broiler pen bedding) was collected from the same broiler pen used by study broilers. fecal samples and environmental samples were placed in individual plastic whirl-bags. all samples were identified using a unique farm letter code, broiler number, or environmental sample number. samples were stored in a cooler with ice packs at study farms and during transportation to a designated laboratory at abg for processing the same day. blood serum samples were tested for detection of antibodies to ibdv, ibv, ndv, and mg by using an idexx enzyme-linked immunosorbent assay (elisa). laboratory tests were conducted at the university of georgia's poultry diagnostic and research center in athens, georgia. samples were classified as seropositive or seronegative to each pathogen of interest using antibody concentration cut-off points (i.e., ibvd s/p ratio = 0.50; ibv s/p ratio = 0.20; ndv s/p ratio = 0.20; mg s/p ratio = 0.20) recommended by the manufacturer (idexx ibd ab test validation data report; idexx ibv ab test validation data report idexx ndv ab test validation data report; idexx mg ab test validation data report). using the hi test as gold standard, the relative sensitivity of the four tests = 100% in 10, 8, 11, and 30 experimentally vaccinated chickens, respectively. similarly, the relative specificity of the four tests = 100% in 45, 45, 31, and 30 specific pathogen free chickens, respectively. fecal samples and environmental samples were processed and analyzed in a designated laboratory at abg on santa cruz or san cristobal. a fecal flotation was performed with approximately 1 g of feces from each sample using sheather's sugar solution (sp. 1.25) [3] . samples were allowed to set at room temperature for two hours with a cover glass. next, samples were analyzed for diagnosis of intestinal parasite eggs under a compound light microscope with a total magnification of 400x. on each study farm, the following data were collected: location (santa cruz or san cristobal), number of broiler pens, number of broilers, breed (ross, cobb), number of employees, duration of production cycle (days), average mortality per cycle (%) in the last 12 months, and main cause of mortality in broilers reported by the farm manager. descriptive statistics (median; minimum, maximum) were calculated for continuous variables, such: number of broilers on study farms, duration (days) of the production cycle, mortality (%) per cycle, and s/p ratios for each investigated pathogen by farm. the proportions of broilers classified as positive to antibody titers to ibdv, ibd, ndv, or mg were calculated by dividing the number of seropositive broilers to each pathogen of interest by the total number of tested broilers. ninety-five percent confident intervals (95% ci) were calculated for each proportion using a statistical software program [4] . the number of pens on each farm varied from two to five (table 1) . pens on each farm had broilers of different age groups. median numbers of broilers on study farms in santa cruz and san cristobal were 1950 (minimum = 1300, maximum = 5200) and 3000 (1050, 6400), respectively. median duration of the production cycle on study farms was 42 days on santa cruz (42, 42) and 42 days on san cristobal (42, 60). median mortality (%) per cycle on study farms was 1.5% on santa cruz (0.9, 2.4) and 3.0% on san cristobal (0.9, 5.0). the main causes of mortality in broilers reported by farm managers were dehydration during the first week of age or heat stress after the first three weeks of age (2/6 farms on santa cruz and 2/7 farms on san cristobal) and heart attack during the fifth and sixth weeks of age (2/6 farms on santa cruz and 3/7 farms on san cristobal). all study farms had ! 1 broilers classified as seropositive to ibdv (table 2) . overall, the frequency of broilers with positive antibody titers to ibdv was 26/60 or 43% (95% ci = 32, 56%) on santa cruz and 48/70 = 69% (57, 78%) on san cristobal. in addition, three of six farms on santa cruz and one of seven farms on san cristobal had ! 1 broilers classified as seropositive to ibv; the frequency of broilers with positive antibody titers to ibv was 22/60 or 37% (26, 49%) on santa cruz and 5/70 = 7% (3, 16%) on san cristobal. one of six farms on santa cruz had one broiler classified as seropositive to ndv (s/p ratio = 0.25). all broilers tested negative to mg antibodies. overall, two of six farms on santa cruz and six of seven farms on san cristobal had evidence of exposure to eimeria spp (table 3 ; fig 1) . in addition, two of 26 harvested broilers had intestinal tract lesions. on santa cruz (farm a), one of two broilers examined contained mild lesions of the proximal duodenal mucosa (i.e., petechiae, white spots); however, no oocysts were observed on fecal flotation. on farm f, one of two broilers examined had presence of blood and mucus in the cecum. this study produced evidence that exposure to ibdv, ibv, and intestinal parasites in broilers on santa cruz island and san cristobal island is important. it is not clear whether the observed exposure to ibdv or ibd indicates natural exposure or vaccination through accidental or illegal means. eimeria spp. infection was common in broilers although this finding does not represent a hazard to native wild birds. in addition, we observed interaction between broilers and wild birds (finches) inside broiler pens, as well as the presence of backyard chickens inside property limits of study farms. study results are relevant because (i) they provide new baseline data on the burden of exposure to avian pathogens in broiler farms, (ii) justify the table 2 need to verify standard operating procedures in hatcheries that supply (non-vaccinated) dayold chicks to the galapagos and (iii) to implement enhanced biosecurity standards on broiler chicken farms to mitigate risk of disease transmission between broilers, backyard poultry, and wild birds on the galapagos. all study farms on santa cruz and san cristobal had ! 1 broilers with positive antibody titers against ibdv. in addition, three of six farms on santa cruz and one of seven farms on san cristobal had ! 1 broilers with positive antibody titers against ibv. possible explanations for the observed exposure to these two pathogens can be: (i) accidental vaccination (e.g., cross-contamination by spray vaccination in hatchery); or (ii) indirect transmission between infected backyard chickens and susceptible broilers (via farm personnel or use of equipment contaminated with field ibdv and/or ibv strains circulating in backyard chickens nearby study farms); although a limitation in this study was that backyard chickens were not investigated. two previous studies have produced serologic evidence of exposure to ibdv and ibv in backyard chickens and broilers on the galapagos. one study on san cristobal reported that exposure to ibdv was high in both backyard chickens (9/25 = 36%; 95% ci = 20, 55%) during 2001-2003 and broilers (30/72 = 42%; 31, 53%) in november 2003 [1] . furthermore, that study reported that exposure to ibv was high in both backyard chickens (6/8 = 75%; 41, 93%) and broilers (33/72 = 46%; 35, 57%) during the same time periods; however, the numbers of farms with one or more broilers classified as seropositive to ibdv or ibv were not reported. another study produced evidence that burden of exposure to ibdv was high in backyard chickens in four flocks (105/119 or 88%; 81, 93%) and broilers on three farms (64/88 or 73%; 63, 81%) located in the same agricultural zone on santa cruz in 2005 [2] . the same study produced evidence that exposure to ibv was higher in study backyard chickens (95/119 or 80%; 72, 86%) compared to study broilers (9/88 or 10%; 5, 18%). in addition, the same study [2] , investigated exposure to ibdv and other pathogens in 236 wild birds. all birds were classified as seronegative to pathogens tested including ibdv, ndv, and mg. in that study, one limitation was that serological testing in wild birds required pooling of samples, an inherent bias that could have produced false negative results if too few samples had positive antibody titers to selected pathogens. in the two previous studies [1, 2] backyard chickens and broilers were sampled for detection of ibdv and ibv antibodies on one occasion. thus, it was not possible to measure seroconversion to these two pathogens and produce serologic evidence of recent infection with ibdv or ibv in either population during the study periods. in this study, one farm on santa cruz had one broiler classified as seropositive to ndv. it is possible this is a false positive result because the antibody concentration against ndv in the study broiler was low (i.e., s/p ratio = 0.25), and no other broilers tested seropositive or showed clinical signs associated with ndv infection. limited funding prevented further testing to reduce false positive results (e.g., testing in series using the hi test, or the use of pcr methods or virus isolation to confirm diagnosis). a previous study produced evidence that exposure to ndv was 16/72 or 22% (14, 33%) on three of five farms in november 2003, and 0/ 27 or 0% (0, 13%) in backyard chickens on san cristobal during 2001-2003 [1] . in that study, a sample size of 27 backyard chickens was not sufficient to detect exposure to ndv if prevalence of exposure was less than 10% [5] . another study produced evidence that exposure to ndv was high both in study backyard chickens (53/119 or 45%; 95% ci = 36, 54%) in four flocks and broilers (32/88 or 36%; 27, 44%) on three farms in santa cruz in 2005 [2] . in that study, exposure to a field strain of ndv explained positive antibody titers detected in backyard chickens, whereas vaccine titers or exposure to a field viral strain explained observed exposure to ndv in broilers. in our study, all broilers were classified as seronegative to mg. one explanation can be that study broilers were not exposed to this pathogen via (i) vertical (transovarian) transmission or (ii) indirect transmission between infected backyard chickens and susceptible broilers (e.g., contaminated feed or water, shoes, or equipment used by farm personnel). another explanation can be that the burden of exposure to mg was low (e.g., < 5%), and both the cross-sectional sampling approach (broilers were sampled one time only) and the sample size of 10 broilers per farm were not sufficient to detect low levels of exposure to mg [5] . previous studies have reported evidence of exposure to mg in broiler chickens on the galapagos. in the study by gottdenker et al. [1] 5/12 or 42% (19, 68%) broilers on one farm were classified as seropositive, and 60 broilers on four farms were classified as seronegative to mg in san cristobal in november 2003. in that same study, 2/19 or 11% (3, 31%) and 12/19 or 63% (41, 81%) backyard chickens on san cristobal and santa cruz, respectively, had positive antibody titers to mg during 2001-2003. in the study by soos et al. [2] on santa cruz, exposure to mg was higher in backyard chickens in four flocks (55/119 or 46%; 38, 55%), compared to broilers on three farms (3/88 or 3%; 1, 10%). two of six poultry farms on santa cruz and six of seven poultry farms on san cristobal had evidence of exposure to eimeria spp. coccidia infections in poultry are not a health hazard to galapagos wild birds due to the parasite high host specificity [6] . although, the source of eimeria spp. infection in study farms is not known, one potential source is indirect transmission between infected backyard chickens (e.g., via farm personnel or use of equipment contaminated with eimeria spp.) and susceptible broilers. in a previous study, eimeria spp. infection was diagnosed in 16/68 (24%) backyard chickens on santa cruz and san cristobal during july 2001-september 2003 [1] . in that study, 100 broilers were included, but they were not examined for intestinal parasites. in another study [2] , 90 broilers, 120 backyard chickens, and 338 wild birds were investigated to measure exposure to multiple pathogens on santa cruz in june 2005. fecal samples were collected from study chickens and wild birds, but samples were not tested for diagnosis of intestinal parasites. a third study [7] included 175 backyard chickens and 274 wild birds to measure exposure to multiple pathogens on floreana island during april-may 2008. in that study, fecal samples were collected from 63 wild birds, and six (10%) were diagnosed with isospora sp. in that study, however, fecal samples were not collected from backyard chickens for diagnosis of intestinal parasites. two of 26 harvested broilers (farm a and farm f) had intestinal tract lesions. one broiler had mild lesions of the proximal duodenal mucosa consistent with eimeria acervulina infection, and the second broiler had presence of blood and mucus in the cecum consistent with eimeria tenella infection. in poultry, eimeria acervulina, eimeria tenella, and eimeria maxima are the most commonly recognized species in broilers [8] . the effects of eimeria spp. infection on poultry include decreased body weight gain, decreased feed efficiency, and increased maintenance energy costs [9, 10] . in addition, infection with eimeria spp. is a predisposing factor for necrotic enteritis and other intestinal infections in poultry due to its ability to cause mucosal damage [11] . pathogenic eimeria spp. have the potential to move quickly through a poultry pen and kill a large number of birds [12] . to our knowledge, this is the first study that has produced evidence of exposure to eimeria spp. in broilers on the galapagos. this study had several limitations. first, the selection of six of 25 broiler farms on santa cruz was not random. thus, the study results on santa cruz apply to study farms only. second, the sample size of 10 broilers per farm was not sufficient to detect one or more broilers as exposed to mg if the burden of exposure per farm was less than 25%. third, study broilers were blood sampled for detection of selected pathogens on one occasion. thus, it was not possible to measure seroconversion to these pathogens and produce serologic evidence of recent infection in study broilers. fourth, detection of antibodies to investigated pathogens was based on one serologic test (idexx elisa) only. testing in series or testing in parallel (using a second serologic test) could have reduce the risk of potential false positive or false negative results, respectively. fifth, diagnosis of eimeria was limited to oocyst morphology. collection of intestinal scrapings would have been useful for a more accurate diagnosis coccidial infection in study broilers. finally, backyard chickens were not included in this study, so it was not possible to measure and compare the burden of exposure to investigated pathogens between broilers and backyard chickens. in order to mitigate the risk of disease transmission between broilers, backyard poultry, and wild birds, galapagos' policymakers can consider implementing a certification program (e.g., green list, red list) that requires poultry products to come from broiler chicken farms that follow best management practices, including enhanced surveillance and biosecurity measures. for example: (i) use of health records to monitor morbidity and mortality events in broilers; (ii) use of written biosecurity protocols; (iii) use of peripheral fence to keep backyard chickens or other animals out; (iv) poultry pens protected against rodents or native birds; and (v) proper disposal of broiler pen bedding material. all-in all-out management practices allow simultaneous depopulation of facilities between flocks, and time for periodic cleaning and disinfection to break the cycle of diseases. the implementation of this practice in small poultry operations on the galapagos, however, can be difficult. poultry farmers are expected to deliver chilled or frozen chicken meat in the local market (e.g., meat shops, grocery stores, restaurants, tourist boats) once or twice every week. the local business environment justifies the implementation of enhanced surveillance and biosecurity measures identified above. conceptualization: jorge a. hernandez. assessing the risks of introduced chickens and their pathogens to native birds in the galapagos archipelago comparison of pathogens in broiler and backyard chickens on the galápagos islands: implications for transmission to wildlife veterinary clinical calculate confidence limits for a population proportion sense and sensitivity-designing surveys based on an imperfect test parasitic diseases of wild birds diseases of poultry and native birds in galapagos: implications for the reintroduction of native species effect of broiler chicken age on susceptibility to experimentally induced cryptosporidium baileyi infection transforming coccidiosis-mediated lesion scores into production and caloific cost. paper presented at the 23rd world's poultry congress the pathogenesis of necrotic enteritis in chickens: what we know and what we need to know: a review key: cord-257077-cdnkk6ou authors: gabor, kristin a.; stevens, chad r.; pietraszewski, matthew j.; gould, travis j.; shim, juyoung; yoder, jeffrey a.; lam, siew hong; gong, zhiyuan; hess, samuel t.; kim, carol h. title: super resolution microscopy reveals that caveolin-1 is required for spatial organization of crfb1 and subsequent antiviral signaling in zebrafish date: 2013-07-09 journal: plos one doi: 10.1371/journal.pone.0068759 sha: doc_id: 257077 cord_uid: cdnkk6ou understanding spatial distribution and dynamics of receptors within unperturbed membranes is essential for elucidating their role in antiviral signaling, but conventional studies of detergent-resistant membrane fractions cannot provide this information. caveolae are integral to numerous signaling pathways and these membrane domains have been previously implicated in viral entry but not antiviral defense. this study shows, for the first time, the importance of spatio-temporal regulation of signaling receptors and the importance of the regulation of clustering for downstream signaling. a novel mechanism for virus evasion of host cell defenses is demonstrated through disruption of clusters of signaling molecules organized within caveolin-rich domains. viral infection leads to a downregulation in caveolin-1b (cav-1b), disrupting clusters of crfb1, a zebrafish type i interferon receptor (–r) subunit. super-resolution microscopy has enabled the first single-molecule imaging of crfb1 association with cav-1b-containing membrane domains. strikingly, downregulation of cav-1b, the major protein component of caveolae, caused crfb1 clusters to disperse. dispersal of crfb1 clusters led to a suppressed antiviral immune response both in vitro and in vivo, through abrogation of downstream signaling. this response strongly suggests that crfb1 organization within cav-1b-containing membrane domains is critical for ifn-mediated antiviral defense and presents a previously undescribed antiviral evasion strategy to alter ifn signaling and the antiviral immune response. the structure and organization of cellular membranes play important roles in a wide range of biological processes. caveolae are specialized membrane nanodomains with a distinct v-shaped morphology in the membrane. caveolae may act as signaling platforms by allowing signaling molecules to cluster together within their ordered domains, facilitating interactions among the components [1] . critical cellular processes associated with caveolae include signal transduction, cholesterol homeostasis, and adaptive immune signaling [2, 3, 4, 5, 6, 7] . caveolin-1 (cav-1) serves as one of the structural components of caveolae and also functions as a scaffolding protein that recruits signaling molecules to caveolae [8] . clustering of proteins in caveolae provides an environment and a mechanism for controlling probabilities of protein interaction and modulating the efficiency of signal transduction. for example, epidermal growth factor receptor (egfr) has been shown to interact with caveolin [9] and changes in receptor clustering may provide a mechanism for regulating egfr signaling [10] . in addition, caveolae are exploited by some viruses to initiate infection [11, 12] , whereas other viruses enter cells without the involvement of caveolae [13, 14, 15, 16, 17, 18] . for example, damm et al. [15] observed that when introduced to cells devoid of caveolae, sv40 exploits an alternative, cav-1-independent pathway in the absence of caveolae. ewers et al. [18] , used transmission electron microscopy to demonstrate that sv40 induced the formation of membrane invaginations in the absence of caveolar coats. it has also been determined that ebola virus can fully infect cell types lacking caveolae [14] and that sars coronavirus entry was mediated by a clathrin-and caveolae-independent mechanism [16] . type 1 interferon (ifn) is crucial for initiation of the innate response to viral infection, and knockout studies in mice have shown that disruption of this response renders the host more susceptible to infection. other studies have demonstrated that mice lacking a functional ifn receptor (ifn-r) were unable to cope with an array of viral infections, including vaccinia virus, vesicular stomatitis virus (vsv), and semliki forest virus [19] . ifn-r knockout mice were highly susceptible to infection with vsv due to high levels of viral replication [20] . the relationship between cell membrane organization and the antiviral immune response is largely unexplored. one of the primary antiviral responses is the generation of ifn, and only recently has the role of lipid rafts in interferon production come under scrutiny [21] . ifn-r and caveolin-1 have both been found in detergent-resistant membrane (drm) fractions [21] , but have not been observed with sufficient spatial resolution to determine their nanoscale distribution in intact cell membranes. such evidence could provide critical insights into the spatial and temporal organization of antiviral receptors and nanodomains. it has previously been shown that zebrafish infected with snakehead rhabdovirus (shrv) produce an ifn response [22, 23] , leading to the binding of ifn to its cognate receptor, ifn-r. the zebrafish ifn-r complex has been recently identified as cytokine receptor family members crfb1, crfb2, and crfb5, which constitute crfb1/crfb5 and crfb2/crfb5 heterodimers [24, 25] . the jak-stat signal transduction pathway is activated upon ifn binding to the receptor and culminates in the expression of ifn-stimulated response element (isre) driven ifn-stimulated genes (isgs) [26] . the ifn pathway and ensuing antiviral response of zebrafish is similar to that described in mammalian systems [24, 25, 27, 28] . many properties of membrane domains cannot be understood solely from drm studies [29] , because drms isolated from cells may not correspond precisely to preexisting rafts in living cells [30] . the small size of caveolae and the spatial proximity of proteins prohibit direct visualization of the dynamic interaction between the host cell membrane nanodomains and antiviral receptors using conventional light microscopy. fluorescence photoactivation localization microscopy (fpalm) ( figure s1 ) [31, 32] is a novel, super resolution technique that extends the resolution of optical microscopy below the diffraction limit, which is on the order of 250 nm, allowing for spatial resolution on the scale of 20-40 nm [31, 32, 33] . three-dimensional fpalm has achieved a lateral resolution of 30 nm and 75 nm axially [34] . fpalm is well suited for investigation, at the single molecule level, of the highly complex molecular structures and mechanisms underlying biological processes. few investigators have focused on the role of caveolae in the immune response. our results suggest that crfb1 interacts with caveolae and that caveolae may be critical for maintaining spatial organization and clustering of crfb1 molecules. the present study demonstrates a novel role for cav-1b-containing membrane domains in the zebrafish response to viral infection. we demonstrate that upon virus infection, cav-1 is downregulated, circumventing the host antiviral ifn response. in vivo knockdown studies showed that disruption of the ifn response by cav-1 depletion renders the host more susceptible to infection. using fpalm, we show that cav-1b-containing membrane domains corral crfb1 molecules together and that this clustering of crfb1 is critical for a robust antiviral immune response. in addition, we determined that the membrane protein cav-1 is responsible for maintaining the crfb1 clustering and that the functional consequence of cav-1 depletion is crfb1 dispersion and abrogation of downstream signaling. by gaining an under-standing of the complex dynamics of membrane domains and the mechanisms through which viruses modulate their function, we will better understand how viruses evade host antiviral mechanisms and can implement this knowledge to develop more targeted therapeutics. we investigated the membrane localization of the crfb1 subunit of the zebrafish ifn-r complex, the components of which are necessary for a functional ifn response in the zebrafish [25] . to test whether crfb1 localizes to cav-1b-containing membrane domains, fpalm was used to simultaneously image crfb1-dendra2 and cav-1b-pamcherry 24 h after transfection of zebrafish liver (zfl) cells. zfl cells express endogenous cav-1b and crfb1 mrna ( figure s2a ), as do rat liver cells [35] , liver sinusoidal cells [36] and primary rat hepatocytes [37] . figure 1 illustrates that at the surface of a single cell, crfb1 colocalizes with cav-1b. acquisition conditions and more details about fpalm imaging and analysis are described in the methods section and figure s1 . these data were acquired in the absence of ligand stimulation and show that clusters of cav-1b molecules are in very close proximity to crfb1 molecules, and in many instances overlap within the estimated spatial resolution of the technique (,20 nm). to quantitatively explore this result, pair correlation analysis was performed for crfb1 and cav-1b ( figure 1c ). pair correlation between the two species had a g(r) value greater than one, which implies that the two molecules are not randomly distributed, and instead, are colocalized. additionally, when cav-1b is knocked down in zfl cells using a previously characterized morpholino oligonucleotide (mo) [2] , the clustering of crfb1 is significantly decreased, with a more random distribution than observed in controls ( figure 1d ). the observation of colocalization between cav-1b-containing membrane domains and crfb1 molecules led to the investigation of whether cav-1 plays a role in the antiviral response to virus infection, since ifn is a critical component of the innate immune response. the roles of both cav-1a and cav-1b in zebrafish development have been previously revealed using mo knockdown technology [2] . further, the presence of caveolae in zebrafish has been confirmed via electron microscopy [2] . compared to cav-1a, cav-1b in the zebrafish is more similar to cav-1b in human and mouse, and in previous studies the two isoforms have been shown to have non-redundant roles [2] . our studies revealed that although cav-1a gene expression was also downregulated after shrv infection ( figure s3a ), the effect was not as pronounced, nor was it as long lasting as the downregulation of cav-1b gene expression ( figure 2a) . furthermore, when compared to controls, knockdown of cav-1b resulted in greater mortality than knockdown of cav-1a after shrv infection ( figure s3b ), leading us to focus our subsequent studies on cav-1b. in embryos infected with shrv, early cav-1b gene expression was shown by quantitative rt-pcr to be significantly dampened at 12, 24, and 48 hpi, with a 3.5-, 2.5-, and 3.8-fold decrease in transcript levels compared to controls, respectively (figure 2a , p,0.05). in order to confirm that during viral infection general suppression of all host gene expression did not occur, zebrafish bactin primers were used to normalize the initial quantity of rna, as previously described [38] . to confirm these results, the 18s housekeeping gene was also used to normalize the gene expression in the rt-pcr experiments. the 18s gene has been previously characterized in the zebrafish and shown to be stable during development and across tissue types [22, 23, 39] . the 18s gene was selected due to its high, relatively stable expression levels. if viral infection globally affected gene expression, then 18s would also be influenced. however, similar results (data not shown) were obtained when 18s primers were used to normalize the quantity of rna. knockdown of cav-1b with mo in embryos has been characterized previously and a reduction in cav-1b protein levels, as well as a reduction in the number of caveolae domains, was demonstrated [2, 40] . we performed knockdown experiments as described [2, 40] after confirming the amount of mo required to knock down cav1b. figure 2d demonstrates that at 24 hpi in control mo-injected embryos cav-1 protein was still detected, while in cav-1b mo-injected embryos no cav-1 protein was found. western blot analysis of age-matched shrv infected embryos detected endogenous cav-1 protein at lower levels than in control mo embryos, but greater than in the cav-1b mo samples. this result shows effective knockdown of cav-1b protein expression by morpholino injection (figure 2d ). these experiments were performed using whole embryo lysates, and so in order to more thoroughly understand the results, we sought to identify tissues in which cav-1b is expressed. we examined cell typespecific pools of cdna from adult zebrafish. of particular interest, cav1b expression was detected in liver, kidney, lymphocyte, and myeloid lineages ( figure s2c ). in addition, cav-1b and crfb1 are also expressed in the liver tissue of embryos when infection studies were performed ( figure s2b ). the cav-1b mo was used to determine whether the observed disruption of cav-1b gene expression would alter the host's susceptibility to virus infection [2] . morphant and control embryos were monitored for survival rates and viral burden. in the absence of virus, knockdown of cav-1b did not affect embryo survival. kaplan-meier curves [41] were constructed showing survival of cav-1b morphants compared to controls after viral challenge, and revealed that cav-1b morphant embryos exhibited a significant increase in mortality (p = 0.009) compared to the controls (figure 2b ). uninfected control morphants had low levels of mortality, similar to that of uninjected controls. cav-1b morphants showed increased mortality throughout the first three days post infection. after just 24 hpi, over 70% of the cav-1b morphants had succumbed to the infection compared to ,40% of control infected embryos. controls and cav-1b morphants that were uninfected both had ,90% or greater survival rates. since the adaptive immune response is not fully developed in zebrafish at the developmental stage selected for these studies [24, 42, 43] , the results are due solely to perturbation of the innate immune response. we sought to determine whether the increase in mortality was a result of increased incidence of viral entry due to cav-1 knockdown, or reduced ability of morphant embryos to clear the infection. preliminary studies suggested that shrv does not utilize cav-1b-containing membrane domains as a means of entry in vitro ( figure s4) ; therefore entry of virus should not be affected by cav-1 knockdown. viral burden assays were conducted to determine if disruption of cav-1b mediated viral entry after infection with shrv. from 0-12 hpi, no significant increase in viral burden was observed between control and cav-1b mo embryos, which were infected at 48 hpf (and therefore 48 h after mo injection). however, by 24-48 hpi, a significant increase in caveolin-1 is critical for antiviral signaling plos one | www.plosone.org viral burden (figure 1c ) was measured. cav-1b morphants showed a 28-fold and 6.5-fold increase in viral titer compared to controls at 24 and 48 hpi, respectively ( figure 2c ). the data were significant (two-way anova, p,0.05) and correlated with the increased mortality shown at 24 hpi and 48 hpi in the cav-1b morphant embryos. if cav-1b-containing membrane domains are being used as a platform for immune signaling through the ifn-r pathway, knockdown of cav-1b should dissipate antiviral signals, such as gene expression of stat1 and subsequent induction of isre. transcript levels of stat1 were assessed at 24 h in both control mo and cav-1b mo embryos with and without shrv infection. a 1.5-fold (60.13) decrease was observed in control mo embryos and a 3.1-fold (60.09) decrease was observed in cav-1b mo embryos ( figure 3a) . to compare the effect of cav-1b depletion on the antiviral response to pathogen, we examined control mo cells, cav-1b mo cells, and control cells after shrv infection in an isre promoterdriven luciferase assay. zfl cells were transfected with cav-1b mo or standard control mo, along with an isre luciferase construct [44] , and subsequently exposed to shrv (0.01 moi for 24 h) ( figure 3b ). cav-1b depletion by cav-1b mo in zfl cells is shown in figure s5 . shrv infected cells displayed a significant decrease in isre activity compared to control mo samples (twotailed student's t test, p,0.001). similarly, depletion with cav-1b mo also resulted in a significant decrease in isre activity compared to control mo samples (two-tailed student's t-test, p,0.001), mimicking the effect of shrv infection. a greater reduction in isre activity was observed in either shrv-infected or cav-1b mo cells when compared to control mo infected cells, a finding that is consistent with the decrease in stat1 gene expression shown in figure 3a . we examined whether disrupted ifn signaling resulting from cav-1b depletion was due to dispersal of crfb1 molecules corralled by cav-1b-containing membrane domains, or to effects on other antiviral components that could exist within cav-1bcontaining membrane domains. covalent crosslinking studies were performed using bis(sulfosuccinimidyl) suberate (bs 3 ) reagent with zfl cells that were transfected with either cav-1b mo or standard control mo and subsequently crosslinked. the crosslinking reagent was employed to ''rescue'' the dispersal of crfb1 that results from cav-1b disruption. if cav-1b depleted cells with crosslinked crfb1 molecules were able to produce an antiviral response, this would indicate that cav-1b depletion and subsequent dispersal of receptor molecules was directly responsible for the abrogated antiviral response. fpalm imaging demonstrated that infection of zfl cells with virus resulted in dispersion of crfb1 molecules ( figure 4 ). similar numbers of crfb1 molecules are seen in the uninfected cell (12, 251) compared to the infected cell (11, 358) , which indicates that there is no overall loss of surface crfb1 as a result of infection. these results demonstrate that virus infection leads to dispersal of ifn receptors. control mo with crosslinking treatment yielded crfb1 molecules that remained clustered together, while cav-1b depletion without crosslinking treatment yielded crfb1 molecules that were dispersed ( figure 5 ). pair correlation analysis quantitatively confirmed the result of our fpalm images, showing that with cav-1 depletion and crosslinking treatment, the receptors remained clustered (figure 5d ). a parallel experiment was conducted in zfl cells that were transfected with control mo, cav-1b mo, or crfb1/crfb2/ crfb5 mo (all subunits of the ifn-r) in order to measure the induction of antiviral genes downstream from the ifn-r. polyinosinic-polycytidylic acid (poly(i:c)) was used in another experiment to mimic an infection and to stimulate the production of ifn by the immune system. poly(i:c) is a synthetic analog of double stranded rna (dsrna) which is associated with viral infection. it is recognized by pattern recognition receptors (prr) [45, 46] and leads to the induction of type i ifn and inflammatory cytokines. cells were crosslinked with bs 3 , exposed to poly(i:c), or both crosslinked with bs 3 and exposed to poly(i:c) (figure 6a ). depletion of cav-1b using the mo in zfl cells is demonstrated in figure s5 . time points shown correlate with the time of an additional experiment was performed with cells transfected together with both mo and cav-1b plasmid to rescue the effect of the cav-1b knockdown. cells were subsequently infected with shrv ( figure 6b ). transcripts of mxa were measured by quantitative rt-pcr. mxa was chosen because its transcripts are produced solely from the ifn-a/b pathway and not the ifn-c pathway [25] . as expected, upon either poly(i:c) exposure ( figure 6a ) or shrv infection (figure 6b ) cav-1b mo samples displayed decreased mxa expression compared to controls (second group of bars, gray), as did crfb1/crfb2/crfb5 mo samples (second group of bars, black). to rescue the effects of cav-1b depletion, cells were also transfected with cav-1b plasmid which resulted in detection of low levels of mxa in the absence of shrv (figure 6b ). cells were then infected with shrv and mxa gene expression was measured. with either crosslinking or cav-1b rescue, mxa gene expression remained comparable to that of control cells (figure 6a and b, fourth group of bars in each, gray bars). we have thus demonstrated that when the depletion of cav1b is rescued, the expression of mxa did not decrease. by developing a more thorough understanding of the mechanisms of the antiviral immune response, we hope to find clues that will aid the development of new therapeutics and vaccine adjuvants capable of augmenting the immune system and providing more effective protection to the host. this study demonstrates an entry-independent mechanism for virus evasion of host cell defenses through disruption of clusters of signaling molecules organized within cav-1b-containing membrane domains. upon viral infection, cav-1b was downregulated (figure 2a and d), leading to a decrease in the number of cav-1b-containing caveolin-1 is critical for antiviral signaling plos one | www.plosone.org membrane domains [2] . we report here the first nanoscale visualization of crfb1 association with cav-1b-containing membrane domains in intact cells and demonstrate the dramatic effect that depletion of cav-1b-containing membrane domains has on the antiviral response. the use of fpalm enabled imaging of the clustering and subsequent dispersal of crfb1 following cav-1 knockdown. the primary focus of the present studies was to investigate the potential abrogation of the antiviral response. the data show that in cav-1b knockdown cells, crfb1 molecules are dispersed and cav-1b-containing membrane domains are disrupted during viral infection, leading to impairment of the antiviral response. this suggests that intact caveolin domains may be crucial for proper clustering and function of crfb1. receptor dispersal from cav-1 knockdown suppressed the antiviral immune response by disrupting downstream signaling, indicating that crfb1 organization within cav-1b-containing membrane domains is critical for ifnmediated antiviral defense. the functional consequences of cav-1 depletion were shown by direct observation with fpalm of crfb1 clustering and identification of the membrane protein responsible for maintaining this clustered state. the crfb1 subunit of the zebrafish ifn receptor complex has been reported to heterodimerize with crfb5 [25] . levraud et al. [25] , assessed several candidates of the crfb family as likely members of the ifn receptor complex and found that knockdown of crfb1 and crfb5 have a dramatic effect on zebrafish ifn responsiveness. the authors postulated that the two should be designated as the heterodimer subunits of the ifn receptor. we have considered this interesting question in light of our current findings. we would like to know where crfb5 is localized and whether or not this receptor subunit also clusters or relies upon cav1b-containing domains for a robust ifn response. such tantalizing questions are currently being investigated. type i ifn belongs to a class of cytokines that play a crucial role in the innate immune response to viral infection [47, 48] . molecular patterns such as viral double stranded rna are detected by prr [49, 50] , resulting in production of ifn and antiviral proteins. in zebrafish, as in mammals, ifn molecules interact with the ifn-r subunits [50, 51] , which exist as heterodimeric complexes [24, 25] . the janus kinase and signal transducer and activator of transcription (jak-stat) signaling pathway is highly conserved evolutionarily, and it is believed that in zebrafish, stat transduces signals through a classical jak-stat pathway [52] . briefly, the jak-stat pathway becomes active upon ifn binding to ifn-r, but two discrete ifn pathways activate jak-stat: ifn-a/b and ifn-c. it is possible to measure components upstream from isre or mxa, such as stat phosphorylation, but nonspecific contributions from the ifn-c pathway can occur, making it difficult to discriminate between the jak-stat contributions of the two pathways. it has been hypothesized that the ifn-c response is attenuated due to reduced levels of stat1 in the ifn-r knockout [53] . to investigate whether a reduced level of stat1 gene expression also contributed to a dampened ifn response in our current studies, stat1 transcripts were measured by qrt-pcr after cav-1b depletion. a decrease in stat1 gene expression was observed at 24 hpi (figure 3a ) as a result of cav-1b depletion. we further demonstrate that crfb1 is dispersed and that downstream mxa signaling can be restored by maintaining crfb1 clusters ( figure 6 ). this indicates that the clustering of crfb1 is critical for an antiviral response. cav-1bcontaining membrane domains appear to corral the receptor molecules, thus providing an environment conducive to efficient signal transduction. previous studies in murine embryonic fibroblasts demonstrated that type i ifn receptors (ifn-r) and type ii ifn receptors (ifngr1 and ifngr2) were associated with caveolae domains after drm isolation [21] . in contrast, ifnar and ifngr distribution in hela cells showed that only ifngr complexes could be found in drms after stimulation [54] . the significance of protein associations with lipid rafts must therefore be reevaluated and interpreted with caution. in our studies, we use zfl cells, in which the endogenous expression levels of cav-1b have been confirmed (data not shown). localization of ifn receptors in cav-1b-containing membrane domains by microscopy is likely to yield less equivocal results than biochemical drm isolation. our study yields images through the use of fpalm, which circumvents the resolution limit imposed by optical diffraction in conventional light microscopy. membrane structure and organization are important for many signaling processes. in this study, crfb1 clustering in cells was examined and the results provided insights into the dynamic behavior of this receptor. super-resolution imaging with fpalm showed that crfb1 clustering is mediated by cav-1b-containing membrane domains and that disruption of such domains results in dispersal of receptor molecules. the consequence of crfb1 dispersal was a dampened antiviral response. studies were performed using either poly(i:c) stimulation or shrv infection, both of which will induce ifn, the ligand that will bind to crfb1 and stimulate it. it is important to note that the crosslinking reagent is non-specific and may impact cellular function because it crosslinks everything on the cell surface, not just cav-1 or crfb1 molecules. we tested for non-specific crosslinking effects by also depleting crfb1/2/5 and demonstrated that nonspecific induction of mxa due to crosslinking of other cellular components does not occur. we also rescued crfb1 clustering through exogenous plasmid expression of cav-1b and found that after shrv infection, expression levels of mxa remained essentially equal to controls (figure 6b ), similar to results seen in figure 6a . mxa was measured because it is a selective and quantitative indicator of antiviral activity that is produced through induction of the ifn pathway. taken together our data demonstrate that viral infection is exacerbated due to the reduced ability of cav-1b morphant embryos to clear the infection resulting from the dispersal of crfb1 and subsequent decrease in stat1 gene expression, isre activation, and mxa induction. our results contrast with the conclusions of many studies demonstrating that viruses use caveolae as a method of entry [55, 56, 57] , but not as a means to alter the host immune response. others have observed virus infections that do not use caveolae as a method of entry [13, 14, 15, 17] , but did not necessarily study the role of caveolae in the antiviral immune response. we took a novel approach and discovered that shrv downregulates cav-1 expression to disrupt the host antiviral response. many viruses in a range of species have developed mechanisms to target and evade the ifn system [58, 59, 60] . the studies outlined here reveal that viruses can escape the antiviral immune response by downregulating cav-1b protein levels, leading to a disruption of antiviral signaling through dispersion of ifn-r and abrogation of downstream signal transduction. we assessed the virus induced downregulation of cav-1b compared to the morpholino depletion of cav-1b and found that viral infection alone is enough to decrease cav-1b protein levels and dampen isre activity (figures 2d and 3 , respectively). taken together, these studies support the hypothesis that cav-1b-containing membrane domains provide the local environment for interaction of critical antiviral receptor molecules. additionally, these studies have demonstrated that cav-1b is responsible for maintaining crfb1 clusters and have shown the functional consequences of cav-1 knockdown. from these observations, it is postulated that cav-1b-containing membrane domains increase crfb1 signaling efficiency by concentrating receptor molecules so that proteins remain at the site of signaling. there have been several studies of immunity in zebrafish that demonstrate similarities to immune function in higher vertebrates [22, 23, 27, 28, 42, 61] . in addition, a publically available microarray database (european bioinformatics institute's gene expression atlas, part of the european molecular biology laboratory; http://www.ebi.ac.uk/gxa/) was used to identify downregulation of cav-1 in humans after infection with viruses such as hsv and hiv. our identification of a cav-1 binding domain in human ifn-r, together with the high degree of functional conservation between the immune system of zebrafish and higher vertebrates, suggests that our studies are relevant to immunity in higher vertebrates. fpalm studies provided critical insight into the mechanisms of viral evasion and modulation of membrane domains that are critical to the host immune response to virus infection. understanding the complex mechanisms through which viruses modulate immune function should provide insight into a range of potential targeted antiviral therapies. zebrafish used in this study were handled in accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health. the protocol was approved by the institutional animal care and use committee (iacuc) at the university of maine (protocol number: a2008-06-03). iacuc approved guidelines for zebrafish care were followed using standard procedures (www.zfin.org). cell culture. epc (epithelioma papulosum cyprinid) cells originated from carp epidermal herpes virus-induced hyperplastic lesions [62] . epc cells have a broad sensitivity for fish viruses and are commonly used for isolation, propagation, and diagnostic assays for fish viruses. epc cells were maintained at 28uc, 4% co 2 in minimum essential medium (mem) (gibco-invitrogen, carlsbad, ca) supplemented with 10% heat-inactivated fetal bovine serum (gibco-invitrogen, carlsbad, ca) and antibiotics. zfl (zebrafish liver) cells were derived from normal adult zebrafish liver [63] . they display an epithelial morphology. ghosh et al demonstrated that the cells exhibit properties in culture that are associated with liver cell function in vivo. zfl cells were maintained at 28uc, 0% co 2 in ldf culture medium (50% leibovitz's l-15 medium, 35% dulbecco's modified eagle's medium, and 15% f-12 medium) supplemented with heatinactivated fetal bovine serum. expression plasmids. a modified pegfp-n1 plasmid (clontech) containing pa-mcherry in place of megfp [64] was digested with xmai and noti (new england biolabs) to linearize the plasmid. cav-1b was cloned from a 30dpf zebrafish cdna library and psti and xmai sites were added by polymerase chain reaction. dendra-crfb1 was generated using a dendra2-ha construct [65] in which psti and xmai restriction sites were added to crfb1 by polymerase chain reaction. crfb1 was subsequently inserted between psti and xmai, replacing ha from the vector. the final constructs were purified by endotoxin free miniprep (omega). cell transfection. zfl cells were transfected by nucleofection according to the manufacturer's protocol (lonza). cells were transfected 24 h prior to fixation. for fixation, cells were removed from the incubator and rinsed three times in dulbecco's pbs (biowhittaker lonza, walkersville, md), and incubated at room temperature for 20 min in 4% paraformaldehyde (sigma-aldrich). immersion water and pbs were both irradiated for ,15 min by 500 w uv-lamp to reduce background fluorescence. during measurements, uv-bleached dulbecco's pbs was used as the imaging medium. luciferase assay. luciferase assays were performed in a manner similar to that described previously [22, 44] . the ifn stimulated regulatory element (isre)-reporter vector isre-luc was provided by r. medzhitov (yale university, new haven, ct) [44] . prior to transfection, cells were allowed to reach 70-80% confluence in a t75 flask, at which point they were resuspended in buffer sf (lonza) at 4610 5 cells/20 ml and mixed with a total of 250 ng of indicated plasmid dna, 250 of either the pb26luciferase or pgl3-ifn reporter construct, and 6.25 ng of prl-cmv renilla luciferase internal control construct. cells were then electroporated using the amaxa 96-well shuttle (lonza) using program ew-158. cells were then plated at 1610 5 cells/well, in triplicate, using fresh medium and incubated at 28uc for 30 hours prior to 0.5 mg/ml polyinosinic-polycytidylic acid (poly(i:c)) exposure for 6 hr. since poly(i:c) resembles the rna of infectious viruses, it was used to mimic an infection and stimulate the immune system to produce ifn and other cytokines. following poly(i:c) exposure or shrv infection, cells were lysed and firefly and renilla luciferase activity was measured using the dual-luciferase reporter assay system (promega). two experiments were performed with three replicates per experiment. the mean of the three replicates was taken for each experiment, and the standard deviation of the means was taken to generate the sem. relative luminescence units (rlu) were measured in a glo-max luminometer (promega). zebrafish care and maintenance. wild-type (strain ab) fish were maintained in the zebrafish facility at the university of maine, orono. the zebrafish facility is maintained according to the institutional animal care and use committee (iacuc) standards. fertilized eggs were collected in petri dishes at the onecell stage before the start of experiments and raised in egg water (60 mg/ml instant ocean sea salts) at 28uc. the translation blocking cav-1 mos were previously published by fang [2] and are targeted to the atg start sites of cav-1a and cav-1b mrnas. cav1 mos and control mo were injected at the same concentration. the cav-1a mo sequence is 59-tcccgtccttgtatccgctagtcat-39 and the cav-1b mo sequence is 59-ttcgttgatgctgtcgttatccatt-39. mos were microinjected into zebrafish embryos at 6 ng/ embryo during the 1-2 cell stage. injected embryos subsequently developed in egg water at 28uc. all crfb mos were previously published [24, 25] . crfb1 mo is a translation blocking mo with the sequence 59-cagtgtat-gatgatgatgtcttcat-39. crfb2 mo is a splice blocking mo with the sequence 59-ctatgaatcctcacctaggg-taaac-39. crfb5 mo is a translation blocking mo with the sequence 59-cagggcacactcctccatgatccgc-39. snakehead rhabdovirus (shrv) was propagated in epc cells. cells were infected at a multiplicity of infection (moi) of 0.1. the supernatant was then collected and filtered to obtain purified virus at a titer of 3.16610 7 50% tissue culture infectious doses (tcid 50 )/ml. for infection and imaging experiments, cell monolayers at ,70-80% confluency were infected 24 h prior to fixation and imaging. shrv infection at 0.1 moi proceeded for 1 h at 28uc before cells were overlain with additional growth medium for another 23 h (24 h total infection time). wild-type and caveolin-deficient zebrafish embryos were infected by static immersion 48 hours post fertilization (hpf) for 5 hours with 1610 6 tcid 50 /ml shrv or maintained as uninfected controls. twenty fish were collected at 24 hr post infection (hpi) for each treatment and homogenized in minimum essential medium (gibco-invitrogen, carlsbad, ca), with 50 mg/ml gentamycin. the homogenate was frozen at 280uc before the tcid 50 assay. tcid 50 is a type of virus quantification method. this endpoint dilution assay enables us to determine how much virus is needed to produce a pathological change (observed as cytopathic effects, or cpe) in 50% of inoculated cells in culture. cpe (i.e. infected cells) was manually observed and recorded for each virus dilution. for our experiments, supernatants previously frozen at 280uc were thawed to be used in tcid 50 assays and subsequently monitored for cytopathic effects (cpe). after seven days, cpe was determined and the tcid 50 /ml of the virus was calculated according to the reed-muench formula [66] . virus infection in cell culture experiments was also performed with shrv propagated in epc cells. for these studies, cells were infected at an moi of 0.01. the virus was allowed to adsorb for 1 h. subsequently, virus was removed and regular cell culture media was replaced. dual luciferase assays were performed as described after 24 hpi. total rna was extracted after cav-1b mo-injected and control mo injected fish were infected with shrv by static immersion for 5 hr. viral samples were collected at 24, 48, and 72 hpi by homogenizing 10 fish from each treatment, per time point in trizol reagent (invitrogen, carlsbad, ca) and subsequently stored at 280uc. rna was extracted according to the manufacturer's protocol. reverse transcription reactions were performed as previously described [38] to synthesize cdna. quantitation of mxa was carried out using an i-cycler iq detection system (bio-rad laboratories, hercules, ca). the cycling parameters used were chosen as described previously [38] . fluorescence measurements were made at each cycle during the annealing step and the copy number was determined based on a standard curve using the icycler software. the value for each sample was normalized to the corresponding b-actin value to determine relative copy number. fold inductions were calculated by dividing the copy number in the virus infected samples by the uninfected samples at the same time point. to identify the cell lineages in which cav-1 is expressed, zebrafish tissues were dissected into trizol (invitrogen, carlsbad, ca) and total rna was purified in preparation for qpcr. lymphoid and myeloid cells (frozen pellets) isolated from zebrafish kidneys and purified by fluorescence-activated cell sorting were generously provided by dr. david traver (university of california, san diego, ca) [28, 67, 68] and resuspended in trizol for rna purification. total rna from tissues (2 ug) and sorted cells (1 ug) was reverse transcribed (superscript tm iii reverse transcriptase, invitrogen) and subjected to thermal cycling with gene-specific primers and titanium tm taq dna polymerase (clontech, mountain view, ca). expression of cav1a and cav1b isoforms was detected using primers previously described [2] and 40 cycles with an annealing temperature of 70uc. pcr conditions and primer sequences for detecting myeloperoxidase (mpx), tcra and b-actin expression were described previously [69] . liver tissue was isolated for detection of cav-1b, crfb1, l-fabp, and b-actin expression in the liver of a 48 hpf embryo according to previously published methods [70] . total rna from liver tissue was extracted and cdna synthesized as described above. pcrs were analyzed by gel (2% agarose) electrophoresis. embryos were prepared in a manner similar to that published previously [2] . embryos were collected, egg water removed, and flash-frozen in a slurry of dry ice prior to storage at 280uc. for use, frozen embryos were solubilized in ripa lysis buffer (pierce, rockford, il) and halt protease/phosphatase inhibitor cocktail (thermo scientific). embryonic zebrafish were incubated on ice for 30 minutes prior to centrifugation at 18,0006g for 15 minutes at 4uc. supernatants were collected as whole cell lysates. zfl cells were transfected with control morpholino (mo) or cav-1b mo to knock down the expression of cav-1b. samples were taken at 6 and 30 h post transfection (hpt), corresponding to the time points used to perform experiments shown in figure 6 . for sampling, cells were centrifuged at 906g for 10 minutes at 4uc. cells were washed twice in dpbs (biowhittaker lonza, walkersville, md) prior to storage at 280uc. for use, cell pellets were solubilized in ripa lysis buffer (pierce, rockford, il) and halt protease/phosphatase inhibitor cocktail (thermo scientific). cells were incubated on ice for 30 minutes prior to centrifugation at 35006g for 10 minutes at 4uc. supernatant was collected for use as the soluble fraction. to determine protein concentrations, a bradford assay was performed using the bio-rad protein assay dye reagent (bio-rad laboratories, hercules, ca). equal volumes of total cell lysate were solubilized in lysis buffer, boiled for 5 minutes, and fractionated by sds-page gel electrophoresis. fractionated proteins were transferred to a nitrocellulose membrane by electrophoresis, blocked with 5% non-fat dry milk, and immunoblotted with the anti-human cav-1 polyclonal antibody (1:500 dilution, bd transduction laboratories). cav-1 protein was visualized using horseradish peroxidase conjugated secondary antibody (santa cruz biotechnology) and the supersignal chemiluminescence system (pierce, rockford, il). membranes were re-probed with antibody against b-actin to control for protein loading. cells were transfected via nucleofection with control mo, cav-1b mo, or combined crfb1/crfb2/crfb5 mo and allowed to recover/adhere to cell culture plates for 6 hr prior to bis(sulfosuccinimidyl)suberate (bs 3 ) cross-linking treatment. cross-linking reactions were performed according to the manufacturer's procedures (pierce, rockford, il). cells were subsequently washed 3 times with 500 ml dpbs and replenished with media and returned to the incubator for 24 hr post exposure (hpe) to bs 3 . for cells that were exposed to 1 mg/ml poly(i:c) (invitrogen), treatments were initiated 18 hpe to bs 3 reagent and proceeded for 6 hr. following exposures, rna samples at sequential time points were taken with trizol according to the manufacturer's procedures. rna extractions, cdna synthesis, and quantitative rt-pcr were performed as described above. in normal fluorescence microscopy, many of the fluorescent molecules are visible at the same time and their images are blurred together by diffraction. since diffraction blurs objects smaller than 200-250 nm, important biological details can be obscured. fpalm circumvents diffraction by limiting the number of visible/fluorescent molecules visualized at once. rather, many small subsets of fluorescently labeled molecules within a sample are imaged separately, such that each molecule is distinct. this is achieved by optical control of molecular transitions between bright and dark states. by limiting the numbers of emitting molecules and activating a subset of molecules, and then imaging and photobleaching them and repeating this process for many subsets of molecules, coordinates of thousands of molecules can be obtained [31, 32] . this iterative process is repeated until sufficient molecules have been localized and the structure of the sample is revealed. the positions of the single molecules can be determined (localized) with a precision better than diffraction limited resolution. the fpalm image is generated by plotting the positions of the localized molecules. single color fpalm imaging and analysis. single color fpalm imaging and analysis were performed as described earlier [31, 32, 33] . a 405 nm diode laser (bcl-405-15, crystalaser,reno, nv) was used to activate labeled molecules in the sample, while a 556 nm (lrs-556-nm-100-10, laserglow, toronto, canada) diode laser was used to read out active molecules. both beams were focused at the back aperture of a 606/1.2na waterimmersion objective lens (uplapo606w, olympus, melville, ny) to produce widefield illumination at the sample. fluorescence from the sample was collected by the objective, separated from laser light by a dichroic mirror (t565lp, chroma technology, rockingham, vt), bandpass filtered (et605/70m, chroma), and imaged by an emccd camera (ixon+ du897dcs-bv, andor scientific, south windsor, ct) operated at an em gain of 200 and frame rate of ,31.5 hz. the camera was controlled using solis software (andor). additional achromatic lenses (f = +60 mm and f = +200 mm, newport corporation, irvine, ca), arranged as a telescope, were mounted in the detection path to provide additional magnification and to produce an effective camera pixel size of ,136 nm. a motorized filter wheel (fw102, thorlabs, newton, nj) containing neutral density filters provided control over the activation intensity to maintain a density of visible molecules of ,1 per mm 2 or less. cells were selected for fpalm imaging by exciting (475/406, chroma) the sample with an hg lamp and searching for green fluorescence (bandpass-filtered, hq535/50m, chroma) to locate cells transfected with crfb1-dendra2. during post acquisition analysis, each frame of an image series (typically 10,000 frames) was background subtracted and positive intensity peaks with at least one pixel above a minimum threshold were fitted to a twodimensional gaussian to determine the x and y coordinates, amplitude (i 0 ), e 2 radius (r 0 ), and an offset. fitted values of i 0 and r 0 were then used to calculate the number of detected photons. fits that yielded n and r 0 consistent with that expected for a single molecule were recorded for further analysis. for each localized molecule the localization precision was calculated using the standard analytical equation from the literature, including an additional 30% [71] . lateral drift of the sample stage has been characterized previously [33] and was assumed to be negligible over the duration of these experiments, compared to the estimated lateral resolution of ,10-30 nm. all analysis was performed using custom software written in matlab (mathworks, natick, ma). two-color fpalm imaging and analysis. two-color imaging of fixed zfl cells transfected with pamcherry-cav1 and dendra-crfb1 was performed at room temperature using the geometry employed in [72] . a dichroic mirror (z568rdc, chroma) and emission filters (ff01-630-92-25, semrock and et605/70m, chroma for transmitted and reflected wavelengths, respectively) were mounted in the detection path between the dichroic mirror and the electron multiplying charge-coupled device camera (emccd) (ixon+du897dcs-bv, andor technology, south windsor, ct). illumination of the sample was achieved by placing a lens f = +350 mm) (thorlabs, newton, nj) near the rear epi-illumination port of an inverted microscope (ix71, olympus america, melville, ny) to focus the beams to the secondary (back) focal plane of a 606, 1.2 na water-immersion objective lens (uplapo606w, olympus). a 405 nm diode laser (bcl-405-15, crystalaser) was used for photoactivation and a 556 nm laser was used for readout. frames were acquired at 31.5 hz (em gain 200) with the emccd camera. images were acquired using labview software (national instruments corporation, austin, tx). analysis for two color imaging was an extension of standard fpalm analysis, described above and previously reported [32, 73] . analysis was performed using matlab software (mathworks, inc. natick, ma) as follows: raw frames containing the two spatially separated images were background subtracted, then correlated and superimposed for localization. each localized molecule is identified by a, the ratio of emission in the red detection channel divided by the sum of the intensity in both red and yellow channels [74] . the species of each localized molecule was assigned as either dendra2-crfb1 (or dendra2-shrv for figure s4a ) or pamcherry-caveolin using a range of a values determined numerically (typically 0.55-0.64 for dendra2 and 0.68-0.75 for pamcherry), such that the error in assignment to either species was ,5%. pair correlation calculations. pair correlation analysis and calculation was performed similar to methods previously described by the hess laboratory [73] . single color pair correlation. coordinates obtained from fpalm imaging were used to calculate pair correlation functions. localizations of the same molecules in consecutive frames were removed from the data set by linking molecules in the i th frame to molecules (i+1) th frame that were separated by less than 3 times the median localization precision. the positions of linked molecules were then averaged for use in pair correlation calculations. values of g(r).1 indicate correlation between species while g(r),1 indicates anti-correlation. for uniform distribution of molecules, g(r) = 1 is expected. calculated values of g(r) were fitted to the analytical correlation function [75] , including a constant offset, where a is the amplitude and r 0 is the correlation length, and g is a number. two color pair correlation. prior to pair correlation calculation using coordinates obtained from two-color fpalm analysis, duplicate localizations of the same molecule in consecutive frames were removed as described above. the crosscorrelation, g(r), of species a with species b was calculated from: where n (i) b (r) is the number of molecules of species b that lie within a dr = 10 nm-thick circular shell of radius r from the i-th molecule of species a, a r is the area of the shell of radius r6(dr/2), n a is total number of species a used in the summation over index i, and r b is the average density of species b. the summation was performed only over molecules of species a that were more than a distance d from the edge of the cell and the imaged region of interest, where d is the maximum length scale of interest for pair correlation analysis such that edge corrections were not required. figure 7 . at 6 hpt there is a marked decrease in cav-1 protein expression compared to control cells, while at 30 hpt a slight decrease in cav-1 protein expression is still observed. membranes were re-probed with antibody against b-actin to control for protein loading. caveolins, a family of scaffolding proteins for organizing ''preassembled signaling complexes'' at the plasma membrane caveolin-1alpha and -1beta perform nonredundant roles in early vertebrate development caveolae, caveolin and caveolin-rich membrane domains: a signalling hypothesis the caveolin proteins caveolin-1-deficient mice show defects in innate immunity and inflammatory immune response during salmonella enterica serovar typhimurium infection the multiple faces of caveolae model systems, lipid rafts, and cell membranes crowded little caves: structure and function of caveolae interaction of a receptor tyrosine kinase, egf-r, with caveolins. caveolin binding negatively regulates tyrosine and serine/threonine kinase activities nanoscale imaging of epidermal growth factor receptor clustering: effects of inhibitors caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the er emerging themes in lipid rafts and caveolae assembly of endocytic machinery around individual influenza viruses during viral entry folate receptor alpha and caveolae are not required for ebola virus glycoprotein-mediated viral infection clathrin-and caveolin-1-independent endocytosis: entry of simian virus 40 into cells devoid of caveolae sars coronavirus entry into host cells through a novel clathrin-and caveolae-independent endocytic pathway characterization of rotavirus cell entry gm1 structure determines sv40-induced membrane invagination and infection antiviral defense in mice lacking both alpha/beta and gamma interferon receptors antiviral protection by vesicular stomatitis virus-specific antibodies in alpha/ beta interferon receptor-deficient mice cross talk between interferon-gamma and -alpha/beta signaling components in caveolar membrane domains molecular and functional analysis of an interferon gene from the zebrafish, danio rerio cloning and characterization of an mx gene and its corresponding promoter from the zebrafish, danio rerio the two groups of zebrafish virus-induced interferons signal via distinct receptors with specific and shared chains identification of the zebrafish ifn receptor: implications for the origin of the vertebrate ifn system signaling through the jak/stat pathway, recent advances and future challenges immunology and zebrafish: spawning new models of human disease the zebrafish as a model organism to study development of the immune system use of detergents to study membrane rafts: the good, the bad, and the ugly lipid rafts, detergent-resistant membranes, and raft targeting signals ultra-high resolution imaging by fluorescence photoactivation localization microscopy imaging biological structures with fluorescence photoactivation localization microscopy dynamic clustered distribution of hemagglutinin resolved at 40 nm in living cell membranes discriminates between raft theories three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples distribution and localization of caveolin-1 in sinusoidal cells in rat liver caveolin-1 mediates endotoxin inhibition of endothelin-1-induced endothelial nitric oxide synthase activity in liver sinusoidal endothelial cells effect of insulin on caveolinenriched membrane domains in rat liver characterization of snakehead rhabdovirus infection in zebrafish (danio rerio) characterization of housekeeping genes in zebrafish: male-female differences and effects of tissue type, developmental stage and chemical treatment caveolin-1 is required for lateral line neuromast and notochord development mycobacterium marinum infection of adult zebrafish causes caseating granulomatous tuberculosis and is moderated by adaptive immunity zebrafish as a model for infectious disease and immune function development and maturation of the immune system in zebrafish, danio rerio: a gene expression profiling, in situ hybridization and immunological study evidence for evolving toll-il-1 receptor-containing adaptor molecule function in vertebrates recognition of double-stranded rna and activation of nf-kappab by toll-like receptor 3 differential roles of mda5 and rig-i helicases in the recognition of rna viruses toll-like receptor signalling regulation of the type i ifn induction: a current view innate immune recognition of viral infection toll-like receptors and type i interferons type i interferon receptors: biochemistry and biological functions the jak/stat pathway in model organisms: emerging roles in cell movement functional crosstalk between type i and ii interferon through the regulated expression of stat1 interfering with interferon receptor sorting and trafficking: impact on signaling endocytosis via caveolae involvement of caveolae in the uptake of respiratory syncytial virus antigen by dendritic cells internalization of echovirus 1 in caveolae innate recognition of viruses mechanisms of inhibition of the host interferon alpha/ beta-mediated antiviral responses by viruses inhibition of interferon-mediated antiviral responses by influenza a viruses and other negative-strand rna viruses animal models of human disease: zebrafish swim into view some properties of the epithelioma papulosum cyprini (epc) cell line from carp cyprinus carpio derivation and characterization of a zebrafish liver cell line photoactivatable mcherry for high-resolution two-color fluorescence microscopy nanoscale imaging of molecular positions and anisotropies a simple method of estimating fifty percent end points structural characteristics of zebrafish orthologs of adaptor molecules that associate with transmembrane immune receptors transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants developmental and tissue-specific expression of nitrs tissue micromanipulation in zebrafish embryos precise nanometer localization analysis for individual fluorescent probes nanoscale imaging of molecular positions and anisotropies superresolution imaging of multiple fluorescent proteins with highly overlapping emission spectra in living cells photochromic rhodamines provide nanoscopy with optical sectioning statistical mechanics of phase transitions we thank dr. paul millard, dr. con sullivan (tif) key: cord-272971-9luzvzsu authors: guo, hainan; zhao, yang; niu, tie; tsui, kwok-leung title: hong kong hospital authority resource efficiency evaluation: via a novel dea-malmquist model and tobit regression model date: 2017-09-08 journal: plos one doi: 10.1371/journal.pone.0184211 sha: doc_id: 272971 cord_uid: 9luzvzsu the hospital authority (ha) is a statutory body managing all the public hospitals and institutes in hong kong (hk). in recent decades, hong kong hospital authority (hkha) has been making efforts to improve the healthcare services, but there still exist some problems like unfair resource allocation and poor management, as reported by the hong kong medical legislative committee. one critical consequence of these problems is low healthcare efficiency of hospitals, leading to low satisfaction among patients. moreover, hkha also suffers from the conflict between limited resource and growing demand. an effective evaluation of ha is important for resource planning and healthcare decision making. in this paper, we propose a two-phase method to evaluate ha efficiency for reducing healthcare expenditure and improving healthcare service. specifically, in phase i, we measure the hkha efficiency changes from 2000 to 2013 by applying a novel dea-malmquist index with undesirable factors. in phase ii, we further explore the impact of some exogenous factors (e.g., population density) on hkha efficiency by tobit regression model. empirical results show that there are significant differences between the efficiencies of different hospitals and clusters. in particular, it is found that the public hospital serving in a richer district has a relatively lower efficiency. to a certain extent, this reflects the socioeconomic reality in hk that people with better economic condition prefers receiving higher quality service from the private hospitals. the hospital authority (ha) is a statutory body established under the hospital authority ordinance in 1990, which manages all the public hospitals and institutes in hong kong (hk). the strategic priorities of hong kong hospital authority (hkha) are to (i) allay staff shortage and high turnover; (ii) better manage growing service demand; (iii) ensure service quality and safety; (iv) enhance partnership with patients and community; (v) ensure adequate resources to meet the service demand; and (vi) enhance the corporate governance. hkha has built a world-class public healthcare infrastructure that handles around 90% of secondary and tertiary medical needs in hk. although hkha has been providing excellent and affordable public healthcare to citizens, it suffers from some problems like long waiting time and low service quality. in order to overcome these limitations, it is important for ha policy makers to evaluate the healthcare efficiency of hospitals, locate the bottlenecks within public healthcare service system, and establish countermeasures accordingly [1, 2] . efficiency measurement is an effective way to evaluate the performance of target configuration in healthcare. since the goals of healthcare services are always multiple, conflicting, intangible, and complex, it is difficult and intractable to set up an overall measurement of performance. regarding the multiple inputs and outputs, data envelopment analysis (dea) has been recognized as an effective nonparametric model to assess the relative efficiencies of a set of decision-making units (dmus) [3] . particularly, in recent decades, dea has been widely applied to improve the productivity of healthcare-related fields in many countries [4] [5] [6] [7] , such as the united states [8] , japan [9] , china [10] and india [11] . as discussed in [12] , dea is more suitable for managerial decision-making, which makes it the best choice to evaluate the efficiencies of hkha hospitals. in dea model, efficiency score is closely related to the quantities of inputs and outputs. an ideal scenario with the highest efficiency is producing the most outputs (e.g. the number of treated patients) using the least inputs (e.g. healthcare expenditure); and such inputs and outputs are called desirable factors. however, the maximum quantities of desirable outputs are usually bounded, given the limited desirable inputs. the reception of the patients beyond the hospital capacity will generate a congestion problem, even resulting in some unexpected mortality. a high patient mortality rate not only reflects a poor medical quality, but also may cause a dispute. thus, the mortality rate of patient, as an undesirable output, is expected to be lower. the measurement of hospital efficiency might be biased if the undesirable factors are ignored. in order to integrate undesirable factors into modeling, one straightforward idea is applying inversion transformation on the value of undesirable factors [13] . however, the transformed values are usually negative, making it difficult for dea modeling. to circumvent this difficulty, one solution is adding a constant to the transformed undesirable factors to keep them positive [14] [15] [16] . however, the determination of this constant has a significant impact on dmu ranking and classification. another widely used transformation is the reciprocal transformation [17] [18] [19] , but the computation complexity of this nonlinear transformation is much higher. färe and grosskopf [20, 21] propose an alternative approach which handles the undesirable factors by using a directional distance function. however, the form of direction vector could affect the dmu ranking. thus, it is a critical issue to design an appropriate transformation function. in some cases, an improper transformation may generate the inverse result as it is expected. to avoid the transformation problem and preserve the original data, liu and sharp [22] treat the undesirable inputs as desirable outputs and vice versa, based on the physical relationship between the input and output. it has been proved as an effective approach which considers computing the operational efficiency by maximizing the undesirable inputs and desirable outputs and minimizing the desirable inputs and undesirable outputs, simultaneously. this idea is further extended by [23] , where a unified model with several different dea approaches dealing with undesirable inputs or outputs is proposed. besides, it is suggested that the weighted additive model [24] can be applied to handle the problem with undesirable inputs or outputs. in [25] , a slacks-based measure (sbm) is proposed, in which the unit invariant, monotone, and reference-set dependent properties are analyzed in details. the proposed sbm model outperforms ccr, bcc and russell model in efficiency evaluation, where both the slacks for the input and the output are calculated. in [26] , a more generalized slacksbased dea model with undesirable factors considering preference is proposed, which combines the advantages of all the factors stated above and makes several dea models unified. the proposed method also outperforms some state-of-the-art dea models on different databases of real applications. in this paper, we focus on evaluating the healthcare efficiency of hkha from 2000 to 2013 by applying the malmquist index [27] based on the updated dea model developed by [26] . the malmquist index can be well applied to track the specific position corresponding to each hospital and to examine changes in productivity and quality [28] [29] [30] [31] . the empirical results reveal that the ha hospitals with low efficiencies may be caused by unfair resource allocation and inefficient resource utilization. additionally, in the context of healthcare measurement, we have found only sparse literature considering the exogenous factors [32, 33] , as they are usually beyond the managers' control. for example, population density and median monthly income could not be reduced in the same way as the healthcare expenditures. in this study, we apply the tobit regression model to explore the impact of these exogenous factors. the experimental results reflect an interesting phenomenon that the ha hospitals serving in the richer clusters have the comparatively lower efficiency values in hk, since people with higher income prefer to accepting treatment from the private hospitals due to the better service quality. dea has been recognized as an effective nonparametric model to assess the relative efficiencies of a set of dmus which consume multiple inputs to produce multiple outputs [34] . dea models evaluate the relative efficiency of dmus by creating a production frontier using the best practice of observed data. a dmu is to be rated as fully efficient on the basis of available evidence if and only if the performances of other dmus does not show that some of its inputs or outputs can be improved without worsening some of its other inputs or outputs. after more than thirty-five years of development, research related to dea is still growing at a very fast rate. liu et al. [35] has summarized that during the period 2010 to 2014, around 2,000 more dea-related papers have been published, in addition to the existing 4,500 collections before 2010 as reported by liu et al. [36] . in this section, we first introduce a novel generalized and slacks-based non-oriented dea model, and then, combine it with the malmquist index. gsbup (generalized slacks-based dea model with undesirable factors considering preference) [26] is a non-oriented dea model, in which the undesirable inputs are regarded as the desirable outputs and vice versa, according to the physical relationship between them [10] . the preferences of different factors for dmus are integrated into the model in terms of the multiplicative weights. the combination of both orientations is proposed to avoid dealing with different results calculated in an input-oriented or an output-oriented manner under variable return scale environment. assume that there are n dmus to be evaluated, and each dmu j (j = 1, 2, . . ., n) is assumed to consume m desirable inputs x d ij ði ¼ (1) can be transformed into equalities by introducing the slacks as follows: thus, eq (2) can be transformed as below: where "á" denotes the element-wise multiplication between vectors, . . . ; g q þ 2 r q þ , and θ ¼ 1 à θ 0 ¼ ðy 1 ; y 2 ; . . . ; y k þ 2 r k þ . in order to take di, do, ui, and uo into account simultaneously, and to integrate efficiency and slacks into a scalar measure, the gsbup model is developed by applying the transformation approach mentioned above: where ω i , μ r , σ l , and ν h (8i, r, l, h) denote the preferences of di, do, ui, and uo, respectively, the decision variables are α i , β r , γ l , and θ h (8i, r, l, h), which also contain the slacks of all the factors. in order to evaluate the relative efficiency of dmu 0 ¼ ðx d 0 ; y g 0 ; x i 0 ; y b 0 þ, we solve the above [gsbup] model. this process is repeated times for 0 = (1, 2, . . ., n). once gsbup identifies the efficient frontier, it can improve the performance of inefficient dmus by either increasing the current do (or ui) levels or decreasing the current di (or uo) levels. thus, the objective of [gsbup] is to minimize the ratio of the weighted efficiency summation of the dis and uos to that of the dos and uis. the details of the linear programming (lp) problem of gsbup is given in appendix a we use two definitions and one theorem to further illustrate the properties of gsbup. theorem 1. though dmu 0 is gsbup-inefficient, its projecteddmu 0 is gsbup-efficient relative to the original set of n dmus. proof. see appendix b. dea-based malmquist productivity index, developed by [27] , provides an evaluation of productivity change over time. to describe the gsbup-based malmquist productivity method, let ðx d;t 0 , y g;t 0 ; x i;t 0 ; y b;t 0 þ denote the input and output levels for a dmu j at any given point in time t. from time t to t + 1, dmu 0 's technical efficiency and empirical production frontier may change. the calculation of malmquist index requires two single period and two mixed period evaluations according to gsbup-dea model (4), shown as follows: þ to the frontier at time t + 1, i.e., calculating þ to the frontier at time t, i.e., calculating then, the malmquist productivity index is defined as follows (the calculation of each period is shown in appendix c): where m 0 measures the productivity change between periods t and t + 1. productivity declines if m 0 > 1, remains the same if m 0 = 1 and improves if m 0 < 1. the m 0 can be divided into two components, the change of technical efficiency (tec) and the movement of the frontier (fs) in terms of a specific dmu 0 : similarly, tec 0 > 1, tec 0 = 1 and tec 0 < 1 indicates that technical efficiency declines, remains unchanged and improves, respectively. and the value of fs 0 > 1 indicates regress in the frontier technology; while fs 0 < 1 means progress in the frontier technology; naturally, fs 0 = 1 indicates no shift in the frontier technology. in this section, we first analyze the hkha data, and then discuss the measurement results of hospital efficiency in hk from 2000 to 2013 by applying malmquist index based on gsbup-dea. it is expected that the results are helpful for hkha policy makers to control healthcare costs and improve healthcare efficiency while ensuing the service quality requirements. to ensure patients in hk receive a continuum of high quality healthcare within the same area, table 1 lists the specific hospitals in each cluster, which are also the dmus evaluated in this study. note that the evaluation of dea performance highly depends on the selection of factors because the discriminatory power may be reduced when the dimensionality of the production space increases [37] . in this study, by reviewing the relevant literature, consulting with hk healthcare professionals and considering data availability and the parametric restrictions, two dis (i.e., the number of full-time equivalent staff and the number of beds), one ui (i.e., inpatient discharge rate), dos (i.e., total patient length of stay, total ed attendances and total outpatient attendances), and one uo (i.e., crude mortality rate) are selected for each dmu, as listed in table 2 . all the data are collected from the hospital authority statistical report [38] . as can be observed in table 3 , regarding the inputs of medical resources, there is a continuous increase of investment in both human resources and bed among hospitals. however, it is worth noting that except for the dis, all the other factors (e.g., the utilization of bed, total patient attendances, and mortality rate) reach the peak values in 2003, especially the mortality rate, which is due to the outbreak of severe acute respiratory syndrome (sars) [39] . in addition, the surge of patient attendances in 2013 is mainly caused by the winter influenza season and avian influenza (h7n9). table 4 shows the curde mortality rate of each ha hospital from 2000 to 2013. the crude mortality rate refers to the standardized hospital death rate covering inpatient and day patient deaths in ha hospitals during a year. the standardized death rate, as a standard statistical measure for temporal comparison, is calculated by applying the ha age-specific hospital death rate in the target year to the "standard" population in mid-2001. we observe that the pok oi hospital, ruttonjee hospital & tang shiu kin hospital, caritas medical centre, north district hospital and yan chai hospital rank top 5 regarding the value of crude mortality rate. the high crude mortality rates can be due to the facts that: (i) pok oi hospital provides the elderly care for the people whose health condition and dependency level are beyond the coping we use the data in 2013 as an example to illustrate the statistical characteristics of the specific input-output factors. it is observed in table 5 that the median value of each factor is significantly different from the average value. furthermore, the standard deviation values are large and the maximum can be 200 times larger than the minimum, indicating that the resource utilization levels and resource allocation of the clusters are seriously unbalanced. thus, the analysis of hkha hospital efficiency and the exploration of the influencing factors are of crucial importance for the policy makers. the correlation coefficients between all the input-output factors are given in table 6 . it is observed that there ia a significantly positive correlation between the dis and dos, satisfying the "isotonicity" production process that dos do not decrease by increasing the dis. crucially, the mortality rate of patients has a significantly positive relationship with the dos, which suggests that an increase in patient attendances is accompanied by an increase in patients' death rate. in addition, the mortality rate of patients has a negative correlation with the amount of medical resources, which suggests that properly increasing the medical resources can relieve a poor service quality in a certain degree caused by resource limitation; while too many resources can increase the fiscal burden of government. thus, it is a trade-off problem that how to determine the resource allocation. evaluation of gsbup-dea model. we evaluate the resource efficiency of hkha in each year by applying the lp of gsbup-dea model (8) . the efficiency scores for all the hospitals during 2000-2013 are listed in table 7 . according to definition 1, the efficient dmus' d ã table 7 that there are only 55% hospitals have higher efficiency values than the average, 0.870, which reveals the fact that there exists unfair resource allocation, inefficient resource utilization and poor management in hkha. for example, pok oi hospital has relatively lower efficiency in the earlier years, because it had no ed service before 2010. queen mary hospital is regarded as one of the best hospitals in hk, but it faces the over-investment problem which lowers the efficiency value. returns to scale (rts) is the variation or change in productivity which describes the outcome from a proportionate increase of all the inputs. in dea, an increasing return to scale (irs) occurs when the output increases by a larger proportion than the inputs. conversely, a decreasing return to scale (drs) occurs when the proportion of output is less than the desired increased input during the production process. in table 7 , the last column displays the ha hospitals' rts in 2013. it reveals that if hkha policy makers can properly increase the input of medical resources for the inefficient hospitals with irs (i.e., tung wah eastern hospital and pok oi hospital), these hospitals will achieve a higher service level than they expected. notably, if hkha policy makers continue to increase the hospital scale for the inefficient hospitals with drs (i.e., queen mary hospital and prince of wales hospital), it will be an uneconomic production process and even introduces unnecessary fiscal burden to the government. we take the year 2013 as an example, table 8 shows the specific input-output factors, efficiency values and rts status of each cluster. we find that kwc receives the most patient attendances; while kec receives the least ones. in practice, hkha allocates the medical resources for each cluster based on the patient attendances. thus, hkha should allocate the most medical resources for kwc and the least medical resources for kec. however, this kind of method ignores the effect of medical case complexity of the patients. in fact, it is more reasonable to consider allocating the resources based on the complexity of the medical cases than purely on the patients' amount. in addition, the last column of table 8 declares that the rts status of kwc is drs, indicating that the hkha should reduce its medical resource inputs. table 9 displays the slacks of each cluster measured by gsbup, which is able to further validate the idea mentioned above. it is observed that the s d− of hkec and hkwc with irs is almost equal to zero; while their s g+ are not null, which means that the inefficient clusters with irs are mainly caused by the inefficient resource utilization. on the other hand, the inefficient hospitals with drs are mainly caused by the unfair resource allocation. this phenomenon can guide the hkha policy makers to better control healthcare costs and improve healthcare efficiency under the service quality requirements. considering all the slacks, the updated clusters' efficiency scores based on gsbup-lp model can be calculated, as shown in the last column of table 9 . the efficiency values of the comparison are displayed in fig 2. it is observed that all the clusters' efficiencies are improved significantly, which further demonstrates that it is necessary to change the ideas for the medical investment standard. evaluation of malmquist productivity change index. table 10 reports hkha hospitals' productivity change during the period 2000-2013. we observe that from 2000 to 2013, all the productivities of hospitals have made significant improvement. there are two major reasons: (i) sars outbreak and (ii) health services fees' reformation. the first diagnosed patient with sars was on 21 february 2003 and the disease had widely spread in hk within just two weeks. the peak of the epidemic started from 24 march 2003, and there was a rapid influx of infected residents in ha hospitals, particularly the united christian hospital which was soon overwhelmed. therefore, its productivity improves by 17.87%, particularly, the tec and fs improve by 4.77% and 13.76%, respectively (see table 11 ). on 29 march 2003, the government and ha decided to designate the princess margret hospital, due to its specialty in treating infectious disease, to receive sars patients after diverting all its existing patients to other hospitals. thus, its fs only improves by 1.81%. however, since the government intensified a strong technical support to the princess margret hospital for resisting sars, its tec improves by 9.87% during 2000-2003 (see table 11 ). besides, the speed of the patient flow increase was unexpected. the additional manpower from other hospitals was deployed to princess margret hospital for help, which is to reduce the medical resources input equivalently for the other hospitals. with the contribution from other hospitals to share the workload, the designation of princess margret hospital officially ended on 11 april 2003. furthermore, with the assistance of the necessary operational and information technology systems, a revised public hospital fees structure was implemented at the beginning of 2003 together with an enhanced waiver mechanism to better provide the available public resources to those in need. along with the soaring sars attendances, there is improvement in tec for almost all the hospitals. not surprisingly, the hkha hospital productivity regresses from 2003 to 2007, since sars has been gradually defeated during this period, and the patient attendances decrease a lot. in the periods, 2007-2010 and 2010-2013, the hkha average productivity has generally increased except st. john hospital, tung wah eastern hospital and north district hospital, which are not the core and mainstream hospitals in hk. in particular, the st. john hospital provides health services for the citizens living on the cheung chau island, which is a geographically isolated place. tung wah eastern hospital and north district hospital were once voted as the one with the highest emergency surgery mortality rates, therefore, their average tec declines by 1.84% and 1.34% during 2007-2013 (see table 11 ). in addition to the observations stated above, it is indicated in table 10 that the productivity and fs of pok oi hospital improve a lot during 2007-2013, as it has provided ed services since 2010. in phase i, we have measured the hkha efficiency changes from 2010 to 2013 by applying the gsbup-malmquist index. in phase ii, we will further explore the impact of some exogenours factors on hkha efficiency by tobit regression model. the hk healthcare system is composed of two sectors: a private track and a government sponsored public track. citizens can access the public healthcare system at facilities operated by the ha. insurance is not necessary for the eligible. however, the public system is geared toward emergency care, which means that if an attending nurse deems a case to be not critical, patients may sometimes spend hours waiting to see a doctor. thus, the citizens with good financial condition sometimes prefer to choose the private hospitals for their efficient and high quality services. private healthcare, however, generally is more expensive than the public healthcare system which mainly comes from the out-of-pocket household payment and commercial medical insurance pay-out. according to the world factbook in 2013, the gini index of hk for households rose from 0.429 in 1976 to 0.557 in 2013, while that for economically active individuals increased from 0.411 in 1976 to 0.497 in 2013. the rising gini index of both household and individual income are often regarded as the evidence of income inequality. therefore, household income inequality has risen naturally because of the population composition change (e.g., more non-working elderly or single-parent household than those in the past) [40] . it is well known that hk is one of the most densely populated areas in the world, with an overall density of around 6300 people per square kilometer. at the same time, hk has one of the world's lowest birth rates-1.11 per woman of child-bearing age in 2013, far below the replacement rate of 2.1. it is estimated that 26.8% of the population will age over 65 in 2033, rising from 12.1% in 2005. therefore, we consider from the aspects of economy, geography, education and demography to further explore the influence of exogenous factors. table 12 provides the details of these factors, which are collected from the hong kong census and statistics department [41] . the most widely used method to model the dea scores against exogenous factors is tobit regression, which is suitable when the dependent variables are either censored or corner solution outcomes [33, 42, 43] . it is validated in [43] that the tobit regression model is powerful in representing the second stage in dea models. the relationship between exogenous factors and cluster efficiencies can be described by the following model: where hace it describes the ith cluster's efficiency in the tth year. the symbol denotes the corresponding exogenous factor's value in the ith cluster of tth year. (β 0 , β 1 , . . ., β 7 ) are the unknown coefficients, and it * n(0, σ) are the independent and identically normally distributed residuals of the observations. in this study, we use the maximum likelihood estimation toolbox in r [44] to obtain the tobit regression results, as presented in table 13 . firstly, the poverty situation of persons with disabilities has a positive influence on ha hospital efficiency; while median monthly domestic household income and education level affect the environmental efficiency negatively. since the median income of each cluster has almost no relationship with population density (i.e., correlation coefficient = 0.03), the empirical results reveal an interesting phenomenon: the public hospitals that serve in a richer district, has a relatively lower efficiency in hk, since people with higher degree of education and income prefer to accepting higher quality service from the private hospitals. secondly, the proportion of population aged over 65 and ha hospital efficiency has a negative correlation, because the elderly are more likely to suffer from life-threatening diseases. many elderly patients are physically too weak to see the doctors. moreover, one out of three elderly hk residents lives under poverty or disabilities, so they are usually unable to take good care of themselves once in sick. in other words, the aged population might increase the complications and the mortality rate. therefore, the aged population have a negative correlation with the ha hospital efficiency. thirdly, the proportion of patients come from the other clusters takes a positive correlation with the efficiency. the ha hospitals in hk are divided into seven clusters according to their geographical locations so that the healthcare service is evenly distributed across hk. although the patients are expected to receive the same level of medical care, wherever they abide in, the quality of medical care are various across different locations. thus, some patients abiding in the relative poverty districts may try to receive the better treatments from the other districts' hospitals, such as queen mary hospital, belonging to the hkwc. these types of patients will increase the workload of some hospitals, which is validated by the regression results. finally, the land area has no significant influence on ha hospital efficiency while population density has a weak positive influence. for example, new territories has the largest land area in hk, while its population density is the lowest. kowloon has the highest population density, at the same time, most of the poor with disabilities abide in this cluster. in addition, the land area and population density of hong kong island lie in the middle, but the citizens are relatively richer. in conclusion, the geography factor has no significant influence on ha hospital efficiency. in dea model, efficiency score is closely related to the quantities of inputs and outputs. an ideal scenario with the highest efficiency in a healthcare setting would be treating the most patients using the least healthcare expenditure. however, the reception of the patients beyond the hospital capacity will generate a congestion problem, even resulting in some unexpected mortality, where a high patient mortality rate not only reflects a poor medical quality, but also may cause a dispute. thus, it is regarded as an uo and is expected to be as low as possible. on the other hand, the in-patient discharge rate is a powerful indicator to represent whether this hospital could provide a good quality of service, which is expected to be as high as possible, as an ui. moreover, the measurement of hospital efficiency might be biased if the undesirable factors are ignored. by integrating the gsbup-dea model and malmquist index, this paper examines the resource efficiency of hkha based on the panel data from 2000 to 2013. then, through the tobit regression model, some exogenous factors' influences are tested. the empirical results show that the hkha hospital efficiencies of each hospital and cluster are significant different. in general, the healthcare efficiency of hospitals belonging to the kec and ntwc are significantly higher than the other hospitals, which has a close correlation with demographic composition, geographic condition and economic position. by analyzing the evaluation of productivity change over time, we observed that the sars outbreak has a strong impact on the hospital efficiency. in addition, we found an interesting phenomenon that the public hospital serving in a richer district with high population density has a relatively lower efficiency, as hk people with better economic condition prefer to accept higher quality service from the private hospital. in recent decades, hkha has been making efforts to improve the healthcare services, but there still exist some problems like unfair resource allocation and poor management, as reported by the hong kong medical legislative committee. one critical consequence of these problems is low healthcare efficiency of hospitals, leading to low satisfaction among patients. in conclusion, we provide two suggestions on how to improve the hkha hospital efficiency in the future: (i) according to the original resource allocation system, the hkha policy makers need to properly increase the input for the inefficient hospitals with irs, such as tung wah eastern hospital and save some investment for the inefficient hospitals with drs, such as tung wah eastern hospital prince of wales hospital. (ii) it is necessary to change the medical investment standard. it may be better to consider patient attendance and hospital current efficiency simultaneously. such a new system can solve the problem of unfair resource allocation effectively. in the future, we will consider the patient service quality and use idea (imprecise data) models to help hkha policy makers find the technology backward hospitals. to further improve the efficiency in resource utilization and meet the need of growing healthcare demand due to aging population, the internal resource allocation system should be personalized and modernized at an individual level by involving heterogeneity of patients need. thus, we will also attempt to investigate the simulation methods for resource optimization by integrating heterogeneous factors at individual level in our future works. by applying the charnes-cooper transformation, [gsbup] can be transformed into a linear programming. suppose t (t > 0) is a scalar variable, let: the linear programming of [gsbup] can be transformed as follows: suppose (λ ã ;α ã ;β ã ;γ ã ;θ ã ) is an optimal solution of eq (9) . according to definition 2, the constraints of eq (9) can be transformed as follows: because we have known that 0 â ã i a ã i 1,ĝ ã l g ã l ! 1,b ã r b ã r ! 1, and 0 ŷã h y ã h 1 (8i, l, r, h). thus, is a feasible solution of eq (4), and then we can get an inequality function as follows: because 0 â ã i 1,ĝ ã l ! 1,b ã r ! 1, and 0 ŷã h 1 (8i, l, r, h), in order to satisfy the inequality eq (10), there must be (α ã , β ã , γ ã , θ ã ) = (1, 1, 1, 1) . thus, we can demonstrate that the projection,dmu 0 ¼ ðx d 0 ;ŷ g 0 ;x i 0 ;ŷ b 0 þ is gsbup-efficient, according to definition 1. in a similar way, using t + 1 instead of t for the models (10) and (11) incorporating quality into data envelopment analysis of nursing home performance: a case study a location-allocation model for service providers with application to not-for-profit health care organizations a 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model with undesirable factors considering preference trabajos de estadistica y de investigacion operativa technological change and efficiency in the presence of quasi-fixed inputs: a dea application to the hospital sector an analysis of hospital efficiency and productivity growth using the luenberger indicator. health care management science productive performance and its components in greek public hospitals. operational research taiwan quality indicator project and hospital productivity growth alternative sbm model for network dea regional environmental efficiency evaluation in china: analysis based on the super-sbm model with undesirable outputs data envelopment analysis (dea)-thirty years on research fronts in data envelopment analysis data envelopment analysis 1978-2010: a citation-based literature survey guidelines for using variable selection techniques in data envelopment analysis hasr. hospital authority statistical report impact of sars on an emergency department in hong kong the world factbook using least squares and tobit in second stage dea efficiency analyses second stage dea: comparison of approaches for modelling the dea score r: a language and environment for statistical computing we thank dandan yu, the associate director of information center of the first affiliated hospital of dalian medical university for sharing suggestions from the perspective of hospital manager. we thank the anonymous reviewers and the department editor for their constructive comments and suggestions, which have greatly improved the exposition of this paper. key: cord-275058-61eof7y8 authors: inoue, hiroyasu; todo, yasuyuki title: the propagation of economic impacts through supply chains: the case of a mega-city lockdown to prevent the spread of covid-19 date: 2020-09-15 journal: plos one doi: 10.1371/journal.pone.0239251 sha: doc_id: 275058 cord_uid: 61eof7y8 this study quantifies the economic effect of a possible lockdown of tokyo to prevent the spread of covid-19. the negative effect of such a lockdown may propagate to other regions through supply chains because of supply and demand shortages. applying an agent-based model to the actual supply chains of nearly 1.6 million firms in japan, we simulate what would happen to production activities outside tokyo if production activities that are not essential to citizens’ survival in tokyo were shut down for a certain period. we find that if tokyo were locked down for a month, the indirect effect on other regions would be twice as large as the direct effect on tokyo, leading to a total production loss of 27 trillion yen in japan or 5.2% of the country’s annual gdp. although the production that would be shut down in tokyo accounts for 21% of the total production in japan, the lockdown would result in an 86% reduction of the daily production in japan after one month. covid-19, a novel coronavirus disease, has been spreading worldwide. by march 31, 2020, the total number of confirmed cases of covid-19 reached 775,306, whereas the total number of deaths was 37,083 [1] . to prevent the spread of covid-19, most countries have implemented unprecedentedly stringent restrictions, such as a shutdown of national borders, limits on public gatherings, and closures of schools, shops, and restaurants. in some cases, whole cities and regions have been locked down. for example, wuhan, the epicenter of the novel coronavirus, was locked down from january 23 to march 27, 2020, including the closure of all public transport and all companies not essential to citizens' survival, including manufacturing plants, for most of the period (reuters, march 11, 2020) . apparently, the lockdown heavily affected wuhan's economy and its population of 11 million people. moreover, because wuhan, known as one of china's "detroits", is a center of the automobile industry and supplies parts and components of automobiles to domestic and foreign plants, the effect of the lockdown propagated to other regions of china and other countries through supply chains. for example, honda, a japanese automobile manufacturer that operates plants in wuhan, reduced the production of automobiles in japan due to a lack of supplies of parts from china in early march 2020 (nikkei newspaper, march 2, 2020) . recently, many studies have empirically confirmed that economic shocks propagate across regions and countries through supply chains. some studies applying econometric analysis to firm-level data show that firms linked with suppliers and customers in areas affected by natural disasters exhibit decreased performance [2] [3] [4] [5] . several others have estimated economic impacts of the spread of covid-19, incorporating propagation through input-output linkages across sectors and countries /citeoecd2020, mckibbin2020, bonadio2020, guan2020, mccann2020. for example, mckibbin and fernando [6] use a hybrid of dynamic stochastic general equilibrium (dsge) models and computable general equilibrium (cge) models assuming international and inter-sectoral input-output (io) linkages and estimate the effect of the spread of covid-19 on the world gdp. guan and others /citeguan2020 utilize international io tables at the country-sector level and estimate impacts of lockdowns of heavily infected countries on production in the world economy. according to their estimates, if europe and the united states imposed a lockdown strategy with a strictness measure of 80% (i.e., 80% of production in most non-essential sectors are shut down) for 2 months, the world value added would decline by 27%. by contrast, inoue and todo [7] apply firm-level data for the actual supply chains in japan, rather than inter-sectoral io tables, to an agent-based model to simulate the production trajectory after the great east japan earthquake in 2011. their results indicate that the propagation effect of an economic shock through supply chains was substantially larger than the direct effect of the earthquake. inoue and todo [7] also show that complex network characteristics of supply chains, such as scale-free properties and complex loops, aggravate the propagation effect. without any network complexity, i.e., if they assume no firm-level inter-linkages but only inter-industry linkages or assume a randomly determined network with no complexity, they find that the propagation effect is quite small. the analytical framework of inoue and todo [7] has several advantages. first, although some studies quantitatively find that supply chains are equipped with complex network characteristics /citeborgatti2009social, battini2007towards, fujiwara10, they do not examine propagation of shocks through supply chains. second, the conclusion of inoue and todo [7] that the structure of networks significantly influences propagation is consistent with recent findings in the network science literature [8] [9] [10] [11] [12] [13] . for example, diffusion is generally promoted when the degree distribution follows a power law and, hence, there are several hub nodes with a large number of links. in such a network, when a shock reaches a hub node, it spreads quickly to many others. third, although the econometric analysis of /citebarrot2016, boehm2019, carvalho2016, kashiwagi2018 quantitatively confirms propagation through supply chains, their analysis cannot estimate the total effect of a shock that can be obtained from the framework of inoue and todo [7] . finally, the studies estimating the effect of covid-19 rely on either a macroeconomic econometric model at the country level [14] or a general equilibrium model assuming international and inter-sectoral io linkages [6, [15] [16] [17] and thus do not incorporate complex inter-firm linkages. as a result, the estimates of the previous studies may be largely undervalued, as suggested by the findings of inoue and todo [7] . therefore, this study utilizes the framework of inoue and todo [7] and examines propagation of the economic effect of lockdown of a city to prevent the spread of covid-19 to other regions through supply chains. in particular, when a large industrial city connected with other regions through supply chains in a complex manner is locked down, the propagation effect may be quite large. therefore, we focus on the economic effect of a lockdown of tokyo on other regions. specifically, by applying the agent-based model developed in inoue and todo [7] to actual supply-chain linkages of approximately 1.6 million firms in japan, we simulate what would happen to production activities outside tokyo if tokyo were locked down or nonessential production activities in tokyo were shut down for a certain period. tokyo is an appropriate case for the purpose of this study because it is one of the largest cities in the world and a hub for global supply chains and was, in fact, locked down in april and may 2020. using this framework, we can obtain a reasonable estimate of the propagation effect of lockdown across regions that cannot be computed from other studies using econometric approaches or io tables and thus can highlight the impact of the propagation through supply chains. the data used in this study are taken from the company information database and company linkage database collected by tokyo shoko research (tsr), one of the largest credit research companies in japan. the former includes information about the attributes of each firm, including the location, the industry, the sales, and the number of employees, whereas the latter includes major customers and suppliers of each firm. because of data availability, we utilize data for firm attributes and supply chains in 2016. the number of firms in the data is 1,668,567, and the number of supply chain links is 5,943,073. that is, our data identify major supply chains of most firms in japan, although they lack information about supply chain links with foreign entities. because the transaction value of each supply chain tie is not available in the data, we estimate sales from a particular supplier to each of its customers and consumers using the total sales of the supplier and its customers and the input-output (io) tables for japan for 2015. in this estimation process, we must drop firms without any sales information. accordingly, the number of firms in our further analysis is 966,627, and the number of links is 3,544,343. although firms in the tsr data are classified into 1,460 industries according to the japan standard industrial classification, we simplify them into 187 industries classified in the io tables. s1 appendix provides details of the data construction process. in the supply chain data described above, the degree, or the number of links, of firms follows a power-law distribution (see s1 fig) , as often found in the literature [12] . the average path length between firms, or the number of steps between them through supply chains, is 4.8. this small average path length indicates that the supply chains have a small-world property, i.e., firms are indirectly and closely connected through supply chains. therefore, we would predict that economic shocks quickly propagate through the supply chains. using the same dataset, previous studies [7, 18] find that 46-48% of firms are included in the giant strongly connected component (gscc), in which all firms are indirectly connected to each other through supply chains. the large size of the gscc prominently shows that the network has numerous cycles and a complex nature, which is unlike the common image of a layered supply chain structure. our simulation employs the dynamic agent-based model of inoue and todo [7, 19] , an extension of the model of hallegatte [20] that assumes supply chains at the firm level. in the model, each firm utilizes inputs purchased from other firms to produce an output and sells it to other firms and consumers. supply chains are predetermined and do not change over time in the following two respects. first, each firm utilizes a firm-specific set of input varieties and does not change the input set over time. the variety of inputs is determined by the industry of the producer, and hence, firms in the same industry are assumed to produce the same output. second, each firm is linked with fixed suppliers and customers and cannot be linked with any new firm over time. furthermore, we assume that each firm keeps inventories of each input at a level randomly determined from the poisson distribution. following inoue and todo [7] , in which parameter values are calibrated from the case of the great east japan earthquake, we assume that firms aim to keep inventories for nine days of production on average. when a lockdown directly or indirectly causes a reduction in the production of particular firms, the supply of products of these firms to their customer firms declines. then, one way for customer firms to maintain the current level of production is to use their inventories of inputs. alternatively, customers can procure input from their other suppliers in the same industry already connected prior to the lockdown if these suppliers have additional production capacity. if the inventories and inputs from substitute suppliers are insufficient, customers have to reduce their production because of a shortage of inputs. in addition, suppliers of the firms directly affected by the lockdown may have to reduce production because of the reduction of demand from affected customers. accordingly, the economic shock propagates both downstream and upstream through supply chains. s2 appendix provides the details of the model. in the simulation, we assume an extreme case of tokyo's lockdown where all production activities that are not essential to citizens' survival (hereafter referred to as non-essential production activities) in the central part of tokyo (23 wards, hereafter simply referred to as tokyo) are shut down for either one day, one week, two weeks, one month, or two months. essential production activities are defined as those in the wholesale, retail, utility, transport, storage, communication, healthcare, and welfare sectors (see s3 appendix for details). after the lockdown period, all sectors immediately resume production at the same level as in the pre-lockdown period. because the inventory target of each firm is randomly sampled from the poisson distribution (see s2 appendix), we run thirty simulations with different sets of inventory targets of firms for each lockdown duration and average over the thirty sets of results. it should be noted that our assumption does not exactly match the experience of japan. in practice, the japanese government announced the state of emergency for some prefectures including tokyo from april 7 to may 25, 2020, and for the whole country from april 16 to may 14. according to the act on special measures for pandemic influenza and new infectious diseases preparedness and response, the japanese government can declare a state of emergency if the spread of a infectious disease, such as covid-19, is rapid and nationwide. in the state of emergency, some economic activities were requested to be closed, and people were requested to reduce their contacts with others by 80%. although there was no legal punishment for disobeying the requests, strong social pressure in japan led people and businesses to voluntarily restrict their activities to a large extent. as a result, production activities including those in sectors not officially restricted were reported to shrink substantially (mainichi newspaper, may 27, 2020). because it is still unclear to what extent economic activities shrank, we assume a complete shutdown of non-essential production activities. in addition, to highlight the propagation of the effect of a lockdown of a mega-city to other regions, we focus on a lockdown of tokyo, rather than the entire economy. when tokyo is locked down, the value-added production of tokyo immediately becomes almost zero. because the daily production of non-essential sectors in tokyo is estimated to be 309 billion yen, or approximately 2.9 billion us dollars, the total direct loss of production in tokyo due to the lockdown is 309 billion yen multiplied by the number of days of the lockdown period. table 1 shows the direct production loss in tokyo for each case in the second column. in addition, the third column shows the production loss outside tokyo (which includes essential sectors in tokyo; hereafter, this area is simply called outside tokyo), which is the propagation effect through supply chains. the indirect loss is calculated from a total loss less the direct damage over the duration of the lockdown and the following three weeks. as shown in fig 1, after the three weeks, the production fully recovers. the total loss is the total gap between the pre-lockdown daily production and the current daily production. the results in table 1 are the averages of the simulations. the results indicate that when tokyo is locked down for only one day, the production loss outside tokyo, though not locked down, is already 252 billion yen, 82% of the production loss in tokyo. when the lockdown continues for a month, the indirect effect on other regions is twice as large as the direct effect on tokyo, and the estimated total production loss is 27.6 trillion yen, or 5.22% of the annual gdp. in fig 1, each line shows the dynamics of the total daily value added in japan in each case assuming different lockdown durations. when the lockdown continues for a month, the daily value added of japan becomes only approximately one-seventh of that before the lockdown. this result implies that even when the initial production loss in tokyo is small, its propagation effect on other regions can become large as the lockdown continues. fig 2 shows temporal and geographical visualizations of the simulation of a lockdown. a video of the visualization is available (see s1 video). the reductions in firm production are averaged in municipalities. the red areas indicate that the production in the area is less than or equal to 20% of firms' capacity on average, whereas the light red and orange areas show firms with a more moderate decline in production. the left figure illustrates that a non-negligible number of areas (firms) distant from tokyo are already affected on the first day of the lockdown. two weeks later, affected areas spread throughout the country, as shown in the right figure. these visualizations indicate that the indirect effect propagates geographically as the lockdown is prolonged. in addition to the benchmark simulations above, we experiment with three alternative sets of simulations. first, we assume that all production activities including essential activities are shut down. then, the daily production loss in tokyo is 471 billion yen, 52% larger than that in the benchmark simulations (309 billion yen). however, we find that the total production loss in japan from a lockdown of tokyo for a month is 31.1 trillion yen, only 13% larger than that in the benchmark (27.6 trillion yen) (see s1 table for details) . second, we assume that industrial demand is prioritized over consumer demand so that production activities outside tokyo will be less affected. for example, computers can be used table 1 . the loss of value added because of tokyo lockdowns. this table shows the results from the simulations assuming the shutdown of all non-essential production activities. these results are based on the average of the simulations. (unit: trillion yen). indirect effect on other regions in japan total effect (% of gdp) both by customer firms for production and citizens for consumption. in the benchmark simulation, when the output of a product is not sufficient because of a lockdown, we assume that the limited output is rationed to customer firms and consumers based on their relative demand prior to the lockdown. however, in this alternative simulation, customer firms are prioritized to maximize production in downstream firms (see s2 appendix for details). then, we find that a one-month lockdown results in a production loss in japan of 27.0 trillion yen. because the production loss does not substantially change from the benchmark result (27.6 trillion yen), we conclude that industry prioritization is not very effective in alleviating the propagation effect of a lockdown. finally, we investigate the assumption of a moderate initial shock. since we only know the addresses of the firms' headquarters, we assign each firm to each headquarters' location in the benchmark and the above alternatives. however, even if the headquarters of a firm is located in tokyo, other business establishments and factories may continue to operate (however, it may not be possible to obtain such detailed trade information between business establishments and factories). to take this situation into consideration, we proportionally assign the production loss to firms that have headquarters in tokyo based on the number of headquarters (i.e., counted individually), business establishments, and factories in tokyo over the number in all areas. this setting may be the other side of the extremely mitigated condition because if the headquarters are closed, the other business establishments and factories probably cannot operate at full capacity. obviously, this assumption shows a much smaller direct shock (107 billion yen per day) than the benchmark shock (309 billion yen per day). however, if the lockdown is prolonged for a month, the total loss is 22.5 trillion yen, a difference of 5.10 trillion yen from the benchmark. we conclude that this difference is not large enough to change the interpretations of the benchmark results. the simulation results clearly show that the effect of a lockdown of tokyo quickly propagates to other regions outside tokyo, leading to a substantial effect on the entire japanese economy. this conclusion is consistent with the previous finding that economic shocks due to natural disasters propagate to non-disaster regions and result in a large total loss /citeinoue2019. although the production of non-essential sectors in tokyo accounts for 21.3% of the total production in japan, a lockdown of tokyo for a month would result in a reduction of the daily production in japan of 86%, or 1.25 trillion yen. in addition, the effect on other regions becomes progressively larger as the duration of the lockdown becomes longer: when the duration doubles, the production loss more than doubles. in the case of a lockdown for one day, the total loss of value added outside tokyo is 82% of the loss in tokyo. however, when the lockdown continues for a month, the loss outside tokyo is twice as large as the loss in tokyo. this result implies that the effect of a longer lockdown can reach firms that are "farther" from tokyo along supply chains in terms of the network. to alleviate the propagation effect through supply chains, one could limit the shutdown of production activities or prioritize producers' use of goods and services over consumers' use. the left and right panels show the first day and the first 2 weeks of the lockdown, respectively. the reductions are aggregated and averaged over firms in municipalities. the red areas, for example, indicate firms in the areas whose actual production is substantially (more than 80%) smaller than their capacity before the lockdown on average. however, our results indicate that these measures would not work well, particularly when the duration of a lockdown is long. our analysis provides several policy implications. first, because the overall effect of a lockdown of a mega-city on the entire economy is extremely large when we take into account its propagation effect through supply chains, lockdowns should be considered a measure of last resort. instead, the spread of covid-19 should be prevented earlier using other means and any lockdown of a mega-city should be avoided. second, because the total effect of a lockdown progressively increases with its duration, a mega-city lockdown, if it cannot be avoided, should be as short as possible. policymakers should be aware that policies to alleviate the propagation effect may not work when the lockdown duration is long. several caveats of this study should be mentioned. first, we assume that firms cannot find any new suppliers when supplies from their suppliers in tokyo are disrupted. second, for simplicity, the model assumes that even service sectors have the inventory mechanism. finally, the model in this study only considers the dynamics of production and ignores possible changes in prices and wages incorporated in the literature [21, 22] . these assumptions may be too strong, leading to an overestimation of the propagation effect. however, our main result that the propagation effect is substantial would hold. this research was conducted as part of a project entitled ''dynamics of economy and conceptualization: hiroyasu inoue, yasuyuki todo. coronavirus covid-19 global cases by the input specificity and the propagation of idiosyncratic shocks in production networks input linkages and the transmission of shocks: firm-level evidence from the 2011 tohoku earthquake supply chain disruptions: evidence from the great east japan earthquake. columbia business school research paper international propagation of economic shocks through global supply chains. winpec working paper the global macroeconomic impacts of covid-19: seven scenarios. cama workding paper firm-level propagation of shocks through supply-chain networks collective dynamics of 'small-world' networks structural holes and good ideas the spread of behavior in an online social network experiment networks: an introduction network science a simple model of global cascades on random networks the world economy at risk (oecd economic outlook global supply chains in the pandemic global economic footprint of the covid-19 pandemic covid-19 and the transmission of shocks through domestic supply chains. central bank of ireland large-scale structure of a nation-wide production network propagation of negative shocks through firm networks: evidence from simulation on comprehensive supply-chain data an adaptive regional input-output model and its application to the assessment of the economic cost of katrina. risk analysis modeling loss-propagation in the global supply network: the dynamic agent-based model acclimate transportation and supply chain resilience in the united republic of tanzania: assessing the supply-chain impacts of disaster-induced transportation disruptions the ministry of internal affairs and communications. the 2015 updated input-output tables for japan methodology: hiroyasu inoue.writing -original draft: hiroyasu inoue, yasuyuki todo. key: cord-279259-eu80ccm6 authors: pandey, aseem; singh, neetu; vemula, sai v.; couëtil, laurent; katz, jacqueline m.; donis, ruben; sambhara, suryaprakash; mittal, suresh k. title: impact of preexisting adenovirus vector immunity on immunogenicity and protection conferred with an adenovirus-based h5n1 influenza vaccine date: 2012-03-14 journal: plos one doi: 10.1371/journal.pone.0033428 sha: doc_id: 279259 cord_uid: eu80ccm6 the prevalence of preexisting immunity to adenoviruses in the majority of the human population might adversely impact the development of adaptive immune responses against adenovirus vector-based vaccines. to address this issue, we primed balb/c mice either intranasally (i.n.) or intramuscularly (i.m.) with varying doses of wild type (wt) human adenovirus subtype 5 (had5). following the development of immunity against had5, we immunized animals via the i.n. or i.m. route of inoculation with a had vector (had-ha-np) expressing the hemagglutinin (ha) and nucleoprotein (np) of a/vietnam/1203/04 (h5n1) influenza virus. the immunogenicity and protection results suggest that low levels of vector immunity (<520 virus-neutralization titer) induced by priming mice with up to 10(7) plaque forming units (p.f.u.) of had-wt did not adversely impact the protective efficacy of the vaccine. furthermore, high levels of vector immunity (approximately 1500 virus-neutralization titer) induced by priming mice with 10(8) p.f.u. of had-wt were overcome by either increasing the vaccine dose or using alternate routes of vaccination. a further increase in the priming dose to 10(9) p.f.u. allowed only partial protection. these results suggest possible strategies to overcome the variable levels of human immunity against adenoviruses, leading to better utilization of had vector-based vaccines. adenoviruses (ad) possess several attributes that make them suitable candidates for vaccine vectors [1, 2] . ad exert an adjuvantlike effect by stimulating the innate immune system through both toll-like receptor (tlr)-dependent and tlr-independent pathways [3, 4] . the effectiveness of ad vector-based vaccines against many infectious diseases, including measles, severe acute respiratory syndrome (sars), human immunodeficiency virus (hiv), hepatitis b and ebola has been evaluated in animal models and clinical trials in humans [5] [6] [7] [8] [9] . previously, we and others have explored the potential of a human ad serotype 5 (had5) vectorbased vaccine strategy for h5n1 influenza [10] [11] [12] . our immunogenicity and protective efficacy studies demonstrated that ad vector-based vaccines provide complete protection against challenge with homologous and antigenically distinct strains of influenza viruses in a mouse model [11] . there is a high incidence of ad infections in the general population due to the circulation of more than fifty ad serotypes. their ubiquitous nature results in the development of ad-specific neutralizing antibodies, popularly known as 'preexisting vector immunity' in the majority of the individuals [13] [14] [15] . adneutralizing antibodies inhibit the vector extracellularly, while ad-specific cd8+ t cells destroy vector expressing cells [16, 17] thereby adversely impacting the duration and levels of transgene expression. experimental studies in animal models have shown that in the presence of extremely high levels of ad-neutralizing antibodies, there is a significant inhibition in the development of immunogen-specific immune responses [18] . a comprehensive analysis of ad seroprevalence found that had5 neutralizing antibody titers in the study's participants varied by geographic location and ranged from 18 to 4690 [19] . according to this study, 26% of the participants had titers below 200, 40% had titers below 1000, and 20% exhibited titers greater than 1000. these studies have underscored the need to further evaluate the role of vector immunity in inhibiting the immunogenicity and efficacy of had vector-based vaccines. to determine the level of vector immunity that can be tolerated without significantly affecting the vaccine efficacy, we primed groups of mice with varying doses of wild type (wt) had5 via intranasal (i.n.) or intramuscular (i.m.) route of inoculation to generate different levels of had5-neutralizing antibody titers. after the development of had5-specific immunity, had-primed mice were immunized i.n. or i.m. with a low or high dose of a had vector (had-ha-np) carrying the hemagglutinin (ha) and nucleoprotein (np) genes of the a/vietnam/1203/04 (h5n1) influenza virus. we also assessed if we could overcome vector immunity by increasing the vaccine dose and changing the route of immunization. our results suggest that a high level (up to a neutralization titer of 2240) of vector immunity can be tolerated or effectively overcome by increasing the vaccine dose or using alternate routes of vaccination. generation and characterization of had vector expressing ha and np of h5n1 influenza virus (had-ha-np) the full coding region of ha under the control of the cytomegalovirus (cmv) immediate early promoter and bovine growth hormone (bgh) polyadenylation signal (polya) and full length coding region of np gene of the a/vietnam/1203/04 virus under the control of the murine cmv promoter and the simian virus 40 (sv40) polya were inserted into early region 1 (e1) of the had genome using the cre-recombinase-mediated site-specific recombination system [20] . both genes in had-ha-np were in the e1-parallel orientation. the recombinant vector, had-ha-np ( figure 1a ) showed visible cytopathic effect (c.p.e.) on the ninth day post-transfection. western blot analysis was done to confirm the expression of ha and np in 293 cells. two distinct polypeptide bands of approximate molecular weights 77 kda and 50 kda, representing the ha precursor (ha0) and a proteolytic cleavage product (ha1), respectively, ( figure 1b) were observed in the had-ha-np infected 293 cell lysate. a single band at approximate molecular weight of 56 kda representing np ( figure 1c ) was visible in the had-ha-np infected 293 cell lysate. to mimic in a mouse model the preexisting immunity against had5 observed in the majority of the human population, groups of animals were inoculated i.n. or i.m. with a single dose of 10 7 , 10 8 , or 10 9 plaque forming units (p.f.u.) had-wt. both i.n. and i.m. primed groups showed a dose-dependent increase in the levels of had-specific neutralizing antibody titers ( figure 2 ). as expected, the highest had-specific neutralizing antibody titers in i.n. inoculated had-primed groups were observed with a 10 9 p.f.u. dose of had-wt (2240) followed by 10 8 p.f.u. (1040) and 10 7 p.f.u. (300) dose ( figure 2) . similarly, the i.m. primed group receiving 10 9 p.f.u. of had-wt developed the highest titer (3040) followed by 10 8 p.f.u. (1480) and 10 7 p.f.u. (520) dose groups (fig. 2) . the i.m. primed groups resulted in the development of higher levels of had-specific neutralizing antibody titers compared to the i.n. primed groups. induction of humoral immune response in had-primed mice immunized with had-ha-np development of a robust ha-specific antibody response is an important indicator of the immunogenicity and protective efficacy of an influenza vaccine [21] . the i.n. or i.m. immunization of had-primed groups indicating that alternating the route of priming and immunization or increasing the vaccine dose can partially overcome the vector immunity. in the 10 9 i.n. had-primed group, i.n. immunization with had-ha-np induced lower serum hi titers (22) . alternating with the i.m. route of immunization resulted in slight improvement in the hi titers (34) . increasing the vaccine dose by five-fold resulted in further improvement in hi titers in mice immunized either i.n. (52) or i.m. (80) indicating that the i.n.-induced (which mimics the natural route of infection in humans) vector immunity can be partially overcome by increasing the vaccine dose (p#0.05). however, in the 10 9 i.m. had-primed groups immunized with had-ha-np, a hi titer of 30 was induced, and there were no significant changes in the titers by either alternating the route of vaccine inoculation or with an increased vaccine dose. these results indicate that the levels of vector immunity induced by i.m. priming with 10 9 p.f.u. of had5-wt negatively impact the development of a humoral immune response against a had vector-based vaccine. cell-mediated immunity (cmi) plays an important role in virus clearance and thus contributes to the recovery from an influenza infection [22, 23] . as anticipated, the had-ha-np vaccine elicited significantly higher percentages of np-147 epitope-specific cd8 t cells in the naïve groups compared to the vector (had-de1e3) control group ( figure 3a & 3b) . the percentages of np-147 epitope-specific cd8 t cells in 10 7 had-primed groups (i.n. or i.m.) was significantly higher than in vector control groups following immunization with had-ha-np by either the i.n. or i.m. route. however, the percentages np-147-specific cd8 t cells were 1.5-2 fold lower in had-primed groups (i.n. or i.m.) compared to naïve groups, suggesting that preexisting vector immunity had a modest effect on the induction of cmi following immunization with had-ha-np ( figure 3a & 3b) . the percentages of np-147 epitope-specific cd8 t cells in 10 8 had-primed groups were two-fold lower compared to the 10 7 had-primed groups (i.n. or i.m.) following immunization with had-ha-np by either route (figure 3a & 3b) . interestingly, increasing the vaccine dose by five-fold resulted in significantly higher percentages of np-147 epitope-specific cd8 t cells in the 10 8 had-primed groups compared to both 10 7 and 10 8 hadprimed groups receiving the lower dose (10 8 p.f.u.) of the vaccine. as expected, a further increase in the level of preexisting vector immunity led to a further decrease in the percentages of np-147 epitope-specific cd8 t cells. furthermore, an increase in the vaccine dose by five-fold resulted in significantly higher percentages of np-147 epitope-specific cd8 t cells in the 10 9 i.n. primed group compared to the 10 9 i.n. primed group receiving the lower vaccine dose (10 8 p.f.u.). however, this increase was not noticeable in the 10 9 i.m. primed group. the functionality of ha-518 and np-147-specific cd8 t cells was assessed by enumerating interferon-c (ifn-c) expressing cells by elispot assay. significantly higher numbers of ifn-c-secreting ha-518-and np-147-specific cd8 t cells were detected in the spleens from the naïve groups immunized i.n. or i.m. with had-ha-np compared to the vector control groups following stimulation with the ha-518 (p#0.0001) or np-147 (p#0.0001) peptide, respectively ( figure 4a-d) . in general, the number of ifn-c-secreting ha-518-or np-147-specific cd8 t cells in the spleens of had-primed groups immunized with the vaccine were lower compared to the naïve immunized groups, and a five-fold increase in the vaccine dose resulted in an increase in the number of ifn-c secreting ha-518-or np-147-specific cd8 t cells in all the primed groups except for the 10 9 i.m. primed groups ( figure 4a-d) . overall, it seems that with the increase in preexisting had5-neutralizing antibodies, there was a titerdependent decline in the cmi response which significantly improved with an increase in the vaccine dose by five-fold. there was excellent correlation between the elispot and np pentamer staining data. protection of had-primed mice immunized with had-ha-np following challenge with a reassortant h5n1 influenza virus log 10 eid 50 /ml). similarly, the 10 7 had-primed groups (i.n. or i.m.) immunized with had-ha-np had lung viral titers at or below the level of detection (1.5 log 10 eid 50 /ml) indicating that the preexisting vector immunity did not adversely impact the protective efficacy. however, the 10 8 had-primed groups (i.n. or i.m.) immunized with had-ha-np by the same route that was used for priming exhibited less efficient virus clearance from the lungs. interestingly, either using a different route of inoculation for priming and vaccination or increasing the vaccine dose by five-fold resulted in lung viral titers at or below the level of detection. in the 10 9 i.n. had-primed groups (i.n. or i.m.) immunized with had-ha-np by the same route that was used for priming, there was only partial (approximately 2-4 logs) virus clearance. by changing the route of priming and immunization, complete protection was observed only in the group where the i.n. priming was followed by a five-fold increase in the vaccine dose administered by either route (i.m. or i.n.). even a five-fold increase in the vaccine dose did not yield complete protection in the 10 9 i.m. had-primed groups immunized with had-ha-np by either route (i.n. or i.m.) suggesting that the level of preexisting vector-neutralizing antibody titer could serve as an indicator for predicting the efficacy of adbased vaccines. to meet the global vaccine demand in a pandemic, various eggindependent vaccine strategies need to be explored to supplement egg-dependent influenza vaccine approaches. ad vector-based influenza vaccines have been shown in clinical studies to be safe and immunogenic in humans [24, 25] . the strong innate and adaptive immune responses induced by ad vectors impart adjuvant-like properties facilitating better immune responses against the transgene product/s. however, several preclinical studies have suggested that the presence of preexisting hadspecific neutralizing antibodies might inhibit the generation of immune responses against the expressed immunogen [1, 14, 18] . in the present study, we evaluated the role of preexisting had5specific neutralizing antibodies in inhibiting the immunogenicity table 1 . hemagglutination inhibition (hi) antibody titers before challenge and lung viral titers 3 days post-challenge with a homologous h5n1 reassortant virus in had-ha-np-immunized naïve and had-primed mice. immunization hi titers (gm) log 10 eid 50 /ml ± s.d. and efficacy of a had5 vector-based h5n1 influenza vaccine in a mouse model. low levels of vector immunity (,520 virusneutralization titer) did not seem to adversely affect the protective vaccine efficacy, while further increases in vector immunity were taken care of by using an alternate route of immunization or by an increase in the vaccine dose. the importance of np-specific cd4 t cells and non-neutralizing antibodies in the virus clearance was not pursued in this study. to mimic the natural exposure of had to the majority of humans, we primed mice with had5 by the i.n. route to establish the state of preexisting vector immunity since many had infect via the mucosal route. the i.m. had5-primed groups represented the development of had-specific immune responses following i.m. immunization with a had vector-based vaccine. we attempted to circumvent the inhibitory effect of high levels of preexisting vector immunity by either a change in the route of vaccine inoculation or an increase in the vaccine dose. in the presence of preexisting neutralizing antibodies (300-520), there was a modest decline in the levels of cmi and hi levels in response to immunization with had-ha-np. this level of immune response was sufficient enough to provide excellent protection against the challenge with a h5n1 reassortant virus. there were lower levels of humoral and cellular immune responses after the i.n. immunization compared to the i.m. immunization which is consistent with earlier findings [11, [26] [27] [28] . further increase in the levels of vector-specific neutralizing antibody response (1000-1480) resulted in a greater decline in influenza virus-specific immune responses with the inhibition more pronounced when the route of inoculation for ad-priming and influenza virus immunization were the same (e.g., i.n. and i.n., i.m. and i.m.). this level of vector immunity could be overcome either by changing the route of priming and immunization or by increasing the vaccine dose by five-fold. to test the upper limit of vector immunity that could be tolerated without adversely figure 3 . np-147 epitope-specific cd8+ t cells in naïve or had5-primed mice immunized with had-ha-np. naïve or had-primed mice were immunized as described in the materials and methods. at four weeks after final immunization, animals were euthanized, and the spleens were collected. single cell suspensions were prepared by passage through screens, and 1610 6 cells were stained with a murine mhc-encoded allele k dspecific pentamer for immunodominant np-147 epitope-conjugated with phycoerythrin (pe) and also with anti-cd8 antibody-conjugated with allophycocyanin (apc) and anti-cd19 antibody-conjugated with flouro-isothiocyanin (fitc). flow cytometeric analysis was done to identify the number of np-147-specific cd8+ t cells. data were collected using bd facscanto ii (bd bioscience, ca) and facsdiva software was used for analysis. affecting the vaccine protective efficacy, the vector-specific neutralizing antibody titer was raised to 2240 by i.n. priming with high doses of had5. the inhibitory effect was partially overcome by the five-fold increase in the vaccine dose, and the resultant immune response was sufficient to provide complete protection. further increase in the level of vector-specific neutralizing antibody titer to 3040 by i.m. priming with had5 only provided partial protection even in the groups receiving a high vaccine dose. alternating the route of priming and immunization was partially successful in overcoming vector immunity thus indicating the potential role of the route of inoculation in developing the level of humoral and cmi responses. it has been suggested that the route of vaccination impacts the magnitude, phenotype and trafficking of antigen-specific cd8 t cells in mice [29, 30] . a hadbased hiv vaccine also showed some inhibition in eliciting immunogen-specific immune responses in the presence of vector immunity, however, this effect was minimized by increasing the vaccine dose [31] . nevertheless, in a clinical trial with a hadbased influenza vaccine, there were no strong correlations between vector immunity levels and a decrease in vaccine efficacy [25] . the inhibitory effect of vector immunity was more pronounced for the humoral immune response compared to the cmi response which is consistent with previous reports [32] [33] [34] . taken together, our data clearly show that the magnitude of humoral and cellular immune responses to ad-vectored vaccine antigens depend upon the levels of preexisting antibodies against the vector. however, vector-specific immune responses can be overcome by increasing the antigen dose or by administering antigen by a different route. in conclusion, based on the present study alone, it will be difficult to predict the range of the vector immunity in humans that can be tolerated or overcome by either increasing the dose or changing the route of administration of the had5 vector-based vaccines. nevertheless, the study does highlight the importance of exploring these strategies in humans to improve the outcome of had5 vector-based vaccines. the 293 (human embryonic kidney cells expressing had5 e1 gene products; obtained from atcc) and 293cre (293 cells that constitutively expresses cre-recombinase enzyme (a gift from merck inc., whitehouse station, nj) [35] were grown as monolayer cultures in eagle's minimum essential medium (mem) (life technologies, gaithersburg, md) and supplemented with 10% reconstituted bovine serum (fetal clone iii; hyclone, logan, vt) and 50 mg/ml gentamycin. all constructs were purified by cesium chloride densitygradient centrifugation and titrated by plaque assays on a hybrid cell line using mdbk and 293 cell lines (bhh2c) [36] as described previously [37] . the construction and propagation of replication defective had-de1e3 (had5 vector having deletions in the e1 and e3 regions) has been previously described [11] . had5-wt virus was purified by cesium chloride density-gradient centrifugation and titrated by plaque assay on bhh2c cells. a cre-recombinase-mediated site-specific recombination technique [20] was used to insert the full-length coding region of the ha gene (with a modified polybasic site) of the a/vietnam/1203/ 04 (h5n1) influenza virus under the control of the cmv promoter and bgh polya. the polybasic cleavage site qrerrrkkrqg present in the ha gene of a/vietnam/1203/04 (h5n1) influenza virus was modified to qretrqg to reduce the rare possibility of genetic exchange between the ha in had-ha-np vaccine and a circulating strains of influenza a virus. the full-length coding region of np gene of the a/vietnam/1203/04 (h5n1) virus under control of the murine cmv promoter and the sv40 polya was similarly inserted into the e1 region of the had genome. both genes in had-ha-np were in the e1-parallel orientation. the recombinant virus was plaque purified, and its genome was analyzed by restriction enzyme digestions to confirm the presence of ha and np gene cassettes and the absence of any other major deletion or insertion. naïve or had5-primed mice were immunized as described in the materials and methods. at four weeks after final immunization, animals were euthanized, and the spleens were collected. single cell suspensions were prepared by passage through screens, and 1610 6 cells were cultured in the presence of ha-518 or np-147 peptide on anti-interferon-c antibody-coated 96-well filter plates and developed according to an elispot protocol. splenocytes cultured in presence of phorbol myristate acetate (pma) and ionomycin served as positive controls in each group. the elispot plates were read using a bioreader 5000 ( development of nonhuman 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bovine x human hybrid cell lines that efficiently support the replication of both wildtype bovine and human adenoviruses and those with e1 deleted porcine adenoviral vectors evade preexisting humoral immunity to adenoviruses and efficiently infect both human and murine cells in culture characterization of bovine adenovirus type 3 e1 proteins and isolation of e1-expressing cell lines dna vaccine expressing conserved influenza virus proteins protective against h5n1 challenge infection in mice detection of anti-h5 responses in human sera by hi using horse erythrocytes following mf59-adjuvanted influenza a/duck/singapore/97 vaccine enhanced antibody and cytokine responses to influenza viral antigens in perforin-deficient mice we are thankful to j. kovach for her excellent secretarial assistance. the findings and conclusions in this report are those of the authors and do not necessarily represent the views of the centers for disease control and prevention, us department of health and human services. conceived and designed the experiments: skm. performed the experiments: ap ns lc svv rd. analyzed the data: skm jmk ss. contributed reagents/materials/analysis tools: rd. wrote the paper: ap skm ss. use of cell line: skm. induction of vector-specific immunity and evaluation of vaccine efficacy [6] [7] [8] week old female balb/c mice (10 animals/group) were inoculated by either the i.m. or i.n. route with a single dose of 10 7 , 10 8 , or 10 9 p.f.u. of had-wt. these groups were referred to as hadprimed groups. the unprimed (naïve) mice were similarly inoculated with phosphate-buffered saline (pbs). four weeks after priming, mice were bled by retro-orbital puncture to evaluate the development of had-specific neutralizing antibody titers. had-primed and naïve (pbs-inoculated) mice were subsequently immunized twice (4 weeks apart) with 1610 8 or 5610 8 p.f.u. of had-ha-np vaccine by either the i.m. or i.n. route. additional groups of had-primed mice were similarly immunized with 5610 8 p.f.u. of had-de1e3 (vector control) to serve as negative controls.four weeks after final immunization, blood samples were collected through retro-orbital puncture to evaluate the development of ha-specific antibodies. five animals from each group were euthanized to collect the spleen cells to evaluate the induction of ha-and np-specific cmi responses. the remaining mice from each group were challenged with 100-fold of 50% mouse infectious dose (mid 50 ) of a reverse genetics derived a/puerto rico/8/ 1934(h1n1) [pr8] containing ha and na gene fragments of a/ vietnam/1203/04 (h5n1) [vnh5n1-pr8/cdc-rg] [24] . since this reassortant virus is not lethal and does not produce clinical disease and weight loss in mice, protection efficacy was monitored by viral clearance in the lungs. three days post-challenge, mice were euthanized, and the lungs were collected to determine viral titers to evaluate protective efficacy. briefly, thawed lung tissues were homogenized in 1 ml of sterile pbs. these lung homogenates were then titrated in 10-day-old embryonated eggs in a 10-fold dilution, and positive eggs were identified by hemagglutination of horse red blood cells with allantoic fluid. values were expressed as log10 eid50/ml 6 sem. the limit of virus detection was set as 1.5 log10 eid50/ml [39] . the np genes are fairly conserved between a/vietnam/1203-04 (in the ad vector) and a/pr8/34 (the backbone of the challenge virus); there is 93% identity at the amino acid level and 85% identity at the nucleotide level. one of the major cd8 t cell epitopes (np147) is 100% conserved in between a/vietnam/1203-04 and a/pr8/34. had neutralizing serum antibody titers were determined as previously described [13] . the virus neutralization titer was the reciprocal of the highest serum dilution that completely prevented the development of c.p.e. pre-challenge serum samples were analyzed for the presence of hi antibody titers using horse red blood cells as described previously [40] . splenocytes were isolated and stained with a murine mhc k dspecific pentamer for immunodominant np-147 epitope (proimmune inc., bradenton, fl) conjugated with phycoerythrin (pe) and an anti-cd8 antibody conjugated with allophycocyanin (apc) (bd pharmingen, san jose, ca.) as described previously [11] . b cells were removed by staining splenocytes with anti-cd19 flouroisothiocyanin (fitc) and gating them out in analysis. flow cytometric analyses were done using bd facscantoii, (bd bioscience, san jose, ca) to identify the percent np-147 epitopespecific cd8+ t cells among the total splenic cd8+ t cells.elispot assay 96-well filter plates (millipore, bedford, ma) were coated with an anti-mouse interferon gamma (ifn-c) antibody (bd bioscience, san jose, ca) and incubated at 4uc overnight. splenocytes (3.3610 5 to 1610 6 cells per well) from each mice were cultured in the presence of ha-518 or np-147 peptides in rpmi medium (gibco, grand island, ny), supplemented with 10% reconstituted fetal bovine serum for 60 h and developed according to an elispot protocol [41] . splenocytes cultured in the presence of phorbol myristate acetate (pma) and ionomycin (sigma-aldrich, inc., st. louis, mo) served as positive control within each group. log-transformation of titer measurement was assessed by shapiro-wilktest, found to be normally distributed and used for the analysis using sas 9.2. tukey's multiple comparison was used for calculation of significance. the significance was set at p ,0.05. key: cord-289555-1z4vbldd authors: mühldorfer, kristin; speck, stephanie; kurth, andreas; lesnik, rené; freuling, conrad; müller, thomas; kramer-schadt, stephanie; wibbelt, gudrun title: diseases and causes of death in european bats: dynamics in disease susceptibility and infection rates date: 2011-12-28 journal: plos one doi: 10.1371/journal.pone.0029773 sha: doc_id: 289555 cord_uid: 1z4vbldd background: bats receive increasing attention in infectious disease studies, because of their well recognized status as reservoir species for various infectious agents. this is even more important, as bats with their capability of long distance dispersal and complex social structures are unique in the way microbes could be spread by these mammalian species. nevertheless, infection studies in bats are predominantly limited to the identification of specific pathogens presenting a potential health threat to humans. but the impact of infectious agents on the individual host and their importance on bat mortality is largely unknown and has been neglected in most studies published to date. methodology/principal findings: between 2002 and 2009, 486 deceased bats of 19 european species (family vespertilionidae) were collected in different geographic regions in germany. most animals represented individual cases that have been incidentally found close to roosting sites or near human habitation in urban and urban-like environments. the bat carcasses were subjected to a post-mortem examination and investigated histo-pathologically, bacteriologically and virologically. trauma and disease represented the most important causes of death in these bats. comparative analysis of pathological findings and microbiological results show that microbial agents indeed have an impact on bats succumbing to infectious diseases, with fatal bacterial, viral and parasitic infections found in at least 12% of the bats investigated. conclusions/significance: our data demonstrate the importance of diseases and infectious agents as cause of death in european bat species. the clear seasonal and individual variations in disease prevalence and infection rates indicate that maternity colonies are more susceptible to infectious agents, underlining the possible important role of host physiology, immunity and roosting behavior as risk factors for infection of bats. bats are among the most successful and diverse mammals on earth. approximately 1230 chiropteran species are found on every continent except antarctica and inhabit a multitude of diverse ecological niches [1] . bats play essential roles in maintaining healthy ecosystems, as they act as plant pollinators, seed dispersers, and predators of populations of insects including harmful forest and agricultural pests [2] . most bat species are listed in the iucn red list of endangered species and almost half of these are considered threatened or near-threatened [3] . to estimate and prevent further population declines, research has been primarily focused on bat biology, ecology and behavior, while disease aspects were largely neglected [4] . in the last two decades, the importance of chiropteran species as potential vectors of significant viral diseases especially in regard to zoonoses has received growing attention. besides bat rabies that has been studied for more than half a century, extensive research efforts identified a large number of microbial agents [5] including important emerging zoonotic viruses detected in bats across the world [6] [7] [8] [9] [10] [11] [12] . however, most studies are limited to the identification of microorganisms detected and investigations regarding infectious diseases and causes of death in bats are sparse [13] [14] [15] [16] . in europe, research is predominantly focused on european bat lyssaviruses [17, 18] and coronaviruses [19, 20] , but first indications of bat-pathogenic bacteria [13, 14, [21] [22] [23] and novel viruses [24, 25] isolated from deceased bats in germany and great britain were found. in this study, we provide new data on infectious diseases in european bat species, considering factors likely to affect the susceptibility of bats to infectious agents including effects of seasonality, individual and species-specific heterogeneities, and possible intra-and inter-species transmission dynamics. all bat species in europe are strictly protected under the flora-fauna-habitat guidelines of the european union (http://ec.europa. eu/environment/nature/legislation/habitatsdirective/index_en.htm) (92/43/eec) and the agreement on the conservation of populations of european bats (www.eurobats.org) that prohibit invasive sampling of bats for research purposes. for the animals investigated in this study, carcasses of deceased bats found in germany were kindly provided by bat researchers and bat rehabilitation centers of different federal states. between 2002 and 2009, a total of 486 deceased bats of 19 european vespertilionid species (i.e., family vespertilionidae) were investigated (fig. 1a , [26] ). the bat carcasses originated from 6 different geographic regions in germany, i.e. berlin greater metropolitan area (n = 223), bavaria (n = 165), brandenburg (n = 38), lower saxony (n = 36), thuringia (n = 21), and baden-wuerttemberg (n = 3), and were collected by bat researchers and bat rehabilitation centers. most animals represented individual cases that were found dead, injured or moribund near human habitation. thus, the species composition in this study predominately reflected the urban and suburban bat fauna, which is characterized by a disproportionate abundance of a few bat species (fig. 1a , [27, 28] ). two groups of 2 and 21 adult noctules (nyctalus noctula), respectively, were collected from tree hibernacula destroyed during wood logging. a further group of 25 deceased adult n. noctula originated from a colony that was trapped in a rain pipe in december. nine dead juvenile pipistrellus pipistrellus were collected from a nursery roost. if bats died in care or had to be euthanized for animal welfare reasons, the carcasses were immediately stored at 220uc and were shipped to the leibniz institute for zoo and wildlife research, berlin, germany, for diagnostic investigations. of all carcasses examined histo-pathologically, about 90% were suitable for bacteriological investigation. a lesser extend (43%) was also examined for selected viral agents at the robert koch institute, berlin, germany. in addition, a brain sample of each animal was submitted to the friedrich-loeffler-institute, wusterhausen, germany, for rabies diagnosis. a full necropsy was performed on each bat and all macroscopic findings including ectoparasite infestation were recorded. for histo-pathological examination, small slices of multiple organ tissues (i.e., lung, liver, heart, kidney, adrenal gland, spleen, intestine, pancreas, brain, tongue, larynx, salivary gland and pectoral muscle) and tissues conspicuous for pathological changes were fixed in buffered 4% formalin, processed using standard methods and embedded in liquid paraffin. sections were cut at 2-5 mm and routinely stained with hematoxylin-eosin (he). in addition, special histological staining methods were used depending on microscopic findings, i.e. for the detection of bacteria (gram or giemsa staining), fungi (periodic acid schiff or grocott's gomori methenamine silver nitrate staining), iron (prussian blue stain), mineralization (von kossa staining), connective and collagen tissue (trichrome staining). details on pathological results are published elsewhere [26] . the causes of mortality were rigorously standardized with the primary cause of death identified for each bat as the most serious injury, disease or event subsequently fatal to the animal. to ensure independence of primary and contributing causes of death, the categorization was based on the severity of pathological findings. samples of lung, liver, heart and kidney, and tissues conspicuous for pathological changes (e.g. enlarged spleen) of 430 bats were plated onto columbia (5% sheep blood), chocolate, gassner, and macconkey agar (oxoid, germany) and were incubated at 37uc (chocolate agar 5% co 2 ) for 24-48 h. specific culture media and conditions for the isolation of yersinia, salmonella and anaerobic bacteria were used if appropriate. primary identification of bacterial strains was based on colony morphology, hemolysis, gram-staining, indol production, catalase and oxidase reaction. bacterial species identification was carried out using the relevant commercial api test system (biomérieux, germany). additional conventional biochemical tests [29, 30] were applied to confirm api test results where necessary. in case of ambiguous biochemical test results, 16s rdna gene analysis was performed for final identification [23] . salmonella isolates were characterized at the national reference laboratory for the analysis and testing of zoonoses (salmonella) at the federal institute for risk assessment, berlin, germany. identification and characterization of yersinia and pasteurella species have been reported earlier [22, 23] . homogenized organ tissue of lung, liver, heart, kidney, spleen, brain and salivary gland of 210 bats were pooled for each individual and used for rna/dna extraction and further molecular analysis by generic pcr assays detecting flavi[31] , hanta[32] , corona[33] , and influenza a-viruses [34] . also, pcr assays specific for 8 previously described herpesviruses [24] from european vespertilionid bats were used. for this purpose, rna/ dna was isolated using the nucleospinh rna ii kit (macherey-nagel, germany) and the nucleospinh tissue kit (macherey-nagel), respectively, according to the manufacturer's instructions. because of limitations in sample volume, for 180 out of the 210 bats pcr assays could only be applied for 4 different bat herpesviruses. internal controls were used for all pcr assays to test for inhibition. for confirmation, all retrieved fragments of bat herpesvirus-specific pcr assays were checked for sequence identity to previously published isolates [24] . for detection of lyssavirus antigen in brain tissue the fluorescent antibody test (fat) using a polyclonal antirabies conjugate (sifin, germany) was used [35] . fat-positive brain tissues were subject of virus isolation in murine neuroblastoma cell culture (na 42/13) using the rabies tissue culture infection test (rtcit) as described elsewhere [36] . lyssaviruses isolated in cell culture were characterized using both a panel of 10 anti-nucleocapsid monoclonal antibodies (mab) [37] and partial sequencing of a fragment of the nucleoprotein gene after rna extraction using trizol (invitrogen, germany) essentially as described [18] . genomic dna was extracted from organ homogenates using the nucleospinh tissue kit (macherey-nagel) according to manufacturer's recommendations. genetic identification of the bat species was performed by amplification and sequencing of a 241 bp fragment of the cytochrome b (cytb) gene [38] using primers fm up (59-ccc chc chc aya tya arc cmg art gat a -39) and fm down (59-tcr acd ggn tgy cct ccd att cat gtt a -39). in addition, for differentiation of the 2 distinct pipistrellus species, p. pipistrellus and p. pygmaeus, a rapid multiplex pcr assay was performed as described by kaňuch et al. the bat data were categorized in regard to different explanatory numeric and factor variables, e.g. bat species, sex and age class. the variable 'age class' ranked between 1 and 4 with increasing age (i.e. neonates, juveniles, subadults, and adults) and was used as numeric variable. for endoparasitic analysis, we defined a 3 level variable 'bat size' according to the body size of a certain bat species to reduce the degrees of freedom of the full model, i.e. large species (n. noctula, eptesicus serotinus, and vespertilio murinus), medium-sized species (e. nilssonii, plecotus auritus, myotis daubentonii, m. nattereri, and p. nathusii) and small species (p. pipistrellus, and m. mystacinus). to detect effects of seasonality, 4 different activity periods were specified according to the date of sampling, i.e. hibernation period (november to march), post-hibernation period (april/may), maternity period (june to august), and swarming period (september/october). as dependent binary variable for the respective models we either classified the mortality cause being disease or not (i.e. trauma), or the presence-absence of bacterial, ecto-and endoparasitic infections. we formulated 4 different hypotheses to test for individual and species-specific differences in disease susceptibility and infection rates: (a) disease-related mortality in bats is influenced by sex, age and species-specific differences, and degree of endoparasitic infection. (b) bacterial infection in bats is influenced by sex, age and species-specific differences, occurrence of traumatic injuries and cat predation. (c) ecto-or (d) endoparasitic infection in bats is affected by age, sex and species-specific differences. seasonal effects were not analyzed because of too many missing data points. because the long-term dataset was highly biased towards sampling procedure, preservation of bat carcasses and following diagnostic investigations, we split and filtered the full data into several subsets reflecting the different analyses (table 1) . all statistical analyses were performed using the r software v. 2.13.1 (r development core team 2011, vienna, austria). we used the chi-square test for given probabilities to evaluate significant differences in the sex ratio among bats of different species. for hypotheses a and b, we used a generalized linear mixed modeling approach (binomial glmm using function lmer in library lme4) with bat species included as random effect. this variable had not been significant as fixed effect (results not shown), but from other studies we can assume that there are speciesspecific differences in susceptibility of bats to certain infectious agents and therefore included it as random effect. we further used generalized linear models (glm with logit link and binomial error structure; for datasets with bat species .10 individuals) to test for individual and species-specific differences in parasite infection rates (hypotheses c and d). we created a full model for each hypothesis (a-d) to examine multiple and interaction effects of the specified variables. to select the final model variables, we used a stepwise backward algorithm (function stepaic in library mass) based on akaike's information criterion (aic) [40] . the daic of the final model was calculated relative to a random intercept model to demonstrate the effect size of the selected variables. results of the diagnostic analyses follow the full data splitting into several subsets (see section 'statistical analysis' in material and methods; table 1 ). all sampled bats belonged to 7 different genera (i.e. pipistrellus, nyctalus, myotis, eptesicus, plecotus, vespertilio, and barbastella) and 19 european vespertilionid species (fig. 1a) . three bat species, the common pipistrelle (p. pipistrellus, n = 138), the noctule bat (n. noctula, n = 92), and the serotine bat (e. serotinus, n = 53) constituted about 60% of all bat carcasses investigated in this study, whereas p. pygmaeus, nyctalus leisleri, myotis brandtii, m. bechsteinii, m. dasycneme, plecotus austriacus and barbastella barbastellus were represented in small numbers of 1 to 4 animals. the overall sex ratio was 1.5 males to 1 female with significant species-specific differences (fig. 1b) . animals in their first year of life (neonates, juveniles, and subadults) represented one third (32.5%, n = 158) of bat samples (fig. 1c) . overall, we were able to assign a cause of death to 70% (n = 304) of bats investigated in this study. two thirds of mortality were due to trauma (n = 145) or disease (n = 144), while almost 4% of bats had died of other non-infectious causes like pulmonary edema, dehydration and hypoglycemia (table 2 ). in 30% (n = 129) no significant pathological findings could be found. among the 145 traumatized bats, additional mild (n = 42), moderate (n = 28) and severe (n = 4) inflammatory organ changes were noted in one half (50.9%) of individuals, and 23% of the bats revealed bacterial (n = 19) and/or parasitic infections (n = 15) ( table 3) . of the 144 bats considered as dying of disease, fatal bacterial (n = 54), viral (n = 5) and parasitic infections (n = 2) were observed in 42%. besides, amniotic fluid aspiration was noted in a neonate noctule bat (n. noctula), and a juvenile common pipistrelle (p. pipistrellus) was euthanized because of severe forearm bone deformation. the remaining 81 bats (56.3%) revealed moderate to severe pathological changes of unknown etiology or unconfirmed bacterial or viral cause ( table 2 ). based on the glmm analysis, significant age-and sexdependent differences (daic = 23.13) were detected between the general causes of mortality, disease and trauma ( table 4 ). the disease presence in bat samples decreased continuously with increasing age. neonates and juveniles of both sexes were significantly more affected by disease than older age classes (table 4 ; fig. 2a) . we also found a significant trend in diseaseassociated mortality between the sexes, with adult females displaying higher disease prevalence (52.5%) than males (36.4%) ( table 4 ). no significant association was observed between a certain cause of mortality (i.e. disease or trauma) and severity of endoparasitic infection (daic = 0.75, result not shown). the seasonal distribution of disease-related mortality cases (fig. 2b ) described a trimodal pattern, with peaks in spring (april), summer (june to august) and winter (december). the proportion of traumatized individuals also increased obviously during the summer months up to and including the swarming period, but was low during the rest of the year. about 90% (n = 430) of bat samples were examined bacteriologically. among these, 42 different bacterial genera with more than 53 bacterial species were identified (table s1 ). predominant bacteria isolated were enterococcus faecalis (14.7%, n = 63), hafnia alvei (11.2%, n = 48), serratia liquefaciens (10%, n = 43), and pasteurella multocida (7.7%, n = 29). in 37% (n = 157) of bats no bacterial growth was observed at all. comparative bacteriologic and histo-pathologic analysis identified 22 different bacterial species that were clearly associated with pathological lesions and/or systemic infection, found in 17% (n = 73) of bats investigated bacteriologically ( table 5) . members of the families pasteurellaceae (above all p. multocida) (41.1%, n = 30), enterobacteriaceae (various bacterial species) (28.8%, n = 21), and streptococcaceae (above all enterococcus spp.) (21.9%, n = 16) were predominant bacteria associated with disease. more than half (54.8%, n = 40) of bacterial infections were observed in bats with traumatic injuries. the glmm analysis revealed low sex-and age-dependent differences in bacterial infection (daic = 1.97, result not shown). female bats (21.9%) and adults (21.6%) showed marginally higher prevalence of bacterial disease compared to males (18.3%) and to other age classes (15.6%), respectively. however, we found a strong influence of cat predation (daic = 16) associated with bacterial infection in bats (table 4 ). testing for human-pathogenic zoonotic viruses, no examined bat sample (0/210) was positive for influenza a virus, corona-, hanta-and flaviviruses, respectively. no inhibition of the pcr assays was notified. out of 486 bats tested for rabies virus infection, 2 serotine bats (e. serotinus) were positive for lyssavirus by fat and rtcit. the viruses were identified as european bat lyssavirus type 1 (eblv-1) sublineage a, both using mabs and sequencing. applying bat herpesvirus-specific pcr assays, 63 out of 210 bats proved to be infected with 7 of the previously described 8 bat herpesviruses ( table 6 ). the highest prevalence of 65% (24/37) was observed for bat gammaherpesvirus 6 (batghv6) in common pipistrelle bats (p. pipistrellus), followed by bat gammaherpesvirus 5 (batghv5, 42.1%) in nathusius' pipistrelle bats (p. nathusii) and bat gammaherpesvirus 4 (batghv4, 33.8%) in noctule bats (n. noctula). co-infection with different bat herpesviruses were recognized in 4 n. noctula (7.4%) infected with batghv3 and batghv4, and in one n. noctula (1.5%) infected with batghv4 and batghv5. batghv5 was not only detected in its initially specific host p. nathusii, but also in 3 other bat species, i.e. n. noctula, myotis myotis and m. mystacinus. although the prevalence of batghv3 (13.0%) and batghv4 (33.8%) differed significantly within its migrating host n. noctula, no difference was observed between the sexes. two juvenile n. noctula were found to be infected with batghv4. interestingly, for the sedentary bat species p. pipistrellus being infected with batghv6, a considerably higher prevalence was observed in 22 juvenile bats (72.7%) resulting in an overall prevalence of 65% also without difference between adult male and female bats. ectoparasites (mites, fleas, and ticks) were noted in 14% (n = 62) of bats, but a potential bias in ectoparasite numbers collected from dead animals in comparison to ectoparasite abundance on live animals has to be taken in account. female bats (17.1%) were slightly more infested by ectoparasites than males (14.7%), whereas in different age classes ectoparasite prevalence was almost balanced. the glm analysis revealed significant species-specific differences in ectoparasite infestation (daic = 14.58, table 4 ). most bat species revealed low ectoparasite prevalence (range 5.3-11.8%), while almost 43% (n = 20) of n. noctula were infested with mites and/or fleas (fig. 3a) . microscopic examination of organ tissues revealed endoparasitic infection in 29% (n = 124) of investigated bats, involving different protozoan (families eimeriidae and sarcocystidae) and helminth parasites (trematodes, cestodes, and nematodes). helminthes were predominantly found in the gastro-intestinal tract of the bats, while in some animals, granulomatous organ lesions were associated with larval migration of nematode species. based on the glm analysis, clear age-and species-specific differences (daic = 24.95) were observed between infected and non-infected bats ( table 4 ). the prevalence of endoparasitic infection in bat samples increased significantly with increasing age, whereas the increase in prevalence was more rapid between juveniles and subadults (8.5%) compared to the older age classes (4.5%). marginal differences were also observed between the sexes, with female bats showing slightly higher (30.4%) endoparasite prevalence than males (24.4%). regarding species-specific differences, large bats like n. noctula, e. serotinus and v. murinus revealed higher endoparasite prevalence compared to individuals of medium-sized or small vespertilionid species (table 4 ; fig. 3b ). this study was based on a passive surveillance sampling strategy that inherently influences the composition of bats sampled for diagnostic investigations [27] and might also effect the data presented on causes of death by ecological and anthropogenic impacts of urban landscapes [41] . trauma and disease represented the most important causes of mortality in deceased bats from germany, which differ from results of previous studies [13] [14] [15] where disease-related mortality often played a subordinate role. young bats and adult females were significantly more affected by disease, indicating that sex-and age-related disease prevalence in table 3 . pathological findings and bacterial, viral and parasitic infections specified for the general causes of mortality, trauma and disease. bats are strongly correlated with the maternal season. this assumption is further supported by the distinct increase of diseaserelated mortality from june to august, which corresponds to the maternity period of central european bat species. similar seasonal prevalence patterns in bats have also been described for parasitic (e.g. [42] [43] [44] [45] ) and viral infections (e.g. [19, 46, 47] ). in contrast, the increase of trauma-associated mortality cases from july to october resembles 4 major behavioral activity patterns of european bat species (i.e. weaning, mating, pre-hibernal fat storage, and migration) [48] and could therefore predispose bats to trauma. however, both seasonal peaks also coincide with time and locations where sick, injured or dead bats are more likely to be discovered as well as with the seasonal roosting behavior of bats adapted on urban habitats [27] . the additional seasonal peaks of disease-associated mortality corresponded to the post-hibernal and the early hibernal period of temperate zone bats. currently, there is a lack of knowledge of bat immunology. it is known for other mammalian species that hibernation reduces the innate and adaptive immune response; likewise an increasing risk of infection could be assumed for hibernating bats [49] . with the start of the hibernation season, large aggregations of bats originating from various colonies might enhance the risk of spreading infectious agents similar to maternity colonies. equally, the post-hibernal increase of disease-related mortality is suggestive for reduced immunity in association with prolonged fasting during hibernation. bacterial diseases have rarely been documented in bats. pasteurella spp., here identified in 7% of bats, were the predominant bacterial pathogens reported in european bats and infection appears to be strongly correlated with cat predation [13, 14, 23, 26] . in our study, bacterial infections were confirmed in 17% of bats investigated bacteriologically. most of these bacterial isolates represented opportunistic pathogens that usually do not harm the host unless the immune system is weakened [50] or preceding injury to natural host barriers (e.g. skin abrasion). primary bacterial pathogens like samonella enterica serovar typhimurium, s. enteritidis and yersinia pseudotuberculosis [22] were identified in almost 12% of affected bats. some of the bacterial species (e.g. burkholderia sp., cedecea davisae and clostridium sordellii) are newly described in bats. nevertheless, bacteriologic analyses can markedly be influenced by post-mortem bacterial invaders, freezing and storage of bat carcasses and the inability to detect certain bacteria by routine culture methods, resulting in some bacterial species that might have escaped detection. we found a strong association between cat predation and bacterial infection in bats as almost one half of bats (44%) caught by cats were affected by bacterial disease. various bacteria can be transmitted via cat bites [51] ; hence bats attacked by cats are likely to succumb to bacterial infection even if non-fatal injuries were present. this relation has been proven for p. multocida infections in european bat species [13, 14, 23, 26] . on the other hand, bats already debilitated by disease might easier fall prey to predators like cats. consequently, bats may also act as vectors for zoonotic pathogens, as domestic cats could pass these infectious agents on to humans. such cross-species transmission events from bats to domestic animals are well documented [9, 52] . for all tested human-pathogenic zoonotic viruses no infected bat could be detected in this study except lyssaviruses. bat rabies is the only bat transmitted zoonosis in europe that is known to have resulted in human cases [53] . unlike in other mammals table 4 . result of the final model variables corresponding to 4 different analyses: (a) disease-vs. trauma-related mortality, and presence-absence of (b) bacterial, (c) ecto-and (d) endoparasitic infection. where lyssaviruses ultimately cause lethal rabies, in bats nonlethal lyssavirus infections may also lead to the development of immunity [47] . with the detection of eblv-1 we confirm that this lyssavirus circulates among e. serotinus as previous studies showed [18] . in germany, bat rabies is also caused by eblv-2 and bokeloh bat lyssavirus (bblv) [54, 55] , but while the latter was recently isolated from m. nattereri, eblv-2 is associated with m. daubentonii and m. dasycneme [56] . the apparent absence of eblv-2 and bblv in the sampled bats is likely due to the fact that lyssavirus infections have a very low incidence in bat populations [18] and that the sample size was too limited, especially concerning the relevant species. there is a high prevalence for herpesviruses in different insectivorous bat species in germany (this study, [24] ). most of the previously described bat herpesviruses have been detected in low numbers in more than one bat species [24] . here, we observed a high species-specific prevalence among herpesvirusinfected bats, indicating that a certain type of european bat herpesvirus is primarily associated with a single bat species. this is supported by batghv6 and batghv7 that were again only identified in their initial hosts p. pipistrellus and p. auritus (both sedentary), respectively, underlining the typical strong speciesspecificity of mammalian herpesviruses. however, species' range overlap and close inter-species contacts in bat roosts may result in cross-species transmission and could explain the observed overcoming of the species barrier (this study batghv5, [24] ). interspecies transmission have also been discussed for other mammalian herpesviruses, i.e. bovine and equine herpesviruses (e.g. [57, 58] ). furthermore, for rna viruses (i.e. rabies virus) phylogenetic distance between different host species and overlap in geographic range have recently been demonstrated as strong predictors of host shifts and cross-species transmission in bats [59] . some of the bat species (i.e. n. noctula, p. pipistrellus, and p. nathusii) in this study appear to be more susceptible to herpesvirus infection. in n. noctula, 3 different gammaherpesviruses (batghv3, 4, 5) with significant prevalence differences were recognized. such type-specific differences in prevalence between the phylogenetically distant viruses batghv3 (13.0%) and batghv4 (33.8%) within one bat species indicates co-evolutionary virus-regulated mechanisms. parasite infestation in wildlife often occurs without clinical effects, but severe infection can reduce host fitness either in terms of survival or reproductive success [60] . most data on infection dynamics in bats came from parasite studies focusing on individual and seasonal variations in ectoparasite prevalence (e.g. [43] [44] [45] 61] ). host density, roost preference and movement pattern seem to be important factors explaining individual and speciesspecific parasite infestation rates in bats [43] [44] [45] . in european vespertilionid species, female-biased parasite loads are most likely associated with host physiology and differences in roosting behavior [42, 44] . we also found species-specific seasonal variations in ectoparasitic infestation, with n. noctula and m. daubentonii showing higher ectoparasite prevalence in spring and autumn compared to the breeding season (data not shown), which is in accordance with zahn and rupp [43] . additional findings of our parasite analyses are distinct variations in ecto-and endoparasite prevalence in relation to bat species. bats primarily roosting in trees or nest boxes were more frequently infested with ectoparasites like n. noctula (43%) and m. daubentonii (25%) compared to other species (range 5-12%) investigated in this study. high ectoparasite loads have generally been described in bats preferring enclosed roosts like burrows and cavities [61, 62] , suggesting that structural characteristics and the microclimate of roosting habitats influence ectoparasite survival and re-infection of bat hosts. this assumption is in accordance with results of pearce and o'shea [63] who found differences in ectoparasite prevalence and intensity in eptesicus fuscus in relation to environmental factors (i.e. temperature and humidity) of different roost sites. in contrast to these results, the endoparasite prevalence in european vespertilionid bats seems to be correlated with the body size of the bat species [26] . species-specific variations in diet and prey selection could possibly effect endoparasite prevalence in insectivorous bats [64] , as larger bats feed on insects of a wider size range including hard-bodied prey [65, 66] . this assumption is supported by the clear prevalence increase in subadult and adult bats compared to low endoparasite infection rates in juveniles primarily feeding on milk. a multitude of publications is restricted to pathogen presence or absence in different chiropteran species; here we demonstrate the impact of diseases and infectious agents on bats themselves. alongside to trauma-associated mortality and undefined mortality cases, disease aspects represented one third of mortality causes in 486 investigated bats of 19 european vespertilionid species. by comparing pathology and bacteriology results, we were able to detect 22 different bacterial species that were clearly associated with disease in bats. at least 12% of all bats had died due to bacterial, viral and parasitic infections. finally, we found clear seasonal and individual variations in disease prevalence and infection rates, indicating an increased susceptibility to infectious agents in female bats and juveniles during the maternity season. our data emphasize and provide the basis for disease related studies in bat species on population level to elucidate the complexity of the ecology of infectious agents and host species likewise. table s1 bacteria isolated from bats found in germany. (doc) the status of the world's land and marine mammals: diversity, threat and knowledge ecosystem services provided by bats a review of the global conservation status of bats emerging diseases in chiroptera: why bats? ecological and behavioral methods for the study of bats. 2 nd edition the isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus isolation of nipah virus from malaysian island flying-foxes fruit bats as reservoirs of ebola virus bats are natural reservoirs of sars-like coronaviruses bats as a continuing source of emerging infections in humans isolation of genetically diverse marburg viruses from egyptian fruit bats bats, emerging infectious diseases, and the rabies paradigm revisited veterinary advances in the investigation of wildlife diseases in britain causes of morbidity and mortality in british bat species and prevalence of selected zoonotic pathogens. thesis for msc in wild animal health infectious and emerging diseases of bats, and health status of bats in new zealand veterinary treatment of evening bats (vespertilionidae) in the czech republic european bat lyssaviruses: distribution, prevalence and implications for conservation epidemiology of bat rabies in germany detection and prevalence patterns of group i coronaviruses in bats identification of sars-like coronavirus in horseshoe bats (rhinolophus hipposideros) in slovenia fatal borreliosis in bat caused by relapsing fever spirochete, united kingdom yersinia species isolated from bats genetic diversity of pasteurella species isolated from european vespertilionid bats discovery of herpesviruses in bats new adenovirus in bats diseases in free-ranging bats from germany habitat preference and flight activity of bats in a city bat ecology and public health surveillance for rabies in an urbanizing region of colorado. urban ecosystem colour atlas for the diagnosis of bacterial pathogens in animals bergey's manual of systematic bacteriology. 2 nd edition generic rt-nested-pcr for detection of flaviviruses using degenerated primers and internal control followed by sequencing for specific identification hantavirus in african wood mouse generic detection of coronaviruses and differentiation at the prototype strain level by reverse transcription-pcr and nonfluorescent low-density microarray development of a real-time reverse transcriptase pcr assay for type a influenza virus and the avian h5 and h7 hemagglutinin subtypes laboratory techniques in rabies. 4 th edition world health organization laboratory techniques in rabies. 4 th edition world health organization application of monoclonal antibodies for epidemiological investigations and oral vaccination studies species determination: the role and use of the cytochrome b gene a rapid pcr-based test for species identification of two cryptic bats pipistrellus pipistrellus and p. pygmaeus and its application on museum and dropping samples information theory as an extension of the maximum likelihood principle urbanization and the ecology of wildlife diseases variation of intensity of a parasitic mite (spinturnix myoti) in relation to the reproductive cycle and immunocompetence of its bat host (myotis myotis) ectoparasite load in european vespertilionid bats relationships between the parasitic mite spinturnix andegavinus (acari: spinturnicidae) and its bat host, myotis daubentonii (chiroptera: vespertilionidae): seasonal, sex-and age-related variation in infestation and possible impact of the parasite on the host condition and roosting behavior host sex and ectoparasites choice: preference for, and higher survival on female host reproduction and nutritional stress are risk factors for hendra virus infection in little red flying foxes (pteropus scapulatus) host and viral ecology determine bat rabies seasonality and maintenance seasonal activity patterns of seven vespertilionid bat species in polish lowlands hibernation: the immune system at rest? bacterial pathogenesis bacteriologic analysis of infected dog and cat bites european bat lyssavirus transmission among cats human rabies due to lyssavirus infection of bat origin first isolation of eblv-2 in germany novel lyssavirus in natterer's bat bat rabies serological survey of herpesvirus infections in wild ruminants of france and belgium new hosts for equine herpesvirus 9 host phylogeny constrains cross-species emergence of rabies virus in bats behavioral adaptations to parasites: an ethological approach relationships between roost preferences, ectoparasite density, and grooming behaviour of neotropical bats roosting habits of bats affect their parasitism by bat flies (diptera: streblidae) ectoparasites in an urban population of big brown bats (eptesicus fuscus) in colorado when parasites become prey : ecological and epidemiological significance of eating parasites the implications of food hardness for diet in bats prey consumed by eight species of insectivorous bats from southern illinois the authors would like to thank berliner artenschutz team-bat-e.v., f. key: cord-294645-yzh8h7zo authors: freeman, david w.; noren hooten, nicole; kim, yoonseo; mode, nicolle a.; ejiogu, ngozi; zonderman, alan b.; evans, michele k. title: association between gdf15, poverty and mortality in urban middle-aged african american and white adults date: 2020-08-07 journal: plos one doi: 10.1371/journal.pone.0237059 sha: doc_id: 294645 cord_uid: yzh8h7zo mortality disparities are influenced by race and poverty. there is limited information about whether poverty influences biologic markers of mortality risk. emerging data suggests that growth differentiation factor 15 (gdf15) is associated with mortality; however, the interplay between gdf15, sociodemographic factors and mortality is not known. we sought to evaluate the interactions between gdf15 and sex, race and poverty status on mortality. serum gdf15 was measured in 1036 african american and white middle-aged men and women above and below 125% of the federal poverty status from the healthy aging in neighborhoods of diversity across the life span (handls) study. multivariable adjusted cox regression models were used to assess the association between log-transformed gdf15 (loggdf15) and 12-year mortality outcomes (all-cause, cardiovascularand cancer-specific outcomes) and interactions with sex, race and poverty status. likelihood ratio tests were used to assess significance of the interaction terms. median gdf15 was 655.2 pg/ml (iqr = 575.1). during 12.2 years of follow-up, 331 died of which 94 cardiovascularand 87 were cancer-specific deaths. one unit of increase in loggdf15 was associated with a hazard ratio for all-cause mortality, cardiovascularand cancer-specific mortality of 2.26 (95% confidence interval [ci], 1.94–2.64), 2.74 (95%ci, 2.06–3.63) and 1.41 (95%ci, 1.00–2.00), respectively. there was an interaction between loggdf15 and poverty status on all-cause mortality (p<0.05). the gdf15×poverty status interaction term improved model calibration for all-cause mortality. our study provides the first evidence that the effect of elevated gdf15 on all-cause mortality is modified by poverty status. although overall mortality rates continue to decline in the united states, recent trends document disproportionate increases in mortality and life expectancy amongst groups in the united states [1] . notably, age-adjusted death rates for non-hispanic african american men increased [2] . additionally, there were increases in the age-specific death rate for all americans younger than 65 years of age; this is particularly true for midlife whites between the ages of 25-64 [3] . low socioeconomic status remains a significant risk factor for morbidity and premature mortality [4] . specifically, african americans (aas) below poverty are particularly vulnerable to early death compared to their white counterparts despite adjustments for various lifestyle cofactors [5, 6] . disentangling the effects of race and poverty on mortality and other negative health related outcomes are difficult given the low median household income and high poverty rate among aas [7] . given this disparity, it is important and useful to evaluate relevant biological markers of mortality in the context of populations at risk for premature mortality in hopes of improving health outcomes for these individuals. mortality is linked to numerous behavioral or physiologic risk factors including tobacco use, alcohol consumption, obesity, physical inactivity, elevated blood pressure and hyperlipidemia [8] . in addition, other biological and clinical measures have been associated with or predict risk of mortality including interleukin-6 [9] , c-reactive protein [9] , albumin, alkaline phosphatase, estimated glomerular filtration rate (egfr), blood urea nitrogen (bun), mean cell volume (mcv) [10] and red cell distribution width (rdw) [11] . several groups have shown recently that growth differentiation factor 15 (gdf15), also known as mic-1, is an independent predictor of all-cause mortality in white elderly (>70 years) individuals [12] [13] [14] [15] , whites with coronary heart disease [16] and also in white middle-aged individuals [17] . data from the dallas heart study suggest that gdf15 may also be associated with mortality in a multiethnic cohort [18] . circulating gdf15 levels are also a biomarker of cardiovascular disease (cvd) and its progression [19] . gdf15 is a multifunctional secreted protein expressed at low levels in most tissues and upregulated in response to tissue stress or injury [20] . gdf15 is an emerging biomarker and a stress response cytokine produced in response to stressors, cellular and systemic injury and inflammation. data suggest that it is a pleiotropic signaling molecule that has both beneficial or advantageous effects on cellular metabolism but may in certain biologic contexts have deleterious consequences as well [21] . under physiological conditions, gdf15 is produced in small amounts by the liver, lung and kidney but may be expressed in large amounts following exposure to external and internal stressors [20] . its production is regulated by various stress responses, senescence and inflammation-related genes such as crp, which regulates gdf15 in a p53 dependent manner [22] , and ire1alpha following endoplasmic reticulum stress [23] . however, these actions of gdf15, which are transduced through the glial-cell-derived neurotrophic factor (gdnf) receptor α-like (gfral) receptor and the tyrosine kinase coreceptor ret [24] [25] [26] [27] , are time and context dependent i.e. organ and system specific. accumulating evidence, largely from animal models, suggests that during early stages of chronic inflammation, cancer and metabolic disorders, the effects of gdf15 are aimed at limiting disease process. however, in advanced cancer, heart failure or renal disease, gdf15 causes the anorexiacachexia syndrome and also may promote metastases [20] . furthermore, recent work indicates that gdf15 plays an important role in energy homeostasis and body-weight regulation [24] [25] [26] [27] . most previous studies on the association between gdf15 and mortality risk (overall and cause-specific) were conducted in highly selected population groups such as randomized controlled trials, hospitalized patients, elderly individuals and critically ill patients who are not representative of the general population. apart from the study by rohatgi et al. [18] , investigations of gdf15 and mortality outcomes in population-based studies were conducted in individuals of european-ancestry with socioeconomically homogeneous individuals. hence the role of gdf15 on the risk of mortality in racially and socioeconomically diverse populations who are at-risk of health disparities and premature mortality has been unclear. at present, it is not known whether gdf15 interacts with any socioeconomic and demographic factors or other biomarkers of mortality. given the persistence of health disparities and disproportionate burden of disease and mortality borne by aas evidenced by the ongoing covid-19 pandemic [28, 29] , it is critical that we examine the utility of mortality and disease biomarkers to identify populations at heightened risk. it is particularly important to examine the potential influence or interaction of poverty, a dominant social determinant of health that drives differential health outcomes. the objectives of the present study were to assess the association between serum gdf15 and all-cause and cause-specific mortality, and to identify interactions between gdf15 and sex, race and poverty status in a large cohort of community-based middle-aged adults recruited from baltimore, maryland. the healthy aging in neighborhoods of diversity across the life span (handls) study of the national institute on aging intramural research program, national institutes of health (nih) is a prospective, community-based longitudinal cohort study based in baltimore, maryland. participants were recruited based on a factorial cross design of sex, race, 5-year age group and poverty status, defined as above or below the 125% of the 2004 us federal poverty guidelines (https://handls.nih.gov/) [30] . a total of 3720 self-identified african american and white participants aged 30-64 who resided in baltimore, md were recruited at baseline. handls has been approved by the institutional review board of the national institute of environmental health sciences, nih. all participants provided written informed consent. mortality was ascertained by matching with the national death index (ndi), national center for health statistics, for all participants from enrollment (2004-2009) through december 31, 2016. ndi data included date and primary cause of death (international classification of diseases 10 th edition). there were 331 deaths (all-causes) after 12.2 years follow-up (median 9.2 years). cvd-specific death was defined as icd10 codes i00-i99 and cancer-specific death (malignant neoplasms) was defined as c00-c96 (www.icd10data.com). power was calculated using the ssizeepi.default function in the powersurvepi r package. sample size calculation revealed, after accounting for five percent assay failure rate, a total of 1037 samples (706 controls and 331 deaths) were needed to detect a hazard ratio of at least 1.75 for all-cause mortality associated with elevated gdf15 at alpha value of 0.05 and a power of 80%. the effect size to estimate the sample size was taken from hagstrom et al. [16] . blood samples were collected at study enrollment (2004) (2005) (2006) (2007) (2008) (2009) in vials with no additives, centrifuged and serum was aliquoted and immediately frozen at -80˚c until use. serum gdf15 (pg/ml) were quantified using the quantikine elisa kit (r&d systems, cat#sgd150, minneapolis, mn) according to the manufacturer's instructions. serum was diluted 1:4 in calibrator diluent rd5-20 and 50 μl was used for the assay. three inter-assay and within assay controls consisting of serum pooled from 6 individuals were present on each plate and an internal standard on each plate was used to calculate gdf15 concentration. inter-assay variability was 9.95% and within assay variability was 4.78%. assays were performed blind. gdf15 level could not be measured in one participant leaving 1036 samples (331 dead and 705 alive) for the current analysis. baseline data were collected using a structured medical history interview and physical examination. data collected included cigarette smoking, body mass index (bmi), waist circumference (wc), waist-hip ratio (whr), diagnoses of hypertension and diabetes mellitus and estimated glomerular filtration rate [31] . biochemical measures that were previously linked with mortality risk were assayed by quest diagnostics (chantilly, va). these variables were low density lipoprotein-cholesterol (ldl), high density lipoprotein-cholesterol (hdl), triglycerides, high-sensitivity c-reactive protein (hscrp), red cell distribution width (rdw), mean corpuscular volume (mcv), albumin, albumin-globulin ratio (agr), alkaline phosphatase (alp) and blood urea nitrogen (bun). ldl concentration was estimated based on total cholesterol, hdl and triglyceride levels using the friedewald equation [32] . continuous variables were plotted using histograms, and skewness and excess kurtosis were calculated to assess their distribution. skewed distribution was defined as skewness � |1| and excess kurtosis � 3. variables with skewed distribution (gdf15, hscrp, hdl, alp and bun) were natural-logarithm transformed to approximate a normal distribution. categorical variables were sex, race (aas and whites), poverty status (above and below the 125% federal poverty line), current cigarette smoking (yes, no), hypertension diagnosis (yes, no) and diabetes mellitus diagnosis (yes, no). unadjusted and multivariable adjusted cox proportional hazards regression models were used to estimate the hazard ratios and 95% confidence intervals for all-cause mortality, cvdand cancer-specific mortality for each unit increase in log-transformed gdf15 (loggdf15). age at study entrance and exit (death or censored date december 31, 2016) in decimal years were used as the measurement of follow-up time [33] . the full cox model was adjusted for sex, race, poverty status and an interaction term between race and poverty status. we included the interaction term between race and poverty status into the full cox model because it was associated with all-cause mortality during initial model selection. we performed supplemental analysis by adding the following variables into the fully adjusted models: current cigarette smoking, hypertension diagnosis, diabetes mellitus diagnosis, bmi, wc, whr, ldl, hdl, triglycerides, hscrp, rdw, mcv, albumin, agr, alp, egfr and bun. these potentially confounding variables were added to the model to assess if they would substantially change the main findings of the paper. we next assessed the interaction between loggdf15 and socioeconomic and demographic factors: sex, race and poverty status. using forward model selection, we built cox regression models for the three outcomes by including possible interaction terms between loggdf15 and sex, race and poverty status in the full cox model described above. significance of an interaction term between loggdf15 and any of the demographic variables was tested by comparing the models with and without the interaction term using a likelihood ratio test and significance value of 0.05. the proportional hazards assumption for all unadjusted and multivariable adjusted cox proportional hazards regression models were explored graphically with the use of log (-log (survival)) plots and formally evaluated using the scaled schoenfeld residuals chi-squared test resulting in global p values � 0.34 indicating the proportional hazards assumption were not violated. we used calibration plots and the modified nam-d'agostino goodness-of-fit test to evaluate the contributions of loggdf15 and the interaction term containing loggdf15 to model performance for prediction of the three outcomes [34] . this study conforms to strengthening the reporting of observational studies in epidemiology-molecular epidemiology (strobe-me) reporting guidelines for molecular epidemiology studies (www.equator-network.org). all data management and statistical data analyses were conducted with r v3.5.1 (www.r-project.org). description of the study participants are shown in table 1 . the age of study participants ranged from 30-64 years with mean age (standard deviation [sd]) of 49.6 (9.2). the proportion of women, aas, above poverty status was 53.2%, 42.8% and 56.5%, respectively. during 12.2 years of follow-up (median = 9.2 years) with 8944.7 person-years, there were 331 deaths due to all-causes. the leading causes of death were cvd 94 (28.4%) followed by cancer 87 (26.3%). the median gdf15 level was 655.2 pg/ml (iqr = 575.1). serum loggdf15 was moderately correlated with age (r = 0.45, p<0.001) and egfr (r = -0.50, p<0.001) and weakly correlated with rdw (r = 0.21, p = <0.001) and bmi (r = -0.11, p<0.001) but not with hscrp (r = 0.04, p = 0.20) or wc (r = -0.06, p = 0.09). correlation coefficients between loggdf15 and other clinical and biochemical measures previously linked with organ-system functions and mortality risk are depicted in s1 fig. the distribution of all-cause mortality events by race, poverty status and gdf15 quartiles is shown in s1 table. unadjusted hazard ratio (hr) for all-cause mortality associated one unit of increase in loggdf15 was 2.41 (95% confidence interval [ci], 2.08-2.79). in the full cox model adjusted for sex, race, poverty status and an interaction term between race×poverty status, the hr for all-cause mortality was 2.26 (95%ci, 1.94-2.64) ( table 2 ). the unadjusted and adjusted hr for cvd-specific mortality were 2.81 (95%ci, 2.13-3.70) and 2.74 (95%ci, 2.06-3.63), respectively. similarly, loggdf15 was associated with cancer-specific mortality (all cancer types): we performed supplemental analysis by further adjusting the full cox model for the following variables: current cigarette smoking, hypertension diagnosis, diabetes mellitus diagnosis, bmi, wc, whr, ldl, hdl, triglycerides, hscrp, rdw, mcv, albumin, agr, alp, egfr and bun. the supplemental analyses did not materially change the association between loggdf15 and all-cause mortality risk and cvd-specific mortality risk (s2 table) . however, the association between loggdf15 and cancer-specific mortality become non-significant. we assessed possible interactions between loggdf15 and sex, race and poverty status. there was a significant two-way interaction between loggdf15 and poverty status on all-cause mortality but not cvd-and cancer-specific mortality ( table 3 ). the increased risk of all-cause mortality associated with elevated loggdf15 was more pronounced in individuals above poverty level. in analyses that stratified participants by poverty status (above and below) and adjusted for sex and race, the hr for all-cause mortality in individuals above poverty was 2.73 (95%ci, 2.14-3.48) and for in individuals below poverty was 2.00 (95%ci, 1.64-2.45) (fig 1) . model calibration test indicated loggdf15 and the interaction term loggdf15×poverty status improved outcome prediction of both all-cause and cvd-specific mortality (goodness-of-fit test p-values � 0.42) (s2 fig). we found no evidence of interaction between loggdf15 and sex or loggdf15 and race on any of the three outcomes. in a community-dwelling cohort of younger urban adults (mean age 49.6) with diverse racial and socioeconomic status, we found that elevated serum gdf15 level was strongly associated with all-cause mortality, cvd-and cancer-specific mortality risk. we also found an interaction between gdf15 and poverty status on all-cause mortality such that the association was more pronounced in individuals above poverty status than those below poverty status. our findings of increased risk of mortality due to all-causes and cvd and elevated gdf15 levels in a diverse cohort are consistent with results of previous studies conducted in apparently healthy, community-dwelling adults [12-15, 17, 18] . in three cohorts of communitydwelling elderly white men and women (mean age ranging from 68-78.6 years) from sweden with baseline median gdf15 from 934.9 to 1393.0 pg/ml [13, 15] , one unit increase in loggdf15 was associated with all-cause mortality with hrs ranging from 2.20 to 4.00 independent of traditional and established markers of mortality. comparable risk estimates of cvd-specific mortality were also reported by these studies. in another swedish male-only elderly cohort (mean age = 71.0 years) that had baseline median gdf15 of 1494.0 pg/ml, one sd increase in loggdf15 was associated with a 48% increase in all-cause mortality risk [14] . in our cohort of socioeconomically diverse aa and white men and women who were markedly younger than the swedish cohort (mean age: 71.0 versus 49.6 years), one sd increase in loggdf15 conferred a 77% increased risk of all-cause mortality (95%ci, 1.59-1.97). in three population-based cohorts from the u.s.a., higher gdf15 levels were associated with increased risk of all-cause and cvd-specific mortality [12, 17, 18] . our findings are also broadly comparable with other studies of gdf15 and mortality risk conducted in clinical trial participants, hospitalized patients and individuals with various chronic diseases. the comparability persists although these cohorts included highly select patient populations with advanced disease or critically ill patients who conceivably have high concentration of circulating gdf15 but are not representative of a general population like the handls cohort [19, 35] . collectively, these associations of gdf15 with mortality risk under diverse pathophysiological scenarios and study designs suggests that gdf15, a stress response cytokine, is a powerful biomarker that captures the body's compensatory response reserve against persistent exposure to stressors and subsequent multi-organ and system failures due to various different diseases and co-morbidities, rather than just a reflection of a single disease burden or an isolated altered pathophysiological process. we note that the median gdf15 concentration in the handls cohort was significantly lower (655.2 pg/ml), almost half as much as previously reported in most studies of mortality risk in european-ancestry community-dwelling adults [12] [13] [14] [15] 17] . however, in the dallas heart study, comprised of multiethnic and younger participants, the median gdf15 value was 670 pg/ml, comparable to the gdf15 value in handls [18] . this may suggest that even lower levels of gdf15 at a relatively younger age and many years before the onset of overt disease predict adverse health outcomes and could be a harbinger of mortality in the general population earlier in the lifespan. given that midlife mortality rates (ages 25-64) have increased across racial/ethnic groups in the u.s.a. compounding the differential death rates already present among aas, native americans and alaska natives, a useful biomarker of mortality risk among those at midlife could be a useful tool [3] . our work provides the first evidence of interaction between gdf15 and poverty status on mortality risk. compared to individuals below poverty, individuals above poverty had higher risk estimates (73%) of all-cause mortality. there are no previous studies that reported interactions between gdf15 and the social determinants of health on mortality risk in any context or study design. previous population-based studies of mortality risk and gdf15 were conducted in largely homogenous population groups [12] [13] [14] [15] 17] and neither measures of socioeconomic status (household income, education status, employment etc.) nor interactions were reported in these studies [12-15, 17, 18] . there is some evidence in animal models that gdf15 plays a role in lifespan. two different transgenic mouse models that overexpress gdf15 showed increased lifespan, decreased adipose tissue weight, enhanced oxidative metabolism and improved insulin sensitivity, especially when challenged with high-fat diet [36] . similarly, overexpression of gdf15 was shown to promote fatty acid oxidation and ketone body production and augmented the effects of prolonged fasting [23] . therefore, it is plausible that gdf15 may reflect compensatory and adaptive response capacity and the ability to successfully mount an integrated organismal response to curb the effects of cellular and systemic stressors. it is possible that gdf15 is part of a cellular stress stimuli feedback loop that may protect from stress or inflammation and repair injury in selected biological settings while in other conditions promotes injury and disease [20] . our observed interaction between gdf15 and poverty status on mortality risk is a bit counterintuitive. the expectation would be that mortality among those below poverty would be most detrimentally influenced by gdf15. this could be explained by differences between above and below poverty status individuals in adaptive response reserve and the ability to adequately respond to a multitude of stressors through increased gdf15 and possible additional molecular mechanisms. indeed, several studies show that compared to high income individuals, those living in poverty and economic hardship across the life course, experience biological weathering evidenced by short leukocyte telomere length, the premature aging phenotype and accelerated biological aging [37] [38] [39] . this suggests that those living in poverty may be in a decompensated pathophysiological state lacking the homeostatic functional reserve necessary to respond to cellular stress and injury. this blunted ability to respond may preclude benefit from the protective effects of elevated gdf15 level. however, our finding that the influence of gdf15 on mortality is stronger among higher income individuals supports the previously described 'diminishing returns hypothesis' applied to various health outcomes among aas including infant birthweight, depression, and self-rated health [40, 41] . the benefits of numerous socioeconomic resources are different for different races and perhaps the protective effects of these socioeconomic resources may also be related to neighborhood. in a recent study, race and living in an urban environment altered the protective effect of education on mortality among aas [42]. our finding is not a perfect fit since the central dogma of the diminishing returns hypothesis holds that aas and, in some studies, hispanics do not reap the expected health benefits of higher economic status perhaps related to the higher cost of coping with discrimination and race-based unequal treatment. in previous studies, whites have beneficial health outcomes from upward social mobility because of decrements in acute and chronic discrimination [43] . our findings suggest that in our cohort diminishing returns holds true equally for younger middle-aged urban whites as well. in the context of the recently reported increasing mortality and declining life expectancy among middle-aged whites, it is possible that young middle-aged whites who are just slightly above the 125% poverty line may also be subject to diminishing returns from the protective benefits of socioeconomic resources and that this correlates with the observed increases in mortality [3] . this could result from changes in economic or social factors that influence health outcomes for whites who are at the lower end of the income scale when compared to other whites in the baltimore city. within race income inequality has increased substantially nationwide among all u.s. racial groups. while it is highest among u.s. asians, since 1970 income inequality among whites has risen by 24%; in 2016 whites at the 90 th percentile income distribution made 7.8 times the income of whites at the 10 th percentile income distribution [44] . baltimore city has the highest income inequality gap in maryland and this may not only influence health outcomes among aas but also whites [45] . in our cohort, two-thirds of those living below poverty status had an annual household income below $30 000. in baltimore the median household income is $44 262: for whites $62 752 and for aas $33 801 [46] . in handls, those living above poverty status were more affluent, but only 25% had an annual household income over $50 000. future studies are required to understand the exact biology behind the interrelationship between gdf15 and stressful life circumstances, as in living in poverty, on adverse health outcomes. it is also important to understand how temporal changes in gdf15 levels vis-à-vis socioeconomic measures relate with mortality risk and aging-related diseases in disadvantaged communities and to assess interaction between changes in gdf15 and the social determinants of health. in a study of elderly individuals of european-ancestry, serum gdf15 levels increased by 11% after five years of follow-up and this rate of change was associated with overall survival [13] . this study highlights the importance of conducting a longitudinal assessment in middleaged individuals to assess rate of change and survival. the limitations of our study are (1) smaller number of mortality events among whites below poverty status precluded three-way interaction analysis between race, poverty and gdf15; (2) similarly smaller number of individuals with specific causes of death limited further disease-specific survival analysis; (3) other important factors such as mental health diagnoses that might be a confounding variable influencing poverty, gdf15, and mortality were not assessed. our study has several strengths including large sample size, contemporary cohort that captures recent mortality trends and their cause and is comprised of younger, socioeconomically and racially diverse adults. previous gdf15 and mortality studies were conducted in predominantly elderly men and of homogenous european-ancestry populations [12] [13] [14] [15] 17] ; of note here is that 4 out of the 7 community-dwelling cohorts were from a single scandinavian country [13] [14] [15] . while we used poverty thresholds, which is based on annual household income used by the u.s. federal government, application of a more nuanced measures of poverty and socioeconomic stress when evaluating the relationship between gdf15, socioeconomic hardship and mortality risk could enhance understanding. in conclusion, elevated gdf15 levels are associated with increased risk of mortality, and that this association was modified by poverty status. this is the first time that a social determinant of health has been shown to enhance the risk of a biomarker of mortality. while gdf15 has independent prognostic value as a single mortality marker, the true value of gdf15 might be enhanced by coupling it with other routinely obtained clinical markers of disease and mortality including hs-crp, rdw and the social determinant of health poverty as well as assessment of mental health status to more accurately define those at highest risk for negative health outcomes. future studies are needed to extend our findings in other population groups. further studies are also required to explore the potential of targeting gdf15 to improve mortality and other 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differentiation factor 15 as a biomarker in cardiovascular disease the mic-1/gdf15-gfral pathway in energy homeostasis: implications for obesity, cachexia, and other associated diseases uniting gdf15 and gfral: therapeutic opportunities in obesity and beyond crp stimulates gdf15 expression in endothelial cells through p53 fasting exacerbates hepatic growth differentiation factor 15 to promote fatty acid beta-oxidation and ketogenesis via activating xbp1 signaling in liver the metabolic effects of gdf15 are mediated by the orphan receptor gfral non-homeostatic body weight regulation through a brainstem-restricted receptor for gdf15 gfral is the receptor for gdf15 and is required for the anti-obesity effects of the ligand gfral is the receptor for gdf15 and the ligand promotes weight loss in mice and nonhuman primates cdc. hospitalization rates and characteristics of patients hospitalized with laboratory-confirmed coronavirus disease 2019-covid-net, 14 states healthy aging in neighborhoods of diversity across the life span (handls): overcoming barriers to implementing a longitudinal, epidemiologic, urban study of health, race, and socioeconomic status estimating glomerular filtration rate from serum creatinine and cystatin c estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge choice of time-scale in cox's model analysis of epidemiologic cohort data: a simulation study tests of calibration and goodness-of-fit in the survival setting plasma inflammatory cytokines and survival of pancreatic cancer patients hnag-1 increases lifespan by regulating energy metabolism and insulin/igf-1/mtor signaling we would like to thank all study participants and the handls medical staff for the excellent medical evaluations of all participants. key: cord-297287-0i4nc353 authors: braun, benjamin; taraktaş, başak; beckage, brian; molofsky, jane title: simulating phase transitions and control measures for network epidemics caused by infections with presymptomatic, asymptomatic, and symptomatic stages date: 2020-09-10 journal: plos one doi: 10.1371/journal.pone.0238412 sha: doc_id: 297287 cord_uid: 0i4nc353 we investigate phase transitions associated with three control methods for epidemics on small world networks. motivated by the behavior of sars-cov-2, we construct a theoretical sir model of a virus that exhibits presymptomatic, asymptomatic, and symptomatic stages in two possible pathways. using agent-based simulations on small world networks, we observe phase transitions for epidemic spread related to: 1) global social distancing with a fixed probability of adherence. 2) individually initiated social isolation when a threshold number of contacts are infected. 3) viral shedding rate. the primary driver of total number of infections is the viral shedding rate, with probability of social distancing being the next critical factor. individually initiated social isolation was effective when initiated in response to a single infected contact. for each of these control measures, the total number of infections exhibits a sharp phase transition as the strength of the measure is varied. the sars-cov-2 virus that has spread throughout the globe has created societal disruption and had a massive impact on global health [1] . with no known treatment, public policy and human behavior are currently the only tools that are available to mitigate the spread [2] . a fundamental characteristic of sars-cov-2 is that after an individual is exposed, that individual passes through an extended presymptomatic stage followed by either an asymptomatic or symptomatic stage [3] . our goal in this work is to construct a theoretical network disease model with these qualities and investigate phase transitions associated with three types of control measures. while many models related to sars-cov-2 are designed to be forecasting tools, our study is intended as a contribution to the theoretical literature regarding qualitative aspects of control measures for viruses with these pathways of disease progression. in the specific case of sars-cov-2, one control measure that has been used is governmentmandated social distancing. different countries, and different states within the us, have implemented different approaches to this [2, 3] . while most government plans include some social distancing, questions have arisen as to the efficacy of social distancing, how long social distancing should last and to what extent it is needed [4] . a second control method involves individually-determined changes to social behavior, which work in concert with mandated social distancing to mitigate viral transmission [5, 6] . individuals who live with an infected individual are being asked or required to quarantine for 14 days prior to interacting in the larger society [7] . one question is whether these individual responses of behavioral modification are sufficient to moderate epidemic spread and whether there are additive or non-additive effects when implemented with top-down government policy on social distancing [5] . a third type of control measure involves use of personal protective equipment to reduce the rate of viral transmission. for example, mask usage has been found to be effective in this regard for sars-cov-2 [8] [9] [10] . these real-world aspects of sars-cov-2 highlight the need for a more thorough understanding of the general behavior of viruses exhibiting multiple progressions of disease development. with this as motivation, we develop a theoretical model in which we investigate how three types of control measures are associated with sharp phase transitions for the total number of infected individuals. while modeling contacts can be done in mean-field, statistical, and metapopulation sir models [1, [11] [12] [13] [14] , we use an agent-based model (abm) on watts-strogatz small world networks [15] [16] [17] . small world networks have connectedness properties that are found in real-world social networks and have been previously considered in epidemiological contexts [18, 19] . the first control measure in our abm is social distancing imposed on the network at a global scale. our model encodes this global social distancing as complete isolation of an agent from other agents. the likelihood of social distancing is applied uniformly to all agents. the second control measure arises when agents have social connections that are infected and symptomatic [2, 8] . in this case, agents temporarily isolate from their contacts in the network if they are in contact with a sufficient number of symptomatic agents. the third type of control measure is to alter the rate of viral spread, which reflects behavior such as use of personal protective equipment, e.g., masks [8, 10] . we examine how each of these measures alone and in concert with each other influence the viral outbreak. for each of these control measures, we ask the following questions: 1. how does varying the strength of the control measure impact the total number of infections in an epidemic? 2. if a control measure impacts the total number of infections, is there a phase transition associated with changes in strength of that control measure? 3. how do these three control measures interact regarding their impact on total number of infections? we develop an sir, network-based, agent-based model where agents pass through various infection states (fig 1) . agents pass through a presymptomatic infection state followed by either an asymptomatic infected stage or a symptomatic infected stage. in our model, each agent carries an individual pathogen level that changes in response to contact with infected agents. initially, this level is set to 0 pathogen units for susceptible agents. at each time step (conceived as a day), if a susceptible agent has no infected contacts then their pathogen level does not change. for each day that a susceptible agent has one or more infected neighbors, their pathogen level increases by a fixed fraction of the pathogen levels of their infected neighbors. there is a global infection threshold that applies to all agents, which we fix at 25 pathogen units; in other words, the day after the pathogen level for an individual agent exceeds 25 units, that agent enters the presymptomatic infected state. once an agent enters the infected state, their pathogen level stays constant until they have reached the resistant/removed state, at which point it is reset to 0 units. model runs are initiated with a small number of infected agents, whose pathogen levels are initially set at 35 pathogen units, and the remainder of the agents are initially deemed susceptible. these initial and threshold values for the pathogen levels in our model are not based on real-world data, but rather were selected for simplicity to investigate general behavior of phase transitions under a mechanism of viral shedding with individualized pathogen levels. because our model does not use a transmission probability for each contact, but rather a viral shedding rate where each individual agent has varying levels of pathogen load, this model is well-suited to abm simulations and less amenable to ode-based deterministic analysis. once the individual pathogen level for an agent exceeds 25 units, that agent enters a presymptomatic infection stage, followed by either a symptomatic or asymptomatic stage (fig 1) . the length of the presymptomatic stage is the same for all agents, and can be set to last one or more days. the lengths of the two possible main infection stages are set independently from each other, but are the same for all agents. following the main infection stage, the agent is either resistant or removed. in addition to the infection stages, each agent is in one of two daily behavior states: socially distanced or not socially distanced. the behavior state is reset each day. if an agent is not socially distanced on a given day, then that agent can interact with any neighboring agent. if an agent is socially distanced, the agent does not interact with any neighboring agents; in our theoretical model, social distancing is equivalent to self-quarantine. agents socially distance in a given day for one of two reasons. a global social distance probability is set, which determines the chance that an agent will socially distance on a given day. a local social distance threshold is set, and this value dictates individual responses to infected symptomatic neighbors. if the number of infected symptomatic neighbors of an agent equals or exceeds this threshold, the agent will social distance independently of the global parameter. this local threshold is the same for all agents. we implement our agent-based model on watts-strogatz (ws) networks. the ws networks can simultaneously demonstrate both high clustering and short average path length, and thus serve as effective approximations of social networks that are neither completely random nor regular [15] . high clustering and short average path length allow for local interactions and more distant interactions to be incorporated [16, 17, 20] , which are properties often found in real-world networks. the ws small world network in our model is characterized by three parameters: number of nodes n, average node degree k and rewiring probability. the rewiring parameter is used to determine the likelihood of rewiring each edge starting from a regular ring lattice. a rewiring parameter of 0 preserves the original ring lattice; a rewiring parameter of 1 simulates a random network. we fix the number of nodes n = 500 and the average degree k = 20, which allows ln(n) � k � n. we then vary the rewiring probability among the values {0.05, 0.10, 0.25, 0.50}. for each of our four rewiring probabilities we construct 10 networks on which to run simulations. we define our three model parameters as follows: 1. social distance probability: the probability that an agent is socially distancing on any given day. 2. social distance threshold: the minimum number of infected symptomatic contacts required to cause an agent to social distance for that day. 3. viral shedding: the fraction of individual pathogen level that an infected agent passes to each of its contacts. we ran two sets of simulations over different parameter spaces. our primary simulation ran through ten networks for each set of parameters given in table 1 . based on the results of this primary simulation, we ran a secondary set of simulations over the refined parameter space given in table 2 to provide a more detailed analysis of the phase transition behavior observed in the primary simulations. the parameters for the secondary simulation were selected based on our analysis of the primary data using regression trees to identify critical variables and on the observed ranges where phase transitions were observed. we used a regression tree to partition the variation in final number of infected nodes across model parameters and runs in our primary simulation [21] . reductions in viral shedding were associated with the primary partition in the regression tree in fig 2. viral shedding below 15% compared to a value of 25% were associated with a mean number of infections of 46 out of 500 agents. reduced viral shedding with social distancing probability over 25% led to overall infection of approximately 2% of the agents. if the overall viral shedding is reduced dramatically to 5%, even without additional social distancing of any type, less than 1% of the population becomes infected. achieving low levels of infections in populations without reducing viral shedding requires significantly higher levels of global social distancing, where each agent has at least a 75% chance of social distancing each day; this results in an approximately 1% infection rate among agents. if each agent has less than a 75% chance of social distancing each day, the total infection rates for the populations are much higher; these range from a low of 21% (if individuals phase transitions and control measures for network epidemics self-isolate in response to one infected social contact) all the way up to 97% with low levels of any type of social distancing. thus, with a higher level of viral shedding, it becomes important to have agents self-isolate when a contact becomes symptomatic. even if this occurs, the infection rate in the population is an order of magnitude higher (10% vs. 1%) than if the viral shedding is reduced. failure to achieve this strict social distancing in response to an infected social contact results in a widespread outbreak with approximately 62% of the agents infected. because our goal is to understand the behavior of phase transitions regarding total number of infections in our model, we conducted secondary simulations on a refined parameter space based on the results of our regression tree analysis. in these simulations, we observed sharp phase transitions in the total number of infections as a function of all three control methods. these transitions are shown in figs 3, 4 and 5. in these figures, the maximum number of possible infections is 500, as there are 500 nodes in our networks. in fig 3, a phase transition exists between viral shedding of 5% and 10%, across all levels of social distance thresholds and social distance probabilities. in fig 4, a phase transition exists at a social distance threshold of 1, across all levels of social distance probabilities and viral shedding. if the social distance threshold parameter is 2 or more, then it is possible to have epidemics that infect the entire population. in fig 5, a phase transition exists around a social distance probability of 73-74%, across all levels of social distance threshold and viral shedding. if the social distance probability is 74% or more, then our simulations end with a small number of infected agents. phase transitions and control measures for network epidemics given the regression tree analysis of our primary simulations, it is clear that viral shedding and social distance probability play key roles. in our secondary simulations over a refined parameter space, this becomes more clear. in fig 6, we observe additional confirmation of the regression tree findings that the main driver of total number of infections is the viral shedding rate, with social distance probability being the next critical factor. specifically, simulations with large total infections cluster to the upper left of the plot, where viral shedding rates are higher and social distancing is enacted by approximately 60% of agents. there is also a clear interaction between the social distance probability and viral shedding parameters and the resulting number of infected agents and the length of the epidemic. these interactions are shown in figs 7 and 8. in fig 7, there is clustering of long epidemics when the probability is near 60% and the viral shedding rate is high. as the social distance probability increases to 80% and the viral shedding rate decreases, there is a phase transition where simulations result in outbreaks of short duration. in fig 8, most infections result in either a limited outbreak (less than 125 out of 500 agents) or almost all agents infected. as the social distance probability is increased from 60% to 80%, the length of the epidemics increase while remaining limited in total number of infections before sharply transitioning to a high number of infections during a return to short epidemic lengths. mathematical modeling can provide tools to better understand epidemic dynamics and can vary from purely theoretical to more data driven and predictive [22] . while a simple model such as this one should not be used to make policy recommendations, it can provide a framework for empirical investigation and specific hypothesis testing related to social networks of smaller size exhibiting small world characteristics, such as those seen in college settings [23] . here we use our theoretical model to investigate how different control methods impact the total number of infections in an epidemic on a network caused by a virus with a presymptomatic stage and both asymptomatic and symptomatic pathways. we specifically examine three main control measures that can be taken to reduce epidemic spread: 1) global social distancing with a fixed probability of adherence. 2) individually initiated social isolation when a threshold number of contacts are infected. 3) reduction of viral shedding. we observe sharp phase transitions in the total number of infected agents as the strength of each of these control measures are varied. to examine the full potential for global social distancing, we consider a wide range of possible scenarios varying from no social distancing to strong adherence to social distancing (90% of agents). when considering the relationship between our theoretical model and real-world contexts, the two extreme scenarios are easy to envision (zero social distancing is business as usual and 90% is all but non-essential businesses closed), while more moderate social distancing scenarios are harder to translate into direct societal actions. nevertheless, we observe in our small-world models a clear phase transition associated with global social distancing. in general, a global social distancing probability below 65% results in a wide-spread epidemic, while a global social distancing probability above 75% limits the epidemic to a dramatically lower number of total infections. we also found that social distance probabilities that approached the threshold from below resulted in prolonged epidemics while with low overall infection rates. for our secondary simulations over a refined parameter space, in the absence of other control measures we observe that there is a phase transition for total infections that occurs as the percentage of agents socially distancing changes from 73% to 74%. individual behavior taken during a pandemic can greatly affect the dynamics of disease spread. for example, for sars-cov-2, the most commonly recommended guideline after contact with an infected individual is 14 days of self-isolation to avoid exposing other individuals [5, 7, 24] . however, despite these official guidelines, self-isolation following exposure requires that infected individuals inform their contacts and that exposed individuals voluntarily comply. thus, from a theoretical perspective it is important to understand how different self-isolation behaviors following contact with an infected agent impact epidemic spread. in our model, we consider self-imposed social distancing as highly responsive to an agent's short-term perceptions regarding infection risks within their community. thus, self-imposed social distancing/isolation occurs only on the days when the agent has sufficiently many symptomatic contacts in the network. interestingly, for self-isolation to significantly decrease the total number of infections in our model, an extreme level of responsiveness was needed by the agents involved; in our model, it was necessary for self-isolation to occur following exposure to only one infected agent. if self-isolation occurred only after contact with two or more symptomatic agents on the same day, the effect on disease spread was minimal. our findings also support the well-known fact that real-world contact tracing following an individual's positive test is critical for limiting the spread of the infection [4] . an important difference between our theoretical model and viruses such as sars-cov-2 [13, 25] is that, in the real world, individuals who have come in contact with an infected individual are not aware of their exposure. our theoretical reduction of viral shedding is motivated by behaviors such as mask wearing or other use of personal protective equipment [10] . while in real-world contexts individual responsiveness to recommended government actions are highly variable [24] , a decrease in viral shedding rate can be achieved through use of protective equipment [9] . when the viral shedding in our model was set at a high shedding rate of 25%, global social distancing was required to be greater than 80% to control the outbreak, resulting in an approximately 1% infection rate in the population. other less stringent social distancing conditions result in a viral infection rate between 25% and 97.5%. with a moderate rate of viral shedding, the social distance threshold at which someone decides to self-isolate after coming into contact with an infected individual becomes much more important. in our model, if the social distance threshold is set to 1 (agents self-isolate after coming into contact with at least one infected agent), then the final total infection rate in the population is approximately 12%. however, if the behaviorally induced social distancing does not take place or takes place at a higher threshold, then the total number of infections is much larger with the overall infection rate in the population approximately 62%. if the viral shedding rate is very low, then the epidemic does not spread and a low total number of infections is observed. thus, there is a sharp phase transition as the viral shedding rate moves from 5% to 10%. an important observation regarding these phase transitions is the relatively extreme values at which they occur, e.g., a high social distancing probability, a low social distancing threshold, and a low viral shedding rate. these values are very high and low both within the context of our model and of real-world epidemics that motivate our model. it would be of interest to investigate whether or not, given an arbitrary set of values, a specific network and selection of parameters could be found for which phase transitions occur near these values. alternatively, if no such network and choice of parameters exist, it would be of interest if a more rigorous theoretical description could be given of the mechanism preventing this occurrence. we develop an agent-based model of epidemic spread on watts-strogatz small world networks, where infected agents pass through a presymptomatic stage followed by either an asymptomatic or symptomatic stage. we consider the impact of three control measures on the total number of infected agents, with regard to both phase transitions and efficacy. the three control methods we consider all generate sharp phase transitions in the total number of infections as the strength of the method varies. social distancing controls in this model exhibit a phase transition regarding total number of infections, either when imposed globally or when based on individual response to infected contacts. individually-enacted social distancing in the form of temporary self-isolation must be immediately enacted if a social contact is known to be infected in order to halt the spread of an epidemic. reductions in viral shedding lead to significant reductions in the size of the final infected population. forecasting the novel coronavirus covid-19 time-to-death approach in revealing chronicity and severity of covid-19 across the world covid-19: the cidrap viewpoint part 1: the future of the covid-19 pandemic: lessons learned from pandemic influenza feasibility of controlling covid-19 outbreaks by isolation of cases and contacts. the lancet global health using social and behavioural science to support covid-19 pandemic response changes in risk perception and protective behavior during the first week of the covid-19 pandemic in the united states the psychological distress and coping styles in the early stages of the 2019 coronavirus disease (covid-19) epidemic in the general mainland chinese population: a web-based survey behavioural change models for infectious disease transmission: a systematic review & covid-19 systematic urgent review group effort (surge) study authors physical distancing, face masks, and eye protection to prevent person-to-person transmission of sars-cov-2 and covid-19: a systematic review and meta-analysis (2020) journey through an epidemic: some observations of contrasting public health responses to sars analysis and forecast of covid-19 spreading in china a simple sir model with a large set of asymptomatic infectives geo-temporal distribution of 1,688 chinese healthcare workers infected with covid-19 in severe conditions-a secondary data analysis machine learning using intrinsic genomic signatures for rapid classification of novel pathogens: covid-19 case study collective dynamics of 'small-world'networks classes of small-world networks disease transmission in territorial populations: the small-world network of serengeti lions network-based analysis of stochastic sir epidemic models with random and proportionate mixing coupled contagion dynamics of fear and disease: mathematical and computational explorations the watts-strogatz network model developed by including degree distribution: theory and computer simulation rpart: recursive partitioning and regression trees mathematics of epidemics on networks the small world network of college classes: implications for epidemic spread on a university campus perceptions of the adult us population regarding the novel coronavirus outbreak data-based analysis, modelling and forecasting of the covid-19 outbreak this project began during the workshop "understanding and exploring network epidemiology in the time of key: cord-312367-24huwt3y authors: coelho, camila; gallo, gloria; campos, claudia b.; hardy, leon; würtele, martin title: biochemical screening for sars-cov-2 main protease inhibitors date: 2020-10-06 journal: plos one doi: 10.1371/journal.pone.0240079 sha: doc_id: 312367 cord_uid: 24huwt3y the severe acute respiratory syndrome corona virus 2 (sars-cov-2) pandemic represents a global challenge. sars-cov-2's ability to replicate in host cells relies on the action of its non-structural proteins, like its main protease (m(pro)). this cysteine protease acts by processing the viruses' precursor polyproteins. as proteases, together with polymerases, are main targets of antiviral drug design, we here have performed biochemical high throughput screening (hts) with recombinantly expressed sars-cov-2 m(pro). a fluorescent assay was used to identify inhibitors in a compound library containing known drugs, bioactive molecules and natural products. these screens led to the identification of 13 inhibitors with ic(50) values ranging from 0.2 μm to 23 μm. the screens confirmed several known sars-cov m(pro) inhibitors as inhibitors of sars-cov-2 m(pro), such as the organo-mercuric compounds thimerosal and phenylmercuric acetate. benzophenone derivatives could also be identified among the most potent screening hits. additionally, evans blue, a sulfonic acid-containing dye, could be identified as an m(pro) inhibitor. the obtained compounds could be of interest as lead compounds for the development of future sars-cov-2 drugs. the agent behind the coronavirus disease 2019 (covid-19) pandemic, sars-cov-2, is an rna virus from the betacoronavirus genus [1, 2] . the genome of this virus has about 88% identity to coronaviruses from bats, but only 79% to sars-cov and 50% to mers-cov viruses [3] . sars-cov-2 shares the typical gene array of coronaviruses. about two thirds of the genome is occupied by orf1ab that encodes the non-structural proteins, while the remaining region next to the 3' end encodes the structural proteins [3] . orf1ab is translated into two polyproteins. they are processed by the virus's main protease m pro (also termed 3cl pro because of its homology to the picornavirus 3c protease) and a second papain-like protease (pl pro ) [4] . the structure of m pro from sars-cov-2, a protein with 96% sequence identity to m pro from sars-cov, was recently solved [5, 6] . it consists of a dimeric 6-stranded β-barrel chymotrypsin-like fold with homology to the monomeric picornavirus 3c protease fold. the enzyme's active site contains a cysteine-histidine catalytic dyad. m pro has an additional cterminal helical domain and an n-terminal chain of amino acids termed the "n-finger". the helical domain, together with the n-finger amino acids, form a dimerization interaction surface for a second m pro protomer. the resulting dimer has an estimated dissociation constant of approximately 2.5 μm [6] . the n-finger chain is important for activity as it stabilizes part of the adjacent monomer's s1 binding pocket. m pro is thought to specifically cleave the viral polyprotein 1ab at 11 cleavage sites. the sequence recognized contains in most cases leu-gln-(ser/ ala/gly) with cleavage occurring after the gln residue [5] [6] [7] . although currently several promising therapeutic strategies against sars-cov-2 are in development [8] , no established covid-19 drug or vaccine exists. by the end of may 2020 worldwide statistics accounted for more than 5.8 million confirmed infections and 360 thousand deaths due to the effects of covid-19 (https://coronavirus.jhu.edu/map.html). as viral proteases, following polymerases, are the most prominent targets for antiviral drug design [9] , here we describe initial biochemical screenings with recombinant purified sars-cov-2 m pro performed in order to define possible candidates which could serve as lead compounds for the design of future covid-19 therapies. in order to contribute to the ongoing worldwide research and development efforts to contain covid-19, we cloned, expressed recombinantly in e.coli bl21(de3) and purified an important drug target of sars-cov-2, its main protease (m pro ). after his-tag cleavage, screens were carried out in concentrations of 1 μm m pro and 10 μm of a previously described fluorogenic substrate-peptide mca-avlqsgfr-k(dnp)-k-nh2 [5] . screens of a library containing 2400 drugs and drug-related molecules as well as natural products led to several interesting hits. as control experiments to validate the screenings, enzyme substrate assays without inhibitors (negative control) as well as enzyme substrate assays with tannic acid, a known inhibitor of sars-cov m pro (positive control) [10] , were used. the relative activity of the assay was defined as the quotient between the initial reaction rates of the experiments and the negative controls. as a result, an average relative activity of 1.0 (standard deviation, sd = 0.08) for the negative and 0.0 (sd = 0.014) for the positive controls was obtained. control experiments thus showed a significant separation of relative activity of the negative and positive controls ( fig 1a) leading to an acceptable hts z' value [11] of 0.72. the average value of the relative activities of the compound screening assays was 0.98 (sd = 0.2, fig 1b) . after the screenings, 13 of the most prominent hits were selected for confirmation and further biochemical characterization based on a cut-off relative activity below 0.2. these compounds, together with their corresponding half-maximum inhibitory concentration (ic 50 ) values are shown in table 1 . for m pro from sars-cov and sars-cov-2, several interesting inhibitors have been reported. inhibitors of sars-cov m pro discovered by high throughput screening include diverse compounds characterized with k i values ranging from 0.5 μm to 75 μm [10, [12] [13] [14] [15] [16] . from these obtained compounds, esculetin-4-carboxylic acid ethyl ester (ic 50 = 46 μm in m pro inhibition assays), a coumarin derivative and natural product, demonstrated an ec 50 of 112 μm (median toxic concentration tc 50 >800μm) in vero-cell sars-cov assays [13] and mp576 (ic 50 = 2.5 μm), a quinolinecarboxylate, demonstrated an ec 50 of 7 μm (tc 50 >50μm) [15, 17] , thus validating the m pro biochemical screening approach for the development of sars-cov drugs. additionally, several other notable sars-cov m pro inhibitors, like tg-0205221 a peptidomimetic covalent inhibitor with a k i value of 53 nm [18] (ec 50 = 0.6 μm, tc 50 >20 μm in cellular assays) and boronic acid derivatives with k i values up to 40 nm [19] , among several others, have been published. regarding sars-cov-2 m pro inhibition, several promising lead compounds have been reported, like e.g. ebselen (ic 50 = 0.67 μm, ec 50 = 4.67 μm, ld 50 > 4,600 mg/kg in rats), tideglusib (ic 50 = 1.55 μm) carmofur (ic 50 of 1.82 μm) [5] ; a peptidomimetic α-ketoamide with an ic 50 value of 0.67 μm for sars-cov-2 m pro and an ec 50 value of 4 to 5 μm in human cell culture experiments that covalently binds to the catalytic cysteine as shown in x-ray diffraction experiments [6] ; two peptidomimetic compounds with an aldehyde reactive group that covalently binds the catalytic cysteine with an ic 50 of 40 nm and 53 nm (ec 50 values of 0.53 μm and 0.72 μm) [20] and atazanavir (ec 50 = 2.0 μm) [21] . in this work, it was possible to confirm thimerosal (1, ic 50 = 0.6 μm, fig 2a) and phenylmercuric acetate (2, ic 50 = 0.4 μm, fig 2b) , both previously described as sars-cov m pro inhibitors [14] , as sars-cov-2 m pro inhibitors. this common mode of inhibition can be expected, as sars-cov-2 and sars-cov m pro share an overall amino acid identity of 96%, with practically all amino acids from the active site being conserved. thimerosal is an organometallic compound originally used as an antiseptic (e.g. merthiolate) and preservative in vaccines, pharmaceutical products as well as cosmetics [22] . phenylmercuric acetate is another organo-mercuric compound, used as preservative in paints and as a disinfectant [23] . thimerosal was initially identified together with phenylmercuric acetate in a hts as a sars--cov m pro inhibitor. this result led to the further identification of four other hg-containing compound as well as several presumably less toxic zn rather than hg-containing compounds, with k i values ranging from 0.17 μm to 1.4 μm [14] . both thimerosal and phenylmercuric acetate and other hg-containing molecules are thought to have antibacterial properties by their capacity to bind thiol groups in proteins [23] , like the catalytic cysteine of m pro . with regards to other viral infections, very low doses of thimerosal have been additionally found to modulate and promote the host's immune response, promoting th2-cell responses and inhibiting proinflammatory cytokines and chemokines [24] , which could provide further benefits in the treatment of covid-19. another compound identified as sars-cov-2 m pro inhibitor in this work is bronopol (3, 2-bromo-2-nitropropane-1,3-diol, fig 2c, ic 50 = 4.4 μm) . bronopol is a wide range antibacterial agent used as a preservative in e.g. cosmetics and pharmaceutical products [25] , which is thought to deactivate enzymes by its oxidative effect on thiol groups [26] . the identification of metal-conjugate inhibitors and thiol oxidizing compounds indicates that approaches that take advantage of the fact that m pro is a cysteine protease are an interesting option to be exploited. we could additionally confirm tannic acid (4, fig 3a) , which has an ic 50 of 3 μm for sars-cov m pro [10] , as a sars-cov-2 m pro inhibitor with an ic 50 of 2.1 μm. tannic acid, a hydrolysable tannin, is a polyphenolic compound formed by a glucose moiety and gallic acid. several enzymes have been shown to be inhibited by tannic acid, including proteases [27] . due to these properties, tannic acid was used successfully in this work as a positive control. surprisingly hematoporphyrin (5, fig 3b, ic 50 = 3.9 μm) was a hit in the sars-cov-2 m pro screens. hematoporphyrin is a derivative of hemoglobin's protoporphyrin ix ring system. consequently, protoporphyrin ix was additionally tested with the m pro assay and an ic 50 value of 23 μm was obtained. hematoporphyrin is used in photodynamic therapy [28] and was formerly used as an antidepressant [29] . although this finding could potentially indicate that m pro mediates a hypothetical link between covid-19 and hematological disorders, like virus induced porphyria [30] and sars-cov-2 induced coagulation disorders [31, 32] the evidence presented here is far too preliminary and speculative. furthermore, both substances have been described as being so called promiscuous compounds in hts [33] . in this sense, hematoporphyrin could catalyze as a photosensitizer through free radical generation inactivation by oxidation of the catalytic cysteine of m pro [34, 35] . thus, further work has to be carried out to corroborate whether hematoporphyrin is indeed a specific inhibitor of sars-cov-2 m pro and eventually a mediator of hematological disorders. two other interesting related m pro inhibitors obtained were 3,4-didesmethyl-5-deshydroxy-3'-ethoxyscleroin (6, iupac-name: (3-ethoxyphenyl)-(2,3,4-trihydroxyphenyl)methanone, fig 4a) and its isomer 2,3,4-trihydroxy-4'-ethoxybenzophenone (7, iupac-name: (4-ethoxyphenyl)-(2,3,4-trihydroxyphenyl)methanone, fig 4b) . whereas the 3-ethoxyphenyl isomer (6) had an ic 50 of 10.6 μm, the 4-ethoxyphenyl (7) isomer had a similar ic 50 of 9 μm. interestingly, other benzophenone derivatives have been reported as inhibitors of protozoan cysteine proteases [36, 37] . the behavior of benzophenones as free radical generators upon uv light stimulation could be a possible explanation for their cysteine protease inhibitory activity [35] . however, the specificity of this effect has to be further elucidated, as other benzophenones present in the library, like 2,3,4'-trihydroxy-4-methoxybenzophenone showed no significant inhibitory activity. two quinones, chloranil (8, fig 4c, ic 50 = 4.1 μm) and plumbagin (9, fig 4d, ic 50 = 17.1 μm) were also identified in the screens. as quinones are mild oxidizing agents, their activity could be related to oxidation of the catalytic cysteine of m pro [38] . another compound, vanitiolide (10, fig 4e, iupac-name: 4-hydroxy-3-methoxyphenyl)(4-morpholinyl) interestingly, both compounds have been previously reported to inhibit human immunodeficiency virus (hiv) in cellular assays as well hiv's reverse transcriptase in biochemical assays [39] and evans blue has been reported as a hepatitis b virus (hbv) inhibitor in cellular assays [40] . for the top inhibiting compounds, thimerosal (1), phenylmercuric acetate (2), bronopol (3), tannic acid (4), hematoporphyrin (5), 3,4-didesmethyl-5-deshydroxy-3'-ethoxyscleroin (6), evans blue (11) and chicago sky blue (12) detailed kinetical experiments were carried out (s1 fig in s1 file) . the resulting k i values are shown in s1 table in s1 file. the values are, as expected, mostly close to the obtained ic 50 values. evans blue (11) , chicago sky blue (12), phenylmercuric acetate (2) and tannic acid (4) show competitive inhibition, as indicated by the dynafit analysis (ssq, summed squared deviation between experimental data and theoretical model, and the related δaic/δbic, second order akeike information criterion/ bayesian information criterion [41] ; s1 table in s1 file). however, thimerosal (1), bronopol (3), hematoporphyrin (5), 3,4-didesmethyl-5-deshydroxy-3'-ethoxyscleroin (6) showed mixed or noncompetitive inhibition. as a whole, the obtained compounds here described are in an ic 50 range from 0.2 to 23 μm ( table 1 ) that would justify further biochemical testing as well as testing in cellular assays which would confirm them as lead compounds for covid-19 drug development. although some are natural products and some have a record as pharmaceutical agents, which may accelerate their development, some have toxicity issues, which have to be carefully evaluated (s2 table in s1 file). emergence of covid-19, with its huge human, social and economic costs and implications has certainly demonstrated the necessity for the development of novel antiviral drugs. the gene of sars-cov-2 m pro (genbank entry mt358641.1) was synthesized (genscript, usa) and cloned into the pet21a expression plasmid. the resulting expression construct contains an n-terminal his-tag followed by a tobacco etch virus (tev) protease cleavage site, so that the resulting protein after his-tag cleavage is the full-length native sars-cov-2 mpro including two additional (gly-ser) n-terminal residues. the protein was expressed in e. coli bl21 (de3) grown in luria bertani broth containing 50 μg/ml ampicillin at 37˚c after induction with 0.5 mm isopropyl-ß-d-1-thiogalactopyranoside (iptg) for 8 hours at 30˚c. after harvesting by centrifugation, cells were disrupted with lysis buffer containing 50 mm tris-hcl ph 8, 1% brij 98, 300 mm nacl, 5 mm imidazole, dnase and lysozyme. the protein was purified from the soluble fraction using an ä ktaprime plus liquid-chromatography system (ge healthcare) by affinity chromatography employing a 5 ml histrap sepharose column (ge healthcare) using a 50 mm tris-hcl ph 7.3, 150 mm nacl buffer and a 5 mm to 500 mm imidazole gradient for elution. a second purification step was performed using size exclusion chromatography with a hiload 26/600 superdex 75 prep-grade column (ge healthcare) using a 50 mm tris-hcl ph 7.3, 150mm nacl buffer. finally, the tev protease [42] was used to cleave the his-tag of the protein in 50 mm tris-hcl ph 8, 1 mm dtt, 0.5 mm edta for 4 hours at 8˚c. the protein was then purified to remove the his-tagged tev protease and the cleaved affinity-tag by a further step of ni-affinity chromatography in 50 mm tris-hcl ph 7.3, 150 mm nacl buffer. the spectrum collection (microsource discovery systems inc.) compound library was screened using a freedom evo 150 liquid handler (tecan group ltd.). assays were performed in 50 mm tris-hcl ph 7.3, 20% glycerol, 1mm edta ph 7.3 and 0.01% triton-x using 1 μm m pro , 40 μm compounds and 10 μm substrate-peptide (mca-avlqsgfr-k(dnp)-k-nh2, biomatik corporation, cambridge, canada) [5] at 30˚c after a compound incubation period of 10 minutes. the reaction was monitored using an excitation wavelength of 330 nm and an emission wavelength of 400 nm on an infinite m200 plate reader (tecan group ltd.). ic 50 values were determined using concentrations from 122 nm to 100 μm compounds and 0.5 μm m pro with 10 μm substrate. all tests were carried out in triplicate and performed on 384 well plates. ic 50 were analyzed by nonlinear regression using a four-parameter dosageresponse variable slope model with the graphpad prism 8.4.2 software (graphpad software, usa). enzyme kinetics experiments were performed using fluorescent peptide concentrations ranging from 1.25 μm to 80 μm and two different inhibitor concentrations. the activity assay was performed using 50 mm tris-hcl ph 7.3, 20% glycerol, 1mm edta ph 7.3 and 0.01% triton-x. final concentrations of 0.5 μm m pro were used. the inner filter effect (ife) was accounted for as described [43] . data were analyzed using dynafit [44] . compounds structures were drawn with acd/chemsketch 2019.2.1 software (advanced chemistry development, canada). supporting information s1 file. (pdf) a new coronavirus associated with human respiratory disease in china a pneumonia outbreak associated with a new coronavirus of probable bat origin genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding from sars to mers: crystallographic studies on coronaviral proteases enable antiviral drug design structure of m pro from sars-cov-2 and discovery of its inhibitors crystal structure of sars-cov-2 main protease provides a basis for design of improved α-ketoamide inhibitors characterization and inhibition of the main protease of severe acute respiratory syndrome coronavirus a review of sars-cov-2 and the ongoing clinical trials approved antiviral drugs over the past 50 years inhibition of sars-cov 3c-like protease activity by theaflavin-3,3'-digallate (tf3). evidence-based complementary and alternative a simple statistical parameter for use in evaluation and validation of high throughput screening assays 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hepatitis c virus and porphyria cutanea tarda covid-19 cytokine storm: the interplay between inflammation and coagulation clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study. the lancet targeting hiv-1 protease autoprocessing for highthroughput drug discovery and drug resistance assessment thiyl free radical production with hematoporphyrin derivative, cysteine and light: a spintrapping study photo-induced antimicrobial and decontaminating agents: recent progresses in polymer and textile applications benzophenone derivatives as cysteine protease inhibitors and biological activity against leishmania(l.) amazonensis amastigotes leishmanicidal therapy targeted to parasite proteases molecular mechanisms of quinone cytotoxicity sulfonic acid dyes: inhibition of the human immunodeficiency virus and mechanism of action evans blue inhibits hbv replication through a dual antiviral mechanism by targeting virus binding and capsid assembly evaluation and comparison of computational models expression and purification of soluble his(6)-tagged tev protease use of a fluorescence plate reader for measuring kinetic parameters with inner filter effect correction program dynafit for the analysis of enzyme kinetic data: application to hiv proteinase the authors thank fastbio, ribeirão preto, brazil for help with reagent importation. key: cord-290773-kgb8r561 authors: ahn, jong gyun; kim, dong soo; kim, ki hwan title: clinical characteristics and cytokine profiles of children with acute lower respiratory tract infections caused by human rhinovirus date: 2018-07-03 journal: plos one doi: 10.1371/journal.pone.0198624 sha: doc_id: 290773 cord_uid: kgb8r561 the clinical profile of human rhinovirus (hrv) with regard to lower respiratory infections remains unclear. we analyzed the clinical features and cytokine responses of hrv isolates in children with respiratory infections. quantitative analysis and genotyping of the hrv-positive samples from 601 nasopharyngeal aspirates (npas) were performed using vp4/vp2 sequencing. to compare t-helper1 (th1) type (ifn-γ, tnf-α) and th2 type (il-4, il-10) cytokine responses between hrv-a, b and c, the levels of the four cytokines were measured. the hrv-positive children had shorter fever duration (p = 0.018), and higher frequencies of chest retraction (p = 0.002) and wheezing (p = 0.022) than did the hrv-negative group. hrv-a was identified in 55 cases (58.5%), hrv-b in 8 (8.5%), and hrv-c in 31 (33.0%). there were no significant differences in the clinical data or npa cytokines levels between patients with hrv-a and hrv-c infections. hrv is an important pathogen of the lower respiratory tract in young children. hrv-a and hrv-c are the dominant species that cause respiratory difficulty in young children. human rhinovirus (hrv) is the most common viral respiratory agent in humans. although it is the predominant cause of the common cold, hrv was recently found to be associated with an extensive range of more severe respiratory illnesses. this virus has been implicated in pneumonia, bronchiolitis, and exacerbation of asthma and chronic obstructive pulmonary disease [1] [2] [3] [4] . hrv is a small, non-enveloped single-stranded rna virus that belongs to the enterovirus genus and picornaviridae family. it is currently classified into three species on the basis of gene sequencing analysis. these include hrv-a, hrv-b, and the recently discovered hrv-c [5, 6] . there are >100 distinct serotypes of hrv species according to their surface proteins. this biological diversity makes it difficult to develop vaccines or antivirals against hrv. understanding the epidemiological, clinical, and immunological features of hrv will help to prevent and treat hrv respiratory diseases. however, these characteristics have yet to be completely assessed. therefore, in this study, we investigated the epidemiological, clinical, and virological characteristics of hrv infections in children with acute lower respiratory tract infections. we also compared the t-helper1(th1)-and th2-related cytokine profiles of hrv infection in nasopharyngeal aspirates (npas) to assess the immune responses of the disease. a total of 601 nasopharyngeal aspirates (npas) were collected from children <18 years old hospitalized with acute lower respiratory tract infections at severance children's hospital in seoul, korea between march 2011 and january 2012. as part of the diagnosis, pneumonia, croup, bronchiolitis, and asthma were included. these diseases were diagnosed through physical examination and chest x-rays. patients were excluded if they were immunocompromised due to hiv infection, malignancy, congenital or acquired immunodeficiency, solid organ transplantation or use of immunosuppressive drugs such as long-term systemic corticosteroids or radiation therapy. npas were obtained by suction using a fine, flexible plastic catheter and syringe. after collection, npas were immediately transported at 4˚c to the laboratory, where they were stored at -70˚c until use. patient demographic and clinical data including medical history, detailed signs and symptoms, physical examination, and laboratory and radiology results were collected from a retrospective review of the medical records and interviews upon admission. disease severity was assessed according to a respiratory symptom scoring system based on previously published studies [7, 8] . nucleic acid from each npa was extracted using a qiaamp viral mini kit (qiagen, hilden, germany) according to the manufacturer's instructions. a one-step multiplex pcr/rt-pcr kit (solgent, daejeon, korea) was used to detect 12 respiratory viruses [9] . these included hrv, rsv, human bocavirus (hbov), influenza a virus (iav), influenza b virus (ibv), human metapneumovirus (hmpv), adenovirus (adv), human coronaviruses (hcov) 229e, oc43, and parainfluenza viruses (piv) 1-3. amplification was performed according to the manufacturer's protocol. the pcr products were run on a 2% agarose gel, stained with ethidium bromide, and visualized with uv light. hrv-positive specimens by multiplex pcr/rt-pcr were subsequently submitted to amplification reaction with the real-time rt-pcr assay using the powerchek™ rhinovirus real-time pcr kit (kogenebiotech, seoul, korea) and abi-7500 fast real-time pcr system (applied biosystems, foster city, ca). rt-pcr was performed according to the manufacturer's instructions under the following conditions: initial denaturation at 50˚c for 30 min and 95˚c for 15 min, followed by 40 cycles at 95˚c for 15 s and 60˚c for 1 min. ninety-four of the 98 hrv-positive pcr samples were sequenced by solgent co. (daejeon, korea). the vp4/vp2 sequences were aligned by clustral w. phylogenetic analysis was conducted using mega 5.0 software. the nucleotide and deduced amino acid sequences of the vp4/vp2 regions were compared with reference sequences of hrv-a, hrv-b and hrv-c strains previously published in the genbank using blast (basic local alignment search tool -http://blast.ncbi.nlm.nih.gov/). we measured the concentration of the th1 (ifn-γ, tnf-α) and th2 (il-4, il-10) cytokines in npa from 94 hrv-positive patients for which genotyping using vp4/vp2 sequencing was undertaken. the concentrations of the four cytokines in npa was measured using commercially available enzyme-linked immunosorbent assay kits (ebioscience, san diego, ca, usa) according to the manufacturer's instructions. statistical analysis was performed using spss (version 18.0, chicago, il, usa). demographic, clinical, and laboratory parameters were compared between hrv-positive and -negative children and also among hrv species. pearson χ 2 or fisher's exact test was applied for categorical variables. the student's t-test or non-parametric mann-whitney u test, anova tests or kruskal-wallis test were used for continuous variables, where applicable. p values <0.05 were considered statistically significant. the study protocol was reviewed and approved by the yonsei university health system institutional review board, seoul, korea (4-2008-0649). the study was conducted in accordance with good clinical practices (national regulations and ich e6) and the principles of the helsinki declaration. written informed consent was obtained from the parents or legal guardians of the patients prior to sample collection following a detailed explanation of schedules and contents of the study. the demographic, clinical and laboratory characteristics of all enrolled patients are shown in table 1 . hrv was detected in 98 (16.3%) of 601 npas. among the hrv-positive samples, 38 (38.8%) were co-detected with other respiratory viruses. hrv-infected children were significantly younger and had a shorter fever duration than the hrv-negative group. in contrast, the frequency of chest retraction and wheezing were significantly higher in the hrv-positive group than in the hrv-negative group. the white blood cell (wbc), hemoglobin (hb) and platelet (plt) counts were within normal range in both groups, although there were significant differences between groups. there were no other significant differences between the laboratory results of hrv-positive and -negative patients. between the hrv mono-and coinfection groups, there were no significant differences in clinical or laboratory data. the median viral load in hrv-positive patients was 2.0×10 4 (interquartile range 4.0×10 3 -9.8×10 4 ) rna copies/ml npa. however, there was no relationship between the hrv viral load and either clinical symptoms or laboratory data. all relevant data, inclusive of particular epidemiologic and clinical data as well as cytokine profiles, are included in a supplemental file (s1 table) . the viral protein vp4/vp2 coding region was sequenced in 94 of 98 hrv-positive samples. phylogenetic analysis demonstrated that there were 55 (58.5%), 8 (8.5%) and 31 (33.0%) cases of hrv-a, -b and -c, respectively. hrv infection occurred throughout the year during the study period, although the peak prevalence of hrv-c was observed in autumn (fig 1) . demographic, clinical, and laboratory findings between rhinovirus genotypes are shown in table 2 . children with hrv-b were significantly older than those infected with hrv-c. clinical manifestations were not associated with any specific rhinovirus. the only exception was that none of the children with hrv-b infections had chest retraction, although the number of infections caused by this virus was low. there were no significant laboratory differences between hrv-a, b and c, except with regard to the wbc count, and percentage of neutrophils and lymphocytes. regardless, these values were all within the normal range in all three groups. the concentrations of ifn-γ, il-4, il-10, and tnf-α in npa of hrv-a-, b-and c-positive patients are shown in table 3 . there were no significant differences in the npa cytokine levels between the three groups. this study demonstrates that hrv is an important cause of lower respiratory infection in young children that is associated with symptoms of respiratory distress, such as chest retraction and wheezing. our findings corroborate several recent studies reporting that hrv, which had previously only been known to cause upper respiratory infection, could play an important role in lower respiratory infection [1, [10] [11] [12] . in our study, the majority of hrv genotypes were hrv-a (58.5%) and hrv-c (33.0%). the results of this study correspond with those of earlier studies, which found that hrv-a and -c were the predominant species, although the detection order depends on the geographic region [1, [13] [14] [15] [16] [17] [18] . previous studies have reported conflicting results with regard to disease severity according to hrv species. most of the earlier studies reported that hrv-c was associated with more severe respiratory disease in children [5, 13, 16, 18, 19] . others found no clinical differences between the three hrv species [20, 21] . further studies showed that both hrv-a and hrv-c were associated with acute respiratory infection hospital admissions, serious disease outcomes or wheezing episodes [13, 15, 22, 23] . in our study, there were no significant differences in the clinical features, laboratory data or npa cytokine levels between hrv-a and -c infections. hrv-b seems somewhat different from hrv-a and -c. in accordance with previous studies [13, [23] [24] [25] , we found that hrv-b was the least frequently detected hrv species, and chest retraction infections caused by this virus was not observed. there are two hypotheses to explain this. it is possible that hrv-b causes a mild form of disease that does not warrant hospitalization, accounting for its lower detection rate in hospital-based studies compared to those of the other hrv species. another possibility is that the genetic and biological fitness of hrv-b leads to its low frequency. one recent article reported that, even in healthy young children, hrv-b was the least frequently detected species of the three [26] . this finding supports the hypothesis that the frequency and virulence of hrv-b may be related to its genetic fitness, rather than selection bias. there have been discrepant results regarding the correlation between the npa hrv viral load and disease severity. most previous studies reported that a high hrv viral load was correlated to clinical severity or wheezing [27] [28] [29] . however, other reports show no correlation between the viral load and severity of the lower respiratory infection [30, 31] . similarly, we did not observe a correlation between the hrv viral load and clinical features or laboratory data. our results suggest that the difference in disease severity may be more related to the host's susceptibility to hrv than to the viral load. viral load can vary according to the sampling process. therefore, the exact relationship between hrv viral load and disease severity merits further investigation. this study has several limitations. first, the study patients were limited to hospitalized children with lower respiratory tract infections at a single center. in addition, the cytokine levels detected in this study were low and the differences were overall minor.these factors may have affected our statistical results. in addition, an asymptomatic control group was not enrolled. including a control group is often a particular challenge in pediatric populations. despite these shortcomings, our results contribute to the understanding of hrv's role and immunopathogenesis in lower respiratory infection. hrv is an important pathogen of the lower respiratory tract in young children. in particular, hrv-a and hrv-c are the dominant species that cause respiratory difficulties including wheezing and chest retraction. hrv-b may cause minor lower respiratory infections in relatively older children. further studies are needed to clarify the pathogenesis of hrv, and to define the specific clinical roles of each species. supporting information s1 table. all relevant data, inclusive of particular epidemiologic and clinical data as well as cytokine profiles in hrv-infected children. (xlsx) human rhinovirus c infections mirror those of human rhinovirus a in children with community-acquired pneumonia rhinovirus bronchiolitis and recurrent wheezing: 1-year follow-up evaluation of respiratory viral pathogens in acute asthma exacerbations during childhood detection of rhinovirus in induced sputum at exacerbation of chronic obstructive pulmonary disease clinical features and complete genome characterization of a distinct human rhinovirus (hrv) genetic cluster, probably representing a previously undetected hrv species, hrv-c, associated with acute respiratory illness in children characterisation of a newly identified human rhinovirus, hrv-qpm, discovered in infants with bronchiolitis relationships among specific viral pathogens, virus-induced interleukin-8, and respiratory symptoms in infancy rhinovirus illnesses during infancy predict subsequent childhood wheezing establishing a surveillance network for severe lower respiratory tract infections in korean infants and young children rhinovirus-associated hospitalizations in young children rhinoviruses are a major cause of wheezing and hospitalization in children less than 2 years of age fatal respiratory infections associated with rhinovirus outbreak clinical and molecular epidemiology of human rhinovirus c in children and adults in hong kong reveals a possible distinct human rhinovirus c subgroup role of rhinovirus c respiratory infections in sick and healthy children in spain human rhinovirus species associated with hospitalizations for acute respiratory illness in young us children high prevalence of human rhinovirus c infection in thai children with acute lower respiratory tract disease molecular epidemiology of human rhinovirus infections in kilifi, coastal kenya human rhinovirus species c infection in young children with acute wheeze is associated with increased acute respiratory hospital admissions patient characteristics and severity of human rhinovirus infections in children human rhinovirus infections in rural thailand: epidemiological evidence for rhinovirus as both pathogen and bystander prevalence of human rhinovirus in children admitted to hospital with acute lower respiratory tract infections in changsha human rhinovirus types and association with respiratory symptoms during the first year of life human rhinovirus species and season of infection determine illness severity novel human rhinoviruses and exacerbation of asthma in children biodiversity and clinicodemographic characteristics of human rhinoviruses from hospitalized children with acute lower respiratory tract infections in malaysia prospective evaluation of rhinovirus infection in healthy young children correlation of rhinovirus load in the respiratory tract and clinical symptoms in hospitalized immunocompetent and immunocompromised patients rhinovirus load and disease severity in children with lower respiratory tract infections impact of rhinovirus nasopharyngeal viral load and viremia on severity of respiratory infections in children enhanced severity of virus associated lower respiratory tract disease in asthma patients may not be associated with delayed viral clearance and increased viral load in the upper respiratory tract clinical and molecular characterization of rhinoviruses a, b, and c in adult patients with pneumonia the authors would like to thank ms. kim heui og for her technical assistance. key: cord-256326-3ebcuzd6 authors: liotta, giuseppe; marazzi, maria cristina; orlando, stefano; palombi, leonardo title: is social connectedness a risk factor for the spreading of covid-19 among older adults? the italian paradox date: 2020-05-21 journal: plos one doi: 10.1371/journal.pone.0233329 sha: doc_id: 256326 cord_uid: 3ebcuzd6 italy was one of the first european countries affected by the new coronavirus (covid-19) pandemic, with over 105,000 infected people and close to 13,000 deaths, until march 31(st). the pandemic has hit especially hard because of the country's demographic structure, with a high percentage of older adults. the authors explore the possibility, recently aired in some studies, of extensive intergenerational contact as a possible determinant of the severity of the pandemic among the older italian adults. we analyzed several variables to test this hypothesis, such as the percentage of infected patients aged >80 years, available nursing home beds, covid-19 incidence rate, and the number of days from when the number of positive tests exceeded 50 (epidemic maturity). we also included in the analysis mean household size and percentage of households comprising one person, in the region. paradoxically, the results are opposite of what was previously reported. the pandemic was more severe in regions with higher family fragmentation and increased availability of residential health facilities. between december 31, 2019 and march 31, 2020, 750,890 cases and 36,405 deaths due to new coronavirus (covid-19) have been reported [1] . as is well known, italy is one of the countries most affected by the pandemic, with 12,428 deaths and 105,792 cases recorded in the months of february and march 2020 [2] . many have wondered why covid-19 has hit so hard in italy, but unfortunately, over the weeks it has become increasingly clear that other eu countries as well as the us are developing similar growth trends. recently some studies [3] stated that age, in both local and national context, as well as the social connectedness of older and younger generations act as powerful determinants in the spread of pandemics. while there is a very clear association between the case fatality rate and age demographics (italy has the second oldest population worldwide and has the highest ageing index in europe [4] with a value of 168.9), we wanted to test the hypothesis that the supposed closeness between younger and older generations in italian families may have played a major role in the pandemic spread. in a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 fact, dowd, et al. stated that "italy is also a country characterized by extensive intergenerational contacts which are supported by a high degree of residential proximity between adult children and their parents." [3] . according to this observation, we expected to find a strong direct association between family size and the spread of covid-19. conversely, social distance should be favored in cities and regions with a high number of residential structures for long term care, usually devoted to older adults. the study is based on the available population data from each italian administrative region. the sources of data used in this paper are the daily situation reports on covid-19 published by the italian ministry of health (28 february-31 march), the national institute of statistics (istat) 2019 data set on households and population, and the 6 th report generated by the non-self sufficiency network (2017-2018). in italy, people aged >80 are 4.33 millions, of which 37.1% are men (mean age 84.7) and 62.9% are women (mean age 85.7). several variables with plausible association with the spread of covid-19 among citizens aged >80 years have been taken into consideration. these include the percentage the italian population aged >80 years [4] , covid-19 incidence rate, and number of days from when the number of confirmed cases exceeded 50 (epidemic maturity) [1] . additionally, we explored the relationship between the proportion of infected patients aged >80 years and social connectedness indicators, such as the percentage of family comprising one members and household size [5] . finally, we considered the availability of beds in nursing homes [6] since the pandemic severely impacted nursing home residents throughout the country caused by difficulties in social distancing in these settings as well as the well documented lack of personal protective equipment (ppe) during the first phase of the pandemic in italy. the population aged >80 years was chosen because they account for about 50% of the deaths due to covid-19 [7] , while representing 7.2% [4] ) of the total population and about 75% [8] of the nursing home residents. univariate (pearson correlation) and multivariate (linear regression) analyses were conducted to test the hypotheses. table 1 reports the proportion of residents aged >80 years among the total number of covid-19 cases, according to the italian administrative regions, that ranges from 4.3% for basilicata to 23.6% for marche region [9] . the deviation from the italian average (18.8%) is +25% for the highest value and -75.5% for the lowest. this cannot be explained by the different percentages of residents aged >80 years among the population that ranges from 7.8 to 11.5%. the maturity of the epidemic (the number of days since the infection exceeded 50 confirmed cases) ranges from 10 to 32 days according to region. this could partially explain the differences, even if other factors seem to be involved. the incidence rate among the general population ranged from 4.09/1,000 residents (valle d'aosta) to 0.27/1,000 residents (sicily and campania) and could explain the spread of the infection among the residents aged >80 years. the mean household size showed a negative correlation with the proportion of infected residents aged >80 years, whereas the percentage of households with one member and availability of nursing homes beds showed a positive correlation with the proportion of infected residents aged >80 years (table 1) . multivariate analyses allowed the comparison of different models of the diffusion of the epidemic to the target population ( table 2 ). the first model included the percentage of the general population aged >80 years and the measurements of the impact of the pandemic at population level. the incidence rate dropped from the stepwise model, showing that the duration of the circulation of the virus among the population, rather than the incidence rate, determined the increased proportion of infections among the population aged >80 years. the second step was to add variables related to social connectedness into the model. at this stage, the percentage of households with only one member was excluded, whereas the mean number of households was included in the model. finally, the percentage of nursing home beds in the total population was included in the model and the mean household size dropped out with a reduction of the adjusted r 2 . this result prompted us to find a combination of the two variables that captured different aspects of a process that linked household size to the percentage of available nursing home beds. a combined index was set up (percentage of nursing home beds/mean household size) that was included in the final model with a slight but significant increase of the adjusted r 2 . fig 1 shows the linear relation between the model and outcome variable. the hypothesis of a relationship between social connectedness and spread of covid-19 was not confirmed by this study. the above mentioned analyses show the spread of the infection among the population aged >80 years was associated with the percentage of households comprising one member (even if it was statistically insignificant in the multivariate analyses), and inversely associated with the mean household size, the latter independently from the population age-structure and spread of covid-19 in the general population. the spread of covid-19 among the older adults was also independently associated with the available nursing homes beds. the model explains more than 70% of the variation of the proportion of infected patients aged >80 years. social relationships in italy have changed dramatically in the last 20 years. the istat reported that, in 2018, one third of italian families (33%) were composed of only one person and only 5.3% had � 5 members [10] . if we include couples without children (20.1%), more than half (53.1%) of the 25 million italian families comprise �2 people [4] . the 2018 istat report stated that more than 25% of the population aged >75 years has no one to count on in case of need, and living alone has become the most common living arrangement, as it is all over in europe (about one third of households comprise only one member) [11] . more than 50% of the population aged >85 years in italy is living alone [12] , and in some regions, as in veneto-northern italy, this percentage increases to 75%, with about 25% of older adults that are not self-sufficient [13] . more than 50% of the residents aged >75 years in lombardy, the most impacted region in italy, live alone; here, in the last 10 years the household size decreased by about 10% [14] , whereas in campania (southern italy with one of the lowest proportions of infected patients aged >80 years) it decreased by <3% [11, 15] . it is likely that in a situation of forced isolation, social relationships represent a powerful tool to reduce the risk of contagion, allowing community-dwelling older adults to receive some assistance (bringing food or drugs home, thus facilitating the older adults to accomplish with social distancing). this kind of support is increasing in many italian cities. the protective power of social connectedness emerged in many crises, mainly because of the increasing prevalence of bio-psycho-social frailty among older adults who have to face repeated "environmental" stresses, such as a pandemic or a heat wave. this was not the case in nursing homes that banned visitors since the early phase of the epidemic, even though this approach did not prevent the spread of the infection. nursing homes are dealing with the same problems as all closed communities, the struggle to ensure social distancing between people who need care, and the lack of ppe, as was the case at national level. in short, social distancing does not necessarily imply social isolation. similarly, social connectedness does not imply physical closeness (which is dangerous in an epidemic) with social contacts. italian nursing homes (called rsas-translated in english as social and health care facilities) house >265,000 older adults in italy. a very recent cross-sectional survey conducted by iss [16] is going to explore the spread of covid-19 in a sample of 2,556 rsas because of several micro epidemics took place in numerous facilities. the role played by residential care facilities in italy as well as in many other countries, due to their lack of preparedness to this kind of events, is progressively emerging [17, 18] . in some cases public policies aimed at discharging covid-19 cases from hospitals to nursing homes in order to improve the capacity of the health systems to face the lack of hospital beds are suspected to have increased also the risk of infection among the nursing homes' hosts [19] . there are some limitations in the present study. first, the study analyzed the proportion of cases in patients aged >80 years instead of analyzing age-specific infection or mortality rates according to regions, as these data are not retrievable from public sources at this stage of the infection. second, the quality of social connectedness parameters is limited, even though this is a limit of each study exploring social relationships based on routine data instead of gathering information ad hoc. finally, the number of performed tests, which varies enormously according to the region (ranging from 1.8 to 18.2 per 1,000 inhabitants) [6] , could affect the proportion of confirmed cases. the policy at the regional level regarding testing varies from an approach aimed at identifying asymptomatic cases as much as possible (veneto) to one aimed at testing only the symptomatic in order to give priority to most severe cases. of course, the most severe cases are more prevalent among the very old, so the differences in the approach could lead to an overestimation of cases in patients aged >80 years in regions where the total number of tests is lower and the number of confirmed cases/number of performed tests ratio (nct/npt � 100) is higher. indeed, this could be the case since the correlation between the nct/npt ratio and the proportion of cases in patients aged >80 years was robust (0.603; p = 0.004), but the multivariate model remained unchanged and the nct/npt ratio was not shown as a statistically significant variable. the association of social connectedness with the spread of covid-19 among older italian adults, hence older adult mortality rate, is not confirmed. paradoxically, it seems that the variables associated with social isolation are risk factors for increase in the proportion of cases in italian patients aged >80 years among the total number of cases. this is consistent with the observation that social relationships are a protective factor against increased mortality rates during a crisis impacting the frailest populations. nursing homes bed rate is one of the determinants of sars-cov-2 infection rate among the individuals aged>80 in italy. conceptualization: giuseppe liotta, maria cristina marazzi, stefano orlando, leonardo palombi. who. coronavirus disease 2019 (covid-19) situation report-71 demographic science aids in understanding the spread and fatality rates of covid-19 popolazione per età, sesso e stato civile aspects of daily life-households size l'assistenza agli anziani non autosufficienti in italia. il tempo delle risposte-sesto rapporto covid-19, aggiornamento nazionale del 30/03/2020 guests of social and health care residential facilities: elderly guests by age class and type of need-reg covid-19, aggiornamento nazionale del 30/03/2020 -appendice annuario statistico italiano annual report 2018 -the state of the nation annuario statistico italiano veneto 208.000 ultraottantenni vivono da soli la condizione degli anziani in lombardia-sintesi active ageing policy in campania (italy) mpra paper 35039 mortality associated with covid-19 outbreaks in care homes: early international evidence report on covid-19 and long-term care in italy: lessons learned from an absent crisis management. article in ltccovid.org, international long-term care policy network, cpec-lse competing interests: the authors have declared taht no competing interest exists. key: cord-307540-dr5m9pfk authors: coelho, flávio c.; lana, raquel m.; cruz, oswaldo g.; villela, daniel a. m.; bastos, leonardo s.; pastore y piontti, ana; davis, jessica t.; vespignani, alessandro; codeço, claudia t.; gomes, marcelo f. c. title: assessing the spread of covid-19 in brazil: mobility, morbidity and social vulnerability date: 2020-09-18 journal: plos one doi: 10.1371/journal.pone.0238214 sha: doc_id: 307540 cord_uid: dr5m9pfk brazil detected community transmission of covid-19 on march 13, 2020. in this study we identified which areas in the country were the most vulnerable for covid-19, both in terms of the risk of arrival of cases, the risk of sustained transmission and their social vulnerability. probabilistic models were used to calculate the probability of covid-19 spread from são paulo and rio de janeiro, the initial hotspots, using mobility data from the pre-epidemic period, while multivariate cluster analysis of socio-economic indices was done to identify areas with similar social vulnerability. the results consist of a series of maps of effective distance, outbreak probability, hospital capacity and social vulnerability. they show areas in the north and northeast with high risk of covid-19 outbreak that are also highly socially vulnerable. later, these areas would be found the most severely affected. the maps produced were sent to health authorities to aid in their efforts to prioritize actions such as resource allocation to mitigate the effects of the pandemic. in the discussion, we address how predictions compared to the observed dynamics of the disease. as of 21 march 2020, the pandemic of covid-19 had reached 184 countries with 266,073 confirmed cases and 11,184 deaths, globally [1] . the first imported case of covid-19 was confirmed in brazil on february 26, 2020, in the city of são paulo [2] , only two months after the alert on covid-19 went off in china. at this date, all twenty seven federative units had reported suspect cases of covid-19 infection, while 16 states and the federal district had confirmed 1128 cases (11, 278 under investigation) and 18 deaths [3] . são paulo and rio de janeiro states identified virus community transmission on march 13, 2020 [4] [5] [6] . just four days later, 240 cases were confirmed in são paulo, with 4 deaths [3] . in rio de janeiro, 45 cases had been confirmed with no reported death. são paulo and rio de janeiro states hold the most populous metropolitan areas of brazil, where a large fraction of the population live in crowded neighborhoods with poor housing and low income. they are the country's main hubs for national and international transportation. it is known that other pathogens such as influenza a h1n1, in 2009, have been introduced in the country through these hubs [7] . in march 2020, when the epidemic was still confined to rio de janeiro and são paulo, we estimated the pattern of covid-19 spreading risk within brazil, taking the states of rio de janeiro and são paulo as the starting points. in our analysis, we considered a worst case scenario in which there would be no implementation of mobility restrictions. at that moment, mobility restrictions and social distance were starting to be adopted, but it was not clear whether the population would adhere to them. brazil is a continental country with strong spatial heterogeneities in terms of demography, age distribution, access to public health, and socioeconomic indexes. because of these inequalities, the covid-19 epidemic should impact these populations differently. to identify regions with high geographical and social vulnerability, we proposed a classification scheme based on three main criteria: population mobility, socio-demographic-economic characteristics, and the available health care infrastructure in terms of hospital capacity. brazil is divided into 558 micro-regional administrative units, with population sizes varying from 13 million people in the metropolitan area of são paulo to 2, 703 in fernando de noronha island, in pernambuco. to measure mobility intensity between micro-regions, we used daily air travel statistics from the official airline guide (oag) [8] . this dataset contains the number of travels per origin-destination airport. data on shorter distance pendular travels motivated by work and study activities were obtained from the 2010 national census (ibge) [9] . demographic data from the 2000 and 2010 national censuses [9] were used to project the population per age group in 2020 using a geometric growth model. socioeconomic indicators were obtained from the same source: infant mortality, life expectancy, gini index, human development index (education, longevity and income), proportion of individuals below the poverty threshold, proportion below the extreme poverty threshold, 25% percentile income, percentage of urban population, percentage of the population in households with piped water, % of population with insufficient water supply and precarious sewage disposal, and percentage of individuals in households without electricity. data on health care capacity per micro-region were obtained from datasus [10] . from these data, we calculated the number of standard hospital beds and number of complementary beds (intensive care unit and intermediate unit) available for each micro-region [10] , aggregating those from the public sector (sus) and private (non-sus) sector. the final indicator is given by 10, 000 inhabitants. number of covid-19 cases notified per day was obtained from the site brasil.io/dataset/ covid19/casofull. this site aggregates official notification data reported by each state. to assess the probability of covid-19 spreading within brazil, in the absence of mobility restrictions, we first calculated the effective distance (e f (i,j)) between micro-regions using the air travel data. due to the continental size of brazil, daily interstate mobility between major urban centers is mainly composed by air travel. therefore, interstate dispersion of the disease during the initial phase of the epidemic is assumed to be driven mainly by air-travel. we computed the effective distance, e f (i,j), between each micro-region and the two covid-19's hotspots, rio de janeiro and sao paulo. e f (i,j) is a measure of proximity between micro-regions i and j created by the flow of travellers. this metric is known to be strongly correlated with the time it takes to import infectious diseases into new territories from a well-defined origin, particularly for diseases with direct transmission [11, 12] . we computed e f from são paulo and rio de janeiro separately in order to assess the potential contribution of each one. to facilitate comparison between different scenarios, we also calculated a relative effective distance (e f ), by dividing e f by the distance to the nearest destination: e f (i,j) = e f (i,j)/min j {e f (i, j)}. this information was mapped into micro-region origin-destination pairs by summing over the corresponding airports serving each micro-region based on its municipality of reference. to calculate the probability of outbreak in each micro-region m, we used the standard expression: [13] . this expression comes from the stochastic sir model, where the probability of extinction of an outbreak is given by ð1=r 0 þ i 0 where i 0 is the initial number of infected when r 0 > 1. thus, the complement of this probability is the probability of the epidemic taking hold in the population (see [13] , p.106 for a derivation). the prevalence of infection, i m , is estimated as is the number of travelers with covid-19 arriving from micro-region i to micro-region m while i m is the product of the fractional prevalence i i /n i times the infection duration τ (assumed equal for all infected) and a scaling parameter k, to account for the number of undetected asymptomatic individuals participating in the transmission. the parameter r 0 is the basic reproduction number [13] . for the purpose of the results shown, we set r 0 = 2.5, which is compatible with previous studies [14] [15] [16] [17] . s1 we computed the outbreak probability per micro-region using two consecutive time windows representing two generations of spread: first generation of outbreaks. we assumed that community transmission has been reached when the count of cases was 100 cases. we selected only rio de janeiro and são paulo as the source municipalities which were the two initial covid-19 hotspots. prevalence of infection was calculated by multiplying the notification count by an expansion factor k = 10 to take into account asymptomatic and unreported cases [18] . this number is then multiplied by the average duration of infection τ = 8 days [19] , resulting in 8000 prevalent infections (infected × infection duration) in each of the two cities of origin. the number of travelers per day between micro-regions was computed by adding the number of air travellers (used to calculate effective distance) and the number of pendular commuters census [9] . the inclusion of pendular mobility was important to allow spreading between geographically close micro-regions, while still preserving data-driven flow estimates. second generation of outbreaks. in this scenario, we assumed that enough time had passed so that all micro-regions with p epi � 0.5 in the first scenario actually had developed community transmission (epidemic). in each of the new hotspots, the prevalence is set to the same level adopted before for rio de janeiro and são paulo micro-regions. then we computed p epi again, to identify the micro-regions most likely to develop outbreaks in this second round. these generations of outbreaks should not be confused with generations of infections within a city. the above mentioned scenarios do not take into account any intervention affecting mobility, nor demographic and environmental effects that may play a role on the magnitude of r 0 . social vulnerability. partitioning cluster analysis (k-medoid) was performed to identify micro-regions with similar social vulnerability. this method is a more robust variation of the standard k-means. the first step was the removal of highly linearly correlated variables (pearson's correlation > 0.8): infant mortality, human development index (longevity and income), proportion of individuals below the poverty threshold, 25% percentile income, percentage of urban population. the following ones remained: life expectancy, gini index, education component of the human development index, proportion of the population living below the extreme poverty threshold, percentage of urban population, percentage of the population in households with piped water, percentage of population with insufficient water supply and precarious sewage disposal, and percentage of individuals without electricity. the elbow method was used to estimate the number of clusters [20, 21] . more information on this analysis can be found in the s2 supplement material. the analysis was done using the r environment [22] , packages cluster [23] , corrplot [24] , factominer [25] , factoextra [20] . recife and salvador, among others. a more complete list is found in the s1 supporting information . fig 1(d) shows the probability of outbreak in a secondary round of propagation, conditioned to the establishment of transmission in the micro-regions with highest risk (p > 0.5) during the first phase. in this second moment, the establishment of covid-19 transmission is very likely along the coast, from porto alegre (in the south) to salvador (northeast). other high risk areas are the neighboring areas of recife and fortaleza (northeast), the neighboring areas of foz do iguaçu, in the western border of paraná state, and the neighboring areas of cuiabá, brasilia, and goiânia in the center-west. we identified five classes of social vulnerability, tentatively ordered from a (least vulnerable) to d-e (most vulnerable). table 1 shows the averages of the socioeconomic indexes for each class, from which we propose the following interpretation. the supporting information file s2 has more detailed description of the analysis. class a. mostly urban micro-regions, with above-average life expectancy, with comparatively less social inequality, less population living in extreme poverty, better access to water supply and sewage disposal services, higher education. they are the largest cities and in the central region. class b. very similar to a in life expectancy. still more urban, but with more population living in extreme poverty (mean = 5%). inequality indexes and infrastructure are worse in comparison to a, but still above average. these are found in the south, southeast, and central regions. class c. mixture or urban and rural populations. in comparison to a and b, they have significantly lower life expectancy, significantly high poverty and less infrastructure. they are the most urbanized areas of the northeast region. manaus, capital of the amazonas state in the north region, is also in this category. class d. predominance of rural populations, high inequality, low hdiedu, poor access to water and sewage services, but with access to electricity. these are mainly located in the dry caatinga biome area of the northeast. class e. predominantly rural regions in the amazon. low hdiedu, poor access to treated water, sewage disposal, and electricity. fig 2 shows four maps that synthesize the main vulnerabilities to covid-19 in brazil in march 21th. fig 2(a) shows a strong difference in age structure, with the proportion of individuals with 60 or more years of age varying from only 3% in the north to more than 15% in the southern part (fig 2(a) . fig 2(c) shows micro-regions with similar social vulnerability indexes. micro-regions in the c, d and e classes are the most vulnerable. they are located mostly in the northeast and north regions. as expected, higher life expectancy is associated with better living conditions, significantly concentrated in the southern portion of the country. hospital capacity is very heterogeneous, the best capacity found in large metropolitan areas. there are under-equipped micro-regions throughout the country but they concentrate in the north and northeast. fig 2(d) highlights the micro-regions with high probability of imminent outbreak (p out > 0.5 on the second wave) and high social vulnerability. they are important targets for immediate attention in terms of local socio-economic factors (s1 file). in the absence of strong mobility restrictions, the fast establishment of covid-19 outbreaks in the larger urban areas of the country was found to be highly likely, with subsequent spread to their vicinity municipalities. the time scale of this spread is not explicitly represented in the model, but it was expected to be a matter of a few weeks considering the short serial interval of life expect. = life expectancy (age), gini, poverty = % living in extreme poverty, water = % individuals without access to piped water, sewage = % population with insufficient water supply and precarious sewage disposal, electricity = % individuals in households without electricity, urban = % living in cities. https://doi.org/10.1371/journal.pone.0238214.t001 this infection. the implementation of mobility restrictions could delay this spread but it was unlikely to change the routes of the travelling wave. the analysis of covid-19 spread using the mobility flows and probabilities of the epidemic taking off, were subjected to uncertainties in both mobility and epidemiological parameters available at the time they were calculated. nevertheless their utility at the time was not diminished by this uncertainty since the parameters were chosen to represent a somewhat extreme scenario, which the country could still strive to avoid. other uncertainties expected to limit the accuracy of the results were the lack of an accurate value for the infectious period of covid-19 and the lack of knowledge about early changes in mobility resulting from news of the pandemic already circulating through the country. social distancing was one of the main strategies put in place to delay transmission. this was expected to be difficult to achieve in areas with high social vulnerability where poor living conditions make it difficult to adhere to hygiene and isolation protocols. even in the a vulnerability class, the success of this strategy requires adaptation to cope with the large inequality within the municipalities of each micro-region. the classification proposed here was developed to help to tailor the mitigation protocols to the needs and possibilities of the different regions. it was clear the great heterogeneity in hospital capacity across the country. the median number of hospital beds was 19 per 10,000, but 5% of the micro-regions have only 6 beds per 10,000. this disparity poses an important challenge for resource allocation, and should be addressed specially in those municipalities combining high probability to early spread, relatively high percentage of individuals above 60 years old, and limited number of hospital beds per 10,000 people. all discussions about the time to overload of the health system should take into consideration the regular level of occupancy of hospital beds in each region, which was not available for this analysis. the regions in the north and northeast of the country were those identified with the highest socioeconomic vulnerabilities. these areas were expected to suffer above average burden if measures were not taken quickly, since the eventual disease spread would add to struggles already present in those populations. all the analyses proposed and applied here focused on being able to rank regions in terms of the seriousness of the health crisis looming over them. as such, their main limitation is that they yield relative results, allowing for optimal resource allocation, but not for accurately predicting number of cases and deaths. further exploration of this model could include monte carlo methods to identify uncertainties regarding different trajectories of disease spread in the first and second round of propagation. when this analysis was sent to public health authorities in the form of a report, we hoped it could help health authorities and decision-makers regarding the best course of action and how to better allocate their resources. one application was the identification of indigenous populations in brazil at imminent risk. in response, the indigenous communities organized themselves and in collaboration with other organizations, prepared a document for the un. these subsequent reports are available in covid-19.procc.fiocruz.br. in 9th july 2020, 110 days after the analysis here presented was finished, the paper was still under revision. during this period, sars-cov-2 spreaded throughout brazil, causing 1.72 million confirmed cases and 68055 deaths. so, we extended this paper, giving an update of the situation, to compare our predictions with what happened. after rio de janeiro and são paulo, the next cities to reach three figures in their reported cases where belo horizonte/mg, brasília/df, porto alegre/rs, salvador/ba and fortaleza/ ce. these cities have small effective distances to sao paulo, and for that reason, with high probability of outbreak according to our models (s1, s3, s4 figs). fast contamination of cities directly connected by road to sao paulo and rio de janeiro was also observed, mainly to the former. one month later, by the end of may, covid-19 activity was already present in many cities along the coast as expected. these patterns had been predicted by the model. we detected that micro-regions in the northeast, in particular ceará and bahia, should receive attention due to the synergistic effect of geographical and social vulnerabilities on the magnitude of the covid-19 public health crisis. ceará was later strongly hit by the epidemic and had to implement lock down measures when hospital beds became unavailable. our model underestimed the spread of the epidemic within the amazon region. this is due to the fact that human mobility by boat, the main mode of travel in the region, was not well represented in our mobility matrix. thus, we did not predict the fast spread to communities along the amazon river between manaus and belem. knowing in advance which regions could potentially suffer the biggest hit first might have allowed authorities to opt for preemptive differential investments to the public health care system (sus) in these regions. unfortunately, the initial efforts by the government to allocate resources in a rational way was not timely enough and was eventually interrupted by political and economic reasons. world health organization. coronavirus disease (covid-19) outbreak linha do tempo coronavírus notificação de casos pelo novo coronavírus (covid-19)-plataforma integrada de vigilância em saúde brasil confirma transmissão comunitária de coronavírus; entenda o que é capitais de sp e rj têm transmissão comunitária do coronavírus ministé rio da saúde confirma transmissão comunitária no rj e em sp the epidemic wave of influenza a (h1n1) in brazil censo demográ fico 2010: resultados gerais da amostra global disease spread: statistics and estimation of arrival times the hidden geometry of complex, network-driven contagion phenomena mathematical epidemiology nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study. the lancet early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia the transmissibility of novel coronavirus in the early stages of the 2019-20 outbreak in wuhan: exploring initial point-source exposure sizes and durations using scenario analysis estimating the risk of sustained community transmission of covid-19 outside mainland china substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (covid-19) transmission dynamics of 2019 novel coronavirus (2019-ncov). biorxiv extract and visualize the results of multivariate data analyses estimating the number of clusters in a data set via the gap statistic r: a language and environment for statistical computing cluster: cluster analysis basics and extensions corrplot": visualization of a correlation matrix factominer: an r package for multivariate analysis we would like to thank marcia triunfol for her help in preparing this manuscript, luiz max carvalho for his revision, and alain barrat for helpful discussions. key: cord-296435-6dergkha authors: wang, tiehua; liu, zhuang; wang, zhaoxi; duan, meili; li, gang; wang, shupeng; li, wenxiong; zhu, zhaozhong; wei, yongyue; christiani, david c.; li, ang; zhu, xi title: thrombocytopenia is associated with acute respiratory distress syndrome mortality: an international study date: 2014-04-14 journal: plos one doi: 10.1371/journal.pone.0094124 sha: doc_id: 296435 cord_uid: 6dergkha background: early detection of the acute respiratory distress syndrome (ards) has the potential to improvethe prognosis of critically ill patients admitted to the intensive care unit (icu). however, no reliable biomarkers are currently available for accurate early detection of ards in patients with predisposing conditions. objectives: this study examined risk factors and biomarkers for ards development and mortality in two prospective cohort studies. methods: we examined clinical risk factors for ards in a cohort of 178 patients in beijing, china who were admitted to the icu and were at high risk for ards. identified biomarkers were then replicated in a second cohort of1,878 patients in boston, usa. results: of 178 patients recruited from participating hospitals in beijing, 75 developed ards. after multivariate adjustment, sepsis (odds ratio [or]:5.58, 95% ci: 1.70–18.3), pulmonary injury (or: 3.22; 95% ci: 1.60–6.47), and thrombocytopenia, defined as platelet count <80×10(3)/µl, (or: 2.67; 95% ci: 1.27–5.62)were significantly associated with increased risk of developing ards. thrombocytopenia was also associated with increased mortality in patients who developed ards (adjusted hazard ratio [ahr]: 1.38, 95% ci: 1.07–1.57) but not in those who did not develop ards(ahr: 1.25, 95% ci: 0.96–1.62). the presence of both thrombocytopenia and ards substantially increased 60-daymortality. sensitivity analyses showed that a platelet count of <100×10(3)/µlin combination with ards provide the highest prognostic value for mortality. these associations were replicated in the cohort of us patients. conclusions: this study of icu patients in both china and us showed that thrombocytopenia is associated with an increased risk of ards and platelet count in combination with ards had a high predictive value for patient mortality. acute respiratory distress syndrome (ards), the most severe form of acute lung injury (ali), is caused by several direct and indirect insults to the lung,life threatening and often lethal. ards usually requires mechanical ventilation and admission to an intensive care unit (icu); ards is a major cause of icu morbidity and mortality worldwide [1] . emerging viral diseases such as severe acute respiratory syndrome (sars) coronavirus, h5n1 avian-origin influenza virus, and h1n1 swine-origin influenza virus not only possess the potential for pandemic spread, but also cause ards [2] [3] [4] .these factors highlight the need for additional research to improve understanding of the pathogenesis of ards, with the ultimate goal of developing specific treatment [5] . ards is associated with several clinical disorders, including direct pulmonary injury from pneumonia and aspiration and extra-pulmonary injury from sepsis, trauma, and multiple transfusions [6] . although low tidal volume ventilation, neuromuscular blockers and prone positioning ventilation have advanced treatments [7] [8] [9] , there are currently no reliable predictive markers for early detection of ards in predisposed individuals. nonetheless, many efforts have been mounted to identify biologic markers, or biomarkers, for ards in critically ill patients, including studies of pulmonary edema fluid, blood, and urine [10] [11] [12] . recent advances on the pathophysiological mechanisms underlying ards have identified several clinical biomarkers to assess disease severity and outcome, including specific cytokines and their receptors (il-6, il-8, soluble tumor factor receptors i and ii), products of epithelial and endothelial injury [receptor for advanced glycation end-products (rage), surfactant protein d, icam-1, and von willebrand factor antigen], and markers of altered coagulation (protein c and plasminogen activator inhibitor-1) [13] . however, no individual biomarker is strongly associated with outcomes and thus cannot provide sufficient discriminating power for either diagnosis or prognosis. biomarker discovery and validation requires patient samples and must be combined with comprehensive clinical data collected from properly designed trials in different populations. given the acute onset and rapid clinical progress of ards, a prospectively enrolled cohort study in multicenter icus is suitable for more complete and unbiased ards/ali research [14] . using a protocol modified from a molecular epidemiology ards study established in boston, ma (boston cohort) [14] , we established a multicenter ards cohort in beijing, china (beijing cohort) in 2009. the overarching objectives of establishing this prospective cohort are to validate relevant biomarkers to ards, as well as genetic polymorphisms, discovered in previous usa studies in chinese population, and discover new biomarkers of ards with a comprehensive sampling protocol. in this report, we present initial results on the clinical factors associated with ards development and mortality in individuals with or at risk for ards. associated clinical factors were replicatedin the boston cohort. this study was approved by the institutional review boards(irbs) of the peking university third hospital, beijing friendship hospital, china-japan friendship hospital, beijing chao-yang hospital, and harvard school of public health and a written informed consent was obtained from each subject or an appropriateproxy of the patient. four medical and surgical icus within four tertiary hospitals participated in the study; hospitals covered the metropolitan area of beijing, china and included peking university third hospital in the northwest (16 beds), beijing friendship hospital in the south (16 beds), beijing chao-yang hospital in the east (14 beds), and china-japan friendship hospital in the northeast (10 beds). as an international collaboration, we used a modified study protocol for recruitment as previously described [14] . briefly, we screened each icu admission for eligible subjects, which were defined as critically ill patients with at least one predisposing condition for ards: 1) sepsis; 2) septic shock; 3) trauma; 4) pneumonia; 5) aspiration; 6) massive transfusion of packed red blood cells (prbc; defined as .8 prbc units during the 24 hours prior to admission); or 7) severe pancreatitis. to avoid interference in biomarker research from certain clinical conditions, exclusion criteria included:1) age ,18 years; 2) history of chronic lung diseases, such as interstitial pulmonary fibrosis or bronchiolitis; 3) history of pneumonectomy; 4) treatment with immunomodulating therapy other than corticosteroids, such as granulocyte colony stimulating factor, cyclophosphamide, cyclosporine, interferon, or tnf-a antagonists; 5) presence of other immunodeficient conditions, such as hiv infection, leukemia, or neutropenia (absolute neutrophil count ,1000/ml); 6) history of solid or bone marrow transplant other than autologous bone marrow transplant; and 7) directive to withhold intubation. sepsis and septic shock were defined by the american college of chest physicians/society of critical care medicine (accp/sccm) consensus conference [15] . after enrollment, subjects were followed daily for the development of ards, as defined by the american-european consensus committee (aecc) as follows [16] : a) evidence of hypoxemia with pao 2 /fio 2 #200 mm hg; b) evidence of bilateral infiltrates on chest radiographs; and c) absence of left atrial hypertension with pulmonary arterial occlusion pressure #18 mm hg or no congestive heart failure. controls were identified as at-risk patients who did not meet criteria for ards during the icu stay and had no prior history of ards. infiltrates on chest radiographs were defined as opacities that could not be explained completely by pleural effusions, mass, body habitus, or collapse. upper zone redistribution and pulmonary vascular congestion were not considered infiltrates. two pulmonary and critical care physicians interpreted daily chest radiographs; any disagreement went to a third intensivist for arbitration. all physicians underwent a consensus training session on the radiologic criteria for ards. all were blinded to the clinical status of the patients. we collected clinical data by chart review, including demographic information of age, gender, race, height, weight and medical history of ards, diabetes, tobacco and alcohol abuse, and liver disease. baseline clinical information, worst vital signs, and laboratory testing results in the first 24 hours of icu admission were collected for calculation of the acute physiological and chronic health evaluation (apache ii) score for severity of illness [17] . we also collected ventilatory parameters including the requirement and mode of mechanical ventilation, pao 2 /fio 2 ratio, positive end-expiratory pressure, tidal volume, and peak and plateau pressures. all enrolled patients were followed until one of the following situations occurred: hospital discharge, death, or 60 days after study entry. starting in late 2006, based on finding from ards network trials, chinese icus universally adopted lower tidal volume for mechanical ventilation [18] . baseline characteristics were compared between groups with fisher's exact test or chi-square test for dichotomous or categorical variables and with student's t test or mann-whitney test for continuous variables. for risk analysis of ards development, we initially used a logistic regression model with a backward stepwise elimination algorithm to select clinical risks or predictors from the univariate analyses with p,0.1; the final logistic regression models also included predictors from backward elimination. for mortality analysis, we used the log-rank test as a univariate measure of association and employed cox proportional hazards models to investigate each clinical variable's effect on clinical outcome. we used the time-dependent receiver operating characteristic (roc) method to determine the best cut-off value of thrombocytopenia in the prediction of prognosis of critically ill patients by exploring the area-under-the-curve (auc) values at 60-day mortality, and selected the maximal sumof sensitivity and specificity [19] . all analyses were performed with the sas statistical software package (version 9.31, sas inc., cary, nc), and p,0.05 was considered statistically significant. between july 2010 and april 2012, we recruited 178 patients with at least one predisposing condition for ards from the participating hospitals in beijing, china. the majority of patients were male (n = 125, 70%) and the mean age was 63 years (median: 69; interquartile range: 51-78 years) ( table 1 ). median apache ii score was 16 (interquartile range: [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] and median length of time in the icu was 10.5 days (interquartile range: 5-17 days). mechanical ventilation was used on 149 patients (84%) for a median length of six days (interquartile range: 2-11 days). thirtynine patients had diabetes, and one patient had chronic kidney disease. during hospitalization, 75 (41%) patients developed ards;among identified cases,31 (41%) and 70 (93%) of the 75 ards patients were diagnosed within the first 24 and 72 hours of icu admission, respectively. there were no significant differences in age, gender, smoking status, and initial apache ii score between ards patients and at-risk non-ards patients (table 1 ). in addition, the major physiological variables during the first 24 hours of icu admission were comparable, except that patients who developed ards had higher respiratory rates (p = 0.025). although not significant, ards patients were in the icu longer (ards median = 13 days; non-ards median = 9 days; p = 0.21) and on mechanical ventilation longer (ards median = 7 days; non-ards median = 5 days;p = 0.53) than non-ards patients. low tidal volume (,7 ml/kg) was used in treating patients with mechanical ventilation. although patient specific data was not available, protocolled low tidal volume ventilation was standardized in study icus. among predisposing conditions for ards in all enrolled patients, sepsis and/or septic shock (n = 149, 83%) were the most [20] were associated with development of ards.respiratory rate (.30 breaths/min), aspiration, and .1 risks for ards were also evaluated in model selection but were eliminated during model selection (not significant). apache ii score (removing age and gender components), age, and gender were forced in as covariatesbut not significant in logistic regression analyses. known factors related to ards, including septic shock, diabetes, and alcohol use, were also tested either by forcing as covariates, individually or combined into the model, and did not change the significant associations of sepsis, direct pulmonary injury, and thrombocytopenia with the development of ards (data not shown).because drinking habits in china differ from those in the u.s.,and it was difficult to develop a comparable criterion for alcohol abuse, we did not including alcohol abuse as risk factor in the analysis. we further conducted a stratified analysis and found that thrombocytopenia was significantly associated with ards in both the beijing cohort (univariate analysis, p = 0.01) and the boston cohort (univariate analysis, p,0.0001) (table s1 ), which has 851 ards and 1,027 non-ards patients recruited at massachusetts general hospital in boston, usa [21] , in the subgroup patients with septic shock, but not in non-septic shock subgroup (p = 0.95 and p = 0.15, respectively). some patients had already developed ards before icu admission, and this subgroup caseswas usually mixed with those patients who were diagnosed ards during the first 24 hours of icu admission, together accounting for a total of 41% ards in the beijing cohort and 40% in the boston cohort (340 of 851 identified cases). since the thrombocytopenia was defined by the lowest platelet counts during the first 24 hours of icu admission in these cohorts, some patients developed thrombocytopenia before the onset of ards, who were difficult to be distinguished within this subgroup ards, and could interfere with the finding that thrombocytopenia was associated with development of ards. we then performed a nested analysis on a clean subgroup patients, by removing ards patients who were diagnosed during the first 24 hours of icu admission, and found that thrombocytopenia was still significantly associated with ards risk (or = 4.04; 95% ci = 1.41-11.59; p = 0.009). because of the small sample size of the beijing cohort, we further conducted the sensitivity test in the boston cohort in 411 ards cases and 1,027 non-ards patients after removing ards patients who were the 60-day mortality rate for all patients was 39%, and the development of ards did not increase mortality risk (table 1) . among predisposing conditions for ards, septic shock was associated with increased mortality (p = 0.014), but pancreatitis was associated with decreased mortality (p = 0.01) in ards patients ( table 2 ). in contrast, pneumonia (p = 0.002) and external pulmonary injury (p = 0.013) had higher mortality rates in non-ards patients. univariate examination of demographic characteristics and physiologic variables in the first 24 hours of icu admission revealed that higher apache ii scores and older age were associated with increased mortality for both ards and non-ards patients ( table 2) . thrombocytopenia was significantly associated with mortalityof ards (p = 0.022) but not non-ards (p = 0.436) patients. in contrast, high serum creatinine levels (.2.0 mg/l) were associated with higher mortality in non-ards (p = 0.004) but not ards (p = 0.997) patients. there were no statistically significant differences between survivors and nonsurvivors for gender, history of diabetes, and tobacco or alcohol use. in multivariate analysis, apache ii score was consistently associated with increased mortality in ards, non-ards, and all patients (table 3) . thrombocytopenia was a mortality covariate for ards and all patients, but not for non-ards patients (table 3) . when replaced with coagulation points of the sequential organ failure assessment score (sofa), thrombocytopenia remained associated with higher mortality in ards [adjusted hazard ratio (ahr) = 1.38; 95% ci = 1.07-1.57; p = 0.04]and all patients (ahr = 1.30; 95% ci = 1.07-1.57; p = 0.008), but not in non-ards patients (ahr = 1.25; 95% ci = 0.96-1.62; p = 0.09). to replicate our findings, we analyzed data from the boston cohort. although univariate analyses identified more physiologic variables in the first 24 hours of icu admission and ards-predisposing conditions significantly associated with mortality (table s1 and s2), multivariate analyses identified apache ii score and thrombocytopenia as major risk factors for mortality in ards, non-ards, and all patients ( table 3) . we also found similar results when thrombocytopenia was replaced with coagulation points of the sofa score (data not shown). we further investigated the interaction between thrombocytopenia and ards on mortality of all patients by creating a combined covariate of the boston and beijing cohorts. in both univariate ( figure 1 ) and multivariate (figure 2 ) analyses, the combination of thrombocytopenia and ards had consistently higher patient mortality. taking advantage of the size of the boston cohort, we conducted a sensitivity analysis to determine the optimal platelet count for prognosis.with adjustmentsfor age, gender, apache ii score, and sepsis, a platelet count of 104610 3 /mlhad the maximal roc value (auc = 0.661; sensitivity = 0.508; specificity = 0.739; p = 0.0007). a sensitivity analysis confirmed a platelet count of 100610 3 /ml by considering a series of stepped (10610 3 /ml)cut this prospective multicenter cohort was established using a modified protocol originally implemented in the boston cohort [14] . among at-risk icu patients, 41% developed ards during icu admission, and a majority of those patients (93%) developed ards within the first 72 hours of admission. these observations are consistent with previous reports in the mostly-caucasian boston cohort [14] . moreover, the profiles of baseline physiologic variables and the major clinical risk factors between ards and atrisk non-ards patients are similar to previous reports from chinese [22, 23] andamerican [14] icus.furthermore, the observation of high baseline respiratory rate (.30 breaths/min) associated with ards cases was consistent withthe findings from several previous studies [24] [25] [26] . in this cohort, in addition to sepsis and direct pulmonary injury, thrombocytopenia was associated with the development of ards.enhanced platelet activation resulting in platelet deposition within the damaged pulmonary microvasculature has been supported by several clinical and preclinical studies of ali [27, 28] , and thrombocytopenia has been reported as a key feature of sars [29] . in the boston cohort, thrombocytopenia (named hematologic failure) was also identified as a risk factor for ards in multivariate analysis [14] . in another cohort of ali in rochester, minnesota (mayo clinic), however, researchers did not observe significant difference of platelet count between ali and non-ali patients with septic shock [18] . since the rochester cohort only focuses on a subgroup icu patients with septic shock, our stratified analysis revealed that thrombocytopenia was significantly associated with ards in both the beijing cohort and the boston cohort in the subgroup patients with septic shock, but not in non-septic shock subgroup. the different results might be explained by that the beijing cohort and the boston cohort focused on ards, which is the most severe form of ali. a major finding of this study is the association ofthrombocytopenia with increased ards mortality. extensive evidence demonstrates that platelet count and function are independently associated with increased icu morbidity and mortality [30] . although thrombocytopenia is a well-established prognostic marker for mortality in patients with sepsis and septic shock [31] , which are risk factors for developing ards, thrombocytopenia has been inconsistently associated with ards mortality in two previous studies with small patient series representing noncontemporary treatment eras [32, 33] . besides apache ii score, thrombocytopenia was the only risk factor for ards mortality identified in the beijing cohort. further, this association was replicatedwitha larger population and different ethnicities in the boston cohort. these results provide strong evidence that thrombocytopenia is a prognostic marker for ards mortality. in both beijing and boston cohorts, the combination of thrombocytopenia and ards further increased risk of 60-day mortality among critically ill patients. thrombocytopenia in icu patients is caused by multiple factors [34] and is considered a marker of illness severity with multiple organ dysfunction scores (mods), simplifiedacute physiology scores (saps), and apache scores.sepsis alone can cause moderate thrombocytopenia, as maladaptive platelet-neutrophil interactionssignificantly increase platelet activation and aggregation, as well as tissue injury [35] . the lung epithelium is central to both the pathogenesis and resolution of ards, and intra-alveolar coagulation changes (e.g., platelet-fibrin deposition and pulmonary vascular thrombi) are hallmarks of pathologic changes in ards [36] . thus, thrombocytopenia likely contributes to the development of ards; in return, the coexistence of ards may aggravate thrombocytopenia to increase mortality of critically ill patients. we used the same platelet count criterion from the boston cohort (,80610 3 /ml)to define thrombocytopenia [14] . although platelet counts are routinely measured daily in the icu, the epidemiology of thrombocytopenia in critically ill patients has not been well studied. further, illness severity scoring systems inconsistently consider platelet count;for example, the sofa score incorporates platelet count, whereas the apache score does not. different platelet count thresholds have been used in epidemiological studies for the prevalence, incidence, risk factors, and consequences of thrombocytopenia among critically ill patients [37] . currently, the rand/ucla appropriateness methodrecommends a platelet count threshold of ,100610 3 / ml, or a .30% decrease in platelet counts, for epidemiological research of thrombocytopenia [20] . taking advantage of a large patient population in the boston cohort, we conducted a sensitivity analysis and determined that platelet count (,100610 3 /ml) was a significant prognostic marker for ards. we further replicated this cut-point value in the beijing cohort. there were some differences between the beijing and boston cohorts. when evaluated individually, there were different association profiles for thrombocytopenia and ards with mortality of all patients with at least one risk factor for ards. thrombocytopenia was significantly associated with higher mortality in the beijing cohort, but not the boston cohort. conversely, ardswas associated with higher mortality in the boston cohort, but not the beijing cohort. it is unexpected that ards did not increase mortality in the beijing cohort, and. it is counter-intuitive that in the univariate analysis thrombocytopenia would be associated with increased mortality in the ards group but not in the non-ards group given that thrombocytopenia is a marker of severity of illness in critical care populations generally. however, the raw numbers were in the direction of higher mortality with lower platelets in the non-ards group, these findings could be explained by the limitation of multiple subgroup analysis in a relatively small dataset. moreover, for several known risk factors or comorbidities of ards [14, [24] [25] [26] , such as septic shock, pneumonia, pancreatitis, trauma, multiple transfusions, and diabetes, we did not observe significant different between ards and non-ards patients in the beijing cohort. due to the relative scarcityand high cost of medical resources to the general chinese population, the participating hospital icus in the beijing cohortmay have admitted more severely ill patients, resulting in less difference in illness severity between ards and non-ards patients. accordingly, although most physiologic variables of the first 24 hours of icu admission, including apache score, were comparable between ards and non-ards beijing cohort patients, thrombocytopenia (platelet counts ,80610 3 /ml) was more common in the beijing cohort than the boston cohort (30.3% vs. 13.9%, respectively; p,0.0001). it is also possible that different ethnicities account for the observed differencesbetween cohorts. based on the boston cohort, our study protocol was modified by the inclusion of severe pancreatitis as a predisposing condition for ards. severe pancreatitis is a well-established risk factor for the development of ards [38] and is frequently observed in critically ill patients in china. in the beijing cohort, we identified 23 (12.9%) cases of severe pancreatitis, similar to a previous report of 15.6% in a large chinese icu survey [39] . about 52% of severe pancreatitis cases eventually developed ards during icu admission. severe pancreatitis was not associated with ards risk, but was associated with lower mortality. however, the beijing study is limited by a small sample size. with the patients' enrollment keeps, we will further evaluate severe pancreatitis as a clinically important factor in the development and outcome of ards. this study describes the successful establishment of a prospective, multicenter cohort study of critically ill patients at-risk for ards in beijing, china. initial characterization of the clinical factors associated with ards risk and mortality revealed an association between thrombocytopenia and ards mortality. we replicated these findings in the larger and more diverse boston cohort, suggesting that the beijing cohort can provide comprehensive data and samples to identify biomarkers for the early diagnosis, prognosis, and treatment of ards. epidemiology of acute lung injury and acute respiratory distress syndrome interactions between influenza and bacterial respiratory pathogens: implications for pandemic preparedness clinical features and outcomes of severe acute respiratory syndrome and predictive factors for acute respiratory distress syndrome newly recognized causes of acute lung injury: transfusion of blood products, severe acute respiratory syndrome, and avian influenza future research directions in acute lung injury: summary of a national heart, lung, and blood institute working group the acute respiratory distress syndrome higher versus lower positive end-expiratory pressures in patients with the acute respiratory distress syndrome prone positioning in severe acute respiratory distress syndrome neuromuscular blockers in early acute respiratory distress syndrome plasma protein c levels in patients with acute lung injury: prognostic significance elevated plasma levels of soluble tnf receptors are associated with morbidity and mortality in patients with acute lung injury higher urine nitric oxide is associated with improved outcomes in patients with acute lung injury the pathogenetic and prognostic value of biologic markers in acute lung injury clinical predictors of and mortality in acute respiratory distress syndrome: potential role of red cell transfusion sepsis change bundles: converting guidelines into meaningful change in behavior and clinical outcome the american-european consensus conference on ards. definitions, mechanisms, relevant outcomes, and clinical trial coordination apache ii-a severity of disease classification system ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury time-dependent roc curves for censored survival data and a diagnostic marker management of thrombocytopenia in the icu (pregnancy excluded) prognostic significance of elevated cardiac troponin-t levels in acute respiratory distress syndrome patients epidemiological investigation on acute respiratory distress syndrome occurring in intensive care units in beijing from retrospective analysis on acute respiratory distress syndrome in icu risk factors for the development of acute lung injury in patients with septic shock: an observational cohort study early identification of patients at risk of acute lung injury: evaluation of lung injury prediction score in a multicenter cohort study early acute lung injury: criteria for identifying lung injury prior to the need for positive pressure ventilation* intravascular coagulation associated with the adult respiratory distress syndrome the risk factors, incidence, and prognosis of ards following septicemia the severe acute respiratory syndrome thrombocytopenia and prognosis in intensive care multiple organ dysfunction score: a reliable descriptor of a complex clinical outcome adult respiratory distress syndrome. prognosis after onset adult respiratory distress syndrome. sequence and importance of development of multiple organ failure. the prostaglandin e1 study group coagulopathy in critically ill patients: part 1: platelet disorders beyond thrombosis: the versatile platelet in critical illness the epithelium in acute lung injury/ acute respiratory distress syndrome the frequency and clinical significance of thrombocytopenia complicating critical illness: a systematic review acute lung injury and ards in acute pancreatitis: mechanisms and potential intervention risk factors for the prognosis of acute kidney injury under the acute kidney injury network definition: a retrospective, multicenter study in critically ill patients the authors would like to thank michelle gong, ednan k. bajwa, and taylor thompson from the molecular epidemiology of ards project for help in study design and preparation of study protocols. key: cord-312678-81gnmxbk authors: elayeh, eman; aleidi, shereen m.; ya’acoub, rawan; haddadin, randa n. title: before and after case reporting: a comparison of the knowledge, attitude and practices of the jordanian population towards covid-19 date: 2020-10-15 journal: plos one doi: 10.1371/journal.pone.0240780 sha: doc_id: 312678 cord_uid: 81gnmxbk coronavirus disease2019 (covid-19) is an emerging contagious infectious disease. it is pandemic and has affected more than 21 million people and resulted in more than 750,000 deaths worldwide (https://www.worldometers.info/coronavirus/#countries; 14/08/20). our research group initiated a study to ascertain the knowledge, attitude and practices (kap) of jordanians toward covid-19 prior to any initial case report in jordan. this project was underway when the first jordanian case was reported. we extended our study to identify how case reporting would alter public kap towards covid-19. this cross-sectional study randomly selected and recruited 2104 jordanian adults. a four-section questionnaire was devised to address the sociodemographic characteristics of the subjects and their kap toward covid-19. the mean knowledge score for the study population was 15.9 ± 2.2 (out of the 20 knowledge questions), with 60.9% of the participants having good knowledge about covid-19. participants’ practices to prevent transmission of covid-19 were adequate in more than 60% of participants. most participants had positive attitudes regarding their role in preventing covid-19 and many of the participants’ attitudes and practices changed to more appropriate ones after reporting the first case of covid-19 in jordan. the percentage of participants who trust the government in confronting covid-19 increased significantly (p value < 0.001). however, one alarming and unexpected finding was that the prevention practice score of participants working in the medical field was similar to those from the general population. this may necessitate stricter training and guidelines for this group who will be in the frontline in combating the disease. impact of this study: the data generated from this study shows that when cases of disease were reported, the public’s attitudes and practices improved in many aspects, and that confidence in the government to contain the disease was boosted. we believe that this study is important in allowing other, international governments to develop an understanding of public kap during pandemic disease outbreaks. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 after reporting 70 cases which were all isolated in dedicated hospitals, the jordanian government applied a nationwide lockdown and activated the defense law, which allows the army and police to ensure the adherence of public to the curfew [16] . under these circumstances, where public health is threatened by the pandemic and since the people's attitudes and practices play a crucial role in limiting or spreading the disease in their community, evaluating the knowledge, attitude, and practice (kap) of the general public towards the covid-19 is crucial. therefore, the initial aim of this study was to evaluate the overall knowledge, attitude and practices (kap) of jordanian citizens to the ongoing international situation regarding the emergence and pandemic nature of covid-19. however, we were lucky that our study was almost complete when the first case of the disease was reported in jordan. hence, we rapidly adapted our study in order to engage more participants and to evaluate how this reported case altered the kap of jordanian citizens. the aim of this report is to aid decision makers in their understanding of the public knowledge and awareness of the disease, the attitudes of the public and their practices to take necessary measures to prevent disease spread. this was a cross-sectional study conducted during the early days of covid-19 pandemic of 2019 and 2020 and over the period from 12 th february to 19 th march 2020. when the study started there were no reported cases in jordan. during the study, the first covid-19 case was reported in jordan, which encouraged the authors to extend the study and include more participants in order to identify how case reporting would alter the public kap towards covid-19. the study targeted adult resident jordanian nationals. it was a questionnaire based and self-selection study. the institutional review board (irb) at the deanship of scientific research, the university of jordan, approved the study protocol and the questionnaire (irb 28/2020). the questionnaire was developed based on extensive literature review [3, [17] [18] [19] [20] . it consisted of four main sections; (a) demographic and general characteristics of the participants, (b) participants knowledge regarding covid-19, its mode of transmission, and its preventative measures (20 questions), (c) participants' attitudes towards covid-19 and its preventative measures (13 questions) , and (d) participants' practices towards prevention of covid-19 transmission (6 questions). the questionnaire content was translated from english into arabic and then critically revised and face-validated by several academic colleagues. since the jordanian general public knows covid-19 as "coronavirus disease", this term was used in the questionnaire. amendments were made according to the notes and comments received after piloting to a sample of 50 adults chosen from the friends, neighbors and coworkers of the authors. the questionnaire was then developed as a google form and disseminated to participants via mobile applications, eg: whatsapp [21, 22] . snow ball sampling technique was used to distribute the google form and enroll participants [22, 23] . the link to the google form was sent to the contact lists of the authors, in addition to neighbors, friends, relatives, coworkers of all levels and departments, friends of friends, different whatsapp groups, who were also asked to complete the form and to disseminate it to all the people they know in jordan. this method of disseminating the questionnaire was selected in view of the need to reduce direct contact with participants under the pandemic situation. participants were asked not to identify themselves in any fashion. also they were informed that completing the questionnaire and returning it were considered as a formal consent to participate in the study since the participant can, at any moment, stop answering questions or choose not to "submit" the final form. a minimum sample of 1050 adults aged 18 and older was estimated based on the following equation [24] : n ¼ where n is the sample size, zα: type one error = 1.96 when α = 5%; zβ: type two error = 1.28 when β = 10%; q = 1-p: expected non-prevalence; p = proportion in the population possessing the characteristic of interest (based on the estimate that 50% of the respondents knew general information about covid-19, its routes of transmission and the main preventative measures), d = one-half of the desired interval of confidence, in this study d = 5%. accordingly, by filling in the equation, n = 0.5x0.5 (1.96+1.28) 2 / 0.05 2 = 1049.76. statistical analysis was performed using spss version 20.0 (spss inc., chicago, il). descriptive statistics were used to describe demographic characteristics of participants. categorical variables were presented as valid percentages to account for missing data with their frequencies, while continuous variables were presented as mean with standard deviation (sd). two scores were calculated for the participants: knowledge about covid-19 and its preventative measures, and participants' practices towards preventing transmission of covid-19. participants' knowledge scores were evaluated using the number of correct questions they answered out of 20 covid-19 knowledge questions. in order to calculate the knowledge score, correct answers were assigned a score of one, while wrong or "i don't know" answers were assigned a score of zero. participants were considered adequately knowledgeable if their knowledge score, for all knowledge questions, was higher than or equal to the sample median of the knowledge score. internal consistency of the knowledge questions was tested using cronbach alpha coefficient as a reliability test. the results showed that the cronbach alpha for knowledge (20 items) was 0.7. cronbach alpha within the range 0.6 to 0.7 is considered adequate and reliable [25] . similarly, participants practice towards preventing transmission of covid-19 score was calculated by assigning a score of one for appropriate practices, while inappropriate practices were assigned a score of zero. the maximum value of practice score is six based on the total number of correct practice items. participants' knowledge, attitudes and practices were compared before and after the reporting of the first confirmed case of coronavirus in jordan which was documented on 2 nd march 2020. by default, 'google forms' identifies the date and time of each participant's response which enabled us to sort the responses submitted into those made before and after case reporting. chi-square test was used for these comparisons. parametric tests including independent sample t-test and one way anova test were used to test the differences among the variables that affect both knowledge and practice scores (bivariate analysis) as specified in the results section (data were first tested for normality using kolmogorov-smirnov (k-s) test and for homogeneity of variances using levene's test). in addition, independent sample t-test was used to test the differences in the scores between the two groups (before and after case reporting). all hypothesis testing was two-sided. a p-value of < 0.05 was considered significant. the total number of participants recruited in the study was 2104. 832 participants were recruited before case reporting and 1272 participants were recruited after case reporting. more than 50% of participants were in the age group of 18-35 (62.7%, n = 1321). females accounted for 75.4% (n = 1586) of the study population. more than 75% of participants had a university or postgraduate degree (81.6%, n = 1717), and 43�6%, (n = 917) had education in the medical field. since the cohort participating in the study after case reporting differed from those participating before case reporting, there were variations in the sociodemographic data of the two groups (p< 0.05, chi square) except for gender and working in the medical field which were similar (p>0.05). participants' sociodemographic characteristics are presented in table 1 . the mean knowledge score for the whole study population was 15.9 ± 2.2 (out of the 20 knowledge questions), with 60.9% (n = 1281) of the participants having a score equal to or higher than the median (16.0) and consequently were considered to have adequate knowledge about covid-19. only 2.4% (n = 51) of the participants were able to correctly answer all of the 20 knowledge questions and 8% (n = 168) of participants were able to correctly answer 19 questions. in addition, 17% (n = 357) of participants knew the general information related to the covid-19, its symptoms and the highest risk group. however, more than 50% of the study population incorrectly thought that death is a common complication of covid-19 (44.2%, n = 929) ( table 2) . regarding covid-19 transmission, a major gap of knowledge regarding routes of covid-19 transmission was identified where only 2.3% (n = 49) of the population correctly recognized all the routes of covid-19 transmission. considering covid-19 preventative measures, more than half of the participants were able to identify the main preventative measures. however, only 18.3% (n = 385) of them knew plos one that a facemask should not be used daily as a preventative measure for covid-19 transmission (which was a recommendation by who and moh at the early days of the pandemic). in general, the mean knowledge scores of participants improved after case reporting (when compared with those exhibited before case reporting) in the three areas studied: definition, signs and symptoms, risk groups and complications (mean score 5.5 ±1.2 vs 5.1 ±1.2, p<0.005); mode of transmission (mean score 3.3 ± 0.8 vs 3.2 ± 0.7, p = 0.037) and prevention of transmission and treatment (mean score 7.6 ± 1.1 vs 6.9 ± 1.1, p<0.005). accordingly, the total knowledge score was improved from 15.2 ± 2.2 before case reporting to 16.3 ± 2.1 after case reporting (p value <0.001, independent sample t-test) ( table 2 ). the detailed frequencies for individual questions are presented in table 2 . the most common source of participants' information about covid-19 was social media, such as facebook, twitter or others (64.3%, n = 1353), followed by internet searching such as google (59.4%, n = 1249), and television (49�7%, n = 1046). a much lower proportion of respondents relied on newspapers (37.3%, n = 784), friends (29.3%, n = 616), brochures (20.2%, n = 426) or physicians' offices (6.6%, n = 138) to get information. a significantly higher knowledge score (p-value < 0.001) was associated with using internet search (score = 16.4) as sources of information (p-value < 0.001; independent sample t-test), while other sources were not associated with significantly higher knowledge score. most participants had positive attitudes regarding their role in preventing covid-19. in particular, 67.2% of participants thought that they could protect themselves against covid-19 and 88.7% of them thought that following advised preventative measures would be effective. however, more than 50% of participants didn't trust the jordanian ministry of health's (moh) approach to confronting covid-19 (57.6%, n = 1211) or the information provided by governmental authorities about the exact number of cases (66.1%, n = 2091). similarly, more than half of participants believed that the occurrence of coronavirus was related to international tension and trade wars (57.9%, n = 1219) and 49.7% (n = 1046) of participants believed that the recent coronavirus was created in a laboratory and was not naturally occurring. on the other hand, most participants were either unsure or didn't believe that herbal remedies (71.7%, n = 1508) or antibiotics (85.9%, n = 1808) were effective in treating or preventing covid-19. interestingly, many of the participants' attitudes had changed after reporting the first jordanian case of covid-19 (2 nd march 2020). most importantly, the percentage of participants who believe that covid-19 was a serious and life threating infection dropped significantly from 81.6% to 52.6% (p value < 0.001; table 3 ). on the other hand, the proportion of participants who trusted moh in confronting covid-19 increased significantly from 35.9% to 46.7% (p value<0.001). similarly, the percentage of participants who thought that they could protect themselves against infection with covid-19 (59.3% vs.72.3%, p value <0.005) and those who thought that treatment approaches were effective also increased significantly (from 31.0% to 42.7%, p value = 0.009). the mean practice score of all the participants was 3.95 ± 1.7 (out of the 6 practice questions) with 66.7% (n = 1403) of the participants having a score equal to or higher than 4 and consequently were considered as having adequate practices to prevent spread of covid-19. respondents' practices are presented in table 4 . participants' practices to prevent transmission of covid-19 in terms of avoiding hand shaking, hugging, kissing and crowded areas, and using disinfectants were appropriate in more than 60% of participants. interestingly, all these practices have increased significantly after reporting the first case of covid-19. on the other hand, the proportion of participants wearing facemasks and avoiding the purchase of chinese table 4 ). overall, the observed practice score improved significantly when compared between respondents before case reporting and after case reporting (3.65 ± 1.9 vs. 4.2 ± 1.4 respectively, p value < 0.001, independent sample t-test; table 4 ). factors affecting participants' knowledge and practice towards preventing covid-19 were determined by bivariate analysis. overall participants' knowledge and practice scores were associated with age, educational level and education in health or medical field. in addition, participants' knowledge score was associated with gender and work in the medical field. participants' practice score was also associated with marital status. for each sociodemographic characteristic, the calculated knowledge score (table 5 ) or practice score ( table 6 ) was improved after case reporting when compared to the value before case reporting. detailed results are shown in tables 5 and 6 . since the who declaration of covid-19 as a public health emergency on 30 th january 2020, health authorities around the world, led by who, have initiated huge campaigns to increase the awareness of the people toward the disease and to disseminate the appropriate practices to prevent its transmission [4] . as a member of these authorities, the moh in jordan started similar campaigns relying on different forms of media. the first case in jordan was reported on 2 nd march 2020 for a citizen returning from italy [28] . our study commenced by distributing the questionnaires on 17 th february before the reporting of the first case of covid-19 in jordan, and the questionnaire process ended on 19 th march. by that time, the measures taken by the country included closure of schools and universities, quarantining thousands of incoming air passengers in hotels, and prohibiting any kind of social gathering (including the closure of all mosques and churches). during that period, the number of cases reported increased to 52 without fatality. this gave our team an opportunity to compare the change in knowledge, attitudes and practices of jordanians towards covid-19 before and after the reporting cases of covid-19 illness. the sample population of this study (2104 participants) was largely well educated with females predominating. the observed skew in the sample toward females and well educated participants has also been seen in previous studies in jordan [29] [30] [31] [32] . in addition, the vast majority of participants were below the age of 55 years, which is representative of the generally younger population of jordan, where at the end of 2019 the estimated proportion of population below 55 years was around 92% [33] . the overall knowledge of the respondents was generally adequate (total knowledge score 15.9 out of 20). they exhibited excellent knowledge of the organs targeted by the virus, its nature as a zoonotic disease, and its signs and symptoms. however, the respondents had an exaggerated idea about the expected death rate of the disease where more than 55% considered death to be a very common outcome. this idea could have been drawn in their minds due to the effect of media focus on death rates and novel cases, rather than recovered cases [34, 35] , thus, giving the audience or readers the impression that this disease is highly fatal. the respondents were well aware of the most common routes of the disease transmission, such as close contact, respiratory droplets or touching contaminated surfaces. the vast majority of participants (> 90%) had an excellent knowledge about the required measures to prevent the disease. however, the more scientific questions related to the role of influenza vaccine in protecting from covid-19 or the one related to use of a specific drug to treat the illness were correctly answered by only around two thirds the participants. the participants' knowledge was gained through multiple sources of information. the electronic sources such as social media (64%) and internet (59%) were the top sources followed by television (50%). these results suggest that in this era, where electronic media sources are predominate at the expense of more traditional media sources, health authorities should focus their healthrelated campaigns on electronic media and they should consider adding social media platforms to their public communication tools, in order to reach the vast majority of population [36] [37] [38] [39] . it is noteworthy that people who obtained their information from internet searches had significantly higher knowledge scores than those using other sources. this is probably due to the fact that people who were keen to get more information about the disease actively sought this information from the internet. however, those relying on social media, such as facebook, twitter and whatsapp may have received inaccurate information since social media platforms often lack fact-checking and editorial control [40, 41] . when investigating the knowledge of the participants the major factors were; the educational level, education background, and the field of work (p value within group <0.005 for each) ( table 5 ). this was anticipated and is in line with previous studies that compare the knowledge of the general population about a specific health-related issue with those of higher levels of education or with a health-related backgrounds [42, 43] . the latter would have much better knowledge. comparing the mean knowledge scores of the respondents before and after case reporting (table 2) , showed an improvement in the respondents' knowledge after case reporting (p value between groups <0.05, independent sample t-test). we are aware that differences in the population characteristics before and after case reporting might contribute to observed changes in the knowledge score. however, statistical analysis of the factors affecting participants' knowledge (table 5 ) exhibits that the observed improvement in the knowledge was significant in all the classes of each characteristic (age, gender, marital status, etc, p value between groups <0.05), indicating a palpable effect of case reporting on the participants' knowledge. apparently, people become more interested in learning about a disease when it is seen to be proximal to their vicinity as has been seen in other similar situations [18] . this suggests the importance of pursuing health campaigns and associated recommendations when case reporting of any outbreak or epidemic commences in a country, as the public seems to become more receptive to that additional information. a significant change in the attitudes of participants in many aspects was noticed after reporting cases of covid-19 illnesses in jordan. unexpectedly, the participants seemed to become more optimistic and comfortable after the reporting of cases of covid-19. this optimism appeared in their perception to the severity of disease as a life threatening one, the effectiveness of preventative measures, their ability to protect themselves, the effectiveness of the treatment approaches and the ability of moh to confront the disease. although this is initially puzzling, it can be explained by the unique situation in jordan. shortly after first case reporting in jordan, there was a sense of panic among the population. in order to calm the public and fight the rumors, the first covid-19 patient was interviewed in hospital where he was isolated, by different national and regional tv and radio channels [44] . the patient tried to comfort the people about his situation, since he only suffered from mild respiratory illness. being a traveler returning back from italy, which was hard hit by the pandemic at that time, he compared the looser measures taken by italian authorities to contain the disease with the tougher measures taken in jordan, and he praised the latter. the same thing happened with the second case who only suffered from mild illness. the situation of both patients was of great public interest until they were discharged from hospital. apparently, the message perceived by the public from both patients was that the disease is mild and can be prevented, and that the measures taken by the authorities are effective. our study suggests that the jordanian government's open approach with the public about the disease, its spread, and the necessity for stringent control measures enhanced the optimistic attitudes of the participants towards the disease and its management after case reporting in the country. on the other hand, the attitudes of the participants in other areas remained the same before and after reporting cases in jordan. more than half the respondents indicated their willingness to buy the vaccine (most pharmaceuticals are paid for by the patient in jordan) and the number increased a little more if the vaccine was offered free of charge. these figures are relatively high compared to the general attitudes of jordanians towards vaccinations other than the mandatory ones at childhood [42, 45] . this improved attitude could be related to the worldwide worries about this disease, which have made the jordanians more willing to protect themselves against coronavirus. as for the emergence of this virus, almost half of the participants believed that the virus was created in a laboratory and more than half believed that its emergence was the result of political reason linked to international tension or trade war. this conspiracy theory is not limited to jordanians, and has been reported worldwide. some americans accused china of bioengineering the virus as a bioweapon and conversely, some chinese have accused the american military of introducing it into china. nevertheless, these theories have been refuted in the media and by scientific studies [46] [47] [48] [49] . the practices of the participants in order to prevent the transmission of the virus were found to be adequate for more than two thirds of the respondents. however, there was a remarkable improvement (p < 0.001) in the participants' practices after the reporting of cases of the disease in jordan. the practices evaluated included; avoidance of shaking hands, avoidance of kissing, avoidance of crowded places, and an increase in the use of disinfectants (table 4 ). this improvement in practices' scores was anticipated because the public was observing the disease having increased proximity to their areas, thus they were more willing to engage with the recommendations of the moh regarding covid-19. the improvement in practices' scores seen among the population after case reporting compared to those before case reporting cannot be attributed solely to differences in sample population, because the increase in practice score was obvious in all the population characteristics studied (table 6 ). after reporting cases in jordan, there was a significant decrease in the number of participants using facemasks when going outside. this contradicts the behavior of chinese people during the epidemic in their region, where 98% of a study population indicated wearing facemasks when leaving homes [50] . nevertheless in this respect, the practices of jordanian respondents exhibited better adherence to the instructions of moh at the time. in the early days of the pandemic who and moh instructions to the general public were not to use facemasks, as it was feared this would lead to shortage of masks for essential health and care workers. many other factors affected the overall practices of the respondents. participants with ages of more than 55 years had statistically better practices than those in the lower age groups (18-25yr and 26-35yr; p value within group <0.005). this implies that older respondents are more cautious about their health than younger participants due to who and moh warnings that people above the age of 60 years were at higher risk of developing severe complications than those of younger ages [51] . these findings suggest that the public is willing to implement the recommendations of the health authorities when continuously and firmly directed to do so. surprisingly, the negative factors affecting the overall practices of the participants towards the prevention of transmission are the education level and having a health-related education. these participants scored lower than their counterparts in terms of the best practices (table 6 ). this finding, albeit odd is not uncommon. in a study among health care workers in saudi arabia to evaluate their kap about middle east respiratory syndrome (mers), physicians, pharmacists, nurses and technicians showed low to average scores on practices, but better knowledge and attitude scores [52] . we speculate that having a better education, particularly in a health-related field, provides the participants with confidence that they are aware of the risks and know better how to protect themselves. another disappointing finding is that the overall participants practice score of those working in the health field is the same as those working outside this field (3.9 for each, table 6 ). this finding should be alarming to the health authorities and health care sector, since these workers will be in the front line in combating the epidemic. being lenient in applying the strictest measures and practices in protecting themselves and preventing the spread of infection could lead to a disaster. therefore, the health sector should immediately intensify education and extensive training and setting guidelines for the proper practices among healthcare workers of different specialties. the findings of the study should be interpreted with the following limitations in mind: • sampling of the study was made through social media, what's app in particular, which could pose some bias to the study where underprivileged people or those having problems in using electronic devices may not be able to participate, thus, will be under-represented in the sample. • females and people with education level of more than 12 years were over-represented in the sample studied when compared to the general jordanian population. • like any other self-reported study, responses (mainly of attitudes and practices) could have been reported based on social desirability, not the actual situation of the participants. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and coronavirus disease-2019 (covid-19): the epidemic and the challenges who [internet]. 2020 centers for disease control and prevention who. events as they happen-rolling updates on coronavirus disease early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia novel coronavirus: where we are and what we know coronavirus disease 2019 treatment: a review of early and emerging options draft landscape of covid-19 candidate vaccines modes of transmission of virus causing covid-19: implications for ipc precaution recommendations q&as on covid-19 for older people refugee population by country or territory of asylum-| data health care and pharmacy practice in jordan higher health council & world health organization government suspends educational system in a bouquet of coronavirus preventive measures government announces 10 decisions to fight against coronavirus jordanians now ponder economic cost of coronavirus. in: al-monitor, the pulse of the middle east covid-19) advice for the public bin saeed a. knowledge, attitude and practice of secondary schools and university students toward middle east respiratory syndrome epidemic in saudi arabia: a cross-sectional study covid-19)-how coronavirus spreads covid-19)-how to protect yourself & others. in: centers for disease control and prevention public knowledge, attitudes and practices towards covid-19: a cross-sectional study in attitudes and practices towards covid-19: an epidemiological survey in north-central nigeria sage research methods foundations parental knowledg attitudes and behaviours towards influenza a/h1n1 in italy global patterns in students' views of science and interest in science who says people receiving packages from china not at risk of contracting coronavirus-society & culture-tass los angeles daily news who. coronavirus disease 2019 (covid-19) situation report-43 promotion of appropriate knowledge and attitude towards medicines among schoolchildren in jordan: the role of teachers knowledge and information sources about covid-19 attitudes and behavior regarding antibiotics use and misuse among adults in the community of jordan. a pilot study albsoul-younes am. knowledge, awareness and practices towards seasonal influenza and its vaccine: implications for future vaccination campaigns in jordan. fam pract table 2: estimated population of the kingdom by sex why coronavirus is killing far more men than women-the washington post. the washington post more covid-19 cases, deaths reported in rest of world than in china-who the use of social media among saudi residents for medicines related information internet and social media use for antibiotic-related information seeking: findings from a survey among adult population in italy taking the paper out of news mers-cov infection: mind the public knowledge gap fake news: spread of misinformation about urological conditions on social media the trump effect: with no peer review, how do we know what to really believe on social media? clin colon rectal surg influenza vaccination coverage rates, knowledge, attitudes, and beliefs in jordan: a comprehensive study hepatitis b knowledge, perceptions and practices in the french general population: the room for improvement jordan free from corona cases as infected citizen left quarantine seasonal influenza vaccination among older adults in jordan: prevalence, knowledge, and attitudes. hum vaccines immunother covid-19 coronavirus was not bioengineered. here's the research that debunks that idea covid-19 coronavirus epidemic has a natural origin-sciencedaily coronavirus did not come from a lab: experts debunk myths that china or usa bioengineered covid-19. mail online evolutionary origins of the sars-cov-2 sarbecovirus lineage responsible for the covid-19 pandemic. nat microbiol knowledge, attitudes, and practices towards covid-19 among chinese residents during the rapid rise period of the covid-19 outbreak: a quick online cross-sectional survey who. coronavirus disease 2019 (covid-19) situation report-51 middle east respiratory syndrome (mers): comparing the knowledge, attitude and practices of different health care workers the authors would like to thank professor phillip collier (visiting professor, faculty of pharmacy and medical technology, university of petra) for his critical review of the paper and proof reading. haddadin. the design and timing of this study could not have been more fortuitous, in that it allowed the authors to rapidly adapt and expand a preliminary study into a fully operational before-andafter study of kap towards covid-19. the findings of this study are necessarily complex, but generally show that the population reacts well to open and honest governmental advice about pandemics and can rapidly adopt safe practices in response to appropriate advice. the international relevance of this work is obvious, in that jordan achieved one of the lowest rates (per million population) of covid-19 infection ranking 186 and 177 out of 213 in the number of cases and related fatality respectively, in the world (https://www.worldometers.info/ coronavirus/#countries; 14/08/20). key: cord-317912-v2wovcqd authors: akmatov, manas k.; gatzemeier, anja; schughart, klaus; pessler, frank title: equivalence of selfand staff-collected nasal swabs for the detection of viral respiratory pathogens date: 2012-11-14 journal: plos one doi: 10.1371/journal.pone.0048508 sha: doc_id: 317912 cord_uid: v2wovcqd background: the need for the timely collection of diagnostic biosamples during symptomatic episodes represents a major obstacle to large-scale studies on acute respiratory infection (ari) epidemiology. this may be circumvented by having the participants collect their own nasal swabs. we compared selfand staff-collected swabs in terms of swabbing quality and detection of viral respiratory pathogens. methodology/principal findings: we conducted a prospective study among employees of our institution during the ari season 2010/2011 (december-march). weekly emails were sent to the participants (n = 84), reminding them to come to the study center in case of new symptoms. the participants self-collected an anterior nasal swab from one nostril, and trained study personnel collected one from the other nostril. the participants self-collected another two swabs (one from each nostril) on a subsequent day. human β-actin dna concentration was determined in the swabs as a quality control. viral respiratory pathogens were detected by multiplex rt-pcr (seeplex rv15 kit, seegene, eschborn, germany). of 84 participants, 56 (67%) reported at least one ari episode, 18 participants two, and one participant three. self-swabbing was highly accepted by the participants. the amount of β-actin dna per swab was higher in the selfthan in the staff-collected swabs (p = 0.008). β-actin concentration was lower in the self-swabs collected on day 1 than in those collected on a subsequent day (p<0.0001). a respiratory viral pathogen was detected in 31% (23/75) of staffand in 35% (26/75) of self-collected swabs (p = 0.36). with both approaches, the most frequently identified pathogens were human rhinoviruses a/b/c (12/75 swabs, 16%) and human coronavirus oc43 (4/75 swabs, 5%). there was almost perfect agreement between selfand staff-collected swabs in terms of pathogen detection (agreement = 93%, kappa = 0.85, p<0.0001). conclusions/significance: nasal self-swabbing for identification of viral ari pathogens proved to be equivalent to staff-swabbing in this population in terms of acceptance and pathogen detection. the need for the timely collection of diagnostic biosamples (such as nasal swabs) during acute symptomatic episodes represents a major obstacle to large-scale studies on acute respiratory infection (ari) epidemiology. this may be circumvented by having the participants collect their own nasal swabs (''selfcollected nasal swabs'' or ''self-swabbing''). this method has been shown to be highly acceptable and feasible in various populations, e.g. among parents who collected nasal swabs from their children [1] or adults who collected swabs from themselves [2] (also reviewed in [3] ). while the mere feasibility of nasal self-swabbing has thus been amply demonstrated, efforts to validate the diagnostic equivalence of self-swabbing compared to staff-swabbing are still ongoing. in a sample of 38 individuals with ari symptoms, larios et al. compared self-collected midturbinate swabs with staff-collected nasopharyngeal swabs (gold standard) that were collected the same day. the self-collected swabs had a sensitivity of 86% for the detection of respiratory pathogens compared to the gold standard [4] . luinstra et al. found similar detection rates for respiratory pathogens between self-and staffcollected midturbinate swabs when one staff-collected and one selfcollected swab were taken from opposite nostrils during the same visit to a campus health center [5] . ip et al. investigated the validity of self-collected nasal (posterior nares) and pharyngeal swabs to detect influenza virus infection and came to the conclusion that self-swabs may be a good alternative [6] . while these results do provide substantial evidence for the validity of selfcollected midturbinate swabs, anterior nasal swabs have not been evaluated in this respect. moreover, self-collected nasal swabs collected on separate days have not been compared with each other. in the present study, we thus compared quality of swabbing and efficiency of viral detection of anterior nasal swabs in the following scenarios: 1) self-vs. staff-collected swabs that were taken during the same visit to the study center (day 1), and 2) self-and staff-collected swabs from day 1vs. self-collected swabs that were obtained at home on a later day. a prospective study was conducted during the 2010/2011 ari season among a convenience sample of employees of our institution, the helmholtz centre for infection research in braunschweig, germany. in december 2010, employees (18 to 69 years old) were sent messages through the internal e-mail system inviting them to participate in the study. this invitation contained a link to the institutional intranet where information about the study was made available in english and german. individuals planning to leave braunschweig during the study period and staff members of the departments of epidemiology and infection genetics were not eligible to participate, the latter due to concern over a potential conflict of interest. at the baseline visit (december 2010-january 2011), the study aims were explained to the participants and informed consent was obtained. a self-administered questionnaire was used to collect basic sociodemographic data. at the end of the ari season (april/ may 2011) the study participants completed a short acceptance questionnaire. all participants received a remuneration of 5 j. the study was approved by the ethics committee of the state board of physicians of the german federal state of lower saxony. during january-march 2011 weekly e-mail messages were sent to the participants reminding them to come to the study center within 7 days of onset of at least one of the following symptoms: sudden onset of stuffy or runny nose, cough, sore throat, headache, malaise, chills, or fever, defined as body temperature .38uc. in the study center, a trained staff member (a.g.) obtained a nasal swab (regular flocked swab, copan, brescia, italy, product number 359c) from the participant's left nostril and instructed him/her how to perform a self-swab. the participants also received written and visual instructions for nasal self-swabbing. the participants then self-collected a swab from the right nostril. briefly, the swab was to be inserted into the nostril to the point where the basal edge of the flocked tip had just entered the nostril, corresponding to a depth of insertion of approx. 1 cm. the swab was then rotated three times, being careful to swab all 360u of the anterior nasal lining and to include the superior recess (''little's area'') and the latero-inferior recess. the swab was then placed into 1 ml universal viral transport medium (copan). a swabbing kit containing written and visual self-swabbing instructions, two nasal swabs and two vials of 1 ml transport medium was then given to the participants with the request to self-collect two nasal swabs (one from each nostril) at home the next day, to place each swab in 1 ml transport medium and to return the swabs to the study center as soon as possible. the swabs were stored at 270uc until analysis. the timeline of the study is shown in fig. 1 . detection of human b-actin coding sequences was used as a measure of sample adequacy, assuming that the amount of b-actin gene dna can serve as proxy for the presence of human epithelial cells [7, 8] , which contains these b-actin gene sequences in a pcdna3.1 backbone, were analyzed in parallel to obtain the standard curve. b-actin dna concentration was determined in all four swabs. the values of the two swabs that were self-collected at home were pooled. the technician performing the laboratory analyses was not blinded as to whether a swab was staff-or selfcollected. detection of viral respiratory pathogens. rna was extracted from 200 ml aliquots of transport medium (utm kit, copan, brescia, italy) with the qiaamp minelute virus spin kit (qiagen gmbh, hilden, germany). cdna was synthesized with the transcriptor first strand cdna synthesis kit (roche diagnostics gmbh, mannheim, germany) and tested by multiplex pcr (seeplex rv15 ace detection kit, seegene germany, eschborn, germany) for the presence of any of 15 human viral respiratory pathogens (adenovirus a/b/c/d/e, human metapneumovirus, enterovirus, bocavirus 1/2/3/4, human coronavirus 229e/nl63 and oc43, parainfluenza virus 1, 2, 3 and 4, influenza virus a and b, respiratory syncytial virus a and b, and rhinovirus a/b/c), following the manufacturer's recommendations except that 4 ml instead of 1 ml cdna was used as input for the pcr reaction. satisfaction and acceptability were assessed using a nine-item questionnaire. participants rated each item on a five-point likert scale with 1 indicating strong disagreement, 2 disagreement, 3 neither disagreement nor agreement, 4 agreement, and 5 strong agreement. some of these items were reverse-phrased to reduce response bias. data were described by percentage for categorical variables and median with interquartile range (iqr) for continuous variables. a staff-collected and a self-collected swab were obtained on day 1 from separate nostrils. the participants were instructed to collect a self-swab from each nostril the next day, but the actual day of self-swabbing ranged from day 2 to day 6, as indicated by the triangle. doi:10.1371/journal.pone.0048508.g001 table 1 . acceptability of nasal swabbing. the collection of the nasal swab by the study personnel was acceptable (n = 56)* 5 collecting the nasal swab myself was acceptable (n = 56)* 5 i felt comfortable when the study personnel collected the swab (n = 56)* 5 (4-5) i felt comfortable when taking the swab myself (n = 56)* 5 i would prefer taking a nasal swab myself and not having it taken by study personnel (n = 56)* 3 (2-3) nasal self-swabbing was easy to perform (n = 56)* 5 the instructions how to take the self-swab were understandable (n = 56)* 5 i would participate in a study where nasal swabs are to be taken by study personnel (n = 77)** 5 i would participate in a study where nasal swabs are to be taken by myself (n = 76)** 5 *only those participants who collected at least one nasal swab. eighty-four participants responded to the invitation e-mail, corresponding to a response rate of 18%. seventy-two participants (86%) were women, the median age was 37 years (range, 28-46). about half of the respondents had a university degree (including university of applied sciences) and about 12% were born outside germany. overall, both staff-and self-collected nasal swabs were highly accepted ( table 1 ). most participants reported that instructions how to collect the swab were easy to understand and that the nasal self-swab was easy to perform. the participants did not make a preference regarding self-or staff-collected swabs ( table 1) . fifty-six of 84 (67%) participants reported at least one ari episode, 18 (32%) participants reported two episodes, and one participant reported three, resulting in a total of 75 ari episodes for the final analysis. the number of ari symptoms reported in a single ari episode ranged from one to seven (median, 3.0). thus, 75 matched pairs of staff-and self-collected nasal swabs, performed on the same day, were available. about 14% of these were taken on the day of symptom onset (day 1), 33% on day 2, 22% on day 3, 12% on day 4, and 19% on or after day 5. the additional set of self-collected swabs, which was to be collected at home the next day, was obtained in the majority of ari episodes (71/75, 95%), for 67/71 (94%) of which swabs from both nostrils were returned, and from only one side in the remaining four. sixty-two percent of these swabs were indeed collected the next day (day 2) and the latest one on day 6 ( fig. 1) . when comparing the staff-and self-swabs collected on day 1, bactin dna concentration was higher in the self-collected swabs (p = 0.008) (fig. 2a) . the b-actin dna concentration was higher in the self-swabs collected on or after day 2 than in the self-swabs from day 1 (p,0.0001). interquartile distance was smallest in the staff-collected swabs and greatest in the swabs that were selfcollected at home, indicating lower variability in the staff-collected than in the self-collected swabs. b-actin dna levels did not correlate with the duration of symptoms preceding the day of swab collection (fig. 2b) . a respiratory viral pathogen was detected in 31% (23/75) of staff-and in 35% (26/75) of self-collected swabs collected on day 1 (p = 0.36, mcnemar test). in both, the most frequently identified pathogens were human rhinoviruses a, b or c (12/27 positive swabs, 44%), human coronavirus oc43 (4/27 swabs, 15%) and parainfluenza viruses 1, 2, 3 or 4 (4/27, 15%). table 2 contains the complete list of detected pathogens, expressed as percentages of all 75 swab pairs collected on day 1 in which a pathogen was detected in at least one of the two swabs. there was nearly perfect agreement between the staff-and self-swabs collected on day 1 in terms of pathogen detection (percent agreement = 93%, k = 0.85, fig. 3 ). the overall sensitivity of self-collected swabs to detect a respiratory pathogen was 96%, compared to the staff-collected swabs (table 3 ). this very good agreement between the staff-and self-collected swabs from day 1 is also evident in the flow diagram shown in fig. 3 . analyzing the results obtained with the self-swabs from day 2 or later, a viral pathogen was detected in 39% (26/67) of the swab pairs when the results of both nostrils were combined. when the swabs from each side were considered separately, a pathogen was detected in 31% (21/67) of the self-collected swab from the right and in 37% (25/67) of swabs from the left nostril. however, this apparent difference was not significant (p = 0.22, mcnemar test). comparing viral detection of these self-swabs with that of the staffcollected swabs from day 1 revealed substantial agreement table 3 . sensitivity and specificity of self-collected swabs, obtained in the study center, to detect viral respiratory pathogens (compared to staff-collected swabs)*. (percent agreement = 85%, k = 0.67). the detection of a viral pathogen was independent of the amount of b-actin dna in both staff-and self-swabs collected on day 1 (fig. 4) . likewise, there was no association between viral positivity status and b-actin dna levels across all samples (p for trend = 0.943). this prospective study comparing staff-and self-collected nasal swabs for the detection of ari pathogens clearly demonstrated the validity of self-swabbing; specifically, self-swabbing was not inferior in terms of acceptance, satisfaction, sample adequacy, and viral detection rate. of note, we observed excellent agreement in viral detection between self-and staff-collected swabs collected the same day. the agreement between the swabs that were self-collected at home and the staff-collected swabs was somewhat less pronounced, but still was classified as substantial (k = 0.67). this lower agreement may be explained by the time lag between collection of the staff-swabs and the self-swabs at home, which spanned up to 6 days and resulted in five additional positive self-swabs, but only two additional negative ones. thus, taken together, the results strongly indicate equivalence of self-collected and staff-collected swabs in this study population. noteworthy differences were detected in b-actin dna concentration, which was used to quantify the amount of host cells and thus served as a measure of sample adequacy [8] . median b-actin dna concentration was significantly higher in the self-collected than in staff-collected swabs, but also in the swabs that were selfcollected at home than in the self-collected swabs from day 1. taken together, these results suggest (1) that the participants applied higher swabbing pressure than the trained staff member, likely due to high confidence in the self-swabbing procedure, and (2) that a training effect resulted in yet greater confidence and more vigorous swabbing when self-swabbing was repeated at home $1 day later. in support of this notion, in a study comparing staffand self-collected swabs that were obtained the same day smieja et al. observed a higher number of epithelial cells and a tendency toward a higher b-actin dna level in a second self-swab that was collected immediately after the first one [8] . alternatively, the presence of leukocytes in purulent secretions may have contributed to b-actin levels in some swabs. however, we do not consider this to be a major contributing factor since most participants did not have purulent secretions. another theoretical reason for the higher b-actin dna levels in the swabs that were self-collected at home might have been the longer disease duration, leading to higher shedding of epithelial cells or higher leukocyte numbers in the secretions. however, as shown in fig. 2b , there was no association between duration of symptoms and b-actin concentration in the swabs, thus ruling out this possibility in the present study. of note, more thorough sampling (as reflected by higher b-actin dna levels) did not improve viral detection in our study, suggesting that the amount of host cells on the swabs was near the optimum in most cases. alternatively, the sampling of host cells may not be a major determinant for the detection of ari viruses due to the presence of sufficient amounts of viral particles in nasal secretions. a criticism of studies comparing staff-and self-collected swabs from the same day has been that the participants might feel more confident in the presence of study personnel [10] and that selfswabs might actually be inferior when collected in the absence of study personnel. we tested this hypothesis by including two additional nasal swabs which were self-collected at home $1 day later. comparison of these swabs with staff-collected swabs collected on day 1 revealed good agreement in terms of viral detection and even better sample adequacy, thus demonstrating the diagnostic equivalence of self-swabbing in an unsupervised setting, provided that the participants have been trained adequately. it should be kept in mind that only two individuals with pcrproven influenza virus infection were detected. therefore, anterior nasal self-swabbing for the detection of influenza virus infection still needs to be validated in dedicated studies. recently, a flocked mid-turbinate swab was developed and turned out to be superior to the gold standard nasopharyngeal swab in terms of ari virus detection [8] . the anterior nasal swab used in the present study has not been compared to this midturbinate swab. this should be done in a future study. our findings are also limited by the fact that the study was conducted in a selected study population recruited within a research institution. however, the population included a mixture of scientific, clerical and support staff. notably, education level spanned a broad range and did not influence viral detection or b-actin dna levels. nonetheless, our findings need to be validated in future studies using random samples drawn from the general population. due to much lower expenses for personnel and travel, selfcollection would be a highly cost-efficient way to obtain diagnostic nasal swabs in medium and large scale population-based studies on ari epidemiology [3] . the presented study adds significantly to the growing body of evidence demonstrating its diagnostic equivalence to staff-collection. nasal self-swabbing proved to be an acceptable, feasible and valid method to identify viral respiratory pathogens in this selected adult population. its applicability in the general population should be tested in future studies. parent-collected respiratory specimens-a novel method for respiratory virus and vaccine efficacy research e-mail-based symptomatic surveillance combined with self-collection of nasal swabs: a new tool for acute respiratory infection epidemiology self-collected nasal swabs to detect infection and colonization: a useful tool for population-based epidemiological studies? self-collected mid-turbinate swabs for the detection of respiratory viruses in adults with acute respiratory illnesses evaluation and clinical validation of an alcohol-based transport medium for preservation and inactivation of respiratory viruses validation of self-swab for virologic confirmation of influenza virus infections in a community setting evidence for altered regulation of i kappa b alpha degradation in human colonic epithelial cells development and evaluation of a flocked nasal mid-turbinate swab for selfcollected respiratory virus diagnostic testing the measurement of observer agreement for categorical data collection by trained pediatricians or parents of mid-turbinate nasal flocked swabs for the detection of influenza viruses in childhood we would like to thank the participants for their kind participation in the study. we also thank gérard krause (helmholtz centre for infection research, braunschweig, and robert koch institute, berlin) and aparna schweitzer (helmholtz centre for infection research, braunschweig) for helpful comments and a critical reading of the manuscript. key: cord-320466-l7017jis authors: akgun, emel; tuzuner, mete bora; sahin, betul; kilercik, meltem; kulah, canan; cakiroglu, hacer nur; serteser, mustafa; unsal, ibrahim; baykal, ahmet tarik title: proteins associated with neutrophil degranulation are upregulated in nasopharyngeal swabs from sars-cov-2 patients date: 2020-10-20 journal: plos one doi: 10.1371/journal.pone.0240012 sha: doc_id: 320466 cord_uid: l7017jis covid-19 or severe acute respiratory syndrome coronavirus 2 (sars-cov-2) appeared throughout the world and currently affected more than 9 million people and caused the death of around 470,000 patients. the novel strain of the coronavirus disease is transmittable at a devastating rate with a high rate of severe hospitalization even more so for the elderly population. naso-oro-pharyngeal swab samples as the first step towards detecting suspected infection of sars-cov-2 provides a non-invasive method for pcr testing at a high confidence rate. furthermore, proteomics analysis of pcr positive and negative naso-oropharyngeal samples provides information on the molecular level which highlights disease pathology. samples from 15 pcr positive cases and 15 pcr negative cases were analyzed with nanolc-ms/ms to identify the differentially expressed proteins. proteomic analyses identified 207 proteins across the sample set and 17 of them were statistically significant. protein-protein interaction analyses emphasized pathways like neutrophil degranulation, innate immune system, antimicrobial peptides. neutrophil elastase (elane), azurocidin (azu1), myeloperoxidase (mpo), myeloblastin (prtn3), cathepsin g (ctsg) and transcobalamine-1 (tcn1) were found to be significantly altered in naso-oropharyngeal samples of sars-cov-2 patients. the identified proteins are linked to alteration in the innate immune system specifically via neutrophil degranulation and netosis. severe acute respiratory distress syndrome-associated coronavirus-2 (sars-cov-2) also known as covid-19 first appeared in wuhan, hubei, china in december 2019, and now it is widespread around the world. the replication of the virus is through the human airway epithelial cells where it targets the receptors of human angiotensin-converting enzyme 2 (ace-2). the high mortality observed in covid-19 is associated with severe acute respiratory distress and systemic coagulopathy. many covid-19 patients show a postponed onset of respiratory issues but then develop into more severe situations. we wanted to investigate the plos one plos one | https://doi.org/10.1371/journal.pone.0240012 october 20, 2020 1 / 10 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 molecular changes in the covid-19 patients' naso-oropharyngeal swab samples via comparison to the proteome of pcr negative cases. our goal is to find pathways associated with the site of infection through proteomics analysis. 17 statistically significant protein alterations lead us specifically to neutrophil degranulation pathways. during airway infections, neutrophils are the first wave of defense that also defines the disease outcome. neutrophils have multiple functions in viral infections such as inactivation of the virus, achieved by phagocytosis, ros production, proteolytic enzyme release, net activation (netosis, neutrophil extracellular traps). neutrophils also interact with immune cells and secreting cytokines participate in eliciting an antiviral response. neutrophil granulocytes express several enzymes linked to controlling host infection. as the cytotoxic molecules are released from the granules they have an impact on the inflammatory response. such adverse molecules cause severe damage to the host where it exhibits itself as perivascular infiltrates around the capillaries in the lungs as observed in sars-cov-2 patients. the identified up-regulated proteins myeloperoxidase, myeloblastin, neutrophil elastase, cathepsin g, and azurocidin (mpo, prtn3, elane, ctsg, and azu1) in nasooropharyngeal swab samples are discussed to highlight the molecular mechanism changes in the site of infection. infected and non-infected study groups were formed from the patients who applied to acıbadem health group hospitals with suspected sars-cov-2 infection. cases were diagnosed on the basis of the interim guidance of the world health organization (who) (world health organization; geneva: 2019. clinical management of severe acute respiratory infection when middle east respiratory syndrome coronavirus (mers-cov) infection is suspected: interim guidance.) and diagnosis and treatment guidelines of covid-19 in turkey (covid-19 (sars-cov-2 infection) guide republic of turkey, ministry of health, april 14th 2020, ankara). selected patients did not have any other known infections at the time of diagnosis. a total of 30 patients were enrolled in the study: 15 patients pcr positive for sars-cov-2 (mean age: 38.9±13.8) and 15 patients pcr negative for sars-cov-2 (mean age: 36.6 ±15.9). infected group was equally distributed regarding gender. most of the non-infected patients were men (60%). as a routine application of our clinical laboratory, wbc count of the patients were also performed (neutrophil count x10 9 /l mean: 0.6±0.5 for non-infected and 5.6±10.2 for infected). samples were collected using a nasal and oropharyngeal (nucliswab) swab (salubris, turkey) and were placed in a tube with universal transport media. after collection the samples were stored at -80˚c before proteomics analysis. no minors were included in the study and a written informed consent was obtained from each patient that was enrolled in the study. ethical approval for the conduct of the study was given by acibadem mehmet ali aydinlar university human scientific and ethical review committee (approval id: 2020-07/9). for molecular testing of sars-cov-2; the extraction of nucleic acids from the samples was performed by a manual liquid phase method using bio-speedy nucleic acid isolation kit (bioeksen, turkey). nucleic acid amplification test (nat) was carried out by bio-speedy covid-19 rt-qpcr detection kit (bioeksen, turkey) according to the manufacturer's instructions on rotorgene (qiagen, germany) real-time pcr instrument. the test briefly funding: acibadem labmed clinical laboratory provided support for this study in the form of salaries for mbt, bs, ck, hnc, and iu. the specific roles of these authors are articulated in the 'author contributions' section. data acquisition was also conducted in acibadem labmed clinical laboratory's proteomics facility. the funders had no further role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. the authors received no other specific funding for this work. the authors have read the journal's policy and have the following competing interests: mbt, bs, ck, hnc, and iu are employees of acibadem labmed clinical laboratory. this does not alter our adherence to plos one policies on sharing data and materials. there are no patents, products in development or marketed products associated with this research to declare. achieves a one-step reverse transcription (rt) and real-time pcr (qpcr) targeting sars--cov-2 specific rdrp (rna-dependent rna polymerase) gene fragment. a shorter sample preparation approach was applied to obtain the tryptic peptides for analyses. 50 ul of the naso-oropharyngeal transport solution was taken and lyophilized. the powder was reconstituted in 20ul of 50 mm ammonium bicarbonate solution with 1 ul of protease inhibitor mixture (thermo scientific). the mixture was centrifuged at 14000 xg for 10 min and the supernatant was transferred to a clean eppendorf tube. dtt was added to 10 mm final concentration and incubated at 60˚c for 10 min. the mixture was alkylated in dark for 30 min with 20mm iaa. to the resulting mixture 1 ug of sequencing grade trypsin (promega gold) was added and incubated at 55˚c for 1.5 hrs. the digest was acidified with 1 ul formic acid and transferred to an lc vial for injection. the samples were analyzed by the protocols in our previous studies [1] . briefly, tryptic peptides were trapped on a symmetry c18 (5μm,180μm i.d. × 20 mm) column and eluted with acn gradient (4% to 40% acn, 0.3 ul/min flow rate) with a total run time of 60 min on a csh c18 (1.7 μm, 75 μm i.d. × 250 mm) analytical nano column. data were collected in positive ion sensitivity mode using a novel data-independent acquisition mode (dia) coined as sonar [2] with a quadrupole transmission width of 24 da. progenesis-qip (v.2.4 waters) was used for data processing. protein-protein interaction networks functional enrichment analysis was carried out using the string (http://string-db.org/, v11.0) database with the highest confidence interaction score level to identify possible pathways related to the identified proteins. textmining, experiments, databases co-expression, neighborhood, gene fusion, and co-occurrence selected as active interaction sources. the minimum interaction score was set to high (high confidence = 0.700). reactome (http://www.reactome.org) pathways analysis tool was also used for processing. proteins were extracted from a small amount of naso-oropharyngeal samples, fast tryptic digestion was applied and followed with a 60 min reverse-phase separation. nanolc-msms analysis provided the identification of 207 protein groups with high confidence (<1% fdr) (s1 and s2 tables). statistical analysis done in progenesis qip software identified 17 proteins to be statistically significantly expressed in patients' naso-oropharyngeal samples (table 1 ). pathway analysis of the significantly altered protein levels between covid-19 positive and negative patients' naso-oropharyngeal swab samples were analyzed using the string online database. the ppi network obtained, contained 17 differentially expressed proteins with 15 nodes (disconnected nodes were not shown) and 14 edges as shown in fig 1a. the main cluster includes neutrophil elastase (elane), azurocidin (azu1), myeloperoxidase (mpo), myeloblastin (prtn3), cathepsin g (ctsg) and transcobalamine-1 (tcn1). the abundance of these proteins were found to be increased in covid-19 positive patient samples compared to negative ones. reactome pathway enrichment analyses of the differentially expressed proteins were performed. nine of the proteins were primarily associated with the immune system pathway. proteins clustered in the ppi network were mainly enriched in the neutrophil degranulation pathway (hsa-6798695, fdr = 2.01e-7) which is a subpathway of the innate immune system (fig 1b) . neutrophils play a vital role in airway infections and may also define the disease outcome and carry out various functions in viral infections ranging from phagocytosis, degranulation to the generation of neutrophil extracellular traps (nets) [3] . neutrophils also interact with the immune cells and secreting cytokines participate in eliciting the antiviral response. degranulation mechanism is part of the innate immune system which is necessary for the fight against infection and provides the neutrophils with the necessary tools. a recent study reported the accumulation of neutrophils in severe covid-19 patients compared to non-severe patients via analyzing 6 different studies [4] . we also observed the same profile among our sars-cov-2 patients where the neutrophils were exceedingly high compared to the healthy group (fig 2) . in addition, our data led us to significant indications that the dysregulation of degranulation and netosis mechanisms may also be involved in sars-cov-2 infection. in sars-cov-2 patients' naso-oropharyngeal samples, we have identified azurophilic granule (ag) proteins like myeloperoxidase (mpo), elastase (elane), cathepsin g (ctsg), azurocidin 1 (azu1) and proteinase 3 (prtn3) to be highly overexpressed. it is known that neutrophils are the first line of defense against the onset of a viral infection and they begin the process of defending against microorganisms by releasing antiviral enzymes and toxins stored in their granule. these azurophilic neutrophils undergo limited exocytosis when activated [5] and their primary role is believed to be killing and degradation of engulfed microbes in the phagolysosome [6] . therefore our observation of upregulated proteins related sars-cov-2 naso-oropharyngeal proteomics to neutrophil degranulation in sars-cov-2 patients' naso-oropharyngeal samples is not suprising. although neutrophil degranulation having a positive impact on clearance of sars-cov-2 from the nasopharynx, there is still a debate in the inflammatory microenvironment the process is dysregulated and may lead to tissue damage by the secreted molecules from the granules [7] . myeloperoxidase (mpo), is the most abundant protein in neutrophils and represents 5% of their total protein content [8] . lau et al. reported the association of mpo and neutrophil activation via cd11b/cd18 integrins which is an indicator of mpo's possible contribution to neutrophil recruitment to the site of inflammation [9] . our data supports this hypothesis for sars-cov-2 patients which we detected significantly high expressions of mpo (�4 fold, p<0.05) at the nasopharynx region. furthermore it is tempting to speculate this neutrophil burst and even more overexpressed mpo may cause production of excess hypochlorous acid (hocl) and other reactive oxidants that also damages the nasopharynx tissue. so mpo may be an important factor where the protective inflammation can become pathological in sars-cov-2 cases. the resolution phase of inflammation is essential to curtail inflammation and restore tissue homeostasis [10] . neutrophil apoptosis and the cytokines released from macrophages during phagocytosis of the neutrophils are necessary in the resolution of inflammation [11] . the resolution of inflammation and tissue repair processes are aided by the cytokines released from macrophages during phagocytosis of the neutrophils. the dysregulation in the macrophage orchestrated phagocytosis and neutrophil apoptosis mechanisms leads to inflammation [12] . prtn3, another azurophilic granule (ag) protein that we identified, is a serine protease enzyme that is involved in granulocyte differentiation and expressed in the neutrophil granulocytes [13] . increased prtn3 was shown to have a negative effect on the resolution of inflammation that causes immune system deregulation [14] the strikingly high prtn3 expression that we observed in sars-cov-2 positive patients compared to negative group (over 29 fold, p<0.05), may be the indicator of such phenomenon. multifunctional protease ctsg is also thought to be critically important in the maintenance of the delicate balance between tissue protection and destruction during inflammatory responses [15] . as a component of neutrophil proteolytic machinery ctsg regulates the inflammatory responses by stimulating the production of cytokines and chemokines, which are responsible for the activation and mobilization of immune cells to the site of pathogen and/or tissue damage [16, 17] . ctsg activates metalloproteases and cleaves extracellular matrix proteins, contributing to neutrophil migration [18] . ctgs upregulation (more than 3 fold, p<0.05) that we observed in sars-cov-2 patients, possibly is another indicator for abnormal neutrophil accumulation in nasopharynx. elane has a physiological function as a powerful host defense, but is also known as one of the most destructive enzymes in the body. an overwhelming release of enzymatically active elane can cause local tissue injury [19] . addition to that it is also reported elane can activate the spike (s) protein of coronaviruses and shift the viral entry to a low ph-independent route [20] . therefore, the highly expressed elane (�3 fold, p<0.05) that we observed in sars-cov-2 infected patients is supporting these findings. sars-cov-2 infected group appeared to be highly expressing the azu1 protein (heparinbinding protein/cationic antimicrobial protein of 37 kd) which is mobilized rapidly from emigrating polymorphonuclear leukocytes (pmn). initially, this inactive serine protease was recognized for its antimicrobial effects. however, it soon became apparent that azurocidin may act to alarm the immune system in different ways and thus serve as an important mediator during the initiation of the immune response. azurocidin, released from pmn secretory vesicles or primary granules, acts as a chemoattractant and activator of monocytes and macrophages. the functional consequence is enhancement of cytokine release and bacterial phagocytosis, allowing for a more efficient bacterial clearance. leukocyte activation by azurocidin is mediated via beta(2)-integrins, and azurocidin-induced chemotaxis is dependent on formyl-peptide receptors. in addition, azurocidin activates endothelial cells leading to vascular leakage and edema formation. ag proteins' active role in neutrophil-associated lung inflammatory and tissue-destructive diseases has been reported [21] . increased expressions of prtn3, elane, and ctsg was reported in copd patients [22] . in a mouse model study elane or prtn3 was introduced to the trachea which caused tissue destruction and enlargement in airspace [23] . elane expression was also implicated in the impairment of host defense resulting in a decrease in mucociliary clearance of bacteria and also pathogens phagocytosis [24] . cd2, cd4, and cd8 can be cleaved on the surface of t-cells by elane and ctsg that dysregulate t-cell function [25] . it was reported that patients that lack alpha-1 antitrypsin (α1-pi) which is the physiological inhibitor of prtn3 and elane carries a high risk of developing emphysema [26] . azurophil granules also carry cathepsin d which was reported to be up-regulated in the pulmonary macrophages in a mouse model of cigarette smoking [27] . severe copd cases exhibited mpo positive cells as a signal of neutrophil activation [28] . on the other hand in a mouse model of influenza it was shown that the inflammation damage was reduced by the absence of mpo [29] . regarding the high expression results of these protein markers we may suggest that during the progression of sars-cov-2 infection the same molecular mechanisms are most likely to be induced. among the ag proteins that we have identified, especially prtn3, elane and ctsg were mostly associated with cytokine driven immune dysregulation. il-32, a proinflammatory cytokine with four isoforms, cleavage by prtn3 propagates cytokine activity and triggers il-1beta, tnf-alpha, il-6, and chemokines. it was argued that the targeted inhibition of pr3 or silencing of il-32 by an inactive form of prtn3 may halt the il-32 driven immune dysregulation [30] . elane, ctsg, and prtn3 can cleave pro-il-1beta to bioactive il-1beta [31] . caspase-1/interleukin-1 converting enzyme (ice) cleaves proteins like precursors of the inflammatory cytokines interleukin 1β and interleukin 18 into their mature biologically active forms [32] and ctsg regulates caspase-1 in this pathway [33] . ice has an active role in cell immunity as an initiator of inflammatory response so once activated it triggers the formation of active il-1beta which is secreted from the cell that induces inflammation in the neighboring cells [34] . it was recently reported that infection in covid-19 patients with acute respiratory syndrome showed release of the pro-inflammatory cytokines like il-1beta and il-6 [35] . netosis is a type of programmed cell death where neutrophil extracellular traps are formed [22] . nets were identified in 2004 and they are often overlooked as drivers of severe pathogenic inflammation [36] . nets have pathogen killing properties and include strands of dna wrapped with histones and are enriched with neutrophil proteins like mpo, elane, prtn3 and azu1 [37] which were also present in our sars-cov-2 (+) samples with high amounts. although netosis is a very powerful mechanism fighting for the infection, the ability of nets to damage tissues is well-documented in infection and sterile disease. nets directly kill epithelial and endothelial cells [11, 38] , and excessive netosis damages the epithelium in pulmonary fungal infection [12] and the endothelium in transfusion-related acute lung injury [39] . through the available literature, we can see that the up-regulation of various proteins observed in the naso-oropharyngeal swab samples of covid-19 patients is tightly interconnected with the immune response. the alterations of various proteins in sars-cov-2 infected patients' naso-oropharyngeal samples depict the molecular changes that govern the host antiviral defense system. the available literature for many respiratory diseases are very helpful in linking altered protein expressions to viral pathogenesis. obtained data provided us an important view of sars-cov-2 molecular changes on the protein level in the infection site. statistically significant protein alterations of prtn3, mpo, elane, ctsg, and azu1 dysregulation is important in the early phases of infection and may be targets for anti-sars-cov-2 therapeutics. further research may show a link between the level of these proteins with disease severity and may be used as prognostic markers. modulating the dysregulated proteins like prtn3 or mpo may promote an anti-inflammatory response to alleviate sars-cov-2 symptoms. also targeting nets to dampen the out-of-control host response as a treatment may increase the survival rate by reducing the number of patients who require mechanical ventilation in icu. we posit here that excess nets may elicit the severe multi-organ consequences of covid-19 via their known effects on tissues and the immune, vascular, and coagulation systems. targeting nsps and nets in covid-19 patients should 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apoptotic cells novel cell death program leads to neutrophil extracellular traps prevention of leukocyte elastase-induced emphysema in mice by heparin fragments critical copd respiratory illness is linked to increased transcriptomic activity of neutrophil proteases genes cleavage of lymphocyte surface antigens cd2, cd4, and cd8 by polymorphonuclear leukocyte elastase and cathepsin g in patients with cystic fibrosis α1-antitrypsin deficiency and chronic respiratory disorders matrix metalloproteinase-12 and cathepsin d expression in pulmonary macrophages and dendritic cells of cigarette smoke-exposed mice cellular and molecular mechanisms in chronic obstructive pulmonary disease: an overview contribution of neutrophil-derived myeloperoxidase in the early phase of fulminant acute respiratory distress syndrome induced by influenza virus infection proteinase 3 is an il-32 binding protein caspase 1-independent activation of interleukin-1β in neutrophil-predominant inflammation naturally occurring genetic variants of human caspase-1 differ considerably in structure and the ability to activate interleukin-1β interaction of mycobacterium tuberculosis with host cell death pathways. cold spring harb perspect med interleukin-1 beta converting enzyme is necessary for development of depression-like behavior following intracerebroventricular administration of lipopolysaccharide to mice induction of pro-inflammatory cytokines (il-1 and il-6) and lung inflammation by coronavirus-19 (covi-19 or sars-cov-2): anti-inflammatory strategies neutrophil extracellular traps kill bacteria. science (80-) a myeloperoxidase-containing complex regulates neutrophil elastase release and actin dynamics during netosis animal models of anca associated vasculitis excessive neutrophils and neutrophil extracellular traps in covid-19 key: cord-312493-wbhji81g authors: tay, ee laine; grant, kristina; kirk, martyn; mounts, anthony; kelly, heath title: exploring a proposed who method to determine thresholds for seasonal influenza surveillance date: 2013-10-11 journal: plos one doi: 10.1371/journal.pone.0077244 sha: doc_id: 312493 cord_uid: wbhji81g introduction: health authorities find thresholds useful to gauge the start and severity of influenza seasons. we explored a method for deriving thresholds proposed in an influenza surveillance manual published by the world health organization (who). methods: for 2002-2011, we analysed two routine influenza-like-illness (ili) datasets, general practice sentinel surveillance and a locum medical service sentinel surveillance, plus laboratory data and hospital admissions for influenza. for each sentinel dataset, we created two composite variables from the product of weekly ili data and the relevant laboratory data, indicating the proportion of tested specimens that were positive. for all datasets, including the composite datasets, we aligned data on the median week of peak influenza or ili activity and assigned three threshold levels: seasonal threshold, determined by inspection; and two intensity thresholds termed average and alert thresholds, determined by calculations of means, medians, confidence intervals (ci) and percentiles. from the thresholds, we compared the seasonal onset, end and intensity across all datasets from 2002-2011. correlation between datasets was assessed using the mean correlation coefficient. results: the median week of peak activity was week 34 for all datasets, except hospital data (week 35). means and medians were comparable and the 90% upper cis were similar to the 95(th) percentiles. comparison of thresholds revealed variations in defining the start of a season but good agreement in describing the end and intensity of influenza seasons, except in hospital admissions data after the pandemic year of 2009. the composite variables improved the agreements between the ili and other datasets. datasets were well correlated, with mean correlation coefficients of >0.75 for a range of combinations. conclusions: thresholds for influenza surveillance are easily derived from historical surveillance and laboratory data using the approach proposed by who. use of composite variables is helpful for describing influenza season characteristics. influenza infection remains a significant public health problem, resulting in considerable global morbidity and mortality [1] [2] [3] . in temperate regions of australia, seasonal influenza outbreaks usually occur between late autumn and early spring and are associated with an increase in disease burden and utilisation of health service [3, 4] . due to differences in circulating viruses, population immunity and environmental factors, the onset, duration and severity of a season may differ from year to year [2, 5] . ongoing monitoring of influenza is therefore needed to determine the onset and severity of seasons and to monitor changes in disease trends. surveillance which involves laboratory testing can add to data on virus characteristics. influenza thresholds have been developed to indicate a level of disease activity that would signal the start or end of a season or provide an alert to an unusually severe or atypical season. the onset of a season may stimulate diagnosis, enhance case detection, promote awareness of the need for patient cohorting or isolation in hospitals, remind people about vaccination and encourage early prescription of anti-viral medication, especially in vulnerable populations [6, 7] . in the setting of a particularly severe season or pandemic, thresholds may inform the appropriate allocation of resources [8] . many influenza surveillance systems around the world have incorporated the use of thresholds. these include australia, new zealand, europe and united states (us) [9] [10] [11] [12] . methods using a variety of surveillance systems have been developed to establish thresholds for influenza activity. the methods vary in their complexity and can use either short-term or longer historical data to create time-varying or fixed thresholds. there is currently no gold standard or consensus for calculating thresholds. the simplest method uses visual inspection of historical data to create a fixed threshold used throughout the year [13] . other methods include regression models [14] [15] [16] [17] , time series methods [15] , calculation of means and medians [18] [19] [20] and adaptation of industrial control processes such as shewhart charts [21] , cumulative sum (cusum) [15, 18, 22] and the exponentially weighted moving average [23] . the us centre for disease control and prevention (cdc) calculates the baseline for their influenzalike-illness (ili) surveillance by adding two standard deviations to the mean percentage of ili visits during non-influenza weeks for the previous three seasons, with non-influenza weeks defined as periods with less than 2% of the year's total positive specimens for influenza for ≥ 2 consecutive weeks [24] . in victoria, the general practice sentinel surveillance (gpss) for ili has historically relied on thresholds determined by inspection [13] . in 2012 a novel but simple method for defining thresholds was proposed by the world health organization (who) as part of the development of global standards for influenza surveillance [7] . the proposed method aligns several years of historical data on the median week of peak activity and assigns thresholds based on means and standard deviations of aligned data. to our knowledge, this method has not yet been field tested. the aim of this study was to explore the feasibility of the who method for the calculation of influenza thresholds using a range of existing surveillance and laboratory data sources in one surveillance system. we used these data sources to compare the onset, duration and intensity of influenza seasons. victoria is a state with a population of over 5 million people located in the south-eastern part of australia. it has a temperate climate with annual seasons of influenza occurring occur between late autumn (may) and early spring (october). it has a well-established influenza surveillance system that monitors influenza activity using syndromic surveillance of ili presentations to sentinel general practitioners (gp) and a medical locum service; laboratory-confirmed influenza; hospital admissions for influenza; and more recently google flu trends and the influenza complications alert network (flucan), which monitors hospitalised patients from sentinel australian hospitals, including four victorian hospitals [9] . four independent surveillance data sources were used: (i) the victorian gpss, (ii) sentinel data from the melbourne medical deputising service (mmds), (iii) routine laboratoryconfirmed influenza (lab data) from the victorian infectious diseases reference laboratory (vidrl) and the (iv) victoria admitted episode dataset (vaed) for hospital admissions. the gpss is an annual surveillance system for ili and laboratory confirmed influenza that was established in 1993, with laboratory support added in 1998 [25] . surveillance extends from week 18 to 44 each year during the influenza season. the number of participating general practitioners (gp) has varied from 40 to 100 since the scheme's establishment. the ili definition used is based on the nationally agreed case definition of cough, fever (measured or reported) and fatigue [13] . approximately 48% of ili patients seen by sentinel gps were swabbed and of these, influenza virus was detected from an average of 34% (18%-47%) of the swabbed ili patients tested from 2003 to 2011 [9, 26] . an alternative source of community sentinel ili surveillance is the mmds, an out-of-hours medical locum service that covers an approximate 45km radius from central melbourne. gps from the deputising service consult with patients in their own home or aged care facility. the diagnosis made by the attending doctor is recorded electronically and de-identified summary data are available on a password protected website within 24 hours. ili data are extracted weekly based on a previously developed search algorithm [27] . there is no laboratory support for the mmds surveillance which has nonetheless been shown to provide equivalent information to surveillance data from sentinel general practitioners [28] . lab data from vidrl consist of laboratory detections of influenza viruses from all routine respiratory samples sent to vidrl, tested using an in-house respiratory multiplex reverse transcriptase polymerase chain reaction (rt-pcr) test that identifies influenza viruses, adenovirus, picornavirus, respiratory syncytial virus, parainfluenza virus, coronavirus and human metapneumovirus [29] . many of the samples are referred from major adult teaching hospitals in victoria [30] . the vaed is a hospitalisation dataset on all patients admitted to public and private acute care hospitals in the state of victoria. the clinical information coded for each episode of care is based on the international classification of diseases and related health problems, tenth revision, australian modification (icd-10-am). we extracted records containing influenza codes j09-11 in primary or secondary diagnostic fields. in addition, a further two composite variables were created from the product of ili and lab data. these were the gpss composite = proportion of ili cases in gpss x proportion of laboratory samples tests positive for influenza in gpss exploring a who method for influenza thresholds plos one | www.plosone.org mmds composite = proportion of ili cases in mmds x proportion of routine laboratory tests positive for influenza in lab data. while the gpss proportion and laboratory testing are part of the same system, the mmds is independent of routine clinical tests referred to vidrl. however, both mmds and routine vidrl clinical testing focus on older age groups and were matched based on age profiles [30, 31] . the metrics used for threshold calculations were the weekly gpss ili proportion per 1000; the weekly mmds ili per 1000; the proportion of laboratory test positive for influenza using the total number of tests for influenza as the denominator (lab test positive influenza); the weekly proportion of influenza admissions in the population using the mid-year estimated resident population in victoria as the denominator and expressed per 100,000 population [32] ; and the product of weekly ili and lab data per 1000. we preferred the use of test positive influenza to count data to compensate for changes in testing behaviour over time [33] . depending on availability, data were extracted from 2002 to 2011 for gpss and mmds, 2003 to 2011 for lab data and 2005 to 2011 for vaed. for mmds, lab data and vaed, data were complete for all 52 weeks but only weeks 18 to 44 were available for gpss. for vaed, only aggregated data containing admissions of more than five counts were provided to protect privacy and confidentiality of individuals. we defined the three levels of influenza threshold based on the terminology used in the who manual and the existing victorian surveillance thresholds that had been adapted from the united kingdom: seasonal, average and alert [13, 34] . seasonal threshold defines the start and end of an influenza season. the two intensity thresholds, termed average and alert thresholds, describe relative seasonal intensity. the who manual did not prescribe a specific method for determining the seasonal threshold, that is, the start of the season. we used the method of inspection of the complete data for the six datasets to determine the seasonal threshold. for each dataset this was done independently by four of the co-authors (et, kg, am, hk) and differences were resolved by discussion. for the four datasets that provided data for the whole year (mmds, lab data, vaed and mmds composite), we calculated the 95% confidence interval (ci) of the metrics used for each dataset for the period defined as out-of-season, that is, the values below the seasonal threshold, that had been determined by inspection. we also explored the seasonal threshold using the 95th percentile of out-of-season data without assuming data were normally distributed. we then compared the seasonal threshold set by inspection with the 95% ci and 95th percentiles of the average out-of-season values. average and alert thresholds were calculated for each dataset using a variation of the who protocol [7] (figure 1 ). we first determined the median week of peak occurrence using historical data, excluding the pandemic year of 2009 which was atypical from both surveillance and testing perspectives [33] . we then aligned the transmission peaks around the median week of peak occurrence ( figure 1a ) and calculated the weekly mean and standard deviations for each week centred on the median week of peak occurrence ( figure 1b and c) . the who protocol suggests the use of the normal distribution to assign thresholds based on the mean and standard deviation of the aligned data for weekly counts. however, we believed data were unlikely to be normally distributed for all years and tested this by inspection and formally using the shapiro-wilks test for normality for gpss, mmds, lab data and the vaed for each year during season [35] . in addition to the mean and standard deviations, we explored the thresholds using the median and 90th and 95th percentiles. the average threshold was determined by a comparison of the peak weekly mean and median, while the alert threshold was determined by a comparison of the peak weekly upper 90% and 95% ci upper limits with the 90th and 95th percentiles. we also performed log transformation of all datasets and calculated the corresponding geometric mean and 90/95 ci upper limit. once the seasonal thresholds were assigned, we determined the start and end of each season independently for all datasets, each defined as the two consecutive weeks where the seasonal threshold was crossed. we used the average and alert thresholds to categorise the influenza seasons, based on the threshold range of peak seasonal activity, specifically seasonal-average, average-alert or alert. comparisons were made for the onset, duration and intensity of a season across all datasets from 2005-2011 based on data availability. we also compared how all seasons compared to the average season created using aligned historical data described above. finally, to determine how correlated the six datasets were, we calculated the correlation coefficient for each year from 2005 to 2011 for a combination of datasets, from which a mean correlation coefficient and its corresponding 95% confidence limits was derived. data were analysed using microsoft® office excel 2003 and stata version 10.0 (stata corp., college station, tx, usa). the study was approved as a quality assurance project by the melbourne health office of research. gpss and mmds data in this study were collected, used and reported under the legislative authorization of the victorian public health and wellbeing act 2008 and public health and wellbeing regulations 2009. during the study period, the highest number of ili or influenza cases annually ranged from 56 to 208 per week for the gpss; 33 to 164 per week for the mmds; 24 to 135 per week for test positive influenza; and 28 to 204 per week for influenza admissions. testing for normality of the weekly count data for each year suggested no seasonal surveillance data had a classical normal distribution graphically (data not shown). data were consistent with a normal distribution by formal testing for 5/10 seasons in the gpss, for 5/10 seasons in the mmds, for 2/9 seasons in lab data and for 2/6 seasons in the vaed. when data were log-transformed, the number of seasons with normal distribution increased (8/10, 8/10, 5/9 and 3/6 respectively). the threshold parameters from the adapted who method are summarised in table 1 . the median week of peak occurrence for all datasets was week 34 except for the mmds composite and vaed. the values assigned for seasonal thresholds by inspection were similar to the 95% ci upper limit and 95 th percentile of the average out-of-season values for all datasets. for the average threshold, we found the peak mean values for the gpss, test positive influenza and vaed were similar to the median but the peak mean was higher for the mmds and mmds composite due to the high call out proportion in 2003. we therefore use the peak mean to define (table 1) . to set the alert thresholds, the peak 90% ci upper limit was used as we found the parameter to be similar to the peak 95 th percentile across all six datasets (table 1 and figure 2 ). the geometric means and 90% ci upper limits from log-transformed data also produced similar parameters (data not shown). using the gpss dataset as an example, figure 3 compares how an annual season compares against the average season calculated using ten years of historical data. onset, end and duration of influenza season across all datasets. for the seven years where data were available for all datasets, gpss assigned seasons tended to start much earlier for most years compared to other datasets. the use of the gpss composite suggested a later start to the season. season onset according to the vaed generally lagged behind other datasets for most years and was variable for the mmds ( table 2) . for most pre-pandemic years, there was generally good agreement for defining the end of a season across datasets. however, a divergent trend, not reflected in other datasets, was noted in the vaed from 2009 onwards ( table 2) . the vaed assigned seasons ended much later, resulting in a longer assigned seasonal duration. category of influenza season. there was agreement in describing the intensity of influenza seasons in 3/7 years prior to 2009 (table 2 and figure 4 ). during the pandemic year, the season intensity varied according to different data sources and from 2009 onwards, peak seasonal influenza activity was between the seasonal and average thresholds (or seasonalaverage) for all datasets except the vaed. datasets were found to be well correlated, with mean correlation coefficients of >0.75 for a range of combinations (table 3) . correlations between the vaed and other datasets improved once the vaed was aligned with other datasets to correspond to the one week lag in median week of peak occurrence. thresholds for influenza surveillance were easily derived using a simple method proposed by the who. the method was adapted to a non-parametric approach that produced similar findings to the suggested protocol based on the normal distribution. log transformation of the data produced comparable findings to both approaches. comparison of thresholds derived from different datasets revealed variations in defining the start of a season but relatively good agreement in describing the end and intensity of influenza seasons, except in the hospital data after the pandemic year. as the who protocol does not prescribe a method for defining the seasonal threshold, we used the simplest method of visual inspection but showed that the levels were consistent with variation in out-of-season virus circulation. numerous other approaches exist, based on more complex statistical techniques but many of these approaches usually require a pre-determined threshold to be nominated [15, 21, 23] , again often by inspection. in the setting of the average and alert thresholds for our datasets, we used the peak mean values to set the intensity thresholds as per the who protocol after observing the data agreement between the peak weekly means and medians and 90% ci upper limit and 95 percentiles. however, the median and 90 or 95 percentiles may be a more appropriate option when data are not normally distributed and no transformation has been performed. in practice, another point of consideration for the setting of the alert threshold may be a level of influenza activity that corresponds to an increased demand on the health care system [7] . this would be dependent on local health care systems. we also incorporated a two week consecutive rule into the definition of the onset and intensity of a season to reduce the number of false positive signals. based on the seasonal thresholds, we found inconsistencies in defining the start and end of a season across the datasets. given the variations in timeliness of influenza reporting [31] , we would expect the onset of ili surveillance to precede laboratory confirmed influenza and hospital admissions, and that both the ili surveillance systems might coincide with one another. by incorporating a laboratory component to the ili measure, the use of the composite variable appeared to improve the specificity and agreement between vaed and ili surveillance data. the finding is consistent with emerging literature that these composite variables may be a better proxy indicator of influenza incidence than either ili or lab data alone [36, 37] . the use of composite variables in surveillance warrants further investigations. in comparing the intensities of an influenza season, there was good agreement across all datasets, except for the vaed after 2009. the number of hospital admissions coded for influenza has increased both in and out of the influenza season, with the duration of the season prolonged due to the late end signal. these changes were not reflected in the ili or lab datasets. while there may be a number of possible explanations, such as changes in testing behaviours or disease coding, a recent study investigating the increase in out-ofseason influenza in australia suggests a genuine increase in influenza activity, combined with increased testing that occurred following the pandemic [38] . this may reflect an increased awareness of influenza in hospitalised patients among health professionals after the pandemic. additionally, surveillance data at vidrl indicate that approximately 40% of patients with an influenza-like illness were swabbed prior to 2009 [26] but this rose to 70% during the 2009 pandemic [39] and has since remained at about this proportion [9] . the measurement of the intensity a season was based on the peak of influenza activity and whether or not the thresholds were exceeded for two weeks. this reflects only a single dimension of measurement and does not take into account how long the influenza activity remained within a particular category or the rate of increase in the number of cases. for example, a short-term acute rise in influenza cases that marginally exceed the alert threshold does not correspond to a gradual or persistent elevated level of activity that may represent a higher disease burden. finally, when we compared the current parameters to the previous threshold based on seven years of historical ili data from 1994-2000 in victoria, the baseline threshold for the gpss was lower at 2 per 1000 cases compared with the revised threshold of 4 and the alert threshold was higher at 35 per 1000 cases compared with the revised threshold of 24 [13] . these differences indicate the need of regular review of surveillance-derived thresholds. in conclusion, this study has shown that the proposed who threshold protocol is simple to implement and could be easily adapted for any influenza surveillance system with adequate historical data. however, the study was based in a region with a temperate climate, and its application in the tropics would require further work. further exploration of the proposed who method in another temperate region would be of interest. while thresholds are useful as a warning system, they should always be interpreted with other available information. influenza (seasonal) factsheet epidemiology of influenza the annual impact of seasonal influenza in the us: measuring disease burden and costs global influenza seasonality: reconciling patterns across temperate and tropical regions how to deal with influenza: worthwhile surveillance system is in action world health organization (2012) who global surveillance standards for influenza. geneva: global influenza programme, surveillance and monitoring team, world health organization resource allocation during an influenza pandemic virological surveillance: influenza weekly updates centers cdcfor disease control and prevention (2013) flu activity & surveillance: weekly us influenza surveillance report weekly influenza surveillance overview in: control establishing thresholds for influenza surveillance in victoria can syndromic thresholds provide early warning of national influenza outbreaks? methods for monitoring influenza surveillance data a routine tool for detection and assessment of epidemics of influenza-like syndromes in france a statistical algorithm for the early detection of outbreaks of infectious disease epidemic features affecting the performance of outbreak detection algorithms influenza surveillance in europe: establishing epidemic thresholds by the moving epidemic method modelling influenza epidemic -can we detect the beginning and predict the intensity and duration? detection of epidemics in their early stage through infectious disease surveillance do cusums have a role in routine communicable disease surveillance? detecting the start of an influenza outbreak using exponentially weighted moving average charts overview of influenza surveillance in the united states laboratory-supported influenza surveillance in victorian sentinel general practices estimation of influenza vaccine effectiveness from routine surveillance data a medical locum service as a site for sentinel influenza surveillance influenza-like illness surveillance using a deputising medical service corresponds to surveillance from sentinel general practices laboratory diagnosis and surveillance of human respiratory viruses by pcr in h1n1 swine origin influenza infection in the united states and europe in 2009 may be similar to h1n1 seasonal influenza infection in two australian states in a comparison of data sources for the surveillance of seasonal and pandemic influenza in victoria influenza surveillance in australia: we need to do more than count the use of thresholds to describe levels of influenza activity an analysis of variance test for normality (complete samples) improving the estimation of influenza-related mortality over a seasonal baseline predicting the epidemic sizes of influenza a/h1n1, a/h3n2, and b: a statistical method the significance of increased influenza notifications during spring and summer of 2010-11 in australia pandemic influenza h1n1 2009 infection in victoria, australia: no evidence for harm or benefit following receipt of seasonal influenza vaccine in 2009 we gratefully acknowledge the ongoing support of general practitioners and their practice staff participating in the general practice sentinel surveillance system and the continued involvement of the melbourne medical deputising service in influenza surveillance in victoria. we thank the victorian government department of health for provision of hospital admissions data and staff at the viral identification laboratory at vidrl for provision of laboratory data. we acknowledge the world health organization global influenza program for the threshold methodology. sentinel surveillance in victoria is supported by the victorian government department of health. ee laine tay is a post-graduate scholar in the master of philosophy in applied epidemiology program, the field epidemiology training program in australia. conceived and designed the experiments: et kg hk am. analyzed the data: et kg mk hk. contributed reagents/ materials/analysis tools: am. wrote the manuscript: et hk. provided critical revisions to the article: et kg mk am hk. key: cord-319256-7pyinx1a authors: jin, xin; duan, yongwei; bao, tengfei; gu, junjuan; chen, yawen; li, yuanyuan; mao, shi; chen, yongfeng; xie, wen title: the values of coagulation function in covid-19 patients date: 2020-10-29 journal: plos one doi: 10.1371/journal.pone.0241329 sha: doc_id: 319256 cord_uid: 7pyinx1a objective: to investigate the blood coagulation function in covid-19 patients, and the correlation between coagulopathy and disease severity. methods: we retrospectively collected 147 clinically diagnosed covid-19 patients at wuhan leishenshan hospital of hubei, china. we analyzed the coagulation function in covid-19 patients through the data including thrombin-antithrombin complex (tat), α2-plasmininhibitor-plasmin complex (pic), thrombomodulin (tm), t-pa/pai-1 complex (t-paic), prothrombin time (pt), international normalized ratio (inr), activated partial thromboplastin time (aptt), fibrinogen (fib), thrombin time (tt), d-dimer (dd), and platelet (plt). result: the levels of tat, pic, tm, t-paic, pt, inr, fib, and dd in covid-19 patients were higher than health controls (p<0.05), and also higher in the patients with thrombotic disease than without thrombotic disease (p<0.05). what’s more, the patients with thrombotic disease had a higher case-fatality (p<0.05). tat, pic, tm, t-paic, pt, inr, aptt, fib, dd, and plt were also found correlated with disease severity. meanwhile, we found that there were significant difference in tat, tm, t-paic, pt, inr, aptt, dd, and plt in the death and survival group. further using univariate and multivariate logistic regression analysis also found that t-paic and dd were independent risk factors for death in patients and are excellent predicting the mortality risk of covid-19. conclusion: most covid-19 patients with inordinate coagulation systems, dynamic monitoring of coagulation parameters might be a key in the control of covid-19 death. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 people are going through a battle against novel coronavirus pneumonia (covid-19) all over the world. by 3 april 2020, more than 1,010,000 people have been infected and more than 50,000 have died worldwide. the 2019 novel coronavirus (sars-cov-2) was confirmed as the pathogen of the covid-19 and belongs to beta coronavirus by the phylogenetic analysis, which is similar to severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov). respiratory symptoms, fever, dry cough, and panting, even acute respiratory distress syndrome (ards) and acute cardiac injury manifested in the covid-19 patients [1] [2] [3] [4] . sars-cov-2 can cause a series of disorders of the coagulation systems including endothelial damage, coagulation activation, and intravascular fibrin deposition. for covid-19 patients in severe, coagulation activation can be leading to the formation of thrombus and even disseminated intravascular coagulation (dic) [5] . and the coagulation activation is more common in patients with pre-existing coagulation disorders. on the other side, although, most severe covid-19 patients treated with extracorporeal membrane oxygenation (ecmo) to temporarily replaces cardiopulmonary function, the coagulation disorder is still one of the most common complications of ecmo, which is also a common cause of disruption of ecmo [6, 7] . therefore, it is significant to monitor the coagulation systems parameters in covid-19 patients to determine the coagulation function. previous studies have suggested that cytokine storms are typical abnormalities caused by sars-cov-2 [8, 9] . nevertheless the underlying reason of the connection between the coagulation function and the disease severity was not completely clear. especially, the index related thrombus such as thrombin-antithrombin complex (tat), α2-plasmininhibitor-plasmin complex (pic), thrombomodulin (tm), t-pa/pai-1 complex (t-paic) are important markers in the process of venous thrombosis and significantly increased before thrombosis, which had not been reported. therefore, this study was executed to evaluate the values of coagulation function for the prediction of severe covid-19 patients. we recruited 147 covid19 statistical analysis was performed with spss version 25.0 software and graphpad prism (version 7.0). all the measurement data were tested for normality, and non-normally distributed data were expressed as median (interquartile range), nonparametric mann-whitney test was used for comparison between the two groups. univariate and multivariate analysis using logistic regression analysis: calculate the odds ratio and 95% confidence interval. the prediction of various indicators for prognosis covid-19 patients were analyzed by the receiver operating characteristic (roc) curves, and the area under the roc curve (auc) was measured to evaluate the predictive ability. positive predictive and negative predictive value analysis by the med-calc software. for all statistical analysis, p<0.05 was considered statistically significant. among the 147 covid-19 patients, 76 were males and 71 were females, the median age was 64 years, while in the control group, 14 subjects were male and 4 subjects were females (s1 table) . there were 105 mild patients, 18 severe patients and 24 critical patients, of which 37 patients with thrombotic disease and 110 patients without thrombotic disease. the levels of coagulation parameters at healthy controls and covid-19 patients were demonstrated in table 1 and s1 table. as the table 1 shown, more than 79% patients with the high level of the tat, pic, tm, t-paic than the normal level, and the positive rate of the tat, pic, tm, t-paic were significant differences in the healthy and covid-19 groups. although the patients with abnormal level of pt, aptt, fib, tt, dd, plt, were less than 50%, the positive rate of the pt, fib, dd were significant differences in the healthy and covid-19 groups. as the table 1 shown, covid-19 patients had significantly higher values of tat, pic, tm, t-paic, pt, inr, fib, and dd than healthy controls, and there were no significant differences in aptt, tt, and plt. among the 147 patients, 37 patients with thrombotic disease, 110 were without the thrombotic disease. there were 7 patients died among the thrombotic disease group, while 3 patients died in the non-thrombotic disease group ( table 2 ). the patients in thrombotic disease group had a higher case-fatality rate than the non-thrombotic disease group (p<0.05). the levels of coagulation parameters at the thrombotic disease group and non-thrombotic disease group were demonstrated in fig 1. covid-19 patients with thrombotic disease had significantly higher levels of tat, pic, tm, t-paic, pt, inr, fib, and dd than those without the thrombotic disease. there were no significant differences in levels of aptt, tt, and plt. among the 147 included patients, 105 patients belonged to mild group, 18 were severe, and 24 were critical. tm, t-paic, pt, inr, dd were significantly different among the three groups, the values of coagulation function in covid-19 patients while tt was no significant difference (fig 2c-2f, 2i and 2j ). the more severe in covid-19 patients, the higher levels of tm, t-paic, pt, inr, and dd. tat and plt were significantly different between the severe and critical groups or mild and critical group, and no significance were found between the mild and severe groups (fig 2a and 2k) . with regard to pic and aptt, there were significant difference between mild and severe or mild and critical patients, and no significant difference in severe and critical group (fig 2b and 2g ). in addition, there was a significant difference of fib in mild group and severe group, however no significant difference was found between severe and critical group or mild and critical patients (fig 2h) . the roc curves and auc (fig 3) demonstrated that tm (auc = 0.85), t-paic (auc = 0.84), pt (auc = 0.90), and dd (auc = 0.92) had good predictive ability in mild vs severe/critical (all p<0.05, fig 3) . the levels of coagulation parameters at death and survival group were demonstrated in fig 4. covid-19 patients in the death group had significantly higher levels of tat, tm, t-paic, pt, inr, aptt, and dd, but lower plt level than the survival group. there were no significant differences in levels of tt, pic, and fib. further analysis using univariate and multivariate logistic regression also found that t-paic and dd were independent risk factors for patients death (table 3) . roc were drawn to analyze the prognostic values of coagulation parameters in covid-19 patients, the auc of t-paic and dd were 0.92 [95% confidence interval (95%ci) = 0.92-1.01], 0.94 [95%ci = 0.90-0.98] (fig 5) . it was showed that the t-paic and dd were excellent in independently predicting the mortality risk of covid-19. the sensitivity and specificity of t-paic in predicting death respectively were 90.0% and 91.2% with the cut-off of greater than 20.6 ng/ml, and the positive predictive value and negative predictive value were 42.73% and 99.20%. and those for dd were 100.0% and 86.0% with the cut-off of greater than 2.78 mg/ l, the positive predictive value and negative predictive were 34.26% and 100.00%. we reported the coagulation function of covid-19 patients in wuhan leishenshan hospital of hubei, china. the world health organization had declared the covid-19 was an international public health emergency at the end of january [10] . within a period of four months, more than 1,010,000 people have been infected and more than 50,000 people have died worldwide. although the values of coagulation function in covid-19 patients the values of coagulation function in covid-19 patients most of the covid-19 patients have mild symptoms with good prognosis and the crude mortality rate was about 2.3%, for patients progressing to severe or critical, mortality rates have increased significantly with crude death rates reaching 49% for critically [11, 12] . early identification of severe patients will help improve recovery rates and reduce mortality. studies have shown that covid-19 patients can develop a variety of secondary diseases that cause pathological changes such as hematology, immunity, biochemistry, etc. [13, 14] , but the changes in coagulopathy are not well understood. in this study, a retrospective analysis was conducted about the coagulation systems parameters of 147 patients. tat, pic, tm, t-paic, pt, inr, aptt, fib, tt, dd, and plt are expected to assess the function of the coagulation systems in patients and indicate the severity of the patients to provide clinical assistance. covid-19, as a life-threatening infectious disease, can cause endothelial damage, coagulation activation, and intravascular fibrin deposition. results of this study indicated that compared with the health controls, the tat, pic, tm, t-paic, pt, inr, fib, and dd higher in covid-19, while the aptt, tt, and plt with no difference. meanwhile, they were also no significant differences in thrombotic disease group and the non-thrombotic disease group. while the levels of tat, pic, tm, t-paic, pt, inr, fib, and dd in thrombotic disease group were higher than non-thrombotic disease group. four items of thrombosis detection (tat, pic, tm and t-paic) can predict the possibility of thrombosis in the patients at an early stage. tat as a marker for activation of the coagulation systems, to determine the optimal period of anticoagulation therapy and early diagnosis of thrombosis and pre-dic [15, 16] . pic as a marker of fibrinolytic system activation, predicting the formation of thrombus, assisting the diagnosis of dic, and guiding antifibrinolytic treatment [17] . the elevated level of tm indicates an impaired vascular endothelial system [18, 19] . t-paic, key marker of fibrinolytic system, suggesting thrombus progression [20, 21] . plasma levels of these sensitive biomarkers of endothelial injury in covid-19 patients may be useful for evaluating the endothelial injury in covid-19. besides, we also found the covid-19 patients with thrombotic disease had a higher case-fatality rate. therefore, it is a situation that requires our vigilance when the covid-19 patient with abnormal coagulation systems parameters, and it is likely to be accompanied by thrombosis, and the risk of death is greater. tat, pic, tm, t-paic, pt, inr, aptt, fib, dd, and plt were also found correlated with disease severity, while the tt was no correlation (fig 2) . tt is the time for plasma to coagulate after adding a prothrombin solution in the plasma, and reflects the presence of anticoagulants. based on clinical practice and roc analysis between mild and non-mild patients (fig 3) , some cut-off values of the test items were obtained. with tm >13.65 tu/ml, dd>1.03 mg/l, progress to severe illness should be closely observed and prevented. among them, the rise of dd concentration is most obvious, similar to wang's [22] and guan [23] study. dd is elevated because inflammation causes coagulation activation. our results suggested that patients' coagulation parameters should be taken seriously to avoid progression of covid-19. as reported, anticoagulant treatment is required in severe and critical patients, and dd � 5 μg/ml is used as an indicator to adjust anticoagulant therapy, while improving the prognosis [24] . to reverse intravascular coagulation, microthrombi formation, fibrin deposition, covid-19 patients might need anticoagulant or fibrinolytic therapy. further, we found the covid-19 patients in death group had significantly higher levels of tat, tm, t-paic, pt, inr, aptt, and dd than the survival group, and the plt decreased. it is shown that the patients have thrombocytopenia, elevated level of dd, and a prolonged aptt, suggesting that covid-19 patients death may be associated with dic [25, 26] . most severe covid-19 patients develop dyspnea or hypoxemia one week after the onset, and their lung is more serious and gas exchange cannot be performed. when ventilator cannot restore lung function, ecmo has become an effective treatment to rescue critical patients, but it is only temporarily replacing cardiopulmonary function, and there are still many complications. among them, the coagulation systems disorder is still one of the most common complications of ecmo, which terminate the ecmo [27, 28] . therefore, safe and scientific monitoring of the ecmo loop and thrombosis in patients, then early intervention can further reduce the harm of these complications to promote effective circulation support for ecmo. tat, pic, tm, and t-paic are the indicators of early prediction and monitoring of dic, and dd is the indicator that a thrombus is occurring or ongoing. what's more, our findings demonstrated that the prediction of the t-paic and dd can be as the excellent independently predicting the mortality risk of covid-19, with an auc of 0.92, 0.94, respectively (fig 5) . coagulation systems have values in covid-19 patients because most patients have coagulopathy. preventing recognition or blocking the occurrence of thrombus or dic in covid-19 patients, dynamic monitoring of coagulation systems parameters might be a key in the control of covid-19 death. supporting information s1 table. comparison of coagulation parameters between covid-19 patients and healthy control. (doc) the continuing 2019-ncov epidemic threat of novel coronaviruses to global health-the latest 2019 novel coronavirus outbreak in wuhan china coronavirus infections-more than just the common cold a new coronavirus associated with human respiratory disease in china genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding using the big data of internet to understand coronavirus disease 2019's symptom characteristics: a big data study preliminary results of routine coagulation parameters during ecmo in an ovine model impact of amicar on hemorrhagic complications of ecmo: a ten-year review the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak-an update on the status discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin world health organization. novel coronavirus (2019-ncov): situation report-15 the epidemiological 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membrane oxygenation: a systematic and narrative review we acknowledge all health-care workers involved in the diagnosis and treatment of patients in wuhan. conceptualization: xin jin, yongfeng chen, wen xie. formal analysis: xin jin. key: cord-319190-esjfhztp authors: lu, xi; yang, xinyi; li, xue; lu, yun; ren, zhitao; zhao, longyin; hu, xinxin; jiang, jiandong; you, xuefu title: in vitro activity of sodium new houttuyfonate alone and in combination with oxacillin or netilmicin against methicillin-resistant staphylococcus aureus date: 2013-07-02 journal: plos one doi: 10.1371/journal.pone.0068053 sha: doc_id: 319190 cord_uid: esjfhztp background: staphylococcus aureus can cause severe infections, including bacteremia and sepsis. the spread of methicillin-resistant staphylococcus aureus (mrsa) highlights the need for novel treatment options. sodium new houttuyfonate (snh) is an analogue of houttuynin, the main antibacterial ingredient of houttuynia cordata thunb. the aim of this study was to evaluate in vitro activity of snh and its potential for synergy with antibiotics against hospital-associated mrsa. methodology: a total of 103 mrsa clinical isolates recovered in two hospitals in beijing were evaluated for susceptibility to snh, oxacillin, cephalothin, meropenem, vancomycin, levofloxacin, minocycline, netilmicin, and trimethoprim/sulfamethoxazole by broth microdilution. ten isolates were evaluated for potential for synergy between snh and the antibiotics above by checkerboard assay. time-kill analysis was performed in three isolates to characterize the kill kinetics of snh alone and in combination with the antibiotics that engendered synergy in checkerboard assays. besides, two reference strains were included in all assays. principal findings: snh inhibited all test strains with minimum inhibitory concentrations (mics) ranging from 16 to 64 µg/ml in susceptibility tests, and displayed inhibition to bacterial growth in concentration-dependent manner in time-kill analysis. in synergy studies, the combinations of snh-oxacillin, snh-cephalothin, snh-meropenem and snh-netilmicin showed synergistic effects against 12 mrsa strains with median fractional inhibitory concentration (fic) indices of 0.38, 0.38, 0.25 and 0.38 in checkerboard assays. in time-kill analysis, snh at 1/2 mic in combination with oxacillin at 1/128 to 1/64 mic or netilmicin at 1/8 to 1/2 mic decreased the viable colonies by ≥2log(10) cfu/ml. conclusions/significance: snh demonstrated in vitro antibacterial activity against 103 hospital-associated mrsa isolates. combinations of sub-mic levels of snh and oxacillin or netilmicin significantly improved the in vitro antibacterial activity against mrsa compared with either drug alone. the snh-based combinations showed promise in combating mrsa. over the past few decades, antimicrobial resistance has been recognized as a major public health problem. since it was first recovered from patients in southern england in 1961 [1] , methicillin-resistant staphylococcus aureus (mrsa) has been reported worldwide and has become one of the leading causes of hospitalassociated and community-acquired infections [2] . in china, infections caused by mrsa in healthcare institutions have increased greatly in the past 20 years. it was reported that mean prevalence of mrsa in 16 medical centers in 12 cities across china had reached 50.4% by 2005 [3] . treatment options for mrsa are limited and less effective than options available for methicillin-susceptible staphylococcus aureus (mssa) infections [4] . this emphasizes the importance of developing more therapeutic options including novel antimicrobials and combinations of existing drugs to combat mrsa. historically, plants have provided us with a good source of antimicrobial agents. drugs of plant origin including artesunate, berberine, and quinine are still effective in some cases in treating infectious diseases [5, 6, 7] . houttuynia cordata thunb (figure 1 . a) is a common vegetable consumed in southwest china as well as an herb used in traditional chinese medicine for hundreds of years [8] . in april 2003, it was approved by the state administration of traditional chinese medicine of the people's republic of china as one of the component herbs in a chinese herb formula to prevent severe acute respiratory syndrome (sars) [9] . recently, several studies also provide scientific data to support and unveil its antibacterial [10, 11] , anti-inflammatory [12] , and antiviral [13] activities. houttuynin (decanoyl acetaldehyde, c 12 h 22 o 2 , mw = 198.3) is the main antibacterial ingredient in volatile oil of houttuynia cordata thunb. as houttuynin is chemically unstable, sodium houttuyfonate (sh, sodium 1-hydroxy-3-oxododecane-1sulfonate, c 12 h 23 nao 5 s, mw = 302.4) and sodium new houttuyfonate (snh, sodium lauroyl-a-hydroxyethyl sulfonate, c 14 h 27 nao 5 s, mw = 330.4), analogue of sodium houttuyfonate, were synthesized and have been approved by china food and drug administration to be used in clinical practice mainly in the formulations of tablets and injection [14, 15] . due to its improved chemical and pharmacological properties, snh has replaced houttuynin, and to a large extent sh, as an effective therapeutic agent for respiratory infections and inflammatory diseases such as acute or chronic bronchitis and pneumonia in clinical settings [16, 17] . the structures of the three compounds are shown in figure 1 . earlier studies indicated that a variety of bacteria could be inhibited by snh with gram-positive bacteria showing a higher sensitivity to the compound than gram-negative bacteria in vitro [18, 19] . however, no report has specially focused on activity of snh against mrsa. in this study, we examined the in vitro activity of snh and its potential for synergy when combined with antibiotics against a collection of hospital-associated mrsa (ha-mrsa) isolates recovered from various clinical samples in recent years. no permits were required for the described study, which complied with all relevant regulations. a total of 103 mrsa strains collected from a variety of clinical samples in two hospitals in beijing between 2005 and 2010 were used in this study. all mrsa isolates were identified by automated antimicrobial susceptibility test systems (bd or biomerieux systems) in hospital laboratories and were further confirmed by vitek 2-compact system (biomerieux, marcy i'etoile, france) and the standard oxacillin agar dilution method recommended by clinical and laboratory standards institute (clsi, formerly nccls) in our laboratory. in addition, genotypic features of these isolates were characterized by multiplex pcr for the meca gene identification and sccmec typing. relevant characteristics of the mrsa isolates used in this study are presented in table 1 . generally, sccmec type iii is the predominant type in ha-mrsa strains in china [20] . two atcc (american tissue culture and collection) mrsa strains, atcc33591 and mu 50 (also referred to as atcc 700699, vancomycin-intermediate s. aureus) were used as quality control strains. sodium new houttuyfonate was provided by beijing standardherbs medical science & technology development co. ltd. eight antimicrobial agents, including oxacillin (oxa), cephalothin (cef), meropenem (mem), vancomycin (van), levofloxacin (lvx), minocycline (min), netilmicin (net), and trimethoprimsulfamethoxazole (sxt) were commercially purchased. snh was dissolved in distilled water. stock solutions of antibiotics were prepared in solvents and diluents recommended by clsi [21] based on their actual purity or potency and sterilized through 0.22 mm filters before use. test solutions with different concentrations of snh and the antimicrobials were obtained by two-fold serial dilutions with cation-adjusted mueller-hinton (camh) broth (bd, cockeysville, md). camh broth was used for all susceptibility testing, checkerboard testing and time-kill analysis. colony counts were determined using tryptic soy agar (tsa; bd, cockeysville, md) plates. the minimum inhibitory concentrations (mics) of the antibacterial agents for all 103 clinical isolates were determined by broth microdilution method according to clsi guidelines [22] . wells of 96-well microtiter plates (nunc, thermo fisher scientific inc., roskilde) were inoculated with 100 ml of camh broth containing serial-diluted antimicrobials and a final inoculum of 5610 5 cfu/ml. the concentrations of snh ranged from 1 mg/ml to 128 mg/ml. the concentration ranges for other antibiotics were as follows: 0.03 mg/ml to 128 mg/ml for mem, van, lvx, min, and net; 0.06 mg/ml to 1024 mg/ml for oxa and cef; 0.008/0.15 mg/ml to 32/608 mg/ml for sxt. testing of oxacillin was performed in camh broth supplemented with 2% nacl. the microtiter plates were incubated at 35uc for 24 h. the mic was defined as the lowest concentration of an antimicrobial agent that prevented turbidity. all mic determinations were performed in duplicate. kill kinetics of snh was determined by time-kill experiments for five mrsa strains (three clinical isolates mrsa 5-20, mrsa 6-29 and mrsa 8-36 and two reference strains atcc 33591 and mu50) according to the method described by verma et al. with slight modifications [23] . an overnight culture was diluted with camh broth in a total volume of 30 ml containing an inoculum of 2610 6 cfu/ml in a 250 ml flask for each strain. distilled water or snh was added to yield concentrations of 06, 1/46, 1/26, 16, 26, and 46mic in the broth at standard inocula. viability counts were performed at 0, 2, 4, 6, 8 and 24 h of incubation at 37uc by plating 0.1 ml undiluted and 10-fold serial diluted samples onto tsa plates in duplicate. drug carryover effect was eliminated by saline and agar dilution. the experiments were performed three times on different days and the results were presented as mean and standard deviation. the time-kill curves were recorded as log 10 reductions in bacterial counts within a specific time period. bactericidal activity was defined as $3 log 10 cfu/ml reduction (99.9% kill) in colony count from the starting inoculum. the detection limit was 2log 10 cfu/ml. checkerboard assay. eight combinations including snh-oxa, snh-cef, snh-mem, snh-van, snh-lvx, snh-min, snh-net, and snh-sxt were evaluated on 10 randomly selected mrsa isolates and two quality control strains (atcc 33591 and mu 50) using microdilution checkerboard technique [23, 24] . in brief, a final inoculum of 5610 5 cfu/ml was added to wells of 96-well microtiter plates containing two-fold diluted snh and the other antimicrobial in camh broth. after incubation at 35uc for 24 h, the combined effect of snh with each antimicrobial was analyzed by calculation of the fractional inhibitory concentration index (fici) using the following equation: fici = (mic of drug a in the combination/mic of drug a alone)+(mic of drug b in the combination/mic of drug b alone). the antimicrobial combination was defined to be synergistic when the fici was #0.5; indifferent when 0.5, fici ,4; antagonistic when fici $4 [23, 24] . the experiments were performed in duplicate on different days. antimicrobial-free inoculation of each strain was included as growth control. snh was combined with oxacillin, meropenem or netilmicin and tested at concentrations below mic of each drug. the concentrations of antimicrobials used for different strains were 1/2 mic or mic for snh, 1/128 to 1/64 mic for oxa, 1/8 to 1/2 mic for net and 1/16 to 1/4 mic for mem. colony counts were determined at 0, 2, 4, 6, 8, 24, 48, and 72 h. synergy was defined as $2 log 10 decrease in cfu/ml between the combination and its most active constituent at 72 h, with the number of surviving organisms in the combination at $2log 10 cfu/ml below the starting inoculum. the susceptibility results of all antimicrobials tested against 103 mrsa isolates and 2 reference strains are presented in table 2 . the time-kill curves of snh for three mrsa clinical isolates and two reference strains are displayed in figure 2 . concentration-dependent killing and significant reductions in the viable bacterial counts were observed after 24 h exposure of the bacteria to snh at concentrations above mic. the maximum reductions in viable counts were 1.5 to 3 log 10 cfu/ml for 46mic of snh when compared with the initial inocula; the antibacterial effect of snh on the five strains was determined to be bacteriostatic or marginal bactericidal at test concentrations. checkerboard assay. the results of the checkerboard analysis are summarized in table 3 (detailed mic and fici values are presented in table s1 ). synergistic interactions were noted between snh and b-lactams as well as snh and net based on fic indices for these combinations. snh-oxa, snh-cef, snh-mem and snh-net demonstrated synergism in 11, 9, 12 figure 3 , 4 and s1, respectively. at the concentrations of sub-mic levels tested, the three antibiotics used alone showed little activity on bacterial growth. snh alone showed weak inhibitory effect on the bacteria but was followed by bacterial regrowth after 8 to24 h of incubation. in contrast, the combinations of snh with oxa, mem or net resulted in potent synergistic effect on all the test strains. the combinations greatly reduced the viable counts of bacteria by $2log 10 cfu/ml when compared with any of the most active agent used individually. the synergistic effect could still be observed at 72 h for some of the strains in certain combinations. in addition, the three combinations reduced the viable count by .3log 10 when compared with the starting inoculum and bactericidal effect was demonstrated. snh contains a hydrophilic sulfinyl head and a hydrophobic alkyl tail with 12 carbon atoms, which is considered to be surfactant-like structure. although antibacterial mechanism of snh remains unknown, it was indicated that snh exerted its antimicrobial effect mainly through binding of non-polar tail group to bacterial hydrophobic membrane proteins or cytoplasmic enzymes [18, 25, 26] . the hydrophobic lipid bilayer of cell membrane might be another target of aliphatic chain of snh, but it seems that a repulsive force between the negatively charged membrane and the anionic hydrophilic moiety of snh may weaken the potential interaction [27] . this might be an explanation of higher susceptibility of gram positive bacteria to snh than gram negative bacteria, which have higher negative charge on the outer membrane due to presence of abundant anion charged phospholipids and lipopolysaccharides [28] . in addition, a latest report showed that treatment of sh, analogue of snh, decreased transcription of some autolysins and inhibited triton x-100-induced autolysis in s. aureus in vitro. although correlation between inhibition of autolysis of s. aureus and antimicrobial effect of sh needs further elucidation, the findings provided a possible explanation of response of s. aureus to snh-like compounds [29] . overall, the enhanced antibacterial effects of snh-based combinations may be attributed to molecular property of snh which disrupted integrity and function of cell membrane. since membrane-mediated processes involved in cell wall biosynthesis, cell permeability and drug efflux are important sources of resistance of mrsa to some antimicrobials, it seems reasonable to hypothesize that mrsa will be sensitized to antimicrobials by disrupting the normal barrier function of the cell membrane. further work is required to identify the exact targets of snh and understand the mechanisms involved in the interactions between snh and b-lactams or aminoglycosides. snh was reported to exhibit potent inhibitory activity against staphylococcus aureus, staphylococcus epidermidis, and klebsiella pneumoniae, etc. [30] . this study evaluated activity of snh against mrsa and its potential for synergistic effect when combined with antibiotics. snh has both mic 50 and mic 90 values of 32 mg/ ml for 103 mrsa strains and displayed growth inhibitory or marginal killing activity against mrsa in a concentrationdependent manner. in another set of experiments, we demonstrated that there was no significant difference between mssa and mrsa on susceptibility to snh (table s1 ) and characteristics of dynamic time-kill curves of snh ( figure s2 ). as a result, it is unlikely that there is cross-resistance between snh and b-lactams. snh in combination with oxa exhibited synergistic effect for all the five mrsa strains tested in time-kill analysis. similarly, it was also demonstrated that the snh-oxa combination showed synergism on four mssa strains tested in another set of time-kill assays conducted to observe its action on mssa ( figure s3 ). besides snh-oxa combination, snh-net and snh-mem combinations also showed synergistic effect on mrsa strains. stressed by snh-based combinations, the viability of the organisms was largely depressed. although regrowth of bacteria was seen for some strains in certain combinations, it was mild and only occurred after 24 h. these findings reflect the gradual and stable antibacterial effect of these drug combinations. as use of mem is reserved to treatment of infections caused by multidrugresistant (mdr) gram negative bacteria in clinical settings, application of snh-mem combination on gram positive cocci including mrsa doesn't seem feasible. it might be interesting to test this combination on mdr gram negative bacteria. considering that oxacillin can combine with other antimicrobial agents to restore its utility against staphylococci [31] , while netilmicin was also reported to be effective on mrsa infection when used in a drug combination [32, 33] , the findings reported here suggest that in vitro combinations of subinhibitory concentrations of snh-oxa and snh-net could be beneficial inhibiting mrsa. however, further investigations, including pharmacokinetic/pharmacodynamic studies need to be conducted to determine if the in vivo activity 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methicillin-resistant staphylococcus aureus clones with a unique geographic distribution in chinese hospitals performance standards for antimicrobial susceptibility testing; seventeenth informational supplement. clsi document m100-s17 methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically clsi document m07-a9 methods for determining bactericidal activity and antimicrobial interactions synergy testing, time-kill curves, and population analysis antimicrobial combinations interaction of houttuyfonate homologues with the cell membrane of gram-positive and gram-negative bacteria antibacterial mechanism of houttuyfonate homologues against bacillus subtilis synthesis of novel analogues on the a-carbon of houttuyfonate and sar analysis of antibacterial activity with mopac antibacterial properties of cationic steroid antibiotics transcriptional and functional analysis shows sodium houttuyfonate-mediated inhibition of autolysis in staphylococcus aureus investigation of efficacy of sythesized sodium neo-houttuyfonate in clinical settings synergy between oxacillin and manuka honey sensitizes methicillin-resistant staphylococcus aureus to oxacillin clinical studies on the time-difference combination therapy with netilmicin and minocycline in methicillin-resistant staphylococcus aureus infections a case of mrsa sepsis treated by the sequential combination therapy netilmycin and minocycline we thank carol mita and gerald b. pier (harvard medical school) for the critical review of this manuscript, as well as jing pang, guoqing li, and congran li (institute of medicinal biotechnology, chinese academy of medical sciences and peking union medical college) for their technical assistance. conceived and designed the experiments: x. you jj x. lu x. yang. performed the experiments: x. lu x. yang x. li yl zr xh. analyzed the data: x. yang x. lu x. you. contributed reagents/materials/analysis tools: lz. wrote the paper: x. lu x. yang. key: cord-308344-ao9z00t7 authors: diep, nguyen van; norimine, junzo; sueyoshi, masuo; lan, nguyen thi; yamaguchi, ryoji title: novel porcine epidemic diarrhea virus (pedv) variants with large deletions in the spike (s) gene coexist with pedv strains possessing an intact s gene in domestic pigs in japan: a new disease situation date: 2017-01-17 journal: plos one doi: 10.1371/journal.pone.0170126 sha: doc_id: 308344 cord_uid: ao9z00t7 since late 2013, after an absence of seven years, outbreaks of porcine epidemic diarrhea virus (pedv) infection have reemerged and swept rapidly across japan, resulting in significant economic losses. in this study, we report the emergence, mixed infection, and genetic characterization of 15 novel field pedv variants with large genomic deletions. the sizes of deletion varied between 582 nt (194 aa) and 648 nt (216 aa) at positions 28–714 (10–238) on the s gene (protein). among 17 pedv samples isolated from individual pigs, all of them contained at least two distinct genotypes with large genomic deletions, and 94.1% of them were found to consist of strains with an intact s gene. these variants were found in eight primary and nine recurrent outbreaks, and they might be associated with persistent pedv infection in the farms. full-length s and orf3 genes of eight variants derived from 2 samples were characterized. this is the first report of mixed infections caused by various genotypes of pedv and would be important for the studies of viral isolation, pathogenesis, and molecular epidemiology of the disease. porcine epidemic diarrhea (ped) is a highly contagious enteric disease characterized by vomiting, acute watery diarrhea, and in particular, a high mortality rate in suckling piglets, resulting in substantial economic losses [1] . the etiologic agent of this disease is the ped virus (pedv), which is an enveloped, positive-sense, single-stranded rna virus that belongs to the order nidovirales, family coronaviridae, and genus alphacoronavirus. the disease was initially reported in england in 1971 [2] and belgium in 1978 [3] . since then, it has been identified in many swine-producing countries such as those in europe and asia, notably belgium, czech a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the samples were obtained from a naturally infected animal in the field by qualified veterinarians as a part of normal veterinary care and diagnostic testing procedures. samples of intestine used were collected from dead piglets and the fecal samples were non-invasively collected immediately after excretion. therefore, no aggressive operation had been conducted against pigs for sampling purpose. no piglets or other animals were sacrificed for the purposes of this study. the university of miyazaki dna recombinant committee approved the protocol for our cloning work of pedv (protocol number 2014-464). amplification of the partial s gene, full-length s gene, and orf3 gene reverse transcription was performed using random primers and oligo-dt primers from reverse transcription system kits (promega, madison, wi, usa). to determine the complete s gene sequence, five primer sets (cs1-cs5, table 1 ) were used to amplify five dna fragments spanning the entire s gene. these primers were designed on the basis of the published sequences of reference pedv strains [5, 9, 33] . emeraldamp max pcr master mix kit (takara bio, japan) containing high fidelity dna polymerases was used for performing the pcr amplifications under the following conditions: denaturation at 94˚c for 2 min; 35 cycles of denaturation at 94˚c for 30 s, annealing at 54˚c for 30 s and extension at 72˚c for 1 min; a final extension step at 72˚c for 10 min. when a partial s gene was amplified using the primer pair cs1-f/cs1-r from generated cdna, one or two distinct bands of unexpected shorter sizes were observed in each pcr product in addition to the predicted band (fig 1) . the same result was observed when one-step rt-pcr tests (accessquick rt-pcr system kit, promega corp, wi, usa) were performed from extracted rna and pcr tests using other kits containing high fidelity dna polymerases (kod fx, kod fx neo, and blend-tag plus kits, toyobo co., japan) were performed from cdna templates to amplify the cs1 fragment. the unexpected and expected dna bands were also observed and distinguished upon performing pcr amplifications with longer fragments containing the first portion of the s gene (primer pairs cs1-f/cs2-r, cs1-f/cs3-r, and cs1-f/cs4-r). the pcr products containing the cs1 fragment were individually gel-extracted and cloned into pcr2.1-topo vectors (thermo fisher scientific, us). the cloning reaction was then chemically transformed into one shot top10 to obtain the sequence of the full-length s gene and orf3 gene of pedv strains coexisting in a single pedv sample, a fragment containing these two contiguous genes was amplified using the primer set fs-f/orf3-r and the emeraldamp max pcr master mix kit (takara bio, japan). the pcr conditions were as follows: denaturation at 94˚c for 2 min; 35 cycles of denaturation at 94˚c for 30 s, annealing at 55˚c for 30 s and extension at 72˚c for 5 min; a final extension step at 72˚c for 10 min. the expected size of this amplified product was 4955 bp. subsequently, the pcr products were gel-extracted and cloned into pcr-xl-topo plasmid vectors (topo xl pcr cloning kit, invitrogen, us). after transformation into competent cells, plasmid dna isolation was performed as described previously. the purified plasmid dna was sequenced using the primer sets cs1-cs5 and the primer set orf3-f/orf3-r [15] . however, when the fragment containing entire s and orf3 genes was amplified, pcr products derived from only two samples jka-295 and jmi-277 were visualized distinctly in agarose gel 0.8%. therefore, these two samples were selected for cloning and sequencing of the fulllength s and orf3 genes. all sequences were determined in both directions, and sequencing reactions were performed using bigdye terminator v3.1 cycle sequencing kits and an abi prism 3130xl genetic analyzer (applied biosystems, us). nucleotide and deduced amino acid sequences were edited, assembled, and analyzed using geneious v9.0.4 software (http://www.geneious.com) and molecular evolutionary genetics analysis (mega) software version 6.0 [34] . the resultant nucleotide sequences were deposited in genbank under the following accession numbers: ku363044-ku363130. phylogenetic trees based on the nucleotide sequences of the full-length s gene and orf3 gene were constructed by the maximum likelihood method using the tamura-nei substitution model with a discrete gamma distribution; then gaps/missing data treatment was done by complete deletion, and bootstrap tests of 1000 replicates in the mega program. a total of 248 field samples (70.7%) collected from animals at 145 farms (82.4%) experiencing acute diarrhea in six prefectures were positive for pedv. the result indicated that pedv has a high prevalence rate in japanese pig herds. notably, ped outbreaks were commonly observed in ped-vaccinated farms, and there was a significant difference in the mortality rate (0-100%) for piglets and durations of diarrhea among pedv-infected farms. this prompted critical questions regarding the molecular characteristics and virulence of circulating pedv as well as efficacy of pedv vaccines that have been used in japan. to monitor heterogeneity among pedv strains, 52 pedv-positive samples were selected for further sequence analyses. however, when cs1-f/cs1-r primers were used for pcr to amplify the first portion of the s gene, one or two distinct bands of unexpected size (approximately 0.2-0.3 kbp) were observed in each pcr product of 17 pedv samples (32.7%) in addition to the predicted band (872 bp). the pcr products were individually gel-extracted, cloned, and sequenced. sequencing results illustrated that the unexpected bands were fragments with large deletions in the s gene, and a total of 15 novel pedv genotypes with large deletions were identified ( table 2 although two genotypes feature 606-nt deletions, the deletions were located at two distinct positions (94-699 and 46-651) in the s gene. furthermore, the genotype with a 203-aa deletion at residues 10-217 featured a 5-aa insertion (yamff) at the position of the deletion. interestingly, the identical nucleotide sequence corresponding to the insertion (tacgccatgttcttt) existed in the middle of the s gene (nt 2091-2105). additionally, all pedv variants of the s protein identified in this study were predicted to have signal peptide cleavage sites using the signalp 4.1 server (http://www.cbs.dtu.dk/services/signalp-4.1). this indicates that the s proteins of the variants are most likely expressed as structural proteins. surprisingly, mixed infection with multiple variants was found in all pedv samples (17/17) collected from individual pigs in primary or recurred ped outbreaks. particularly, in sample jka-295, we found nine coexisting genotypes including a genotype with an intact s gene and eight genotypes with deletions of 194, 197, 201, 204, 205, 211, 215, and 216 aa (table 3) . identical genotypes were also isolated from different farms of various prefectures that are geographically distant from each other. in addition to the variants, pedv strains with an intact s gene were detected in 94.1% of the samples (16/17), with no intact gene detected in sample jao-56. these results revealed that complex mixed-genotype infection of pedv had occurred in individual pigs and farms. full-length s and orf3 genes of pedv strains derived from samples jka-295 and jmi-277 were cloned and sequenced. identical nucleotide sequences of both s and orf3 genes were distinguished and excluded, resulting in the identification of 11 individual strains (table 3) . three strains that were obtained had an intact s gene (4161 nt, 1386 aa), whereas eight other variants possessed deletions of 582 (194 aa), 591 (197 aa), and 645 (215 aa) nt. notably, the variants with a 197-aa deletion were identical to that of us strain pc177-p2, which was proposed to be obtained during adaptation of the virus in cell culture [17] . in addition, the 194-aa deletion from the variants in this study was similar to that of the tottori2 strain that has been recently isolated from domestic pigs in japan [19] . however, apart from tottori2, all variants with a 194-aa deletion in our study were found to coexist with other variants as well as strains with an intact s gene. the pedv s protein is a type i glycoprotein [35] . its identified epitope regions include the coe domain (499-638 aa) and epitopes ss2 (748-755 aa), ss6 (764-771 aa), and 2c10 (1368-1374 aa) [36] [37] [38] . it was demonstrated that the large deletions found in the s gene of the variants did not occur in these four regions, and there was no significant difference in the epitope regions between the pedv variants and pedv strains with an intact s gene (s1 fig) . sequencing analysis indicated that strains found in a single pedv sample displayed genetic diversity in their s genes. in sample jmi-277, three strains with an intact s gene had 99.64-99.78% nucleotide sequence identity with each other and 99.64-99.89% nucleotide sequence identity with three variants possessing the 197-aa deletion when pairwise distances were measured using mega v6.05 software. within jka-295, three variants with the 197-aa deletion shared 99.72-99.92% nucleotide sequence identity with each other but had 99.38-99.55% nucleotide sequence identity with jka-295fsde204co25, which possessed the 204-aa deletion (s1 table) . on the contrary, all strains found in jka-295 and jmi-277 had the greatest [39] . furthermore, phylogenetic analysis based on the full-length s gene classified these japanese strains into the north american (highly virulent) type as shown in fig 2a. it is suggested that the variants originated from north american type of pedv. the sequencing results illustrated that the orf3 gene of the 11 strains in jka-295 and jmi-277 had the same length of 675 nt, and it did not possess any deletion found in known pedv vaccine strains [15, 27, 40] . all of the variants with large deletions have the same orf3 nucleotide sequence excluding jka-295fsde194co25. nucleotide identity of the orf3 gene between the variants and intact s strains was 98.81-99.41% which was lower than the 28 field isolates (99.6-100%) collected from 5 prefectures in japan during 2103-2104 [15] . eight distinct snps between the variants and the strains with an intact s gene were also identified at positions 136, 154, 178, 228, 302, 561, 645, and 657 in the orf3 gene (s2 fig) . phylogenetic analysis revealed that the strains with an intact s gene and the variants fell into two distinct clades (fig 2b) . the result indicated that the variants were not vaccine-related, and might have evolved independently as compared to the intact s strains which coexisted in the individual samples. in this study, variants with large deletions in the s gene were found in eight primary and nine recurrent outbreaks from 16 pig farms, and they mostly (94.1%) coexisted with pedv strains with an intact s gene. in four farms (te, sa, is, and at), the variants were not found in the primary outbreaks, but they appeared in subsequent pandemics. this indicated that pedv variants could be transmitted from farm to farm and/or that the proportion of the variants in samples collected at the primary outbreaks was insufficient to permit detection of the variants by pcr. additionally, ped outbreaks in japan since late 2013 were proposed to be caused by the invasion of recent overseas strains [15, 31] . taken together, it is possible that in the recent pandemic, both the pedv variants and the strains with an intact s gene were introduced together from other countries, after which they spread across japan. however, the earliest ped outbreak of the variants possessing the large s deletions occurred on kagoshima on december 2013 (sample jka-292) while the first ped case in japan was reported earlier in okinawa on october 2013. presently, no pedvs possessing the large deletions in the n-terminus of the s gene analogous to the japanese variants was reported in other countries except pc177-p2 which was concluded to derive from cell adaptation in vitro. therefore, the variants might have arisen spontaneously during the pedv spreading in japan. further studies are required for the confirmation. in this study, due to the coexistence of the variants and pedv strains with an intact s gene in all of the samples excluding jao-56, it might not be accurate to evaluate the virulence of individual strains based solely on the information from clinical signs and mortality rates recorded from the pig farms. in jao-56, we could not identify any pedv strain with an intact s gene. in the farm from which this sample was collected, the sows had never been vaccinated with modified live pedv. piglets in the farm manifested various severities of diarrhea from mild to severe, and the mortality rate reached 72.4% (670/926). the mortality rate was much higher than the mortality rate recorded in the epidemic during which tottori2 was isolated (0%) [19] . therefore, the large deletion in the variants observed in this study might not necessarily lower the virulence of this virus. further investigations are needed to elucidate the virulence of variants in ped outbreaks. after the primary outbreaks occurred in 2013 and the first half of 2014, there was a sharp decrease of ped cases that were reported during the second half of 2014 and 2015 in japan. among the few recurrent ped outbreaks reported in 2014 and 2015, all the samples collected from pig farms in miyazaki (6 farms) and aichi (2 farms) were found to contain the pedv variants. in those farms, diarrhea in piglets was observed for a long time (up to three months), and outbreaks of ped were reported to frequently recur in intervals from three weeks to several months (s2 table) . although many strict measures for establishing a high level of security were implemented such as quarantine of reposition, strict visitor policies, banning of unwashed vehicles, controlling of carcasses, movement of the caretakers on the farms and using virucidal disinfectants; ped still recurred in the farms. therefore, it is suggested that mixed infection of pedv variants with large genomic deletions may play an important role in the persistence of pedv in pig farms. defective interfering (di) particles are a common observation in many virus families [41] . di particles are defective for replication in the absence of the product of the deleted gene, and its replication requires the presence of the complete functional virus particle to co-infect a cell with it, in order to provide the lost factors [42] . the pedv variants wherein large genomic deletions were observed may be di particles. however, we speculated otherwise because the reported pedv strains pc177-p2 and tottori2 possessing the same deletions found in the variants of this study could replicate well in cell culture and cause cytopathic effect [17, 19] . besides, the mutant strain pc177-p2 can propagate well and induce diarrhea in piglets [43] . a deletion of 215 aa (position in the n-terminal domain of the s protein analogous to the deletion observed in the variants revealed a not impaired propagation of pedv in vivo [44] . moreover, only variants with large s deletions were found in the field sample jao-56, which indicated that the variants were able to independently propagate well in pigs. additionally, all the deletions found in the variants are in-frame and the s proteins were predicted to express as a functional structure protein. taken together, it is suggested that the variants with the large s deletions in this study were not di particles. functions of the spike protein n-terminal domain of pedv and other alphacoronaviruses were previously elucidated [44] . the loss of this domain occurred in a number of alphacoronaviruses correlates with a loss or reduction of enteric tropism. the most obvious example of this phenomenon is transmissible gastroenteritis virus (tgev) that has a dual tropism targeting the respiratory and gastrointestinal tract of the pig. meanwhile, porcine respiratory coronavirus (prcv) is a natural variant of tgev which lacks the s protein n-domain. it has reduced considerably its enteric tropism and primarily replicated in the respiratory tract then, produced an attenuated disease in comparison with parental tgev strains. another typical example is feline coronaviruses that have 2 pathotypes: feline enteric coronavirus (fecv) causing a clinical mild or asymptomatic disease and feline infectious peritonitis (fipv) inducing a highly pathologic disease. fipv is thought to be derived from fecv by natural mutation. however, compared to fecv, pipv has a large deletion in the s protein n-domain which results in the loss of tropism for enterocytes [44, 45] . these examples revealed an important role of alphacoronavirus spike n-domain for viral replication in the enteric tract. the n-terminus (residues 1-249) of the s protein was demonstrated to be responsible for the sialic acid binding activity of pedv [44] . the loss of enteric tropism observed in tgev was specifically associated with the loss of the sialic binding activity that located in the n-domain. however, how the sialic acid binding activity affects the tropism of these alphacoronaviruses remains unclear. the s protein n-domain of some pedv strains were thought to be dispensable for replication of pedv in vitro [44] [45] [46] . the large deletions in the s protein of the pedv variants in this study indicated that this domain might also be dispensable in vivo. in the present study, the characteristics of large deletions found in the japanese variants were highly similar to those observed in the prcv that have a 224 or 227-aa deletion in the n-terminal domain of the s protein [47] . prcv can persist in pig herds throughout the entire year, or it can disappear temporarily from a herd for several months and reappear subsequently. the emergence and widespread prevalence of prcv lessened the clinical impact of tge in the united states and europe due to cross-protective immunity with tgev [48] . thus, the pedv variants identified in this study might have biological characteristics analogous to prcv and may persist in the pig farms of japan. unfortunately, we did not collect lung tissues from pedv-infected pigs in these outbreaks, and virus isolation for the individual variants has not yet been completed. therefore, it remains unclear whether the pedv variants found in this study exhibited altered virulence, pathogenicity, and tissue tropism from the strains with an intact s gene. in future studies, we will continuously seek to answer this question. this is the first report describing ped outbreaks with mixed infections of various genotypes. our study identified 15 field pedv genotypes with large deletions in the s gene that have been circulating in japan from 2013 to 2015. the full-length s gene and orf3 gene of eight variants and three strains with an intact s gene were characterized. the variants coexisted with intact s strains in high frequency and they might be associated with persistent pedv infection in pig farms in japan. such a reemerging and mixed infection of the variants in this study have put ped in a new situation indicating this disease has become more complex in terms of viral isolation, pathogenesis, and epidemiology. the mechanism by which the pedv variants were generated and evolved remains unclear. hence, further studies are required to elucidate biological properties such as changes in tissue tropism and the virulence patterns of the new variants. table. nucleotide sequence identity based on the full-length spike genes of 11 japanese pedv field strains identified in this study and pedv reference strains ã . (docx) s2 table. status of pig farms where the pedv samples using in this study were collected. (docx) porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines an apparently new syndrome of porcine epidemic diarrhoea a new coronavirus-like particle associated with diarrhea in swine porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis heterogeneity in spike protein genes of porcine epidemic diarrhea viruses isolated in korea porcine epidemic diarrhea virus variants with high pathogenicity new variants of porcine epidemic diarrhea virus, china emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences distinct characteristics and complex evolution of pedv strains comparison of porcine epidemic diarrhea viruses from germany and the united states complete genome sequence of a porcine epidemic diarrhea s gene indel strain isolated in france in complete genome sequence of a porcine epidemic diarrhea virus from a novel outbreak in belgium pubmed central outbreak-related porcine epidemic diarrhea virus strains similar to us strains, south korea us-like strain of porcine epidemic diarrhea virus outbreaks in taiwan us-like isolates of porcine epidemic diarrhea virus from japanese outbreaks between new variant of porcine epidemic diarrhea virus, united states cell culture isolation and sequence analysis of genetically diverse us porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene novel porcine epidemic diarrhea virus variant with large genomic deletion new porcine epidemic diarrhoea virus variant with a large deletion in the spike gene identified in domestic pigs detection and molecular diversity of spike gene of porcine epidemic diarrhea virus in china cloning and further sequence analysis of the spike gene of attenuated porcine epidemic diarrhea virus dr13 porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus genetic diversity of orf3 and spike genes of porcine epidemic diarrhea virus in thailand molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) samples from field cases in fujian differentiation of a vero cell adapted porcine epidemic diarrhea virus from korean field strains by restriction fragment length polymorphism analysis of orf 3 pedv orf3 encodes an ion channel protein and regulates virus production molecular epidemiology of porcine epidemic diarrhea virus in china isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate an immunohistochemical investigation of porcine epidemic diarrhoea porcine epidemic diarrhea: its diagnosis and control molecular characterization of pig epidemic diarrhoea viruses isolated in japan from differential detection of transmissible gastroenteritis virus and porcine epidemic diarrhea virus by duplex rt-pcr sequence determination of the nucleocapsid protein gene of the porcine epidemic diarrhoea virus confirms that this virus is a coronavirus related to human coronavirus 229e and porcine transmissible gastroenteritis virus mega6: molecular evolutionary genetics analysis version 6.0 coronavirus spike proteins in viral entry and pathogenesis identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus identification of two novel b cell epitopes on porcine epidemic diarrhea virus spike protein the gprlqpy motif located at the carboxy-terminal of the spike protein induces antibodies that neutralize porcine epidemic diarrhea virus mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo genetic variation analysis of reemerging porcine epidemic diarrhea virus prevailing in central china from defective viral particles and viral disease processes defective interfering viruses and their potential as antiviral agents evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains cellular entry of the porcine epidemic diarrhea virus establishment of feline intestinal epithelial cell cultures for the propagation and study of feline enteric coronaviruses manipulation of the porcine epidemic diarrhea virus genome using targeted rna recombination genetic evolution and tropism of transmissible gastroenteritis coronaviruses diseases of swine, tenth ed the work was conducted with funding from the university of miyazaki (grant number 2014-70). key: cord-280471-cqypwi5d authors: sun, hua-bao; zhang, yi-ming; huang, li-gui; lai, qi-nan; mo, qun; ye, xin-zhou; wang, tao; zhu, zhong-zhen; lv, xiao-lin; luo, yan-ji; gao, shi-ding; xu, jin-song; zhu, hao-hao; li, ting; wang, zhan-ke title: the changes of the peripheral cd4+ lymphocytes and inflammatory cytokines in patients with covid-19 date: 2020-09-25 journal: plos one doi: 10.1371/journal.pone.0239532 sha: doc_id: 280471 cord_uid: cqypwi5d to investigate the clinical value of changes in the subtypes of peripheral blood lymphocytes and levels of inflammatory cytokines in patients with covid-19, the total numbers of lymphocytes and cd4+ lymphocytes and the ratio of cd4+/cd8+ lymphocytes were calculated and observed in different groups of patients with covid-19. the results show that the lymphocytopenia in patients with covid-19 was mainly manifested by decreases in the cd4+ t lymphocyte number and the cd4+/cd8+ ratio. the decreased number of cd4+ t lymphocytes and the elevated levels of tnf-α and il-6 were correlated with the severity of covid-19 disease. novel coronavirus pneumonia (ncp) is mainly transmitted through respiratory droplets and close contact. the world health organization (who) lists ncp as a public health emergency of international concern [1] and officially named the disease caused by the novel coronavirus disease 2019 (covid-19) [2] . characteristic chest ct imaging patterns, positive nucleic acid detection in nasal and throat swab samples, normal or decreased numbers of peripheral white blood cells, decreased numbers of lymphocytes and increased levels of inflammatory cytokines are the key factors in the diagnosis of covid-19 [3] . there are many kinds of lymphocytes in human peripheral blood, including cd3+cd4+ helper t lymphocytes (cd4+ t lymphocytes) and cd3+cd8+ cytotoxic t lymphocytes (cd8+ t lymphocytes). the percentage of cd4+ t lymphocytes and the ratio of cd4+/cd8+ lymphocytes are decreased in hiv-infected patients. to investigate whether the peripheral blood lymphocytopenia in covid-19 patients was mainly caused by the decrease in cd4+ t lymphocytes, in this study, patients admitted to our hospital with different severities of covid-19 were examined as subjects. the total number of lymphocytes, the percentages of lymphocyte subtypes and the levels of inflammatory cytokines (tnf-α and il-6) secreted by cd4+ helper t lymphocytes in the peripheral blood were detected by hematology counter and flow cytometer, respectively. the number of cd4 + lymphocytes and the ratio of cd4+/cd8+ lymphocytes were calculated and observed, and a new method for studying the mechanism of 2019-ncov-induced lymphocyte reduction was provided. a total of 35 patients (26 men and 9 women; age range: 8-70 years) with covid-19 who were admitted to the affiliated infectious diseases hospital of nanchang university were evaluated. according to the diagnostic and clinical classification criteria of the novel coronavirus infection prevention and control plan (2nd edition) issued by the national health commission of the people's republic of china, all the patients were divided into the general covid-19 group (n = 13, 10 men and 3 women), severe covid-19 group (n = 12, 9 men and 3 women) and critical covid-19 group (n = 10, 8 men and 2 women). the normal control group (n = 20, 15 men and 5 women) included normal physical examination personnel. there were no significant differences in the ratio of men to women and age among the groups (p>0.05). the clinical symptoms of patients with covid-19 include fever, cough, sore throat and muscle ache. the results of the 2019-ncov nucleic acid tests performed on throat swabs were all positive in all the patient groups, and ct showed the changes characteristic of viral pneumonia. laboratory examination showed that the white blood cell counts in the peripheral blood were normal or decreased, and the total number of lymphocytes was decreased. all the patients with covid-19 had a history of travel to wuhan, hubei province, or to an epidemic area outside wuhan, hubei province. none of the patients died during the study, and the results of the 2019-ncov nucleic acid tests in the normal control group were all negative. the study protocol was approved by the ethics committee of the affiliated infectious diseases hospital of nanchang university (approval no. 202005, nanchang, china), and written informed consent was obtained from all participants when the participants were awake or from their next of kin when the participants were minors or/and comatose. none of the patients or normal controls suffered from aids, influenza virus a or b infection, hepatitis b virus infection, tuberculosis infection, autoimmune diseases or other types of pneumonia. throat swabs from the patients were tested for the presence of 2019-ncov nucleic acids by rt-pcr on an abi7500 pcr amplification instrument (applied biosystems inc., california, usa). the conserved gene sequences of open reading frame (orf1a/1b) and nucleocapsid protein (n) in the 2019-ncov genome were used as targets. when the orf1a/1b and n target fragments were both positive at the same time, and the rising peaks were obvious, the nucleic acid test result was determined to be positive. the 2019-ncov nucleic acid rt-pcr test kit (including the amplification primers) was produced by shanghai biogerm biotechnology co., ltd (shanghai). the national machinery registration number of the test kit is 20203400065. the catalog number of the rt-pcr test kit is zc-hx-201-2. the total numbers of peripheral white blood cells and lymphocytes in the patients were detected by impedance combined with laser on a bc-6900plus automatic hematology analyzer (shenzhen mindray biomedical electronic co., ltd., shenzhen, china). the detection reagent for peripheral blood cell analysis was purchased from shenzhen mindray biomedical electronic co., ltd (shenzhen, china). the name of the cell staining solution used for blood cell analysis is m-60fn staining solution (mindray), which product number is 105-012183-00; the name of hemolysin for hematology analysis is m-60ld hemolysin (mindray), which product number is 105-012177-00; the name of the diluent used for blood cell analysis is ds diluent (mindray), which product number is 105-005707-00. the blood cell control materials were produced by beckman coulter co., ltd. the name of control materials of blood cells used for blood cell analysis is coulter 5c cell control (beckman coulter), which product number is 7547001. the quality control data of the blood cell control materials were used as the control. the total number of lymphocytes was one of the results obtained by routine blood cell analysis. the detection of the subtypes of lymphocytes in the peripheral blood of the patients was analyzed on a beckman coulter dxflex flow cytometer (suzhou saijing biotechnology co., ltd, suzhou, china). the lymphocyte population was gated based on brightly positive cd45 staining and low ssc in the cd45 vs ssc dot plot. then, the cd3 vs ssc dot plot shows the lymphocyte population, and the t lymphocyte population was gated based on brightly positive cd3 staining. then, suppressor/cytotoxic (cd3+cd8+) and helper/inducer (cd3+cd4+) lymphocytes were identified in the cd8 vs cd3 dot plot and the cd4 vs cd3 dot plot, respectively. ten microliters of anti-cd molecule antibodies labeled with different fluorescent dyes and 50 μl of edta anticoagulant-treated peripheral blood were fully mixed, protected from light, and incubated at room temperature (19-25˚c) for 15-20 minutes. then, 500 µl of hemolysin(beckman) was added to the solution, and the solution was vortexed for 10 seconds, mixed well, protected from light, and incubated at room temperature (19-22˚c) for 15-20 minutes until the solution changed from turbid to clear. then, 200 µl of saline was added, and the solution was mixed well for detection by flow cytometry. the test kits used to analyze the lymphocyte subtypes, including anti-cd molecule antibodies labeled with different fluorescent dyes, were produced by beckman coulter co., ltd. (740 west 83rd st., hialeah, fl 33014, usa). the production approval document number (china) for the lymphocyte subtype test kits was guoxiezhujin20173401372. the medical device production license number (china) of the beckman coulter dxflex flow cytometer is sushiyaojianxieshenchanxu20130069. the catalog number of the test kits named cyto-stat tetra chrome for detection of lymphocyte subtype(beckman coulter co., ltd), which including cd45, cd3, cd4 and cd8 antibodies is 6607013. the name of hemolysin reagent is opti lyse c(beckman), which catalog number is a11895. all antibodies were undiluted for use in this study. the absolute number of cd4+ t lymphocytes was calculated as the total number of lymphocytes × the percentage of cd4+ t lymphocytes. the total number of lymphocytes in the patients was determined from the results of routine blood cell analysis. the levels of tnf-α and il-6 in the plasma of the patients were simultaneously determined by cytometric bead array (cba) via flow cytometry. then, 3-5 ml of heparin anticoagulant-treated blood from the patients was centrifuged to obtain plasma. twenty-five microliters of immune microspheres and 25.0 μl of patient plasma were mixed and incubated in the dark at room temperature for 2.5 hours. then, 25 μl fluorescent detection reagent was added. after washing the microspheres, the fluorescence intensity of the washed microspheres was quantitatively measured by a beckman coulter dxflex flow cytometer (suzhou, china). different concentrations of tnf-α and il-6 standards were analyzed together with the plasma to be tested. the fluorescence intensity of the microspheres was determined by flow cytometry, and the fluorescence intensity was in direct proportion to the concentrations of tnf-α and il-6 examined. the test kit used for the detection of tnf-α and il-6 in plasma by cba was produced by jiangxi saiji biotechnology co., ltd. (nanchang, china), which national machinery registration number is 20180010. the human plasma tnf-α and il-6 detection kit is named human th1 and th2 subgroup detection kit(jiangxi saiji), which product number is p010001. the antibodies of tnf-α and il-6 were undiluted for use in this study. all the data are expressed as the mean ± standard deviation (sd). comparison of continuous variables between two groups was performed using student's t-test. p < 0.05 was considered to be statistically significant. all the statistical analyses were performed using ibm spss statistics 23.0 software for windows (spss inc., chicago, il, usa) on a computer. in the covid-19 patients in the general, severe and critical groups, the numbers of peripheral lymphocytes and cd4+ t lymphocytes and the ratio of cd4+/cd8+ lymphocytes were significantly lower than those in the normal control group. the levels of peripheral lymphocytes and cd4+ t lymphocytes and the ratio of cd4+/cd8+ lymphocytes in the general group were lower than those in the normal control group (p = 0.000441252, 0.000404213, and 0.00361 3912, respectively, all <0.01), and the levels in the severe group were lower than those in the general group (p = 0.009585116, 0.000294487, and 0.004389093, respectively, all <0.01). the levels in the critical group were lower than those in the severe group (p = 0.00011258, 0.001189364, and 0.00764968, respectively, all <0.01). all the differences were significant, with p<0.01 (fig 1) . the correlation coefficient of the number of peripheral blood cd4+ t lymphocytes and the number of total lymphocytes in the 35 covid-19 patients was 0.9051 (p = 0.000002, <0.01). in the patients with covid-19, the decreased number of cd4+ t lymphocytes was positively correlated with the decreased number of total lymphocytes. the lower the number of cd4+ t lymphocytes was, the lower the number of total lymphocytes was. in the patients with covid-19, the total number of peripheral blood lymphocytes decreased mainly as the number of cd4+ t lymphocytes decreased. the levels of peripheral cd8+ t lymphocytes in the general group were lower than those in the normal control group (p = 0.02, <0.05), and the levels in the severe group were lower than those in the general group (p = 0.007, <0.01). the levels in the critical group were lower than those in the severe group (p = 0.002, <0.01). all the differences were significant, with p<0.05 (fig 2) . the tnf-α and il-6 levels in the peripheral blood of covid-19 patients in the general, severe and critical groups were significantly higher than those in peripheral blood of the subjects in the normal control group. the levels of tnf-α and il-6 in the general group were higher than the total numbers of lymphocytes and cd4+ lymphocytes and the ratio of cd4+/cd8+ lymphocytes in the patients in the general covid-19 group were significantly lower than those in the normal control group. the levels in the severe covid-19 group were significantly lower than those in the general covid-19 group, and the levels in the critical covid-19 group were significantly lower than those in the severe covid-19 group. the total number of lymphocytes (10 9 cells/l) and cd4+ lymphocytes (cells/µl) and the ratio of cd4+/cd8+ lymphocytes of the patients in the normal control group were all set to 1.0. �� p<0.01. https://doi.org/10.1371/journal.pone.0239532.g001 the total number of cd8+ lymphocytes in the patients in the general covid-19 group was significantly lower than that in the normal control group, the levels in the severe covid-19 group were significantly lower than those in the general covid-19 group, and the levels in the critical covid-19 group were significantly lower than those in the severe covid-19 group. the total number of lymphocytes (10 9 cells/l) and cd4+ lymphocytes (cells/µl) and the ratio of cd4+/cd8+ lymphocytes of the patients in the normal control group were all set to 1.0. � p<0.05; �� p<0.01. https://doi.org/10.1371/journal.pone.0239532.g002 those in the normal control group (p = 0.00000209 and 0.00001729, respectively), and the levels in the severe group were higher than those in the general group (p = 0.00007539 and 0.00004138, respectively). the levels in the critical group were higher than those in the severe group (p = 0.00003602 and 0.00006652, respectively). all the differences were significant, with p<0.01 (fig 3) . coronaviruses (cov) are divided into four genera, including α−/β−/γ−/δ-cov. the 2019-ncov that causes covid-19 is a β-coronavirus, which is an enveloped, positive singlestranded rna (ssrna) coronavirus. the shape of 2019-ncov is round or oval with a diameter of 60-140 nm, as observed by electron microscopy. the human-to-human transmission of 2019-ncov is clear. it was found that the genomic sequence of 2019-ncov shares more than 85% identity with that of sars-cov [4] . ace2 (angiotensin-converting enzyme 2), which is expressed in the lower respiratory tract of humans, is confirmed as a cellular receptor for 2019-ncov [5] ; this receptor mediates cellular entry of the virus and causes severe and potentially fatal respiratory tract infections. the incubation period is 1-14 days, mostly 3-7 days. the main clinical manifestations are fever, headache, dry cough, fatigue and muscle ache. some patients have symptoms such as nasal congestion, pharyngeal pain and diarrhea. the clinical types of covid-19 include the general type, severe type and critical type. patients with severe and critical conditions can rapidly develop respiratory failure, septic shock and multiple organ failure after approximately 1 week [6] . all the patients in our study exhibited typical clinical manifestations, such as fever, cough, sore throat and muscle ache. critical patients require mechanical ventilation with a ventilator, and severe patients have dyspnea, along with the characteristics of severe viral pneumonia observed on ct images. the clinical symptoms of general type patients are mild, and the lung ct showed characteristic images of mild viral pneumonia. pulmonary ct imaging can diagnose viral pneumonia, but determining the cause of the disease requires 2019-ncov nucleic acid detection. all of our subjects were positive the tnf-α and il-6 levels of the patients in the general covid-19 group were significantly higher than those in the normal control group. the levels in the severe covid-19 group were significantly higher than those in the general covid-19 group, and the levels in the critical covid-19 group were significantly higher than those in the severe covid-19 group. the tnf-α and il-6 levels in the peripheral blood of patients in the normal control group were all set to 1.0. �� p<0.01. https://doi.org/10.1371/journal.pone.0239532.g003 according to a throat swab 2019-ncov nucleic acid test, suggesting that the diagnosis of covid-19 was clear and that there was no misdiagnosis. it was reported that the total number of peripheral blood (pb) lymphocytes in patients with covid-19 is decreased [7] . to study which type of lymphocyte exhibits the greatest decrease, we analyzed the lymphocyte subtypes in the pb of patients with covid-19. the total number of lymphocytes was tested by routine blood tests, the percentage of lymphocyte subtypes was determined by flow cytometry, and the absolute number of lymphocytes in each subtype was calculated. the results showed that the lymphocytopenia in patients with covid-19 was mainly manifested by the decrease in cd4+ helper t lymphocytes (the absolute number); in addition, the ratio of cd4+/cd8+ lymphocytes decreased, which showed a typical cellular immune dysfunction similar to that observed in hiv patients. a cd4+ t lymphocyte count below 200 cells/µl in the peripheral blood is the basis for antiviral treatment in hiv patients. although there is no direct relationship between the genomes of the two viruses [8] , the results of this study suggest that for covid-19 patients with decreased cd4+ t lymphocyte numbers in their peripheral blood, the protease inhibitors used to treat human immunodeficiency virus (hiv) infection, such as lopinavir and ritonavir, are also worth trying and studying to improve the outcome of patients with covid-19. cd4+ t lymphocytes are mainly helper t lymphocytes (th) and include th1 and th2, which can secrete inflammatory cytokines. at present, the specific mechanism by which cd4 + t lymphocyte numbers decrease in the peripheral blood of patients infected with sars--cov-2 is unclear. there are 4 possible mechanisms. (1) 2019-ncov directly damages cd4+ t lymphocytes but does not invade cd4+ t lymphocytes. (2) 2019-ncov directly invades cd4 + t lymphocytes and takes cd4+ t lymphocytes as host cells. (3) patients with covid-19 develop viremia and progress to systemic inflammatory response syndrome (sirs). cd4+ t lymphocytes or other immune cells secrete a large amount of inflammatory cytokines, resulting in excessive consumption of cd4+ t lymphocytes. (4) sars-cov-2 inhibits the differentiation and production of cd4+ t lymphocytes. at present, there is no evidence that 2019-ncov can directly invade peripheral blood cd4+ t lymphocytes, and there is no evidence that a 2019-ncov receptor exists on the cd4+ t lymphocyte membrane. the maturation and formation of cd4+ t lymphocytes depend on the function of human thymus tissue cells, which can express the ace2 receptor [9] , suggesting that the damage to human thymus cells caused by 2019-ncov may be a cause of the decrease in peripheral blood lymphocytes in patients with covid-19. the inflammatory cytokine storm plays an important role in the development of covid-19 in patients [10] . tnf-α and il-6 are important inflammatory cytokines, and we found that the levels of tnf-α and il-6 in the plasma of patients with covid-19 were significantly increased and positively correlated with the severity of the disease. the significant increase in the levels of tnf-α and il-6 in the plasma of patients with covid-19 is related to many kinds of inflammatory immune cells that secrete inflammatory factors. the secretion of inflammatory factors may require the assistance of cd4+ cells, which can secrete a large amount of inflammatory cytokines themselves. our results suggest that the decrease in the number of cd4+ t lymphocytes in patients with covid-19 may be related to the excessive consumption of cd4+ t lymphocytes. how to effectively increase the number of cd4+ t lymphocytes in the peripheral blood of patients with covid-19 still requires further study. we found that the cd4+ t cells in the peripheral blood were decreased in individuals infected with covid-19 but that the levels of tnf-α and il-6 in the plasma, which can be produced by cd4+ t cells, were significantly increased. the reason may be that in addition to cd4+ th cells in the peripheral blood, a large number of monocytes in the blood and macrophages in human tissues, including the liver and intestine, can secrete large amounts of tnf-α and il-6 into the blood in humans. the number of cd4+ th cells in the peripheral blood decreased; however, this decrease does not mean that the abilities single cd4+ th cells to secrete tnf-α and il-6 decreased. the number of cd4+ th cells in the peripheral blood significantly decreased, but the plasma levels of inflammatory cytokines significantly increased in patients with covid-19; this finding may be related to the increased secretion of tnf-α and il-6 by a large number of immune cells other than cd4+ th cells in the peripheral blood or the enhanced ability of single cd4+ th cells to secrete tnf-α and il-6 during 2019-ncov infection. we detected a decrease in the number of cd4+ th cells in the peripheral blood, but this decrease does not mean that the number of cd4+ th cells in human tissues, such as lymph nodes or spleen, has decreased. the number of cd4+ th cells in the peripheral blood significantly decreased, but the plasma levels of inflammatory cytokines significantly increased in patients with covid-19, which may also be related to the fact that cd4+ t cells are sequestered in tissues and therefore are not detected in the blood. the targeting of thymus tissue by 2019-ncov may be an important reason for the decrease in cd4+ th cells in the peripheral blood of covid-19 patients. many immune cells in the blood secrete tnf-α and il-6, especially during infection and inflammation, and monocytes and macrophages in the peripheral blood and tissues mainly secrete large amounts of tnf-α and il-6. this phenomenon is also the main reason why although the number of cd4+ th cells in the peripheral blood significantly decreased, the plasma levels of tnf-α and il-6 significantly increased in patients with covid-19. the secretion of tnf-α and il-6 by cd4 + th cells in the peripheral blood may not be the main reason for the significant increase in tnf-α and il-6 in the peripheral blood of covid-19 patients. our study also found that not only the absolute number of cd4+ th cells but also the absolute number of cd8+ tc cells decreased in the peripheral blood. however, the decrease in the number of cd8+ tc cells is not as substantial as that of cd4+ th cells, resulting in a trend of decreasing ratios of cd4+/cd8+ lymphocytes in the peripheral blood with the severity of the disease. cd8+ tc cells are cytotoxic t lymphocytes and belong to one of the subtypes of t lymphocytes. the differentiation and maturation of all t lymphocytes in the body, including cd4+ and cd8 + t lymphocytes, is dependent on thymocytes; therefore the targeting of thymus tissue by 2019-ncov may be an important reason for the decrease in the number of cd8+ tc cells in the peripheral blood of covid-19 patients. further study is needed to investigate the mechanism by which cd4+ and cd8+ t cell numbers decrease in the peripheral blood of covid-19 patients. in patients with covid-19, the number of peripheral blood lymphocytes decreased, mainly manifesting as a decrease in the number of cd4+ t lymphocytes, a decrease in the ratio of cd4+/cd8+ lymphocytes, and a decrease in the number of cd8+ lymphocytes; the degrees of these reductions was significantly correlated with the severity of disease. the levels of tnf-α and il-6 in the peripheral blood were significantly increased in covid-19 patients, the degree of this elevation was significantly correlated with the severity of disease. the significantly decreased levels of cd4+ t lymphocytes and ratio of cd4+/cd8+ lymphocytes and the significantly increased levels of tnf-α and il-6 in the peripheral blood can be used as important laboratory indicators to assess the severity of covid-19 in patients. supporting information s1 table. the experimental data of the numbers of lymphocytes, cd4+ t lymphocytes, and cd8+ lymphocytes and the ratio of cd4+/cd8+ lymphocytes in the peripheral blood of patients with different severities of covid-19. compared with the normal control group, � p<0.05, �� p < 0.01. (doc) s2 table. the experimental data of the levels of tnf-α and il-6 in the peripheral blood of patients with different severities of covid-19. compared with the normal control group, �� p < 0.01. (doc) the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak-an update on the status genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding clinical features of patients infected with 2019 novel coronavirus in wuhan, china a novel coronavirus from patients with pneumonia in china analysis of angiotensin-converting enzyme 2 (ace2) from different species sheds some light on cross-species receptor usage of a novel coronavirus 2019-ncov clinical findings in a group of patients infected with the 2019 novel coronavirus (sars-cov-2) outside of wuhan, china: retrospective case series clinical and biochemical indexes from 2019-ncov infected patients linked to viral loads and lung injury hiv-1 did not contribute to the 2019-ncov genome tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis considerations for statin therapy in patients with covid-19. pharmacotherapy all the authors would like to express great gratitude to dr. qin zhang, department of clinical laboratory, the second xiangya hospital of central south university, for the guidance and help. we would also like to acknowledge the professional manuscript services of american journal experts conceptualization: hua-bao sun, yi-ming zhang, zhan-ke wang. writing -original draft: zhan-ke wang. key: cord-312817-gskbu0oh authors: witte, carmel; hungerford, laura l.; rideout, bruce a.; papendick, rebecca; fowler, james h. title: spatiotemporal network structure among “friends of friends” reveals contagious disease process date: 2020-08-06 journal: plos one doi: 10.1371/journal.pone.0237168 sha: doc_id: 312817 cord_uid: gskbu0oh disease transmission can be identified in a social network from the structural patterns of contact. however, it is difficult to separate contagious processes from those driven by homophily, and multiple pathways of transmission or inexact information on the timing of infection can obscure the detection of true transmission events. here, we analyze the dynamic social network of a large, and near-complete population of 16,430 zoo birds tracked daily over 22 years to test a novel “friends-of-friends” strategy for detecting contagion in a social network. the results show that cases of avian mycobacteriosis were significantly clustered among pairs of birds that had been in direct contact. however, since these clusters might result due to correlated traits or a shared environment, we also analyzed pairs of birds that had never been in direct contact but were indirectly connected in the network via other birds. the disease was also significantly clustered among these friends of friends and a reverse-time placebo test shows that homophily could not be causing the clustering. these results provide empirical evidence that at least some avian mycobacteriosis infections are transmitted between birds, and provide new methods for detecting contagious processes in large-scale global network structures with indirect contacts, even when transmission pathways, timing of cases, or etiologic agents are unknown. avian mycobacteriosis is a bacterial disease that has long been considered contagious, passing indirectly between birds through the fecal-oral route [1, 2] . however, recent long-term studies in well-characterized cohorts have found low probabilities of disease acquisition among exposed birds [3, 4] and multiple strains and species of mycobacteria associated with single outbreaks [5] [6] [7] [8] . these findings suggest pre-existing environmental reservoirs of potentially san diego zoo global houses one of the largest, breeding bird populations in the world, historically averaging over 3,000 birds at any given time across two facilities, the san diego zoo and san diego zoo safari park (collectively referred to as san diego zoo global, sdzg). birds are frequently moved among enclosures for breeding, behavior or other management reasons, as well as imported from or exported to other institutions. this creates a dynamic network of contacts over time that varies individual exposure to environments and other birds. the source population included 16,837 birds present at sdzg between 1 january 1992-1 june 2014 that were at least 6 months old and present for at least seven days. all birds in this population were under close keeper observation and veterinary care during the entire study period and received complete post-mortem exams if they died. birds in this population had documented dates of hatch, acquisition, removal, and death. we excluded a small number of birds (n = 437) because they had incomplete information on enclosure histories. the 16,430 remaining birds had near-complete enclosure tracking over time with move-in and move-out dates for each occupied enclosure. all management data were stored in an electronic database. thus, the population represents a group of birds for which 1) a near-complete social network could be assembled from housing records that tracked dynamic movement over time, and 2) avian mycobacteriosis disease status could be determined for any bird that died. all historic data in these retrospective analyses were originally collected for medical activities and animal management purposes unrelated to the present study. for these reasons, the san diego zoo global institutional animal care and use committee exempted our study from review for the ethical use of animals in research. if a bird in the source population died, a board-certified veterinary pathologist conducted a thorough post-mortem exam that included histopathology on complete sets of tissues, unless advanced autolysis precluded evaluation. if lesions suggestive of avian mycobacteriosis were observed, then special stains (ziehl-neelsen or fite-faraco) were used to confirm presence of acid-fast bacilli. occasionally, clinical presentation permitted antemortem diagnosis based on tissue biopsy. for this study, any bird with acid-fast bacilli present in tissues was considered positive for avian mycobacteriosis at the date of diagnosis. birds were classified as 'infected' on their date of diagnosis or 'uninfected' on their date of death if the post-mortem examination showed no evidence of disease. birds were also classified as 'uninfected' on their date of export if they were still apparently healthy. birds that were still alive on the study end date of 6/1/2014, were followed for up to the assumed minimum incubation period (further described below; e.g., six months or through 11/28/2014) to determine final disease status. the network was defined based on the subset of birds that qualified as subjects and their friends (network nodes), and the connections between them (network edges). study subjects included all birds from the source population with complete information on history of exposure to other birds. this included both birds that hatched in the population, as well as birds imported from elsewhere. if a bird was imported, then it must have been present for a duration equal to or greater than the maximum incubation period (further defined below); those that were present for less time were not included as a study subject because they could have been infected prior to importation. any bird that directly shared an enclosure with a subject for at least seven days was considered a "friend". thus, the same bird could serve as both a subject as well as a friend for other birds, as illustrated in fig 1. spatial connections between subjects and friends were determined through cross-referencing enclosure move-in and move-out dates of all birds. contact occurring in a few enclosures, including hospital and quarantine enclosures, could not be determined and was therefore excluded. exposures that could lead to potential transmission of mycobacteriosis would be those which occurred within the incubation period of the subject (fig 1) . however, the distribution of the true incubation period for avian mycobacteriosis is unknown. as a starting point, minimum incubation period, i.e., the minimum time for an exposure to result in detectable disease, was set to six months. this was based on early literature from experimental studies that mimicked natural transmission [27, 28] . this is also consistent with our own data where the earliest case in the population occurred at 182 days of age [3] . the maximum incubation period was set to two years. early studies reported deaths occurring up to 12-14 months after infection [27] [28] [29] ; however, some authors reviewed by feldman [1] considered it possible that the disease progression could take years. for subjects that were classified as non-infected, this same interval (two years to six months prior to death or censoring) was used to identify contact with friends. for example, if a subject died on january 1, 2005, it would be connected to all friends with which it shared an enclosure for at least seven days within the time window of two years until six months prior to the subject's death, or between january 1, 2003 and july 1, 2004. exposures of subjects to friends that could lead to potential disease transmission would also be those which occurred within the friends' infectious periods when the bacteria could spread to other birds (fig 1) . the period of shedding during which a bird is infectious for other birds is unknown and no estimates were available for a naturally occurring disease course. therefore, friends were assumed to be infectious for the maximum incubation time, or two years, as diagram of potential transmission relationships and connectivity of birds in the network. the figure represents three example birds, assessed for the potential for each to have acquired infection from the other. each bird, or "subject", was defined to have an incubation period, initially set to the period occurring six to 24 months before the bird's final date in the study. any other bird that shared an enclosure with the subject during its incubation period was defined as a "friend" if the two birds shared the space during the second bird's infectious period. a friend's infectious period was initially set to the period occurring two years prior to its final date in the study. thus, the figure shows the incubation and infectious periods for each bird in the larger bars while the smaller bars show the overlapping period when the other two birds would be defined as its infectious"friends". the network edges were created from identifying the spatial and temporal overlap of potential incubation and infectious periods of subjects and friends in the study population. https://doi.org/10.1371/journal.pone.0237168.g001 a starting point. exposure of the subject to friends that were not infected was considered for the same two-year period prior to the friend's final date in the study. fig 2a and 2b illustrates network assembly over time for an example subject and its friends. the transmission network was graphed using the kamada-kawai [30] algorithm and all visualizations and analyses were performed using r software, package: igraph [31] . an initial clustering of disease associated with all indirect contacts that could influence the subject's disease status (based on timing of contact). friends-of-friends: same environment. clustering of disease associated with influential indirect contacts that were exposed to the same enclosure/environment. friends-of-friends: contagion. clustering of disease associated with influential indirect contacts that were never exposed to the same environment. this evaluation is key for removing the confounding effects of the environment and testing for a contagious process. friends-of-friends: homophily. clustering of disease associated with friends of friends that were never exposed to the same environment and could not have transmitted disease to the subject based on the timing of the connection. this reverse-time placebo test evaluates our data for the presence of homophily, or whether disease clustering can be explained by similarities among connected birds. https://doi.org/10.1371/journal.pone.0237168.g002 network was structured to include all connections of seven days or more between birds that occurred during their lifetimes. from this, the transmission network used in the analyses was constructed by refining connectivity based on the subjects' incubation periods and friends' infectious periods as described above. network topology was characterized by size (number of nodes and edges), average path length, and transitivity (probability that two connected birds both share a connection with another bird). to evaluate statistically whether or not disease status of a subject is predicted by the disease status of its friend, we calculated the probability of mycobacteriosis in a subject given exposure to an additional infected friend relative to the probability of mycobacteriosis in a subject exposed to an additional non-infected friend, i.e., the relative risk (rr). to determine significance of the rr, the observed rr was compared to the distribution of the same rr calculation on 1000 randomly generated null networks where the network topology and disease prevalence were preserved, but the disease status was randomly shuffled to different nodes [15, 32] . if the observed rr fell outside the range of permuted values between the 2.5 th and 97.5 th percentiles, i.e., the null 95% confidence interval (ci), then we rejected the null hypothesis that the observed relationship was due to chance alone. reported p-values were estimated from the null 95% ci. we evaluated the relative risk of disease transmission through five types of shared relationships between subjects and their friends ( fig 2b) . each evaluation targeted different groups of subject-friend pairs that varied in degrees of separation as well as spatial and temporal characteristics of network edges. risk (also referred to as "clustering") of disease associated with directly connected birds, or "friends". this analysis examined all pairs of birds where the subject was in direct contact with its friend during the subject's defined incubation period and the friend's infectious period (illustrated in fig 1) . the rr estimate includes the combined risk from direct exposure to both other infected birds and a common environmental source. this analysis examined whether associations persisted among the indirectly connected friends, as observed in other contagious processes [15] . to identify these friends of friends, we constructed a matrix of shortest paths between all subject-friend pairs that never directly shared an enclosure but were indirectly connected through an intermediary bird. before estimating the rr and conducting the random permutation tests, the data were limited to each subject's set of "influential" nodes, or the friends of friends connected by pathways that respect time ordering along which disease could propagate [33] . in other words, the friend of friend shared an enclosure with an intermediary bird before the intermediary bird contacted the subject. the estimated rr includes the indirect risk of disease from both contagion and exposure to a common environmental source. risk of disease transmission associated with influential friends of friends sharing an environment with their subject. this analysis examined associations with the subset of all influential friends of friends, where both birds were in the same enclosure but not at the same time. for example, bird a shares an enclosure with c. if a moves out and b subsequently moves in, then b is exposed to a via c. importantly, both a and b also were exposed to the same environment. associations in this group would reflect a combination of risk due to common environmental exposure and contagion. for contagion, we evaluated associations with the influential friends of friends that were never in the same enclosure as their subject. from the earlier example, if bird c is moved from an enclosure with bird a to an enclosure with bird b, then b is exposed to a via c. that is, a can transmit infection to b, even though they never shared an enclosure. case clustering could not be attributed to exposure to the same environment because the subject and its friends of friends were never housed in the same enclosure. this evaluation also ensured correct temporal alignment between exposure to an infectious agent and disease outcome in the subject. this comparison was key for removing confounding effects of environmental exposure and testing for a contagious process. although disease clustering among friends of friends could represent a contagious process, there is a possibility that some of the association could be explained by homophily, i.e., that connected birds could be more alike than the general bird population in terms of species, behavior, susceptibility, enclosure characteristics, etc. [19] . this could make both birds more likely to acquire infection from any source and manifest as clustering on a network at degrees of separation. we tested the network for the presence of homophily using a reverse-time placebo test. for this test, we evaluated disease clustering between a subject and its friends of friends from different enclosures that could not have transmitted infection based on the timing of the contact. for the tests of contagion, we described how b could be exposed to a via c; however, in that same example, the reverse would not be true. b could not transmit infection to a because disease transmission is time-dependent. for our reverse-time placebo test, we evaluated whether the infection status of b predicted the infection status in a. if so, then it would suggest homophily is present and driving disease clustering. sensitivity analyses were performed to compare differences in rr estimates while varying model assumptions. we varied subjects' incubation time (testing a minimum of three months and a maximum of one, three, four and five years) and friends' infectious time (two years, one year, and six months). we also refined network edges to evaluate associations in subsets of data where biases were minimized. this included limiting the friends to those whose exposure to the subject was exclusively outside of the two-year infectious window. it also included refining network edges to contact between subjects and friends that occurred only in small enclosures where enclosure sharing may be a better proxy of true exposure. finally, we limited analyses to subjects and friends that died and received a post-mortem examination. the 16,430 birds in the source population consisted of 950 species and subspecies housed across 848 enclosures. mycobacteriosis was diagnosed in 275 of these birds (1.7%). the subset that qualified as study subjects included 13,409 of the birds, which represented 810 species and subspecies. subjects were housed across 837 different enclosures that varied in size, housing anywhere from one to over 200 birds at any given time. in total, 203 (1.5%) subjects developed mycobacteriosis. subjects were present in the study population for variable amounts of time with the median follow-up being 3.4 years (iqr: 1.4-7 years). on average, subjects moved between enclosures 4.4 times (sd: 4.1; range: 0-71), and were housed in three separate enclosures (sd: 2.5; range 1-26). the average time a subject spent with each friend was about ten months (314 days; sd: 201 days). the full network that included all subject-friend connections contained 2,492,438 edges, but we focused on the transmission network limited to plausible fecal-oral transmission routes based on sharing an enclosure for at least 7 days during the subjects' incubation periods and its friends' infectious periods. this transmission network included all 16,430 birds with 905,499 connections linking their temporal and spatial location. the median number of friends each subject contacted (network degree centrality), was 105 (iqr: 21-303; range: 0-1435). the network exhibited small world properties [34] with short paths (average path length = 3.8) and many cliques where groups of birds were all connected to each other (transitivity = 0.63). a portion of the network diagram that includes subjects infected with avian mycobacteriosis and their directly connected friends is shown (fig 3) . results from all five associations are shown in fig 4 and rr estimates with p-values are reported in table 1 . when we performed our test between the subject and its directly connected friends we found significant clustering of cases based on social network ties; the risk of mycobacteriosis given exposure to an infected friend was 7.0 times greater than the risk of mycobacteriosis given exposure to an uninfected friend (p<0.001). significant associations persisted among the friends of friends. the rr of disease given exposure to any influential, infected friend of friend, compared to exposure to an uninfected friend of friend, was 1.35 (p<0.001). when subset to just the influential friends of friends that shared the same environment, the rr was 1.47 (p = 0.004). importantly, the friends-of-friends contagion model identified a significant 31% increase in risk of infection among subjects that were exposed to an infected friend of friend compared to those exposed to an uninfected friend of friend (rr: 1.31, p = 0.004). we found no evidence of homophily with our reverse-time placebo test; i.e., there was no significant association when the friends of friends were limited to those who may have correlated traits, but could not have influenced the subject's disease status based on location and timing of their indirect connection (rr: 0.95; p = 0.586). results of sensitivity analyses for all five evaluated relationships are shown in table 1 . the sensitivity analyses did not yield drastically different findings than the analyses of the main network and the significance of most associations remained. generally, as the subjects' incubation periods increased, the magnitude of the rrs with the friends and friends of friends decreased. this same pattern was observed when connectivity was limited to that occurring two years prior to the friends' removal dates (i.e., outside of the friends' incubation windows). patterns of significance were mostly unchanged when the network was limited to just animals with post-mortem exams, and just birds housed in small enclosures. importantly, significant disease clustering in the test for contagion persisted in most examined network variations. the exception to this is when the subjects' maximum incubation periods or the friends' infectious periods became more narrowly defined. homophily was detected only when network edges were restricted to exposures outside friends' incubation periods when long time spans were present (rr: 1.10; p = 0.014). our friends-of-friends network analysis suggests that avian mycobacteriosis can spread through bird social networks. although connected birds may acquire infection from exposure to common environmental sources and may share features that make them more likely to acquire disease through the environment, our friends-of-friends method detected statistically significant bird-to-bird transmission. one of the biggest challenges in determining if bird-to-bird contagion is present for infectious agents that are present in the environment, such as mycobacteria, is distinguishing the role of the environment. in one scenario, the environment serves as an intermediate collection place for mycobacteria being passed via (mostly) fecal contamination from an infected bird to one or more other birds, leading to infection spread in chain-or web-like patterns across a network [35] . alternatively, the environment may serve as the natural, independent reservoir of mycobacteria (e.g., biofilms in the water [36] ) giving rise to opportunistic infection among birds that share the location. spatial and temporal disease clustering could represent either or both of these two infection routes. homophily, where connected individuals tend to be more alike in species or habitat needs than the general population, and, therefore, may share the same disease susceptibility, could occur in both scenarios. for the directly connected birds in our study, the significantly elevated rr represented a combination of these three effects. examining the friends of friends rather than directly connected birds provided a means to disassociate exposure to another bird from exposure to that bird's environment. at two degrees of separation, the characteristics of network edges were more distinct, with temporal separation in potential transmission pathways and spatial separation in location. we exploited these pathways in a stepwise approach to calculate the rr of disease given exposure to friends of friends with different types of network ties. the subset of all influential friends of friends were temporally aligned to pass infection, but this group again represented a combined effect of multiple transmission pathways. because there was no evidence of significant homophily (further discussed below), we could use the network structure to test for the presence of contagion. among subjects who were connected to infected friends of friends in a different enclosure, the significant increase in risk for mycobacteriosis represents contagion. while this very specific subset of network edges allowed us to disentangle environmental and contagious transmission, it required two consecutive infections among a chain of related birds. this ignored most subjects and their friends of friends that shared enclosures where both processes were possible and completely confounded. while our extensive, long-term set of connections = 16,430) . the estimated relative risk (rr) for each of five different relationships between subjects and friends that were directly and indirectly connected. evaluated relationships are described in the methods and fig 2b. significance of the estimate was determined by comparing conditional probability of mycobacteriosis in the observed network with 1000 permutations of an identical network (with the topology and incidence of mycobacteriosis preserved) in which the same number of infected birds were randomly distributed. error bars show the null 95% confidence intervals generated from the random permutations. rrs that were outside of the null and significant are indicated with � . https://doi.org/10.1371/journal.pone.0237168.g004 in this network allowed detection of disease transmission using just this subset, the relative risks likely underestimate the true magnitude of bird-to-bird contagion. our data show significant, directional clustering along the pathways on which disease could propagate; however we did not find clustering when we reversed these pathways-where birds were connected, but disease could not be transmitted because passing an infection cannot move backwards through time. we applied our test of directionality, which is similar to those used by others [15] , to evaluate whether homophily could be driving the observed associations. in this bird population, similar species with comparable habitat needs have always tended to be housed together. therefore, we would expect biases due to homophily would exert similar effects along all pathways of connectivity, regardless of time. it is well documented that homophily and contagion are confounded in social networks [37, 38] and we could not specifically adjust the rrs for unobserved homophily; however many of the psychosocial factors that lead to homophily in human networks [19, 38] are not directly applicable to birds. while homophily table 1 , 1992-2014 (n = 16,430) . subjects network edges the five evaluated relationships are described in detail in the methods and fig 2b. the calculated statistic is the probability that a subject has disease, given that its friend has disease, compared to the probability that a subject has disease given that its friend does not (i.e., rr). to determine whether the observed rr falls within the 2.5 th and 97.5 th percentile of the null distribution, the disease status was randomly shuffled in 1000 network permutations where the network structure and prevalence of mycobacteriosis was preserved. significant p values indicate that the observed rr fell outside of the null 95% ci and we reject the null hypothesis that the observed rr is due to chance alone. https://doi.org/10.1371/journal.pone.0237168.t001 might still be present, our data strongly suggest that it is not driving the observed clustering of disease between a subject and its friends of friends. historically, in experimental infection studies, birds have been shown to be susceptible to the infectious bacilli when directly administered, i.e., introduced intravenously, intramuscularly, intraperitoneally, subcutaneously, or orally [39] [40] [41] [42] . yet, the relevance of direct inoculation to natural transmission has always been tenuous. some studies have shown little to no transmission when healthy chickens were placed in contact with either diseased birds or their contaminated environments [43] . therefore, our study provides new evidence, which supports bird-to-bird transmission in natural settings. our results also suggest that avian mycobacteriosis is not highly contagious, which is consistent with early experimental studies that conclude the bacteria must be given repeatedly over long periods of time to ensure infection [1] . the small world network structure that we identified for birds in the study population would predict epidemic-style outbreaks for diseases with facile and rapid transmission [34, 44] ; however, most birds did not acquire infection even when directly linked to other positive birds for long periods. over time, we have not seen epidemics and the incidence of disease in this population is consistently low (1%) [3] . our network approach was elucidating in this particular scenario, enabling us to uncover subtle patterns of a contagious process. environmental mycobacteria are recognized as the cause for ntm infections in humans and other animals [9] [10] [11] . limited genetic and speciation data from managed avian populations have found multiple strains and species of mycobacteria attributed to single outbreaks [6] [7] [8] . in our bird population, several different species and genotypes of mycobacteria have also been identified [5, 45] . consequently, we know that some birds could not have passed the infection to each other. genetic data from mycobacterial isolates would be a more definitive method of identifying the transmission of infection within a shared environment. for the present study, our approach was to isolate and test for contagion when there is missing information on the specific etiologic agents and transmission pathways. additional studies using genetic data could refine relevant transmission pathways or highlight important environmental sources within the network. we took care in assembling our network to ensure that the edge construction between subjects and friends adhered to general recommendations for disease networks [26, 46, 47] . this included incorporating biologically meaningful time-periods relevant to mycobacterial disease ecology and the type of exposure needed for transmission. generally, mycobacteriosis is considered a chronic disease, with an incubation period that can last for months and possibly years [1, 2] . it is also thought that animals can insidiously shed the organisms for long periods of time and those organisms can potential stay viable in the environment for years [48, 49] . we know there is misclassification of exposure in this network, because the true extents of incubation and infectious periods are wide, variable, and unknown. in sensitivity analyses, our rr estimates were generally similar when we varied incubation and infectious periods (table 1) . we did find a significant rr when limiting network edges to those occurring before the friends' 2-year incubation period, which suggests that some contagious processes may occur before the 2-year window. we also found that evidence for contagion was lost when either the subject incubation period or friend infectious period was short (less than six months and less than one year, respectively). it is likely that the shorter incubation times did not allow sufficient overlap of risk periods between subjects and friends. the duration of exposure needed for transmission is also unknown, but birds can be housed together for a year or more and not acquire infection [1, 4] . generally, mathematical models show that increasing the intensity or duration of contact between individuals with an infectious disease increases the probability of a transmission event and this can be reflected in weighted networks [35, 50] . in the present study, we required a minimum of seven days together to establish a network link that could capture relevant, short-duration exposure; however, the majority of birds were together for longer, with the mean contact-days being about 10.5 months (314 days). further exploration of contact heterogeneity on network associations may provide additional insight into clinically relevant exposure, infectious periods, and incubation times. inferring contagion by testing for disease clustering in subsets of the network requires quite complete network ascertainment, very good information on location over time, knowledge of disease outcomes, and a large number of subjects and their connected friends over time. our zoo data were unique in this respect and represent an example of how network substructures can inform global disease processes. many of the issues that cause bias in network measures, such as node censoring [51] , network boundary specification [52] , or unfriending [53] are unlikely to have affected our findings due to the completeness of our data. while such data may currently be rare, large datasets with similar network resolution may become widely available in the future as the world becomes increasingly connected by technology. for example, many new public and private contact-tracing initiatives are taking advantage of mobile phone technology to digitally track covid-19. eventually, these may allow near-complete human disease transmission networks to be assembled. this makes our friends-of-friends social network approach using network substructures a viable option for informing indirect covid-19 transmission pathways and public policy. most epidemiologic studies that use a network approach focus on directly transmitted, infectious diseases [47] . social networks to investigate diseases transmitted through the environment are assembled less often because defining contact in the presence of environmental persistence or other important transmission routes, such as fomites or insects, can be challenging [26] . to our knowledge, this is the first application of a friends-of-friends method to determine whether global patterns of connectivity support a contagious process. similar approaches could be useful to investigate diseases of humans or animals when the network is complete and mobility patterns are known, but the disease etiology or transmission pathways are unknown. avian tuberculosis infections. baltimore: the williams & wilkins company diseases of poultry investigation of characteristics and factors associated with avian mycobacteriosis in zoo birds investigation of factors predicting disease among zoo birds exposed to avian mycobacteriosis molecular epidemiology of mycobacterium avium subsp. avium and mycobacterium intracellulare in captive birds pcr-based typing of mycobacterium avium isolates in an epidemic among farmed lesser white-fronted geese (anser erythropus) mycobacterium 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heterogeneity on tb reproduction ratio r 0 in a free-living brushtail possum trichosurus vulpecula population infectious disease transmission and contact networks in wildlife and livestock untersuchungen uber die tuberkulinkehllappenprobe beim huhn die empfanglichkeit des huhnes fur tuberkulose unter normalen haltungsbedingungen seasonal distribution as an aid to diagnosis of poultry diseases an algorithm for drawing general undirected graphs the igraph software package for complex network research a guide to null models for animal social network analysis network reachability of real-world contact sequences dynamics, and the small-world phenomenon mathematical models of infectious disease transmission surrounded by mycobacteria: nontuberculous mycobacteria in the human environment homophily and contagion are generically confoudnded in observational social network studies origins of homophily in an evolving social network contribution to the experimental infection of young chickens with mycobacterium avium morphological changes in geese after experimental and natural infection with mycobacterium avium serotype 2 a model of avian mycobacteriosis: clinical and histopathologic findings in japanese quail (coturnix coturnix japonica) intravenously inoculated with mycobacterium avium experimental infection of budgerigars (melopsittacus undulatus) with five mycobacterium species bulletin north dakota agricultural experimental station mathematical studies on human disease dynamics: emerging paradigms and challanges whole-genome analysis of mycobacteria from birds at the san diego zoo network transmission inference : host behavior and parasite life cycle make social networks meaningful in disease ecology networks and the ecology of parasite transmission: a framework for wildlife parasitology avian tuberculoisis: collected studies. technical bulletin of the north dakota agricultural experimental station field manual of wildlife diseases: general field procedures and diseases of birds. usgs-national wildlife health center epidemic processes in complex networks censoring outdegree compromises inferences of social network peer effects and autocorrelation social networks and health: models, methods, and applications the "unfriending" problem: the consequences of homophily in friendship retention for causal estimates of social influence we thank the many people from sdzg that made this work possible, including the disease investigations team and veterinary clinical staff for ongoing disease surveillance, and the animal management staff for tracking housing histories of birds. we thank caroline baratz, dave rimlinger, michael mace, and the animal care staff for assistance with historical enclosure data. we thank richard shaffer, florin vaida, and christina sigurdson for thoughtful comments on manuscript preparation. key: cord-313506-6bb4q7nv authors: sano, akiko; matsushita, hiroaki; wu, hua; jiao, jin-an; kasinathan, poothappillai; sullivan, eddie j.; wang, zhongde; kuroiwa, yoshimi title: physiological level production of antigen-specific human immunoglobulin in cloned transchromosomic cattle date: 2013-10-24 journal: plos one doi: 10.1371/journal.pone.0078119 sha: doc_id: 313506 cord_uid: 6bb4q7nv therapeutic human polyclonal antibodies (hpabs) derived from pooled plasma from human donors are food and drug administration approved biologics used in the treatment of a variety of human diseases. powered by the natural diversity of immune response, hpabs are effective in treating diseases caused by complex or quickly-evolving antigens such as viruses. we previously showed that transchromosomic (tc) cattle carrying a human artificial chromosome (hac) comprising the entire unrearranged human immunoglobulin heavy-chain (high) and kappa-chain (higk) germline loci (named as κhac) are capable of producing functional hpabs when both of the bovine immunoglobulin mu heavy-chains, bighm and bighml1, are homozygously inactivated (double knockouts or dko). however, b lymphocyte development in these tc cattle is compromised, and the overall production of hpabs is low. here, we report the construction of an improved hac, designated as cksl-hacδ, by incorporating all of the human immunoglobulin germline loci into the hac. furthermore, for avoiding the possible human-bovine interspecies incompatibility between the human immunoglobulin mu chain protein (higm) and bovine transmembrane α and β immunoglobulins (bigα and bigβ) in the pre-b cell receptor (pre-bcr) complex, we partially replaced (bovinized) the higm constant domain with the counterpart of bovine igm (bigm) that is involved in the interaction between bigm and bigα/igβ; human igm bovinization would also improve the functionality of higm in supporting b cell activation and proliferation. we also report the successful production of dko tc cattle carrying the cksl-hacδ (cksl-hacδ/dko), the dramatic improvement of b cell development in these cattle and the high level production of hpabs (as measured for the human igg isotype) in the plasma. we further demonstrate that, upon immunization by tumor immunogens, high titer tumor immunogen-specific human igg (higg) can be produced from such tc cattle. immunotherapy with human polyclonal antibodies (hpabs) or immunoglobulins (higs) prepared from the plasma of normal and convalescing human donors have been shown to be an effective treatment for several human diseases [1] . for example, hpabs derived from the plasma donated from the general population have been widely used to treat autoimmune and immunodeficiency diseases [2] , and hpabs from recovering patients or pre-vaccinated individuals are effective in treating infections caused by the same pathogens [3, 4] . human polyclonal antibodies are also used in medical emergency crisis situations such as severe acute respiratory syndrome (sars) outbreaks [5] . however, the availability and applications of hpabs are currently limited. a paucity of voluntary human donors has resulted in a severe shortage of hpabs, and donor-patient disease transmission concerns as well as a lack of reproducibility restrict their usage [6] . to address the medical demand for hpabs, different alternatives have been explored to substitute or even replace human plasma-derived hpabs. for example, igg or fabs (fragments of antigen binding), prepared from the plasma of immunized horses or sheep has been widely used to treat severe envenomation resulting from snake and spider bites [7, 8] . however, animal derived igg tends to cause immune reactions from treated patients. for example, it has been reported that rabbit derived anti-thymocyte globulin (atg) causes serum sickness in patients in several clinical applications [9, 10] . recently, an in vitro antibody production system based on recombinant dna and mammalian cell culture technologies has been in the development by symphogen a/s [11] . this approach tries to mimic polyclonal nature of humoral immune response by producing mixtures of monoclonal antibodies (mabs) that recognize multiple epitopes of an antigen. such an approach, if successful, has the potential to produce antibody mixtures in large quantities within a well defined system, allowing for improved reproducibility and elimination of the risks associated with human plasma-derived hpabs. however, these antibody mixtures do not fully take advantage of the vastness of antibody diversity generated by natural immune responses. additionally, as pre-defined antigens are needed to identify the mabs and a lengthy process is needed to engineer cell lines expressing the recombinant mabs, this system may not be useful for treatment of diseases in which antigens are not well characterized, such as in autoimmunity, nor in dealing with sudden outbreaks of infectious diseases such as the 2002 sars epidemic [5] . to harness the power of natural humoral immune response not only for its unparalleled diversity but also for its capability to respond rapidly after antigen exposure, we have been developing a transchromosomic (tc) bovine system that quickly produces diverse hpabs in large quantities [12] . previously, we reported the generation of tc cattle carrying a human artificial chromosome (hac) comprising the entire unrearranged germline loci of human immunoglobulin heavychain (high) and kappa light-chain (higk) with homozygous double knockouts (dko) of the two bovine ig mu heavy-chain loci (bighm -/-and bighml1 -/-) [12] . in these cattle, the production of serum hpabs, as measured for its higg isotype, was only up to 1.5 g/l which is significantly lower than the physiological serum igg levels of approximately 5-10 g/l in normal humans. the level of fully higg (higg kappa; higg/ higκ) was even lower, at only approximately 0.65 g/l [12] . further analysis of the tc cattle showed suboptimal development of b cells, indicating that the low higg production in these tc cattle is the result of poor b cell development [12] . the pre-b cell receptor (pre-bcr), comprising of igm and invariant surrogate light-chains vpreb and lambda5 (λ5), plays critical roles for pre-b cell proliferation and maturation during b lymphogenesis [13] . it transmits b cell proliferation and maturation signals to the nucleus through its interaction with the b cell transmembrane proteins α and β immunoglobulins (igα and igβ) [14] [15] [16] . we reasoned that the poor b cell development and low higg production in the previously produced tc cattle could be due to a compromised function of pre-bcr, as in the hac/dko cattle the bigm in the pre-bcr complex is replaced by higm. specifically, we speculated that, due to the difference in protein sequences between human and bovine igms, higm may not interact with bovine surrogate lightchains effectively; similarly, the functional interaction between the ch-tm domains of higm with the bovine igα and igβ proteins for transmitting the cellular signals from a pre-bcr to the nucleus may also be compromised. therefore, in an effort to improve b cell development and higg production in tc cattle, we sought to enhance pre-bcr function by engineering a new hac into which, in addition to the high, higk and higl chromosome loci that carry the entire human immunoglobulin gene repertoire, the human vpreb (hvpreb1) and λ5 (higll1) genomic loci from human chromosome 22 (hchr22) was incorporated, and part of ch and tm domains, ch2-tm, of highm gene, was replaced by the corresponding bovine gene sequence (bovinization of the ch2-tm domains of highm). as igm interacts with surrogate light-chains and igα/igβ via its variable (v) and ch-tm domain, respectively, such modifications would restore the natural intra-species proteinprotein interactions in the pre-bcr ( figure 1 ). we also envisioned that the bovinized ch-tm domain would also improve the functionality of higm in interacting with bovine b cell proteins that are involved in b cell activation and proliferation. our results showed that dko tc cattle carrying the modified hac have greatly improved b cell development and produced up to 9 g/l of higg in the plasma. through immunization studies, we further demonstrated that these tc cattle are responsive to immunization with the tested tumor antigens and produce high titer tumor-specific higg. for deleting the nonimmunoglobulin genes on hchr14, intact hchr14 was transferred to chicken dt40 cells by using micro-cell mediated chromosome transfer (mmct) for chromosomal modification as described previously [17] . we employed a cre/loxp mediated site-specific recombination strategy to delete the dna sequences on hchr14 that are irrelevant to immunoglobulin locus. specifically, we integrated two lox511 sequences into hchr14, one at the al512355 locus (about 300 kb centromeric to the high locus) and the other at al391156 locus (about the 85 mb centromeric to the al512355 locus), through homologous recombination for deleting the 85 mb sequences on hchr14 between these two loci ( figure 2a ). in order to facilitate the identification of the correctly deleted dt40 cell clones, we also integrated a cag promoter and a hisd selection cassette along with the lox511 sequence at locus al512355 and the promoter-less puromycin (puro) gene along with the second lox511 sequence and a hygromycin selection cassette at locus al391156, respectively (figure 2a ; for the details, see materials and methods). such strategy ensured that only the correctly deleted hchr14 acquired puromycin resistance and was sensitive to hygromycin and histidinol (hisd) (figure 2a ). post electroporation of the dt40 cells with a cre-expressing vector; the correctly deleted colonies were screened by genomic pcr and confirmed by fluorescence in situ hybridization (fish) (data not shown). to prepare the correctly shortened hchr14 (labeled d) as one of the building blocks for the construction of the cksl-hacδ, it was further modified by integrating a loxp sequence and a gfp reporter cassette at the rnr2 locus as described in materials and methods and previously [12] . through extensive genomic pcr analysis (data not shown) and fish ( figure 2b ), a dt40 clone, 14d, was confirmed to have the loxp integration at the desired locus and selected for the bovinization of the ch2-tm2 domain of higm (see below). 2. bovinization of higm ch2-tm domain. in order to improve the functional interactions between the higm and bigα/ igβ proteins in the pre-bcr, as well as the overall functionality of higm in tc bovine b cells, we constructed a gene-targeting vector to bovinize the ch2-tm2 domain of higm that is involved in interacting with bigα/igβ [18] . the bovine genomic dna used for the gene targeting vector construction were cloned from an isogenic bovine genomic phage library (see materials and methods). we employed a positive and negative selection for this gene targeting event: a zeocin (zeo) expression cassette flanked by loxp sequences (floxed-zeo; for eventually removing of the zeo cassette from the bovine genome) was inserted into the intron between ch 4 and tm1 for positive selection and a dt-a selection cassette was added outside of the 3'-homologous arm for negative selection ( figure 3 ). a dt40 cell clone, ch2d4, was confirmed by extensive genomic pcr genotyping experiments (data not shown) to carry the correctly targeted 14d and was selected for the final hac construction. to isolate the human chromosome fragment that contains the entire higl gene cluster and the hslc locus (hvpreb1/ higll1), we set out to modify hchr22 as outlined in figure 4 . specifically, a dt40 cell line, 52-18, carrying the intact hchr22 was first truncated at the ap000350 locus with the targeting vector ptelcagzeoslfr and then was further modified with the targeting vector p553cag lox2272 bsrdt to integrate the lox2272 and the cag promoter at the locus ap000553. through extensive genomic pcr screening, a dt40 clone, stl54, was identified to carry the correctly modified hchr22 ( figure 4 ). we previously engineered a hchr2 fragment containing the entire higk locus in dt40 cells [12] . we further modified this hchr2 fragment carried by a dt40 clone (named as κtl1) with the targeting vector ptel'hisdpuro lox2272 f9r9 to both truncate the hchr2 fragment and integrate the lox2272 and the promoter-less puro gene at the ac104134 locus ( figure 5 ). locus ac104134 is about 300 kb telomeric to the higk constant region cκ gene, igkc, and this truncation event deleted all the non-immunoglobulin chromosomal sequences between this locus and the telomere of the p-arm on hchr2. a lox511 sequence along with the promoter-less puro cassette was integrated at the al512355 locus with gene targeting vector p14cen(fr)hygpuro lox511 dt, and a lox511 sequence along with a cag promoter and a hygromycin (hyg) selection cassette was integrated at locus al512355 with gene targeting vector psc355cag lox511 hisddt. following cre expression, the ~85 mb genomic sequence was removed rendering puro expression. a loxp sequence and a gfp reporter cassette was then integrated at the rnr2 locus to generate 14d. (b) fish analysis of a dt40 clone, 14d, containing the correctly modified hchr14. doi: 10.1371/journal.pone.0078119.g002 dt40 colonies were screened with genomic pcr (data not shown) for the correctly modified hchr2, and clone k53 was identified and selected for the final hac construction ( figure 5 ). with the chromosome cloning system we described previously [12] , we translocated the hslc and higl loci on hchr22 to the ac104134 locus adjacent to higk locus on hchr2 through cre/loxp mediated site-specific chromosome recombination ( figure 6 ). specifically, a dt40 clone k53 carrying the hchr2 fragment with the previously inserted hisd-lox2272-promoter less puro and hyg-loxp cassettes, and a dt40 clone stl54 carrying the hchr22 fragment with the previously inserted bsr cassette and lox2272, were fused via whole cell fusion (wcf) to generate dt40 hybrid cells. colonies derived from wcf were screened for the presence of both hchr2 and hchr22 with genomic pcr and fish by using human cot-1 dna as the probe as described in materials and methods. clone slk2 was identified as a positive clone ( figure 7) . finally, to translocate the hslc and higl loci on hchr22 to the higk locus at ac104134 on hchr2 through site-specific recombination, slk2 was transfected with the cre expression plasmid to induce the recombination between the two lox2272 sites that were previously inserted at the ac104134 locus on the hchr2 and ap000553 locus on the hchr22, respectively ( figure 6 ). puromycin resistant colonies (conferred by reconstitution of the cag promoter-lox2272-puro cassette at the translocation site) were screened by genomic pcr with cagpuro-f3r3 primers, followed by direct sequencing of the pcr product. clone slkh6 was identified to contain the correctly generated slk hac ( figure 6 ). we subsequently transferred the slk hac from slkh6 to plain dt40 cells by mmct. puromycin resistant and blasticidin s sensitive colonies were screened by extensive genomic pcr (data now shown); candidate clones were further confirmed by two-color fish using the hchr2 painting probe labeled with rhodamine and the hchr22 painting probe labeled with fluorescein ( figure 7 ). we chose clone slkd18 for subsequent hac engineering. the cksl-hacδ was constructed in chicken dt40 cells as outlined in figure 8 . dt40 clones, ch2d4 containing the ch2d hac and slkd18 containing the slk hac established above were fused by wcf to generate dt40 hybrid clone cksld22. after confirming with genomic pcr (data not shown) and fish with human cot-1 dna as the probe (figure 8 ), the selected clone cksldh22 was induced for site-specific translocation between the two loxp sites at the cos138 locus on the slk hac and another at the rnr2 locus on the ch2d hac by cre expression. cre expression also resulted in the deletion of the floxed cag promoter-zeo cassette within the cigm (ch2) domain. recombinants were enriched by sorting of gfp positive dt40 cells, as gfp expression was conferred by reconstitution of the pgk promoter-loxp-gfp cassette at the translocation site (data not shown). cksldh22 was finally identified as a dt40 hybrid cell line retaining the cksl-hacδ and subjected to extensive genomic pcr (data not shown) and three-color fish by dna probes recognizing the high, higk and higl loci, respectively, for structural confirmation. the constructed cksl-hacδ was transferred to chinese hamster ovary (cho) cells by mmct to establish cho-based master cell banks. the structural integrity of the constructed cksl-hacδ was further examined by extensive genomic pcr, array comparative genomic hybridization and three-color fish (data not shown). to our knowledge, this is the first report of construction of an artificial chromosome composed of structurally defined human chromosome fragments from three different chromosomes. to clone cksl-hacδ/dko cattle, we established cksl-hacδ/dko bovine fibroblast donor cells by transferring the newly constructed cksl-hacδ from cho cells into the dko (bighm -/-and bighml1 -/-) bovine fibroblast cells via mmct. cells from the fibroblast colonies derived from mmct were used as donors for somatic cell chromatin transfer (ct) as previously described [19] . cloned embryos developed to the blastocyst stage were singly transferred into recipient cows (one cloned blastocyst to one recipient cow) either for establishing gestation day 40 fetal cell lines or directly for tc cattle production. some of the established cell lines were also used for cksl-hacδ/dko cattle cloning. in total, 314 embryo transfers were conducted, with 114 from cell colonies (col) and 200 from fetal cell lines, and 17 healthy calves were produced (table 1) . we confirmed the cksl-hacδ/dko genotype of these tc calves with extensive genomic pcr with primers amplifying all of the junction points created during the cksl-hacδ construction process ( figure 9a and b). such analyses were performed on the genomic dnas isolated both from peripheral blood lymphocytes samples and ear biopsies collected from tc calves ( figure 9b shows a representative gel image of pcr products amplified from the genomic dnas isolated from peripheral blood lymphocytes). the retention of cksl-hacδ and its structural integrity in the produced tc calves were in order to investigate the impact of hac modification on b cell development in tc cattle, we analyzed the peripheral blood mononuclear cells (pbmcs) in newborn animals with flow cytometry. we used anti-higm antibody to detect igm + b cells in cksl-hacδ/dko animals, as the anti-higm antibody can still recognize the retained ch 1 domain of the bovinized higm. in comparison to the κhac/dko animals [12] , cksl-hacδ/dko calves demonstrated higher percentage igm-single positive (igm + ) and igm/cd21-double positive (igm + /cd21 + ) b cells ( figure 10 ). we measured the serum concentrations of total higg, i.e. all the higg regardless of whether it pairs with human lambda or kappa (higg/higλ/κ) light-chains or bovine lambda or kappa (higg/bigλ/κ) light-chains, as well as fully higg (higg/higλ/κ only), when the tc calves reached to 5-6 months of age. even though the total higg levels varied among these tc calves, the overall total higg production was found to be dramatically improved compared to the κhac/dko tc animals, at levels comparable to the physiological higg levels found in healthy humans ( figure 11a ). nevertheless, the average level of fully higg (higg/higλ/κ) produced by cksl-hacδ/dko calves was about 0.383 g/l which is only about 8.5% of the total higg. this is not unexpected, as the bovine immunoglobulin light-chain genes, lambda and kappa, were not genetically knocked out in such tc cattle, the expression of human light-chain loci on the hac would be easily overwhelmed by the endogenous bovine light-chain locus resulting in the produced higg mostly to be chimeric (higg/bigk/l). we also analyzed the igg subclass distribution in the plasma of such tc animals and showed that igg1 is the dominant subclass (higg1/higg2 ratio >1) which is similar to what is observed in the plasma of healthy humans ( figure 11b ). therefore, we produced tc cattle expressing physiological levels of higg with a subclass distribution profile similar to that of healthy humans. as our ultimate goal is to produce high titer antigen-specific hpabs from the tc cattle, we conducted an immunization study by immunizing two tc cattle with a human oral squamous cell carcinoma cell line as an antigen. to evaluate antibody titers specific to the tumor cells, we collected sera from each of the immunized tc cattle at 14 days post second vaccination (v2) and performed flow cytometry-based immunofluorescence assay. as shown in figure 11c , tc calves readily responded to immunization and generated high higg titer against human oral squamous cell carcinomas post v2 as demonstrated by the mean fluorescence intensity (mfi) of tumor cells stained with the sera from immunized cattle. furthermore, when mfi was measured by using anti-human kappa antibodies (to detect fully higg staining of the tumor cells, see materials and methods), measurable mfi was detected even after a 10,000-fold dilution of the sera. however, the titer against human tumor cells from fully higg is significantly lower than that from total higg. this was expected due to the fact that bovine immunoglobulin lightchain genes have not been genetically inactivated in these tc cattle and only roughly 8% fully higg is produced. there are numerous technical challenges in producing fully hpabs totally independent of the human immune system. first, as the human immunoglobulin genes and gene segments exist as gene clusters spanning large regions on three chromosomes, the only feasible way currently of delivering such a large size of human genomic dna into a biological system for hpabs production is through the engineering of hacs that comprise all or most of the human ig genes. we report here the successful engineering of a hac comprising the entire hig gene repertoire that reside on three different human chromosomes, i.e. the igh locus from hchr14, the igk locus from hchr2, and the igλ and surrogate light-chain loci from hchr22. the second challenge for fully hpabs production by a recombinant system is to have a biological mechanism to efficiently tap into the genetic information provided by the immunoglobulin gene repertoires for generating the seemingly unlimited diversity of hpabs. the mammalian humoral immune system is currently the only conceivable biological system that has such functionality. therefore, we set out to develop a tc animal system for fully hpabs production. we chose bovine for the large body size, which makes it feasible to collect large volumes of plasma, a prerequisite for producing large amounts of therapeutic hpabs. however, our initial success in producing antigen-specific fully hpabs in the tc cattle system was limited by the relatively low levels of total hpabs production and the low level of fully hpabs production [12] . in this report, we addressed this issue by improving the pre-bcr function through incorporating the human surrogate light-chain genomic locus into the hac and by bovinizing the hac in the higm domain that is involved in interacting with bovine igα and igβ signal molecules in the pre-bcr complex. our results showed that restoring intra-species interactions between proteins in pre-bcr had a positive impact on tc bovine b cell development as demonstrated by the increased development of igm + /cd21 + b cells in newborn tc calves ( figure 10) . very importantly, a dramatic improvement in total higg production was shown in these tc calves as compared to our previously reported κhac/dko tc animals, likely due to improved b cell development. finally, we demonstrated that tc cattle developed here are responsive to antigen immunization and generate high titer antigen-specific human polyclonal antibodies ( figure 11 ). therefore, we developed an improved tc bovine system to produce large quantities of hpabs. there are apparently some limitations that still exist in this tc bovine system, as the endogenous bovine immunoglobulin light-chain genes are intact and 80% of total serum higg produced is chimeric composing the higg heavy and big lightchains. while such chimeric higg are fully functional in recognizing antigens (our unpublished data) and are likely much less immunogenic than fully animal derived antibodies for therapeutic applications, it is obvious that fully human antibodies are more desirable as a biotherapeutic. this is especially true for long-term, repetitive treatments. therefore, we have recently knocked out the bovine lambda light genes by deleting the entire bovine lambda light-chain gene cluster (manuscript in preparation) and are in the process of knocking out the bovine kappa light-chain genes. accompanying our efforts to genetically improve our tc bovine system, we also have developed robust immunization protocols in tc bovine for producing high titer immunogen-specific fully hpabs. the fact that high titer antigen-specific hpabs can be produced from tc animals through hyper-immunization will offer great advantages over the human plasma derived hpabs in that the titers to certain pathogens from human plasma derived hpabs tends to be low and sources of convalescent plasma are often difficult to obtain. in summary, our improved tc bovine system for the production of fully hpabs has further advanced achieving the goal of producing large quantities of therapeutic hpabs as an alternative for plasma derived hpabs. the animal protocols contained in the study were approved by the hematech (previous name of sanford applied biosciences) institutional animal care and use committee (iacuc) (usda research facility 46-r-0008, olaw #a4438-01, aaalac #001114). care of all vertebrate animals is subject to regular review by the iacuc and complies with animal welfare laws and regulations of the united states. periodic health evaluations (blood profile, weight, etc.) are made by veterinary service to ensure that tc cattle are healthy. all tc bovine receive adequate housing, feed, access to water and bedding. daily observations are made by herdsmen to ensure that appropriate standards of animal care are being met. sanford applied biosciences uses the guide for the care and use of laboratory animals and the guide for the care and use of agricultural animals used in agricultural and research teaching for animal care standards. animal care and use and facilities are inspected by the iacuc on a semiannual basis. animals may have to be euthanized due to unfortunate events like terminal illness and trauma. methods of euthanasia must follow the guidelines given by the american veterinary medical association (avma) 2013. these are acceptable methods by the avma and they have been approved by the iacuc. all protocols and procedures used in the animal care and use program that may cause discomfort, distress and pain, and injury are reviewed by the iacuc for appropriate management practices. analgesic, anesthetics and tranquillizing drugs are used when appropriate under supervision of veterinary services. veterinarians and herdsmen use cattle chute for restraining when needed. cattle are not restrained for more than four hours per day. genomic dna isolated from primary bovine fibroblast cell lines established from holstein dairy cattle was used to construct the bovine genomic library. each λ phage-based genomic library was constructed using λfix ii vector through a custom library construction service (lofstrand labs ltd., gaithersburg, md). library screening and λ phage dna extraction/purification was done as described previously [12] . ptel'hisdpurolox2272f9r9: a previously described plasmid ptelpuro [12] was modified by replacing the puro gene with the hisd gene, eco ri site with srf i, and spe i site with pme i site to generate plasmid ptel'hisdpm. a ppurlox2272 plasmid was constructed by cloning the double strand lox2272containing oligo dna (oligo dna pair 1) into hindiii site of plasmid ppur (bd bioscience clontech, mountain view, ca). in parallel, a 7.3kb homologous arm (amplified by using a pcr primer pair, kd-f9 and kd-r9, in 40 cycles of 98 °c for 10 s and 68 °c for 9 min) was subcloned into bam hi site of plasmid ptel'hisdpurolox2272. then the bam hi fragment from the ppurlox2272 was blunted and cloned to pme i site of the ptel'hisdpm to generate the ptel'hisdpurolox2272f9r9 vector. ptelcagzeoslf2r2: plasmid ptelpuro was modified by converting the eco ri site to pme i and by replacing the puro gene with cagzeo gene to generate vector ptelcagzeo(sr)pm. then the second homologous arm (7.4kb) (amplified by using a pcr primer pair, sl-f2 and sl-r2, in 40 cycles of 98 °c for 10 s and 68 °c for 9 min) was cloned into bam hi site of the plasmid ptelcagzeo(sr)pm to generate ptelcagzeoslf2r2. p553caglox2272bsrdt: a previously described targeting vector phcf2loxphyg [17] was modified by replacing the homology arm sequence of the hcf2 gene with that of the ap000553 (amplified by using a pcr primer pair, 553-f3 and 553-r3, in 40 cycles of 98 °c for 10 s and 68 °c for 15 min) to generate p553loxphyg(f). this plasmid was not i-digested and self-ligated to remove loxphyg cassette, followed by cloning of dt-a fragment into srf i site. in parallel, pdrive-cag (invivogen, san diego, ca) was modified by replacing the lacz fragment (bsr gi-eco ri) with the loxp-containing oligo dnas (oligo dna pair 2). then sda i-swa i fragment was liberated from this plasmid and cloned into pst i/sma i-digested pbluescript sk − (stratagene, la jolla, ca) to generate pcagloxp. the loxp sequence was then replaced by the lox2272-containing sequence that was generated after annealing oligo dna pair 3. then the bsr gene was added to spe i site to generate pcagloxp2272bsr. finally, the not i-kpn i fragment (cag-lox2272-polya-bsr) was cloned into not i site to complete p553caglox2272bsrdt. psc355caglox511hisddt: a genomic dna fragment (10.2 kb) as one of the homologous arms was amplified by using a pcr primer pair, sc355-f3 and sc355-r3, in 40 cycles of 98°c for 10 s and 68 °c for 15 min. this pcr product was cloned into spe i site of a plasmid pbluescript where the kpn i site was converted to srf i site to generate psc355f3r3. the pcagloxp plasmid was similarly modified by replacing the loxp sequence with the lox511-containing sequence (oligo dna pair 4) and by adding the hisd gene to spe i site to generate pcaglox511hisd. the not i-kpn i fragment (cag-lox511-polya-hisd) from pcaglox511hisd was cloned into the eco rv site of psc355f3r3. finally, the dt-a cassette was cloned into not i site to complete psc355caglox511hisddt. p14cen(fr)hygpurolox511dt: a genomic dna fragment (10.2 kb) for another homologous arm was amplified by using a pcr primer pair, 14cen-f and 14cen-r, in 40 cycles of 98 °c for 10 s and 68 °c for 15 min. this pcr product was cloned into bam hi site of a plasmid pbluescript where the kpn i site was converted to pme i site to generate p14cen(fr). the modified lox511-containing oligodna (oligo dna pair 5) was cloned into hin diii site of a plasmid ppur (bd bioscience clontech, mountain view, ca) to generate plasmid ppurlox511. the bam hi fragment from the ppurlox511 was cloned to bam hi site of pbluescript sk − (stratagene, la jolla, ca), followed by cloning of the hyg gene to eco rv, to generate phygpurolox511. the not i-kpn i fragment (puro-lox511-hyg) was cloned into the hpa i site of p14cen(fr). finally, the dt-a cassette was subcloned into pme i to complete p14cen(fr)hygpurolox511dt. prnr2loxpbsrdt: the previous vector prnr2loxpbsr [17] was modified to construct the prnr2loxpbsrdt by simply adding the dt-a cassette. pch1cagzeo(r)dt(f): a genomic λ phage library was constructed from cho cells containing the κhac using λfix ii vector through a custom library construction service (lofstrand labs ltd., gaithersburg, md). the genomic library was screened for highm constant region by using a probe that was a pcr product (with pcr primer pair, hcμ-fr). a 1.7 kb of pml i fragment from one of the positive phage clones was cloned into sma i site of pbluescript to generate pch1s (f). then a 1 kb of sac i-pml i bovine ighm genomic fragment from plasmid pbcμay37-95 (a clone containing ighm bovine genomic fragment derived from lambda phage based genomic library [12] was subcloned into pst i site of the pch1s (f) to generate pch1ssp (f). a 7.4 kb of the sma i-eco ri fragment from another positive phage clone was cloned into eco rv/eco ri-digested pch1ssp (f) to generate pch1sl. in parallel, a 3.5 kb of sac i fragment from pbcμay37-95was cloned into pbluescript and then the xho i fragment of floxed cagzeo was cloned into van91 i site to generate pmaysazeo (f). the sac i fragment from the pmaysazeo (f) was then cloned into blunted eco ri site of pch1sl resulting in pch1zeo (f). as a final step, the dt-a cassette was cloned into not i site of the pch1zeo (f) to complete pch1cagzeo(r)dt(f). pch2cagzeodt: an annealed oligo dna pair, sesp, which contains human and bovine junction region at ch2 flanked by sexai and sph i site, was cloned into blunted pst i site of pbluescript. from the pbcμay37-95, a 2 kb of sph i-bam hi fragment was cloned into sph i-bam hi site of the plasmid generated by cloning sesp into the pst i site, generating pmayspb. similarly, a 2 kb of bam hi-pml i fragment from the pbcμay37-95 was cloned into bam hi-pme i site (converting the original spe i site) resulting in pmayspbpml. a 0.6 kb of eco ri-sex ai fragment from the above phage clone was cloned into eco ri-sex ai site of the pmayspbpml to generate prise. then the floxed cagzeo was cloned into van91 i site of the prise to generate prisecagzeo (r), of which not i site was converted to eco ri site, generating prisecagzeoe. meanwhile, 1.7 kb of pml i fragment from the above clone 4 was subcloned into sma i site of pbluescript of which eco rv site was converted to mlu i site, generating pch2s (f). 6.6 kb of mlu i-eco ri fragment from the above clone 1 was cloned into mlu i-eco ri of the pch2s (f), generating pch2ls. then, the eco ri fragment from the prisecagzeoe was subcloned into eco ri site of the pch2ls, generating pch2cagzeo (f). as a final step, the dt-a cassette was subcloned into not i site of the pch2cagzeo (f) to complete the pch2cagzeodt. hac vector construction was carried out as previously described [12, 17, 20] . briefly, dt40 (japan collection of research bioresources, #9130, japan) cells containing each hchr fragment were electroporated by using gene pulser ii (550 v, 25 μf) with ~25 μg of each targeting vector. dt40 cell colonies were selected by the appropriate drugs based the experimental design. the concentrations of drugs used are the following: g418 (2 mg/ml), puromycin (0.5 μg/ml), hygromycin b (1.5 mg/ml), blasticidin s (15 μg/ml), histidinol (0.5 mg/ml) or zeocin (1 mg/ml). genomic dnas isolated from selected cell colonies were subjected to pcr screening to identify the colonies with the correct gene targeting events. mmct was done with each hac vector as described previously [12, 17, 20] . these analyses were implemented as previously described [12, 17, 20, 21] . the pcr primer sequences used are listed in table s1 (oligo dna information). all the pcr products were resolved on 0.8 % agarose gels for analysis. primer sequences are available from table s1 (oligo dna information). array probe design, chip hybridization, and data analysis were performed by roche nimblegen (www.nimblegen.com). human cot-1 fish and hchr-specific multi-color fish were performed as previously described [12] . to specifically stain the high, higk and higl loci, probes were synthesized from dna derived from bac clones rp11-417p24, rp11-316g9 and rp11-22m5 (mbl-ebi and sanger institute: http:// www.ensembl.org/index.html), respectively. cloned fetuses and calves were produced using chromatin transfer procedure as described previously [19] . flow cytometry analysis on b cell development in newborn tc calves was performed as previously described [12] , except for some of the detection antibodies used. to detect surface higg on tc bovine b cells, goat anti-higg (life technologies, grand island, ny) directly labeled with af 488 was used. to label surface higκ or higλ on tc bovine b cells, mouse anti-higκ antibody directly labeled with pe (biolegend, san diego, ca) or mouse anti-higλ antibody directly labeled with pe (southern biotech, birmingham, al) was used. to label surface bigλ or bigκ on the b cells, mouse monoclonal anti-bigλ (in-house clone 132d7) or mouse monoclonal anti-bigκ (in-house clone 132b10) followed by zenon mouse igg1pe labeling (life technologies, grand island, ny) were used. staining was done by a standard protocol and then analyzed by facsaria flow cytometer (bd biosciences, san jose, ca). briefly, 10 6 cells were incubated with primary antibody on ice for 20 min. after washing twice, the cells were incubated with appropriate secondary antibody for 20 min on ice if the primary antibody is not directly labeled with a fluorochrome. finally, the cells were washed followed by re-suspend in facs stain media for reading. total higg elisa assay was performed as previously described [12] . for fully higg/higκ or higg/hlgλ detection, goat anti-higκ affinity-purified or goat anti-hlgλ affinity-purified (bethyl, montgomery, tx) as a capture and goat anti-higg fc-hrp (bethyl, montgomery, tx) as a detection antibody were used. for higg/bigκ detection, mouse monoclonal anti-bigκ (inhouse clone 132b10) as a capture and mouse anti-higg fcγ-hrp (jackson immunoresearch, west grove, pa) as a detection antibody was used. for detection of higg1 or higg2, mouse anti-higg1 fc or mouse anti-higg2 fc (hybridoma reagent laboratory, baltimore, md) as a capture and mouse anti-higg hrp (southern biotech, birmingham, al) as a detection antibody were used. cksl-hacδ/dko calves were immunized with x-rayirradiated human oral squamous cell carcinoma (dsmz) antigen at 2 x10 8 cells/dose formulated with montanide isa 25 adjuvant (seppic, puteaux, france) as water-in-oil emulsion plus quil a (accurate chemical & scientific corp, westbury, ny) as immune stimulant. the tc calves were immunized two times at 3-week intervals (primary immunization followed by the booster after 3 weeks). vaccine was administered by intramuscular injection in the neck region. serum samples were collected as previously described [5] before each immunization (v1 and v2) and 10 days and 14 days after each immunization for antibody titer analysis. anti-human oral squamous cell carcinoma antibody titers were determined by flow cytometry analysis. sera collected from tc calves immunized with human carcinoma cells were used as the primary antibody to stain the human carcinoma cells. pre-immune tc calf serum (v1, day 0) was used as the negative controls. af488-conjugated goat anti-higg fc (invitrogen, grand island, ny) at 1:80 dilution and pe-conjugated mouse anti-higκ (biolegend, san diego, ca) at 1:8 dilution were used to detect bound higg/higκ antibody. the assay was performed in pbs supplemented with 4 % horse serum, 0.1% sodium azide and 2 mm edta. the results were expressed as mean fluorescence intensity (mfi) as measured by facsaria flow cytometer (bd biosciences, san jose, ca). table s1 . oligo dna information. (xlsx) preparation and use of therapeutic antibodies primarily of human origin using intravenous immunoglobulin (ivig) to treat patients with primary immune deficiency disease using high titer west nile intravenous immunoglobulin from selected israeli donors for treatment of west nile virus infection preparation of commercial quantities of a hyperimmune human intravenous immunoglobulin preparation against an emerging infectious disease: the example of pandemic h1n1 influenza experience of using convalescent plasma for severe acute respiratory syndrome among healthcare workers in a taiwan hospital purification of intravenous immunoglobulin g from human plasma--aspects of yield and virus safety antibody production: polyclonalderived biotherapeutics intravenous immunoglobulin in the treatment of snake bite envenoming: a pilot study serum sickness after treatment with rabbit antithymocyte globulin in kidney transplant recipients with previous rabbit exposure serum sickness following rabbit antithymocyte-globulin induction in a liver transplant recipient: case report and literature review recombinant antibody mixtures: production strategies and cost considerations antigen-specific human polyclonal antibodies from hyperimmunized cattle the pre-b-cell receptor basal b-cell receptor signaling in b lymphocytes: mechanisms of regulation and role in positive selection, differentiation, and peripheral survival basal igalpha/igbeta signals trigger the coordinated initiation of pre-b cell antigen receptor-dependent processes signaling proteins and transcription factors in normal and malignant early b cell development manipulation of human minichromosomes to carry greater than megabase-sized chromosome inserts structural and functional studies of igalphabeta and its assembly with the b cell antigen receptor cloned calves from chromatin remodeled in vitro cloned transchromosomic calves producing human immunoglobulin sequential targeting of the genes encoding immunoglobulinmu and prion protein in cattle we thank trans ova genetics for performing embryo transfer and the genetic advancement center for tc calf delivery and care. key: cord-317058-anvmj4li authors: liu, xinkui; yue, xinpei; liu, furong; wei, le; chu, yuntian; bao, honghong; dong, yichao; cheng, wenjie; yang, linpeng title: analysis of clinical features and early warning signs in patients with severe covid-19: a retrospective cohort study date: 2020-06-26 journal: plos one doi: 10.1371/journal.pone.0235459 sha: doc_id: 317058 cord_uid: anvmj4li coronavirus disease 2019 (covid-19) was first identified in wuhan, china, in december 2019. although previous studies have described the clinical aspects of covid-19, few studies have focused on the early detection of severe covid-19. therefore, this study aimed to identify the predictors of severe covid-19 and to compare clinical features between patients with severe covid-19 and those with less severe covid-19. patients admitted to designated hospital in the henan province of china who were either discharged or died prior to february 15, 2020 were enrolled retrospectively. additionally, patients who underwent at least one of the following treatments were assigned to the severe group: continuous renal replacement therapy, high-flow oxygen absorption, noninvasive and invasive mechanical ventilation, or extracorporeal membrane oxygenation. the remaining patients were assigned to the non-severe group. demographic information, initial symptoms, and first visit examination results were collected from the electronic medical records and compared between the groups. multivariate logistic regression analysis was performed to determine the predictors of severe covid-19. a receiver operating characteristic curve was used to identify a threshold for each predictor. altogether,104 patients were enrolled in our study with 30 and 74 patients in the severe and non-severe groups, respectively. multivariate logistic analysis indicated that patients aged ≥63 years (odds ratio = 41.0; 95% ci: 2.8, 592.4), with an absolute lymphocyte value of ≤1.02×10(9)/l (odds ratio = 6.1; 95% ci = 1.5, 25.2) and a c-reactive protein level of ≥65.08mg/l (odds ratio = 8.9; 95% ci = 1.0, 74.2) were at a higher risk of severe illness. thus, our results could be helpful in the early detection of patients at risk for severe illness, enabling the implementation of effective interventions and likely lowering the morbidity of covid-19 patients. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 in december 2019, a local cluster of patients with pneumonia of unknown etiology was observed; subsequently, similar cases were observed nationally. on january 7, 2020, the virus strain causing pneumonia was successfully isolated by researchers [1, 2] . this virus is genetically related to the virus responsible for the severe acute respiratory syndrome-related coronavirus (sars-cov) outbreak in 2003 [3] ; consequently, the new virus was named the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) by the international committee on taxonomy of viruses on february 11. on the same day, the world health organization (who) proclaimed the official name of the disease caused by the new virus as coronavirus disease (covid-19) [4] . as covid-19 continued to spread and developed into an epidemic, the who declared covid-19 a "public health emergency of international concern" [5] . as of march 9, there were 80,754 confirmed cases reported in china [6] , of which 1,272 cases were in the henan province [7] . a study by zhou et al. reported the risk factors for mortality of covid-19 inpatients in wuhan [8] , but the thresholds for each risk factor was not given. to our knowledge, there have been no studies to predict patients with severe covid-19 from the results of their first visit. the objective of this study was to determine the risk factors for severe covid-19 based on the test results from the patient's first visit, and describe the clinical features in patients with severe covid-19. our study population consisted of confirmed covid-19 patients who were either discharged or died prior to february 15, 2020. our study was launched on march 1, 2020. participants with the following missing information were excluded from the study: age, date of first symptom, contact date, or examination data. all demographic and clinical information was collected retrospectively from the electronic medical records (emr). patients who underwent at least one of the following procedures were assigned to the severe group: continuous renal replacement therapy (crrt), high flow oxygen absorption, noninvasive and invasive mechanical ventilation, and extracorporeal membrane oxygenation (ecmo). the remaining participants were assigned to the non-severe group. data obtained from the emr included age, sex, contact history, latest contact date, primary symptoms and date of occurrence, comorbidities, and the results of routine blood tests as well as liver function, kidney function, c-reactive protein (crp), and procalcitonin tests from the initial visit. confidentiality of information was maintained by removing personal identifiable information. the medical research ethics committee of the first affiliated hospital of zhenghzou university approved this study. the following comorbidities were noted among our participants: respiratory diseases such as chronic obstructive pulmonary disease (copd), asthma, and interstitial pneumonia; metabolic diseases such as diabetes mellitus; cardiovascular disease (cvd) such as hypertension and coronary heart disease; and neurological diseases such as cerebral hemorrhage and infarction. a clustered onset was defined as two or more cases with fever or respiratory symptoms in a confined area, such as a home, office, school class, or similar setting, within the previous two weeks. the incubation period was defined as the time between the date of occurrence of primary symptoms and latest contact date with known infected individuals. data from the emr were entered using epidata and confirmed twice. every case was entered independently by two different data entry clerks and consistency was verified by a supervisor. three members of our research group were responsible for reviewing each data entry for which conflicting data had been entered. the doi link is dx.doi.org/10.17504/ protocols.io.bfpejmje. all data were entered and managed in epidata. spss 19.0 was utilized for cleaning and analyzing the data. as the quantitative data did not follow a normal distribution, the data were expressed as medians and interquartile ranges (iqrs) after applying the mann-whitney u test for comparison between the groups. qualitative data were expressed using counts and percentages and compared using the chi-squared or fisher's exact test. thresholds were calculated using receiver operating characteristic (roc) curves. independent effects were shown using multivariate logistic regression. based on the univariate analysis results and clinical experience, five variables were chosen for the multivariable analysis including age, comorbidity, lymphocyte count, crp levels, and procalcitonin levels. the significance level was set at 0.05. in the henan province, there were a total of 410 confirmed covid-19 patients with a final outcome of discharge or death prior to february 15. after excluding cases with missing values, 74 participants were assigned to then on-severe group and 30 participants were assigned to the severe group. the median age of the study participants was 42.0(iqr = 31.0-55.0) years, ranging from 5 to 85 years. there were 63 male patients, accounting for 60.6% of all participants and 41 female patients, accounting for 39.4% of all participants. of the 104 participants, 84 (80.8%) had a history of traveling or residing in either wuhan or the surrounding areas, or other communities with reported covid-19 cases, while 38 participants (36.5%) had contact with individuals who were experiencing fever or respiratory symptoms and had recently been to wuhan or its surrounding areas, or other communities with reported cases. furthermore, 17 (16.3%) patients had a clustered onset, 24 (23.1%) had a history of contact with a covid-19 patient, and 6 (5.8%) had an unknown history of exposure. the median incubation period for the entire group was 5.0 (iqr = 3.0-9.0) days, ranging from1 days to14 day. an incubation period of less than 7 days was observed in 70.5% of the patients. further, 29 (27.9%) participants had underlying diseases, with cardiovascular disease being the most common underlying disease, followed by metabolic and respiratory disease. the main symptoms of the participants included fever, cough, and fatigue. fever was the most common initial symptom, manifesting in 79 patients (76.0%) on admission ( table 1) . the median age in the non-severe group (55 years) was significantly higher than in the severe group (40 years). underlying diseases manifested in 13 (43.3%) patients in the severe group, which was higher than in the non-severe group, and the difference was statistically significant (p<0.05). the proportion of patients with cardiovascular disease was also greater in the severe group than in the non-severe group, and the difference was statistically significant (p <0.05). with regard to symptoms, the proportion of patients with dyspnea was significantly higher in the severe group than in the non-severe group (p <0.05). the proportions of patients with fever, cough, asthma, and chest distress were greater in the severe group; however, these differences were not significant. other symptoms demonstrated no quantifiable difference between the severe and non-severe groups ( table 1) . the majority of factors examined at first admission indicated a non-significant difference between the two groups; however, direct bilirubin, lactate dehydrogenase, crp, and procalcitonin levels were significantly higher in the severe group than in the non-severe group (p <0.05). additionally, the absolute value of lymphocyte count was also significantly lower in the severe group than in the non-severe group (p <0.05) as shown in table 2 . as demonstrated by the roc curves using a single predictor, age, the absolute lymphocyte value, crp, and procalcitonin are valuable predictors for detecting severe conditions in patients; the area under the curve (auc) for these predictors were 0.676, 0.708, 0.734, and 0.773, respectively. further, the thresholds for these factors were 63 for age, 1.02×10^9/l for lymphocyte, 65.08mg/l for crp, and 0.12μg/l for procalcitonin (figs 1-4 and table 3 ). the results of the multivariate logistic regression indicate that age, lymphocyte, and crp are independent predictors for an increased risk of severe covid-19. patients who are at least 63 years old, with an absolute lymphocyte value less than 1.02×10^9/l, and a crp greater than 65.08 mg/l are at greater risk of developing severe covid-19 with corresponding odds ratios of 41.0, 6.1, and 8.9, respectively (table 4 ). an analysis of the roc curves with multiple predictors is shown in fig 5. these results have a high degree of accuracy as implied by an auc of 0.858 (95%ci = 0.754, 0.962). our study indicates that age, the absolute lymphocyte count at initial visit, and crp may be used as predictors during the early stage of diagnosis in patients who are at risk of developing severe covid-19. in many severe cases, patients have cvd as a comorbidity, dyspnea, and higher concentrations of direct bilirubin, lactate dehydrogenase, and procalcitonin. in our study, the median age of the 104 participants was 42.0 years (iqr = 31.0-55.0) ranging from 5 to 85 years of age. in addition, for 70.5% of the patient, the incubation period was less than seven days, which indicates that covid-19 has a short incubation period and may impact both children and adults. the same result shave been reported in previous studies [9] . the median age in the severe group was 55.0 years (iqr = 31.0-72.0), which is higher than the median age in the non-severe group, (median = 40.0; iqr = 32.0-55.0). the threshold for age was 63 years, as indicated in the roc curve analysis. the threshold age is a predictor for an increased risk of severe covid-19. in addition, our multivariate logistic regression analysis implied that patients who are older than 63 years are at higher risk of developing severe covid-19 (or: 41.0). the trends indicated in our study are similar to those indicated in previous studies [10] . moreover, studies show that immune responses in older adults are slower, less coordinated, and less efficient, rendering older adults more susceptible to emerging infections [11] . a study by shahid et al. indicates that the probability of having multiple comorbidities increased the risk of mortality from sars-cov-2 in older adults [12] . our data indicate that 27.9% of patients had an underlying disease, especially cardiovascular and metabolic disorders. the percentage of patients suffering from cvd was higher in the severe group than in the non-severe group. these results have been verified by epidemic reports from the chinese center for disease control and prevention [13] . as in previous studies, the principal symptoms of covid-19 are respiratory symptoms. some patients also suffer significant cardiovascular damage from covid-19. coupled with underlying cvd, these complications may increase the risk of death for some patients. covid-19 results from a spike protein in the virus binding with the angiotensin-converting enzyme 2 (ace2) which is highly expressed in the heart and lung tissue. a contributing factor to severe covid-19 in patient with cvd may be the increased concentration of ace2 in these patients. as the middle east respiratory syndrome-related coronavirus (mers-cov) and severe acute respiratory syndrome coronavirus (sars-cov) may result in acute myocarditis and heart failure, acute coronary syndrome patients infected with the novel coronavirus are at an increased risk of cardiac insufficiency that might lead to severe covid-19 or death [14] . the most common symptoms of covid-19 in our study were fever (92.3%), cough (63.5%), and fatigue (48.1%). some patients also experienced dizziness (8.7%), a runny nose (8.7%), nasal obstruction (6.7%), and diarrhea (6.7%). these numbers are similar to those described in previous studies [15, 16] . our study indicates that in 76.0% of the patients, the initial symptom was fever. in addition, 92.3% of the patients had fever during the inpatient period; of those, all patients with severe covid-19 had fever. as shown in previous studies, although only 43.8% of the patients had fever on admission, 88.7% of all patients had fever during hospitalization [17] . this implies that fever did not present as an initial symptom in all covid-19 patients; nonetheless, fever occurred as pneumonia symptoms developed. fever that developed as a result of critical pulmonary infection was frequently observed in patients with severe covid-19; therefore, temperature monitoring should not be the only screening measure for covid-19. we suggest that in addition to temperature monitoring, the patient's contact history and symptoms should be examined to identify individuals who require observation. a higher number of patients showed signs of dyspnea in the severe group than in the non-severe group in our study, indicating that dyspnea was one of the main symptoms among patients with severe covid-19; thus, a daily evaluation for dyspnea should be conducted among covid-19 patients. wang et al. suggest that more frequent chest ct scans should be performed for patients with severe dyspnea to understand the changes and indications in pulmonary imaging [18] . all cases in our covid-19 study had a decreased lymphocyte count on admission; patients in the severe group had even lower absolute lymphocyte values, as indicated by a median of 0.91 (iqr = 0.59-1.1)×10^9/l. similar changes in the lymphocyte counts were observed in patients with sars [19, 20] . a previous study regarding lymphocyte subsets indicated that covid-19 patients had reduced count of total lymphocytes, cd4 + t cells, cd8 + t cells, b cells, and natural killer cells. furthermore, severe covid-19 cases had lower counts than non-severe cases [21] . a study by qin et al. suggests that covid-19 might damage lymphocytes, especially t lymphocytes; consequently, the immune system is impaired during the course of the disease [22] .the coefficients from our multivariate regression analysis indicated that patients with an absolute lymphocyte value of �1.02×10^9/l have an increased risk of experiencing severe covid-19 (or = 6.1). this implies that a decrease in the lymphocyte count should be a key predictor in the early diagnosis of severe covid-19. research on sars has shown similar results; a decrease in the lymphocyte count could be an early warning of severe disease [23, 24] . this study indicates that patients with elevated concentrations of serum crp on admission are at an increased risk of experiencing severe covid-19. the risk of developing severe covid-19 in patients with a serum crp of �65.08 mg/l is 8.9 times than in patients with a serum crp�65.08 mg/l. at elevated concentrations, crp, which is an acute-phase protein, is correlated with an increased risk of organ failure and death for patients admitted to the icu. further, prolonged periods of high crp concentrations are associated with adverse outcomes [25] . previous research in wuhan indicated that increased levels of crp were indicative of a sustained inflammatory response subsequent to infection with sars-cov-2. additionally, patients with severe covid-19 had more prominent inflammation [26] . similar trends of elevated serum crp were also found in sars patients [27] ; a previous study shows that a crp concentration of > 47.5mg/l is correlated with an increased risk of death for sars patients (or = 5.8) [28] . our study has several limitations. first, due to the limited ability to collect data and the relatively low morbidity and mortality of covid 19, the sample size in this study is small, which resulted in relatively few variables being included in the multivariate analysis; it is possible that some significant variables may have been neglected. second, as this study is a retrospective study, the data collected from the electronic medical records are limited; thus, data about additional significant factors may have not been available. for example, we were unable to analyze the possible causes of higher procalcitonin levels because we lacked data on bacterial infections. in this study, we summarized the clinical features of covid-19 patients and identified the early warning signs of severe covid-19, which will help physicians determine which patients require further observation. although this study has some limitations, including a small sample size, few variables included in the multivariate analysis, a retrospective cohort design, and limited data collected from medical records, the results of our study indicate that older age, a decreased lymphocyte count on admission, and an increased concentration of serum crp could serve as early warning signs in patients who are at risk of developing severe covid-19. consequently, we suggest that patients with these clinical characteristics be monitored closely. further studies should be conducted to confirm the results of our study. conceptualization: xinkui liu, xinpei yue. the novel coronavirus originating in wuhan, china: challenges for global health governance severe acute respiratory syndrome-related coronavirus: the species and its viruses-a statement of the coronavirus study group a pneumonia outbreak associated with a new coronavirus of probable bat origin world health organization. who director-general's statement on ihr emergency committee on novel coronavirus (2019-ncov) update on the epidemic situation of covid 19 as of 24:00 on march 9 update on the epidemic situation of covid 19 in henan province as of 24:00 on march 9 clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study epidemiology and transmission of covid-19 in shenzhen china: analysis of 391 cases and 1,286 of their close contacts neutrophil-to-lymphocyte ratio predicts severe illness patients with 2019 novel coronavirus in the early stage sars-cov-2 and covid-19 in older adults: what we may expect regarding pathogenesis, immune responses, and outcomes covid-19 and older adults: what we know epidemiology working group for ncip epidemic response, chinese center for disease control and prevention. the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) in china covid-19 and the cardiovascular system clinical features of patients infected with 2019 novel coronavirus in wuhan clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan clinical characteristics of coronavirus disease 2019 in china insights on diagnosis and treatment of coronavirus disease 2019 a major outbreak of severe acute respiratory syndrome in hong kong clinical features and short-term outcomes of 144 patients with sars in the greater toronto area characteristics of peripheral lymphocyte subset alteration in covid-19 pneumonia dysregulation of immune response in patients with covid-19 in wuhan, china measurement of subsets of blood t lymphocyte in 93 patients with severe acute respiratory syndrome and its clinical significance changes of blood cells in patients with severe acute respiratory syndrome c-reactive protein levels correlate with mortality and organ failure in critically ill patients clinical characteristics of 140 patients infected with sars-cov-2 in wuhan sars in singapore-predictors of disease severity hematological and biochemical factors predicting sars fatality in taiwan key: cord-305274-mcsdem7y authors: beniac, daniel r.; devarennes, shauna l.; andonov, anton; he, runtao; booth, tim f. title: conformational reorganization of the sars coronavirus spike following receptor binding: implications for membrane fusion date: 2007-10-24 journal: plos one doi: 10.1371/journal.pone.0001082 sha: doc_id: 305274 cord_uid: mcsdem7y the sars coronavirus (sars-cov) spike is the largest known viral spike molecule, and shares a similar function with all class 1 viral fusion proteins. previous structural studies of membrane fusion proteins have largely used crystallography of static molecular fragments, in isolation of their transmembrane domains. in this study we have produced purified, irradiated sars-cov virions that retain their morphology, and are fusogenic in cell culture. we used cryo-electron microscopy and image processing to investigate conformational changes that occur in the entire spike of intact virions when they bind to the viral receptor, angiotensin-converting enzyme 2 (ace2). we have shown that ace2 binding results in structural changes that appear to be the initial step in viral membrane fusion, and precisely localized the receptor-binding and fusion core domains within the entire spike. furthermore, our results show that receptor binding and subsequent membrane fusion are distinct steps, and that each spike can bind up to three ace2 molecules. the sars-cov spike provides an ideal model system to study receptor binding and membrane fusion in the native state, employing cryo-electron microscopy and single-particle image analysis. viral membrane fusion proteins are responsible both for binding to cellular receptors, and the subsequent fusion of viral and cellular membranes. the paradigm for class i fusion proteins consists of two heptad repeat regions, and a hydrophobic fusion peptide [1] . this motif is present in sars-cov [2] and other coronaviruses [3] , as well as the hemagglutinin (ha) of influenza [4] , gp21 of human t-cell leukemia virus type 1 [5] , gp41 of hiv [6] , gp2 of ebola virus [7, 8] , and the fusion protein of paramyxovirus [9] [10] [11] [12] . class i viral fusion proteins can also be divided into two sub-types; those whose fusion mechanism is low ph-dependent such as influenza ha, and those that are ph-independent like the retroviral fusion proteins. in retroviruses, receptor binding itself can trigger fusion, with temperature and redox conditions also influencing the fusion mechanism [13, 14] . the sars spike appears to be insensitive to redox conditions [15] . for sars-cov, it is proposed that the virus is internalized in the cell by endocytosis, and is then exposed to a low ph environment, and it is postulated that proteolytic cleavage between the s1 and s2 domains initiates the membrane fusion process [16] . although the factors which trigger fusion (endocytosis, ph sensitivity, single receptor vs. primary and co-receptor binding, redox change) differ amongst diverse virus families, all viral fusion proteins are thought to share the same basic fusion mechanism [1, 4, [17] [18] [19] [20] . a notable feature of the sars spike is its large mass (,500 kd per trimer) and striking, club-shaped appearance, from the end-on, this appears like a three-bladed propeller with a radius of 90 å [21] . despite the structural differences, the sars spike performs the same fundamental task in viral entry to the host cell as other class i viral fusion proteins, such as the influenza ha (,220 kd per trimer). the sars spike can be subdivided into four structural domains (from n to c terminus); the two large external domains s1 and s2 are largely responsible for receptor binding and membrane fusion, respectively. in many class i viral fusion proteins the analogous peptides are generated by proteolysis of the spike precursor during the maturation process in the host cell, resulting in two peptides with the fusion peptide on the n-terminus of s2. in sars-cov the s1/s2 assignment is given based on sequence homology to other viral fusion proteins, however there is no peptide cleavage during maturation. the final two small domains are comprised of a transmembrane domain, and a carboxyterminal cytoplasmic domain, which principally anchor the spike to the viral envelope. the cell-surface molecule ace2 is the receptor for the sars spike protein [22] and is a relatively large macromolecule with a diameter of 70 å . in comparison, the receptor for influenza ha, sialic acid, is much smaller with a 10 å diameter. the precise mechanisms by which class i viral fusion proteins gain access to the host cell remain unknown. the hypothetical entry process includes several steps that take place in sequence: receptor binding, fusion core re-arrangement, fusion peptide insertion in host cell membrane, refolding of heptad repeats, membrane fusion, and finally viral nucleocapsid transfer [23] . the structures of ace2 bound to a fragment of the sars spike containing the receptor-binding domain and the pre-and postfusion configurations of the fusion core heptad repeats of the spike have been solved to atomic resolution [2, 3, [24] [25] [26] . in addition, the atomic resolution structures of two neutralizing antibodies bound to the sars spike receptor-binding domain have been solved [27, 28] showing that blocking of the receptor binding domain, preventing attachment of virions to cell-surface ace2, is the likely mechanism of virus neutralization by these antibodies. the aim of the present study was to delineate any possible structural changes in the sars spike that accompanied receptor-binding, and to precisely localize the receptor binding domains (which are only 14% of the total mass of the spike) within the overall structure, employing cryo-em and image processing of intact virions bound to soluble ace2. we demonstrate the structural dynamics which accompany spike-receptor binding, which may be involved in triggering membrane fusion. to study the structure of the spike-ace2 complex, we infected vero e6 cells with sars-cov and purified supernatant virus by iodoxanol density gradient centrifugation. previously, we have shown that specimens could be c-irradiated with a sufficient dose (2mrad) for viral inactivation, while still preserving protein antigenicity. inactivation was verified by passage in cell culture and testing by quantitative pcr [29] . purified c-irradiated sars-cov preparations had a fusogenic activity when added to vero e6 cells at high multiplicity (,500-3000 virions per cell), causing the formation of syncytia in the absence of replication or cytopathic effects ( figure 1a ). these syncytia had identical morphology to those observed in cytopathic studies of sars-cov in tissues and in tissue cultured cells; syncytia have also been observed upon expression of coronavirus s protein, or following addition of cells expressing the s-protein to cells with surface-expressed ace2. [22, [30] [31] [32] [33] [34] . the same inactivated c-irradiated virus preparations were incubated with a human ace2-human fc chimeric protein, for analysis of the complex by cryo-em, 3d image processing, and immuno-electron microscopy (immuno-em) ( figure 1 , movie s1). the ace2-fc construct was selected since it is soluble and dimeric, and it was anticipated that the additional mass was would be beneficial in locating the receptor attached to the spike. immuno-em confirmed that recombinant ace2 protein bound to virions, and that binding affinity was not affected by c-irradiation ( figure 1b) . we did not observe any structural changes associated with prior irradiation. image averaging further suppressed any possible random radiation induced structural changes. cryo-em coupled with 3d single particle image analysis was employed to investigate the binding of ace2 to the spike of sars-cov. although sars-cov is approximately spherical when observed by cryo-em, both its size and shape vary slightly, and thus it is not amenable to single-particle averaging techniques. however, individual spikes on the surface envelope of the virus do provide a repetitive structure that is ideal for single particle techniques. spikes on the surface of virus particles are readily imaged by cryo-em in the frozen-hydrated state ( figure 1c ). three-dimensional image processing was carried out on 11,153 selected spike images taken at various defocus levels, using routine single-particle image processing [35, 36] . the resolution of the final 3d structure was evaluated by a fourier shell correlation of 0.5 to be 18.5 å (figure 2 ). the structure of the unbound spike was compared with that of the spike-ace2 complex ( figures 1e, 1f , 3, movie s2). in both cases the spike portion of the 3d structure is fairly similar, indicating that ace2 binding does not result in a fundamental structural unfolding of the spike. however, the overall height of the spike was reduced from 160 å to 150 å following ace2 binding. when viewed from the end-on perspective and the spike undergoes a rotation of ,5u following binding (movie s2), and the mass at the center of the axis of symmetry on the distal end of the spike redistributes itself from one small central blob to three blobs or nubs (figure 3 the precise location of ace2 binding on the distal end of the spike is centered at 70 å from the central axis of the spike, with a 30 å gap between the axis of symmetry and ace2. one ace2 molecule was bound to each of the three propeller-like blades of the spike, making a structure 220 å high ( figure 1f ). the density of these ace2 molecules is high in the 3d density map, indicating a high occupancy of binding sites. in addition, the structure shows that binding of one ace2 to the spike would not sterically hinder binding of additional ace2 molecules on the other two propellerblades of each trimer. the cryo-em 3d structures of the spike and the spike-ace2 complex were combined with the atomic resolution structures of the sars spike receptor-binding domain-ace2 complex [24] and the heptad repeat pre-and post-fusion cores [2, 25] to interpret the cryo-em structure. the receptor-binding domain-ace2 data were docked with a correlation score of 0.965 using the situs software package ( figure 4 ) [37] . as expected the receptor-binding domain docked to the distal end of the spike with ace2 filling the extra mass on the spike (shown by the color violet in figure 4 ). the empty upper region of the mass is likely composed of the fc component of the chimeric protein. although this chimeric molecule is dimeric, only one leg of the ace-2 is able to bind to each of the three receptor binding domains of the trimer. we anticipate the mass distal to the hinge region to be flexible, and therefore components were blurred out in the averaging process, leaving only a portion of the additional mass in the 3-d map. the c-terminus of the docked ace2 is in a location consistent with this interpretation. previously we modeled the putative location of the receptor-binding domain and ace2 in the cryo-em structure of the unbound spike using solely the receptor-binding domain atomic resolution data [21] . in the present study, we have shown that our previous prediction of the location of the receptor-binding domain was only partially correct. the actual location of the receptor binding domain and ace2 are in fact shifted 29 å towards the 3-fold axis, corresponding to about 40% of the diameter of ace2. the results from the present study will aid in the precise location of future atomic resolution structure fragments of the sars spike, as well as in the structural mapping of neutralizing antibody-binding epitopes (which are in progress). the coordinates of the three dimensional reconstruction of the sars spike (emd-1423) and the sars spike-ace2 complex (emd-1425) have been deposited in the macromolecular database (http://www.ebi.ac.uk/ msd). our cryo-em results demonstrate that there is a structural transition of the spike that occurs upon receptor binding (figure 3 , movie s2). the overall height of the spike is reduced by 10 å due to a shift in the mass in the s1 domain (figure 3 d,i) and the three s1 blades of the spike twist ,5u along the axis of symmetry. on the distal end of the spike, a small blob feature is replaced by three small nubs which appear radially. (figure 3 f,j) . we propose that this structural re-arrangement represents an initial ''priming'' of the spike for membrane fusion, which brings the cellular and viral membranes 10 å closer to each other to facilitate fusion. the coronavirus s protein is known to mediate both virion-cell membrane fusion, and intercellular membrane fusion [22, [30] [31] [32] [33] and in the present study we have shown that purified inactivated virions are also fusion-competent. we show that the addition of sars-cov virions to cultured cells that express ace-2 can induce fusion, presumably from the fusion activity of spikes on virions bridging neighboring cells. similar observations on the induction of ''fusion-from-without'' were made when cells surfaceexpressing the spike protein were mixed with cells expressing ace-2 [35] . the trigger that initiates membrane fusion varies amongst class i fusion proteins, with some activated by receptor binding, others by ph, and others by redox conditions. in most proposed models of membrane fusion it is postulated that the s1 domain or analogous receptor binding domains dissociate from the spike during the membrane fusion process. this dynamic process was demonstrated for influenza ha by kemble et al. [38] in their investigation where they engineered intermonomer disulfide bonds between the ha s1 subunits. the result of this was that fusion activity was impaired; however it could be restored under reducing conditions. it is likely that the sars spike shares a similar mechanism, and the structural changes that we have detected represent the initial step in this process. by analogy with other class i viral fusion proteins, we anticipate that the fusion core of the sars spike must undergo similar structural re-arrangements during fusion. the receptor-binding domain is localized in a position on the distal end of the molecule, closer to the 3-fold axis than anticipated, yet still in a position that would not impede these structural re-arrangements. putative mechanisms by which class i viral fusion proteins achieve membrane fusion have been proposed [1, 4, [17] [18] [19] , but complete structural evidence for the role of intermediate structures in these mechanisms has yet to be obtained. the structural biology of this process has been best characterized for the influenza hemagglutinin, and paramyxovirus fusion (f) protein, for which the prefusion and membrane fusion ph structures have been determined by x-ray crystallography [4, 11, 12, 39, 40] . all of the subsequent models for class i viral fusion proteins are based on the structural data of these two fusion proteins. a drawback in all of these models is that they are based on recombinant ectodomains that are not proven to exist as a component in the complete molecule, and they lack both membrane-interacting residues, and lipids [4] . the cryo-em structures presented in this investigation are of intact sars spike bound to native virion lipid envelopes. the cryo-em data are very instructive when atomic resolution fragments are docked within the overall molecule, especially as the entire sars spike has proven to be a difficult subject for x-ray crystallography, and atomic resolution data exist for only a few fragments of the sars spike. our cryo-em results show that it is possible for the spike to attach to three ace2 receptors at once; this may serve to hold on to the host membrane like a tripod so as to accurately orientate the fusion core. in addition the 30 å gap between the axis of symmetry and ace2 provides sufficient space for fusion core rearrangement. damico et al., [14] demonstrated that the kinetics of binding of the rous sarcoma virus envelope protein to its receptor suggested that binding of each spike molecule to multiple receptor monomers occurred. it may therefore be possible that other structurally homologous viral envelope proteins can also bind multiple receptors, and this may be a general adaptation that provides the correct temporal and spatial arrangement to bring about membrane fusion. the observation that the sars spike could bind three soluble ace2 receptors provides three possible binding states with one, two or three membrane-bound receptors attached to the spike. with one or two receptors bound, the spike and virus have a wide range of movement possible. whereas, in the case of three bound receptors the spike and its fusion core will be arranged perpendicular to the cell surface with minimal movement possible. it is interesting to note that binding three receptors is the minimum number of binding events required to achieve this perpendicular orientation in three dimensional space. this observation matches up with the conserved trimeric structures of class i fusion proteins which are common amongst enveloped viruses, thus indicating a possible conserved structural-functional relationship may exist. another line of reasoning is that oligomerisation favors a better ''chance'' of successful receptor binding and subsequent insertion of the fusion peptide into the host membrane. according to this model, oligomeric multiples higher then three would be favored, whereas the ''tripod'' model of receptor binding favors the conserved trimeric structures of class i viral fusion proteins. in figure 3 we have we have presented the ace2-sars structures as they were solved in this cryo-em investigation. for other fusion proteins like influenza ha1 and hiv gp120 it has been modeled that the re-arrangements upon membrane fusion are dramatic involving a shedding of the above mentioned domains. in this investigation we have detected structural movement of s1 upon ace2 binding. this could represent the initial phase of this dramatic process that is followed by fusion core re-arrangement, fusion peptide insertion in the host cell membrane, refolding of heptad repeats, and finally membrane fusion. analyzing the structure of the spike-receptor complex is essential to understanding how viruses can adapt to utilize receptors from different species and how they may evolve to gain specificity for new receptor types. rna viruses have a high rate of mutation and recombination [41] . in sars coronaviruses, the spike is able to retain specific binding affinity for the ace2 of more than one host species, and rapid evolution to gain specificity for novel ace2 species has been demonstrated [42, 43] . the structure and large size of the spike of coronaviruses appears to be an adaptation related to utilization of large host cell-surface molecules such as ace2 as specific receptors. amongst the coronavirus family, specific cell surface receptors for the spike protein are all in the range of 60-110 kd [44] . these large host receptor molecules are in turn functionally constrained and thus conserved across species barriers. in utilizing binding to a large receptor molecule, it may be important that the spike s1 domain also acts as a ''spacer arm'' holding the receptor far enough away from the three-fold axis of symmetry of the spike s2 domain to permit fusion core re-arrangement and subsequent membrane fusion. such a property necessitates having a large spike molecule. moreover, multiple receptor binding may have functional significance, enhancing the binding and entry of viruses. crosslinking of adjacent host receptor molecules could increase the affinity of the virus for its target cell, as well as improving the kinetics of fusion. the sars spike-ace2 complex is an ideal model system for the investigation of class i viral fusion protein dynamics, utilizing cryo-em to investigate fusion protein binding and structural changes in the native state, within virion membranes that are fusogenic. the presence of a common cell fusion mechanism shared by diverse virus families holds the prospect of developing broad-spectrum anti-virals that target these conserved mechanisms, as well as the potential use of these fusogenic proteins in drug delivery systems. sars-cov (tor3 strain) was inoculated onto vero e6 cells grown in dulbecco's modified eagle medium supplemented with 10% foetal bovine serum, 100 u/ml penicillin, 100 mg/ml streptomycin, and 0.22 mg/ml l-glutamine at 37uc with 5% co 2 . cell supernatant was harvested (300 ml) with a sars-cov tcid 50 titre of 5610 7 /ml and virus was pelleted using a beckman sw32 rotor at 28000 rpm for 90 minutes. the virus pellet was resuspended in pbs buffer, layered onto of a 12-30% iodoxanol gradient (optiprep) and centrifuged in a sw40 rotor at 38000 rpm for 2.5 hours. fractions (0.7 ml) were collected from the gradient with an auto densi-flow gradient collector (labcono) and dialyzed against pbs. sars-cov-enriched fractions were checked by sds-page and western blotting, and rendered noninfectious by irradiation in a gamma cell on dry ice with a 2mrad exposure for 90 minutes. irradiated specimens were tested for infectivity by inoculation onto vero e6 cells, and examined for cytopathogenic effects for 10 days, followed by blind passage of the cells and testing for the growth of sars-cov by pcr [29] . to test for spike fusogenic activity vero e6 cells were cultured in 96 well plates (corning). once the cells were confluent the medium was removed from each well and 20 ml of gamma irradiated sars-cov containing 10 10 pfu/ml diluted 1:1 and 1:9 in culture media. the virus dilutions were incubated with the cells for 1 hour at 37uc. after the incubation the plates were washed three times with fresh culture medium to remove unbound virus. the vero e6 cells were observed by light microscopy 2 and 4 days post treatment for the presence of syncytia. virus growth was not detected at either dilution of irradiated virus, and no cytopathic effect was observed. the presence of nucleic acid in syncytia was investigated by propidium iodide staining and observation by fluorescence microscopy. the human ace2-fc (human igg1) recombinant protein (adipogen) had an initial concentration of 1 mg/ml in pbs and was diluted 1:10 in pbs for incubation with sars-cov, for the remainder of this document this is referred to as ace2. 2 ml of sars-cov were injected into 4 ml of pbs on a formvar-carbon coated 400-mesh nickel grid, and incubated for 1 minute. all incubations were carried out at 20uc. grids were then washed in pbs six times for 1 minute, followed by a 10-minute block in pbs-g-bsa (pbs ph7.2, 0.2% glycine, 2% bsa). grids were then washed in pbs-g (pbs ph7.2, 0.2% glycine) six times for 1 minute, followed by a 1-hour incubation on a 20 ml drop of ace2. grids were then washed in pbs-g (pbs ph7.2, 0.2% glycine) six times for 1 minute, followed by a 1-hour incubation with rabbit anti-human ace2 polyclonal igg (adipogen; diluted 1:200 in pbs). grids were then washed in pbs-g (pbs ph7.2, 0.2% glycine) six times for 1 minute, followed by a 30 minute incubation with goat anti-rabbit igg (conjugated to 10 nm gold, sigma; diluted 1:10 in pbs). grids were then washed in pbs-g (pbs ph7.2, 0.2% glycine) three times for 1 minute. grids were fixed for 2 minutes (1% paraformaldehyde, 2% glutaraldehyde in pbs), washed in deionised water and negatively stained with 2% methylamine tungstate (nanoprobes), and observed by transmission electron microscopy in a tecnai 20 g2 transmission electron microscope (fei) operated at 200 kv, digital images were collected using an amt advantage xr-12 digital camera. sars-cov was incubated with ace2 for one hour, and 4 ml samples were applied to glow-discharged holey carbon films supported on 400-mesh copper grids. after blotting immediately for 2-5 seconds with filter paper, grids were plunged into liquid ethane cooled by liquid nitrogen, using a custom built gravityoperated freezing device. specimens were transferred to a tecnai 20 g2 transmission electron microscope (fei) operated at 200 kv, equipped with a gatan 626.dh low-temperature specimen holder. observations were made at temperatures of ,2185uc and images recorded at 29,0006 magnification on kodak so-163 electron image film at a dose of 10-20 electrons/å 2 with an exposure of 1-2 seconds. film was developed in kodak d19 for 12 minutes at room temperature, rinsed for 2 minutes in water, and fixed for 10 minutes with kodak fixer. the exact magnification in the microscope that sars-cov was imaged at was determined to be 29,9686 using a calibration grid (pelco international). images of sars-cov were digitized on a nikon super coolscan 9000 ed scanner at a pixel size of 2.125å . processing was carried out using the eman and spider/web image processing program packages [35, 45] on sgi fuel and tezro workstations running irix 6.5, or a dell pe6850 with 4way 64-bit xeon dual core 3.6 ghz cpus and 32gb ram running linux (fedora core 5). images were corrected for contrast transfer function (ctf) using the ''ctfit'' function in the eman software package, which estimates defocus and corrects for ctf by phase-flipping. the data set composed of images centred on spikes-ace2 (n = 11,153; recorded at 2.9-11.9 å defocus) were processed using spider by the projection matching approach [36] which incorporated three-fold symmetry into the search function and reconstruction procedure. after each refinement cycle the new reference volume was either theresholded or segmented using the ''floodfill'' function in the situs software package [37] to select the spike-ace2 structure and minimize the influence of neighbouring spikes in the reference structure that was used in the subsequent iteration of the projection match alignment. the initial reference model used in this procedure was the structure of the sars spike that we previously solved by cryo-electron microscopy [21] . the population of spike images was composed of both side view projections as well as end-on projections, with the latter being easily identified in the images with greater defocus values. these high-defocus/end-on images were essential to eliminate the ''missing cone'' from the reconstruction. the resolution of the cryo-em reconstruction was estimated by fourier shell correlation to be 18.5å using the 0.5 fsc criteria. the structures of the pre-fusion hr2 domain and the ace2-receptor-binding domain were docked into the cryo-em reconstruction using the ''floodfill'' and ''colores'' functions in the situs software package. the resultant docking generated one distinct location for the ace2-receptor-binding domain, which placed ace2 in the additional mass that was not present in the reconstruction of the spike alone. the docking of the entire 2fxp.pdb [2] structure was previously conducted [21] . three-dimensional cryo-em reconstructions, and the atomic resolution structures 2ajf.pdb [24] , 2fxp.pdb [2] , were visualized using ucsf chimera (computer graphics laboratory, university of california, san francisco, supported by nih p41 rr-01081) [46] . the spike component of the spike-viral envelope reconstruction was segmented from the entire reconstruction using the ''floodfill'' algorithm in the situs software package. movie s1 of the rotating sars spike-ace2 complex was generated on a silicon graphics workstation using ucsf chimera, and the ''mediarecorder'' and ''mediaconvert'' software provided with the silicon graphics irix 6.5 operating system. movie s2 showing the morphing of the spike between the sars spike and sars spike-ace2 complex was generated using the ucsf chimera software package with the ''morph map'' morphing tool plug-in. both reconstructions were masked with a cylindrical mask, which when applied masked the peripheral boundaries of the sars-cov envelope in both reconstructions. movie s1 three-dimensional reconstruction of the of the sars spike-ace2 complex. this movie supplements figs. 1, 4 showing both the reconstruction of the spike-ace2 complex as well as the location of the docked atomic structures. the movie starts showing the spike-ace2 complex viewed from an end-on perspective. the reconstruction then tilts 90o, followed by a 120o rotation along the three-fold axis of symmetry of the spike. in the second section of the movie the cryo-em reconstruction becomes transparent revealing the location of the docked atomic structures, followed by a final rotation of 90o back to the initial end-on perspective. the following color scheme was used: cryo-em surface; ace2, violet; spike, green; stalk, blue; envelope, beige; nucleocapsid, red. atomic structures: pdb id code, 2ajf, (sars spike receptorbinding domain, red; ace2, white; ace2 c-terminus, blue); and pdb id code, 2fxp, hr2 fusion core, yellow. found at: doi:10.1371/journal.pone.0001082.s001 (7.24 mb mov) movie s2 three-dimensional morphing of the sars spike, and ace2 bound structures. this movie supplements figs. 1, 3 showing the putative structural re-arrangement of the spike as it binds to ace2. the movie starts showing the sars spike and then gradually morphs to the sars spike-ace2 complex, and back to the sars spike. the morphing reconstruction is shown from the side and end-on perspectives, and the mass attributed to the nucleocapsid is presented without morphing. as one watches the move play back and forth (this can be done by selecting ''loop'' in a quicktime player) one can see the spike twist by ,5o when viewed from the end-on perspective, and the spike becomes squatter by ,10 &#x00c5; when viewed from the side perspective. the following color scheme was used: cryo-em surface; ace2, violet; spike, green; stalk, blue; envelope, beige; nucleocapsid, red. virus membrane fusion proteins: biological machines that undergo a metamorphosis solution structure of the severe acute respiratory syndrome-coronavirus heptad repeat 2 domain in the prefusion state crystal structure of severe acute respiratory syndrome coronavirus spike protein fusion core receptor binding and membrane fusion in virus entry: the influenza hemagglutinin crystal structure of human t cell leukemia virus type 1 gp21 ectodomain crystallized as a maltosebinding protein chimera reveals structural evolution of retroviral transmembrane proteins atomic structure of the ectodomain from hiv-1 gp41 core structure of the envelope glycoprotein gp2 from ebola virus at 1.9-a resolution crystal structure of the ebola virus membrane fusion subunit, gp2, from the envelope glycoprotein ectodomain the structure of the fusion glycoprotein of newcastle disease virus suggests a novel paradigm for the molecular mechanism of membrane fusion structural characterization of the human respiratory syncytial virus fusion protein core structure of the uncleaved ectodomain of the paramyxovirus (hpiv3) fusion protein structure of the parainfluenza virus 5 f protein in its metastable, prefusion conformation activation of a retroviral membrane fusion protein: soluble receptor-induced liposome binding of the alsv envelope glycoprotein receptor-triggered membrane association of a model retroviral glycoprotein cell entry by enveloped viruses: redox considerations for hiv and sars-coronavirus inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry structural basis for paramyxovirus-mediated membrane fusion the structural biology of type i viral membrane fusion virus entry: molecular mechanisms and biomedical applications cellular entry of the sars coronavirus architecture of the sars coronavirus prefusion spike angiotensinconverting enzyme 2 is a functional receptor for the sars coronavirus the many mechanisms of viral membrane fusion proteins structure of sars coronavirus spike receptor-binding domain complexed with receptor structure of a proteolytically resistant core from the severe acute respiratory syndrome coronavirus s2 fusion protein central ions and lateral asparagine/glutamine zippers stabilize the post-fusion hairpin conformation of the sars coronavirus spike glycoprotein structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody structural basis of neutralization by a human anti-severe acute respiratory syndrome spike protein antibody, 80r detection of airborne severe acute respiratory syndrome (sars) coronavirus and environmental contamination in sars outbreak units intracellular complexes of viral spike and cellular receptor accumulate during cytopathic murine coronavirus infections monoclonal antibodies to murine hepatitis virus-4 (strain jhm) define the viral glycoprotein responsible for attachment and cell-cell fusion the secret life of ace2 as a receptor for the sars virus the sars-cov s glycoprotein: expression and functional characterization palmitoylation of the cysteine-rich endodomain of the sars-coronavirus spike glycoprotein is important for spike-mediated cell fusion spider and web: processing and visualization of images in 3d electron microscopy and related fields the ribosome at improved resolution: new techniques for merging and orientation refinement in 3d cryo-electron microscopy of biological particles situs: a package for docking crystal structures into low-resolution maps from electron microscopy intermonomer disulfide bonds impair the fusion activity of influenza virus hemagglutinin binding of influenza virus hemagglutinin to analogs of its cell-surface receptor, sialic acid: analysis by proton nuclear magnetic resonance spectroscopy and xray crystallography structure of influenza haemagglutinin at the ph of membrane fusion the population genetics and evolutionary epidemiology of rna viruses receptor and viral determinants of sars-coronavirus adaptation to human ace2 animal origins of the severe acute respiratory syndrome coronavirus: insight from ace2-s-protein interactions molecular determinants of species specificity in the coronavirus receptor aminopeptidase n (cd13): influence of n-linked glycosylation eman: semiautomated software for high-resolution single-particle reconstructions ucsf chimera-a visualization system for exploratory research and analysis conceived and designed the experiments: tb db. performed the experiments: db sd aa rh. analyzed the data: tb db. contributed reagents/materials/analysis tools: aa rh. wrote the paper: tb db. key: cord-270647-vn4kirrx authors: romero-espinoza, jose a.; moreno-valencia, yazmin; coronel-tellez, rodrigo h.; castillejos-lopez, manuel; hernandez, andres; dominguez, aaron; miliar-garcia, angel; barbachano-guerrero, arturo; perez-padilla, rogelio; alejandre-garcia, alejandro; vazquez-perez, joel a. title: virome and bacteriome characterization of children with pneumonia and asthma in mexico city during winter seasons 2014 and 2015 date: 2018-02-15 journal: plos one doi: 10.1371/journal.pone.0192878 sha: doc_id: 270647 cord_uid: vn4kirrx background: acute asthma exacerbations and pneumonia are important causes of morbidity and mortality in children and may coexist in the same children, although symptom overlap may lead to difficulties in diagnosis. microbial and viral diversity and differential abundance of either may play an important role in infection susceptibility and the development of acute and chronic respiratory diseases. objectives: to describe the virome and bacteriome present in the upper respiratory tract of hospitalized children with a clinical diagnosis of asthma and pneumonia during an acute exacerbation and an acute respiratory illness ari episode respectively. methods: during the winter seasons of 2013–2014 and 2014–2015, 134 nasopharyngeal swabs samples of children <15 years of age with ari hospitalized at a referral hospital for respiratory diseases were selected based on clinical diagnosis of asthma or pneumonia. the virome and bacteriome were characterized using whole genome sequencing (wgs) and in-house bioinformatics analysis pipeline. results: the asthma group was represented mainly by rv-c, bov-1 and rsv-b and the pneumonia group by bacteriophage ej-1 and ttmv. ttv was found in both groups with a similar amount of reads. about bacterial composition moraxella catarrhalis, propionibacterium acnes and acinetobacter were present in asthma and veillonella parvula and mycoplasma pneumoniae in pneumonia. streptococcus pneumoniae and haemophilus influenzae were mostly found with both asthma and pneumonia. conclusions: our results show a complex viral and bacterial composition in asthma and pneumonia groups with a strong association of rv-c presence in asthmatic children. we observed streptococcus pneumoniae and haemophilus influenzae concurrently in both groups. acute respiratory infection (ari), both from the upper and lower tract (urti/lrti), represent a major public health problem worldwide especially in developing countries [1, 2] . besides being one of the most common diseases, it is a leading cause for health care use, and mortality, particularly due to the presence of pneumonia [3] [4] [5] . in the clinical practice, acute asthma exacerbations and pneumonia have an overlap of symptoms, and may also coexist, leading to misdiagnosis. for example over-diagnosis of pneumonia and under-diagnosis of asthma may contribute to significantly untreated respiratory morbidity and mortality in children less than five years old in low-income countries [6] . both groups differ with respect to the associated virus and bacteria: while asthma exacerbations have been associated to a specific rhinovirus infection, pneumonia can be related to a wide range of bacterial, fungal and viral agents, with a high prevalence of respiratory syncytial virus (rsv) [2, 7] . it has been proposed that microbial composition plays an important role in infection susceptibility and the development of acute and chronic respiratory diseases [8] . recent studies describe the community of microorganisms that reside in the respiratory tract, referred to also as the microbiome, by using next generation sequencing (ngs), and in most cases by evaluating bacterial components based only for specific gene (16s) [9, 10] . on the other hand, the whole genome shotgun (wgs) approach provides enough information to identify all viral and bacterial species not only for a specific gene [11] . while bacterial composition of the microbiome is called bacteriome, the virome (or viral metagenome) refers to the viral fraction comprising viruses responsible for acute, persistent, or latent infections, and viruses integrated into the host genome such as endogenous retroviruses [12] . a limited number of studies have attempted to characterize the virome (yang et. al. [13] ; zoll et.al. [14] ; moesker et.al. [15] ; wang et.al. [16] ; bal et.al. [17] ) and bacteriome (hilty et. al. [10] ; taboada et.al. [18] ; teo et.al. [19] ) of pediatric patients with aris. a few others studies have used well-defined clinical parameters of asthma exacerbation and pneumonia (moreno-valencia et.al. [2] ; vazquez-perez et.al. [20] ). here we describe the virome and bacteriome present in the upper respiratory tract of hospitalized children clinically diagnosed with asthma and pneumonia, during an acute exacerbation and an ari episode respectively, at the national institute of respiratory diseases (iner, mexico city) during 2014 and 2015 winter seasons. the science and bioethics committee of the national institute of respiratory diseases revised and approved the protocol and the consent procedure (b2613). for all children, the corresponding legal guardians provided written informed consent. nasopharyngeal swabs (ns) were collected from children less than 15 years of age with clinical signs of aris hospitalized at the national institute of respiratory diseases (iner) in mexico city, a referral center for respiratory diseases, which primarily provided services for uninsured individuals, during the winter season 2013-2014 and 2014-2015. all subjects enrolled were classified with an asthmatic crisis (characterized by wheezing and difficult breathing especially in previously known asthmatics or with atopic symptoms) or pneumonia (with the presence of new lung opacities, with changes in the white blood count). maximum sample number for each group was defined after each season, resulting in 42 for asthma, and 78 for pneumonia (table 1) . for all samples, automated nucleic acids isolation was performed on magna pure lc 2.0 using the total nucleic acids kit (roche diagnostics). an in-house rt-qpcr respiratory viral panel (moreno-valencia et al., 2015) was used as previous screening and for virus identification (s1 table and s1 appendix). nucleic acids were isolated directly from stored samples. for each one, 250 μl of transport media was centrifuged at 5,000xg for 5min to remove cellular debris. the supernatant was filtered through 0.2 μm-pore-size disc filters (syringe filter, corning). samples were pooled in a 15ml concentrator tube (amicon ultra -15, merck millipore) and centrifuged at 4,000xg until volume was reduced to 500 μl. nucleic acids were isolated from the concentrated sample pool using the purelink viral rna/dna kit according to the manufacturer's instructions (invitrogen, waltham, ma). nucleic acids were eluted in 60 μl of nuclease free water and stored in aliquots at -80˚c. to improve viral detection, one aliquot of 20 μl for each extraction was processed for rna viruses and another aliquot for dna viruses. the aliquot for rna viruses was treated with rnase-free dnase i, (thermo scientific) for 30 min at 37˚c and immediately chilled on ice. to reduce the amount of human dna, the aliquot for dna viruses was treated with nebnext microbiome dna enrichment kit, according to the manufacturer's instructions (new england biolabs inc.). for rna viruses, reverse transcription was performed on 10 μl de rna using a m13-random hexamer primer with transcriptor first strand cdna synthesis kit (roche diagnostics) next double strand cdna (dscdna) was generated by two rounds of synthesis using m13-random hexamer primer with klenow fragment (thermo scientific). for dna viruses and bacteria, dna previously enriched was labeled using m13-random hexamer primer. dna from each process was amplified with platinum taq dna polymerase high fidelity (thermo scientific) using m13 primers and the following program: 2 min 95˚c, 30 cycles of 15 sec at 95˚c, 30 sec at 50˚c, 3 min at 68˚c and a final step of 5 min at 68˚c. dna was quantified by qubit dsdna hs assay kit (thermo scientific), and length fragment was evaluated with agilent high sensitivity dna kit (agilent technologies). a whole genome approach (shotgun) was used for both viral and bacterial genomes. libraries were built with 1 ng of amplified dna from each sample of rna viruses and dna viruses/ bacteria, and processed with nextera xt dna library preparation kit according to the manufacturer's instructions (illumina). libraries were labeled with nextera xt index kit (illumina) according the s1 table and pooled into one. the pooled library was loaded in a flow cell and sequencing was performed in a miseq desktop sequencer (illumina) to obtain paired-end reads of 250 bp in length (2x250). an in-house pipeline was developed to perform the bioinformatics analysis for all files (fig 1 step 3) . datasets of human, bacteria and virus sequences (downloaded from the ncbi ftp server in march 2015) were downloaded to build smalt, bwa index, local blastn and blastx databases. all data from miseq instrument were trimmed through a phred-like q20 < 20 using fastqc software. reads were aligned to databases described above using a standalone blastn for direct assignment of each read with an evalue of 1e-30. for viruses, blastn with an e-value of 1e-30 and blastx with an e-value of 1e-01, on trimmed reads and after human, bacterial and viral mapping, were used respectively. velvet algorithm was used for contig assembly. to reduce data complexity for bacterial detection, human sequences were removed by mapping with human smalt index before the pathogen identification process (fig 1, step 3 ). bacterial species were identified using local blastn with an e-value of 1e-30 and the first 10 hits were obtained for each sequence. megan [21] 5.2.2 software was used to assign reads to the most appropriate taxonomic level, by assigning a read to the lowest common taxonomic ancestor of the organisms corresponding to the set of significant hits. for the rhinovirus analysis, full-length genome of human rhinovirus a, b c (rv-a, rv-b, rv-c), enterovirus 68 and 71 (ev68, ev71) were retrieved from the genbank database. for phylogenetic analysis of parvovirus b19 (pvb19), bocavirus (bov) and respiratory syncytial virus b (rsv-b), full-length genomes of representative sequences of human strains were used. analyses also were performed for three different anellovirus, torque teno virus (ttv), torque teno midi virus (ttmdv) and torque teno mini virus (ttmv). alignments were created and manually edited with mega [22] 6.0. unrooted maximum likelihood tree with 1,000 bootstrap replicates was constructed using the tajima-nei model with 5-parameter gamma distributed rates. one hundred and twenty respiratory samples were grouped into seven pools: asthma 2014 (a2014), asthma 2015 (a2015), pneumonia 2014 (p2014) and pneumonia 2015 (p2015) each one with qpcr positive (qpcr+) and qpcr negative (qpcr-) results, except qpcr-for a2015 season since we did not have enough samples to analyze (s1 table) . the average amount of sequences obtained from each group after quality filtering was 2,247193 within a range of 3,634,710 to 519,470 reads. from those reads, 0.18 to 0.35% belonged to viral or bacterial sequences, and as much as 28.08% belonged to endogenous retrovirus and sequences without certain identification. to describe the virome of the two groups of children, we grouped reads from both seasons 2014 and 2015 into one. the most represented viruses were in order of number of reads rhinovirus c (rv-c), bocavirus 1 (bov-1), rsv-b and parvovirus b19 (pvb19) in patients with asthma while bacteriophage ej-1, ttmv, streptococcus phage, rsv b and rv a were in subjects with pneumonia. ttv, influenza virus and staphylococcus phage were present in similar levels for both groups of patients ( table 2 ). most of the viral reads were classified into six virus families picornaviridae, parvoviridae, paramyxoviridae, anelloviridae, orthomyxoviridae and herpesviridae (fig 2a and 2b) . bacteriopaghes also had high abundance of reads ( fig 2b) . the viral composition in samples was complex and dominated by the most common respiratory four representative viruses were used to compare the genome coverage and depth. contigs were ensembled to generate partial consensus genomes of rv-c (97% coverage, 6,824 bp) (s1 the bacterial composition in samples was also complex. streptococcus pneumoniae and haemophilus influenzae were the species with more reads in both groups. staphylococcus aureus, mycoplasma pneumoniae veillonella parvula and different streptococcus spp had more reads in the pneumonia group (fig 3a and 3b) . while moraxella catarrhalis, acinetobacter and propionibacterium acnes had more reads in the asthma group. other bacteria genus detected of clinical interest with low frequency were: pseudomonas, neisseria and prevotella. we conducted a metagenomic analysis of the virome and bacteriome of children with asthma and pneumonia. this is the first report to describe both communities in a well-defined group of pediatric patients. previously, our group reported the role of specific viral infections on asthma exacerbations in hospitalized children using rt-qpcr detection [2, 20] . although rt-qpcr is the gold standard for viral and bacterial diagnosis, it is still limited to specific target assays. by contrast, ngs is a platform that generates millions of reads without the need of previous knowledge of the sequences present in a sample. it is also a powerful tool to confirm and discover microbial etiologies in clinical samples. whole genome shotgun sequencing (wgs) provides enough information to identify microbes to the species level as a result of a more accurate alignment with genome reference sequences, not just in particular for specific genes such as 16s in bacteria [11] . in our analyses, most of the bacterial reads were classified into a species level, implying that wgs can be a useful tool to describe the microbiome in a clinical sample. in our study, 90% of reads were classified as q30, and only 5% were discarded due to low sequencing quality. after being analyzed, human reads ranged from 71 to 91% in each group despite the use of an experimental strategy to reduce human dna. these values are found in the same range for equivalent sample types previously reported by yang et al. [13] and nakamura et al. [23] : 76-95% and 90-94.6%, respectively. however, the percentage range obtained for bacterial and viral reads (0.10-0.32%) was lower than the quantities previously reported by yang et al.(1.03%) [13] , nakamura et al. (1.33-3 .48%) [23] and taboada et al. (9.4%) [18] . all viruses detected by the qpcr panel were identified through ngs except coronavirus 229e in the asthma group and coronavirus 229e and adenovirus in the pneumonia group, probably owing to a low number of reads and the dilution effect of sample-pooling. however, the ngs method combined with the bioinformatics workflow showed greater sensitivity for detection of infa h1n1, piv-1 and bov-2 compared with qpcr detection, probably as a result of the identification of less conserved sequences. moreover, other viruses such as ttv, ttmv, ttmdv and different phages like propionibacterium phage, streptococcus phage, staphylococcus phage and bacteriopage ej-1 were also detected without having knowledge of the viruses. a diverse viral community was found in the respiratory tract of children with asthma and pneumonia; rv-c and rsv-b were the predominant species. viruses found in these respiratory samples belonged to different families, mainly picornaviridae, orthomyxoviridae and paramyxoviridae. members of the anelloviridae family were found in both groups of pediatric patients; although members of this family are usually considered ubiquitous and benign, a potential role of ttmv in pneumonia pathogenesis has been suggested [24] , other studies have implicated ttv as contributing elements in lung impairment and it has been associated with asthma [25] and chronic obstructive pulmonary disease (copd) [26] . even so, more studies are needed to elucidate the role of respiratory pathogenicity of the torque teno viruses. evd68 has been associated with asthma exacerbation and pneumonia in children [20] , in this study, ev-d68 was detected in the 2014-2015 winter seasons, confirming the presence of this virus in mexico city. with respect to cmv, some studies have found evidence of its implications in asthma pathogenesis and asthmatic inflammation [27] . infa h1n1pdm09 was found in most of the studied groups, its participation and association with bacterial pneumonia [7] are well known, but a strong association with asthma exacerbation has been reported as well [28] . among other of the found viruses, adenovirus (adv) has been associated with pneumonia [29] , and bov has been deemed as a cause of sari in the absence of viral and bacterial co-infections [15] . finally, wiersbitzky et al. [30] suggested that pvb19 can cause acute respiratory diseases with obstructive ventilatory disturbances of the upper or lower airways in children with a specific endogenous predisposition. other components detected in the viruses were bacteriophages, viruses commonly excluded in the final analysis of the viral community, yet, these highly suggest the presence of their specific bacterial hosts in the sample. we compared our results to previous reports that have used metagenomics in children [17] , and shown strategies that use pooling of samples (lysholm et.al. [31] and wang et.al. [16] ), where greater diversity has been found. in our study, we detected 25 different species of viruses using this same strategy. this is most likely due to a greater ability of analyzing more samples in the same library, thereby increasing the probability to detect more viruses. in our study, we found the preferred virus associated with respiratory diseases, rhinovirus, and consistent with others reports (lysholm et.al. [31] and wylie et.al. [12] ), however in contrast with a report from wang et.al. [16] that found a higher prevalence of rsv-b. furthermore, few reports included healthy individuals as a control group (wang et.al [16] ), detecting lesser viral diversity with a high prevalence of viruses belonging to the anelloviridae family. according to this report, the torque teno virus and torque teno mini virus that we found in children with asthma and pneumonia in our study, may be otherwise normal residents in children free of respiratory disease. further data is lacking concerning the prevalence of viruses in the respiratory tracts of healthy children, which may be a limitation of our study. in performing the bacteriome analysis, we found streptococcus pneumoniae and haemophilus influenzae had the greater amount of identified reads in both groups with a significant presence of moraxella and staphylococcus aureus in asthma and pneumonia groups respectively (fig 3a and 3b ). these findings are consistent with data reported by bisgaard et al. [32] and hilty et al. [10] for children with asthma. before rendering judgment, is important to remember that the individual presence of any virus or bacteria does not determine a direct cause for disease. interactions between invaders and host commensals may offer a better understanding about respiratory diseases [33] . identifying coinfections between different microorganisms either between viruses or viruses and bacteria is essential in determining the etiology of the respiratory disease. identifying the etiologic agents present on each disease may assist in developing better treatments and thereby improving patient care. metagenomic and bioinformatic analyses are very powerful tools to describe the microbiome, which allow discerning the etiologic agents involved, and eventually may lead to the discovery of new respiratory viruses. molecular detection of human rhinoviruses in respiratory samples: a comparison of taqman probe-, sybr green i-and boxtobased real-time pcr assays detection and characterization of respiratory viruses causing acute respiratory illness and asthma exacerbation in children during three different season (2011-2014) in mexico city. influenza other respir viruses respiratory severity score separates upper versus lower respiratory tract infections and predicts measures of disease severity characterization of acute respiratory infections among 340 infants in wuxi human pharyngeal microbiome may play a protective role in respiratory tract infections simple predictors to differentiate acute asthma from ari in children: implications for refining case management in the ari control programme the role of respiratory viruses in the etiology of bacterial pneumonia: an ecological perspective host-microbiome interactions in acute and chronic respiratory infections the lung microbiome and exacerbations of copd disordered microbial communities in asthmatic airways strengths and limitations of 16s rrna gene amplicon sequencing in revealing temporal microbial community dynamics sequence analysis of the human virome in febrile and afebrile children unbiased parallel detection of viral pathogens in clinical samples by use of a metagenomic approach direct multiplexed whole genome sequencing of respiratory tract samples reveals full viral genomic information human bocavirus infection as a cause of severe acute respiratory tract infection in children metagenomic analysis of viral genetic diversity in respiratory samples from children with severe acute respiratory infection in china metagenomic analysis of the respiratory virome associated with acute respiratory illness of unknown etiology in infants is there still room for novel viral pathogens in pediatric respiratory tract infections? the infant nasopharyngeal microbiome impacts severity of lower respiratory infection and risk of asthma development ev-d68 infection in children with asthma exacerbation and pneumonia in mexico city during 2014 autumn. influenza other respir viruses microbial community analysis using megan mega: molecular evolutionary genetics analysis software for microcomputers direct metagenomic detection of viral pathogens in nasal and fecal specimens using an unbiased high-throughput sequencing approach potential implication of new torque teno mini viruses in parapneumonic empyema in children associations between nasal torquetenovirus load and spirometric indices in children with asthma the presence of torque teno virus in chronic obstructive pulmonary disease cytomegalovirus dna is highly prevalent in the blood of patients with asthma and is associated with age and asthma traits prevalence of respiratory viral infection in children hospitalized for acute lower respiratory tract diseases, and association of rhinovirus and influenza virus with asthma exacerbations characteristics of adenovirus pneumonia in korean military personnel characterization of the viral microbiome in patients with severe lower respiratory tract infections, using metagenomic sequencing childhood asthma after bacterial colonization of the airway in neonates viral and bacterial interactions in the upper respiratory tract key: cord-310061-nro623aa authors: valitutto, marc t.; aung, ohnmar; tun, kyaw yan naing; vodzak, megan e.; zimmerman, dawn; yu, jennifer h.; win, ye tun; maw, min thein; thein, wai zin; win, htay htay; dhanota, jasjeet; ontiveros, victoria; smith, brett; tremeau-brevard, alexandre; goldstein, tracey; johnson, christine k.; murray, suzan; mazet, jonna title: detection of novel coronaviruses in bats in myanmar date: 2020-04-09 journal: plos one doi: 10.1371/journal.pone.0230802 sha: doc_id: 310061 cord_uid: nro623aa the recent emergence of bat-borne zoonotic viruses warrants vigilant surveillance in their natural hosts. of particular concern is the family of coronaviruses, which includes the causative agents of severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and most recently, coronavirus disease 2019 (covid-19), an epidemic of acute respiratory illness originating from wuhan, china in december 2019. viral detection, discovery, and surveillance activities were undertaken in myanmar to identify viruses in animals at high risk contact interfaces with people. free-ranging bats were captured, and rectal and oral swabs and guano samples collected for coronaviral screening using broadly reactive consensus conventional polymerase chain reaction. sequences from positives were compared to known coronaviruses. three novel alphacoronaviruses, three novel betacoronaviruses, and one known alphacoronavirus previously identified in other southeast asian countries were detected for the first time in bats in myanmar. ongoing land use change remains a prominent driver of zoonotic disease emergence in myanmar, bringing humans into ever closer contact with wildlife, and justifying continued surveillance and vigilance at broad scales. infectious diseases are considered to be "emerging" if they appear in a new population or geographic region or are occurring with greater frequency than the expected background rate [1] [2] [3] . emerging infectious diseases (eids) are capable of causing debilitating health effects and financial instability, especially in less developed countries with insufficient capacity to mount health interventions, and thus pose a significant global public health challenge in the 21 st [4] . an estimated 60-75% of eids are comprised of zoonotic diseases; of these, more than 70% have purportedly originated in wildlife species [3] [4] [5] . spillover has been largely attributed to changes in anthropogenic activity subsequent to exponential human population growth since the latter half of the 20 th century. large-scale land use change, such as deforestation and land conversion for agriculture, can alter host-pathogen relationships and increase human encounter rates with wildlife and their pathogens, making cross-species transmission events more likely [6, 7] . for established pathogens, human-mediated biodiversity loss often leads to reduced populations of suboptimal host species and increased numbers of competent or amplifying hosts, potentially precipitating higher infection rates in people [8] . in addition, intensification of livestock and poultry production systems results in artificially dense populations of domestic animals, which can lead to pathogen amplification and spillover to humans [7] . approximately two-thirds of human pathogens occupy complex, multi-host systems, and pathogens with multiple animal hosts, including some wildlife species, are more likely to become emergent [9] . bats are increasingly recognized as the natural reservoirs of viruses of public health concern [10] [11] [12] [13] . the capacity of bats to carry and transmit zoonotic pathogens has been hypothesized to be due to their unique life history traits, including their ability for sustained flight, potential for long-distance dispersal, aggregation into densely populous colonies, and adaptation to peri-urban habitats [11, 12] . historically, bats have been linked to highly pathogenic viruses that pose a serious threat to human health, including the coronaviruses responsible for severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers), the hemorrhagic ebola and marburg filoviruses, and paramyxoviruses such as nipah virus [10, 11, [13] [14] [15] [16] [17] [18] . more recently, a pandemic of an acute respiratory syndrome originating in wuhan, china in december 2019 was linked to a coronavirus (designated "sars-cov-2") that shared 96% identity with a bat-borne coronavirus at the whole-genome level [19] . in some cases, these viruses can subsequently spread through person-to-person contact following spillover from animals, increasing their epidemic potential [10, 11, 19] . the 2002-2003 sars epidemic, the emergence of mers in people in 2012, and the ongoing covid-19 pandemic have prompted substantial interest in detecting coronaviruses of bat origin due to public health concern and their pandemic potential [10, [13] [14] [15] [16] [17] [18] . coronaviruses (cov) are a family of enveloped, single-stranded rna viruses that commonly infect the respiratory and gastrointestinal tracts of their mammalian and avian hosts [10] . the alphacoronaviruses and betacoronaviruses are of particular importance to human health, with sars-cov, sars-cov-2, and mers-cov-which have caused the most severe disease in humans to datebelonging to the latter group [10, 20, 21] . mounting evidence indicates that bats are the evolutionary hosts and origin for these cov lineages [10, [19] [20] [21] [22] . in addition to human-associated covs, bats are also hosts of coronaviruses that infect production animals, and have been implicated in the emergence and origin of swine acute diarrhea syndrome (sads), transmissible gastroenteritis virus (tgev) in pigs, and porcine epidemic diarrhea (ped), which can cause considerable losses [23] [24] [25] [26] . thus, bat-borne covs can pose a significant threat to human health and food production. in spite of these infectious disease threats, bats are an indisputably essential component of ecosystems. they provide critical services such as seed dispersal, pollination, control of insect populations (including crop pests and disease vectors), and fertilization via guano, making them invaluable assets to agricultural industries and small-holder farming [27] . the importance of bats to ecosystems and human communities while being the natural reservoirs of many zoonotic pathogens presents a challenge for disease control. the potential threats posed by bat-borne coronaviruses to human and livestock health necessitate the identification and characterization of these viruses at high-risk interfaces among humans, domestic animals, and wildlife. particular attention is needed in developing regions of high biodiversity, where eids are most likely to arise, and where substantial losses in agricultural production may be a source of financial insecurity [28] [29] [30] [31] [32] . myanmar is a particularly vulnerable country due to the interplay of ecological and human factors, which increase opportunities for viral spillover. the nation is situated in the heart of the southeast asia region, a hotspot for eids, including some neglected tropical diseases and some of pandemic potential like sars and h5n1 influenza [31, 32] . a combination of biological, ecological, socioeconomic, and anthropogenic factors renders the region particularly susceptible to emerging zoonoses that could impart a considerable public health and economic burden [31, 32] . our study aimed to detect coronaviruses in free-ranging bats living in close proximity to human communities. (fig 1) . these sites were targeted as potential high-risk human-animal interfaces due to land use change increasing human proximity to wildlife and potential human exposures through livelihood, recreational, commercial, and religious or cultural activities. two of these sites also featured popular cave systems where people were routinely exposed to bats through guano harvesting, religious practices, and ecotourism. sites 1 and 2 consisted of several smaller sub-sites where bat capture and sampling events occurred. all surveillance activities were conducted in collaboration with three of myanmar's government ministries: (1) the ministry of livestock, agriculture, and irrigation; (2) the ministry of health and sports; and (3) the ministry of natural resources and environmental conservation. all work conducted was approved through a letter of agreement, ethical review committee, and memorandum of understanding, respectively. bat sampling was performed by trained field personnel in collaboration with myanmar's ministry of agriculture, livestock and irrigation (moali) and ministry of natural resources and environmental conservation (monrec). all bats were captured using mist nets, with each individual manually restrained for species identification, morphometric evaluation, and sample collection. no anesthetic or immobilization agents were used during capture or handling. oral and rectal swabs were collected when possible using sterile polyester-tipped applicators (animal size often precluded rectal swab collection). naturally voided guano samples consisting of combined urine and feces were also collected from the environment using plastic tarps. at site 2, the tarps were placed on the floor of the caves and left overnight, with sample collection occurring the following morning. at sites 1 and 3, the tarps were placed at cave entrances and under roosting areas in the evening as the bats emerged to forage, and samples were collected immediately. guano pellets were collected randomly from the tarps and pooled. tarps were disinfected between each use and gloves were changed in between each sampling event. pooled guano samples were attributed to a presumptive host species based on field identification of species in caves when possible, otherwise were designated as "unidentified chiropterans" when multiple species were present. all sample types were collected into 500 μl viral transport medium (thermoscientific microtest tubes, fisher scientific, pittsburgh, pa, usa) or 500 μl trizol reagent (invitrogen trizol reagent, fisher scientific, pittsburgh, pa, usa), transported from the field in liquid nitrogen, and transferred to a -80˚c freezer within five days and stored until time of testing. bats were humanely trapped, handled, and sampled from according to protocols approved by the institutional animal care and use committee of the university of california at davis (protocol 19300) and smithsonian institution (protocol 16-05) and with approvals from moali and monrec. bats were released within 1 km of the sample testing was performed at the uc davis one health institute laboratory and the veterinary diagnostic laboratory, livestock, breeding, and veterinary department (lbvd) in myanmar. 250 μl was used from each sample for rna extraction per kit instructions, and to ensure availability of an additional aliquot should a second extraction or other downstream analyses be needed. rna was extracted using direct-zol rna columns (zymo research corp), and 8 μl rna was used for cdna transcription using superscript iii (invitrogen). samples were screened for coronaviruses using two broadly reactive consensus conventional polymerase chain reaction (pcr) assays targeting two non-overlapping fragments (434 bp and 332 bp) of the rna-dependent rna polymerase (rdrp) of orf1ab of covs [33, 34] . bands of the expected size were cloned (pcr4-topo vector; invitrogen corp.) and sanger sequenced (abi 3730 capillary electrophoresis genetic analyzer; applied biosystems, inc., foster city, ca). sequences were analyzed and edited using geneious prime (version 2019.1.3), uploaded to genbank (s1 table) , and compared with known sequences in the database. coronavirus sequences were classified as belonging to viral taxa according to established cut-offs and methods [28] . virus sequences that shared less than 90% identity to a known sequence were labelled sequentially as predict_cov-1, -2, -3 etc; while groups sharing �90% identity to a sequence already in genbank were given the same name as the matching sequence. based on these criteria, the cov sequences detected were assigned to discrete viral taxa. viral culture and isolation were not attempted for any positive samples. bat samples positive for a cov-including positive pooled guano samples-were barcoded to confirm the host species using pcr assays targeting fragments of the cytochrome b gene (cytb) and the cytochrome oxidase subunit 1 genes (co1) [35] . one pcr amplicon was selected for sequencing and compared to reference sequences in genbank using blast tools. a threshold of 97% sequence identity was used to confirm the species. sequences with <95% sequence identity were classified to the genus. dna barcoding was also performed on a subset of the cov-negative pooled guano samples. pooled guano samples were assigned a presumptive origin species based on host barcoding. a total of 464 bats representing at least 11 species across eight genera from six families were captured and sampled (table 1 ). both insectivorous microbats and fruit bats were represented in our study population. a total of 759 samples were collected and tested (464 oral swabs, 140 rectal swabs, 155 guano samples). a total of 461 samples were collected in the dry-season sampling (244 oral swabs, 117 rectal swabs, and 100 guano samples) and 298 samples (220 oral swabs, 23 rectal swabs, and 55 guano samples) in the wet season. covs were detected in 48 samples: one oral swab and seven rectal swabs from seven individual bats and 40 pooled guano samples (table 1 ). viral fragments were detected from one unidentified tomb bat (taphozous sp.), three horsfield's leaf-nosed bats (hipposideros larvatus), and three greater asiatic yellow house bats (scotophilus heathii). thirty-six of the 40 positives detected in guano were attributed to h. larvatus, while the host species for the remaining four positive pooled guano samples was identified as wrinkle-lipped free-tailed bats (chaerephon plicatus). overall viral prevalence across all bat taxa and all coronaviral genotypes was approximately 1.5%. the vast majority of positive detections (83.3%) were made from pooled guano samples, while oral swabs had the lowest yields. positive detections were made from 40 samples collected during the dry season (83.3%), while wet-season sampling resulted in positive detections from eight samples (16.7%). both sites 1 and 2 accounted for positive detections, while no coronaviral sequences were detected at site 3. fifty-four total sequences were recovered, clustering within seven distinct coronaviral genotypes. using established cut-offs and methods [28] , we detected four alpha coronaviruses (predict_cov-35, 47, 82, and 90) and three betacoronavirues (predict cov-92, 93, and 96). of these, the alphacoronavirus predict_cov-35 was previously known, having been found in scotophilus kuhlii, unidentified myotis, and other unspeciated host bats in the neighboring countries of cambodia and vietnam from 2013 to 2017 [36] . the remaining six coronaviruses were novel (three alphacoronaviruses and three betacoronaviruses). predict_cov-92 was the most commonly detected coronavirus, found in 36 pooled guano samples attributed to h. larvatus (table 1) . interestingly, three coronaviruses were only found as co-infections: predict_cov-90 was detected with predict_cov-35, predict_cov-93 with -96, and predict_cov-96 also with -92. 3 indicates at least one instance of co-infection. 4 did not meet the 95% nt identity threshold for identification to the taxonomic level of species. https://doi.org/10.1371/journal.pone.0230802.t001 three new alphacoronaviruses, three new betacoronaviruses, and one previously described alphacoronavirus were detected in bats in myanmar. none of the viruses appeared to be closely related to sars-cov, mers-cov, or sars-cov-2. guano samples accounted for the majority of positives, suggestive of an important transmission route for cov shedding from bats [29, 28, 29] and a possible risk to people during the act of guano harvesting [37, 38] . viral detection in guano also has implications for future surveillance, as our study demonstrates the value of non-invasive collection of guano for viral surveillance, potentially obviating the need for handling individual bats for coronaviral detection. our findings supplement those of he et al., who profiled the virome of insectivorous bats from northern myanmar but did not detect coronaviruses in that study [40] . a difference was found in positives for cov by species, as samples from h. larvatus represented 83% of positives. a wide diversity of covs has been found in hipposiderid bats [28, 34, 39, 41] , and our study is consistent with those findings. four covs detected in our surveillance study were found in a single host species each: predict_cov-90 was found only in s. heathii; and predict_cov-92, -93, and -96 were found only in h. larvatus (table 1) . these findings may possibly suggest limited host-switching and viral sharing for certain viruses within our study populations, a pattern consistent with prior observations that viral groups are likely significantly associated with host taxa at the family level [28] . however, further evidence is needed to elucidate host-viral relationships and ecology in the region. our findings also likely reflect a bias in our sampling effort. although h. larvatus samples accounted for the most positives, these were largely detected in guano samples collected from the environment, as individuals were not frequently caught by mist net. overall in our study, the numbers of individual bats handled and sampled per species were relatively low, ranging from one to 218 (table 1 ). viral prevalence may vary widely with the species of host and pathogen. anthony et al. suggested a sample size of at least 154 individuals per species in order to maximize our ability to detect covs. targeting more host species, specific taxa (hipposideridae), and larger sample sizes might have improved our detection rate in the species where no covs were found [28, 29] . currently, active pathogen surveillance at human-wildlife interfaces in myanmar is limited. despite relatively small sample sizes, our study detected several coronaviruses in insectivorous bats, suggesting that more may remain to be uncovered. given the potential consequences for public health in light of expanding human activity, continued surveillance for coronaviruses is warranted, especially in other species and human-wildlife interfaces. anthony et al. estimated that over 3,200 covs occur in bats, most of which remain undiscovered [28] . enhancing our sampling effort to incorporate more diverse bat families and larger sample sizes may enable us to identify more covs in bats in myanmar. additionally, because only short fragments of the conserved rdrp gene (328 bp and 434 bp) were amplified in this study, protein sequence and phylogenetic analyses were not pursued, and identification of recombination events was not possible. while this is an inherent shortcoming of our methodology, the purpose of this study was not to fully characterize specific viruses, but to broadly screen for viruses in bats living in proximity to human communities to better understand potential sources of zoonotic transmission in the context of these human-wildlife interfaces. further studies may consider complete genomic sequencing for more comprehensive profiling of the bat viromes in this ecosystem. in particular, evaluation of the spike gene sequences may provide insights into host range, including potential viral host-sharing or host-switching events [42] . land use change will likely continue bringing people into closer proximity with bats, raising encounter rates and opportunities for spillover, facilitating the emergence of zoonotic viruses, and supporting the need for surveillance [12, 43] . historically, human activities have arguably played a significant role in interspecies transmission events. following the sars outbreak, coronaviruses have since been detected in numerous bat species globally, including in asia, africa, europe, the americas, and the australasian region [28, [44] [45] [46] [47] [48] [49] . mounting evidence supports the role of bats in the transmission of viruses of public health concern-including sars--cov and mers-cov-and the zoonotic potential of unknown bat-borne coronaviruses warrants vigilant, continued surveillance [10] . understanding their ecology and prevalence in their natural hosts can improve our ability to detect, prevent, and respond to potential public health threats. finally, given the essential ecosystem services provided by bats, public health efforts should advocate for preventative measures to protect people against disease transmission while enabling human communities and bats to coexist on a shared landscape. supporting information s1 fauci as the challenge of emerging and reemerging infectious diseases factors in the emergence of infectious diseases host range and emerging and reemerging pathogens global trends in emerging infectious diseases risk factors for human disease emergence human ecology in pathogenic landscapes: two hypotheses on how land use change drives viral emergence ecology of zoonoses; natural and unnatural histories impacts of biodiversity on the emergence and transmission of infectious diseases diseases of humans and their domestic mammals: pathogen characteristics, host range and the risk of 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fertilizer coronaviruses in guano from pteropus medius bats in peradeniya diversity of coronavirus in bats from eastern thailand virome profiling of bats from myanmar by metagenomic analysis of tissue samples reveals more novel mammalian viruses circulation of alphacoronavirus, betacoronavirus and paramyxovirus in hipposideros bat species in zimbabwe recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission bats, coronaviruses, and deforestation: toward the emergence of novel infectious diseases? front microbiol detection of group 1 coronaviruses in bats in north america detection of novel sars-like and other coronaviruses in bats from kenya detection and prevalence patterns of group i coronaviruses in bats coronavirus infection and diversity in bats in the australasian region coronaviruses in bats from mexico detection of coronaviruses in bats of various species in italy we also thank the livestock breeding and veterinary department (lbvd) within the ministry of agriculture, livestock, and irrigation (moali); ministry of natural resources and environmental conservation (monrec); and the department of medical research (dmr) within the ministry of health and sports (mohs), myanmar, with whom we collaborated closely on surveillance activities. thanks also to the invaluable field and laboratory staff who provided technical skill and expertise and were critical in the research process. key: cord-269453-30l6rzgo authors: yang, po; qi, jun; zhang, shuhao; wang, xulong; bi, gaoshan; yang, yun; sheng, bin; yang, geng title: feasibility study of mitigation and suppression strategies for controlling covid-19 outbreaks in london and wuhan date: 2020-08-06 journal: plos one doi: 10.1371/journal.pone.0236857 sha: doc_id: 269453 cord_uid: 30l6rzgo recent outbreaks of coronavirus disease 2019 (covid-19) has led a global pandemic cross the world. most countries took two main interventions: suppression like immediate lockdown cities at epicenter or mitigation that slows down but not stopping epidemic for reducing peak healthcare demand. both strategies have their apparent merits and limitations; it becomes extremely hard to conduct one intervention as the most feasible way to all countries. targeting at this problem, this paper conducted a feasibility study by defining a mathematical model named semcr, it extended traditional seir (susceptible-exposed-infectious-recovered) model by adding two key features: a direct connection between exposed and recovered populations, and separating infections into mild and critical cases. it defined parameters to classify two stages of covid-19 control: active contain by isolation of cases and contacts, passive contain by suppression or mitigation. the model was fitted and evaluated with public dataset containing daily number of confirmed active cases including wuhan and london during january 2020 and march 2020. the simulated results showed that 1) immediate suppression taken in wuhan significantly reduced the total exposed and infectious populations, but it has to be consistently maintained at least 90 days (by the middle of april 2020). without taking this intervention, we predict the number of infections would have been 73 folders higher by the middle of april 2020. its success requires efficient government initiatives and effective collaborative governance for mobilizing of corporate resources to provide essential goods. this mode may be not suitable to other countries without efficient collaborative governance and sufficient health resources. 2) in london, it is possible to take a hybrid intervention of suppression and mitigation for every 2 or 3 weeks over a longer period to balance the total infections and economic loss. while the total infectious populations in this scenario would be possibly 2 times than the one taking suppression, economic loss and recovery of london would be less affected. 3) both in wuhan and london cases, one important issue of fitting practical data was that there were a portion (probably 62.9% in wuhan) of self-recovered populations that were asymptomatic or mild symptomatic. this finding has been recently confirmed by other studies that the seroprevalence in wuhan varied between 3.2% and 3.8% in different sub-regions. it highlights that the epidemic is far from coming to an end by means of herd immunity. early release of intervention intensity potentially increased a risk of the second outbreak. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 throughout human history, infectious diseases (id), also known as transmissible diseases or communicable diseases, are considered as serious threats to global public health and economics [1] . from the 1918 influenza pandemic in spain resulting in nearly 50 million deaths in 1920s, to recent ongoing global outbreaks of corona-virus disease 2019 (covid-19) killing over 11 thousands people in all over the world [2] , infectious disease is a leading contributor to significant mortality and causes huge losses to society as well as personal family burden. among a variety of factors leading to emergence and outbreaks of id, the key issues are population density and human mobility where in these cities with developed transportation systems, pathogens can be spread to large geographic space within a short period of time. for instance, the ongoing global epidemic outbreak of covid-19 has spread to at least 146 countries and territories on 6 continents in 2 months. in order to give an accurate prediction of outbreaks, many researchers have been working in traditional id propagation models [3] [4] [5] [6] [7] like sir, seir, etc, for understanding covid-19 transmission with human mobility and predicting outbreak process of epidemics. meanwhile, as realizing a long period of this battle against covid-19, many of them recently focus on intervention strategies [8] [9] [10] that can balance a trade-off between limited human mobility and potential economic loss in covid-19 control. it poses an important research area that explores how and when to take what level of interventions in light of multiple natures and capabilities of countries. in traditional compartmental models paradigm in epidemiology, sir (susceptible-infectious-recovered) [3] and seir (susceptible-exposure-infectious-recovered) [4] are two popular approaches to simulate and predict how infectious disease is transmitted from human to human. these two models have defined several variables that represent the number of people in each compartment at a particular time. as implied by the variable function of time, these models are dynamic to reflect the changes and fluctuations of these numbers in each compartment over time. for covid-19 control in wuhan, zhong, et.al [11] introduced a modified seir model in prediction of the epidemics trend of covid-19 in china, where the results showed that under strong suppression of "lockdown hubei", the epidemic of covid-19 in china would achieve peak by late february and gradually decline by the end of april 2020. some other extended models [8] [12] are also proposed for predicting the epidemics of covid-19 in wuhan and give some similar forecasts. while above methods demonstrate good performance in prediction of covid-19 outbreak by taking strong public intervention, also named as suppression strategy [13] that aims to reverse epidemic growth, one important challenge is that taking suppression strategy only is to treat disease controls as single-objective optimization of reducing the overall infectious populations as soon as possible, and require strategic consistency in a long term. in real-world, taking public health intervention strategies is actually a multiple-objective optimization problem including economic loss and society impacts. thus, most countries have taken different intervention strategies, like enhanced surveillance and isolation to affected individuals in singapore [14] , four-stage response plan of the uk [15] [16] , mitigation approaches [13] and even multiple interventions taken in many eu countries [17] [18] [19] [20] [21] . due to the fact that standalone intervention strategy has apparent merits and limitations, it becomes highly necessary to study the feasibility of intervention strategies to certain country in light of its multiple natures and capabilities. targeting at this problem, this paper conducts a feasibility study that analyses and compares mitigation and suppression intervention strategies for controlling covid-19 outbreaks in wuhan and london. as shown in fig 1 with wuhan as a simulated case using data from [6] , we demonstrated performance of taking different intervention strategies: a) no interventions: the peak of daily infections would be up to 2.1 million, but will be completed in 150 days. the epidemics lasts a relatively shorter period of 140-150 days, but lead to more death. b). mitigation intervention from the 32 nd day: the peak of daily infectious populations increased to 27.7 thousand, but the period of maintenance extended to 150 days. it implied there would be growing death, but less economic loss compared to suppression. c). suppression intervention from the 32 nd day: the peak of daily infections greatly reduced to 16 thousand, but it had to be followed at least 200 days. nearly 3 months suppression may potentially lead to economic loss even crisis. d). hybrid intervention of taking both suppression and intervention every 2 weeks: the epidemics of covid-19 appeared a long-term multimodal trend where the peaks of daily infectious populations were within a range of 40-60 thousand. this might lead to less daily critical cases and offer more time to hospital for releasing their resources. e) contain phase: taking 90% effectiveness of surveillance and isolation from the 2 nd day of confirmed case potentially enables controlling a new outbreak of covid-19, but it needs to be maintained over 300 days. above analysis demonstrates the complexity of controlling covid-19 outbreaks that how and when to take what level of interventions. in this paper, we proposed a mathematical model: semcr to study this problem. the model extended traditional seir (susceptible-exposed-infectious-recovered) model [3, 4] by adding one important fact: there has been a direct link between exposed and recovered population. we found that the number of confirmed diagnoses reported by governments of various countries is actually much smaller than the actual number of infected persons. although many exposed people have been infected, they will not show symptoms and transformed into infected people after the incubation period of the virus, and directly become recovered people after a certain self-healing cycle [22] [23] . because they are asymptomatic, they will not take the initiative to go to the hospital for treatment testing, so they will not be counted as the actual number of infected people. if we make a statistic on the cumulative number of cured people and the cumulative number of infected people, the difference between them is the number of asymptomatic patients who are directly transformed into cured people without going through the infection stage. then, it defined parameters to classify two stages of covid control: active contain by isolation of cases and contacts, passive contain by suppression or mitigation. the model was fitted and evaluated with public dataset containing daily number of confirmed active cases including wuhan, london, hubei province and the uk during january, 2020 and march 2020. for each point, we design and set up experimental protocols for comparison and exploration, highlighting following contributions: • immediate suppression taken in wuhan significantly reduced the total exposed and infectious populations, but it has to be consistently maintained at least 90 days (by the middle of april 2020). its success heavily relied on sufficiently external support from other places of china. this mode was not suitable to other countries that have no sufficient resources. • in london, it is possible to take a hybrid intervention of suppression and mitigation for every 2 or 3 weeks over a longer period to balance the total infections and economic loss. while the total infectious populations in this scenario would be possibly 2 times than the one taking suppression, economic loss and recovery of london would be less affected. • both in wuhan and london, one important issue of fitting practical data was that there were a large portion (probably 62.9% in wuhan) of self-recovered populations that were asymptomatic or mild symptomatic. these people might think they have been healthy at home and did not go to hospital for covid-19 tests. early release of intervention intensity potentially increased a risk of the second outbreak. • one limitation of our model was that our prediction of infections and deaths depended on a parameter estimation of intervention intensity that presented by average-number contacts with susceptible individuals as infectious individuals in a certain region. it assumed that each intervention had equivalent effects on the reproduction number r in different regions over time. the measures of strong intervention in different countries and regions are similar, so the culture or other issues of different countries will not change the impact of strong intervention on the basic regeneration number. the remainder of this paper is arranged as follows. section 2 introduces the model. in the section 3, the materials and implementation of experiment are reported. section 4 provides detailed experimental evaluation and discussion. the conclusion and future directions are given in section 5. we modified a seir model to account for a dynamic susceptible fig 2. here, we assume that susceptible population e represents susceptible population of a certain region; and β represents effectiveness of intervention (strict isolation) in contain phase. if effectiveness of intervention in contain phase is not sufficiently strong, susceptible individuals may contract the disease with a given rate when in contact with a portion of exposed population (asymptomatic but infectious) e. after an incubation period, the exposed individuals become the infectious population i (symptomatic) at a ratio α 1 . notably, infectious population starts from mild cases m to critical cases c at a ratio α 2 . finally, a portion d of critical cases lead to deaths; the rest of infectious population will be recovered. there are two enhanced features in our model in comparison to popular seir models [6, 8, 11] . the first one is a straightforward relationship between exposed and recovered population. we find that in the early outbreaks of covid-19, some portion of exposed people may have no obvious symptoms or only develop as mild cases, but they cannot get a test due to lack of testing kits. this group of populations might be self-recovered in some days, but will not realize they were infected. and we can calculate the approximate proportion of the number of asymptomatic or mild symptoms in the total number of infected people by counting the cumulative number of each population. the second feature in our model is that we separate infectious population into mild and critical cases in light of their symptoms. according to the curve of the number of critical cases and the number of deaths, we can make a certain explanation for the relatively high mortality rate of early outbreaks of covid-19 after wuhan city took immediate inhibitory interventions on january 23, 2020. introducing above two features, it is helpful for evaluating real effects of different interventions. if we assumed the overall population of a certain region is n, the number of days is t, the dynamic transmissions of each components of our model are defined as follow: regarding mild cases, critical cases and death, the dynamic transmission is as below: lastly, we define a benchmark in semcr model to reflect the strength of intervention over time, as m t . it is presented by average number of contacts per person per day in a region. we estimated changes in covid-19 transmissibility over time via the effective reproduction number(r t ), which represents the mean number of secondary infections that result from a primary case of infection at time t. values of r t exceeding 1 indicate that the epidemic will tend to grow, whereas values below 1 indicate that the epidemic will tend to decline. we estimated the time-varying reproduction numbers from serial intervals and incidence of covid-19 cases over time. in practical cases, it also needs to estimate the defined parameters including α 1 , α 2 , β, and γ 1 , γ 2 , γ 3 , b, where β is the product of the people exposed to each day by confirmed infected people (k) and the probability of transmission (b) when exposed (i.e., β = r t /γ = kb) and σ is the incubation rate which is the rate of latent individuals becoming symptomatic (average duration of incubation is 1/α 1 ). according to recent report [24] , the incubation period of covid-19 was reported to be between 1 to 14 days, we chose the midpoint of 6 days. preliminary data suggests that the time period α 2 from onset to the development of severe disease, including hypoxia, is 1 week [24] . γ is the average rate of recovery or death in infected populations. using epidemic data from [11] , we used semcr model to determine the probability of transmission (b) which was used to derive β and the probability of recovery or death (γ). the number of people who stay susceptible in each region was similar to that of its total resident population. other transmission parameters were estimated with early prediction of hubei cases in [11] on january 23 2020 using monte carlo simulation, as shown in the table 1 notably, as for the strength of intervention m, it was related to the population density in a region. we used a benchmark reported in [11] that assumes hubei province with no intervention as m = 15, and after suppression intervention, m reduced to 3. when applying semcr model into other simulated cases, m was initialized according to the population density and human mobility in these places. also, after taking any kind of interventions, the change of m would follow a reasonable decline or increase over few days, not immediately occur at the second day. following previous assumptions, the implementation of dynamic transmission of semcr model follows steps as below: in order to utilize our proposed semcr model into practical cases, we design an evaluation protocol to access multiple effects of taking different intervention strategies to control outbreak of covid-19 in 4 typical cases, including hubei province, wuhan city, the uk and london, as shown in feasibility study of mitigation and suppression strategies for controlling covid-19 outbreaks the first stage is initial parameters estimation using covid-19 data from four cases: hubei, wuhan, uk and london. in this stage, a preliminary qualitative assessment of each case is performed, by comparing their similarity and dissimilarity on area, transportation, population density, migration flows, date of the first confirmed case, etc. we would determine value of initial parameters in the semcr including n, m t , and date of the first confirmed case. notably, in wuhan, the date of the first confirmed case is not officially released. the work [8] estimates the first confirmed case is by the end of nov 2019; and zhong [11] points out the first confirmed case in wuhan is on 23 nd december 2019. here, we take the same settings of first confirmed case on 23 nd december 2019. the next step is to estimate and normalize other parameters in the model. assuming that covid-19 has similar transmission ratio and incubation rate in all four cases, we use parameter values fitted from [11] , where incubation rate is 1/6; the rate of transmission for the i to s is 0.157; the rate of transmission for the e to s is 0.787. as for estimation of other parameters, we follow the covid-19 official report from who [24] , including the proportion of mild, severe and critical cases, the probability of death, etc. thirdly, how to take intervention strategies needs to be evaluated by tuning parameters in semcr model. the key tuning operation is to adjust the level of m t over a period. for instance, we assume that no intervention strategies result in unaltered internal mobility of a region, taking suppression strategy in wuhan means a reduction of m to 3. but in other cases with larger area, it is extremely difficult to take a complete suppression strategy. so the reduction of m will be relatively adjusted to 4 or 5. final stage, we perform quantitative analysis of effectiveness of different intervention strategies, including: strict surveillance and isolation, suppression strategy, mitigation strategy, and multiple intervention. the evaluation metric of cross-validation is employed to evaluate the performance of covid-19 progression model. the final two evaluation indicators are the length of intervention and the peak time. the length of intervention is calculated due the date that confirmed cases are nearly clear to zero. the most recent epidemiological data in hubei province, china based on daily covid-19 outbreak numbers reported by the national health commission of china were retrieved [25] . the dataset used to analyse several european countries comes from the statistics of worldmeters [26] . specifically, we used confirmed cases, new cases, recovery cases and deaths since 22nd january 2020 to 25th march 2020. all the datasets used in this paper are anonymised. also regarding the daily update from world meter, we record the number of confirmed cases and death each day in four cases. in order to simulate four cases, we require the exact confirmed cases in the first 2-5 days to initialise parameters of our model. we simulated four cases that predict covid-19 outbreaks without taking any interventions. the initial populations were given as london (9.3 million), wuhan (14.18 million), uk (66.49 million) and hubei (58.9 million). the parameter m representing average number of contacts per person per day was given as 15 to london, wuhan and hubei; 12 to the uk. the simulation results were given in fig 4. the results showed that in the peak time, there would be up to 1.16 million, 2.1 million, 4.5 million and 8.7 million exposed population (infection but no symptoms) at london, wuhan, uk and hubei. this implied that: 1) the total infectious population of these four cases would be 7.76 million in london, 12.27 million in wuhan, 50.95 million in hubei and 48.46 million in the uk. 2) the total death of these four cases would be 76 thousand in london, 120 thousand in wuhan, 493 thousand in hubei and 475 thousand in the uk. it equalled to about over 80% of total population of each region will be infectious, with the mortality rate nearly 1%. it showed that without intervention, the outbreak of covid-19 would lead to huge infections and deaths. the main reason was that covid-19 was estimated as relatively high production number r 0 up to 2-2.5 [24] , where the transmission ratio β 2 from susceptible to exposed is up to 78%. thus, in some regions with high migration and dense population, it would easily lead to an outbreak. but, one notable issue was that the initialization of parameter m seems to impact on the occurrence of peak time and the length of overall period. in the cases of london, wuhan and hubei with m = 15, their peak date were all roughly on the 60 th day from the date of first confirmed case; but uk with m = 12, their peak time was delayed to the 100 th day from the date of first confirmed case. that meant to regions with similar total population, low population density potentially reduced overall infectious population, but delayed the peak time of outbreak as a result of longer period. as for the strategy of taking surveillance and isolation intervention in the contain phase, recent study [15] developed a stochastic transmission model parameterised to the covid-19 outbreak. it proved that highly effective contact tracing and case isolation can control a new outbreak of covid-19 within 3 months, where for a production number r 0 of 2-2.5, more than 90% contacts had to be traced. we transferred this finding as a tuning parameter β 2 to evaluate if the outbreak of covid-19 could be controlled. considering that 98% of contacts had to be traced, it implied that the surveillance and isolation was effective to scale down the group of contacts. in other words, we simulated a situation that from the second day of receiving confirmed case, only 5% of the overall population would be possible to contact the infectious ones. then, the β 2 = 0.05. the results were shown in the fig 5. the results show that in all four cases, the outbreaks of covid-19 were successfully controlled, and had no peak time. there would be only more than 40 people that were infection and finally recovered at london, wuhan, uk and hubei. the overall period of covid outbreak was less than 100 days. this finding was as similar as the proof in [15] . notably, the simulated situation of wuhan, london and hubei were same, it was mainly because we initialised the same value of confirmed cases as 3, and m = 15 in all cases. when taking highly effective surveillance and isolation, the transmission of covid-19 was limited and controlled within a small group of population. the population difference would not affect the total infectious ones. but in the uk case with m = 12, low population density limited the effectiveness of surveillance and isolation, as a result of more infectious population. it implied another fact that to these countries with low population density, it was challenging to take high quality of intervention like surveillance and isolation. feasibility study of mitigation and suppression strategies for controlling covid-19 outbreaks then, we evaluated when the outbreak of covid-19 could occur by adjusting the value of parameter β 2 in the uk case. as shown in table 2 , we recorded the total recovered population, and if there would be a peak of outbreak. as the increased β 2 , the total recovered population was dramatically increased and generated a peak as β 2 = 0.075. that meant that if we cannot guarantee at least 92.5% of uk population being not contacted by infectious people, it would be indispensable to have an outbreak of covid-19. if the isolation intervention was less effective; the more population would be infectious; and the peak day was brought forwarded. in the early stage, taking high effective surveillance and isolation in any regions is necessary to avoid the outbreak of covid-19. the suppression strategy was recognized as the most effective solution to reduce the infectious population, where it was taken in wuhan on 23 rd january 2020. in the work [11] , zhong has reported that taking suppression strategy has successfully limits the overall infectious population in hubei on 22 nd february 2020 to 50k. and if the suppression strategy was taken one week earlier, this figure would reduce to 18k. thus, we would like to simulate the situation of taking suppression strategy in london. the parameter m was given as 15 to 4 in london, in comparison to wuhan from 15 to 3. the simulation results were given in fig 6. after taking intensive suppression on 23rd march in the uk, the change trend of the basic regeneration number in the uk and wuhan is consistent. a rapid decline in r has occurred in later march, from 2.81[1. 16-5.19 ] at the 24th day (1st march 2020) to 0.68[0.58-0.79] at the 51st day (28th march 2020). it implied implementing suppression in the uk performed significantly impact on reduction of infections. the results showed that taking suppression strategy, the cases in wuhan and london appeared a similar trend that daily exposed and infectious population would be greatly reduced. the outbreak of covid-19 was controlled by the 100 th days, and can be nearly ended by the 150 th days. the difference was that the daily infectious population of london was nearly double to the ones in wuhan. it was probably because we simulated the date of taking suppression strategy in wuhan (the 32 nd day) is 3 days earlier than london (the 35 th day). another possible reason was that considering the impact of culture difference, the value of m was only limited to 4 in london, but not as lower as 3 in wuhan. in fact, the suppression in wuhan was actually applied to limit mobility in community level with very high intensity, which was hard to be followed by london. we considered another two situations of taking suppression intervention 1 week earlier or 1 week later in london, as shown in fig 6. the results showed that the overall infectious population would be greatly reduced to 3.5 thousand if taking actions one week earlier; oppositely it would increase to 178 thousand infection if taking actions one week later. we used our model to estimate the impacts of suppression on controlling infections of other 6 eu countries (italy,spain, germany, france, belgium and switzerland), as shown in table 3 . most suppression in other countries began around 10 th -17 th march (the 28 th -40 th day from first confirmed case).for each country, we model the number of infections, the number of deaths, and r, the effective reproduction number over time. specific interventions are assumed to have the same relative impact on r in each country when they were introduced there and are informed by mortality data across all countries. fig 6. (a, c) wuhan and (b, d, e, f) london by taking suppression intervention. note that a,b; c,d,e and f have different scales. and the orange dotted line on the x axis represents the date of implementation of the intervention, a, c is the 32nd day, b, d, e, f is the 35th day. https://doi.org/10.1371/journal.pone.0236857.g006 as shown in table 3 , in italy from 8th february 2020, the total number of infections is about 1.9 million, the total number of deaths is about 41 thousand, the true mortality rate is 2.17%. in spain from 1st february 2020, the total number of infections is 2 million, the number of deaths is 41 thousand, the true mortality rate is 2.07%. in germany from 11th february, the total number of infections is about 0.77 million, and the number of deaths is about 11 thousand. the true mortality rate is 1.5%. in france from 15th february 2020, the total number of infections is about 1.6 million, and the number of deaths is about 33 thousand. the true mortality rate is 2.09%. in belgium from 15th february 2020, the total number of infections is about 0.62 million and the number of deaths is about 12 thousand. the true mortality rate is 1.93%, and. lastly, in switzerland 19th from february 2020, the total number of infections is 0.23 million people, the number of deaths is about 3.4 thousand people, the real mortality rate is 1.47%. the time of intervention in these six european countries is similar, but from the analysis of the infections in these six european countries can be seen that the intervention effects of germany, belgium and switzerland are better, and the mortality rate of germany and switzerland is less than four other countries. from the data we collected can be seen that germany and switzerland have adequate medical resources, while italy, spain, france and belgium have insufficient medical resources. this corresponds to the difference between the number of deaths and mortality we predicted. the above also confirms the previously mentioned point of view. when medical resources are insufficient, premature intervention may cause strain on medical resources, leading to more deaths and higher mortality. another two important issues of taking suppression strategy were intervention length and the peak time. in order to effectively control the outbreak, the intervention length of taking actions at above three timing points all required at least 100 days. it potentially led to significant side effects to economic and society, including job loss, mental health, etc.also, the peak time arrived earlier than its nature transmission if taking actions one week earlier. this might increase a shorter time to government for preparing sufficient resources, and causing more difficulties in control the outbreak of covid-19. the mitigation strategy was recently highlighted by researchers from imperial college [27, 28] , where this strategy was initially taken by uk government. the aim of mitigation was to use other strategies to help individuals so that not to interrupt transmission completely, but to reduce the health impacts of an epidemic. in this cases, population immunity built up through the epidemic, leading to an eventual rapid decline in case numbers and transmission dropping to low levels. thus, we simulated the situation of the uk by taking mitigation strategy with different level of strengths. the parameter m representing average number of contacts per person per day was given as 12 to the uk, and gradually reduced to 10 and 8 from the 32 nd day of first confirmed case. the simulation results were given in fig 7a and 7c . the simulation of basic regeneration numbers(r) compared to suppression strategy, mitigation strategy taken in the uk gave a slower decline in r in march, from 2.73[0.97-5.40] on the 24th day (1st march 2020) to 0.98[95% ci 0.88-1.09] on the 110th day (27th may 2020). it implied that before r drops below 1, there were still much growth of infections in the uk. and it can be seen from the fig 7 that the more relaxed the intervention, the longer it takes r to fall below 1. the results showed that taking mitigation strategy was effective to reduce daily infectious population, further lead to huge reduction of total infectious population. by reducing the parameter of m from 12 to 10 or 8, the peak of daily infectious population reduced from 4.5 million to 2.5 million or 1 million. on the other hand, the peak time of infectious population would be delayed as taking mitigation intervention, where the peak dates of infectious population at m = 12, 10 and 8 were roughly on the 80 th , 110 th and 170 th days. the overall period of outbreak would be extended from 180 days to 200 or 250 days. above simulated figures appeared a similar trend as findings from [25] . taking mitigation intervention in the uk was capable of reducing the impact of an epidemic by flattening the curve, reducing peak incidence and overall death. while total infectious population may increase over a longer period, the final mortality ratio may be minimized at the end. but as similar as taking suppression strategy, the mitigation interventions need to remain in place for as much of the epidemic period as possible. however, the timing of introducing this mitigation intervention was important, where too early execution may allow transmission to return once they were lifted and sufficient "herd immunity" has not been developed. in terms of above discussion, the effectiveness of taking any one intervention (either suppression or mitigation) is likely to be limited. it is highly necessary to consider the possibility of taking multiple interventions to be combined to have a substantial impact on social and economic cost reduction. we simulated one simple situation of taking multiple strategy in london from the 35 th day, by giving a hybrid of suppression and mitigation strategies every 2 weeks. the m was given as a pattern of 6-4.5-3-4.5-6 in london, where it meant mitigation and suppression strategies were taken in an every 2 weeks roll. for minimizing side effects of taking interventions on human mobility, the application of first mitigation to reduce m from 12 to 6 spends 2 days. the simulation results were given in fig 7b and 7d the results showed that the epidemic appeared a multi-modal decline trend over 500 days. the first peak of infectious population occurred on the 53 rd days with 1017 infections after taking suppression intervention to reduce m from 12 to 3. after two weeks, mitigation strategies were taken so that the second peak of infectious population raised up to 750 infections. the total infectious population was 53075 over 500 days; the deaths was limited to 520. apparently, taking multiple intervention in the uk is capable of reducing the impact of an epidemic by fluctuating the curve, reducing overall infections and death. while the total period will be extended, final mortality ratio may be minimised at the end. but the longer period of limiting human mobility might increase economic risks and reduce employment ratio. there will be plenty of choices to taking multiple interventions through adjusting the strength and length of intervention. the consequence possibility show a similar multimodal curve but with different peak incidence. as we mentioned in section 1, the question on how and when to take what level of interventions to control an epidemic is highly challenging, particularly in light of multiple natures and capabilities of countries. in many cases, it is even hard to evaluate effectiveness until the end of epidemic, and there is always controversy on taking any one intervention. however, our findings contribute to several useful suggestions on controlling covid-19 outbreak. the first point is that highly effective surveillance and isolation strategy is necessary to control an epidemic in early stage. ideally, if this strategy can be executed in excellent level, there will be no huge outbreak later on. considering the area, transportation, migration flows and population density of a region, most countries cannot achieve excellent level of isolation in contain phase, probably only in fine or good level. the outbreak of an epidemic is inavoidable. but considering its low social and economic cost, this strategy is still a cost-effective option. secondly, the cases in wuhan and london approve high effectiveness of suppression strategy to reduce the overall infection. probably in the uk or similar countries, suppression will minimally require a combination of social distancing of the entire population, home isolation of cases and household quarantine of their family members. but its practical effectiveness is not possible to achieve as same as wuhan, as the success of suppression strategy in wuhan is based on locking down human mobility to community level and sufficient resource support from other cities or provinces in china. if there are no sufficient external support, it will be risky to take intensive suppression to entire country due to huge impacts on its economics. also, taking such intensive intervention to control an epidemic will need to be maintained until vaccine released (up to 12 months or more). if intensive interventions are relaxed at any time points, the transmission will quickly rebound. this is more like a multi-modal curve when taking multi-intervention strategies. thirdly, we also find out that while covid-19 is estimated as a high production rate (r = 2-2.5) [24] , experimental evaluation results show that in either wuhan or london cases fitted with real data in the last 6 weeks, high percentage of exposed or infectious population (at least 62.9% of infectious population in wuhan) are actually self-recovered. these people may have no or mild symptoms but been not checked as confirmed cases. this is one important issue that zhong's seri model [11] has ignored. it will answer the model [8] predicts practical infectious population in wuhan that ten times over figures in [11] . similarly, it could explain the estimation of practical mortality ratio can be varied in [11] and [8] . lastly, one limitation of our model is that its prediction of infections and deaths depends on a parameter estimation of intervention intensity that presented by average-number contacts with susceptible individuals as infectious individuals in a certain region. we assume that each intervention has the same effect on the reproduction number in different regions over time. the measures of suppression intervention in different countries and regions are similar, so the culture or other issues of different countries will not change the impact of suppression intervention on the basic regeneration number. as implementing hybrid intervention, the policy needs to be specific and well-estimated at each day according to the number of confirmed cases, deaths, mortality ratio, health resources, etc. this paper conducts a feasibility study by defining a mathematical model named semcr that analyses and compares mitigation and suppression intervention strategies for controlling covid-19 outbreaks in london and wuhan cases. the model not only fits and evaluates through public data sets containing the number of daily confirmed active cases in wuhan, london, hubei province and the united kingdom, but also uses the trained model to predict and analyze six other european countries at the end. the experimental findings show that the optimal timing of interventions differs between suppression and mitigation strategies, as well as depending on the definition of optimal. in the future, we can expand our model to realize the comparative analysis of the number of severe cases and the number of medical resources by adding medical resources, and optimize the timing and intensity of interventions to reduce the demand for medical resources. conceptualization: po yang, gaoshan bi. world health organization infectious diseases citation patterns: mapping the literature 2008-2010 a 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covid-19 epidemic early estimation of the case fatality rate of covid-19 in mainland china: a data-driven analysis key: cord-282303-idh7io9v authors: hassan, md. zakiul; sturm-ramirez, katharine; rahman, mohammad ziaur; hossain, kamal; aleem, mohammad abdul; bhuiyan, mejbah uddin; islam, md. muzahidul; rahman, mahmudur; gurley, emily s. title: contamination of hospital surfaces with respiratory pathogens in bangladesh date: 2019-10-28 journal: plos one doi: 10.1371/journal.pone.0224065 sha: doc_id: 282303 cord_uid: idh7io9v with limited infection control practices in overcrowded bangladeshi hospitals, surfaces may play an important role in the transmission of respiratory pathogens in hospital wards and pose a serious risk of infection for patients, health care workers, caregivers and visitors. in this study, we aimed to identify if surfaces near hospitalized patients with respiratory infections were contaminated with respiratory pathogens and to identify which surfaces were most commonly contaminated. between september-november 2013, we collected respiratory (nasopharyngeal and oropharyngeal) swabs from patients hospitalized with respiratory illness in adult medicine and paediatric medicine wards at two public tertiary care hospitals in bangladesh. we collected surface swabs from up to five surfaces near each case-patient including: the wall, bed rail, bed sheet, clinical file, and multipurpose towel used for care giving purposes. we tested swabs using real-time multiplex pcr for 19 viral and 12 bacterial pathogens. case-patients with at least one pathogen detected had corresponding surface swabs tested for those same pathogens. of 104 patients tested, 79 had a laboratory-confirmed respiratory pathogen. of the 287 swabs collected from surfaces near these patients, 133 (46%) had evidence of contamination with at least one pathogen. the most commonly contaminated surfaces were the bed sheet and the towel. sixty-two percent of patients with a laboratory-confirmed respiratory pathgen (49/79) had detectable viral or bacterial nucleic acid on at least one surface. klebsiella pneumoniae was the most frequently detected pathogen on both respiratory swabs (32%, 33/104) and on surfaces near patients positive for this organism (97%, 32/33). surfaces near patients hospitalized with respiratory infections were frequently contaminated by pathogens, with klebsiella pneumoniae being most common, highlighting the potential for transmission of respiratory pathogens via surfaces. efforts to introduce routine cleaning in wards may be a feasible strategy to improve infection control, given that severe space constraints prohibit cohorting patients with respiratory illness. with limited infection control practices in overcrowded bangladeshi hospitals, surfaces may play an important role in the transmission of respiratory pathogens in hospital wards and pose a serious risk of infection for patients, health care workers, caregivers and visitors. in this study, we aimed to identify if surfaces near hospitalized patients with respiratory infections were contaminated with respiratory pathogens and to identify which surfaces were most commonly contaminated. between september-november 2013, we collected respiratory (nasopharyngeal and oropharyngeal) swabs from patients hospitalized with respiratory illness in adult medicine and paediatric medicine wards at two public tertiary care hospitals in bangladesh. we collected surface swabs from up to five surfaces near each case-patient including: the wall, bed rail, bed sheet, clinical file, and multipurpose towel used for care giving purposes. we tested swabs using real-time multiplex pcr for 19 viral and 12 bacterial pathogens. case-patients with at least one pathogen detected had corresponding surface swabs tested for those same pathogens. of 104 patients tested, 79 had a laboratory-confirmed respiratory pathogen. of the 287 swabs collected from surfaces near these patients, 133 (46%) had evidence of contamination with at least one pathogen. the most commonly contaminated surfaces were the bed sheet and the towel. sixty-two percent of patients with a laboratory-confirmed respiratory pathgen (49/79) had detectable viral or bacterial nucleic acid on at least one surface. klebsiella pneumoniae was the most frequently detected pathogen on both respiratory swabs (32%, 33/104) and on surfaces near patients positive for this organism (97%, 32/33). surfaces near patients hospitalized with respiratory infections were frequently contaminated by pathogens, with klebsiella pneumoniae being most common, highlighting the potential for transmission of respiratory pathogens via surfaces. efforts to introduce routine cleaning in wards may be a feasible strategy to improve infection control, given that severe space constraints prohibit cohorting patients with respiratory illness. pathogens present in ill patients' respiratory secretions can contaminate nearby hospital surfaces, such as floors, walls, bedrails and mattresses, through coughing, sneezing and touching [1] [2] [3] [4] . respiratory viral and bacterial pathogens, including staphylococcus aureus, streptococcus pyogenes, influenza viruses, respiratory syncytial virus, adenovirus, rhinoviruses and novel coronavirus strains, can survive on hospital surfaces for days, weeks or even months. furthermore, touching contaminated surfaces may lead to nosocomial transmission of pathogens between patients, family caregivers, visitors, and healthcare workers [1, 5, 6] . patient care areas in bangladeshi hospitals are open wards with multiple beds in a room and are frequently overcrowded with patients, family caregivers, and visitors [7, 8] . a previous study by rimi et al. found a median of four people per 100 sq. feet of floor space in hospital wards in bangladesh and observed a median of five uncovered coughs or sneezes per 100 sq feet per hour [7] . due to shortage of health care workers, family caregivers (family members who provides 24 hour hands on care to sick patient, including bedside nursing and cleaning) are integral part of inpatient care in bangladeshi public hospitals, contributing crowding of hospital wards [8, 9] . the world bank estimated that in 2014 only $32 of public funds per capita were spent annually on health infrastructure in bangladesh. thus, resources for infection control are severely limited in bangladeshi hospitals [7, 10] , making it difficult to implement international infection control guidelines [11] . the lack of routine infection control practices, including no regular surface cleaning, may increase the transmission of respiratory pathogens via hospital surfaces [3, 5, 6, 8] . family caregivers, visitors, and hospital staff may acquire respiratory infections either through direct contact with infected patients or via droplets, aerosols or contaminated surfaces. contaminated hospital surfaces can pose a serious risk of infection for patients, health care workers, caregivers and visitors. within this context of scarce resources, describing the magnitude of surface contamination in bangladeshi hospitals, particularly identifying priority areas for decontamination, could influence infection control policy and practice. our objective was to assess the frequency with which patients hospitalized for respiratory illnesses in bangladeshi public hospitals contaminate nearby surfaces, to identify commonly contaminated surfaces, and to determine which pathogens are detected most frequently. we conducted the study in two public tertiary care teaching hospitals in rajshahi and jessore, bangladesh between september and november 2013. rajshahi medical college hospital contains approximately 1,200 beds with eight adult medicine wards and four pediatric wards. jessore medical college hospital is a 250-bed hospital with two adult medicine wards and one pediatric ward. paediatric wards typically admit patients <14 years of age and older patients are admitted to adult medicine wards. the mean bed occupancy proportion in these hospital wards are consistently >100% with patients being treated on the floor and in hallways when beds unavailable [8, 12] . study physicians in adult medicine and pediatric wards identified patients aged �5 years who met the severe acute respiratory illness (sari) case definition of subjective or measured fever (�38 c˚) within the past seven days with cough or sore throat [13] . in pediatric wards, physicians identified children <5 years of age who met the severe pneumonia (sp) case definition: cough or difficulty breathing and at least one danger sign (i.e. chest indrawing, stridor while calm, history of convulsions, inability to drink, lethargy or unconsciousness and/or intractable vomiting) with onset of symptoms within the last seven days [13] . since case-patients were identified immediately after admission, their illnesses were mostly community acquired. study physicians collected respiratory swabs (nasopharyngeal and oropharyngeal) from identified cases using the world health organization's laboratory safety manual protocol [14] and pooled them into a single cryovial containing viral transport media (vtm). trained research assistants in each hospital collected one swab sample from five different surfaces near each enrolled case patient: the wall next to the patient's bed, bed rail, bed sheet, clinical record files, and a multipurpose towel. the multipurpose towel is a cloth brought from home by family caregivers and used to clean patient respiratory secretions, wiping the patient's face or head, and to dry caregivers' hands and face [8] . we selected these surfaces because patients, caregivers, and healthcare workers (hcws) frequently come into contact with them in the hospital wards [6, 7, 15] . the research assistants collected surface swabs between 12-72 hours after the case-patients' admission to the hospital. this allowed for adequate time for hospital surfaces to be exposed to possible contamination by respiratory pathogens, while also making sure that surfaces were swabbed before the enrolled patients were discharged or died, as patient turnover in wards was high with a median hospital stay of three days [16] . the risk for infection from these potentially contaminated surfaces between patients within a room, between patients and healthcare workers, or between patients and family caregivers would vary based on the particular surface and how these different risk groups interacted with the surface. with one sterile rayon swab stick per surface, the research assistant swabbed the area of the wall in contact with the bed 45 cm high from the level of the bed sheet, all surfaces of the bed rail located in the area near the patients' head, half of the bed sheet where the patient's head was including underneath the patient, front and back cover of the patient file and both sides of the multi-purpose towel. not all patients had a wall or bedrail nearby as some patients were cared for on the floor, due to overcrowding. swab samples from each surface area were put into individual cryovials containing vtm and kept in a cool box for up to 30 minutes with a temperature between 2˚-8˚c. both the respiratory swabs and surface swab samples were labelled, packaged, stored in a nitrogen dry shipper (-150˚c) and sent to the icddr,b virology laboratory by batch twice a month. the swab samples were thawed and the cryovials containing the sample were vortexed. about 200 μl of the swab supernatant was used for nucleic acid extraction using invimag1 pathogen kit/kf 96 (stratec molecular, berlin, germany) and the final eluted volume of nucleic acid solution was 200 μl, as per the manufacturer's instruction [17] . the real-time multiplex pcr assay was performed as per the manufacturer's instructions using an agpath-id ™ one-step rt-pcr kit (ambion) with the fast track diagnostic (ftd) respiratory pathogens 33 kit (fast track diagnostics, luxembourg) for 19 different viruses and 12 bacterial pathogens [18] . casepatients with at least one pathogen detected in their respiratory swab had corresponding surface swabs tested for those same pathogens. detection of nucleic acid of at least one similar pathogen on respiratory swab and a nearby surface was defined as contamination of that surface. to investigate wider hospital contamination, we also tested the surface swabs collected near casepatients with no pathogens detected in their respiratory swabs for the most commonly detected pathogens identified on surfaces near patients with detected respiratory pathogens. we summarized the data using descriptive statistics. we assessed the difference in proportion of pathogen detection in respiratory swabs and surface swabs between adult and paediatric ward patients using chi-square test considering fisher exact test where appropriate. any association with a p value <0.05 was considered statistically significant. study participants (aged �18 years) or their legal guardians (if aged <18 years) provided informed written consent. the institutional review board of icddr,b reviewed and approved the study protocol. the institutional review board at the centers for disease control and prevention (atlanta, ga, usa) deferred to icddr,b's approval. we collected and tested respiratory swabs from 104 patients hospitalized with respiratory illness: 50 sari cases from adult medicine wards and 54 severe pneumonia cases from paediatric medicine wards. the median age of patients in the adult wards was 32 years (iqr 25-48) and in paediatric wards three months (iqr 2-6). the male-to-female ratio was 2.6:1 (table 1) . of the 104 patients, 79 (75%) had detectable viral and/or bacterial nucleic acid in their respiratory swabs. paediatric patients more frequently had one or more detectable pathogen in their respiratory swabs than adult patients (91% versus 60%, p = 0.001). bacterial pathogens were identified in 76% of adult respiratory swabs. klebsiella pneumoniae, streptococcus pneumoniae and human cytomegalovirus 0(0) 21(39) human parainfluenza viruses 2(4) 3(5.5) other viruses b 3(6) 6(11) 18 ( staphylococcus aureus were most commonly detected during the study period. in contrast, viral pathogens were commonly detected among paediatric patients, including human cytomegalovirus, respiratory syncytial viruses, and human rhinoviruses (table 1) . clinical features, mean duration of symptom onset to sample collection (3.2 days vs 3 days) did not vary between patients with a detectable viral and a detectable bacterial nucleic acid in respiratory swabs. two patients, one with klebsiella pneumoniae and one with streptococcus pneumoniae detected in their respiratory swabs, had been hospitalized at other facilities within two weeks prior to admission to the study hospital, suggesting that these organisms could have been hospital-acquired. both these patients had abnormal chest x-rays and were diagnosed with severe pneumonia in the study hospital. we tested the surrounding hospital surface swabs for each of the 79 patients with evidence of respiratory pathogens for the same viral/bacterial nucleic acid detected in their respiratory swabs. we collected and tested 287 surface swabs near the 79 patients as not all these patients had a wall or bedrail near them. nearly half of the hospital surface swabs (46% [133/287]) had evidence of contamination by at least one pathogen included in the testing panel. the most commonly contaminated surfaces were the bed sheet, the multipurpose towel, and the bed rail. we infrequently detected bacterial or viral nucleic acid on wall surfaces or on patients' clinical record files (fig 1) . sixty-two percent of patients (49/79) had detectable viral and/or bacterial nucleic acid on at least one (range: 1-5) nearby surface, including 60% of adults (18/30) and 63% (31/49) of pediatric patients. the most common bacterial pathogen detected on surface swabs was klebsiella pneumoniae and 97% (32/33) of patients positive for klebsiella pneumoniae had at least one surface with detectable dna. the most frequently detected viral pathogen on surfaces was human cytomegalovirus and 86% (18/21) of patients positive for human cytomegalovirus had detectable dna on nearby surfaces ( table 2) . we tested nearby surfaces for 22 patients (18 patients from adult wards and 4 from pediatric wards) without detectable viral/bacterial nucleic acid in their respiratory swabs. we tested these surfaces for six frequently identified pathogens in respiratory swabs of case-patients: klebsiella pneumoniae, streptococcus pneumoniae, staphylococcus aureus, human cytomegalovirus, respiratory syncytial viruses and human rhinoviruses. klebsiella pneumoniae was detected on at least one nearby surface in 95% (21/22) of these patients, staphylococcus aureus in 18% (4/22), and streptococcus pneumoniae in 14% (3/22) patients. viruses, including, human cytomegalovirus (4/22), respiratory syncytial viruses a and b (1/22) and human rhinoviruses (0/22), were rarely detected nearby these patients. nearly two-thirds of the patients hospitalized with laboratory-confirmed acute respiratory infection had at least one nearby contaminated surface. klebsiella pneumoniae was the most commonly detected pathogen in patients' respiratory swabs, and was detected in nearly every environmental swab testing, suggesting widespread hospital contamination from current and previously hospitalized patients. with the ability to spread rapidly in the hospital environment, klebsiella pneumoniae has been linked to several nosocomial outbreaks [19, 20] . the most frequently contaminated surfaces were the bed sheet, towel, and bed rail, further highlighting the perils of no routine surface cleaning practices in these hospitals. studies in tertiary care hospitals of bangladesh have shown that one in 20 patients with a hospital stay greater than three days developed a hospital-acquired respiratory infection, and that only 21% of those infections had a viral aetiology, suggesting a large proportion of these infections might be bacterial [21, 22] . since, future work should further investigate the role of klebsiella pneumoniae may be an important nosocomial pathogen in these hospitals. several other studies have identified multidrug resistant klebsiella pneumoniae in hospital environments and its association with severe infections, prolonged hospital stays and increased mortality rates, particularly in debilitated and immunocompromised patients [23, 24] . a 2004 study in an urban tertiary care hospital in dhaka, bangladesh, reported that 40% of the clinical specimens (sputum, pus, urine, throat, and vaginal swabs) collected from hospitalized patients were drug resistant [25] . moreover, our study findings were consistent between the two hospitals despite being located in different geographical areas and may suggest surface contamination with klebsiella pneumoniae as a wider public health problem. the predominant bacterial pathogens we identified, klebsiella pneumoniae, streptococcus pneumoniae, and staphylococcus aureus, can survive on surfaces from a few days to a few months [5, 26, 27] . bacteria, in the presence of low humidity, forms biofilms protecting microorganisms from harsh environmental influences and are difficult to eradicate [28, 29] . in bangladesh, hospital surfaces are not adequately cleaned and hospitals report insufficient supplies of cleaning agents [7, 8, 30] . to remove and prevent biofilm formation, hospital decontamination protocols should include strategies such as daily cleaning of surfaces with disinfectant (e.g. 0.3% sodium hypochlorite or 2% chlorhexidine solutions). among the most commonly detected viral pathogens, rsv was prevalent in respiratory swab of paediatric patients and nearby surfaces. rsv has been a major nosocomial hazard on pediatric wards and has been linked with hospital outbreaks [31, 32] . with reported higher case fatality among patient with nosocomial rsv infections (or 4.46, 95% ci 1.1-18), widespread surface contamination with rsv is concerning for low income hospitals (34, 35) . we identified, that towel, was frequently contaminated with respiratory secretions. [8] . islam et al. reported that family caregivers frequently used a multipurpose towel for patient secretions and for their own use, without cleaning it in between these uses [8] .this suggests that the towel may act as a potential vehicle for transmission of respiratory viral and bacterial pathogens from patient to caregiver [33] . infection control could target care giving practices associated with the use of the towel and should test feasible low-cost interventions such as the supply of low cost disinfectant by hospitals that encourage caregivers to clean the towels more frequently and to improve hand washing practices, including the use of hand sanitizer. an important limitation of our study is that we only identified the presence of viral or bacterial nucleic acid on different hospital surfaces, and cannot be sure that the pathogens we detected were viable. a second limitation is that we conducted the study in only two public hospitals, so the findings may not be representative of all public hospitals. however, our findings were consistent between the two typical tertiary care hospitals we studied, located in completely different parts of the country. a third limitation is that we only sampled surfaces once, which limits our ability to comment on duration of contamination, and did not have the ability to observe contamination from seasonal infections. influenza, for example, has a known seasonal pattern in bangladesh, circulating usually between may and september, potentially explaining the low detection rate in our study [34, 35] . lastly, we did not investigate drug resistance patterns of the bacteria we detected due to resource constraints. based on evidence from other studies, however, it is likely that many of the pathogens we detected on surfaces were drug resistant and future studies should consider including investigations about drug resistance [25] . this study identified that hospital surfaces in these bangladeshi hospitals, were frequently contaminated with respiratory pathogens and pose a potential threat for fomite-borne transmission of respiratory infections to patients, healthcare workers and family caregivers. to prevent the spread of klebsiella and other infections between patients, healthcare personnel must follow specific infection control precautions including strict adherence to hand hygiene and the use of gloves. in addition, our data clearly indicate that efforts to regularly disinfect environmental surfaces and ensure clean towels for patient caregiving could reduce risk of exposure to patients, healthcare staff and visitors. the government of bangladesh has taken a number of initiatives to improve infection control in hospitals [36] . despite this, a 2013 nationally representative survey of healthcare facilities showed that healthcare workers performed recommended hand hygiene in only 9% of 919 opportunities, suggesting low adherence to international standards [30] . barriers include awareness, training, accountability and appropriate infrastructure (among 875 health facilities, 10% handwashing locations had no water, 20% had no soap and 50-80% had no alcohol based sanitizer) to support these behaviours [30, 37, 38] this study highlighted the gaps in practice, as well as the substantial barriers to improvement that will require widespread investments to address. in 2018, the directorate of hospital infection control, directorate general of health services (dghs) at the ministry of health and family welfare in bangladesh has communicated the intention to form infection control committees in each district and tertiary care hospital across the country to improve the safe care [39] . further investigation to identify the true contribution of fomites in the transmission of respiratory pathogens within hospital settings could be useful to help these committees prioritize efforts to improve hand and surface cleaning. supporting information s1 file. dataset. (dta) significance of fomites in the spread of respiratory and enteric viral disease environmental contamination makes an important contribution to hospital infection contamination, disinfection, and cross-colonization: are hospital surfaces reservoirs for nosocomial infection? clinical infectious diseases nipah virus contamination of hospital surfaces during outbreaks how long do nosocomial pathogens persist on inanimate surfaces? a systematic review hand-touch contact assessment of high-touch and mutual-touch surfaces among healthcare workers, patients, and visitors infrastructure and contamination of the physical environment in three bangladeshi hospitals: putting infection control into context family caregivers in public tertiary care hospitals in bangladesh: risks and opportunities for infection control the health workforce crisis in bangladesh: shortage, inappropriate skill-mix and inequitable distribution universal health coverage assessment people's republic of bangladesh international nosocomial infection control consortium report, data summary of 50 countries for 2010-2015: device-associated module incidence of and risk factors for hospital-acquired diarrhea in three tertiary care public hospitals in bangladesh. the american journal of tropical medicine and hygiene who interim global epidemiological surveillance standards for influenza manual for the laboratory diagnosis and virological surveillance of influenza a quantitative approach to defining "high-touch" surfaces in hospitals economic burden of influenza-associated hospitalizations and outpatient visits in bangladesh during 2010. influenza and other respiratory viruses maternal vitamin d supplementation during pregnancy and lactation to prevent acute respiratory infections in infancy in dhaka, bangladesh (mdari trial): protocol for a prospective cohort study nested within a randomized controlled trial ftd respiratory pathogens 33 luxemburg: fast track diagnostics an outbreak of hospital-acquired klebsiella pneumoniae bacteraemia, including strains producing extended-spectrum β-lactamase outbreak of klebsiella pneumoniae producing a new carbapenem-hydrolyzing class a β-lactamase, kpc-3, in a new york medical center incidence and viral aetiology of hospital-acquired respiratory infections at three tertiary care hospitals in bangladesh rates of hospital-acquired respiratory illness in bangladeshi tertiary care hospitals: results from a low-cost pilot surveillance strategy detection and treatment options for klebsiella pneumoniae carbapenemases (kpcs): an emerging cause of multidrug-resistant infection predictors of hospital surface contamination with extended-spectrum β-lactamase-producing escherichia coli and klebsiella pneumoniae: patient and organism factors. antimicrobial resistance and infection control prevalence of extended-spectrum βlactamase-producing escherichia coli and klebsiella pneumoniae in an urban hospital in dhaka, bangladesh survival of enterococci and staphylococci on hospital fabrics and plastic survival of enterococci and staphylococci on hospital fabrics and plastic survival strategies of infectious biofilms efficacy of disinfecting solutions in removing biofilms from polyvinyl chloride tracheostomy tubes. the laryngoscope healthcare worker and family caregiver hand hygiene in bangladeshi healthcare facilities: results from the bangladesh national hygiene baseline survey risk of nosocomial respiratory syncytial virus infection and effectiveness of control measures to prevent transmission events: a systematic review. influenza and other respiratory viruses the clinical and phylogenetic investigation for a nosocomial outbreak of respiratory syncytial virus infection in an adult hemato-oncology unit nipah virus contamination of hospital surfaces during outbreaks influenza in outpatient ili case-patients in national hospital-based surveillance estimates of seasonal influenzaassociated mortality in bangladesh bangladesh: dghs, ministry of health & family welfare behaviour change intervention to reduce caregivers' exposure to patients' oral and nasal secretions in bangladesh health worker and family caregiver hand hygiene in bangladesh healthcare facilities: results from a nationally representative survey bangladesh: health economics unit, health sevices division we would like to thank authorities of participating hospitals, all the study participants, the study physicians, field staff and laboratory staff for their contribution. we acknowledge gladys leterme for reviewing and editing this manuscript.icddr,b acknowledges with gratitude the commitment of cdc to its research effort. icddr,b is also grateful to the governments of bangladesh, canada, sweden and the uk for providing core/unrestricted support. key: cord-281665-6n7aq4k9 authors: qiu, sangsang; pan, hongqiu; zhang, simin; peng, xianzhen; zheng, xianzhi; xu, guisheng; wang, min; wang, jianming; lu, hui title: is tuberculosis treatment really free in china? a study comparing two areas with different management models date: 2015-05-20 journal: plos one doi: 10.1371/journal.pone.0126770 sha: doc_id: 281665 cord_uid: 6n7aq4k9 objective: china has implemented a free-service policy for tuberculosis. however, patients still have to pay a substantial proportion of their annual income for treatment of this disease. this study describes the economic burden on patients with tuberculosis; identifies related factors by comparing two areas with different management models; and provides policy recommendation for tuberculosis control reform in china. methods: there are three tuberculosis management models in china: the tuberculosis dispensary model, specialist model and integrated model. we selected zhangjiagang (zjg) and taixing (tx) as the study sites, which correspond to areas implementing the integrated model and dispensary model, respectively. patients diagnosed and treated for tuberculosis since january 2010 were recruited as study subjects. a total of 590 patients (316 patients from zjg and 274 patients from tx) were interviewed with a response rate of 81%. the economic burden attributed to tuberculosis, including direct costs and indirect costs, was estimated and compared between the two study sites. the mann-whitney u test was used to compare the cost differences between the two groups. potential factors related to the total out-of-pocket costs were analyzed based on a step-by-step multivariate linear regression model after the logarithmic transformation of the costs. results: the average (median, interquartile range) total cost was 18793.33 (9965, 3200-24400) cny for patients in zjg, which was significantly higher than for patients in tx (mean: 6598.33, median: 2263, interquartile range: 983–6688) (z = 10.42, p < 0.001). after excluding expenses covered by health insurance, the average out-of-pocket costs were 14304.4 cny in zjg and 5639.2 cny in tx. based on the multivariable linear regression analysis, factors related to the total out-of-pocket costs were study site, age, number of clinical visits, residence, diagnosis delay, hospitalization, intake of liver protective drugs and use of the second-line drugs. conclusion: under the current “free of diagnosis and treatment” policy, the financial burden remains heavy on tuberculosis patients. policy makers need to consider appropriate steps to lessen the burden of out-of-pocket costs for tuberculosis patients in china and how best to improve service delivery for poor patients. tuberculosis (tb) is a global health problem and remains a major cause of morbidity and mortality in developing countries [1] . china has the world's second largest tuberculosis epidemic, accounting for 12% of the total number of cases. in 2012, there were 1 million new cases and 44000 tuberculosis-related deaths in china [1] . the rising multidrug-resistant (mdr) tuberculosis is increasing an already heavy burden on china's health system [2, 3] . tuberculosis has been regarded as a "poverty-related disease" due to the association with poverty and malnutrition, which are more prevalent in developing countries. for example, in south africa, tuberculosis is referred to as a "barometer of poverty" [4] . tuberculosis-affected patients and their family members face many economic and social problems, such as high medical costs, loss of productivity, stigmatization and social isolation [5, 6] . in 1992, china initiated its modern national tuberculosis control program (ntp) with directly observed treatment, short-course (dots) [7] . recently, especially after the outbreak of severe acute respiratory syndrome in 2003, the chinese government has taken a series of measures to strengthen its public health system with great efforts towards tuberculosis control. by 2005, china achieved the global targets for tuberculosis control with 100% dots coverage and over 90% treatment success [8] . each year in china, more than 1 million tuberculosis patients receive dots therapy [9] . to reduce the financial barriers to and burdens on patients seeking essential healthcare, a "free-tb service policy" has been implemented gradually throughout the country [10, 11] . under this policy, tuberculosis suspects are provided a free diagnosis and anti-tuberculosis treatment, including a free chest x-ray examination, sputum smear test and designated firstline anti-tuberculosis drugs [12] . initially, the free-service policy was only performed for sputum smear-positive patients. now it has expanded to sputum smear-negative patients. moreover, the government has taken more measures to reduce the patient burden, including the establishment of universal health coverage and increasing the reimbursement rate for patients with tuberculosis [13] . the central government's spending on tuberculosis control increased from 40 million chinese yuan (cny) in 2001 to 580 million cny in 2010 [14] . china has established universal health coverage for 830 million rural residents through the rapid expansion of the new cooperative medical scheme (ncms). moreover, a free-service policy has been gradually adopted in order to lighten the economic burden of patients with tuberculosis. despite these policy changes, previous studies have revealed that patients still bear a high financial burden, which could affect their health care-seeking behaviors and treatment outcomes [10, [15] [16] [17] [18] . the revenue-driven practices in some health facilities, such as over-prescription of medication, poor referral and high hospitalization rates not only influence a patient's economic burden but also affects the entire tuberculosis control program [19, 20] . there are three common types of tuberculosis management models in china: the tuberculosis dispensary model, specialist model and integrated model [21] . for the dispensary model, patients are diagnosed and treated in tuberculosis dispensaries, which are usually affiliated with local cdc (center for disease control and prevention). the specialist model is similar to the dispensary model, but a specialized hospital is also responsible for treating the patients. for the integrated model, tuberculosis diagnosis and treatment is integrated into a general hospital which is referred to as a "designated hospital" [22] . this study describes the economic burden on patients with tuberculosis, identifies related factors by comparing two areas with different management models, and provides a policy recommendation for the tuberculosis control system in china. the institutional review board (irb) of nanjing medical university approved the study. written informed consent was obtained from all of the participants. ethical practices were used throughout the study period. a descriptive study was performed in two counties of jiangsu province, china. one county, zhangjiagang (zjg), is located in the southern part of jiangsu, which is a relatively rich area and ranked as the third most developed county of china in 2013. another county, taixing (tx), is located in the middle of jiangsu, which is a moderately developed county. in 2010, the per capita gdp was 129535 chinese yuan (cny) in zjg and 36994 cny in tx (http://www. jssb.gov.cn/). in the same year, the per capita gdp was 30567 cny in china (http://www.stats. gov.cn/tjsj/ndsj/). zjg adopted the integrated model, and the diagnosis and treatment of tuberculosis are provided by the designated county-level general hospital. tx implemented the dispensary model, and tuberculosis patients are diagnosed and treated at the local cdc (tuberculosis dispensary). in zjg, the free service covered the first-line anti-tuberculosis drugs, four liver function tests, three chest x-ray examinations and all sputum smear microscopy tests. moreover, each patient could receive a reimbursement of 150 cny for liver protection drugs and each migrant patient with tuberculosis could receive an additional subsidy of 120 cny. in tx, the free service policy covered the first-line anti-tuberculosis drugs, two liver function tests, one chest x-ray examination and all sputum smear microscopy tests. hospitalization and second-line anti-tuberculosis drugs were not free of charge in either area. baseline characteristics of the two study settings were listed in table 1 . in zjg, 23 villages were selected as study sites based on a random cluster sampling method. three hundred and forty patients who were diagnosed with pulmonary tuberculosis since january 2010 were recruited for the investigation. the patients had already completed the standard anti-tuberculosis treatment prior to march 2013. in tx, we randomly selected 34 villages as study sites. three hundred and ninety patients who were diagnosed with pulmonary tuberculosis since january 2010 were enrolled. patients with mdr-tb were excluded from the study. due to the gradually evolving tuberculosis management models in tx since 2012, the recruited patients were limited to those who had finished anti-tuberculosis treatment prior to 2012. a total of 590 patients (316 patients from zjg and 274 patients from tx) were successfully interviewed and involved in the analysis with a response rate of 81%. with the patients' informed consent, trained investigators interviewed them at their homes using a structured questionnaire to gather information, including demographic characteristics, socioeconomic status, health insurance, health care-seeking history and costs related to tuberculosis diagnosis and treatment. the economic burden of the patients with tuberculosis consisted of direct and indirect costs. the direct costs were medical costs related to tuberculosis diagnosis and treatment (including outpatient and inpatient expenses), transportation, accommodation and food [23, 24] . the indirect costs were calculated as a loss of income due to an inability to work due to the disease. we examined the loss of income (or decreased earning ability) for both patients of the labor force and their caregivers [23] . the total out-of-pocket costs were calculated by excluding reimbursement through medical insurance. the out-of-pocket direct costs were direct costs calculated by excluding reimbursement through medical insurance. the costs are expressed in the chinese yuan (cny). in 2014, 1 cny was equal to 0.16 usd. in order to calculate the costs for outpatient visits to the doctor, the number and costs of the visits made for tuberculosis diagnosis and treatment were obtained from the patients and their family members. if the patients could not remember the costs of the doctor's visits, medical records and invoices were checked. the hospitalization cost of the patients was computed based on the cost that they paid when being discharged from the hospital. non-medical, direct costs included transportation; commuting and food costs (of patients and their families) during their visit to health facilities; the cost of purchasing extra health products that were required due to the tuberculosis; the cost of residing in other cities for treatment and nursing patients at home. indirect costs depended on daily income; the number of sick leaves; the average daily income of each patient's companion; and the duration of absence from work resulting from nursing and caring for the patient. the individuals' wages were used to calculate lost income. for patients who were not willing to declare their income and also for housekeepers, the local average daily wage was used. we used epidata 3.1 (http://epidata.dk) software to input data with double entry for consistency. statistical analyses was performed using stata 12.0 software (college station, texas, usa). considering the distribution of costs, we used the mean and median (interquartile range, iqr) to describe a patient's economic burden. the mann-whitney u test was used to compare the cost difference between the two groups. the cost was logarithmically transformed as the dependent variable, and a multivariate linear regression model was performed to analyze potential factors related to the out-of-pocket costs of patients with tuberculosis. we included product terms in our model to individually account for each possible two-way interaction, considering as statistically significant those interactions with a p-value < 0.05. this study recruited 590 tuberculosis patients, including 425 men (72.0%) and 165 women (28.0%). among them, 316 (53.6%) resided in zjg, and 274 (46.4%) resided in tx. in zjg, there were many young, migrant patients with tuberculosis, and fewer patients had a treatment history. the proportion of hospitalized patients and patients taking liver protective drugs were also higher in zjg than tx (table 2 ). as shown in table 3 , the average (median, iqr) total costs was 18793.33 (9965, 3200-24400) cny for patients in zjg, which was significantly higher than for patients in tx (mean: 6598.33, median: 2263, iqr: 983-6688) (z = 10.42, p < 0.001). after excluding the expenses covered by health insurance, the total out-of-pocket costs was 14304.4 cny in zjg and 5639.2 cny in tx. the median (iqr) ratio of total out-of-pocket cost to the annual family income was 20.5% (7.5%-58.7%) in zjg and 10.3% (3.9%-31.2%) in tx (table 3 ). in zjg, the average direct costs was 11936.9 cny with a median (iqr) of 4590 (2024-14600). in tx, the average direct costs was 3983.1 cny with a median (iqr) of 1200 (520-2845). when we excluded the expenses covered by health insurance, the average out-of-pocket direct costs was 7448. table 3) . the common factors contributing to the total out-of-pocket costs in these two areas were diagnosis delay, hospitalization, and intake of liver protective drugs and second-line drugs. a univariate analysis also identified several peculiar features related to the total out-of-pocket costs in tx, including the patients' age, treatment history, adverse drug reactions and the number of clinical visits (table 4) . we further performed a step-by-step linear regression analysis by including multiple variables in the model. significant factors related to the total out-of-pocket costs were study setting (t = -3.10, p = 0.002), age (t = -4.04, p < 0.001), number of clinical visits (t = 4.46, p < 0.001), residence (t = 3.19, p = 0.002), diagnosis delay (t = 3.47, p = 0.001), hospitalization (t = 15.04, p < 0.001), intake of liver protective drugs (t = 2.78, p = 0.006) and intake of second-line drugs (t = 2.87, p = 0.004) ( table 5) . a significant interaction was found between the number of clinical visits and study setting (p interaction = 0.001). we then performed a multivariate linear regression analysis stratified by study setting. as shown in table 6 , age, residence, diagnosis delay and hospitalization entered into the model in zjg. the number of clinical visits, age, diagnosis delay, hospitalization, intake of liver protective drugs and intake of second-line drugs entered into the model in tx. tuberculosis has historically been endemic in china, primarily due to limited health resources and government neglect [14] . in recent years, the political commitment to public health has significantly increased [25] . china is moving towards primary health care based on community services [14] . for example, the government pays sustainable funds per person for the prevention of disease, and some funds are allotted to tuberculosis control. financial concern is the most important factor guiding health care seeking behavior among the chinese population [26] [27] [28] . however, as demonstrated in the current study and previous reports [10, 13, 16, 20] , most of the patients have complained about paying a major part of the treatment cost through out of pocket payments. to modify the current tuberculosis control strategies, policy makers must identify the factors related to patient economic burden. in this study, we selected two counties from the jiangsu province using different tuberculosis management models. zjg adopted the integrated model. one advantage of this model is that it can shorten the delay time of diagnosis and treatment [22] . previous studies have demonstrated that multiple clinical visits and a delay of diagnosis can increase the financial burden on patients and influence the successful control of tuberculosis [29] . patient delay also occurs due to a lack of knowledge or negligence in seeking health care services until their symptoms deteriorate [30] [31] [32] . the inability of doctors to diagnose tuberculosis during the patient's early visits leads to an institutional delay [15] . suspected patients are usually provided symptomatic treatment resulting in increased expenses. the who recommends outpatient treatment for non-complicated tuberculosis patients. however, unnecessary hospitalization has been reported, which not only leads to higher inpatient expense, but also results in extra costs related to accommodation and transportation. the integrated tuberculosis management model may increase the hospitalization rate of suspected patients in the designated hospital. a study demonstrated that in tuberculosis patients without concomitant disorders, the initiation of treatment at a hospital adversely influenced the outcome of treatment, as reflected by the percentage of completers [33] . however, for patients with complicated or severe symptoms, hospitalization can be advantageous. the intake of liver protective drugs and second-line anti-tuberculosis drugs were two common factors influencing a patient's economic burden. one of the most adverse reactions caused by anti-tuberculosis drugs is drug-induced liver damage. to avoid this reaction, clinical doctors in china usually prescribe protective drugs. these drugs include herbals, manufactured herbal products, and combinations of vitamins and other non-herbal substances (although their preventive effects have not been proven) [34, 35] . liver-protecting drugs are not free. over-prescription of these medications also increases the patients' out-of-pocket costs [10] . currently, in many settings in china, the prescription of second-line drugs primarily depends on the treatment history of the patients rather than the drug susceptibility test. as reported in one cross-sectional study conducted in five provinces within china, approximately 23.8% of the patients had a history of taking second-line drugs [36] . even worse, there was no significant association between the prescription of second-line drugs and the drug resistant statuses of the patients. abuse of second-line drugs merely based on clinical judgments not only increases the risk of mdr-tb and xdr-tb but also results in extra economic burden on the patients [17] . in addition to the medical costs due to the disease, the loss of working time experienced by patients and their family members should not be ignored. a dutch study demonstrated that patients lose (on average) 2.7 months (81 days) of productive days due to tuberculosis [37] . in developed areas, the indirect costs would be much higher. for example, in our study, patients in zjg had nearly four times higher indirect costs compared with patients in tx, which might be attributed to a higher level of economic development and per capita income in zjg. besides the common factors related to the total out-of-pocket costs in zjg and tx, the univariate analysis revealed that age, treatment history, adverse drug reactions and number of clinical visits were significant factors in tx but not in zgj. these particular factors may play different roles in a patient's economic burden in different areas. for example, a significant interaction was found between the clinical visits and study setting. the number of clinical visits was more important in tx as compared with zjg. also, the relatively small sample size in zjg may reduce the statistical power, resulting in nonsignificant findings. some limitations existed in this study. first, we selected zjg and tx as the study sites representing areas using the integrated model and tb dispensary model. however, the socioeconomic status and local health system may also influence a patient's direct and indirect costs. the cost differences between these two areas could not be completely attributed to the disparity of tuberculosis management models. second, although we estimated the expenses by interviewing patients and their family members and checking medical records, recall bias should not be neglected. third, the mean costs may not be reflective of the median. considering the non-normal distribution of the costs, we used the mean and median (interquartile range, 25%-75%) to describe a patient's economic burden. we also applied a non-parametric test to compare the cost differences between groups. a logarithmic transformation was performed to normalize the distribution when we performed a step-by-step multivariate linear regression analysis. in this study, we revealed several common and specific factors affecting the economic burden of patients' with tuberculosis. health policy makers should consider these factors by enhancing financial support, strengthening health facilities and involving human resources to achieve success in tuberculosis control. tuberculosis control in china is a long-term, public health challenge and needs the support of affordable and sustainable health resources. community health resources within a strengthened health system might be the best approach [38] . evidence-based measures to improve healthcare-seeking behavior, reduce patient and detection delays, address financial and system barriers, improve the quality of direct observed therapy and increase the access to health promotion are urgently needed [39] . policy makers need to further document their challenges when implementing tuberculosis management models [21] . under the current "free diagnosis and treatment" policy, the financial burden remains heavy on tuberculosis patients. policy makers need to consider appropriate steps to lessen the burden of out-of-pocket costs for tuberculosis patients in china and how best to improve service delivery for poor patients. global tuberculosis report 2013: world health organization multidrug-resistant tuberculosis around the world: what progress has been made? epidemiology of anti-tuberculosis drug resistance in a chinese population: current situation and challenges ahead economic support to improve tuberculosis treatment outcomes in south africa: a pragmatic cluster-randomized controlled trial economic impact of pulmonary tuberculosis on patients and their families of dharan municipality, nepal perception and social consequences of tuberculosis: a focus group study of tuberculosis patients in sialkot dots in china-removing barriers or moving barriers? progress in tuberculosis control and the evolving public-health system in china adverse reactions due to directly observed treatment strategy therapy in chinese tuberculosis patients: a prospective study analysis of the economic burden of diagnosis and treatment of tuberculosis patients in rural china barriers to tb care for rural-to-urban migrant tb patients in shanghai: a qualitative study tuberculosis patient expenditure on drugs and tests in subsidised, public services in china: a descriptive study effective reimbursement rates of the rural health insurance among uncomplicated tuberculosis patients in china tuberculosis control in china: striving for sustainability how affordable are tuberculosis diagnosis and treatment in rural china? an analysis from community and tuberculosis patient perspectives perceptions and experiences of health care seeking and access to tb care-a qualitative study in rural jiangsu province adherence to anti-tuberculosis treatment among pulmonary tuberculosis patients: a qualitative and quantitative study why india should become a global leader in high-quality, affordable tb diagnostics revenue-driven in tb control-three cases in china patient medical costs for tuberculosis treatment and impact on adherence in china: a systematic review china tuberculosis policy at crucial crossroads: comparing the practice of different hospital and tuberculosis control collaboration models using survey data comparing patient care seeking pathways in three models of hospital and tb programme collaboration in china tuberculosis and poverty: the contribution of patient costs in sub-saharan africa-a systematic review patients are paying too much for tuberculosis: a direct cost-burden evaluation in burkina faso early appraisal of china's huge and complex health-care reforms perceptions of tuberculosis and health seeking behaviour in rural inner mongolia equity in health and health care: the chinese experience persistent problems of access to appropriate, affordable tb services in rural china: experiences of different socio-economic groups factors affecting patient delay of diagnosis and completion of direct observation therapy, short-course (dots) among the migrant population in shandong delays in diagnosis and treatment of pulmonary tuberculosis in india: a systematic review factors associated with patient, and diagnostic delays in chinese tb patients: a systematic review and meta-analysis can hospitalization provide better compliance in smear positive tuberculosis patients? perspectives on tuberculosis among traditional chinese medical practitioners in new york city's chinatown drugs and herbs given to prevent hepatotoxicity of tuberculosis therapy: systematic review of ingredients and evaluation studies experiences in anti-tuberculosis treatment in patients with multiple previous treatments and its impact on drug resistant tuberculosis epidemics direct and indirect costs of tuberculosis among immigrant patients in the netherlands a sustainable agenda for tuberculosis control and research are we doing enough to stem the tide of acquired mdr-tb in countries with high tb burden? results of a mixed method study in chongqing i would like to thank dr. stefanie röhrs (institute of tropical medicine & international health, charité -universitätsmedizin berlin) for her editorial support on this manuscript. key: cord-311941-0dpm35dd authors: jones, bryony a.; sauter-louis, carola; henning, joerg; stoll, alexander; nielen, mirjam; van schaik, gerdien; smolenaars, anja; schouten, matthijs; den uijl, ingrid; fourichon, christine; guatteo, raphael; madouasse, aurélien; nusinovici, simon; deprez, piet; de vliegher, sarne; laureyns, jozef; booth, richard; cardwell, jackie m.; pfeiffer, dirk u. title: calf-level factors associated with bovine neonatal pancytopenia – a multi-country case-control study date: 2013-12-02 journal: plos one doi: 10.1371/journal.pone.0080619 sha: doc_id: 311941 cord_uid: 0dpm35dd bovine neonatal pancytopenia (bnp), a high fatality condition causing haemorrhages in calves aged less than 4 weeks, was first reported in 2007 in germany and subsequently observed at low incidence in other european countries and new zealand. a multi-country matched case-control study was conducted in 2011 to identify calf-level risk factors for bnp. 405 bnp cases were recruited from 330 farms in belgium, france, germany and the netherlands by laboratory confirmation of farmer-reported cases. up to four calves of similar age from the same farm were selected as controls (1154 calves). risk factor data were collected by questionnaire. multivariable modelling using conditional logistic regression indicated that pregsure®bvd (pregsure, pfizer animal health) vaccination of the dam was strongly associated with bnp cases (adjusted matched odds ratio amor 17.8 first lactation dams; 95% confidence interval – ci 2.4, 134.4; p = 0.005), and second or more lactation pregsure-vaccinated dams were more likely to have a case than first lactation vaccinated dams (amor 2.2 second lactation; ci 1.1, 4.3; p = 0.024; amor 5.3 third or more lactation; ci 2.9, 9.8; p = <0.001). feeding colostrum from other cows was strongly associated with bnp if the dam was not pregsure-vaccinated (amor 30.5; ci 2.1, 440.5; p = 0.012), but the effect was less if the dam was pregsure-vaccinated (amor 2.1; ci 1.1, 4.0; p = 0.024). feeding exclusively dam’s milk was a higher risk than other types of milk (amor 3.4; ci 1.6, 7.5; p = 0.002). the population attributable fractions were 0.84 (ci 0.68, 0.92) for pregsure vaccination, 0.13 (ci 0.06, 0.19) for feeding other cows’ colostrum, and 0.15 (ci 0.08, 0.22) for feeding dam’s milk. no other calf-level factors were identified, suggesting that there are other important factors that are outside the scope of this study, such as genetics, which explain why bnp develops in some pregsure-colostrum-exposed calves but not in others. bovine neonatal pancytopenia (bnp) was first reported in 2007 in germany and subsequently observed at low incidence in several other european countries and new zealand [1] [2] [3] [4] . it affects calves aged up to 4 weeks, causing skin and internal haemorrhages, prolonged haemorrhage from wounds or orifices, and high case fatality (90%) [5] [6] [7] . no evidence of an infectious, toxic or genetic aetiology has been found [2, [5] [6] [7] . in 2010 an association was suspected between affected calves and vaccination of their dams with pregsurehbvd (pregsure, pfizer animal health) [8] . bnp was induced experimentally by feeding calves colostrum from unrelated pregsure-vaccinated dams that had previously given birth to bnp calves [9] . it was hypothesized that maternal antibodies in colostrum were destroying calf blood and bone marrow cells. pregsure is an inactivated bovine viral diarrhoea (bvd) virus type 1 vaccine, which was first marketed in 2004 [10] . the manufacturer, pfizer animal health, voluntarily stopped sales to wholesalers in europe in 2010, and new zealand in 2011, pending investigations into the cause of bnp. the marketing authorisations in all concerned european union (eu) member states were suspended in august 2010 following an eu commission decision based on recommendations from the european medicines agency committee for veterinary medicinal products (cvmp), with recall of product at wholesale level [11] . by the end of august 2012, 6913 suspected bnp cases had been reported by farmers, veterinarians and laboratories via the pharmacovigilance system in each european member state to the marketing authorization holder, pfizer animal health. the numbers of reported suspected cases decreased in 2011 and 2012 compared with previous years. this case-control study was conducted to identify potential risk factors for bnp occurrence at the calf level. all procedures on animals used in this study were in concordance with the ethical conditions for animal experimentation as mentioned in the european legislation (directive 86/609/ eec). the blood samples collected from calves on the farms were taken for diagnostic purposes at the request of the owner as part of clinical veterinary practice and therefore were not considered to be experimental, so no formal approval of the protocol by an ethical committee in any of the four countries was required. the study was conducted between january and december 2011 in four countries that had experienced a high number of bnp cases since 2007; belgium, france, germany and the netherlands. the target was to recruit 400 cases and match them to 2-4 control calves of a similar age from the same farm. a multi-country design was required to obtain sufficient cases and to recruit cases from different management systems. a standard procedure for recruitment of cases and controls was developed jointly by the country research teams. affected farms were identified from suspected cases reported voluntarily by farmers and veterinarians to the research teams in the respective country. case-reporting was encouraged via notices in the national veterinary and farming press in each country, asking for cases of calves aged less than one month old with one or more signs of bleeding from the skin (either spontaneously or from injection or ear tag sites), mucosal petechial haemorrhages, blood in diarrhoea, or death with internal or external bleeding. free diagnostic testing and/or post mortem examination were offered. all farms that reported suspected cases were visited by a veterinarian, either from the research team or their own veterinarian on behalf of the research team, who conducted a clinical examination. if the calf was less than 29 days of age and had one or more clinical signs of bnp; multiple skin haemorrhages, melena, petechiation of mucous membranes, or sudden death with internal haemorrhage, then it was included in the study as a suspected case. in order to confirm the diagnosis a whole blood sample was collected if the calf was alive, or if the calf was dead then a post mortem examination and bone marrow histopathology were performed. during the same visit, up to four calves without bnp clinical signs and aged 10-28 days were selected from the same farm to be matched controls. whole blood samples were collected to verify that they did not have abnormal haematology. the veterinarian collected data on the characteristics of suspected case and the unaffected calves by face-to-face questionnaire-based interview (questionnaire s1). a case was therefore defined as a calf that had developed one or more bnp clinical signs on or before 28 days of age and had bone marrow depletion on histopathology and/or had thrombocytopenia (,150610 9 /litre) and leucopenia (,5610 9 /litre). a control was defined as a calf on the same farm as a case, aged 10-28 days at the time of case reporting, no clinical signs of bnp up to 28 days of age, and normal blood results (thrombocytes . = 300610 9 / litre, leucocytes . = 5610 9 /litre). farmers were contacted again when control calves were 28 days of age to confirm that they had not developed bnp signs. if the laboratory results subsequently indicated that a suspected case did not meet the case definition then the case and its matched controls were excluded from the study, and if a control calf did not have normal blood results then it was excluded from the study. the questionnaire was developed in english, translated into french and german, and field-tested by researchers with experience of bnp cases from the four countries. in the netherlands and belgium the english questionnaire was used and the interview conducted in dutch. there were 91 questions, of which 36 collected descriptive data on calf, dam and sire identification, calf characteristics, clinical signs and laboratory results, and 55 collected data on potential risk factors related to colostrum and milk feeding, dam and sire characteristics, and dam vaccination history. data were entered into an internet-based form created in open source software (lime-survey http://www.limesurvey.org/), exported to microsoft excel and then to stata ic 12.1 for coding, cleaning and analysis. thirty (6 descriptive, 24 potential risk factor) questions were dropped due to a low number of responses or differences in interpretation between countries. five variables identified the calf, dam and sire. twenty two variables describing clinical and post-mortem signs and laboratory results were used to define cases and controls. thirty four variables were used in the statistical analysis and an additional 15 variables were created by recoding, to give a total of 49 exposure variables (3 descriptive and 46 potential risk factor). due to the matched design, conditional logistic regression with farm as the matching variable was used for univariable analysis to obtain matched odds ratios (mor), 95% confidence intervals (ci) and wald test p values. it was first conducted on the dataset of 1559 calves, but due to missing observations the sample size for each variable was different. variables with greater than 30% missing observations were excluded. the number of calves in the final multivariable model was 1296 due to missing observations in the retained variables, so univariable analysis was repeated with the smaller dataset. variables with p values greater than 0.2 in univariable analysis were excluded from multivariable analysis. pair-wise associations between the exposure variables were examined by the chi-squared test, and polychoric correlation was used to check for collinearity. multivariable analysis was conducted using conditional logistic regression with farm as the group variable. the model was built using forward stepwise regression starting with the variable with the highest odds ratio and lowest p value on univariable analysis. variables were retained if the likelihood ratio test indicated that inclusion led to a better model fit (p,0.05). for variables from the same risk factor group (e.g. colostrum management, dam vaccination) that were likely to be collinear, the variable with the highest number of observations was added first. it was then removed and the other collinear variables were added and removed one by one. if more than one of the collinear variables improved the model then the one with the most observations and/ or that was most biologically relevant was retained, as described below. the population attributable fraction, paf (proportion of cases in the total population that would be avoided if the exposure was removed, assuming the exposure is causal), was estimated using the punafcc package for stata [12] . data were collected for 502 suspected cases and 1583 potential controls from 410 farms. however, 62 suspected cases did not fit the case definition, 122 potential controls had incomplete blood results, and 210 potential controls had ''atypical'' blood results (thrombocytes ,300610 9 /litre and/or leucocytes ,5610 9 /litre). exclusion of these led to the loss of 97 confirmed controls with no matching case and 35 confirmed cases with no matching control. the remaining 405 confirmed cases were matched to 1154 confirmed controls from 330 farms with an overall case control ratio of 1:2.8 ( table 1 ). the proportions of dairy, beef and mixed farms that were included in the study from each country approximately reflected the proportions of those types of farms in each country. in the netherlands, dairy farms predominate so almost all suspected calves came from dairy farms. in germany a high proportion of cases recruited were from the south where beef farms are few so the high proportion of dairy farms reflects the regional distribution. the number of cases per farm varied from 1 to 3 and the number of controls from 1 to 9 (table s1 ). the proportion of males amongst cases (41%) was higher than amongst controls (34%). in the netherlands, cases were more likely than controls to be male probably due to the sale of male calves before one month of age so fewer were available to be selected as controls. this difference was not observed in the other countries. the most common breed of calf was holstein-friesian or red holstein-friesian (56.0%), followed by fleckvieh (13.7%), belgian blue (7.0%), charolais (5.9%), other pure beef or dairy breeds (7.8%), and crossbreeds (9.6%) (table s2 ). there was no evidence of a difference in breed distribution between cases and controls. colostrum management ( table 2, table s3 ). there was no evidence of an association between case/control status and the following variables: suckled dam within 12 hours of birth, time to first colostrum administration, number of times colostrum received in first 24 hours, artificial colostrum used. there were increased odds of exposure of cases compared with controls for total colostrum received, colostrum obtained from cow(s) different from dam, pooled colostrum, pooled colostrum includes colostrum of dam, and frozen colostrum. these variables were associated with each other so colostrum obtained from cow(s) different from dam was included first in the multivariable model from this group because it had the highest number of observations, and pooled colostrum, pooled colostrum includes colostrum of dam and frozen colostrum were nested within it. milk feeding ( table 2, table s3 ). there were increased odds of exposure of cases compared with controls for feeding milk powder, raw milk from dam, raw milk from dam only, and type of milk fed. there was no evidence of an association between case/control status and feeding raw milk, bulk milk, milk from cows with high somatic cell count/clinical mastitis or withdrawn/discarded milk. type of milk fed was included first in the multivariable model from this group because it provided the most information. dam and sire characteristics ( table 3, table s4 ). there was no evidence of an association between case/control status and dam breed, dam was born on farm, dam was reared at another farm, or source of bull. case dams (dams of cases) had increased odds of being in second or more lactation rather than first lactation compared with control dams (dams of controls). case dams had 12 times the odds of previously giving birth to a bnp calf compared to control dams (mor 12.02; ci 5.44, 26.57; p = ,0.001) since previously giving birth to a bnp calf was considered to be on the causal pathway between other exposures and the outcome, it was not included in the multivariable model. lactation number was correlated with many of the vaccination variables and was a potential confounder of the association between dam vaccination and case/control status. cases had increased odds of having a fleckvieh rather than a holstein-friesian or red holstein-friesian sire, compared with controls. it was not possible to obtain estimates for all breeds because of low numbers of observations. dam bvd vaccination (table s5) . case dams had increased odds of being vaccinated against bvd compared with control dams, and increased odds of having received more doses of bvd vaccine. there was no evidence of any difference in the timing of last bvd vaccination before calving between case and control dams. bvd vaccination variables (all types of vaccine) were correlated with each other, as well as with specific bvd vaccine variables. they were not used in the multivariable model because the specific bvd vaccine variables provided more information. dam pregsure vaccination (table 4, table s5 ). case dams had increased odds of being pregsure-vaccinated compared with control dams. case dams were more likely to have received their last dose of pregsure longer before calving, and to have received more doses of pregsure, compared with control dams. pregsure vaccination variables were correlated with each other so they were added to the model separately. the responses to dam pregsure-vaccinated were more reliable than no. doses pregsure because some farmers reported the initial two doses as a single dose, and for some dams the number of doses was unknown. first lactation cows were less likely to have received pregsure (49%) compared with second (14%) and third or more lactation cows (12%), and were more likely to have received fewer doses of pregsure than second or third or more lactation cows (table s6 ). the polychoric correlation coefficient between pregsure doses and lactation number was 0.61. other dam bvd vaccinations ( table 4, table s5 ). after pregsure, bovilis bvd (msd animal health) was the most commonly used bvd vaccine, followed by rispoval 3 (containing bvd, respiratory syncytial virus -rs, and parainfluenza type 3, pfizer animal health) and bovidec bvd (novartis animal health). case dams were more likely to have received bovilis bvd, bovidec bvd and mucosiffa (merial) than control dams. there was no evidence of an association between case/control status and rispoval bvd, rispoval rs-bvd, rispoval 3 or mucobovin (merial) vaccination of dams. some of these variables were correlated with bvd vaccination (all types of vaccine) and pregsure variables. there were 26 bvd vaccine combinations and 34 bvd vaccination sequences (tables s7, s8 ). the most common combination was pregsure and bovilis bvd (22%) and the most common sequence of bvd vaccines was pregsure followed by bovilis bvd (21%). when the bvd vaccine combinations were regrouped into five categories (table s5) , case dams were more likely to have received pregsure in combination with other bvd vaccines than pregsure only, compared with control dams. when the bvd vaccine sequence variable was regrouped into eight categories (table s5) , case dams had increased odds of having received one or more other bvd vaccine then pregsure, or receiving one or more other bvd vaccine, then pregsure, then one or more other bvd vaccine, rather than pregsure only, compared with control dams. these variables were correlated with the other bvd vaccination variables so they were added separately to the model. other dam vaccinations (table 4, table s5 ). case dams had increased odds of having received bluetongue or ibr vaccination, and being vaccinated against more diseases, compared with control dams. there was no association between case/ control status and dams receiving rota/coronavirus or other vaccines. these variables were correlated with each other and with some of the other vaccine variables. four variables were retained in the final model: dam pregsurevaccinated, colostrum from different cow(s), lactation number, and raw milk from dam only ( table 5, table s9 ). no. doses pregsure, and combinations and sequences of bvd vaccines were alternatives to dam pregsure-vaccinated, and frozen colostrum, pooled colostrum and pooled colostrum including dam's were alternatives to colostrum from different cow(s), but they had fewer observations. there was evidence of interaction between dam pregsurevaccinated and lactation number, between dam pregsure-vaccinated and colostrum from different cow(s), and between lactation number and colostrum from different cow(s). including two-way interactions between dam pregsure-vaccinated and lactation number and between dam pregsure-vaccinated and colostrum from different cow(s) was a better fit than the models with single interactions or all three interactions. the odds of a case having a second lactation dam were five times the odds of having a first lactation dam, if the dam was pregsurevaccinated (interaction term adjusted matched odds ratio -amor 4.8; ci 1.1, 20.7; p = 0.034), and the odds of having a vaccinated third or more lactation dam were 7 times the odds of having a vaccinated first lactation dam (interaction term amor 7.4; ci 1.9, 28.9; p = 0.004). the odds of a case having received colostrum from different cow(s) were 90% lower than the odds of not having received colostrum from other cows, if the dam was pregsurevaccinated (interaction terms amor 0.1; ci 0.01, 0.95; p = 0.046). there was very strong evidence of an association between case/ control status and having a pregsure-vaccinated dam. a case had 18 times the odds of being born to a dam that was pregsurevaccinated rather than unvaccinated compared with a control, if the dam was first lactation and the calf did not receive colostrum from other cows, adjusting for type of milk fed (amor 17.8; ci. 2.4, 134.4; p = 0.005). for calves that received colostrum from , and a case with a third or more lactation dam that did not receive colostrum from other cows had 132 times the odds of having a pregsure-vaccinated dam rather than an unvaccinated dam (amor 132.0; ci 9.9, 1764.7; p = ,0.001), adjusting for type of milk fed. for calves with unvaccinated dams, there was no evidence of a difference in the odds of a case being born to a dam in first, second or third or more lactation compared with a control, adjusting for source of colostrum and type of milk fed (second lactation amor 0.5; ci 0.1, 1.6; p = 0.23, third or more lactation amor 0.7; ci 0.2, 2.4; p = 0.60). however, if the dam was pregsure-vaccinated, a case had twice the odds of having a second lactation dam (amor 2.2; ci 1.1, 4.3; p = 0.024) and 5 times the odds of having a third lactation dam (amor 5.3; ci 2.9, 9.8; p = ,0.001) compared with a first lactation dam, adjusting for source of colostrum and type of milk fed. a case had 30 times the odds of having received colostrum from another dam compared with a control, if its dam was not pregsure-vaccinated (amor 30.5; ci 2.1, 440.5; p = 0.012), but had only twice the odds of having received colostrum from another dam if its dam had been pregsure-vaccinated, adjusting for lactation number and type of milk fed (amor 2.1; ci 1.1, 4.0; p = 0.024). a case had 3 times the odds of having been fed raw milk only from its dam rather than other types of milk (with or without dams milk) compared with a control (amor 3.4; ci 1.6, 7.5; p = 0.002) when adjusting for dam pregsure-vaccination, lactation number and source of colostrum. the population attributable fraction (paf) for dam pregsure vaccination was 0.84 (ci 0.68, 0.92) indicating that if no pregsurevaccinated cows had been used for breeding then 84% of cases would have been avoided. if calves had been fed colostrum from their own dams only, rather than colostrum from other cows, then 12% of cases would have been avoided (paf 0.13; ci 0.06, 0.19). if calves had not been fed exclusively on their dam's milk, then 15% of cases would have been avoided (paf 0.15; ci 0.08, 0.22). these estimates are based on the assumption of a causal relationship between each variable and case/control status. of the 440 confirmed bnp cases in this study, 20 cases (4.5%) had dams that were not pregsure-vaccinated and had not previously had a bnp calf, had received only dam's colostrum, and came from farms with no history of pregsure vaccination or bnp cases. our results show that pregsure vaccination of a cow was strongly associated with her having a bnp calf, and that older pregsure-vaccinated cows were more likely to have a bnp calf than younger vaccinated cows. this was partly explained by the increased odds of bnp with increasing doses of pregsure and correlation between pregsure doses and lactation number. feeding colostrum from other cows, in addition to or instead of dam colostrum, was strongly associated with bnp if the dam had not been pregsure-vaccinated, but its effect was less if the dam had been vaccinated. when calves received colostrum from other cows, most farmers were unable to identify which cows the colostrum came from, so their pregsure vaccination status was unknown. these findings suggest that if a dam had been pregsurevaccinated and the calf received colostrum from its dam then there was an increased odds of bnp. but if the calf received some colostrum from other cows (which may or may not have been vaccinated) then the dilution effect of receiving some ''non-bnp colostrum'' reduced the odds. however, for calves of unvaccinated cows, feeding colostrum from other cows was strongly associated with bnp, presumably because feeding colostrum from multiple cows increased the chance of the calf ingesting some colostrum from a pregsure-vaccinated cow. the strong association between bnp and pregsure vaccination, and consumption of colostrum from pregsure-vaccinated dams, was consistent with singlecountry case-control studies with small sample sizes conducted in the uk and germany [13, 14] . we also found that exclusively feeding dam's raw milk was associated with an increased odds of bnp, compared to feeding milk from one or more other sources. early lactation milk contains some antibodies that can still be absorbed up to 48 hours after birth [15] . feeding milk powder or bulk tank milk instead of, or to supplement, dam's milk will reduce the amount of antibody ingested. alternatively the observed effect could be due to an association between feeding dam's milk and other management factors that affect the risk of bnp. in our study, calves that suckled their dam within 12 hours of birth were more likely to be fed only dam's raw milk. we hypothesize that calves that suckle could receive a larger volume of colostrum earlier in life leading to higher levels of antibody absorption, which could increase the risk of bnp. the estimated population attributable fractions indicate that the most effective intervention would have been to avoid breeding from pregsure-vaccinated cows (86% case avoided). not exclusively feeding dam's milk would have avoided 15% cases and not feeding colostrum from other cows would have avoided 12%. twenty cases in this study had no apparent exposure to the identified risk factors. they could have been misclassified with respect to dam vaccination status, colostrum feeding or dam's bnp history due to incorrect farmer recall, or they could have developed pancytopenia due to other causes. prior to the identification of bnp there were sporadic cases of unexplained pancytopenia in young calves [16] [17] [18] [19] , so some of our 20 calves could represent a background incidence of pancytopenia that is unrelated to ingestion of colostrum from pregsure-vaccinated cows. further research is required to determine whether sporadic unexplained pancytopenia cases have the same pathogenesis as pregsure-associated bnp cases, and therefore whether the introduction of pregsure vaccination has increased the incidence of an existing but rare syndrome. despite widespread use of pregsure vaccine prior to its withdrawal, bnp incidence has been low, suggesting that consumption of colostrum from a pregsure-vaccinated dam is not a sufficient cause of bnp. we did not identify any other important calf or management-related risk factors. research into bnp pathogenesis is on-going with a prevailing hypothesis that it is a neonate-maternal incompatibility phenomenon related to pregsure-induced maternal alloantibodies against bovine cell surface molecules due to bioprocess-related impurities from the cell line used for virus propagation [20] [21] [22] . bastian et al. [20] showed that sera of dams that had previously had a bnp calf contained alloantibodies that bound to bovine leucocytes, and foucras et al [23] reproduced bnp in healthy calves by transferring serum antibodies from pregsure-vaccinated dams. animals vaccinated with three doses of pregsure had higher alloantibody titres compared with animals receiving a single pregsure dose or other bvd vaccines [10] . this supports our finding that the odds of bnp increase with the number of pregsure doses given to the dam. bridger et al [24] showed that maternal alloantibodies to surface antigens of neonatal leucocytes are transferred via colostrum from bnp dams to neonatal calves, and higher antibody titres in the dam led to more severe clinical signs in the calf. various proteins found in both pregsure vaccine and the cell line used to produce the vaccine have been implicated as possible alloantigen candidates [21] [22] [23] 25] . bell et al. [26] suggest that feeding bnp colostrum from multiple cows increases the likelihood that the colostrum will contain antibodies that will react with most calf allotypes, which fits with our finding that calves from unvaccinated dams were at increased risk of bnp if they were fed colostrum from other cows. they suggest that the unique adjuvant in pregsure could amplify production of alloantibody against vaccine antigens as well as boosting pregnancy-induced maternal alloantibodies against paternallyderived foetal mhc antigens [26] . the latter mechanism may explain the aetiology of some of the sporadic cases of unexplained pancytopenia that are not linked to the ingestion of colostrum from pregsure-vaccinated cows. it was anticipated that bnp incidence would decline during the study period due to withdrawal of pregsure from distribution in 2010, and advice given to some farmers to avoid feeding colostrum from cows that had previously had bnp calves. a multi-country study was therefore necessary to obtain sufficient bnp cases to have the statistical power to detect important risk factors. a casecontrol study is the most appropriate design to investigate a rare disease, and for the investigation of calf-level risk factors it was necessary to match by farm. this meant that most management factors were the same for both cases and controls on the same farm, and, even where there were differences, the farmer could have reported routine practices rather than what had happened to individual calves, leading to misclassification and an underestimation of effect. the multi-country design meant that there was variation between countries in case-reporting, farm visits, sample collection, post-mortem examination, laboratory testing and questionnaire interpretation. there were also differences between countries in production methods, cattle breeds, and policies on bvd control and vaccination programmes. the matched design minimised the effect of these country differences on our results. case recruitment relied on passive reporting by farmers and veterinarians, strengthened by a communication campaign to encourage reporting of cases and provision of free post mortem examination and laboratory testing. the reasons for a farmer or veterinarian not reporting a case might include; being unaware of the invitation to report cases or too busy to report, or having reported cases prior to the study and therefore seeing no benefit in reporting further cases. it is possible that farmers who report disease are more likely to take preventive measures such as vaccination. there is therefore a potential bias in selection of case farms in that the study population might not be representative of all bnp-affected farms in the four countries. but even if these biases were present, they are unlikely to have biased the particular inferences reported here. strict definitions for case and control calves were applied due to variations between countries in time elapsed before blood analysis and use of multiple laboratories, to minimise the inclusion of false positive cases. using the thrombocyte and leucocyte values of the netherlands control calves, a 95% reference interval was calculated based on the mean and standard deviation, where values below the lower limit of the interval were considered abnormal for the current study. using these results the blood values for controls were set at .300610 9 /litre thrombocytes and .5610 9 /litre leucocytes. for case calves, the thrombocyte value was set at ,150610 9 /litre, the lower threshold of the reference range in the netherlands. the number of questions in the questionnaire was high, to capture management practices in the diverse farming systems of the four countries, which increased the risk of detecting an association by chance when there was no true association. as the questionnaire had been translated into three languages and administered by a range of veterinarians and researchers, prior to data analysis the research team discussed in detail how the questionnaire had been administered and differences in interpretation, and there was regular consultation during analysis to inform the interpretation of results. in conclusion, this multi-country study provides strong evidence that receiving colostrum from a pregsure-vaccinated cow is a major risk factor for bnp. if calves are only given colostrum from unvaccinated cows then it is highly unlikely that a calf will develop bnp. the study design and sample size provided adequate statistical power to investigate many hypotheses related to farming practices such as colostrum management, calf feeding and vaccination, but no other important calf management-related risk factors were identified. this suggests that there are other important factors, such as genetics, that were outside the scope of this study, which explain why bnp develops in some pregsure-colostrumexposed calves but not in others. these require further investigation. questionnaire s1. (docx) sera from dams of calves with bovine neonatal pancytopenia contain alloimmune antibodies directed against calf leukocytes a genetic predisposition for bovine neonatal pancytopenia is not due to mutations in coagulation factor xi bovine neonatal pancytopenia (03): new zealand archive number proceedings of satellite symposium on haemorrhagic diathesis in calves increased incidence of haemorrhagic diathesis as a result of bone marrow damage in young calves) bone marrow depletion with haemorrhagic diathesis in calves in germany: characterization of the disease and preliminary investigations on its aetiology haemorrhagic diathesis in neonatal calves: an emerging syndrome in europe bovine neonatal pancytopenia -germany: vaccinal etiology suspected. archive number ingestion of colostrum from specific cows induces bovine neonatal pancytopenia (bnp) in some calves effect of the vaccination scheme on pregsure(r) bvd induced alloreactivity and the incidence of bovine neonatal pancytopenia opinion following an article 78 procedure for pregsure bvd and associated names 2013 in press) attributable and unattributable risks and fractions and other scenario comparisons factors associated with bovine neonatal pancytopenia (bnp) in calves: a casecontrol study case control study to investigate risk factors for bovine neonatal pancytopenia (bnp) in young calves in southern germany colostral immunoglobulin transfer in calves i. period of absorption bone marrow aplasia with pancytopenia and hemorrhage in a japanese black calf pancytopenia associated with bone marrow aplasia in a holstein heifer pancytopenia with bleeding tendency associated with bone marrow aplasia in a holstein calf presumptive bovine neonatal pancytopenia in a holstein calf in quebec bovine neonatal pancytopenia: is this alloimmune syndrome caused by vaccineinduced alloreactive antibodies? vaccineinduced antibodies linked to bovine neonatal pancytopenia (bnp) recognize cattle major histocompatibility complex class i (mhc i) bovine neonatal pancytopenia -comparative proteomic characterization of two bvd vaccines and the producer cell surface proteome (mdbk) alloantibodies against mhc class i: a novel mechanism of neonatal pancytopenia linked to vaccination detection of colostrum-derived alloantibodies in calves with bovine neonatal pancytopenia demonstration of early functional compromise of bone marrow derived hematopoietic progenitor cells during bovine neonatal pancytopenia through in vitro culture of bone marrow biopsies reproduction of bovine neonatal pancytopenia (bnp) by feeding pooled colostrum reveals variable alloantibody damage to different haematopoietic lineages the authors gratefully acknowledge the contributions of the farmers and veterinarians who participated in this study. key: cord-323433-9km824uh authors: van den wijngaard, cees c.; van asten, liselotte; van pelt, wilfrid; doornbos, gerda; nagelkerke, nico j. d.; donker, gé a.; van der hoek, wim; koopmans, marion p. g. title: syndromic surveillance for local outbreaks of lower-respiratory infections: would it work? date: 2010-04-29 journal: plos one doi: 10.1371/journal.pone.0010406 sha: doc_id: 323433 cord_uid: 9km824uh background: although syndromic surveillance is increasingly used to detect unusual illness, there is a debate whether it is useful for detecting local outbreaks. we evaluated whether syndromic surveillance detects local outbreaks of lower-respiratory infections (lris) without swamping true signals by false alarms. methods and findings: using retrospective hospitalization data, we simulated prospective surveillance for lri-elevations. between 1999–2006, a total of 290762 lris were included by date of hospitalization and patients place of residence (>80% coverage, 16 million population). two large outbreaks of legionnaires disease in the netherlands were used as positive controls to test whether these outbreaks could have been detected as local lri elevations. we used a space-time permutation scan statistic to detect lri clusters. we evaluated how many lri-clusters were detected in 1999–2006 and assessed likely causes for the cluster-signals by looking for significantly higher proportions of specific hospital discharge diagnoses (e.g. legionnaires disease) and overlap with regional influenza elevations. we also evaluated whether the number of space-time signals can be reduced by restricting the scan statistic in space or time. in 1999–2006 the scan-statistic detected 35 local lri clusters, representing on average 5 clusters per year. the known legionnaires' disease outbreaks in 1999 and 2006 were detected as lri-clusters, since cluster-signals were generated with an increased proportion of legionnaires disease patients (p:<0.0001). 21 other clusters coincided with local influenza and/or respiratory syncytial virus activity, and 1 cluster appeared to be a data artifact. for 11 clusters no likely cause was defined, some possibly representing as yet undetected lri-outbreaks. with restrictions on time and spatial windows the scan statistic still detected the legionnaires' disease outbreaks, without loss of timeliness and with less signals generated in time (up to 42% decline). conclusions: to our knowledge this is the first study that systematically evaluates the performance of space-time syndromic surveillance with nationwide high coverage data over a longer period. the results show that syndromic surveillance can detect local lri-outbreaks in a timely manner, independent of laboratory-based outbreak detection. furthermore, since comparatively few new clusters per year were observed that would prompt investigation, syndromic hospital-surveillance could be a valuable tool for detection of local lri-outbreaks. the sars epidemic in 2003, the bioterrorism attacks in 2001, and the ongoing threat of new infectious disease outbreaks have prompted many countries to invest in their capacity to respond timely to emerging infectious disease outbreaks, as early outbreakdetection may well mitigate their impact. as a result, new surveillance systems for earlier detection have been implemented, often labeled ''syndromic surveillance'' [1] [2] [3] [4] [5] [6] . these systems use increased reporting of critical symptoms or clinical diagnoses as early indicators of infectious disease outbreaks. this not only allows monitoring of clinical syndromes before laboratory diagnoses have been made, but also allows detection of outbreaks of diseases for which no diagnostics were requested or available (including emerging pathogens). geographic analysis methods -such as space-time scan statistics -may further increase the sensitivity of syndromic surveillance for detection of local outbreaks or of regional differences in regular seasonal epidemic diseases [2, 6] . in the sars outbreak in hongkong in 2003, it is believed that a near real-time space-time analysis would have detected the highly unusual clustering of severe acute respiratory syndrome cases much sooner [7] . however, concerns exist about the specificity of space-time syndromic surveillance, i.e. that it might generate many false signals [8, 9] . the objective of this study was to evaluate to what extent syndromic surveillance detects local outbreaks of lower-respiratory infections (lris) without swamping true signals by false alarms. using retrospective hospitalization data, we simulated prospective space-time syndromic surveillance for lri-elevations. the two largest outbreaks of legionnaires' disease in the netherlands in the last decade were used as ''positive controls'' to test whether these known outbreaks would have been detected by space-time signals in lri data. to assess other (likely) causes for detected lrielevations, we examined regional increases in the reported incidence of influenza-like-illness (ili), hospital discharge diagnoses for respiratory illnesses and age group distributions for lri cases. we also evaluated whether the number of generated spacetime signals can be reduced by restricting the time and spatial windows for the analyses. since we only used anonymous data from existing medical research and surveillance registries, neither formal ethics committee approval nor informed consent from the patients were required. lri-syndrome data (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) hospitalization data were collected from the dutch national medical register (discharge and secondary diagnoses by date of hospitalization for 1999-2006). in 1999-2004 this registry had a 99% coverage (16 million pop.) and in 2005/6 approximately 80%, after exclusion of hospitals with incomplete data for those years. we included all records on hospitalizations with any kind of lri as either discharge or secondary diagnosis, under the assumption that this reflects prospective classification of patients with a lower respiratory infection in a ''lri-syndrome'' on the day of hospitalization. icd-9-cm (international classification of diseases, 9th revision, clinical modification) codes for a lri syndrome were selected from the cdc respiratory syndrome codes-list (centers for disease control and prevention, usa, http://www.bt.cdc.gov/surveillance/syndromedef/; and see appendix s1). after excluding duplicate hospitalizations of the same patient within 6 weeks (5% excluded), 222638 records were included for 1999-2004, and 68124 for 2005-2006. data were aggregated by hospitalization date, postal-code and age group (0-4, 5-19, 20-49, 50-64, $65 years). since higher levels of spatial resolution can result in more sensitive detection of outbreaks [10, 11] we used 4-digit postal-codes (4023 areas in a 16 million population), which provide the highest level of spatial resolution available within privacy regulations. regional ili-surveillance data ili-data were collected from a sentinel network of general practitioners (gps, continuous morbidity registration centres, cmr sentinel stations, 1% population coverage) [12] ). the ilicounts and underlying gp-practice populations were aggregated by region and week. the gp-practice populations were corrected for weeks that specific gp-practices did not supply data. due to the small number of gp-practices in some parts of the country, the weekly ili-data were aggregated in 4 major regional groups instead of postal codes. two large outbreaks of legionnaires' disease were used as ''positive controls'' for emerging lri-outbreaks [13, 14] : 1) in march 1999, a large legionnaires' disease outbreak occurred among persons who had visited a flower show [13] . ten patients with pneumonia were admitted to one hospital between march 7 th to 11 th . by march 11 th , six patients were diagnosed with legionnaires' disease and an alarm notice was given to hospitals and gps in the region. follow-up investigation detected a total of 188 cases, of whom 167 (87%) were hospitalized and 21 (11%) died. 2) between july 6 th -28 th 2006, 30 legionnaires' disease cases were identified in amsterdam, 2 of which were fatal [14] . on july 7 th an alarm notice was given. a cooling tower in the town centre was later identified as the outbreak-source. for the lri-data, we used a space-time permutation scan statistic which compared the observed number of cases in circular areas with variable radii in flexible time periods vs the expected number of cases, based on the geographic distribution of cases in the whole dataset [15] . in this way, only the case data is needed to estimate the expected number of cases in each space-time window, and population density and time trends in the case data are automatically adjusted for. we used satscan software [16] and the satscan macro accessory for cartography (smac [17] , applied in sas version 9.1, sas institute inc., cary, nc, usa) to run the scan-statistic and visualize the results. we simulated a prospective surveillance by running the scan-statistic on data from the year preceding each time unit (day or week) in the analysis period. thus, weekly or daily space-time signals were generated, each time that the observed number of cases in a certain space and time window exceeded the defined significance threshold. since such analysis consumes a lot of computation time, we performed weekly analysis (instead of daily) over the whole study period. daily analyses were also performed in the years that the test-case outbreaks occurred (1999 and 2006), to assess the earliest possible detection date. for all analyses, we chose to use a time-aggregation level of 7-days length. for the daily analyses, these 7-day aggregation windows shifted one day forward for each daily run. thus we both reduced the computation time and adjusted for day-of-week effects (both purely temporal and spatial day-of-week effects). to indicate the significance of detected space-time signals, we used recurrence intervals, which indicate how often a signal of the observed significance would be observed by chance under the hypothesis of no outbreak [18] . i.e. if the recurrence interval of a signal is say 1 year, 1 signal of the observed significance is expected in 1 year. two thresholds levels were used: signals with recurrence interval $1 and $5 years. we assessed whether successive signals overlapped in space and time, which suggests the same cause. for the sake of readability, we indicated a group of such overlapping space-time signals as ''cluster'' and an individual space-time signal as ''cluster-signal''. we evaluated how many lri-clusters and signals were detected over the whole study period (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) and looked for explanations guided by the two-step criteria in figure 1 . in step one, we assessed likely causes for the cluster-signals by looking for significantly higher proportions of specific hospital discharge diagnoses (e.g., legionnaires' disease [19, 20] ). in step two we assessed overlap with regional ili clusters (appendix s2), as (local) influenza activity might be reflected in local lri-elevations. since other pathogens than influenza might cause some ili fluctuations, influenza activity was only considered to be a likely cause if space-time overlap between lri and ili-clusters coincided with the annual influenza season (figure 1 ). if a specific cause was defined for one or more signals within one cluster, we considered that to be a likely cause for the whole cluster. we also evaluated the timeliness of detection for the clusters related to the known legionnaires' disease outbreaks. a sensitivity analysis was used to evaluate the impact of time and spatial window settings on the number of clusters and signals detected. for the initial analyses, we put only minor constraints on the maximum temporal and spatial windows of the scan-statistic, to avoid wrongful assumptions about time, geographical location and size of an outbreak. we then repeated these weekly analyses with a temporal window of maximum 7 weeks and also with a spatial window of maximum 25 km radius, to assess the impact of these parameters on the number of signals generated. see appendix s2 for further details on use and settings of the scan-statistics. between feb 1 st 1999 and sept 30 th 2006, a total of 35 lriclusters with 221 cluster-signals were detected by weekly analysis (table 1 , non-restrictive parameter settings, recurrence interval $1 year). by raising the threshold (recurrence interval $5 years), we observed only 24 clusters with 146 cluster-signals (respectively 31% and 34% decrease). figure 2a shows all lri-clusters and signals on a timescale for the different recurrence interval levelsas detected with the initial non-restrictive parameter settings for space and time windows. the time between the first and the last signal within one cluster ranged from 0 to 26 weeks. by daily analysis, in 1999 and 2006 a total of 194 cluster-signals were detected (compared to 75 signals by weekly analysis with a $1 year recurrence level, both with non-restrictive parameter settings). however, the number of clusters was lower (10 clusters by daily analysis vs 12 by weekly analysis in 1999 and 2006). figure 2a and table 1 also show the likely causes for the detected lri-clusters (according to the criteria in figure 1 , see methods section). the known legionnaires' disease outbreaks in 1999 and 2006 were detected by lri-clusters, since cluster-signals were generated with an increased proportion of patient discharge diagnoses for legionnaires' disease in both outbreak areas and periods (table 1, figure 2a and 3a-b) (proportions differed between successive signals: 44-65% in 1999, and 21-63% in 2006; p:,0.0001). the 1999 legionnaires' disease related cluster-signals included a higher proportion of persons 50-64 years of age (37-48%; p:,0.0001). we compared the earliest detection dates for these outbreaks for daily and weekly analysis. daily analysis signaled the outbreak 4 days earlier than weekly analysis, 2 days before the national alarm was given during the 1999 legionnaires' disease outbreak. the 2006 legionnaires' disease outbreak was detected by weekly analysis on 2006 july 15 th , and could have been detected by daily analysis 5 days earlier, 3 days after the national alarm was given. many of the other clusters and signals seemed to be related to local rsv and/or influenza activity (70% of cluster-signals and 60% of clusters, table 1 ). some of the influenza and rsv related clusters tended to persist over longer periods (figure 2a) . young children (0-4 years old) were overrepresented in 82 of the 99 cluster-signals that we scored as rsv related (table 1 ; p:,0.05). in 2000, a cluster was detected with an unusually high number of patients diagnosed with aspergillosis, which was traced to a registration error (one patient was accidentally registered under 28 different anonymous identifiers). for 46 cluster-signals we did not find a ''likely cause'' according to the criteria in figure 1 . of these, 6 belonged to influenza and/ or rsv related clusters (figure 2a when repeating the weekly analyses with restricted time or spatial windows, both legionnaires' disease outbreaks were still detected with the same timeliness. table 1 and figure 2b and 2c also show the clusters and signals that were still detected with a temporal window of maximum 7 weeks, and with a spatial window of maximum 25 km respectively (as compared to the signals detected with the initial non-restrictive settings). with a time window of maximum 7 weeks, 129 of the 221 initial cluster-signals and 30 of the initial 35 clusters were still detected (respectively 42% and 14% decline, table 1 ). of the 5 clusters not detected -as compared to the initial analyses -2 had been scored as likely due to rsv, 1 to influenza and for the other 2 no likely cause had been scored (table 1 and figure 2a-b) . with a maximum 25 km radius, 165 of the 221 initial clustersignals and 33 of the 35 clusters were still detected (respectively 25% and 6% decline, table 1 ). one of the 2 undetected clusters figure 1 . two-step criteria to define (likely) causes for lri hospitalization clusters detected in 1999-2006. * as evaluated by the rightsided fisher's exact test for 262 tables (alpha#0.01) of hospitalizations within vs hospitalizations outside of the cluster-signal. the proportion of hospitalizations with a specific characteristic (e.g. legionnaires' disease as discharge diagnoses, or age 20-49 yrs) can be significantly higher among hospitalizations within the cluster-signal than the proportion outside of the cluster-signal. ** for the ili-cluster-signals we could only use 4 major regions as spatial resolution. overlap in time between lri and ili-cluster-signals was defined as occurrence of weekly ili-cluster-signals within 2 weeks (+/2) around lri-cluster-signals. ***the annual influenza season was defined as all weeks with a national weekly ili-incidence $3 per 10.000 pop. **** possibly unreported/undetected local lri-outbreaks by undetected pathogens. doi:10.1371/journal.pone.0010406.g001 had been scored as likely due to rsv, for the other no likely cause had been scored (table 1, figure 2a and 2c) . some of the cluster-signals detected with restrictive time/spatial windows had not been detected with the initially detected signals (data not shown). with the restrictive time window 2 borderline significant cluster-signals were detected, that had been nonsignificant in the initial analysis. this was due to the fact that the restrictive settings limited the adjustments for taking into account the multiple testing (stemming from the many potential cluster locations and sizes evaluated) [15] . with the restrictive spatial window 3 extra cluster-signals were detected due to the same mechanism, and 2 other extra cluster-signals were detected due to the fact that initial cluster-signals that geographically overlapped with them had dropped out. in this study, prospective surveillance of hospitalization data was simulated using retrospective data, to evaluate whether syndromic surveillance can effectively detect local outbreaks of lowerrespiratory infections (lris). over 1999-2006 (400 weeks), 35 space-time lri-clusters were detected by weekly analysis, with a total of 221 generated cluster-signals. this represents an average rate of approximately 5 new clusters per year, or 3 per year using a threshold recurrence interval $5 years. the number of clusters detected per year differed over the study period, reflecting substantial annual variation in influenza epidemics. two clusters were related to the legionnaires' disease ''testcase'' outbreaks and would have been detected around the same time as the outbreaks were actually detected. this indicates that syndromic surveillance will pick up similar outbreaks of severe respiratory disease in a timely manner. note that the legionnaires' disease outbreaks are used here as ''positive controls'' (or gold standard) for realistic severe respiratory outbreaks by uncommon pathogens that may not be (timely) detected by traditional surveillance, such as the dutch q-fever outbreak in 2007, for which the initial diagnoses were delayed by several weeks [21, 22] . as 17 out of the total 35 lri clusters probably reflected local rsv and/or influenza activity, many signal investigations could be limited to checking their concurrence with local rsv and/or influenza activity. the 3 clusters with ''unknown cause'', that concur with local ili-elevations outside the influenza season, possibly represent very early local influenza activity or local activity of another respiratory pathogen reflected in both gp-ilidata and hospital lri-data. for these 3 clusters and the other 8 clusters for which no likely cause was defined, it would have been interesting to investigate possible causes in a truly prospective setting (e.g., by additional diagnostics). some of these clusters possibly represent unreported and/or undetected local lrioutbreaks. as a threshold value for the significance of cluster signals, we used a threshold of recurrence intervals $1 year, and only evaluated the lri clusters that were above this threshold. to illustrate the impact of changing the threshold we repeated the analyses for recurrence intervals $5 years. at both threshold levels, two lri clusters showed a higher proportion of legionnaires' disease cases (p:,0.0001, see also results section) overlapping with the known outbreak areas, which made us conclude that these lri clusters indeed detected the legionnaires' disease outbreaks. the results of the sensitivity analysis show that the test outbreaks are still detected with the restricted time and spatial windows (at both threshold levels), without loss of timeliness and with less signals generated in time. to limit the computation time we only performed a modest sensitivity analysis. in this study, the restrictions on the time window almost halved the number of signals (42% decline), whereas the clusters in time to investigate declined much less (14% decline). the spatial restrictions resulted in less decline in generated signals (25% decline in signals and 6% decline in clusters). this indicates that with little loss of sensitivity, the restricted time window would be most appropriate to limit the number of generated signals. local rsv and influenza activity n/a n/a 4 3 n/a n/a 4 3 n/a n/a 3 2 other specific pathogen** the total number of detected clusters and signals is presented, for the non-restrictive parameter settings on space and time (a), for the settings with a maximum time window of 7 weeks (b), and for the settings with a maximum radius of 25 km (c). the distribution of (likely) causes according to the criteria in figure 1 is also presented in the table. * a cluster is defined by a set of successive cluster-signals that overlap in space and time. ** the cluster-signals in this category formed only one cluster, which appeared to be caused by a data artifact. *** possibly unreported/undetected local lri-outbreaks by undetected pathogens. doi:10.1371/journal.pone.0010406.t001 to our knowledge this is the first study that evaluates the performance of syndromic surveillance with nationwide high coverage data (80-99% of hospitalizations) over a longer period (8 years) with all detected clusters analyzed and (if possible) explained in a systematic way. feasibility of localized outbreak detection is demonstrated without swamping true signals by excessive false alarms. some other studies evaluating the performance of spacetime syndromic surveillance have concluded differently, but these studies were based on shorter periods, had lower coverage or lacked comparable outbreaks which could be tested [8, 23, 24] . cooper et al. tracked the spatial diffusion of influenza and norovirus, using space-time analysis on syndromic data from a telephone help line system in the uk, but did not test space-time detection for more localized outbreaks [23] . using syndromic surveillance for detection of local gastro-intestinal outbreaks in new york city, balter et al. found numerous cluster-signals in time, but these could not be used for effective surveillance because of insufficient comparable diagnostic data [8] . respiratory disease outbreaks could not be evaluated in the nyc study, because no local respiratory outbreaks had been reported in the study period. nordin et al. used simulated anthrax attack data injected in true physician's visit data to confirm that a respiratory outbreak initiated by bioterrorism will be detected in a timely manner by syndromic surveillance [24] . however, no results on the number of possibly false alarms were presented. these studies present space-time cluster detection analyses over relatively few years and are therefore prone to miss the effects of annual variation. furthermore, sensitivity for local outbreaks is reduced by using data with relatively low coverage levels. for such data sources with low coverage, methods other than space-time scan statistics seem more appropriate to generate useful information for public health practice (like aberration detection in time). we performed weekly analyses (instead of daily) over the whole study period, because these analyses consume considerable computation time. daily analyses in 1999 and 2006 detected fewer clusters than weekly analyses because the threshold level for recurrence intervals ($1 year) is more strict (see appendix s2). daily analyses would therefore probably not detect more epidemiological events but would yield more timely signals. hospital based syndromic surveillance could be a helpful tool in detecting local lri-outbreaks, complementing outbreak detection by laboratory surveillance or astute clinicians. syndromic surveillance might be most valuable for outbreaks due to uncommon or novel pathogens (like the sars outbreak), as these seem more likely to be missed by the laboratory and clinicians. furthermore, outbreaks due to more common pathogens could also be missed, as for community acquired pneumonia often no causative pathogen is detected [25, 26] . apart from that, under-notification can complicate outbreak detection through laboratories and clinicians [20] . a prerequisite for prospective syndrome surveillance is the realtime availability of hospitalization data, including clinical diagnoses and symptoms by date of hospitalization. although at present not available in the netherlands, such real-time syndromic data collection may become feasible after the nationwide implementation of electronic health-care information exchange. in this light, the results of our study justify further development of these methods, including retrospective evaluation of other types of documented health events than the ones presented in our study. besides that, further research should focus on prospective application of these methods. in a prospective setting, sustaining reliable data with high coverage and few data artifacts might be more challenging, thus possibly leading to higher numbers of false alarms. in addition, it should be evaluated to what extent 3 to 5 new syndromic clusters per year would indeed be manageable in a prospective setting. responding to such clusters is complicated, because the cause and thus possible threat will initially often be unknown. for each new cluster, it should first be verified whether plausible explanations can be found in epidemiological or laboratory data. for example, lri clusters need to be interpreted in relation to local influenza or rsv activity similar as we did in our study, and provided the age distribution of cases reflects the usual pattern, further investigation would seem unnecessary. internet-based ilimonitoring [27] combined with virological self -sampling (at home) [28] could increase the microbiological base for interpreting syndromic surveillance data. age stratified syndromic surveillance with a multivariate space-time scan statistic [29] may further facilitate quick interpretation of clusters by revealing the affected age groups. this retrospective study shows that space-time syndromic surveillance on hospitalizations can timely detect local lrioutbreaks independent of detection of the causative pathogen. the frequency of cluster detection, when interpreted in the light of available epidemiological and microbiological data, does not give rise to excessive levels of further investigations. consequently, we recommend real-time syndromic surveillance as an additional tool for detection of local lri outbreaks, but only if syndromic data with sufficient quality and coverage can be collected, coupled with epidemiological and microbiological data. public health responses can be based on a combination of syndromic surveillance data, reports by astute clinicians and early diagnostic test results, which all could generate the first alarm for different kinds of disease events. future research on prospective syndromic surveillance should therefore focus on practical methods for integrating syndromic surveillance alarms with clinical reports and laboratory information for effective public-health responses. appendix s1 detailed syndrome definition for hospitalizations with lower-respiratory infection syndrome. syndromic surveillance and bioterrorism-related epidemics using automated medical records for rapid identification of illness syndromes (syndromic surveillance): the example of lower respiratory infection surveillance of the bioterrorist threat: a primary care response evaluation of australia's national notifiable disease surveillance system experimental surveillance using data on sales of over-the-counter medications-japan syndromic surveillance in public health practice understanding the spatial clustering of severe acute respiratory syndrome (sars) in hong kong three years of emergency department gastrointestinal syndromic surveillance in new york city: what have we found? syndromic surveillance: is it a useful tool for local outbreak detection? privacy protection versus cluster detection in spatial epidemiology early detection of tuberculosis outbreaks among the san francisco homeless: trade-offs between spatial resolution and temporal scale continuous morbidity registration sentinels netherlands a large outbreak of legionnaires' disease at a flower show, the netherlands a space-time permutation scan statistic for disease outbreak detection satscantm v7.0: software for the spatial and space-time scan statistics a satscan macro accessory for cartography (smac) package implemented with sas software a generalized linear mixed models approach for detecting incident clusters of disease in small areas, with an application to biological terrorism 260201002: rivm incidence and completeness of notification of legionnaires' disease in the netherlands: covariate capture-recapture analysis acknowledging regional differences an outbreak of q fever in the netherlands-possible link to goats tracking the spatial diffusion of influenza and norovirus using telehealth data: a spatiotemporal analysis of syndromic data simulated anthrax attacks and syndromic surveillance improved diagnosis of the etiology of community-acquired pneumonia with real-time polymerase chain reaction value of intensive diagnostic microbiological investigation in low-and high-risk patients with community-acquired pneumonia internet-based monitoring of influenza-like illness (ili) in the general population of the netherlands during the 2003-2004 influenza season linking syndromic surveillance with virological self-sampling multivariate scan statistics for disease surveillance prospective time periodic geographical disease surveillance using a scan statistic we thank dutch hospital data for providing data from the dutch national medical register (lmr) and we thank douglas fleming for reading and commenting on the manuscript. conceived and designed the experiments: ccvdw lva wvp njdn mpgk. performed the experiments: ccvdw. analyzed the data: ccvdw. contributed reagents/materials/analysis tools: gd gad. wrote the paper: ccvdw. helped drafting and reviewed drafts of the paper: lva wvp njdn gad wvdh mpgk. helped interpreting the analysis results: lva wvp njdn mpgk. reviewed drafts of the paper: gd. key: cord-286065-x0g67pnb authors: metzgar, david; frinder, mark w.; rothman, richard e.; peterson, stephen; carroll, karen c.; zhang, sean x.; avornu, gideon d.; rounds, megan a.; carolan, heather e.; toleno, donna m.; moore, david; hall, thomas a.; massire, christian; richmond, gregory s.; gutierrez, jose r.; sampath, rangarajan; ecker, david j.; blyn, lawrence b. title: the iridica bac bsi assay: rapid, sensitive and culture-independent identification of bacteria and candida in blood date: 2016-07-06 journal: plos one doi: 10.1371/journal.pone.0158186 sha: doc_id: 286065 cord_uid: x0g67pnb bloodstream infection (bsi) and sepsis are rising in incidence throughout the developed world. the spread of multi-drug resistant organisms presents increasing challenges to treatment. surviving bsi is dependent on rapid and accurate identification of causal organisms, and timely application of appropriate antibiotics. current culture-based methods used to detect and identify agents of bsi are often too slow to impact early therapy and may fail to detect relevant organisms in many positive cases. existing methods for direct molecular detection of microbial dna in blood are limited in either sensitivity (likely the result of small sample volumes) or in breadth of coverage, often because the pcr primers and probes used target only a few specific pathogens. there is a clear unmet need for a sensitive molecular assay capable of identifying the diverse bacteria and yeast associated with bsi directly from uncultured whole blood samples. we have developed a method of extracting dna from larger volumes of whole blood (5 ml per sample), amplifying multiple widely conserved bacterial and fungal genes using a mismatchand background-tolerant pcr chemistry, and identifying hundreds of diverse organisms from the amplified fragments on the basis of species-specific genetic signatures using electrospray ionization mass spectrometry (pcr/esi-ms). we describe the analytical characteristics of the iridica bac bsi assay and compare its pre-clinical performance to current standard-of-care methods in a collection of prospectively collected blood specimens from patients with symptoms of sepsis. the assay generated matching results in 80% of culture-positive cases (86% when common contaminants were excluded from the analysis), and twice the total number of positive detections. the described method is capable of providing organism identifications directly from uncultured blood in less than 8 hours. disclaimer: the iridica bac bsi assay is not available in the united states. bloodstream infection (bsi) and associated clinical sepsis represent a major source of mortality in the developed world, ranking as the 3 rd leading cause of death in germany [1] and the 11 th in the united states [2] . with increasing numbers of elderly and immunocompromised patients as well as increased use of implanted medical devices, the incidence of sepsis is rising rapidly-by 75% between 1993 and 2003 in france [3] . with mortality rates ranging as high as 50% [3, 4] , this situation constitutes a public health disaster. average total costs associated with individual cases in germany and the united states have been estimated at €23,297 and $22,100 respectively, with annual national burdens of €3.6-7.9 billion and $16.7 billion [4, 5] . the primary factor affecting the clinical outcome and financial burden of bsi is prompt treatment with appropriate antibiotics [1, 6, 7, 8, 9] . mortality risk doubles with a 24 hour delay in provision of appropriate antibiotics in cases of bacteremia [10] , and with a 12 hour delay in provision of antifungals in cases of candidemia [11] . current culture-based methods used to detect and identify agents of bloodstream infection are inadequate. incubation times of up to 5 days may be necessary to capture the majority of cultureable bacteria and fungi associated with bsi, though many infections can be detected after 24 to 48 hours [12, 13] . further time is required to obtain identifications via maldi--tof, dna sequence, or traditional biochemical analysis. these temporal delays leave practitioners with little choice but to treat all patients with suspected bsi empirically using broadspectrum antibiotics. this strategy results in 15-30% of septic patients receiving inappropriate antibiotic therapies, which is, in turn, associated with a 2 to 5-fold higher mortality risk [6, 8, 14] . this risk derives from the empirical use of antibiotics that are ineffective for rarer organisms such as vancomycin-resistant enterococci (vre) and candida [14] . standard culture-based methods are hampered by treatment with inhibitory antibiotics prior to sampling [7, 15] and are insensitive to non-cultivable or fastidious organisms such as coxiella burnetii, tropheryma whipplei, and species of genera such as bartonella, rickettsia, mycobacterium, and nocardia [16, 17] . many of these organisms have been identified as causal agents of bsi by culture-independent molecular methods such as single-analyte pcr and 16s ribosomal gene sequencing; however, such methods lack either the coverage necessary to identify the diverse agents of bsi [9] or the sensitivity to consistently detect those agents directly from blood in the majority of cases [18, 19, 20] . despite the low apparent sensitivity of existing broad-spectrum molecular methods with respect to culture-approximately 50% for many technologies [18, 19, 20] -such methods frequently yield additional positive results in culture-negative blood samples. these positive molecular detections are often confirmed in later cultures performed on samples taken from the same patients [1, 9, 12] , are strongly correlated with sepsis-associated biomarkers [7, 21, 22] , and have been shown to improve patient outcome when used to guide antibiotic therapy [1] . molecular detection of bacteria in culture-negative specimens is correlated with antibiotic pretreatment [9, 23] and primarily identifies species known to be causal agents of bsi. these correlations all suggest that many culture-negative, pcr-positive detections represent culture insensitivity rather than a lack of specificity or clinical relevance on the part of molecular methods. this is supported by literature which widely reports that blood culture is positive in only 50% of cases where bsi is strongly suspected from a clinical standpoint [16, 24, 25] . there is a clear unmet need for rapid and sensitive molecular detection and identification of bsi agents directly from blood samples [2, 7, 20, 26, 27, 28, 29] . to meet this need, we have developed a universal lysis and dna extraction method capable of sampling 5 ml of whole blood [24] . this is paired with conserved-site pcr primers capable of generating amplicons from >95% of the eubacteria and candida species associated with human infection [30, 31] and pcr chemistry and cycling conditions compatible with high concentrations of background human dna [24, 32, 33] . the method utilizes an automated desalting and dna debulking platform to prepare amplicons for mass spectrometry [24, 34] and an electrospray ionization mass spectrometry (esi-ms) platform capable of discriminating amplicon sequence variants from one or more different species present in a sample [35, 36, 37] . an onboard analysis computer is used to parse and report detections of 673 species of bacteria and candida on the basis of multi-locus amplicon base composition signatures and quality controls including an external lysis control, internal pcr controls, external mass spectrometry standards, and signal strength and quality metrics [24, 31, 35] . in this study we explore the analytical sensitivity, specificity, robustness, reproducibility, and breadth of coverage of the iridica bac bsi assay as performed on the iridica system, and compare its performance to that of traditional culture-based methods in a collection of prospectively collected clinical whole blood specimens. culture and quantification of microbial stocks. microbial stocks were obtained from atcc (manassas, va) or clinical laboratories, grown on appropriate media and quantified by standard dilution and colony-count methods [31] at ibis biosciences (carlsbad, california) or zeptometrix (buffalo, new york). stocks were stored frozen in 15% glycerol at -70°c, thawed and diluted into test matrices, and again stored at -70°c or tested immediately. clinical sample collection and testing. two hundred and eighty-five 5 ml whole blood samples were prospectively collected from consenting patients who presented to the johns hopkins hospital emergency department and met at least two of the sirs criteria for sepsis [38] . samples were collected by phlebotomists in edta blood tubes following blood draws taken for standard-of-care culture analysis, using the same venipunctures. samples were then blinded by replacement of identifying information with numerical identifiers by study coordinators, frozen, and transported to ibis biosciences on dry ice. chart data and standard-of-care culture results were collected thereafter at johns hopkins hospital by the study coordinators, associated with the respective blind sample identities, and then also blinded with respect to patient identity. ibis biosciences performed the iridica bac bsi assay testing in the absence of chart and culture data. paired specimens taken for clinical testing were processed per standard of care in the clinical microbiology laboratory at the johns hopkins hospital, with no knowledge of the iridica bac bsi assay results, and the results reported by the clinical microbiology laboratory were used here as the comparator. the two independently generated datasets (iridica and culture) were brought together for comparison only after both methods had been performed and the data traceably documented. clinical laboratory culture and identification. the clinical microbiology laboratory used the bactec fx (bd diagnostics, sparks, md) continuous monitoring blood culture instrument. the bottles routinely used for clinical care included the bactec lytic/10 anaerobic bottle; the bactec plus aerobic/f bottle containing resins for antibiotic neutralization (used for patients on antibiotics at the time of blood draw); and the bactec standard/10 aerobic bottle (an all-purpose medium that does not contain resins recommended for patients not on antibiotics at the time of blood draw). the laboratory blood culture procurement policy recommended that clinicians send a minimum of two sets of blood cultures with each set consisting of an aerobic bottle (either standard or aerobic plus) and an anaerobic lytic bottle, each inoculated with 10 ml of blood. aerobic gram-positive cocci and gram-negative rods were routinely identified using the phoenix system (bd diagnostics, inc., sparks, md). anaerobes were identified following recovery on routine media using either maldi-tof mass spectrometry (bruker, billerica, ma) or the rapid ana ii system (thermoscientific, waltham, ma). gram-positive rods were identified using either the phoenix system, maldi-tof ms, or cell wall fatty acid analysis using gas liquid chromatography, depending upon the genera isolated. candida species were identified using recovery on chromagar, morphology, and the phoenix system yeast panels. phenotypic resistance-typing methods were performed on isolates using the phoenix system and the associated gram-negative (nmic/id-132) and gram-positive (pmic/id-105) panels (bd diagnostics, sparks, md), extended-spectrum beta-lactamase e-tests (biomerieux, durham, nc), and the modified hodge test. molecular testing for meca, vana and vanb was performed using the verigene bc-id microarray system (nanosphere, northbrook, il). the iridica system. pcr/esi-ms testing was performed using the iridica system components and their associated reagents (abbott molecular, des plaines, il), including a high-volume bead-beating platform (iridica bb), automated 5ml dna extraction and pcr set-up platform (iridica sp), pcr thermocycler (iridica tc), automated amplicon desalting and dna debulking platform (iridica ds), automated electrospray ionization mass spectrometer (iridica ms), and a control and analysis computer (iridica ac). functionality of these components has been previously described [24] . the system is capable of running 6 samples simultaneously in batches, and subsequent batches may be started 2.5 hours following initiation of the first. most testing described here was performed by running multiple overlapping 6-sample batches on each testing day on each instrument. each sample requires approximately 30 minutes of hands-on time for a single technician when run in batch mode (~2.5 hours hands-on time for a batch of 6 samples), involving reagent preparation, sample loading, transfer between instruments, and instrument decontamination. hands-on requirements are interspersed temporally throughout the first 5 hours of the process and require a trained laboratory technician with competence in sterile technique, metered fluid transfer, vortexing, pipetting, use of a clinical centrifuge, and bar code scanning to track samples, equipment, users, reagents, and results. time to first result averages approximately 7 hours. the iridica bac bsi assay. all testing utilized the iridica bac bsi assay kit, which includes a pre-filled pcr reaction strip encompassing 18 primer pairs in 16 wells. the primers target broadly conserved bacterial and candida genes, 4 specific antibiotic resistance markers, meca, vana, vanb and bla kpc , and an extraction control target [31] . reactions and cycling conditions were optimized to function in samples with a high human dna background, and have been described previously [24] . mass spectrometry signals from unfragmented amplicons were processed using analysis software and database elements designed to translate raw spectral data into base composition signatures for each detected amplicon. the software then performed signature matching between detected base compositions and multilocus species-specific signatures derived through analysis of type strains or surveys of wholegenome data from genbank [35, 39] . core test organisms. the iridica bac bsi assay detects and identifies all target bacteria using a common set of primers and analysis algorithms. the same is true for all target candida. on the basis of this unified functionality, a tiered approach to validation was followed [27, 31] . four "core" organisms, which together utilize all primer pairs of the assay, were used for analytical studies designed to challenge the assay's ability to detect target organisms at concentrations near the limit of detection (lod) in various circumstances. the core organisms included meca+ staphylococcus aureus (mrsa), vana+/vanb+ enterococcus faecium (vre), bla kpc + klebsiella pneumoniae (kpc), and candida albicans. analytical studies. the limit of detection (lod) of the assay was characterized for the 4 core organisms in 5 ml aliquots of both edta whole blood and sterile buffer (iridica negative control, abbott molecular, des plaines, il). based on the resulting demonstration of comparable sensitivity in these 2 matrices, the lods of 19 additional bacteria and candida species were determined and confirmed in the sterile buffer. all lods were measured by initial testing of 5 replicates at each of several 2-fold dilution steps, with the "determined lod" being defined as the lowest concentration at which all 5 replicates were detected and correctly identified, including both the organism and any known drug resistance markers. this was followed by confirmation testing using 20 replicates at the determined lod. the final (reported) lod was defined as the lowest concentration at which at least 19 of 20 replicates were detected and correctly identified. another 47 species were tested at single concentrations in sterile buffer to confirm the designed breadth of coverage of the assay. robustness was challenged through studies of potentially interfering substances (in edta whole blood), potentially cross-reacting organisms and carryover (in sterile buffer), and an in-house reproducibility analysis using multiple instruments, reagent lots and users (in edta whole blood, alongside samples prepared in sterile buffer, body fluids, and tissues). carryover and cross-reactivity studies were performed at high titer while other studies were performed using the 4 core organisms at 3x lod. clinical sample performance was evaluated by comparing iridica bac bsi assay results to culture results using 285 prospectively collected edta whole blood specimens from consented subjects with suspected bsi. assay controls. all analytical and clinical sample testing was carried out with the same control scheme using integrated and automated positive controls. these controls included both an extraction control target added to each sample and internal pcr calibrants formulated in each reaction well. pcr calibrants are synthetic competitive dna constructs that contain the assay primer binding sites for one of the assay primer pairs contained in the well. the constructs are designed to produce unique base composition signatures which can be readily discriminated from the expected amplicons produced from target analytes. the calibrants are included in the pcr wells at defined concentrations and are used both to compete with low levels of background template and to gauge template input levels. negative controls consisting of sterile buffer were run as a part of every set of 1-6 samples run concurrently on the platform. for positive detections in negative controls, any associated test results yielding the same detection were excluded. the rate of test validity was 96% over the course of the experiments described here. during analytical spiked sample studies, negative results paired with valid secondary detections of unspiked organisms (contaminants) were excluded on the basis of potential competitive interference. as part of the standard of care, the collecting facility generally drew and tested two independent blood samples, each of which was inoculated into a blood culture bottle set consisting of one aerobic and one anaerobic bottle. performance of multiple cultures is common practice to increase sensitivity and to discriminate between contamination events and clinically relevant detections. contamination is generally characterized by single, unrepeated detections of common contaminants such as coagulase-negative staphylococci, viridans group streptococci, and species of the corynebacterium, bacillus, micrococcus, and propionibacterium genera. bsi is generally characterized by single or repeated detections of organisms strongly associated with bsi, such as mrsa, kpc, or vre, or repeated detection of the same potential contaminant organism [40, 41, 42, 13, 43, 44] . in this study, culture results from a single blood culture bottle set were compared to iri-dica bac bsi assay results from a single test sample, as only one sample was collected from each subject for testing on the iridica system. in cases where the test sample was obtained from the same venipuncture as a sample used to inoculate a specific blood bottle set and that pairing was clearly documented, the associated culture result was used as the comparator. in cases where the specific link between test sample and clinical sample could not be confirmed (approximately one third of cases), the first reported culture result from the same day was used as the comparator. when available, results from secondary culture bottle sets and other species-specific pathogen identification results from patient chart data were used to assess iri-dica bac bsi assay-positive, culture-negative discrepancies. as discussed above, single positive detections of common contaminant organisms are of questionable clinical relevance, and the specificity of culture for these organisms is known to be low [40, 41, 42, 13, 43, 44] . therefore, comparisons between culture and the iridica bac bsi assay were made both with and without consideration of common contaminant organisms. when both culture and the iridica bac bsi assay reported the presence of a bacterial species associated with one of the drug resistance markers targeted by the iridica bac bsi assay, iridica bac bsi assay resistance marker results were compared to phenotypic and/ or genotypic resistance data reported by the clinical laboratory as part of post-culture standard-of-care isolate characterization. in cases where either the iridica bac bsi assay or culture were negative for these bacteria, no resistance comparisons could be made because both methods report resistance markers or phenotypes only in association with specific bacterial detections. limits of detection were measured for the four core organisms in both edta whole blood and sterile buffer matrices (see fig 1) . all four core organisms' lods were within 2-fold of each other between these two matrices, and further analytical studies were performed in sterile buffer except as noted. the confirmed limits of detection of the iridica bac bsi assay for 19 additional bacteria and candida representing the breadth of coverage of the assay are shown in fig 1. the geometric mean (log 2 ) of the lods for these species was 18.5 cfu/ml, with a range of 0.25-128 cfu/ ml. this general limit of detection supports the theoretical ability of the assay to detect bacteria directly from the blood of most bsi patients, as the literature suggests that bsi patients carry approximately 1000 pcr-detectable genome copies of infecting organisms per ml of blood, in contrast to the very low levels of cultureable bacteria (1-10 cfu/ml) that can be grown directly from the blood of such patients [24] . the only species for which the assay lods were < 4 cfu/ml were streptococci, which occur in chains and therefore have a high and variable genome:cfu ratio. the assay successfully detected 47 additional diverse bacteria and candida species in 2/2 replicates when tested at single concentrations. these organisms are shown in fig 1, and include the species isolated by culture in >95% of bsi cases [30] , organisms representing the full phylogenetic breadth of the assay [27, 31, 35] , and five organisms identified as "bioinformatic worst-case" organisms (those to which the assay primers are most poorly matched) [27, 31] . cross-reactivity was characterized with samples containing 10 5 cfu/ml, copies/ml, or tcid 50 /ml of a variety of fungi, bacteria, and viruses which the assay was not designed to report. these included aspergillus flavus, aspergillus niger, clavispora lusitaniae, candida kefyr, caulobacter segnis, pedobacter heparinus, shewanella oneidensis, streptomyces griseus, influenza, parainfluenza, adenovirus, parvovirus, coronavirus, and herpesvirus. no cross-reactivity was observed. the assay was able to detect and identify 3x lod spikes of the four core organisms in edta whole blood supplemented with the potentially interfering substances listed in table 1 . for all but one substance, the test concentration corresponded to the recommendation in clsi organisms tested in analytical studies. the four core organisms are shown in bold, with lods in blood and buffer shown in parentheses (blood lod in cfu per ml / buffer lod in cfu per ml). the lod of 19 further organisms in buffer are also shown in parentheses. remaining organisms shown in blue and purple were tested and detected in 2/2 replicates at either 100 cfu/ml (all except bacteroides fragilis) or 200 cfu/ml (bacteroides fragilis). *indicates bioinformatic "worst-case scenario" organisms, for which the broad-spectrum primers used in the iridica bac bsi assay are the least wellmatched. standards guidance [45] . the exception was bilirubin, for which all tested preparations were heavily contaminated with bacterial and fungal dna. despite the contamination, which prevented detection of one spiked analyte in one replicate through competitive interference at the original concentration (342 μmol/l), the assay still detected 11 of 12 spiked test organisms. all 12 spiked analytes were detected at 171 μmol/l bilirubin. the same contaminants were detected at both concentrations, indicating that extraction and pcr were fully functional and not directly affected by the bilirubin. carryover testing was performed by testing adjacent negative and high positive samples. positives were spiked with 10 7 cfu/sample of either kpc, vre, or both mrsa and candida albicans. the test configuration provided 104 independent opportunities to observe carryover events between adjacent high titer and negative samples. no carryover events were observed. performance in samples containing multiple analytes was characterized by testing all 6 possible mixtures of two of the four core organisms at a 1:1 ratio at 3x lod in triplicate. because the iridica bac bsi assay uses shared primers to detect related targets, competitive interference may prevent simultaneous detection of multiple targets. for this reason, this study was not intended to demonstrate that all analytes could be simultaneously detected and identified in mixtures, but rather that the presence of multiple organisms did not prevent the iridica bac bsi assay from detecting and correctly identifying at least one of the components of a mixture. the assay successfully detected and accurately identified at least one of the two spiked analytes in 18/18 instances, and was able to detect and identify both spiked analytes in 15/18 instances. the three cases in which only one of the two spiked analytes were detected were all mixtures of mrsa and vre-in two of these only mrsa was detected, and in one case only vre was detected. the within-laboratory reproducibility of the iridica bac bsi assay bead-beating, sample prep, and pcr components (bb, sp, tc, assay strips, and related reagents) was characterized tables 3, 4 and 5, while antibiotic resistance marker/phenotype results are shown in table 6 . both culture and the iridica bac bsi assay utilize single reagents to detect many or all targets. therefore, the presence of detected organisms can mask the detection of other organisms through competitive interference. in culture, faster growing organisms can readily outcompete slower-growing organisms in culture bottles and obviate isolation of slower-growing organisms. in broad-spectrum pcr-based systems like iridica, which use conserved-site primer pairs to amplify partially conserved regions from multiple species, targets with better matches to specific primer pairs may outcompete other species for amplification [27, 35] . this obscures meaningful interpretation of negative results in samples that are already positive for at least one analyte. for the analysis presented here, results from both culture and the iridica bac bsi assay were considered negative only if the sample yielded negative results for all analytes, while positive samples were considered positive for the reported analytes and indeterminate for all others. positive detections which thereby had indeterminate comparators were assigned to the "additional detection" category in the final comparison (see table 5 ). two hundred and seven samples yielded matched negative results for all analytes by both the iridica bac bsi assay and culture methods. the iridica bac bsi assay matched 32 of the 40 comparable detections reported by culture (80% positive agreement) and detected an additional 46 organisms in culture-negative samples. when potential contaminant organisms (listed at the bottom of tables 3 and 4) were excluded from analysis, the iridica bac bsi assay results matched 30 of 35 comparable detections reported by culture (86% positive agreement), and detected an additional 34 organisms not identified in culture. the majority of the iridica bac bsi assay-positive, culture-negative detections identified either species commonly associated with bsi, including escherichia coli, enterococcus faecium, klebsiella pneumoniae, and staphylococcus aureus; or clinically relevant species unlikely to be detected by common culture bottle methods, including ehrlichia chaffeensis [33] and mycoplasma hominis [46] . others were of fastidious species which occur as both pathogens and common environmental contaminants, such as mycobacterium simiae [47] . eleven of the 46 iridica bac bsi assay-positive, culture-negative detections were supported by chart data showing positive detections of the same organisms and identifying them as causal pathogens in subsequent standard-of-care analyses of blood, biopsy, or respiratory specimens from the same patients. these are noted in tables 3 and 4 . the overall results obtained in this clinical sample study were consistent with those obtained in similar studies comparing the performance of the iridica bac bsi assay with culture in separate populations of patients with suspected sepsis [48, 49] . eight propionibacterium acnes detections were made by the iridica bac bsi assay and one by culture, none of which were matched. it is assumed that these represent contaminants introduced during venipuncture or sample preparation and reagent handling [40] . the iri-dica bac bsi assay may be more sensitive to p. acnes contamination than culture methods. the organisms listed as potential contaminants in table 4 , along with other common blood culture contaminants, are notated as potential contaminants on iridica bac bsi assay reports. it has been recommended that detection of these and other well-recognized common contaminant organisms in blood samples, whether through culture or molecular means, be confirmed with secondary samples rather than assuming clinical relevance [40, 41, 43] . during the clinical sample study, performed following the sterility and personal protective equipment recommendations of the manufacturer, 61 negative controls were tested and yielded no other reportable organisms excluding potential contaminants (n = 550) 0 0 0 207 a these 11 culture-negative, iridica bac bsi assay-positive detections were supported by later organism-specific id data which identified the same species as agents of infection (as noted on the subjects' charts). numbers in parentheses indicate how many such cases were supported. table 6 . the data supports the ability of the iridica bac bsi assay to identify diverse bacteria and candida species directly from uncultured edta whole blood specimens, detecting 86% of the clinically relevant organisms isolated using traditional blood culture assays. the iridica bac bsi assay is capable of providing such identifications within eight hours of sample collection, potentially allowing for the timely provision of appropriate targeted antibiotic therapy in cases of suspected bloodstream infection and sepsis. the iridica bac bsi assay cannot determine antibiotic resistance phenotypes, and is limited in terms of detecting resistance-associated genotypes to four common broad-spectrum antibiotic resistance elements-meca, vana, vanb, and kpc. inclusive determination of resistance remains dependent on successful culture and subsequent phenotypic resistance typing of infecting organisms. for this reason, the iridica bac bsi assay should be considered an additional tool in the diagnostic regime, not as a replacement for culture-based methods. similar to culture-based methods, the iridica bac bsi assay is sensitive to both infecting pathogens and environmental microorganisms introduced during sample collection and preparation, necessitating rigorous phlebotomy technique, sterile laboratory handling, and use of negative controls. iridica bac bsi assay results should be interpreted in the context of associated symptoms, risk factors, and other laboratory findings, in the same manner that current standard-of-care culture identifications are utilized. the iridica bac bsi assay yielded approximately twice as many positive detections as culture across the described set of 285 clinical blood specimens from patients with symptoms of sepsis. in the majority of cases, the iridica bac bsi assay-positive, culture-negative detections were either of species commonly associated with bsi or of clinically relevant human pathogens that would be difficult to grow in blood culture bottles (e.g. ehrlichia chaffeensis). common contaminants (e.g. propionibacterium acnes) were also observed in culture-negative specimens. despite having access to only partial chart data, 11 of the 46 unmatched iridica bac bsi assay detections were specifically supported by subsequent identifications and/or diagnostic data from independent specimens showing infection of the patient by the species initially detected by the iridica bac bsi assay. many of the iridica bac bsi assay-positive, culture-negative detections thereby represent clinically relevant bloodstream infections which were missed by culture in the original paired specimen. this is consistent with literature suggesting that the sensitivity of culture is suboptimal [16, 24, 25] . the broad-spectrum nature of the iridica bac bsi assay primers, paired with a signal analysis method capable of sensitive and specific detection and identification of one or more species signatures in samples with high background levels of human dna, make it uniquely suited as a molecular test for bacterial and candida dna in blood samples. clinical blood samples and associated clinical chart and microbiology data were collected under irb protocol na_00013251, "evaluation of universal diagnostic assays for rapid and accurate pathogen identification in acute care settings", approved by the johns hopkins medical institution internal review board, irb-x. verbal consent was obtained from all subjects or their designated surrogates, either in person or by telephone. verbal consent was documented on copies of the consent script by the consenting physician or consent designee, and consent records were stored in a secure location. this procedure was approved by the jhmi irb based on minimal risk of harm. healthy subject blood used as matrix was provided by biomed supply llc, carlsbad, california and collected under fda license. pcr-based rapid sepsis diagnosis effectively guides clinical treatment in patients with new onset of sirs laboratory detection of sepsis: biomarkers and molecular approaches episepsis: a reappraisal of the epidemiology and outcome of severe sepsis in french intensive care units burden of illness imposed by severe sepsis in germany epidemiology of severe sepsis in the united states: analysis of incidence, outcome, and associated costs of care initiation of inappropriate antimicrobial therapy results in a fivefold reduction of survival in human septic shock the potential for pcr based testing to improve diagnosis and treatment of sepsis community-acquired bloodstream infection in critically ill adult patients: impact of shock and inappropriate antibiotic therapy on survival multiplex real-time pcr and blood culture for identification of bloodstream pathogens in patients with suspected sepsis benefit of appropriate empirical antibiotic treatment: thirty-day mortality and duration of hospital stay delaying the empiric treatment of candida bloodstream infection until positive blood culture results are obtained: a potential risk factor for hospital mortality improved detection of blood stream pathogens by real-time pcr in severe sepsis blood cultures: key elements for best practices and future directions the influence of inadequate antimicrobial treatment of bloodstream infections on patient outcomes in the icu setting antibiotic use in thailand: quantifying impact on blood culture yield and estimates of pneumococcal bacteremia incidence molecular diagnosis of bloodstream infections caused by non-cultivable bacteria then and now: use of 16s rdna gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories dnaemia detection by multiplex pcr and biomarkers for infection in systemic inflammatory response syndrome patients evaluation of a commercial multiplex pcr test (septifast) in the etiological diagnosis of community-onset bloodstream infections comparison of three different commercial pcr assays for the detection of pathogens in critically ill sepsis patients the detection of microbial dna in the blood: a sensitive method for diagnosing bacteremia and/or bacterial translocation in surgical patients new approaches to sepsis: molecular diagnostics and biomarkers direct amplification of rrna genes in diagnosis of bacterial infections improved sensitivity for molecular detection and identification of bacteria and candida in blood surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock molecular diagnosis of sepsis: new aspects and recent developments the value and validation of broad spectrum biosensors for diagnosis and biodefense management of sepsis pcr-electrospray ionization mass spectrometry: the potential to change infectious disease diagnostics in clinical and public health laboratories new technology for rapid molecular diagnosis of bloodstream infections broad-spectrum biosensor capable of detecting and identifying diverse bacterial and candida species in blood genotypic variation and mixtures of lyme borrelia in ixodes ticks from north america and europe detection and identification of ehrlichia species in blood by use of pcr and electrospray ionization mass spectrometry comprehensive biothreat cluster identification by pcr/electrospray-ionization mass spectrometry microbial identification by pcr/electrospray ionization-mass spectrometry molecular genotyping of microbes by multilocus pcr and mass spectrometry: a new tool for hospital infection control and public health surveillance tiger: the universal biosensor definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis updated review of blood culture contamination what is the relevance of obtaining multiple blood samples for culture? a comprehensive model to optimize the strategy for diagnosing bacteremia the clinical and prognostic importance of positive blood cultures in adults blood culture contamination: persisting problems and partial progress a critical appraisal of the role of the clinical microbiology laboratory in the diagnosis of bloodstream infections interference testing in clinical chemistry; approved guideline. clsi document ep7-a2 detection of mycoplasma hominis septicemia by radiometric blood culture mycobacterium simiae complex infection in an immunocompetent child evaluation of the broad-range pcr/esi-ms technology in blood specimens for the molecular diagnosis of bloodstream infections rapid diagnosis of infection in the critically ill, a multicenter study of molecular detection in bloodstream infections, pneumonia, and sterile site infections disclaimers: the described system and assay are not available in the united states.portions of the data presented in this paper were previously presented in a poster entitled "performance characterization of the iridica bac bsi assay for detection and identification of bacteria and candida in direct specimens from patients with suspected blood stream infections" at the 25 th european congress of clinical microbiology and infectious diseases (ecc-mid 2015), copenhagen, denmark, april 2015.the authors acknowledge dr. pam lipsett, the director of the surgical intensive care unit, and roy brower, the director of the medical intensive care unit, both at johns hopkins hospital, for facilitating study process and enrollments. key: cord-299852-t0mqe7yy authors: janssen, loes h. c.; kullberg, marie-louise j.; verkuil, bart; van zwieten, noa; wever, mirjam c. m.; van houtum, lisanne a. e. m.; wentholt, wilma g. m.; elzinga, bernet m. title: does the covid-19 pandemic impact parents’ and adolescents’ well-being? an ema-study on daily affect and parenting date: 2020-10-16 journal: plos one doi: 10.1371/journal.pone.0240962 sha: doc_id: 299852 cord_uid: t0mqe7yy due to the covid19 outbreak in the netherlands (march 2020) and the associated social distancing measures, families were enforced to stay at home as much as possible. adolescents and their families may be particularly affected by this enforced proximity, as adolescents strive to become more independent. yet, whether these measures impact emotional well-being in families with adolescents has not been examined. in this ecological momentary assessment study, we investigated if the covid-19 pandemic affected positive and negative affect of parents and adolescents and parenting behaviors (warmth and criticism). additionally, we examined possible explanations for the hypothesized changes in affect and parenting. to do so, we compared daily reports on affect and parenting that were gathered during two periods of 14 consecutive days, once before the covid-19 pandemic (2018–2019) and once during the covid-19 pandemic. multilevel analyses showed that only parents’ negative affect increased as compared to the period before the pandemic, whereas this was not the case for adolescents’ negative affect, positive affect and parenting behaviors (from both the adolescent and parent perspective). in general, intolerance of uncertainty was linked to adolescents’ and parents’ negative affect and adolescents’ positive affect. however, intolerance of uncertainty, nor any pandemic related characteristics (i.e. living surface, income, relatives with covid-19, hours of working at home, helping children with school and contact with covid-19 patients at work) were linked to the increase of parents’ negative affect during covid-19. it can be concluded that on average, our sample (consisting of relatively healthy parents and adolescents) seems to deal fairly well with the circumstances. the substantial heterogeneity in the data however, also suggest that whether or not parents and adolescents experience (emotional) problems can vary from household to household. implications for researchers, mental health care professionals and policy makers are discussed. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 since march 2020, the coronavirus disease 2019 is referred to as a pandemic by the world health organization [1] . to slow the spread of covid-19, national governments have taken radical measures to minimize social interactions by closing public places, demanding people to keep physical distance and stay at home and-in some countries-by enforcing 'full lockdown'. in the netherlands, at march 15 th 2020, measures of social distancing enforced all dutch citizens to stay home and work remotely as much as possible, public spaces (e.g. schools, offices, parts of public transport, theatres) were closed and public gatherings were prohibited (see fig 1 for a timeline) . these measures of social distancing (a so-called 'lockdown') created drastic changes in daily social life; distinct domains such as family life, school, and work suddenly coincided and families faced an unforeseen increase in hours spent together under the same roof. adolescents and their families may be particularly affected by this enforced proximity, as adolescents strive to become independent and focus more on socializing and spending time with friends rather than with their families [2, 3] . to that end, this study aimed to investigate well-being of adolescents and their parents and parenting behaviors during the covid-19 pandemic and explored daily difficulties and helpful activities during the covid-19 pandemic linked to their well-being. for some families, spending more time together during a lockdown may bring family members closer towards each other and foster a sense of well-being. however, several factors that are emblematic for the covid-19 crisis, such as financial insecurity, concerns about own and others' health, uncertainty about quarantine duration, lack of social and physical activities, and boredom have all frequently been shown to negatively affect a person's mood and mental wellbeing [4] [5] [6] [7] [8] . moreover, parents and adolescents may also experience stress because they are faced with more daily hassles (e.g. a suboptimal work or school environment) and additional tasks (e.g. parents homeschooling their children or caring for significant others). previous studies have shown that the impact of these quarantine related factors on mental health outcomes (e.g. depressive symptoms, anxiety, and ptsd) can be wide-ranging, substantial and long-lasting (see review of brooks et al. [9] ). as a consequence, these confinements may also lead to more tension, irritability, family conflicts, and at worse, domestic violence or child abuse [10] . one of the key questions that have been raised by governmental agencies and health care workers is to what extent the covid-19 pandemic and the associated distancing measures affect families' well-being and parenting behaviors. in this study, dutch adolescents and their parents filled in 14 days of ecological momentary assessments (ema; [11] ) twice, before the covid-19 outbreak (2018-2019) and also during the covid-19 pandemic (14-28 april 2020). in addition, we asked parents and adolescents about daily difficulties and helpful activities during the covid-19 pandemic that possibly influenced their affect in positive and negative ways. this enabled us to investigate how and to what extent well-being and parenting behaviors in daily life were impacted by the covid-19 pandemic and the related social distancing measures. gaining more insight into these processes, our findings can contribute to formulating recommendations for policy makers and mental health professionals. individuals' affect states are not one-dimensional and static in nature, but can fluctuate from moment to moment in response to other individuals and external circumstances (e.g., [12] ). positive and negative affect reflect a persons' momentary mood state. both positive and negative affect have implications for health and well-being over time for adults and adolescents [13] [14] [15] [16] [17] [18] . positive affect predominantly generates action, motivation, social connectedness and cognitive flexibility, whereas negative affect might result in actions such as avoidance, attack, or expel [19, 20] . using momentary assessments enabled us to identify the potential impact of the pandemic on parents' and adolescents' positive and negative affect in daily life without the potential bias of retrospective recall. the covid-19 pandemic and the related social measures might also impact parenting behaviors, such as the amount of expressed warmth and criticism. parental warmth is typically considered as one of the primary dimensions of sensitive parenting behavior and can include acceptance, support, and positive involvement towards the child [21] . parental criticism can be defined as expressing negativity, disapproval, or dissatisfaction to a child [22] . psychological distress related to the covid-19 pandemic may influence parenting behaviors, with parents being more emotionally withdrawn or critical and irritated, instead of being supportive, sensitive and encouraging to the child [23] . previous studies have shown that especially positive mood of family members is closely related to warm family interactions, whereas negative mood is related to withdrawal from interactions [19, [24] [25] [26] . however, no prior studies have examined the effects of a situation comparable to the current covid-19 pandemic on parenting. therefore, in addition to its impact on affect, we also aimed to investigate the impact of the covid-19 pandemic and its consequences on parental warmth and criticism in daily life. since parenting is a dynamic process [16] , we will examine day-to-day parental warmth and criticism. furthermore, as perspectives from parents and adolescents on parenting might differ (e.g., [27] ), we examined both the parent and adolescent perspective on parental warmth and criticism. a crucial aspect of unforeseen stressful situations, such as the covid-19 pandemic, is uncertainty. uncertainty is one of the key determinants of experienced levels of stress [28] [29] [30] . moreover, the ability to deal with uncertainty varies widely. while some people can tolerate uncertainty very well, others have difficulties tolerating uncertainty and try to avoid it at best [31] [32] [33] . intolerance of uncertainty (iu) is described as a predisposition to negatively perceive and respond to uncertain information and situations, irrespective of its probability and outcomes [34, 35] . as the worldwide covid-19 pandemic influenced daily life for all people, escaping from the accompanied uncertainty is deemed impossible. consequently, parents and adolescents with higher levels of iu might experience greater distress under the current circumstances, which might in turn also impact their affect and parenting behaviors. no prior studies have investigated the relation between iu and daily affect and parenting behavior within the family context. this was pursued in the present study. in the light of the pandemic, it is also examined to what extent iu is related to a change in affect and parenting behaviors. in the present study, we examined the impact of the covid-19 pandemic on daily affect and parenting of both dutch parents and adolescents. the aims were: (1) to explore parents' and adolescents' daily difficulties and helpful activities during the covid-19 pandemic, (2) to examine and compare positive and negative affect of both parents and adolescents during 2 weeks of the covid-19 pandemic and a similar 2-week period pre-pandemic (from now on referred to as baseline), (3) to examine and compare (perceived) parenting behaviors in terms of parental warmth and criticism towards the adolescent (as assessed by both the adolescent and the parent) during 2 weeks of the covid-19 pandemic and a similar 2-week period prepandemic, (4) to examine whether parents' and adolescents' levels of iu at baseline are associated with affect and parenting behaviors in general, and (5) as well as with the hypothesized changes in affect and (perceived) parental warmth and criticism. we expect an increase of negative affect and a decrease in positive affect for both parents and adolescents during the covid-19 pandemic as compared to baseline. regarding parenting behaviors, we expect lower levels of parental warmth and higher levels of parental criticism during the covid-19 pandemic as compared to baseline, both from the perspective of parents and adolescents. with respect to iu, we expect that higher levels of iu predict higher levels of negative affect and lower levels of positive affect in parents and adolescents at both time points, as well as a greater increase in negative affect and decrease in positive affect during the covid-19 pandemic compared to baseline. the current study was based on baseline data of the ongoing dutch multi-method two-generation re-pair study: 'relations and emotions in parent-adolescent interaction research' and on the follow-up assessment 're-pair during the covid-19 pandemic'. in re-pair, we examine the relation between parent-child interactions and adolescent mental well-being. the study design and in-and exclusion criteria of the baseline assessment can be found in s1 text. the current study included data from adolescents without psychopathology and their parents (i.e., healthy control families). inclusion criteria for the adolescents to participate in the current study at baseline were: being aged between 11 and 17 years, living at home with at least one primary caregiver, going to high school or higher education, and a good command of the dutch language. adolescents were excluded if they had a current mental disorder, a life-time history of major depressive disorder or dysthymia, or a history of psychopathology in the past two years. adolescent psychopathology was assessed at baseline during a face-to-face interview using the structured interview of the kiddie-schedule for affective disorders and schizophrenia-present and lifetime version (k-sads-pl [36] ). for parents, no in-or exclusion criteria were specified, except for a good command of the dutch language. to participate in the follow-up during the covid-19 pandemic the adolescent had to still live at home with at least one caregiver. adolescents and parents were allowed to sign up individually. from the 80 adolescents and 151 parents who were contacted for the follow-up assessment during the covid-19 pandemic, 51 individuals (14 adolescents and 37 parents) did not respond to any of the attempts of contact from the researchers. of the individuals who did respond, 76 (31 adolescents and 45 parents) were not willing to participate. reasons were: being busy and having other priorities (i.e., work, school, taking care of children or parents). the remaining 104 participants gave consent to participate. two participants did not start the ema and one participant did not complete the measures and hence, the final sample of the current study included 101 participants, consisting of 34 adolescents and 67 parents. descriptive statistics of the current sample are described in the result section and in table 1 . recruitment of the participants was done via social media, advertisements, and flyers, with a specific focus on the inclusion of both parents (i.e., mothers and fathers). the focus was on primary caregivers, so not only biological parents could participate, but also stepparents and guardians, as long as they played an important role in the upbringing of the adolescent. does the covid-19 pandemic impact parents' and adolescents' well-being interested families could sign-up for the study via the website or mail and received information letters. approximately two weeks later families were contacted by phone by one of the researchers to provide them with more information and check the inclusion criteria. if all criteria were met, families could participate in the study. all participants signed informed consent (including consent to contact them to request to participate in follow-up research). in addition, for adolescents younger than 16 years of age, both parents with legal custody signed informed consent. the families completed the ema in the period between september 2018 and november 2019 with ema not taking place during holidays and exam weeks of the adolescent. instructions on the ema were given face-to-face prior to the baseline assessment and researchers assisted with installing the ethica app [37] on the smartphone of the adolescent and both parents. each family member also received written instructions and their individual account information. for participation in the ema, parents received €20,and adolescents €10,-. in addition, four gift vouchers of €75,were raffled based on compliance. all families who participated at baseline were invited for the follow-up in april 2020. the follow-up assessment was announced in a newsletter followed by a personal e-mail, and reminders were sent to parents and adolescents who had not responded yet. parents and adolescents who agreed to participate were sent an online questionnaire on demographic characteristics and general mental well-being. thereafter, participants received written instructions on how to download and reinstall the ethica app. ema data collection took place one month into the lockdown, from april 14 th to april 28 th . for participation in the follow-up assessment, parents received €20,and adolescents €10,in gift vouchers. the current study focusses on the ema data of the baseline assessment (2018-2019) and the follow-up assessment (2020). the re-pair study was approved by the medical ethics committee of leiden university medical center (lumc) in leiden, the netherlands (nl62502.058.17) and the follow-up assessment 're-pair during the covid-19 pandemic' was approved by the psychology research ethics committee of leiden university in leiden, the netherlands (2020-03-30-b.m. elzinga-v2-2334). ema. the ema procedures and set-ups were almost entirely similar at baseline and during the covid-19 pandemic and consisted of filling out questionnaires at four timepoints per day, for 14 consecutive days on parents' and adolescents' own smartphones using the mobile app ethica (ethica data, 2019). at all timepoints participants completed questions about their affect and how they experienced contact with the last person they interacted with. detailed information on the concepts in the questionnaires, triggering schedules, differences in set-up, number of items and completing time, and monitoring process can be found in s2 text. compliance. the overall response rate at baseline was 81.0%. adolescents affect. momentary affect states of parents and adolescents were assessed four times per day with a slightly adapted and shortened four-item version of the positive and negative affect schedule for children (panas-c; [38, 39] ). at each timepoint participants were asked "how do you feel at the moment?" followed by two positive affect states "happy" and "relaxed", and two negative affect states "sad" and "irritated". each affect state was rated on a 7-point likert scale, ranging from 1 (not at all) to 7 (very). a mean score of the positive affect state was calculated per moment to create a momentary pa scale and a mean score of the negative affect state was calculated per moment to create a momentary na scale. a higher score represented higher levels of pa or na. daily parenting. in the last questionnaire of each day, adolescents were asked to indicate with whom they spoke during that day (i.e., mother, father, stepmother, stepfather), and if so, to rate each parent's warmth and criticism by answering the questions "throughout the day, how warm/loving was your parent towards you?" and "throughout the day, how critical was your parent towards you?" on a 7-point likert scale ranging from 1 (not at all) to 7 (very). if adolescents only reported on mother and stepfather for instance throughout the ema, scores about stepfathers were recoded as father. this was the case for two adolescents during the baseline and three adolescents during the covid-19 pandemic. one adolescent reported on four caregivers (i.e. biological parents and stepparents) during both periods and we included scores about biological parents because these were mostly rated. in the questionnaire at the end of each day parents also had to indicate whether they spoke to their child (i.e., the participating adolescent) and if so, to rate their own behavior towards their child by answering the questions "how warm/loving were you towards your child?" and "how critical were you towards your child?" on a 7-point likert scale ranging from 1 (not at all) to 7 (very). both for adolescent and parent report, a higher score represented more warmth and more criticism. daily difficulties and helpful activities. to assess the difficulties and helpful activities during the covid-19 pandemic, at the end of each day, participants were asked to choose items from a list of potential activities. parents and adolescents could select almost similar activities and it was possible to give multiple answers. the list of potential daily difficulties consisted of: boredom, fights/conflicts, work (for parents)/homework (for adolescents), irritations with family members, noise disturbance, loneliness, missing social contact with friends, worries about own health, worries about health of others, concerns about the coronavirus in general, coronavirus-related news items or 'anything else, namely. . .'. the list of potential helpful activities consisted of: work (for parents)/homework (for adolescents), watching series/television, listening to music, gaming, social media, reading a book, sports, chilling, online contact with relatives or friends, being together with the family, card or board games, diy or crafts, cooking/dining, 'anything else, namely'. based on the total number of observed responses a top 5 of daily difficulties and helpful activities was composed. percentages were calculated by dividing the number of observed responses on one activity by the total of given answers. intolerance of uncertainty. the 12-item version of the intolerance of uncertainty scale (ius; [40] ) was used to assess iu of parents and adolescents. participants completed this questionnaire online prior to baseline. the 12 items of the ius (e.g., "uncertainty makes me uneasy, anxious, or stressed." or "i should be able to organize everything in advance.") were answered on a 5-point likert scale ranging from 1 (strongly disagree) to 5 (strongly agree). a higher sum score represents higher levels of intolerance of uncertainty. both the original and the 12-item version of the ius appear to have satisfactory concurrent, discriminant, and predictive validity [41] . internal consistency of the scale was good with a cronbach's alpha of .81 for adolescents and .83 for parents. depressive symptoms. the patient health questionnaire (phq-9; [42] ) was used to screen for the presence of depressive symptoms during the past two weeks. depressive symptoms were assessed at both timepoints. the items are based on nine dsm-iv criteria for depression and are scored as 0 (not at all) to 3 (nearly every day). the phq-9 has been validated for use in primary care. sum scores range from 0 to 27 and a score above 10 is suggestive of the presence of depression [43] . for parents, the cronbach's alpha at baseline was .79 and during the covid-19 pandemic .73. for adolescents, cronbach's alpha at baseline was .53 and during the covid-19 pandemic .76. parents and adolescents reported repeatedly on positive affect, negative affect, parental warmth, and parental criticism at baseline and during the covid-19 pandemic. these repeated measures (level 1) were nested within individuals (level 2). given this nested structure of the data, multilevel modelling [44] was used for the main analyses. models were specified in r version 3.6.1 [45] , using the multilevel version 2.6 [46] package to test our hypotheses with maximum likelihood (ml) estimation. level 2 predictors were grand-mean centered, following guidelines proposed by hoffman [47] and bolger and laurenceau [48] . to evaluate within-person change in positive affect, negative affect, parental warmth, and parental criticism from baseline to the covid-19 pandemic, a series of models were tested. separate models were tested per outcome and per informant (adolescents and parents), resulting in a total of 8 models. per model, several similar steps were taken. first, we specified an unconditional random intercept model with covariance structure (model 1). for more information on the selection of covariance structure and results see s3 text. second, we added period as predictor (model 2), which was scored 0 (baseline) and 1 (during the covid-19 pandemic) to model change. for example, to model change in positive affect, we specified period as the predictor and positive affect as the outcome. the intercept of the model estimates is positive affect score at baseline and the slope of the model is the estimated change from baseline to during the covid-19 pandemic. fourth, we added a random effect (model 3) indicating that the change from baseline to during the covid-19 pandemic could vary between persons. significant changes in model fit were tested with likelihood ratio tests (following guidelines of hox [44] ). fifth, we examined whether the changes were predicted by iu by adding a main effect of iu (model 4). in the models on parental warmth and parental criticism gender of parents was also added to the model as main effect to test for possible gender differences. in the final model (model 5), we also added an interaction term of iu with period to test the possible moderating role of iu. since two parents of a same family could participate in the study, a third level (family) was specified in all models including parents (model 1b). to not overcomplicate our models, we tested whether adding family level (level 3) to model 1 for parents improved the model fit based on the likelihood ratio tests. only if these tests were significant, the third level remained in the model. since adolescents could report on parenting of fathers and mothers, family was specified as extra level in the models concerning parental warmth and parental criticism reported by adolescents (model 1b). for adolescents, answers on father and mother (level 2) are nested within adolescents (level 3). we tested whether adding parent level (level 2) to model 1 for adolescents improved the model fit based on the likelihood ratio tests. if these tests were significant, the second level remained in the model. we used two-tailed tests with an α = 0.05. the analytic plan for this study was uploaded to open science framework prior to the analyses (preregistered at april 27 th , osf.io/34ycu). in the current study, 67 dutch parents (age range during the covid-19 pandemic: 36.25-71.04 years) and 34 adolescents (age range during the covid-19 pandemic: 14.66-19.01 years) participated. participant characteristics can be found in table 1 . the sample reported little to none depressive symptoms as measured with the phq-9. phq-9 scores of adolescents ranged between 0-9 at baseline and between 0-16 during the covid-19 pandemic. phq-9 scores of parents ranged between 0-16 at baseline and between 0-16 during the covid-19 pandemic. levels of depressive symptoms did not differ between the two periods for adolescents (t = 1.11, df = 33, p = .275) and parents (t = 1.24, df = 67, p = .221). information on household composition of participating families can be found in s3 text. correlations between study variables (gender, age, affect, parenting behavior, and iu) can be found in s1 parents. of all parents, 91% (n = 61) were currently employed, 6% (n = 4) were unemployed and 3% (n = 2) were unable to work or lost their job due to the covid-19 pandemic. during the 14 days of ema, 53.7% of the parents who were employed worked more from home, 7.5% worked less from home and 38.8% worked just as much from home as compared to the period before the covid-19 pandemic. all parents indicated owning a house with a garden and having a living surface >100m2. of our sample, 17.9% (n = 12) of the parents reported having covid-19 related symptoms during the 14 days of ema. during the covid-19 pandemic, the most reported daily difficulties across the 14 days of ema for parents were (1) missing social contact with friends (14.6%), (2) concerns about the coronavirus in general (13.5%), (3) irritations with family members (12.8%), (4) worrying about health of others (8.3%), and (5) coronavirus-related news items (8.0%). it was also asked daily which activities were helpful during the day. the top 5 of helpful activities reported by parents was (1) being together with family (20.0%), (2) cooking/dining (14.4%), (3) watching television/series (9.9%), (4) work (7.4%), and (5) online contact with relatives or friends (6.2%). adolescents. due to the covid-19 pandemic all national final school exams were canceled and some high schoolers already graduated (or not) based on their prior school exams, 5 (21.7%) adolescents graduated promptly in march 2020 prior to the 14 days of ema. of our adolescent sample, one person reported having covid-19 related symptoms during the 14 days of ema. for adolescents (n = 34) the top 5 daily difficulties was (1) boredom (22.9%), (2) missing social contact with friends (17.7%), (3) irritations with family members (13.1%), (4) homework (12.3%), and (5) worry about the health of others (6.4%). the top 5 helpful activities for adolescents were (1) chilling (12.9%), (2) watching television/series (11.4%), (3) online contact with relatives or friends (11.0%), (4) listening to music (10.8%), and (5) being together with the family (9.6%). affect: parent reports. first, an unconditional means model of negative affect with the intercept only was built (referred to as 'model 1'-complete model results of parents can be found in s3 table, model fit statistics of parents can be found in s4 table) . the intraclass correlation coefficient (icc) was .31 on the person level, indicating that moderate concordance of negative affect across time points within persons existed. next, family was added as level to the unconditional means model (model 1b). the icc of the family level was .11, which indicates that some concordance of negative affect existed within families. however, the model fit did not improve significantly (χ2(1) = 1.581, p = .209) and family level was therefore removed from the model. next, in model 2, we tested change in negative affect from baseline to during the covid-19 pandemic by adding period to the model. parents reported more negative affect during covid-19 pandemic as compared to the baseline (b = 0.096, se = .025, df = 5982, t = 3.900, p < .001). adding individual variance in model 3 improved the model fit significantly (χ2(2) = 56.613, p < .001). in model 4, we added iu which was significantly associated with negative affect (b = 0.022, se = .010, df = 62, t = 2.075, p = .042) indicating that more iu was related to more negative affect (main effect). lastly, we added iu as moderator in model 5 and results of this final model are presented in table 2 . no moderating effect of iu was found (b = 0.002, se = .007, df = 5752, t = 0.225, p = .822) and iu was no longer significantly associated with negative affect (b = 0.021, se = .011, df = 62, t = 1.960, p = .054), but period remained significantly associated with negative affect. results are shown in fig 2. for positive affect, the same steps were followed. model 1 showed an icc of .32 and adding family level (model 1b) did not significantly improve the model fit (χ2(1) = 0.738, p = .390). results of model 2 showed that parents' positive affect did not differ across the two periods (b = 0.012, se = .028, df = 5986, t = 0.404, p = .686). adding individual variance in model 3 improved the model fit significantly (χ2(2) = 122.186, p < .001). in model 4 iu was added as a main effect, but no significant association with positive affect was found. lastly, iu was added as moderator in model 5, but no moderating effect of iu was found (b = -0.008, se = .009, df = 5756, t = -0.823, p = .411). results of this final model are presented in table 2 . affect: adolescent reports. in model 1, the icc of negative affect on the person level was .32 (complete model results of adolescents can be found in s5 table, model fit statistics of adolescents can be found in s6 table) . results of model 2 showed that there was no significant change in adolescent negative affect (b = 0.016, se = .027, df = 2618, t = 0.595, p = .552). adding individual variance in model 3 improved the model fit significantly (χ2(2) = 39.759, p < .001). in model 4, we added iu as a main effect which was significantly associated with negative affect (b = 0.030, se = .011, df = 30, t = 2.737, p = .010) indicating that more iu was related to more negative affect. iu was added as moderator in model 5 and iu remained significantly associated with negative affect, but no moderating effect of iu was found (b = -0.006, se = table 2 . results of final model 5 on the relation between period and affect and the moderating role of intolerance of uncertainty in parents. table 3 . parenting: parent reports. in model 1, the icc of parental criticism on the person level was .39 (complete model results of parents can be found in s3 table, model fit statistics of parents can be found in s4 table) . adding family level (model 1b) did significantly improve the model fit (χ2(1) = 5.430, p = .020) with an icc of .20 at the family level and 'family' remained in the model. results of model 2 showed that no difference in parental criticism between baseline and during the covid-19 pandemic was found (b = 0.126, se = .064, df = 1530, t = 1.963, p = .050). adding individual variance in model 3 improved the model fit significantly (χ2(4) = 39.527, p < .001). in model 4, we added iu and gender of the parent as main effects. both were not significantly associated with parental criticism. iu was added as table 3 . results of final model 5 on the relation between period and affect and the moderating role of intolerance of uncertainty in adolescents. table 4 . for parental warmth in model 1, the icc on the person level was .46 and adding family level (model 1b) did not significantly improve the model fit (χ2(1) = 0.761, p = .383). no significant change in parental warmth (b = 0.010, se = .038, df = 1530, t = 0.255, p = .799) was found in model 2. adding individual variance in model 3 improved the model fit significantly (χ2(2) = 22.499, p < .001). in model 4, we added iu and gender of parent and both were not significantly associated with parental warmth. iu was added as moderator in model 5, but no moderating effect of iu was found (b = 0.004, se = .008, df = 1466, t = .489, p = .625). results of this final model are presented in table 4 . parenting: adolescent reports. in model 1, the icc of parental criticism on the person level was .45 (complete model results of adolescents can be found in s5 table, model fit statistics of adolescents can be found in s6 table) . adding family level (model 1b) did not significantly improve the model fit (χ2(1) = 2.925, p = .087). results of model 2 showed that the change in reports on parental criticism between baseline and during the covid-19 pandemic was not significant (b = 0.036, se = .062, df = 1350, t = 0.576, p = .565). adding individual variance in model 3 improved the model fit significantly (χ2(2) = 53.931, p < .001). in model 4, we added iu and gender of parent as main effects. gender of parent was significantly associated with reports on parental criticism (b = -0.121, se = .058, df = 1268, t = -2.099, p = .036), indicating that adolescents reported more parental criticism of mothers than fathers. iu was not significantly associated with parental criticism. iu was added as moderator in model 5, but no moderating effect of iu was found (b = 0.028, se = .021, df = 1267, t = 0.083, p = .934). results of this final model are presented in table 5 . gender of parents remained significantly associated with parental criticism. table 4 . results of final model 5 on the relation between period and daily parenting behavior and the moderating role of intolerance of uncertainty in parents. does the covid-19 pandemic impact parents' and adolescents' well-being in model 4, we added iu and gender of parent and both were not significantly associated with parental warmth. iu was added as moderator in model 5, but no moderating effect of iu was found (b = 0.002, se = .021, df = 1267, t = 0.083, p = .934). results of this final model are presented in table 5 . post hoc analyses on increase in parents' negative affect during the covid-19 pandemic. as iu did not explain why parents reported more negative affect during covid-19 pandemic as compared to the baseline, we did some post hoc analyses to examine whether characteristics related to the lockdown and the covid-19 pandemic were associated with the increase of parents' negative affect. living surface, income, having suffered from covid-19 symptoms, helping children with school at home, working from home, going to work, daily difficulties during the past two weeks of covid-19, and working with covid-19 patients were examined (see s7 and s8 tables for description of the ema items). none of these characteristics were related to the increase of parents' negative affect during the covid-19 pandemic as compared to the baseline (all p-values < .001). in this study we (1) explored parents' and adolescents' daily difficulties and helpful activities during the covid-19 pandemic (2) examined positive and negative affect of both parents and adolescents during 2 weeks of the covid-19 pandemic and compared them to a 2-week baseline period pre-pandemic, (3) examined parenting behaviors (assessed by both the adolescent and the parent) and compared parental warmth and criticism towards the adolescent during 2 weeks of the covid-19 pandemic and a 2-week baseline period, (4) examined whether parents' and adolescents' levels of iu at baseline are associated with affect and parenting in general, and (5) as well as with the hypothesized changes in affect and (perceived) parental warmth and criticism. most importantly, both parents and adolescents were bothered by a lack of social contact with friends, by irritations with family members, and worried about the health of others. this might table 5 . results of final model 5 on the relation between period and daily parenting behavior and the moderating role of intolerance of uncertainty in adolescents. does the covid-19 pandemic impact parents' and adolescents' well-being be a logical consequence of the lockdown and social distancing. remarkably, adolescents struggled with boredom whereas this was not the case for parents. parents worried about the coronavirus in general, while this did not bother adolescents that much. in response to social distancing, online contact with relatives or friends aided both parents and adolescents to cope with the situation. in addition, watching tv-shows was also mentioned as a helpful activity by parents and adolescents. other activities that helped to cope with the situation varied across parents and adolescents. while parents reported to benefit from being together with family and cooking and dining, adolescents reported chilling and listening to music. previous studies have shown that quarantine and quarantine-related issues (i.e., financial insecurity, fear of infection, uncertainty about duration) in general have a negative influence on adult mood and mental well-being [9] . therefore, it was expected that the covid-19 pandemic and lockdown would increase negative affect and decrease positive affect as compared with a period before the lockdown. our results show that, indeed, parents' negative affect increased as compared to the period before the lockdown. important to note is that we collected data during 5 th and 6 th week of the lockdown in the netherlands with only minor prospects of easing regulations. we also explored whether other pandemic-related characteristics (i.e. living surface, income, relatives with covid-19, hours of working at home, helping children with school and contact with covid-19 patients at work) were linked to the increase of negative affect in parents. this was not the case. our findings suggested however the presence of heterogeneity among individuals. all our models improved significantly when allowing the associations between period (2 weeks of the covid-19 pandemic versus a similar 2-week baseline period) and affect and parenting behavior to vary across individuals, which is in line with the theoretical notion of differential susceptibility (e.g., [49] ). whether or not parents and adolescents experience (emotional) problems during lockdown can clearly vary from household to household, suggesting that in general families seem to be able to adapt to the circumstances, but that some families struggle. this is important to keep in mind for potential future measures of social distancing. it was expected that the forced social distance during the covid-19 pandemic and particularly the physical distance from friends and peers and the school closure would result in an increase of negative affect and decrease of positive affect in adolescents (see also loades et al. [50] ). yet, in our study, no differences in adolescent reports on negative affect were found during the covid-19 pandemic as compared to a baseline period. as for adults, the opportunities for adolescents of online social interaction might have buffered feelings of isolation or loneliness and bolstered mental well-being during the covid-19 pandemic [51] . moreover, it should be noted that our sample is considered healthy on average, based on the phq-9 scores, and lived in relatively favorable circumstances (e.g., high socioeconomic status). affect of adolescents with (subclinical) mental health issues (e.g. depressive or anxiety symptoms) or living under less fortune circumstances might be more influenced during the covid-19 pandemic. therefore, it is important to examine the effect of the covid-19 pandemic in clinical samples to elucidate its effect on psychopathology. moreover, it should be noted that our assessments were in the rather poignant phase of social lock down, when school closings may also have yielded relief for some adolescents. even though individuals thrive to become independent during adolescence and start to explore the environment outside family household [2, 3] this period of enforced proximity did not seem to affect adolescents on the short-term. potentially, the endurance of the lockdown may have more detrimental effects on adolescent well-being. not for parents nor for adolescents, a change in positive affect was found. despite the increase of stress and uncertainty around the covid-19 pandemic, disasters such as a pandemic also might increase the sense of social connectedness and morality [10] . this sense of shared social identity and the feeling of 'we are all in this together' can be related to positive affect [20] , which could explain why positive affect did not decrease in the present study. in families, as in our sample, no one was home alone, and one could still have online social interactions with others outside the household. to that end, 'physical distancing' might be a better term for the imposed social isolation or social distance, as was previously suggested in literature [10] . as mentioned before, the covid-19 pandemic and the related lockdown may lead to more tension, irritability, and family conflicts or worse [10] . notably, parent' affect and parenting behavior are interrelated and are both involved in giving comfort, expressing approval or expressing criticism [52, 53] . for instance, parents who worry more, express more criticism towards their adolescents, indicating that a negative affect promotes insensitive and in more extreme cases abusive parenting behavior, whereas positive affect strongly relates to supportive parenting [52, 53] . regarding parenting behaviors, we therefore expected higher levels of parental criticism and lower levels of parental warmth during the covid-19 pandemic as compared to baseline. we found, however, that parental warmth and criticism from both parent and adolescent perspective, did not differ between before and during the covid-19 pandemic. interestingly, even though negative affect of parents increased compared to the period before lockdown, this did not seem to affect parenting behavior (self-report and perceived by the adolescent). it should be noted that, in general, adolescents perceived their mothers as more critical compared with fathers, unrelated to measurement period. this might be due to the unique roles of mothers and fathers in caregiving and setting rules and boundaries [54, 55] . results showed that iu was related to more negative affect in both parents and adolescents, independent of the period of assessment. furthermore, in adolescents, iu was also linked to a decrease in positive affect, while for parents no link between iu and positive affect was found. it was expected that people with elevated iu levels might experience even greater distress under the covid-19 circumstances as compared to baseline, however our results do not support this. iu is often described as a predisposition to negatively perceive and respond to uncertain information and situations, irrespective of its probability and outcomes [34, 35] . apparently, it is negatively associated with affect in daily life, regardless of whether there are major threats and uncertainties, or more daily hassles. future research could elucidate why iu may particularly dampen positive affect in adolescents and not in adults. even though iu seems to relate to affect of parents and adolescents, it did not seem to spill over into parenting behaviors. these results give a first indication that iu also relates to more micro processes in daily life, for both adolescents and parents. firstly, the intensive longitudinal study design with multiple assessments per day enabled us to gain more fine-grained insights in affect and parenting behaviors in daily life and to consider individual differences. secondly, assessment during two periods, before and during the covid-19 pandemic, allowed us to detect changes due to the covid-19 pandemic. next to the strengths, it should be acknowledged that the sample (67 parents and 34 adolescents) was relatively small. second, it should be noted that the study sample consisted of overall healthy, well-functioning parents and adolescents. that is, adolescents were screened at baseline and were excluded if they had a current mental disorder, a history of psychopathology in the past two years, or a lifetime history of major depressive disorder or dysthymia. moreover, the phq-9 scores of adolescents and parents indicated few depressive symptoms. therefore, findings might not be applicable to adolescents and parents with (sub)clinical mental health problems or at-risk populations (e.g. refugees, low socioeconomic status), since these groups might be at increased risk of problems such as loneliness, negative affect or negative parenting practices during the covid-19 pandemic. lastly, it should be noted that information on long-term consequences of lockdown during the covid-19 pandemic is lacking. prior research has suggested that the impact of stress can be altered by mindsets and appraisals of stressful events [10, 56, 57] . these factors could possibly explain the individual variations we found. for instance, people with low expectations of the course of events might adapt relatively well to new situations and, therefore, experience little emotional problems. moreover, adaptive mindsets about stressful events might increase positive emotions and reduce negative health symptoms [58] . considering these factors in future studies might be useful to elucidate individual differences in risk and resilience. in our study parents, but not adolescents, showed an increase of negative affect in a twoweek period (14-28 april 2020) during the covid-19 pandemic compared with a similar two-week baseline period pre-pandemic. positive affect and parenting behaviors 'warmth' and 'criticism' did not change. it can be concluded that, on average, parents and adolescents in our sample seem to deal fairly well with the circumstances. individuals and families differed however to what extent the covid-19 pandemic influenced their affect and (perspective of) parenting behavior. living surface, income, having suffered from covid-19 symptoms, helping children with school at home, working from home, going to work, difficulties during covid-19, and working with covid-19 patients did not explain the increase of parental negative affect. policy makers and mental health professionals working to prepare for potential disease outbreaks should be aware that the experience of being quarantined might affect individuals differently. each parent and adolescent could therefore benefit from a different coping strategy, as 'one size does not fit all'. providing easily accessible and safe ways to increase online contact for all ages and layers of society, recommending to search for distraction such as listening to music or watching television, and helping to accept the uncertain situation are for instance potential coping strategies. in this way, individuals can find ways that suit their own personal needs in order to benefit their well-being in times of a lockdown and social distancing measures. table a : model results on the relation between period and negative affect, and the moderating role of intolerance of uncertainty in parents. table b : model results on the relation between period and positive affect, and the moderating role of intolerance of uncertainty in parents. table c : model results on the relation between period and parental criticism, and the moderating role of intolerance of uncertainty in parents. table a : model results on the relation between period and negative affect, and the moderating role of intolerance of uncertainty in adolescents. table b : model results on the relation between period and positive affect, and the moderating role of intolerance of uncertainty in adolescents. table c : model results on the relation between period and parental criticism, and the moderating role of intolerance of uncertainty in adolescents. who announces covid-19 outbreak a pandemic handbook of parenting: children and parenting cognitive and affective development in adolescence the experience of quarantine for individuals affected by sars in toronto sars control and psychological effects of quarantine depression after exposure to stressful events: lessons learned from the severe acute respiratory syndrome epidemic posttraumatic stress disorder in parents and youth after health-related disasters. disaster medicine and public health preparedness mental health status of people isolated due to middle east respiratory syndrome the psychological impact of quarantine and how to reduce it: rapid review of the evidence. the lancet using social and behavioural science to support covid-19 pandemic response. nature human behaviour ecological momentary 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cut-off score for diagnosing depression with the patient health questionnaire (phq-9): a meta-analysis multilevel analysis: techniques and applications r: a language and environment for statistical computing austria: r foundation for statistical computing. retrieved from package 'multilevel longitudinal analysis: modeling within-person fluctuation and change. routledge intensive longitudinal methods: an introduction to diary and experience sampling research differential susceptibility to parenting and quality child care rapid systematic review: the impact of social isolation and loneliness on the mental health of children and adolescents in the context of covid-19 helping others regulate emotion predicts increased regulation of one's own emotions and decreased symptoms of depression the affective organization of parenting: adaptive and maladaptative processes relations between parental affect and parenting behaviors: a meta-analytic review. parenting father-child relationships. handbook of father involvement: multidisciplinary perspective +father-child+relationships.+handbook+of+father +involvement:+multidisciplinary+perspectives the role of fathers' versus mothers' parenting in emotion-regulation development from mid-late adolescence: disentangling between-family differences from within-family effects optimizing stress responses with reappraisal and mindset interventions: an integrated model arousal and physiological toughness: implications for mental and physical health the role of stress mindset in shaping cognitive, emotional, and physiological responses to challenging and threatening stress we are grateful to all the families that have invested their time by participating in this study. we furthermore thank ethica data for offering their services (i.e., use of the ethica app and platform) free of charge during the covid-19 pandemic. key: cord-299491-8rfm0jxh authors: xiao, shenglan; li, yuguo; wong, tze-wai; hui, david s. c. title: role of fomites in sars transmission during the largest hospital outbreak in hong kong date: 2017-07-20 journal: plos one doi: 10.1371/journal.pone.0181558 sha: doc_id: 299491 cord_uid: 8rfm0jxh the epidemic of severe acute respiratory syndrome (sars) had a significant effect on global society in the early 2000s and the potential of its resurgence exists. studies on the modes of transmission of sars are limited though a number of outbreak studies have revealed the possible airborne route. to develop more specific and effective control strategies, we conducted a detailed mechanism-based investigation that explored the role of fomite transmission in the well-known ward 8a outbreak. we considered three hypothetical transmission routes, i.e., the long-range airborne, fomite and combined routes, in 1,744 scenarios with combinations of some important parameters. a multi-agent model was used to predict the infection risk distributions of the three hypothetical routes. model selection was carried out for different scenarios to compare the distributions of infection risk with that of the reported attack rates and select the hypotheses with the best fitness. our results reveal that under the assumed conditions, the sars coronavirus was most possible to have spread via the combined long-range airborne and fomite routes, and that the fomite route played a non-negligible role in the transmission. the severe acute respiratory syndrome coronavirus (sars-cov) was a substantial global threat associated with significant morbidity and mortality in the early 2000s [1] . since its emergence in november 2002, the sars-cov had induced 8,096 cases, including 774 deaths, in 37 countries within 8 months [2] . although no new outbreaks have been reported since 2004 [3] , reported biosecurity breaches of sars-cov specimens in research facilities [4] [5] [6] [7] and continuous findings of sars-like coronaviruses in wild animals [8] [9] [10] suggest the distinct potential for a resurgence of sars [11] [12] [13] . like many other respiratory viruses, the sars-cov is suspected to spread from an infected person to the susceptible via three basic transmission routes, i.e., the long-range airborne, close contact and fomite routes [14] [15] [16] , as shown in fig 1. understanding of the relative importance of the three routes is limited, so the recommended infection control measures (standard, contact, droplet and airborne precautions [12, 17] ) have been vague and unfocused. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 due to safety and ethical concerns, experiments on human subjects are not appropriate [18] . several studies have proposed probable evidence for the airborne spread of the sars-cov based on the consistencies between bio-aerosol concentration distributions and reported attack rates [19] [20] [21] , but no mechanism-based investigations exist for the fomite route. nevertheless, the detection of positive environmental samples in sars outbreak hospitals [22] [23] [24] , infections caused by intranasal instillation in animal experiments [25, 26] and findings that hand washing reduces the infection rate [27] [28] [29] all reveal that the fomite route might have played a non-negligible role in transmission. to investigate the role the fomite route plays in sars-cov transmission, we conducted a detailed modelling study of the largest hospital outbreak in hong kong [20] , in which the distribution of reported attack rates of inpatients showed a statistically significant spatial pattern. since the index inpatient was weak and bedridden [30] , we excluded the possibility of the close contact route from the index patient to other inpatients and identified three hypotheses, namely the single-route long-range airborne transmission (hypothesis 1 [long air]), the single-route fomite transmission (hypothesis 2 [fomite] ) and the two-route combination (hypothesis 3 [long air + fomite]). based on a typical 3-shift rotation over 24 hours, six routine round patterns of healthcare workers (hcws) were considered. a multi-agent model ( fig 2) was developed to simulate the possible spread of the viruses from the index patient to the susceptible by air flow and surface touching, and to calculate the possible exposure doses and infection risks for each hypothesis. model selection was carried out in 1,744 scenarios with various combinations of 4 important parameters. the results reported as follows provide probable evidence for the additional fomite transmission of the sars-cov under assumed conditions. as shown in fig 3a, the outbreak occurred in a general medical ward, ward 8a, in the prince of wales hospital in early march 2003 [31] . the index patient was a 26-year-old man who developed fever and cough on february 24, 2003 and was admitted to bed 11 in ward 8a on march 4 [32] . as his condition deteriorated with difficulty in expectorating sputum, he was treated with salbutamol via a jet nebulizer four times a day from march 6 to march 12 to facilitate mucociliary clearance [20] . on march 13, 2003 , after he was identified as the index patient for the outbreak, he was transferred to an isolation room [20] . thus, the period of march 4-12, 2003 was taken as the suspected exposure period. seven groups of people are assumed to be involved in transmission during the exposure period, including inpatients, visitors, doctors, nurses, health assistants, cleaners and medical students. the infection patterns of inpatients were studied because their behaviour was simpler than that of hcws and visitors, and they presented more available data than medical students [20] . during the hospitalisation of the index patient, 30 of 74 inpatients were infected. as shown in fig 3a, the distribution of infected inpatients exhibited a clear pattern (p = 0.0015, pearson chi-square test), with the highest attack rate (0.6500, 13 of 20 inpatients) in the source cubicle (including beds 9x, 9-16 and 16x), a little lower (0.5238, 11 of 21 inpatients) in the adjacent cubicle (including beds 17x, 17-24 and 24x) and lowest (0.1818, 6 of 33 inpatients) in the remote cubicles (including beds 1x, 1-8, 25x, 25-32 and 32x) [20] . as the information for this outbreak and the studies on properties of sars-cov are not sufficient, we made three major assumptions to build our model as follows. first, six representative hcws' routine round patterns were considered, and the contact modes between hcws and different patients were assumed to be the same. as shown in fig 3c and figure d (iii) in s1 file, doctors and nurses were responsible for all of the inpatients, and they examined all of the patients in the ward in clockwise and anticlockwise directions in patterns 1 and 2, respectively. as shown in fig 3e and figure d (v) in s1 file, each doctor and each nurse was responsible for inpatients in a cubicle, and examined them in clockwise and anticlockwise directions in patterns 3 and 4, respectively. in addition, in the scenario in which doctors or nurses allocated the patients in the order in which they checked in, the inpatients for whom a doctor or a nurse took responsibility might have been random. in that scenario, each doctor and each nurse examined a random nine or ten inpatients (e.g., circles of four colours in fig 3g and figure d (vii) in s1 file) in the ward in clockwise and anticlockwise directions in patterns 5 and 6, respectively. second, the uncertain parameters related to the properties of sars-cov were assumed and individual differences were not considered, as listed in s1 file. these parameters included surface areas ( table b in s1 file), transfer rate between surfaces (table c in s1 file), virus inactivation rates on surfaces (table d in s1 file), virus loads (table e in s1 file), dose-response parameters (table f in s1 file) and the largest virus-containing droplet size (table i in s1 file) . third, for all susceptible patients, the length of exposure period were assumed to be same (march [4] [5] [6] [7] [8] [9] [10] [11] [12] 2003 ) and the index patient was assumed to be the only source. since the information about admission and discharge timing of patients were vague in the outbreak related reports and researches [20, 30, 32] , we could not estimate the exposure period for every patient. as for the virus source, although there were 13 normal patients getting infected during the exposure period [20] , the viral load was still low compared to the index patient [33] . therefore, we did not consider the transmission from early-onset cases to the later cases. a multi-agent model was used to model the spread of the sars-cov from the index patient to the susceptible and predict the infection risk distributions from the three hypotheses. fig 2 shows the system architecture of the modular-based model, which includes four parts: the initialization generator, simulation engine, global database and data processing module. initialization generator had two branches, namely geometric generator and agents generator. geometric generator was used to build the virtual physical environment and produce surfaces. eighteen kinds of representative surfaces were identified (table a in s1 file) and categorised into five types of material: porous surfaces, non-porous surfaces, toilet surfaces, skin and mucous membranes, which differed in their properties (tables b and c in s1 file). in this study, 'mucous membranes' refers in particular to the exposure site for the fomite route, namely the mucous membranes of eyes, noses and mouths [15] . agents generator was used to create representative individuals in the outbreak. agents in seven representative roles (inpatients, visitors, doctors, nurses, health assistants, cleaners and medical students) were identified as study objects, (table h in s1 file) and each agent corresponded to a person in the outbreak. the core of the model, the simulation engine, including seven behaviour models, was used to set behaviour rules and simulate the behaviour of agents. the frequencies and the touching sequences for different types of behaviour are shown in tables f and g in s1 file. the heterogeneity was retained for every agent, so agents behaved independently. after every time step, the information about the agents was sent to update the global database, which temporarily recorded the agents' state information and the contamination situations of surfaces and air. the data processing module was used to calculate the exposure dose and infection risk. for the long-range airborne route, the multi-zone model [21, 34, 35] and long-range airborne route exposure model [36] were used to acquire the aerosol concentrations in the six zones of ward 8a and exposure doses in the respiratory tract, respectively. for the fomite route, a surface contamination model was used to calculate the number of viruses exchanged between surfaces in every touching process and the exposure doses on the mucous membranes. the infection risk of every agent for the three hypothesised transmission modes was calculated by the dose-response relationship model [36, 37] . details of these mathematical models are provided in s1 file. with the multi-agent modelling framework, we calculated the average infection risk for every region (source cubicle, adjacent cubicle and remote cubicles). in this study, maximizing fit was selected as the approach to model selection [38] . in this approach, the residual sum of squares (rss), as a measure of fit [39] , was calculated for every hypothesis. since a small rss indicates a good fit of the model to the data, the hypothesis with the minimum rss was selected. in this study, since several uncertain parameters related to the properties of sars-cov were assumed, we investigated some important ones and discussed their value ranges. as suggested by gao [36] , the largest virus-containing droplet size, dose-response parameters in respiratory tracts and on mucous membranes and viral load all greatly influence infection risk, but the related measurements are lacking in the literature. in this study, the viral loads during the exposure period (march 4-12, 2003) were assumed to vary according to the measured data of peiris et al. [33] , increasing at first, reaching a peak on the 10th day after the onset of symptoms and then decreasing. therefore, the viral load coefficient was defined as the ratio of the viral load in the computation to the average values in [33] . the coefficient was assumed to be constant for the total exposure period. to reduce the number of variables, η r , η m and c l were combined as the products η r c l and η m c l , defined as the dose effects of introducing c l mrna copies of sars-cov to the respiratory tract and mucous membranes, respectively. in summary, the ranges of three parameters were investigated in the study, namely the largest virus-containing droplet size d g (four values; 50, 100, 150 and 200 μm) ; products of the viral load coefficient and dose-response parameters in respiratory tracts η r c l (26 values, 10 −1 -10 4 / mrna copy) and on mucous membranes η m c l (26 values, 10 −4 -10 1 /mrna copy). as η r and η m were assumed to be 10 −1 -10 1 and 10 −4 -10 −2 , respectively, the ratio of η r to η m was in the range of 10 1 -10 5 , and thus the ratio of η r c l to η m c l should have been in the range of 10 1 -10 5 . with several unqualified scenarios excluded, 1,744 scenarios were considered in the study. for efficient computations and accurate predictions, we ran simulations 1,000 times for each scenario. spatial distributions of infection risks fig 3b, 3d , 3f and 3h) shows average infection risk distributions of 1,000 simulations at the end of the exposure period via the long-range airborne route and fomite route (patterns 1, 3 and 5), respectively. correspondingly, figure d (iv, vi and viii) in s1 file shows those of the fomite route (patterns 2, 4 and 6). for fair comparison, the parameters were set to be the same for the aforesaid distributions (fig 3b, 3d , 3f and 3h and figure d (iv, vi and viii) in s1 file). for the long-range airborne route, the spatial distribution of infection risk (fig 3b) was similar to that of the reported attack rates (fig 3a) , i.e., highest in the source cubicle, lower in the adjacent cubicle and lowest in the remote cubicles. virus-containing airborne droplets were generated by the index patient in the source cubicle, leading to the highest virus concentration in the air in the source cubicle (figure c in s1 file) and thus the highest infection risk (fig 3b) . due to the small temperature differences between zones, two-way airflow occurred at each inner opening in the ward [35] , so some virus-containing airborne droplets spread to other cubicles by airflow. as the remote cubicles were farther away from the source than the adjacent cubicle was, the airborne droplet concentrations in the former were further diluted than that in the latter (figure c in s1 file), leading to lower infection risk (fig 3b) . with the high mechanical ventilation rates in ward 8a, the results from both cfd simulations [20] and multi-zone modelling methods ( [35] and figure c in s1 file) show that the aerosol concentration in the source cubicle was much higher than that in the adjacent cubicle. therefore, the difference between infection risks (fig 3b) in the source and adjacent cubicles was very large (1:0.43 in this scenario), which was inconsistent with the small difference (1:0.80) in the reported attack rate distribution (fig 3a) . although several studies showed the very probable evidences for the airborne transmission of sars such as in the amoy gardens outbreak [19] , the inconsistence suggests that the outbreak might not merely be induced by the long-range airborne route. for the fomite route, on the whole, the infection risk distributions were influenced by hcws' hands and common environmental surfaces, which were important mediums to transfer viruses from the index patient to other inpatients. as hcws' hands usually contact patients in a certain sequence, viruses received by normal inpatients vary with their positions in the ward. in fig 3d, 3f and 3h and figure d (iv, vi and viii) in s1 file, the infection risk always reaches its highest value in inpatients visited by hcws after the index patient, and then decreases in the direction of the hcws' routine rounds. however, inpatients had the same opportunities to contact common surfaces, such as common toilets in ward 8a, so common surfaces reduced the difference between viruses received by each inpatient from hcws and contributed to a uniform infection risk distribution. except for a few visited by hcws after the index patient, the inpatients share a similar infection risk of 0.07. for the six routine round patterns considered here, infection risk distributions vary. in patterns 1 and 2 ( fig 3d and figure d (iv) in s1 file), as more hcws examined each patient in a routine round, the transmission of viruses was enhanced and the infection risks were generally higher than those in other patterns (fig 3f and 3h and figure d(vi and viii) in s1 file). in patterns 3 and 4 ( fig 3f and figure d(vi) in s1 file), as different groups of hcws were responsible for different cubicles, hcws did not transmit viruses across cubicles, and thus the infection risks for inpatients in the adjacent cubicle and remote cubicles were nearly the same. in patterns 5 and 6 ( fig 3h and figure d(viii) in s1 file), the inpatient subsequently visited by hcws after the index patient was not necessarily inpatient 10 or 12. therefore, although the infection risks still decreased in the direction of routine rounds, the reduction was small compared with other patterns (fig 3d and 3f and figure d (iv and vi) in s1 file). among the six infection risk distributions, only those of patterns 1 and 5 (fig 3d and 3h ) were highest in the source cubicle, lower in the adjacent cubicle, and lowest in the remote cubicles, similar to that of the reported attack rates (fig 3a) . nevertheless, as for pattern 1, the difference between infection risks (fig 3d) in the source and adjacent cubicles was too large (1:0.35 in this scenario) and not consistent with the small difference (1:0.80) in the reported attack rate distribution (fig 3a) . in contrast, for pattern 5, the difference between infection risks (fig 3h) in the source and remote cubicles was too small (1:0.52 in this scenario), not consistent with the large difference (1:0.28) in the reported attack rate distribution (fig 3a) . respectively. since the distribution of reported attack rates in this sars outbreak exhibited a statistically significant pattern (p = 0.0015, pearson chi-square test), in many scenarios the minimum rss were larger than 2.5525 (small black dots in fig 4) , indicating large deviations from the outbreak data. therefore, these scenarios were regarded as less probable ones. in fig 4, when the product of dose-response parameters in mucous membranes and viral load coefficient η m c l was very large, the prediction of hypothesis 1 [long air] (red dots) fitted best with the reported data. as the exposure for this hypothesis occurred only in the respiratory tract, the value of rss did not vary with dose-response parameters on mucous membranes η m . thus, large η m values would have led to overly high infection risks under hypotheses 2 and 3, but would not have influenced the long-range airborne route. similarly, when the product of dose-response parameters in respiratory tracts and viral load coefficient η r c l was very large, the prediction of hypothesis 2 [fomite] (orange and green dots) fitted best with the reported data. when η r c l and η m c l were relatively small, the prediction of the hypothesis 3 [long air + fomite] (cyan, blue and purple dots) fitted best with the reported data. in fig 4, the viral load coefficient is larger than 1 in over 95% of more probable scenarios (non-black dots), meaning that the viral load for the index patient in the computation was very probable to be higher than the measured data of ordinary sars patients [33] . assuming that the dose-response parameter on mucous membranes η m was 3.2 × 10 −3 /mrna copy [40] and that parameter in respiratory tracts η r was 10 3 times higher than that on the mucous membranes, i.e., 3.2/mrna copy [41] , all of the viral load coefficients for more probable scenarios (non-black dots) were larger than 1.97. the high viral load coefficients support other studies suggesting that the index patient was a super-spreader [42] [43] [44] . as shown in tables 1 and 2 , the minimum rss of other patterns in hypotheses 3 [long air + fomite (p1, p2, p3, p4, p5 and p6)], 0.7092, 0.9762, 0.7790, 0.7514, 0.5105 and 0.7675, are smaller than those of the two single-route modes, 1.0394 at least. thus, hypothesis 3 [long air + fomite] was more possible than the two single-route hypotheses. moreover, among the 6 patterns in hypothesis 3, the minimum rss of pattern 5 was smallest (0.5105), indicating that it was the most possible. in table 2 , in scenarios with the minimum rss for hypothesis 3 [long air + fomite], the fomite route plays a non-negligible role in transmission, contributing at least 37% to the infection risk. except for pattern 1, the long-range airborne route was predominant, which is consistent with several findings of the similarity between bio-aerosol concentrations and reported attack rates distributions in sars outbreaks [19] [20] [21] . this study had three main limitations. first, most of the human behaviour was assumed in the multi-agent model (tables g and h in s1 file) because relevant descriptions were not available in the literature. the information of human behaviours were very important for our model. as shown in this study, the routine round patterns of doctors and nurses influenced the infection risk patterns (fig 3 and figure d in s1 file) and hypothesis probabilities (fig 4, tables 1 and 2 ). in addition, the diversity of modes of hcws visiting patients was not considered in this study. different patients might have required different frequencies, intensities, or hcws visiting patterns, which might bring in more deviations in infection risk distributions. in future, more detailed information about human behaviours of representative people such as patients, hcws and visitors in healthcare settings should be reported for outbreaks of infectious diseases. second, some parameters for the biological properties of sars-cov in the multi-agent model were not available, such as the transfer rates between surfaces (table c in s1 file) and the first-order inactivation rates in the air and on surfaces (table d in s1 file), and were estimated or replaced by those of other viruses or even bacteria. moreover, several parameters such as dose response parameters might be variable from patient to patient, but the individual differences were not considered in this study, which decreases the diversity of predicted infection risk distributions. experimental investigations of sars-cov are lacking mainly because of safety considerations [18] ; and several authors have suggested 229e, a low-virulence human coronavirus [45] , as a surrogate [18, 40] . in future, more experimental measurements of parameters for the biological properties of sars-cov or the surrogate, 229e, are needed. third, due to the lack of information, the length of exposure period were assumed to be the same for all susceptible patients, and the index patient was assumed to be the only source. however, different patients might have different timings of admission, discharge, or symptom onset, and thus different exposure periods. ignoring the individual differences in the exposure periods reduces the diversity of infection risk distributions, and leads to omission of several possible scenarios. moreover, the transmission from early-onset cases to the later cases might have occurred, according to the illness onset dates reported by li et al. [20] . neglecting the exposure doses caused by these early-onset cases results in underestimation of infection risk for other cases, which affects the distribution patterns of infection risk in the ward. in future, more detailed information about the timing for patients should be recorded in outbreak reports of infectious diseases. in this study, a mechanism-based investigation was conducted to explore the role of the fomite route in the transmission of sars-cov infection. the results could help to recommend appropriate infection control measures in a focused manner. based on the simulation results and analyses, the following conclusions can be drawn under our assumed conditions. • in our investigated scenarios, for most of the routine round patterns, sars-cov was less probable to transmit via the fomite route alone. the virus might have spread via the long-range airborne route alone, but it was more probable that the virus could transmit in combined routes, especially when the viral loads and dose-response parameters were relatively small. • it's found that the index patient was very probable to generate more viruses than ordinary sars patients, which supported the perception that the patient was a super-spreader. • in the very probable combined routes, the fomite route played a non-negligible role. for most patterns, the airborne route was predominant. • doctors and nurses were found to be the most possible to conduct their routine rounds following pattern 5 (examining inpatients randomly in the clockwise direction). supporting information s1 file. the following information is described in detail, e.g. details of the mathematical models, parameter selections for the mathematical models and supplemental figures. a novel coronavirus associated with severe acute respiratory syndrome summary of probable sars cases with onset of illness from 1 world health organization. situation updates-sars severe acute respiratory syndrome (sars) in singapore: update 2 severe acute respiratory syndrome (sars) in taiwan, china world health organization. china's latest sars outbreak has been contained, but biosafety concerns remain: update 7 vials of deadly sars virus 'go missing' in france. the daily telegraph bats are natural reservoirs of sars-like coronaviruses genomic characterization of severe acute respiratory syndrome-related coronavirus in european bats and classification of coronaviruses based on partial rna-dependent rna polymerase gene sequences isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor world health organization. alert, verification and public health management of sars in the post-outbreak period severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection where has sars gone? the strange case of the disappearing coronavirus transmission of influenza a in human beings relative contributions of four exposure pathways to influenza infection risk transmission of sars and mers coronaviruses and influenza virus in healthcare settings: the possible role of dry surface contamination clinical management and infection control of sars: lessons learned environmental survival and microbicide inactivation of coronaviruses. coronaviruses with special emphasis on first insights concerning sars. birkhäuser advances in infectious diseases baid evidence of airborne transmission of the severe acute respiratory syndrome virus role of air distribution in sars transmission during the largest nosocomial outbreak in hong kong multi-zone modeling of probable sars virus transmission by airflow between flats in block e severe acute respiratory syndrome coronavirus on hospital surfaces detection of airborne severe acute respiratory syndrome (sars) coronavirus and environmental contamination in sars outbreak units lethal infection of k18-hace2 mice infected with severe acute respiratory syndrome coronavirus pathogenicity of severe acute respiratory coronavirus deletion mutants in hace-2 transgenic mice effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars). lancet factors associated with transmission of severe acute respiratory syndrome among health-care workers in singapore sars transmission, risk factors, and prevention in hong kong cluster of sars among medical students exposed to single patient index patient and sars outbreak in hong kong outbreak at the prince of wales hospital clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study prediction of natural ventilation in buildings with large openings role of two-way airflow owing to temperature difference in severe acute respiratory syndrome transmission: revisiting the largest nosocomial severe acute respiratory syndrome outbreak in hong kong relative effectiveness of ventilation in community indoor environmentsfor controlling infection. doctoral dissertations. the university of hong kong airborne spread of measles in a suburban elementary school model selection in ecology and evolution supplemental information: dose response parameters for gain of function pathogens quantifying the routes of transmission for pandemic influenza transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions super-spreaders in infectious diseases the role of super-spreaders in infectious disease differences in neutralizing antigenicity between laboratory and clinical isolates of hcov-229e isolated in japan in 2004-2008 depend on the s1 region sequence of the spike protein key: cord-316703-8kxx3034 authors: parera, mariona; martrus, gloria; franco, sandra; clotet, bonaventura; martinez, miguel angel title: canine hepacivirus ns3 serine protease can cleave the human adaptor proteins mavs and trif date: 2012-08-01 journal: plos one doi: 10.1371/journal.pone.0042481 sha: doc_id: 316703 cord_uid: 8kxx3034 canine hepacivirus (chv) was recently identified in domestic dogs and horses. the finding that chv is genetically the virus most closely related to hepatitis c virus (hcv) has raised the question of whether hcv might have evolved as the result of close contact between dogs and/or horses and humans. the aim of this study was to investigate whether the ns3/4a serine protease of chv specifically cleaves human mitochondrial antiviral signaling protein (mavs) and toll-il-1 receptor domain-containing adaptor inducing interferon-beta (trif). the proteolytic activity of chv ns3/4a was evaluated using a bacteriophage lambda genetic screen. human mavsand trif-specific cleavage sites were engineered into the lambda ci repressor. upon infection of escherichia coli cells coexpressing these repressors and a chv ns3/4a construct, lambda phage replicated up to 2000-fold more efficiently than in cells expressing a chv protease variant carrying the inactivating substitution s139a. comparable results were obtained when several hcv ns3/4a constructs of genotype 1b were assayed. this indicates that chv can disrupt the human innate antiviral defense signaling pathway and suggests a possible evolutionary relationship between chv and hcv. the origin of hepatitis c virus (hcv) infections in humans has remained unknown, because related animal virus homologs had not been identified [1, 2] . recently, a flaviviridae rna genome that was isolated from domestic dogs with respiratory illness was found to be the virus most closely related to hcv [3] . this flaviviridae agent is known as canine hepacivirus (chv). the discovery of chv may shed light on the origin of hcv, and may also serve as a new model system with which to study this deadly human virus. chv-like viruses (also known as nonprimate hepaciviruses [nphv] ) were also recently identified in horses [4] . hcv belongs to the genus hepacivirus, one of the four genera in the family flaviviridae [2] . hcv infects more than 170 million people worldwide (http://www.who.int/mediacentre/factsheets/fs164/ en/) and is one of the leading causes of liver cirrhosis and failure [5] . although the discovery of the close homology between chv and hcv is intriguing, there are barriers preventing viral transmission across species. cross-species transmission of chv to humans would most likely require evasion of the human cellular innate immune response, which leads to type i interferon production through rna composition-dependent activation of retinoic acid inducible gene-i (rig-i) and toll-like receptor (tlr) [6] . a number of host-encoded restriction factors can protect human cells from viral infections. a well characterized model is retroviruses. the human genome has evolved a constellation of antiviral genes with the potential to target retroviruses. these genes are part of the innate immune system, and in the retrovirus literature, they are often called restriction factors. well-character-ized restriction factors are apobec3g [7] , trim5-a proteins [8] , tetherin (bst2) [9] , and samhd1 [10, 11] , which inhibit human immunodeficiency virus (hiv) propagation at various steps in its replication cycle. the evolution of the genes encoding these restriction factors affected the adaptation of hiv to humans in the current pandemic, and the adaptation of other primate lentiviruses to their modern hosts. in the case of hcv, signalling pathways leading to type i interferon production are the first line of defence employed by the host to combat viruses and represent a barrier that invading viruses must overcome in order to establish infection [12, 13] . a major strategy employed by hcv to subvert the host innate immune response is disruption of the rig-i and tlrsignalling pathway through hcv ns3/4a protease cleavage of the adaptor proteins mavs and trif [14, 15] . mavs from multiple primates are resistant to inhibition by the hcv ns3/4a protease [16] . this resistance maps to single changes within the protease cleavage site in mavs, which protect mavs from getting cleaved by the hcv ns3/4a protease. these changes were likely driven by ancient hepaciviruses, providing insights into hepaciviral infections over the course of primate evolution [16] . because the propagation of chv and nphv in humans would most likely have had to overcome the human innate host defence mediated by type i interferon, we hypothesized that chv ns3/ 4a protease could specifically process the human adaptor proteins mavs and trif. to test this hypothesis, we coexpressed the chv ns3/4a protease with specific mavs and trif cleavage sites that are known to be processed in vivo by the hcv ns3/4a protease. using this approach, we demonstrated that the chv ns3/4a protease can cleave human mavs and trif. the catalytic activity of chv ns3/4a protease was assayed using a bacteriophage lambda-based genetic screen. we previously demonstrated that the bacteriophage lambda-based genetic screen employed here can be used to characterize site-specific proteases, including hcv ns3/4a, hiv, and respiratory syndrome (sars) coronavirus proteases [17, 18, 19, 20, 21, 22, 23, 24] . this genetic screen is based on the bacteriophage lambda ci-cro regulatory circuit, in which the viral repressor ci is specifically cleaved to initiate the switch from lysogeny to lytic infection [25] . this genetic screen is a simple alternative approach to the purification and characterization of expressed proteases by in vitro classical methodologies. the specific cleavage sites engineered in the lambda ci repressor and assayed in this study are depicted in fig. 1 . these sites included specific mavs and trif cleavage sites known to be processed in vivo by the hcv ns3/4a protease, as well as chv and hcv ns5a/ns5b viral polyprotein cleavage sites. a mutated version of the trif cleavage site was also included as a control. the target specificity for the mavs and trif cleavage sites was tested by coexpressing them with chv or hcv ns3/4a protease constructs. the ns3/ 4a protease constructs contained ns4 residues 21-34 fused in frame via a short linker to the amino terminus of the ns3 protease domain (residues 1-181) (fig. 2) . the chv ns3/4a protease construct was chemically synthesized following the nucleotide sequence reported by kapoor et al. [3] . upon infection of e. coli cells coexpressing the lambda ci repressor with either mavs or trif cleavage site and a chv ns3/4a construct, lambda phage replicated up to 2,000-fold more efficiently than in cells expressing a chv protease variant that included a substitution in catalytic residue s139 ( fig. 3a and 3b). equivalent results were obtained when four hcv ns3/4a of genotype 1b were assayed. three assayed hcv ns3/4a proteases were isolated from three treatment-naïve hcv-infected patients [18, 24] . the fourth hcv ns3/4a protease was amplified from the hcv subgenomic replicon i389/ns3-3 [26] . to investigate the influence of the ns4 protein on the catalytic efficiency of chv ns3 protease, the chv ns4 residues i25 and v29 were replaced with a residues (fig. 1 ). hcv ns4 residues 25 and 29 are critical for ns4 activation of hcv ns3 protease [18, 27] . similar to hcv ns3 protease, a mutated version of chv ns4 reduced the catalytic efficiency of the chv ns3 protease, indicating that the chv ns4 peptide is essential for efficient processing of mavs and trif cleavage sites ( fig. 3a and 3b) . nevertheless, the effect of the mutated ns4 residues 25 and 29 on chv ns3/4a protease activity was less drastic than that observed on hcv ns3/4a protease ( fig. 3b ) (see below). we further tested the target specificity of the mavs and trif cleavage sites by analyzing a control mutant trif target site. a mutant trif cleavage site was constructed in which the c residue at the p1 position was substituted with a y residue (fig. 1 ). as shown in fig. 3c , the mutant trif cleavage site prevented phage replication, indicating that trif processing was specifically mediated by chv ns3/4a protease. western blot analysis also demonstrated ( fig. 4 ) that the expression of the chv ns3/4a protease resulted in nearly complete cleavage of the lambda ci repressor with either mavs (fig. 4a ) or trif cleavage site (fig. 4b ). expression of ns3/4a proteases that included a substitution in catalytic residue s139 (fig. 4) completely abolished the cleavage of lambda ci repressor with either mavs or trif cleavage site. expression of ns3/4a proteases with a mutated version of ns4 partially abolished wildtype mavs or trif repressor cleavage (fig. 4) ; specifically, mutations in ns4 had less impact in the activity of chv ns3/4a protease than in the activity of hcv ns3/4a protease (fig. 4b) . as expected, a control mutant trif target site ( fig. 1) was not cleaved by wild-type ns3/4a proteases. overall, these results demonstrate that chv ns3/4a protease is able to specifically cleave the human adaptor proteins mavs and trif. we next investigated whether chv and hcv ns3/4a proteases could specifically process heterologous viral polyprotein cleavage sites. to do so, the chv and hcv ns5a/ns5b cleavage sites were engineered ( fig. 1 ) and tested against both proteases. interestingly, the chv and hcv ns3/4a proteases processed the heterologous viral cleavage sites with comparable efficiency (fig. 5 ), suggesting that chv and hcv ns3/4a proteases may share the same evolutionary linage. finally, two macrocyclic hcv ns3/4a protease inhibitors, 25a [28] and danoprevir [29] , were assayed to test their ability to inhibit chv ns3/4a protease. macrocyclic inhibitors were chosen because they have a higher potency against different hcv genotypes compared to linear inhibitors [30] . the inhibitory capability of these two macrocyclic inhibitors was assessed with the mavs and trif cleavage sites. as expected, 25a and danoprevir inhibited hcv ns3/4a protease (fig. 6 ) when tested with either cleavage site. in contrast, no significant inhibition was observed when either of the two inhibitors was assayed with the chv ns3/4a protease. of note, the positions associated with resistance to macrocyclic hcv ns3/4a protease inhibitors, hcv residues q80, r155, a156, and d168 [31] , are mutated in the chv ns3/4a protease sequence (fig. 2) . the specificity of this inhibition was tested by including an hcv ns3/4a protease mutant carrying the protease inhibitor resistance substitution d168v; this substitution reduced the inhibitory capacity of the two tested protease inhibitors (fig. 6 ). recent studies have identified viruses, in domestic dogs and horses, which are genetically very closely related to hcv [3, 4] . these findings suggest that hcv could have been introduced into the human population through contact with dogs and/or horses. nevertheless, adaptation of chv and nphv to human cells would likely require these viruses to evade the human cellular innate immune response, which responds to viral pathogens by rna composition-dependent activation of rig-i and subsequent signalling via interferon regulatory factor (irf)-3 [6] . the response to hcv infection is regulated by hepatic immune defences triggered by cellular rig-i [14, 32] . in human liver cells, hcv interferes with rig-i-dependent signalling to irf-3 by ns3/4a-dependent cleavage of the crucial adaptor protein mavs [14] . likewise, tlr-3-dependent signalling to irf-3 is ablated by cleavage of trif by the hcv ns3/4a protease [15] . in this study, we tested the ability of chv ns3/4a protease to specifically cleave the human adaptor proteins mavs and trif. we found that chv ns3/4a protease could process, with comparable efficiency, the specific mavs and trif cleavage sites known to be processed in vivo by hcv ns3/4a protease [14, 15] . these findings extend previous reports [3, 4] , confirming a possible evolutionary relationship between chv and hcv. the results of our study indicate that chv can disrupt the human innate antiviral defense signaling pathway. in addition, our study also showed that, similar to hcv ns3/4a protease, chv ns3 protease efficiency is regulated by the viral ns4 protein [33] , suggesting that the mechanism of action and substrate specificity is similar between the two proteases. chv ns4 polypeptide improved cleavage of mavs-and trif-specific cleavage sites. however, chv ns3/4a protease was not susceptible to macrocyclic inhibitors of hcv ns3/4a protease. important hcv genotype-specific differences in the efficacy of ns3/4a protease inhibitors were reported recently [29] . most of the hcv ns3/4a protease inhibitors now in clinical development were identified using hcv replicon systems of genotype 1 and are being licensed for treatment of genotype 1 infection. in vitro studies performed with hcv chimeras have shown that genotypes 2a, 5a, and 6a had similar sensitivity, whereas genotype 3a was comparatively resistant to macrocyclic hcv ns3/4a protease inhibitors [30] . chv ns3/4a protease has amino acid changes at positions associated with resistance to macrocyclic hcv ns3/4a protease inhibitors [31] , which would explain why this protease is resistant to macrocyclic hcv ns3/4a protease inhibitors. 1-181) . it was previously demonstrated that the kinetic parameters of a single-chain protein containing the ns4a cofactor and the hcv ns3 protease are identical to those of the bona fide ns3/4a (ns3 residues 1 to 631 and ns4a residues 1 to 54 protein complex generated in eukaryotic cells (5, 38) . asterisks represent the stop codons. mutated residues generated by site-directed mutagenesis are marked in red. doi:10.1371/journal.pone.0042481.g002 gb virus b (gbv-b) is another hepacivirus that before the discovery of chv/nhv was the virus phylogenetically most closely related to hcv. gbv-b does not infect humans or chimpanzees, but it causes acute hepatitis in experimentally infected tamarins (a type of new world monkey) [2] . although there are important differences in the outcomes of hcv and gbv-b infections (gbv-b rarely causes persistent infections), gbv-b has evolved a strategy similar to that employed by hcv, resulting in the disruption of rig-i signalling. indeed, gbv-b ns3/4a proteolytically cleaves human mavs. more distantly related hepacivirus such as gbv-c, which infects humans and chimpanzees, and gbv-a are also able to antagonizing mavs [16] . in contrast, proteases from the more distantly related pestivirus bovine viral diarrhea virus and flavivirus yellow fever virus do not antagonize mavs [16, 34] . overall these data suggest that the ability of ns3/4a proteases to antagonize mavs is a phylogenetically discrete characteristic exclusive to hcv and the three gbv viruses. based on these observations, chv ns3/4a should be able to cleave the canine's mavs and trif proteins. canine orthologs of human mavs and trif differ in sequence at the cleavage site processed by hcv ns3/4a protease; therefore, they were not tested in this study. inspection of the canine mavs and trif proteins did not reveal the presence of apparent substrate specificities for hcv ns3/4a protease. although chv was initially found in dogs, subsequent efforts to find similar viruses in canids has remained largely unsuccessful [4] . recently, evidence of a chv-like virus infection was confirmed by the detection of diverse viral genomes, termed nphv, in horses [4] . horses can support replication of several other flaviviruses, including the vector-borne west nile, japanese encephalitis, dengue, and st. louis encephalitis viruses. these flaviviruses can be transmitted among several mammalian species, including humans. most of the nphv strains detected in horses are genetically distinct from chv; however, one variant is almost expression of the protease was induced with iptg for 3 h. the western blot proved that the lambda ci repressor with either mavs or trif cleavage site was not cleaved by ns3/4a proteases that included a substitution in catalytic residue s139. similarly, a control mutant trif target site (fig. 1) was not cleaved by wild-type ns3/4a proteases. the hcv 1 ns3/4a protease was also tested without iptg. doi:10.1371/journal.pone.0042481.g004 identical to chv, suggesting that chv/nphv viruses may be able to jump species [4] . this is the first study to our knowledge to demonstrate that at least parts of the identified and sequenced chv/nphv rna genomes are biologically active. initial attempts to culture chv were not successful [3, 35] . only a single strain of hcv, the genotype 2a strain jfh1 from a japanese patient, has been found to grow robustly in culture, in human hepatoma cell lines [36] . however, viable jfh1-based recombinants with gene elements specific to other hcv genotypes have been developed [30, 37, 38] . given the difficulties in culturing hcv, our results suggest that it may be possible to generate hcv/chv chimeras that can be cultivated. our study has some limitations that are worth noting. first, this study only tested the functionality of a small chv genomic region; other chv/nphv genomic regions that have been previously described [3, 4] should be analyzed to guarantee the viability of future hcv/chv chimeras. second, the in vitro approach used here to measure the ability of the protease to cleave human adaptors mavs and trif only partially mimics what occurs in vivo. third, it has not been determined here whether chv ns3/ 4a protease can efficiently cleavage the mavs and trif proteins equivalent of horses and canines, their primary hosts. nevertheless, the similarity observed between hcv and chv ns3/4a proteases in their capability to process mavs and trif, as well as hcv/chv ns5a/ns5b cleavage sites, strongly supports the hypothesis that chv might be able to disrupt the human innate antiviral defense signaling pathway. further work should include an evaluation of ability of the chv ns3/4a protease to process both the human and canine adaptor proteins mavs and trif in human and canine hepatoma cell lines, as well as the construction of hcv/chv chimeras to explore this hypothesis or to perform other functional studies related to hepacivirus pathogenesis and treatment. insertion of the mavs and hcv ns5a/ns5b-specific cleavage sites (fig. 1) into the lambda ci repressor has been described elsewhere [20, 24] . in this study, trif and chv ns5a/ns5b-specific cleavage sites (fig. 1) were inserted into the lambda ci repressor. briefly, the nucleotide sequence for trif and chv ns5a/ns5b-specific cleavage sites (fig. 1 ) was chemically synthesized (integrated dna technologies). the constructs included two restriction sites, nsii and hindiii, at the ends, which are present in the lambda ci repressor [39] . after digestion with nsii and hindiii, the construct was ligated into pci.hcvns5a/ns5bcro [20] , previously digested with nsii and hindiii, to generate the pci.trifcro and pci.chvns5a/ ns5bcro plasmids sequence analysis of this plasmid confirmed the accuracy of the synthetic construct. e. coli jm109 cells containing plasmid pci.mavscro, pci.trifcro, pci.hcvns5a/ ns5bcro or pci.chvns5a/ns5bcro were then transformed with the plasmid expressing the corresponding protease. transformed cells were grown overnight at 30uc in the presence of 0.2% maltose, 12.5 mg/ml tetracycline, and 20 mg/ml ampicillin; harvested by centrifugation; and resuspended to an optical density at 600 nm of 2.0/ml in 10 mm mgso4. to induce expression of the protease, 20 ml of cells were incubated in 100 ml of luria-bertani (lb) medium containing 12.5 mg/ml tetracycline, 20 mg/ ml ampicillin, 0.2% maltose, 10 mm mgso4, and 0.1 mm isopropyl-b-d-thiogalactopyranoside (iptg) for 1 h. the cell cultures were then infected with 10 5 plaque-forming units (pfu) of phage. after 3 h at 37uc, the titer of the resulting phage was determined by coplating the cultures with 200 ml of e. coli xl-1 blue cells (adjusted to an optical density at 600 nm of 2.0/ml in 10 mm mgso4) on lb plates using 3 ml of top agar containing 12.5 mg of tetracycline/ml, 0.2% maltose, and 0.1 mm iptg. after incubation at 37uc for 6 h, the resulting phage plaques were counted in order to score growth. western blots were performed as previously described [19] . briefly, e. coli jm109 cells containing plasmid pci.mavscro or pci.trifcro were transformed with the plasmid expressing the corresponding protease. transformed cells were then grown overnight at 30uc in the presence of 0.2% maltose, 12.5 mg/ml tetracycline, and 20 mg/ml ampicillin; harvested by centrifugation; and resuspended to an optical density at 600 nm of 2.0/ml in 10 mm mgso4. to induce expression of the protease, 200 ml of cells were incubated in 1 ml of lb medium containing 12.5 mg/ml tetracycline, 20 mg/ml ampicillin, 0.2% maltose, 10 mm mgso4, and 0.1 mm iptg for 3 h. the ods of the cultures after 3 h (in the presence of iptg) were measured to assure that equivalent amounts of total cell protein were blotted. no significant differences were observed when the ods of the different cultures were compared, suggesting that the expression of the ns3/4a proteases did not affect the growth of the bacteria. cultures were lysed in sodium dodecyl sulfate (sds)-polyacrylamide gel electrophoresis sample buffer, resolved in 12% gradient sds polyacrylamide gels (invitrogen), transferred to nitrocellulose membranes, and blocked in phosphate-buffered saline-0.1% tween 20-10% nonfat dry milk. for immunochemical detection of the lambda repressor, membranes were subsequently incubated with rabbit serum containing polyclonal anti-ci antibodies (anti-ci sera; invitrogen). bound antibodies were visualized with peroxidaselinked anti-rabbit antybody hrp-linked igg (cell signaling technology) and the supersignal west pico chemiluminescent substrate (thermo scientific). the nucleotide sequence for chv ns3/4a protease (fig. 2 ) was chemically synthesized (integrated dna technologies) following the nucleotide sequence reported by kapoor et al. [3] . the construct included two restriction sites, ecori and xhoi, at the ends. after digestion with ecori and xhoi, the construct was cloned into pbluescript sk-(agilent technologies) to generate a b-galactosidase-chv ns3/4a protease fusion protein. sequence analysis of this plasmid confirmed the accuracy of the synthetic construct. control mutant constructs (red residues in figs. 1 and 2) were generated by site-directed mutagenesis using the quik-change kit (agilent technologies) and following the manufacturer's instructions. three hcv ns3/4a proteases used in this study, 1, 50, and 51, were obtained from hcv genomic rna extracted from the plasma of three different hcv-infected patients. the nucleotide sequences and amplification procedures of these proteases have been published previously [18, 24] . the fourth hcv ns3/4a protease employed in this study was amplified from the hcv subgenomic replicon i389/ns3-3 [26] . macrocyclic protease inhibitor 25a [28] was kindly provided by merck, sharp and dohme. donoprevir was purchased from selleck chemicals (houston, tx, usa). genetic diversity and evolution of hepatitis c virus-15 years on the gb viruses: a review and proposed classification of gbv-a, gbv-c (hgv), and gbv-d in genus pegivirus within the family flaviviridae characterization of a canine homolog of hepatitis c virus serology enabled discovery of genetically diverse hepaciviruses in a new host hepatitis c virus: virology, diagnosis and management of antiviral therapy evasion of intracellular host defence by hepatitis c virus isolation of a human gene that inhibits hiv-1 infection and is suppressed by the viral vif protein the cytoplasmic body component trim5alpha restricts hiv-1 infection in old world monkeys tetherin inhibits retrovirus release and is antagonized by hiv-1 vpu samhd1 is the dendritic-and myeloid-cell-specific hiv-1 restriction factor counteracted by vpx vpx relieves inhibition of hiv-1 infection of macrophages mediated by the samhd1 protein evasion and disruption of innate immune signalling by hepatitis c and west nile viruses induction and evasion of innate antiviral responses by hepatitis c virus cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus immune evasion by hepatitis c virus ns3/4a protease-mediated cleavage of the toll-like receptor 3 adaptor protein trif convergent evolution of escape from hepaciviral antagonism in primates a bacteriophage lambda-based genetic screen for characterization of the activity and phenotype of the human immunodeficiency virus type 1 protease genetic screen for monitoring hepatitis c virus ns3 serine protease activity genetic screen for monitoring severe acute respiratory syndrome coronavirus 3c-like protease genetic and catalytic efficiency structure of an hcv protease quasispecies fitness landscape of human immunodeficiency virus type 1 protease quasispecies hiv-1 protease catalytic efficiency effects caused by random single amino acid substitutions epistasis among deleterious mutations in the hiv-1 protease complexity and catalytic efficiency of hepatitis c virus (hcv) ns3 and ns4a protease quasispecies influence responsiveness to treatment with pegylated interferon plus ribavirin in hcv/hiv-coinfected patients a genetic switch replication of subgenomic hepatitis c virus rnas in a hepatoma cell line identification of the sequence on ns4a required for enhanced cleavage of the ns5a/5b site by hepatitis c virus ns3 protease molecular modeling based approach to potent p2-p4 macrocyclic inhibitors of hepatitis c ns3/4a protease preclinical characteristics of the hepatitis c virus ns3/4a protease inhibitor itmn-191 (r7227) differential efficacy of protease inhibitors against hcv genotypes 2a, 3a, 5a, and 6a ns3/ 4a protease recombinant viruses treatment failure and resistance with direct-acting antiviral drugs against hepatitis c virus innate immunity induced by composition-dependent rig-i recognition of hepatitis c virus rna multiple determinants influence complex formation of the hepatitis c virus ns3 protease domain with its ns4a cofactor peptide gb virus b disrupts rig-i signaling by ns3/4a-mediated cleavage of the adaptor protein mavs hepatitis c homolog in dogs with respiratory illness production of infectious hepatitis c virus in tissue culture from a cloned viral genome development and characterization of hepatitis c virus genotype 1-7 cell culture systems: role of cd81 and scavenger receptor class b type i and effect of antiviral drugs efficient culture adaptation of hepatitis c virus recombinants with genotype-specific core-ns2 by using previously identified mutations dna sequence of the bacteriophage gama ci gene we thank merck, sharp and dohme for providing the 25a protease inhibitor. key: cord-292475-jrl1fowa authors: abry, patrice; pustelnik, nelly; roux, stéphane; jensen, pablo; flandrin, patrick; gribonval, rémi; lucas, charles-gérard; guichard, éric; borgnat, pierre; garnier, nicolas title: spatial and temporal regularization to estimate covid-19 reproduction number r(t): promoting piecewise smoothness via convex optimization date: 2020-08-20 journal: plos one doi: 10.1371/journal.pone.0237901 sha: doc_id: 292475 cord_uid: jrl1fowa among the different indicators that quantify the spread of an epidemic such as the on-going covid-19, stands first the reproduction number which measures how many people can be contaminated by an infected person. in order to permit the monitoring of the evolution of this number, a new estimation procedure is proposed here, assuming a well-accepted model for current incidence data, based on past observations. the novelty of the proposed approach is twofold: 1) the estimation of the reproduction number is achieved by convex optimization within a proximal-based inverse problem formulation, with constraints aimed at promoting piecewise smoothness; 2) the approach is developed in a multivariate setting, allowing for the simultaneous handling of multiple time series attached to different geographical regions, together with a spatial (graph-based) regularization of their evolutions in time. the effectiveness of the approach is first supported by simulations, and two main applications to real covid-19 data are then discussed. the first one refers to the comparative evolution of the reproduction number for a number of countries, while the second one focuses on french departments and their joint analysis, leading to dynamic maps revealing the temporal co-evolution of their reproduction numbers. the ongoing covid-19 pandemic has produced an unprecedented health and economic crisis, urging for the development of adapted actions aimed at monitoring the spread of the new coronavirus. no country remained untouched, thus emphasizing the need for models and tools to perform quantitative predictions, enabling effective managements of patients or an optimized allocations of medical ressources. for instance, the outbreak of this unprecedented pandemic was characterized by a critical lack of tools able to perform predictions related to the pressure on hospital ressources (number of patients, masks, gloves, intensive care unit needs,. . .) [1, 2] . as a first step toward such an ambition goal, the present work focuses on the pandemic time evolution assessment. indeed, all countries experienced a propagation mechanism that is basically universal in the onset phase: each infected person happened to infect in average more than one other person, leading to an initial exponential growth. the strength of the spread is quantified by the so-called reproduction number which measures how many people can be contaminated by an infected person. in the early phase where the growth is exponential, this is referred to as r 0 (for covid-19, r 0 * 3 [3, 4] ). as the pandemic develops and because more people get infected, the effective reproduction number evolves, hence becoming a function of time hereafter labeled r(t). this can indeed end up with the extinction of the pandemic, r(t)!0, at the expense though of the contamination of a very large percentage of the total population, and of potentially dramatic consequences. rather than letting the pandemic develop until the reproduction number would eventually decrease below unity (in which case the spread would cease by itself), an active strategy amounts to take actions so as to limit contacts between individuals. this path has been followed by several countries which adopted effective lockdown policies, with the consequence that the reproduction number decreased significantly and rapidly, further remaining below unity as long as social distancing measures were enforced (see for example [4, 5] ). however, when lifting the lockdown is at stake, the situation may change with an expected increase in the number of inter-individual contacts, and monitoring in real time the evolution of the instantaneous reproduction number r(t) becomes of the utmost importance: this is the core of the present work. monitoring and estimating r(t) raises however a series of issues related to pandemic data modeling, to parameter estimation techniques and to data availability. concerning the mathematical modeling of infectious diseases, the most celebrated approaches refer to compartmental models such as sir ("susceptible-infectious-recovered"), with variants such as seir ("susceptible-exposed-infectious-recovered"). because such global models do not account well for spatial heterogeneity, clustering of human contact patterns, variability in typical number of contacts (cf. [6] ), further refinements were proposed [7] . in such frameworks, the effective reproduction number at time t can be inferred from a fit of the model to the data that leads to an estimated knowledge of the average of infecting contacts per unit time, of the mean infectious period, and of the fraction of the population that is still susceptible. these are powerful approaches that are descriptive and potentially predictive, yet at the expense of being fully parametric and thus requiring the use of dedicated and robust estimation procedures. parameter estimation become all the more involved when the number of parameters grows and/or when the amount and quality of available data are low, as is the case for the covid-19 pandemic real-time and in emergency monitoring. rather than resorting to fully parametric models and seeing r(t) as the by-product of their identification, a more phenomenological, semi-parametric approach can be followed [8] [9] [10] . this approach has been reported as robust and potentially leading to relevant estimates of r(t), even for epidemic spreading on realistic contact networks, where it is not possible to define a steady exponential growth phase and a basic reproduction number [6] . the underlying idea is to model incidence data z(t) at time t as resulting from a poisson distribution with a time evolving parameter adjusted to account for the data evolution, which depends on a function f(s) standing for the distribution of the serial interval. this function models the time between the onset of symptoms in a primary case and the onset of symptoms in secondary cases, or equivalently the probability that a person confirmed infected today was actually infected s days earlier by another infected person. the serial interval function is thus an important ingredient of the model, accounting for the biological mechanisms in the epidemic evolution. assuming the distribution f to be known, the whole challenge in the actual use of the semi-parametric poisson-based model thus consists in devising estimatesrðtþ of r(t) with satisfactory statistical performance. this has been classically addressed by approaches aimed at maximizing the likelihood attached to the model. this can be achieved, e.g., within several variants of bayesian frameworks [5, 6, 8, 10] , with even dedicated software packages (cf. e.g., https://shiny.dide.imperial. ac.uk/epiestim/). instead, we promote here an alternative approach based on inverse problem formulations and proximal-operator based nonsmooth convex optimisation [11] [12] [13] [14] [15] . the questions of modeling and estimation, be they fully parametric or semi-parametric, are intimately intertwined with that of data availability. this will be further discussed but one can however remark at this point that many options are open, with a conditioning of the results to the choices that are made. there is first the nature of the incidence data used in the analysis (reported infected cases, hospitalizations, deaths) and the database they are extracted from. next, there is the granularity of the data (whole country, regions, smaller units) and the specificities that can be attached to a specific choice as well as the comparisons that can be envisioned. in this respect, it is worth remarking that most analyses reported in the literature are based on (possibly multiple) univariate time series, whereas genuinely multivariate analyses (e.g., a joint analysis of the same type of data in different countries in order to compare health policies) might prove more informative. for that category of research work motivated by contributing in emergency to the societal stake of monitoring the pandemic evolution in real-time, or at least, on a daily basis, there are two classes of challenges: ensuring a robust and regular access to relevant data; rapidly developing analysis/estimation tools that are theoretically sound, practically usable on data actually available, and that may contribute to improving current monitoring strategies. in that spirit, the overarching goal of the present work is twofold: (1) proposing a new, more versatile framework for the estimation of r(t) within the semi-parametric model of [8, 10] , reformulating its estimation as an inverse problem whose functional is minimized by using non smooth proximal-based convex optimization; (2) inserting this approach in an extended multivariate framework, with applications to various complementary datasets corresponding to different geographical regions. the paper is organized as follows. it first discusses data, as collected from different databases, with heterogeneity and uneven quality calling for some preprocessing that is detailed. in the present work, incidence data (thereafter labelled z(t)) refers to the number of daily new infections, either as reported in databases, or as recomputed from other available data such as hospitalization counts. based on a semi-parametric model for r(t), it is then discussed how its estimation can be phrased within a non smooth proximal-based convex optimization framework, intentionally designed to enforce piecewise linearity in the estimation of r(t) via temporal regularization, as well as piecewise constancy in spatial variations of r(t) by graph-based regularization. the effectiveness of these estimation tools is first illustrated on synthetic data, constructed from different models and simulating several scenarii, before being applied to several real pandemic datasets. first, the number of daily new infections for many different countries across the world are analyzed independently. second, focusing on france only, the number of daily new infections per continental france départements (départements constitute usual entities organizing the administrative life in france) are analyzed both independently and in a multivariate setting, illustrating the benefit of this latter formulation. discussions, perpectives and potential improvements are finally discussed. datasets. in the present study, three sources of data were systematically used: • source1(jhu) johns hopkins university provides access to the cumulated daily reports of the number of infected, deceased and recovered persons, on a per country basis, for a large number of countries worldwide, essentially since inception of the covid-19 crisis (january 1st, 2020 time series. the data available on the different data repositories used here are strongly affected by outliers, which may stem from inaccuracy or misreporting in per country reporting procedures, or from changes in the way counts are collected, aggregated, and reported. in the present work, it has been chosen to preprocess data for outlier removal by applying to the raw time series a nonlinear filtering, consisting of a sliding-median over a 7-day window: outliers defined as ±2.5 standard deviation are replaced by window median to yield the pre-processed time series z(t), from which the reproduction number r(t) is estimated. an example of raw and pre-processed time series is illustrated in fig 3. when countries are studied independently, the estimation procedure is applied separately to each time series z(t) of size t, the number of days available for analysis. when considering continental france départements, we are given d time series z d (t) of size t each, where 1 � d � d = 94 indexes the départements. these time series are collected and stacked in a matrix of size d × t, and they analyzed both independently and jointly. model. although they can be used for envisioning the impact of possible scenarii in the future development of an on-going epidemic [3] , sir models, because they require the full estimation of numerous parameters, are often used a posteriori (e.g., long after the epidemic) with consolidated and accurate datasets. during the spread phase and in order to account for the on-line/on-the-fly need to monitor the pandemic and to offer some robustness to partial/ incomplete/noisy data, less detailed semi-parametric models focusing on the only estimation of the time-dependent reproduction number can be preferred [8, 9, 16] . let r(t) denote the instantaneous reproduction number to be estimated and z(t) be the number of daily new infections. it has been proposed in [8, 10] that {z(t), t = 1, . . ., t} can be modeled as a nonstationary time series consisting of a collection of random variables, each drawn from a poisson distribution p p t whose parameter p t depends on the past observations of z(t), on the current value of r(t), and on the serial interval function f(�): the serial interval function f(�) constitutes a key ingredient of the model, whose importance and role in pandemic evolution has been mentioned in introduction. it is assumed to be independent of calendar time (i.e., constant across the epidemic outbreak), and, importantly, independent of r(t), whose role is to account for the time dependencies in pandemic propagation mechanisms. for the covid-19 pandemic, several studies have empirically estimated the serial interval function f(�) [17, 18] . for convenience, f(�) has been modeled as a gamma distribution, with shape and rate parameters 1.87 and 0.28, respectively (corresponding to mean and standard deviations of 6.6 and 3.5 days, see [5] and references therein). these choices and assumptions have been followed and used here, and the corresponding function is illustrated in fig 1. in essence, the model in eq (1) is univariate (only one time series is modeled at a time), and based on a poisson marginal distribution. it is also nonstationary, as the poisson rate evolves along time. the key ingredient of this model consists of the poisson rate evolving as a weighted moving average of past observations, which is qualitatively based on the following rationale: whenr is above 1, the epidemic is growing and, conversely, when this ratio is below 1, it decreases and eventually vanishes. non-smooth convex optimisation. the whole challenge in the actual use of the semiparametric poisson-based model described above thus consists in devising estimatesrðtþ of r (t) that have better statistical performance (more robust, reliable, and hence usable) than the direct brute-force and naive form defined in eq 2. to estimate r(t), and instead of using bayesian frameworks that are considered state-of-the-art tools for epidemic evolution analysis, we propose and promote here an alternative approach based on an inverse problem formulation. its main principle is to assume some form of temporal regularity in the evolution of r(t) (we use a piecewise linear model in the following). in the case of a joint estimation of r(t) across several continental france départements, we further assume some form of spatial regularity, i.e., that the values of r(t) for neighboring départements are similar. univariate setting. for a single country, or a single département, the observed (possibly preprocessed) data {z(t), 1 � t � t} is represented by a t-dimensional vector z 2 r t . recalling that the poisson law is pðz ¼ njpþ ¼ p n n! e à p for each integer n � 0, the negative log-likelihood of observing z given a vector p 2 r t of poisson parameters p t is where r 2 r t is the (unknown) vector of values of r(t). up to an additive term independent of p, this is equal to the kl-divergence (cf. section 5.4. in [15] ): given the vector of observed values z, the serial interval function f(�), and the number of days t, the vector p given by (1) reads p = r � fz, with � the entrywise product and f 2 r t�t the matrix with entries f ij = f(i − j). maximum likelihood estimation of r (i.e., minimization of the negative log-likelihood) leads to an optimization problem min r d kl (zjr � fz) which does not ensure any regularity of r(t). to ensure temporal regularity, we propose a penalized approach usinĝ r ¼ argmin r d kl ðz j r � fzþ þ oðrþ where o denotes a penalty function. here we wish to promote a piecewise affine and continuous behavior, which may be accomplished [19, 20] using o(r) = λ time kd 2 rk 1 , where d 2 is the matrix associated with a laplacian filter (second order discrete temporal derivatives), k�k 1 denotes the ℓ 1 -norm (i.e., the sum of the absolute values of all entries), and λ time is a penalty factor to be tuned. this leads to the following optimization problem: spatially regularized setting. in the case of multiple départements, we consider multiple vectors (z d 2 r t , 1 � d � d) associated to the d time series, and multiple vectors of unknown (r d 2 r t , 1 � d � d), which can be gathered into matrices: a data matrix z 2 r t�d whose columns are z d and a matrix of unknown r 2 r t�d whose columns are the quantities to be estimated r d . a first possibility is to proceed to independent estimations of the (r d 2 r t , 1 � d � d) by addressing the separate optimization problemŝ which can be equivalently rewritten into a matrix form: is the entrywise ℓ 1 norm of d 2 r, i.e., the sum of the absolute values of all its entries. an alternative is to estimate jointly the (r d 2 r t , 1 � d � d) using a penalty function promoting spatial regularity. to account for spatial regularity, we use a spatial analogue of d 2 promoting spatially piecewise constant solutions. the d continental france départements can be considered as the vertices of a graph, where edges are present between adjacent départements. from the adjacency matrix a 2 r d�d of this graph (a ij = 1 if there is an edge e = (i, j) in the graph, a ij = 0 otherwise), the global variation of the function on the graphs can be computed as ∑ ij a ij (r ti − r tj ) 2 and it is known that this can be accessed through the so-called (combinatorial) laplacian of the graph: [21] . however, in order to promote smoothness over the graph while keeping some sparse discontinuities on some edges, it is preferable to regularize using a total variation on the graph, which amounts to take the ℓ 1 -norm of these gradients (r ti − r tj ) on all existing edges. for that, let us introduce the incidence matrix b 2 r e�d such that l = b > b where e is the number of edges and, on each line representing an existing edge e = (i, j), we set b e,i = 1 and b e,j = −1. then, the ℓ 1 -norm krb > k 1 = kbr > k 1 is equal to p t t¼1 p ði;jþ:a ij ¼1 jr ti à r tj j. alternatively, it can be computed as krb > k 1 ¼ p t t¼1 kbrðtþk 1 where rðtþ 2 r d is the t-th row of r, which gathers the values across all départements at a given time t. from that, we can define the regularized optimization problem: optimization problems (6) and (7) involve convex, lower semi-continuous, proper and non-negative functions, hence their set of minimizers is non-empty and convex [11] . we will discuss right after how to compute these using proximal algorithms. by the known sparsity-promoting properties of ℓ 1 regularizers and their variants, the corresponding solutions are such that d 2 r and/or rb > are sparse matrices, in the sense that these matrices of (second order temporal or first order spatial) derivatives have many zero entries. the higher the penalty factors λ time and λ space , the more zeroes in these matrices. in particular, when λ space = 0, no spatial regularization is performed and (7) is equivalent to (6) . when λ space is large enough, rb > is exactly zero, which implies that r(t) is constant at each time since the graph of départements is connected. optimization using a proximal algorithm. the considered optimization problems are of the form where f and g m are proper lower semi-continuous convex, and k m are bounded linear operators. a classical case for m = 1 is typically addressed with the chambolle-pock algorithm [22] , which has been recently adapted for multiple regularization terms as in eq. 8 of [23] . to handle the lack of smoothness of lipschitz differentiability for the considered functions f and g m , these approaches rely on their proximity operators. we recall that the proximity operator of a convex, lower semi-continuous function φ is defined as [24] prox φ ðyþ ¼ arg min in our case, we consider a separable data fidelity term: as this is a separable function of the entries of its input, its associated proximity operator can be computed component by component [25] : where τ > 0. we further consider g m (�) = k.k 1 , m = 1, 2, and k 1 (r) ≔ λ time d 2 r, k 2 (r) ≔ λ space rb > . the proximity operators associated to g m read: where (.) + = max(0,.). in algorithm 1, we express explicitly algorithm 161 of [23] for our setting, considering the moreau identity that provides the relation between the proximity operator of a function and the proximity operator of its conjugate (cf. eq. (8) of [23] ). the choice of the parameters τ and σ m impacts the convergence guarantees. in this work, we adapt a standard choice provided by [22] to this extended framework. the adjoint of k m , denoted k � m , is given by the sequence ðr ðkþ1þ þ k2n converges to a minimizer of (7) (cf. thm 8.2 of [23] ). input: data z, tolerance � > 0 ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi p m¼1;2 kk m k to assess the relevance and performance of the proposed estimation procedure detailed above, it is first applied to two different synthetic time series z(t). the first one is synthesized using directly the model in eq (1), with the same serial interval function f(t) as that used for the estimation, and using an a priori prescribed function r(t). the second one is produced from solving a compartmental (sir type) model. for such models, r(t) can be theoretically related to the time scale parameters entering their definition, as the ratio between the infection time scale and the quitting infection (be it by death or recovery) time scale [26, 27] . the theoretical serial function f associated to that model and to its parameters is computed analytically (cf., e.g., [28] ) and used in the estimation procedure. for both cases, the same a priori prescribed function r(t), to be estimated, is chosen as constant (r = 2.2) over the first 45 days to model the epidemic outbreak, followed by a linear decrease (till below 1) over the next 45 days to model lockdown benefits, with finally an abrupt linear increase for the last 10 days, modeling a possible outbreak at when lockdown is lifted. additive gaussian noise is superimposed to the data produced by the models to account for outliers and misreporting. for both cases, the proposed estimation procedure (obtained with λ time set to the same values as those used to analyze real data in the next section) outperforms the naive estimates (2), which turn out to be very irregular (cf. fig 2) . the proposed estimates notably capture well the three different phases of r(t) (stable, decreasing and increasing), with notably a rapid and accurate reaction to the increasing change in the 10 last days. the present section aims to apply the model and estimation tools proposed above to actual covid-19 data. first, specific methodological issues are addressed, related to tuning the hyperparameter(s) λ time or (λ time , λ space ) in univariate and multivariate settings, and to comparing the consistency between different estimates of r(t) obtained from the same incidence data, yet downloaded from different repositories. then, the estimation tools are applied to the estimation of r(t), both independently for numerous countries and jointly for the 94 continental france départements. estimation of r(t) is performed daily, with t thus increasing every day, and updated results are uploaded on a regular basis on a dedicated webpage (cf. http://perso.ens-lyon.fr/patrice. abry. regularization hyperparameter tuning. a critical issue associated with the practical use of the estimates based on the optimization problems (5) and (7) lies in the tuning of the hyperparameters balancing data fidelity terms and penalization terms. while automated and data-driven procedures can be devised, following works such as [29] and references therein, let us analyze the forms of the functional to be minimized, so as to compute relevant orders of magnitude for these hyperparameters. let us start with the univariate estimation (5). using λ time = 0 implies no regularization and the achieved estimate turns out to be as noisy as the one obtained with a naive estimator (cf. eq (2)). conversely, for large enough λ time , the proposed estimate becomes exactly a constant, missing any time evolution. tuning λ time is thus critical but can become tedious, especially because differences across countries (or across départements in france) are likely to require different choices for λ time . however, a careful analysis of the functional to minimize shows that the data fidelity term (9), based on a kullback-leibler divergence, scales proportionally to the input incidence data z while the penalization term, based on the regularization of r(t), is independent of the actual values of z. therefore, the same estimate for r(t) is obtained if we replace z with α × z and λ with α × λ. because orders of magnitude of z are different amongst countries (either because of differences in population size, or of pandemic impact), this critical observation leads us to apply the estimate not to the raw data z but to a normalized version z/std(z), alleviating the burden of selecting one λ time per country, instead enabling to select one same λ time for all countries and further permitting to compare the estimated r(t)'s across countries for equivalent levels of regularization. considering now the graph-based spatially-regularized estimates (7) while keeping fixed λ time , the different r(t) are analyzed independently for each département when λ space = 0. conversely, choosing a large enough λ space yields exactly identical estimates across départments that are, satisfactorily, very close to what is obtained from data aggregated over france prior to estimation. further, the connectivity graph amongst the 94 continental france départements leads to an adjacency matrix with 475 non-zero off-diagonal entries (set to the value 1), associated to as many edges as existing in the graph. therefore, a careful examination of (7) shows that the spatial and temporal regularizations have equivalent weights when λ time and λ time are chosen such that the use of z/std(z) and of (10) above gives a relevant first-order guess to the tuning of λ time and of (λ time , λ space ). estimate consistency using different repository sources. when undertaking such work dedicated to on-going events, to daily evolutions, and to a real stake in forecasting future trends, a solid access to reliable data is critical. as previously mentioned, three sources of data are used, each including data for france, which are thus now used to assess the impact of data sources on estimated r(t). source1(jhu) and source2(ecdpc) provide cumulated numbers of confirmed cases counted at national levels and (in principle) including all reported cases from any source (hospital, death at home or in care homes. . .). source3(spf) does not report that same number, but a collection of other figures related to hospital counts only, from which a daily number of new hospitalizations can be reconstructed and used as a proxy for daily new infections. the corresponding raw and (sliding-median) preprocessed data, illustrated in fig 3, show overall comparable shapes and evolutions, yet with clearly visible discrepancies of two kinds. first, source1(jhu) and source2(ecdpc), consisting of crude reports of number of confirmed cases are prone to outliers. those can result from miscounts, from pointwise incorporations of new figures, such as the progressive inclusion of cases from ehpad (care homes) in france, or from corrections of previous erroneous reports. conversely, data from source3 (spf), based on hospital reports, suffer from far less outliers, yet at the cost of providing only partial figures. second, in france, as in numerous other countries worldwide, the procedure on which confirmed case counts are based, changed several times during the pandemic period, yielding possibly some artificial increase in the local average number of daily new confirmed cases. this has notably been the case for france, prior to the end of the lockdown period (mid-may), when the number of tests performed has regularly increased for about two weeks, or more recently early june when the count procedures has been changed again, likely because of the massive use of serology tests. because the estimate of r(t) essentially relies on comparing a daily number against a past moving average, these changes lead to significant biases that cannot be easily accounted for, but vanishes after some duration controlled by the typical width of the serial distribution f (of the order of ten days). confirmed infection cases across the world. to report estimated r(t)'s for different countries, data from source2(ecdpc) are used as they are of better quality than data from source1(jhu), and because hospital-based data (as in source3(spf)) are not easily available for numerous different countries. visual inspection led us to choose, uniformly for all countries, two values of the temporal regularization parameter: λ time = 50 to produce a strongly-regularized, hence slowly varying estimate, and λ time = 3.5 for a milder regularization, and hence a more reactive estimate. these estimates being by construction designed to favor piecewise linear behaviors, local trends can be estimated by computing (robust) estimates of the derivativeŝ bðtþ ofrðtþ. the slow and less slow estimates ofrðtþ thus provide a slow and less slow estimate of the local trends. intuitively, these local trends can be seen as predictors for the forthcoming value of r:rðt þ nþ ¼rðtþ þ nbðtþ. let us start by inspecting again data for france, further comparing estimates stemming from data in source2(ecdpc) or in source3(spf) (cf. fig 4) . as discussed earlier, data from source2(ecdpc) show far more outliers that data from source3(spf), thus impacting estimation of r and β. as expected, the strongly regularized estimates (λ time = 50) are less sensitive than the less regularized ones (λ time = 3.5), yet discrepancies in estimates are significant, as data from source2(ecdpc) yields, for june 9th, estimates of r slightly above 1, while that from source3(spf) remain steadily around 0.7, with no or mild local trends. again, this might be because late may, france has started massive serology testing, mostly performed outside hospitals. this yielded an abrupt increase in the number of new confirmed cases, biasing upward the estimates of r(t). however, the short-term local trend for june 9th goes also downward, suggesting that the model is incorporating these irregularities and that estimates will return to unbiased after an estimation time controlled by the typical width of the serial distribution f (of the order of ten days). this recent increase is not seen in source3(spf)based estimates that remain very stable, potentially suggesting that hospital-based data are much less affected by changes in testing policies. this local analysis at the current date can be complemented by a more global view on what happened since the lifting of the lockdown. considering the whole period starting from may 11th we end up with triplets [5th percentile; median; 95th percentile] that read as given in table 1 : source2(ecdpc) provides data for several tens of countries. figs 5 to 8 reportrðtþ and bðtþ for several selected countries. more figures are available at perso.ens-lyon.fr/patrice.abry. as of june 9th (time of writing), fig 5 shows that, for most european countries, the pandemic seems to remain under control despite lifting of the lockdown, with (slowly varying) estimates of r remaining stable below 1, ranging from 0.7 to 0.8 depending on countries, and (slowly varying) trends around 0. sweden and portugal (not shown here) display less favorable patterns, as well as, to a lesser extent, the netherlands, raising the question of whether this might be a potential consequence of less stringent lockdown rules compared to neighboring european countries. fig 6 shows that whiler for canada is clearly below 1 since early may, with a negative local trend, the usa are still bouncing back and forth around 1. south america is in the above 1 phase but starts to show negative local trends. fig 7 indicates that iran, india or indonesia are in the critical phase withrðtþ > 1. fig 8 shows that data for african countries are uneasy to analyze, and that several countries such as egypt or south africa are in pandemic growing phases. phase-space representation. to complement figs 5 to 8, fig 9 displays a phase-space representation of the time evolution of the pandemic, constructed by plotting one against the other the local average (over a week) of the slowly varying estimated reproduction numberrðtþ and local trend, ð � rðtþ; � bðtþþ, for a period ranging from mid-april to june 9th. country names are written at the end (last day) of the trajectories. interestingly, european countries display a c-shape trajectory, starting with r > 1 with negative trends (lockdown effects), thus reaching the safe zone (r < 1) but eventually performing a u-turn with a slow increase of local trends till positive. this results in a mild but clear reincrease of r, yet with most values below 1 today, except for france (see comments above) and sweden. the usa display a similar c-shape though almost concentrated on the edge point r(t) = 1, β = 0, while canada does return to the safe zone with a specific pattern. south-american countries, obviously at an earlier stage of the pandemic, show an inverted c-shape pattern, with trajectory evolving from the bad top right corner, to the controlling phase (negative local trend, with decreasing r still above 1 though). phase-spaces of asian and african countries essentially confirm these c-shaped trajectories. envisioning these phase-space plots as pertaining to different stages of the pandemic (rather than to different countries), this suggests that covid-19 pandemic trajectory resembles a clockwise circle, starting from the bad top right corner (r above 1 and positive trends), evolving, likely by lockdown impact, towards the bottom right corner (r still above 1 but negative trends) and finally to the safe bottom left corner (r below 1 and negative then null trend). the lifting of the lockdown may explain the continuation of the trajectory in the still safe but. . . corner (r below1 and again positive trend). as of june 9th, it can be only expected that trajectories will not close the loop and reach back the bad top right corner and the r = 1 limit. continental france départements: regularized joint estimates. there is further interest in focusing the analysis on the potential heterogeneity in the epidemic propagation across a given territory, governed by the same sanitary rules and health care system. this can be achieved by estimating a set of localrðtþ's for different provinces and regions [5] . such a study is made possible by the data from source3(spf), that provides hospital-based data for each of the continental france départements . fig 4 (right) already reported the slow and fast varying estimates of r and local trends computed from data aggregated over the whole france. to further study the variability across the continental france territory, the graphbased, joint spatial and temporal regularization described in eq 7 is applied to the number of confirmed cases consisting of a matrix of size k × t, with d = 94 continental france départements, and t the number of available daily data (e.g., t = 78 on june 9th, data being available only after march 18th). the choice λ time = 3.5 leading to fast estimates was used for this joint study. using (10) as a guideline, empirical analyses led to set λ space = 0.025, thus selecting spatial regularization to weight one-fourth of the temporal regularization. first, fig 10 ( top row) maps and compares for june 9th (chosen arbitrarily as the day of writing) per-département estimates, obtained when départements are analyzed either independently (r indep using eq 6, left plot) or jointly (r joint using eq 7, right plot). while the means of r indep andr joint are of the same order ('0.58 and '0.63 respectively) the standard deviations drop down from '0.40 to '0.14, thus indicating a significant decrease in the variability across departments. this is further complemented by the visual inspection of the maps which reveals reduced discrepancies across neighboring departments, as induced by the estimation procedure. in a second step, short and long-term trends are automatically extracted fromr indep and r joint and short-term trends are displayed in the bottom row of fig 10 (left and right, respectively) . this evidences again a reduced variability across neighboring departments, though much less than that observed forr indep andr joint , likely suggesting that trends on r per se are more robust quantities to estimate than single r's. for june 9th, fig 10 also indicates reproduction numbers that are essentially stable everywhere across france, thus confirming the trend estimated on data aggregated over all france (cf. fig 4, right plot) . video animations, available at perso.ens-lyon.fr/patrice.abry/deptregul.mp4, and at barthes.enssib.fr/coronavirus/ixxi-sisyphe/., updated on a daily basis, report further comparisons betweenr indep andr joint and their evolution along time for the whole period of data availability. maps for selected days are displayed in fig 11 ( with identical colormaps and colorbars across time). fig 11 shows that until late march (lockdown took place in france on march 17th),r joint was uniformly above 1.5 (chosen as the upper limit of the colorbar to permit to see variations during the lockdown and post-lockdown periods), indicating a rapid evolution of the epidemic across entire france. a slowdown of the epidemic evolution is visible as early as the first days of april (with overall decreases ofr joint , and a clear north vs. south gradient). during april, this gradient rotates slightly and aligns on a north-east vs. south-west direction and globally decreases in amplitude. interestingly, in may, this gradient has reversed direction from south-west to north-east, though with very mild amplitude. as of today (june 9th), the pandemic, viewed hospital-based data from source3(spf), seems under control under the whole continental france. estimation of the reproduction number constitutes a classical task in assessing the status of a pandemic. classically, this is done a posteriori (after the pandemic) and from consolidated data, often relying on detailed and accurate sir-based models and relying on bayesian frameworks for estimation. however, on-the-fly monitoring of the reproduction number time evolution constitutes a critical societal stake in situations such as that of covid-19, when decisions need to be taken and action need to be made under emergency. this calls for a triplet of constraints: i) robust access to fast-collected data; ii) semi-parametric models for such data that focus on a subset of critical parameters; iii) estimation procedures that are both elaborated enough to yield robust estimates, and versatile enough to be used on a daily basis and applied to (often-limited in quality and quantity) available data. in that spirit, making use of a robust nonstationary poisson-distribution based semiparametric model proven robust in the literature for epidemic analysis, we developed an original estimation procedure to favor piecewise regular estimation of the evolution of the reproduction number, both along time and across space. this was based on an inverse problem formulation balancing fidelity to time and space regularization, and used proximal operators and nonsmooth convex optimization. this tool can be applied to time series of incidence data, reported, e.g., for a given country. whenever made possible from data, estimation can benefit from a graph of spatial proximity between subdivisions of a given territory. the tool also provides local trends that permit to forecast short-term future values of r. the proposed tools were applied to pandemic incidence data consisting of daily counts of new infections, from several databases providing data either worldwide on an aggregated percountry basis or, for france only, based on the sole hospital counts, spread across the french territory. they permitted to reveal interesting patterns on the state of the pandemic across the world as well as to assess variability across one single territory governed by the same (health care and politics) rules. more importantly, these tools can be used everyday easily as an onthe-fly monitoring procedure for assessing the current state of the pandemic and predict its short-term future evolution. updated estimations are published on-line every day at perso.ens-lyon.fr/patrice.abry and at barthes.enssib.fr/coronavirus/ixxi-sisyphe/. data were (and still are) automatically downloaded on a daily basis using routines written by ourselves. all tools have been developed in matlab™ and can be made available from the corresponding author upon motivated request. at the methodological level, the tool can be further improved in several ways. instead of using o(r) ≔ λ time kd 2 rk 1 + λ space krb > k 1 , for the joint time and space regularization, another possible choice is to directly consider the matrix d 2 rb > of joint spatio-temporal derivatives, and to promote sparsity with an ℓ 1 -norm, or structured sparsity with a mixed norm ℓ 1,2 , e.g., kd 2 rb > k 1,2 = ∑ t k(d 2 rb > )(t)k 2 . as previously discussed, data collected in the process of a pandemic are prone to several causes for outliers. here, outlier preprocessing and reproduction number estimation were conducted in two independent steps, which can turn suboptimal. they can be combined into a single step at the cost of increasing the representation space permitting to split observation in true data and outliers, by adding to the functional to minimize an extra regularization term and devising the corresponding optimization procedure, which becomes nonconvex, and hence far more complicated to address. finally, when an epidemic model suggests a way to make use of several time series (such as, e.g., infected and deceased) for one same territory, the tool can straightforwardly be extended into a multivariate setting by a mild adaptation of optimization problems (6) and (7), replacing the kullback-leibler divergence d kl (zjr � fz) by p i i¼1 d kl ðz i j r � fz i þ. finally, automating a data-driven tuning of the regularization hyperparameters constitutes another important research track. factors determining the diffusion of covid-19 and suggested strategy to prevent future accelerated viral infectivity similar to covid pooling data from individual clinical trials in the covid-19 era expected impact of lockdown in ile-de-france and possible exit strategies estimating the burden of sars-cov-2 in france the impact of a nation-wide lockdown on covid-19 transmissibility in italy measurability of the epidemic reproduction number in data-driven contact networks mathematical models in epidemiology a new framework and software to estimate time-varying reproduction numbers during epidemics the r0 package: a toolbox to estimate reproduction numbers for epidemic outbreaks improved inference of time-varying reproduction numbers during infectious disease outbreaks convex analysis and monotone operator theory in hilbert spaces image restoration: total variation, wavelet frames, and beyond proximal splitting methods in signal processing proximal algorithms. foundations and trends ® in optimization wavelet-based image deconvolution and reconstruction different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures epidemiological parameters of coronavirus disease 2019: a pooled analysis of publicly reported individual data of 1155 cases from seven countries epidemiological characteristics of covid-19 cases in italy and estimates of the reproductive numbers one month into the epidemic nonlinear denoising for solid friction dynamics characterization sparsest continuous piecewise-linear representation of data the emerging field of signal processing on graphs: extending high-dimensional data analysis to networks and other irregular domains a first-order primal-dual algorithm for convex problems with applications to imaging proximal splitting algorithms: relax them all! fonctions convexes duales et points proximaux dans un espace hilbertien. comptes rendus de l'acadé mie des sciences de paris a douglas-rachford splitting approach to nonsmooth convex variational signal recovery on the definition and the computation of the basic reproduction ratio r0 in models for infectious diseases in heterogeneous populations reproduction numbers and sub-threshold endemic equilibria for compartmental models of disease transmission figs 10 and 11 are produced using open ressources from the openstreetmap foundation, whose contributors are here gratefully acknowledged. mapdata©openstreetmap contributors. conceptualization: patrice abry, pablo jensen, patrick flandrin. key: cord-300859-nqklx0vn authors: henderson, kelley c.; benitez, alvaro j.; ratliff, amy e.; crabb, donna m.; sheppard, edward s.; winchell, jonas m.; dluhy, richard a.; waites, ken b.; atkinson, t. prescott; krause, duncan c. title: specificity and strain-typing capabilities of nanorod array-surface enhanced raman spectroscopy for mycoplasma pneumoniae detection date: 2015-06-29 journal: plos one doi: 10.1371/journal.pone.0131831 sha: doc_id: 300859 cord_uid: nqklx0vn mycoplasma pneumoniae is a cell wall-less bacterial pathogen of the human respiratory tract that accounts for > 20% of all community-acquired pneumonia (cap). at present the most effective means for detection and strain-typing is quantitative polymerase chain reaction (qpcr), which can exhibit excellent sensitivity and specificity but requires separate tests for detection and genotyping, lacks standardization between available tests and between labs, and has limited practicality for widespread, point-of-care use. we have developed and previously described a silver nanorod array-surface enhanced raman spectroscopy (na-sers) biosensing platform capable of detecting m. pneumoniae with statistically significant specificity and sensitivity in simulated and true clinical throat swab samples, and the ability to distinguish between reference strains of the two main genotypes of m. pneumoniae. furthermore, we have established a qualitative lower endpoint of detection for na-sers of < 1 genome equivalent (cell/μl) and a quantitative multivariate detection limit of 5.3 ± 1 cells/μl. here we demonstrate using partial least squaresdiscriminatory analysis (pls-da) of sample spectra that na-sers correctly identified m. pneumoniae clinical isolates from globally diverse origins and distinguished these from a panel of 12 other human commensal and pathogenic mycoplasma species with 100% cross-validated statistical accuracy. furthermore, pls-da correctly classified by strain type all 30 clinical isolates with 96% cross-validated accuracy for type 1 strains, 98% cross-validated accuracy for type 2 strains, and 90% cross-validated accuracy for type 2v strains. the cell wall-less prokaryote mycoplasma pneumoniae is a major cause of respiratory disease in humans, accounting for 20% to 40% of all community acquired pneumonia (cap). m. pneumoniae is the leading cause of cap in older children and young adults, while the incidence of infection in the very young and the elderly is on the rise [1] [2] [3] [4] [5] . for adults alone the annual economic burden of cap is > $17 billion [6] . macrolide resistance is a growing concern, particularly in children [5] , and extra-pulmonary sequelae occur in up to 25% of infections. finally, evidence continues to indicate a contributing role for m. pneumoniae infection in the onset, exacerbation, and recurrence of asthma [5] . an area of growing interest is the role of m. pneumoniae strain type in pathogenesis and disease epidemiology. genetic diversity is relatively limited among m. pneumoniae strains, which can be categorized into two major groups (type 1 or type 2) based on variation within sequence of the p1 (mpn141) gene, although variant strains of the two are increasingly more common [7] . the p1 protein is an important virulence factor and immunogen in m. pneumoniae infection [8] [9] [10] . p1 must complex with several other proteins in order to localize to the tip of the terminal organelle, where it mediates receptor binding for attachment to the respiratory epithelium, an essential step in successful colonization of the airways [9, 11] . variation in the p1 gene sequence is used to distinguish between type 1 and type 2 strains of m. pneumoniae, but little is known about phenotypic differences arising from this genetic variation. perhaps notable in this regard is the periodicity of type-switching that occurs between the two major genotypes in regular patterns of four to seven years [12] . m. pneumoniae infection is transmitted through aerosolized respiratory secretions and spreads slowly but efficiently through close living quarters, with incubation periods up to three weeks [13, 14] . symptoms tend to be non-descript, often with complex and variable clinical presentations, which makes definitive diagnosis challenging [2, 6, 15] . as a result, diagnosis is often presumptive and relies heavily on the combination of physical findings and the elimination of other possible causes [4, 5, 14] . the success rate for laboratory culture is poor, even for experienced labs, while serologic testing, historically considered the foundation for diagnosis of m. pneumoniae infection, has limited sensitivity and specificity, a high tendency for false-negatives, requires paired sera resulting in retrospective diagnosis, and must often be paired with another diagnostic method [2, 4, 5, 10, 14] . of the currently existing alternatives, the most efficient means for detection is quantitative polymerase chain reaction (qpcr). at present, the only fda-approved tests for the clinical detection of m. pneumoniae are the illumigene automated detection system (meridian bioscience, inc., cincinatti, ohio) and the filmarray respiratory panel (biofire diagnostics inc., salt lake city, utah). the illumigene platform uses loop-mediated isothermal amplification and is capable of detecting m. pneumoniae in both throat and nasopharyngeal swab specimens with a high degree of sensitivity and specificity. the filmarray respiratory panel employs nested, multiplex qpcr with endpoint melt curve analysis on nasopharyngeal swabs to test for 21 different viral and bacterial respiratory pathogens, and is capable of detecting m. pneumoniae as low as 30 colony-forming units (cfu)/ml [16] . the current standard for m. pneumoniae genotyping is pcr-restriction fragment length polymorphism, but can also be done by nested pcr and sequencing, multilocus variable-number tandem-repeat analysis, or by qpcr and high resolution melt curve analysis [15, [17] [18] [19] . these methods for detection and genotyping exhibit high sensitivity and specificity for all known strain variants, can allow for detection in the early stages of infection, and can be performed in hospitals and reference laboratories [2, 4, 5] . however, the requirement for separate tests for detection and genotyping, as well as the cost, complexity, and expertise required, limits the practicality for widespread, point-of-care use [2, [4] [5] [6] 14] . these limitations create a critical barrier to the accurate and timely diagnosis of m. pneumoniae infection and epidemiological tracking, and a rapid, simple, diagnostic platform capable of simultaneous detection and genotyping would greatly improve the control of m. pneumoniae disease. vibrational spectroscopy has an inherent biochemical specificity that led to its consideration as a next-generation platform for the rapid detection, characterization, and identification of infectious agents [20] [21] [22] [23] . raman spectroscopy in particular has several advantages for application to biological samples, including narrow bandwidths, good spatial resolution, and the ability to analyze aqueous samples due to the absence of interference by water molecules [20, 21, 24] . furthermore, raman spectra provide detailed structural information on the chemical composition of a sample and can serve as a characteristic molecular fingerprint for pathogen identification [23, 24] . despite these advantages, standard raman spectra are inherently limited by weak signals for detection. as a result, the application of traditional raman spectroscopy for biosensing applications was impractical and inefficient [13, 21, 24] until the discovery that sample adsorption onto nanoscopically roughened metallic surfaces results in significant enhancements in raman signal and spectral intensity [23] [24] [25] . this enhancement, by factors up to 10 14 -fold, is attributed to the increased electromagnetic field for molecules in close proximity to the metallic surface [20, 21] . surface-enhanced raman spectroscopy (sers) retains the advantages of standard raman spectroscopy, in addition to markedly improved sensitivity, allowing for considerable success at whole organism molecular fingerprinting [20, 24, 26, 27] . inconsistency and lack of reproducibility in the preparation of sers-active substrates has hindered its widespread use for biosensing applications [20, 21, 24] . however, highly ordered silver nanorod array (na) substrates fabricated using oblique angle deposition (oad) yield consistent sers enhancement factors of around 10 8 , with less than 15% variation between substrate batches [21] . the reproducibility of na-sers substrates can be improved further when patterned into a multiwell format with polydimethylsiloxane (pdms) [20] . the highly reproducible detection capabilities of na-sers have been well demonstrated for multiple infectious agents, including rsv, rotavirus, influenza, hiv, adenovirus, sars coronavirus, and m. pneumoniae [13, 22, [28] [29] [30] . hennigan et al. described an na-sers-based platform capable of detecting m. pneumoniae with statistically significant sensitivity and specificity in both simulated and true clinical throat swabs, with the potential to detect and type m. pneumoniae within a single test [13] . we recently determined the sensitivity of na-sers for m. pneumoniae detection to be < 1 genome equivalent (cell/μl) qualitatively, and to have a quantitative multivariate detection limit of 5.3 ± 1 cells/μl [31] . initial evaluation of this biosensing platform's capabilities indicates the potential for application as a next-generation diagnostic tool for the clinical detection of m. pneumoniae, but a more comprehensive analysis is needed prior to proceeding with clinical validation. in the present study we further explored the specificity of na-sers for m. pneumoniae detection with a panel of 30 m. pneumoniae isolates collected from representative global outbreaks and spanning clinically relevant genotypes. furthermore, since na-sers has inherent biochemical specificity, we analyzed a panel of 12 other human commensal and pathogenic mycoplasmas to demonstrate that this biosensing platform could distinguish m. pneumoniae from its clinically relevant closest phylogenetic relatives. finally, we evaluated the ability of the na-sers platform to correctly type the 30 m. pneumoniae clinical isolates relative to known reference strains of m. pneumoniae. wild type m. pneumoniae reference strains m129 (type 1) and fh (type 2) were grown, harvested, and prepared at the university of georgia (uga) for this study. a panel of 30 additional clinical isolates consisting of 13 type 1 strains, 11 type 2 strains, and six type 2 variant strains were grown, harvested, and prepared for sers and quality control analysis at the pneumonia response and surveillance laboratory at the centers for disease control and prevention (cdc) in atlanta, georgia. p1 genotype groups were determined by the pneumonia response and surveillance laboratory at the cdc in atlanta, ga using dna sequence analysis, qpcr in combination with high resolution melt curve analysis, and rflp sequencing analysis. all mycoplasma isolates and controls were cultured in sp4 medium [2, 30] in tissue culture flasks with a 1μl/ml inoculation and incubated at 37°c. strains grown at uga were harvested at log phase when the phenol red indicator turned an orange color upon reaching a ph of~6.5. strains grown at the cdc were harvested 14 days from the date of inoculation to ensure adequate growth for all isolates. at time of harvest, the spent growth medium was decanted for each flask and 0.1× volume of sterile pbs (ph 7.2) was added to wash the adherent mycoplasmas. the pbs wash was then decanted and the pbs wash repeated 3× before the cells were scraped into 1 ml sterile pbs. cells were then syringe-passaged 10× with a 25 gauge needle and aliquots made for determination of protein content, plating on pplo agar [32] for cfu determination (for select isolates and controls), dna extraction for genome equivalent determination, and sers analysis. m. pneumoniae samples for sers analysis were syringe-passaged 10× with a 25-gauge needle to disperse clumps, fixed with the addition of one volume of 8% formaldehyde in sterile pbs (ph 7.0), and stored at 4°c. growth medium negative control samples were prepared in parallel under the same conditions as the m. pneumoniae reference strains as described previously [31] . at the time of sers analysis, mycoplasma and medium-only negative control samples were diluted in sterile di water to a concentration of 10 5 cells/μl, which falls within the sers detectable range for m. pneumoniae and was found to be dilute enough to ensure that the spectra adequately represent sers raman spectra arising from the ag nanorod substrate [31] . samples were loaded onto the na-sers substrate immediately following this dilution. preparation of non-m. pneumoniae human commensal and pathogenic species for na-sers analysis twelve human commensal and pathogenic mollicutes species closely related [33] to m. pneumoniae were grown and harvested at the university of alabama at birmingham (uab). these . mycoplasma buccale, mycoplasma lipophilum, and mycoplasma faucium were originally intended to be included in the panel, but attempts at culturing these organisms were unsuccessful. for each culture, 500 μl to 1 ml of stock culture was inoculated into approximately 30 ml of sp4, hayflick's, or 10b depending on the individual species' growth requirements, and incubated until the ph indicator changed color, indicative of microbial growth and utilization of the metabolic substrate in the media, i.e., glucose, arginine, or urea. at the time of harvest the cells and spent media were poured into 50 ml polycarbonate tubes and centrifuged at 8,000 rpm for 15 min, except for ureaplasma species, which were centrifuged for 1 hr. the supernatants were decanted and the pellets suspended in 30 ml sterile pbs. the cells were washed by centrifugation at 8,000 rpm for 15 min as above, or 10,000 rpm for 1 hr for ureaplasma species. the supernatants were then decanted and the pellets suspended in 1 ml sterile pbs, transferred to a 1.5 ml vial, and centrifuged at 14,000 rpm for 20 min. the supernatants were again decanted and the pellets suspended in 1 ml sterile pbs and syringe-passaged using a 26-gauge needle to disperse clumps. aliquots were made for spotting onto a blood agar plate to test for contamination, and plating for cfu and color-changing unit (ccu) determination. two 400 μl aliquots for each were centrifuged at 14,000 rpm for 20 min, the supernatant was removed, and the pellets were frozen for shipment to uga, where they were stored at -80°c. for sers and quality control analysis, cell pellets were suspended in 1 ml sterile pbs (ph 7.2) and syringe-passaged 10× with a 25 gauge needle to disperse clumps. aliquots were then made for dna extraction and genome equivalent determination, protein assay, and na-sers analysis. sers samples were prepared by fixing 500 μl of suspended cells with 500 μl of 8% formaldehyde in sterile pbs (ph 7.0), and stored at 4°c until time of sers analysis. at that time the samples were diluted in sterile di water to a concentration of 10 3 to 10 4 cells/μl and then immediately loaded onto the na-sers substrate. a growth medium only negative control and m. pneumoniae strain m129 samples were prepared as described above for comparison. all samples were analyzed for protein content via the bicinchoninic acid assay [34] . dna was extracted by the qiaamp dna blood minikit (qiagen, valencia, ca) using the blood and body fluids protocol, including rnase a treatment. 200 μl of sample were used for dna extraction, with a final elution volume of 200 μl for use to quantify dna content and genome equivalents. genomic dna concentration and absorbance measurements for the bicinchoninic acid assay were performed on a nanodrop instrument (model nd-1000, thermo scientific, wilmington, de) using software v3.5.2. genome equivalents of m. pneumoniae samples were calculated from the dna concentration obtained from this analysis and using the previously determined mass of the m. pneumoniae genome, 5.3x10 7 daltons [35] . genome equivalents for all non-m. pneumoniae samples were determined from dna concentrations obtained from this analysis and a genome mass calculated for this study based on published genome lengths and known g+c contents from the genbank database. na-sers substrates were prepared by oad as described [21, 29, 36, 37] . prior to their use, substrates were cleaned for 5 min in an ar+ plasma using a plasma cleaner (model pdc-32g, harrick plasma, ithaca, ny) to remove any surface contamination [38] and then patterned into 40 3mm diameter pdms-formed wells. 1,2-bis(4-pyridyl)ethylene (bpe; 10 -4 molar in methanol) was used as an external control to ensure consistency between substrates. raman spectra were acquired using a renishaw invia reflex multi-wavelength confocal imaging microscope (hoffman estates, il). a leicha apochromatic 5× objective (na 0.12) illuminated a 1265 μm 2 area on the substrate, which allows spatial averaging and minimizes the effect of potential random hot spots. a 785-nm near-infrared diode laser (renishaw) operating at 10% power capacity (28 mw) provided the incoming radiation, and spectra were collected in 3 10-sec acquisitions. an internal silicon standard measurement was obtained at the beginning of each sers analysis as an internal control for instrument performance. all samples were applied in duplicate to the na substrates at the concentrations specified, in a volume of 1 μl per well, and allowed to dry overnight. spectra were collected from five random locations within each sample spot for analysis, for a total of 10 spectra per sample, and m. pneumoniae reference strain and growth medium controls were independently prepared and analyzed for each substrate. two wells per substrate were intentionally left blank to obtain a background sers reading on the naked nanorod substrate only. a total of three separate na substrates were used for these experiments: two for analysis of m. pneumoniae isolates with n = 390 spectra, and one for analysis of other human and commensal mollicutes species with n = 150 spectra, resulting in a total of n = 540 spectra. spectra were deliberately collected from multiple locations within a single substrate, as well as from multiple substrates, to ensure any inherent variance present in the substrates did not impact the results. raman spectra between 400-1800 cm -1 were acquired using renishaw's wire 3.4 software. instrument settings were optimized to maximize signal and minimize saturation or sample degradation arising from laser stimulation [13, 20] . raman spectra were first averaged using grams32/a1 spectral software package (galactic industries, nashua, nh) in order to assess signal-to-noise quality, and baseline-corrected using a concave rubberband algorithm which performed 10 iterations on 64 points to aid in preliminary evaluation of the spectra and peak assignment (opus, bruker optics, inc., billerica, ma). chemometric analysis was carried out with matlab version 7.10.0 (the mathworks, inc., natick, ma) using pls-toolbox version 7.5.1 (eigenvector research inc., wenatchee, wa). raw spectra were pre-processed using the 1 st derivative of each spectrum and a 15-point, 2 nd order polynomial savitsky-golay algorithm. each dataset was then vectornormalized and mean-centered. due to the inherently complex nature of spectral data, multivariate statistical analysis of the datasets was performed using principal component analysis (pca) and partial least squares-discriminatory analysis (pls-da), using the pls toolbox software. unless otherwise specified, all pls-da models were cross-validated using a venetian blinds algorithm with 10 data splits. all pls-da models in this study, excluding those for individual sample analysis, were generated using between 110-495 total spectra per model. we analyzed 32 clinical isolates, including reference strains m129 (type 1) and fh (type 2), alongside a growth medium control prepared in parallel with the m. pneumoniae samples. full details regarding isolate origin and year, p1 genotype, macrolide susceptibility, protein and dna content, and genome equivalents for m. pneumoniae strains are given in table 1 . cfu values were determined for both reference strains and six randomly chosen additional isolates to assess cell viability at time of fixation and ranged from 1x10 5 to 1x10 7 cfu/ml. due to the propensity for mycoplasma cells to clump, a confounding factor in using cfu values as a metric for sample content is the potential discrepancy between cfu value and actual cell number, which can differ by as much as 10 3 -fold [39] . therefore, protein content and genome equivalents were determined in order to better define the content of the samples at the concentration analyzed by sers. these values fell within comparable ranges and were consistent with published values for bacterial cells [40] . protein concentration per cell was higher for m. pneumoniae isolates harvested during stationary phase relative to those harvested during log phase (growth phase based on the color of the ph indicator in the sp4 medium), but no notable differences in genome equivalents or sers spectra were observed between m. pneumoniae samples relative to growth phase at time of harvest (data not shown). average sers spectra of the nanorod substrate background, growth medium control, and m. pneumoniae samples are shown in fig 1, with each class exhibiting a distinct band pattern, as expected. pls-da was applied here to determine statistically significant detection of m. pneumoniae by na-sers. pls-da is a full-spectrum, multivariate, supervised statistical method whereby prior knowledge of classes is used to yield more robust discrimination by minimizing variation within classes while emphasizing latent variables arising from spectral differences between classes [41, 42] . a pls-da model was generated to discriminate between three classes: the nanorod substrate background (fig 2a) ; the growth medium control ( fig 2b) ; and all m. pneumoniae strains (fig 2c) . the inclusion of substrate background and growth medium controls allowed us to ensure that any differences in growth medium and nanorod background signal within the substrate did not affect the ability of the model to discriminate between the presence or absence of m. pneumoniae. two nanorod substrates were used for these experiments, with each containing duplicate wells of the bare nanorod substrate, independently prepared m129, fh, and growth medium controls, and 15 additional clinical isolates of m. pneumoniae. a total of n = 390 pre-processed na-sers spectra collected from both substrates were included in the model, consisting of 20 nanorod substrate background spectra, 20 growth medium control spectra, 25 m129 spectra, 25 fh spectra, and 10 spectra per additional clinical isolate. the cross-validated statistics for the model show that na-sers correctly classified all 32 clinical isolates as m. pneumoniae regardless of global origin, year isolated, genotype, or macrolide susceptibility phenotype, and distinguished them from the substrate background and the growth medium control with 100% cross-validated sensitivity and specificity. a critical question for clinical detection platforms is specificity for the pathogen of interest, particularly in the context of other organisms potentially present in a clinical sample. sers is a structure-based technique that generates a raman fingerprint or barcode based on the unique molecular content of the sample, and as such, the most likely organisms to generate false positives would be those most closely resembling m. pneumoniae structurally. to evaluate the specificity of the na-sers biosensing platform, 12 human commensal and pathogenic mollicutes species closely related to m. pneumoniae in rpob β-subunit nucleotide and amino acid sequence phylogenies and 16s rdna phylogeny were chosen for analysis alongside m. pneumoniae strain m129 and a growth medium control [33] . in order to best define the content of the sample at the concentration used for sers, analyses were done to determine total protein and dna content, the latter allowing calculation of genome equivalents based on known genome sizes and g+c content ( table 2 ). these fell within comparable ranges and were consistent with published values for bacterial cells [40] . our goal here was to develop a pls-da model that distinguished m. pneumoniae from a panel of closely-related other mollicutes species that might be found in humans. a total of n = 150 pre-processed na-sers spectra were collected on a single nanorod substrate consisting of n = 10 substrate background spectra, n = 10 growth medium control spectra, n = 10 m. pneumoniae spectra, and 10 spectra each per other mollicutes species. an initial pls-da model was generated to discriminate between two classes, the nanorod substrate background and all other biological samples, which it did with 100% cross-validated sensitivity and dotted line indicates the classification threshold line for positive class prediction, and the black-dotted line indicates the 95% confidence interval. cross-validated sensitivity, specificity, and class error for the panels were as follows: (a) nanorod substrate background: 1.00, 1.00, and 0, respectively; for (b) growth medium control: 1.00, 1.00, and 0, respectively; and for (c) m. pneumoniae: 1.00, 1.00, and 0, respectively. crossvalidated statistics were obtained using venetian blinds with 10 data splits to represent the prediction performance of the pls-da model for m. pneumoniae detection. doi:10.1371/journal.pone.0131831.g002 specificity (data not shown). the purpose of this model was to ensure that the nanorod substrate background signal was significantly different than all other samples in order to exclude the background spectra from our future models. once we determined that the nanorod substrate background class could be excluded, a second pls-da model was generated using the same spectra to distinguish among three classes; the growth medium control, m. pneumoniae, and the other mollicutes species. this model had a total of n = 140 pre-processed na-sers spectra, consisting of n = 10 growth medium control spectra, n = 10 m. pneumoniae spectra, and 10 spectra each per other mollicutes species. this model distinguished the three classes with 100% cross-validated sensitivity and specificity (data not shown). upon the successful development of a pls-da model to distinguish between the growth medium control, m. pneumoniae, and all other mollicutes species, a final pls-da model was generated using pre-processed na-sers spectra from all three nanorod substrates analyzed during these experiments. this model contained a total of n = 495 spectra, consisting of 25 growth medium control spectra, 25 m129 spectra, 25 fh spectra, 10 spectra each per other m. pneumoniae clinical isolates (30 isolates total), and 10 spectra each per other mollicutes species (12 species total). this model was also categorized into three classes: the growth medium control ( fig 3a) ; all m. pneumoniae clinical isolates, including reference strains ( fig 3b) ; and all other mollicutes species ( fig 3c) . pls-da distinguished all m. pneumoniae strains from all 12 other human mollicutes species and the growth medium control with 100% cross-validated sensitivity and specificity (fig 3a-3c ). a key advantage of na-sers for biosensing is the potential to detect and type an organism in a single test, especially of interest here since there is currently no existing platform capable of the simultaneous detection and typing of m. pneumoniae. to evaluate this capability we applied pls-da to the m. pneumoniae strain spectra above. our panel of clinical isolates contained three distinct and clinically relevant genotypes of m. pneumoniae: 13 type 1 strains, 11 type 2 strains, and six type 2 variant (2v) strains. m. pneumoniae strains m129 (type 1) and fh (type 2) were used as reference strain controls, as they have been previously applied in this manner for evaluation of m. pneumoniae genotyping assays [19] . for the type 1 strains a pls-da model was generated using 180 pre-processed na-sers spectra consisting of the 25 m129 spectra and the 25 fh spectra as controls, and all spectra from the 13 other type 1 clinical isolate samples (10 spectra per isolate, 130 total spectra). the model was built to discriminate between 2 classes, either type 1 or type 2. pls-da was able to correctly classify all other 13 type 1 strains with the type 1 reference strain with 96.8% sensitivity and 96% specificity (fig 4a) . for the type 2 strains a second pls-da model was generated using 160 pre-processed na-sers spectra consisting of the 50 type 1 and 2 reference strain control spectra and all spectra from the 11 other type 2 clinical isolate samples (10 spectra per isolate, 110 total spectra). this model was likewise built to discriminate between 2 classes, either type 1 or type 2. pls-da was able to correctly classify all 11 other type 2 isolates with the type 2 reference strain control with 99.3% sensitivity and 100% specificity (fig 4b) . for type 2v clinical isolates, a third pls-da model was generated using 110 pre-processed na-sers spectra consisting of the 50 type 1 and 2 reference strain control spectra and all spectra from the type 2v clinical isolate samples (10 spectra per isolate, 110 total spectra). however, this model was built to discriminate between 3 classes, type 1 reference strain control, type 2 reference strain control, or type 2v clinical isolate spectra. a third class was necessary for classification of this genotype, as existing methods are capable of identifying variant strains as unique from type 1 and 2 isolate strains [18] , and as such for clinical purposes na-sers typing should be able to do the same. pls-da correctly classified the type 1 reference strain control as distinct from the type 2 control and the type 2v clinical isolates with 100% cross-validated sensitivity and 98.8% cross-validated specificity (fig 5a) . furthermore, pls-da distinguished the type 2 reference strain control from the type 1 control and 2v clinical isolates with a crossvalidated sensitivity and specificity of 92% and 90.6%, respectively ( fig 5b) . lastly, pls-da correctly classified all six type 2v strains as distinct from the type 1 and 2 reference strain controls with 100% cross-validated sensitivity and specificity (fig 5c) . the drop in sensitivity and specificity observed for the type 2 reference strain control is likely due to the fact that these are variant strains of the type 2 parent strain, and variant strains tend to be more similar to their respective parent strains genetically than either are to the opposite strain type [18, 37] . to further evaluate the strain typing capabilities of na-sers, pls-da models were generated using the m129 and fh reference strains alongside each clinical isolate individually. thirty pls-da models were built using the 25 type 1 m129 spectra and 25 type 2 fh spectra as reference strain control classes, and 10 clinical isolate spectra treated as an unknown class. for type 1 and 2 clinical isolates, two categories were used for cross-validation of the model, while for type 2v isolate strains, three categories were incorporated to cross-validate the model, as described above. for all clinical isolate types the model was cross-validated by using a venetian blinds algorithm with seven data splits. these pls-da models were incorporated to simulate a potential strategy for future application of na-sers for m. pneumoniae genotyping wherein known strain type controls are used to predict the genotype of an unknown clinical sample. full cross-validated statistics for all 30 pls-da models are given in table 3 for all type 1 and 2 clinical isolates, and table 4 for all type 2v clinical isolates. overall, pls-da performance was consistent with the models shown in figs 4 and 5. the only notable difference in performance was a decrease in cross-validated specificity in the individual modeling for type 1 clinical isolates k20, nm2, and fl1, but this likely arises due to the decreased sample size (n = 60) used to build the individual pls-da models. additionally, we compared averaged, baseline-corrected, and normalized spectra of all three genotypes to look for any differences in band pattern between the three genotypes that could be contributing to the classification capabilities demonstrated in the pls-da modeling (fig 6) . the majority of the spectral fingerprint was identical for all three strain types, which is to be expected since they are all the same species and classify as such in the pls-da models shown in figs 2 and 3. however, several visible differences in band pattern were present in the spectra for each genotype of m. pneumoniae, which could account for the ability of na-sers to distinguish between the three genotypes with statistically significant sensitivity and specificity. the averaged type 1 spectrum had two unique peaks, one at 1636 cm -1 that does not appear in the averaged type 2 or 2v spectra, and one at 959 cm -1 which appeared as more distinct and shifted slightly right in the type 1 spectrum when compared to the type 2 spectrum, and did not appear in the type 2v spectrum. the averaged type 2 strain spectrum was very similar to the type 1 strain spectrum aside from the differences mentioned above and the presence of a doublet at 767 and 778 cm -1 , which appeared as more distinct than that present in the type 2v spectrum pneumoniae spectra by open shapes that differ by cluster to indicate the different individual strains and isolates, and the human commensal and pathogenic mollicutes species are represented by light gray shapes that differ by cluster to indicate the individual species. the red-dotted line indicates the classification threshold line for positive class prediction, and the black-dotted line indicates the 95% confidence interval. cross-validated sensitivity, specificity, and class error for the panels were as follows: (a) growth medium control: 1.00, 1.00, and 0, respectively; for (b) all m. pneumoniae samples: 1.00, 1.00, and 0, respectively; and for (c) all 12 mollicutes species: 1.00, 1.00, and 0, respectively. cross-validated statistics were obtained using venetian blinds with 10 data splits to represent the prediction performance of the pls-da model for m. pneumoniae detection. and as a broad singlet in the type 1 spectrum. the averaged type 2v spectrum appeared to be the most distinct of the three, with a doublet at 875 and 890 cm -1 that appeared as a single peak at 890 in type 1 and 2 spectra, and a small peak at 521 that was also absent in type 1 and 2 spectra. while these spectral differences were extremely subtle, chemometric analysis is highly capable of discerning differences such as these with substantial discriminatory classification power [43] . although little is known about the phenotypic effects of strain type beyond observable differences in biofilm formation [44] , the genotypic differences between them are well characterized. briefly, homologous recombination within the p1 gene of repetitive element sequences located both in and outside the p1 gene contributes to sequence variation between strain types indicate the individual clinical isolates, and the strain/isolate designation is indicated above the brackets for each cluster. the red-dotted line indicates the classification threshold line for positive class prediction, and the black-dotted line indicates the 95% confidence interval. the cross-validated sensitivity, specificity and class error for panels a-c were obtained using venetian blinds with 10 data splits to represent the prediction performance of (a) type 1 strains: 1.00, 0.988, and 0.006, respectively; for (b) type 2 strains: 0.92, 0.906, and 0.08, respectively; and for (c) type 2v strains: 1.00, 1.00, and 0, respectively. doi:10.1371/journal.pone.0131831.g005 two categories were used for cross-validation of the model, either type 1 or type 2. clinical isolates were treated as an unknown class and cross-validated sensitivity, specificity, and class error were based on their classification prediction score with their respective reference strain control class. cv, crossvalidated. doi:10.1371/journal.pone.0131831.t003 [7] . nucleotide and amino acid sequencing of 60 m. pneumoniae isolates indicates that trinucleotide short sequence repeats (ssr's) coding for serine can be found in all strain types anywhere from 5-14 times, but appear to be most prevalent in type 1 strains [7] . serine repeats may form a hinge structure and lead to downstream conformational differences in the p1 protein between the different strain types, which could potentially affect its interaction with the host as a surface antigen [45, 46] . in addition, 14 of the 60 isolates in the zhao et al. study had point mutations in several variant strains corresponding to amino acid changes in p1 to glutamine, proline, asparagine, and isoleucine residues [7] . in our study, the peaks unique to the type 1 spectrum are commonly associated with vibrational mode bonds present in lysine (959 cm -1 ) and amide i or alpha helix (1636 cm -1 ) molecular structures [47] [48] [49] . the peaks unique to the type 2 spectral fingerprint located at 767 and 778 cm -1 are commonly associated with vibrational modes found in histidine, tryptophan, or carbohydrate bonds [48] [49] [50] . finally, the peaks unique to the type 2v clinical isolates found at 521, 875 and 890 cm -1 are frequently associated with bonds present in histidine, tryptophan, ribose, indole, asparagine, methionine, glutamine, and s-s and c-c stretching vibrational modes [48] [49] [50] . interestingly, the unique peaks present in the average spectra for the strain types analyzed in this study are predominately associated with protein backbone, amino acid residue, and dna bond vibrations. furthermore, spectral features in the averaged spectrum of the 2v variant strains are consistent with the point mutations identified in the zhao et al. study [7] , and our overall spectral interpretation of the averaged spectra for each strain type is consistent with what is known about the differences between strain types of m. pneumoniae infection. unsupervised chemometric analysis of m. pneumoniae strain types and mollicutes species we applied pca to supplement the pls-da modeling of sample spectra and evaluate the total variance present in our m. pneumoniae typing and other human commensal and pathogenic mollicutes datasets. pca is an unsupervised form of chemometric analysis which reduces the dimensionality of the dataset and facilitates establishing patterns and grouping of similar spectra without a priori knowledge of sample class [43] . pca explains successively smaller proportions of the variance, with the first few principal components explaining the greatest percentage of total variance present in the dataset [51] . pre-processed sers spectra from m. pneumoniae reference strain type 1 and 2 controls and all other type 1 clinical isolates were used to generate a pca plot comparing principle components 1, 2, and 3, which captured 54.3% of the total variance present in the 180 spectra used to build the model (fig 7a) . type 2 control strain fh clustered in the bottom right corner, and the clustering pattern for all type 1 strains was predominately below and to the left, though some overlap between the two strain types was present. the pca model of the type 1 clinical isolates supports the pls-da modeling of the spectra shown in fig 4a. fig 6. comparison of averaged, baseline-corrected, and normalized sers spectra for type 1, type 2, and type 2v genotypes. raw spectra of all type 1 (n = 155), type 2 (n = 135), and type 2v (n = 60) clinical isolates and controls were averaged, baseline-corrected, and normalized using grams32/a1 spectral software package (galactic industries, nashua, nh). red, average spectrum of all type 1 m. pneumoniae strains; green, average spectrum of all type 2 m. pneumoniae strains; blue, average spectrum of all type 2v m. pneumoniae strains. peaks unique to a specific genotype of m. pneumoniae are indicated by arrows and identified above the spectral fingerprint. type 1 peaks, red arrows; type 2 peaks, green arrows; and type 2v peaks, blue arrows. inset at top right of image depicts zoomed-in view of the type 2 doublet at 767 and 778 cm -1 . doi:10.1371/journal.pone.0131831.g006 a second pca model was built using pre-processed sers spectra (n = 160) consisting of type 1 and 2 reference strain controls and all other type 2 clinical isolates of m. pneumoniae. principal components 1-3 captured 58.0% of the total variance and when plotted orthogonally showed a distinct separation between the type 1 reference strain control and all type 2 reference strain and other isolates, with very little overlap of clusters (fig 7b) . pca modeling for the type 2 clinical isolate dataset was consistent with the pls-da modeling of the data shown in fig 4b. a third pca model was built using the pre-processed sers spectra from the type 2v clinical isolate dataset (n = 110). principal components 1-3 captured 54.1% of the total variance and when plotted orthogonally showed distinctly separated clusters for the type 1 control, the type 2 control, and the type 2v clinical isolates, with some overlap present between the type 2 and type 2v clusters (fig 7c) . this clustering pattern further supports the pls-da classification performance shown in fig 5. finally, a pca model was built using the full m. pneumoniae and mollicutes species dataset consisting of pre-processed spectra from all 3 nanorod array substrates (n = 495). principal components 1-3 captured 50.1% of the total variance and when plotted orthogonally showed three distinctly separated clusters for growth medium control spectra, all m. pneumoniae spectra, and all other mollicutes species spectra, with no overlap between clusters (fig 7d) . this supports the pls-da model of the data shown in fig 3. our final question of interest was the ability of the platform to discriminate among the 13 different species analyzed in this study. however, pls-da classification performance diminishes as the number of classes in question increases, due to the underlying algorithms applied by the table 5 . cross-validated pls-da modeling statistics for the prediction performance for species discrimination between m. pneumoniae and nine other human commensal and pathogenic mycoplasma species individually. cv sensitivity cv specificity cv class error for species discrimination, three categories were incorporated to cross-validate the model: either category 1: growth medium control (gmc); category 2: m. pneumoniae (m129); or category 3: other mycoplasma species in question. eight spectra from each category were of known class and two spectra from each category were treated as unknowns to generate a cross-validated prediction model. cross-validated sensitivity, specificity, and class error were based on the classification prediction score for each category. modeling, and as such, attempts to classify all 13 species within a single pls-da model failed to yield statistically significant accuracy. in order to overcome this limitation and address the question in a clinically relevant classification system, individual pair-wise pls-da modeling was employed. for this analysis, we chose to focus on the nine other human commensal and pathogenic mycoplasma species most closely related phylogentically to m. pneumoniae. individual pls-da models were built for each of the nine mycoplasma species to distinguish among three categories: the growth medium control (1), m. pneumoniae (2), or the mycoplasma species in question. each model contained a total of n = 30 spectra, with 10 spectra representing each category. furthermore, for each category eight of the 10 spectra were of known sample class, whereas two out of the 10 spectra were treated as unknowns and classified based on their resemblance to category 1, 2, or 3 spectra. cross-validation of the prediction capability for each model was done using a venetian blinds algorithm with five data splits. models were designed this way to simulate the prediction of a potential unknown clinical sample as mycoplasma-negative, m. pneumoniae-positive, or positive for one of the other human commensal or pathogenic mycoplasma species. the cross-validated sensitivities and specificities for all nine models are given in table 5 . overall, the cross-validated sensitivity and specificity was 90% or greater for all nine models, which is very promising for further application of na-sers for species-level discrimination and prediction. m. pneumoniae is a significant human respiratory tract pathogen in both incidence and public health impact, but diagnostic strategies are complicated by the atypical and complex presentation of disease, non-descript symptoms, the requirement for separate tests for detection and genotyping, and the challenges posed by direct culture. serologic testing was historically the gold standard for diagnosis but suffers from severe limitations that make it both unreliable and impractical for rapid detection. advances in qpcr technologies have overcome many issues with sensitivity and reliability, but the cost of reagents and requirement for technical expertise are still high, and independent tests must be done for detection and genotyping, limiting diagnosis by qpcr to hospital or advanced laboratory facilities and making it impractical for pointof-care use. we previously established that this na-sers biosensing platform is capable of statistically significant detection of m. pneumoniae in true and simulated throat swabs and has a qualitative endpoint of detection for m. pneumoniae of < 1 cell/μl, a sensitivity exceeding that of qpcr [13, 31] . here, na-sers showed statistically significant specificity for m. pneumoniae detection regardless of clinical isolate origin, year of isolation, macrolide susceptibility phenotype, or strain type, and was also able to distinguish all m. pneumoniae clinical isolates and control strains from 12 other human commensal and pathogenic mollicutes species. furthermore, na-sers discriminated between the two major strain types of m. pneumoniae with a high degree of statistically significant accuracy and correctly identified variant strains as different from the two major genotypes. most importantly, na-sers was capable of detecting and strain-typing m. pneumoniae within a single test and thus has the potential to facilitate tracking epidemiological trends, such as type-switching and outbreak periodicity [12] . studies with clinical samples are ongoing, and the effect of the presence of a clinically relevant sample background, for example from a patient's throat swab, on the ability of this platform to identify strain types or distinguish m. pneumoniae from other human commensal and pathogenic mollicutes species remains to be determined. furthermore, assessment of alternative methods of chemometric analysis to account for the increased number of classes and modeling complexity for species-level discrimination by na-sers is necessary. in addition, future clinical application of this technology will require collection of a larger spectral library of isolates and background controls. however, from a point-of-care clinical standpoint, the ability to detect m. pneumoniae rapidly is critical to informing appropriate treatment regimens consistent with the responsible use of antimicrobials. this feature is underscored by the availability of handheld raman instruments having the potential for point-of-care use [52] [53] [54] . in combination with the minimal sample preparation requirements and expedient detection, na-sers shows great promise as a platform for future application to point-of-care m. pneumoniae diagnostics. increased detection of mycoplasma pneumoniae infection in children in england and wales laboratory diagnosis of mycoplasma pneumoniae infection seroprevalence of mycoplasma pneumoniae in healthy adolescents in taiwan acute respiratory infection due to mycoplasma pneumoniae: current status of diagnostic methods new insights into the pathogenesis and detection of mycoplasma pneumoniae infections atypical pathogens and challenges in community-acquired pneumonia sequence analysis of the p1 adhesin gene of mycoplasma pneumoniae in clinical isolates collected in beijing in 2008 to topological mapping of the p1-adhesin of mycoplasma pneumoniae with adherence-inhibiting monoclonal antibodies immunodominant epitopes of the adhesin of mycoplasma pneumoniae comparison of laboratory diagnostic procedures for detection of mycoplasma pneumoniae in 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multivariate approaches in genome-based cancer research: identification of candidate genes for new diagnostics by pls discriminate analysis type 1 and type 2 strains of mycoplasma pneumoniae form different biofilms analysis of distribution indicates diverse functions of simple sequence repeats in mycoplasma genomes hinge atlas: relating protein sequence to sites of structural flexibility characterization of thermophilic bacteria using surface-enhanced raman scattering adsorption of s-s containing proteins on a colloidal silver surface studied by surface-enhanced raman spectroscopy surface-enhanced raman spectroscopy of amino acids adsorbed on an electrochemically prepared silver surface detection and identification of aqueous saccharides by using surfaceenhanced raman spectroscopy chemometrics: a practical guide development of a compact, handheld raman instrument with no moving parts for use in field analysis portable raman explosives detection critical evaluation of a handheld raman spectrometer with near infrared (785 nm) excitation for field identification of minerals key: cord-309010-tmfm5u5h authors: dietert, kristina; gutbier, birgitt; wienhold, sandra m.; reppe, katrin; jiang, xiaohui; yao, ling; chaput, catherine; naujoks, jan; brack, markus; kupke, alexandra; peteranderl, christin; becker, stephan; von lachner, carolin; baal, nelli; slevogt, hortense; hocke, andreas c.; witzenrath, martin; opitz, bastian; herold, susanne; hackstein, holger; sander, leif e.; suttorp, norbert; gruber, achim d. title: spectrum of pathogenand model-specific histopathologies in mouse models of acute pneumonia date: 2017-11-20 journal: plos one doi: 10.1371/journal.pone.0188251 sha: doc_id: 309010 cord_uid: tmfm5u5h pneumonia may be caused by a wide range of pathogens and is considered the most common infectious cause of death in humans. murine acute lung infection models mirror human pathologies in many aspects and contribute to our understanding of the disease and the development of novel treatment strategies. despite progress in other fields of tissue imaging, histopathology remains the most conclusive and practical read out tool for the descriptive and semiquantitative evaluation of mouse pneumonia and therapeutic interventions. here, we systematically describe and compare the distinctive histopathological features of established models of acute pneumonia in mice induced by streptococcus (s.) pneumoniae, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, legionella pneumophila, escherichia coli, middle east respiratory syndrome (mers) coronavirus, influenza a virus (iav) and superinfection of iav-incuced pneumonia with s. pneumoniae. systematic comparisons of the models revealed striking differences in the distribution of lesions, the characteristics of pneumonia induced, principal inflammatory cell types, lesions in adjacent tissues, and the detectability of the pathogens in histological sections. we therefore identified core criteria for each model suitable for practical semiquantitative scoring systems that take into account the pathogenand model-specific patterns of pneumonia. other critical factors that affect experimental pathologies are discussed, including infectious dose, time kinetics, and the genetic background of the mouse strain. the substantial differences between the model-specific pathologies underscore the necessity of pathogenand model-adapted criteria for the comparative quantification of experimental outcomes. these criteria also allow for the standardized validation and comparison of treatment strategies in preclinical models. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 as one of the most frequent infectious diseases, pneumonia causes a tremendous socioeconomic burden in industrialized countries [1] and is the leading infectious cause of death in children worldwide [2] . numerous classes of pathogens can cause acute pneumonia [3] and the risk of pneumonia is greatly enhanced under conditions of impaired pulmonary host defense, including preceding viral infections [4] , mechanical ventilation [5] and sepsis [6] . the leading causative pathogen of community acquired pneumonia (cap) is the gram-positive bacterium streptococcus (s.) pneumoniae [7, 8] which accounts for the majority of bacterial upper and lower respiratory tract infections and is responsible for millions of deaths annually [9, 10] . as another cause of cap influenza a virus (iav) infection leads to rapid progression of lung failure with limited treatment options and frequent fatal outcome [3, 11, 12] . moreover, iav infections are commonly complicated by bacterial superinfection, mostly caused by s. pneumoniae, resulting in severe progressive pneumonia associated with increased mortality [13] . in contrast, the gram-negative and facultatively intracellular bacterium legionella (l.) pneumophila is the causative agent of the severe cap legionnaires' disease, and the second most commonly detected pathogen in pneumonia in patients admitted to intensive care units (icu) in industrialized countries [14, 15] . however, in addition to cap, ventilator-associated pneumonia (vap) is also a major cause of hospital morbidity and mortality in icus [16] and the spectrum of pathogens is shifted in these forms of pneumonia. here staphylococcus (s.) aureus, klebsiella (k.) pneumoniae, acinetobacter (a.) baumannii, and escherichia (e.) coli have been isolated with varying prevalences [17] [18] [19] . more specifically, the gram-negative k. pneumoniae is a significant opportunistic pathogen causing severe life-threatening hospitalacquired respiratory tract infections [20] [21] [22] while s. aureus, a gram-positive bacterium, is one of the most prevalent pathogens of community-and hospital-acquired lower respiratory tract infections in humans and accounts for a significant health and economic burden [23] [24] [25] . a. baumannii and e. coli are ubiquitous gram-negative bacteria which have recently emerged as major causes of community-associated, nosocomial [26, 27] and ventilator-associated pneumonia [19, 28] as well as septicemia induced acute lung injury (ali) [29, 30] . in addition, more recently discovered pulmonary pathogens indicate that novel emerging diseases may add to the list of highly relevant pneumonias that may also be of interest to be studied in animal models. for example, the middle east respiratory syndrome coronavirus (mers-cov) which is transmitted by dromedary camels as vectors [31] has emerged as the cause of severe human respiratory disease worldwide [32, 33] with elderly and immunocompromised individuals particularly in saudi arabia being at highest risk [34] [35] [36] . the various forms of pneumonia have been successfully reproduced in specific murine models of experimentally-induced acute pneumonia [37] [38] [39] . these models have substantially contributed to our understanding of the pathogenesis of community-and hospital-acquired pneumonia as well as emerging lung infections worldwide and are indispensable for the development of novel therapeutic strategies [40] [41] [42] . histopathology has been a powerful, reliable, and reproducible read-out tool for the evaluation of morphological changes in animal lung infection experiments for many decades [43, 44] . qualitative diagnoses are based on a summation of microscopically observable changes in the morphology and cellular composition of the tissue and cell types involved. for a more comparative inclusion of histopathologic information in biomedical research, scoring systems have been widely applied which allow for a first semiquantitative assessment of lesions compared to controls [44, 45] . moreover, all preclinical models used for the development of novel treatment strategies and acceptance by regulatory agencies need to be assessed histopathologically by board certified pathologists as gold standard for qualitative and semiquantitative evaluation of tissue alterations in experimental animals [46] [47] [48] . previous studies have revealed first fundamental differences in histopathologic lesions caused by different pathogens in mouse lungs [38, 41, 42] . however, scoring schemes for acute murine pneumonia existing to date are very superficial, addressing only a few, rather unspecific parameters [45, 49, 50] . more importantly, they hardly allow for a differentiating perspective between distinct pathogens or for group comparisons, e.g., infections of wild type versus genetically modified mice. clearly, there is a strong need for more precise and pathogen-as well as model-specific parameters to allow for an accurate description and semiquantification of the inflammatory phenotype for reliable and reproducible comparisons between experimental groups within each model. therefore, we have recently adapted more specific scoring criteria for s. pneumoniae and s. aureus-induced pneumonia [38, 42] . however, such pathogenspecific scoring criteria have not been employed for other lung pathogens in mice. here, we systematically describe and compare the histopathologies at their peaks of inflammation and injury of nine previously established acute lung infection models induced by s. pneumoniae, s. aureus, k. pneumoniae, a. baumannii, l. pneumophila, e. coli, mers-cov, iav and superinfection with iav and pneumococci. we provide model-specific criteria that can be used for appropriate histological quantitative comparisons, e.g., when different therapeutic interventions are evaluated within these established models. whole mouse lung sections were used to obtain complete overviews, particularly of the distributions of lesions and inflammatory patterns. on the basis of the different and oftentimes quite pathogen-and model-specific changes we identified the most suitable evaluation criteria for each model that will allow for more accurate semiquantitative assessments of the severities and distributions of pneumonic lesions. the lung tissues examined here were derived from experiments primarily conducted for purposes other than this study and most have been published elsewhere [38, 41, 42, [51] [52] [53] , except for the a. baumannii and e. coli experiments that will be published elsewhere. all animal procedures and protocols were approved by institutional ethics committees of charité-university of berlin, justus-liebig university of giessen, philipps university of marburg, university hospital of jena and local governmental authorities (landesamt für gesundheit und soziales (lageso) berlin, regierungspräsidium (rp) gießen and darmstadt, landesamt für verbraucherschutz (tlv) thüringen), respectively. permit numbers were g 0356/10, a-0050/15 (s. pneumoniae), g 0358/11 (s. aureus), g 75/2011, g 110/2014 (k. pneumoniae), a 0299/15 (a. baumannii), g 0175/12 (l. pneumophila), 02-049/12 (e. coli), 114/2012 (mers-cov), g 0152/ 12, and g 0044/11 (iav and superinfection). all animal studies were conducted in strict accordance with the federation of european laboratory animal science associations (felasa) guidelines and recommendations for the care and use of laboratory animals, and all efforts were made to minimize animal discomfort and suffering. all mice, except for mers-cov infected mice, were monitored at 12 hour intervals throughout the experiment to assess appearance, behavior, grooming, respiration, body weight, and rectal temperature. humane endpoints were defined (body temperature <30˚c, body weight loss = 20%, cumbersome breathing, accelerated breathing in combination with staggering, pain or paleness) but not reached by any of the mice at the indicated time points of termination of the experiments. mers-cov infected mice were monitored once daily and appearance, behavior, grooming, respiration and body weight were protocolled. here, a single humane endpoint (loss of body weight of >15%) was defined but not reached by any of the mice employed due to their favourable clinical outcome at the infection dose used. for all experimental infection models, except of k. pneumoniae, female mice (aged 8-12 weeks and weighing 17-22 g) were randomly assigned to groups (n = 2-4) per cage whereas in the k. pneumoniae model female and male mice (aged 23-25 weeks and weighing 22-24 g) were used for model specific reasons [41] . furthermore, for all experimental infection models, specificpathogen-free (spf) mice on c57bl/6 (all except for mers-cov) or balb/c (as previously used for the mers-cov model [52, 54] ) background were used and housed in individually ventilated cages under spf conditions with a room temperature of 22 ± 2˚c and a relative humidity of 55 ± 10%. a 12 hour light/ 12 hour dark cycle was maintained and the animals had unlimited access to standard pellet food and tap water. all experimental details of the infection models compared here were applied following previously published and well established protocols that partly vary in terms of infection doses, routes of infection and time point of examination due to pathogen-or model specific reasons, as given below. for bacterial infections, except for e. coli, mice were anesthetized intraperitoneally with ketamine (80 mg/kg) (ketavet; pfizer, berlin, germany) and xylazine (25 mg/kg) (rompun; bayer, leverkusen, germany). for experimental viral infections, mice were anesthetized using inhalation of isoflurane (forene; abbott, wiesbaden, germany). for lung histology, all mice except of the mers-cov model were humanely euthanized by exsanguination via the caudal vena cava after anesthesia by intraperitoneal injection of premixed ketamine (160 mg/kg) and xylazine (75 mg/kg). mers-cov infected mice were humanely euthanized by cervical dislocation after isoflurane anesthesia. s. pneumoniae (serotype 3 strain, nctc 7978), s. aureus newman (nctc 10833), k. pneumoniae (serotype 2, atcc 43816), a. baumannii (ruh 2037), l. pneumophila (serogroup 1 strain, jr 32) were cultured as described [37, 38, 40, 55, 56] and resuspended in sterile pbs. mice were anesthetized intraperitoneally (i.p.) with ketamine (80 mg/kg) and xylazine (25 mg/kg) and transnasally inoculated with 5 x 10 6 cfu s. pneumoniae (n = 14 mice), 5 x 10 7 cfu s. aureus (n = 4), 5 x 10 8 cfu a. baumannii (n = 8), in 20 μl pbs. mice transnasally infected with l. pneumophila (n = 8) received 1 × 10 6 cfu in 40 μl pbs. mice infected with k. pneumoniae (n = 16) received 3.5 x 10 5 cfu intratracheally in 50 μl nacl (0.9%) via microsprayer1 aerosolizer (model ia-1b, penn-century, inc., wyndmoor, pa) using intubation-mediated intratracheal instillation through intact airways [57] which has previously been optimized for this model [41, [57] [58] [59] . e. coli (atcc 25922) from -80˚c glycerol stock was added to lb broth (carl roth, karlsruhe, germany) and incubated for 12 hours at 200 rpm and 37˚c with 5% co 2 . optical density of 0.03 was adjusted in lb broth followed by incubation until midlogarithmic phase for 1.5 hours at 200 rpm and 37˚c. after centrifugation, the pellet was resuspended in sterile 0.9% nacl at 8 x 10 5 cfu e. coli / 200 μl and administered intraperitoneally (n = 10). for initial transduction of human dpp4 for subsequent infection of balb/c mice with mers-cov (hcov emc) viruses were cultured and prepared as described [52, 54, 60] . mice were transduced transnasally with 20 μl of an adenovirus vector encoding human dpp4 and mcherry with a final titer of 2.5 x 10 8 pfu per inoculum (adv-hdpp4; viraquest inc.), resulting in hdpp4 expression in the epithelial compartment of the lung [60] and transnasally infected with a final titer of 7 x 10 4 tcid 50 of mers-cov in 20 μl dmem (n = 17) under isoflurane anesthesia (forene; abbott, wiesbaden, germany). influenza a/pr/8/34 virus (h1n1; pr8) was grown as described [42] and mice were transnasally infected with 100 pfu pr8 in 50 μl pbs (n = 4) under isoflurane anesthesia. for superinfection experiments, the iav infection procedure was applied as described above with 40 pfu pr8 in 50 μl pbs. 8 days after viral infection, s. pneumoniae was cultured as described [37] and resuspended in sterile pbs. mice were anesthetized intraperitoneally and transnasally inoculated with 1 x 10 3 cfu s. pneumoniae in 20 μl pbs (n = 4). mice were humanely euthanized at model-specific time points as indicated ( table 1 ). between 2 and 6 repetitions of the entire experimental procedures were performed in each model with similar group sizes in each repetition. lungs were carefully removed after ligation of the trachea to prevent alveolar collapse, immersion-fixed in formalin ph 7.0 for 24 to 48 hours (mers-cov for 7 days), embedded in paraffin, cut in 2 μm sections and stained with hematoxylin and eosin (he) after dewaxing in xylene and rehydration in decreasing ethanol concentrations. bacteria were visualized using the giemsa and gram (modified by brown and brenn) stains. for the display of whole lung overviews, he stained slides of entire lung sections were automatically digitized using the aperio cs2 slide scanner (leica biosystems imaging inc., ca, usa) and image files were generated using the image scope software (leica biosystems imaging inc.). three evenly distributed whole-organ horizontal sections throughout the entire lungs were microscopically evaluated to assess the distribution and character of pathologic alterations, generating a modified panel of specific lung inflammation parameters adapted to each pathogen used (table 1 and table 2 ). all examinations were performed by trained veterinary experimental pathologists. for immunohistochemical detection of s. pneumoniae and iav (h1n1), antigen retrieval was performed with microwave heating (600 w) in 10 mm citric acid (750 ml, ph 6.0) for 12 minutes (min). lung sections were then incubated with a purified rabbit antibody polyclonal to s. pneumoniae (1:2,000, kindly provided by s. hammerschmidt) or with a purified goat antibody polyclonal to iav h1n1 (1:4,000, obt155, bio-rad, puchheim, germany) at 4˚c overnight. incubation with an immuno-purified rabbit or goat antibody at the same dilution served as negative controls. subsequently, slides were incubated with a secondary, alkaline phosphataseconjugated goat anti-rabbit (1:500, ap-1000, vector, burlingame, ca) antibody for 30 min at room temperature. the alkaline chromogen triamino-tritolyl-methanechloride (neufuchsin) was used as phosphatase substrate for color development. all slides were counterstained with hematoxylin, dehydrated through graded ethanols, cleared in xylene and coverslipped. transnasal infection of mice with s. pneumoniae, serotype 3 resulted in a broad spectrum of tissue lesions and immune cell infiltrations that are typical of aerogenic bacterial pneumonia. specific for this model, lesions widely expanded down to the periphery of the lung lobes (fig 1a) with inflammation closely surrounding the airways and blood vessels. pneumococcal spread led to an early immune response which was mainly characterized by predominantly intrabronchial ( fig 1b) and intraalveolar ( fig 1c) infiltrations of neutrophils provoking a lobular, suppurative bronchopneumonia with consolidation of affected lung areas. large areas of coagulation and liquefaction necrosis (fig 1d, arrowhead) as indicated by cellular fragmentation, decay, and loss of cellular details, accumulation of cellular and karyorrhectic debris as well as karyorrhexis, karyopyknosis, and karyolysis with consecutive hemorrhage were also present. the perivascular interstitium was widely expanded by edema due to vascular leakage [53] with massive extravasation of neutrophils recruited into perivascular spaces ( fig 1e) . furthermore, suppurative and necrotizing vasculitis accompanied by hyaline thrombi within small-sized blood vessels were occasionally present, indicating early histological evidence of incipient septicemia. increased pulmonary vascular permeability [53] also led to expanded areas of protein-rich alveolar edema which presented as homogenous, lightly pink material within the alveolar spaces in the he stain (fig 1f, asterisk) . a distinctive histopathological feature of pneumococcal pneumonia was the occurrence of massive suppurative to necrotizing pleuritis (fig 1g, arrowhead) and steatitis ( fig 1h) with widespread dispersion of bacteria into the thoracic cavity, likely accounting for the painful and morbid clinical behavior with rapid progression in affected mice. myriads of pneumococci were clearly visible as bluish to purple dots of approximately 1 μm in size in the standard he stain, mostly located on the pleural surface, in the mediastinal adipose tissue or within perivascular spaces in the lungs. + + ++ ++ ++ abscess formation ++ ++ + granuloma formation ++ alveolar edema ++ + ++ + + ++ ++ perivascular edema ++ ++ + + + + perivascular lymphocytic cuffing ++ + ++ ++ ++ + + ++ + vasculitis ++ + + + ++ + fibrinoid degeneration of vascular walls ++ vascular thrombosis +/++ ++ ++ ++ + + + ++ + + pleuritis ++ ++ ++ ++ ++ ++ in contrast, transnasal infection with s. aureus resulted in multifocally extensive but non-expansive bronchopneumonia predominantly located near the lung hilus (fig 2a) , affecting the bronchi, alveoli and interstitium. the main inflammatory cell population consisted of neutrophils, leading to mainly suppurative (fig 2b and 2c ) lesions with a tendency towards abscess formation. in contrast to pneumococci, macrophages were also present albeit at lower numbers than neutrophils. (fig 2c) . similar to klebsiella and streptococci, large areas of necrosis and hemorrhage ( fig 2d) were present. the perivascular areas were predominantly infiltrated by lymphocytes and fewer neutrophils (fig 2e) . compared to the s. pneumoniae model mentioned above [53] , vascular permeability seemed only slightly increased as reported before [38] and perivascular edema (fig 2e) as well as protein-rich alveolar edema (fig 2f, asterisk) were also present albeit to a lesser extent. neither pleuritis nor steatitis were observed consistent with a rather favorable clinical outcome under the conditions used. furthermore, staphylococci were largely undetectable by he stain which was possibly due to the low bacterial spread within the lungs. intratracheal infection of mice with k. pneumoniae resulted in severe widely expansive bronchopneumonia with increased lesion severity in the lung periphery ( fig 3a) . recruited immune cells predominantly consisted of neutrophils, leading to suppurative ( fig 3b) to abscessing ( fig 3c) bronchopneumonia with hemorrhage and necrosis as well as neutrophilic interstitial pneumonia (fig 3d) in less affected areas. increased vascular permeability as reported [40] was associated with massive alveolar (fig 3d, asterisk) and perivascular edema (fig 3e) , admixed with myriads of bacteria easily recognizable as purple dots in the he stain. suppurative to necrotizing vasculitis, pleuritis ( fig 3f, arrowhead) , and steatitis were also present and associated with marked bacterial spread and the rapid lethal clinical outcome. after transnasal infection with a. baumannii, mice developed a widely expansive ( fig 4a) bronchopneumonia with predominantly infiltrating neutrophils causing a suppurative ( fig 4b) to abscessing inflammation with areas of hemorrhage within alveoli and interstitium and large areas of parenchymal necrosis as well as alveolar edema. perivascular spaces had mild to moderate edema and infiltration of lymphocytes and neutrophils ( fig 4c) . vascular thrombosis was a common change (fig 4d, arrowhead) in small-sized blood vessels. similar to staphylococci, acinetobacter was invisible by he stain and neither pleuritis nor steatitis were present. transnasal infection of mice with l. pneumophila resulted in slightly different lesion patterns depending on the time point of examination after infection. at 48 hours after infection, nonexpansive interstitial pneumonia was found in close proximity to the hilus (fig 5a) with comparative histopathology of mouse models of acute pneumonia prominent alveolar wall necrosis (fig 5b) . at the 6 day-time point, specifically the numbers of infiltrating macrophages were clearly increased, leading to accentuated perivascular granuloma formation (fig 5c, arrowhead) . here, marked lymphocytic cuffing of most blood vessels as well as highly activated endothelial cells (fig 5d, arrowhead) were observed. at both time points, neither pleuritis nor steatitis were present. bacteria were invisible in the he stained sections. after intraperitoneal infection, the hematogeneous spread of e. coli to the lungs had resulted in diffuse, interstitial suppurative pneumonia, diffusely affecting the entire lungs, modelling sepsis-induced ali (fig 6a) . the interalveolar interstitium was heavily infiltrated with neutrophils ( fig 6b) with most prominent aggregation around blood vessels (fig 6c) , consistent with bacterial entry via the circulation. numerous hyaline thrombi were present within small-sized blood vessels (fig 6d, arrowhead) , suggestive of disseminated intravascular coagulopathy (dic) due to bacterial septicemia. large, rod-shaped bacteria were easily detectable only outside the lungs, mostly present in the adipose tissue surrounding the esophagus, possibly due to local spread of e. coli via the abdominal cavity. transnasal infection with mers-cov following adenoviral transduction of human dpp4 yielded an expansive, (fig 7a) interstitial pneumonia with severe alveolar epithelial cell necrosis and infiltration of mainly macrophages, lymphocytes, and fewer neutrophils (fig 7b) . only comparative histopathology of mouse models of acute pneumonia moderate peribronchial ( fig 7c) and perivascular (fig 7d) lymphocytic infiltrations were present while most venous blood vessels had marked fibrinoid degeneration and necrosis of comparative histopathology of mouse models of acute pneumonia vascular walls (fig 7d, asterisk) . additional hallmarks of mers-cov infection were large areas of protein-rich alveolar edema (fig 7e, arrowhead) , pronounced hemorrhage within perivascular and alveolar spaces, and interstitium (fig 7f, arrowhead) , and the formation of hyaline thrombi within small-sized blood vessels. after transnasal infection with iav, mouse lungs displayed a diffusely distributed bronchointerstitial pneumonia restricted to single lung lobes only (fig 8a) . alveolar necrosis was prominent and alveolar septae were diffusely distended by infiltrating inflammatory cells (fig 8b) . bronchial epithelial cells were markedly necrotic (fig 8c, arrowhead) and extensively scaled off into the bronchial lumen. alveoli and interstitium were filled with macrophages and lymphocytes as major effector cells ( fig 8d) and prominent perivascular lymphocytic cuffing ( fig 8e) was a characteristic change. furthermore, large areas of alveolar edema (fig 8f, asterisk) and, albeit to a much lesser extent, areas of hemorrhage within alveoli and interstitium were present, suggesting vascular damage and increased permeability. when mice had been infected with iav prior to infection with s. pneumoniae, a combination and exponentiated phenotype of both models was observed 24 hours later. lesions were widely expansive to the lung periphery but restricted to single lung lobes pre-damaged by iav ( fig 9a) . the character of pneumonia included massive infiltration of neutrophils into alveoli ( fig 9b) and bronchi, typical features of severe, suppurative bronchopneumonia. bronchial epithelium was almost entirely necrotic and bronchi were filled up with pus ( fig 9c) . perivascular spaces were edematous and infiltrated by neutrophils and lymphocytes (fig 9d) whereas only mild lymphocytic perivascular cuffing (fig 9e) was present. a severe proteinrich alveolar edema was seen, similar to that seen in the s. pneumoniae mono-infection (fig comparative histopathology of mouse models of acute pneumonia 9f). pneumococci were difficult to visualize by he stain, possibly due to the lower infectious dose used here when compared to the mono-infection. prior to processing for histopathology, small tissue samples from experimentally infected mouse lungs are commonly removed for molecular analyses of gene and/ or protein expression or other readout systems to receive additional information. to obtain representative data from such samples that can be correlated with the histological changes, it is crucial to know about the homogeneity of the distribution of lesions. also, some experimental protocols recommend to use the left and right halves of the lungs, respectively, for different analytical procedures, again anticipating lesion homogeneity and symmetry. however, when we analyzed the distributions and bilateral symmetry of lung lesions for each of the infection models, we found a wide spectrum of distinct distributions and asymmetries (fig 10) . in principle, lesion distributions followed the route of pathogen entry into the lungs. however, the tendencies to spread towards the periphery of the lobes after aerogenous infection varied between different pathogens despite similar aerogenous infection routes. mostly centrally focused lesions induced by s. aureus and l. pneumophila remained close to the hilus with no trend towards peripheral expansion. infection with s. pneumoniae, a. baumannii and mers-cov resulted in lesions closely surrounding major airway segments with centrifugal expansion towards the periphery. in contrast, lesions induced by k. pneumoniae were mostly located in the periphery of the lobes and airways and much weaker adjacent to the hilus despite aerogenous infection. hematogenously-induced sepsis with e. coli was associated with an entirely diffuse distribution of lesions affecting the whole lung with myriads of inflammatory hot spots, commonly surrounding blood vessels. iav-induced lesions were restricted to individual lung lobes only with a rather homogeneous distribution within affected lobes. superinfection of s. pneumoniae into an iav-pneumonia resulted in a pattern virtually identical to that seen after iav infection alone. except for blood borne e. coli pneumonia which was consistently and evenly distributed throughout the entire lungs, affected areas in all other models tested here were randomly distributed more or less asymmetrically between the right and left halves of the lungs and also between adjacent lobes (fig 10) . for more than 100 years, a wide range of special stains have been used for the histological visualization of pathogens and other relevant structures, based on their more or less specific affinities to certain dyes. here, gram stain modified by brown and brenn was used for the visualization of gram-positive bacteria, including s. pneumoniae (fig 11a, arrowhead) , as easily recognizable, dark blue cocci. in contrast, giemsa stain was conducted predominantly for the detection of gram-negative bacteria such as k. pneumoniae (fig 11b, arrowhead) which then turned into light blue to greenish rods. for more specific pathogen detection on slides, particularly for viruses, immunohistochemistry is the method of choice. here, s. pneumoniae and iav were detected by immunohistochemistry using specific anti-s. pneumoniae or anti-iav antibodies, respectively. s. pneumoniae-positive signals were obtained as myriads of red cocci predominantly in the perivascular interstitium (fig 11c) , within neutrophils in alveoli and interstitium, and on pleural surfaces as well as in mediastinal adipose tissue. in addition, pneumococci were also visualized in the marginal sinuses of tracheal lymph nodes, both in macrophages and extracellularly. iav antigen was localized to the apical surface and cytosol of intact and necrotic bronchial epithelial cells (fig 11d) and within alveolar macrophages. the diversity of lesions and in particular the presence or absence of specific patterns in several of the models used ( table 1 ) strongly suggested that a uniform scoring scheme for the pneumoniae were mostly located in the periphery of the lobes and airways and much weaker adjacent to the hilus. hematogenous infection with e. coli was associated with entirely diffuse distribution of lesions affecting the whole lungs with myriads of inflammatory hot spots, commonly surrounding blood vessels. iav-induced lesions were restricted to individual lung lobes with a rather homogeneous distribution within affected lobes. which lobes were affected followed a rather random and inconsistent pattern. superinfection of s. pneumoniae into an iav-pneumonia resulted in a pattern virtually identical to that seen after iav infection alone. except for e. coli induced pneumonia, virtually all lung lesions were distributed asymmetrically between the left and right lung halves with no tendency of either half to be more often or more strongly affected. https://doi.org/10.1371/journal.pone.0188251.g010 comparative histopathology of mouse models of acute pneumonia semiquantification of mouse pneumonia is inconceivable. instead, scoring systems should take into account the more or less pathogen-specific lesion patterns that can be distilled from the comparative characterizations given above. for this purpose, we carved out the most characteristic lesion patterns that appear suitable for the development of specific scoring schemes for each model (table 2 ). different mouse models of acute pneumonia differ widely, with an obvious strong dependence on pathogen-specific features of virulence and spread, route of infection, infectious dose and other factors. here, we provide a detailed descriptive overview of histopathological features and distributions of lesions within infected lungs and compare them between nine relevant and commonly used infection models at their peaks of injury and inflammation. the models employed here all represent well established protocols that have been optimized and successfully used in previous studies, with model-specific variations in infection doses, routes of pathogen administration and analyzed time points [37-42, 51, 52, 54-56] . our model-specific description parameters (table 2 ) provide a rational for the selection of histopathological quantification criteria, in order to best reflect the model-specific lesion and distribution characteristics, which appear to be most relevant. clearly, the severity of lesions in terms of outcome of quantification systems will depend on several other factors that will have to be addressed separately in each model, such as the infection dose, time point of examination or therapeutic interventions. comparative histopathology of mouse models of acute pneumonia the model-associated characteristics of tissue lesions and immune cell infiltrates are widely consistent with well-established properties of the different pathogens used. for example, the destructive tissue damage with mostly neutrophilic infiltrations as seen in s. pneumoniae, s. aureus, and a. baumannii, are typically seen with extracellular bacteria that express cytotoxic virulence factors, such as pneumolysin and hydrogen peroxide [61] from s. pneumoniae or immunogenic cell wall components such as lipoteichoic acid (lta) from s. aureus [62, 63] . on the other hand, the intracellular pathogen l. pneumophila which primarily infects macrophages [64] resulted in a histiocytic infiltrate at 48 h that developed into granulomatous inflammation at 6 days after infection, typical of a t h 1-response [65, 66] . however, several of the pathogens used were associated with additional distinctive features. for example, histology revealed marked pleuritis and steatitis due to pathogen invasion into adjacent extrapulmonary tissues after infections with s. pneumoniae and k. pneumoniae. this massive bacterial spreading throughout the thoracic cavity was exclusively present in these two models and most likely associated with sepsis, responsible for the rapid clinical progression and unfavourable outcome [41, 67] . however, only k. pneumoniae had the tendency of abscess formation which was not seen in pneumococcal pneumonia. infection with s. aureus and a. baumannii also resulted in similar lesion patterns, except for acinetobacter-induced lesions widely expanding to the lung periphery while staphyloccocus-induced pneumonia was restricted to the lung hilus. a second difference between the two was the presence of prominent vascular thrombosis in a. baumannii-induced pneumonia which was absent from staphylococcus pneumonia. the clinical outcome of mice infected with a. baumannii and s. aureus was more favourable when compared to infection with s. pneumoniae or k. pneumoniae [38] which may be explained by the lack of bacterial spreading throughout the thoracic cavity and adjacent tissues, and possibly sepsis. e. coli infection was included here as a model for sepsis-associated ali [29, 30, 68] and consequently induced wide spread vascular thrombosis and vasculitis, most likely due to its blood borne entry into the lungs and concurrent septicemia with disseminated intravascular coagulopathy and associated vascular lesions. vascular thrombosis with or without vasculitis was also observed in other models, including s. pneumoniae, a. baumannii and mers-cov, however, to a much lesser extent and only within the most strongly affected areas. mers-cov and iav-associated lesions clearly reflected the known cellular tropisms of these viruses with necrosis of alveolar walls or bronchial epithelial cells, respectively, being the most characteristic histopathologic features [69] [70] [71] [72] . typical of virally induced lesions, the inflammatory cell infiltrates in mers-cov and iav infections were dominated by lymphocytes with no or only few neutrophils. nevertheless, the two viral models could be clearly distinguished from each other by additional histological characteristics. only the mers-cov infection led to a marked vascular phenotype with necrosis and degeneration of blood vessels, vasculitis, and consecutive vascular thrombosis as well as pronounced hemorrhages [69, 73] . in contrast, iav-induced pneumonia did not display any of these features, but was dominated by marked perivascular lymphocytic cuffing and alveolar edema [42, 74] . subsequent superinfection with low-dose s. pneumoniae potentiated the severity of the iav-induced lesions and aggravated the course of pneumonia. however, it did not alter the principal histological characteristics of iav-pneumonia. the patterns seen after single infection with s. pneumonia were not repeated in this superinfection model, likely owing to the much lower inoculation dose which is usually rapidly cleared from virus-naive lungs. when the distributions of lesions were compared among the 9 models tested, four distinct patterns could be clearly distinguished. the most common pattern, where lesions were focused around central airways and blood vessels close to the lung hilus with the periphery less or not affected can likely be explained by the aerogenous route of infection and pathogen entry. the opposite pattern characteristic of k. pneumoniae infection where the periphery of the lobes was more strongly affected than their hilus areas despite a similar aerogenous route of infection may be due to the aerosolic intratracheal application [75] of these bacteria which is typical of and necessary in this model [41, [57] [58] [59] . these differences are therefore more likely attributable to the model-specific route of infection rather than pathogen-specific properties. similarly, the very homogenous distribution of e. coli induced pneumonia likely followed the diffuse blood borne entry of the pathogen into the lungs after intraperitoneal infection. again unique among the pathogens tested here, the iav-associated pattern affected entire but only select lung lobes with almost complete sparing of others. this distribution probably followed a random spread of the virus along major airways but why some lobes remained virtually unaffected after transnasal infection remains hard to explain. apart from helping to understand differences in pathogen spread, the uneven and often quite asymmetrical distributions have a tremendous impact in practical terms when acute mouse pneumonia is sampled for molecular studies. when quantitative data on mrna or protein expression levels or other biochemical information are to be compared with one another or with tissue lesions, it is imperative that only identically affected areas are compared. since this is impossible to predict or recognize on the macroscopical level for most models, the practice of sampling different regions of such lungs for different readout systems appears highly problematic. another implication of the distinct lesion characteristics, immune cell reactions and distributions among the different models appears highly relevant for histological scoring systems that aim at first quantitative comparisons [45] . to narrow down the list of parameters appropriate for each pathogen and exclude features that are likely irrelevant for some of the models, we selected 23 single histopathologic criteria for the design of semiquantitative scoring systems suitable for each model. these criteria are partly composed of standard parameters such as the determination of the affected lung area, the distribution of lesions or the type of pneumonia induced. however, numerous other and more model-specific parameters were identified which precisely describe particular aspects and allow for a differentiation between the models, such as the presence of perivascular edema, vascular thrombosis, pleuritis or steatitis. appropriate scoring systems may thus encompass more general parameters when different pathogens are compared to one another or more pathogen-specific parameters in case specific pathogen features are in the focus of an experiment. for example, some of the parameters selected here have proven helpful in the discovery and semiquantification of different phenotypes of mouse pneumonia following genetic engineering of pathogens or mice [38, 51, 76] . however, scoring systems that claim universality for all mouse models of acute pneumonia seem neither generally applicable nor meaningful for all specific experimental goals. even the list of 23 parameters selected here may become inappropriate or insufficient when genetic changes on the pathogen or host side may result in different types of lesions, immune cell responses, time courses or other relevant features. in those cases, the list selected here may have to be adjusted or extended to better meet the specific challenges of each new study. as standard hematoxylin and eosin (he) staining of tissue sections failed to visualize most pathogens, traditional special stains as well as immunohistochemical techniques were employed, depending on specific staining properties of the pathogens and the availability of appropriate antibodies. while s. pneumoniae, k. pneumoniae and e. coli were easily visible in he stained tissue sections in areas with low density of inflammatory cells, e.g., in perivascular spaces, they were very difficult to identify in heavily infiltrated and consolidated lung parenchyma. in contrast, s. aureus, a. baumannii, l. pneumophila and both viruses were entirely invisible by he staining and thus had to be visualized by appropriate histotechnical stains or immunohistochemistry. both approaches will likely also allow for a rough quantification of pathogen numbers in tissues when appropriate image analysis tools are used. in this first comparative study of its kind, we examined previously established models with their optimized routes of infection, time points, and infection doses and volumes specific for each model to reach peaks of lung injury and inflammation. variations of such factors can be expected to result in different lesion severities, composition of the cellular infiltrates, and for some models in different expansions of lesions within the lung. still, the conditions used here are all based on observations that have evolved during extensive previous establishment studies of these models [37-39, 41, 42, 51, 52, 54, 56-58, 60, 77-79] . among the most important reasons, most human pathogens are not pathogenic for mice under non-experimental conditions and the decisive factor for obtaining a useful pneumonia model appears to be the determination of the appropriate infection dose and route of infection. in addition, the exact time points of tissue analysis after infection had to be determined for virtually all models with care to obtain a useful model, including a precise definition of the strain or variant of the pathogen used [51, 53, 80, 81] . another variable to consider is the mouse strain used. except for balb/c mice which were used in the mers-cov infection model here for model-specific reasons [60] , all models were conducted with c57bl/6 mice which is among the most commonly used mouse strain in infection research and therefore allows for comparisons with similar studies. however, variations of the strain or genetic background may have a dramatic impact on the type, severity and outcome of inflammation, particularly in innate immune responses [82] [83] [84] . again, the criteria suggested here for scoring procedures should allow to recognize and quantify such differences related to changes in infection dose and volume, time point of examination, strain and age of mice used, pathogen variant and other variables. histopathology of the lungs may be complex and requires fundamental knowledge in species-specific anatomy, physiology, organ-specific immunology, pathology, and histotechnical procedures. furthermore, various background lesions in mice, including strain specific spontaneous degenerative or inflammatory conditions and the possibility of accidental infections unrelated to the experiment should not be confused with experimental outcome. thus, despite our efforts to specify and simplify the criteria relevant for model-specific assessment and quantification of lesions, it appears crucial that trained histopathology experts be involved in the microscopical examination of mouse lungs [46, 85] . clearly, in addition to descriptive or semiquantitative histology, a number of other parameters may be useful for quantitative comparisons between experimental groups to determine the role of specific cell types, molecules, and therapeutic interventions, depending on the strategy and goal of the study [44] . such parameters could include flow cytometric immune cell identifications and quantifications, elisa or quantitative rt-pcr for the probing of cytokines, chemokines or matrix proteins involved in lung pathology and remodeling, and plaque/colony forming assays for the identification or quantification of pathogens, as previously published for most of the models used here [38, 41, 42, 51-53, 86, 87] . all of these methods, however, lack the spatial resolution that only histological assessments offer. only the combination of these techniques will lead to a better understanding of the disease in the complex context of the entire lung pathology. in conclusion, we have identified a spectrum of pathogen-and model-specific lesion characteristics in mouse models of acute pneumonia. our findings underscore the necessity of model-specific criteria for the accurate histopathological characterization and quantitative assessments of experimental pneumonia. 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subunit expression and regulation in pneumococcal pneumoniacomparison to chlamydial lung infection influenza h3n2 infection of the collaborative cross founder strains reveals highly divergent host responses and identifies a unique phenotype in cast/eij mice strain differences in a murine model of air pollutant-induced nonatopic asthma and rhinitis. toxicologic pathology murine strain differences in inflammatory angiogenesis of internal wound in diabetes the ecvp/esvp summer school in veterinary pathology: high-standard, structured training for young veterinary pathologists miniaturized bronchoscopy enables unilateral investigation, application, and sampling in mice il-37 causes excessive inflammation and tissue damage in murine pneumococcal pneumonia the authors thank charlene lamprecht and angela linke for excellent technical assistance and nancy a. erickson for helpful discussions. key: cord-306135-pt4jsr6d authors: chan, kamfai; wong, pui-yan; yu, peter; hardick, justin; wong, kah-yat; wilson, scott a.; wu, tiffany; hui, zoe; gaydos, charlotte; wong, season s. title: a rapid and low-cost pcr thermal cycler for infectious disease diagnostics date: 2016-02-12 journal: plos one doi: 10.1371/journal.pone.0149150 sha: doc_id: 306135 cord_uid: pt4jsr6d the ability to make rapid diagnosis of infectious diseases broadly available in a portable, low-cost format would mark a great step forward in global health. many molecular diagnostic assays are developed based on using thermal cyclers to carry out polymerase chain reaction (pcr) and reverse-transcription pcr for dna and rna amplification and detection, respectively. unfortunately, most commercial thermal cyclers are expensive and need continuous electrical power supply, so they are not suitable for uses in low-resource settings. we have previously reported a low-cost and simple approach to amplify dna using vacuum insulated stainless steel thermoses food cans, which we have named it thermos thermal cycler or ttc. here, we describe the use of an improved set up to enable the detection of viral rna targets by reverse-transcription pcr (rt-pcr), thus expanding the ttc’s ability to identify highly infectious, rna virus-based diseases in low resource settings. the ttc was successful in demonstrating high-speed and sensitive detection of dna or rna targets of sexually transmitted diseases, hiv/aids, ebola hemorrhagic fever, and dengue fever. our innovative ttc costs less than $200 to build and has a capacity of at least eight tubes. in terms of speed, the ttc’s performance exceeded that of commercial thermal cyclers tested. when coupled with low-cost endpoint detection technologies such as nucleic acid lateral-flow assay or a cell-phone-based fluorescence detector, the ttc will increase the availability of on-site molecular diagnostics in low-resource settings. the ability to make technologies for the rapid diagnosis of infectious disease broadly available in a portable, low-cost format would mark a revolutionary step forward in global public health [1, 2] . access to decentralized molecular diagnostic testing can enable faster diagnostics, treatments, and subsequent control of infectious diseases. a critical challenge to efforts in decentralizing molecular diagnostic testing is that a large segment of the population in need of these advances resides in low-resource settings (lrs) that offer extremely limited laboratory infrastructure [3, 4] . while many well-characterized molecular assays have been developed around polymerase chain reaction (pcr) [5, 6] , including pathogen and infectious disease detection [7] [8] [9] , food and water safety [10] [11] [12] , forensics [13, 14] , population-scale polymorphisms [15, 16] , and mutation studies [17] , it remains largely a laboratory technique requiring expensive equipment and trained personnel. thus, pcr-based devices for molecular diagnostics have not been appropriately commercialized for geographical areas and demographics that would benefit most from the technology. besides the cost and temperature-sensitivity of reagents, thermal cyclers are generally much too expensive to be purchased for users in these areas. in addition, the timescales required to perform a typical pcr remain slow, generally about an hour or more. this is partially due to the large thermal masses of most commercial thermal cyclers that make the process very inefficient. to this end, many innovative methods aimed at performing pcr faster have been reported [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] . although these approaches provide valuable scientific innovation, most of them are difficult to implement in lrs. in short, there remains an unmet need for a rapid and low-cost thermal cycler that can carry out molecular diagnostics in rural areas and developing countries in a rapid and low-cost manner. we recently reported a practical solution for bringing a low-cost, simple-to-operate, and rapid thermal cycler to underserved and developing populations [37] . we demonstrated fast pcr thermal cycling at a rate of 15 to 30 s per cycle using a device that can be assembled for $130 using commercial off-the-shelf items [37] . this thermos thermal cycler (ttc) uses a very simple design that performs pcr amplification based on the "archaic" method of hand-transferring reaction tubes through a series of water baths, minimizing the temperature ramping time needed for pcr tubes to reach thermal equilibrium (fig 1) . the automation of the pcr reactions by an arduino microcontroller is performed so that the pcr tubes are mechanically transferred by the actions of servomotors in order to carry out pcr steps. our ttc does not need active thermal control, so it is highly suitable for use in lrs, where the continuous supply of electricity is often unreliable. furthermore, our water-based ttc can accommodate all industry-standard pcr tubes, including 200 to 500 μl pcr tubes and plastic/glass capillary tubes (20 μl capacity) [38, 39] . in this paper, we extend the use of ttc to carry pcr and reverse-transcription pcr (rt-pcr) for the detection of infectious diseases. to carry out a rt-pcr for an rna target, we added a thermos that is maintained between 40 to 50°c to perform reverse transcription. the use of an additional thermos increases the overall cost to slightly under $178. de-identified clinical urine specimens were collected from patients who signed a written consent form from an earlier irb approved study and who agreed to participate in the study and have their de-identified achieved sample be utilized in future research for the development of new molecular tests for stis. original written consent forms were stored in a binder in a locked filing cabinet with access provided to study team coordinator only. both the consent process and study protocol were written in accordance with the approved guidelines set forth by johns hopkins university school of medicine and institutional review board protocols (irb numbers: na_00012998 and na_00023037). for our typical reactions involving a two-step pcr, two 16-or 24-oz vacuum-insulated thermoses (thermos, catalog number ns340tl4 and sk3020mbtri4, purchased from a local target store) were used, with each thermos being prepared with water and oil at denaturation (95 to 97°c) or annealing/extension temperatures (60 or 65°c). a third thermos (50°c for reverse transcription) was also set up when rt-pcr was performed (fig 1) . the temperature of the water baths was measured using k-type thermocouples (model no. sc-tt-k-30-36, omega engineering, stamford, ct) connected to a data logger thermometer (hh147u, omega engineering, stamford, ct). a portable immersion heater can be used to quickly reheat water in thermoses, allowing consecutive pcr runs. detailed procedures on setting up the thermoses to the desired temperatures and automating the ttc for pcr runs can be found in s1 file and our earlier publication [37] . speeding up pcr reactions when polypropylene tubes are used while polypropylene plastic tubes are commonly used in most commercial thermal cycles, they are not ideal for achieving a fast pcr reaction time because of the slow heat transfer between the plastic and the heating element. this is especially true when the tubes are cooled from denaturation temperature to annealing/extension temperature; we have found that most thermal cycles need 10 to 12 s to switch between 95 and 60°c. to reduce the time needed to cool the reagent for annealing in the ttc setup, we added a room-temperature bath (rtb) between the denaturation and annealing/extension bath during down-ramping. the rtb major components include three 24-oz thermoses and a panand-tilt servo set to control the up, down, and rotational motion to shuttle pcr vessels in and out of the thermoses. also included are the battery pack, the arduino electronic controller, and a breadboard. to reduce costs, the pan-and-tilt setup is constructed using a soup can for a fixed height, a wood stick, and a pcr tube holder made with metal wire. doi:10.1371/journal.pone.0149150.g001 rapidly lowered the temperature of the reagents inside the pcr tubes from over 90°c to just above 60°c in 2.5 s before lowering the tubes into the 60°c bath (figure c in s1 file). all commercial thermal-block-based pcr runs were performed in a cfx96 touch real-time thermal cycler (bio-rad) or simpliamp thermal cycler (life technologies). polypropylene plastic tubes and glass capillary tubes were also used to demonstrate the ttc's flexibility in terms of accepting various tubes to deliver fast heat transfer in water. while there is no heated lid in our design, and we noticed that some evaporation and condensation occurred, we found that the quality of the pcr amplification was not significantly affected when checked by gel electrophoresis. in this manuscript, the template concentrations used in pcr reactions were purposely chosen to be very realistic, with most of them giving a cq of 25 to 34, which allows us to access and demonstrate the efficiency of the ttc. table 1 summarizes the targets, primer sequences, product sizes, and components of the pcr reactions. the individual pcr and rt-pcr run protocols will be described in the corresponding data sections. stock primer concentrations were 10 μm and were diluted to a final concentration of 500 nm after the master mixes were prepared. unless specified otherwise, reaction volumes for the commercial cycler and ttc were 20 μl when polypropylene tubes were used. the sample volume for glass capillary tubes was 17 μl. after amplification by the ttc or the commercial unit, the pcr amplicons were typically evaluated using a 2.2% pre-casted gel (flash gel by lonza) at 275 v for 7 min (4 μl mix added to 1 μl of loading dye). the resulting gel images were captured using a cell phone camera. the typical ladder sizes used in the gel were 50, 100, 150, 200, 300, 500, 800, and 1500 bp. to demonstrate the speed of the ttc system, a 45-bp fragment of the single-copy gene kcne1 (potassium channel, voltage-gated isk-related subfamily e regulatory beta subunit 1) was amplified from human genomic dna by the ttc using primers reported in another study [25] . we note that this is a very small fragment, so we expect that pcr can be completed relatively fast. using the ttc with glass capillary tubes, we empirically reduced the time spent in the thermos with a 30-s hot-start, followed by 40 cycles of 97°c (8 s) and 60°c (10 s). the shortest time we tried that obtained pcr amplicons was 2-s denaturation and 4-s annealing/ extension (~10 cycles per min, or 4 min for 40 pcr cycles). after the reactions performed with sybr premix ex taq (takara/clonetech), the tubes were checked by viewing with a blue led gel illumination box (lonza), with the fluorescent signal captured by an orange filter placed in front of a cell phone camera. to demonstrate the ttc's ability to carry out multiplexed reactions (6-plex) and multiple reactions (eight) in a single run, a commercially available multiplexed pcr kit (isohelix dqc kit) was purchased, and the positive control human dna template was used in pcr reactions. the isohelix kit typically yields six proprietary amplicons at 100, 200, 300, 400, 500, and 600 bp. the 500-bp fragment is derived from an internal control and is present even in no-template controls. commercial pcr reactions were performed in the following conditions: 5 min of hotstart at 95°c, followed by 35 cycles of 95°c (30 s), 65°c (30 s), and 72°c (45 s). the total run time was 84 min. eight ttc-based reactions were performed simultaneously using three water baths. the first water bath was for denaturation, while the second water bath was maintained at room temperature to help quickly cool the reaction tubes close to 65°c. the tubes were then transported to the third bath, maintained at 65°c. unlike the manufacturer's suggested protocols, we opted to combine the annealing and extension step at 65°c. we shortened the incubation time in each water bath: a 3-min hot-start at 97°c, followed by 35 cycles of 95°c (15 s), 25°c (2.5 s), and 65°c (20 s). we referred to this protocol as 180s/35x(15s/2.5s/20s), which took the ttc 28 min to complete. to demonstrate the practical value of the ttc, we used it to amplify genomic bacterial dna extracted from clinical samples of previously collected urine samples found to be positive with ct. serially diluted ct positive urine samples 1x, 1/10, 1/100, and 1/1000 were extracted by our in-house-developed, magnetic-particle-based extraction protocol. the eluted dna templates were prepared in premix ex taq (probe qpcr) polymerase master mix (takara/clontech) and amplified in glass capillary tubes with a taqman hydrolysis probe assay. commercial pcr reactions were performed with the following conditions: 30 s of hot-start at 95°c, followed by 40 cycles of 95°c (5 s) and 60°c (15 s). this took 43 min to complete 40 cycles. the ttc-pcr was performed with the following conditions: a 30-s hot-start at 97°c, followed by 35 or 40 cycles of 95°c (5 s) and 60°c (10 s). the pcr amplicon (165 bp) was checked with gel electrophoresis. the progress of the real-time pcr using ttc can also be monitored by using blue leds for dye excitation and digital or cellphone camera with orange filter to take photos of the glass capillary tubes after each cycle. the green fluorescence from samples with higher template concentrations should rise up faster than the ones with less templates. to demonstrate that our ttc is robust and versatile, we performed rt-pcr with extracted hiv rna. cultured hiv-1 type b virus (8e5) in hiv-1 rna negative, defibrinated human plasma was used as our sample (accurun 315 series 500, seracare). the 8e5 virus contains an intact but defective viral genome. this control is formulated for use with in-vitro diagnostic test methods that detect and quantitate hiv-1 rna. the 500 series contains 130,000-300,000 copies/ml of sample. rna extraction was carried out by a nuclisens protocol or by using a spin-column extraction method by qiagen. the extracted template was tested by qrt-pcr using a superscript iii platinum one-step quantitative rt-pcr system (life technologies) or a one step primescript rt-pcr kit (takara/clontech). a commercial thermal cycler was used to perform real-time rt-pcr, with a 5-min reverse-transcription step followed by 30 s of reverse transcriptase inhibition and polymerase actuation at 95°c. the cdna was amplified with 45 cycles of 95°c (5 s) and 60°c (15 s). this 300s/30s/45x(5s/15s) reaction took~45 min to complete. primers and probe information are listed in table 1 . with the ttc, we performed numerous reactions in both propylene pcr tubes and glass capillary tubes. for reactions carried out in thin-walled polypropylene pcr tubes (cat. no. 16950, sorenson bioscience), we used the following typical protocol: 300s/10s/45x or 50x (10s/30s) of rt process, followed by 10 s of rt inhibition, and then 45 or 50 cycles of 10 s of denaturation and 30 s of annealing/ extension. the shortest protocol we tried and that gave a high performance of target amplification was 300s/30s/45x(5s/10s). we used 45 instead of 40 cycles in performing rt-pcr because we wanted to show that the ttc can handle very low template copies (<10 copies per reaction) since the threshold cycles of commercial pcr run were over 30 (32 to 36 in most cases). when rt-pcr was performed, an additional thermos was needed for the reverse-transcription step. a ttc with pan-and-tilt servos was used to rapidly and precisely shuttle the reaction tubes between the rt bath, rt inhibition/cdna denaturation bath, rtb bath, and annealing/ extension bath (four containers total). outbreaks of new viral communicable diseases and emergence of new viral strains, such as the recent outbreak of the ebola 2014 strain in guinea, can represent public health emergencies. rapid and sensitive pcr assays can help diagnose and treat the infected while helping to contain a rapid outbreak. therefore, we used a positive reference material to demonstrate our ability to amplify rna in low-resource settings using the ttc. rna from recombinant ebola virus (accuplex rebola gp/np, seracare) was obtained for rt-pcr by the ttc after an inhouse 15-min extraction was processed. the synthetic constructs were designed targeting the glycoprotein gene (gp), nucleoprotein gene (np), and vp24 of ebola zaire isolate (h.sapienswt/gin/2014/gueckedou-c05; genbank accession number kj660348.2). the capped rna was introduced into baby hamster kidney cells. the recombinant virus was cultured and purified, heat-inactivated, and then diluted into defibrinated human plasma and 0.09% sodium azide as a preservative. primers and probe information are listed in table 1 . the extracted rna was tested using a lab-developed research-use-only assay developed by primerdesign (southampton, united kingdom) and seracare (milford, ma). the ttc rt-pcr was performed using protocols similar to the hiv test, with pcr tubes transferred between three thermoses (reverse transcription, denaturation, and annealing/extension) and an optional room-temperature water bath. the ttc-based pcr conditions involved 5 min of rt processing followed by 10 s of rt enzyme inhibition and cdna denaturation. forty-five to 50 cycles of pcr reactions (with various incubation times) were then performed. the cdc has recently developed a new diagnostic test, "cdc denv-1-4 real time rt pcr assay," for the detection and serotype identification of dengue [40] . this is the first fdaapproved molecular test for dengue that detects evidence of the virus itself. the test can identify all four dengue virus types [40, 41] . this new test will help diagnose dengue within the first 7 days after symptoms of the illness appear; which is the period when people are most likely to see a health care professional. we used the positive samples in this kit, which include heat-inactivated dengue virus rna in human serum, to extract rna for rt-pcr reactions. the extracted total rna samples were amplified by a commercial cycler and the ttc. the commercial rt-pcr condition is listed in table 1 . a 45-bp fragment of the single-copy gene kcne1 was amplified from human genomic dna by the ttc. with a normal concentration of primers (500 nm), 40 cycles of rapid pcr can generate pcr amplicons for gel identification using the 30s/40x(2s/4s) (under 5.5 min) protocol, although the result is slightly more consistent with 30s/40x(4s/6s) (under 7.5 min). in contrast, commercial pcr needs 40 min for completion. fig 2a is an image of the glass capillary tubes after 30s/40x(4s/6s) pcr reactions (total run time of 7.5 min). the capillary tube on the left did not undergo pcr, while the two tubes on the right show reactions after ttc-pcr. it is clear that the fluorescent signal at the conclusion of pcr was higher due to the hydrolysis of the probes. the gel electrophoresis shown in fig 2b confirms that the correct amplicons were produced with both the 5.5-and 7.5-min protocols using glass capillary tubes. these results confirm that the performance of our ttc was very impressive in terms of the cost-to-build and the speed with which reaction can be accomplished. based on a recent report by the wittwer group, we expect extremely fast pcr (15 to 60 s per run) can be achieved using the ttc if 20-fold more concentrated primers and enzymes are used [25] . using the isohelix dna quality check (dqc) kit, we ran the pcr using ttc and used gel electrophoresis to confirm that multiple targets could be amplified. the gel photo in fig 3 shows that the ttc can produce multiplexed amplicons with the correct sizes and that the yield is similar to a three-step reaction performed in the commercial cycler with same number of pcr cycles. in addition, all eight samples that were run simultaneously produced very good amounts of products, indicating that the ttc can accept up to eight samples and provide homogenous conditions within the thermos for pcr. the 28-min reaction time needed by the ttc to perform 35 cycles is much shorter than the protocol performed with a commercial thermal cycler using the manufacturer's suggestions (85 min for 35 cycles). to demonstrate ttc's practical value, we used it to amplify genomic dna extracted from chlamydia trachomatis (ct) positive urine samples. we extracted serially diluted ct) from positive urine samples at 1, 1/10, 1/100, and 1/1,000 of the original concentration using a protocol developed in house, and the template was then amplified in glass capillary tubes with a taqman hydrolysis probe assay. the ttc-pcr reactions that produce 132-bp amplicons were performed with the following condition: 30-s hot-start at 97°c, followed by 35 or 40 cycles at 95°c (5 s) and 60°c (10 s). the amplicons were checked by gel electrophoresis (fig 4) . photos of the glass capillary tubes after each cycle can be recorded to monitor the progress of increasing green fluorescence in tubes with positive target template. an example pcr reaction was recorded and photos were compiled into a gif file as supporting information (s1 video). ttc was able to amplify clinical, sample-derived bacterial dna in a <12-min (40-cycle) protocol, even though a 10.5-min (35-cycle) protocol already can produce enough amplicons to be visible by gel electrophoresis imaging. in contrast, commercial pcr reactions took 45 min to complete a 40-cycle reaction (not shown). while we have successfully demonstrated the use of ttc for dna, the amplification of rna is also critical and, in general, more complicated for point-of-care or low-resource settings since it needs an additional reverse-transcription process prior to pcr. also, many infectious disease diagnostics rely on the detection of viral rna (e.g., hiv, ebola, dengue viruses), so it is critical that the ttc can handle rna templates by performing one-step rt-pcr, especially for emerging rna viruses (such as middle east respiratory syndrome coronavirus-specific rna) [42, 43] . using our ttc, which is equipped with a pan-and-tilt servo kit, users can move pcr tubes into the 3 thermoses/containers needed to optimally run this reaction. for traditional onestep rt-pcr, four water baths for reverse transcription, denaturation, annealing, and extension can be prepared. alternatively, annealing and extension steps can be combined, so users can perform reverse transcription in one thermos, followed by denaturation in another thermos, and then use a third thermos for annealing/extension. when polypropylene tubes are used, we can perform reverse transcription in a thermos, then perform denaturation in another thermos, then use a third room-temperature water bath (a thermos is not necessary) to quickly drop the temperature from over 90°c to slightly above annealing/extension temperature, and finally use a fourth thermos for annealing/extension. this helps speed up the reactions due to faster heat transfer. using a three-bath system and before optimizing the run protocol for speed, we were able to amplify and detect recombinant ebola rna in glass capillary tubes in under 32 min. the gel picture in fig 5a shows that the yield from rapid rt-pcr is about the same as commercial products. however, the rt-pcr reactions in plastic tubes can be completed (including a 5-min reverse-transcription process) within 44.2 min vs. 69 min in the commercial unit. our results support the notion that high-quality, low-resource setting molecular diagnostics can be achieved with a low-cost device. it is important to note that the cq of the pcr reaction demonstrated by the real-time rt-pcr plot was over 35 cycles, meaning that the number of targets in each pcr reaction is likely below 10 copies (fig 5b) . the ability of the ttc to produce enough amplicons for gel electrophoresis means that the efficiency of the ttc is quite high. while others have reported fast pcr reactions using various approaches, seldom were the systems challenged with pcr reactions containing low numbers of template per pcr reaction. this supports our belief that the efficiency of pcr performed by the ttc is very close to that of a commercial unit. the ability of this low-cost ttc to perform highly sensitive rt-pcr amplification of <10 template copies of rna is quite remarkable. we also used the ttc for hiv virus detection by amplifying rna extracted from hiv-1 in serum. the expected product size of the reaction is 85 bp. the commercial pcr thermal cyclers showed that rt-pcr can be completed in about 84 min with a cq of 35 in a 50-cycle reaction. fig 6a shows that rt-pcr of hiv rna can be amplified in polypropylene pcr tubes using the ttc. the two upper tubes contain target rna, while the bottom two tubes are samples that did not go through the reactions. this reaction was performed using the following protocol: 420s/10s/45x(15s/2.5s/30s). during the ttc rt-pcr run (46 min), the denaturation bath dropped from 97.4°c to 92.7°c, while the annealing/extension bath dropped from 61.6°c to 59.2°c. the temperature drops were not severe enough that they affected pcr results severely. fig 6b is the representative gel data showing the amplicons after pcr reactions in propylene tubes. the first two samples in the gel picture are results of commercial rt-pcr using a 50-cycle protocol (total run time of 84 min). the next two samples (lanes 4 and 5) are amplicons from a 45-cycle ttc rt-pcr run using a protocol of 330s/10s/45x(15s/30s) and a total run time of 41 min. in another run, ttc was able to complete 45 cycles of rt-pcr in 41.3 min using a protocol of 330s/20s/45x(15s/30s). during the run, the temperature of the denaturation thermos dropped 4.2°c, while the annealing/extension thermos dropped 1.5°c. therefore, ttc can produce the correct-sized amplicons in about half the reaction time of a commercial unit. we note that because the commercial run included five more cycles than the ttc runs, it is expected that the gel bands' intensities are higher than those from the ttc. fig 6c is the realtime pcr plot of the commercial run (50 cycles). next, glass capillary tubes were used to perform rt-pcr for hiv detection. fig 7 summarizes the results with a range of reaction conditions. in these runs, the rt period (300 s) and the rt inactivation period (10 s) were identical, and only the annealing and extension time were modified. using a protocol of 9 s denaturation and 21 s annealing/extension, ttc completed 45-cycle runs (protocol at 300s/10s/45x[9s/21s]) in 27.7 min. even when the protocol was reduced to 300s/10s/40x(5s/10s) with a total run time of 16.4 min for a 45-cycle rt-pcr reaction (lane 10 and 11), amplicons were still being produced, even though the overall efficiency seems to have dropped slightly. overall, the gel data demonstrate that the ttc was able to complete 45 cycles of rt-pcr using the three-bath ttc next, we used the ttc to demonstrate possible dengue diagnostics by rt-pcr using the rna extracted from inactivated dengue in human serum. similar to the amplification of rna from hiv and recombinant ebola virus, the ttc can carry out effective dengue rna amplification in both plastic and glass capillary pcr tubes. the product size range of the dengue amplicons (i to iv) in the cdc multiplex rt-pcr reactions is between 74 to 112 bp, but only primer and probe targeting denv-1 was used in this study. the gel electrophoresis data (not shown) demonstrated that ttc can be effectively performed with the following protocol: 300s/10s/45x(8s/ 16s) (total run time of 25 min) in glass capillary tubes and 300s/15s/40x(12s/18s) (total time of 26.3 min) in plastic tubes. the commercial rt-pcr used a protocol of 300s/10s/45x(8s/16s), which took 53 min to complete and had a cq of 33.4. besides the gel electrophoresis of amplicon post-pcr, we monitored the progress of amplification by running multiple pcrs that each received a different number of cycles after the same reverse-transcription process. we chose a low template concentration (cq of 33.4 from a commercial run) to ensure that the pcr did not reach the plateau phase too early and mask the real-time intensity difference between runs. fig 8a is the gel electrophoresis data of a series of 300s/10s/45x(8s/16s) reactions showing the amplicons at cycle 0, 15, 20, 25, 30, 35, 40, and 45 . it is clear that the amplicon gel band did not show up until the cycler number reached 35; therefore, the cq is likely between 30 and 35. next, we converted the cycler number information to a time-scale in order to illustrate the speed of the ttc (fig 8b) . the fluorescence signal from the ttc (measured by a portable fluorescent detector [tubescanner, qiagen]) and the commercial run was plotted against the reaction time. it is very clear that the ttc signal rose above the background signal much faster (1068 s, or 17.8 min, after rt started) than the commercial run (2424 s, or 40.4 min), even though the cq values were similar. the results clearly indicated that the ttc is a much faster thermal cycler for generating a positive signal in the real-time reactions. while pcr performed by the ttc has been conducted without using any enclosure, it is expected that in order for it to perform well in low-resource settings with extreme temperature range, an enclosure should be added. we note that, while our setup is more than enough to perform pcr rapidly and with great results, we can further stabilize the temperature by simply adding a styrofoam box to enclose the ttc, placing a small metal can inside, and filling it with boiling water. this adds little cost and complexity to the ttc runs. the heat released by the near-boiling water quickly warms the air mass inside of the box. with the increased air temperature, the thermal gradient between the water and the air becomes smaller, which can lead to a reduced temperature drop in both thermoses. we also tested the ttc's water baths' ability to maintain temperature by placing them inside a refrigerator (8°c) and in the cargo space of an suv under a hot sun (47°c) for 1 hr. when placed inside a refrigerator, the temperature of the denaturation and annealing/extension baths dropped 5.8 and 2.4°c in 60 min. when placed inside a hot suv, the temperature of the denaturation and annealing/extension baths dropped only 3.5 and 1.0°c in 60 min. the smaller temperature drop in a hot environment was expected due to the smaller thermal gradient between the water and the environment. while energy input (electricity, propane gas, or even wood fire) is needed to heat water for the first run, subsequent reheating of water uses very little energy since the temperature in each thermos only drops a few degrees by the end of each run. we have calculated that 10 normal pcr runs in the ttc would use only 0.44 kwh worth of electricity. we also have calculated that the total cost of electricity for 10 consecutive runs can be as low as 5.3 or 8 cents in the united states or nigeria, respectively. in contrast, at least 1.6 kwh will be consumed if pcr runs are performed using a regular thermal cycler (e.g., the simpliamp from life technologies). the combined use of mineral oil and multiple vacuum-insulated, stainless steel thermoses in our ttc enables the capability to maintain water temperatures stable enough to perform rapid pcr and rt-pcr without any active temperature control during the reactions. this simple platform can be used in resource-limiting settings where stable or continuous power supply is not guaranteed. specifically, we demonstrated that our innovative ttc can potentially deliver high-speed and sensitive pcr and rt-pcr for the amplification and detection of infectious diseases such as sexually transmitted diseases (chlamydia/gonorrhea), hiv/aids, ebola hemorrhagic fever, and dengue fever. we also demonstrated that the ttc easily accommodates eight samples simultaneously, and we successfully performed highly multiplexed reactions. by designing the reaction to produce short strands of amplicons (45 bp), we also demonstrated that a 6-s per cycle pcr is feasible without using high concentrations of pcr reagents to speed up the reactions, thus making it one of the fastest and most economical thermal cycling units reported in the literature. in addition, we demonstrated that, when using propylene tubes, the duration of pcr runs can be further shortened by employing an extra room-temperature water bath to rapidly cool the pcr reagents from the denaturation temperature to near the annealing temperature. this simple step can cut 10 s off each pcr cycle without compromising pcr efficiency. since the volume and pcr tubes we used are typical to lab-based pcr, little assay optimization is needed to run pcr using the ttc. with the advantages we have demonstrated, the ttc system could increase the availability of on-site molecular diagnostics in low-resource settings when coupled with low-cost, endpoint-detection technologies such as nucleic acid lateralflow assay or a smartphone-based fluorescence detector. we are working toward equipping the ttc with real-time capability by adding a fluorescence detection system using leds for fluorescent excitation and using a smartphone for fluorescent signal monitoring. supporting information s1 file. detail description on how to construct and program the ttc, arduino codes for the 4-bath setup to run rt-pcr with room temperature bath, and room-temperature bath can speed up pcr when plastic tubes are used. (docx) s1 video. this is a gif file showing the progress of real-time pcr in glass capillary tubes. the left tube has the highest template concentration (1x), the second tube has 1/10 diluted template, the third tube has 1/1000 diluted template and last tube on the right is the no-template-control sample. as expected, it took the least number of cycles for fluorescence signal in the left tube to rise above the background signal. the fluorescence in no-template control tube remained low throughout the reaction. 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cycle number selection continuous flow thermal cycler microchip for dna cycle sequencing transformation of personal computers and mobile phones into genetic diagnostic systems a rapid and low-cost pcr thermal cycler for low resource settings automated polymerase chain reaction in capillary tubes with hot air plastic versus glass capillaries for rapid-cycle pcr analytical and clinical performance of the cdc real time rt-pcr assay for detection and typing of dengue virus serotype-specific detection of dengue viruses in a fourplex realtime reverse transcriptase pcr assay real-time reverse transcription-pcr assay panel for middle east respiratory syndrome coronavirus isolation of a novel coronavirus from a man with pneumonia in saudi arabia disclaimer: the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health.we thank dr. jorge l. muñoz-jordán of us centers for disease control and prevention's dengue branch in puerto rico for providing the cdc denv-1-4 rt-pcr assay used in this manuscript. key: cord-287761-73qgx58i authors: aly, mahmoud; elrobh, mohamed; alzayer, maha; aljuhani, sameera; balkhy, hanan title: occurrence of the middle east respiratory syndrome coronavirus (mers-cov) across the gulf corporation council countries: four years update date: 2017-10-13 journal: plos one doi: 10.1371/journal.pone.0183850 sha: doc_id: 287761 cord_uid: 73qgx58i the emergence of the middle east respiratory syndrome coronavirus (mers-cov) infections has become a global issue of dire concerns. mers-cov infections have been identified in many countries all over the world whereas high level occurrences have been documented in the middle east and korea. mers-cov is mainly spreading across the geographical region of the middle east, especially in the arabian peninsula, while some imported sporadic cases were reported from the europe, north america, africa, and lately asia. the prevalence of mers-cov infections across the gulf corporation council (gcc) countries still remains unclear. therefore, the objective of the current study was to report the prevalence of mers-cov in the gcc countries and to also elucidate on its demographics in the arabian peninsula. to date, the world health organization (who) has reported 1,797 laboratory-confirmed cases of mers-cov infection since june 2012, involving 687 deaths in 27 different countries worldwide. within a time span of 4 years from june 2012 to july 2016, we collect samples form mers-cov infected individuals from national guard hospital, riyadh, and ministry of health saudi arabia and other gcc countries. our data comprise a total of 1550 cases (67.1% male and 32.9% female). the age-specific prevalence and distribution of mers-cov was as follow: <20 yrs (36 cases: 3.28%), 20–39 yrs (331 cases: 30.15%), 40–59 yrs (314 cases: 28.60%), and the highest-risk elderly group aged ≥60 yrs (417 cases: 37.98%). the case distribution among gcc countries was as follows: saudi arabia (1441 cases: 93%), kuwait (4 cases: 0.3%), bahrain (1 case: 0.1%), oman (8 cases: 0.5%), qatar (16 cases: 1.0%), and united arab emirates (80 cases: 5.2%). thus, mers-cov was found to be more prevalent in saudi arabia especially in riyadh, where 756 cases (52.4%) were the worst hit area of the country identified, followed by the western region makkah where 298 cases (20.6%) were recorded. this prevalence update indicates that the arabian peninsula, particularly saudi arabia, is the hardest hit region regarding the emerging mers-cov infections worldwide. gcc countries including saudi arabia now have the infrastructure in place that allows physicians and scientific community to identify and immediately respond to the potential risks posed by new outbreaks of mers-cov infections in the region. given the continuum of emergence and the large magnitude of the disease in our region, more studies will be required to bolster capabilities for timely detection and effective control and prevention of mers-cov in our region. immediately respond to the potential risks posed by new outbreaks of mers-cov infections in the region. given the continuum of emergence and the large magnitude of the disease in our region, more studies will be required to bolster capabilities for timely detection and effective control and prevention of mers-cov in our region. the emergence of mers-cov dates back to july 2012 when an elderly patient of age 60 years died from an acute pneumonia in saudi arabia, and a new coronavirus strain was isolated from his lung tissue [1] . another case of acute respiratory disease was diagnosed in a 49-year old male in london who was from qatar and a new strain of coronavirus was isolated from this patient as well [2] . shortly after, the entire genome of the new coronavirus was sequenced and deposited in the genebank database under the number jx869059, kc164505.2. the phylogenetic analysis of the new virus genome revealed that homology of the nucleotide sequence between two cases was 99.5% and the isolates were closely related to bat coronavirus (bat-cov) which belongs to group 2c of β-coronavirus [3] . according to the recommendations by the international committee on taxonomy of viruses (ictv), the new coronavirus was named as 'middle east respiratory syndrome coronavirus' (mers-cov) [4] . although, initially reported from the middle east, mers-cov exported cases have also been observed worldwide. with regard to viral origin and transmission, the first case of mers-cov infection did not relate it to any particular contact with animals before the disease onset; however, other studies did link it to dromedary camels [5] [6] [7] [8] . beta-coronaviruses are strongly associated with bats serving as reservoir host, particularly, the african neoromicia bats were speculated to be the natural reservoir of mers-cov [9] [10] [11] [12] . notably, serological evidence suggests that mers-cov has been in the circulation for at least 2-3 decades in dromedary camels [13] . given that, the ancestral origin of mers-cov links it to african bats whereas, dromedary camels have been functioning as an intermediate host for this virus for a significantly long period of time [14] [15] [16] . health facilities, hospitals and households with mers patients are considered to be the epidemic centers of mers-cov outbreaks. mers-cov is mainly spreading across the geographical region of the middle east while only sporadic cases are reported in the europe, north america, africa, and lately asia. this may be because of the widespread population of dromedary camels in the middle east; however, the scientific proof of evidence that camel farms are a potential source of mers-cov infections still remains to be established. besides, the typical seasonality pattern is not seen in case of mers-cov infections and only one report links it to camel breeding season. the modes of mers-cov transmission by droplet, contact, or airborne are not yet confirmed as well and thus its transmission among animals and from animals to human and human to human remains unclear. there is also no documentation available regarding mers-cov transmission during airplane flights. therefore, standard infection prevention and control procedures are followed including droplet and airborne precautions. the viral incubation period is from 2 days to 2 weeks. the viral cytopathic effects clearly show prominent syncytium formation in humans as well as non-human primates. mers-cov targets directly the lower respiratory tract (pneumocytes) in dromedary camels and continues to replicate preferentially in the airway cells of the upper respiratory tract. the clinical manifestations of mers-cov infections represent a wide spectrum ranging from asymptomatic cases to the ones with severe respiratory indexes. according to the who, mers-cov infection is an acute respiratory infection involving pyrexia of 38˚c or more, cough with radiologic pulmonary presentation and also the history of the patients originating from or travel to the arabian peninsula and its neighboring countries within 10 days of symptoms. mers-cov cases have been identified as both community-and hospital-acquired, mainly among the aged population and in patients with multiple comorbidities such as acute pneumonia, upper respiratory tract infections, influenza-like illness, or asymptomatic infection(s) in children and immunocompromised hosts. moreover, the common extra-pulmonary symptoms include diarrhea and acute renal failure. the early clinical diagnostic changes include the impaired liver and renal functions, lymphopenia, leukopenia and thrombocytopenia whereas leukocytosis, and neutrophilia are linked to progressive infections. the gold standard for diagnosis is detection of viral rna by rt-pcr in compliance with the who guidelines for positive case criteria. the virus is found to be present in different diagnostic specimens such as the lower respiratory tract, sputum, endotracheal aspirate, bronchoalveolar lavage; upper respiratory tract, nasal or nasopharyngeal swabs, urine, feces, and blood. nevertheless, positive biopsy and autopsy tissue specimens still remain to be reported. direct or indirect contact seems to explain a part of the transmission kinetics observed between dromedary camels and humans. in the general population, transmission is rather inefficient (r0<0.7) and it was reported that mers-cov mortality rate is 35% [17] . however, once the virus is introduced into hospital setting with large numbers of susceptible patients at risk, the virus appears to be transmitted very efficiently among such vulnerable host populations. regarding viral evolution and natural reservoir, dromedary camels are the natural reservoir for mers-cov and given the multitude of different clades found in both dromedary camels and human outbreaks, it appears that virus evolution takes place in the reservoir host rather than in humans. the study objective was to report the prevalence of mers-cov infections in the gcc countries and to also investigate its demographics in the arabian peninsula. the data for the last 4 years were collected form the king abdulaziz medical city, riyadh, ksa. further data were collected from who s1 table and also from ministry of health portals of the gcc countries as follows: bahrain http://www.moh.gov.bh/, kuwait www.moh.gov.kw, oman www.moh.gov.om, qatar www.moph.gov.qa, united arab emirates http://www.moh. gov.ae/, and saudi arabia http://www.moh.gov.sa/. we also consulted the gcc countries' reports and websites for the incidence of mers-cov infections between june 2012 and july 2016. furthermore, we searched pubmed database for articles form gcc countries reporting mers-cov infections. epidemiological data including age, sex, symptoms, date of onset, and date of sampling were collected and entered into excel worksheets. descriptive analysis, frequencies and percentages were calculated using spss vr. 20 statistical software. between june 2012 and july 2016, a total of 1797 confirmed mers-cov cases were reported worldwide with a mortality rate of 38.2% (n = 687). the regional distributions of mers-cov were as follows: middle east had the highest number cases (88.4%), followed by asia (10.7%), europe (0.8%) and usa with only 2 cases officially reported (0.1%) the data are summarized in table 1 and fig 1a. one hundred fifty five patients out of the total 1797 confirmed cases (8.6%), reported their exposure to animals, of which, 130 out of 155 cases (83.9%) were exposed to camels, while 25 out of 155 (16.1%) stated exposure to other animals including sheep, cows and poultry ( table 2 ). despite the fact that 674 out of 1797 mers-cov cases (37.5%) were health careassociated infections and 284 out of 1797 cases (15.8%) involved contact with an infected family member, 147 cases (8.2%) were still reported with no exposure to any of the above. the exposure data were found missing for the remaining 537 (29.9%) patients. the distributions of mers-cov infections among 6 gulf countries are illustrated in table 3 and fig 1b. the majority of mers-cov infections (93%) reported between the time period from june 2012 to july 2016, were from saudi arabia. while, the remaining 5 gcc countries contributed only 7% of the cases with the distributions as follows: united arab emirates (5.0%), qatar (1.0%), oman (0.5%), kuwait (0.2%) and bahrain (0.06%). gender analysis shown in table 3 reveals that 60% of the patients were male and 31% were female. moreover, saudi arabia. next, we sought out the detailed demographic distributions of reported cases among 14 governorates in saudi arabia. as shown in table 4 and fig 1c, most of the cases (52%) were reported in al riyadh region, making it the worst hit area in the country followed by makkah (20%), ash sharqiyah (11%), al madinah (4%) and najran (3%). the remaining 9 regions together contributed to 5% of the cases. to date, cases are still logged from ksa and newly-diagnosed positive cases are on the rise. bahrain. to date, there was only one case reported from bahrain in manama region. herein, a 61-year-old saudi male was admitted on the 29 th of march, 2016 to a health care facility in bahrain for an unrelated medical condition. this person was later on tested as positive for mers-cov ( table 1) . state of kuwait. according to kuwait ministry of health, a total of 4 cases were confirmed as mers-cov infections. the first case was reported form the capital (kuwait city) on and an 85-year-old female from abu dhabi were traced to another confirmed mers-cov patient with no history of exposure to camels or other risk factors. finally, a 37-year-old expat male from abu dhabi developed symptoms on the 9 th of june, 2016 and was later tested positive for mers-cov. finally, we searched for the pattern of mers-cov infections over the months in order to identify seasonality relationship. as shown in fig 2, the average of the reported cases during this 4 years period shows that 60-80 cases are reported in the period between february and may, while about 90-100 are reported in august and september. in addition, a low point of infection occurs in the period of october-january and another one in june. the highest number of cases were reported overall during the summer time. over the past 4 years or so, increasing numbers of mers-cov infections have been reported from the middle eastern region [1] . herein, we present a prevalence update on the current status of mers-cov infections in the gcc countries. the data collected over a time period from june 2012 and july 2016 show that the highest number of cases (1441) were reported from saudi arabia (93%) among a total of 1797 cases reported worldwide. overall, a total of 1550 cases were reported only from the gcc region. the saudi arabian capital city of riyadh with 756/1441 (52.4%) cases remained the hardest hit area for mers-cov outbreaks. the incidence of mers-cov infections was found to be highest among the elderly population aged 60 yrs or above. moreover, the gender analysis showed that there were twice more number of males infected (871/1317) than females (446/1317). there is no evidence that mers-cov has gender predisposition [18] . the observed gender-related rates could be simply due to the higher probability of male exposure to camel population than females in the region [18, 19] . furthermore, over one third (37.5%) of mers-cov patients received intensive care among all hospitalized cases [20] . one could argue that the hot climate shared by arabian peninsula and sub-saharan african region could contribute to the spread of mers-cov infections across these geographical regions. although lesser in number, there are still numerous mers-cov infections recorded in saudi arabia during the winter time as compared with summer. the seasonality pattern analysis identifies a 2-phase annual cycle wherein the outbreaks occur during the winter and summer months. altogether, the summer time represents the peak season for mers-cov infections and transmission. the evolutionarily related bat virus might have undergone modifications and adaptation in order to be able to successfully infect and multiply in the camel as an intermediate host before transmission to the human host [21] . although, camel is a well-known animal that is widely colonized in the gulf region, it is also reared and maintained in other parts of the world. we speculate that there might be certain conditions or factors involved with regard to camel herding and shepherding exclusively in saudi arabia that would have facilitated and contributed to the survival of pathogen and fast spread of mers-cov infections from camels to humans across all over the country. there are some reports showed that human consumption of unpasteurized camel milk and or other camel products maybe a reason for the zoonotic transmission of mers-cov in the region [22] [23] [24] . on the other hand, there is strong evidence that mers-cov has been circulating in the dromedary camel population for more than 2 decades [25] . yet, the reason that first human infected case was identified in 2012 remains unclear. we found that mortality rates were higher among the elderly group for both genders which was also concordant with a previous study [26] . the possible explanation for the enhanced mortality in aged patients could be the presence of senescence-associated immune vulnerability in these individuals and suboptimal immune reactivity following a systemic challenge by exposure to mers-cov natural infection. other gulf countries show a few sporadic cases which may be due to the missing data that still have to be set straight or it could possibly be due to small size of camel populations is wide spread across vast geographical region. there may be still other factors involved that remain hidden at present but contribute significantly to the survival, transmission and pathogenesis of this relatively newly identified pathogen in this region of the world. notably, it appears as if coronaviruses are able to cause serious viral infections when transferred from their reservoir (wild bat) host to the human host as observed previously for ebola virus as well which was transmitted from wild bats to humans in africa. actually, many of mers-cov cases are initiated in rural areas and following hospitalization, further cases were reported. the majority of the mers-cov outbreak cases took place at health facilities; index cases are very crucial and they raise the question of the route of transmission of this zoonotic virus. this warrants caution that strict healthcare protocols and guidelines need to be followed and practiced by health care personnel in order to prevent the new outbreaks of mers-cov. in conclusion, mers-cov infections were reported to occur in saudi arabia during the whole year whereas the incidence of human outbreaks peaked in winter and summer months. the disease incidence was also highest among the elderly population aged 60 yrs and above. besides, the fact that majority of these cases were due to human to human interaction i.e. especially among the hospitalized icu patients and not due to camel to human transmission, the local health sectors need to be made aware to mandate implementation of effective control strategies and stringent compliance with better standards of health and hygiene nationwide. despite all whistle blowing efforts aimed at raising awareness of the magnitude of the problem at home, further efforts are still needed for proper treatment and care of mers-cov-infected patients in this country. last but not least, the availability detailed reports of each and every case of mers-cov infection globally, and the gcc region particularly will provide valuable information to the scientific community that may be used to track, contain, and eradicate this disease more effectively. supporting information s1 table. isolation of a novel coronavirus from a man with pneumonia in saudi arabia genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia tropism and replication of middle east respiratory syndrome coronavirus from dromedary camels in the human respiratory tract: an invitro and ex-vivo study prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate infection control and mers-cov in health-care workers mers coronovirus has probably been present in bats for many years, research shows link to mers virus underscores bats' puzzling threat genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus in bats, saudi arabia mers coronavirus neutralizing antibodies in camels animal models of middle east respiratory syndrome coronavirus infection mers-cov an emerging viral zoonotic disease: three years after and counting. recent pat antiinfect drug discov middle east respiratory syndrome prevalence of comorbidities in the middle east respiratory syndrome coronavirus (mers-cov): a systematic review and meta-analysis middle east respiratory syndrome coronavirus: review of the current situation in the world mers-cov geography and ecology in the middle east: analyses of reported camel exposures and a preliminary risk map characteristics and outcomes of middle east respiratory syndrome coronavirus patients admitted to an intensive care unit in jeddah, saudi arabia middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir human infection with mers coronavirus after exposure to infected camels, saudi arabia mers-cov in upper respiratory tract and lungs of dromedary camels, saudi arabia middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels mers coronaviruses in dromedary camels a lesson learned from middle east respiratory syndrome (mers) in saudi arabia the authors thank the kaimrc members and infectious disease research unit for their help and support. this study was approved by the irb and supported by funds from king abdullah international medical research center kaimrc (grant #rc15/128). conceptualization: mahmoud aly. key: cord-321624-z2mntwef authors: kowitdamrong, ekasit; puthanakit, thanyawee; jantarabenjakul, watsamon; prompetchara, eakachai; suchartlikitwong, pintip; putcharoen, opass; hirankarn, nattiya title: antibody responses to sars-cov-2 in patients with differing severities of coronavirus disease 2019 date: 2020-10-09 journal: plos one doi: 10.1371/journal.pone.0240502 sha: doc_id: 321624 cord_uid: z2mntwef background: a greater understanding of the antibody response to sars-cov-2 in an infected population is important for the development of a vaccination. aim: to investigate sars-cov-2 iga and igg antibodies in thai patients with differing severities of covid-19. methods: plasma from the following patient groups was examined: 118 adult patients with confirmed sars-cov-2 infections, 49 patients under investigation (without confirmed infections), 20 patients with other respiratory infections, and 102 healthy control patients. anti-sars-cov-2 enzyme-linked immunosorbent assay (elisa) from euroimmun was performed to assess for iga and igg antibodies. the optical density (od) ratio cutoff for a positive result was 1.1 for iga and 0.8 for igg. additionally, the association of the antibody response with both the severity of disease and the date after onset of symptoms was analyzed. results: a total of 289 participants were enrolled and 384 samples analyzed from march 10 to may 31, 2020. patients were categorized, based on their clinical manifestations, as mild (n = 59), moderate (n = 27), or severe (n = 32). the overall sensitivity of iga and igg from the samples collected after day 7 of the symptoms was 87.9% (95% ci: 79.8–93.6) and 84.8% (95% ci: 76.2–91.3), respectively. compared to the mild group, the severe group had significantly higher levels of spike 1 (s1) antigen-specific iga and igg. all patients in the moderate and severe groups had s1-specific igg, while 20% of the patients in the mild group did not have any igg detected after two weeks after the onset of symptoms. interestingly, in the severe group, the sars-cov-2 igg level was significantly higher in males than females (p = 0.003). conclusion: the serological test for sars-cov-2 has a high sensitivity more than two weeks after the onset of illness. additionally, the serological response differs among patients based on sex as well as the severity of infection. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 in late december 2019, an outbreak of initially undiagnosed pneumonia was reported in wuhan, hubei province, china [1] . the causative pathogen was later identified as a novel beta coronavirus closely related to the severe acute respiratory syndrome (sars) coronavirus (cov) family and was recently termed sars-cov-2 [2] . as of july 30, 2020, more than 17 million people were infected with sars-cov-2, and there were up to 670,000 sars-cov-2-associated deaths [3] . the first case in thailand was reported on january 12, 2020 and was a traveler from wuhan [4] . on july 30, 2020, there were 3,304 confirmed sars-cov-2 cases in thailand, with an epicenter in the bangkok metropolitan area. real-time reverse transcription polymerase chain reaction (rt-pcr) diagnostic assays are a goal standard for case ascertainment and diagnosis [5] . however, validated serological tests provide evidence to compliment virological diagnoses, particularly in or after the second week of infection [6] . a greater understanding of the antibody response in an infected population is beneficial for the development of a vaccine. enzyme-linked immunosorbent assay (elisa) is commonly used to access viral-specific antibodies in a quantitative manner, and for decades has been widely accepted as a diagnostic test for antibodies. the sensitive, quantitative measurements of elisa make it suitable to assess dynamic changes in viral-specific antibodies. in principle, antigen-specific igm and iga should be detected in approximately the second week of infection, followed by antigen-specific igg after the second week of infection. there are several serology platforms currently available, which use various antigens. one large nucleocapsid-based elisa study assessing 208 samples reported that igm and iga were detected 3-6 days after the onset of symptoms with a sensitivity of 85.4% and 92.7%, respectively, while igg was detected later, 10-18 days after the onset of symptoms, with a sensitivity of 77.9% [7] . interestingly, another study showed that the seroconversion if igg against the sars-cov-2 nucleocapsid and a peptide from the spike region was detected as early as that of igm and reached its peak within six days after seroconversion [8] . compared to patients with severe cases, a weaker and more rapidly declining antibody response was observed in asymptomatic patients and in those with milder symptoms [9] . the euroimmun anti-sars-cov-2 elisa was one of the first ce-marked (european conformity) diagnostic assays developed and available worldwide. it assesses the response of iga and igg to the spike 1 (s1) protein and has been reported to correlate well with the plaque reduction neutralization test (prnt) [10, 11] . the euroimmun igg assay received emergency use authorization (eua) from the united states (us) food and drug administration (fda). thus far, most of the results have been reported from europe and the us. the objective of this study was to investigate the response of iga and igg antibodies to sars--cov-2 in serial blood samples collected from a population of thai patients with confirmed covid-19, and the association of these responses with the severity of the illness. the present study was conducted at the thai red cross emerging infectious diseases clinical center (trc-eidcc) and the faculty of medicine at chulalongkorn university. the study present was reviewed and approved by the institutional review board of the faculty of medicine (irb number 242/63) and the national blood center, thai red cross society (coa no. nbc 5/2020). confirmed covid-19 cases were defined as those that tested positive for sars-cov-2 rna using real-time reverse transcription-polymerase chain reaction (rt-pcr) testing of combined nasopharyngeal and throat swab (nt) samples. rt-pcr testing was performed in the department of microbiology of the faculty of medicine at chulalongkorn university. sars--cov-2 rna was detected using the cobas1 sars-cov-2 kit (roche diagnostics, basel, switzerland) on a fully automated cobas1 6800 system (roche diagnostics, basel, switzerland) according to the manufacturer's recommendations. nucleic acid was automatically extracted from 400 μl of the nt specimens in viral transport medium (vtm) along with added internal control rna (rna ic). subsequent real-time rt-pcr was performed automatically by the system, targeting orf1a/b and e genes specific to sars-cov-2 and pan-sarbecovirus, respectively. classification of the confirmed case was as follows, according to the covid-19 management guideline of the thai ministry of public health: 1) mild-asymptomatic or upper respiratory tract infection (uri), 2) moderate-pneumonia without hypoxia, and 3) severepneumonia with hypoxia, of which the antiviral treatment was given. the date of the onset of symptoms, disease severity, hospitalization time, and personal demographic information were obtained from hospital medical records. the control group included three subgroups. the first subgroup included 20 plasma samples collected from healthy volunteers in the laboratory and 82 plasma samples leftover from healthy blood donors prior to february 2020. the second subgroup included 49 plasma samples collected from may 1 to may 31, 2020, from patients under investigation (pui) for covid-19 with rt-pcr results that were negative for sars-cov-2. the third control subgroup included 20 serum specimens collected from may 1 to may 31, 2020 from patients with other infections (dengue, hbv, hcv, hiv, mumps, measles, rubella, ebv, cmv, vzv, hsv, and treponema). plasma and serum were aliquoted and stored at -20˚c prior to serological testing. plasma samples of 10 μl were diluted to 1:101 in sample buffer in order to perform sars--cov-2 s1-specific iga and igg assays using anti-sars-cov-2 elisa igg/iga kits (euroimmun, lubeck, germany) according to the manufacturer's instructions. semi-quantitative results were evaluated by calculating the ratio of extinction at 450 nm of each sample over the calibrator. a cutoff ratio of 1.1 was used for sars-cov-2 iga, as suggested by the package insert. the borderline cutoff ratio of 0.8 for sars-cov-2 igg was assigned as positive. demographic characteristics were described for each patient. continuous variables were expressed as the median with an interquartile range (iqr). differences in continuous and categorical variables between the two groups were assessed using the wilcoxon rank-sum test and chi-square test or the fisher exact test, respectively. sensitivity, specificity, positive predictive value (ppv), and negative predictive value (npv) were also calculated. there were 118 confirmed sars-cov-2 infections from march 10 to may 31, 2020: 59 with mild (upper respiratory symptoms), 27 with moderate (pneumonia without hypoxia), and 32 with severe (pneumonia with hypoxia), with a median age of 38 years (iqr: 27-48). a total of 213 samples collected from 118 patients were tested for antibodies against sars-cov-2, with 36 patients having 1 sample, 69 patients having 2 samples, and 13 patients having 3 samples. a total of 99 samples were collected seven days after the onset of symptoms. there were 49 pui who were negative for sars-cov-2, with a median age of 47 years (iqr: 28-65 years), 25 males and 24 females. the baseline clinical characteristics are summarized in table 1 . there were significant differences in age and sex between the groups, with the patients in the severe group being mostly male (66%) and 40-59 years old. among the 118 confirmed sars-cov-2 patients, 99 had blood samples collected at least once more than 7 days after the onset of symptoms. the overall seroconversion of antibodies after the 7 th day of symptoms is summarized in the specificity, however, varied between the control subgroups. the raw data for all controls are shown in s1 table. there were two false-positive iga and one false-positive igg results out of 102 healthy controls. we were able to obtain and re-analyze seven samples, with an od ratio � 0.8 after two months. the od ratio was quite similar to the initial results, confirming the healthy control group's limited background. of the 49 pui, there were five positive iga and two positive igg results for covid-19 with negative rt-pcr results for sars-cov-2. two of these patient's tests were repeated after 2-4 weeks, and the od ratios returned to normal. of 20 serum specimens collected from patients with other infections, there were two samples with both iga and igg cross-reactivity to cmv-and ebv-positive samples. the seroconversion of the antibodies, stratified by the day of illness, is shown in table 3 . the sensitivity for serological testing within seven days of the onset of symptoms was only 29.7-30.6% for iga and 10.2-16.2% for igg. the iga positivity rate increased to 60% during the 2 nd week and 100% during the 3 rd -4 th weeks, and then declined to 81.9% in the 2 nd month. the igg positivity rate increased to 90% during the 3 rd -4 th weeks of diseases. to investigate the association of antibody levels to the severity of the disease, the antibody levels at the first time point were expressed using the specified cutoff value, stratified by disease severity. the severe group had a significantly higher level of s1-specific iga and igg antibodies compared to the mild group (fig 1) . it should be noted that the two patients in the severe group who did had no detectable s1-specific iga were tested only once, at 31 and 40 days after the onset of symptoms, and therefore it was likely that the iga levels had already declined in these patients. to see the dynamics of each group, we plotted the average antibody level from mild, moderate, and severe groups at five intervals (fig 2) . there were 103, 52, and 58 samples from the mild, moderate, and severe groups, respectively (table 3) . a clear pattern emerged, showing that the severe and moderate groups had significantly higher iga and igg levels 15 days post-symptoms compared to the mild group. of the group with mild symptoms, 20% (7/35) of the samples had no detectable igg antibodies more than 2 weeks after the onset of symptoms. only 1 out of 15 patients from the moderate group had no detectable igg antibodies, while all 15 patients with severe symptoms had high igg levels after the second week (table 3) . since age and sex were associated with the disease outcome, we analyzed the correlation between antibody level and age in the severe group, as shown in fig 3a and 3b ; however, no significant correlation was found. we also compared the antibody levels between males and females within the severe group. interestingly, the levels of both s1-specific iga and igg to were higher in males than in females, with igg being statistically significant (fig 4a and 4b) . the median age of males (51, iqr: 43-59) was also higher than that of females (41, iqr: 24-46) in the severe group. the results of the present study have demonstrated that during the first week of covid-19 infection, the sensitivity of the antibody response to acute viral infection is low. the antibody response seen in the present study started with iga, followed by igg. because it is difficult to compare results using different serological analyses, we have only used data from studies that tested with euroimmun for comparison. the results from 15 studies using euroimmun assays are summarized in s2 table. previous studies have mostly reported that the sensitivity of iga within the first week was less than 60% [12, 13] . in the present study, 30% of covid-19 patients developed positive iga antibodies very early, within 3 days after the onset of symptoms. therefore, the presence of positive iga antibodies might help identify some covid-19 patients in the early stage, however, negative results cannot be used to exclude infection. the seroconversion of iga was 100% in 21 patients at 15-28 days after the onset of symptoms. in a study from france, a 100% sensitivity of iga seroconversion was reported in 82 cases after the second week of symptoms [13] , and in 91 patients after the third week [14] . interestingly, in the present study we noticed a decline in iga after one month, with the sensitivity decreasing to 80%. in regard to igg, it should be noted that for the present study, we used the borderline cutoff to define a positive result to increase the sensitivity of igg. this cutoff did not change the specificity of the test. igg antibodies specific to the sars-cov-2 s1 antigen developed later than iga. the sensitivity of igg was 90% after the second week of symptoms, which is comparable to other studies [13, [15] [16] [17] . in the present study, 20% of the patients with mild symptoms did not develop any igg antibodies specific to covid-19, even after 2 weeks after the onset of symptoms. other studies have found up to 20-30% of cases to be negative for igg [18, 19] . when we analyzed the correlation of antibody levels with clinical severity, it was clear that patients with more severe clinical manifestations had higher antibody levels, for both iga and igg, than patients in the mild group. this observation has been consistently reported in other study populations as well [10, 19, 20] . the explanation behind these findings is not yet clear, however, one current hypothesis is that the elevated inflammatory response in the severe patients might produce a more robust immune response, including antibody production from b-lymphocytes. it also raises concerns about the role of antibody-mediated severity, although there is no evidence to support it. moreover, several studies have reported that there were higher rates of severity and mortality in male patients [21] . in the present study, more females were infected with covid-19 than males (60% female vs. 40% male); however, there were significantly more males (66%) in the severe group. interestingly, we found a significantly higher level of igg in males than in females in the severe group, similar to recent results found by klein et al. [19] . the median age of males was higher than that of females in the severe group. it is possible that higher levels of antibodies might be associated with greater illness severity in male patients. however, there is a speculation that biological sex might affect immunity through various mechanisms [21] . although women seem to have greater antibody responses and are more susceptible to autoimmune diseases than men, other factors, including innate immunity, regulatory t cells, expression of angiotensin-converting enzyme 2 (ace2), or other mechanisms related to sex hormones might explain the greater severity and higher antibody levels observed in male patients. further studies are needed to elucidate the impact of sex on disease severity, which might lead to a better understanding of this challenging disease. although the assessment of specificity was not the main objective of this study, our results confirmed those from previous studies, that the specificity of euroimmun anti-sar-cov-2 iga is lower than that of igg. as summarized in s2 table, the specificity of euroimmun anti-sar-cov-2 iga ranged from 68.3-94.6%, while anti-sar-cov-2 igg ranged from 85-100%. a higher background was observed in the control group with respiratory symptoms. as previously stated, of the 49 pui, there were 5 positive iga and 2 positive igg results that had corresponding negative rt-pcr results for sars-cov-2. two of these patients were repeated after 2-4 weeks, and the od ratio returned to normal. it should be noted that the positive results in these patients could be the result of either a false positive or a true positive case with negative rt-pcr results. however, there was no evidence to support covid-19 infection in these patients. we also observed two samples with both iga and igg cross-reactivity with cmv-and ebv-positive samples. since we did not have serological results for other coronaviruses in these two samples, we did not know definitively if they were directly cross-reactive with cmv and ebv, or the result of cross-reactivity with another coronavirus. however, based on results from other studies summarized in s2 table, there were reports of false positives with various infections, including ebv and cmv seropositives [12, 17] . in summary, the present study extensively reported the serological responses of covid-19 patients in thailand up to 60 days after the onset of symptoms. although most of the samples were tested at two time points, blood samples were collected from patients at different stages and at various intervals. therefore, we did not determine a median time for positive results, which might have been subject to bias. supporting information s1 clinical features of patients infected with 2019 novel coronavirus in wuhan a novel coronavirus outbreak of global health concern early transmission patterns of coronavirus disease 2019 (covid-19) in travellers from wuhan to thailand detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr sars-cov-2 antibody testing-questions to be asked profiling early humoral response to diagnose novel coronavirus disease (covid-19) antibody responses to sars-cov-2 in patients with covid-19 clinical and immunological assessment of asymptomatic sars-cov-2 infections severe acute respiratory syndrome coronavirus 2-specific antibody responses in coronavirus disease patients clinical performance of different sars-cov-2 igg antibody tests evaluation of two automated and three rapid lateral flow immunoassays for the detection of anti-sars-cov-2 antibodies assessment of sars-cov-2 serological tests for the diagnosis of covid-19 through the evaluation of three immunoassays: two automated immunoassays (euroimmun and abbott) and one rapid lateral flow immunoassay (ng biotech) performance characteristics of four high-throughput immunoassays for detection of igg antibodies against sars-cov-2 clinical performance of two sars-cov-2 serologic assays diagnostic performance of seven rapid igg/igm antibody tests and the euroimmun iga/igg elisa in covid-19 patients neutralizing antibodies responses to sars-cov-2 in covid-19 inpatients and convalescent patients sex, age, and hospitalization drive antibody responses in a covid-19 convalescent plasma donor population. medrxiv different longitudinal patterns of nucleic acid and serology testing results based on disease severity of covid-19 patients considering how biological sex impacts immune responses and covid-19 outcomes we would like to thank the health care team for at king chulalongkorn memorial hospital, thai red cross, particularly dr. kampol suwanpimolkul, dr. leilanee paitoonpong, and dr. suvaporn anulgulreungkitt. special thanks for the advice from dr. parvapan bhattarakosol and statistical analysis by miss jiratchaya sophonphan. key: cord-295536-dbpt4dhr authors: shook, natalie j.; sevi, barış; lee, jerin; oosterhoff, benjamin; fitzgerald, holly n. title: disease avoidance in the time of covid-19: the behavioral immune system is associated with concern and preventative health behaviors date: 2020-08-20 journal: plos one doi: 10.1371/journal.pone.0238015 sha: doc_id: 295536 cord_uid: dbpt4dhr the coronavirus disease 2019 (covid-19) poses a serious global health threat. without a vaccine, behavior change is the most effective means of reducing disease transmission. identifying psychological factors that may encourage engagement in preventative health behaviors is crucial. the behavioral immune system (bis) represents a set of psychological processes thought to promote health by encouraging disease avoidance behaviors. this study examined whether individual differences in bis reactivity (germ aversion, pathogen disgust sensitivity) were associated with concern about covid-19 and engagement in recommended preventative health behaviors (social distancing, handwashing, cleaning/disinfecting, avoiding touching face, wearing facemasks). from march 20 to 23, 2020, a us national sample (n = 1019) completed an online survey. germ aversion and pathogen disgust sensitivity were the two variables most consistently associated with covid-19 concern and preventative health behaviors, while accounting for demographic, health, and psychosocial covariates. findings have implications for the development of interventions intended to increase preventative health behaviors. in december 2019, there was an outbreak of a novel coronavirus originating in wuhan, china. the virus and its resultant infection, coronavirus disease 2019 (covid19) , spread rapidly across the globe, leading the world health organization (who) to label the outbreak a pandemic on march 11, 2020 [1] . as of may 18, 2020, there were over 4.5 million confirmed cases of covid-19 worldwide and over 300,000 deaths [2] . in order to decrease transmission of the virus and reduce likelihood of illness, health organizations recommended a number of preventative health behaviors, such as washing hands frequently and thoroughly, avoiding touching one's face, disinfecting and cleaning frequently touched surfaces, engaging in social distancing (i.e., avoiding close contact with others), and wearing facemasks [3, 4] . on march 13, 2020, the us declared covid-19 a national emergency [5] , which prompted many-but and would more frequently engage in social distancing, avoidance of face touching, handwashing, cleaning or disinfecting commonly used surfaces, and wearing a facemask. of note, at the time of data collection, health organizations did not recommend wearing facemasks by the general public, except if an individual had covid-19 [23] . to isolate these associations, we accounted for a wide variety of demographic, health, social, and personality characteristics. data were collected on march 20 and 23, 2020, from a nationally representative sample of 1023 individuals residing in the us, recruited through the panel provider qualtrics for a larger longitudinal study about the effects of covid-19. sample size was determined based on monte carlo simulations (n = 10,000) of the most conservative models for the data analysis plan associated with the larger longitudinal project. a minimum sample of 500 was estimated to provide sufficient power (>95%) to detect anticipated effects (β = .15 to .20) based on previous bis research assuming α = .05. to account for missing or unusable data, we aimed to recruit a panel of at least 1000 us individuals. the current study used the first wave of data from the longitudinal study. four participants were excluded from analyses due to problematic response patterns (e. table 1 for a full description of the sample). this project was approved by the university of connecticut irb (protocol #l20-0018). participants provided electronic consent before completing the study. after electronically agreeing to be part of the study, participants completed the primary study measures and other questionnaires in a random order, except for perceived health, illness recency, health history, and demographics which appeared last (see s1 appendix for all measures). upon completion, participants were given monetary compensation in an amount established by the panel provider. the online survey took approximately 27 minutes to complete. bis indicators. the 15-item perceived vulnerability to disease questionnaire [13] is an individual difference measure that consists of two subscales: germ aversion and perceived infectability. the germ aversion subscale includes eight items and measures individual discomfort with situations that imply high likelihood of pathogen transmission (e.g., "it really bothers me when people sneeze without covering their mouths"; α = .70). germ aversion is a component of the bis. the perceived infectability subscale includes seven items and measures perceived personal susceptibility to infectious disease (e.g., "i am more likely than the people around me to catch an infectious disease"; α = .79). although involving some level of subjective assessment, the perceived infectability subscale does not necessarily assess psychological disease avoidance processes; rather, responses to the items are in large part based on health history and biological susceptibility to infection [24, 25] . as previous likelihood of contracting infectious disease may influence covid-19 concern and preventative health behaviors, we included perceived infectability as a covariate in the primary analyses. all ratings of items were made on a scale ranging from 1 ("strongly disagree") to 7 ("strongly agree"). a composite score for each subscale was created by taking the average of the items. higher scores reflect greater germ aversion or perceived infectability. the 7-item pathogen disgust subscale from the three domains of disgust scale [12] was used to assess individual differences in disgust sensitivity specifically related to pathogens. participants rated how disgusting they found each item (e.g. "stepping on dog poop") on a 7-point scale from 0 ("not disgusting at all") to 6 ("extremely disgusting"). a composite variable was created by taking the average of the items (α = .85). higher scores reflect greater pathogen disgust sensitivity. covid-19 concern. at the time of data collection, there were no existing, validated measures of covid-19 concern. we developed six items to assess the degree to which participants were concerned about covid-19. one item explicitly asked participants to indicate how concerned they were about covid-19 on a 5-point scale from 1 ("not at all concerned") to 5 ("very concerned"). for the other five items, participants indicated their agreement with statements regarding the seriousness of the coronavirus (e.g., "the coronavirus is just the flu or a common cold," "people are not doing enough to prevent the spread of the coronavirus") on a scale 1 ("strongly disagree") to 6 ("strongly agree"). necessary items were reverse scored, and all items were standardized. a composite measure was created by taking the average of the standardized items (α = .81). higher scores indicate more concern towards covid-19. preventative health behaviors. participants were asked to indicate how frequently they had engaged in 11 behaviors in the past week on a scale from 1 ("not at all") to 6 ("multiple times a day"). a single item assessed the extent to which participants avoided touching their faces. a single item assessed how often participants washed their hands for at least 20 seconds. a single item assessed frequency of wearing an antiviral mask. three items assessed frequency of disinfecting and cleaning frequently touched surfaces (i.e., "clean and disinfect surfaces in your home with antibacterial wipes," "clean your laptop," and "clean your mobile phone"). a composite score was created by taking the average of the items (α = .86). higher scores indicated engaging in each behavior more frequently. five items assessed the extent to which participants avoided contact with others (e.g., "avoid shaking someone's hand for greeting," "avoid going to school/job"). participants were also asked to indicate the extent to which they were engaging in social distancing on a scale from 1 ("not at all") to 5 ("a great deal"). all six items were standardized and a composite measure was created by taking the average of the standardized items (α = .76). higher scores indicate more social distancing. personality. a 10-item short version of the big five inventory (bfi-10) [26] was used to assess the big five personality characteristics. each personality trait was assessed with two items. participants rated their agreement on a scale from 1 ("disagree strongly") to 5 ("agree strongly") to statements for openness to experience (e.g., "has an active imagination"; r = .07), conscientiousness (e.g., "does a thorough job"; r = .37), neuroticism (e.g., "gets nervous easily"; r = .39), agreeableness (e.g., "is generally trusting"; r = .18), and extraversion (e.g., "is outgoing, sociable"; r = .28). a composite score for each personality characteristic was created by taking the average of the items. higher values indicate stronger identification with that trait. participants indicated if they thought they have or had covid-19 by indicating as "yes", "maybe", or "no". for the analyses, the variable was dichotomized. responses of "yes" or "maybe" were coded as 1, and "no" was coded as 0. perceived health. participants rated their overall health in general on a scale from 1 ("poor") to 5 ("excellent"). recent illness. illness recency was assessed by asking the participants their agreement with four statements [27] . using a scale from 1 ("strongly disagree") to 7 ("strongly agree"), participants indicated their agreement to statements, such as "over the past couple days, i have not been feeling well". a composite score was created by taking the average of the items. higher scores indicate more recent illness (α = .93). health history. participants were presented with 46 medical conditions and were asked to indicate whether they and/or a family member ("e.g., your mother, father, sister, brother, aunt, uncle, etc.") had each condition. participants were also asked to indicate if they were taking any immunosuppressive medication and for females if they were pregnant. according to the centers for disease control and prevention (cdc), some individuals are at higher risk for severe illness from covid-19 [28] . twenty-two of the medical conditions in the health history questionnaire have been identified by the cdc as placing individuals at risk for severe complications from covid-19 (e.g., diabetes, dialysis/transplant, cirrhosis), as well as pregnancy and immunosuppressive medication. from this information, a dichotomous variable indicating risk of complications from covid-19 was created. if participants indicated they had at least one of the medical conditions identified by the cdc, were taking immunosuppressive medication, or were pregnant, they were coded 1 as a participant with high risk of complication from covid-19. participants who did not meet any of these criteria were coded 0. based on health history information, a variable was also created to indicate whether participants had a family member at risk for complications from covid-19. if participants indicated that a family member had at least one of the conditions identified by the cdc, they were coded 1 as a participant with a family member at high risk for complications from covid-19. participants who had no family members with any of the conditions were coded 0. political orientation. participants indicated their political orientation on a 5-point scale from 1 ("very conservative") to 5 ("very liberal"). religiosity. participants indicated the extent to which they were religious on an 11-point scale from 0 ("not at all religious") to 10 ("extremely religious"). demographics. participants reported their age, race, sex, education level, annual family income, size of town they lived in, and if they were working in a healthcare field. means and standard deviations for all primary study variables are presented in table 1 . bivariate correlations were estimated between the individual characteristics (i.e., demographics, health, social, personality, and bis indicators) and concern about covid-19, as well as the preventative health behaviors (see table 2 ). respondents who were older, white, female, more table 2 . bivariate correlations between respondent characteristics and covid-19 concern and preventative health behaviors. highly educated, at high risk for complications from covid-19, had a family member at high risk for complications from covid-19, had not recently been ill, never had covid-19, were less religious, were more liberal, were more agreeable, conscientious, or open to experience, and were higher in perceived infectability, germ aversion, or pathogen disgust sensitivity reported greater covid-19 concern. in general, younger age, higher income, more populated location of residence, more recent illness, better perceived health, having/had covid-19, greater religiosity, greater extraversion, greater conscientiousness, greater perceived infectability, greater germ aversion, and greater pathogen disgust sensitivity were associated with engaging in most (at least three) of the preventative health behaviors more frequently. of note, however, germ aversion and conscientiousness were negatively associated with wearing an antiviral facemask. also, younger age, more recent illness, having/had covid-19, and greater perceived infectability were associated with less frequent handwashing. greater concern about covid-19 was associated with more frequent engagement in all of the preventative health behaviors, except for wearing an antiviral facemask. concern about covid-19 was negatively associated with wearing an antiviral facemask. to determine whether bis indicators were uniquely associated with concern about covid-19 and preventative health behaviors, a series of regression analyses were conducted. bis indicators (germ aversion and pathogen disgust sensitivity) were entered as primary predictors. concern about covid-19 and preventative health behaviors (social distancing, avoidance of face touching, wearing a facemask, hand washing, cleaning/disinfecting) were entered as primary outcomes. demographic and health characteristics (i.e., age, race, sex, education, income, size of hometown, whether or not one works in healthcare, one's risk of severe illness from covid-19, family's risk of severe illness from covid-19, illness recency, perceived health, perceived infectability, and history of covid-19 infection), religiosity, political orientation, and personality (extraversion, openness, conscientiousness, neuroticism, agreeableness) were entered as covariates. for analyses with preventative health behaviors as outcomes, covid-19 concern was also entered as a covariate. multicollinearity was checked and found not to be a problem (all vif < 5, tolerance > 0.20). standardized estimates from regression analyses are presented in table 3 (see s1-s6 tables for unstandardized estimates, standard errors, and confidence intervals for all models). concern about covid-19. overall, the model examining individual differences in concerns about covid-19 was significant, adjusted r 2 = 0.21, f(22, 906) = 12.35, p < .001. older age, higher education, greater perceived infectability, and not having been recently ill were each significantly associated with greater covid-19 concern. being more liberal, greater conscientiousness, and greater neuroticism were also each significantly associated with greater concern for covid-19. after accounting for demographic, health, social, and personality factors, greater germ aversion and greater pathogen disgust sensitivity were each significantly associated with greater concern about covid-19. social distancing. overall, the model examining individual differences in social distancing was significant, adjusted r 2 = 0.23, f(23, 905) = 13.08, p < .001. higher income, more recent illness, and better perceived health were each significantly associated with greater social distancing. greater religiosity, greater extraversion, and greater covid-19 concern were also each significantly associated with greater social distancing. after accounting for demographic, health, psychosocial, and personality factors, greater germ aversion and greater pathogen disgust sensitivity were each significantly associated with greater social distancing. avoidance of face touching. the overall model examining individual differences in avoidance of face touching was significant, adjusted r 2 = 0.16, f(23, 927) = 8.52, p < .001. greater religiosity, more liberal political ideology, and covid-19 concern were each significantly associated with greater avoidance of touching one's face. after accounting for demographic, health, social, and personality factors, greater germ aversion was significantly associated with avoidance of touching their face. wearing a facemask. overall, the model examining individual differences in wearing a facemask was significant, adjusted r 2 = 0.27, f(23, 904) = 15.69, p < .001. younger age, being male, more populated location of residence, being at high risk for complications from covid-19, not having a family member who is at high risk for complications from covid-19, more recent illness, and better perceived health were each significantly associated with greater extent of wearing an antiviral facemask. greater religiosity, less conscientiousness, and table 3 . regression model results for covid-19 concern and preventative health behaviors. less openness were also each significantly associated with greater extent of wearing an antiviral facemask. after accounting for demographic, health, psychosocial, and personality factors, we did not find evidence of a significant association between any bis indicators and wearing a facemask. handwashing. overall, the model examining individual differences in handwashing was significant, adjusted r 2 = 0.19, f(23, 903) = 10.33, p < .001. being female and less recent illness were each significantly associated with more handwashing. greater conscientiousness and greater covid-19 concern were also significantly associated with more handwashing. after accounting for demographic, health, social, and personality factors, greater germ aversion and greater pathogen disgust were each significantly associated with more handwashing. cleaning/disinfecting. overall, the model examining individual differences in cleaning or disinfecting surfaces was significant, adjusted r 2 = 0.21, f(23, 905) = 11.51, p < .001. younger age, higher income, being at high risk, not having a family member who is at high risk, more recent illness, and better perceived health were significantly associated with more cleaning/disinfecting. additionally, greater religiosity, greater extraversion, and greater covid-19 concern were each significantly associated with more cleaning/disinfecting. after accounting for demographic, health, social, and personality factors, greater germ aversion and greater pathogen disgust were each significantly associated with greater cleaning/disinfecting. the covid-19 pandemic represents an unprecedented infectious disease threat to people around the world. the bis is a collection of psychological characteristics thought to serve a disease-avoidance function. the primary goal of the current study was to determine the extent to which individual differences in the bis were uniquely associated with covid-19 concern and covid-19-related preventative health behavior. when demographic, health, social, personality, and bis variables were considered simultaneously, greater germ aversion and pathogen disgust sensitivity were most consistently associated with covid-19 concern and preventative behaviors. these findings are consistent with bis theory and suggest potential targets for health promotion interventions. both germ aversion and pathogen disgust were uniquely associated with greater concern for covid-19. additionally, greater germ aversion was connected with greater engagement in all preventative health behaviors, except wearing a facemask, and greater pathogen disgust sensitivity was associated with more social distancing, handwashing, and cleaning/disinfecting. notably, the effect sizes of the associations between germ aversion and preventative health behaviors were comparable to the effect sizes of the relations between covid-19 concern and preventative health behaviors. these findings are consistent with recent work linking bis indicators with covid-19 related behavioral intentions and policy attitudes [14, 15] . in general, these findings support theory suggesting the protective function of psychological diseaseavoidance mechanisms during a time of real disease threat [29] . the pattern of findings were relatively consistent across preventative health behaviors, except for wearing an antiviral facemask. recommendations regarding facemasks have varied during the pandemic. at the time of data collection, the cdc only recommended wearing a facemask if individuals thought they had covid-19, and people were dissuaded from wearing antiviral (e.g., n95) facemasks in order to prevent shortages of such personal protective equipment at hospitals and for healthcare workers [23] . on april 3, 2020 (after data collection), the cdc recommended that everyone should wear a cloth facemask in public or when around others [3] . the lack of significant associations between facemask wearing and bis indicators or covid-19 concern, as well as significant negative associations with other variables (e.g., conscientiousness, age), may reflect that wearing facemasks, especially antiviral facemasks, was not recommended at the time. the present study also provided a comprehensive test of the extent to which a large range of demographic, health, and psychosocial variables were related to covid-19 responses. older age was associated with more concern about covid-19, which is understandable as older adults are at higher risk of complications from covid-19 [28] . however, this concern did not translate into greater engagement in preventative health behaviors. in fact, older age was associated with less cleaning/disinfecting behavior and utilization of antiviral facemasks. potentially, older adults were self-isolating more and thus had less need to engage in these behaviors. greater income was associated with more social distancing and cleaning/disinfecting behavior. greater financial resources may facilitate the ability to engage in these practices, such as working from home and having access to disinfecting supplies. recent illness and general perceived health were also associated with many preventative health behaviors. well-being may be more salient for these individuals, but the underlying motivations may differ. for the former, engaging in preventative health behaviors may be motivated by wanting to protect others from getting sick. indeed, those who had been ill recently were less concerned about covid-19, but recent illness was the variable most strongly associated with wearing antiviral facemasks. individuals who perceive themselves as having generally good health may be motivated to maintain their health and engage in preventative health behaviors to reduce the likelihood of getting sick themselves. unlike previous work [19] , political orientation was not reliably associated with preventative health behavior in this nationally-representative sample. although those who were more liberal endorsed greater concern about covid-19, we did not find evidence that political ideology was independently associated with greater engagement in preventative health behaviors, other than greater avoidance of touching one's face. in our study, we considered a broader range of demographic, health, and psychosocial factors than kushner gadarian et al. [19] , which may account for the differences in results. greater religiosity was also associated with engaging in all of the preventative health behaviors, except for handwashing. although the empirical evidence is mixed, religiosity has been proposed to encourage prosocial behaviors [30] , so these results could reflect prosociality if engaging in preventative health behaviors is for the benefit of others. religiosity has also been associated with greater social conformity [31] . thus, these findings may be due to religious individuals' greater sensitivity to social norms. consistent with the existing literature, conscientiousness and neuroticism were associated with greater concern about covid-19. however, personality traits were not consistently related to engaging in preventative health behaviors when accounting for demographics and other psychosocial variables. in times of infectious disease threat and the unique situation of a pandemic, traditional personality traits may be a lesser determinant of behavior. rather, personality traits may be more strongly associated with regular daily activities and lifestyle choices (e.g., physical activity, alcohol consumption). the behavioral immune system is theorized to promote the avoidance of pathogen threats. some cross-cultural research indicates group differences in bis reactivity due to differences in historic and contemporary parasite or disease prevalence rates associated with geographic locations [32, 33] . however, most of the bis research has been conducted in lab-based settings using artificial tasks designed to prompt disease threat [22] . the current study extends this body of evidence and further supports bis theory by demonstrating that individual differences in germ aversion and pathogen disgust sensitivity were associated with responses to a real-world, contemporary pathogen threat. such an extension provides important ecological validity to lab-based and historical research. findings from this study have implications for policy and public response to future epidemics or pandemics. our results identify a variety of demographic and psychosocial characteristics that may place individuals at-risk for contracting and spreading disease during pandemics. designing efficacious messaging that targets these populations may help optimize alterations in human behavior necessary to prevent the spread of infectious diseases. for instance, in addition to focusing on the severity of infection, it may be beneficial for messages about covid-19 to emphasize aspects of the virus and disease that activate the psychological disease-avoidance processes. for example, messages could incorporate images or language that induce feelings of disgust or make salient the presence of germs or contamination. although individuals reliably differ in these psychological traits, there are malleable components to the diseaseavoidance processes that can be activated, leading to behavior change [29] . thus, it may be important for future research to examine whether temporarily increasing disgust or germ aversion promotes pandemic-related health behaviors. results from this study should be taken in light of certain limitations. the data are cross-sectional preventing causal claims; however, the wide range of demographic and psychosocial variables considered reduces the possibility of third variables. of course, other variables not assessed in the current study may still account for the findings. theoretically, germ aversion and pathogen disgust sensitivity motivate concern about infectious disease and engagement in preventative health behaviors. experimental evidence indicates that inducing disgust results in greater avoidance behavior, reducing potential contact with pathogens [34] . however, we cannot rule out the opposite causal relation with the current data. greater concern about covid-19 may lead to increased germ aversion and pathogen disgust sensitivity. experimental or longitudinal data are necessary to determine the causal direction. all variables were assessed with self-report measures, which raises concerns of social desirability or biased responding. at the time of data collection, there were no publically available measures of covid-19 concern, so we developed our own measure. although reliable and the items were deemed to be face valid by the research group, the covid-19 concern measure was not validated. also, data were collected online. although a nationally representative sample was recruited, only individuals with reliable internet access, computer, or smartphone were able to complete the study, thus limiting generalizability. our study has highlighted several factors associated with concern about covid-19 and engagement in preventative health behaviors. further research is needed to test these associations prospectively. these findings identify those who may be at greater risk of contracting and spreading covid-19, or future infectious diseases, as well as 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differences conceptualization: natalie j. shook. key: cord-302962-qw6s1t7j authors: hause, ben m.; collin, emily a.; anderson, joe; hesse, richard a.; anderson, gary title: bovine rhinitis viruses are common in u.s. cattle with bovine respiratory disease date: 2015-03-19 journal: plos one doi: 10.1371/journal.pone.0121998 sha: doc_id: 302962 cord_uid: qw6s1t7j bovine rhinitis viruses (brv) are established etiological agents of bovine respiratory disease complex however little research into their epidemiology and ecology has been published for several decades. in the u.s., only bovine rhinitis a virus 1 (brav1) has been identified while bovine rhinitis a virus 2 (brav2) and bovine rhinitis b virus (brbv) were previously only identified in england and japan, respectively. metagenomic sequencing of a nasal swab from a bovine respiratory disease (brd) diagnostic submission from kansas identified contigs with approximately 90% nucleotide similarity to brav2 and brbv. a combination of de novo and templated assemblies using reference genomes yielded near complete brav2 and brbv genomes. the near complete genome of bovine rhinitis a virus 1 (brav1) was also determined from a historical isolate to enable further molecular epidemiological studies. a 5’-nuclease reverse transcription pcr assay targeting the 3d polymerase gene was designed and used to screen 204 archived brd clinical specimens. thirteen (6.4%) were positive. metagenomic sequencing of six positive samples identified mixed brav1/brav2, brav1/brbv and brav2/brbv infections for five samples. one sample showed infection only with brav1. seroprevalence studies using a cell culture adapted brbv found immunofluorescence assay-reactive antibodies were common in the herds analyzed. altogether, these results demonstrate that brv infections are common in cattle with respiratory disease and that brav1, brav2 and brbv co-circulate in u.s. cattle and have high similarity to viruses isolated more than 30 years ago from diverse locations. along with equine rhinitis virus (erv) and foot and mouth disease virus (fmdv), bovine rhinitis a and b viruses (brav and brbv, respectively) are species in the genus aphthovirus, family picornaviridae [1] . two serotypes of brav have been identified, brav1 and brav2, while brbv consists of a single serotype. the brav1 strain sd-1 was isolated in germany in 1962 from nasal secretions from a calf with rhinitis [2] . additional brav1 strains were subsequently isolated from both healthy and diseased bovines in england, japan, italy and the u.s. and shown to cross react in serum neutralization assays [3] [4] [5] [6] . the sole brbv isolate ec-11 was isolated in england in 1964 by reed from the lung of a specific pathogen free calf with respiratory disease [7] . likewise, brav2 consists of a single specimen, strain h-1, isolated from an outbreak of respiratory disease in cattle in 1984 [8] . despite numerous studies on bovine rhinitis viruses (brv) in the 1960's through mid-1980's, little work has been published on their epidemiology and ecology the past several decades. bovine respiratory disease complex (brdc) is the most economically significant disease of the cattle industry, leading to losses due to mortality, morbidity, treatment costs and feed inefficiency in excess of $750 million dollars per year in the u.s. alone [9] . brdc has a multifactorial etiology involving a variety of bacteria and viruses in addition to host and environmental factors [10] . numerous commercial vaccines including both killed and attenuated live bacteria are available. viruses commonly included in commercial vaccine include bovine viral diarrhea virus (bvdv), bovine herpes virus 1 (bhv1), parainfluenza virus 3 (pi3) and bovine respiratory syncytial virus (brsv). despite their widespread use, brdc incidence has increased over the past 20 years [11, 12] . brdc pathogenesis often involves a primary viral infection which damages respiratory mucosa and alters host immune responses leading to secondary bacterial pneumonia caused by commensal bacteria already present in the respiratory tract [13] . both brav and brbv are established but rarely studied etiologic agents of brdc. experimental inoculation of calves with brav1 via intranasal (in) or intratracheal (it) routes, either singly or in combination, resulted in variable clinical signs of respiratory disease and histologic lesions consistent with pneumonia [14] . brav1 was also recovered from nasal swabs of in inoculated animals and all animals inoculated or exposed by contact seroconverted to brav1 by day seven post inoculation. a similar experiment using a different brav1 strain (rs 3x) and colostrum deprived calves failed to reproduce clinical disease but was successful in isolating brav1 from nasal swabs post inoculation and found histological lesions of focal rhinitis and a neutralizing antibody response in all inoculated calves [15] . brbv pathogenesis was investigated using intranasal inoculation of gnotobiotic calves [16] . clinical signs including fever, nasal discharge and increased respiration rate were observed. foci of epithelial necrosis were observed histologically in the turbinates and trachea and interstitial pneumonia was evident in the lungs. virus was isolated from multiple tissues and was neutralized by convalescent antiserum. in addition to controlled studies, numerous investigations of acute respiratory disease in cattle resulted in the isolation of bovine rhinitis viruses where paired acute and convalescent sera suggested a causative role for bovine rhinitis virus [6, 8, 17, 18] . metagenomic sequencing on nasal swabs obtained from brdc diagnostic submissions were performed to survey viruses present. contigs with high identity to brav2 and brbv were identified in one swab. to further our understanding of the epidemiology and ecology of bovine rhinitis viruses in brdc, a more comprehensive survey was performed. bovine clinical samples used in this study were submitted to ksvdl for routine diagnostic testing. the samples were obtained from naturally infected animals in the field by licensed veterinarians as a part of normal veterinary care and diagnostic investigations. clinical samples (nasal and pharyngeal swabs and lung tissue) from bovine respiratory disease submissions to ksvdl were screened by a brdc pcr panel which detects bvdv, bhv-1, brsv and bovine coronavirus (bcv) [19] . a total of 204 samples were screened. samples were collected in years 2011-2014 from twelve states: nebraska (n = 48), kansas (n = 112), colorado (n = 6), missouri (n = 1), mississippi (n = 7), texas (n = 4), oklahoma (n = 2), idaho (n = 2), montana (n = 6), oregon (n = 4), washington (n = 11) and virginia (n = 1). samples were also screened for influenza d virus (idv) using quantitative real time reverse transcription pcr as previously described [20] . a 5'-nuclease reverse transcription pcr assay was designed to detect bovine rhinitis viruses targeting the 3d polymerase gene: probe, 5'-fam-cgg cag tcc agg tcc agt gt-iowa black-3'; forward: 5'-ctt ttc ggt gtg att ggc ag-3'; reverse: 5'-gaa atc tat cag ggc agg tct g-3'. viral rna was extracted using the mag-max-96 viral rna isolation kit (life technologies) according to the manufacturer's instructions. real time reverse transcription pcr was performed using qiagen quantitect rt-pcr with brv primers and probe as follows: 50°c, 30 minutes; 95°c, 15 minutes; followed by 40 cycles of 94°c for 15 seconds and 60°c for 60 seconds. the pcr assay specificity was confirmed using bovine rhinitis virus positive samples as determined by metagenomic sequencing as well as with cultures of common brdc pathogens bvdv, bhv-1, brsv, bcv, mannheimia haemolytica, histophilus somni, pasteurella multocida and mycoplasma bovis. sample preparation for metagenomic sequencing was performed similar to previously described [21] . in brief, swabs or tissue homogenate were clarified by centrifugation followed by filtration through a 0.2 micron syringe filter. samples (180 μl) were treated with a cocktail of nucleases to degrade host or unprotected environmental nucleic acids at 37°c for 90 minutes. viral nucleic acids were isolated using the minelute virus spin filter kit (qiagen) according to the manufacturer's instructions. reverse transcription was performed using primers consisting of a known 20 nt sequence followed by (n) 6 at the 3' end [22] using a superscript iii reverse transcription kit (life technologies). second strand synthesis was performed using sequenase 2.0 (affymetrix). double stranded cdna was purified using a qiagen minelute pcr spin column and subsequently amplified using primers identical to the known 20bp region of the random hexamer containing primer. amplicons were purified using a qiagen minelute pcr spin column and quantified using a qubit fluorimeter (life technologies). samples were diluted to 0.2 ng/μl and 5μl was used for sequencing library preparation using the nextera xt library preparation kit (illumina) according to manufacturer's instructions. pooled barcoded libraries were sequenced on an illumina miseq instrument using paired 150bp reads. reads from each sequencing library were parsed into individual folders based on barcoded sequences. reads were imported into the clc genomics software package (qiagen). reads were mapped to the host genome (bos taurus) and unmapped reads were collected. de novo assembly was performed on unmapped reads and assembled contigs were analyzed by blastn. brv genomes were assembled using a combination of de novo and templated assembly with reference brav2 and brbv genomes (genbank jn936206 and eu236594, respectively). raw sequencing reads were submitted to the national center for biotechnology information sequence read archive under bioproject prjna273738 (accession number srp052864). accession numbers for the individual samples are srr1776513 (sample 140032-4), srr1776531 (sample 5-52), srr1776533 (sample 6-17), srr1776541 (sample 20-76), srr1776564 (sample 21-22), srr1776524 (sample 4-26) and srr1776537 (sample [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] . the genomes for brav1 sd-1, brav2 140032-1 and brbv 140032-2 were submitted to genbank under accessions kp236128, kp236129 and kp236130, respectively. phylogenetic analyses were performed by using mega6.06 software using the maximum likelihood algorithm with tree topology verified by performing 1000 bootstrap replicates [23] . fetal bovine kidneys, sourced from an abattoir, were purchased from innovative research. primary embryonic bovine kidney (ebk) cells were cultured by treatment of finely minced kidneys with 0.25% trypsin at 37°c for 4 hours following transfer to cell culture flasks with mem with l-glutamine and 100 units/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin b. madin-darby bovine kidney (mdbk) (atcc ccl-22) were maintained in mem with l-glutamine and 5% fetal calf serum at 37°c with 5% co 2 . brbv (atccvr-1806) was first passaged on ebks before being expanded on mdbk cells. virus was grown in mem at 33°c with 5% co 2 . indirect immunofluorescence was performed using 59 bovine serum samples submitted for diagnostic testing at ksvdl. the samples were originated from kansas (n = 36), illinois (n = 5), missouri (n = 9), texas (n = 2), colorado (n = 2) and idaho (n = 5) from beef and dairy herds. antiserum from a gnotobiotic calf hyperimmunized with bcv was used as a negative control (national veterinary services laboratories catalog #320-bdv). sera from two high health cows housed in isolation at the kansas state university veterinary health center were also used as negative controls. non-infected cells were also stained to determine level of background fluorescence. mdbk cells were infected with brbv at a dilution of 1:15 and incubated overnight. once the onset of cytopathic effect (cpe) was evident in approximately 20% of the cells, plates were washed with pbs and fixed in 80% aqueous acetone. serum was added at a 1:10 dilution and seven serial two-fold dilutions were made in pbs, with a final serum dilution of 640. plates were incubated for 1 hour at 37°c, then washed three times with pbs. fitc-conjugated rabbit anti-bovine igg (jackson immunoresearch) was added at a dilution to 1:50, along with evan's blue at 1:500, and incubated for 1 hour at 37°c. plates were then washed three times with pbs and 100 μl of 50% glycerol in pbs was added to each well and subsequently read on a fluorescence microscope. in september, 2013, nasal swabs were submitted to the kansas state veterinary diagnostic laboratory (ksvdl) from an approximately 6-month old calf in kansas with acute respiratory disease. aerobic culture identified mannheimia haemolytica. a bovine respiratory disease pcr panel was also performed and was positive for mycoplasma bovis [cycle threshold (c t ) = 27.8] and bovine coronavirus (bcv, ct = 20.0). metagenomic sequencing was next performed on the sample to investigate bcv genetic diversity for an unrelated project. the nasal swab sample was filtered through a 0.2 micron syringe filter and treated with a cocktail of nucleases before nucleic acid isolation. reverse transcription, second strand synthesis and amplification were performed using sequence independent single primer amplification (sispa) and sequencing libraries were prepared from the resulting amplicons [22] . approximately 4 million reads were generated on an illumina miseq instrument. reads mapping to host dna were subtracted (*3.6m reads) and the remaining sequences (*430k reads) were assembled de novo into 192 contigs and classified based on best blastn expectation (e) scores. besides the expected bcv, contigs with similarity to bovine adenovirus 4 (badv4), brav2 and brbv were identified with e values of 0. templated assembly was next performed using reference genomes identified by blast analysis. a near complete bcv genome was assembled from 15,500 reads to yield a consensus sequence of 31,017 nucleotides (nt) (missing the 5'-terminal 4 nt) with 99% identity to strain e-ah65 [24] . significantly fewer reads mapped to badv4 (238 reads) which resulted in *25% genome coverage with 91% identity to strain tht/62 [25] . assembly using a brav2 reference genome mapped 5,924 reads which spanned the 5'-non coding region and the complete polyprotein open reading frame with the exception of a 16 nt gap approximately 150 nt from the 3'-end of the polyprotein. the determined brav2 sequence was 89% identical to the brav2 strain h-1. similarly, assembly using the brbv reference genome identified 2,505 reads which spanned the entire reference sequence with the exception of 48 and 13 nt at the 5' and 3'-termini. the consensus sequence was 87% identical to brbv strain ec11 [1] . as only a single genome each of brav2 and brbv have been published and neither of the viruses have previously been identified in u.s. cattle, further investigation was warranted. genome sequence brav1 strain sd-1 there are two serotypes of brav, 1 and 2. while the genome sequence of brav2 strain h-1 is available in genbank, no sequence for brav1 is available. to enable further molecular epidemiology studies on bovine rhinitis viruses, the genome of brav1 strain sd-1 was determined. brav1 sd-1 was obtained from american type culture collection (atcc) and sequenced using metagenomic sequencing methodology as described above directly from cell culture harvests of brav1 sd-1 obtained from atcc. genome assembly was performed using a combination of de novo and templated assemblies using the brav2 strain h-1 reference. a 7,245 bp contig with 82% identity to brav2 h-1 was determined from *200,000 reads mapping to brav2 spanning the entire brav2 h-1 reference sequence with the exception of the 5'-terminal 4 nt. similar to all picornaviruses, the genome of brav1 consists of a long 5' non-translated region (ntr), a single large orf encoding a polyprotein and a short 3'-ntr followed by a poly-a tail. the region of the 5' ntr sequenced encompasses 537 nt and is 89% identical to brav2 h-1. the 5' ntr contained three insertions (g334, c350, c527) and one deletion (a345) as compared to brav2 h1. the aphthovirus 5'ntr contains a type ii internal ribosome entry site [26] [27] [28] . the polyprotein initiation codon is typically preceded by a polypyrimidine tract by approximately 20 nt's. a polypyrimidine tract is present in brav1 from nt 514-522 (uuuuccuuu) followed by an aug initiation codon at nt 538-540. a second initiation codon is present closely downstream and in the same reading frame at nt 553-555. aphthoviruses typically express two different forms of the leader polypeptide (lab and lb) from alternate initiation codons [29] [30] . fmdv favors translation from the second initiation codon while erav favors the first aug. aug codons favored for translation are located in the context of kozak sequences [31] . for brav1, the second aug from nt 553-555 is in the context of a strong kozak sequence and expression of a 2,213 amino acid polyprotein is expected to be dominant. the complete orf of brav1 sd1 encodes a predicted 2,218 amino acid polypeptide with 93% identity to brav2 and 50% identity to brbv ( table 1 ). the polyprotein is processed by viral proteases into l, four structural (vp4, vp2, vp3, vp1) and seven nonstructural (2a, 2b, 2c, 3a, 3b, 3c, 3d) proteins. the non-structural proteins of brav1 were very similar to those of brav2 with over 90% identity. the most divergent proteins were the capsid proteins vp1, vp2 and vp3 with 84.9-87.5% identity. vp1, vp2 and vp3 are the only surface-exposed viral proteins and consequently are antigenic determinants [32, 33] . as brav1 and brav2 are serologically distinct, the sequence divergence observed between vp1, vp2 and vp3 represents serotypic differences [8] . the region of the 5'ntr of brav2 strain 140032-1 sequenced (487 nt) was 90% identical to brav2 strain h-1 ( table 2 ). the 5'ntr contained four deletions (t195, t197, c343, a345) and four insertions (t198, g199, c339, c341). a large orf was identified from nt 488-7,219 encoding a predicted 2,243 amino acid polypeptide with 97% identity to brav2 h-1 that was preceded by a polypyrimidine tract of ccccucuuu at nt 468-476. the l protein of brav2 strain 140032-1 was unusual such that it had four initiation codons in frame with the polypeptide within the first 100 bp of the orf (nt's 488-490, 539-541, 554-556 and 578-580). the latter initiation codon at 578-580 however was the only one associated with a kozak sequence. further experimentation is required to determine which initiation codon(s) is used by brav2. the l protein initiated at nt 488 is predicted to encode 204 amino acids and has only 82.4 and 82.8% identity to brav1 and brav2 strain h-1, respectively, and is the most divergent protein of 140032-1. the lab proteins of brav1 and brav2 strain h-1 consist of 179 amino acids while the full length l protein of 140032-1 is more similar in length to that of brbv (207 amino acids). the l protein autocatalytically cleaves the polyprotein at the l/p1 junction and also catalyzes the cleavage of the p220 subunit of the cap binding complex eif4g resulting in inhibition of cellular cap-dependent translation initiation [34] . further studies are required to determine if translation initiation of 140032-1 occurs at all four codons and if these proteins have similar biological properties. the structural and non-structural proteins of 140032-1 all had >95% identity to brav2 h-1. a 566 nt region of the 5'ntr was sequenced for 140032-2 that was 93% identical to brbv strain ec-11 ( table 3 ). the 5' ntr had a single insertion of a c at position 59 and a single deletion of c529. a large orf from nt 567-7,409 was identified that codes for a predicted 2,280 amino acid polypeptide with 94.6% identity to ec-11. similar to ec-11, two initiation codons in frame with the polypeptide were identified near the 5'-terminus (nt's 567-569 and 639-641). translation from the latter codon beginning at nt 639 is predicted to be more favorable as it is in the context of a kozak sequence. with the exception of proteins vp1 and 2a, all proteins had greater than 92% identity to ec-11. interestingly, vp1 showed only 86.8% amino acid identity to ec-11. vp1 is exposed on the virion surface and at least three antigenic regions are targeted by neutralizing antibodies [35] . as vp1 from the two serotypes of brav share approximately 85% amino acid identity, 140032-2 may represent a new serotype of brbv however serological experiments are required to confirm this hypothesis. to determine the evolutionary relationship between brav1, brav2 and brbv, phylogenetic analysis was performed on the p1 protein encoding capsid proteins vp1-4, as well as the highly conserved 3d polymerase protein (fig. 1) . analysis of p1 found that brav2 140032-1 and brbv 140032-2 formed well-defined clades with brav2 h-1 and brbv ec-11, respectively, while brav1 sd-1 represented as an outlier most closely related to brav2. phylogenetic analysis of 3d polymerase also found well-defined clades for brbv and brav2 with brav1 as an outlier closely related to brav2. as metagenomic sequencing of a nasal swab from a calf with acute respiratory disease identified concurrent infection with two bovine rhinitis virus species, we designed a 5'-nuclease reverse transcription pcr (rtpcr) assay targeting the conserved 3d polymerase gene to investigate the incidence of brv in brdc diagnostic submissions. a subset of six samples positive on this assay were further confirmed as brv by metagenomic sequencing (below). assay specificity was verified using pure cultures of common brdc pathogens; all were negative. clinical samples from bovine respiratory disease submissions to ksvdl were previously screened by a brdc pcr panel which detects bvdv, bhv-1, brsv and bcv [19] . samples were separately analyzed for idv using a previously published method [20] . a total of 204 samples were screened. thirteen of 204 samples (6.4%) were positive for brv with ct values 18.0-29.4. positive samples originated from kansas (n = 12) and nebraska (n = 1). nine samples were positive for brv in addition to at least one other virus tested. four samples were only positive for brv. incidences for other viruses involved in brdc pathogenesis were 36% for bcv (n = 75), 4.8% for idv (n = 10), 7% for bhv1 (n = 15), 5% for bvdv (n = 11) and 3% for brsv (n = 7). these results demonstrate that brv are present in brdc clinical samples with a similar incidence to other established brd etiological agents. to further characterize a subset of the brv positive samples, six samples were analyzed by metagenomic sequencing in addition to the original sample 140032 from which brav2 and brbv genomes were assembled. following subtraction of reads mapping to the host genome, sequences were assembled de novo and the resulting contigs were analyzed by blastn against a database of the brv genomes (brav1 sd-1, brav2 h-1, brav2 140032-1, brbv ec-11, brbv 140032-2). an e value of 1x10 -5 is generally considered significant for organism identification by metagenomic sequencing [36] . one sample, 6-17, had four contigs with best blast hits only to brav1 (table 4 , e values 8.9x10 -78 -6.1x10 -146 ). four samples, 5-52, 6-19, 20-76 and 21-22, showed evidence of co-infection with two different brv (brav1 and brav2, brav1 and brbv, brav2 and brbv). sample 4-26 had multiple individual contigs with high similarity to all three species/serotypes. resequencing of 140032 found contigs mapping to brav2 and brbv similar to the initial analysis. likewise, reanalysis of the raw data obtained for the reference brav1 sd-1 found contigs with blastn hits solely to brav1. similar analysis of metagenomic sequences from a brv pcr-negative nasal swab submitted for brd diagnostic testing failed to identify any contigs with e values >10 -5 to brv. together, these results suggest that co-infections with multiple species and/or serotypes of brv are common in cattle. similar observations were made during the discovery of brbv, where analysis of viral isolates from cattle with acute respiratory disease found 32 out of 38 isolated viruses were brav1 using serum neutralization tests [8] . the remaining six isolates were not neutralized by brav1 or brbv antiserum and were subsequently shown to represent a new serotype, brav2. the six brv-positive samples were also analyzed by blastn at the national center for biotechnology information using the nucleotide collection database. reads mapping to the host bos taurus were subtracted and the remaining reads were assembled de novo and the resulting contigs were analyzed by blastn. contigs with similarity to brv were identified for all six samples. no other bovine viruses were identified in four of the samples (4-26, 5-52, 6-19 and 20-76; table 4 indirect immunofluorescence assays were performed on a collection of bovine sera using cell culture adapted brbv to determine seroprevalence. sera represented 15 herds in six states. all samples were positive with a titer of 160-640. where age of animal was known (n = 44), the minimum age was 9 months (n = 4) and the majority of animals were adults >2 years of age. controls including gnotobiotic calf antiserum, sera from high health animals held in isolation and uninfected cells were negative. attempts to perform ifa using brav1 and primary embryonic bovine kidney cells were unsuccessful due to non-specific fluorescence. ifa was not performed with brav2 as we were unable to acquire the virus. these results suggest that brbv infections are common in cattle however additional serological testing is needed to confirm these results. in particular, while brav and brbv are serologically distinct from one another in serum neutralization assays, cross reactivity between brav and brbv in our ifa assay was not performed [7] . similar detection rates and seroprevalence were recently reported for the newly described bovine idv [37] . idv was found in 4.8% of brd diagnostic submissions and serosurveys found 87.5% of herds and individuals had hemagglutination inhibition assay titers greater than 40. previous serological studies also found evidence for widespread brv infections [4, 7, 17, 38, 39] . while brv are established etiological agents for brdc, little work has been published on these viruses over the past several decades. this is likely in part due to difficulty in isolating these viruses in cell culture. nearly all reports of brv isolation utilized primary cells and the titers obtained in vitro were relatively low. also, controlled animal inoculation with brv resulted in mild respiratory disease and histopathology. combined, these results likely contribute to the lack of research on these viruses. advances in sequencing technology now allow researchers to readily characterize metagenomic samples in a sequence-independent manner, enabling detection and characterization of both established and novel or neglected organisms. the serological evidence for brbv and molecular data for bovine rhinitis viruses herein suggest that brv infections are common components of brdc. these results, combined with previous controlled inoculation studies for brav1 and brbv, suggest brv are pathogens involved in brdc. while our results do not demonstrate a definitive role for brv in brdc pathogenesis, we were unable to identify any other viruses present in over half of brv-positive brdc clinical samples using metagenomic sequencing. further research is needed to differentiate the possibility of brv being a non-pathogenic commensal organism. this first report of brav2 and brbv in u.s. cattle and the occurrence of concurrent mixed brv co-infections further expands our knowledge on the epidemiology of these viruses. molecular and phylogenetic analyses of bovine rhinovirus type 2 shows it is closely related to foot-and-mouth disease virus ein rhinovirus des rindes rhinoviruses of bovine origin isolation of a bovine rhinovirus isolation and characterization of rhinoviruses from calves isolation of a bovine rhinovirus from calves with respiratory disease studies on a rhinovirus (ec11) derived from a calf. i. isolation in calf tracheal organ cultures and characterization of the virus isolation of a new serotype of bovine rhinovirus from cattle economic impact associated with respiratory disease in beef cattle prevention and control of bovine respiratory disease brd in 2014: where have we been, where are we now, and where do we want to go? anim control, management, and prevention of bovine respiratory disease in dairy calves and cows review of brd pathogenesis: the old and the new experimental exposure of calves to a bovine rhinovirus studies with a rhinovirus of bovine origin studies on a rhinovirus (ec11) derived from a calf rhinoviruses: isolation from cattle with acute respiratory disease isolation of rhinovirus from cattle in outbreaks of acute respiratory disease co-circulation of two distinct genetic and antigenic lineages of proposed influenza d virus in cattle isolation of a novel swine influenza virus from oklahoma in 2011 which is distantly related to human influenza c viruses simultaneous rapid sequencing of multiple rna genomes cloning of a human parvovirus by molecular screening of respiratory tract samples mega6: molecular evolutionary genetics analysis version 6.0 quasispecies of bovine enteric and respiratory coronaviruses based on complete genome sequences and genetic changes after tissue culture adaptation analysis of the hexon gene sequence of bovine adenovirus type 4 provides further support for a new adenovirus genus (atadenovirus) topological organization of picornaviral genomes: statistical prediction of rna structural signals conservation of the secondary structure elements of the 5'-untranslated region of cardio-and aphthovirus rnas rna determinants of picornavirus cap-independent translation initiation two initiation sites for foot-and-mouth disease virus polyprotein in vivo internal ribosomal entry site-mediated translation initiation in equine rhinitis a virus: similarities to and differences from that of foot and mouth disease virus the scanning model for translation: an update the structure and antigenicity of a type c foot and mouth disease virus antigenic sites on foot and mouth disease virus type a10 the two species of the foot and mouth disease virus leader protein, expressed individually, exhibit the same activities epitopes on foot-and-mouth disease virus outer capsid protein vp1 involved in neutralization and cell attachment the fecal virome of pigs on a high-density farm characterization of a novel influenza virus in cattle and swine: proposal for a new genus in the orthomyxoviridae family bovine rhinoviruses studies with a rhinovirus of bovine origin iv. neutralizing activity against strain rs 3x in bovine sera the authors would like to thank the molecular diagnostics group at kansas state veterinary diagnostic laboratory for assistance with pcr and erin schirtzinger for assistance with sequencing. conceived and designed the experiments: bmh rah. performed the experiments: bmh eac ja. analyzed the data: bmh eac. contributed reagents/materials/analysis tools: rah ja. wrote the paper: bmh ga rah. key: cord-302529-43pd2qsp authors: el moussi, awatef; pozo, francisco; ben hadj kacem, mohamed ali; ledesma, juan; cuevas, maria teresa; casas, inmaculada; slim, amine title: virological surveillance of influenza viruses during the 2008–09, 2009–10 and 2010–11 seasons in tunisia date: 2013-09-19 journal: plos one doi: 10.1371/journal.pone.0074064 sha: doc_id: 302529 cord_uid: 43pd2qsp background: the data contribute to a better understanding of the circulation of influenza viruses especially in north-africa. objective: the objective of this surveillance was to detect severe influenza cases, identify their epidemiological and virological characteristics and assess their impact on the healthcare system. method: we describe in this report the findings of laboratory-based surveillance of human cases of influenza virus and other respiratory viruses' infection during three seasons in tunisia. results: the 2008–09 winter influenza season is underway in tunisia, with co-circulation of influenza a/h3n2 (56.25%), influenza a(h1n1) (32.5%), and a few sporadic influenza b viruses (11.25%). in 2010–11 season the circulating strains are predominantly the 2009 pandemic influenza a(h1n1)pdm09 (70%) and influenza b viruses (22%). and sporadic viruses were sub-typed as a/h3n2 and unsubtyped influenza a, 5% and 3%, respectively. unlike other countries, highest prevalence of influenza b virus yamagata-like lineage has been reported in tunisia (76%) localised into the clade b/bangladesh/3333/2007. in the pandemic year, influenza a(h1n1)pdm09 predominated over other influenza viruses (95%). amino acid changes d222g and d222e were detected in the ha gene of a(h1n1)pdm09 virus in two severe cases, one fatal case and one mild case out of 50 influenza a(h1n1)pdm09 viruses studied. the most frequently reported respiratory virus other than influenza in three seasons was rsv (45.29%). conclusion: this article summarises the surveillance and epidemiology of influenza viruses and other respiratory viruses, showing how rapid improvements in influenza surveillance were feasible by connecting the existing structure in the health care system for patient records to electronic surveillance system for reporting ili cases. identification and characterization of circulating influenza viruses is essential to detect the emergence of antigenic drift variants causing influenza epidemics and novel a subtypes with the potential to cause an influenza pandemic. thus, virological surveillance of influenza provides a basis for selection of the virus strains to be included in the annual formulation of influenza vaccines [1] . surveillance data from the african continent has increased substantially in the past five years [2] [3] [4] [5] , but they are still insufficient to allow for a thorough understanding of influenza virus circulation patterns on the continent and their associated morbidity and mortality, or to inform influenza control strategies [6, 7] . the primary objective of this study was to develop or strengthen influenza sentinel surveillance systems in line with who standards in selected north african countries. in the past decade, information of epidemic strains from tunisia was largely unknown due to lack of any systemic study. in this present study we are reporting the activity and circulation of influenza viruses during three seasons (2008-2009, 2009-2010 and 2010-2011 ) as a part of global influenza surveillance network, which was expanded to tunisia since 1980. a subset of sentinel primary care physicians participating in virological surveillance schemes in the community submits respiratory samples for virological testing from patients presenting in primary health care with an ili, as well as all regional emergency centres and hospitals that take on surveillance of influenza from community, hospitalized and fatal cases. the surveillance of influenza and other respiratory viruses is undertaken by 268 primary care centres for adult and pediatric patients ( fig. 1 ) distributed in 24 governorates covering 2.7% of general tunisian population (table 1) . sentinel physicians report weekly the total number of patient visits to their facilities for influenza-like illness (ili) and acute respiratory infection (ari) within four age categories (0-4 years, 5-14 years, 15-64 years, and 65+ years). sentinel physicians are asked to collect respiratory specimens from patients with symptoms of ili or ari. ili was defined as an outpatient with fever (38uc) and cough or sore throat with onset less than five days prior to presentation in the absence of a specific diagnosis. ari was defined as an outpatient with sudden onset of respiratory signs including cough, difficulty breathing, rhinitis and general symptoms such as fever, headache, fatigue, and myalgia less than five days prior to presentation. the physicians sent specimens for influenza testing and basic demographic data from a subset of patients with ili each year during october through may to the national influenza centre (nic) situated at the charles nicolle's hospital tunis, provided that the number of consultants is 10% above the total number of consultants in the sentinel centre. in addition, according to world health organization (who) recommendations, severe acute respiratory infection (sari) related to influenza viruses has been added to existing outpatient surveillance systems to fully describe the spectrum of disease related to influenza and to identify individuals at highest risk for severe disease. as a result, various existing routine influenza surveillance systems in tunisia were enhanced or supplemented to gain a rapid understanding of this novel virus, to monitor its spread and impact, and to evaluate the uptake, impact and effectiveness of the various countermeasures that were implemented. during each season, oro-pharyngeal and naso-pharyngeal swabs were collected from ili and sari patients enrolled under the virological surveillance system and placed in viral transport medium (vircell, spainh). oro-pharyngeal and nasopharyngeal swabs collected from the same patient were placed in one cryovial, stored at 4uc at the sentinel site, and transported daily to the nic. the samples taken from severe cases were from children and/or adult hospitalised in the care unit or patients hospitalized with severe acute respiratory infection were additionally tested for other respiratory viruses. for all participants, respiratory specimens collection were performed after informed consent, under the supervision of local sanitary authorities. it is just an informal statement of the study and members of the ethic committee have previously always approved the work of the nic. in fact, patients included in this study were of all ages and consulted the sentinel clinics or were hospitalized in state or private hospitals for influenza like symptoms infection. the consent was verbal because the patients, or parents in the case of minors, accept the test for influenza viruses since it is free and safe. until now, written consent is judged not necessary by the ethics committee. samples were analysed for diagnostic purposes using the real time pcr cdc protocol [8] for detection of influenza viruses and using xtagh respiratory viral panel fast (abbott molecular, germany) and the luminexh technology for other respiratory viruses. all influenza positive samples were sub-typed using specific real-time pcrs for influenza a(h1n1)pdm09, a/h3n2 and for influenza b viruses using ''influenza virus b real time rt-pcr kit'', ''subtype h1 of influenza virus a real time rt-pcr kit'' and ''subtype h3 of influenza virus a real time rt-pcr kit''(shanghai zj bio-tech co., ltd). every year, we realized the exchange of influenza strains with who collaborating centre for influenza in london. in the pandemic year, we succeeded to cultivate some tunisian strains of influenza a(h1n1)pdm09 and influenza b in national influenza centre madrid spain. in fact, cell culture virus isolation will be implemented in the national influenza centre in tunisia in the next few years. a representative number of influenza viruses were genotypically characterized by analysis of the nucleotide sequence of partial haemagglutinin ha1 chain (931 nucleotide residues) and partial neuraminidase (836 nucleotide residues) genes in order to know if circulating viruses were well-matched with vaccine viruses and check for the most frequent amino acid key changes related to neuraminidase inhibitors resistance respectively. all viruses analysed were amplified and sequenced according to the protocol of national influenza centre madrid [9] . tunisian sequences were aligned with other sequences from reference influenza a viruses available at the ncbi influenza virus resource (http://www.ncbi.nih.gov/genomes/flu/ swineflu.html) and global initiative on sharing avian influenza data database (http://www.platform.gisiad.org) using clustal w program implemented in mega version 4 under default conditions [10] . the nucleotide sequence data reported in this work were deposited in the genbank nucleotide sequence database with accession numbers jn037697 to jn037779. were associated with influenza a/h3n2 infection. despite the importance of these preliminary results, our surveillance system had limitations in season 2008-2009, and the rate of influenza virus detection remained low (,10%). in fact, the specimen collection and storage techniques may not always have been optimal. in addition, the identification of influenza viruses was performed primarily using immunofluorescence assays which are less sensitive for the detection of influenza viruses than viral culture the curve of cases of influenza viruses spread out over 11 weeks (from wk 1 to wk 11). there is a decrease of this curve during two weeks (wk 5 and wk 6) (in january 24-30 th and january 31 st -february 6 th ). this descent of curve is probably due to the disturbances in the schooling, the transport and the work in this period. two peaks have been reported in the wk 4 and wk 9 (58.3% and 69.2%).we observe the decrease of influenza a (h1n1)pdm09 in the sentinel samples since week 11. nine influenza-associated deaths were confirmed (ranging 19 to 57 years old) in an ante mortem or post mortem specimen. five of these fatal cases were pregnant women with an underlying clinical risk factor (mean 32 year old). it is important to note, that all deaths were associated with influenza a(h1n1)pdm09 infection. in fact, pregnancy was identified as a particular risk group for adverse influenza outcome during previous pandemics, and also during seasonal influenza [11, 12] . two respiratory disease outbreaks in closed settings were reported during the 2010-2011 season; one in the intensive care unit of charles nicolle's hospital concerning eight members of the medical staff and the second in the intensive care unit of rabta's hospital of tunis when a patient 67 year old diabetic died after infection. ten deaths were virologically confirmed in two separate outbreaks with influenza a(h1n1)pdm09 detected. it was expected that the virus would behave as a seasonal virus and continue to circulate in the population. its behaviour, however, could not be reliably predicted [13] , although it was considered likely that the virus would continue to cause serious disease in a minority of those infected in younger age groups and people in high-risk groups [14] . severe cases were defined as any condition or clinical presentation requiring hospital admission for clinical management according to who guidance criteria [15] . overall, influenza activity in tunisia in 2010-2011 reached a level higher than that seen in the winter of the 2008-2009 season, but lower than during the first wave of the pandemic in the summer of 2009. in fact, in season 2010-2011 over 50% of sentinel specimens were tested positive for influenza. this intensity of influenza activity was similar to that observed during the peak of the 2009-2010 'pandemic' season. this may reflect a greater intensity of influenza circulation resulting from the introduction of a novel virus into naïve human populations, and/or improvements in the sensitivity of laboratory diagnostic methods to detect influenza in use in tunisia. however the percentage of consultants for ili or ari in the sentinel centres in tunisia in 2010-2011 season was lower (8.3%) than the percentage of consultants in the pandemic year (30.3%) (fig. 3) . clinical consultation rates for ili or/and ari were declining overall, whereas the percentage of consultants in the next year of the pandemic was not correlated to virological analysis of influenza viruses in tunisia. despite the increased number of samples obtained in season 2010-2011 under the enhanced surveillance system, there were limitations to our ili surveillance system. this is probably due to the participating clinicians may have not correctly identified all ili or sari cases. also it is possible due to a sub-declaration of the clinical services in the sentinel centres in comparison with the last year in which the medical crow was more motivated by the ministry of health because of the pandemic. the same situation has been reported in england by who european influenza network [16] . in fact, both the burden of severe respiratory infection and the proportion due to viral etiologies including influenza are largely undocumented in africa highlighting the need for continued development of respiratory illness surveillance in this continent [17] . during the 2008-2009 season, 420 specimens were collected from patients at sentinel sites. of those, 80 (19%) were positive for influenza viruses. 45 influenza a/h3n2 (perth lineage) (56.25%), rsvs (26.8%) and 32 hmpvs (21%). in season 2010-2011, a total of 160 specimens were tested positive for respiratory virus and the most frequent respiratory viruses were: rsvs (97/160; 60.6%) and rhinovirus/enterovirus (37/60; 61.6%). the most common nonflu pathogen circulating in three seasons causing the lower respiratory tract infections leading to hospitalisation especially in children was rsv (207/457; 45.29%). in 2010 in chile there have been more cases of acute ari in children but this is attributable to epidemics of respiratory syncytial virus infections (rsv) rather than influenza [18] . this emphasises the importance of countries being able to test for a suite of respiratory pathogens, not just influenza. this data contribute to a better understanding of the circulation of influenza viruses and other respiratory viruses especially in north-africa. phylogenetic analysis of the ha1 nucleotid sequence of 23 influenza a(h1n1)pdm09 viruses from mild, severe (patients hospitalized with severe pneumonia and severe acute respiratory syndrome) and fatal cases, shows that all viruses characterised in tunisia during season 2009-2010 were outside the seven genetic groups described in the european centre for disease prevention and control (ecdc) report [19] . a total of 27 ha genes of influenza a(h1n1)pdm09 viruses regardless of whether they were from 2011 from mild, severe cases and from influenza cases admitted to intensive care units were also sequenced and analysed [20] . this genetic diversity of tunisian strains compared to a/california/7/2009 was consistent with expected patterns of virus evolution. additional substitutions in the position 222 of ha gene were found in 4 tunisian sequences out of 50 viruses (8%). d222g substitution observed in a total of three viruses analysed (6%). most of viruses with this mutation were found in severe cases (2/3), one of them from a fatal case [21] . although most of studies have demonstrated the presence of d222g substitution in severe cases, it was also reported in mild cases [22] [23] [24] [25] . d222e substitution was found in one out of 50 viruses studied (2%). this sample was taken from a patient with severe clinical syndrome. in fact, d222g mutation has been considered relevant for the acquisition of a hypervirulent phenotype during the 1918 influenza pandemic [26] , while the role of d222e in virulence has been ruled out [27] . the clinical significance of d222e mutation is still unclear. analysis of sequences of neuraminidase gene of influenza a(h1n1)pdm09 from 18 severe cases, not received any antiviral treatment, did not show presence of any of the known mutations associated to neuraminidase inhibitors resistance. influenza b viruses were grouped as victoria lineage or yamagata lineage on the basis of the ha gene sequence (fig. 6 [28] . most of yamagata strains were detected in severe cases (9/13; 69%). inspite of lower mutation rate and lower pathogenicity than influenza a, influenza b infection contributes to significant proportion of acute respiratory infection among tunisian people. notably, in season 2008-2009 influenza a/h3n2 viruses, followed by influenza b, have been the predominant influenza viruses circulating in tunisia. in season 2009-2010, pandemic influenza a(h1n1)pdm09 viruses were the predominant circulating viruses but, in contrast to the 2010-2011 season, there is a higher rate of co-circulation with influenza b viruses. the circulation of other winter viruses such as human respiratory syncytial virus and the particularly cold weather were also identified during three seasons in tunisia. unlike the vast majority of influenza b viruses circulating in the world during season 2010-2011, which were from the b/victoria lineage, most of influenza b tunisian strains belong to the b-yamagata lineage, not included in the 2010-2011 vaccine. therefore a virus strain belonging to the b-yamagata lineage was indeed recommended for the vaccine composition of the northern hemisphere for the season 2012-2013. appearance of d222g substitution in ha of a(h1n1)pdm09 viruses circulating in tunisia might be related with severe respiratory disease. mutations associated with resistance to neuraminidase inhibitors oseltamivir and zanamivir were not detected in the neuraminidase gene. to this end proper surveillance systems should be set up in already existing and wellestablished national influenza centres to understand the epidemiology of influenza and other respiratory viruses in north africa, which in turn may help the processes of decision making regarding influenza vaccination on the continent, which may have a high impact on health in africa. pandemic h1n1 and seasonal h3n2 influenza infection in the human population show different distributions of viral loads, with substantially effect the performance of rapid influenza tests influenza surveillance among outpatients and inpatients in morocco one health concept for strengthening public health surveillance and response through field epidemiology and laboratory training in ghana afriflu-international conference on influenza disease burden in africa surveillance and management of influenza on the african continent improving influenza surveillance in sub-saharan africa world health organization. cdc protocol of realtime rtpcr for swine influenza a(h1n1) substitutions in position 222 of haemagglutinin of pandemic influenza a (h1n1)2009 viruses in spain mega 4: molecular evolutionary genetics analysis (mega) software version 4.0 severe forms of influenza a (h1n1) 2009 in pregnant women: experience of the university hospital of fez, morocco and literature review pandemic influenza a(h1n1) 2009 virus in pregnancy world health organization. weekly epidemiological record 18 world health organization. who recommendations for the post-pandemic period clinical management of human infection with pandemic (h1n1)2009: revised guidance world health organization. epidemiological and virological situation update of the 2010/2011 influenza season in the who european region seasonal influenza epidemiology in sub-saharan africa: a systematic review global influenza epidemiology overview for europe -week 30 &id = 919&rootfolder = %2fen%2factivities%2fsciadvice%2flists%2fecd c%20reviews. accessed summary for europe. community network of reference laboratories (cnrl) for human influenza in europe genetic diversity of influenza a(h1n1)2009 virus circulating during the season 2010-2011 in spain haemagglutinin d222g mutation found in a fatal case of pandemic (h1n1) flu in tunisia virulence associated substitution d222g in the hemagglutinin of 2009 pandemic influenza a(h1n1) virus affects receptor binding altered receptor specificity and cell tropism of d222g hemagglutinin mutants isolated from fatal cases of pandemic a(h1n1) 2009 influenza virus observed association between the ha1 mutation d222g in the 2009 pandemic influenza a(h1n1) virus and severe clinical outcome evolutionary trends of a(h1n1) influenza virus hemagglutinin since 1918 molecular and phylogenetic analysis of the haemagglutinin gene of pandemic influenza h1n1 2009 viruses associated with severe and fatal infections transmission of hemagglutinin d222g mutant strain of pandemic (h1n1) 2009 virus european surveillance centre for disease prevention and control. surveillance report. weekly influenza surveillance overview. main surveillance developments in week the authors gratefully acknowledge who centre for influenza in london and centers for disease control and prevention (cdc) for the collaboration. we also thank dorra arab ennigrou and ines laaribi in the national influenza centre-tunis; mónica gónzalez-esguevillas, nieves cruz, ana calderón, noelia reyes, manuela lopez-valero, mar molinero and silvia moreno in the national influenza centre-madrid for excellence technical assistance. key: cord-311074-j3fw4dfc authors: alviset, sophie; riller, quentin; aboab, jérôme; dilworth, kelly; billy, pierre-antoine; lombardi, yannis; azzi, mathilde; ferreira vargas, luis; laine, laurent; lermuzeaux, mathilde; mémain, nathalie; silva, daniel; tchoubou, tona; ushmorova, daria; dabbagh, hanane; escoda, simon; lefrançois, rémi; nardi, annelyse; ngima, armand; ioos, vincent title: continuous positive airway pressure (cpap) face-mask ventilation is an easy and cheap option to manage a massive influx of patients presenting acute respiratory failure during the sars-cov-2 outbreak: a retrospective cohort study date: 2020-10-14 journal: plos one doi: 10.1371/journal.pone.0240645 sha: doc_id: 311074 cord_uid: j3fw4dfc introduction: because of the covid-19 pandemic, intensive care units (icu) can be overwhelmed by the number of hypoxemic patients. material and methods: this single centre retrospective observational cohort study took place in a french hospital where the number of patients exceeded the icu capacity despite an increase from 18 to 32 beds. because of this, 59 (37%) of the 159 patients requiring icu care were referred to other hospitals. from 27th march to 23rd april, consecutive patients who had respiratory failure or were unable to maintain an spo2 > 90%, despite receiving 10–15 l/min of oxygen with a non-rebreather mask, were treated by continuous positive airway pressure (cpap) unless the icu physician judged that immediate intubation was indicated. we describe the characteristics, clinical course, and outcomes of these patients. the main outcome under study was cpap discontinuation. results: cpap was initiated in 49 patients and performed out of icu in 41 (84%). median age was 65 years (iqr = 54–71) and 36 (73%) were men. median respiratory rate before cpap was 36 (30–40) and median spo2 was 92% (90–95) under 10 to 15 l/min oxygen flow. median duration of cpap was 3 days (iqr = 1–5). reasons for discontinuation of cpap were: intubation in 25 (51%), improvement in 16 (33%), poor tolerance in 6 (12%) and death in 2 (4%) patients. a decision not to intubate had been taken for 8 patients, including the 2 who died while on cpap. two patients underwent less than one hour cpap for poor tolerance. in the end, 15 (38%) out of 39 evaluable patients recovered with only cpap whereas 24 (62%) were intubated. conclusions: cpap is feasible in a non-icu environment in the context of massive influx of patients. in our cohort up to 1/3 of the patients presenting with acute respiratory failure recovered without intubation. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the pandemic of novel coronavirus disease 2019 (covid19) began in wuhan, china in december 2019. as of august 2th 2020, the who reported a total of 17 660 523 covid-19 cases globally, including 680 894 deaths. in a large uk cohort, death from covid-19 was strongly associated with being male, older age, deprivation, uncontrolled diabetes and severe asthma [1] . the nature of the pulmonary lesions triggered by sars-cov-2 is still a matter of debate. some histopathological studies suggest that diffuse alveolar damage is not the single pattern [2, 3] . disorders of the pulmonary circulation (thrombosis, endothelial injury) and organizing pneumonia may also be present. the classical clinical features of ards after intubation such as low pulmonary compliance are not found in all patients [4, 5] . in terms of clinical management, initial recommendations suggested early intubation and ards-type ventilator settings [6] . although some studies suggest a role for non-invasive ventilation (niv) in mild ards [7] [8] [9] [10] , including a recent meta-analysis [11] , invasive mechanical ventilation remains the standard of care, especially for severe cases. while cpap in cardiogenic pulmonary oedema has been shown to reduce intubation rate [12] , a randomized trial in acute hypoxemic respiratory failure, showed no effect of cpap in reducing intubation rate and mortality, despite improved oxygenation [13] . however, during the chinese and european covid-19 outbreaks, a number of critical care teams proposed using high flow oxygen through nasal cannula (hfonc) or niv at least for initial management [14] [15] [16] [17] [18] . optimal respiratory support for covid-19 patients presenting with acute hypoxemic respiratory failure, however, remains unknown. the district of seine saint denis has been the worst affected area during the 2020 sars-cov-2 outbreak in parisian region, with a mortality in excess of 168.7% as compared to the same period in 2019 [19] . it is densely populated and has a high-deprivation index. from mid-march until end of april 2020, the delafontaine hospital, a large public hospital in saint denis, experienced a massive influx of patients requiring invasive ventilation. both intensive care unit (icu) and emergency department (ed) were quickly overwhelmed. the number of patients admitted in the wards (210 non-icu beds, for 585 covid-19 admissions) exceeded our icu capacity (18 beds, increased to 32 during the crisis, for 100 admissions). fifty-nine (37%) out of the 159 patients requiring icu care during this period had to be referred to other hospitals (fig 1) . therefore, we had an urgent need to delay the course of respiratory failure in the less severe patients in order to manage the flow of patients in the icu and the ed resuscitation room. to achieve this, we considered face-mask cpap because it does not require a ventilator. from 27th march onwards, patients with signs of respiratory failure despite 10 to 15 l/min of oxygen delivered by non-rebreather mask (nrm) were systematically assessed for face-mask cpap or immediate intubation. in this single centre retrospective observational cohort study, we describe the characteristics and outcomes of patients supported with cpap in our hospital during the sars-cov-2 outbreak. we reviewed the characteristics, clinical course and outcomes of all consecutive adults with proven covid-19 treated with face-mask cpap in icu or in wards between 27th march and 23 april. during this 4 weeks-period, patients receiving 10-15 l/min oxygen through nrm who had clinical signs of respiratory failure or were unable to maintain an spo2 > 90% were treated with face-mask cpap unless the icu physician judged that immediate intubation was indicated. every patient included had a thoracic ct scan suggestive of covid-19 pneumonia and/or a positive sars-cov-2 pcr on naso-pharyngeal swab or broncho-alveolar lavage. the primary outcome under study was reason for cpap discontinuation (poor tolerance, intubation, death or improvement). poor tolerance was defined as a refusal by the patient to do more cpap sessions, because of breathing discomfort. the following baseline patient characteristics were retrieved from patient electronic medical record: sex, age, comorbidities, body mass index (bmi), withholding / withdrawal of lifesustaining therapies, associated covid-19 therapies administered before the primary outcome under study occurred (antivirals, corticosteroids, immuno-modulating therapies, prone positioning), oxygen flow rate and spo2 before and after starting cpap treatment, duration of cpap treatment, medical unit where cpap treatment was performed, duration of invasive mechanical ventilation, saps2 score for patients admitted in icu, driving pressure and p/f ratio on first day of mechanical ventilation. the clinical outcomes (i.e. discharges from hospital, mortality) were recorded until the final day of follow-up on june 24 th . cpap of 5 to 10 cm h2o was delivered via a face-mask dedicated to niv (performa track1) with one of 2 types of cpap valve (boussignac™ or cpap-o-two™) or alternatively, an icu ventilator (servo i1 or evita infinity v5001). treatment was undertaken in a medical ward, the ed short-stay unit or the icu. an electrostatic heat and moisture exchanger filter (dar™) was placed between the mask and the cpap valve to prevent aerosolization of virus through expired gases. all patients were admitted to a single room with implementation of contact and airborne precautions. medical and nursing staff in wards, unfamiliar with niv, were trained by the intensivist who was initiating the cpap treatment. patients received an initial prolonged session lasting at least 4 hours before being reassessed of their need of invasive mechanical ventilation. if the patient could be temporarily taken off cpap without an immediate fall of spo2 below 90% (on o2 15l/min via nrm) or recurrence of clinical signs of acute respiratory failure, cpap treatment was resumed for 2 hours every 4 hours. progressive weaning of cpap was performed according to clinical signs, pulse oximetry and arterial blood gases. when possible, patients were managed in the icu (nurse/patient ratio 1:2). if no icu bed was available (as in over 80% cases), patients with cpap were shifted to the ed short-stay unit (8 beds) adjacent to the icu (nurse/patient ratio 1:4) which allowed frequent re-evaluation of the patient's state by the intensivist on duty. in the eventuality of no available bed in the ed short stay unit, cpap treatment was instituted and managed in the medical ward were the patient had been admitted (nurse/patient ratio 1:7 during the outbreak). ward patients on cpap were systematically reviewed overnight by the resident on duty responsible for the covid-19 medical wards. no a priori statistical sample size calculation was performed. sample size was equal to the number of patients treated during the study period. quantitative values are expressed as the median (interquartile range, iqr), and qualitative values are presented as numbers (percentages). univariate analysis was performed using fisher exact test or wilcoxon test, as appropriate. all tests were two-sided and a p value <0.05 was considered statistically significant. because of alpha inflation due to multiple comparisons, findings should be interpreted as exploratory. a cox hazard proportional model was fit for time to intubation, controlling for potential confounders in the cohort of 39 patients analysed. all variables available at baseline and associated with intubation in univariate analysis with a p-value <0.10 were selected. variables selected are: ct-scan severity (<50% vs �50% of lung involved), spo 2 at the time of cpap initiation, dose of anticoagulant (simple, double or curative) and time between hospital admission and cpap initiation. because of the important differences in the proportion of patients on corticosteroids in the 2 groups (though statistically non-significant in univariate analysis) and the impact on mortality of corticosteroid treatment found in the recovery trial [20] , we included it as an additional variable in the model. variables with more than 10% missing values were not implemented in the multivariate analysis. the analyses were carried out using r version 3.6.2 (the r project for statistical computing, vienna, austria; http://www. r-project.org). the study was approved by the national ethics review board (cnriph-commission nationale des recherches impliquant la personne humaine) under the number 2020-a01396-33. the ethic committee waived the requirement for informed consent: patients or their next-ofkin were informed by mail about the data collection process and their right to oppose. the database was declared to the commission nationale informatique et libertés (cnil) under the number 2217928. electronic medical records of the delafontaine hospital (saint-denis, france), concerning patients who sought care between march and april 2020, were accessed between may and june 2020. statistical analyses were conducted on anonymised data. forty-nine consecutive patients were treated with cpap between 27 th march and 23 rd april 2020 (fig 2) . initiation of cpap occurred throughout the entire study period and followed the epidemic curve (fig 1) . sars-cov-2 pneumonia was confirmed by pcr in 39 (79%) patients and by thoracic ct scan in all patients. twenty-six (53%) patients were eventually intubated and a total of 18 (37%) died. patients' characteristics are presented in table 1 . the median age was 65 years (iqr = 54-71) and 36 (73%) were men. forty-one (84%) patients had at least one comorbidity. the most frequent were hypertension (31 patients, 63%), obesity (13 patients, 34%) and diabetes (16 patients, 33%). the median duration of symptoms before hospital admission was 6 days (iqr = 5-9). thoracic ct-scan at admission showed mild (10 to 25%), moderate (25 to 50%) or severe (>50%) lung involvement in 13 (27%), 23 (46%) and 13 (27%) patients respectively. modalities of cpap therapy and associated interventions are described in table 2 . cpap was performed out of icu in 41 (84%) cases. median duration of cpap therapy was 3 days (iqr = 1-5). reasons for discontinuation of cpap were intubation for invasive mechanical ventilation in 25 (51%) patients, improvement in 16 (33%), poor tolerance in 6 (12%) and death in 2 (4%). a decision not to intubate had been taken with the patient and their family for the 2 patients who died while on cpap. all patients received at least once daily prophylactic anticoagulation. twice daily (thus double dose) prophylactic anticoagulation, typically enoxaparin 40mg every 12 hours, was administered in 19 (39%) patients while 14 (29%) received therapeutic anticoagulation. hydroxychloroquine was administered in 17 (35%) patients, lopinavir/ritonavir in 4 (8%), corticosteroids in 29 (59%) and anakinra in 7 (14%). awake prone positioning was used in 7 (14%) patients. two of those were eventually intubated. eight patients had a withdrawal or limitation of life-sustaining therapies ("do-not intubate" decision). of the 41 other patients, 2 had poor tolerance of cpap, resulting in its discontinuation within less than one hour. we did not consider these patients as being significantly treated, which left 39 patients suitable for analysis of outcome. fifteen patients (38%) out of 39 showed sustained clinical improvement with cpap therapy and never required intubation, including 1 patient (25%) in the 40-49 years age category, 4 patients (40%) in the 50-59 years, 8 patients (47%) in the 60-69 years and 2 patients (29%) in the 70-79 years. the other 24 patients (62%) eventually required invasive ventilation (fig 2) . in this group, median time from cpap initiation to intubation was 1 day (iqr = 1-2), median p/f immediately after intubation was 100 (iqr = 80-139), and median duration of mechanical ventilation was 17 days (iqr = [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] . patients who improved with cpap were compared to patients who required invasive mechanical ventilation. characteristics regarding age, sex, comorbidities and disease presentation were similar in both groups. patient who improved with cpap were treated later in their hospital stay, had higher oxygen saturation before cpap initiation, longer duration of cpap and received more often concomitant double dose prophylactic anticoagulation. in multivariate analysis, only low oxygen saturation before initiation of cpap was independently associated with a higher risk of intubation (fig 3) . twelve (46%) of the 26 intubated patients had a fatal outcome. median saps 2 score of ventilated patients was 57 (iqr = 38-64), resulting in a standardized mortality ratio of 0.75. at the time of final follow up, 18/49 (37%) patients were dead, 30 (61%) were discharged (14 from the group of patients who improved with cpap), 1 (2%) was still hospitalized in intensive care unit but weaned from mechanical ventilation. our hospital experienced a massive influx of hypoxemic patients during the covid-19 outbreak, 59 (37%) of the 159 patients requiring icu care had to be referred to other hospitals for lack of icu beds. in this context we tried cpap as a temporizing treatment in the management of acute respiratory failure. we choose not to use bi-level pressure niv for several reasons. first, the number of ventilators available could not ensure surge capacity in the context of massive patients influx, and the cpap devices were cheap enough to be bought in a large amount (28€ for a boussignac™, 62€ for a cpap-o-two™). second, bi-level pressure modes could have exposed patients who already have increased respiratory drive to the risk of ventilation induced lung injury through excessive tidal volume [21] . third, the increase in positive pressure during inspiration was suspected to carry a greater risk of aerosolization of virus particles, hence increasing the risk of contamination of health care workers [22, 23] . the final reason was to keep pressure support ventilation as an option for pre-oxygenation before intubation when indicated [24] . using bi-level pressure modes would have also required more intensive training of ward staff unfamiliar with niv techniques. hfonc was also not considered because of the lack of high flow oxygen delivery devices during the outbreak period. in addition, there was also a concern about a greater risk of aerosolization, especially in non icu settings were strict compliance with airborne precautions was more difficult to achieve. in this single center retrospective observational study, overall mortality of this cohort was 37% (18/49). sixteen (33%) patients improved with face-mask cpap, and eventually did not require invasive ventilation though they were very hypoxemic (11 (73%) of them required 15l/min oxygen). apart from 2 patients with a do-not-intubate orders, no death occurred during cpap therapy. mortality rate was 46% in the patient group requiring invasive mechanical ventilation, which was consistent with a large cohort of 20133 patients from 208 hospitals in uk (international severe acute respiratory and emerging infections consortium-isaric). among 3001 admitted to critical care (high dependency unit or intensive care unit), mortality was 32% while 41% continued to receive care at the date of reporting. in the 1658 patients receiving invasive ventilation mortality was 37% while 46% were still in hospital [25] . this mortality rate was related to the severity of illness (median saps2 score of 57) but may also be due to delayed intubation. reports are emerging on the use of cpap in situations similar to ours during the sars-cov-2 pandemic. in a single center in uk, cpap was initiated in patients requiring more than 28% fio2 or 4l/mn oxygen in combination with awake proning [26] . when compared to the isaric cohort [25] , there were reduced icu admissions (7.2 versus 16.5%) and invasive ventilation rates (4.8 versus 9.8%), with comparable hospital mortality (33.3 versus 36.8%). another single center retrospective study in uk suggest a positive effect of cpap therapy, with a favourable outcome (i.e. survival without mechanical ventilation) in 14 (58%) patients, and an intubation rate of 38%, which was similar to our results [27] . a french two period retrospective study favours cpap over oxygen: intubation-free survival was 77% (29/38) with cpap compared to 43% (6/14) with oxygen (p = 0,043). however it was performed in the absence of shortage of icu beds, and cpap was initiated in patients less hypoxemic than our population (oxygen flow > 6 l/min for sp02 > 92%), those two points may be the reasons of a lower intubation rate compared to ours [28] . in an italian prospective cohort, 157 patients underwent helmet cpap with a 55% survival without mechanical ventilation. in this study, patients were less hypoxemic than in our cohort at cpap initiation (pao2/fio2 < 300 mmhg). the helmet interface was well tolerated with discontinuation in only 4 patients [29] . on the whole, despite the fact that non-invasive ventilation techniques (hfonc, bi-level pressure ventilation or cpap) have already been used in several respiratory virus outbreaks (sars, mers, h1n1), we lack strong evidence on their efficiency because studies were mainly retrospective, with inappropriate control for selecting or confounding bias, or without any control group [30, 31] . as a consequence, recommendations from scientific societies concerning non-invasive oxygen therapy in covid-19 are heterogenous, some favouring hfonc, cpap, or bi-level pressure, depending on the country [32] . it is not possible to infer from our study any definite conclusion on the role of cpap to avoid invasive ventilation because of the small sample size and because we were unable to identify a population of patients that could have been a comparator. our study has several other limitations. first, due to its retrospective design, we were unable to collect additional data that could have contributed to a better understanding of the role of cpap in managing hypoxemic respiratory failure in covid-19. data on actual pressure levels delivered to each patient and the number of hours per day of cpap could not be collected. it was also not possible to ascertain in all patients whether vital signs (spo2, respiratory rate) and arterial blood gases were taken while on cpap or while on nrm, hence a high rate of missing values. patients with profound hypoxemia and high respiratory rate on cpap may be exposed to self-induced lung injury. we attempted to collect the values for driving pressures immediately after intubation and positive expiratory pressure levels during cpap therapy but these data were unfortunately only available in a few cases. this should be investigated in further studies. secondly, due to small sample size, the observed effect of cpap in avoiding invasive mechanical ventilation within a sub-group of patients could be biased by concomitant treatments (drugs and/or prone positioning during spontaneous breathing) administered to spontaneously breathing-patients. however, in the multivariate analysis, corticosteroid treatment, the main therapy that has been shown to impact mortality [20] , was not associated with the success of cpap treatment. the absence of a control group does not allow us to make any firm conclusion on the role of cpap in avoiding intubation. in addition, some patients treated with cpap may simply have received higher fio2 because the seal of the mask is better and the o2 flow higher as compared to patients on nrm: for example with a boussignac™ cpap, o2 flow was usually set between 20 to 30 l/mn to reach the target pressure of 5 to 10 cm h2o. this could have contributed to their clinical improvement. third, there might be several selections bias. we chose to include all patients with findings highly suggestive of covid-19 on thoracic ct scan among which only 79% had a positive pcr on respiratory samples. however, sars cov2 rt pcr on naso pharyngeal swabs is known to have an imperfect sensibility, and we considered that at the peak of the outbreak, alternative diagnosis were improbable [33] . the group of patients who did not need mechanical ventilation may have been less severe. this may be an important bias for their favourable outcome, which may not be due to the effect of cpap only. this is suggested by the higher levels of spo2 at initiation of cpap in the group of patients who improved compared with the group of patients who progressed to intubation. response to cpap could be used to identify patients who do not require intubation despite being profoundly hypoxemic [34] . however, cpap could also have worsened the condition of patients whose intubation was delayed. the high saps2 scores of the intubated patients in the study provide some evidence to this effect. fourth and finally, contamination of health care workers was not evaluated. expired gases dispersion during cpap seems to be limited if there is good mask interface fitting [35] , but leaks do occur incidentally and niv is considered an aerosol-generating procedure [23] . potential benefit from face-mask cpap should be weighed against the risk of contamination of health care workers, especially in settings were infection prevention and control precautions are difficult to maintain. choosing the appropriate interface is critical to decrease leaks and minimize aerosolization and there may be some advantages to select full face masks [22, 36] . helmet is another option but is more difficult to handle in a non-icu setting [37] . the role of face-mask cpap in managing acute hypoxemic respiratory failure in covid-19 patients warrants further investigation in larger prospective studies and comparison with other ways to manage hypoxemic respiratory failure, such as high flow nasal oxygen cannula [14, 16, 17] . the simplicity and practicality of cpap in a number of contexts, including massive patient influx and resource limited settings, is appealing [38] . however, the likely increased risk of contamination of health care workers, notably if personal protective equipment is inadequate, must be taken in account. cpap could also be considered as a first-line respiratory support strategy in less hypoxemic patients without significant respiratory failure in association with other strategies to improve oxygenation, such as awake prone positioning [17, 26, [39] [40] [41] [42] [43] [44] . we found that treatment with face-mask cpap was feasible in a non-icu environment and in the context of a massive influx of patients. in our situation, it was useful to post-pone intubation and to manage the flow of patient requiring invasive ventilation. we also found, that among patients who have low spo2 and /or signs of respiratory failure while on 15l/min o2 via nrm more than one third eventually did not need invasive mechanical ventilation. given the limitations of our study, the role of face-mask cpap in managing patients with hypoxemic respiratory failure should be investigated in further research. supporting information s1 dataset. (xlsx) opensafely: factors associated with covid-19-related hospital death in the linked electronic health records of 17 million adult nhs patients facing covid-19 in the icu: vascular dysfunction, thrombosis, and dysregulated inflammation time to consider histologic pattern of lung injury to treat critically ill patients with covid-19 infection covid-19 pneumonia: different respiratory treatments for different phenotypes? management of covid-19 respiratory distress surviving sepsis campaign: guidelines on the management of critically ill adults with coronavirus disease 2019 (covid-19) predictors of failure of noninvasive positive pressure ventilation in patients with acute hypoxemic respiratory failure: a multicenter study a multiple-center survey on the use in clinical practice of noninvasive ventilation as a first-line intervention for acute respiratory distress syndrome* is there a role for noninvasive ventilation in acute respiratory distress syndrome? a meta-analysis can non-invasive positive pressure ventilation prevent endotracheal intubation in acute lung injury/acute respiratory distress syndrome? a meta-analysis association of noninvasive oxygenation strategies with all-cause mortality in adults with acute hypoxemic respiratory failure treatment of severe cardiogenic pulmonary edema with continuous positive airway pressure delivered by face mask treatment of acute hypoxemic nonhypercapnic respiratory insufficiency with continuous positive airway pressure delivered by a face mask high flow nasal cannula is a good treatment option for covid-19 efficacy and safety of early prone positioning combined with hfnc or niv in moderate to severe ards: a multi-center prospective cohort study the experience of high-flow nasal cannula in hospitalized patients with 2019 novel coronavirus-infected pneumonia in two hospitals of chongqing early awake prone position combined with high-flow nasal oxygen therapy in severe covid-19: a case series severity of 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trial features of 20 133 uk patients in hospital with covid-19 using the isaric who clinical characterisation protocol: prospective observational cohort study reduced icu demand with early cpap and proning in covid-19 at bradford: a single centre cohort is continuous positive airway pressure (cpap) a new standard of care for type 1 respiratory failure in covid-19 patients? a retrospective observational study of a dedicated covid-19 cpap service continuous positive airway pressure to avoid intubation in sars-cov-2 pneumonia: a two-period retrospective case-control study helmet cpap treatment in patients with covid-19 pneumonia: a multicenter, cohort study noninvasive respiratory support in acute hypoxemic respiratory failure associated with covid-19 and other viral infections ventilation techniques and risk for transmission of coronavirus disease, including covid-19: a living systematic review of multiple streams of evidence covid-19 pandemic and non invasive respiratory management: every goliath needs a david. an evidence based evaluation of problems diagnostic performance between ct and initial real-time rt-pcr for clinically suspected 2019 coronavirus disease (covid-19) patients outside wuhan, china a plea for avoiding systematic intubation in severely hypoxemic patients with covid-19-associated respiratory failure exhaled air dispersion distances during noninvasive ventilation via different respironics face masks noninvasive mechanical ventilation in high-risk pulmonary infections: a clinical review helmet cpap to treat acute hypoxemic respiratory failure in patients with covid-19: a management strategy proposal frugal innovation for critical care respiratory parameters in patients with covid-19 after using noninvasive ventilation in the prone position outside the intensive care unit use of prone positioning in nonintubated patients with covid-19 and hypoxemic acute respiratory failure prone positioning in awake, nonintubated patients with covid-19 prone positioning in awake, nonintubated patients with covid-19 hypoxemic respiratory failure is the prone position helpful during spontaneous breathing in patients with covid-19? early self-proning in awake, non-intubated patients in the emergency department: a single ed's experience during the covid-19 pandemic we thank the doctors, residents and nursing staff of the delafontaine hospital who managed patients under cpap therapy during the sars-cov-2 outbreak. key: cord-294912-xl0wzi16 authors: alteri, claudia; cento, valeria; antonello, maria; colagrossi, luna; merli, marco; ughi, nicola; renica, silvia; matarazzo, elisa; di ruscio, federica; tartaglione, livia; colombo, jacopo; grimaldi, chiara; carta, stefania; nava, alice; costabile, valentino; baiguera, chiara; campisi, daniela; fanti, diana; vismara, chiara; fumagalli, roberto; scaglione, francesco; epis, oscar massimiliano; puoti, massimo; perno, carlo federico title: detection and quantification of sars-cov-2 by droplet digital pcr in real-time pcr negative nasopharyngeal swabs from suspected covid-19 patients date: 2020-09-08 journal: plos one doi: 10.1371/journal.pone.0236311 sha: doc_id: 294912 cord_uid: xl0wzi16 since sars-cov-2-based disease (covid-19) spreads as a pandemic, the necessity of a highly sensitive molecular diagnosis that can drastically reduce false negatives reverse transcription pcr (rtpcr) results, raises as a major clinical need. here we evaluated the performance of a ddpcr-based assay to quantify sars-cov-2 titer in 55 suspected covid-19 cases with negative rtpcr results thanks to in-house ddpcr assay (targeting rdrp and host rnasep). samples were collected at asst-gom niguarda between february and may 2020 at hospital admission. clinical and imaging data were obtained for clinical staging and definition of disease severity. patients were mainly female (45.5%) with a median age of 73 (57–84) years. ddpcr-based assay detected sars-cov-2 genome in nasopharyngeal samples of 19 (34.5%) patients (median viral-load: 128 copies/ml, iqr: 72–345). in 15 of them (78.9%), chest ct showed a classical covid-19 bilateral interstitial pneumonia; 14 patients (73.7%) showed severe covid-19 manifestations. ddpcr did not identify any trace of sars-cov-2 genome in the respiratory samples of the remaining 36 patients. the serological assay performed in a subgroup of 34 patients at the later stage of illness (from 3 days to 90 days after) confirmed the presence of sars-cov-2 antibodies in all patients tested positive for sars-cov-2 in ddpcr (100%). contrariwise, negative tests were observed in 95.0% ddpcr negative patients (p<0.001). thanks to a ddpcr-based assay, we achieved a rapid and accurate sars-cov-2 diagnosis in rtpcr-negative respiratory samples of individuals with covid-19 suspect, allowing the rapid taking care and correct management of these patients. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the pandemic of covid-19, caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), has posed a serious threat to global public health, calling for the development of reliable diagnostic tests, able to identify (and possibly quantify) sars-cov-2. currently recommended diagnostic tests for sars-cov-2 detection are real-time pcr (rtpcr) assays [1, 2] , which are able to detect viral rna through the amplification of 2 or 3 distinct genomic regions. despite the availability of these methods, the diagnostic optimum is far to be reached. the dynamic range of these tests is limited, and their diagnostic sensitivity on nasopharyngeal and oropharyngeal swabs has been shown to be insufficient in a number of sars-cov-2 related pneumonia cases [3] [4] [5] . first evidences suggest false negative results in 20%-30% of cases [3, 6] . the persistently negative rtpcr results in patients with clinical suspects of covid-19, and no alternative diagnosis, could be related to the lower viral titers that characterize the most widely used nasopharyngeal samples [7] in comparison to other respiratory specimens, such as sputum [8] , as well as to the replication kinetics of the virus. initial data are indeed suggesting that viral loads in throat swab and sputum samples peak at around 5-6 days after symptoms onset [9] , with consequent risk of uncontrolled sars-cov-2 transmission in the time period preceding the viral load peak. the prompt diagnosis of covid-19 patients is therefore a clinical need that is only partially met, which advocates for more sensitive and accurate diagnostic technologies. droplet digital pcr (ddpcr) is a highly sensitive assay for the direct detection and quantification of dna and rna targets. it has been increasingly used in infectious disease settings, especially thanks to its ability to consistently and reliably detect down to few copies of viral genomes [10] [11] [12] [13] [14] . whether the detection of low-level and/or residual viral presence is required, quantitative data obtained by ddpcr are far more informative than those provided by standard rtpcr assays [13] . standing the necessity of a limitation (as much as possible) of false negative results in covid-19 diagnosis, the use of ddpcr could provide a critical support [8] . its use for sars-cov-2 detection, however, is still very poorly investigated in clinical settings, and no data are currently available for european patients. in this study, the presence of sars-cov-2 genome was evaluated in 55 sars-cov-2 rtpcr negative nasopharyngeal swabs from covid-19 suspected patients thanks to a quantitative ad hoc designed assay based on ddpcr. negative nasopharyngeal swabs tested for sars-cov-2 by rtpcr (genefinder tm covid-19 plus realamp kit, elitech; allplex tm 2019-ncov assay, seegene) were collected during february-april 2020 from 55 covid-19 suspected patients at asst gom niguarda admission. for each patient, demographic and clinical information such as age, gender, clinical manifestations and symptoms were retrieved and stored in an anonymous database ad hoc built for the study. the severity of the covid-19 was classified into mild, moderate, or severe, if showing i) mild clinical symptoms without sign of pneumonia on imaging, ii) fever and respiratory symptoms with radiological findings of pneumonia, iii) respiratory distress, with oxygen saturation �93% at rest, mechanical ventilation, or presence of multiorgan failure (septic shock) and/or admission to intensive care unit (icu) hospitalization. the authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. medical records were registered and processed by using pseudonymization measures and assuring that it was not (and it is no longer) permitted the identification of data subjects. the study protocol was approved by the asst grande ospedale metropolitano niguarda research ethics committee (prot. 92-15032020). signed informed content was obtained for each participant. this study was conducted in accordance with the principles of the 1964 declaration of helsinki. total rna was extracted from 280 ul of nasopharyngeal swabs using qiaamp viral rna mini kit (qiagen) following manufacturer's instruction. sars-cov-2 genomic rna was quantified by means of the qx200™ droplet digital™ pcr system (ddpcr, biorad) using an home-made protocol targeting the rna dependent rna polymerase (rdrp) of sars-cov-2 (forward: to verify the correct performance of the sars-cov-2 rna quantification and housekeeping gene quantification, two independent experiments using different dilutions of the sars-cov-2 rna from a reference patient (rtpcr cycle n. 20 corresponding to 10 7 copies/ml in ddpcr) were performed. in particular, six serial dilutions were performed in order to deposit 10 5 , 10 4 , 10 3 , 10 2 , 10, 2 copies per reaction. all samples were repeated in duplicate, with exception of the last point repeated in quadruplicate. coefficient of determination (r 2 ) of sars-cov-2 quantification was assessed by linear regression analysis by plotting the standards' measured copies and comparing them with expected values of serial dilutions. negative and positive controls, consisting of 40 rna samples obtained from human nasopharyngeal swabs collected before september 2019, and 60 rna samples obtained from human sars-cov-2 positive nasopharyngeal swabs collected during the pandemics were included in each experiment. negative samples for sars-cov-2 were retrospectively selected on the basis of their availability, the storage at -80˚c in order to preserve eventual presence of rna, and the collection date (september 2019-january 2019). an alternative confirmed diagnosis was available for most of these samples (influenza a, n = 15; bacterial infections, n = 9; influenza b, n = 3, rsv, n = 2; hcov-oc43, n = 1). in order to further confirm the performance of the ddpcr-based assay, igg against sars--cov-2 were tested by using a commercial chemiluminescent microparticle immunoassay igg against sars-cov-2 (https://www.corelaboratory.abbott/us/en/offerings/segments/ infectious-disease/sars-cov-2, sensitivity:99.9% specificity:100%) [16] in a subgroup of 34 patients for those serum samples at the later stage of illness (from 3 days to 100 days after) were available (sars-cov-2 ddpcr positive: 14; sars-cov-2 ddpcr negative: 22). for ddpcr methods characterization, coefficient of determination (r2) was assessed by linear regression analysis, whereas the limit of detection (lod), defined as the lowest concentration at which 95% of positive samples were detected, was determined by probit regression analysis. reproducibility of sars-cov-2 quantification methods was assessed by intra-and inter-run tests using serial standard dilutions. the coefficient of variation (cv) was calculated as the standard deviation (sd) of sars-cov-2 copies/reaction divided by replicates mean. descriptive statistics are expressed as median values and interquartile range (iqr) for continuous data and number (percentage) for categorical data. to assess significant differences, fisher exact test and wilcoxon test were used for categorical and continuous variables, respectively. a p-value <0.05 was considered statistically significant. statistical analyses were performed with spss software package for windows (version 23.0, spss inc., chicago, il). baseline demographic and clinical characteristics of the 55 patients included in the study are reported in table 1 . patients were mainly female (45.5%) with a median age of 73 (iqr: 57-84) years. all patients were symptomatic with exception of one (covid-19 contact) and thus immediately hospitalized and tested for sars-cov-2 rtpcr, whose result was negative. thirty-five patients reported fever (63.6%) and 36 had pulmonary involvement (65.5%). further chest ct showed a bilateral interstitial pneumonia in 16 patients (29.1%). for the remaining 20, other lung diseases were diagnosed ( table 1) . the method showed a good linear correlation between expected and observed sars-cov-2 quantification (r2 = 0.996) (fig 1a and s1a fig) . by intra-run tests, the differences between the expected and observed sars-cov-2 copy number per reaction in the two experiments were 121,200±566 and 124,400±1,697 for 10 5 copies, 10,080±113 and 12,560±2,715 for 10 4 copies, 1,035±26 and 1,580±254 for 10 3 copies, 108 ±11 and 120±3 for 10 2 copies, 16±3 and 26±3 for 10 copies, 2.2±3.3 for 2 copies. for inter-run tests, the mean (+sd) sars-cov-2 copy number per reaction was 122,800 ±2,262 for 10 5 copies, 11,320±1,753 for 10 4 copies, 1,307±385 for 10 3 copies, 114±8 for 10 2 copies, 21±7 for 10 copies, 1.1±1.5 for 2 copies (fig 1b) . mean cv was 0.09 and 0.15 for intrarun and inter-run tests, respectively. probit analysis predicted a limit of detection (lod) of 2.9 (95% ic 2.0-11.5) copies per reaction. no signal was detected in any of the 40 sars-cov-2 negative samples tested, all collected between june and september 2019, before sars-cov-2 pandemic (s1b the rtpcr-negative nasopharyngeal swab samples obtained at first visit from the 55 patients included in the analysis were tested with ddpcr in blind. the quality of all swab samples was confirmed by rnasep quantification (median, iqr, copies/ml: 136,286 [84,548-256,714]). our home-made ddpcr assay detected sars-cov-2 in nasopharyngeal swab samples belonged to 19 patients (table 1 and s1 table) . median viral load was 128 copies/ml (iqr: 72-345); the highest value detected was 1800 copies/ml (s1c and s1d fig) . of note, for 12 patients the subsequent nasopharyngeal swabs gave multiple negative rtpcr results (median number of negative swabs: 3 [3] [4] ). for the remaining 7 patients sars-cov-2 was later on detected also by rtpcr, during the follow-up investigation performed within 3 days since the initial negative test. ddpcr provided negative results in the remaining 36 nasopharyngeal swabs. table 1 reported demographic and clinical characteristics respect to ddpcr results. patients with a ddpcr confirmed sars-cov-2 infection were more recently admitted to the hospital (march 18 vs. april 28, p = 0.008), were more frequently affected by cough and dyspnea sars-cov-2 detection by ddpcr (p = 0.047 and <0.001, respectively), and showed more frequently a classical bilateral interstitial pneumonia (p<0.001) respect to patients with a negative ddpcr result. of note, a severe covid-19 manifestation characterized 14 of the 19 patients with a ddpcr confirmed sars--cov-2 infection (73.7%, s1 table) . median time from symptoms-onset to hospital admission was not significantly different between the two groups (p = 0.425). in order to further confirm the performance of the ddpcr-based assay in sars-cov-2 detection, igg against sars-cov-2 were tested by using a commercial chemiluminescent immunoassay [16] . sars-cov-2 was detected at later time points also by standard qualitative rtpcr for three of these five patients (id 1, 4, 8, s1 table) . contrariwise, negative antibody findings were observed in 19/20 (95.0%) patients tested negative for sars-cov-2 in ddpcr ( table 1 ). the single patient tested negative for sars-cov-2 by ddpcr, but positive at the serological assay was characterized by a time from symptoms-onset to nasopharingeal swab of 28 days, and a time from symptomsonset to serological assay of 48 days, thus suggesting a late-stage disease at hospital admission, when probably the viral clearance already occurred. this proof-of-concept study shows that an in-house ddpcr-based assay can allow an efficient detection of sars-cov-2 at low copy number in symptomatic cases resulted negative by standard rtpcr. the effective prevention, treatment and control of covid-19 cannot be achieved without an early, sensitive, and reliable diagnosis. the laboratories play a critical role in confirming the initial clinical suspicion of this disease, as confirmation of sars-cov-2 presence is essential to ensure the prompt initiation of containment and treatment protocols. this is of utmost importance to avoid further spread of the pandemic, and to assure the best clinical and therapeutic management of the infected patients in the hospital setting. unfortunately, currently used rtpcr assays lack of the necessary sensitivity to identify all cases of sars-cov-2 infection (20% of false negative results [5, 6] ). complementary laboratory assays are therefore strongly needed. here, we applied an in-house ddpcr-based assay for sars-cov-2 detection in rtpcr negative swab samples. thanks to this rdrp-based assay, sars-cov-2 genome could be accurately identified and quantified down to only 2.9 (95% ic 2.0-11.5) copies per reaction corresponding to a viral load of 28 (95% ic 13-118) copies/ml, highlighting the great sensitivity of the method. moreover, being the quantification per ml highly dependent on the quality of sampling and the extraction procedure, the presence in this assay of an internal reference gene (rnasep) as a quality control, excludes any eventual pcr inhibition and confirms successful rna extraction, thus dramatically reducing the risk of false negative results. rnasep showed a quantification close to 10 5 copies/ml in the majority of samples, with few peaks of 10 6 , suggesting a relatively stable expression of this gene in our population. of note, when the sars-cov-2 amount was normalized according to the rnasep quantification (per 100,000 copies of rnasep) viral load results did not differ significantly respect to quantifications per ml in the majority of patients (73.6%, 14/19, s1 table) . a 1 log decrease was observed only for 5 samples, all characterized by an rnasep expression at least 0.5 log higher respect to the mean value (s1 table) . by using this reliable, reproducible and highly sensitive assay, we could improve the efficiency of covid-19 diagnosis on nasopharyngeal swabs even at a very early stage of viral replication, or in anatomical districts where the virus is present at lower levels, such as the oropharyngeal or the nasopharyngeal samples [8, 9] . this bridges a significant diagnostic gap left by commercial rtpcr systems used to date as a bulwark of molecular diagnostics of sars--cov-2. this provides a significant clinical advantage in terms of prompt identification of subjects with sars-cov-2 infection, and for the consequent implementation of all appropriate containment and therapeutic measures. indeed, all the 19 patients tested positive by ddpcr were characterized by generally low viral loads (never above the 2,000 copies/ml) that potentially justified the negative contextual rtpcr results. the sars-cov-2 detection by ddpcr in these patients was also confirmed by the presence of sars-cov-2 igg at the later stage of illness (from 3 days to 90 days after, 14/14 patients with a serum sample available). on the other side, ddpcr did not detect sars-cov-2 genomes in 36 patients hospitalized during the pandemic and for whom (out one, probably hospitalized at a late-stage disease, when the viral clearance was already occurred) subsequent serological assays excluded a previously contact with sars-cov-2. taken together, our results support the superiority of our ddpcr-based assay to currently used rtpcr tests for the molecular detection of sars-cov-2. the ability of ddpcr to generate accurate quantitative data has had a huge impact on the study of viral agents of infectious disease [8, 10-12, 14, 18] . thanks to an extremely high sensitivity, this next generation pcr platform is providing a consistent help in all those clinical scenarios in which is of great importance to identify even the smallest amount of viral particles [10, 12, 14] . in the context of covid-19 diagnosis, two recent studies highlight the best performances of ddpcr in detecting low viral load samples [19, 20] . in particular, by targeting orf1ab and n genes, these papers demonstrated that negative rtpcr samples could be identified as positive by the optimized ddpcr (4/96 samples in [19] and 25/27 samples in [20] ). in the suo et al paper, the sars-cov-2 positivity by ddpcr was confirmed by rtpcr in subsequent follow-up investigations for all the 25 patients [19] . differently, our study showed the ddpcr advantages over currently available rtpcr approaches also in the setting of persistently and repeatedly negative rtpcr results. in particular, without this assay, no molecular laboratory confirmation of sars-cov-2 would have been available at hospitalization for 12 patients, all characterized by a severe covid-19 manifestation and with evidence of bilateral interstitial pneumonia, and for two patients affected by pneumonia with pleuritis (n = 1), and lung cancer (n = 1), for whom the mere clinical evaluation excluded the covid-19. our study has some limitations. a) the sample size was small (the study has been designed as a proof of concept, though); b) the correlation of the sars-cov-2 viral load with the timing of infection was not feasible; c) the infectivity of the virus was not assessed. moreover, the ddpcr turnaround time (longer than for qpcr), and the difficulty to convert this system in a high throughput individual assay makes it necessary to apply this system only in specific clinical scenarios. as this assay could potentially be used on different samples, the evaluation of its performances in different laboratories will be necessary for the inter-laboratory reproducibility, and thus the cross-validation of the methods. overall, ddpcr should be a complement to the current standard rtpcr-based diagnosis, in order to improve as much as possible the rapid identification of sars-cov-2 infection (making possible the diagnosis long before the viral load peak is reached and antibodies appear) and thus the definitive containment of the pandemics. even more relevant, this test could be used for treatment monitoring, or for all supposed covid-19 patients, who are negative for nasopharyngeal swab nucleic acid tests twice by rtpcr, but probably at risk of carrying sars--cov-2. in conclusion, we have provided preliminary results to prove that ddpcr is a promising molecular diagnostic tool to detect low levels of sars-cov-2 rna. it possesses all the critical characteristics that would allow its use to improve and accelerate covid-19 diagnosis in clinical samples. as its use could be foreseen in routine clinical practice, additional data on a larger number of clinical samples, and possibly from multicenter studies, are required to further confirm its sensitivity, specificity, and reliability. diagnosis and clinical management of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection: an operational recommendation of peking union medical college hospital (v2.0). emerging microbes & infections 2020 laboratory testing for coronavirus disease (covid-19) in suspected human cases covid-19 disease with positive fecal and negative pharyngeal and sputum viral tests negative nasopharyngeal and oropharyngeal swab does not rule out covid-19 false-negative results of real-time reverse-transcriptase polymerase chain reaction for severe acute respiratory syndrome coronavirus 2: role of deep-learning-based ct diagnosis and insights from two cases antibody responses to sars-cov-2 in patients of novel coronavirus disease sars-cov-2 viral load in upper respiratory specimens of infected patients quantitative detection and viral load analysis of sars-cov-2 in infected patients viral load of sars-cov-2 in clinical samples a sensitive and accurate quantification method for the detection of hepatitis b virus covalently closed circular dna by the application of a droplet digital polymerase chain reaction amplification system clinical utility of droplet digital pcr for human cytomegalovirus highly precise measurement of hiv dna by droplet digital pcr absolute quantification by droplet digital pcr versus analog real-time pcr quantification of hiv-dna and residual viremia in patients starting art by droplet digital pcr: their dynamic decay and correlations with immunological parameters and virological success performance characteristics of the abbott architect sars-cov-2 igg assay and seroprevalence in use of digital droplet pcr to detect mycobacterium tuberculosis dna in whole blood-derived dna samples from patients with pulmonary and extrapulmonary tuberculosis ddpcr: a more sensitive and accurate tool for sars-cov-2 detection in low viral load specimens quantitative detection and viral load analysis of sars-cov-2 in infected patients we thank bio-rad italia for providing technical support, particularly dr. laura sard, dr. marilisa marinelli and dr. marco tronconi and medical affairs bio-rad team, for their valuable assistance in the implementation of the study. the authors also thank dr. silvia nerini and all the staff of the microbiology and virology laboratory of asst grande ospedale metropolitano niguarda for outstanding technical support in processing swab samples, performing laboratory analyses and data management. conceptualization: luna colagrossi, chiara vismara, carlo federico perno. key: cord-322533-adqqm0n9 authors: sha, dexuan; miao, xin; lan, hai; stewart, kathleen; ruan, shiyang; tian, yifei; tian, yuyang; yang, chaowei title: spatiotemporal analysis of medical resource deficiencies in the u.s. under covid-19 pandemic date: 2020-10-14 journal: plos one doi: 10.1371/journal.pone.0240348 sha: doc_id: 322533 cord_uid: adqqm0n9 coronavirus disease 2019 (covid-19) was first identified in december 2019 in wuhan, china as an infectious disease, and has quickly resulted in an ongoing pandemic. a data-driven approach was developed to estimate medical resource deficiencies due to medical burdens at county level during the covid-19 pandemic. the study duration was mainly from february 15, 2020 to may 1, 2020 in the u.s. multiple data sources were used to extract local population, hospital beds, critical care staff, covid-19 confirmed case numbers, and hospitalization data at county level. we estimated the average length of stay from hospitalization data at state level, and calculated the hospitalized rate at both state and county level. then, we developed two medical resource deficiency indices that measured the local medical burden based on the number of accumulated active confirmed cases normalized by local maximum potential medical resources, and the number of hospitalized patients that can be supported per icu bed per critical care staff, respectively. data on medical resources, and the two medical resource deficiency indices are illustrated in a dynamic spatiotemporal visualization platform based on arcgis pro dashboards. our results provided new insights into the u.s. pandemic preparedness and local dynamics relating to medical burdens in response to the covid-19 pandemic. coronavirus disease 2019 (covid19) was first identified in december 2019 in wuhan, china as an infectious disease, and has quickly resulted in an ongoing pandemic. just before the global pandemic covid-19, a report by the global health security index was released, which is the first-ever comprehensive ranking of 195 countries based on their pandemic preparedness, with six categories of 140 questions and 34 indicators [1] . although national health security is fundamentally weak across the globe, the u.s. scored 83.5/100 and ranked no.1 in a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the report. as evidence, there were 34.7 critical care beds per 100,000 inhabitants in the u.s. by 2009, which is higher than that of any other country [2, 3] . however, the u.s. has fewer hospital beds (2.8), and practicing physicians (2.6) per 1,000 capita compared to other similar large and wealthy countries [4] . since the covid-19 outbreak, it has been estimated that a significant percentage of the u. s. population would test positive for covid-19 even given a conservative estimation [5] . for example, a recent aha (american hospital association) webinar on covid-19 projected that 30% (96 million) of the u.s. population would test positive, with 5% (4.8 million) being hospitalized, 2% (1.9 million) would be admitted to the intensive care unit (icu), and 1% (960,000) would require ventilators [6] . this projection is generally compatible with the characteristics of covid-19 in wuhan, china, where 5% of patients required the intensive care unit and 2.3% required a ventilator [7] . based on a recent cdc survey, the actual weekly hospitalization rate in april 2020 was around 5.8-7.5% for 100 counties across 14 states [8], which means a large number of infected patients will swarm into hospitals and icus. as a matter of fact, the u.s. had the highest number of confirmed cases of covid19 (82,404) in the world on march 26, 2020, and surpassed italy for the highest national death toll (20, 413) on april 11, 2020 [9, 10] . are u.s. medical resources enough to handle the worst scenario during this crisis? the society of critical care medicine (sccm) released a report regarding the medical resources both available and needed for a potentially overwhelming number of critically ill patients [6] . in this report, three fundamental elements or features, i.e. ventilators, icu beds, and critical care staff (ccs) were identified as medical resources to plan for or manage a covid-19 pandemic, and it would be wise to consider the interconnections among these factors in a spatiotemporal data analysis framework. specifically, the medical resource distribution should be correlated with covid-19 pandemic statistics in space (2d) and time (1d). so medical resource burden or deficiency can be identified through feature selection, visualization, monitoring, and cluster analysis [11] . among the three elements mentioned above, an inventory of ventilators is difficult to quantify for estimating critical supply shortages. based on a 2009 aha survey, a total of 5,752 u.s. acute care hospitals were estimated to have 62,188 full-featured mechanical ventilators and 98,738 ventilators with limited features [12] . the strategic national stockpile (sns) had an estimated 8,900 ventilators for emergency deployment in 2010, and between 12,000 and 13,000 ventilators by march 13, 2020 [13-15] . based on these numbers, the ventilator inventory was approximately 173,000-174,000 in the u.s. a model-based analysis suggested that us hospitals could absorb between 26,200 to 56,300 additional ventilators at the peak of a national pandemic with robust pre-pandemic planning [16] . since sns can deliver ventilators within 24-36 hours after being requested by states and approved by federal organizations, and no reliable database for ventilator inventory exists at county or state level, we will not consider this factor in our spatiotemporal analysis. a recent model-driven study simply assumes one ventilator per critical care bed [17] and we use this same assumption in our analysis. hospital beds, especially icu beds, are an important factor in evaluating medical resource deficiency during the covid-19 pandemic, and quantity of beds has been used as a major factor in model-driven predictions of local critical care capacity limit [17, 18] . however, safe use of ventilators in icu requires trained personnel. in a previous study, the number of trained medical personnel is assumed to correlate with the number of staffed beds maintained by hospitals [16] . this assumption is perhaps unrealistic at county level without considering the geographic disparity. for this research, we assumed that a realistic measurement of the medical burden at county level should consider both icu beds and critical care staff (ccs), which will provide reasonable evidence for stakeholder (e.g., hospital, county and state governments policy and decision-making). in this study, we (1) conduct a medical data analysis, and re-evaluate the spatial distribution of medical resource features (hospital beds, icu beds, and ccs) at county level; (2) develop two medical resource deficiency indices (mrdi and mrdi d ) by linking positive covid-19 infections and local medical resources to measure local medical burden; and (3) develop a data-driven dynamic spatiotemporal framework to visualize and analyze the mrdi /mrdi d trends at the county level. our results provided a new dimension of insight into the u.s. pandemic preparedness and local dynamic medical burden during covid-19 pandemic. the dataset is open sourced and hosted on github (https://github.com/stccenter/ covid-19-data/tree/master/us), and are visualized through arcgis dashboards at: http:// mrd-dashboard.stcenter.net/. a total of 3,143 counties and county-equivalents in the u.s. are used as the primary unit of this study, since they are manageable in a gis system and small enough to reflect local geographic discrepancies. the base map was downloaded from the 2019 tiger/line products from the u.s. census bureau, which is the most comprehensive spatial dataset designed for gis platforms [19] . the county vector layer delineates the administrative boundary with land/water area without any demographic data, but it provides geographic entity codes (geoids) for joining with other socio-economic data such as census data. based on the attributes of our collected medical-related datasets, we also prepared state and zip code boundaries for data fusion and integration at county level. in this study, two fundamental features of medical resources in the u.s. were extracted, i.e., hospital beds and critical care staff. besides, the population and 60+ senior population data was extracted at county level from khn online database [20] , which is used to normalize the local medical data in the subsequent analysis. 2.2.1. hospital beds. national public and private online datasets were used to prepare county-level hospital bed counts. hospital data were collected from definitive healthcare [21] . definitive healthcare consulting services share their hospital dataset to the entire health research community through arcgis online, which cover information of nationwide bed capacity and average yearly bed utilization of hospitals. although it is not a real-time dataset that reflects each hospital's bed capacity during covid-19, it can be used as a baseline to estimate the geographic disparity of local health resources. a hospital is defined as a healthcare institution providing inpatient, therapeutic, or rehabilitation services under the supervision of physicians with the capability of inpatient care [21] . all types of hospitals are included in our study. five types of hospital beds are clearly identified in the definitive healthcare dataset. in our study, two hospital bed capacities were selected and used in the analysis. the first one is the number of licensed beds, which is the potential or maximum number of beds for which a hospital holds a license to operate. the second type of capacity refers to the number of adult icu beds that could be used for covid-19. during this crisis, hospitals could use additional intensive care beds to supplement an influx of patients. therefore, adult icu beds include not only internal medical icu beds, but also burn, surgical, and trauma icu beds. however, pediatric, premature or neonatal icu beds are not included because they are mainly for a different target patient population, which has a much lower incidence rate of covid-19. two other independent data sources of hospital beds are compared with the data from definitive healthcare. one is from kaiser health news (khn) based on reports of icu beds in 2018-2019 [20] , and the other is from homeland infrastructure foundation-level data (hifld) for licensed hospital beds updated on october 7, 2019 [22] . we conducted a regression analysis comparing khn with definitive healthcare in terms of icu beds, and comparing hifld with definitive healthcare in terms of licensed beds, and the coefficients of determination (r 2 ) are 0.94 and 0.97, respectively. the results validated the quality of the definitive healthcare dataset. a dataset of critical care staff (ccs) was extracted from the weekly updated national provider identifier registry (npi) database (~7.1 gb) through structured query language (sql) [23] . the npi is a unique 10-digit identification number for each health-care provider issued by the centers for medicare medicaid services through the national plan and provider enumeration system. each health-care provider could have multiple taxonomy codes, which indicate areas of specialization. through consulting with medical researchers and front-line physicians, we extracted detailed ccs data from the npi database released on april 15, 2020 as a medical resource feature (table 1 ). our study identifies 197,061 health care providers by searching unique npi records and removing duplicate records. with the development of covid-19 in the u.s., all these icu-related staff (emergency medicine physician, critical care physicians, anesthesiologists, hospitalists, pulmonologist, infectious disease physician, surgery, anesthesiologist assistant, critical care nurses, nurse anesthetist, and respiratory therapists trained in mechanical ventilation) would become valuable but limited asset for critically ill ventilated patients [6] . the u.s. centers for disease control and prevention (cdc) published daily covid-19 confirmed cases on february 25, 2020. each state got involved soon after and began to report covid-19 data, including the daily and accumulated test and confirmed case numbers, hospitalization data, and death numbers at state level. however, numbers of discharged or released patients from hospitals are less widely available, e.g., only a few states, such as maryland, colorado, and new york provide some (incomplete) statistics on recovered patients from both hospital and home. this study mainly uses the data collected by the nsf spatiotemporal innovation center (stc) at george mason university. this dataset uses a datacube structure for spatiotemporal data aggregation from multiple sources. the data is cleaned, standardized, and updated daily to solve any data conflicts, and a time-series summary at state and county level is provided for the u.s. [10, 24] . the numbers of county-level confirmed positive cases as well as deaths were originally extracted from usa facts based on cdc data [25] , and compared with local public health agencies for verification. the confirmed and death cases reflect cumulative statistics since january 22, 2020, the day after the first confirmed cases were reported in washington state. furthermore, state level test and hospitalization data were extracted from the covid tracking project [26] . however, the current and accumulated hospitalization cases from state health departments are largely incomplete. by april 29, 2020, a total of 22 states reported both current and accumulated hospitalized patient numbers, 17 states reported only current hospitalized numbers, and 10 states only reported accumulated hospitalized numbers, while washington, d.c., nevada and nebraska did not provide information on the number of hospitalized cases. our analysis was mainly based on the publicly available data of the new confirmed daily cases reported for the u.s. from the 25th of february until the 1st of may, 2020. all data were fully anonymized. raw datasets in this study were collected from multiple sources with heterogeneous formats and structures. all data were processed and aggregated at county level based on county federal information processing standard (fips). several aggregation methods were used for each raw dataset, as summarized in fig 1. first, the hospital data was originally presented as a point location in a coordinate format, and its attribute table includes five types of hospital beds. the spatial point aggregation algorithm was used to integrate the numbers of licensed beds and adult icu beds at county level. the bed numbers per 1,000 residents were also calculated at county level. the primary practice addresses of ccs were imported from the npi database, and 5-digital zip codes were extracted. the total number of ccs within a county was counted based on the county's zip codes through geocoding and the point/ polygon aggregation algorithm. the number of ccs per 1,000 residents were also calculated at county level. the accumulated covid-19 confirmed case numbers were extracted at county level. we used existing hospitalization data to estimate the average length of stay (alos) in acute care, since it is key for estimating the daily hospitalized patients. for a given state, the current hospitalized patients should be equal to the accumulation of hospitalized patients minus the accumulation of deaths and discharged patients within the most recent alos. since no patient discharge data was available, we assumed that the number of discharged patients was zero. therefore, we estimated alos by matching (1) the accumulation of hospitalized patients minus the accumulation of deaths in most recent days, and (2) the current number of hospitalized patients, and finally interpolating by two nearest days or accumulation periods. it turns out to be an optimization problem to find a parameter (n) to match the two data sources, as shown in eq (1). where n h,n is the accumulated number of hospitalized patients in the past n days, n death,n is the accumulated number of deaths in the past n days, and n ch is the number of currently hospitalized patients. state hospitalization data were only available recently (starting from march 17, 2020 in ny) with numerous missing data. by may 1, 2020, among 22 states that have both current and accumulated numbers of hospitalized patients, eight states (colorado, massachusetts, maine, minnesota, montana, north dakota, new york, oklahoma) had complete data for the most recent 20 days; 12 states (oklahoma, wisconsin, mississippi, maryland, new hampshire, new mexico, oregon, south dakota, virginia, wyoming, rhode island, kentucky) only had data in the most recent 5-15 days; and data from arkansas, arizona, and connecticut were abandoned due to poor quality. we calculated the daily alos for these 19 states and pooled the results in fig 2. the state alos ranges from 8.8 (new mexico)-28.5 (mississippi) days. the overall national alos weighted by state hospitalized patients is 15.5 days, which is longer than a previous estimation that the alos in acute care were 11 days [18] . it is worth noting that alos is likely to be underestimated since we assumed no discharged patients. furthermore, alos is subject to change when more hospitalization data become available in the future. finally, we define the covid-19 hospitalized rate as the ratio of the number of current hospitalized patients and the accumulated confirmed case numbers during the most recent alos. if the hospitalized rate remains the same within a state, the daily hospitalized patient number in a county can be estimated by using the accumulated covid-19 confirmed case numbers minus deaths in the most recent alos, multiplied by the state average hospitalized rate. if no state alos is available, we use the overall national average alos of 15.5 days. this daily hospitalized patient number can be used to evaluate the daily medical burden at county level. the medical resource deficiency indices (mrdi) are defined as an indicator of medical resource burden at county level. we define two forms of mrdi: general mrdi, and local daily mrdi (mrdi d ). where n c is the accumulated number of confirmed covid patients, n death is the accumulated number of deaths, n licbed is the total number of licensed beds, and n ccs is the number of critical care staff. we assumed that n licbed and n ccs were relatively independent at county level, and the product of them represents the interconnection of these two medical resource features or factors. therefore, the mrdi represents the number of accumulated active confirmed cases normalized by the local maximum potential medical resources (total licensed beds and total ccs). mrdi d is represented as where n ca is the accumulated confirmed case numbers during a most recent alos, n da is the accumulated death numbers during the same alos, r h is the state hospitalized rate derived based on arcgis dashboard, we designed a comprehensive operational dashboard for monitoring, analyzing, visualizing, and sharing our medical data and analyzed results. a multistacked map is built at the center of the interface (fig 3) , which represents the spatial distributions of covid-related statistics such as mrdi, death rate, and infection rate at county level over the u.s. in addition to visualizing the macro spatial distribution pattern of those statistics results, two lists of counties are displayed. those counties are dynamically filtered by the current map extent in map view and are ranked in real-time by hospitalized rate and death rate to represent the spreading of covid-19 and the outbreak situation in the selected study area. focusing on a specific county, an indicator and two pie charts are applied to display for each county (fig 4) : 1) the comparison of active covid-19 cases and the number of overall beds; 2) the percentage of icu beds in overall beds; and 3) the proportion of each type of ccs. from the temporal analysis perspective, a time series chart is designed to demonstrate the dynamics of medical resource deficiencies for each county on a daily basis during the pandemic. in the following section, we will use the dashboard components to analyze spatiotemporal distributions of medical resource deficiencies. we will further explore the possible factors relating to the medical resource deficiencies for specific counties and areas as well as the medical resource capacity for non-severe covid-19 patients, the supplies needed for severe cases, and proportion of each type of ccs. the icu beds per 1,000 residents ( fig 5a) and ccs per 1,000 residents ( fig 5b) are mapped at county level. both maps show that these two medical resources are not homogeneously distributed across the u.s. some midwestern states, such as north dakota, south dakota, nebraska, kansas, and montana have more icu beds, but less ccs. the spatial distribution of ccs shows a checker board pattern, with many gaps or low numbers across the country. the product of icu beds and ccs per 1,000 residents is shown in fig 6a. the darkest green zones represent counties with higher quantities of medical resources including icu beds and ccs. a total of 19 major medical centers represent top ranking healthcare facilities in the u.s. (table 2 ) [27] . medical centers are conglomerations of health care facilities including hospitals and research facilities that could be affiliated with a medical school. overlaying the locations of these 19 medical centers on the map (purple circles on the map), it seems these counties and medical centers are spatially highly correlated (fig 6a) . since senior people (aged 60+) are vulnerable to covid-19, we also produced a map of the product of icu beds and ccs per 1,000 senior residents (fig 6b) . this map represents locations where the supply of medical resources for seniors is higher. a regression analysis was conducted to examine the correlation between ccs and adult icu beds at county level (fig 7) . if all 3,143 counties are included, the coefficient of determination (r 2 ) is 0.90. however, this high r 2 value is quite misleading, since it is heavily influenced by several large counties with rich medical resources (blue dots). removing the top 30 counties, causes the coefficient of determination (r 2 ) to drop to 0.78, which better represents the geographic disparity of these two factors in most (3113) of the u.s. counties, as shown in fig 5a and 5b . a total of 671 counties have neither icu beds nor ccs, and are shown in fig 8. these counties are mainly distributed in less-populated rural areas across the u.s., and they are not included in mrdi or mrdi d calculation to avoid a divide-by-zero error. during the covid-19 pandemic, individuals requiring a higher level of care in these areas would be sent to the spatiotemporal dynamics of general mdri across the u.s. is illustrated at: http://mrddashboard.stcenter.net/. the general mdri represents the number of accumulated active confirmed covid-19 cases normalized by local maximum potential medical resources, while the dynamic view provides an insightful alternative visualization of covid-19 u.s. cases by county. six snapshot maps are illustrated in fig 9a-9f , which demonstrate six time-stamped frames taken on february 15, march 15, april 15, may 15, june 15, and july 15, 2020. a proportional symbol map is used with semi-transparent red circles to represent the general mdri. this visualization technique enhances clustering patterns, and there is a clear trend where the general medical burden shifted from the east coast of the u.s. to midwestern states. as of july 2020, it would seem that louisiana, mississippi, georgia, tennessee, indiana, and iowa are possibly suffering a new wave of medical resource deficiencies due to the rapid increase of accumulated active confirmed cases in some counties. furthermore, the spatiotemporal dynamics of local daily mrdi d is also illustrated in the dashboards. since hospitalization data has been available only recently, we illustrate two frames taken on may 1, and august 1, 2020 (fig 10a and 10b) . the red circle symbols are semi-transparent, and county-level medical resource deficiencies are visually enhanced by searching the reddest clustering patterns in the map. during this covid-19 infection period, it seems that mississippi, louisiana, tennessee, and indiana were suffering from medical resource deficiencies, which would have required special attention when relocating medical resources if necessary. these hotspots have been partially confirmed from local news reports. for example, there were 5,153 known presumptive cases with the total death toll of 201 in mississippi on april 23, 2020 [28] ; new cases of covid-19 rose sharply on may 1 in east baton in the center of the dashboard, several map layers could be selected to show the general spatial distribution of mrdi, death rate, infection rate and active cases over licensed beds per capita. after interactive map scaling (by zooming in/out) and moving (by dragging) operations, or using the polygon selection tool, the charts and rank list are linked and self-adapted to the analysis region of interest to a user. by clicking the polygon of a selected county, attribute information about medical resources and covid-19 related data would popup and the relevant chart is automatically updated in the dashboard. northern tennessee state is presented as a use case to show the possible interactive analysis (fig 4) . since western and east coast regions have more medical resources than central regions (fig 6a) , and the states along the mississippi river in the southern u.s. show a high risk (fig 10) , we zoom in on the map and select the nearest region with the largest red bubble in tennessee (fig 4) . thirty counties are selected as a result, and relevant numbers are calculated and presented in dashboard charts. the medical bed pie chart shows icu beds are 10.89% in overall licensed beds, and the medical staff pie chart shows the nurses group is the highest (55.77%) followed by physicians (44.01%), physician assistants (0.18%) and therapists (0.04%). the line chart shows a time-series trend for mrdi in the northern tennessee area, and we find the index varied greatly between april 30, 2020 to may 1, 2020, which could be explained by the possible tracing of the virus to a correction center outbreak in trousdale county [32] . on the right column of the dashboard, the risk factors of medical resource and infection rate is ranked by the selected region. trousdale, davidson, and sumner county are the top 3 with highest infection risks, while trousdale also shows the highest medical resource risk in this region. the case study in fig 4 demonstrates the potential of our developed dashboard for interactive and visual analysis of specific regions of interest for policy makers, other stakeholders, and the general public. in this study, a data-driven approach has been used to estimate the medical resource deficiencies or medical burden at county level during the covid-19 pandemic across the u.s. specifically, spatiotemporal data analysis methods including feature extraction, database structured query (sql), data fusion or aggregation, linear regression analysis, and spatial statistics were used to extract medical resource features and patient statistics, such as hospital beds, ccs, local population, covid-19 confirmed case numbers, and hospitalization data at county level. the average length of stay (alos) was then estimated from hospitalization data at state level, and the hospitalized rate were calculated at state and county level. based on these datasets, we developed two medical resource deficiency indices mrdi and mrdi d that measure the local medical burden from two different perspectives. the first index represents the number of accumulated active confirmed cases normalized by local maximum potential medical resources; and the second one represents the number of hospitalized patients that can be supported per icu beds per critical care staff. the related medical resource data, mrdi and mrdi d were visualized and analyzed using a dynamic spatiotemporal platform created through arcgis pro dashboards, which is a convenient way to enhance the clustering patterns and trends. our analysis showed that (1) the spatial distribution of medical resources (hospital beds, icu beds, and ccs) at county level is highly heterogeneous across the u.s., and icu beds and ccs are not spatially highly correlated; (2) mrdi and mrdi d can provide new insights into the u.s. pandemic preparedness and local dynamics relating to medical burdens during a peak period in the covid-19 pandemic; and (3) a data-driven dynamic spatiotemporal framework is a powerful data visualization tool to illustrate the trends of mrdi / mrdi d and other medical-related statistics. it is worth noting that we have not considered the number of discharged patients due to lack of data, leading to a possible slight underestimate of alos during the covid-19 rapid infection period. as a result, mrdi d may also be slightly underestimated. we also did not consider the ratio of icu patients and acute hospitalized patients due to lack of data, and assumed all hospitalized patients were treated as icu cases. as a result, mrdi d was possibly overestimated, and the values calculated here should be viewed as the upper limit of local medical burdens. some other uncertainties include (1) the numbers of registered hospital beds and ccs could be incomplete or not up-to-date, although the most recent definitive healthcare and npi databases have been used, so the medical resources could be underestimated, (2) critically ill patients in counties without icu beds and ccs would be sent to neighboring counties with sufficient medical resources, (3) some numbers of experienced icu staff may become ill, (4) the number of trained professionals may have increased based on emergent recruiting, and (5) the capacity in icus and emergency rooms may have been expanded during the crisis. however, mrdi d can still serve as a useful indicator to measure the county-level medical resource deficiencies, and this index can be improved once more public health data are available in the future. furthermore, it could provide reasonable evidence for policy makers in local and state governments to assess their medical inventories and staff resources, and provide preparedness for decision of re-opening the economies and public life. in the future, our work can be combined with epidemic models to either provide driving parameters or calibrate the models and predict the local medical burdens. the spatiotemporal analysis used in this study can be extended to include remote sensing data, social media data, and mobile traffic flow data to estimate severity of pandemic or predict the outbreak cases in the u.s. and other counties. conceptualization: dexuan sha, xin miao, yuyang tian, chaowei yang. the variability of critical care bed numbers in europe critical care bed growth in the united states. a comparison of regional and national trends how prepared is the us to respond to covid-19 relative to other countries? estimating covid-19 prevalence in symptomatic americans availability for covid-19 clinical characteristics of coronavirus disease 2019 in china an interactive web-based dashboard to track covid-19 in real time taking the pulse of covid-19: a spatiotemporal perspective big spatiotemporal data analytics: a research and innovation frontier mechanical ventilators in us acute care hospitals. disaster medicine and public health preparedness stockpiling ventilators for influenza pandemics ventilator stockpiling and availability in the us strategic national stockpile: overview and ventilator assets assessing the capacity of the us health care system to use additional mechanical ventilators during a large-scale public health emergency flattening the curve before it flattens us: hospital critical care capacity limits and mortality from novel coronavirus (sars-cov2) cases in us counties estimating the maximum capacity of covid-19 cases manageable per day given a health care system's constrained resources. annals of internal medicine tiger/line shapefiles and tiger/line files where the icu beds are definitive healthcare: usa hospital beds spatiotemporal patterns of covid-19 impact on human activities and environment in mainland china using nighttime light and air quality data. remote sensing coronavirus locations: covid-19 map by county and state the covid tracking project: most recent data medical centers in the united states mississippi covid-19 cases now number more than 115 new confirmed coronavirus cases in east baton rouge, part of sharp rise; see latest statewide data covid-19 update: april saw rural america's infection rate increase 8-fold. the daily indiana passes 1,000 covid-19 deaths; lake county reports four more deaths, brings total to 81 large spike in coronavirus cases traced to prison in trousdale county key: cord-302355-3se1wp8o authors: chen, yi-shiuan; fan, yi-hsin; tien, chih-feng; yueh, andrew; chang, ruey-yi title: the conserved stem-loop ii structure at the 3' untranslated region of japanese encephalitis virus genome is required for the formation of subgenomic flaviviral rna date: 2018-07-26 journal: plos one doi: 10.1371/journal.pone.0201250 sha: doc_id: 302355 cord_uid: 3se1wp8o flaviviruses accumulate abundant subgenomic rna (sfrna) in infected cells. it has been reported that sfrna results from stalling of host 5’-to-3’ exoribonuclease xrn1 at the highly structured rna of the 3’ untranslated region (utr). although xrn1 digestion of a 3’-terminal 800-nt rna could stall at a position to generate the sfrna in vitro, we found that knocking out xrn1 had no effect on the accumulation of sfrna in japanese encephalitis virus (jev) infected cells. mutagenesis studies revealed that the stemloop ii (slii) at the 3’ utr is required for the accumulation of sfrna. according to the results of an in vitro rna-dependent rna polymerase (rdrp) assay, the (-)10431-10566 rna fragment, containing the putative promoter on the antigenome for the sfrna transcription, binds to rdrp protein and exhibits a strong promoter activity. taken together, our results indicate that the jev sfrna could be transcribed initially and then be trimmed by xrn1 or other unidentified exoribonucleases. japanese encephalitis virus (jev) belongs to the family flaviviridae and is a mosquito-borne zoonotic pathogen that causes human encephalitis and meningitis. the 10,976 nucleotides (nts) genome contains a single open reading frame (orf) encoding a polyprotein which is subsequently processed into three viral structural proteins and seven nonstructural proteins [1] . the orf is flanked by 5' and 3' untranslated regions (utrs) which contain many important cis-acting elements involved in the regulation of viral translation, replication, and pathogenesis [2] [3] [4] [5] . during virus infection, all arthropod-flaviviruses generate abundant amounts of a noncoding subgenomic rna (sfrna) representing the 3'-terminal highly conserved region of the 3' utr [6] [7] [8] [9] . the sfrna engages in multiple functions in order to control viral replication and antagonize host antiviral responses [4, 10, 11] . these functions include (i) the involvement of cytopathicity and pathogenicity in mammals [8] ; (ii) the down-regulation of plos antigenome synthesis and translation [12] ; (iii) the dysregulation of host mrna stability [13, 14] ; (iv) the antagonizing of host innate immune response [15] [16] [17] ; (v) the suppression of rna silencing [18, 19] ; (vi) the induction of apoptosis through the bcl-2-mediated pi3k/akt signaling pathway [20] ; and (vii) the determination of infection and transmission rates of west nile virus (wnv) and dengue virus (denv) in mosquitoes [21, 22] . how the sfrna is made in the infected cells is intriguing. several studies have shown that the sfrna is a product of the incomplete degradation of the viral genome by cellular 5'-to-3' exoribonuclease xrn1; a pseudoknot or a three-helix junction structure in the 3' utr is responsible for stalling xrn1 degradation [8, [23] [24] [25] [26] . this cleavage happened within a few minutes when carried out in vitro, and there were no intermediate byproducts during the exoribonuclease degradation process of the viral genome to the sfrna in the virus-infected cells, which indicated that the degradation process is extremely efficient or that another biogenesis mechanism might be involved. the reason for these findings is not clear because degradation from the full-length genome seems uneconomic for viruses from an evolutionary point of view. furthermore, being infected with rna viruses induces double membrane vesicles to form replication/transcription complexes that are protease-and nuclease-resistant [27] [28] [29] . the reason for such abundant levels of viral genome within the membrane-protected complex subjected to rna degradation remains unclear. although rdrp has no proofreading activity, considering the high molar ratio of the sfrna to the genome (1. 25-5. 14 in mosquito cells and 0.25-1.5 in mammalian cells) [9] , the possibility that large portions of the synthesized genome are dysfunctional and subjected to degradation pathways is elusive. it may just be that the rna turnover by xrn1 exoribonucleases is quite normal but that sfrna accumulates because it is degradation resistant. in this study, we found that either knocking down or knocking out of exoribonuclease xrn1 had no effect on the accumulation of sfrna in jev-infected cells. in contrast, mutation of gdd motif in rdrp and disruption of the slii structure halted accumulation of the sfrna. furthermore, the minus-strand templates covering the putative promoter region used for an in vitro rdrp assay gave rise to synthetic products, suggesting that the jev sfrna could be initially transcribed from the antigenome and may be further trimmed by xrn1 or other unidentified exoribonucleases. bhk-21 and a549 cells were grown at 37˚c in rpmi 1640 medium supplemented with 2% and 10% fetal bovine serum (fbs, gibco), respectively. a549 xrn1 knock-out cells were kindly provided by dr. bernard moss (niaid, nih) and were propagated as described previously [30] . human embryonic kidney (hek293t) cells were grown in dulbecco's modified eagle's medium (dmem, gibco) supplemented with 10% fbs (gibco). jev strain rp9 (genbank accession number af014161) and dengue virus type 2 (denv-2) strain 16681 were used in this study. detailed methods for the construction of clones are described in the supporting information s1 text. the majority of plasmids used for this study were constructed by rt-pcr using synthetic oligonucleotides, as listed in s1 table. the cdna encoded rdrp domain from amino acids at position 274 to 905 of the jev ns5 protein was amplified by rt-pcr and cloned into pet28a (novagen). the lentiviral vectors expressing the short hairpin rna (shrna) (trcn-296739) targeting human xrn1 gene (genbank accession no. nm_019001.3) or a negative control shrna targeting gfp (shgfp) were obtained from the taiwan national rnai core facility. to knockdown xrn1 expression, 5 × 10 5 hek293t and a549 cells were transduced with the shxrn1 or shgfp lentiviruses, respectively, and selected with puromycin (1 μg/ml). at one-week posttransduction, cells were seeded on 60-mm plates (approximately 2 × 10 6 cells per plate) and infected with jev rp9 (or denv-2 as a control) at an moi of 5 (for hek293t) or 10 (for a549). the cells were harvested with pbs at 48 h post-infection, and the precipitates were then divided into two parts. one part was analyzed for western blotting and the other part was used for northern analysis. equivalent amounts of proteins (20 μg/sample) were resolved by 8% sds-page, followed by western blotting using anti-xrn1 (1:1000, sigma) or anti-β actin (1:5000, sigma) antibodies. rna extraction and northern analyses were conducted as described previously [9, 15] . in some cases, the blots were hybridized with the ird 700-labeled jev(-) 10950-10976 oligonucleotide, and the signals were scanned on a typhoon 9000 imaging scanner (ge healthcare); otherwise, the blots were hybridized with the strand-specific digoxigenin (dig)-labeling riboprobe detecting jev nt 10454-10976 or denv-2 nt 10270-10732 made by in vitro transcription (roche molecular biochemicals) and then visualized using luminescent image analyzer (las-3000, fujifilm), as described in a previous study [12] . the relative intensities of rna bands on the membrane were estimated using multi gauge software (fujifilm). plasmid pgemt-jev-800 was linearized with sal i and transcribed in vitro using t7 rna polymerase (promega) according to the manufacturer's protocol. dna template used for runoff transcription to generate 3'-terminal 800 nts of denv was amplified from ptight-denv plasmid [31] using oligonucleotides f10 and r10 as shown in supplemental s1 table. dna template was removed by dnase i. approximately 60 pmol of triphosphated rnas were incubated with 20 units of rna 5' pyrophosphohydrolase (rpph, neb) at 37˚c for 2 hours to generate 5'-monophosphated rna. the rpph-treated rna was purified by phenol-chloroform extraction and ethanol precipitation. next, 500 ng of the monophosphated rna was digested with the indicated amount of xrn1 (neb) or rnase a (qiagen) for 1 hour at 37˚c. 200 ng of rna was then separated in a formaldehyde-containing 1.5% agarose gel and hybridized to a riboprobe as indicated. the procedure for dna-based infectious clone transfection has been described previously [31] . briefly, bhk-21 cells (3 × 10 4 cells/well) in 12-well plates were transfected with 0.25 μg of ptight-jev (or mutant) plasmid, 0.25 μg of ptet-off (bd bioscience), and 1 μl of lipofectamine 2000 (invitrogen) in 200 μl of opti-mem medium per well at 37˚c for 4 hours, and the cells were refed with fresh medium. total rnas were extracted at 5 days post-transfection (dpt), and the supernatant fluids were collected for measuring the virus titers. the transfected cells were passaged every 5 days if no apparent cytopathic effect (cpe) was observed. rnas were extracted at the time when the cells showed a severe cpe. the titration of infectious virus was performed as previously described [32] . briefly, serial 10-fold dilutions of virus were made in serum-free alpha-mem medium, and 0.1 ml of each dilution was added per well to a monolayer of bhk-21 cells in a 12-well plate. after 1 hour of absorption at 37˚c, the cells were washed with pbs and overlaid with growth medium containing 0.3% le agarose (lonza), and the plaque was visualized with naphthol blue-black solution (0.1% naphthol blue-black, 1.36% sodium acetate and 6% glacial acetic acid) at 4 days postinfection. viral titers were determined as plaque forming units per milliliter. riboprobe was made by in vitro transcription with t7 rna polymerase (promega) and dig-utps (roche molecular biochemicals). the dig-labeled rnas (0.25 pmol) were denatured at 80˚c for 10 min and then quick cooling on ice. binding reactions were set at room temperature in binding buffer (10 mm hepes ph 7.1, 20 mm kcl, 1 mm mgcl 2 , 1 mm dtt, 10 units rnaseout) with purified recombinant rdrp protein or bovine serum albumin (bsa) at different concentrations (0.25, 0.5, 1, and 2.5 μm) in a volume of 20 μl for 30 min, followed by adding 2 μl heparin (25 mg/ml) for 10 min. the rna-protein complexes were resolved by non-denaturing 5% polyacrylamide gel at 4˚c, transferred to a positively charged nylon membrane (amersham), and then cross-linked using uv stratalinker (stratagene). detection of dig-labeled rna was performed using dig luminescent detection kit (roche) according to the manufacturer's instructions. to demonstrate the specificity of rna-protein complex formation, competition assays were performed using various amounts of unlabeled rnas added to the binding reaction. a recombinant jev rdrp was expressed in e. coli rosetta (novagen). protein expression was induced with isopropyl-β-d-thiogalactopyranoside (iptg) at a final concentration of 0.5 mm and induced culture was grown at 22˚c for 15 hours. expressed rdrp was purified by metal affinity chromatography on a nickel-chelate column according to the manufacturer's instructions (invitrogen). the rna templates used for the rdrp assay and the rna size markers were synthesized by in vitro transcription (promega) in the absence or presence of α-32 p-ctp, respectively. the in vitro rdrp assay was performed with 500 ng of the recombinant rdrp protein and 200 ng of rna templates or as indicated in a total volume of 25 μl of the reaction buffer containing 35 mm tris-hcl (ph 7.9), 50 mm nacl, 25 mm potassium glutamate, 5 mm mgcl 2 , 1 mm dtt, 5 mm mncl 2 , 10% glycerol, 20 units of rnase inhibitor (promega), 5 mm (each) atp, utp, and gtp, 0.5 mm ctp, and 10 mci of α-32 p-ctp (3,000 ci/mmole; mp biomedicals). the reaction mixture was incubated at 30˚c for 2 hours. rdrp reaction product was denatured in 2x loading buffer (95% formamide, 0.025% xylene cyanol, 0.025% bromophenol blue, 18 mm edta, 0.025% sds) and resolved on 6% polyacrylamide gel containing 6m urea. the gel was dried and visualized by autoradiography. to investigate whether the jev sfrna is an exoribonuclease xrn1 cleavage product, we analyzed the effect of xrn1 depletion caused by rna interference on sfrna generation in virusinfected cells. knocking-down (kd) the xrn1 expression by up to 95% in hek293t cells continued to increase the abundance of sfrna throughout the 48-h infection period. more sfrna (121%) was observed in xrn1 knocked-down cells than in the wild-type (wt) cells ( fig 1a) . it should be noted that there were no decay intermediates between viral genome and the sfrna even in the xrn1-deficient cells. similar results were also observed in a549 cells infected with jev (fig 1b) , whereas denv infection caused a slight reduction of sfrna accumulation in the xrn1-kd cells (fig 1c) . statistical analysis of jev sfrna accumulation in xrn1-kd cells among three independent experiments showed about 112.5% ± 8.5% at 48 h post infection. no statistically significant differences in the amount of the sfrna were detected in the infected cells at any time in relation to the depletion of xrn1. these results indicated that either the exoribonuclease xrn1 in jev-infected cells could be extremely active, such that the residual xrn1 (4-5%) might be sufficient to digest the jev genome or that another nuclease or mechanism might be involved in the formation of sfrna. to rule out the possibility that the residual xrn1 may contribute to the formation of sfrna, we performed the experiments using xrn1 knock-out (ko) cells. as shown in fig 1d, sfrna accumulated abundantly in xrn1-ko cells infected with either jev or denv indicating that xrn1 does not play a direct role for the formation of sfrna in vivo. to determine whether purified exoribonuclese xrn1 is able to stall at a position to generate the sfrna in vitro, a 3'-terminal 800-nt monophosphate rna derived from genome of jev or denv was incubated with 0.01, 0.1, or 1 unit of xrn1, respectively, and analyzed by northern blot analysis for sfrna production. the denv sfrna is readily formed when treated with xrn1 at concentration as low as 0.01 units (fig 1e, lane 7) , whereas the 800-nt jev rna was relatively resistant to low concentrations of xrn1 (0.01 and 0.1 unit) (fig 1e, lanes 2 and 3) . in contrast, incubation with 1 unit of exoribonuclease xrn1 resulted in the production of the sfrna (fig 1e, lane 4) , indicating that a higher concentration of exoribonuclease xrn1 is required for stalling at a position to generate jev sfrna. the 800-nt rna treated with rnase a was rapidly and completely degraded (data not shown). several studies suggest the existence of xrn1-resistant structure (xrrna) for the production of sfrna. to investigate the structure requirements for the production of jev sfrna in vivo, we performed mutagenesis studies in the context of a jev infectious clone (in the s1 text and s1 table) . the structure of the jev 3' utr is diagramed in fig 2a. although equal amounts of cdna (250 ng) for all mutants were transfected into bhk-21 cells, according to immunofluorescence assay (ifa) and northern hybridization results at 5 days post-transfection (dpt), the replication efficiency of the cdna from different mutants varied (data not shown). these results suggest that the mutations made in these regions delayed the completion of the viral life cycle. we therefore collected the supernatant and measured the virus titers at the time when the cells showed strong cpe that varied from 4 to 11 dpt for different mutants (table 1) . we then used equal amounts of moi at 0.1 for each recombinant virus to infect bhk-21 cells and analyzed the rna synthesis at 48 h post-infection (hpi). a deletion that spanned nt 10423-10729 (δ307) abolished the viral replication (fig 2c, lane 3) , while the deletion of the au-rich region (δau-rich) or the 5'-stemloop (δ5'-sl) slightly delayed viral replication but had no lasting effect on the viral replication overall or on the sfrna accumulation (fig 2c, lanes 4-5 and table 1 ). genome cyclization has been shown to play an important role in viral replication [33] . beside the canonical 3'-cyclization (3'cyc) motif (cagcauauuga) at 10863-10873 that is complementary to 5'cs (ucaauaugcug) at nt 136-146, we found another motif located at the initiation site of jev sfrna that is very similar to the 3'cyc motif (named cyc-like motif), the sequence of which is caugcauaugga, where the mismatches with the 3'cyc motif are underlined). however, deletion of the cyc-like motif had no effect on the synthesis of the sfrna and the genome (fig 2c, lane 6) , whereas deletion of the entire slii abolished the production of sfrna (fig 2c, lane 7) . meanwhile, deletion of the lower left stemloop of the slii (δslii.1), the upper stemloop (δslii.2) or the lower left stem (δslii.3) all impaired sfrna synthesis (fig 2c, lanes 8-10) , indicating that the slii structure is essential for sfrna accumulation. three point mutations disrupted base pairing of the first pseudoknot at the 3' utr (pk1' or pk1") prevented sfrna formation (fig 2c, lanes 11-12) , while compensatory changes restoring the pk pairing (pk1'1") recovered the sfrna synthesis ( fig 2c, lane 13) suggesting that pseudoknot structure is also required for the sfrna formation. to further demonstrate that the production of sfrna requires the pk structure but does not rely on the activity of exoribonuclease xrn1, pk1'1" and pk1" mutants were used to infect wt or xrn1-kd hek293t cells, and then the resulting effects were compared. disruption of the pk structure (pk1") abolished the sfrna production in wt as well as in xrn1-kd cells, while compensatory changes that maintained the pk structure (pk1'1") made no difference in the production of sfrna in xrn1-kd cells or in wt cells (fig 2d) , indicating that the requirement of the pk structure for the production of sfrna does not rely on the activity of exoribonuclease xrn1. all mutations made at the slii region apparently resulted in relatively smaller plaques ( fig 2e) , suggesting that the structure or sequences of the slii might play a role in determining the plaque size. viral titers measured at 12-h intervals showed about 2-to 120-fold reductions in comparison with the wt control for all the mutants except for the slii deletion mutant, which exhibited slow growth at earlier time points but eventually reached to the wt-virus growth level at 72 hpi ( fig 2f) . mutations resulted in the reduction of virus titers, suggesting that these mutations delayed virus maturation and release rather than affecting viral replication because the rna levels of mutants measured at 48 hpi by northern hybridization ( fig 2c) did not show apparent differences in comparison with the wt control. to test whether the formation of jev sfrna requires viral replication, the replication-deficient mutant, which had the essential polymerase motif gdd mutated (ns5mt), was compared to the wild-type (wt) infectious clone. the sfrna in wt dna-transfected cells were visible at 24 hpt and continued to increase to a very high level at 72 hpt (fig 3, lanes 3-6) . the ns5mt clone, on the other hand, completely abolished the viral genome replication while barely any sfrna accumulated, indicating that the formation of the jev sfrna correlates with viral replication (fig 3, lanes 7-10) . although efficient rna replication is required for the detection of any flaviviral rnas despite which mechanism used for the sfrna formation, our results were clearly different from the observations from wnv that bhk-21 cells transfected with replicon constructs containing various deletions had no effect on the accumulation of sfrna when compared to the wt [8] . to explore the possibility that the jev sfrna could be a transcription product, we assumed that its promoter would be located on the antigenome adjacent to the 5' initiation of the sfrna. to test whether viral rdrp binds to the putative promoter and initiates sfrna synthesis, we cloned the cdna fragment corresponding to the nucleotide at position 10431-10566 of the genome, and performed emsa experiments using the minus-strand rna as the riboprobe. purified rdrp protein (0.25, 0.5, 1, and 2.5 μm) retarded the migration of the (-)10431-10566 rna in a dose-dependent manner, whereas no band shift was observed with non-specific rna or bsa control (fig 4a) . it should be noted that the additional band above the rna/protein complex (rpc) (fig 4a, lanes 1-3) is inversely correlated to the occurring of rpc suggesting that the highly ordered rna secondary structure may have intermolecular interactions when separated by non-denaturing gel electrophoresis. this band is an indirect evidence of rdrp binding at this region. the intermolecular interactions were disrupted with increasing amounts of rdrp (fig 4a, left panel) . in contrast, the band remained the same when incubated with increasing amounts of bsa control (fig 4a, right panel) . competition assays were performed to test the specificity of the interaction between the rdrp and the (-) 10431-10566 rna. increasing amounts of unlabeled rna out competed the rpc (fig 4b, lanes 3-5) . in contrast, neither trna (lanes 6-8) nor nonspecific rna (lanes 9-11) competed away the rpc. these results suggested that jev rdrp binds specifically to the (-)10431-10566 rna. studies have shown that the first 160 nucleotides containing the 5' utr and 5'cs of the plus-strand rna in denv [34] and wnv [35] contain promoter activity. we first tested the enzymatic activity of purified recombinant jev rdrp using (+)1-160 rna as a template for an in vitro rdrp assay. the result showed that the (+)1-160 rna is a functional template for rna synthesis whereas the 5' 226-nt rna from bovine coronavirus (as a nonspecific template control) could not direct rna synthesis, nor the reaction without adding the rdrp (fig 4c) , indicating that the purified recombinant jev rdrp has specific enzymatic activity. we then used the (-)10431-10566 rna as the template for the rdrp assay. two major synthetic products were detected, the larger band with 136 nt in length same as the size marker driven by t7 rna polymerase, and the smaller band with 113 nts in length presumably a transcription product initiated from the putative promoter (fig 4d, lane 7) . we then constructed various templates for in vitro rdrp assays (fig 4e and 4f and s1 table) . interestingly, all constructs showed a de novo synthesis product same as the template size and a smaller band with approximately 113 nts in length except for the (-)10454-10566 rna (fig 4d and 4e ). although some nonspecific bands were observed, these results indicated that the recombinant jev rdrp could recognize the promoter and synthesize the corresponding sequences. several studies have demonstrated that the accumulation of the sfrna results from incomplete degradation of viral genome by host exoribonuclease xrn1 [24] [25] [26] . the crystal structure of the xrrnas in the 3' utr that resist the degradation process has been determined [7, 23] , and the function of the stalling has been studied in several flaviviruses [26, [36] [37] [38] . although stalling of xrn1 by the jev sfrna has been reported previously [14] , detailed mechanism of the sfrna formation has not yet been elucidated. the results of this study showed that either knocking down or knocking out of xrn1 gave rise to more sfrna accumulation in virusinfected cells (fig 1a, 1b and 1d ). it has also been reported that xrn1 could be redundant with other exonucleases in producing zika virus sfrnas [7] . these controversial results may be explained by several reasons including (i) exonucleases other than xrn1 can contribute to sfrna formation, (ii) depletion of xrn1 causes changes in total cellular rna level that may include products of endonucleolytic cleavage, and (iii) reduction in xrn1 may contribute to differences in susceptibility to viral infection [7] . although we cannot vehemently rule out these possibilities, theoretically, exoribonuclease xrn1 depletion should reduce the production of the sfrna, resulting in a reduction of the molar ratio of the sfrna to the genome, yet an increase rather than a decrease in molar ratio was observed. furthermore, no intermediate degradation byproducts from viral genome to the sfrna were detected during infection even in the xrn1-deficient cells (fig 1a-1d) . morevoer, several unsolved mysteries including the unreasonably high rate of nonsense-mediated decay to generate the sfrna, the question of how the viral genome within the membrane-protected complex is subjected to rna degradation, and the lack of economy in degradation from the full-length genome from an evolutionary point of view, led us to reconsider other possible mechanisms of the jev sfrna formation. mutation of the gdd motif of rdrp in jev halted the accumulation of sfrna (fig 3) , suggesting that abundant accumulation of sfrna depends on viral replication. by transfecting replicon constructs containing various deletions pijlman et al. demonstrated that sfrna does not require viral rna replication, viral proteins, or 5' utr since all deletion mutants showed similar levels of sfrna accumulation [8] . the sfrna was also detected in bhk-21 cells electroporated with nonreplicating replicon denv-2 containing the catalytic gdd motif of the ns5 replaced with aaa [20] . the initial rna transcripts either from replicon or infectious clone are all driven by cmv promoter. if formation of the jev sfrna were not required for rna replication, the accumulation of sfrna could be detected in the gdd-mutant infected cells. although efficient rna replication is required for the detection of any flaviviral rnas no matter the sfrna is made either by degradation or by transcription, our results are clearly different from those of previous studies of the wnv and denv. although the wnv and jev belong to the same serotype, different viruses may have evolved to have different strategies for survival in infected cells. it is intriguing that, according to in vitro rdrp assay results, the ns5 protein binds to the putative promoter and produces a de novo synthesis product and a presumable transcription product of approximately 113 nts in length (fig 4) . we attempted to clarify the initiation site of the rdrp products by applying 5' rapid amplification of cdna ends (5' race) assays but failed to observe any specific sequences due to low amount of the radiolabeled products. efforts had also been made to demonstrate that the putative promoter is functional in infected cells by transfecting rna construct containing unrelated sequences under the control of the promoter. unfortunately, our numerous attempts to make such a construct failed probably because the transfected molecule may not be accessible as a template to the viral rdrp at the right place and right time. it has been shown that the recombinant jev ns5 exhibited a weak terminal transferase activity only when substrate ribonucleotides were limited [39] . sufficient amounts of ribonucleotides were supplied in the in vitro rdrp assay (fig 4c and 4d) , thus the products are least likely resulting from the terminal transferase activity. furthermore, in vitro digestion of the 3' utr containing fragment (a 800-nt rna) by xrn1 revealed that xrn1 could stall at a position to generate the sfrna (fig 1e) . these results let us to propose the model that the jev sfrna is likely made by transcription initially and then could be further trimmed by xrn1 or other unidentified exoribonucleases (fig 5) . several sequences and structures were determined to define the cis-acting elements for sfrna formation. it has been shown in previous studies with wnv that the deletion of the 5' half of the 3' utr does not affect the genome replication but does halt the sfrna formation [8] . however, a similar deletion at the 3' utr of jev (δ307) totally killed virus replication ( fig 2c, lane 3) . whether a partial deletion of the dumbbell structure resulted in the lethal phenotype remains to be determined. it has been suggested that the subgenomic rna promoter sequences often form a stem-loop structure, which is proposed to facilitate the interaction with the transcriptase as well as other host factors [40] . we found that deletion of the entire or any part of the slii all impaired sfrna synthesis (fig 2c, lanes 7-10) , indicating that the slii structure is essential for sfrna accumulation and could be a potential promoter for the sfrna synthesis. furthermore, pseudoknot structure on slii is required for the formation of sfrna in wnv, yellow fever virus (yfv), and zikv [7, 24, 25] . our result also showed that pseudoknot structure is essential for the formation of sfrna in jev (fig 2c, lanes 11-13) . interestingly, mutations made at the slii region showed relatively smaller plaques and delayed viral maturation or release (fig 2e and 2f ). this observation may correlate with the report that sfrna-deficient wnv displays significantly decreased infection and transmission rates in mosquitoes [21] . our results revealed from in vivo mutagenesis studies (fig 2) and in vitro rdrp assays ( fig 4) are quite consistent except for the template nt 10454-10566. this template contains the entire slii region that is essential for the formation of sfrna in vivo (fig 2) but has no product by the in vitro rdrp assay (fig 4d) . this difference may due to that there is not enough space on the template for rdrp binding because rdrp is considered as a large protein so both de novo products and transcripts vanished when performed the in vitro rdrp assay (fig 4d, lane 6) . deletion of the slii in wnv led to downstream xrn1 stalling and the accumulation of smaller sfrna species (sfrna2, sfrna3 and sfrna4) [8, 25] . previous studies of yfv have shown that the sfrna2 is indeed truncated at the 3'-end [24] . it has been reported that the sfrna2 only exists in certain isolates of the denv-2 [6] . filomatori et al. reported that the accumulation of different species of sfrnas in denv-2-infected vertebrate and invertebrate cells due to selective pressures act on the 3' utr during host adaption [41] . although smaller sfrna species were found in jev, sequences of these rnas have not yet been determined. nevertheless, deletion mutants made on the slii structure abolished all sfrna formation but did not give rise to smaller rna species (fig 2c) . it is still unclear whether the downstream sliv or the dumbbell structure on jev genome plays a role in stalling of the xrn1. the mechanism for the formation of smaller jev rna species needs to be further characterized. genome cyclization has been shown to play an essential role in the replication of flaviviruses (for review see [33] ). a promoter element known as stemloop a (sla) at the 5' end of the genome has been well studied with regard to its function in denv and wnv [34, 35, 42] . the initiation of minus-strand rna synthesis includes the rdrp binding at the sla at the 5' end of the genome and the relocation of the rdrp at the 3' initiation site by long-range rna-rna interactions [34, 43, 44] . in this study, we identified the slii that showed promoter activity as good as the sla promoter (figs 2 and 4) . we also found that the initiation site of jev sfrna located at a 3'-cyclization-like motif. this cyc-like motif complementary to the 5' cyclization sequences may be involved in genome cyclization during antigenome synthesis and may cause template switching to occur, as reported by maeda et al. [45] . however, deletion of the cyc-like motif had no effect on sfrna accumulation in vivo (fig 2c, lane 6) . a potential role of this cyc-like motif remains to be studied. although viruses in the same family share many common strategies for completion of their life cycles, some differences in those strategies make certain viruses unique within the given family. in the present study, we provide evidence that the jev sfrna is likely synthesized by transcription and could be further trimmed by exoribonuclease xrn1 and/or other unidentified enzymes (fig 5) . the slii structure is responsible for sfrna synthesis. our observations expand the current knowledge about the mechanism of sfrna formation in jev-infected cells. supporting information s1 japanese encephalitis virus: from genome to infectome. microbes and infection rna structure duplications and flavivirus host adaptation specific requirements for elements of the 5' and 3' terminal regions in flavivirus rna synthesis and viral replication the 5' and 3' untranslated regions of the flaviviral genome 3' cis-acting elements that contribute to the competence and efficiency of japanese encephalitis virus genome replication: functional importance of sequence duplications, deletions, and substitutions identification and characterization of small sub-genomic rnas in dengue 1-4 virus-infected cell cultures and tissues zika virus produces noncoding rnas using a multi-pseudoknot structure that confounds a cellular exonuclease a highly structured, nuclease-resistant, noncoding rna produced by flaviviruses is required for pathogenicity accumulation of a 3'-terminal genome fragment in japanese encephalitis virus-infected mammalian and mosquito cells inhibition of type i interferon induction and signalling by mosquito-borne flaviviruses functional rna during zika virus infection small noncoding rna modulates japanese encephalitis virus replication and translation in trans a noncoding rna produced by arthropod-borne flaviviruses inhibits the cellular exoribonuclease xrn1 and alters host mrna stability xrn1 stalling in the 5' utr of hepatitis c virus and bovine viral diarrhea virus is associated with dysregulated host mrna stability japanese encephalitis virus non-coding rna inhibits activation of interferon by blocking nuclear translocation of interferon regulatory factor 3 west nile virus noncoding subgenomic rna contributes to viral evasion of the type i interferon-mediated antiviral response dengue subgenomic rna binds trim25 to inhibit interferon expression for epidemiological fitness noncoding flavivirus rna displays rna interference suppressor activity in insect and mammalian cells flavivirus sfrna suppresses antiviral rna interference in cultured cells and mosquitoes and directly interacts with the rnai machinery. virology dengue virus subgenomic rna induces apoptosis through the bcl-2-mediated pi3k/akt signaling pathway noncoding subgenomic flavivirus rna is processed by the mosquito rna interference machinery and determines west nile virus transmission by culex pipiens mosquitoes dengue subgenomic flaviviral rna disrupts immunity in mosquito salivary glands to increase virus transmission the structural basis of pathogenic subgenomic flavivirus rna (sfrna) production an rna pseudoknot is required for production of yellow fever virus subgenomic rna by the host nuclease xrn1 rna structures required for production of subgenomic flavivirus rna rna structures that resist degradation by xrn1 produce a pathogenic dengue virus rna the endoplasmic reticulum provides the membrane platform for biogenesis of the flavivirus replication complex sars-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro architecture of the flaviviral replication complex. protease, nuclease, and detergents reveal encasement within double-layered membrane compartments opposing roles of double-stranded rna effector pathways and viral defense proteins revealed with crispr-cas9 knockout cell lines and vaccinia virus mutants a novel approach to propagate flavivirus infectious cdna clones in bacteria by introducing tandem repeat sequences upstream of virus genome defective interfering rnas of japanese encephalitis virus found in mosquito cells and correlation with persistent infection genome cyclization as strategy for flavivirus rna replication a 5' rna element promotes dengue virus rna synthesis on a circular genome rna elements within the 5' untranslated region of the west nile virus genome are critical for rna synthesis and virus replication new hypotheses derived from the structure of a flaviviral xrn1-resistant rna: conservation, folding, and host adaptation functional non-coding rnas derived from the flavivirus 3' untranslated region noncoding subgenomic flavivirus rna: multiple functions in west nile virus pathogenesis and modulation of host responses biochemical characterization of a recombinant japanese encephalitis virus rna-dependent rna polymerase analyses of subgenomic promoters of hibiscus chlorotic ringspot virus and demonstration of 5' untranslated region and 3'-terminal sequences functioning as subgenomic promoters dengue virus genomic variation associated with mosquito adaptation defines the pattern of viral non-coding rnas and fitness in human cells functional rna elements in the dengue virus genome terminal structures of west nile virus genomic rna and their interactions with viral ns5 protein we thank dr. bernard moss for kindly providing the a549 xrn1 knock-out cells and dr. muthukumar nadar and dr. yu-pin su for constructive comments on this manuscript. conceptualization: yi-shiuan chen, yi-hsin fan, ruey-yi chang.investigation: yi-shiuan chen, yi-hsin fan, chih-feng tien. writing -original draft: yi-shiuan chen, yi-hsin fan, ruey-yi chang. yi-shiuan chen, yi-hsin fan, ruey-yi chang. key: cord-289017-vwye3pk9 authors: comach, guillermo; teneza-mora, nimfa; kochel, tadeusz j.; espino, carlos; sierra, gloria; camacho, daria e.; laguna-torres, v. alberto; garcia, josefina; chauca, gloria; gamero, maria e.; sovero, merly; bordones, slave; villalobos, iris; melchor, angel; halsey, eric s. title: sentinel surveillance of influenza-like illness in two hospitals in maracay, venezuela: 2006–2010 date: 2012-09-11 journal: plos one doi: 10.1371/journal.pone.0044511 sha: doc_id: 289017 cord_uid: vwye3pk9 background: limited information exists on the epidemiology of acute febrile respiratory illnesses in tropical south american countries such as venezuela. the objective of the present study was to examine the epidemiology of influenza-like illness (ili) in two hospitals in maracay, venezuela. methodology/principal findings: we performed a prospective surveillance study of persons with ili who presented for care at two hospitals in maracay, venezuela, from october 2006 to december 2010. a respiratory specimen and clinical information were obtained from each participant. viral isolation and identification with immunofluorescent antibodies and molecular methods were employed to detect respiratory viruses such as adenovirus, influenza a and b, parainfluenza, and respiratory sincytial virus, among others. there were 916 participants in the study (median age: 17 years; range: 1 month – 86 years). viruses were identified in 143 (15.6%) subjects, and one participant was found to have a co-infection with more than one virus. influenza viruses, including pandemic h1n1 2009, were the most frequently detected pathogens, accounting for 67.4% (97/144) of the viruses detected. adenovirus (15/144), parainfluenza virus (13/144), and respiratory syncytial virus (11/144) were also important causes of ili in this study. pandemic h1n1 2009 virus became the most commonly isolated influenza virus during its initial appearance in 2009. two waves of the pandemic were observed: the first which peaked in august 2009 and the second higher than the preceding that peaked in october 2009. in 2010, influenza a/h3n2 re-emerged as the most predominant respiratory virus detected. conclusions/significance: influenza viruses were the most commonly detected viral organisms among patients with acute febrile respiratory illnesses presenting at two hospitals in maracay, venezuela. pandemic h1n1 2009 influenza virus did not completely replace other circulating influenza viruses during its initial appearance in 2009. seasonal influenza a/h3n2 was the most common influenza virus in the post-pandemic phase. acute respiratory infection (ari) remains a leading cause of global burden of disease, and is the second most common cause of illness worldwide, with an annual global incidence exceeding 400 million [1] [2] [3] . a prerequisite of public health planning to reduce global disease burden from ari is to examine data on its epidemiology in order to better define environmental factors as well as target populations for preventive interventions [4] . respiratory viruses are predominant causes of aris, and the epidemiology of acute viral respiratory illnesses in developed countries with temperate climates has been well-characterized [5] [6] [7] . in countries such as the united states, children have been shown to carry a large burden of viral respiratory diseases [5] . recent prospective studies, which utilized more sensitive methods for detecting respiratory viruses such as multiplex polymerase chain reaction (pcr), have similarly demonstrated that the highest rates of viral respiratory infection occur among children and the frequency of infection tends to decrease with age due to increasing acquired immunity [8] . respiratory syncytial virus (rsv), influenza virus, parainfluenza virus, and rhinovirus have long been identified as common causes of ari [9] . recent improvements in molecular detection techniques have allowed the identification of multiple new respiratory viruses such as human metapneumovirus (hmpv), human bocavirus (hbov) and human coronavirus nl63 [8] . while the body of literature describing the epidemiology of acute viral respiratory diseases in developed countries has rapidly expanded, knowledge of the distribution of these diseases in regions such as tropical south america remains limited. influenza viruses are among the most impactful acute respiratory pathogens in terms of morbidity and mortality. despite developed public health intervention programs, the estimated annual average number of influenza-related hospitalizations in the united states exceeds 200,000, and 36,000 deaths are attributable to influenza infections yearly [10, 11] . information on the contribution of influenza viruses to the global burden of disease due to acute respiratory illness is incomplete. data on the epidemiology of influenza viruses in developed countries are derived from multiple sources to include laboratory-based surveillance, sentinel surveillance, as well as hospitalization and outpatient records. in developing countries, where resources are sparse, sentinel surveillance methods may be more readily accessible and more cost-effective than laboratory-based or population-based surveillance for determining the viral etiology of influenza-like illness (ili) in these regions. better identification of the viral causes of ili will enable clinicians in resource-limited settings to appropriately treat and manage patients; more importantly, it will allow public health officials to formulate more effective prevention and control strategies, including monitoring of influenza vaccine efficacy in their communities [12] . studies on the epidemiology of ili in the tropical south and central american countries of peru, brazil, ecuador, nicaragua, honduras, and el salvador have been published [13] [14] [15] [16] . a prospective study of adults with ili in sao paulo, brazil, revealed that while influenza viruses were the predominant cause of ili, rhinoviruses and other respiratory viruses were detected in 19.6% and 13.7% of subjects, respectively [14] . this observation illustrated that a significant proportion of patients who are clinically diagnosed with influenza virus infection may have symptoms indistinguishable from other respiratory viruses. in a prospective study of ili in ecuador, the regional distribution of influenza virus infections varied; a higher detection rate of influenza a occurred in quito, located in the highlands where the level of absolute humidity is lower, whereas influenza a detection rate was lower in the coastal city of guayaquil, which has a more humid tropical climate [15] . additionally, an expanded sentinel surveillance of ili conducted at 31 health centers and hospitals located in 13 peruvian cities showed that the distribution of ili-causing viruses varied by region [13] . these studies illustrate the importance of conducting baseline and continuing ili surveillance in other countries of central and south america because ili can be caused by pathogens other than influenza viruses. the distribution of respiratory infections may vary within the region or within a particular country, depending on climate and topography. in venezuela, ari is the primary cause of weekly notifiable diseases registered by the national epidemiological surveillance system (ness) [17] . nonetheless, very low numbers of respiratory samples were collected (16,664 from 2006 to 2010) and even lower numbers (5, 167) were confirmed by the national system for virological surveillance of ari [17] [18] [19] [20] [21] . furthermore, the epidemiology, clinical characteristics, and viral causes of ili have been poorly characterized in venezuela; to our knowledge, only one longitudinal study, carried out during a limited period of time (february 2005-july 2006) and with a low number of patients (n = 102) with ari, has been published [22] . the objective of this paper is to describe the epidemiology of ili using data from a sentinel surveillance system at two major hospitals in maracay, venezuela. a total of 916 subjects participated in this study. six hundred and thirty seven (69.5%) were recruited at the hospital central de maracay (hcm). there was a slightly larger proportion of females compared with males who enrolled in the study (55.2% vs 44.8%), and the gender distributions at the two sites were similar. subject ages ranged from 1 month to 86 years with a mean age of 19.2 years (s.d. = 14.3 years) and a median of 17 years. a significant percentage of the subjects (17.6%) were under 5 years of age, while less than 1.0% were 60 years or older. the 15-29 year old group was the most predominant group comprising 32.8% of the study population, followed by the 5-14 year old age group (27.2%). the subjects who presented to hcm were significantly older (mean age = 21.2 years; s.d. = 13.2 years) compared with those who were seen at the hospital del instituto venezolano de los seguros sociales jose maria carabano tosta (ivss-jmct) (mean age = 14.4 years; s.d. = 15.6 years). a large proportion (37.8%) of the subjects at hcm belonged to the 15-29 age group, while nearly half of the patients (42.3%) seen at ivss-jmct were younger than 5 years old. approximately 5% of the subjects reported having received influenza vaccination within the last year. a small percentage of patients (2.0%) received antibiotic treatment for their acute respiratory illness prior to enrollment in the study. no hospitalized cases with ili were recruited in this study; only outpatient subjects participated. demographic information is summarized in table 1 . among the 916 patient samples obtained from the sentinel surveillance, at least one viral organism was detected by viral isolation and/or reverse transcriptase-polymerase chain reaction (rt-pcr) in 143 (15.6%) subjects ( table 2 ). only one participant with a co-infection was observed. influenza viruses were the most frequently detected organisms in this passive surveillance, consisting of 67.4% (97/144) of all the viruses detected by viral isolation and/or rt-pcr. of the 916 samples collected, 78 (8.5%) were positive for influenza a viruses and 19 (2.1%) for influenza b viruses by viral isolation and/or rt-pcr. in addition, noninfluenza viruses were detected only by virus isolation; they were: adenovirus (1.6%), parainfluenza virus (1.4%), rsv (1.2%), and other viruses (0.8% ), which included hbov (0.1%), enterovirus (0.2%), hmpv (0.1%), rhinovirus (0.1%), and herpes simplex virus (hsv, 0.2%). the only co-infection was observed in a 22 month old toddler in whom adenovirus and hsv were isolated. the virus etiology and detection rates varied with age ( table 2 ). the most commonly detected viral pathogens in the 0-4 year old group were influenza a (11.1%) and adenoviruses (5.6%). influenza a and b were the most commonly detected viruses in the school-age group (5-14 year old: 6.8% and 4.0%, respectively) and the late adolescent and young adult group (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) year-old: 9.7% and 1.7%, respectively). influenza a was the only detectable virus among patients who were 60 years or older. of the 78 influenza a cases, h3 subtype was detected in 33, and was observed in all the age groups but in subjects aged 45-59 year old ( table 2) . six cases had the h1 subtype (non-pandemic), and 18 influenza a viruses were isolated but not subtyped by one-step rt-pcr; of the latter, 11 can be classified as seasonal because they were isolated from patients samples collected before the start of the h1n1 pandemic outbreak of 2009. twenty-one cases of pandemic h1n1 2009 influenza virus (ph1n1) infections were identified in this sentinel surveillance; thirteen occurred in the 15-29 year old group. there were no cases of ph1n1 infections among adults 45 years old or higher. genetic analysis based on partial hemagglutinin gene sequences (approximately 900 bp) of 27 influenza isolates is shown in figure 1 (see material and methods for genbank accession numbers). this analysis shows that the circulating seasonal a/h1n1 strains in venezuela ( figure 1a ) were similar to the ones previously described in central and south america [15, 16] and all of these samples can be grouped with the a/ brisbane/56/07 like 2008-2009 genotype. the ph1n1 samples were part of only one cluster similar to previously reported strains in latin america [23] . influenza a/h3n2 samples possessed more genetic variability ( figure 1b) ; isolates from 2007 revealed two genotypes, a/brisbane/10/07-like and a/ california/7/04-like, while the more recent isolates were closer to the a/perth/16/09-like genotype. finally, in figure 1c , the genetic analysis for influenza b isolates disclosed the presence of two different genotypes, b/florida4/06-like and b/malaysia/ 2506/07-like, in agreement to what was previously found in the region [13, 16] . adenovirus was detected more frequently from december to april. rsv infection occurred more frequently from june to november. parainfluenza viruses were detectable throughout the year but without distinct seasonality. the passive surveillance illustrated the impact of ph1n1 on the distribution of respiratory viruses associated with ili in 2009 ( figure 3 ). during the ili peaks from 2006-2010, the percentage of ili cases attributable to influenza viruses ranged from 10% to 44%. the first cases of ph1n1 in this surveillance system were detected in july 2009, three months after the swine origin influenza outbreak began in mexico [24] . during the first wave in july 2009, the monthly virus detection rate rose to 33%, and 57% (4/7) of all the viruses detected were of the pandemic strain. the first wave simulated a typical ili peak activity similar to those observed in other years. however, during the larger second wave in october 2009, the virus positivity rate exceeded 40%, and the pandemic strain was identified in greater than 73% (11/15) of the viruses detected. figure 3 demonstrates that ph1n1 did not completely replace seasonal influenza a viruses. the peak pandemic activity in october 2009 was followed by a rapid decline in the rate of pandemic strain detection one month later. meanwhile, seasonal influenza a viruses remained in circulation throughout the pandemic period comprising 27% of all the influenza a viruses detected in october 2009. in 2010, 20% of influenza a specimens obtained via oropharyngeal swabs were randomly selected and tested for the pandemic strain via rt-pcr, and no cases of ph1n1 were detected ( figure 3 ). however, seasonal influenza a/h3n2 continued to be detected by our surveillance system with monthly positivity rates ranging from 16% to 30%. the clinical features of subjects in our surveillance are summarized in table 3 . compared with those whose respiratory secretions tested negative, subjects in whom virus was identified were more likely to have sore throat, headache, pharyngeal congestion, and ear pain. there were no significant differences in the symptoms of individuals who had seasonal influenza a when compared with those who suffered from ph1n1 influenza, except that a higher proportion of the latter subjects had expectoration, myalgias, and lymphadenopathy. the symptoms of influenza a (either seasonal or pandemic) and influenza b were clinically indistinguishable. when compared with patients who had ili due to other viruses, a higher percentage of those with confirmed influenza virus infection experienced sore throat, myalgias, and headache. there is scarce information about the epidemiology of acute febrile respiratory illness in venezuela and, to our knowledge, only one longitudinal study has been published [22] . this investigation, however, was limited to few number of subjects (n = 102) recruited during a short period of time (17 months) and did not describe the transmission seasonality of the viral infections. thus, our study is the first to fully describe the epidemiology and viral etiology of ili in venezuela and provides baseline levels of ili activity in a typical highly-populated urban city. our study demonstrated that influenza viruses are a main cause of ili at hcm and ivss in maracay in agreement with findings reported by venezuelas ness [17] [18] [19] [20] [21] . nevertheless, the rate of confirmed influenza virus infections found in our surveillance (10.6%, 97/916; tables 2 and 3) during the study period was lower than the one (27.7%) reported by the ness for the same period [17] [18] [19] [20] [21] . these differences in detection rates may be attributable to different strategies for capturing ari patients, especially those with influenza, used by the ness and by this study protocol (flores e, director of epidemiology, corposalud 2011, personal communication). the ness randomly selected a sample of ari cases (including those with influenza) with emphasis on severe hospitalized cases, whereas in our protocol we recruited ili subjects in an outpatient setting where the majority had symptoms that were not severe. on the other hand, the percentage of influenza viruses (not including ph1n1) detected in our study during a similar period of time, but in different years accounted for the significant differences found in both studies: a) the collection, preservation and further processing of respiratory samples, and b) the type of cells and ifa reagents used for virus isolation and identification. the proportion of influenza cases was significantly different when comparing non-pandemic and pandemic periods. before the h1n1 2009 pandemic, the ness [21] detected influenza virus in 64.7% of subjects in whom a virus was isolated; a similar proportion to the 55% (data not shown) found in our study. during the height of the pandemic (from july through dec 2009), the ness confirmed 97% of all ari cases with a virus as having either influenza a or b virus compared with 87% observed in our study. the predominance of influenza viruses as etiological agents of ili in maracay, venezuela, is consistent with observations of surveillance studies in other tropical central and south american countries [13, 15, 16] . while the overall virus detection rate (143/ 916, 15.6%; table 2 ) was lower compared with other studies, the positive rates for influenza a (8.5%) and influenza b (2.1%) in this surveillance were comparable to those observed in a similar study in el salvador, honduras, and nicaragua [16] . our findings were also consistent with a study in indonesia, which identified influenza a or b in 11% of all respiratory samples using viral isolation and rt-pcr [25] . our findings were consistent with those from a prospective study of outpatient children in northern taiwan, in which influenza a or b were isolated in 12.2% of subjects with ili, using madin-darby canine kidney (mdck) cell cultures and hemagglutinin inhibition assay for antigen detection [26] . however, our detection rate for all respiratory viruses, as well as influenza a and b viruses, was generally lower compared to other studies conducted in south america. in an expanded sentinel surveillance study in peru, which utilized a similar methodology for viral detection as our study, the virus positivity rate was 42.1%, and influenza a and b rates were 25.1% and 9.7%, respectively [13] . in a prospective surveillance study of ili in two ecuadorian cities using similar methods, at least one virus was detected in 35% of the participants; influenza a was detected by pcr in 21.6% while 6.4% tested positive for influenza b [15] . the lower virus detection rates found in our study is unclear because we used the same operative protocol described in the mentioned studies [13, 15] . nevertheless, the different skills to collect respiratory tract specimens from ili patients, by the health personnel employed in their studies and ours may have accounted for the lower virus detection rates found in our study. while influenza viruses were observed to be the most prevalent viral pathogen, adenovirus, parainfluenza virus, and rsv were important causes of ili at the two hospitals (table 2 ). in venezuela, the ness reported rsv as the most frequently detected non-influenza respiratory virus followed by parainfluenza virus and rhinovirus [17] [18] [19] [20] [21] . the study performed in zulia, venezuela, also reported rsv as the most common detected virus, followed by adenovirus, parainfluenza virus and influenza viruses [22] . as previously mentioned, the differences in the detection rates may be attributed to the different procedures used by the ness, other venezuelan researchers. [22] and us. these viral pathogens were also frequently detected in other surveillance studies in central and south america [14] [15] [16] . the highest detection rate of respiratory viruses was observed in the 0-4 year old group (29.2%; table 2 ). the rate of viral positivity generally decreased with age as acquired immunity increased in older subjects. a slight increase can be seen between the 30-44 year old group (14.1%) and 60 years or older group (14.3%). subjects who were 60 years old or older comprised less than 1% of the study population, and thus, the prevalence in this age group may be unreliable. contrary to our study, the other study from. venezuela had higher detection rates in both the 0-6 and $41 year old groups (48.2% and 57.1%, respectively) [22] . on the other hand, our findings are consistent with observations in the tecumseh study, a community-based surveillance in michigan, in which children were shown to have the highest rate of viral respiratory diseases [5, 6] . the tecumseh study further illustrated the variable impact of influenza viruses among the different age groups depending on the influenza virus subtype. for example, influenza a/h3n2 affected a wide range of age groups while influenza a/h1n1 and influenza b virus infections occurred more frequently among older children and young adults [5, 6] . our findings were similar being seasonal influenza a/h1n1 viruses and influenza b detected primarily in children and young adults, and seasonal influenza a/h3n2 found in all age groups. as expected, ph1n1 was the most common influenza a subtype identified among the subjects with ili in 2009 ( figure 3 ). in 2009, 37.5% (figure 3 ) of ili cases were due to ph1n1; this detection rate was lower than the 52.2% detection rate reported by the venezuela's ness [21] . nevertheless, this finding was similarly demonstrated in respiratory illness surveillance networks in other tropical and temperate south american countries such as guatemala [27] , peru [28] , argentina [29] , and brazil [30] . in 2010, non-pandemic influenza viruses continued to circulate in venezuela, and ph1n1 was not detected in our surveillance study, suggesting that ph1n1 did not displace seasonal influenza a viruses. during the same year, 1.6% of the ari cases reported to venezuela's ness were due to ph1n1 [21] . infection with ph1n1 may have stimulated immunity among the residents of this community during its initial arrival in 2009. this observation suggests that the circulating ph1n1 in 2010 may not have significantly mutated relative to the strain in 2009, so that antibodies stimulated by the natural infection during its initial arrival may have still been highly efficient in protecting the influenza-like illness in maracay, venezuela plos one | www.plosone.org community from another wave of ph1n1 outbreak. in 2010, the infection rate of influenza a/h3 was higher than the rates observed during previous years, further illustrating the lack of cross protection between ph1n1 and influenza a/h3. our study shows that hmpv and hbov were not commonly associated with ili, based on the low detection rates observed in our surveillance. in a study from ecuador which utilized methods similar to those employed in our study, a low detection rate for hbov (0.2%) was reported [15] . the latter study, as well as another from central america which used methods similar to ours, reported rare detection of hmpv (,0.2%) [15, 16] . in contrast, a prospective study of ili among brazilian adults, which utilized viral isolation and rt-pcr testing on respiratory samples, detected rhinoviruses in 19.6% of patients [14] . although rhinoviruses are typically associated with milder illness, they can contribute to the misdiagnosis of influenza based on clinical case definition alone. a cohort study of vietnamese children hospitalized for acute febrile respiratory illness, which applied multiplex-pcr assays on respiratory samples, revealed slightly higher prevalence rates of hbov (2%) and hmpv (5%) infections [31] . it is important to note that our method of identification (culture on three cell lines), compared to molecular diagnostic methods, substantially lacked sensitivity for detecting rhinoviruses, hmpvs, and hbovs. our study shows that patients with ph1n1 infections were more likely to have myalgias, productive cough with expectoration, and lymphadenopathy than with those infected with seasonal influenza a virus (table 3 ). in contrast, clinical manifestations in guatemalan subjects hospitalized for ph1n1 and seasonal influenza a infections did not significantly differ [27] . our study had limitations worth noting. data collection at only two hospitals in an urban area limits our ability to generalize our findings to the population. the low virus detection rates may be attributable to variations in the skills of the health staff employed to collect the respiratory specimens. the sampling method may have a significant effect on the proportion of respiratory viruses identified, and nasopharyngeal washes may yield higher sensitivity over nasopharyngeal or oropharyngeal swabs [32] . study participants were exclusively seen in the outpatient setting, thus limiting our ability to examine the impact of respiratory viruses in hospitalized cases. a large proportion of ili cases were not associated with any pathogen, and the impact of bacteria on this clinical syndrome cannot be determined from this study, since the respiratory samples were not cultured for bacteria. two methods of detection were used for identification of influenza (pcr and culture) whereas only one method of detection was used for the other viruses (culture). twenty-two percent (9/40) of the respiratory samples which were positive for influenza viruses by rt-pcr were negative by viral isolation illustrating that viral detection by culture underestimated the true prevalence. viral culture may not be the ideal way of isolating organisms such as rsv, hmpv, hbov and rhinoviruses leading to significant underestimation of their detection rates [33] . despite these limitations, our study contributes information on the distribution and etiology of ili at two hospitals in maracay, venezuela. this knowledge can serve as a baseline for future, more expansive population-based surveillance studies of influenza and other respiratory viruses in this region. maracay is located in the central northern region of venezuela (10u 159 n, 67u 399 w) , approximately 27 miles from the caribbean coast (figure 4) . the climate is tropical with two seasons: dry (december-april) and rainy (may-november). the monthly averages for temperature, relative humidity and rainfall are 25.1uc (range = 23.4uc-27.6uc), 75% (range = 66%-82%) and 56 mm (range: 0 mm-187 mm), respectively. maracay is comprised of two main urban municipalities, named girardot and mario briceñ o iragorry, and had an estimated total population of 677,359 in 2010. the hcm and the ivss-jmct are located approximately 4 miles apart. both are major county hospitals and referral health centers with adult and pediatric departments including emergency services and 241 (ivss-jmct) to 433 (hcm) hospitalization beds. they provide services to people in maracay as well as those from the adjacent city of aragua and three neighboring states. the study population included every outpatient with ili, regardless of age, who sought attention at the two study sites between october 2006 and december 2010, and agreed to participate in the study. at each site, trained medical personnel were responsible for properly identifying and classifying patients with ili. each person with ili was asked to enroll in the study. a person was defined as having ili if he or she had a sudden onset of fever ($38uc) and either cough or sore throat for less than five days in duration, with or without general symptoms such as myalgias, prostration, headache, or malaise [13] . data on gender, age, previous treatments, medical attention before enrollment, influenza vaccination status, and days of work/ school lost at the time of acute illness were collected utilizing a case report form (crf) from all participants who met the case definition criteria. temporal distribution of the results were recorded by month during the study period, taking into account the number of ili cases identified and the number of confirmed cases of influenza a and b in each study site. monthly reports of enrolled ili participants and laboratory results were sent to the venezuelan ministry of health. regular personnel training in protocol procedures were conducted as part of the strategy to improve sampling, storage,and shipping procedures. this protocol was approved as less than minimal risk research by the naval medical research center (nmrc), silver spring, maryland. institutional review board (irb; protocol nmrcd.2002.0019) authorization was given to perform the study using an information sheet approved and stamped by the irb. as this was part of clinical care and routine surveillance benefiting the ministry of health, verbal consent was obtained from all participants. this method of consent was accepted by the nmrc irb as well as the venezuelan institutions involved. a verbal consent was approved by both irbs following cioms and 45cfr46 (the common rule), 1) it was a minimal risk study, and 2) local health installations would not require a written consent for the procedures required in this study. additionally, this document included all the information that a written consent would require; a copy was provided to each study subject; and study personnel responsible for administering the process of informed consent were trained at each site on human research protection issues and were certified through the collaborative institutional training initiative (citi). finally, the frequent monitoring visits conducted by the study team found no problems in the process nor complaints from study participants. sample collection. nasal (807 of 916, 88.1%) or oropharyngeal (109 of 916, 11.9%) swabs were obtained from each subject for viral isolation and identification. the swabs were placed in viral transport media and stored at -70uc until they were delivered on dry ice to namru-6 in lima, perú, for laboratory analysis. duplicate swabs were processed and analyzed at the laboratorio regional de diagnostico e investigacion del dengue y otras enfermedades virales/instituto de investigaciones biomedicas de la universidad de carabobo (lardidev/ biomed-uc) in maracay, venezuela, following the same operative protocols used in namru-6. virus isolation and identification. nine-hundred and sixteen nasal or oropharyngeal swabs were processed for viral isolation and identification following the procedure described by laguna-torres et al. [13] . briefly, patient specimens were inoculated onto four cell lines: madin-darby canine kidney (mdck; atcch number ccl-34), african green monkey kidney (vero76; atcch numbercrl-1587) and veroe6 atcch number crl-1586), and rhesus monkey kidney (llc-mk2 atcch number ccl-7). upon the appearance of cytopathic effect or after ten days of culture (or thirteen days in the case of vero cells), the cells were spotted onto microscope slides. cell suspensions were dried and fixed in chilled acetone for 15 minutes. virus isolates were identified using direct fluorescence antibody (dfa) assays. the respiratory virus screening and identification kit (d3 dfa respiratory virus diagnostic hybrids; athens, oh) was utilized for the identification of adenoviruses, influenza a virus, influenza b virus, parainfluenza viruses (types 1, 2, and 3), and rsv. the d3 dfa herpes simplex virus (hsv) identification kit and the d3 ifa enterovirus id kit (diagnostic hybrids; athens, oh) were utilized for the identification of hsv (both hsv-1 and hsv-2) and enteroviruses, respectively. for isolation of hmpv, we used vero e6 and llc-mk2 cell lines. for detection of hmpv antigens by direct fluorescence assay, we used an anti-hmpv mouse monoclonal antibody from diagnostic hybrid (athens, oh). all assays were performed following the manufacturers' instructions. hbov was identified using the methods described by salmon-mulanovich, et al [34] . since duplicate swab samples were analyzed in different laboratories (lardidev/biomed-uc and namru-6) with the same diagnostic kit and standard operating procedures, a virus isolation result was considered positive if the specific virus was isolated and identified at either site. a subset of 254 specimens (222 nasals and 32 oropharyngeals) was analyzed by one-step rt-pcr and/or real time rt-pcr in order to sub-type influenza a/h1n1 (including ph1n1), influenza a/h3n2, and influenza b viruses. one-step rt-pcr and/or real time rt-pcr were not used to identify noninfluenza viruses because the specific primers and protocols were available only for sub-typing influenza viruses; thus, only virus isolation was used to detect and identify non-influenza viruses. of the 254 specimens, 48 (18.9%) were randomly selected from repository samples collected before the 2009 pandemic and tested for influenza a sub-typing by one-step rt-pcr and/or real time rt-pcr. at the onset of the 2009 pandemic, 150 of 254 (59.1%) respiratory samples were tested for ph1n1 by real time rt-pcr at the request of the venezuelan ministry of health. after the peak of the second wave of the pandemic, the percentage of respiratory samples tested by real time rt-pcr was reduced to 22% (56 of 254). one-step rt-pcr was performed according the procedure and influenza primers described below. real time rt-pcr was these protocols are available from cdc upon request. for the purpose of this study, an ili case with a confirmed viral respiratory infection was one in which viral isolation and/or rt-pcr identified a virus. rna extraction and one-step rt-pcr. viral rna extraction was performed from the supernatant of infected mdck cells using a qiaamp viral rna kit (qiagen; valencia, ca) following the manufacturer's protocol. the onestep rt-pcr was performed following a procedure described previously [13] with primers that amplified the hemagglutinin (ha) gene of influenza a and influenza b viruses using the superscript iii one-step rt-pcr system kit (invitrogen; san diego, ca). the following primers were used for the amplification of h1 influenza a viruses: h1f-6 (59-aagcaggggaaaa-taaaa-39) and h1r-1193 (59-gtaatcccgttaatggca-39); for h3 influenza a viruses: h3f-7 (59-actat-cattgctttgagc-39) and h3r-1184 (59-atggctgctt-gagtgctt-39); for influenza b viruses: bhaf-36 (59-gaagg-caataattgtact-39) and bhar-1140 (59-accagcaatagctccgaa-39). five ml of the extracted rna was added to 20 ml of master mix containing the enzyme mixture (superscript iii rt/platinum taq), 2x reaction mixture (containing 0.4 mm of each dntp and 3.2 mm of mg 2 so4) and 20 mm of each primer. cycling conditions included a reverse transcription step at 50uc for 30 minutes and a denaturation step at 94uc for 2 minutes. cycling conditions of the pcr were 40 cycles of 94uc for 15 seconds, 52uc for 30 seconds, and 68uc for 75 seconds, followed by a final incubation step at 68uc for 5 minutes. dna sequencing and phylogenetic analysis. for confirmation of serotype and genotype of the circulating influenza viruses, 27 samples were sequenced. as part of the respiratory surveillance protocol in venezuela, these 27 viruses were randomly selected from approximately 10% of the positive samples. only the ha gene region of influenza viruses was analyzed routinely for genotyping the one-rt-pcr products amplified with the primers described before were purified using centri-sep columns (princeton separation; englishtown, nj) and sequenced using the bigdye terminator v. 3.1 cycle sequencing kit (applied biosystems; foster city, ca) following the manufacturers' instructions. sequences were analyzed and edited using the sequencer 4.8 software (applied biosystems; foster city, ca). phylogenetic trees were constructed by the neighbor-joining method and bootstrap analysis to determine the best-fitting tree for the gene using mega software (version 4). the statistical significance of the tree topology was tested by bootstrapping (1,000 replicas). pairwise distances between and within the genotypes at the nucleotide level were calculated with kimura 2 parameters and with poisson correction at the amino acid level with mega software. genbank accession numbers are listed in table s1 . information on the crfs was entered into a database created in microsoft office access 2003. the chi square and fisher exact tests were used to compare means and associations using spss software version 10.0 (spss inc.; chicago, il) and r version 2.8.0 (r development core team; vienna, austria). projections of global mortality and burden of disease from 2002 to 2030 acute respiratory infections are the leading cause of death in children in developing countries the global burden of disease occurrence of respiratory virus: time, place and person viral respiratory infections in the community: epidemiology, agents, and interventions the tecumseh study of respiratory illness epidemiology of viral respiratory infections community epidemiology of human metapneumovirus, human coronavirus nl63, and other respiratory viruses in healthy preschool-aged children using parent-collected specimens viral pneumonia influenza-associated hospitalizations in the united states mortality associated with influenza and respiratory syncytial virus in the united states cdc (2010) prevention and control of influenza with vaccines: recommendations of the advisory committee on immunization practices influenza-like illness sentinel surveillance in peru acute respiratory infection and influenza-like illness viral etiologies in brazilian adults sentinel surveillance of influenza-like illness in two cities of the tropical country of ecuador influenza and other respiratory viruses in three central american countries boletin epidemiologico semanal semana epidemiologica 52 available boletin epidemiologico semanal semana epidemiologica 52 boletin epidemiologico semanal semana epidemiologica 52 boletin epidemiológico semanal semana epidemiologica 52 boletin epidemiologico semanal semana epidemiologica 52 etilogía viral de las infecciones respiratorias agudas genetic analysis of influenza a/h1n1 of swine origin virus (soiv) circulating in central and south america outbreak of swine origin influenza a (h1n1) virus infection -mexico influenza surveillance in indonesia surveillance of respiratory viral infections among pediatric outpatients in northern taiwan a comparison of the epidemiology and clinical presentation of seasonal influenza a and 2009 pandemic influenza a(h1n1) in guatemala pandemic influenza in a southern hemisphere setting: the experience in peru from pandemic (h1n1) 2009 cases epidemiology of human infection with the novel virus influenza a(h1n1) in the hospital das clinicas, sao paolo, brazil viral pathogens associated with acute respiratory infections in central vietnamese children respiratory viruses in adults with community-acquired pneumonia role of cell culture for virus detection in the age of technology frequency of human bocavirus (hbov) infection among children with febrile respiratory symptoms in argentina we would like to express our gratitude to all personnel working at the two sentinel health centers (hcm and ivss-hjmct) in venezuela for supporting this surveillance study. we thank mr. eduardo guerra, the informatics superior technician of lardidev/biomed-uc, for his irreplaceable technical work in venezuela. we also thank the professional staff of the virology department of namru-6 for invaluable laboratory and technical support in the execution of the study.disclaimer: the views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the department of the navy, department of defense, nor the u.s. government. the corresponding author had full access to all data in the study and final responsibility for the decision to submit this publication.additional key: cord-314651-e4uaw5fy authors: zhao, guangyu; jiang, yuting; qiu, hongjie; gao, tongtong; zeng, yang; guo, yan; yu, hong; li, junfeng; kou, zhihua; du, lanying; tan, wenjie; jiang, shibo; sun, shihui; zhou, yusen title: multi-organ damage in human dipeptidyl peptidase 4 transgenic mice infected with middle east respiratory syndrome-coronavirus date: 2015-12-23 journal: plos one doi: 10.1371/journal.pone.0145561 sha: doc_id: 314651 cord_uid: e4uaw5fy the middle east respiratory syndrome coronavirus (mers-cov) causes severe acute respiratory failure and considerable extrapumonary organ dysfuction with substantial high mortality. for the limited number of autopsy reports, small animal models are urgently needed to study the mechanisms of mers-cov infection and pathogenesis of the disease and to evaluate the efficacy of therapeutics against mers-cov infection. in this study, we developed a transgenic mouse model globally expressing codon-optimized human dipeptidyl peptidase 4 (hdpp4), the receptor for mers-cov. after intranasal inoculation with mers-cov, the mice rapidly developed severe pneumonia and multi-organ damage, with viral replication being detected in the lungs on day 5 and in the lungs, kidneys and brains on day 9 post-infection. in addition, the mice exhibited systemic inflammation with mild to severe pneumonia accompanied by the injury of liver, kidney and spleen with neutrophil and macrophage infiltration. importantly, the mice exhibited symptoms of paralysis with high viral burden and viral positive neurons on day 9. taken together, this study characterizes the tropism of mers-cov upon infection. importantly, this hdpp4-expressing transgenic mouse model will be applicable for studying the pathogenesis of mers-cov infection and investigating the efficacy of vaccines and antiviral agents designed to combat mers-cov infection. all procedures involving animals were approved by the laboratory animal center, state key laboratory of pathogen and biosecurity, beijing institute of microbiology and epidemiology iacuc's (the permit number is bime 2014-0017). animal studies were carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals. all experimental operations to mice were performed under sodium pentobarbital anesthesia, and mice were euthanized with overdose inhalational carbon dioxide. all efforts were made to minimize suffering of animals. considering the species differences, the hdpp4-coding sequence was codon-optimized and synthesized (genscript, nanjing, china) according to the mrna sequence (accession number nm_001935.3) and cloned into the multiple cloning site (mcs) of a pcaggs plasmid, leading to strong expression of the gene. the recombinant plasmid pcaggs-hdpp4 contained the hcmv ie enhancer (hcmviee), the cag promoter (chicken β-actin promoter/intron), a codon-optimized open reading frame (orf) of hdpp4 and rabbit β-globin polya ( fig 1a) . expression of the codon-optimized hdpp4 was achieved by transfection of the pcaggs-hdpp4 plasmid into cos-7 cells, followed by confirmation of expression using anti-human cd26 monoclonal antibody (r&d systems, minneapolis, mn, usa). to confirm the binding of hdpp4 to the mers-cov receptor-binding domain (rbd), transfected cos-7 cells were incubated with a recombinant protein expressing the rbd of mers-cov fused to human igg fc (rbd-fc) [18] , followed by addition of dylight 549-conjugated goat anti-human igg to detect binding. the purified 5861bp fragment generated from apal1 digestion of pcaggs-hdpp4 was used as the transgene for injection into the pronuclei of fertilized f1 (c57bl/6 × c57bl/6) mouse eggs to generate transgenic embryos. the mice used in this studywere backcrossed two to three times onto a c57bl/6 background. all animal studies were performed in strict accordance with the recommendations outlined in the guide for the care and use of laboratory animals. genomic dna from each transgenic founder line and from wild-type c57bl/6 mice was isolated from the liver tissue using dnazol reagent (qiagen, valencia, ca, usa) according to the manufacturer`s instructions. the hdpp4 dna copy numbers were determined by quantitative pcr using power sybr 1 green pcr master mix (life technologies, carlsbad, ca, usa). total hdpp4 dna was normalized using a single-copy reference gene (pkd1) as an endogenous control. the primers specific for hdpp4 were forward 5' ctacagctcctggg caacgtgctg 3' and reverse 5' agatgtcgtaactggcggtgtaag 3'. the primers specific for mpkd1 were forward 5' ggctgctgagcgtctggta 3' and reverse 5' ccaggtcctgcgtgtctga 3'. to detect the expression of optimized hdpp4 in the tissues of our transgenic mice model, samples were harvested and stored immediately in rnalater rna stabilization reagent (qiagen, valencia, ca, usa) until total rna was extracted and purified using an rneasy extraction kit (qiagen, valencia, ca, usa). for each sample, 2 μg of total rna was used as template for first-strand cdna synthesis. the resulting cdna was subjected to quantitative pcr using power sybr 1 green pcr master mix (life technologies, carlsbad, ca, usa) to determine the relative abundances of hdpp4 in the tissues. the forward and reverse primers for hdpp4 amplicons were as the same as those noted above. the relative amount of hdpp4 in different tissues was obtained by normalizing mrna expression to that of the endogenous control gene gapdh [19] . mers-cov (hcov-emc/2012 strain) was propagated and titered on vero cells in an approved biosafety level 3 laboratory. following intraperitoneal anesthetization with sodium pentobarbital (5 mg/kg of body weight), mice under virus infection were intranasally inoculated with mers-cov (10 4.3 tcid 50 ) in 20 μl dulbecco's modified eagle's medium (dmem), and mice in sham group were treated with the same volume of dmem. mice were monitored for weight loss and survival for 12 days. we have taken special precautions and followed standard guidelines outlined in the guide for the care and use of laboratory animals to ensure that ample food and water as well as sanitary cage conditions with enrichment devices were present in order to maximize the animal's comfort. humane endpoints were used during the survival experiments. after virus infection, mice were closely monitored by daily observation and weighing of trained animal caretaker, and the monitoring will raise to twice per day once mice lost more than 10% of their initial body weight, or exhibited lethargy, ruffled hair coat, or hunched posture. animals were deemed gravely ill and were euthanized by overdose inhalation of carbon dioxide if they lost more than 25% of their weight, or lost ability to ambulate and access food or water. sera were collected on day 5 and inactivated at 56°c for 30 min before the levels of cytokines and chemokines were measured. to assess viral replication and histopathologic damage following mers-cov infection, mice were euthanized with overdose inhalational carbon dioxide, and tissues included lungs, kidneys, livers, spleens, intestines and brains were harvested on indicated time points. the viral rna copies in tissues were determined by qrt-pcr according to the protocol described elsewhere [20] . briefly, total rna was extracted from 20mg of collected tissues using rneasy extraction kits (qiagen, valencia, ca, usa) following the manufacturer's instructions. mers-cov rna was quantified in a 25 μl mixture containing 5 μl rna using the invitrogen superscript™ iii one-step rt-pcr system with platinum 1 taq (life technologies, carlsbad, ca, usa). the primers and probes specific for the upe envelope gene of the mers-cov virus were as follows: forward 5' gcaacgcgcgattcagtt 3'; reverse 5' gcctctac acgggacccata 3'; fluorescence probe /56-fam/ ctcttcacataatcgccccgagctcg cg/36-tamsp/ [21] . mouse gapdh was used as a housekeeping gene control. a standard curve was generated for pcr reaction using 10-10 7 copies of quantified rna transcripts to calculate copy numbers for each reaction. the results were considered positive at c t values below 35 for primer and probe set, and were calculated as viral rna copies per gram of tissues. the tissues of infected mice were harvested aseptically at indicated time points and homogenized in minimal essential medium (mem) plus antibiotics to produce 10% (w/v) suspensions. tissue homogenates were centrifuged and titered on the monolayer of vero cells. the cytopathic effects (cpe) were daily observed under phase-contrast microscropy for 3 days. the viral titer was determined as tcid 50 by cpe-based assay, and calculated using the reed and muench method. the viral titer in tissue was expressed as log 10 tcid 50 /g of tissue. collected tissue samples were immediately fixed in 10% neutral buffered formalin, sectioned at 4 μm thickness, and stained with hematoxylin and eosin (h&e) to examine histopathology as described previously [22] . the expression of virus antigens and infiltration of neutrophils and macrophages in organs after mers-cov infection was detected by immunohistochemistry. briefly, formalin-fixed, paraffin-embedded lung sections were deparaffinized and hydrated using graded alcohols. expression of virus antigen and inflammatory cell infiltration was assessed using rabbit polyclonal antibody to mers-cov nucleoprotein (np) (sino biological inc., beijing, china), neutrophil marker nimp-14, (santa cruz biotechnology, paso robles, ca, usa) and cd68 (abcam, cambridge, ma, usa). the reaction was detected using a standard streptavidin-biotin detection system (beijing zhongshan biotechnology co., ltd., beijing, china) and visualized using dab with hematoxylin counterstaining. cytokines and chemokines in mouse sera were quantified using a commercial milliplex mouse cytokine/chemokine magnetic panel kit (merck millipore, usa.). a panel of inflammatory cytokines and chemokines (ifn-γ, ip-10, il-1β, il-17, il-6, tnf-α, gm-csf, kc, mcp-1, mip-1ɑ, mip-1β and rantes) was measured according to the manufacturer's protocols. statistical analyses were performed using graphpad prism version 5.01. to compare the groups in terms of hdpp4 copies, inflammatory cytokine/chemokine levels, and tissue viral rna copies, student's t test with welch's correction was used. the significance between survival curves was analyzed by kaplan-meier survival analysis with log-rank test. p values lower than 0.05 were considered statistically significant. dpp4 is constitutively expressed in human parenchyma cells in tissues including the liver, intestines and kidneys as well as in t and b cells [7] . we developed transgenic mice in which hdpp4 expression was driven by the chicken β-actin promotor in the pcaggs plasmid ( fig 1a) . expression of hdpp4 in vitro was achieved by transfection of the pcaggs-hdpp4 plasmid into cos-7 cells and detected by immunofluorescence analysis. the results showed that hdpp4 was expressed in cos-7 cells and that it effectively bound mers-cov (fig 1b-1e ). after microinjection of linearized dna, founder lines that transferred the hdpp4 gene to their progeny were established. the levels of transgenic dna in the founder lines ranged from 2 to 6 copies per genome, as determined by qpcr (fig 1f) . in addition, the mrna levels of hdpp4 in the trachea, lung, liver, spleen, kidney, intestine and brain in two of the founder lines were measured by qrt-pcr. the data showed that hdpp4 expression was higher in the trachea, lung, spleen, kidney and brain in both lines, and the expressions were relative higher in trachea and kidney in line 4 compared with that in line 2 ( fig 1g) . in order to determine the effective infection of mers-cov in different lines of transgenic mice, 6 mice in each line were infected with mers-cov and the changes of body weight were monitored. the results showed that all mice in line 1, 2 and 3 survived with no significant changes in body weight but all mice in line 4 died by day 10 following mers-cov inoculation (fig 1h) . in order to confirm the virus replications in transgenic mice, three mice of each line were sacrificed on day 5 after mers-cov infection for the detection of viral titer and viral antigen expression in lungs. as expected, compared with high level of lung viral titers in line 4 mice, no viral titer were detected (fig 1i) in lungs of line 1, 2 and 3 mice, which also confirmed by the negative results of viral antigen expression detected by immunohistochemistry in lung (data not shown). wild-type mice, such as balb/c, 129/svev and stat1 mice, are not permissive to mers-cov infection, and syrian hamsters do not support replication of mers-cov [9] [10] [11] [12] . macaques have been confirmed to develop mild to marked interstitial pneumonia upon mers-cov infection [23] , but fail to progress to the extent observed in human subjects. marmoset model, which has the same interaction characteristic between its dpp4 and mers-cov spike protein, was partially lethal and developed a progressive severe pneumonia after mers-cov infection [14] . in this study, we found that hdpp4 transgenic mice infected with mers-cov exhibited decreased activity and significant weight loss from day 6 after infection, and all of the infected mice died by day 10 (fig 2a and 2b) . importantly, symptoms of neural defects with paralysis were observed on day 9 in the infected transgenic mice. compared to the sham-infected group (fig 3a, 3d, 3g and 3j) , the histopathological analysis of the tissues in transgenic mice on days 5 and 9 after mers-cov infection showed the presence of inflammatory tissue damage in the kidney, liver and spleen, with mild inflammatory responses in the lungs but no significant changes in the intestines. on day 5 after inoculation, the hdpp4 transgenic mice exhibited mild inflammation in the lungs with focal exudation and hemorrhage (fig 3b) , and by day 9, the damage was more severe, with evidence of diffused alveolar damage, alveolar septal thickening, hemorrhage and activated macrophage infiltration (fig 3c) . in the kidneys, mild interstitial inflammation with inflammatory cell infiltration was observed in the interstitium and exudates of renal tubules on day 5 (fig 3e) . by day 9, more renal tubular epithelial cells were injured, with evidence of focal hemorrhage in the renal interstitium (fig 3f) . in the liver, mild to moderate liver damage was seen in the transgenic mice infected with mers-cov on day 5, including scattered hepatocyte necrosis and numerous activated kupffer cells and macrophage infiltrates in hepatic sinusoid (fig 3h) , and on day 9, fatty change in hepatocytes with less hepatocyte necrosis was observed (fig 3i) . in spleens, necrosis of splenic cells and increased reticulum cells in red pulp with significant hemosiderin deposition was observed on both day 5 and day 9 (fig 3k and 3l) . typical characteristics of viral encephalitis were observed in the brain at day 9, with perivascular cuffs ( fig 3m) and neuronal cell necrosis in the cerebral cortex (fig 3n) , including hippocampal neurons (fig 3o) . there was no apparent damage in the intestines after mers-cov infection on day 5 or day 9 (data not shown). to evaluate mers-cov infection in the hdpp4 transgenic mice, distribution of the viral antigen np protein was assessed in mouse tissues by immunohistochemical staining. the results showed that type i and type ii pneumocytes in the lungs and renal tubular epithelial cells in the kidneys expressed viral antigen on day 5 after mers-cov infection and that the expression was more abundant on day 9, and no viral antigen was detected in sham-infected group ( fig 4a-4f) . strong viral antigen expression was evident not only in neuronal cell bodies, but also in dendrites, axons and some microglia in the cerebral cortex (fig 4h) including the hippocampus (fig 4i) . no viral antigen expression was observed in the brains of mice in the shaminfected group (fig 4g) . viral rna copies in lung and brain were detectable on day 5 postinfection and increased to a higher level on day 9 (fig 4j) . lower level of viral rna copies can be also detected in kidney collected on day 9. in order to further confirm the effective replication of mers-cov, the dynamic changes of viral titer in organs were detected. the results showed that on day 3 after infection, only lower viral titer was detected in lung and not detectable in other organs. however, on day 5, viral titer was also detected in brain. on day 7 and day 9, increased viral titers were detected in both lung and brain, and lower level viral titer in kidney was detectable (fig 4k) . these results indicate that the transgenic mice had been successfully infected and that mers-cov exhibited cell and tissue tropism especially for pneumocytes and neurons. furthermore, synapses may be one of the structures by which viruses diffuse through the brain after mers-cov infection. to date, only limited data on the immune response of mers patients are available [24] . because of the clinical similarity between the symptoms of mers-cov and those of patients infected with sars-cov [1] , the aberrant immune response may be related to the pathogenesis of mers-cov infection. in our mouse model, an aberrant inflammatory response was confirmed by infiltration of neutrophils in the lung, liver and spleen (fig 5a-5f ) and macrophages in the lung, liver and kidney (fig 5g-5l ). in addition, deposition of hemosiderin in the spleen (fig 3k and 3l ) indicated an increase in activated macrophages, and a systemic inflammatory response after virus infection was demonstrated by a significant increase in cytokines such as ifn-γ, ip-10, and il-17 in the serum on day 5 after mers-cov infection (fig 5m) . although several species, such as rhesus macaques and common marmosets are reported to be sensitive to mers-cov infection and to develop mild to marked broncho-interstitial pneumonia, small animal models are urgently needed to study the pathogenesis of this disease and evaluate the effects of vaccines and antiviral agents. a mers-cov-infection mouse model was generated by transducing mice with a recombinant non-replicating adenovirus expressing hdpp4; however, while the mice exhibit viral antigen expression and progress to interstitial pneumonia, there is no mortality after virus infection [15] . although a transgenic mouse model expressing human dpp4 was also established, and its immune response was studied after infection with mers-cov [16] , the transgenic mice in the study died on day 6 with only progressive pneumonia and mild perivascular cuffing in brain, and no neurological disorder or other multi-organ damage was observed. different from the aforementioned transgenic mice, our hdpp4 transgenic mice experienced a longer incubation period post-infection and developed progressive pneumonia and neurological disorders accompanied by histological damage to the lungs, brain, spleen and liver, which more closely resembles the clinical cases. the results from this study indicate that mers-cov infection was lethal in hdpp4 transgenic mice with higher transgene copy number, while lines with lower levels of hdpp4 expressed especially in trachea had no significant clinical symptoms (data not shown), suggesting that the copy number of the transgene was closely related to the efficiency of mers-cov infection. although the expression level of optimized hdpp4 was not highest in the brains, the tissues had higher viral titers following mers-cov infection, revealing the tropism of mers-cov for both lungs and brains, tissues in which pneumocytes and neurons may be the main target cells (fig 4) . however, mers-cov infection in brain via blood or olfactory nerves needs to be further studied due to the strong expression of viral antigens in dendrites and axons. although few autopsy reports exist on fatal mers-cov cases and atypical pneumonia and respiratory failure are suspected the causes of death [24] , other types of extrapulmonary organ dysfunction have also been documented in mers critically ill patients with abnormal clinical manifestations [25] [26] [27] . for example, acute renal failure was described in a number of mers cases [28] [29] [30] [31] [32] . in addition, renal epithelial cells may produce almost 1000-fold more infectious mers-cov progeny than bronchial epithelial cells [33] . more recently, severe neurologic syndrome including altered level of consciousness from confusion to coma, ataxia and focal motor deficit was also reported in three critically cases by balkhy h et al [4] . according to the neurologic manifestations and distinct imaging patterns, an important question was raised on the pathogenic mechanisms that underlie the occurrence of neurologic injury in patients. it has been studied that corona virus such as sars-cov are generally known for causing respiratory illness, and the strong tropism of sars-cov to cns and causing neuronal injury had also been demonstrated both in clinical and experimental studies [34] [35] [36] [37] [38] , which suggested the possible relations of mers-cov infection with brain injury. as to the extensive expression of hdpp4 in the lung, kidney, placenta, liver, skeletal muscle, brain, endothelium, pancreasour and t cell in human, our globally expressed hdpp4 transgenic mouse manifested with multiorgan damage infected with mers-cov could be a suitable model for the pathogenic mechanisms study. systemic inflammation is believed to be a primary reason for the severe outcome in mers--cov infections [14, 24] . although the transgenic mice model died after mers-cov infection with multi-organ damages, it is still uncertain whether these mice are mainly dying from lung damage. in our transgenic mice, aberrant immune response such as elevated ip-10, ifn-γ and il-17, which are closely related to acute virally-mediated lung injury [39] [40] [41] [42] , was also a specific manifestation after mers-cov infection. so, as to the mechanism of lethal which was due to the virus replication directly or induced by the aberrant inflammatory response need to be further studied. the mechanism of mers-cov infection caused death is complex and complicated. aberrant immune response after mers-cov infection may be one of main reasons which need to be further studied. we have added more discussions on this issue in the revised manuscript. in summary, the transgenic mouse model developed in this study was efficiently infected by mers-cov and exhibited severe acute respiratory injury and considerable extrapulmonary organ damage. this model will be useful for studying the pathogenesis of mers-cov infection and evaluating the efficacy of mers vaccines and therapeutic agents. molecular pathology of emerging coronavirus infections state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans hospital outbreak of middle east respiratory syndrome coronavirus severe neurologic syndrome associated with middle east respiratory syndrome corona virus (mers-cov) host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc the multifunctional or moonlighting protein cd26/dppiv middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd wild-type and innate immune-deficient mice are not susceptible to the middle east respiratory syndrome coronavirus reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase 4-mediated entry of the middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques infection with mers-cov causes lethal pneumonia in the common marmoset rapid generation of a mouse model for middle east respiratory syndrome generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection a conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in middle east respiratory syndrome coronavirus spike protein analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method real-time reverse transcription-pcr assay panel for middle east respiratory syndrome coronavirus detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction inhibition of complement activation alleviates acute lung injury induced by highly pathogenic avian influenza h5n1 virus infection treatment with interferon-alpha2b and ribavirin improves outcome in mers-cov-infected rhesus macaques pathogenesis of middle east respiratory syndrome coronavirus epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection isolation of a novel coronavirus from a man with pneumonia in saudi arabia family cluster of middle east respiratory syndrome coronavirus infections clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection latest outbreak news from promed-mail: novel coronavirus-middle east clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission in-vitro renal epithelial cell infection reveals a viral kidney tropism as a potential mechanism for acute renal failure during middle east respiratory syndrome (mers) coronavirus infection possible central nervous system infection by sars coronavirus detection of severe acute respiratory syndrome coronavirus in the brain: potential role of the chemokine mig in pathogenesis multiple organ infection and the pathogenesis of sars pathology and pathogenesis of severe acute respiratory syndrome susceptibility of human and rat neural cell lines to infection by sars-coronavirus early and sustained innate immune response defines pathology and death in nonhuman primates infected by highly pathogenic influenza virus severe acute respiratory syndrome in children interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome therapy of h7n9 pneumonia: current perspectives key: cord-328633-c31xsyeo authors: moser, michael j.; difrancesco, robert a.; gowda, krishne; klingele, audrey j.; sugar, darby r.; stocki, stacy; mead, david a.; schoenfeld, thomas w. title: thermostable dna polymerase from a viral metagenome is a potent rt-pcr enzyme date: 2012-06-04 journal: plos one doi: 10.1371/journal.pone.0038371 sha: doc_id: 328633 cord_uid: c31xsyeo viral metagenomic libraries are a promising but previously untapped source of new reagent enzymes. deep sequencing and functional screening of viral metagenomic dna from a near-boiling thermal pool identified clones expressing thermostable dna polymerase (pol) activity. among these, 3173 pol demonstrated both high thermostability and innate reverse transcriptase (rt) activity. we describe the biochemistry of 3173 pol and report its use in single-enzyme reverse transcription pcr (rt-pcr). wild-type 3173 pol contains a proofreading 3′-5′ exonuclease domain that confers high fidelity in pcr. an easier-to-use exonuclease-deficient derivative was incorporated into a pyroscript rt-pcr master mix and compared to one-enzyme (tth) and two-enzyme (mmlv rt/taq) rt-pcr systems for quantitative detection of ms2 rna, influenza a rna, and mrna targets. specificity and sensitivity of 3173 pol-based rt-pcr were higher than tth pol and comparable to three common two-enzyme systems. the performance and simplified set-up make this enzyme a potential alternative for research and molecular diagnostics. reverse transcription pcr (rt-pcr) is a powerful analytical and preparative method for detecting, quantifying and analyzing gene expression and rna viruses. most rt-pcr protocols rely on two dna polymerase (pol) enzymes; a retroviral reverse transcriptase (rt) to copy rna into cdna and a thermostable dna pol to amplify the target sequence. we describe a unique single-enzyme alternative to the traditional format based on the innate reverse transcriptase activity of the thermostable 3173 pol, which was recently isolated from a viral metagenomic library [1, 2] . we believe this is the first report of a reagent enzyme produced from a viral metagenomic library, the first viral pol shown to be fully thermostable in vitro and the first single-enzyme rt-pcr protocol with high sensitivity and specificity comparable to two-enzyme systems. despite their wide use and general reliability, existing twoenzyme rt-pcr systems have several documented performance problems attributed to deficiencies inherent in retroviral rts: 1) poor reagent stability, 2) low fidelity, 3) frequent rearrangements during cdna synthesis, 4) secondary enzymatic activities (i.e. rnase h and strand switching), 5) bias for specific primers and templates, and 6) inhibition of pcr pol enzymes [3, 4, 5, 6, 7] . these deficiencies are associated with cloning errors, amplification bias, poor concordance between and within testing labs, and target dependent variation in amplification efficiency [8, 9, 10, 11, 12, 13, 14, 15, 16, 17] . the two-enzyme systems require an initial low temperature reverse transcription step that reduces specificity, increases reaction time, and impairs synthesis through complex secondary structures. the limited shelf stability in solution of retroviral rts has precluded development of complete rt-pcr enzyme premixes popular for standard pcr. alternative chemistries based on an improved rt-pcr enzyme are a means of addressing these shortcomings. numerous thermostable dna polymerases have been described and commercialized for pcr [18, 19] . all of these fall into one of two groups of high molecular identity and biochemical similarity; bacterial pol i-type enzymes and archaeal pol ii-type enzymes [20] . remarkably, no truly thermostable viral replicase-type pol has ever been described. the pool of useful rts consists mainly of retroviral moloney murine leukemia virus (mmlv) rt and its derivatives and avian myeloblastosis virus (amv) rt. substantial effort has been devoted to engineering mmlv rt. truncating the mmlv rt protein to eliminate rnase h activity [21] fortuitously increased thermostability [22] . however, none of the engineered retroviral rts are thermostable enough for pcr. the alternative bacterial and archaeal pols also do not fulfill the goal of a facile single-enzyme rt-pcr reagent. thermus thermophilus (tth) pol i was originally induced to reverse transcribe by inclusion of manganese ions in the reaction buffer [23] . however, tth pol in the presence of manganese is highly inaccurate and much less sensitive than the two-enzyme systems and therefore not widely used. notably absent is a thermostable rt suitable for single enzyme rt-pcr that matches or exceeds the performance of two-enzyme systems with regard to fidelity, sensitivity, specificity and low bias. viral pols are interesting alternatives to bacterial or archaeal pol i and pol ii enzymes, differing significantly in their biologic roles as replicases rather than as short patch repair and lagging strand polymerases [24] . these enzymes possess many important and highly useful biochemical characteristics. bacteriophage t5, t7 and phi29 dna polymerases are highly processive enzymes [25, 26, 27] and the latter two are less prone to slippage [28] . t4 bacteriophage dna polymerase is extremely accurate [29] . while all of these properties make viral pols very useful as molecular biology reagents, none is suitable for thermocycling based amplification due to limited thermostability. the need for new pols that combine the practical advantages of viral enzymes with improved thermostability has motivated the metagenomic screens of viral sequences from thermal springs described in this report. viral metagenomes are an unexplored source of sequence diversity for the development of new enzymes. a screen of hot spring viral metagenomes identified thousands of open reading frames [2] including many encoding putative thermostable viral pols. we describe the discovery and biochemical attributes of one of these, 3173 pol, its inherent rt activity and its incorporation into a single-enzyme pyroscripth 2x rt-pcr master mix. the sensitivity, specificity and overall performance of this mix were compared to available one-and two-enzyme systems using a control ms2 rna bacteriophage template, the clinically-relevant influenza a rna and commonly used reference mrna transcripts. unless indicated otherwise, standard molecular methods were used [30] . primers and other oligonucleotides (table 1) were synthesized by idt (coralville, ia). except where noted, the 3173 pol reaction buffer used throughout was 20 mm tris-hcl ph 8.8 at 25uc, 10 mm (nh 4 ) 2 so 4 , 10 mm kcl, 2 mm mgso 4 , 0.1% triton x-100, and 200 mm of each dntp (n = a,c,g,t). construction, sequencing and blastx analysis of viral metagenomic libraries have been described [2] . clones identified by blastx analysis as encoding likely pol genes were functionally screened to detect expression of thermostable pol activity. for these screens, viral proteins were constitutively expressed in the original clones by growth to saturation in 2 ml luria broth. cells were pelleted at 2,800 rcf and suspended in 50 mm tris-hcl ph 7.5,1 mm edta, 0.5 mm dtt, 0.1% triton x-100, 10% (v/v) glycerol. cells were lysed by sonication and host proteins were denatured by incubation at 70uc for 10 minutes. soluble proteins were collected from the supernatant after centrifugation at 11,000 rcf for 10 minutes and assayed for dna pol activity based on their ability to extend a 59 fluorescently labeled oligonucleotide primer. the labeled assay primer was annealed at room temperature to the assay template and incubated for 10 minutes at 70uc with 5 ml of each clarified lysate. primer extension was detected using an abi 310 genetic analyzer (applied biosystems, foster city, ca) in genescan mode. for preparative expression, the coding sequence of 3173 pol was inserted into pet28 and used to transform bl21(de3) cells according to the manufacturer (emd bioscience, san diego, ca). pol proteins were expressed, extracted and heattreated as described for the functional screen and purified using heparin-agarose and q-sepharose chromatography. pol units were determined by a radioactive nucleotide incorporation assay [19] as the amount of enzyme that incorporates 10 nmol of deoxynucleotides per 30 minutes at 70uc. enzyme dilutions were incubated for 30 minutes at 70uc with reaction buffer supplemented with 10 mg/ml activated calf thymus dna and 10 mci/ml [ 33 p] dctp and unit activity was determined based on counts adhering to a de81 filter (whatman, piscataway, nj). single-stranded exonuclease activity was determined by incubating the polymerase in standard buffer supplemented with a [ 33 p] dctp radiolabeled pcr product. this substrate was heated to 95uc for 5 minutes and then cooled to 4uc for 10 minutes prior to incubation with 3173 pol in reaction buffer. counts due to free nucleotides were measured after precipitation of polynucleotide substrate with 10% trichloroacetic acid (tca) for 10 minutes on ice. site directed mutagenesis was performed using the quick-changeh site-directed mutagenesis kits (agilent, santa clara, ca). kinetics and thermal profiles were determined using the radioactive incorporation assay under pseudo-first order conditions of substrate excess [19] . thermal stability (half-life) was determined by pre-incubating the enzyme in reaction buffer for varying times and measuring the remaining activity by the same assay. time points were determined in triplicate and decay kinetics were calculated by least squares linear regression of the inverse natural log of the remaining activity at the time points. standard pcr was performed using cycling conditions described for pyrophage 3173 dna pol by the manufacturer (lucigen, middleton, wi). the processivity assay is a modification of published methods [31] . m13f primer (table 1 ) was 59 end labeled with rhodamine. mix a contained 50 nm primer, 50 nm m13mp18 single strand dna, and 0.5 nm of pol in the standard buffer. mix b contained 0.25 mm each dntp (n = a,c,g,t) and 0.6 mg/ml activated calf thymus dna in reaction buffer. an aliquot of mix a was incubated at room temperature to anneal primer. the reactions were pre-incubated with enzyme at 70uc and an equal volume of mix b preheated to 70uc was added. reactions were stopped at 0, 3, 5 and 10 minutes by addition of 50% formamide, 1 mm edta. the extension products were resolved on an abi prism 310 instrument using data collection software and peaks were identified and integrated by genescan software (applied biosystems, foster city, ca). processivity was calculated by the following equation: where i = area of each peak, n = number of nt added. strand displacement was demonstrated by the ability of 3173 pol to extend the m13f primer on an m13mp18 ssdna template for greater than the length of the phage genome (7,249 nt) as determined by 1% agarose gel electrophoresis. extension from nicks was demonstrated by pre-incubating puc19 plasmid with nt.bstnbi nicking enzyme (new england biolabs, ipswitch, ma) and incubating the plasmid with 5 units of 3173 pol for two hours at 55uc. synthesis was detected by agarose gel. the pol fidelity assay was a modification of the laci q reversion assay [32] . the template for this assay was constructed by inserting pcr-amplified laci q coding dna into the cloning site of psmart hckan vector (lucigen), creating psmiq. primers fid-f and fid-r (table 1) were used to amplify a sequence containing the laci q and kan genes. 3173 wild-type and exo-pols were compared to pfu (agilent), phusion (new england biolabs) and taq (lucigen) pols. each of the pol enzymes was tested according to the respective manufacturer recommendations. the amplicons were digested with eco0109 i restriction enzyme and ligated to dephosphorylated, eco0109 i-digested puc19 vector. the resulting construct was used to transform 10g supreme cells (lucigen) that were plated on yt agarose plates containing 0.02% (w/v) x-gal, 0.3 mm iptg, 100 mg/ml carbenicillin, and 30 mg/ l kanamycin. the plates were incubated 20 hours at 37uc and the number of blue and white colonies was determined visually. fidelity was calculated using the published formula [32] : fidelity = 2lnf/d * t, where f = fraction white colonies, d = number of duplications during pcr (log 2 of fold amplification) and t is the effective target size (t = 349 for laci q ). the fluorogenic rt assay was performed by incubating 500 ng/ml polya (sigma-aldrich, st. louis, mo) with 25 ng/ml reactions lacking dttp were preincubated at 37uc to equilibrate secondary structures of the substrate and reduce high initial fluorescence background. next 37uc dttp was added to start the reaction. one hundred fluorescence reads were performed every six seconds at 37uc followed by an additional one hundred fluorescence reads at 65uc. direct incubation at 65uc does not detect rt activity because the reaction temperature is greater than the melting temperature of the oligo dt primers on the polya template. data analysis was performed by linear least squares regression of a plot of fluorescence data in rfu versus reaction time in seconds using data from 30 to 150 seconds of incubation at 37uc and data from 30 to 90 seconds of incubation at 65uc. rt primer extension assays were performed using the same conditions as the fluorogenic rt assay. reactions with polya template employed hexachlorofluorescein (hex) labeled dt 20 oligonucleotide instead of oligo dt primer. the rt primer extension assay reactions with ms2 rna template and cal-fluor560-labeled (biosearch technologies, novato, ca) ms2specific primer (ms2 160-r, table 2 ) were incubated for 10 minutes at 37uc, and then 30 minutes at 65uc. for page analysis, reactions were stopped by incubation for 5 minutes at 95uc in 1m urea and held on ice prior to electrophoresis on denaturing 5 or 10% polyacrylamide 1x tbe gels (biorad). hex and calfluor560 fluorescence was detected by a pharos fx fluorescence scanner (biorad). ms2 rna bacteriophage (accession number nc_001417) was cultivated using published procedures [33] . the ms2 phage particles were precipitated from 0.5 m nacl and 10% peg-8000, purified by isopycnic centrifugation in 1.40 g/ml cscl and dialyzed into 10 mm tris-hcl ph 7.4, 100 mm nacl, 0.1 mm mgso 4 . phage preparations were adjusted to 50% glycerol and stored frozen. rna was isolated from thawed aliquots with either the qiaamp minelute virus spin kit (qiagen, valencia, ca) or the tri reagent ls reagent (molecular research center, inc., cincinnati, oh) according to manufacturer instructions. influenza a rna was isolated from cultures of mcdk cells infected with influenza a strain a/puerto rico/8/1934 (h1n1). infected cells were clarified by centrifugation and rna isolated by qiaamp minelute virus spin kit was frozen immediately. no dnase treatment was used for either preparation. for detection of transcripts, total human liver rna (ambion, austin, tx) was used. for quantification, ms2 rna was re-suspended in 100 mm edta and the rna concentration was estimated by absorbance at 260 nm with an extinction coefficient of 40 mg ml21 od21. the estimated ms2 rna copy number was calculated from the determined concentration using an average molecular weight for an rna base of 340 g mole21 and the ms2 genome length of 3,569 nt. two-step rt-pcr reactions were performed using either 5 units 3173 pol, exonuclease negative mutant or 200 units mmlv rt (neb). rna was combined with primers and annealed in water at 70uc for 5 minutes followed by incubation on ice. primers used were oligo dt 12-18 mer, random hexamers, random nonamers, and gene specific primers. no primer and no rt controls were performed. first strand synthesis was performed in manufacturer recommended buffer with 0.5 mm dntps for 5 minutes at 25uc and then for 30 minutes at 37uc for the oligo dt, random hexamer, random nonamer and control reactions. gene specific rt reactions were incubated for 30 minutes at 42uc for mmlv and 60uc for 3173 pol. reactions were terminated by incubation at 95uc for 5 minutes. following reverse transcription a tenth of the reaction was pcr amplified by taq polymerase (lucigen) in 40 cycles of pcr. for one-step rt-pcr reactions the following conditions were used. the pyroscripth rt-pcr 2x master mix (lucigen) containing 2.5 units of 3173 pol, was used in reactions at 1x concentration with primers at 200 mm each. the superscripth iii one-step rt-pcr system with platinumh taq dna polymerase (life technologies, carlsbad, ca), the qscript tm one-step sybrh green qrt-pcr kit (quanta biosciences, gaithersburg, md), the transcriptorh one-step rt-pcr kit (roche applied science, mannheim, germany) and tth dna polymerase (epicentre technologies, madison, wi) were used according to manufacturer instructions. pcr and real-time pcr were performed using an icyclerh myiq tm thermal cycler (biorad) on sample sizes of 25 ml employing the cycling conditions specified by the respective rt-pcr kit manufacturers (table 2 ). for qpcr, amplification data was acquired during the pcr extension step, a thermal melt was performed from 70-95uc. for the roche and the lucigen reagents, a fluorescent dna-binding dye, evagreen (biotium, hayward, ca), was added at 0.56. data acquisition used the iq optical system software version 2.1 (bio-rad) and analysis was performed using multicode-rtx analysis software version 1.6.2.10 (eragen biosciences, madison, wi). a viral metagenomic library was constructed from octopus hot spring (93uc) in yellowstone national park and 21,198 sanger sequence reads were analyzed [2] . blastx alignment [34] to the genbank protein sequence database identified hundreds of potential pol genes. analysis of paired end reads of individual metagenomic clones suggested 59 complete pol genes. all of these were tested for expression of pol activity using a primer extension assay, and ten clones displayed detectable thermostable pol activity. the most thermostable of these activities was from clone number 3173, encoding 3173 pol ( figure 1 ). this enzyme belongs to a family of thermostable viral pols identified in this and other screens that have strongest sequence similarity to pol i-type enzymes from the aquificales family. the 3173 pol (genbank acc. no. adl99605.1) shares 32% amino acid identity with thermocrinis albus pol i (genbank acc. no. adc89878.1), but no significant sequence similarity to any previously described viral protein. the 3173 pol was over-expressed in e. coli and purified. its biochemical attributes are summarized in table 3 . protein sequence alignment identified a pol domain and a 39-59 exonuclease domain, but no detectable 59-39 exonuclease domain. the primer extension pol assay also detected 39-59 exonuclease activity in the purified pol preparation ( figure 1 ) and this activity was further confirmed by digestion and release of acid soluble counts from a radiolabeled dna fragment (table s1 ). the identification of a proofreading exonuclease domain suggested high fidelity synthesis. a variant of the laci q forward mutation fidelity assay [35] was used to determine the fidelity of 3173 pol in pcr amplification of a dna target ( table 3 ). the wild-type 3173 pol had a fidelity of 6.7610 4 . proofreading exonuclease activity can complicate pcr by degrading unmodified primers and templates [36] . since fidelity of incorporation is less important for detection and quantification, the exonuclease activity of the 3173 pol was eliminated to create a more robust enzyme for routine rt-pcr. sequence alignment to the 39-59 exonuclease domains of known pols [37] predicted that aspartate 49 and glutamate 51 of 3173 pol would be required for exonuclease activity. substitution of either acidic residue with alanine eliminated measurable exonuclease activity. as would be expected, disabling the proofreading exonuclease reduced pcr fidelity to 0.9610 4 . the d49a mutant of 3173 pol (pyrophage 3173 dna polymerase, exonuclease minus, lucigen) was used for all of the remaining work. we determined the processivity of the 3173 enzyme using a variant of the ''enzyme trap'' method [38] , in which pol was preloaded onto a fluorophore-labeled primer/template complex. excess activated calf thymus dna was added simultaneously with figure 2) shows peak activity at 77uc, with approximately half maximal activity at 55uc. both radioactive and fluorogenic incorporation assays indicated strong rna-dependent dna synthesis (reverse transcription) activity for 3173 pol in buffers containing either magnesium or manganese (not shown). we used two assays (figure 3 ) to compare the rt activities of the wild-type and exonuclease deficient 3173 pols to those of amv and mmlv rts at 37uc or 65uc on an oligo dt primed poly a substrate. the amv and mmlv had higher rt activity at 37uc while the 3173 pol rt was much more active at 65uc using the fluorogenic incorporation assay ( figure 3a ). the taq polymerase and no enzyme controls had no detectable rt activity at either temperature. extension products from a 59fluorophore-labeled dt20 primer were resolved by denaturing polyacrylamide gel to further demonstrate rt activity and to assess the relative lengths of the extension products of the 3173 pol and mmlv rt ( figure 3b ). both rts were able to efficiently extend the primer when polya rna template was provided. the length distribution of the 3173 pol cdnas was visibly shorter than that produced by the mmlv rt, although a subset of the 3173 extension products appeared to be so large that they barely entered the gel. incubation of the dna primer:rna template complex with the taq pol negative control resulted in a structure-dependent 59-39 exonuclease cleavage product that migrated at the dye front [39] . as an additional test to compare the rt activity of the 3173 pol to that of mmlv-rt on a complex rna substrate, a primer specific to bases 714 to 690 of the negative sense rna ms2 genome [40] was 59-fluorophore-labeled. the labeled cdna primer was extended using extracted ms2 rna as a template ( figure 3c ). the 3173 pol and mmlv rt were both able to extend the primer to produce faint, nearly full-length products although the 3173 pol product was detectably longer than that of mmlv rt. the 3173 pol also synthesized a larger amount of several shorter length extension products from 175 to 300 bases in length. the mmlv rt formed a visible template-independent product in the absence of added rna template that was not resolved by the gel, while the 3173 pol did not. we compared the first strand cdna synthesis by 3173 pol to that by mmlv rt using biological rna templates. production of cdna was detected by two-step pcr amplification in which cdna synthesis was primed by random, target-specific, oligo dt or no primers and detected by pcr with target-specific primers ( figure 4) . the rna targets were ms2 bacteriophage and a human mrna. the 3173 pol readily synthesized 77 bp ms2 [40] (lanes a1-7) and 144 bp beta-actin cdnas (lane b2, 3, 5, 6, 7) but generally failed to synthesize cdna targets longer than ,400 bp (panel c). of the two longer target sequences tested, only the 821 bp beta-actin sequence (lane c3) was reverse transcribed by the 3173 pol and this synthesis appeared less efficient than that of mmlv rt. interestingly, both enzymes appeared to reverse transcribe with primers that would not be expected to prime near the target. (a1, a11, b14) and even in the absence of primers (lane a7, b17, c17). in the case of 3173 pol, it is likely that both cdna synthesis and amplification occur during the pcr step so the presence or absence of primers during cdna synthesis may be inconsequential. the basis of the product in the mmlv rt reaction is not known. under favorable conditions 3173 pol did reverse transcribe mrna transcripts ( figure 5 ). the 3173 pol was compared to mmlv rt for the detection of three shorter target sequences in common high-abundance reference genes using the two-step rt-pcr protocol. both enzymes appear to transcribe the targets with similar efficiency and specificity. the amount of pcr product for all three transcripts appeared visibly greater in the 3173 pol reactions, although we cannot rule out the contribution of residual thermostable 3173 pol to the pcr reaction yield. to facilitate rt-pcr, the exonuclease deficient 3173 pol was combined with buffer and deoxynucleotides to formulate pyro-scripth rt-pcr 2x master mix (lucigen) for single-enzyme, one step rt-pcr. preliminary testing indicated that an initial lower temperature rt extension prior to thermal cycling did not improve results with the pyroscript enzyme (not shown). therefore this step was eliminated from pyroscript rt-pcr protocols. in contrast to the typical rt-pcr primers designed for the lower extension temperatures of mmlv or amv rts, primers used with melting temperatures of about 72uc significantly improved rt-pcr performance of the pyroscript enzyme mix. to assess sensitivity and specificity of the pyroscript master mix reagent in one step rt-pcr, a quantitated control target was prepared from rna bacteriophage ms2 [41] . we used the oneenzyme pyroscript rt-pcr mix with nine primer sets (table 1) to amplify regions of the ms2 rna genome [40] up to 362 bp. the mix proved effective for these primer sets and this range of target sizes ( figure 6a ). amplification efficiency for longer target lengths was poor as judged by a substantial increase in qpcr cycle threshold with amplicons greater than about 400 bp (not shown). to demonstrate quantitation and sensitivity, the 160 bp ms2 primer set from figure 6a was chosen as well suited for both qpcr and electrophoresis analysis and combined with pyroscript to amplify a ten-fold dilution series from 1,200,000 to 1.2 target copies of ms2 rna ( figure 6b ). the estimated limit of detection was between one and ten rna copies. the water-only control gave a negative response demonstrating high specificity, which is supported by the melt-curve analysis ( figure 6c ) and agarose gel electrophoresis of the product (not shown). linear quantitation was seen over the full six-log dilution series ( figure 6d ) suggesting a broad quantitation range. the most common single enzyme rt-pcr method uses tth pol [23] . we compared the rt-pcr sensitivity, specificity and quantitation of the pyroscript mix with tth pol. the 160 bp ms2 target from figure 6 was amplified by each enzyme over a dilution range of 10 22 to 10 28 (estimated at 120,000 to 0.12 copies) using manufacturer recommended conditions for each (figure 7) . the near single copy sensitivity and six-log linear detection range seen with pyroscript 3173 rt contrasts with the ,120,000 copy detection limit and absence of a linear quantitation range seen with tth pol. a small amount of false product was detectable in the negative control by rt-qpcr but not by agarose gel. significant false background pcr products generated by the tth pol system were readily detectable by both agarose gel and melt-curve analysis. figure 5 . rt-pcr detection of human transcript rnas. betaactin, beta2-microglobulin and cyclophilin target sequences of the indicated sizes were amplified from human liver total rna using the primers described in table 1 . shown are products of two step reactions where either mmlv rt or 3173 pol were used for first strand cdna synthesis, as indicated. taq pol was used for pcr. products were resolved on a 1% agarose gel. doi:10.1371/journal.pone.0038371.g005 since two-enzyme systems using mmlv rt derivatives and taq pol are far more commonly used than single-enzyme systems, we compared the performance of single-enzyme pyroscript mix to three widely used mixes that are based on the two enzyme mmlv rt plus taq pol combination, but are referred to as ''one-step'' systems. the comparators were: superscripth iii one-step rt-pcr system with platinumh taq dna polymerase (life technologies), the qscript tm one-step sybrh green qrt-pcr kit (quanta), and the transcriptorh one-step rt-pcr kit (roche). ms2 rna extract was amplified using primers targeting the 160 bp product from figure 6 . three dilutions of ms2 rna (the lower dilutions from figure 6d ) and a water-only control were amplified by 40 cycles of rt-qpcr using each of the respective reagents ( figure 8a ). all of the reagents appeared to have similar limits of detection and amplified the expected product as seen by electrophoresis. all of the reagents produced a weak background amplification product of about 60 bp, from both the lowest rna dilution and the water-only control. the transcriptor kit reproducibly amplified more false product than did the other three. similar slopes from plots of qpcr cycle threshold versus fold target dilution show that all four reagent mixes amplified the ms2 target with similar efficiency although the qscript reagent appeared to amplify the target a few cycles later than the other three mixes. the ability of these four reagents to amplify human influenza a virus rna was also compared. cultured influenza a puerto rico/ 8/1934 (h1n1) rna was extracted from cell medium and amplified by 40 cycles of one-step rt-pcr ( figure 8b ). the results with influenza a were similar to those seen for the ms2 target. all four reagents appeared to have similar limits of detection for the influenza a rna extract and amplified the intended 60 bp target from the second lowest dilution of rna. the pyroscript rt-pcr reaction was largely free of extraneous bands. in contrast, the transcriptor and the superscript-based mixes produced spurious bands, primarily of a size greater than the expected amplicon size. the transcriptor kit also produced false products in the negative control reaction. again the slopes of plots of qpcr cycle threshold versus fold dilution show that all four reagent mixes amplified the ms2 target with similar efficiency. in contrast with ms2, both the qscript and the pyroscript reagent were found to amplify influenza a several cycles later than the superscript and transcriptor master mixes did. the 3173 pol based pyroscript rt-pcr master mix represents a practical alternative to two-enzyme (e.g. mmlv rts/taq pol) rt-pcr systems and provides both theoretical and demonstrated advantages. no truly viable substitute for the two-enzyme systems has been described previously. among bacterial dna pols that can be induced to use rna templates, only the tth pol is thermostable enough for pcr, but its performance in rt-pcr in general has not proven competitive with the two-enzyme mixes. since the upper limit for eukaryotic life is around 62uc, it seems unlikely that retroviruses will ever provide rts thermostable for single-enzyme rt-pcr. while the two-enzyme systems are widely used and generally reliable, deficiencies inherent in these systems have restricted certain improvements in rt-pcr. for example, secondary activities, including rnase h and terminal transferase, are associated with strand switching [4, 6] and insertion errors [42] . replication of native retroviral genomes depends on specific sequences within the terminal repeats [43] , which may be related to a significant bias seen with certain combinations of primer table 1 and 3173 pol. a. products from 89 to 362 bp in length were amplified using one-step singleenzyme rt-pcr cycling conditions: 15 sec @ 94uc, (10 s @ 94uc, 30 s @ 72uc)*40. products were resolved by 2% agarose gel electrophoresis. b. the ms2 rna was diluted from 10 1 to 10 7 -fold and amplified using a primer pair corresponding to the 160 bp fragment in panel a. real-time pcr fluorescence in rfu (relative fluorescence units) vs. pcr cycles. c. post-amplification thermal melt in -drfu/dtemperature vs. temperature (uc). light blue region indicates melt curves for specific products. d. standard curve pcr cycle threshold vs. log 10 rna copy number in triplicate with linear least squares best fit line. doi:10.1371/journal.pone.0038371.g006 sequences and reverse transcriptases [44] used in vitro. in particular, the two 39-terminal nucleotides of the primers can account for a 35,000-fold range in the frequency of misincorporation, a measured km variation of 100-fold and vmax range of several-fold when used with mmlv or amv rts [5] . these preferences are possible causes of amplification errors, amplification bias [8] , poor concordance between tests [9, 45] and sequences that are completely refractory to reverse transcription [44, 45] . extensive effort has been directed at engineering retroviral rts to disable or eliminate the rnase h domain implicated in rtdependent rearrangement [21] . although such rts produce fewer rearrangements, inactivation of rnase h also increases misincorporation and bias due to impaired amplification of specific sequences [3, 4, 46] . additional mutations incorporated into superscript iii rt (life technologies) to increase thermostability may have resulted in lower sensitivity [10] and exacerbated the interference with taq pol [16] , but still have not provided adequate thermostability for single-enzyme rt-pcr. alternative approaches of evolving or engineering thermostable pols to use rna templates [47, 48] have shown promise, but, to our knowledge, have not yet provided a commercial rt-pcr reagent. to discover new thermostable enzyme activities, we investigated the previously unexplored resource encoded in the genomes of viral populations in thermal springs. viruses are a highly abundant and diverse source of genetic variation [2, 49] and a promising source of new reagent enzymes [1] . a viral metagenomic library originating from a thermal hot spring provided a new enzyme, 3173 pol, with efficient reverse transcription activity and thermostability for pcr. the physiological role of the rt activity of 3173 pol is not clear. lacking a cultivated virus/host combination, the replication mechanism of the source virus can only be inferred from sequence data. based on the method of library construction, the virion has a double-stranded dna genome. thus, the overall viral replication mechanism is distinct from retroviruses. in our experiments, the half-life of 3173 pol at 94uc was 11 minutes compared to more than two hours for taq pol when assayed under the same conditions. a previously reported half-life of taq pol at 95uc is 20 minutes [50] . although the thermostability of 3173 pol is significantly lower than taq and most other commonly used thermostable pols, it is clearly adequate for pcr since product continues to accumulate up to forty cycles (figures 6 and 7) . the combination of thermostability and reverse transcriptase activity in one enzyme has practical implications. because the two enzyme rt systems contain a thermolabile protein component, the use of hot start technologies to improve specificity of reverse transcription is not practical. the 3173 pol should allow ''hot start'' methods to function during reverse transcription as well as amplification, which should improve specificity (data not shown). the thermal profile of 3173 pol ( figure 2 ) shows a peak of activity at 77uc, similar to taq pol, but nearly half of its activity remains at 55uc, significantly higher than the 10-20% reported for taq pol [50] . the higher reverse transcription temperature, combined with the strand displacement activity, should improve specificity and allow synthesis through difficult, structured and g/ c rich rna templates and may have been the basis of the lower amount of spurious products in pyroscript reactions seen in figure 8 . this broad thermal profile, strand displacement and initiation at nicks have enabled certain isothermal amplification schemes [51, 52] (manuscript in preparation). one advantage of a thermostable rt is that the initial lower temperature incubation step can be eliminated, reducing overall reaction time and potentially increasing priming specificity. the high stability of 3173 pol in solution compared to mmlv rt also allows the formulation of a complete pyroscript rt-pcr master mix, which lacks only analyte-specific primers and target. this formulation is stable for over a year at 220uc and simplifies reaction set up and reduces the potential for formulation errors. a drawback of the two-enzyme systems is reduced efficiency during early rounds of rt-pcr amplification of low abundance targets when taq pol is used with mmlv rt [7, 12, 13, 14, 15, 16] . this inhibition has some sequence specificity [12, 16] , which presumably biases amplification and may compromise the measurement of differential gene expression levels and the reliability of internal and external quantification standards. one explanation for this effect is that heating eliminates rt activity without fully disrupting dna binding and this interferes with the efficiency of pcr amplification. the result is an underestimation of low abundance target concentration. if this is true, the availability of a single enzyme that reverse transcribes and amplifies should eliminate this effect. we report other biochemical attributes likely to affect rt-pcr. affinity for template influences the sensitivity and specificity of amplification, but has not been widely described for other pols. this affinity can have an important impact on certain applications. for example, bst pol has a higher template affinity for dna than taq pol, allowing use of lower template concentrations when dna sequencing [53] . also important is affinity for nucleotides. the nucleotide dissociation constants for pol i enzymes from t. thermophilus and three thermostable bacillus species were reportedly between 115 and 85 mm [54] . processivity is probably related to affinity for template. the phi29 pol has a processivity value of greater than 70,000 nt [27] . the processivity of 3173 (47 nt) is comparatively modest but still higher than either bst or taq pols (37 and 9 nt, respectively). while processivity measurements are highly dependent on reaction conditions, the measured result for taq is comparable with previously published values [50] . although it is not as important for detection and quantification applications, fidelity is critical for preparative cdna synthesis methods and for transcriptome sequencing. published methods of fidelity measurement use dna templates [35] . using a variant of these methods, the wild-type 3173 pol had a fidelity of 6.7610 4 similar to our measurements for the most accurate pcr enzymes, pfu and phusion pols (5.8610 4 and 7.5610 4 respectively) when assayed in parallel. an exonuclease deficient mutant of 3173 pol had a pcr fidelity of 0.9610 4 , similar to the value measured for taq pol (1.4610 4 ) and slightly below the reported range of 2.5 to 5.0610 4 for taq pol [32, 35] . published in vitro fidelity measurements for mmlv rt are especially difficult to compare since the assay conditions and temperatures are quite different; however, the reported fidelity for mmlv rt is between 1.7 and 3.0610 4 [55] . measurement of the fidelity of 3173 pol on rna templates will require extensive studies beyond the scope of this report. if the fidelity on rna is similar to the fidelity on dna, 3173 pol could prove especially valuable as an rna sequencing enzyme for transcriptomics research. thus, the determination of 3173 pol fidelity is the basis of ongoing study. the pyroscript mix was comparable in sensitivity to three leading commercial two-step rt-pcr kits when used to detect either ms2 phage or influenza a rna. background amplification in the absence of target, especially after 40 cycles of pcr, is problematic in clinical diagnostic tests where rna target copies may approach single molecule levels. this problem is exacerbated in two-enzyme, one-step rt-pcr since the retroviral rts are not thermostable and background reduction during reverse transcription using hot start methods for these rts are not possible. we found that both the ms2 and the influenza one-step rt-pcr amplification reactions exhibited some propensity for non-specific product formation. however, all of the one-step kits produced background at similar or higher levels. furthermore, these background products were often generated in earlier cycles than the 3173 enzyme false products. the yield of end product formed by the transcriptor and by the superscript reagents appeared greater than those of the quanta and the pyroscript mixes ( figure 5 ), likely due to higher recommended amounts of primers used by these reactions (500 nm vs. 200 nm). however, the higher concentration of primers probably resulted in increased background observed with the transcriptor and superscript reagents. with additional effort, reaction parameters for any of the enzymes could undoubtedly be optimized for specificity or for yield. this higher yield of end product does not appear to affect qpcr results. while the pyroscript enzyme mix shows utility for a range of detection applications, we noted some limitations. the use of 3173 pol for amplification of targets greater than about 350 nt is not reproducible although both the 3173 wild-type and exo-mutants generate pcr products from dna targets up to 5 kb and higher (data not shown). this is consistent with the shorter length of cdna products that we observed in the labeled primer extension experiments ( figure 3b) . figure 3c indicates a small amount of full-length, 714 nt cdna product, although the bulk of product is less than 300 nt. each of these shorter products terminates within a region of secondary structure of the ms2 rna associated with rnase sensitivity [56] so these apparent size limits may reflect labile sites in target rna and may have to do with rna stability at the high extension temperatures used with 3173 pol (72uc) than with inherent properties of the enzyme. most detection modes amplify much shorter targets, but preparative rt-pcr with 3173 pol will likely be affected by this observed limitation. in contrast to the two-enzyme mixes, use of 3173 pol in rt-pcr was significantly more reliable when the primers were designed to anneal at the higher (72uc) annealing/extension temperature of the two step pcr protocol. throughout the rt-qpcr studies, we used dye binding as the detection mode. an alternative detection chemistry uses hydrolysis probes commonly known as taqmanh probes (life technologies) [57] . this chemistry was not tested since the 3173 pol lacks the 59-39 exonuclease activity required to cleave a taqman probe. finally, while the 3173 pol reliably detected high abundance transcripts, as shown in figure 6 , it was noticeably less consistent with less abundant targets. the reason for this inconsistency is not fully understood and is under investigation. one explanation may be that the enzyme is sensitive to high abundances of non-target sequences, typical in total rna extracts. this effect has been seen to a lesser extent with mmlv rt-based rt-pcr [10] and with optimization may be ameliorated. such abundant non-target rna is generally absent in viral rna preparations and the 3173 pol has proven especially useful for detection and quantification of rna viruses. for detection of rna viruses the pyroscript mix appears to be competitive with two enzymes systems that use a retroviral rt and a thermostable taq pol. since rna viruses including influenza, hiv, dengue, west nile and sars coronavirus represent a substantial portion of emerging pathogens worldwide, an improved means of detecting and quantifying these viruses could have an important impact on global health care. table s1 exonuclease assay. the indicated number of units of enzyme were incubated with [33p]-labeled pcr product for 10 minutes at 70uc as described in methods. shown are the percent counts released with background (water-only control) subtracted. not detected is indicated when counts are not significantly above background counts, i.e. ,10%. (doc) functional viral metagenomics and the next generation of molecular tools assembly of viral metagenomes from yellowstone hot springs development of an in vivo assay to identify structural determinants in murine leukemia virus reverse transcriptase important for fidelity rnase h activity is required for high-frequency repeat deletion during moloney murine leukemia virus replication reverse transcriptases and genomic variability: the accuracy of dna replication is enzyme specific and sequence dependent mutations in the rnase h primer grip domain of murine leukemia virus reverse transcriptase decrease efficiency and accuracy of plus-strand dna transfer reverse transcriptase inhibits taq polymerase activity quantitative comparison of est libraries requires compensation for systematic biases in cdna generation evaluation of the cobas hepatitis c virus (hcv) taqman analyte-specific reagent assay and comparison to the cobas amplicor hcv monitor v2.0 and versant hcv bdna 3.0 assays detection limits of several commercial reverse transcriptase enzymes: impact on the low-and high-abundance transcript levels assessed by quantitative rt-pcr comparison of qualitative (cobas amplicor hcv 2.0 versus versant hcv rna) and quantitative (cobas amplicor hcv monitor 2.0 versus versant hcv rna 3.0) assays for hepatitis c virus (hcv) rna detection and quantification: impact on diagnosis and treatment of hcv infections reverse transcriptase (rt) inhibition of pcr at low concentrations of template and its implications for quantitative rt-pcr reverse transcriptase can inhibit pcr and stimulate primer-dimer formation reverse transcriptase can block polymerase chain reaction improved quantitative real-time rt-pcr for expression profiling of individual cells pcr inhibition by reverse transcriptase leads to an overestimation of amplification efficiency variability in the levels of pml-rar alpha fusion transcripts detected by the laboratories participating in an external quality control program using several reverse transcription polymerase chain reaction protocols thermostable dna polymerases dna polymerases from hyperthermophiles compilation, alignment, and phylogenetic relationships of dna polymerases isolation of cloned moloney murine leukemia virus reverse transcriptase lacking ribonuclease h activity reverse transcriptase. the use of cloned moloney murine leukemia virus reverse transcriptase to synthesize dna from rna reverse transcription and dna amplification by a thermus thermophilus dna polymerase cellular dna replicases: components and dynamics at the replication fork the highly processive dna polymerase of bacteriophage t5. role of the unique n and c termini escherichia coli thioredoxin confers processivity on the dna polymerase activity of the gene 5 protein of bacteriophage t7 highly efficient dna synthesis by the phage phi 29 dna polymerase. symmetrical mode of dna replication replication slippage of different dna polymerases is inversely related to their strand displacement efficiency interacting fidelity defects in the replicative dna polymerase of bacteriophage rb69 molecular cloning: a laboratory manual methods of analyzing processivity high-fidelity amplification using a thermostable dna polymerase isolated from pyrococcus furiosus dissecting the role of protein-protein and protein-nucleic acid interactions in ms2 bacteriophage stability basic local alignment search tool pcr fidelity of pfu dna polymerase and other thermostable dna polymerases phosphorothioate primers improve the amplification of dna sequences by dna polymerases with proofreading activity genetic and crystallographic studies of the 39,59-exonucleolytic site of dna polymerase i escherichia coli thioredoxin stabilizes complexes of bacteriophage t7 dna polymerase and primed templates comparison of the 59 nuclease activities of taq dna polymerase and its isolated nuclease domain realtime fluorogenic reverse transcription-pcr assays for detection of bacteriophage ms2 use of bacteriophage ms2 as an internal control in viral reverse transcription-pcr assays base mispair extension kinetics. binding of avian myeloblastosis reverse transcriptase to matched and mismatched base pair termini retroviral reverse transcriptase: synthesis, structure, and function a one-tube method of reverse transcription-pcr to efficiently amplify a 3-kilobase region from the rna polymerase gene to the poly(a) tail of small round-structured viruses (norwalklike viruses) evaluation of the cobas amplicor hbv monitor assay and comparison with the ultrasensitive hbv hybrid capture 2 assay for quantification of hepatitis b virus dna structural determinants of murine leukemia virus reverse transcriptase that affect the frequency of template switching evolving thermostable reverse transcriptase activity in a dna polymerase scaffold chimeric thermostable dna polymerases with reverse transcriptase and attenuated 39-59 exonuclease activity marine viruses-major players in the global ecosystem high-level expression, purification, and enzymatic characterization of full-length thermus aquaticus dna polymerase and a truncated form deficient in 59 to 39 exonuclease activity isothermal reactions for the amplification of oligonucleotides loop-mediated isothermal amplification of dna bst dna polymerase permits rapid sequence analysis from nanogram amounts of template purification and characterization of dna polymerases from bacillus species fidelity of two retroviral reverse transcriptases during dna-dependent dna synthesis in vitro aprotein gene of bacteriophage ms2 oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting pcr product and nucleic acid hybridization induction of p16/ink4a gene expression and cellular senescence by toyocamycin akt and mapk signaling in kgf-treated and uvb-exposed human epidermal cells chemotactic properties of angiopoietin-1 and -2, ligands for the endothelialspecific receptor tyrosine kinase tie2 effect of iodide on human choriogonadotropin, sodium-iodide symporter expression, and iodide uptake in bewo choriocarcinoma cells regional differentiation of retinoic acid-induced human pluripotent embryonic carcinoma stem cell neurons key: cord-290833-m0wodqr3 authors: yuan, lvfeng; zhang, shuai; peng, jie; li, yuchen; yang, qian title: synthetic surfactin analogues have improved anti-pedv properties date: 2019-04-11 journal: plos one doi: 10.1371/journal.pone.0215227 sha: doc_id: 290833 cord_uid: m0wodqr3 surfactin has antiviral activity against various enveloped viruses by inhibiting viral membrane fusion. however, the potential utility of surfactin as an antiviral drug is limited by its cytotoxicity. in this study, 10 surfactin analogues were obtained by chemical synthesis and evaluated to determine their anti-pedv activities, hemolytic activities, and critical micelle concentrations. the main goal of our study was to develop a safer drug; a surfactin analogue with high anti-pedv activity and low hemolytic activity. compared with surfactin, one of the analogues we developed, slp5, has lower hemolytic activity, with the same antiviral activity. the selectivity index of slp5 is 52, while the si for surfactin is 4, in other words, the safe and effective concentration range of slp5 is 12 times greater than that of surfactin. like surfactin, slp5 has a direct antiviral effect on pedv. structurally, slp5 is a linear lipopeptide with three carboxyl groups. surfactin derivatives similar to slp5 could be obtained by lactone bond hydrolyzation of surfactin, as well as total synthesis. surfactin has antiviral activity against a variety of enveloped viruses, including herpes simplex virus (hsv-1, hsv-2), vesicular stomatitis virus (vsv), simian immunodeficiency virus (siv) and newcastle disease virus (ndv) [1, 2] . we recently demonstrated that surfactin exerts its antiviral effects by inhibiting viral membrane fusion [3] . membrane fusion between the viral envelope and the cell membrane is essential for enveloped viruses to invade host cells. surfactin can act directly on virus particles by insertion into the viral envelopes' lipid bilayer and thereby reduce the membrane fusion rate. in addition, since the lipid components of viral envelopes are provided by the host cell, their composition, structure, and function are widely similar in the enveloped viruses. surfactin has antiviral activity against numerous enveloped viruses, and it has promise as a broad-spectrum antiviral reagent, however, the effective dose range of surfactin is narrow, merely 4 x the antiviral concentration causes hemolysis and cytotoxicity. in this study we compared chemically synthesized surfactin analogues to determine future directions for surfactin modification. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 surfactin is a cyclic lipopeptide naturally produced by various strains of bacillus subtilis, the structure consists of a seven amino acid peptide loop and a hydrophobic fatty acid chain. the production of designer surfactins, made by changing the number and composition of amino acids and fatty acids has proven to be an effective strategy for screening large numbers of lipopeptides for biological activity, but most current research focuses on their anticancer [4] , antimicrobial [5] and insulin delivery [6] properties but not on their antiviral potential. fatty acid chain length is critical to the antimicrobial effect of synthetic polymyxin analogues [7] , while the specific antimicrobial spectrum of these analogues depends the amino acids in the lipopeptide [8] . additionally, in synthetic daptomycin analogues, the introduction of aromatic groups in the fatty acid moiety also affects antibacterial activity [9] . in this study, a series of similar lipopeptides were designed and synthesized using surfactin as a template. these analogues differ from each other in the number of hydrophobic amino acids, number of hydrophilic groups, charge properties, amino acid chirality, position of hydrophilic amino acids, and aromatic groups in the fatty chain. porcine epidemic diarrhea virus (pedv), a coronavirus, can infect pigs of all ages, but is especially virulent in newborn piglets, causing diarrhea, dehydration and even death [10] . outbreaks of pedv have been reported in many countries [11] [12] [13] [14] , and have caused immeasurable losses to the global swine industry. recent research in our lab showed that surfactin acts directly on the pedv envelope and inhibits the fusion process with the host cell membrane. in this study, pedv was used as a target to screen chemically synthesized surfactin analogues for enhanced antiviral properties. our results provide a theoretical basis for the development of new surfactin-derivative antiviral drugs. all lipopeptides were synthesized by synpeptide co., ltd (shanghai, china). mass spectrometry was used to determination molecular weight and to confirm product sequences. the purity of all synthetic lipopeptide samples was over 90% by hplc. ten synthetic lipopeptide samples were named slp1 to slp10. and their sequences as table 1 . cercopithecus aethiops kidney epithelial cells (vero, atcc, ccl-81) were cultured in high glucose dmem (gibco, us), supplemented with 10% fetal bovine serum (fbs, gibco), at 37 c in a 5% co 2 humidified atmosphere. cells were routinely seeded at a density of 2×10 5 /ml vero cells were seeded at 2 × 10 5 cells/well in 24-well tissue culture plates and incubated 18-24 h at 37˚c until approximately 95% confluency was reached. 100 pfu of pedv mixed with an equal volume of slp in dmem or dmem alone were incubated 10 minutes at 37˚c, then added into the wells of the vero cells and incubated 30 minutes at 4˚c. cells were washed 3 times with dmem then overlaid with dmem/1% agar and incubated 72 hours at 37˚c. the cells were fixed with 4% formaldehyde, then stained with 0.1% crystal violet after the agar overlay was removed. the data are representative of 3 biological replicates and each plaque assay was performed in triplicate and values are expressed as means ± the standard deviation. curve fitting and ec 50 were calculated with graphpad prism 6 using the log[inhibitor] vs. response equation. hemolytic activity was measured according to the methods described in jingdan [15] , with some modifications. briefly, serial dilutions of slps were added to 200 ul of a 1% suspension of porcine rbcs in pbs, followed by incubation for 1 h at 37˚c. cells were centrifuged at 1000 g for 10 min, then 100 ul of each supernatant was transferred to wells of a 96-well plate. the optical density at 540 nm was measured using a microplate reader (infinite 200 pro, tecan group ltd., switzerland) and % hemolysis was calculated using the formula: where od s , od b , and od p represent the optical density of the slp-treated samples, negative control and positive control, respectively. 1% triton x-100 was used for the positive control. each sample was run in triplicate. surface tensions were measured using the ring method [16] . samples were freshly prepared in a testing flask and allowed to stand for 30 minutes at 22˚c. surface tension measurements were made using a surface tensiometer (hld-lst-ii, henlida co., ltd, china). linear regressions of the drop and the flat area were performed separately for the surface tension-concentration curve. the concentration at the intersection of the two lines is the critical micelle concentration (cmc). time of addition assay was performed according to the procedures described [17] , with modifications. confluent vero cells in 12-well plates were infected with 1000 pfu of pedv and incubated for 1 hr at 4˚c to synchronize infection. the inoculum was removed and 1 ml of 37˚c dmem was added to each well, cells were then placed at 37˚c in a humidified incubator. at the indicated time points, slp5, surfactin, or dielaidoyl-phosphatidylethanolamine (depe, avantipolarlipids, alabaster, al) was added dropwise to cells or virus to a final concentration of 50 μg/ml, 20 μg/ml, or 5 μg/ml and incubated a further 12 hours at 37˚c. viral nucleic acid and protein levels in the cells were measured by qrt-pcr and western-blot respectively. the data shown is representative of 3 independent experiments. total rna was extracted from cells using trizol reagent (invitrogen) according to the manufacturer's instructions. cdna was generated by reverse transcription using hiscript tm qrt supermix for qpcr (vazyme) according to the manufacturer's instructions. pedv nucleic acid levels were assessed by measuring the viral nucleoprotein (n) using qrt-pcr with the takara sybr green qpcr kit (takara). primer sequences were as follows: gene level was calculated using the comparative ct method and normalized to the endogenous levels of gapdh. boiled cell lysates were subjected to sds-page then transferred to pvdf membrane (roche, basel, switzerland) using a semi-dry transfer apparatus (ge, little chalfont, buckinghamshire, uk). membranes were blocked in 5% non-fat milk in tbs containing 0.01% tween-20 (tbst) then incubated overnight at 4˚c with anti-pedv n-protein monoclonal antibody (median diagnostics, south korea) diluted in tbst with 1% bsa. membranes were washed 30 min in tbst followed by incubation for 1 h at rt with 1:5000 hrp goat anti-mouse igg antibody (abgent, san diego, ca, usa) then thrice washed for 15 min in tbst. membranes were incubated with ecl reagent (thermo, waltham, ma, usa) and imaged using a chemidoc™ system (bio-rad, hercules, ca, usa). native surfactin is an amphiphilic cyclic lipopeptide, it consists of a heptadine interlinked with fatty acid. using the native structure as a template, we designed 10 surfactin analogues that were then commercially synthesized ( fig 1a) . slp1 consists of a heptadiene identical to the peptide of native surfactin, and a fatty acid chain the same length as in native surfactin. the n' of heptadine is linked to the fatty acid and the c'-is aminated. slp2 and slp 3 have one and two fewer hydrophobic amino acids respectively, than slp1. relative to spl3, slp4 has one fewer hydrophilic amino acids, while slp5 has a free carboxyl at its c'-end that acts as an additional hydrophilic group. slp6 replaces the two acidic amino acids of slp 3 with lysine (a basic amino acid). slp7 is a version of slp3 that contains only l-amino acids. the two hydrophilic amino acids of slp8 are distant from each other, but are in close proximity in slp9. slp 10 is identical to slp3 except for a diphenyl group in the fatty acid moiety. the mass spectra of the slps are shown in fig 1b and are consistent with the expected molecular weights. in all cases, slp purity exceeded 90%. the slps were tested for anti-pedv activity using a plaque reduction assay. serial dilutions of slps were incubated with an equal volume of pedv then aliquoted onto precooled cells in 12-well plates. after the virus has adsorbed to the cells at 4˚c, slps were washed away. the adsorbed virus particles were then counted by plaque forming assays (fig 2a) . the results were fitted to a sigmoid-curve and plotted fig 2b. all slps have potent anti-pedv effects above 50 μg/ml, but differences in their anti-pedv effect. slp2, slp4, slp6, and slp8 have a higher potency than surfactin. hemolysis is commonly used to assess the biosafety of lipopeptides [18, 19] . as shown in fig 3, slps have a range hemolytic activities. those of slp1, slp2, slp3, and surfactin are similar, suggesting that the number of hydrophobic amino acids has little effect on hemolytic activity. however, other modifications significantly affect activity; slp4, slp3 and slp5 have one, two and three carboxyl groups respectively, and the hemolytic activity is reduced in turn. in addition, the chiral differences that distinguish slp7 and slp3 at two amino acid residues, and the differences in the arrangement of amino acids between slp8 and slp9 also affect hemolytic activity. these results indicate that amino acid composition by itself does not completely determine the characteristics of slps. it is noteworthy that slp8 has an unexpected effect on porcine erythrocytes. although slp8 has a low hemolytic activity, it prevents the precipitation of red bloods under our assay conditions at concentrations above 50 μg/ml. the selectivity index (si) was determined as the ratio of the 50% cytotoxicity concentration (cc 50 ) to the 50% effective concentration (ec 50 ). as shown in table 2 , slp8 and slp5 have the highest and second-highest si values respectively. given the anti-sedimentation effect of slp8 on porcine blood cells, slp5 was selected as the most promising candidate. although slp4 and slp6 have stronger antiviral activity than surfactin, their si is lower due to their high hemolytic activity. the cmc was calculated by measuring the surface tension of each slp over a range of concentrations. as shown in fig 4, surface tension decreases as slp concentration increases, and as it approaches its lowest value, the curve exhibits an inflection point when the slp concentration reaches cmc. linear regressions were performed independently using the data on the descending part of the curve below the cmc and the equilibrium part above the cmc. the intersection of the linear regression lines corresponds to the cmc (table 3) . in order to explore the relationship between cmc, anti-pedv activity, and hemolytic activity, scatter plots for pairs of assays (fig 5) . each plot contains 11 points representing the characteristic indices of slp1 to 10 and surfactin. the distribution of points has no obvious regularity in any of the three plots, and the linear regression results are not significant. we conclude that the anti-pedv and hemolytic activities of the slps examined in this study are not related to their surfactant activity (represented by the cmc). in addition, there is no significant correlation between anti-pedv activity and hemolytic activity. synthetic lipopeptides that combine greater antiviral activity with lower cytotoxicity are still to be found, and the specific structure-activity relationship needs to be further elucidated. since slp5 was the most promising surfactin analogue, we chose it for further study. time of addition assays were performed to determine whether the slp5 exerts its anti-pedv effect at the same stage during infection as surfactin. since surfactin acts directly on virions, the experiment was designed to examine events early in the infection process. fig 6a summarizes the experimental plan and the eight treatments tested. briefly, slp5, surfactin, or depe was added to virus alone, cells alone, or to cells and virus together prior to infection, during virus adsorption, during virus invasion (1 h post infection at 37˚c), or during replication (1-12 hpi). samples were harvested 12 hours after infection, and analyzed to measure cellular levels of viral protein (fig 6b and 6c ) and viral rna ( fig 6d) . as expected for a normal component of the cell membrane, depe did not affect pedv replication at any stage, while slp5 and surfactin exhibited antiviral activity at specific stages. for example, when slp5 or surfactin are present throughout the experiment (treatment group 2), little pedv replication occurs, as indicated by nucleic acid and protein levels. the same is true in treatment group 5, indicating that both compounds act directly on the virus. other antiviral mechanisms of slp5 and surfactin cannot be ruled out since pedv replication is inhibited to different degrees in some of the other treatment groups. wedge-shaped lipids in which the hydrophilic head has a larger cross-sectional area than the hydrophobic tail, are potential membrane fusion inhibitors [20, 21] . in a recent study of rigid amphipathic fusion inhibitors, compounds with deoxyribose or acetate as hydrophilic moieties had anti-hsv effects [22] . in addition, a recent study on antimicrobial activity of synthetic lipopeptides reported that lipopeptides with two to four positive charges and 16 carbon atoms in the lipid chain have potent antimicrobial activity [23] . fatty acid chain length from 8 to 16 carbon atoms is positively correlated with antimicrobial activity, but is also positively correlated with hemolytic activity and membrane selectivity [24] . these factors must be considered in the design of new lipopeptides, and the structure-function relationships warrant further study. here we investigated lipopeptides containing two and three negative charges, and two positive charges, all with a fatty acid chain of 16 carbon atoms. all of these lipopeptides had antiviral activity. in a previously published study we demonstrated that surfactin has antiviral activity as membrane fusion inhibitor [3] . in order to improve the viral envelope selectivity of surfactin, we designed 10 analogues with altered peptide amino acids, but kept the length of fatty acid chain of 16 carbon atoms. compared with slp3, slp5 has one additional carboxyl group at the end of the peptide, but the antiviral effect is similar. although slp4 has one less hydrophilic amino acid than slp3, its antiviral activity increases about 3-fold. this result indicates that the hydrophilic portion of the wedge-shaped lipid needs to be sized appropriately for better antiviral function. in addition, slp8 differs from slp3 only in the order of two amino acids, but the antiviral potency of slp8 is 6.5 times greater, indicating the complexity of the relationship between molecular structure and antiviral effects. the cationic properties of synthetic lipopeptides are thought to be related to their antibacterial and hemolytic activities [25] . slp6, the only cationic lipopeptide examined in this study, has the strongest hemolytic activity, consistent with this hypothesis. cyclic lipopeptides are reported to more readily lyse bacterial membranes than linear or branched lipopeptides [26] . in agreement, we found that the hemolytic activity of slp1 (a linear molecule, identical in amino acid sequence to surfactin) is slightly weaker than that of surfactin, which has a cyclic structure. it has also been reported that the total hydrophobicity of synthetic lipopeptides is inversely related to hemolytic activity, although this conclusion was from data on only 3 related samples [27] . our results do not support this conclusion. the relative hydrophobicity of slp1, 2, and 3 is slp1>slp2>slp3, as indicated by their hplc retention times (17.6, 16.7, and 14.5 min, respectively). however, their hemolytic activity is not significantly related to the total hydrophobicity. in summary, we synthesized 10 surfactin analogues and characterized them to identify those with enhanced anti-viral properties. slp5 equaled and slp8 exceeded surfactin's anti-pedv activity and both compounds had greatly reduced hemolytic activity, making their selection indexes 13 and 21 times larger respectively than surfactin's. however, slp8 exhibited a peculiar inhibition of red blood cell sedimentation. slp5 is therefore a more promising synthetic surfactin analogue. compared with surfactin, the structure of slp5 is distinguished by its linear lipopeptide and the additional carboxyl group at the c' of the peptide. slp5 also has two fewer hydrophobic amino acids than surfactin, this reduces the cost of synthesis while having little effect on antiviral activity. since surfactin has been shown to protect piglets from pedv challenge [3] , the in vivo antiviral properties of surfactin analogues needs to be tested. furthermore, with respect to the future use of synthetic surfactins to control or ameliorate infection in pigs, the broader spectrum physiological activities of surfactin analogues needs further study and side effects beyond hemolytic toxicity need to be explored. our study suggests that slp5, and other surfactin analogues, should be the object of in-depth study in order to develop a safer broad-spectrum family of surfactin antiviral agents. synthetic surfactin analogues have improved anti-pedv properties conceptualization: lvfeng yuan, qian yang. the pedv genome was detected by qrt-pcr. the experiment was repeated three times, normalized against group 1, and plotted as mean ± sd. � , p < 0.05; �� , p < 0.01 in two-tail t-test, for each reagent, compared with group 1. https://doi.org/10.1371/journal.pone.0215227.g006 synthetic surfactin analogues have improved anti-pedv properties mechanism of inactivation of enveloped viruses by the biosurfactant surfactin from bacillus subtilis antiviral activity of antimicrobial lipopeptide from bacillus subtilis fmbj against pseudorabies virus, porcine parvovirus, newcastle disease virus and infectious bursal disease virus in vitro surfactin inhibits membrane fusion during invasion of epithelial cells by enveloped viruses a multicomponent macrocyclization strategy to natural product-like cyclic lipopeptides: synthesis and anticancer evaluation of surfactin and mycosubtilin analogues semisynthetic lipopeptides derived from nisin display antibacterial activity and lipid ii binding on par with that of the parent compound surfactin variants for intra-intestinal delivery of insulin probing the penetration of antimicrobial polymyxin lipopeptides into gram-negative bacteria a bioinspired peptide scaffold with high antibiotic activity and low in vivo toxicity discovery and development of surotomycin for the treatment of clostridium difficile outbreak of porcine epidemic diarrhea in suckling piglets, china. emerging infectious diseases porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus porcine epidemic diarrhea virus and discovery of a recombinant swine enteric coronavirus, italy. emerging infectious diseases pig jejunum protein profile changes in response to a porcine epidemic diarrhea virus challenge comparison of porcine epidemic diarrhea viruses from germany and the united states a potential biopreservative: chemical composition, antibacterial and hemolytic activities of leaves essential oil from alpinia guinanensis surface tension examination of various liquid oral, nasal, and ophthalmic dosage forms discovery of cyclosporine a and its analogs as broad-spectrum anti-influenza drugs with a high in vitro genetic barrier of drug resistance improving the biological activity of the antimicrobial peptide anoplin by membrane anchoring through a lipophilic amino acid derivative interaction of acylated and substituted antimicrobial peptide analogs with phospholipid-polydiacetylene vesicles. correlation with their biological properties. chemical biology & drug design 5-(perylen-3-yl)ethynyl-arabino-uridine (auy11), an arabino-based rigid amphipathic fusion inhibitor, targets virion envelope lipids to inhibit fusion of influenza virus, hepatitis c virus, and other enveloped viruses rigid amphipathic fusion inhibitors, small molecule antiviral compounds against enveloped viruses antivirals acting on viral envelopes via biophysical mechanisms of action cationic net charge and counter ion type as antimicrobial activity determinant factors of short lipopeptides characterization of antimicrobial and hemolytic properties of short synthetic cationic lipopeptides based on qsar/qstr approach roles of hydrophobicity and charge distribution of cationic antimicrobial peptides in peptide-membrane interactions short cationic lipopeptides as effective antibacterial agents: design, physicochemical properties and biological evaluation identification of novel cyclic lipopeptides from a positional scanning combinatorial library with enhanced antibacterial and antibiofilm activities key: cord-334218-bkjfy66e authors: lin, jung-da; lin, chuen-fu; chung, wen-bin; chiou, ming-tang; lin, chao-nan title: impact of mated female nonproductive days in breeding herd after porcine epidemic diarrhea virus outbreak date: 2016-01-15 journal: plos one doi: 10.1371/journal.pone.0147316 sha: doc_id: 334218 cord_uid: bkjfy66e porcine epidemic diarrhea virus (pedv) is an important pathogen that has a significant economic impact on the swine industry by imposing a high rate of mortality in suckling piglets. however, limited information on the productivity values of gilts and sows infected with pedv is available. here, we evaluate the productivity index in gilts and sows during the 1-year period before (19 january 2013 to 18 january 2014) and after (19 january 2014 to 18 january 2015) a pedv outbreak from a 2000-sow breeding herd in taiwan. the farrowing rate (fr), return rate (rr), total pigs born per litter (tb), pigs born alive per litter (ba), weaning pigs per litter (wpl), pre-weaning mortality, percentage of sows mated by 7 days after weaning, weaning to first service interval (wfsi), mated female nonproductive days (npds), replacement rate of sows and sow culling rate were compared using productive records. the fr (-9.6%), rr (+9.8%), tb (-1.6), ba (-1.1), wpl (-1.1), sows mated by 7 days after weaning (-6.9%), wfsi (+0.8 days), npds (+6.9 days) and sow culling rate (+7.2%) were significantly different between the 1-year pre-pedv outbreak period and the post-pedv outbreak period. impacts of the pedv infection on the reproductive performance were more severe in pregnant gilts than in sows. in conclusion, these findings indicate that the outbreak of pedv caused an increase in the rate of npds in breeding herds. porcine epidemic diarrhea (ped) is an important swine disease that causes a significant impact in most pig-producing countries [1] . the causative agent, the ped virus (pedv), belongs to the genus alphacoronavirus, family coronaviridae, and order nidovirales [1] . although pedv was first observed in europe in the early 1970s [2] , it has become an increasing problem worldwide, including in the americas [3] [4] [5] , asia [6] [7] [8] [9] [10] [11] and europe [12, 13] . the devastating effect of pedv infection is mainly due to the acute watery diarrhea and dehydration induced in infected pigs that not only leads to high (80-100%) mortality in neonatal piglets [3, 9] but also impairs the health and performance of the surviving pigs [14] . the impact of pedv infection on the reproductive performance of gilts and sows depends on the period of pregnancy, during which females are exposed to the pathogen and the parity number [15] . the farrow rate (fr) (-3.8%), percentage of stillborn piglets per litter (+1.8%), and percentage of mummified fetuses per litter (+1.1%) were significantly different during the 4-month period of the pedv outbreak compared with the same period in the year before the outbreak [15] . however, limited information on the productivity index of the gilts and sows that were exposed to the pedv during the 1-year period of the pedv outbreak is available. mated female nonproductive days (npds) in the herd with the pedv outbreaks have not been reported. npds are the days that a mated sow or gilt is present in the herd and is neither gestating or lactating [16] . the formula for calculating npd is npd = 365−[(litter/female/year) × (gestation days + lactation days)]. several factors affect the npds [16] : i) replacement gilt days, entry to first service, entry to culling and entry to death; ii) weaning-to-first service days (the number of days from weaning until a female is mated again); iii) first to repeat service interval (days to find re-cycling females after breeding); iv) weaning to removal period; and v) death loss and gestation days that do not result in farrowing. therefore, npds represent key performance indicators of breeding herd performance. the objectives of the present study were to investigate the effects between a 1-year period before and after pedv outbreak on a sow's reproductive traits on a commercial pig farm in taiwan. this was a retrospective study that did not require direct intervention, retrieval of clinical specimens or animal experiments. the pig owners provided written consent for data collection and publication. no specific permissions were required for the location of the data because the data were collected with swine management software. the present study was conducted on a 2000-sow commercial pig farm in the central region of taiwan. the name of the pig farm is mai-chung pig farm with the following geographical coordinates (latitude/ longitude): 23°46 0 14.2@n and 120°14 0 56.0@e. on average, the productivity index of the study herd before the outbreak of pedv was superior to that recorded in other breeding herds in taiwan. the health of the herds was monitored by the herd veterinarian and the animal disease diagnostic center (addc), national pingtung university of science and technology (npust). the majority of the females were crossbreed landrace × yorkshires that were produced from their own grandparent stock. the veterinarian recommended vaccinating gilts against foot-and-mouth disease virus (fmdv), classical swine fever virus (csfv), aujeszky's disease (adv), porcine parvovirus (ppv), porcine circovirus type 2 (pcv2) and atrophic rhinitis between 24 and 30 weeks of age in replacement gilts. mass vaccinations for adv, pcv2 and fmdv were conducted in the sows every 4 months, 6 months and year, respectively. vaccinations of sows against csfv and porcine reproductive and respiratory syndrome virus (prrsv) were conducted on weaning day. no sows exhibited adv, pcv2, ppv, prrsv or bacterial abortion (streptococcus suis, erysipelothrix spp, leptospira interrogans) in this breeding herd during the study period as monitored by the herd veterinarian and the addc, npust through molecular diagnosis and serological surveillance. the target replacement rate of the sows by gilts was approximately 48% annually. sow culling due to age was planned to occur after the sixth parity, after the second return or vulva discharges at 14-21 days postservice. on 18 january 2014, pedv infection was confirmed from this pig farm by addc, npust [9] . farm immunization was performed using twice feedback with a 2-week interval for gilts and sows with approximately 10 ml of the homogenized intestines collected from pedv-infected suckling piglets (1 piglet for 20 sows on average). after the first feedback, more than 90% of the gilts and sows showed clinical signs of anorexia, diarrhea and vomiting. stool specimens were positive for pedv using real-time pcr assays conducted by addc, npust. less than 5% of the gilts and sows showed clinical signs of mild diarrhea and vomiting in the second feedback. suckling piglet mortality reduced and pedv-signs of gilts and sows stopped within 4 weeks after the first feedback immunization. until early august 2014, pig owners worried about the reemergence of pedv. therefore, feedback of gilts and sows was conducted with approximately 10 ml of the homogenized frozen intestines from pedv-infected suckling piglets (1 piglet for 100 sows on average) for three consecutive days. after this feedback event, more than 80% of the gilts and rarely sows showed clinical signs of mild anorexia, diarrhea and vomiting. no feedback immunization has been performed in this pig herd since august 2014. there were 20 and 8 sows (approximately 60% of them are primiparous sows) that were observed to have endemic pedv infections in early december 2014 and september 2015, respectively. these pedv infections were confirmed by addc, npust. during the post-pedv period, all of the gilts and sows were exposed to pedv by natural or/and feedback routes. data on gilt and sow reproductive traits were obtained from the swine management software of the herd from 19 january 2013 to 18 january 2015 (porcitec 2009 version, agritec). the collected data included sow identities, mating dates, mating results, number of days until the sows returned to estrus after mating, fr, return rates (rr), litters/mated female/year (lmfy), percentage of sows mated by 7 days after weaning, weaning to first service intervals (wfsi), farrowing intervals (fi), npds, replacement rate of sows, sow culling rate, total pigs born per litter (tb), pigs born alive per litter (ba), weaning pigs per litter (wpl) and pre-weaning mortality. the reproductive data from before and after the pedv outbreak were collected during periods from 19 january 2013 to 18 january 2014 and from 19 january 2014 to 18 january 2015, respectively. npds of the different parities (1st, 2nd and >2nd) during the 1 year pre-(2013) and post-(2014) pedv outbreak were compared using a two-way anova for multiple comparisons. the average number of mated females, average parity of farrowed sows, number of matings, number of farrowings, fr, rr, number of abortions, lmfy, percentage of sows mated by 7 days after weaning, wfsi, fi, npds, replacement rates of sows and sow culling rates of preand post-pedv outbreak periods were compared using a mann-whitney test. p values < 0.05 and <0.01 were considered to be statistically significant and highly significant, respectively. the productivity index of the sows in the herd during the 1-year period pre-and post-pedv outbreak is presented in table 1 . the number of matings showed a 58-point increase during the post-pedv outbreak period; however, the number of farrowings showed a 214-point decrease during this period. the average parity of the farrowed sows was significantly higher post-pedv outbreak compared with the period before the pedv outbreak (3.8 vs. 3.5). one year before the pedv outbreak, the fr, rr and number of abortions in the herd were 90.5%, 8.1% and 20, respectively (table 1) . however, a 9.6 percentage point decrease in fr (p<0.001) ( table 1 and fig 1a) , 9.8 percentage point increase of rr (p<0.001) ( table 1 and fig 1a) and a 4.8% increase of abortion rates (p = 0.0288) ( table 1) were observed after the pedv outbreak (table 1) . additionally, accounting for parity, the influence of the pedv outbreak on the fr and rr was more pronounced in pigs having their initial pregnancies (fig 1b and 1c ). interestingly, the number of abortions rapidly increased during the pedv outbreak period (19 january to 18 february 2014) (fig 2) . the abortion rate (ar) after the pedv outbreak was significantly higher than the ar before the pedv infection (+4.8%, p = 0.028) ( table 1) . together, these results indicated that the reduction in reproductive performance was more severe in pregnant gilts than in pregnant sows during the post-pedv outbreak period. litter size at birth and weaning tb, ba, total weaning pigs, wpl and pre-weaning mortality during the 1-year period pre-and post-pedv outbreak are shown in table 2 . tb (p<0.001), ba (p<0.001) and wpl (p<0.001) decreased significantly after the pedv outbreak compared with the 1-year period before pedv infection. the suckling piglets infected with pedv during the disease outbreak period (19 january to 18 february 2014) had a 41.7 percentage point increase in pre-weaning mortality (17.3% vs. 59%) compared with the same period in the year before the outbreak (19 january to 18 february 2013) (data not shown). * and ** were considered statistically significant and very highly significant, respectively. npds, replacement rate of sows and culling rate the percentage of sows mated within 7 days post-weaning, wfsi, fi, npds, lmfy, replacement rates of sows and sow culling rates are listed in table 1 . one year after the pedv outbreak, we recorded a 6.9 percentage point decrease in the sows mated within 7 days after weaning (p = 0.0121) ( table 1) , 0.8 percentage point increase in wfsi (p = 0.0131) ( table 1) , 3-day increase in fi (p = 0.0035) and 6.9-day increase in npds (p = 0.0126) ( table 1) , whereas the influence of the pedv outbreak on the lmfy (p = 0.0681), replacement rate of sows (p = 0.9206) and sow culling rate (p = 0.0575) was not significantly different between the preand post-outbreak periods. in addition, when parity was taken into account, the influence of the pedv outbreak on the npds was more pronounced in pigs with initial pregnancies (fig 3) . the percentage of sows mated within 7 days post-weaning was related to the wfsi, which is one of the factors that affected the npds. interestingly, the percentage of sows mated within 7 days post-weaning declined (from 86.7% to 45.6%) during the pedv outbreak period (19 january to 18 february 2014) compared with the same period in the year before the pedv outbreak (19 january to 18 february 2013) (fig 4) . the wfsi was highly variable during the post-pedv outbreak period compared with the period 1 year before the outbreak (fig 5) . in addition to this pig farm, we also analyzed the productivity index during the 1-year period before and after pedv outbreaks from one taiwanese farrow-to-finish herd (500 sows). the results showed that there were impacts on reproductive performance after pedv infection. we recorded a 6.2-day increase in npds during the post-outbreak periods (data not shown). taken together, these results indicate that there was a significant increase of the npds after the pedv outbreaks occurred, especially in pregnant gilts. the devastating effect of pedv infection is primarily due to the acute watery yellowish diarrhea and dehydration, with mortality rates ranging from 80 to 100% in suckling piglets under 2 weeks of age [9] . however, only a few studies have attempted to assess the impact of pedv infection on the reproductive and growth performance of sows [15] and surviving pigs [14] , respectively. in the present study, we compared the productivity index of gilts and sows between 1 year pre-and post-pedv outbreak in a taiwanese breeding herd. comparison of the fr (80.9% vs. 87.5%), rr (17.9% vs. 5.0%), ar (6.5% vs. 2.7%), tb (-1.6 vs. +0.3), ba (-1.1 vs. -0.1) and pre-weaning mortality (59% vs. 49.2%) revealed that post-pedv period effects were more severe than those observed in a study conducted in thailand [15] . this finding may be due to the following: i) the different observation periods (a 1-year period in the present study vs. a 4-month period in the thailand study); ii) the endemic pedv outbreak was present in this herd after the pandemic outbreak of pedv and iii) the different strains of pedv (us-like strain in taiwan vs. chinese-like strain in thailand) [9, 15, 17] . overall, these two studies consistently observed that the influence of the pedv outbreak on fr and rr was more pronounced in pigs that are early in their pregnancy. npds are key performance indicators of breeding herd performance. some factors that may affect npds include the following: i) replacement gilt timing, ii) weaning-to-first service days, iii) first service to repeat service intervals, iv) weaning to removal intervals, and v) death losses. to our knowledge, this is the first report showing the influence of pedv on npds in gilts and sows. factors that contributed to prolonging npds include increases in rr, number of abortions, percentage of sows mated within 7 days after weaning, wfsi and fi. in general, lactation levels declined during the pedv outbreak, especially in infected herds with high suckling mortality. incomplete uterine involution and tissue repair in early weaned sows contributed to the increased embryo loss in infected herds. the percentage of sows mated within 7 days after weaning rapidly declined (86.7% from 19 january to 18 february 2013 vs. 45.6% from19 january to 18 february 2014) during the pedv outbreak period, resulting in an increase in wfsi. a significant increase in ar was also observed by pijpers et al. [18] and olanratmanee et al [15] , although the mechanism underlying rr and fr is not known. previously, studies have shown that lactation intervals not only affect the average number of days from weaning to estrus but also the pregnancy rates and number of live embryos per female [19] . this fact explains why the fr and tb significantly decreased after the pedv outbreak in the present study. our results revealed that the influence of the pedv outbreak on npds was more pronounced in primiparous sows. the primiparous sows exhibited severe clinical signs of anorexia, diarrhea and vomiting when infected with pedv, while young sows were still utilizing nutrients for both growth and maintenance of the reproductive function. previous studies have reported that i) increasing feed intake during lactation can increase luteinizing hormone secretion and reduce the weaning-to-estrous and farrowing-to-estrous intervals in primiparous sows [20] , ii) protein (lysine) restriction throughout lactation alters circulating concentrations of somatotropic hormones and insulin at the end of lactation and has a negative impact on the post-weaning ovulation rate in primiparous sows [21] , iii) low lysine levels in primiparous lactating sows impaired follicular development and reduced the ability of follicles to support oocyte maturation [22] and iv) low-parity sows were more sensitive to lactational feed intake than high-parity sows in terms of wfsi [23] . we recorded a 0.8-day increase in wfsi post pedv infection (table 1) , occurring prominently in primiparous sows (fig 5) . these facts may explain why the influence of the pedv outbreak on npds was more pronounced in primiparous sows. additionally, maximizing feed intake during lactation is critical to improve the overall sow reproductive performance including productivity and longevity [24] . until now, pedv has not been considered as direct cause of reproductive problems. therefore, inadequate feed and nutrient intake when pedv infection may be cause the excessive body weight loss that can lead to short-term reproductive problems such as extended wfsi and smaller subsequent litter size. in the long run, problems such as higher culling rate of the breeding herd will result in low average parity; reduced pigs weaned per reproductive lifetime and increased production cost. in general, all phases of the reproductive cycle are related [24] . overall, these facts may provide an explanation to why pedv infection has such long-term impact on sow reproductive performance. the impact of pedv on npds in gilts and sows may be controlled or improved by the following methods: i) reducing the abortion rate (performance of the feedback immunization must be avoided when the gilts and sows are in their first month of pregnancy [15] ), ii) decreasing lactation length [25] , iii) decreasing the percentage of gilts in the breeding herd inventory [25] , iv) decreasing the female culling rate [25] [26] [27] and v) increasing the percentage of multiple matings [25] [26] [27] . the pig owners did not alter the gilt and sow management, including lactation length (25.6 days vs. 25.7 days)( table 1) , percentage of multiple matings (99.7% vs. 98.7%)( table 1) , parity of culled sows (sow culling due to age was planned to occur after the sixth parity), and percentage gilts in the breeding-female inventory (gilt pool: 1019 vs. 971), etc. (table 1) . therefore, the reproduction indices were normal until the second year after the first pedv outbreak. we recorded a 91.7 percentage of sows mated within 7 days post-weaning, 12.9 percentage of rr, 5.8-day of wfsi and 40.5-day of npds during the second year of the pedv outbreak (19 january 2015 to 18 november 2015) (s1 table) . overall, the control and improvement of impact of pedv on npds in gilts and sows may alter the management of females. outbreaks of pedv not only lead to high mortality in neonatal piglets and a poorer performance of surviving pigs but also impair the productivity index in gilts and sows. this is the first report to show the influence of pedv on npds in gilts and sows. these findings should contribute to an understanding of the effects of pedv outbreak post-infection on sow herds and to an identification of ways to curtail losses as a result of this disease. porcine epidemic diarrhea: a review of current epidemiology and available vaccines letter to the editor emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences investigation into the role of potentially contaminated feed as a source of the first-detected outbreaks of porcine epidemic diarrhea in canada distinct characteristics and complex evolution of pedv strains molecular characterization of the porcine epidemic diarrhea virus tw4/2014 in taiwan comparative genome analysis and molecular epidemiology of the reemerging porcine epidemic diarrhea virus strains isolated in korea outbreak-related porcine epidemic 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blood concentrations of glucose, insulin and luteinizing hormone in primiparous sows protein (lysine) restriction in primiparous lactating sows: effects on metabolic state, somatotropic axis, and reproductive performance after weaning impact of dietary lysine intake during lactation on follicular development and oocyte maturation after weaning in primiparous sows feed intake pattern during lactation and subsequent reproductive performance of sows nutrition of the sow management factors associated with swine breedingherd productivity in the united states six component intervals of nonproductive days by breeding-female pigs on commercial farms by-parity nonproductive days and mating and culling measurements of female pigs in commercial breeding herds key: cord-333413-8buawes0 authors: liebing, j.; völker, i.; curland, n.; wohlsein, p.; baumgärtner, w.; braune, s.; runge, m.; moss, a.; rautenschlein, s.; jung, a.; ryll, m.; raue, k.; strube, c.; schulz, j.; heffels-redmann, u.; fischer, l.; gethöffer, f.; voigt, u.; lierz, m.; siebert, u. title: health status of free-ranging ring-necked pheasant chicks (phasianus colchicus) in north-western germany date: 2020-06-16 journal: plos one doi: 10.1371/journal.pone.0234044 sha: doc_id: 333413 cord_uid: 8buawes0 being a typical ground-breeding bird of the agricultural landscape in germany, the pheasant has experienced a strong and persistent population decline with a hitherto unexplained cause. contributing factors to the ongoing negative trend, such as the effects of pesticides, diseases, predation, increase in traffic and reduced fallow periods, are currently being controversially discussed. in the present study, 62 free-ranging pheasant chicks were caught within a two-year period in three federal states of germany; lower saxony, north rhine-westphalia and schleswig-holstein. the pheasant chicks were divided into three age groups to detect differences in their development and physical constitution. in addition, pathomorphological, parasitological, virological, bacteriological and toxicological investigations were performed. the younger chicks were emaciated, while the older chicks were of moderate to good nutritional status. however, the latter age group was limited to a maximum of three chicks per hen, while the youngest age class comprised up to ten chicks. the majority of chicks suffered from dermatitis of the periocular and caudal region of the head (57–94%) of unknown origin. in addition, intestinal enteritis (100%), pneumonia (26%), hepatitis (24%), perineuritis (6%), tracheitis (24%), muscle degeneration (1%) and myositis (1%) were found. in 78% of the cases, various mycoplasma spp. were isolated. mycoplasma gallisepticum (mg) was not detected using an mg-specific pcr. parasitic infections included philopteridae (55%), coccidia (48%), heterakis/ascaridia spp. (8%) and syngamus trachea (13%). a total of 8% of the chicks were avian metapneumovirus (ampv) positive using rt-pcr, 16% positive for infectious bronchitis virus (ibv) using rt-pcr, and 2% positive for haemorrhagic enteritis virus (hev) using pcr. all samples tested for avian encephalomyelitis virus (aev), infectious bursal disease virus (ibdv) or infectious laryngotracheitis virus (iltv) were negative. the pool samples of the ten chicks were negative for all acid, alkaline-free and derivative substances, while two out of three samples tested were positive for the herbicide glyphosate. pheasant chick deaths may often have been triggered by poor nutritional status, probably in association with inflammatory changes in various tissues and organs as well as bacterial and parasitic pathogens. theses impacts may have played a major role in the decline in pheasant populations. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the original distribution area of the ring-necked pheasant (phasianus colchicus) ranged from the black sea over the dry areas of central asia to the east of asia to south korea and siberia [1] . the romans introduced the pheasant to europe around 500 ad, from where it spread through regular release throughout central and western europe [2] . according to current published data, the pheasant mainly prefers structurally semi-open land, using trees and hedges as cover, and also occupies adjacent sparse forests and reedy areas [3] . most pheasants seek shelter under trees to be protected from natural predators. however, some subspecies spend the night on the ground or among dense reeds. their resting places during the day are usually well-hidden hedges, where sand-baths are taken in carved hollows [1] . adult pheasants mainly feed on plants, consuming different parts of the plant such as seeds, berries, tubers, root shoots and leaves, as well as green sprouts. however, on occasions, their diet is supplemented by animal protein, preferably in the form of insects [1] . for chicks, smaller ground-level insects are especially important during the first weeks of life. they feed on a variety of species of insects such as spur cicadas (delphacidae), bugs (heteroptera), sawfly wasps (tenthredinidae) and butterfly caterpillars (lepidoptera larvae) [1, 4, 5] . this diversity is particularly important for a healthy growth [6, 7] . for example, a diet based only on aphids can lead to delayed plumage development due to inadequate amino acid supply [8] . in germany, the pheasant is a typical soil-breeding bird of the agricultural landscape. the main part of the german population is found in southwest lower saxony, north rhine-westphalia and schleswig-holstein. the population level reached its plateau between 1960 and 1970 in lower saxony. during this period, the hunting bag statistics (state registered numbers of hunting animals, in this case pheasants), i.e. the absolute number of pheasants killed, amounted to approximately 300,000 pheasants in germany [9] . in the severe winter of 1970 and the following wet spring of 1971, the population of pheasants and many other wild living animals declined [9, 10] . the hunting bag was reduced to an average of about 80,000 pheasants and declined further. not only was the pheasant population subjected to this decline, but also that of many other farmland birds [11, 12, 13] . around 2007/2008, the population showed another severe decline of unknown cause. in germany, the renewable energy sources act (erneuerbare-energie-gesetz: renewable energy sources act describes the implementation of ecological energy generation in germany) amendment of 2004 with an advancement in biogas, triggered the doubling of corn cultivation. consequently, huge areas of fallow land disappeared in lower saxony [14] . the contributory factors to the ongoing decline in the pheasant population, such as the effects of pesticides, infectious agents, predation, increasing traffic and human populations as well as reduced fallow periods, are currently the subject of controversial discussion among different stakeholders [15, 16, 17, 18] . some authors see a correlation between the changes in agriculture and the decline in the populations of many farmland birds [19, 20] . in the third week of life, 70% of the chicks' diet consists of insects. gradually, the insect percentage is reduced. from the sixth week of life, the diet is similar to that of adult birds. previous studies [21, 22] associated the decline in the number of many farmland birds with the use of insecticides. if chicks are unable to find a sufficient number of insects during the first weeks of life, they have to search a larger range of their habitat, which can lead to malnutrition and weakening. thus, harmless ubiquitous pathogens may have negative effects on chicks [23, 24, 25, 26, 27, 28] . investigations carried out led to the assumption that there is no specific epidemic infectious agent currently circulating in the adult pheasant population [29] . many hunters report that especially the number of chicks has declined, with more older birds making up the hunting bag. however, the authors found serological evidence of certain viruses (infectious bronchitis virus (ibv), avian encephalomyelitis virus (aev) and infectious bursal disease virus (ibdv)) which typically cause chick mortality. these pathogens infected adult and young pheasants, but the pathogenicity in chick and subadult populations is considerably more serious than in adult birds [29, 30] . in addition, other factors may weaken the population and pathogens become more important. based on these findings, our study focused on pheasant chicks up to eleven weeks of age. previous studies on pheasants indicated that the most sensitive age class for infectious diseases was pheasant chicks, possibly due to a higher susceptibility [30, 31] . the aim of our research was to assess the health state of free-living pheasant chicks in order to check the animals for lesions indicative of infections or toxic substances. the findings should contribute to understanding the causes for the decline in the pheasant population in north-western germany. in 2014 and 2015, the institute for terrestrial and aquatic wildlife research (itaw), university of veterinary medicine hannover, foundation, hannover and the wildlife research institute, state office for nature, environment and consumer protection of north rhine-westphalia caught free-living ring-necked pheasant chicks from lower saxony (cuxhaven, grafschaft bentheim, emsland, osnabrück, vechta), north rhine-westphalia (coesfeld, warendorf) and schleswig-holstein (dithmarschen) to assess the health state by means of pathological, microbiological, virological, parasitological and toxicological investigations. the caught chicks were grouped into three age classes (ac) based on the feather markings of the hand-wings. age class one (ac1) included chicks up to three weeks of age, ac2, chicks from four to six weeks of age and ac3, chicks older than six weeks and up to 11 weeks. an animal experiment permit was obtained from the responsible veterinary office of the lower saxony state office for consumer protection and food safety (laves) (permit number: 33.14-42502-04-14/1486). the study areas comprised 11 hunting regions with 15 traps in lower saxony (hemmoor, meppen, neuenkirchen, osten, strücklingen, vechta, wilsum,)11 regions with 10 traps in north rhine-westphalia (ahlen, dülmen, lippstadt, welte) and 4 districts with 4 traps in schleswig-holstein (warwerort) (fig 1) . the catching period lasted from may until august. in 2014, the investigated chicks were three to 11 weeks old. in 2015, the age of the chicks varied from one-day-old to eleven-week-old chicks. at the age of 11 weeks, the young pheasants were considered as sexually mature. after the catch, the mother hen was released and at maximum, half of the chicks in the trap were taken for analysis (mostly onethree chicks at random). in 2014, the traps had a size of 2.3 m 2 and were covered with iron bars with a mesh-size of 1 cm 2 . in 2015, the traps were slightly adapted based on experience from 2014, using a cover made of loose polyethylene netting with a mesh-size of 1 cm 2 . a piece of string was used as a trigger so that both trap doors closed when the chicks moved forward. corn was used to attract the hen and her chicks. afterwards, the chicks were transported alive to the university of veterinary medicine hannover for examination. the time span from catch to examination took on average about five hours. in 2014, the chicks were stunned by a head blow and killed by exsanguination. in 2015, the chicks were euthanised with an intravenous injection of pentobarbital-sodium (boehringer-ingelheim, ag & co. kg, ingelheim, germany). the nutritional condition score was evaluated macroscopically by the thickness of the pectoral muscles and the body fat percentages as good, moderate, poor or cachectic. as described by curland et al. [29] , animals in a good body condition revealed a vast amount of fatty tissue within the thoracic and abdominal regions, whereas animals with a moderate body condition demonstrated reduced amounts of body fat tissue. animals in a poor body condition possessed only low amounts of fat reserves, these frequently associated with pectoral muscle atrophy. in contrast, cachectic animals lacked fat reserves and exhibited serous atrophy of the coronal myocardial fatty tissue. the necropsy was carried out in accordance with the standard protocol [31] . representative samples of the following tissues and organs were collected, fixed in 10% neutral-buffered formalin and routinely embedded in paraffin wax: the skin of the head and abdomen, skeletal muscle (musculus pectoralis, musculus quadriceps), ischiadic nerve, brachial plexus, nose with infraorbital sinus, eye with lacrimal gland, bone with bone marrow, trachea, thymus, thyroidal gland, lung, heart, liver, pancreas, spleen, kidney, crop, proventriculus, gizzard, intestine, adrenal gland, gonads, bursa of fabricius and brain. paraffin sections of 3-5 μm were stained with haematoxylin and eosin (he) for histological examination. in selected cases, periodic acid schiff (pas) reaction, ziehl-neelsen stain, brown-brenn stain and turnbull's blue stain were performed [32] . for parasitological examinations, samples of the small intestine were collected from all 62 chicks at necropsy. at the same time, skin and plumage of the chicks were macroscopically examined for ectoparasites. for coproscopical examination, the combined sedimentation-flotation method was performed: the faecal sample was filled into a tea strainer (mesh size 1 mm) and rinsed in a beaker with a jet of water. the filtrate containing helminth eggs and protozoan oocysts was allowed to sediment for 30 min. afterwards, the supernatant was decanted and the sediment transferred to a 15-ml centrifuge tube filled with saturated zinc sulphate solution (znso 4 , specific gravity 1.30) and centrifuged at 450 x g for 5 min. the liquid surface was transferred onto a slide with a wire eyelet and examined microscopically. if at least one egg or oocyst was detected, the sample was classified as positive. a semiquantitative classification was applied using the following key: one-two eggs or oocysts were categorised as mild, six-ten eggs or oocysts as moderate, 11-20 eggs or oocysts as severe; if more than 20 eggs or oocysts were detected, the shedding intensity was classified as by mass [29] . the fresh samples, consisting of brain, trachea and caecal tonsils, as well as the bursa of fabricius were placed in rnalater1 (sigma-aldrich chemie gmbh, münchen, germany). samples were analysed by rt-pcr for avian metapneumovirus (ampv), infectious bronchitis virus (ibv), avian encephalomyelitis virus (aev) and by pcr for infectious bursal disease (ibdv) and infectious laryngotracheitis as described in [33, 34] . serum was taken from all birds to check for antibodies against avian influenza virus (aiv) subtypes h5, h7 and h9. eight additional liver samples from chicks with hepatitis were analysed for the presence of haemorrhagic enteritis virus (hev) by pcr [29] . for microbiological investigations for mycoplasma, 13 tracheal swabs, six tracheal tissue samples and three periorbital skin tissue samples were taken [29] . the samples were directly transferred to mycoplasma cultivation medium (sp4). detection of mycoplasma by pcr. for dna extraction, swabs were soaked and rubbed in 350 μl phosphate buffered saline (pbs). using the dneasy 1 blood & tissue kit (qiagen gmbh, hilden, germany) in accordance with the manufacturer's instructions, 100 μl of the liquid was taken for dna extraction. for dna extraction of tissue samples and the single colony subcultures, the fluid medium from culturing (2 ml) was centrifuged at 4000 x g for 45 minutes. the remaining pellet was incubated with 180 μl lysis buffer (atl buffer, qiagen, gmbh) and 20 μl proteinase k (qiagen gmbh) for two hours at 56˚c. all samples and single colony subcultures were screened via mycoplasma-genus-specific pcr (target: 16s rrna gene sequence) for dna of mycoplasma spp. as described by [35] and modified [36] . from all single colony subcultures, an additional pcr (target: 16s-23s rrna sequence (intergenetic transcribed spacer region)) was performed [37] . furthermore, all samples were examined via mycoplasma gallisepticum-specific pcr [38] . the pcr products were sequenced by a commercial dna sequencing service (lgc genomics gmbh, berlin, germany). the sequences of the pcr products were aligned with the 16s rrna gene and 16s-23s rrna isr sequences of mycoplasma spp. in the ncbi database using blast (ncbi, bethesda, md, usa) algorithm [39] . mycoplasma culture. the samples were cultured using sp4 liquid and agar media produced in house as described previously [34] . each sample was immersed in the sp4 broth and afterwards removed and stored for further investigations. the broth was diluted (ten-fold dilution up to 10 −2 ) and an aliquot of 50 μl each was transferred onto agar media. both, liquid and solid media were incubated at 37˚c with 5% co 2 in a humidified environment for up to ten days. broth was examined for colour change and agar plates for colony growth daily. in case of colour change, or after five days, an additional "subculture" on agar media was performed. in case of mycoplasma growth, several single colony subcultures were performed at least twice in order to ensure pure species cultures. each third single colony subculture was stored at -80˚c until further investigation by molecular biological methods [36, 40] . liver samples of nine pheasants were screened for herbicide glyphosate and other pollutants (for details see s1 table) . of these nine samples, one sample was taken from a ten-chick ratchet in ac1, while the remaining eight samples were single-samples from ac3 with liver or kidney inflammation. the samples were stored directly after autopsy at -80˚c. toxicological samples (n = 10), 7 g liver pool samples, were used to detect substances by means of the gas chromatography-mass spectrometry ( during the two-year-period, a total of 62 chicks were caught: 29 birds in lower saxony, 27 in north rhine-westfalia and six in schleswig-holstein. fourteen chicks were allocated to ac1, 16 to ac2 and 32 to ac3. of the investigated animals, 34 were female, 17 male and for 11 chicks, macroscopic gender estimation was unknown; histological samples were not taken. the nutritional status in ac1 was predominantly poor (n = 12; 85.7%) or cachectic (n = 2, 14.3%). in ac2, approximately nine (56.3%) of the chicks were well fed and seven (43.8%) were moderately fed. the majority of birds in ac3 were well fed (n = 25, 78.1%), some were moderately fed (n = 6, 18.8%) and one chick (3.1%) was in a poor body condition (table 1) . to an excessive amount, mild to severe cutaneous abrasions with feather loss, lacerations and/ or subcutaneous haemorrhages of the head were noticed in one out of two chicks (50%) in ac1, nine out of 16 chicks (56%) in ac2 and 11 out of 20 chicks (55%) in ac3 trapped with the tt1. one out of 12 animals (8%) in ac1 and nine out of 12 individuals (75%) in ac3 that had been trapped with tt2 were more mildly affected by those lesions. histological examination of the skin from the head revealed various types of inflammatory alterations which occurred solely or concurrently in one individual (table 2 ). in all age classes and independent of trap type, mainly perivascular predominantly lympho-histiocytic dermatitis admixed with occasional heterophils and plasma cells of varying degrees was present (fig 2) . this type of inflammation was in some cases accompanied by follicular aggregations of lymphocytes sometimes with secondary follicle formation (ac3, tt2). in addition, ulcerative (fig 3) , occasionally necrotising, suppurative and pustular inflammatory changes were found more often in chicks trapped with tt1 than with tt2, these in many cases being associated with dermal and/or subcutaneous haemorrhages of varying degrees. only a few animals showed no cutaneous alteration in this localisation. the abdominal skin of the chicks was rarely affected by perivascular dermatitis; single individuals were mainly affected by lymphohistiocytic or pustular dermatitis. crops, glandular stomachs and gizzards were variably filled (table 3 ). however, it should be noted that the chicks had spent up to five hours in the traps. the mentioned parts of the digestive tract contained grains, green food, and, inside the gizzard, grit stones. in one chick (7%) in ac1, in seven chicks (44%) in ac2 and in ten chicks (31%) in ac3, the quality of the intestinal content was associated with hyperaemic intestinal mucosa and perianal attachment of faeces suggestive of catarrhal enteritis. histologically, one animal (3%) in ac3 showed focally severe ulcerative stomatitis at the gums and one chick in ac3 focally moderate lympho-histiocytic ingluvitis. in single chicks in ac3, nematodes without reactive inflammatory changes were found in the crop. furthermore, single individuals showed erosive and heterophilic inflammation of the gizzard, occasionally associated with intralesional nematodes which were not differentiated here. the intestinal mucosa in all animals showed a mild to moderate infiltration with eosinophils, lymphocytes and a few plasma cells. in eight out of 16 chicks (50%) in ac2 and in four out of 32 chicks (13%) in ac3, reproduction stages of protozoal organisms, most likely coccidia sp., were found histologically within the epithelium (fig 4) . predominantly mild focal lymphohistiocytic hepatitis was observed in one out of 14 chicks (7%) in ac1, in four out of 16 chicks (25%) in ac2, and in ten out of 32 chicks (31%) in ac3. single individuals in ac3 showed multifocal granulomatous hepatitis with severe acute coagulation necrosis (fig 5) . in eight out of 14 chicks (57%) in ac1, a mild to severe diffuse fatty change in hepatocytes was present. nematodes with the morphology consistent with syngamus trachea were found in the trachea of none of the 14 chicks in ac 1, in five out of 16 chicks (31%) in ac2 and in ten out of 32 (31%) chicks in ac3. histologically, tracheal parasitism in most animals was associated with multifocal lympho-histiocytic, occasionally granulomatous or ulcerative tracheitis of variable extent. in single animals, subepithelial lymphoid follicles were found. in the lung, focal or multifocal interstitial, mild to moderate lymphohistiocytic pneumonia was observed in one out of 14 chicks (7%) in ac1, in three out of 16 chicks (19%) in ac2, and in six out of 32 chicks (19%) in ac3. focal or multifocal, mild to moderate granulomatous, occasionally necrotising pneumonia was present in three individuals (19%) in ac2 and in two individuals (6%) in ac3. one animal in ac3 suffered from severe suppurative to necrotising pneumonia. there was no evidence of viral, bacterial, fungal or parasitic agents in these lungs, even in the histological special stains. hyperplasia of bronchus-associated lymphoid tissue was noticed in two chicks (13%) in ac2 and in three chicks (9%) in ac3. numerous lungs showed acute haemorrhages. the kidneys displayed focally mild interstitial infiltrations consisting mainly of lymphocytes and macrophages in two chicks (13%) in ac2 and five chicks (16%) in ac3. independent of the age class, a mostly moderate diffuse infiltration with plasma cells was observed in almost all examined lacrimal glands. miscellaneous findings included focally mild lymphocytic myocarditis in one chick (3%) in ac3 (fig 6) , focally moderate lymphohistiocytic perineuritis (n. ischiadicus) in one chick in both ac2 (19%) and ac3 (3%), severe subacute hyaline degeneration of skeletal muscles with histiocytic infiltration in one chick (3%) in ac3, focal chronic suppurative myositis in another chick (3%) in ac3, and single protozoal cysts, most likely sarcosporidia sp., in the skeletal musculature of two individuals (6%) in ac3 without inflammatory changes. in many brains, perivascular and parenchymatous haemorrhages were observed without reactive changes. of all 62 chicks tested, 8% of the chicks were positive for avian metapneumovirus (ampv) using rt-pcr, 16% positive for infectious bronchitis virus (ibv) using rt-pcr, and 2% for haemorrhagic enteritis virus (hev) using pcr. none of the 37 chicks tested for hev were positive using pcr. tracheae of 33 chicks and caecal tonsils of ten birds were tested by the coronavirus-rt-pcr and were negative for the respective virus. all samples tested for avian encephalomyelitis virus (aev), infectious bursal disease virus (ibdv), or infectious laryngotracheitis virus (iltv) were negative. the pool samples of the ten chicks were completely negative for all acid, alkaline-free and derivative substances as listed in s1 table which summarises the substances tested and the detection limits. two out of three samples tested for the herbicide glyphosate were positive (0.044 mg/kg, 0.095 mg/kg). since the 1970s, a population decrease in ring-necked pheasants has been observed. especially in 2007/2008, the population decline intensified [41] . the present investigation revealed that the randomly trapped pheasant chicks displayed inflammatory lesions in different organs. in association with environmental stressors and a depleted nutritional status, these health changes may increase pheasant chick mortality, thus contributing to the population decrease. the dermatitis detected was often of a non-purulent character, mostly perivascularly accentuated with different cellular compositions of gradual variable infiltrations by lymphocytes, plasma cells and macrophages. especially on the head, this alteration additionally displayed pustular and lymphocytic inflammation. it was possibly itch-or parasite-induced. avian pox was excluded due to lack of pathognomonic and histological changes [42] . these alterations occurred in chicks as young as one or two days of age. six out of 12 chicks (50%) in ac1 already showed these alterations, with different types and degrees of inflammation. m. gallisepticum (mg) is an important etiological differential diagnosis, especially inducing periocular dermal swelling with lymphocytic inflammation [43] . however, this pathogen was ruled out by the investigations. nevertheless, various mycoplasma spp. were isolated in 15 out of 21 (71.4%) of the investigated chicks. however, the role of these mycoplasma spp. as a potential cause of periorbital skin alterations in pheasants is still unclear, but should be considered in following investigations in pheasants. as some birds were rt-pcr positive for ibv or ampv, it has to be elucidated further whether these viruses may have contributed to these lesions, as they are known to be respiratory disease associated. the inflammations might be itch induced following insect or tick bites. furthermore, the head injuries with lacerations and haemorrhages resulted from catching caused by the chicks jumping against the iron bars of the traps. these injuries did not appear anymore after exchanging these bars for loose nylon mesh. a total of 65% of the 26 pneumonia cases were of an eosinophilic character and were most likely caused by syngamus trachea in ten cases. all cases of granulomatous inflammation were free of acid-fast bacteria as shown by the ziehl-neelsen stain. therefore, the cause of this granulomatous inflammation remains unknown. other possible agents able to induce pneumonia, bronchopneumonia, tracheitis and bronchitis were not detected. a prevalent eosinophilia tracheitis (94%) occurred in almost all cases in connection with detected parasites at different stages including coccidia, heterakis/ascaridia spp. and syngamus trachea. the degrees of inflammation were mainly mild up to moderate so that the clinical relevance is rather subordinate. the proventriculitis can have many origins. a histologically similar disease, that of gizzard erosion in broilers is often caused by an interaction between vitamin deficiency, fungal infections and stress situations after consuming mycotoxins. with periodic acid schiff reaction (pas) and brown-brenn stain, fungi, gram-positive and gram-negative bacteria were excluded [44] . as ampv, ibdv, coronavirus and siadenovirus were excluded by pcr, a viral cause is relatively unlikely. marek's disease is doubtful as well due to the lack of other typical organ changes [45] . it is possible that the birds may have been exposed to mycotoxins or pesticides that caused proventriculitis. based on localisation, size and shape of the eggs found in the proventriculus, a nematode-infection with dyspharynx nasuta probably resulted [46] . the inflammation of the livers showed lymphocytic and lymphohistiocytic characters. the causes for these inflammatory changes are manifold and may include infectious as well as noninfectious agents. in three cases, the inflammation was granulomatous and necrotising. using ziehl-neelsen stain, acid-resistant bacteria were excluded. differential diagnoses for granulomatous and necrotising hepatitis include toxic, ischemic or infectious causes [47, 48] . in the presented investigations, only a limited number of samples could be investigated for pesticides. therefore, it is difficult to directly link pathological findings to any of the investigated chemicals. further investigations are needed to elucidate the role played by pesticides in the declining pheasant populations as their habitat is regularly exposed to different chemicals used in agriculture. the main findings in the study were the poor nutritional status in the younger age groups and the increasing occurrence of various inflammation when the birds were ageing. as no direct cause for the inflammation was found and the inflammation affected various organs, it might be more a sign of various pathogens affecting the chicks. this seems to be more a sign of a weakened immune system, unable to defeat facultative pathogenic organisms. this is in line with the poor nutrition status, which triggers the development of diseases. no suspected virus infection was detected though. virus infections cannot be ruled out completely as a cause as viruses obviously circulate in the adult pheasant population and infected chicks die quickly. therefore, such cases were not among the sampled animals as the study focused on live chicks which still followed the hen. due to predation, decomposition and vegetation in the field, diseased pheasants are difficult to retrieve for health examinations and therefore were not included here. concerning parasites, low coccidian infections can be regarded as desirable to build up a protective immunity against reinfections. however, intestinal changes of the chicks show that coccidia sometimes considerably damage the intestinal mucosa due to severe infections, which may lead to a reduction in nutrient uptake. furthermore, a severe syngamus sp. infection can occlude the tracheal lumen, resulting in suffocation of the chicks, or their general condition deteriorates to such an extent that they become easy prey for predators. all these findings point to an effective complexity that either chicks die of starvation or their immune system becomes weakened. it seems that when the effects of maternal antibodies slowly diminish and the chicks have to mobilise their own immune system, the chicks become weakened as their immune system is not sufficiently developed. also, it is known from poultry that the development of the immune system is influenced by nourishment and that malnutrition can negatively influence the immune system [31, 49, 50] . this hypothesis has not been confirmed yet in pheasants. not only pheasants, but also many other farmland birds have to cope with the intensive agricultural landscape. this change in habitat and the use of pesticides make it increasingly difficult to find insects that are vital for the chicks during their first weeks of life. a whole concatenation of circumstances could be explained by this connection; namely, the rather poor nutritional status, a possibly weakened immune system and the increased susceptibility to diseases. additionally, it is possible that the chicks are easier prey for predators due to various inflammations or poor physical condition, too. also, the weather can have a greater influence. supporting information s1 table. toxicological investigation of substances and limits of detection. highlighted in bold are the substances found in the pheasant samples. 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lymphoid organs and immune competence in neonatal chickens: a review romeis mikroskopische technik kompendium der geflü gelkrankheiten modified live infectious bursal disease virus (ibdv) vaccine delays infection of neonatal broiler chickens with variant ibdv compared to turkey herpesvirus (hvt)-ibdv vectored vaccine experimental haematobiochemical alterations in broiler chickens fed with t-2 toxin and co-infected with ibv prevalence of mycoplasmas in eggs from birds of prey using culture and a genus-specific mycoplasma polymerase chain reaction genus-and species-specific identification of mycoplasmas by 16s rrna amplification high inter-species and low intra-species variation in 16s-23s rdna spacer sequences of pathogenic avian mycoplasmas offers potential use as a diagnostic tool das vorkommen von mykoplasmen bei storchnestlingen in brandenburg und sachsen-anhalt basic local alignment search tool recovery of mycoplasmas from birds wild und jagd-landesjagdbericht 2010/2011. ni ministerium für ernä hrung, landwirtschaft, verbraucherschutz und landesentwicklung a retrospective studie of skin lesions in wild turkeys (meleagris gallopavo) in the eastern usa, 1975-2013 mycoplasma gallisepticum in pheasants and the efficacy of tylvalosin to treat the disease hard af segerstad c. mycotic proventriculitis in gray partridges (perdix perdix) on two game bird farms causes of gizzard erosion and proventriculitis in broilers first report of five nematode species in phasianus colchicus linnaeus (aves, galliformes, phasianidae) in brazil / primeiro registro de cinco espécies de nematóides em phasianus colchicus linnaeus (aves, galliformes, phasianidae) no brasil necrotizing hepatitis in a domestic pigeon (columba livia) hepato nephropathology associated with inclusion body hepatitis complicated with citrinin mycotoxicosis in a broiler farm early nutrition programming (in ovo and posthatch feeding) as a strategy to modulate gut health of poultry nutrition-mechansims of immunosuppression we wish to thank the hunting associations of lower saxony, north rhine-westphalia and schleswig-holstein for supporting the study. furthermore, special thanks go to the laboratory personnel for their excellent technical assistance in the laboratory investigations. key: cord-298131-zolwjl9u authors: xiao, shuqi; jia, jianyu; mo, delin; wang, qiwei; qin, limei; he, zuyong; zhao, xiao; huang, yuankai; li, anning; yu, jingwei; niu, yuna; liu, xiaohong; chen, yaosheng title: understanding prrsv infection in porcine lung based on genome-wide transcriptome response identified by deep sequencing date: 2010-06-29 journal: plos one doi: 10.1371/journal.pone.0011377 sha: doc_id: 298131 cord_uid: zolwjl9u porcine reproductive and respiratory syndrome (prrs) has been one of the most economically important diseases affecting swine industry worldwide and causes great economic losses each year. prrs virus (prrsv) replicates mainly in porcine alveolar macrophages (pams) and dendritic cells (dcs) and develops persistent infections, antibody-dependent enhancement (ade), interstitial pneumonia and immunosuppression. but the molecular mechanisms of prrsv infection still are poorly understood. here we report on the first genome-wide host transcriptional responses to classical north american type prrsv (n-prrsv) strain ch 1a infection using solexa/illumina's digital gene expression (dge) system, a tag-based high-throughput transcriptome sequencing method, and analyse systematically the relationship between pulmonary gene expression profiles after n-prrsv infection and infection pathology. our results suggest that n-prrsv appeared to utilize multiple strategies for its replication and spread in infected pigs, including subverting host innate immune response, inducing an anti-apoptotic and anti-inflammatory state as well as developing ade. upregulation expression of virus-induced pro-inflammatory cytokines, chemokines, adhesion molecules and inflammatory enzymes and inflammatory cells, antibodies, complement activation were likely to result in the development of inflammatory responses during n-prrsv infection processes. n-prrsv-induced immunosuppression might be mediated by apoptosis of infected cells, which caused depletion of immune cells and induced an anti-inflammatory cytokine response in which they were unable to eradicate the primary infection. our systems analysis will benefit for better understanding the molecular pathogenesis of n-prrsv infection, developing novel antiviral therapies and identifying genetic components for swine resistance/susceptibility to prrs. porcine reproductive and respiratory syndrome (prrs), also called ''blue ear'' disease due to a typical, but not often observed hallmark of ''blue ears'', is widely accepted as being one of the most economically important diseases affecting swine industry. since its first appearance in the late 1980s in the us and europe, prrs has spread worldwide [1, 2, 3] . prrs is characterized with high mortality in piglets, reproductive failure (late-term abortions and stillbirths, premature farrowing, mummified pigs) in pregnant sows and respiratory disease (interstitial pneumonia, respiratory difficulties) in nursery and grower/finishing pigs, causing highly significant economic losses to the swine industry worldwide, resulting in .$ 560.32 million losses each year in the us alone [4] . the etiologic agent of prrs is prrs virus (prrsv), a small enveloped, linear, single, positive-stranded rna virus, which is a member of the family arteriviridae which includes lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus and enters in the newly established order delayed and their levels remain low, which can not eliminate effectively prrsv-infected cells [12, 13] . because of these features of prrsv infection, prrs has been one of the most challenging subjects of research in veterinary viral immunology [12] . regulation of immune responses and genetic resistance to infectious viral diseases is an area of concern for human and swine [14] . prrsv strongly modulates the host's immune responses, and changes the host's gene expression. studies showed that prrsv inhibits type i interferons (ifn-a/b, spi ifn), especially ifn-a [15] , and induces interleukin-10 (il10) [16, 17] . because the primary cellular target of prrsv is the porcine alveolar macrophages (pams) of the lung, several studies have analysed the immune responses of pams to prrsv infection. one group [18] used differential display reverse-transcription pcr to identify molecular genetic changes within prrsv-infected pams over a 24 h pi period. their results suggest that myxovirus resistance 1 (mx1) and ubiquitin specific proteases (usp) genes may play important roles in clinical disease during prrsv infection. notably, one recent paper on genome-wide transcriptional response of pams following infection with the lelystad prrsv strain (european type, eu prrsv) using affymetrix microarrays has been published during the preparation of our manuscript [19] . they found that the expression of beta interferon 1 (ifn-b) was strongly upregulated while the expression of il-10 and tnf-a was weakly upregulated. almost in the same time, the other group employed serial analysis of gene expression (sage) to examine the global expression of genes in vr-2332 prrsv strain (north american type, na prrsv)-infected pams. they identified over 400 unique tags with significantly altered expression levels [20] . in vitro studies will be useful for investigating how prrsv modifies genes expression in primary target cells, such as pams. however, many of the outstanding issues will be answered only in the context of prrsv-infected animals. hence, the characterization of host immune response under in vivo environment to prrsv is still an area in urgent need of investigation. lung pathogenesis is a major feature of prrsv infection. moreover, in addition to serving as a source of protein in the human diet, the pig is also an excellent biomedical model for humans because of the similarity in size and physiology, and in organ development and disease progression [14] . thus, understanding the host's immune response to prrsv infection is important not only for swine production but also for human consumption. however, to date, the immune response to prrsv in porcine lung has not been analyzed by transcriptome profiling. next generation high-throughput sequencing technology has been adapted for transcriptome analysis because of the inexpensive production of large volumes of sequence data [21, 22, 23, 24] . the technology developed by illumina (formerly solexa sequencing) [25] , which is also referred to as digital gene expression (dge) tag profiling, allows identification of millions of short rnas in a sample and of differentially expressed genes without the need for prior annotations. here we employed the illumina genome analyzer platform to perform a digital gene expression analysis of the porcine lung transcriptome response to n-prrsv infection, and used histopathology examination to analyze the pulmonary pathological changes of the infected-porcine lungs. the relationship between pulmonary gene expression profiles after n-prrsv infection and infection pathology was systematically analyzed. the comprehensive analysis of the global host response induced by n-prrsv suggested an inflammatory response, mediated by multiple inflammatory molecules early during infection that induced tissue injury, an immunosuppressive state, mediated by apoptosis of infected cells, which caused depletion of immune cells and induced an anti-inflammatory cytokine response in which they were unable to eradicate the primary infection. our systems analysis will benefit for better understanding the molecular pathogenesis of n-prrsv infection, developing novel antiviral therapies and identifying genetic components for swine resistance/susceptibility to prrs. our study had been approved by animal care and use committee of guangdong province, china. all animal procedures were performed according to guidelines developed by the china council on animal care and protocol approved by animal care and use committee of guangdong province, china. nine conventionally-reared, healthy 6-week-old, crossbred weaned pigs (landrace6yorkshire) were selected from a highhealth commercial farm that has historically been free of all major pig diseases, such as prrsv, porcine circovirus type 2, classical swine fever virus, porcine parvovirus, pseudorabies virus, swine influenza virus and mycoplasma hyopneumoniae infections. all pigs were prrsv-seronegative determined by elisa (herdchek prrs 2xr; idexx laboratories) and absence of prrsv tested by rt-pcr. pigs were randomly assigned to two groups in the experiment and raised in isolation rooms. six pigs were inoculated with 6 ml viral suspension (4 ml intranasally and 2 ml intramuscularly) of classical north american type prrsv (n-prrsv) strain ch 1a, isolated from china in 1996, gifted by dr. zhang guihong, south china agricultural university) at a dose of 10 6.0 tcid50 ml 21 on day 0. three uninfected negative control (unc) pigs were treated similarly with an identical volume of dmem culture media from uninfected marc-145 cells 1 day prior to experimental infection, and were immediately necropsied. n-prrsv-inoculated pigs were clinically examined daily and rectal body temperatures were recorded from days 22 to 7 pi. viral reisolates were performed after the pigs were killed. the infected group showed positive, and the unc group was negative. tissue homogenates and serum were examined by n-prrsv-specific quantitative pcr (qpcr). the oligonucleotide primers used were nsp2f(59-gtgggtcggcaccagtt-39) and nsp2r , designed in the gene segment encoding for nsp2. the taqman probe, 59 fam-cacagttctacgcggtgcagg -tamra 39, was synthesized. three infected pigs randomly chosen were necropsied at each time point of 96 h pi and 168 h pi. lung samples were collected from unc group (c), three pigs at 96 h pi (n96), three pigs at 168 h pi (n168) and immediately frozen in liquid nitrogen for rna isolation or fixed in 10% neutralized buffered formalin for histological processing. lungs of unc and experimentally infected pigs were processed routinely for haematoxylin and eosin (h&e) and immunohistochemistry staining, as described previously [26] . total rna was extracted from frozen lungs using standard protocols (trizol) and then treated with dnase to remove potential genomic dna contamination according to the manufactures's protocols. rna integrity and concentration were evaluated by agilent 2100 bioanalyzer (agilent technologies). for rna library construction and deep sequencing, equal quantities of rna samples from three unc individual lungs were pooled, rna samples from the three infected pig lungs (n96) were pooled, and rna samples from the three infected individual lungs (n168) were pooled. approximately 6 mg of rna representing each group were submitted to solexa (now illumina inc.) for sequencing. sequence tag preparation was done with illumina's digital gene expression tag profiling kit according to the manufacturer's protocol. in brief, mrna was isolated from 6mg total rna by binding the mrna to a magnetic oligo bead. first-and secondstrand cdna were synthesized while the mrna was attached to the beads. the double stranded cdnas were digested with nlaiii to wash away all fragmens other than the 39 catg fragment attached to the oligo bead. then gex nlaiii adapter 1 was ligated at the site of nlaiii cleavage. in addition, gex nlaiii adapter 1 contains the sequence for the restriction enzyme mmei, subsequently, we applied the restriction enzyme mmei to create the 17 bp tag. the gex adapter 2 was ligated at the site of mmei cleavage. a pcr with 12 cycles was performed with two primers that anneal to the ends of the adapters to enrich the adapterligated cdna construct. the resulting 85 bp fragments were purified from 6% novex tbe page gel. subsequently, the purified cdna tags were sequenced on the illumina cluster station and genome analyzer. image recognition and base calling were performed using the illumina pipeline. all data is miame compliant. the raw data (tag sequences and counts) has been submitted to gene expression omnibus (geo) under series gse19970. for the raw data, we filtered adaptor tags, low quality tags and tags of copy number = 1 to get clean tags. subsequently, we classified the clean tags according their copy number in the library and show their percentage in the total clean tags and analyzed saturation of the library. the preprocessed database of all possible catg +17-nt tag sequences was created, using sus scrofa unigene (http://www.ncbi. nlm.nih.gov/unigene/ugorg.cgi?taxid = 9823, unigene build #35 sus scrofa, nov, 7th, 2008) from ncbi. for monitoring the mapping events on both strands, both the sense and the complementary antisense sequences were included in the data collection. information on the position of polyadenylation signals was also collected from the transcript dababase. then we aligned all clean tags to the reference sequences, and unambiguous tags were annotated. we counted the clean tag number corresponding to each gene. to compare the de of gene across samples (n96/c, n168/c, n168/n96), the number of raw clean tags in each library was normalized to tags per million (tpm) to obtain normalized gene expression level. de detection of gene or tag across samples was performed according to the previous description [27] . genes were deemed significantly differentially expressed with a p-value , 0.005, a false discovery rate (fdr) ,0.01 and an estimated absolute log2-fold change .0.5 in sequence counts across libraries. in order to verify the dge results, we used qpcr analysis. the rna samples used for the qpcr assays were both the same as for the dge experiments and independent rna extractions from biological replicates. qpcrs were done on the lightcycler480 (roche), with sybr-green detection (sybr primescript rt-pcr kit, takara biotechnology co., ltd.), according to the manufacture's instruction. each cdna was analyzed in triplicate, after which the average threshold cycle (ct) was calculated per sample. the relative expression levels were calculated with the 2 2ddct method. the results were normalized to the expression level of hprt1 and relative to the c sample. through browsing all health traits of pig quantitative trait locus (qtl) database (pigqtldb, http://www.animalgenome. org/qtldb/pig.html) by trait classes, we obtained mapping details of qtl on the corresponding pig chromosome. then pig affymetrix elements corresponding to health trait qtl regions were downloaded to an excel file. by matching the id of de genes to all genes in the qtl regions, we obtained de genes of the corresponding qtl region. pathway analysis was mainly based on the kyoto encyclopedia of genes and genomes (kegg) database. two-side fisher's exact test with a multiple testing and x2 test were used to classify the pathway category. the false discovery rate (fdr) was used to correct the p-value. we chose only pathway categories that had a p,0.05. within the significant category, the enrichment re was , where n f is the number of flagged proteins within the particular category, n is the total number of proteins within the same category, nf is the number of flagged proteins in the protein reference database list, n is the total number of proteins in the gene reference database list. stc is implemented entirely in java. the clustering algorithm first selects a set of distinct and representative temporal expression profiles. these model profiles are selected independent of the data. the clustering algorithm then assigns each gene passing the filtering criteria to the model profile that most closely matches the gene's expression profile as determined by the correlation coefficient. since the model profiles were selected independent of the data, the algorithm can then determine which profiles have a statistically significant higher numberthan genes assigned using a permutation test. this test determines an assignment of genes to model profiles using a large number of permutations of the time points. it then uses standard hypothesis testing to determine which model profiles have significantly more genes assigned under the true ordering of time points compared to the average number assigned to the model profile in the permutation runs. significant model profiles can either be analyzed independently, or grouped together based on similarity to form clusters of significant profiles [28, 29] . stc-go supports gene ontology enrichment analyses for sets of genes having the same significant temporal expression pattern. we select random samples of s a (s a is the number of genes assigned to the same model temporal expression profile r.) genes at each iteration and compute fisher's exact test p-values for the selected genes in all go biological categories [30] . the two-sided fisher's exact test p-value for a category reflects a test of the null hypothesis that the category is enriched in genes assigned to profile r with respect to what would have been expected by chance alone. to decide whether or not to follow up a category that appears enriched in these genes, we would know the statistical reliability of the apparent enrichment. to assess the significance of a particular category, we need to know the distribution of p-values that would occur by random chance. the percentage of false positives to be tolerated will generally depend on the relative costs of false positives and false negatives in whatever follow-up study is to be done. this way of framing the question leads us to specify the false discovery rate (fdr) for a set of categories, rather than significance level (p-value) for each category. with the significance at the 0.05 level, for a given category, the enrichment r e is given by r e~( i=m)=(s a =n) where i is the number of genes assigned to profile r within the go category of interest, m is the total number of genes within the go category of interest, and n is total number of unique genes in the gene reference database list. after n-prrsv infection, the affected pigs exhibited the following clinical symptoms within 3-7 days: fever of 40.08-40.8uc, depression, anorexia, rough hair coats, dyspnoea, reddening of skin, oedema of the eyelids, conjunctivitis, mild diarrhoea, shivering. those unc pigs did not show any obvious changes in body temperature and clinical signs. qpcr assay showed that n-prrsv virus was present in each of the 6 infected pigs. but n-prrsv nsp2 gene was not differentially expressed at 96 h pi and 168 h pi (table s1 ). histopathology examination of n-prrsv-affected pigs showed interstitial pneumonia in lungs with thickening of alveolar septa accompanied with infiltration of immune cells ( figure 1b ). most viral antigen was detected in alveolar cells and bronchiolar epithelial cells in lesions ( figure 1c ). to investigate the regulation of the host response to the n-prrsv virus, we considered the global gene expression profiles in lungs using solexa/illumina's dge system, a tag-based transcriptome sequencing method. we sequenced three porcine lung dge libraries from c, n96, n168 using massively parallel sequencing on the illumina platform. major characteristics of these three libraries were summarized table 1 . we obtained approximately 6.9 million total sequence tags per library with 515885 distinct tag sequences. prior to mapping these tag sequences to reference sequences, we filtered adaptor tags, low quality tags and tags of copy number = 1, producing approximately 6.6 million total clean sequence tags per library with 179589 distinct clean tag sequences. the c library had the highest number of both total sequence tags and distinct sequence tags; this was followed by the n168, n96 libraries. moreover, the c library had the highest ratio of number of distinct tags to total tags and the lowest percentage of distinct clean high copy number tags. the data showed that more genes were detected in the c library than other two libraries and more transcripts were expressed at lower levels in the c library. saturation analysis of capacity of libraries showed that new emerging distinct tags were gradually reduced with increasing of total sequence tags when the number of sequencing tags was big enough. when the number of sequencing tags reached 3 million, library capacity approached saturation ( figure s1 ). for tag mapping, we preprocessed one reference tag database that included 51670 sequences from sus scrofa unigene. to get the reference tags, we used nlaiii to digest all the samples and took all the catg+17 tags in the gene as the gene's reference tags, not only the 39 most one. we obtained 194664 total reference tag sequences with 172119 unambigous tag sequences. considering polymorphism across samples, tolerances were set to allow one mismatch in each alignment. by the criteria, 47.71%,51.04% of distinct clean tags mapped to the unigene virtual tag database, 39.42%,42.11% of the distinct clean tags mapped unambiguously to the unigene, and 52.29%,48.96% of the distinct clean tags didn't map to the unigene virtual tag database ( table 1 ). the occurrence of unknown tags was probably due to the incompleteness of pig genome sequencing. most solexa experimental tags matched to the 1st or 2nd 39 catg site in high-confidence transcripts ( figure s2 ). for solexa sequencing can distinguish between transcripts originating from both dna strands, employing the strand-specific nature of the sequencing tags obtained, we found evidence for bidirectional transcription in 6368 to 8271 of all detectable unigen clusters and 3889 to 4043 antisense-stand specific transcripts (table s2 ). by comparison, the ratio of sense to antisense strand of the transcripts was approximately 1.7:1 for all libraries. this suggests that in spite of the high number of antisense mapping events detected, the transcriptional regulation in the n-prrsv-induced immune response acts most strongly on the sense strand. to analyze the depth of transcriptome sampling in the dge libraries, we studied the rate of increase of the number of genes (sense+antisense strand) identified as the size of the corresponding library increases. when the library size reached one million, we could identify 45% and 30% all genes and genes identified by unambigous tags, respectively ( figure s3 ). at this time, library capacity approached saturation. to gain the global transcriptional changes in n-prrsv infected porcine lungs, we applied the method described previously [27] to identify de genes from the normalized dge data by pairwise comparisons between all differential time points (n96/c, n168/c, n168/n96) during infection. results showed that 5430 genes had p values ,0.005, false discovery rate (fdr) ,0.01 and estimated absolute log2-fold change .0.5 in at least one of the pairwise comparisons, which were declared to be differentially expressed during infection course (table s3) . to characterize the functional consequences of gene expression changes associated with infection with n-prrsv, we performed pathway analysis of de genes based on the kegg database by two-side fisher's exact test. we chose only significant pathway categories that had a p-value of ,0.05 and an fdr of ,0.05. as shown in figure s4 , the significant signaling pathways include cell adhesion molecules (cams), t cell receptor signaling pathway, antigen processing and presentation, toll-like receptor signaling pathway, biosynthesis of unsaturated fatty acids, pantothenate and coa biosynthesis, etc (table s4) . to validate de genes identified by solexa sequencing, we selected 8 genes for qpcr confirmation. the set included two down-regulated genes (epithelial chloride channel protein (aecc) and hyaluronan and proteoglycan link protein 1 (hapln1)) and six up-regulated genes (inflammatory response protein 6 (irg6), dead (asp-glu-ala-asp) box polypeptide 58 (ddx58), usp18, cxcl10, cytochrome p450 (cyp3a88), and cd209). data were presented as fold changes in gene expression normalized to the hprt1 gene and relative to the c sample. pearson's correlation coefficient (r) showed that both the dge and qpcr data (pooling samples) were highly correlated, for the genes modulated by n-prrsv had a high consistency and r values ranging from 0.781 (cyp3a88) to 0.997 (aecc) between the two methods ( figure 2 ). qpcr analysis (both pooling samples and independent rna extractions from biological replicates) confirmed the direction of change detected by dge analysis. this correlation indicated the reliability of dge results. qtl play a central role in linking genomic information with phenotypes. the ultimate goal of qtl studies is to identificate the actual gene(s) that are responsible for the phenotypic variation observed in a particular trait [31] . in the present paper, we mapped the de genes to pig qtl regions of health traits in pig qtldatabase (pigqtldb). our search found that 240 de genes were distributed in 18 different known qtl regions related to pig health traits ( figure s5 ; table s5 ). among the 240 de genes, 122 and 114 were located in qtl regions of the cd4-positive leukocytes and cd2-positive leukocytes, respectively; 53 were distributed in the qtl region of the band-formed neutrophils and cd8-positive leukocytes. immune responses against pathogens depend in part on the generation of fully differentiated 'killer' (or effector) and memory cd8 + t cell. effective priming and maintenance of cd8 + t cell responses to viral infection require 'help' from cd4 + t cells, the latter play also a critical role in programming cd8 + t cell memory development [32] . moreover, recent study showed that cd4 + t cells guide effector cytotoxic t lymphocytes (ctls) to virally infected tissues where they can destroy infected cells [32, 33] . in order to profile gene expression time series and search for the most probable set of clusters generating the observed time series, we used stc algorithm of gene expression dynamics, which explicitly took into account the dynamic nature of temporal gene expression profiles during clustering and identified the number of figure s6 ; table s3 ) with 4 (profile 1,6,0,7) significant cluster profiles which have significantly more genes assigned under the true ordering of time points compared to the average number assigned to the model profile in the permutation runs ( figure s6 ). then gene ontology (go) based on biological process (bp) enrichment analyses for sets of de genes having significant cluster profiles was performed by two-side fisher's exact test (table s6 and table s7 ; figures s7, s8 , s9 s10). we chose only significant go categories that had a p-value of ,0.05. the most prominently overrepresented go terms of significant cluster profile 1 (0,21,21) and profile 0 (0, 21, 22) , which are down-regulated genes, involved in regulation of lipid, cholesterol biosynthetic and metabolic process; regulation of skeletal muscle development, muscle cell differentiation; digestion; negative regulation of neuron apoptosis and neurological system process (table s6 ; figures s7 and s8) . the most prominently overrepresented go terms of significant cluster profile 6 (0,1,1) and profile 7 (0,1,2), which are up-regulated genes, included negative regulation of fibroblast proliferation; natural killer cell, macrophage, lymphocyte, mononuclear cell, leukocyte and t cell proliferation, differentiation and activation; complement activation, immune response, inflammatory response, defense response, and apoptosis; response to stimulus(stress); lipid and fatty acid metabolic process and oxidation; positive regulation of ubiquitinprotein ligase activity and protein proteolysis, protein targeting to mitochondrion (table s7 ; figure s9 and s10). these results are consistent with these genes and their associated processes playing important roles in n-prrsv replication and pathogenesis. viral infection of host leads to the initiation of antiviral innate immune responses, which results in the induction of expression of the type i interferons [34] . meanwhile, many viruses have also developed strategies to evade and subvert the immune response. as shown in figure 3a , transcripts of the ifn c was significantly induced in n-prrsv-infected pigs at days 4 through 7 pi, but short type i interferon (spi ifn) gene expression was suppressed, and interferon alpha 5 (ifna5) gene expression was markedly down-regulated. lipid rafts, lipid microdomains of the cell membrane enriched in sphingolipids, cholesterol and associated proteins, play critical roles in the life cycle of many viruses [35] . some viruses enhance their replication by modulating host cell lipid metabolism [36] . dge analysis of pigs infected with n-prrsv showed significant increase of transcript abundance in many genes involved in lipid metabolism, including those for apolipoprotein b48 receptor (apob48r), apolipoprotein-e (apoe), low density lipoprotein b (ldlb), phosphatidylinositol 3-kinase catalytic subunit type 3 (pik3c3) ( figure 3b ). perhaps n-prrsv alters hosts' lipid metabolism to create a lipid-rich intracellular environment to facilitate its own multiplication. moreover, we also observed that n-prrsv induced upregulation expression of anti-apoptotic genes in n-prrsv infected lungs, including myeloid cell leukemia sequence 1 (bcl2-related) (mcl1), nuclear factor kappa-b 1 (nfkb1), nfkb2, adrenomedullin (adm), and interleukin 10 (il10), and downregulation expression of pro-apoptotic genes, including bak protein (bak), (apoptosis-related protein 1) (apr1) ( figure 3d ) to inhibit apoptosis, which might prolong cell life and increase the yield of progeny virions. n-prrsv infection caused anorexia and subsequent slow growth. accordingly, we observed that transcript abundance of genes involved in digestion, such as gastric mucin (muc5ac) and cytochrome p450 (cyp39a1), was significantly decreased ( figure 3e ). simultaneously, transcript abundance of the genes associated with cell, muscle and cartilage development was markedly decreased ( figure 3f ). these genes include insulin-like growth factor binding protein 3 (igfbp3), collagen, type ii, alpha 1 isoform 2 (col2a1), connective tissue growth factor (ctgf), epidermal growth factor (egf). fever and heat shock fever is frequently the host's initial response to infection [37] . after viral infection, pathogen-associated molecular patterns (pamps) in viral proteins and nucleic acids were recognized by host pathogen-recognition receptors (prrs), such as toll-like receptors (tlrs), which trigger gene expression and synthesis of the il-1b precursor. active caspase-1 (casp1) cleaves the il-1b precursor into mature, bioactive il-1b, which is an inflammatory cytokine most responsible for fever [37, 38, 39] . as shown in figure 4a , transcript abundance of tlr1, 2, 4, 6, il-1b and casp1 was significantly increased in n-prrsv infected porcine lungs. moreover, transcript abundance of genes involved in the activation of casp1 and il-1b secretion including apoptosisassociated speck-like protein containing a card (asc), prostaglandin e synthase 2 (pge2) and phospholipase a2, group vii (pla2g7) was significantly increased ( figure 4a ). the expression of heat shock proteins (hsps), known as stress proteins, can be markedly upregulated by all cells under conditions of stress, such as increased temperature (fever) and viral infection [40] . transcript abundance for most of these heat shock genes, including 90-kda hsp (hsp90), hsp70, and heat shock protein beta-1 (hsp27) was significantly elevated in n-prrsv infected lungs relative to unc lungs ( figure 4b ). viral infection results in an inflammatory response, which is an essential component of the antiviral innate immune response [41] . after recognizing the pamps, either surface or intracellular prrs trigger intracellular signaling cascades that results in the activation of transcription factors, including nuclear factor-kb (nf-kb), interferon-regulatory factors (irfs), and signal transducer and activator of transcription (stats). as shown in figure 4a , transcripts of the toll-like prrs tlr1, tlr2, tlr4, tlr6, were significantly increased in n-prrsv-infected pigs at days 4 through 7 pi, but no change in tlr3 which specializes in the recognition of viral dsrna was detected. cytoplasmic prrs ( figure 5a ), retinoic-acid-inducible protein i (rig-i, ddx58) and melanoma differentiation-associated gene 5 (mda5), the two most relevant for defense against viruses, were expressed at a high level after n-prrsv infection. cell surface prrs such as cd14, md-2 protein (md2) and cd163 (which is probably involved in prrsv entry during uncoating [42] ) were likewise up-regulated expression after n-prrsv infection ( figure 5a ). after binding to n-prrsv viral pamps, prrs initiate intracellular signaling cascades that activate transcription factors, including irf1, irf7, irf9, but not irf3 and stat1, stat3, stat6 ( figure 5b ). activated transcription factors and stats in turn induce the transcription of specific sets of interferon-stimulated genes (isgs) [34, 43] , and expression of multiple inflammatory genes [44] , which induce a pro-inflammatory response and attract cells, such as neutrophils and macrophages, to sites of infection. accordingly, we observed significant increase of transcript abundance in many genes involved in isgs ( figure 5c ), pro-inflammatory cytokines (such as il1b, il8) ( figure 5d ), chemokines (ccl2, cxcl9, cxcl10) ( figure 5e ), adhesion molecules (vcam, icam1, sell), and other inflammatory molecules (such as mmp-2,) ( figure 5f ). moreover, immunoglobulin (such as igg2b, igg3) ( figure 5g ), three categories of fc receptors and mannose receptor c1 (mrc1) ( figure 5h ), and complement proteins ( figure 5i ) were also significantly induced in the n-prrsv-infected lungs. however, several complement inhibitors that possess inhibitory and/or decay-accelerating acitivity, such as decay-accelerating factor cd55, complement component 4 binding protein, alpha (c4bpa), c4bpb, were significantly repressed in the n-prrsvinfected lungs ( figure 5i ). cytotoxic t lymphocytes (ctls) detect cells infected with a virus and destroy them through perforin-mediated apoptosis [45] . cd8 + t cells activation require t cell receptors (tcrs) to recognize cognate antigenic peptides for presentation on mhc class i molecules displayed on the surface of antigen presenting cells (apcs) [32] . accordingly, we observed that transcript abundance of ubiquitin specific peptidase (usp) and ubiquitin enzyme ( figure 6a ), 16 proteasomes, and aminopeptidases ( figure 6b ) was significantly increased in n-prrsv-infected lungs. the transcript abundance of b2m, mhc class i antigen 2 (sla-2), sla-3, tap2, and chaperones (such as grp78) was markedly increased after infection with n-prrsv while the transcript abundance of sla-b was significantly decreased ( figure 6c ). in addition to recognization of cognate peptides presented by mhc class i molecules, cd8 + t cells activation needs also to receive 'costimulatory' signals and help by helper cd4 t + cells [33] . as shown in figure 6d and 6e, 8 cathepsins and 5 mhc class ii antigens were significantly induced in n-prrsv-infected lungs. the transcript abundance of costimulatory molecules (such as cd86, icos), cams, and tcrs/cd3 complex as well as co-receptor molecules (such as cd8a, table s3 for full gene names. doi:10.1371/journal.pone.0011377.g006 cd8b) was remarkably increased after infection with n-prrsv ( figure 6f and 6g) . activated ctls release perforin (pfr) and granzymes, which two effectors act collaboratively to induce apoptosis of target cells. as shown in figure 6h , prf1 and granzyme b (gzmb) transcript abundance was significantly elevated in n-prrsv infected lungs relative to unc lungs. in addition to cytotoxins released from ctls, the transcript abundance of other pro-apoptotic members ( figure 6i ), such as nfkbia, growth arrest and dna-damage-inducible protein alpha (gadd45a), bh3 interacting domain death agonist (bid), xiap-associated factor 1 (xaf1), cytochrome c (cycs), casp10, was also significantly increased after infection with n-prrsv, which can induce apoptosis of virus-infected cells. in addition, we also identified the upregulated expression of cytochrome b245 heavy chain (gp91-phox), a critical component of the membrane-bound oxidase of phagocytes (macrophages and neutrophils), and the downregulated expression of heme oxygenase 1 (hmox1) during n-prrsv infections, which might result in the oxidative stress response and subsequent oxidative damage of tissues ( figure 6j ). from the data presented in the paper, a model for the relationship between pulmonary gene expression profiles and infection pathology can be surmised in figure 7 , n-prrsv virus replicates and spreads by subverting host innate immune response and hijacking host lipid metabolism as well as inducing an antiapoptotic and anti-inflammatory state, as indicated by suppression expression of spi ifn, ifn-a, down-regulation expression of proapoptotic genes for bak, apr-1, sarp3, high levels expression of genes involved in lipid metabolism, such as apoe, ldlb, pik3c3, anti-apoptotic genes for mcl1, bcl2a1, chfr, adm, nfkb, il10, and anti-inflammatory molecule pge2 as well as cd163. infections of n-prrsv viruses resulted in fever and inflammatory response, as indicated by high expression of proinflammatory cytokines and chemokines, adhesion molecules, inflammatory enzymes and receptors, such as il-1b, il8, sell, icam, ccl2, cxcl9, cxcl10, b2m, proteasomes, cathepsins. this was compounded by cell death and elevated expression of nfkbia, xaf1, gadd45a, perforin, granzymes, and cytochrome c, coupled with increased ros-mediated oxidative stress, as indicated by by up-regulation expression of cytochrome b245. taken together, the n-prrsv infections may have resulted in an excessively immune and inflammatory response that contributed to tissue damage. infection of pigs with n-prrsv caused fever, dyspnoea, reddening of skin, oedema of the eyelids, conjunctivitis, depression, anorexia, mild diarrhoea. histopathology examination showed interstitial pneumonia in lungs with thickening of alveolar septa accompanied with infiltration of immune cells [46] (figure 1 ). although great efforts have been made by many researchers, the molecular basis of n-prrsv infection is largely unknown. here we report on the first genome-wide host transcriptional response to n-prrsv infection using solexa/illumina's digital gene expression (dge) system, a tag-based novel high-throughput transcriptome deep sequencing method. given the nature of the methodology of illumina's dge system, we have pooled biological replicates from three pigs for each group to make representative samples for deep sequencing analysis. we could reach a sequencing depth of 6.3-7.9 million tags per library (table 1) and found over 5000 genes to be differentially expressed during n-prrsv infection processes (table s3) . although others studies have also pooled biological replicates for library construction and deep sequencing [47, 48] , resulting in the lack of biological replicate, one may blur the impact of variations in pooling samples. because of the variations of pigs in response to prrsv infection, it is possible that one pig could significantly affect results without independent libraries. but we performed the qpcr validation both on the same pooled material that was used for deep sequencing and on independent rna extractions from each pig, which all confirmed the direction of change detected by dge analysis (figure 2 ). our dge analysis showed massive changes in the transcript abundace of known immune response genes and of genes that have been implicated in prrsv infection [18, 19, 20] . we also identified many interesting genes that had not been linked to prrsv infection in previous studies. for example, transcript abundace of lipid metabolism-related genes including apob48r, apoe, pik3c, was significantly increased during n-prrsv infection processes. alterations in lipid metabolism, perhaps including those with significant upregulation in this study, have been observed in response to infection by a range of viruses including sars-cov, hcv, influenza a virus, or dengue virus [35, 36, 49, 50] . although in vitro studies have investigated how prrsv modifying genes expression in pams [18, 19, 20] , many of the outstanding issues will be answered only in the context of prrsv-infected animals [51] . hence, we characterized the genome-wide transcriptome response to prrsv infection in porcine lung by deeping sequencing. but studies of transcript abundance in lung tissues have also their intrinsic limitations. for example, the transcriptome of lung tissues is actually a merging transcriptional responses of a wide range of cell types, some of which are infected, some of which are responding directly to the infectious process and others of which are bystanders. moreover, increased cellularity of tissues may be confused as biologically important increased transcript abundance. despite such limitations, our dge study offers a broad, system-wide window into molecular processes that regulate gene expression and also provides new leads for functional studies of candidate genes involved in host-virus interaction, as illustrated in this paper. the induction of expression of type i interferons (ifns; including ifn-a and ifn-b) is a well-known innate antiviral immune reaction in the virus-infected cells [52, 53] . however, n-prrsv infection suppressed spi ifn gene expression and decreased the transcript abundance of ifn-a ( figure 3a ). previous studies [19, 54, 55] , both in vitro and in vivo, have also showed that prrsv elicited only a minimal ifn-a production or even suppressed it's expression. the suppression of spi ifn, in particular of ifn-a, is probably a crucial step in the pathogenesis, because ifn-a has been shown to inhibit prrsv replication [11] . other viruses infection, such as the 1918 influenza virus [56] , hepatitis c virus (hcv) [57] , ebola virus [58] , also suppressed type i ifn gene expression which led to extensive viral replication and increased pathogenesis. irf3 plays an important role for type i ifn gene expression. the transcript abundance of irf3 was decreased intensively in n-prrsv-infected pigs by 168 h pi ( figure 4b ). one study [59] indicated that prrsv nsp1b inhibited irf3, and then down-regulated ifn-b gene expression. it is worth mentioning that the nsp1 of the influenza a can also suppress innate immunity by inhibiting irf3 activation, and subsequently disrupting the induction of a/b-interferon [60] . research has indicated that the expression of cd163, a prrsv receptor [61] , on macrophages in different microenvironments, in vivo, may determine the replication efficiency and subsequent pathogenecity of prrsv [62] . transcript abundance of cd163 was significantly increased after n-prrsv infection ( figure 5a ). the internalization of prrsv via cd163 in the target cells may induce the expression of il10, and in turn induce the expression of cd163 on neighboring undifferentiated monocytes and increased overall prrsv susceptibility [62] . moreover, infected pigs develop a strong and rapid humoral response but these initial antibodies do not confer protection and can even be harmful by mediating an ade [12] . these antibodies enhance prrsv viral replication by coating the virus and enhancing the internalization of viral particles into macrophages [12] . as shown in figure 5g and 5h, igg and fcc receptors were significantly induced during n-prrsv infection processes. interestingly, the presence of antibodies during feline enteric coronaviruses (fecvs) infection does not also provide sterilizing immunity, instead, these antibodies opsonize virus particles and facilitate their entry to monocytes and/or macrophages through fcc receptors, resulting in disease enhancement [51] . the activation of pro-inflammatory transcription factor nf-kb induces robust activation of the casp1 inflammasome and subsequent release of il-1b that cause fever and inflammation [63, 64, 65, 66] . accordingly, we identified upregulation expression of casp1, nf-kb, and il-1b genes during n-prrsv infection processes ( figure 4a ). nf-kb activation also enhanced the expression of matrix metalloproteinases (mmp2) and mmp9, two cytotoxic substances in prrsv-infected cells [11, 67] . similarly, transcript abundance of mmp2 and ngal (25 kda alpha-2-microglobulin-related subunit of mmp-9) was significantly increased in the lungs of n-prrsv-infected pigs ( figure 5f ). upregulation expression of mmps would likely facilitate infiltration of inflammatory cells and increase inflammation. upregulation expression of il8 (also known as cxcl8), which is an attractant for neutrophils and other polymorphonuclear leukocytes produced after acute infection, in prrsv-infected pams [68, 69] and lungs of n-prrsv-infected pigs ( figure 5d ), was observed. other chemokines such as ccl2 (also known as mcp1 ), cxcl9, cxcl10 (also known as ip10), which were significantly increased ( figure 5d ), may be also crucial for lymphocyte and macrophage infiltration into the sites of n-prrsv infection. ccl2, il8 and ip10 expression were upregulated during sars-cov [70, 71] , and murine coronavirus [72] infections process, which may recruit monocytes and/or macrophages to sites of infection and be a major cause of lung pathology. although the present study indicates that upregulation expression of pro-inflammatory molecules contributes to the pathogenesis of n-prrsv, increased transcript abundance of anti-inflammatory molecules, such as il10, pge2, was also detected in the study. upregulation of il10 gene expression was found previously in prrsv-infected porcine leukocytes, pams, dcs, and in vivo in prrsv infected pigs [16, 17, 19, 73] . perhaps an increase in pro-inflammatory molecules followed by increased anti-inflammatory molecules is the normal progression of events in inflammation [74] . the upregulation expression of il10 might skew the immune response away from a protective th1-cell response towards a non-protective th2-cell response, therefore impairing clearance of virus, which benefits viral infections [51] . upregulation expression of anti-inflammatory molecules and proinflammatory molecules occurring concurrently was also observed after sars-cov and fipv infection [75, 76] . antibodies might also contribute to immunopathogenesis through increasing the uptake of virus by macrophages, resulting in activation of these macrophages and secretion of pro-inflammatory cytokines and chemokines. antigen-antibody complexes might increase transcript abundance of complement ( figure 5i ), which leads to generation of the classical inflammatory response through the production of potent proinflammatory molecules [77] . furthermore, complement activation might also contribute to the development of pulmonary edema and oedema of the eyelids. further understanding the roles complement plays in the hostpathogen interactions may help to develop more effective pharmacological agents against infection. moreover, damage to the lungs of n-prrsv-infected pigs seems to occur directly by viral destruction of alveolar and bronchial epithelial cells and macrophages ( figure 1c) , as well as indirectly through production of immune mediators. activated ctls and nk cells release perforin (pfr) and granzymes, which two effectors act collaboratively to induce apoptosis of target cells. transcript abundance of pfr1 and granzymes increased in the lungs of n-prrsv infected pigs ( figure 6h ). pro-apoptotic molecules xaf1, bid, cyto c, casp10, aifm2, were significantly up-regulated after infection with n-prrsv, which may induce apoptosis of virus-infected cells ( figure 6i ). simultaneously, we also observed upregulation expression of anti-apoptotic genes in n-prrsv infected lungs, including bcl2a1, mcl1, chfr, nfkb, adm, il10 etc ( figure 3d ). upregulation expression of anti-apoptotic genes and pro-apoptotic genes occurring concurrently after n-prrsv infection seems in contradiction of each other. however, this may reflect a balance between apoptotic and anti-apoptotic mechanisms. perhaps prrsv actively induces an anti-apoptotic state to complete its virus replication cycle through delaying cell death while induces apoptosis of virus-infected cells after completion of virus replication to increase rate of spread of virus [19, 78, 79] . anti-apoptotic and pro-apoptotic activaties were also observed in prrsv-infected marc-145 cells, in which prrsv stimulated anti-apoptotic pathways early in infection while caused apoptosis of prrsv-infected cells late in infection [80, 81] . infection with n-prrsv also increased transcript abundance of nfkbia ( figure 6i ), an inhibitor of the tnf receptor activated transcription factor nf-kb. loss of nf-kb activity has been shown to increase the cytotoxic effects of tnf which resulted in increased cell death [82] . an increase of transcript abundance in proapoptotic genes might result in disruption of the mitochondria transmembrane potential, thereby inducing release of cyto c from mitochondrial membranes to induce apoptosis and secondary necrosis [83] . the production of ros, especially superoxide radicals, and the subsequent oxidative damage of cells and tissues are recognized as key contributors to the viral pathogenesis [82, 84] . ros-mediated oxidative stress might also be involved in inducing apoptosis by prrsv [81] . accordingly, we identified the remarkable upregulation of cytochrome b245 heavy chain (gp91-phox) ( figure 6j ), a critical component of the membrane-bound oxidase of phagocytes (macrophages and neutrophils), after infection with n-prrsv, that generated superoxide radicals that killed both infected and normal cells at sites of infection, which would further exacerbate the immunopathological response. infection of macrophages, monocytes and dcs that are essential for immune function, is likely to be a key component in n-prrsv-induced pathogenesis [19, 85, 86, 87] . apoptosis of infected cells causes immune suppression by two mechanism: apoptosis either induces a decrease in the numbers of immune cells that compromises both the innate and adaptive immune response in which they are unable to eradicate the primary infection, or impairs immunity by inducing immunosuppressive effects in the surviving cells [88] . for example, uptake of apoptotic cells by normal macrophages and dcs stimulates immune tolerance by inducing the release of anti-inflammatory cytokines, such as il10, and suppressing the release of pro-inflammatory cytokines [89] . histopathological analysis of the lymphnodes of pigs infection with n-prrsv revealed a profound depletion of immune cells compared with those of unc (data not shown). in summary, the data presented in this study suggest that n-prrsv appears to utilize multiple strategies for its replication and spread in infected pigs, including subverting host innate immune response, inducing an anti-apoptotic and anti-inflammatory state as well as developing ade. after infection of macrophages and possibly dcs, prr-pamp interactions triggered signaling cascades that increased the transcript abundance of multiple inflammatory molecules, including cytokines, chemokines, adhesion molecules and inflammatory enzymes that induce a proinflammatory response, activate and recruit immune cells, such as macrophages and neutrophils, to sites of infection for virus elimination and thereby produce the clinical symptoms of viral infection, such as fever, dyspnoea, interstitial pneumonia in lungs. further, antibodies and complement activation might exacerbate inflammatory response. n-prrsv might induce an immunosuppressive state, mediated by apoptosis of infected cells, which caused depletion of immune cells and induced an anti-inflammatory cytokine response in which they were unable to eradicate the primary infection. figure s4 signaling pathways of de genes. pathway analysis was mainly based on the kegg database. a p-value of ,0.05 and an fdr of ,0.05 in the two-side fisher's exact test were selected as the significant criteria. the vertical axis is the pathway category and the horizontal axis is the log10(p value) of these significant pathways. found at: doi:10.1371/journal.pone.0011377.s004 (0.66 mb tif) figure s5 genes that distributed in the known pig qtls of health traits. the x axis represents the qtl symbol, and the y axis indicates the number of genes associated with health traits. see table s4 for full qtl names. figure s7 biological process go terms of profile 1. functional classification of the de genes was performed according to go biological processes. a p-value of ,0.05 in the two-side fisher's exact test were selected as the significant criteria. these de genes were sorted by the enrichment of go categories. the vertical axis is the go category and the horizontal axis is the enrichment of go. found at: doi:10.1371/journal.pone.0011377.s007 (1.53 mb tif) figure s8 biological process go terms of profile 0. functional classification of the de genes was performed according to go biological processes. a p-value of ,0.05 in the two-side fisher's exact test were selected as the significant criteria. these de genes were sorted by the enrichment of go categories. the vertical axis is the go category and the horizontal axis is the enrichment of go. found at: doi:10.1371/journal.pone.0011377.s008 (2.78 mb tif) figure s9 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their antigen-presenting ability apoptosis and caspases regulate death and inflammation in sepsis death-defying immunity: do apoptotic cells influence antigen processing and presentation? we thank the beijing genomics institute (bgi) shenzhen and genminix informatics ltd.,co for their providing us with technical assistance in dge and bioinformatics analysis. key: cord-312033-iarl77n0 authors: lópez barreda, rodrigo; guerrero, alonso; de la cuadra, juan cristóbal; scotoni, manuela; salas, wilbaldo; baraona, fernando; arancibia, francisca; uriarte, polentzi title: poverty, quality of life and psychological wellbeing in adults with congenital heart disease in chile date: 2020-10-08 journal: plos one doi: 10.1371/journal.pone.0240383 sha: doc_id: 312033 cord_uid: iarl77n0 the objective of this study was to assess the quality of life and psychological wellbeing of adults with congenital heart disease (chd) in chile, and to identify other associated factors. the study enrolled 68 patients aged between 18 and 72 (median 29), 35 being females. they completed a questionnaire, which included a quality of life assessment tool (the medical outcome study 36-item short form health survey), a number of psychological scales (the general health questionnaire, the basic psychological needs scales and the beck hopelessness scale), a socioeconomic survey, and some clinical data. chd patients reported worse scores in those scales assessing physical dimensions of quality of life (physical function (70.5), physical role functioning (64), vitality (65.3)), and general quality of life (58.6), than in emotional or social dimensions. female gender was associated with lower scores in physical function (59.12 versus 82.66; p<0.01) and physical role functioning (53.68 versus 75; p<0.05); poverty was associated with worse results in physical function (61.92 versus 82.96; p<0.01), role physical (53.21 versus 79.63; p<0.01), vitality (60.89 versus 71.67; p<0.05), social role functioning (70.19 versus 82.87; p<0.05) and bodily pain (65.77 versus 81.2; p<0.05). furthermore, we found that psychological scales had an association with quality of life, but clinical variables did not show significant correlations to any dimension. poverty has an impact on the quality of life of chd patients. this population only has a decrease in the quality of life physical dimensions, suggesting that quality of life depends on many different factors. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 congenital heart diseases (chd) include a wide range of cardiac disorders that are present at birth, ranging from simple conditions to severe structural abnormalities. it is estimated that approximately 1% of children are born with chd and a number of them die during the first year of life [1] . however, mortality has sharply decreased in the last decades thanks to improvements in technology and medical care. nowadays, more than 85% of these patients reach adulthood [2] . healthcare for these patients has become a challenge, not only because of medical issues, but also because patients have quality of life (qol) and psychological functioning needs that should be met [3] . for a better understanding of the impact of disorder and therapy on patients' lives, surveying qol and psychological functioning is essential [4] . for this reason, the qol of chd patients has been extensively surveyed. remarkably, in this regard, the literature is mostly focused on clinical features and shows ambiguous results. while some studies in children and adolescents suffering from chd showed a decrease in qol [5, 6] , others found these trends only in specific life domains [5, 7] , and other reports claimed that patients with chd enjoy a better qol than the general population [8] [9] [10] . it is not surprising that studies aiming at identifying those patients at risk for low qol provided contradictory results. older age has been identified as a risk factor [11] [12] [13] , but silva found the opposite [9] . gender makes a difference according to fteropoulli [11] , but not according to apers [4] . severity of the chd has been related to low qol by a number of studies [7, [9] [10] [11] , but a recent survey with a large sample size did not find this correlation [4] ; moreover, jackson [12] reported that patients suffering from moderate chd lesions had the best qol, in comparison to those with most severe conditions who had the worst qol, and patients with mild abnormalities were in between those two groups. qol has been claimed to depend on many other factors beyond the clinicals and physicals [5] , and this could explain some of those discrepancies. personality traits [14] as well as social support [9, 10] may play a protective role. cultural background, on the other hand, does not affect patients' qol [4] . a recent study assessing the effect of standard of living and healthcare system characteristics, by means of national gross domestic product per capita and total health expenditure per capita, showed that these variables do affect qol [13] . however, this study used aggregated information, such as gross domestic product per capita, and did not collect individual data, such as personal income, which allegedly may have an effect on these patients' qol. regarding psychological wellbeing, the available evidence is also contradictory. some studies report a higher rate of depression and anxiety, especially in patients with complex lesions [15] , while other studies have more positive results, identifying a normal level of anxiety [3, 16] and hopelessness [17] in daily life. as was the case for qol, socioeconomic factors have been related to anxiety and depression [18] . the primary aim of this study was to assess the qol and psychological wellbeing of a population of adults suffering from chd in chile. in addition, it explores whether socioeconomic factors, such as poverty, are associated with the qol of this population. this is a quantitative cross-sectional study. all procedures contributing to this work comply with the ethical standards of the relevant national guidelines on human experimentation (law nº20120, september 22nd 2006) and with the helsinki declaration of 1975, as revised in 2008, and have been approved by the institutional committees: pontificia universidad católica de congenital heart diseases and being followed in a particular hospital), even if personal information was deleted, the participants would be easily identifiable; moreover, the scales used in this study collect information that has been considered highly sensitive. therefore, there are ethical reasons that impede the publication of our data set. the irb that assessed this study and imposed this restriction is the comité ético científico de ciencias de la salud. data access requests may be made to the irb at its email: eticadeinvestigacion@uc.cl or its phone: (+56) 223548173 / (+56) 22354-2397 funding: the author received no specific funding for this work. chile faculty of medicine committee and hospital del tórax committee. patients agreed to participate and signed the informed consent form. a group of adult patients with chd who are being followed at the 'instituto nacional del tórax', the main national reference center for adult congenital heart disease, were invited to participate. inclusion criteria were: males or females over 18 years old with any type of congenital heart disease (repaired or unrepaired), a stable clinical condition (no recent acute decompensations or hospitalizations for surgical or percutaneous procedures). exclusion criteria were: patients under 18 years old or unable to consent, illiterate, unwilling to participate or receiving more than two psychotropic drugs. a researcher interviewed the participants in a private room while patients were waiting for a medical appointment. an informed consent form was signed beforehand and subjects were asked to complete a selection of psychological self-report scales and a socioeconomic survey questionnaire. the researcher remained available to provide clarification if needed. the scales and survey required 20 to 30 minutes approximately. medical history was reported by patients and then compared with our local database. in order to assess qol and wellbeing, the following questionnaires were used: the medical outcome study 36-item short form health survey (sf-36) [19] . as suggested by gill [20] , before selecting a scale to assess qol, it is important to define the meaning of qol used in the study. we decided to use the definition coined by him, namely "a reflection of the way that patients perceive and react to their health status and to other, nonmedical aspects of their lives" [20] (p.619). using this definition, we selected the medical outcome study 36-item short form health survey (sf-36). this scale is one of the most used to subjectively assess health status in biomedical studies. it contains 36 likert questions and has 8 subscales: physical functioning, physical role functioning, bodily pain, general health perception, vitality, social role functioning, emotional role functioning, and mental health. scores range from 0 to 100; higher scores indicate better subjective health status. the psychometric properties of this scale have been assessed in several countries and on different groups of patients, proving its accuracy. although it does not consider all relevant dimensions to comprehensively assess qol, it is the most used in this type of population [21] . the spanish version was validated by alonso [22] . the general health questionnaire (ghq-12) [23] . this scale explores non-severe psychiatric symptoms in the general population and it evaluates mental health more than general health [24] . the selected version includes 12 likert questions, scores between 0 and 36, and higher scores indicate worse mental health. it was validated in spanish by humphreys [25] . the basic psychological needs scales (bpn) [26] . the self-determination theory states that there are three universal psychological needs that must be met in order for people to experience psychological wellbeing, namely autonomy, competence and relatedness. they allow people to act according to their intrinsic motivations [27] . this scale assesses the degree to which these needs are met. the scale selected for this study was published by gillet [26] , and it consists of 15 items rated with a 5-point likert scale, and scores from 15 to 75; higher scores indicate better fulfilment of psychological needs. the validated spanish version has adequate internal consistency [28] . [29] . hopelessness refers to a cognitive schema with negative expectations about the future. this scale is focused on pessimism and the belief that problems cannot be solved, both characteristics that could be related to depression. this is a 10-question true-or-false scale, and its results are expressed with a numeric value from 0 to 20, higher scores show more hopelessness; scores above 8 are considered abnormal and further assessment is advised. some studies have assessed hopelessness in this population using the bhs [17] . the spanish version was validated by aguilar [30] . socioeconomic level (sel), an important variable to be taken into account, was measured by a sample of questions from the survey regularly conducted by the chilean ministry of social development [31] . the selected dimensions and indicators were: age, marital status, education (highest degree obtained), work status (employment, formal contract), health insurance, and housing/neighborhood (number of people living in the house, social participation). these socioeconomic data were analyzed independently and also transformed into a dichotomous variable (poverty) following the multidimensional poverty approach proposed by aplablaza for the chilean population [32] . this account has been used in a similar study [33] . clinical variables. the severity of the disease was assessed using american college of cardiology/american heart association guidelines [34] . other variables included age and total number of past hospitalizations up to one year before the survey. data on demographic variables and socioeconomic status was presented using descriptive parameters such as median and interquartile range because these variables' sample values had a non-normal distribution; data on qol and psychological scales was shown using mean and standard deviation, as the distribution passed normal distribution tests. associations between qol and clinical and social measures, as well as with psychological scales, were analyzed with the pearson or spearman correlation, and t-tests. when the distribution did not pass normal distribution tests, the kolmogorov-smirnov test was applied. stata v.13 (statacorp, 4905 lakeway dr. college station, tx 77845) was used to carry out the statistical analysis. data was collected from june to august 2019. during that period, 85 patients were contacted, 75 of whom were invited to participate as they met the inclusion criteria. 67 accepted and completed the questionnaires. the age of the participants ranged from 18 to 72 years old, median 29 (interquartile ranges (iqr) [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] . 51.5% were female, 57.4% were single and 77.9% had completed secondary education. the total number of hospitalizations ranged from 1 to 30, median 2 (iqr 2-7) and 64.7% had a moderate chd. more demographic characteristics of the participants are shown in table 1 . the results of the sf-36 and the psychological tests are shown in table 2 . 11 participants (16.2%) showed a hopelessness level considered abnormal (>8) and were referred for mental health evaluation at their local healthcare provider. clinical dimensions did not show a significant correlation to any of the qol dimensions. the pearson's correlation coefficients range for these variables were: age from -0. table 3 . poverty, quality of life and psychological wellbeing in adults with congenital heart disease in chile gender had a mild effect on qol and no effect on psychological wellbeing, with the remarkable exception of physical function (males 82.66 and females 59.12; p<0.01) and physical role functioning (males 75 and females 53.68; p<0.05). on the contrary, poverty was associated with low results in a number of the qol dimensions, but without effect on psychological wellbeing. poor participants scored worse than nonpoor participants in physical function ( as was commented beforehand, research in qol studies shows ambiguous results [11, 21, 35] . this may be explained by heterogeneous measuring methods (e.g. study design, different populations, disease severity class, etc.) as well as a lack of methodological and conceptual rigor [11, 35] . qol is a vague concept that opens up a large margin of interpretation [36, 37] . diversion in perception and inappropriate use of the term qol lead inevitably to inconclusive findings. with their review of qol measurements, gill and feinstein set a milestone by developing 10 criteria that aim to support the evaluation of qol measurements [35, 36] . yet, more than 40 years later, there has been only a slight improvement in methodological and conceptual accuracy in qol publications [21] . despite this issue, our study has similar findings as some of those reported in the literature. we found a difference in qol according to gender [11] , and there was no association with chd severity [4, 7, 13] . more important are the differences across the different dimensions of qol. as other authors reported, chd patients showed low scores in the physical domains of qol, but the results in the social and emotional domains are comparable to those of the poverty, quality of life and psychological wellbeing in adults with congenital heart disease in chile general population [5, 11, 38] . our findings support the notion that qol is a complex concept, which is related to factors beyond physical functioning and clinical facts (such as age, chd severity or number of hospitalizations) [4, 5, 13] , highlighting the importance of social support [10] . despite the fact that an association between chd and socioeconomic burden has been described [39] , this variable is frequently neglected when surveying this population's qol. aiming at assessing this factor, jackson et al. surveyed individual income of chd patients and showed that earning less than us$30,000 per year explained 23% of the variability in the qol [12] . a recent study in chinese children suffering from chd showed that there is an association between qol and socioeconomic status [40] , assessed by means of household income, parental occupation and educational level. nevertheless, the mechanism by which socioeconomic level influences qol is still not fully characterized. it has been suggested that chd causes poverty or that inability to pay makes access to healthcare more problematic [12] , which is not the case for chilean patients. other plausible explanations are an eventual link between poverty and low health literacy, affecting patients' adherence and healthcare consulting behavior [40] , and the effect of long-term and comprehensive rehabilitation therapy, which is often expensive. the association between socioeconomic status and these patients' qol should be carefully explored, as well as the underlying mechanisms. this population has an increased risk of suffering from economic hardships [39] and any effort to improve their qol that neglects socioeconomic factors would be rather ineffective. qol includes different areas, and the subjective perceived qol may be associated with psychological wellbeing. an appropriate psychological functioning is crucial to all human beings. as was the case for qol, several studies with chd patients have been conducted assessing psychological wellbeing, revealing inconsistent findings. whereas some studies describe higher rates of anxiety and depression [15, 38, 41] , others report no differences compared to healthy controls [3, 16, 17] and outline independent variables like social support and socio-economic factors that affect people's level of anxiety and depression [18] . the importance of the individual psychological wellbeing is highlighted by the fact that it influences the adherence to medical treatment and hence affects patient's recovery [42] . moreover, poor emotional (and physical) qol might result in struggles with important tasks like physical activities or attending medical appointments [12] . in order to evaluate the relation between qol and psychological wellbeing of our study patients, we made use of the ghq-12 and the bpn scale. results from the ghq-12 questionnaire revealed a good negative correlation with different qol factors like vitality, social functioning and general health, whereas bpn showed positive correlations to almost all dimensions. neither gender nor poverty had a significant effect on psychological functioning. this is a very interesting finding, as arguably both issues have been related to low psychological wellbeing [30] . patients suffering from chd, however, are a particular population, and the psychological resources they use to face their disease might be applied to cope with gender and socioeconomic issues as well. this explanation should be further explored. providing the appropriate care to these patients is still a challenge. diagnosis and medical treatment frequently puzzle healthcare teams, and structural abnormalities are often difficult to repair. despite these issues, mortality has importantly decreased and, according to our results, psychological wellbeing is also preserved. however, if we want to offer these patients good qol, attention should be paid to those factors that compromise physical functioning and global qol. our study suggests that socioeconomic factors need to be taken into account. our study has many limitations that have to be acknowledged. the sample size is small and we recruited patients from only one public center. however, the 'instituto nacional del tórax' is the main reference center treating these patients, and receives patients from the whole country. this fact, added to a high response rate (89.3%), enhanced our outcomes' representativeness. having a control group and comparing the results of both populations would have provided very useful information to have a better understanding of the phenomenon. we intended to extend the project in this way, but the sociopolitical events that happened in chile from october 18th 2019 onwards and the covid-19 pandemic thwarted our intention, as the levels of anxiety and hopelessness skyrocketed in the chilean population and the results would not have been comparable. the proportion of patients classified as poor is remarkably high (58.8%). this fact could be considered as a limitation, due to lack of representativeness. however, in this study we surveyed socioeconomic factors using a multidimensional approach, considering dimensions beyond household income, such as education, working status, health insurance and housing, with specific cutoff values, according to chilean standards [32] . multidimensional poverty indexes usually classify more people under the line of poverty than pure income approaches, but they provide a well-grounded method to identify people suffering from socioeconomic deprivation, rendering them vulnerable. furthermore, this possible overrepresentation of the poor does not challenge the differences found when comparing this group with the non-poor. regarding the methodological issues that have been identified in the studies exploring qol of chd patients, our study meets a number of the rigor criteria, and those that were not fulfilled were left as such after extensive discussions. despite the relevance of this topic, and the huge amount of available literature, there is not much evidence regarding qol and the psychological wellbeing of adults with chd in latin america. to our knowledge, this is the first study exploring qol and the psychological wellbeing of chilean adult chd patients. in conclusion, this study provides evidence that poverty is associated to low qol in chilean patients suffering from chd. these patients show low qol in the physical dimensions, but this phenomenon is not seen in other qol dimensions, suggesting that it depends on factors beyond physical functioning and clinical tests. further studies should be done in order to have a better understanding of this phenomenon, such as longitudinal studies and qualitative research. this knowledge will allow us to design effective strategies, aiming at improving the qol for this vulnerable population. cardiovascular health and disease in children: current status. a special writing group from the task force on children and youth congenital heart disease never goes away, even when it has been 'treated': the adult with congenital heart disease general anxiety of adolescents and adults with congenital heart disease is comparable with that in healthy controls quality of life of adults with congenital heart disease in 15 countries: evaluating country-specific characteristics health related quality of life and health status in adult survivors with previously operated complex congenital heart disease psychological adjustment and quality of life in children and adolescents following open-heart surgery for congenital heart disease: a systematic review quality of life in adults with congenital heart disease is sense of coherence a pathway for improving the quality of life of patients who grow up with chronic diseases? a hypothesis quality of life of patients with congenital heart diseases living with chd: quality of life (qol) in early adult life quality of life of adult congenital heart disease patients: a systematic review of the literature medical factors that predict quality of life for young adults with congenital heart disease: what matters most? patient-reported outcomes in adults with congenital heart disease: inter-country variation, standard of living and healthcare system factors personality traits, quality of life and perceived health in adolescents with congenital heart disease depression and anxiety in adults with congenital heart disease: a pilot study psychosocial functioning of the adult with congenital heart disease: a 20-33 years follow-up hopelessness among adults with congenital heart disease: cause for despair or hope? anxiety, depressive and somatic symptoms in adults with congenital heart disease the mos 36-item short-form health survey (sf-36): i. conceptual framework and item selection quality of quality-of-life measurements forty years of quality-of-life research in congenital heart disease: temporal trends in conceptual and methodological rigor la versió n española del sf-36 health survey (cuestionario de salud sf-36): un instrumento para la medida de los resultados clínicos user's guide to the general health quest estrés ocupacional en personal de salud validació n preliminar en chile de una versión abreviada del cuestionario general de salud de goldberg (ghq-12). iii chilean congress of neurosciences and psychiatry and xlvi meeting of the chilean society of neurology dé veloppement d'une é chelle de satisfaction des besoins fondamentaux en contexte sportif human autonomy in cross-cultural context: perspectives on the psychology of agency, freedom and well-being traducció n y validació n de la versión española de la é chelle de satisfacció n des besoins psychologiques en el contexto educativo the measurement of pessimism: the hoplessness scale estudio prospectivo de la desesperanza en pacientes psicó ticos: características psicomé tricas de la escala de desesperanza de beck encuesta casen chronic multidimensional poverty or multidimensional chronic deprivation. resource document well-being and agency in parents of children with congenital heart disease: a survey in chile acc/aha 2008 guidelines for the management of adults with congenital heart disease: a report of the american college of cardiology/american heart association task force on practice guidelines (writing committee to develop guidelines on the management of adults with congenital heart disease) caliber of quality-of-life assessments in congenital heart disease: a plea for more conceptual and methodological rigor a critical appraisal of the quality of quality-of-life measurements quality of life: a concept analysis quality of life, health status, and depression: comparison between adolescents and adults after the fontan procedure with healthy counterparts social burden and lifestyle in adults with congenital heart disease impact of family socioeconomic status on health-related quality of life in children with critical congenital heart disease mental disorders in adults with congenital heart disease: unmet needs and impact on quality of life physical wellness, health care and personal autonomy the authors wish to thank margarita bernales phd, guillermo lema md and justine robertson for their invaluable comments and suggestions during the writing of this manuscript. key: cord-309043-dlmx12vt authors: von brunn, albrecht; teepe, carola; simpson, jeremy c.; pepperkok, rainer; friedel, caroline c.; zimmer, ralf; roberts, rhonda; baric, ralph; haas, jürgen title: analysis of intraviral protein-protein interactions of the sars coronavirus orfeome date: 2007-05-23 journal: plos one doi: 10.1371/journal.pone.0000459 sha: doc_id: 309043 cord_uid: dlmx12vt the severe acute respiratory syndrome coronavirus (sars-cov) genome is predicted to encode 14 functional open reading frames, leading to the expression of up to 30 structural and non-structural protein products. the functions of a large number of viral orfs are poorly understood or unknown. in order to gain more insight into functions and modes of action and interaction of the different proteins, we cloned the viral orfeome and performed a genome-wide analysis for intraviral protein interactions and for intracellular localization. 900 pairwise interactions were tested by yeast-two-hybrid matrix analysis, and more than 65 positive non-redundant interactions, including six self interactions, were identified. about 38% of interactions were subsequently confirmed by coip in mammalian cells. nsp2, nsp8 and orf9b showed a wide range of interactions with other viral proteins. nsp8 interacts with replicase proteins nsp2, nsp5, nsp6, nsp7, nsp8, nsp9, nsp12, nsp13 and nsp14, indicating a crucial role as a major player within the replication complex machinery. it was shown by others that nsp8 is essential for viral replication in vitro, whereas nsp2 is not. we show that also accessory protein orf9b does not play a pivotal role for viral replication, as it can be deleted from the virus displaying normal plaque sizes and growth characteristics in vero cells. however, it can be expected to be important for the virus-host interplay and for pathogenicity, due to its large number of interactions, by enhancing the global stability of the sars proteome network, or play some unrealized role in regulating protein-protein interactions. the interactions identified provide valuable material for future studies. the observation of atypical pneumonias in the chinese province guangdong in november 2002 led to the identification of the severe acute respiratory syndrome (sars). within a few months, the disease spread to a large number of countries and caused more than 8,000 cases and almost 800 deaths. the causative pathogen identified was shown to be a new human coronavirus designated the sars-cov [1] . tight intervention strategies limited the further spread of the pathogen. sequence analysis of the first isolates of the newly identified sars-cov revealed characteristic features typical of the known three coronavirus groups [2] [3] [4] . according to several phylogenetic analyses the virus is grouped either a novel group iv or an early split-off of group ii coronaviruses [5, 6] . the genome of the sars-cov consists of a positive-stranded rna of approximately 29,700 nt in length. the replicase genes span the first two-thirds of the genome containing the two overlapping orf1a and orf1b, which are connected by a ribosomal frameshift. the two polyproteins expressed are predicted to encode and to be cleaved by a papain-like proteinase 2 (pl-pro = part of nsp3) and a 3c-like proteinase (3cl-pro = nsp5) to 16 mature replicase proteins [1, 6] . they are well conserved between sars-cov and other coronaviruses. it is suggested that they are required for the synthesis of the full-length genome and subgenomic rna synthesis as well as for virus replication [6, 7] . functions of the processed proteins include a single-stranded rna-binding protein (nsp9) an rna-dependendent rna polymerase (rdrp = nsp12) as well as a non-canonical rdrp (nsp8) synthesizing short primers for nsp12, a superfamily 1like helicase (hel1 = nsp13), and a uridylate-specific endoribonuclease (nendou = nsp15) [7] [8] [9] [10] [11] [12] [13] [14] [15] . nsp3, nsp14, nsp16 are thought to have adp-ribose 19-phosphatase, 39-.59 exonuclease and 29-o-ribose methyltransferase activities, respectively [6] . but many of the functions of the nsps are still unknown. at their 59terminus the subgenomic mrnas share a common leader sequence encoded at the 59-end of the genome, which is joined to the respective gene sequences at specific transcription regulatory sequences. their common 39-ends extend to the end of the genome. the last third of the genome encodes the s, e, m and n structural genes with the group-specific genes interspaced among them. former are encoded by mrnas 2, 4, 5, and 9, latter by transcripts 3, 6, 7, 8, and 9, respectively. these genes, orf3a/b, orf6, orf7a/b, orf8a/b, and orf9b are not found in other coronaviruses and their functions with respect to replication and pathogenesis are not well understood. there is evidence that some of the accessory orfs can be deleted individually or in combination with almost no impact on in vitro growth, rna synthesis, or on in vivo virus replication in a murine model [16] . also, the nsp2 replicase protein of murine hepatitis virus (mhv) and sars-cov is dispensable for virus replication in cell culture. its deletion results in attenuation of viral growth and rna synthesis [17] . there are reports that a number of mhv and sars-cov replicase proteins colocalize and eventually interact in cytoplasmic membrane bound complexes, in which viral rna synthesis occurs [18, 19] . direct interactions of nsp7 and nsp8 in a hexadecameric supercomplex could be demonstrated by crystallography [20] . interactions of the structural n and m proteins were demonstrated by a mammalian two-hybrid system [21] . for the elucidation of molecular mechanisms during the course of viral growth and propagation there is a need to systematically examine possible interactions of all viral proteins. we therefore cloned the sars-cov orfeome by recombinatorial cloning (gateway technology) and performed a genome-wide analysis for viral protein interactions by yeast-two-hybrid (y2h) matrix screen. we have designed a set of nested pcr primers to amplify all viral non-structural, structural and accessory orfs at the predicted protease cleavage sites or at the respective start and stop codons ( table 1) . for cloning reasons, nsp3 was subdivided into a nterminal (nsp3n, nt positions 2719-4431) and a c-terminal (nsp3c, nt positions 4885-8484) fragment containing the adpribose-1''monophosphatase domain and the papain-like proteinase, respectively. an accessory orf14 described only by marra et al. was also included [3] . primers were designed such that they contained gene-specific sequences for the amplification of the respective orfs. overhanging sequences made them compatible to the gatewayh recombinatorial cloning system allowing the cloning into a so-called pdonr207 vector with the subsequent subloning into the destination vectors pgadt7-dest (prey) and pgbkt7-dest (bait). the y2h bait and prey vectors pgadt7-dest and pgbkt7-dest containing the sars orfs were transformed into the haploid yeast strains ah109 and y187, respectively, and mated and grown under selective conditions on media lacking leucine, tryptophane and histidine. all orfs were tested pairwise against each other and 900 individual interactions were tested in quadruplicates. interaction of the viral proteins was indicated by colony growth on the selective plates. the result of the matrix screen is shown in figure 1 . positive interactions are indicated by (black and patterned squares). positive y2h interactions were validated by co-immunoprecipitation (coip) in mammalian 293 cells as a second interaction test (double-lined red squares). of 65 interactions detected by y2h 25 were corroborated by coip. four coips were detected in both directions: non-structural proteins nsp12 (rdrnap) and nsp13 (c/h, ntpase, dntpase, 59-to 39 rna helicase, dna helicase, rna 59-triphosphatase), nsp8 and accessory protein orf9b, nsp14 (c/h, 39-to 59 exoribonuclease) and orf9b, and accessory proteins orf8a and orf8b. six of the proteins, including nsp7, nsp8, nsp13, ''e'', orf9b and orf14 interacted with themselves indicating the formation of dimeric or multimeric complexes. four of the self-interactions including nsp8, ''e'', orf9b and orf14 self-interactions were also found in the coip assay. two non-structural proteins nsp2 and nsp8, and the accessory protein orf9b showed a rather large number of interactions (8, 14 and 15 interactions, respectively). six interactions of the non-structural proteins nsp2 could be confirmed by coip including the non-structural proteins nsp3n, nsp6, nsp8, nsp11 and nsp16 and orf 3a, which only recently had been described to be a novel structural protein [22, 23] . figure 2a shows ip and coip results with anti-ha and anti-c-myc antibodies: 293 cells were transfected with ha-tagged nsp2 and cmyc-tagged nsp2-, nsp3n-, nsp6-or nsp8. the lysates were split into two and immunoprecipitated in the presence of protein g with the anti-ha (left upper panel) or with the anti-c-myc (right upper panel) antibody. the bound proteins were separated by 12.5% sds-page western blot analysis. expressed ha-tagged proteins are indicated by stars and coprecipitated proteins by arrows. although the nature of the additional protein species with higher molecular weight in the anti-ha wb of the anti-c-myc coip is unclear, they might reflect a multimeric nsp2 band that is not present in the other lanes, further supporting the nsp2-nsp2 interaction. but they could also be a result of post-translational modification upon binding to other proteins. in the reciprocal analysis (lower left and right panel) nsp2 proteins coprecipitated with itself and with nsp3n. the second-most connected protein of the replicase complex is nsp8 ( figure 2a, 2b) . of the 14 interacting proteins (nsp2, nsp5, nsp6, nsp7, nsp8, nsp9, nsp12, nsp13, nsp14, e protein, orf8, orf8a, orf9b, orf14) found in the y2h system eight could be confirmed by coip (nsp2, nsp7, nsp8, nsp9, nsp12, nsp13, nsp14, e protein). nsp 8 and nsp12 proteins interacted with orf9b and nsp13, respectively, in both directions in the y2h. this was also the case for the interaction between nsp14 and orf9b, which could also be confirmed by coip. of the ''classical'' structural proteins s, e, m and n, only the e protein showed a number of interactions with the non-structural proteins nsp1, nsp8, nsp11, as well as with the accessory proteins orf3b, orf7b and orf9b. interestingly, m and s reacted with the only recently described accessory structural proteins orf 3a and orf7a [22, 24] . self-interaction of the e protein was seen in both assays. the interaction between the structural proteins n and m, which has been described in two recent reports [21, 25] , was found positive by coip only. the most dominant interactor of the accessory proteins was the orf9b. of the 16 orf9b y2h interactions detected, four could be confirmed by coip ( figure s1 ). 10 of the non-structural and five of the other accessory proteins showed interactions with other proteins. self interactions were seen for orf9b and orf14. these could be confirmed by coip in both directions as well as the reactivity of orf7b with the two e and orf 6 proteins. orf 8a and orf 8b were also reactive in both directions in the y2h system. the dominant interactor proteins nsp2 [17] and nsp8 (deming et al., submitted) are dispensable or essential for viral replication in vitro, respectively. to determine the importance of the dominant interactor orf9b accessory protein for viral growth a deletion mutant was made by synthesizing a dna fragment that included changes that ablate each of the orf9b atg start sites while maintaining the primary sequence of the n protein ( figure 3a ). the recombinant virus had normal plaque sizes and grew like wildtype virus in vero cells after infection at a moi of 0.1 pfu/ cell ( figure 3b ). thus, deletion of orf9b is not lethal. y2h matrix and coip interaction screens of the present study were performed similarly as in our recently published study on herpesviral protein networks describing intra-viral protein interactions in kaposi sarcoma-associated herpesvirus (kshv) and varicella-zoster virus (vzv) [26] . table 2 shows the comparison of network parameters of sars to kshv as well as other cellular protein interaction networks. the average degree and characteristic path length are slightly smaller than in the kshv network and significantly smaller than in the cellular networks due to smaller network size. the fraction of pairwise interactions confirmed among those tested is higher in the sars network with 65 out of ,450 possible non-redundant pairwise interactions ( = 14.4%) than in the kshv network with 123 out of ,4050 possible interactions ( = 3%). furthermore, the clustering coefficient is significantly higher than in all of the networks analyzed. when comparing against random networks of the same size, we found that the clustering coefficient is approximately as high as expected at random given the degree distribution. interestingly for the kshv network it is actually smaller, whereas for the cellular networks it is much higher. known virus-host and intraviral interactions of sars proteins were identified by a literature screen (see tables s1 and s2). based on ten known sars-host interactions as well as two interactions predicted from homologous proteins, the viral network was connected to the human network assembled from large-scale y2h screens [27, 28] , ortholog predictions [29] and literature mining ((ref-hprd) and [27] ). the sars-cov-host interaction network is shown in figure 4 . human proteins are included which are distant from the sars-cov proteins by at most two or three interactions as well as the interactions between these proteins. on the basis of the small number of known virus-host interactions the intraviral sars-cov network is separated from the bulk of the human interactome. proteins targeted by sars either directly or via other proteins are involved in various molecular functions and pathways such as apoptosis, cell communication and signalling pathways. the literature screen for intraviral sars interactions identified 23 interactions. 3 of these (13%) were also determined by the y2h screen. to systematically study the subcellular localization of viral proteins within eukaryotic hela cells the sars-cov orfs were transfected in eukaryotic vectors with either n-or c-terminal flag tags and detected with an anti-flag antibody. since artificial tagging of proteins often leads to an aberrant expression in cellular compartments, we tagged the sars-cov protein on both the nand c-terminus and only considered the cellular localization correct if consistent with both tags [30] . some of the proteins were not expressed if tagged either n-or c-terminally (see figure s2 ), and some were not expressed at all, and were not included in the analysis. the table shows network parameters for two viral and two cellular protein interaction networks. the sars network contains 65 interactions between 31 nodes with 6 of those interactions being self-interactions. for comparison purposes, network parameters are also shown for kshv [26] , s. cerevisiae and h. sapiens. interactions for the cellular networks were derived from the following sources: s. cerevisiae from dip (the database of interacting proteins) [45] , the yeast two-hybrid interactions of h. sapiens from the studies of stelzl et al. [28] and literature interactions from rual et al. [27] , predicted human interactions (core) from lehner and fraser [29] and interactions taken from the hprd (human protein reference database) [46] . parameters shown include the number of nodes and edges in the networks, the average degree, characteristic path length (average shortest path), diameter (maximum shortest path), clustering coefficient and for the clustering coefficient enrichment values compared to appropriate random networks (er and es). both types of random networks contain the same number of nodes and edges as the original network. er networks [47] are created by connecting edges randomly, whereas es networks are created by an edge swapping strategy which preserves the degree distribution (see [26, 48] orf3a, orf7a and m were detected in the golgi, orf3b in the nucleus, orf6, orf7b, nsp3n and nsp16 in the er ( figure 5 ). orf8b and orf14 showed a vesicular staining, and nsp2 was found both in cytoplasm as well as in the nucleus. orf3a and m were found to be interacting by y2h, and were both detected in the golgi. similarly, orf6 and orf7b were both found in the er. in this study we report the cloning of the complete orfeome of sars-cov and the results of a matrix-based yeast two-hybrid screen of pairwise viral protein-protein interactions. from a number of recent structural studies it is clear that during the viral life cycle large replication complexes are formed, which involve a large number of viral proteins [31] . sars-cov is a representative of the coronaviridae, the largest rna viruses known (27 to 32 kb, plus-stranded). sars-cov expresses at least 16 non-structural replicase proteins which are cleaved co-and post-translationally from two precursor polyproteins by two viral proteinases, four structural proteins and a set of eight accessory proteins specific for the individual virus groups [6] . since the polypeptide processing sites of non-structural proteins are well defined, we chose the strategy to subclone all individual orfs predicted, and not to use the precursor polyproteins. using this approach we expected to avoid problems in the expression, folding or targeting of the polypeptides due to incorrect processing. such problems had been reported for yeast two-hybrid assays performed with the plus-stranded rna viruses hepatitis c virus [32] and wheat streak mosaic virus (wsmv) [33] , where interactions had been observed only when random fragments, not mature proteins were used. but there are also reports on potato virus a (pva) and pea seed-borne mosaic virus (psbmv), which belong to the potyvirus (+) strand rna virus family similar to wsmv [34] , as well as on a subset of poliovirus proteins [35] , where interactions have been detected among cloned mature proteins. in our screen approximately 14% of the 450 possible nonredundant protein interactions tested were positive and approximately 38% of which were confirmed by coip. this result is in the same order of magnitude as the outcome of similar y2h matrix screens in kshv and vzv, indicating that this approach can also be applied for plus-strand rna viruses. the low numbers of y2h interactions detected in two directions are a common phenomenon in y2h assays and are probably due to steric constraints of either bait or prey fusion proteins. coronavirus replication complexes consist of intricate macromolecular structures in which many of the non-structural replicase proteins are involved. one of the most interesting interactor proteins found in our study is nsp8, which interacts with replicase proteins nsp2, nsp5, nsp6, nsp7, nsp8, nsp9, nsp12, nsp13, nsp14. the importance of this protein is supported by recently reported crystallization studies, which described the multimeric association of various of the non-strucutural proteins. nsp 8 seems to be one of the proteins involved in these complexes. nsp8 deletion or irreversible fusion to nsp7 or nsp 9 by mutagenesis of the corresponding cleavage site results in a lethal phenotype supporting the idea that nsp8 is absolutely essential for virus replication (deming et al., submitted). evidence has been presented for interaction with nsp9, a ssrna-binding protein, by analytical ultracentrifugation experiments and by a decrease of the disorder of the nsp8 n-terminal region after the addition of nsp9 [36] . furthermore, a hexadecameric nsp7-nsp8 supercomplex was described which was suggested to encircle rna where it may serve as a general processivity factor for the rna-dependent rna polymerase (rdrp) nsp12 (19) . a very recent report described nsp8 as a second rdrp of sars-cov. it was shown to initiate the synthesis of complementary oligonucleotides of ,6 residues in a low fidelity reaction which eventually might serve as primers for the primer-dependent nsp12 rdrp [15] . for mhv it was shown that rdrp co-immunoprecipitates with nsp8, nsp9, nsp5 and the helicase nsp13 [37] , and that it also colocalizes with nsp7, nsp9 and nsp10 [18, 37] . thus, the nsp8 interactions found by us are confirmed by a number of different studies and it seems to play an important role in the viral replication complex. in this manuscript, we demonstrate interactions between rdrp (nsp12) and nsp8, and with the helicase nsp13 in both directions of the y2h screen. it is likely that the rdrp interacts with more nsps than were found, but these interactions may require mediator proteins like nsp8. nsp2 interacted with seven other nsps including nsp8 and with one of the newly described structural proteins orf3a. as shown by coip, it also self interacts to a dimeric or multimeric complex. the relatively large number of interactions might imply a crucial role of nsp2 in the viral life cycle. however, it was shown by deletion mutants of sars-cov and mhv that neither the encoding genomic rna sequences nor the nsp2 proteins are necessary for the generation of infectious viruses in cell culture [17] . since these viruses displayed slightly reduced phenotypes in growth, rna synthesis but not protein processing, it was speculated that nsp2 might play a role in global rna synthesis, and possibly in virus-cell interactions or viral pathogenesis. the reported subcellular localization of individually expressed nsp2 in delayed brain tumor (dbt) cells [17] is similar to the diffuse cytoplasmic and nuclear immunofluorescence staining pattern found with our n-or c-terminally tagged nsp2 proteins. thus, the exogenously expressed nsp2 does not target specific membranes in the absence of infection. however, after coinfection with a mhv mutant virus lacking nsp2, the protein expressed in trans was reported to be recruited into distinct viral replication complexes. this relocalization of nsp2 to small vesicular foci in the cytoplasm was also confirmed in sars-cov-infected vero cells by immunofluorescence staining with anti-nsp2 antibodies [19] . in our study, only few interactions were found for the structural proteins, which might be biased by transmembrane sequences preventing the transfer of expressed prey (containing the gal4 activating domain) and/or bait (containing the gal4 dnabinding domain) fusion proteins to the nucleus of the yeast cell where protein-protein interaction leads to transcription. only the e and orf3a proteins showed a number of associations whose relevance is unclear. interactions of orf3a -m and orf7a-s fit to the recent finding that the two accessory proteins display structural functions as has been described [24, 38] . for the group-specific accessory proteins it has recently been shown that deletion of five of the eight orfs (orfs 3a, orf3b, orf6, orf7a and orf7b) alone or in combination did not influence dramatically the level of rna or the replication efficiency in vitro or in an in vivo mouse model [16] . the most interesting accessory protein with respect to interactions in our study turned out to be orf9b. y2h interactions with nsp8 and nsp14 were found bi-directionally and the self-interaction could also be confirmed by coip. latter result is confirmed by recent structure data [39] . the orf9b protein, which is encoded within the nucleocapsid gene, is an intertwined dimer with an amphipathic outer surface and a long hydrophobic lipid binding tunnel. this suggests that orf9b is targeted to er-golgi compartments via an unusual anchoring mechanism and acts as an accessory protein during virion assembly. although most of the accessory proteins do not seem to play pivotal roles in viral replication, they might still be important for the virus-host interplay and for pathogenicity. currently, there is no reasonable explanation for the large number of interactions found for orf9b by the y2h screen. as deletion of orf9b does not seriously reduce virus replication in vitro consistent with a luxury function, the 9b protein may function to enhance the global stability of the sars proteome network and play some unrealized role in regulating virus-host protein-protein interactions. it thus might be more important for enhancing in vivo virulence. immunofluorescence localization of flag-tagged viral proteins corresponded in most cases to published data on sars-cov and other coronaviruses. we found nsp2 proteins in the cytoplasm and to some extent in the nucleus which is in accordance with anti-nsp2 antibody stainings of stably dbt-nsp2 (mhv) expressing cells [17] . many of the non-structural proteins are involved in the replication of the virus and locate to virus-induced cytoplasmic double-membrane vesicular complexes as the sites of viral replication. it is therefore important to take into account that the localization patterns of nsps might be quite different when expressed individually in cells as compared to the situation of viral infection where various viral proteins might help to recruit each other to the sites of active replication. accessory protein 3a, for which a number of effects on cellular functions were described [40] , we located in our flag-tagged versions to the golgi complex as yuan et al. [41] observed using egfp-tagged constructs. as a structural protein orf3a interacts with the m protein [23] which was also clearly found in the golgi as flag fusion proteins. the nuclear localization of orf3b is also reasonable because it induces cell cycle arrest at the g0/g1 phase and apoptosis [42] . proteins orf6 and orf7b, interacting in y2h and coip, were both found in the er. to our knowledge this localization has not been described for orf7b before. not much is known about orf9b other than it is expressed in infected cells [43] and that antibodies to it are found in infected patients [44] . as opposed to meier et al. [39] , who located orf9b to intracellular vesicular structures (293t cells), we found it to be diffusely distributed within cytoplasm and nucleus (hela cells). analysis of network statistics showed that despite high clustering coefficients the sars interaction network is not higher clustered than expected at random. it, thus, appears as a single module such as the kshv network and is not subdivided into separate functional modules as cellular networks. based on currently known and predicted host-virus interactions, a joint virus-human network was derived in which the viral part of the network appears to be separated from the main host network. in this respect, the sars network differs from the kshv viral network which is incorporated into the host interactome. however, this may be due to the small number of virus-host interactions identified so far for sars. indeed for kshv, the predicted virus-host network was based on about twice as many interactions to the host. to better understand the role of the intraviral protein interactions it is necessary to gain more knowledge on the sars-cov with it's host during infection. we certainly missed a considerable number of intraviral protein interactions in our y2h screen as can be seen for m-n and nsp2-nsp2, nsp5-nsp5 self-interactions, which we could only detect by coip. although, it is generally acknowledged and certainly has to be taken into account that y2h assays are error-prone by producing false positives and false negative results, we identified a large number of interactions which have not been reported previously and which could be confirmed biochemically. these interactions will be of great help for further studies which are aiming at the elucidation of sars-cov replication and pathogenesis. future experiments with the mutant viruses lacking nsp2, nsp8 or orf9b will show the relevance of the interactions detected for virus replication, growth and pathogenicity in vitro and in vivo model systems. experimental procedures viral nucleic acids sars-cov orfs were derived from subcloned cdnas described by yount et al. [16] and thiel et al. [7] . orfs were amplified by nested polymerase chain reaction (pcr) using plasmids ptopo xl containing fragments a (nt 1-4436) and b (nt4344-8712), psmart containing fragments c (8695-12070), d (12055-18924), e (18907-24051) and f (24030-29736) as well as plasmids pmal-scov-mpro 5 (nsp5), pmal-scov-nsp8, pet-scov-pol 8 (nsp12), pmal-shel-nsp13, pet-scov-exon 3 (nsp14), pmal-scov-nsp15-7, pet-scov-mtr 12 (nsp16)and pbs-sarscov-s30. the nucleotide sequences of sars-cov urbani (genbank accession ay278741), frankfurt (genbank accession ay291315) and tor2 (genbank accession nc_004718) isolates were used to design primers for subcloning of all putative orfs and making them compatible to gatewayh recombinatorial cloning system (invitrogen). nested pcrs were performed with two separate sets of primers. for the first pcr internal forward (aaaaagcaggctccgccatgn [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] ) and reverse (agaaagctgggtcn [13] [14] [15] [16] [17] [18] [19] [20] primers containing the internal attb1 and attb2 recombination sites were used. the gene-specific 59 forward sequence (n) introduced an aug start codon prior to the predicted protease cleavage sites with further 14-27 nucleotides downstream, while the 39 reverse sequence (n) matched 13-20 nucleotides. there was no stop codon introduced at the specific ends of the predicted cleavage sites in order to allow the c-terminal inframe fusion of tag sequences. the second pcr was performed using forward (59-ggggacaagtt-tgtacaaaaaagcaggct-39) and reverse (59-ggggac-cactttgtaca agaaagctgggt-39) primers which included the external parts of the attb1 and attb2 recombination sites. for the putative peptide nsp11 two oligos were synthesized as the coding (59-aaaaagcaggctccgccatgtctgcgg atgcatcaacgtttttaaacgggtttgcggtggacc-cagctttct-39) and non-coding (59-agaaagctggg-tccaccgcaaacccgtttaaaaacgttgatgcatccg cagacatggcggagcctgcttttt-39) strands. primers for orfs n and s were designed such that the complete attb1 and attb2 sites were added to the gene specific sequences. pcr conditions were 20 mm of dntps, 0,2-0,4 mm forward and revers primers, 20 ng of template and 1 u of long expand taq polymerase enzyme (roche diagnostics gmbh). depending on size and nucleotide composition of the amplificates two standard conditions were used for amplification. pcr fragments were separated by agarose gel electrophoresis and purified utilizing nucleospin extraction kits (macherey& nagel). the resulting pcr-fragments, flanked by complete attb1 and attb2 sites, were cloned by gatewayh recombinatorial cloning into the entry vectors pdonr207 or pdonr221 (invitrogen) via the bp clonase reaction as described by the manufacturer (invitrogen). the overlaps of the vector and sars-cov-orf sequences were confirmed by dna sequencing using the big dye terminator kit (perkin elmer) on a 377 dna sequencer, a 310 genetic analyser (both applied biosystems) or the genome lab dtcs-quick start kit on the ceq tm 8800 sequencer (beckman coulter). two eukaryotic destination vectors were constructed allowing the inframe fusion of a his-flag tag to the n-terminus and of a flag-his tag to the c-terminus of a the viral orfs. for the nterminal tag a dna oligo adaptor molecule was synthesized (coding strand: 59-ttagtcaagcttgaaggagatagagc-caccatggcacaccatcaccatcaccatgactacaag-gacgacgatgacaaggcgatatcttaatctagatgat-a-39) and sub-cloned via hind iii and xbai restricition sites into plasmid pcr3. the c-terminal oligo adaptor molecule (coding strand: 59-tttatatgatatcgactacaaggacgacgatga caaggcacaccatcaccatcaccattaactcgagatt-aata-39) was subcloned via ecorv and xhoi into pcr3. both plasmids were converted to destination vectors by ligating an ecorv -ecorv dna fragment, containing the gate-wayh conversion cassette reading frame b (rfb cassette) into their individual ecorv sites. in the 59-and 39-tag vectors an inframe stop codon was introduced immediately after the ecorv site or after the his tag sequence, respectively. the y2h destination vectors pgbkt7-dest (bait) and pgadt7-dest (prey) were derived from pgbkt7 and pgadt7 (clontech) by introducing a gatewayh rfb conversion cassette into the smai sites as described recently (23) . into these vectors the sars-cov orfs were transferred from the donor plasmids via lr reaction. clones were checked by restriction enzyme analysis using ecorv for the his-/flag tag vectors and ecori and bamhi (neb) for the yeast vectors. the haploid saccharomyces cerevisiae strains ah109 (mata, trp1-901 m, leu2-3, 112, ura3-52, his3-200, gal4d, gal80d, lys::ga-l1 uas -gal1 tata -his3, mel1 gal2 uas -gal2 tata -ade2, ura3::mel1 uas -mel1 tata -lacz) and y187 ( mata, his3-200, trp1-901, ade2-101, ura3-52, leu2-3, 112, gal4d, met, gal80d, ura3::gal1 uas -gal1 tata -lacz, mel1) were chosen for the yeast-two hybrid assay. ah109 and y187 were transformed using 1 mg of prey (pdest-gadt7) or bait vector (pdest-gbkt7), respectively. yeast cells were incubated for 1 h in 750 ml peg/bicine solution (40% peg 1000, 200 mm bicine ph 8.35) at 30uc, followed by 5 min at 45uc. cells were pelleted and resupended in 1 ml np-buffer (0.15 m nacl, 10 mm bicine ph 8.35), pelleted a second time and resuspended in 200 ml npbuffer and plated on to sd medium (+2% agar) lacking either leucine (prey) or tryptophane (bait). colonies were visible after 2-3 days. the yeast strains ah109 and y187 containing proteins in prey and bait were arrayed in a 96-deep-well plates with sd liquid media lacking leu or trp according to the interactions to be tested. the liquid cultures were transferred on to sd medium plates lacking leu or trp using a 384-pin replica tool (nunc). colonies were grown for 2 days at 30uc and used directly for mating on ypd medium plates. each mating was performed in quadruplicates, and after 2 days at 30uc the colonies were stamped onto sd medium (-leu-trp) plates. the interactions were assessed by transfer to sd-leu-trp-his plates, and interactions considered positive if at least three out of four possible colonies grew. viral proteins acting as self-activating baits were analyzed on increasing amounts of +3 mm 3-amino-1,2,4-triazole (3at, sigma) (3 mm, 10 mm, 50 mm and 100 mm), and excluded from the results if no clear positive interactions could be determined. 293 cells were infected with recombinant vaccinia virus vtf-7 expressing the t7 rna polymerase (nih aids repository) at a moi of 10 in dmem/1% fcs. one hour post infection, viruscontaining medium was removed and substituted by dmem/1% fcs. ten micrograms (per dish) of the respective pgbkt7-sars-cov-orf and pgadt7-sars-cov-orf plasmids were then transfected into two 10 cm dishes of 293 cells using the calcium phosphate method. after 20 to 24 h, cells were lysed by incubation in np-40 lysis-buffer (1% np-40, 140 mm nacl, 5 mm mgcl 2 , 20 mm tris ph 7.6, 1 mm pmsf, one tablet of complete protease inhibtor cocktail (roche) per 50 ml on ice for 30 min. lysates were centrifuged at 20.5006 g for 10 min and precleared using 50 ml preequilibrated protein g-sepharose (amersham pharmacia). lysates were precipitated using either 5 ml (200 mg/ml) mouse monoclonal anti-myc (santa cruz biotechnology) or 10 ml (100 mg/ml) rat monoclonal anti-ha (roche diagnostics gmbh) antibodies in the presence of 50 ml protein g sepharose beads and incubated on at 4uc by overhead rotation. the beads were washed 3 times in ice-cold np-40 buffer and resuspended in 26sds protein sample buffer. precipitates were separated by sds-page using 12.5% or 15% polyacrylamide gels. proteins were transferred to nitrocellulose (schleicher & schuell) membranes in western blot chambers on at 4uc. filters were blocked with 5% milk powder in tbst (50 mm tris, ph7.6, 150 mm nacl, 0,05% tween-20) for 1 h. they were then incubated with the anti-myc and anti-ha antibodies at dilutions of 1:1000 on at 4uc. filters were washed three times for 10 min with tbst. incubation with the secondary, peroxidase-conjugated antimouse igg or anti-rat igg (1:3000 each) antibodies (jackson) was carried out for two to three hours. after three further washing steps filters were developed using the ecl tm western blotting detection kit (amersham biosciences). the coip was scored positive if a coprecipitate was detected in at least one direction. recombinant sars-cov technology was done as described by yount et al. [16] . the sequence of the mutated sars-cov orf9b knockout (icsarsdorf9b) was confirmed by sequencing cdna isolated from recombinant virus. immunofluorescence subcellular localization analysis of orfs was carried out in hela cells (atcc ccl-2). cells were transfected with the plasmids using fugene6 (roche) according to the manufacturer's instructions, for a total of 24 h. cells were then fixed in ice cold methanol prior to processing for immunofluorescence. primary mouse anti-flag (m2) (sigma) and anti-mouse-alexa488-conjugated secondary antibodies (molecular probes) were used to detect transfected cells, and coverslips were mounted in mowiol. images were acquired on a zeiss axiovert 200 microscope with a 636/1.4 na oil objective and standard filter sets. figure s1 coips of accessory proteins. 293 cells were infected with vaccinia virus vtf-7 and subsequently co-transfected with ha-and c-myc-tagged plasmids carrying the respective sars-cov orfs. after 20 hours half of the cell lysates was immunoprecipitated with anti-c-myc, the other half with anti-ha antibody (left panel). bound proteins were subjected twice to 15% sds-page and western blot transfer, and probed crosswise with the two antibodies. co-precipitated proteins are indicated in the right panel. ha and c-myc tags are are expressed as n-terminal fusions with the corresponding sars-cov orf in plasmids pgadt7 and pgbkt7, respectively. a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome severe acute respiratory syndrome coronavirus phylogeny: toward consensus unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage mechanisms and enzymes involved in sars coronavirus genome expression coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs rna recognition and cleavage by the sars coronavirus endoribonuclease coronavirus genome: prediction of putative functional domains in the non-structural polyprotein by comparative amino acid sequence analysis identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity major genetic marker of nidoviruses encodes a replicative endoribonuclease the human coronavirus 229e superfamily 1 helicase has rna and dna duplex-unwinding activities with 59-to-39 polarity virus-encoded proteinases and proteolytic processing in the nidovirales a second, non-canonical rna-dependent rna polymerase in sars coronavirus severe acute respiratory syndrome coronavirus group-specific open reading frames encode nonessential functions for replication in cell cultures and mice the nsp2 replicase proteins of murine hepatitis virus and severe acute 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of protein networks we are very grateful to dr. christian drosten and prof. j. ziebuhr for providing materials and a number of plasmids, and to prof. ulrich koszinowski for critically reading the manuscript. key: cord-311065-ie3gty6e authors: gaddi, pamela j.; crane, meredith j.; kamanaka, masahito; flavell, richard a.; yap, george s.; salazar-mather, thais p. title: il-10 mediated regulation of liver inflammation during acute murine cytomegalovirus infection date: 2012-08-03 journal: plos one doi: 10.1371/journal.pone.0042850 sha: doc_id: 311065 cord_uid: ie3gty6e various cell types in both lymphoid and non-lymphoid tissues produce the anti-inflammatory cytokine interleukin (il)-10 during murine cytomegalovirus (mcmv) infection. the functions of il-10 in the liver during acute infection and the cells that generate this cytokine at this site have not been extensively investigated. in this study, we demonstrate that the production of il-10 in the liver is elevated in c57bl/6 mice during late acute mcmv infection. using il-10 green fluorescence protein (gfp) reporter knock-in mice, designated il-10-internal ribosomal entry site (ires)-gfp-enhanced reporter (tiger), nk cells are identified as major il-10 expressing cells in the liver after infection, along with t cells and other leukocytes. in the absence of il-10, mice exhibit marked elevations in proinflammatory cytokines and in the numbers of mononuclear cells and lymphocytes infiltrating the liver during this infection. il-10-deficiency also enhances liver injury without improving viral clearance from this site. collectively, the results indicate that il-10-producing cells in the liver provide protection from collateral injury by modulating the inflammatory response associated with mcmv infection. an effective immune response against microbial pathogens is dictated by a fine equilibrium between host defense and inflammatory tissue damage. interleukin (il)-10, one of the most immunosuppressive cytokines, maintains this balance by dampening aspects of both innate and adaptive immunity [1] [2] [3] [4] . il-10 blocks the production of proinflammatory cytokines during the resolution period of infection and consequently reducing the immunopathology caused by these cytokines [3, 5] . il-10 also alters the function of antigen-presenting cells such as dendritic cells and macrophages by limiting expression of mhc class ii and costimulatory molecules, and the production of chemokines, thereby indirectly inhibiting t cell responses and controlling cellular accumulation [1, 4, 6, 7] . in addition to its immunosuppressive effects, il-10 can stimulate the proliferation of b cells [1] , the proliferation and activation of nk cells and cytotoxic cd8+ t cells [1, 8] , as well as promote the differentiation of regulatory t cells [4] . in the context of viral infections, the presence of il-10 may be considered either harmful or beneficial to the host. il-10 is harmful to the host as it promotes immunosuppression and viral persistence [3, 9, 10] . in these instances, the genetic deletion of il-10 or the blockade of its receptor augments proinflammatory and antiviral cytokine responses facilitating more efficacious viral clearance and preventing viral persistence [2, 3, [10] [11] [12] [13] . while enhanced immune responses and viral eradication achieved in the absence of il-10 may be desirable outcomes, the absence of il-10 may promote unwanted immune mediated pathology with or without improved viral elimination depending on the site of infection [12] [13] [14] [15] [16] . therefore, in these occasions, il-10 may be necessary to reduce collateral damage due to inflammatory antiviral responses. these varied outcomes not only imply the central role of il-10 in regulating the magnitude of immune responses, but also underscore the necessity for detailed studies of how il-10 regulates immunity to infection for the benefit or detriment of the host. murine cytomegalovirus (mcmv) is a cytopathic herpesvirus that replicates at high levels in the spleen and liver [17, 18] . early acute mcmv infection induces a rapid mobilization of nk cells and monocyte/macrophages into infected liver sites [19, 20] . nk cell cytotoxicity and production of antiviral cytokines such as ifnc and tnf-a limits mcmv infection in the liver [21, 22] . during late acute infection, between 4 and 7 days following mcmv challenge, cd8+ t cells [23] and to a lesser extent, cd4+ t cells [24, 25] accumulate and participate in antiviral responses in the liver. cd8+ t cell cytotoxicity and the release of ifn-c and tnfa are associated with viral elimination from this organ [17, 18, 23, 26] . collectively, these immune responses are protective; however, they contribute to liver pathology and impaired liver function if inadequately regulated [17, 18, 22, 27, 28] . many studies have characterized the robust proinflammatory immune responses in the liver during acute mcmv infection. by contrast, the negative regulation of these immune responses has not been extensively addressed. due to its immunosuppressive properties, il-10 has been considered a factor affecting the outcome of mcmv infection and associated pathologies [28] [29] [30] [31] [32] [33] . the suppression of macrophage and dendritic cell costimulatory properties by il-10 has been shown to limit cd4+ t cell priming during acute mcmv infection [29] . b cell derived il-10 diminishes cd8+ t cell activation in lymphoid organs, thereby controlling the magnitude of mcmv-specific cd8+ t cell responses [30] . additionally, il-10 production by cd4+ t cells hinders viral elimination in the salivary glands, consequently promoting persistence of mcmv replication within this organ [31] . further work has described a role for il-10 in reducing systemic ifn-c production, cd4+ and cd8+ t cell cytokine responses and viral elimination in the spleen during mcmv infection [28] . nk cells have been implicated as sources of il-10 in mcmv-infected perforin-deficient mice and have been suggested to restrain exaggerated cd8+ t cell responses in this model [32] . finally, a recent study has demonstrated the impact of il-10 in the regulation of cytokine responses and inflammation in the liver during mcmv infection, through il-10 repletion experiments in mcmv-infected il-10 2/ 2 mice [33] . nevertheless, it remains unclear what endogenous cellular sources of il-10 modulate inflammatory processes in the liver in normal, wild type mice and what cellular effectors are affected by il-10 functions in the liver during acute mcmv infection. in this study, we investigated the kinetics and cellular sources of il-10 in the liver during acute mcmv infection and determined that while multiple cell types produce il-10, nk cells emerge as a prominent source of this cytokine. the absence of il-10 during acute mcmv infection results in elevated levels of systemic and local proinflammatory cytokines and chemokines, as well as increased inflammatory cell infiltration into infected livers. these enhanced inflammatory responses were associated with increased liver pathology. however, despite augmented immune responses in infected il-10-deficient livers, viral clearance is not improved. together, our studies illustrate a role for il-10-producing cells in regulating the extent of liver inflammation and injury during acute mcmv infection. to establish the time course of il-10 production during acute mcmv infection in the liver, conditioned media were prepared from total liver leukocytes isolated from c57bl/6j (wt) mice that were uninfected or infected with mcmv for 3, 4, 5, and 7 days. il-10 protein levels were slightly increased on day 3, but peaked significantly at day 4 after infection ( fig. 1) . by days 5 and 7 post infection, the levels of il-10 produced were reduced as compared to day 4 ( fig. 1) . because the rise in il-10 protein levels coincided with a period of nk cell expansion [34, 35] and t cell recruitment [23] [24] [25] [26] 36] in the livers of mcmv-infected mice, we evaluated the relative contributions of nk cells and t cells to liver il-10 expression. to identify the cells expressing il-10 on day 4 after mcmv infection, liver leukocytes were prepared from il-10-(ires)-gfp-enhanced reporter (tiger) mice infected for 4 days. furthermore, liver leukocytes from uninfected or day 4 mcmv-infected non-reporter c57bl/6j (wt) mice were included in the analysis to indicate background fluorescence. analyses show that liver leukocytes from uninfected tiger mice marginally expressed il-10/gfp, whereas after infection, 10%61% of the gated leukocytes were il-10/ gfp+ ( fig. 2a) . il-10/gfp+ liver leukocytes were then characterized by their expression of nk1.1 and tcrb and delineated as nk cells (nk1.1+ tcrb2), t cells (nk1.12 tcrb+), nk1.1+ t cells (nk1.1+ tcrb+) and non-nk1.1+ non-tcrb+ cells (nk1.12 tcrb2) (fig. 2b ). on day 4 after infection, we found that higher frequencies of il-10/gfp+ cells were nk1.1+ tcrb2 (nk cells), as compared to nk1.1+ tcrb+, nk1.12 tcrb2, and nk1.12 tcrb+ cells (nk cells 56%63% compared to nk1.1+ t cells: 5%61%, and non-nk non t cells: 12%61%, and t cells: 27%62%) (fig. 2c ). these trends were also reflected in the absolute numbers of il-10/gfp+ cells, with nk cells being the most numerous of the il-10/gfp+ cells within the day 4 infected liver leukocyte population relative to other examined cell types (fig. 2d ). further characterization of il-10/ gfp+ nk cells by flow cytometry revealed that il-10/gfp+ nk cells (defined as nkp46+ cd3e2) expressed known markers of maturation, dx5/cd49b, cd122, cd11b, klrg1, and cd43 (table s1 ). more than 50% of these cells were ly49h+, cd69+, and skewed to a cd27+ cd11b+ nk cell phenotype (table s1 ). additionally, characterization of il-10/gfp+ tcrb+ cells demonstrated that these t cells were comprised of both cd8+ and cd4+ cells (data not shown). these results demonstrate that during acute mcmv infection, several cell types contribute il-10 in the liver, most of which are mature and activated nk cells. systemic and liver cytokine and chemokine production is amplified in the absence of il-10 during mcmv infection since il-10 can influence inflammation by regulating the expression of cytokines and chemokines, the effects of il-10 deficiency on systemic and local production of key inflammatory mediators during mcmv infection were evaluated. ifn-c and tnf-a are produced systemically and locally during this infection and limit viral replication and dissemination [17, 37, 38] . furtherfigure 1 . il-10 production by liver leukocytes after mcmv infection. liver leukocyte conditioned media were prepared from the livers of c57bl/6j mice that were uninfected (0) or infected with mcmv for 3, 4, 5, and 7 days. concentration of il-10 protein in liver leukocyte conditioned media was determined by standard sandwich elisa. results are combined data of three independent experiments (n = 5-13 mice for each time point tested). the means of il-10 protein produced 6 se are shown. # denotes statistically significant differences between infected wt groups, where p values are #0.05 (one-way anova, tukey's multiple comparisons test). doi:10.1371/journal.pone.0042850.g001 more, cxcl9/mig, which is produced in the liver as a result of ifn-c responses, directs the trafficking of virus-specific cd8+ t cells to this site [23] . considering the importance of these cytokines in mediating antiviral defense, the levels of these cytokines were assessed in wt and il-10 2/2 mice left uninfected or infected with mcmv for 4, 5, and 7 days. sera were collected and protein levels of ifn-c, tnf-a, and cxcl9 were measured using elisa (fig. 3 , a, c and e). serum levels of all three cytokines peaked at day 4. though levels of these cytokines declined after 4 days post infection in wt and il-10 2/2 mice, they remained elevated in il-10 2/2 mice over those in wt mice at most time points analyzed (fig. 3 , a, c and e). considering these observations, we next evaluated whether cytokine production by liver leukocytes during mcmv infection is similarly augmented in the absence of il-10. elisa was used to assess the levels of ifn-c, tnf-a, and cxcl9 proteins in liver leukocyte conditioned media supernatants prepared from uninfected or infected wt and il-10 2/2 mice (fig. 3 , b, d and f). the production of ifn-c and cxcl9 by wt and il-10 2/2 liver leukocytes was increased as infection progressed. the levels of ifn-c were significantly higher on days 4 and 7 post infection in il-10 2/2 liver leukocytes compared to wt (fig. 3b ). further-more, while the kinetics of cxcl9 production were similar between wt and il-10 2/2 mice on days 4 and 5 post infection, cxcl9 levels produced by il-10 2/2 leukocytes were greater 7 days post infection as compared to levels measured in wt leukocyte conditioned media (fig. 3f ). in contrast, tnf-a levels in infected il-10 2/2 liver leukocytes were elevated above wt over the course of infection, though these differences were not statistically significant (fig. 3d) . collectively, these observations indicate that il-10 clearly suppresses the production of systemic inflammatory cytokines during mcmv infection, while the effects of il-10 in the liver appear limited to suppression of ifn-c and cxcl9. mcmv infection in the liver results in increased infiltration of nk cells, t cells and macrophages that contribute to viral clearance through cytokine and chemokine production [20, 23, 27, [38] [39] [40] . the findings in figure 3 show that il-10 deficiency results in increased local proinflammatory cytokine production. therefore, we hypothesized that this cytokine response could be attributed to an augmented presence of inflammatory effector leukocytes in infected il-10 deficient livers. to assess whether il-10 deficiency alters the infiltration of these critical immune effectors, total liver leukocyte numbers from uninfected and infected wt and il-10 2/2 mice were compared. as shown in figure 4a , the numbers of infiltrating leukocytes in il-10 2/2 mice were increased on days 5 and 7 after infection compared to wt, implying il-10 regulates the magnitude of leukocyte infiltration. given the increased total leukocyte infiltration in the absence of il-10 during this infection, we investigated whether this rise in cell number was due to elevations in the numbers of a few or all of these effector cell populations. the numbers of nkp46+ tcrb2 nk cells in wt and il-10 2/2 livers at 4 days post infection were nearly comparable (fig. 4b) . however, nk cell numbers were higher in il-10 2/2 livers than in wt livers at 5 and 7 days post infection. despite differences in the numbers of infiltrating nk cells, nk cells from il-10 2/2 mice exhibited similar kinetics in accumulation and contraction as those in wt mice. like nk cells, the numbers of f4/80+ cd11b+ monocyte/macrophages were also increased on day 7 following infection ( fig. 4c ) in the absence of il-10. furthermore, total numbers of cd4+ t cells (fig. 4d ) and cd8+ t cells (fig. 4e , day 5 only) were elevated in infected il-10 2/2 livers as compared to their wt counterparts 5 and 7 days post mcmv infection. these findings prompted further analysis of the persistence of these cells beyond day 7. by day 9 post infection the numbers of cd8+ t cells found in il-10 2/2 livers were comparable to wt (fig. 5d) , whereas the numbers of macrophages, cd4+ t cells, and nk cells in these same il-10 2/2 livers were still higher as compared to day 9 wt livers (fig. 5 , a-c). these observations suggest that il-10 regulates the persistence of effector cells in the liver following mcmv infection. virus-specific cd8+ t cells are recruited to the liver within 4 days of infection and control viral replication through release of cytotoxic molecules and production of cytokines such as ifn-c and tnf-a [18, [21] [22] [23] 26] . given the augmented accumulation of cd8+ t cells in infected il-10 2/2 livers noted above, we evaluated whether il-10 deficiency affected the infiltration of cytokine-producing cd8+ t cells activated by an immunodominant epitope of mcmv, an h-2d b viral peptide derived from the mcmv m45 protein [41] [42] [43] . the frequencies and total numbers of antigen-specific ifn-c and tnf-a producing cd8+ t cells were determined by intracellular staining for these cytokines after restimulation with this viral peptide. the results shows that the frequency of ifn-c+ (fig. 6a ) and tnf-a+ (fig. 6c ) cd8+ t cells on day 5 post infection were comparable between wt and il-10 2/2 mice. interestingly, il-10 2/2 mice exhibited decreased frequencies of both ifn-c+ (fig. 6a ) and tnf-a+ (fig. 6c ) cd8+ t cells on day 7 post infection. the total numbers of ifn-c+ (fig. 6b ) and tnf-a+ (fig. 6d ) cd8+ t cells in day 5 infected il-10 2/2 livers were increased as compared to wt mice. despite these differences on day 5, the numbers of both ifn-c+ and tnf-a+ cd8+ t cells in il-10 2/2 livers from mice infected for 7 days were comparable to those numbers in wt (fig. 6, b and d) . taken together, these observations indicate that il-10 limits the number of activated virus-specific cd8+ t cells in the liver. our previous observations demonstrated that il-10 deficiency leads to inflated cellular and cytokine responses in the liver during this infection. we next sought to determine whether such inflammatory events exacerbated liver damage in infected il-10 2/2 mice. the formation of inflammatory clusters in the liver after mcmv infection has been correlated with the onset of damage as well as the establishment of antiviral responses at this site [20, 27, 44] . to dissect the consequences of il-10 deficiency on liver inflammation after infection, we first quantitated the number of inflammatory foci in livers using hematoxylin and eosin (h&e) stained liver sections taken from uninfected and mcmv-infected wt and il-10 2/2 mice. we noted no differences in the numbers of inflammatory clusters counted in wt and il-10 2/2 livers infected for 4 days (table 1) . however, by days 5 and 7 post infection, there were increased numbers of inflammatory foci observed in il-10 2/2 livers as compared to wt. by day 9 post infection, the number of foci in il-10 2/2 livers was nearly comparable to wt. furthermore, the number of nucleated cells within each focus was greater in il-10 2/2 livers than in wt livers infected for 5 and 7 days (table 1) , thus, corroborating our previous observations of increased leukocyte infiltration (fig. 4a) . further evaluation of liver pathology and damage in the absence of il-10 during this infection was performed using periodic acid schiff stained (pas) liver sections taken from uninfected and infected wt and il-10 2/2 mice. pas staining has been utilized as an indicator of liver function or glycogen storage, whereby reductions of staining denote impaired liver function. using this approach, we revealed no distinct differences in pas staining between the livers of uninfected wt (fig. 7, a and b ) and il-10 2/2 (fig. 7 , e and f) mice. on day 4 post infection, both wt and il-10 2/2 livers had comparable reductions in pas staining and an increased presence of inflammatory foci (data not shown). on day 5 after infection, wt mice exhibited reduced liver pas staining (fig. 7, c and d) , though this reduction in positive pas staining was markedly more apparent in il-10 2/2 mice (fig. 7, g and h) . in contrast, the extent of positive pas staining in liver sections from day 7 infected il-10 2/2 and wt mice were comparable (data not shown), indicating recovery of glycogen synthesis. concurrent with recent reports [33] , day 5 infected il-10 2/2 livers revealed more focal necrosis (fig. 7 , g and i) and inflammatory foci (fig. 7 g and h ) as compared to wt livers. furthermore, day 5 infected il-10 2/2 livers had more councilman bodies (apoptotic hepatocytes) (fig. 7h ) and demonstrated increased tunel staining (fig. 7 , k and l) as compared to day 5 infected wt livers (fig. 7j) . the extent of liver damage in infected il-10 2/2 mice was also assessed by measurement of serum alanine aminotransferase levels (alt) in uninfected and infected wt and il-10 2/2 mice. wt and il-10 2/2 mice exhibited elevated serum alt on day 4 and 5 after infection, with serum values in the il-10 2/2 mice higher than wt on day 5 post infection, though these differences did not attain statistical significance (table 2) . on day 7 after infection, serum alt levels had declined in both wt and il-10 2/2 mice, although the levels in il-10 2/2 mice remained elevated compared to wt levels ( table 2) . finally, viral titers of infected wt and il-10 2/2 livers were examined to determine whether the inflated immune responses and resultant immunopathology in infected il-10 2/2 livers coincided with improved viral clearance. despite the increased immune responses and pathology during infection in the absence of il-10, viral titers in the il-10 2/2 livers remained comparable to those in wt livers at all time points analyzed (table 3) . taken together, these results indicate that il-10 minimizes immunopathology during acute mcmv infection without affecting viral clearance in the liver. this study evaluated the production and the function of il-10 in the liver during acute mcmv infection. our results show il-10 is produced in the liver at a key time point before the accumulation of t cells and after the early effector activities of nk cells [23, 24, 34, 35, 38] . furthermore, they highlight nk cells we identified mature, activated nk cells as prominent il-10 expressing cells during acute mcmv infection. these results concur with recent studies demonstrating the presence of il-10 producing nk cells during various microbial infections [32, 45, 46] . during mcmv infection, il-10 production by nk cells has been implicated in modulating over-exaggerated cd8+ t cell responses [32] . our studies suggest that through the production of il-10, nk cells may participate in dampening downstream effector responses and subsequent immunopathology in the liver during acute mcmv infection. notably, although nk cells appear to be predominant il-10 expressers, t cell and non-nk, non t cell populations also comprise the il-10+ leukocyte population in the liver after mcmv infection. macrophages and dendritic cells [29] , b cells [30] and cd4+ t cells [31, 47] have been shown to produce il-10 during this infection, though their contributions to il-10 production in acutely infected livers remain to be examined. virus-specific cd8+ t cells have been identified as important il-10 producers during infections of influenza a [48] , respiratory syncytial virus [15] and coronavirus [49] . it is possible that mcmv-specific, effector cd8+ t cells and cd4+ t cells contribute il-10 in the liver during this infection, although further characterization of il-10+ t cells at this site is necessary as regulatory subsets of cd4+ t cells [50] and cd8+ t cells [51, 52] have been shown to also generate il-10 in various contexts. finally, our study cannot rule out the contributions of specific nk1.12 tcrb2 cells, which may include macrophages and dendritic cells, to il-10 mediated regulation in the liver. peritoneal and splenic macrophages and dendritic cells produce il-10 after mcmv infection [29] and it is known that monocyte/macrophages accumulate in the liver during infection [18, 38, 39] . therefore, further studies are necessary to delineate il-10+ cells table 2 . serum alanine aminotransferase (alt) levels of uninfected and mcmv infected c57bl/6 (wt) and il-10 2/2 mice. within the nk1.12 tcrb2 population and the tcrb+ population, as these may also contribute to the regulation of inflammation in the liver during infection. the immune response to mcmv infection involves the mobilization and activation of nk cells, t cells, and monocyte/ macrophages, which are sources of proinflammatory cytokines and chemokines [17, 18, 23, 38, 44, 53] . consistent with previous observations [28, 33] , we found that in the absence of il-10 there is augmented cellular infiltration of livers and increased levels of ifn-c and cxcl9. though tnf-a production is involved in the cytokine response during mcmv infection, we note that tnf-a protein levels, though increased systemically in the absence of il-10, remained unchanged in the liver during acute mcmv infection, corroborating with reported transcript data [33] . elevated levels of ifn-c and cxcl9 may partly account for the increased cd8+ t cell accumulation in mcmv-infected il-10 2/ 2 livers as studies have associated liver production of ifn-c and cxcl9 with recruitment of mcmv-specific cd8+ t cells to the liver during mcmv infection [23, 38] . additionally, the increased cellular accumulation in mcmv-infected il-10 2/2 livers may also reflect the broad immunosuppressive impact of il-10 on cellular activation and proliferation [1] [2] [3] [4] [5] . il-10 dampens macrophage proliferation and t cell priming [1, 4, 6, 7] , and during acute mcmv infection, il-10 limits cd8+ t cell numbers in lymphoid organs [28, 30, 32] , thereby, perhaps restricting the numbers of cells recruited to the liver. immune responses to mcmv precipitate the development of liver pathology during late acute mcmv infection [17, 22, 27, 33] , and our studies and others [33] suggest that in the absence of il-10, liver damage is enhanced. although the cells and cytokines that mediate liver damage in il-10 2/2 mice have yet to be clearly identified, it has been established that overproduction of ifn-c and tnf-a can promote the development of severe hepatitis during acute mcmv infection [18, 22, 27] . moreover, excess production of these cytokines by activated monocytes/macrophages, nk cells, cd4+ and cd8+ t cells have been associated with mcmv-induced liver disease in immunodeficient mice [22] . it is probable that increased cellular accumulation and the elevated production of inflammatory mediators such as ifnc and cxcl9 as well as others not evaluated by this study accounts for the extent of liver disease in observed il-10 deficient livers. our findings support the conclusions of recent studies in mcmv infection [33] and other viral infections [16, 48] that demonstrate il-10 as a key cytokine in limiting immune mediated pathology during viral infection. despite increased effector cell infiltration and cytokine production in virally infected il-10 deficient livers, we did not detect any significant differences in viral titers between wt and il-10 2/2 livers, concurring with a recent report [33] . these findings contrasted other studies, which demonstrated il-10 suppresses mcmv elimination in the spleen [28] and salivary glands [31] . these differences may reflect compartmental differences in viral elimination and in regulation of antiviral immune responses. lack of improved clearance in il-10 deficient livers could be attributed to the activity of other immunoregulatory mechanisms such as programmed death-1(pd-1)-pd-l1 (pd ligand-1) or tim-3galectin-9, which are known to negatively regulate t cell responses. interestingly, multiple cell types in the liver express the ligands to these molecules [54] [55] [56] [57] , and pd-1 and tim-3 have been shown to be upregulated on cd8+ t cells during both acute [58] [59] [60] [61] and chronic persistent viral infections [10, [61] [62] [63] [64] . further work exploring the roles of inhibitory receptor-ligand interactions during acute mcmv infection in the liver in the absence of il-10 may elucidate mechanisms underlying compartmental differences in viral elimination. in conclusion, the current studies demonstrate a role for il-10 in regulating inflammatory responses involved in the elimination of mcmv infection in the liver. nk cells are identified as prominent expressers of il-10, although other cell populations also contribute to il-10 in the liver during this infection. the absence of il-10 mediated suppression during mcmv infection results in amplified cellular and cytokine responses that ultimately result in liver injury. in spite of a robust immune response in infected il-10-deficient livers, viral clearance is not improved. therefore, our results highlight the role for il-10 and cellular sources of il-10 in the protection of the liver from damage due to infection induced inflammatory responses. this study was carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health. all animal work was approved by the brown university institutional animal care and use committee (protocol number: 0903035). pathogen-free c57bl/6j and il-10 2/2 mice were obtained from the jackson laboratory (bar harbor, me, usa). b6-il-10internal ribosome entry site (ires) green fluorescence protein (gfp) knockin (il-10-ires-gfp) reporter tiger mice were generated as described in [65] and bred in pathogen-free mouse facilities at brown university. all mice were maintained in pathogen-free mouse facilities at brown university. age-(6-8 week old) and sex-matched mice were used in all experiments. infections were initiated on day 0 by intraperitoneal (i.p) injection of 5610 4 pfu of salivary gland-passaged mcmv smith strain prepared as previously described [40, 66] . in vivo responses were examined at indicated times post infection. viral titer quantification was done as described (66) . briefly, duplicate samples of serially diluted homogenates were plated on bone marrow stromal cell (m2-10b4) (american type culture collection, manassas, va) monolayers for one hour at 37uc, 5% co 2 . after 1 h incubation, inocula were removed from monolayers. monolayers were overlaid with 16 dmem/0.5% low melt agarose solution and further incubated for 7 days. infected monolayers were fixed with 10% buffered formalin and stained with crystal violet to allow for visualization of plaques. plaques were counted to calculate viral titers as previously described [23, 39, 66] . livers harvested from infected wt and il-10 2/2 mice were weighed and then processed to isolate single-cell liver suspensions as previously described [19, 23, 38] . for generation of leukocyte conditioned media, isolated leukocytes were plated without additional stimulation in round-bottom microtiter plates at 10 6 cells/well in rpmi 1640 (invitrogen), supplemented with 10% heat-inactivated fetal calf serum (atlanta biologicals). after 24 h of incubation at 37uc, cell free supernatants were collected and stored at 220uc until used in cytokine analyses. blood serum was collected following centrifugation of whole blood collected into heparin-containing tubes and stored at 280uc until further use in cytokine analyses or alanine aminotransferase assays. liver leukocytes were incubated with anti-cd16/32 antibody (clone 2.4g2) (bd biosciences/pharmingen, san diego, ca, usa) prior to staining to block non-specific binding. to identify inflammatory leukocytes, the following antibodies were used in surface staining: pe-conjugated anti-f4/80 (abd serotec, raleigh, nc, usa), apc-conjugated anti-cd11b, pecy7 or percp-cy5.5-conjugated anti-cd8a, fitc-or apc-conjugated anti-tcrb, percp-conjugated anti-cd4, pecy7 or pe-conjugated anti-nk1.1, and efluor-710-or percp-cy5-conjugated anti-nkp46. for phenotypic characterization of il-10/gfp+ nk cells, in addition to nk specific antibodies for nk1.1 and nkp46 listed above, alexa 647 conjugated anti-ly49h, apc-conjugated anti-dx5/cd49b, anti-cd43, anti-klrg1, and -anti-cd69, pacific blue-conjugated anti-cd122, pacific blue-conjugated anti-cd11b, and pecy7-conjugated anti-cd27, and pe-conjugated anti-cd3e antibodies were utilized. all antibodies, except for anti-f4/80 (abd serotec), were obtained from bd biosciences or ebioscience (san diego, ca, usa), unless otherwise noted. isotype control antibodies were used to set analysis gates. where indicated, gfp expression in leukocytes from tiger mice was analyzed in the fitc/fl1 channel. approximately 70,000-100,000 events per sample were acquired using a facscalibur or facsaria and data was analyzed using bd cellquest software. where indicated, positive gfp gates were set using c57bl6j negative controls. for phenotypic characterization of il-10/gfp+ nk cells, a lymphoid gate was used in analysis and nk cells were defined as nkp46+ cd3e2. to evaluate the cytokine production by mcmv specific cd8+ t cells, total liver leukocytes from uninfected or mcmv-infected mice were cultured in complete media and 100 ng/ml of the immunodominant peptide from the m45 viral protein [23, [41] [42] [43] for a total of 5 h with brefeldin a (ebioscience), which was added within the last 3 h of this incubation period. after 5 h of incubation, leukocytes were stained with pe-cy7-or percpconjugated anti-cd8a, fixed, permeabilized (cytofix/cytoperm; bd biosciences), and incubated with pe-conjugated anti-mouse ifn-c (clone xmg1.2), apc-conjugated anti-tnf-a (clone mp6-xt22), or isotype control igg1 (bd biosciences). isotype control antibodies were used to correct for background fluorescence and set analysis gates. cells were acquired and analyzed using facscalibur and cellquest-pro software. serum and leukocyte-conditioned media were tested for il-10, ifn-c, tnf-a and cxcl9 using duosets (r&d systems, minneapolis, mn). the limits of detection were 15-32 pg/10 6 cells. liver lobes were fixed in 10% buffered formalin and paraffinembedded for sectioning. tissue sections of 5 mm in thickness were cut and stained with hematoxylin and eosin (h&e) and periodic acid schiff (pas) stain for microscopic evaluations. for in situ detection of apoptotic cells, tdt-mediated dutp-biotin nick end labeling (tunel) assay was used as described by manufacturer (millipore, temecula, ca, usa). inflammatory foci, which are defined as discrete clusters of 6-60 nucleated cells [27] were quantified by analysis of eight 50 mm 2 fields of view on h&e stained liver sections under a magnification of 200. the number of nucleated cells per inflammatory focus was determined by counting cells within 20 random foci per representative h&e stained liver section at a magnification of 400. images were photographed with a dp70 digital camera and software (optical analysis corporation, nashua, nh, usa). serum alt levels were measured by marshfield labs (marshfield, wi, usa). statistical differences in experimental results were determined using graphpad prism version 5 and excel. statistical differences for the frequencies and numbers of il-10/gfp+ groups and in il-10 levels measured in the conditioned media of multiple wt infection groups were assessed by one way analysis of variance (anova) test, accompanied by tukey's multiple comparisons post hoc test. for these multiple comparisons tests, p values of #0.05 were considered significant and denoted with a ''#''. furthermore, differences between wt and il-10 2/2 groups in cell number, cytokine levels for serum and liver leukocyte conditioned media, foci number, and serum alt levels were determined by student's t tests, where p values of #0.05 were significant. for comparisons made between phenotyped il-10/ gfp+ nk cells versus il-10/gfp-nk cells, differences were also evaluated using student's t tests, where p values of #0.05 were significant. table s1 expression of maturation and activation markers on il-10/gfp+ nk cells from day 4 infected livers. 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cd8 t cell regulation during viral infection interleukin-10 repletion suppresses pro-inflammatory cytokines and decreases liver pathology without altering viral replication in murine cytomegalovirus (mcmv)-infected il-10 knockout mice expansion and contraction of the nk cell compartment in response to murine cytomegalovirus infection specific and nonspecific nk cell activation during virus infection expansion of protective cd8+ t-cell responses driven by recombinant cytomegaloviruses distinct organ-dependent mechanisms for the control of murine cytomegalovirus infection by natural killer cells a chemokine-to-cytokineto-chemokine cascade critical in antiviral defense ifn-alphabeta-mediated inflammatory responses and antiviral defense in liver is tlr9-independent but myd88-dependent during murine cytomegalovirus infection monocyte chemoattractant protein-1 and ccr2 interactions are required for ifn-alpha/ beta-induced inflammatory responses and antiviral defense in liver the murine cytomegalovirus immunomodulatory gene m152 prevents recognition of infected cells by m45-specific ctl but does not alter the immunodominance of the m45-specific cd8 t cell response in vivo four distinct patterns of memory cd8 t cell responses to chronic murine cytomegalovirus infection genome-wide analysis reveals a highly diverse cd8 t cell response to murine cytomegalovirus cytomegalovirus hepatitis: characterization of the inflammatory infiltrate in resistant and susceptible mice posttranscriptional regulation of ii10 gene expression allows natural killer cells to express immunoregulatory function systemic but not local infections elicit immunosuppressive il-10 production by natural killer cells cutting edge: murine cytomegalovirus induces a polyfunctional cd4 t cell response effector t cells control lung inflammation during acute influenza virus infection by producing il-10 highly activated cytotoxic cd8 t cells express protective il-10 at the peak of coronavirus-induced encephalitis natural and adaptive foxp3+ regulatory t cells: more of the same or a division of labor? cd8+cd122+ regulatory t cells (tregs) and cd4+ tregs cooperatively prevent and cure cd4+ cell-induced colitis cutting edge: cd8+cd122+ regulatory t cells produce il-10 to suppress ifn-gamma production and proliferation of cd8+ t cells cellular and molecular basis of the protective immune response to cytomegalovirus infection tolerogenic maturation of liver sinusoidal endothelial cells promotes b7-homolog 1-dependent cd8+ t cell tolerance inhibition of tcell responses by hepatic stellate cells via b7-h1-mediated t-cell apoptosis in mice chronically inflamed livers up-regulate expression of inhibitory b7 family members a crucial role for kupffer cell-derived galectin-9 in regulation of t cell immunity in hepatitis c infection temporal analysis of early immune responses in patients with acute hepatitis b virus infection dynamic programmed death 1 expression by virus-specific cd8 t cells correlates with the outcome of acute hepatitis b galectin-9/tim-3 interaction regulates virus-specific primary and memory cd8 t cell response t cell immunoglobulin and mucin protein-3 (tim-3)/galectin-9 interaction regulates influenza a virus-specific humoral and cd8 t-cell responses role of tim-3/ galectin-9 inhibitory interaction in viral-induced immunopathology: shifting the balance toward regulators negative immune regulator tim-3 is overexpressed on t cells in hepatitis c virus infection and its blockade rescues dysfunctional cd4+ and cd8+ t cells tim-3 expression on pd-1+ hcv-specific human ctls is associated with viral persistence, and its blockade restores hepatocyte-directed in vitro cytotoxicity expression of interleukin-10 in intestinal lymphocytes detected by an interleukin-10 reporter knockin tiger mouse unc93b1 mediates innate inflammation and antiviral defense in the liver during acute murine cytomegalovirus infection we thank paula weston and melinda golde from the molecular pathology core facility at brown university for their assistance with histological sample preparations. we would also like to acknowledge stephanie terrizzi for her assistance with flow cytometry on facsaria, in the brown university flow cytometry facility. key: cord-309621-6jj19xpr authors: yu, pin; xu, yanfeng; deng, wei; bao, linlin; huang, lan; xu, yuhuan; yao, yanfeng; qin, chuan title: comparative pathology of rhesus macaque and common marmoset animal models with middle east respiratory syndrome coronavirus date: 2017-02-24 journal: plos one doi: 10.1371/journal.pone.0172093 sha: doc_id: 309621 cord_uid: 6jj19xpr middle east respiratory syndrome (mers), which is caused by a newly discovered coronavirus (cov), has recently emerged. it causes severe viral pneumonia and is associated with a high fatality rate. however, the pathogenesis, comparative pathology and inflammatory cell response of rhesus macaques and common marmosets experimentally infected with mers-cov are unknown. we describe the histopathological, immunohistochemical, and ultrastructural findings from rhesus macaque and common marmoset animal models of mers-cov infection. the main histopathological findings in the lungs of rhesus macaques and common marmosets were varying degrees of pulmonary lesions, including pneumonia, pulmonary oedema, haemorrhage, degeneration and necrosis of the pneumocytes and bronchial epithelial cells, and inflammatory cell infiltration. the characteristic inflammatory cells in the lungs of rhesus macaques and common marmosets were eosinophils and neutrophils, respectively. based on these observations, the lungs of rhesus macaques and common marmosets appeared to develop chronic and acute pneumonia, respectively. mers-cov antigens and viral rna were identified in type i and ii pneumocytes, alveolar macrophages and bronchial epithelial cells, and ultrastructural observations showed that viral protein was found in type ii pneumocytes and inflammatory cells in both species. correspondingly, the entry receptor ddp4 was found in type i and ii pneumocytes, bronchial epithelial cells, and alveolar macrophages. the rhesus macaque and common marmoset animal models of mers-cov can be used as a tool to mimic the oncome of mers-cov infections in humans. these models can help to provide a better understanding of the pathogenic process of this virus and to develop effective medications and prophylactic treatments. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the middle east respiratory syndrome coronavirus (mers-cov) was identified in 2012 in a cell culture taken from a patient who died of pneumonia in saudi arabia [1] . more men than women have become infected with this virus, and the median age of those affected is 47 years old (range: 9 months-94 years), with most of the fatalities occurring in patients over 60 years old [2, 3] . the respiratory symptoms of this infection are primarily related to severe lower respiratory tract complications (e.g., dyspnoea and coughing associated with a fever) that may become fatal, while there is generally little involvement of the upper respiratory tract. a large proportion of severely ill patients require mechanical ventilation [4, 5] . complications that have been described in fatal cases include hyperkalaemia with associated ventricular tachycardia, disseminated intravascular coagulation leading to cardiac arrest, pericarditis and multiorgan failure [6] . mers-cov seems to be widely present in dromedary camels in the middle east and in some parts of africa [7, 8] . zoonotic transmission is likely to have originated from this species and is expected to continue indefinitely in these regions. the entry receptor for mers-cov, dipeptidyl peptidase 4 (ddp4), also named cd26, shows a high similarity in both humans and dromedary camels. most mers patients acquire the infection in the middle east, which subsequently leads to limited human-to-human transmission in local groups and healthcare workers and eventually to travel-related cases outside the region, all of which can result in a mild to severe or even fatal respiratory disease [2] . finding a suitable animal model is a major challenge for understanding the pathogenesis of mers-cov infection, evaluating the safety and efficacy of mers-cov vaccine candidates and developing therapeutic interventions. experimental infections with mers-cov in rhesus macaques (macaca mulatta) [9] , common marmosets (callithrix jacchus) [10] , rabbits (oryctolagus cuniculus) [11] and mice (mus musculus) [12, 13] have been reported in studies on the pathological changes that occur as a result of this viral infection. however, little is known about the pathological changes in the lungs of humans infected with mers-cov, which makes it difficult to interpret data from experimental mers-cov animal models. overall, based on the known clinical aspects of mers-cov infection in humans, useful experimental animal models of mers-cov infection should exhibit a life-threatening lower respiratory tract disease. although there have been several studies in animal models on the pathogenic mechanisms of mers-cov infection, little is known about the comparative pathology and inflammatory cell response in rhesus macaques or common marmosets infected with this virus. therefore, it is vital to study comparative pathology on the association of the mers-cov antigen with its receptor, ddp4, or the histopathological changes in nonhuman primate (nhp) models of mers-cov infection. here, we comprehensively describe the histopathological features of the disease and the distribution of the mers-cov antigen and ddp4 in rhesus macaque and common marmoset models. our findings may contribute to a better understanding of the pathogenic process of mers-cov infection and help in evaluating the suitability and efficacy of the animal models used in the development of effective medications and prophylactic treatments for this disease. this research on the mers-cov virus was discussed among the staff members of the department of pathogen biology at the institute of laboratory animal science (ilas) of the chinese academy of medical sciences and peking union medical college (pumc). the experiments and protocols for this nhp models of mers-cov infection were discussed explicitly and extensively among the staff members of the department of pathogen biology. these discussions were followed by consultations with biosafety officers and facility managers at the ilas of pumc, as well as with numerous specialists in the fields of sars-cov and general infectious disease research throughout china. all research procedures were approved by the ilas institutional animal care and use committee and the laboratory safety committee (lsc). the committee recommended that the number of animals be reduced to comply with the 3r (reduction, replacement, refinement) principles. therefore, our experiment was designed to include three rhesus macaques and three common marmosets to test their effectiveness as animal models of mers-cov infection. two rhesus macaques and two common marmosets were infected with the virus and one individual of each species was left uninfected to serve as a control. the animals were planned to be euthaniazed when they were suffering from fatal respiratory symptom, impending death or 20% of body weight loss, which included fatal dyspnea and infectious shock. the approved registration number for this study is ilas-pc-2013-004. all experiments were conducted within an animal biosafety level 3 (absl-3) facility, which was constructed and accredited based on national standard gb19489 at the ilas of pumc, beijing, china. rhesus macaques and common marmosets were housed in accordance with chinese national standards, which are consistent with the standard set forth in the 8th edition of the nrc guide for the care and use of laboratory animals. because of the infectious nature of this study, nhps were housed individually instead of the generally recommended group or social housing. stainless steel cages measuring 0.5-0.75 m 2 and 0.5-0.6 m depending on the weight of the individual animal, consisted of wire flooring and resting boards or perches. rooms have natural lighting and the photoperiod is supplemented during the winter months with an artificial lighting source to provide a 12: 12 light cycle. temperature and humidity in animal holding rooms are maintained in accordance with recommendations in the chinese national standards for animal care. drinking potable water is obtained from the city of beijing and delivered to the animals via automated watering system (aws). the aws is checked daily to ensure proper operation i.e., water pressure, free flowing exits and absence of leakages. pans were cleaned daily and cages were washed every week by hand. all animals have individual cage id cards which contain the following basic information: study no., sex, weight, principal investigator's name and study protocol number. nhps were fed a measured amount of a commercially available nhp diet (beijing hfk bioscience co., ltd) offered twice daily. fresh fruit (apples, bananas and oranges) are supplemented on alternating days. additional environmental enrichment consists of toys, stainless steel mirrors and heavy-duty dog chew toys (nyla bones or similar), which are provided on a rotating basis. toys are left inside the cages when these are transported out of the room for washing and are sanitized at this time. damaged toys are removed from circulation. soft background music, plants, as well as pictures and photos hung on the animal room walls are provided for relaxation. opportunities for limited social interaction with compatible nhps are also provided at every other cage change when cages of compatible animals are placed in close proximity to each other while avoiding direct physical contact between animals. two rhesus macaques, 2-3 years old, were anesthetised with ketamine hydrochloride (30 mg/kg, i.m) prior to the procedures and intratracheally inoculated with 1 ml of hcov-emc (6.5 ×10 7 tcid 50 /1 ml) diluted in dmem. one mock-infected rhesus macaque was intratracheally inoculated with tissue culture media dmem for use as control. the rhesus macaques were observed twice daily, and clinical signs were recorded. the infected and mock-infected rhesus macaques were anesthetized with pentobarbital sodium (60 mg/kg, i.m) prior to the procedures, and while under deep anesthesia, the animals were sacrificed through femoral artery bloodletting at 3 days post-infection. tissue specimens, including samples from lung, trachea, heart, spleen, kidney, brain, liver, and colon tissue, were collected for various pathological, virological, and immunological tests. two common marmosets, 2-3 years old, were anesthetised with ketamine hydrochloride (120 mg/kg, i.p) prior to the procedures and intratracheally inoculated with 1 ml of hco-v-emc (5 ×10 6 tcid 50 /0.5 ml) diluted in dmem. one mock-infected common marmoset was intratracheally inoculated with tissue culture media dmem for use as control. the common marmosets were observed twice daily, and clinical signs were recorded. the infected and mock-infected common marmosets were anesthetized with pentobarbital sodium (40 mg/kg, i.m) prior to the procedures, and while under deep anesthesia, the animals were sacrificed through femoral artery bloodletting at 3 days post-infection. tissue specimens, including samples from lung, trachea, heart, spleen, kidney, brain, liver, and colon tissue, were collected for various pathological, virological, and immunological tests. none of the infected animals were euthanized or died without euthanasia prior to their sacrifice at 3 days post-infection. the fixed samples were dehydrated and dewaxed according to conventional procedures, and 4-μm sections were prepared with a microtome. some sections were stained with haematoxylin-eosin (he) using routine methods. two independent pathologists observed all slides and were blinded to the experimental design. lungs were fixed in glutaraldehyde and prepared for ultrastructural observations. transmission electron microscopy was performed essentially as previously described [14] . briefly, serial sections were dewaxed and rehydrated in graded ethanol, and a standard avidinbiotin immunoperoxidase technique was performed [15] . table 1 lists the primary antibodies used for ihc. optimal antibody dilutions were determined in experiments on positive control tissues. negative control sections were prepared using the same steps as described above, but the primary antibodies were derived from an irrelevant sera. sections were dewaxed and rehydrated in a graded ethanol series. ish was carried out using the enhanced sensitive ish detection kit i (boster, china) according to the manufacturer's instructions. endogenous peroxidase activity was quenched with 0.5% hydrogen peroxide in methanol at room temperature for 30 minutes. proteinase k digestion was performed at 37˚c for 20 min. then, pre-hybridization was performed at 37˚c for 3 hours. after removing excess pre-hybridization buffer, 2 μg/ml digoxin (dig)-modified oligo-nucleotide antisense probes (table 2) in the hybridization solution were applied to the sections, followed by incubation at 37˚c overnight. after washing the slides in 2× saline-sodium citrate (ssc), 0.5×ssc, and 0.2×ssc buffer, the sections were incubated in a blocking buffer at 37˚c for 30 min. the sections were then incubated with biotinylated mouse anti-dig at 37˚c for 60 min and with streptavidin biotin peroxidase and biotinylated peroxidase for an additional 20 min, with each incubation followed by three washes in phosphate-buffered saline (pbs). the sections were treated with 3, 3-diaminobenzidine for 2 min, counterstained in haematoxylin for 5 min, dehydrated, and mounted with neutral gum. sections for the negative controls were prepared using the same steps described above, but the antisense or sense probes were replaced with pbs at ph 7.4. rhesus macaques were observed twice daily for clinical signs. the rectal temperature of the infected rhesus macaques increased to 40.5˚c at 1-2 days post-infection, and thereafter turned to normal. the infected common marmosets showed manifest symtoms of viral infection, including severe respiratory symtoms, drastical water intake decrease and overt weight loss, and the maximum body weight loss were about 11%. none of the mock infected nhps showed abnormal clinical signs or died during the expriment. pathological findings in the rhesus macaque tissues he stained tissues from rhesus macaques experimentally infected with mers-cov demonstrate that mers-cov induces lesions that are primarily observed in the lungs, with varying degrees of inflammation, interstitial pneumonia (fig 1a) , pulmonary oedema (fig 1b) , haemorrhaging, degeneration and necrosis of pneumocytes and bronchial epithelial cells (fig 1c) , and the infiltration of inflammatory cells. focal interstitial pneumonia and pulmonary oedema were observed in different parts of the pulmonary lobes, as was mild haemorrhaging. the most prominent pathological effect observed in the lungs of rhesus macaques was diffuse and focal eosinophil infiltration in the thickened alveolar septum and oedematous alveolar cavities, around the bronchus, and among the necrotic bronchial epithelial cells. no significant pathological changes induced by viral infection were observed in the other organs, and no obvious pathological changes were identified in any tissues examined from the control rhesus macaque (s1a fig) . pathological findings in common marmoset tissues a histopathological analysis detected numerous lesions in the lungs of the infected marmosets. exudative pathological changes were found, exhibiting haemorrhage, widespread pulmonary oedema and a large number of inflammatory cells. fibrinous exudates were observed in the oedematous alveolar cavities (fig 1d) . diffuse and focal neutrophil infiltration was found in the oedematous alveolar cavities (fig 1e) , bronchial lumen, and mildly thickened alveolar septum, around the bronchus, and among the necrotic bronchial epithelial cells (fig 1f) . no significant pathological changes induced by viral infection were observed in the other organs, and no obvious pathological changes were identified in any tissues examined from the control common marmoset (s1b fig) . to investigate the infiltration of specific inflammatory cells, ihc was carried out to identify cd68+ macrophages, cd15+ neutrophils, cd57+ natural killer cells, cd20+ b lymphocytes, cd138+ plasma cells, and cd3+, cd4+, cd8+ t lymphocytes. in the lungs of both species, the diffuse infiltration of numerous macrophages (fig 2b and 2d ) was observed in the expanded alveolar septum and the oedematous alveolar cavities. however, in the lungs of rhesus macaques, a large number of diffusely and focally infiltrating eosinophils (fig 2a) were found in the thickened alveolar septum and oedematous alveolar cavities, around the bronchus, and among the necrotic bronchial epithelial cells. however, in the lungs of common marmosets, numerous neutrophils ( fig 2c) infiltrated into the oedematous alveolar cavities. in both of the nhp models, other types of inflammatory cells were rarely observed. using immunohistochemical techniques and an ish analysis, we confirmed that mers-cov protein and viral rna were distributed in the lungs of rhesus macaques and common marmosets and that they were primarily located in the pneumocytes and inflammatory cells. in the lungs of rhesus macaques, mers-cov antigens were extensively distributed in type i and ii pneumocytes, alveolar macrophages (fig 3a) , eosinophils and bronchial epithelial cells ( fig 3b) . from the microscopic characteristics, the cuboidal type ii pneumocytes are located on the alveolar cavities, and smaller than macrophages. viral rna was also distributed in pneumocytes and inflammatory cells in the lungs of rhesus macaques (fig 3c) . in the lungs of common marmosets, a moderate level of mers-cov-positive antigens were detected in pneumocytes, and antigens were found more extensively in alveolar macrophages (fig 3d) , especially in the inflammatory cells around the bronchus (fig 3e) . viral rna was massively distributed in pneumocytes and inflammatory cells in the lungs of common marmosets (fig 3f) . no mers--cov-positive antigens or viral rna was detected in the lungs of the control nhps (data not shown). pathological lesions and virus distribution in rhesus macaque and common marmoset animal models are summarized and shown in table 3 . to further determine the effects of mers-cov infection and replication in the lungs of common marmosets, ultrastructural observations were performed on lesions in infected lung samples and on mock-infected samples. virus particles were found in type ii pneumocytes ( fig 4a-4c ) and in inflammatory cells (fig 4d-4f) . under the electron microscope, the characteristic of type ii pneumocytes is lamellar bodies (s2 fig). no viral particles were observed in the lungs of mock-infected common marmosets (data not shown). to elucidate the relationship between mers-cov and its entry receptor, ddp4, we determined the expression pattern of ddp4 in the lungs of rhesus macaques and common marmosets using immunohistochemical techniques. we found that in the lungs of rhesus macaques, ddp4 was strongly expressed in type i and ii pneumocytes, bronchial epithelial cells (fig 5a) , and inflammatory cells, primarily alveolar macrophages (fig 5a) . similarly, in the lungs of common marmosets, ddp4 was widely expressed in type i and ii pneumocytes and alveolar macrophages (fig 5b) . however, ddp4 was only weakly expressed in the bronchial epithelial cells, mainly in basal and ciliated cells (fig 5b) . in the present study, we analysed the histopathological features of mers-cov infection in rhesus macaques and common marmosets. moreover, we compared the distribution of mers-cov antigens, viral rna and ddp4 expression in the infected lungs of these species. we found that the lungs of both species exhibited varying degrees of lesions, including pneumonia, pulmonary oedema, haemorrhaging, degeneration and necrosis of the pneumocytes and bronchial epithelial cells, and inflammatory cell infiltration. comparing the different trends in the two nhp models, it can be seen that varying degrees of inflammation, especially interstitial pneumonia, were found in the lungs of rhesus macaques, indicating mild disease and trend of chronic pneumonia; however, in the lungs of common marmosets, exudative pathological changes were found, exhibiting pulmonary oedema, inflammatory cell infiltration and fibrinous exudates, suggesting acute pneumonia. similar to our results, previous study have also reported that rhesus macaques developed mild disease, and common marmoset exhibited potentially lethal disease [16] . however, in our study we found that the prominent inflammatory cells in the two nhp models were different, which may be the causality of process in mers-cov infection. in our study, the diffuse infiltration of numerous macrophages was observed in the expanded alveolar septa and oedematous alveolar cavities of both species. however, the most prominent pathological effect observed in the lungs of rhesus macaques was a diffuse and focal eosinophil infiltration in the thickened alveolar septum and oedematous alveolar cavities, around the bronchus, and among the necrotic bronchial epithelial cells. in contrast, in the lungs of common marmosets, diffuse and focal neutrophil infiltration occurred in the oedematous alveolar cavities, bronchial lumen and mildly thickened alveolar septum, around the bronchus, and among the necrotic bronchial epithelial cells. these differences in inflammatory cell infiltration suggest that inflammatory cells may function in the development of mers-cov infection. additionally, it is worth noting that eosinophils and neutrophils play important roles in rhesus macaques and common marmosets, respectively, in the development of pulmonary lesions and the pathogenesis of mers-cov infection. in the lungs of common marmoset, pulmonary oedema exhibited much more severe than that in the lungs of rhesus macaques, which may be due to the difference of inflammatory cells in the lungs of nhp models. similar to our results, previous studies have also reported that common marmosets infected with mers-cov exhibit acute bronchointerstitial pneumonia centred at doi:10.1371/journal.pone.0172093.g003 table 3 . pathological lesions and virus distribution in rhesus macaque and common marmoset animal models. pathology of mers-cov infection the terminal bronchioles, with an influx of inflammatory cells, a thickening of alveolar septa, oedema, haemorrhaging and fibrosis in lung tissues [10] . previous studies on rhesus macaques have shown that histological lesions induced by mers-cov infection were limited to the lungs, which exhibited interstitial pneumonia with a thickening of alveolar septa caused by oedema and fibrin accumulation, and small to moderate numbers of macrophages and even fewer neutrophils. in addition, alveoli tissue samples contained moderate numbers of pulmonary macrophages and neutrophils [17] . previous studies on common marmosets infected with mers also showed that marmosets developed a progressive severe pneumonia or interstitial lymphohistiocytic pneumonia, exhibiting hypoproteinemia consistent with high protein pulmonary effusions resulting from alveolar oedema and interstitial lymphohistiocytic pneumonia with type ii pneumocyte and bronchial associated lymphoid tissue hyperplasia [18, 19] . however, rhesus macaque model did not develop breathing abnormalities and showed no-tovery mild radiographic evidence of pneumonia [20] . similar to our study, the common marmoset model of mers-cov infection mimics the acute and severe pathological process, yet the rhesus macaque model represents the mild infection of mers-cov. thus, the nhp models meet different needs of mers-cov researches. fatal human cases of mers-cov infection cause upper respiratory tract illness, severe pneumonia and multiorgan failure. these cases also include exudative-phase diffuse alveolar damage with the denuding of bronchiolar epithelia, the prominent formation of hyaline membranes, alveolar fibrin deposits, type 2 pneumocyte hyperplasia, the occurrence of rare multinucleated syncytial cells, changes in alveolar septa related to oedema and increases in lymphocytes, with fewer plasma cells, neutrophils, and macrophages [21] . these findings provide evidence for the pulmonary tropism of mers-cov infection. the pathological features of mers-cov are shared by other similar respiratory illnesses, such as severe acute respiratory syndrome (sars)-cov [22] . previous studies that have evaluated fatal human cases of sars--cov have described diffuse alveolar damage at various stages as the most characteristic feature pathology of mers-cov infection of the disease, with sars-cov antigens primarily found in alveolar epithelial cells. in this study, we examined the histopathological features and the inflammatory cell response that occurs in the lungs of rhesus macaques and common marmosets experimentally infected with mers-cov. our findings indicate that inflammatory cells may play a crucial role in fatal mers-cov infections and that the progression of this disease in our animal models may mimic the infection in humans, making these models useful for further study of the pathogenesis, prevention and treatment of mers-cov infections. in this study, we analysed the distribution of mers-cov protein and viral rna in the lungs of rhesus macaques and common marmosets. we found that pneumocytes and inflammatory cells were positive for mers-cov antigens and viral rna. similarly, in the lungs of both species, mers-cov antigens were identified in type i and ii pneumocytes, alveolar macrophages and bronchial epithelial cells, viral rna was distributed in pneumocytes and inflammatory cells, and viral protein were found in type ii pneumocytes and inflammatory cells. based on our observations, we therefore propose that mers-cov may proliferate and spread from the lungs through an inflammatory cell migration pathway. it has been reported that in fatal human cases of mers-cov infection, viral antigens were identified in both unremarkable and necrotic bronchial submucosal glands and in pneumocytes and epithelial syncytial cells. however, macrophages showed no localization with mers-cov antigens in these cases [23] . findings of pulmonary consolidation, diffuse alveolar damage, and pleural effusion are consistent with the clinical features with mers-cov infection [2, 4, 24] . in this study, we also detected mers-cov protein and viral rna in type i and ii pneumocytes, alveolar macrophages and bronchial epithelial cells. therefore, our models of mers-cov infection using rhesus macaques and common marmosets may be suitable for use in the development of effective medications and prophylactic treatment and may serve as a tool to improve our understanding of the pathogenic process of mers-cov infection. dpp4 is a single-pass type ii transmembrane glycoprotein with a short n-terminal cytoplasmic tail. the structural residues comprising the receptor-binding domain have been defined via the co-crystallization of the mers-cov spike glycoprotein and dpp4. dpp4 is typically found in type i and ii cells and alveolar macrophages. it has only rarely been detected in the surface epithelia of the nasal cavity and has also been found in a subset of mononuclear leukocytes and serous cells from submucosal glands [25] . in fatal human cases, dpp4 has been observed in scattered pneumocytes and syncytial cells, bronchiolar epithelia and endothelia, and alveolar macrophages [23, 26, 27] . in this study, we found that in the lungs of the nhps infected with mers-cov, ddp4 was expressed in type i and ii pneumocytes, bronchial epithelial cells, and inflammatory cells, primarily alveolar macrophages. in conclusion, we established animal models of mers-cov infection using rhesus macaques and common marmosets, which mimic the oncome of mers-cov infection in humans and provide a tool that may help in better understanding the pathogenic process of this disease. they may also be suitable models for aiding in the development of effective medications and prophylactic treatments for mers-cov infections. fouchier ra isolation of a novel coronavirus from a man with pneumonia in saudi arabia epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients clinical course and outcomes of critically 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ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia dipeptidyl peptidase 4 distribution in the human respiratory tract: implications for the middle east respiratory syndrome emerging human middle east respiratory syndrome coronavirus causes widespread infection and alveolar damage in human lungs tropism and replication of middle east respiratory syndrome coronavirus from dromedary camels in the human respiratory tract: an invitro and ex-vivo study key: cord-335272-jypxi99z authors: sharma, anupam joya; subramanyam, malavika a. title: a cross-sectional study of psychological wellbeing of indian adults during the covid-19 lockdown: different strokes for different folks date: 2020-09-03 journal: plos one doi: 10.1371/journal.pone.0238761 sha: doc_id: 335272 cord_uid: jypxi99z the psychological impacts of the lockdown due to the covid-19 pandemic are widely documented. in india, a family-centric society with a high population density and extreme social stratification, the impact of the lockdown might vary across diverse social groups. however, the patterning in the psychological impact of the lockdown among lgbt adults and persons known to be at higher risk of the complications of covid-19 (such as persons with comorbidities or a history of mental illness) is not known in the indian context. we used mixed methods (online survey, n = 282 and in-depth interviews, n = 14) to investigate whether the psychological influence of the lockdown was different across these groups of indian adults. we fitted linear and logistic regression models adjusted for sociodemographic covariates. thematic analysis helped us identify emergent themes in our qualitative narratives. anxiety was found to be higher among lgbt adults (β = 2.44, ci: 0.58, 4.31), the high-risk group (persons with comorbidities) (β = 2.20, ci:0.36, 4.05), and those with a history of depression/loneliness (β = 3.89, ci:2.34, 5.44). persons belonging to the lgbt group reported a greater usage of pornography than the heterosexuals (β = 2.72, ci: 0.09, 5.36) during the lockdown. qualitative findings suggested that lgbt adults likely used pornography and masturbation to cope with the lockdown, given the limited physical access to sexual partners in a society that stigmatizes homosexuality. moreover, both qualitative and quantitative study findings suggested that greater frequency of calling family members during lockdown could strengthen social relationships and increase social empathy. the study thereby urgently calls for the attention of policymakers to take sensitive and inclusive health-related decisions for the marginalized and the vulnerable, both during and after the crisis. the coronavirus disease , caused by the novel coronavirus sars-cov-2, first emerged in wuhan, china during late 2019 and was labeled a public health emergency by the world health organization [1] . the recent and rapid increase in the number of covid-19 cases, 16 ,523,815 as on 29 th july 2020 globally [2] , has increased panic across countries [3] . every country affected by the virus adopted several measures in order to curb its spread. india, plos one | https://doi.org/10.1371/journal.pone.0238761 september 3, 2020 1 / 23 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 home to 1.3 billion people, announced a nationwide "lockdown" on 25 th march 2020 [4] . the lockdown restricted citizens' physical mobility, advocated social distancing norms, and limited a majority of public services while allowing the essential ones. however, these measures of sheltering-in-place, equivalent to an extended quarantine, likely created a stressful environment for the citizens, given the sudden disruption in their daily routines [5] , [6] . these disruptions could contribute towards adverse psychological outcomes such as post-traumatic stress symptoms [7] and aggressive behaviors [8] . for instance, one indian study by gautam & sharma [9] , highlighted that the lockdown could increase the psychological toll on the indian academic fraternity because of the disruption in their work, which additionally brings financial instability to the contractual staff. however, the impact of a lockdown might vary across diverse social groups. individuals who are living alone or away from family (or loved ones), those suffering from economic losses, or having a history of negative psychological states, could be at a higher risk of depression, loneliness, and anxiety disorders during the lockdown. in addition to these sources of stress that could get exacerbated during the lockdown, certain social groups may have to deal with stress due to identifying as a member of a minority community in their society. meyer's minority stress model [10] explains this minority stress as arising from the risk of social shunning, discrimination based on identity, or the consequent efforts to hide minority status. one such minority group that could be vulnerable to the effects of the lockdown is the lgbt community. according to meyer's minority stress model, lgbt individuals are exposed to unique stressors rooted in societal structure and related to their minority identity, which could combine with other stressors to impact their psychological wellbeing [10] . therefore, we posit that persons belonging to the lgbt community could suffer from increased stress during lockdown. further, the restrictions on physical mobility might have not only disrupted the social lives of many individuals but also paused their sexual lives [11, 12] . to cope with these disruptions in sexual lives, in addition to the on-going stress and boredom due to the lockdown, individuals could also rely on pornography [11, 13] . reports revealed a 33% average increase in pornwatching during the lockdown period (march 2020) in india [14] . moreover, the time spent indoors during the lockdown could also enable adults to explore their body and experience pleasure through masturbation [15] . pornography use and masturbation are recommended ways of meeting sexual needs without the risk of contracting sars-cov-2 infection during the pandemic [12] , although prolonged and habitual usage of pornography and masturbation could have negative consequences on sexual satisfaction [16] . the psychological impact of the lockdown could also be higher among individuals with a known higher, versus lower, risk of experiencing complications of covid-19, such as the elderly or those with co-morbid conditions (for example, persons with chronic respiratory illnesses and diabetes). the extent of daily exposure to the pandemic may also matter: a study from wuhan, china, found a high prevalence of depressive symptoms among frontline healthcare workers [17] . similarly, another study from australia showed that people living in high risk infection zones reported greater psychological distress than those living in uninfected areas [18] . these findings suggest that a higher perceived risk of covid-19 could increase anticipatory fear and anxiety. this fear, depression, loneliness, and anxiety during the time of crisis not only could affect mental health but also adversely affect one's lifestyle and diet, ultimately impacting physical health [19] . previous studies have shown that depression (or anxiety) worsens sleep disorders [20] and eating disorders [21] . despite these risks, several personal and social resources could be available for individuals to cope with the adverse effects of the crisis. in a family-centric country such as india, family is regarded as a vital social support [22] , especially during a crisis. living with family/relatives (or regular virtual interactions through phone or online media) could act as social support which could result in lowering stress during the lockdown. of note, an opportunity to spend extended time with family members could strengthen family bonds and enhance work-family balance, leading to a better quality of life [23] . while the lockdown period may be spent in fulfilling varied responsibilities, it could also have created opportunities for many to spend time with family (and loved ones) and potentially improve the quality of family relationships through physical or virtual proximity. nevertheless, these opportunities might act as a situational coercion for a few individuals (who have a history of family maladjustment or family conflict) and induce additional stressors, further increasing their vulnerability to adverse psychological outcomes during a lockdown. in addition to these social resources, several individual-level characteristics such as the nature of employment, access to material resources; and, psychological resources such as resilience-coping and optimism might be beneficial in minimizing the effect of this crisis [24, 25] . findings of a recent study from china suggest that positive personal-level characteristics such as emotional-control and optimism could also help minimizing the negative effects of the covid-19 crisis [26] . although these concerns warrant attention in the indian context, we could locate only a few studies reporting the prevalence of depression and anxiety during the covid-19 crisis in india, including a comparison across age and gender groups [27, 28] . moreover, these studies did not examine the prevalence of these outcomes across other social groups, including the vulnerable and the hidden group of lgbt adults. the rapid increase in the number of covid-19 cases in india and the disruptions due to the lockdown, warrant investigating the processes explaining any social patterning in the psychosocial wellbeing of indian citizens during this crisis. in response, our study of indian adults unpacks how social factors such as sexual orientation, relationship status, and residence in high-infection areas, could be linked with several psychological outcomes during the lockdown. we also investigated whether a higher risk of covid-19 complications and a history of depression or loneliness worsens the mental health impact of the lockdown. we further explored the complex processes explaining if and how anxiety or depressive symptoms were related to sleeping and eating habits during the lockdown. we also investigated the role individual-level resources played in coping with the effects of the crisis. because the lockdown likely changed the nature of social interactions, we additionally examined if this brought any change in how individuals viewed the world and in their social empathy, which could be an important psychological resource for overall wellbeing and quality of life. the primary research questions explored in this study were: in addition to the above primary questions, inspired by the initial two qualitative interviews, we also addressed the following in our study: 1. how are sharing vulnerabilities (stress and depression) with loved ones, and the frequency of interaction with family related to strengthening of social bonds and social empathy during the lockdown? we followed a convergent mixed methods approach [29, 30] in our study. first, two exploratory qualitative in-depth interviews were conducted to refine our research questions. the narratives of the two participants (one male and one female participant) guided us in identifying key factors affecting their mental wellbeing during the time of the lockdown. these participants shared their frustrations related to the lockdown, the disruption in their routine work, the chaos around them regarding the increase in number of cases, their challenges in general, and their overall feeling about the entire crisis situation. based on the data from these two interviews, we constructed our online quantitative survey. data for the study were simultaneously collected through the online survey and qualitative interviews. however, whenever the data from the quantitative survey revealed an interesting picture, we dug deeper about it in our qualitative interviews to understand its context and complexity. we carried out an online survey from 9 th may to 15 th may 2020. our survey questionnaire in the form of an anonymous google form was circulated through several facebook groups as well as whatsapp and instagram contacts. we further used a snowball sampling procedure to increase the number of responses. the authors requested their family members, friends, colleagues, and professional networks to further spread the form among their networks. the introductory passage in the google form briefed the participants about the broad objective of the study and requested their voluntary participation. further, the passage also promised anonymity and confidentiality to the participants. once the participants read the introductory passage, they were requested to proceed to fill up the survey. the participants' agreement to take part in the survey after they were given an opportunity to carefully and unhurriedly review the information about all relevant information about the study, the voluntary nature of participation, their right to withdraw at any time, and the confidentiality of their data, was considered to be implied consent. due to the online nature of the survey, we could not limit its spread to a specific geography. however, we specified our eligibility criteria (indian citizen, presently residing in india, aged 18 years or above, and willing to fill the form in english) in the introductory passage to maximize the chance that we only got responses from india. we received responses from 282 participants. response variables. anxiety. we measured anxiety using the general anxiety disorder (gad-7) scale [31] . this widely used scale includes items such as "over the past 2 weeks how often have you been bothered by the following problems: feeling nervous, anxious or on edge?" the responses were recorded on a 4-point likert scale ranging from "not at all (0)" to "nearly every day (3) ." we found a good internal consistency of the gad-7 scale in our sample (cronbach's alpha = 0.91). the aggregate of the item scores reflected the total anxiety score. depressive symptoms. we assessed depressive symptoms of our participants using the short version of the cesd-d scale, a 10-item scale [32] . the scale includes items such as "in the past week how often have you felt any of these: i had trouble keeping my mind on what i was doing." two items were reverse scored. the responses to all the items varied from "less than a day" (0) to "5-7 days" (3) . we later discovered that responses to one item (i was bothered by things that usually do not bother me) did not get recorded possibly due to some technical error in the google form. however, following siddiqui [33] and hawthorne et al. [34] , we imputed the personmean score for the missing item. we found good internal consistency of the scale (including the imputed score) in our sample (cronbach's alpha = 0.86). the item total was used as the depressive symptom score. symptoms of the internet addiction. we used the internet addiction test (iat) scale [35] to measure symptoms of addiction of the internet. the scale includes 12 items such as "over the past 2 weeks, how often have your felt: find yourself saying "just a few more minutes" when online?" responses varied from rarely (1) to always (5) . the cronbach's alpha was found to be 0.92. the total score of all 12 items yielded the internet addiction score. compulsive consumption of pornography. the compulsive pornography consumption (cpc) scale [36] was used to assess the symptoms of usage of pornography. the 6-item scale included items such as "please indicate how these statements described you during the past 2 weeks: i thought of pornography (porn) when i was trying to focus on other things." the responses were recorded on a 7-point likert scale ranging from never (1) to very frequently (7) . the cronbach's alpha was 0.87 in our sample. the sum total of the scores of all items resulted in the pornography consumption score. experiences of hostility. a single item was used to measure experiences of hostility during the lockdown: have you been facing the following problems in the last 2 weeks? you faced a hostile situation (including emotional, physical, and mental violence) from anyone in the place you are currently in. responses were dichotomized to yes and no. change in food habits (time and consumption). we assessed any change in the participants' food habits using a single item: have you been facing the following problems in the last 2 weeks? food patterns (type of foods consumed/timings) have changed. responses varied from not at all (1) to always (5) . sleeping problems. sleeping problems were measured using a combination of two items, have you been facing the following problems in the last 2 weeks? your sleep cycle has changed drastically, and you have difficulty in falling asleep. the responses varied from not at all (1) to always (5) . the additive score yielded the level of sleeping problem with scores ranging from 2 to 10. frequency of masturbation. frequency of masturbation was assessed using a single item, how often are you engaging yourself in masturbation activities in the last 4 weeks? the responses varied from never (1) to multiple times a day (6) . social empathy and quality of social relationships. social empathy was operationalized based on the participants' choice of several options offered. the selection of any of the following options: you have become more socially responsible; you have become more active in neighborhood associations/groups or other social groups near your residence; you have been thinking about the vulnerable in our society and tried to do at least something for them (donating or helping in other ways) indicated increased social empathy coded as 1 (otherwise 0). similarly, the quality of social relationships was recorded as 1 (improved), if the participants selected even one of the following options: you have started liking to spend time with your closed ones more than before; you have strengthened your relationship with your friends; and you have strengthened your relationship with your family/partner, otherwise as 0. predictors. sharing stress and anxiety with loved ones. if participants selected "yes" to the question have you shared your stress and vulnerability with loved ones during the lockdown, it was coded as 1, else as 0. resilience coping. we assessed resilience coping of the participants using the 4-item brief resilience coping scale (brcs) [37] . the scale included items such as i look for creative ways to alter difficult situations, with responses varying from does not describe me at all (1) to describes me very well (5) . the aggregated score of all items reflected the participants' resilience. the cronbach's alpha was 0.82. optimism. optimism was measured using the 10-item revised life orientation test (lot-r) scale [38] . it included items such as in uncertain times, i usually expect the best while responses ranged from, i disagree a lot (1) to i agree a lot (5) . four items were fillers and were removed from the analysis. the aggregate of all item scores resulted in the optimism score. we found moderate internal consistency of the scale in our sample (cronbach's alpha = 0.55). change in frequency of calling family members. we compared the frequency of calling family members during the lockdown with that during october 2019-march 2020. we treated this as an indicator of the change in frequency of calling family members during the lockdown. we coded it as 1 if the frequency increased, otherwise as 0. high-risk group. individuals who reported having any of the following: chronic respiratory illnesses, diabetes, heart disease, hypertension, or a weakened immune system, were categorized as belonging to the "high-risk group (1)", else as "low-risk group (0)." history of depression/loneliness. we grouped the participants who reported having a history of depression or loneliness as "group with history of depression/loneliness (1)", otherwise "group with no history of depression/loneliness (0)." categories of state exposed to covid-19. we referred to data from ministry of health and family welfare, india [39] for categorizing the states as per the counts of covid-19 cases. we coded maharashtra (with cases more than 25000 during the data collection) as "highest exposure;" tamil nadu, gujarat and new delhi (with around 10,000 cases) as "high exposure;" rajasthan, madhya pradesh, and uttar pradesh (near to 5000 cases) as "moderate exposure"; and rest of the states as "low exposure." sociodemographic characteristics. we also collected information on age (18-29/30-44/45-59/and above 60 years), gender (male/female/others), sexual orientation (straight/queer), relationship status (opposite-sex relationship/same-sex relationship/single/complicated), place of residence (rural/urban), educational qualification (postgraduate/graduate or diploma/12 th or lower), and annual income in indian rupees (0-3,00,000 (approximately, $0-$4000)/ 3,00,000-10,00,000 ($4000-$13500)/10,00,000-20,00,000($13500-$27000)/above 20,00,000 ($27000 and above)), and the state of residence. we conducted 14 in-depth interviews from 10 th may through 17 th may 2020. we circulated an advertisement inviting participants for telephonic interviews through social media (facebook, instagram, and twitter) and personal contacts of the authors. the advertisement included a brief introduction about the study, contact information of the first author (also the interviewer) the nature of the interviews, and about the approximate length of the interview. the introduction also informed the participants about the sensitive nature of the topic (which included questions on their personal/intimate lives) and asked their preferences of the gender of the interviewer. however, none of the participants shared concerns being interviewed by ajs (a man). interested participants contacted ajs through email/facebook/whatsapp showing their willingness to participate. at that time, ajs briefed the participants about the study; about their anonymity and confidentiality of data; and that the interview would be terminated at any point the participant showed discomfort. ajs and the participants mutually agreed on a time for the telephonic interview. before beginning the interview, ajs once again sought informed verbal consent. in addition to verbal consent, ajs also sought consent to audiotape the interviews. four participants were reluctant to get the interviews audiotaped. detailed notes (including several quotes) were taken during these interviews. all interviews began with broader questions such as "how do you feel about the entire situation (of covid-19 and lockdown)?" ajs was cautious while asking personal questions, especially about romantic and sexual lives of the participants. ajs ensured participants' comfort, not only while asking sensitive questions, but throughout the interview process, by taking a pause and asking, "should we proceed?" however, there were no instances where there arose the need to terminate any interview. all participants shared their emotions, vulnerabilities, moods, challenges, change of lifestyle, and perceived wellbeing during the lockdown (and the covid-19 crisis). specific comments regarding the mood and context were noted by ajs to give rigor to the analysis. at the end of the interviews, the participants were requested to share about the study with their peers and network, seeking their participation. the length of the interviews ranged from 28 minutes to 1 hour 40 minutes. eight participants were recruited through the advertisement while 2 participants were recruited through snowball sampling. additionally, we allowed the participants of the online quantitative survey to express interest for a follow-up telephone call. we recruited 4 participants through this method. different methods of recruitment helped us get a socially diverse sample in a short time. in addition to the interviews, we collected data through an open-ended question in the online quantitative survey. this allowed the participants to share their concerns related to the pandemic situation (and lockdown). extracted quotes were used in our qualitative analysis. quantitative analysis. the distribution of all continuous variables was checked for normality [40] . next, we fitted separate multivariable linear regression models to estimate the association of the independent variables (sexual orientation, relationship status, high-risk group, and living in a state with high number of cases) with psychological outcomes (anxiety, depressive symptoms, internet addiction and pornography consumption) adjusted for the sociodemographic covariates-age, gender, annual income, educational qualification, place of residence-and for individual personal resources (optimism and resilience). we fitted separate logistic regression models to estimate the associations of sexual orientation and relationship status with the binary variable indicating the experience of hostility, adjusting for all sociodemographic variables. we also fitted multivariable linear regression models to estimate the association of anxiety and depressive symptoms with changes in sleep and food cycles (separate models), adjusted for the sociodemographic covariates and personal resources. additionally, we fitted separate logistic regression models to estimate the association of increased frequency of calling family members with social empathy and the quality of social relationships adjusted for sociodemographic covariates. for all our analyses, alpha was set at 0.05. all statistical models were run in stata version 12 [41] . qualitative analysis. we chose a thematic analysis approach [42] to analyze the qualitative data. the analysis began with ajs (who also conducted all the interviews) familiarizing himself with the data by spending prolonged time in re-listening to the audiotaped interviews and reviewing the transcript excerpts. the participants' narratives about their emotional responses to the situation; their description of how the lockdown affected their routine, relationships, and social responsibilities; their sense of self (including their body); their perspective on life; their coping mechanisms; and, views towards a "new world" guided the coding process. four themes emerged from these indexed codes [43] and the detailed comments. additionally, nvt (an external researcher) categorized the themes emerging from the codes. the coding scheme was discussed among the two authors (and nvt), and after critical analysis, the themes were confirmed with a high inter-coder reliability [44] . follow-up interviews with two participants were carried out separately for respondent validation [45] . additionally, several quotes from the open-ended section of the online survey were included in the themes that emerged from the qualitative interviews allowing better representation of all the voices heard. both quantitative and qualitative findings carried equal weight in this study. the qualitative themes that emerged gave richer context to the quantitative results during the interpretation phase. the study was motivated by previous work of ajs and mas on the queer community, and their understanding of the community's unique vulnerabilities. apart from this, the lockdown has severely restricted the ability of ajs and mas, not only in terms of physical mobility but also in terms of distance from loved ones and has affected their productivity. interviews by ajs were conducted with this frame of reference. the study was approved by the institutional ethics committee, iit gandhinagar, india. utmost precaution was taken by ajs while conducting the telephonic interviews. the participants were informed about the sensitive nature of the questions and were informed that they could skip any question. ajs constantly monitored the mood of the conversation and frequently asked the participants about their willingness to continue. names of all the participants have been changed in this study to protect anonymity. we analyzed a sample of 282 indian adults who responded to the online survey (table 1) . a majority (~75%) of our participants were 30 years or younger. around 60% identified themselves as male, and about 77% reported to be heterosexuals. only a small proportion (~12%) of our participants had education less than 12 th standard (high school). greatest proportion (~81%) of the participants resided in urban areas. anxiety and depressive symptoms across social groups. our fully adjusted models (adjusted for gender, age, educational qualification, income, and place of residence) (see table 2 ) found that gad scores were higher, on average, in lgbt adults (β = 2.44, ci: 0.58, 4.31) versus heterosexuals, high-risk group (β = 2.20, ci:0.36, 4.05) versus low-risk group, and participants with history of depression/loneliness (β = 3.89, ci:2.34, 5.44) versus participants with no history of depression/loneliness. however, gad scores were lower for single participants (β = -2.35, ci: -4.30, -0.39) than those who were in opposite-sex relationships. we could not find statistically significant associations of living in a state reporting a high count of covid-19 cases with anxiety symptoms. unsurprisingly, we found a statistically significant association of a history of depression/ loneliness with increased depressive symptoms during the lockdown (β = 4.34, ci: 2.38, 6.30), independent of other covariates. however, we could not find any evidence linking sexual orientation, relationship status, living in a state reporting a high count of covid-19 cases, and belonging to a high-risk group, with depressive symptoms. addiction to the internet, consumption of pornography, and frequency of masturbation across groups. we found that a history of depression/loneliness was statistically significantly associated with higher internet-addiction symptoms (β = 4.55, ci: 1.47, 7.63), independent of all other covariates. however, we could not find evidence that other predictors were associated with internet addiction. moreover, our fully adjusted models showed greater symptoms of pornography usage, on average, in lgbt adults (β = 2.72, ci: 0.09, 5.36) versus heterosexuals, in the high-risk group (β = 2.80, ci: 0.22, 5.39) versus low-risk group, in participants in same-sex relationships (β = 9.15, ci: 0.93, 17.38) versus opposite-sex relationships, and among those with a history of depression/loneliness (β = 2.63, ci: 0.41, 4.86) versus no such history. additionally, our adjusted models showed that lgbt adults (β = 1.39, ci:0.94, 1.86) and participants in same-sex relationships (β = 2.07, ci = 0.50, 3.63) reported a higher frequency of masturbation during the lockdown compared to their heterosexual peers. experiences of hostility. we did not find statistical evidence that experiences of hostility differed across sexual orientation and relationship status. although statistically not significant, lgbt adults (versus heterosexuals) had higher odds of experiencing hostility (aor = 1.63, ci: 0.82, 3.23) during the lockdown independent of the sociodemographic covariates. association of anxiety and depressive symptoms with food and sleep habits. our fully adjusted models (adjusted for sociodemographic variables and positive resources) (see table 3 ) showed that higher depressive symptoms and anxiety symptoms were associated with greater reports of self-reported sleep disorders (β = 0.16, ci: 0.11, 0.20 and β = 0.19, ci: 0.14, 0.25, respectively) and self-reported changes in food pattern (β = 0.05, ci: 0.03, 0.08 and β = 0.08, ci: 0.05, 0.11). social empathy and quality of relationships. our fully adjusted logistic regression models (see table 3 ) showed that participants who increased the frequency of calling their family members during the lockdown (compared to 6 months earlier) had higher odds of enhancing the quality of their social relationships (aor = 2.56, ci: 1.19, 5.52), and reporting increased social empathy (aor = 2.27, ci: 1.06, 4.88), independent of all sociodemographic covariates. moreover, our models found that sharing vulnerabilities (stress/depression) with loved ones was associated with higher odds of being socially empathetic (aor = 3.99, ci:1.95, 8.14), and enhancing social relationships (aor = 2.96, ci: 1.52, 5.74), after accounting for all sociodemographic covariates. fourteen participants shared with us the slices of their lives during the lockdown. of the 14 participants, 8 were male, 5 were female, and 1 identified themselves as a non-binary transgender. six of the 14 identified themselves as lgbt adults. four were students, 5 worked in private/public sectors (hereafter "service"), and 5 engaged in business/entrepreneurship. the thematic analysis of the narratives of these 14 participants revealed four broad themes. not all participants' narratives highlighted all the themes; however, each participant's narrative was reflected in at least one theme. theme 1: emotional responses to "distance from the real world". all 14 participants expressed their unique concerns about the lockdown situation. words such as "frustrated," "stressed," "angry," and "suffocated," were frequently used to describe their emotions. although the intensity of the negative impact of the lockdown varied across participants, most participants (9/10) shared how the lockdown disrupted their lives causing frustration and agitation. for instance, ashok (male, heterosexual, service, 27 years old) shared, similar responses of frustration were shared by several students who had mostly enjoyed an outdoorsy life, be it spending time on their college campuses or with friends outside. however, a few shared a different reason for their anxiety: living in a place with a high number of covid-19 cases. rajini's (female, heterosexual, homemaker, 39 years old) narrative is an example: we are in a containment (severe movement restriction) zone, and since the last 12 while several respondents shared the fear they felt currently due to a high number of covid-19 cases in their areas, two respondents, tulika (female, belonged to lgbt group, service, 23 years old) and salma (transgender (non-binary), belonged to lgbt group, service, 43 years old) shared how this "worse" time had forced them to revisit their past trauma. tulika, who had gone through a break-up one year earlier and was recovering with the help of therapy elaborated, another instance of past trauma being triggered was in the case of salma (they/their/them), who had always managed situations of discrimination (against their transgender identity) calmly but recently lost their temper during such an event. the policewoman stopped us (them and their partner), and asked me "what do you think you are," [. . .] i have always tried to be calm in such situations, but this time, i just lost all my calmness. it was a mix of so many things, my frustration at work during the crisis, my mother falling ill just a week before the incident, all these acted together. their stories revealed that the stress and anxiety developed during the lockdown had revived old memories of trauma. thus, in response, tulika chose to go through emergency sessions with her therapist, while salma failed to stay calm and burst out when faced with genderbased discrimination. while tulika and salma revisited trauma, anurag (male, belonged to lgbt group, businessman, 37 years old) who moved to his parental home during the lockdown felt distant from his "real" world. he shared, anurag's feeling of distance from his "real" world highlighted how uncomfortable he was at his old home with his parents. he shared how things around his parents' house reminded him of how uneasy he felt while growing up as a gay man. anurag was conflicted by a dilemma: while the lockdown brought him closer to his parents at a time of their need, it also placed on him an additional psychological burden. the suffocation potentially felt by lgbt adults forced due to the lockdown to stay with others to whom they were not out was evident in this quote shared via the online survey: while a majority of the participants (9/14) shared mostly about their negative states of mind, a few took a moment to share the positive impact of the lockdown on their lives. for instance, rumi (female, heterosexual, businessperson, 33 years old) described how she saw the lockdown as an opportunity to introspect about her own life. the narrative of ashish (male, belonged to lgbt group, service, 23 years old, hiv positive) highlighted his equanimity during the crisis. he shared that he did not have any trouble during the lockdown, mostly because he was an introvert who had always loved spending time indoors. however, he mentioned that he and his mother (whom he was living with) had been adhering to the usual precautions to avoid the virus. he said that he had always been protective about his health, as was his mother. theme 2: impact of the lockdown/covid-19 on lifestyle. almost all participants (11/14) described how their daily routines had changed because of the lockdown. while many of them were trying to keep themselves healthy by striving to live a "normal" life, a few mentioned about drastic changes in lifestyle, especially in their eating and sleep patterns. for instance, tulika shared how she lost motivation to stick to a routine during the lockdown. rumi however understood this "bad time" as an opportunity to work on herself, a point shared by three other participants. she believed that she could explore a completely different side of hers because of this long break from her busy work. she narrated, i have been experimenting with my life these days. i wake up early and do yoga and then meditation, and then helped my mother with some household chores. currently, i think i feel physically very light, maybe because of exercise. the free time has helped me a lot to explore these, which otherwise was not really possible given the busy life that i had. theme 3: coping with challenges. each participant shared unique stories of coping with the crisis. while most adapted themselves to the "smaller world," a few struggled with it and found alternate ways to negotiate their challenges. a few other participants were positive about the crisis, spending time relaxing or pursuing long-held passions. for instance, rumi described her introspective exploration and enhanced ability to connect with the society through solitude and meditation. on the other hand, tulika kept her mind distracted from the "outside chaos" by immersing herself in social media. for instance, tulika shared, the number of hours i am awake, i am using social media. even if i am going to sleep, i will mindlessly keep scrolling until i fall asleep. because these are the places currently where you see people. otherwise it is quite just you. i think it is a good place to connect. it keeps me engaged. tulika's narrative indicates that the online activities have connected her with the outside world, which created an avenue for her to share her vulnerabilities with others through online interactions (messaging and commenting on posts). notably, more than 50% of the participants reported perceiving the internet as a way to reduce stress and anxiety during the lockdown. in fact, most of them referred the internet as the easiest way to keep them distracted from thinking (or overthinking). a few of the participants who shared their frustration about disruption in their sex lives, reported finding solace in watching pornography and masturbating. sujoy (male, belonged to lgbt group, student, 22 years) quoted, i think it is the only thing you could right now. i mean i see people online in grindr (a dating app for lgbt adults), and surprisingly people are still looking for sex. and very honestly, i completely understand this desperateness. most of them, and that includes me, were having our fun days. and all of a sudden this happens. initially, even i had the thought, ghar ke paas to jake hookup kar sakte hain (it should be okay to have a hookup close to my place). but i realized immediately that it is not wise enough to meet people during this time, especially when you know that it (covid-19) could also not show any symptoms[. . .] what i do right now is watch porn and jerk off. while sujoy showed high self-control and overcame his behavioral impulse of going out to have sex, his initial inclination to go out of his home for sex highlights the repercussions of forced abstinence due to the lockdown. similarly, a participant (male, heterosexual, student, 18-25 years old) answering the online quantitative survey shared via the open-ended question: my sexual desires are making me feel more anxious to masturbate, as a single (man), very often during this lockdown period. theme 4: new perspectives on self, life, and society. ten of the 14 participants believed that their lives were no longer the same. they believed that they had changed significantly in terms of how they viewed their selves and society in general. for instance, tulika said, earlier i used to be worried about very little things, and now it has changed drastically, maybe after spending so much time with myself. i am clearer about my life. i feel i got clarity and i see a huge change in myself. i feel spending time with myself has been the best thing. similar observation was made by rumi, who pointed out that, [. . .] my mental state is also is very different than before. earlier i used to get irritated, worried, angry frequently, but now i feel that i am quite easy, surprisingly, even with my husband. we used to fight (giggles) but now i see that i have been very much calm even with him. it is more because of a lot of things, like about spending time with myself. the universal vulnerability, extended time to reflect on their lives, and sharing their vulnerabilities with others had increased the participants' level of compassion and social empathy, which further strengthened their relationships with their loved ones. this was reflected in what tulika shared, what i have learnt from this entire situation is that you do not take things for granted anymore, even for like interacting with people. i connected with a lot of friends lately, made a couple of new friends as well, and i feel the conversations are no longer superficial but they seem very real, even though we are not in the same physical space but i feel closer to the people more than when we were closer physically [. . .]people have become more vulnerable and have started sharing. everyone is going through some upheaval right now with this feeling and everyone is trying to connect with others. we are in our mid 20s and everyone is going through this time in pretty much (a) similar way, a huge disruption in our lives i would say. so, we become more vulnerable, now i guess these feelings of vulnerability comes out, when you are sharing. and especially when you know that the other person is going through the same as well, and it actually also allows you to connect to the society at large. . .to a wide range of people. similarly, rajan (male, heterosexual, student, 30 years old) reflected, rumi considered the lockdown situation as an opportunity to reflect upon her own life, tried to connect with people around her, spent longer time in spiritual, motivational, and meditational activities-all of which had helped her find meaning in her life and optimism about her marriage. using quantitative data from 282 indian adults and qualitative narratives of 14 adults, our mixed methods study found that even though the covid-19 crisis indiscriminately affected everyone, its psychological effects were disproportionate among diverse social groups in india. our quantitative and qualitative findings both suggest that lgbt adults, compared to the heterosexuals, are at a higher risk of developing anxiety, depressive symptoms, and using pornography during the lockdown. moreover, higher levels of anxiety and depressive symptoms were associated with greater disruption in sleep and food cycles. lastly, our findings unpacked how sharing vulnerability with loved ones, and frequently talking to family members, strengthened social relationships and social empathy among indian adults during the covid-19 lockdown. the higher risk of anxiety in our survey among the lgbt adults than heterosexuals was corroborated by our qualitative findings. several reasons might explain this. first, previous research suggests that the lgbt community have a higher prevalence of anxiety and depressive symptoms compared to heterosexuals [46] , independent of any crisis. this could be explained using the meyer's minority stress model [10] during the lockdown, their minority stress (such as sexual orientation-based discrimination and internalized homonegativity) could interact with the lockdown-related stress, thereby increasing their anxiety much more than that experienced by heterosexuals. second, the lockdown had likely paused their social as well as sexual lives (which connected them with their own community) which likely restricted their access to a safe space, and limited the social support they received from the community [47] . subhash and sujoy's narratives are good examples that show how abstinence from active sex life could make the lgbt adults more anxious during the lockdown. while sujoy's example reflects the high demand of self-control during the covid-19 lockdown [48] , research suggests that the application of self-control is effortful and aversive [49] . therefore, individuals with low self-control could be at a higher risk of succumbing to their behavioral impulses during the pandemic. this also explains our qualitative finding which suggests a higher likelihood of compulsive consumption of pornography and greater frequency of masturbation among lgbt adults (and people in same-sex relationships) during the lockdown compared to heterosexuals (and people in opposite-sex relationships). this disruption in sexual life could explain our quantitative finding which suggested greater anxiety among heterosexuals who were in (opposite-sex) relationships. the lockdown could have resulted in restrictions in physical interactions and romantic dates with their partners, and reduced the social support received, thus increasing their anxiety. however, the higher consumption of pornography and frequency of masturbation might suggest a healthy sexuality. notably, these practices may enable individuals to stay away from seeking physical intimacy outside the home during the lockdown which could contribute towards curbing the spread of covid-19 [12] . lastly, during the lockdown, it is likely that most adults would move closer to their families for support and to avoid loneliness [50] , especially in a family-centric country such as india. however, for many lgbt adults moving in with their parents, to whom they were not out or who disapproved of their sexuality, could be challenging, increasing their risk of experiencing hostility during the lockdown. previous studies have shown that parental support and familial environment play crucial roles in self-acceptance among lgbt adults [51, 52] . the lack of such familial and/or parental support could hinder self-acceptance among lgbt adults, see for instance, anurag's narrative of how he could accept his sexuality only after he moved out of his parental home. moreover, moving away from his "safe space" to a place which brought memories of discomfort likely increased his anxiety. this could be true for several of the lgbt adults who had gone through interpersonal and familial conflict earlier. our quantitative findings also found that individuals at greater, versus lower, risk of the complications of covid-19 showed higher levels of anxiety. a previous study suggested that patients with existing risk factors to covid-19 such as cardiovascular disease (cvd) were more also more likely have worse health outcomes if infected [53] . irrespective of worse health outcomes, belonging to a group with increased risk of covid-19 complications, given that covid-19 has no known cure and unpredictably causes mortality, could potentially induce additional stress and anxiety. this corroborates findings from previous studies suggesting a higher prevalence of stress among front-line health workers [17] , elderly persons [54] , and people living with hiv [55, 56] during the global public health crisis. however, our qualitative findings are in contrast to this finding. ashish (who was living with hiv) showed no added concern (or anxiety) due to the lockdown. one explanation for this could be that ashish was among those who adopt optimism and, in combination with constant precaution, show stronger resilience to adverse situations. findings from a previous study found that people living with hiv could develop resilience despite their physical and psychological challenges [57] . in fact, our study found that optimism and resilience coping were negatively related to anxiety and depressive symptoms. moreover, ashish enjoyed spending time indoors, which likely reduced any frustration related to not being able to enjoy regular life, in addition to the lower likelihood of contracting covid-19. we did not find quantitative evidence supporting the hypothesis that living in a state with a higher count of covid-19 cases predicted greater anxiety and depressive in indian adults. this is in contrast to previous research in australia which found that respondents living in areas with a high number of influenza cases were at much greater risk of stress than those living in uninfected areas [18] . however, our qualitative results supported our hypothesis. the lack of evidence in our quantitative findings could be because of the operationalization of the concept of area. our study operationalized area-level risk at the state level. it is possible that anxiety was higher among people living in a neighborhood (and not the state) with higher number of covid-19 cases. also, in addition to just the count of covid-19 in the neighborhood (or state) including the infection fatality rate in the operationalization could have given a reliable estimate of the influence of place. our quantitative findings suggest that a past history of depression or loneliness could increase anxiety and depressive symptoms during the lockdown. our qualitative findings corroborate this. tulika and salma's narratives suggest that stress during lockdown could revive past trauma. previous study findings also support this interpretation [58] . our qualitative and quantitative findings also suggested that increased depressive symptoms in this group could also increase their internet consumption leading to internet addiction during the lockdown. depressed individuals could use the internet as way to cope with their negative psychological state during the lockdown, which risks addiction during a restrictive state such as a lockdown and could affect their quality of life even after the lockdown. anxiety and depressive symptoms during lockdown were found to predict disruptions in sleep and food schedules, corroborating findings from a previous study [59] . qualitative data found that an increase in anxiety and a lack of motivation to lead a routine life increased the risk of an unbalanced sleep cycle, which also impacted food consumption and its timings. additionally, the inaccessibility to quality food due to the restrictions in physical mobility during the lockdown could affect the balanced diet among indian adults. beyond any immediate health effects which could further worsen mental health, such prolonged changes in timing and consumption of food could impact their overall food eating patterns even after the lockdown, resulting in poorer physical and mental health in the longer term as well. our quantitative findings suggest that sharing about stress with loved ones and an increase in frequency of interacting with family members likely strengthened social bonds and also increased social empathy among indian adults. our qualitative findings elucidate this. tulika's narrative highlighted that the universal vulnerability due to the global pandemic and her sharing about it with others in a similar situation improved her connectedness with the people thereby strengthening her social relationships. similarly, interacting and knowing the vulnerability of rajan's parents (more vulnerable) made him more empathetic, and increase his connection with the society at large. this fits with the findings from a previous study that highlighted this argument-sharing and expressing emotions (and vulnerabilities) could make people more empathetic [60] . such increased social empathy could also be a positive response to the pandemic (and lockdown). for instance, a recent study from the west found that higher empathy towards the more vulnerable could induce motivation to maintain and promote social distancing [61] . there are several limitations of this study that need to be noted while interpreting the results. we used convenience sampling in our study that limits the generalizability of the findings. generalizability is also limited due to our use of an english language questionnaire, a small sample size, and choosing the online mode of administering it. however, despite the modest sample size and the sampling design, we were able to show several interesting findings with statistical confidence. we were also constrained in our ability to seek non-english speakers and administer the questionnaires using hard copies due the lockdown situation. notably, our use of a qualitative strand strengthened the interpretation of the quantitative findings by allowing us to unpack several complex processes. furthermore, we speculate that the associations under study are stronger among those sub-groups who have a limited access to the internet that plays a crucial role in ensuring social connectedness during the lockdown. we used psychological scales to measure anxiety, depressive symptoms, internet addiction, and compulsive consumption of pornography, instead of clinical interviews which would have yielded medical diagnoses. however, our use of widely cited, reliable scales are informative and could indicate symptoms of the psychological outcomes we explore. additionally, due to scarcity of available validated scales, we could not use scales that were validated for use in the indian population. however, we have used scales that have been frequently administered in the indian context earlier. we also found high internal consistency of the scales in our study suggesting their reliability. another limitation of our quantitative study is the use of several shorter and single-item scales (such as the brief resilience coping scale). longer scales could have yielded robust results. because our pretest suggested that the length of the questionnaire was perceived as "a lot" we chose to use shorter scales. for instance, we used a single item to measure self-reported change in food consumption patterns and used two items to measure self-reports of sleep disturbances. however, the items used in our study captured the perception of the participants about the change in their food and sleep cycles during the lockdown, which adds an informative nuance. additionally, our qualitative data around these measures add more details and increase our confidence in interpreting these findings. lastly, the cross-sectional nature of the study limits our ability to make causal claims. longitudinal studies with frequent follow-ups during the lockdown could have shed light on causal processes. however, we were grateful to be able to recruit a diverse sample for our quantitative and qualitative strands, which allowed us to explore the differences in the psychological outcomes during the lockdown across different groups in india. despite these limitations, our mixed methods findings highlight the additional psychological burden that the lockdown has brought to an invisible group, the lgbt adults. to our knowledge this is the first study to look at the differential psychological impact of the lockdown across different social groups (including sexual orientation) in india. moreover, our use of qualitative narratives allowed us to understand the processes linking several social factors to the psychological outcomes in a nuanced manner. our study also highlights a few positive aspects of the lockdown, underscoring the increase in social empathy and strengthened social bonds among indian adults. our findings echo balagos' argument that the marginalization of lgbt adults would be heightened during disasters, because existing inequalities are magnified at such times [62] . while the indian supreme court decriminalized homosexual acts in 2018, indian policies are not yet inclusive of lgbt adults, who remain socially invisible. our findings call for the attention of counsellors and health professionals in understanding the specific psychological needs of the lgbt adults during such crises and providing services accordingly. this study highlights the need for regular interaction and emotional support from friends, family, partners, and caregivers of lgbt adults, individuals with a history of depression or loneliness, a higher risk of developing complications if they contract covid-19. a recent study [63] highlighted the promise of delivering psychological support through online-and telecounselling. this study warrants the use of such technologies in an inclusive manner. the study also opens avenues for researchers to further investigate the extent and nature of the psychological impact in such marginalized groups during crises or disasters. lastly, our study findings provide evidence for mental health policymakers to begin designing inclusive policies to address the concerns of marginalized groups during and in the aftermath of the covid-19 global crisis. all in all, our study highlights the differential psychological effect of the covid-19 pandemic among lgbt adults, groups with history of depression, and those with high-risk of covid-19 complications. the study thereby urgently calls for the attention of policymakers to take sensitive and inclusive health decisions for the marginalized and the vulnerable, both during and after the crisis. supporting information s1 file. covid-19 data. 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meta-analysis gay, lesbian, and bisexual youth and young adults: social support in their own words too bored to bother? boredom as a potential threat to the efficacy of pandemic containment measures task duration and task order do not matter: no effect on self-control performance coronavirus drives some millennials home to their parents-vox the influence of family environment factors on self-acceptance and emotional adjustment among gay, lesbian, and bisexual adolescents gay, lesbian, and bisexual youths coming out to their parents: parental reactions and youths' outcomes cardiovascular disease and covid-19 physical distancing in covid-19 may exacerbate experiences of social isolation among people living with hiv notes from the field symptoms, stress, and hiv-related care among older people living with hiv during the covid-19 pandemic motivation, management, and mastery risk for recurrence in depression sleep and anxiety disorders predictors of empathy in women social workers the emotional path to action: empathy promotes physical distancing during the covid-19 pandemic the warias of indonesia in disaster risk reduction: the case of the 2010 mt merapi eruption in indonesia the need for a mental health technology revolution in the covid-19 pandemic ajs and mas thank nilesh thube (nvt) for his diligent contribution in data management and representation of results. ajs conveys special thanks to dipankar dutta for being extremely patient during the work. ajs and mas thank harvansh dandelia, rakshit verma, and several other friends and colleagues who helped in the circulation of the online survey in a short time. ajs thanks his bula da. ajs is grateful to all the participants for filling up the form and sharing their emotions during this crisis. conceptualization: anupam joya sharma, malavika a. subramanyam.data curation: anupam joya sharma. key: cord-318008-4s9eoae3 authors: parsons leigh, jeanna; fiest, kirsten; brundin-mather, rebecca; plotnikoff, kara; soo, andrea; sypes, emma e.; whalen-browne, liam; ahmed, sofia b.; burns, karen e. a.; fox-robichaud, alison; kupsch, shelly; longmore, shelly; murthy, srinivas; niven, daniel j.; rochwerg, bram; stelfox, henry t. title: a national cross-sectional survey of public perceptions of the covid-19 pandemic: self-reported beliefs, knowledge, and behaviors date: 2020-10-23 journal: plos one doi: 10.1371/journal.pone.0241259 sha: doc_id: 318008 cord_uid: 4s9eoae3 introduction: efforts to mitigate the global spread of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) causing corona virus disease-19 (covid-19) have largely relied on broad compliance with public health recommendations yet navigating the high volume of evolving information can be challenging. we assessed self-reported public perceptions related to covid-19 including, beliefs (e.g., severity, concerns, health), knowledge (e.g., transmission, information sources), and behaviors (e.g., physical distancing) to understand perspectives in canada and to inform future public health initiatives. methods: we administered a national online survey aiming to obtain responses from 2000 adults in canada. respondent sampling was stratified by age, sex, and region. we used descriptive statistics to summarize responses and tested for regional differences using chi-squared tests, followed by weighted logistic regression. results: we collected 1,996 eligible questionnaires between april 26(th) and may 1(st), 2020. one-fifth (20%) of respondents knew someone diagnosed with covid-19, but few had tested positive themselves (0.6%). negative impacts of pandemic conditions were evidenced in several areas, including concerns about healthcare (e.g. sufficient equipment, 52%), pandemic stress (45%), and worsening social (49%) and mental/emotional (39%) health. most respondents (88%) felt they had good to excellent knowledge of virus transmission, and predominantly accessed (74%) and trusted (60%) canadian news television, newspapers/magazines, or non-government news websites for covid-19 information. we found high compliance with distancing measures (80% reported self-isolating or always physical distancing). we identified associations between region and self-reported beliefs, knowledge, and behaviors related to covid-19. discussion: we found that information about covid-19 is largely acquired through domestic news sources, which may explain high self-reported compliance with prevention measures. the results highlight the broader impact of a pandemic on the general public’s overall health and wellbeing, outside of personal infection. the study findings should be used to inform public health communications during covid-19 and future pandemics. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 since the emergence of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in december 2019 [1] , the global public has been inundated with information related to the rapidly evolving corona virus disease 2019 (covid-19) pandemic [2] . organizations such as the world health organization (who) [3] , worldwide public health networks [4] , and government public health agencies [5] have used multiple media platforms (e.g., internet, television, radio, print) in attempts to keep the public informed of emerging details and public health recommendations. in canada, this messaging has included mitigation strategies such as appropriate hand and face hygiene practices [5] , physical distancing policies including closing non-essential business and public spaces [5] , restrictions and limitations on visitation in hospitals and long-term care facilities [6] , and travel restrictions [5] . effective and transparent communication of evolving information related to covid-19 is needed to ensure the public understands how and why to adapt their behaviors to bolster public safety [7] . however, the influx of covid-19 information and widespread circulation and exchange of misinformation (i.e., false or inaccurate information) [8, 9] have been linked to increased public fear [10] , under-use of health services [11] , and distrust in government messaging [12] -a phenomenon the who has characterized as an 'infodemic' (a term originally coined in 2003 by david rothkopf in the washington post during sars), to describe when the proliferation of information about a problem detracts from possible solutions) [9] . effective pandemic management is dependent on understanding public views and behaviors, including concerns, frequently used and trusted sources of information, and reasons to observe or violate public health mandates [7, 13] . countries around the world have used online crosssectional surveys to rapidly assess public awareness, understand health behaviors, and identify sources of information and misinformation during covid-19 [13] [14] [15] [16] [17] . a survey of the american and british public very early in the pandemic indicated adequate public awareness of disease transmission but a lack of understanding of appropriate preventative measures as well as high uptake of common misconceptions which were circulating on social media [18] . evidence collected from public surveys [14, [16] [17] [18] [19] may inform the development of targeted public health messaging and track uptake of new information [20] ; however, to date no comprehensive canadian-based surveys investigating public perspectives related to covid-19 and potential variations by geographic region have been published. this may be particularly important given the variation across provinces and territories in covid-19 case burden and local government response [21] . we conducted a national survey of adults residing in canada to gain a better understanding of public perceptions in several important domains-beliefs (e.g. severity of pandemic, concerns, impact on health), knowledge acquisition (e.g. sources, topics), and behaviors (e.g. isolation and physical distancing)-related to the covid-19 pandemic. this benchmarking data will help inform future public health messaging and initiatives. we developed a cross-sectional, online, anonymous survey and contracted ipsos incorporated (https://www.ipsos.com/en-ca), a world-wide market research and polling firm, to administer it across canada. we first iteratively synthesized a comprehensive list of questions based on broad content areas reported in previously published survey research on pandemics [22] [23] [24] [25] and in current covid-19 public opinion polls [15] . we subsequently invited seven members of the research team (co-investigators, research assistants, and patient partners) to provide feedback on question format, comprehensiveness, clarity, and flow [26] . we refined the questionnaire based on feedback. the questionnaire domains and sub-domains are illustrated in s1 fig. question types included 5-point unipolar scales (e.g., 1 = not at all/poor, 5 = extremely/excellent), 7-point bipolar agreement scales (1 = strongly disagree to 7 = strongly agree), single-response multiple choice, and multiple response multiple choice. we randomized the order of the response options to reduce response selection bias [26] . we compared respondents' retrospective ratings of five domains of overall health (mental/emotional, physical, social, economic, spiritual) at the start of 2020 to ratings of their health status at the time of data collection, with differences categorized into 'worse', 'same', or 'better'. we provided respondents with definitions for self-isolation and social/physical distancing. self-isolation was defined as "separating yourself from others, including those within your home, with the purpose of preventing the spread of the virus (whether diagnosed or undiagnosed, with or without symptoms" and social/physical distancing defined as "limiting your time in spaces occupied by others, including reducing trips to visit others in person and reducing time spent in public spaces." to ascertain whether the questionnaire could be completed within 15-minutes, we piloted it with a sample of 104 canadian residents. no changes to the questionnaire were made and we therefore included the pilot responses in the final data set. the questionnaire was optimized for 'device agnosticism' to ensure its compatibility across most systems (e.g., mobile phone, computer, tablet). the final questionnaire (see s1 appendix) was formatted in english and french and consisted of 21 demographic and 46 covid-19-related questions covering three overarching domains of self-reported perceptions: beliefs, knowledge acquisition, and behaviors. dalhousie university (#2020-5121) and university of calgary (#20-0538) research ethics boards approved this study. prior to entering the questionnaire, respondents reviewed an informed consent page; consent was implied by completing the questionnaire. the questionnaire was distributed electronically through ipsos' proprietary isay panel of approximately 250,000 canadians using direct email and social media posts. panelists were eligible to complete the survey if they were adults (�18 years), lived in canada, and were able to read english or french. we screened respondents by age (18-34, 35-55, >55) , sex at birth (female/male), and provincially defined regions (british columbia, alberta, saskatchewan/ manitoba, ontario, québec, and atlantic provinces) to ensure population representation based on 2016 census data [27] . respondents received ipsos reward points after completing the questionnaire; points are accumulated and redeemed for gift cards and merchandise. we derived a minimum sample size estimate of 385 based on a normal approximation to the binomial distribution with a finite population correction applied [28] (assuming an observed proportion of respondents selecting a specific response option of 50%) that incorporated population size (~36.3 million in canada), a 95% confidence level and a margin of error of 5%. we elected to collect 2,000 questionnaires to allow for regional subgroup analyses and calculated the associated margin of error to be +/-2.2% at a 95% confidence level. we used descriptive statistics (frequencies (percent) or means (standard deviation)) to summarize respondent characteristics. we weighted responses by age, sex, and regional population estimates derived from 2016 census data [27] . likert scales were reported as frequencies with percent for each point on the scale. we tested for overall differences between regions using weighted chisquared tests. if p was less than 0.05, we followed with post-hoc comparisons using weighted logistic region to quantify differences between regions with odds ratios (or) using ontario as the comparison group. we conducted all quantitative data analyses using spss, version 23 and r, version 3.5.1 [29] . we used the r package "survey" version 3.36 [30] to obtain weighted descriptive statistics, chi-squared tests, and or estimates. statistical significance was set at α = 0.05. we collected data from april 26 th to may 1 st , 2020. we excluded four respondents who reported being unaware of the current covid-19 pandemic, resulting in a final sample of 1,996 respondents. on the last date data was collected (may 1 st ) there were 56,158 confirmed cases of covid-19 in canada; 83% of the cases were in the two most populated provinces, québec (51%) and ontario (32%). of the 1,996 respondents, 135 respondents (6.8%, 95% confidence interval (ci) 5.7%-7.9%) reported that they currently or previously had an illness that they believed was covid-19. only 12 (0.6%, 95%ci 0.3%-0.9%) of these reported ever testing positive for covid-19, 41 (2.1%, 95% ci 1.4%-2.7%) tested negative, and 82 (4.1%, 95% ci 3.2%-5.0%) were not tested. most (n = 1,858, 93.2%, 95%ci 92.1%-94.3%) were either uncertain (n = 96, 4.8%, 95% ci 3.9%-5.8%) or believed they had not contracted covid-19 (n = 1762, 88.4%, 95%. ci 87.0%-89.8%); one-fifth of all respondents (n = 404, 20.3%, 95% ci 18.5%-22.0%) reported personally knowing someone diagnosed with covid-19. our survey sample is proportionally similar to the canadian population [27] in terms of sex (female, 54vs51), age distribution (18-29 years, 15vs19; 30-44 years, 25vs24; 45-64 years, 32vs35), marital status (single, 25vs24; married/living together, 58vs58; separated/divorced/widowed, 14vs14), college or university educated (56vs54), and housing (detached dwelling, 55vs54). the percentage of respondents 65 years and older was somewhat higher in our sample (28%) than reported in the national census (21%). just over one-half (n = 563, 50.1%) of the 1001 employed respondents in our survey were working in a job deemed essential and 14 percent (n = 143) of unemployed respondents (n = 995) reported their unemployment being a direct result of covid-19. respondent characteristics are summarized in table 1 as unweighted results. a majority (n = 1,236, 62.1%, 95%ci 59.9%-64.2%) of respondents perceived covid-19 to be a very serious problem in canada though only a small proportion (n = 268, 13.5%, 95% ci 11.9%-15.0%) rated it to be slightly more or much serious than in other countries ( table a in s2 appendix). more respondents were moderately or extremely concerned about a family member contracting covid-19 (n = 889, 45.3%, 95%ci 43.0%-47.5%) than were concerned about themselves contracting the disease (568/1,885 reported not believing they had contracted covid-19, 30.1%, 95%ci 28.1%-32.2%) (fig 1) . in rating concerns about the impacts of covid-19 on the health system , a greater proportion of respondents were moderately or extremely concerned that there would be insufficient personal protective equipment (ppe) for hospital staff to stay safe (n = 1,024, 51.7%, 95%ci 49.5%-53.9%) compared to concerns about access to healthcare and availability of equipment to care for covid-19 patients (fig 1) . just under half (n = 898, 45.2%, 95%ci 43.0%-47.4%) of respondents agreed or strongly agreed that the pandemic was stressful; however, fewer (n = 566, 28.5%, 95%ci 26.5%-30.5%) agreed or strongly agreed that it was something that made them feel helpless (fig a in s2 appendix). when asked to sequentially rate their past (start of 2020) and present health (physical, mental/emotional, social, economic, spiritual), respondents expressed experiencing declines in all dimensions of health with the largest decreases reported for social health (n = 964, 48.5%, 95%ci 46.3%-50.7%) and mental/emotional health (n = 778, 39.1%, 95%ci 36.9%-41.2%) (fig 2) . the majority of respondents (n = 1,741, 87.5%, 95%ci 86.1%-89.0%) rated their understanding of how the virus was spread as good (n = 629, 31.6%, 95%ci 29.5%-33.7%), very good (n = 793, 39.9%, 95%ci 37.7%-42.0%), or excellent (n = 319, 16.1%, 95%ci 14.4%-17.7%). fig 3 shows respondents' level of agreement to a series of statements about the transmission of the virus that causes covid-19. the highest consensus among respondents was in agreeing or strongly agreeing that people can be infected with covid-19 and not show any symptoms (n = 1,713, 86.5%, 95%ci 84.9%-88.0%). there was greater variability across respondents in their degree of agreement to other knowledge-based statements (fig 3) . when respondents were asked how often they search for information about covid-19, over half (n = 1,345, 67.9%, 95%ci 65.8%-69.9%) reported searching once per day or more and predominantly accessing and trusting canadian over american or other international sources for their information. the top accessed source was canadian news-based television, print, or websites (n = 1,488, 75.6%, 95% ci 73.6%-77.5%) (fig 4) . the lowest rated sources for covid-19 information included social media posts from influencers or celebrities (n = 1,039, 54.8% selected as least trusted, 95% ci 52.5%-57.1%) and american news television, print, and websites (n = 711, 50.4% selected as source of misinformation, 95% ci 47.7%-53.0%). consistent with valuing canadian sources, respondents most frequently reported going directly to government or health authority sources (n = 979, 50.6%, 95% ci 48.4%-52.8%) to verify information (fig b in s2 appendix) . half of respondents surveyed (n = 1,017, 51.3%, 95% ci 49.1%-53.5%) agreed or strongly agreed that they were able to find the kind of information they want about covid-19 ( fig c in s2 appendix) . information about covid-19 infection rates dominated respondent's searches (n = 1,414, 71.5%, 95% ci 69.5%-73.5%) (fig d in s2 appendix) , while information about vaccines and treatments were most frequently (n = 933, 48.9%, 95% ci 46.7%-51.2%) cited as topics of misinformation (fig d in s2 appendix) from those who reported having seen or heard incorrect or misleading information related to covid-19 during the previous two weeks (n = 1,520, 75.3%, 95% ci 73.4%-77.3%). yet, only half (n = 937, 47.4%, 95% ci 45.2%-49.6%) of respondents felt moderately or extremely confident that they could identify incorrect or misleading information about covid-19 (fig e in s2 appendix) , and comparable numbers reported being uncertain (n = 455, 23.0%, 95% ci 21.2%-24.9%) or agreeing (n = 634, 32.1%, 95% ci 30.0%-34.2%) that they find it hard to determine if an information source was trustworthy or not (fig c in s2 appendix) . https://doi.org/10.1371/journal.pone.0241259.g001 just under half of respondents indicated they were in self-isolation (n = 842, 43.4%, 95% ci 41.2%-45.6%). of those who were not self-isolating (n = 1,144), the vast majority (n = 1,083, 95.1%, 95% ci 93.8%-96.4%) reported that they practiced physical distancing always (n = 783, 68.8%, (95% ci 66.0%-71.5%) or often (n = 300, 26.3%, 95% ci 23.7%-28.9%). furthermore, many (n = 814, 41.0%, 95% ci 38.9%-43.2%) respondents felt that they could reasonably sustain their current level of physical distancing longer than six months (or as long as needed) (fig 5) . self-reported distancing behaviors were consistent with respondent perceptions of 'self' as effective agents to prevent the spread of the virus, with most (n = 1,380, 69.7%, 95% ci 67.7%-71.8%) agreeing or strongly agreeing that they were doing a good job at preventing the spread of the virus with changes to their behavior; about one-third (n = 677, 34.9%, 95% ci 32.7%-37.0%) agreed or strongly agreed that they were doing a better job than other people (fig c in s2 appendix) . respondents (mean age of 50) most commonly perceived teenagers as least consistently practicing physical distancing (n = 855, 43.2%, 95% ci 41.0%-45.4%) while identifying middle-aged adults (n = 786, 39.6%, 95%ci 37.4%-41.8%) and seniors (n = 744, 37.5%, 95%ci 35.4%-39.6%) as most consistently practicing physical distancing (fig f in s2 appendix) . the most frequently selected reasons (fig 6) for self-isolating or physical distancing were to protect oneself (n = 1,602, 81.0%, 95% ci 79.2%-82.7%), to protect other people in one's household (n = 970, 49.1%, 95% ci 46.8%-51.3%) and to protect other members of the general public (n = 962, 48.6%, 95% ci 46.4%-50.8%). three-quarters (n = 1,436, 75.8%, 95% ci 73.9%-77.8%) of respondents reported that they would get vaccinated for the virus when a vaccine became available. regional differences. overall analyses based on the chi-squared tests revealed several associations between the region in which respondents resided and their self-reported perceptions (see table a in s3 appendix). post-hoc multiple comparisons presented as odds ratios are in table b in s3 appendix. in comparison to respondents in ontario, respondents in all other regions were less likely to believe that covid-19 was a very serious problem in canada (0.50 to 0.74 times as likely to report covid-19 was a very serious problem) and were more likely to be "not at all" concerned about the impact of covid-19 on hospitals (e.g. 1.86 to 2.50 times as likely to report being "not at all" concerned at lack of ppe) and patients (e.g. limited access to necessary services, 1.72 to 2.84 times as likely to report being "not at all" concerned). a complete lack of concern about themselves or a family member contracting the virus also was more likely to be expressed by respondents in regions outside of ontario, compared to those in ontario. this included respondents in québec in spite of being more likely to have close friends who tested positive for covid-19 (or 2.39, 95% ci 1.39-4.11) and having the highest rate of confirmed covid-19 cases at the time. respondents in british columbia, québec, and manitoba/saskatchewan were less stressed by the pandemic than were respondents in ontario, although québec respondents also were more likely to strongly agree that the pandemic made them feel helpless (or 2.38, 95%ci 1.71-3.32). there were no statistically significant associations between region and self-reported ratings of physical, mental/emotional, social, or economic health. respondents in québec felt least knowledgeable about how the virus is spread, reporting "fair" more often than respondents in ontario (or 1.53, 95%ci 1.08-2.18), while respondents in manitoba/saskatchewan were more likely to report "very good" understanding than did respondents in ontario (or 1.65, 95% ci 1.13-2.40) but less likely to report "excellent" (or 0.37, 95% ci 0.19-0.73). respondents in alberta and the atlantic provinces were less likely to access canadian news than respondents from ontario, and along with respondents from québec, were less likely to access american news. respondents from all regions but british columbia were less likely to access international sources than respondents from ontario. respondents in québec were less likely to agree (15%) or strongly agree (36.9%) that they would get vaccinated compared to those in ontario (or 0.71, 95% 0.52-0.98 and or 0.68, 95% ci 0.53-0.87, respectively) while respondents from alberta were more likely to strongly disagree (9.1%) that they will get vaccinated (or 1.91, 95% 1.08-3.40). with changes to their behavior, respondents in quebec are more likely to strongly agree that they are doing a good job at preventing the spread of covid-19 compared to respondents from quebec (or 1.31, 95% ci 1.02-1.69). the covid-19 pandemic has substantially altered many aspects of public life, yet little is known about the perspectives and experiences of broader populations. our study provides a national cross-sectional description of public perceptions, knowledge and behaviors related to covid-19 in the context of the evolving pandemic, adding to survey data published early in the outbreak [16] [17] [18] . our data suggest that canadians are concerned about the threat of covid-19 to the healthcare system, to themselves and their family members, and that they consider the ongoing pandemic a serious problem on both national and international levels. there are three main findings of the survey: (1) the negative impact of the pandemic on canadians' perceptions of their health, (2) the frequent searching for up-to-date information about covid-19 (largely via canadian based sources), and (3) current and future perceived desire and ability of the public to comply with public health recommendations (e.g. physical distancing, vaccination for covid-19 if/when available). to our knowledge, this is the first national survey in canada to comprehensively assess multiple domains of public perceptions important to understanding the public's response to the ongoing pandemic. we found that overall health has been markedly impacted by pandemic conditions, and that this is largely irrespective of personal infection with covid-19. in fact, very few of our respondents reported ever testing positive for covid-19, yet many perceived that aspects of their overall health had deteriorated, particularly mental/emotional and social health. this is further evidenced in high agreement among our respondents that the pandemic is stressful, which may be unassociated with case burden. although respondents in ontario, a province with a high number of confirmed covid-19 cases, were more likely to report stress than those in other provinces, respondents in québec, also a province with high cases, did not. the need to assess and respond to health impacts beyond infection with sars-cov-2 has been increasingly recognized as a critical part of pandemic response [31] [32] [33] [34] [35] [36] [37] . for example, dramatic shifts in routines, livelihoods and behaviors during quarantine, coupled with the unfulfilled basic need for human connection [33] , have been described as significant threats to mental health and well-being. in addition, findings from surveys commissioned by the uk academy of medical sciences (ams) and the charity mq: transforming mental health through research reported widespread public concern about isolation, loneliness, practical aspects of life (e.g. finances), and general negative feelings, and provided groundwork for the collaborative development of sweeping research priorities to improve these conditions [34] . in our survey, fewer respondents reported that the pandemic makes them feel helpless, suggesting some resiliency to the detrimental circumstances the pandemic has produced. the media's role in disseminating information that will concurrently educate and motivate public behaviors in accordance with recommended guidance and avoid creating undue stress, skepticism, or rebuff of guidelines is a critical factor in navigating pandemic response [32, 34, 38] . our study found that the public frequently searches for information about covid-19 and is primarily getting information from domestic news sources, including television, print, and websites that are not government or public health agency websites. respondents in our study also view news sources as equally credible to national government and public health websites. this finding suggests that public health officials should view mainstream media, and in particular television, as important promoters or messengers of covid-19-related information. given this, it is crucial for mainstream media to take this responsibility seriously to ensure accurate information is conveyed. at the same time, perceptions of trust may be moderated by other factors not accounted for in this survey, such as perceived congruence between government guidelines and impact reducing virus spread. in our survey, respondents from ontario and québec reported the least amount of trust in canadian government and news sources and these were also the same provinces with the highest number of confirmed covid-19 cases in canada (32% and 51%, respectively). much attention has been paid to the proliferation of information about covid-19, raising concerns about parallel increases in misinformation. we found that a substantial proportion of respondents value science-based sources (e.g. government websites) which may explain high rates of self-reported behavior change to prevent virus spread. this correlates with other public opinion data [39] ; however, about half of our respondents still expressed only moderate levels of confidence in being able to identify misleading information (fig e in s2 appendix) or determine if an information source is trustworthy (fig c in s2 appendix) . of note, many respondents indicated that they do not view american news sources as trustworthy, and more specifically, see it as a source of misinformation. familiarity with and interest in context-specific information may influence respondents' perceptions of credibility. social media posts were also commonly identified as untrustworthy, however, these perceptions ranged depending on who was sharing the information. posts from family and friends or influencers were viewed as less trustworthy than posts from government or public health agencies. as a quickresponse platform with open posting and limited moderation, misinformation is easily spread on social media [39] [40] [41] . while some social media platforms (e.g. facebook, twitter and instagram) have increased efforts to monitor and remove incorrect or harmful information related to covid-19 in an attempt to reduce public consumption of misinformation, the effectiveness of these efforts is currently unknown [42] . we found that our respondents most frequently fact checked their information using government and public health websites (51%) and scientific articles (30%), but greater efforts to better understand how individuals may proactively limit their exposure to misinformation, identify misinformation, and fact check information are needed. the vast majority of respondents in our study reported practicing a high level of physical distancing, and a surprisingly high number felt that they could maintain this for a long period of time (6 months or more) if necessary. this finding is somewhat unexpected given the high level of reported self-isolation amongst our respondents. although it may be that not all respondents clearly understood the difference between self-isolation and physical distancing, it is evident that most were motivated to limit social and physical interactions as a means to protect themselves and others from becoming infected with covid-19. the lower than predicted infection rates in many countries has been credited largely to the high public compliance of mandated preventative measures. however, this comes at a price, including significant global economic losses [43] . in our survey, 14% of respondents reported unemployment as a result of the pandemic, and 34% of all respondents reported worse economic health. in contrast to respondents' positive association to physical distancing recommendations, we report slightly lower numbers of respondents who intend to receive a covid-19 vaccine once available as compared to other recent surveys [44] . while this is another somewhat unanticipated finding given the reported propensity of respondents to access and trust sources considered 'reputable' (e.g., public health agencies), individual and social determinants of vaccination are wide-ranging [45] [46] [47] [48] . previous research has highlighted that the media can both hinder [49, 50] and enhance [50] vaccination uptake. to optimize potential future vaccine uptake, public health agencies should align key messaging with public perceptions, concerns, and information needs (e.g. preferred sources) [51, 52] , tailoring by jurisdiction. for example, in our study, respondents from the province with the highest number of covid-19 cases (québec) were significantly less likely to report that they plan to get vaccinated (table b in s3 appendix) and reported the least amount of trust in canadian government and news sources. such complexities must be taken seriously if we are to ensure that public health recommendations are effectively communicated. our survey has limitations. although providing a broad snapshot of population, cross-sectional surveys capture relevant data only at a single moment in time on specific topics. in a rapidly changing landscape, it is expected that self-reported perceptions and behaviors would change with new information. the use of serial surveys [13, 14, 44] is one strategy to strengthen cross-sectional survey designs. at the same time, our study provides useful descriptive data at a pandemic peak in canada. subsequent qualitative methodologies will further enrich our understanding of public actions and reactions to the covid-19 pandemic. second, as we elected to set a survey response quota, we are not able to determine a response rate. while there is a risk of non-response bias, the rapid collection of responses to reach our 2,000 quota (five days) and methodological strengths in our design (rigorous development including pre-testing and device agnosticism, large sample size, population representation and weighting by age, sex at birth, and region) outweigh this limitation. third, differences in public perceptions that may be associated with socio-demographic factors such as age and gender were not addressed in this manuscript but will be the focus of future investigation. finally, though overall results may be affected by larger numbers of respondents from canada's two largest provinces (ontario and québec), the weighting ensures results accurately reflect the actual regional populations within canada. at the same time, regional differences should be cautiously interpreted as we did not adjust for multiple comparisons. we conducted a national survey including a representative sample of the canadian public to assess overall perceptions, knowledge, and behaviors related to the covid-19 pandemic. our results highlight the impact of the pandemic on individual perceptions of health which may be further exacerbated by salient concerns around risks of infection, healthcare safety, and access. we found that knowledge about covid-19 is largely acquired through domestic news sources, which may explain high self-reported compliance 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on-line comments during the 2009 h1n1 pandemic communicating during a pandemic: information the public wants about the disease and new vaccines and drugs key: cord-321438-llnqzkqt authors: ma, ruili; zhang, yanming; liu, haiquan; ning, pengbo title: proteome profile of swine testicular cells infected with porcine transmissible gastroenteritis coronavirus date: 2014-10-21 journal: plos one doi: 10.1371/journal.pone.0110647 sha: doc_id: 321438 cord_uid: llnqzkqt the interactions occurring between a virus and a host cell during a viral infection are complex. the purpose of this paper was to analyze altered cellular protein levels in porcine transmissible gastroenteritis coronavirus (tgev)-infected swine testicular (st) cells in order to determine potential virus-host interactions. a proteomic approach using isobaric tags for relative and absolute quantitation (itraq)-coupled two-dimensional liquid chromatography-tandem mass spectrometry identification was conducted on the tgev-infected st cells. the results showed that the 4-plex itraq-based quantitative approach identified 4,112 proteins, 146 of which showed significant changes in expression 48 h after infection. at 64 h post infection, 219 of these proteins showed significant change, further indicating that a larger number of proteomic changes appear to occur during the later stages of infection. gene ontology analysis of the altered proteins showed enrichment in multiple biological processes, including cell adhesion, response to stress, generation of precursor metabolites and energy, cell motility, protein complex assembly, growth, developmental maturation, immune system process, extracellular matrix organization, locomotion, cell-cell signaling, neurological system process, and cell junction organization. changes in the expression levels of transforming growth factor beta 1 (tgf-β1), caspase-8, and heat shock protein 90 alpha (hsp90α) were also verified by western blot analysis. to our knowledge, this study is the first time the response profile of st host cells following tgev infection has been analyzed using itraq technology, and our description of the late proteomic changes that are occurring after the time of vigorous viral production are novel. therefore, this study provides a solid foundation for further investigation, and will likely help us to better understand the mechanisms of tgev infection and pathogenesis. porcine transmissible gastroenteritis coronavirus (tgev) is an animal coronavirus that causes severe gastroenteritis in young tgev-seronegative pigs. various breeds of pigs, regardless of age, are susceptible to tgev; however, the mortality rate for piglets under 2 weeks of age is the highest, reaching almost 100%. diseased pigs often present with vomiting, dehydration, and severe diarrhea. further, the disease is known to affect pigs in many countries throughout the world and an outbreak can cause enormous losses in the pig industry [1, 2] . the pathogen, tgev, which belongs to the alphacoronavirus genus of the coronavirinae subfamily within the family coronaviridae, is an enveloped, nonsegmented, single-stranded positive-sense rna virus [3, 4] . the envelop, core, and nucleocapsid of the tgev virion contain four major structural proteins: the nucleocapsid (n) protein, the membrane (m) glycoprotein, the small envelope (e) protein, and the spike (s) protein [5] . the tropism and pathogenicity of the virus are influenced by the s protein, which has four major antigenic sites, a, b, c, and d, with site a being the major inducer of antibody neutralization [3, 5] . the m protein, which plays a central role in virus assembly by interacting with viral ribonucleoprotein (rnp) and s glycoproteins [6] , is embedded within the virus membrane and interacts with the nucleocapsid, forming the core of tgev virion. in addition, the n-terminal domain of the m protein is essential for interferon alpha (ifn-a) induction [7] , which is involved in the host's innate immune response. the e protein, a transmembrane protein that acts as a minor structural component in tgev and affects virus morphogenesis, is essential for virion assembly and release [8] . tgev rna, along with the n protein, is infectious and invades the organism through the digestive and respiratory tracts, resulting in infection of the small intestinal enterocytes, villous atrophy, and severe watery diarrhea. these changes in intestinal health are known to be important during the pathogenesis of tgev infection [9] . furthermore, corresponding to these pathologic changes observed in vivo, tgev can also propagate and cause cytopathic effects (cpes) in multiple types of cultured cells, such as swine testicular (st) cells, pk-15 cells, and villous enterocytes. notably, st cells are more susceptible to tgev, and higher levels of virus replication have been observed in this cell line [10, 11] . the full rna genome of tgev is approximately 28.5 kb in length and has a 59-cap structure and a poly(a) tail at the 39 end. the 9 open reading frame (orf) genes included in the tgev genome are arranged in the following order 59-la-lb-s-3a-3b-e-m-n-7-39. the first gene at the 59 end consists of two large orfs, orf la and orf lb, which constitute the replicase gene, known for its rna-dependent rna-polymerase and helicase activities, as well as other enzymes, such as endoribonuclease, 39-59exoribonuclease, 29-o-ribose methyltransferase, ribose adp 1'' phosphatase, etc. [12] . orf2, orf4, orf5, and orf6 encode the s, e, m, and n proteins, respectively, while orf3a, orf3b, and orf7 encode non-structural proteins [13] . some investigators have suggested that orf3 may be related to viral virulence and pathogenesis [12] , while orf7 may interact with host cell proteins and play a role in tgev replication [14] . in fact, a recent study indicates that plasmid-transcribed small hairpin (sh) rnas targeting the orf7 gene of tgev is capable of inhibiting virus replication and expression of the viral target gene in st cells in vitro [15] . although we have some knowledge concerning the translation and function of these viral proteins, the interactions that occur between these proteins and host cell proteins are not fully understood. importantly, recent advances in proteomic technology have allowed for more in depth investigation of virus-host interactions, and different techniques have been successfully applied to identify altered proteins in infected host cells and tissues. for example, sun et al. [16] have identified 35 differentially expressed proteins in pk-15 cells infected with classical swine fever virus (csfv) using two-dimensional polyacrylamide gel electrophoresis (2d page) followed by matrix-assisted laser desorption-ionization time-offlight tandem mass spectrometry (maldi-tof-ms/ms). in addition, two-dimensional fluorescence difference gel electrophoresis (2d-dige) and ms/ms proteomic approaches have been applied to characterize protein changes occurring in host cells in response to porcine circovirus type 2 (pcv2) infection [17] . the same methods have also been studied for many other pathogenic animal viruses, including porcine reproductive and respiratory syndrome virus (prrsv) [18] , coronavirus infectious bronchitis virus (ibv) [19] , severe acute respiratory syndrome-associated coronavirus (sars-cov) [20] , and tgev [21] . however, these conventional approaches based on 2d gel electrophoresis are not suitable for detecting low abundance, hydrophobic, or very acidic/ basic proteins. on the other hand, the isobaric tags for relative and absolute quantitation (itraq) technique, in association with liquid chromatograph (lc), is a more advanced method for proteomic research, and is capable of detecting a much larger number of proteins, even those with low abundance, in addition to identifying and quantifying the proteins simultaneously [22] . to this end, lu et al. [23] previously used the itraq method to identify 160 significantly altered proteins in pulmonary alveolar macrophages (pams) infected with prrsv. similarly, this method has been used to investigate influenza virus infection in primary human macrophages [24] , human immunodeficiency virus 1 (hiv-1) infection in cd4 + t cells [25] , and epstein-barr virus (ebv) infection in nasopharyngeal carcinoma cell line [26] . here, we report the first differential proteomic analysis of tgev-infected and uninfected st cells using itraq labeling followed by 2d-lc-ms and bioinformatic analyses. the proteomic data obtained in this study will help to enhance our understanding of the host response to tgev infection, but also provide new insights on the mechanisms of disease onset. cell culture and viral replication st cells were obtained from the american type culture collection (atcc). the cells were cultured in high-glucose dulbecco's modified eagle's medium (dmem; gibco, uk) containing 1% l-glutamine and 10% fetal bovine serum (fbs) (hyclone, logan, ut) at 37uc in 5% co 2 . culture medium was replaced two to three times per week. the tgev th-98 strain was isolated from a suburb of harbin, heilongjiang province, china. the virus was propagated in st cells and preserved at 2 70uc in our laboratory. the monolayer of confluent st cells was dispersed with 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid (edta) and seeded in 6-cm cell culture flasks. after a 24 h incubation period, the culture medium was removed and the st cells were washed with phosphate buffered saline (pbs, ph 7.4). the cells were then infected with the tgev th-98 strain at a 50% tissue culture infectious dose (tcid 50 to determine the extent of tgev infection, conventional rt-pcr and qrt-pcr assays were performed to detect the viral n gene. monolayers of st cells were infected with tgev as described above. cells were collected from 24 to 80 hpi at 8 h intervals, and the total rna of the infected cells was extracted using trizol (invitrogen). rna samples were reverse-transcribed using primescript rt reagent kit (takara bio, dalian, china), according to the manufacturer's instructions. the rt reaction was incubated at 37uc for 15 min followed by 85uc for 5 s. a mixture of oligo dt primers and random 6 mers was used in the rt step. the cdna was stored at 220uc until further use. pcr was performed for the tgev n gene in a 25 ml reaction mixture containing 1 ml of the cdna, 0.5 ml of each forward (f) and reverse (r) primer, 12.5 ml of premix taq (takara bio, dalian, china), and 10.5 ml depc water, starting with a 5 min denaturation at 95 c followed by 32 cycles of 30 s denaturation at 95 c, 30 s annealing at 56 c, and 40 s extension at 72 c. a final extension step was carried out at 72 c for 10 min. rt-pcr products were resolved on a 15 g/l agarose gel. the following pcr primers were used in this study: tgev n (f, 59-gagc-agtgccaagcattaccc-39 and r, 59-gacttctat ct-ggtcgccatcttc-39) and b-actin (f, 59-gcaaggacctc-tacgccaa-39 and r, 59-ctggaaggtggacagcgag-39). the mrna expression level of the tgev n gene was quantified using a sybr green assay on a bio-rad iq5 real time pcr detection system as described previously [27] . we used the same primers listed above for qrt-pcr. reactions were carried out in 50 ml volumes containing 0.5 ml of 20 6 sybr green i, 2 ml of cdna template, 1 ml of each f and r primer, 25 ml of 2 6 pcr buffer, and 20.5 ml depc water. the cycling conditions were 94uc for 4 min, followed by 35 cycles of 94uc for 20 s, 60uc for 30 s, 72uc for 30 s, and then a final extension of 10 min at 72uc. the relative gene expression was determined with the 2 (2ddct) method [28] , and the tests were performed in triplicate. following st cell infection, cells were collected at 48 and 64 hpi by centrifugation at 3,000 rpm for 5 min at 4uc, washed twice with pbs, and 1 ml of itraq lysis solution (8 m urea, 1% (w/v) dithiothreitol (dtt)) containing protease inhibitor was added. then, the cells were put in an ice bath and broken up by sonication. the solution was then mixed for 30 min at 4uc. the soluble protein fraction was harvested by centrifugation at 40,000 6 g for 30 min at 4uc and the debris was discarded. the protein concentration was determined with the bradford protein assay (2-d quant kit, bestbio, china). a 100 mg aliquot of protein from each sample was reduced, alkylated, and trypsin-digested as described in the itraq protocol (ab sciex, american), followed by labeling with the 4-plex itraq reagents multiplex kit according to the manufacturer's instructions (ab sciex, american). two virus-free samples at 48 h and 64 h were labeled with itraq tags 114 and 115, while two tgev-infected samples at 48 h and 64 h were labeled with tags 116 and 117. the labeled digests were then pooled, dried using a vacuum freeze drier (christ rvc 2225, germany), and preserved at 220uc for later use. the combined peptide mixtures were separated by reversed phase high-performance liquid chromatography (hplc) (ekspert ultralc 100, ab sciex, usa) on a durashell-c18 reverse phase column (4.6 mm 6 250 mm, 5 mm 100 å , agela). the mobile phases used were composed of 20 mm ammonium formate (ph 10) in water (labeled mobile phase a) and 20 mm ammonium formate (ph 10) in acetonitrile(acn) (mobile phase b). the flow rate was 0.8 ml/min, and the elutant was collected into 48 centrifuge tubes at each minute after the first 5 min. each aliquot was then dried by vacuum freezing. the peptides were then analyzed with a nanoflow reversedphase liquid chromatography-tandem mass spectrometry (nano-rplc-ms/ms) system (tripletof 5600, ab sciex, usa). the above 48 tubes were merged into 10 components dissolved in 2% acn and 0.1% formic acid (fa), then centrifuged at 12,000 6 g for 10 min. the supernatant (8 ml) was used for loading at a rate of 2 ml/min, with a separation rate of 0.3 ml/min. the mobile phase a used in this analysis was composed of 2% acn and 0.2% fa, while mobile phase b was composed of 98% acn and 0.1% fa. the following ms parameters were utilized: source gas parameters (ion spray voltage: 2. protein identification and quantification were performed with the proteinpilot software (version 4.0, ab sciex) using the paragon algorithm. each ms/ms spectrum was searched against a database of sus scrofa protein sequences (ncbi nr, released in march 2011, downloaded from ftp://ftp.ncbi.nih.gov/genomes/ sus_scrofa/protein/). the following search parameters were used: itraq 4-plex (peptide labeled), cysteine alkylation with methyl methanethiosulfonate(mmts), trypsin digestion, biological modifications allowed, a thorough search, a detected protein threshold of 95% confidence (unused protscore $1.3), and a critical false discovery rate (fdr) of 1%. the peptide and protein selection criteria for relative quantitation were performed as described previously, whereby only peptides unique for a given protein were considered [29] . in addition, proteins with an itraq ratio higher than 20 or lower than 0.05 as well as proteins in reverse database were removed [30] . to assign enriched gene ontology (go) terms to the identified proteins, the differentially expressed proteins identified from itraq experiments and all of the 4,112 measured proteins were classified based on their go annotations using quickgo (http:// www.ebi.ac.uk/quickgo/), with uniprot id (http://www. uniprot.org/?tab=mapping) as the data source. go enrichment analysis of the differentially regulated proteins was evaluated using all of the 4,112 quantified proteins as background with hypergeometric distribution [31] . categories belonging to biological processes, molecular functions, and cellular components that were identified at a confidence level of 95% were included in the analysis. the protein-protein interaction network for a select group of proteins was analyzed using the string 9.1 database (http://string-db.org/). network analysis was set at medium confidence (string score .0.4). following st cell infection with tgev, the culture medium was removed after incubating for 48 h and 64 h; then, the cells were washed with cold pbs and collected after centrifugation at 3,000 rpm for 10 min. cells were then lysed in ripa lysis buffer with protease inhibitors (applygen technologies inc., china). cellular debris was removed by centrifugation at 12,000 6 g for 5 min at 4uc, and the protein concentration was measured by coomassie blue g250 staining. an equal amount (20 mg) of cell lysate from each sample was separated using 10% sds-page and then transferred to polyvinyl difluoride (pvdf) membranes (millipore, bedford, usa). the pvdf membranes were then blocked with 5% (w/v) de-fatted milk powder dissolved in tris buffered saline and tween 20 (tbst) buffer (150 mm nacl, 50 mm tris, 0.05% tween 20) for 1 h at 37uc. after blocking, membranes were incubated with anti-glyceraldehyde 3-phosphate dehydrogenase (gapdh) mouse monoclonal antibody (1:3000; western biotechnology, china), anti-heat shock protein 90 alpha (hsp90a/hsp90aa1) antibody (1:300; abcam, cambridge, uk), anti-caspase 8 antibody (1:300; abcam, cambridge, uk), or antitransforming growth factor b 1 (tgf-b1/tgfb1) antibody (1:300; abcam, cambridge, uk) overnight at 4uc, followed by hrp-conjugated secondary antibody (1:5000; western biotechnology, china) for 1.5 h at 37uc. the membranes were then washed four times in tbst buffer for 5 min each time. protein band detection was performed using ecl reagents (applygen technologies inc., china), and the band intensities were analyzed using labworks 4.6 software. after introducing tgev into the st cells, we observed the induction of typical cpes, including cell rounding, swelling, granular degeneration of the cytoplasm, cell detachment, and severely diseased cell morphology, from 40 to 64 h after inoculation ( to further identify the extent of tgev infection, the mrna expression levels of viral genes in infected cells were determined using qrt-pcr. comparative threshold (ct) cycle values in three independent experiments were calculated and the results indicated that the average ct value for the tgev n gene ranged from 25.2 to 27.5. correspondingly, the average ct value observed for the bactin control gene ranged from 19.6 to 21.0. the relative expression of tgev n mrna was calculated using the 2 (-ddct) method [28] , and the change in expression at each time point is indicated in figure 2b . these data show that, following infection, the viral mrna levels increased gradually over time, and reached a peak at 48 hpi. following this time point, the viral mrna levels appear to decrease. in the infected st cells, a total of 29,214 peptides and 4,364 proteins were detected (table s1) ; however, only 4,112 proteins were quantified reliably (table s2) . notably, the abnormal proteins, such as the proteins with itraq ratio higher than 20 or lower than 0.05, which are not quantifiable [30] , were removed and only proteins with reasonable ratios across all channels were investigated further. figure 3a depicts the scatter plots for the log 10 116/114 and log 10 117/115 ratios in the itraq experiment. linear regression analysis showed that correlation (r 2 ) was 0.58, with a p-value less than 0.05. these results suggest that the alterations in protein abundance due to virus infection were nearlinear dependency between the two time points. in order to identify the proteins that were significantly different at each time point (infected/uninfected) or between the different time points, we analyzed the distribution of ratios for the identified proteins as shown in the figure 3b . for the distribution range of the differentially expressed proteins identified at 48 hpi, shown in figure 3c , a ratio higher than 3.35 or lower than 21.35 was defined as a statistically significant difference in protein expression. at 64 hpi, a ratio higher than 4.55 or lower than 22.15 was defined as a statistically significant difference in protein expression. according to analyses, the differentially expressed proteins identified were considered to show a significant upward or downward trend if their expression ratios were greater than 4.0 or less than 0.25 compared to the control group. using the criterion listed above, the expression of 146 proteins was significantly changed at 48 hpi (95 upregulated and 51 downregulated), while 219 proteins were significantly changed at 64 hpi (172 upregulated and 47 downregulated). further, 72 proteins were identified to be significantly different between the two time points (54 upregulated and 18 downregulated), resulting in a total of 316 unique proteins being significantly altered during tgev infection, including 162 predicted proteins (table s3 and table 1 (excluding the predicted proteins)). because the current pig genome database is poorly annotated compared to the human genome database, there were numerous proteins that were unassigned or uncharacterized, resulting in a large number of predicted proteins in our analysis. however, our ability to detect the unannotated proteins by ms demonstrates that they do existence in this species, and additional research concerning their function is warranted. biological process-based enrichment analysis of the differentially expressed proteins revealed that six common go terms were significantly enriched in this set of proteins (p,0.05). thus, it appears that in tgev-infected st cells at 48 and 64 hpi there are expression changes in proteins that are related to cell adhesion, neurological system processes, extracellular matrix organization, locomotion, cell junction organization, and cell-cell signaling. moreover, at the later time point, 64 hpi, our go term analysis also indicated that a significant number of the differentially expressed proteins were related to cellular stress (p = 8.18e-4), generation of precursor metabolites and energy (p = 2.74e-3), cell motility (p = 6.71e-3), protein complex assembly (p = 4.69e-2), growth (p = 3.87e-2), developmental maturation (p = 1.53e-2), and immune system processes (p = 4.67e-2) ( table 2) . to further investigate the localization pattern of these differentially expressed genes, a cellular component-based enrichment analysis was performed. at 48 hpi, we observed the significant enrichments in extracellular region (p = 1.29e-4), proteinaceous extracellular matrix (p = 1.62e-4), and extracellular space (p = 1.52e-2) (table s4 ). in addition, 37 differentially expressed proteins were also significantly enriched (p = 8.65e-3) in mitochondrion at 64 hpi (table s5 ). the final step of our go enrichment analysis consisted of investigating the mechanistic role these genes play in the cell. to do so, we performed a molecular function-based enrichment analysis. this analysis showed that two go terms, unfolded protein binding (p = 2.67e-2) and transmembrane transporter activity (p = 3.55e-2), were significantly enriched at 64 hpi (table s5) . further go analysis of the differentially expressed proteins between the two time points indicated that there were no significant enriched terms. in order to understand the interactions between tgev and host cell proteins, we further analyzed the differentially expressed proteins by searching the string 9.1 database (http://string-db. org/) for protein-protein interactions (figure 4) . in this string analysis, the interactions (edges) of the submitted proteins (nodes) were scored according to known and predicted protein-protein interactions. we created three protein network maps: one for proteins changed significantly at 48 hpi (30 nodes and 15 edges; figure 4a ), one for proteins changed significantly at 64 hpi (66 nodes and 70 edges; figure 4b ), and one for the proteins that were significantly changed when the viral infection was prolonged from 48 to 64 h (24 nodes and 9 edges; figure 4c) . notably, the protein network constructed for the 64 hpi time point is clearly much more extensive than the two other networks, and these proteinprotein interactions suggest the existence of reported functional linkages. go enrichment analysis for the string protein network at 64 hpi showed that several biological processes were significantly affected (p,0.05 based on the fdr correction) in this network, including the regulation of viral genome replication, the innate immune response, negative regulation of viral genome replication, positive and negative regulation of viral processes, and atp biosynthetic processes (table 3) . however, at 48 hpi, the most enriched biological process was related to cell recognition during phagocytosis(p = 8.02e-1). in figure 4c , we have shown that the majority proteins in these protein networks, such as radical s-adenosyl methionine domain containing protein 2 (rsad2), mx dynamin-like gtpase 1 (mx1), 29-59-oligoadenylate synthetase 1 (oas1), mx dynamin-like gtpase 2 (mx2), are involved in the innate immune response. these data suggest that some entirely different host proteins, interactions, or processes, including the immune response, were perturbed at these times during tgev infection. edges. network analysis was set at medium confidence (string score = 0.4). seven different colored lines were used to represent the types of evidence for the association: green, neighborhood evidence; red, gene fusion; blue, co-occurrence; black, co-expression; purple, experimental; light blue, database; yellow, text mining. doi:10.1371/journal.pone.0110647.g004 western blot confirmation of altered expression for three of the differentially expressed proteins to further confirm the proteomic data for three of the proteins, western blot analysis was performed to investigate the changes in the expression of hsp90a, caspase 8, and tgf-b1. the proteins were selected based on three criteria: 1) the expression of the protein was increased or decreased during tgev infection according to our proteomics data; 2) the protein is known to be relevant during viral infection; and 3) each protein analyzed needs to be involved in a special biological process as determined by our go enrichment analysis [32] . hsp90a, caspase 8, and tgf-b1 all filled these criteria and their protein expression was analyzed via western blot analysis of the cell lysate. as shown in figure 5 , the expression of hsp90a was significantly downregulated in tgevinfected cells at 64 hpi, while the expression of caspase-8 was upregulated from 48 to 64 hpi in these cells. the expression of tgf-b1 was also significantly induced in tgev-infected cells following infection. thus, these results confirm the altered expression observed in the proteomic data for these three representative proteins during tgev infection. the interactions between a virus and a host cell during a viral infection are complex, involving numerous genes and signaling pathways. st cells are known to be sensitive to tgev, resulting in increased viral multiplication and cpes [15] . in order to better understand the interactions between the host proteome and tgev, we adopted an itraq quantitative proteomic approach to investigate the altered cellular proteins of the st cells during tgev infection in vitro. compared with the 2-de and 2d-dige methods often used, the 2d-lc-ms/ms method utilized here provides more quantitative and qualitative information about the proteins, and can also detect membrane proteins, hydrophobic proteins, higher molecular weight proteins, and low-abundance proteins, which are often missed by other methods. itraq also has more advantages compared to isotope-coded affinity tags (icat) and stable isotope labeling by amino acids in cell culture (silac) methods, which both allow multiple labeling and quantitation of four to eight samples simultaneously with high sensitivity [22, 33, 34] . further, the itraq technique has been widely used for quantitative proteomics, including protein expression analysis and biomarker identification [23] [24] [25] [26] 35] . prior to proteomic analysis, we determined which time points to investigate following infection by observing the morphological changes and analyzing viral gene expression dynamics in the tgev infected cells. the results indicated that tgev induced significant cpes from 40 to 64 hpi in infected cells compared to the mock infected cells. at 40 hpi, less than 50% of the infected cells were morphologically altered, while at 48 hpi more than 80% infected cells showed rounding and granular degeneration. further, the mrna level of the viral n gene in st cells continuously increased in the infected cells until 48 h, at which time we observed the highest viral replication level. at 64 hpi, the morphological effects observed were much more pronounced, characterized by even more cellular rounding and detachment. however, the mrna levels of the viral n gene decreased rapidly from 48 to 64 h, a phenomenon we believe may be attributed to the host's immune response or a decrease in infected cell viability as the tgev infection progressed. based on our qrt-pcr and cpe analyses, we choose to more deeply investigate the proteomic changes occurring in the tgev-infected st cells at 48 hpi and 64 hpi using a 4-plex itraq analysis. in our analysis, we observed a statistically significant change in the expression of 316 proteins during tgev infection in vitro. this number includes protein changes that were unique for a specific time point as well as those shared at these different time conditions. for example, the expression level of hsp90a expression was unchanged at 48 hpi, but decreased at 64 hpi, note: the significance of the go biological process is derived from the network in figure 4b and was determined using the fdr correction (p,0.05). doi:10.1371/journal.pone.0110647.t003 making this change unique for the latter time point. on the other hand, tgf-b1 was observed to increase at both of the time points, and was thus labeled a shared protein change. moreover, the 316 altered proteins also includes proteins that changed from 48 hpi to 64 hpi, rather than one of these time points compared to noninfected cells. for example, mitochondrial aldehyde dehydrogenase 2 (aldh2) and mhc class i antigen (pd1) were not changed at 48 or 64 hpi compared to the control group, but increased at 64 hpi compared with 48 hpi. we also observed a larger proteomic shift at 64 hpi compared to the 48 hpi time point in the infected st cells. further, some proteins previously reported to play a role in virus-induced host cell death, such as caspase-8, caspase-3, caspase-9, and porcine aminopeptidase-n (papn) [36] [37] [38] , were also identified using this itraq technique. these caspase proteins are known to be involved in tgev-induced cell apoptosis processes, while papn is the cell receptor for tgev. our results indicate that tgev infection caused significant upregulation of caspase-8 expression at two time points (approximately 7-fold at 48 hpi and 16-fold at 64 hpi) in the virus-infected st cells, and this change was verified by western blotting analysis. however, the expression of caspase-3, caspase-9, and papn was not significantly altered, indicating that the pathways involving these genes are not altered or that other proteins are compensating for their lack of change. in this regard, we identified an additional 15 proteins involved in cell death pathways that had significantly altered expression levels (p = 4.46e-2) (table s6) , including melanoma differentiation associated protein-5 (mda5), monocyte chemoattractant protein 1 (ccl2), thioredoxin-dependent peroxide reductase, mitochondrial (prdx3), peroxiredoxin-2 (prdx2), predicted protein cyr61 (cyr61), keratin, type ii cytoskeletal 8 (krt8), predicted bcl-2-like protein 13 (bcl2l13), predicted integrin alpha-5 isoform 1 (itga5), tgf-b1, amyloid beta a4 protein (app), clusterin (clu), c-c motif chemokine 5 (ccl5), heat shock 70 kda protein 1b (hspa1b), alpha-crystallin b chain (cryab), voltage-dependent anion-selective channel protein 1 (vdac1), all of which, with the exception of prdx2 and bcl2l13 were upregulated at one or two time points. regulation of cell death is known to be important for replication and pathogenesis in various coronaviruses [39] , and we believe that further research on these proteins will lead to a better understanding of cell death regulation during tgev infection. in order to determine what other processes, in addition to cell death, were affected by tgev infection, we performed a go enrichment analysis for the different temporal conditions. this analysis indicated that six biological processes were significantly affected at 48 and 64 hpi, and the differentially expressed proteins involved in these processes were almost the same. the large overlap between the two time points suggests that some of the same sets of host proteins or processes were disturbed at these times. however, it is also likely that some processes were affected solely at one time point or the other. at 48 hpi, serine/threonineprotein phosphatase pp1-beta-catalytic subunit (ppp1cb), scavenger receptor class b member 1 (scarb1), transforming growth factor-beta-induced protein ig-h3 (tgfbi), and predicted inositol 1,4,5-trisphosphate receptor type 3 (itpr3) were uniquely altered, likely indicating changes in cell adhesion and/or cell-cell signaling processes. at 64 hpi, on the other hand, calreticulin (calr), predicted tumor-associated calcium signal transducer 2-like (tacstd2), vascular cell adhesion molecule, galectin-3 (lgals3), glutamate dehydrogenase 1 (glud1), and c-x-c motif chemokine 16 (cxcl16) were uniquely changed, also indicating changes in cell adhesion and/or cell-cell signaling as well as extracellular matrix organization and locomotion. we believe that these uniquely altered proteins reflect changes in specific/specialized processes at each time point that are tightly linked to the temporal changes observed in the host cell morphology and gene/protein expression after tgev infection. the most significantly enriched go category related to the differentially expressed proteins was stress, which included 12 differentially expressed proteins at 48 hpi and 27 different proteins at 64 hpi. the increased number of proteins association with this go term at 48 hpi likely highlights the initial upregulation of the cellular stress response, while the higher number at 64 hpi indicates that the stress response to tgev infection is likely more fully induced at this later stage. hsps, also known as stress proteins, are often involved in the cellular response to stress, influencing changes in the state or activity of the cell or organism. hsp90, which has two isoforms (hsp90a and hsp90b), is one of the most abundant molecular chaperones that is induced in response to cellular stress, and it functions to stabilize proteins involved in cell growth and anti-apoptotic signaling [40] . the expression of hsp90a has been reported to play an important role in the replication of some viruses, such as ebola virus (ebov) [41] , hepatitis c virus (hcv) [42] , influenza virus [43] , and japanese encephalitis virus [44] . on the other hand, the reduction of hsp90b has been reported to decrease the correct assembly of human enterovirus 71 viral particles [40] . in this study, hsp90a and heat shock 90kd protein 1, beta (hspcb/hsp90b) were significantly downregulated at 64 hpi in the tgev-infected st cells, but were unchanged at 48 hpi, indicating that they may play a similar role in tgev infection. interestingly, a member of the hsp70 protein family, heat shock 70 kda protein 1b (hspa1b), as well as mitochondrial 60 kda heat shock protein (hsp60) were both upregulated in infected st cells at 48 and/or 64 hpi. hsp60 is a mitochondrial chaperonin protein involved in protein folding and a number of extracellular immunomodulatory activities. elevated expression of hsp60 is associated with a number of inflammatory disorders [45] . hsp70 plays an important role in multiple processes within cells, including protein translation, folding, intracellular trafficking, and degradation. a previous study has revealed that hsp70 is involved in all steps of the viral life cycle, including replication, and is highly specific in regards to viral response, differing from one cell to another for any given virus type [46] . for example, silencing hsp70 expression has been associated with an increase in viral protein levels, while an increase in hsp70 has been suspected to be the initial cellular response to protect against viral infection in rotavirus-infected cells [47] . further, a recent study showed that hsp70 is an essential host factor for the replication of prrsv as the silence of hsp70 significantly reduced prrsv replication [48] . our results provide new experimental evidence relating the expression of hsp90, hsp70, and hsp60 to tgev infection, and we speculate that these proteins play a potential role in tgev replication. additional work is required to investigate the detailed role of these proteins during tgev infection. furthermore, another significantly enriched go process we observed that 11 significantly altered proteins was immune system processes. most of these proteins were significantly upregulated at 64 hpi in response to the viral infection, while some were first upregulated at 48 hpi, including ccl5 and tgf-b1. chemokines, such as ccl2, ccl5, and cxcl16, whose main function is macrophage recruitment and activation, are potentially involved in host-mediated immunopathology. a recent study showed that coronavirus infection of transgenic mice expressing ccl2 led to a dysregulated immune response without effective virus clearance and enhanced death [49] . in additional, tgev-infection can induce the expression of proinflammatory genes, including ccl2, ccl5, and probable atp-dependent rna helicase ddx58 (ddx58/rig-1), in cell culture and in vivo in the absence of viral protein 7 [50] . in this study, we observed an upregulation of ccl2, ccl5, cxcl16, tgf-b1, and ddx58 expression. tgf-b1 is a multifunctional cytokine, secreted from various cells, and, in immunology, it regulates cellular proliferation, differentiation, and other cellular functions for a variety of cell types, especially regulatory t cells [51] . some research has indicated that sars-cov papain-like protease (plpro) increases tgf-b1 mrna expression and protein production in human promonocytes [52] . further, gomez-laguna et al. [53] inferred that the upregulation of the tgf-b may impair the host immune response during prrsv infection by limiting the overproduction of proinflammatory cytokines necessary to decrease prrsv replication. in response to viral infection, ddx58 plays important roles in the recognition of rna viruses in various cells, and has been identified as a candidate for a cytoplasmic viral dsrna receptor [54] . further, upregulation of this gene activates cells to produce type i interferons, which may increase the antiviral status of cells to protect against viral infection. in this regard, we found that interferon-inducible antiviral proteins, rsad2, oas1, were also upregulated in the period of late infection, suggesting that many of the proteins identified in this study are associated with inflammation, ifn activation, and the innate immune response. increased expression of these proteins may help the virus enter the cell as well as potentially enhance tgev replication or the host response 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hun4 infection by twodimensional differential gel electrophoresis proteomics analysis of differentially expressed proteins in chicken trachea and kidney after infection with the highly virulent and attenuated coronavirus infectious bronchitis virus in vivo quantitative analysis of severe acute respiratory syndrome (sars)-associated coronavirus-infected cells using proteomic approaches implications for cellular responses to virus infection identification of cellular proteome using two-dimensional difference gel electrophoresis in st cells infected with transmissible gastroenteritis coronavirus comparative study of three proteomic quantitative methods, dige, cicat, and itraq, using 2d gel-or lc-maldi tof/tof two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (itraq) labeling approach revealed first proteome profiles of pulmonary alveolar macrophages infected with porcine reproductive and respiratory syndrome 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of genes: which test? high-throughput quantitative proteomic analysis of dengue virus type 2 infected a549 cells multiplexed protein quantitation in saccharomyces cerevisiae using aminereactive isobaric tagging reagents using silac and quantitative proteomics to investigate the interactions between viral and host proteomes identification of candidate biomarker proteins released by human endometrial and cervical cancer cells using two-dimensional liquid chromatography/tandem mass spectrometry transmissible gastroenteritis virus infection induces apoptosis through fasl-and mitochondria-mediated pathways transmissible gastroenteritis coronavirus induces programmed cell death in infected cells through a caspase-dependent pathway aminopeptidase n is a major receptor for the enteropathogenic coronavirus tgev regulation of cell death during infection by the severe acute respiratory syndrome coronavirus and other coronaviruses heat shock protein-90-beta facilitates enterovirus 71 viral particles assembly inhibition of heat-shock protein 90 reduces ebola virus replication hepatitis c virus rna replication is regulated by fkbp8 and hsp90 identification of hsp90 as a stimulatory host factor involved in influenza virus rna synthesis identification of heat-shock protein 90 beta in japanese encephalitis virus-induced secretion proteins heat shock protein 10 inhibits lipopolysaccharide-induced inflammatory mediator production hsp70 protein positively regulates rabies virus infection hsp70 negatively controls rotavirus protein bioavailability in caco-2 cells infected by the rotavirus rf strain inhibition of hsp70 reduces porcine reproductive and respiratory syndrome virus replication in vitro transgenic ccl2 expression in the central nervous system results in a dysregulated immune response and enhanced lethality after coronavirus infection alphacoronavirus protein 7 modulates host innate immune response transforming growth factor-beta 1 pathways in inflammatory airway diseases correlation between tgf-b1 expression and proteomic profiling induced by severe acute respiratory syndrome coronavirus papain-like protease enhanced expression of tgfb protein in lymphoid organs and lung, but not in serum, of pigs infected with a european field isolate of porcine reproductive and respiratory syndrome virus mda5/rig-i and virus recognition we thank qiangqiang zhao, chen lou, wulong liang, and helin li for their technical support, and shuo chen for his valuable advice. key: cord-319675-mwy3t1ny authors: gu, li; qu, jiuxin; sun, bing; yu, xiaomin; li, hui; cao, bin title: sustained viremia and high viral load in respiratory tract secretions are predictors for death in immunocompetent adults with adenovirus pneumonia date: 2016-08-17 journal: plos one doi: 10.1371/journal.pone.0160777 sha: doc_id: 319675 cord_uid: mwy3t1ny the predictors for fatal adenovirus (adv) pneumonia among immunocompetent adults are unclear. laboratory-confirmed, hospitalized adv pneumonia adults were prospectively enrolled in beijing chao-yang hospital from march to june 2013. clinical data and serial whole blood and respiratory tract secretions from such patients were collected. quantitative real-time polymerase chain reaction was performed to quantify the viral load. a total of 14 adv pneumonia cases were consecutively enrolled, and four of them were fatal. ten cases were caused by adv-55, three by adv-7 and one by adv-3. there were no differences in age, gender or underlying diseases between the patients in the fatal cases and surviving cases. at admission (on day 5–7 after illness onset), the patients in fatal cases presented higher initial viral loads in respiratory tract secretions (8.578 ± 2.115 vs 6.263 ± 1.225 log(10) copies/ml, p = 0.023). all patients in fatal cases presented with viremia on day 12–14 (100% vs 66.7%, p = 0.017). a higher initial viral load in the respiratory tract and sustained viremia (more than 2 weeks) may be predictors for fatal clinical outcomes. severe adenovirus (adv) infections causing significant acute respiratory distress syndrome have raised concerns for immunocompetent adults [1] . most severe cases were previously reported to be associated with adv-3, 4, 7 and 21 [2, 3] . a new strain, adv-55 (formerly known as adv-11a), has been a major adv pneumonia pathogen in immunocompetent adolescents and adults in china since 2008 [4] . our previous study identified a fatal adv-55 infection case with a patient who presented with systemic infection and high-level viremia [5] . however, even though adv-55 as well as adv-3, 4, 7 and 21 can cause severe cases, those strains are not necessarily associated with bad outcomes, and the pathogenesis of fatality is still unknown. we have already ascertained that adv can be detected in whole blood specimens of severe cases, but we still lack the knowledge about the viral shedding history and the relationship between the viral clearance in the respiratory tract and viremia duration and clinical outcome. in this study, we focus on the dynamic virological changes in whole blood and respiratory tract secretions to see if lethal cases experienced a high viral load and longer duration of viral shedding compared to the non-lethal cases. we hypothesize that sustained virus shedding in the blood and/or respiratory tract can appear in severe immunocompetent adult cases, and it may be a risk factor for fatal outcome. the study was reviewed and approved by the institutional review board of beijing chao-yang hospital (the project approval number is 10-ke-49). written informed consent was provided by all adults and the parents of patients aged less than 18 years. adults with communityacquired pneumonia (cap) (age14yrs) admitted to beijing chao-yang hospital from march to june 2013 were prospectively included. patients with hiv infection or neutropenia; receiving immunosuppressive chemotherapy or steroids equivalent to prednisone >15 mg/d for 30 days; who were pregnant or breast feeding women; or who were known or suspected to have active tuberculosis were excluded. methods of etiological evaluation followed the standard for adults suspected with cap [4] [5] [6] . in short, sputum or respiratory tract aspiration, blood and urine were collected at admission and submitted to the infectious disease and clinical microbiology laboratory. microbiological methods were based on the following tests: (1)sputum specimens for gram stain and cultures considered valid only if microscopy showed > 25 neutrophils and <10 epithelial cells per low field microscopy; (2)urine specimens for the rapid detection of s. pneumonia and legionella pneumophila antigen; (3) blood culture; (4) sputum and tracheal aspiration for virus real-time polymerase chain reaction (pcr) detection, including rhinovirus, influenza a and b, respiratory syncytial virus a and b, adenovirus, parainfluenza 1-4, coronavirus oc43 and 229e, and metapneumovirus; and (5) sputum for mycoplasma pneumoniae pcr detection. only subjects with positive adenovirus results who were negative for other etiologies during the study period were enrolled. the results of pcr testing for respiratory viruses were reported to clinicians within 6 hours after sputum collection. for those with positive adv pcr testing, serial whole blood and respiratory tract samples were collected until death or discharge. in this study, we tried to collect the same type of respiratory tract samples from each patient to carry out serial viral load testing, to lower bias from different types of samples. for patients with mechanical ventilation, we collected serial tracheal aspirations, and for patients without mechanical ventilation, we collected sputum samples. we also collected data regarding age, gender, co-morbidities, clinical symptoms, vital signs, antimicrobial treatment, chest radiographic findings, and laboratory results. recorded complications included the following: use of mechanical ventilation and extracorporeal membrane oxygenation (ecmo), and second bacterial or fungal infection. patients were also followed up to discharge or death. respiratory tract samples, including sputum and tracheal aspirations, were processed by equal volume 1% trypsin digestion. the viral dna was extracted from 1 milliliter (ml) digested respiratory sample and 1 ml whole blood sample using a qiaamp dna mini kit (qiagen, valencia, ca, usa). first, we amplified the entire hexon genes of the samples by pcr. the primer sequences are listed in table 1 [7] . then, the adv type was determined using blast (http:// blast.ncbi.nlm.nih.gov/blast.cgi). regarding the quantification of adv in the samples, we used a commercial fq-pcr kit (daan gene, cat. #da-b067, guangzhou, china) targeting the dna polymerase gene [genebank: kf279555.1] two-tailed independent samples t-test or mann-whitney u-test (on condition of non-normal distributions) was used to compare continuous variables between the two groups. for the categorical data, univariate analysis was performed using the chi-square test or fisher's exact test. significance was fixed at p value < 0.05. data analysis was performed using spss 15.0 (spss inc; chicago, il). from march 2013 to june 2013, a total of 14 admitted cases were confirmed with adenovirus as the only pathogen of pneumonia; four of the patients died in the icu. the mean age was 30 years old. males predominated in number over females, with a sex ratio of approximately 6:1. only two patients had co-morbidities, one with tuberculous pleuritis for 2 months and one with congenital heart disease. there were no differences in age, gender or underlying diseases between fatal cases and surviving cases. there are three types of adv found in this study, adv-55 (n = 10), adv-7 (n = 3) and adv-3 (n = 1). the adv-55 ratio in the fatal group was similar to that in the surviving group (100% vs 60%, p = 0.251) ( table 2) . clinical features: comparison between surviving and fatal cases most clinical symptoms and signs noted in the surviving cases and fatal cases had no differences, except for the pneumonia severity index (psi) score and dyspnea. the fatal cases had a higher psi score (101.3 ± 8.0) and dyspnea incidence (4/4) compared to the psi score (46.5±26.7) and dyspnea incidence (2/10) in surviving cases (p = 0.002 and 0.015, respectively) ( table 2 ). all of the fatal cases described bilateral involvement on chest radiography, and 75% noted pleural effusion (table 2) . for the surviving cases, 40% (4/10) noted bilateral involvement, and 20% (2/10) described pleural effusion. there were no significant differences between the two groups in bilateral involvement or pleural effusion. the adv load tracking in the respiratory tract samples and whole blood the mean time from disease onset to initial pcr test for respiratory tract samples and whole blood was 6.23 days, and there were no significant difference between the fatal group (7.5±1.8days) and surviving group (5.5±1.3days). we measured the adenoviral load in respiratory tract samples and whole blood, expressed as log 10 dna copies per ml samples. for initial viral load in respiratory tract samples on day 5-7 after disease onset, the results showed that, compared to the surviving cases, the fatal cases had a higher initial viral load (8.578 ± 2.115 vs 6.263 ± 1.225, p = 0.023) (fig 1a) . however, for the initial viral load in whole blood, there was no significant difference between the two groups (3.71±2.67 vs 5.978±1.610, p = 0.162, fig 1b) . we also investigated viral shedding duration evaluated by positive ratio on day 5-7 and on day 12-14 after illness onset. for respiratory tract samples, there was no significant difference of viral positive ratio between the two groups either on day 5-7 (100% vs 100%) or on day 12-14 after onset of disease (60% vs 100%, p = 0.126) (fig 1c) . viremia was common at admission (on 5-7 day after onset of illness), as noted in 100% (4/4) of the fatal cases and 71.4% (5/7) of the surviving cases (p = 0.491). on day 12-14 after onset of illness, however, it persisted in 100% (4/4) of the fatal cases with 33.3% (2/6) of the surviving cases (p = 0.017) (fig 1d) . for the surviving severe patients, we found that the clinical manifestation recovered gradually with a downward trend in viral load in respiratory tract and whole blood samples. as shown in fig 2, a 25 -year-old male with acute respiratory distress syndrome (ards) had a high initial viral load (10 8.32 copies/ml) in tracheal aspiration. his condition improved, with the sputum viral load going down on day 14, and ecmo was withdrawn on day 14. with the viral load in blood going down to negative on day 22, the clinical condition was significantly improved, and the patient did not need oxygen therapy; the chest x-ray was also remarkably resolved. as shown in fig 3, the respiratory tract viral load of a 34-year-old male with ards also had a high initial viral load (10 9.25 copies/ml) in tracheal aspiration. the viral load in tracheal aspiration gradually decreased during the first 20 days, and the lung infiltrate absorbed partially. however, the viral load trended up again and the patient died. he also maintained a viral load in whole blood before death. antibiotics were given to all of the patients empirically before and after confirmed diagnosis. there is currently no formally approved antiviral therapy for the treatment of severe lifethreatening adenovirus infection in china. acyclovir, ganciclovir or ribavirin is commonly chosen by the physician to treat adenoviral infection. in this study, all of the fatal patients were administered antiviral drugs-one was treated with ganciclovir, two with acyclovir and one with ribavirin. in surviving patients, 50% were treated with antiviral drugs-three with ganciclovir, one with acyclovir and one with ribavirin. all of the fatal patients (4/4) were complicated with ards and admitted to the icu. they needed mechanical ventilation, and three of them received ecmo to maintain oxygenation. for surviving patients, one of them (1/10) was admitted to the icu due to ards, and had mechanical ventilation and ecmo; one patient with respiratory failure was treated with non-invasive ventilation, and one patient was treated for myocarditis. the myocarditis patient presented with peak levels of creatine kinase isoenzyme (ck-mb) and cardiac troponin i (ctni) on day 7-8 after disease onset, and eight days later, with viral load going down to negative, ck-mb and ctni went down to normal levels in parallel (fig 4) . for the four fatal patients, the times of death were on days 14, 16, 22 and 28 after disease onset, respectively. there was no significant difference in length of stay in-hospital between the two groups (13.5±6.5 days vs 13.3±9.2 days, p = 0.969) ( table 2 ). there were three patients complicated with bacterial or fungal infections among the fatal cases. the first patient presented with consecutive aspergillus fumigatus in tracheal aspirate cultures, the second patient had a cavity present on chest ct, and the third patient had acinetobacter baumannii in tracheal aspirate and pleural effusion cultures. there were only two patients complicated with superinfections in surviving patients. one patient (a 16-year-old male) presented with a typical crescent sign and halo sign with chest ct follow-up; voriconazole was administered to resolve this condition (fig 5) . another patient with ecmo presented with acinetobacter baumannii in tracheal aspirates. we investigated the relationship between the virological factors and clinical outcomes in a cohort of 14 hospitalized adults with adv pneumonia. our results suggest that a higher initial viral load (10 8 copy/ml) in the respiratory tract samples on day 5-7 after disease onset is a predictor for fatal clinical outcome. we also reported that viremia is common and sustained viremia for 14 days or more may be associated with mortality the pathogenesis of mortality in adv pneumonia is still unknown. virological factors, e.g., a new strain with new genetics, viral load, slow virus clearance and systemic infection with viremia likely play key roles for severe adv [2] [3] [4] [5] [6] [7] [8] . however, no study has evaluated the viral shedding history among immunocompetent adults with adv pneumonia. this study first monitored the consecutive viral load in respiratory tract samples and whole blood samples. our previous clinical study demonstrated that on day 5-7 after disease onset, the peak stage of illness presented for patients with shortness of breath or severe dyspnea [5] . again, in this study, we showed that the viral load on day 5-7 could also provide an insight into the severity of illness. evidence even proved that a higher level of viral load in respiratory tract samples on day 5-7 after disease onset was significantly associated with fatal outcome. we have noted that viremia is quite common on day 5-7 after disease onset, when 9 out of 11 (81.8%) patients had viremia. adenovirus viremia has been found in hematopoietic stem cell transplantation recipients and associated with adv disease [8] . compared with previous reports of viremia and clinical outcomes, another novel finding is that we demonstrated that fatal outcomes could be predicted by sustained viremia, but not by viremia itself. in this study, we showed that 100% (4/4) of patients in fatal cases presented with viremia on day 12-14 after disease onset, compared with 60% (p = 0.126) of the patients in surviving cases. in one case, as shown in fig 2, even though the patient presented with a higher viral load (10 8.32 copies / ml) in tracheal aspiration, which may be associated fatal outcome, his clinical manifestation recovered gradually with a downward trend in the viral load in respiratory tract and whole blood samples. compared to this case in fig 3, the patient described in fig 3 not only had a higher viral load (10 9.25 copies/ml) in tracheal aspiration but also presented with sustained elevated viral copies, especially in whole blood. shike et al. also reported a 6-monthold infant with systemic infection by adenovirus who had high-level viremia and showed reduction in viral load paralleling her clinical recovery [9] . therefore, in severe cases, dynamic monitoring of viral shedding, especially in whole blood, could help predict the clinical outcome. patients might have bad outcomes if the viral load in whole blood does not present a significant downward trend around two weeks after disease onset. there is currently no formally approved antiviral therapy for the treatment of severe lifethreatening adenovirus infection in china. cidofovir is considered the medicine of choice for severe infection in immunocompromised patients. cidofovir is not available in most hospitals in china, including our hospital. acyclovir, ganciclovir or ribavirin is usually prescribed in china. in this study, antiviral drugs were administered in all of the fatal cases-one patient was treated with ganciclovir, two with acyclovir and one with ribavirin. in surviving patients, 50% were treated by antiviral drugs-three with ganciclovir, one with acyclovir and one with ribavirin. the choice of the antiviral medicine was decided by the patient's physician. as none of these three medicines have been confirmed to be effective for adv infection, the relationship between viral shedding and clinical outcomes in this study was not associated with anti-adenoviral treatment effect. our study has two limitations. as adv 55 was the most common infection type (10/14, 71.4%) in this study, results might be more significant in adv 55-associated pneumonia and might not be generalizable to other types of adv pneumonia. in our previous study, adults infected with adv 55 were 10 years older and presented with higher psi scores compared with adults infected with other serotypes [4] . another limitation of this descriptive work may be the small number of analyzed patients, especially in the group of fatal cases (n = 4). more cases are needed to confirm our findings. in conclusion, our data provide new insight into the virology of adv pneumonia. a higher initial viral load (10 8 copy/ml) in the respiratory tract on day 5-7 after disease onset and sustained viremia for 2 weeks or more may be associated with fatal clinical outcomes. two fatal cases of adenovirus-related illness in previously healthy young adults-illinois severe adenovirus pneumonia in immunocompetent adults: a case report and review of the literature severe pneumonia due to adenovirus serotype 14: a new respiratory threat? emergence of community acquired adenovirus type 55 as a cause of community-onset pneumonia severe community-acquired pneumonia caused by adenovirus type 11 in immunocompetent adults in beijing viral and mycoplasma pneumoniae community-acquired pneumonia and novel clinical outcome evaluation in ambulatory adult patients in china outbreak of acute respiratory disease in china caused by b2 species of adenovius type 11 adenovirus viremia and disease: comparison of t celle depleted and conventional hematopoietic stem cell transplantation recipients from a single institution quantitation of adenovirus genome during acute infection in normal children we thank drs. ran li, chen ma, yudong yin, lin wu, and yiqun guo for their contributions to specimen collection.notation of publication: this work has been accepted as an oral presentation at the third isirv-avg conference on influenza and other respiratory virus infections: advances in clinical management on june 4 th -6 th , 2014 at the keio plaza hotel, tokyo, japan. conceived and designed the experiments: bc. lg jxq bs xmy.analyzed the data: lg hl.contributed reagents/materials/analysis tools: jxq bc.wrote the paper: lg jxq bc. competing interests: the authors have declared that no competing interests exist. key: cord-335505-s013j5ex authors: zhang, chen; zhu, na; xie, zhengde; lu, roujian; he, bin; liu, chunyan; ma, xuejun; tan, wenjie title: viral etiology and clinical profiles of children with severe acute respiratory infections in china date: 2013-08-22 journal: plos one doi: 10.1371/journal.pone.0072606 sha: doc_id: 335505 cord_uid: s013j5ex background: no comprehensive analysis is available on the viral etiology and clinical characterization among children with severe acute respiratory infection (sari) in china during 2009 h1n1 pandemic and post-pandemic period. methods: cohort of 370 hospitalized children (1 to 72 months) with sari from may 2008 to march 2010 was enrolled in this study. nasopharyngeal aspirate (npa) specimens were tested by a commercial assay for 18 respiratory viral targets. the viral distribution and its association with clinical character were statistically analyzed. results: viral pathogen was detected in 350 (94.29%) of children with sari. overall, the most popular viruses were: enterovirus/rhinovirus (ev/rv) (54.05%), respiratory syncytial virus (rsv) (51.08%), human bocavirus (boca) (33.78%), human parainfluenzaviruse type 3 (piv3) (15.41%), and adenovirus (adv) (12.97%). pandemic h1n1 was the dominant influenza virus (ifv) but was only detected in 20 (5.41%) of children. moreover, detection rate of rsv and human metapneumovirus (hmpv) among suburb participants were significantly higher than that of urban area (p<0.05). incidence of vsari among suburb participants was also significant higher, especially among those of 24 to 59 months group (p<0.05). conclusion: piconaviruses (ev/rv) and paramyxoviruses are the most popular viral pathogens among children with sari in this study. rsv and hmpv significantly increase the risk of sari, especially in children younger than 24 months. higher incidence of vsari and more susceptibilities to rsv and hmpv infections were found in suburban patients. acute respiratory infection (ari) is the leading causes of children death globally [1] [2] [3] [4] [5] . even in developed country, severe acute respiratory infection (sari), with its high mortality and morbidity among children younger than 5, impose great burden on the society [2, 3] . lacking of etiologic diagnosis is the main reason that more than 50% of ari cases suffered unnecessary or inappropriate prescription of antibiotics, since most acute respiratory tract infections are caused by viruses [3] . this often leads to severe consequence such as high rate of resistance [6] , especially in those with severe acute respiratory infection (sari), which frequently happens in virus-infected children [3] . therefore etiology studies, especially those of viruses, have been performed to help diagnosis and proper antiviral treatment of sari [7, 8] , as well as prevention of nosocomial infections among in-patients [9] . diagnosis of ari is complicated by the wide range of potential pathogens that can present with similar clinical symptoms [10, 11] . in recent years, the introduction of nucleic acid based diagnostic tests has markedly improved our ability in understanding viral etiology among ari patients [12] . xtag® rvp fast, a test based on multiplex pcr and luminex molecular diagnostics universal array that detects 18 most commonly seen viral targets in respiratory infection, is approved by the us food and drug administration (fda) and has shown improved viral detection ability as compared to traditional methods like direct fluorescent antibody (dfa) and culture methodology [13] [14] [15] [16] [17] [18] [19] . shortly after the advent of severe acute respiratory syndrome (sars) and the avian influenza, the emergence of the influenza virus a (h1n1) 2009 pandemic caused significant vibrations to the public health authorities and stressed the health systems worldwide. this highlighted our weaknesses regarding the diagnosis and assessment of sari. china is the biggest developing country with largest population of child. however, we have found no published case-control studies regarding comprehensive viral etiology and clinical characterization of children sari using commonly acknowledged reliable test in china. to better understand the role of respiratory viruses in children with sari during 2009 h1n1 pandemic and postpandemic era, and help diagnosis and antiviral treatment, we conducted a comprehensive evaluation of viral etiology and clinical characterization among hospitalized children with sari admitted to the beijing children hospital from may 2008 to march 2010. this study has increased our knowledge on the management of sari and community-acquired pneumonia. the study protocol was approved by the institutional review board of national institute for viral disease control and prevention, china cdc, and the scientific committee of the beijing pediatric research institute, and the ethical review committee of beijing children's hospital. individual written informed consent was obtained from the parents or guardians of all participants. all participants in this study were inpatients from beijing children's hospital between may 2008 and march 2010, diagnosed as sari based on clinical grounds recommended by the world health organization (who) (20) . eligibility and classification of the clinical syndromes of sari were determined from individual's original record of medical history and examination. the criteria of hospitalized patient inclusion were: sudden onset of fever >38 o c and cough or sore throat and difficulty breathing (dyspnea, oxygen saturation < 90%). additional criteria were a normal or low leukocyte count, or lower chest wall indrawing. among these sari cases, 120 were defined as very severe acute respiratory infection (vsari) cases with any of the following criteria: 1) incidence of complication such as vomit or diarrhea, 2) respiratory failure or anhelation or heart failure, 3) icu admissions. nasopharyngeal aspirate (npa) or blood or induced sputum (is) was collected from the patients at the first day of admission and transferred into virus transport medium and stored at -70 o c until tested. demographic information and medical test result were collected with standardized forms. nucleic acid was extracted from 200 µl of the virus transport medium using qiaamp minelute virus spin kit (qiagen, mississauga, ontario, canada) according to the manufacturer's instructions. 10 µl of the nucleic acids were tested using xtag® rvp fast assay according to the manufacturer's instructions (abbott molecular inc., usa) and analyzed on bio-plex 200 system (bio-rad laboratories, inc., usa). the rvp fast assay simultaneously detects the following viruses: respiratory syncytial virus (rsv); influenza(ifv) a (h1, h3, and h5) and b viruses; parainfluenza viruse (piv) 1, 2, 3, and 4; human metapneumovirus (hmpv); adenovirus(adv); piconavirus(pic) which includes enterovirus (ev) and rhinovirus (rv); human coronaviruse(hcov) nl63, hku1, 229e, and oc43; and human bocavirus(boca). the assay also includes an internal positive control added to each specimen at the extraction stage (escherichia coli phage ms2 rna) and a positive run control that is added to each run (bacteriophage lambda dna). comparisons between urban and suburb patients, as well as severe and very severe infection, were performed using chisquare test. correlation of virus detected and clinical signs or diagnosis was performed using binary logistic regression of spss statistics 17.0. comparison of continuous variables like body temperature was conducted using variance analysis. the clinical symptoms of all 370 participants presented with different severe respiratory infection signs including convulsion, shock, pleural effusion, cough, wet rale, dry rale, expectoration, vomiting, respiratory failure, and heart failure. the three most commonly observed symptoms were cough (97.57%), wet rale (62.43%), and expectoration (54.32%). 341 participants showed parenchymal infiltration in chest radiography (data not shown). viral prevalence is also presented in table 1 .5-16.2%), orthomyxoviruses (7.84%, ifva,7.03%; ifvb, 0.81%) and hcovs (6.76%: hcov-oc43, 4.32%; hcov-229e, 0.81%; hcov-nl63, 1.08%; and hcov-hku1, 1.08%). pandemic h1n1 was the dominant influenza virus(ifv) but only detected in 20 (5.41%) of children enrolled in this study. we noticed that all pandemic h1n1 infection took place during 2009(from january through august). figure 1 shows the seasonal distribution of few dominant respiratory viruses and incidence of sari. ev/rv was the most frequently discovered pathogen in this study; it was active throughout whole year, with a flat line of infection rate around 50% ( figure 1a) . rsvb, in contrast to ev/rv, only prevailed in winter. it infected 60-100% patients in winter, but almost disappeared in summer ( figure 1b) . hmpv showed similar distribution as rsvb, but the infection rate was much lower ( figure 1d ). of all the other 3 dominant pathogens: both piv3 ( figure 1c ) and boca( figure 1e ) showed a peak in the august of 2009, however adv ( figure 1f ) infection was always active with a flat line from september of 2008 to december of 2009. patients from urban area are obviously older than suburb/ rural ones, average age 15.34 vs. 8.72 months ( table 2 ). further analysis of age distribution based on chi-test showed that ratio of young children (24 to 59 months) is obviously higher in rural area (21.6% vs. 3.9%, p < 0.001, figure 2a) . several viruses are more frequently detected among suburb/ rural patients( to find the reason that causes severe infection, we performed complete comparison between vsari patients and the sari, including clinical signs, number of viral target, gender, and age(table 3, figure 2b ). no difference in gender or average age was discovered between vsari and sari patients. also multiple infection does not have significant impact in severity of infection (p=0.11). however, comparison of age group showed some difference (p = 6.93e-05). judging from detailed list of figure 2b , it is obvious that patients older than 24 months are more resistant to very severe infection (3.16% vs. 19.64%). to find the association between virus infection and clinical signs in sari, binary logistic regression was performed between 4 commonly diagnosed respiratory abnormality, including anhelation, respiratory failure, heart failure and pleural effusion, and the viral target detected by xtag® rvp fast. as shown in table 4 association between commonly diagnosed respiratory disease and virus detection was also performed by binary logistic regression. as shown in table 5 , both rsva (p=0.00, or=11.11; 95%ci, 2.08-50) and rsvb (p=0.00, or=10; 95%ci, 2.38-50) is associated with bronchiolitis and hmpv associated with pneumonia/bronchopneumonia (p=0.00, or=14.29, 95%ci, 2.78-100). in this study, we performed systematic study of viral etiology and clinical profiles among children with sari admitted in beijing children's hospital between may 2008 and march 2010. unlike most previous studies in china, which primarily focused on common acute respiratory infections in children [20] [21] [22] [23] , we exclusively studied the in-hospital patients with deeper infection in the respiratory tract and severer symptoms. 32.34% of the cases in this study suffered very severe symptoms such as heart failure and respiratory failure, as well as complications like diarrhea and vomiting. another result that is different from previous study is a co-infection rate of 64.32%. besides the special focus on in-patients, a broader viral spectrum and higher sensitivity of xtag® rvp fast test may also give rise to this [15] [16] [17] [18] 24] . the most supportive evidence is an unprecedented high infection rates of ev/rv (54.05%), which is consistent with previous report that xtag rvp fast is extremely sensitive in ev/rv detection compared to other molecular based methods [19, 25] . virus infection in children with sari plos one | www.plosone.org who and the pan american health organization recommend hospital-based surveillance of severe acute respiratory infections (sari) as a tool to monitor severe disease caused by influenza. although the study was performed during 2009 h1n1 pandemic period, our data showed that the special cohort of sari patients resulted in relatively low incidence of influenza viruses (7.03%). pandemic h1n1 was the dominant influenza virus(ifv) but was only detected in 20 (5.41%) of children. as reported in previous study [5, 26] , children infected with influenza viruses are usually older than those suffered from rsv, while more than 80% patients in this study were under 24 months old. paramyxoviruses, especially rsv and hmpv, are might prevalence and might be the main reason of sari outbreak during 2008 winter to 2009 spring in this study. similar to previous studies [5, [27] [28] [29] , rsv played an important role in children of sari. it showed obvious seasonal distribution and was obviously related to several respiratory symptoms. infection of rsvb increased and peaked with increase of sari cases during september 2008 through march 2009. both rsva and rsvb increased the risk of bronchiolitis with high odds ratio ( table 5 ). the obvious relationship between rsv and severe respiratory symptoms coincides with a similar retrospective study performed on kenya children of severe pneumonia, in which rsv, among all commonly discovered chi-square test respiratory viruses, was found to be the only associated virus with children pneumonia [7, 30] . another paramyxoviruse, hmpv, showed similar seasonality as rsvb, although the infection rate was not high among all cases. what's more important is that infection of hmpv is obviously related to all three vsri-related symptoms: anhelation, heart failure, and respiratory failure ( table 4) . the third commonly detected paramyxovirus was piv3, a viral target infects about 1-13% of children with pulmonary diseases. infection rate varies with age and severity of cohort among different studies [5, 31] . piv3 is the predominant subtype among parainfluenza viruses, and was responsible for 83.8% parainfluenza infection in this cohort. to our knowledge, piv3 was more related with immunocompromised children than common sari patients in previous studies [32] [33] [34] [35] . it has frequently been reported that infection of rsv and hmpv result in same or similar symptoms and were indistinguishable on clinical basis [9] [10] [11] 36, 37] . however, our analysis of association produced somehow different results: hmpv was more directly related with vsari and thus improved risk of pneumonia, while rsv was more related to risk of bronchiolitis. in contrast to paramyxoviruses, infection rate of ev/rv distributed relatively even throughout the whole year, in spite of its high total detection rate (50.4%). this assumption is supported by association study, in which no confident relationship was found between ev/rv and any severe symptoms (table 4 ) or any diagnosis (table 5 ). in reference to previous studies, ev/rv in respiratory tract is frequently detected by pcr among children of upper respiratory infection and asymptomatic controls. however, few evidence has been found supporting association between ev/rv detection and severe lower respiratory symptoms [36, 37] . further studies are needed to address the relevance of the ev/rv single and coinfections on clinical outcomes. great difference was discovered between urban and suburb patients in this study. children from suburb area are apparently younger, with higher incidence of sari, and were more susceptible to rsv, hmpv, and ev/rv. bronchiolitis and asthmatic bronchitis also happened more frequently in suburb area ( table 2) . for each of the five most popular pathogens: boca, adv, rsv, ev/rv (piconaviruses), and piv3, we performed binary logistic regression between existence of co-infection and all clinical signs. unfortunately, no statistical significance was observed in any run of regression data not shown). to our knowledge, this is the first report on the viral etiology, epidemiological and clinical profiles of hospitalized children with sari using a commercial assay for 18 respiratory viral targets in asia. improved laboratory test highlighted the significance of viral etiology and its distribution among children with sari in china. our data is similar to the recent surveillance report on sari in south africa by use of the validated rt-pcr multiplex assay(39), which is also consistent with several studies in other parts of the world (3, 7, 30) . secondary, comparison analysis was firstly performed between urban and suburban/rural patients in china; in addition, this is also the first study on association analysis estimates of mortality in children younger than 5 years for burkina faso a molecular epidemiological study of respiratory viruses detected in japanese children with acute wheezing illness respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology clinical and epidemiological comparison of human metapneumovirus and respiratory syncytial virus in seoul epidemiology and seasonality of respiratory viral infections in hospitalized children in kuala lumpur, malaysia: a retrospective study of 27 years the global problem of antibiotic resistance a preliminary study of pneumonia etiology among hospitalized children in kenya etiology and epidemiology of viral pneumonia among hospitalized children in rural mozambique: a malaria endemic area with high prevalence of human immunodeficiency virus the burden of respiratory syncytial virus infection in young children human metapneumovirus and severity of respiratory syncytial virus disease detection of human metapneumovirus in infants with acute respiratory tract infection detection of respiratory viruses by molecular methods development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbead-based assay 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tract infection in children from clinical study on interstitial lung disease in children of china performance of the luminex xtag respiratory viral panel fast in a clinical laboratory setting comparison of three multiplex pcr assays for the detection of respiratory viral infections: evaluation of xtag respiratory virus panel fast assay, respifinder 19 assay and respifinder smart 22 assay etiology and incidence of viral and bacterial acute respiratory illness among older children and adults in rural western kenya molecular monitoring of causative viruses in child acute respiratory infection in endemo-epidemic situations in shanghai respiratory viruses in hospitalized children with acute lower respiratory tract infections in harbin, china viral etiology of bronchiolitis among pediatric inpatients in northern taiwan with emphasis on newly identified respiratory viruses viral etiology of severe pneumonia among kenyan infants and children impact of parainfluenza virus infection in pediatric cancer patients parainfluenza 3 virus and other common respiratory pathogens in children with human immunodeficiency virus infection parainfluenza and influenza virus infections in pediatric organ transplant recipients parainfluenza virus infection in adult lung transplant recipients: an emergent clinical syndrome with implications on allograft function comparison of results of detection of rhinovirus by pcr and viral culture in human nasal wash specimens from subjects with and without clinical symptoms of respiratory illness persistence of rhinovirus and enterovirus rna after acute respiratory illness in children we are grateful to the staff and patients of the pediatric wards of beijing children hospital, and to the clinical, information and communication technology, and laboratory staff who contributed to this study. we thank dr lyna zhang, cdc of usa, for their proofreading of this manuscript. no compensation was received for participation in this research. conceived and designed the experiments: cz zx xm wt. performed the experiments: cz nz xz rl bh cl. analyzed the data: cz nz zx wt. contributed reagents/materials/ analysis tools: rl. wrote the manuscript: cz nz wt. key: cord-335441-bj3me7p8 authors: jourdain, elsa; gunnarsson, gunnar; wahlgren, john; latorre-margalef, neus; bröjer, caroline; sahlin, sofie; svensson, lovisa; waldenström, jonas; lundkvist, åke; olsen, björn title: influenza virus in a natural host, the mallard: experimental infection data date: 2010-01-28 journal: plos one doi: 10.1371/journal.pone.0008935 sha: doc_id: 335441 cord_uid: bj3me7p8 wild waterfowl, particularly dabbling ducks such as mallards (anas platyrhynchos), are considered the main reservoir of low-pathogenic avian influenza viruses (lpaivs). they carry viruses that may evolve and become highly pathogenic for poultry or zoonotic. understanding the ecology of lpaivs in these natural hosts is therefore essential. we assessed the clinical response, viral shedding and antibody production of juvenile mallards after intra-esophageal inoculation of two lpaiv subtypes previously isolated from wild congeners. six ducks, equipped with data loggers that continually monitored body temperature, heart rate and activity, were successively inoculated with an h7n7 lpai isolate (day 0), the same h7n7 isolate again (day 21) and an h5n2 lpai isolate (day 35). after the first h7n7 inoculation, the ducks remained alert with no modification of heart rate or activity. however, body temperature transiently increased in four individuals, suggesting that lpaiv strains may have minor clinical effects on their natural hosts. the excretion patterns observed after both re-inoculations differed strongly from those observed after the primary h7n7 inoculation, suggesting that not only homosubtypic but also heterosubtypic immunity exist. our study suggests that lpai infection has minor clinically measurable effects on mallards and that mallard ducks are able to mount immunological responses protective against heterologous infections. because the transmission dynamics of lpaivs in wild populations is greatly influenced by individual susceptibility and herd immunity, these findings are of high importance. our study also shows the relevance of using telemetry to monitor disease in animals. influenza a viruses (iavs) have a wide range of host species, including humans, pigs, horses, wild mammals, and birds. their classification relies on two antigenic surface proteins, the hemagglutinin (ha) and the neuraminidase (na) for which 16 and 9 different subtypes are known, respectively. because many combinations of ha and na have been found in wild waterfowl and the prevalence in these species is high worldwide, they are considered the natural reservoir of iavs [1] . prevalence is particularly high in dabbling ducks (i.e. ducks that feed by tipping into the water to graze on aquatic vegetation or feed on small aquatic preys), probably because their feeding behavior favors ingestion of viral particles. in ducks, iavs usually replicate in the epithelial cells of the intestinal tract and are excreted at high concentrations from the cloaca into water [2] [3] [4] . the main transmission route in waterfowl is oro-fecal [5] but indirect contamination by ingestion of contaminated water may also play a role in yearly infection dynamics [6] . the peak of iav isolation occurs during or just prior to the autumn migration, a time when many immunologically naïve juveniles share water with adult birds from different breeding areas [1, 7] . understanding the ecology of iavs in their natural hosts is essential for several reasons. first, low pathogenic avian influenza virus (lpaiv) strains of the h5 and h7 subtypes, which are known to circulate in wild birds, may become highly pathogenic (hpai) if introduced into poultry and cause high morbidity and mortality with severe economic consequences [1] . second, wild birds represent a reservoir of iavs that may reassort with human viruses in pigs or other so-called ''mixing vessel'' hosts and ultimately generate strains with pandemic potential [8] . finally, it has been suggested that wild birds might be involved in the spread and transmission to humans of iav strains with direct zoonotic potential [9] . influenza a virus transmission dynamics in waterfowl may vary depending on whether (1) hosts are affected by infection and (2) herd immunity exists in the population [10] . these two questions were addressed by using an experimental infection approach. the wild type mallard (anas platyrhynchos) was chosen as an experimen-tal model because it is the most widespread and abundant migratory dabbling duck from which iavs are frequently isolated both in eurasia and north america [11, 12] . previous experimental infection studies on mallards or closely related domestic ducks (i.e. pekin ducks, anas platyrhynchos domesticus) revealed that infection by iavs generally does not adversely affect this natural host [3, 4, [13] [14] [15] [16] [17] [18] . however, a recent field study found a lower body mass in mallards positive for iav compared to negative mallards [19] indicating that there might be an ecological cost of infection despite the absence of obvious clinical signs. previous successive experimental studies reported the existence of a relative protection against ha homologous [3, 18] and heterologous [18] re-infections. in this study, we tested the hypotheses that lpai infection in mallards (1) may have clinical effects on its host; (2) provides shortterm homosubtypic immunity (i.e. immunity against re-infection by the same lpai strain); (3) provides short-term heterosubtypic immunity (i.e. cross-protective immunity to a heterologous lpaiv subtype). six juvenile male mallards caged individually and continuously monitored by telemetry for body temperature, heart rate, and activity, were successively inoculated with an h7n7 lpai isolate (day 0), the same h7n7 virus again (day 21) and an h5n2 lpai isolate (day 35) (figure 1 ). we found that birds infected by a lpaiv (1) may develop a slight and transient increase in body temperature, (2) are immune to homosubtypic reinfection and (3) may be immune to heterosubtypic re-infection. the six mallards appeared to be unaffected by the successive virus inoculations. they were active, ate and drank normally, and gained weight during the course of the study. baseline values for body temperature, heart rate and activity were recorded for each duck during the control period and used as pre-challenge references (table 1) . after the first h7n7 inoculation, day to day comparisons were performed using general linear models (glms) and tukey's post hoc test, each duck being used as its own control. for four ducks (m1-m4), day-to-day comparison showed a significant (p,0.001) increase in body temperature on the day their viral rna excretion started (table 2 ). this increase in body temperature was recorded by both dsi and ibutton data loggers (mean = 0.5uc and sd = 0.1uc for both data loggers). a few other significant (p,0.05) day-to-day changes in body temperature, heart rate and activity were recorded but the timing (i.e. days) of these changes was not consistent among ducks ( figure 2 ). the six implanted ducks gained weight throughout the study period (paired t-test: t = 212.2, df = 5, p,0.001) with a mean figure 3 ). day-to-day comparisons did not reveal an effect of infection on body mass. throughout the study period, the total number of samples positive for the matrix gene by real-time reverse transcription polymerase chain reaction (rrt-pcr) was 53 for oral swabs, 65 for water samples, 75 for feces, and 80 for cloacal swabs. the proportion of positive differed among sample types (x2 = 8.46; df = 3; p = 0.037) and pair-wise comparisons with bonferroni-corrected p-values showed that oral swabs were less frequently positive than cloacal swabs (x2 = 7.35; df = 1; p = 0.021). primary h7n7 inoculation. five of the six ducks excreted viral rna in their feces on the first day post-inoculation (pi) and all samples (feces, cloacal and oral swabs) from all birds were positive on the second day pi (figures 4 and s1 ). finally, viral rna was detected in all sample types (fecal, cloacal, oral, and water) three days pi. considering all sample types, continuous shedding was recorded on average for 12.0 days (sd = 1.1) and intermittent shedding for another 3.7 days (sd = 3.1). table 2 . mean daily body temperature values (in uc) recorded by the data loggers after the three successive challenges. secondary h7n7 inoculation. intermittent and moderate (high ct-values) viral rna shedding was detected for all birds in water, fecal or cloacal samples between day 1 and 7 after h7n7 re-inoculation ( figure 4 ). conversely, the control duck (m7) became infected and shed viral rna with a pattern similar to that observed for the six implanted ducks after h7n7 primary inoculation ( figures 5 and s2 ). h5n2 inoculation. no viral rna shedding was recorded for two birds (m1 and m2) whereas low viral rna excretion (high ctvalue) was detected in a single swab for three birds (m4, m6 and m5). these samples were low-positive by the rrt-pcr method targeting the matrix gene but negative by the h5 and h7 rrt-pcr methods (which are less sensitive). it was therefore not possible to determine if the viral rna excreted by these three ducks was from the h5 or h7 isolate. one duck (m3) excreted h5 viral rna from day 3 to 6 after h5n2 inoculation (figures 4 and s1) with a pattern similar to the h5-inoculated control bird (m8) ( figures 5 and s2 ). all samples from this duck were negative by h7 rrt-pcr. primary h7n7 inoculation. antibodies were detected in all birds both by np-and h7-elisa one week after the first h7n7 inoculation ( figure 6 ). h7-specific hemagglutination inhibition (hi) antibodies titers were ,20 except for m1 at 13, 16 and 20 days pi (titer 20) and m3 at 20 days pi (titer 40). secondary h7n7 inoculation. for all birds, h7n7 reinoculation was followed by an increase in h7-and, to a lesser extent, np-elisa inhibition values ( figure 6 ). h7-specific hi antibodies at titers $20 were detected in four birds (m1, m3, m4 and m6). h5n2 inoculation. antibodies remained detectable by npand h7-elisa in all birds ( figure 6 ) but only m3 had h7-specific hi antibodies at titers $20. h5-specific antibody production was only detected in m3 by h5-elisa, which was in accordance with the h5n2 viral rna detection in this bird. h5-specific hi antibodies titers were ,20 in all birds. the six implanted ducks (m1-m6) were euthanized at the end of the study (51 days pi) whereas the two control ducks (m7 and m8) were euthanized 7 days post-challenge. no lesion potentially associated with lpaiv infection was detected grossly or histologically in any of the ducks and no iav antigen was detected by immunohistochemistry in any of the tested organs. two birds had a mild proctitis and colitis with infiltration of mononuclear cells (primarily lymphocytes) in the lamina propria, likely a result of the presence of spirochetes. varying amounts of spirochetes were seen in the caecum of all mallards, which confirmed the positive screening previously observed on feces. a subcutaneous granulomatous inflammation was observed around the transponder in four birds. the overall response to infection was minimal. all six implanted mallards remained alert with neither clinical nor pathological signs of disease both after the lpaiv h7n7 primary inoculation and after the successive re-inoculations. their activity (i.e. movements per minutes) monitored by dsi transponders was not modified after inoculation and heart rate values remained in agreement with mean values reported for mallards [20] . only a brief (,2 days) and small (,0.5uc) increase in body temperature was recorded in four of the implanted birds on the day they started shedding viral rna in their feces. because fever is known to be monophasic in the closely related pekin duck [21] , it is possible that the four individuals which showed an increased body temperature developed a slight short-term fever during the h7n7 primary infection. the minimal response detected in this study does not allow concluding whether infection with lpaivs may have a significant impact on mallard populations. ecologic studies in a natural environment are needed to assess if the increase in body temperature observed during early infection is associated with ecological costs. developing fever and mounting an immunologic response are physiologically costly and, in situations in which animals are resource limited and exposed to predators, these costs may have to be balanced against other expenses such as reproduction, growth, molt, or migration [22, 23] . previous studies reported that female mallards experimentally infected with a lpaiv isolate significantly decreased egg production during the following week [24] and field investigations showed that lpaiv infected mallards have a lower body mass than negative mallards [19] . a correlation between iav infection and migration success has also been suspected in bewick swans [25] but field investigations on mallards captured in southeast sweden failed to show a correlation between infection and migration speed or distance [19] . during autumn migration, juvenile mallards get in contact with congeners coming from other areas and may successively be exposed to various iav subtypes [7, 12] . this study had a design to reflect these relatively short-term (i.e. within season) re-exposures. because the samples were only tested by rrt-pcr, it is not known for how long infective viral particles were excreted but we may speculate from previous studies that viral isolation would likely have been successful for samples with low ct-values (i.e. ct-values ,30) [26, 27] . positive oral swabs and water samples were likely the result of an environmental contamination by viral particles shed in feces. homologous re-inoculation of the h7n7 isolate 21 days after primo-inoculation induced a weak secondary antibody response and the shedding pattern was very different from that observed after primo-inoculation, with only intermittent detection of viral rna in feces, pool water or cloacal swabs. as previously reported in experimental infections of mallards with lpaiv strains, hi antibody titers were low, confirming that elisa sensitivity exceeds that of hi test and that protection against homologous re-infection exists despite the absence of significant hi titers. heterologous challenge with an h5n2 isolate from a wild mallard was performed 14 days after the homologous h7n7 re-inoculation. active h5 infection was confirmed only in one duck, by expression of h5-specific antibodies and detection of viral rna in the various sample types (feces, water, oral and cloacal swabs) with a pattern similar to the h5-inoculated control bird. in all other ducks, h5-specific antibodies were not detected and no or only intermittent viral rna shedding was observed after the heterologous challenge. in these ducks, prior infection with h7n7 lpaiv seems to have provided protection against infection by h5n2 lpaiv. this result supports speculations from sharp et al. [28] who suggested that, if infections with two welladapted heterosubtypic viruses occur at different times, the first infectant may prevent the replication of a later infectant. another study on mallards recently reported a protective effect of infection by an h4n6 lpaiv strain prior to exposure to an hp h5n1 [18] . further investigations with other viral strains and different challenge timings are needed to better assess the importance of heterologous immunity in ducks. in chickens, gut immune responses induced by exposure to lpaivs are suspected to play an important role in protection from the lethal effects of hpai strains [29] . likewise, local immunity may influence the outcome of duck infections and provide protection against further infections. in mallards, hi antibodies were detected in the bile and peaked at about 12 days pi, i.e. approximately the moment when cloacal shedding ceased [30] . additionally, cellular immune response was shown to play a role in the protection of chickens [31] and turkeys [32] against heterologous infections and may be important in the protection of ducks [29, 33] . inter-individual variations in immunity also likely exist, as revealed in our study by replication of the heterologous re-infectant virus in one duck. because the heterologous challenges were separated by a short interval (2 weeks), rna excretion may have been influenced both by the host protective immune responses and by the impact of co-infection on virus replication. further investigations about viral interactions, cellular and local immunity are needed to understand when heterologous re-exposure leads to co-infection compared to when elimination of the re-infectant occurs. because concomitant infections may lead to genetic reassortment and emergence of new viral strains, these studies are essential in understanding the overall ecology of aivs. this study showed that in mallards (1) a mild transient increase in body temperature may occur during lpaiv infection, (2) infection by a lpaiv is limited by prior infection with a homologous strain and (3) may be prevented by prior infection with a heterologous strain. the study also showed that (4) individual heterogeneity exists, both in the susceptibility to infection and the ability to develop heterosubtypic immunity. finally, the study showed that (5) viral rna intermittent shedding occurs and that (6) the daily shedding pattern differs among sample types (water, feces, cloacal and oral swabs). further investigations are needed to better assess the ecological costs of iavs on wild waterfowl populations because such costs may deeply influence the transmission dynamics of the viruses [10] . field studies based on frequent sampling of the same individuals are encouraged to gain a better knowledge on how frequently re-infections occur in nature and how they influence viral shedding patterns. experimental infections with longer time intervals between challenges (e.g. several months) would also be useful to investigate long-term immunity. such data will be essential to optimize virus transmission models and better understand iav ecology in their natural hosts. the animal experiment procedures were approved by the committee for laboratory animal science of the swedish board of agriculture. eight 3-month-old male wild-type mallards (anas platyrhynchos) of approximately the same body mass and size (measurement of the left wing, the right tarsus length and the distance from bill tip to back of the skull) were selected from a swedish duck farm known from previous successive sampling to be free from iav infection. the eight mallards were isolated from the other ducks for ten days before they were transferred to the animal house. absence of active shedding of influenza viruses was checked three times, at one-week intervals, by testing oral and cloacal swabs using rrt-pcr (see below for methods). absence of previous exposure to influenza viruses was also checked by np-elisa on sera sampled three successive times at one-weekintervals. the eight mallards were also checked for the presence of intestinal pathogens known to occur in mallards, i.e. intestinal parasites [34] , coronaviruses [35] , chlamydophila sp. [36] , and brachyspira sp. spirochetes [37] . they were free from infection, except for brachyspira sp., which were found in all ducks. culture of fecal material showed that the bacterial flora were normal [34] . the ducks were individually identified by unique color combinations of plastic rings placed around their right tarsus and randomly separated in two groups (figure 1 ). the six mallards assigned to the first group (referred to as m1, m2, etc… m6) were surgically implanted with data loggers (see below) and successively infected with two strains of iavs. the other two ducks (m7 and m8) were used as controls for virus infectivity during a 7-dayperiod. the study took place within the animal facility of the swedish institute for infectious disease control (smi) in stockholm, sweden. the mallards were kept in individual cages with access to an individual pool and a shelter and were fed an equal mixture of chicken food and crushed wheat and oat ad libitum. the cages were cleaned and the water from each pool changed every morning. all ducks were caged in the same room, except the controls for virus infectivity which were kept separately until the day they were challenged: m7 was introduced in the room on day 21 (and euthanized on day 28) whereas m8 was introduced in the room on day 35 (and euthanized on day 42). a dsi transmitter (data sciences international, model ta11-cta-f40), consisting of a plastic body and two electrodes, was surgically implanted under the skin of six ducks (m1 to m6). twice every minute during the whole study period, the dsi transmitter stored data on body temperature (uc), heart rate (beats per minute) and activity (movements per minute of the implanted device relative to the receiver) in a computer using dataquest a.r.t. data acquisition software, gold package. each transmitter was in contact with a receiver (model rpc-1) placed under the cages, and all six receivers were connected through two data exchange matrixes connected to the computer. details about dsi data acquisition system can be found in savory and kostal [38] . a preliminary study using lipopolysaccharide (as in maloney and gray [21] ) revealed that the system allowed detection of increased body temperature and heart rate (jourdain et al., unpublished data). as previously reported, handling of the ducks resulted in increased body temperature [21] and heart rate [20] , and body temperature was lower when the light was off [39] ; therefore, only data recorded during this time (7.00 pm to 6.00 am) were used in the analyses. in total, 76,061 data points were analyzed for each of the three dsi parameters (n m1 = 14099; n m2 = 7224; n m3 = 13837; n m4 = 27557; n m5 = 4668; n m6 = 8676;), corresponding to only 34% of the recording potential of the system. the remaining 66% of data were not recorded due to signal loss (i.e., cage area was larger than receiver range, which was ,40 cm). because dsi transmitters have not been used on mallards before, and to cover possible technical failures, a thermochron ibutton (maxim integrated products; model ds1922l), which records temperature into an internal memory [40] , was also surgically implanted under the skin of the six transmitterimplanted birds (m1 to m6). the ibuttons were programmed (using 1-wire drivers software, version 4.00) to measure body temperature (uc) every half hour throughout the study period. the ibuttons were recovered from the ducks after euthanasia and data were downloaded using 1-wire drivers software. an ibutton placed in the experiment room monitored the room temperature at the same frequency as for the duck body temperature and showed that room temperature was stable throughout the experiment (21.5uc, sd = 0.1). in total, the ibuttons recorded 6,216 body temperature data points, i.e. about 1,300 data points for each duck except m3 (n = 794) and m6 (n = 222) which lost their respective ibuttons before the end of the experiment (days 28 and 2 pi, respectively). surgery under general anesthesia was performed on six ducks (m1 to m6) to equip them with a dsi transmitter and an ibutton. food was withdrawn the night before surgery to avoid false deglutition during anesthesia. before cutting the feathers from the incision areas, each duck was sedated by intramuscular administration of meloxicam (metacamh, 0.5 mg/kg), butorphanol (turbogesich, 1 mg/kg) and xylazine (paxmanh, 1 mg/kg). anesthesia was induced using 3-4% isoflurane (foreneh) with an oxygen flow of 2-3 l/min administered via a small-dog narcomask. a local anesthetic cream (emlah) was applied on the different incision sites and an eye gel (lubrithalh) was applied in both eyes. during surgery, 2-2.5% isoflurane was administered with an oxygen flow of 1-2 l/min. after surgery, an oxygen flow of 4 l/min was used. once awake, the duck was placed back into its cage under an infrared light. to prevent infection of the wounds, a long-lasting antibiotic (amoxicillin la, vetrimoxinh, 15 mg/kg) was injected in the pectoral muscle both after surgery and two days later. meloxicam (0.5 mg/kg) was re-administrated one day after surgery to minimize inflammation. access to the pool was prevented until 2-3 days after surgery. the body of the transponder and the electrocardiogram leads were positioned in a base-apex configuration as described by harm and colleagues [41] . body feathers above and around the incision areas were cut with bended scissors. incision areas were then cleaned and disinfected with povidone iodine, and an anesthetic cream was applied. a dorsal skin incision of approximately 3 cm was made at the base of the neck, between the shoulders. the ibutton was inserted through this incision and, after blunt dissection, was placed subcutaneously on the left part of the back. the body of the dsi transmitter was inserted through the same incision and attached to the skin by two stitches using non-absorbable surgical suture (supramid 3/0, braun). a second incision was made at the base of the right wing. the negative electrode was then tunneled with a trocar until it reached the wing incision. it was fixed to the muscular fibers by 3-4 stitches of absorbable surgical suture (vicryl 3/0, ethicon). the positive electrode was similarly fixed to the left pectoralis major muscle after being tunneled to the abdomen. all skin incisions were sewn up with an absorbable surgical suture (vicryl rapid 3/ 0, ethicon). the ducks were allowed to recover from surgery for at least 10 days prior to starting the monitoring of individual data (figure 1 ). the experiment was divided into four successive periods, during which the six implanted mallards were monitored continuously (body temperature, heart rate, activity) and weighed and sampled daily (figure 1 ). the first period (1 week) allowed monitoring baseline body temperature, heart rate and activity levels for each mallard. the second period (3 weeks) aimed at studying the effects of primo-infection with an h7n7 lpaiv strain inoculated in the esophagus (10 8.7 eid 50 in a 1 ml inoculum). this three-week-period corresponds to the maximum time during which iavs are usually excreted by infected ducks [3, 17, 30, 42] . the third period (2 weeks) investigated the impact of re-inoculation with the same h7n7 lpaiv strain administered through the same route and at the same dose. a naïve mallard (m7) was simultaneously inoculated (through the same route and at the same dose to serve as a positive control) and necropsied 7 days later to search for lesions associated with infection by the h7n7 isolate. the fourth period (2.5 weeks) allowed studying the effects of heterologous inoculation in the esophagus with an lpaiv h5n2 strain (10 8.7 eid 50 in a 1 ml inoculum). as previously, a naïve mallard (m8) was used as a positive control. it was inoculated along with the other ducks and necropsied 7 days later to search for lesions associated with infection by the h5n2 isolate. the six implanted ducks were euthanized 51 days after the first inoculation and necropsied. two lpaiv strains isolated in 2004 from wild mallards at ottenby, southern sweden, were used: a/mallard/sweden/7206/ 2004 (h7n7) and a/mallard/sweden/6566/2004 (h5n2). new viral stocks were grown by inoculating 200 ml of the selected isolates (dilution 1:50 in pbs) in the allantoic cavity of 10-day-old embryonated chicken eggs. the corresponding allantoic fluid was harvested three days later, centrifuged and pooled. viral titers were determined by 50% embryo infectious dose (eid 50 ) using the method of reed and muench [43] . water samples, feces, oral and cloacal swabs were collected daily and blood samples bi-weekly throughout the study, from day 27 to 51 ( figure 1 ). every morning, before the cages were cleaned, 40 ml of water was sampled from each pool and stored directly at 280uc. the mallards were placed in individual single-use paper boxes for a few minutes before being sampled. they were swabbed from the cloaca and oral cavity and fecal samples were collected by rolling a sterile cotton swab in the fresh droppings left in the paper box. the swabs were placed in 1 ml of virus transportation medium (hanks balanced salt solution) as described in wallensten et al. [7] and kept on ice until they were stored at 280uc. the ducks were bled biweekly, alternating between the right and left brachial veins, for serological analyses. after centrifugation, sera were stored at 220uc. biosafety precautions were used between handling the ducks by spraying the gloves, table and lab coats with an alcoholic solution. before their inclusion in the study (on day 21 and 35, respectively), the control ducks m7 and m8 were handled before the other ducks and in a separate room. matrix gene rrt-pcr for fecal samples, cloacal and oral swabs. after thawing, the tubes were thoroughly vortexed and 150 ml were removed and mixed with 450 ml trizol reagent (invitrogen, paisley, uk) for virus inactivation. cold chloroform (160 ml) was added to yield an excess of 300 ml needed for rna extraction. after vortexing, the water and organic phases were allowed to separate for 1-2 minutes after which the tubes were centrifuged at 14000 g for 15 minutes. the water phase (300 ml) was then removed and rna extracted using the m48 biorobot (qiagen, hilden, germany) with the magattract viral rna m48 extraction kit (qiagen) according to the manufacturer's specifications and eluted in 65 ml. a rrt-pcr system targeting the matrix gene was used for screening and quantification [44] . the pcr was run in a one-step fashion using quantitect probe rt-pcr kit (qiagen) according to the manufacturer's specifications for abi 7900ht pcr machine (applied biosystems, carlsbad, usa) and 5 ml rna was used as template. cycle threshold (ct-) values obtained were normalized by setting threshold value to 0.1 for all runs. a ct-value of ,40 was considered positive for lpaiv antigen. matrix gene rrt-pcr for water samples. a slightly different protocol was used for water samples. after thawing, the tubes were thoroughly vortexed and 400 ml were removed for direct rna extraction using the magattract viral rna m48 extraction kit with the biorobot m48 set to obtain 75 ml of elution volume. a lightcycler 1.5 (roche diagnostics gmbh, germany) was used to perform the thermo-cycling. a ct-value of ,40 was considered positive for lpaiv antigen. h5 and h7 rrt-pcr. only water and swab samples collected from the implanted ducks after day 35 (h5n2 inoculation) and positive for the matrix gene (n = 13) were further tested for the presence of h5 or h7-specific viral rna. rna was extracted as before and rrt-pcr performed using a taqman probes system as described by eu community reference laboratory protocols [45] . serum samples were tested for antibodies targeting iav nucleoprotein (np) with a commercial elisa kit (np-elisa, pourquier avian influenza a blocking elisa, montpellier, france). the presence of h7-and h5-specific antibodies was studied by using the id screen influenza h7 antibody competition and id screen influenza h5 antibody competition (id vet innovative diagnostics, montpellier, france). according to the manufacturers' instructions, sera were considered positive if the calculated value was $65% (np-elisa) or $50% (h7-and h5-elisa). in order to detect hi antibodies specific for h5 and h7 respectively, hi test was performed following standard procedures [11] , using red blood cells from specific pathogen-free chickens and four ha units of the viral strains used for the experimental challenge. only samples with a titer $20 were considered. the two mallards that served as positive controls (m7 for h7n7 infection and m8 for h5n2 infection) were euthanized 7 days pi with h7n7 or h5n2, respectively, whereas the six implanted mallards were euthanized at the end of the study (51 days pi). the birds were euthanized with 100 mg/kg sodium pentobarbital (pentobarbital veth, 100 mg/ml i.v.). routine necropsies were carried out directly after euthanasia. tissue samples (brain, lungs, trachea, air sacs, heart, liver, spleen, kidneys, pancreas, adrenal glands, duodenum, jejunum, colon, caecum, ventriculus, proven-triculus, and testicles) were fixed in 10% neutral buffered formalin for histopathology and immunohistochemistry (ihc). after formalin fixation, the tissue samples were processed routinely, sectioned at 4-5 mm, stained with hematoxylin and eosin, and examined for pathologic changes. in order to investigate the presence of viral antigen, duplicate sections of liver, lung, trachea, air sacs, pancreas, duodenum, jejunum, colon, and caecum from all ducks, as well as all other collected tissues from the two control ducks, were processed for ihc using a commercial anti-influenza a nucleoprotein primary monoclonal antibody [46] . each immunostain included a positive reference control and a negative control. each section was also accompanied by a primary antibody-omitted control. sections from all levels of intestine were further stained with warthin-starry silver stain in order to investigate the presence of spirochetes. statistical analyses of the physiological parameters were done individually for each implanted mallard. multivariate general linear models (glms) were used with mean values for each hour and day of dsi body temperature, activity, and heart rate as dependent variables, and day and hour as independent variables. the latter variable was included to control for the fact that the physiological variables varied over time. additional univariate glms were run with ibutton body temperature as the dependent variable and the same predictors as above. for three ducks (m1, m3 and m4), data were transformed to obtain normally distributed residuals. day was a significant variable (p,0.001) for all ducks and dependent variables (except for the heart rate of m2). because it was biologically relevant to compare values obtained on successive days, we used tukey's post hoc test to compare the means from the control period with the means from day-one-pi, day-one-pi with day-two-pi, etc… until the end of the experiment. body mass changes were analyzed on a daily basis by comparing each day with the previous one as described above. because body mass is naturally dependent on preceding values, paired t-tests with bonferroni-corrected p-values were used. in addition, paired t-tests were used to get an overview of how the body mass of each duck changed over the study period. in order to show whether there was any difference in virus detection between the four sample types (oral and cloacal swabs, feces and pool water), the proportion of positive samples for the different techniques was analyzed with x2-statistics. only days for which all sample types from a given duck had been tested were included in this analysis. global patterns of influenza a virus in wild birds intestinal influenza: replication and characterization of influenza viruses in ducks duck influenza lacking evidence of disease signs and immune response virus replication in the digestive tract of ducks exposed by aerosol to type-a influenza evolution and ecology of influenza a viruses water-borne transmission drives avian influenza dynamics in wild birds: the case of the 2005-2006 epidemics in the camargue area surveillance of influenza a virus in migratory waterfowl in northern europe the role of swine in the generation of novel influenza viruses wild ducks as long-distance vectors of highly pathogenic avian influenza virus (h5n1) evolutionary biology, community ecology and avian influenza research mallards and highly pathogenic avian influenza ancestral viruses influenza a viruses of migrating wild aquatic birds in north america avian influenza virus infections. iv. response of pheasants, ducks, and geese to influenza a-turkey-wisconsin-1966 virus antigenic and phenotypic variants of a virulent avian influenza virus selected during replication in ducks detection of influenza a virus by rt-pcr and standard methods in experimental infection of ducks pathological lesions in the lungs of ducks infected with influenza a viruses experimental exposure of franklin's gulls (larvus pipixcan) and mallards to a turkey 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of wild ducks by influenza a viruses: distribution patterns and biological significance immunology of avian influenza virus: a review bile immunoglobulin of the duck (anas platyrhynchos). ii. antibody response in influenza a virus infections protective cross-reactive cellular immunity to lethal a/goose/guangdong/1/96-like h5n1 influenza virus is correlated with the proportion of pulmonary cd8(+) t cells expressing gamma interferon cellular immune response in turkeys inoculated with two antigenically indistinguishable avian influenza-a viruses effect of an h5n1 avian influenza virus infection on the immune system of mallard ducks fecal shedding and antimicrobial susceptibility of selected bacterial pathogens and a survey of intestinal parasites in free-living waterfowl molecular identification and characterization of novel coronaviruses infecting graylag geese (anser anser), feral pigeons (columbia livia) and mallards (anas platyrhynchos) chlamydial infections in duck farms associated with human cases of psittacosis in france brachyspira hyodysenteriae and other strongly beta-haemolytic and indolepositive spirochaetes isolated from mallards (anas platyrhynchos) application of a radiotelemetry system for chronic measurement of blood pressure, heart rate, eeg, and activity in the chicken avian febrile response thermochron ibuttons: an inexpensive method for long-term recording of core body temperature in untethered animals a technique for dorsal subcutaneaous implantation of heart rate biotelemetry transmitters in black ducks: application in an aircraft noise response study role of domestic ducks in the propagation and biological evolution of highly pathogenic h5n1 influenza viruses in asia a simple method of estimating fifty percent endpoints development of a real-time reverse transcriptase pcr assay for type a influenza virus and the avian h5 and h7 hemagglutinin subtypes veterinary laboratory agency. pcr protocols for the detection of avian influenza virus pathology of natural highly pathogenic avian influenza h5n1 infection in wild tufted ducks (aythya fuligula) we thank christine leterrier (inra tours, france), jordi altimiras (linköping university, sweden) and géraldine bruyère for advice about data loggers and surgery; olov rosendahl and his colleagues from the animal house (smi stockholm, sweden) for excellent animal care and sampling; jenny olofsson, jorge hernandez and conny tolf (linnaeus university, sweden) for participation in laboratory analyses; kerstin falk (smi, sweden), anders larsson and patrik ellström (uppsala university, sweden), dolores gavier-widen (sva uppsala, sweden) and leslie reperant (princeton university, nj, usa) for relevant advice regarding experimental set up; aleksija neimanis (sva uppsala, sweden) for assistance with the necropsies. we thank nicole nemeth and two anonymous reviewers for their comments on the manuscript. key: cord-338067-vjyad10p authors: hao, yan; xu, ting; hu, hongping; wang, peng; bai, yanping title: prediction and analysis of corona virus disease 2019 date: 2020-10-05 journal: plos one doi: 10.1371/journal.pone.0239960 sha: doc_id: 338067 cord_uid: vjyad10p the outbreak of corona virus disease 2019 (covid-19) in wuhan has significantly impacted the economy and society globally. countries are in a strict state of prevention and control of this pandemic. in this study, the development trend analysis of the cumulative confirmed cases, cumulative deaths, and cumulative cured cases was conducted based on data from wuhan, hubei province, china from january 23, 2020 to april 6, 2020 using an elman neural network, long short-term memory (lstm), and support vector machine (svm). a svm with fuzzy granulation was used to predict the growth range of confirmed new cases, new deaths, and new cured cases. the experimental results showed that the elman neural network and svm used in this study can predict the development trend of cumulative confirmed cases, deaths, and cured cases, whereas lstm is more suitable for the prediction of the cumulative confirmed cases. the svm with fuzzy granulation can successfully predict the growth range of confirmed new cases and new cured cases, although the average predicted values are slightly large. currently, the united states is the epicenter of the covid-19 pandemic. we also used data modeling from the united states to further verify the validity of the proposed models. infectious diseases are caused by various pathogens that can be transmitted from person to person, animal to animal, or person to animal. they can be transmitted in various ways, and the speed of transmission is fast. early diagnosis of infectious diseases is crucial, and prevention and control are paramount. in people infected with the 2019-ncov will experience varying symptoms, including fever, mild cough, or pneumonia, occasionally leading to death. the mortality rate of covid-19 is approximately 2% to 4%, although this is an extremely early percentage and may change as more information becomes available. meanwhile, this does not mean that the virus is not serious, it simply means that not everyone infected with it will face the worst outcome. we are currently in a tense state of prevention and control, and are concerned about a global pandemic. 2019-ncov is an intractable and unexpected virus with the following characteristics. first, it has a strong ability to camouflage itself. the virus can be present asymptomatically, making an infected person appear healthy, or can manifest with various other symptoms in which an infected person seems to be suffering from a common respiratory disease. it also has a long latency period. according to a report from lancet, the median latency time of the new coronavirus is approximately 20 days, reaching up to 37 days for some patients. in addition, it is transmitted in various ways. the transmission method of the 2019-ncov is similar to that of various other infectious diseases. in addition to a traditional droplet transmission, it also includes contact, air, fecal-oral, and blood transmissions. a high relapse rate has also been seen. since the initial outbreak of covid-19, there have been many cases of recovered patients testing positive again after a reexamination, even leading to death after a relapse. the virus has also shown variability. as a foreboding aspect of the future development of novel coronaviruses, their rapid genetic recombination results in a mutation into new strains. each recombination can increase the toxicity and infectivity, invalidating original treatment methods and drugs. forty variants of novel coronaviruses have been discovered by scientists in iceland. finally, it is highly infectious. scientific data have shown that the affinity between the sprotein of 2019-ncov and angiotensin converting enzyme 2 (ace2) is 10 to 20 times that of sars, which means that the infectivity of covid-19 is significantly higher than that of sars. there are currently no specific treatments for covid-19. however, many symptoms can be treated, and treatment must be given according to the clinical status of the patient. in addition, supplementary care for those infected may be effective. self-protection includes maintaining basic hand and respiratory hygiene, adhering to safe eating habits, and avoiding close contact with anyone who shows symptoms of a respiratory disease (such as coughing or sneezing). at present, owing to the widespread nature of covid-19, factories are being shut down, schools are being suspended, and people are isolating in their own homes, significantly disrupting daily life. it is therefore extremely important to reasonably predict and analyze the development trend of this pandemic. in previous studies, the prediction methods for the occurrence, spread, and change in infectious diseases mainly included regression prediction models [1, 2] , markov chain models [3] , bayesian networks [4] [5] [6] , and other machine learning methods [7] [8] [9] . most of the previous studies have been based on research related to influenza, with a few studies conducted on human immunodeficiency virus (hiv) infection [10, 11] and sars [12, 13] . for example, ray [14] used ensemble methods with three component models to predict the seasonal timing of influenza and severity in the united states. xue et al. [15] established five models for predicting and assessing influenza outbreaks across 10 regions of the united states. experiments on google flu trends (gft) and centers for disease control (cdc) data have shown that gft and historical influenza data are partially complementary and that gft +cdc regression is a preferable model. in addition, alkouz et al. [16] proposed a system called tweetluenza, which can predict the spread of influenza in real time. many scholars have published reports on covid-19. for example, in [17] [18] [19] [20] , its clinical features are described, and in [19] and [20] , the authors demonstrated that covid-19 has a high comorbidity with hypertension, diabetes, and cardiovascular diseases. anastassopoulou et al. [21] estimated the case fatality and case recovery ratios based on a susceptible infectiousrecovered-dead (sidr) model, along with their 90% confidence intervals as the outbreak evolves. in [22] [23] [24] [25] [26] [27] , the authors use machine learning methods to diagnose covid-19 based on x-ray and ct images. randhawa et al. [28] also used machine learning-based methods to achieve ultra-fast, scalable, and highly accurate classification of whole covid-19 virus genomes based on the covid-19 intrinsic virus genomic signature. in [29] [30] [31] , other characteristics related to covid-19 were studied. to date, few studies related to the use of quantitative prediction methods to predict the development trend and growth range of covid-19 have been conducted. in the present study, three methods, namely, an elman neural network, lstm, and svm are applied to predict and analyze covid-19 data from january 23, 2020 to april 6, 2020 in wuhan, hubei province, china, including cumulative confirmed cases, confirmed new cases, cumulative deaths, new deaths, and cumulative cured cases and new cured cases. first, the elman neural network, lstm, and svm were used to predict the development trend of cumulative confirmed cases, cumulative deaths, and cumulative cured cases. next, the svm with fuzzy granulation was used to predict the growth range of confirmed new cases, new deaths, and new cured cases. experimental results showed that the elman neural network and svm adopted in this study can accurately predict the development trend of covid-19, whereas lstm is more suitable for the prediction of cumulative confirmed cases. the svm with fuzzy granulation is effective for the growth range prediction of confirmed new cases, new deaths, and new cured cases, despite the larger averages. the same models were also used on data in the united states to verify the robustness of the models. the rest of this paper is organized as follows. section 2 introduces the methods applied. section 3 describes the experiments and analysis results. finally, some concluding remarks and areas of future research are provided in section 4. there are two main research topics in this paper. the first is the development trend prediction and analysis of the cumulative confirmed cases, deaths, and cured cases. the second is the growth range prediction of the confirmed new cases, new deaths, and new cured cases. because the present state of cumulative confirmed cases, deaths, and cured cases is related to the state of the previous day, and shows a non-linear growth, recurrent neural networks (rnns) are applicable. in this study, we mainly apply the elman neural network and lstm. in addition, the svm has a better prediction effect on non-linear data, therefore, the svm model is also introduced for development trend prediction. because there are many factors affecting the growth of new cases, the growth trend fluctuates significantly. here, only the growth range of the new cases is predicted. to predict the change space of the three datasets more accurately, this paper introduces a svm model with fuzzy granulation. the elman neural network is generally divided into four layers: input, hidden, receiving, and output layers. the receiving layer is also called the context layer or state layer, and is used to memorize the output value of the hidden layer unit at the previous moment and return it to the network input. it can be considered a one-step delay operator. as the characteristic of the elman neural network, the output of the hidden layer is self-connected to the input of the hidden layer through the delay and storage of the receiving layer. this self-connected method makes it sensitive to historical data. the addition of an internal feedback network enhances the ability of the network to process dynamic information, thereby achieving the purpose of dynamic modeling. the network structure of the elman neural network is shown in fig 1. the connections of the input layer, hidden layer, and output layer are similar to those of a feedforward network. unlike a feedforward network, the elman neural network contains a receiving layer, which takes the hidden layer state of the previous moment together with the network input of the current moment as the input of the hidden layer. here, x c (k) = x(k − 1) indicates that the feedback state vector is equal to the hidden layer state at the previous moment. the hidden layer state is expressed as follows: the output layer state is expressed as where u is the input vector, x c is the feedback state vector, w 1 is the weight of the receiving layer to the hidden layer, w 2 is the weight of the input layer to the hidden layer, w 3 is the weight of the hidden layer to the output layer, f( � ) is the activation function of the hidden layer, and g( � ) is the activation function of the output layer. lstm networks are variants of rnns. general rnns only have short-term memory owing to a disappearance of the gradients. lstm networks combine short-term and long-term memory through a subtle gate control and solve the problem of disappearing gradients to a certain extent. lstm was proposed by hochreiter and schmidhuber [32] in 1997. it has three special structures: a forget gate, an input gate, and an output gate. lstm networks can delete or add information to a cell state through a structure called a gate. fig 2 shows a diagram of the lstm network. the diagram shows the details of the forget gate, input gate, and output gate. the first step is to decide what information to discard from the cell state, which is done through a layer called the forget gate. the forget gate reads the hidden state of the previous moment h t−1 and the current input data x t , and then outputs a vector between zero and 1. the value between zero and 1 in this vector indicates how much information is retained or discarded in the cell state c t . a value of zero means all information is discarded, and 1 means all information is retained. the next step is to decide how much new information is added to the cell state. two steps are needed to achieve this. first, we use h t−1 and x t to decide which information to update through an input gate operation. we then use h t−1 and x t to obtain a new candidate cell state z through a tanh layer. next, the cell state is updated as follows: finally, we need to determine the output value. after updating the cell state, we need to determine which part of the cell state will be output according to the input h t−1 and x t . here, we need to pass the input through a sigmoid layer called the output gate to obtain the judgment conditions. we then need to pass the cell state through the tanh layer to obtain a vector between -1 and 1, which is multiplied by the judgment conditions obtained by the output gate to obtain the output. where z i , z f , and z o are the gate control state of the forget gate, input gate, and output gate, respectively. in addition, z is the input through a tanh layer, which is a so-called candidate cell state. finally, � represents a multiply operation of the corresponding elements of the matrix. svm is a classic model that can be used not only for classification but also for regression. we do not delve into its theoretical derivation here, however. unlike with a classification problem, the output of the regression problem is no longer a discrete value, but a continuous value. in reality, it is often impossible to accurately predict the value of the covid-19, and to this end, it is particularly important to predict the development trend and change space for the important parameters of this disease. without considering other factors, time is clearly an important independent variable affecting covid-19. to more accurately predict the change space of each group of data on this disease, in this paper, a fuzzy granulation model is introduced. first, we divide the original data into multiple granulation windows and then apply fuzziness to each window. the so-called fuzzy granulation means that the information granulation adopted is based on the model of fuzzy set theory. the key to fuzzy granulation is fuzzification, here, we use the pedrycz's granulation method [33] . for a given time series, we consider the entire time series x as a window and then apply fuzziness to it. fuzzification is a task used to establish a fuzzy particle p on x, that is, a fuzzy concept g that can reasonably describe x. fuzzy particles can reasonably represent the original data and have certain specialties. a fuzzy particle p can be described simply as p = a(x), where a is a membership function of the fuzzy concept g. commonly used fuzzy particles include triangular, ladder, gaussian, and parabolic particles. in this study, we choose triangular fuzzy particles, the membership function of which is as follows: here, m is the kernel of triangular fuzzy particles, and a and b are the lower and upper bounds of the support, respectively. we studied covid-19 data from january 23, 2020 to april 6, 2020, which were published by the wuhan municipal health commission (http://wjw.wh.gov.cn/) [34] . we used the elman neural network, lstm, and svm to predict and analyze the development trend of cumulative confirmed cases, cumulative deaths, and cumulative cured cases. as an additional study, we also used the svm with fuzzy granulation to predict and analyze the growth range of confirmed new cases, new deaths, and new cured cases. to further demonstrate the robustness of the models, we used the same models on data from the united states from march 23, 2020 to june 5, 2020. the data were obtained from the world health organization (who) [35] . the data from the united states contains confirmed cases, deaths while without cured cases. all experiments were conducted in matlab (r2014a) . fig 3(a) shows a line chart of the confirmed new cases. from fig 3(a) , it can be seen that, from january 23, 2020 to february 11, 2020, the number of confirmed new cases showed a fluctuating upward trend. confirmed new cases fluctuated sharply on february 12, showing a peak, the reason for which was the announcement of 12,364 clinically confirmed cases for the first time on that day, with a sharp increase in confirmed new cases. fig 3(b) shows a line chart of the new deaths and cured cases. the number of new cured cases showed an increasing trend before february 27, after which the growth trend of the cured cases slowed. moreover, the number of new deaths showed a significant downward trend after february 24, demonstrating a promising future recovery. in this section, the elman neural network, lstm, and svm were used to predict and analyze the development trends of the cumulative confirmed cases, deaths, and cured cases. in the elman neural network and lstm, the experimental datas are in the form of the first three days to the fourth (i.e., using data from the first three days to predict the data on day four). thus, the original 75 groups of data are converted into 72 groups, and the last group of data are used as the test sample. according to the data, both the elman neural network and the lstm in this paper contain only one hidden layer. because the initial weights of the elman neural network and lstm were randomly initialized, the average of 20 experimental results is given here as the final outcome. in addition, the number of hidden layer neurons is an important factor. we chose 7, 11, 14, and 18 hidden layer neurons according to experience. fig 4 shows the error in the elman neural network based on these different numbers of hidden layer neurons. fig 4 shows that the effect of the number of hidden neurons on the error is not too obvious. by comparison, with 18 hidden neurons, the prediction result is slightly better. in addition, the prediction error of the cumulative confirmed cases is larger than that of the cumulative deaths and cumulative cured cases because the number of confirmed cases increased dramatically owing to the clinically confirmed cases announced on february 12, which has had a significant impact on the prediction results. fig 5 shows the error rates of the elman neural network and lstm when the number of hidden layer neurons is 18. the prediction errors of the two methods are listed in table 1 . as can be seen from fig 5 and table 1 , the prediction results of the lstm are worse than those of the elman neural network, particularly for cumulative cured cases. as is well-known, lstm is a powerful model for time-series prediction. however, the novel coronavirus pneumonia data have no obvious periodicity, and lstm has a relatively poor prediction effect on data with a weak periodicity. regarding the poor result of the cumulative cured cases, no effective treatment has been found, and fewer people were cured during the initial stage of the pandemic. in addition, it takes time between treatment and recovery. the treatment period varies from person to person. with continuous research, the number of cured cases continues to increase nonlinearly. for irregular growth, neural networks tend to fall into the local optimum, making it difficult to achieve an accurate prediction, which is also a major drawback of a neural network. in the svm experiment, we used a one-day-to-the-next approach, that is, we used the data from the previous day to predict data for the next day. in this study, data of 61 days from january 23 to march 23 were used as training samples, and data from march 24 to april 6 were used as test samples. the radial basis function was used in the experiments. fig 6 shows the prediction results of the cumulative confirmed cases, deaths, and cured cases. the prediction mean square error and square correlation coefficient of the cumulative confirmed cases, deaths, and cured cases are shown in table 2 . it can be seen from the results that the svm can predict the three sets of data well, and the square correlation coefficients of the cumulative deaths and cumulative cured cases between the predicted output and the actual output are all above 99%. although the mean square error of the cumulative confirmed cases is relatively large and the square correlation coefficient is relatively small, it can be seen from fig 6(a) that the fluctuation range of the cumulative confirmed cases from march 24 to april 6 is extremely small, and only two of the 14-day prediction results are incorrect. similar to the aforedescribed experiments, herein we predict and analyze the development trend of cumulative confirmed cases and deaths from the data in the united states. here, 12 hidden layer neurons of the elman neural network and lstm are applied. table 3 shows the prediction error of the two methods on data from the united states. in the svm experiment, data of 61 days from march 23 to may 22 were used as training samples, and data from may 23 to june 5 were used as test samples. the prediction results of the cumulative confirmed cases and deaths are shown in fig 7. the mean square error and square correlation coefficient of the cumulative confirmed cases and deaths are shown in table 4 . comparing the experimental results of wuhan and the united states, the elman neural network and svm can well predict the development trend of wuhan and the united states, whereas the prediction result of lstm for the united states is clearly worse than that of wuhan. this occurs because the data from the united states used in this study are national data, not data from a certain city, and the data growth is more irregular and without any periodicity. in addition to predicting the development trend of covid-19, we also used a svm with fuzzy granulation to predict the growth range of the confirmed new cases, new deaths, and new cured cases. taking 5 days as a granulation window, the data from january 23, 2020 to april 1, 2020 were used as the training samples, and the growth range of the next 5 days was predicted, that is, the growth range of the confirmed new cases, new deaths, and new cured cases from april 2 to april 6. to reduce the impact of the peak value on the experimental results, we first smoothed the data of confirmed new cases and new cured cases. table 5 shows the predicted growth range of confirmed new cases, new deaths, and new cured cases. table 5 shows that the growth ranges predicted in this paper are basically reasonable, although the growth range of new deaths is slightly large. however, the average predicted values are larger than actual average values. this is because the algorithm depends on the data, regardless of the actual situation. owing to continuous medical research and strict control, there are increasingly fewer confirmed new cases and existing confirmed cases in wuhan, resulting in increasingly fewer new deaths and cured cases. in the middle of the epidemic, the numbers of confirmed new cases and new cured cases increased rapidly, leading to a higher predicted average value. in addition, the number of confirmed cases had steadily increased from january 23 to february 11. however, on february 12, 12,364 clinically confirmed cases were announced for the first time and included in the confirmed new cases. the subsequent growth trends were completely inconsistent, which severely affected the prediction results. the data from march 23, 2020 to may 31, 2020 were used as the training samples, and the growth range of the confirmed new cases and new deaths from june 1 to june 5 were predicted. table 6 shows the results. the predicted growth range of confirmed new cases and new deaths is smaller than the actual growth. the predicted average for confirmed new cases is larger than the actual average of 19,648.6, whereas the predicted average for new deaths is smaller than the actual average of 879. the negative value in the public data has a significant influence on the prediction results (the number of confirmed new cases on may 10 is −99, and the number of new deaths on may 4 is −1696). there is no obvious downward trend in confirmed new cases and new deaths, and strict control is still required. individuals should therefore strengthen their awareness of prevention and control and be responsible for themselves and others. the emergence of covid-19 has been a heavy burden. during the past 2 months, however, significant progress has been made in the diagnosis and treatment of this disease. in this study, machine learning was used to predict and analyze the development trend and growth range of covid-19. the results using data from wuhan showed that the elman neural network and svm can better predict the development trend of cumulative deaths and cumulative cured cases, whereas the prediction error of cumulative confirmed cases is relatively large. in comparison, the lstm model has a worse predictive effect on cumulative confirmed cases, deaths, and cured cases owing to the aperiodic data. moreover, the prediction results of the two recurrent neural network models used in this study are unstable because neural networks tend to fall into a local optimum for data with irregular growth. this is a major drawback of neural networks, and needs to be improved in the future. for the prediction of the growth range of confirmed new cases, new deaths, and new cured cases, the svm with fuzzy granulation introduced in this paper was shown to be effective. however, the predicted average values are larger than the actual average values, which indicates that the model is less robust to data with large fluctuations and still needs to be improved. the experimental results on data from the united states verified the robustness of the models, although the prediction result of lstm was worse than that of wuhan. in future research, we will improve the models based on the aforementioned problems and continue to improve the generalizability of the models. data curation: ting xu, hongping hu. prediction of infectious disease spread using twitter: a case of influenza. international symposium on parallel architectures the utility of lasso-based models for real time forecasts of endemic infectious diseases: a cross country comparison a novel approach to real-time risk prediction for emerging infectious diseases: a case study in avian influenza h5n1 a personalized infectious disease risk 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density ensembles influenza activity surveillance based on multiple regression model and artificial neural network predicting flu trends from twitter data. big data mining and analytics clinical presentation and virological assessment of hospitalized cases of coronavirus disease 2019 in a travel-associated transmission cluster clinical features of patients infected with 2019 novel coronavirus in wuhan clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan clinical characteristics of 2019 novel coronavirus infection in china data-based analysis, modelling and forecasting of the covid-19 outbreak iteratively pruned deep learning ensembles for covid-19 detection in chest x-rays covid-19: automatic detection from x-ray images utilizing transfer learning with convolutional neural networks deep learning system to screen coronavirus disease 2019 pneumonia a framework of deep learning classifiers to diagnose covid-19 in x-ray images covidiagnosis-net: deep bayes-squeezenet based diagnosis of the coronavirus disease 2019 (covid-19) from x-ray images covid-19) classification using ct images by machine learning using intrinsic genomic signatures for rapid classification of novel pathogens: covid-19 case study a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster the epidemic of 2019-novel-coronavirus (2019-ncov) pneumonia and insights for emerging infectious diseases in the future clinical and computed tomographic imaging features of novel coronavirus pneumonia caused by sars-cov-2 long short-term memory a granulation of linguistic information in ahp decision-making problems. information fusion coronavirus disease (covid-2019) situation reports from the world health organization(who) key: cord-342133-khrljehj authors: principi, nicola; piralla, antonio; zampiero, alberto; bianchini, sonia; umbrello, giulia; scala, alessia; bosis, samantha; fossali, emilio; baldanti, fausto; esposito, susanna title: bocavirus infection in otherwise healthy children with respiratory disease date: 2015-08-12 journal: plos one doi: 10.1371/journal.pone.0135640 sha: doc_id: 342133 cord_uid: khrljehj to evaluate the role of human bocavirus (hbov) as a causative agent of respiratory disease, the importance of the viral load in respiratory disease type and severity and the pathogenicity of the different hbov species, we studied all hbov-positive nasopharyngeal samples collected from children who attended an emergency room for a respiratory tract infection during three winters (2009–2010, 2011–2012, and 2013–2014). human bocavirus was detected using the respiratory virus panel fast assay and real-time pcr. of the 1,823 nasopharyngeal samples, 104 (5.7%) were positive for hbov; a similar prevalence was observed in all three periods studied. among hbov-infected children, 53.8% were between 1–2 years old, and hbov was detected alone in 57/104 (54.8%) cases. all of the detected hbov strains belonged to genotype 1. the median hbov load was significantly higher in samples containing strains with both the n546h and t590s mutations compared to other samples (p<0.05). children with a single hbov-1 infection more frequently had upper respiratory tract infections (urtis) than those who were co-infected (37.0% vs 17.8%, respectively, p = 0.04). the duration of hospitalization was longer among children with high viral loads than that observed among children with low viral loads (8.0 ±2.2 days vs 5.0 ±1.5 days, respectively, p = 0.03), and the use of aerosol therapy was more frequent among children with high viral loads than among those with low viral loads (77.1% vs 55.7%, respectively, p = 0.04). this study shows that hbov is a relatively uncommon but stable infectious agent in children and that hbov1 seems to be the only strain detected in italy in respiratory samples. from a clinical point of view, hbov1 seems to have in the majority of healthy children relatively low clinical relevance. moreover, the viral load influences only the duration of hospitalization and the use of aerosol therapy without any association with the site of the respiratory disease. human bocavirus (hbov) is a recently identified viral agent that belongs to the family parvoviridae and contains a single linear positive-sense or negative-sense single-stranded deoxyribonucleic acid genome [1] . this virus has been detected mainly in younger children, in nasopharyngeal secretions, in sera and blood samples of patients with upper (urti) and lower (lrti) respiratory tract infections and in faecal specimens of subjects with gastroenteritis [2] . currently, hbovs are classified into species 1 through 4; hbov1 is predominantly found in the respiratory tract, and hbov2, hbov3, and hbov4 are found mainly in stool [3] . despite there are studies suggesting that hbov is able to infect the lower airways causing severe infections in both children and adults, the role of hbov as a causative agent of respiratory disease is frequently questioned due to its common detection with other potential pathogens [4] and the evidence that in some studies co-infections can have a significantly greater clinical and socioeconomic impact on infected children and their households than hbov infection alone [5] . moreover, the importance of the viral load in determining the type and severity of respiratory disease as well as the pathogenicity of the different hbov species [6] are not precisely defined. the main aim of this study was to contribute to resolving these problems. the circulation of hbov during several winter seasons in italy was investigated, and a phylogenetic analysis of detected strains was performed. in addition, correlations between different hbov strains and the severity of disease in cases with infections due to hbov alone or due to co-infections were studied. finally, the role of the viral load was analysed. to evaluate the circulation of the different hbov types and the possible relationships between viral load, virus genetic characteristics, and the severity of infection, nasopharyngeal swabs were collected from otherwise healthy children attending the emergency room of the fondazione irccs ca' granda ospedale maggiore policlinico, university of milan, italy, due to a respiratory tract infection arising between november 1 and march 31 during 3 winters (2009-2010, 2011-2012, and 2013-2014) . the study was approved by the ethics committee of the fondazione irccs ca' granda ospedale maggiore policlinico, milan, italy. written informed consent of a parent or legal guardian was required, and children 8 years of age were asked to give their written assent. patients' demographic characteristics and medical histories were retrieved from hospital charts and were systematically recorded before and after the first visit to the emergency room using standardized written questionnaires [7] . the study patients were classified into disease groups (i.e., acute otitis media, rhinosinusitis, pharyngitis, croup, infectious wheezing, acute bronchitis, pneumonia) on the basis of signs and/or symptoms using well-established criteria and were finally subdivided into two subgroups: upper (urtis) and lower respiratory tract infections (lrtis) [8] . nasopharyngeal secretions were collected from all of the children immediately after admission to the emergency room using a paranasal flocked swab (1 swab per child), which was stored in a tube containing 1 ml of universal transport medium (kit cat. no. 360c, copan italia, brescia, italy). viral nucleic acids were extracted from each swab by means of a nuclisens easymag automated extraction system (biomeriéux, craponne, france), and the extract was tested for respiratory viruses using the respiratory virus panel xtag rvp fast v2 (luminex molecular diagnostics, inc., toronto, canada), which simultaneously detects influenza a virus (subtypes h1 or h3); influenza b virus; respiratory syncytial virus (rsv) types a and b; human parainfluenza virus types 1-4 (hpiv1-4); adenovirus (adv); human metapneumovirus (hmpv); coronaviruses (hcov) 229e, nl63, oc43 and hku1, enterovirus/rhinovirus (ev/hrv); and hbov, in accordance with the manufacturer's instructions [9, 10] . samples that were positive for hbov were stored at -80°c. hbov real-time polymerase chain reaction (pcr) viral nucleic acid extracts previously testing positive for hbov were re-tested for confirmation by two different singleplex real-time pcrs using taqman universal master mix ii (applied biosystems, california, usa). amplification and detection of viral dna were performed with a 7900ht real-time pcr system machine (applied biosystems, california, usa). conserved regions for rt-pcr primers and probes were identified in the hbov ns-1 and np-1 genes from the nucleotide sequence alignments available from genbank (for ns1, dq206700-08, dq000495-96, and dq200648, and for np-1, dq000495-96, ab243566-72, dq296618-35, dq353695-99, dq299885, dq267760-75, dq284856, dq295844, and am109958-66; http:// www.ncbi.nlm.nih.gov/genbank/). each 25 μl singleplex reaction mixture consisted of 0.5 μl of forward primer 5'-tgcagacaacgcytagttgttt-3' and reverse primer 5'-ctgtcccg cccaagataca-3' for the 88 base pair ns-1 target or forward primer 5'-agcatcgctcctacaaaagaaaag-3' and reverse primer 5'-tcttcatcacttggtctga ggtct-3' for the np-1 target, 0.125 μl of probe 5'-ccaggattgggtggaacctgcaaa-3' or 5'-aggctcgggctcatatcatcaggaaca-3', and 2.5 μl of sample viral dna. pcrs were conducted at 50°c for 2 min and then at 95°c for 10 min, followed by 45 cycles at 95°c for 15 sec and at 60°c for 1 min. taqman probes were labelled at the 5' ends with the reporter molecule 6-carboxyfluorescein and at the 3' ends with black hole quencher 1 (biosearch technologies, inc., novato, ca). each run included one synthetic template control and one no-template control for each target. specimen extracts were also tested by real-time pcr for the human rnase-p gene to monitor specimen quality and the presence of pcr inhibitors. a positive test for both the ns1 and np-1 targets or for a single target confirmed from a second extraction from a new sample aliquot was considered definitive evidence of hbov infection. the viral load was obtained using real-time pcr with the ns1 primers and probe previously described and a dna plasmid used as the standard calibrator. the amplified target fragment of the plasmid was verified by sequencing. plasmid dna concentrations were detected with an nd-1000 spectrophotometer (nanodrop products, wilmington, de, usa). each run included plasmid and negative controls. standard precautions were taken throughout the pcr process to avoid cross-contamination. negative controls and serial dilutions of the positive controls were included in every pcr assay. finally, quantitative results were reported as dna copies/ ml of respiratory samples. the viral load was defined as low for values 10 6 log (copies/ml) and as high for values >10 6 log (copies/ml). for genotyping, the viral vp-1/2 gene was amplified using a conventional pcr assay. briefly, 4 sets of forward and reverse primers (5'-cacagacagaagcagacgagat-3' and 5'-ggtg agaagtgacagctgtattg-3'; 5'-ttcagaatggtcacctctaca-3' and 5'-ctgtgc ttccgttttgtctta-3'; 5'-aactttgactgtgaatgggtta-3' and 5'-aaatagtgcc tggaggatgat-3'; 5'-ctatcaccagagaaaatccaatc-3' and 5'-gagacggtaaca ccacta-3') were used in pcr amplification and the quantitect probe master mix (qia-gen, venlo, netherlands) was used as the basis for the reaction mix. viral products were analysed by electrophoresis on a 1.5% agarose gel and purified with the qiaquick gel extraction kit (qiagen, venlo, netherlands). sequencing reactions were set up with purified dna, one of the specific primers used in the pcr and bigdye terminator v3.1 cycle sequencing kit (applied biosystems, california, usa) according to the protocol recommended by the manufacturer. sequencing and sequence analysis were performed on a 3130 genetic analyser (applied biosystems, california, usa). all alignments were performed using clustalx 2.1 and bioedit (version 7.1.3.0) software (ibis biosciences, carlsbad, ca). phylogenetic trees of the vp-1/2 protein gene were generated using the neighbour-joining method and p-distance model of the molecular evolutionary genetics analyses (mega) software, version 5.05 [11] . bootstrap probabilities for 1,000 iterations were calculated to evaluate confidence estimates. the graphs were made using graphpad prism version 5.01 for windows (graphpad software, san diego, ca). all genotyped sequences of the hbov vp-1/2 gene were submitted to genbank (accession numbers kr014412-kr014516). tests for positive selection were conducted on the datamonkey server [12] using the singlelikelihood ancestor (slac), and the fixed-effects likelihood (fel) [13] , the internal branch fixed-effects likelihood (ifel) [14] , the mixed effects model of evolution (meme) [15] , and fast unconstrained bayesian approximation methods (fubar). the dn/ds ratios were calculated using the slac and fel codon-based maximum likelihood approaches. the slac approach counts the number of non-synonymous changes per non-synonymous site (dn) and tests whether it is significantly different from the number of synonymous changes per synonymous site (ds). the fel approach estimates the ratios of non-synonymous to synonymous changes for each site in an alignment. the ifel method is similar to the fel, but tests site-bysite selection along only the internal branches of the phylogeny. in order to avoid an excessive false-positive rate, sites with slac, fel, ifel and meme p-values of <0.1 and a fubar posterior probability of >0.90 were accepted as candidates for selection. descriptive statistics of the responses were generated. continuous variables were presented as mean values and standard deviations (sds) and categorical variables as numbers and percentages. for categorical data, comparisons between groups were performed using a contingency table analysis with the χ 2 or fisher's exact test when appropriate. for ordered categorical data, a cochran-armitage test for trend was used to compare the groups. continuous data were analysed using a two-sided student's t-test after ensuring the data were normally distributed (based on the shapiro-wilk statistic) or using a two-sided wilcoxon's rank-sum test if the data were non-normal. all analyses were two tailed, and p-values of 0.05 or less were considered to be statistically significant. all analyses were conducted using sas version 9.2 (cary, nc, usa). during the three study periods, 1,823 nasopharyngeal samples were collected in the emergency room. of these, 104 (5.7%) tested positive for hbov (table 1) . among hbov infected children, 53.8% were between 1-2 years old, whereas 28.8% and 17.3% were aged <1 and 3 years, respectively. the prevalence of hbov detection was quite similar in the three studied periods; hbov was the only virus detected in 57/ 104 (54.8%) cases and was detected in association with one (89.5%) or more (10.5%) viruses in 47 (45.2%) cases. ev/hrv and rsv were the most common co-infecting viral agents and were found, respectively, in 20 and 18 samples. subjects with co-infection were younger than those without (p = 0.03). considering 10 6 dna copies/ml as a cut-off, the viral load was classified as low in 66 (63.5%) cases and as high in 38 (36.5%) cases. the phylogenetic tree constructed using the vp1/vp2 sequences showed that all of the italian hbov strains detected during the three study periods belonged to hbov genotype 1 (fig 1) . no unusual clustering was observed among the identified strains; hbovs circulating in 2009 were closely related to strains circulating in 2014. the sequence identity matrix of the vp1/vp2 gene showed minimum to maximum identity ranges of 97.8-100% between the italian strains and 98.4-99.7% with respect to hbov st1 reference strains (dq000495). in comparison to the reference strain, 8/105 (7.6%) strains had only one amino acid difference, 32/105 (30.4%) strains had two amino acid differences, and the remaining strains (65/105; 61.9%) had at least three amino acid changes. a total of 61/672 (9.1%) amino acid positions were observed to have at least one change in the vp1/vp2 sequence alignments (fig 2) . of these, 7/61 (11.5%) changes occurred within the vp1 unique (vp1u) region corresponding to the first 129 amino acids at the n-terminus of the vp1 gene. specifically, the following changes were observed: r17k, g28d, e29k, l40s, h43q, d72n, and g126e (fig 2) . the vp1u region includes the conserved phospholipase a 2 (pla 2 ) motif (nt 21-63). the vp1u sequences of all hbov isolates identified in this study revealed conserved yxgxg (nt [16] [17] [18] [19] [20] and hdxxy (nt 41-45) motifs in the catalytic site of secreted pla 2 . in addition, the amino acid residues at positions 21, 41, 42 and 63 have been hypothesized to form the catalytic network for enzymatic activity. in our hbov strains, all the sequences had amino acids associated with efficient enzymatic activity (p21, h41, d42, and d63). of note, two hbov strains (mi-267-jan2014 and mi-272-jan2014) had a peculiar amino acid sequence in the 19-amino acid segment starting at amino acid 411 (kvptrrvqpyirqtnwkhr), which has not been previously reported in hbov strains included in the genbank database (in red, fig 1) . overall, these two strains had 13 and 14 amino acid changes compared to the hbov st1 strain, and almost all these changes were included in the region described below. the origin of this highly divergent region, which occurred in spite of the conservation displayed in the rest of the hbov dna genome, remains to be defined. a global analysis of selective pressure made using the slac model indicated an estimated overall dn/ds ratio of 0.18. overall, the site-specific analyses identified three sites (411, 536, and 546) as under positive selection by at least two methods used (slac, fel, fubar, and meme). the ifel model was used to determine the selection pressure acting on the vp1/vp2 codons along the internal branches of the tree. two positively selected codons (411 and 546) were identified. the selected sites, highlighted with arrows in fig 2, were presumably located in of these strains, 6/13 (46.1%) were also characterized by the n534k, q535p, and q535d mutations. several negatively selected sites were identified by different methods (table 2 ). regarding the viral load, a wide range of hbov dna levels from 3.5x10 2 to 7.5x10 9 copies/ml were found in the clinical samples. in fig 2 (right side) , the viral load of each italian hbov strain is reported near the aligned mutations. in the group of strains (n = 70; 66.6%) harbouring at least 2 mutations in addition to a149t, the values of the hbov load greater than 1x10 8 dna copies/ml are reported in grey. a total of 16 strains had a very high viral load, and 13/16 (81.3%) harboured the t590s mutation. this percentage is nearly significantly different than the overall frequency (42/70; 53.8%) of t590s in the group of strains described below (p = 0.08). seven of the 13 strains with the t590s mutation had an additional mutation in one of the sites under positive selection (reported in fig 2 with an asterisk) . in detail, 4/13 (30.8%) had the n546h change, 2/13 (15.4%) had the a411d change, and 1/13 (7.7%) had the a411t change. based on the observed data, we hypothesize that the double mutation of n546h with t590s may positively affect viral replication and specific immune response. as shown in table 3 , the median hbov load was significantly higher in samples of strains with the n546h and t590s changes than that in samples of wild type t590 strains, strains with only the t590s mutation, and strains with only the n546h mutation (p-values of 0.0078, 0.016 and 0.018, respectively). finally, the two divergent strains (mi-267-jan2014 and mi272-jan2014) with unusual amino acid changes were observed with viral loads lower than 10 6 dna copies/ml. this could suggest that these mutations do not confer a replicative advantage in these virus strains. in table 4 , data regarding demographic, clinical and laboratory characteristics of children infected by hbov-1 alone or co-infected with hbov-1 and one or more other respiratory viruses are reported. because a preliminary evaluation did not find any differences among subjects co-infected with ev/hrv, rsv or other viruses, all co-infections were considered together. as shown, children infected with only hbov-1 had urtis more frequently than those with a co-infection (37.0% vs 17.8%, respectively, p = 0.04). moreover, a similar illness within the family in the 7 days since patient enrolment was significantly more common among co-infected children than among those with a single infection (48.9% vs 26.5%, respectively, p = 0.02). no other significant differences between the groups were observed. table 5 shows the demographic, clinical, and laboratory characteristics of the enrolled subjects according to the hbov load. subjects with low and high viral loads were quite similar. the only significant differences were found in the duration of hospitalization, which was longer among children with a high viral load than among those with a low viral load (8.0 ±2.2 days vs 5.0 ±1.5 days, respectively, p = 0.03), and in the use of aerosol therapy, which was more frequent among children with a high viral load than among those with a low viral load (77.1% vs 55.7%, respectively, p = 0.04). moreover, mutations leading to a high or low viral load were not associated with atypical clinical characteristics. this study shows that in italy during the winter periods 2009-2010, 2011-2012, and 2013-2014, the incidence of hbov infection among children with respiratory disease was relatively low, limited to approximately 5% of cases, and did not significantly vary from year to year. the phylogenetic analysis showed that all of the strains detected in this study belonged to hbov genotype 1 and were closely related to the prototype strain identified by allander et al. [1] . this was expected because this genotype is the most common among hbovs associated with respiratory infections [1] . most of the patients in whom hbov1 was identified were younger than 3 years of age, further highlighting that younger children are the individuals most frequently infected by this viral agent [1] . serological studies have shown evidence that the number of subjects positive for anti-hbov1 antibodies continuously increases with increasing age group from the ages of 6 months to 6 years, and by the age of 2 years approximately 80% of children have been infected with hbov1 [16, 17] . more than 50% of the children infected by hbov1 in this study were coinfected with at least one other respiratory virus. moreover, co-infected patients had lrti more frequently than those infected by hbov alone. these findings are not surprising because simultaneous detection of hbov1 and other viruses in children with respiratory disease and greater severity in co-infected cases have been already reported in studies in which it was also demonstrated that hbov1 can frequently be identified in the respiratory secretions of asymptomatic subjects [18] [19] [20] [21] [22] [23] . recently, it has been reported that hbov1 can be shed for several days or months after a previous infection [24] , which could explain the simultaneous identification of hbov1 and other respiratory viruses, the frequency of asymptomatic infections and the generally greater severity of infections in co-infected individuals compared to those with hbov1 alone. in most of the co-infected cases, detection of hbov in the respiratory secretions with the new sensitive molecular methods able to identify very low viral loads might be a consequence of a previous clinically resolved disease, and a virus other than hbov was therefore the real cause of the disease. however, reports of severe clinical manifestations in patients infected with only hbov have been published [25] , highlighting that the assessment of the real importance of hbov infection in a single patient remains very difficult. studies on children with severe pneumonia, acute wheezing, asthma and/or bronchiolitis suggested that hbov1 is able to infect the lower airways down to the bronchioles [26] [27] [28] [29] [30] [31] [32] . moreover, hbov1 has been found as the only infectious agent in adult lung transplant recipients with severe lrti, whereas it was not detected in respiratory secretions of asymptomatic transplanted subjects [33] . this would indicate that hbov1 is not always a bystander or the cause of mild respiratory problems but rather a real, although relatively rare, causative agent of severe disease in both children and adults, particularly when they are immunocompromised. on the other hand, hbov can cause serious neurological infections [34] and contribute to chronic disease in adult patients mainly because it can persist after childhood infection and reactivate [35] . evaluation of the viral load has been considered a possible method to define when this virus is the real cause of a respiratory disease and when it is only a secondary infection. unfortunately, this approach has had no success because although some studies have shown evidence for a strict correlation between high viral load and severe lrti in children with a single hbov infection [36] [37] [38] , others, including the present study, did not show a clear relationship between these two variables [39] . however, the evolution of virulence appears to involve a variety of mechanisms in different viral systems, including mutations in regulatory regions and viral adaptation for utilization of alternative or expanded repertoires of cellular receptors. an alternative hypothesis to evaluate the importance of hbov1 concerns the correlation between viral load levels and the presence of specific mutations. however, mutations associated with increased or reduced replication are rarely reported for hbov. recently, hao et al. have reported that few nucleotide changes were correlated with a lower viral load [40] . in the present study, a double mutant (n546h and t590s) was observed in samples with a significantly higher viral load. however, further phenotypic validation studies are required to draw major conclusions regarding the impact of these mutations on viral replication. furthermore, as reported by qu et al. [41] , it seems that nucleotide changes in the vp1u region could affect the replication efficiency of hbov. likewise, in our strains all the amino acids of the catalytic site were conserved, and no mutations that affect spla 2 activity were identified. in agreement with others [42] , the phylogenetic analysis of this study confirmed the very low degree of variability in the hbov genomic region encoding proteins that are exposed to the virus surface and are therefore under immunologic pressure. only 9% (62 codons) of amino acids were found to have at least one change in the vp1/vp2 gene, a finding not substantially different from that reported by hao et al. in a different geographic area [40] . in our study, several amino acid changes were observed in strains circulating in almost all the respiratory seasons. this finding provides evidence that the selection of those variants best adapted to each particular environment might select for variants with an evolutionary advantage. seven of these mutations were located in a genomic region (i.e., vp1u) previously reported to be involved in the mechanisms of virus replication. for instance, the vp1u amino acid variations r17k and l40s have been previously reported [43] , whereas the remaining variations have not yet been described. interestingly, two hbov strains identified in respiratory samples collected in january 2014 had unusual amino acid sequences in a somewhat conserved genomic region. the reason for this genetic diversity is still undefined. however, as described for hbov, other parvoviruses [44] , and enteroviruses, a series of α-helices and β-barrels in the vp2 protein were intercalated by an external loop, in which the majority of the genetic variability accumulated. nevertheless, these two divergent strains were found in samples with low viral loads, and we might hypothesize a loss of replication advantage for these strains. in the present study, the dn/ds ratios for all pairwise comparisons were <1, which is in line with previous results showing that positive selection was extremely limited in parvoviruses [45] . in fact, selective pressure analyses have identified 3 codons under positive selection. in a previous paper, a different codon (l40) was identified as being under positive selection [46] . nevertheless, the great majority of codons were under negative or neutral selection, which has also been confirmed by others [47] . this finding suggests that only a few amino acids of the vp1/vp2 proteins present on the surface of the virion are potentially subjected to a strong selective pressure by the host immune response. in conclusion, this study confirms that hbov is less common than other respiratory viruses but that the frequency of its detection in children with respiratory disease is in time stable. it was detected with a prevalence of about 5% in several consecutive seasons and no unusual clustering was observed among identified strains, with strains circulating in 2009 being closely related to those circulating in 2014. moreover, only a minority of virus sites were found to be under positive selective pressure, and all the strains detected in respiratory tract infections of this italian study belonged to genotype 1. from a clinical point of view, this study highlights that in otherwise healthy children, hbov1 seems to have relatively low clinical relevance, because patients infected with hbov alone mainly suffered from an urti. the viral load was not associated with clinical characteristics of the infection, and viral mutations, despite affecting viral replication, did not affect the conditions or severity of the clinical presentation. further studies are needed to clarify the clinical relevance of hbov in children, particularly in those at risk for severe chronic underlying disease, and to evaluate the role of viral modification in conditioning the degree of viral virulence and the specific immune response. conceived and designed the experiments: np se. performed the experiments: ap az as. analyzed the data: fb. contributed reagents/materials/analysis tools: sbi gu as sbo ef. wrote the paper: np ap se. cloning of a human parvovirus by molecular screening of respiratory tract samples human bocavirus infections human bocaviruses are highly diverse, dispersed, recombination prone, and prevalent in enteric infections impact of viral infections in children with community-acquired pneumonia: results of a study of 17 respiratory viruses impact of human bocavirus on children and their families the human bocavirus role in acute respiratory tract infections of pediatric patients as defined by viral load quantification clinical and socioeconomic impact of different types and subtypes of seasonal influenza viruses in children during influenza seasons textbook of pediatric infectious diseases comparison of the luminex respiratory virus panel fast assay with in-house realtime pcr for respiratory viral infection diagnosis comparison of the luminex xtag 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vp1 unique region human bocavirus amongst an allages population hospitalised with acute lower respiratory infections in cambodia complete coding sequences and phylogenetic analysis of human bocavirus (hbov) human bocavirus capsid structure: insights into the structural repertoire of the parvoviridae evolutionary relationships among parvoviruses: virus-host coevolution among autonomous primate parvoviruses and links between adeno-associated and avian parvoviruses epidemic and molecular evolution of human bocavirus in hospitalized children with acute respiratory tract infection rapid molecular evolution of human bocavirus revealed by bayesian coalescent inference key: cord-316853-vaea6siv authors: xie, nanzhen; qin, yan; wang, taiwu; zeng, ying; deng, xia; guan, li title: prevalence of depressive symptoms among nurses in china: a systematic review and meta-analysis date: 2020-07-07 journal: plos one doi: 10.1371/journal.pone.0235448 sha: doc_id: 316853 cord_uid: vaea6siv background: depression is one of the most common mental disorders, profoundly impacting an individual’s performance and quality of life. due to their unique working conditions, nursing is counted among the occupational groups at high risk for developing depression. because of the shortage of nursing resources in china, chinese nurses suffer from heavy daily workloads more than those in many other countries. therefore, this study aimed to evaluate the overall prevalence of depressive symptoms and analyse the potential risk factors of depressive symptoms in chinese nurses. methods: a systematic literature search in pubmed, embase, web of science, the chinese biomedical literature database (cbm), the china national knowledge infrastructure (cnki), and the weipu and wanfang databases up to dec 31st, 2019 was performed regarding the prevalence of depressive symptoms in chinese nurses. eligibility assessment and data extraction were performed independently by 2 researchers, and meta-analysis was used to synthesize the data. heterogeneity was evaluated using cochran’s q test and quantified using the i(2) statistic. to explore the potential source of heterogeneity, subgroup analyses were also performed. in addition, both funnel plot and egger’s tests were adopted to assess publication bias. results: a total of 102 studies published from 1996 to 2019 covering 22 provinces were included for further analysis. the total number of participants was 52,592, with a range of 46 to 7205 per study. the overall prevalence of depressive symptoms in chinese nurses was 43.83% (95%ci: 40.26%-47.42%), and 31.12% (95%ci: 27.30%-35.07%) were classified as mild degrees of depressive symptoms. the prevalence of depressive symptoms may be significantly affected by region, province or municipality and department marital status. moreover, an increasing trend in the prevalence of depressive symptoms was observed in recent years. conclusion: the results presented a high prevalence of depressive symptoms among chinese nurses, which suggests interventional programmes by health decision-makers to improving the mental state of nurses is needed urgently, especially in nurses with high risk factors for depressive symptoms. furthermore, the nationwide investigation of depressive symptoms prevalence should be performed with a standard diagnostic tool, which may be more useful for policy makers and planners. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the results presented a high prevalence of depressive symptoms among chinese nurses, which suggests interventional programmes by health decision-makers to improving the mental state of nurses is needed urgently, especially in nurses with high risk factors for depressive symptoms. furthermore, the nationwide investigation of depressive symptoms prevalence should be performed with a standard diagnostic tool, which may be more useful for policy makers and planners. depression is one of the most commonly diagnosed mental disorders or statuses, sometimes resulting in serious damage to the patient's work ability [1, 2] , performance [3, 4] , interpersonal communications, physical health [5, 6] , and quality of life [7] ; some cases of depression may even result in the patient committing suicide [8] . according to the world health organization (who), approximately 300 million people of all ages suffer from depression worldwide, with an increase of more than 18% between 2005 and 2015. the global point, one-year and lifetime prevalence of depression are 12.9%, 7.2% and 10.8% respectively [9] . depression is one of the biggest sources of disability and imposes a considerable economic burden on society [10] . in addition, more women are affected by depression than men [11] . it has been reported that doctors and nurses are one of the highest risk groups for developing depression [12] . special working conditions, such as burnout [13, 14] , high tension, overloaded clinical work, and occupational stress, seriously threaten the mental health of nurses. in addition, nurses often have to witness many different life events, such as disease, trauma, and even death, which imposes further physical and psychological effects on them. because of the shortage of resources for nurses in china, chinese nurses suffer from heavy daily workload more than those in any other country. the psychological status of nurses not only directly affects their own health but also affects the quality of medical care provided for their patients in a hospital setting [15] . some studies have shown that the most common psychological problems experienced by nurses are anxiety and depression [16] , and the incidence of depression in nurses has been showing an increasing trend [17, 18] . at present, relevant studies at home and abroad have found that there is a very high prevalence of depression in the nurse population [19, 20] . for example, studies from usa, taiwan, and south korea found the depressive symptoms prevalence in nurses population ranged from 18% to 61.7% [21] [22] [23] [24] [25] . furthermore, a total of 46 cases of nurse suicide were reported or published from 2007 to 2016 [26] . although various studies have been published in different regions in chinese mainland, there has been no systematic comprehensive study about the prevalence of depressive symptoms. therefore, the primary aim of this study is to quantitatively assess the prevalence of depressive symptoms in nurses from chinese mainland and its primary related influencing factors by systematic review and meta-analysis. all potential articles from pubmed, embase, web of science, the chinese biomedical literature database (cbm), the china national knowledge infrastructure (cnki), and the weipu and wanfang databases were obtained by electronic search. the last search for all databases was performed on dec 31st, 2019. the keywords used for relevant studies were ("prevalence" or "frequency" or "epidemiology") and ("depression" or "mental health disorder" or "major depression disorder" or "mood disorder" or "affective disorder") and ("nurses" or "nurse") and ("china" or "chinese"). each keyword was searched individually or in combination to avoid missing relevant articles and maximize outputs. manuscripts that fulfilled all the following criteria were included for further analysis: (1) crosssectional study, or cohort studies that reported the prevalence of depressive symptoms; (2) targeted objects were nurses in chinese mainland; (3) data available for depressive symptoms prevalence and corresponding depression scale; (4) the depression measuring scales adopted for depression assessment were well recognized internationally, for example, zung's self-rating depression scale (sds) [28] . studies that met the following criteria were excluded: (1) not an original study, such as a review or editorial; (2) non-peer-reviewed local or government report or conference abstract; (3) studies from regions of china other than chinese mainland (including hong kong, macao, and taiwan); (4) duplicate published studies; (5) nurses that were in specific training stages: students, standardized training or rotation; (6) nurses with specific characteristics, including pregnancy, perimenopause, and nurses suffering from trauma following an earthquake; (7) studies with small sample sizes (n<45). to evaluate the selected articles, the 'ahrq cross-sectional/prevalence study quality checklist' [29, 30] was used as a research instrument. which is the most widely accepted quality assessment tool for a cross-sectional study [31] . this instrument is available at http://www. ncbi.nlm.nih.gov/books/nbk35156/ and also can be found in the s1 table. the instrument includes 11 items, which are answered with "yes", "no" and "unclear" respectively. 1 point will be given if one item is satisfied, and 0 point will be given for items not involved or unclear in the study. article quality was assessed as follows: low quality = 0-3; moderate quality = 4-7; high quality = 8-11. the evaluation was conducted independently by two authors, and possible disagreements were settled through discussions with a third author. the following information was extracted from all included studies: title, year of publication, province, sample size, number of positive cases, diagnostic methods and other potential factors that may affect the prevalence of depressive symptoms in nurses and that was provided in the studies. some of studies did not contain all the above-mentioned variables. point estimates and 95% confidence intervals (95%cis) for the prevalence rate of depressive symptoms in nurses were calculated for each study. to avoid having a confidence interval (ci) outside of the 0-1 range as well as studies with large weightings when the prevalence proportion becomes too small or too large, we calculated prevalence estimates with the variance-stabilizing double arcsine transformation [32] . statistical heterogeneity was evaluated by cochran's chi-squared test (with p < 0.10 indicating statistically significant heterogeneity) and the statistic i 2 [33] . heterogeneity with an i 2 of 0 to 40% was treated as not important, while an i 2 of 30 to 60% was treated as moderate heterogeneity, i 2 of 50 to 90% was treated as substantial heterogeneity and i 2 of 75 to 100% was treated as considerable heterogeneity [34] . if obvious heterogeneity existed (with p < 0.10), a random effects model was adopted for pooled results; otherwise, a fixed effects model was adopted. fixed-effect models assume that the population effect sizes are the same for all studies [35] . in contrast, random-effects model attempted to generalize findings beyond the included studies by assuming that the selected studies are random samples from a larger population [36] . furthermore, to identify potential influential studies, sensitivity analysis was performed by sequentially removing individual studies and evaluating the effect on the overall estimate. in addition, subgroup analysis was performed based on other potential sources of heterogeneity, such as province, regions (northwest, southwest, northeast, south, central, east and north china), severity of depressive symptoms, department, gender, age, job title, marriage, education background, shift work and hospital grade (if available). furthermore, meta-regression was also performed to identify the causes of heterogeneity or examine the impact of moderator variables on study effect size of the prevalence. publication bias was examined by funnel plots, and statistical significance was assessed by egger's test. in addition, for the meta-analysis, we assumed that the included studies were a random sample from each study population. meta-analysis was carried out with the "meta" package version 4.11-0 [37] and graphical representation with ggplot2 package version 3.3.0 [38] of r language version 3.6.3 [39] . through the initial search, a total of 3142 potentially relevant citations were identified. a total of 264 duplicate papers were removed first, and 2734 papers were excluded after scanning their titles and abstracts. after screening the full texts of the included articles, 42 studies were excluded for the following reasons: prevalence unreported(n = 28); intervention study of small samples(n = 5); no mention of psychological scale(n = 4); duplicates (n = 3); wrong data (n = 1); after psychological intervention(n = 1). finally, a total of 102 studies meeting the inclusion and exclusion criteria were included for further analysis (fig 1) , of which 3 were published in english-language journals, and the others were published in chinese-language journals. the basic characteristics of the final included studies are shown in s1 table. these studies were published ranging from 1996 to 2019, covering 22 provinces, autonomous regions and municipalities. the scales used for depression assessment were listed as follows: zung's self-rating depression scale (sds) [28] , centre for epidemiologic studies-depression scale (ces-d) [40] , beck depression inventory (beck) [41] , beck depression inventory (2nd ed) (bdi-ii) [42] , hospital anxiety and depression scale (hads) [43] , hamilton depression rating scale (hamd) [44] , and patient health questionnaire (phq-9) [45] . the total number of participants was 52,592, with a range of 46 to 7205 per study. the ahrq cross-sectional/ prevalence study quality checklist was applied to evaluate the study quality (s1 and s2 tables). among the selection items, the evaluation results ranged from 2 to 8, with the median score was 4. overall, 65 of 102 studies have moderate or high quality, indicating a medium quality of the studies included. a total of 28,382 cases among the 52,592 nurses in the studies were found to have different degrees of depressive symptoms; the overall prevalence of depressive symptoms in nurses was 43.83% with 95%cis of 40.26%-47.42%, with significant heterogeneity (i 2 = 98.50%, p < 0.01). a total of 37 studies reported different degrees of depressive symptoms. after we pooled the results based on the severity of the depressive symptoms, the pooled prevalence and 95%cis were 31 geographic analysis based on provinces and regions was performed. we found that the highest prevalence of depressive symptoms was in nurses from the northeast (54.69%, 95%ci: 50.51%-58.84%) and the lowest was in south china (37.59%, 95%ci: 32.75%-42.56%) ( table 1 ). the prevalence of depressive symptoms in nurses among different provinces is shown in table 1 . the highest and lowest prevalence of depressive symptoms were found in hubei province (58.62%, 95%ci: 49.55%-67.41%) and in inner mongolia (22.41%, 95%ci: 5.39%-46.20%), respectively. overall, the geographic location could significantly affect the prevalence, regardless of whether it was broken down by region or province. we also performed subgroup analysis by year. as shown in fig 3, the lowest and highest prevalence were 26.64% (95%ci: 21.27%-32.38%) in 1999 and 62.99% (95%ci: 53.22%-72.26%) in 2017, respectively. concerning the results of subgroup differences for prevalence in different years, a significant difference in terms of the prevalence trends was also found (p < 0.01). other factors that may affect the prevalence of depressive symptoms in nurses were also analysed. the pooled estimates by potential risk factors associated with depressive symptoms in nurses are presented in tables 2 and 3 . of all factors analysed in our study, the prevalence of depressive symptoms was significantly affected by department (table 2) ; the lowest prevalence in addition, we also found that marriage, educational background, age, job title, hospital grade, and shift work did not significantly affect the prevalence of depressive symptoms in nurses (table 3) . publication bias was examined by funnel plot and egger's test. a funnel plot shows that publication bias may exist (fig 4) , which was also confirmed by the result of egger's test (t = -6.20, p < 0.01). a sensitivity analysis for the pooled results was conducted by sequentially removing individual studies, and no significant differences before and after pooling were found, indicating stability in the pooled results. along with the rapid development of the economy in china, psychological problems have become increasingly more common. nurses, as an important role in hospitals, have been increasingly demonstrating depressive symptoms. although many articles have been published to assess the prevalence of depressive symptoms in chinese nurses, a comprehensive study on this population is still absent. in our study, a total of 102 studies with 52,592 participants were obtained to assess the prevalence of depressive symptoms in mainland chinese nurses. to our knowledge, this is the most comprehensive report to date to estimate that estimates this statistic, which may provide useful and valuable information for health decision-makers, helping them to properly implement interventional programmes and prevention activities. in our study, the overall prevalence of depressive symptoms in mainland chinese nurses was 43.83%, with 95%ci of 40.26%-47.42%, and obvious heterogeneity was demonstrated. we also found that the prevalence may be affected by regions/provinces, hospital department, and time, and may not be affected by educational background, age, job title, marriage, hospital grade, or shift work. because of the high prevalence of depressive symptoms in chinese nurses, which may result in large problems for society overall, we suggest that decision-makers should take actions to aid nurses in safeguarding their psychological wellbeing. in a previous meta-analysis [46] , the prevalence of depression in nursing students worldwide was 34.0% and was affected by age and geographical regions, with asian nursing students experiencing a higher prevalence (43.0%). we may see that both student and professional nurses, especially in asia, have a very high prevalence of depression; this prevalence is higher than even that of older patients with diseases such as stroke, hypertension, diabetes and coronary heart disease [47] , and is similar to that of empty-nest elderly individuals [48] . in addition, the prevalence of depression among chinese nurses is higher than that of nurses in iran [49] and chinese hong kong [50, 51] , australian midwives [52] , and hungarian [53] and australian [54] nurses. however, to our surprise, only 13.2% of nurses in vietnam have depression [55] , as well as 24.9% of iranian nurses working in military hospitals [56] . in addition, the chinese nurses even seem to have higher prevalence of depression than some special populations, such as people living with hiv with 38% [57] , outpatients with 27.0% [58] , and indian elderly population with 34.4% [59] . therefore, we may conclude that chinese nurses were at a particularly high risk of having depressive symptoms. moreover, based on the time trend shown in fig 3, the prevalence of depressive symptoms among chinese nurses may have increased in recent years, especially in large hospitals with a low ratio of doctors to nurses and of nurses to patients. we also found that the prevalence of depressive symptoms was significantly different based on geographic distribution and hospital department. in total, we could see that nurses from the hubei province and the northeast region had the highest prevalence of depressive symptoms. this may be because of the occupational environment and policies in each region. from table 2 , we can see that the departments with the highest prevalence of depressive symptoms for nurses are infectious diseases, paediatrics, haemodialysis, icu, and oncology. this may be due to the heavy workload and time pressures inherent in working in these departments. in addition, we also found that, in terms of marital status, despite no significant difference was found, divorce/widowhood/separation had higher prevalence than the others, which may be due to the sample size. to our surprise, we found that the prevalence of depressive symptoms in department of psychiatry wasn't that high as we expect, which may be due to as follows [60] : 1) more professional education about mental health was obtained, 2) the workload and difficulty of nurses in department of psychiatry were easier than others, 3) as closed-off plos one management was adopted in most department of psychiatry, they didn't face the trouble from family members of patients, 4) psychopaths often didn't have physical disease, 5) more medical disputes existed in general hospitals than psychiatric hospitals. in china, there is a large shortage of resources for nurses, the ratio of the nurse population to the total population is 1:1750, which is much lower than that of some developed countries (1:140-1:320) [61] . nurses are faced with heavy workloads, especially in the grade 3a hospitals in the city. however, the present situation cannot be changed in a short period of time. due to the recent covid-19 pandemic, the impact on mental health on healthcare workers is tremendous and more nurses suffer from depression [62, 63] . it is suggested that hospital managers should pay attention to the physical and mental state of their nurses, establish mechanism for the prevention and control of negative emotions such as depressive symptoms, formulate feasible measures to reasonably reduce the workloads of nurses, improve the working environment and the sense of occupational identity, improve and maintain the quality of life while ensuring the quality of medical service, and ensure the physical and mental health of the nurses. these steps may play a role in saving resources and improving nurses' quality of life and work efficiency [64] . the strengths of this review include a comprehensive analysis of the literature to identify all potential articles related to the topic, a robust methodology in conducting the systematic review, and combining estimates generated from the meta-analyses. the meta-analysis results also have some limitations that should be acknowledged: 1) all studies used a cross-sectional observational study design; 2) most of the literature included in this study was published in chinese-language journals, with very few in english-language journals, the overall quality of included studies; 3) the criteria and cut-off for diagnosis varied with studies, which may have led to the heterogeneity observed; 4) only 22 provinces in chinese mainland have been covered with regards to the prevalence of depressive symptoms in their nurses, which may have led to deficiencies or inaccuracies in estimating the overall prevalence; 5) some potential confounding factors were analysed to try and understand the high heterogeneity, but the main reason is still unknown; 6) as the limitation of sample size in some groups, such as department of infectious diseases, anhui and jilin provinces, some results still 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systematic review depression and other common mental disorders-global health estimates key: cord-338594-wft7yy6j authors: winkler, michael; gärtner, sabine; wrensch, florian; krawczak, michael; sauermann, ulrike; pöhlmann, stefan title: rhesus macaque ifitm3 gene polymorphisms and siv infection date: 2017-03-03 journal: plos one doi: 10.1371/journal.pone.0172847 sha: doc_id: 338594 cord_uid: wft7yy6j interferon-induced transmembrane proteins (ifitms) have been recognized as important antiviral effectors of the innate immune system, both in cell culture and in infected humans. in particular, polymorphisms of the human ifitm3 gene have been shown to affect disease severity and progression in influenza a virus (fluav) and human immunodeficiency virus (hiv) infection, respectively. rhesus macaques (macaca mulatta) are commonly used to model human infections and the experimental inoculation of these animals with simian immunodeficiency virus (siv) is one of the best models for hiv/aids in humans. however, information on the role of ifitm3 in siv infection of rhesus macaques is currently lacking. we show that rhesus macaque (rh) ifitm3 inhibits siv and fluav entry in cell culture, although with moderately reduced efficiency as compared to its human counterpart. we further report the identification of 16 polymorphisms in the rhifitm3 gene, three of which were exonic and synonymous while the remainder was located in non-coding regions. employing previously characterized samples from two cohorts of siv-infected rhesus macaques, we investigated the relationship between these rhifitm3 polymorphisms and both aids-free survival time and virus load. in cohort 1, several intronic polymorphisms were significantly associated with virus load or survival. however, an association with both parameters was not observed and significance was lost in most cases when animals were stratified for the presence of mhc allele mamu-a1*001. moreover, no significant genotype-phenotype associations were detected in cohort 2. these results suggest that, although ifitm3 can inhibit siv infection in cell culture, genetic variation in rhifitm3 might have only a minor impact on the course of siv infection in experimentally infected animals. interferon-induced transmembrane proteins are encoded by a gene family of diverse function in vertebrates [1] [2] [3] . a subgroup of immunity-related ifitms (ifitm1-3) has been recognized recently to inhibit multiplication of a broad range of viruses including, but not limited to, a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 influenza a viruses (fluav), flaviviruses, coronaviruses, filoviruses and lentiviruses [4] [5] [6] [7] [8] [9] [10] [11] . ifitm proteins are type ii transmembrane proteins and consist of a variable cytoplasmic nterminus followed by a conserved intramembrane domain, an intracellular loop, a transmembrane domain and a short variable and luminal c-terminus [12] [13] [14] . it is well established that ifitms inhibit viral entry [11] . while the exact mechanism still has to be elucidated, ifitms seem to act through the arrest of virus-host cell membrane fusion at the hemifusion step by altering membrane fluidity [15, 16] . despite their broad antiviral activity, ifitms can also promote entry or assembly of selected viruses [17, 18] , indicating that they belong to a pathway that can positively and negatively regulate viral infection. all immunity-related ifitms (ifitm1-3) have been shown to affect early stages of human and simian immunodeficiency virus (hiv, siv) replication, with ifitm2 and ifitm3 inhibiting virus entry [8, 19, 20] . in addition, ifitms may target later stages of the viral replication cycle such as gag expression and proteolytic processing of env [8, 21, 22] . the antiviral activity of ifitm1 can restrict hiv-1 cell-to-cell spread, and mutations in env and vpu have been identified that rescue the virus from inhibition by ifitm1 at the cost of reduced viral fitness [21, 23] . moreover, ifitms are incorporated into lentiviral particles thereby reducing viral infectivity even though inhibition by ifitms and virion incorporation of ifitms were not found to be intimately related [21, 24, 25] . finally, unlike other antiviral effector proteins of innate immunity, ifitms do not seem to be antagonized by viral factors [22] . different lines of evidence suggest that ifitms can limit viral spread and reduce disease burden in the infected host. thus, absence of ifitm3 expression was found to be associated with reduced fluav spread and pathogenesis in experimentally inoculated mice [26, 27] . moreover, a polymorphism in the human ifitm3 gene (rs12252 t>c) was found in several studies to be associated with severe influenza [16, [27] [28] [29] , although one study reported an association with mild influenza only [30] . the rs12252 t>c polymorphism is believed to alter ifitm3 mrna splicing [27] and to result in the expression of an ifitm3 variant lacking the first 21 amino acids, which displays reduced antiviral activity against fluav upon directed expression in cell culture [31] . conversely, the truncated protein exerts increased anti-hiv-1 activity in transfected cells, due to preferential localization at the cell surface [32] , the site of hiv-1 entry and exit from target cells. however, an association between rs12252-c and more rapid progression to aids, but not risk of hiv-1 infection, has been demonstrated [33] , and the reason for the discrepancy between these results and the increased anti-hiv-1 activity of truncated ifitm3 in cell culture is at present unclear. finally, a recent study demonstrated that hiv-1 variants sexually transmitted between individuals are resistant against blockade by ifitm proteins indicative of a strong selection pressure exerted by ifitms [34, 35] . moreover, ifitm expression was shown to be a major contributor to the blockade of hiv-1 by treatment of cells with ifn [34] , suggesting that ifitms constitute important defenses against hiv-1 infection. non-human primates are an important animal model for infectious diseases in humans. in particular, experimental siv infection of rhesus macaques has contributed substantially to our current understanding of hiv/aids. polymorphisms in several immune-relevant gene loci such as mhc or kir are associated with the transmission and course of disease in siv infected rhesus macaques and hiv-1 infected humans [36, 37] . some primate ifitm gene products were recently shown to have antiviral activity against siv and hiv-1 [19, 20] . however, these analyses did not include rhesus macaque so that it is presently unknown whether rhesus macaque (rh) ifitm3 also inhibits siv infection in this species. moreover, no rhifitm3 gene polymorphisms have been described so far or their modulatory effect upon infection by siv or other viruses studied. therefore, we systematically searched for polymorphisms in rhifitm3 and assessed their association with viral load at set point and disease progression in the context of siv infection. human embryonal kidney (hek) 293t cells (atcc crl-3216) were cultivated in dulbeccos modified eagles medium (dmem), supplemented with 10% fetal calf serum (fcs), l-glutamine and penicillin/streptomycin in a humidified atmosphere containing 5% co 2 . the cells were obtained from collaborators and their identity was verified by short tandem repeat (str) analysis. plasmids encoding machupo virus glycoprotein (macv-gpc), murine leukemia virus envelope protein (mlv-env), fluav hemagglutinin (ha) and neuraminidase (na), vesicular stomatitis virus glycoprotein (vsv-g) and siv envelope protein (siv-env) were previously described [6, 7] . the mlv-based vector pqcxip (clontech, heidelberg, germany) encoding human and rhesus macaque ifitm3 proteins, chloramphenicol-acteyltransferase (cat) or firefly luciferase as well as the mlv gag-pol encoding plasmid were also reported before [6, 7] . for the production of mlv particles encoding ifitm proteins, we followed a published protocol [6, 7] . in brief, 293t cells were cotransfected with vector pqcxip harboring rhifitm3, huifitm3 or cat, an mlv gag-pol-encoding plasmid and a plasmid encoding vesicular stomatitis virus glycoprotein (vsv-g). for the production of reporter particles required for the analysis of ifitm3-mediated inhibition of viral entry, 293t cells were cotransfected with vector pqcxip encoding firefly luciferase, an mlv gag-pol encoding plasmid and a plasmid encoding the viral glycoprotein to be tested. medium was changed at 8 h and supernatants were harvested at 48 h post transfection, passed through a 0.45 μm filter, aliquoted and stored at -80˚c. the antiviral activity of rhifitm3 was analyzed as described previously [6] . in brief, 293t cells seeded at 10 4 cells per well in a 96-well plate were spin-oculated [38] at 4,000×g for 30 minutes with ifitm3 or cat encoding vectors. after incubation for 48h at 37˚c, the medium was replaced by 50μl of fresh culture medium followed by the addition of 50μl of mlv vectors harboring the viral glycoproteins to be analyzed and normalized for equal luciferase activity upon transduction of control cells. after incubation for 8h, medium was replaced by fresh culture medium and luciferase activities in cell lysates were analyzed at 72h post infection, employing a microplate reader, plate chameleon v (hidex, turku, finland), and a commercial luciferase assay system (promega, mannheim, germany) or beetle-juice (pjk, kleinblittersdorf, germany) kits. genetic and phenotypic data from two cohorts of siv-infected rhesus macaques (macaca mulatta) of indian origin, comprising a total of 94 individuals (19 female, 75 male), were screened retrospectively for rhifitm3 polymorphisms ( table 1) . samples of mhc-typed animals were selected that were infected intravenously, intrarectally or via tonsils through a single dose of sivmac239, sivmac251 or sivmac239/32h and which displayed the whole spectrum of disease progression, from rapid progression to long-term non progression, for each combination of virus and infection route. all macaques were either bred at the german primate centre and had known ancestry, or were from breeders in france, united kingdom or the united states. the definition of early aids-defining illness was based on clinical, necropsy and histopathological findings as described [39] . the 39 animals included in the first cohort were previously used as a 'screening cohort' to search for polymorphisms influencing aids-free survival time and viral load in siv infection [39] . it should be noted that no viral load data were available for some of the animals included in the survival analysis. the second cohort of 55 animals consisted of macaques more recently infected, and viral load data were available for all animals. the animals were housed and treated at the german primate centre under standard conditions which are in accordance with the german animal protection act and the european union guidelines on the use of non-human primates for biomedical research as described [40] . briefly, all experiments were approved by an external ethics committee authorized by the lower saxony state office for consumer protection and food safety (project licenses: 509.42502/08-02.95, 509.42502/08-13.98, 504.42502/08-03.90 (v+ä ), 33.9-42502-04-12/0820, 509-42502/08-04.03, 33.9.42502/04/017/07, 33.9.42502/04/72/08). according to §11 of the german animal welfare act, the dpz is permitted to breed and house non-human primates under license number 392001/7 issued by the local veterinary authorities. all macaques were under daily surveillance by veterinarians and animal caretakers. during quarantine, the animals were usually housed in groups of 2-3 animals per cage, with a minimum enclosure height of 180 cm and a cage volume of 3 to 4.5 m 3 . animals in experiments had to be housed singly (cage dimensions [in cm]: 190h × 90w × 90d), but with constant visual, olfactory and acoustic contact to their roommates. procedures for animal welfare and to minimize discomfort and suffering were undertaken in accordance with the recommendations of the weatherall report "the use of nonhuman primates in research". monkeys were fed with dry monkey biscuits supplemented with fresh fruit or vegetables twice daily and fresh water access ad libitum. for environmental enrichment, animals were offered feeding puzzles, varying toys and wood sticks for gnawing. in addition, music was played. for dna preparation, blood samples were drawn under anesthesia with 10 mg ketamine i.m. per kg body weight. animals were humanely euthanized by an overdose of pentobarbital-natrium (narcoren 1 , merial, hallbergmoos, germany) under anesthesia either for experimental reasons without exhibiting clinical symptoms or in cases of suffering predefined by a scoring system of termination criteria that was approved by the external ethics committee and corresponds to the iacuc endpoint guidelines as described [40] . for the purpose of this study, no monkey was sacrificed since archival samples were used. the rhifitm3 gene was amplified from genomic dna by pcr and screened for polymorphisms by re-sequencing. primers for amplification were based on the macaca mulatta chromosome 14 scaffold (mmul_051212) [41] . we used this scaffold before to successfully amplify cdnas of rhifitm genes [6] . amplification of genomic ifitm3 sequences (loc697829) was performed using primers mamuifitm3gen5-1fora (5'-tttgttccgccctcatctgg-3') and mamuifitm3gen5-1rev (5'-tctgagatccacgctcagga-3'). primers were designed using primer-blast [42] to specifically avoid amplification of rhifitm3(2) (loc697564) or rhifitm3 pseudogenes. reaction mixtures contained 100 ng genomic dna, 50 pmol of each primer, 100μm dntp mix, 10% betain, 1x hf synthesis buffer and 1 u phusion dna polymerase. pcr cycling started with an initial denaturation step at 95˚c for 5 min, followed by 40 cycles with denaturation at 95˚c for 30 s, annealing at 68˚c for 30s and elongation at 72˚c for 1:40 min followed by a post amplification step at 72˚c for 10 min. pcr products were gel purified and sequenced using abi big dye chemistry and primers mamuifitm3gen5-1fora, mamuifitm3gen5-1rev, if3gi-for (5'-gtgcccacgtc agtagcttta-3') or if3gi-for2 (5'-tgtgcccacgtcagtagcttt-3') and if3gi-rev (5'-cttcctcactcaggctcagac-3') or if3gi-rev2 (5'-caaactctgagcc agccagc-3'). amplicons were assembled on a scaffold sequence using vector nti contigexpress and polymorphisms identified by visual inspection. aids-free survival analysis, including a log-rank test for rhifitm3 genotype-related differences, was performed using the lifetest procedure of the sas software version 9.4 (sas institute inc., cary, nc). the association between rhifitm3 polymorphisms and virus load was assessed for statistical significance by means of either a kruskal-wallis test (three genotypes) or a mann-whitney test (two genotypes), as appropriate, using the graphpad prism software. all p-values were two-sided. the level of statistical significance was depicted as follows, ã , p<0.05; ãã , p<0.01; ããã , p<0.001. one ifitm1 gene and two ifitm3 genes have been identified in rhesus macaques. for the purpose of the present study, we will distinguish between the two rhifitm3 genes as rhifitm3 (loc697829) and rhifitm3(2) (loc697564) because rhifitm3(2) shows a slightly higher sequence homology to the human ifitm2 gene than rhifitm3. here, we investigated whether rhifitm3 inhibits viral entry in a way similar to human (hu) ifitm3, employing a pseudotyping approach. expression of huifitm3 in 293t cells did not appreciably inhibit entry driven by the mlv envelope protein (env) or the machupo virus glycoprotein (macv-gpc) whereas entry driven by the fluav-hemagglutinin (ha) was robustly inhibited (fig 1) , in keeping with published findings [4] [5] [6] . entry driven by siv-env was also reduced upon hui-fitm3 expression (fig 1) , again in agreement with published data [19, 20] . a similar tendency was observed for rhifitm3 even though inhibition of siv-env-and fluav-ha-driven entry was less robust than in the case of huifitm3 (fig 1) , despite comparable expression levels [6] . thus, rhifitm3 exerts antiviral activity but does so less efficiently than huifitm3. we next aimed to characterize polymorphisms in the rhifitm3 gene based upon a previously characterized cohort of 39 siv infected animals (cohort 1) (table 1) , where relationships between animals and confounding factors had been minimized [39] . to obtain results from a larger collection of animals, we randomly selected additional 55 animals for inclusion into a second cohort (cohort 2) ( table 1 ). the rhifitm3 gene has two exons leading to a mature mrna of 636 bp (xm_001085567.2) whereas the genomic region encoding the primary transcript comprises 1202 bp. we devised a pcr strategy to selectively amplify the genomic region of the rhifitm3 gene so as to yield a 1340 bp amplification product (fig 2) . the dna sequence was determined directly from purified pcr products, and sequence variations were identified at 16 positions (fig 3 and table 2 ). three polymorphisms were located in the coding region but did not change the amino acid sequence. twelve polymorphisms (including one single nucleotide deletion) were intronic and one polymorphism was located upstream of the coding sequence. seven polymorphisms had a low level of heterozygosity ( 10%) but, with few exceptions, the two cohorts were found to be characterized by similar major allele table 2 ). polymorphism g.488 c>a was only detected in the second cohort. polymorphisms g.279 g>a and g.678 g>t showed higher heterozygosity in cohort 2, while the opposite was true for g.693 dela, g.708 a>g, g.709 g>a and g.747 t>g. finally, a polymorphism in the 5´region of the coding sequence that corresponds to rs12252 t>c in the human ifitm3 gene was not identified in our sample. to assess whether the detected sequence variations had an influence on the course of siv infection, we first analyzed their effect on aids-free survival time (fig 4, table 3 ). polymorphisms g.270 c>t (genotype gt) and g.678 g>t (genotype gg) showed a nominally significant association with faster disease progression when all animals from both cohorts were considered. however, these associations were not observed when each cohort was analyzed separately and were not significant when animals with mhc allele mamu-a1 ã 001 were excluded from the analysis (not shown). polymorphism g.279 g>a showed a highly significant association of the aa genotype with faster disease progression (log-rank test < 0.0001) and this association was also detected when carriers of mamu-a1 ã 001 (log-rank test < 0.0001) were excluded (fig 4, table 3 ). however, this variant was only little polymorphic (maf: 0.95), and the significance of the association hinged heavily on a single aa animal in cohort 1. for a subset of 81 animals, virus load data were available that allowed the potential association between rhifitm3 polymorphism and siv amplification to be analysed. however, no statistically significant association with plasma viral rna copies at 20 weeks after infection or at time of death was noted for any of the polymorphisms (fig 5, table 4 ). only in cohort 1, we found a nominally significant association of polymorphisms g.693 dela (genotype -/-), g.708 a>g (genotype gg) and g.709 g>a (genotype aa) with increased viral load and of polymorphism g.678 g>t (genotype gt) with lower virus load (table 4 ). however, these findings were not replicated in cohort 2 and were not detected when the full set of animals was analyzed. immune-related ifitm proteins have been established as important antiviral effectors of the interferon response, and a polymorphism in the human ifitm3 gene has been found to be associated with disease severity and progression in fluav and hiv-1 infection [27, 33] . rhesus macaques are important animal models for human infections but it was hitherto unknown whether the rhifitm3 gene is polymorphic and whether possible polymorphisms would impact upon the course of siv infection. we demonstrated that rhifitm3 displays antiviral activity and identified 16 polymorphisms in rhifitm3. some of the polymorphisms were associated with either disease progression or viral load. however, none of the polymorphisms impacted on both parameters, and significant associations were not observed in cohort 2. moreover, most associations failed to attain statistical significance when mamu-a1 ã 001 animals were excluded from the analysis, arguing against a major impact of rhifitm3 polymorphisms on siv infection. recently, a comprehensive characterization of the antiviral activity of ifitm proteins of non-human primates revealed that these proteins, like their human counterparts, can block viral infection [20] . however, ifitm proteins from rhesus macaques were not included in these studies. our results demonstrate that rhifitm3 and huifitm3 target the same viral glycoproteins, but with somewhat different efficiency. thus, entry driven by fluav-ha and siv-env appears to be more efficiently inhibited by huifitm3 than by rhifitm3. these results are in agreement with our previous analysis demonstrating that huifitm3 inhibits entry driven by filovirus glycoproteins with greater efficiency than rhifitm3, despite comparable expression in cells [6] . in summary, rhifitm3 inhibits entry of siv in cell culture which raised the question whether rhifitm3 is polymorphic and whether possible polymorphisms would impact the course of siv infection. our sequence analysis identified variations at 16 positions present in at least two out of 94 animals. six of these polymorphisms were highly polymorphic (maf <75%) and homozygous animals could be detected for both alleles. three of the polymorphisms identified in our study were located within the coding sequences, but led to silent changes. this is noteworthy since ifitm3 polymorphisms in siv-infection the n-terminal residues of ifitm3 control intracellular localization and spectrum of antiviral activity [32, 43, 44] and were suggested to be shaped by selective pressure during primate evolution [32] . one polymorphism was located upstream of the coding sequence; all other polymorphisms were identified within the intron. we found no evidence for a polymorphism that would lead to the production of a truncated protein similar to rs12252-c in human ifitm3, a variant that is associated with the clinical severity of influenza disease [27] . our results were in keeping with a previous report suggesting that ifitm1 and ifitm3 in non-human primates probably underwent purifying selection [20] . thus, we did not find any non-synonymous polymorphisms, indicating that maintenance of the primary amino acid sequence is essential for the function of the protein in rhesus macaques. our analysis was based upon dna sequences derived from assembly mmul_051212 [41] . in the meantime, a new genomic framework (mmul_8.0.1) has been published [45] where the sequences at the 3' end of the coding regions of rhifitm3 (loc697829) and rhifitm3(2) (loc697564) have been exchanged in comparison to the previous assembly (mmul_051212). however, our previously reported cloning and sequencing of cdnas from both loci [6] and the analysis presented here show better agreement with the rhifitm3 locus as annotated in the older genomic assembly. therefore, we continued to use mmul_051212 throughout this study. a reconstruction of haplotypes using phase (v. 2.1) [46] based upon our results revealed 16 haplotypes for the rhifitm3 gene, all of which clustered together and were clearly distinct from rhifitm3(2) (data not shown). this reinsures us that all detected polymorphisms are true rhifitm3 polymorphisms. silent mutations may affect translational efficiency and protein folding [47] whereas polymorphisms in untranslated regions or introns may alter mrna stability or splicing [48] . in consequence, we analyzed the association between the polymorphisms and survival time and viral load in siv-infected rhesus macaques. for this, we used a sample that previously allowed detection of an association between tlr7 polymorphisms and survival time (cohort 1, [39] ) and a sample of randomly selected animals (cohort 2). when the full set of animals was considered, we identified three polymorphisms (g.270 c>t, g.279 g>a, g.678 g>t) with a nominally significant association between a genotype and aids-free survival, but not with virus ifitm3 polymorphisms in siv-infection load at set point (20 weeks post infection). however, for two of these polymorphisms the association was not significant if the two cohorts were analyzed separately or when animals with mhc allele mamu-a1 ã 001 were excluded from the analysis. the polymorphism g.279 g>a genotype aa showed a nominally significant association with aids progression even after adjustment for mhc alleles. however, this position is only modestly polymorphic and the association observed was dependent on a single animal with aa genotype. collectively, these results suggest that rhifitm3 polymorphism might not play a major role in siv infection. in summary, our study not only demonstrated that rhifitm3 displays antiviral activity, but also led to the identification of polymorphisms in the coding and non-coding region of the ifitm3 gene of rhesus macaques. notably, all polymorphism in the coding region were silent and strong evidence for an association of rhifitm3 polymorphisms with disease progression and viral load in siv infected animals was not obtained. we cannot exclude that the analysis of a substantially larger number of animals might reveal such associations. however, the frequency of potentially affected animals and/or the effect of the polymorphisms in terms of viral inhibition would be very low and are therefore not expected to significantly influence results 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required for both ifitm protein association and inhibition of influenza a virus and dengue virus replication the interferon-induced transmembrane proteins, ifitm1, ifitm2, and ifitm3 inhibit hepatitis c virus entry a new rhesus macaque assembly and annotation for next-generation sequencing analyses a comparison of bayesian methods for haplotype reconstruction from population genotype data exposing synonymous mutations the dark matter of the cancer genome: aberrations in regulatory elements, untranslated regions, splice sites, non-coding rna and synonymous mutations we thank heidi meyer for technical assistance and christiane stahl-hennig for the support of our work and for providing access to material and data that were generated under her supervision including the blood samples from the experimental animals. key: cord-309981-4p3ybrn1 authors: dai, ling-ling; wang, xi; jiang, tian-ci; li, peng-fei; wang, yu; wu, shu-jun; jia, liu-qun; liu, meng; an, lin; cheng, zhe title: anxiety and depressive symptoms among covid-19 patients in jianghan fangcang shelter hospital in wuhan, china date: 2020-08-28 journal: plos one doi: 10.1371/journal.pone.0238416 sha: doc_id: 309981 cord_uid: 4p3ybrn1 fangcang shelter hospitals were established in china during the coronavirus disease 2019 (covid-19) pandemic as a countermeasure to stop the spread of the disease. to our knowledge, no research has been conducted on mental health problems among patients in fangcang shelter hospitals. this study aimed to determine the prevalence and major influencing factors of anxiety and depressive symptoms among covid-19 patients admitted to fangcang shelter hospitals. from february 23, 2020, to february 26, 2020, we obtained sociodemographic and clinical characteristics information of covid-19 patients in jianghan fangcang shelter hospital (wuhan, china) and assessed their mental health status and sleep quality. data were obtained with an online questionnaire. the questionnaire consisted of a set of items on demographic characteristics, a set of items on clinical characteristics, the self-rating anxiety scale, self-rating depression scale, and pittsburgh sleep quality index. three hundred seven covid-19 patients who were admitted to jianghan fangcang shelter hospital participated in this study. the prevalence of anxiety and depressive symptoms were 18.6% and 13.4%, respectively. poor sleep quality and having ≥ two current physical symptoms were independent risk factors for anxiety symptoms. female sex, having a family member with confirmed covid-19, and having ≥ two current physical symptoms were independent risk factors for depressive symptoms. anxiety and depressive symptoms were found to be common among covid-19 patients in fangcang shelter hospital, with some patients being at high risk. originating as a cluster of unexplained cases of pneumonia, coronavirus disease 2019 (covid19) was first identified in wuhan, hubei province, china in december 2019 [1] . spreading rapidly worldwide, the covid-19 pandemic is a global health threat with devastating consequences that can potentially impact the citizens of all nations [2] . widespread outbreaks of infectious diseases, such as ebola virus disease and severe acute respiratory syndrome, are not only associated with physical illness but also with psychological distress and symptoms of mental illness [3, 4] . results from prospective studies have consistently suggested that psychological distress is a predictor of future health and disease outcomes [5] . as with other infectious diseases, preliminary evidence suggests that covid-19 also causes public panic and mental health stress; symptoms of anxiety and depression are common psychological reactions to the covid-19 pandemic, and may be associated with sociodemographic factors and sleep quality [6] [7] [8] [9] . however, previous studies have focused mainly on covid-19-related mental health issues among the general population, medical staff, children, pregnant women with their husbands, people with mental illness and individuals in self-isolation [10] [11] [12] [13] . we know very little about the psychological effects of the disease on patients with covid-19 [14] . to control the spread of infection and save lives, china has implemented covid-19 countermeasures, including the establishment of fangcang shelter hospitals in hubei province [15] . fangcang shelter hospitals are a novel public health concept. these hospitals were established in china to assist in the country's management of covid-19 [16] . the fangcang shelter hospitals are large, temporary hospitals built by converting public venues, such as stadiums and exhibition centers, into healthcare facilities to receive non-seriously ill individuals with positive sars-cov-2 tests from their families and communities, while providing disease monitoring, medical care, food, shelter, and social activities [16, 17] . fangcang shelter hospitals have been crucial to the quick containment of covid-19 in china, relieving some of the enormous pressure on the healthcare system, and providing an encouraging example for other countries [18] . as the covid-19 pandemic spreads globally, some countries, such as serbia, have also built fangcang shelter hospitals. moreover, other countries, such as iran, the united states, the united kingdom, and spain, have implemented similar measures [16] . based on the evidence from previous research on the general population and on medical staff, we speculate that the mental health of the fangcang shelter hospital patients is affected by the covid-19 pandemic. to our knowledge, no previous studies have been conducted on the mental health of fangcang shelter hospital patients during the covid-19 pandemic. a better understanding of the psychosocial problems of fangcang shelter hospital patients can provide important guidance in carrying out timely psychological interventions for targeted populations in need and in the management of the fangcang shelter hospitals in any future outbreaks. therefore, this study aimed to determine the prevalence and major influencing factors of anxiety and depressive symptoms among covid-19 patients admitted to fangcang shelter hospitals. this was a cross-sectional study performed via an anonymous online questionnaire from february 23, 2020, to february 26, 2020, in jianghan fangcang shelter hospital in wuhan, china. the study was designed and conducted by doctors who worked at the hospital, and was approved by the ethics committees of the first affiliated hospital of zhengzhou university (no.2020-ky-169). all study respondents were made aware that participation in the study was voluntary and provided prior written informed consent online. the inclusion criterion was a covid-19 diagnosis based on the guidelines of the national health commission of the people's republic of china [19] . exclusion criteria included a previously diagnosed severe psychiatric illness (e.g., schizophrenia, bipolar disorder, anxiety disorder, or depression disorder), inability to complete or failure to complete the online questionnaire, currently taking oral medication for a chronic disease (e.g., metoprolol, reserpine, prednisone, and methylprednisolone) that can cause side effects associated with anxiety, depression, and insomnia [20] [21] [22] . the participants scanned the quick response codes with their mobile phones and completed the questionnaires. the study questionnaire consisted of five main components: a set of items on demographic characteristics, a set of items on clinical characteristics, the self-rating anxiety scale (sas), the self-rating depression scale (sds), and the pittsburgh sleep quality index (psqi). using the questionnaire, we collected data on the physical symptoms and comorbidities that had been discussed in previous literature [23] . participants were asked to indicate whether they were currently experiencing any of the 14 listed physical symptoms (fever, coughing, sputum production, shortness of breath, chest pain, fatigue, soreness or discomfort in the throat, nasal congestion, conjunctival congestion, hemoptysis, headache, diarrhea, abdominal pain, myalgia, and arthralgia) or had any of the 9 listed comorbidities (chronic bronchitis or chronic obstructive pulmonary disease, asthma, hypertension, diabetes, coronary heart disease, cerebrovascular disease, connective tissue diseases, chronic renal disease, and cancer). anxiety and depressive symptoms were assessed using the chinese versions of the sas and sds. the chinese versions of the sas and sds have been proven to be effective instruments and have good psychometric properties when used in china (cronbach's α coefficient was 0.82 and, 0.83 respectively) [24, 25] . the sas and sds both contain 20 items, with responses based on a 4-point scale. for both scales, each question is based on mood experiences in the previous 7 days. an aggregate score of 20 is multiplied by 1.25, with higher scores indicating more severe levels of anxiety and depression [26] . cutoff scores of �50 in sas and �53 in sds represent a positive screen for depression and anxiety symptoms [26] . sleep quality was assessed using the chinese version of the psqi. the chinese version of the psqi has adequate reliability, with an internal consistency cronbach's α of 0.77 to 0.84 [27] . the psqi consists of seven domains: sleep quality, sleep duration, sleep latency, habitual sleep efficiency, sleep disturbance, use of sleeping medications, and daytime dysfunction [28] . responses to each item are based on a 3-point scale, with the total score ranging from 0 to 21. higher scores indicate lower sleep quality. poor sleep quality was defined as a total score �6 [29]. all statistical analyses were performed using ibm spss version 25 (ibm corporation, armonk, ny, usa). continuous data that were normally distributed were presented as mean ± standard deviation, non-normally distributed data were described as median and first, third quartile: m (q1, q3); t-tests were used to compare the normal distribution of continuous data. univariate analyses of anxiety and depression symptoms were performed using the chisquared (χ 2 ) test. covariates with p <0.10 in the univariate analyses were included in the multivariate analyses. the multivariable logistic regression models were built using the forward lr variable selection method to identify independent factors associated with anxiety and depression. a p-value <0.05 was considered statistically significant. a total of 307 patients participated in the study. among them, 57 (18.6%) experienced anxiety symptoms, and 41 (13.4%) experienced depressive symptoms. two hundred sixty (84.7%) had poor sleep quality, as determined with the psqi. the three most common coexisting illnesses were hypertension (16.0%), chronic bronchitis or chronic obstructive pulmonary disease (13.0%), and diabetes (4.6%), as shown in fig 1a. there were 20 currently asymptomatic patients. the three most common current physical symptoms were coughing (26.4%), shortness of breath (24.4%), and soreness or discomfort in the throat (17.9%) (see fig 1b) . data on the demographic and clinical characteristics of the patients, and on the differences in incidence of anxiety and depressive symptoms among different groups are shown in table 1 . based on covariates with p < 0.10 as screening covariates, having a family member who has been confirmed with covid-19, number of current physical symptoms, symptoms change after hospitalization, and poor sleep quality were the related factors of anxiety symptoms, and gender, education level, smoking history, drinking history, having a family member who has been confirmed covid-19, number of current physical symptoms, symptoms change after hospitalization, and poor sleep quality were the related factors of depressive symptoms (see table 1 ). results of the multivariate logistic regression analyses to determine the risk factors for anxiety and depressive symptoms are presented in table 2 . poor sleep quality (odds ratio [or], 3.655, table 2 ). this cross-sectional study examined the prevalence of anxiety, depression, and poor sleep quality among 307 patients in jianghan fangcang shelter hospital in wuhan, china, 2 months after the start of the covid-19 pandemic. of all the participants, 18.57%, 13.36%, and 84.69% had anxiety symptoms, depressive symptoms, and poor sleep quality, respectively. in addition, using one-sample-tests, it was determined that both sas (42.92±7.30) and sds (39.77±10.11) scores of the participants of our study were higher than chinese norms (sas, 29.78±10.07, n = 1158; sds, 33.46±8.55, n = 1340) (both p<0.001) [26] , indicating more severe levels of anxiety and depressive symptoms among covid-19 patients admitted to fangcang hospitals, compared with the general public. clearly, anxiety and depressive symptoms were common responses to the covid-19 outbreak, and patients in the jianghan fangcang shelter hospital had severe levels of anxiety and depressive symptoms. the reason for this may be related to many factors: differing viewpoints on mask wearing, misperceptions in society, shortage of b, partial regression weight, se, standard error, or, odds ratio; ci, confidence interval; multivariate logistic regression analyses with forward stepwise variable selection personal protective equipment, uncertainty about the progression of the pandemic, and fear of a difficult recovery from the disease [30] [31] [32] [33] . in one previous chinese study conducted in the initial stage of the pandemic, which reported a high prevalence of moderate to severe depressive symptoms, anxiety symptoms were found to be 16.5% and 28.8% among the general population, respectively [8] . in another chinese study, the prevalence of anxiety and depressive symptoms among healthcare workers treating patients with covid-19 was 44.6% and 50.4%, respectively [34] . this is in sharp contrast to the low prevalence of anxiety and depressive symptoms found in the present study. however, one chinese study conducted within the same study period as that of the present study reported prevalence rates of anxiety and depressive symptoms among all the participants (including medical health workers and nonmedical health workers) of 10.4% and 10.6%, respectively [35] . therefore, differences in prevalence rates may be due to differences in study periods. with the covid-19 pandemic, the government of china has provided appropriate information and knowledge in a timely manner. transparency and open communication can efficiently lower fear, anxiety, stigmatization, and discrimination [36] . moreover, the national health commission of china has performed psychological crisis intervention through the general deployment of disease prevention and mental health professionals and expert groups providing psychological intervention for different subpopulations, including patient isolation in fangcang shelter hospitals [16, 37] . early psychological crisis intervention has reduced the prevalence of negative psychological outcomes caused by the covid-19 outbreak. results of the multivariate logistic regression analyses indicated that poor sleep quality and having more current physical symptoms were risk factors for anxiety symptoms among patients in fangcang shelter hospitals. sleep provides time for the recuperation and rejuvenation of the brain. a substantial body of literature has shown that stressful life events and outbreaks of infectious diseases, including covid-19, can affect sleep quality [34, [38] [39] [40] [41] , and 84.69% of the participants in the present study had poor sleep quality. syntheses of longitudinal studies suggested that sleep quality was bidirectionally related to anxiety [42] . there is a large amount of data on the effects of sleep quality on anxiety symptoms in other populations, such as shift workers, firefighters, paramedics, pregnant females, and older adults. poor sleep quality was found to be associated with higher risk for anxiety symptoms, and greater anxiety was found to be associated with poorer sleep quality [43] [44] [45] [46] [47] . similarly, anxiety affects sleep quality because anxious people find it hard to fall asleep and wake up frequently [42] . in addition, the present study demonstrated that patients with more physical symptoms of covid-19 were more vulnerable to anxiety symptoms. possible reasons are as follows: first, common symptoms of covid-19, such as fever, shortness of breath, and headache can induce anxiety symptoms [48] . second, patients with more symptoms are generally more serious than asymptomatic patients, and the prevalence of anxiety is also related to the severity of the disease [49, 50] . last, patients with more symptoms are more concerned about the progression of the illness. another finding from the present study was that females, patients with family member with confirmed covid-19, and patients with more current physical symptoms were more likely to have anxiety symptoms. as early as the 1970s, weissman underscored the gender differences in depression, and noted that females were more likely to experience depression than males [51] . since then, there has been a proliferation of research and theories on gender differences in depression. one recent meta-analysis showed that females are more vulnerable to depression disorders and depression symptoms [52] . there is now a consensus that gender differences in depression have a multifactorial etiology; for example, there is a confluence of hormonal and neurodevelopmental changes that vary by sex during the pubertal transition and may influence gender differences in depression [52] . in addition, patients with family members diagnosed with covid-19 were more vulnerable to depressive symptoms, owing to greater family burden and psychological distress [53, 54] . compared with patients with less physical symptoms, patients with more physical symptoms were more likely to have depressive symptoms because they were more severe and the prevalence of depressive symptoms in relation to the severity of the disease [50] . this study had some limitations. first, this was a cross-sectional study, conducted within a short time frame. therefore, future longitudinal studies are needed for follow-up and intervention. second, psychological assessment was based on an online survey with self-report tools that had not been specifically designed for use with covid-19 patients [55] [56] [57] . the use of clinical interviews and instruments designed specifically for covid-19 patients is encouraged in future studies, in order to produce more comprehensive findings. third, this was not a multinational, multicenter study, and therefore, further research is needed to produce more data on anxiety and depressive symptoms among patients in fangcang shelter hospitals. this study identified the prevalence rates and risk factors of anxiety and depressive symptoms among patients in fangcang shelter hospitals. anxiety and depressive symptoms were found to be common among the covid-19 patients in the hospitals. those with more physical symptoms and poor sleep quality demonstrated more vulnerability to anxiety symptoms. females, patients with family members who had been diagnosed with covid-19, and patients with more current physical symptoms were more vulnerable to depressive symptoms. the poorer the sleep quality, the more serious the symptoms of anxiety and depression. our findings can aid in the development of interventions to reduce the adverse psychological impact of the covid-19 pandemic on patients in fangcang shelter hospitals in the future. the extent of transmission of novel coronavirus in wuhan, china, 2020 ayurveda and covid-19: where psychoneuroimmunology and the meaning response meet post-ebola psychosocial experiences and coping mechanisms among ebola survivors: a systematic review psychological 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the fear of covid-19 scale: 57. development and initial validation the authors would like to thank all the patients who participated in this study and editage (www.editage.com) for english language editing. key: cord-312002-4qhvljpv authors: pacheco-barrios, kevin; cardenas-rojas, alejandra; giannoni-luza, stefano; fregni, felipe title: covid-19 pandemic and farr’s law: a global comparison and prediction of outbreak acceleration and deceleration rates date: 2020-09-17 journal: plos one doi: 10.1371/journal.pone.0239175 sha: doc_id: 312002 cord_uid: 4qhvljpv the covid-19 outbreak has forced most of the global population to lock-down and has put in check the health services all over the world. current predictive models are complex, region-dependent, and might not be generalized to other countries. however, a 150-year old epidemics law promulgated by william farr might be useful as a simple arithmetical model (percent increase [r1] and acceleration [r2] of new cases and deaths) to provide a first sight of the epidemic behavior and to detect regions with high predicted dynamics. thus, this study tested farr’s law assumptions by modeling covid-19 data of new cases and deaths. covid-19 data until april 10, 2020, was extracted from available countries, including income, urban index, and population characteristics. farr’s law first (r(1)) and second ratio (r(2)) were calculated. we constructed epidemic curves and predictive models for the available countries and performed ecological correlation analysis between r(1) and r(2) with demographic data. we extracted data from 210 countries, and it was possible to estimate the ratios of 170 of them. around 42·94% of the countries were in an initial acceleration phase, while 23·5% already crossed the peak. we predicted a reduction close to zero with wide confidence intervals for 56 countries until june 10 (high-income countries from asia and oceania, with strict political actions). there was a significant association between high r(1) of deaths and high urban index. farr’s law seems to be a useful model to give an overview of covid-19 pandemic dynamics. the countries with high dynamics are from africa and latin america. thus, this is a call to urgently prioritize actions in those countries to intensify surveillance, to re-allocate resources, and to build healthcare capacities based on multi-nation collaboration to limit onward transmission and to reduce the future impact on these regions in an eventual second wave. change of events nor acceleration; iv) phase d-small change of events but higher acceleration; and v) phase e-both small change of events and acceleration. farr's law has not been widely used as other models using differential-equations were instead employed, such as the susceptible-infectious-removed (sir) model [10] . the main reason is that it does not consider other important variables as population characteristics (immunity and susceptibility), public health interventions, and political actions against the pandemic. however, it may still be valuable and especially for new outbreaks where there is a lack of knowledge on parameters of disease such as ebola [11] , chikungunya [11] , opioid abuse [12] , and indeed the current covid-19 pandemic. in the study of 1990, bregman et al. [9] showed a good prediction model for the cases of aids, showing that the peak was close, and it would happen a rapid deceleration which it did take place in the following years. santillan et al. [11] compared the farr's model with the incidence decay with exponential adjustment (idea) and sir models. they reported farr's law mathematical approach to resemble solutions of sir model in an asymptotic limit, where changes of transmission respond both to control policies and depletion of susceptible individuals. moreover, they suggested the concept of the reproduction number (r 0 ) and the effects of control of epidemics (via behavior change or public health policy intervention) were implicitly captured in farr's work (pre-microbial era). in this study, we will model covid-19 current data (until april 10, 2020) of new confirmed cases and deaths, from 210 countries as to test the assumptions of the 1840 farr's law, to describe the epidemic dynamics, and also to make predictions to identify areas with high dynamic and suggest preparation and actions of health system in those regions. we extracted the covid-19 data of total and new confirmed cases and deaths from all countries available in "worldometer" website (210 countries, see table 1 ) [13] , until april 10, 2020. this website provides a real-time update on the actual number of covid-19 cases worldwide, including the total confirmed cases, daily new confirmed cases, and severity of the disease (recovered, critical condition, or confirmed death) by country. worldometer is composed of a team of researchers, developers, and volunteers with no political, governmental, or corporate affiliation to provide time relevant world statistics. as general information, it has been voted as one of the best free reference websites by the american library association, and its data has been used in the united nations conference rio+20 [14] . in the context of covid-19 pandemic, it has been a provider for important governmental institutions such as the uk, thailand, pakistan, sri lanka, and vietnam governments as well as for the covid-19 dashboard by the center for systems science and engineering (csse) at john hopkins university [15] . based on its webpage, they obtain the data directly from official reports from the government's communication channels or indirectly through local media sources when they are considered reliable. we downloaded the data on new cases and deaths, separately, per day and country since the first available record (january 23, 2020). we extracted directly from the webpage code source from each plot using a standardized extraction spreadsheet to prevent losing data. additionally, we extracted data of income, urban index, population density, population size, and proportion of the older population (>65 years old) from the world bank data repository [16] of the included countries; we estimated the number of cases and deaths per 1000 and 100,000 inhabitants by country, respectively. finally, we performed a post-hoc extraction of the new cases for june 10, 2020 (one point extraction), and the political actions against covid-19 per country, from the university of washington health index and evaluation c: both ratios are equal to 1; d: first ratio less than 1�0 but second ratio higher than 1�0; e: both ratios are less than 1�0� � no cases for more than 10 days�� data belong to june 09, on june 10 there was no cases reported�� the countries with end date beyond june 10 are countries having more than zero predicted cases or deaths after we ended our simulation (june 10, 2020), meaning they could continue with the pandemic wave beyond the end our simulation. https://doi.org/10.1371/journal.pone.0239175.t001 center (ihme) database [17] . we used this analysis to compare current data with our predictions on the covid-19 worldwide trend, including the countries with reduced cases near zero, and the countries with high predicted dynamics. we exported the data to a standardized spreadsheet in excel microsoft 2019 to performed data cleaning. according to farr's law, the intrinsic epidemic's behavior could be described as the relation of two arithmetic ratios: 1. the first ratio (r1) represents the change of cases or deaths (first level dynamic) comparing one time against the immediately before time (could be in days or months, based on the natural history of the disease). thus, by subtracting 1�0 from this ratio, we can calculate the percent increase of cases or deaths, in physical terms, this measure could be understood as the "velocity of spread" of the epidemic. 2. the second ratio (r2) measures the rate of change of r1s. it compares the r1 of one time against the r1 from the immediately before time. in physical terms, we can interpret this ratio as the acceleration of the epidemic (new cases or deaths). we calculated the farr's ratios based on prior reports [9, 11, 12] , the first ratio (r 1 ) was the division of given cases or deaths (i) at t time over that estimate of t time before (formula 1). then, dividing the first ratio of one time over the immediately before resulted in the second ratio (r 2 )(formula 2). we predefined the sum of events over five days of consecutive data as a time length to calculate the ratios, as was suggested in prior studies to use clustered data to stabilize the data distribution [11] . we constructed the epidemic curves of all the 210 countries using those clustered data on new cases and deaths. the normality of the data was examined using skewness and kurtosis without data transformations to assure the estimates' interpretability. the countries with disease data less than 15 days (to calculate at least one r 2 ) were excluded from the ratio's calculation analysis. we fit a normal curve to these data to use farr's law as a predictive model of epidemic dynamics. first, similar to previous studies [9, 11, 12] , we assumed the future r 1 and r 2 values would be the same as the mean of the past three-time intervals (five days), then we predicted the future first ratio and daily confirmed new cases and deaths, for the next two months (until june 10, 2020), by back-calculation, using the mean of the last three calculated r 2 [9] . to fit a normal curve, we set two assumptions [9, 11] : i) the r 2 was constant, having a value between 0�0 and 1�0, which signifies a constant deceleration in the rate of change (r 1 ). ii) each included country should report data of three-time intervals at minimum. the countries which do not fulfill these criteria were excluded from the predictive analysis [11] . additionally, we performed ecological correlation analyses to assess the relationship between r 1 and r 2 of new cases and deaths, with urban index, population density, cases/1000 inhabitants, and deaths/100,000 inhabitants. the predefined hypothesis was higher r 1 , and r 2 ratios are correlated with higher numbers of cases [9, 11] adjusted by population size, and these ratios are related to the number of urban areas and population density in the country. we calculated 95% confidence intervals using the standard error of the mean of the last three calculated r 2 . two-sided p<0�05 was considered statistically significant. the analyses were performed in microsoft excel 2019 and stata 15 (statacorp llc: college station, tx). for the report of this study, we followed the guidelines for accurate and transparent health estimates reporting (gather) to define best reporting practices for studies that compute health estimates for multiple populations in different times and spaces [18] . the checklist is reported in s1 table. this study is exempt from institutional review board's review due to the use of publicly available and de-identified information. we obtained information from 210 countries, including reported new confirmed cases and deaths until april 10, as well as their approximate first report date of covid-19 (table 1) . from them, it was possible to calculate the r 1 and r 2 of covid-19 cases and deaths of 170 countries; it was not possible to estimate neither of the ratios in 40 countries due to limited data available for these countries. the r 1 and r 2 ranged from 0�20 to 6�64 and from 0�17 to 22�06, respectively. our calculation showed that 73 countries (42�94%) are in the epidemic phase a (initial accelerated phase), 57 (33�52%) were decelerating getting closer to the peak (phase b), and 40 (23�5%) were already on the other side of the epidemic curve (phase d and e). for deaths, the r 1 was calculated for 116 countries with a range between 0 and 6, while for the r 2 only in 96 (range, 0 to 32�7). the majority of them, 74 (66�07%), showed an increase in mortality ratios (r 1 and r 2 ). due to absent data on mortality, 75 and 112 countries lacked r 1 and r 2 calculation, respectively. as an overall, the world is in a and b epidemic phases and is increasing death rates (45�8%) ( table 1) . from the 20 countries with highest r 1 (range, 2�38 to 6�64) and r 2 (range 2�20 to 22�06) for covid-19 new cases the majority were from africa (around 40%, fig 2) and had a middleincome economy (70% for r 1 and 35% for r 2 ). in case of deaths the range of r 2 were from 2�73 to 6 and for r 2 from 1�33 to 32�70. the predominant region for both ratios was europe, while high income countries were predominant for the r 1 and middle-income for r 2 ( table 2 ). in general terms, from all the countries with high r 1 low income countries represented 9�92% (13 countries) and 3�75% (3 countries) for cases and deaths respectively, whereas for high r 2 12�79% (11 countries) for cases and 5�71% (2 countries) for deaths. we then calculated the median r 1 and r 2 rates for new cases according to the epidemic phase (fig 1) . while r 1 median rate starts with 1�69 (for phase a) and then decreases to 0�79 in phase e; r 2 median rate starts with 1�42 for phase a and decreases to 0�81 in phase b but then increases again in phase d to 1�23 to then decrease in phase e to 0�75 thus being aligned with farr's law. correlational analyses. regarding the correlation analysis, the r 1 for mortality was positively correlated with urban index (rho = 0�2, p = 0�03), and with deaths per 100 000 inhabitants (rho = 0�3, p = 0�001). no significant correlations were found for the rest of the ratios. predictive analyses. for the prediction of new cases, we included 69 countries out of 210 countries, the rest of them did not meet the assumption criteria or have enough data. on the other hand, for the prediction of mortality, 64 countries were included in the modeling (in s2 table and in s3 table) . worldwide we predict 1 284 553�6 (ci 95%, 935 337�5-1 988 290�9) of new cases (43�1% of the total cases) and 221 329�3 (ci 95%, 155 105�3-371 461�1) new deaths (68�1% of the total deaths) during the period after april 10 to june 10. the peak of new cases would reach around april 11 to 15th with approximately 432 4843�7 new cases (ci 95%, 400 294�6-464 672�7) and the peak of mortality around april 16 to 20th with approximately 46 051�7 deaths during this period (ci 95%, 39 846�2-52 870�2). following a bell-shape curve, regardless neither of the new cases and new deaths reach zero until june 10, the lowest number of new cases would be around 1�2 (ci 95%, 0-321�3) new cases and 38 (ci 95%, 0�9-1 378�8) new deaths during the lowest peak on june 6th -10th. (fig 3, s2 and s3 tables) . regarding the prediction of individual countries, we divided them into quartiles based on the number of daily cases and deaths trends (figs 4 and 5) . the highest quartile of new cases has a range of 1 863 to 165 364 daily cases and included 18 countries (56% from europe, 22% from america, 17% from asia and 6% from oceania). the highest quartile for mortality includes values from 141 to 3 2867 daily deaths with 16 countries (10 [62%] from europe, 4 [25%] from america, 1[6%] from asia, and 1[6%] from africa). regarding new cases, from 69 countries, 56 will reach zero, and 13 will continue beyond june 10, 2020 (see s2 table) . for new deaths, from 64 countries, 58 will reach zero deaths and 6 will still be continued beyond june 10, 2020 (in s3 table) . the countries with higher predicted cases are the usa, uk, and spain, and higher predicted mortality are the usa, france, and sweden (figs 4 and 5 and s2 and s3 tables). comparison with updated data. we found in our post-hoc comparison with updated data (june 10, 2020) that from the countries we predicted a higher number of cases and deaths worldwide (using data till april 10, 2020), 70% and 100% are actually among the first 20 countries with more cases and deaths, respectively, by june 2020. our model predicted high dynamics in us, uk, brazil, italy, spain, france (table 1) , and this was confirmed with current data. additionally, we found 55 (26.2%) countries reported strict restriction strategies as part of political actions against the pandemic, the most common were stay-home policies, gathering table) . however, there is important missing information for several countries in the available dataset (ihme). regarding the worldwide curve for new cases and deaths of in june 2020 (s1 fig) showed a pseudo-normal distribution (negative kurtosis), with a steep slope by the second semester of march with a plateau by the beginning of april which last one month. by the beginning of may the curve started to rise again but gradually. similarly, the curve of death had a steep slope during the last 10 days, from march reaching a peak by mid-april, and then a gradual descent until the end of may, where it reached a plateau. by june 10, 36 countries (64.6%) of the 56 countries we predicted to be around no new cases before june 10 are decreasing or around zero new daily cases (table 1) . moreover, as we predicted countries as new zealand, greenland, macao, saint martin, and faeroe islands report no new cases for more than ten days up to june 10. concerning the 20 countries with high r1 for new covid-19 cases (table 2) , we found that five countries (25%) were still having an increment on their curve with one of them (uzbekistan) increasing a second wave. two countries (10%) were in a plateau; for seven (35%), the descend on the curve was still not clear, while three (15%) has a clear descend and other three (15%) has not reported new cases for more than ten days. among the three countries with the highest r1, congo and gabon are still reporting cases with a heterogeneous pattern, which hinders the determination of a clear dynamic on the curve. on the other hand, angola reported its peak on june 10 and still in an accelerated phase of the pandemic ( table 2 ). in the case of the countries with high r2 for cases, three countries (15%) had an increasing curve, one (5%) was in a plateau, three (15%) had a not clear descend, seven (35%) were descending and six (30%) had not reported cases for more than ten days. among the countries with the highest r2, angola and bangladesh showed a clear increment on its curve, reporting more new cases on june 10 ( table 2 ). farr's law is a simple arithmetical model that provides useful and important insights on epidemic dynamics. the findings from our modeling suggest that most of the countries over the world (76�43%) are in the early stage of the epidemic curve (phase a and b of our theoretical framework). the countries with higher epidemic dynamics (acceleration of cases and death numbers) are in africa (around 40%) and had middle-income economies. based on our model, the pandemic curve will reduce significantly until june 10, 2020, for both new cases and deaths, in the overall worldwide model and for 56 countries (in s2 and s3 tables). the countries with higher predicted cases (adjusted for population) are usa, uk, and spain, and higher predicted mortality are usa, france and sweden; however, 60% of the countries could not enter to the predictive modeling due to lack of data or instability of r 2 estimate (higher than 1). the percentage of countries on phases a and b and with higher dynamics from low and middle-income sectors are higher (mainly from africa and latin america). this is a potential risk due to the limited health resources in those countries that could lead to a high rate of mortality and burden for the health system but also could generate a devastating socioeconomic, political, and inequality impact [19, 20] . recent studies are reporting the lack of preparedness and high vulnerability of african countries against an eventual increase of covid-19 cases [5] . also, moore et al. reported a ranking of countries based on the infectious disease vulnerability index [21] , which considered a number of socioeconomic and health factors, several countries from the top of their list, such as angola, niger, guinea, congo, togo, and ivory coast are in our ranking using the farr's ratios, indicating a higher epidemic dynamics in those countries, yet with a small number of cases, currently. thus, this is a call to prioritize actions in those countries to intensify surveillance, to re-allocate resources, and to build healthcare capacities based on multi-nation collaboration [22] to limit onward transmission and to reduce the future impact on these regions. based on our prediction, the worldwide trend will reach values near to zero at the beginning in june, and approximately 56 countries (s2 table) will reach values near zero before june 10, 2020. compared with the current trend at june 10, 2020, we can see a pseudo-normal distribution with low kurtosis (more pronounced in the curve of new cases-s1 fig) . this could be explained by the heterogeneity of the clusters (countries) included in the model, with different pandemic start date, different socioeconomic characteristics, and public health and political actions against the pandemic; therefore, this produces potentially an overlap of multiple normal distributions curves. this also could be true for large countries with independent states such as usa (implementing multiple political actions and public health strategies at different moments) [23] . however, for more homogenous clusters (such as new zealand, australia, and some asian countries), the predicted curves were accurate. similarly, the prediction estimates were also accurate-most of the countries (70%) that we predicted a higher number of cases and deaths (till june 10) were confirmed in our post-hoc extraction, as well for the predicted countries with high dynamics (higher predicted r1 and r2). thus, we should consider that our estimates using the farr's law depend on the precision of the reported data, the cluster heterogeneity, and the current acceleration (r 2 ) of the epidemic dynamics (i.e. higher values of r 2 produces an exponential function of the fitted values). similar behavior was reported in previous studies [11, 12] . although, santillana et al. suggested a potential use of these higher r 2 ratios, not to use them as predictive measures but rather as sentinel index for the change in epidemic dynamic, which could indicate the start of a new wave of cases [11] . previous studies have reported behavior predictions for the covid-19 pandemic; most of them focus on specific countries, such as china [24] , chile [25] , italy [24] , france [24] , and usa [23, 26] . the estimation of end date varies from may 12 (for chile) to june 15 (for italy), these dates from more complex models (most of them from a sir model) are consistent with our predictions for those countries (table 1) , suggestion an acceptable accuracy to describe the epidemic dynamics with a simpler model. none of the previous studies used the farr's law to model the current pandemic behavior, and the available models reported prediction for highincome countries with better health system infrastructure and data registration; however, we could not identify published models from low-and middle-income countries, especially from africa, those who are paradoxically more at risk due to high pandemic dynamic. thus, farr's law approximation will be a valid option for scenarios with low resources and to identify countries at risk. moreover, it has been reported farr's law is an adequate model to assess the behavior of epidemics more than predict the exact number of cases accurately [2] . however, under certain assumptions (in epidemic phases with relative deceleration), its estimates are near to the sir and idea models [11] . besides, it seems to apply across different outbreaks types-because it relies on the intrinsic natural history of epidemics-and allow as to model fast with simple assumptions and limited data. the r 1 and r 2 ratios are variable across countries and epidemic phases, allowing us to classify the epidemic behavior over the world. besides, it does seem that a higher r 1 for mortality is associated with a high urban index and a higher number of deaths per 100,000 inhabitants, which is along with the literature on the impact of urbanization on the transmission of respiratory infection diseases [11] . therefore, countries could also use r 1 and r 2 ratios to monitor the first deceleration phase (phase b). interestingly, the median r 2 ratio is similar to the past aids epidemic reports [9] , thus reflecting perhaps the behavior of an outbreak without immune protection. the sociodemographic characteristics and the political actions against the pandemic are important factors within countries to describe pandemic behavior. from our model, we predicted 43 countries (table 1) would reach near to zero in june. most of them are middle to high-income countries, implement early and strict restriction policies; it seems no particular sociodemographic characteristics (population size, density, or proportion of older people) are predominant in these groups. these findings are aligned with previous studies showing the positive effects of strict restriction policies [27] . from our model and post-doc extraction, countries as new zealand reached no new cases around may, while australia has less than 20 new daily cases by june 10. the normality of these curves might be related to different explanations. first of all, both countries are high-income countries with a high human capital index (0.8 and 0.7 respectively) that have invested in health since 1990 [28] . germany, with 21.5% of the population more than 65 years old, has a smaller number of deaths per million people than other countries like us, italy, spain. all these countries established political and health social regulations during the pandemic and widespread testing even before reaching the peak. however, the key factors to successfully implement restrictive measurements could be adequate health literacy and socioeconomic equity [29] . for example, in our model, we found a high dynamic in peru; it was one of the first countries in america to close its borders, established a strict lock-down with a curfew, and even provided financial aid for vulnerable people during the lock-down [30] . however, high socioeconomic disparities, great urban low-income conglomerations with limited health services accessibility, high levels of business informality [31] , and even corruption have played an important role in jeopardizing the fight against the covid-19 outbreak. despite that, the peruvian government's quarantine policies might have prevented a major sanitary catastrophe considering the fragile and fragmented peruvian public health system [32] . indeed, our results suggested that restrictive measurements (social-distancing, restricting gatherings and non-essential travels) and widespread testing are critical to ending the ongoing covid-19 pandemic. however, it is necessary to consider the sociodemographics and health literacy characteristics of the population to implement these measures in the mid-to long-term successfully. one limitation in our modeling is the probable high rate of underreported cases all over the world, especially in low and middle economies either because of population size, weak health systems, geographical issues, inequity or lack of health access [33] . additionally, the migration of people between countries is an important covariate for epidemic modeling [34] we did not include in our model due to data availability. in fact, this variable could contribute to underestimating the real dynamic; however, the utility for public health decision still valid as a similar limitation was founded in the previous influenza h1n1 pandemic [35] . finally, another limitation is the potential heterogeneity on the criteria to identify cases in the worldometer dataset, since they used a confirmation status based on public health policies and available test in each country. therefore, there is a possibility of underreporting and false negatives cases [36] , especially in low-and middle-income countries. finally, even though our model predicts a significant reduction of cases and deaths worldwide, a second wave of cases is possible. currently, there are examples of countries with these patterns, such as south korea and china, countries where the restriction strategies and political actions were applied early and rigorously. this highlights the importance of other factors such as viral reintroduction, particularly international importation from countries with higher epidemic dynamics, as well as a rebound of viral transmissibility due to the gradual resumption of economic activities and normal levels of social interaction [37] . in this scenario, our modeling approach could be a potential tool to assess the pandemic dynamics in a simple manner, especially in regions with already compromised health and socioeconomic systems. in conclusion, to develop a global health perspective on a pandemic, the first step could be to use of simple modeling techniques to depict a broad global picture of the disease's dynamics, that allow us to properly identify areas with high-risk due to high dynamic of the disease. farr's law seems to be a useful model to give an overview of covid-19 pandemic dynamics. the regions with high dynamics are countries from africa and latin america. thus, this is a call to urgently prioritize actions in those countries to intensify surveillance and re-allocate resources based on multi-nation collaboration to limit onward transmission and to reduce the future impact on these regions. close monitoring of epidemic dynamics is needed to ensure correct worldwide policy interventions and to be prepared for an eventual covid-19 second wave. supporting information s1 coronavirus disease 2019 (covid-19) situation report-83. world health organization prediction models for diagnosis and prognosis of covid-19 infection: systematic review and critical appraisal data-based analysis, modelling and forecasting of the covid-19 outbreak centre for mathematical modelling of infectious diseases covid-19 working group. early dynamics of transmission and control of covid-19: a mathematical modelling study preparedness and vulnerability of african countries against importations of covid-19: a modelling study feasibility of controlling covid-19 outbreaks by isolation of cases and contacts modelling covid-19 transmission: from data to intervention historical note on farr's theroy of the epidemic farr's law applied to aids projections a systematic review of studies on forecasting the dynamics of influenza outbreaks. influenza other respir viruses relatedness of the incidence decay with exponential adjustment (idea) model applying farr's law to project the drug overdose mortality epidemic in the united states from rio to rio+ 20: the changing role of local governments in the context of current global governance dashboard by the center for systems science and engineering (csse) at johns hopkins university (jhu) institute for health metrics and evaluation. covid-19 projections the gather working group. guidelines for accurate and transparent health estimates reporting: the gather statement health inequalities and infectious disease epidemics: a challenge for global health security managing covid-19 in low-and middle-income countries. jama. 2020. epub ahead of print identifying future disease hot spots. california: rand corporation looming threat of covid-19 infection in africa: act collectively, and fast forecast and evaluation of covid-19 spreading in usa with reducedspace gaussian process regression analysis and forecast of covid-19 spreading in china, italy and france proyección epidemioló gica de covid-19 en chile basado en el modelo seir generalizado y el concepto de recuperado reconstructing and forecasting the covid-19 epidemic in the united states using a 5-parameter logistic growth model modelling the covid-19 epidemic and implementation of population-wide interventions in italy measuring performance on the healthcare access and quality index for 195 countries and territories and selected subnational locations: a systematic analysis from the global burden of disease study covid-19: health literacy is an underestimated problem. the lancet public health how poorer countries are scrambling to prevent a coronavirus disaster empobrecimiento de los hogares y cambios en el abastecimiento de alimentos por la covid-19 en lima the national health system in peru. revista peruana de medicina experimental y salud publica estimation of covid-19 outbreak size in italy pandemics, migration and global health security. handbook on migration and security estimates of the prevalence of pandemic (h1n1) laboratory diagnosis of covid-19: current issues and challenges projecting the transmission dynamics of sars-cov-2 through the postpandemic period key: cord-326011-5rmhjbri authors: cui, dawei; feng, luzhao; chen, yu; lai, shengjie; zhang, zike; yu, fei; zheng, shufa; li, zhongjie; yu, hongjie title: clinical and epidemiologic characteristics of hospitalized patients with laboratory-confirmed respiratory syncytial virus infection in eastern china between 2009 and 2013: a retrospective study date: 2016-11-01 journal: plos one doi: 10.1371/journal.pone.0165437 sha: doc_id: 326011 cord_uid: 5rmhjbri respiratory syncytial virus (rsv) is a leading cause of morbidity and mortality worldwide in children aged <5 years and older adults with acute lower respiratory infections (alris). however, few studies regarding the epidemiology of hospitalizations for rsv infection have been performed previously in china. here, we aimed to describe the clinical and epidemiologic characteristics of hospitalized patients with laboratory-confirmed rsv infection in eastern china. active surveillance for hospitalized alri patients using a broad case definition based on symptoms was performed from 2009–2013 in 12 sentinel hospitals in eastern china. clinical and epidemiologic data pertaining to hospitalized patients of all ages with laboratory-confirmed rsv infection by pcr assay were collected and analyzed in this study. from 2009 to 2013, 1046 hospitalized patients with laboratory-confirmed rsv infection were enrolled in this study, and 14.7% of patients had subtype a, 24.2% of patients had subtype b, 23.8% of patients with subtype not performed, and 37.3% of patients had rsv coinfections with other viruses. rsv and influenza coinfections (33.3%) were the most common coinfections noted in this study. moreover, young children aged <5 years (89.1%, 932/1046), particularly young infants aged <1 year (43.3%, 453/1046), represented the highest proportion of patients with rsv infections. in contrast, older adults aged ≥60 years (1.1%, 12/1046) represented the lowest proportion of patients with rsv infections among enrolled patients. the peak rsv infection period occurred mainly during autumn and winter, and 57% and 66% of patients exhibited symptoms such as fever (body temperature ≥38°c) and cough separately. additionally, only a small number of patients were treated with broad-spectrum antiviral drugs, and most of patients were treated with antimicrobial drugs that were not appropriate for rsv infection. rsv is a leading viral pathogen and a common cause of viral infection in young children aged <5 years with alris in eastern china. effective vaccines and antiviral agents targeting rsv are needed to mitigate its large public health impact. respiratory syncytial virus (rsv) is a leading cause of viral infection in children and older adults worldwide, particularly young children aged <5 years [1] . studies indicate that rsv infection is associated with high morbidity and mortality rates in children with acute lower respiratory infections (alris) in both industrialized and developing countries [1] [2] [3] [4] . approximately 34 million rsv-related alris occur in children under 5 years of age, resulting in at least 3.4 million hospitalizations and 66,000-199,000 rsv-related deaths, 99% of which occur in developing countries [1] . rsv infection is responsible for approximately 1/4 of hospitalizations in children aged <5 years in china [2, [5] [6] [7] [8] [9] in areas such as jingzhou [6] , lanzhou [7] , hangzhou [8] , suzhou [9] , and hong kong [2] . accumulating evidence indicates that rsv is a common cause of severe alris, such as bronchiolitis and pneumonia, particularly in young children (aged <5 years) and older adults [10] [11] [12] . safe and effective vaccines are needed to combat the high morbidity and mortality rates associated with rsv infection, and some candidates are in development. previous reports have estimated the prevalence of rsv infection in hospitalized children and adults and determined that rsv seasons vary by geographic region and that rates of hospitalizations vary by age [13] [14] [15] [16] . however, more data regarding the clinical and epidemiologic characteristics of rsv infection are needed to develop strategies to prevent and control the disease. in this study, we aimed to evaluate the epidemiology, clinical characteristics and management of alris caused by rsv infection among hospitalized patients of all age groups in eastern china between 2009 and 2013 in a hospital-based surveillance study. ethics statement rsv data collection from patients with alris was considered public health surveillance by the national health and family planning commission of the people's republic of china. brief verbal informed consent was obtained from all individuals enrolled in this study, according to the declaration of helsinki (1964), and this study was approved by the ethics committee of the chinese center for disease control and prevention (china cdc, beijing, china). all subjects with laboratory confirmed rsv infection were hospitalized at 12 sentinel hospitals located in 6 provinces in eastern china between january 2009 and december 2013. detailed data pertaining to these patients, including data regarding patient demographic and clinical characteristics and laboratory results, were obtained from an online data management system established by the china cdc [17] [18] . patient enrollment, sample collection, laboratory testing, and case reporting were conducted by all sentinel hospitals and laboratories according to a national surveillance protocol [17] . patients were admitted to surveillance wards presided over by the departments of pediatrics, internal medicine, and infectious diseases or to the intensive care units (icus) of the abovementioned sentinel hospitals and screened by physicians and nurses for alri, which was diagnosed according to the following criteria: (1) at least one of the following manifestations of acute infection: fever (!38°c), abnormal white blood cell (wbc) differentials, leukocytosis (wbc count more than 10,000/ml) or leukopenia (wbc count less than 4,000/ml), and chills; and (2) at least one of the following signs/symptoms of respiratory tract infection: cough, sputum production, shortness of breath, lung examination abnormalities (crackles or wheeze), tachypnea, and chest pain [17] [18] . clinical samples, such as nasopharyngeal swabs, aspirates and sputum, were collected and immediately placed in viral transport medium (vtm) before being stored at 4-8°cat each sentinel hospital. these specimens were subsequently transferred to the nearest influenza network laboratory (provincial or prefecture center for disease control and prevention [cdc]) for diagnostic testing. most of the specimens were tested within 24 hours of collection, and any specimens not tested within 24 hours were stored in vtm at -70°c. all specimens were tested for rsv (a and b subtypes), human influenza virus, human parainfluenza virus (hpiv), human bocavirus (hbov), adenovirus (adv), rhinovirus (rv), coronavirus (cov), human metapneumovirus (hmpv), and enterovirus (ev)by real-time polymerase chain reaction (pcr), reverse transcriptase pcr (rt-pcr) or real-time rt-pcr, as previously described [17, 19, 20] . detailed data regarding patient demographic and clinical characteristics and laboratory results were collected by the staff of the sentinel hospitals and laboratories using standardized case reporting forms and were uploaded into the online data management system. descriptive statistics were used to analyze the demographic, epidemiological and clinical characteristics of the patients with rsv infection. statistical analysis was performed with spss software (v18.0, spss, chicago, il, usa). wilcoxon tests were used to compare the medians of continuous variables, and chi-square tests were performed to analyze frequency data. p<0.05 was considered statistically significant. from january 2009 to december 2013, 1046 hospitalized patients with laboratory-confirmed rsv infections were treated at 12 sentinel hospitals in 6 provinces in eastern china (fig 1) . of these patients, 154 (14.7%) were diagnosed with rsv-a, 253 (24.2%) were diagnosed with rsv-b, 249 (23.8%) were subtyping not performed, and 390 (37.3%) were diagnosed with coinfections with rsv and other respiratory tract viruses (fig 2a) . influenza virus (48.7%, 190/ 390) was the most common infectious agent noted in patients mixed infections, and coinfections with rsv/influenza (33.3%, 130/390) were the most frequent coinfections noted in this study, as 13 patients were diagnosed with rsv-a and influenza, 9 patients were diagnosed with rsv-b and influenza, and 108 patients were diagnosed with rsv subtyping not performed and influenza. furthermore, 61 (15.6%) and 52 patients (13.3%) were diagnosed with rsv and hpiv or rsv and adv coinfections, respectively ( fig 2b) . in this study, 671 (64.1%) of the enrolled patients were male, and no significant differences were observed between men and women with the respect to the incidences of rsv-a and rsv-b infections and coinfections (table 1) . rsv infection was more common in young children aged <5 years (89.1%, 932/1046) than in patients aged !5 years (11.9%, 114/1046). rsv-a (85.1%, 131/154), rsv-b (90.9%, 230/253), subtyping not performed (87.1%, 217/249) and rsv coinfection (83.3%, 325/390) were also more common in young children than in other patients. moreover, rsv infection was more common in young infants aged <6 months (26.8%, 280/1046) than in other patients. rsv-a (40.0%, 60/154), rsv-b (39.5%, 100/253), and subtyping not performed (22.1%, 55/249) were also more common in this age group than in other age groups. additionally, the median age of patients with rsv single-infections (8-12 months) was considerably lower than that of patients with coinfections (24 months) (p = 0.000, mann-whitney test). rsv infection exhibited a gradually decreasing trends with increasing age, as did rsv-a, rsv-b, and subtyping not performed and coinfections. rsv infection exhibited clear seasonal trends during this study. the majority of rsv infections occurred between september 2010 and february 2011 (fig 3a) . rsv-a infections were most frequently observed in spring, autumn and winter from january to march 2010, july 2011 to may 2012, and january to february 2013 (fig 3b) . similarly, rsv-b infections were most frequently observed between january and april in 2009, 2010 and 2013 ( fig 3c) . moreover, rsv with subtyping not performed and rsv coinfections were most frequently observed between october 2010 and february 2011 ( fig 3d) and between september 2010and february 2011 ( fig 3e) compared with other seasons, respectively. a temperature of !38°c was documented in 61.0% of patients with rsv infection, and 59.0% of patients with rsv coinfection with other viruses at the time of physical examination. a moderately significant difference in temperature was observed between patients with rsv coinfections and patients with rsv single-infections (χ2 = 4.825, p = 0.028). cough was the most common symptom exhibited by rsv patients (66.3%), and runny nose (22.7%) and sputum production (18.6%) were also common among affected patients. moreover, significant differences between patients with rsv coinfections with other viruses and patients with rsv single-infections were observed with respect to symptoms such as cough (χ2 = 5.774, p = 0.016), runny nose (χ 2 = 8.749, p = 0.003), sore throat (χ 2 = 10.357, p = 0.001), sputum production (χ2 = 8.063, p = 0.005), shortness of breath (χ2 = 27.700, p = 0.000), and difficulty breathing (χ2 = 5.920, p = 0.015), as well as wbc count (χ2 = 0.015, p = 0.903). moreover, the patients with rsv and influenza coinfection had highest rate in clinical symptoms as sore throat (18.4%), shortness of breath (8.4%) and/or difficulty breathing (11.1%). of 803 patients who underwent a chest x-ray, 168 (20.9%) were found to have radiographic evidence of pneumonia, although the proportion of the patients with pneumonia/rsv coinfection was not significantly different from that of patients with rsv single-infection (χ2 = 0.653, p = 0.419) ( table 2 ). currently, only a few drugs exist effective against rsv infections or other viral infections. many antimicrobial drugs were used by doctors to treat patients in this study. of 147 patients with rsv infection, 102 were treated with one drug. amoxicillin-clavulanate (23.5%, 24/102) was the most frequently prescribed antibacterial drug, and cefuroxime (17.6, 18/102) was the second most frequently prescribed antibacterial drug. seventy patients were treated with two drugs with azithromycin (16.7%, 12/72) and cefuroxime (13.9%, 10/72) that were the most frequently prescribed drugs. moreover, multiple other drugs were also prescribed for patients. however, broad-spectrum antiviral agents, such as ribavirin and potassium sodium dehydroandroandrographolide succinate, were not commonly prescribed to patients with rsv infection. additionally, some drugs were used excessively and administered for longer time periods than recommended, such as azithromycin (whose course is normally limited to 5 days) and cefuroxime (whose course is normally limited to 7 days) ( table 3 ). this study is a part of a hospital-based alri surveillance study in china and documented the etiologies, epidemiology and clinical characteristics of alris caused rsv, which was diagnosed via laboratory testing, using data from a sentinel surveillance system in eastern china. from 2009 to 2013, a total of 1046 hospitalized alri patients (children and adults) were treated at 12 sentinel hospitals in eastern china. we found that most patients had rsv single-infections and that 37.3% had mixed viral infections caused by rsv and other respiratory tract viruses. additionally, we observed that coinfections with rsv and influenza were the most frequently diagnosed mixed infections among our patient population, a finding consistent with those of previous studies in china [17] [18] [19] [20] [21] and other countries [15, [22] [23] [24] [25] [26] . taken together, these findings indicate that coinfections may influence severe illnesses in patients with rsv infections, especially in young children aged <5 years. although many studies regarding rsv have focused on children aged <5 years and elderly adults, among whom the morbidity and mortality rates of rsv infection are high, little is known regarding the effects of rsv among patients of all ages. we analyzed the overall age distributions of the patients with rsv infections. our results indicated that children aged <5 years represented the largest proportion of patients with rsv infections (86.4%) and coinfections (83.3%), particularly young children (total: 62.3%; coinfections: 48.7%) aged <2 years, compared to patients aged !5 years. these observations indicate that an association exists between rsv prevalence and age, a finding consistent with those of previous reports in china, japan, thailand and other countries [17] [18] [19] [20] [21] [24] [25] [26] [27] . our surveillance data indicate that rsv infection occurred almost year-round from 2009 to 2013 and peaked mainly during autumn and winter in areas of eastern china with subtropical climates, results similar to those reported previously in china and other countries [19] [20] [21] [22] [23] [24] but different from those reported in tropical countries [16] . these findings indicate that rsv infection is characterized by a seasonality that may be associated with regional climate and demographic factors [6, 12, 22] . the seasonality of rsv is different in tropical and subtropical countries; thus, hospitals in these countries can utilize seasonally timed interventions and specific vaccination and hospital infection control strategies [1, 9, 13] . most of the patients enrolled in this study had symptoms of fever (temperature !38°c) and cough, and moderately significant differences in temperature and cough were observed between patients with rsv coinfections and patients with rsv single-infections. these observations are consistent with those of previous reports [17] [18] [19] . a few patients experienced shortness of breath or difficulty breathing, the majority of whom suffered from rsv coinfections, indicating that rsv coinfections with other viruses can cause more severe disease than rsv single-infections. however, the proportions of patients with pneumonia were not significantly different between patients with rsv coinfection and patients with rsv single-infection, indicating that pneumonia development was likely related to other factors, such as patient age, patient physical condition, and viral load [23] [24] [25] [26] [27] [28] . generally, viral infections do not significantly affect common laboratory parameters (such as wbc, po 2 , and pco 2 ), particularly during their early stages [11, [29] [30] . our data indicated that there were no significant differences in laboratory parameters between patients with rsv single-infections and patients with coinfections. currently, only a few antivirals exist that are effect against rsv infections and other viral infections, and delays in viral testing have important effects on infection severity, particularly among patients with rsv, influenza virus and enterovirus infections [1, 11, 30] . in our study, only a few patients with rsv infections were treated with broad-spectrum antiviral drugs (ribavirin and potassium sodium dehydroandroandrographolide succinate) that were not specific for rsv. most of the antimicrobial drugs administered to patients in this study were not appropriate for patients with rsv infection but are commonly used in such patients in china. it is well known that antimicrobial drug overuse selects for highly resistant organisms, causes immune dysfunction, and increases viral loads, which may explain the high morbidity and mortality rates associated with rsv infection [12, [26] [27] [28] . our study had some limitations. first, patients were tested for rsv and other respiratory tract viruses, but not bacterial pathogens; thus, our data regarding pathogens causing alris were not comprehensive. second, we tested for rsv-a and rsv-b to determine the relationships between rsv subtype and age, seasonality and clinical characteristics, but some of total rsv infections did not test for rsv subtypes. third, some of our clinical and laboratory data were not comprehensive, which affected our ability to estimate rsv disease severity. in summary, this study provided important epidemiologic information regarding rsv and associated respiratory viral infections in patients with alris in eastern china. rsv infection is common in young children, and implementing disease control strategies (such as safe vaccination) during the appropriate seasons may have a significant impact on public health. clinical and laboratory data pertaining to patients with rsv infection may be helpful for optimizing patient treatments. timely and accurate diagnosis of rsv infection in patients with alris is necessary to prevent large-scale rsv spread and to reduce the burden imposed by the disease and the misuse of antimicrobial drugs. these preliminary results indicate that more surveillance data are needed to estimate the rsv disease burden and to determine whether geographic locations, climate, other environmental factors and rsv coinfections impact the severity ofrsv-related epidemics. global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis respiratory syncytial virus morbidity, premorbid factors, seasonality, and implications for prophylaxis viral etiology of hospitalized acute lower respiratory infections in children under 5 years of age-a systematic review and meta-analysis. croatian medical journal respiratory syncytial virus infection in elderly and high-risk adults. the new england journal of medicine a 4 year prospective study to determine risk factors for severe community acquired pneumonia in children in southern china clinical and epidemiologic characteristics of respiratory syncytial virus infection among children aged <5 years clinical microbiology and infection: the official publication of the european society of clinical microbiology and infectious diseases human respiratory syncytial virus in children with lower respiratory tract infections or influenza-like illness and its co-infection characteristics with viruses and atypical bacteria in hangzhou epidemiological and clinical profiles of respiratory syncytial virus infection in hospitalized neonates in suzhou, china. bmc infectious diseases modelling estimates of the burden of respiratory syncytial virus infection in adults and the elderly in the united kingdom global and regional burden of hospital admissions for severe acute lower respiratory infections in young children in 2010: a systematic analysis respiratory syncytial virus and other respiratory viral infections in older adults with moderate to severe influenza-like illness. the journal of infectious diseases predictors of the duration of the respiratory syncytial virus season. the pediatric infectious disease journal clinical characteristics and risk factors of severe respiratory syncytial virus-associated acute lower respiratory tract infections in hospitalized infants respiratory syncytial virus-associated mortality in hospitalized infants and young children respiratory syncytial virus infection in tropical and developing countries. tropical medicine & international health viral etiologies of hospitalized acute lower respiratory infection patients in china characterizing the epidemiology, virology, and clinical features of influenza in china's first severe acute respiratory infection sentinel surveillance system viral etiology of acute respiratory infection in gansu province, china clinical microbiology and infection: the official publication of the european society of clinical microbiology and infectious diseases human respiratory syncytial virus in children with acute respiratory tract infections in china hospitalizations for acute lower respiratory tract infection due to respiratory syncytial virus in thailand prevalence and incidence of respiratory syncytial virus and other respiratory viral infections in children aged 6 months to 10 years with influenza-like illness enrolled in a randomized trial. clinical infectious diseases: an official publication of the infectious diseases society of america impact of respiratory syncytial virus infection as a cause of lower respiratory tract infection in children younger than 3 years of age in japan the burden of hospitalized lower respiratory tract infection due to respiratory syncytial virus in rural thailand respiratory syncytial virus infections in hospitalized infants: association between viral load, virus subgroup, and disease severity epidemiology of respiratory syncytial virus infection in rural and urban kenya. the journal of infectious diseases high viral load and respiratory failure in adults hospitalized for respiratory syncytial virus infections. the journal of infectious diseases incidence of hospitalization for respiratory syncytial virus infection amongst children in ontario, canada: a population-based study using validated health administrative data challenges and opportunities in developing respiratory syncytial virus therapeutics we thank susan gerber from division of viral diseases, national center for immunization and respiratory diseases, centers for disease control and prevention, atlanta, usa for comments on the manuscript. writing -review & editing: hy. key: cord-324405-6uanhe2p authors: burke, rachel m.; balter, sharon; barnes, emily; barry, vaughn; bartlett, karri; beer, karlyn d.; benowitz, isaac; biggs, holly m.; bruce, hollianne; bryant-genevier, jonathan; cates, jordan; chatham-stephens, kevin; chea, nora; chiou, howard; christiansen, demian; chu, victoria t.; clark, shauna; cody, sara h.; cohen, max; conners, erin e.; dasari, vishal; dawson, patrick; desalvo, traci; donahue, matthew; dratch, alissa; duca, lindsey; duchin, jeffrey; dyal, jonathan w.; feldstein, leora r.; fenstersheib, marty; fischer, marc; fisher, rebecca; foo, chelsea; freeman-ponder, brandi; fry, alicia m.; gant, jessica; gautom, romesh; ghinai, isaac; gounder, prabhu; grigg, cheri t.; gunzenhauser, jeffrey; hall, aron j.; han, george s.; haupt, thomas; holshue, michelle; hunter, jennifer; ibrahim, mireille b.; jacobs, max w.; jarashow, m. claire; joshi, kiran; kamali, talar; kawakami, vance; kim, moon; kirking, hannah l.; kita-yarbro, amanda; klos, rachel; kobayashi, miwako; kocharian, anna; lang, misty; layden, jennifer; leidman, eva; lindquist, scott; lindstrom, stephen; link-gelles, ruth; marlow, mariel; mattison, claire p.; mcclung, nancy; mcpherson, tristan d.; mello, lynn; midgley, claire m.; novosad, shannon; patel, megan t.; pettrone, kristen; pillai, satish k.; pray, ian w.; reese, heather e.; rhodes, heather; robinson, susan; rolfes, melissa; routh, janell; rubin, rachel; rudman, sarah l.; russell, denny; scott, sarah; shetty, varun; smith-jeffcoat, sarah e.; soda, elizabeth a.; spitters, christopher; stierman, bryan; sunenshine, rebecca; terashita, dawn; traub, elizabeth; vahey, grace m.; verani, jennifer r.; wallace, megan; westercamp, matthew; wortham, jonathan; xie, amy; yousaf, anna; zahn, matthew title: enhanced contact investigations for nine early travel-related cases of sars-cov-2 in the united states date: 2020-09-02 journal: plos one doi: 10.1371/journal.pone.0238342 sha: doc_id: 324405 cord_uid: 6uanhe2p coronavirus disease 2019 (covid-19), the respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), was first identified in wuhan, china and has since become pandemic. in response to the first cases identified in the united states, close contacts of confirmed covid-19 cases were investigated to enable early identification and isolation of additional cases and to learn more about risk factors for transmission. close contacts of nine early travel-related cases in the united states were identified and monitored daily for development of symptoms (active monitoring). selected close contacts (including those with exposures categorized as higher risk) were targeted for collection of additional exposure information and respiratory samples. respiratory samples were tested for sars-cov-2 by real-time reverse transcription polymerase chain reaction at the centers for disease control and prevention. four hundred four close contacts were actively monitored in the jurisdictions that managed the travel-related cases. three hundred thirty-eight of the 404 close contacts provided at least basic exposure information, of whom 159 close contacts had ≥1 set of respiratory samples collected and tested. across all actively monitored close contacts, two additional symptomatic covid-19 cases (i.e., secondary cases) were identified; both secondary cases were in spouses of travel-associated case patients. when considering only household members, all of whom had ≥1 respiratory sample tested for sars-cov-2, the secondary attack rate (i.e., the number of secondary cases as a proportion of total close contacts) was 13% (95% ci: 4–38%). the results from these contact tracing investigations suggest that household members, especially significant others, of covid-19 cases are at highest risk of becoming infected. the importance of personal protective equipment for healthcare workers is also underlined. isolation of persons with covid-19, in combination with quarantine of exposed close contacts and practice of everyday preventive behaviors, is important to mitigate spread of covid-19. in late december 2019, a cluster of patients with pneumonia of unknown etiology was identified in wuhan, china [1] . this disease, now termed coronavirus disease 2019 (covid19) , spread rapidly within china and quickly became pandemic [1] . phylogenetic analyses of severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the causative agent of covid-19, suggest zoonotic origin [2] , with subsequent rapid spread indicative of person-to-person transmission [3, 4] . on january 20, 2020, the first case of covid-19 was confirmed in the united states [5] ; through the end of january, nine more travel-related cases were identified. to interrupt transmission and facilitate early identification of secondary cases (i.e., transmissions of sars-cov-2 from the original travel-related case patient to a close contact), public health authorities at the state, county, and local levels, in consultation with subject-matter experts from the u.s. centers for disease control and prevention (cdc), mobilized rapidly to place the patients under appropriate isolation and identify contacts exposed to these patients. some of the information from the contact investigations of the first 10 travel-associated case patients was briefly and rapidly reported through a morbidity and mortality weekly report [6] . enhanced contact investigations were conducted centered on nine of these first 10 patients, and included in-depth interviews to better characterize exposure type, exposure duration, and contact medical history, in addition to collection of respiratory specimens to identify asymptomatic or pre-symptomatic infections. the present manuscript details the results from these nine enhanced contact investigations, including information on exposures, active monitoring, laboratory testing, and secondary attack rates among close contacts. these investigations included the close contacts of nine travel-related covid-19 patients (hereafter referred to as "travel-associated case patients") first identified as suspected covid-19 patients between january 19 and january 30, 2020, and represent collaboration among state and local health department staff, cdc personnel, and healthcare facility staff (hereafter referred to as "covid-19 outbreak response teams"). the aims of these investigations were to interrupt transmission, investigate risk factors for transmission, and identify both symptomatic and asymptomatic infections among contacts of travel-associated case patients. after diagnosis confirmation, these nine travel-associated case patients were interviewed to determine the date of symptom onset and to collect information on movements and interactions with other individuals during the presumed infectious period of the travel-associated case patient. individuals with potential exposure to a travel-associated case patient were contacted by covid-19 outbreak response teams to determine their exposure risk level, whether they met the definition of a close contact, and whether they met criteria for active monitoring (terms defined below in "classification of contacts and exposures"). individuals not meeting the definition of a close contact are not included in this report. to better understand sars-cov-2 transmission, covid-19 outbreak response teams selected a convenience sample of the close contacts who met active monitoring criteria to target for collection of additional, detailed exposure and demographic information using standardized forms; some sites were able to collect information on all close contacts, while others collected information only on those close contacts they classified as having had high-or medium-risk exposures. to understand the prevalence of asymptomatic or pre-symptomatic infection, a convenience sample of actively monitored close contacts was selected from whom to request respiratory (nasopharyngeal [np] and oropharyngeal [op]) samples outside of diagnostic specimen collection procedures (i.e., while contacts were asymptomatic or, in some cases, symptomatic with � 1 previous negative sars-cov-2 result); some sites were able to request at least one set of samples from all close contacts, but most sites targeted sample collection mainly to close contacts determined to have had high-risk exposures, such as household members and some healthcare personnel. definitions of the infectious period, close contact, and exposure risk, as well as decisions regarding which types of contacts to monitor and how to manage their movement, were made at the site level by the covid-19 outbreak response teams. in one site, the presumed infectious period began on the date of symptom onset in the travel-associated case patient; however, in other sites, investigators also considered between one and three days before symptom onset in the travel-associated case patient. in all sites, the presumed infectious period ended when the travel-associated case patient was released from isolation. travel-associated case patients were released from isolation after consultation with subject matter experts and following the cdc test-based criteria that were in use at that time (requiring two consecutive negative rt-pcr tests from samples collected at least 24 hours apart). generally, close contacts were defined as persons having frequent or more than brief contact (>1-2 minutes within 6 ft) with a travel-associated case patient during the travel-associated case patient's presumed infectious period. most sites also considered persons sharing the same airspace as the travel-associated case patient for more than 10 minutes to be close contacts. for persons whose exposure occurred in the healthcare setting, the definition of close contact also depended on usage of personal protective equipment (ppe) by the exposed person during their interaction with the travel-associated case patient. close contacts were grouped by type. close contacts were classified as "household contacts" if they were family members or friends of a travel-associated case patient who spent at least one night in the same residence as the travel-associated case patient during the presumed infectious period. close contacts were classified as "healthcare personnel" (hcp) if they were healthcare or public health personnel working in healthcare settings who had the potential for exposure to a travel-associated case patient or to infectious materials from a travel-associated case patient. close contacts who were defined as neither household contacts nor hcp were classified as "community contacts"; this category was broken down into contact occurring in a healthcare setting (e.g., waiting room close contacts) and contact occurring outside a healthcare setting (e.g., coworkers). "community contacts" also included flight-related close contacts of travel-associated case patients whose infectious periods included plane travel; these were defined as passengers seated in the same row as the travel-associated case patient, or in the two rows in front of or behind the travel-associated case patient. for the purposes of analyses of hcp ppe data, "all recommended ppe for airborne and contact precautions" is defined as gloves, gown, eye protection (goggles or a disposable face shield that covers the front and sides of the face), and a powered air-purifying respirator (papr) or n95 filtering facepiece respirator (ffr) with fit-testing in the past year"; "all recommended ppe for droplet and contact precautions" is defined as gloves, gown, eye protection, and a face mask [7] . any encounter during which an hcp was wearing any combination of ppe not meeting either of the above definitions was considered to be "less protected." risk stratification varied by site, but all sites considered household contacts as well as hcp who provided patient care without all recommended ppe to have had high-risk exposures. community contacts less frequently met the criteria for having high-risk exposure, given that their interactions with the travel-associated case patient tended to be shorter and less frequent. however, there were some community contacts (including workplace and social contacts) across sites that did meet the high-risk exposure criteria and were treated accordingly. close contacts meeting criteria for active monitoring were contacted daily via phone, text message, email, or in person, and were asked to report temperature and any symptoms. close contacts selected for collection of detailed exposure information were interviewed using forms that were standardized within but not across jurisdictions. information collected included dates of exposure to the travel-associated case patient; basic demographics; duration and types of exposure (e.g., face-to-face contact, direct physical contact); and, among hcp close contacts, use of ppe and interactions with or procedures performed on travel-associated case patients. information was entered into standardized forms, spreadsheets, or databases maintained by the covid-19 outbreak response teams. close contacts selected for biological sample collection agreed to provide np and op samples for sars-cov-2 testing. samples were collected by trained personnel. most sites aimed to collect samples from close contacts with high-risk exposures every 2-3 days through 14 days following last exposure to the travel-associated case patient. close contacts living outside the response jurisdictions were forwarded to their respective health jurisdictions for assessment and monitoring. some flight-related close contacts were forwarded to cdc's division of migration and quarantine for follow-up. close contacts meeting criteria for active monitoring were monitored through 14 days following their last exposure to a travel-associated case patient. close contacts not meeting criteria for active monitoring were asked to self-monitor and to contact the local health department if they developed any symptoms. close contacts who developed new or worsening symptoms that covid-19 outbreak response teams deemed possibly consistent with covid-19 (e.g., fever, cough) were considered to be suspect covid-19 patients and had respiratory samples (and serum samples, when possible) collected and sent for testing at the time that determination was made, regardless of whether previous samples had been collected. household contacts of some non-hospitalized travel-associated case patients were quarantined in the same home with their respective travel-associated case patient following the travelassociated case patient's diagnosis; this quarantine lasted through the period of case isolation. this situation is hereafter referred to as "co-habitation during case isolation." to prevent household transmission, the travel-associated case patients were instructed to stay in a separate bedroom, use a separate bathroom if possible, and to wear a face mask if they had to be in the same room as cohabitants [8] . all specimen samples were collected according to cdc guidance then packaged and shipped to cdc [9] . specimens were refrigerated at 2-8˚c before shipping on icepacks. cdc performed real-time reverse transcriptase-polymerase chain reaction (rrt-pcr) to detect three separate genetic markers of sars-cov-2 in the n1, n2, and n3 regions [10] . respiratory specimens were considered positive if all three genetic markers were positive by rrt-pcr, negative if all three genetic markers were negative, and inconclusive otherwise. demographic, clinical, exposure, and testing results for contacts are summarized and described. because the exact information that was collected varied by site, denominators are presented throughout. collection of respiratory specimens is described by sample type and days since last exposure to the travel-associated case patient. the secondary attack rate by close contact type was calculated as the percent of close contacts with at least one positive sars--cov-2 test result among close contacts for whom �1 set of respiratory samples was tested; 95% confidence intervals were calculated using the wilson score interval. differences in continuous variables were tested by the mann-whitney u test given non-parametric distributions. data were entered into access, excel, or redcap databases. data were merged and analyzed using the r environment for statistical computing. these activities were reviewed by cdc's human research protection office and determined to be exempt from human participants' research regulations as the activities involved identification, control or prevention of disease in response to an immediate public health threat. patient consent was waived. a total of 553 (range 18 to 222 close contacts per case) individuals were identified as close contacts of the first nine patients with travel-related covid-19 in the united states (fig 1) . four hundred and four (73%; range: 1 to 206 per confirmed case) met criteria and participated in local active monitoring. most of the 149 close contacts who did not participate in local active monitoring were determined to have had low-risk exposures that did not require active monitoring. other close contacts who did not participate in local active monitoring were either unable to be reached or were managed by other jurisdictions. among those close contacts actively monitored, 338 (84%) had either limited demographic and exposure type information (hereafter referred to as "at least basic exposure data") or demographic plus detailed exposure information collected; the type and amount of information for different types of close contacts varied by study site depending on their protocol. of these 338 close contacts, 15 (4%) were household contacts, 163 (48%) were hcp, 95 (28%) were community contacts exposed in a healthcare setting, and 65 (19%) were community contacts exposed in another setting ( table 1 and fig 1) . among the actively monitored close contacts with demographic information, the majority were female (n = 179/283; 63%) and aged 18-44 years (n = 152/251; 61%). of these 338 close contacts, only nine (3%) were exposed prior to symptom onset in the travel-associated case patient, including seven household members who were also exposed while the travel-associated case patient was symptomatic. a total of 49 actively monitored close contacts with at least basic exposure data (14%) developed new or worsening respiratory symptoms and had a set of diagnostic respiratory samples table 1 . demographic, clinical, exposure, and investigation characteristics among actively monitored close contacts with at least basic data on exposures to 9 confirmed covid-19 patients, united states, january-february 2020. private collected; 19 of these 49 (39%) had additional respiratory samples taken as part of enhanced contact investigations or case follow-up. there were 289 (86%) actively monitored close contacts with at least basic exposure data who remained without new or worsening symptoms, of whom 110 (38%) had at least one set of respiratory samples collected and 19 had more than one set of respiratory samples collected (fig 1) . a histogram of samples collected by type, contact category, and days since last exposure is shown in s1 and s2 figs. household contacts. the nine travel-related travel-associated case patients had a total of 15 household contacts, of whom all were actively monitored. two household contacts, both spouses of travel-associated case patients, became symptomatic and tested positive for sars--cov-2 ( table 2 ). all others repeatedly tested negative. the secondary attack rate among all household members of travel-associated case patients was 13% (95% ci: 4-38%). the secondary attack rate among significant others of travel-associated case patients was 25% (95% ci: 7-59%). travel-associated case patients who transmitted sars-cov-2 to their household contacts had an older average age (mean age 60 vs. 45 years; p = 0.07), and both were hospitalized for clinical management (as compared to 3 of the 7 travel-associated case patients who did not transmit to their household contacts; p = 0.6). there were two couples ("a" and "b") in which secondary transmission occurred (i.e., transmission of sars-cov-2 from the travel-associated case patient to their spouse); neither spouse shared a travel history with their respective travel-associated case patient. in couple a, the travel-associated case patient developed symptoms one day after returning from travel and enrolled in active monitoring with the local health department; the spouse, who had no reported comorbidities, developed symptoms three days after the travel-associated case patient's symptom onset. couple a reported the use of some isolation precautions during the period of exposure: the travel-associated case patient wore a face mask and used a separate bedroom and bathroom even prior to developing symptoms, although the couple did share a meal and spent extended time in the car together. both persons in couple a were diagnosed with covid-19 at the same time and were instructed to co-isolate at home until released by public health officials; however, eight days after symptom onset in the travel-associated case patient, couple a had to be hospitalized due to worsening conditions in both patients. in couple b, as in couple a, the travel-associated case patient's symptoms began the day after returning from travel. six days after symptom onset, the travel-associated case patient was hospitalized; the next day, the travel-associated case patient was identified as having suspect covid-19, and the spouse entered active monitoring. symptom onset in the couple b spouse could not be precisely determined, as this person has underlying conditions, but could have been as early as three days after the travel-associated case patient's symptom onset. couple b did not report any isolation precautions prior to the diagnosis of the travel-associated case patient (which occurred 8 days after symptom onset) and had frequent unmasked face-to-face and direct contact. in both couples, the spouse tested positive in the first respiratory samples collected. in general, the length of exposure to the travel-associated case patient was lower among the 13 household contacts who did not develop covid-19. among these 13, eight (including 3 significant others) were completely separated from the travel-associated case patient once the patient was diagnosed and isolated; these persons maintained separate living space throughout the period of case isolation. two (25%) had <12 hours of exposure to the symptomatic travelassociated case patient before isolation, five (63%) had 1-3 days of household exposure to the symptomatic travel-associated case patient before isolation, and one (13%) had eight days of household exposure to the symptomatic travel-associated case patient before isolation. among these eight, three household members of two travel-associated case patients shared a travel history with the travel-associated case patient. all eight household members who were separated from the travel-associated case patients following diagnosis had serial specimens collected (median: 6.5 sets; range: 3-7), and all persistently tested negative; the date of last specimen collection ranged from five to 14 days following last exposure. five household members (including 4 significant others) across four households co-habited with the travel-associated case patient during the period of case isolation; none shared a travel history with the travel-associated case patient. none of these travel-associated case patients were hospitalized at any time. when following up with these four co-habiting households via phone and in-person visits, the covid-19 outbreak response teams noted moderate to very high adherence to recommended home isolation procedures: in two out of four households, the travel-associated case patient was able to use a separate bathroom, and in only one of four households did the covid-19 outbreak response team ever directly observe or learn of the travel-associated case patient spending time in the same room as their household member(s). all five household members undergoing co-habitation had multiple respiratory samples collected for testing (median: 5 sets; range: 3-7) and remained sars-cov-2-negative throughout the period of cohabitation, with the last samples collected within seven days of the end of case isolation or the end of co-habitation. these samples are shown in s3 fig. healthcare personnel (hcp) . the nine travel-related travel-associated case patients made a total of 16 visits to healthcare facilities (3 outpatient clinics, one urgent care clinic, 12 hospital visits), resulting in 163 hcp contacts who met criteria for active monitoring. there were 126 hcp (77%) for whom data were available to describe patient contact and ppe usage in more detail. among 49 hcp who provided care to or came into contact with the infectious fluids of travel-associated case patients and who had at least one set of respiratory samples collected and tested for sars-cov-2, the secondary attack rate was 0% (95% ci: 0-7%). two hcp (<1%) reported performing a possibly aerosol-generating procedure on a travelassociated case patient (nebulizer therapy: 1; spirometry: 1); both reported using all recommended ppe during the procedure. laboratory personnel reported using recommended ppe when processing samples from travel-associated case patients. fourteen hcp (11%) reported collecting diagnostic respiratory specimens, of whom six (43%) reported always using all recommended ppe for airborne and contact precautions, one (7%) reported always using all recommended ppe for droplet and contact precautions, and seven (50%) reported using less ppe than recommended on at least one occasion (i.e., ppe meeting neither airborne/contact nor droplet/contact precautions; table 3 ). among the seven hcp using less ppe than recommended, five reported the details of their ppe use: all five wore gloves, one wore a gown, one wore eye protection, and one wore only a face mask; one wore an ill-fitting n95 ffr. all occasions during which "less-protected" hcp collected respiratory specimens from a travel-associated case patient were at the travel-associated case patient's first presentation to care-before covid-19 was confirmed. there were 76 (60%) hcp who reported providing patient care, touching the patient, or coming into contact with the patient's infectious fluids ( table 3) . of these 76 hcp, 33 (43%) reported always using all recommended ppe for airborne and contact precautions, two (3%) reported always using all recommended ppe for droplet and contact precautions, and 41 (54%) reported using less ppe than recommended on at least one occasion. among hcp using less ppe than recommended, 38 described ppe usage in detail. eye protection was most frequently missing (34; 90%), followed by gown (28; 74%), and less commonly ffr or mask (13; 33%). all but three of these less-protected encounters occurred before covid-19 was suspected in the travel-associated case patient. during all but seven of these less-protected encounters, the patient wore a mask for source control and removed it only for specimen collection and temperature measurement. community contacts. there were 78 community contacts (24 exposed in a healthcare setting and 54 exposed in another setting) for whom information was sufficiently detailed to classify at least one specific type of exposure ( table 4 ). the secondary attack rate for community contacts of travel-associated case patients was 0% (95% ci: 0-6%) among the 58 contacts with some detailed exposure information and at least one set of respiratory samples tested. for community contacts exposed in the healthcare setting, some exposure information was missing as the patient was not always identified to these persons. out of 19 who provided information, only one (5%) recalled having face-to-face contact with the travel-associated case patient. as compared to community contacts in the healthcare setting, community contacts exposed outside the healthcare setting had more interaction with the travel-associated case patient, and included 17 airport quarantine station screeners, 17 workplace contacts, 13 flight contacts, four rideshare drivers, and three friends. of those with data, many reported having face-to-face contact (27/35; 77%) with the travel-associated case patient or spending time within 6 feet of the patient (34/38; 90%), and nearly all (43/45; 96%) could remember being in the same room as the travel-associated case patient. fewer (8/28; 29%) reported being within 6 feet of the patient while the patient was coughing. airborne & contact precautions � a total of 24 community contacts exposed in the health care setting and 55 exposed in other settings had information sufficiently detailed to classify at least one specific type of exposure. sample sizes within each exposure do not sum to these totals because data was either not collected by the covid-19 outbreak response team or was not reported by the participant. �� zero samples tested positive for sars-cov-2. the enhanced contact tracing investigations undertaken by the covid-19 outbreak response teams around nine early travel-related covid-19 cases in the united states resulted in identification of two transmissions from two travel-associated case patients to their spouses. we did not find evidence of secondary transmission of sars-cov-2 among the other 47 actively monitored close contacts who developed symptoms suspicious for covid-19 nor among the 110 close contacts who submitted respiratory specimens for virologic testing and who did not develop new symptoms during the 14-day active monitoring period. no clear patterns of differences were noted between travel-associated case patients who transmitted the virus as compared to those for whom no evidence of secondary transmission was observed. the results of these investigations suggest that the risk of becoming infected with sars--cov-2 is high among close household contacts of confirmed covid-19 patients, especially significant others. additional detail on one of these transmissions has been previously published [11] , and a brief summary of key findings from the overall contact investigations of the first 10 travel-associated patients in the u.s. was previously published as an mmwr [6] . overall, the average exposure durations among household contacts were longer among those who developed covid-19 compared with those who did not. the higher transmission risk observed for household contacts is consistent with published research on other coronaviruses as well as sars-cov-2 [3, 12, 13] . interestingly, we did not detect any sars-cov-2 infections among household members who were co-habiting with their confirmed sars-cov-2-positive household members during the period of case isolation. since the majority of these co-habiting case patients and close contacts adhered very well to home isolation precautions, it is possible that careful adherence to isolation protocols mitigated the transmission risk to household members co-habiting with travel-associated case patients during the period of case isolation. we did not detect any symptomatic secondary cases of covid-19 among the 389 nonhousehold contacts who completed active monitoring, including 77 hcp contacts and 67 community contacts from whom respiratory samples were tested for sars-cov-2. this finding is similar to findings from early contact investigations in europe and asia [14] [15] [16] [17] , but in contrast to other data suggesting a much higher secondary attack rate [18] . it is possible that asymptomatic secondary cases could have developed in persons from whom respiratory samples were not collected [19, 20] , or that secondary cases could have developed without being detected in respiratory samples (for instance, if the timing of shedding did not align with the timing of sample collection) [12] ; follow-up serology could help to rule out some of these possibilities [21] . the low number of secondary cases could also be related to the quick isolation of most of these travel-associated case patients, as found in studies of mers-cov [22, 23] . out of 41 hcp with higher-risk exposures to a travel-associated case patient, all but three reported these exposures to have occurred prior to confirmation of the patient's diagnosis. once the patients were classified as suspected covid-19 cases, contact and transmission-based precautions were begun and maintained by the majority of hcp throughout the patient's care. in contrast to the first community-acquired cases identified in the united states, it is also important to consider that most if not all of these first travel-related cases were likely cognizant of the possibility that they were infected, and engaged in practices such as wearing face masks, practicing social distancing in some cases, and seeking care [5, 11, 24] ; these practices have the potential to have reduced secondary transmission from these cases. further, these investigations were taking place at a very early stage in the u.s. outbreak, and many sites employed a low threshold for defining close contacts, potentially inflating our denominator. this report is subject to several limitations. because individual covid-19 outbreak response teams were operating out of distinct locations with unique considerations, the procedures of the investigation were heterogeneous across sites: different teams made different decisions regarding how to define close contacts, how to categorize exposure risk, which close contacts to actively monitor, which types of exposure information to collect, and how often (and from whom) to attempt collection of respiratory samples. although available information was standardized across sites for the purposes of analysis, this heterogeneity led to missing data, limiting our ability to assess certain types of exposure in this population. relatedly, only 47% of actively monitored close contacts provided any respiratory specimens, and fewer provided serial respiratory specimens. incomplete sample collection could have biased secondary attack rates, and also raises the possibility of a missed asymptomatic infection. symptomatic infections occurring after the end of the 14-day monitoring period also could have been missed. although some sites attempted to reach individuals with close contact prior to symptom onset in the travel-associated case patient, many of these exposures occurred during the travel of the travel-associated case patient and therefore were managed by other jurisdictions. as a result, only nine such persons are included in the present analysis (of whom seven were household members with continued exposure). thus, secondary transmissions occurring prior to symptom onset in the travel-associated case patient may have been missed. missed transmissions (either asymptomatic infections, transmissions occurring outside the presumed infectious period, or transmissions with an incubation period outside the 14-day active monitoring period) may have contributed to community spread, as suggested by some genetic analysis of sars-cov-2 circulating in washington [25] . lastly, although all sites endeavored to interview and sample the close contacts with the highest-risk exposures, the selection of the sample for interview and respiratory specimen collection was not systematic or consistent across sites. this may have introduced bias in the characteristics of the close contacts described as well as in the secondary attack rates. further, the relatively small sample of primary cases (travel-associated case patients) and the low observed incidence of secondary transmission makes it difficult to identify any characteristics of the travel-associated case patients that might have been associated with an increased risk of secondary transmission. the findings of these enhanced contact investigations, consistent with other research, suggest that household members of covid-19 patients, particularly significant others, who are likely to have prolonged close contact with the patients, are at excess risk of developing covid-19 [3, 13] . further, the lack of observed transmission to hcp underlines the importance and effectiveness of rapid identification and isolation of suspected cases of covid-19 and appropriate use of ppe in caring for these patients [26, 27] . careful adherence to infection prevention procedures within the home and the healthcare setting may help to reduce the risk of transmission of covid-19, while monitoring and quarantine of household contacts may help interrupt transmission. close contacts with high-risk exposures (such as household contacts or hcp providing patient care without ppe) should continue to be quarantined and actively monitored, as data from covid-19 clusters have raised the concern of transmission from asymptomatic or pre-symptomatic individuals infected with sars-cov-2 [19, 20, 28, 29] , which may be contributing to the widespread community transmission that is currently occurring in the united states [30] . it is important for hcp and public health professionals to remain vigilant to the possibility of sars-cov-2 transmission from confirmed cases to household, community, and hcp contacts, and continue to emphasize isolation and other precautions [7, 8] . further, individuals exposed to sars-cov-2 should self-quarantine, and all persons should practice everyday behaviors to limit the spread of covid-19 such as proper hand hygiene, covering coughs and sneezes, wearing cloth face coverings, and social distancing [31, 32] . supporting information s1 fig. histogram of specimens collected by day since last exposure, grouped by type of contact and days per person. specimens (n = 448) collected from 149 contacts with known exposure periods are shown by the days following last exposure to the confirmed covid-19 patient on the x axis. excludes specimens from five household contacts with ongoing household exposure after the diagnosis of the travel-associated case patient, one household member diagnosed concurrently as the travel-associated case patient, and four community contacts without a known exposure period or date of collection. specimens from contacts submitting only a single set of respiratory specimens are shown in navy, while specimens from those submitting multiple sets of respiratory specimens are shown in lighter blue. household contacts (hh) are shown in the first row (excluding 5 who were co-habiting with the case patient following diagnosis and 1 who was concurrently diagnosed), healthcare personnel (hcp) contacts are shown in the second row, and community contacts are shown in the bottom row. the first column shows the number of nasopharyngeal (np) swabs collected and tested, and the second column shows the number of oropharyngeal (op) swabs collected and tested. some hh members and hcp have specimens collected at negative days from last exposure-these represent specimens collected during a period of ongoing exposure to the covid-19 patient. samples collected relative to the first day of exposure are shown in s1 fig patient on the x axis. excludes specimens from five household contacts with ongoing household exposure after the diagnosis of the travel-associated case patient, one household member diagnosed concurrently as the travel-associated case patient, and four community contacts without a known exposure period or date of collection. specimens from contacts submitting only a single set of respiratory specimens are shown in navy, while specimens from those submitting multiple sets of respiratory specimens are shown in lighter blue. household contacts (hh) are shown in the first row, healthcare personnel (hcp) contacts are shown in the second row, and community contacts are shown in the bottom row. the first column shows the number of nasopharyngeal (np) swabs collected and tested, and the second column shows the number of oropharyngeal (op) swabs collected and tested. only the initial specimens are shown for the secondary cases, as these specimens tested positive. (tiff) s3 fig. histogram of specimens collected by day since first exposure, among household contacts who were co-habiting with travel-associated covid-19 case patients during the period of case isolation. specimens (n = 48) collected from 5 contacts who were co-habiting with a covid-19 patient following the travel-associated case patient's diagnosis are shown by the days following first exposure to the confirmed covid-19 patient on the x axis. the first column shows the number of nasopharyngeal (np) swabs collected and tested, and the second column shows the number of oropharyngeal (op) swabs collected and tested. a pneumonia outbreak associated with a new coronavirus of probable bat origin a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster. the lancet early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia first case of 2019 novel coronavirus in the united states active monitoring of persons exposed to patients with confirmed covid-19-united states interim infection prevention and control recommendations for patients with suspected or confirmed coronavirus disease 2019 (covid-19) in healthcare settings 2020 phylogenetic analysis of sars-cov analysis of the epidemic growth of the early 2019-ncov outbreak using internationally confirmed cases longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of sars-cov-2 infected patients first known person-toperson transmission of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in the usa middle east respiratory syndrome coronavirus transmission in extended family, saudi arabia case management team kcfdc, prevention. early epidemiological and clinical characteristics of 28 cases of coronavirus disease in south korea case management team kcfdc, prevention. coronavirus disease-19: summary of 2,370 contact investigations of the first 30 cases in the republic of korea investigation of three clusters of covid-19 in singapore: implications for surveillance and response measures first cases of coronavirus disease 2019 (covid-19) in france: surveillance, investigations and control measures, january 2020 serological and molecular findings during sars-cov-2 infection: the first case study in finland secondary attack rate and superspreading events for sars-cov-2 presumed asymptomatic carrier transmission of covid-19 asymptomatic and presymptomatic sars-cov-2 infections in residents of a long-term care skilled nursing facility investigation and serologic follow-up of contacts of an early confirmed case-patient with covid-19 clinical and epidemiologic characteristics of spreaders of middle east respiratory syndrome coronavirus during the 2015 outbreak in korea transmission of middle east respiratory syndrome coronavirus infections in healthcare settings initial public health response and interim clinical guidance for the 2019 novel coronavirus outbreak-united states cryptic transmission of sars-cov-2 in washington state escalating infection control response to the rapidly evolving epidemiology of the coronavirus disease 2019 (covid-19) due to sars-cov-2 in hong kong risk of nosocomial transmission of coronavirus disease 2019: an experience in a general ward setting in hong kong public health responses to covid-19 outbreaks on cruise ships-worldwide presymptomatic transmission of sars-cov-2-singapore epidemiological and clinical features of the 2019 novel coronavirus outbreak in china estimation of the time-varying reproduction number of 2019-ncov outbreak in china the authors would first like to acknowledge the covid-19 patients and close contacts described in this manuscript. we also gratefully acknowledge the assistance and contributions of glen key: cord-336843-c0sr3six authors: gerritsen, m. g.; willemink, m. j.; pompe, e.; van der bruggen, t.; van rhenen, a.; lammers, j. w. j.; wessels, f.; sprengers, r. w.; de jong, p. a.; minnema, m. c. title: improving early diagnosis of pulmonary infections in patients with febrile neutropenia using low-dose chest computed tomography date: 2017-02-24 journal: plos one doi: 10.1371/journal.pone.0172256 sha: doc_id: 336843 cord_uid: c0sr3six we performed a prospective study in patients with chemotherapy induced febrile neutropenia to investigate the diagnostic value of low-dose computed tomography compared to standard chest radiography. the aim was to compare both modalities for detection of pulmonary infections and to explore performance of low-dose computed tomography for early detection of invasive fungal disease. the low-dose computed tomography remained blinded during the study. a consensus diagnosis of the fever episode made by an expert panel was used as reference standard. we included 67 consecutive patients on the first day of febrile neutropenia. according to the consensus diagnosis 11 patients (16.4%) had pulmonary infections. sensitivity, specificity, positive predictive value and negative predictive value were 36%, 93%, 50% and 88% for radiography, and 73%, 91%, 62% and 94% for low-dose computed tomography, respectively. an uncorrected mcnemar showed no statistical difference (p = 0.197). mean radiation dose for low-dose computed tomography was 0.24 msv. four out of 5 included patients diagnosed with invasive fungal disease had radiographic abnormalities suspect for invasive fungal disease on the low-dose computed tomography scan made on day 1 of fever, compared to none of the chest radiographs. we conclude that chest radiography has little value in the initial assessment of febrile neutropenia on day 1 for detection of pulmonary abnormalities. low-dose computed tomography improves detection of pulmonary infiltrates and seems capable of detecting invasive fungal disease at a very early stage with a low radiation dose. neutropenic fever is one of the most important complications in cancer patients. [1] it is critical to rapidly localize infections and identify the organism involved, especially for fungal infections, which need a specific treatment approach. despite a standard diagnostic workup including chest radiograph (cxr) and microbiological screening, no focus is identified in up to 44% of patients. [2] this can partly be explained by the low sensitivity of radiographs for diagnosing pulmonary infections in neutropenic patients. [3] acquiring cxrs in respiratory asymptomatic patients with febrile neutropenia is therefore controversial. the esmo guidelines recommend performance of a cxr in every neutropenic patient with fever, whereas the idsa guidelines suggest its use only in patients with respiratory signs or symptoms. [1, 4] in febrile neutropenia a substantial part (~10%) of infections is caused by invasive fungal disease (ifd). [5] ifd usually presents as a pulmonary infection, which is rarely visible on cxr and is therefore often missed in the early phase of neutropenic fever. notably, a delayed start of appropriate treatment has a negative impact on clinical outcome. [6] in order to improve the early detection of pulmonary infiltrates in febrile neutropenia, other imaging tools have been investigated as an alternative to cxr. high-resolution computed tomography (hrct) improved pulmonary focus detection: pulmonary abnormalities were found in 60% of the neutropenic patients with persistent fever (>48 hours) and a normal cxr. [7] furthermore, direct initiation of effective antifungal treatment after the early detection of a halo sign on hrct images, had a positive effect on treatment response and survival in case of ifd, emphasizing the importance of early diagnosis. [8] however, due to costs and high radiation doses of approximately 7 msv average, hrct scanning is usually not incorporated in the initial febrile neutropenia workup. [9] another imaging modality that could be used for the evaluation of febrile neutropenia is low-dose ct scanning (ldct). ldct is performed with low mean radiation doses below 1.5 msv and without the use of contrast. two studies comparing ldct to cxr in patients with persistent febrile neutropenia demonstrated an increased detection of pulmonary abnormalities. [10, 11] therefore, we hypothesized that ldct already acquired on day 1 of febrile neutropenia would improve detection of pulmonary infections. the primary aim of our study was to compare the diagnostic performance of ldct and 2-view cxr for early detection of pulmonary infiltrates. the secondary aim was to explore its performance for early ifd detection. the study was approved by the local institutional review board (approval number nl41415.041.12) of the university medical center utrecht. written informed consent was obtained from all patients. this prospective study was conducted at the haematology ward of the university medical center utrecht, a tertiary care oncology center, between march 2013 and december 2014. patients with febrile neutropenia treated with intensive chemotherapy for haematological malignancies or receiving an autologous or myelo-ablative allogeneic stem cell transplant (sct) were eligible for inclusion. febrile neutropenia was defined as a single temperature measurement of !38.3˚c or a temperature of !38˚c for more than 1 hour, accompanied by an absolute neutrophil count of <0.5×10 9 /l or a neutrophil count <1.0×10 9 /l with a predicted decline to <0.5×10 9 /l within 3 days. patients were excluded in case of a known focus of infection unrelated to the lower respiratory tract at inclusion, active possible or probable fungal infection at inclusion, or concomitant participation in clinical research in which the subject was exposed to additional radiation. patients could participate only once in case of multiple febrile episodes. after conformation of neutropenic fever the diagnostic workup was started according to the esmo guidelines [1] and patients received broad spectrum antibiotics (imipenem) within 2 hours. all patients received selective digestive tract decontamination at the start of chemotherapy, comprising of ciprofloxacin 2dd 500 mg and fluconazole 1dd 150 mg. an additional ldct scan was made within 24 hours after the start of fever for research purposes only. all ldct scans remained blinded during the study. the study patients were monitored until they were 24 hours without fever. after this follow-up period and when all culture results were available a predefined consensus diagnosis of the cause of the fever episode was made by an expert panel consisting of 2 haematologists, a microbiologist and a radiologist. diagnostic workup for febrile neutropenia. the diagnostic workup included a clinical examination, a 2-view cxr and microbiological screening. standard microbiological screening included evaluation of urine and blood cultures. [1, 4] a respiratory microbiological evaluation was performed in case of a suspected pulmonary infection (clinical symptoms of dyspnea or cough, sputum production, or an oxygen saturation level <93%). for the respiratory evaluation a throat swab was taken and tested for viral pathogens with pcr pack 1 (influenza virus, respiratory syncytial virus (rsv), coronavirus, rhinovirus) and pcr pack 2 (adenovirus, human metapneumovirus (hmpv), parainfluenzavirus type 1-4, bocavirus and mycoplasma pneumoniae). in case of a possible atypical pneumonia the swab was additionally tested for atypical pathogens with pcr pack 3 (legionella pneumoniae, chlamydophila pneumoniae, chlamydophila psitacci, coxiella burnetti). if sputum was available this was tested for bacteria (gram stain, culture) and fungal pathogens (blancophore stain, culture). other microbiological testing was only performed if judged appropriate by the attending physician. furthermore, serum samples were taken twice weekly for galactomannan (gm) testing at the end of inclusion. if fever persisted for 4 days, a routine hrct scan was acquired to detect possible ifd. in case of abnormalities suspect for (fungal) pulmonary infection a broncho-alveolar lavage (bal) was performed. bal fluid was routinely tested for bacteria (gram, stain, culture) including haemophilus influenza, and fungal pathogens (blancophore stain, gm, microscopy and culture). on indication bal fluid was also tested for nocardia (culture), mycobacteria (culture, microscopy, pcr), pneumocystis jiroveci pneumonia (pcr, microscopy) and viral pathogens: pcr pack 1,2 and 3. in case of ifd anti-fungal treatment was started and a hrct was repeated after six weeks. consensus diagnosis. the consensus diagnosis of a fever episode was defined according to predefined categories as either pulmonary infection or non-pulmonary causes of fever such as line infection, mucositis, other infections (i.e. sinusitis), unknown focus of fever or any combination of the above. the expert panel diagnosis was based on information obtained from the clinical charts, microbiology results and imaging results: the 2-view cxr and the hrct. hrct was only available in case of persistent (4 days) fever, and ldct results were not used for the consensus diagnosis. clinical criteria for a pulmonary infection included either one of the following symptoms: coughing, sputum production, dyspnea or an oxygen saturation level <93%. microbiological criteria were a positive culture or pcr for bacterial, viral and fungal pathogens known to cause pulmonary infections, or in case of invasive aspergillosis a positive gm in bal (!0.8) or serum (!0.5). in case of abnormal imaging results without additional clinical or microbiological criteria these were considered false positive. fungal infections were classified as either possible, probable or proven in accordance with the revised european organization for research and treatment of cancer/mycosis study group (eortc/msg) criteria. [12] ifd was considered to be ruled out in all patients recovering from neutropenic fever within 3 days without ever receiving mould-active antifungal therapy, given the very low likelihood of spontaneous recovery during neutropenia, without appropriate treatment. central venous catheter infections were defined by criteria adapted from the dutch surveillance network of nosocomial infections (prezies). [13] mucositis was diagnosed based on clinical and radiological (typhlitis) criteria. clinical criteria included pain when swallowing, nausea, vomiting, abdominal pain/cramping and diarrhea, physical examination and the absence of a positive microbiological test. any other focus of infection was mainly determined by the judgment of the attending physician combined with the results of the diagnostic workup. fever of unknown origin was defined as fever without any focus or etiology identified by clinical, radiological or microbiological examination. the ldct scan was made during inspiration at the lowest achievable radiation dose. ldct images were acquired at a tube potential of 80 kvp and a tube current-time product of 10 mas or 20 mas depending on patient's weight using a 256-slice ct system (ict, philips healthcare, best, the netherlands), or at a tube current-time product of 15 mas or 30 mas with a 64-slice ct system (brilliance 64, philips healthcare, best, the netherlands). at the end of the study, anonymized cxr and ldct images were first independently evaluated by a board certified radiologist and a radiology resident who were not involved in the consensus diagnosis. the observers used a standard imaging scoring table (table 1) . second, cxr and ldct images were re-analysed if one or two observers gave a positive score. re-evaluation was performed during consensus reading of the study exams, for which additional historical radiological images were available to prevent false-positives as a result of pre-existing abnormalities. the primary objective was to investigate whether ldct is a better diagnostic tool than cxr for detection of pulmonary infections on the first day of neutropenic fever. we hypothesized that ldct could increase the detection rate of pulmonary infiltrates by 80%; from 15% with cxr (unpublished data from our institute) to 27%, considering an expected incidence of pulmonary focus of infection in febrile neutropenia of 30%. [14] we expected a proportion of discordant pairs of 15% with a two-sided test (alpha 0.05 and power 0.80). a sample size of 68 patients was esteemed sufficient according to a power calculation based on the mcnemar test. sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) of both tests were calculated using contingency tables, the consensus diagnosis served as reference standard. an uncorrected mcnemar test was applied to determine the differences between the two modalities. [15] a p-value of 0.05 was considered significant. parametric data are presented as means ± standard deviation (sd) and non-parametric data as medians (range). cohen's kappa (κ) was used to assess the inter observer agreement and defined as excellent with κ > 0.80, good with κ between 0.61 and 0.80, moderate with κ between 0.41 and 0.60, and poor with κ 0.40. ibm spss statistics software version 22.0 (ibm, somers, ny, usa) was used for statistical analyses. the secondary objective was to explore the performance of ldct for detection of pulmonary lesions indicating ifd on day 1 of neutropenic fever. a total of 72 consecutive patients were recruited between march 2013 and december 2014. five patients were excluded: one because of a known infection unrelated to the respiratory tract at inclusion, one as a result of active fungal infection, one because the ldct was acquired more than 24 hours after the cxr, one was not able to undergo ldct scanning and for one patient cxr was not available (fig 1) . baseline characteristics of the 67 patients are listed in table 2 . according to the consensus diagnosis 11 patients (16.4%) had a pulmonary infection, of which 5 had ifd (2 possible, 3 probable), 1 had rhinovirus and 5 pneumonia of unknown aetiology. five of the patients with pneumonia underwent bronchoscopy due to persistent fever and abnormalities on hrct. in 3 of those patients a probable pathogen was identified after evaluation of bal-fluid, which was probable ifd in all 3. furthermore in 35 (55.2%) patients, overall nonspecific mucositis was the most likely cause of fever. in four of these patients abdominal imaging (ct) was performed showing no signs of typhlitis. in 15 patients (22.4%) a focus of infection was not found. the other consensus diagnosis results are listed in table 3 . the mean interval between cxr and ldct was 3.1 ± 6.3 hours. the mean radiation dose for ldct exams was 0.24 ± 0.15 msv, which is comparable to 2-3 chest radiographs (average dose posteroanterior and lateral chest radiograph: 0.1 msv). [9] none of the patients experienced adverse events as a result of ldct scanning or chest radiography. eight cxrs (11.9%) were indicative of pneumonia, 4 cxrs were false positive and 7 false negative as compared to the consensus diagnosis (table 4 ). this resulted in a sensitivity of 36%, a specificity of 93%, a ppv of 50% and a npv of 88% for cxr. none of the cxrs were suspect for ifd. thirteen ldct scans (19.4%) were suggestive of pneumonia. compared to the consensus diagnosis, 5 ldct scans were false positive and 3 false negative. sensitivity, specificity, ppv and npv were 73%, 91%, 62% and 94% respectively. (table 4) consensus diagnosis of the 5 false positive ldct scans were phlebitis and in 4 cases fever of unknown origin. no hrct scans were made because in all 5 patients the fever subsided within 48 hours after receiving broad spectrum antibiotics, therefore cxr was the only available imaging tool for the consensus meeting. only one of the patients with a false positive ldct had respiratory symptoms (cough), however a microbiological respiratory evaluation was negative. an uncontrolled mcnemar's test showed no statistically significant difference in the proportion of scans positive for pulmonary infection between cxr and ldct (p = 0.197), which was our primary endpoint. inter-observer agreement was moderate for both cxr and ldct (κ = 0.52 for cxr and κ = 0.52 for ldct, respectively). based on the consensus diagnosis 5 patients in this cohort were classified as either probable (n = 3) or possible (n = 2) ifd. the diagnosis of probable ifd was based on a positive bal gm test in all cases and 2 out of 3 also had a positive serum gm test. only 1 patient had a positive bal culture (a. versicolor). four of the patients with ifd (3 probable, 1 possible) had a ldct scan suspect for ifd on the first day of fever. in all these patients the abnormalities were also seen on the hrct performed as part of the diagnostic workup for persistent fever (mean 3.5 days later than ldct). (fig 2) none of the patients diagnosed with pulmonary ifd had abnormalities on their cxr on the first day of fever, and only one had respiratory signs or symptoms. the diagnosis of possible ifd in the patient with a negative ldct scan was based on abnormalities on hrct made on day 4 of fever. serum gm values were negative and bronchoscopy was not performed because of severe thrombocytopenia. finally one ldct scan with abnormalities suspect for ifd was considered false positive: the fever episode was classified as "fever of unknown origin". we conducted a prospective study to evaluate whether pulmonary focus detection would improve using a ldct scan instead of cxr on the first day of febrile neutropenia. we established an improved detection rate from 11.9% of radiographs to 19.4% of the ldct scans but our primary endpoint was not met. as a result of the lower than expected incidence of pulmonary infections in this study, the study was underpowered. [14] nevertheless, we demonstrated a clinically significant increased sensitivity of ldct (73% versus cxr 36%) in detecting a pulmonary focus on day 1 of neutropenic fever. the limited value of cxr as a standard diagnostic procedure is in line with previous results. [3] in a retrospective study 1083 adult sct patients were evaluated, but in none of the 242 cxrs performed in asymptomatic patients with febrile neutropenia pulmonary abnormalities indicative of infection were detected. in contrast, in 76 patients with respiratory symptoms 24 cxrs showed evidence of pneumonia. [3] in our study 4 patients had a true positive cxr; 1 of these patients had respiratory symptoms on the first day of fever. two previous studies have compared the use of cxr with ldct in febrile neutropenic patients. in the present study a much lower mean radiation dose of 0.24 msv was used compared to 0.6 msv and 1.5 msv in the other studies. [10, 11] still, reconstructed images were of diagnostic quality in all patients. the ldct scans in the previous studies were acquired using older generation ct systems (4 slice ct scanner by patsios et al. [10] and 16 slice ct scanner by kim et al. [11] .) we used two newer generation ct systems (64 slices and 256 slices) with newer x-ray tubes, detectors and software, resulting in the potential to further reduce the radiation dose without compromising on image quality. both previous studies demonstrated that ldct increased the detection rate but they differ in several aspects from our study. patsios et al. [10] compared cxr with ldct in neutropenic aml patients with already clinically suspected pneumonia, and found abnormal imaging results in 31 of 40 cxrs, whereas 38 out of 40 patients had abnormalities on ldct. instead of only focusing on pulmonary abnormalities suggestive of pulmonary infection, this study also included radiological signs of cardiac failure or fluid overload, which might explain the high number of patients with abnormalities in both cxr and ldct. [10] in a prospective study kim et al. [11] evaluated the use of ldct in a selection of patients with persistent neutropenic fever (>48 hours) regardless of the presence of respiratory symptoms. of the 207 included patients 150 were diagnosed with pneumonia (72%). differences between sensitivity of cxr and ldct for correctly diagnosing infectious pneumonia were a little less pronounced than in our study: cxr 39% and ldct 63%. to our knowledge this is the first study to evaluate ldct in all patients with febrile neutropenia on day 1 of fever. we demonstrated an increase in sensitivity and an improved npv when performing ldct in the detection of a pulmonary infection in febrile neutropenia. this is important because detection of a focus of infection in neutropenic fever is difficult and patients often undergo several diagnostic tests and uncertain treatments which can have negative side effects. therefore it can be expected that every improvement in the diagnostic workup will eventually lead to improved patient care. a major advantage of our study is the completely independent assessment of both index and reference test (ldct and cxr by independent radiologists) and the short time interval between cxr and ldct (3.1 ± 6.3 hours). however, the inter-observer agreement was moderate and should be improved when moving forward with the ldct technique. this points out that evaluation of ldct images in this neutropenic population at high-risk of developing pulmonary infections requires expert-thoracic radiologists, which may not yet be available in every hospital. a limitation of the study is the use of the reference standard (consensus diagnosis) which may have contributed to the lower test performance of ldct. in 38 of the 67 patients a cxr was the only imaging modality available for the consensus meeting, since hrct was only performed in case of persistent fever. considering the low sensitivity of cxr in this population, pulmonary abnormalities could have been missed, this may have led to an underestimation of the amount of patients with pneumonia, and ldcts might have incorrectly be judged as false positives. furthermore clinical symptoms of cough, sputum production and dyspnea are not always evident in a (bedridden) neutropenic patient. [16] an extensive microbiological evaluation for respiratory causes of fever, which was only performed upon clinical indication, might therefore not have been performed in all patients that had abnormalities suspect for pulmonary infection on ldct. several studies report on the limited diagnostic yield of a respiratory microbiological evaluation in neutropenic patients. in up to 70% of the lower airway infections a pathogen can not be identified. [17, 18] sputum cultures (which are often not available) only reveal a possible pathogen in less than 50% of cases. [18] bal seems to be the diagnostic procedure with the highest yield. despite the low detection rates of a probable pathogen in approximately 50% of cases, it is the most sensitive procedure for detecting ifd. [19] however, since routinely performance of bal in the initial assessment of fever is not performed due to its invasive nature, a consensus diagnosis as was used in our study is the best available option and complies with established guidelines. [1, 4] we were able to identify a probable pathogen in 36% of the patients with pneumonia (2 cases of possible ifd excluded), which is consistent with reports in literature.[17, 18] however we did not establish any case of bacterial pneumonia. this may be explained by the low amount of patients with sputum available for culture (36%, all culture-negative), or a possible treatment response to broad spectrum antibiotics, resulting in defervescence within 3 days, in which case hrct and bal were not performed. we included a heterogenic population with patients with prolonged neutropenia as well as patients with shorter neutropenic episodes. this could have had an effect on the incidence of pulmonary infections, because the risk of developing pneumonia (especially ifd) increases in case of prolonged neutropenia. the use of ldct scans for detection of pulmonary lesions indicating ifd at day 1 of neutropenic fever seems promising. out of 5 fever episodes classified as either possible or probable ifd, 4 patients already had abnormalities suspect for ifd on their ldct whereas these where not seen on cxr. importantly, only one of these patients had respiratory signs or symptoms and therefore we think that omitting chest imaging in patients without respiratory symptoms can lead to a delayed diagnosis of ifd. incorporation of ldct in the diagnostic workup of all patients with neutropenic fever will increase costs when compared to cxr. however, initiation of early and targeted treatment of ifd may reduce overall costs, for example by reducing length of hospital stay, intensive care unit admittance rates and the amount of diagnostic procedures required. therefore the performance of ldct in all patients with febrile neutropenia might still be cost-effective. this issue should be evaluated in future research. performance of cxr in the initial assessment of febrile neutropenia is of limited value for detection of pulmonary abnormalities. the introduction of ldct improved the detection of pulmonary infiltrates and there was a clear signal that ldct scanning is capable of detecting invasive fungal infections at a very early stage. therefore, the use of ldct in the initial assessment of febrile neutropenia is promising, and should be further evaluated in a larger study powered on ifd detection. furthermore it would be interesting to see whether ldct could replace hrct as imaging tool in patients with suspected ifd in order to decrease radiation exposure. management of febrile neutropenia: esmo clinical practice guidelines a prospective survey of febrile events in hematological malignancies the utility of routine chest radiography in the initial evaluation of adult patients with febrile neutropenia patients undergoing hsct clinical practice guideline for the use of antimicrobial agents in neutropenic patients with cancer: 2010 update by the infectious diseases society of america. clinical infectious diseases: an official publication of the infectious diseases society of america comparison of the occurrence of mold infection among patients receiving chemotherapy for acute leukemia versus patients undergoing stem cell transplantation delay of active antimicrobial therapy and mortality among patients with bacteremia: impact of severe neutropenia pneumonia in febrile neutropenic patients and in bone marrow and blood stem-cell transplant recipients: use of highresolution computed tomography imaging findings in acute invasive pulmonary aspergillosis: clinical significance of the halo sign. clinical infectious diseases effective doses in radiology and diagnostic nuclear medicine: a catalog chest low-dose computed tomography in neutropenic acute myeloid leukaemia patients. respiratory medicine ultra-low-dose chest ct in patients with neutropenic fever and hematologic malignancy: image quality and its diagnostic performance. cancer research and treatment: official journal of korean cancer association revised definitions of invasive fungal disease from the european organization for research and treatment of cancer/invasive fungal infections cooperative group and the national institute of allergy and infectious diseases mycoses study group (eortc/msg) consensus group definities ziekenhuisinfecties: lijnsepis -2014 comparison of infectious complications during induction/consolidation chemotherapy versus allogeneic hematopoietic stem cell transplantation. bone marrow transplantation interval estimation for the difference between independent proportions: comparison of eleven methods clinical presentation of infection in granulocytopenic patients the authors would like to thank all of the patients who participated in this study and all the consulting physicians on the ward. key: cord-305393-96mrxt8a authors: lai, yvonne; yi, guanghui; chen, alice; bhardwaj, kanchan; tragesser, brady j.; rodrigo a. valverde,; zlotnick, adam; mukhopadhyay, suchetana; ranjith-kumar, c. t.; kao, c. cheng title: viral double-strand rna-binding proteins can enhance innate immune signaling by toll-like receptor 3 date: 2011-10-10 journal: plos one doi: 10.1371/journal.pone.0025837 sha: doc_id: 305393 cord_uid: 96mrxt8a toll-like receptor 3 (tlr3) detects double-stranded (ds) rnas to activate innate immune responses. while poly(i:c) is an excellent agonist for tlr3 in several cell lines and in human peripheral blood mononuclear cells, viral dsrnas tend to be poor agonists, leading to the hypothesis that additional factor(s) are likely required to allow tlr3 to respond to viral dsrnas. tlr3 signaling was examined in a lung epithelial cell line by quantifying cytokine production and in human embryonic kidney cells by quantifying luciferase reporter levels. recombinant 1b hepatitis c virus polymerase was found to enhance tlr3 signaling in the lung epithelial beas-2b cells when added to the media along with either poly(i:c) or viral dsrnas. the polymerase from the genotype 2a jfh-1 hcv was a poor enhancer of tlr3 signaling until it was mutated to favor a conformation that could bind better to a partially duplexed rna. the 1b polymerase also co-localizes with tlr3 in endosomes. rna-binding capsid proteins (cps) from two positive-strand rna viruses and the hepadenavirus hepatitis b virus (hbv) were also potent enhancers of tlr3 signaling by poly(i:c) or viral dsrnas. a truncated version of the hbv cp that lacked an arginine-rich rna-binding domain was unable to enhance tlr3 signaling. these results demonstrate that several viral rna-binding proteins can enhance the dsrna-dependent innate immune response initiated by tlr3. viral nucleic acids are agonists for innate receptors that can activate anti-viral responses [1, 2, 3, 4] . the receptors include the rig-i-like receptors that are localized to the cell cytoplasm and several membrane-associated toll-like receptors [5, 6] . mutations in these receptors have been correlated with pathologies in viral infections in humans [7, 8, 9, 10] . understanding how the innate immune receptors can interact with agonists will be important for the detection and response to viral infections. toll-like receptor 3 (tlr3), the focus of this study, is composed of a large ligand-binding ectodomain, a transmembrane helix, and a signaling toll/il-1 receptor homology (tir) domain [11, 12] . the ectodomain contains 23 leucine-rich repeats flanked with cysteine-rich caps and a bipartite motif, both of which are critical for ligand binding [13, 14] . tlr3 localizes to either the plasma membrane or acidic endosomes, the latter was presumed to be the site of high affinity binding to dsrna [15, 16] . ligand binding induces the tlr3 dimer to orient the two tir domains to recruit adaptor proteins and activate signal transduction [6, 14] . karioko et al. [17] have observed the activation of tlr3 signaling in cultured cells with complex ligand mixtures, such as nucleic acids fractions from necrotic cells. however, we have anecdotally observed that highly purified rnas, including necrotic rnas, are poor activators of tlr3 in cultured cells when compared to the synthetic dsrna analog, poly(i:c). these observations suggest that intrinsic features within dsrnas can influence signaling by tlr3. also, component(s) in addition to the dsrnas are needed to activate tlr3. the antimicrobial peptide ll37 and other peptides that can bind dsrna were recently found to enhance the tlr3 response to poly(i:c) and viral dsrnas [18] . since viral rnas exist as complexes with proteins, we wanted to determine whether viral proteins could act to modulate tlr3 signaling. in this work, we determined that viral dsrna-binding proteins could substitute for ll37 in activating signaling by tlr3. furthermore, the proteins promoted more efficient recognition of viral dsrnas. the recombinant polymerases from the hepatitis c virus (hcv) and three viral capsid proteins were characterized in this study. hcv, a member of the hepacivirus genus in the flaviviridae family, infects approximately 2% of the world's population, with up to 80% of untreated individuals progressing to chronic infection and severe liver damage [19, 20] . the hcv-encoded polymerase, ns5b, is a validated drug target and has been extensively characterized [21] . as with polymerases in general, ns5b resembles a closed right hand that contains thumb, palm and finger subdomains [19] . in vitro, recombinant ns5b proteins can bind and initiate rna synthesis by a de novo initiated mechanism or by extension from a template annealed to a primer [22, 23, 24, 25, 26, 27, 28] . de novo initiated rna synthesis uses a single-stranded (ss) rna template and a purine nucleotide as the first nucleotide, while primer extension uses a partially duplexed rna [28] . the de novo initiated versus primer-extended modes of rna synthesis is regulated by a flexible loop named d1 that extends from the fingers subdomain to contact hydrophobic residues in the thumb subdomain [27] . the release of the d1 loop is needed to sterically accommodate the ternary complex during elongative synthesis. the polymerase from the 2a genotype of hcv exists in a structurally more closed conformation than that of the 1b polymerase in part due to a tighter contact between the d1 loop and the thumb subdomain [27, 29, 30] . this feature has been proposed to allow the 2a polymerase to be more efficient at rna synthesis by the de novo initiated mechanism since it cannot initially accommodate a partially duplexed rna. two of the viral capsid proteins we examined are from members of the alphavirus-like superfamily with positive-stranded rna genomes: ross river virus (rrv) and brome mosaic virus (bmv) [31, 32] . the third is from the orthohepadnavirus, hepatitis b virus (hbv), which has a partially dsdna genome and replicates by reverse transcription [33, 34] . the capsids of all three viruses contain a positively-charged arginine-rich region that is required for rna binding [35, 36, 37, 38, 39] . we have anecdotally observed that cultured cell lines that express tlr3 respond poorly to viral dsrnas when compared to the response to poly(i:c). to document this observation, beas-2b cells that endogenously express tlr3, tlr4, and rig-i were examined for their response to several dsrnas. beas-2b cells have been used extensively to study the effects of viral infection and activation of innate immunity on cytokine production, including the production of interleukin 6 (il6) [40, 41, 42] . the dsrnas tested include the reovirus genomic rna, the s4 dsrna made by annealing in vitro transcribed positive-and negative-sense strands of the reovirus s4 rna, and the bell pepper endornavirus dsrna, bpev, purified from bell pepper plants [43] , and other viral and synthetic dsrnas. transcripts of the jfh-1 genome were used as a ssrna control. all the rnas were added to the media of beas-2b cells and the amount of cytokine il6 secreted into the medium quantified by elisa (fig. 1a) . at 20 h, il6 levels induced by poly(i:c) (0.13 mg/ml) was at least six-fold higher than that of dsrnas or ssrna (0.3 mg/ ml; fig. 1a and fig. s1a ). the production of il6 in response to poly(i:c) was rapid and could be observed within 2 h of poly(i:c) addition (fig. s1b ). in contrast, cells treated with s4 dsrna did not yield level of il6 significantly above the background even after 20 h (fig. s1b) . hek 293t cells do not detectably express tlr3 but can be transfected to express either wild-type or mutant tlr3 [44] . transfection of two plasmids, one containing an interferon stimulated response element (isre) promoter-driven firefly luciferase and a second encoding a constitutively expressed renilla luciferase allow the analysis of tlr3 activation by different rnas. hek 293t cells expressing wt tlr3 responded to poly(i:c) (1-2 mg/ml), better than viral dsrnas (1-2 mg/ml) purified from reovirus and bpev (fig. 1b) . annealed s4 reovirus dsrna, ssrnas from jfh-1 (fig. 1b) , endornavirus dsrna from rice plants and other viral ssrnas (fig. s1c ) also had no effect on tlr3 signaling. none of rna tested induced reporter activity in hek 293t cells expressing a tlr3 mutant h539e that had a substitution at the c-terminal ligand-binding domain [16, 45] ( fig. 1b) or in cells transfected with the empty vector (data not shown), demonstrating that the observed responses are from tlr3. the results from 293t cells are consistent with those from the beas-2b cells and support our observation that viral dsrnas are poor agonists in comparison to poly(i:c). to determine whether tlr3 was responsible for the increased il6 levels, we treated cells with sirnas specific to tlr3, rig-i, or a nonspecific control for 48 h prior to the addition of bpev dsrna to the cells (fig. 1c) . rig-i is a suitable control in this experiment since it also recognizes dsrna [46] . only cells treated with sirnas to tlr3 had reduced il6 levels in the presence of bpev dsrna (fig. 1c) . with poly(i:c)-induced cells, sirnas to tlr3 also reduced il6 production by 5163% (n = 4) (data not shown; [18] ). rt-pcr confirmed that the sirnas did knock down tlr3 mrna levels in both untreated and poly(i:c) treated cells (fig. 1d) . odn2006, which we previously shown to inhibit tlr3 signaling [47] reduced the il6 levels in the presence of poly(i:c) to less than 20% of the control reaction (data not shown) while egcg, an inhibitor of rig-i [48] had no effect on il-6 production in the presence of poly(i:c) (data not shown). thus, while the viral dsrnas are poor tlr3 agonists, the low level of cytokine production by beas-2b cells was primarily due to the activation of tlr3. these results form the basis of the hypothesis to be tested in this work: the activation of tlr3 by viral dsrna requires additional factors. several viral dsrna-binding proteins can enhance poly(i:c)-dependent signaling addition of the peptide ll37 to the cell culture media along with poly(i:c) or viral dsrnas was found to enhance signaling by tlr3 [18] . ll37 binding to the dsrna was correlated with enhancement of tlr3 signaling [18] . since rnas in cells exist in complex with proteins, we postulate that viral rna-binding proteins could also enhance the tlr3 response to the viral dsrnas. several highly purified rna-binding proteins were selected to test this idea. these include the recombinant nsp15 protein from the sars coronavirus that binds ssrnas [49] , two versions of the hepatitis b virus capsid protein (h-cp149 and h-cp183) [38] , the capsid protein from the ross river virus (r-cp) [39] , the capsid from the plant-infecting brome mosaic virus (b-cp) [50] and polymerases from the 1b and 2a genotypes of the hepatitis c virus (1bd21 and 2ad21) [27] . except for the b-cp, which was extracted from cesium-chloride purified bmv virions, the other proteins were produced in e. coli. while the cytokine induction by the various rna-binding proteins in the absence of poly(i:c) varied depending on individual experiments, fold inductions of il6 above basal across multiple experiments showed no statistically significance in paired t-tests ( fig. 2 and table s1 ). these results, including those from b-cp that was produced in plants also suggest that common contaminants such as lipopolysaccharides which would induce tlr4 signaling, is not a significant factor in the assays. incubation of poly(i:c) with the corresponding buffers in which the proteins were purified also did not result in a significant change of cytokine production (data not shown). the presence of poly(i:c) (0.13 mg/ ml) alone induced il6 and il8 levels at least five-fold above background (fig. 2) . several of the proteins added to a final concentration of 0.15 mm along with poly(i:c) resulted in additional increases in il6 and il8 levels of two-to five-fold ( fig. 2a and 2b ; table s1 ). the level of enhancement by the recombinant proteins were comparable to that observed with ll37 (3 mm). the viral proteins that did not significantly increase cytokine production include the polymerase from hcv genotype 2a figure 1 . a comparison of viral dsrnas and poly(i:c) for activation of tlr3. a) effects of various potential tlr3 ligands on il6 production by beas-2b cells. all of the rnas were added to the culture media. poly(i:c) (pic) was added to 0.13 mg/ml and the other rnas were added at 0.13 to 0.3 mg/ml. il6 was quantified in an aliquot of clarified cell medium, and the total amount of il6 was normalized to the total medium volume. the data shown is the mean 6 sem, with the number of assays in parentheses: pic (n = 23), reovirus dsrna (reov, n = 9), reovirus s4 dsrna (s4; n = 28), bpev (n = 6), jfh1 (n = 3). b) a comparison of the effects of viral dsrnas and poly(i:c) in hek 293t cells transfected to express the wt tlr3 or a ligandbinding mutant, h539e. activation of tlr3 was measured by the ratio of the firefly luciferase driven by an isre promoter element to a constitutivelyexpressed renilla luciferase [47] . each bar represents the mean value of three replicates, with the range for one standard error shown above the bars. c) tlr3 is required for the increase in il6 production in response to bpev dsrna. beas2b cells were transfected with a control nonspecific sirna (nsrna), a set of three sirnas to tlr3 (sitlr3), or a sirna to rig-i (sirig-i), grown for 48 h before addition of bpev dsrna (0.3 mg/ml) to the culture media. d) rt-pcr results demonstrating that the sirna targeting tlr3 decreased the abundance of the tlr3 message. beas2b cells were transfected with either control nonspecific sirna or sirnas to tlr3. after 48 h, cells were exposed to media alone (f) or to poly(i:c) at 0.13 mg/ml. rt-pcr was performed to determine fold induction of tlr3 mrna above media 18 h later. data is presented as a percent of the result from the nonspecific sirna. doi:10.1371/journal.pone.0025837.g001 (2ad21), the truncated hbv cp (h-cp149), and the sars-cov nsp15 ( fig. 2a and 2b ). the nsp15 used in this experiment is a catalytic-inactive version of an endoribonuclease that binds and preferentially cleaves ssrnas [49] . identical results were obtained with the wt nsp15, hence the enzymatic activity of nsp15 is not a factor in the response (data not shown). these results show that a subset of the rna-binding proteins is more competent in enhancing tlr3 signaling. the activities of the hcv polymerases and the viral capsids will be analyzed in turn below. the hcv 1b polymerase can enhance dsrna-induced signaling by tlr3 we seek to determine whether the observed il6 production in the presence of the 1b hcv polymerase was due to signaling by tlr3. sirnas to tlr3, to rig-i, or to a nonspecific target were transfected into beas-2b cells prior to the addition of either poly(i:c) alone or poly(i:c) and 1bd21 (fig. 3a) . only the cells transfected with the sirnas to tlr3 had reduced il6 production compared to cells transfected with nsrna ( fig. 3a ; p,0.05). we reproducibly observed using rt-pcr that tlr3 mrna treated with the sirnas were reduced to between 8 and 26% of the level in cells treated with the nsrna control (table s2 ). the presence of the polymerase did not change lps-induced tlr4 response or change the response from cells treated with only polyinosinic acid, suggesting specific induction of tlr3 (fig. 3b) . moreover, odn2006, an inhibitor of tlr3 signaling [47] reduced il6 levels to 4067% of control while egcg, an inhibitor of rig-i [48] had no effect, further suggesting that the enhancement of dsrna induced signaling is dependent on tlr3 (data not shown). the hcv polymerase also enhanced the response to poly(i:c) lengths that ranged from 45 to 125 bp (fig. s2a ). these results indicate that tlr3 is required for 1bd21 and poly(i:c) to enhance cytokine production. 1bd21 is less active for rna synthesis in vitro than is the 2ad21 [27, 29, 30] . therefore, it is unlikely that rna synthesis by 1bd21 is responsible for the induction of tlr3 signaling. however, to establish this directly, we tested a catalytic mutant named gda wherein the divalent metal-binding motif required for polymerization was mutated. we also tested three additional mutants that are defective for de novo initiation, including a five-residue deletion of the d1 loop name m26-30 [26] . the locations of the key mutations in the 1b polymerase structure are shown in fig. 3c . since m26-30 has a major change in conformational to 1bd21, we tested it for the ability to bind a dsrna of ca. 900 bp derived from reovirus genome and found that retained the ability to bind dsrna (fig. 3d ). all of these mutant polymerases enhanced il6 production over the level of poly(i:c) alone by at least two-fold ( fig. 3e and fig. s2b ). figure 3 . the 1b hcv polymerase can enhance il6 production by beas-2b cells. a) enhanced il6 production in the presence of 1bd21 can be knocked down by sirna to tlr3. sirnas specific to tlr3 (sitlr3), rig-i (sirig-i), or a nonspecific control (nsrna) were transfected into at a concentration of 30 nm into cells 48 h prior to the addition of 1bd21 (0.15 mm) and poly(i:c) (0.13 mg/ml) to the culture medium. il6 in the medium was collected 24 h later and quantified by elisa. the data from 3 to 4 sets of samples are presented as a percentage of the il6 in the sample treated with nsrna (100%). b) il6 production by beas-2b cells in response to stimulation by the single-stranded polyinosinic acid (0.13 mg/ml), poly(i:c) (0.13 mg/ml) or lipolysaccharide (lps) (1 mg/ml), the agonist for tlr4 in the absence (ã�) or presence of 1bd21 (0.15 mm). c) a model of the 1b hcv polymerase that illustrates the location of the d1 loop (in light blue), the gdd active site (in yellow), and the short helix deleted in mutant m26-30 (in red). d) the 1bd21 polymerase and 1b.m26-30 can form a complex with the double-stranded s4 rna. the s4 dsrna was labeled with a-32 p-ctp and used in an electrophoretic mobility shift assay. the gel image shown is from a non-denaturing stacked polyacrylamide gel of 5 and 20%. e) effects of mutations in 1bd21 on il6 production induced by poly(i:c). f denotes a reaction with no added proteins. proteins 1bd21, 1b.m26-30, and the active site mutant gda were added to the culture media (final concentrations 0.01, 0.1 or 0.5 mm) in the absence or presence of 0.13 mg/ml poly(i:c). ll37 was added to a final concentration of 3 mm. f) the 1b hcv polymerase (1bd21, 0.1 mm), but not the 2a polymerase (2ad21, 0.1 mm), allowed tlr3 to produce cytokines in response to viral dsrnas. in addition to poly(i:c), the rna ligands used were those extracted from reovirus virions (reov), from the endornavirus bpev, and the annealed transcripts of the sense and antisense strands made from the s4 cdna of reovirus (s4), and the transcript of the hcv 2a jfh-1. doi:10.1371/journal.pone.0025837.g003 next, we examined whether 1bd21 could enhance il6 production in concert with viral rnas (fig. 3f ). in the absence of 1bd21, the single-stranded jfh-1 rna and three different viral dsrnas were poor inducers of il6 production in beas2b cells when compared to poly(i:c). this result is consistent with our initial observation that tlr3 does not respond efficiently to viral rnas ( fig. 1a & 3f, fig. s1a ). 1bd21 (0.1 mm) increased il6 levels induced by the s4 dsrna and the bpev dsrna by 4 to 6fold (fig. 3f) . 1bd21 had only a modest increase when jfh-1 ssrna was used as a ligand. in contrast, the activation of signaling was approximately 20-fold higher with dsrna than with jfh-1 (fig. 3f) . consistent with our previous observation, 2ad21 (0.1 mm) was unable to enhance il6 production when added to cells along with either the ssrna or dsrna (fig. 3f ). the conformation of 2ad21 can affect dsrna-dependent tlr3 signaling 2ad21 differs from 1bd21 in the relative amounts of product generated from de novo initiated rna synthesis or extension from a primed template (fig. 4a ). 2ad21 produced high levels of de novo initiated rna when compared to the primer extension products while 1bd21 produced similar amounts of the two products (fig. 4a) . in this assay, de novo initiated rna synthesis was determined with template le19p, whose 39 terminal puromycin prevents primer extension, while primer extension used template pe46, which contains a partially double-stranded hairpin structure that can be extended by the polymerase. the products of the two modes of rna synthesis demonstrate whether a single or a partially duplexed rnas are bound in the template channel of the polymerase [24] [25] [26] . furthermore, products from the two templates are associated with two distinct polymerase conformations: a structurally closed conformation can initiate better by a de novo mechanism or a more open conformation that favors primer extension [26] . notably, the crystal structure of 2ad21 has a more closed conformation than that of 1bd21 due to additional interactions between the d1 loop and the thumb subdomain of the polymerase [29] . if the closed conformation of 2ad21 decreases its ability to bind dsrna and to induce tlr3, a five-residue deletion at the tip of the d1 loop which results in a more open conformation should the templates used, le19p and pe46, can, respectively, produce de novo initiated products (dn) as well as those extended from a partial duplex rna (labeled as ''pe'') as described in chinnaswamy et al. [27] . b) the denaturation profile of the proteins (5 mm) analyzed by differential scanning fluorimetry. the reactions were performed in a stratagene mx3005p realtime pcr machine in the presence of sypro orange, which fluoresces when complexed to denatured proteins. the ramp for temperature increase was at 0.5uc per min. c) the mutant hcv 2a polymerase, 2am26-30, can enhance poly(i:c) (0.13 mg/ml)-induced il6 production by beas-2b cells. d) the sequence and likely structure of a fluorescently-labeled rna, sl26, used to analyze polymerase-dsrna interaction. e) binding of sl26 by the three hcv polymerases. the proteins were mixed together with a 1:1 molar ratio of sl26 then crosslinked with a stratalinker prior to analysis by sds-page. the gel image on the left shows the fluorescently-labeled rna shifted above the free sl26 at the bottom of the gel. the complexes that contain a monomer or a dimer of the hcv polymerase are labeled to the left of the gel image. the same gel was then stained with coomassie blue to allow visualization of the proteins, the image of which is shown to the right. doi:10.1371/journal.pone.0025837.g004 increase dsrna binding and thus the rna synthesis from a primed template. a protein with such a deletion named 2a.m26-30 was found to be debilitated in de novo initiated rna synthesis while retaining the ability to extend from a primed template (fig. 4a ). 2a.m26-31 also showed a distinct thermal denaturation profile compared to that of 2ad21, when analyzed by differential scanning fluorimetry, consistent with the two proteins having different conformation(s) (fig. 4b) . protein 2a.m26-30 added to beas-2b cells had no effect on il6 production (fig. 4c) . when added along with poly(i:c) to beas-2b cells, 2a.m26-30 dramatically enhanced il6 production to levels comparable to reactions containing 1bd21 (fig. 4c, n = 4) . these results clearly demonstrate that the conformations of the hcv polymerase that impact interaction with single or double-stranded rnas affect the ability of the polymerase to enhance signaling by tlr3. the deletion of the d1 loop in the 1b polymerase makes the template channel more accessible to dsrnas [27] . to determine whether 2a.m26-30 was altered in rna binding in comparison to 2ad21, we performed a uv-crosslinking assay with a fluorescently-labeled rna sl26, which contains a gnra tetraloop with an 11-bp stem (fig. 4d ). the 26-nt sl26 was used to minimize non-specific interaction by the polymerase so as to better analyze rna binding potential of polymerase mutant. the rna-protein complex was visualized with a phosphorimager and the position of the protein identified by staining with coomassie blue (fig. 4e) . the purified hcv polymerase usually exists as a mixture of monomers and higher order oligomers [27] . both the monomeric and dimeric forms of 2a.m26-30 bound sl26 better than those from 2ad21 (fig. 4e) . taken together, these results show that the changes in 2a polymerase to increase its binding to a largely dsrna is correlates with increased enhancement of tlr3 signaling. we sought to determine whether the recombinant hcv polymerase could enter cells and co-localize with tlr3 in endosomes. 1bd21 added to the medium of beas-2b cells in the absence of poly(i:c) was localized to punctate spots within 30 min. of addition (fig. 5a) . some of these spots co-localized with lysotracker that stains acidic compartments in the cell where tlr3 is located ( fig. 5b ; [15] ). the observation that not all of the dsrna co-localized with tlr3 is consistent with previous reports that dsrna could traffic into cells independent of tlr3 [47, 51] . to determine whether 1bd21 co-localized with tlr3, cells exposed to 1bd21 for 6 h were stained with the antibodies to tlr3 and the hcv polymerase. signals to both proteins overlapped significantly, although not at every location where tlr3 was detected (fig. 5c) . the co-localization of the hcv polymerase to endosomes containing tlr3 may factor into the mechanism to enhance tlr3 signaling. the localization of 1bd21 in beas-2b cells suggests that it is taken up by endocytosis. arginine-rich cell penetrating peptides and vaccinia virus had been reported to enter cells by several pathways, including macropinocytosis [52, 53, 54] . to examine whether macropinocytosis is likely involved in the uptake of 1bd21, we tested the inhibitor 5(n-ethyl-n-isopropyl)-amiloride (eipa) on tlr3 signaling in the presence of poly(i:c) and either ll37 or recombinant 1bd21 (fig. s3) . eipa dose-dependently inhibited tlr3 signaling by either poly(i:c) alone, or poly(i:c) added to the cell culture medium along with ll37 or 1bd21. these results are consistent with macropinocytosis playing a role in the uptake of either the dsrna and/or the recombinant protein into endosomes. to extend the analysis of viral protein-rna interaction further, we characterized the effects of three capsid proteins (cps) that enhanced the dsrna-specific innate immune signaling (fig. 2) . knockdowns of tlr3, rig-i, or a nonspecific target prior to stimulation with poly(i:c) and the cps (50 nm) showed that tlr3 was the receptor primarily responsible for il6 production for h-cp184 and r-cp (fig. 6a) . sirnas to tlr3 also decreased b-cp induced il6 production (data not shown). in addition, all three wt viral cps increased il6 production in a concentrationdependent manner (fig. s4) . h-cp149 that lacks the c-terminal sequence of h-cp183 (fig. s4a ) was unable to modulate il6 production (fig. s4b) . we examined whether the cp can enhance signaling by viral ssrna or dsrna. il6 level were enhanced when the reovirus genomic rnas and the s4 dsrna (all at 0.15 mg/ml) were added to the medium in the presence of h-cp183. the single-stranded jfh-1 rna was not significantly enhanced by h-cp183. furthermore, h-cp149 did not result in statistically significant enhancement of il6 in the presence of viral dsrnas or the jfh1 ssrna (fig. 6b, fig. s4b ). the amount of enhancement with the 150 nm of h-cp183 was comparable to that of 3 mm ll37. the rna-binding is required to enhance dsrna-dependent signaling by tlr3 the differential effects of h-cp183 and h-cp149 should reveal a requirement important for the enhancement of dsrna-dependent signaling by tlr3. the hbv cp contains sixteen arginines in its c-terminal thirty-four residues (fig. s4a ). h-cp149 is competent for the formation of virus-like particles, but is defective for binding to rna [38] . it is therefore likely that the altered interaction with rna is responsible for the inability of h-cp149 to enhance tlr3 signaling when compared to the other capsid proteins. again, to reduce the effects of nonspecific rna binding inherent to many rna-binding proteins, sl26 containing an 11-bp dsrna was used to examine whether h-cp183 and h-cp149 are distinguishable in their ability to bind rna. sl26 bound h-cp183 as well as b-cp, and the r-cp (indicated by shifts to higher molecular mass smears). however, no shift occurred with h-cp149 (fig. 6c) . these results provide another set of example wherein a viral protein's ability to bind dsrna is linked to the enhancement of tlr3-dependent signaling. the hcv polymerase 1bd21 co-localized with tlr3 in endosomes (fig. 5) . the bmv cp has also been shown to enter barley cells through macropinocytosis and localize to punctate spots within the cytoplasm that are suggestive of endosomes [53] . the hbv capsid can also enter cells, although permeabilization of the cells with digitonin was required [55] . we seek to determine whether the capsid proteins can localize to endosomes that contain tlr3, using the r-cp as an example. 2 h after the addition of rcp to the media of beas-2b cells, r-cp was found close to the plasma membrane, with a small proportion of the signal localizing in endosomes (fig. s5 ). at 6 h after r-cp addition, the majority of the signal was within endosomes (fig. 7) . immunostaining to detect tlr3 revealed an extensive co-localization of r-cp (fig. 7) . the effects of the hcv polymerases and the viral capsid proteins have thus far been examined in beas-2b cells. we seek to determine whether they could enhance signaling in hek 293t cells transiently transfected to express tlr3. tlr3specific activation of luciferase production from an isre promoter element was used to assess signal transduction. reporter production in the presence of poly(i:c), and reovirus genomic dsrna were all reproducibly enhanced in the presence of 1bd21 or 2a.m26-30, but not with 2ad21 (fig. 8a) . as with the beas-2b cells, jfh1 rna was unable to induce tlr3 in hek 293t cells. the activation of reporter production was dependent on the concentration of 1bd21 added to the 293t cell culture media (fig. 8b) . the effects of the viral cps on tlr3 signaling in hek cells were also examined using the reovirus dsrna as the tlr3 ligand. b-cp, h-cp183, and the r-cp all increased reporter levels in a concentration-dependent manner when compared to cells transfected with only the empty plasmid vector (fig. 8c ). this result shows that all five recombinant proteins that enhanced tlr3 signaling with viral dsrnas in beas-2b cells enhanced tlr3-dependent signaling in hek 293t cells. while tlr3 binds to dsrna to activate innate immune responses, viral dsrnas tend to be poor tlr3 agonists, especially when compared to the synthetic dsrna analog, poly(i:c). in a companion manuscript [18] , we demonstrated that ll37 is a potent enhancer of dsrna-dependent tlr3 signaling. herein, we demonstrate that a subset of viral proteins can significantly enhance both poly(i:c) and viral dsrna-induced tlr3 signaling. these viral proteins include polymerases from two genotypes of hepatitis c virus and three viral capsid proteins. requirements for the polymerases and capsid proteins (1bd21, 2a.m26-30, b-cp, rcp, and h-cp183) to activate tlr3 signaling were examined and a common property is the ability to bind dsrnas. two proteins (2ad21 and h-cp149) that bind dsrna less well are also less effective in enhancing tlr3 signaling. these results suggest that features that affect dsrna binding modulate the ability of proteins to activate tlr3 signaling. a second requirement elucidated with the 1b hcv polymerase and the rrv cp is the ability of the proteins to enter cells and co-localize with tlr3 in endosomes. the focus of the discussion below will be on the requirements for activating tlr3 signaling, with a final section on the conformation of the hcv polymerase. the chemical features in dsrna that are recognized by tlr3 are poorly understood. the difference between what makes poly(i:c) a good tlr3 agonist in the absence of rna-binding proteins and what makes the viral dsrnas inferior ones will require in-depth analysis. we believe that this work identifies conditions that will facilitate these efforts. based on our current results, reovirus s4 dsrna made by in vitro transcription is as potent a tlr3 agonist as the genomic dsrna extracted from reovirus virions, suggesting that no obvious posttranscriptional modifications are required to activate tlr3 signaling. another feature of poly(i:c) that may obviate the need for viral proteins or ll37 may be that the inosine-cytosine base pair has only two hbonds. we observed that poly(a:u) is also an excellent agonist for tlr3 in the absence of ll37 and viral protein (fig. s1a) . however, weaker base pairing alone is unlikely to provide the full explanation for poly(i:c) being such a potent agonist; poly(g:u), which also has two h-bonds per base pair, is a poor agonist for tlr3 signaling in the absence of viral dsrna proteins (fig. s1a) . we can offer the following observations on proteins and peptides that can enhance tlr3 signaling. the proteins can be produced in e. coli or extracted from virions (as in the case of bmv), suggesting that post-translational modifications are not required for the enhancement of the tlr3 response. similarly, peptides such as ll37 that are potent enhancers of tlr3 signaling can be synthesized chemically without specific modifications [18] . in addition, both enzymes and structural proteins can be potent enhancers of tlr3 signaling as long as they interact with either dsrna or partially duplexed rnas. the sars-cov nsp15 preferentially binds ssrna and not dsrna [49] , and could not efficiently enhance tlr3 signaling. a deletion that increased interaction between the 2a hcv polymerase and a partially duplexed rna template made the polymerase a better enhancer for tlr3 signaling (fig. 4c) . the second feature of the tlr3 enhancers is that they have the ability to form oligomers. this is intuitive with the viral capsid proteins, but viral rdrps have surfaces that mediate oligomerization that may facilitate rna binding [56, 57, 58] . indeed, oligomerization of the hcv polymerase can affect the mode of rna synthesis [27] . ll37 also exists in higher order complexes in the absence of rna and is required at micromolar concentrations in contrast to the viral proteins that are active in our assays at nanomolar concentrations. this is likely due to multiple ll37 molecules being needed to bind and/or coat the dsrnas and mediate tlr3 activation [18] . the fact that bmv and rrv encapsidate ssrnas may initially seem to incompatible with their ability to enhance signaling by dsrnas. however, the encapsidated viral rnas are highly compacted and contain a high percentage of dsrnas with non-watson-crick base pairing, close to 70% [59, 60] . this likely explains how the cp can bind the dsrna needed to activate tlr3. the cp's of bmv and rrv are capable of binding to the double-stranded s4 rna in an electrophoretic mobility shift assay (fig. s4c ). in the context of a viral infection, however, we note it is likely that fully assembled virions may not result in activation of tlr3 signaling if the dsrnas are not exposed. we have added highly purified bmv virions and reovirus virions to beas-2b cells and 293t cells expressing tlr3 and have not observed induction of cytokines or tlr3-specific reporters (kao and ranjith-kumar unpublished results). we also demonstrated that two dsrna-binding proteins that can enhance tlr3 signaling can colocalize with tlr3 in endosomes. whether the dsrna-binding proteins facilitate binding of the dsrna by the tlr3 or the uptake of dsrna to endosomes remain to be determined. however, it is known that tlr3 binds to linear dsrna [61] . tlr3, unlike the rig-i-iike receptors [62] , lacks helicase activity to unravel the tertiary interactions that could stabilize the dsrna. the dsrna-binding proteins could disrupt the tertiary structure in the dsrnas to facilitate recognition of the dsrna by tlr3. electron microscopy revealed that ll37 decreases the particle size of the primarily globular poly(i:c) and results in more filamentous structures [18] . with the second scenario, the dsrna-binding proteins could facilitate entry of the viral dsrnas into endosomes, perhaps by increasing the interaction with trafficking chaperones, such as scavenger receptors [51] , but these rna-binding proteins likely need to complex and bring dsrna to endosome in order for tlr3 activation to occur. the net effect is an increase in the concentration of ligand available to tlr3. further studies will be required to unravel the complex requirements whereby the dsrna-binding protein activate tlr3 signaling. our demonstration that ll37 and viral dsrna-binding proteins can enhance tlr3 signaling raises the possibility that inappropriately expressed cellular dsrna-binding proteins could activate tlr3 signaling from cellular rnas. autoimmunity has been linked to antibodies that recognize rnp complexes [63, 64] , and tlr signaling can affect the production of autoantibodies associated with lupus erythematosis [65] . the results in this work are also informative for the function of the hcv polymerase. we note that the enhancement of tlr3 signaling by hcv polymerase is independent of rna synthesis since the active site mutant is capable of enhancing tlr3 signaling (fig. 3d ). all polymerases undergo structural transitions between open and closed conformations in response to the stages of nucleic acid synthesis. indeed, the monomer of the hcv polymerase contains a template channel that can sterically accommodate ssrna, but not dsrna [26, 66] . during the formation of a nascent rna from the template, however, the polymerase needs to release the interaction between the d1 loop and the thumb subdomain in order to accommodate the elongating nascent rna-template rna duplex. the 1bd21 can both extend from a primed template as well as initiate from the 39 terminus of a single-stranded rna. we believe that this is a manifestation of the 1b polymerase existing in an equilibrium between the open and closed forms in vitro, likely accounting for better dsrna-binding by the 1b polymerase and its enhanced ability for dsrna-induced signaling by tlr3. in contrast, the 2a polymerase exists predominantly in a closed conformation and thus is less able to bind to dsrna and enhance signaling by tlr3 [29] . increasingly, the open and closed conformations of the hcv polymerases are being linked to polymerase function. for the 1b hcv polymerase, the open conformation also exposes a binding pocket for the cell cycle regulator retinoblastoma and mediates the ubiquitinylation and degradation of retinoblastoma to increase cell cycle progression during hcv infection [67, 68] . during viral infection, viral polymerases and rnas are expressed inside the cell and depending on the cellular locations of the polymerase protein and viral or cellular dsrnas they recognize, could activate cytoplasmic innate immunity receptors such as rig-i or endosomal tlrs. previous study by naka et al [69] found that immortalized liver cells stably transfected with hcv ns5b and hek 293 cells stably transfected with both ns5b and tlr3 genes show increased activation of tlr3-depedent ifnb signaling, although the activation in hek 293 cells was only 2-fold. in a recent study from our laboratory, transient overexpression of ns5b and rig-i resulted in 8 to 14-fold induction of ifnb promoter-driven luciferase activity in huh7 and in hek 293t cells while overexpression of ns5b and tlr3 had no effect, suggesting that expression of ns5b in these two cell lines predominantly activates cytoplasmic innate immunity receptors such as rig-i and mda5 [70] . in both these studies, the ns5b polymerase was expressed from transfected plasmids and catalytic activity of the polymerase is required. in the present study, exogenously added hcv polymerase protein activates tlr3 without requiring rna synthesis since polymerase mutated at the catalytic site still activates tlr3. in addition three viral capsid proteins as well as ll37 that bind rna but do not have catalytic activity also activate tlr3 signaling. addition of both dsrna and the rna-binding protein are required for this activation. rnabinding protein alone has no effect since rnas synthesized by the hcv polymerase in cells may not enter efficiently into acidic endosomes, the site of tlr3 signaling [15] . thus the effect and mechanism of tlr3 activation by rna-binding protein are likely different from that observed by naka et al. [69] or ranjith-kumar et al. [70] . during viral infection and subsequent cell lysis, viral proteins and rnas may be released to the cell media and these viral proteins and rnas may be taken up by cells to activate innate immunity. exogenously added hcv core and ns3 proteins enters cells to activate tlr2 signaling in the absence of any added ligand, although the mechanism(s) of activation is not known [71] . in the present study, addition of ns5b protein to beas2b or hek293t cells has minimal effect on its own but significantly activates tlr3 signaling in combination with dsrna ligands, including viral dsrnas (table s1 ). indeed, ns5b and the rrv capsid protein have the ability to enters beas-2b cells within 30 min and colocalize with endosomal tlr3 by 6 h. the cellular entry of released viral proteins and rnas during viral infection may contribute to the cellular innate immune responses and could provide a point for therapeutic intervention. recombinant genotype 1b (con1 strain) and 2a (jfh-1) hcv polymerases lacking the c-terminal transmembrane helix were purified from e. coli as described by chinnaswamy et al. [28] . the preparations were devoid of detectable contaminating rna, dna, or lps. the hbv and rrv capsid proteins were expressed in e. coli and purified according to porterfield et al. [38] and mukhopadhyay et al., [39] . poly(i:c) was purchased from amersham biosciences (piscataway, nj) and reconstituted in phosphate-buffered saline. rna from purified reovirus virions was extracted with trizol, followed by ethanol precipitation and a wash with 70% ethanol. the purified rna was stored in rnasefree water. s4 dsrna from reovirus was synthesized by in vitro transcription from cdna (kind gift of p. danthi, indiana university, indiana) that has been engineered to have t7 promoters at termini of the two complementary strands. the annealed rnas were then analyzed for quantity and concentration before use. bpev dsrna was purified according to the protocol of valverde and gutierrez [43] and stored in rnase-free water. beas2b and hek293t cells were from atcc and cultured in begm media with supplements (lonza, basel, switzerland). culture media from beas-2b cells were collected 24 h after treatments, centrifuged at 2000 g for 2 min, and the supernatants were assayed for il6 secretion using a human il6 elisa kit along with quantification controls (bd systems) as per the manufacturer's protocol. hek293t cells were plated in costar white 96-well plates in dmem amended with 10% fbs at 4.4610 4 cells per well and transfected at ,85 to 90% confluency with a mixture of the lipofectamine 2000 and plasmids pisre-luc, puno-hutlr3 and phrl-tk as described in sun et al. [44] . the transfected cells were induced 18 to 22 h later by the addition of poly(i:c) or viral dsrnas to the culture medium to a final concentration of 1 mg/ ml in the presence or absence of 100 nm of rna binding proteins. the cells were harvested for analysis of luciferase 14 to 18 h after poly(i:c) addition. rdrp assays were performed as 20 ml reactions containing 20 mm sodium glutamate (ph 8.2), 12.5 mm dtt, 4 mm mgcl 2 , 1 mm mncl 2 , 0.5% triton x-100, 0.2 mm gtp, 0.1 mm atp and utp, 250 nm [a-32 p] ctp (mp biomedicals), 2 pmol pe46 and 1 pmol le19p as templates [26] . the reaction mixture was incubated at 30uc for 1 h and terminated by phenolchloroform extraction, followed by precipitation of the rna in the presence of two volumes of ethanol, 5 mg glycogen and 0.3 m naoac (ph 5.2). rna products were separated by 20%-7.5 m urea polyacrylamide gels. the signals were detected and quantified by using a phosphorimager. each reaction contained 400 ng of purified protein mixed with 1 pmol cy5-labeled sl26 or s4 dsrna radiolabled by a polynucleotide kinase reaction in a buffer containing 50 mm tris-hcl (ph 7.5), 4 mm mgcl 2 , 50 mm nacl, and 1 mm dithiothreitol. the reaction was irradiated with uv at 1200 mj for 3 min. and then subjected to sds-page. the signals were detected and quantified with a phosphorimager. the gel was then stained with coomassie blue to visualize the locations of proteins. dsf was performed in a stratagene mx3005p real-time pcr machine according to the protocol of niesen et al. [72] . each sample (5 mm final concentration) was prepared in a total volume of 50 ml containing sypro orange (molecular probes) in 100 mm tris, ph 7.0, 50 mm kcl, and 5 mm mgcl 2 ). the ramp condition was from 25 to 75uc, at increments of 0.5uc/min and the change in fluorescence intensity used to determine the t m values with the use of kaleidagraph software (synergy, reading, pa). beas-2b cells were seeded at 1.0610 5 cells/well in begm amended with supplements (lonza, switzerland) in a 48-well tissue culture plate or 1.0610 4 cells/well in a 96-well plate. 6 h later, the cells were transfected with a pool of three sirnas specific to tlr3, nonspecific control sirna (santa cruz biotechnology inc.), or sirna to rig-i (qiagen, valencia ca). 30 nm sirnas were transfected using lipofectamine rnaimax (invitrogen, carlsbad ca) according to the manufacturer's protocol. the cells were incubated for 48 h prior to treatment with ligands and proteins. culture media was collected 24 h later and assayed for il6 production using elisa. rt-pcr analysis of the tlr3 mrna to confirm the effects of the sirna knockdown was performed. after treatments total rna samples pooled from either six wells of a 96-well plate or three wells of a 48-well plate were isolated from beas2b cells using rneasy kit (qiagen, valencia, ca). 0.5 mg of total rna was then reverse transcribed to cdna with mmlv reverse transcriptase (ambion, austin, tx) using random decamers (new england biolabs, ipswich, ma). cdna generated from 16 ng of total rna was amplified using sybr green supermix (bio-rad, hercules, ca) with an initial 3 min denaturing temperature of 95uc, followed by a total of 40 cycles of 30 s of denaturation at 95uc and 30 s of annealing at 55uc and elongation at 72uc. tlr3-specific primers were previously used by homma et al. [73] : (forward: 59-gatct-gtctcataatggcttg-39; reverse: 59-gacagattcc-gaatgcttgtg-39; and gapdh primers (59-gagtcaacg-gatttggtcgt-39; reverse: 59-tgggatttccattgat-gaca-39) were used. for each sample, tlr3 ct values were subtracted from corresponding gapdh ct values. tlr3 mrna levels for each treatment were calculated as %tlr3 mrna for samples treated with control nonspecific sirna. beas-2b cells were seeded on coverslips in 6 well plates. highly purified recombinant ns5b (0.2 mm) that was free of pyrogen was added to the cells at the same time as the dsrnas and incubated for 30 min at 37uc. after washing with pbs, cells were fixed with 4% paraformaldehyde at room temperature for 30 min. after two additional washes with pbs, they were permeabilized with 0.5% triton x-100 at room temperature for 10 min. the cells were then washed twice more with pbs amended with 0.02% tween-20 (tbs-t) and incubated with blocking buffer (1% bsa in pbs) for 1 h at room temperature. after washing three times with pbs-t, slides were incubated with anti-ns5b antibody (mab; 5b-3b1 from alexis biochemicals) and goat anti-tlr3 (r&d systems, minneapolis, mn) overnight at 4uc. slides were then probed with a texas red-labeled bovine anti-goat mab (santa cruz biotechnology) for 1 h at rt, washed, then incubated with a goat anti-mouse secondary antibody conjugated with alexa fluor 488 (santa cruz biotechnology) for 1 h at room temperature. coverslips were washed with pbs-t three times, air dried for 1 h and mounted in vectashield mounting medium with dapi (vector laboratories). the images were obtained on a leica tcs sp5 scanning confocal microscope with an hcx pl apo lambda blue 63 x 1.4 oil objective lens (leica microsystems). excitation was at 20% of the maximum laser power. images were captured with a scanning speed of 200 hz and image resolution of 5126512 pixels and then analyzed using leica application suite 2.02. ross river virus capsid protein (0.2 mm) was added the culture media of beas-2b cells seeded on coverslips for up to 6 hours at 37uc. the cells were washed and fixed as described above. the cells were incubated with anti r-cp rabbit polyclonal antibody (custom prepared by cocalico biologicals, reamstown, pa) and anti-tlr3 (mab, a kind gift of l. san mateo of centocor inc) diluted in pbs+0.1% bsa+0.01% tx100 overnight at 4uc. slides were then probed with a goat anti-mouse secondary antibody conjugated with alexa fluor 488 (santa cruz biotechnology) for tlr3 and bovine anti-rabbit texas red (santa cruz biotechnology) for r-cp for 1.5 h at room temperature. the slides were imaged and the results analyzed as described above. figure s1 effects of various rnas on il6 production. a) results from beas-2b cells. the endornaviral dsrnas labeled a-c were extracted from several rice isolates collected in lousiana and have distinct migrations in agarose gels. however, the genomes have not been determined. pic, pau, and pgu are three homopolymeric rnas. the single-stranded viral rnas were all extracted from purified virions. all rnas used were at a final concentration of 0.5 mg/ml. b) a time course of il6 production by beas-2b cells in response to poly(i:c) and s4 dsrna. the addition of ll37 to 3 mm final concentration resulted in tlr3 enhancing signaling in response to the s4 dsrna or poly(i:c). c) results from hek 293t cells expressing recombinant tlr3. all rnas were used at 1 mg/ml. the ratios denote the ratio of the isre-driven firefly luciferase to the renilla luciferase produces in the same cells. (tif) figure s2 additional characterization of the 1b hcv polymerase that could affect il6 production in beas-2b cells. a) effects of poly(i:c) of different lengths on il-6 production. the poly(i:c) fragments were separated on a denaturing gel and eluted from the gel fragments. the eluted fragments were annealed and their lengths determined by comparison to an dna ladder. b) effects of several recombinant hcv polymerase proteins on tlr3 induction of il6. the final concentrations of the proteins added are shown on the horizontal axis. the amount of poly(i:c) in each reaction was at 0.13 mg/ml. c) a demonstration that the mutant hcv polymerases retain the ability to bind the sl26 rna. dsrna. the gel is from a nondenaturing stacked polyacrylamide gel of 5 and 20%. the s4 dsrna was radiolabeled during in vitro transcription of the plus-and minus-strands and then annealed at a 1:1 ratio. (tif) figure s5 localization of the r-cp in beas-2b cells 2 h after addition into the cell culture medium. r-cp is stained red. the boxed area in the upper right is enlarged to show that small punctate spots of r-cp can be found in the cell's cytoplasm. 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rna-dependent rna polymerase oligomeric interaction of hepatitis c virus ns5b is critical for catalytic activity of rnadependent rna polymerase ordered duplex rna controls capsid architecture in an icosahedral animal virus comparison of the native ccmv virion with in vitro assembled ccmv virions by cryoelectron microscopy and image reconstruction structural basis of toll-like receptor 3 signaling with double-stranded rna cytosolic viral sensor rig-i is a 59-triphosphate-dependent translocase on doublestranded rna two novel classes of small ribonucleoproteins detected by antibodies associated with lupus erythematosus anti-5s rna/protein (rnp) antibody levels correlate with disease activity in a patient with systemic lupus erythematosus (sle) nephritis endosomal tlr signaling is required for anti-nucleic acid and rheumatoid factor autoantibodies in lupus structural analysis of the hepatitis c virus rna polymerase in complex with ribonucleotides hepatitis c virus induces e6ap-dependent degradation of the retinoblastoma protein down-regulation of the retinoblastoma tumor suppressor by the hepatitis c virus ns5b rnadependent rna polymerase hepatitis c virus ns5b delays cell cycle progression by inducing interferon-beta via toll-like receptor 3 signaling pathway without replicating viral genomes a cell-based assay for rna synthesis by the hcv polymerase reveals new insights on mechanism of polymerase inhibitors and modulation by ns5a hepatitis c core and nonstructural 3 proteins trigger toll-like receptor 2-mediated pathways and inflammatory activation the use of differential scanning fluorimetry to detect ligand interactions that promote protein stability corticosteroid and cytokines synergistically enhance toll-like receptor 2 expression in respiratory epithelial cells we thank members of the kao lab and jarrat jordan and lani san mateo of centocor inc. for helpful discussions and jing wang for some assays for il-8. we thank laura kao for editing the manuscript and the indiana university metacyte microscopy center and jim powell for the use of the facility and help with confocal microscopy. key: cord-340718-amfs4zay authors: zhu, gengping; peterson, a. townsend title: potential geographic distribution of the novel avian-origin influenza a (h7n9) virus date: 2014-04-01 journal: plos one doi: 10.1371/journal.pone.0093390 sha: doc_id: 340718 cord_uid: amfs4zay background: in late march 2013, a new avian-origin influenza virus emerged in eastern china. this h7n9 subtype virus has since infected 240 people and killed 60, and has awakened global concern as a potential pandemic threat. ecological niche modeling has seen increasing applications as a useful tool in mapping geographic potential and risk of disease transmission. methodology/principals: we developed two datasets based on seasonal variation in normalized difference vegetation index (ndvi) from the modis sensor to characterize environmental dimensions of h7n9 virus. one-third of well-documented cases was used to test robustness of models calibrated based on the remaining two-thirds, and model significance was tested using partial roc approaches. a final niche model was calibrated using all records available. conclusions/significance: central-eastern china appears to represent an area of high risk for h7n9 spread, but suitable areas were distributed more spottily in the north and only along the coast in the south; highly suitable areas also were identified in western taiwan. areas identified as presenting high risk for h7n9 spread tend to present consistent ndvi values through the year, whereas unsuitable areas show greater seasonal variation. in march and early april 2013, a new avian-origin influenza a (h7n9) virus emerged in eastern china, this h7n9 is causing disease in humans. people infected with h7n9 usually showed symptoms including fever, coughing, and severe acute respiratory illness, and the virus has now infected at least 240 and killed 60 people in china, causing global concern regarding potential pandemic threats [1] [2] . since the first case was reported, researchers focused on improving diagnosis, understanding location of origin, and methods of cure; however, little is known about the geographic potential of h7n9 or environmental correlates of its transmission, except butler [3] and he and chen [4] , who presented ideas based on lessons from the previous avian influenza threat (i.e. h5n1), and shi [5] and fang [6] , who mapped the spread potential of h7n9 using spatial regression method. the first known human h7n9 infections were reported on 31 march 2013, with two cases in shanghai and one in the neighboring province of anhui. by 22 april 2013, the world health organization (who) tallied 104 confirmed cases, over an expanded area including neighboring jiangsu and zhejiang provinces [3] . most human h7n9 cases remain concentrated around shanghai, but cases have been detected in beijing in the north, henan and anhui provinces in the interior, and hunan, jiangxi, fujian and guangdong provinces in the south (figure 1 ). the source of h7n9 human infections is unclear, but it appears to be carried by poultry, in their secretions or excretions [7] [8] . ecological niche modeling (enm) offers a means by which to characterize environmental conditions suitable for species or phenomena such as disease transmission, and identify where those suitable environments are manifested in geographic space [9] . although applications of enm to understanding the geography and ecology of disease transmission are in early stages of exploration and development, it has been successfully applied to transmission of several diseases (e.g. [10] [11] ), including avian influenza (e.g. h5n1; [12] ). this contribution aims to present a first range-wide enm-based analysis of h7n9 in china. h7n9 cases have been documented in detail since the initiation of the outbreak; in particular, a real-time h7n9 reporting system was launched (http://goo.gl/maps/zsvw8; [1] ), from where we obtained 87 georeferenced occurrences for analysis; an additional 9 points were obtained from the literature or internet (table s1 in supporting information). only the laboratory confirmed cases were used in this study. the 96 occurrence points varied in terms of spatial clumping, so we subsampled them to reduce sampling bias and effects of spatial autocorrelation, as follows. we first generated models using all available occurrence points, and measured spatial autocorrelation among model pseudoresiduals (1 -probability of occurrence generated by model) by calculating moran's i at multiple distance classes using sam v4.0 [13] ; significance was determined using permutation tests. a minimum distance of 13 km was detected, after which we created a grid with cell dimensions of 0.11760.117u, and selected the occurrence point closest to the centroid of each grid cell; this procedure reduced the number of occurrences to 77 points for final model calibration. we sought environmental data that would match the occurrence data in spatial resolution and temporal coverage, and provide a rich characterization of environmental landscapes for model calibration. the best match was multitemporal normalized difference vegetation index (ndvi) data from the modis (moderate resolution imaging spectroradiometer) satellite. sixteen-day ndvi composites were obtained for january 2012 to june 2013 at a resolution of 1 km 2 (http://modis.gsfc.nasa.gov/ data/). we used two approaches by which to reduce dimensionality from the initial 34 data layers. the first was to extract the first 10 principal component axes to summarize major axes of variation among the 34 ndvi layers (97.5% of overall variation); this dataset captured major dimensions of environmental variation across eastern china. the second 'biondvi' was based on all biweekly images from january 2012 -june 2013, transformed into data layers parallel to 'bioclimatic' variables (i.e. mean, maximum, minimum, range, and standard deviation) over the entire period, plus a parallel data set (mean, maximum, minimum, range, standard deviation) over spring 2013 (march-may) only, the period which we believe most relevant to the h7n9 outbreak. we used a maximum entropy algorithm (maxent; [14] ) to estimate an ecological 'niche' for h7n9 virus. maxent follows the principle of maximum entropy, spreading out probabilities as uniformly as possible, subjects to the constraints of observed values (i.e. known occurrences). we thresholded model predictions to produce binary maps by assessing the highest suitability value at which 90% of input occurrence points were included in the predicted suitable area (i.e. maxent's 10% training presence threshold). this approach is conservative in estimating ecological niches, as it eliminates extreme values that may result from erroneous identifications or georeferencing [15] . to evaluate niche models, we used a spatial subsetting approach to erect a rigorous test of model predictions: we sorted occurrences by latitude and separately by longitude, and set aside the central third as evaluation data, using the remaining two-thirds for model calibration. hence, we developed two evaluation data sets (i.e. subsetting by latitude and subsetting by longitude) for models produced in relation to the two ndvi datasets, for a total of four tests. significance was assessed by means of a cumulative binomial test, comparing success in anticipating the spatial distribution of known occurrences against null expectations based on the proportional area identified as suitable. a final niche model was calibrated using all of the 77 records. most h7n9 cases have been reported from shanghai, zhejiang, and jiangsu provinces, from where occurrences were aggregated, whereas occurrences in beijing, shandong, henan, hunan, jiangxi, and fujian provinces were more sporadic and scattered ( figure 1 ). environmental variation across the range of h7n9 is quite dramatic (figure 1) , with highest ndvi values observed in southern china; the independent variation in the principal components can be appreciated in figure 1 . in model evaluations, our niche models showed good performance in anticipating the spatial distribution of independent testing records in three of the four tests (p,0.05; table 1; figure 2 ). omission rates in models with longitude-based subsetting were higher than in models with latitude-based subsetting (table 1) , which might result from the broader geographic spread in longitude ( figure 2 ). in final model predictions, environmental variables with highest gain [14] in isolation were principal component 5 and standard deviation of the entire period; environmental variables that decreased gain most when omitted were principal component 1 and standard deviation of the entire period. in final models, models based on both environmental datasets identified a particularly suitable zone in east-central china, including shanghai, zhejiang, southern jiangsu, northwestern fujian and jiangxi, and parts of anhui provinces (figure 3) . the model based on the 'biondvi' dataset was somewhat more conservative in predictions. hubei province was identified as highly suitable, although no h7n9 cases have been reported there ( figure 3 ). suitable areas in the north were distributed less continuously, whereas suitable areas in the south were only along the coast. western taiwan was also identified as highly suitable by models based on both environmental datasets ( figure 3 ). attempting to visualize conditions under which models identified suitability, suitable areas tended to present conditions that were generally constant through the year (i.e. not much seasonal variation), with only gentle seasonal fluctuations. unsuitable areas, in contrast, showed much more seasonal variation: ndvi values rose sharply in june-august and declined during september-december (figure 4 ). limitations on the materials employed in this study have to be addressed here. the general distributional pattern of h7n9 has not been understood in detail to date, such that the distributional pattern so far has manifested sporadic and small clusters (figure 1 ). knowledge about the main virus reservoirs and the extent and distribution of the virus in animals remains limited, in spite of considerable efforts (e.g. [8] ), it is likely that most human cases were exposed to the h7n9 virus through contact with infected poultry or contaminated environments, including markets that sell live poultry [16] . however clear links between infections in poultry and human cases have been difficult to establish, because this virus does not appear to cause clinical signs in infected poultry. information to date also does not support sustained human-tohuman transmission [17] . effective and predictive risk maps can provide a useful means by which to design targeted surveillance efforts [3] [4] [5] [6] , and ecological niche modeling approaches are offering novel views of the geography of potential for disease transmission [9, 18] . many aspects of the natural history, geography, and ecology of h7n9 virus remain poorly known or unknown; in such cases, niche modeling can only be applied to the outputs of the overall cycle as a ''black box'', attempting to capture the integration of relevant niche components in h7n9 virus. consisting with the regression model, which integrated agroecological, environmental and meteorological factors to map the spread potential of h7n9 [5] [6] , our niche model also identified the central coastal areas in guangdong province as high suitable, which were validated by the new emerging cases in 2014. the first law of geography was introduced by tobler [19] , stating that everything is related to everything else, but that near things are more related than distant things; recent extension was to add dimensions of time in understanding and expressing distance and adjacency, resulting in a concept of spatiotemporal proximity proposed during the sars (severe acute respiratory syndrome) outbreaks [20] . here, we developed multitemporal vegetation index datasets to match the h7n9 outbreaks in spatiotemporal coverage; the hope was to characterize ecological requirements of h7n9 optimally, although many questions concerning the sporadic outbreaks of h7n9 remain up in the air. using these datasets, models showed good performance in anticipating the known geographic distribution of independent occurrence records (including the new emerging cases in january 2014); however, given that h7n9 is likely still in the process of spreading out to its full distributional and environmental potential, we are most confident in our result as near-term and local solution rather than as global summaries. for h5n1, researchers integrated large data sets combining information on poultry trade routes, numbers of birds being transported, distribution of live-bird markets, supply routes, waterfowl numbers, land use, and human population densities to trace h5n1 transmission (e.g. [21] [22] ). for h7n9, such analyses are only just beginning [3] [4] [5] [6] , particularly as sources of data on the virus and routes by which it infects humans remain unclear. birds at live poultry markets have been suspected as a source, but broad testing of poultry and other animals have not turned up significant infection rates [3] ; only isolates from shanghai live poultry markets showed high homology with viruses causing human infections [8] . based on experience with h5n1, butler [3] suggested that high suitability areas for h7n9 might include shandong province and a belt extending around the bohai sea to liaoning province in the north. however, h5n1 dynamics offer only a starting point for identifying areas at risk, since ecological requirements and natural history of h5n1 and h7n9 might well be different. our models identified high-risk zones for h7n9 in east-central china. continued vigilance therefore is needed within affected and neighboring areas to detect infections in animals and humans, as the current distributional pattern suggest that h7n9 would be in previously affected and possibly neighboring areas, and potentially in travelers from these areas returning to other countries. attention should be paid to transmission of h7n9 between the mainland and taiwan, since highly suitable areas were observed along both the eastern seaboard of mainland china and in western taiwan. origins and evolutionary genomics of the novel 2013 avian-origin h7n9 influenza a virus in china: early findings. arxiv: 1304 global concerns regarding novel influenza a (h7n9) virus infections mapping the h7n9 avian flu outbreaks predicting the global transmission potential of h7n9 avian flu inferring the potential risks of h7n9 infection by spatiotemporally characterizing bird migration and poultry distribution in eastern china mapping spread and risk of avian influenza a (h7n9) in china emerging risk of h7n9 influenza in china isolation and characterization of h7n9 viruses from live poultry markets-implication of the source of current h7n9 infection in humans ecological niches and geographic distributions ecologic niche modeling of blastomyces dermatitidis in wisconsin ecology and geography of plague transmission areas in northeastern brazil continent-wide association of h5n1 outbreaks in wild and domestic birds in europe towards an integrated computational tool for spatial analysis in macroecology and biogeography maximum entropy modeling of species geographic distributions rethinking receiver operating characteristic analysis applications in ecological niche modelling relationship between domestic and wild birds in live poultry market and a novel human h7n9 virus in china preferential recognition of avian-like receptors in human influenza a h7n9 viruses biogeography of diseases: a framework for analysis a computer movie simulating urban growth in the detroit region the first law of geography and spatial temporal proximity mapping h5n1 highly pathogenic avian influenza risk in southeast asia spatial distribution and risk factors of highly pathogenic avian influenza (hpai) h5n1 in china key: cord-298078-uqrwq5qk authors: kwak, hoyun; park, min woo; jeong, sunjoo title: annexin a2 binds rna and reduces the frameshifting efficiency of infectious bronchitis virus date: 2011-08-30 journal: plos one doi: 10.1371/journal.pone.0024067 sha: doc_id: 298078 cord_uid: uqrwq5qk annexin a2 (anxa2) is a protein implicated in diverse cellular functions, including exocytosis, dna synthesis and cell proliferation. it was recently proposed to be involved in rna metabolism because it was shown to associate with some cellular mrna. here, we identified anxa2 as a rna binding protein (rbp) that binds ibv (infectious bronchitis virus) pseudoknot rna. we first confirmed the binding of anxa2 to ibv pseudoknot rna by ultraviolet crosslinking and showed its binding to rna pseudoknot with anxa2 protein in vitro and in the cells. since the rna pseudoknot located in the frameshifting region of ibv was used as bait for cellular rbps, we tested whether anxa2 could regulate the frameshfting of ibv pseudoknot rna by dual luciferase assay. overexpression of anxa2 significantly reduced the frameshifting efficiency from ibv pseudoknot rna and knockdown of the protein strikingly increased the frameshifting efficiency. the results suggest that anxa2 is a cellular rbp that can modulate the frameshifting efficiency of viral rna, enabling it to act as an anti-viral cellular protein, and hinting at roles in rna metabolism for other cellular mrnas. ribosomal frameshifing is a recoding process of translation where a specific messenger rna (mrna)-mediated signal directs a ribosome to shift its reading frame and to continue in the new frame. programmed-1 ribosomal frameshifting is the most widely used translational recoding mechanism of many viruses [1] . many viruses employ frameshifting during replication and generate the frameshifted protein critical for efficient viral replication [2, 3, 4] . in the case of retroviruses such as hiv-1, ribosomal frameshifting is required for the expression of protease, reverse transcriptase and integrase. for most other viruses, including coronavirus avian infectious bronchitis virus (ibv), frameshifting generates rna dependent rna polymerases. since translation is a complex process involving many regulatory factors in addition to the ribosome, binding of rna binding protein (rbp) at a nearby rna signal could affect rna conformation and somehow redirect the translational machinery [5, 6] . therefore, the identification of rbps involved in frameshifting is important in elucidating the regulatory mechanism for rna translation. viral frameshifting signals are present in coding regions of mrna, and are generally composed of a ''slippery'' sequence, a stimulatory downstream rna hairpin or pseudoknot structure and an intervening spacer region [7, 8, 9] . rna pseudoknots are structural elements found in almost all classes of rna, which are now recognized as a widespread motif with diverse biological functions [2] . for example, viral pseudoknots in non-coding regions act in the regulation of translation initiation and in template recognition by viral replicase. in contrast, when pseudoknots are present in coding regions, they regulate translation elongation and termination, leading to ribosomal frameshifting [10] . it is not clear whether ribosomal frameshifting occurs through the interaction of ribosome or any rbps, or due to the intrinsic nature of the pseudoknot rna. annexin a2 (anxa2) is a multi-functional protein that has been implicated in a number of cellular functions, including calcium dependent regulation of exocytosis, dna synthesis and cell proliferation [11] . recently, its potential role in rna metabolism was proposed because it was found to bind some mrna transcripts, such as c-myc, collagen prolyl 4-hydroxylasea (i) and its own mrna [12, 13, 14, 15, 16] . since anxa2 binds 39untranslated region (utr) of these mrnas, it was suggested to be involved in mrna localization and translational regulation [15, 17] . interestingly, anxa2 is reported to be the most abundant proteins in cytoskeleton bound polyribosome fraction and also associated with mrnas [17] . these findings led to the suggestion that anxa2 may bind to some rna elements involved in translational regulation. in fact, aberrant expression of anxa2 is somehow related to the carcinogenesis of human cancers, because it has been shown to be clearly absent in some prostate cancers and highly overexpressed in other human cancers [18, 19, 20] . to identify the proteins that might recognize frameshifting signals, we presently utilized ibv pseudoknot rna as a model transcript. we identified anxa2 as a rbp that can recognize viral pseudoknot rna and reduce viral frameshifting. we confirmed the rna binding activity of anxa2 by various methods and tested its role in the regulation of frameshifting. by overexpression and knockdown approaches, we showed it to be an efficient modulator of viral frameshifting. we speculate that cellular anxa2 protein may act as an anti-viral protein that can reduce detrimental viral frameshifting and successful replication of the virus. the present results offer a clue for the role of anxa2 as a rbp for other cellular rna transcripts. since ibv pseudoknot rna was shown to induce ribosomal frameshifting during rna translation, we utilized ibv frameshifting rna sequences as the model transcript [3] . ibv genomic rna contains the frameshifting rna pseudoknot as well as the slippery site and the spacer. sequences of wild-type and mutant ibv pseudoknot rna (72 nts) shown in figure 1a were generated as in vitro transcribed transcript. rna structure of wild-type ibv rna was predicted to fold into pseudoknot structure, whereas mutant ibv rna was not able to form a pseudoknot structure (pknotsrg: http://bibiserv.techfak.uni-bielefeld.de/pknotsrg/submission.html) [21, 22] . these predicted structure are largely consistent with the structure previously reported for ibv pseudoknot rna by brierley et al. (1992) , albeit with minor differences [9] . to search for cellular proteins that directly interacted with ibv pseudoknot rna, a rna pull down assay was performed in the presence of cell extracts (figure 2a ). wild-type and mutant ibv pseudoknot rnas were end-labeled with biotin to serve as bait for cellular proteins that might precipitate with streptavidin-coated beads. in addition, rna was in vitro transcribed in the presence of 4-thio utp that could serve as crosslinker for directly interacting proteins. ibv pseudoknot rna was incubated with cell extracts from human lung fibroblast imr90, which is a natural host cell for the infection of ibv. the complex was irradiated with 365 nm uv light to directly crosslink interacting proteins to 4-thio utp incorporated ibv pseudoknot rna. rna-protein complexes were precipitated with streptavidin-coated magnetic beads to pulldown any proteins bound to biotin-labeled ibv pseudoknot rna. proteins were eluted from the beads, fractionated by sds-page and analyzed by silver staining. comparison of the bands from wild-type pseudoknot rna bound to those from the mutant demonstrated that most of the bound proteins were common for two rna molecules ( figure 2a ). however, a couple of protein bands seemed to present more in wild-type than mutant pseudoknot rna, so we eluted those bands from the gel. identification of the protein by maldi-tof-tof revealed that one of such proteins was annexin a2 (anxa2). the previous experiment indicated the possible interaction of anxa2 to ibv pseudoknot rna. to confirm whether anxa2 could bind wild-type ibv pseudoknot rna but not mutant ibv rna, we performed uv crosslinking assay to show that radiolabeled wild-type ibv pseudoknot rna bound to anxa2 protein ( figure 2b ). after rnase treatment on rna-protein complex, radiolabeled ibv pseudoknot rna bound protein bands were characterized by western blot analysis with anti-anxa2 antibody. radiolabeled rna was found near the size of the cytosolic extracts were prepared from imr90 cells, and the extracts were incubated with either wild-type or mutant ibv pseudoknot rna and precipitated with streptavidin-coated magnetic beads. bound proteins (pellet) were eluted from the beads, fractionated using 10% sds-page and stained with silver. unbound proteins (sup) were also analyzed as a control. input refers to 10% input of imr90 cell extract. bands isolated from the gel are also shown on the right with higher magnification. maldi-tof-tof analysis identified the wild-type bound protein as anxa2, indicated with asterisk. (b) uv crosslinking assay (left). imr90 cell extracts were incubated with the [a-32 p] labeled wild-type (wt) or mutant ibv pseudoknot rna. after crosslinking with uv, rna-protein complexes were untreated (-) or treated (+) with rnase a and fractionated in polyacrylamide gel. two protein-rna complex bands from imr90 cell extract (shown with an arrow on the right side of the gel, an asterisk indicating non-specific band) were shown to be specific to wild-type (wt) ibv rna. competition assay (middle). cell extracts were incubated with the [a-32 p] labeled wild-type (wt) or mutant (mt) ibv rna and non-labeled competitor (16, 106 and 1006, respectively). western blot analysis of uv-crosslinked rna-protein complexes (right). the same gel as shown on the left was blotted and incubated with anti-anxa2 antibody. all data were generated by the results from at least three independent experiments. doi:10.1371/journal.pone.0024067.g002 anxa2 protein, which suggests an interaction between radiolabeled ibv pseudoknot rna and cellular anxa2 protein. significantly, this band specifically competed with wild-type ibv pseudoknot rna but not with mutant ibv rna, also supporting the binding specificity of anxa2 to wild-type pseudoknot rna ( figure 2b ). identity of the rna bound protein was also confirmed by western blot analysis of the same gel ( figure 2b ). these data clearly demonstrated binding of anxa2 to the frameshifting rna pseudoknot in the ibv genome. next, we tested whether anxa2 could bind directly to ibv pseudoknot rna. to explore this, gst-fused recombinant anxa2 protein was purified and used for gst pull-down assay. more radioactivity was recovered after precipitating with gst-anxa2 in comparison to gst, suggesting that recombinant anxa2 could associate with ibv pseudoknot rna. moreover, wild-type ibv pseudoknot rna tended to bind more than mutant ibv rna did ( figure 3a ). we tested whether such binding was dependent on the presence of calcium, given that anxa2 is a calcium binding protein. however, calcium did not cause any change in the binding of anxa2 to ibv pseudoknot rna (data not shown). we performed a rna-emsa to analyze the anxa2 binding pattern ( figure 3b ). rna-protein complexes were resolved using native polyacrylamide gels. only a single shifted band was generated in the presence of anxa2 protein but not with the same amount of the gst protein ( figure 3b ). we also confirmed whether anxa2 bound to wild-type ibv pseudoknot rna in the cells. through the rna-immunoprecipitation assay, we showed that anxa2 specifically interacted with wild-type ibv pseudoknot rna but not with mutant ibv rna in lncap and hek293t cells ( figure 3c and 3d). these data clearly demonstrated the specific binding of anxa2 to ibv pseudoknot rna. anxa2 regulates ribosomal frameshifting of ibv pseudoknot rna since we found that anxa2 bound ibv pseudoknot rna, we next tested whether it could control the efficiency of ribosomal frameshifting on ibv pseudoknot rna in the cells. we constructed dual luciferase reporters controlled by ibv frameshifting sequences with or without pseudoknot rna ( figure 4a ). frameshifting of the dual luciferase reporter expressed firefly luciferase as well as renilla luciferase ( figure 4b ), so the frameshifting could be assessed by the ratio between these two proteins as well as by control reporters. control reporters with either in frame or no expression of firefly luciferase used as positive or negative controls, respectively. to test how anxa2 regulates the frameshifting efficiency of ibv pseudoknot rna, we first overexpressed anxa2 protein in the presence of the reporters and measured the luciferase activities. transfection of flagtagged anxa2 in hek293t cells reduced the relative frameshifting efficiency of ibv pseudoknot rna up to 40% ( figure 5a ). since hek293t cells possess an endogenous protein, overexpression of flag-anxa2 might generate additional exogenous anxa2 protein. to assess more definitively the inhibitory effect of anxa2 on the frameshifting of ibv pseudoknot rna, we used a prostate cancer cell line, lncap, which does not express endogenous anxa2 protein in the cells. more significant reduction of the relative frameshifting efficiency was shown in lncap cells, which likely resulted from the overexpression of anxa2 without any basal level expression of the protein ( figure 5b ). conversely, we showed that the downregulation of the anxa2 by three independent knockdown greatly increased the relative frameshifting efficiency of ibv pseudoknot rna in hek293t cells ( figure 5d and 5e). targeting sequences of shrna and sirna in anxa2 mrna were diagrammed in figure 5c . by independent experiments either with shrna expression vector or with sirna duplex, we clearly demonstrated that frameshifting efficiency was dramatically increased by anxa2 knockdown. these data clearly indicated that anxa2 controls the frameshifting efficiency in the cells, which could act as the cellular inhibitor against the frameshifting of viral rna translation. translation is the complex rna metabolism regulated by diverse factors, such as ribosome, trna and many ribosome associated proteins. here, we identify anxa2 as the regulator of ribosomal frameshifting. since it was previously reported that anxa2 binds rna and regulates rna localization [13, 14, 15, 16, 17] , our proteomic identification of anxa2 as an ibv pseudoknot rna binding protein leads us to propose that it might regulate the frameshifting efficiency of viral rna translation. as shown in figure 5 , knockdown of anxa2 increased the frameshifting efficiency up to seven times, whereas overexpression of anxa2 significantly down-regulated the frameshifting efficiency of ibv pseudoknot rna. these results clearly show that anxa2 reduces ibv viral frameshifting in some way, probably by binding to pseudoknot rna. since the frameshifting of ibv generates rna dependent rna polymerase, which is critical for successful viral replication, the protein that inhibits frameshifting must be a major antiviral regulator in eukaryotic cells. however, many questions remain to be answered. these include the exact mechanism of anxa2 action on ibv frameshifting, possible involvement of anxa2 on frameshifting of other viral rna and unexplored functions of rna metabolism on many other pseudoknot containing cellular rna. rna pseudoknots are structural elements formed upon basepairing of a single-stranded region to a stretch of complementary nucleotides elsewhere in the rna chain. since the first discovery of pseudoknot structure in 39-utr of turnip yellow mosaic virus (tymv), numerous viruses have been reported to contain pseudoknot rna [7, 8, 9] . many other cellular rnas, such as rrna, mrna, tmrna, catalytic rna, telomerase rna, rna components of ribonucleoprotein complex and many artificially selected rna aptamers also form pseudoknot structures [23, 24, 25, 26, 27] . a pseudoknot is not only a structural element of rna, but also a functional regulator of rna. as mentioned previously, rna pseudoknot located in the coding region of virus is involved in the -1 ribosomal frameshifting of viral rna translation, and pseudoknot in 59-utr in some cases regulates the initiation of viral rna replication [28] . since mutational analysis revealed drastic changes of ribosomal frameshifting efficiency upon disruption of pseudoknot structure, this structure is likely to be kept intact in addition to the slippery site and spacer region for efficient frameshifting [29] . recent advances in rna bioinformatics broaden the possibility of finding pseudoknot structure from rna sequences and predicting threedimensional structures of the rna molecules [30, 31, 32] . the detailed mechanism of pseudoknot function on frameshifting is being intensively studied. recent data suggests that the mechanical strength of the pseudoknot structure is correlated with ribosomal frameshifting, with pausing of ribosome itself is not sufficient for frameshifting [33, 34, 35, 36] . while still contentious, several reports describe pseudoknot binding protein factors. for example, 102-kda rna binding protein and eukaryotic elongation factor 1 (eef1) bind rna pseudoknot of 39-utr in the tobacco mosaic virus genome [37, 38] . recently, it was also reported that replicase gene product of mouse coronavirus interacts with its own rna pseudoknot [29] . also, we present data showing that anxa2 ; lanes 4-7 contain gst-anxa2 protein (0.5, 2, 5 and 10 mm, respectively). (c) rna immunoprecipitation assay. lncap cells were cotransfected with reporters (wild-type or mutant ibv pseudoknot plasmid wild-type or mutant) and anxa2 expression plasmids (flag-vector or flag-anxa2). after formaldehyde fixation, immunoprecipitations were performed with flag-m2 agarose beads. bound rna was extracted from the immune complexes and analyzed by rt-pcr (left panel) and qrt-pcr (right panel). pcr products were resolved by electrophoresis in agarose gel and visualized by staining with ethidium bromide. anxa2 mrna and ibv pk rna were also shown as an expression and input controls. immunoprecipitation of flag-anxa2 was confirmed by immunoblotting (ib) using anti-flag antibody. in the qrt-pcr data, wild-type pk rna (wt) in binds pseudoknot rna and inhibits ribosomal frameshifting of ibv. even though it is necessary to determine the exact mode of action for anxa2 on ribosomal frameshifting, evidence is accumulating concerning the role of anxa2 in translational regulation. for example, anxa2 is associated with active and inactive mrnas, polyribosomes and many ribosomal subunit proteins [12, 19] . in addition, the receptor for activated c-kinase (rack1), which resides in ribosome acting as the signal regulator of translation, also interacts with anxa2 [39] . deregulated expression of anxa2 is implicated in many human tumors and diseases [20, 40] . especially, anxa2 has an important role in angiogenesis and tumor progression, and, thus, might be a potential therapeutic target [41] . furthermore, the gene linked to optiz bbb/g syndrome, mid1, is associated with microtubule-associated ribonucleoprotein complex including ef-1a, rack-1, small ribosomal subunit proteins and anxa2, which again suggests a possible role of anxa2 in rna translation and in pathogenesis of the cell [42] . since viral frameshifting is critical for many pathogenic viruses including coronavirus, anxa2-pseudoknot rna interaction might be a novel target for anti-viral drug discovery [43, 44] . plasmids containing wild-type (pfs-cass5) and mutant (pfs-cass5.15) pseudoknot sequences from ibv genomic rna were generous gifts form dr. ian brierley (university of cambridge) [3] . bovine anxa2 cdna was kindly provided by dr. anni vedeler (university of bergen) [12] . human embryonic kidney cell line hek293t were purchased from the american type culture collection (atcc) and maintained in dulbecco's modified eagle's medium (dmem) containing 10% fetal bovine serum (fbs; brl life technology). human prostate cancer cell line lncap were purchased from the atcc and cultured in rpmi1640 with 10% fbs. human lung fibroblast imr90 and human lung adenocarcinoma sk-lu i cells were purchased from atcc and cultured in mem containing 10% fbs using standard techniques. cells were harvested and washed twice with ice-cold phosphate buffered saline (pbs). cell pellets were resuspended in two volumes of buffer a (20 mm hepes, ph 7.9; 10 mm nacl; 1 mm edta; 1 mm dtt) supplemented with protein inhibitor cocktail (1 mm pmsf, sigma-aldrich). after incubating 15 min on ice, cells were lysed with 0.5% nonidet p-40 and centrifuged at 4,000 rpm at 4uc for 30 sec. clarified cytosolic extract was quantitated for protein concentration and stored in aliquots at 280uc. wild-type and mutant ibv rnas were transcribed in the presence of 4-thio utp (ambion) by in vitro transcription with t7 rna polymerase. phosphate was removed from 59-end by calf intestinal alkaline phosphatase (new england bio lab) and c-s 32 was added by incubating with c-s 32 atp (10 mm) with t4 polynucleotide kinase (new england bio lab). biotin was added at the 59-end of the prepared rna by incubating one and half hours with 10 mm peo-iodoacetyl biotin (pierce) in the dark. cytosolic extracts were pre-cleared with streptavidin-coated magnetic beads (new england bio lab) and beads were preblocked with trna. biotin and 4-thio labeled rna was incubated with pre-cleared cell extract in binding buffer (20 mm hepes, ph 7.5; 50 mm kcl; 1 mm dtt; 0.1 mm edta; 5% glycerol; 40 u rnase inhibitor) for one hour in room temperature. ultraviolet radiation (365 nm) was performed to induce binding between 4-thio utp and interacting proteins. pre-blocked magnetic beads were added and incubated for one hour to pellet rna-protein complexes. two volumes of washing buffer (20 mm hepes, ph 7.5; 500 mm nacl, 1 mm dtt, 0.1 mm edta, 5% glycerol) was added and washed seven times, followed by elution with boiling in 26 sample buffer for 5 min. proteins were separated by 10% sds-page and visualized by silver staining. after pulling-down interacting proteins by a biotin pull-down assay as described below, protein identification was performed in the functional proteomics center, korea institute of science and technology. silver stained bands were excised and an automated in-gel tryptic digestion was performed on a mass prep station (micromass). the gel pieces were destained, reduced with dtt, alkylated with iodoacetamide and digested with sequencing grade modified trypsin (promega). resulting peptides were extracted from the gel and analyzed via maldi-tof and maldi-tof-tof. proteins were identified from the mass spectrometry data using mascot (matrix science) and searching the ncbi data base. ibv pseudoknot rna was transcribed and labeled in vitro with t7 rna polymerase (ambion) with a-[ 32 p]ctp. 32 p-labeled ibv pseudoknot rna (1.1610 5 c.p.m) was incubated with 30 mg of cell extract with 2 mg of trna, 0.2 mg of poly d(i-c) and 40 u of rnase inhibitor. the rna-protein binding reaction was carried out in a 30 ml reaction mixture containing 20 mm hepes ph 7.5, 50 mm kcl, 1 mm dtt, 0.1 mm edta and 5% glycerol. the mixtures were incubated at room temperature for 30 min, after which they were uv-irradiated (120 mj/cm 2 ) on ice for 30 min with an uv cross linker (fisher scientific). rnas were digested with 1 mg rnase a, 25 u rnase s1 and 0.1 u rnase v1 at 37uc for 10 min and analyzed by 10% sds-page. anti-anxa2 antibody (bd biosciences) was used for western blot analysis. proteins in sample buffer were resolved by 10% sds-page, transferred to a polyvinylidene difluoride membrane, immunostained by specific antibodies and visualized using a chemiluminescent substrate. gst-pull down assay was performed for confirmation of direct rna-protein interaction. radiolabeled rna transcript (5610 4 c.p.m) and purified proteins were incubated at room immune complex was normalized with input pk rna level and presented as relative enrichment in comparison to mutant pk rna (mt). (d) rna immunoprecipitation assay in hek293t cells. reporter plasmids (wild-type or mutant ibv pseudoknot plasmid) were transfected into hek293t cells and fixed with formaldehyde. sonicated lysates were then incubated with the antibodies as indicated (anti-igg or anti-anxa2). rna-protein complexes were precipitated with protein-g beads. bound rna was extracted from the immune complexes and analyzed by rt-pcr and qrt-pcr. immunoprecipitated anxa2 protein was shown by immunoblotting (ib). relative enrichment of binding was shown as in (c). doi:10.1371/journal.pone.0024067.g003 temperature for 1 h in a binding buffer containing 25 mm hepes ph 7.5, 100 mm nacl and 1 mm mgcl 2 . rna-protein complexes were pulled down by glutathione sepharose 4b (ge healthcare) pre-blocked with trna for 30 min. following washing using binding buffer, the radioactivity in the recovered pellets was measured by scintillation counting. construction of anxa2 expression clone and rna interference cdna for anxa2 was polymerase chain reaction (pcr)amplified and ligated into pcmv-tag2b vector (stratagene). anxa2 was amplified with primers 59 -caggatccatgtc-taccgttca-39 and 59 -ccgaattctcagtcatcccca-c-39. to knock-down anxa2 mrna, small hairpin rna (shrna) was designed using the ambion small interfering rna (sirna) converter website. the anxa2 target sequence was 59-ugcauaugggucugucaa-39 corresponded to coding region nucleotides 66-83. to make psuper-anxa2 (shanx2-1), specific oligonucleotides were synthesized (bioneer, korea) and ligated to psuper vector as previously reported [45] . also, two different sirnas corresponding to coding region nucleotides 109-129 (sianx2-2: 59-cgggaugcuuugaacauugaa-39) and 772-792 (sianx2-3: 59-aaccugguucagugcauucag-39) were designed. sirna duplex were chemically synthesized and contain dtdt 39 overhangs (bioneer, korea). purification of recombinant anxa2 protein cdna of the anxa2 protein was pcr amplified and inserted into the pgex-4t-1 vector (ge healthcare), followed by the transformation of the vectors into the protease deficient strain bl21. gst-anxa2 and gst protein was purified according to manufacturer's instructions. the basic procedure for emsa was previously described [46] . briefly, rna was in vitro transcribed and labeled in vitro with t7 rna polymerase (ambion) with [a-32 p]atp. 32 p labeled ibv pseudoknot rna (1.1610 5 c.p.m) and purified gst-anxa2 or gst protein were incubated at room temperature for 30 min in a binding buffer containing 25 mm hepes ph 7.5, 100 mm nacl, 1 mm mgcl 2 and 4% glycerol. rna-protein complexes were resolved on 5% native polyacrylamide gels. gels were dried and analyzed by autoradiography. the basic procedure for rna-ip was previously described [46] . briefly, lncap and hek293t cells were transiently cotransfected with wild-type of mutant ibv pseudoknot reporters and pcmv tag2b vector (flag-vector) or flag-anxa2 plasmids, and incubated with 1% formaldehyde for crosslinking. sonicated lysates were immunoprecipitated with flag m2 agarose beads (sigma aldrich) or with antibodies as indicated (anti-normal mouse igg or anti-anxa2). pellets were subsequently incubated at 70uc for 1 h to reverse the crosslinks, and the rna was purified with tri reagent (ambion) and treated with dnase i (ambion) to remove reporter plasmid dna. after reverse transcription, cdna was amplified by using primer pairs for ibv pseudoknot rna (59-gtcgactttaaactgatacggggt-atc-39 and 59-gaaggatcccagctgaaaggc-39). qrt-pcr was performed with stepone real-time pcr system (applied biosystems). reactions were amplified using selective primers as described above with power sybr master mix (applied biosystems) according to the manufacturer's instructions. quantification was carried out with the stepone tm software (ver 2.2). percentages of pk rna binding to anxa2 were expressed as the to make ibv dual luciferase reporters, a frameshifting sequence (72 bp) was amplified from pfs-cass 5 and pfs-cass 5.15. the sequence was inserted between renilla and firefly luciferase genes of a dual reporter, p2luc vector (kindly provided by dr. yang kyun kim, sungkyunkwan university) [47] . wildtype reporter is composed of slippery site, stop codon, spacer linker and pseudoknot region of ibv genomic sequences. the mutant reporter is as same as the wild-type reporter except for a disrupted pseudoknot. the in-frame reporter has wild-type pseudoknot but not frameshifting regulatory elements, where no-frame reporter has wild-type frameshifting regulatory elements except for the slippery site. wild-type and mutant ibv pseudoknot sequences were amplified by pcr with primers 59-aatgtcgactt-taaactgatacggg-39 and 59-taaggatcccagctga-aaggctc-39 from of pfs-cass 5 and pfs-cass 5.15, respectively. in frame sequence was amplified with primers 59-aa-tgtcgacacggggtatcagtc-39 and 59-taaggatcc-cagctgaaaggctc-39. no frame sequence was amplified with primers 59-aatgtcgacctgatacggggtat-39 and 59-taaggatcccagctgaaaggctc-39. cells were cultured and transfected with frameshifting reporters using lipofectamine tm (invitrogen). for the luciferase assay, cells were scraped into 100 ml of passive lysis buffer (promega). luciferase activity in the lysate was determined with a dualluciferase reporter assay system (promega), according to the manufacturer's instruction and measured with a turner luminometer td-20/20. firefly luciferase activity was normalized to the activity of renilla luciferase. frameshifiting efficiency was calculated by the following formula: where fe = frameshifting efficiency, f = wild-type or mutant luc activity, fn = no-frame luc activity and fp = in-frame luc activity. programmed ribosomal frameshifting in hiv-1 and the sars-cov viral rna pseudoknots: versatile motifs in gene expression and replication decreasing the frameshift efficiency translates into an equivalent reduction of the replication of the human immunodeficiency virus type 1 a three-stemmed mrna pseudoknot in the sars coronavirus frameshift signal trading translation with rna-binding proteins translation matters: protein synthesis defects in inherited disease an efficient ribosomal frame-shifting signal in the polymerase-encoding region of the coronavirus ibv characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot mutational analysis of the "slipperysequence" component of a coronavirus ribosomal frameshifting signal structure, stability and function of rna pseudoknots involved in stimulating ribosomal frameshifting annexins -unique membrane binding proteins with diverse functions annexin a2 is a novel rna-binding protein annexin a2 binds to the localization signal in the 39 untranslated region of c-myc mrna annexin a2 recognises a specific region in the 39-utr of its cognate messenger rna translational control of collagen prolyl 4-hydroxylase-(i) gene expression under hypoxia the mrnabinding site of annexin a2 resides in helices c-d of its domain iv an au-rich stem-loop structure is a critical feature of the perinuclear localization signal of c-myc mrna medicationrelated problem type and appearance rate in ambulatory hemodialysis patients annexin ii is associated with mrnas which may constitute a distinct subpopulation loss of annexin ii heavy and light chains in prostate cancer and its precursors equal loading of proteins were confirmed by anti-b-actin antibody. (b) luciferase reporter assay after overexpression of anxa2 in lncap cells. relative frameshifting efficiency was measured for the vector and flag-tagged anxa2 transfected cells. expression of anxa2 was confirmed by western blot analysis with anti-anxa2 antibody. anti-b-actin antibody was used as a loading control. (c) diagram of sirna or shrna target sequences (dashed line) in the coding regions anxa2 mrna (filled line). locations of a shrna and two sirnas were indicated (shanx2-1: 66-83 nt, sianx2-2: 109-129 nt, sianx2-3: 772-792 nt). (d) dual luciferase reporter assay following knockdown of anxa2 in hek293t cells. relative frameshifting efficiency was measured with psuper vector (vector) and with psuper-anxa2 (shanx2-1) transfected cells. western blot analysis was performed after shrna transfection. anti-b-actin antibody was used as a loading control. (e) luciferase reporter assay after knockdown of anxa2 in hek293t cells. relative frameshifting efficiency was measured from control sirna (sigfp) or two independent anxa2 sirnas (sianx2-2 and -3) transfected cells. western blot analysis was performed after sirna transfection. anti-b-actin antibody was used as a loading control pknotsrg: rna pseudoknot folding including near-optimal structures and sliding windows design, implementation and evaluation of a piratical pseudoknot folding algorithm based on thermodynamics structure and function of telomerase rna structural basis of a ribozyme's thermostability: p1-l9 interdomain interaction in rnase p rna folding of the sam aptamer is determined by the formation of a k-turn-dependent pseudoknot endogenous expression of a high-affinity pseudoknot rna aptamer suppresses replication of hiv-1 rna aptamers that bind the nucleocapsid protein contain pseudoknots genetic interactions between an essential 39 cis-acting rna pseudoknot, replicase gene products, and the extreme 39 end of the mouse coronavirus genome mutational study reveals that tertiary interactions are conserved in ribosomal frameshifting pseudoknots of two luteoviruses beyond mfold: recent advances in rna bioinformatics the mc-fold and mc-sym pipeline infers rna structure from sequence data knotseeker: heuristic pseudoknot detection in long rna sequences the 9-ã� solution: how mrna pseudoknots promote efficient programmed -1 ribosomal frameshifting correlation between mechanical strength of messenger rna pseudoknots and ribosomal frameshifting a mechanical explanation of rna pseudoknot function in programmed ribosomal frameshifting ribosomal pausing at a frameshifter rna pseudoknot is sensitive to reading phase but shows little correlation with frameshift efficiency isolation and characterization of the 102-kilodalton rna-binding protein that binds to the 5 and 3 translational enhancers of tobacco mosaic virus rna eukaryotic elongation factor 1a interacts with the upstream pseudoknot domain in the 39 untranslated region of tobacco mosaic virus rna regulation of eukaryotic translation by the rack1 protein: a platform for signalling molecules on the ribosome the annexinopathies: a new category of diseases the role of annexin ii in angiogenesis and tumor progression: a potential therapeutic target the opitz syndrome gene product mid1 assembles a microtubule-associated ribonucleoprotein complex coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus identification of novel ligands for the rna pseudoknot that regulate -1 ribosomal frameshifting specific inhibition of gene expression using a stably integrated, inducible small-interfering-rna vector b-catenin stabilizes cyclooxygenase-2 mrna by interacting with au-rich elements of 39-utr from the cover: specific mutations in a viral rna pseudoknot drastically change ribosomal frameshifting efficiency we appreciate drs. brierly (university of cambridge), vedeler (university of bergen) and yang kyun kim (sungkyunkwan university) for plasmids. we especially thank dr. myeong-hee yu (director of functional proteomics center, korea institute of science and technology) for allowing us to use proteomics facility and providing us excellent analysis. key: cord-340627-xyvzgkxl authors: ornaghi, sara; callegari, clelia; milazzo, roberta; la milia, laura; brunetti, federica; lubrano, chiara; tasca, chiara; livio, stefania; savasi, valeria maria; cetin, irene; vergani, patrizia title: performance of an extended triage questionnaire to detect suspected cases of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection in obstetric patients: experience from two large teaching hospitals in lombardy, northern italy date: 2020-09-15 journal: plos one doi: 10.1371/journal.pone.0239173 sha: doc_id: 340627 cord_uid: xyvzgkxl objectives: 1. to assess the performance of an extended questionnaire in identifying cases of sars-cov-2 infection among obstetric patients. 2. to evaluate the rate of infection among healthcare workers involved in women’s care. study design: a prospective cohort study of obstetric patients admitted to mbbm foundation and buzzi hospital (lombardy, northern italy) from march 16(th) to may 22(nd), 2020. women were screened on admission by a questionnaire investigating major and minor symptoms of infection and high-risk contacts in the last 14 days. sars-cov-2 assessment was performed by rt-pcr on nasopharyngeal swabs. till april 7(th), a targeted sars-cov-2 testing triggered by a positive questionnaire was used; from april 8(th), a universal testing approach was implemented. results: there were 1,177 women screened by the questionnaire, which yielded a positive result in 130 (11.0%) cases. sars-cov-2 rt-pcr was performed in 865 (73.5%) patients, identifying 51 (5.9%) infections. during the first period, there were 29 infected mothers, 4 (13.8%) of whom had a negative questionnaire. after universal testing implementation, there were 22 (3%, 95% ci 1.94% 4.04%) infected mothers, 13 (59.1%) of whom had a negative questionnaire; rate of infection among asymptomatic women was 1.9%. six of the 17 sars-cov-2-positive women with a negative questionnaire reported symptoms more than 14 but within 30 days before admission. isolated olfactory or taste disorders were identified in 15.7% of infected patients. rate of infection among healthcare workers was 5.8%. conclusions: an exhaustive triage questionnaire can effectively discriminate women at low risk of sars-cov-2 infection in the context of a targeted and a universal viral testing approach. in 15.7% of infected women, correct classification as a suspected case of infection was due to investigation of olfactory and taste disorders. extension of the assessed time-frame to 30 days may be worth considering to increase the questionnaire’s performance. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 of whom had a negative questionnaire. after universal testing implementation, there were 22 (3%, 95% ci 1.94% -4.04%) infected mothers, 13 (59.1%) of whom had a negative questionnaire; rate of infection among asymptomatic women was 1.9%. six of the 17 sars-cov-2-positive women with a negative questionnaire reported symptoms more than 14 but within 30 days before admission. isolated olfactory or taste disorders were identified in 15.7% of infected patients. rate of infection among healthcare workers was 5.8%. the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has caused a new, unexpected public health emergency. italy has been particularly affected, with lombardy being the country epicenter of the sars-cov-2 outbreak [1, 2] . obstetric patients and their caring physicians face unique challenges for their need of inperson visits and hospital admission for delivery. identification of suspected cases of sars-cov-2 infection at admission is essential for correctly applying isolation measures and use of personal protective equipment (ppe), thus protecting the women, their newborns, and the healthcare workers (hcws). universal screening by reverse transcription-polymerase chain reaction (rt-pcr) of nasopharyngeal swabs has been proposed as the optimal approach [3] [4] [5] [6] . however, limited testing supplies and laboratory workforce may prevent its application in some clinical settings [7] . in addition, although a rapid laboratory testing has been developed [6] , most hospitals rely on standard tests with a 5 to 24 hour-turn-around time [8, 9] . this may be a problem when caring for a laboring woman in whom delivery can occur before the rt-pcr result is available. a targeted screening guided by a structured questionnaire may represent a feasible and valid alternative [5, 10, 11 ]. yet, this approach has been questioned due to evidence of high rates of asymptomatic sars-cov-2 infection in the obstetric population [4, 6, 12, 13] . however, only major respiratory symptoms were assessed in these studies. since minor symptoms, such as loss of smell or taste, have been described at earlier stages of infection and also as isolated symptoms in milder forms of the coronavirus disease (covid-19) [14] [15] [16] [17] [18] [19] [20] , the possibility of some women being erroneously classified as asymptomatic in these reports has to be considered. here we report our data on the use of a comprehensive admission questionnaire for obstetric patients, including both major and minor symptoms of infection as well as high-risk contacts and living environment. accuracy of the questionnaire in the context of both a targeted and a universal sars-cov-2 screening by rt-pcr on nasopharyngeal swabs in two consecutive periods of the outbreak is assessed and discussed herein. secondary objective of the study was the assessment of the sars-cov-2 infection rate among the hcws involved in patients' management. this was a prospective cohort study of all women admitted to the obstetric unit of mbbm foundation at san gerardo hospital and vittore buzzi hospital during pregnancy or the postpartum period from march 16 th to may 22 nd , 2020. these hospitals are located in the milan area, lombardy region, northern italy, and perform approximately 5,600 deliveries per year. since the beginning of march, strict lockdown measures were in place in this geographic area, which entered a deceleration phase of the outbreak in mid-april [1] . starting on march 16 th , a comprehensive questionnaire including both major and minor symptoms of infection and high-risk contacts in the last 14 days as well as a high-risk living environment (i.e., immigration centers, drug rehabilitation centers) was administered to all women at hospital admission (fig 1) . the questionnaire was deemed positive when at least one positive answer was present. support persons (one for each woman) were also screened by means of the questionnaire, and refused hospital access in case of a positive result. both patients and their support persons were given surgical masks and asked to wear them during their hospital stay; they were also instructed to practice frequent hand sanitization. in addition, all hcws involved in women's care underwent questionnaire assessment (section a) at the beginning of every shift and wore a surgical mask for the entire shift duration, unless different ppe were required, as means of source control [21, 22] . initially, a targeted sars-cov-2 screening approach triggered by a positive questionnaire and based on rt-pcr testing of nasopharyngeal swabs was used in women with hospital admission after accessing the emergency department. in turn, a universal screening was applied to all patients with scheduled admission (i.e., elective pre-labor cesarean section). on april 8 th , we changed our policy and started testing all women for sars-cov-2 infection independent of the type of hospital admission and the questionnaire result, in agreement with a disposition of the lombardy region health care authority. cases with scheduled admission underwent rt-pcr testing 24-48 hours in advance in a designated drive-through testing center so results would be available at the time of hospital access to guide isolation measures and use of ppe. instead, questionnaire results were used for this purpose in cases of unscheduled admission: women with a positive questionnaire were classified as persons under investigation (pui) and managed accordingly while nucleic acid test results were pending, whereas women with a negative questionnaire were considered not at risk until the result of the rt-pcr test was available. nasopharyngeal sampling was performed by a trained resident physician or midwife in appropriate ppe using dedicated swabs. samples were transferred to the laboratory and processed by rt-pcr testing sars-cov-2 with the automated elite ingenius system and the genefinder covid-19 plus realamp kit assay, according to manufacturer's instructions. this assay targets three genes, rna-dependent rna polymerase, nucleocapsid protein, and envelope membrane protein, with high specificity. test results were available in 5 to 24 hours and scored as "positive" or "negative" in both hospitals [9] . viral testing was also performed in all hcws involved in patients' management. the accuracy of the questionnaire to predict sars-cov-2 infection in both study periods (march 16 th to april 7 th and april 8 th to may 22 nd ) was tested by constructing a 2x2 table and calculating sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio (sensitivity/1-specificity), and negative likelihood ratio (1-sensitivity/specificity). the study was approved by the irb of the university of milan-bicocca and the university of milan (#15408/2020). a written informed consent was obtained for all women involved in the study. a total of 1,177 women were assessed at hospital admission by the questionnaire during the study period (n = 447 at mbbm foundation at san gerardo hospital and n = 730 at vittore buzzi hospital). nine-hundred and forty-five (80.3%) women were admitted to the l&d unit, whereas 196 (16.7%) and 36 (3.0%) women to the antepartum and postpartum unit, respectively. of the 1,177 patients assessed, 865 (73.5%) were tested for sars-cov-2 by rt-pcr on nasopharyngeal swab and 51 (5.9%) were positive. between march 16 th and april 7 th , rt-pcr testing was performed in 129 out of 441 patients. questionnaire was positive on admission in 63 (14.3%) women. among the 319 patients with unscheduled admission and a negative questionnaire, 7 (2.2%) were tested for sars-cov-2 during hospitalization because of onset of fever. all of them were negative. sars-cov-2 infection was diagnosed in 29 mothers, 4 (13.8%) of whom had a negative questionnaire. one of these 4 patients failed to report high-risk contacts (i.e., fever and cough in close family members a few days before admission). an additional 2 women revealed symptoms suggestive of sars-cov-2 infection occurring more than 2 weeks but within one month before admission. none of the 4 patients developed symptoms during hospitalization. after implementing universal viral screening, we identified 67/736 positive questionnaires. nasopharyngeal swab analysis by rt-pcr recognized 22 (3%, 95% ci 1.94% -4.04%) cases of sars-cov-2 infection. questionnaire was negative in 13 (59.1%) of them, for a rate of infection among asymptomatic women of 1.9%. four out of these 13 women reported loss of taste or smell more than 14 days but within one month before admission. none of the 13 patients developed symptoms during hospitalization. accuracy of the questionnaire to predict sars-cov-2 infection in both study periods is shown in table 1 . detailed assessment of positive questionnaires showed that fever � 37.5˚c was the most common positive item (42.3%), followed by high-risk contacts/living environment (30.8%), cough (25.4%), gastrointestinal symptoms (13.8%), and loss of smell or taste (11.5%) (fig 2a) . fever and cough were more commonly identified during the first study period compared to the second one (57.1% versus 23.4% and 30.2% versus 20.9%, respectively), whereas gastrointestinal symptoms displayed an opposite trend (7.9% versus 19.4%). rate of olfactory and taste disorders, as well as of high-risk contacts/living environment, remained stable over time. of the fifteen women who reported loss of smell or taste within 14 days before admission, this was the only positive questionnaire item in three. also, when the time-frame of investigation was extended to the last 30 days before admission, an additional five mothers were identified with isolated olfactory or taste disorders. all these eight patients tested positive for sars-cov-2. frequency and time-trend of positive questionnaire items among sars-cov-2 infected women is shown in fig 2b. there were 307 hcws involved in patients' management in both hospitals during the overall study period. eighteen of them (5.8%) tested positive for sars-cov-2. there were no cases of moderate or severe covid-19. our study investigated the accuracy of a comprehensive questionnaire thoroughly assessing obstetric patients upon hospital admission to identify cases suspected for sars-cov-2 infection. differently from previous reports [4, [6] [7] [8] 13] , our questionnaire evaluated the presence of not only major respiratory symptoms of sars-cov-2 infection, including fever, cough, and shortness of breath, but also minor symptoms, such as loss of smell or taste, as well as high-risk contacts during the last two weeks preceding admission. we observed a negative predictive value for sars-cov-2 infection of 93.2% and 98.1% in the context of a targeted and a universal viral screening, respectively, in two consecutive periods of the outbreak. in addition, we identified a low rate of viral infection among the hcws involved in women's care [23] [24] [25] . the ability of accurately discriminating women at low risk for infection is pivotal in case of both a targeted and a universal sars-cov-2 screening. in clinical settings with no ability to perform a universal testing, a well-performing admission questionnaire can adequately guide a targeted screening approach and still allow protection of the patients and the hcws. on the other hand, when a universal screening approach is feasible, the questionnaire allows appropriate patients' cohorting and application of isolation measures and contact precautions while rt-pcr results are pending. this is particularly important to prevent potential viral spread to other patients and hcws and mother-to-child transmission when caring for laboring women, in whom delivery can occur before nucleic acid test results are available. in fact, turn-around time of rt-pcr testing is usually >5 hours in most facilities [8, 9] and availability of rapid testing is limited [6] . much of the push for implementation of a universal screening with sars-cov-2 rt-pcr hinges on avoiding unintended exposures to hcws when caring for an asymptomatic patient, especially in geographic areas with a high community prevalence of covid-19 [26, 27] . our hospitals are located in the epicenter of sars-cov-2 outbreak in italy [1, 2] . nonetheless, we had only 1.9% asymptomatic, sars-cov-2 positive women during the universal screening period. this rate is 7 times lower than that reported by other centers in similarly hardly hit table 1 . accuracy of the admission questionnaire in the two study periods. positive questionnaire 25 • positive likelihood ratio = 5.13; negative likelihood ratio = 0.64 � one patient failed to report exposure to high-risk contacts a few days before admission, and two patients reported fever and dry cough (n = 1) and loss of smell and taste (n = 1) more than 14 days but within 30 days before admission. # four patients reported loss of smell or taste more than 14 days but within 30 days before admission. https://doi.org/10.1371/journal.pone.0239173.t001 sars-cov-2 triage questionnaire in obstetric patients areas, such as new york city [4, 6] . a different phase of the outbreak during the study period may have contributed to this difference [1, 28] . however, our detailed investigation of minor sars-cov-2 triage questionnaire in obstetric patients symptoms, such as loss of smell or taste, may also have played an important role [3, 29] . olfactory and taste disorders have been reported as the only symptoms of infection in up to 17% of sars-cov-2 positive individuals, and to display a higher predictive ability of having the virus than fever or persistent cough [16, 18, 19] . isolated loss of smell or taste was identified in 15.7% of our virally infected patients. in addition, frequency of olfactory and taste disorders did not differ between the two study periods, whereas that of fever and cough displayed a substantial reduction (fig 2a and 2b) . these data suggest that patients' assessment upon admission during an outbreak by a novel air-tract pathogen should include not only major respiratory but also minor symptoms, especially in the more advanced phases of the outbreak. prolonged sars-cov-2 rt-pcr positivity at more than 2 weeks from symptom onset has been reported in infected individuals [30, 31] . overall, we identified 51 women with sars--cov-2 infection, and in 17 (33.3%) questionnaire upon admission was deemed negative. however, 6 (35.3%) of these patients reported symptoms suggestive of infection more than 14 but within 30 days before hospital admission. had the questionnaire investigated a one-month time-frame, negative predictive values would have increased to 98.2% and 98.6% in the targeted and the universal viral screening period, respectively. of note, whether such patients would have been infectious and thus able to spread the virus at the time of admission is still a matter of debate [32] [33] [34] [35] [36] . similarly, the infectiousness of fully asymptomatic women with positive sars-cov-2 rt-pcr results is unclear [31, 33, [36] [37] [38] . unfortunately, we could not address this issue in our patients since we did not perform viral culture experiments [33, 34] . nonetheless, independent of the infectiousness potential of these women, widespread use of face masks and frequent hand sanitization have likely contributed to successful viral spread control in our units [21, 22] . strengths of our study are the following. first, it was conducted in two large teaching hospitals in the italian epicenter of the outbreak, thus providing useful data for equally affected areas. second, it investigated the questionnaire's performance in the context of both a targeted and a universal viral screening approach in two consecutive periods of the outbreak. third, it assessed the universal screening approach over a 6-week time period, which may have allowed to better capture the real trend of infection over time among obstetric patients than much shorter study periods [4, 6, 8] . admission questionnaires may have limitations since they rely on honest answering. the possibility of patients being not completely sincere due to fear of isolation measures, especially in laboring women, has to be considered. this event occurred in at least one woman in our cohort. addition of objective, point-of-care parameters, such as lymphocyte count and lung ultrasound, to the admission screening procedure to increase its accuracy may be worth exploring [7, 10, 27, 31, [39] [40] [41] . another limitation of our study is that sars-cov-2 testing by rt-pcr on nasopharyngeal swabs was performed in a targeted manner during the first study period, thus leading to 312 untested women. this also prevented a meaningful comparison of infection rates between the two study periods with a targeted and a universal sars-cov-2 testing approach, respectively. with recognition that a "one-size-fits-all" approach is unlikely to be justifiable [26] , decisions regarding universal viral testing should be made in the context of regional prevalence of sars-cov-2 infection as well as financial and human resources and ppe availability in each obstetric unit. our data show that thorough assessment of obstetric patients upon hospital admission by means of an exhaustive questionnaire is feasible and effective in discriminating women at low risk of sars-cov-2 infection in the context of both a targeted and a universal screening dipartimento della protezione civile. covid-19 italia-monitoraggio della situazione critical care utilization for the covid-19 outbreak in lombardy, italy: early experience and forecast during an emergency response clinical implications of universal severe acute respiratory syndrome coronavirus 2 (sars-cov-2) testing in pregnancy. obstetrics and gynecology universal screening for sars-cov-2 in women admitted for delivery. the new england journal of medicine effectiveness of a covid-19 screening questionnaire for pregnant women 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information for healthcare professionals covid-19 infection among asymptomatic and symptomatic pregnant women: two weeks of confirmed presentations to an affiliated pair of new york city hospitals. american journal of obstetrics & gynecology mfm severe acute respiratory syndrome coronavirus 2 in pregnancy: symptomatic pregnant women are only the tip of the iceberg. american journal of obstetrics and gynecology a new symptom of covid-19: loss of taste and smell clinical infectious diseases: an official publication of the infectious diseases society of america presentation of new onset anosmia during the covid-19 pandemic defining the burden of olfactory dysfunction in covid-19 patients. european review for medical and pharmacological sciences the importance of olfactory and gustatory disorders as early symptoms of coronavirus disease (covid-19). the british journal of oral & maxillofacial surgery quantifying additional covid-19 symptoms will save lives real-time tracking of selfreported symptoms to predict potential covid-19. nature medicine estimating the effects of non-pharmaceutical interventions on covid-19 in europe physical interventions to interrupt or reduce the spread of respiratory viruses. the cochrane database of systematic reviews presymptomatic sars-cov-2 infections and transmission in a skilled nursing facility. the new england journal of medicine first experience of covid-19 screening of health-care workers in england covid-19 screening of health-care workers in a london maternity hospital. the lancet infectious diseases is universal testing for severe acute respiratory syndrome coronavirus 2 (sars-cov-2) needed on all labor and delivery units? obstetrics and gynecology isuog interim guidance on coronavirus disease 2019 (covid-19) during pregnancy and puerperium: information for healthcare professionals-an update new york city department of public health. coronavirus disease 2019 (covid-19) daily data summary postpartum care during the covid-19 pandemic. medscape. 2020. 30. control kcfd. findings from investigation and analysis of re-positive cases comparison of clinical characteristics of patients with asymptomatic vs symptomatic coronavirus disease detection of sars coronavirus in patients with suspected sars. emerging infectious diseases heatlh emergencies preparedness and response who global. advice on the use of masks in the context of understanding covid-19: what does viral rna load really mean? the lancet infectious diseases variation in false-negative rate of reverse transcriptase polymerase chain reaction-based sars-cov-2 tests by time since exposure. annals of internal medicine virological assessment of hospitalized patients with covid-2019 we don't actually have that answer yet': who clarifies comments on asymptomatic spread of covid-19 viral rna load as determined by cell culture as a management tool for discharge of sars-cov-2 patients from infectious disease wards. european journal of clinical microbiology & infectious diseases: official publication of the european society of clinical microbiology lung ultrasound and computed tomographic findings in pregnant woman with covid-19 how to perform lung ultrasound in pregnant women with suspected covid-19 covid-19 outbreak: less stethoscope, more ultrasound. the lancet respiratory medicine key: cord-313107-6cfenpxm authors: singh, anirudh k.; nema, ram kumar; joshi, ankur; shankar, prem; nema, shashwati; raghuwanshi, arun; patankar, chitra; mathew, bijina j.; shrivas, arti; pandey, ritu; tripathi, ranu; biswas, debasis; singh, sarman title: evaluation of pooled sample analysis strategy in expediting case detection in areas with emerging outbreaks of covid-19: a pilot study date: 2020-09-22 journal: plos one doi: 10.1371/journal.pone.0239492 sha: doc_id: 313107 cord_uid: 6cfenpxm timely diagnosis of covid-19 infected individuals and their prompt isolation are essential for controlling the transmission of sars-cov-2. though quantitative reverse transcriptase pcr (qrt-pcr) is the method of choice for covid-19 diagnostics, the resource-intensive and time-consuming nature of the technique impairs its wide applicability in resource-constrained settings and calls for novel strategies to meet the ever-growing demand for more testing. in this context, a pooled sample testing strategy was evaluated in the setting of emerging disease outbreak in 3 central indian districts to assess if the cost of the test and turn-around time could be reduced without compromising its diagnostic characteristics and thus lead to early containment of the outbreak. from 545 nasopharyngeal and oropharyngeal samples received from the three emerging districts, a total of 109 pools were created with 5 consecutive samples in each pool. the diagnostic performance of qrt-pcr on pooled sample was compared with that of individual samples in a blinded manner. while pooling reduced the cost of diagnosis by 68% and the laboratory processing time by 66%, 5 of the 109 pools showed discordant results when compared with induvial samples. four pools which tested negative contained 1 positive sample and 1 pool which was positive did not show any positive sample on deconvolution. presence of a single infected sample with ct value of 34 or higher, in a pool of 5, was likely to be missed in pooled sample analysis. at the reported point prevalence of 4.8% in this study, the negative predictive value of qrt-pcr on pooled samples was around 96% suggesting that the adoption of this strategy as an effective screening tool for covid-19 needs to be carefully evaluated. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the transmission of covid-19 is difficult to contain owing to the dual factors of high transmissibility of sars-cov-2 (r0 = 2.1-3.3) and asymptomatic/ mildly symptomatic individuals serving as sources of infection [1] [2] [3] [4] . extensive testing of suspected cases and asymptomatic direct and high-risk contacts is, therefore, recommended as key to controlling the ongoing pandemic [5] . however, the testing modality of choice viz. quantitative reverse transcriptase pcr (qrt-pcr), is too technically demanding, time-consuming and resource-intensive to be widely adaptable in low-and middle-income countries as well as remote locations and thus it often fails to inform early identification and quick isolation of the infected patients. innovative methods for expediting the qrt-pcr results, without compromising the diagnostic sensitivity and specificity, are therefore urgently needed. the central indian state of madhya pradesh (mp), with a total of 52 districts, reported the first case of covid-19 on march 20 th 2020 and till 8 th april 2020 majority of the reported cases from the state (78.2%) were restricted to the 2 districts of indore (district a) and bhopal (district b). from the second week of april 2020, cases started being reported from district dhar (a1), which borders district a, and from districts raisen (b1) and hoshangabad (b2), both of which are adjacent to district b (fig 1) . this prompted massive contact tracing in each of these 3 districts in an effort to contain the spread of infection. since the samples collected from these emerging districts converged on a laboratory system that was already over-burdened with unprecedented workload, a need was felt to evaluate strategies for testing additional samples without compromising the diagnostic characteristics and turn-around time of the test. we hypothesized that testing of pooled respiratory samples, collected from potentially infected individuals, could lead to faster laboratory confirmation and quicker containment of the emerging infection in these districts and, thus, undertook this study to evaluate the diagnostic concordance between the strategies of pooled vs. individualized testing and estimate the gain in turn-around time (tat) and resources that could be achieved through pooling. this study was approved by the all india institute of medical sciences, bhopal institutional human ethics committee with the waiver of consent as per the "national ethical guidelines for biomedical and health research" set forth by indian council of medical research, which is an apex government body responsible for making and enforcing policies of medical research in india. as per the guidelines, the institutional ethics committee can grant waiver of consent if the research is done on anonymized biological samples and/or the primary purpose of the research is refinement and improvement of the public health programs. as our study met both these criteria it was approved with the waiver of consent. nasopharyngeal and oropharyngeal swabs were collected by trained healthcare workers from suspected covid-19 patients belonging to the districts a1, b1 and b2 in vials containing viral transport medium (vtm) during april and may 2020. samples were transported at 2-8˚c to the testing laboratory within 24 hours. the relevant clinical and epidemiological details of the patients were entered in a standard form approved by the indian council of medical research, which is spearheading the nationwide laboratory network for covid-19 testing. aliquots of the original consecutive clinical samples were anonymized and processed in parallel for individualized and pooled analysis in a blinded manner. a pool size of 5 was chosen as per the advisory of the indian council of medical research for pooled sample testing for covid-19. for pooled analysis, 200 μl from each of 5 consecutive samples were collected in a single 1.5 ml centrifuge tube and processed for rna extraction using qiaamp viral rna mini kit (qiagen, hilden, germany) as per manufacturer's instructions. rna extraction for individualized testing was also performed using the same kit. the extracted rna samples were subjected to diagnosis using real-time fluorescent rt-pcr kit for detecting sars-cov-2 (bgi, hong kong) as per the manufacturer's protocol on a biorad cfx96 thermal cycler. the kit targets orf1ab for detection of the virus and human β-actin as the internal control. as recommended by the manufacturer, a sigmoidal curve with a ct value � 35 was considered as the criterion for considering a sample as positive for sars-cov-2. all the necessary controls namely no-template control, extraction control and positive control were tested in parallel with every batch of samples, as part of the quality control of the procedure. the data was entered in ms excel in wide data format. it was checked for missing values, redundancies and outliers. the descriptive summarization of variables of interest was done by median and iqr for ordinal and interval data (non-parametric distribution) and counts for nominal data. the diagnostic accuracy of pooled strategy, in reference to individual qrt-pcr, for correctly classifying the pool was determined by point and interval estimates of sensitivity, specificity and by likelihood ratios. as positive predictive value (ppv) and negative predictive value (npv) are prevalence-dependent parameters, we ran a simulation to estimate the ppv and npv values with a varying pre-test probability from 1%-5% in addition to group prevalence. further a kappa statistic was calculated between the two strategies in order to detect the agreement beyond chance. we also calculated the nonparametric spearman correlation to check the magnitude and direction of the relationship between pooled ct values and number of positive samples in the same pool. all the analyses were done by base r software which is in open domain and associated epi-r package. the choropleth map was drawn with the aid of gg plot2 package in r software. the bland altman (ba) plot was drawn and the relevant ba statistic was calculated with 'bland altman leh' and 'ggplot2' r package. a total of 545 samples were collected, with 140, 270 and 135 of them belonging to districts a1, b1 and b2, respectively. both, the individual samples and their pools were processed in parallel for testing. a total of 109 pools were created from 5 consecutive samples received from each district and the diagnostic performance of the 2 strategies were compared. considering the individualized qrt-pcr technique as the gold standard, we observed pcr-positivity of 7.1% (10/140), 3.7% (10/270) and 4.4% (6/135) in the districts a1, b1 and b2 respectively. the 25 samples that tested positive on individualized testing got sorted into 16 pools and 12 of these 16 pools were detected as positive during pooled sample testing. majority of these 12 pools contained 1 positive sample (n = 7). while all 5 pools containing more than one positive sample could be detected on pooling, 4 pools each containing single positive sample were missed by this strategy. although all the pools with more than one positive sample tested positive, we observed a weak negative and nondiscordant pools. the statistical significance of this difference could not however be determined by mann whitney u test, due to the statistical constraint of having less than 5 observations in the discordant group (table 1) . similarly, 93 pools contained samples that tested negative on individualized rt-pcr and 92 of these pools were also found to be negative on pooled analysis. one of the 93 pools tested positive on pooled analysis with a ct value of 33.3; though on deconvolution the individual samples tested negative. the prevalence-dependent and prevalence-independent diagnostic characteristics of the pooled sample analysis strategy are depicted in table 2 (this table in a different format is included as s2 table) , with ppv and npv simulated at 1% to 5% background positivity. we observed 75.0% (47.6% to 92.7%) sensitivity, 98.9% (94.2 to 100%) specificity, 95.8% (90.8% to 98.2%) npv and 0.3 (0.1-0.6) negative likelihood ratio values for pooled analysis. the extent of agreement between pooled versus individual sample strategy was estimated further by kappa statistic (κ = 0.8; 95% ci = 0.6-1.). the pooled strategy expectedly led to a significant gain in turn-around time. the total laboratory processing time required for the analysis of 545 samples through individualized for individual and pooled ct values refer s1 table. https://doi.org/10.1371/journal.pone.0239492.t001 sampling strategy was 67 hours, while the same was found to be 23 hours for pooled sampling including the testing of individual samples after deconvolution of the positive pools. similarly, the pooled testing strategy led to 68% savings in reagent costs with the total cost of individualized testing being $3137 whereas the pooled analysis including the deconvoluted samples was $1002. in this study we report that the pooled sample analysis strategy, if applied in the scenario of an emerging outbreak of covid-19, offers a significant reduction of laboratory turn-around time and reagent requirement at the cost of compromised diagnostic sensitivity. at the reported point prevalence of 4.8% among the individuals tested in this study, the npv of pooled sample strategy was around 96%; thereby suggesting the possibility of missing infected cases and risking community transmission from undiagnosed individuals. particularly, presence of a single infected individual with relatively low viral load (ct value of � 34), in a pool of 5, was likely to be missed in pooled sample analysis. one of the reasons for this could be the dilution of sample beyond the limit of detection of the assay used. since pcr-positivity is a function of the net viral load in the pooled sample, we understand that the success of the pooling strategy as a sensitive screening tool would be dependent on the number of infected patients in the pool and the viral load in the individual samples. while the latter is likely to be influenced by host-specific factors like immune competence, comorbidities and age, the former is likely to be a reflection of the background prevalence of infection in the community. while pooled sample testing offers the advantage of cost effectiveness and timely reporting, the strategy becomes less useful in communities where the prevalence of the disease is high. with increased prevalence, number of positive pools are likely to increase and more pools are to be deconvoluted for identifying the positive individuals negating the advantages such as cost and time saving. furthermore, with the increase in the prevalence of a disease, npv of the diagnostic test for the disease decreases leading to higher false negative results. based on this premise, national advisories have suggested testing of 5 sample pools for covid-19. in communities with prevalence of upto 5% [6] . however, our data reflects the lack of a robust relationship between the former factor and the ct value of the pooled sample; thus, implying the greater role of viral titers in individual samples in influencing the pcr results. the relatively low sensitivity and resultant negative predictive value of the pooled sample analysis strategy hint at its weakness as an effective screening tool in reliably "ruling out" the diagnosis of covid-19. in our study, pooled sampling led to false negative results in 4 of the 545 patients; all of whom were direct household contacts of known covid-19 patients and two of them were clinically symptomatic with cough, sore throat and fever. despite being clinically and epidemiologically suggestive, three of these 4 patients had ct values above 34; thereby suggesting that pooled analysis is likely to miss the detection of samples with low viral loads. these instances of missed case detection pose the risk of community transmission of infection from undetected sources. earlier studies evaluating the utility of pooled sample testing for surveillance of bovine viral diarrhea virus and porcine reproductive and respiratory syndrome virus demonstrated that sensitivity of rt-pcr tests reduced when run on pooled samples, due to a dilution effect [7, 8] . in a recently published study lohse et al., value of 34 were detected as positive in this study as a cut-off value of 45 was used by the authors [9] . of the 93 pools created from samples testing negative on individualized testing, 92 were negative on pooled analysis and one pool tested positive with ct of 33.3 for orf1ab gene. however, on deconvolution, each of the 5 constituent samples in that pool tested negative. we observed that this pool comprised of 3 patients with severe acute respiratory infection. we surmise that non-specific inhibitors of pcr amplification, co-habiting the viral genomic target in any of these patients, might have suppressed the amplification of low-level viral target in individual samples beyond the detectable threshold. pooling the samples could potentially dilute these inhibitors and lead to a detectable signal with a relatively delayed ct on poled analysis. in a recent study hogan and co-workers made a similar observation while assessing the utility of pooled sample testing strategy to detect community transmission of sars-cov-2 in san francisco bay area, ca, usa. one of the pools was positive in their study, however, upon deconvolution all the individual sample tested negative [10] . our study suffered from several limitations. apart from having a relatively small sample size, as mentioned above, the 3 districts did not have similar background prevalence. hence the diagnostic characteristics like positive and negative predictive values are likely to be dissimilar between the districts. furthermore, pooling of consecutive samples was done in the study. while family members of infected cases were likely to be included in the same pool which may have decreased the possibility of false negative results. adopting a strategy of random pooling could lead to a different proportion of infected individuals in the pools, the effect of which was not explored in the study. we also did not have pools in which multiple infected samples had ct values of � 34 and, hence, the diagnostic performance of such pools could not be assessed. we therefore, conclude that though the pooling strategy could be an alternative in resource-constrained settings with overwhelmed laboratory infrastructure, its adoption could miss the detection of pools having single infected individuals with low viral loads. our study, which is probably the first report on pooled testing in areas experiencing the early phase of emergence of covid-19 outbreaks, shows that 15.4% of the infected pools could be missed by this strategy. it is therefore recommended that adoption of the pooled sample testing strategy, as a cost effective screening tool for early containment of transmission in emerging outbreaks, has to be tailored to the prevalence of covid-19 in the geographical area concerned. supporting information s1 presymptomatic sars-cov-2 infections and transmission in a skilled nursing facility presumed asymptomatic carrier transmission of covid-19 delivery of infection from asymptomatic carriers of covid-19 in a familial cluster the basic reproduction number of novel coronavirus (2019-ncov) estimation based on exponential growth in the early outbreak in china from 2019 to 2020: a reply to dhungana covid-19 strategy update advisory on feasibility of using pooled samples for molecular testing of covid-19 factors affecting sensitivity and specificity of pooled-sample testing for diagnosis of low prevalence infections evaluation of the sensitivity of reverse-transcription polymerase chain reaction to detect porcine reproductive and respiratory syndrome virus on individual and pooled samples from boars pooling of samples for testing for sars-cov-2 in asymptomatic people sample pooling as a strategy to detect community transmission of sars-cov-2 key: cord-317244-4su5on6s authors: maganga, gael d.; bourgarel, mathieu; obame nkoghe, judicael; n'dilimabaka, nadine; drosten, christian; paupy, christophe; morand, serge; drexler, jan felix; leroy, eric m. title: identification of an unclassified paramyxovirus in coleura afra: a potential case of host specificity date: 2014-12-31 journal: plos one doi: 10.1371/journal.pone.0115588 sha: doc_id: 317244 cord_uid: 4su5on6s bats are known to harbor multiple paramyxoviruses. despite the creation of two new genera, aquaparamyxovirus and ferlavirus, to accommodate this increasing diversity, several recently isolated or characterized viruses remain unclassified beyond the subfamily level. in the present study, among 985 bats belonging to 6 species sampled in the belinga caves of gabon, rna of an unclassified paramyxovirus (belinga bat virus, belpv) was discovered in 14 african sheath-tailed bats (coleura afra), one of which exhibited several hemorrhagic lesions at necropsy, and viral sequence was obtained in two animals. phylogenetically, belpv is related to j virus and beilong virus (beipv), two other unclassified paramyxoviruses isolated from rodents. in the diseased belpv-infected c. afra individual, high viral load was detected in the heart, and the lesions were consistent with those reported in wild rodents and mice experimentally infected by j virus. belpv was not detected in other tested bat species sharing the same roosting sites and living in very close proximity with c. afra in the two caves sampled, suggesting that this virus may be host-specific for c. afra. the mode of transmission of this paramyxovirus in bat populations remains to be discovered. members of the paramyxoviridae family are pleomorphic enveloped viruses [1] divided into two subfamilies, paramyxovirinae and pneumovirinae. paramyxovirinae has recently been subdivided into seven genera: aquaparamyxovirus, avulavirus, ferlavirus, henipavirus, morbillivirus, respirovirus, and rubulavirus (http://ictvonline.org/virustaxonomy. asp?version52012). viruses of this family affect a wide range of animals, including primates, birds, carnivores, ungulates, snakes, cetaceans and humans, and cause a wide variety of infections, such as measles, mumps, pneumonia and encephalitis in humans, and distemper, peste des petits ruminants, newcastle disease and respiratory tract infections in animals. however, several paramyxoviruses (pvs) have not been classified into any of these seven genera, including nariva virus (narpv) [2] , mossman virus (mospv) [3] , beilong virus (beipv) [4] , j virus (jpv) [5, 6] , tupaia paramyxovirus (tuppv) [7] and tailam virus [8] , all of which belong to a group of novel paramyxoviruses isolated from wild animals, as well as salem virus isolated from horses [9] . among them, only jpv has been shown to be pathogenic, causing extensive haemorrhagic lesions in rodents [6] . horizontal transmission is the principal mode of intraspecies pv infection, suggesting that contaminated faeces, urine or saliva may be responsible for spillover to other species [10] . bats have a close evolutionary relationship with several genera of mammalian paramyxoviruses [11] . otherwise, bat-borne paramyxoviruses are in close relationship to known paramyxoviruses of mammalian. these small mammals are known to harbour a broad diversity of pvs, including emergent henipaviruses (nipah virus and hendra virus) and rubulaviruses [menangle virus, tioman virus, mapuera virus, and tuhoko virus 1, 2 and 3 (thkpv-1, thkpv-2 and thkpv-3)]. a very broad diversity of paramyxoviruses, including henipa-, rubula-, pneumo-and morbilli-related viruses, have been detected in six of ten tested bat families [11] . whereas most of the viruses identified in bats do not seem to cause clinical disease in these animals, there have been reports of rabid bats [12, 13] and of unusually large numbers of animals succumbing to infection by rabies virus [14] . as part of a large-scale investigation of viral diversity in bats and of associated zoonotic risks, we have previously detected a bat paramyxovirus in one insectivorous african sheath-tailed bat (coleura afra) [11] , exhibiting several hemorrhagic lesions at necropsy. we therefore examined occurrence of this bat paraymxovirus in other bats. the study was conducted in the belinga mountains (northeast gabon), where ebola outbreaks occurred in 1994-1996 and 2001-2002 . all the capture events, animal handling, euthanasia and transfer of samples across country borders were gabon. there are no patents, products in development, or marketed products to declare. this does not alter the authors' adherence to all the plos one policies on sharing data and materials, as detailed in the online guide for authors. performed in accordance with the guidelines of the american society of mammalogists (http://www.mammalsociety.org/committees/animal-care-anduse) [15] : bats were captured following recommendations by kunz and parsons [16] and identified by trained field biologist. captured bats were removed carefully from nets as soon as possible to minimize injury, drowning, strangulation, or stress. safe and humane euthanasia was achieved through the use of inhalant anaesthetic (halothane) prior to autopsy. all work (capture, euthanasia and autopsy) was carried out with authorization from the gabonese ministry of water and forestry (département de la samples of liver, spleen, kidney, lung, heart, gut, brain and salivary glands were collected, stored in liquid nitrogen and transferred to the cirmf laboratory (centre international de recherches médicales de franceville, gabon), where they were stored at 280˚c until analysis. blood samples were also collected, except from the smallest individuals (body mass ,12 g). a total of 985 bats (table 1) interestingly, at autopsy, only one c. afra out of the 26 individuals, from batouala cave, exhibited diarrhea and severe hemorrhagic lesions in both thoracic and abdominal organs, along with lung congestion and pleurisy. virological screening of the diseased bat was performed to identify the cause of these lesions. the screening process included the whole paramyxoviridae family and also filoviruses [marburg virus (marv) and zaire ebola virus (ebov)], given the nature of the lesions and the known filovirus tropism for bats. briefly, approximately 100 mg each of this animal's liver and spleen were pooled and crushed in 600 ml of cold pbs in a ball-mill tissue grinder (genogrinder 2000, spex centripep). total rna was extracted using a biorobot ez1 and the ez1 rna tissue mini kit (qiagen) according to the manufacturer's guidelines. the rna was then tested for paramyxoviruses, using three heminested reverse transcription-pcr (hnrt-pcr) assays targeting the polymerase gene [17] , and also for marburg virus [18] and zaire ebola virus [19] . however, the screening was extended to arenaviruses, flaviviruses, alphaviruses, polyomaviruses, orthopoxviruses, parapoxviruses, influenza viruses, lyssaviruses and rhabdoviridae family. to further investigate the presence of the virus in bat populations, a strain-specific real-time rt-pcr assay (primers: gb09-478-f, 59-ggcggctcttaaaagt-gaatg-39; gb09-478-r, 59-gcggggtcaaattggtcat-39; probe: gb09-478-p, 59-tccagcacaaacatatccgagaaggctag-39) was designed within the initial pcr fragment and was used to test total rna extracted from mixed liver and spleen samples from each of all the other bat species. the amplification was performed in a final volume of 25 ml, containing, 12.5 ml taqman r 2x pcr master mix (applied biosystems), 0.5 ml each primer and probe (10 mm), 1 ml bovine serum albumin (1 mg/ml) (invitrogen), 5 ml cdna and rnase-free water (invitrogen). amplification generally involved 2 min at 55˚c, 10 min at 95˚c followed by 45 cycles of 15s at 95˚c and 1 min at 58˚c. in order to determine the organ distribution of this virus in infected bats, total rna was extracted from heart, liver, spleen, kidney, lung, intestine and brain samples from all 14 real-time rt-pcr-positive bats, as described previously, and screened, using the same strain-specific real-time rt-pcr assay shown above. transmission by hematophagous arthropods was studied. hematophagous arthropods were collected inside faucon and zadié caves. mosquitoes were sampled using cdc light traps whereas bat-flies were manually collected on bats (coleura afra, miniopterus inflatus, hipposideros cf. ruber and rousettus aegyptiacus) trapped in the faucon and zadié caves between november and december 2009, june 2010, and january, march and april 2011. after the morphologic species determination, insects were crushed by monospecific pools (up to 10 specimens for mosquitoes, and between 1 and 5 for bat-flies) in 300 ml pbs. total rna was extracted using 100 ml of the supernatant from each pool by using the rneasy mini kit (qiagen) and then was tested with the specific realtime rt-pcr assay described above. the viral sequences obtained were first compared to those available in the public database using the algorithm "blastn" from blast program [20] and then aligned with homologous sequences of paramyxoviridae reference strains from genbank, using the mega program version 5 [21] . bayesian inference of phylogeny was done using mrbayes v.3.2 software and the gtr+g+i nucleotide substitution model [22] for two million generations with a burn-in of 25%. one of three hnrt-pcr, respiro-, morbilli-and henipaviruses pcr (rmh-pcr) assays yielded an amplicon of 559 base pairs (bp) from diseased bat, in collaborative work with the bonn institute of virology bonn (germany). the pcr product was sequenced with dye terminator chemistry (applied biosystems). no other virus was detected in the diseased bat. blast and phylogenetic analysis confirmed that the sequence from diseased bat, designated batpv gb09 478 (genbank accession number hq660155), belonged to the paramyxoviridae family. it showed 65% and 66% nucleotide identity, respectively, with jpv and beipv, and had pairwise nucleotide identities of 36-42% with the henipavirus group and 37-40% with the morbillivirus group. phylogenetic analysis indicated that this bat pv, that we named belinga bat virus (belpv), clustered with unclassified paramyxoviruses and was located between the genera morbillivirus and henipavirus (fig. 1) . in this study, among all animals tested by the strain-specific real-time rt-pcr assay only 14 c. afra bats, including the diseased animal, were positive. bat samples positive by specific real-time rt-pcr were then tested with rmh-hemi nested-pcr for molecular characterization. another 439-bp pcr product was amplified from one liver-spleen pool from a second c. afra specimen, and was sequenced. these two phylogenetically related sequences (fig. 1) , designated batpv gb09 478 and batpv gb09 450, displayed 100% of nucleotide identity. belpv-specific rna was detected only in c. afra, with a prevalence of 14.9% (14/ 94 of the c. afra individuals sampled from the faucon and batouala caves) ( table 1) . interestingly, no bats belonging to the other five species tested positive. belpv rna was detected in the heart (8/12) and liver (5/14) , at low and variable loads ( table 2 and fig. 2 ). no significant difference in the belpv rna detection rate was found between the two organs (x 2 52.476, df51, p.0.20). in the bat exhibiting severe hemorrhagic lesions (gb09 478), both the liver and heart were belpv-positive, with a higher viral load in the heart (ct value 528). in total, 432 arthropods were collected inside faucon and zadié caves, including culicidae (10 uranotaenia nigromaculata and 320 culex wigglesworthi), nycteribiidae (51 eucampsipoda africana, 26 nycteribia schmidlii scotti and 21 penicillidia fulvida) and streblidea (brachytarsina allaudi and 1 raymondia huberi huberi) ( table 1) . no arthropods tested were positive for belinga bat virus (table 1 ). the belpv nucleotide sequence obtained showed similarity with the jpv and beipv sequences. belpv has previously been reported to hold a phylogenetic position between the genera henipavirus and morbillivirus. the same phylogenetic position had been observed with mospv and j-v [4] . in this study, organs with high belpv concentrations are different from those found with high paramyxovirus concentrations in pteropodids and microchiroptera bats. in microchiroptera bats from brazil, spleen has been found more positive than the others organs with highest viral load, as in eidolon helvum (megachiroptera bat) in africa [11] . however, in our study majority of spleen were not available. the within-host belpv distribution tended to be organ-specific. belpv seemed to be restricted to the heart and liver. in contrast, jpv has been isolated from blood, lung, liver, kidney and spleen of experimentally infected laboratory mice [6] but not in heart. the belpv distribution for the heart and liver, together with the high viral load in heart tissue, could suggest that this virus is likely to be present in the bloodstream and might thus be transmitted during aggressive contacts between bats, or by blood-sucking vectors. nethertheless, viremia was not proven. belpv rna was not searched from blood because in these small species of bats blood was difficult to collect in the field. we detected belpv only in coleura afra and not in other bat species sharing the same roosting sites and living in very close proximity in the two caves sampled. however, it has been shown that bats of different species occupying the same roosting sites can share the same viruses. marburg virus had been detected in rousettus aegyptiacus and hipposideros sp. bats living in kitaka cave in uganda [23] and miniopterus inflatus and rousettus aegyptiacus bats caught in goroumbwa mine in the democratic republic of the congo [24] . these bat species are known to live in close proximity. thus, virus transmission between different bat species is possible [25] . thus, we can speculate that the failure to detect belpv in other bat species sharing the same caves would suggest that this virus has strong host specificity for c. afra, as well as restricted intraspecies transmission. henipaviruses occur naturally in fruit bats belonging to the genus pteropus [26] , and this also appears to be true of severe acute respiratory syndrome-like coronaviruses in rhinolophus bats [27, 11] . in view of our data we can assume that belpv might have pathogenic potential for its host c. afra. indeed, high viral load was detected in the heart of the diseased bat, and the lesions were consistent with those reported in wild rodents and mice experimentally infected by jpv [6] . although belpv rna was also detected in asymptomatic bats, pathogenicity may appear in long term under some immunological and/or ecological conditions. indeed, virus must not induce pathology to persist or adapt within its reservoir host. many authors suggested that persistence in the absence of pathology or disease appears to be a common characteristic of bat viruses in their natural host population [28, 29] . however, a severe immunodepression for instance, may increase the risk of infection with opportunistic pathogens. under some environmental conditions (cool environments for example), some avirulent pathogens, such as geomyces destructans, causative agent of white-nose syndrome, may become pathogenic in hibernating bats in north america [30, 31] . nevertheless, infection by belpv may be mild for bats and thus the pathology observed not directly related. otherwise, it may also be that this animal had an underlying disease or infection with a different pathogen. even in this case, we might not draw any conclusions neither establish a link with lesions seen. therefore, the pathogenicity of the belpv should be demonstrated by experimental animal infection. otherwise, viral antigens or rnas should be detected histologically in the lesions of naturally-infected bats. however, the unavailability of biological tissues from the diseased bat failed to perform these analyzes. consequently, other captures of coleura afra species are considered in order to find belpv again for further studies (pathogenicity to its host, isolation and complete genome characterization). however, coleura afra is a migratory species living in colonies of several hundred individuals. in gabon, this species, which has been recently described, is not present all year round in the caves of the north-east of the country, making the studies on this species difficult and thereof partly explaining the lack of virological studies. some viruses appear to cause clinical disease in wild-living bats; these include lyssaviruses and an ebola-like filovirus named lloviu virus [32, 33, 34] . bats are the natural reservoirs for many viruses, including emerging zoonotic viruses such as sars-cov [27] , hendra and nipah viruses [26, 35] , ebola virus [36] , marburg virus [23, 37] , rabies virus and other lyssaviruses [11] . in general, humans are infected through an intermediate amplifying host such as palm civets for sars-cov, horses for hendra virus and pigs for nipah virus [38] . however, in humans nipah virus outbreaks linked to bats exposure have been reported [39] . it remains to be shown whether the belpv reported here presents a zoonotic risk. nonetheless, like most rna viruses, for example coronaviruses, characterized by high mutation and/or recombination rates [40] , pvs may adapt to novel hosts, including humans. a serological test capable of detecting antibodies to this virus in human populations living in the vicinity of these animals is needed to assess zoonotic potential. all the blood-sucking arthropods collected from bats, as well as mosquitoes collected in the caves where bat sampling took place, were negative for belpv, in keeping with the lack of known pv vectors [41] . however, belpv transmission by blood-sucking vectors within the gabonese population of c. afra cannot be ruled out. indeed, a haemosporidian parasite (polychromophilus) was found in a blood parasite vector (penicillidia fulvida) in faucon cave in gabon in 1977 [42] and also in its host m. inflatus (greater long-fingered bat) from the same cave in 2010 and 2011 [43] . in addition, the methodology used to collect flying hematophagous insects (based on light traps) possibly introduced a bias by selecting only those attracted by light. therefore, we can not exclude that additional sampling techniques could increase the number of mosquitoes species or groups known to colonize caves such as sandflies or biting midges. hence, the natural mode of transmission of this unclassified paramyxovirus in bat populations, through batbat aggression for example, remains to be determined. this association between c. afra and belpv could serve as an interesting model, (i) to evaluate modes of transmission within host populations, (ii) to study hostvirus interactions (pathogenesis and host specificity), and (iii) to evaluate the zoonotic risk of a newly identified virus. further studies of c. afra populations and a broader diversity of arthropod vectors, spanning larger areas and time scales, are needed to confirm this apparent host-virus specificity, and to determine the modes of belpv transmission. further studies are needed to characterize complete belpv genome and demonstrate the pathogenicity of this virus for its host coleura afra. conceived and designed the experiments: gdm mb eml. performed the experiments: gdm jon nn. analyzed the data: gdm jfd. contributed reagents/ materials/analysis tools: gdm mb cd cp jfd eml. wrote the paper: gdm cp eml sm. paramyxoviridae: the viruses and their replication nariva virus, a hitherto undescribed agent isolated from the trinidadian rat, zygodontomys b. brevicauda (j.a. allen and chapman) mossman virus, a paramyxovirus of rodents isolated in queensland beilong virus, a novel paramyxovirus with the largest genome of non-segmented negative-stranded rna viruses studies on a virus isolated from wild mice (mus musculus) a new mouse paramyxovirus (j virus) isolation and molecular characterization of a novel cytopathogenic paramyxovirus from tree shrews complete genome 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bayesian inference of phylogenetic trees isolation of genetically diverse marburg viruses from egyptian fruit bats studies of reservoir hosts for marburg virus genomic characterizations of bat coronaviruses (1a, 1b and hku8) and evidence for co-infections in miniopterus bats isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus bats are natural reservoirs of sars-like coronaviruses bats: important reservoir hosts of emerging viruses novel astroviruses in insectivorous bats bat white-nose syndrome: an emerging fungal pathogen? inoculation of bats with european geomyces destructans supports the novel pathogen hypothesis for the origin of white-nose syndrome pathogenesis studies with australian bat lyssavirus in grey-headed flying foxes (pteropus poliocephalus) bats and lyssaviruses discovery of an ebolavirus-like filovirus in europe nipah virus infection in bats (order chiroptera) in peninsular malaysia fruit bats as reservoirs of ebola virus is marburg virus enzootic in gabon? bats as a continuing source of emerging infections in humans risk factor for nipah virus encephalitis in bangladesh severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats paramyxovirus and pneumovirus diseases of animals and birds: comparatives aspects and diagnosis dionisia bunoi n. g. n. sp., haemoproteidae parasite du microchiroptè re hipposideros cyclops au gabon the chiropteran haemosporidian polychromophilus melanipherus: a worldwide species complex restricted to the family miniopteridae we thank all the persons involved in sample collections, and especially andré délicat, peggy motsch, dieudonné nkogué, tabea binger, peter vallo, and nil rahola. we acknowledge heïdi lançon for improving the english. cirmf is supported by the government of gabon, total-fina-elf gabon, and ministère de la coopération française. key: cord-323330-ghwhgkdm authors: ekundayo, temitope cyrus; okoh, anthony i. title: a global bibliometric analysis of plesiomonas-related research (1990 – 2017) date: 2018-11-29 journal: plos one doi: 10.1371/journal.pone.0207655 sha: doc_id: 323330 cord_uid: ghwhgkdm plesiomonas shigelloides is an emerging pathogen with damaging effects on human health such as gastroenteritis and extraintestinal infections. here, we carried out a bibliometric survey that aimed to examine publication trends in plesiomonas-related research by time and place, international collaborative works, identify gaps and suggest directions for future research. the search term “plesiomonas shigelloides” was used to retrieve articles published between 1990 and 2017 from the web of science database. only primary research articles were included in the analysis. a total of 155 articles were published within the survey period, with an average of 5.54±2.66 articles per year and an annual growth rate of −0.8%. research output peaked in 2000 and 2006 (each accounting for 7.7% of the total). the united states ranked first in terms of numbers of articles (n = 29, 18.1%) and total citations (n = 451). cameroon, canada, cuba, switzerland and turkey co-shared the 10(th) position each with 2 articles (1.3%). research collaboration was low (collaboration index = 3. 32). in addition to plesiomonas shigelloides (n = 82, 52.9%), the top authors keywords and research focus included lipopolysaccharide and nuclear magnetic resonance (n = 13, 8.4%). diarrhea (n = 43, 27.7%), aeromonas species (n = 41, 26.5%) and infections (n = 31, 20.0%) were also highly represented in keywords-plus. authors’ collaborations and coupling networks formed two mega-clusters which nodes were shared solely by authors from high-income countries. the common conceptual framework in retrieved articles determined by k-means clustering revealed three clusters with sizes of 7, 16, and 29, representing research responses focused on extraintestinal and gastroenteritis, p. shigelloides lipopolysaccharide structure, and co-infections, respectively. our bibliometric analysis revealed a global diminishing research in plesiomonas; greater research outcomes from high-income countries compared to others and low collaboration with developing countries. plesiomonas shigelloides is a bacterium that has been labeled as an emerging pathogen for over three decades. there are many outstanding questions regarding its pathogenic potential, a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 despite evidence for its detrimental effects on human health such as gastroenteritis and extraintestinal diseases [1] [2] [3] [4] [5] [6] [7] . also, some food and waterborne outbreaks have been traced to p. shigelloides [8] [9] [10] [11] [12] [13] , and the incidence of plesiomonas infections linked to immunocompromised health [14] is increasing, especially in light of present-day lifestyles [15] . climate change and global warming are also predicted to contribute to increased incidence of waterborne infectious diseases including plesiomonas infections [16] [17] [18] [19] . accurate estimates of the incidence of plesiomonas-related gastroenteritis and extraintestinal infections both globally and at the level of individual countries remain unknown [1, 2, [4] [5] [6] [7] 10, [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] . retrospective reviews of infections due to p. shigelloides in china and hong kong have been published [38] , and cases of p. shigelloides co-infection with viral and bacterial diarrheal pathogens are common in the literature [39] . prevalence of p. shigelloides gastroenteritis varies considerably across regions, with lower rates reported from north america and europe and higher estimates from southeast asia and africa [40] . nonetheless, there is a general underestimation of p. shigelloides infection, in part because it shares some clinical manifestations with other pathogens [38] . p. shigelloides is not routinely examined in clinical settings, and as such, awareness regarding this pathogen remains limited. bibliometric analysis is a statistical method for assessing both the quantitative and qualitative scope and adequacy of research efforts attained in an area of interest [41] . it can be used to determine national and international research focus and evaluate research performance in order to identify future research priorities, funding sources, and interdisciplinary collaborations [42] [43] [44] . it also provides a resource to policy-makers for implementing necessary prophylactic measures in case the analysis reveals a sharp increase in case reports or articles regarding a health issue in a particular geographic area [45, 46] . bibliometric reviews can additionally help international health agencies to identify priorities (e.g. by nations) for disbursing aid [47] and awarding research grants. there have been a few recent reviews on p. shigelloides [2, 14, 48] but no comprehensive surveys of published studies on p. shigelloides have been yet conducted. on the other hand, bibliometric analyses have been applied to global disease research on viral agents such as dengue virus [49] , ebola virus [50] , john cunningham virus [51] , mayaro virus [52] , middle east respiratory syndrome coronavirus [53] [34] , yellow fever virus [54] , west nile virus [55] , and zika virus [56] ; and bacterial agents such as campylobacter [45] , leishmania species [57] , and mycobacterium tuberculosis [58] . other bibliometric analyses have addressed plasmodium species and resistant malaria vectors [59, 60] , toxocara species [43] , and antifungal triazole resistance (especially in candida and aspergillus species) [46] . here we carried out a bibliometric analysis of studies on p. shigelloides published between 1990 to 2017. the articles were evaluated in terms of annual and country-specific output, theme, domain clusters, international collaboration networks, citations, topical evolution related to keywords and co-occurrence networks, co-authorship, and funding. the aim of the survey was to evaluate international participation in p. shigelloides research-with a special interest in regions where plesiomonas infections have higher prevalence rates (i.e., africa and southeast asia)-in order to address knowledge gaps and provide a resource that can help identify present and future research priorities. publication dataset (e.g.; authors, words, countries, references, keywords) for coupling, cocitation, collaboration, conceptual framework and multiple correspondence analyses. bibliographic coupling occurs between two articles ⅈ and ⅉ when their reference lists cited at least one common source [62] . but, in a collaboration network, the nodes comprise authors and the links co-authorships [63] . the number of bibliographic coupling that occurs between articles ⅈ and ⅉ or co-authorship in scientific collaboration network denotes the strength of the network [61] . a network depicts relationships in a system as a set of nodes (components) and links (relationships) [64] . co-word or conceptual framework analysis explore k-means clustering and other dimensionality reduction techniques to identify clusters of common concepts known in a bibliographic collection. it relies on word co-occurrences in a publication dataset [61, 64] . scientific productivity or an author's contributions in a field is evaluated in term of lotka's law [65] . the lotka's law is an inverse square law that describes how often authors published in a field [65] . published peer-reviewed articles on p. shigelloides were retrieved from the web of science (wos) database on august 19, 2018. the wos is among the most reliable and comprehensive databases for bibliometric studies and hosts a wide range of quality and high-impact scientific studies (12 million articles in over 12,000 journals) [44] . we used the search term "plesiomonas shigelloides" to identify primary research articles published between 1990 and 2017. all available information was retrieved. to obtain subject-specific results and for the sake of accuracy (in order to avoid false-positive results), only article titles were searched. a title-specific search has been reported to increase recovery and specificity with a minimal loss of sensitivity compared to a topic search [44, 45, 66] . articles were downloaded in the bibtex file format. in order to account for variations in country population on rate of scientific production, the world population was retrieved from the world bank website (https://data.worldbank.org/indicator/sp.pop.totl) and the midperiod population corresponding to the top 19 countries was extracted for calculation of article per million populations. england population data was retrieved from office for national statistics website (https://www.ons.gov.uk/peoplepopulationandcommunity/populationandmigration/ populationestimates/timeseries/enpop/pop). we analyzed the retrieved data for bibliometric indicators using rstudio v.3.4.1 software (2017-06-30) with bibliometrix r-package (http://www.bibliometrix.org) [61] . data were imported into rstudio and converted to a bibliographic data frame and normalized for duplicate marching. the duplicated article was reduced to one record in the analysis. the data frame as typical columns named after the standard isi wos field tag codify. further, authors' names, authors' keywords (de), and keywords-plus (id) were extracted for standardization. authors' names were extracted twice as two different sets (a and b). each set was checked for variant names, spelling errors and matched with affiliations. we achieved normalized authors' names when |a \ b| � |a [ b|. for keywords (de) and keywords-plus (id), a primary term was assigned to words with similar meanings (e.g., "plesiomonas shigelloides", "plesiomonas", "shigelloides", "aeromonas-shigelloides", and "plesiomonas-shigelloides" were allotted to "plesiomonas shigelloides"). multiple occurrences of a keyword or a similar keyword in an article were regarded as one. co-occurrence of a term in authors' keywords (de set) and keywords-plus (id set) in the dataset was assessed as a set made of the intersect of the two sets (de \ id||). all set-based test was performed using a venn diagram software (http://bioinformatics.psb.ugent.be/webtools/venn/). an annual number of articles and total citations were also graphed. data were analysed for descriptive output, citation analysis, authors' h-index and scientific productivity using the relevant functions of the bibliometrix r-package. bibliometric networks (e.g., citation, author, country, author keyword, and keywords-plus networks) and bibliographic coupling (co-citation and keyword co-occurrences) were computed and visualized from bibliometric two-way (bipartite) network of rectangular matrices of articles × attributes. a typical bibliometric network is expressed as network(n) = x × n t where x is a bipartite network matrix composed of articles × attribute (e.g. authors, keywords, citations, and countries) and n is a symmetrical matrix n = n t . we created a graphic model of all networks using force-directed algorithms (fruchterman) implemented in the networkplot function of the bibliometrix r-package. all networks were standardized using the simpson's coefficient (inclusion index), proximity index (association strength), the jaccard's similarity index, and the salton's cosine coefficient among nodes of a network [61] . in addition, k-means clustering were performed on keywords to evaluate concepts in plesiomonas field of research using the function conceptualstructure of the package. the function implements porter's stemming algorithm [67] to modulate inflected words to their root form. for detailed search boolean for articles identification from wos, see supplementary file (s1 appendix). other bibliometric indicators such as language and affiliation were determined using the content search of bibtex file. a total of 155 articles were published within the survey period; their attributes are presented in table 1 . the studies involved 493 authors, with 0.31 article/author (3.18 authors/article), 4.34 co-authors/article, and a collaboration index of 3.32. with the exception of two authors publishing solo, all 491 authors were involved in multi-author articles. an average of 11.49 citations/article was recorded during the study period. the scientific output related to p. shigelloides research by lotka's law showed a beta coefficient and constant of 2.30 and 0.44, table 2 shows the top 20 most productive authors in the field. k. krovacek (sweden) ranked first, co-authoring 12 (7.7%) articles; and i. ciznar (slovak republic) was second with 11 (7.1%) articles. the h_index (total citations) was 7 (162) for k. krovacek, and 6 (148) for i. ciznar. it is worth noting that the topmost active authors were affiliated with institutions in developed nations, including sweden (n = 6), usa (n = 5), japan (n = 3), spain (n = 5), poland (n = 3), czech republic (n = 1), and slovak republic (n = 1). the 20 top cited articles on p. shigelloides are listed in s1 table. these studies spanned the fields of infection, immunity, clinical microbiology, and biochemistry. the total no. of citations of the top-cited articles ranged from 35 to 111; most of these were the result of funded research. research output related to p. shigelloides for the top 20 most active countries is shown in table 3 . united states ranked first in terms of total number of articles (n = 29, 18.7%) and citations (n = 451), followed by sweden (n = 14, 9.0%) and germany (n = 10, 6.5%). the frequency of publication varied among the top countries from 1.3 to 18.7%. sweden had highest productivity (1.563 article/million population) when normalized for population size using mid-period population (2003) . the rank order of these countries changed when productivity was meathe top 20 journals with the most published articles on p. shigelloides are listed in s2 table. these journals cover a range of subjects including carbohydrates, microbiology, food science, infectious disease, immunology, and biochemistry, reflecting active areas in plesiomonas research. carbohydrate research ranked first (n = 9, 5.8%), followed by journal of clinical microbiology, folia microbiologica and food biotechnology each with 6 articles (3.9%). table 4 shows the most relevant keywords related to plesiomonas studies, including both author keywords (de) and keywords-plus (id). both author keywords (de) and keywords-plus (id) have 10 keywords in common (lipopolysaccharide, aeromonas species, antigens, oligosaccharide, disease, children, diarrhea, shigella, virulence, and water). fourteen keywords were unique to author keywords (plesiomonas shigelloides, nuclear magnetic resonance ranking based on the number of articles; tc, total citations. (nmr), pcr, structure, maldi-tof, fish, pathogenicity, media, meningoencephalitis, resistance, enterotoxin, gastroenteritis, serotyping, and sepsis), and 13 keywords were unique to keywords-plus (infections, escherichia, septicemia, environments, in-vitro, polysaccharide, vibrio species, bacteria, biological repeating unit, iron, meningitis, humans, and strains). the unique author keywords primarily described medium of transmission (fish) and methods involved in isolation and characterization of the microorganism (nmr, maldi-tof, pcr, enterotoxin, resistance, pathogenicity, serotyping, and structure) and specific infections (meningoencephalitis, gastroenteritis, and sepsis). author keyword terms associated with identification methods of p. shigelloides included polymerase chain reaction (pcr, n = 8, septicemia (n = 24, 15.5% (id)), meningoencephalitis (n = 4, 2.6 (de)), sepsis (n = 3, 1.9% (de)), and meningitis (n = 10, 6.5% (id)), which ranked 5th, 11th, and 11th respectively. the common conceptual frames in retrieved articles determined by k-means clustering with three clusters of 8, 14, and 39 elements showed research responses focused on neonates (children) extraintestinal infections and gastroenteritis, elucidation of cell wall structure (lipopolysaccharides, core oligosaccharide, o-specific polysaccharide etc.), and co-infections of p. shigelloides with other pathogens, respectively (fig 2) . the 33-element cluster explained the co-occurrence and co-infection of p. shigelloides with other bacteria. other indicators of frequently represented concepts and frameworks related to p. shigelloides included co-occurrence of terms and keywords. s1 fig shows the co-occurrence network of the top 20 terms associated with p. shigelloides studies, while s2 fig shows the co-occurrence networks of keywords. these concept-related frameworks or terms included virulence, meningoencephalitis, aeromonas, newborn, antigen, pathogenicity, sepsis, diarrhea, infections, escherichia coli, septicemia, biological repeating unit, diarrheal disease/gastroenteritis, lipopolysaccharide, water, iron, polysaccharide, bacteremia, meningitis, septicemia, lipid-a, strains, and aquatic environments. the top 20 authors' collaboration and coupling networks on p. shigelloides studies were divided into two mega-clusters or spheres with nodes occupied solely by researchers from high-income countries (fig 3a and 3b ). the first sphere of the authors' network comprised 13 nodes (authors) with no fewer than 10 linkages, while the second sphere included 10 nodes (authors) with the number of collaboration linkages ranging from nine to 10. similarly, the two separate authors' coupling network spheres included 13 and 17 authors' networks, respectively; collaborative conjugation ranged from eleven nodes (authors) in the former and 18 in the latter. fig 4 shows 50 countries' collaboration networks on p. shigelloides studies. collaboration pathways ranged from 1 to 9. the sweden had a high number of collaborations (n = 9), followed by united states (n = 8), slovakia (n = 8), and italy (n = 7). other countries had no collaboration networks. prominent network color code: green, usa network; purple, spain network; light green, sweden network; pink, cuba-brazil network; blue, japan-china network. the present bibliometric analysis of p. shigelloides examined global research trends between 1990 and 2017 based on data retrieved from wos. we found that the number of research articles on p. shigelloides increased non-linearly from 5 to 155 articles. however, a negative trend in rate of increase was noted (−0.8%) suggesting that research on p. shigelloides has not been of broad interest in the past 27 years, likely due to discontinuation of plesiomonas-related research by certain authors and differences in regional distribution of the microorganism. furthermore, the function estimates and goodness-of-fit indicated that scientific output on p. shigelloides does not follow lotka's law, suggesting that the number of articles related to plesiomonas research will further decline in the future. in general, emerging and re-emerging bacterial pathogens are not accorded the same degree of attention as their viral counterparts. health emergencies (e.g., outbreaks of infection) relating to emerging viral pathogens including zika and chikungunya viruses have driven the generation of new scientific knowledge, resulting in a significant increase in the number of research articles on these subjects [68] . for instance, in 2005 only eight articles were published on chikungunya virus, but by 2014, the number had reached 302 [69] . similarly, only 43 articles on ebola virus were published in 2013 prior to the ebola outbreaks in west africa, but this increased to more than 600 articles in 2014 [70] . as is the case with other research areas, most of the leading authors in the plesiomonas research field were from developed nations such as the united states, sweden, austria, japan, spain, poland, czech republic, and the slovak republic, with few from low-income countries, thus follow the similar trend of low productivity of the region in other research areas. it has been suggested that the economic strength (growth) of a nation influences the research output [43, [71] [72] [73] . the higher prevalence of plesiomonas infections in developing countries [74] should motivate researchers in countries most affected to carry out more studies on this pathogen. some of the most frequently cited studies pertained to health and identification of the pathogen-for example, efforts to produce a multivalent vaccine using o-specific polysaccharides from shigella spp. and p. shigelloides; iron uptake in plesiomonas shigelloides; and comparative analysis of p. shigelloides and e. coli (shigella) sonnei o-antigen gene clusters. others focused on p. shigelloides hemolytic expression; serological analysis of the plesiomonas core; and multilocus sequence typing of plesiomonas and its pathogenic potential. the united states and sweden dominated the list of top 20 countries most actively researching plesiomonas in terms of numbers of articles and citations. in addition to economic strength and availability of research facilities and funding [43, [71] [72] [73] , this productivity can be ascribed to a high level of intranational and possibly multinational collaboration with other institutions, which can impact research visibility and citation frequency [43, 73, 75] . in particular, the dominance of the united states has been noted in other fields of research [42, 54, 76, 77] . also, authors' multiple affiliations influence country collaboration network. conversely, the low contributions from developing countries including countries from africa characterized by a high frequency of selffunded or independent studies [78] , mirror the situation of research in other fields. the shift in rank among the top 20 nations most active in the plesiomonas research field when productivity was measured based on total citation per country ought not be regarded as a precise measure of productivity. citation rate does not reflect publication output of an author or country [76] , since the smaller the number of articles used for estimation, the larger the impact of a few frequently cited articles [76] . self-citations and inaccurate citations can also provide false quality metrics [76] . the most frequently mentioned keywords and research areas (including publication outlets) associated with plesiomonas studies reflect the research hotspot during the survey period, which included cell wall (carbohydrate research), co-infection, and extraintestinal infections such as septicemia, bacteremia, and meningitis. these findings reveal the most pressing health issues related to plesiomonas-induced gastroenteritis and extraintestinal infections, and coinfections with other pathogens and effort to gain an understanding of the structural architect of the pathogen's cellwall; this was supported by other conceptual framework indicators such as co-words or keyword co-occurrence networks. however, important topics such as strainbased delineation and identification, including detection of pathogenic and non-pathogenic strains, that are necessary for infection management were lacking and were not evident from the bibliometric analyses. newer research themes such as molecular and genomic-level studies as an alternative or complementary to traditional experimentation (which has some limitations) necessary to clarify the pathogenesis of plesiomonas infection were not apparent throughout this study. a bibliometric survey complemented with a narrative review or metaanalysis may be beneficial in plesiomonas research. future research is needed to answer questions related to what particular strain of the microorganisms are pathogenic and how to differentiate pathogenic variants from non-pathogenic ones. the two mega-clusters or spheres in the top authors' collaboration and coupling networks on p. shigelloides studies showed collaboration pathways mainly among authors from highincome countries, which is similar to trends observed in collaboration network analyses of human immunodeficiency virus and human papilloma virus studies [78] . alliances between developing and developed countries are rare in a number of scientific areas [78] . among researchers in the united states, collaboration pathways were largely intra-national, as suggested by a large number of publications but 6 multiple country publications. in contrast, collaborations by authors in cuba, finland, czech republic, italy, slovakia, and sweden tended to be multi-national, which is more valuable for the epidemiological control of pathogens. the absence of collaboration pathway in venezuela, india, nigeria, and other african nations is consistent with the low number of publications from these countries. intra-and international collaborations between developed and developing nations could provide opportunities for the division of labor and resources to address important scientific questions. there were some limitations related to the bibliometric survey adopted in this study, including the use of a single database (the wos), the low sensitivity and strictness of the search terms and search strategy used, and the exclusions of other document types (e.g., meeting abstract, note and proceeding papers etc.) and articles published in non-english language (e.g in chinese). also, the current analysis did not allow for a narrative review and judgment on contents and results of the articles [79] . as noted earlier, emerging themes and recent research focus in plesiomonas are not easily recognized in bibliometric studies due to low frequency of appearance in keywords. notwithstanding these limitations, this is the first bibliometric study on plesiomonas-related research contributing to the evidence base and would help direct future research. also, the wos has a larger coverage compared to other database, reliable indexing technology that minimizes the ''indexer effect" and is well accepted among scientific communities [80] . our bibliometric analysis revealed a global diminishing research in plesiomonas, greater research output from high-income countries compared to low-and middle-income countries and limited collaboration with developing countries. the low productivity in developing countries in plesiomonas research mirror the state of affairs in other research fields. a better understanding of the clinical features, epidemiology and plesiomonas-associated diseases is needed in countries with high infection rates. emerging themes and recent research focus in plesiomonas research are not easily recognized in bibliometric studies due to low frequency of appearance in keywords and, hence, the need for future studies guided by narrative reviews. supporting information s1 fig. co-occurrence network of top 20 terms associated with p. shigelloides studies. each node in the network represents a different term. the node's diameter corresponds to the frequency of co-occurrence with other terms. lines depict co-occurrence pathways between terms. (tif) s2 fig. keyword co-occurrence networks related to p. shigelloides studies. each node in the network represents one of the top 20 keywords. the node's diameter corresponds to the keyword's frequency of co-occurrence with other keywords. lines depict co-occurrence pathways between keywords. (tif) s1 treating spontaneous and induced septic abortions plesiomonas shigelloides periprosthetic knee infection after consumption of raw oysters isolation, genome sequencing and functional analysis of two t7-like coliphages of avian pathogenic escherichia coli wound infection with plesiomonas shigelloides following a freshwater injury susceptibility of bacteria isolated from acute gastrointestinal infections to rifaximin and other antimicrobial agents in mexico plesiomonas shigelloides: an extremely rare cause of spontaneous bacterial peritonitis plesiomonas shigelloides meningitis in an adult in the ed diarrheal disease caused by plesiomonas shigelloides oyster-associated out-break of diarrhoeal disease possibly caused by plesiomonas shigelloides. the lancet occurrence of plesiomonas shigelloides in surface water: relationship with faecal pollution and trophic state an acute foodborne outbreak due to plesiomonas shigelloides in yaounde, cameroon plesiomonas shigelloides and salmonella serotype hartford infections associated with a contaminated water supply-livingston county plesiomonas shigelloides revisited listeriosis: a resurgent foodborne infection. clinical microbiology and infection impact of regional climate change on human health the ecology of climate change and infectious diseases climate change and infectious diseases: from evidence to a predictive framework non-cholera vibrios: the microbial barometer of climate change fatal plesiomonas shigelloides septicaemia in a splenectomised patient cellulitis and compartment syndrome due to plesiomonas shigelloides: a case report spontaneous bacterial peritonitis due to plesiomonas shigelloides a case of plesiomonas shigelloides cellulitis and bacteraemia from northern europe plesiomonas shigelloides infection in hong kong: retrospective study of 167 laboratory-confirmed cases plesiomonas shigelloides sepsis and splenic abscess in an adolescent with sickle-cell disease not always a sexual transmitted 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biodivers conserv plesiomonas shigelloides infection in southeast china bibliometric analysis on global parkinson's disease research trends during 1991-2006 influenza: a scientometric and density-equalizing analysis world-wide architecture of osteoporosis research: density-equalizing mapping studies and gender analysis international scientific collaboration in hiv and hpv: a network analysis policy and the mapping of scientific change: a co-word analysis of research into environmental acidification knowledge discovery through co-word analysis the authors thank the south africa medical research council (samrc) and the national research foundation, the world academy of science (nrf-twas) for financial support. conclusions arrived at and opinions expressed in this article are those of the authors and are not necessarily to be attributed to samrc or nrf-twas. key: cord-327534-f2wvh6la authors: zhou, peng; cowled, chris; mansell, ashley; monaghan, paul; green, diane; wu, lijun; shi, zhengli; wang, lin-fa; baker, michelle l. title: irf7 in the australian black flying fox, pteropus alecto: evidence for a unique expression pattern and functional conservation date: 2014-08-06 journal: plos one doi: 10.1371/journal.pone.0103875 sha: doc_id: 327534 cord_uid: f2wvh6la as the only flying mammal, bats harbor a number of emerging and re-emerging viruses, many of which cause severe diseases in humans and other mammals yet result in no clinical symptoms in bats. as the master regulator of the interferon (ifn)-dependent immune response, ifn regulatory factor 7 (irf7) plays a central role in innate antiviral immunity. to explore the role of bat irf7 in the regulation of the ifn response, we performed sequence and functional analysis of irf7 from the pteropid bat, pteropus alecto. our results demonstrate that bat irf7 retains the ability to bind to myd88 and activate the ifn response despite unique changes in the myd88 binding domain. we also demonstrate that bat irf7 has a unique expression pattern across both immune and non-immune related tissues and is inducible by double-strand rna. the broad tissue distribution of irf7 may provide bats with an enhanced ability to rapidly activate the ifn response in a wider range of tissues compared to other mammals. the importance of irf7 in antiviral activity against the bat reovirus, pulau virus was confirmed by sirna knockdown of irf7 in bat cells resulting in enhanced viral replication. our results highlight the importance of irf7 in innate antiviral immunity in bats. bats have been implicated in the spillover of many deadly viruses including rabies, henipaviruses (hendra and nipah), ebola virus, and the coronaviruses (cov): severe acute respiratory syndrome (sars-cov) and the recently emerged middle eastern respiratory syndrome virus (mers-cov), all of which impose a significant threat to human health [1, 2, 3, 4, 5, 6] . as natural hosts, bats rarely show clinical signs of disease during infection [7] . how bats co-exist with viruses and the role of the bat innate immune system in controlling viral replication remain poorly understood [8] . identifying the mechanisms responsible for controlling viral replication in bats has profound implications for the development of therapeutic strategies targeting viral infections in humans and other species. one of the most important early anti-viral defenses in mammals is the ifn system, which not only provides pivotal protection immediately following infection but also shapes the adaptive immune response [9] . of the three ifn families discovered, type i (including a and b) and type iii (l) ifns respond directly to viral infection. due to the importance of ifns in controlling viral replication, the regulation of the ifn response has been extensively studied in humans and other mammals. key to the regulation of ifn production and signaling is the ifn regulatory factor (irf) transcription factor family. the irf family consists of nine members which share functional and structural characteristics. however, only irf1, irf3, irf5 and irf7 have been implicated as positive regulators of type i ifn transcription, and only irf3 and irf7 are designated as antiviral irfs [10, 11] . since their first discovery within the biological context of epstein-barr virus latency, irf7 was identified as the master regulator of the type i ifn-dependent immune response, and perhaps that of type iii ifn as well [12, 13, 14] . irf7 is expressed only at low levels in most cells but is constitutively expressed in certain immune cells such as plasmacytoid dendritic cells (pdc) which specialize in ifn production. correspondingly, the tissue distribution of human irf7 is restricted to immune tissues which contain large numbers of specialized immune cells including spleen, thymus and peripheral blood lymphocytes whereas non-immune tissues including the intestine and colon express almost undetectable levels of irf7 [12] . although irf7 is expressed at low levels in most cell types, it is induced strongly by type i ifn mediated signaling in all cells [15] . interestingly, multiple fish species (japanese flounder, crucian carp, mandarin fish, snakehead fish and atlantic salmon) have been demonstrated to express irf7 constitutively in all tissues including both immune and non-immune tissues [16, 17, 18, 19, 20] . viral sensing either by toll like receptors (tlrs) or retinoic acid-inducible gene 1 (rig-i)-like receptors can result in the activation of irf7 and subsequent induction of ifns [21, 22] . all tlrs with the exception of tlr3 activate irf7 through the adaptor protein, myd88 (myeloid differentiation primary response gene 88) through the myd88-dependent pathway. myd88 forms a complex with the kinases irak-4 (interleukin 1 receptor associated kinase 4), irak-1 and traf-6 (tnf receptor associated factor). this complex binds directly to irf7 leading to ubiquitination by traf-6 and phosphorylation by irak1 or ikk-1 (ikb kinase-1) and translocation from the cytosol to the nucleus where irf7 binds to promoter elements inducing ifn production [21, 23] . tlr3 and tlr4 activate irf7 through the myd88-independent pathway through the adaptor molecule trif (tir-domain-containing adapter-inducing ifn-b) which forms a complex with tbk1 (tank binding kinase 1), ikk-e (inhibitor of nuclear factor-kb kinase e) and irf7. in this case the phosphorylated irf7 forms a homodimer or heterodimer with irf3 and translocates to the nucleus where it binds to the ifn promoter via its dna-binding domain to induce type i or type iii ifn [24] . in irf7 knockout mice, viral induced ifn production through the tlr3 (myd88-independent) pathway is greatly impaired. as a result, mice become more susceptible to viral infection [13] . although irf3 has been reported to preferentially activate ifn-b over ifn-a genes, irf7 is believed to efficiently activate both ifn-a and ifn-b [25] . the human ifn-b promoter region contains four positive regulatory domains (prds 1 to iv) that serve as binding sites for irfs. in the human ifn-a promoter region there may be two or three prd modules depending on the ifn-a subtype [26] . due to the importance of irf7 in the innate immune response, it is an active target for viruses to evade the host immune response [27] . a role for irf7 in immunosurveillance has also been identified in breast cancer [28] . using the australian black flying fox (p. alecto) as a model species we have begun to explore the role of the ifn system in the control of viral replication in bats. we have demonstrated that tlrs, rig-ilike receptors, and some ifn stimulated genes (pkr, mx1 and oas1) appear to be conserved in sequence compared to other mammals [29, 30, 31] . however, bats appear to have relatively higher expression of type iii ifn and wider distribution of type iii ifn receptors consistent with a role for type iii ifns in antiviral immunity [32, 33] . bat genome analysis has also provided evidence for positive selection of genes within the ifn pathway, including tlr7, c-rel, tbk-1, ifn-c, isg15 and rig-i [34] . these changes may have occurred in response to the co-evolution of bats with viruses and may have consequences for the clearance of viral infections and the ability of bats to coexist with viruses. due to the central role of irf7 in the regulation of the ifn response, we performed sequence and functional analysis of p. alecto irf7. our results provide the first description of irf7 in any species of bat and evidence for conserved irf7 functional activity despite variation at the sequence level in the bat irf7 gene. all animal experiments were approved by the australian animal health laboratory (aahl) animal ethics committee (protocol number 1389). immortalized and cloned p. alecto kidney (pakit03) and lung (palut02) cells established previously [35] were cultured in dmem/f12-hams (sigma), supplemented with 10% foetal calf serum (fcs, hyclone), 100 units/ml penicillin, 100 mg/ml streptomycin and 50 mg/ml gentamycin (sigma). human embryonic kidney hek293t cells were cultured in dmem supplemented with 10% fcs (hyclone), 15 mm lglutamine, 100 mg/ml penicillin, neaa/na-py/fungizone. all cells were maintained in a humidified atmosphere of 5% co 2 at 37uc. sendai virus (sev, cantell strain) was prepared in chicken embryos as described previously [29] . pulau virus (pulv) was prepared and titered as described previously [33] . for infection of cells, virus was incubated with cells for one hour at 37uc, then replaced with normal cell culture medium for the indicated time. full-length irf7, irf3 and myd88 open reading frames (orfs) were identified in the p. alecto genome (ncbi id p. alecto asm32557v1) [34] using blastx. for comparative purposes, sequences were obtained from the current genome assemblies from the ensembl database for the following species: ensp00000380697, homo sapiens (human); ensmusp00000095565, mus musculus (mouse); ensecap00000007698, equus caballus (horse); enssscp00000013664, sus scrofa (pig); ensbtap00000056564, bos taurus (cow). the microbat myotis davidii irf7 amino acid sequence was deduced from irf7 orf annotated from the published m. davidii genome (ncbi id m. davidii asm32734v1) [34] . the p. alecto irf7 sequence has been submitted to genbank under accession number kj534586. sequence alignment was performed using clustalx and visualized using genedoc (http://www.nrbsc.org/gfx/genedoc/ index.html). alignment files were visualized using emboss plotcon to determine the conservation of irf7 proteins among different species. genomic intron-exon maps of the genes were drawn using fancy gene v1.4 by comparing individual irf7 orfs of p. alecto, horse and human (http://host13.bioinfo3.ifomieocampus.it/fancygene/). phylogenetic trees were constructed using the neighbour joining method and mega4.1 program with 1000 bootstrap replicates [36] . primers listed in table 1 were designed based on the p. alecto genomic sequences and used in rt-pcr to amplify irf7, irf3 and myd88 from rna extracted from freshly isolated bat splenocytes. to construct expression plasmids, pcr products corresponding to full-length irf3 and irf7 were ligated directly to vivid colors pcdna 6.2/emgfp topo vector (life technologies) with an n-terminal gfp tag. the myd88 orf was ligated to the pflag-cmv2 expression vector (sigma) using restriction enzymes noti and sali with an n-terminal flag tag for detection. to generate a truncated bat irf7 (tirf7) that lacked the myd88 binding region at amino acids (aa) 234-298, overlapping pcr was performed [37] . the resulting tirf7 pcr product was ligated to the pcdna 6.2/emgfp topo vector. the human irf7 (hu-irf7) and hu-myd88 plasmids have been described previously [38] . hu-irf7 is in the pegfp-n1 vector and hu-myd88 is in the pef-bos vector with an n-terminal flag tag. mouse ifn-a4, ifn-a6 and human ifn-b promoter plasmids, abbreviated as mu_ifn-a4p, -a6p and hu_ifn-bp respectively, have been described previously [21] . the bat ifn-b promoter plasmid was constructed using sequence 1000 bp upstream from the start codon of ifn-b orf from the p. alecto genome. promoter prediction was performed using the online transcriptional start site prediction tool, matinspector in the genomatix software suite (http://www.genomatix.de/cgi-bin//matinspector_prof). regions containing putative irf3 or irf7 binding sites were identified from 2221 to 270 bp from the atg of the bat ifn-b gene by comparison with human ifn promoters and cloned into the pgl4.1 expression vector (promega). a transfection control prl-tk plasmid containing renilla luciferase was obtained from promega. details of primers used during plasmid construction can be found in table 1 . hek293t cells were transfected using fugene 6 (promega) according to the manufacturer's instructions. approximately 2610 5 cells per well in a 24-well plate were co-transfected with 100 ng of the relative ifn promoter plasmids, 50 ng of prl-tk (promega) served as an internal control. where indicated, expression plasmids for bat irf7, human irf7 or bat myd88 were included in the transfection mix. cells were harvested 30 h post-transfection and lysed using passive lysis buffer provided in the following kit. luciferase activities were determined using the dual-luciferase assay system (promega) using a thermo fluoroskan ascent fl machine. for promoter experiments in pakit03 cells, similar transfections were performed using lipofectamine 2000 (life technologies). a smartpool consisting of four small interfering rnas (sirnas) targeting bat irf7 (siirf7) was designed based on the full-length irf7 orf sequence using dharmacon custom services (thermo). information of the four siirf7 can be found in table 1 . transfection of siirf7 was performed in pakit03 cells using the neon transfection system (life technologies) according to the manufacturer's instructions. briefly, 20 nm of each of the four siirf7 was used for every 10 5 cells. cells were harvested into rlt lysis buffer (rneasy kit, qiagen) 48 h post-transfection and stored at 280uc prior to rna extraction. rna was extracted from cell lysate from the sirna knockdown experiment using a qiagen rneasy kit and converted to cdna using the quantitect reverse transcription kit for real-time pcr (qiagen). all experiments were performed according to manufacturer's protocols. preparation of cdna from 12 p. alecto tissues including brain, kidney, liver, lung, lymph nodes, spleen, heart, small intestine, wing, salivary gland, thymus and testis from three individual bats and from polyi:c stimulated palut02 cells has been described previously [33] . for each sample, 1 mg rna was applied to reverse transcription using the quantitech reverse transcription kit (qiagen). irf7 qrt-pcr primers were designed using primer express 3.0 (applied biosystems) with default parameter settings and are listed in table 1 . primers for ifn-b and 18s rrna have been described previously [33] . reactions were carried out using express sybr greener qpcr supermix universal (life technologies) in an applied biosystems 7500 fast real-time qrt-pcr instrument. for each reaction from cdna, 2 ml of 1:5 diluted cdna were used with a final concentration of 200 nmol of each primer. the cycling profile for cdna samples consisted of an initial denaturation at 94uc for 2 minutes followed by 40 cycles of 94uc for 15 seconds, 60uc for 1 minute, followed by melt curve analysis. expression levels of target genes were calculated using the standard curve method after normalisation to the housekeeping gene 18s rrna. pakit03 cells were seeded onto glass coverslips in 24-well plates at 10 5 cells per well one day before transfection. they were transfected with 200 ng each of gfp-irf7 and flag-tagged myd88 (human or bat) using lipofectamine 2000 (life technologies). at 16 h post-transfection, cells were fixed in 4% paraformaldehyde in pbsa at room temperature for 40 minutes. after removal of fixative, cells were washed three times with pbsa, followed by treatment with 0.1% triton x-100 for 10 minutes and blocked with 0.5% bsa in pbsa for 30 minutes. mouse anti-human myd88 antibody (santa cruz, cat. sc11356) was diluted 1:1000 in 0.5% bsa and applied to cells and incubated at 37uc for 1 h. cells were then incubated with alexa 488 conjugated goat anti-mouse secondary antibody at 37uc for 1 h and washed three times in pbsa. nuclei were labelled with dapi and coverslips were mounted on glass slides for analysis. all slides were examined under a leica confocal microscope (leica, germany). the immunoprecipitation method for analysis of irf7 and myd88 has been described previously [38] . in brief, 2610 6 hek293t cells were seeded 24 h prior to co-transfection with 1.25 mg of both flag-myd88 and gfp-irf7 (human or bat) using fugene 6 (promega). cells were then lysed 24 h later with lysis buffer (50 mm tris (ph7.4), 1.0% triton x-100, 150 mm nacl, 1 mm edta, 2 mm na 3 vo 4 , 10 mm naf, 1 mm pmsf and protease cocktail inhibitor mixture; promega). cell lysates were cleared by centrifugation (9000 g, 10 min, 4uc), and then pre-cleared with protein g-sepharose beads for 30 min at 4uc and flag-myd88 immune complexes were immune-precipitated from supernatant using anti-flag m2-agarose (sigma) conjugated beads for 2 h at 4uc. beads were washed with 3 x lysis buffer and eluted by boiling beads in 5 volumes of sds page sample buffer. lastly, sds page and western blot analysis were performed using the samples obtained. for the blotting, antihuman myd88 rabbit antibody and anti-human irf7 goat antibody (both from santa cruz) were used at 1:1000 dilution during primary antibody incubation and ap-conjugated goat antirabbit or rabbit anti-goat secondary antibody (both from life technologies) were used at 1:2000 dilution. to explore possible differences in the bat antiviral immune system which may influence the association between bats and viruses, irf family members were chosen as primary targets due to their importance in ifn induction and signaling [10] . all irf members from irf1 to irf9 were identified in the bat genome, indicating their relative conservation at the family level. we next performed sequence analysis on the four known positive regulators of type i ifn transcription in humans; irf1, irf3, irf5 and irf7. comparison of the sequence similarity of the deduced protein sequence of human and bat irfs demonstrated significant conservation of irf1 (96%), irf3 (88%) and irf5 (87%). in contrast, bat irf7 shares only 64% and 55% amino acid similarity to human and mouse respectively. to determine whether the relatively low sequence conservation of the bat irf7 gene compared to human and mouse affects the functional activity of bat irf7, this gene was chosen for further functional analysis. the recently published p. alecto whole genome sequence and transcriptome data were used to identify the irf7 gene [39, 40] . primers based on the genomic irf7 sequence were used to amplify full-length irf7 by pcr from bat spleen cdna resulting in the identification of a single full length irf7 transcript. by aligning the bat irf7 cdna sequence with the corresponding region in the p. alecto genome, we were able to determine the intron and exon structure of this gene. as shown in figure 1 , bat and horse irf7 have nine exons compared to the ten exon structure of human irf7. horse was included in this comparison due to the close phylogenetic relationship between bats and horses (figure s1 and [34] ). nine exons were also identified in irf7 genes from other laurasiatheria species, including pig, cow and dog. of the sequences available in the ensembl database, the ten exon structure appears to be typical only among primate irf7 genes (data not shown). analysis of the putative bat irf7 promoter region around 1000 bp upstream of the start site of the orf resulted in the identification of two ifn stimulated response elements (isres) and one nuclear factor kappa b (nf-kb) binding site. to determine whether the presence of two isre sites was unique to bats, the irf7 promoters of other species (human, mouse, horse, cow, dog, cat and rat) were also examined using the publicly available databases. all species examined contain two isre sites (one isre and one irf binding site) with the exception of human which has a single isre (data not shown). based on the deduced protein sequence, p. alecto irf7 was aligned with six other species: human, mouse, pig, cow, horse and the microbat, david's myotis (m. davidii) available from the recently completed genome sequence [40] . these species were chosen because human and mouse have been well studied, while all other species are phylogenetically close to p. alecto [34] . fulllength irf7 contained multiple functional domains including an n-terminal dna-binding domain (dbd), followed by a constitutive activation domain (cad), virus-activated domain (vad), inhibitory domain (id), and c-terminal serine-rich region ( figure 1 ; [22, 37] . the vad is responsible for binding to the upstream activators of irf7: myd88, traf6 or tbk-1, while the serine-rich region is the target for virus-inducible phosphorylation [21, 41] . from the alignment, bat irf7 appears to have a conserved dbd ( figure s2 ), c-terminal serine-rich domain which is the target of phosphorylation, and auto-inhibitory domain [37] . however, the region between amino acids 200-300, which corresponds to the vad and contains the myd88 binding site is not conserved in sequence among all species ( figure 1b) . furthermore, in the putative myd88 binding region, both bats are less conserved not only to human but also to other laurasiatheria species shown here (pig, cow and horse). as the myd88-irf7 pathway is critical to ssrna-induced human ifn production, alignment was performed between bat myd88 and myd88 from human, mouse, pig, cow and horse. no significant change that would potentially alter the functionality of bat myd88 was identified based on sequence analysis (figure s3). to determine the tissue distribution of bat irf7, its transcription was examined in a range of immune and non-immune associated bat tissues. the tissues were from three apparently healthy bats caught from the wild in which the irf7 level should represent the normal expression pattern in wild bats. as shown in figure 2a , bat irf7 is widely expressed among all bat organs at the mrna level, with spleen, small intestine and lung having the highest irf7 expression and wing and salivary gland the lowest. with the exception of the wing, all other tissues showed similar expression levels of irf7 with around 10-fold difference between spleen which had the highest expression and salivary gland with the lowest. this pattern differs from the transcription pattern of bat tlr7, 8 and 9, which appear to be predominantly expressed in immune tissues [30] . next, the inducibility of irf7 by a known virus mimic was explored by testing irf7 transcription using our cloned and immortalized bat lung cell line (palut02 cells) following stimulation with the double stranded rna (dsrna) ligand, alecto irf7 with irf7 from human, mouse, cow, pig, horse and m. davidii using a 20 amino acid sliding window and created using emboss plotcon (http://emboss.bioinformatics.nl/cgi-bin/emboss/plotcon). the similarity across the whole orf between these species is shown by scores. the lower the score the lower the sequence conservation. the least conserved region is boxed. doi:10.1371/journal.pone.0103875.g001 polyi:c. the palu02 cell line has previously been demonstrated to produce ifn in response to polyi:c in a dose dependent manner [42] . as shown in figure 2b , stimulation by either treatment or transfection with polyi:c, which mimics ifn production through tlr3 or rlh pathways respectively resulted in strong induction of irf7. notably, irf7 mrna was induced at the earliest time point of 3 h following stimulation, to a peak level of around 1000 times higher than mock treated cells, highlighting the importance of irf7 in early antiviral defense in bats. these data are consistent with bat irf7 being inducible by both tlr and rlh pathways. in summary, bat irf7 is constitutively transcribed in all tissues and cell lines tested (palut02 cells and pakit03 cells, below) and can be strongly induced by dsrna. due to the limitations of the human irf7 antibody used in this study, the expression of irf7 at the protein level awaits the development of a suitable bat specific reagent. to test the ability of irf7 to induce ifn-b production a bat ifn-b promoter assay was used. the putative promoter region of the bat ifn-b gene was examined and predicted to contain irf7 and irf3 binding modules ( figure s4 ). this region was ligated to the pgl4.1 luciferase reporter vector. interestingly, the bat ifn-b promoter contains one residue difference in its prdi which may abolish its ability to bind to irf3 or irf7 [43] . in contrast, the second prd (prdiii) is almost identical to the corresponding domain of the human ifn-b promoter region and was therefore predicted to be functional [44] . comparison of the prdi domain of other closely related species available in the ensembl database and the microbat, m. davidii, revealed the disruption of prdi is unique to p. alecto ( figure s4 ). in the bat immortalized kidney cell line (pakit03 cells), a plasmid encoding bat irf7 was cotransfected with the bat ifn-b promoter plasmid for 24 h and cells were then infected with sev for another 6 h before performing the luciferase test. the pakit03 cells were chosen for these experiments due to their ability to be successfully transfected and sev was chosen due to its potent ability to induce ifns and other cytokines in the absence of viral products that block the ifn response [45] . results clearly indicate that irf7 alone can induce ifn-b activation and sev induces enhanced activation of irf7 in a dose dependent manner ( figure 3a) . to examine whether the poorly conserved myd88-binding region in bat irf7 influenced its transactivation potential, bat irf7 was compared with that of human irf7 using ifn promoter assays. this experiment was designed to test the hypothesis that the sequence differences identified in the bat irf7 region do not affect its ability to activate ifn transcription. this response was tested using mouse ifn-a4, mouse ifn-a6 or human ifn-b promoter plasmids co-transfected with increasing doses of bat or human irf7 expression plasmids in hek293t cells. as shown in figures 3b-d , bat irf7 activates all three promoters in a dosedependent manner. together with the results described above using the bat ifn promoter, these results demonstrate that bat irf7 is capable of activating ifn in bat cells following stimulation with sev or in human cells co-transfected with human or mouse ifn promoters. cells transfected with mock or empty vector failed to activate the ifn promoters significantly. although identical doses of human and bat irf7 plasmid were used in these experiments, the results are not statistically comparable due to the difference in irf7 protein expression from the two plasmids. of note, we used human hek293t cells and mouse ifn-a and human ifn-b promoters in this assay because they are a well established system for testing the activation of ifn [21] . given the high conservation in the dna-binding domain of bat irf7, it was predicted to be capable of binding to the human and mouse ifn promoters followed by activation by downstream factors. to explore the importance of bat irf7 in the ifn production pathway, a knockdown approach was used in our p. alecto pakit03 cells. transfection of sirna targeting bat irf7 (siirf7) for 24 h resulted in a reduction of native irf7 mrna expression to approximately 20% compared to mock transfected cells. the statistical analysis of the knock down effects was calculated by comparing mrna expression in the knock down samples to mock transfected cells. to exclude the possibility of off-target effects of sirna transfection, the expression of a closely related gene, irf3 was examined in transfected cells. as shown in figure 4a , there was a decrease in irf3 transcription in siirf7 transfected cells due to possible toxic effects and/or off target effects of the sirna smartpool but this change was not statistically significant. having confirmed the knockdown effect of siirf7, we then explored the downstream effect of reduced irf7 on ifn-b production and viral replication. two experiments were performed; firstly, 24 h after siirf7 transfection, cells were stimulated with sev for 6 h and ifn-b mrna was detected by qpcr. knockdown of irf7 impaired the induction of ifn-b by sev by 2.5 fold relative to untransfected cells ( figure 4b) . notably, bat cells maintained some ifn-b induction in siirf7 cells, a result which likely reflects insufficient knockdown of irf7 or ifn-b induction through alternative (irf3 or nf-kb) pathways. to examine the effect of ifn knockdown on the replication of a bat-borne virus, pulv, a dsrna reovirus originating from pteropid bats, was used to infect siirf7-transfected bat cells [46] . a dose of 10 moi was used to infect pakit03 cells one day after siirf7 transfection (or mock transfection). cell supernatant containing virus was collected 24 h after infection and applied to a tcid 50 test. figure 4c shows that when bat irf7 was knocked down, pulv replicated to a titer more than four-fold higher than in mock-transfected cells. these data demonstrate that bat irf7 is functionally important in sev induced ifn-b production and antiviral defense against pulv infection of bat cells. we next wanted to determine whether bat irf7 is involved in the production of ifn-a and ifn-b by the myd88 despite the divergent nature of its myd88 binding domain. the transactivation activity of bat irf7 was compared to that of human irf7 using expression plasmids containing bat or human myd88 and irf7 co-transfected with mouse ifn-a4 or ifn-a6 promoter plasmids. in mice, ifn-a4 is the earliest ifn-a induced by viral infection, while ifn-a6 is induced later in the response. a dose of 10 ng or 100 ng of irf7 was co-transfected with myd88 for the ifn-a4p and ifn-a6p promoter assay respectively in hek293t cells. these doses of irf7 were chosen as they do not result in huge induction of the native ifn promoter. as shown in figure 5a , the activation of ifn-a4p by both human and bat irf7 was increased by co-transfection with myd88. co-transfection of cells with bat myd88 and irf7 resulted in a higher response compared to co-transfection with the corresponding human plasmids. our results demonstrate that even with a significant difference in its myd88 binding region, bat irf7 is still capable of inducing ifn-a transcription via myd88 ( figure 5 ). some differences were observed between ifn-a4 and ifn-a6 inducibility which may be due to differences in their irf or nf-kb binding motifs. no ifn-a4 activation occurred following cotransfection of bat myd88 with bat irf3 confirming that irf3 is myd88 independent ( figure s5a) . a similar experiment was performed to confirm that bat irf3 is capable of activating the human ifn-b promoter confirming the activity of bat irf3 ( figure s5b ) [21] . thus, only ifna production by irf7 is dependent on myd88. to confirm that bat irf7 interacts with bat myd88, experiments were performed to examine the interaction between the two proteins. firstly, hek293t cells were co-transfected with plasmids encoding flag-tagged myd88 (either human or bat, as indicated) and gfp-tagged irf7 (human or bat). cells were lysed and protein immunoprecipitated with anti-flag antibody conjugated beads followed by immunoblotting with anti-flag or anti-human irf7 antibody. human irf7 protein was successfully captured by human myd88 which was detected in ip samples demonstrating protein interaction between human myd88 and human irf7 ( figure 6a, panel 1) . the detection of only a faint band may be due to the relatively low expression of the input human irf7 and myd88 proteins from these expression plasmids. clear signals were detected for irf7 and myd88 following co-ip of bat myd88 with bat or human irf7 ( figure 6a ). there is a slight difference in the molecular weights of human and bat myd88 and irf7 which are reflected on the blot (human and bat myd88 are 33.3 and 33.6 kd respectively and human and bat irf7 have a molecular weight of 54.2 and 55.6 kd respectively). these results clearly demonstrate that bat myd88 protein is capable of binding both bat and human irf7 proteins. confocal microscopy was used to determine the colocalisation of the two proteins, to further confirm protein interaction. bat kidney pakit03 cells or human kidney hek293t cells were used to examine colocalisation of irf7 with myd88. a dose of 200 ng/ well of either human or bat myd88 and irf7 plasmids were used to transfect cells grown overnight on coverslips in 24-well plates. sixteen hours later, cells were fixed and stained with anti-human myd88 antibody and examined under the confocal microscope. myd88 transfection alone resulted in the formation of very large condensed aggregates in the cytoplasm of both human and bat cells. human myd88 and human irf7 colocalised in a manner similar to previous studies ( figure 6b ) [21, 41] . similarly, bat myd88 and irf7 proteins also demonstrated clear co-localisation. as shown in figure 6b , bat irf7 appeared to be surrounded by myd88 in an aggregated form, which is the typical myd88 structure [47, 48] . in addition, bat myd88 also colocalised with human irf7, which is consistent with our ip results ( figure 6a ). as expected, no such aggregated structure was observed following co-expression of bat myd88 with bat irf3, ruling out the possibility of interaction between these two proteins. having confirmed the functional reliance and binding capability of bat irf7 to bat myd88, it appeared that the poorly conserved myd88-binding region (between aa 200-300) retains a similar function to the corresponding human domain. to further confirm this was the case, an internal deletion mutant of bat irf7, tirf7 was constructed ( figure 7a ). this form of bat tirf7 contained a deletion of aa 225-331 which has been shown to abolish the ability of human irf7 to transactivate the ifn-a1 promoter [22] . the mouse ifn-a4 promoter transactivation activity by tirf7 was compared to the full-length irf7 protein using a luciferase assay. having confirmed successful expression, figure 7b clearly demonstrates that tirf7, was unable to activate the mouse ifn promoter, even in the presence of myd88 ( figure 7b ). these data are consistent with functional conservation of the region corresponding to aa 233-298 in bats with that of human irf7 despite significant sequence variation. irf7 is a master regulator of ifn expression in mammals and is therefore central to the innate antiviral immune response. in humans, irf7 acts predominately in pdcs via activation of tlr7/9 and the myd88 dependent signaling pathway [49] . regulation of the ifn response may play an important role in the ability of bats to coexist with viruses in the absence of clinical signs of disease. this report describes the analysis of irf7 from our model bat species, the australian black flying fox, p. alecto, an important reservoir for viruses including hendra virus, which has resulted in the deaths of numerous horses and humans since its discovery in 1994 [4, 46] . our results support the conservation of functional activity of irf7 in p. alecto but provide evidence of a wider tissue distribution which has implications for broader activation of the ifn response in bats. our results provide the first functional characterization of irf7 in any species of bat and contribute to our understanding of the function and evolution of irf7 in mammals. bat irf7 was identified from the bat genome and bat transcriptome data [39, 40] , together with rt-pcr results from spleen cdna resulting in the identification of a single full length variant of irf7. in humans and mice, irf7 expression is very low in most tissues and cells with the exception of pdcs and cells that have been activated by ifn [12, 15] . in contrast, the transcription of p. alecto irf7 was detected not only in immune-related tissues but comparable expression was observed in many other organs as well. although there is a lack of data on the tissue distribution of irf7 in mammals besides human and mouse, there have been several studies on fish irf7. interestingly, at least five species of fish including crucian carp, mandarin fish, snakehead fish, atlantic salmon and japanese flounder express irf7 constitutively in a wide variety of tissue types although different irf7 transcripts were expressed in each species. these tissues were neither primarily immune-related nor serve as portals for microbial infection, where the immune response is easily initiated. since these fish irf7s can also be induced by dsrna, they were hypothesised to play an important role in fish immunity [16, 17, 18, 19, 20] . although further analysis of the cell types responsible for constitutive irf7 expression in bats is required, a constitutively expressed irf7 in a broad range of cells and organs may result in faster and stronger ifn production upon viral infection [49] . this observation is similar to the pattern of type iii ifn receptor expression which has a wide distribution in bats but only limited distribution in other mammals [50] . thus, bats may maintain the potential to rapidly activate the innate immune response in a broader subset of tissues and cells than other mammals. induction of irf7 by treatment or transfection of our bat kidney cell line with the dsrna ligand, polyi:c resulted in a peak in the induction of irf7 at 9 h post-treatment, which is 3 h later than the peak in bat type i and type iii ifns but similar to that of isgs mx1, oas1 and pkr described previously in bat cells [29, 33] . this result is consistent with the induction of irf7 through type i ifn feedback similar to other species. in humans, irf7 is generated through multiple pathways following ifn induction. following the production of ifn and binding to the . bat irf7 interacts with bat myd88. (a) binding of bat irf7 to bat myd88. hek293t cells were transfected with gfp-tagged irf7 and flag-tagged myd88 (human or bat, as indicated). at 24 h post-transfection, whole cell lysate was prepared and immunoprecipitated with anti-flag antibody m2. immunoprecipitated complexes (ip) were analysed by immunoblot for irf7 and myd88 expression using an anti-human irf7 antibody (top panel), and an anti-flag antibody m2 for detection of flag tagged myd88 (middle panel). whole cell lysate (10 ul) was also run on an sds-page gel and subsequently analysed for irf7 expression using anti-human irf7 antibody (bottom panel). (b) bat irf7 co-localizes with bat myd88. pakit03 cells were transfected with bat gfp-irf7/irf3 and bat flag-myd88 and hek293t cells were transfected with human gfp-irf7 and human or bat flag-myd88. 16 hours post-transfection (or 24 h), cells were fixed for indirect immunofluorescence assay using an anti-human myd88 antibody and red fluorescence-conjugated secondary antibody. pictures show co-localisation of bat irf7 or bat irf3 with bat myd88 in pakit03 cells and human irf7 or human irf3 with human myd88 in hek293t cells. doi:10.1371/journal.pone.0103875.g006 ifn-ar, a complex consisting of activated stat1, stat2 and irf9, called the ifn stimulated gene factor 3 (isgf3) is formed, which in turn binds to the isre on the irf7 promoter and induces irf7 transcription. the human irf7 promoter region contains an nf-kb binding site and a single functional isre approximately 1.3-kb upstream from the atg start site, both of which are important in the induction of irf7 [49, 51] . analysis of the putative bat irf7 promoter region resulted in the identification of two isres and one nf-kb binding site indicating that multiple mechanisms for irf7 activation may also exist in bats. however, two isre sites were also identified in the irf7 promoters of other species examined (mouse, horse, cow, dog, cat and rat). thus, whether the broad distribution of constitutively expressed irf7 is the result of the presence of a more efficient irf7 promoter region driven by transcription factors other than irfs, or simply due to enriched immune-related cells in all tissues will require further study. sequence differences in the myd88 binding domain of bat and human irf7 led to the hypothesis that there may be functional differences in the activation of bat irf7 and the regulation of the ifn response that may contribute to the ability of bats to resist the clinical outcomes of viral infection. our results demonstrate that these sequences differences do not appear to affect irf7 function either in ifn transactivation activity or activation by myd88. bat irf7 was capable of activating both ifn-a and ifn-b promoters and the levels of transactivation were equivalent to or higher than that of human irf7. similarly, bat myd88 and bat irf7 maintained binding capability similar to their human counterparts. deletion of the myd88-binding region of bat irf7 impaired its ability to activate ifn, demonstrating functional conservation of the myd88 binding domain with that of human irf7. collectively, these data demonstrate that bat irf7 is capable of inducing ifn and myd88 binding in a similar manner to human irf7. although the myd88 binding domain of bat irf7 has low sequence conservation with the equivalent region in human irf7, experimental data demonstrate a fully functionally irf7 exists in pteropid bats. our results describing the experimental knockdown of irf7 using sirna is to our knowledge the first description of the use of sirnas in bat cells. the successful knockdown of irf7 is consistent with the presence of an rna-silencing mechanism in bats similar to that in other mammals. irf7 knockdown resulted in impaired ifn-b induction in sev infected cells and enhanced pulv replication. although further work will be required to determine whether irf7 is the master regulator of the bat ifn response, these results confirm that irf7 plays an important role in anti-viral defense and the early innate immune response in bats [13] . analysis of the bat ifn-b promoter also revealed one residue difference in the prdi module, known to be associated with activation by irf3 or irf7 [43] . a similar change in the human ifn-ap impairs its inducibility by irfs [26] . although bat ifn-b is strongly inducible in bat cells following either stimulation or viral infection, further work will be necessary to determine whether this mutation affects the induction of ifn-b under conditions other than those described here [29] . the presence of this mutation may also indicate that transcription factors other than irf3 and irf7 are involved in the regulation of ifns in bats. therefore, future work focusing on ifn promoters (including ifn-a and ifn-l) will be necessary to explore whether the bat irf-ifn induction pathway is as critical to ifn induction as it is in other species. in humans, there are around 1400 transcription factors that have been recognized [52] . whether these factors play similar roles in bats or whether they perform different functions resulting in differences in the expression of downstream genes remains to be determined. understanding the mechanisms responsible for the regulation of the ifn response will assist in discovering how bats successfully coexist with viruses. in conclusion, our data clearly demonstrate that bat irf7 exhibits a constitutive expression pattern across a broad range of immune and non-immune related organs, intact ifn transactivation function, and binds to and is activated by bat myd88. these findings not only provide 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genomes provides insight into the evolution of flight and immunity role of a transductional-transcriptional processor complex involving myd88 and irf-7 in toll-like receptor signaling type iii ifns in pteropid bats: differential expression patterns provide evidence for distinct roles in antiviral immunity regulation of virusinduced interferon-a genes evidence for a nuclear factor(s), irf-1, mediating induction and silencing properties to human ifn-beta gene regulatory elements sendai virus defective-interfering genomes and the activation of interferon-b pulau virus; a new member of the nelson bay orthoreovirus species isolated from fruit bats in malaysia regulation of myd88 aggregation and the myd88-dependent signaling pathway by sequestosome 1 and histone deacetylase 6 distinct roles of tir and non-tir regions in the subcellular localization and signaling properties of myd88 irf7: activation, regulation, modification and function type iii ifn receptor expression and functional characterisation in the pteropid bat, pteropus alecto regulation of the promoter activity of interferon regulatory factor-7 gene. activation by interferon snd silencing by hypermethylation a census of human transcription factors: function, expression and evolution we thank drs prasad paradkar and glenn marsh for critical review of the manuscript. key: cord-339392-2ocz784l authors: sharma, kulbhushan; tripathi, shashank; ranjan, priya; kumar, purnima; garten, rebecca; deyde, varough; katz, jacqueline m.; cox, nancy j.; lal, renu b.; sambhara, suryaprakash; lal, sunil k. title: influenza a virus nucleoprotein exploits hsp40 to inhibit pkr activation date: 2011-06-15 journal: plos one doi: 10.1371/journal.pone.0020215 sha: doc_id: 339392 cord_uid: 2ocz784l background: double-stranded rna dependent protein kinase (pkr) is a key regulator of the anti-viral innate immune response in mammalian cells. pkr activity is regulated by a 58 kilo dalton cellular inhibitor (p58(ipk)), which is present in inactive state as a complex with hsp40 under normal conditions. in case of influenza a virus (iav) infection, p58(ipk) is known to dissociate from hsp40 and inhibit pkr activation. however the influenza virus component responsible for pkr inhibition through p58(ipk) activation was hitherto unknown. principal findings: human heat shock 40 protein (hsp40) was identified as an interacting partner of influenza a virus nucleoprotein (iav np) using a yeast two-hybrid screen. this interaction was confirmed by co-immunoprecipitation studies from mammalian cells transfected with iav np expressing plasmid. further, the iav np-hsp40 interaction was validated in mammalian cells infected with various seasonal and pandemic strains of influenza viruses. cellular localization studies showed that np and hsp40 co-localize primarily in the nucleus. during iav infection in mammalian cells, expression of np coincided with the dissociation of p58(ipk) from hsp40 and decrease pkr phosphorylation. we observed that, plasmid based expression of np in mammalian cells leads to decrease in pkr phosphorylation. furthermore, inhibition of np expression during influenza virus replication led to pkr activation and concomitant increase in eif2α phosphorylation. inhibition of np expression also led to reduced irf3 phosphorylation, enhanced ifn β production and concomitant reduction of virus replication. taken together our data suggest that np is the viral factor responsible for p58(ipk) activation and subsequent inhibition of pkr-mediated host response during iav infection. significance: our findings demonstrate a novel role of iav np in inhibiting pkr-mediated anti-viral host response and help us understand p58(ipk) mediated inhibition of pkr activity during iav infection. influenza a viruses (iav) are negative sense segmented rna genome viruses [1, 2] which can rapidly develop resistance to the drugs available against them [3] . these viruses pose a continuing threat of pandemics, thus it is imperative to develop novel strategies to prevent their infection and spread [4] . interactions between viral proteins and host factors are often crucial for successful replication of the virus in host cells [5] . many of these interactions are aimed at overcoming the early innate immune response of infected cells against the virus [6] . mammalian cells respond to viral infections through several innate immune mechanisms [7] . one such crucial antiviral mechanism is activation of pkr (a dsrna dependent protein kinase) which is phosphorylated upon encountering viral dsrna [8] . activated pkr has several downstream substrates, one of which is the eukaryotic translation initiation factor 2 alpha subunit (eif2a) [9] [10] [11] . phosphorylation of eif2a by activated pkr renders it unable to participate in translation initiation leading to translation arrest and inhibition of protein synthesis from viral mrnas [12, 13] . another effector function of pkr is activation of transcription factor irf3, which leads to the expression of ifn b and inhibition of virus replication [14, 15] . being such a crucial component of the host innate immune system, pkr is tightly regulated by cellular inhibitors [16] and very often targeted by viral proteins [17] [18] [19] [20] . for example, the non-structural protein 1 (ns1) of influenza virus directly binds to pkr and prevents its activation [21, 22] . apart from pkr inhibition, ns1 is also involved in the inhibition of other cellular signaling cascades, which lead to the activation of anti-viral interferon response [23, 24] . pkr activity is also inhibited by a cellular 58 kda protein, p58 ipk , which promotes influenza viral replication [25, 26] . in naïve cells, p58 ipk exists in an inactive state in a complex with heat shock protein 40 (hsp40), which becomes active upon release from this complex [27] . influenza virus infection leads to the dissociation of p58 ipk from hsp40 and suppression of the pkr response [28, 29] . however, neither the viral component nor the mechanism responsible for this event is known to-date. segment 5 of the influenza virus genome encodes for 498 amino acids nucleoprotein (np) whose primary function is viral genome encapsidation [30] . apart from that, np is also known to interact with several viral and host factors and play additional roles in the viral life cycle [31] [32] [33] [34] [35] [36] [37] . we were interested in identifying new cellular interactors of np from a highly virulent a/h5n1 bird-flu isolate {a/hatay/2004(h5n1)}, which may facilitate viral replication. for this a h5n1 np was used as bait to search for novel interactors in a yeast two-hybrid system based screen of human lung cdna library. in the screen, we identified that iav np interacts with human chaperone heat shock protein 40 (hsp40) [38] . considering the known role of hsp40 in regulation of pkr activity during influenza a virus infection [27] , we explored the possibility of np playing a regulatory role in this process. we observed that expression of iav np in mammalian cells lead to reduced phosphorylation of pkr and its substrate eif2a. we thus hypothesize that influenza np is the viral factor that facilitates the inhibition of pkr activation by releasing p58 ipk from hsp40-p58 ipk complex. consistent with this hypothesis, we observed that during iav infection, the association of np with hsp40 coincided with the release of p58 ipk from hsp40. also, rnai-mediated inhibition of np expression in iav infected cells enhanced the phosphorylation of pkr and its downstream substrate eif2a. np inhibition also led to enhanced irf3 phosphorylation and ifn b production which may be mediated by pkr activation. collectively, these findings identify a novel role for influenza a virus np in blocking the pkr-dependent antiviral response in influenza a virus infected cells. a human lung cdna library was screened using iav np as bait, in gal4 based matchmaker yeast two-hybrid system (clontech). yeast cells (ah-109) were co-transformed with bait and prey plasmids, and selected for growth on selective l -t -hplates supplemented with 50 mm aminotriazole. b-galactosidase positive colonies were further analyzed (fig. s1a ). plasmids from positive colonies were isolated and subjected to dna sequencing followed by blast analysis to identify their cdna insert. the mammalian chaperone heat shock protein 40 (hsp40/ dnajb11) was thus identified as an interacting partner of np. the strength of the np-hsp40 interaction was determined using a quantitative b-galactosidase assay, and was found to be comparable to the positive control used in the assay (fig. s1b , bars 6 and 7 respectively, p-value = 0.0668). the np-hsp40 interaction in mammalian cells was ascertained using co-transfection of plasmids coding h5n1 np and hsp40 in the hek293t cells. transfected cells were metabolically labeled with s 35 , and co-immunoprecipitation was performed using the lysates with either np-or hsp40-specific antibodies. these results showed that np co-precipitated with hsp40 and vice-versa (fig. 1a , lane 2 and 4). these results were confirmed using a549 lung epithelial cells, which were transfected with np expressing plasmid, followed by immunoprecipitation. it was observed that ectopically expressed np could immunoprecipitate endogenous cellular hsp40 and vice-versa (fig. 1b, panel 1 and 2) . a direct interaction between np-hsp40 was further confirmed using a co-immunoprecipitation assay in which 35 s labeled np and hsp40 proteins were expressed from plasmids using in-vitro coupled transcription-translation rabbit reticulocyte lysate system (tnt, promega, inc) (data not shown). collectively, these results showed that iav np directly interacts with hsp40. to validate the interaction between iav-np and hsp40 in virus-infected cells, we investigated the kinetics of expression of np and hsp40 in a549 cells. a549 cells were infected with a figure 1 . detection of iav np-hsp40 interaction in mammalian cells tranfected with np expressing plasmid by co-immunoprecipitation. a. hek293t cells were pcdna3.1-np and pcdna3.1-hsp40 plasmids alone or in combination, followed by metabolic labeling with s 35 . 48 hours post-transfection cells were harvested and ip was setup using anti-np-specific antibody and anti-hsp40-specific antibody followed by autoradiography. lanes 2 and 4 show co-ip of hsp40 with np and vice-versa. lanes 1 and 3 show anti-np and anti-hsp40 antibodies were not cross reacting with hsp40 and np, respectively. b. a549 cells were transfected with pcdna3.1-np plasmid or control pcdna3.1 plasmid. cells were harvested 48 hours post-transfection and immunoprecipitation was setup using anti-myc tag antibody and anti-hsp40 antibody, followed by western blotting. although np expression was maximal at 24 h post-infection, hsp40 expression levels remained unchanged throughout the course of infection ( fig. 2a) . therefore, in subsequent experiments, a549 cells were infected with pr8 at an moi of 1 for 24 h and lysates were prepared. a co-immunoprecipitation assay was performed using infected and control cell extracts. pr8 np was able to co-precipitate endogenous hsp40 (fig. 2b, panel 2 ). conversely, hsp40 was able to co-precipitate np (fig. 2b, panel 1) . the np-hsp40 interaction was also observed when a549 cells were infected with influenza virus isolates belonging to various subtypes (table 1) . co-immunoprecipitation of proteins from a549 cells infected with these select viral isolates showed that np co-precipitated with hsp40 in all cases without exception (fig. 2c , panel 1). a phylogenetic analysis of influenza np genes used in the experiment was constructed using the neighbor-joining method, nucleotide model tamura-nei, in mega version 4 (fig. s2) . the diversity of changes in np amino-acid sequences of the strains used in this study are shown in fig. s3 . the np genes of iav used in the study had amino-acid sequence divergence in the range of 1% to 10% from hatay/h5n1/2004 isolate. these results clearly indicated that the np-hsp40 interaction was conserved among seasonal human, avian h5n1 and the 2009 h1n1 pandemic influenza a viruses. it is known that under stress conditions the expression level of hsp40 is enhanced and its cellular localization changes from cytoplasmic to nuclear [38] , however its distribution in influenza virus infected cells was not studied. thus we investigated the cellular localization pattern of iav np in context to hsp40 in mammalian cells. we transfected a549 cells with iav np expressing plasmid for 24 h, and an immunofluorescence staining was performed with specific antibodies. results showed that np and cellular hsp40 colocalize primarily in the nucleus (fig. 3a , lower right panel). similar results were obtained with a549 cells infected with pr8 virus. confocal microscopy revealed that np and hsp40 were present primarily in the nucleus. however there was significant amount of np present in the cytoplasm at 24 h post-infection (fig. 3c , lower right panel). we also observed that figure 3 . co-localization of iav np and hsp40 in nucleus of mammalian cells. a and b. a549 cells were transfected with pcdna3.1-np or control pcdna3.1 plasmid for 24 hours, and cells were fixed and processed for immunostaining. np was stained using anti-myc tag specific primary antibody and alexa488 conjugated secondary antibody (green). hsp40 was stained using hsp40 specific primary antibody and alexa 594 conjugated secondary antibody (red). nuclei were stained with dapi. a shows pcdna3.1-np transfected cells whereas b shows control pcdna3.1 transfected cells. panels are labeled for their respective staining. lower right panel shows nuclear colocalization of np and hsp40. c and d. a549 cells were infected with pr8 influenza a virus at 1 moi for 24 hours, and cells were fixed and processed for immunostaining. np was stained using anti-np monoclonal primary antibody and alexa488 conjugated secondary antibody (green). hsp40 was stained using hsp40 specific primary antibody and alexa 594 conjugated secondary antibody (red). nuclei were stained with dapi. panels are labeled for their respective staining. c shows pr8 infected cells whereas d shows control uninfected cells. lower right panel in c shows primarily nuclear colocalization of np and hsp40. doi:10.1371/journal.pone.0020215.g003 hsp40 cellular levels were elevated after iav infection, as compared to uninfected cells ( fig. 3 c and d, upper right panels). hsp40 is known to negatively regulate eif2a phosphorylation through pkr (27) . therefore, we next assessed if changes in pkr and eif2a phosphorylation occurred during the course of iav infection of a549 cells. we found that the phosphorylation of both pkr and eif2a increased initially between 1-2 h post-infection and subsequently declined between 4-8 h post-infection ( during iav infection p58 ipk activity was increased as it was released from hsp40 binding [27] . we hypothesized that np might disrupt the p58 ipk -hsp40 complex too and liberate p58 ipk . to investigate this possibility, we monitored changes in the cellular levels of p58 ipk and np associated with hsp40 during iav infection. a549 cells were infected with the pr8 virus, harvested at different time points after infection and immunoprecipitation was conducted using equal amounts of total protein and anti-hsp40 antibody. western blot analysis revealed that between 4 and 8 h post-infection, the levels of np associated with hsp40 continued to rise (fig. 4b , panel 1) with a concomitant decline in p58 ipk associated with hsp40 (fig. 4b, panel 2) . during this period, total amounts of hsp40 and p58 ipk remained constant (fig. 4b , panel 5, 6). these results were consistent with the hypothesis of replacement of p58 ipk from hsp40, by np. these findings indicate that the dissociation of p58 ipk -hsp40 complex occurs around 4 to 8 h post-infection, and is associated with downregulation of eif2a phosphorylation. taken together, these results suggest that during iav infection, np induces the dissociation of the p58 ipk -hsp40 complex leading to an inhibition of pkr activation and downregulation of eif2a phosphorylation. during iav infection, the ns1 protein inhibits pkr activation by directly interacting with it, and thereby ensuring continued viral mrna translation [21, 22] . results from our study indicated that np may also play a role in inhibiting pkr activity by intercepting this pathway at the level of hsp40. to examine this aspect, we performed a time course study in hek293t cells transfected with the plasmid expressing iav h5n1 np by monitoring p-eif2a and p-pkr levels. western blot analysis of transfected cell lysate showed downregulation of both pkr and eif2a phosphorylation, which began as early as 12 h and was most prominent at 36 h post-transfection ( fig. s4 a fig. 5d ). to confirm the pkr inhibitory action of np, we transfected hek293t cells with np-gfp expressing plasmid and inhibited np expression using specific sirna ( table 2) against it (fig. s5a) . inhibition of np expression led to an increase in p-pkr and p-eif2a levels as compared to control np-gfp transfected cells (fig. s5b) . these results suggest that the expression of np leads to inhibition of pkr activity in a ns1 independent manner. this action of iav np is likely to be mediated through its interaction with hsp40. we suppressed np expression pr8 iav infected a549 cells, using a pool of gene specific sirnas (table 2) , and determined its effect on pkr activity. expression of ns1 was also blocked using sirna against ns1 alone and in combination with np sirna to establish their exclusive and combined contribution to pkrinhibition. a549 cells were first transfected with the indicated sirnas, and 6 hours later were infected with pr8 influenza virus. infected cells were harvested 24 h post-infection and cell lysates were subjected to western blot analysis. we observed that inhibition of np expression led to upregulation of pkr phosphorylation, as compared to the control (fig. 6a, panel 1, fig. 6b ). increased pkr activity resulted in enhanced phosphorylation of eif2a and irf3 (fig. 6a, panel 2, 4, fig. 6c, d) . inhibition of ns1 had a similar effect, whereas inhibition of both np and ns1 had a cumulative effect on upregulation of pkr, eif2a, and irf3 phosphorylation (fig. 6b, c, d) . pkr mediated activation of irf3 should lead to an increased ifn response. previous results confirmed the involvement of np in the inhibition of pkr and irf3. to check the further downstream effect of np, we suppressed the expression of np using sirnas as mentioned earlier. 24 hours post-infection, the cells were harvested and rna was isolated to determine ifnb and viral rna levels by real-time pcr using gene-specific primers. the inhibition of np expression led to increased ifnb production as compared to control (fig. 7a, bar 2, 3) . furthermore, the inhibition of ns1 had greater impact on ifnb production as compared to that of np, and there was a synergistic effect when both ns1 and np were inhibited (fig. 7a, bar 4, 5) . increased ifnb production should lead to reduced virus replication and reduced production of influenza vrna. to confirm this, influenza vrna levels in the above mentioned samples were measured by real-time pcr. inhibition of np or ns1 led to reduced vrna production as compared to control (fig. 7a, bar 2, 3, 4) , and inhibition of both np and ns1 together had greater synergistic effect (fig. 7b, bar 5 ). heat shock proteins are stress response factors which also regulate several cellular processes [39] . the hsp40 family chaperones are known to play important roles in protein folding, translocation, cell signaling and apoptosis [40] [41] [42] . very often they are targeted by viral components for successful virus replication. for example, hsp40 is known to interact with hiv type 2 vpx protein and facilitate nuclear import of the pre-integration complex [43] . hiv type 1 nef protein interacts with hsp40 to enhance viral gene expression [44] . hsp40 is also known to interact with the hbv core protein and affect viral turnover [45] . heat shock proteins are known to affect the viral replication of influenza viruses also. for example hsp90 is known to interact with influenza virus polymerase components and aid in viral rna synthesis [46] . hsp70 is also known to be involved in the nuclear export of the rnp complex and play a role in temperature dependence of iav replication [47, 48] . likewise, hsp40 is also known to regulate pkr signaling in influenza virus infected cells [25] . similarly, the iav np is also a multifunctional protein that interacts with a wide variety of viral and cellular macromolecules, including rna, pb1 and pb2 subunits of the viral rnadependent rna polymerase and the viral matrix protein [30] [31] [32] [33] . it also binds to several host factors which include crm1, uap56, alpha-importin 1 and nf90 [33] [34] [35] [36] [37] . through these interactions, iav-np is known to encapsidate the viral genome, regulate virus transcription and replication, contribute towards pathogenicity of virus, and help in interspecies transmission of the virus [30] . however, so far iav np is not reported to play any role in modulating the host antiviral response. a key component of mammalian antiviral response mechanism is dsrna dependent protein kinase pkr, which is activated by viral dsrna [8] . upon activation, pkr gets dimerized and autophosphorylated at multiple serine and threonine residues. activated pkr phosphorylates eukaryotic translation initiation factor eif2a, which in phosphorylated state cannot participate in mrna translation [12] . this is an important strategy of the host to arrest translation of viral mrnas thereby limiting viral replication [9, 13] . another crucial host pathway which is activated by pkr is irf3mediated ifnb production. activation of pkr is known to enhance irf3 phosphorylation and nuclear movement where it drives expression of interferon b production and built up of antiviral host response [14] . similarly, pkr also has other substrates such as mapk and ikkß which upon phosphorylation trigger various signaling pathways leading to apoptosis or interferon response [10, 11] . being such a crucial molecule, pkr is very often the target of viral factors [15] [16] [17] [18] . in case of influenza virus infection, viral ns1 protein is known to bind directly to pkr and inhibit its activation [20, 21] . ns1 also inhibits the function of retinoic acid inducible gene-i (rig-i), a cytosolic pathogen sensor involved in the antiviral response [49] . apart from that, pkr activity is controlled by another mechanism where p58 ipk , the cellular inhibitor of pkr is activated in influenza virus infected cells [25, 28] . further, p58 ipk itself is inhibited by hsp40 and is present as p58 ipk -hsp40 complex under normal conditions. however upon influenza virus infection, it is released from the hsp40 complex and inhibits pkr activation [24] . in a recent report, it was shown that m2 protein of influenza a virus stabilizes the p58 ipk -hsp40 complex and activates pkr phosphorylation, probably during later stage of infection [50] . however the mechanism of dissociation of hsp40-p58 ipk complex and concomitant pkr inhibition during influenza virus infection remain unknown. here, we report that iav np interacts with the human chaperone hsp40 and employs this interaction to mitigate pkrmediated antiviral response of the host. np-hsp40 interaction was identified through a yeast two-hybrid screen and confirmed in a cell-free translation system, in transfected cells and in influenza virus infected cells. the interaction was found to be conserved across different influenza a viruses, ranging from seasonal, avian h5n1 virus and the 2009 h1n1 pandemic virus despite substantial amino acid differences that range from 0-5% within a subtype/group and 6-10% between the subgroups in np amino-acid sequence. our findings demonstrate that iav np is the viral component that dissociates p58 ipk from the p58 ipk -hsp40 complex during influenza virus infection in mammalian cells. it was observed that during the course of influenza virus infection in lung epithelial cells, a gradual increase in the association of np with hsp40 coincided with a concomitant decrease in p58 ipk association with hsp40. increased activity of p58 ipk , promoted by np, should lead to the inhibition of pkr activation and subsequent downstream effects (fig. 8 ). in accordance with the above hypothesis, we observed that ectopic expression of iav np in mammalian cells substantially reduced the phosphorylation levels of pkr and eif2a. furthermore, sirna-mediated inhibition of np expression during influenza virus infection led to increased phosphorylation of pkr and eif2a, confirming the role of np in the negative regulation of pkr. although eif2a is phosphorylated by other kinases also, namely, hri, gcn2 and perk which are activated during stress condition, only pkr is known to be targeted by viral inhibitors [12] . in line with this, np and ns1 had similar effects on pkr mediated eif2a phosphorylation; however their synergistic effect was higher than their individual effects (fig. 6 b) . activation of pkr signaling during virus infections is known to result in irf3 phosphorylation and concomitant ifnb production. however irf3 is not a direct substrate of pkr and it can get activated by the rig i pathway, nfkb pathway and other unknown mechanisms [14, 15] . influenza ns1 protein is known to inhibit pkr, rig i and nfkb pathways, thus it is expected to have greater impact on irf3 phosphorylation as compared to np, which may inhibit only pkr mediated irf3 phosphorylation [23, 24] . in line with this, we observed that np inhibition during iav infection led to enhanced irf3 phosphorylation, ifnb production and reduced viral replication; however inhibition of ns1 had greater impact on these events. as expected the synergistic effect of np and ns1 inhibition on irf3 activity was higher than their individual effects. the effect of np on ifnb production is also reflected on virus replication as sirna-mediated inhibition of np led to reduced vrna production. this effect may also be attributed to the essential requirement of np for proper functioning of influenza virus polymerase. however the inhibitory action of np on pkr-mediated host response may also contribute to the reduced virus replication in case of sirna-mediated inhibition of np. based on our findings, we proposed a model for pkr inhibition by influenza virus nucleoprotein as shown in fig. 7 . according to this model, iav np interacts with hsp40 and facilitates the release of p58 ipk from it, which in turn inhibits pkr activation (fig. 8) . reduced pkr activity, on one hand leads to reduced eif2a phosphorylation and ensures continued translation from viral mrnas and on the other hand leads to reduced irf3 mediated ifnb production. therefore, apart from the ns1 protein which is already known to inhibit pkr activation and irf3 phosphorylation [21, 24] , np also participates in this process, but through a different mechanism involving hsp40. with structure information of both np and hsp40 being available [30, 42] , it would be interesting to see which domains and key amino-acid residues are involved in this interaction. since the np-hsp40 interaction is conserved across influenza viruses of various subtypes including the 2009 pandemic h1n1 virus, it serves as an important target for developing anti-viral strategies. yeast two-hybrid screening gal4 based matchmaker (clontech) yeast two-hybrid system was used for screening human lung cdna library, as described previously [51] . h5n1 np (a/hatay/2004) gene cloned in pgbk vector (clontech) was used as bait and a mammalian cdna library cloned in pgad (clontech) vector was used as prey. the ah109 strain of yeast was used for co-transformation of bait and prey plasmids. the full-length hsp40 gene was cloned into pgad vector and used in yeast two-hybrid assays. colonies which grew on l -t -hplates (leucine, tyrosine and histidine dropout standard dextrose media) supplemented with 50 mm aminotriazole were considered positive. ß-gal assays (liquid and filter) were performed as per manufacturer's protocols. all dna transfections were done using lipofectamine 2000 (invitrogen) and cells were maintained in dmem medium devoid of serum and antibiotics. six hours post-transfection, culture medium was supplemented with 5% fcs and 24 h post-transfection the medium was replaced with fresh culture medium. all virus infections were done at multiplicity of infection (moi) of 1 for 1 h in dmem medium supplemented with 2% bsa (gibco). after 1 h incubation, the cells were washed with dmem once and then grown with dmem supplemented with 0.2% bsa and 1 mg/ml n-p-tosyl-1phenyl alanine chloromethyl ketone (tpck) (sigma aldrich). the virus strains used in infection experiments are listed in table 1 . cells were lysed using a buffer (20 mm hepes, ph 7.5, 150 mm nacl, 1 mm edta, 10% glycerol, 1% triton x-100) supplemented with protease-inhibitors (roche diagnostics) and the lysates were subject to sds page. anti-np antibodies were obtained from abcam and the immunology and pathogenesis branch, influenza division, centres for disease control and prevention, atlanta, ga, usa. antibodies against pkr, p-pkr, eif2a, p-eif2a, p58 ipk and hsp40 were obtained from cell signaling. anti-ß-actin antibody was purchased from sigma-aldrich. anti-myc tag and anti-ns1 antibodies were purchased from santa cruz. cellular lysates were incubated with primary antibody overnight followed by incubation with protein a dyna beads (invitrogen) for 2 hours. beads were washed thrice and the ip products were subjected to western blotting. np was immunoprecipitated using anti-np monoclonal antibody (immunology and pathogenesis branch/ipb, cdc, atlanta) in case of infection or anti-myc tag antibody in case of transfection. hsp40 was immunoprecipitated using anti-hsp40 monoclonal antibody (cell signaling). after infection or transfection for 24 h, a549 cells were fixed with 2% paraformaldehyde for 30 min at room temperature. they were permeabilized with 0.5% triton x-100 for 5 min at room temperature and blocked with pbs containing 2% bovine albumin. immunostaining was performed using rabbit anti-hsp40 (cell signaling) and mouse anti-np (ipb, cdc, atlanta) antibodies. unbound antibodies were washed away with pbs and cells were incubated with alexa488 tagged goat anti-rabbit antibodies and alexa594 tagged goat anti-mouse. nuclei were stained with dapi. photomicrographs were captured at 1006 magnification using a leica dm6000b confocal microscope. images were processed using nis elements ar 3.0 software (nikon). control (non-targeting) and np-and ns1-specific sirnas of pr8 were purchased from dharmacon and the cells were transfected using the dharmafect 1 transfection reagent (dharmacon). in each case, a pool of three specific sirnas capable of targeting different regions of np or ns1 were used (table 2) . a549 cells at a density of 10 6 /well of a 6-well plate were transfected with 90 nm of the indicated sirna for 6 h prior to infection with a/ pr/8/34 at a moi of 1. lysates were prepared 24 h postinfection and analyzed for the expression of np, ns1 and other cellular proteins by western blotting. total rna was isolated from cells using the rnaeasy kit (qiagen, valencia, ca, usa) and real-time rt-pcr was conducted using a stratagene q3005 pcr machine for expression of ifnb, b-actin mrna and np vrna. for each sample, 2 mg of rna was reverse transcribed using superscript ii reverse transcriptase (invitrogen, carlsbad, ca, usa) according to the manufacturer's directions. oligo dt primers were used for ifnb and b-actin cdna synthesis. for np vrna, cdna was synthesized as described by ge et al [52] . (parallel reactions without reverse transcriptase were included as negative controls. reverse transcription reactions (1/50 th of each reaction) were analyzed in using syber green q-pcr reagents (stratagene, la jolla, ca, usa). interacts with hsp40, thereby displacing p58 ipk from the hsp40-p58 ipk complex. as a result, there is an increased amount of free p58 ipk available in the cell which prevents pkr activation. downregulation in pkr activity ensures less eif2a phosphorylation and continued translation from viral mrnas. on the other hand reduced pkr activity also leads to reduced irf3 activation and subsequent ifnb production. doi:10.1371/journal.pone.0020215.g008 pcr condition was kept as 94uc for 15 s, annealing at 56uc for 30 s, and extension at 72uc for 30 s for a total of 45 cycles. the threshold cycle number for cdna was normalized to that of b-actin mrna, and the resulting value was converted to a linear scale. data from three independent experiments were taken account for analysis. all data points fell into a normal distribution and there were no outliers. primer sets used for these studies are provided in table 3 . figure s1 human heat shock protein 40 was found to interact with influenza a nucleoprotein in yeast twohybrid system. a. yeast two-hybrid screen was performed to find the host interacting partners for h5n1 iav np. results with one of the positive co-transformants (later found to be hsp40 by blast analysis) are shown. ah109 yeast strain cotransformed with np-gbk bait plasmid and hsp40-gad prey plasmid grew in minimal synthetic ypd media devoid of leucine, tryptophan and histidine amino-acids. positive colonies grew on plates supplemented with up to 50 mm aminotriazole (at). a filter b-gal assay was performed to confirm the interaction. blue colored colonies indicate positive clones. b. np-hsp40 interaction was confirmed by liquid ß-gal assay and was found to be statistically comparable to the positive control used (p-value = 0.0668). in the bar-graph, bar 1 represents untransformed ah109 yeast cells; bars 2 and 3 represent control prey plasmids, bars 4 and 5 represent prey plasmids expressing full-length hsp40 and np, respectively; bar 6 represents the co-transformation of hsp40 and np plasmids; bar 7 is a positive control (sars coronavirus np both as bait and prey self-associating to form oligomers) [53] . (tif) figure s2 phylogenetic analysis of np sequence used in the study. a phylogenetic tree was constructed using neighbor-joining method, nucleotide model tamura-nei, in mega version 4 [54] . np gene sequences from selected human seasonal, avian, swine and 2009 pandemic influenza viral isolates were used. the tree shows evolutionary distances between various strains of influenza. the 2009 pandemic h1n1 np belongs to the classical swine lineage which had previous limited introductions into humans and is more distantly related to the np of seasonal or h5 influenza viruses. the h5n1 virus used in this study is shown in red and other iavs used in infection assays are boxed. (tif) figure s3 amino acid sequence comparison of np sequence used in the study. the number and percent difference in amino acids of the iav subtypes used in the infection assays including seasonal h1n1 and h3n2, avian h5n1 and 2009 h1n1 pandemic are compared to a/puerto rico/8/1934 (h1n1) virus. analyses were conducted using the dayhoff matrix based method in mega4 [48] . orthomyxoviridae: the viruses and their replication fields virology fourth edition structures of influenza a proteins and insights into antiviral drug targets h5n1 avian influenza: preventive and therapeutic strategies against a pandemic cellular networks involved in the influenza virus life cycle inverse interference: how viruses fight the interferon system cytoplasmic nucleic acid sensors in antiviral immunity molecular cloning and characterization of the human double-stranded rna-activated protein kinase induced by interferon constitutive expression of human double-stranded rna-activated p68 kinase in murine cells mediates phosphorylation of eukaryotic initiation factor 2 and partial resistance to encephalomyocarditis virus growth protein kinase r (pkr) interacts with and activates mitogen-activated protein kinase kinase 6 (mkk6) in response to double-stranded rna stimulation pkr stimulates nf-kappab irrespective of its kinase function by interacting with the ikappab kinase complex coping with stress: eif2a kinases and translational control inhibition of host and viral translation during vesicular stomatitis virus infection. eif2a is responsible for the inhibition of viral but not host translation multiple signaling pathways leading to activation of interferon regulatory factor 3 irf3 and irf7 phosphorylation in virus-infected cells does not require double-stranded rnadependent protein kinase r or ikb kinase but is blocked by vaccinia virus e3l protein cellular inhibitors of the interferon induced, dsrnaactivated protein kinase molecular mechanisms of interferon resistance mediated by viral-directed inhibition of pkr, the interferon-induced protein kinase inhibition of pkr by rna and dna viruses the dsrna protein kinase pkr: virus and cell control influenza virus regulates protein synthesis during infection by repressing autophosphorylation and activity of the cellular 68,000-mr protein kinase binding of the influenza virus ns1 protein to double-stranded rna inhibits the activation of the protein kinase that phosphorylates the elf-2 translation initiation factor influenza virus ns1 protein counteracts pkr-mediated inhibition of replication influenza a virus ns1 protein prevents activation of nf-kappab and induction of alpha/beta interferon the ns1 protein of a human influenza virus inhibits type i interferon production and the induction of antiviral responses in primary human dendritic and respiratory epithelial cells the p58 cellular inhibitor complexes with the interferon-induced, double-stranded rnadependent protein kinase, pkr, to regulate its autophosphorylation and activity double-stranded rna-independent dimerization of interferon-induced protein kinase pkr and inhibition of dimerization by the cellular p58ipk inhibitor the molecular chaperone hsp40 regulates the activity of p58ipk, the cellular inhibitor of pkr the cellular protein p58ipk regulates influenza virus mrna translation and replication through a pkr-mediated mechanism p58ipk: a novel ''cihd'' member of the host innate defense response against pathogenic virus infection the mechanism by which influenza a virus nucleoprotein forms oligomers and binds rna the influenza virus nucleoprotein: a multifunctional rna-binding protein pivotal to virus replication structure and sequence analysis of influenza a virus nucleoprotein influenza virus nucleoprotein interacts with influenza virus polymerase proteins interaction of the influenza virus nucleoprotein with the cellular crm1-mediated nuclear export pathway cellular splicing factor raf-2p48/npi-5/bat1/uap56 interacts with the influenza virus nucleoprotein and enhances viral rna synthesis interaction of polymerase subunit pb2 and np with importin alpha1 is a determinant of host range of influenza a virus nuclear factor 90 negatively regulates influenza virus replication by interacting with viral nucleoprotein a stressinducible 40 kda protein (hsp40): purification by modified two-dimensional gel electrophoresis and co-localization with hsc70(p73) in heat-shocked hela cells dual role of heat shock proteins as regulators of apoptosis and innate immunity structure, function and evolution of dnaj: conservation and adaptation of chaperone function the diversity of the dnaj/hsp40 family, the crucial partners for hsp70 chaperones heat shock protein 40: structural studies and their functional implications hsp40 facilitates nuclear import of the human immunodeficiency virus type 2 vpx-mediated preintegration complex heat shock protein 40 is necessary for human immunodeficiency virus-1 nef-mediated enhancement of viral gene expression and replication turnover of hepatitis b virus x protein is facilitated by hdj1, a human hsp40/dnaj protein involvement of hsp90 in assembly and nuclear import of influenza virus rna polymerase subunits heat shock protein 70 is related to thermal inhibition of nuclear export of the influenza virus ribonucleoprotein complex molecular chaperone function of mammalian hsp70 and hsp40-a review ns1 protein of influenza a virus inhibits the function of intracytoplasmic pathogen sensor, rig-i interaction of hsp40 with influenza virus m2 protein: implications for pkr signaling pathway the orf3 protein of hepatitis e virus interacts with hemopexin by means of its 26 amino acid n-terminal hydrophobic domain ii rna interference of influenza virus production by directly targeting mrna for degradation and indirectly inhibiting all viral rna transcription the nucleocapsid protein of the sars coronavirus is capable of self-association through a c-terminal 209 amino acid interaction domain mega4: molecular evolutionary genetics analysis (mega) software version 4.0 key: cord-343973-n5ogyxz7 authors: ip, andrew; berry, donald a.; hansen, eric; goy, andre h.; pecora, andrew l.; sinclaire, brittany a.; bednarz, urszula; marafelias, michael; berry, scott m.; berry, nicholas s.; mathura, shivam; sawczuk, ihor s.; biran, noa; go, ronaldo c.; sperber, steven; piwoz, julia a.; balani, bindu; cicogna, cristina; sebti, rani; zuckerman, jerry; rose, keith m.; tank, lisa; jacobs, laurie g.; korcak, jason; timmapuri, sarah l.; underwood, joseph p.; sugalski, gregory; barsky, carol; varga, daniel w.; asif, arif; landolfi, joseph c.; goldberg, stuart l. title: hydroxychloroquine and tocilizumab therapy in covid-19 patients—an observational study date: 2020-08-13 journal: plos one doi: 10.1371/journal.pone.0237693 sha: doc_id: 343973 cord_uid: n5ogyxz7 hydroxychloroquine has been touted as a potential covid-19 treatment. tocilizumab, an inhibitor of il-6, has also been proposed as a treatment of critically ill patients. in this retrospective observational cohort study drawn from electronic health records we sought to describe the association between mortality and hydroxychloroquine or tocilizumab therapy among hospitalized covid-19 patients. patients were hospitalized at a 13-hospital network spanning new jersey usa between march 1, 2020 and april 22, 2020 with positive polymerase chain reaction results for sars-cov-2. follow up was through may 5, 2020. among 2512 hospitalized patients with covid-19 there have been 547 deaths (22%), 1539 (61%) discharges and 426 (17%) remain hospitalized. 1914 (76%) received at least one dose of hydroxychloroquine and 1473 (59%) received hydroxychloroquine with azithromycin. after adjusting for imbalances via propensity modeling, compared to receiving neither drug, there were no significant differences in associated mortality for patients receiving any hydroxychloroquine during the hospitalization (hr, 0.99 [95% ci, 0.80–1.22]), hydroxychloroquine alone (hr, 1.02 [95% ci, 0.83–1.27]), or hydroxychloroquine with azithromycin (hr, 0.98 [95% ci, 0.75–1.28]). the 30-day unadjusted mortality for patients receiving hydroxychloroquine alone, azithromycin alone, the combination or neither drug was 25%, 20%, 18%, and 20%, respectively. among 547 evaluable icu patients, including 134 receiving tocilizumab in the icu, an exploratory analysis found a trend towards an improved survival association with tocilizumab treatment (adjusted hr, 0.76 [95% ci, 0.57–1.00]), with 30 day unadjusted mortality with and without tocilizumab of 46% versus 56%. this observational cohort study suggests hydroxychloroquine, either alone or in combination with azithromycin, was not associated with a survival benefit among hospitalized covid-19 patients. tocilizumab demonstrated a trend association towards reduced mortality among icu patients. our findings are limited to hospitalized patients and must be interpreted with caution while awaiting results of randomized trials. trial registration: clinicaltrials.gov identifier: nct04347993 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the global pandemic caused by a novel coronavirus [severe acute respiratory syndrome (sars)-cov-2] and its disease, covid-19, has led to infection in over 15.8 million individuals and more than 640,000 deaths as of july 25, 2020 [1, 2] . as there are no approved treatments, management of covid-19 is largely supportive [3, 4] . one empirical treatment for covid-19 which has received attention is hydroxychloroquine, an antimalarial drug repurposed in recognition of its anti-inflammatory properties in the treatment of autoimmune conditions. hydroxychloroquine and its analogue, chloroquine, demonstrate suppression of sars-cov-2 replication in vitro, with hydroxychloroquine demonstrating greater potency [5, 6] . studies from the original sars-cov virus suggest a mechanism of action involving impairment of the terminal glycosylation of angiotensin converting enzyme 2 (ace2), inhibition of sars-cov viral entry, and rapid elevation of endosomal ph that prevents endosome-mediated viral entry [7] [8] [9] [10] . the immunomodulatory effects are thought to be due to the accumulation of the drug in lymphocytes and macrophages leading to reduction of proinflammatory cytokines, including type i interferons, tumor necrosis factor alpha, and interleukin-6 [9] . other anti-inflammatory effects may be related to inhibition of signaling pathways [11] . several early small clinical reports have shown conflicting evidence regarding the efficacy of hydroxychloroquine in covid-19 [12, 13] . subsequently, an observational cohort study of 1376 hospitalized patients from a new york hospital using propensity modeling found no significant association between hydroxychloroquine use and intubation or death (hazard ratio, 1.04, 95% confidence interval, 0.82 to 1.32) [14] . a second observational cohort study of 1438 hospitalized patients throughout the new york metropolitan region also found a lack of survival association with hydroxychloroquine with or without concomitant azithromycin (hr 1.35 and 1.08 respectively) [15] . a recently reported randomized brazilian trial enrolling 504 hospitalized sars-cov-2 confirmed patients with mild-to-moderate disease (defined as not requiring significant supplemental oxygen support) found that a 7-day course of hydroxychloroquine either with azithromycin or alone did not result in better clinical outcomes as measured by a seven-level ordinal scale at 15 days [16] . as the clinical course of covid-19 progresses, patients enter a hyperinflammatory phase with dysregulation of adaptive immune responses and a cytokine storm with elevation in plasma levels of pro-inflammatory cytokines including interleukins (il) 2,6, 7, and 10, granulocyte-colony stimulating factor (g-csf), interferon-gamma-inducible protein-10 (ifngamma, il-10), and tumor necrosis factor alpha (tnf-alpha). this cytokine storm results in a pro-thrombotic milieu, cardiomyopathy, and ultimately multi-organ failure [17, 18] . tocilizumab, a monoclonal antibody against membrane bound il-6 receptor inhibiting binding of soluble il-6 and subsequent signal transduction, has been proposed as a therapeutic candidate for impeding cytokine storm [19] . small single institution series have suggested benefit among severely ill patients [20, 21] . preliminary results from a press release for the french corimu-no-toci trial (nct04331808), an open-label randomized trial of hospitalized patients with covid-19 (n = 129), noted a reduction in the proportion of participants who died or needed we have included in the supporting information the statistical output that may assist interested readers in understanding the data more fully and could be utilized to assist in independent confirmation. ventilation in the tocilizumab group, although full results of the trial have not yet been released [22] . an italian observational study involving 544 covid-19 patients (with 16% requiring mechanical ventilator support) reported that tocilizumab treatment was associated with a reduced risk of subsequent invasive mechanical ventilation or death [23] . a michigan usa observational study also noted an associated reduction in mortality among intubated covid-19 patients [24] . in the absence of rcts, observational studies may provide useful early insights into effective treatment strategies [25, 26] . however in an observational study, treatment allocations are based upon physician judgement, rather than random assignment, increasing the risk of bias and not accounting for known and unknown risk factors. thus, causal inferences on effectiveness of treatments are challenging, but confounding effects can be partially mitigated via statistical methods [27, 28] . understanding these limitations, but with the urgency for evaluating potential therapeutic approaches during the current covid-19 pandemic, we established an observational database within a 13-hospital network spanning new jersey using an integrated electronic health record (ehr) system (epic; verona, wi). in this observational cohort study we report our survival outcomes with hydroxychloroquine and tocilizumab among hospitalized patients with covid-19. this retrospective, observational, multicenter cohort within the hackensack meridian health network (hmh) used ehr-derived data to study hospitalized covid-19 patient outcomes. our primary objective was to analyze the effect of hydroxychloroquine in hospitalized patients. a secondary, exploratory objective was to investigate the effect of tocilizumab in the icu population. patients were included in the database based on the following inclusion and exclusion criteria: 1) positive sars-cov-2 diagnosis by reverse-transcriptase polymerase chain reaction, 2) hospitalized within the time frame of march 1, 2020 until may 5, 2020, 3) non-pregnant, 4) not on a randomized clinical trial, and 5) did not die during first day of hospitalization, and 5) were not discharged to home within 24 hours (fig 1) . hackensack meridian health institutional review board approved this study on march 27, 2020 under study # pro2020-0342. waiver of consent and hipaa authorization was granted as this retrospective research was a non-interventional protocol utilizing electronic health records to abstract data. no patients were contacted for this study. protected health information was not made available except to investigators. de-identified information was provided to statisticians. the study period was march 1, 2020 until may 5, 2020. we collected data from hmh's ehr (epic) which is utilized throughout the network. hospitalized patients throughout hmh were flagged by the ehr if sars-cov-2 testing was positive. these ehr-generated reports served as our eligible cohort to sample. demographic, clinical characteristics, treatments, and outcomes were manually abstracted by research nurses and physicians from the john theurer cancer center at hackensack university medical center. assigning patients to our data team occurred in real-time, and not randomized. to reduce sampling bias, we abstracted 3325 of the 3438 (97%) possible hospitalized patients by april 22, 2020 (with follow up until may 5, 2020), and performed stratification as discussed in our analytic approach. data abstracted by the team was entered using redcap (research electronic data capture) hosted at hmh [29, 30] . data abstraction occurred daily from march 28, 2020 until may 5, 2020. quality control was performed by physicians (ai, slg) overseeing nurse or physician abstraction. demographic information was collected by an electronic face sheet, with gender, race or ethnicity self-reported. academic centers were defined as quaternary referral centers with accredited residency, fellowship, and medical student programs. nursing home or rehabilitation patients, if diagnosed prior to hospital admission, were defined as ambulatory patients. comorbidities were defined as diagnosed prior to hospitalized for covid-19. history of hypertension, diabetes, chronic lung disease (copd or asthma), hypertension, cancer, coronary artery disease, cerebrovascular disease, renal failure, and rheumatologic disorder were abstracted from provider notes or medical history sections found within the ehr. if not listed in the patient's record, the comorbidity was recorded as absent. presenting clinical data was abstracted from thorough review of unstructured notes as well as structured data. hospital readmissions were counted as the same admission, with baseline data used from the initial hospitalization. if multiple positive or indeterminate results were found in a patient's record for sars-cov-2, the first initial positive test was used as the date of diagnosis. for the effect of hydroxychloroquine, we separated patients into 4 different groups-1) hydroxychloroquine, 2) hydroxychloroquine in combination with azithromycin, 3) azithromycin alone, and 4) neither drug. exposure to hydroxychloroquine or azithromycin was defined as documentation of drug administration in the ehr. dosing, duration, and timing in relation to symptom onset and admission were also collected. if no evidence of administration of drug was found, this was recorded as not having received the drug. for tocilizumab, exposure was defined as receipt of the drug within the icu setting as found in the ehr. if no date of administration was found, this was labeled as insufficient missing data for analysis. if no evidence of administration of the drug was found, this was recorded as not having received the drug. the primary outcome measurement was death with follow-up through may 5, 2020. mortality was identified on chart review by a provider note announcing time of death or if the ehr labeled the patient as deceased. as death certificates were not readily available, cause of death was identified using the ehr by identifying the most immediate cause(s) documented [31] . respiratory cause of death included any hypoxic condition related to covid-19. cardiac cause of death included cardiac arrest, myocardial infarction, or arrhythmias. infectious cause of death included bacterial sepsis or secondary infections not including covid-19. other cause of death included multi-organ failure as well as alternative causes. follow-up occurred until the study cut-off date of may 5. adverse drug events related to hydroxychloroquine were also described, including discontinuation due to arrhythmia or qt prolongation. this was obtained by provider documentation as well as ekg reports within the ehr. the statistical plan is available in the (s1 appendix). descriptive analyses of baseline characteristics by hydroxychloroquine exposure were performed using chi-square tests for categorical variables. dose, frequency, timing, and duration of treatment were also summarized. we used propensity-score stratification for the remaining statistical analyses [32, 33]. we fit a logistic regression model to the probability of being assigned to the experimental arm (tocilizumab, hydroxychloroquine, or hydroxychloroquine plus azithromycin) compared with the control population (not assigned to the respective treatment). patients are stratified into propensity-score quintiles and these strata are used to adjust treatment effects in a proportional hazards model. the model for selecting factors to be included in propensity scores was a two-stage backward selection approach. we evaluated each of the factors as univariate predictors with factors having p-value less than 0.10 included for further consideration. we removed factors sequentially and one at a time from the multivariate model if their p-values were less than 0.50, with largest p-values considered first. we fit the final propensity-scores model using multivariate logistic regression of the selected factors. we then stratified the propensity scores for the entire population into quintiles and used these quintiles as an ordinal (4-degree-of-freedom) variable to adjust the relative treatment comparison in a proportional hazards model (see s2 appendix, for output). we evaluated the following factors for all propensity-score models: gender, coronary disease, stroke, heart failure, arrhythmia, african american, copd, renal failure, rheumatologic disorder, inflammatory bowel disease, advanced liver disease, age, diabetes mellitus, insulin use prior to hospitalization, asthma, hiv/hepatitis, any cancer, and log ferritin. the final propensity-score model for hydroxychloroquine included the first 15 of these factors. that for hydroxychloroquine plus azithromycin included the first 15 of these factors plus cancer. that for tocilizumab included age, gender, copd, and renal failure. tocilizumab was assigned preferentially for patients in the icu. tocilizumab patients included in our analyses received their first dose of the drug in the icu. control patients were those who were admitted to the icu and who never received tocilizumab either before or after admission to the icu. the start time for analysis was the day of admission to the icu. hydroxychloroquine and hydroxychloroquine plus azithromycin were evaluated from day of hospital admission, whether initially in the icu or not. the control population consisted of patients who never received the respective treatment. we also sought to address the factorial nature of treatment with combination hydroxychloroquine and azithromycin. propensity scores are based on predicting particular an individual therapy and so do not naturally generalize to a factorial setting. for these analyses we averaged the two propensity scores calculated separately for hydroxychloroquine and hydroxychloroquine plus azithromycin. we then stratified into propensity quintiles based on that average and proceeded as indicated above. the raw results of our proportional hazards analyses adjusting for propensity scores is provided (s2 appendix). patients still alive and in the hospital were censored as of may 5, 2020. patients who had been discharged from the hospital were censored as of day 36 following hospital admission. we provide hazard ratios of treatment in comparison with control together with corresponding p-values and confidence intervals based on wald tests. we show the unadjusted survival data using kaplan-meier plots from which we identify 30-day mortality for each treatment. statistical calculations used jmp 1 pro 15.0.0. confidence intervals and p-values in this study are descriptive measures of distance between outcomes of treatment groups or distance from hazard ratio 1.00. these measures do not have the same inferential interpretations that are possible for primary end point analyses of rcts. there were 3,438 patients flagged in our ehr with positive covid19 infection within the 13-hosptial network spanning new jersey, and data was abstracted on 3,325 (97%). 2512 hospitalized patients met inclusion criteria for this study (fig 1) . as indicated in table 1 , the median age of the cohort was 64 years (iqr 52-76) with a male predominance (62%). nursing home and rehabilitation patients comprised 16% of the cohort. co-morbidities were common with 55% having hypertension, 41% obesity (bmi �30), 32% diabetes, 16% coronary arterial disease, 15% copd/asthma, 12% cancer, and 31% having 3 or more chronic conditions. at the time of hospital presentation, fever was present in 72%, 69% with dyspnea, 68% with cough, 23% gastrointestinal complaints and 12% had altered mental status. the median time from self-reported onset of symptoms to hospitalization was 5 days (iqr 3-7). 611 (24%) patients required intensive care unit support during their hospitalization, of which 150 patients were admitted to the icu within the first day. oxygen saturation below 94% was identified in 44%. when measured and recorded in the electronic health record, inflammatory markers were elevated with serum ferritin >1500 ng/ml in 23% and d-dimer >1 mcg/ml in 16% of patients. in this non-randomized observational cohort 1914 hospitalized patients (76%) received at least one dose of hydroxychloroquine during the study timeframe (23% as single agent and 77% in combination with azithromycin) (fig 1) . dosing and duration was at prescribers' discretion. the majority of patients received 800 mg on day 1, and 400 mg on day 2-5 (80%, n = 1533), followed by 200 mg tid (4%, n = 71) and other (15%, n = 299), and missing dosing information (1%, n = 11). median duration of hydroxychloroquine was 5 days (iqr 4-5). the median days of symptoms before hydroxychloroquine administration was 5 (iqr 2-9), and median days in hospital before first dose 1 was 1 (iqr 0-2). patients receiving hydroxychloroquine at any time during their hospitalization were younger, less likely to live in a nursing home, but presented later in their clinical course (5 days vs 3 days of symptoms prior to hospital admission) with more symptomatic disease (higher incidences of fever, cough, dyspnea, and lower oxygen saturation) ( table 1) discontinuation of hydroxychloroquine due to prolongation of qtc or arrhythmias was recorded in the electronic health records in 76 (4%) and 33 (2%) patients. during the entire hospital course, arrhythmias were noted in 101 (5%) hydroxychloroquine patients, compared to 22 (4%) in the non-hydroxychloroquine cohort. cardiomyopathy was described in 20 (1%) treated patients compared to 7 (1%) patients without hydroxychloroquine. among 2512 hospitalized patients with covid-19 there have been 547 deaths (22%), 1539 (61%) discharges and 426 (17%) remain hospitalized as of the study cut-off date. in the entire cohort, causes of death included 319 (58%) respiratory, 108 (20%) cardiac, 42 (8%) other, 41 (7%) infectious, and 37 (7%) other. within the hydroxychloroquine treated cohort, 89 of 432 (21%) of deaths were attributed to cardiac causes, compared to 19 of 115 (16%) deaths in the non-hydroxychloroquine cohort. as shown in table 2 , using propensity modeling as described above, there was no significant association between survival and any use of hydroxychloroquine during the hospitalization ( azithromycin alone, the combination or neither drug was 25%, 20%, 18%, and 20%, respectively (fig 2) . toci tocilizumab; hcq hydroxychloroquine; azi azithromycin. toci in icu includes all patients whose first dose of tocilizumab was in the icu or controls who were admitted to the icu and known to not have received tocilizumab during their hospital stay. "in hospital" includes all patients who received the drug at any time during their stay in the hospital. "main effect" is the estimated benefit of the drug whether added to the other drug or not. a drug given "only" means not with the other drug. 30-day mortality rates are estimated using the corresponding kaplan-meier survival curve at 30 days ( see figs 2 and 3 ). 198 (8%) patients received tocilizumab therapy during their hospitalization. table 3 shows baseline characteristics. 11 patients received their first dose prior to admission to the icu and 53 patients had unknown timing or insufficient data. thus 134 patients with documentation of their first dose of tocilizumab within an icu setting represented the exploratory treatment cohort. 413 patients in the icu never received tocilizumab as of may 5, 2020 and serve as the control cohort. tocilizumab was administered as a single dose in 104 (78%), with the majority receiving 400 mg (96%), followed by 800 mg (1%), 8 mg/kg (1%), 4 mg/kg (1%), and missing dosing (1%). secondary bacteremia occurred in 44 of the 413 (11%) patients in the non-treated group, compared to 18/134 (13%) in the treated group. secondary pneumonia occurred in 25 of the 413 (6%) patients in the non-treated group, compared to 12 of the 134 (9%) in the treated group. as shown in table 2 , in the analysis using propensity modeling, there was a trend association between survival and treatment with tocilizumab within the icu setting (hr, 0.76 [95% ci, 0.57-1.00]). the unadjusted 30-day mortality favored tocilizumab (46% versus 56%) (fig 3) . this retrospective observational cohort study of 2512 hospitalized covid-19 patients within a 13-hospital network did not find the empirical use of hydroxychloroquine with or without co-treatment with azithromycin to be associated with a reduction in mortality (adjusted hr, 0.99 for any hydroxychloroquine during hospitalization [95% ci, 0.80-1.22]). our multi-center findings confirm the recent observational studies from new york [14, 15] . our results are also in agreement with the results of a recent randomized controlled study that enrolled patients with only mild-to-moderate disease, a cohort with less severe than our study [16] . collectively these studies do not support the routine use of hydroxychloroquine outside a clinical trial. furthermore, none of the reported observational studies have addressed the role of hydroxychloroquine among individuals with minimally symptomatic disease in the pre-hospital setting. unlike randomized controlled trials, observational studies have inherent biases in patient allocations that cannot be fully adjusted for during statistical analyses. although we utilized propensity modeling to mitigate known imbalances it is possible that unmeasured confounding factors may still be important. for example, in our series we observed a change in the prescribing patterns of hydroxychloroquine during the study timeframe. similarly dosing and timing of hydroxychloroquine varied throughout the 13-hospital network. these factors a potentially clinically important finding from an exploratory analysis revealed a favorable association between tocilizumab administration and survival among covid-19 patients requiring icu support. our study represents one of the largest report of tocilizumab during this pandemic but is subject to all of the concerns of observational studies. however, if confirmed by ongoing randomized clinical trials, tocilizumab would represent the first successful therapy to reduce mortality. multiple ongoing rct's will ultimately determine the efficacy of hydroxychloroquine and tocilizumab in covid-19. this observational study has limitations. first, observational studies cannot draw causal inferences given inherent known and unknown confounders. we attempted to adjust for known confounders using our propensity model approach. second, misclassification is possible as we performed manual abstraction of ehr data. third, hydroxychloroquine is a drug that likely has a therapeutic window that requires appropriate dosing, duration, and timing of administration [35] . we acknowledge our results should be interpreted with caution as significant heterogeneity existed within these factors, although a majority of our patients received the same dose, completed 5 days of treatment, and were given the drug within 24 hours of hospitalization. fourth, low sample size limited our exploratory tocilizumab analysis. fifth, our study focused on patients in new jersey, limiting the applicability to other geographic regions, although the state's population is diverse, and the network included 13-hospitals (both academic and community) all with differing covid-19 treatment protocols. lastly, we acknowledge the possibility of sampling bias as we collected data from a convenience sample in attempts to conduct the investigation quickly during this pandemic. this observational cohort study from a multi-hospital system spanning new jersey, when taken together with other studies, does not support the routine (off label and outside of clinical 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caring for their patients assisted in the abstraction of the clinical data. key: cord-337913-eu2gn4bl authors: stojanov, ana; bering, jesse m.; halberstadt, jamin title: does perceived lack of control lead to conspiracy theory beliefs? findings from an online mturk sample date: 2020-08-17 journal: plos one doi: 10.1371/journal.pone.0237771 sha: doc_id: 337913 cord_uid: eu2gn4bl it is widely believed that conspiracy theory beliefs are the product of perceived lack of control. however, to date there is mixed evidence, at best, to support this claim. we consider the reasons why conspiracy theory beliefs do not appear to be based in any straightforward way on control beliefs, interrogating existing findings and presenting new data that call the relationship into question. across six studies conducted online using mturk samples, we observed no effect of control manipulations on conspiracy theory beliefs, while replicating previously reported correlational evidence of their association. the results suggest that conspiracy beliefs are not suitable for compensating for threats to control. we discuss possible reasons for the discrepancy between experimental and correlational effects and examine the limitations of the studies. the world is ruled by a secret cabal. the us government was involved in the 9/11 attacks. hiv is a man-made virus. such statements, which are promoted on the internet and elsewhere, are examples of conspiracy theories: implausible, unwarranted claims that important social events are caused by malevolent clandestine groups, that usually run in contradiction to the explanations offered by the relevant epistemic authorities, and that are embedded in a more general worldview [1] . although researchers have tried to understand conspiracy theory beliefs-which to nonbelievers appear unwarranted by evidence, if not entirely irrational-from many perspectives, including personality theory [2, 3] , intergroup identity [4] [5] [6] , and cognitive bias [7] [8] [9] , arguably the most frequent accounts rely on some notion of "control." the claim that perceived lack of control is the natural breeding ground for conspiracy theory beliefs is a recurring theme in popular science (e.g. [10] [11] [12] [13] [14] ), and many academics agree (e.g., [15] [16] [17] [18] ). however, on closer inspection, neither the theoretical role of control in conspiratorial thinking, nor the empirical relationship between the two constructs, is entirely convincing. although specific accounts differ in their emphasis, control accounts of conspiracy theories generally assume that the perceived loss of personal control is threatening to one's need for order (e.g., [19] ) or one's schemas about the world (e.g., [20] ). conspiracy beliefs help compensate for these threats by ascribing control to external entities or organizations (e.g. [21] [22] [23] ), or by creating new meaning frameworks via novel connections among unrelated events. for example, perceiving a connection between a tornado and the unusually long contrails observed the previous day, provides a basis for believing that the government (or some other entity) has deliberately sprayed chemicals in the sky to cause the tornado. however, it seems equally plausible that conspiracy theories would have the opposite effect on the perception of control and order. a common theme among conspiracy theories is that reality is not what it seems; they are counter-normative narratives that suggest institutions and explanations that we take for granted are illusions-a revelation that, if taken seriously, seems bound to challenge the larger meaning-making systems individuals have come to rely on. furthermore, although people desire order, they seek to optimize, not to maximize it, and it could be argued that conspiracy theories provide "too much" order, structure, and meaning by accounting for errant data, connecting seemingly unconnected events and leaving nothing unexplained [24, 25] . finally, even if conspiracy theories represent an effective means of restoring order and structure, they are not the only means [19, [26] [27] [28] , and, it would seem, a means of last resort. while control accounts do not require that sources of control or meaning be benevolent, people theoretically favour control systems that are both "culturally accessible" and "socially acceptable" [27, 29] . in most-if not all-conspiracy theories, however, the alleged conspirators are malevolent [30] , thus making them poor candidates for compensatory processes when numerous effective and socially sanctioned alternatives exist. the experimental evidence for conspiracies as compensatory sources of personal control is as ambivalent as its theoretical rationale. in a typical experiment, researchers ask participants to write about a time they did not have control, and to compare these participants' conspiracy beliefs with a control group that is asked to describe an event when they felt in complete control (or about a neutral event). some studies report stronger conspiracy beliefs in the low/neutral control group compared to the high control group (e.g. [31] [32] [33] ), but others report weaker ones (e.g. [34] ), and yet others (e.g. [35, 36] ) report no effects of control on conspiracy beliefs at all. one factor in the variability of results is the corresponding variability-and sometimes dubious validity-of how belief in conspiracy theories is operationalized and measured. for example, whitson and galinsky [33, study 4] found that participants recalling uncontrollable situations were more likely to perceive patterns in ambiguous stimuli (e.g., images in visual noise) and links between events (e.g., an increase in e-mail correspondence and a lack of a promotion). however, while conspiracy theories are related to illusory pattern perception [37] , they are not reducible to it. and while novel connections between seemingly unrelated events can, of course, be the building blocks of conspiracy theories, such theories tell a more complex story that taps into a broader worldview. for example, seeing a connection between the "umbrella man" (a man who opened and lifted an umbrella when kennedy's limousine approached) and the assassination of kennedy is a starting point for building a conspiracy theory, but it is not a conspiracy theory per se (e.g., that kennedy's assignation was an organized soviet-backed plot carried out for political purposes). in another operationalization of conspiracy belief, van prooijen and acker [32] asked participants about the construction of the north-south metro line in amsterdam, by posing situations that were technically conspiracies, but also plausible abuses of power warranting legitimate skepticism. accusations such as, "the city council transferred parts of the budget to the bank accounts of others", and "members of the city council received money from construction companies to set this plan in motion," describe political corruption, which differ from "conspiracy theories" in scope, plausibility, official endorsement, and psychological interest. indeed, recent evidence indicates that belief in conspiracy theories and belief in corruption are two distinct psychological constructs, predicted by different aspects of a larger construct ("conspiracy mentality"; [1] ). even studies that explicitly ask about beliefs in specific conspiracy theories may not be well suited for assessing the construct of conspiracy theory belief. for one, participants can easily recognize such items as "conspiracy theories," whose negative connotations are likely to produce socially desirable responses [1] . moreover, using specific conspiracy beliefs limits the generalizability of the findings to very particular and culturally specific claims, and may say little about a person's general tendency to employ conspiratorial logic. more relevant, we argue, is an individual's tendency to engage in conspiracy thinking, rather than their belief in any particular claim. given the significance and potential consequences of widespread conspiracy beliefs [38, 39] , and the plausible but largely unsubstantiated role of control in their appeal, we here report three studies to test the effects of lack of control on conspiracy theory beliefs using a standard priming paradigm and a validated measure of conspiracy ideation, which reflects the belief that a powerful entity lies behind significant social or political events and that the conventional (official) truth is not the "real" truth. a fourth study confirms the results with a measure of specific claims, and two final studies examine two of the most likely moderators of control effects: the degree of control that a particular conspiracy claim affords; and the effectiveness of alternative means of restoring control. although we conceptually replicate previous correlational work-lower control beliefs were associated with stronger conspiracy ideation-we find no evidence for a causal effect. this and subsequent studies were approved by the university of otago human ethics committee. before the beginning of the study participants read an information page and gave written consent for participation; after the study they were debriefed online. participants. in this and subsequent studies, sample size was determined a priori. detecting an effect of f = 0.2, the average effect size reported in psychology research [40] with alpha = 0.05 and power of 80%, required 246 participants. expecting some attrition, we recruited 301 mechanical turk (mturk) workers (105 male, 195 female, 1 other), who completed an online questionnaire designed and presented in the qualtrics survey environment. the mean age of the sample was m = 36.20 (range 18-79 years, sd = 12.24). the majority had bachelor's degree (41.2%), while 27.9% had a high school degree, 16.6% master's degree, 3% doctoral degree, 2.7% no degree; 8.6% reported their education level as "other". materials and procedure. participants first read a general information sheet, which explained the tasks that they would be completing, but did not reveal the hypotheses being tested; the passive deception was revealed and explained in post-experimental debriefing. the experiment was described as a study of memory, in which, participants would be asked to recall and describe an event twice, once at the beginning of the study and once "after a break interval," during which they completed the key dependent variables. following whitson and galinsky [31] , we used the to-be-recalled event as our manipulation of control. the low control group described an event in which they "did not have any control over a situation," while the high control group described an event in which they "were in complete control of the situation." both groups were instructed to include "what happened, how you felt, etc.," and to write at least six lines of text. a neutral group was asked to recall and write about their dinner the previous night. the wording of the low and high control manipulation was taken verbatim from whitson and galinsky [31] with the exception of the instruction for the length of the text. to assess the effectiveness of the manipulation we measured participants' feelings of control on pearlin and schooler's [41] mastery scale. it consists of seven items (e.g., i have little control over the things that happen to me) that capture the extent to which people see themselves as being in control over their life [42] , and which has been used in numerous studies as a measure of perceived control [43, 44] . the scale was anchored at 1 (strongly disagree) and 4 (strongly agree). five items were reverse scored, such that higher scores indicate higher levels of control. in the current study cronbach's alpha was 0.84. conspiracy beliefs were measured on the conspiracy theory ideation subscale of stojanov and halberstadt's [1] conspiracy mentality scale. the subscale consists of seven items capturing the abstract premises of conspiratorial thinking, such as the belief in a powerful entity that controls significant social or political events. (a second dimension of the scale, measuring rational scepticism, is not relevant to the current study.) the subscale has good psychometric characteristics (in the current sample cronbach's alpha was 0.91) and convergent, divergent and predictive validity, and its construct validity has been confirmed in united states, new zealand and north macedonia populations [1] . participants rated their agreement with each statement on a 1-to-7 scale anchored at "strongly disagree" and "strongly agree." each subscale was presented in a separate block, always in the same order (with the dimension of interest, conspiracy theory ideation, presented first). an attention check item ("to make sure you read attentively please select strongly agree"), was included in the second block. after completing the dependent measure, participants were asked, in line with the cover story, to describe their event a second time (memory was not analyzed). finally, they were asked to speculate about the study's hypotheses, to answer basic demographic questions, and to assess the quality of their data ("did you honestly answer the question in this survey? you'll be paid regardless of how you answer"), before being debriefed. we report all measures, manipulations, and exclusions in this and subsequent studies. twenty-nine participants were excluded from the analysis: sixteen because they failed the attention check question (low control = 5, neutral = 4, high control = 7); two because they assessed their data as unreliable (high control = 2) and 11 (low control = 7, high control = 4) because they expressed suspicion about the research aims (in studies 1-5, two research assistants coded whether the participants were naïve to the research aims. the inter-rater reliability ranged from 0.8 to 0.97, and disagreements were settled by the first author) leaving 272 participants in total (including such participants does not change the pattern of results in any of the studies reported in this paper). a sensitivity analysis indicated that the study was able to detect a minimum effect size of f = 0.18. the effectiveness of the control manipulation was tested with a one-way anova on mastery scores, which revealed a significant effect of experimental condition f (2, 269) = 7.69, p < 0.001, f = 0.23. post hoc bonferroni comparisons indicated that the low control group (m = 2.64, sd = 0.50, n = 79) differed from both the neutral (m = 2.96, sd = 0.58, p < 0.001, n = 108) and the high control group (m = 2.89, sd = 0.58, p = 0.02, n = 85), but the high control group did not differ from the neutral group (p = 1.0). this is in line with other unsuccessful attempts to induce higher feelings of control compared to baseline levels [45, 46] . contrary to the primary hypothesis, an anova on conspiracy ideation indicated that the low control group (m = 3.66, sd = 1.17) had descriptively lower conspiracy beliefs than both the neutral (m = 3.99, sd = 1.27) and high control groups (m = 3.84, sd = 1.35), although the effect was not significant, f (2, 269) = 1.55, p = 0.215, f = 0.11. excluding the high control group (in which the manipulation was ineffective), revealed a marginally significant difference t (185) = -1.815, p = 0.07, d = 0.27; the low and high groups did not differ, t (162) = -0.925, p = 0.36, d = 0.14. finally, we calculated the pearson's correlation coefficient between conspiracy beliefs and the mastery score, finding a significant negative relationship, r = -0.237, p < 0.0001. in sum, despite using a standard and empirically successful manipulation of personal control [31, 47, 48] , we did not find that those whose control was threatened expressed greater conspiracy ideation; if anything, the tendency was for the opposite to be true. however, weaker feelings of control (independent of experimental condition) predicted stronger conspiracy ideation, as in previous research [32, 34, 49] . in study 2, we attempted to replicate the results of study 1 with a conceptually similar but arguably stronger manipulation of control. participants. three hundred mturk participants (137 male, 163 female) completed a questionnaire in the online survey platform qualtrics. the mean age of the sample was 37.28 years (sd = 11.66, age range 18-71 years). the majority had an undergraduate degree (45%), a third (30.3%) had a high school degree, 14.4% had a graduate degree, 9.7% other and 0.7% no degree. none of the participants had taken part in study 1. procedure and materials. after providing informed consent, participants were randomly assigned to one of three groups. those in the high control group were informed that we were interested in the types of situations that people experience as controllable (or in the low control group, as uncontrollable). for additional clarity, the instructions defined the construct of control, noting that "a person is 'in control' of something to the extent that they are able to direct or influence it if they want to." participants were asked to take a moment to think about some of the situations in their life where they have control (or no control), and to briefly describe ten such situations in a separate box. after providing ten examples they were administered the mastery scale as a manipulation check (see study 1). the neutral group proceeded immediately to this scale without engaging in any recall activity. as in study 1, after completing the dependent measures, participants were invited to speculate about the experimental hypothesis before providing demographics and assessing their data quality. twenty-nine participants were excluded from analysis: nine (low control = 2, neutral = 2, high control = 3) because they failed the attention check question, one (low control) because they self-assessed their data as unreliable and 19 (low control = 4, high control = 14, neutral = 1) because they were not naïve to the research aims. a sensitivity analysis indicated that the study was able to detect a minimum effect size of f = 0.19. next, we checked the effectiveness of the manipulation. a one-way anova revealed a marginally significant effect, f (2,268) = 2.610, p = 0.075, f = 0.14. (2,268) = 0.707, p = 0.494, f = 0.08. as in study 1, there was significant negative correlation between the mastery and conspiracy beliefs scores, r = -0.217, p < 0.001. in sum, despite successfully (albeit weakly) manipulating control and using a conceptually strong measure of conspiracy thinking, neither of two well-powered studies detected any causal evidence for the compensatory hypothesis, though both revealed a correlational relationship. it is worth considering, however, whether the manipulation check itself might be contributing to the null findings, by providing an opportunity to restore control, and thereby eschewing participants' need for further efforts in the form of conspiracy belief change. hauser, ellsworth and gonzalez [50] argue that manipulation checks as simple as asking participants about their feelings of control may serves the purpose of restoring their control. we note that, if this were true, it supports our contention that there are far easier ways to restore control than to endorse extreme, counter-normative belief systems. nevertheless, we considered this possibility in study 3. if the presence of the self-report measure of control was responsible for the null effect of the control manipulation on conspiracy ideation, then removing the manipulation check should produce the hypothesized relationship. participants. two hundred and two mturk workers (79 males, 123 females) participated. half (51.5%) had undergraduate degree, a third (28.7%) high school degree, an eighth (12.9%) had a graduate degree, while 5.9% selected "other" as their highest level of education, and 1% reported they did not have a degree. the mean age of the sample was 39.14 years (sd = 12.62, range 20-71 years). no participants had taken part in previous studies reported in this paper. procedure and materials. the procedure was identical to study 1, except that the manipulation check was omitted from the procedure, and that control was only manipulated downward. seven participants were excluded from the analysis: one (neutral group) because they self-assessed their data as unreliable, four (neutral = 3, low control = 1) because they failed the attention check question and two (both low control group) because they were not naïve to the research aims. a sensitivity analysis, indicated that the study is able to detect a minimum effect size of f = 0.2. the low control group scored numerically higher on the conspiracy theory ideation (m = 3.33, sd = 0.96, n = 86) than the neutral group (m = 3.19, sd = 1.09, n = 109), but the difference was not significant, t (193) = 0.936, p = 0.350, f = 0.06. thus, it does not appear that the inclusion of the manipulation check interfered with participants' hypothesized motivation to restore control via conspiratorial thinking. studies 1-3, using a novel (but, we argue, more theoretically appropriate) operationalization of conspiracy beliefs, revealed no empirical support for the control hypothesis. to explore whether similar results would be obtained with a more typical operationalization of conspiracy beliefs (i.e. as specific claims), and therefore exclude the possibility that the null effects are unique to the conspiracy ideation scale, we replicated the studies again, this time using the beliefs in conspiracy theories inventory [3] . participants. two hundred mturk workers (72 male, 126 female, 2 other) participated. a third had an undergraduate degree (39.5%), a third a high school degree (29.5%), while 19.5% had a graduate degree, 1.5% did not have a degree, and 9% had an "other" degree. the mean age of the sample was 40.85 years (sd = 13.44, range 20-82 years). no participant had taken part in the previous studies reported here. procedure and materials. the procedure was identical to study 3 with the exception of the dependent measure. instead of conspiracy ideation, we measured belief in fifteen specific conspiracies (e.g., a powerful and secretive group, known as the new world order, are planning to eventually rule the world through an autonomous world government, which would replace sovereign government), using the belief in conspiracy theories inventory (bcti), developed and later modified by swami and colleagues [3, 51] . (we used the modified version) participants rated the conspiracy theories on a 1 (completely false) to 9 (completely true) scale, with higher scores indicating higher conspiracy theory beliefs. eight participants (all low control group) were excluded from the analysis, one because they self-assessed their data as unreliable and seven because they were not naïve to the research aims. a sensitivity analysis indicated that the study was able to detect a minimum effect size of f = 0.20. the neutral group scored numerically higher on conspiracy theory beliefs (m = 3.31, sd = 1.57, n = 109) than the low control group (m = 3.27, sd = 1.65, n = 83), but the difference was not significant t (190) = 0.157, p = 0.88, f = 0.01. ostensibly, then, the null effects in studies 1-3 do not appear to be due to the instrument used to measure conspiracy theory beliefs. although our four studies show little evidence for a main effect of control priming on conspiracy beliefs, it is entirely possible that additional factors moderate the effects of controlthat control motivates conspiracy beliefs in some circumstances but not others. in the final two studies, we explore two of the most plausible possibilities: the degree to which a conspiracy theory itself implies a controlling entity (study 5); and the availability of other means of restoring control when it is threatened (study 6). if people turn to conspiracy theories as a compensatory control mechanism, it may be that some theories are more effective at restoring perceptions of order than others. in particular, threats to control may only prompt belief in conspiracies involving a controlling entity (e.g., the theory that the u.s.s.r. started the "hippie" movement in the united states in order to undermine and weaken america's traditional values), because the belief that someone is in control is sufficient to satisfy the higher order need for structure and meaning. other theories in which a controlling entity is absent or only implicit (e.g., the theory that osama bin laden died prior to the 9/11 terrorist attacks but was used as a scapegoat) may not restore control, or do so to a lesser extent. consistent with this proposal, kay et al. [27] found that participants whose control was threatened increased their belief in god, but only when god was described as controlling, rather than as a creator. thus, in study 5, we developed a new stimulus set to test the hypothesis that participants whose control was threatened would be inclined to vest more belief in specific conspiracy theories about controlling entities, but not in other conspiracy theories. participants. the participants were three hundred mturk workers (108 males, 192 females, m = 37.52, sd = 12.001). the majority (44%) had an undergraduate degree, while 29% had a high school degree, 13% had a master's degree, 2% a doctoral degree and 1% did not have a degree. the mean age was 37.54 years (range 18 to 73 years). none had taken part in our previous studies. materials. the dependent measure was developed in several stages. first, an independent group of mturk participants (n = 210) were asked to write five conspiracy theories that they had heard before, from which we identified 112 unique exemplars. next, two research assistants were asked to code these theories on a 9-point scale anchored at 1 ("the goal of this conspiracy is not control") and 9 ("the goal of this conspiracy is control"). the intraclass correlation coefficient was r = 0.84. the final rating comprised the average of the ratings of the two research assistants. next, a second group of 85 mturk participants was asked to rate the conspiracy theories in terms of their prototypicality, using a seven point scale anchored at 1 ("a bad fit" with their idea of a conspiracy theory) and 9 (an "excellent fit" with their idea of a conspiracy theory). a third group of 96 mturk participants was asked to rate the same conspiracies for likely veracity, using a nine point scale anchored at 1 ("definitely false") and 9 ("definitely true"). the intraclass correlation coefficient for prototypicality ratings was r = 0.89 and for the veracity r = 0.96. the prototypicality and veracity scores for each conspiracy theory were obtained by averaging ratings, respectively across all participants. these extensive pretest data were used to create seven pairs of conspiracy theories that were equated on veracity (t (95) = 0.604, p = 0.55, d = 0.06) and prototypicality (t (84) = 0.439, p = 0.66, d = 0.05), but that each differed by at least 5 scale points in terms of control (ms = 8.21 vs. 1.71). for the full list of the selected conspiracies, along with their control, prototypicality and veracity rating (obtained from the pre-test) see table 1 , for the full list of all pretest conspiracies see the online supplementary materials. procedure. the procedure was identical to that of study 3, except for the use of two types of specific conspiracy theories, varying in the control they imply, and the addition of a high control group as additional comparison group. participants rated their agreement with the 14 conspiracy statements, presented in a random order, on a 7-point scale (1 = strongly disagree, 7 = strongly agree). mixed among these statements was an attention check question (i.e., "as a response to this question select strongly agree."). nineteen participants were excluded from the analysis: nine (low control = 3, neutral = 6) participants due to failures of attention and ten (low control = 4, high control = 6) because they were not naïve to the research aims. a sensitivity analysis (α = 0.05, 1β = 0.8, correlation among repeated measures = 0.8) indicated that the study is able to detect a minimum effect size of f = 0.06. a mixed anova with control manipulation (low control vs. neutral vs. high control) as between subject factor, and type of conspiracy theory (control related theory or non-control related theory) as a repeated measure, revealed no main effect of the manipulation f . crucially for our purposes, there was no conspiracy theory type x control manipulation interaction f (2, 278) = 1.144, p = 0.32, f = 0.08 (the pattern of results was unchanged when we analyzed the low control vs. neutral groups only). we also analysed each pair separately. the interaction was not significant for any individual pair. in sum, we found no support for the hypothesis that control threat motivates belief in theories that hypothesize a controlling entity. in addition, participants rated the theories as more believable when they did not afford control, an unexpected result given that the two types of theories had been matched for believability in pretesting. to explore this discrepancy further, we compared our participants to the pretest sample on key demographic variables: age, gender, education and political ideology. the analyses indicated a marginal difference in the gender distribution, χ 2 = 4.98 (2), p = 0.08; there were a greater proportion of women in the main study compared to the pretest. in addition, women rated the control unrelated conspiracy theories as more believable than the control related, t(159) = -2.77, p<0.01, d = 0.21. when we reran the repeated measures anova, with gender as a covariate, the main effect of conspiracy theory type was not present, f (1, 277) = 0.045, p = 0.83, f = 0.01 and neither were the main effect of group, f (2, 277) = 0.225, p = 0.8, f = 0.04, nor the interaction effect f (2, 277) = 1.15, p = 0.318, f = 0.09. thus, the significant effect of conspiracy theory type was most likely due to the higher proportion of women in the main study. another plausible moderating factor determining the value of conspiracy theories for restoring threatened control, is the availability of alternative means of control. as noted above, while compensatory control processes do not require that sources of control be benevolent, people unsurprisingly favour sources of control that are not themselves threatening in other ways. furthermore, there is a plethora of alternative control and meaning-making systems that are proven substitutes for personal control, such as god [52] , government institutions [48] , scientific [53] , and even more abstract beliefs such as in meritocracy [54] and societal progress [55, 56] . even if these alternatives are not all equally effective at establishing a sense of control, conspiracy theories would seem to be an option of last resort, given the essentially malevolent worldview they imply, and the social stigma that can accompany their endorsement [57] . consequently, conspiracy theorizing may be a secondary strategy for restoring control, avoided when one has other means of affirming that the world is ordered [58] . thus, in study 6, we tested the hypothesis that threats to control would increase belief in conspiracy theories to a greater degree (or only) when the effectiveness of a second salient source of control-the government [29] -was called into question. participants. six hundred and five mturk volunteers participated (267 male, 337 female, 1 "other"). the majority (42.6%) had an undergraduate degree, 24.5% had a high school degree, while 21.3% had a graduate degree, 9.3% an "other" degree and 2.3% had no degree. the mean age was 33.96 years (sd = 11.11 age range . none had participated in our previous studies. materials and procedure. participants were randomly assigned to a low control or a neutral group, as described in previous studies, and, independently, to a "competent government" or "incompetent government" condition. in the former, participants read a passage (about which they expected to answer questions) describing the u.s. government's response, generally considered competent and effective [59] , to hurricane irma which most severely struck florida, georgia and south carolina in 2017; in the latter, they read a similar passage about the government's response to hurricane katrina, generally considered incompetent and ineffective [60] , which struck florida and louisiana in august 2005. for example, hurricane irma's response was described as "timely, well managed, planned and coordinated. residents were ordered to a shelter of last resort with sufficient provision of food, water, security and sanitary conditions. no one was thirsty, exhausted or victim to violence." hurricane katrina's response was described as "delayed, mismanaged, unprepared and uncoordinated. residents were ordered to a shelter of last resort without provision of adequate food, water, security or sanitary conditions. several citizens died of thirst, exhaustion and violence, days after the storm had passed." (the full text of both scenarios appear in the appendix.) afterwards, consistent with the cover story, participants answered several multiple choice questions about the scenario (which were not analyzed). afterwards, all participants completed the conspiracy mentality scale, as described in study 1, including an attention check, followed by demographic questions and self-assessment of data quality. one hundred and four participants were excluded from the main analysis: 74 due to failures of attention and 30 due to failure to following instructions. this left a total of 501 participants. a sensitivity analysis (α = 0.05, 1β = 0.8) indicated that the study is able to detect a minimum effect size of f = 0.12. table 2 for descriptive statistics). thus, the study, again, provides no evidence that control priming influences conspiracy beliefs, regardless of whether an alternative source of control has been compromised. although studies 1-6 varied somewhat in methodology, their consistent inclusion of low control and neutral conditions permitted a meta-analysis, which maximizes both power and accuracy of our effect size estimates. because in study 2 the low control manipulation did not lower perceived control compared to the neutral group we excluded study 2 from the analysis. for studies 5 (low control compared to neutral) and 6 we looked at the effect at each level of the moderator. because the effect sizes in studies 5 and 6 were not independent we used the robumeta [61] package in r, which takes the dependency into account, to conduct the meta-analysis. a test of heterogeneity suggested consistency between the studies, i 2 = 11. the cumulative effect size did not suggest effect of condition, d = -0.03, 95% ci [-0.20, 0.13], p = 0.60 (fig 1) . to investigate support for the null hypothesis relative to the alternative hypothesis that control priming influences conspiracy theory beliefs, we used a bayesian information criterion (bic), using the calculator by masson [62] , based on wagenmakers [63] . the analysis requires transformation of the sum of squares generated by an anova output and provides graded level of evidence for the null and alternative hypothesis. according to raftery [64] , the strength of evidence for the posterior probability of a hypothesis given the data (p bic (h 1 |d) within the range of 0.50-0.75 is "weak", between 0.75-0.95 is "positive", between 0.95-0.99 "strong" and above 0.99 "very strong". for those studies containing three groups we analysed the low and neutral (study 1 and 5) or low and high (study 2). for studies 5 and 6 we looked at each level of the moderator. table 3 . shows the posterior probability of the null hypothesis given the data, p bic (h 0 |d), and the probability of the alternative hypothesis given the data, p bic (h 1 |d). there is positive evidence for the null hypothesis in five out of the six studies. in study 1, there is only weak evidence for the null hypothesis, but the support for the alternative hypothesis is even lower (unsurprisingly, given that the effect is in the "wrong" direction). six studies, conducted online and using mturk participants, tested the hypothesis that lack of control motivates belief in conspiracies. while the hypothesis is plausible, there was in fact little support for it in the (small) literature on the subject. the current findings offer no further evidence, and some positive evidence for the null hypothesis, at least when the most common experimental manipulation of control is used with online samples. participants who were asked to recall instances in which they felt out of control were no more likely to endorse either general conspiracy beliefs, or specific conspiracy theories, relative to participants who recalled neutral memories, or nothing at all. plausible moderating variables-the extent to which conspiracies provide evidence of control (study 5), and the effectiveness of an alternative means of control (study 6)-had no effect, and a meta-analysis of the studies determined a cumulative effect size not statistically different from zero. on the contrary, a bayesian analysis indicated that the null hypothesis (i.e., that there is no causal relationship between lack of control and conspiracy theory beliefs) should be retained. how to explain the inconsistency between previous positive results and our null findings? one explanation, of course, is that previous "positive" findings were simply type 1 errors, which is not farfetched given social psychology's historical inattention to power [65, 66] combined with publication bias. indeed, assuming the "actual" effect size of control threat on conspiracy beliefs is 0.2 (the average effect size reported in psychology research [67] ), then previous studies reporting positive results may have included chance findings given their low [35] or nyhan & zeitzoff [36] , have higher power, 0.52 and 0.88, respectively. another possibility, however, is that some previous studies measured constructs other than "pure" conspiracy ideation (e.g., pattern perception, paranoid beliefs, corruption beliefs), which are susceptible to control threats. indeed, studies that examine more typical conspiracy theories and have larger samples tend to report null results. for example, hart and graether [35] , found no effects of control on the generic conspiracist beliefs scale (another psychometrically validated measure of conspiracy beliefs, with items such as, "experiments involving new drugs or technologies are routinely carried out on the public without their knowledge or consent"). similarly, nyhan and zeitzoff [36] found no effect of control priming on middle eastern and north african participants' conspiracy beliefs, using a list of conspiracy theories consisting of items such as "jewish leaders are secretly conspiring to achieve world domination." it is also worth repeating that the current results were obtained entirely with online samples, while most studies in this area (including the majority of those with positive effects of control) have been conducted in the laboratory (typically with student samples). in principle, online manipulations of control could be weaker than those in the laboratory, accounting for the lack of support for the control hypothesis. as an empirical matter, however, there is no evidence that effects in this paradigm depend on sample type [68] . in a recent meta-analysis of experimental manipulations of control on conspiracy beliefs [68] conducted on 45 effect sizes across 23 studies (including those reported here), we found no moderating effect of sample type (mturk vs. students vs. other). thus, although it is important to continue to examine the role of control threats in diverse samples and contexts, the current data, despite being collected online, nevertheless challenge the hypothesis that such threats account for conspiracy beliefs in any significant way. although there was no evidence in the current studies for a causal effect of control on conspiracy beliefs, studies 1 and 2 provided evidence for a correlational relationship. such correlational findings are consistent with previous research [32, 34, 49, 69, 70] , suggesting that chronic, rather than acute, feelings of low control are related to higher conspiracy theory beliefs. while longitudinal studies may help shed light on the effects of chronic lack of control on conspiracy beliefs, it is also possible that the link is spurious. there are any number of covariates of both lack of control and conspiracy theory belief, including stress [71, 72] , selfesteem [73, 74] , anxiety [75, 76] or uncertainty [77] , that could account for their association. also, the correlation may be due to conspiracy beliefs' shared variance with paranoia (see for example imhoff & lamberty, [78] ). alternatively, the causality may run in the opposite direction: conspiracy theories themselves may reduce feelings of control. jolley and douglas [79] , for example, found that participants exposed to conspiracy theories about vaccines reported greater feelings of powerlessness compared to participants exposed to anti-conspiracy material, suggesting that conspiracy beliefs threaten rather than satiate one's sense of control. furthermore, it is not clear what type of correlational relationship should be taken as evidence for satiation; if conspiracy theories compensate for compromised personal control, should the relationship between control and conspiracy beliefs in cross-sectional studies be positive or negative? thus, correlational evidence in the absence of experimental evidence is not a strong foundation for claiming that lack of control is a precursor to conspiracy beliefs. even if our present null results are confirmed in subsequent research, it is important to acknowledge their limitations. priming is only one method of threatening (or bolstering) a sense of personal control, and while we found no evidence for changes in conspiracy beliefs, more potent or qualitatively distinct experimental manipulations could conceivably produce effects that we were unable to detect here. furthermore, we could not assess the effects of chronic lack of control: it may be that prolonged periods of control deprivation do indeed produce reliance on other, even malevolent sources of control, including conspiracy theories. another unexplored area is whether lack of control in a specific domain will lead to belief in conspiracy theory in that domain. for example, people diagnosed with hiv or covid-19 might be particularly inclined to believe in conspiracy theories involving those viruses, rather than conspiracy theories in general. indeed, in a recent naturalistic study [80] , we found a domain-specific link between threat to control and conspiracy beliefs. for example, after a series of tornados, those who experienced such natural disasters were more likely to endorse weather-related conspiracy beliefs than they were other types of conspiracy beliefs. moreover, the strength of these endorsements subsided over time (i.e., three months after tornado season). such findings seem to suggest that the link between perceived control and conspiracy beliefs is both nuanced and transitory, and that there is no "one size fits all" explanation for lack of control and conspiracy-theory endorsement in general. finally, we relied exclusively on laboratory manipulations of control (or lack thereof). while such empirical techniques are widely used in the literature, the effectiveness of the "recall task" in threatening participants' sense of control has been questioned [34] , and its effects were weak in our studies. "real life" events, such as natural disasters, terrorist attacks, and pandemics, may produce stronger threats to personal control (and indeed, evidence suggests that traumatic events are followed by an increase in conspiracy discourse [81] ). findings from our own naturalistic study seems to confirm this. however, such naturalistic "manipulations" may confound perceptions of control with other variables, such as threats to identity, uncertainty about the future, and stress. thus, any naturalistic observations need to be accompanied by rigorous experimental research to triangulate the mechanism(s) responsible for any changes in conspiracy beliefs. on their own, both empirical approaches are problematic, in that experimental manipulations of control may be too "weak" to evoke conspiracy-belief endorsements, and perceived loss of control under naturally occurring conditions are confounded with other variables. the conspiracy mentality scale: distinguishing between irrational and rational suspicion anxious attachment and belief in conspiracy theories conspiracist ideation in britain and austria: evidence of a monological belief system and associations between individual psychological differences and real-world and fictitious conspiracy theories they will not control us': ingroup positivity and belief in intergroup conspiracies the effect of intergroup threat and social identity salience on the belief in conspiracy theories over terrorism in indonesia: collective angst as a mediator the role of social identification, intergroup threat, and out-group derogation in explaining belief in conspiracy theory about terrorism in indonesia belief in conspiracy theories and susceptibility to the conjunction fallacy intention seekers: conspiracist ideation and biased attributions of intentionality someone is pulling the strings: hypersensitive agency detection and belief in conspiracy theories suspicious minds here's why people believe in conspiracy theories producer) & alpert d. 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hans title: feline leukemia virus and other pathogens as important threats to the survival of the critically endangered iberian lynx (lynx pardinus) date: 2009-03-09 journal: plos one doi: 10.1371/journal.pone.0004744 sha: doc_id: 319921 cord_uid: uxtydu60 background: the iberian lynx (lynx pardinus) is considered the most endangered felid species in the world. in order to save this species, the spanish authorities implemented a captive breeding program recruiting lynxes from the wild. in this context, a retrospective survey on prevalence of selected feline pathogens in free-ranging lynxes was initiated. methodology/ principal findings: we systematically analyzed the prevalence and importance of seven viral, one protozoan (cytauxzoon felis), and several bacterial (e.g., hemotropic mycoplasma) infections in 77 of approximately 200 remaining free-ranging iberian lynxes of the doñana and sierra morena areas, in southern spain, between 2003 and 2007. with the exception of feline immunodeficiency virus (fiv), evidence of infection by all tested feline pathogens was found in iberian lynxes. fourteen lynxes were feline leukemia virus (felv) provirus-positive; eleven of these were antigenemic (felv p27 positive). all 14 animals tested negative for other viral infections. during a six-month period in 2007, six of the provirus-positive antigenemic lynxes died. infection with felv but not with other infectious agents was associated with mortality (p<0.001). sequencing of the felv surface glycoprotein gene revealed a common origin for ten of the eleven samples. the ten sequences were closely related to felv-a/61e, originally isolated from cats in the usa. endogenous felv sequences were not detected. conclusions/significance: it was concluded that the felv infection most likely originated from domestic cats invading the lynx's habitats. data available regarding the time frame, co-infections, and outcome of felv-infections suggest that, in contrast to the domestic cat, the felv strain affecting the lynxes in 2007 is highly virulent to this species. our data argue strongly for vaccination of lynxes and domestic cats in and around lynx's habitats in order to prevent further spread of the virus as well as reduction the domestic cat population if the lynx population is to be maintained. the iberian lynx (lynx pardinus) is native to the iberian peninsula and is considered the most endangered felid species in the world [1, 2, 3] . at present this species is especially threatened due to the decline of its basic prey (the wild european rabbit, oryctolagus cuniculus), fragmentation and loss of its habitat, and nonnatural mortality [4] . iberian lynxes are confined to two isolated populations in southern spain in the doñ ana and sierra morena areas, and only 40-50 and 150-200, respectively, are estimated to remain [5, 6, 7] . to save this species from extinction, an eu life nature project is underway that includes habitat preservation, lynx population monitoring, and rabbit population management. in addition, cryopreservation of lynx genetic material and a captive ex situ breeding project were initiated to preserve the genetic diversity of the species and produce new specimens for future reintroduction. despite extensive information concerning the field ecology of this species [8, 9, 10, 11, 12, 13] , only limited information is available regarding the diseases that affect the iberian lynx in the wild or in captivity. infections due to cytauxzoon felis (a theileria-like agent), toxoplasma gondii, hemotropic mycoplasmas, mycobacterium bovis, and several parasitic diseases have been described in some individuals [14, 15, 16, 17, 18, 19, 20, 21] . bovine tuberculosis was reported as the cause of death for five iberian lynxes between 1998 and 2007 [22, 23, 24, 25, 26, 27] . histopathological studies revealed generalized immune depletion in these animals, apparently unrelated to infectious agents or malnutrition [24] , and glomerulonephritis [28] . furthermore, the presence of feline leukemia virus (felv) provirus was recently reported in six samples originating from both the doñ ana and sierra morena areas in southern spain between 1994 and 2003 [29] . a recent study documented the limited genetic diversity of the iberian lynx population [30] . similarly to the cheetahs, where limited genetic diversity had been shown to be associated with disease by an otherwise harmless coronavirus [31, 32] , the restricted genetic basis of iberian lynxes may contribute to render this endangered species particularly susceptible to pathogens and possibly even to opportunistic infectious agents. therefore, a retrospective study, aimed to systematically analyze the prevalence and importance of known feline pathogens in free-ranging iberian lynxes, was initiated. animals were screened for the following viral agents: feline herpesvirus (fvh), feline calicivirus (fcv), feline parvovirus (fpv), feline coronavirus (fcov), feline leukemia virus (felv), feline immunodeficiency virus (fiv), and canine distemper virus (cdv). these viral infections are known to affect also wild felids [33, 34, 35] . in addition, animals were tested for c. felis, anaplasma phagocytophilum, hemotropic mycoplasmas, bartonella henselae, and chlampydophila felis. thus, in the present study, we report on the prevalence of the aforementioned pathogens and we describe a dramatic felv epidemic, which most likely led to the death of 6 iberian lynxes within a 6-months period in 2007, its possible origin, and its relationship to other infectious agents. as a consequence of this study, we hope to provide insight on how to reduce danger originating by these infections and to raise public awareness about the critical situation of the iberian lynx. sixteen of the 77 iberian lynxes studied died. seven lynxes were found dead after being stuck by vehicles, one due to illegal hunting, and eight died for reasons presumed to be related to infectious diseases. one of the latter eight lynxes exhibited high viral loads of cdv, suggesting that cdv infection was the cause of death (data not shown). in another, young animal, m. bovis was found in the tracheal wash. one of the animals struck by a vehicle had been found to be felv provirus-positive in 2004. the remaining six dead lynxes were all positive for felv provirus and felv p27 antigen, and all died within six months during the spring-summer of 2007 in the northern part of the doñ ana area (fig. 1) . in a routine check of animals (n = 16) in this area in early december 2006, an adult free-ranging male was found to be felv provirus-and p27-positive. the same animal was negative for felv when sampled one year before. this animal demonstrated no clinical signs and was released. in may 2007, the same lynx was found dead. in december 2006, the other 15 lynxes from the northern part of the doñ ana area were felv-negative. beginning in march 2007, five of these 15 lynxes died: all five were positive for felv provirus and felv p27. in the same period additional seven lynxes were found to be felv-positive and survived. felv p27-positive animals were captured and transported to the los villares rescue center in order to reduce infectious pressure in the field [36] . relationships and geographical distribution of the infected lynxes are depicted in figure 1 . the majority of the remaining lynxes (n = 61) were clinically healthy. to determine the association of felv infection with mortality, the proportions of felv infection in dead and surviving lynxes were analyzed for significant differences by fisher's exact test. both, provirus positivity and antigenemia, were significantly correlated with mortality (p fisher = 0.0138 for provirus; p fisher = 0.0187 for antigenemia). the significance level even increased when the traumatic deaths (n = 8) were omitted from the comparison (p fisher = 0.0007 for provirus; p fisher = 0.0002 for antigenemia). provirus positivity was less frequent in lynxes that died from traumatic reasons than in the total population of the doñ ana area (p fisher = 0.041). the quantitative real-time pcrs specific for endogenous felv sequences were weakly positive in samples obtained from 5 of 77 lynxes. four animals tested positive in only one of the three assays used, one was positive for the enu3-1, enu3-2 and env assays. however, the enfelv copy numbers were extremely low, with less than 1 copy per 10'000 cells, suggesting that the weak positive results may be the consequence of sequences cross reacting with the assay. fourteen lynxes (13 from the doñana area and one from sierra morena) tested positive for felv-a provirus ( table 1 ), but negative for felv-b and felv-c (data not shown). felv provirus was amplified and sequenced from eleven positive lynxes from the doñana area. nucleotide sequence analysis of the surface unit (su) of the env gene revealed a common origin for the provirus found in doñana lynxes in 2007 (99.5-100% identity). the sequences clustered with and were 97.9-98.2% identical to the felv-a/61e strain ( fig. 2a) [37, 38] , while the sequence obtained from the lynx found to be provirus-positive in 2004 (arena) was only 94.8-95% identical to the other sequences and was more related to felv-a/ rickard (97.4% identity, fig. 2a table 1 . fecal samples tested negative for fcov (0/68) and positive for fpv in 1/ 62 (1.6%) and cdv in 1/45 (2.2%) samples; the latter was obtained from the only animal in which cdv was found in the blood. only six fecal samples from the 14 felv provirus-positive animals were available. five fecal samples originating from antigenemic animals tested positive for felv dna, whereas one sample from a p27negative animal tested negative by pcr (data not shown). statistically significant associations (based on pcr results) were found between felv infection and infections with m. haemofelis (p fisher = 0.0004) and 'candidatus m. turicensis' (p fisher = 0.0205). the other pathogens did not show any significant association with felv infection. in view of the endangered situation of the iberian lynx, the central government of spain in conjunction with the autono-mous government of andalusia has initiated an ex situ conservation program comprising captive breeding centers. animals that were selected as founders for the captive breeding project had been caught in the wild and tested for the absence of pathogens prior to introduction into the captive breeding program. of note, some of the founder lynxes were found to be infected with c. felis, hemotropic mycoplasmas and b. henselae, and also to exhibit antibodies to other infectious agents. however, these animals were clinically healthy. between late 2003 and 2007, substantial information was obtained regarding the prevalence and importance of infectious agents in iberian lynx populations of the doñ ana and sierra morena habitats. with the exception of fiv, evidence for all tested feline pathogens was found. interestingly, the seroprevalence of fhv, fcv, fpv, and fcov was substantially higher than that observed in free-ranging eurasian lynxes (lynx lynx) from sweden [39] . the increased seroprevalence of these feline pathogens in iberian lynxes probably reflects higher population density and closer contact with domestic cats as compared to lynxes in the swedish population. it is clearly desirable that the infectious pressure by these feline pathogens arising from domestic cats should be kept as low as possible. [29] , six of 21 lynxes sampled between 1993 and 2003 were found to be felv provirus-positive, but not antigenemic. although no clinical data were reported in that study, there was no indication that felv infection would represent a major risk for the lynx population. this situation changed acutely when, in 2007, several animals with severe clinical signs were found to be infected with felv and eventually died, the statistical association between felv infection and death being highly significant. interestingly, presence of felv-provirus in lynxes that died from traumatic incidents (1 shot, 7 hit by cars) was significantly lower than in the lynxes with felv infection found dead. from this it was concluded that felv provirus positivity was not a predisposing factor for death by accidents. the pathogenicity of the felv strain involved in the outbreak, with six of 13 infected animals succumbing within six months, was rather unexpected. five of the felv-infected lynxes that had died were living in the coto del rey nucleus, where three adult pairs of lynxes and their offspring normally only use 700-1000 ha [13] . consequently, contact between individuals may be frequent in this area [40] . the other felv-positive dead lynx was living in the gato nucleus, where animals have relatively high rates of contact with those living in the coto del rey nucleus via dispersal [9] . several of the felv-infected lynxes showed clinical signs and/or hematologic abnormalities, such as anemia, lymphopenia or neutropenia, compatible with felv infection also observed in domestic cats. in contrast, the rapid onset of the clinical signs leading to death are not a typical feature of felv-a infection in cats, where some felv infected animals can survive more than 10 years [41, 42] . the presence of co-infections could also have contributed to the high pathogenicity of felv in the lynxes. from the observation that a significant association exists between felv and hemotropic mycoplasma infections, one is tempted to argue that these coinfections may lead to increased pathogenicity. however, although the association is statistically significant, co-infection by felv and hemotropic mycoplasms cannot be the cause of death in all cases, as all lynxes that survived felv infection were also positive for hemotropic mycoplasms. it is possible that pre-existing infection by these agents may favor felv infection. the increased frequency of felv infection in lynxes positive for hemotropic mycoplasms could also reflect simultaneous transmission of these agents with felv during cat-to-lynx contact. sequencing of the complete felv env gene revealed that the virus present in lynxes affected in 2007 was clearly distinct from the virus detected in the felv-positive lynx found dead in 2004. this suggests that felv was introduced into the iberian lynx population on at least two occasions. nonetheless, all analyzed felv sequences were closely related to felv-a and, in particular, to felv-a/61e, a member of a highly conserved family representing prototype viruses of the horizontally transmitted, minimally pathogenic felv-a. these felv-a prototype viruses are assumed to be present in all naturally occurring felv infections in domestic cats. felv-a/61e in conjunction with felv-a/61c was found to cause severe immunodeficiency in domestic cats (felv-faids) and was originally isolated from a cat in colorado [43] . although we were not able to detect envelope variants similar to felv-a/61c in the blood, the presence of additional felv variants in different organs cannot be excluded. thus, important minority genomes harboring changes in the env gene sequence may have been present but were not detected. to exclude this possibility, we not only sequenced the majority of felv variants present in the peripheral blood, but also cloned felv from four lynxes and analyzed several clones from each animal, without finding additional sequences. in domestic cats with felv-faids, the second virus variant (felv-a/61c) was mainly found in the intestines and was not present in the blood [37] . thus, further investigations should include such tissue samples from infected lynxes. alternatively, mutations, deletions, or repeats in other parts of the felv genome that were not characterized in the present study (e.g., in the ltr) could have contributed to the pathogenicity of the virus. felv variants with 21-bp tandem repeats in the ltr have been isolated from domestic cats with naturally occurring lymphomas and, more recently, from florida panthers [44, 45, 46] ; the ltr transcriptional enhancer repeats were described in t-cell tumors [47] . these 21-bp repeats confer a replicative advantage to the virus rather than modify the disease spectrum [44, 47] ; they were not investigated in the present study. furthermore, the high pathogenicity observed in felv-infected iberian lynxes might be due to the host, rather than solely to viral factors. the genetic diversity of the doñ ana lynx population is lower than that of the sierra morena population [30] , and therefore the pathogenic potential of felv could have been enhanced by inbreeding. in addition, an immune-mediated systemic disease of unknown origin has been recently postulated [28] . cheetahs, another wild felid species with reduced genetic diversity, also demonstrate increased susceptibility to some infectious disease agents [48] . endogenous felv-related sequences (enfelv) could be another host factor that might contribute to the high pathogenicity of felv in lynxes [49, 50] . these enfelv exist as multiple, nearly full-length proviral sequences (1 to 100 copies/cell) in domestic cats, as well as in closely related wild cats of the genus felis, e.g., european wildcats (felis silvestris silvestris) [51] . however, endogenous felv sequences related to those of domestic cats are apparently not present in iberian lynxes: only 5 of the 77 lynxes tested displayed weak signals by quantitative realtime pcr, which is not compatible with presence of enfelv sequences. as endogenous retroviruses are integrated in the germ line and by consequence should be present in at least one copy per cell, the results obtained can only be explained by the presence of either endogenous sequences distantly related to the enfelv of domestic cats or of a co-infection with another gammaretrovirus, both showing a weak cross-reactivity to the real-time pcr systems used. the absence of felv-b, which is the result of a recombination between exogenous and endogenous felv, is also in agreement with the absence of enfelv in the iberian lynxes, since the incidence of felv-b in the domestic cat is usually higher than 50% in diseased cats [52] . absence of enfelv sequences suggests that the mechanisms inducing disease in lynxes must be distinct from those in felv-b-infected cats. as only a few cases of felv infection were observed prior 2007 [29] , it is likely that felv infection in lynxes is rare, is not readily carried within the lynx population, and most likely originates from domestic cats, which are increasingly frequent in doñ ana. a felv prevalence of 15.6% was reported in spanish cats [53] ; this might be even higher around doñ ana (f. martinez, personal communication). therefore, a higher risk of transmission from domestic cats to lynxes is likely. since some felv variants seem to be highly pathogenic in the iberian lynx, it would seem important for the survival of lynxes to protect them against further felv transmission from domestic cats. thus, it is important that felv infection in free-ranging lynxes be controlled as quickly as possible. to this end, conservation authorities have started to vaccinate lynxes against felv infection using a canarypox-based felv vaccine [54] . from what is known about the control of felv infection in domestic cats, vaccination is highly effective. although vaccination did not induce sterilizing immunity in domestic cats, it was nonetheless able to stimulate the immune system to a degree that allowed the cats to overcome the infection rapidly and to clear most of the viral rna from the blood [55] . even if immunization does not completely protect lynxes against transient infection, there is justified hope that the outcome of felv infection will be less severe. in addition to vaccination, it is probably also important to decrease the infectious pressure on lynxes by preventing domestic cats from entering the lynx habitats and by vaccinating them against felv infection to reduce the risk of felv transmission to lynxes. from late 2003 until september 2007, edta-anticoagulated blood, serum, and fecal samples from 77 free-ranging iberian lynxes were collected in both the doñ ana (n = 45) and sierra morena (n = 32) areas in the south of spain. some of these animals were later included in the ex situ breeding program. only samples collected at the time of capture (free-ranging status) were included in this study. blood samples were collected from the vena cephalica of animals that had been caught and immobilized with a mixture of ketamine-medetomidine for radiocollaring, sanitary checking, allocation to the captive breeding, or other management programs. blood or serosanguineous samples were also collected from the thoracic cavity or the heart of animals found dead. nucleic acid extraction from blood, fecal, and tissue samples total nucleic acids (tna) were extracted from 200 ml of edta-blood, serosanguineous fluid, or fecal samples using the magna pure lc total nucleic acid isolation kit (roche diagnostics, rotkreuz, switzerland), or from tissues collected upon necropsy using dneasy and/or rneasy tissue kits (qiagen, hombrechtikon, switzerland) according to the manufacturer's instructions. serological methods. serum samples from 75 animals were available for the serological assays. the presence of antibodies against fhv, fcv, fpv, fcov, and cdv was determined by an immunofluorescence assay (ifa), and antibodies to fiv were detected by elisa and western blot, as previously described [33, 56] . felv p27 antigen was detected and quantified by elisa as previously described [57] . antibodies against a. phagocytophilum were determined by an immunofluorescence assay using commercially available ifa slides (vmrd, inc. pullman, wa, usa). detection and quantification of dna and rna. dna specific for fhv, fpv, fiv, and felv provirus, b. henselae, ch. felis, a. phagocytophilum, and hemotropic mycoplasmas was detected and quantified by real-time taqman pcr, as previously described [58, 59, 60, 61, 62, 63, 64, 65, 66] . the presence of c. felis was detected by conventional pcr using the forward primer cytfelis.203f ( rna specific for fcv and fcov was detected and quantified by real-time taqman rt-pcr, as previously described [59, 67, 68] ; for cdv the rt-pcr reaction was set up with 12. specific felv-b and -c subtype detection. felv-b was detected as previously described [69] . for felv-c, pcr was designed to amplify a 2 kb region encoding the envelope and the 39ltr of felv-c. the specificity of the system was validated as described for felv-b [69] . the pcr reactions contained 0.4 units of phusion tm high fidelity dna polymerase (finnzymes, keilaranta, finland), final concentrations of 500 nm each for the forward primer 5944f and reverse primer 8041r (table 2) , 200 nm dntps (sigma), 4 ml 56 pcr hf buffer (finnzymes), and 5 ml of template in a final volume of 20 ml. an initial denaturation of 30 s at 98uc was followed by 35 cycles of 98uc for 20 s, 64uc for 30 s, 72uc for 2 min, and a final extension at 72uc for 5 min on a biometra tpersonal thermal cycler (biolabo). full-length felv-a env amplification, subcloning, and sequencing. tna from blood or dna from tissue samples was used for amplification of the complete felv provirus env gene. to discover specific primers, a multiple sequence alignment (msa) of all genbank felv env sequences (available as of june 2006) was compared with the msa of endogenous felv (enfelv) and felv-c env entries. primers that anneal to felv-a, but not enfelv or felv-c, were used (table 2 ). msas were constructed using the multiple alignment editor jalview [70] . the primer melting temperature and degree of self-annealing were determined using the gcg software package (accelrys, san diego, ca, usa). for amplification of full env, pcr reactions comprised 0.4 units of phusion tm high-fidelity dna polymerase (finnzymes), final concentrations of 500 nm each for primers 5617f and 8197r, 200 mm dntps, 4 ml of 56 phusion tm hf buffer, and 2 ml of template in a final volume of 20 ml. pcr was performed using a biometra tpersonal pcr thermal cycler (biolabo). after an initial denaturation at 98uc for 30 s, amplification was performed using 35 cycles of 98uc for 20 s, 64uc for 30 s, and 72uc for 2 min 30 s, and a final extension of 72uc for 8 min. pcr products were either sequenced directly after purification using the genelute pcr clean-up kit (sigma) or separated by agarose gel electrophoresis, excised from the gel, purified using the genelute gel extraction kit (sigma), and cloned into the pcrii-topo vector (invitrogen, basel, switzerland) according to the manufacturer's instructions; the obtained clones were then sequenced. sequencing was carried out by synergene biotech, zurich, switzerland using primers 5617f, 5847f, 6123f, 6123r, 6382f, 6382r, 6956f, 6956r, 7266r, and 8197r ( table 2 ). the resulting iberian lynx felv-a env surface glycoprotein (su) sequences were deposited in the genbank database under accession numbers eu293175 to eu293194. msas of iberian lynx enfelv and felv subtypes env su (genbank accession numbers: m18247, m18246, m12500, af052723, m14331, ay662447, ab060732, m23025, m25425, ay354318) were constructed using clustalw [71] and refined using t-coffee [72] . phylogenetic trees were constructed using mega3 software [73] . boostrap support (% of 1,000 bootstrap replicates) was calculated using the neighbor-joining, minimum evolution, and maximum parsimony methods. endogenous felv (enfelv) detection and quantification. the enfelv dna loads were determined as described using three different systems (enu3-1, enu3-2, and enenv) [51] that detect enfelv sequences present in the recently annotated cat genome [74] . a feline glyceraldehyde-3-phosphate dehydrogenase (fgapdh) pseudogene, of which one copy is present in the genomic dna of feline cells [64] , was quantified by real-time taqman pcr assay [75] . copy numbers of enfelv were divided by fgapdh copy numbers to calculate the number of copies per cell. phylogenetic trees were constructed using mega3 [73] . bootstrap support (1000 replicates) was calculated by the neighbor-joining/minimum evolution/mp methods and considered significant when .70% [76] . mp trees were obtained using the close-neighbour-interchange algorithm [77] with search level 3 [76] , in which initial trees were obtained by random addition of sequences (10 replicates). all alignment gaps were treated as missing data. branch lengths were calculated using the average pathway method [77] . pid values for dna and predicted protein sequences were calculated using the software package matgat (matrix global alignment tool) version 2.02 [78] . to assess associations between felv and other infectious agents, infection frequencies were compared using the fisher's exact test (graphpad prism version 3.00 for windows, graphpad software, san diego, usa). p-values,0.05 were considered as significant. group comparison was performed using fisher's exact test (analyse-it for microsoft excel version 2.03, analyse-it software, ltd., leeds, uk). iucn red list of threatened species wild cats: status survey and conservation action plan revision of the felidae red list of threatened species action plan for the conservation of the iberian lynx (lynx pardinus) in europe faecal genetic analysis to determine the presence and distribution of elusive carnivores: 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fluorogenic real-time detection system prevalence of bartonella infection in wild african lions (panthera leo) and cheetahs (acinonyx jubatus) use of real-time quantitative pcr to detect chlamydophila felis infection quantitative real-time pcr for detection of members of the ehrlichia phagocytophila genogroup in host animals and ixodes ricinus ticks melting curve analysis of feline calicivirus isolates detected by real-time reverse transcription pcr one-tube fluorogenic reverse transcription-polymerase chain reaction for the quantitation of feline coronaviruses association between endogenous feline leukemia virus loads and exogenous feline leukemia virus infection in domestic cats the jalview java alignment editor clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice t-coffee: a novel method for fast and accurate multiple sequence alignment mega3: integrated software for molecular evolutionary genetics analysis and sequence alignment initial sequence and comparative analysis of the cat genome rapid detection of feline leukemia virus provirus integration into feline genomic dna confidence limits on phylogenies: an approach using the bootstrap molecular evolution and phylogenetics matgat: an application that generates similarity/identity matrices using protein or dna sequences subtle mutational changes in the su protein of a natural feline leukemia virus subgroup a isolate alter disease spectrum the authors would like to thank y. gahler, e. gönczi, t. meili prodan, b. pineroli, e. rogg, e. schuler, and b. weibel for excellent laboratory assistance and helpful support. the authors are indebted to the environmental council of the government of andalusia, southern spain, for providing the iberian lynx samples. we are grateful to our collaborators in the iberian lynx life project 2000-2005, life project 2006-2011, the doñ ana national park, and the ex situ breeding program for sampling the free-ranging iberian lynxes, and to laura peñ a from the departamento de patologia for her expertise in pathology. key: cord-307934-84zfabti authors: lai, chao-kuen; saxena, vikas; tseng, chung-hsin; jeng, king-song; kohara, michinori; lai, michael m. c. title: nonstructural protein 5a is incorporated into hepatitis c virus low-density particle through interaction with core protein and microtubules during intracellular transport date: 2014-06-06 journal: plos one doi: 10.1371/journal.pone.0099022 sha: doc_id: 307934 cord_uid: 84zfabti nonstructural protein 5a (ns5a) of hepatitis c virus (hcv) serves dual functions in viral rna replication and virus assembly. here, we demonstrate that hcv replication complex along with ns5a and core protein was transported to the lipid droplet (ld) through microtubules, and ns5a-core complexes were then transported from ld through early-to-late endosomes to the plasma membrane via microtubules. further studies by cofractionation analysis and immunoelectron microscopy of the released particles showed that ns5a-core complexes, but not ns4b, were present in the low-density fractions, but not in the high-density fractions, of the hcv rna-containing virions and associated with the internal virion core. furthermore, exosomal markers cd63 and cd81 were also detected in the low-density fractions, but not in the high-density fractions. overall, our results suggest that hcv ns5a is associated with the core of the low-density virus particles which exit the cell through a preexisting endosome/exosome pathway and may contribute to hcv natural infection. hepatitis c virus (hcv) is a major causative agent of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. hcv is an enveloped virus with a 9.6-kb positive-strand rna genome. this genome encodes a large polyprotein, which is processed by host and viral proteases into 10 viral proteins that consist of three structural proteins, six nonstructural proteins, and a small hydrophobic peptide, p7 [1, 2] . the structural proteins, core protein and two envelope glycoproteins e1 and e2, are derived from the n terminal portion of the polyprotein and constitute physical virion components. the nonstructural (ns) proteins, ns2, ns3, ns4a, ns4b, ns5a, and ns5b, are derived from the c terminal portion of the polyprotein. most of the ns proteins (with the exception of ns2) are involved in hcv replication [3, 4] . hcv rna is synthesized in the replication complex (rc), which exists in the membranous web derived from altered er membranes [5, 6] . the hcv rc is transported on microtubules and this transport is facilitated by the interaction of ns3 and ns5a with tubulin [7] . the intact microtubule network also is directly involved in hcv rna replication [8] [9] [10] and virus release [10, 11] . following hcv rna replication, core protein and ns5a serve as central regulators of virus assembly [12] . core protein forms multimers [13] and interacts with the viral rna [14] to form the viral nucleocapsid. the core protein is localized mainly on the surface of the lipid droplets (lds) [15, 16] , which is essential for the production of infectious hcv particles [15] . further, core protein promotes the accumulation of lds to facilitate virus assembly [11, 17] and recruits viral rcs to ld-associated membranes [15] . thereby, viral rna interacts with core protein in juxtaposition to ld for virus packaging. moreover, the interaction between ns5a and core protein is essential for the recruitment of the viral rcs to lds and plays an important role in virus assembly [18, 19] . however, how viral rcs and core protein target to ld remains unclear. in addition to ns5a, other ns proteins, including ns2, ns3, and ns4b, have also been shown to influence the production of infectious virus [12] . up to now, it is not known whether the ns proteins are incorporated into infectious virions. previous studies have indicated that cell culture[20] [21] [22] [23] [24] and patients' serum-derived [25] [26] [27] [28] [29] hcv particles display heterogeneous diameters (from 35 to 145 nm) and have a broad range of buoyant density (between 1.01 g/ml and 1.17 g/ml). the main peak of both viral core protein and rna exhibited at a density of 1.15 to 1.17 g/ml in the cell culture derived-hcv (hcvcc) [30, 31] , and the highest specific infectivity of extracellular virion was observed at a density of 1.14 g/ml [20] . notably, the lowdensity fraction (density of ,1.1 g/ml) displays exosome-like structures and also contains infectivity [20] , but the nature and origin of their properties are still unknown. many types of cell continuously secrete a large number of microvesicles, called exosomes, which have a diameter of approximately 50-150 nm and have a buoyant density between 1.08 g/ml and 1.22 g/ml [32] . exosomes are released into the extracellular space from late endosomes/multivesicular bodies (mvbs) fusion with the plasma membrane [33] . more recently, the exosomes derived from cells containing hcv subgenomic replicon have been demonstrated to contain hcv rna, but not viral ns proteins [34] . our previous results [10] have shown that hcv core proteins are transported from early to late endosomes/mvb in hcv-infected cells. however, it is not known whether any hcv proteins are incorporated into the released exosomes from hcv-infected cells. in this study, the trafficking mechanism of the ns5a and core proteins is defined further. both ns5a and core proteins are found to be closely associated with and co-transported along the microtubules from the perinuclear region of cells via the lds and endosomes to the plasma membrane. this association of ns5a-core proteins implicated them in virus assembly as well as release. interestingly, we found that both ns5a and core, in addition to exosomal proteins cd63 and cd81, were detected in the lowdensity hcv particles (1.083 to 1.098 g/ml) with low-grade infectivity. ns5a appeared to be incorporated into hcv particles through interaction with core protein and microtubules during intracellular transport. our data suggest that ns5a-containing, low-density hcv particles were released in the form of exosome. huh7.5 cells, a mutant line of huh7 cells that support hcv replication at high efficiency [35] were cultured in dulbecco's modified eagle's medium (dmem) containing either 10% fetal bovine serum (fbs) or 10% dialyzed fbs [36] for a general subculturing and for preparation of purified hcv, respectively. rep 1.1 cells [7, 37] are huh7 cells that harbor a genotype 1b hcv subgenomic replicon. they were grown in the aforementioned medium containing 0.5 mg/ml of g418. plasmid puc-jc1, which encodes a chimera genome of hcv j6cf/jfh1, was constructed as previously described [38] . antibodies used in this study included anti-ns5a (austral biologicals), anti-core (affinity bioreagents inc.), anti-bromodeoxyuridine (sigma-aldrich), anti-eea1 or -cd63 or -lamp-1 (santa cruz biotechnology), mouse anti-cd81 (bd pharmingen), rabbit anti-cd81 (santa cruz biotechnology), anti-e2 (genetex, inc.), anti-calnexin (assay designs/stressgen), and anti-mouse orrabbit colloidal gold conjugates (jackson immunoresearch inc.). rabbit polyclonal abs against core (rr8) and ns4b (rr12) were described previously [15] . cy3-conjugated primary ab to btubulin and nonspecific normal mouse immunoglobulin g (igg) were obtained from sigma-aldrich. secondary abs against mouse, rabbit and goat were purchased from invitrogen molecular probes. reagents used were nocodazole (sigma-aldrich), taxol (paclitaxel; sigma-aldrich), bodipy 493/503 (invitrogen molecular probes), 4',6-diamidino-2-phenylindole (dapi; invitrogen molecular probes) and wheatgerm agglutinin (wga) alexa fluor 647 conjugate (invitrogen molecular probes). the jc1 viruses were produced and titrated in huh7.5 cells based on a previously described method [7] . the same batch of culture supernatant from virus-infected and the control cells, including hcv subgenomic replicon cells (hcv assembly-defective replicon cells) and uninfected cells, was used in all experiments. the concentrated hcv jc1 and controls were subjected to 10 to 50% sucrose gradient sedimentation centrifugation as previously described [39] . a total of 15 fractions of 0.75 ml each was collected from the bottom to the top of the sucrose gradient and monitored for hcv rna by using quantitative pcr (qpcr) [10] . fractions containing the hcv rna signal (fractions 7 to 8 and fractions 11 to 13) and their uninfected counterparts were pooled and dialyzed against tne buffer (10 mm tris, 150 mm nacl, 2 mm ethylene diamine tetraacetic acid) overnight at 4uc. the virus-containing fractions were then concentrated by 10-fold in ultracel-3k concentration devices (millipore) and further processed for electron microscopy applications or infection. infectivity of each fraction was determined by quantifying the amounts of intracellular hcv rna levels at day 3 postinfection (p.i.). huh7.5 cells in six-well plates were infected with concentrated hcv from each fraction of the sucrose gradient suspended in dmem at 37uc for 3 h. cells were washed with pbs and incubated with 2 ml of dmem containing 10% fbs. at 3 days postinfection, total rna was isolated from cell lysates using a high pure rna isolating kit (roche). viral rna was isolated from cell culture supernatants using a qiaamp viral rna kit (qiagen). qrt-pcr was performed as described previously [10, 40] . the procedure of em was carried out exactly as described previously [40] . for immuno-em, samples were first incubated with an anti-ns5a, anti-core or anti-e2 mouse mab, and followed by incubation with colloidal gold particles of 18 nm conjugated to antimouse immunoglobulin g. for immuno-em of the purified viruses, two microliters of purified virus were adsorbed onto carbon-coated grids. grids were fixed with 2% paraformaldehyde for 10 min and blocked in a solution of 0.5% bovine serum albumin (bsa) for 20 min. the grids were incubated with primary abs and normal mouse igg for overnight at 4uc. grids were then washed and incubated with goat anti-mouse and/or goat anti-rabbit colloidal gold particles (6-and 12-nm diameter) for 1 h at room temperature. after extensive washing, the grids were stained with 1% uranyl acetate for 1 min. for analysis of hcv core particle, the purified virus was treated with 0.01% saponin in pbs for 20 min before processing. all samples were analyzed under a tecnai spirit transmission electron microscope (fei co) at 120 kv. cell labeling with 5-bromouridine 5'-triphosphate (brutp) was performed according to the methods described previously [7] . hcv-infected cells (at day 10 p.i.) were grown on 4-well chamber slides. one day after seeding, cells were incubated with actinomycin d (10 mg/ml) for 30 min. then, 20 ml of brutp/ fugene 6 (roche molecular biochemicals) mixture were added to each well containing 500 ml medium with actinomycin d (10 mg/ ml). after 30 min of incubation at 37uc, cells were treated with 10 mm nocodazole or taxol for 1 h, and then fixed and processed for immunofluorescence staining as described below. cells were gown on glass chamber slides. cell fixation and immunostaining were performed by the methods described by listenberger l. l. and brown d. a. [41] . photographs of the cells were taken with a confocal microscope (zeiss confocal laser scanning microscope lsm 510meta-nlo). the procedure for quantitative colocalization analysis used in this study followed the published method [10] . the weighted colocalization coefficient is the sum of intensities of colocalization pixels relative to the overall sum of pixel intensities above the threshold. it was used to determine the relative levels of figure 1 . colocalization of ns5a with core protein and microtubules in lipid droplets. hcv-infected cells (at day 10 p.i.) were co-stained with anti-tubulin (green), -ns5a (red) (a) and/or -core (red, b; blue, c) antibodies. lipid droplets (lds) were stained with bodypi 493/503 (blue, a and b; brown, c) and nuclei with dapi (gray). stained cells were examined by confocal fluorescence microscope. merging of the images in the green, red, blue, and gray channels generated the pictures in fig. a and b. fig. c was generated by merging the images in the green, red, blue, and brown channels. yellow indicates overlapping localization of the green and red channels, cyan indicates overlapping localization of the green and blue channels, magenta indicates overlapping localization of the red and blue channels, and white indicates overlapping localization of the red, green, and blue channels. the upper third panels in fig. a shown for core (red) or ns5a (red) and with dapi (blue) in the lower panels of fig. a and c, respectively. colocalization efficiency between ld and core protein (b) and between ld and ns5a (d) was analyzed by using zeiss lsm zen software. (e) analysis of cellular proliferation and survival by mts assay. (f, g) quantitation of confocal microscopic fluorescent signals of core and ns5a in cells. the total fluorescence intensities of core protein and ns5a were measured using metamorph integrated morphometry analysis. a total of 20 cells were used for calculation of colocalization efficiency and total fluorescence intensity from two independent experiments and error bars represent standard deviations of the mean. noc, nocodazole. bars, 10 mm. (h) in parallel, the cell lysates were collected and then immunoblotted with antibodies against core and ns5a. results were quantified by phosphorimager counting. doi:10.1371/journal.pone.0099022.g002 were transfected with brutp in the presence of actinomycin d for 1 h and then treated with either 10 mm nocodazole or 10 mm taxol for 1 h. the cells were co-stained with mouse mab against bromodeoxyuridine (green) and rabbit polyclonal antibodies against core (rr8) (red). lds and nuclei were stained with bodypi 493/503 (blue) and dapi (cyan), respectively. enlarged views of parts of every image are shown (insets). (b) the number of brutp-labeled viral rna was counted manually using an original magnification of 6630 and followed by a quantitation analysis performed by an observer blinded to the experimental treatment. (c) the average distance between the center of the signal emitted by the brutp-labeled viral rna colocalization between core protein, ns5a or ns4b with the protein of interest (e.g., calnexin, ld). the shortest and longest average distances between the brutp-labeled viral rna signal center and the nearest edge of ld were calculated manually using zen software (zen 2009 light edition; carl zeiss inc). the number of brutp-labeled viral rna and antibody-labeled signals of viral proteins at the plasma membrane was manually counted in an original magnification of 630. total fluorescence intensity values of core protein and ns5a for individual cells were measured using the metamorph software (universal imaging corporation), using 8 bit images. the gray-scale of the 8-bit images ranged from 0 (black) to 256 (white). image analysis was carried out using the integrated morphometry analysis program provided by metamorph. fluorescence intensity is expressed as the integrated value of all pixels per cell that exceed the inclusive threshold value set at 40. a total of 20 cells were used in each experimental condition from two independent experiments. a celltiter96a queous one solution cell proliferation assay kit (promega) was used to evaluate cell viability, which was performed as described previously [40] . our previous studies have shown that microtubule provides the track for the movement of hcv rcs through ns5a-microtubule interaction [7] and that this transport is required for virus release [10] . we propose that microtubules also provide tracks for the transport of ns5a or the ns5a-containing rc and core protein to reach the ld, where virus assembly occurs. to test this possibility, we first investigated whether core-containing lds colocalized with ns5a and associated with microtubules in hcvinfected huh7.5 cells [at day 10 postinfection (p.i.)]. immunofluorescence staining revealed that ns5a and core protein together (fig. 1c ), or either one alone ( fig. 1a and 1b) , is colocalized on the surface of ld and is closely associated with tubulins. the microtubule network is required for the trafficking of ns5a or the ns5a-containing replication complexes and core protein to the lipid droplet we used nocodazole (which induces microtubule depolymerization) or taxol (which stabilizes tubulin polymerization) to examine whether intact microtubules are required for the transport of core protein, ns5a or viral rcs to the ld. huh7.5 cells were first infected with hcv, and treated with either nocodazole or taxol at 3 hr p.i. for 2 days. the colocalization coefficient of ld with core protein or ns5a was then calculated. under these conditions, cell viability was not affected, as revealed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2h-tetrazolium salt (mts) assay (fig. 2e) . under the control conditions [dimethyl sulfoxide (dmso)], ld colocalized with core protein throughout the entire cytoplasm, including the perinuclear region; the proportion of ld that colocalized with core protein was 69%. when the cells were treated with increasing concentrations of nocodazole, a dose-dependent decrease in the colocalization coefficient of ld with core protein was observed ( fig. 2a upper panels and 2b) . the colocalization coefficient of ld with ns5a also decreased correspondingly (fig. 2c upper panels and 2d) . taxol did not have effects on this coefficient. in order to rule out the possibility that the dose-dependent decrease in the colocalization of ld with core or ns5a by nocodazole treatments might have been the consequence of a decrease in the amounts of core and ns5a proteins, a further analysis of core protein ( fig. 2a and 3a , lower panels) and ns5a ( fig. 2c and 3c , lower panels) labeling in cells was carried out using the metamorph integrated morphometry analysis. in nocodazole-treated cells, there was no significant change on the total fluorescence intensities of core and ns5a relative to control cells ( fig. 2f and 2g ). immunoblot analysis also showed that the levels of ns5a and core proteins in the cells were not significantly affected by the low concentration (up to 4 mm) of nocodazole or taxol used (fig. 2h,) . this was in contrast to the effects of nocodazole at 10 or more mm used in most of the published studies, which inhibited hcv rna replication and thereby hcv protein level [8, 10, 42] . these data support the conclusion that the nocodazole treatment at low concentrations affected the colocalization of core and ns5a with ld ( fig. 2b and fig. 2d ). taken together, these results indicated that microtubules are involved in the transport of core protein and ns5a to the ld. newly synthesized membrane proteins generally leave the endoplasmic reticulum (er) and transported to other destinations in the cells. therefore, we next investigated whether ns5a and core proteins are transported from er to lds along the microtubules. in the presence of dmso or taxol, core protein was colocalized with the er marker protein calnexin throughout the entire cytoplasm, including the perinuclear region, with a colocalization coefficient of 32% ( fig. 3a and 3b ). in contrast, the nocodazole treatment caused a dose-dependent increase of colocalization coefficient to 75% and 84% at 2 and 4 mm nocodazole, respectively (fig. 3a, upper panels and 3b) . similarly, the colocalization coefficient of ns5a with calnexin increased from 41% to 69% and 75%, respectively (fig. 3c , upper panels and 3d). thus, the nocodazole treatment caused an increased accumulation of core protein and ns5a in the er and corresponding decrease in the lds. taken together, these results suggested that ns5a and core protein are translocated from er to lds in a microtubule-dependent manner. we further confirmed the mode of transportation of the hcv rc to the ld by immunofluorescent labeling of newly synthesized hcv rna with brutp [43] . in the dmso control and taxoltreated cells, the number of brutp-labeled speckles averaged 30 per cell, and the average distance between speckle and ld was 0.43 mm (fig. 4) . in the presence of nocodazole, the number of speckles was reduced to an average of 11 per cell (fig. 4b) , indicating that the hcv rna replication is partially suppressed by nocodazole treatments, consistent with the previous reports [8] [9] [10] . correspondingly, the average distance between the speckle center and the nearest edge of ld increased from 0.43 mm in the dmso controls and taxol-treated cells to 1.63 mm in the nocodazole-treated cells (fig. 4c) , suggesting that the hcv rcs are normally delivered to the vicinity of the lds through microtubule and that this transport is disrupted by nocodazole. taken together, these data suggest that ns5a or the ns5a-and the nearest edge of ld (hcv rna-ld distance) were analyzed by using zeiss lsm zen software. a total of 20 cells were used for quantitation and calculation of the hcv rna-ld distance and the number of brutp-labeled viral rna from two independent experiments and error bars represent standard deviations of the mean. n.s., non-significance; *, p,0.01; noc, nocodazole. bars, 10 mm. ns5a and core protein complex is cotransported from the perinuclear region to the plasma membrane via microtubule ns5a, together with core protein, is involved in virus assembly; the assembled virions are presumably transported to the plasma membrane to be released off the cell. therefore, we next analyzed whether ns5a is involved in the post-assembly processes of viral life cycle. for this purpose, we examined the colocalization of ns5a with core protein in two regions of the cytoplasm, viz. the perinuclear (region just around the nucleus) and the peripheral (region just underneath the plasma membrane) regions. hcvinfected cells were co-stained with fluorescent (alexa 647) wga (which binds glycoproteins on the cell membrane), which serves as a membrane marker, anti-core, and either anti-ns5a or -ns4b abs. as shown in fig. 5a, ns5a and core protein colocalized both in the perinuclear region and at the cell periphery. in contrast, ns4b colocalized with the core protein only in the perinuclear region of cytoplasm, but not in the peripheral region. analysis of a large number of cells indicated that core protein was colocalized with ns5a throughout the entire cytoplasm, but not with ns4b (fig. 5b, upper panels) . the total fraction of ns5a that colocalized with core protein was 76%, while the corresponding ns4b was 51% (fig. 5b, lower panel) . further, the ns5a-core complex colocalized in the peripheral region in 33% of the cells, whereas ns4b-core did not colocalize at all in the same region (fig. 5c) . we next examined the possibility that the core-ns5a complexes are also transported along microtubule to the cell periphery. in the dmso-treated cells, ns5a colocalized with core protein in both the perinuclear and peripheral regions (fig.5d ). after treatment with either nocodazole or vinblastine, the core-ns5a complexes clustered almost exclusively in the perinuclear region (fig. 5d) . these results suggested that microtubules are required for the transport of core-ns5a complexes to the cell periphery. this observation further suggests that ns5a, but not ns4b, is involved in the post-assembly transport of virus particles. to further evaluate the involvement of the ns5a-core complex in hcv assembly and release, we investigated the localization of this complex with respect to lds [15] , early and late endosomes [10, [44] [45] [46] [47] , and microtubules [10, 11] , all of which are involved in the various steps of hcv assembly and exit. the hcv-infected cells were co-stained with the ld marker (bodipy 493/503), the early endosome marker (early endosome antigen 1; eea1), the late endosome marker (lysosome-associated membrane protein-1; lamp-1), or the microtubule marker (b-tubulin). in the perinuclear region of cytoplasm, both the ns5a-core and ns4b-core complexes colocalized with the lds and the early endosomes ( fig. 5e and 5f ). in contrast, in the peripheral region, only the ns5a-core complex, but not the ns4b-core, colocalized with the late endosomes (fig. 5g) . immunoelectron microscopy (immuno-em) further showed that the late endosome/mvb contained ns5a, but not ns4b, the latter being mainly in the perinuclear regions (fig. 5h) . these results suggested strongly that ns5a-core complexes, but not ns4b, are transported through early-to-late endosomes following virus assembly at ld. furthermore, ns5a and core protein, but not ns4b, colocalized with the microtubules in the peripheral region of cytoplasm, especially at the microtubule end that is closest to the cell periphery (fig. 6a) . finally, we characterized the relationship of ns5a-core complexes with the plasma membrane. hcvinfected cells were co-stained with fluorescent (alexa 647) wga, anti-core, and anti-ns5a or -ns4b abs. as shown in fig. 6b , ns5a and core protein colocalized at the plasma membrane, particularly at the membrane curvature, which has been reported to be essential for virus exit [48] . quantitative analysis showed that the number of anti-core and anti-ns5a antibodies-labeled signals at the plasma membrane averaged 63 and 26 per cell, respectively, whereas that for anti-ns4b was less than 1. interestingly, some signals containing both ns5a and core (5 per cell on average) were also detected at the plasma membrane, whereas no ns4b-core signals were found (fig. 6c) . furthermore, immuno-em showed that ns5a (fig. 6d) , core protein (fig.6e ) and the core-ns5a complex (fig. 6f ) localized mainly to patches or clusters on the plasma membrane. taken together, these results again suggest that at least some of ns5a-core complexes (or the assembled virions) are transported from ld through early-to-late endosomes to the plasma membrane via microtubules. to confirm that ns5a is released from the plasma membrane as a component of some virion particles, we cultured hcvinfected huh7.5 cells with dialyzed serum to reduce nonspecific binding of irrelevant proteins to virus particles. the culture supernatant was subject to sucrose gradient sedimentation, and the distribution of the viral rna and infectivity of hcv were figure 5 . core-ns5a complexes are transported from perinuclear region to early and late endosomes. (a) the hcv-infected cells (at day 10 p.i.) were labeled with antibodies specific for core protein (red) and ns5a (green) (upper row) or ns4b (green) (lower row). plasma membrane and nuclei were stained with wga alexa fluor 647 conjugate (blue) and dapi (gray), respectively. in parallel with panel a, the hcv-infected cells were labeled with antibodies to core protein (green) and ns5a (red) or ns4b (red) (b, upper rows). nuclei were stained with dapi (blue). colocalization efficiency between core protein and ns5a or ns4b was analyzed by using zeiss lsm zen software (b, lower panel). error bars represent standard deviations of the mean from 20 cells in two independent experiments. the images were analyzed by using metamorph, and the proportion of cells (of 200 counted) in which the ns5a or ns4b colocalized with core protein at the cell periphery was calculated (c). (d) effects of nocodazole (noc) and vinblastine (vbl) on movement of core-ns5a protein complex to the cell periphery. the hcv-infected cells (at day 10 p.i.) were treated with nocodazole, vinblastine, or dmso for 3 h. the cells were labeled with antibodies to core protein (green), ns5a (red). nuclei were stained with dapi (blue). images with differential interference contrast (dic) and fluorescence images were then merged. an enlarged view of part of image is shown (inset). in parallel with panel a, the hcv-infected cells were labeled with antibodies to core protein (red) (e, f, g), ns5a (green, e, f, g) (upper rows), ns4b (green, e, f, g) (lower rows), eea1 (blue) (f) and lamp-1 (blue) (g). lds and nuclei were stained with bodypi 493/503 (blue) (e) and dapi (gray), respectively. the second and third panels in each row are magnified views, marked with a white box in the panel at the extreme left, of the perinuclear and the peripheral regions of cytoplasm, respectively (a, e, f, g). colocalization of core protein with ns5a or ns4b is depicted as yellow (a, b, d, e, f, g) . colocalization of core-ns5a or -ns4b complexes with lds (e), early endosomes (f) or late endosomes (g) is depicted as white. bars, 10 mm. (h) in parallel, the hcv-infected cells was labeled with anti-ns5a or -ns4b ab. bound antibodies were detected using anti-mouse or -rabbit secondary antibodies conjugated to 18-or 12-nm gold particles, respectively. sections were visualized by em. arrows, gold-labeled ns5a (18 nm) and ns4b (12 nm) are indicated. mvb contains several intravesicular vesicles (white arrows). an enlarged view of the perinuclear region of lower left image is shown (inset). arrows, gold-labeled ns5a and ns4b. pm, plasma membrane; mvb, multivesicular bodies; n, nucleus; nm, nuclear membrane; bars, 500 nm (left panels) and 100 nm (right panels). doi:10.1371/journal.pone.0099022.g005 determined. two peaks, one from fractions 7-8, with density of 1.083 to 1.098 g/ml sucrose (low-density fractions; ldf), and another from fractions 11-13 with density of 1.145 to 1.178 g/ml sucrose (high-density fractions; hdf), were found to contain distinct hcv rna signals (fig. 7a ). fraction 12, at 1.162 g/ml sucrose, contains the largest amount of viral rna, consistent with the previously reported density of free hcv virions [27, 31] . each fraction was further analyzed for its infectivity on naive huh7.5 cells. the results showed that both the ldf and hdf from culture supernatant contained infectivity (fig. 7b) , with hdf having approximately 153-fold higher specific infectivity. thus, both the ldf and hdf are infectious, but ldf contained only 0.65% of the total infectivity; thus, the possibility that the apparent infectivity of ldf may have come from contaminations of hdf cannot be ruled out. further, we analyzed the various fractions for the possible presence of core and ns5a. as shown in fig. 7c , the hdf contained abundant core protein, but no ns5a or ns4b. in contrast, the low-density fraction 7 (1.083 g/ml) contained ns5a in addition to core protein, but no ns4b. recent studies have revealed that hcv virion release requires late endosome (or multivesicular body; mvb) [10] , the functional endosomal sorting complex required for transport (escrt) [46, 47] and hepatocyte receptor tyrosine kinase substrate (hrs) [45] , all of which are involved in the biogenesis of mvb and exosome secretion. in addition, hcv particles are associated with circulating exosomes in hepatitis patients' serum [49] . we therefore investigated whether hcv virion release from cell membrane occurs through the exosome pathway. we probed each sucrose density fraction for the presence of two exosomal markers viz. cd81 and cd63. interestingly, both cd81 and cd63 were detected in ldf (fig. 7c) , but not hdf, suggesting that the exosome is involved only in ldf virus release. since exosomes are derived from endosomes and are also secreted from normal cells to participate in a variety of cellular functions [50] , we also analyzed the sucrose gradient fractions of extracellular medium obtained from naã¯ve huh7.5 cells and cells harboring an hcv subgenomic replicon. none of the fractions obtained from these cells contained hcv ns5a protein (data not shown), consistent with the previous report [34] . these data suggest that ldf contained hcv particles released through exosome-like structure, and contained ns5a. the morphology and composition of virus particles in both the ldf and hdf were further characterized. the diameter of particles ranged from 105 to 132 nm (n = 60) and 52 to 86 nm (n = 90) in the low-and high-density pools, respectively ( fig. 7d and 7g ). the size heterogeneity of hcv particles has been described previously [20, 25, 31] . correspondingly, particles of ,130 nm and ,70 nm in diameter, probably representing the low-and high-density virus particles, respectively, were observed in the extracellular space surrounding the hcv-infected cells (fig. s1 ). after treatment with saponin (0.01%), which removed the viral envelope, the size of the particles was reduced to 35-41 nm for both the ldf and hdf viruses (fig. 7e and 7f) , consistent with its being the internal viral core [20, 51] . next, the purified virions were characterized for the presence of viral proteins and exosomal marker cd81 by immuno-em. the particles in the low-density pool could be stained with anti-cd81 and -e2 mabs, but not with a purified normal igg or an anti-ns5a mab (fig. 7d, upper panels) or an anti-ns4b ab (data not shown). in contrast, only e2 protein, but not cd81, was detected in the high-density pool (fig. 7d, lower panels) . the association of hcv envelope proteins with exosomes has previously been reported [49] . following the treatment of virus samples with detergent (0.01% saponin), the virion core of both the low-and high-density pools was stained with anti-core mab (fig. 7e) . interestingly, the virion core of ldf, but not the hdf, could be stained with anti-ns5a antibody, but neither normal igg nor anti-ns4b antibody (fig. 7e) . in parallel, the saponin-treated virus of ldf could be stained with both core and ns5a simultaneously (fig. 7f) . the specificity of staining was confirmed by staining under various treatment conditions (fig. 7h ). in all virus samples, normal igg or anti-ns4b antibody yielded only background staining. in hdf, anti-cd81 and -ns5a antibodies also yielded very little staining with or without the saponin treatment. in contrast, in ldf, anti-cd81 (40%) and -e2 (25%) stained intact virus samples strongly, while anti-core (70%) and -ns5a (40%) stained saponin-treated samples strongly. the above results prompted us to further characterize the localization of the exosomal proteins relative to ns5a and core protein on the plasma membrane. the results indicated that ns5a or core protein colocalized with cd81 at the plasma membrane by immunofluorescence staining (fig. 8a ) and immuno-em ( fig. 8b and 8c ), but not ns4b. these results again suggest that some ns5a-containing hcv particles exit the cells via fusion of mvb with the plasma membrane. taken together, these data suggest that a minor population of hcv virion exits cells via exosomes, as ns5a-containing, low-density particles, but the majority of virions do not contain ns5a and exit cells through an exosome-independent pathway. in this study, we found that in the presence of a microtubuledisrupting drug, both ns5a and core proteins failed to be transported to the ld (fig. 2) , resulting in the accumulation of ns5a and core protein in the er (fig. 3 ). in addition, ns5a and core protein colocalized with microtubule throughout the entire figure 6 . core-ns5a complexes are transported from perinuclear region to the plasma membrane via microtubules. the hcvinfected cells (at day 10 p.i.) were labeled with antibodies to core protein (red) (a, b), ns5a (blue, a; green, b) (upper rows), ns4b (blue, a; green, b) (lower row) or tubulin (green) (a). plasma membrane and nuclei were stained with wga alexa fluor 647 conjugate (blue) (b) and dapi (gray), respectively. the second and third panels in each row are magnified views, marked with a white box in the panel at the extreme left, of the perinuclear and the peripheral regions of cytoplasm, respectively (a). colocalization of core with ns5a or ns4b is depicted as magenta (a) or yellow (b). colocalization of core-ns5a or -ns4b protein complexes with microtubules (a) or plasma membrane (b) is depicted as white. the microtubule end (white arrow) is closest to the cell periphery (a). (b) at the right is an enlarged area, marked with a white box, from the merged image. colocalization of ns5a with core protein was observed in the membrane curvature (white arrow). pm, plasma membrane; bars, 10 mm. (c) quantitation of antibody-labeled signals of viral proteins at the plasma membrane. images from 20 cells were counted manually using an original magnification of 6630 and followed by a quantitation analysis performed by an observer blinded to the experimental treatment. (d, e) ns5a and core are localized in clusters or patches on the plasma membrane. in parallel, the hcv-infected cells were labeled with anti-ns5a (d) or anti-core (e) antibodies. bound antibodies were detected using anti-mouse secondary antibodies conjugated to 18-nm gold particles. sections were visualized by em. arrows, gold-labeled ns5a (d) or core protein (e). (f) immuno-em of core-ns5a complex colocalized at the plasma membrane. the hcv-infected cells were co-labeled with antibodies against ns5a (6 nm) and core (18 nm). shown is a view of the plasma membrane. arrowhead, gold-labeled ns5a. arrow, gold-labeled core. pm, plasma membrane; bars, 100 nm (d, f) and 200 nm (e). doi:10.1371/journal.pone.0099022.g006 cytoplasm, mostly also associated with lds ( fig. 1) and at the microtubule end near the plasma membrane (fig. 6a) . these results suggest that the intact microtubule network served as a transport highway for the movement of hcv ns5a (or viral rcs) and core proteins from the er towards the ld and plasma membrane for virus assembly and exit. additionally, it has been shown that the interaction between ns5a and core protein on lds plays an important role for virus assembly [18, 19] , and that core protein induces redistribution of ld to the perinuclear region for virus assembly in a microtubule-dependent manner figure 7 . association of ns5a and exosomal proteins with the low-density hcv particles. hcv virion populations were analyzed by sucrose density gradient ultracentrifugation. (a) concentrated culture medium collected from hcv jc1-infected cells was fractionated using a continuous 10-50% sucrose density gradient. hcv rna levels were determined in each fraction. (b) infectivity of each density gradient fraction. an aliquot of each fraction was used to infect naive huh7.5 cells. intracellular hcv rna copy number per mg of total rna was determined 3 days after infection by qrt-pcr. (c) western blot analysis of each density gradient fraction, shown in panel a, by using antibodies indicated at the left. (d, e and f) electron micrograph of the viral spherical structures shown by immunogold labeling. hcv particles were purified from two pooled fractions; the low-density particles (from fractions 7 to 8), and the high-density particles (from fractions 11 to 13), after dialysis and concentration. the virus samples were untreated (d) or treated with 0.01% saponin (e, f), as described in materials and methods. grids were incubated with an untreated or saponintreated purified hcv and then with antibodies against cd81, e2, or ns5a (d) or with antibodies against core protein, ns5a or ns4b (e), respectively. bound antibodies were detected using anti-mouse or -rabbit secondary antibodies conjugated to 12-nm gold particles. (f) core-ns5a complex localized in the virion core of the low-density particles. grids were incubated with a saponin-treated purified hcv and then co-labeled with antibodies against ns5a (6 nm) and core (12 nm). to determine antibody specificity, primary antibodies were replaced with nonspecific normal mouse igg. bars, 50 nm. (g) the average diameter of the low-(n = 60) and high-density (n = 90) particles was obtained from the combined analysis of two independent viral preparations and error bars represent standard deviations of the mean. (h) statistics were performed by counting hcv particles and immunogold labeled hcv particles from the combined analysis of two independent viral preparations. relative proportion of hcv particles that were labeled with antibodies (n = 60). doi:10.1371/journal.pone.0099022.g007 [11] . these studies explain previous finding that the extracellular virus production was decreased by nocodazole [10] . by immunofluorescence staining and em, we found that ns5a is closely associated with core protein in almost every step of hcv life cycle, whereas ns4b is not involved in the late steps. however, only a small number (5 signals/cell) of the ns5a-core complexes were detected at the plasma membrane of the cells, in contrast to core protein alone (,63 signals/cell) (fig. 6c) . this finding may explain why the amount of hcv rna in ldf (,1610 4 copies/ ml) was significantly lower than that of hdf (1.3610 5 copies/ml) (fig. 7a) . we suggest that ns5a-containing, low-density hcv particles constitute a minor population of the released hcv virions. nonetheless, this population was apparently infectious, though with slightly lower specific infectivity than the bulk of virus particles in hdf (fig. 7b) . the result is similar to a published report, which showed that less than 2% of all infectivity was in the low-density pool (density of ,1.1g/ml) [20] . however, we cannot exclude the possibility that ldf with very weak infectivity may have come from virus contamination from hdf. notably, we demonstrated that low-density hcv particles utilize a host exosome biogenesis pathway for the production of ns5a-containing virions. the low-density hcv particles (density of 1.08 g/ml) were also found in hepatitis c patients' serum [26, 52, 53] . most importantly, ns5a and exosomal markers coincided with core protein in the low-density fractions (fig. 7c) , which also contained hcv rna (fig. 7a ). ns5a-core complexes (fig. 7f ) and exosomal markers (fig. 7d) were detected in purified ldf virions. correspondingly, exosomal markers and ns5a or core protein were colocalized at the plasma membrane, where virus release occurs, as revealed by immunofluorescence staining and immuno-em (fig. 8) . taken together, the results indicated that the low-density, ns5a-containing virions are released off the cell via exosomes, whereas the high-density particles exit the cell via some alternative mechanism not involving the exosomal pathway. consistent with this hypothesis, hcv particles have been found to be associated with circulating exosomes in serum of hcv-infected patients [49] . combined with our previous results [10] showing that hcv particles are transported from early to late endosomes/mvb, our current studies suggest that the ns5a-containing hcv particles most likely exit the cells via fusion of mvb to the plasma membrane. exosomes have been demonstrated to facilitate the budding of human immunodeficiency virus (hiv) [54] and hepatitis a virus (hav) [55] . exosome have been known to play important roles in intercellular communications. in hiv and hav infection, exosomes are required for trans-infection of cd4 + t cells [56] and are likely to promote virus spread within the liver [55] , respectively. similarly, exosome-containing hcv virions might fuse to uninfected cell in a mechanism independent of viral envelope proteins and may contribute to hcv natural infection. interaction of ns5a with core protein plays an important role in hcv particle production [19] , but its mechanism of involvement in infectious virus production is still unclear. the association of viral ns proteins with viral genomic rna and their incorporation into viral particles is common among rna viruses, such as the ns2 of influenza virus [57] , the v protein of simian virus 5 [58] , the ns3 of coronavirus [59] , and the vpx (viral protein x) of hiv-1 [60] . however, in contrast to our observation, merz et al. [24] could not observe any ns5a in the virus preparation after affinity purification. the reason for this discrepancy may lie in the sensitivity of detection methods and the method of virus purification, as ns5a was detected only in a minor population of low-density particles in our study. it was also possible that the affinity tag used in the previous study [24] could not be detected in the low-density hcv particles. such virion-associated ''nonstructural proteins'' probably do not play structural roles in virus particles, but may participate in some yet unknown functions required at different steps of the viral life cycle. recently, an amphipathic a-helical peptide (ah) containing the n-terminal 31 amino acids of ns5a was shown to induce swelling of the vesicles leading to vesicle lysis [61] ; in addition, the infectivity of hcv particle was reduced when hcv virions were first treated with the ah peptide, causing the disruption of hcv envelope [62] . taken together, the data presented here and elsewhere show that ns5a may induce fusion and lysis of lipid vesicles as well as virus particles during the virus entry step. since ns5a is present inside the virus particles, it is likely that these membrane-altering functions of ns5a could function at a postbinding step, namely, after the virus envelope has been disrupted. indeed, a postbinding fusion step within an acidic endosomal compartment is required for hcv entry [63] . this will be an interesting topic to study in the future. in summary, our data demonstrated that a minor population of low-density hcv virions was released in the form of exosome from the infected cells, and that its virion core contains ns5a via close interaction with core protein during intracellular transport. our studies provide an additional target for designing new therapeutic approaches by possible use of exosome inhibitor to block the spread of hcv infection. figure s1 the hcv-infected cells (at day 10 p.i.) were fixed and processed for em. at the right is an enlarged area. the large-diameter (a, white arrows) and small-diameter (b, black arrows) hcv-like particles were released from plasma membrane. pm, plasma membrane; bars, 500 nm (left panels) and 100 nm (right panels). (tif) processing pathways of the hepatitis c virus proteins expression and identification of hepatitis c virus polyprotein cleavage products efficient initiation of hcv rna replication in cell culture replication of subgenomic hepatitis c virus rnas in a hepatoma cell line expression of hepatitis c virus proteins induces distinct membrane alterations including a candidate viral replication complex identification of the hepatitis c virus rna replication complex in huh-7 cells harboring subgenomic replicons association of hepatitis c virus replication complexes with microtubules and actin filaments is dependent on the interaction of ns3 and ns5a cytoskeletal requirements for hepatitis c virus (hcv) rna synthesis in the hcv replicon cell culture system initiation of hepatitis c virus infection requires the dynamic microtubule network: role of the viral nucleocapsid protein hepatitis c virus egress and release depend on endosomal trafficking of core protein hepatitis c virus core protein 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c virus entry depends on clathrin-mediated endocytosis we thank dr. takaji wakita of national institute of infectious disease, tokyo, japan, for plasmid pjfh1, dr. charles m. rice from center for the study of hepatitis c, rockefeller university, new york, usa, for huh7.5 cells. key: cord-292537-9ra4r6v6 authors: liu, fenglin; wang, jie; liu, jiawen; li, yue; liu, dagong; tong, junliang; li, zhuoqun; yu, dan; fan, yifan; bi, xiaohui; zhang, xueting; mo, steven title: predicting and analyzing the covid-19 epidemic in china: based on seird, lstm and gwr models date: 2020-08-27 journal: plos one doi: 10.1371/journal.pone.0238280 sha: doc_id: 292537 cord_uid: 9ra4r6v6 in december 2019, the novel coronavirus pneumonia (covid-19) occurred in wuhan, hubei province, china. the epidemic quickly broke out and spread throughout the country. now it becomes a pandemic that affects the whole world. in this study, three models were used to fit and predict the epidemic situation in china: a modified seird (susceptible-exposed-infected-recovered-dead) dynamic model, a neural network method lstm (long short-term memory), and a gwr (geographically weighted regression) model reflecting spatial heterogeneity. overall, all the three models performed well with great accuracy. the dynamic seird prediction ape (absolute percent error) of china had been ≤ 1.0% since mid-february. the lstm model showed comparable accuracy. the gwr model took into account the influence of geographical differences, with r(2) = 99.98% in fitting and 97.95% in prediction. wilcoxon test showed that none of the three models outperformed the other two at the significance level of 0.05. the parametric analysis of the infectious rate and recovery rate demonstrated that china's national policies had effectively slowed down the spread of the epidemic. furthermore, the models in this study provided a wide range of implications for other countries to predict the short-term and long-term trend of covid-19, and to evaluate the intensity and effect of their interventions. novel coronavirus pneumonia (coronavirus disease 2019, covid-19) break out firstly in wuhan, hubei province, china in december 2019, then the epidemic became prevalent in the rest of the world. with the research on covid-19 so far, through the comparison of the gene sequence of the virus with that of the mammalian coronavirus, some studies found that its source may be related to bat, snake, mink, malayan pangolins, turtle and other wild animals [1] [2] [3] [4] . covid-19 can also cause severe respiratory diseases such as fever and cough [5] , and there is a possibility of transmission after symptoms of lower respiratory diseases [6] . however, unlike sars-cov and mers-cov, covid-19 is separated from airway epithelial cells of patients [6] , yet the mechanism of receptor recognition is not consistent with sars [7] . therefore, the pathogenicity of covid-19 is less than that of sars [8] , and its transmissibility is higher than that of sars [9] . in addition, this new coronavirus presents human-to-human transmission [10] , and close contact could lead to group outbreaks [11] . as of july 7th, 2020, 85,359 confirmed cases and 4,648 deaths had been reported in china [12] . in addition to china, there are over 200 countries and regions in the world with a total of 11,630,898 of confirmed cases and 538,512 of deaths [12] . the outbreak of covid-19 happened right before the lunar new year, which is typical chinese spring festival transportation period. with a population of over 11 million, wuhan is one of the major transportation hubs in china as well as a core city of the yangtze river economic belt. the time and location of the outbreak further led to the rapid spread of the epidemic in china [13] . since there is still no vaccine or antiviral drug specifically for covid-19, the government's policies or actions play an important role in flatting the epidemic curve [14] . from the perspective of public health, the interventions of wuhan government have achieved the purpose of reducing the flow of people and the risk of exposure to the diagnosed patients, and also effectively slowed down the spread of the epidemic [15] . nevertheless, covid-19 can be transmitted by asymptomatic carriers [16] , and some of the recovered patients may still be virus carriers [17] . in order to implement non-pharmaceutical interventions more effectively, we used a combination of epidemiological methods, mathematical or statistical modeling tools to provide valuable insights and predictions as benchmarks. for the study of infectious diseases like covid-19, sars, and ebola, most of the literature used descriptive research or model methods to assess indicators and analyze the effect of interventions, such as combining migration data to evaluate the potential infection rate [18, 19] , understanding the impact of factors like environmental temperature and vaccines that might be potentially linked to the diseases [20, 21] , using basic and time-varying reproduction number (r 0 & r t ) to estimate changeable transmission dynamics of epidemic conditions [22] [23] [24] [25] [26] [27] , calculating and predicting the fatal risk to display any stage of outbreak [28] [29] [30] , or providing suggestions and interventions from risk management and other related aspects based on the results of modeling tools or historical lessons [31] [32] [33] [34] [35] [36] [37] [38] [39] . some literature only used one kind of model to simulate and predict the course of diseases. for instance, to use relatively common epidemiological dynamics models like seir or sird to forecast epidemic trends and peaks in certain provinces, even the world [9, [40] [41] [42] [43] [44] ; to apply some other types of statistical models such as the logistic growth models or time series approaches to analyze the epidemic situation [45, 46] , or to develop new models to support more complex trajectories of epidemics or to predict the number of confirmed cases and the spatial progression of outbreaks [47] [48] [49] . several studies were further expanded based on the basic epidemic dynamic models. for example, joining the border protection mechanism with the seir model to better identify high-risk groups and infected cases [50] ; adding the effect of media or awareness into basic models to assess whether these outside influences would possible change the transmission mode of infectious diseases [51, 52] ; or according to transmission routes contained in dynamic models, using a multiplex network model or transmission network topology to analyze the outbreak scale and epidemic spread more accurately [53, 54] . a small number of studies combined the analysis capabilities of two types of models, like seir model and the recurrent neural networks model (rnn), to determine whether certain interventions could affect the results of outbreak control [55] . however, we did not find any analysis method using geographically weighted regression (gwr) on covid-19 study based on our literature research. there is also a lack of understanding the model efficacy of predicting the epidemic curve among different algorithms. in this study, an seir's extended model seird was used to simulate the epidemic situation in china and to predict the number of confirmed and cured cases in each province and several major chinese cities. an lstm model combined with traffic data and a gwr model were used to predict the number of confirmed patients. specifically, gwr model showing geographical differences was used to predict the development of epidemic situation and analyze the impact of geographical factors. this paper also compares the characteristics and prediction ability of these models. in the absence of vaccines and drugs for covid-19, it makes sense to use multiple models to show the situation and intensity of non-pharmaceutical interventions needed to simulate and guide the control of outbreaks. daily updated covid-19 epidemiological data used in this study were retrieved from national health commission of china [12] and accessed via https://github.com/wybert/openwuhan-ncov-illness-data. the daily number of outbound from wuhan city and relevant migration indice from january to march were collected from an online platform called baidu qianxi [56] . the demographic data and medical resources data were from china urban statistical yearbook published by the national bureau of statistics as shown in s1 table. this study used seird model and the changes in the status of the susceptible (s), exposed (e), infected (i), recovered (r) and dead (d) population in the total population (n) are shown in fig 1. according to the medical characteristics and clinical trials of covid-19, both confirmed patients and asymptomatic carriers have the ability to transmit the virus. therefore, susceptible people have a certain chance to become infected after they come into contact with exposed or infected individuals [43] . carriers in the exposed status may develop obvious symptoms after the incubation period and become diagnosed or they may be recovered. the final status of individuals can be basically divided into two categories: one is the recovery from the combined effects of treatment in hospital and autoimmunity, and the other is the death without effective treatment. in the model formula, the infectious rate β needs to be adjusted in real time to adapt to the trend of disease development. in the middle and late stages of the epidemic, the number of daily new cases decreased significantly due to the positive influence of government policies. thus, to better fit the model, we added an attenuation factor desc to β. based on the basic seird model formulas [57, 58] , our modified model was shown as eqs (1) (2) (3) (4) (5) (6) . here, the parameter t denotes the time; β is the infectious rate; α is the rate for the exposed to be infected; γ 1 is recovery rate for the exposed; γ 2 is the recovery rate for the infected; k is the mortality rate; "desc" is the attenuation factor for β, so that β decays exponentially when 0<desc<1, and β is a constant when desc = 1. lstm (long short-term memory) architecture for recurrent neural networks was first proposed in 1997 [59] . a lstm block is illustrated in fig 2. it features three gates (input, forget, and output), a block input and an output. the output of the block is recurrently connected to the input of the block. the vector formulas for a lstm layer forward pass are given below in eqs (7) (8) (9) (10) (11) (12) . here, z t , i t , f t , c t , o t and h t denote the block input, input gate, forget gate, cell state, output gate and block output, respectively. and x t represents the input vector at time t, ⊙ is the pointwise multiplication operator of two vectors, the w z , w i , w f , and w o are input weight matrices, is used as the activation function of the gates and relu is used as the activation function of the block input and output. epidemic situations and medical resources in different geographic situations may have different extents of influence on the development of the epidemic. ordinary least squares fitting method for regression may not be applicable in this case. geographically weighted regression model (gwr) was proposed in 1996 [60] , which extended the ordinary linear regression model and embedded the geographic location data into the regression parameters as shown below: where y i is the i th dependent variable, x ik is the k th independent variable in location i, p is the total number of independent variables, β i0 is the intercept parameter in location i, β ik is the regression coefficient for the k th independent variable in location i, which varies with the geographical location, and ε i is the error term in location i. the spatial weight matrix in this study uses the bi-square kernel function shown below: if d ij <b, otherwise w ij = 0, where b is the bandwidth, a non-negative attenuation parameter and d ij denotes the distance between the i th and j th observation points. the bandwidth is calculated by optimizing the root mean square prediction error of cross-validation [61, 62] . in this study, we used the modified seird model to make predictions of the number of cumulative confirmed cases in the next day for all provinces, province-level municipalities and autonomous regions in china as well as wuhan city. the parameters were adjusted daily in our dynamic seird model based on the daily updated epidemic data. the comparison of the actual data on february 14 th and february 25 th with the forecast results of our models is shown in table 1 . the percent error was calculated using the formula: (predicted number-actual number)/ actual number × 100%. on february 14 th , the absolute percent errors of all provinces were < 5%. the percent error for wuhan city, hubei province and china were -3.00%, -1.60% and 1.00%, respectively. on february 25 th , the absolute percent error of prediction of cumulative confirmed cases in china was < 0.10%. the absolute percent errors of most provinces were < 0.10%, among which the absolute percent errors in wuhan city was < 0.10% and that of hubei province was less than 0.10%. regarding the number of recovered cases, wuhan city and hubei province had percent errors of -6.03% and -3.12%, respectively. the overall prediction of recovered of the whole country was consistent with the actual situation with percent error of -2.46%. the predicted number of deaths in hubei province was off by 1.40% (forecast 2,599 vs. actual 2,563). actual and predicted number of confirmed cases using the modified seird model for china, hubei province and wuhan city are shown in fig 4 ( hubei province and wuhan city adjusted the criteria for diagnosis on february 13 th , and the number of confirmed cases increased by about 10,000 on that day [63] . in order to smooth the sudden change, the number of cumulative cases before february 12 th in hubei city and wuhan province was proportionally enlarged according to the new criteria. the same for fig 5) . the actual and calculated values of these three regions provided satisfying fitting curves, indicating that the situation simulated by the model was basically in line with the actual situation of the epidemic development. in this study, the inflection point was defined as the date when the number of existing confirmed cases has the largest slope. according to the seird dynamic model, the inflection points of all provinces appeared generally in february, while the specific time varied from region to region. the results of model simulation revealed that the inflection point in wuhan city and hubei province showed up in early february, and that of the whole country roughly in the first half of february, which basically conformed to the spread of covid-19 in china. using data on march 5 th , the model predicted the long-term trends in the number of confirmed, cured and deaths for china, hubei province and wuhan city (fig 5) . again, the model used adjusted historical data as discussed above. under the various social non-pharmaceutical interventions and not allowing for the imported cases from foreign countries, the cumulative number of confirmed nationwide was expected to reach about 83,000 at the end of the epidemic. hubei province was expected to have a total of about 70,000 confirmed cases and wuhan city about 50,000. data from four regions, zhejiang, guangdong, beijing, and shanghai were selected to train the lstm neural network to predict the number of cumulative confirmed cases of the next day. since the lstm model had a memory function, the first feature included in the model was the number of cumulative confirmed cases on the previous day. considering that the number of migrants from wuhan also affected the studied city, thus the number of migrants from wuhan was also included in the analysis. there was a certain probability that some migrants from wuhan may be patients because of the virus's incubation period, and the inference of this probability was based on the number of confirmed cases in wuhan. therefore, the second feature considered the number of migrants from wuhan on the previous day, and the confirmed number of patients in wuhan on the previous day. the feature was calculated as the cumulative number of immigrants from wuhan multiplied by the incidence of covid-19 in wuhan on the previous day. this lstm architecture was designed into 4 layers: an input layer, an lstm layer (hidden layer), a fully-connected layer and an output. each lstm neuron had 10 hidden features, and the activation function was relu. the loss function was mse, and the optimizer was "adam". the model structure diagram is as fig 6. this study used the grid search method to set different hyperparameters for data in different regions. the model was trained and the predicted results for latest 8 consecutive days as shown in in this study, the data of 220 cities that had confirmed cases on february 2 nd were selected to predict the number of confirmed cases on february 3 rd . the number of confirmed cases, the number of deaths and the number of cured cases are main indicators for the epidemic. among them, the number of confirmed cases was the mostly used and reflected the severity of covid-19 epidemic. therefore, this study used the cumulative number of confirmed cases in different places released by the national health commission as dependent variable. in this study we select the population of each city, the number of hospitals per 10,000 people, the number of doctors per 10,000 people, the number of inpatient beds per 10,000 people, the number of confirmed cases, the number of cured cases, and the number of deaths one day and 2 days ago as independent variables. the gwr model was fitted using the data of february 2 nd , and we further made forecast for the number of the confirmed cases on february 3 rd . the r 2 of gwr regression on february 2 nd was 99.98% and the r 2 of the prediction of the data on february 3 rd was 97.95%. the percent errors of fitting and prediction varied for different cities: for beijing were 11.67% and 3.95%, respectively; for shanghai were 2.24% and -5.88%, respectively, for xiaogan in hubei province were -1.27% and 1.70%, respectively, and for wuhan were 0.00% and 14.57%, respectively. the summary of the intercept and coefficients of the independent variables were listed in table 4 . it shows that the coefficients of the demographic data, and the medical resources data have larger variations than those of epidemic data. the coefficients of population, number of hospitals per 10,000 people, number of doctors per 10,000 people, dead_lag1, confirmed_lag2, cured_lag2 were negative, showing that these factors have negative influence on the dependent variable. while the other independent variables, number of inpatient beds per 10,000 people, confirmed_lag1, cured_lag1, dead_lag2 have positive coefficients, indicating positive influence on the dependent variable as shown in table 4 . as of mid-march 2020, more than 60,000 people had been cured in 31 provinces, provincelevel municipalities, and autonomous regions in china, and new cases of infection were mainly led by overseas imports. although the covid-19 epidemic was not over, the traffic in the low-and medium-risk areas in hubei province had been gradually resuming, indicating that the government's non-pharmaceutical interventions had significantly positive effects. in this study, the modified seird model was used to conduct parameter sensitivity analysis of β, desc, and γ 2 based on data before march 5 th , so as to simulate the impact of prevention and control measures on real-time infections for china, hubei province, wuhan city, and beijing city (fig 9) . the decrease of the infectious rate β would promote the reduction of infections during the entire epidemic stage with other conditions being equal (fig 9a) . the shape of the epidemic curve was basically unchanged, but the duration of the epidemic increase as the infectious rate itself increases. the number of cases increased obviously, and the peak of real-time infections was postponed as the infectious rate increases. when the infectious rate increased to 125%, the epidemic size doubled with the delay of the peak of real-time infections by about 10 days ( fig 9a) . moreover, increasing the attenuation factor of infectious rate could lead to a significant slowdown in the spread of the epidemic and the shape of the epidemic curve changed (fig 9b) . in the beginning, the growth of attenuation factor changed the number of confirmed cases little, but the number had changed dramatically over time, the peak of the epidemic moved forward with the increase in the attenuation factor (fig 9b) . the duration of the epidemic also advanced correspondingly. a combination of the changes in the infectious rate β itself and the changes in the attenuation factor of β could reflect the effects of the measures such as timely isolation of confirmed or suspected patients and reduction of population mobility. coupled with the community containment measure, the number of exposed, infected and susceptible individuals outside were greatly reduced, so that the extent of the epidemic in china had been under control. implemented metropolitan-wide quarantine of wuhan city itself could also interfere with the change of infectious rate. the decrease in the number of daily new confirmed cases since late february showed that the corresponding policies had effectively blocked the spread of the epidemic. the change in the recovery rate of infected γ 2 had little effect in the early stage of the epidemic. as time went by, the growth of recovery rate could significantly raise the number of recovered, thus advancing the peak time of the real-time confirmed cases (fig 9c) . when the recovery rate raised from 75% to 125%, the whole country, hubei province, wuhan city and beijing city could reach the time of maximum real-time infections about 6-15 days in advance, and the scale of the epidemic could be reduced as well (fig 9c) . in fact, china transported advantage medical resources of more than 20,000 people to hubei province [5] in order to achieve the goal of early detection, early reporting, early diagnosis, and early isolation. besides, the measure of "one province helping one city" established provincial counterparts to support the rescue work in hubei province except wuhan [5] , so as to rationally allocate advanced resources. these interventions could improve the treatment and medical level of key provinces and cities, thereby increasing the recovery rate of infected and reducing the mortality rate. by march 13 th , 2020, more than a thousand people each day have been cured and discharged for 29 consecutive days [6] , indicating the effectiveness of related policies. although the covid-19 has been effectively controlled in china, it has spread rapidly in other countries. italy, the united states and spain have become the focused areas of the outbreak. by may 2 nd , 2020, the united states, as the country with the largest number of confirmed cases, has over 1.1 million cases, and spain had 216,582 cases, and italy ranked the third with 207,428 confirmed patients [12] . in order to control the spread of coronavirus, america took measures to reduce the mobility of the population, built hospitals and facilitate the treatment of the coronavirus [64] [65] [66] [67] . similar to the us, italy and spain also tried to limit the movement and gathering of the crowds, improve the protection level and provide more medical resources [64, [68] [69] [70] . in conclusion, all three countries have implemented various interventions to slow down the spread of the covid-19 disease. the measures could be basically divided into two categories: reducing the infection rate and increasing the recovery rate. however, according to the recent large-scale outbreak in the united states and spain, it could be found that a part of the people in these two countries might have insufficient awareness of prevention and control of the epidemic [64] . the supervision of those prevention and control measures needs further improvement. thanks to the joint efforts of the people across the italy, while the number of confirmed cases in italy is still large, this country, which was called "the second hubei province" in the early stage of the epidemic, has a trend of declining new cases of infection and death [12] . in order to test the capability of the seird model in foreign countries, data before june 29 th , 2020 of italy were used to calculate the epidemic curve. the results of the model also fitted well with the actual data as shown in fig 10. although some other countries successfully controlled the epidemic using similar measures with china [71] , they may not always work in other countries because the effect depends on the public attitudes towards the measures and commitment to the intervention as debated in [72] . therefore, in the face of the same epidemic situation and similar crises, our seird dynamics model can be potentially applied to other countries to evaluate the intensity and effect of policies implemented by simulating and forecasting the situation of the epidemic, but the effect may be limited by the attitudes and action of the public. to better understand the spatial distribution of the coefficients of the independent variables in the gwr model, four parameters and their correlations in the model of february 2 nd have been studied to evaluation the heterogeneity of their coefficients in space. there was a strong negative correlation between the number of hospitals per 10,000 people and the number of confirmed cases (fig 11a) . this can be explained as that the isolation of confirmed cases in the hospital can prevent contagion. from the perspective of the spatial distribution of the regression coefficients, it has a trend of gradual decline from the northeast to the southwest and northwest of china (fig 11a) . the most influenced areas are located in the northeast of china, while the least influenced areas are in southwest and northwest of china. there was a negative correlation between the number of doctors per 10,000 people and the number of confirmed cases (fig 11b) . from the perspective of the spatial distribution of regression coefficient, it shows a gradually decreasing trend from the northeast and northwest of china to the south (fig 11b) . the regions that are influenced the most are concentrated in northeast and northwest of china, while the least influenced regions are in the south. there was a positive correlation between the number of confirmed cases and the confirmed cases one day ago (fig 11c) . this suggests that the more cases confirmed the day before, the more confirmed cases would emerge the next day. effective local quarantine measures can be used to prevent a pandemic. from the perspective of the spatial distribution of the regression coefficient, it shows a trend of gradual decline from the northeast to the southwest and northwest of china (fig 11c) . this trend is not significant, which shows a universal pattern across the country. there was a positive correlation between the number of cured case and the number of confirmed cases one day ago (fig 11d) . from the perspective of the spatial distribution of regression coefficient, it shows a gradually decreasing trend from the northeast and northwest of china to the south, with the most influenced areas in the northeast and northwest, and the least influenced areas in the south (fig 11d) . by comparing the prediction capabilities of these three types of models, the modified seird, lstm and gwr model could effectively predict the epidemic data for the next day generally. the percent errors of the seird model to predict confirmed cases were within ±5.0% in all of these four selected regions (beijing, wuhan, hubei and china) shown in table 5 . the lstm model also fit well to the real curve by incorporating traffic big data, indicating good simulation and prediction effects. the average percent error of lstm model predictions for the four selected provinces and cities was within ±1.0% on february 14 th (table 5) . gwr model could reflect spatial heterogeneity but larger percent errors showed than the other two models in some cases ( table 5 ). the mape (mean absolute percentage error) for the seird, lstm and gwr models in the selected areas were 1.70%, 1.51%, 3.44%, respectively. in order to compare the ape (absolute percent error) of the three models, we ran wilcoxon signed rank test for the paired observations in table 5 . the p-values for the hypotheses: the ape of gwr> that of lstm, the ape of gwr > that of seird and the ape of seird> that of lstm were 0.173, 0.187 and 0.459, respectively, thus not significant at the level of 0.05. overall, the prediction efficacy of gwr model was inferior to those of seird and lstm models according to the mape and p-values. in this study, the modified seird model, the lstm model with traffic data and the gwr model reflecting the geographical environment were used to make forecasts for the development of covid-19 in china. these three types of models all showed remarkable prediction capabilities. the parameter sensitivity analysis reflected the effectiveness of nonpharmaceutical interventions. now the epidemic quickly spread abroad, in the absence of targeted pharmaceutical treatment such as vaccines, the interventions implemented in various countries were basically similar to those in china, which were based on the two aspects: reducing the infectious rate and improving the recovery rate. as the number of daily new cases continues to increase globally, models in this study shows potential being used for epidemic curve prediction and prevention of covid-19 in other countries. supporting 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networks long short-term memory geographically weighted regression: a method for exploring spatial nonstationarity a simulation-based study of geographically weighted regression as a method for investigating spatially varying relationships geographically weighted regression: the analysis of spatially varying relationships dxy. 2019-ncov daily 24 coronavirus news active alerts in parks fda will allow doctors to treat critically ill coronavirus patients with blood from survivors italy under lockdown to fight coronavirus italy implements more measures in response to coronavirus epidemic spain has nationalized all of its private hospitals as the country goes into coronavirus lockdown active case finding with case management: the key to tackling the covid-19 pandemic we thank our colleagues of taikang pension & insurance co., ltd. for their support, especially dr. ying han, who helped improve english and ms. jieyu wang, who helped in discussion and material preparation. key: cord-330110-pamxy4av authors: teissier, elodie; zandomeneghi, giorgia; loquet, antoine; lavillette, dimitri; lavergne, jean-pierre; montserret, roland; cosset, françois-loïc; böckmann, anja; meier, beat h.; penin, françois; pécheur, eve-isabelle title: mechanism of inhibition of enveloped virus membrane fusion by the antiviral drug arbidol date: 2011-01-25 journal: plos one doi: 10.1371/journal.pone.0015874 sha: doc_id: 330110 cord_uid: pamxy4av the broad-spectrum antiviral arbidol (arb) inhibits cell entry of enveloped viruses by blocking viral fusion with host cell membrane. to better understand arb mechanism of action, we investigated its interactions with phospholipids and membrane peptides. we demonstrate that arb associates with phospholipids in the micromolar range. nmr reveals that arb interacts with the polar head-group of phospholipid at the membrane interface. fluorescence studies of interactions between arb and either tryptophan derivatives or membrane peptides reconstituted into liposomes show that arb interacts with tryptophan in the micromolar range. interestingly, apparent binding affinities between lipids and tryptophan residues are comparable with those of arb ic50 of the hepatitis c virus (hcv) membrane fusion. since tryptophan residues of membrane proteins are known to bind preferentially at the membrane interface, these data suggest that arb could increase the strength of virus glycoprotein's interactions with the membrane, due to a dual binding mode involving aromatic residues and phospholipids. the resulting complexation would inhibit the expected viral glycoprotein conformational changes required during the fusion process. our findings pave the way towards the design of new drugs exhibiting arb-like interfacial membrane binding properties to inhibit early steps of virus entry, i.e., attractive targets to combat viral infection. distinct from specific antiviral compounds that target key viral functions are a group of broad-spectrum medicinal drugs that were originally designed for other treatments [1] [2] [3] or targeted toward a number of viruses ( [4] ; reviewed in [5] ). the advantage of this group of antivirals is that they have already met the pharmacological criteria for medicinal drugs and are already approved for clinical use in some countries. among these molecules, antiviral agents targeting viral entry of enveloped viruses are of major interest since they seize an early step in the viral life cycle, before damages have occurred to cells (recently reviewed in [6, 7] ), and since they can be incorporated into combinations of multiple drugs with different targets. one of these compounds, arbidol [arb; 1hindole-3-carboxylic acid, 6-bromo-4-[(dimethylamino)-methyl]-5hydroxy-1-methyl-2-[(phenylthio)methyl]-, ethyl ester, monohydrochloride; cas registry number 131707-23-8 ( figure 1 )], is already licensed in russia and china, and is described as an antiinfluenza drug with immunostimulant properties. arb is in use for several years as prophylaxis and treatment for influenza a and b infections. it inhibits influenza virus-induced membrane fusion and may have the capacity to induce serum interferon [8] . recent studies extended its inhibitory activity to other human viruses such as the respiratory syncytial virus, parainfluenza virus 3, rhinovirus 14 , and hepatitis b virus (reviewed in [5, 9] ). we demonstrated that it also inhibits hepatitis c virus (hcv) infection in vitro, and hcv replication [10] , hcv cell entry and membrane fusion using hcv pseudoparticles (hcvpp) and hcv grown in cell culture (hcvcc) [11, 12] . most recently, ciliberto and coworkers demonstrated the efficacy of arb derivatives at inhibiting hcv entry and replication into hepatoma cells in the low micromolar range [13] . hcv infection is a leading cause of liver diseases, including hepatocellular carcinoma, and therapeutic options are still limited (for recent reviews, see [14] and refs therein). there is thus an urgent need to develop efficient and well tolerated drugs to combat this virus. arb demonstrated a propensity to enter into hydrophobic interactions with membranes, and with membrane-like environments such as detergent micelles [12] . here we further characterize the mechanism of action of arbidol, and analyze at the molecular and atomic level the interactions of arb with membranes, tryptophan-rich derivatives and peptides. we first examined how arb inhibits hcv entry and membrane fusion using hcvpp of different genotypes, and found that arb inhibition was genotype-independent. by combining surface plasmon resonance, fluorescence and nmr spectroscopy approaches, we showed that arb directly interacts with the phospholipid membrane interface, with an affinity in the micromolar range, comparable to the concentration inhibiting hcvpp membrane fusion by 50% (ic50). arb also displayed micromolar affinity toward aromatic components of proteins such as tryptophan and derivatives, and toward peptides containing tryptophans and derived from hcv envelope glycoproteins. altogether our results demonstrate that arb interacts with the polar head of phospholipid membranes and protein motifs enriched in aromatic residues, suggesting that the inhibitory activity of arb on hcv entry and fusion could involve both types of interactions. phosphatidylcholine from egg yolk (pc, 99% pure), dimyristoylphosphatidylcholine (dmpc, 99% pure), cholesterol (chol, 99% pure), lyso-phosphatidylcholine (lysopc), dodecyl-phosphocholine (dpc), triton x-100, tryptophan octyl ester hydrochloride (toe) and n-acetyl-l-tryptophanamide (nata) were purchased from sigma. octadecyl rhodamine b chloride (r 18 ) was from molecular probes. the peptides used were part of the sequence of structural or non structural (ns) proteins of hcv and of the bovine viral diarrheal virus (bvdv). the amphiphilic helix of bvdv ns5a [15] and the transmembrane domain of hcv ns4a [16] were obtained as described previously. the peptides identified as important for hcv fusion [17] were purchased from clonstar biotech (90% purity) or sigma genosys (70% purity), respectively, and dissolved in dmso before preparation of lipid:peptide mixtures. arbidol [arb, 1h-indole-3-carboxylic acid, 6-bromo-4-[(dimethylamino)methyl]-5-hydroxy-1-methyl-2-[(phenylthio)methyl]-, ethyl ester, monohydrochloride ( figure 1 )] was a kind gift from stephen j. polyak. arb was readily soluble in ethanol, and soluble in the mm range in water. ethanol stock solutions of arb were diluted to a 1.88 mm final concentration in milliq water (the final stock solution contained 10% ethanol). for spr experiments, one mg of arb was resuspended in water, followed by centrifugation (160006g, 15 min, 4uc). arb concentration in solution was measured at 280 nm in the supernatant (arb extinction coefficient = 9510 m 21 .cm 21 ). after solvent evaporation, samples were resuspended in phosphate-buffered saline (pbs, ph 7.4) or water, and underwent 5 freeze/thaw cycles (liquid nitrogen and 37uc, respectively). liposomes were prepared by extrusion over a stack of avestin polycarbonate filters (100 nm), as described [18] . huh-7 cells [19] were maintained in dmem containing 4.5 g/ l d-glucose and 4 mm l-glutamine (invitrogen, cergy-pontoise, france), supplemented with 100 u/ml penicillin, 100 mg/ml streptomycin and 10% fcs (lonza). productions of pseudotyped viruses were obtained by the transient transfection of 293t cells by the calcium-phosphate method. for the genotype study, hcvpp of genotypes 1a (h77; af011752), 1b (con1; aj238799), 2a (jfh1; ab047639), 2b (ukn2b 2.8, ay734983), 3a (ukn3a 1.28, ay734984), 4a (ukn4 21.16, ay734987), 5a (ukn5.14.4, ay785283) and 6a (ukn6.5.340, ay736194) were produced as described previously [20] from 293t cells co-transfected with a murine leukemia virus (mlv) gag-pol packaging construct, an mlv-based transfer vector encoding gfp as a marker protein, and the e1-e2 expression constructs. supernatants were collected 48h post-transfection and filtered on 0.45 mm. for genotypes 5a and 6a, pseudoparticles were concentrated 100-fold after ultracentrifugation through a 20% sucrose cushion at 75,0006g for 2h at 4uc. pellets were resuspended in the regular medium of huh-7 cells. for infection experiments, huh-7 cells were seeded at 4000 cells/well in 96-well plates. the following day, cells were infected in the presence of increasing arb concentrations for 6 h. arbidol effect on viral infectivity was evaluated by assaying gfp activity 72 hours after infection using flow cytometry (facscalibur). pseudoparticles harbouring at their surface the influenza hemagglutinin (happ) and the envelope glycoprotein of the rd114 feline oncovirus (rd114pp) were prepared as described in [18] and [12] , respectively. lipid mixing between pseudoparticles and pc:chol:r 18 liposomes was monitored by fluorescent spectroscopy, as the dequenching of r 18 [18] . in brief, r 18 note that numbering in panel a refers to proton numbers, as identified in nmr (cf figure 5 and table 1 ). doi:10.1371/journal.pone.0015874.g001 ay734987 (4a) 9.10e 4 ; ay785283 (5a) 3.10e 4 ; ay736194 (6a) 5.10e 3 ; ha 8.10e 8 ; rd114 2.10e 6 ], and incubated 2 min. fusion was initiated by acidification to ph 5 with hcl, and recorded on an slm aminco 8000 spectrofluorimeter over a 10-min time period, at excitation and emission wavelengths of 560 nm and 590 nm, respectively. maximal r 18 dequenching was measured after the addition of 0.1% triton x-100 (final concentration) to the cuvette to lyse liposomes. the same procedure was used to follow pseudoparticle fusion in the presence of arb; in this case, after a 1-min incubation of pseudoparticles with liposomes, arb (11.3 mm final concentration) was added and incubated for 1 min, and fusion initiated by acidification. indole emission fluorescence spectra of tryptophan derivatives were recorded at excitation wavelength of 286 nm (spectral zone of lowest absorption of arb), under various conditions: nata (5 mm final) in pbs at ph 7.4 or 5.0; toe at 5 mm in lyso-pc micelles (toe:lysopc molar ratio 1:800), and in pc and pc:chol liposomes (toe:lipid molar ratio 1:20); peptides at 5 mm in pc:chol liposomes (peptide:lipid molar ratio 1:20). spectra were obtained in the absence or presence of increasing concentrations of arb (0 to 100 mm). samples were incubated 2 min at 37uc prior to recording. emission spectra were collected in the 300-400 nm region (with 2 nm-increments), with blanks substracted, using a black flat-bottom, low-binding 96-well microplate (greiner bioone). measurements were recorded on a tecan infiniteh m1000 spectrofluorimeter. k d app values were calculated from the difference between the areas under the spectra in the absence or presence of arb (da), at various arb concentrations, by nonlinear fitting using the equation da = da max c/(k d +c). fluorescence measurements were repeated three times to obtain averaged values of k d app. guvs were made by the electroformation method [21] . the flow chamber (warner instruments, connecticut, usa) used for vesicle preparation was equipped with two glass coverslips, each coated with optically transparent and electrically conductive indium tin oxide (ito) (philips, eindhoven, nl). mixtures of lipids [pc:chol:r 18 (65:30:5, molar ratio)] were prepared at 0.1 mm in chloroform. the lipid mixture (2 nmoles) was spread into a thin and uniform film on the conductive face of ito-coated slide. after chloroform evaporation, the dried lipid film was hydrated by adding water into the chamber (,400 ml) and an alternative electrical field (10 hz and 1.2v) was applied at room temperature for 3 hours. guvs in the absence or presence of increasing amounts of arb solubilized in water (0 to 40 nmoles), were observed by epifluorescence microscopy. interaction of arb with dmpc layers was investigated with a biacore 3000h using a l1 sensor chip at 30uc. the sensor chip surface was washed with a mixture of 50 mm naoh and isopropanol (6:4, v:v) , at a flow rate of 20 ml/min for 1 min. the running buffer was milliq water. the influence of liposome concentrations on the final spr signal was tested; we assayed lipid concentrations from 0.5 to 5 mm and measured the resulting resonance units (ru). we obtained a well detectable, reproducible and stable signal from 2 mm, and further increasing this concentration did not improve the signal. we therefore chose the 2 mm concentration for our experiments. dmpc liposomes were resuspended in milliq water and captured on sensor chip at 2 ml/min for 5 min. the flow rate was increased to 30 ml/min and the liposome surface was then washed with 10 mm naoh for 1 min. liposomes immobilized on the chip surface gave approx. a 5000 ru signal. to calculate arb affinity for lipids, its association to and dissociation from dmpc layers were studied at different arb concentrations in water, from 0.5 to 10 mm, at a flow rate of 20 ml/min. after each binding cycle, the sensor surface was regenerated to the original matrix by injecting 50 mm naoh/ isopropanol (6:4, v:v). the sensor surface was then coated with a fresh liposome suspension for the next binding cycle. k d values were calculated from the equilibrium resonance signal (r eq ) as a function of the analyte concentration. r eq values were estimated by extrapolation to infinite time using plots of resonance signal as a function of the reciprocal of time. apparent k d were then calculated by nonlinear fitting to the expression r eq = r max c/ (k d +c), where r max is the maximum binding capacity of the surface and c is the analyte concentration, using the sigmaplot software. for the nmr studies, the bicellar system was prepared by another sample with similar lipid concentration and higher arb content (molar ratio 1/15) was also prepared. the amount of free arb in arb:dmpc mixture was estimated after rapid separation of lipids on ultrafiltration membrane (cutoff 5000 da) and measure of arb concentration in the ultrafiltrate at 280 nm. for arb:dmpc molar ratio of 1:4 at neutral ph, free arb was found to be lower that 0.2%. we thus concluded that the amount of free arb in nmr samples was negligible. 1 h nmr experiments were performed on a bruker dmx 400 spectrometer operating at a proton frequency of 400 mhz. spectra were recorded with a 5 mm triple resonance inverse txi probe equipped with z-gradient. the p/2 pulse was 10.3 ms, the recycle delay was 3 s and solvent suppression with presaturation was used. 1d 1 h spectra were measured acquiring 120 scans. these spectra were used for the assignment of the drug signals together with 2d noesy and 1 h/ 13 c hsqc spectra (data not shown). the assignment of the lipid resonances was derived from the comparison with data in the literature [22] . to times were measured by using inversion recovery experiments with an interpulse delay ranging between 5 ms and 13 s. each measurement was repeated 3 times, adding 240 scans with a delay time between scans of 15 s. all the spectra were processed using matnmr [23] . the 1 h frequency scale is given in terms of chemical shift relative to the acetone signal used as an external reference (2.218 ppm). we have previously shown that arb could inhibit cell entry and membrane fusion of hcvpp of genotypes 1a, 1b and 2a [10, 12] , and hcvcc of genotype 2a [11] . here we sought to investigate the effect of arb on other major hcvpp genotypes as well. hcvpp infectivity toward huh-7 cells, objectifying hcvpp entry, was assayed by counting cells positive for gfp (as the marker protein), incubated with or without increasing arb concentrations for 6h (see materials and methods). a representative data set is shown in figure s1a , and inhibition obtained for the highest concentration of arb (11.3 mm) is presented in figure 2a . the inhibitory effect of arb on hcvpp cell entry depends on hcvpp genotype. indeed, within biological intrinsic variability of hcvpp preparation and samples, three cases could be distinguished: entry of hcvpp of genotypes 2a and 3a was inhibited by ca. 60%, while 1a, 1b and 2b exhibited a 40%-inhibition, but entry of genotypes 5a and 6a was weakly affected by the presence of arb ( fig. 2a) . the influence of arb on hcvpp-mediated lipid mixing was assayed by fluorescence spectroscopy using fluorescent liposomes, as previously described [18] . lipid mixing between hcvpp and liposomes was only observed at low ph and optimal at ph 5.0 [18] . in the presence of increasing arb concentrations, lipid mixing was inhibited in an arb dose-dependent manner ( figure s1b for hcvpp genotype 4a). in contrast to what was observed for hcvpp infectivity, the effect of 11.3 mm arb on hcvppmediated membrane fusion ( figure 2b ) was similar for all tested genotypes, with about 50% inhibition of membrane fusion activity. this indicates that membrane fusion inhibition by arb is not genotype-dependent. these data suggest that the differential inhibitory effect of arb on hcvpp infectivity of various genotypes is likely due to a genotype-dependent modulation of hcv glycoproteins interaction with the cellular proteins (e.g. hcv receptors) involved in hcv cell entry. conversely, arb inhibition of hcvpp membrane fusion, as assessed by a in vitro model system where the only proteins present are the viral glycoproteins, could merely reflect the interaction of arb on lipids and/or on motifs present in hcv glycoproteins of any genotype. to test these hypotheses, we further investigated arb interaction properties with lipids and protein fragments using the approaches described in the following. we previously showed that arb could interact with liposomes and membrane-like environments such as detergent micelles [12] . we further investigated this feature by studying the interactions of arb with giant unilamellar liposomes (guv) by optical microscopy ( figure 3 ). guv are pure lipid bilayers, intrinsically flexible and unstable due to their very large size (in the range of tens of mm) [24] . increasing arb concentrations were added to the chamber where guv composed of pc:chol were electroformed (see methods section), with arb-to-lipid molar ratios of 1:40, 1:20, 1:10, 1:1, 10:1 and 20:1. the guv bilayer was unaffected by the presence of arb up to a 1:20 arb-to-lipid ratio, with occasional membrane flickerings ( fig. 3c and asterisk in fig. 3e ). at higher ratios, membrane inhomogeneities and invaginations appeared (fig. 3f , asterisks in fig. 3d ), and a major overall membrane reorganization was observed at a 20:1 arb-to-lipid ratio (fig. 3g ). note that no lysis or membrane dislocation of guv was observed happ are presented as control pseudoparticles sensitive to arbidol (cf also [10] ), and rd114pp insensitive to arbidol (cf also [12] ). * 1 , the mutant hcvpp w529a (cf [17] ) are presented as a negative control of entry, displaying very low infectivity. b, membrane fusion between hcvpp and r 18 -labeled liposomes was measured by recording the kinetics of lipid mixing by fluorescence spectroscopy (excitation and emission wavelengths were 560 and 590 nm, respectively), as described in the materials and methods section. values of the last 30 s of fusion kinetics (final extent of fusion) were used to calculate the percentage of fusion in the presence of arb, relative to fusion kinetics without arb (100%). results are the mean +/2 sem of 4 separate experiments. happ and mutant hcvpp w529a were taken as controls. * 2 : no fusion was observed for rd114pp. doi:10.1371/journal.pone.0015874.g002 for any condition, even at the highest ratio (data not shown). these results reveal that only very high concentrations of arb with respect to lipids could significantly perturb the lipid organization of these bilayers. this also indicates that the direct interaction of arb to lipid bilayers at the concentrations used to inhibit hcvpp infectivity and membrane fusion (panel e) do not perturb lipid organization. in addition, hcvpp pre-incubated at neutral or acidic ph with arb, even at very high concentrations (100 mm), displayed similar morphology (visualized by transmission electron microscopy) as those observed in the absence of the drug (data not shown). indeed we counted over 160 hcvpp for each condition, and no difference in hcvpp morphology could be observed between the parameters assessed. this indicates that arb inhibition of hcvpp fusion is not due to viral particle disruption/damage. to gain insight into the molecular details of the interaction of arb with lipid membranes, we next investigated the lipid binding properties of arb by using surface plasmon resonance (spr, biacoreh technology). we used a biacore's l1 sensor chip to capture dmpc liposomes. this sensor chip displays lipophilic groups attached on the surface of a carboxymethylated dextran layer, and was shown to provide a quick and reproducible method for the preparation of bilayer-mimetic systems [25] . we first tested whether arbidol per se could bind or not to the chip. arb at 11.3 mm (the highest concentration relevant in the biological context) was injected onto the chip devoid of liposomes. this led to approx. 60 resonance units (ru, see methods section). dmpc liposomes (2 mm) captured onto the sensor chip reached about 5000 ru, and a further ,600 ru was seen when arb was pulsed onto the liposome-coated chip. the binding of arbidol alone on the l1 chip remains therefore negligible. measures of arb/dmpc association and dissociation were performed with various arb concentrations ranging from 0.5 to 11.3 mm. after passage over the surface of the sensor chip, arb bound to immobilized dmpc in a concentration-dependent manner ( figure 4 ). arb initial binding was fast, but then slowed down without reaching saturation equilibrium (from 0 to 240 s). after stopping the arb flow onto the sensor chip (from 240 s), bound arb was rapidly but incompletely dissociated from dmpc membranes. indeed, for all arb concentrations tested, about 50% of arb remained bound to dmpc. this demonstrates that arb is capable of interacting with lipid membranes, in a stable association between arb and dmpc. however the behaviour of arb binding to membranes rendered difficult the fitting of a kinetic model to the data, and hence the determination of reliable on-and off-rates. indeed using global fitting, binding curves could not be fitted properly with the different models included in the biaevalution 3.0 software (1:1 langmuir binding, bivalent analyte, heterogeneous ligand, heterogeneous analyte, conformational change), with or without mass transport. furthermore, because equilibrium was not reached during the association phase, the direct use of scatchard analysis to calculate the apparent equilibrium dissociation constant was not allowed. instead, the apparent equilibrium dissociation constant k d was calculated from the equilibrium resonance signal (r eq ) as a function of analyte concentration, r eq values being estimated by extrapolation to infinite time using plots of resonance signal as a function of the reciprocal of time [26, 27] . apparent k d was then calculated by nonlinear fitting to the expression r eq = r max c/(k d +c), where r max is the maximum binding capacity of the surface and c is the analyte concentration, using sigmaplot software. this calculation, performed on 4 separate experiments, gave an apparent k d of 6.860.4 mm (see inset to figure 4 for a representative experiment). this dissociation constant is in the same order as the ic50 of hcvpp fusion (11.3 mm) . this result indicates that the inhibitory effect of arb on hcvpp membrane fusion is at least in part deriving from arb association to lipid membranes. nmr spectroscopy was used to characterize the arb insertion in a model membrane system. the 1 h nmr spectrum of arb recorded at 305 k in deuterated water is shown in figure 5a (black spectrum), and the assignment of the proton signals deduced from 2d spectra analysis (data not shown) are reported in table 1 . in order to study arb in a membrane-mimetic environment, we used isotropic phospholipid bicelles consisting of a mixture of dmpc/dhpc in water. long-chain phospholipid molecules of dmpc self-assemble into planar bilayers, while the short-chain molecules of dhpc segregate to edge regions of high curvature [28] . bicelles with [dmpc]/[dhpc] molar ratio ,1 form fast and isotropically tumbling aggregates, amenable to solution nmr studies. still, isotropic bicelle systems are used as a phospholipid bilayer mimetic, since dmpc has been shown to form a flat bilayered surface [29] [30] [31] . the 1 h nmr spectrum of arb in this bicellar phase is shown in figure 5a (red spectrum). in this system, the spectral crowding due to the presence of phospholipid resonances allowed only the observation of protons denoted 1, 2, 3, 4, 5 and 6 of the arbidol molecule (see fig. 1a ) where only 2 signals can be distinguished for the three protons 2, 3, 4. additional 1 h resonances could be resolved from 2d 1 h noesy spectra and 1 h/ 13 c hsqc spectra ( figure s2 ) and the corresponding chemical shifts are shown in table 1 together with the assignment of the proton lines for arb in water. arbidol interaction with lipids induce chemical-shift changes in the arb resonances when compared to that observed in water. in order to investigate the immersion depth of arb in the membrane, we monitored the proton longitudinal relaxation rate of arb protons upon the addition of the soluble paramagnetic agent gadolinium-diethylenetriamine pentaacetic acid-bismethylamide gd(dtpa-bma) [32] . this paramagnetic contrast agent stays soluble in the water surrounding the membrane and induces a paramagnetic relaxation enhancement (pre) on the spin of the atoms close to the surface of the membrane. recently, pre effects due to gd(dtpa-bma) were used to probe the immersion depth and orientation of an anti-microbial peptide [33, 34] . here we measured the proton t 1 relaxation times of both arb and phospholipid protons to probe the immersion depth of arb in the membrane, using the pre values of the phospholipids as an approximated yardstick. a titration of arb with gd(dtpa-bma) was performed at increasing concentrations of the paramagnetic agent, from 2.0 to 9.7 mm. at each step of the titration, the proton t1 relaxation times for the protons 1, 2, 3, 4, 5 and 6 of arb, and for the protons alpha, beta, gamma, g1, g2, g3, c2, c3 and omega14 of the phospholipids, were measured. the plot in figure 5b shows the relaxation times as a function of the gd(dtpa-bma) concentration. this titration leads to a curve for each proton whose slope corresponds to the pre values. as shown in fig. 5c , pre measured on the arb-bicelle system range from 0.90 sec 21 to 0.03 sec 21 for the phospholipid protons and from 0.90 sec 21 to 0.20 sec 21 for arb protons. the pre observed for each spin can be described as an overall relaxation enhancement [35] , due to all the paramagnetic agents in solution. for a planar membrane surrounded by a buffer containing a non-interacting paramagnetic probe, the total pre of a nucleus with immersion depth d is given by the equation [33, 34] : pre = z/d 3 , where d is the immersion depth of a specific nucleus within the membrane plus the radius of the magnetic probe, and where the constant z is a combination of various parameters, among them a correlation time, itself a combination of the electron relaxation time, the lifetime of the intermolecular adduct bicelle-gd(dtpa-bma), and the rotational correlation time. in order to determine the immersion depth of arb, instead of determining z, we used the phospholipids as a yardstick by comparing their pre with the one of arb. the pres of the resolved signals of arb and phospholipids are reported and compared in figure 5c . this procedure is based on two assumptions: (i) the amount of free arb in solution in the presence of lipids is negligible (see nmr sample preparation in experimental procedure section), and it does therefore not affect significantly the pre of arb in the membrane ; ii) the constant z is the same for lipids and arb. figure 5c shows that methyl protons 6 of arb are at the lipid/ water interface as the gamma-proton of the hydrophilic headgroup of the lipids. protons 5 and 1 of arb are at the level of respectively protons alpha and g1 of the phospholipids. the aromatic protons 2 and 3 are the most buried protons of arb, close to the beginning of the hydrophobic chain (protons c2). the validity of assumption (ii) is supported by the noe crosspeaks detected between proton 1 of arb and the glycerol moiety of phospholipids. in addition, the maximum pre measured for arb (protons 6) and phospholipids (c protons) are about the same, suggesting that the corresponding molecule regions are the most exposed to water. this estimation of the immersion depth of arb protons relative to the phospholipids protons enables us to propose a model for the positioning of arb in the membrane, shown in figure 5d . these nmr data clearly demonstrate that arb interacts at the membrane interface, mainly at the level of phospholipid polar head. this result supports the assumption that the arb inhibitory effect on hcvpp membrane fusion is dependent, at least in part, from this interaction. a second possibility regarding arb activity is that arb might interact with key motifs present in viral proteins, thereby impeding their structural reorganization at the onset of fusion and thus leading to fusion inhibition. a first set of experiments was designed to investigate whether the order of addition of fusing partners would affect arb-induced fusion inhibition. for this purpose, we measured fusion after preincubation of hcvpp or liposomes or both in the absence or presence of 11.3 mm arb. as shown in table 2 , when arb was pre-incubated with both partners before fusion was initiated by lowering the ph, fusion inhibition was ca. 50%. in contrast, only ca. 10% fusion inhibition was observed when arb was preincubated with either hcvpp or liposomes. the greater inhibitory effect of arb when it has simultaneously access to both viral and target membranes suggests that arb could also act by interacting with selective residues of the hcv glycoprotein sequences. this assumption was tested by studying the interaction behaviour of arb with tryptophan (trp) derivatives, as tryptophan is a constituent of proteins often found in regions located close to membrane interfaces, such as stem regions in several viral fusion proteins (e.g. hiv gp41 [36] ). we also reasoned that arb, being an indole derivative, might interact with tryptophan and tyrosine residues through aromatic ring stacking. for this purpose, we tested the effect of increasing concentrations of arb on the fluorescence of n-acetyl tryptophanamide (nata) as a water-soluble trp derivative, and tryptophan octyl-ester (toe) as a membranotropic molecule ( figure 1b-c) . the fluorescence of nata and toe was recorded between 300 and 400 nm, using an excitation wavelength of 286 nm, which corresponds to an absorption minimum of arb [12] . results are presented in table 3 and figure 6 . (table 3) . similarly, the k d of arb for toe in lyso-pc micelles was in the 50 mm range at both ph. this indicates that arb is able to interact with indole rings, but with a relatively low affinity. in contrast, when arb was added to toe associated to liposomes (1:20, toe/lipid molar ratio), a marked increase in affinity was observed (table 3 , compare toe/micelles and toe/ liposomes), reaching k d values in the 10 mm range. note that toe fluorescence could not be measured in dpc micelles, due to a great intrinsic fluorescence of the dpc used for our experiments. interestingly, these k d values are comparable to arb ic50 inhibition of hcvpp fusion (see above and discussion section). indole fluorescence decreased when arb concentration increased, with virtually no measurable fluorescence for 100 mm arb ( figure 6 ). this further confirms that arb interacts with indole rings, but with a higher affinity when indole is incorporated into lipid membranes. this affinity was higher for pc than for pc:chol liposomes (table 3) , and at neutral than at acidic ph (table 3 and figure 6 ). at acidic ph, arb is most likely protonated in the lipid environment [12] , as is probably toe as well. arb affinity for toe under these conditions might then be lower than that of uncharged arb at neutral ph, because of repulsive electrostatic interactions. a third set of experiments was designed to assess the behaviour of arb in the presence of aromatic residues into protein sequences, more specifically toward trp present in peptides. for this purpose, we used synthetic membrane-binding peptides of known structure and containing only one tryptophan residue, expected to be localized at the membrane interface: the transmembrane helix of hcv ns4a protein [16] and the n-terminal amphipathic helix of bvdv ns5a protein interacting in-plane of the membrane interface [15] . intrinsic tryptophan fluorescence of both peptides was monitored in the presence of increasing concentrations of arb; this is illustrated in figure 7 for hcv ns4a peptide inserted into pc:chol liposomes (and in figure s3 for bvdv ns5a, 1:20 peptide-to-lipid molar ratio). the arb dose-dependent quenching of tryptophan fluorescence at both neutral and acidic ph clearly indicates arb interaction with both peptides (insets in figure 7 and fig. s3 ). for the ns4a peptide at both ph, a red shift of the spectral maximum, proportional to arb concentration, accompanied the fluorescence quenching; this effect was more pronounced at acidic ph (6 nm at ph 7.4 for 5 mm arb, and 12 nm at ph 5.0 for 100 mm arb). this red shift suggests that arb, when interacting with ns4a peptide, relocates its tryptophan residue to a more shallow zone of the membrane, where the trp environment would be more hydrophilic. the measure of the apparent affinity of arb for these peptides inserted into pc:chol liposomes was performed as described above. interestingly arb displayed an apparent k d toward peptide trp between 3.3 and 5.6 mm (table 4 ), twice lower than that observed for toe in pc:chol liposomes. since both peptides contain one or two tyrosine residues (hcv ns4a and bvdv ns5a, respectively) in addition to the trp, interaction of arb molecules with these aromatic residues might account for a higher affinity of arb for peptides than for a small molecule such as toe. since arb is an inhibitor of hcv membrane fusion, we reasoned that it might interact with the regions of e1 and e2 described as important for hcv fusion [11, 17] . these peptides were described as membranotropic on model membranes [37] and contain aromatic residues. we therefore analyzed the effect of increasing concentrations of arb on the fluorescence quenching of two peptide sequences derived from hcv e2 (positions 415-432 and 606-625, see aa sequences in table 5 ), and inserted into pc:chol liposomes (1:20 peptide-to-lipid molar ratio). note that we also tested a third peptide located at position 270-283 of e1, containing only one tyr; but its fluorescence quantum yield was too low to monitor any interpretable fluorescence signal (data not shown). we then calculated the k d values as described above. as shown in table 5 , e2 415-432 contains only one trp, whereas e2 606-625 contains one trp and 3 tyr. the apparent affinity was in the 15 mm range at ph 7.4 for both peptides, reminiscent to arb ic50 of hcvpp fusion. this indicates that arb is able to interact with the aromatic residues of both peptides in the membrane, and lends further support to our hypothesis that arb could interact with key residues/motifs in viral fusion proteins, which would constitute a possible (partial) explanation to its inhibition of hcvpp fusion. strikingly this affinity decreased at acidic ph for both peptides, and even drastically to 70 mm for e2 606-625. interestingly, this relatively high k d value is reminiscent of that observed for arb interaction with nata in solution (table 3) . this suggests that the interaction between the e2 peptide and the membrane would be weak at acidic ph, and that most of the peptide could be in solution. moreover an histidine residue, located in the immediate vicinity of trp in the sequence of both peptides, is expected to be charged at ph 5.0. since arb is also protonated at that ph value, this could create repulsive forces affecting the interaction between trp and arb. moreover, as protonation of the histidine cycle is expected to decrease the free energy of partition from lipids to water, the peptide could be released from the membrane at acidic ph, possibly in relation with peptide conformational change(s). this behavior in not in favor with their direct role as fusion peptides of hcv, a virus dependent on low ph for its membrane fusion activity. however, due to their membranotropism [37] , and since our and other studies showed their involvement in hcv membrane fusion [17, 38] , it is possible that the conformational changes they might undergo at low ph would lead to a proper relocation of the actual fusion peptide/loop toward the target membrane [39] (and see discussion section). this study aimed at further investigating the molecular mechanism of action by which arbidol (arb) inhibits virus cell entry and membrane fusion, using hcvpp as a model of an enveloped virus. we showed that arb displayed a dual binding capacity, on lipid membranes interface on one hand and on the aromatic residue table 4 ). doi:10.1371/journal.pone.0015874.g007 tryptophan of proteins on the other hand. it therefore appears plausible that the observed inhibitory effect of arb on viral entry and membrane fusion might result from a combined effect of binding of arb on membranes and on (fusion) proteins. from a physico-chemical point of view, arb displayed tropism for membranes or membrane-like environments such as detergent micelles, particularly prominent at low ph [12] . by combining several biochemical approaches, we show here that arb has the propensity to bind to and incorporate into lipid bilayers, with calculated apparent affinities in a similar range as the ic50 value for fusion, i.e. ca. 10 mm. our nmr studies of arb interaction with dmpc leads to a model where arb binds at the membrane interface and establishes contacts mainly with the polar heads of phospholipids (fig. 5d ). altogether these data suggest that at least part of arb inhibitory activity could be explained by its membranotropism. this physicochemical property has been further emphasized in a recent work by villalain [40] , using fourier-transform infrared spectroscopy. arb interaction with phospholipids would disturb membrane fluidity crucial to the fusion process, thereby rendering the lipid bilayer less prone to fusion. such a model is consistent with the behavior of other indole derivatives, that were shown to exhibit a preference for membrane interfaces [41, 42] , due to the flat rigid structure of these molecules and to their aromaticity, which allows them to establish cation-p interactions with the positively charged quaternary ammonium lipid headgroups [41, 43] . at low ph, the optimal ph for hcv fusion, these interactions would be favored due to the protonation of the amino groups. as was described for other substituted indoles [44] , it is possible that protonation of the carbon bearing the ester group of arb could displace this group out of the indole plane, and place it in a better position to bond with neighboring molecules. this could in turn lead to a better membrane association. arb might therefore have the propensity to intercalate into lipids of the viral and target membranes while adopting a consistent orientation by filling the gaps between lipid molecules. the interfacial region of the lipid bilayer provides a suitable environment for a wide range of chemical groups, as long as they possess a large enough hydrophobic moiety and a group capable of forming hydrogen bonds with the lipid carbonyl groups. several compounds with antiviral pharmacological properties belong to this category, in particular adamantanes active against influenza a viruses [45, 46] and against some hcv clones but not all [47, 48] , the natural triterpene glycyrrhizin efficient in the treatment of chronic viral hepatites [49] and the flavonolignan molecules composing silymarin, an herbal extract with potent anti-hcv activities [1] [2] [3] 50, 51] . in a previous study, we noticed that arb inhibition of cell entry concerned hcvpp and pseudoparticles bearing the influenza hemagglutinin (happ), but not pseudoparticles bearing the envelope glycoprotein of a feline oncogenic retrovirus (rd114pp) [12] . these data suggest that arb might display selectivity for the recognition of key motifs inside envelope proteins. this hypothesis was tested by assessing the influence of arb on the fluorescence properties of aromatic compounds derived from tryptophan (trp) and of peptides containing trp. trp is a component of proteins with interfacial properties [41, 42] , often located at the lipid/water interface and grouped into so-called tryptophan-rich motifs crucial to protein/membrane association [52] , and found in the envelope (fusion) proteins of the sars coronavirus or hiv-1 [36, 53, 54] . trp is also enriched at protein/protein binding interfaces of the small envelope protein of the hepatitis b virus [55] and of membrane proteins in general [56] . we demonstrated here that arb was able to alter/quench the fluorescence properties of small trp derivatives in solution (nata), in detergent micelles and in liposomes (toe, [57] ), in a dose-dependent manner. this occurred most likely through stacking of the aromatic rings of both molecules which is often involved in stabilization of inter-cations. interestingly the apparent affinity of the arb/trp derivative interaction was in the order: lipid bilayers.micelles.solution, indicating that arb binding strength for trp could increase in membrane environments where both molecules accomodate and get packed. indeed arb apparent affinity for toe in liposomal membranes was in the 10 mm range, a value comparable to the ic50 of fusion. arb affinity was even greater for membrane peptides containing trp and tyrosine (tyr) residues (ca. 4 mm). due to its indole group, it is conceivable that arb might display selectivity not only for indole rings (trp) but more generally for aromatic groups, as the phenol ring of tyr. a greater number of arb molecules could therefore interact with aromatic residues in peptide sequences, leading to some cooperativity in the quenching effect and to an overall larger apparent affinity. the nmr structures of synthetic peptides hcv ns4a * and bvdv ns5a peptides have been reported in references [16] and [15] , respectively. the solubilization tags kkgg and ggkk at the n-and c-terminal ends, are indicated in italic. aromatic residues trp and tyr are indicated in bold, his is underlined. b peptide-to-lipid molar ratio was 1:20. although hcv entry inhibition by arb was found genotypedependent, hcv membrane fusion was inhibited by arb in a genotype-independent manner. hcv entry and fusion are early steps in the life cycle of the virus [58, 59] . hcv first interacts through its envelope glycoproteins with a set of coreceptors at the plasma membrane level (recently reviewed in [60, 61] ) and eventually becomes endocytosed [62] [63] [64] . due to a combined action of acidification in the endosome and particular lipids like cholesterol and sphingomyelin [11, 18] , viral fusion occurs over a broad spectrum of ph's ranging from 6.3 to 5.0 [11, 18, 65] . hcv binding to the hepatocyte membrane followed by endocytosis therefore requires several cellular proteins, and most likely involves several levels of interactions (interactions between viral proteins, between cellular and viral proteins, between viral/cellular proteins and lipids). these features might explain the differential effect exerted by arb on entry of various hcv genotypes: indeed subtle differences in protein sequences could translate into modified interactions with several partners and/or at several levels. conversely some common principles of action apply to all fusion reactions, viral fusion and cellular fusion processes alike [66] . indeed all fusion processes involve two partners: lipids and the fusion protein(s). this might account for the similar inhibitory effect of arb on hcv fusion observed for all genotypes. this is in line with the observations that arb displayed potent antiviral activity against some antigenic serotypes of influenza viruses, but not against all [9] . previously we noticed that arb inhibition of primary infection of huh-7.5.1 cells with hcv (clone jfh-1) was efficient only when cells were preincubated with arb 24 or 48h before infection [10] ; in addition, inhibition of hcvpp and happ cell entry was most efficient when arb was pre-incubated with both viral and cell membranes [12] . here, using our in vitro fusion assay, we observed that arb inhibition of hcvpp fusion was maximal when both viral and target membranes were incubated with arb, before fusion was initiated. this suggests that a certain level of membrane impregnation and/or saturation with arb must be achieved to efficiently inhibit viral infection. membranes might therefore act as ''concentrators'' of arbidol, and high concentrations of the molecule might be locally achieved. this could explain why arb, exhibiting an apparent (medium to low) affinity for membranes in the mm range, exerts a relevant antiviral activity without noticeable membrane damages. along these lines, in spite of its marked membranotropism, arb displays only low toxicity [9, 10] . arb exhibited a comparable micromolar apparent affinity for aromatic residues present in membrane peptides in a membrane environment. altogether, these observations lead us to propose a mechanistic model of the way arb would inhibit hcv entry and fusion. through its membranotropism, arb is able to freely interact with viral and target membranes, and could locally get highly concentrated. arb is also able to interact with aromatic residues within viral proteins involved in membrane interactions and membrane destabilization necessary for fusion. through this dual binding capacity, arb could then locally impede the structural rearrangements required for the fusion protein to adopt its fusion conformation. the fact that arb is active in the mm range suggests that arb would act by reducing the overall speed of the fusion reaction rather than by blocking a specific protein conformation. this could therefore explain the broad antiviral spectrum of arb, and the genotype independence of its inhibitory effect on hcv fusion, since hcv envelope proteins contain well-conserved aromatic residues in all genotypes. mechanistically, the key point is the relative accessibility of these residues to arb at the membrane interface. a cooperative effect between arb and several aromatic residues might therefore occur. also the local environment of these aromatic aa is important, since the presence of residues such as histidines (his) in their vicinity could modify their accessibility with respect to ph. interestingly enough, in the sequence of both hcv e2 peptides studied here (table 5 ) and shown to be involved in hcv fusion [17] , his is contiguous to trp, and in the 606-625 peptide, his is surrounded by three tyrosines. the concept of his as a critical ph sensor at a key intramolecular domain interface in a viral fusion protein has recently emerged [67, 68] . indeed, the protonation of a sole his in the e protein of the tick-borne encephalitis flavivirus (tbev) triggers large-scale conformational changes leading to viral fusion. concerning hcv, rey and coworkers recently proposed a model of the 3d arrangement of the e2 ectodomain [39] . in this model, the fusion loop/peptide would lie within the poorly structured domain ii, and the e2 606-625 peptide would be found in the globally unstructured domain iii, where a critical his residue is disposed at the interface with domain i. the putative fusion loop contains a phenylalanine and a tyrosine [39] . at low ph, the optimal ph for hcv membrane fusion, key histidine(s) could become protonated. this could result in conformational rearrangements and, in the context of arb fusion inhibition, aromatic residues might consequently become more or less accessible to arb molecules present in their vicinity. we noted that the apparent affinity of arb for hcv peptides was weaker at ph 5.0 than at ph 7.4. at low ph, arb is also protonated, and this protonated form could exhibit a greater preference for the interfacial region of the lipid bilayer than the deprotonated form, as demonstrated for adamantanes [45] . combined with the notion that key aromatic and his residues would also display interfacial (re)localization at low ph, this would in turn explain the higher efficiency of arb at inhibiting fusion at acidic ph [12] . in conclusion our data reveal that arb directly interacts with the lipid membrane-water interface, and is able to bind to aromatic residues present in hcv glycoproteins, in their membraneassociated form. through a subtle binding interplay between arb, lipids, viral and cellular proteins, arb might efficiently block hcv entry and membrane fusion interacting with the main actors of the early steps of viral entry. most interestingly, arb inhibition of these processes demonstrated an affinity in the mm range, although the membranotropic properties of arb suggest that it could become locally more concentrated in membranes. together, these findings suggest that arb could increase the strength of viral glycoprotein's interactions with the membrane due to a dual binding mode, involving aromatic residues and phospholipids. the resulting complexation would inhibit the expected viral glycoprotein conformational changes required during the membrane fusion process. the antiviral mechanism of arb therefore opens promising perspectives for the development of small membranotropic low affinity molecules, that would become locally concentrated in membranes and would mainly act on the kinetics of the conformational rearrangements of the viral fusion protein. figure s1 arb inhibits infectivity and membrane fusion in a dose-dependent manner. a, infectivity. results are the mean 6 sem of 5 separate experiments. black, no arb; blue, 1.9 mm; green, 5.6 mm and red, 11.3 mm arb, respectively. b, membrane fusion between hcvpp of genotype 4 (4.11.21) and r18-labeled liposomes. the lipid mixing kinetic was followed by fluorescence spectroscopy using excitation and emission at 560 and 590 nm, respectively. fluorescent liposomes (12.5 mm final lipid concentration) were added to 40 ml of hcvpp in pbs ph 7.4 at 37uc, in the absence or presence of the indicated concentrations of arb. after a 2 min-equilibration, lipid mixing was initiated by decreasing the ph to 5.0 with diluted hcl, and r18 dequenching was recorded. maximal fluorescence was obtained after addition of 0.1% final triton x-100. average value of the last 30 s of fusion (i.e. final extent of fusion) was used to calculate the percentage of fusion in the presence of arb, relative to 100% fusion without arb ( figure 2) . black, no arb; blue, 1.9 mm green, 5.6 mm and red, 11.3 mm arb, respectively. (tif) of increasing concentrations of arb (2, 5, 10, 25 and 100 mm from top to bottom). trp emission fluorescence was measured between 300 and 400 nm, with excitation at 286 nm. the apparent affinity of arb toward trp was calculated from the plot of the difference da between areas under the curve (auc) of peptide without arb (da = auc no arb 2auc with arb ) as a function of arb concentration (insert). (tif) identification of hepatoprotective flavonolignans from silymarin multiple effects of silymarin on the hepatitis c virus lifecycle silibinin and related compounds are direct inhibitors of hepatitis c virus rnadependent rna polymerase synthesis and in vitro anti-hepatitis b virus activities of some ethyl 6-bromo-5-hydroxy-1h-indole-3-carboxylates antiviral chemotherapeutic agents against respiratory viruses: where are we now and what's in the pipeline? hepatitis c virus entry: an intriguing challenge for drug discovery screening of small-molecule compounds as inhibitors of hcv entry characteristics of arbidolresistant mutants of influenza virus: implications for the mechanism of antiinfluenza action of arbidol arbidol: a broadspectrum antiviral compound that blocks viral fusion arbidol: a broad-spectrum antiviral that inhibits acute and chronic hcv infection low ph-dependent hepatitis c virus membrane fusion depends on e2 integrity, target lipid composition, and density of virus particles biochemical mechanism of hepatitis c virus inhibition by the broad-spectrum antiviral arbidol synthesis and anti-hepatitis c virus activity of novel ethyl 1h-indole-3-carboxylates in vitro hepatitis b and hepatitis c in 2009 nmr structure and molecular dynamics of the in-plane membrane anchor of nonstructural protein 5a from bovine viral diarrhea virus structural determinants for membrane association and dynamic organization of the hepatitis c virus ns3-4a complex characterization of fusion determinants points to the involvement of three discrete regions of both e1 and e2 glycoproteins in the membrane fusion process of hepatitis c virus hepatitis c virus glycoproteins mediate low ph-dependent membrane fusion with liposomes growth of human hepatoma cells lines with differentiated functions in chemically defined medium characterization of host-range and cell entry properties of hepatitis c virus of major genotypes and subtypes dna-induced endocytosis upon local microinjection to giant unilamellar cationic vesicles switchedangle spinning applied to bicelles containing phospholipid-associated peptides matnmr: a flexible toolbox for processing, analyzing and visualizing magnetic resonance data in matlab entropic stabilization and equilibrium size of lipid vesicles surface plasmon resonance in protein-membrane interactions interaction properties of the procollagen c-proteinase enhancer protein shed light on the mechanism of stimulation of bmp-1 studies of protein interactions by biosensor technology: an alternative approach to the analysis of sensorgrams deviating from pseudo-first-order kinetic behavior magnetically-oriented phospholipid micelles as a tool for the study of membrane-associated molecules micelle-induced curvature in a water-insoluble hiv-1 env peptide revealed by nmr dipolar coupling measurement in stretched polyacrylamide gel structural evaluation of phospholipid bicelles for solution-state studies of membrane-associated biomolecules isotropic solutions of phospholipid bicelles: a new membrane mimetic for high-resolution nmr studies of polypeptides identification of protein surfaces by nmr measurements with a pramagnetic gd(iii) chelate mapping the orientation of helices in micelle-bound peptides by paramagnetic relaxation waves positioning of micelle-bound peptides by paramagnetic relaxation enhancements nmr of paramagnetic substances membrane-induced conformational change during the activation of hiv-1 gp41 the membrane-active regions of the hepatitis c virus e1 and e2 envelope glycoproteins mutagenesis of a conserved fusion peptide-like motif and membrane-proximal heptad-repeat region of hepatitis c virus glycoprotein e1 the disulfide bonds in glycoprotein e2 of hepatitis c virus reveal the tertiary organization of the molecule membranotropic effects of arbidol, a broad anti-viral molecule, on phospholipid model membranes interfacial tryptophan residues: a role for the cation-pi effect? the preference of tryptophan for membrane interfaces cation-pi interactions in ligand recognition and catalysis the protonation of indoles. basicity studies. the dependence of acidity functions on indicator structure distribution and dynamics of adamantanes in a lipid bilayer structure of the amantadine binding site of influenza m2 proton channels in lipid bilayers the p7 protein of hepatitis c virus forms an ion channel that is blocked by the antiviral drug nmr structure and ion channel activity of the p7 protein from hepatitis c virus glycyrrhizin, the main active compound in liquorice, attenuates proinflammatory responses by interfering with membrane-dependent receptor signalling ground and excited state proton transfer and antioxidant activity of 7-hydroxyflavone in model membranes: absorption and fluorescence spectroscopic studies groundand excited-state proton transfer and antioxidant activity of 3-hydroxyflavone in egg yolk phosphatidylcholine liposomes: absorption and fluorescence spectroscopic studies the nterminal region of comparative gene identification-58 (cgi-58) is important for lipid droplet binding and activation of adipose triglyceride lipase a conserved tryptophan-rich motif in the membrane-proximal region of the human immunodeficiency virus type 1 gp41 ectodomain is important for env-mediated fusion and virus infectivity important role for the transmembrane domain of severe acute respiratory syndrome coronavirus spike protein during entry a tryptophan-rich motif in the carboxyl terminus of the small envelope protein of hepatitis b virus is central to the assembly of hepatitis delta virus particles a study of the membrane-water interface region of membrane proteins tryptophan octyl ester in detergent micelles of dodecylmaltoside: fluorescence properties and quenching by brominated detergent analogs replication of hepatitis c virus hepatitis c virus entry the hepatitis c virus and its hepatic environment: a toxic but finely tuned partnership virology: final entry key for hepatitis c time-and temperature-dependent activation of hepatitis c virus for low-ph-triggered entry hepatitis c virus entry requires a critical postinternalization step and delivery to early endosomes via clathrincoated vesicles rna interference and single particle tracking analysis of hepatitis c virus endocytosis functional analysis of hepatitis c virus envelope proteins, using a cell-cell fusion assay viral and developmental cell fusion mechanisms: conservation and divergence identification of specific histidines as ph sensors in flavivirus membrane fusion the ph sensor for flavivirus membrane fusion the xplor-nih nmr molecular structure determination package this work was presented at the 16 th international symposium on hepatitis c and related viruses, (nice, france, october 3-7, 2009).the authors gratefully acknowledge sylvie ricard-blum for spr analysis expertise. fluorescence experiments were performed on the platform ''production et analyse des protéines'' of the ifr 128 biosciences gerland-lyon sud. key: cord-328627-cf8f71dr authors: jando, julia; camargo, simone m. r.; herzog, brigitte; verrey, françois title: expression and regulation of the neutral amino acid transporter b(0)at1 in rat small intestine date: 2017-09-15 journal: plos one doi: 10.1371/journal.pone.0184845 sha: doc_id: 328627 cord_uid: cf8f71dr absorption of neutral amino acids across the luminal membrane of intestinal enterocytes is mediated by the broad neutral amino acid transporter b(0)at1 (slc6a19). its intestinal expression depends on co-expression of the membrane-anchored peptidase angiotensin converting enzyme 2 (ace2) and is additionally enhanced by aminopeptidase n (cd13). we investigated in this study the expression of b(0)at1 and its auxiliary peptidases as well as its transport function along the rat small intestine. additionally, we tested its possible shortand long-term regulation by dietary proteins and amino acids. we showed by immunofluorescence that b(0)at1, ace2 and cd13 co-localize on the luminal membrane of small intestinal villi and by western blotting that their protein expression increases in distal direction. furthermore, we observed an elevated transport activity of the neutral amino acid l-isoleucine during the nocturnal active phase compared to the inactive one. gastric emptying was delayed by intragastric application of an amino acid cocktail but we observed no acute dietary regulation of b(0)at1 protein expression and l-isoleucine transport. investigation of the chronic dietary regulation of b(0)at1, ace2 and cd13 by different diets revealed an increased b(0)at1 protein expression under amino acid-supplemented diet in the proximal section but not in the distal one and for ace2 protein expression a reverse localization of the effect. dietary regulation for cd13 protein expression was not as distinct as for the two other proteins. ring uptake experiments showed a tendency for increased l-isoleucine uptake under amino acid-supplemented diet and in vivo l-isoleucine absorption was more efficient under high protein and amino acid-supplemented diet. additionally, plasma levels of branched-chain amino acids were elevated under high protein and amino acid diet. taken together, our experiments did not reveal an acute amino acid-induced regulation of b(0)at1 but revealed a chronic dietary adaptation mainly restricted to the proximal segment of the small intestine. proteins in ingested food are hydrolyzed to small oligopeptides and amino acids on their way from the oral cavity to the small intestine where they are absorbed across the mucosa. more a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 precisely, they are transported mostly across epithelial cells by being first imported through their luminal membrane and sequentially exported across their basolateral membrane in order to be distributed to other body tissues. also prevention of loss of amino acids via urine is necessarily important and is ensured by their reabsorption in the renal proximal tubules [1] . intestinal absorption and renal reabsorption across membranes requires membrane-spanning transporters. these are classified according to structural similarity in groups of transporters which form solute carrier families (slc) [2] . the slc6 family for example first included sodium and chloride dependent neurotransmitter transporter, but later it has been shown that a cluster of formerly "orphan" transporters within this family are actually amino acid transporters including b 0 [3] . most neutral amino acids are transported across the apical brush-border membrane of the small intestine and the renal proximal tubules by the luminal broad neutral amino acid transporter b 0 at1 (slc6a19), which was first identified in 2004 in mice [1, 3, 4, 5] . b 0 at1 function has been investigated by electrophysiological experiments in xenopus laevis oocytes, which revealed an electrogenic sodium co-transport for all neutral proteinogenic amino acids with the highest affinity for branched-chain amino acids and l-methionine [5, 6] . malfunction of this transporter due to mutations in the slc6a19 gene causes hartnup disorder, an autosomal recessive condition which is characterized by a urinary loss of neutral amino acids [7, 8] . in slc6a19 nullizygous mice the sodium-dependent uptake of l-leucine was completely abolished in kidney and intestine and even the sodium-dependent glucose uptake was reduced [9] . expression and function of b 0 at1 have also been shown to depend on co-expression of members of the renin angiotensin system (ras), namely tmem27 (collectrin) in kidney and angiotensin converting enzyme 2 (ace2) in small intestine [10, 11] . in ace2 nullizygous mice, b 0 at1 was absent in the intestinal but present in the renal brush-border membrane and in collectrin nullizygous mice b 0 at1 was absent in the renal but present in the intestinal brush-border membrane, demonstrating a tissue-specific necessity of either of these auxiliary proteins for b 0 at1 trafficking to the apical membrane and surface expression [11] . ace2 is an abundant carboxypeptidase of the small intestine and has been identified as the severe acute respiratory syndrome receptor [12, 13] . it is also well known for its important angiotensin iidegrading function in the context of the renin-angiotensin system and has been shown to be involved in heart and kidney pathologies [14] . immunoprecipitation and native electrophoresis of intestinal brush-border membrane proteins revealed that b 0 at1 forms not only complexes with ace2 but also with aminopeptidase n (cd13) [15] . experimental evidence suggesting the association of peptidases and transporters in the brush-border was already reported earlier, for instance the significant reduction of sodium-dependent l-alanine transport upon removal of cd13 in bovine renal brush-border membrane vesicles (bbmv) by papain treatment [16] . cd13 is the most abundant peptidase in the mammalian small intestine [17] . it is a zinc metalloprotease that hydrolyses small oligopeptides to single amino acids at the brush-border membrane with a broad specificity for neutral amino acids [18, 19] . b 0 at1 apparent affinity for its substrate amino acids was shown to be increased by cd13 possibly by increasing the local substrate concentration [15] . as ferraris and diamond postulated already in 1989, transporters should be repressed if costs exceed their benefits [20] . in general, nutrient transporters have to adapt to various dietary conditions, development and disease states [21, 22] . there is however yet not much known about the dietary regulation of amino acid transporters in contrast to that of the diand tripeptide transporter pept1 (slc15a1). this proton/peptide co-transporter is transporting small oligopeptides and additionally a range of peptide-like drugs through the brush-border membrane of intestinal epithelial cells [23, 24] . previous studies revealed different mechanism of regulation including changes on transcriptional level, mrna stability or trafficking to the apical membrane [22] . in vivo studies with mice exposed to different protein diets illustrated that pept1 function is highest in the jejunum but its strongest dietary regulation takes place in duodenum and jejunum [25] . in contrast, highest regulation of pept1 mrna levels was observed in the distal region of the small intestine [26, 27] . investigation of the dietary regulation of pept1 revealed that increased dipeptide transport activity is caused by transcriptional activation of the pept1 gene by selective amino acids and dipeptides. furthermore, it was shown that not only the peptide transport is increased during high protein diet but also activities of brush-border membrane enzymes and neutral aminopeptidases [28] . the few published studies about dietary regulation of amino acid transporters have revealed for instance that a high protein diet leads to an increased uptake of some amino acids and to an increased mrna level of the apical excitatory amino acid transporter 3 (eaat3, slc1a1) [26, 29] . also a glutamine-enriched diet was shown to increase glutamine uptake in jejunal bbmv from rats compared to a control diet [30] . some experiments in which high concentrations of amino acids were administered in small intestinal loops of rats also suggested the possibility of a short-term (20 min) amino acid-induced regulation of amino acid absorption [31] . rodents are nocturnal animals with their active phase in the night. studies investigating a possible diurnal rhythm of pept1 expression and function revealed variations in mrna, protein and transport function in duodenum and jejunum, but not in ileum [32] . based on the fact that protein and neutral amino acid availability fluctuates and that not much is yet known about the dietary regulation of intestinal amino acid transporters, we tested the hypothesis that b 0 at1 function and expression in rat small intestine is acutely regulated by amino acid loads and/or chronically by diets. furthermore, we tested the potential co-regulation of the associated peptidases ace2 and cd13 along the small intestine also taking a possible diurnal regulation into account. all experiments were performed with male wistar rats (janvier, france and charles river, germany) that were handled in accordance with the swiss federal and cantonal law and with the approval of the cantonal veterinary office zürich. rats were group-housed and adapted to housing conditions for at least 1 week prior to experiments. the first centimeter of the small intestine after the stomach was discarded and the next 10 cm taken as duodenum. similarly, the last centimeter before the caecum was discarded and the 10 cm before defined as ileum. the middle 10 cm of the remaining small intestine was taken as middle jejunum, whereas 10 cm in the middle of the remaining proximal and distal parts were defined as proximal and distal jejunum, respectively. measurements of gastric emptying by in vivo ct were performed as previously described elsewhere [33] . briefly, 4 h food-deprived rats received an intraperitoneal zoletil injection (20 mg/ kg, virbac; leuven, belgium) and subsequently an intragastric application of 2 ml of sodium diatrizoate hydrate (200 mg/ml, sigma-aldrich, steinheim, germany) solubilized in water or in an amino acid cocktail. these 2 ml isomolar amino acid cocktail contain 0.34 mmol of each of the proteinogenic amino acids which in the case of glutamic acid corresponds to 50 mg and in terms of total amino acids to approximately 25% of the mean daily intake. rats were imaged immediately and every 10 min for 90 min using quantum fx 2.2 micro-ct (perkin elmer, waltham, ma, usa). image analysis was performed using caliper analyse 11.0 (analyse direct, stilwell, ks, usa). rats were fed ad libitum a standard diet (3436 kliba nafag, kaiseraugst, switzerland). on the day of experiment, animals were either starved for 4 h, 16 h or fed ad libitum followed by an intragastric application of the amino acid cocktail (as described before) or water. after 1 h, rats were anesthetized with attane tm isoflurane ad us. vet. (piramal healthcare, india) and euthanized by heart cut. amino acid uptake experiments of the middle jejunum have been performed as described below. brush-border membrane vesicles (bbmv) were prepared from 2 cm scraped mucosa of the proximal and distal jejunum for western blot analysis as described below. rats were housed under artificial 12:12-h light-dark cycle and fed ad libitum a standard diet (3436 kliba nafag, 18.5% protein content, kaiseraugst, switzerland). three hours after light onset (zeitgeber time 3, zt3) and 3 h after light offset (zeitgeber time 15, zt15), rats were anesthetized with attane tm isolfurane ad us. vet. (piramal healthcare, india) and euthanized by heart cut. quantitative real-time pcr of rna extracted from duodenal and ileal scraped mucosa was performed as well as western blot analysis of proximal and distal jejunal bbmv proteins and amino acid uptake experiments with proximal and distal sections of the jejunum. rats were fed ad libitum a standard normal protein diet (np, 18% casein (ain-93g)), a high protein diet (hp, 45% casein) or a standard diet with supplemented single amino acids (aa) in the same ratio as in the hp diet. all diets were isocaloric via adjustment of the starch content and produced by kliba nafag (kaiseraugst, switzerland). experiments were performed after 7 days of diet treatment. brush-border membrane vesicles (bbmv) were prepared following the microscale preparation protocol published by s.p. shirazi-beechey [34] with few modifications. scraped mucosa from 2 cm of the small intestine was homogenized in 600 μl buffer 1 containing 100 mm mannitol and 2 mm hepes/tris ph 7.1 supplemented with protease inhibitor cocktail (sigma aldrich, steinheim, germany). homogenization was performed at 6 000 rpm for 20 sec with magna lyser green beads (roche diagnostics, mannheim, germany) using the precellys24 (bio-labo scientific instruments, chatel-st-denise, switzerland). immediately after homogenization, 600 μl of chilled buffer 1 was added to the samples followed by magnesium precipitation (10 mm final concentration) at 4˚c on a rotary mixer. after centrifugation at 3 000 g for 15 min at 4˚c, supernatant was transferred to a new tube and centrifuged at 30 000 g for 30 min at 4˚c. the pellet was suspended in 200 μl buffer 2 containing 300 mm mannitol, 20 mm hepes/tris, ph 7.4 supplemented with protease inhibitor cocktail (sigma aldrich, steinheim, germany) and again centrifuged at 30 000 g for 30 min at 4˚c. the final pellet was resuspended in 100 μl buffer 2 and homogenized by passing at least 10 times through a 25-gauge needle. bbmv were either used immediately for protein quantification or snap frozen and stored at -80˚c. alkaline phosphatase activity was analyzed in bbmv and the homogenized tissue with the alkaline phosphatase assay kit (colorimetric) (abcam, cambridge, uk) according to the manufacturer's instructions. protein concentration of bbmv was determined by pierce 1 bca protein assay (thermo scientific, rockford, il, usa), following the manufactures guidelines. bbmv (3 μg protein) were diluted in 4x laemmli buffer supplemented with 10% β-mercaptoethanol and resolved by sds-page on 8% polyacrylamide gels. after electrophoretic transfer of proteins to a polyvinylidene difluoride (pvdf) membrane (immobilion 1 -p, merck millipore, darmstadt, germany), nonspecific binding sites were blocked for 1 h at room temperature with 2% top-block tm (lubioscience, lucerne, switzerland) in tris-buffered saline supplemented with 0.1% tween-20 (tbs-t). pvdf membranes were incubated overnight at 4˚c with the primary antibody diluted in 2% top-block tm -tbs-t. after washing the membranes with tbs-t, secondary antibodies diluted in 2% top-block tm -tbs-t were applied for 1 h at room temperature. antibody binding was detected with luminata tm classico western hrp substrate (merck millipore, darmstadt, germany) or cdp-star (roche diagnostics, mannheim, germany) and visualized with fujifilm las-4000 camera (ge healthcare, glattbrugg switzerland) according to the manufacturer's instructions. image j software (national institutes of health, bethesda, md, usa) has been used for densitometric analysis of western blots. after diet treatments, rats were anesthetized with attane tm isolfurane ad us. vet (piramal healthcare, india) and euthanized by a heart cut. the small intestine was harvested, washed and flushed with phosphate buffered saline (pbs). sections of 1 cm were incubated overnight at 4˚c in 3% paraformaldehyde solution. after washing with pbs, samples were incubated in 30% sucrose for 8-12 h at 4˚c and then for 6 h in a 1:1 mix of 30% sucrose and oct embedding matrix (cellpath, biosystems, muttenz, switzerland). subsequently, samples were embedded in oct embedding matrix and stored at -80˚c. serial sections of 5 μm were cut on a cryostat, collected on superfrost plus slides (gerhard menzel b.v. & co. kg, braunschweig, germany) and stored at -80˚c. sections were incubated in pure methanol for 90 sec at -20˚c and then rehydrated for 15 min at room temperature. after washing in phosphate-buffered saline (pbs), blocking was performed with pbs-2% bsa-0.04% triton x-100 for 1 h followed by incubation with the primary antibodies (diluted in pbs-2% bsa-0.04% triton x-100) overnight at 4˚c. sections were then washed three times with pbs and subsequently incubated with the secondary antibodies (diluted in pbs-2% bsa-0.04% triton x-100) for 1 h at room temperature. to counterstain the nuclei, 4',6-diamidino-2-phenylindole dihydrochloride (dapi, thermo fischer scientific, waltham, ma, usa) was added to the secondary antibody mixture (1:5 000 dilution). after being washed three times with pbs, sections were mounted using dako glycergel mounting medium (dako north america, capinteria, ca, usa). analysis has been performed with the automated upright confocal laser scanning microscope sp8 (leica) and image analysis has been performed with imaris (bitplane) software at the center for microscopy and image analysis (zmb) of the university of zürich. the following primary antibodies were used: rabbit anti-mouse b 0 at1 antibody ( [3] , 1:100 for if, 1:2 000 for wb), goat anti-mouse ace2 (r&d systems, minneapolis, mn, usa; dilution: 1:100 for if, 1:1 000 for wb), rabbit anti-human cd13 (abcam, cambridge, uk; dilution: 1:100 for if, 1:2 000 for wb), and mouse anti-β-actin (sigma aldrich, steinheim, germany; dilution: 1:100 for if and 1:5 000 for wb). secondary antibodies were anti-rabbit igg hrp conjugate, anti-mouse igg (h+l) ap conjugate (promega, dübendorf, switzerland) or donkey anti-goat igg-hrp (santa cruz biotechnology, dallas, texas, usa) for western blot analysis (1:5 000 dilution) and alexa fluor 1 594 donkey anti-rabbit igg (h+l) (life technologies, carlsbad, california, usa) and dnk pab to goat-igg (dylight 1 488) (abcam, cambridge, uk) for immunofluorescence (1:2 000 dilution). compared to na + -dependent glucose transport, intestinal bbmv displayed little and variable na + -dependent neutral amino acid transport activity. therefore amino acid transport studies were performed using inverted sections (rings) of small intestine. rats were anesthetized with attane tm isolfurane ad us. vet. (piramal healthcare, india) and euthanized by heart cut. the small intestine was harvested, washed and flushed with pbs and subsequently inverted. to investigate the acute regulation of b 0 at1 ring uptake of radiolabeled l-isoleucine was performed as previously described [35] . briefly, one to two centimeter inverted rings of the middle jejunum were incubated for 5 min at 37˚c in bubbling (oxycarbon) krebs-tris buffer (ph 7.4) containing l-isoleucine (0.1 μci 14 c-l-ile/ml) with or w/o sodium. for these experiments 1 mm l-isoleucine was used (a concentration slightly below its published k m for b 0 at1 transport [6, 9] ) in order to minimize the relative contribution of the basolateral high-and lowaffinity transporters to the measured uptake rate. rings were washed on a filter system, dried o/n at 55˚c and weighed. lysis was performed for 6 h on a shaker in 1 ml solvable tm (perkin elmer, waltham, ma, usa) followed by 1 h of bleaching with h 2 o 2 . ultimate gold tm scintillation fluid (perkin elmer, waltham, ma, usa) was added followed by determination of radioactivity using the liquid scintillation analyzer (packard tri-carb 2900tr, perkinelmer, waltham, ma, usa). to investigate the chronic effect of diets on b 0 at1 function, 1 cm ring sections of the proximal and distal jejunum were used for transport studies. ring sections were slid onto 2 ml-serological pipettes (sarstedt, nümbrecht, germany) which were placed in tubes filled with sodium-free krebs-tris buffer (ph 7.5) stored on ice. uptake of 1 mm l-isoleucine (0.1 μci 14 c-l-ile/ml) was performed in krebs-tris buffer (ph 7.5) in the presence and absence of sodium at 37˚c for 2 min. the uptake was stopped by washing the ring sections in ice-cold sodium-free krebs-tris buffer (ph 7.5). after lysis of the tissue overnight on a shaker in 1 ml solvable tm (perkin elmer, waltham, ma, usa), ultimate gold tm scintillation fluid (perkin elmer, waltham, ma, usa) was added followed by determination of radioactivity using the liquid scintillation analyzer (packard tri-carb 2900tr, perkinelmer, waltham, ma, usa). total rna from scraped mucosa was isolated using rneasy mini kit (qiagen, hilden, germany), following the manufacturer's instructions. reverse transcription of the isolated rna was performed with taqman 1 reverse transcription kit (thermo fischer scientific, waltham, ma, usa) followed by quantification of the gene expression by quantitative real-time pcr using sybr green jumpstart tm taq readymix tm (sigma-aldrich, steinheim, germany) according to manufacturer's instructions. primers are listed in table 1 . after diet treatments, rats were starved for 12 h during the day. three hours after light offset they received an intragastric application (1 ml per 100 g) of an amino acid mixture of all proteinogenic amino acids with a final concentration 10-fold higher than the plasma concentration (gavage-amino acid mixture, table 2 ). the mixture was supplemented with 0.5 μci/ml 3 h-mannitol and 0.05 μci/ml 14 c-radiolabeled l-isoleucine. after 1 h, rats were anesthetized with attane tm isolfurane ad us. vet (piramal healthcare, india) and euthanized by heart cut. the blood was collected in heparin-coated tubes (braun medical ag, sempach, switzerland), plasma concentration according to [36] . plasma was purified and 20 μl were used for ultra performance liquid chromatography amino acid measurements. the small intestine was harvested and each 10 cm of the proximal, middle and distal part were washed with 1 ml pbs. the content was collected and sections were inverted followed by mucosa scraping. the intestinal content, scraped mucosa and plasma samples were lysed overnight on a shaker in 1 ml solvable tm (perkin elmer, waltham, ma, usa), followed by addition of ultimate gold tm scintillation fluid (perkin elmer, waltham, ma, usa) and determination of radioactivity using the liquid scintillation analyzer (packard tri-carb 2900tr, perkinelmer, waltham, ma, usa). analysis of the amino acid concentration was performed at the functional genomic center zurich (fgcz) using the mass track amino acid analysis application solution by acquity ultra performance liquid chromatography (uplc; waters, milford) according to the manufacturer's protocol. plasma samples were diluted 1:1 with 10% sulfosalicylic acid for deproteinization prior to uplc. analysis of experimental data was performed with graphpad prism 5.0 and ms office excel. statistical significance between mean values of groups was tested using paired two-tailed student's t-test, one-way anova or two-way anova and a bonferroni post test. differences were considered significant at p < 0.05. data are expressed as mean ± sem. the localization of b 0 at1, ace2 and cd13 in rat small intestine was investigated by immunofluorescence. in duodenum and ileum, antibodies directed against b 0 at1, ace2 and cd13 showed a strong apical staining along the intestinal villi as it was shown before for mouse tissue [3, 11] . as shown for all three proteins in ileum (fig 1a) , their signal was absent in the crypts and displayed a similar gradient along the villi with an increasing intensity towards the tips. the relative abundance of mrnas encoding b 0 at1 and ace2 was measured in duodenal and ileal mucosa by quantitative real-time pcr using 18s rrna as an internal control. fig 1b shows the relative abundances of these transcripts in rats euthanized 3 h after light-onset (zt3) or 3 h after light-offset (zt15). three independent experiments have been performed in which rats were fed either a normal protein diet (np), a high protein diet (hp) or a normal protein diet supplemented with amino acids (aa) and data were subsequently pooled, because no dietary effect on the expression of b 0 at1 and/or ace2 mrna was observed. both transcripts showed at both times of the day a higher level in duodenum compared to ileum. protein abundance of b 0 at1, ace2 and cd13 along the small intestine was measured by western blotting performed on brush-border membrane vesicles (bbmv) prepared from duodenum, proximal-, middle-and distal jejunum as well as ileum and quantified relative to the microvilli cytoskeletal protein β-actin,. surprisingly, as shown in representative western blots of fig 1c (left panels) , the abundance of these proteins displayed an axial gradient opposite to the one of the corresponding mrnas. the enrichment of luminal proteins in bbmv used for western blot analysis was verified by alkaline phosphatase activity measurement that indicated an 8-fold increase compared to total lysate (data not shown). additionally, rats fed different diets (np, hp and aa) were euthanized 3 h after light-onset (zt3) or 3 h after light-offset the three proteins tested in these bbmv showed a clearly increasing expression level in distal direction along the small intestine, as shown for distal versus proximal jejunum at both time points (fig 1c, right panels) . indeed, the protein expression levels of b 0 at1, ace2 and cd13 were at least two-fold higher in distal than proximal jejunum and their maximal increases were 5-, 9-and 13-fold, respectively. rats belong to the category of nocturnal animals with their active phase in the night and their inactive phase during the day. to investigate whether there is a diurnal change of b 0 at1 mrna, protein and/or function, rats fed different diets (np, hp or aa diet) were euthanized 3 h after light-onset (zt3) or 3 h after light-offset (zt15). b 0 at1 protein expression in bbmv (same preparations as in fig 1) was measured in proximal and distal jejunum at zt3 and zt15 by western blotting and signals standardized to β-actin were normalized to zt3 values (fig 2) . as shown in fig 1c, the distal b 0 at1 protein expression was higher than the proximal one, independent of the diet or time point. this effect is not visible in fig 2 due to the normalization to zt3 in order to highlight the difference between the active and inactive phase. these experiments revealed that luminal b 0 at1 protein expression was not significantly different between the active and inactive phase except in the proximal jejunum of rats fed a np diet in which it was significantly increased during the active phase (zt15) compared to the inactive one (zt3) (fig 2, left panel) . it is not clear why this difference was observed under np diet and not under hp or aa diet as well. interestingly, compared to the protein expression the mrna expression of b 0 at1 measured in duodenum and ileum appeared to follow a time-shifted circadian regulation with a significantly higher mrna expression at zt3 compared to zt15 in duodenum that was observed with all diets (fig 1b) . it might be that this timing difference represents a phase shift of the mrna regulation that is adapted to the timing of protein translation and half-life. the transport function of b 0 at1 was tested by measuring the uptake rate of l-isoleucine in intestinal rings. l-isoleucine belongs as a large aliphatic neutral amino acid to the preferred substrates of b 0 at1 [5] . the results of these experiments performed with rings of proximal and distal jejunum and in the presence and absence of sodium are shown in fig 2. in the proximal jejunum (fig 2, left panel) , the uptake rate was significantly higher at zt15 compared to zt3 under np and aa diet. the same trend was shown for hp diet and also in the distal jejunum (fig 2, right panel) independent of the diet but without significance. both, lowest and highest transport rates were observed in proximal jejunum at zt3 and zt15, respectively, suggesting that the function of b 0 at1 is more regulated in early than late segments of the small intestine. microscopy. upper panel: low-and high magnification images of protein of interest; lower panel: low-magnification images of protein of interest (green) and dapi (blue). b: relative mrna abundance of b 0 at1 and ace2 in duodenum versus ileum. the mrna expression levels of b 0 at1 and ace2 were measured by quantitative real-time pcr. rats were either euthanized 3 h after light-onset (zt3) or 3 h after light-offset (zt15). scraped mucosa of duodenum and ileum was used for analysis. the level of tested mrnas standardized to 18s rrna and normalized to the zt15 value in the ileum is shown. the values represent mean levels from 27 rats pooled from three independent experiments in which rats were fed either a np, hp or aa diet. values are expressed as means ± sem; n = 27; two-way anova and bonferroni's post test; *** p < 0.001, ** p < 0.01. c: protein expression of b 0 at1, ace2 and cd13 along the small intestine. western blotting experiments of bbmv with antibodies directed against b 0 at1, ace2, cd13 and β-actin were performed. representative western blotting images are shown in the left panels; d = duodenum, jp = proximal jejunum, jm = middle jejunum, jd = distal jejunum, i = ileum; molecular weight markers are indicated in kda. the intensity of the immunoreactive bands was quantified, standardized to β-actin and normalized for each time point to jp in order to highlight the differences between jp and jd. data shown in the right panels represent mean levels from 18-27 rats pooled from three independent experiments in which rats were fed either a np, hp or aa diet. values are expressed as means ± sem; n = 18-27; two-way anova and bonferroni's post test; *** p < 0.001. to determine the time-point at which to sacrifice animals after amino acid gavage, we examined the time-course of gastric emptying and small intestinal transit upon amino acid gavage. we administered a hydrophilic contrast agent together with water or with an isomolar amino acid cocktail and measured its impact on gastric emptying by in vivo ct imaging in rats ( fig 3a) . quantification of the contrast agent in the stomach over time revealed a delay of gastric emptying of about 20 min with the amino acid cocktail compared to pure water (fig 3b) . the majority (three-quarter) of the contrast agent was released into the small intestine 60 min after intragastric application, a time at which no measurable amount had reached the caecum yet. for following experiments, animals were therefore euthanized at 60 min after gavage. to investigate the possible acute regulation of b 0 at1, rats food-deprived for 16 h were gavaged with water or an isomolar amino acid cocktail containing 0.34 mmol of each of the proteinogenic amino acids. one hour after gavage, analysis of l-isoleucine transport in ring sections of the middle jejunum was performed. sodium-dependent uptake of radiolabeled l-isoleucine was clearly demonstrated but no difference in transport between animals receiving water or amino acid cocktail was observed (fig 4a) . analysis of protein expression in bbmv of proximal and distal jejunum from the same rats was in line with this result displaying no obvious differences between the two groups (data not shown). additional quantitative examinations of the b 0 at1 protein expression in bbmv from animals starved for 4 h followed by intragastric application of water or the amino acid cocktail also showed similar values without any significant differences (fig 4b) . furthermore, animals fed ad libitum showed similar values of b 0 at1 protein expression. we tested these different conditions because previous studies had revealed food-deprived rats were gavaged with contrast agent (sodium diatrizoate hydrate, sdh) and water or an isomolar amino acid cocktail that fasting itself can have an impact on transport rate and transporter expression. it had been shown that one day of fasting increases dipeptide transport in rat intestine by increasing the pept1 protein expression in the brush-border membrane and in addition 16 h of starvation of mice had been shown to cause a significant upregulation of intestinal pept1 protein expression [37, 38] . taken together, our results did not support the hypothesis of an acute regulation of intestinal b 0 at1 by dietary amino acids. to investigate the impact of different diets on b 0 at1 regulation, rats were fed with a np, hp or aa diet for 7 days. three hours after light-onset (zt3) or 3 h after light-offset (zt15) rats were euthanized, tissues were harvested and transport measurements by intestinal ring uptakes and the impact on gastric emptying was measured with computed tomography (ct). three-dimensional volume renderings of representative stomachs are shown immediately (0 min), 20, 40 and 60 min after gavage (a). gastric sdh content was quantified over time which reflects the gastric emptying (b). values are expressed as means ± sem; n = 5. https://doi.org/10.1371/journal.pone.0184845.g003 immediately performed. the results of these experiments that aimed at revealing the impact of the different diets on mrna and protein expression of b 0 at1 and its auxiliary peptidases as well as on b 0 at1-mediated amino acid transport were not straight forward but clearly demonstrated significant effects that are summarized here. as regards the transport function, uptake was significantly higher (~3-fold) under hp diet compared to np and aa diet in the proximal jejunum at zt3 (figs 2b and 5a ). in contrast, at zt15 the transport rate (that in all conditions was higher than at zt3) was slightly increased under aa diet compared to hp or np diet (by about 20%). in the distal jejunum, in the inactive (zt3) as well as in the active (zt15) phase, transport under aa diet was increased by approximately 30-40% compared to np and hp diet (fig 5a) , indicating that also in the distal jejunum transport was adapted to the diet. in summary, the transport rate of l-isoleucine was slightly increased effect of different diets on l-isoleucine transport and b 0 at1, ace2 and cd13 membrane expression. animals fed np, hp or aa diet for 7 days were euthanized 3 h after light-onset (zt3) or offset (zt15). a: the transport of l-isoleucine into everted rings of the proximal and distal jejunum was measured in the presence and absence of sodium and sodium-dependent uptake was calculated. data represent mean values of 3 intestinal ring uptakes of 9 different animals tested in 3 independent experiments. b-d: western blotting experiments with antibodies directed against b 0 at1, ace2, cd13 and β-actin were performed in bbmv of the proximal and distal jejunum (same preparations as in fig 1) . the intensity of the immunoreactive bands was quantified, standardized to β-actin and normalized to np diet. data represent mean values of 9 different animals. all values are expressed as means ± sem, one-way anova and bonferroni's post test was performed for each time point; * p < 0.05, ** p < 0.01, *** p < 0.001. https://doi.org/10.1371/journal.pone.0184845.g005 along the jejunum under aa and/or hp diet compared to np diet. since this increase was measured per unit gut length, it is not clear to what extent it may be due to tissue hypertrophy. to evaluate whether there is also an adaptation of the expression of proteins involved in the luminal transport of neutral amino acids, protein expression of b 0 at1, ace2 and cd13 in bbmv (same samples as in fig 1) was analyzed (fig 5b-5d) . regarding the luminal b 0 at1 protein, its expression in the rat proximal jejunum was significantly increased under hp and aa diet in the active (zt15) as well as in the inactive phase (zt3) (fig 5b) . however, in the distal jejunum there was no significant change in b 0 at1 protein expression under hp or aa diet compared to np diet, neither at zt3 nor at zt15. these data suggest that there is an adaptation of b 0 at1 protein expression to the diet in the proximal part of the jejunum but interestingly not in its distal part. in summary, luminal b 0 at1 protein expression was increased in the proximal section of the small intestine under aa diet. since the two peptidases ace2 and cd13 were shown to form functional complexes with b 0 at1 and to alter its function [15] , protein expression analysis in bbmv was performed ( fig 5c and 5d ). regarding the luminal ace2 protein, its expression in the proximal jejunum was not significantly different between the diets, neither at zt3 nor at zt15 (fig 5c) . only marginal changes with a slightly higher expression of ace2 under hp diet and aa diet was found at zt15. on the other hand, in the distal part of the jejunum ace2 protein expression in bbmv was significantly increased under aa diet compared to hp or np diet at both times correlating with the transport measurements of l-isoleucine. in summary, luminal ace2 protein expression was increased in the distal section of the small intestine under aa diet. furthermore, analysis of cd13 protein expression in bbmv revealed that in the proximal jejunum at zt3 it was only marginally increased under aa diet whereas at zt15 it was significantly increased under hp diet compared to np diet and to a lesser extent also compared to aa diet (fig 5d) . in the distal jejunum at zt3, protein expression of cd13 was significantly increased under aa diet compared to np and even more compared to hp diet. however, at zt15 protein expression was significantly decreased under hp diet compared to np diet and slightly decreased under aa diet compared to np diet. in summary, luminal cd13 protein expression revealed no distinct expression pattern regarding the different diets. taken together, these results suggest that a diet with elevated free amino acid levels increases b 0 at1 protein expression in the proximal jejunum and ace2 protein expression as well as the transport by b 0 at1 in the distal jejunum. to characterize the impact of a np, hp or aa diet on neutral amino acid absorption along the small intestine, the luminal content in different intestinal segments, lysates of the scraped mucosa and plasma were analyzed 1 h after intragastric application of a mixture containing all proteinogenic amino acids (table 2) supplemented with a radioactive tracer for l-isoleucine (fig 6) . the remaining amount of gavaged radiolabeled l-isoleucine in the content of the proximal and the distal part of the small intestine showed similar values under the different diets ( fig 6a) . however, the remaining amount of radiolabeled l-isoleucine in the middle jejunum was significantly decreased under hp or aa diet compared to np diet. this suggested the possibility that rats on a hp or aa diet may have expressed more functional b 0 at1 along the small intestine and performed a more efficient absorption of neutral amino acids like l-isoleucine. however, analysis of radiolabeled l-isoleucine in cell lysates of the scraped mucosa revealed no differences between the different diets. interestingly, the amount of radiolabeled l-isoleucine that accumulated in mucosal cells was substantially higher in the proximal part of the small intestine compared to the middle or distal part (fig 6b) . this latter observation supports the idea that free amino acids were to a large extent absorbed within the first section of the small intestine. the amount of radiolabeled l-isoleucine found in the plasma 60 min after intragastric application did not show significantly different values between the diets but a trend of higher l-isoleucine levels under hp and aa diet compared to a np diet (fig 6c) . this trend was supported by uplc measurements of unlabeled amino acids in plasma shown in fig 6d for the amino acids with significant differences between diets. not only the concentration of l-isoleucine but also that of l-leucine and l-valine was significantly increased under hp and aa diet compared to np diet implying that not only the newly absorbed neutral amino acids are taken up more efficiently but also the plasma concentration of branched-chain amino acids was elevated under hp and aa diet. on the other hand, the plasma concentration of glycine, l-serine and l-threonine was significantly decreased under hp and/or aa diet compared to np diet suggesting that they were possibly consumed by increased amino acid metabolism and gluconeogenesis and/or by increased basolateral uptake into small intestinal cells in exchange for absorbed bcaas. taken together, these results supported the hypothesis that a diet with either elevated free amino acids or elevated protein increases the expression of functional b 0 at1 and thereby the efficient absorption of neutral amino acids along the small intestine and also increases the branched-chain amino acid plasma levels. the impact of nutrition on function and expression of the neutral amino acid transporter b 0 at1 and its auxiliary peptidases in small intestine was investigated in this study. we could show that there was a chronic dietary effect but not an acute one. interestingly, there are many studies about regulation of sugar or peptide transporters, but there is not much known about the dietary regulation of amino acid transporters. with this study, we shed more light on the dietary regulation for b 0 at1. we show here that the luminal membrane expression of b 0 at1 and of its associated peptidases ace2 and cd13 on villi of the small intestine of rats displays an axial gradient toward the villi tips, as previously shown in mus musculus intestine [3] . additionally, our data indicated that along the small intestine there is an axial gradient of b 0 at1 protein expression in distal direction with highest expression in the ileum, whereas its mrna expression displays an opposite gradient. as regards the protein expression, we specifically show that in brush-border membrane vesicles (bbmv) made from intestinal mucosa the protein level of b 0 at1 relative to β-actin increases towards the ileum. it appears that β-actin was an adequate reference for western blot analysis because it is a highly abundant cytoskeletal protein with a "housekeeping" structural function in brush-border microvilli. the observed axial gradient of b 0 at1 along the small intestine might make sense considering that the proportion of single amino acids relative to peptides is higher in the distal section of the small intestine compared to the proximal one after proteins have been digested and cleaved by peptidases. concerning the mrna levels of b 0 at1 and ace2 that showed a reverse pattern with lower expression in the ileum compared to duodenum, measurements were done using scraped mucosal cells and quantifying the mrna levels relative to the housekeeping 18s ribosomal rna. we had first used villin mrna as housekeeping reference, but this turned out to be more variable between samples and segments. also analysis of these preliminary experiments revealed that the mrna expression of b 0 at1 and ace2 decreased towards the ileum when quantified relative to villin. our observation in rat small intestine contrasts with results obtained previously in mice where b 0 at1 mrna expression had been quantified relative to gapdh and had shown an increase in distal direction along the small intestine [3] . on the other hand, in humans, mrna expression of b 0 at1 and ace2 relative to villin was not significantly different, rather slightly decreased in the ileum compared to duodenum [39] , and in cancer patients it was slightly increased in distal direction relative to total rna [40] . we consider it as likely that the discrepancies between mrna and protein levels along the small intestine may be explained by axial differences in translational efficiency and protein stability. however, it is also possible that the different references used for quantification played a role. rats are nocturnal animals and the intestinal expression of some transporter proteins has been shown to vary diurnally. it appeared therefore likely that b 0 at1 expression and function would be higher in the evening, when more than 70% of the food intake takes place and the animals have their active phase. therefore we investigated transporter function and expression at two different time points, 3 h after light onset (inactive phase) and 3 h after light offset (active phase). it was for example shown in previous studies that the mrna level of pept1 peaks three hours before onset of the dark cycle and is followed by an increased transport function just before dark-onset [32] . on the other hand, the protein level of pept1 showed only minimal diurnal changes. for intestinal hexose transporters it was shown that mrna expression peaks 6-12 h earlier than protein expression [41] . furthermore, it was shown that protein expression and uptake of glucose goes together with a peak at the beginning of the dark cycle. our present experiments show on the one hand an increased transport rate in the evening (zt15) and on the other hand an increased mrna expression of b 0 at1 and ace2 in the morning (zt3). the increased mrna levels measured in the morning could, depending on the half-life of the gene products, participate to an upregulation of the corresponding protein for the evening. however, b 0 at1 protein expression was upregulated in the evening only under normal protein diet in the proximal part of the small intestine. nevertheless, we observed an increased l-isoleucine uptake in the active phase (zt15) compared to the inactive phase (zt3) independent of the diet or section of the small intestine. short-term dietary effects have been demonstrated in previous studies for the regulation of glucose and l-alanine transport. for instance, the jejunal glucose absorption rate was shown to be significantly increased when either glucose or maltose was present in the lower ileum [42] . in addition, it was shown that glucose transporters respond to changes in intestinal luminal glucose concentration within one hour [43] . also the amino acid l-alanine was shown to exert an inhibitory effect on its jejunal absorption when injected in the ileum or in an adjacent distal jejunal segment. its absorption from the ileum was shown to be similarly decreased by its presence in the jejunum [31] . because of the description of such substrate-mediated shortterm regulatory effects, we tested whether b 0 at1 was also acutely regulated in vivo by its own substrates using intragastric application of an amino acid cocktail. to determine the optimal sampling time point after gavage, we analyzed gastric emptying of the amino acid cocktail compared to water and could demonstrate a delayed gastric emptying after intragastric application of the amino acid cocktail similar to the effect already shown previously in our laboratory for specific amino acids [44] . based on these experiments, one hour delay was considered as an optimal time point for our measurements. however, we did not observe alterations in luminal b 0 at1 protein expression or in the l-isoleucine uptake rate one hour after amino acid application. we did not observe an effect either because potentially regulatory changes were not maintained during the experimental procedure or because such a short-term regulation does not exist. previous studies about acute dietary regulation of nutrient transporters mentioned above have been performed by in situ perfusion techniques using high, probably supraphysiological substrate concentrations. thus, it might be that the lack of effect observed in our studies was due to the more physiological experimental conditions used. as our experiments had not revealed an acute regulation of b 0 at1 expression or transport function in the small intestine by dietary amino acids, we then tested the hypothesis that diets containing different amounts of amino acids would in a longer-term exert such a regulatory action. for glucose and oligopeptide transporters an adaptive regulation to the diet had been described previously. specifically in rats, mice and sheep intestinal glucose transport had been shown to be increased in response to a high carbohydrate diet and also the peptide transporter pept1 had been shown to be upregulated by dietary proteins at the transcriptional level [27, 45, 46] . our current experiments showed that in the proximal part of the small intestine, protein expression of b 0 at1 in the brush-border membrane was significantly increased under aa diet (fig 5b) , an effect that in ex vivo ring uptake experiments translated only in a non-significant increase in uptake of the b 0 at1 substrate l-isoleucine. however, absorption experiments of l-isoleucine in vivo were consistent with a more efficient absorption of this b 0 at1 substrate from the proximal and the middle part of the jejunum under hp and aa diet (fig 6a) . it is possible that the lack of significant transport increase in ring uptakes was due to a methodological limitation of this assay that might have been not precise enough to register the difference in l-isoleucine uptake. interestingly, ring uptake experiments in contrast showed an increase of l-isoleucine uptake in the distal part of the small intestine where b 0 at1 protein expression was not upregulated, in contrast to its accessory protein ace2. these data suggest the possibility that ace2 might besides its role for b 0 at1 trafficking also impact on its function. that associated peptidases positively impact on the function of the b 0 at1 transporter was for instance reported by fairweather et al., who showed that the intestinal peptidase cd13 forms functional complexes with b 0 at1 and ace2 [15] . since the protein expression of luminal cd13 was increased in the distal part of the small intestine under aa diet, it might also have contributed to the observed elevation of l-isoleucine absorption. similarly, it was reported by others that intestinal brush-border membrane enzymes like neutral aminopeptidases and γ-glutamyl-transferase displayed a higher activity when rats were fed a high protein diet [28] . in the present study, we observed an increased expression of cd13 protein under hp diet at zt15 only in the proximal section of the jejunum. the sensing and regulatory mechanisms underlying the observed dietary adaptation of small-intestinal neutral amino acid transport are not yet understood and need further investigation. interestingly, the hp and aa dietinduced increase of in vivo absorption of the neutral amino acid l-isoleucine correlated with a slightly elevated postprandial l-isoleucine plasma level and a significantly increased general plasma concentration of the branched-chain amino acids l-leucine, l-isoleucine and l-valine together with a decreased concentration of glycine, l-serine and l-threonine. indeed, the concentration of amino acids in plasma depends, next to their uptake from nutrition, on multiple other factors controlling their fluxes within the body, for instance on their metabolism and on their loss via urine, sweat and feces [47, 48] . taken together, the results of the present study indicate that the expression and function of luminal amino acid transporter b 0 at1 in small intestinal enterocytes is controlled by a series of superposed regulatory effects impacting on its mrna and protein expression levels as well as on its functional state. specifically we show here that b 0 at1-mediated absorption of neutral amino acids is adapted not only to the diurnal rhythm but also in the long-term to the amount of dietary proteins and amino acids, particularly at the level of b 0 at1 expression and function in proximal segments of the small intestine. novel renal amino acid transporters the abcs of solute carriers: physiological, pathological and therapeutic implications of human membrane transport proteinsintroduction luminal kidney and intestine slc6 amino acid transporters of b0at-cluster and their tissue distribution in mus musculus molecular cloning of mouse amino acid transport system b0, a neutral amino acid transporter related to hartnup disorder characterization of mouse amino acid transporter b0at1 (slc6a19) steady-state kinetic characterization of the mouse b(0)at1 sodium-dependent neutral amino acid transporter mutations in slc6a19, encoding b0at1, cause hartnup disorder hartnup disorder is caused by mutations in the gene encoding the neutral amino acid transporter slc6a19 impaired nutrient signaling and body weight control in a na+ neutral amino acid cotransporter (slc6a19)-deficient mouse essential role for collectrin in renal amino acid transport tissue-specific amino acid transporter partners ace2 and collectrin differentially interact with hartnup mutations angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury angiotensin-converting enzyme 2 is an essential regulator of heart function intestinal peptidases form functional complexes with the neutral amino acid transporter b(0)at1 a role for aminopeptidase n in na(+)-dependent amino acid transport in bovine renal brush-border membranes cd13-not just a marker in leukemia typing cell-surface peptidases the moonlighting enzyme cd13: old and new functions to 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lumen regulate their own absorption from a distant intestinal site diurnal expression and function of peptide transporter 1 (pept1) simultaneous assessment of gastric emptying and secretion in rats by a novel computed tomography-based method preparation and properties of brush-border membrane vesicles from human small intestine defective intestinal amino acid absorption in ace2 null mice profile of plasma amino acid levels in rats exposed to acute hypoxic hypoxia functional and molecular expression of intestinal oligopeptide transporter (pept-1) after a brief fast influence of fed-fasted state on intestinal pept1 expression and in vivo pharmacokinetics of glycylsarcosine in wild-type and pept1 knockout mice human intestine luminal ace2 and amino acid transporter expression increased by ace-inhibitors expression profiles of various transporters for oligopeptides, amino acids and organic ions along the human digestive tract hexose transporter expression and function in mouse small intestine: role of diurnal rhythm adaptation of hexose uptake by the rat jejunum induced by the perfusion of sugars into the distal ileum rapid enhancement of brush border glucose uptake after exposure of rat jejunal mucosa to glucose specific amino acids inhibit food intake via the area postrema or vagal afferents crypt-villus site of glucose transporter induction by dietary carbohydrate in mouse intestine effect of dietary carbohydrate on monosaccharide uptake by mouse small intestine in vitro plasma amino acid levels with a note on membrane transport: characteristics, regulation, and metabolic significance interorgan amino acid transport and its regulation we would like to thank the zurich integrative rodent physiology facility, the zurich center for microscopy and image analysis and the functional genomics center zurich for help and equipment. key: cord-308261-hxlebas8 authors: broekhuis, femke; madsen, emily k.; keiwua, kosiom; macdonald, david w. title: using gps collars to investigate the frequency and behavioural outcomes of intraspecific interactions among carnivores: a case study of male cheetahs in the maasai mara, kenya date: 2019-04-03 journal: plos one doi: 10.1371/journal.pone.0213910 sha: doc_id: 308261 cord_uid: hxlebas8 intraspecific interactions between individuals or groups of individuals of the same species are an important component of population dynamics. interactions can be static, such as spatial overlap, or dynamic based on the interactions of movements, and can be mediated through communication, such as the deployment of scent marks. interactions and their behavioural outcomes can be difficult to determine, especially for species that live at low densities. with the use of gps collars we quantify both static and dynamic interactions between male cheetahs (acinonyx jubatus) and the behavioural outcomes. the 99% home-ranges of males overlapped significantly while there was little overlap of the 50% home-ranges. despite this overlap, male cheetahs rarely came into close proximity of one another, possibly because presence was communicated through frequent visits to marking posts. the minimum distance between individuals in a dyad ranged from 89m to 196m but the average proximity between individuals ranged from 17,145 ± 6,865m to 26,367 ± 11,288m. possible interactions took place more frequently at night than by day and occurred mostly in the 50% home-range of one individual of a dyad or where cores of both individuals overlapped. after a possible encounter male cheetahs stayed in close proximity to each other for up to 6 hours, which could be the result of a territory defence strategy or the presence of a receptive female. we believe that one of the encounters between a singleton and a 5-male coalition resulted in the death of the singleton. our results give new insights into cheetah interactions, which could help our understanding of ecological processes such as disease transmission. intraspecific interactions, or interactions between members of the same species, are an important component of population dynamics as they play a role in sociality [1] , mating events [2] , of movement behaviour and mortalities. based on previous research we predict that males will overlap spatially but that there will be little overlap of the core areas [29] . we also predict that marking posts are frequently visited by both individuals in a dyad, i.e. pair of cheetahs, and that occasions where individuals of the dyad are in close proximity to each other are infrequent. because encounters between males can be aggressive [27] we predict that the movement behaviour after a possible encounter would indicate avoidance behaviour (moving away from the encounter location, moving away from one another, increased distance travelled and decreased path tortuosity). the study was conducted in the maasai mara, in the southwest of kenya (centred at 1˚s and 35˚e), which is part of the larger serengeti-mara ecosystem. the study area (~2,600 km 2 ) included the maasai mara national reserve and the surrounding wildlife conservancies. the area experiences one wet season spanning from november to june and one dry season spanning from july to october [30] . after the wet season, the long grass attracts large numbers of migratory ungulates, including the white-bearded wildebeest (connochaetes taurinus) and the common zebra (equus quagga), from the serengeti in tanzania. throughout the year there is an abundance of cheetah prey including resident white-bearded wildebeest, thomson's gazelle (eudorcas thomsonii), grant's gazelle (nanger granti) and impala (aepyceros melampus) [31] . the habitat in the maasai mara varies, ranging from open grasslands and shrubland, to riverine forests found along the major rivers and their tributaries [32] . the open grassland plains, which are dominated by red oat grass (themeda triandra), are mostly found toward the south and west of the study area, while the north and north-east consist mostly of croton thickets (croton dichogamous) and vachellia woodlands (vachellia drepanolobium and v. gerrardii). global positioning system (gps) satellite collars (african wildlife tracking -www.awt.co.za) were fitted on four adult male cheetahs between 19 th october 2016 and 9 th february 2018. in compliance with kenyan law, all immobilizations for deployment/removal of collars were performed by a kenya wildlife service veterinarian. cheetahs were free-darted and immobilized using a combination of ketamine (2-2.5 mg/kg) and medetomidine (0.07 mg/kg), remotely administered by a dan-inject co 2 rifle (dan-inject, denmark), and reversed with atipamezole (0.3 mg/ml; following [33] ). sedation time was kept to a minimum, typically less than 1 hour. after immobilization all cheetahs recovered fully, showing no signs of distress and no apparent side effects were observed in the short-and long-term. collars, which were only fitted on adults, weighed 400 grams which is the recommended weight for cheetah collars [34] . all collars were removed if they malfunctioned or if the batteries were low. the animal handling protocols used conformed to the standards of the american society of mammalogists [35] and permissions to deploy collars were provided by the kenya wildlife service (permit no.: kws/ brm/5001) and the national commission for science, technology and innovation (permit no.: nacosti/p/16/69633/10821). the collared males were all singletons, except for one male (m03), who was part of a fivemale coalition. over an 18 month period, the five-male coalition were sighted on 73 occasions and only on one occasion did the coalition separate for a period of <24 hours. we therefore collected data on a total of eight individuals in four social groups. while this is a relatively small sample size, it is a quarter of the entire population as there are approximately 32 adults within the study area [36] . the collars collected gps coordinates every three hours (00h, 03h, 06h, 09h, 12h, 15h, 18h and 21h) and when there was satellite communication, data were uploaded on a daily basis at 06h. on average, the collars were deployed for 285 days ranging from 115 to 349 days (table 1) . for each pair of cheetahs (hereafter referred to as a dyad), we only used simultaneously collected data for the analyses. static interactions. to determine the static interactions between male cheetahs we calculated their space use and the amount of overlap for each dyad to determine the possibility that individuals could encounter each other either directly or indirectly. space use for each individual per dyad was based on their utilisation distributions, which is the distribution of an individual's locations over time [37] , using the adehabitat package [38] in r [39] . to calculate the utilisation distributions we used a fixed kernel density estimate using a bivariate normal kernel. we used the reference bandwidth parameter (h ref ) as the smoothing factor unless h ref > 1000 then we used 80% of the h ref to minimise over-smoothing of the data. using the resulting utilisation distribution, we determined both the 99% and 50% kernels which respectively represent an individual's total space use and their core areas. for each dyad we then calculated the amount of overlap of the 99% kernels and the 50% kernels. marking posts are used by male cheetahs to communicate their presence to conspecifics. using the methods described by [29] , we located marking posts based on a cluster analysis using data from the gps collars and opportunistically when conducting fieldwork. for each dyad, we determined how many marking posts were found within the 99% kernel overlap. we used the recurse package [40] to calculate 1) how many marking posts were visited by each individual and 2) how many marking posts were visited by both individuals in a dyad (hereafter referred to as mutual marking posts). we classified a visit when a cheetah came within 500m (half the average step-length, see dynamic interaction for details) of a marking post. for the mutual marking posts we calculated 1) the time between individual visits and 2) the time spent within 500m of a mutual marking post by each individual and tested whether there were differences between individuals. in addition, we calculated the time between visits from two different individuals i.e. we calculated how long it took for individual x to visit a mutual marking post once individual y had visited and vice versa. we then tested whether the time it took for an individual to visit a marking post once the other individual had been there differed between the individuals. we tested the data for normality using the shapiro-wilk test and used a t-test if the data were normally distributed and a wilcoxon test if they were not. if the data were not normally distributed then we provided the median ± median absolute deviation in addition to the mean ± standard deviation. dynamic interaction. we first explored the movement of the different individuals using the movevis package [41] . then, to determine whether interactions between two individuals in a dyad were likely to occur, we calculated the proximity between simultaneous locations using the wildlifedi package [42] . the 3hr resolution of the data is quite coarse so we used different proximity thresholds to group possible encounters based on the average 3-hour step-length which was 1,021m ± 1,487m (mean ± standard deviation). we used four proximity thresholds: <500m, <1000m, <1500m and <2000m which correspond to 0.5, 1, 1.5 and 2 times the average step-length. we then determined whether these possible encounters took place at night (fixes at 21hr, 00hr, 03hr or 06hr) or during the day (fixes at 09hr, 12hr, 15hr or 18hr), whether they occurred within 50% kernels and the distance to the nearest known marking post. the half-way points between the two individuals were used as the estimated location where a possible encounter occurred. encounter outcomes. the gps data were examined once every few days. if any unusual behaviour was detected, such as no movement after a possible encounter, the field team would investigate to establish whether any injuries or deaths occurred as a result. in addition, for each possible encounter we compared the behaviour before to the behaviour after at four different time lags; 3hrs, 6hrs, 12hrs and 24hrs. first, we calculated the distance between two individuals before and after a possible encounter to determine whether males moved away from one another after an encounter. then, per dyad, we calculated 1) the distance to the encounter location for each individual, 2) the distance travelled per individual and 3) the path tortuosity, or straightness. path tortuosity was calculated by dividing the net displacement by the total distance travelled. a value of around 1 would indicate that the individual travelled in a straight line whereas a value <1 would be indicative of a tortuous path. we compared the proximity of the two individuals within a dyad, distance to encounter location, distance travelled and tortuosity before and after a possible encounter. we tested the data for normality using the shapiro-wilk test and used a t-test if the data were normally distributed and a wilcoxon test if they were not. in total, we secured simultaneous data on four male cheetah dyads ranging from 113 to 329 days per dyad. one individual (m01) was part of three of the four dyads, two individuals (m02 and m03) were part of two dyads and one individual (m04) was part of one dyad ( table 2 ). across the four dyads the 99% kernels ranged from 253 km 2 to 1,903 km 2 and the 50% kernels ranged from 11 km 2 to 359 km 2 ( table 2) . for all the dyads, the 99% kernels overlapped but the amount of overlap ranged from 10% to 100% where in dyad 2 the 99% kernel of individual m01 fell completely within the 99% kernel of individual m03 (fig 1) . only the core areas (50% kernel) of dyad 3 had an extensive area of overlap (28% and 48%), whereas the cores of the other dyads did not overlap or the area of overlap was minimal (< 4%). we found 125 marking posts in the study area and the number of mutual marking posts per dyad ranged from 4 to 37 with the average number of visits to these marking posts ranging from 1 to 46.16 (table 3) . for the three dyads that had the most extensive spatial overlap and the largest number of mutual marking posts (dyads 1, 2 and 3) the average time that an individual was within 500m of a mutual marking post did not vary significantly between individuals in the same dyad, ranging from 5.06 ± 4.56 hours to 7.95 ± 5.85 hours (mean ± standard deviation; table 3 ). the data were not normally distributed and the median for the same three dyads ranged from 2.52 ± 2.35 hours to 7.02 ± 6.01 hours (median ± median absolute deviation). for all the dyads, the average time between visits of a mutual marking post varied significantly between the individuals (table 3 ). in the case of dyad 1 and 2, individual m01, who we classified as territorial, visited mutual marking posts more frequently compared to individuals m02 and m03, neither of which were strictly territorial ( table 1) . the time between different individuals visiting the same mutual marking post ranged from 3.11 ± 4.01 days to 13.64 ± 8.51 days and did not differ significantly across the dyads (table 3) . for three of the four dyads (dyads 1, 2 and 3) the possibility of individuals within each dyad encountering each other was high as they overlapped extensively in space and had a large number of mutual marking posts. for these three dyads we explored their simultaneous movements and calculated the proximity between each individual within a dyad. the individuals within the three dyads did on occasions come into close proximity to one another as can be seen in the animation provided in the s1 movie. the minimum distance between individuals in a dyad ranged from 89 m to 196 m but the average proximity between individuals ranged from 17,145 ± 6,865 m to 26,367 ± 11,288 m (fig 2) . possible encounters were classified according to four different thresholds and we detected four possible encounters with a proximity threshold of <500m, 11 with a proximity threshold of <1000m, 21 with a proximity threshold of <1500m and 25 with a proximity threshold of <2000m (table 4 ). possible encounters were more likely to occur at night than during the day (χ = 8, df = 1, p = 0.005) and occurred most frequently at 21hr and midnight. of the 25 possible encounters, 64% (n = 16) occurred within the core area of one individual, 28% (n = 7) occurred where the 50% kernels overlapped and two possible encounters in dyad 3 did not occur in any of the core areas. eleven (44%) of the possible encounters occurred within 500m of a known marking post. for possible encounters with proximity thresholds of <500m, <1000m and <1500m the distance between males was overall significantly less during the period 3 to 6 hours after a possible encounter, compared to the 3 and 6 hours before a possible encounter (table 5 ). in other words, rather than moving away from each other, male cheetahs stayed in close proximity to each other for up to 6 hours after a possible encounter. for the distance to the encounter location, distance travelled and tortuosity we wanted to determine whether there was individual variation within each dyad. however, because of the paucity in the number of possible encounters that were detected per dyad we were only able to carry out the analysis for possible encounters with a proximity threshold <2000m. in general, cheetahs were closer to the encounter location after a possible encounter compared to before for all four time lags, apart from individual m03 in dyad 3 where the opposite trend was intraspecific interactions among carnivores: a case study of male cheetahs observed, however none of the results were significant (s1 table) . similarly, cheetahs travelled less after a potential encounter compared to before, apart from individual m03 in dyad 3 where the opposite trend was observed. some of the results, especially at the 12hr and 24hr lag were significant for dyad 1 and 2 (s1 table) . on the 11 th february 2017 the collar on m04 stopped transmitting but when the team visited the last location sent by the collar, neither cheetah nor collar could be found and the individual has not been seen since. on the 1 st october 2017 the collar on m01 stopped transmitting data after the collar data showed that individuals m01 and m03 had come within 89m of each other. the team went to the last gps coordinate that was transmitted by the collar and found the remains of m01 70m from the last gps fix sent by the collar. upon inspecting the carcass, a puncture wound was found on the left side of the skull. based on the circumstantial evidence, we believe that the death of m01 was either a direct or an indirect result of an aggressive interaction between him and the 5-male coalition (m03). interestingly, three months prior to this encounter m03 and his coalition mates started establishing a territory approximately 25km southwest from m01's territory. two days before the encounter the coalition travelled 25 km to the encounter location, spent 12 hours within 150 m of m01 and then travelled 19km straight back to the core of their territory. m03 did not return to the vicinity of the encounter between the 1 st october 2017, when the encounter took place, and 3 rd february 2018, when m03's collar was removed. the closest they came to m01's territory during that time was approximately 10 km (fig 3 and the animation in s2 movie). using gps collar data we documented static and dynamic interactions between male cheetahs in kenya's maasai mara and investigated the outcomes of these interactions in terms of movement behaviour and mortalities. as we predicted, male cheetahs showed extensive spatial overlap of the 99% kernels. this high degree of overlap observed in the maasai mara could be related to the pattern of prey availability [43] , although we do not have the data to test this. however, apart from one dyad, there was little overlap of core areas (50% kernels) and it could be that core areas are defended more intensively than the peripheral areas [29] . similar to observations in other areas, marking posts were frequently visited by males [29, 44] and this could indicate the mechanism that results, despite the extensive spatial overlap, in the rarity of occasions when members of a dyad were in close proximity [27] . interestingly, our results show that possible encounters were most likely to take place in the core area of one individual of a dyad or where cores of both individuals overlapped. we also found that, similar to african wild dogs (lycaon pictus), possible encounters occurred more at night than during the day [17] . while cheetahs, like african wild dogs, are predominantly diurnal they can be active at [45] and nocturnal activity for males has been found to be considerably higher than for females [46] . data from camera traps set at marking posts found that visits occurred more at night than during the day ( [44] ; kk unpublished data) suggesting that male nocturnal activity is partly driven by patrolling behaviour which is probably why encounters predominantly took place at night. in some species, including african wild dogs and white-faced capuchins (cebus capucinus), avoidance behaviours, characterised by an increase in distance and speed travelled postencounter, were observed as a result of interactions between different groups [17, 18] . however our results, in contrast to our predictions, did not show avoidance behaviour postencounter as males stayed in close proximity to each other 3-6 hours after a potential encounter. it is possible that males stayed in close proximity to each other, as part of a territorial defense strategy, if a recent scent of a conspecific was detected. this behaviour has been observed in dwarf mongoose (helogale parvula) groups, who moved slower and covered shorter distances in the hour following the encounter of rival faeces at a latrine site within their territory [16] and red fox (vulpes vulpes) males who spent more time in scent-marked areas [47] . alternatively, males could come into close proximity to one another if they are attracted to a resource, such as a female in oestrus [27, 48] . cheetahs exhibit a high rate of multiple paternity [49] so it is possible that multiple males stay in the vicinity of a receptive female with the hope of getting a chance to mate. these encounters could however result in fatalities if the removal of competition increases future mating opportunities [50] . if encounters occur as a result of access to a receptive female rather than to a static, long-term resource such as a territory then this could explain why the five-male coalition did not take-over the territory of individual m01 after he died. aggressive interactions with fatal consequences are not uncommon in cheetahs. caro [22] reported three cases in serengeti where singletons were killed by coalitions (all three-male coalitions). similarly, mills and mills [27] found that 50% of male-male encounters recorded in the kgalagadi transfrontier park in botswana/south africa resulted in death. to our knowledge, fatal interactions have not been observed between female cheetahs. this could explain why male mortality is higher and life expectancy lower for males compared to females [51, 52] resulting in a female biased sex ratio [52] . for some species, such as voles (microtus oeconomus), lions and grizzly bear (ursus arctos), the removal of males, through either displacement or mortality, has a negative effect on population growth as a result of increased infanticide [53] [54] [55] . infanticide has however not been observed amongst cheetahs [56] possibly because it rarely occurs in predominantly solitary species [57] . the removal of males could however have other population-level consequences [58] but the impact of male mortality on population dynamics in cheetahs is unclear. static and dynamic interactions can play a role in disease transmission [3, 4] . in the mara-serengeti ecosystem there is a relatively high prevalence of mange [59, 60] and in southern africa cheetahs have been positively tested for feline coronavirus (fcov) and feline panleukopenia virus (fpv), which can be highly contagious and fatal [61, 62] . pathogens such as these can easily spread through faeces and other bodily fluids, which are deposited and investigated by male cheetahs at marking posts. this could explain why in 2015 several males in the maasai mara, who overlapped spatially, died of a yet unknown disease within a short space of time [63] . we suggest that future epidemiological research should investigate the role of scent marking posts and movement in disease transmission [64] . here we give a descriptive analysis of the static and dynamic interactions between male cheetahs and the outcomes of these encounters. despite the clear patterns that were observed, there are several caveats that warrant discussion. firstly, we were only able to use data from four collared males, one of which was part of a 5-male coalition. it is therefore possible that other uncollared individuals, including the other members of the 5-male coalition, could have influenced the results. secondly, because of the resolution of the collar data we might have missed visits to marking posts and we inferred when interactions took place rather than being able to detect actual interaction (apart from one occasion). our results are therefore likely to be on the conservative side and we suggest that future studies use higher resolution data and/ or proximity loggers to investigate actual interactions between individuals (e.g. [17, 65] ). however, even with a relatively coarse resolution of data and only a small number of individuals we managed to investigate interactions and subsequent outcomes between males giving a first detailed insight into intraspecific interactions in cheetah. table. summaries for each dyad of the distance to the encounter location, distance travelled and tortuosity before and after a possible encounters with proximity threshold of <2000m. 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free-ranging namibian cheetahs (acinonyx jubatus) disease outbreak thresholds emerge from interactions between movement behavior, landscape structure, and epidemiology wildlife contact analysis: emerging methods, questions, and challenges we would like to thank the mara cheetah project and mara lion project teams for assisting with fieldwork. key: cord-319845-oob2ktnz authors: proença-modena, josé luiz; gagliardi, talita bianca; escremim de paula, flávia; iwamoto, marisa akiko; criado, miriã ferreira; camara, ataíde a.; acrani, gustavo olszanski; cintra, otávio augusto leite; cervi, maria célia; de paula arruda, luisa karla; arruda, eurico title: detection of human bocavirus mrna in respiratory secretions correlates with high viral load and concurrent diarrhea date: 2011-06-20 journal: plos one doi: 10.1371/journal.pone.0021083 sha: doc_id: 319845 cord_uid: oob2ktnz human bocavirus (hbov) is a parvovirus recently identified in association with acute respiratory infections (ari). despite its worldwide occurrence, little is known on the pathogenesis of hbov infections. in addition, few systematic studies of hbov in ari have been conducted in latin america. therefore, in order to test whether active viral replication of human bocavirus is associated with respiratory diseases and to understand the clinical impact of this virus in patients with these diseases, we performed a 3-year retrospective hospital-based study of hbov in outpatients and inpatients with symptoms of acute respiratory infections (ari) in brazil. nasopharyngeal aspirates (npas) from 1015 patients with respiratory symptoms were tested for hbov dna by pcr. all samples positive for hbov were tested by pcr for all other respiratory viruses, had hbov viral loads determined by quantitative real time pcr and, when possible, were tested by rt-pcr for hbov vp1 mrna, as evidence of active viral replication. hbov was detected in 4.8% of patients, with annual rates of 10.0%, 3.0% and 3.0% in 2005, 2006 and 2007, respectively. the range of respiratory symptoms was similar between hbov-positive and hbov-negative ari patients. however, a higher rate of diarrhea was observed in hbov-positive patients. high hbov viral loads (>10(8) copies/ml) and diarrhea were significantly more frequent in patients with exclusive infection by hbov and in patients with detection of hbov vp1 mrna than in patients with viral co-infection, detected in 72.9% of patients with hbov. in summary, our data demonstrated that active hbov replication was detected in a small percentage of patients with ari and was correlated with concurrent diarrhea and lack of other viral co-infections. the discovery of human bocavirus (hbov) was the result of a viral metagenomic study of respiratory secretions from swedish children with symptoms of acute respiratory infection (ari) reported in 2005 [1] . hbov is classified in the family parvoviridae, which includes small non-enveloped, icosahedral viruses with 5.3 kb single-stranded dna genome containing three open reading frames (orfs). the first two sequential orfs encode non-structural proteins ns1 and np-1, whereas the third downstream orf encodes two viral capsid proteins, vp1 and vp2 [1] . hbov circulates worldwide [2] and more recent phylogenetic analyses revealed that three novel hbov species, different from the respiratory hbov1, named hbov2, 3 and 4, are frequently detected in stools from children with acute gastroenteritis [3, 4, 5, 6] . hbov is often detected in patients with ari [7, 8, 9] , including those with wheezing, croup, cough, rhinorrhea and fever [2, 10] . the most frequent clinical diagnoses associated with respiratory hbov are general upper respiratory tract infections (urti), bronchiolitis, pneumonia, bronchitis and exacerbation of asthma [2] . symptoms associated with hbov usually last 1-2 weeks [11, 12] , but the agent has also been detected in association with prolonged fever [13] . an association of hbov with respiratory disease has been based mostly on the significantly higher frequencies of detection of the agent in ari patients than in control subjects without respiratory symptoms [8, 10, 14] . hbov is also detected in feces from children with diarrhea with frequencies ranging from 0.8% to 9% [6, 15, 16] . in addition, hbov dna has been detected in tonsil tissue in children with chronic tonsillitis undergoing surgical resection [17, 18] . although hbov has been propagated in primary cultures of human respiratory cells [19] , isolation of the agent from clinical samples has not been achieved in common cell lines. moreover, no experimental animal model of hbov infection has been developed, which hampers fulfillment of koch's postulates for a definitive association of hbov with disease. this scenario is further complicated by the possibility that hbov may persist in a way similar to other viruses of the family parvoviridae [20] , with consequent prolonged detection in human secretions. in addition, detection of hbov dna by pcr is commonly associated with detection of genomes of other viruses whose roles in pathogenesis of human diseases is already established [21] . this article reports a cross-sectional study of hbov in ari patients from ribeirão preto, brazil, in which the shedding of vp1 mrna in respiratory secretions was used as surrogate marker for active hbov replication, to look for correlations with viral load, and presence of particular clinical manifestations and simultaneous detection of other respiratory viruses. hbov genome was detected by pcr in 48 out of 1015 (4.8%) respiratory samples from ari patients. out of the 48 hbov positive samples, 40 (83.3%) were from patients under or equal to 2 years of age (#2 years old) and only 2 (4.1%) were from adult patients. hbov was detected in 2.8% of 70 adult patients enrolled in the study. the patient demographics and clinical information are summarized on figure 1 ). it was noticeable that hbov circulated roughly along with hrsv ( figure 1 ). in this 3year study period, there was a trend for an increase in the total number of respiratory samples from children submitted for viral testing, hrsv and hbov positivity, in association with dry season and declining average montly temperatures [22] . it is noteworthy that hbov was not detected in stored samples obtained from 50 patients without respiratory symptoms, seen at the emergency room for reasons unrelated to the respiratory tract from 1999 to 2000 [23] . for the purpose of the analysis, the patients were divided into two groups: those with age equal or less than 2 years of age (410 males and 335 females; median and mean age 6 sd: 5 and 6.4565.49 months), and those older than 2 years (125 males and 145 females; 63.5 and 145.156187.75 months). in general, the clinical findings were more severe in patients younger than 2 years (table s2 ). the most frequent respiratory symptoms/signs (present in .50%) seen in hbov-positive patients were cough, fever, dyspnea and coryza, but wheezing, nasal obstruction and sneezing were also recorded. no significant differences were noted in the frequencies of specific respiratory symptoms/signs between hbov positive and negative patients (table 1) . of 1015 studied patients, 721 (71.0%) were diagnosed with lrti and 294 (28.9%) with urti. the complications of aom and gerd were diagnosed in 77 (7.6%) and 44 (4.4%) patients, respectively (table s2) . over one third of the patients had illness of low or moderate severity and 37.0% of them had an ics = 0 or 1,and only 5.7% had ics = 6 or 7 (table 1) . significantly higher frequencies of lrti (85.4% vs. 72.2%, p = 0.01), requirement for hospitalization (83.3% vs. 67.5%, p = 0.02), and diarrhea (20.8% vs. 5.5%, p,0.01) were noted in the 48 hbov-positive patients, in comparison with the 967 hbov-negative ones (p,0.05) ( table 1) . no significant association was noted between hbov positivity and the presence of specific signs or symptoms (table 1) . also, no significant differences were noted in length of hospital stay, rates of requirements for o 2 and positive airway pressure, aom, and illness severity as measured by ics, between hbov positive and negative patients (table 1) . while the frequency of gerd was roughly twice as high in hbov-positive, than in hbov-negative patients (p = 0.04), only 4 hbov patients had gerd (table 1) , a low number of cases that restricts the power of the comparison. logistic regression analyses of the relationship between hbov detection and disease, after adjustment for patient age, gender and concurrent hrsv infection, showed that the clinical features more significantly associated with hbov were diarrhea (odds ratio [ all samples positive for hbov by conventional pcr were tested for the other respiratory viruses, including hrsv, hrv, hmpv, flu, hpiv, hcov and hadv. of 48 hbov-positive patients, 35 (72.9%) had at least one additional respiratory virus detected (table s3 ). the viruses most frequently detected simultaneously with hbov were hrsv in 22 (45.8%), hrv in 16 (33.3%), hmpv in 9 (18.8%), and hadv in 7 (14.5%) patients. the frequencies of co-detection of one, two, three and four additional viruses in patients with hbov were respectively 35.4%, 27.1%, 4.1% and 6.3% (table s3) . to assess whether this high frequency of co-detection of other respiratory viruses was a finding specific of hbov-infected patients, a subset of 48 samples from hbov-negative ari patients, collected in the same season as those from hbovpositive ones, was randomly selected for testing by pcr for the other respiratory viruses, without knowing the result of the hrsv or influenza screening for which the samples had been originally submitted. such strategy was adopted since it would not be possible to test all samples for all respiratory viruses. a single respiratory virus different from hbov was detected in 26 of 48 (54.1%) patients, and co-detections with more than one agent were found in an additional 13 (27.1%) (table s3) . there were no significant differences in frequencies of specific respiratory symptoms between the patients with ari in whose samples only hbov was detected, and those with hbov and codetection of other viruses. however, significantly lower frequencies of diarrhea (or = 0.08; 95% ci = 0.01-0.40; p,0.01) and complication of gerd (or = 0.10; 95% ci = 0.01-1.05; p = 0.05) were noted in patients with co-detection of other viruses in addition to hbov (table s4) . absolute quantification of dna by qpcr revealed that the hbov viral load in hbov-positive patients varied very broadly, over a range that reached 10 decimal orders of magnitude, with median value of 2.37610 6 copies/ml ( figure 2 and table s5 ). importantly, the median viral load in samples from patients in whom hbov was the only virus detected (1.91 6 10 9 copies/ml), was significantly higher than that in the patients with hbov in coinfection with other respiratory viruses (4.41 6 10 5 copies/ml) (p,0.001) (figure 2a) . a comparison of demographics and clinical data between patients shedding very high hbov viral loads (.10 8 copies/ml of npa) and those shedding lower loads, evidenced significantly higher frequencies of diarrhea (p,0.01) in patients shedding higher viral loads (table 2) . remarkably, detection of other respiratory viruses simultaneously with hbov was significantly less frequent in samples from patients shedding very high loads of hbov (p,0.01) ( table 2) . logistic regression analyses confirmed that diarrhea (or = 6.53; 95% ci = 1.41-30.26; p = 0.02) were directly related, whereas simultaneous detection of other viruses in the npas was inversely related (or = 0.01; 95% ci = 0.00-0.13; p,0.01) to the shedding of very high hbov loads. the association of shedding very high loads of hbov (.10 8 copies/ml) with lack of co-detection of other respiratory viruses suggested that such high viral loads may result from early acute hbov infections with active viral replication, as opposed to long term shedding unrelated to current clinical symptoms. in order to confirm this hypothesis, an assay was developed to detect hbov vp1 mrna in npas by real time rt-pcr done on total rna extracts previously treated with dnase i. as an internal control, real time rt-pcr was done for the housekeeping gene of b-actin. when simultaneously positive for both hbov vp1 and bactin mrnas, this assay was considered a marker of active hbov replication. positivity for hbov vp1 or b-actin by pcr done directly on dnase-treated material without previous reverse transcription were considered indicative of remaining genomic dna after incomplete dnase treatment, and samples were discarded. of the 48 samples positive for hbov, 40 had sufficient remaining volume left for testing. of those, only 10 (25.0%) tested positive for vp1 mrna and, remarkably, 9 of these had very high hbov loads of greater than 10 8 copies of dna/ml. the median hbov viral load in patients shedding vp1 mrna was 8.85 6 10 9 copies/ml, which was significantly higher than that in patients negative for vp1 hbov mrna, 3.82 610 5 copies/ml (p,0.01) (figure 2b , table 3 ). the association of high viral loads importantly, a significant inverse association was noted between shedding of hbov vp1 mrna and having another respiratory virus detected in the npa. four of 10 (40.0%) patients who shed hbov vp1 mrna also had another respiratory virus detected in their npas, whereas 28 of 30 (93%) of the patients in whose samples shedding of vp1 mrna was not present, had another respiratory virus detected in their npas (p,0.01) ( table 3) . such inverse association was confirmed by logistic regression analysis (or = 0.05; 95% ci = 0.01-0.32; p,0.01). a comparison of clinical features between hbov patients with and without shedding of vp1 mrna revealed an association of active viral replication with diarrhea (or = 6.00; 95% ci = 1.15-34.14; p = 0.04) and a clinical diagnosis of urti (or = 19.33; 95% ci = 1.82-294.96; p = 0.01). the frequencies of diarrhea and diagnosis of urti in patients shedding vp1 mrna were 40%, while in patients without detectable vp1 mrna they were respectively 10% and 6.6% (table 3 ). the results of this cross-sectional study of hbov in ari patients from ribeirão preto, brazil, indicate that shedding of vp1 mrna in respiratory secretions, as a marker of hbov replication, correlates positively with high viral load, presence of diarrhea, and lack of co-infection by other respiratory viruses. during the last five or six years since its discovery, hbov has been detected in association with respiratory and gastrointestinal infections, mostly of children, in many countries in all continents [2, 24] . rates of detection of hbov in ari studies have varied from 1.5% to 19.0%, depending on patient age, case definition, and geographic location [2, 24] . in the present study, the overall detection rate of hbov in ari patients was 4.8%, similar to rates in other regions of the world. however, it should be kept in mind that this study, like most others published on hbov, was a hospital-based surveillance, subject to selection bias. therefore, rates of hbov in ari in the community at large could be different. to the best of our knowledge, this has been the largest study of hbov in ari conducted in brazil and south america. a few previous studies conducted in the region reported hbov infection rates of 6.0% to 10.7% in patients with ari [9, 25, 26, 27] and in 2.0% of patients with gastroenteritis [15] . importantly, the present study revealed a significantly higher rate of concurrent diarrhea in patients with ari positive for hbov as compared to those with ari but without detectable hbov, suggesting a causative role for hbov in gastrointestinal manifestations observed in ari patients. in general, parvoviruses replicate more efficiently in tissues with high cellular turnover, such as respiratory and digestive epithelia [28] . several members of the family parvoviridae are known to cause gastroenteritis, such as canine parvovirus (cpv), feline panleukopenia virus (fpv) and canine minute virus (cnmv). in addition studies previously published by others have reported hbov detection in stools from patients with gastroenteritis [29] . although the mechanisms of pathogenesis and routes of infection of hbov are not yet understood, some information is available for bovine parvovirus (bpv), another member of the genus bocavirus. bpv causes respiratory and enteric diseases in calves following initial replication in the tonsils and intestinal epithelium, from where it spreads into the blood, lymphoid tissues and respiratory epithelium. bpv induces atrophy of microvilli in the small intestine and death (by apoptosis and necrosis) of lymphoid cells and ciliated cells of the respiratory epithelium [28] . it is interesting that novel species of hbov (hbov 2, 3 and 4) associated with gastrointestinal infections have recently been identified [3, 4, 5] and two of those have been detected in stools from brazilian patients with acute diarrhea, at rates varying from 0.6% for hbov 3 to 20.8% for hbov 2 [6] . however, a causative role for hbov in human diarrhea still awaits confirmation [30] . interestingly, in this study a trend was noted for an increase in respiratory infections, positivity for hrsv and hbov, in association with drier and cooler months. while the reasons for this are not entirely understood, one possibility is that the increased respiratory virus activity in march and april could be favored by the initiation of school term one or two months before, when assembly of susceptible children could provide conditions for viral spread. the frequency of detection of hbov in 2005 was greater than three times that observed in the two following years, evidence that circulation of this agent in this region of brazil may vary greatly from year to year, in a way similar to that previously documented in italy [31] . even though hbov has been regarded as an infectious agent present worldwide, its pathogenic role in respiratory illness is still debatable. this virus is frequently detected in co-infection with other respiratory viruses of well-established pathogenic role [2, 24] . in addition, hbov is fastidious to propagate in conventional cell cultures and an experimental infection model is lacking, what limits advances in understanding pathogenesis. therefore, the search for an association of hbov infections with possible molecular markers of active viral replication is a useful approache to investigate pathogenesis of this emerging virus. in the present study, hbov dna was detected by pcr in samples from ari patients, but not in nasopharyngeal secretions collected from asymptomatic children approximately 5 years before (1999) (2000) in the same hospital. the significance of this finding is limited by the relatively small number of control samples available for testing, and by the fact that they were collected years before. however, the lack of detection of hbov dna by a sensitive pcr assay in samples collected from children carefully scrutinized for the presence of respiratory symptoms, adds support for a pathogenic role of hbov in ari, in agreement with previous studies [20, 32, 33] . high frequencies of co-infection by other respiratory viruses in patients with ari and hbov were confirmed in this study with a rate of 72.9% (35 of 48) of co-infections, mostly caused by hrsv, hrv, hmpv and hadv, and frequently by more than one additional agent. this was similar to the 75% reported by allander et al., in sweden [10] , and higher than the 50% reported by esposito et al., in italy [34] . while viral co-infections have long been recognized in ari studies, the advent of highly sensitive pcr assays magnified their observation. in the particular case of hbov, frequencies of co-infections have consistently been reported to be higher than those observed for other respiratory infections, reaching rates similar to those reported for hadv [35] , a tendency confirmed by the present study. yet unproven, this may suggest that long term shedding of hbov dna, unrelated to current illness may regularly occur, as it is known to happen with hadv [36] . indeed, hbov dna was found by pcr in lymphocytes from palatine tonsils of 32% of children undergoing tonsillectomy [18] , suggesting that this virus can establish latent or persistent infection in pharyngeal lymphoid tissue. another important piece of information befitting a systemic nature of hbov infections is the finding of viremia, reported by tozer et al. in children with ari [37] . viremia was also reported by allander et al [10] in hbov-exclusive infections, a feature strongly suggestive of a causal role for this agent in acute symptomatic infections. in addition, those authors reported that patients with hbov without concurrent infections by other respiratory viruses, tended to shed higher hbov loads [10] . in this regard, an association of high viral loads in npas with hbov acute infections, as evidenced by igm seroconversion in wheezing children, was clearly documented in a study conducted in finland by söderlund-venermo [38] . these results prompted us to determine hbov loads by qpcr in all hbov-positive npas of the study. the results revealed hbov viral loads to be significantly higher in patients without other viral co-infections than in those with multiple viruses detected. moreover, rates of diarrhea were significantly higher in patients with very high hbov viral loads in their npas. such finding, in the absence of respiratory viral coinfections, suggests a causal role for hbov in the genesis of the diseases presented by these patients. nevertheless, detection of a direct evidence of active replication of hbov in the secretions from patients was required to strengthen the causality link between the virus and disease. in order to do that, an assay to detect hbov vp1 mrna by real time rt-pcr in nasopharyngeal samples was developed. production of vp1 mrna is unequivocal evidence of hbov replication, but its shedding in nasopharyngeal secretions does not indicate the possible sources of virus production. because rna is very highly unstable in ribonuclease-rich respiratory secretions, one can speculate that vp1 mrna more likely originates from disruption of sloughed whole hbov-infected epithelial cells directly into the rnaseinhibiting extraction reagent. unfortunately, preparation of cytology slides that would have enabled detection of hbov on sloughed cells by in situ hybridization was not possible given the nature of the study. the presence of extremely high viral loads of hbov in secretions, argues in favor of abundant sources of virus, either on or adjoining the mucosal surfaces. this observation alone highlights the extremely productive replication levels reached by hbov in the permissive upper respiratory tract. however, in situ studies will be needed to provide definitive localization of viral replication sites. to the best of our knowledge this is the first report of an assay for mrna to confirm replication of hbov in clinical samples, and its application revealed that shedding of vp1 mrna in nasopharyngeal secretions was associated with high viral loads, presence of concurrent diarrhea, and lack of demonstrable coinfections by other respiratory viruses. conversely, in the absence of a marker of current viral replication, hbov was also often detected in nasopharyngeal secretions, but in much lower viral loads and frequently associated with shedding of other pathogenic respiratory viruses. it should be pointed out that vp1 mrna was found only in 25% of the hbov infected ari patients, with a significant part of whom with diarrhea. this may be interpreted to mean that only one quarter of the hbov respiratory infections are associated with genuinely acute viral replication, likely involving the gastrointestinal tract. conversely, in most cases of hbov detection in respiratory secretions, it may be the result of viral persistence/latency, with co-detection of another viral agent. a retrospective cross-sectional study was carried out with nasopharyngeal aspirates (npas) collected from 1015 patients with ari (535 males and 480 females) with 1 month to 66 years of age (median age of 8 months). the samples were from outpatients and inpatients brought for health care at the hospital of the university of são paulo school of medicine, ribeirão preto, sp, brazil, from january 2005 through december 2007. the study included all samples from patients with symptoms of ari seen at any of the hospital clinics and wards, submitted to the virology laboratory for testing. the vast majority of the samples had been originally sent to the laboratory for respiratory syncytial virus and influenza screening and no exclusion criteria were applied. clinical information obtained retrospectively from patient records upon discharge was entered on study forms. clinical data collected included the presence of wheezing, cough, fever, sneezing, dyspnea, coryza, nasal obstruction, diarrhea, requirement for oxygen therapy and length of hospitalization. npas were collected in saline solution from all patients [9] and sent on ice to the virology laboratory within four hours. in addition to the 1015 samples from ari patients, a convenience set of 50 control samples from patients without any respiratory symptoms who were participants in a previous study [23] were used as negative controls. this group consisted of 26 males, with median age of 19.5 months, mean of 37 months (sd = 44 months). after routine virology sample processing, remaining samples were split into backup aliquots, including two in trizol reagent (invitrogen carlsbad, ca), which were kept in storage at 270uc [9] . this study was approved by the internal review board of the hospital das clínicas de ribeirão preto (hcfmrp-usp) under approval number 4856/2004. all patients or the parents or guardians of the patients in the study signed an informed consent form. illnesses presented by all patients in this study were classified as lower respiratory tract infections (lrti) and upper respiratory tract infections (urti), with or without acute otitis media (aom) and gastro-esophageal reflux disease (gerd). patients with urti had only symptoms of the upper respiratory tract, such as coryza, sneezing, nasal obstruction and sore throat, without wheezing, and dyspnea, with or without fever. patients with lrti had diagnosis of bronchiolitis, bronchiolitis obliterans, bronchodysplasia or pneumonia. they had wheezing and/or dyspnea, with or without fever, cough, coryza, sneezing or nasal obstruction. all patients were assigned to a single clinical definition, and could not be allocated to more than one group. in order to assess disease severity, a numeric index of clinical severity (ics) was adapted from walsh et al (1997) [39] , based on the presence of clear-cut criteria that indicate higher risk for severe disease: wheezing, requirement for hospitalization, oxygen therapy and positive airway pressure. for all patients the ics was obtained by attributing 1 point each for the presence of wheezing, requirement for hospitalization, length of hospital stay longer than 5 days, requirement for o 2 , and use of o 2 for longer than 5 days; and 2 extra points attributed in case of requirement for positive airway pressure, for a maximum ics equal to 7. dna and rna were extracted from 250 ml of npa using, respectively, wizardh genomic dna purification kit (promegah, madison, wi, usa) and trizolh (invitrogenh, carlsbad, ca, usa), following manufacturers protocol. detection of hbov and other respiratory viruses was conducted by conventional pcr, as previously described [9] . for detection of hbov and human adenovirus (hadv), 100 ng of extracted dna was used, whereas for other respiratory viruses, including human respiratory syncytial virus (hrsv), human metapneumovirus (hmpv), human rhinovirus (hrv), human influenza virus (flu), human parainfluenza viruses (hpiv) and human coronaviruses (hcov), pcr was done with cdna generated by reverse transcription (rt) of 1 mg of rna, primed with 10 pmol of random hexamers, using improm-ii reverse transcriptase (promega, madison, wi) following manufacturer's instructions. prior to reverse transcription, rna was denatured at 95uc for 5 minutes, then immediately placed on ice while reverse transcriptase was added and then incubated at 42uc for 2 hours. pcr was done with150 ng of dna, 10 pmol forward and reverse primers (table s1 ), 50 mm mgcl 2 , 10 mm deoxynucleoside triphosphate (dntp), 2.5 ml of supplied 10 x buffer, 1u of taq dna polymerase (invitrogen carlsbad, ca) and water to complete the volume of 25 ml. the cycling conditions were denaturation at 95uc for 5 min, followed by 35 cycles of 94uc for 1 min, 48uc to 54uc for 1 min, 72uc for 2 min, and then a final extension step of 72uc for 10 min [9] . all pcr products were analyzed by electrophoresis in ethidium bromide-stained 1.5% agarose gels. pcr products of all tested viruses were cloned into the plasmid pgem-t easy (promega, madison, wisconsin) and used to determine detection limits for each assay. all clones were sequenced with bigdye terminator v3.1 cycle sequencing (applied biosystems, foster city, ca) using the abi prism 3100 genetic analyzer. the detection limits for the pcr assays were determined by testing serial decimal dilutions of the clones, and results varied from 1 to 100 copies of plasmid dna. the limit of detection for hbov was determined to be 40 copies. all applicable measures to prevent contamination of pcr reactions were taken in this study. the quantitative pcr (qpcr) for hbov was targeted to the viral np1 gene according to previously published procedures [40] . briefly, the reaction was performed in triplicate in a final volume of 10 ml, containing 150 ng of template dna, 5 ml of taqman universal pcr master mix (applied biosystems foster city, ca), 300 nmol/l of each primer and 150 nmol/l of probe (table s1 ). the amplification was performed on a 7300 real time pcr system (applied biosystems foster city, ca) with the following settings: 50uc for 10 min, 95uc for 3 min and 45 cycles of 95uc for 15 s and 60uc for 1 min. a standard curve of amplification was produced using serial decimal dilutions of a plasmid (pgem-full4hbov) in which a fragment of the hbov np1 gene (2270 nt to 3280 nt) was cloned. a positive qpcr for hbov was considered when the threshold was reached before the 40 th cycle with fluorescence count higher than 0.5. the detection limit was 4.6 copies of hbov plasmid, the slope and the r2 of the standard curve were 23,386 and 0,994, respectively. all qpcr results were normalized by the amplification of the b-actin gene, included in duplicate in all tested batches, using 300 nmol/l of each primer and 150 nmol/l of the probe (table s1 ). with this approach, viral loads were determined as the number of copies of hbov dna per ml of npas. shedding of hbov vp1 mrna was tested by real-time rt-pcr in npas to ascertain the presence of active viral replication, as opposed to shedding of viral genome. to ensure target-specific amplification total rna extraction products were treated with dnase i (invitrogen carlsbad, ca) 2 h prior to pcr. as a control, the same pcr was done in parallel directly with the rna extraction product without previous rt. samples were considered positive for hbov vp1 mrna only when they were simultaneously negative for amplification directly from the extracted rna without previous rt. rt was performed on 1 mg of rna extracted by trizol using10 pmol of random primers, according to the protocol provided by the manufacturer. after rna denaturation at 95uc for 5 min reverse transcriptase was added and incubation went on for 2 h at 42uc. pcr was then carried out with 150 ng of cdna, with 300 nmol/l of each primer (vp1-f and vp2-r), 150 nmol/l of the probe (vp1-s, table s1), and 5 ml of taqman universal pcr master mix (applied biosystems foster city, ca). amplification was done using the 7300 real time pcr system (applied biosystems foster city, ca) with the following settings: 50uc for 10 min, 95uc for 3 min and 45cycles of 95uc for 15 s and 60uc for 1 m. all samples, including all cdnas and rnas pre-treated with dnase, were tested by qpcr for b-actin mrna, following the protocol described above. samples found to be positive for b-actin by amplification directly from the rna not pre-treated with dnase, were considered to be false positives. as above mentioned, positive reactions were considered when the threshold was reached before the 40 th cycle, with a fluorescence higher than 0.5. the detection limit of this assay was found to be 3 copies of dna, as determined using the decimal serial dilutions of viral cdna obtained from in vitro transcription of a plasmid containing thevp1 gene, according to manufactures' instructions (riboprobe in vitro transcription systems, promega madison, wi). correlation between positivity for hbov and clinical conditions was assessed by the chi-square test. statistical analysis was conducted using the bioestat 3.0 software (cnpq, brazil). logistic regression was done to examine the relationship of hbov shedding (including positivity for hbov dna, hbov loads, lack of other viral co-infections and detection of hbov vp1 mrna) and clinical data adjusted for age, gender and detection of hrsv in the same samples [41] . to analyze the hbov loads obtained by qpcr, data in two unpaired groups of patients were compared with the mann-whitney test. these analyses were performed using the sash 9.0software (sas institute inc, raleigh, nc). a p value of #0.05 was chosen for significance. table s1 primers and probes used in conventional and real-time pcr for respiratory viruses and b-actin. (doc) cloning of a human parvovirus by molecular screening of respiratory tract samples human bocavirus: passenger or pathogen in acute respiratory tract infections? a novel bocavirus associated with acute gastroenteritis in australian children human bocaviruses are highly diverse, dispersed, recombination prone, and prevalent in enteric infections a newly identified bocavirus species in human stool human bocavirus species 2 and 3 in brazil human bocavirus infection human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand human bocavirus respiratory infections in children human bocavirus and acute wheezing in children isolation of human bocavirus from swiss infants with respiratory infections viral etiology of acute respiratory infections with cough in infancy: a communitybased birth cohort study human bocavirus in febrile children epidemiological profile and clinical associations of human bocavirus and other human parvoviruses human bocavirus infection in children with gastroenteritis detection of human bocavirus in children hospitalized because of acute gastroenteritis human bocavirus infections in hospitalized children and adults human bocavirus in tonsillar lymphocytes human bocavirus can be cultured in differentiated human airway epithelial cells frequent and prolonged shedding of bocavirus in young children attending daycare frequent detection of viral coinfection in children hospitalized with acute respiratory tract infection using a real-time polymerase chain reaction monitoramento climático anterior risk factors for wheezing in a subtropical environment: role of respiratory viruses and allergen sensitization the human bocaviruses: a review and discussion of their role in infection novel respiratory virus infections in children human bocavirus in very young infants hospitalized with acute respiratory infection in northeast brazil hospitalacquired human bocavirus in infants animal bocaviruses: a brief review human bocavirus, a respiratory and enteric virus human bocavirus in children hospitalized for acute gastroenteritis: a case-control study human bocavirus in italian patients with respiratory diseases human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus low de high prevalence of asymptomatic bocavirus in daycare: is otitis media a confounder? impact of human bocavirus on children and their families detection of a broad range of human adenoviruses in respiratory tract samples using a sensitive multiplex real-time pcr assay transmission dynamics and prospective environmental sampling of adenovirus in a military recruit setting detection of human bocavirus in respiratory, fecal, and blood samples by realtime pcr clinical assessment and improved diagnosis of bocavirus-induced wheezing in children severity of respiratory syncytial virus infection is related to virus strain realtime pcr for diagnosis of human bocavirus infections and phylogenetic analysis on the existence of maximum likelihood estimates in logistic regression models the authors thank the expert technical support of maria lucia silva, ariane matioli saranzo and luana sella delcaro. we thank also the staff responsible for the collection of the clinical samples on hcrp and the ''centro de métodos quantitativos'' (cemeq-fmrp), particularly mayara piani luna da silva sicchieri, for the statistical analysis and suggestions. key: cord-340387-ohkjheat authors: wynne, james w.; di rubbo, antonio; shiell, brian j.; beddome, gary; cowled, christopher; peck, grantley r.; huang, jing; grimley, samantha l.; baker, michelle l.; michalski, wojtek p. title: purification and characterisation of immunoglobulins from the australian black flying fox (pteropus alecto) using anti-fab affinity chromatography reveals the low abundance of iga date: 2013-01-07 journal: plos one doi: 10.1371/journal.pone.0052930 sha: doc_id: 340387 cord_uid: ohkjheat there is now an overwhelming body of evidence that implicates bats in the dissemination of a long list of emerging and re-emerging viral agents, often causing illnesses or death in both animals and humans. despite this, there is a paucity of information regarding the immunological mechanisms by which bats coexist with highly pathogenic viruses. immunoglobulins are major components of the adaptive immune system. early studies found bats may have quantitatively lower antibody responses to model antigens compared to conventional laboratory animals. to further understand the antibody response of bats, the present study purified and characterised the major immunoglobulin classes from healthy black flying foxes, pteropus alecto. we employed a novel strategy, where igg was initially purified and used to generate anti-fab specific antibodies. immobilised anti-fab specific antibodies were then used to capture other immunoglobulins from igg depleted serum. while high quantities of igm were successfully isolated from serum, iga was not. only trace quantities of iga were detected in the serum by mass spectrometry. immobilised ligands specific to iga (jacalin, peptide m and staphylococcal superantigen-like protein) also failed to capture p. alecto iga from serum. igm was the second most abundant serum antibody after igg. a survey of mucosal secretions found igg was the dominant antibody class rather than iga. our study demonstrates healthy p. alecto bats have markedly less serum iga than expected. higher quantities of igg in mucosal secretions may be compensation for this low abundance or lack of iga. knowledge and reagents developed within this study can be used in the future to examine class-specific antibody response within this important viral host. bats represent approximately one fifth of the world's mammalian species and are among the most diverse and geographically dispersed mammals. frugivorous and nectivorous pteropid bats (family pteropodidae, suborder megachiroptera) constitute approximately 170 species and are known colloquially as fruit bats or flying foxes. their capacity for long-distance dispersal through flight has undoubtedly contributed to their widespread distribution throughout tropical and sub-tropical asia and australia, and on islands of the indian and western pacific oceans. the black flying fox, pteropus alecto, is distributed mainly across the northern and eastern coasts of australia [1] . since 1994, this species has gained particular attention after it was identified as the natural host of hendra virus (hev) [2, 3] . a body of evidence exists implicating bats as a major source of zoonotic viruses [4] [5] [6] . bats have been shown to harbour and disseminate highly pathogenic viruses including ebola, sars-like coronavirus and the henipaviruses (hev and nipah) [4] . spillover events from bats to other species -including humans -have increased. in 1998, a spillover event of nipah virus from pteropid bats to farmed pigs caused a major disease outbreak in malaysia. more than 1 million pigs were culled, and of the 265 reported human cases, 105 were fatal [7, 8] . while the incidence of hev in australia has been more sporadic, an increase in spillover events from pteropid bats to horses has occurred. between 1994 and 2010, 14 hev outbreaks occurred in australia involving 48 confirmed equine cases (75% fatality rate) and seven human cases (60% fatality rate). in contrast, between june 2011 and january 2012, 19 hev outbreaks occurred [9] . the increasing incidence of hev, combined with its high fatality rate, means hev has the potential to cause further significant disease outbreaks in australia and abroad. recently, a novel paramyxovirus that is closely related to the henipaviruses, named cedar virus, was isolated from fruit bats in australia [10] . however unlike hev, cedar virus caused no clinical disease in experimentally infected ferrets and guinea pigs [10] . evidence of henipavirus infection in multiple bat species has also been identified in continental africa [11] . despite the risk of zoonotic diseases originating in bats, little is known regarding the bat immune system and very few diagnostic reagents are commercially available. despite their ability to carry highly pathogenic viruses such as hendra, nipah and ebola, bats appear asymptomatic and rarely show signs of disease following both experimental and natural infection with these agents [12] [13] [14] [15] [16] . the notable exceptions are rabies-like viruses, which are capable of causing clinical disease in bats and humans [17, 18] . anecdotal observations raise the possibility that the bat immune system may have atypical and possibly unique characteristics that permit asymptomatic virus persistence and spillover events. immunoglobulins, namely igg, iga and igm, are important and diverse secretory components of the mammalian immune system. antibodies of the igg class are the most abundant immunoglobulins in mammalian serum and consequently provide the majority of immunity against blood-borne infectious agents [19] . in contrast, iga is most abundant in mucosal secretions including milk, tears, and secretions of the upper respiratory tract and digestive tract. the abundance of iga in normal human serum is approximately one fifth of the igg level and accounts for about 15-20% of total immunoglobulins in serum [20] . in humans, serum iga is predominantly monomeric whereas iga within mucosal secretions is found as a dimer complex. polymerisation of the iga (and igm) is facilitated by the joining (j) chain which is expressed by immunocytes of various secretory tissues [21] . polymeric iga is secreted into the mucosa through its interaction with the polymeric ig receptor (pigr) which is expressed at the basolateral surface of secretory epithelial cells. the iga/pigr complex is transported via transcytosis to the cell surface where it is ultimately secreted in to the lumen [22] . mammalian iga plays a crucial role in mucosal immunity while in serum it interacts with the iga fc receptor (fcar) to control the inflammatory response [20] . indeed, in the absence of antigen, iga down-regulates igg mediated phagocytosis, chemotaxis, cytokine release and oxidative burst activity [23] [24] [25] [26] . igm is the first antibody produced following microbial invasion and exists predominantly as either a monomeric antigen receptor on b cells or as a soluble pentamer. igm can also exist as tetramer and dimers, however these forms are significantly less abundant. in healthy humans serum igm is slightly less abundant compared to serum iga. variation in the nature of n-linked glycosylation provides further diversity to immunoglobulins. these glycans play a vital role in maintaining structural integrity and in some cases act as ligands for immunologically important serum lectins such as mannan-binding lectin [27] . few studies have examined the immunoglobulin system of bats. recently baker et al. [28] sequenced mrna encoding the heavy chains of igg, igm and iga of p. alecto, revealing a diverse repertoire of variable heavy chain transcripts. transcripts resembling ige have also been indentified in different bat species, while igd appears specific to only insectivorous bats [29] . wild-caught bats are certainly capable of generating neutralising antibodies to viruses such as hendra, ebola and sars-like coronavirus [2, 30, 31] , but early studies have demonstrated pronounced differences in both the magnitude and duration of antibody responses in bats compared to other mammals [32] [33] [34] . the large frugivorous bat, p. giganteus, demonstrated a delayed primary antibody response following immunization with sheep red blood cells [32] . similarly, the magnitude and duration of the neutralising antibody response of the big brown bat (eptesicus fuscus) to the model antigen phix174 bacteriophage was lower than that of rabbits and guinea pigs [33] . the likelihood that bats have an atypical immunoglobulin response compared to other animals requires further attention. specifically, the abundance, tissue distribution and post-translational modification of the different immunoglobulin classes must be examined. given the importance of bats as viral reservoirs, understanding the bat immune system may facilitate development of methods to mitigate spillover events in the future. as a first step, this study aimed to purify and characterise the major immunoglobulin classes from healthy p. alecto biological specimens. considering that in other mammalian species, immunoglobulins igg, igm and iga are present in relatively high abundance in serum and tissues, we anticipated that bats would possess a similar immunoglobulin profile. however, while igg and igm appeared abundant in p. alecto serum, iga was not. iga was detected in the mucosal secretions of the small and large intestine lavages, milk and tears. diverse isoforms of igg and igm, suggestive of multiple subclasses, were identified. reagents developed within this study will aid future studies of this unique immunoglobulin repertoire, particularly in response to viral infection. all animal experimentation and sample collection was conducted following guidelines approved by the aahl animal ethics committee (permit no. 1302). p. alecto bats were captured in southern queensland, australia as described previously [35] and transported live by air to the csiro australian animal health laboratory (aahl). the animals were bled for serum and plasma and then euthanized for dissection of tissues. tissues were stored at 280uc in rnalater (ambion) for rna analysis or snap frozen in liquid nitrogen for downstream mass spectrometry (ms) analysis. lungs, small and large intestines were washed with 15-20 ml of cold phosphate buffered saline (pbs). washes (lavages) and tissues were stored at 280uc. faeces samples were collected from bat cages within 1-2 hours of being excreted and immediately resuspended in pbs containing protease inhibitors as previously described [36] . where indicated, serum was extracted from plasma according to the protocol described by salvador-morales et al. [37] . all chemical reagents were of the highest analytical grade available from merck, unless otherwise specified. the purification strategy employed in this study exploited the molecular characteristics of isotypes of mammalian immunoglobulins with the assumption that bat immunoglobulins would resemble those of other mammals. serum and plasma samples from p. alecto bats were used as a source of immunoglobulins and human serum was used as a control. immobilised proteins a, g and l were used in this study to capture igg from p. alecto serum. fab fragments, derived by papain digestion of purified p. alecto igg, were used to generate fab-specific antibodies in rabbits. in turn, immobilised anti-fab-specific antibodies were employed to capture remaining p. alecto immunoglobulins, from igg-depleted bat serum (fig. 1) . the final separation of igm from iga was attempted by size exclusion chromatography (sec) exploiting significant molecular mass difference between the two molecules or by reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) separation of respective heavy chains. the identity of separated proteins was determined by comparing tandem mass spectrometric data with available protein sequence databases. isolation of igg from human, rabbit and p. alecto sera affinity chromatography purification of igg from mammalian sera using immobilised protein a, g or l was performed according to a method modified from that described by bjorck and kronvall [38] . protein a and protein g affinity columns (ge healthcare) or protein l affinity columns (thermo) were connected to a 280 nm uv detector and a chart recorder. serum samples were diluted 5-fold in binding buffer (20 mm phosphate buffer, ph 7.4) and loaded on to columns equilibrated with the binding buffer at a flow rate of 0.5 ml/min. columns were washed with 10 volumes of binding buffer and igg was eluted in elution buffer (100 mm glycine hcl, ph 2.7). collected fractions were immediately ph neutralised with 1 m tris-hcl, ph 9.0. protein concentration was determined using the micro bca protein assay (thermo) and protein samples were analysed by reducing sds-page. digestion of purified p. alecto igg with papain purified p. alecto and rabbit igg samples (1 mg) were diluted with pbs and concentrated 5-fold to 200 ml using 4 ml ultra-4 centrifugal filter devices with a 5 kda molecular weight cut-off (mwco) membrane (amicon). the concentrate was diluted with 100 mm sodium acetate buffer, ph 5.8, containing 50 mm cysteine and 0.5 mm ethylenediaminetetraacetic acid (edta). fifty microlitre aliquots were digested with increasing concentrations of papain (ranging from 0.01 to 0.05 mg/ml or 1:100 to 1:20 enzyme to substrate ratios, respectively) over different incubation periods (6 to 22 h) at 37uc. the reactions were stopped by addition of 30 mm iodoacetamide (sigma) and incubated at room temperature for 30 min. the extent of the digestion was assessed by reducing sds-page. the optimal digestion conditions obtained for bat igg (1:20 enzyme to substrate ratio, 18 h) were used for digestion of 20 mg of igg in scaled-up experiments. following large scale digestion, the papain digest was concentrated 5-fold to 200 ml using ultra-4 centrifugal filter devices with a 5 kda mwco membrane and buffer exchanged with protein g affinity chromatography binding buffer. this fraction contained igg fragments (fab and fc) and was diluted 25-fold with the binding buffer prior to fractionation on an immobilised protein a affinity column. p. alecto igg fc fragments bound to the column whilst fab fragments were not retained by the column. the fab and fc fraction was tested for purity by reducing sds-page and used to immunise rabbits to generate fab-specific antiserum. the igg fraction of this rabbit antiserum was obtained by purification on an immobilised protein a affinity column as described above. the purified p. alecto fab fragments (5 mg) obtained by digestion of p. alecto igg with papain were concentrated and buffer exchanged with 0.5 m nacl in 0.2 m sodium bicarbonate buffer, ph 8.3, using amicon ultra-4 centrifugal filter devices. coupling to a 1 ml hitrap nhs-activated hp column (ge healthcare) was accomplished according to the manufacturer's instructions. rabbit anti-fab antibodies were purified by affinity chromatography by applying the igg fraction to the immobilised-fab column using the procedure described for the purification of igg. the purified antibodies, designated anti-fab-specific antibodies (4 mg), were coupled to a hitrap nhs activated hp column as described above. igg-depleted p. alecto serum and plasma samples were obtained by repeated (2-3 times) affinity chromatography on immobilised protein g affinity columns. the flow through was assessed for the absence of igg by reducing sds-page and western blot analysis with protein g or whole p. alecto anti-igg rabbit serum as detecting agents. igg-depleted samples were fractionated by affinity chromatography on immobilised anti-fab-specific antibodies adopting the same procedure as that described for immobilised protein a and g except that the binding and washing buffer consisted of 0.3 m nacl in 50 mm phosphate buffer, ph 7.4. all fractions were assayed for protein concentration and analysed by reducing sds-page. affinity chromatography media reported to specifically interact with mammalian iga, including jacalin [39] , peptide m [40] and staphylococcal superantigen-like protein 7 (ssl7) [41] , were also used in attempts to purify p. alecto iga from serum, plasma and tissue lavages. the affinity procedures were first tested on human serum and plasma samples and proved suitable for isolation of human iga. the efficiency of purification steps was assessed by reducing sds-page and mass spectrometry. affinity-purified igg, fab and fc fragments from papain digests, and sec-purified igm fractions (250 mg) were separated by preparative reducing sds-page using large well 4-12% gradient bis-tris gels (invitrogen, carlsbad, ca, usa) under reducing conditions. the protein bands corresponding to heavy chain subunits of immunoglobulins were electroeluted from the gel using a bio-rad mini whole gel eluter according to the manufacturer's instructions with 0.01% sds, 60 mm tris, 40 mm n-cyclohexyl-3-aminopropanesulfonic acid (caps), ph 9.5, as eluting buffer or by passive elution overnight in pbs buffer containing 0.1% sds [42] . the eluted fractions were analysed by reducing sds-page, pooled and used for production of specific antiserum in rabbits. purified antigens (50 mg igg; fc fragment; fab fragment; igm h and igg h ) were emulsified with csiro triple adjuvant (montanide/quil a/deae-dextran; [43] ) to a final volume of 500 ml and injected intramuscularly at two sites at approximately 3 weekly intervals for 10 weeks. protein fractions were analysed using reducing sds-page under reducing conditions in precast gels (4-12% bis-tris gels in a mops/sds buffer or in 12% gels in tris-glycine/sds, invitrogen). protein bands were visualized after staining with either coomassie brilliant blue (cbb) or silver nitrate. serum samples (15 mg) were analysed by 2-de as described previously [44] . proteins were either visualized by silver nitrate or western transferred onto polyvinylidene fluoride (pvdf) membranes in 10 mm caps buffer, ph 11, containing 10% methanol for immunoblot analysis. membranes were blocked with 5% skim milk overnight at 4uc, then probed with specific rabbit antiserum diluted 1:10,000 or 1:20,000 followed by goat anti-rabbit igg horseradish peroxidase (hrp) conjugates diluted 1:20,000 or 1:40,000, respectively. visualization of reactive protein bands was achieved by enhanced chemiluminescence (ecl, ge healthcare) and exposure to x-ray film (fuji). samples of purified igg and igm from p. alecto and human sera were denatured with 0.25% triton x-100 (icn biomedicals) and treated with n-glycosidase f (pngasef, roche) or neuraminidase (from arthobacter ureafaciens, roche; relative rate of cleavage is a2r6.a2r3.a2r8) at 37uc overnight. samples were separated by reducing sds-page and western transferred onto low fluorescence pvdf transfer membranes (fluorotrans, pall) as described above and then blocked overnight in 50 mm tris-hcl, 150 mm nacl and 0.25% tween 20, ph 7.4 (tbs-t). after washing 3 times with tbs-t, membranes were incubated for 1 h with biotinylated lectins (vector labs burlingame, ca, usa): datura stramonium agglutinin (dsa; 1:3,000 dilution), galanthus nivalis agglutinin (gna; 1:3,000), maackia amurensis agglutinin ii (maa ii; 1:1,000), sambucus nigra agglutinin (sna; 1:30,000), peanut agglutinin (pna; 1:1,000) and ulex europaeus agglutinin i (uea i; 1:1,000). after lectin incubation, the blots were washed three times in tbs-t for 10 min and then incubated for 1 h with streptavidin-hrp conjugate (twice the dilution of lectin; dako, nsw, australia) in tbs-t followed by three 10 min washes, two in tbs-t then one in tbs. visualization of reactive protein bands was achieved by 5 min incubation with enhanced chemiluminescence substrate (ecl plus tm ) and scanning on a typhoon fla 9000 gel imaging scanner (ge healthcare). preparative sec was performed on a hrlc system (bio-rad) with uv monitoring of the eluate at 280 nm. the column used for separation was a bio-gel 40xl column (bio-rad) and the mobile phase consisted of 50 mm na 2 po 4 , 300 mm nacl, ph 7.4. eluted fractions were manually collected and subjected to analysis by reducing sds-page and mass spectrometry. purified proteins were analysed by mass spectrometry of bands excised from cbb-stained bands from reducing sds-page gels and subjected to in-gel digestion with trypsin [45] . resultant peptides were analysed by liquid chromatography tandem mass spectrometry (lc-ms/ms) using a surveyor high-performance liquid chromatography (hplc) system connected directly to the nano-spray ion source of an lcq classic quadrupole ion-trap mass spectrometer (thermo) as described previously [46] . identification of proteins was obtained using the turbosequest program [45] (v3.2) (thermo). ms/ms spectra of hplcseparated peptides from the in-gel tryptic digests was matched with theoretical mass spectra produced by in silico tryptic digestion of protein sequences from the ensembl pteropus vampyrus protein database (http://asia.ensembl.org/info/data/ftp/index.html) and published sequences [15] . search results were filtered by a single threshold where matches to peptides were reported only if the sequest cross correlation factor (xcorr) was .1.5 for a singly charged peptide ion, .2.0 for a doubly charged peptide ion or .2.5 for a triply charged peptide ion. furthermore, a protein was reported as identified only if 2 or more different peptide matches were found for that protein. total rna was extracted from lymph node, spleen, liver, lung, heart, kidney, small intestine, brain and salivary gland as previously described [47] . for each sample, 500 ng total rna was used as template in a 20 ml cdna synthesis reaction. quantitative pcr primers were designed using primer express 3.0 (applied biosystems) with default parameter settings. . qpcr reactions were preformed in triplicate as described previously [47] . copy numbers of target sequences were calculated using standard curves and normalised relative to 18 s ribosomal rna. purification of igg and production of antiserum. commercially available immobilised proteins g and a successfully captured whole igg from p. alecto serum and plasma. it appeared that p. alecto igg had greater binding affinity to protein g than to protein a ( fig. 2a and b) . washing removed significantly more igg from the immobilised protein a column compared to protein g. igg binding to protein l was negligible (data not shown). igg h was approximately 50-52 kda. a light chain immunoglobulin of approximately 25 kda was also visible in protein g purified fractions ( fig. 2a) . papain fragmentation of protein g purified igg revealed that the p. alecto immunoglobulin was significantly more resistant to protease cleavage compared to rabbit igg. the rabbit igg was completely digested after 12 h incubation (data not shown) while almost complete fragmentation of p. alecto igg could only be achieved after 18 h incubation with 5-fold higher enzyme concentration (fig. 2c) . following digestion, the fab and fc fragments were separated by protein a affinity chromatography. the fc (fcc) fragment was bound to the column and later eluted, whilst the fab fragment did not bind and was collected in the flow through fraction. this method isolated approximately 5.4 mg fab from 1 ml of serum. purified whole igg, igg h , fab fragments and fcc fragments were used as antigens for production of specific polyclonal antibodies in rabbits. igm enrichment by anti-fab affinity chromatography. immobilised anti-fab-specific antibody was used to capture non-igg immunoglobulins (igm and iga) from igg-depleted p. alecto serum and plasma samples. two major bands were detected by reducing sds-page in the eluate from both serum and plasma samples; a 66-70 kda band representative of igm h , and a 25 kda band representative of immunoglobulin light chain ( fig. 3a and 3b ). no band representative of iga h (predicted 52-55 kda from mrna sequence [28] ) was observed. to confirm the identities of these bands and others, the putative igm h band, its associated immunoglobulin light chain, and other minor bands (highlighted by arrows, fig. 3b lanes 1 and 3) were excised and subjected to lc-ms/ms (table 1) . bands 1 to 6 in serum and plasma contained protein corresponding only to cm, immunoglobulin light chain and/or j chain. however, band 4 in serum samples also contained sequence corresponding to ca. a similar result was obtained when the igm/iga enriched fractions from serum and plasma were pooled and subjected to sec ( fig. s1a and s1b). four fractions from each serum and plasma were analysed by reducing sds-page and lc-ms/ms. all fractions from plasma were found to correspond to cm, immunoglobulin light chain and/or j chain. one fraction from serum was also shown to correspond to ca (table s1 ). gel eluted igm h was used as antigen for production of specific polyclonal antibodies in rabbits. distribution and expression. the distribution of igg, igm and iga in healthy p. alecto was examined using lc-ms/ms analysis of gel-separated proteins from various tissue extracts and secretions (fig. s2) . no proteins of high molecular weight were detected in faeces (data not shown) and these samples were excluded from further analysis. igg h was detected in all samples with the exception of tears. in contrast, igm h was only detected in lung lavage while iga h was identified in the small and large intestine lavages, milk and tears ( table 2 ). due to the apparent low abundance of iga in tissues and secretions where it may be expected to be found, we examined the ighg, igha, ighm, igj and pigr mrna expression levels in ten tissues collected from three individual wild-caught p. alecto fruit bats by qpcr (fig. 4) . igha mrna was highly transcribed in the small intestine, lung and salivary gland. ighm was highly expressed in lymph node and spleen, and moderately transcribed in peripheral blood mononuclear cells (pbmc), small intestine and lung. igj and pigr were strongly expressed in small intestine, lung and salivary gland. brain, heart, kidney and liver expressed very low levels of all three mrnas. ighg was most abundant in the lymph node, spleen and pbmc. glycosylation. the glycosylation status of whole p. alecto igg and igm was assessed by treatment with glycosidases and lectin binding. human immunoglobulins were used as controls. electrophoretic analysis of immunoglobulin subunits following deglycosylation revealed greater glycan content associated with igm molecules than igg which was observed as a greater mass differential following deglycosylation ( fig. 5a and 5b ). this observation was confirmed by staining of igg and igm with a glycan specific stain (data not shown). following deglycosylation with pngasef, two bands of igm h were observed in p. alecto. it can be estimated from the mass differences that bat igg h and igm h have approximately 1-2 and 3-4 occupied glycosylation sites, respectively. glycan composition was analysed through lectin binding and revealed that the overall structure of igg and igm glycans was most likely n-linked di-antenary. reactivity with dsa and gna indicated the presence of 1,4-glcnac and 1,3-mannose component residues (fig. s3 ) and the reactivity with maa ii and sna indicated the presence of sialic acid residue(s), most likely in an a2r3 configuration (fig. s3c and s3d) . treatment with pna and uea i indicated the presence of galactose and fucose residues; however, these tests were less conclusive, possibly due to incomplete deglycosylation or nonspecific binding of the lectins. diversity of igg h and igm h isoforms. antiserum generated against igg h reacted predominantly with the two bands representative of igg h in p. alecto serum (fig. 6a ). the antiserum serum bands 1-8 from figure 3b are denoted ms1 to ms8, plasma bands 1-8 from figure 3b are denoted mp1 to mp8. doi:10.1371/journal.pone.0052930.t001 generated against igm h reacted predominantly with a single band representative of igm h in p. alecto serum (fig. 6a) . the anti-fab antiserum reacted only with immunoglobulin light chains (fig. 6a) . immunoblotting of 2-de separated p. alecto serum revealed the presence of a diverse range of igg h and igm h isoforms (fig. 6b, 6c and 6d) . igg h antiserum reacted with a variety of igg h isoforms ranging in pi from 5-9 (fig. 6d) . in contrast, the igm h antiserum reacted with a less complex igm h repertoire (fig. 6c) . reactivity of igg h and igm h antiserum. serum proteins for a number of species were separated by reducing sds-page (fig. 7a) . antiserum generated against p. alecto igg h showed reactivity to igg h of the fruit bats p. conspicillatus and, to a lesser extent, rosettus megaphylus (fig. 7b, lanes 2 and 3 respectively) . reactivity was also detected with horse igg h (lane 6). antiserum generated against p. alecto igm h reacted with p. conspicillatus and very weakly with ferret igm h (fig. 7c ). consistent with previous reports [39] [40] [41] , human iga was purified from igg-depleted human serum and plasma using immobilised ligands (jacalin, peptide m and ssl7) (data not shown). immobilised-peptide m and ssl7 bound no protein from p. alecto plasma and serum, and immobilised jacalin bound approximately 19 bands with varying molecular masses (fig. s4a, s4b, s4c) . protein fractions eluted from the jacalin column were analysed by reducing sds-page and 19 distinct protein bands were tested for the presence of iga by lc-ms/ms (fig. s4d) . none of these proteins contained peptides representative of iga (table s2) iga is the second most abundant immunoglobulin class in normal human serum, accounting for approximately 15-20% of the total immunoglobulin [20] . the immobilised anti-fab-specific antibody used within this study clearly enriched igm from igg depleted p. alecto serum. however, no visible enrichment of iga was observed. trace quantities of serum iga were detected only by lc-ms/ms. parallel experiments with anti-human fab antibody successfully enriched iga from human serum,. furthermore, affinity chromatography with ligands known to be specific to iga [39] [40] [41] failed to enrich this immunoglobulin from p. alecto serum. it must be kept in mind the approaches utilised in this study to isolate iga are based on the assumption that p. alecto iga would have similar characteristics to mammalian iga and the variable heavy chain regions would be shared between the major immunoglobulin classes. previous mrna sequencing studies of the p. alecto immunoglobulin repertoire would suggest this assumption is sound [28] . however, if p. alecto contains additional atypical iga-like molecules, our approaches may have failed to detect such variants. various iga-like equivalents have been identified across a range of species including teleost fish, amphibians and reptile [48] [49] [50] , highlighting the divergent evolutionary pathways that contribute to mucosal immunity. previous studies on a range of bat species found that the bat iga gene sequence more closely resembles human iga2. this similarity is due, in part, to the absence of a cysteine in the constant heavy 1 domain that is part of the covalent disulfide bond to the light chain [29] . the iga h sequence previously obtained for p. alecto is also missing this cysteine [28] . in humans, iga2 is the dominant iga subclass in mucosal secretions, whereas iga1 is more common in serum. if bats contain only a single iga2-like class, we may expect to find a higher concentration of iga in mucosal secretions compared to that found in serum. the apparent scarcity of serum iga led us to further investigate the abundance of iga in various mucosal secretions and tissues. lc-ms/ms analysis detected iga in the small and large intestine lavages, milk and tears. the absence of iga in the lung lavage and salivary gland may be the result of differential regulation of either iga itself or the j-chain or pigr. real-time pcr revealed both the igj and pigr transcripts co-localised with igha across most tissues. pigr and igj were most abundantly expressed in the small intestine where both iga transcript and protein was detected. in contrast the expression of pigr was markedly lower in the lung and salivary gland which may have contributed to the lower abundance of iga protein in these tissues. as expected ighg was the most highly expressed immunoglobulin mrna in the lymph node, spleen and pbmc. interestingly, while ighg mrna was not highly expressed in the mucosal tissues, lc-ms/ms revealed igg was the dominant immunoglobulin in these tissues and secretions. this result suggests the majority of igg protein within the mucosal tissues and secretions is not locally produced and is more likely transported to these sites. in humans, the transportation and regulation of igg across the mucosal epithelium is driven by the neonatal fc receptor [51] . the importance of mucosal igg in the defence against epithelium associated pathogens has been demonstrated previously and is now recognised as playing a key role in mucosal immunity along with iga [52] . the relative abundance of igg over iga within the milk of p. alecto may have implications for the transfer of material immunity. in humans the majority of igg is transferred to the fetus via the placenta and only trace quantities of igg are found in lacteal secretions [53, 54] . other species, such as cows, sheep and horses do not transfer igg across the placenta and the majority of igg, and a smaller proportion of iga, is provided to the newborn through the colostrum and milk [53, 54] . the data presented herein would suggest p. alecto, unlike humans and rabbits, transfers the majority of igg through the lacteal secretions rather than through the placenta. selective iga deficiency is a common condition in humans affecting approximately 1 in 600 individuals [55] . in most cases individuals deficient in iga show no clinical symptoms. however, severe allergies, autoimmune and gastrointestinal disorders have been reported in some patients [55] . studies in iga knockout mice samples were run on separate gels and bands were excised from the region predicted to comprise the immunoglobulin heavy chains . gel images are presented in figure s2 . [56] . interestingly, however, the iga deficient mice produced higher quantities of systemic igg. similar findings have also been reported in humans [57, 58] . indeed, significantly higher levels of igm and igg were found in the saliva of iga deficient individuals compared to normal healthy controls [58] . in light of these studies, we hypothesise that the dominance of igg in the mucosal secretions of p. alecto may be an evolutionary compensation for an inherent lack of iga. further research is required to determine what functional relevance this would have on the immune response. 2-de resolution of p. alecto serum demonstrated the existence of multiple igg h isoforms. the immunoblot reactive spot train spanned a wide pi range of 5 to 9. this range is in agreement with the predicted pi range of 15 separate ighg mrna previously sequenced (range of 6.3 to 8.3) [28] . a doublet of slightly different molecular weights for the igg h chain was also observed after 1-de immunoblotting. most mammalian species contain multiple igg subclasses and therefore it is not surprising that p. alecto contained multiple subclasses of igg. indeed, butler et al. [29] reported the presence of multiple ighg subclass transcripts in a number of bat species including myotis lucifugus, eptiscus fuscus and cynopterus sphinx. in contrast to igg h , a much smaller number of reactive igm h isoforms were detected in serum following 2-de western blotting. however, following deglycosylation with pnga-sef, two bands of igm h were observed in p. alecto. this result was not observed in human igm. this finding suggests the possible existence of a subclass of igm h in p. alecto that is absent from humans. to our knowledge the existence of multiple igm subclasses has not been previously described in bats. considering that igm provides a first line of defence against microbial invasion, diversification of this immunoglobulin class may confer a distinct immunological advantage to the species. indeed, different igm subclasses may confer an enhanced and diverse classical complement pathway which, to some extent, may compensate for the lack of serum iga in p. alecto. further studies are required to elucidate the functional significance of putative igm subclasses within p. alecto. p. alecto igg from serum and plasma binds to both protein a and protein g but has significantly greater binding affinity to protein g. protein g recognises most igg subclasses in many species [59] whilst protein a binds only certain igg subclasses and in some species does not bind igg (e.g. rat and goat). this finding may reflect the presence of different p. alecto igg subclasses with different binding affinity to protein a. p. alecto igg h antiserum cross reacted strongly against two other bats (p. conspiculatus and r. megaphylus) and horse igg h . a close evolutionary relationship between chiroptera bats and horse has been reported on a number of occasions and may have contributed to this cross reactivity [60, 61] . the fact that p. alecto igm h antiserum did not cross react with horse suggests this immunoglobulin class has diverged more rapidly since the separation of horse and bats compared to igg. divergence of igm may have contributed to the rise of different igm subclasses as described above. given that serum iga is drastically reduced in p. alecto, diversification of igm may have functional significance for the early antibody response. the novel method involving the use of immobilised anti-fabspecific antibody described herein allows for the simultaneous purification of immunoglobulin classes from serum and production of class-specific antiserum without prior knowledge of the species' immunoglobulin repertoire. igg and igm were purified from p. alecto serum, and rabbit antiserum specific to both molecules were produced. while igg and igm were successfully isolated from serum, no visible enrichment of serum iga was achieved. trace quantities of iga were detected only by mass spectrometry in serum. iga was also detected in the mucosal secretions of the small and large intestine lavages, milk and tears. the glycosylation pattern of p. alecto igg and igm appears typical of that described for other mammalian immunoglobulins. furthermore deglycosylation of igm h and 2-de immunoblotting of igg h revealed the possible existence of multiple igm h and igg h subclasses in p. alecto. taken together, this study suggests healthy p. alecto bats have considerably less serum iga than expected. diversification of igm and igg subclasses may be an evolutionary compensation for this lack of iga. further research is now required to investigate the functional relevance of this unique antibody repertoire, particularly in response to viral infection. the knowledge and tools developed here will facilitate this research. (table s2 ) (tif) table s1 protein identification by lc-ms/ms analysis of igm affinity purified fractions. the four fractions were separated by sec from p. alecto serum and plasma (see figure s1 ). (docx) table s2 protein identification by lc-ms/ms analysis of 19 excised bands. proteins were purified by jacalin affinity chromatography from igg depleted p. alecto serum (see figure s3d ). (docx) flying-foxes: fruit-and blossom-bats of australia isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus serologic evidence for the presence in pteropus bats of a paramyxovirus related to equine morbillivirus bats: important reservoir hosts of emerging viruses bats, emerging infectious diseases, and the rabies paradigm revisited bats as a continuing source of emerging infections in humans fatal encephalitis due to nipah virus among pig-farmers in malaysia nipah virus infection of pigs in peninsular malaysia henipaviruses:an updated review focusing on the pteropid reservoir and features of transmission cedar virus: a novel henipavirus isolated from australian bats henipavirus neutralising antibodies in an isolated island population of african fruit bats human ebola outbreak resulting from direct exposure to fruit bats in luebo, democratic republic of congo experimental nipah virus infection in pteropid bats experimental inoculation of plants and animals with ebola virus transmission studies of hendra virus (equine morbillivirus) in fruit bats, horses and cats experimental hendra virus infection in pregnant guinea-pigs and fruit bats (pteropus poliocephalus) australian bat lyssavirus infection in a captive juvenile black flying fox pathogenesis studies with australian bat lyssavirus in grey-headed flying foxes (pteropus poliocephalus) iga and the iga fc receptor p (1974) presence of j-chain in human immunocytes containing various immunoglobulin classes role of j chain in secretory immunoglobulin formation dual function of human-iga antibodies -inhibition of phagocytosis in circulating neutrophils and enhancement of responses in il-8-stimulated cells suppression by iga of igg-mediated phagocytosis by human polymorphonuclear leukocytes human serum iga down-regulates the release of inflammatory cytokines (tumornecrosis-factor-alpha, interleukin-6) in human monocytes antiinflammatory properties of human serum iga: induction of il-1 receptor antagonist and fc alpha r (cd89)-mediated down-regulation of tumour necrosis factor-alpha (tnf-alpha) and il-6 in human monocytes the impact of glycosylation on the biological function and structure of human immunoglobulins immunoglobulin heavy chain diversity in pteropid bats: evidence for a diverse and highly specific antigen binding repertoire the two suborders of chiropterans have the canonical heavy-chain immunoglobulin (ig) gene repertoire of eutherian mammals severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats fruit bats as reservoirs of ebola virus antibody mediated immune response in the bat immune response in chiroptera to bacteriophage phix174 studies of arthropod-borne virus infections in chiroptera .4. immune response of big brown bat (eptesicus fuscus) maintained at various environmental temperatures to experimental japanese b encephalitis virus infection establishment, immortalisation and characterisation of pteropid bat cell lines vaccine-induced protection against gastrointestinal bacterial infections in the absence of secretory antibodies complement activation and protein adsorption by carbon nanotubes purification and some properties of streptococcal protein-g, protein-a novel igg binding reagent separation of human iga1 and iga2 using jacalin agarose chromatography isolation and detection of human iga using a streptococcal iga-binding peptide the staphylococcal superantigen-like protein 7 binds iga and complement c5 and inhibits iga-fc alpha ri binding and serum killing of bacteria molecular cloning : a laboratory manual a new adjuvant comparative 2d dige analysis of the depleted serum proteome for biomarker discovery mass spectrometric identification and characterisation of the nucleocapsid protein of menangle virus generation of tioman virus nucleocapsid-like particles in yeast saccharomyces cerevisiae molecular characterisation of toll-like receptors in the black flying fox pteropus alecto a novel iga-like immunoglobulin in the reptile eublepharis macularius discovery of a unique ig heavy-chain isotype (igt) in rainbow trout: implications for a distinctive b cell developmental pathway in teleost fish a third immunoglobulin class in amphibians neonatal fc receptor for igg regulates mucosal immune responses to luminal bacteria beyond iga: the mucosal immunoglobulin alphabet immunoglobulins and immunocytes in the mammary gland and its secretions perspectives on immunoglobulins in colostrum and milk iga deficiency mucosal immunity to influenza without iga: an iga knockout mouse model compensatory levels of salivary igm anti-streptococcus mutans antibodies in iga deficient patients immunoglobulin levels in saliva in individuals with selective iga deficiency -compensatory igm secretion and its correlation with hla and susceptibility to infections purification of igg using protein a or protein g pegasoferae, an unexpected mammalian clade revealed by tracking ancient retroposon insertions the immune gene repertoire of an important viral reservoir, the australian black flying fox the authors thank susanne wilson for the management of the bat colony and rabbits at aahl, and for assistance with sample collection. for serum and plasma donation we acknowledge dr kristen glenister from the australian red cross blood bank, dr hume field from queensland primary industries and fisheries, dr kaku yoshi from the national institute of infectious diseases (tokyo), jennifer barr and meagan gillespie from aahl. we are also grateful to mary tachedjian and dr kim blasdell for critical reading of this manuscript. key: cord-329727-h47q76y8 authors: sisó-almirall, antoni; kostov, belchin; mas-heredia, minerva; vilanova-rotllan, sergi; sequeira-aymar, ethel; sans-corrales, mireia; sant-arderiu, elisenda; cayuelas-redondo, laia; martínez-pérez, angela; garcía-plana, noemí; anguita-guimet, august; benavent-àreu, jaume title: prognostic factors in spanish covid-19 patients: a case series from barcelona date: 2020-08-21 journal: plos one doi: 10.1371/journal.pone.0237960 sha: doc_id: 329727 cord_uid: h47q76y8 background: in addition to the lack of covid-19 diagnostic tests for the whole spanish population, the current strategy is to identify the disease early to limit contagion in the community. aim: to determine clinical factors of a poor prognosis in patients with covid-19 infection. design and setting: descriptive, observational, retrospective study in three primary healthcare centres with an assigned population of 100,000. method: examination of the medical records of patients with covid-19 infections confirmed by polymerase chain reaction. logistic multivariate regression models adjusted for age and sex were constructed to analyse independent predictive factors associated with death, icu admission and hospitalization. results: we included 322 patients (mean age 56.7 years, 50% female, 115 (35.7%) aged ≥ 65 years): 123 (38.2) were health workers (doctors, nurses, auxiliaries). predictors of icu admission or death were greater age (or = 1.05; 95%ci = 1.03 to 1.07), male sex (or = 2.94; 95%ci = 1.55 to 5.82), autoimmune disease (or = 2.82; 95%ci = 1.00 to 7.84), bilateral pulmonary infiltrates (or = 2.86; 95%ci = 1.41 to 6.13), elevated lactate-dehydrogenase (or = 2.85; 95%ci = 1.28 to 6.90), elevated d-dimer (or = 2.85; 95%ci = 1.22 to 6.98) and elevated c-reactive protein (or = 2.38; 95%ci = 1.22 to 4.68). myalgia or arthralgia (or = 0.31; 95%ci = 0.12 to 0.70) was protective factor against icu admission and death. predictors of hospitalization were chills (or = 5.66; 95%ci = 1.68 to 23.49), fever (or = 3.33; 95%ci = 1.89 to 5.96), dyspnoea (or = 2.92; 95%ci = 1.62 to 5.42), depression (or = 6.06; 95%ci = 1.54 to 40.42), lymphopenia (or = 3.48; 95%ci = 1.67 to 7.40) and elevated c-reactive protein (or = 3.27; 95%ci = 1.59 to 7.18). anosmia (or = 0.42; 95%ci = 0.19 to 0.90) was the only significant protective factor for hospitalization after adjusting for age and sex. conclusion: determining the clinical, biological and radiological characteristics of patients with suspected covid-19 infection will be key to early treatment and isolation and the tracing of contacts. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 on 31 december 2019, the health authorities of wuhan city (hubei province, china) reported a cluster of 27 cases of pneumonia of unknown aetiology with onset of symptoms on 8 december, including 7 severe cases, with a common exposure identified in a city market [1], which was closed on january 1, 2020. on 7 january 2020, the chinese authorities identified a new coronaviridae family virus, initially named coronavirus 2019-ncov and later coronavirus sars-cov-2 as the causal agent [2] . the genetic sequence was shared by the chinese authorities on 12 january 2020. on january 19, the first case was detected in the usa, in washington state [3] . on 30 january 2020, the world health organization declared the sars-cov-2 outbreak in china a public health emergency of international concern [4] . subsequently, the outbreak has spread outside china, with europe especially affected [5] . the first positive case diagnosed in spain was confirmed on january 31, 2020 on the island of la gomera, while the first death occurred on february 13 in valencia city (the date was confirmed twenty days later). the first confirmed case in barcelona was on 24 february, and from then until 29 june 2020, there have been 248,970 confirmed cases in spain [6] . the most common signs of infection are respiratory symptoms: fever, cough and shortness of breath. in more severe cases, the infection may cause pneumonia, severe acute respiratory syndrome, renal failure and death [7] . transmission appears to be mainly person-to-person via the airway through respiratory droplets measuring > 5 microns when the patient has respiratory symptoms (cough and sneezing) and contact with fomites [8] . most estimates of the incubation period of covid-19 range from 1 to 14 days, with most around five days. evidence on the transmission of the virus before symptom onset is unclear. there is currently no specific treatment for covid-19 infections. to date, the most important scientific efforts have focused on three areas: strategies to contain the spread of the disease, the initiation of clinical trials with antivirals and multiple therapies, and the design of a new vaccine, which is still unclear. these strategies include some of a community nature, where primary healthcare plays a central role in disease prevention and control [9] . few studies have described the clinical characteristics of the disease, fewer the predictive factors, and virtually none have described the mediterranean population compared with the rest of the world. therefore, this study aimed to describe the clinical, biological and radiological manifestations, the evolution, treatments and mortality rate of patients with covid-19 infection in the population of barcelona city and determine the most important predictors of a poor prognosis. a multicentre, observational descriptive study was carried out in three urban primary healthcare centres serving an assigned population of 100,000, with one reference hospital. the study included all consecutive adult patients with covid-19 confirmed by polymerase chain reaction (pcr) from nasal and pharyngeal samples during the study period of 29 february to 4 april 2020. diagnostic confirmation was made in the hospital laboratories, as pcr is not available in primary healthcare centres. signs and symptoms, the main available haematological and biochemical data and the results of imaging tests were recorded, as were comorbidities, the evolution, the hospitalization rate, intensive care unit (icu) admission and the treatments received. the study population was divided into four age groups: 15-30 years, 31-49 years, 50-64 years and �65 years. other variables recorded were the type of follow-up, the need for temporary work disability, and the source of possible contacts. the time to first medical visit was defined as the difference (in days) between symptom onset and medical visit by a family physician. the factors that determined a poor prognosis (hospitalization, icu admission, death) were collected. the data were obtained from the electronic medical record. missing data were collected by telephone interviews with patients when possible. patients from nursing homes were excluded, as the rate of infections and mortality has been shown to be much higher than in the non-institutionalized population. the study was approved by the ethics committee of the hospital clinic of barcelona (registration number hcb/2020/0525). the study was conducted according to the helsinki declaration and spanish legislation on biomedical studies, data protection and respect for human rights. categorical variables are presented as absolute frequencies and percentages (%) and continuous variables as means and standard deviations (sd). predictors of death, icu admission and hospitalization were determined using the student's t test for continuous variables and the chisquare test for categorical variables. logistic multivariate regression models adjusted for age and sex were constructed to analyse independent predictive factors associated with death, icu admission and hospitalization. odds ratios (or) and their 95% confidence intervals (95%ci) obtained in the adjusted regression analysis were calculated. forest plots were used to represent or and 95%ci. values of p<0.05 were considered statistically significant. the statistical analysis was performed using the r version 3.6.1. for windows. we included 322 patients (mean age 56.7 years, 50% female, 115 (35.7%) aged � 65 years). the mean time from symptom onset to the medical visit was 3.9 (sd 4.6) days. clinical characteristics are shown in table 1 2) . comorbidities were presented by 212 (65.8%) patients: the most common were hypertension in 109 (33.9%), diabetes mellitus in 46 (14.3%), and obesity in 46 (14.3%) ( table 2) . heart disease (19.6% vs 5.3%, p = 0.001), autoimmune disease (16.1% vs 4.5%, p = 0.004), diabetes (32.1% vs 10.5%, p < 0.001), hypertension (60.7% vs 28.2%, p < 0.001) and chronic kidney disease (17.9% vs 7.9%, p = 0.042) were the comorbidities significantly associated with icu admission and death (table 2) . autoimmune disease was the only significant predictive comorbidity for icu admission and death after adjusting for age and sex (or = 2.82; 95% ci = 1.00 to 7.84) (fig 1) . depression was the best predictor of hospitalization among all comorbidities (or = 6.06; 95%ci = 1.54 to 40.42) (fig 2) . having � 1 comorbidity was associated with icu admission and death (or = 3.43; 95%ci = 1.30 to 10.83) and hospitalization (or = 2.05; 95%ci = 1.13 to 3.75) independently of age and sex. chest x-ray was necessary in 227 patients (70.5%) and showed lobar pulmonary infiltrates in 35 (15.4%), bilateral pulmonary infiltrates in 129 (56.8%) and an interstitial pattern in 48 (21.1%) ( table 3) . chest ct was required in 28 patients and pulmonary ultrasound in 10 (3.1%). biologically, 171 (81.4%) of 210 patients had lymphopenia (< 1,000 mm3). likewise, 60.8% had a lactate dehydrogenase (ldh) > 250 u/ml and liver test alterations were common: elevated ast/got in 41.4% and alt/gpt in 32.4%. in 86 (52.1%) of 165 cases d-dimer was elevated (> 500mg/l). the most important factors for icu admission and death were bilateral pulmonary infiltrates (or = 2.86; 95%ci = 1.41 to 6.13), elevated lactate-dehydrogenase (or = 2.85; 95%ci = 1.28 to 6.90), elevated d-dimer (or = 2.85; 95%ci = 1.22 to 6.98) and elevated c-reactive protein (or = 2.38; 95%ci = 1.22 to 4.68) (fig 1) . significant predictive factors associated with hospitalization, after adjusting for age and sex, were lymphopenia (or = 3.48; 95%ci = 1.67 to 7.40) and elevated c-reactive protein (or = 3.27; 95%ci = 1.59 to 7.18) (fig 2) . this study summarizes the clinical, biological and radiological characteristics, evolution and prognostic factors of patients with covid-19 disease in primary and community healthcare. to date, we are aware of three published spanish studies [10] [11] [12] . the first reported data from 48 patients on icu admissions in a region where the pandemic was reported early [10] . the study by borobia et al [11] describes the first 2226 adult patients with covid-19 consecutively admitted to a university hospital in madrid. the third focuses on the differences by agedependent categories in the clinical profile, presentation, management, and short-term outcomes [12] . although there have been two systematic reviews and meta-analysis that analyse the clinical characteristics of covid-19, they are limited to chinese cohorts or case series [13, 14] and a large usa cohort [15] that did not analyse clinical predictors of a poor prognosis. clinically, the same main symptoms of cough and fever are reported in all series. however, in barcelona city, we have observed diarrhoea, anosmia and dysgeusia, which is hardly reported in the chinese series [7] which, unlike ours comes principally from hospitals: diarrhoea occurred in 23.8% of cases, very similar to the 23% in new york [16] and clearly higher than the 3.8% reported in china. nearly 20% of patients had anosmia and dysgeusia, similar to the results obtained in french patients [17] . in contrast, expectoration was found in only 9%, compared with 33.7% in the chinese series. in bold, statistically significant independent predictive factors associated with hospitalization, death, or icu admission (logistic multivariate regression adjusted for age and sex). † comorbidities with a frequency of < 5 patients were: bronchiectasis (n = 4), fibromyalgia (n = 4), anaemia (n = 3), arthritis (n = 3), hiv (n = 2), syphilis (n = 1) and tuberculosis (n = 1). https://doi.org/10.1371/journal.pone.0237960.t002 in bold, statistically significant independent predictive factors associated with hospitalization, death or icu admission (logistic multivariate regression adjusted for age and sex). † 227 (70.5%) patients had a chest x-ray. the alterations with a frequency < 5 patients were: pneumothorax (n = 2) and pleural effusion (n = 1). chest x-ray results were not available in 5 patients. ‡ 28 (8.7%) patients had a chest cat scan. alterations with a frequency of < 5 patients were: pulmonary thromboembolism (n = 4), emphysema (n = 2), lobar pulmonary infiltrates (n = 2), pneumonia (n = 2), atelectasis (n = 2) and pleural effusion (n = 1). cat scan results were not available in five patients. https://doi.org/10.1371/journal.pone.0237960.t003 chinese patients had a mean age of 47 years, ten years lower than our series, and 35.7% of our patients were aged � 65 years, compared with 15%, 29% and 31% in china, germany and the usa respectively, but > 40% in italy [7, [18] [19] [20] . older age and male sex predisposed to a higher mortality rate in our and all large series [7, 15] . in our patients, comorbidities were three times higher than in the chinese cohort [7] and were similar to the findings of the new york study [15] . any comorbidity was a risk factor for hospitalization, icu admission and death. depression was an independent risk factor for hospitalization, which has not been observed in other cohorts studied. depression was often accompanied by a vulnerable social situation, which may have justified hospitalization. likewise, autoimmune diseases were independent risk factor for icu admission and death. various hypotheses have been postulated on possible autoimmune alterations in the pathogenic evolution of the disease. with respect to in bold, statistically significant independent predictive factors associated with hospitalization, death, or icu admission (logistic multivariate regression adjusted for age and sex). † complications in < 5 patients were: sepsis (n = 3), multiorgan failure (n = 2), electrolyte alterations (n = 2), hematologic alterations (n = 2) and lung cancer (n = 1). ‡ treatments with a frequency of < 10 patients, except remdesivir, were: amoxicillin (n = 6), interferon (n = 5), rituximab (n = 5), darunavir (n = 2) and entecavir (n = 1). treatment, no drug has proved effective against covid-19 until now. moreover, many treatments were unavailable in the outpatient setting. currently, we are only certain that treatment with tocilizumab showed better survival rates in retrospective cohorts [21] , although its efficacy has not been tested in randomized clinical trials. therefore, the results on the outcomes associated with treatment should be interpreted with caution. the same comorbidities were identified, with hypertension and diabetes being the two most common, while in the usa and italy, obesity seems to be higher. our results show that obesity was close to being an independent risk factor for hospitalization (or = 2.05; 95% ci = 0.99 to 4.46). strikingly, 38.2% of our patients were healthcare workers, compared with 3.5% in wuhan and 5.2% in germany [7, 18] . although these studies recognized an important degree of underreporting of cases in health workers, the difference remains important. there are at least two possible explanations: first, the lack of personal protective equipment in the initial phase of the epidemic, a constant revindication of health professionals, who felt undersupplied. secondly, many cases were health professionals from primary healthcare or the reference hospital who reside in the same area where they work. in all reported series, bilateral pneumonia was the most common radiological finding, was present in more than half the cases [22] and was a factor of a poor prognosis and mortality. in contrast, an interstitial radiological pattern did not confer an increased risk of mortality. the wuhan study reported a cat scan use of 88.7%, compared with 8.7% in barcelona. in contrast, chest x-rays were carried out in 59.1% and 70.5%, respectively: the availability of diagnostic means was higher in china. a recent international consensus states that radiological assessment is not necessary in asymptomatic patients or those with mild disease but is required in patients with moderate or severe disease, regardless of whether a definite diagnosis of covid-19 has been made [23] . in addition, simple chest x-rays are preferable in a resourceconstrained environment with difficulties in accessing cat scans [23] . the possible use of pulmonary ultrasound for the point-of-care diagnosis of covid-19 pneumonia has not been sufficiently analysed but might be an efficient alternative due to its portability and reliability [24] . in fact, the regional catalan government has recently acquired 90 ultrasound machines to enable family physicians to make doctors can make point-of-care (home or nursing home) diagnoses of pneumonia [25] . biologically, lymphopenia and increased crp, ldh and ddimer were usually constant and similar in all series and were associated with an increased risk of mortality. a differential variable in our series is a greater number of alterations in liver tests, which was present in 30-40% of patients, data similar to the usa and italian cohorts, but different from the chinese cohort, where it was 22% [7] . we also found hypokalaemia in 20.5% of patients, a factor not reported in other studies. we found a hospitalization rate of 48.7%, compared with 20-31% in the usa and 93.6% in china, and an icu admission rate of 13%, which was similar to the chinese (15%), usa (5-11.5%) and german (10%) results. while the protocols of action and admission are similar and depend on the level of clinical involvement, the therapeutic protocols differ between hospitals, cities, and countries. there remain many unknowns in the treatment of covid-19. the only truth is that we do not have a vaccine, an etiological treatment or a treatment with sufficient scientific evidence to generalize its use. currently, the systematic review of antiretroviral treatments has not offered conclusive results [26] and despite in vitro results for hydroxychloroquine, covid-19 infections are currently intractable [27, 28] . the mortality rate in our study was 5.6%, compared with 10.2% in new york (21% in hospitalized patients), 1.4% in china, 3.1% in germany and 6.8% in italy. different information and recording systems, the availability of diagnostic tests, and above all, the organization of national health systems may have contributed to the differences observed. the study had some limitations due to the observational, retrospective design. however, it is sufficiently representative of the population with confirmed covid-19 to permit better identification of the factors of a poor prognosis of the disease from a clinical perspective. we cannot rule out some heterogeneity in data codification due to observers' interpretations of the medical records. however, this bias is minimal, as most clinical factors included are clearly defined in the electronic medical record. another limitation of this study is the percentage of patients without laboratory parameters (more than 30%). even though in real clinical practice these percentages may be expected, the results corresponding to laboratory parameters should be interpreted with caution. four months after the declaration of the pandemic, there is not a sufficiently reliable, available and generalizable diagnostic test that can analyse the seroprevalence of covid-19, even in the most industrialized countries. given this lack, determining the clinical, biological and radiological characteristics of probable cases of covid-19 infection will be key to the initiation of early treatment and isolation, and for contact tracing, especially in primary healthcare. visualization: antoni sisó-almirall, belchin kostov, jaume benavent-àreu. novel coronavirus (2019-ncov) situation summary first case of 2019 novel coronavirus in the united states world health organization. novel coronavirus situació n del covid-19 en españa clinical characteristics of coronavirus disease 2019 in china enteric involvement of coronaviruses: is faecal-oral transmission of sars-cov-2 possible? ready for a long fight against the covid-19 outbreak: an innovative model of tiered primary health care in taiwan sars-cov-2 in spanish intensive care: early experience with 15-day survival in vitoria a cohort of patients with covid-19 in a major teaching hospital in europe clinical presentation and outcome across age categories among patients with covid-19 admitted to a spanish emergency department clinical characteristics of coronavirus disease 2019 (covid-19) in china: a systematic review and meta-analysis clinical, laboratory and imaging features of covid-19: a systematic review and meta-analysis presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new york city area clinical characteristics of covid-19 in new york city utility of hyposmia and hypogeusia for the diagnosis of covid-19 covid-19)-united states the italian coronavirus disease 2019 outbreak: recommendations from clinical practice impact of low dose tocilizumab on mortality rate in patients with covid-19 related pneumonia the role of imaging in 2019 novel coronavirus pneumonia (covid-19) the role of chest imaging in patient management during the covid-19 pandemic: a multinational consensus statement from the fleischner society thoracic ultrasound and sars-covid-19: a pictorial essay generalitat de catalunya. darreres mesures systematic review of the efficacy and safety of antiretroviral drugs against sars, mers or covid-19: initial assessment should chloroquine and hydroxychloroquine be used to treat covid-19? a rapid review chloroquine and hydroxychloroquine in covid-19 the authors thank david buss for editorial assistance. conceptualization: antoni sisó-almirall, belchin kostov, jaume benavent-àreu. key: cord-346819-11fkgzaa authors: khan, mohd imran; khan, zainul a.; baig, mohammad hassan; ahmad, irfan; farouk, abd-elaziem; song, young goo; dong, jae-jun title: comparative genome analysis of novel coronavirus (sars-cov-2) from different geographical locations and the effect of mutations on major target proteins: an in silico insight date: 2020-09-03 journal: plos one doi: 10.1371/journal.pone.0238344 sha: doc_id: 346819 cord_uid: 11fkgzaa a novel severe acute respiratory syndrome-related coronavirus-2 (sars-cov-2) causing covid-19 pandemic in humans, recently emerged and has exported in more than 200 countries as a result of rapid spread. in this study, we have made an attempt to investigate the sars-cov-2 genome reported from 13 different countries, identification of mutations in major coronavirus proteins of these different sars-cov-2 genomes and compared with sars-cov. these thirteen complete genome sequences of sars-cov-2 showed high identity (>99%) to each other, while they shared 82% identity with sars-cov. here, we performed a very systematic mutational analysis of sars-cov-2 genomes from different geographical locations, which enabled us to identify numerous unique features of this viral genome. this includes several important country-specific unique mutations in the major proteins of sars-cov-2 namely, replicase polyprotein, spike glycoprotein, envelope protein and nucleocapsid protein. indian strain showed mutation in spike glycoprotein at r408i and in replicase polyprotein at i671t, p2144s and a2798v,. while the spike protein of spain & south korea carried f797c and s221w mutation, respectively. likewise, several important country specific mutations were analyzed. the effect of mutations of these major proteins were also investigated using various in silico approaches. main protease (mpro), the therapeutic target protein of sars with maximum reported inhibitors, was thoroughly investigated and the effect of mutation on the binding affinity and structural dynamics of mpro was studied. it was found that the r60c mutation in mpro affects the protein dynamics, thereby, affecting the binding of inhibitor within its active site. the implications of mutation on structural characteristics were determined. the information provided in this manuscript holds great potential in further scientific research towards the design of potential vaccine candidates/small molecular inhibitor against covid19. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 in the last two decades, three coronaviruses viz. severe acute respiratory syndrome coronavirus (sars-cov) [1] , middle-east respiratory syndrome coronavirus (mers-cov) [2] and sars-cov-2 have crossed the species barrier to cause deadly pneumonia in humans. in 2002, sars-cov emerged in the guangdong province of china and spread to five continents, infecting 8,098 people with 774 deaths. in 2012, mers-cov emerged in the arabian peninsula, transmitted to 27 countries, infecting a total of~2,494 individuals and claiming 858 lives. the current outbreak of coronavirus disease caused by sars-cov-2, was first reported in december 2019 in wuhan, hubei province of china [3, 4] and spread across 200 countries, infecting over 2.5 million people and killed more than 1.5 lakh as of april, 23, 2020. sars-cov-2 was declared a pandemic by the world health organization on march 12, 2020. sars-cov-2 belongs to the family coronaviridae of genus betacoronavirus, having positive sense strand rna genome of 26-32 kb size. sars-cov-2 genome has six major open reading frames (orfs) viz. replication enzyme coding region (orf 1a and 1b), e gene (envelope protein), m gene (membrane protein), s gene (spike protein), and n gene (nucleocapsid protein) that are common to coronaviruses and a number of other accessory genes (orf 3a, 6, 7a, 7b and 8) (fig 1) [3] . the structural proteins: envelope protein, nucleocapsid protein, spike protein and membrane protein are essential for producing the structurally complete viral particle [5] [6] [7] [8] . entry of coronavirus into host cells is guided by spike glycoprotein. orf 1a and 1b encode replication enzyme consisting 16 non-structural proteins (nsp1-16) that are highly conserved among the coronaviruses. main protease (mpro, also known as 3clpro) is one of the important nsp encoded by orf 1a and 1b, play an essential role in the processing of polyproteins and control the replication of coronavirus [9, 10] . rna-dependent rna polymerase (rdrp) also known as nsp12, another important replicase catalyze the replication of rna using viral genomic rna template [11] . reports showed that mers-cov originated from bats, but the reservoir host fueling spillover to humans is unequivocally dromedary camels [12, 13] . both sars-cov and sars-cov-2 are originated from bats, which serve as reservoir host for these two viruses [3, 14, 15] . raccoon dogs and palm civets have been identified as intermediate hosts for zoonotic transmission of sars-cov between bats and humans [16] [17] [18] , however, the intermediate host of sars-cov-2 remains unknown. mutation rate is very high in rna viruses, up to a million times higher than their host, which enhance their virulence and evolvability (formation of new species) [19] . coronavirus replication is error prone as compared to other rna viruses and the estimated mutation rate is 4x10 -4 nucleotide substitutions/site/year [20] . the rate of sars-cov-2 mediated disease spread and the mortality varies from country to country. of several reasons affecting the rate of disease spread and mortality, mutations within the sars-cov-2 strains is also considered one of the major factors. this study was conducted to gather additional information on the sars-cov-2 sequences from different geographical locations infected with covid-19. the genome analysis of the sars-cov-2 strains from 13 different countries showed a large number of mutations within the major structural proteins. this is the first time we have comprehensively investigated these mutations and also discussed their potential roles in the pathogenicity, replication and entry of virus particle. this study provides a deeper insight into the emergence of these mutations within the major structural as well as nsp encoded by the sars-cov-2 genome from different countries. here, molecular dynamics and other in silico studies were also performed to investigate the effect of mutations on the dynamics of mpro. the findings of this study provide a clue for the futuristic development of potential vaccine candidate or therapeutic design against covid19. the genome sequence of orf1ab for sars with reference sequence id: nc_004718.3 and protein sequence with genbank id: aap41036. and visualization of sars and sars-cov-2 sequences from 13 different countries was performed using molecular evolutionary genetics analysis (mega) version 10.1.8. to delineate and analyze the mutation across different countries, an in-house script written in perl and python was used. mupro server was used to determine the effect of mutation on various sars-cov-2 proteins [21] . the crystal structure of mpro protein from sars-cov-2 in complex with boceprevir (pdb id: 7brp) was taken as a wildtype (wt). the structure of r60c was generated by inserting the point mutation and modelled using modeler 9v13 [22] . boceprevir was retrieved and redocked within the structure of mpro using ccdc gold. the rmsd for the crystal and redocked conformation of boceprevir were compared. further, boceprevir was subjected to dock within the active site of r60c mutant. the poses were visualized on pmv viewer [23] . the structure of boceprevir in complex with mpro (wt) and r60c mutant was subjected to energy minimization using gromacs-5 with the charmm27 all atom force field [24] [25] [26] . the models were solvated with a spc/e water model in a cubic periodic box with 1 nm distance from the edge of the complex atoms. the solvated system was neutralized by seven chloride ions. the system was, thereafter, minimized using steepest descent algorithm with convergence criteria of tolerance value 1000 kj mol −1 nm −2 . the complete simulation of minimized solvated proteins was performed under periodic boundary condition with time step of 2 fs. particle mesh ewald was used for long range electrostatic interactions with an interpolation order of 4 and a fourier spacing of 0.16. the first phase simulation was conducted under an nvt ensemble for 500 ps by keeping all bonds constrained using the lincs algorithm for temperature equilibration. the system was heated to 300 k using leap-frog integrator while pressure coupling was set off. a v-rescale thermostat was used to maintain constant temperature for each system, followed by pressure equilibration at 300 k using parrinello-rahman pressure coupling algorithm under an isothermal-isobaric ensemble for another 500 ps at 1.0 bar. isothermal compressibility of the solvent was set to 4.5e -5 bar −1 . further, simulation of 50000 ps production run was carried out at 300 k and 1 atm pressure for trajectory analysis. the final models obtained at the end of md were validated and taken for structural analysis. the complete nucleotide sequences of 13 sars-cov-2 reported from 13 different countries showed~82% sequence identity with sars-cov. also, all 13 sequences shared more than 99% sequence identity to each other. replicase polyprotein (orf 1ab) of 13 isolates, which are most conserved in all coronaviruses shared maximum identity (87%) with sars-cov (nc_0047180), which is less than the threshold value (90%) for demarcation of betacoronavirus species [27, 28] . phylogenetic analysis revealed that all 13 sars-cov-2 identified from different geographical locations clustered together in a single clad as compared to sars-cov (fig 2a and 2b ). further, we checked the mutation in all major proteins of 13 sars-cov-2 sequences and compared with sars-cov. orf 1a and 1b showed 11 changes among all 13 sars-cov-2. indian sars-cov-2 sequence showed three changes at 671 (isoleucine to threonine), 2144 (proline to serine) and 2798 (alanine to valine) compared to all other 12 isolates. here, we also noted two amino acid mutations (in orf1ab) in each sars-cov-2 sequences isolated from china (2708: asparagine to serine; 2908: phenylalanine to isoleucine), south korea (902: methionine to isoleucine; 6891: threonine to methionine) and sweden (818: glycine to serine; 4321: phenylalanine to leucine). brazil and vietnam isolate showed only one change at 3606 (leucine to phenylalanine) and 3323 (arginine to cystine), respectively (table 1) . 996 changes have been reported when orf 1a and 1b amino acid sequences of all 13 sars-cov-2 was compared with sars-cov (s1 file). the viral mpro controls the replication of coronavirus and is a key protein responsible for its life cycle [29] [30] [31] . mpro is an attractive drug discovery target. the analysis of mpro reveals that there was only one point mutation (r60c) in the vietnam strain of sars-cov-2 ( fig 3a) . rdrp, which is another important target for antiviral drugs functions by catalyzing the viral rna synthesis [32] . only one mutation (a406v) was observed in the rdrp of indian sars-cov-2 isolate (fig 3b) . spike proteins are the key surface glycoproteins and are well reported for their prominent role in interaction with host cell receptors [33, 34] . here, we analyzed the mutations in the spike protein of sars-cov-2 from different countries. it was found that this glycoprotein carried five different amino acid mutations at various positions within the investigated sars-cov-2 isolates. for instance, india, finland, australia, south korea and sweden sars-cov-2 isolates showed one amino acid change at 408 (arginine to isoleucine), 49 (histidine to tyrosine), 247 (serine to arginine), 221 (serine to tryptophan) and 797 (phenylalanine to cysteine), respectively ( table 2 and fig 3c) . the value of δδg show that the mutant r408i (0.49732107 kcal/mol) mutation was having stabilization effect on spike protein. it was found that the mutation on the receptor binding domain (rbd) of spike protein increases the stability. when these 13 sars-cov-2 isolates were compared to sars-cov sequence, 1338 changes have been reported (s1 file). the analysis of orf3a showed 3 mutations within different sars-cov-2 strains: w128l (south korea), l140v (japan), g251v (australia, south korea, brazil, italy, sweden) ( table 3) . one amino acid change occurred in each envelope protein of south korea sars-cov-2 isolate at 37 (leucine to histidine) and nucleocapsid protein of japan sars-cov-2 isolate at 344 (proline to serine) when compared among 13 sars-cov-2 isolates (tables 4 and 5 i i i i i i i i i i i i 818 t t m t t t t t t t t t t https://doi.org/10.1371/journal.pone.0238344.t001 when compared to sars-cov. mem glycoprotein did not show any amino acid change among 13 sars-cov-2 isolates, while 24 changes occurred when compared to sars-cov (s1 file). all the other point mutations occurring within the structural proteins of sars-cov-2 isolates from different countries were found to decrease protein stability (table 6 ). in the present study, we performed the md simulations for the boceprevir bound complexes of sars-cov-2 mpro and its r60c mutant to study the effect of mutation on the protein dynamics. the root mean square deviations of the backbone were calculated to analyze the trajectories of mpro from sars-cov-2 and its r60c mutant. in the complex form, a slight fluctuation in the comparative genome analysis of novel coronavirus (sars-cov-2) from different geographical locations comparative genome analysis of novel coronavirus (sars-cov-2) from different geographical locations comparative genome analysis of novel coronavirus (sars-cov-2) from different geographical locations comparative genome analysis of novel coronavirus (sars-cov-2) from different geographical locations backbone rmsd was also noticed (fig 4a) . it was found that the rmsd of mutant was comparatively more stable than its wt. this variation in the average rmsd values suggests that this mutation was affecting the dynamic behavior of mpro. rmsf values of native as well as r60c mutant mpro were calculated to determine the impact of mutation on dynamic behavior of protein at residue level. rmsf plot clearly indicates the fluctuation in residues and showed the existence of higher degree of flexibility in r60c mutant mpro. it was found that the maximum amino acid fluctuation was in the region 50-76 and 127-222 ( fig 4d) . the binding studies also confirm that the residues falling within this region were very much involved in accommodating the inhibitor within the active site of mpro [29, 31] . throughout the md trajectory, the interaction energy of ligand in complex with the surrounding protein residues of wt and mutant mpro were calculated. the lennard-jones shortrange (lj-sr) and coulombic short-range (coul-sr) potential energies were calculated throughout the course of 50 ns of md simulation (fig 4e) . the average interaction energy for 50 ns are shown in table 7 . it was found that the binding of inhibitor within the active site of mpro (wt) was stronger as compared to the r60c mutant. the analysis of hydrogen bond network revealed that the r60c mutation also cause disturbance in the interactions with inhibitor as well as other surrounding active site residues of mpro. a large fluctuation was noticed in the hydrogen bond network of mpro and its r60c mutant (fig 5a) . it was found that r60c mutation results in the changes in local environment that cascade further to the short helix and loop of the catalytic active site of mpro. till date (april 23, 2020), 2.6 million cases of covid-19 have been reported worldwide. in this study, we analyzed 13 complete sequences of sars-cov-2 reported from 13 different countries and compared with sars-cov. phylogenetic analysis showed that sars-cov-2 sequences clustered together in a single clad irrespective of their geographic origin, whether from the same continent or neighboring countries. replicase polyprotein, which are most conserved among coronaviruses, shared 87% amino acid sequence similarity to sars-cov, less than the threshold value (90%) for demarcation of betacoronavirus species [27] . they belong to new virus species severe acute respiratory syndrome-related coronavirus of genus betacoronavirus [28] . among all known rna viruses, coronaviruses consist of the largest genome (26.4 to 31.7 kb) [36, 37] . the large genome size provides more plasticity in accommodating and modifying genes [36] [37] [38] . mutation frequency is very high in rna viruses, which enhances virulence and responsible for the formation of new species [19] . the high frequency of mutation within the viral genome at different geographical locations may be one of the reasons that sars-cov-2 is responsible for change in mortality rate and symptom of the disease [39] . the comparison of amino acid sequences of replicase polyprotein of 13 sars-cov-2 showed mutations in india, china, south korea, sweden, vietnam and brazil strains at different amino acid locations. earlier report showed the similar result i.e. a single mutation in replicase polyprotein at 3606 (l to f) [40] . we could identify few more single amino acid mutations at different positions in above mentioned sars-cov-2 strains ( table 1 ). the replicase polyprotein codes for nsp2 and nsp3 and it has been suggested in previous research that the mutation in nsp2 and nsp3 play a key role in infectious capability and are responsible for the differentiation mechanism of sars-cov-2 [39] . the rbd of spike protein is the region which specifically interact with ace2 leading to viral entry into the host cell [41] [42] [43] . the indian isolate of sars-cov-2 showed mutation within this region where at 408 position, arginine is replaced by isoleucine. for several years, the prediction of protein stability via theoretical or experimental approaches has been a profound area of research [44] . earlier findings suggest that a single point mutation at rbd is responsible for disrupting the antigenic structure, thereby, affecting the binding of rbd to ace2 [45, 46] . the mutation within this region of spike protein may affect the binding of rbd to its receptor, thus, affecting the viral entry within the host cells. further, in silico studies revealed that this point mutation within the rbd of spike glycoprotein was having stabilization effect on spike protein and found to increase the protein stability (δδg 0.49732107 kcal/mol). single amino acid mutation was observed in both mpro (r60c) of sars-cov-2 vietnam isolate and rdrp (a408v) of sars-cov-2 india isolate. the in silico findings revealed that the mutations in both strains decrease the stability of protein. the md simulation studies on mpro further confirmed that the point mutation on mpro affects the stability of proteins as well as the binding of inhibitor. our in silico study found that the catalytic active site of mpro is surrounded by amino acid residues of a loop (142-145, 175-200), short helix (40) (41) (42) (43) (46) (47) (48) (49) (50) , and beta sheet regions (25) (26) (27) (164) (165) (166) (167) . the r60c mutant lies at helix adjacent to the short helix (h2) that forms the catalytic channel (fig 5) . substitution of an amino acid with charged side chain to uncharged cysteine residue leads to loss of conserved ionic bond interaction and the effect cascades to other conserved ionic interactions. loss of conserved ionic interaction was observed between amide nitrogen of arginine and carboxylic oxygen atom of aspartic acid at position 48 of the catalytic channel. it was found that the short helix h2, that form the catalytic channel, have attained a more flexible loop like conformation in the mutant protein. conserved hydrogen bonded interactions that stabilizes the catalytic channel l1 loop between tyr54 oh� � �asp187 oδ1, tyr54 oh� � �asp187 o and leu50 o� � �arg188 ne were lost in the mutant enzyme, thereby, increasing the flexibility of structure forming the binding pocket ( fig 5b) . therefore, the local change of an ordered secondary structure to a more disordered loop like structure have increased the overall flexibility of the secondary structure elements forming the catalytic pocket, thereby, effecting the binding of ligand to residue in the catalytic channel. this is quite evident from the reduced lj-sd and coulombic-sr interaction energies between the enzyme and ligand in wild and mutant complexes ( fig 4e and table 7 ). the role of important active site residues, discussed in this study, has been reported before [47, 48] . the rmsf plot also reveals the key role of these residues in accommodating the inhibitor within the active site of mpro. envelop protein plays an important role in the assembly of viral genome and the formation of ion channels (ic), responsible for virus-host interaction, which is mainly associated with pathogenesis [5, 49] . we detected one amino acid mutation l37h in transmembrane domain (tmd) of envelop protein of sars-cov-2 south korea isolate. the tmd is hydrophobic in nature consisting mainly hydrophobic amino acids, while the mutation in tmd at 37 position changes hydrophobic to hydrophilic amino acid which changes the integrity of tmd. earlier report showed that mutations within the tmd domain of envelop protein completely disrupted ic activity [50] . this might be one of the reasons for slow spreading/low pathogenicity of sars-cov-2 in south korea. nucleocapsid protein of coronavirus is necessary for rna replication, transcription and genome packaging [51, 52] . a mutation p344s in nucleocapsid protein has been detected in sars-cov-2 japan strain. the p344s mutation on nucleocapsid was found to decrease the protein stability (δδg -1.2252261). this mutation is located in carboxy-terminal rna-binding domain (ctd) of nucleocapsid protein. earlier studies showed that ctd is responsible for oligomerization [53] . it was also revealed that among all the genomes studied in this study, the indian sars-cov-2 isolates were carrying maximum mutation. the indian isolates were carrying the r408i on the spike protein while a406v on the rdrp and several mutations on the replicase polyprotein of sars-cov-2. it is expected that these large number of mutations among the sars-cov-2 may affect the vaccine/inhibitor development against these isolates. to conclude, sars-cov-2 complete sequences from 13 countries were analyzed and compared with sars-cov. we identified country specific mutations in major proteins (replicase polyprotein, spike protein, envelop protein and nucleocapsid protein). further, molecular dynamics and other in silico studies revealed that mutations decrease the stability of protein and also hinders the binding of inhibitor. mutation r408i in spike protein of indian strain has significant influence on rbd domain of spike protein and this point mutation has a stabilization effect on the spike protein. the findings of the present study could help for the design of potential vaccine candidates/small molecular inhibitor against covid19. supporting information s1 file. (zip) identification of a novel coronavirus in patients with severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia a pneumonia outbreak associated with a new coronavirus of probable bat origin a novel coronavirus from patients with pneumonia in china coronavirus envelope protein: current knowledge the molecular biology of coronaviruses mers-cov virus-like particles produced in insect cells induce specific humoural and cellular imminity in rhesus macaques efficient assembly and release of sars coronavirus-like particles by a heterologous expression system the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme fused-ring structure of decahydroisoquinolin as a novel scaffold for sars 3cl protease inhibitors the rna polymerase activity of sars-coronavirus nsp12 is primer dependent middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus in bats, saudi arabia isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor discovery of a rich gene pool of bat sars-related coronaviruses provides new insights into the origin of sars coronavirus isolation and characterization of viruses related to the sars coronavirus from animals in southern china molecular evolution analysis and geographic investigation of severe acute respiratory syndrome coronavirus-like virus in palm civets at an animal market and on farms sars-cov infection in a restaurant from palm civet why are rna virus mutation rates so damn high? severe acute respiratory syndrome coronavirus sequence characteristics and evolutionary rate estimate from maximum likelihood analysis prediction of protein stability changes for single-site mutations using support vector machines comparative protein structure modeling using modeller python: a programming language for software integration and development systematic validation of protein force fields against experimental data are protein force fields getting better? a systematic benchmark on 524 diverse nmr measurements charmm additive all-atom force field for carbohydrate derivatives and its utility in polysaccharide and carbohydrate-protein modeling international committee on taxonomy of viruses coronaviridae study group of the international committee on taxonomy of v. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs prediction of the sars-cov-2 (2019-ncov) 3c-like protease (3cl (pro)) structure: virtual screening reveals velpatasvir, ledipasvir, and other drug repurposing candidates structure of m(pro) from covid-19 virus and discovery of its inhibitors structure of the rna-dependent rna polymerase from covid-19 virus structure of sars coronavirus spike receptor-binding domain complexed with receptor genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding insight into the effect of inhibitor resistant s130g mutant on physico-chemical properties of shv type beta-lactamase: a molecular dynamics study comparative analysis of complete genome sequences of three avian coronaviruses reveals a novel group 3c coronavirus identification of a novel coronavirus from a beluga whale by using a panviral microarray coronavirus genomics and bioinformatics analysis covid-2019: the role of the nsp2 and nsp3 in its pathogenesis the establishment of reference sequence for sars-cov-2 and variation analysis characterization of the receptor-binding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine the spike protein of sars-cov-a target for vaccine and therapeutic development angiotensin-converting enzyme 2: a functional receptor for sars coronavirus can one predict protein stability? an attempt to do so for residue 133 of t4 lysozyme using a combination of free energy derivatives, profec, and free energy perturbation methods structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor unravelling lead antiviral phytochemicals for the inhibition of sars-cov-2 m(pro) enzyme through in silico approach in silico screening of natural compounds against covid-19 by targeting mpro and ace2 using molecular docking the coronavirus e protein: assembly and beyond severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis nucleocapsid protein-dependent assembly of the rna packaging signal of middle east respiratory syndrome coronavirus coronavirus genomic rna packaging oligomerization of the carboxyl terminal domain of the human coronavirus 229e nucleocapsid protein the authors are very grateful to prof. indranil dasgupta, university of delhi south campus, new delhi, india, for giving suggestions to improve the manuscript. key: cord-308480-t2vukbwp authors: liang, zhongjie; li, lianchun; wang, yuanyuan; chen, limin; kong, xiangqian; hong, yao; lan, lefu; zheng, mingyue; guang-yang, cai; liu, hong; shen, xu; luo, cheng; li, keqin kathy; chen, kaixian; jiang, hualiang title: molecular basis of ndm-1, a new antibiotic resistance determinant date: 2011-08-24 journal: plos one doi: 10.1371/journal.pone.0023606 sha: doc_id: 308480 cord_uid: t2vukbwp the new delhi metallo-β-lactamase (ndm-1) was first reported in 2009 in a swedish patient. a recent study reported that klebsiella pneumonia ndm-1 positive strain or escherichia coli ndm-1 positive strain was highly resistant to all antibiotics tested except tigecycline and colistin. these can no longer be relied on to treat infections and therefore, ndm-1 now becomes potentially a major global health threat. in this study, we performed modeling studies to obtain its 3d structure and ndm-1/antibiotics complex. it revealed that the hydrolytic mechanisms are highly conserved. in addition, the detailed analysis indicates that the more flexible and hydrophobic loop1, together with the evolution of more positive-charged loop2 leads to ndm-1 positive strain more potent and extensive in antibiotics resistance compared with other mbls. furthermore, through biological experiments, we revealed the molecular basis for antibiotics catalysis of ndm-1 on the enzymatic level. we found that ndm-1 enzyme was highly potent to degrade carbapenem antibiotics, while mostly susceptible to tigecycline, which had the ability to slow down the hydrolysis velocity of meropenem by ndm-1. meanwhile, the mutagenesis experiments, including d124a, c208a, k211a and k211e, which displayed down-regulation on meropenem catalysis, proved the accuracy of our model. at present, there are no effective antibiotics against ndm-1 positive pathogen. our study will provide clues to investigate the molecular basis of extended antibiotics resistance of ndm-1 and then accelerate the search for new antibiotics against ndm-1 positive strain in clinical studies. the new delhi metallo-b-lactamase (ndm-1) was first reported in 2009 in a swedish patient, who travelled to new delhi and acquired a urinary tract infection caused by klebsiella pneumonia [1] . a recent study reported that klebsiella pneumonia ndm-1 positive strain or escherichia coli ndm-1 positive strain was highly resistant to all antibiotics tested except tigecycline and colistin [2] . since august 2010, the spreading and dissemination of ndm-1 positive strain has occurred, with cases being globally reported by medias from countries including united states, canada, sweden, united kingdom, austria, belgium, france, netherlands, germany, africa, oman, australia, japan and china [3] . although ndm-1-positive cases are not currently prevalent in the worldwide, it can spread through renal or bone marrow transplantation, dialysis, cerebral infarction, chronic obstructive pulmonary disease, pregnancy, burns, road traffic accidents, and cosmetic surgery. in addition, ndm-1 positive strain can destroy carbapenem antibiotics such as meropenem, imipenem, doripenem and ertapenem by breaking down the carbapenem groups of antibiotics, which have been serving as the basis for the treatment of antibiotic-resistant bacterial infections and now can no longer be relied on for this purpose due to the emergence of nmd-1 positive strain. therefore, the spread of the pathogenic microorganisms carrying ndm-1 gene (also been called ''super bugs'') now becomes potentially a major global health threat. ndm-1 belongs to the metallo-b-lactamase (mbl, class b) family containing zn 2+ and other divalent cations as cofactors. it inactivates almost all classes of b-lactams antibiotics including carbapenems by catalyzing the hydrolytic cleavage of the substrate amide bond. on the basis of the protein sequence similarities, three different lineages, named as subclass b1, b2 and b3 have been characterized. a number of experimental and theoretical studies have also been devoted to understanding structural and mechanistic properties of mbls [4, 5, 6, 7] . however, since this novel mbl-ndm-1 is likely more potent and extensive than known mbls in inactivating b-lactams antibiotics, it is urgent for us to address the ligand binding properties and catalytic mechanism of ndm-1 in order to light up the road for the development of novel antibiotics to combat emerging ndm-1 positive pathogen. to explore the molecular basis for antibiotics hydrolysis by ndm-1, homology modeling method was performed to obtain the 3d structure. then molecular docking method was applied to obtain the binding modes with antibiotics and the comparison of ndm-1 with other mbls complexes was also investigated. in addition, ndm-1 catalyzed hydrolysis of various antibiotics substrates was monitored by following the absorbance variations resulting from the opening of the b-lactam ring, which suggested that ndm-1 displays different resistant abilities to different kinds of antibiotics. based on the modeling results, four point mutants, including d124a, c208a, k211a and k211e, were made and their enzymatic activities were measured comparing with the wildtype. the significantly decreased activity of mutants validated the accuracy of our model. on the other hand, it shed light on the catalytic mechanism of the novel ndm-1 from the molecular basis for the first time. furthermore, enzymatic activities toward different antibiotics tested in our study would provide clues to accelerate the new antibiotics design against ndm-1 positive strain in further studies. overall structure of ndm-1 from homology modeling as the multiple sequences alignment shown in figure 1a , among vim-4 (b1 subclass), cpha (b2 subclass) and fez-1 (b3 subclasses), ndm-1 is highly homologous to the b1 subclass (vim-4, sequence identity is 37%). in addition, sequence alignment indicates that ndm-1 contains the identical coordinating residues (his-his-his) in the zn 2+ (i)-binding site and (asp-cys-his) in the zn 2+ (ii)-binding site of the b1 subclass, which implies that ndm-1 belongs to the b1 subclass of mbls. accordingly, the crystal structure of vim-4 (pdb id: 2whg) was used as the template in homology modeling [8] . as shown in figure 1b , the overall structure of ndm-1 shares the common characteristic folding of ab/ba sandwich, which resembles the architectural features in other b1 subclass. the active site of ndm-1 is highly homologous to that of vim-4. in the active site (figure 2a ), zn 2+ (i) is coordinated with three conserved histidine residues 120, 122 and 189, while zn 2+ (ii) is coordinated with the conserved residues asp124, cys208 and his250, which is exactly consistent with the x-ray crystal structures [9, 10] . a water molecule bridging both zinc ions acts as the nucleophile during the b-lactam hydrolysis. two mobile loops in the active site shown in figure 1b are crucial for substrate recognition, binding and catalysis in mbls [11, 12] . in ndm-1, the loop1, also called the flapping loop, is composed of amino acids ldmpgfgava (residues 65-74). compared with the loop1 (qsfdgavyp) in vim-2 and vim-4, the size of the active site cavity is enlarged with less bulky amino acids, suggesting a broader substrate profile. besides it is worth noting that it is more hydrophobic than others [9] . there is only one benzene ring of phe70 in the middle of the loop1 of ndm-1, and the side-chains of asp66 and met67 pointed to the solvent, the less bulky amino acids, especially for the glycines, are proposed to make the loop1 more flexible for substrate binding. in the crystal structure of ndm-1 in complex with hydrolyzed ampicillin (pdb id: 3q6x) [9] , the loop1 represents a more open state compared with our model, while in the crystal structure of apo ndm-1 (pdb id: 3s0z) [10] , the loop1 displays a semi-closed state. consequently, it is proposed to undergo significant conformational changes during the substrate binding, in different states with or without substrate. regarding the loop2, arg185 and asn190 in vim-2 was proved to play important roles for substrate binding, catalysis and inhibition through h-bond interactions [12, 13] . while in ndm-1, the arginine is mutated to ala215, which also enlarges the size of active site cavity. moreover, lys211 instead of tyr181 in vim-2, probably plays the similar role of arg185 in vim-2 by forming electrostatic interaction with the carboxyl of substrate. the detailed comparison of the two loops in ndm-1 and vim-2 is shown in figure 2b . furthermore, two residues, lys125 and tyr229, were pointed to play crucial roles in stabilizing the conformation of the active site, through h-bond network (with residues asn76, asp90, thr91, his122 and ser249) and hydrophobic interactions (with residues leu209, leu218, leu221 and leu269) [9] . besides the unique residues in the two loops, the n terminus of ndm-1 is longer than other analogues. even without modeled in our structure, it is speculated to assist in loop1 packing so as to affect the hydrolytic catalysis. ndm-1 also contains an additional insert between residues 162 and 166, which is not present in other mbls. since the insert is in the opposite side of the active site, with a distance of around 20 å , its role in the hydrolysis reaction is still unknown. in summary, the unique structural characteristics probably contributes to the more potent hydrolysis and broader substrate range of the novel mbl-ndm-1, especially for those bulkier antibiotics, which are not degraded by other mbls. after the 3d structure of ndm-1 was modeled, molecular docking study was performed. two reported effective antibiotics (tigecycline and colistin) against ndm-1 positive strains [2] and other 13 well-known antibiotics including carbapenem, which were reported to be destroyed by ndm-1 positive pathogen, were docked into the active site of ndm-1. the molecular docking data indicates that the latter 13 antibiotics fit the active site of ndm-1 quite well. taking two typical antibiotics, imipenem and carbapenem as example, the docked complex structures revealed that although the antibiotics adopted diverse conformations in the active site, the lactam motifs were positioned in the same orientation by coordinating with zinc ions tightly ( figure 2c ), which suggested that the catalytic mechanisms were highly conserved among b1 subclass enzymes, as shown in figure 3 . substrate-binding polarizes the lactam bond due to coordination of the carbonyl oxygen with zn 2+ (i) and the lactam nitrogen with zn 2+ (ii). attack of the water oxygen leads to oxyanion stabilized by zn 2+ (i), which is followed by subsequent cleavage of the c-n bond. then, the lactam nitrogen is expelled as an anion and stabilized by coordination with zn 2+ (ii) acting as a general acid. the last step is the protonation of the nitrogen, which is considered as the rate-limiting and the most controversial step to date [4] . compare to other known b1 subclass enzymes such as vim-2 and vim-4, the loop2 of ndm-1 harbors a lysine-rich positive-charged region, which is more favorable for protonation, probably accounting for its potent catalytic activity at least in part. notably, colistin, the reported antibiotic susceptible for ndm-1 positive pathogens, was not able to fit the active site well probably due to its bulky volume. comparison the complex structure of ndm-1/ meropenem with vim-2/meropenem and fez-1/ meropenem to gain the structural insight into the mechanism of the potent hydrolysis of ndm-1, the intermolecular interactions of three models of ndm-1, vim-2 and fez-1 in complex with antibiotics meropenem were compared and analyzed in details ( figure 4a -c). in ndm-1 complex structure, the lactam oxygen and the carboxyl oxygen atoms coordinate with zn 2+ (i) and zn 2+ (ii) with distances of 3.56 å and 2.64 å respectively. in addition, the h-bond between the atom n d2 of asn220 and the lactam oxygen atom with distance of 2.96 å also contributes to the polarization of the lactam bond. meanwhile, the water molecule, which bridges with the two zinc ions with distances of 1.88 å and 2.40 å respectively, can serve as the nucleophile to attack the atom c of b-lactam. the carbonyl methyl of meropenem forms h-bonds with the atom n e2 of gln123. together with the fact that the relatively bulky active site cavity caused by the residue variations in the flexible loop1, ndm-1 is potent to hydrolyze bulkier antibiotics. different with ndm-1/meropenem, the vim-2/ meropenem has a strong h-bond between the carboxyl of meropenem and arg185 in the loop2. moreover, phe61 and try67 in the loop1 form hydrophobic interactions with meropenem, and dramatically reduce the size of the active site cavity. structurally, the hydrophobic interactions make the lactam ring rotate to the loop1 and elongate the distance between the lactam oxygen and zn 2+ (i) to 4.3 å . (figure 4b ) consequently, the shallow active site makes the lactam more difficult to be positioned in a proper orientation for catalysis, which probably explains its weaker activity to catalyze antibiotics compared with ndm-1. in contrast, fez-1 shares little structural similarity with ndm-1 and vim-2, especially for that the flapping loop is not conserved in fez-1, which leads to a flat active site cleft [14] . obviously,meropenem mediates no other interactions with fez-1 except for the lactam motif ( figure 4c ), suggesting the low binding affinity for substrates. and the fez-1/meropenem flexible characteristic makes the antibiotic little access to proper orientation for hydrolysis. in agreement with our intermolecular interaction models of enzymes with antibiotics, the reported minimal inhibitory concentrations of meropenem for vim-2 and fez-1 positive e.coli strains are both 0.25 mg/ml, while for ndm-1 positive e.coli strain, it is 32 mg/ml [2, 15] , suggesting that ndm-1 is probably more potent in antibiotics hydrolysis. besides, the binding modes of hydrolyzed antibiotics were also investigated. taking the hydrolyzed meropenem for example ( figure 5) , it is noticed that the original carboxyl formed electrostatic interactions with lys211. the hydrophobic residues such as leu65, val73 and ala74 in the loop1 formed stable hydrophobic interactions with the dimethylamino group. it appears that the conformations of antibiotics undergo slight exchange during the catalysis. to validate the results extracted from the in silico study, we went ahead to perform ''wet'' experiments to further explore the detailed molecular basis for the catalytic mechanism of ndm-1. firstly, we constructed a 66his and sumo (small ubiquitin-related modifier) tagged ndm-1 expression plasmid and overexpressed sumo-ndm-1 in e.coli bl21(de3) strain; then we purified fusion protein by ni-column affinity chromatography, the ndm-1 protein was released from fusion protein by ulp-1 (ubiquitin-like protein-specific protease 1) protease cleavage. more than 95% pure ndm-1 protein was obtained by final gel filtration chromatography ( figure 6a, b and c) . secondly, the ndm-1 catalyzed hydrolysis of various antibiotics substrates was monitored by following the absorbance variations resulting from the opening of the b-lactam ring. the susceptibilities of seven typical antibiotics were assessed. the chemical structures of these antibiotics are displayed in figure 7 . as shown in figure 8a , ndm-1 rapidly hydrolyzes the carbapenem antibiotics including meropenem and imipenem, and it harbored moderate catalytic hydrolysis ability against cephalosporin antibiotics, including ceftazidime, cefotaxime and cefpirome. in contrast, ndm-1 is susceptible to tigecycline and monobactam-aztreonam, which is similar with the catalytic ability of vim-4 and ndm-1-positive enterobacteriaceae [2, 8] . to explore the detailed mechanism for the susceptibility of the two antibiotics against ndm-1 positive strain, 100 mm meropenem was used as ndm-1 substrate, then 50 mm and 150 mm tigecycline or aztreonam were mixed with the reaction system as the inhibitors. surprisingly, tigecycline probably displayed a certain degree of inhibition against ndm-1. the hydrolysis velocity of meropenem by ndm-1 is partially slowed down when different concentrations of tigecycline were added into the reaction system ( figure 8b ). for another ndm-1 positive strain susceptive antibiotic aztreonam, we didn't see any inhibitory effects on ndm-1 favorite substrate (data now shown). in short, our data revealed the molecular basis of the novel mbl-ndm-1, of which ndm-1 positive strain possessed potent resistance to carbapenems. on the other hand, we identified a weak inhibitortigecycline, which was previously reported to inhibit the growth of ndm-1 harbored klebsiella pneumonia [2] . our work may pave the road in designing inhibitors and new antibiotics susceptible against ndm-1. meanwhile, based on our 3d model, we designed the following point mutants, including, d124a, c208a, k211a and k211e. then the activity of the mutants for meropenem was measured and the result is shown in figure 8c . it is obvious that the mutants almost completely lose their activity in antibiotics hydrolysis. this result in turn verified the accuracy of our 3d model in which the zn 2+ (ii) is coordinated with the conserved residues asp124 and cys208, in spite that it is still intriguing whether the effect is structural or functional. however, the decreased catalytic activity implied the mechanism should be zn 2+ (ii) assisted, supporting the proposal that zn 2+ (ii) ion is crucial for stabilizing the development of a negative charge on the b-lactam nitrogen atom(as show in figure 3 ). regarding k211a and k211e mutants, the significantly decreased hydrolysis activity indicated that the electrostatic interaction between the original carboxyl group of hydrolyzed antibiotics and positive-charged side-chain of k211 probably afford one of the driving force for the catalysis. while with respect to loop1 (ldmpgfgava), we did the loop displacement with qsfdga-vyp in vim-2 and vim-4. different with the point mutants, the hydrolytic catalysis didn't decrease compared with the wild-type, which means only the loop1 change wouldn't affect the hydrolytic catalysis very much. moreover, it was reported that a121f, q123d and a121f/q123d mutants strongly weakened the imipenem hydrolysis activity of ndm-1, proving the crucial roles of the unique ha(121)hq(123)d motif compared with hfhdd motif in other mbls [10] . furthermore, since loop1 and loop2 are critical for substrate binding and catalysis, more attention would be paid on the glycines in loop1 and lysines in loop2. nowadays, the antibiotics resistance in gram-negative bacteria has already been a great risk to the public health. the newly emergent ndm-1, called ''superbug'', possesses more potent hydrolysis ability toward almost all antibiotics and hence becomes a new threat in clinical surgery. this is one typical example of the remarkable ability of bacteria to adapt and eventually become resistant to new antibiotics. it is made much easier by the existence of plasmids, which can transmit easily from bacterium to bacterium. in this study, the structural models of ndm-1/ antibiotics complex were obtained from homology modeling and molecular docking methods. the detailed analysis indicates that the loop1 of more flexibility and hydrophobic, together with the loop2 of more positive-charged, leads to ndm-1 more potent in antibiotics hydrolysis compared with other mbls. meanwhile, the experiments proved that ndm-1 was highly resistant to carbapenems and cephalosporins and susceptible to aztreoname and tigecycline, which was firstly implied to slow down the hydrolysis velocity of meropenem by ndm-1 in our study. moreover, the mutant results displayed the molecular basis for the catalytic mechanism. at present, there are no effective antibiotics against ndm-1 positive pathogen. an appreciated strategy is to identify drug candidates from the existing antibiotics, such as tigecycline, based on the 3d model of ndm-1 by using structurebased virtual screening (e.g., molecular docking and ligand-based, receptor-based and pharmacophore-based drug design) in conjunction with bioassay. this strategy has been used successfully in the discovery of the compound cinanserin against sars (severe acute respiratory syndrome) [16] . taken together, our study provided clues to investigate the molecular basis of extended antibiotics resistance of ndm-1 and shed light upon the discovery of new antibiotics against ndm-1 positive strains in clinical studies. the protein sequence of ndm-1 from enterococcus faecium (hq256747) was retrieved from ncbi (http://www.ncbi.nlm.nih. gov) protein database. blastp program was then performed to search for its homologues from the rcsb protein databank [17] . accordingly, the crystal structure of vim-4 (pdb id: 2whg) was selected as the template [8] , whose sequence identity with ndm-1 is 37%. to analyze the sequence conservation, sequences alignment of ndm-1, vim-4, cpha and fez-1 was performed and gaps were inserted into the sequences to find an optimal alignment as shown in figure 1 . the 3d structure of ndm-1 was then modeled by using the insightii software (accelrys inc., san diego, ca, usa) and optimized by energy minimization using the amber force field implemented in the sybyl software package. it was minimized gradually (hydrogens, side-chains, all) using the constraints in heavy or backbone atoms to alleviate any remaining bad steric contacts. the optimized structure of ndm-1 was then subjected to evaluation by procheck and profiles-3d to examine the stereochemical quality and the structural rationality [18, 19] . after verified the rationality, the 3d structure of ndm-1 was subjected to the subsequent study. glide calculations were performed with maestro v7.5(schrodinger, inc.) [20] . hydrogen atoms and charges were added during a brief relaxation performed using the protein preparation module in maestro with the ''preparation and refinement'' option, and a restrained partial minimization was terminated when the root-mean-square deviation (rmsd) reached a maximum value of 0.3 å in order to relieve steric clashes. the grid-enclosing box was centered on the zn 2+ (i) and defined so as to enclose residues located within 10 å , and a scaling factor of 1.0 was set to van der waals (vdm) radii of those receptor atoms with the partial atomic charge less than 0.25. in the docking process, extra-precision (xp) docking was adopted to generate the minimized pose, and the glide scoring function (g-score) was used to select the final 20 poses for each antibiotic. together with reported study, the reasonable poses were used for the binding mode analysis. all antibiotics used in this study were purchased from j&k scientific ltd., except for kanamycin purchased from sangon biotech(shanghai) co., ltd.. the ndm-1 gene, which was obtained as a 900 bp pcr product from huashan hospital. a new pcr product with restriction sites (ndei on forward site and xhoi on reverse site) encoding truncation protein from amino acid q37 to the last amino acid (designated as ndm-1 37-270 ) of ndm-1 were constructed into a modified pet28 vector. as a result, ndm-1 37-270 were expressed as fusion protein with a n-terminal 66his -sumo (small ubiquitin-related modifier) tag. the quickchange ii site-directed mutagenesis kit (stratagene, la jolla, ca) was used to introduce all the mutations, including d124a,c208a,k211a,k211e, replacing residues of loop1(65-74, ldmpgfgava) with vim-2 corresponding residues (40-48,qsfdgavyp), into the gene of ndm-1 37-270 . the expression vectors were transformed into e.coli strain bl21(de3). a 5-ml overnight culture of these transformed bacteria in luria-bertani (lb) medium containing 50 mg/ml kanamycin was used to inoculate 1 liter of lb medium containing 50 mg/ml kanamycin and 20 mm zncl 2 . bacteria were cultured at 37uc with shaking, until reach an optical density at 600 nm of 0.6, then transferred to 16uc, induced by 0.4 mm iptg overnight. the bacteria were collected by centrifugation and resuspended in 20 ml lysis buffer containing 20 mm tris, ph 8.0, 200 mm nacl, 0.1% b-mercaptoethanol per liter culture. the bacteria were ruptured by sonication, and the bacteria debris was removed by centrifugation at 18000 rpm for 30 min. the cleared supernatant was load onto a ni-column, which pre-equilibrated by lysis buffer, and washed with lysis buffer supplemented with 10 mm imidazole. then ulp-1 (ubiquitin-like protein-specific protease 1) was add into the ni-column, digest the fusion protein at 4uc overnight. collect the flow through, concentrated and load onto a superdex 75 column, which pre-equilibrated by 20 mm tris, ph 8.0, 150 mm nacl, 5 mm dtt. fractions containing ndm-1 37-270 and corresponding mutation proteins were pooled and concentrated, then stored at 280uc freezer. protein purity was more than 95%, estimated by sodium dodecyl sulfate (sds)polyacrylamide gel electrophoresis. the final proteins concentration were determined by using the molar extinction coefficient at 280 nm of 27,880 m 21 cm 21 . the ndm-1 catalyzed substrate hydrolysation reaction were carried out in uv-starh 96 well microplates (greiner bio-one ltd.) at room temperature within reaction buffer 50 mm hepes, ph 7.5. the antibiotics hydrolysation were monitored by tecan infinite 200 multimode and absorbance microplate readers (tecan group ltd.) in the peak absorbance of various antibiotics. characterization of a new metallo-lactamase gene, blandm-1, and a novel erythromycin esterase gene carried on a unique genetic structure in klebsiella pneumoniae sequence type 14 from india emergence of a new antibiotic resistance mechanism in india, pakistan, and the uk: a molecular, biological, and epidemiological study new delhi metallo-beta-lactamase (ndm-1): towards a new pandemia metallo-beta-lactamase: structure and mechanism the mechanism of catalysis and the inhibition of the bacillus cereus zinc-dependent beta-lactamase on the mechanism of the metallo-betalactamase from bacteroides fragilis hybrid qm/mm and dft investigations of the catalytic mechanism and inhibition of the dinuclear zinc metallo-beta-lactamase ccra from bacteroides fragilis biochemical and structural characterization of the subclass b1 metallo-{beta}-lactamase vim-4 crystal structure of ndm-1 reveals a common {beta}-lactam hydrolysis mechanism a structural view of the antibiotic degradation enzyme ndm-1 from a superbug analysis of the importance of the metallo-beta-lactamase active site loop in substrate binding and catalysis crystallographic investigation of the inhibition mode of a vim-2 metallo-betalactamase from pseudomonas aeruginosa by a mercaptocarboxylate inhibitor mercaptophosphonate compounds as broad-spectrum inhibitors of the metallobeta-lactamases three-dimensional structure of fez-1, a monomeric subclass b3 metallo-betalactamase from fluoribacter gormanii, in native form and in complex with dcaptopril metallo-beta-lactamases (classification, activity, genetic organization, structure, zinc coordination) and their superfamily why are oseltamivir and zanamivir effective against the newly emerged influenza a virus (a/h1n1)? basic local alignment search tool procheck: a program to check the stereochemical quality of protein structures} a method to identify protein sequences that fold into a known three-dimensional structure glide: a new approach for rapid, accurate docking and scoring. 1. method and assessment of docking accuracy we gratefully thank dr. xin liu from stanford university for his helpful comments. we deeply appreciate dr. fupin hu from huashan hospital giving us the ndm-1 pcr fragment. key: cord-335880-m8gecsf0 authors: peci, adriana; winter, anne-luise; warshawsky, bryna; booth, tim f.; eshaghi, alireza; li, aimin; perusini, stephen; olsha, romy; marchand-austin, alex; kristjanson, erik; gubbay, jonathan b. title: epidemiology of enterovirus d68 in ontario date: 2015-11-23 journal: plos one doi: 10.1371/journal.pone.0142841 sha: doc_id: 335880 cord_uid: m8gecsf0 in august 2014, children’s hospitals in kansas city, missouri and chicago, illinois notified the centers for disease control and prevention (cdc) about increased numbers of pediatric patients hospitalized with severe respiratory illness (sri). in response to cdc reports, public health ontario laboratories (phol) launched an investigation of patients being tested for enterovirus d-68 (ev-d68) in ontario, canada. the purpose of this investigation was to enhance our understanding of ev-d68 epidemiology and clinical features. data for this study included specimens submitted for ev-d68 testing at phol from september 1, 2014 to october 31, 2014. comparisons were made between patients who tested positive for the virus (cases) and those testing negative (controls). ev-d68 was identified in 153/907 (16.8%) of patients tested. in the logistic regression model adjusting for age, sex, setting and time to specimen collection, individuals younger than 20 years of age were more likely to be diagnosed with ev-d68 compared to those 20 and over, with peak positivity at ages 5–9 years. cases were not more likely to be hospitalized than controls. cases were more likely to be identified in september than october (or 8.07; 95% ci 5.15 to 12.64). routine viral culture and multiplex pcr were inadequate methods to identify ev-d68 due to poor sensitivity and inability to differentiate ev-d68 from other enterovirus serotypes or rhinovirus. testing for ev-d68 in ontario from july to december, 2014 detected the presence of ev-d68 virus among young children during september-october, 2014, with most cases detected in september. there was no difference in hospitalization status between cases and controls. in order to better understand the epidemiology of this virus, surveillance for ev-d68 should include testing of symptomatic individuals from all treatment settings and patient age groups, with collection and analysis of comprehensive clinical and epidemiological data. in august 2014, children's hospitals in kansas city, missouri and chicago, illinois notified the centers for disease control and prevention (cdc) about increased numbers of pediatric patients hospitalized with severe respiratory illness (sri) [1] . laboratory testing identified enterovirus d68 (ev-d68) in respiratory specimens from most of these children. from mid-august 2014 to january 15, 2015, 1,153 ev-d68 cases in 49 us states were confirmed by cdc. reports indicated many of these children had a history of asthma or wheezing [2] . a total of 214 ev-d68 positive patients were confirmed in canada from august to october, 2014. as of december 9, 2014, cases were reported from alberta, british columbia, manitoba, new brunswick, nova scotia, ontario, quebec, prince edward island and saskatchewan [3] . ev-d68 is a member of the picornaviridae family [1] . although ev-d68 is an enterovirus, it shares biological characteristics with rhinoviruses such as improved growth at low temperatures and instability at low ph [4, 5] 5. in addition, similar to rhinoviruses, ev-d68 has been isolated primarily from respiratory specimens rather than from stool specimens of patients with aseptic meningitis, which is characteristic of most other enteroviruses [4, 5] . ev-d68 has primarily been associated with respiratory disease since its discovery in 1962 [4, 5] . in the literature, the spectrum of illness attributed to ev-d68 ranges from asymptomatic to severe respiratory infection requiring hospitalization particularly in children with previous history of asthma or wheezing; however the full spectrum of disease is not yet known [4, [6] [7] [8] . a possible association with neurological syndromes such as acute flaccid paralysis was noted during the 2014 increase in the us of ev-d68, and some individuals presenting with acute flaccid paralysis (now classified as acute flaccid myelitis) had ev-d68 detected in respiratory specimens. a recent investigation of hospital patients presenting with acute flaccid myelitis [9] reported an association of ev-d68 infection and acute flaccid myelitis; however, the connection between ev-d68 infection and neurological syndromes remains unclear and causality has not been determined [10, 11] . it is difficult to know the true burden of disease related to ev-d68 since ev infections are not reportable in north america and some patients infected with the virus don't seek medical care or even when they do, routine testing does not differentiate this serotype [12] . thus, the reported incidence of infection is likely an underestimation of the true incidence [1] . while more recent investigations reported on ev-d68 infection in children who present to hospital [13] , the virus has also been identified in both children and adults seeking community-based care [9, 14] . consistent with previous reports [15] , a higher proportion of individuals in whom the virus was identified were male. in response to more recent reports from the us, public health ontario laboratories (phol), in ontario, canada launched an investigation to document clinical and laboratory data on patients being tested for ev-d68. the purpose of this investigation was to enhance our understanding of ev-d68 epidemiology and associated clinical features. specimens are collected from patients by submitters and sent to phol for testing as part of routine clinical service. these data are also used for routine laboratory surveillance, which is a mandate of public health ontario. therefore, consultation with our organization's privacy office or ethics committee was not required. to protect patient privacy and confidentiality, data are reported in an aggregated anonymized format. data for this study included specimens submitted for ev-d68 testing at phol from patients who presented with respiratory symptoms in different health care settings across ontario, from september 1, 2014 to october 31, 2014. for the purpose of this investigation, cases were individuals who tested positive for the virus whereas controls were individuals who tested negative for ev-d68; the term "patients" refers to both cases and controls. phol has had the capacity to test respiratory specimens for ev-d68 since september 24, 2014. prior to that time, all ev-d68 testing on behalf of phol was performed at the national microbiology laboratory, (nml) canada's reference laboratory. at both laboratories, primary specimens underwent total nucleic acid extraction using the biomérieux nuclisens 1 easy-mag 1 protocol (biomérieux inc. marcy i'etoile, rhone, france). screening of clinical specimens for ev-d68 at phol was performed using a real time pcr assay targeting the 5'ntr region) [16] . subsequently, all positive specimens were subjected to a semi-nested reverse transcriptase polymerase chain reaction (rt-pcr) targeting the vp1 gene coding for the capsid protein followed by sanger sequencing with bigdye v3.1 kit (life technologies inc. california, usa) to amplify of a 375 bp partial vp1 region for serotype identification) [17] . a similar testing method was performed at nml, with sequencing performed by the genomics core facility at nml [18] . sequences were assembled using sequencer 5.2.4 and compared to vp1 sequences in genbank using blast. nml also employed an ev-d68 strain-specific real-time reverse transcription pcr test [19] . testing at phol continued until december 1, 2014 and it was also performed retrospectively for some patients from whom specimens were submitted before september. a selection of specimens included in this investigation were also tested by viral culture and/ or multiplex pcr as per phol's routine laboratory testing algorithm. for virus culture testing, rhesus monkey and wi-38 (and /or mrc5) cell lines were used to isolate viruses from different specimens including respiratory specimens. growth of viruses (cytopathogenic effects) were confirmed using fluorescent monoclonal antibodies (mabs). multiplex pcr (seeplex1 rv, seeplex1 rv15 ace; seegene, usa) were used to test for adenovirus, coronavirus, enterovirus/ rhinovirus influenza a and b, human metapneumovirus, parainfluenza and rsv. statistical analyses were performed at the patient and specimen level using stata/se version 10.0 (statacorp lp, college station, tx, usa). clinical and demographic information provided on phol's laboratory requisition was used for this investigation; patient charts were not available for review as phol is the provincial reference laboratory and as such has no direct access to patient records. specimen collection date was used for these analyses and in the event that this was missing, it was replaced by the date when the specimen was received at phol. data were transformed from specimen to patient level by probabilistic patient linkage; when discrepant results (both a positive and a negative result) were identified for the same patient, the positive result took priority over the negative one. in the event more than one specimen was submitted per patient, specimen collection date of the first specimen submitted was used and the setting that indicated the highest clinical severity (e.g. hospitalized vs. community dwelling) was used to describe disease severity. percent positivity and descriptions of ev-d68 cases by timing of specimen collection, age, sex, setting, symptoms and local health unit of residence were analysed at the patient level. reports of other viruses identified in addition to ev-d68 and specimen type were described at the specimen level. bivariate and multivariate logistic regression analyses were performed at the patient level to compare cases and controls in terms of age, sex, settings, symptoms and timing of specimen collection. p-values and odds ratios with a 95% confidence interval (ci) were reported. there were a total of 907 patients tested for ev-d68 at phol from september to october 2014; of these patients 153 (16.9%) tested positive for the virus. the date for which the first ev-d68 positive specimen from a case was collected was september 4th, 2014. september 16 th , 2014 was the specimen collection date for the greatest number of cases and highest test positivity, with 18/35 (51.4%) cases (fig 1) . after september 16 th , the number of ev-d68 cases begins to decline; the specimen collection date for the last case was october 17 th , 2014. ev-d68 was not identified in any of the patients tested before september (n = 18 patients) or after october (n = 168 patients); therefore, only data from september and october were included in the final analyses. in bivariate analysis, compared to controls, ev-d68 cases were more likely to occur in september than october (or 9.47; 95% ci 5.99 to14.36) ( table 1 ). the median age of patients (i.e. cases and controls) tested was 3 years (range 3 weeks to 94 years). more than half of patients tested (686/907; 75.6%) were less than 10 years old (table 1) . in bivariate analysis, using patients 20 years of age and over as the reference, cases were more likely than controls to be less than 20 years of age; the effect peaked at ages 5-9, (or 7.58; 95% ci 3.30 to 17.40). of those patients for whom sex was reported, 525 (58.0%) were male; sex was not reported for three patients. cases and controls did not differ by sex (p>0.05). in patients for whom a setting type was reported, most patients (710/880; 80.6%) were hospitalized and (12/880; 1.4%) were admitted to an intensive care unit (icu). the remainder of patients were either seen in a community physician's office (140/880;15.9%), in the emergency department (14/880; 1.6%) or were in an institution (4/880;0.2%). in bivariate analysis when compared to controls, cases were more likely to be hospitalized as opposed to seeking care at a physician's office (or 2.51; 95%ci 1.35 to 4.68). among all patients tested for ev-d68, 609/907 (67.1%) reported at least one symptom; 463 (76%) reported undefined respiratory symptoms (i.e. when the "respiratory symptom" option was selected on the laboratory requisition without providing further clinical details), 277 (45.5%) reported fever or chills, 51(8.3%) reported cough, 37 (6.1%) reported shortness of breath, 9 (1.5%) reported headache and six (0.9%) reported vomiting. in addition, 20 (3.3%) were reported to have pneumonia and 50 (8.2%) reported as having asthma. comparing cases to controls in regards to symptoms, cases were more likely to report at least one symptom (75.8% versus 67.1%, respectively; (p-value <0.05)). in comparison to controls, undefined respiratory symptoms were reported more frequently in cases than controls, (81% versus 60.5%, respectively, (p-value <0.001)). in the logistic regression model adjusting for age, sex, setting and time from symptom onset to specimen collection, individuals younger than 20 years of age were more likely to be diagnosed with ev-d68 compared to those 20 and over (table 1) ; this effect peaked at ages 5-9 (or 5.67; 95% ci 2.14 to 15.05). in addition, cases were more likely to be identified in september than october (or 8.07; 95% ci 5.15 to 12.64). the local health unit of residence where the greatest numbers of patients tested for ev-d68 resided were peel regional with 124/907 (13.6%) patients, city of toronto 121/907 (13.3%), windsor essex county (wec) 80/907(8.9%) and halton regional 63/907 (7%). the remaining 32 health units each represented less than 5% of the other patients tested. the greatest number of cases resided in south western ontario; the city of toronto was the health unit of residence for 26/153 (17%) of cases, wec for 20/153 (13.1%), york regional 13/153 (8.5%), peel 13 (8.5%), halton regional 13/153 (8.5%), niagara regional 11/153 (7.2%) and chatham-kent (chk) for 10/153 (6.5%) cases (fig 2) . in proportion to each health unit's population in ontario, the highest incidence rate was observed in chk with 9.1 cases per 100, 000 population followed by wec with 5 cases per 100,000 population. however, when comparing the proportion of patients tested by local health units proportionate to ontario's population, both health units tested more specimens relative to their proportion of ontario's population (p-value<0.001). of the 959 respiratory specimens tested, ev-d68 was identified in 155. thirty-eight patients had more than one specimen submitted; two patients had two positive specimens identified. of all specimens tested for ev-d68, 408 (42.5%) were tested by nml only, 489 (50.9%) were specimen source for ev-d68 positive cases was either nasopharyngeal swab, respiratory aspirate, throat swab or nasal swab. percent positivity by specimen source was 146/869 (16.8%) for nasopharyngeal swab, 5/10 (50%) for respiratory aspirate, 3/17 (17.7%) for throat swab, and 1/20 (5%) for nasal swab. ev-d68 was not detected in any of the five cerebrospinal fluid (csf) specimens submitted for testing. similarly, ev-d68 was not detected in any specimens collected from bronchoalveolar lavage (bal) (n = 9), sputum (n = 3), or stool (n = 9). the other specimen types submitted for ev-d68 testing included poorly defined specimen types e.g. fluid or swab or aspirate (n = 27). of all 155 ev-d68 positive specimens, 154 were also tested by viral culture and / mrvp as per routine laboratory testing at phol. of the 148 specimens tested by viral culture, 4 (2.7%) specimens were classified as "entero-like virus", 2(1.4%) specimens as "enterovirus, probably rhinovirus" and 1(0.7%) specimen as parainfluenza 2 virus. the remaining 141(95.3%) specimens were negative for any respiratory virus. of 89 ev-d68 positive specimens tested by mrvp, 29 (32.6%) specimens were classified as rhinovirus, 1(1.1%) specimen as rhino and enterovirus, 3(3.4%) specimens as rhino and parainfluenza 4 virus, 1 (1.1%) specimen as parainfluenza and 1(1.1%) specimen as adenovirus. more than half 54 (60.7%) of ev-d68 specimens tested by mrvp were negative for any virus. in total, co-infection of ev-d68 with adenovirus or parainfluenza viruses was detected in 6 specimens. ev-d68 virus activity was identified from september to october, 2014 in ontario, canada. ev-d68 was detected in 17% of patients on whom testing was performed, with peak activity occurring in september. testing of specimens submitted prior to september 2014 and after october 2014 did not identify ev-d68. from 1987 until 2014, ev-d68 had been rarely reported in the us [20] , however the number of cases reported in 2014 increased sharply, and was much higher than previously reported [21] . in ontario, ev-d68 may have circulated prior to 2014 not all enteroviruses are typed and not all test methods are able to distinguish between rhinovirus and enterovirus. routine viral culture and mrvp testing did not detect enterovirus and ev-d68 viruses in 95.9% and 60.3% of pcr-positive specimens, respectively. in addition neither viral culture nor mrvp could distinguish ev-d68 from rhinovirus in 1.3% and 36.4% of specimens tested by each method, respectively. hence it is likely that ev-d68 has been underdiagnosed when only viral culture or mrvp test methods were used [22] . consistent with previous reports, in ontario ev-d68 was most commonly found in younger children as more than 50% of cases were identified in children less than 5 years of age (7, (9) (10) (11) (12) (13) (14) . when adjusted for age, sex, setting and date of specimen collection, the likelihood of having ev-d68 was higher for individuals less than 20 years of age compared to over 20, peaking for the 5-9 year old age group. while this may be indicative of the population in whom ev-d68 is most frequently detected, but may also represent a testing bias as three-quarters of patients tested for ev-d68 were less than 10 years of age. consistent with what has been reported elsewhere, males were more likely to be cases than controls [2] , however this did not achieve statistical significance. higher detection of ev-d68 in hospitalized patients was reported by other studies [23] . in our investigation, most patients tested for ev-d68 were hospitalized and this may reflect the fact that hospitalized patients are more likely to be tested than patients seeking community based care. however, in multivariate analysis when adjusted for age, sex, setting and date of specimen collection, cases were not more likely to be hospitalized than controls. symptoms reported among patients tested for ev-d68 included fever, chills, cough and shortness of breath. when we compared cases and controls in regards to reported symptoms, undefined respiratory symptoms were more likely to be reported for ev-d68 cases as opposed to controls. neurological symptoms such as muscle weakness or acute flaccid paralysis were not reported. a wide range of disease severity has been reported in the literature, varying from mild to more severe illness particularly among children [7, 9] . based on the limited information available to phol through the laboratory requisition, we could not determine disease severity for ev-d68 cases. in addition, neither cases nor controls were followed up to determine disease trajectory, therefore we could not determine if clinical patient's condition deteriorated after specimen collection. all ev-d68 viruses were detected in respiratory specimens including nasopharyngeal, respiratory aspirate, throat swabs and nasal swabs; however this should be interpreted with caution as the number of non-respiratory specimens submitted for testing was small (n = 14 patients). infection with ev-d68 predominantly resulted in respiratory symptoms. ev-d68 was not detected in stool, csf, sputum or bal. this is congruent with what has previously reported elsewhere [7] ; notably ev-d68 has been seldom reported in csf [13, 24] , or stool [25] . we also found cases who were co-infected with ev-d68 and parainfluenza or adenovirus, which has not been previously described in other studies. there are some limitations to this investigation. firstly, phol was not the only laboratory that performed ev-d68 testing in ontario since the regional virology laboratory in hamilton ontario and the children's hospital of eastern ontario in ottawa also performed ev-d68 testing. as well, some hospitals may have forwarded their specimens directly to nml. hence, these results do not represent all ev-d68 detections in ontario. in addition, physicians may not routinely test patients presenting with respiratory symptoms and some patients with ev-d68 with mild symptoms likely did not present for care; therefore cases reported in this investigation underestimate the true incidence of this virus. secondly, for the purposes of this investigation, testing for ev-d68 was performed by phol and nml. different test methods for enterovirus detection were used by each laboratory, which may have different sensitivity and specificity and consequently may have affected the number of positive results reported in this study. thirdly, information about at-risk groups (e.g. children, particularly those with asthma) requiring hospitalization as a result of ev-d68 had been communicated broadly to clinicians. hence, it is not possible to determine if requests for testing for ev-d68 by health care providers was requested more frequently for these specific population groups as opposed to broadly testing individuals of any age regardless of setting type. in addition, some clinicians may have been more likely to test than others, which would impact the number of ev-d68 positive specimens attributed to any given health unit. finally, reviewing patients' charts would have been the optimal method by which to obtain clinical information such as patients' symptoms and clinical outcomes. at phol, we are reliant on information supplied by clinicians on the laboratory requisition. unfortunately, symptoms and patient's setting were not consistently reported on the requisition. in addition, even when reported, they reflect one specific period during the clinical course of disease. as a result, we are unable to report on clinical severity of disease, and/ or clinical outcome. presence of ev-d68 was identified among young children during september-october 2014 in ontario, with positive cases occurring more frequently in september. although most of the patients were hospitalized there was no difference in hospitalization status between cases and controls. most cases reported respiratory symptoms, and the virus was only detected in respiratory specimens. in order to better understand the epidemiology of this virus, surveillance for ev-d68 should include testing of symptomatic individuals regardless of treatment setting (including ambulatory care settings) patient age, along with collection and analysis of comprehensive clinical and epidemiological data. supporting information s1 dataset. ontario patients who tested for enterovirus d68, phol, 2014. ontario patients who tested for evd-68 at phol are included in this dataset. to protect the patient's privacy, the data were aggregated and de-identified from all personal identifiers including (health card number, birth date, health unit and address). a generic patient id was created to identify unique patients. (csv) conceived and designed the experiments: tb ae. performed the experiments: al sp. analyzed the data: ap alw jbg. contributed reagents/materials/analysis tools: ama ek. wrote the paper: ap alw jbg bw ro tb ae. that other evd: enterovirus-d68-whats all about? can cdc. enterovirus d68 in the united states detection and whole genome sequence analysis of an enterovirus 68 cluster enterovirus 68 is associated with respiratory illness and shares biological features with both the enteroviruses and the rhinoviruses mmwr.clusters of acute respiratory illness associated with human enterovirus 68-asia, europe, and united states genetic diversity of human enterovirus 68 strains isolated in kenya using the hypervariable 3'-end of vp1 gene acute flaccid paralysis in a child infected with enterovirus d68: a case report a novel outbreak enterovirus d68 strain associated with acute flaccid myelitis cases in the usa (2012-14): a retrospective cohort study cdc. notes from the field: acute flaccid myelitis among persons aged 21 years-united states cdc. summary of findings: investigation of acute flaccid myelitis in u.s. children clinical disease due to enterovirus d68 in adult hematologic malignancy patients and hematopoietic cell transplant recipients outbreaks of enterovirus d68 continue across the usa continued seasonal circulation of enterovirus d68 in the netherlands lineages, sub-lineages and variants of enterovirus 68 in recent outbreaks comparative evaluation of taqman real-time pcr and semi-nested vp1 pcr for detection of enteroviruses in clinical specimens sensitive, seminested pcr amplification of vp1 sequences for direct identification of all enterovirus serotypes from original clinical specimens upsurge of human enterovirus 68 infections in patients with severe respiratory tract infections ev-d68) 2014 outbreak strain-specific real-time reverse transcription / polymerase chain reaction (rrt-pcr) assay instructions-version 10/14/2014 enterovirus surveillance united states multi-centre study in the netherlands examining laboratory. ability to detect enterovirus 68, an emerging respiratory pathogen that other evd: enterovirus-d68-whats all about? ped infect dis notes a fatal central nervous system enterovirus 68 infection acute flaccid paralysis following enterovirus d68 associated pneumonia key: cord-307036-n44yml79 authors: ng, oi-wing; keng, choong-tat; leung, cynthia sau-wai; peiris, j. s. malik; poon, leo lit man; tan, yee-joo title: substitution at aspartic acid 1128 in the sars coronavirus spike glycoprotein mediates escape from a s2 domain-targeting neutralizing monoclonal antibody date: 2014-07-14 journal: plos one doi: 10.1371/journal.pone.0102415 sha: doc_id: 307036 cord_uid: n44yml79 the severe acute respiratory syndrome coronavirus (sars-cov) is the etiological agent for the infectious disease, sars, which first emerged 10 years ago. sars-cov is a zoonotic virus that has crossed the species barriers to infect humans. bats, which harbour a diverse pool of sars-like covs (sl-covs), are believed to be the natural reservoir. the sars-cov surface spike (s) protein is a major antigenic determinant in eliciting neutralizing antibody production during sars-cov infection. in our previous work, we showed that a panel of murine monoclonal antibodies (mabs) that target the s2 subunit of the s protein are capable of neutralizing sars-cov infection in vitro (lip km et al, j virol. 2006 jan; 80(2): 941–50). in this study, we report our findings on the characterization of one of these mabs, known as 1a9, which binds to the s protein at a novel epitope within the s2 subunit at amino acids 1111–1130. mab 1a9 is a broadly neutralizing mab that prevents viral entry mediated by the s proteins of human and civet sars-covs as well as bat sl-covs. by generating mutant sars-cov that escapes the neutralization by mab 1a9, the residue d1128 in s was found to be crucial for its interaction with mab 1a9. s protein containing the substitution of d1128 with alanine (d1128a) exhibited a significant decrease in binding capability to mab 1a9 compared to wild-type s protein. by using a pseudotyped viral entry assay, it was shown that the d1128a substitution in the escape virus allows it to overcome the viral entry blockage by mab 1a9. in addition, the d1128a mutation was found to exert no effects on the s protein cell surface expression and incorporation into virion particles, suggesting that the escape virus retains the same viral entry property as the wild-type virus. the severe acute respiratory syndrome (sars) first emerged as an infectious disease ten years ago, manifesting itself as a severe form of pneumonia. its etiological agent was identified as a then novel coronavirus known as the sars coronavirus (sars-cov) [1, 2] . within a short span of time from december 2002 to july 2003, the newly emerged virus spread quickly to infect more than 8000 people across 25 countries with an overall fatality rate of approximately 10% [3] . sars-cov is a zoonotic virus that has crossed the species barrier to infect humans. small animals such as palm civets (paguma larvata) and raccoon dogs (nyctereutes procynonoides) sold in live-animal wet markets in guangdong province of southern china are believed to be the zoonotic source of the virus transmitted to humans [4] . in 2005, the complete sequences of sars-like coronaviruses (sl-covs) of genetic homology of 87-92% to sars-cov were identified from horseshoe bats of the genus rhinolophus in china [5, 6] . however, these sl-covs display significant differences in sequences at the receptor-binding domain (rbd) compared to sars-cov and are unable to use the sars-cov receptor, the human angiotensin-converting enzyme 2 (ace2), for cellular entry [7] , rendering them unlikely to be the immediate progenitor of sars-cov. more recently, a bat sl-cov capable of using the human ace2 receptor for cellular entry was characterized and isolated from chinese horseshoe bats, providing strong evidence that bats are the natural reservoirs of sars-cov [8] . the sars-cov is classified as a virus from the genus betacoronavirus (lineage b), family coronaviridae and order nidovirales. it is an enveloped, positive-sense, single-stranded rna virus of genome size of approximately 29.7 kb, encoding for 16 non-structural proteins, 8 [9, 10, 11] . every surface spike of the sars-cov is composed of a trimer of s protein of 1255 amino acids in length. the s protein is a type 1 glycoprotein and a class 1 fusion protein responsible for viral attachment and entry into host cells, and is therefore the principal determinant of host range [12] . it consists of 2 functional subunits: the n-terminal s1 subunit (amino acids ) and the c-terminal s2 subunit (amino acids 680-1255). the s1 subunit contains the rbd [13, 14] that recognizes the sars-cov receptor, ace2 [15] , allowing the attachment of the virus to its host cell. the s2 subunit contains the putative fusion peptide and two heptad repeats, hr1 and hr2, important in sars-cov fusion with target host cells [16, 17] . upon the association of the rbd with the ace2 receptor, a conformational change of s2 is triggered, resulting in the insertion of the fusion peptide into the target cell membrane [18] , followed by the interaction of the hr1 and hr2 domains in an anti-parallel manner to form a stable six-helical bundle fusion core [17, 19] . this allows the viral membrane and target cell membrane to come into close proximity, facilitating membrane fusion and viral entry [20, 21] . besides its roles in viral entry, the s protein is also a major antigenic determinant in eliciting humoral immune responses in infected humans [22] and is therefore an important target in the development of vaccines and therapeutic intervention against sars [23, 24] . numerous human and murine monoclonal antibodies (mabs) binding to the s1 or s2 regions of the s protein have been identified and were shown to confer neutralizing activities in vitro and protection in vivo against sars-cov infection [25, 26, 27, 28] . the s1 subunit of the s protein, especially the rbd, is highly variable among coronaviruses, resulting in a wide range of tissue tropism, while the s2 subunit is a well-conserved domain, indicating the highly conserved nature of the fusion process [29] . as a result, anti-s2 mabs have broadly neutralizing characteristics against a wider range of sars-cov variants, including human and zoonotic sars-cov strains, through the recognition of highly-conserved epitopes [28, 30] . in our previous study, it has been shown that a panel of murine mabs targeting the hr2 domain and the region upstream of hr2 of the s protein are capable of neutralizing sars-cov infection in vitro [31] . in this study, the cross-neutralization ability of one of these mabs, termed as 1a9, was investigated by studying its ability to prevent viral entry mediated by the s protein of sars-cov strain from civet as well as sl-cov strains from bats. mab 1a9 binds to the loop region between the hr1 and hr2 domains and this region is highly conserved but has no known function ( figure 1 ). in addition, escape mutants against mab 1a9 were generated to identify critical residues important for mab 1a9 binding to s protein. we found that mab 1a9 has broad cross-neutralizing activity and identified a single amino acid (aspartic acid) at position 1128 in s to be crucial for the interaction with mab 1a9. by using a pseudotyped viral entry assay, it was shown that the substitution of this residue with alanine (d1128a) mediates escape from mab 1a9 neutralization. in addition, the d1128a mutation exerts no effects on the expression of s on the cell surface and its incorporation into virion particles, suggesting that the escape virus retains the same viral entry property as the wild-type virus. ascites were produced by injecting hybridoma cells into the peritoneal cavities of pristine-primed balb/c mice. the protocol was approved by the institutional animal care and use committee (iacuc) of the biological resource centre, a*star, singapore (protocol number: 110694). all the procedures were carried out in strict accordance with the recommendations of the national advisory committee for laboratory animal research (naclar) guidelines in singapore. all efforts were made to minimize suffering and euthanasia was performed using carbon dioxide. vero e6 (american type culture collection) and 293 ft cells (invitrogen) were grown in dulbecco's modified eagle's medium (invitrogen) supplemented with 10% fetal bovine serum (hyclone), nonessential amino acids (gibco) and penicillin (10,000 units/ml)streptomycin (10 mg/ml) solution (sigma aldrich). cho cell line stably expressing the human ace2, known as cho-ace2, was established previously [31] , and cultured in the same medium. all cell lines were maintained at 37uc with 5% co 2 . the human sars-cov strain hku39849 was used in this study. antibodies were purified from the ascites by using affinity chromatography. briefly, a 1 ml hitrap protein g hp beads column (ge healthcare) was pre-washed using ,20 ml of 20 mm sodium phosphate buffer, ph 7.0 at a constant flow-rate of 1 ml/ min using a peristaltic pump. 5 ml of ascites fluids were mixed with equal volume of 40 mm sodium phosphate buffer and passed through a 0.45 mm filter. the filtered ascites fluids were passed through the column at the same flow-rate of 1 ml/min. extensive washing was performed using the 20 mm sodium phosphate buffer. elution buffer (0.1 m glycine-hcl, ph 2.7) was then passed through the column at a flow-rate of 1 ml/min and the flow-through was collected at 0.5 ml fractions in 1.5 ml microtubes containing 20 ml of neutralization buffer (1 m tris-hcl, ph 9.0). the concentration of the purified monoclonal antibodies in each tube was determined using the coomassie plus protein assay reagent (thermo scientific). the generation of escape mutants was performed in a biosafety level 3 (bsl-3) laboratory. using 100 tcid 50 of sars-cov (strain hku39849) for infection of vero e6 cells in the presence of different concentrations of mab 1a9, the concentration of mab 1a9 that reduced the virus titers by about 4 logarithms (log) was determined to be 0.25 mg/ml and used for the generation of virus escape mutants. serial dilutions of sars-cov ranging from 10 21 to 10 28 were incubated in the presence of 0.25 mg/ml of mab 1a9 for 1 h at 37uc and 5% co 2 . the virus-mab mixtures were then incubated with vero e6 cells in a 96-well plate for 1 hour at 37uc and 5% co 2 , after which the virus-mab mixtures were removed and the cells were washed twice with medium. the cells were further incubated for 2 days in the presence of mab 1a9 at concentration 0.25 mg/ml. the supernatant from the wells containing cells that exhibited cytopathic effect (cpe) at the highest dilution of sars-cov was harvested. the percentage cpe was determined by visual counting of floating cells and attached cells. wells with more than 80% floating cells are considered to have cpe. this supernatant was again incubated in the presence of 0.25 mg/ml of mab 1a9 for 1 h at 37uc before the virus-mab mixture was used to infect vero e6 cells in the presence of mab 1a9 at concentration 0.25 mg/ml. this was performed 3 times. the final virus sample was added to vero e6 cells in a 6-well plate and incubated for 1 hour at 37uc and 5% co 2 before the wells were overlaid with agarose containing 0.25 mg/ml mab 1a9 and incubated for 3-5 days at 37uc and 5% co 2 . five plaques were picked using a pasteur pipette, freezethawed once and further amplified in vero e6 cells. neutralization tests were then performed on all the virus clones to confirm that they could escape neutralization by mab 1a9. viral rna of the 5 escape virus clones was isolated using the qiaamp viral rna mini kit (qiagen) and converted into cdna by standard reverse transcription (superscript ii reverse transcriptase, invitrogen). the cdna was then amplified by pcr using specific primers targeting the s gene to generate a long fragment (amino acids 1 to 1003) and a short fragment (amino acids 969 to 1255). these gene fragments were cloned into pcr2.1-topo vector (invitrogen) and five colonies were sequenced. respectively. western blot analysis was performed on the cell lysates using mab 1a9 to determine if it can bind to different s proteins. mab 7g12, which binds to the s1 of the s protein, was used as a control antibody to detect protein expression. (c) s-pps containing s of human hku39849, civet sz3, bat rp3 or rf1, were pre-incubated with different concentrations of mab 1a9 at 100, 150 and 200 mg/ml for 1 hour before infecting cho-ace2 cells. cells were harvested 48 hours post-infection and luciferase activities were measured. viral entry, as indicated by the luciferase activity measured in relative light units (rlu), was expressed as a percentage of the reading obtained in the absence of antibody, which was set at 100%. data shown represents that obtained from 3 independent experiments. bars represent sd of the experiment carried out in triplicates. doi:10.1371/journal.pone.0102415.g002 the s gene of the human sars-cov hku39849 strain was obtained from viral rna after reverse transcription and pcr and cloned into the pxj39 expression vector using the bamhi and xhoi restriction sites (pxj39-s). chimeric s genes of civet sars-cov sz3 strain, bat sl-cov rp3 and rf1 strains were designed by replacing their receptor-binding domain (rbd) with that of the human sars-cov hku39849 strain. for sz3, the arginine (r) residue at position 344 was substituted with lysine (k); serine (s) residue at position 360 was substituted with phenylalanine (f); lysine at position 479 was substituted with asparagines and serine at position 487 was substituted with threonine (t). for rp3 and rf1, the rbd domain of amino acids 322-496 was replaced with the rbd domain (amino acids 318-510) of the human hku39849. they were then chemically synthesized (genscript usa inc., piscataway, nj) and cloned into the same expression vector by using the same restriction sites. to generate plasmids for the expression of mutant s, specific primers were designed for two-round pcr site-directed mutagenesis of wild-type s gene using the expand high fidelity pcr system (roche). the pcr products were then cloned into the pxj39 expression vector using bamhi and xhoi restriction sites to form pxj39-s-n1056k, pxj39-s-d1128a, pxj39-s-d1128a/ n1056k, pxj39-s-d1128e and pxj39-s-d1128n plasmids. expression plasmids expressing wild-type and mutant human sars-cov s, chimeric civet sars-cov sz3 s, bat sl-cov rp3 and rf1 s genes were transiently transfected into 293 ft cells using lipofectamine 2000 reagent (invitrogen) according to manufacturer's protocol. the cells were harvested at 24 hours post-transfection by scrapping and cells were spun down by centrifugation and washed with cold pbs twice. cell were then resuspended in lysis buffer (50 mm tris [ph 8.0], 150 mm nacl, 0.5% np40, 0.5% deoxycholic acid, 0.005% sds and 1 mm phenylmethylsulfonyl fluoride) and subjected to freeze-thaw five times followed by spinning down at 13,000 rpm to remove cell debris. cell lysate protein concentrations were quantitated using the commassie plus protein assay reagent (thermo scientific) for use in western blot analysis and immunoprecipitation (ip). in western blot analysis, proteins were separated on a 7.5% polyacrylamide gel by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) and transferred onto nitrocellulose membranes. the membranes were blocked in 5% skimmed milk in tris-buffered saline (tbs) with 0.05% tween 20 (tbst) and incubated with primary antibodies, mabs 1a9 and 7g12 [31] overnight at 4uc. the membranes were then washed in tbst before incubation with goat anti-mouse horseradish peroxidase (hrp)-conjugated antibody (pierce) as the secondary antibody at room temperature for 1 hour. the membranes were washed in tbst again followed by the addition of enhanced chemiluminescence substrate (pierce) for film development. in ip, mabs 1a9 and 7g12 were used to pull down wild-type and mutant s proteins in the cell lysates for 1 hour at 4uc, followed by the addition of protein a beads (roche) and incubation at 4uc overnight. the beads were then washed in lysis buffer three times and subjected to western blot analysis for the detection of s proteins using the rabbit anti-sd1 antibody (binds to amino acids 48-358 of the s1 subunit) [32] as primary antibody and goat anti-rabbit hrp-conjugated antibody (pierce) as secondary antibody. a fragment of s(1030-1188 aa) was expressed as a gst (glutathione-transferase) fusion protein using the pgex6p1 vector (ge healthcare). this fragment is located in the s2 subunit at amino acids 1030-1188 and contains the mab 1a9 binding site. the wild-type and mutant (n1056k and d1128a) s fragments were expressed in escherichia coli bl21-de3. cultures were grown in terrific broth and on reaching an optical density at 600 nm (od600 nm) of 0.8, cells were cooled to 16uc and induced with isopropyl b-d-thiogalactopyranoside (iptg) at a final concentration of 0.5 mm. after an incubation period of 24 hours, cells were harvested. bacterial pellets were resuspended in lysis buffer containing 20% sarkosyl and subjected to sonication. the lysate was cleared by centrifugation and incubated with gluthathione (gsh) sepharose beads (ge heathcare) overnight at 4uc. after several washes, the gst-tagged s fragments were eluted from the beads in 10 mm reduced gluthathione solution (sigma aldrich). purified s fragments were then subjected to sds-page on a 12% gel and stained using coomassie blue to visualize the purity of proteins. purified wild-type and mutant (n1056k and d1128a) gst-s(1030-1188 aa) proteins were coated onto 96-well elisa plates (nunc) overnight at 4uc at 100 ng/well. the wells were blocked in 5% skimmed milk in phosphate-buffered saline (pbs) with 0.1% tween 20 (pbst) for 1 hour at room temperature, and primary antibodies (mab 1a9 and mouse anti-gst antibody [santa cruz]) were added as primary antibodies at 4-fold dilutions and incubated at 37uc for 2-3 hours. the wells were then washed in pbst followed by the addition of goat anti-mouse hrp-conjugated antibody (pierce) as secondary antibody and incubated at 37uc for 1 hour. tetramethylbenzidine substrate (pierce) was added and reaction was stopped using 0.2 m sulphuric acid. optical density at 450 nm (od450 nm) was obtained using an absorbance reader (tecan infinite m200). statistical difference in binding of mab 1a9 to wild-type s and mutant s was analysed using unpaired ttest. significance was indicated by p-value of ,0.01. the ability of sars-cov containing mutant s to infect cells and the resulting effect in mab neutralization in sars-cov entry were studied using a pseudotyped virus system. based on this pseudotyped virus system, replication incompetent lentiviral particles expressing s proteins on the surface and containing the firefly luciferase reporter gene were used in replacement of live sars-covs. viral entry into permissive cell lines will be reflected in the luciferase activity of the infected cells. to generate spseudotyped particle (s-pp), lentiviral vector pnl43-r -e -luc and plasmids expressing s genes were co-transfected in 293 ft cells using lipofectamine 2000 reagent (invitrogen) according to manufacturer's protocol. 48 hours post-transfection, the viral supernatant was collected and spun down in a centrifuge to remove cell debris. p24 elisa (quicktiter lentivirus titer kit, cells biolabs) was used according to manufacturer's protocol to quantify viral titres. mab 7g12, which binds to the s1 of the s protein, was used as a control antibody to detect protein expression. (b) cell lysates containing s-wt, s-d1128a, s-n1056k or s-d1128a/n1056k were subjected to immunoprecipitation (ip) using mab 1a9 or 7g12 and protein a beads. s proteins immunoprecipitated by mabs 1a9 and 7g12 were detected using rabbit anti-sd1 antibody in western blot analysis (wb). the rabbit anti-sd1 antibody binds to amino acids 48-358 of the s1 subunit. doi:10.1371/journal.pone.0102415.g005 in vitro s-pp neutralization assay all s-pp neutralization assays were carried out in 24-well plates. cho-ace2 cells were grown in 500 ul of growing media per well for 24 hours before each experiment. in s-pp neutralization assays, 16 ng of s-pp (as quantified using p24 elisa) were preincubated with mab 1a9 or mab 1g10 at 0, 25, 50, 100, 150 and 200 mg/ml for 1 hour at room temperature. the mab-virus mixtures or virus alone were used to infect cho-ace2 cells and incubated at 37uc. a non-neutralizing anti-s1 antibody that binds to the rbd of s, mab 7g12 [31] , was used as a control antibody at 200 mg/ml. at 48 hours post-infection, cells were harvested using the luciferase assay system (promega) and luciferase expressions of the cells were determined according to manufacturer's protocol. percentages of viral entry were then calculated based on the luciferase readings obtained. all experiments were carried out in triplicates. statistical difference in viral entry between wild-type and mutant s-pp was done using unpaired ttest. significance was indicated by p-value of ,0.01. s-pps were coated onto 96-well elisa plates (nunc) at 16 ng/ well (as quantitated p24 elisa) overnight at 4uc. the wells were blocked in 5% skimmed milk in pbs with 0.1% tween 20 (pbst), and primary antibodies (mab 7g12 [31] and mouse anti-p24 antibody) were added as primary antibodies at 4-fold dilutions and incubated at 37uc for 2 hours. the wells were then washed in pbst followed by the addition of goat anti-mouse hrpconjugated antibody (pierce) as secondary antibody and incubated at 37uc for 1 hour. tetramethylbenzidine substrate (pierce) was added and reaction was stopped using 0.2 m sulphuric acid. od450 nm was obtained using an absorbance reader (tecan infinite m200). difference in s protein level in wild-type and mutant d1128a s-pp was evaluated using unpaired t-test. fluorescence-activated cell sorting (facs) analysis for surface expression of s protein 293 ft cells were seeded in 6-cm dishes 24 hours prior to transfection. the cells were transfected with pxj39 empty vector, figure 6 . neutralization of wild-type, mutant d1128a, n1056k and d1128a/n1056k s-pps by mab 1a9 and mab 1g10. s-pp containing wild-type (s-wt-pp) or mutant s (s-d1128a-pp, s-n1056k-pp and s-d1128a/n1056k-pp) were pre-incubated with different concentrations of (a) mab 1a9 or (b) mab 1g10 at 25, 50, 100 and 200 mg/ml for 1 hour before infecting cho-ace2 cells. an anti-s1, nonneutralizing mab 7g12 was used as control antibody at 200 mg/ml. cells were harvested 48 hours post-infection and luciferase activities were measured. viral entry, as indicated by the luciferase activity measured in relative light units (rlu), was expressed as a percentage of the reading obtained in the absence of antibody, which was set at 100%. data shown represents that obtained from 3 independent experiments. bars represent sd of the experiment carried out in triplicates. *indicates statistically significant difference of p,0. 01 when compared to s-wt-pp. doi:10.1371/journal.pone.0102415.g006 pxj39-s and pxj39-s-d1128a plasmids using lipofectamine 2000 reagent (invitrogen) according to manufacturer's protocol and harvested at 72 hours post-transfection. the cells were first detached using the cell dissociation solution (sigma), washed twice in 1x pbs and incubated with purified mouse mab 7g12 [31] (that binds to the s1 subunit) in 1x pbs containing 1% bovine serum albumin (bsa) for 3 hours at 4uc on a nutator. the cells were washed 3 times using 1x pbs containing 1% bsa and then incubated with fluorescein isothiocyanate (fitc)-conjugated goat anti-mouse igg (santa cruz) secondary antibody for 1 hour at 4uc on the nutator. cells were washed again 3 times and immediately used for facs analysis using the cyan flow cytometer (beckman coulter). all facs data was analysed using the flowjo software application. as described in our previous publication, we have a panel of neutralizing mabs largely grouped into type i, ii, iii and iv based on their binding sites on the s protein. by membrane fusion experiment, we found that mab 1a9 belonging to type ii was the most effective in cell-cell membrane blocking and bound to residues 1111-1130 which are immediately upstream of the hr2 domain ( figure 1a ) [31] . as the contribution of the mab 1a9 binding site to the structure and function of s has not been defined, we chose mab 1a9 for further investigation in this study in order to gain a better understanding of the neutralizing mechanism of mab 1a9. sequence alignment shows that residues 1111-1130 is a highly conserved region within the s2 subunit of human, civet sars-cov and bat sl-cov strains ( figure 1b ). it has been demonstrated by ren et al. [7] that the bat sl-cov rp3 uses a different unknown receptor for entry and thus, could not infect cells expressing human ace2 receptor. in order to evaluate the viral entry properties of the civet sars-cov sz3, bat sl-cov rp3 and rf1 strains, the receptor-binding domain (rbd) of s in the civet sars-cov sz3 strain and bat sl-cov rp3 and rf1 strains were replaced with the rbd of s in the human sars-cov to create chimeric s proteins (figure 2a) to allow viral attachment step through the binding of the rbd of s to human ace2 receptor. by western blot analysis, mab 1a9 was found to be able to bind to the s2 subunits of the civet sars-cov sz3 and bat sl-cov rp3 and rf1 strains to similar extent as the homologous human sars-cov hku39849 ( figure 2b ). an antibody targeting the s1 domain of the human sars-cov s protein, mab 7g12 [31] , was used to determine the relative expressions of the chimeric s proteins ( figure 2b) . next, to determine if mab 1a9 exhibits cross-neutralizing activity, s-pseudotyped virus particles, or s-pps, carrying the human sars-cov s or the various rbd-modified chimeric s of civet sars-cov sz3 strain and bat sl-cov rp3 and rf1 strains were generated and used to infect cho-ace2 cells in the absence or presence of different concentrations (100, 150 and 200 mg/ml) of mab 1a9. in this pseudotyped virus system, replication incompetent lentiviral particles that express s proteins on the surface were used in replacement of live sars-covs and sl-covs. this system has been successfully employed in the study of highly pathogenic viruses including the influenza virus [33] and sars-cov [34, 35, 36] . neutralizing antibody titers measured using pseudotyped sars-cov correlated well with the use of replication competent sars-cov [37] , as such, this system has been widely used in the evaluation of sars-cov neutralizing antibodies [30, 38, 39, 40, 41] . in this study, the s-pps expressing human sars-cov s or rbd-modified chimeric s of civet sars-cov sz3 strain and bat sl-cov rp3 and rf1 strains were able to infect and enter cho-ace2 cells at similar extent ( figure s1a in file s1). the results obtained from the mab 1a9 neutralization assay ( figure 2c , figure s2 in file s1) showed that mab 1a9, apart from being able to neutralize s-pp expressing human sars-cov hku39849 s, was able to cross-neutralize the s-pp expressing chimeric s of civet sars-cov sz3 and bat sl-cov rp3 and rf1. this indicates that mab 1a9 has broad neutralizing capability. to identify critical residue(s) required for mab 1a9 interaction with s, mab 1a9 escape mutants were generated. sars-cov hku39849 strain was cultured in vero e6 cells at a sub-optimal level of mab 1a9 (0.25 mg/ml). supernatant from the wells containing cells that exhibited cytopathic effect (cpe) at the highest dilution of sars-cov was harvested as passage 1 and passaged 3 times in the presence of mab 1a9 (0.25 mg/ml), after which the virus titres gradually increased ( figure 3 ) and a plaque assay was done to isolate 5 individual sars-cov escape mutant clones. the wild-type and escape mutant viruses were used at 1000 tcid 50 and subjected to neutralization assays by mab 1a9 and mab 1g10 in vero e6 cells to determine cytopathic effects (cpe). all 5 escape mutant clones were resistant to neutralization by mab 1a9 and the result of one representative clone is shown in figure 4 . the percentage of cpe in vero e6 cells infected with wild-type virus decreased significantly at mab 1a9 concentrations .1 mg/ ml ( figure 4a ). in contrast, 100% cpe was still observed in escape mutant virus-infected cells even in the presence of 2 mg/ml of mab 1a9 (figure 4b) , indicating that the mutant virus is resistant to neutralization by mab 1a9. however, both the wild-type and mutant virus were equally sensitive to neutralization by another mab 1g10 ( figure 4c and d) , which binds to an epitope (residues 1151-1192) in s2 different from that of mab 1a9 [31] . after confirming the escape ability of the escape mutant clones, the viral rna was extracted and the s gene was sequenced. two escape mutations, n1056k and d1128a, were identified in the mab 1a9 escape mutants. 2 out of 5 clones had d1128a mutation and 3 out of 5 clones had n1056k mutation. none of the clones had both mutations. wild-type s, substitution s mutants, namely d1128a, n1056k, and that containing both d1128a and n1056k, were then expressed in 293 ft cells and western blot analysis was performed to determine the effects of these mutations on the binding of the s protein to mab 1a9. as shown in figure 5a , s protein containing mutation d1128a (s-d1128a) showed a reduced binding to mab 1a9 compared to the wild-type s protein (s-wt), while s protein with the n1056k mutation (s-n1056k) did not show a reduction in mab 1a9 binding compared to s-wt. while western blot reveals the binding of mab 1a9 to denatured epitope on s, immunoprecipitation (ip) was performed to compare the mab 1a9 binding to native forms of s. consistent with the results obtained in western blot analysis, s-d1128a exhibited a decrease in mab 1a9 binding compared to s-wt while s-n1056k did not ( figure 5b ). similar results were also observed in elisa ( figure s3 in file s1) using wild-type and mutant d1128a and n1056k gst-s(1030-1188) fragments that were expressed from bacteria cells ( figure s4 in file s1). these results suggest that residue 1128 is important in the binding of mab 1a9 to the s protein. in addition, s protein containing both mutations was evaluated for synergistic effects in the reduction of mab 1a9 binding by western blot and immunoprecipitation. as shown in figure 5a and b, no significant synergistic effect was observed. resistance of s-pp expressing mutant d1128a s protein to neutralization by mab 1a9 both d1128a and n1056k are mutations identified in escape sars-cov mutant clones generated against mab 1a9. to verify if these two mutations contribute to the escape from neutralization by mab 1a9 in an in vitro pseudotyped virus assay, s-pps expressing the wild-type, mutant d1128a, mutant n1056k and mutant d1128a/n1056k s proteins were generated. as seen in figure s1b in file s1, all s-pps were able to infect and enter cho-ace2 cells, with the mutant s-pps showing a slightly lower infectivity compared to wild-type. they were then used to infect cho-ace2 cells in the absence or presence of different concentrations (25, 50, 100 and 200 mg/ml) of mab 1a9 and the neutralization activities of mab 1a9 against wild-type s-pp, s-d1128a-pp, s-n1056k-pp and s-d1128a/n1056k-pp were compared. mab 7g12, an anti-s1, non-neutralizing mab, was used as the control antibody at 200 mg/ml. as shown in figure 6a and figure s5a in file s1, at the highest concentration of 200 mg/ ml, mab 1a9 prevented the viral entry of wild-type s-pp and s-n1056k-pp in cho-ace2 cells by 36% and 35% respectively, while the entry of s-d1128a-pp was not significantly affected. at lower concentrations of mab 1a9 (25, 50 and 100 mg/ml), similar results were obtained, suggesting that s-d1128-pp was resistant to mab 1a9 neutralization. this further indicates that through a reduction in binding to mab 1a9 (as observed in western blot, ip and elisa), the d1128a mutation in s protein is sufficient to mediate the escape from neutralization by mab 1a9. in addition, s-pp containing both d1128a and n1056k mutations was investigated for its resistance to mab 1a9 neutralization. s-d1128a/n1056k-pp was also resistant to mab 1a9 neutralization at a similar extent as the s-d1128a-pp ( figure 6a and figure s5a in file s1), indicating no synergistic effects between the d1128a and n1056k mutations in conferring mab 1a9 resistance to the viral particles. no resistance to neutralization was observed for all s-pp with mab 1g10, another neutralizing mab that binds to a different epitope within the s2 subunit at amino acids 1151-1192 ( figure 6b and figure s5b in file s1). mutations within the coronavirus s protein can have profound effects on the synthesis and maturation process of the s protein, resulting in decreased cell surface expression as well as defects in its incorporation into matured virion particles. to evaluate the possible effects of d1128a mutation on the maturation process of the s protein during its synthesis, facs analysis was performed to compare the cell surface expression of wild-type (wt) and mutant d1128a s proteins in transfected 293 ft cells. vector-transfected, wild-type s-transfected and mutant d1128a s-transfected cells were incubated with mab 7g12 (which binds to the s1 subunit of s) followed by an fitc-conjugated anti-mouse secondary antibody. a positive shift in fluorescence was observed in the wild-type s-expressing cells when compared to the vectortransfected cells ( figure 7a ) because of the specific binding of mab 7g12 to the native form of the s protein expressed on the cell surface. in comparison, the mutant d1128a s-expressing cells showed similar degree of shift in fluorescence as those expressing wild-type s ( figure 7a) . thus, the d1128a mutation did not reduce the surface level expression of the s protein, suggesting that this substitution at residue 1128 did not hamper the synthesis and processing of the s protein. to determine if the mutant d1128a s protein is incorporated into the s-pps as efficiently as wild-type s, equal amount of wildtype s-pp and s-d1128a-pp was coated onto an elisa plate and mab 7g12 was used to compare the amount of s protein in the spps. it was observed that there was no significant difference in the expression of wild-type and mutant d1128a s protein in the s-pps ( figure 7b , top). this indicates that the mutation did not cause any change in the efficiency of s protein incorporation into viral particles. when an anti-hiv-1 p24 mab was used instead of mab 7g12, the od450 nm readings obtained were similar, confirming that equal amounts of s-pp viruses were successfully coated on the elisa plate ( figure 7b , bottom). although there has been no reported case of sars-cov infection in humans since 2004, the development of anti-sars-cov treatments and vaccines remains crucial as the threat of a reemergence of sars exists till today. human and civet sars-covs are believed to have originated from sl-covs residing in bats [42] . as coronaviruses are known to be capable of frequent cross-species transmission [12] , the continual persistence of sl-covs in animal hosts and reservoirs poses a threat to humans should a cross-species transmission occurs. the development of broadly neutralizing mabs that confer cross-protection not only against human sars-cov, but also zoonotic strains of sars-cov and sl-cov, is therefore important. the putative s1 subunit of bats sl-covs has a low sequence homology of about 63% to that of sars-cov, especially in the rbd, indicating the usage of different host cell receptors and different tissue tropisms [43] . on the other hand, the high sequence homology in the s2 subunit of about 92-96% suggests that the fusion mechanism during viral infection is well-conserved [44] . broadly neutralizing mabs usually target conserved epitopes required for highly conserved process, such as the post-attachment fusion process [45] . a majority of sars-cov-neutralizing mabs reported bind and target the s1 protein at the rbd region [46] . nonetheless, neutralizing mabs that bind to the s2 subunit have been reported. the epitopes of these mabs are found to be located at the s2 subunit upstream of hr1 (residues 787-809, 791-805) [47, 48] , within the loop region in between hr1 and hr2 (residues 1023-1189) [31, 40] and within the hr2 domain (residues 1151-1192) [31] . it has also been shown that anti-s2 mabs that bind to the highly conserved hr1, hr2 and ectodomain of the sars-cov s protein were able to neutralize a wider range of clinical isolates, including human and zoonotic strains of sars-covs [28, 30] . in this current study, mab 1a9, an anti-s2 mab that binds to the s2 subunit at the highly conserved loop region at residues 1111-1130, was demonstrated to be able to cross-neutralize pseudotyped s-pp viruses of the human sars-cov, civet sars-cov and bat sl-cov strains. this is consistent with the sequence conservation of the mab 1a9 binding epitope in s. in addition, sequence alignment (not shown) revealed that the mab 1a9 binding site is also conserved in other bat covs such as the bulgarian sarsrelated cov strain [49] , indicating the potential cross-protective effect of mab 1a9 against not only bat sl-cov from china, but also from other parts of the world such as europe. several cross-neutralizing mabs against human and civet sars-cov strains targeting the rbd have been described, with ic50 values ranging from 0.01 to 0.5 mg/ml [38, 39, 50, 51] . as observed in this study, the ic50 value of mab 1a9 is between 25-50 mg/ml, which is higher than that of rbd-targeting mabs, indicating the lower potency of mab 1a9. higher ic50 values and lower potency have also been observed in several other anti-s2 mabs [40, 52] . this can possibly be attributed to the inaccessibility of the s2 subunit, which constitutes the stalk region of s, as compared to the s1 subunit that is exposed on the viral surface [53] . however, sars-cov is capable of attaining mutations in the rbd region without affecting viral infectivity [54] , leading to escape in mab neutralization, while mutations within the highly conserved s2 subunit region are more likely to be detrimental to the mutant virus. therefore, the characterization of anti-s2 mabs remains important in the development of antibodies as anti-viral therapies against sars-cov. through the generation of mab 1a9 escape virus, it was found that the escape mutation d1128a in the s protein resulted in diminished binding to mab 1a9 and s pseudotyped viral particles containing the d1128a mutation could not be neutralized by mab 1a9. in addition, the substitution of d1128 by either n (having same side-chain as d) or e (having same charge as d) also reduced the interaction with mab 1a9 to similar extent as the a substitution ( figure s6 in file s1). thus, it appears that the d residue at position 1128 in the s protein plays an essential role in the interaction with mab 1a9. another mutation, n1056k, also identified in mab 1a9 escape virus, was found to have no effects on mab 1a9 binding and neutralization, indicating that this is most likely a random mutation that arose during the generation of escape mutants. no significant synergistic effect was observed between the d1128a and n1056k mutations in decreasing mab 1a9 binding and conferring resistance to mab 1a9 neutralization. as there is no information on the possible functional role of the mab 1a9 binding epitope or the residue d1128, the cell surface expression of mutant s-d1128a and its incorporation into virallike particles was compared to that of wild-type s. the results showed that s-d1128a is similar to wild-type s in these aspects, suggesting that viral entry property of the escape virus has not changed. in summary, we characterized mab 1a9, a sars-cov neutralizing mab that binds to a novel epitope located within the loop region located in between hr1 and hr2, at a position directly upstream of hr2 at residues 1111-1130 of the s protein that has not been previously identified and characterized. the aspartic acid at residue 1128 is crucial for the interaction of s protein with mab 1a9 and a substitution to alanine in the escape virus is sufficient to abolish neutralization by mab 1a9 but has little effect on the viral entry property. consistently, while a detailed study on the fitness of the escape virus has not been performed, it was observed that the virus titre of the escape virus after 3 passages in presence of mab 1a9 reached similar level as the wild-type virus (see figure 3 ). however, we cannot rule out the possibility that the mutation may affect viral virulence and further work is needed to address this. the loop region in between hr1 and hr2 in the s2 subunit is believed to be a region required for viral-cell membrane fusion, as peptides analogous to the loop region were found to inhibit sars-cov infection [55] . although d1128 residue has not been shown to be directly involved in membrane fusion, it is probable that the binding of mab 1a9 to the d1128 residue in the loop region causes steric hindrance that prevents the association of hr1 and hr2 to form the six-helical fusion bundle core. hence, structural analysis of the interaction between mab 1a9 and s is actively being pursued in order to define the inhibition mechanism of mab 1a9. file s1 supporting information figures s1-s6. figure s1 . infectivity of pseudotyped viruses expressing s protein (s-pps). (a) s-pp expressing s protein of humans sars-cov hku39849, civet sars-cov sz3, bat sl-cov rp3 and rf1 and (b) s-pp containing wild-type or mutant d1128a, n1056k or d1128a/ n1056k s were generated and used to infect cho-ace2 cells at equal amount (as quantitated using p24 elisa). cells were harvested 48 hours post-infection and luciferase readings were measured. pnl43-r-e-luc virus, which do not express s protein, was used as negative control. error bars represent sd of experiment carried out in triplicates. figure s2 . neutralization of human sars-cov hku39849, civet sars-cov sz3, bat sl-cov rp3 and rf1 s-pps by mab 1a9 (data presented using absolute luciferase readings). s-pps containing s of human sars-cov hku39849, civet sars-cov sz3, bat sl-cov rp3 or rf1, were pre-incubated with different concentrations of mab 1a9 at 100, 150 and 200 mg/ml for 1 hour before infecting cho-ace2 cells. cells were harvested 48 hours post-infection and luciferase activities were measured. data shown represents that obtained from 3 independent experiments. bars represent sd of the experiment carried out in triplicates. figure s3 . binding of mab 1a9 to wild-type, mutant d1128a and n1056k gst-s(1030-1188) fragments by elisa. in elisa, gst, gst-s(1030-1188) wild-type, gst-s(1030-1188)-d1128a and gst-s(1030-1188)-n1056k proteins were coated on a 96-well plate at 100 ng/ well and detected using (c) mab 1a9 and (d) mab gst at 4-fold serial dilutions. optical density (od) was measured at 450 nm. bars represent sd of the experiment carried out in triplicates. *indicates statistically significant difference (p,0.01) when compared to wild-type. mab gst was used as a control antibody to ensure that equal amounts of gst-tagged proteins were coated onto each well. a significant reduction of mab 1a9 binding was observed for s fragment containing the d1128a mutation (p, 0.01). figure s4 . expression and purification of wild-type and mutant gst-s(1030-1188) fragments. purified gst, gst-s(1030-1188) wild-type, gst-s(1030-1188)-d1128a and gst-s(1030-1188)-n1056k fragments (lanes 1-4) were separated on a 12% gel by sds-page and stained using commassie blue. molecular weight markers in kda are indicated on the left. expected size of each gst-s(1030-1188) fragment is around 43 kda as indicated by the arrow. figure s5 . neutralization of wild-type and mutant d1128a, n1056k and d1128a/n1056k s-pps by mab 1a9 and mab 1g10 (data presented using absolute luciferase readings). s-pp containing wild-type (s-wt-pp) or mutant s (s-d1128a-pp, s-n1056k-pp and s-d1128a/ n1056k-pp) were pre-incubated with different concentrations of (a) mab 1a9 and (b) mab 1g10 at 25, 50, 100 and 200 mg/ml for 1 hour before infecting cho-ace2 cells. an anti-s1, nonneutralizing mab 7g12 was used as control antibody at 200 mg/ ml. cells were harvested 48 hours post-infection and luciferase activities were measured. data shown represents that obtained from 3 independent experiments. bars represent sd of the experiment carried out in triplicates. *indicates statistically significant difference of p,0. 01 when compared to s-wt-pp. figure s6 . binding of mab 1a9 to wild-type, mutant d1128a, d1128e, d1128n and n1056k s proteins. 293 ft cells were transfected with no plasmid (mock) or with plasmids expressing full length wild-type s (s-wt) or full length mutant s (s-d1128a, s-d1128e, s-d1128n and s-n1056k). western blot analysis was performed on the cell lysates using mab 1a9 to determine its binding to the various s proteins. mab 7g12, which binds to the s1 of the s protein, was used as a control antibody to detect protein expression. 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syndrome (sars) coronavirus s protein neutralizes the virus in a rhesus macaque sars model b-cell responses in patients who have recovered from severe acute respiratory syndrome target a dominant site in the s2 domain of the surface spike glycoprotein genomic characterization of severe acute respiratory syndrome-related coronavirus in european bats and classification of coronaviruses based on partial rna-dependent rna polymerase gene sequences structural basis for potent cross-neutralizing human monoclonal antibody protection against lethal human and zoonotic severe acute respiratory syndrome coronavirus challenge potent crossreactive neutralization of sars coronavirus isolates by human monoclonal antibodies a single amino acid substitution in the s1 and s2 spike protein domains determines the neutralization escape phenotype of sars-cov the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex the sars coronavirus s glycoprotein receptor binding domain: fine mapping and functional characterization inhibition of severe acute respiratory syndrome-associated coronavirus (sars-cov) infectivity by peptides analogous to the viral spike protein key: cord-336615-jfnj6l41 authors: wong, sarah sze wah; kao, richard yi tsun; yuen, kwok yong; wang, yu; yang, dan; samaranayake, lakshman perera; seneviratne, chaminda jayampath title: in vitro and in vivo activity of a novel antifungal small molecule against candida infections date: 2014-01-22 journal: plos one doi: 10.1371/journal.pone.0085836 sha: doc_id: 336615 cord_uid: jfnj6l41 candida is the most common fungal pathogen of humans worldwide and has become a major clinical problem because of the growing number of immunocompromised patients, who are susceptible to infection. moreover, the number of available antifungals is limited, and antifungal-resistant candida strains are emerging. new and effective antifungals are therefore urgently needed. here, we discovered a small molecule with activity against candida spp. both in vitro and in vivo. we screened a library of 50,240 small molecules for inhibitors of yeast-to-hypha transition, a major virulence attribute of candida albicans. this screening identified 20 active compounds. further examination of the in vitro antifungal and anti-biofilm properties of these compounds, using a range of candida spp., led to the discovery of sm21, a highly potent antifungal molecule (minimum inhibitory concentration (mic) 0.2 – 1.6 µg/ml). in vitro, sm21 was toxic to fungi but not to various human cell lines or bacterial species and was active against candida isolates that are resistant to existing antifungal agents. moreover, sm21 was relatively more effective against biofilms of candida spp. than the current antifungal agents. in vivo, sm21 prevented the death of mice in a systemic candidiasis model and was also more effective than the common antifungal nystatin at reducing the extent of tongue lesions in a mouse model of oral candidiasis. propidium iodide uptake assay showed that sm21 affected the integrity of the cell membrane. taken together, our results indicate that sm21 has the potential to be developed as a novel antifungal agent for clinical use. candida is the most common fungal pathogen of humans and the fourth leading pathogen in nosocomial bloodstream infections [1] . it causes a wide range of infections, from invasive to superficial, at various anatomical sites [2] . invasive candidiasis is associated with high mortality among immunocompromised populations, and superficial mucosal candidiases are highly prevalent and persistent, especially among immunocompromised patients. the most common superficial candidiases are oral candidiasis and candidaassociated denture stomatitis [3] [4] [5] . candida-associated denture stomatitis could affect up to 70% of denture wearers [6] . the most prevalent candidiasis-causing species is candida albicans. it has a versatile morphogenesis, existing in three forms (i.e., yeasts, pseudohyphae and hyphae), and has two lifestyles (planktonic and biofilm), permitting c. albicans to thrive under diverse environmental conditions. in particular, the yeast-to-hypha (y-h) transition is a major virulence attribute that facilitates tissue invasion by c. albicans [7, 8] . in addition to y-h transition, biofilm formation on host tissue or abiotic devices is also a significant basis for candida infections [5] , examples include candida-associated denture stomatitis [9] . the formation of candida biofilms, which are more resistant to antifungal agents than planktonic cells, is directly associated with treatment failure [5, 10] . although candida infections have always posed a heavy burden on public health, this situation has recently worsened. first, the worldwide incidence of candidiasis has been increasing over the past few decades [11] , which may be attributable to increased numbers of immunocompromised patients and the use of broad-spectrum antibiotics [12] . second, the increased incidence of invasive candidiasis caused by non-albicans candida (nac) species, such as candida glabrata, candida tropicalis, candida krusei or candida parapsilosis, has been a major concern [13, 14] , because these infections are often associated with higher mortality and antifungal resistance than those caused by c. albicans [15, 16] . lastly, the emergence of antifungal resistance and treatment side effects have further restricted treatment options because of the already limited arsenal of current antifungal agents [17, 18] . only a few classes of antifungal drugs (polyenes, azoles, echinocandins, allylamines and dna analogues) are available for candidiasis treatment [19] , but these drugs are far from ideal. polyenes, for example, have dose-related toxicity, particularly nephrotoxicity (even though the recent introduction of lipid formulations has now improved the risk-benefit ratio) [20] . in addition, rising drug resistance is an inevitable problem. this is pertinent for fluconazole, a drug of choice for treating aids patients with candida infections. while c. glabrata and c. krusei are intrinsically resistant to fluconazole, the emergence of fluconazoleresistant c. albicans strains is also on the rise [21, 22] . similarly, emerging resistance has also been reported for the more recently introduced echinocandins [23] [24] [25] [26] . therefore, the situation is likely to be ameliorated only by the discovery and introduction of safe and new antifungal agents. small molecules are an invaluable source of novel antifungal agents [27] . previous screening of the same small-molecule library has resulted in the identification of novel compounds effective for severe acute respiratory syndrome-associated coronavirus [28] . here, we sought small molecules with anti-candida properties (with the overall strategy depicted in figure 1 ). we first screened a collection of over 50,000 small molecules for inhibitors of the y-h transition in c. albicans. the identified hits were further assessed for antifungal and anti-biofilm activity, which led us to discover a novel antifungal small molecule, which we designated sm21. we examined the in vitro antifungal activity of sm21, as well as its in vivo efficacy in mouse infection models of oral and systemic candidiases. last, the potential mechanism of action of sm21 was also investigated. high-throughput screening was performed at the chemical genetics unit, department of microbiology, research center of infection and immunology, li ka shing faculty of medicine, university of hong kong, on a library with 50,240 small molecules (chembridge, san diego, ca, usa) to identify inhibitors of y-h transition in c. albicans sc5314, as was previously described [29] . c. albicans sc5314 were seeded at 5610 3 cells per well in complete yeast peptone dextrose (ypd) (1% yeast extract, 2% peptone, 2% glucose/dextrose) supplemented with 20% heat-inactivated foetal bovine serum (invitrogen, carlsbad, ca, usa) in a total volume of 50 ml in 384-well microtitre plates. the small molecules were dissolved in dimethyl sulfoxide (dmso) and were added to the wells at final concentration of 20 mg/ml, whereas the controls contained the same amount of dmso but without small molecules. assay plates were incubated at 37uc in 5% co 2 for 12 h. morphologies of the candida were scored using a leica dmil inverted microscope equipped with dc300f digital imaging system (leica microsystems, heidelberg, germany). small molecules with lower scores than that of the control (yeast-to-hypha transition inhibitors) were selected as primary hits. dose-dependent y-h inhibition by the primary hits was further examined using the reference strain c. albicans sc5314 and ten clinical isolates of c. albicans (strains cl1 -cl10, of oral origin from the archival collection of oral biosciences, faculty of dentistry, university of hong kong) under robust hypha-inducing conditions (lee's medium [30] and 37uc incubation). each sample contained 100 ml of candida suspension (1610 6 cfus/ml) and 100 ml of the small molecules of different final concentrations (3.13 -25 mg/ml). after incubation for 24 h, cell morphologies were observed by light microscopy (olympus bh-2, tokyo, japan). the degree of y-h inhibition was quantified by calculating the percentage of hyphal cells in one hundred cells in a single sample [29] . counting was performed in quadruplicate for each sample. the minimum inhibitory concentration (mic) of y-h transition (mic y-h ) was determined as the minimum concentration at which no hyphae were observed. the assay was performed on three different occasions in duplicate. the antifungal activity of small molecules was assessed by disk diffusion and broth dilution assays (see below) using four groups of candida isolates. the first group consisted of atcc strains: c. albicans atcc 90028 (quality control strain), c. glabrata atcc 90030, c. krusei atcc 6258, c. parapsilosis atcc 22019 and c. tropicalis atcc 13803. the other three groups were clinical isolates: 92 bloodstream isolates (queen mary hospital and queen elizabeth hospital, hong kong), 46 oral isolates from nasopharyngeal carcinoma patients (queen mary hospital, hong kong) and 10 denture stomatitis clinical isolates (from the archival collection of oral biosciences, faculty of dentistry, university of hong kong). all of the isolates were subcultured on sabouraud dextrose agar (sda) and were incubated aerobically at 37uc overnight before the figure 1 . strategy for screening for novel antifungal small molecules. we screened for y-h inhibitors in a library containing 50,240 small molecules and found 20 active compounds. these 20 primary hits were further validated by assessing their activity in a dosedependent manner, which led to the identification of eight potent y-h inhibitors. the antifungal properties of the eight compounds were analysed in antifungal susceptibility tests, and the four most potent molecules were selected. finally, the anti-biofilm activity of the four hits was evaluated, and sm21 was chosen for comprehensive in vitro and in vivo assays. hts, high-throughput screening; ast, antifungal susceptibility test; abt, anti-biofilm test. doi:10.1371/journal.pone.0085836.g001 assay. all of the assays were performed on three different occasions in duplicate per isolate. the standard disk diffusion assay (clinical and laboratory standards institute (clsi) m44-a2) was used. in brief, a 1610 6 cfus/ml yeast suspension was prepared for each isolate in phosphate-buffered saline (pbs) and distributed evenly by a spiral plater on mueller-hinton (m-h) agar supplemented with 2% glucose and 0.5 mg/ml methylene blue. antifungal tablets (10 mg amphotericin b, 5 mg caspofungin, 15 mg ketoconazole and 25 mg fluconazole) (neo-sensitabs, rosco diagnostica, taastrup, denmark) were used as positive controls. paper coupons were placed on the agar, and either 2 mg of the small molecule or pbs (as a negative control) was added onto the coupons. the m-h agar plates were then incubated aerobically at 37uc for 24 h, and diameters of the resultant inhibition zones were measured. the standard protocol for broth microdilution assay (clsi m27-a3) was followed with modifications [31] . yeast suspensions (approximately 1610 3 colony forming units per millilitre (cfus/ ml)) were prepared in rpmi 1640 medium (life technologies, new york, usa) and were added to a 96-well microtitre plate (iwaki, tokyo, japan). serial dilutions of the small molecules were prepared with the medium and added to the wells. amphotericin b was used as a positive control. the plate was incubated at 37uc for 48 h, and then candida growth on the well bottoms was visually observed to determine the minimum inhibitory concentration (mic). activity against cryptococcus neoformans, aspergillus fumigatus and penicillium marneffei was similarly assessed after 72 h of incubation. candida biofilms were formed according to a previously described method [32] . in brief, yeast cells were subcultured on sda and incubated at 37uc aerobically for 18 h. they were then transferred to yeast nitrogen base (ynb) liquid medium supplemented with 50 mm glucose, and incubated aerobically at 37uc, 75 rpm overnight. the harvested cells were washed twice with 10 ml pbs and were resuspended to a density of 1610 7 cfus/ml in ynb medium supplemented with 100 mm glucose. each of the wells of the microtitre plate (iwaki) was inoculated with 100 ml yeast suspension. the cells were allowed to adhere to the well bottoms by incubating at 37uc, 75 rpm for 1.5 h (adhesion phase). then, the biofilm was washed with 100 ml pbs to remove the nonadherent cells, and 200 ml fresh medium was added to each well. the biofilm was then incubated at 37uc, 75 rpm for 24 h. for the screening assay, the small molecules (0.2 -100 mg/ml) were added to the biofilm before or after the adhesion phase. the effect of sm21 on mature biofilms was further investigated by adding sm21 (0.2 -100 mg/ml) to the biofilm at 24 h and 48 h. cell viability of the treated biofilms was measured by the xtt reduction assay [33] . the medium in each well was discarded, and 200 ml xtt solution was added. the xtt solution consisted of 40 ml xtt stock solution (1 mg/ml in pbs), 2 ml menadione (0.4 mm in acetone) and 158 ml pbs. the microtitre plate was incubated at 37uc in the dark for 3 h. then, 160 ml xtt solution from each well was transferred to a new microtitre plate, and the absorbance was measured at 490 nm. the minimum inhibitory concentration for biofilm (mic biofilm ) was determined as the concentration that caused a 50% reduction in cell viability. all of the assays were performed on three different occasions in duplicate. anti-biofilm assay using an in vitro denture stomatitis model cold-cured polymethylmethacrylate (pmma) discs were prepared according to the manufacturers' instructions (vertex rs, vertex-dental bv, zeist, the netherlands) and as described previously with some modifications [34] . in brief, transparent selfpolymerising acrylic powder was mixed with monomer liquid at a 1.5:1 ratio (w/v). this mixture was then immediately spread onto and pressed tightly between two aluminium foil-covered glass slides that had been secured with two binder clips to ensure similar thickness and surface of the resultant acrylic strips. after curing for 15 min at 40uc, 2 -6 bar, the strips were carefully retrieved and cut into 1-cm 2 squares. the strips were immersed in distilled water for 7 days to remove excess monomers, and then disinfected in 70% alcohol for 1 min and washed thrice with sterile distilled water. to simulate conditions present in the mouth and to promote candida adhesion, the strips were treated with saliva for 30 min at 37uc [35] . the saliva had been collected from five healthy volunteers and centrifuged at 12,0006 g for 15 min at 4uc, and the resulting supernatant was stored at 270uc until use [36] . c. albicans isolate bf-1, which was identified as a stronger biofilm former in a previous study [36] , was subcultured on sda one day before the assay. the yeast inoculum was standardised to 1610 7 cfus/ml in ynb supplemented with 100 mm glucose. the acrylic strips were placed in 24-well plates (iwaki), and 1 ml yeast inoculum was added to each well, completely covering the acrylic strips. sm21 was added at mic biofilm (1.56 mg/ml) before, after, or both before and after the adhesion phase. the adhesion phase was assessed 1.5 h after inoculation, and the plate was then placed in a shaking incubator at 37uc. the cell viability of the biofilm on the acrylic strips was quantified by the xtt reduction assay and was also observed by confocal laser scanning microscopy (olympus fluoview fv1000) after staining using a live/dead baclight viability kit (invitrogen). engineered c. albicans strain p hwp1 -gfp, which expressed gfp under the control of either the hwp1 promoter, was gift from dr. b. p. krom [37] . the p hwp1 -gfp strain was used to test the effect of sm21 on hwp1 expression. the strains were cultivated overnight at 37uc in lee's medium containing sm21 at mic (0.2 mg/ml) or 26mic (0.4 mg/ml). gfp expression was then observed using confocal laser scanning microscopy (olympus fluoview fv1000). the disk diffusion assay (clsi m44-a2) was also used to assess the potential activity of sm21 against six bacterial species (escherichia coli, lactobacillus acidophilus, streptococcus mutans, streptococcus mitis, streptococcus sanguinis and aggregatibacter actinomycetemcomitans). the bacteria were subcultured one day before the assay. bacterial cell suspensions were prepared for each sample at a density of 1610 6 cfus/ml and were then distributed evenly on sheep blood agar plates with a spiral plater. paper coupons inoculated with 10 ml of drugs or controls (0.2 mg/ml sm21, 1 mg/ ml amphotericin b and 0.5 mg/ml caspofungin) were placed on the agar. chlorhexidine gluconate (0.2%) was used as a positive (antibacterial) control, and pbs as a negative (harmless) control. the plates were incubated anaerobically (except for the e. coli plate) at 37uc for 24 h, and the diameters of inhibition zones were measured. the efficacy of sm21 against 16 antifungal-resistant candida strains was evaluated by antifungal susceptibility tests (broth microdilution and disk diffusion assays) as described above. the strains were clinical blood isolates belonging to various candida spp., which had been obtained from helsinki university central hospital (helsinki, finland) and that had been confirmed to have resistance to one or more antifungal agents by standard clsi antifungal susceptibility tests in our previous study [25] . primary human oral keratinocytes (hoks) (sciencell research laboratories, carlsbad, ca, usa) at passage three were seeded into a 96-well plate (iwaki) in oral keratinocyte medium (sciencell research laboratories). the serum-free medium contained 1% oral keratinocyte growth supplement, 1% penicillin and 1% streptomycin. each well initially contained approximately 1610 4 hoks, which were incubated in the presence of 5% co 2 at 37uc (medium was changed every day) until the cells reached confluence. a dilution series of sm21 (0.05 -54.8 mg/ml) was then supplied to the wells so that each well contained 200 ml of medium in total. the plate was incubated at 37uc for 24 h, and then the viability of the hoks was determined by mtt assay (see below) . t-75 flasks were also used to assess cell viability in the presence of sm21. besides hoks, human gingival fibroblasts (hgfs) and monocyte cell lines (sciencell research laboratories) were also used. each cell line was seeded in t-75 flasks, and after confluence was reached, 2 mg sm21 in 10 ml of growth medium (0.2 mg/ml final concentration) was added to each flask. for comparison, amphotericin b was used at the same concentration. after 24 hincubation at 37uc, mtt assay was performed to measure the cell viability. mtt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is a yellow tetrazolium dye that is converted into a purple compound by mitochondrial enzymes. mtt solution (5 mg/ml) was prepared in growth medium and added to the wells or flasks. after incubation at 37uc for 3 h in dark, the mtt solution was discarded. the converted dye was solubilised with an equal amount of dmso. the absorbance of the converted dye was measured at 570 nm with background subtraction at 650 nm. the concentration at which the cell viability had dropped by 50% was recorded as the cytotoxic concentration (cc 50 ) [38] . hok culture was performed as mentioned above. hoks at passage three were seeded into an 8-well plate (ibidi, martinsried, germany), which was compatible for the observation with confocal laser scanning microscope. the cells were incubated at 37uc in the presence of 5% co 2 until confluence was reached, with the medium changed every day. each well contained approximately 1610 5 hoks at confluence. the cells were washed once with pbs, and fresh medium without antibiotics (as antibiotics could inhibit candida growth) was added. a suspension of c. albicans sc5314 (1610 4 cfus/ml obtained by subculturing on sda plates and incubating at 37uc one day before inoculation) was prepared in the growth medium (without antibiotics), and 100 ml of this cell suspension was added to each well. sm21 was the animal studies strictly followed the recommendations in ''guide for the care and use of laboratory the mice used were 8-week-old male c57bl/6 mice that were obtained from the laboratory animal unit, university of hong kong. mouse model of systemic candidiasis was established according to a previously described method [39] , with modifications. the mice were injected via the tail vein with 100 ml cell suspension of c. albicans sc5314 (1610 6 cfus/ml) 3 h before the start of antifungal treatment. drugs were given to the mice twice a day for 5 days via intraperitoneal injection. the test group (n = 5) was treated with 0.01, 0.1, 1 and 10 mg/kg sm21 in 100 ml pbs, while the control group (n = 5) was treated with pbs. the mice were weighed every day. all of the mice were sacrificed after 5 days, and the kidneys were harvested (as most of the candida cells in systemic candidiasis murine models are found in the kidneys [39] ). the kidneys were divided into equal portions for fungal burden determination and histopathological evaluation. for fungal burden determination, the kidneys were weighed and then homogenised in pbs, and the homogenates were serially diluted before plating on sda. the plates were incubated at 37uc, and fungal burden was expressed as the ratio of colony forming units (cfus) to the organ weight. for histopathological analysis, organs were fixed in 4% paraformaldehyde, embedded with paraffin wax and stained with periodic acid-schiff (pas). the murine model of oral candidiasis was established according to a previously described method [40, 41] . one day before infection, c57bl/6 mice were immunosuppressed by subcutaneous injection of two doses of prednisolone (100 mg/kg body weight), and the antibiotic tetracycline hydrochloride (0.83 mg/ ml) was administered in the drinking water. immediately before infection, the mice were anesthetised with 50 ml chlorpromazine chloride (2 mg/ml) via intramuscular injection on each femur. the oral cavities of the anesthetised mice were swabbed with a sterilised cotton swab that had been dipped in a cell suspension of c. albicans sc5314 (2.5610 7 cfus/ml; from cultures grown overnight at 37uc on sda). subsequently, 10 ml sm21 (200 mg/ ml; n = 3), 10 ml nystatin (10 mg/ml; n = 3) or 10 ml pbs (as a control; n = 3) was administered to the mice by pipetting into their mouths; this was done five times, at 3, 6, 12, 24 and 36 h after inoculation. the mice were sacrificed 72 h after infection and were immediately observed macroscopically for tongue lesions. the tongue lesion degree was evaluated using a scoring system of 0 -3 [40] , with 0 denoting a healthy tongue surface and 3 the most severe stage. the entire oral cavities of the mice were swabbed with sterilised cotton swabs, which were then submerged in 500 ml pbs and vortexed vigorously. the resultant suspension was spiral plated on sda, and the number of cfus was counted after incubation at 37uc for 24 h. in addition, the invasion of tongue tissues by candida was evaluated histologically by pas staining, as described above. propidium iodide, a membrane-impermeable fluorescent dye that binds to nucleic acids, is widely used to differentiate cells that have damaged plasma membranes from healthy ones [42, 43] . to evaluate the effect of sm21 on the fungal plasma membrane, c. albicans sc5314 cells (approximately 1610 3 cfus/ml) were obtained from logarithmic phase cultures and suspended in rpmi 1640 medium, as described above. the cells were then exposed to sub-mic of sm21 (0.1 mg/ml) for 1 h at 30uc with gentle shaking. subsequently, cells were harvested, incubated with propidium iodide using a live/dead baclight viability kit one-way analysis of variance (anova) was employed to evaluate the mean difference between the control and the test groups. statistical significance of the data was analyzed using spss software package (spss version 20; spss inc, chicago, il, usa). data was considered significant if p-values are less than 0.05. to obtain small molecule inhibitors of candida spp., we first screened 50,240 small molecules for inhibitors of the y-h transition in c. albicans, and 20 active compounds were identified. of these, eight compounds displayed strong dose-dependent y-h inhibition under hypha-inducing conditions for the reference strain c. albicans sc5314 and ten clinical isolates of c. albicans (figure 2 ). of these eight compounds, four (sm10, sm12, sm16 and sm21) formed inhibition zones in disk diffusion assays using the standard reference strain c. albicans atcc 90028, indicating fungicidal activity (the result of sm21 is shown in figure s1 ). in addition, sm21 had the most potent anti-biofilm activity out of the four compounds ( figure 3) . therefore, sm21 (molecular weight = 438 g/mol, chembridge id# 6633321), which had not figure 8 . effect of sm21 on c. albicans biofilm formation on denture acrylic. (a) confocal images of c. albicans biofilm after treatment with sm21 and staining with fluorescent labels that distinguish between live and dead cells. all cells were labeled with the green fluorescence, while only the dead cells were labeled with red fluorescence. (b) biofilm cell viability, quantified by xtt reduction assay, was reduced by 85% and 66%, respectively, when sm21 was added before or after the adhesion phase. the reduced biofilm viability was maximised (97%) when sm21 was added both before and after the adhesion phase. the standard deviations of each sample are shown in the graph, and all the mean differences between the control and test (sm21) were statistically significant (p-value,0.05). doi:10.1371/journal.pone.0085836.g008 been previously identified as an antifungal agent, was chosen for further study (figure 4 ). to further examine the y-h inhibition of sm21, the phenotype of a panel of candida isolates, including reference strain c. albicans sc5314 and ten c. albicans clinical isolates of oral origins was observed under robust hypha-inducing condition in the presence of sm21. in the control, where sm21 was not added, extensive hyphae could be observed after 24-h incubation. in contrast, only yeast cells, and not hyphae, could be observed in samples laced with sm21 of concentration 0.43 mg/ml or higher ( figure 5a ). the mic y-h of sm21 was 0.43 mg/ml (equivalent to 0.98 mm) (for 1610 6 cfus/ml c. albicans sc5314; figure 5b ), which was similar to the average mic y-h for ten clinical isolates (0.5 mg/ml) ( table 1) . furthermore, the mic y-h was directly proportional to the cell density (table 1 ). in addition, we used a c. albicans p hwp1 -gfp strain to test whether sm21 affected hwp1 expression. gfp was expressed in candidal hyphae under untreated conditions (as observed by confocal fluorescence microscopy; figure 6 ). by contrast, no trace of fluorescence could be detected from the sm21-treated cells at both mic y-h and 26mic y-h , suggesting that sm21 influenced hwp1 expression. an ideal antifungal agent should be active against fungal species but not against bacteria. the antifungal activity of sm21 was exhibited by disk diffusion assay and broth microdilution assay. sm21 formed inhibition zones with c. albicans sc5314, c. albicans atcc 90028 and all of the 148 clinical isolates tested. the average diameter of the inhibition zones of sm21 against these isolates was 27.7 mm (range = 25 -31 mm) (the growth inhibition zones of sm21 against c. albicans sc5314 and c. albicans atcc 90028 were shown in figure s1 .). for the broth microdilution assay, the mic of sm21 against c. albicans atcc 90028 was found to be 0.2 mg/ml, which was similar to that of the conventional antifungal amphotericin b, suggesting a comparable potency ( table 2 ). the mic of sm21 for a wide range of candida spp. (including clinical isolates) and other fungal species (except a. fumigatus) ranged from 0.2 to 1.6 mg/ml (table 2) . lower susceptibility to sm21 was noted with the fungus a. fumigatus (mic = 6.25 mg/ml), as was also observed for amphotericin b (mic = 1.56 mg/ml). sm21 did not inhibit the growth of any of the six bacterial species evaluated in disk diffusion assay, implying the fungal specificity of the novel molecule (figure 7) . we then assessed the activity of sm21 against candida biofilm formation. we first used a microtitre-plate biofilm model in which sm21 was added to the 24-h and 48-h biofilm of c. albicans, c. glabrata and c. krusei. sm21 demonstrated a lower mic biofilm than table 3 ). in an in vitro model of denture stomatitis, sm21 effectively prevented candidal adhesion and biofilm development on denture acrylic surfaces ( figure 8a ). in this model, biofilm viability was reduced by 85%, 66% and 97%, when sm21 was added before, after, or both before and after the 1.5 h adhesion phase respectively ( figure 8b ). summarized from the two assays, sm21 was effective against mature candida biofilm and inhibited biofilm formation on denture acrylic surfaces. sm21 is active against antifungal-resistant candida spp. the emergence of antifungal-resistant candida strains has led to the urgent need of new antifungal agents. all of the drug-resistant isolates tested were susceptible to sm21. for instance, a clear sm21 inhibition zone was observed for t-1549, a multidrugresistant c. guilliermondii strain (figure 9 ). the mic of sm21 against drug-resistant candida isolates in broth microdilution assays ranged from 0.5 to 1 mg/ml (table 4 ), which was marginally higher than the mic for c. albicans atcc 90028. cytotoxicity assays performed in a 96-well plate revealed the cc 50 of sm21 to be 3.4 mg/ml (7.8 mm) for hoks. in assays conducted in t-75 flasks, 0.2 mg/ml sm21 reduced the viability of hoks by 12% compared with the untreated controls, which is lower than the 22% reduction observed for amphotericin b. treatment with sm21 or amphotericin b caused, respectively, a 20% or 5% reduction in monocyte viability compared with the controls. neither sm21 nor amphotericin b affected the viability of hgfs (figure 10 ). to test whether sm21 was able to prevent c. albicans invasion of human cells, a co-culture of hoks and c. albicans sc5314 was exposed to various concentrations of sm21. in the co-culture control (in which no sm21 was present), many hyphae were observed. the hyphal invaded and caused the death of most of the hoks (figure 11 ). at 0.5 mg/ml sm21, candida hyphae could still be observed, however, the proportion of live hoks increased. at 2 mg/ml and 4 mg/ml sm21, most of the hoks were alive, and no candida were observed. therefore, this sm21 concentration was safe to hoks and was sufficient to prevent candida from invading the human cells. a mouse model of systemic candidiasis was used to assess the in vivo activity of sm21. infection was established in mice by injecting 100 ml of 1610 6 cfus/ml c. albicans sc5314 via tail vein. the test group was treated with 0.01, 0.1, 1 and 10 mg/kg sm21, while control group was treated with pbs. at day 5 (the experimental end point) after infection with c. albicans, all of the untreated mice with systemic candidiasis had died, whereas all of the sm21-treated mice were alive ( figure 12a ). the fungal burden in the kidneys was significantly lower in the sm21-treated mice than in the untreated ones ( figure 12b ). moreover, white candida lesions were visible covering the surfaces of the kidneys of the untreated mice, while the kidneys of the sm21-treated mice appeared healthy ( figure 12c ). clusters of candida hyphae were easily identified in pas-stained kidneys of the untreated mice, whereas very few candida cells were observed in the kidney parenchyma of the sm21-treated mice ( figure 12d ). these results indicated that sm21 was effective in preventing systemic spread and invasion of candida in the mouse model. we then assessed the effects of sm21 and nystatin treatments in a mouse model of oral candidiasis established by inoculating c. albicans sc5314 (2.5610 7 cfus/ml) in the oral cavities of the mice. treatment was given to the mice at five time intervals. immediately after sacrifice (72 h after inoculation), scores were assigned for tongue lesions from 0 (healthy tongue surface) to 3 (most severe stage). thick oral thrush was observed in the untreated mice (score = 3; figure 13a ). tongue lesions in the nystatin-treated mice were less severe than in the untreated controls, although considerable oral thrush was noted at the back of the tongue (score = 2). compared with the other two groups, figure 10 . cytotoxicity of sm21 against three primary cell lines. hok -human oral keratinocyte, hgf -human gingival fibroblast, ambamphotericin b. both sm21 (0.2 mg/ml) and amphotericin b (0.2 mg/ml) reduced the viability of hoks and monocytes. sm21 caused a reduction of 12% in cell viability compared with the untreated controls, which is lower than the 22% reduction observed for amphotericin b. treatment with sm21 or amphotericin b caused, respectively, a 20% or 5% reduction in monocyte viability compared with the controls. neither sm21 nor amphotericin b affected the viability of hgfs. doi:10.1371/journal.pone.0085836.g010 sm21-treated mice displayed the least severe tongue lesions (score = 1). moreover, candida cell counts from tongue swabs of the sm21-treated mice (4.5610 2 cfus/ml) were consistently lower than those of the control mice (4.8610 3 cfus/ml) and the nystatin-treated animals (6.0610 2 cfus/ml). numerous candidal hyphae were noted in pas-stained tongue sections of the untreated mice, whereas less profuse candidal growth was observed in samples from the mice treated with sm21 or nystatin ( figure 13b ). to shed some light on the mechanism of action of sm21, we performed propidium-iodide uptake assays. candida exposed to sub-mic of sm21 (0.1 mg/ml) displayed abundant propidium iodide uptake, indicating cell membrane damage ( figure 14) . therefore, the molecular target of sm21 might be a component of the cell membrane. a handful of small molecules have been reported to possess activity against c. albicans [29, [44] [45] [46] [47] . however, the therapeutic potential of these small molecules is unclear, because they have been evaluated mainly for the ability to inhibit c. albicans virulence properties such as y-h transition or biofilm formation in vitro. it has been hypothesised that arresting candida in the unicellular yeast form would result in reduced virulence. this hypothesis is supported by the finding that non-filamentous c. albicans mutants are not virulent in a mouse infection model [48] , however, this is only relevant to c. albicans. some nac species (which do not form true hyphae) are also virulent. moreover, the strategy of inhibiting virulence factors could facilitate the emergence of resistant strains because it only inhibits the growth of virulent strains but does not kill them. complete elimination of pathogen is clearly a safer option; therefore, fungicidal drugs are preferred. figure 11 . effect of sm21 treatment in candida-hok co-culture model. the viability of co-cultured hoks and yeast cells in the presence of sm21 was assessed by confocal laser scanning microscopy using fluorescent probes. in the untreated control, many hyphae were observed, and most of the hoks were dead (the nuclei of the dead cells were labeled with red fluorescence). samples containing 0.5 mg/ml sm21 still included many hyphae, but they contained more live hoks than the untreated control. samples containing 1 mg/ml or 2 mg/ml sm21 included few yeast cells and no hyphae, and most of the hoks were alive. magnification = 206 (unless specified otherwise). doi:10.1371/journal.pone.0085836.g011 for all of the above reasons, we set out to identify new antifungal compounds with both fungicidal and anti-virulence properties. to this end, we first screened for y-h inhibitors a library of 50,240 small molecules. it should be noted that the y-h inhibitor screening assay selects for molecules that block the change from the yeast form of c. albicans to the hyphal form, as assessed by morphology. therefore, this assay does not discriminate between molecules that inhibit the growth of c. albicans, which would also prevent the y-h transition, and true inhibitors of the y-h transition. moreover, conventional antifungal agents such as fluconazole, amphotericin b and 5-fluorocytosine, whose mechanisms are not directly related to y-h inhibition, also inhibit the formation of hyphae [49] . nevertheless, this ambiguity of the screening assay was favourable for increasing the hit rate in our initial screening. after several in vitro analyses of the antifungal properties of 20 small molecules identified by our initial screening, we found that one of them, sm21, is a potent y-h inhibitor that is effective at [46] , which is much higher than the sm21 mic y-h (0.98 mm for 1610 6 cfus/ml). the number of candida decreased after sm21 treatment in vitro, suggesting that this compound is fungicidal. fungicidal drugs inhibit y-h transition at sub-mic, whereas fungistatic drugs inhibit y-h transition only at much higher concentrations than their mic [50] . it has been suggested that fungicidal drugs inhibit hyphal development and the budding process, whereas fungistatic drugs inhibit only the budding process [50] . the in vitro antifungal activity of sm21 is comparable to that of commonly used antifungal drugs such as amphotericin b (a polyene). in disk diffusion assays, sm21 forms clear inhibition zones that resemble those produced by polyenes and echinocandins, and unlike the partial inhibition zones formed by azoles, for which trailing endpoint is observed [51] . the mic of sm21 in broth microdilution assays for a wide range of c. albicans strains (0.2 -1.6 mg/ml) is similar to the mic of amphotericin b, which has been considered to be a gold standard of antifungal agents [52] . moreover, the activity of sm21 against c. albicans biofilms grown on microtitre plates is similar or more potent than that of figure 13 . effect of sm21 treatment on a mouse model of oral candidiasis. (a) the degree of tongue lesions in the oral candidiasis mouse model was evaluated by a scoring system of 0 -3 (0 denotes healthy tongue surface and 3 denotes the most severe stage). a thick oral thrush (score = 3) was observed in untreated mice. nystatin-treated mice displayed less oral thrush on the tongue surface than the untreated controls, but a considerable amount of oral thrush was observed at the back of the tongue (score = 2). sm21-treated mice displayed the least severe tongue lesions (score = 1). (b) pas staining of tongue sections. abundant hyphae (indicated by black arrows) covered most of the tongue surface in the untreated mice, whereas comparatively fewer hyphae were observed in nystatin-and sm21-treated mice. doi:10.1371/journal.pone.0085836.g013 amphotericin b or caspofungin ( table 3 ). the mic biofilm of sm21 increases as the biofilm matures, a common phenomenon among antifungal agents [53] . attachment to mucosal tissue and biofilm formation are the basis of mucosal candidiases such as oral candidiasis and candidaassociated denture stomatitis [4, 9] . sm21 is effective at reducing candidal attachment to denture acrylic surfaces and inhibiting biofilm development in an in vitro model of denture stomatitis. these promising data indicate that sm21 might have potential as a denture disinfectant or as the active ingredient in an oral drug delivery system. we also analysed the in vivo effects of sm21 treatment on mouse models of oral and systemic candidiasis. we showed that oral rinses containing 200 mg/ml sm21 significantly reduced tongue lesions in the oral candidiasis mouse model. moreover, the efficacy of sm21 was better than that of nystatin, which is the most commonly used antifungal for oral candidiasis [4] . in particular, oral thrush was observed at the back of the tongues in nystatintreated mice (indicating oropharyngeal candidiasis) but not in sm21-treated animals. however, very little is known about the pharmacokinetics and pharmacodynamics of nystatin in murine models of oral candidiasis, and therefore the concentration used in our experiments (10 mg/ml) might not be high enough to produce an optimal outcome. in addition, the duration of an oral rinse cannot be controlled in a mouse model. we evaluated sm21 toxicity against human cell lines in vitro. in contrast to bacteria, the cells of fungi and humans are eukaryotic and, therefore, very similar. this similarity is a major hurdle in antifungal development, which requires drug targets to be fungusspecific to avoid undesirable toxicity [54] . we found that the cc 50 of sm21 for hoks is 7.8 mm (3.4 mg/ml) in a 96-well plate assay. although this value is lower than the minimum cc 50 (16 mm) reported for 14 antifungal compounds that were identified by lafleur et al (tested against human fibroblasts in 96-well plates; [45] ), the cc 50 of sm21 is still higher than its mic (0.2 mg/ml), which indicates that there is a range of concentrations at which sm21 could be used as an antifungal agent without causing significant toxicity to human cells. indeed, no detrimental effects were observed in mice that had been injected with sm21 doses as high as 10 mg/kg twice daily. in addition, sm21 does not seem to have antibacterial properties, indicating that its target is present in fungi but not in bacteria. we demonstrated that intraperitoneal administration of sm21 is an effective treatment for systemic candidiasis in a mouse model. an sm21 dose of 0.1 mg/kg or higher twice daily beginning 3 h post-infection significantly reduced fungal burden in non-neutropenic mice. the in vivo efficacy of sm21 is similar to that of conventional antifungal agents in comparable non-neutropenic mouse models of systemic c. albicans candidiasis. for example, the intraperitoneal ed 50 (50% effective dose) of fluconazole is 4.56 mg/kg when administered to the mice as a single dose 5 h after infection; the animals were sacrificed 24 h later [55] . intraperitoneal amphotericin b (1 mg/kg dose daily) decreases fungal burden in mouse kidneys from day 1 to day 3 after infection (with treatment beginning 24 h after infection) [56] . in addition, intravenous micafungin treatment (0.125 mg/kg or higher daily dose, starting 1 h after infection for 4 days) prolongs survival of infected mice [57] . however, it is difficult to compare the effective doses from different studies because there is no standard protocol, and differences in the candida strain, inoculum size, mouse strain, mouse immune status and the start and interval of antifungal therapy could interfere with the outcome. in addition, there are differences in the in vivo drug efficacy among normal and neutropenic murine models, which may be due to the possible interactions between the drug and host defence mechanisms [56] . detailed pharmacodynamics-pharmacokinetics studies would need to be performed in the future to determine the time/concentration relationship of the drug at the infection sites and, thus, to generate the dosing regimens that produce the best treatment outcome in vivo [58] . sm21 is effective against antifungal-resistant clinical isolates, indicating that its mechanism of action is distinct from those of conventional antifungals. our preliminary results described here indicated that sm21 could be targeting a component of the fungal cell membrane. nevertheless, extensive assays are needed to elucidate the mechanism of action of this new drug. in conclusion, in the present study we have characterised novel antifungal small molecule, sm21 using a comprehensive set of in vitro and in vivo assays. our findings support sm21 as a promising compound for development of novel antifungal agent for treating local and systemic candidiasis, which could bring enormous benefit to the patients suffering from this recalcitrant fungal pathogen. supporting information figure s1 antifungal susceptibility test (disk diffusion assay) of sm21 against c. albicans sc5314 and c. albicans atcc 90028. 21 -sm21, a -amphotericin b, ffluconazole, k -ketoconazole. growth inhibition zones were observed for sm21 against the two c. albicans strains tested. the growth inhibition zones produced by sm21 were clear, similar to those produced by amphotericin b, but dissimilar to those produced by fluconazole. this phenomenon suggested that sm21 is, like amphotericin b, fungicidal in nature. 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need new patented drugs apart from new strategies? pharmacodynamics of fluconazole in a murine model of systemic candidiasis comparison of the efficacies of amphotericin b, fluconazole, and itraconazole against a systemic candida albicans infection in normal and neutropenic mice efficacy of fk463, a new lipopeptide antifungal agent, in mouse models of disseminated candidiasis and aspergillosis animal model pharmacokinetics and pharmacodynamics: a critical review we acknowledge the editorial assistance by nature publishing group (npg) accredited msc editorial service. key: cord-333208-tibtngy8 authors: muñoz-moreno, raquel; cuesta-geijo, miguel ángel; martínez-romero, carles; barrado-gil, lucía; galindo, inmaculada; garcía-sastre, adolfo; alonso, covadonga title: antiviral role of ifitm proteins in african swine fever virus infection date: 2016-04-26 journal: plos one doi: 10.1371/journal.pone.0154366 sha: doc_id: 333208 cord_uid: tibtngy8 the interferon-induced transmembrane (ifitm) protein family is a group of antiviral restriction factors that impair flexibility and inhibit membrane fusion at the plasma or the endosomal membrane, restricting viral progression at entry. while ifitms are widely known to inhibit several single-stranded rna viruses, there are limited reports available regarding their effect in double-stranded dna viruses. in this work, we have analyzed a possible antiviral function of ifitms against a double stranded dna virus, the african swine fever virus (asfv). infection with cell-adapted asfv isolate ba71v is ifn sensitive and it induces ifitms expression. interestingly, high levels of ifitms caused a collapse of the endosomal pathway to the perinuclear area. given that asfv entry is strongly dependent on endocytosis, we investigated whether ifitm expression could impair viral infection. expression of ifitm1, 2 and 3 reduced virus infectivity in vero cells, with ifitm2 and ifitm3 having an impact on viral entry/uncoating. the role of ifitm2 in the inhibition of asfv in vero cells could be related to impaired endocytosis-mediated viral entry and alterations in the cholesterol efflux, suggesting that ifitm2 is acting at the late endosome, preventing the decapsidation stage of asfv. upon infection with pathogens such as bacteria or viruses, the host cell activates the innate immune response as a first line of defense. the group of cytokines known as interferons (ifn) plays a major role in the cell immunity by inducing a cascade of interferon-stimulated genes (isgs) that encode for several antiviral innate immune effectors. among isgs, the interferoninduced transmembrane proteins (ifitms) are known to inhibit entry of a wide variety of enveloped rna viruses [1] . this group of proteins is present across a wide range of species: from amphibians, fish and birds to mammals. ifitms in humans were identified 26 years ago as interferon-stimulated genes upon induction of type-i and type-ii ifn [2, 3] . human ifitm1, ifitm2 and ifitm3 are expressed in almost every cellular type, whereas ifitm5 is expressed primarily in osteoblasts, as it is required for bone mineralization [4] . ifitms are found mainly distributed at the plasma membrane and/or at endosomal membranes. the ifitm1, 2, 3 and 5 genes are clustered on chromosome 11, and they encode for relatively small proteins (about 130 amino acids) with both extra-cytoplasmic termini separated by two transmembrane domains (tm1 and tm2) and a cytoplasmic loop (cil) [3] [5] . tm1 and the cil are well conserved between the ifitm proteins and a large group of members of the cd225 protein family. ifitm 1, 2, and 3 are currently known to inhibit the replication of multiple rna viruses that enter the host cell via endocytosis, including influenza a virus (iav), west nile virus (wnv), dengue virus (denv) [6] , severe acute respiratory syndrome coronavirus (sars cov) and hepatitis c virus (hcv) [7] . in contrast, ifitms do not inhibit the entry process of mouse leukemia virus (mlv), machupo virus (mach), lassa virus (lasv) or lymphocytic choriomeningitis virus (lcmv) [4] . little is known about the ifitm-mediated antiviral activity against dna viruses. only ifitm1 has been recently described to inhibit rana grylio virus (rgv), a frog/fish iridovirus, at the entry stage [8] . on the other hand, ifitm1, 2 and 3 have been reported not to affect the replication of other dna viruses, such as human papillomavirus (hpv), human cytomegalovirus (hcmv) and adenovirus 5 (ad5) [9] . the antiviral effect of ifitms is mainly exerted through their effects on the endocytic pathway and would affect viruses entering the cell through a late endosomal compartment [4] . to further expand our understanding on the antiviral activity of ifitms against dna viruses, we investigated the role of these proteins in the replication cycle of the african swine fever virus (asfv), belonging to the nucleocytoplasmic large dna virus (ncldv) superfamily [10] . asfv infection is strongly dependent on the endocytic pathway [11, 12] , thus a possible ifitm-mediated inhibition of the virus could likely occur in the endosomal compartments. asfv is the only member of the asfarviridae family and is responsible of a highly lethal and hemorrhagic disease affecting domestic swine, which often results in important economic losses in many countries due to the high rate of mortality associated with the illness and the lack of an effective vaccine [13] . an epidemic outbreak is currently affecting east europe and is slowly spreading between neighboring countries [14] [15] [16] . we previously reported that asfv enters into the host cell by dynamin-dependent and clathrin-mediated endocytosis [12, 17] . thus, our goal in the current work was to test whether the ifitm family of proteins affected early entry steps of asfv infection in vero cell cultures using the cell-adapted ba71v isolate. vero cells were obtained from the american type culture collection (atcc, richmond, va, usa) and maintained in dulbecco modified eagle medium supplemented with 5% fetal bovine serum (fbs), 100 lu/ml penicillin, 100ug/ml streptomycin and 2mm l-glutamine at 37°c at 5% co 2 . cells were pretreated with 1,000 or 10,000 u/ml of universal type-i ifn (pbl assay science) for 24 h, as indicated. the tissue culture-adapted asfv isolate ba71v was used in most experiments [18] . for flow cytometry analyses, a recombinant virus expressing the viral protein p54 fused to the green fluorescent protein (gfp) was used (b54gfp) [19] . preparation of viral stocks, titrations and infection procedures were carried out in vero cells as previously described [18] . when synchronization of viral infection was required, the adsorption phase took place at 4°c to allow viral attachment to the cell surface but impeding its internalization. when indicated, asfv was semi-purified by sucrose cushion (40%) in pbs at 40,000xg for 50 min at 4°c. to generate stable cell lines expressing different proteins, the commercially available lentiviral expression vector plvx-puro (clontech) was used to clone the proteins of interest. 293t cells were transfected at 100% confluency using lipofectamine 2000 (life technologies) with opti-mem (life technologies) in 10-cm 2 plates. plates were previously pretreated with poly-l-lysine (sigma-aldrich) at a final concentration of 0.1 mg/ml to avoid cell detachment. co-transfection of plvx-puro expression vector together with vesicular stomatitis virus glycoprotein (vsv-g) and the human immunodeficiency virus (hiv) gag-pol expressing plasmids was performed to produce pseudotyped lentiviral vectors. supernatants containing the pseudotyped lentiviruses were collected twice at 48 h and 72 h postransfection. cell debris was removed by brief centrifugation at 1,000 rpm for 5 min and cleared supernatants were 0.22 μm-filtered and stored at -80°c until use. sub-confluent vero cells were transduced with the pseudotyped lentiviruses expressing the gene of interest and supplemented with 1 μg/ml of polybrene (emd millipore). 24 h later, transduced cells were selected by adding 8 μg/ml of puromycin (life technologies). optimal puromycin working concentration was previously titrated in non-transduced cells. finally, protein expression levels of vero stable-cell lines were determined by western blot (wb). cells were seeded and grown on glass coverslips. mock-infected and infected cells were fixed with 4% paraformaldehyde in pbs for 15 min at room temperature (rt). following cell fixation, aldehyde fluorescence was quenched by incubation of cells with 50 mm nh 4 cl in pbs for 10 min. then, cells were permeabilized with pbs-0.1% triton x-100 or saponin (sigma) for 10 min at rt. after blocking with bovine serum albumin (bsa; sigma) or normal goat serum (sigma), cells were stained with primary and secondary antibodies and then incubated with topro-3 (molecular probes) in pbs at a 1:1,000 ratio for dna staining. after washing, coverslips were finally mounted on glass plates and cells were observed under a leica tcs sp2-aobs confocal microscope (leica-microsystems, wetzlar, germany) using a 63x immersion oil objective. to detect cholesterol distribution we used filipin staining (sigma), as previously described [20] . cholesterol was visualized in a conventional leica dm rb microscope by combining a 63x immersion oil objective and a uv filter set. images were captured with leica application suite advanced fluorescence software (las af) and imagej software. finally, digital images were processed with adobe photoshop 8.0. the primary antibodies used for immunofluorescence assays included the following: rabbit polyclonal antibodies to ifitm1, ifitm2 and ifitm3 (proteintech), 1:200; anti-asfv p30 mouse monoclonal antibody, 1:100 (kindly given by dr. j.m. escribano, inia); asfv mouse monoclonal antibodies anti-p72 (clone 1bc11 for immunofluorescence 1:1,000 or clone 18bg3 for wb 1:2,000) and anti-p150 (clone 17ah2, ingenasa), 1:1,000; mouse monoclonal to cd63 (developmental studies hybridoma bank, university of iowa, clone h5c6), 1:200; rabbit polyclonal to lamp1 (abcam), 1:50; rabbit polyclonal to eea1 and rab7 (cell signalling), 1:50. secondary antibodies were purchased from molecular probes and diluted 1:200. specificity of labeling and absence of signal crossover were determined by examination of single labeled control samples. cells were harvested in sample buffer laemmli 2x concentrate (sigma). then, the samples were incubated for 5 min at 95°c and resolved by sds-page in 12% or 7% polyacrylamidebisacrylamide gels. afterwards, separated proteins were transferred to a nitrocellulose membrane (bio-rad) and the non-specific antibody binding sites were blocked with skimmed milk diluted in pbs and then incubated with the specific primary and hrp (horseradish peroxidase)-conjugated secondary antibodies. antibodies used for western blotting included: rabbit polyclonal to ifitm1, ifitm2 and ifitm3 (proteintech), 1:2,000; anti-asfv p30 mouse monoclonal antibody, 1:500; anti-asfv p72 mouse monoclonal antibody (clone 1bc11, ingenasa), 1:1,000, and mouse monoclonal to tubulin (sigma-aldrich), 1:2,000. as secondary antibody, anti-mouse igg (ge healthcare) or anti-rabbit igg (bio-rad) conjugated to horseradish peroxidase was used at a 1:5,000 dilution. precision protein streptactin-hrp conjugate (bio-rad) was used to reveal the ladder precision plus protein westernc (bio-rad). as a loading control an anti-mouse antibody against β-tubulin (sigma) was used, 1:2,000. finally, bands obtained after development with ecl reagent (ge healthcare, piscataway, nj, usa) were detected using a chemidoc xrsplus imaging system (bio-rad). band densitometry was performed with image lab software (bio-rad) and normalized to control values. the quantitation of the number of copies of asfv genome was achieved by quantitative realtime pcr (qpcr) using specific oligonucleotides and a premix extaq (tm) (2x; takara) probe. fluorescent hybridization probes were used to amplify a region of the p72 viral gene, as described previously [21] . dna from cells mock-infected or infected with asfv ba71v at moi of 1 pfu/cell was extracted at 16 hpi and purified with a dnaeasy blood and tissue kit (qiagen). dna concentration was measured using nanodrop. the amplification mixture was prepared on ice as follows: 250 ng template dna diluted in mq h 2 o to a total volume of 7μl, 1 μl oligonucleotide oe3f (50 pmol), 1 μl oligonucleotide oe4r (50 pmol), 10 μl pcr premix ex taq(tm) (2x; takara), 1 μl taqman probe se2 (5 pmol) [21] . positive amplification controls included dna purified from asfv purified virions at different concentrations as standards. negative amplification controls consisted in dna from mock-infected cells. each sample was included in triplicates and values were normalized to standard positive controls. reactions were performed using the abi 7500 fast real-time pcr system (applied biosystems) with the following parameters: 1 cycle at 94°c for 10 min, 45 cycles at 94°c for 15 s, and 45 cycles at 58°c for 1 min. to study virion decapsidation in asfv, a protocol was adapted from a previous publication [22] . briefly, stable cell lines vero-ifitm1, ifitm2, ifitm3 or control cells containing the empty vector were infected at moi of 10 pfu/cell after viral synchronization at 4°c for 90 minutes to enable virus attachment to the cell but restricting viral entry. infection was allowed to proceed for 75 minutes at 37°c, 5% co 2 . cells were then washed with cold pbs 1x and treated with 0.05% trypsin-edta (gibco) for 10 minutes at 37°c to remove the membrane-bound virus. finally, cells were placed in media containing fcs to quench trypsin activity and washed with pbs. after this treatment, only internalized virions were observed in an immunofluorescence assay as described above. we used specific antibodies to detect the major viral capsid protein p72 and the viral core protein p150, and staining was analyzed by confocal microscopy. decapsidated virions were single labeled for p150 and were counted for each condition and normalized to the total number of fully encapsidated virions which were double labeled for p72 and p150. stable cell lines vero-ifitm1, ifitm2, ifitm3 or control cells containing the empty vector were infected with ba71v or b54gfp at the indicated moi. recombinant b54gfp is a recombinant asfv expressing green fluorescent protein as a fusion protein of viral p54 [19] . samples infected with b54gfp at 16 hpi were just fixed and washed with facs buffer three times before analysis. vero cells infected with ba71v at 6 hpi or 16 hpi (early or late postinfection times respectively), were harvested by trypsinization, washed with facs buffer (containing pbs, 0,01% sodium azide, and 0,1% bovine serum albumin), fixed and permeabilized with perm2 (bd science) for 10 min at rt and finally incubated with specific primary antibodies against p30 and p72 for 30 min at 4°c. the secondary antibody used was phycoerythrin (pe) conjugated (dako) 1:50 diluted in facs buffer for 30 min at 4°c. after repeated washes, 10,000 cells/ tube were analyzed in the facscalibur flow cytometer (bd science) in triplicates. the obtained infection rates were always normalized to the corresponding control. bonferroni's multiple-comparison test was used to compare different experimental groups. prism software (graphpad software, inc.) and instat3 software were used for the statistical analysis. values were expressed in graph bars as mean ±sd of at least three independent experiments unless otherwise noted. metrics were normalized to control values and represented in graphics. asterisks denote statistically significant differences ( ããã p<0.001, ãã p<0.01 and ã p<0.05). to determine the effect of interferon on asfv infection, vero cells were pretreated with 1,000 or 10,000 u/ml of universal type-i ifn (pbl assay science) and 24 h later, they were infected with recombinant asfv b54gfp at a moi of 5 pfu/cell (fig 1) . viral infection was quantified by analyzing the number of gfp-positive cells by flow cytometry at 16 hpi (fig 1a) . pretreatment of vero cells with universal type-i ifn at both concentrations completely abrogated asfv infectivity when compared to untreated control cells. a sample flow cytometry profile is shown (fig 1a) . ifn treatment induces expression of ifitm proteins ifitm proteins are located in different cellular compartments and their antiviral properties strongly correlate with their capacity to alter the fluidity and fusion ability of the membranes in which they reside [23, 24] . then, we analyzed ifitms expression and distribution in vero cells upon ifn treatment. to this end, cells were incubated with either 1,000 or 10,000 u/ml of universal type-i ifn for 24 h and ifitms 1, 2 and 3 distribution was analyzed by confocal microscopy ( fig 1b) . although basal levels of ifitm2 and 3 were detected prior to treatment with to further analyze the expression of ifitm1, 2 and 3 upon ifn treatment, vero and 293t cells were incubated with universal type-i ifn for 24 h and analyzed by wb (fig 2) . while ifitm1 and ifitm2 were induced by ifn in both cell lines (fig 2a and 2b) , ifitm3 showed the highest induction (fig 2c) . in order to analyze the possible impact of ifitms in asfv infection, we generated vero cells stably expressing the human ifitm1, 2, 3 (hereinafter referred to as vero-ifitm1, 2 or 3 cells respectively) or control cells containing the empty vector. to generate the stable cell lines we used a lentiviral transduction system. our proteins of interest were cloned into the plvx vector (see material and methods section for detailed experimental procedures). positively transduced vero stable cells were selected with 8 μg/ml of puromycin. once these cells were established, the expression of different ifitm proteins was confirmed by wb analysis ( fig 3a) . as shown in the corresponding wb densitometry (fig 3b) , the highest expression levels within the ifitm family members corresponded to ifitm3, followed by ifitm2 and ifitm1. after assessing the expression levels of the vero-ifitm cells, we next wanted to ascertain the subcellular distribution of each ifitm. expression of ifitm1, 2 and 3 in vero-ifitm cells was compared with the distribution of ifitms in vero cells with the empty vector. confocal microscopy experiments revealed that, ifitm1 was mainly distributed at the plasma membrane and to a lesser extent in perinuclear compartments, resembling endosomal structures (fig 3c, lower left panel) , while endogenous ifitm1 was barely detected in vero cells containing the empty vector (fig 3c, upper left panel) . in vero-ifitm2 cells, overexpression led to a high accumulation of the protein in the perinuclear region, colocalizing with vesicular structures that resembled endosomes (fig 3c, medium lower panel). consistent with fig 1b, there was a significant expression of endogenous ifitm2 in control vero cells containing the empty vector, which displayed a mitochondrialike pattern together with vesicular-like structures. finally, overexpressed ifitm3 was found predominantly accumulated in the perinuclear region, showing a pattern consistent with localization at clustered endosomal structures ( fig 3c, lower right panel) . endogenous ifitm3 was barely detected in vero cells containing the empty vector (fig 3c, upper right panel) . analysis of endogenous ifitm2 expression in control vero cells suggested a mixed mitochondrial and vesicular pattern. to confirm this observation, we analyzed subcellular localization of ifitm2 using mitotracker red as a specific marker for mitochondria. confocal images revealed a distribution of ifitm2 to mitochondrial structures in cells containing the empty vector (fig 4) . however, in vero-ifitm2 cells, ifitm2 labelling localized to endosomal-like structures and we found few areas of colocalization between ifitm2 and mitochondria (fig 1b) . then, we investigated the possible mechanism underlying the inhibition caused by ifitm in the viral infection. to further analyze the vesicular localization of ifitms, we studied the cell. data are expressed as mean±sd of three independent experiments. statistical significance was evaluated by one-way anova followed by bonferroni's multiple comparison test. differences are marked with asterisks as indicated (***p<0.001). an example of a typical facs profile is shown. (b). confocal fluorescence images of ifitm1, 2 and 3 subcellular distribution in untreated vero cells or upon increasing universal ifn concentrations (1,000 or 10,000u/ml) for 24 h. bar = 10μm. (fig 5a) . endosomes are normally distributed through the cytoplasm and this dispersed distribution was found for the different maturation stages of endosomes in controls and in vero-ifitm1 cells. however, in vero-ifitm2 and ifitm3 cells dispersed distribution changed and endosomes aggregated around the nucleus (fig 5a) . this endosome redistribution was analyzed by confocal microcopy in x, y, z planes by measuring the mean distance between each endosomal marker and the cell nucleus, using the "distance measure" imagej plug-in (fig 5b) . a total of 30 cells were analyzed for each condition. we concluded that overexpression of ifitm2 and ifitm3 altered endosome distribution by accumulating these vesicles to the perinuclear region, similar to the pattern previously found after ifn treatment of vero cells (fig 1b) and this redistribution might reflect alterations in endo-lysosomal maturation and function. next, we analyzed the colocalization rate between ifitms and endosomal structures. cd63 remains mainly associated with intracellular vesicular membranes and it is particularly abundant in endosomal structures called multivesicular bodies (mvbs), which constitute a late and acidic endosomal compartment filled with intraluminal vesicles (ilvs). these ilvs are filled with cholesterol and are crucial for endosomal membrane backfusion and necessary for late endosome maturation. co-staining of ifitms and cd63 by immunofluorescence assay revealed a clear colocalization of ifitms and cd63 in vero-ifitm cells, with 75% colocalization for vero-ifitm2 cells and 40% in vero-ifitm1 and in ifitm3 cells (fig 6a) . then, ifitm2, and to a lesser extent ifitm1 and 3, were located primarily in endosomal compartments as indicated by higher colocalization with endosomal marker cd63 (fig 6c) when compared to the empty vector ( fig 6b) . ifitm proteins could reduce curvature of cell membranes for fusion pore formation [24, 25] at the outer endosomal membrane, also called endosomal-limiting membrane, to differentiate it from the membranes of intraluminal vesicles inside multivesicular bodies (mvb/le). this would lead to an alteration of membrane fusion and impaired cholesterol endosomal efflux. changes in endosomal distribution such as those found in ifitm stably expressing cells (fig 5) is a characteristic phenotype of an altered cholesterol endosomal efflux [23, 26] . conversely, a conserved endosomal cholesterol efflux is required for a correct endosomal function. therefore, we analyzed intracellular and intra-endosomal cholesterol levels by using the cholesterol marker filipin. vero-ifitm2 and ifitm3 cells showed intense intracellular cholesterol accumulation at the perinuclear area ( fig 7a) that was absent in control cells containing the empty vector (fig 7b) . this cholesterol accumulation also colocalized with the ifitm-labeled endosomal vesicles. in contrast, in vero-ifitm1 cells, only discrete colocalization areas between ifitm1 and cholesterol were found at the plasma membrane. collectively, these findings indicate that ifitm2 and ifitm3 overexpression in vero cells results in cholesterol accumulation in endosomal compartments, and as a result it might be responsible of an altered endosomal function possibly altering viral entry through this pathway. next, we investigated whether overexpression of ifitms could restrict asfv infection or not. asfv is known to require acidic endosomal compartments for entry into host cells. successful asfv entry and egress from endosomes depends on the acidic ph of late endosomes [11] . previous experiments in the laboratory revealed that the inhibition of acidification impaired viral decapsidation and viral particles were retained in rab7-positive late endosomes, thus blocking viral infection progression [11] . given that ifitm restriction could be mediated at the endocytic pathway [1, 4] , we hypothesized that ifitm overexpression may affect virus entry process and subsequent asfv infection. acidic ph of late endosomal compartments is required for viral decapsidation, which is the first step required for uncoating previous to endosomal escape of the virus and the start of replication [11] . the asf virion is composed of several concentric domains. the viral capsid is formed by major capsid protein p72 organized in capsomers. hence, after decapsidation, p72 staining of virions is lost. the internal core is composed by the nucleoid containing genome coated by the core shell, a thick protein layer that can be labeled with antibodies against p150, a core shell protein derived from processed core shell polypeptides [27] . . (b) . the change in distribution was quantified by measuring the mean distance to the nucleus of the different endosomal markers in x, y and z planes as described in materials and methods section. as shown in graphics, distance was reduced in vero-ifitm2 and 3 cells. graphics depict mean±sd of n = 30 cells per condition. statistical significance was evaluated by a one-way anova followed by bonferroni's multiple comparison test. differences are marked with asterisks as indicated (*p<0.05; **p<0.01). doi:10.1371/journal.pone.0154366.g005 we used antibodies against the major viral capsid protein p72 to detect viral capsids and against viral core protein p150 to detect viral cores (fig 8) . we analyzed the number of encapsidated viral particles, double labeled for p72 and p150, and compared with the number of successfully decapsidated viral cores positive for p150 at 75 minutes postinfection (mpi). at this time point, most virions undergo uncoating and progress to replication in normal conditions, otherwise, encapsidated virions would accumulate. confocal microscopy revealed that the number of viral cores was severely decreased in vero-ifitm2 cells when compared to control cells containing the empty vector (fig 8b) . double-labeled encapsidated viral particles were counted and the ratio of decapsidated viral cores compared to the total number of virions per individual cell was calculated and expressed in percentages (fig 8a) . the percentage of decapsidated virus severely decreased under ifitm2 expression. however, ifitm3 expression produced an accumulation of virions leading to higher numbers of total virions (fig 8c) . this increase in the number of total virions might be the result of an impaired progression of the asfv replication cycle. these virions would neither proceed with infection nor be degraded and this would be consistent with an inhibition of the membrane fusion capacity at the endosomal level. altogether, these results indicate that viral entry was the rate-limiting step in vero-ifitm2 and ifitm3 cells for asfv infectivity. finally, we analyzed the impact of ifitm expression on other infection parameters to find reduced number of copies of asfv genome at 16 hpi (fig 9a) . ifitm overexpression induced a consistent reduction in infectivity as measured by early protein p30 expression by flow cytometry (fig 9b) . however, these reductions were even more marked when analyzing infected cell percentages by late p72 protein expression (fig 9c) . finally, we also analyzed viral protein expression by wb (fig 9d-9f) . the expression of protein p30 at 6 hpi ( fig 9d) and p72 at 16 hpi (fig 9e) resulted significantly reduced as other infection parameters. in the present work, we have studied the antiviral effect of the ifitm family of proteins in the context of cell-adapted asfv infection in vero cells. while different ifitm proteins have been repeatedly described as inhibitors of a broad spectrum of rna viruses [6] , their antiviral role involving dna viruses is poorly studied and only reported in the rana grylio virus (rgv), blocking the virions at the entry stage into the host cell [8] . asfv ba71v infection in vero cells is significantly affected upon ifn treatment [28, 29] . in general, the sensitivity towards the induction of the innate immune response of the host cell has led viruses to acquire different strategies to regulate the ifn pathway for its own benefit. such is the case of asfv viral gene a276r, which negatively regulates the induction of ifn through targeting irf3 in a nfκb-independent manner [30] . another example is the asfv gene i329l, which codifies for a viral tlr3 homolog with inhibitory activity against ifn [31] . finally, the myxovirus resistance gene a (mxa), which is another interferon stimulated gene (isg), also inhibits the replication and the late gene expression of asfv [32] . we report here that upon ifn treatment of vero cells, the distribution of ifitm proteins changes into a perinuclear vesicular pattern resembling endosomes. our analysis of endogenous ifitm2 expression in the absence of ifn induction showed colocalization with mitochondrial structures. interestingly, ifitm2 underwent vesicular pattern redistribution around the cell nucleus when overexpressed and upon ifn treatment as well. our characterization of the cellular distribution of ifitm1, 2 and 3 also unveiled specific localization patterns linked to the endosomal pathway upon overexpression, particularly in the late endosomal compartments. this distribution is coincident with previously reported data of other groups, which described localization of endogenous ifitm1 at the plasma membrane and early endosomes, and of ifitm2 and ifitm3 in late endosomes and lysosomes [33, 34] . interestingly, overexpression of ifitm1 has been recently described to delay the proteolytic degradation of human papilloma virus (hpv) capsids in keratinocytes [9] . this, however, did not affect the replication of the virus. our analysis of asfv ba71v uncoating correlated the expression of ifitm2 with a decrease in the numbers of decapsidated asf virions in vero cells. this suggests a possible role for ifitm2 in inhibiting asfv exit from late endosomes. in contrast, ifitm3 did not modify the ratio between encapsidated and decapsidated virions. instead, ifitm3 apparently increased the accumulation of virions that are probably retained and do not proceed to degradation and/ or to a productive infection. these results may also suggest that the presence of ifitm3 affects the release of the virions from the late endosomal compartments. asfv enters into the host cell by dynamin-dependent and clathrin-mediated endocytosis [12, 17] and macropinocytosis [35] . in fact, endosomal pathway integrity is known to be important for asfv infection, both for culture-adapted isolates in cell lines [11] or for virulent and attenuated isolates in primary macrophages [12] . hence, it is not surprising that infection could be impaired by these restriction factors acting at the endosomal membrane. the aforementioned ifitm2-and ifitm3-mediated inhibition of asfv entry has been previously reported in other viruses, including iav, flaviviruses (denv and wnv) [6] , filoviruses (marv and ebov) and coronaviruses (such as sars) [34] . in contrast, infection with alphaviruses, arenaviruses and mlv (a retrovirus) seems to be unaffected by ifitm protein expression [4] . in general, ifitm-mediated viral inhibition has been related to impaired viralhost membrane fusion subsequent to viral binding and endocytosis [33, 34] . ifitm3 has also been reported to modulate the fluidity and the bending modulus of the cell membrane, thus making it resistant to viral fusion machinery [36] . we also studied whether the inhibition of asfv entry could be due to an alteration of the endosomal compartments. analysis of endosome distribution upon ifitm overexpression revealed that ifitm2 and ifitm3 altered the normal distribution of early and late endosomes and lysosomes. however, this alteration was not found in the presence of ifitm1. a collapse of endosomes to the perinuclear area is also a characteristic phenotype of an alteration of endosomal cholesterol efflux [26] . also, recent publications from our laboratory demonstrated that accumulation of cholesterol in endosomes caused by a chemical inhibition of cholesterol flux, causes virion retention inside endosomes and inhibition of infection progression [37] . there are currently two proposed models trying to explain the ifitm-mediated inhibition of viral entry. the first one, known as the "tough membrane" model, postulates that intramembranous interactions between adjacent ifitms alter the fluidity and bending of the host cellular membrane, making it resistant to the viral fusion machinery [36] . the second model suggests that ifitms can induce the accumulation of cholesterol in the endosomal membrane and membrane fusion defects [38] disturbing intracellular cholesterol homeostasis that finally blocks the viral release into cytosol [23] . ifitm2 and ifitm3 have been previously reported to alter the cholesterol homeostasis at the late endosome, leading to cholesterol accumulation and blocking the viral release [23] . in the present study, we have described accumulation of cholesterol upon overexpression of ifitm2 and ifitm3. we have recently reported that inhibition of cholesterol exit from endosomes using chemical inhibitors causes retention of virions inside these vesicles, thus impairing progression of the infection [37] . altogether these data could suggest that the antiviral action of ifitms may affect to a higher extent to those viruses that require the endosomal pathway during the early stages of infection. collectively, our results illustrate a close relationship between the ifitm protein family and the endosomal pathway, leading to the inhibition of asfv infection. the antiviral action of ifitms could rely on alterations of the endosomal physiology and ongoing studies in our laboratory will be focused on antiviral targets at the molecular regulation of late endosomes. also, a role in cell-to-cell viral transmission has been postulated for ifitms, which are incorporated into hiv-1 virions [39] . we have performed this study in a cellular system using the cell-adapted ba71v isolate in vero cells but these results will be next extended to other asfv isolates with porcine macrophages. further studies will be required for better understanding the relevance of ifitms in the context of asfv infection. in summary, ifitms represent a broad and previously unappreciated class of restriction factors that degrade invading enveloped viruses and may therefore be considered as potential antiviral components to protect the host cell. ifitms restrict the replication of multiple pathogenic viruses transcriptional and posttranscriptional regulation of interferon-induced gene expression in human cells 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entry by blocking the formation of fusion pores following virus-endosome hemifusion alteration of dynein function affects alpha-synuclein degradation via the autophagosome-lysosome pathway phosphatidylinositol 4-kinases: hostages harnessed to build panviral replication platforms african swine fever virus morphogenesis. virus research effect of interferon-alpha, interferon-gamma and tumour necrosis factor on african swine fever virus replication in porcine monocytes and macrophages. the journal of general virology interferon cures cells lytically and persistently infected with african swine fever virus in vitro identification and utility of innate immune system evasion mechanisms of asfv. virus research a novel tlr3 inhibitor encoded by african swine fever virus (asfv) inhibition of a large double-stranded dna virus by mxa protein 00781-08 pmid: 19109387 ifitm3 inhibits influenza a virus infection by preventing cytosolic entry distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus african swine fever virus uses macropinocytosis to enter host cells the cd225 domain of ifitm3 is required for both ifitm protein association and inhibition of influenza a virus and dengue virus replication cholesterol flux is required for endosomal progression of african swine fever virions during the initial establishment of infection the diverse functions of oxysterol-binding proteins ifitm proteins incorporated into hiv-1 virions impair viral fusion and spread distance measure software plug-inn of imagej was kindly provided by dr. esteban veiga and giulia morlino from institute for health research "hospital universitario la princesa", cnb, madrid.funding sources for ca: ministerio de economía y competitividad (es) http://www. mineco.gob.es/portal/site/mineco/idi agl2012-34533 and agl2015-69598-r and for ag-s national institute for allergy and infectious diseases (niaid); http://www.niaid.nih.gov/; u19ai106754. key: cord-348055-azlb1zy1 authors: patel, mira c.; wang, wei; pletneva, lioubov m.; rajagopala, seesandra v.; tan, yi; hartert, tina v.; boukhvalova, marina s.; vogel, stefanie n.; das, suman r.; blanco, jorge c. g. title: enterovirus d-68 infection, prophylaxis, and vaccination in a novel permissive animal model, the cotton rat (sigmodon hispidus) date: 2016-11-04 journal: plos one doi: 10.1371/journal.pone.0166336 sha: doc_id: 348055 cord_uid: azlb1zy1 in recent years, there has been a significant increase in detection of enterovirus d-68 (ev-d68) among patients with severe respiratory infections worldwide. ev-d68 is now recognized as a re-emerging pathogen; however, due to lack of a permissive animal model for ev-d68, a comprehensive understanding of the pathogenesis and immune response against ev-d68 has been hampered. recently, it was shown that ev-d68 has a strong affinity for α2,6-linked sialic acids (sas) and we have shown previously that α2,6-linked sas are abundantly present in the respiratory tract of cotton rats (sigmodon hispidus). thus, we hypothesized that cotton rats could be a potential model for ev-d68 infection. here, we evaluated the ability of two recently isolated ev-d68 strains (vanbt/1 and mo/14/49), along with the historical prototype fermon strain (atcc), to infect cotton rats. we found that cotton rats are permissive to ev-d68 infection without virus adaptation. the different strains of ev-d68 showed variable infection profiles and the ability to produce neutralizing antibody (na) upon intranasal infection or intramuscular immunization. infection with the vanbt/1 resulted in significant induction of pulmonary cytokine gene expression and lung pathology. intramuscular immunization with live vanbt/1 or mo/14/49 induced strong homologous antibody responses, but a moderate heterologous na response. we showed that passive prophylactic administration of serum with high content of na against vanbt/1 resulted in an efficient antiviral therapy. vanbt/1-immunized animals showed complete protection from vanbt/1 challenge, but induced strong pulmonary th1 and th2 cytokine responses and enhanced lung pathology, indicating the generation of exacerbated immune response by immunization. in conclusion, our data illustrate that the cotton rat is a powerful animal model that provides an experimental platform to investigate pathogenesis, immune response, anti-viral therapies and vaccines against ev-d68 infection. picornaviruses of the genus enterovirus (ev) comprise many human pathogens that cause most common infections in humans, such as ev a-d and rhinovirus (rv) a-c [1] . the evs are small, single-stranded, positive-sense rna viruses with a genome of~7.5 kb, encapsidated into an icosahedral capsid, forming a non-enveloped virion of around 30 nm diameter. there are total 5 types of ev-d species: ev-d70, associated with acute hemorrhagic conjunctivitis [2, 3] , ev-d94, causative agent of acute flaccid paralysis [4, 5] , ev-d111 and d120, identified in non-human primates [6, 7] , and ev-d68. ev-d68 was first isolated from four hospitalized pediatric patients with pneumonia and bronchiolitis in california in 1962 [8] , indicating that its initial tropism targets the respiratory tract. there are three major clades of ev-d68, designated as a, b and c, which are circulating worldwide [9, 10] . the ev-d68 genome consists of single open reading frame (orf), encoding four structural proteins (vp1-vp4) and seven nonstructural proteins (2a-2c and 3a-3d), flanked by a long 5' untranslated region (utr) with a hairpin-loop secondary structure and a short 3'utr with a poly(a) tract [11] . since its discovery in 1962, ev-d68 infections were among the most rarely reported until the early 2000's [12] . however, an upsurge in detection of ev-d68 has been documented in the last decade among patients with acute respiratory infections of various severities, ranging from mild upper respiratory tract infections to severe pneumonia, including fatalities in pediatric and adult patients [9] [10] [11] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] . in 2014, the largest outbreak of ev-d68 infection in usa was reported. from mid-august 2014 to january 2015, a total of 1,153 cases of respiratory illness caused by ev-d68 in 49 states and in the district of columbia were reported, which were confirmed by either the centers for disease control and prevention (cdc) or different state public health laboratories [23, 24] . most cases were children, with a large percentage of them requiring pediatric intensive care, and some cases were fatal [25] . previously, ev-d68 was detected in the cerebrospinal fluid (csf) in a 5 year-old boy who died due to meningomyeloencephalitis and pneumonia [26] . during the 2014 usa outbreak, a geographically and temporally defined cluster of cases with acute flaccid paralysis and cranial nerve dysfunction was also reported in 12 children, where the virus was detected sporadically in nasopharyngeal samples [27] . in addition, 3 cases of pediatric ev-d68 infections associated with acute flaccid paralysis were also reported in europe in 2014 [28, 29] . in 2016, a total of 50 cases of acute flaccid myelitis were confirmed in 24 states (cases reported up to august 31), while limited sporadic cases of ev-d68 have been detected across usa [30] . these reports have raised concerns that genetic changes in ev-d68 could be contributing to the increased detection of the virus in human respiratory infections and the increase in disease severity and neurological symptoms. thus, ev-d68 is now recognized as a re-emerging pathogen [11] . currently, there is no specific antiviral therapy against ev-d68 available and treatment is primarily supportive. furthermore, until now, there has been no suitable animal model available to develop and test therapeutics against ev-d68 virus and to obtain comprehensive understanding of its pathogenesis. over the years, ev-d68 genome has undergone many deletions in the spacer region of the 5' utr between the end of the internal ribosome entry sites (ires) and the polyprotein orf. the significance of these deletions is not clear; however, such mutations might influence translational efficiency and thereby affect viral virulence. clades a and b are further divided into subclades: a1 and a2 and b1 and b2, on the basis of amino acid substitutions in both structural and nonstructural proteins [11] . compared to clades a and b, clade c is geographically restricted and circulated in japan during 2005 to 2010 and in italy during 2008 [9, 14, 17] . subclades a1 and b2 are considered endemic and were found in many countries before the 2014 outbreak, such as thailand from 2006 to 2011 [19] , the united kingdom from 2009 to 2010 [18] , china from 2009 to 2012 [20] , the philippines from 2009 to 2014 [13, 15, 16] , and the netherlands from 1994 to 2014 [21, 22] . during the 2014 outbreak, subclade b1 was dominant among usa cases (specimens collected from kansas city, mo) [31], while another report showed that the majority of ev-d68 strains circulating in the 2014 outbreak (specimens collected from the lower hudson valley of new york) differ significantly from prior ones, mostly having three nucleotide variables, c1817t, c3277a and a4020g, and belong to a new clade [32] . sialic acids (sas) are receptors for ev-d68 [33, 34]. using glycan array and enzymatically modified erythrocytes, it was shown that ev-d68 has a stronger affinity for α2,6-linked sas than α2,3-linked sas [33]. the sa receptor induces a cascade of conformational changes in the ev-d68 virus that prime viral uncoating and facilitate cell entry [34] . lectin-based staining showed that both α2,3-linked and α2,6-linked sa receptors are present in the respiratory tract of cotton rats; α2,6-linked sa receptors were found on ciliated cells, whereas α2,3-linked sa receptors were more associated with mucin-producing cells in the cotton rat trachea. cotton rat lung parenchyma showed a consistent staining of type i and type ii pneumocytes with α2,6-linked sa, but undetectable levels of α2,3-linked sa [35] . consistent with these observations, we have shown that influenza a of human and avian origin, as well as influenza b isolates replicate without the need for "adaptation" in cotton rat upper and lower respiratory tracts [35, 36] . in addition, we reported that intranasal infection of cotton rats with another picornavirus, human rhinovirus (hrv) type 16, resulted in isolation of infective virus, lower respiratory tract pathology, mucus production, and expression of interferon-activated genes without any genetic modification of either the host or the virus [37] . in contrast to other evs, ev-d68 is biologically more similar to hrvs [38]. in fact, hrv87, discovered in 1963, was subsequently reclassified as ev-d68 based on molecular analysis [39, 40] . similar to rvs, ev-d68 grows optimally at 33°c compared to 37°c preferred by other evs, and is both heat and acid labile [38] . in the present study, we evaluated three strains of ev-d68, belonging to different clades, for their abilities to infect cotton rats. we report that intranasal (i.n.) infection of cotton rats with ev-d68 (vanbt/1) resulted in isolation of infective virus from the nose and lung tissues, expression of lung inflammatory cytokines, and marked lung pathology. infection and immunization of cotton rats with live ev-d68 generated various levels of protection from virus challenge that correlated with the production of different levels of serum na. furthermore, we demonstrate that this model could be an excellent tool to decipher cross-reactive immunity among different ev-d68 clades, which is relevant for the generation of an efficacious ev-d68 vaccine with broad protection. we conclude that ev-d68 infection in cotton rats can provide novel insights that will enable the molecular dissection of immune responses to ev-d68 and thus develop effective intervention and prevention strategies. for this work, three different strains of ev-d68 were used to encompass both clades a and b, representing their historical appearance relevant to usa. we classified the three strains as: (1) classical atcc (accession # kt725431, referred as atcc), which is the prototype fermon virus strain purchased from atcc. the fermon strain is the oldest ev-d68 sequence in gen-bank and it was collected in 1962 in california [8] . the sequence of the atcc strain is clustered near the root of phylogenetic tree, reflecting its sampling date in the 1960s (fig 1a) . (2) pre-outbreak isolate vanbt/1 (accession # kt347280, referred to as vanbt) was collected in 2012 from nashville, tn at vanderbilt university medical center, and belongs to subclade a1 ( fig 1a) ; and, (3) outbreak isolate mo/14/49 (accession # km851227, referred to as mo/ 49), was collected in kansas city, mo during the 2014 outbreak, and belongs to subclade b1 ( fig 1a) . all the three strains were propagated in hela h1 cells at 34°c and their titers were determined by standard endpoint dilution assay and expressed as tcid 50 /ml. we infected groups of 10 adult cotton rats i.n. with 10 6 tcid 50 of each of the 3 strains of ev-d68. five animals per group were euthanized at 10 and 24 h post infection (p.i.) for determination of viral titers in the nose and lung of each animal (fig 1b and 1c) . atcc virus was recovered from the lung, but not from the nose at 10 h p.i., whereas detection of this strain was almost negligible in both tissues at 24 h p.i. (only one animal exhibited virus in the nose). vanbt was consistently recovered from both nose and lung tissues at 10 and 24 h p.i. the amount of vanbt virus recovered was generally higher in the lung than in the nose at both 10 and 24 h p.i. (fig 1b and 1c ). the decrease in recovered infectious vanbt titer was around 1 log 10 in both the tissues at 24 h p.i. compared to 10 h. mo/49 titers in both nose and lung tissues were comparable among all 5 animals at 10 h p.i. (3.5 to 4 log 10 tcid 50 /g tissue), but isolation was less consistent at 24 h p.i. (fig 1b and 1c) . overall, these results show that vanbt replicates more strongly than mo/49 or atcc in cotton rats and, thus, we used vanbt for subsequent infection experiments. adult cotton rats were infected i.n. with 10 6 tcid 50 of vanbt or inoculated i.n. with an identical amount of uv-inactivated vanbt virus (uv-vanbt). groups of 4 animals were euthanized at 0.5, 2, 4, 6, 8, 10, 24, 48, and 96 h p.i. to measure the profile of virus replication in nose and lung tissues (fig 1d and 1e , respectively). vanbt virus was detected in the nose at 0.5 h, which showed a brief but defined drop at 2-4 h p.i. ( ã p<0.05 for 4 h compared to 0.5 h p. i. titer) that corresponds to the viral eclipse, and a subsequent increase at 6 h, reaching a peak in this tissue at 10 h p.i. (#p<0.05 for 10 h compared with 4 h p.i. titer) ( fig 1d) . infectious virus was almost undetectable at 48 h p.i. in the nose. in the lung, virus titers remained constant between 0.5 to 10 h p.i. and subsequently decreased to undetectable levels by 48 h (fig 1e) . as expected, no infectious virus was detected in the lung and nose of animals inoculated with uv-vanbt (fig 1d and 1e , uv-6). to determine whether there was a sex-bias in the replication of ev-d68 in cotton rats, we compared virus yields at 10 h p.i. in nose and lung tissues from both male and female cotton rats. vanbt titers recovered from these tissues were essentially identical between animals of different sexes (s1 fig). for vanbt, the extent of viral replication was also assessed by determining the amount of negative strand viral rna [(-) vrna] (replication intermediates) by qrt-pcr. (-) vrna strands were detected in lungs of infected animals up to 48 h p.i. (fig 1f, and insert) . the levels of (-) vrna in the lung briefly peaked at 4 h, maintained a brief plateau between 6-10 h, followed by a decrease and final clearance of viral intermediate by 96 h p.i. similar to (-) vrna, quantification of total vanbt vrna by qrt-pcr showed an almost comparable pattern (s2 fig) . animals inoculated with uv-vanbt showed undetectable levels of (-) vrna and total vrna ( fig 1f and s2 fig) . a set of animals infected i.n. with the different ev-d68 strains (atcc, vanbt, and mo/ 49) were followed 21 days after challenge to determine whether infection by the three strains induced immunologic responses in the form of na titer. paralleling their various levels of replication in cotton rats, vanbt infection generated the highest na titer (8 of 10 infected animals showed detectable homologous na titers), mo/49 infection showed only one animal with detectable na titer, and atcc infection resulted in no detectable na response ( fig 1g) . moreover, the na titer against vanbt persisted with the comparable strength through 9 weeks p.i. (s3 fig) . to determine effect of the ev-d68 infection on the induction of an inflammatory response, we focused on the lung tissue and measured the expression of cotton rat mrna for several chemokines, type i and type ii interferons (ifns), cytokines, and select ifn-inducible genes following vanbt infection. cotton rats were infected with vanbt or uv-vanbt and euthanized at the indicated time points to measure levels of gene expression by qrt-pcr (fig 2) . vanbt infection dramatically induced early production of the neutrophil chemoattractant chemokine gro and monocyte chemotactic protein 1 (mcp-1), peaking within 4 h and sharply decreasing to basal levels by 10 h of infection. a different profile was found for chemokines ip-10 and rantes that were induced with a more delayed kinetic, peaking within 10-24 h p.i. expression of ifn-mrna was gradual, peaking by 10 h and remaining high at 48 h p.i., whereas expression of the ifn-inducible genes, mx-1 and mx-2, were consistently induced following the peak of ifn-β, peaking by 24 h and remaining detectable until 96 h p.i. (fig 2) . in addition, vanbt infection induced the proinflammatory cytokines interleukin-6 (il-6) and ifn-γ with different peak times. il-6 peaked at 4 h, while ifn-γ showed a late expression, remaining higher at 24-48 h p.i. (fig 2) . we additionally measured the expression of th2 cytokines (e.g., il-4, il-5, il-13, and il-10), but none of them showed significant expression in the lung tissues upon vanbt infection (see below). consistent with the results of pulmonary inflammatory gene expression, lung pathology was moderate and peaked at 48 h p.i., and was characterized by the presence of a mild, but significant, increase in cumulative pathology that included bronchiolitis, perivasculitis, interstitial pneumonia, and alveolitis ( fig 3a) . examination of h&e-stained lung sections revealed areas of extensive peribronchial and alveolar cellular infiltration in vanbt-infected animals, which were not evidenced in the lungs of control uv-vanbt inoculated animals ( fig 3b) . as na are pivotal for prevention of viral infection or blocking viral replication, we next investigated the effect of i.m. immunization with live ev-d68 strains on generation of humoral protective immune responses by measuring serum na against different ev-d68 strains. we immunized groups of adult cotton rats i.m. with each of the three ev-d68 strains, either live or uv-inactivated, at the dose of 10 6 tcid 50 at day 0 and boosted 3 weeks after the first immunization with the same dose of virus. serum samples were collected from animals at 3 (before boosting) and 7 weeks after the first immunization and homologous and heterologous na levels were determined by in vitro neutralization assay. no signs of clinical disease were seen in animals immunized i.m. with either inactivated or live viruses. immunization with live atcc, vanbt, or mo/49 induced homologous na titers in sera at 3 weeks post-immunization, which were further increased at 7 weeks due to boosting (fig 4a) . at 7 weeks, serum samples from animals immunized with live atcc showed the lowest na titer (log 2 8.7±0.5), followed by the group immunized with mo/49 (log 2 12.7±0.5), whereas animals immunized with live vanbt showed the highest titer (log 2 13.8±0.3) (fig 4a and table 1 ). the intensity of induction of homologous na titer followed i.m. immunization with live virus mirrors their respective infectivity in cotton rats (fig 1b and 1c ). similar to naïve animals, i.m. immunization with uv-vanbt did not induce detectable na titer ( fig 4a, table 1 ). next, we tested whether vanbt replicates at the site after i.m. inoculation and thus enhance na production. groups of cotton rats were mock-inoculated i.m. with 1x pbs, or inoculated i.m. with live vanbt and sacrificed at 2, 5, and 15 h post inoculation. vanbt (-) vrna was measured in rna samples extracted from inguinal and lumbar lymph nodes, draining from the site of inoculation. (-) week (before boosting) and 7 weeks after the first immunization and homologous serum na titers were determined. the sera were assayed in duplicate and na titer is expressed as log 2 geometric mean ± se. n = 10-15 from two independent experiments, ***p< 0.001 for 3 weeks na titer compared with 7 weeks na. (b) passive transfer of vanbt immune sera protects animals from vanbt challenge. animals were treated intraperitoneally with 0.5 ml 1 x pbs (mock) or serum from animals immunized i.m. with either uv-vanbt, vanbt, or mo/49. the following day, animals were challenged i.n. with 10 6 tcid 50 of vanbt and euthanized 10 h later to determine nose and lung viral titers. n = 4-5 per group. * p<0.05 for each group compared with mock group. vrna strands were detected in lymph nodes of vanbt inoculated animals (s4 fig). the levels of (-) vrna at 2 and 5 h were almost comparable, and decreased by 15 h post inoculation. next, we sought to determine the capacity of the ev-d68-immune serum samples to neutralize viruses of different clades used for immunization. for testing cross-reactivity, an immune serum against one virus strain was used to measure its ability to neutralize the other two virus strains in vitro. as shown in table 1 , atcc immune sera generated measurable homologous na titers, but negligible na titers against vanbt and mo/49. both vanbt and mo/49 immune sera showed strong homologous na responses and also moderate heterologous response against the other two strains. however, vanbt appeared to be the strongest inducing na responses against all the three strains (table 1) . serum samples from mock (pbs) or from animals immunized with uv-vanbt showed no neutralizing activity against all the ev-d68 strains. these data strongly suggest that immunization with vanbt substantially induces a broader na response that could be protective against heterologous ev-d68 challenge. as vanbt and mo/49 both generated a strong and a moderate na response, respectively, against all three viruses, we next tested the efficacy of the prophylactic administration of serum from ev-d68 immunized (vanbt and mo/49), uv-vanbt immunized, or mock treated animals to protect against i.n. vanbt challenge. all animals that received immune sera with high na antibodies against vanbt intraperitoneally prior to challenge (average na titer of vanbt immune sera = 13.7 log 2 ) showed a significant reduction in the nose and the lung viral titers by~1 log 10 at 10 h p.i., whereas animals that were mock-treated or that received uv-vanbt or mo/49 sera prophylactically, showed no significant protection (fig 4b) . these data demonstrate that passive transfer of antibodies can be considered an effective prophylactic therapy against ev-d68 infection. we tested whether the na responses induced by ev-d68 immunization were sufficient to confer protection against the virus challenge. groups of cotton rats were mock immunized i.m., infected with vanbt i.n., or immunized i.m. with uv-vanbt, or live atcc, live vanbt, or live mo/49 ( fig 5a) . all animals were immunized on day 0 and boosted at 3 weeks, whereas the vanbt-infected group was inoculated on day 0. at week 7, all animals were challenged i. n. with vanbt and sacrificed at 10 h p.i. to measure nose and lung viral load. as shown in fig 5b, i.m. immunization with live vanbt resulted in complete and rapid virus clearance in both nose and lung tissues. no protection was detected in mock-immunized animals, animals previously infected with vanbt, animals immunized with uv-vanbt, or animals immunized with live atcc (fig 5b) . however, mo/49-immunized animals showed a moderate reduction in viral titer in nose and lung tissues by~0.7 log 10 and~1.7 log 10 , respectively (fig 5b) , correlating with the generation of the cross-protective na titers in this model (table 1) . next, we examined the effect of vanbt i.m. immunization on the type and kinetics of pulmonary cytokines and ifn responses after vanbt challenge. we analyzed animals mockimmunized i.m. (as primary infection), previously infected with vanbt i.n. (as secondary infection), or immunized i.m. with either uv-vanbt or live vanbt. as shown for primary infection, vanbt induced strong ifn-β mrna expression, peaking at 10 h p.i. in the lungs (fig 2) . we measured ifn-β and mx-2 expression at 10 and 48 h p.i. in the immunized groups (fig 6a and 6b) . immunization with live vanbt significantly reduced expression of ifn-β and mx-2 at 10 h p.i. compared to mock-immunized group, which is consistent with complete protection seen in this group (fig 5b) . in addition, animals previously infected with vanbt i. n. or immunized with uv-vanbt i.m. showed decreased expression of ifn-β, but not of mx2 ( fig 6a and 6b) . moreover, immunization with live vanbt showed a dramatic increase in the expression of chemoattractant ip-10 at 10 h p.i., which remained elevated even at 48 h p.i. (fig 6c) . immunization with live vanbt elicited a strong th1 (ifn-γ) cytokine response in the lung tissues at 10 h, which remained even higher at 48 h p.i. (fig 6d) . animals previously infected with vanbt i.n. or immunized i.m. with uv-vanbt showed higher expression of ifn-γ compared to mock-immunized group, although the magnitude of ifn-γ induction in live vanbt-immunized group was higher than these two groups (#p<0.05 when vanbt/i. m. compared to vanbt/i.n. and ⦁p<0.05 when vanbt/i.m. compared with uv-vanbt/i. m. at 10 h p.i.). furthermore, both uv-vanbt and vanbt-immunized groups showed significant induction of signature th2 cytokines il-6, il-4, il-5, and il-13 mrna expression at both 10 and 48 h p.i. (fig 6e-6h) . we determined the extent of lung pathology in all the four groups by scoring h&e-stained lung sections. as shown in fig 7, immunization with both uv-vanbt and live vanbt i.m. showed significantly increased scores for vasculitis, interstitial pneumonia and alveolitis, compared to mock-immunized, or the previously infected with vanbt i.n. group, which suggests an exacerbated inflammatory response in these groups. in the past decade, the detection frequency of ev-d68 in respiratory infections has been on the rise worldwide and the 2014 usa outbreak was the largest and most widespread ev-d68 epidemic investigated to date [9, 10, [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] . increasing numbers of ev-d68 cases showing acute flaccid paralysis and cranial nerve dysfunction in children raised concerns about its potential impact on public health [27-29, 41, 42]. however, the underlying causes for this upsurge of ev-d68 strains circulating in recent years have been divided into three distinct clades a, b, and c based on vp1 phylogenetic tree [9, 10] . since the first isolation of the virus, mutations have accumulated in the bc and de loops of the vp1 sequence, which corresponds to region of the protein on the viral surface that are associated with antigenic epitopes, indicating that unique sequence variations in these loops may cause a shift in the antigenicity [19, 22, 43 ]. in the current study, we used three different strains, (1) classical atcc (prototype fermon strain) that was used as a reference strain before the more abundant circulation of the virus [8, 33] ; (2) pre-outbreak isolate vanbt collected in 2012 that belongs to subclade a1, which is considered to be endemic and isolated in many different geographical regions [13, 15, 16, [18] [19] [20] [21] [22] rat model has led to our conclusion that their replication and immunological profiles differ significantly. it has been reported that ev-d68 binds to α2,6-linked sas; however, the lack of a suitable animal model has severely limited the study of ev-d68 infection, immunity, and pathogenesis to test methods of intervention and prevention strategies [33, 34] . the species of the cotton rat (genus sigmodon) has been shown to be a preferred model for an impressive list of human pathogens including respiratory syncytial virus (rsv), influenza (a and b serotypes), adenoviruses (several serotypes), parainfluenza virus (type 3), measles, herpes simplex (types 1 and 2), and human metapneumovirus [35, 36, [44] [45] [46] [47] [48] . for many of these viruses, the cotton rat represents a reliable model that better mimics human disease [49] . in fact, picornaviruses were the first group of viruses studied in cotton rats. initial studies of poliovirus demonstrated that the lansing strain of poliomyelitis was selected for replication in mice by a process of adaptation involving serial passage of viruses in cotton rats [50]. moreover, it was shown that ev-a71 strain, isolated from samples of the brain and feces of children during an outbreak in bulgaria in 1975, induced a paralytic disease in newborn and adult cotton rats [51] . we recently showed that cotton rats are permissive to infection with hrvs, and i.n. infection resulted in recovery of infectious viral load in the lung until 2 days p.i., accompanied by significant pathology, mucus production, and expression of inflammatory mediators [37] . as there are no current animal models for ev-d68 and information regarding relevant disease-related outcomes from human cases is limited, we herein characterized ev-d68 infection in cotton rats by measuring viral load and vrna (negative or total) in either nose or lung tissues until 4 days p.i., assessing mrna expression of various chemokines, ifns, and proinflammatory cytokines, and lung histology. we focused on analysis of the nose and lung tissues since most recent cases of ev-d68 showed the respiratory tract as the most evident target for infection with this virus [10, 15, 22] . our data clearly shows that cotton rats are permissive to ev-d68 and that it replicates in respiratory airways, although with differing infection profiles among three strains, atcc being the weakest, while vanbt showed the strongest infection and replication profile (fig 1b) . these differences in viral replication could represent different evolutionary stages of the three strains that can affect at various levels of replication of these viruses or they may differentially use an alternative, nonsialylated receptor to infect the cells [52] . although the replication cycle of vanbt appeared to be short-lived, its profile of viral titer in the nose over time (showing clear virus eclipse at 4 h, rising quickly and reaching a peak by 10 h p.i.), and their differences with the output virus obtained after infection with different strains demonstrate that vanbt replicates in upper respiratory tract of cotton rats. replication of vanbt was studied further by measuring viral replication intermediate, (-) vrna, which showed early production (showing a peak at 4 h p.i.) of replicative rna in the lungs. as viral genome replicating intermediates, (-) vrna showed peak expression prior to detection of peak infectious virus titer, it confirms that we are detecting early products of viral replication, which eventually increased the viral titer at~10 h p.i. since we could detect vanbt primarily in the nose and lung tissues and infectious virus decreased after 10 h p.i., it is possible that vanbt could infect only a limited amount or type of cells in the respiratory tract of cotton rat with reduced transmission to other tissues. the 2014 usa outbreak of acute flaccid paralysis occurred at the same time as a far larger outbreak of ev-d68 across the usa. the vast majority of confirmed cases of ev-d68 mostly showed development of only respiratory illness and viral presence was confirmed in only a handful of the paralyzed children, which emphasize that the ev-d68 tropism is primarily directed to the upper and lower respiratory tract [11, 53] . although we do not rule out potential changes in tropism in our model, these events could be, as in humans, very rare and need further investigation. our data shows that vanbt infection causes temporally defined expression of lung cytokines and elicits lower respiratory tract pathology in cotton rats. the absence of pathologic, inflammatory, or antiviral responses (induction of ifn-β and ifn-inducible genes mx-1 and mx-2) in rats inoculated with uv-vanbt indicates that these responses were replicationdependent. mx proteins possess gtpase activity and the ability to form oligomers, which are important to mediate ifn-dependent antiviral activity [54] . induction of timely antiviral responses in cotton rats by fully functional mx genes may underlie the short-lived replication cycle of vanbt in cotton rats. as we observed mild inflammation in the lung of infected animals, there is a possibility that only limited cells could be infected by vanbt in cotton rats. however, it has been suggested that healthy adults usually show a milder range of respiratory symptoms upon ev-d68 infection, similar to that described here in cotton rats [18, 53, 55] . interestingly, ev-d68 infection was reported in adult patients with hematologic malignancy and hematopoietic cell transplant recipients, showing mild upper respiratory symptoms to respiratory failure [56] . thus, it would be important to measure vanbt replication in immunocompromised (cyclophosphamide treatment induced) cotton rats or blocking ifn-β production in cotton rat upper respiratory tract cells in vitro [57] . as children are at higher risk for severe respiratory symptoms due to ev-d68 infection, it would be pertinent to evaluate ev-d68 replication in younger cotton rats or cotton rat pups that are only a few days old [53, 58] . in a tight correlation with the significant differences in the infection profile by three strains of ev-d68 in cotton rats, we observed that the strains differ in their capacity to induce na followed by i.n. infection or i.m. immunization. atcc induced the lowest titers, while vanbt elicited the highest homologous na response (fig 1f, fig 4a and table 1 ). on the basis of hemagglutination inhibition (hi) and na titers, it was previously shown that the fermon strain had lower hi and na titers than recently detected ev-d68 strains, which may explain that atcc has lower antigenicity leading to induction of lower na titers. the hi and na titers were also shown to be significantly different between strains of different genetic clades among recently detected ev-d68 strains [33] . compared to vanbt and mo/49, atcc elicited essentially negligible heterologous na titers (table 1) . a recent study using the bayesian markov chain monte carlo approach have estimated that genetic diversity in the vp1 region increased after the late 1990s, which may have resulted in the emergence of the three clades [22] . this suggests that atcc, being the prototype strain, and isolated in 1962, could differ vastly in terms of its antigenic characteristics, and even replication in humans, and as shown here, in cotton rats, when compared with the more recent vanbt and mo/49 strains. previously, we showed that i.n. infection of cotton rats with hrv1b or hrv16 at comparable doses, only hrv16 showed high levels of virus replication in nose, trachea, and lungs, whereas hrv1b showed significantly lower infectious virus titers in these tissues [37] . furthermore, we reported that i.m. live hrv16 vaccination of cotton rats generated high titers of na in all vaccinated animals, while hrv1b did not elicit detectable homologous na [37] . with hrvs, we have found a strong correlation between production of high titers of serum na against certain hrvs and the ability of these hrvs to replicate in the airways of cotton rats. as there were vast differences among the three strains of ev-d68 in infectivity profile and na titer generation followed by infection, we think that it correlate to rv infection in cotton rats. our data showed that vanbt replicates in the lymph nodes draining from the site of i.m. inoculation (s4 fig), thus increasing the chance of antigen processing and presentation by macrophages and dendritic cells to initiate the robust immune response. our results with passive transfer of ev-d68 immune sera showed that prophylactic administration of vanbt hyper-immune serum protects both nose and lung tissues of naïve animals against vanbt challenge, indicating that passive antibody transfer could be a potentially effective prophylactic therapy against ev-d68 infection during outbreaks or for treatment of individuals at high-risk (fig 4b) . prophylactic antibody therapy for infectious diseases has become an important alternative in the absence of a vaccine, such as respigam tm and synagis tm for rsv and recently zmapp tm for ebola virus [59] [60] [61] . we further assessed different immunization using live and uv-inactivated ev-d68 against vanbt challenge. we found that i.m. immunization with live vanbt and mo/49, but not live atcc, resulted in a decrease of viral loads in nose and lung tissues (fig 5b) . though i.n. infection with vanbt resulted in induction of na titer (fig 1g) , animals previously infected with vanbt did not show any significant protection against re-infection (fig 5b) , which suggests that infection with ev-d68 fails to provide the degree of immunity induced by infection with other respiratory viruses in this model, i.e., influenza and rsv, where infection induces complete protection to re-infection [62] [63] [64] [65] . however, similar results were seen for i.n. infection with hrv that replicate in cotton rats, but does not protect against re-infection [37]. the moderate protection achieved by live mo/49 immunization against vanbt challenge (fig 5b) is consistent with its capacity to generate detectable heterologous na against vanbt (table 1) . our results clearly demonstrate generation of cross-strain na response conferred by immunization with live ev-d68 belonging to different clades, which could serve as a tool to define conserved antigenic features to use as candidates to induce broad-spectrum immunity against different clades of ev-d68 and thus warrants further investigation. on the basis of pulmonary cytokine analysis on ev-d68 immunized animals after challenge, we conclude that live i.m. vanbt-immunized animals mount both strong th1 and th2 responses and uv-vanbt i.m. immunized group showed excessive th2 response that could predispose to the development of allergic responses. similarly, it was previously shown that uv-inactivated viruses used for immunization have the propensity to develop th2-biased responses and enhanced respiratory disease [66, 67] . for the first time, this study assessed the effect of immunization with crude vaccine preparations for ev-d68 given i.m. despite the efficacy of i.m. immunization with vanbt to reduce viral titers in vivo, animals vaccinated i.m. with uv-vanbt and vanbt enhanced cytokine responses and lung histopathology, which raises the possibility that pre-existent immunity to these viruses could have deleterious effects. association of ev-d68 with acute flaccid paralysis in children definitively raises safety issues for vaccine developers. our data serve to emphasize that a live ev-d68 vaccine generates high titer na and could be useful to define mechanisms of immunity against the virus. future studies to develop either live-attenuated vaccine, inactivated ev-d68 whole virus vaccine, viruslike particle-based vaccines, recombinant vp1 protein based vaccines with varied dosage, or inclusion of adjuvants to formulate safe vaccines for ev-d68 are logical next steps to reduce potential adverse effects. in this regard, we have previously shown that a formalin-inactivated rsv vaccine, shown to induce "vaccine enhanced disease" in clinical trials in the mid-1960's, could be rendered safe in this model by inclusion of the tlr4 agonist adjuvant, monophosphoryl lipid a [68] . we used ev-d68 clinical isolates from human patients without any adaptation to infect cotton rats. this is one of the biggest advantages of our model because it is clear that virus adaptation introduces a considerable number of nucleotide variations in viral genome that may dramatically alter the course of natural infection [35, 69] . despite the variety of geographical sources for ev-d68, the strains detected in recent years have similar vp1 sequences as long as they belong to the same genetic clade [33] . previous studies have shown that viruses of the clades a to c circulated or co-circulated during different time periods in different geographic locations [9, 10, 14-17, 19, 21, 22] . thus, the possibility of antigenic differences of strains belonging to same clade/subclade but different geographical distance is minimal [18] . therefore, we hypothesize that the cotton rat model could be an important tool to infect other current or future ev-d68 isolates detected in other geographical locations to assess vaccine strategies and antiviral molecules against ev-d68. all animal work presented in this paper was conducted under strict accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health. the animal protocols (protocol # 2 and 7) were approved by the institutional animal care and use committee (iacuc) of sigmovir biosystems inc. (sbi) (olaw assurance #a4642-01). sbi has usda breeding and research licenses (51-a-0031 and 51-r0091, respectively), and is accredited by the association for assessment and accreditation of laboratory animal care (aaalac). four to six week old female cotton rats were obtained from the inbred colony maintained at sbi. cotton rats in the colony were seronegative for ev-d68 by neutralization assay, and seronegative by elisa to other adventitious respiratory viruses (e.g., pneumonia virus of mice, rat parvovirus, rat coronavirus, sendai virus). animals were housed in large polycarbonate cages, and fed a diet of standard rodent chow and water ad libitum. cotton rats were infected i.n. or immunized intramuscularly (i.m.) with three different strains of ev-d68 under isoflurane anesthesia by inoculation of 100 μl of virus preparation [10 6 50% tissue culture infective dose (tcid 50 )] per rat. serum samples were obtained by retro-orbital blood collection under isoflurane anesthesia. animals were euthanized by carbon dioxide asphyxiation. three strains of ev-d68 were studied: (1) atcc (accession # kt725431), which is the prototype fermon virus strain purchased from american type culture collection (atcc, manassas, va), (2) vanbt/1 (supplied by dr. tina v. hartert; accession # kt347280) collected in 2012 from nashville, tn at vanderbilt university medical center as a pre-outbreak isolate, and (3) mo/14/49 (bei resources, niaid; accession # km851227) collected in kansas city during the 2014 outbreak in usa [8, 31, 32] (fig 1a) . virus stocks were produced in hela h1 cells (atcc, manassas, va) grown in dmem (lonza) with 10% fbs (seradigm) at 34°c and 5% co 2 . five days p.i., cells were freeze-thawed 4-5 times and vortexed vigorously to release the cell associated viruses and centrifuged at 800 x g before determination of tcid 50 . replication-deficient virus was generated by exposing the virus stocks to ultraviolet light for 30 min. tissue samples (left lung lobe and entire nose) were homogenized in 3 ml of homogenization buffer (emem, 1x spg, 1% fungizone, 0.1% gentamycin) at different time-points p.i. infectious virus titers were determined by standard endpoint dilution assay and expressed as tcid 50 /g of tissue. briefly, after overnight incubation, hela h1 cells in 96 well plates were washed with serum free medium and infected with serial 10-fold diluted freeze-thawed tissue homogenates and cell viability was assessed by crystal violet staining. each assay was repeated at least three times with three to five replicates per assay. virus neutralization was performed by incubation of serial 2-fold dilution of immunized and control cotton rat sera with 200 tcid 50 virus/well in 96 well plate. after 5 days of incubation at 34°c, cell viability was assessed. each assay was repeated at least three times with four replicates per assay. each run contained cell control plate and back titration plate for each ev-d68 virus. rna was isolated from the lung lingular lobe using the rnaeasy kit (qiagen sciences). cdna was prepared by quantitect reverse transcription kit (qiagen sciences) or superscript1 ii reverse transcriptase (thermo fisher scientific). each cdna reaction was prepared from 1 μg of initial rna and diluted to 100 μl of the final volume. three μl of cdna was subsequently used for each pcr reaction. ev-d68 specific qrt-pcr was developed using primers that target the vp1 of ev-d68 (s1 table) . to quantify viral replication, cdna for the negative strand viral rna (as an indicator of active rna transcription) was synthesized by priming with a forward primer flanking at the start of the vp1 sequence (evd68 vp1 f1/1979 primer). the pcr reaction was performed using internal nested primers (vanbt vp1 f3/1983 and vanbt vp1 r3/1984). the entire vp1 pcr amplicon (using primers evd68 vp1 f1/1979 and evd68 vp1 r1/1980) was gel purified and diluted to generate a copy number standard curve, which was used to quantify copies/g tissue of negative viral rna. the assessment of cotton rat cytokines mrna expression was carried out using primers as previously described [70, 71] . lungs were dissected en bloc and right lobe was inflated with 10% neutral buffered formalin to their normal volume, and submersed in the same fixative solution. following fixation, lungs were embedded in paraffin, sectioned, and stained using hematoxylin and eosin (h&e). four parameters of pulmonary inflammation were evaluated: peribronchiolitis (inflammatory cell infiltration around the bronchioles), perivasculitis (inflammatory cell infiltration around the small blood vessels), interstitial pneumonia (inflammatory cell infiltration and thickening of alveolar walls), and alveolitis (cells within the alveolar spaces). slides were scored blindly on a 0 to 4-severity scale (absent, minimal, mild, moderate and marked). viral titers, na titers, expression of cytokine genes and total vrna or (-) vrna were calculated as geometric means ± standard error (se) for all animals in a group at a given time p.i. student t-test was used to determine statistically significant differences between two groups, using an unpaired, two-tailed test with significance set at p<0.05. pulmonary pathology scores were expressed as the arithmetic means ± se for all animals in a group. rhinoviruses and respiratory enteroviruses: not as simple as abc unusual type of epidemic conjunctivitis in ghana pandemic of new type of conjunctivitis new enteroviruses, ev-93 and ev-94, associated with acute flaccid paralysis in the democratic republic of the congo enterovirus 94, a proposed new serotype in human enterovirus species d detection and genetic characterization of enteroviruses circulating among wild populations of chimpanzees in cameroon: relationship with human and simian enteroviruses co-circulation of enteroviruses between apes and humans a probable new human picornavirus associated with respiratory diseases phylogenetic characterization of enterovirus 68 strains in patients with respiratory syndromes in italy worldwide emergence of multiple clades of enterovirus 68 global reemergence of enterovirus d68 as an important pathogen for acute respiratory infections centers for disease c, prevention. enterovirus surveillance-united states molecular epidemiology of enterovirus d68 from 2013 to 2014 in philippines acute respiratory infections due to enterovirus 68 in yamagata enterovirus 68 among children with severe acute respiratory infection, the philippines molecular evolution of enterovirus 68 detected in the philippines enterovirus 68 in children with acute respiratory tract infections lineages, sub-lineages and variants of enterovirus 68 in recent outbreaks molecular epidemiology and evolution of human enterovirus serotype 68 in thailand detection of enterovirus 68 as one of the commonest types of enterovirus found in patients with acute respiratory tract infection in china continued seasonal circulation of enterovirus d68 in the netherlands emergence and epidemic occurrence of enterovirus 68 respiratory infections in the netherlands in 2010 distinct cellular immune responses following primary and secondary influenza virus challenge in cotton rats evidence of a cross-protective immune response to influenza a in the cotton rat model induction of type i interferons and interferoninducible mx genes during respiratory syncytial virus infection and reinfection in cotton rats lack of antibody affinity maturation due to poor toll-like receptor stimulation leads to enhanced respiratory syncytial virus disease enhanced pulmonary pathology in cotton rats upon challenge after immunization with inactivated parainfluenza virus 3 vaccines the tlr4 agonist, monophosphoryl lipid a, attenuates the cytokine storm associated with respiratory syncytial virus vaccine-enhanced disease coxsackievirus a16 induced neurological disorders in young gerbils which could serve as a new animal model for vaccine evaluation the cotton rat: an underutilized animal model for human infectious diseases can now be exploited using specific reagents to cytokines, chemokines, and interferons interferon-inducible mx gene expression in cotton rats: cloning, characterization, and expression during influenza viral infection the authors would like to thank mr. charles smith, ms. martha malache, mr. freddy and ms. ana rivera for their technical support with the cotton rats. key: cord-310947-aqau2n7q authors: pan, ji'an; peng, xiaoxue; gao, yajing; li, zhilin; lu, xiaolu; chen, yingzhao; ishaq, musarat; liu, dan; dediego, marta l.; enjuanes, luis; guo, deyin title: genome-wide analysis of protein-protein interactions and involvement of viral proteins in sars-cov replication date: 2008-10-01 journal: plos one doi: 10.1371/journal.pone.0003299 sha: doc_id: 310947 cord_uid: aqau2n7q analyses of viral protein-protein interactions are an important step to understand viral protein functions and their underlying molecular mechanisms. in this study, we adopted a mammalian two-hybrid system to screen the genome-wide intraviral protein-protein interactions of sars coronavirus (sars-cov) and therefrom revealed a number of novel interactions which could be partly confirmed by in vitro biochemical assays. three pairs of the interactions identified were detected in both directions: non-structural protein (nsp) 10 and nsp14, nsp10 and nsp16, and nsp7 and nsp8. the interactions between the multifunctional nsp10 and nsp14 or nsp16, which are the unique proteins found in the members of nidovirales with large rna genomes including coronaviruses and toroviruses, may have important implication for the mechanisms of replication/transcription complex assembly and functions of these viruses. using a sars-cov replicon expressing a luciferase reporter under the control of a transcription regulating sequence, it has been shown that several viral proteins (n, x and sud domains of nsp3, and nsp12) provided in trans stimulated the replicon reporter activity, indicating that these proteins may regulate coronavirus replication and transcription. collectively, our findings provide a basis and platform for further characterization of the functions and mechanisms of coronavirus proteins. interactions between viral proteins play pivotal roles in many processes during the viral infection cycle. this is the case in the formation of virus replication complexes, coordinated functions between different viral proteins, assembly of virions, and counterdefense of host immune responses. analysis of protein-protein interactions is essential to understand protein functions and the molecular mechanisms underlying biological processes. as the viral genomes are of limited sizes, they are particularly well suited for genome-wide analysis of all possible protein-protein interactions. however, the viral protein interaction maps have been generated until now only for a limited number of viruses, including t7 bacteriophage [1] , vaccinia virus [2] , potato virus a [3] , pea seed-borne mosaic virus [3] , wheat steak mosaic virus [4] , hepatitis c virus [5, 6] , porcine teschovirus [7] , kaposi sarcoma-associated herpesvirus [8] , and very recently severe acute respiratory syndrome coronavirus (sars-cov) [9, 10] . although a large variety of methods have been developed to detect protein-protein interactions, only a few of them are suited for largescale and high throughput protein interaction analysis. until now, all the genome-wide analysis of protein interaction networks for viruses and cells have been carried out mainly with the yeast two-hybrid systems, in combination with glutathione-s-transferase (gst) pulldown assays to verify major interactions [8, 9] . considering that protein modifications that are likely to influence protein interactions may be different for certain proteins in the context of yeast and mammalian cells, the mammalian two-hybrid system may better reflect genuine protein interactions for human viruses. accordingly, we adopted the mammalian two-hybrid system for detecting genomewide protein-protein interactions of sars-cov. the coronaviruses are classified into the family coronaviridae in the order nidovirales and possess the largest rna genomes known. the genome of sars-cov contains a single-stranded, plus-sense rna of approximately 29.7 kb in length. fourteen open reading frames (orfs) have been identified, of which 12 are located in the 39 end of the genome [11, 12] . the two large orfs (1a and 1b) in the 59proximal two-third of the genome encode the viral replicase and are translated directly from the genomic rna, while orf 1b is expressed by 21 ribosomal frameshifting. the large polypeptides encoded by 1a and 1b are considered to be cleaved into 16 functional replicase proteins by two proteinases, a papain-like proteinase 2 encoded by nsp3 and a 3c-like proteinase (or main proteinase) encoded by nsp5 [12] . a number of functions or characteristics have been identified for the 16 non-structural proteins (nsps). nsp1 was proved to be able to suppress host gene expression by promoting host mrna degradation and was involved in cellular chemokine deregulation [13, 14] . nsp2 seems not to play a crucial role in the generation of infectious viruses in cell culture [15] . nsp3 is involved in many activities including papain-like proteinase activity, deubiquitinating activity, and adp-ribose-10-phosphatase activity [16] [17] [18] 60] , which are essential for viral replication and transcription. nsp5 encodes a 3c-like proteinase which is considered to be an important target for antiviral drug design [19] . coronavirus nsp4 and nsp6 are transmembrane proteins that could anchor the replication complexes to double membrane vesicles [20] . based on the structural analysis, hexadecamer of nsp7 and nsp8 may possess dsrna-binding activity [21] . nsp8 was shown to have rna-dependent rna polymerase (rdrp) activity that could be involved in producing primers utilized by nsp12 which is normally accepted to be the rdrp for sars-cov [22, 23] . nsp9 is a single-stranded rnabinding protein [24] . though the structure of nsp10 is resolved, its function is still poorly understood, except that of nsp10 of mhv, homologous to that of sars-cov, which regulates viral rna synthesis [25] [26] [27] . nsp13 is shown to be rna helicase and 59-triphosphatase that may play a crucial role in the viral rna capping [28, 29] . nsp14 of coronaviruses possesses a 39-.59 exoribonuclease activity which may be involved in the proof-reading ability during the viral rna replication and transcription [30] [31] [32] . besides the exoribonuclease activity, sars-cov also possesses the endoribonuclease activity that is rendered by nsp15 [33] . according to the bioinformatic prediction, nsp16 and sud domain of nsp3 of sars-cov may function as 29-o methyltransferase and guanine n7 methyltransferase, respectively [34, 35] . very recently, the 29-o methyltransferase activity was confirmed experimentally for nsp16 of feline coronavirus [36] . nsp14, 15 and 16 were shown to be essential for efficient replication of coronavirus [30, 37, 38] . the 39 one-third of genome encodes 12 viral proteins including the structural and accessory proteins, which are translated from about 10 subgenomic rnas [39, 40] . proteins s, e, m, and n are four well described structural proteins, and 3a, 6, 7a, and 7b were also reported to be virion-associated proteins or viral structural proteins [41] [42] [43] [44] . multiple functions and activities have been identified for the structural and accessory proteins, including apoptosis induction, interference with the innate immunity response, and regulating the cellular protein expression [45] . however, these proteins are not essential for viral replication and transcription at least in cell culture and tested animal models, except nucleocapsid (n) protein [46] [47] [48] [49] . previous studies on sars-cov focused mostly on the molecular characterization and functional analysis of individual proteins encoded by sars-cov and limited information is available on viral protein-protein networks of coronaviruses [50] . to provide more insights into the functions of individual proteins, we analyzed interactions between all sars-cov-encoded proteins. by using mammalian two-hybrid assays, 40 different interactions between viral proteins have been identified, and six novel interactions could be confirmed in vitro by biochemical assays. moreover, a sensitive replication and transcription reporter system of sars-cov was established in this study, and, based on this system, we examined the impacts of all the individual viral proteins on the viral replication and transcription and found that the n protein played an important role at the early stage of sars-cov genome replication. in this study, a mammalian two-hybrid system was adopted to analyze the protein-protein interactions of sars-cov as it was assumed that the viral proteins expressed in mammalian cells were prone to be in their native conformations and therefore the interactions detected were more likely to be biologically relevant in comparison with other in vitro biochemical methods and assays performed in yeast cells [51] . for analysis of genome-wide protein interactions of sars-cov, all known orfs were amplified by pcr from viral cdnas of sars-cov isolate whu [39] and cloned into pgem-t vector ( table 1 ). the large polyprotein encoded by orf 1a/b was split into 18 domains according to the proteinase cleavage sites, except for nsp3 which was divided into 3 parts based on predicted functional domains. for structural and accessory proteins, the intact orfs including start and stop codons were amplified, except for s protein which was divided into s1 and s2 as these domains were proved to be two separate domains with distinct functions [52] . all the primers used for amplification were designed by inserting appropriate restriction sites which could be used for subcloning all the fragments from pgem-t cloning vectors into mammalian two-hybrid vectors pm (bait) and pvp16 (prey) and other protein expression vectors (see below) in correct reading frames. in total, 1024 interaction combinations between all sars-cov proteins were examined in a pairwise matrix. as a result, 40 different interactions were detected using the mammalian twohybrid assays (fig. 1a) . all the interactions shown ( fig. 1) were most likely specific for the viral protein domain of the fusion proteins as the activities of the reporter genes were reduced to background levels when one interacting partner in any of the combinations was replaced with non-relevant fusion protein (negative control) (data not shown). to further show the specificity of the interactions (fig. 1) , a quantitative assay for a typical positive interaction exemplified by nsp10-nsp14 and various controls were shown (fig. 1b) . to test the specificity of individual interactions, competitive assays were performed by co-expression of viral proteins using a different vector within the cells transformed with the bait and prey constructs. the co-expressed partner proteins interfered with the nsp10-nsp14 interaction, leading to reduced reporter activity (fig. 1c) , indicating that the same proteins with different fusion domains competed with each other and impaired the specific interactions needed for activation of the reporter genes. most interactions showed directionality, indicating the influence of fusion domains on the interacting sites. nevertheless, three pairs of interactions were detected in both directions: nsp10 and nsp14, nsp10 and nsp16, and nsp7 and nsp8. nsp11 is a small polypeptide containing only 13 amino acids and no interaction was detected with it in various assays but nsp11 in the fusion with nsp10 (nsp10/ 11) could significantly enhance the binding capability of nsp10 with either nsp14 or nsp16, indicating that the small nsp11 may also play important roles in viral protein interactions and replication. self-interactions of nsp3, nsp5, nsp8, nsp15 and n were observed, suggesting that these proteins could form dimeric or multimeric complexes by interacting with themselves. furthermore, 8 interactions between non-structural proteins and accessory proteins (3b, 7b, 8b, 9b) and one interaction between nonstructural protein nsp16 and structural protein n were detected, indicating that some of the accessory proteins might be involved in viral replication and transcription processes. nevertheless, that being the case, these interactions could have a minor effect on modulating virus replication as the virus-specific proteins are not strictly essential for viral replication at least in cell culture and mouse models [48, 49] . to confirm the interactions which were newly detected by mammalian two-hybrid assays, pull-down assays were performed. proteins that were related to the interactions not reported previously were expressed in different bacterial systems including pet30a (his-tagged), pgex-6p-1 (gst fusion) and pmal-c2x (mbp fusion). using pull-down assays that were described in materials and methods section, six protein-protein interactions were confirmed, including those between: nsp10 and nsp14, nsp10 and nsp16, nsp13 and 3b, nsp8 and 3b, nsp16 and n, and 8b and n (fig. 2 ). due to difficulties in bacteria expression, the interactions involving nsp12, nsp3.2 and nsp3.3 could not be confirmed. although a large number of protein-protein interactions were detected for sars-cov in virtue of the large-scale screening analysis in mammalian two-hybrid system, the roles of these interactions in the viral replication and transcription were still not clarified. to obtain more clues to the general roles of individual proteins, we constructed a sars-cov replicon (rep-scv-luc/ neo) that expresses the firefly luciferase gene, a sensitive reporter, [19, 68, 69] , nsp5-nsp7 and nsp8 [9] , nsp7-nsp7 [21, 70] , nsp7-nsp8 [21] , nsp7-nsp9 [9] , nsp8-9b [9] , nsp10-nsp14 and nsp10-nsp16 [10] , nsp15-nsp15 [54, 56, 71, 72] , nsp7-e and 7a-m [73] , n-n [55, 57] and n-n [55, 57] . (b) a typical result for a positive interaction with the example of nsp10-nsp14. the combination of pm-53 and pvp16-t represents a positive control. (c) a typical interaction inhibition assay performed to confirm that the interaction was not resulted from self-activation. error bars represent standard deviations from three independent experiments. doi:10.1371/journal.pone.0003299.g001 under the control of m gene transcription regulatory sequence (trs) as described in the materials and methods section (fig. 3a) . the effects of individual proteins or protein domains provided in trans on the replication/transcription of the sars-cov replicon were evaluated. any effect of the protein provided in trans on the luciferase expression levels could in principle be due either to changes in the extent of the replication, the transcription, or a combination of both. to evaluate the reporter gene expression of the replicon generated in this work, the bac plasmid encoding rep-neo/luc (pbac-rep-scv-neo/luc) was transfected into bhk21 cells. a significant increase in luciferase activity, in comparison with that of the parental replicon plasmid pbac-sars-cov-rep was observed ( fig. 3b ). as the luciferase gene is located in the 39-end of the genome and under the control of a viral trs, it can only be expressed from subgenomic rnas but not from the viral genome rna. therefore, expression of luciferase was expected to reflect the genome replication and transcription of sars-cov replicon. to further clarify whether the luciferase expression of rep-scv-luc/ neo derived from the viral replication and transcription processes but not from nuclear splicing products of the large viral replicon rna during the cmv promoter-driven transcription in nucleus, the positive and negative subgenomic rnas were examined according to the strategy described in our previous work [39] and the existence of correct subgenomic rnas was confirmed (data no shown). moreover, when the gene segment between two mlui restriction sites in viral genome, which encodes nsp4-nsp11 and part of nsp12, was deleted, the construct pbac-rep-scv-dmluluc/neo produced much lower levels of luciferase expression although it still retained detectable level of luciferase activity (fig. 3b) . to analyze the luciferase expression from the mlui deletion mutant, the identity of the luciferase mrna was sequenced. the results showed that a low level of luciferase mrna was generated either by cryptic promoters or by splicing of cryptic introns within the replicon transcripts in nucleus but not in the mrnas derived from the cytoplasmic replication and transcription of the replicon (data not shown). taken together, these results showed that the reporter-containing sars-cov replicon could be used as a model system for analyzing the functions of viral proteins. to study the effect of sars-cov proteins on replication and transcription, individual viral proteins were co-expressed in trans with the sars-cov replicon. renilla luciferase was employed as an internal control reporter to normalize the transfections among different wells (fig. 4) . though most of the viral proteins did not exert significant impact on the replication and transcription of the replicon, the nsp3.1 containing x and sud domains, and the rna polymerase nsp12 (fig. 4a) , and nucleocapsid n (fig. 4b ) significantly increased the luciferase activity, whereas nsp3.2 containing the papain-like (pl) proteinase domain reduced the viral replication and transcription. other proteins, such as nsp10/11, also showed a minor but significant increased expression. these results confirmed that the function of n gene fragment was not resulted from protein 9b that is internally nested in n sequence, as 9b showed no obvious impact on the replication and transcription of replicon when it was co-expressed (fig. 4b ). in addition, these results indicate that nucleocapsid protein, nsp3 and rna polymerase may possess a dominant function in trans, while the pl proteinase is involved in important cis functions like cisprocessing of the large viral polyprotein. trans-activation activity of n protein at the early stage of genome replication or transcription of sars-cov to make further analysis on the enhancement effect of n protein on the viral replication and transcription, we examined the dynamic changes of the luciferase activity expressed from the reporter replicon rep-scv-luc/neo. when the replicon plasmid pbac-rep-scv-luc/neo was transfected alone, the luciferase activity could be detected 13 h post transfection (more than 10 times compared with background levels) while the highest value of activity was reached around 32 h post transfection (fig. 5a) . the luciferase activity became stable 72 h post transfection and the activity level was as low as that of 14 h post transfection (fig. 5a) . to study the role of n protein provided in trans, we next transfected cells with equal amount of the reporter replicon together with either n protein expression construct (pcdna3.0-n) or the empty vector pcdna3.0. luciferase activity became detectable at 7 h post transfection when n protein was provided in trans (fig. 5b) , which was 9 h earlier than similar transfection without additional n protein (fig. 5b) . it was also observed that the luciferase activity was significantly enhanced in the first 40 h post transfection (fig. 5b) . the ratios of luciferase activities with and without co-expression of n protein at different time points post transfection were shown in fig. 5c . the ratios became lower step by step from over 50 times at 16 h post transfection to around 4 times at 38 h post transfection (fig. 5c) . the corresponding ratios for nsp3.2, nsp3.1 and nsp12 were also investigated and no similar phenomena were observed (data not shown). collectively, these data showed that the nucleocapsid n protein provided in trans could enhance the efficiency of sars-cov genome replication and transcription at early stages. coronaviruses have the largest rna genome known, which encodes a large number of proteins that are involved in viral replication, assembly, and other important functions that are essential to viral amplification in host cells. except for some viral proteins that might perform their activities individually, most of the viral proteins could associate with other proteins or themselves to carry out their functions, indicating that the interactions between these proteins may play a crucial role during the viral life cycle. for sars-cov, at least 17 proteins, including 16 nonstructural proteins and the structural nucleocapsid protein, are most likely involved in the replication process [12, 53] . in this study, a mammalian two-hybrid system was used to determine the genome-wide matrix-based protein-protein interactions of sars-cov. in total, 40 different interactions for 28 predicted mature proteins were observed, and most of them have not been previously reported. interestingly, 32 percent of the interactions tested could be confirmed by pull down assays. to our knowledge, this is the first genome-wide protein-protein analyses for an intact viral orfeome by using mammalian two-hybrid system. in our screen, 1.4 protein interactions per viral protein on the average was detected, and this result is in the upper range of the detection rates of viral protein interactions obtained by yeast two-hybrid systems [58] . therefore, this study indicates that mammalian two-hybrid system can also serve as a convenient system to detect viral protein interactions of the whole orfeomes. in addition, mammalian two-hybrid system may have some advantages over yeast two-hybrid system for detecting genuine viral protein interactions due to the native posttranslational modifications and folding of the proteins. very recently, two large-scale analyses of protein interactions of sars-cov were carried out by using yeast two-hybrid systems [9, 10] . in the report by von brunn et al, 70 pairs of interactions were revealed, among which 30% could be verified by coimmunoprecipitation [9] . in the work by imbert et al, 17 pairs of interactions were observed for non-structural proteins, about half of which were related to nsp3 [10] . surprisingly, none of the interactions revealed in the two studies was overlapping although they both employed the yeast two-hybrid system for the screening. in contrast, overlapping interactions could be identified between that detected by mammalian two-hybrid system in current work and that by different yeast two-hybrid systems. there are 9 pairs of interactions found in our work that are overlapping with that of von brunn et al, and they are nsp5 with nsp5, nsp7 and nsp8, nsp7 with nsp7, nsp8 and nsp9, nsp8 with nsp8, nsp9 and 9b. there are 4 pairs of interactions found in this study that are identical to that of imbert et al. and they are nsp10 with nsp14 and nsp16 in both directions. interestingly, all the overlapping interactions represented strong interactions in the mammalian two-hybrid assays and could be verified by biochemical assays. in the two previous studies with yeast two-hybrid system, four proteins (nsp2, nsp3, nsp8 and 9b) were shown to have a wide range of interactions with other viral proteins, but these phenomena could not be observed in mammalian two-hybrid assays. in contrast, a number of interactions including n-n and nsp15-nsp15 were detected in current study and proved previously by other studies [54] [55] [56] [57] but they could not be revealed by the yeast two-hybrid systems [9, 10] . intriguingly, the two strong interactions nsp10-nsp14 and nsp10-nsp16 identified in mammalian two-hybrid system was also revealed in a yeast two-hybrid system by imbert et al. [10] but not by von brunn et al [9] . although it is difficult to judge which system offers more biologically relevant results, great caution should be taken when interpreting the interaction data as both two-hybrid screenings as well as biochemical methods may generate false positive and false negative interactions. nevertheless, the above comparative analysis of overlapping interactions of different systems may suggest that mammalian two-hybrid system could provide more reliable assays for detecting human viral protein interactions. in other scenario, the profiles of sars-cov protein interactions revealed by mammalian two-hybrid system (in this work) and yeast systems [9, 10] , respectively, may be complementary to each other and jointly serve as a framework for further characterization of protein-protein interactions and their biological functions on coronavirus replication cycle. similar to that of yeast two-hybrid system, the detectable protein interactions all take place in the nucleus whereas, on the contrary, all positive-stranded rna viruses replicate in cytoplasm, the natural location for viral proteins. thus, the two-hybrid systems may have obvious limitation on detecting proteins that contain transmembrane domains or change to abnormal conformations in acidic conditions, and this may represent one reason for generating false negative and false positive interactions. in current screen, only few interactions were detected for the sars-cov membrane proteins such as nsp4, m, e and 3a proteins, and no interactions could be detected for nsp1, nsp6, spike and the small transmembrane protein 6, possibly reflecting the limitation of the two-hybrid system. therefore, using shorter or random cdna fragments by removing the transmembrane and other inhibitory domains may help detect more interactions in this system. in this study, we have detected several interactions between accessory proteins and replicase proteins, for example, 3b with nsp8, nsp12, nsp13 or nsp14, 7b with nsp9 or nsp16, 8b with nsp9, and 9b with nsp8. interestingly, among these interactions those between 3b with nsp8 and nsp13 were confirmed by pull-down assays. however, previous studies showed that deletions of orfs 3b, 7b, 8b and 9b alone or in combination with other virus-specific proteins did not significantly influence the level of viral rna and replication efficiency in cell culture and mouse models [48, 49] , suggesting either that these interactions may not play significant roles in viral replication or that these systems are not sensitive enough to detect minor differences. in any case, it would be reasonable to speculate that such interactions may contribute to the virus-host interplay and hence to the viral pathogenicity. the interactions between nsp10 with nsp14 and nsp16 showed bi-directionality in the mammalian two-hybrid analyses, which could be confirmed in pull-down assays and were also revealed in one of the two yeast-hybrid analyses [10] . recently, nsp10 was shown to form a dodecameric rna-binding protein complex [26, 27] involved in regulation of rna synthesis and polyprotein processing [25, 59] . nsp14 and nsp16 were proved to play an essential role in viral replication and transcription as shown by mutational analysis [37] . considering that nsp14 has exoribonuclease activity and could play an essential role in rna proofreading activity [30] [31] [32] , and that nsp16 is involved in the viral methyltransferase activity and rna capping [34, 36] , the interactions of nsp10 with nsp14 and nsp16 may indicate that nsp10 could play an important role in the formation of replication complex bound to rna (i.e., nsp10 could mediate the binding of nsp14 and nsp16 to the rna) and in the replication/transcription processes. although no interactions were detected for nsp11, which was predicted to be small polypeptide with 13 amino acids, fusion of nsp11 with nsp10 strengthened the respective interactions of nsp10 with nsp14 and nsp16. these observations may imply that nsp11 could act as a cofactor for nsp10 and the predicted cleavage site between nsp10 and nsp11 may be inefficient in vivo, thus resulting in the production of nsp10/nsp11 fusion protein in cells. indeed, no evidence for existence of the fully processed nsp11 has been presented in published researches. however, such speculation needs to be confirmed by further investigations. several interactions detected in current work have also been observed in previous studies. for example, nsp7 and nsp8 could associate with each other and thus form a hexadecameric supercomplex with a central channel that has dimensions and positive electrostatic properties favorable for nucleic acid binding [21] . consistent with our results, self-interactions of n protein and nsp15 have also been revealed and the results indicate that these proteins have the propensity to oligomerize [56, 57, 61] . in this study, a sars-cov replicon containing a sensitive luciferase reporter was constructed, with its expression under the control of m gene trs. in this situation, the reporter activity would indicate the efficiency of both genome replication and transcription. when individual viral proteins were provided in trans to the reporter replicon, most proteins did not exert significant influence on the reporter activity, with exception of nsp3, nsp12 (polymerase), and nucleocapsid protein n. as protein nsp3 is a large multidomain protein, different domains were tested separately in this system. while nsp3.1 that covers domains predicted for adenosine diphosphate-ribose 10phosphatase [12] and methyltransferase [35] could enhance the reporter activity, the nsp3.2 had a negative effect, indicating the domains in nsp3.2 may play structural roles in the formation of replication/transcription complex and could behave in a dominantnegative manner when separated from other domains. in accordance with this hypothesis, nsp3.2 was found to associate with itself, nsp4 and nsp12 in this work. n protein of coronaviruses increases the rescue efficiency of coronaviruses from infectious rna transcripts [37, 49, 62] and is required for efficient genome replication [53, 63] . in this study, we showed that n protein was not absolutely essential for starting the replication but may play important roles at the early stages of genome replication and/or transcription because the reporter replicon in absence of additional n protein provided in trans also resulted in obvious replication and transcription but in lower efficiency and delayed manner. the enhancement effects of n protein phased out at late stage probably when the n protein expressed from the genome had accumulated to certain level. although the exact mechanisms for n-mediated stimulation activity at early stages are not known, several scenarios could be envisaged, such as protecting viral genome rna, increasing the translation efficiency of viral rna, stabilizing replication/ transcription protein complex and inhibition of cellular innate immune responses, as is supported by the recent reports on diversified functions of n protein [64, 65] . in summary, current work constructed an interaction network of sars-cov proteins and established a sensitive replicon for studying the functions of individual viral proteins. the intraviral protein interactions identified in this study, in combination with the data obtained by using other systems, could serve as a basis for further studies of viral protein functions and molecular mechanisms of the genome replication/transcription processes of coronaviruses. african green monkey kidney (vero e6) cells, baby hamster kidney (bhk21) cells and 293t cells were grown and maintained in dulbecco's modified eagle medium and modified eagle medium (gibco invitrogen), respectively, supplemented with 10% heat-inactivated fetal bovine serum and 100 u/ml of penicillin and 100 mg/ml of streptomycin (gibco invitrogen). viral cdnas encoding individual sars-cov orfs were generated and described in our previous work [39, 66] . the primers for individual orfs were designed according to the genome sequence of sars coronavirus strain whu (genbank accession number: ay394850) with restriction sites which were compatible for the downstream subclonings. the reagents for pcr were 0.2 mm dntps, 0.4 mm forward and reverse primers, ,10 ng template dna and 1 u of kod dna polymerase (toyobo) in 50 ml reaction system. the amplification conditions were 94uc for 2 min and 30 cycles of 94uc for 15 sec, 52uc for 15 sec and 68uc for 3 min, followed by 68uc for 10 min and 4uc for 10 min. after the separation by agarose gel electrophoresis, pcr fragments were recovered from the gels by dna extraction kit (omega bio-tech) and cloned into pgem-t vector (promega) after a-tailing reaction according to standard protocols. the sequences of positive clones examined by restriction and pcr analyses were confirmed by dna sequencing on abi 3730 dna analyzer (applied biosystems) and ceq tm 8000 sequencer (beckman coulter). subsequently, the coding sequences were cut from pgem-t constructs with appropriate restriction enzymes and cloned into the mammalian two-hybrid vectors pm and pvp16 (clontech), escherichia coli protein expression vectors pet30a (novagen), pgex-6p-1 (amersham biosciences) and pmal-c2x (new england biolabs), and mammalian expression vectors pcdna3.0 (invitrogen corporation), pcmv-tag2b (stratagene corporation) and pcih that was generated by adding an atg-ha-tag oligo at the xhoi-ecori sites of vector pci-neo (promega). details of the cloning processes and various constructs can be provided upon request. the reporter construct pg5-luc was reconstructed from pg5cat (promega) by replacing chloramphenicol acetyl transferase (cat) gene with luciferase gene sequence. for protein interaction analysis, 0.3 mg of dna-binding domain fusion constructs in plasmid pm, 0.3 mg of transcriptional activation domain fusion constructs in plasmid pvp16, 0.15 mg of reporter construct pg5-luc and 0.1 mg of vector prl-tk (promega) as internal control were transfected into 293t cells by calcium phosphate transfection method. one day before transfection, 293t cells were seeded in 24-well plate with ,1610 5 cells per well. on the following day, the dna of specified amount and 2.5 ml of 2.5 m cacl 2 were diluted with deionized h 2 o to reach a final volume of 25 ml. after an incubation for 5 minutes, 25 ml of 26hbs solution (50 mm hepes, 10 mm kcl, 12 mm dextrose, 280 mm nacl, 1.5 mm na 2 hpo 4 , ph 7.05) was added and mixed. the calcium phosphate-dna solution was added dropwise to the cell culture medium while swirling the plate gently. after incubation for 6 h at 37uc with 5% co 2 , the medium was replaced with fresh one. 24 h later, the luciferase assays were performed by dual-luciferase reporter assay system (promega corporation) according to the provider's instructions. several control constructs were adopted in the analysis, including pm-53 which encodes the p53 protein, pvp16-t which encodes the sv40 large t antigen, and the empty vectors pm and pvp16. the known interaction between p53 and t antigen was used as positive control, and the combinations of test construct and one control plasmid were assigned as negative controls. the assays on each pair of plasmid combinations were repeated at least 4 times and 3 wells were examined per assay. recombinant glutathione s-transferase (gst)-fusion proteins and maltose-binding protein (mbp)-fusion proteins were expressed in e. coli bl21 and dh5a, respectively. bacterial cells harvested were resuspended in phosphate-buffered saline (pbs, 10 mm sodium phosphate, 150 mm nacl, ph 7.5) and lysed by sonication in ice. the lysate was subsequently clarified by centrifugation at 12,000 g for 30 min at 4uc. gst fusion proteins and gst protein immobilized on glutathione-sepharose were coated firstly with mbp protein and were subsequently incubated with the lysate for mbp-fusion proteins in pbs containing protease inhibitor cocktail. after incubation with rotation for 2 h, the resin of glutathione-sepharose was precipitated by brief spin and washed by pbs at least for 5 times. the resin was re-suspended in sds-page loading buffer and heated at 100uc for 5 min. the samples were separated in 10% sds-page and transferred to pvdf membrane following the standard procedures of westernblotting. anti-mbp rabbit serum (new england biolabs, dilution 1:5000) was used in the immunoblot analysis to identify mbpfusion proteins. construction of a replication and transcription report system for sars-cov the reporter system was based on the primary sars-cov replicon (pbac-sars-cov-rep) [37] . to construct a reportercontaining replicon including a selection gene for mammalian cells, the luciferase and neomycin fusion gene cassette was amplified by pcr from a hepatitis c virus replicon provided by dr. ralf bartenschlager [67] . the luciferase-neomycin gene sequence was fused with and thus controlled by the transcription regulation sequence (trs) of sars-cov m gene. the fused sequence containing trs/m-luciferase-neomycin cassette was inserted between asci and bamhi sites in pbac-sars-cov-rep, resulting in the reporter replicon construct pbac-rep-scvluc/neo. the cloning details can be provided upon request. several transfection strategies and reagents were examined for the transfection of pbac-rep-scv-luc/neo and the relatively higher transfection efficiency was obtained with fugene hd transfection reagent (roche applied science). several cell lines were also examined and bhk21 turned out to better support the replication/transcription of rep-scv-luc/neo and produced consistent results. a protein linkage map of escherichia coli bacteriophage t7 genome-wide analysis of vaccinia virus protein-protein interactions towards a protein interaction map of potyviruses: protein interaction matrixes of two potyviruses based on the yeast two-hybrid system multiple interactions among 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infectious cdna of severe acute respiratory syndrome coronavirus selective replication of coronavirus genomes that express nucleocapsid protein ribonucleocapsid formation of severe acute respiratory syndrome coronavirus through molecular action of the n-terminal domain of n protein severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists isolation of virus from a sars patient and genome-wide analysis of genetic mutations related to pathogenesis and epidemiology from 47 sars-cov isolates enhancement of hepatitis c virus rna replication by cell culture-adaptive mutations dissection study on the severe acute respiratory syndrome 3c-like protease reveals the critical role of the extra domain in dimerization of the enzyme: defining the extra domain as a new target for design of highly specific protease inhibitors the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor structural genomics of the severe acute respiratory syndrome coronavirus: nuclear magnetic resonance structure of the protein nsp7 the structure of the endoribonuclease xendou: from small nucleolar rna processing to severe acute respiratory syndrome coronavirus replication crystal structure and mechanistic determinants of sars coronavirus nonstructural protein 15 define an endoribonuclease family severe acute respiratory syndrome coronavirus protein 7a interacts with hsgt we thank yu chen for valuable discussion and comment on the manuscript and nian xiang for excellent technical assistance. key: cord-331652-oc5s1if2 authors: trudeau, michaela p.; verma, harsha; sampedro, fernando; urriola, pedro e.; shurson, gerald c.; mckelvey, jessica; pillai, suresh d.; goyal, sagar m. title: comparison of thermal and non-thermal processing of swine feed and the use of selected feed additives on inactivation of porcine epidemic diarrhea virus (pedv) date: 2016-06-24 journal: plos one doi: 10.1371/journal.pone.0158128 sha: doc_id: 331652 cord_uid: oc5s1if2 infection with porcine epidemic diarrhea virus (pedv) causes diarrhea, vomiting, and high mortality in suckling pigs. contaminated feed has been suggested as a vehicle of transmission for pedv. the objective of this study was to compare thermal and electron beam processing, and the inclusion of feed additives on the inactivation of pedv in feed. feed samples were spiked with pedv and then heated to 120–145°c for up to 30 min or irradiated at 0–50 kgy. another set of feed samples spiked with pedv and mixed with ultracid p (nutriad), activate da (novus international), kem-gest (kemin agrifood), acid booster (agri-nutrition), sugar or salt was incubated at room temperature (~25°c) for up to 21 days. at the end of incubation, the virus titers were determined by inoculation of vero-81 cells and the virus inactivation kinetics were modeled using the weibull distribution model. the weibull kinetic parameter delta represented the time or ebeam dose required to reduce virus concentration by 1 log. for thermal processing, delta values ranged from 16.52 min at 120°c to 1.30 min at 145°c. for ebeam processing, a target dose of 50 kgy reduced pedv concentration by 3 log. all additives tested were effective in reducing the survival of pedv when compared with the control sample (delta = 17.23 days). activate da (0.81) and kem-gest (3.28) produced the fastest inactivation. in conclusion, heating swine feed at temperatures over 130°c or ebeam processing of feed with a dose over 50 kgy are effective processing steps to reduce pedv survival. additionally, the inclusion of selected additives can decrease pedv survivability. porcine epidemic diarrhea virus (pedv) is a pleomorphic, enveloped rna virus, classified as a coronavirus under the family coronaviridae [1] . the virus was first identified during an outbreak of diarrhea on a belgian swine breeding farm in 1978 [2] . upon infection with pedv, suckling pigs experience diarrhea, vomiting, and high mortality [3] . since the initial identification of pedv, it has been reported in canada, korea, china, thailand, italy, hungary, and vietnam [4] . it is important to note that china and vietnam are two of the top swine producing countries in asia and that they were both impacted by pedv [5] . in the united states, the virus was first detected in april 2013 and has since caused high piglet mortality in over 17 states [6] . some have suggested that pedv is transmitted via contaminated feed [7] . although research has been conducted to show the survival of pedv in feed and feed ingredients [8] [9] [10] there is limited data comparing feed processing treatments and their efficacy in inactivating the virus in complete swine feed. in the feed and ingredient industry, temperature and time conditions vary with the processing method used e.g., pelleting, rendering, pasteurization, or spray drying. moisture content of feed and ingredients is also impacted by the type of processing method used. the interactions between temperature, time, and moisture play a role in virus inactivation in feed. ingredients of porcine origin have been thought to have the greatest risk for disease transmission [9] . when processing rendered ingredients for animal feed, the processing conditions vary based on the composition of the raw material. in general, the national renderers association suggests temperatures between 115°c and 145°c for 40 to 90 min for most rendering systems. such temperatures are deemed effective in inactivating pathogens such as salmonella spp. [11] . similarly, the enveloped classical swine fever rna virus is readily inactivated in pasteurization processes after only 1 min at 71°c [12] . high temperatures (around 100°c) are also used in other processes including micronization to prepare cereal grains [13, 14] . the high temperatures used in processing rendered products and cereal grains may also be effective in inactivating pedv in contaminated feed, but no research has been conducted to compare pedv survival at temperatures greater than 80°c. other processing procedures such as ionizing irradiation have been shown to reduce the survivability of pathogens in feed, specifically listeria monocytogenes in poultry feed [15] . more recent applications of ionizing radiation technology such as electron beam (ebeam) are now used to routinely pasteurize foods and decontaminate animal feeds from pathogens [16, 17] . the main characteristics of ebeam technology include its non-thermal nature (thereby reducing potential nutrient losses), utilization of commercial electricity (does not rely on radioactive isotopes), high speed processing, and the ability to precisely control the dose delivered [17, 18] . the us food and drug administration (fda) has recognized this process as a treatment method for food and animal feed for doses up to 50 kgy [19] . all of these attributes make the ebeam technology attractive for a wide variety of applications, including pasteurization of animal feed. in addition to thermal and ebeam treatments, certain organic acid feed additives including propionic, formic, and butyric acid have been used in the past for their antimicrobial properties. these additives have previously been used as a method of controlling pathogens such as salmonella spp. and escherichia coli in poultry feed and other matrices [20, 21] . however, the effects of organic acids and other additives including sugar and salt in feed have not been investigated. therefore, the objective of this study was to determine if thermal and non-thermal methods of microbial inactivation, as well as the use of selected feed additives, are effective in reducing the survival of pedv in experimentally contaminated swine feed. the nvsl (national veterinary service laboratory, ames, ia) strain of pedv was propagated and titrated in vero-81 (african green monkey kidney, atcc 1 ccl-81™) cells. we have found that both vero-76 and vero-81 cells are susceptible to pedv. we chose to use vero-81 in this study. the cells were grown in dulbecco's modified eagle medium (dmem; mediatech, herndon, va, usa) containing 8% fetal bovine serum (fbs; gibco, life technologies, grand island, ny, usa), 50 μg/ml gentamicin (mediatech), 150 μg/ml neomycin sulfate (sigma, st. louis, mo, usa), 1.5 μg/ml fungizone (sigma), and 455 μg/ml streptomycin (sigma). the cells were washed three times with phosphate buffered saline (pbs; ph 7.2). after virus inoculation, the cells were incubated at 37°c for 1 h, allowing virus adsorption using maintenance medium (dmem with gentamicin, neomycin sulfate, fungizone, streptomycin and 10 μg/ml trypsin). the cells were washed again 60 minutes after inoculation. the washing medium was hanks' balanced salt solution containing no trypsin. however, the maintenance medium did contain trypsin. inoculated cells were incubated at 37°c under 5% co 2 . cells were examined daily under an inverted microscope for the appearance of virus-induced cytopathic effects (cpe) for up to five days post-infection. the virus was harvested by subjecting the infected cells to three freeze-thaw cycles (-80°c/25°c) followed by centrifugation at 2500 × g for 15 min at 4°c. the supernatant was collected and aliquoted into 50 ml tubes followed by storage at -80°c until used. in all experiments, the surviving virus after a certain treatment was recovered in an eluent consisting of a 3% solution of beef extract solution (lab scientific, highlands, nj) in 0.05m glycine (sigma, st. louis, mo), ph 7.5. following elution, the eluate was centrifuged for 10 min at 2500 x g to remove organic matter/debris. the supernatants were used to determine the amount of surviving virus, if any. for titration of pedv in virus stock and other samples, serial ten-fold dilutions of the eluates prepared in dmem (maintenance medium) were inoculated into vero-81 monolayers contained in 96-well microtiter plates (nunc, ny, usa) using 100 μl/well and three wells per dilution. inoculated cells were incubated at 37°c under 5% co 2 for up to seven days until the cpe appeared. the highest dilution showing cpe was considered the end point. virus titers were calculated as a median tissue culture infectious dose (tcid 50 /ml) by the karber method [22] . the amount of surviving virus was compared with the starting virus titer to calculate the amount of inactivated virus and was expressed in log scale (log 10 tcid 50 /ml). a complete phase ii pig starter feed (cgi, enhanced np-nt, batch no. 831458) was obtained and confirmed to be pedv negative by rt-pcr. the proximate analysis of this feed sample is displayed in table 1 . aliquots of feed (5 g amounts) were prepared in glass beakers, which were the survival of porcine epidemic diarrhea virus (pedv) in complete feed then placed into drying ovens set at 120, 130, 140, and 145°c. the feed aliquots remained in the ovens for 30 min to reach oven temperature. once the feed reached the indicated temperature, the beakers were immediately removed and spiked with 1 ml of pedv (6.8x10 3 tcid 50 / ml) that was previously tempered at room temperature (~25°c). after mixing, beakers were placed back in the ovens at the appropriate temperatures and incubated for 0, 5, 10, 15, 20, 25, and 30 min. after the incubation period, the samples were removed from the oven, actively cooled with a fan for 15 min, the virus eluted from the feed using the 3% beef extract eluent solution, and the number of surviving viral particles titrated. triplicate samples of each temperature were combined for a single titration and inoculation into cells. the experiment was performed in duplicate. preliminary dose mapping experiments were performed to calculate the dose uniformity within the samples. a dose-uniformity ratio was calculated by maximum dose/minimum dose for each experiment. under ideal circumstances, the dose-uniformity ratio (dur) should be about 1.0. during commercial processing of animal feed using ebeam technology, the size of the final package needs to be optimized to assure dose uniformity. the optimization involves adjusting the dimensions and weight to ensure adequate penetration of the ebeam electrons as well as dose uniformity within the package. under commercial processing conditions, the goal is to try to attain as uniform a dose as possible. at the ebeam facility on the tamu campus, the dur of a commercial pet food product is 1.78. aliquots of complete phase ii pig starter feed (5 g amounts) were prepared in several 50 ml plastic centrifuge tubes followed by the addition of 1 ml of pedv to each tube. after mixing, the virus-spiked feed was placed into individual plastic bags. the bags were flattened to remove all of the air and create a thin, even layer of the feed sample. the bags were triple-sealed to meet the biosafety procedures at the ebeam irradiation facility. the sealed bags were shipped on ice via overnight shipping to the national center for electron beam research at texas a&m university, college station, tx. preliminary dose delivery trials were performed to determine the appropriate conveyor speed and other specifications to achieve the target doses. on the day of arrival, the samples were exposed to target ebeam doses of 10, 20, 30, and 50 kgy. a control sample was also shipped with the irradiated samples, but it was not exposed to ebeam irradiation at the facility. after treatment, the samples (including the controls) were shipped back to the university of minnesota (st. paul) on ice via overnight shipping. upon arrival, the samples were immediately eluted using the aforesaid eluent solution. this experiment was performed only once. aliquots of feed in 5 g amounts were placed in plastic scintillation vials. the amount of feed additive used in the 5 g sample was determined based on the recommended dosage from the manufacturers. the following additives were added to separate aliquots of feed: 0.015 g ultracid p (nutriad, dendermonde, belgium), 0.02 g activate da (novus international, st. charles, mo), 0.01 g of acid booster (agri-nutrition, deforest, wi), 0.01 g kem-gest (kemin agrifoods, des moines, ia), 0.02 g of granulated sugar (shoppers value, eden prairie, mn) and 0.02 g of commercial salt (essential everyday, eden prairie, mn). another set of vials was used as paired controls without the addition of any feed additive. after the additives were added to the appropriate vials, 1 ml of pedv was added to each vial followed by vortexing. all vials were incubated at room temperature (around 25°c during the season which this experiment was performed). after incubation for 0, 1, 3, 5, 7, 14, and 21 days, the surviving virus was eluted from the samples using the 3% beef extract eluent solution. the experiment was performed in duplicate. each experiment used a set of triplicate samples that were combined and used for a single titration and inoculation into cells. after completion of the trial, the ph of each feed sample solution with and without the additive was measured. the ph was measured by adding 50 ml of deionized water to 5 g of feed followed by mixing continuously on a magnetic stirrer for 15 min at room temperature. after mixing, a ph probe was used to measure the ph of the liquid. the ph measurement experiment was performed in triplicate. in addition to measuring ph, the active ingredients for activate da (2-hydroxy-4-methylthiobutanoic acid), kem-gest (phosphoric, fumaric, lactic, and citric acids), sugar (sucrose), acid booster (phosphoric, citric, and lactic acids), salt (sodium chloride), and ultracid p (orthophosphoric, citric, fumaric, and malic acids) were compared. inactivation kinetic data (log tcid 50 /ml) were analyzed by ginafit, a freeware add-in for microsoft excel developed by geeraerd et al. [23] . the traditional log-linear model that assumes a linear relationship between the virus concentration and processing time was developed by bigelow and esty [24] and used to characterize the survival curves of pedv by using the following equation: where n is the surviving virus titer after the treatment (expressed as tcid 50 /ml), n 0 is the initial virus titer (tcid 50 /ml), k is the kinetic parameter (min -1 or day -1 ), and t is the treatment time (min or days). the kinetic parameter k is usually expressed as d, also known as the decimal reduction time (time required at a certain temperature to reduce 90% or 1 log of the initial virus titer) and was calculated as follows: the weibull distribution function has been used to describe non-linear inactivation patterns of various microorganisms after different thermal and non-thermal processing [25] . assuming that the temperature resistance of the virus is governed by a weibull distribution, mafart et al. developed the following weibullian equation: where n is the surviving virus after the treatment expressed as (tcid 50 /ml), n 0 is the initial virus titer (tcid 50 /ml), δ is the time of the first logarithm decline for the virus titer population (min or days), and n is the shape parameter. the n value provided a general description of the form of the curve; if n > 1, the curve is convex (it forms shoulders), if n < 1, the curve is concave (it forms tails), and if n = 1, the curve is a straight line and can be described by a linear model. two valid replicates were used to evaluate how well the model fit with the experimental data by calculating the adjusted r 2 (adj. r 2 ) as follows: where m is the number of observations, j is the number of model parameters, and ssq is the sum of squares. an anova test using the mixed procedure of sas (sas inst., cary, nc) was used to determine statistical differences across treatments. least squared means with tukey adjustment was used to determine differences between treatment means, with p < 0.05 considered to be significantly different. the experimental unit was the value obtained from the combined triplicate vials. the fixed effects were temperature or feed additive used. table 2 . the pedv showed a high thermal resistance in the dry feed samples and it was completely inactivated (3.0 log reduction) at each of the tested temperatures within 30 min. the original moisture content of the feed sample was 8.57% (w/v). after adding 1 ml of virus media the moisture increased to 23.8% (w/v). this change in moisture content potentially increased the ability of heat to reduce virus concentration, as heat inactivation is more efficient in higher moisture content as previously demonstrated by the higher sensitivity of bovine parvovirus to moist heat vs. dry heat [26] . the shape parameter (n) indicates the shape of the curve with a value n > 1 forming shoulders and being convex, n < 1 forming tails and being concave, and n = 1 being linear. the virus concentration in feed (control sample) and feed with additives is shown in table 3 . the addition of the additives to the feed sample decreased the initial virus titer. the d and delta values of the log-linear and weibull models respectively, are shown in table 4 . the use of the weibull model resulted in greater adj. r 2 values than the log-linear model, and therefore, was used to characterize the survival of pedv with the addition of additives during storage at room temperature. delta values for all the additive-containing samples, except ultracid p and salt, were significantly lower than the control sample (p < 0.05) indicating faster inactivation kinetics (table 4 ). after 21 days of incubation at room temperature (25°c), a 3 log reduction was observed in samples containing kem-gest, sugar, and salt. in the same 21-day incubation period, a 2 log reduction was observed in activate da, and <2 log reduction was observed in the control, ultracid p, and acid booster. the comparison of all feed additives in terms of active ingredient and ph value is shown in table 5 . in general, the additives containing phosphoric acid were more effective at reducing virus concentration. the only additive containing 2-hydroxy-4-methylthiobutanoic acid (activate da) was the most effective in reducing virus concentration. there were differences in the ph values of the feed samples after the addition of additives. feed was suggested as a vehicle of transmission of pedv when the first canadian swine farm tested positive for the virus [9] . a follow-up epidemiological investigation suggested that a the shape parameter (n) indicates the shape of the curve with a value n > 1 forming shoulders and being convex, n < 1 forming tails and being concave, and n = 1 being linear. doi:10.1371/journal.pone.0158128.t004 batch of spray-dried porcine plasma (sdpp) used in swine nursery feed was the potential source of pedv infection. in a bioassay experiment, a contaminated sdpp sample was able to infect pigs, however, when added to the complete feed no additional pigs were infected. this was potentially caused by a dilution effect or an extended time period between virus contamination and bioassay [9] . a similar conclusion that feed or feed ingredients could be spreading pedv between farms was made during an investigation of a pedv outbreak in ohio. in this situation, virus was detected in the feed through rt-pcr on a farm with infected sows. in a follow up bioassay, none of the pigs developed an infection after consuming the contaminated feed [7] . in this study, the source of feed contamination was not found, although the rt-pcr test was positive for pedv. dee et al. [10] showed that feed experimentally contaminated with pedv rna could cause an active infection in pigs when feed was consumed via natural feeding behavior. the collection of references indicates that if complete feed happens to be contaminated with pedv, it is possible for the contaminated feed to cause an active infection on a farm. in summary, these results suggested that additional research is needed to evaluate potential mitigation strategies to control transmission of pedv through feed processing methods. the survival of pedv in feed that was thermally treated at 120 to 145°c for 0 to 30 minutes was first evaluated. previous research has focused on the survival of pedv at temperatures below 80°c. one study found that a cell culture-adapted strain of pedv was moderately stable at 50°c for 30 min, with only a reduction of 0.4 log 10 pfu/ml at 50°c when compared to the control [1] . a further investigation indicated that treating pedv at temperatures between 60 to 80°c for 30 min caused a complete loss of infectivity, but the log reduction in virus concentration was not reported [1] . in another study, the survival of a wild type strain of pedv in fecal samples required only 10 min of exposure at 71°c to be inactivated to the extent that it was not able to cause infection in live pigs [27] . this variability in virus inactivation could be possibly due to differences in moisture content between the different samples tested. the moisture content will ultimately have an impact on virus survivability during heat treatment. although the exposure times and temperatures varied in these studies, their results correspond with previous data indicating that pedv is a low thermally stable virus [4] . our hypothesis was that the thermal treatment of feed would reduce the survival of pedv in swine feed. this hypothesis was confirmed by our experimental results which demonstrated that a 3 log (99.9%) reduction of the pedv concentration is achieved within 25 min of exposure at 120°c. these results also highlight the finding that temperatures over 130°c don't increase the inactivation of pedv. this is an important observation because excessive heating of high protein feed ingredients results in reductions of amino acid digestibility through maillard reactions [28] . in addition to the log reduction of pedv in feed at high temperatures, the production of a shoulder in the inactivation curve produced by the weibull model also help determine pedv survival at high temperatures. the observed shouldering phenomenon observed in the inactivation curves may indicate that virus is resistant to a certain temperature-time level combination and a threshold needs to be surpassed to see a significant level of inactivation. another possible reason for this shoulder in the curve could be the possibility that the feed was cooled when the room temperature inoculum was added. this would mean the first few min the feed was placed back in the oven was spent heating back up to oven temperature, which could explain the plateau in virus concentration. overall, the results from our experiment confirm that the concentration of pedv in complete feed can be reduced through the application of heat. when exploring non-thermal treatment processes to reduce pedv in feed, an ebeam irradiation dose of 50 kgy was found to be effective in reducing virus concentration by 3 log (99.9%). currently, the food and drug administration has stipulated an upper dose limit of 50 kgy of ionizing irradiation doses in animal feeds [19] . the use of ebeam processing of animal feeds and diets has been shown to be an effective process for inactivating pathogens in the us [16, 17] . animal feed and bags (weighing about 20 kg) are routinely treated using ebeam processing. process controls are in place to measure the maximum and minimum doses in these sample bags to meet customer needs and adhere to regulatory limits. though we have shown that ebeam treatment is effective in reducing the concentration of pedv in swine feed, followup studies are needed to verify the results seen in this study with different types of feed. our experimental data, suggesting that a target dose of 50 kgy will achieve approximately 3 log 10 reduction in pedv, are important in that it highlights the criticality of ensuring low viral bioburden during feed formulation by adhering to good manufacturing processes. these results suggest that if the initial pedv bioburden is greater than 4 log 10 tcid 50 , the fda mandated 50 kgy dose upper limit may not be effective in complete inactivation of virus in these feeds. furthermore, if the maximum titers of pedv in commercial swine feed are known, then the dose can be calibrated to theoretically achieve the total elimination of pedv in commercial swine feed. in addition to more research investigating maximum pedv titers, it may also be necessary to investigate the use of irradiation on individual feed ingredients. our experiment only measured the impact of irradiation on complete swine feed, and so it cannot be confirmed if the same results would be seen in individual feed ingredients. finally, we evaluated the survival of pedv in feed when various feed additives were included at manufacturers' recommended doses. the additives experiment was only performed at room temperature. on a swine farm, feed can be stored at a variety of temperatures based on season, barn layout, bagged feed vs. bulk feed, barn temperature, and other variables. room temperature was used in this experiment as a feasible average temperature, though it may not accurately apply to every situation. feed is suggested to be stored in a cool, dry place and so it is likely some swine feed will be stored at temperatures below the approximately 25°c tested in this experiment. the addition of organic acid feed additives in the diet has been shown to increase nutrient digestibility by lowering ph and modifying gut microflora, which ultimately improve growth performance and provide an alternative to antibiotic use in weaned pig diets [29] . organic acids have been used to reduce the prevalence of bacterial pathogens such as salmonella in poultry feed [21] . in an additional experiment, the antimicrobial product salcurb, which is used to control salmonella in feed, was tested for its ability to decrease the presence of viable pedv in feed. in this experiment, a bioassay showed that the feed treated with the salcurb product was not able to cause an infection in naïve pigs [30] . a proposed method for how the salcurb inactivated the pedv was not reported. differences in pedv survival in feed has not only been observed in the presence of additives, but also in the presence of different feed ingredients. in an experiment testing 18 different feed ingredients, it was determined that the survival of pedv in these ingredients was ingredient specific with an extended survival being observed in soybean meal [31] . this indicates that variation as small as changes in ingredients could change virus survival in feed. it has been reported that pedv is stable at a ph of 6.5 to 7.5 at a temperature of 37°c [32] . because intestinal viruses must survive the acidic conditions of the stomach, they are generally more resistant to acidic ph. with this being said a change in ph outside of ideal conditions for an extended period could cause an increase in virus inactivation. a previous review reported that the ph of the diet was decreased from an average ph of 5.96 to 4.71 when organic acids (fumaric, propionic, formic, citric, sodium citrate, hydrochloric, sodium fumaric, or malic acid) were added to feed [33] . this ph decrease of the diet was hypothesized to decrease the survival of pedv in the presence of additives. the results from our study did not necessarily support this hypothesis. activate da and kem-gest were the most effective in reducing virus survivability and also had the most acidic ph values when added to feed, which confirm the original hypothesis. however, ultracid p did not result in a different diet ph compared with kem-gest, yet it had the highest delta value of all additives evaluated. in addition to this, the feed sample would require a type of liquid to be added to the feed for ph to even play a role, as a dry substrate will not have a ph. in the experiment, 1ml of virus media was added to the sample and mixed. this means it could be possible for the ph of the media to change because of the additive when the virus was initially added. if this single change were to explain the differences in survival for our experiment, there would be a more prominent pattern between the ph of the feed solution and the delta value observed. with a lack of a clear pattern between ph and virus survival, and in some cases no difference between the ph of the solution with the control and with some of the additives tested, there is another aspect causing the changes in virus survival between the feed additives used. there are other components of feed additives that have the capability to reduce pedv survivability, as suggested by the inability to explain the decrease in pedv survivability due to change in ph alone. we hypothesized that specific active ingredients play a role in differences in virus survivability among additives. many of the less effective additives contained citric acid and lactic acid as active ingredients. it has been previously shown that citric and lactic acids only have moderate antimicrobial activity because they are solely effective at a low ph in the environment [34] . because most swine feed samples have a neutral ph, it is not surprising that additives containing citric acid as an active ingredient may not have been as effective in inactivating pedv. in contrast, fumaric acid has been shown to be a very effective antimicrobial, and is extremely effective in reducing the survivability of e. coli [35] . no studies have been published to show that fumaric acid is an effective anti-viral compound, but its presence in kem-gest, which resulted in the second lowest pedv survivability in our study, implies it may also be an effective ingredient in reducing survivability of pedv. the active ingredient of activate da, the most effective additive in our study, is 2-hydroxy-4-methylthiobutanoic acid. this ingredient has been researched exclusively as a cost effective supplement in low methionine diets for swine [36] . although it has not been approved for use as an antimicrobial or antiviral agent, the results from our study suggest it could have potential in reducing virus survivability. overall, careful selection of additives containing the most effective active ingredients is necessary for achieving reduced pedv survivability in complete swine feeds. further research is needed to confirm the relative effectiveness of each active ingredient and how they may interact with each other. one limitation in this experiment is the unknown variable of water activity. by adding liquid media to the samples the moisture content was already dramatically increased. this increase in moisture potentially caused changes in the inactivation of the virus when exposed to heat that may not be observed in the low moisture conditions of animal feed. it has been demonstrated that pedv inactivation using heat treatments and changes in ph differs in liquid media compared with a dried plasma product [37] . because our experiments investigated both change in ph through an additive and change in heat treatment, our increased moisture from inoculating the sample could cause different inactivation kinetics than what would be seen in dry feed without media. in addition to this, the humidity in the environment was not controlled and the water activity of the samples were not measured. without measuring these variable there is no way to tell the role they played in the inactivation of pedv in the feed samples. in addition to unknown water activity, this experiment also had the limitation of low starting virus titers. this made it impossible to reach the standard of complete inactivation (typically 7 log reduction) because the starting virus titers were only 4 logs. our results show that pedv survivability can be reduced using ebeam processing or thermal processing at temperatures greater than 130°c. the use of some feed additives was also effective in reducing virus levels, but future investigations are needed to determine the prevalence and maximum titers of pedv in commercial swine feeds to determine the most appropriate treatment parameters (time and temperature of thermal processing, dose of ebeam processing, and feed additive selected) to be most effective in inactivating pedv. feed has been suggested as a possible vehicle of pedv transmission. this has created a need to evaluate new and existing feed processing methods to reduce pedv survivability in feed. results from this study have shown that both thermal (130°c for at least 15 min) and non-thermal (50 kgy ebeam dose) feed processing technologies can eliminate 99.9% of pedv infectivity when working with low virus titers (6.8x10 3 tcid 50 /ml). additionally, the survivability of pedv can be reduced by the use of selected acidifiers and organic acids in swine feeds. quantitation, biological and physicochemical properties of cell culture-adapted porcine epidemic diarrhea coronavirus (pedv) a new coronavirus-like particle associated with diarrhea in swine emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences porcine epidemic diarrhea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines pig production in subtropical agriculture origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states investigating the introduction of porcine epidemic diarrhea virus into an ohio swine operation risk assessment of feed ingredients of porcine origin as vehicles for transmission of porcine epidemic diarrhea virus (pedv) investigation into the role of potentially contaminated feed as a source of the first-detected outbreaks of porcine epidemic diarrhea in canada an evaluation of contaminated complete feed as a vehicle for porcine epidemic diarrhea virus infection of naive pigs following consumption via natural feeding behavior: proof of concept real and perceived issues involving animal proteins survival and inactivation of classical swine fever virus performance and carcass characteristics of growing pigs and broilers fed diets containing micronised barley, ground barley, wheat and maize effects of micronization on the digestibility of whole soya beans and rapeseeds for the growing pig radiation sensitivity of listeria monocytogenes in phosphate buffer, trypticase soy broth, and poultry feed applications of ionizing irradiation for phytosanitary treatment and food safety for fresh produce. in: global safety of fresh produce: a handbook of best-practice examples, innovative commercial solutions and case studies electron beam pasteurization and complimentary food processing methods sterilization methods and the comparison of e-beam sterilization with gamma radiation sterilization food and drug administration. 21 c.f.r. 579 feed additives to control salmonella in poultry the use of organic acids to combat salmonella in poultry: a mechanistic explanation of the efficacy fifty percent endpoint calculation ginafit, a freeware tool to assess non-log-linear microbial survivor curves the thermal death point in relation to typical thermophylic organisms on calculating sterility in thermal preservation methods : application of the weibull frequency distribution model further studies on thermal resistance of bovine parvovirus against moist and dry heat methods for inactivating pedv in hog trailers. in: twenty-second annual swine disease conference for swine practitioners amino acid digestibility of heat damaged distillers dried grains with solubles fed to pigs acidifier as an alternative material to antibiotics in animal feed an evaluation of a liquid antimicrobial (sal curb 1 ) for reducing the risk of porcine epidemic diarrhea virus infection of naïve pigs during consumption of contaminated feed an evaluation of porcine epidemic diarrhea virus survival in individual feed ingredients in the presence or absence of a liquid antimicrobial diseases of swine critical review of acidifiers microbial ecology of foods addition of fumaric acid and sodium benzoate as an alternative method to achieve a 5-log reduction of escherichia coli o157:h7 populations in apple cider the relative effectiveness of 2-hydroxy-4-(methylthio) butanoic acid and dl-methionine in young swine 1 sensitivity of porcine epidemic diarrhea virus (pedv) to ph and heat treatment in the presence or absence of porcine plasma we thank mickey speakmon for his assistance with the ebeam irradiation experiment. key: cord-346586-fxxceffl authors: razanajatovo, norosoa harline; guillebaud, julia; harimanana, aina; rajatonirina, soatiana; ratsima, elisoa hariniaina; andrianirina, zo zafitsara; rakotoariniaina, hervé; andriatahina, todisoa; orelle, arnaud; ratovoson, rila; irinantenaina, judickaelle; rakotonanahary, dina arinalina; ramparany, lovasoa; randrianirina, frédérique; richard, vincent; heraud, jean-michel title: epidemiology of severe acute respiratory infections from hospital-based surveillance in madagascar, november 2010 to july 2013 date: 2018-11-21 journal: plos one doi: 10.1371/journal.pone.0205124 sha: doc_id: 346586 cord_uid: fxxceffl background: few comprehensive data exist regarding the epidemiology of severe acute respiratory infections (sari) in low income countries. this study aimed at identifying etiologies and describing clinical features of sari-associated hospitalization in madagascar. methods: it is a prospective surveillance of sari in 2 hospitals for 3 years. nasopharyngeal swabs, sputum, and blood were collected from sari patients enrolled and tested for viruses and bacteria. epidemiological and clinical information were obtained from case report forms. results: overall, 876 patients were enrolled in the study, of which 83.1% (728/876) were tested positive for at least one pathogen. viral and bacterial infections occurred in 76.1% (667/876) and 35.8% (314/876) of tested samples, respectively. among all detected viruses, respiratory syncytial virus (rsv) was the most common (37.7%; 348/924) followed by influenza virus a (flua, 18.4%; 170/924), rhinovirus (rv, 13.5%; 125/924), and adenovirus (adv, 8.3%; 77/924). among bacteria, streptococcus pneumoniae (s. pneumoniae, 50.3%, 189/370) was the most detected followed by haemophilus influenzae type b (hib, 21.4%; 79/370), and klebsiella (4.6%; 17/370). other streptococcus species were found in 8.1% (30/370) of samples. compared to patients aged less than 5 years, older age groups were significantly less infected with rsv. on the other hand, patients aged more than 64 years (or = 3.66) were at higher risk to be infected with flua, while those aged 15–29 years (or = 3.22) and 30–64 years (or = 2.39) were more likely to be infected with flub (influenza virus b). conclusion: the frequency of influenza viruses detected among sari patients aged 65 years and more highlights the need for health authorities to develop strategies to reduce morbidity amongst at-risk population through vaccine recommendation. amongst young children, the demonstrated burden of rsv should guide clinicians for a better case management of children. these findings reveal the need to develop point-of-care tests to avoid overuse of antibiotics and to promote vaccine that could reduce drastically the rsv hospitalizations. samples. compared to patients aged less than 5 years, older age groups were significantly less infected with rsv. on the other hand, patients aged more than 64 years (or = 3.66) were at higher risk to be infected with flua, while those aged 15-29 years (or = 3.22) and 30-64 years (or = 2.39) were more likely to be infected with flub (influenza virus b). the frequency of influenza viruses detected among sari patients aged 65 years and more highlights the need for health authorities to develop strategies to reduce morbidity amongst at-risk population through vaccine recommendation. amongst young children, the demonstrated burden of rsv should guide clinicians for a better case management of children. these findings reveal the need to develop point-of-care tests to avoid overuse of antibiotics and to promote vaccine that could reduce drastically the rsv hospitalizations. severe acute respiratory infections (sari) are among the leading cause of hospitalization and deaths worldwide [1] . it is estimated that about 4.2 million of deaths are attributed to sari annually [2] . estimation in 2010 have shown that 11 million of children aged less than 5 years were admitted for sari in developing countries, with an estimated case fatality ratio of 2.3%, compared to 570 000 cases and a case fatality ratio of 0.6% in developed countries [3] . in low income countries, the annual incidence of pneumonia is estimated at 151 million children of whom 13% are severe enough to warrant hospitalization [1, 4] . nearly 2 million children die from pneumonia every year, 70% of which occur in africa and southeast asia [5, 6] . sari may be caused by various pathogens. while bacterial infections play a critical role in causing life-threatening pneumonia [1, [7] [8] [9] , viral infections are associated with significant proportion that range from mild to severe infections [10, 11] . however, due to the lack of gold standard methods to rapidly differentiate between viral and bacterial infections, most of the patients may be treated empirically with antibiotics [12] . rapid etiologic diagnosis is therefore needed to select the most appropriate treatment protocol and to avoid the development of bacterial resistance. following the a/h1n1/2009 influenza pandemic that was associated with a high morbidity and an increased risk of mortality among particular groups [13] , a number of countries have strengthened vigilance for the surveillance of severe diseases and deaths in order to rapidly detect new viruses and to provide information in assessing the impact on the population and having operational preparedness plans. so far, only few sub-saharan african countries are collecting data on hospitalization associated to acute respiratory illness [14] [15] [16] . in madagascar, the sentinel syndromic surveillance has been collecting sari data since 2010. however, hospitals only collect few data that are not exploitable for analysis. thus, data are still scarce regarding the epidemiology of sari in this country. during the last influenza pandemic in 2009, an excess of mortality was observed among inhabitant of the main capital city of antananarivo [17] . following the pandemic, health authorities requested to develop an active surveillance of sari in order to better understand the epidemiology of this disease in the context of madagascar. the present study aimed to identify etiologies of sari and to assess clinical features of hospitalization linked to sari in 2 hospitals during 3 years of surveillance. a prospective hospital-based sari surveillance was conducted from november 2010 to july 2013 in 2 selected sites: the soavinandriana hospital of antananarivo and the secondary level public hospital (chd ii) of moramanga. the cenhosoa is a national referral hospital and is among the 4 largest hospitals that deserve antananarivo, the main capital city of madagascar which has around 2.5 million inhabitants. the chd ii in moramanga is the only local referral hospital for the health district of moramanga that has 250 000 inhabitants. it is located 115 km east from the capital city. antananarivo and moramanga present the same climatic profile: hot and rainy in summer and cold and dry in winter, but moramanga encompasses both semiurban and rural areas. all patients presenting sari symptoms at admission were enrolled. sari case definition was defined according to the who case definition as fever (t�38˚c) or history of fever and cough that required hospitalization. for children less than 5 years, the eligibility criteria were suspected sepsis or sari diagnosed by physician including bronchiolitis, pneumonia, bronchitis, pleural effusion, and cough or difficult breathing as previously published [18] . the onset of illness should be less than 7 days before hospitalization. for each consented patient, demographic, socio-economic, clinical, and epidemiological data were recorded in case report forms (crfs). nasopharyngeal, blood, and sputum specimens were collected for each patient enrolled from monday to friday and shipped the same day to laboratories located at the pasteur institute of madagascar where they were immediately processed or stored at 4˚c until testing (test were performed within 48 hours post-sampling). all procedures for the biological analyses have been previously described [18, 19] . briefly, nasopharyngeal swabs were screened for 14 respiratory viruses using in-house multiplex real-time pcr [19] . sputum was collected for cytobacteriologic testing while blood samples were used for cell blood count. single infection was defined as an infection caused by one pathogen (virus or bacteria) and multiple infection as an infection caused by at least 2 pathogens (virus/virus, virus/bacteria or bacteria/bacteria) in a single sample. patients were divided into 5 age groups: infants and young children (� 5 years old); children (5-14 years old); adolescent and young adults (15-29 years old); adults , and elderly (� 65 years old). demographic, clinical characteristics, and etiologies were compared by study sites and by age groups. univariate analysis and logistic regression were performed using r software. in univariate analysis, qualitative variables were compared with fisher's exact test and quantitative variables by non-parametric tests (wilcoxon test). furthermore, logistic regressions were performed to adjust odds-ratio (or) found via maximum-likelihood estimation according to age group for the different variables as dependent variables and to compare each of them according to age group by wald test. the age group less than 5 years has been considered as reference group. statistical differences were considered significant for two-sided p-values < = 0.05. although sari surveillance in madagascar is not considered as research, this prospective study requested extra sampling and the collection of personal information. for that reason, the present study was submitted and approved by the national ethics committee from the ministry of health in madagascar (authorization n˚068-msanp/ce). adult participants or parents were fully informed of the study's objectives and procedures. written informed consent was obtained from patients enrolled in the study. young participants aged from 7 to 18 years old were asked to sign an informed assent form if they were willing to take part in the study. for all patients aged less than 18 years, consent was obtained from the parents or legal guardians. after obtaining the consent/assent form, the team survey proceeded to collect samples and fill out the crfs. refused consent and prior hospitalization within 2 weeks before consultation were reasons for exclusion. from november 2010 to july 2013, 876 hospitalized patients completed the established sari case definition of whom 657 cases (75.0%) were from cenhosoa and 219 cases (25.0%) from chd ii moramanga. the mean age was 8.2 years ranging from 1 day to 89 years and 54.8% of patients were male cases. the majority of recruited patients were children aged less than 5 years and accounting for 81% of total inclusion. adult aged more than 65 years represented 2% of cases ( table 1) . differences of mean age (2.8 years vs. 9.9 years; p<0.001; wilcoxon test) and age repartition (p<0.001) of included patients were observed between the 2 study sites with patients hospitalized in chd ii moramanga being younger than those hospitalized in cenhosoa (table 1) . with exception of dyspnea and fever which are among the principal inclusion criteria, runny nose (69.7%), intercostal recessions (69.9%), productive cough (53.0%), movement of nose wings (52.6%), and anorexia (50.3%) were the most recorded at the time of examination. bronchiolitis (44.5%) was the most clinical diagnostic made by clinicians at admission followed by bronchopneumonia (15.0%) and pneumonia (8.8%). other low respiratory tract infections (bronchoalveolitis/exacerbation of chronic obstructive pulmonary disease (copd), pleuropneumonia, and acute lobar pneumonia) were observed in 14.8% of inpatients (s1 table) . comparison of clinical symptoms between the 2 sites showed that dry cough (p = 0.026), asthenia (p = 0.001), runny nose (p<0.001), and anorexia (p = 0.001) were statistically frequently observed in patients hospitalized in chdii moramanga whereas productive cough (p = 0.026), sore throat (p<0.001), headache (p = 0.014), thrill (p = 0.001), and cyanosis (p<0.001) were significantly associated with patients hospitalized in cenhosoa (table 1) . at least one pathogen was found in 83.1% (728/876) of tested patients of which 38.8% (340/ 876) were single infection and 44.3% (388/876) were multiple infections. the highest rate of infection was obtained in patients aged less than 5 years (85.2%; 605/710). for this age group, single and multiple infections were reported in 79.1% (269/340) and 86.6% (336/388) of cases, respectively. the type of infection (mono-infection vs. multiple infection) differed significantly between age groups (p = 0.03, fisher's exact test). indeed, patients aged 30-64 years were significantly less likely at risk for being co-infected (or = 0.4; p = 0.003) compared to those aged less than 5 years (s2 table) . overall, viral and bacterial infections occurred in 76.1% (667/876) and 35 comparison of the prevalence of pathogens by study sites revealed that flua (p = 0.04), s. pneumoniae (p = 0.003), and hib (p = 0.04) were statistically relatively common in patients hospitalized in cenhosoa (s3 table) . in respect to multiple infections, flua (or = 4. ) . surprisingly, 118 patients were co-infected with 4, 5, and 6 different pathogens (92, 20, and 6, respectively) ( table 3) . comparison of demographic and clinical characteristics of sari patients by age group. analysis of data by the age group showed that the frequency of clinical symptoms significantly varied across groups. indeed, by multivariate analysis adjusted by age, patients aged �5 years appeared to suffer chest pain compared to patients aged <5 years while dry cough and gpd were more often observed in the elderly. dyspnea and cyanosis were significantly more common in adults aged 30-64 years. headache, thrill, sweats, anorexia, weight loss, and asthenia were more often observed in the older age groups (15-29 years, 30-64 years and �65 years). sore throat was frequently reported in the age groups 5-14 years, 15-29 years and 30-64 years. runny nose and intercostal recession appeared to be relatively infrequent in the older age groups (15-29 years, 30-64 years and �65 years) ( table 4 and s4 table) . regarding infection, prevalence of rsv (p<0.001) and influenza viruses (p<0.001) substantially varied depending the age group. as expected, rsv accounted for the highest proportion of sari in young children (45.9%; 326/710). in fact, the older age groups were significantly less at risk to develop an rsv infection compared to the age group less than 5 years (table 5) 2) and between 30-64 years (or = 2.4) were more likely to catch flub infection. by multivariate analysis, the age group between 30-64 years were less likely to suffer from s. pneumoniae infection (or = 0.5) than the age group less than 5 years ( table 5) . pattern of circulation of respiratory pathogens. sari cases were detected all year around during the 3 years of surveillance with distinct peaks of detection observed each year: between january and march in 2011 and 2013 and between may and july in 2012 (fig 1) . these peaks coincided with an active circulation of rsv and influenza viruses. nevertheless, peaks of sari appear to be more correlated to rsv circulation. unlike other respiratory pathogens that are detected all year along, rsv seems to circulate once a year and being responsible of an epidemic every year. to the best of our knowledge, this study is the first description of etiologies associated to hospitalized sari patients from both urban and peri-urban areas in madagascar. during the 3 years of surveillance, 867 consented patients meeting the established case definition of sari were enrolled. the observed different clinical spectrum across age groups may be helpful to avoid misclassification of patients presenting with respiratory illness at the triage level when no standardized protocol is available. the finding that about 82% of inpatients were tested positive for a pathogen is consistent with those reported elsewhere which showed etiologies ranging from 40% to 85% of hospitalized sari cases depending on case definition or methodology design [14, 20, 21] . most of the included patients were children less than 5 years old (78.5%). this study confirms that young children are the most vulnerable group to suffer from sari [22, 23] . nevertheless, these results could also be explained by a socio-economic behavior of the malagasy population. indeed, parents are keener to seek care for their children while older individuals try to cure themselves at home using self-medication or traditional medicine. in the present study, about 43% of inpatients presented multiple infections by at least 2 pathogens. here, viral infections were commonly found in 75.4% of tested patients which is similar to what was observed within the community during the influenza-like illness surveillance [19] . taken together, the present study clearly demonstrates that respiratory viruses are the leading cause of severe acute respiratory illnesses in madagascar. overall, rsv was the most commonly detected followed by s. pneumoniae and influenza viruses. similar to other studies, substantial rate of rsv infection was observed in young population [14, 24, 25] . a meta-analysis of data from africa reported that the incidence of rsv in lower acute respiratory infections that required hospitalization ranged from 10-18 per 1000 person year for infants and 3-9 per 1000 person year for children under 5 years of age [26] . the increased circulation of rsv that coincided with the peak of sari cases may be informative for clinicians to predict an epidemic due to rsv and to choose the most appropriate care for patients in order to avoid overuse of antibiotics. in addition, the epidemic pattern of this virus reveals the need to develop rapid diagnostic tests to rapidly manage patients and reduce the number of hospitalization, and to promote effective vaccine to prevent severe infections in high-risk populations. the role of influenza viruses in sari-associated hospitalization is increasingly defined because of available data from active influenza sentinel surveillance system initiated in several countries including sub-saharan africa [27] [28] [29] [30] . the proportion of influenza viruses in young population obtained here (20.4%) is much higher than in other african countries were sari attributable to influenza are around 7% [16] . as seen elsewhere, the highest rate of hospitalization is obtained with the subtype a/h3n2 virus during its circulation [31, 32] . in madagascar despite decades of reliable influenza surveillance, no national policy is implemented regarding vaccination. moreover, influenza vaccines are not widely accessible and affordable for the whole population. in the present study, about 35.2% of sari cases were likely due to bacterial infection. nevertheless, it is not clear what this high proportion of bacteria means due to the notion of carriage. the substantial rate of s. pneumoniae detected might be partly explained by the very low pneumococcal vaccination rate among the younger population (estimated at 0.4%) because most of the positive cases were detected before the introduction of pneumococcal vaccine into the expanded program on immunization (epi) in madagascar in 2012 [33] . this vaccine is believed to reduce substantially the burden of pneumococcal disease. thus, it would be interesting to evaluate the proportion of sari associated to s. pneumoniae more than 5 years after the introduction of the vaccine in madagascar. on the other hand, the role of atypical bacteria in hospitalized ari is poorly understood. one assumes that certain bacteria predispose to bacterial super-infections [34] . while rv is considered as the common cold virus, the present study showed that this virus accounted for 14% of total sari cases. this finding highlights the potential role of rv in public health like other authors already postulated [35] [36] [37] . our study presents some limitations. first, data were collected only from 2 sites that are not representative of the whole population preventing us to real estimate the prevalence at the national level. indeed, prevalence of each pathogen may differ for regions having different bioclimatic or demographic patterns, access to healthcare, and connectivity. since children are more at risk to develop severe infection, our sampling was skewed toward the younger group and likely underestimate the prevalence in adult. indeed, few adult cases had been identified at the adult ward mainly because the duration of symptoms prior to admission exceeded the 7 days limit of our sari case definition. it was also observed that severe cases were treated on external consultation or admitted at the emergency department for a few hours and then discharged. patients that did not recover, worsened at home, and returned to the hospital were then admitted resulting in prolonged duration of illness (usually more than 7 days) at the time of admission. another limitation is to attribute the pathogen(s) that is (are) the main cause that lead to severe illness. indeed, we could not exclude that some pathogens may be present due to carriage. to conclude, the high prevalence of etiology associated to severe infections of relevant pathogens highlights the importance of sustaining national surveillance of sari to clearly estimate the role of associated pathogens and establish the burden of disease. further challenges in such program include efforts to implement strategies that capture adult cases as it is relevant to measure the impact of sari in this population. since vaccines play an important role in preventing sari, vaccination against important pathogens, including rsv, should be accessible at least for high-risk population in developing countries. indeed, although use of synthetic antibodies against rsv can be provided to certain at-risk infants, this treatment only provides a short-term protection, is not always effective, and is also expensive, putting it beyond the reach of developing countries like madagascar. supporting information s1 table. clinical diagnosis of patients hospitalized for sari, madagascar, november 2010 to july 2013. � lrti: low respiratory tract infection including bronchoalveolitis/exacerbation of chronic obstructive pulmonary disease (copd), pleuropneumonia, and acute lobar pneumonia; �� other: neonatal infection, asthma, laryngitis, influenza-like illness etc. . . n = number of included patients. n = number of patients that responded by "yes" or "no" for a given symptom. only bronchiolitis, pneumonia, and bronchopneumonia were statistically analyzed. statistical analyses were performed using fisher's exact test. (docx) s2 table. univariate analysis of the distribution of monoinfection and multiple infection detected in sari adjusted by age groups, november 2010 to july 2013. the age group less than 5 years was considered as reference group. (docx) s3 epidemiology and etiology of childhood pneumonia the global burden of disease: 2004 update. geneva: world health organization global and regional burden of hospital admissions for severe acute lower respiratory infections in young children in 2010: a systematic analysis global estimate of the incidence of clinical pneumonia among children under five years of age global, regional, and national causes of child mortality in 2008: a systematic analysis estimates of world-wide distribution of child deaths from acute respiratory infections burden of disease caused by haemophilus influenzae type b in children younger than 5 years: global estimates burden of disease caused by streptococcus pneumoniae in children younger than 5 years: global estimates community-acquired pneumonia in children childhood community-acquired pneumonia the association of newly identified respiratory viruses with lower respiratory tract infections in korean children study of community acquired pneumonia aetiology (scapa) in adults admitted to hospital: implications for management guidelines transmission characteristics of the 2009 h1n1 influenza pandemic: comparison of 8 southern hemisphere countries severe acute respiratory infection in children in a densely populated urban slum in kenya etiology and incidence of viral and bacterial acute respiratory illness among older children and adults in rural western kenya severe acute respiratory illness deaths in sub-saharan africa and the role of influenza: a case series from 8 excess mortality associated with the 2009 a(h1n1)v influenza pandemic in antananarivo outcome risk factors during respiratory infections in a paediatric ward in antananarivo viral etiology of influenza-like illnesses in antananarivo etiology of communityacquired pneumonia in 254 hospitalized children respiratory viral coinfections identified by a 10-plex real-time reverse-transcription polymerase chain reaction assay in patients hospitalized with severe acute respiratory illness-south africa viral co-infections in pediatric patients hospitalized with lower tract acute respiratory infections viral and atypical bacterial etiology of acute respiratory infections in children under 5 years old living in a rural tropical area of madagascar viral etiology of severe pneumonia among kenyan infants and children incidence of medically attended respiratory syncytial virus and influenza illnesses in children 6-59 months old during four seasons global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and metaanalysis viral etiology of severe acute respiratory infections in hospitalized children in cameroon sentinel surveillance system for early outbreak detection in madagascar unfinished business: severe acute respiratory infection in sub-saharan africa the national burden of influenza-associated severe acute respiratory illness hospitalization in rwanda the impact of influenza epidemics on mortality: introducing a severity index influenza-associated hospitalizations in the united states the burden of acute respiratory infections in crisis-affected populations: a systematic review epidemiology and clinical profile of pathogens responsible for the hospitalization of children in sousse area epidemiology of multiple respiratory viruses in childcare attendees we would like to express our gratitude to all clinicians and nurses involved in this study: emma rasoanirina, roland randriamirado, harimboahangy rakotoarison, lucien radozahana, rani razafindrakoto, domohina rakotovao, durandalle ny hanitrin'ny ala razana, dominique razafimandimby, françois de paul razanakoto, and saholiarisandy ndremihavana. we are deeply indebted to all the staff involved in the hospital surveillance program on a daily basis. we also would like to thank the laboratory technicians at the virology unit and the cbc for their tremendous work. the views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the ministry of health or of the us centers for disease control and prevention. key: cord-319706-2e9jrv0s authors: ebinger, joseph e.; achamallah, natalie; ji, hongwei; claggett, brian l.; sun, nancy; botting, patrick; nguyen, trevor-trung; luong, eric; kim, elizabeth h.; park, eunice; liu, yunxian; rosenberry, ryan; matusov, yuri; zhao, steven; pedraza, isabel; zaman, tanzira; thompson, michael; raedschelders, koen; berg, anders h.; grein, jonathan d.; noble, paul w.; chugh, sumeet s.; bairey merz, c. noel; marbán, eduardo; van eyk, jennifer e.; solomon, scott d.; albert, christine m.; chen, peter; cheng, susan title: pre-existing traits associated with covid-19 illness severity date: 2020-07-23 journal: plos one doi: 10.1371/journal.pone.0236240 sha: doc_id: 319706 cord_uid: 2e9jrv0s importance: certain individuals, when infected by sars-cov-2, tend to develop the more severe forms of covid-19 illness for reasons that remain unclear. objective: to determine the demographic and clinical characteristics associated with increased severity of covid-19 infection. design: retrospective observational study. we curated data from the electronic health record, and used multivariable logistic regression to examine the association of pre-existing traits with a covid-19 illness severity defined by level of required care: need for hospital admission, need for intensive care, and need for intubation. setting: a large, multihospital healthcare system in southern california. participants: all patients with confirmed covid-19 infection (n = 442). results: of all patients studied, 48% required hospitalization, 17% required intensive care, and 12% required intubation. in multivariable-adjusted analyses, patients requiring a higher levels of care were more likely to be older (or 1.5 per 10 years, p<0.001), male (or 2.0, p = 0.001), african american (or 2.1, p = 0.011), obese (or 2.0, p = 0.021), with diabetes mellitus (or 1.8, p = 0.037), and with a higher comorbidity index (or 1.8 per sd, p<0.001). several clinical associations were more pronounced in younger compared to older patients (p(interaction)<0.05). of all hospitalized patients, males required higher levels of care (or 2.5, p = 0.003) irrespective of age, race, or morbidity profile. conclusions and relevance: in our healthcare system, greater covid-19 illness severity is seen in patients who are older, male, african american, obese, with diabetes, and with greater overall comorbidity burden. certain comorbidities paradoxically augment risk to a greater extent in younger patients. in hospitalized patients, male sex is the main determinant of needing more intensive care. further investigation is needed to understand the mechanisms underlying these findings. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the severe acute respiratory syndrome coronavirus-2 (sars-cov-2) is now well recognized as the cause of the coronavirus disease 2019 (covid-19) global pandemic [1] [2] [3] . the rate of rise in covid-19 infection and its associated outcomes in the united states is now comparable to rates observed in other severely affected countries such as china, italy, and spain [4] [5] [6] [7] [8] [9] [10] . the spread of covid-19 in the united states has been especially pronounced in the states of california, new york, michigan, louisiana, and washington [11] . consistently reported across all regions is the observation that, of all individuals who become infected with sars-cov-2, a majority tend to have mild or no symptoms; however, an important minority will develop predominantly respiratory disease that can lead to critical illness and death [12] [13] [14] [15] . multiple, reports suggest that certain demographic and clinical characteristics may predispose infected persons to more severe manifestations of covid-19, such as older age, male sex, and pre-existing hypertension, pulmonary disease, or cardiovascular disease [4, [16] [17] [18] [19] . given that these traits tend to cluster among the same persons, the relative contribution of each trait to the risk for developing more severe presentations of covid-19 illness remains unclear. we conducted a comprehensive investigation of the pre-existing demographic and clinical correlates of covid-19 illness severity observed among patients evaluated for covid-19 within our multi-site healthcare system in los angeles, california. we deliberately focused our study on pre-existing characteristics for two main reasons: first, we recognize that patients with covid-19 illness can present early or late in the disease course, causing many clinical features to vary at the time of initial clinical encounter; and, second, we anticipate that ongoing public health efforts can be informed and augmented by understanding which predisposing factors may render certain segments of the population at higher risk for the most morbid sequelae of sars-cov-2 infection. the cedars-sinai health system is located in los angeles, california with a diverse catchment area of 1.8 million individuals, 33% of whom are over the age of 45 years and 80% identify as a racial or ethnic minority. the cedars-sinai health system includes cedars-sinai medical center (csmc), marina del rey hospital (mdrh), and affiliated clinics. for the current study, we included all patients who were found to have a laboratory confirmed diagnosis of sars-cov-2 infection while being evaluated or treated for signs or symptoms concerning for covid-19 at csmc or mdrh, beginning after the first confirmed case of community transmission was reported in the u.s. on february 26, 2020. subsequently, the first laboratory confirmed covid-19 case in our health system was on march 8, 2020. all laboratory testing for sars--cov-2 has been performed using reverse transcriptase polymerase chain reaction of extracted rna from nasopharyngeal swabs. all patient testing was performed by the los angeles department of public health until march 21, 2020, at which time the csmc department of pathology and laboratory medicine began using the a � star fortitude kit 2.0 covid-19 real-time rt-pcr test (accelerate technologies pte ltd, singapore). for the minority of patients in our study who had sars-cov-2 testing performed at an outside facility (3.6%), documentation of a positive test was carefully reviewed by our medical staff and considered comparable for accuracy. for all patients considered to have covid-19, based on direct or documented laboratory test result and suggestive signs and/or symptoms, we obtained information from the electronic health record (ehr) and verified data for the following demographic and clinical characteristics: age at the time of diagnosis; sex; race; ethnicity; smoking status defined as current versus prior, never, or unknown; comorbidities, including obesity, as clinically assessed and documented by a provider with icd-10 coding; and, chronic use of angiotensin converting enzyme (ace) inhibitor or angiotensin ii receptor blocker (arb) medications. chronic use of ace or arb medications was verified by confirming presence of documented ongoing medication use in an outpatient provider's clinic note along with presence of an active outpatient prescription for the medication, both dated from prior to covid-19 testing. we conducted iterative quality control and quality assurance analyses on all information extracted from the ehr; all data variables included in the main analyses were verified for completeness and accuracy through manual chart review, to avoid variable missingness or potential impact of inappropriate outliers in statistical modeling. because presenting clinical measures such as vital signs and laboratory values can be highly variable, based on timing of the original clinical presentation, we elected to focus on pre-existing traits that may predispose to covid-19 illness severity in a manner less dependent on the timing of patients presenting to medical care. to capture variation in relative comorbid status, in a way that is not captured by distinct medical history variables alone, we calculated the elixhauser comorbidity index (eci) with van walraven weighting for all patients based on all available clinical data [20] [21] [22] [23] . the eci uses 31 categories to quantify a patient's burden of comorbid conditions and has been shown to outperform other indices in predicting adverse outcomes (s1 table) [22] [23] [24] [25] [26] [27] [28] . for patients admitted to the hospital, length of stay, admission to an intensive care unit (icu) and death were ascertained from time stamps recorded for admission, unit transfers, and discharge. interventions such as intubation and prone positioning were identified through time stamped orders in the ehr and verified by manual chart review. dates and times of onset for reported or observed relevant signs and/or symptoms were also determined via manual chart review. all care was provided at the discretion of the treating physicians. our outcomes for this study included: severe illness (defined as requiring any kind of hospital admission), critical illness (defined as the need for intensive care during hospitalization), and respiratory failure (defined as the need for intubation and mechanical ventilation). the csmc institutional review board approved all protocols for the current study and waived the requirement for informed consent. for the total sample of covid-19 patients, we used parametric tests to compare normally distributed continuous variables and non-normally distributed or categorical variables, respectively. we also used histograms to display age and sex distribution for the total cohort, the patients admitted but not requiring intensive care, and patients requiring intensive care at any time during hospitalization, and the patient requiring intubation and mechanical ventilation at any time during hospitalization. we used ordinal logistic regression to examine the associations between pre-existing characteristics (based on clinically relevant, non-missing data) and a primary outcome measure of illness severity, defined as an illness severity score. we constructed the illness severity score, with higher values assigned to needing more intensive levels of clinical care, based on the following stepwise categories: 0 = clinically deemed to not require admission; 1 = required hospital admission but never required intensive care; 2 = required intensive level care but never intubation; and, 3 = required intubation during hospitalization. we constructed age-and sex-adjusted models, from which significantly associated covariates (based on p<0.20) were selected for inclusion in the final multivariable-adjusted models, where appropriate (i.e. smaller sample sizes). race was treated as a binary covariate: african american and non-african american. this approach was selected given the recently reported concerns of excess risk for african americans [29] , along with limited understanding of whether or not comorbidities contribute to this risk, in addition to the sample size for other race groups being too small for certain comparisons. because hypertension and diabetes are not calculated as substantial contributors to the elixhauser comorbidity index, we included each of these traits as separate additional covariates in all multivariable-adjusted analyses. we calculated the variance inflation factor (vif) for each of the predictor variables to confirm absence of any substantial multicollinearity. in secondary analyses, we analyzed the associations of pre-existing patient characteristics with the distinct outcomes of needing any hospital admission (severe illness) and, in the cohort of all hospitalized patients representing an especially vulnerable population, the need for intensive care (critical illness) or intubation (respiratory failure). all analyses were performed using r, version 3.5.1 (r foundation for statistical computing) and stata, version 15 (statacorp). for all final models, p values were 2-sided and considered significant at threshold level of 0.05. regional analyses showed that patients presented to our healthcare system from across a broad geographic catchment area in los angeles county (s1 fig) . the demographic and clinical characteristics of all patients in our study sample are shown in table 1 . of all patients with pharmacologically treated hypertension, a minority were taking ace inhibitor or arb class agents and a majority were taking anti-hypertensive medications from alternate classes. overall, almost half of patients (n = 214, 48%) were clinically assessed to require hospital admission, of whom over a third (n = 77; 36%) required intensive care and almost a quarter (24.3%) required intubation. in unadjusted analyses, the patients who were more likely to require higher levels of care tended to be older, male, african american, and with known hypertension, diabetes mellitus, higher elixhauser comorbidity index, and have prior myocardial infarction or heart failure ( table 1 ). the number of men with confirmed covid-19 infection outnumbered women in nearly all age groups; this sex difference was more pronounced among patients requiring hospitalization and particularly among patients requiring intensive care or intubation (fig 1) . we also observed a consistently higher rate of greater illness severity among african americans compared to persons of other racial groups (fig 2) . for the primary outcome of illness severity, categorized by escalating levels of care (i.e., hospitalization, intensive care, intubation), the pre-existing characteristics that demonstrated statistical significance in age-and sex-adjusted models included older age, male sex, african american race, obesity, hypertension, diabetes mellitus, and the elixhauser comorbidity score ( table 2 ; fig 3) . the associations that remained significant in the fully-adjusted multivariable model included older age (odds ratio [or] 1.49 per 10 years, 95% confidence interval [ci] 1.30-1.70, p<0.001), male sex (or 2.01, 95% ci 1.34-3.04, p = 0.001), african american race (or 2.13, 95% ci 1.19-3.83, p = 0.011), obesity (or 1.95, 95% ci 1.11-3.42, p = 0.021), diabetes mellitus (or 1.77, 95% ci 1.03-3.03, p = 0.037) and the comorbidity score (or 1.77 per sd, 95% ci 1.37-2.28, p<0.001). we also observed a trend towards lower severity of illness among patients chronically treated with ace inhibitor therapy, with or 0.48 (95% ci 0.22-1.04; p = 0.06). each estimated or value represents the increment in higher (or lower) odds of a patient requiring a next higher level of care, for every unit difference in a continuous variable (e.g. per 10 years of age) or for presence versus absence of a given categorical variable (e.g. male sex). in effect, every 10 years of older age was associated with~1.5-fold higher odds of requiring a higher level of care, and being male versus female was associated with a~2-fold higher odds of requiring higher level care. we used the brant method to test the proportional odds assumption for consistency of associations across our ordinal outcome; these analyses revealed no substantial qualitative violations, but did indicate that the elixhauser score was predominantly associated with the specific outcomes of admission versus non-admission (or 4.34, p<0.001) and need for intensive care versus no intensive care need (or 1.55, p = 0.008) that with the less frequent outcome of needing intubation versus no need for intubation (or 1.24, p = 0.25). for the specific outcome of needing any hospital admission, the pre-admission characteristics that demonstrated statistical significance included older age, male sex, african american race, obesity, hypertension, diabetes mellitus, the elixhauser comorbidity index, and prior myocardial infarction or heart failure (s2 table) . in the multivariable model adjusting for all key covariates, the pre-existing traits that remained significantly associated with needing any hospital admission were older age, diabetes mellitus, and higher comorbidity index. among the patients whose illness severity required hospitalization, male sex was associated with the outcome of requiring further escalating levels of care (i.e., intensive care and intubation) ( table 3 ; fig 3) . in the multivariable model adjusting for key covariates, male sex remained the single most important risk marker of requiring higher-level care (or 2.53, 95% ci 1.36-4.70, p = 0.003). the results for male sex were similar for the individual outcomes of requiring intensive care or intubation (s3 table) . we again observed a trend towards lower need for admission to the intensive care unit among patients chronically taking an ace inhibitor (or 0.38, 95% ci 0.13-1.17, p = 0.09), and greater need for intubation among african americans patients (or 2.14, 95% ci 0.99-4.64, p = 0.053). in secondary analyses, we used multiplicative interaction terms to assess for effect modification for associations observed in the main analyses (s4 table) . while considered exploratory pre-existing traits associated with covid-19 illness severity or hypothesis generating analyses, we found several interactions of potential interest (fig 4) . in particular, the associations of hispanic ethnicity, obesity, diabetes, and elixhauser comorbidity index with the primary outcome appeared paradoxically more pronounced in younger compared to older individuals (s5 table) . by contrast, the primary outcome was more pronounced among older compared to younger african americans. also paradoxically, hypertension appeared associated with greater risk in non-obese patient and with lower risk in obese patients. we repeated all main analyses with additional adjustment for smoking status in the subset of patients with available data on smoking; in these models, all significant results remained unchanged (s6 and s7 tables). we examined the pre-existing characteristics associated with severity of covid-19 illness, as observed thus far in our healthcare system located in los angeles, california. we found that almost half of patients presenting for evaluation and then confirmed to have covid-19 were clinically assessed to require hospital admission. these higher risk individuals were more likely to be older, male, african american, obese, and have diabetes mellitus in addition to a greater overall burden of medical comorbidities. notably, chronic use of an ace inhibitor appeared related to lower illness severity, in the absence of a similar finding for arb use. among all individuals requiring inpatient care for covid-19, male patients had a greater than 2.5-fold odds of needing intensive care and a 3.0-fold odds of needing intubation. all of our findings were observed even after accounting for co-existing risk factors and chronic medical conditions. recognizing that patients with covid-19 illness can present with clinical features that vary based on timing of the index encounter, we sought to identify the pre-existing traits that render some individuals at highest risk for developing the more severe forms of covid-19 illness once contracted. in our u.s. based metropolitan community, we observed that both obesity and diabetes mellitus are predisposing factors associated with a greater odds of needing hospital admission for covid-19 but not of requiring further escalation of care; this finding is consistent with emerging reports of obesity and diabetes mellitus each being associated with a greater risk for pneumonia due to covid-19 as well as other community-acquired viral agents-particularly in areas of the world where obesity is prevalent [30] [31] [32] [33] [34] [35] . also consistent with worldwide reports, we observed that older age is a significant predisposing risk factor for greater covid-19 illness severity in multivariable-adjusted models; this finding may represent an age-related immune susceptibility that is not completely captured by even a comprehensive comorbidity measure such as the elixhauser index. notwithstanding an overall age association in the expected direction of risk, we also found a paradoxical age interaction for certain key correlates. in effect, presence of obesity, diabetes, or an elevated overall comorbidity index were each associated with greater covid-19 illness severity in younger (i.e. <52 years) compared to older age groups. while unexpected, this finding is actually consistent with the known reduction of ace2 expression with advancing age, a phenomenon that has been proposed as a major contributor to the broad susceptibility to covid-19 seen in younger to middle aged individuals across the population at large [36] . consistent with worldwide reports, we found that the association of male sex with greater odds for every metric of covid-19 illness severity was especially prominent-and this was not explained by age variation, risk factors, or comorbidities [37] . reasons for the male predominance of illness severity remain unclear. although ace2 genetic expression is on the x chromosome, evidence to date would suggest relatively comparable expression levels between sexes relative risks associated with illness severity score are shown for all associations observed in the total sample (n = 442), stratified by subgroups defined by age (younger vs. older than median age 52 years), sex, and obesity (bmi �30 kg/m 2 ). � the primary outcome of covid-19 illness severity score in the total sample was defined as an ordinal variable wherein: 0 = referent, 1 = required admission but never icu level care, 2 = required icu level care but never intubated, 3 = required intubation. �� p for interaction values were calculated from likelihood ratio test between models with and without the interaction term. for each variable in the list, age (<versus �median age of 52 years), sex, and obesity interaction terms are implemented in multivariable adjusted models, with other covariates representative of the entire cohort. https://doi.org/10.1371/journal.pone.0236240.g004 [38, 39] , albeit with some potential for variation in relation to differences in sex hormones; select animal studies have shown increased ace2 activity in the setting of ovariectomy and the opposite effect with orchietomy [40, 41] . while there remains scant data currently available to explain sex differences for covid-19, male sex bias was also observed for sars and mers [42, 43] . similar to the findings in our study, this increase risk was not attributable to a greater prevalence of smoking among men. notably, prior murine studies have also demonstrated male versus female bias in susceptibility to sars-cov infection, which may be related to the effects of sex-specific steroids and x-linked gene activity on modulation of both the innate and adaptive immune response to viral infection [44] . further research specific to the sexual dimorphism seen in sars-cov-2 susceptibility is needed. in our u.s. based metropolitan community, we also observed racial and ethnic patterns of susceptibility to greater covid-19 illness severity. specifically, we found that african americans were at greater risk for needing higher levels of care overall, and this vulnerability appeared more pronounced in older age and among men. although an overall risk association was not seen for hispanic ethnicity, there was a trend towards greater covid-19 illness severity in younger aged compared to older aged hispanic/latino persons. a recent national report from the cdc also suggests overall higher rates of covid-19 susceptibility in african americans, and our findings confirm this trend exists even after adjusting for age, risk factors, and comorbidities [45] . in addition to the effects of unmeasured socioeconiomic and healthcare access variables, racial/ethnic disparities in covid-19 illness severity may relate to yet unidentified host-viral susceptibility factors that could also be contributing to heterogeneity of community transmission seen across regions worldwide and populations at large [29] . the use of ace inhibitor or angiotensin receptor blocker (arb) medications has been a focus of attention given that these agents may upregulate expression of ace2, the viral point of entry into cells [46] and alveolar type 2 epithelial cells in particular [47] . alternatively, these agents may confer benefit, given that sars-cov-2 appears to reduce ace2 activity and lead to potentially unopposed excess renin-angiotensin-aldosterone activation [36, 46, 48] . although we observed a non-significant trend in association of chronic ace inhibitor treatment with lower covid-19 illness severity, we found evidence of neither risk nor benefit with arbs. together, our findings are supportive of current recommendations to not discontinue chronic ace inhibitor or arb therapy for patients with appropriate indications for these medications. several limitations of our study merit consideration. our cohort included all individuals who underwent laboratory testing for covid-19 and not individuals who did not undergo testing; thus, our study results are derived from individuals presenting with symptoms that were deemed severe enough to warrant testing. all data including past medical history data were collected from the ehr and, thus, subject to coding bias and variations in reporting quality. to minimize the potential effects of these limitations that are inherent to ehr data, we performed iterative quality checks on the dataset and conducted manual chart review to verify values for key variables. we recognize that the illness severity outcomes defined as clinically ascertained need for hospital admission, icu level care, and intubation, may vary from practice to practice. as in many other u.s. medical centers affected by the covid-19 pandemic, our clinical staff have been practicing under institutional guidance to conserve resources and we anticipate that the thresholds for escalating care are likely comparable; thresholds for admission, transfer to intensive care, and intubation may be different in more resource constrained environments. given the relatively small number of observed in-hospital deaths (n = 11), and thus limited statistical power to detect associations, we deferred analyses of pre-existing characteristics and mortality risk to future investigations. the modest size of this early analysis of our growing clinical cohort may have limited our ability to detect potential additional predictors of covid-19 illness severity, as well as potential interactions or effect modification relevant to the outcomes; thus, further investigations are needed in larger sized samples. finally, our results are derived from a single healthcare system, albeit multi-center and serving a large catchment of the diverse population of los angeles, california. additional studies are needed to examine the extent to which our findings are generalizable to other populations affected by covid-19. in summary, we found that among patients tested and managed for laboratory confirmed covid-19 in our healthcare system to date, approximately half require admission for inpatient hospital care. these individuals are more likely to be older, male, african american, obese, and with known diabetes mellitus as well as a greater overall burden of medical comorbidities. well over a third of hospitalized patients require intensive care, with a substantial proportion needing intubation and mechanical ventilation for respiratory failure. among hospitalized patients, the highest risk individuals were more likely to be predominantly men of any age or race-for reasons not explained by comorbidities. further investigations are needed to understand the mechanisms underlying these associations and, in turn, determine the most optimal approaches to attenuating adverse outcomes for all persons at risk. (docx) s1 fig. los angeles county regional distribution of all patients with covid-19. the patients treated in our healthcare system for covid-19 illness presented from across a diverse regional distribution of residential locations across los angeles county. the map shown was generated using arcgis software by esri. 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male and female rats the pattern of middle east respiratory syndrome coronavirus in saudi arabia: a descriptive epidemiological analysis of data from the saudi ministry of health do men have a higher case fatality rate of severe acute respiratory syndrome than women do? sex-based differences in susceptibility to severe acute respiratory syndrome coronavirus infection hospitalization rates and characteristics of patients hospitalized with laboratory-confirmed coronavirus disease 2019-covid-net, 14 states renin-angiotensin-aldosterone system inhibitors in patients with covid-19 sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor acute respiratory distress syndrome in critically ill patients with severe acute respiratory syndrome we are grateful to all the front-line healthcare workers in our healthcare system who continue to be dedicated to delivering the highest quality care for all patients. key: cord-321834-n5w88l23 authors: huang, cheng-yang title: inhibition of a putative dihydropyrimidinase from pseudomonas aeruginosa pao1 by flavonoids and substrates of cyclic amidohydrolases date: 2015-05-19 journal: plos one doi: 10.1371/journal.pone.0127634 sha: doc_id: 321834 cord_uid: n5w88l23 dihydropyrimidinase is a member of the cyclic amidohydrolase family, which also includes allantoinase, dihydroorotase, hydantoinase, and imidase. these metalloenzymes possess very similar active sites and may use a similar mechanism for catalysis. however, whether the substrates and inhibitors of other cyclic amidohydrolases can inhibit dihydropyrimidinase remains unclear. this study investigated the inhibition of dihydropyrimidinase by flavonoids and substrates of other cyclic amidohydrolases. allantoin, dihydroorotate, 5-hydantoin acetic acid, acetohydroxamate, orotic acid, and 3-amino-1,2,4-triazole could slightly inhibit dihydropyrimidinase, and the ic(50) values of these compounds were within the millimolar range. the inhibition of dihydropyrimidinase by flavonoids, such as myricetin, quercetin, kaempferol, galangin, dihydromyricetin, and myricitrin, was also investigated. some of these compounds are known as inhibitors of allantoinase and dihydroorotase. although the inhibitory effects of these flavonoids on dihydropyrimidinase were substrate-dependent, dihydromyricetin significantly inhibited dihydropyrimidinase with ic(50) values of 48 and 40 μm for the substrates dihydrouracil and 5-propyl-hydantoin, respectively. the results from the lineweaver−burk plot indicated that dihydromyricetin was a competitive inhibitor. results from fluorescence quenching analysis indicated that dihydromyricetin could form a stable complex with dihydropyrimidinase with the k (d) value of 22.6 μm. a structural study using patchdock showed that dihydromyricetin was docked in the active site pocket of dihydropyrimidinase, which was consistent with the findings from kinetic and fluorescence studies. this study was the first to demonstrate that naturally occurring product dihydromyricetin inhibited dihydropyrimidinase, even more than the substrate analogs (>3 orders of magnitude). these flavonols, particularly myricetin, may serve as drug leads and dirty drugs (for multiple targets) for designing compounds that target several cyclic amidohydrolases. thus, whether the substrate and inhibitor of each enzyme in this family competitively inhibit dihydropyrimidinase remains unclear. few therapies are effective against the six antibiotic-resistant eskape pathogens. the increased prevalence of beta-lactamases in p. aeruginosa and other bacteria has begun to reduce the clinical efficacy of beta-lactams against the most common opportunistic pathogen [16] . to date, over 800 beta-lactamases have been identified, of which at least 120 beta-lactamases have been detected in p. aeruginosa [17] . the development of clinically useful small-molecule antibiotics and identification of new targets in microorganisms are seminal events in the field of infectious diseases [18] . flavonols belong to flavonoids, the most common group of plant polyphenols that is responsible for most of the flavor and color of fruits and vegetables [19] . over 5,000 different flavonoids have been identified, many of which display structure-dependent biological and pharmacological activities [20, 21, 22] , including antimicrobial agents [23, 24] . these natural products are safe as pharmaceuticals because they have fewer side effects for human use. in this study, we investigated the effects of the substrates and inhibitors of allantoinase and dihydroorotase, including the flavonols myricetin, quercetin, kaempferol, and galangin, on inhibiting the catalytic activity of a putative dihydropyrimidinase from p. aeruginosa pao1. the derivatives of myricetin, namely, dihydromyricetin and myricitrin, were further used to test the structure-inhibition relationship of dihydropyrimidinase. construction of the dihydropyrimidinase expression plasmid pa0441, the gene encoding the putative dihydropyrimidinase, was amplified by pcr using genomic dna of p. aeruginosa genomic dna as the template. the forward (5 0 -cgcggcata tgtttgatttactcctgc-3 0 ) and the reverse (5 0 -tcgcactcgagaaaatcgaaggc atgt-3 0 ) primers were designed to introduce unique ndei and xhoi restriction sites (underlined), permitting the insertion of the amplified gene into the pet21b vector (novagen inc., madison, wi, usa). the dna fragment was then inserted into pet21b to produce the plasmid pet21b-padht for p. aeruginosa dihydropyrimidinase expression. the expected gene product expressed by pet21b-padht has a c-terminal his tag, useful for purifying the recombinant protein. the dihydropyrimidinase mutants (h59a, h61a, k150a, y155a, h183a, h239a, s289a, d316a, and n337a) were generated according to the manufacturer's protocol (stratagene, lajolla, ca). the presence of the mutation was verified by dna sequencing in each construct. the oligonucleotide primers for the preparation of mutants are shown in table 1 . recombinant wild-type and mutant dihydropyrimidinases were expressed and purified using the protocol described previously for hydantoinase [12] , allantoinase [11, 15] , dihydroorotase [11, 15] , prib [25, 26] , dnab [27] , dnat [28, 29] , and ssb [30, 31] . the protein purified from the soluble supernatant by ni 2+ -affinity chromatography (hitrap hp; ge healthcare bio-sciences, piscataway, nj, usa) was eluted with buffer a (20 mm tris-hcl, 250 mm imidazole, and 0.5 m nacl, ph 7.9) and dialyzed against a dialysis buffer (20 mm hepes and 100 mm nacl, ph 7.0; buffer b). protein purity remained >97% as determined by sds-page. a rapid spectrophotometric assay was used to determine the enzymatic activity according to a previously described protocol for hydantoinase [12] , allantoinase [11, 15] , dihydroorotase [11, 15] , and imidase [3, 4, 5] . dihydrouracil, 5-propyl-hydantoin, and phthalimide were used as substrates. unless explicitly stated otherwise, dihydrouracil (2 mm) was used as the substrate in the standard assay of dihydropyrimidinase. briefly, the decrease in absorbancy at 230, 248, and 298 nm was measured upon hydrolysis of dihydrouracil, 5-propyl-hydantoin, and phthalimide as the substrate at 25°c, respectively. to start the reaction, the purified dihydropyrimidinase (10-70 μg) was added to a 2 ml solution containing the substrate and 100 mm tris-hcl (ph 8.0). substrate hydrolysis was monitored with a uv/vis spectrophotometer (hitachi u 3300, hitachi high-technologies, tokyo, japan). the extinction coefficient of each substrate was determined experimentally by direct measurement with a spectrophotometer. the extinction coefficients of dihydrouracil, 5-propyl-hydantoin, and phthalimide were 0.683 mm -1 cm -1 at 230 nm, 0.0538 mm -1 cm -1 at 248 nm, and 3.12 mm -1 cm -1 at 298 nm, respectively. the initial rates of change were a function of enzyme concentration within the absorbance range of 0.01-0.18 min -1 . a unit of activity was defined as the amount of enzyme catalyzing the hydrolysis of 1 μmol substrate/min, and the specific activity was expressed in terms of units of activity per milligram of enzyme. the kinetic parameters k m and v max were determined from a non-linear plot by fitting the hydrolyzing rate from individual experiments to the michaelis-menten equation (enzyme kinetics module of sigma-plot; systat software, chicago, il, usa). dissociation constants determined by fluorescence spectrophotometer the dissociation constants (k d ) of dihydropyrimidinase was determined using the fluorescence quenching method as previously described for allantoinase [15] , dihydroorotase [15] , and dnab helicase [27, 32] . an aliquot of each compound was added into the solution containing dihydropyrimidinase (0.8 μm), 50 mm hepes at ph 7.0. the decrease in intrinsic fluorescence of protein was measured at 334.5 nm upon excitation at 279 nm and 25°c with a spectrofluorimeter (hitachi f-2700; hitachi high-technologies, tokyo, japan). at least seven data points were used to calculate each k d . each data point was an average of 2-3 determinants, and the difference of the determinants was within 10%. the the amino acid sequences of 497 sequenced dihydropyrimidinase homologues were aligned using consurf [33] . the model of p. aeruginosa dihydropyrimidinase was built using human dihydropyrimidinase (pdb entry: 2vr2) as a template by swiss-model (http://swissmodel. expasy.org) [34] and (ps) 2 (http://140.113.239.111/~ps2v2/docs.php) [35] . the coordinate and topology file of the flavonoids was found in drugbank (http://www.drugbank.ca/) [36] . myricetin and dihydromyricetin were computationally docked into the three-dimensional model of dihydropyrimidinase by using patchdock (http://bioinfo3d.cs.tau.ac.il/patchdock/) [37] . the dihydrouracil-complexed structure model of p. aeruginosa dihydropyrimidinase was directly constructed by superimposing the crystal structure of the dihydrouracil-yeast dihydropyrimidinase complex (the coordinate of 2fvk). the structures were visualized by using the program pymol. expression and purification of a putative dihydropyrimidinase from p. aeruginosa pao1 the gene pa0441 encoding putative dihydropyrimidinase was pcr-amplified using genomic dna of p. aeruginosa pao1 as a template. the amplified gene was then ligated into the pet21b vector for protein expression. p. aeruginosa dihydropyrimidinase was hetero-overexpressed in e. coli and purified from the soluble supernatant using ni 2+ -affinity chromatography. pure protein was obtained in this single chromatographic step with an elution of buffer a. approximately 50 mg of purified protein was extracted from 1 l of a culture of e. coli cells. the mutant dihydropyrimidinases were also purified according to the same protocol used for the wild-type proteins, and yielded very similar purification results. the catalytic activity of purified dihydropyrimidinase (without any metal supplement in the culture) was not high, so some metal ions were added to the reaction mixture. table 2 shows that the addition of 1 mm cocl 2 , zncl 2 , or mncl 2 activated dihydropyrimidinase activity, and followed the order co 2+ > zn 2+ > mn 2+ ; cdcl 2 , nicl 2 , mgcl 2 , and cacl 2 were not useful. we also added 1 mm cocl 2 , the best supplement, into the bacterial culture for dihydropyrimidinase expression, and the resultant dihydropyrimidinase was purified and analyzed. the specific activity of this dihydropyrimidinase toward dihydrouracil was 5.9 ± 0.4 μmol/mg/min, a value very similar to that of the co 2+ -activated enzyme (5.8 ± 0.5 μmol/mg/min). thus, dihydropyrimidinase (1 mm cocl 2 supplemented into the bacterial culture) was used for all analyses in this study, unless explicitly stated otherwise. this enzyme from microorganisms is commonly known as hydantoinase. although the gene product of pa0441 has been described as a dihydropyrimidinase, the substrate specificities of dihydropyrimidinase and hydantoinase may differ. for example, the recombinant hydantoinase from agrobacterium radiobacter prefers 5-leucinyl-hydantoin to phthalimide and dihydrouracil (~two orders of magnitude), as revealed by the catalytic efficiencies [12] . to ensure that the gene product of pa0441 is suitably identified as a dihydropyrimidinase, we analyzed the substrate specificity of this dihydropyrimidinase toward dihydrouracil (fig 1b) , phthalimide ( fig 1c) , 5-propyl-hydantoin ( fig 1d) , allantoin, and dihydroorotate, which are typical substrates for dihydropyrimidinase, imidase, hydantoinase, allantoinase, and dihydroorotase, respectively (table 3) . unlike a. radiobacter hydantoinase, the catalytic efficiency of this enzyme toward dihydrouracil was higher than that toward phthalimide (fourfold) and 5-propyl-hydantoin (100-fold). therefore, this bacterial enzyme was suitably identified as a dihydropyrimidinase, not a hydantoinase. substrate analogs for any enzyme are usually potential inhibitors. members of the cyclic amidohydrolase family have highly similar active sites, and may use the same catalytic mechanism for substrate hydrolysis [38] . this condition raises an interesting question as to whether the substrate and inhibitor of allantoinase and dihydroorotase can be a potential inhibitor of dihydropyrimidinase because of their structural similarity. allantoin and dihydroorotate are not substrates for dihydropyrimidinase-catalyzed reactions (table 3) . allantoin and dihydroorotate are structurally similar to hydantoin and dihydrouracil, except for the 5' side chain ( fig 1a) . to assess whether allantoin and dihydroorotate are dihydropyrimidinase inhibitors, allantoin and dihydroorotate were individually included in the standard assay using 5-propylhydantoin (fig 2a) or dihydrouracil ( fig 2b) as a substrate. allantoin and dihydroorotate only slightly inhibited the activity of dihydropyrimidinase. thus, substrates of other cyclic amidohydrolase may not be strong inhibitors of dihydropyrimidinase. to assess whether inhibitors or substrate analogs of allantoinase and dihydroorotase can be inhibitors of dihydropyrimidinase, 5-hydantoin acetic acid, acetohydroxamate, and orotic acid were used in this study. similarly, 5-hydantoin acetic acid and acetohydroxamate (an inhibitor of allantoinase) could only slightly inhibit the activity of dihydropyrimidinase (fig 2) . we also tested whether inhibitors of the metalloenzyme glutaminyl cyclase, namely, n-ω-acetylhistamine, 3,5-diamino-1,2,4-triazole, and 3-amino-1,2,4-triazole [39, 40] , are potential inhibitors of dihydropyrimidinase, in which 3-amino-1,2,4-triazole at 100-750 mm affected enzyme activity (fig 2) . high concentrations of these compounds were required to obtain significant inhibition of dihydropyrimidinase. thus, inhibitors or substrate analogs of allantoinase, dihydroorotase, and glutaminyl cyclase were not potent inhibitors of dihydropyrimidinase. to compare the inhibitory capability of these compounds on dihydropyrimidinase, their ic 50 values, the inhibitory concentration required to reduce the activity of the enzyme by 50%, were determined and compared. the ic 50 values of dihydroorotate, 5-hydantoin acetic acid, and acetohydroxamate were around 30, 50, and 500 mm, respectively. the ic 50 values of these substrate and inhibitor analogs of other cyclic amidases for dihydropyrimidinase were within the millimolar range. substrate analogs for any enzyme are usually potential inhibitors, but this common assumption was not present in this study of dihydropyrimidinase. given broad substrate specificity, the active site of dihydropyrimidinase can accommodate many different substrates. therefore, substrate analogs cannot be specifically recognized and cannot significantly inhibit its activity. although we found that dihydroorotate, 5-hydantoin acetic acid, and acetohydroxamate inhibited dihydropyrimidinase activity, their ic 50 values were at the millimolar range and higher than the k m values of dihydropyrimidinase, which were insufficient as potent inhibitors. to determine whether a naturally occurring product is an inhibitor of dihydropyrimidinase, the inhibitory capabilities of the flavonols myricetin (with three hydroxyl substituents on the aromatic ring), quercetin (with two hydroxyl substituents on the aromatic ring), kaempferol (with one hydroxyl substituent on the aromatic ring), and galangin (without hydroxyl substituent on the aromatic ring) were tested. furthermore, to study the structure-inhibition relationship with dihydropyrimidinase, the derivatives of myricetin, namely, dihydromyricetin (the ring does not contain a double bond but has additional two hydrogen atoms) and myricitrin (the ring is fused to an additional sugar block), were further used and tested (fig 3a) . identification of flavonol inhibition of dihydropyrimidinase flavonols are known to have antioxidant [20] , antiradical [22] , and antibacterial properties [23, 24] . myricetin, quercetin, kaempferol, and galangin at different concentrations were included in the standard assay, and their ic 50 values were determined and compared to determine whether flavonols inhibit dihydropyrimidinase. generally, the activities of dihydropyrimidinase toward dihydrouracil ( fig 3b) and 5-propyl-hydantoin (fig 3c) continued to decrease as the concentrations of the compound increased. however, these compounds caused distinct effects when different substrates were used. when dihydrouracil was used as a substrate, the inhibitory effects of myricetin and kaempferol on the activity of dihydropyrimidinase were significant; however, kaempferol only slightly inhibited the activity of acetohydroxamate and 3-amino-1,2,4-triazole solutions, whose ph values, were pre-adjusted to ph 8. when using dihydrouracil as a substrate, some compounds at high concentrations were difficult to determine the inhibitory effect on the activity of dihydropyrimidinase using the spectrophotometric assay at 230 nm. dihydropyrimidinase toward 5-propyl-hydantoin (fig 3c) . the effect of quercetin was insignificant. overall, myricetin was found to strongly inhibit the hydrolysis of both dihydrouracil and 5-propyl-hydantoin catalyzed by dihydropyrimidinase. we further tested whether dihydromyricetin and myricitrin (fig 3a) , derivatives of myricetin, can be inhibitors of dihydropyrimidinase. no effect was found when myricitrin was added into the standard assay using dihydrouracil and 5-propyl-hydantoin as substrates of dihydropyrimidinase. however, opposite to myricitrin, dihydromyricetin exhibited a significant inhibitory effect on the activities of dihydropyrimidinase for both substrates, even more than myricetin did (fig 3b and 3c) . the ic 50 values of dihydromyricetin for dihydropyrimidinase determined from the titration curves using dihydrouracil and 5-propyl-hydantoin were 48 ± 2 and 40 ± 2 μm, respectively; myricetin yielded higher values of 83 ± 3 and 63 ± 3 μm, respectively. based on the ic 50 values, the order of the inhibitory capability of the flavonoids for dihydrouracil was as follows: dihydromyricetin > kaempferol > myricetin > galangin > quercetin > myricitrin. moreover, the order of the inhibitory capability of the flavonoids for 5-propyl-hydantoin was as follows: dihydromyricetin > myricetin > galangin > kaempferol > quercetin > myricitrin. thus, for the first time, flavonols, especially dihydromyricetin, were established as novel inhibitors of dihydropyrimidinase. in addition, the inhibitory capabilities of these flavonols (at the μm range) were significantly higher than those of the substrate and inhibitor analogs (at the mm range) of dihydropyrimidinase. to study the structure-inhibition relationship between dihydropyrimidinase and the flavonoids in silico, the structure of dihydropyrimidinase was modeled and used for docking experiments. the crystal structure of p. aeruginosa dihydropyrimidinase has yet to be determined. we modeled the p. aeruginosa dihydropyrimidinase structure (s1 fig) by homology modeling using swiss-model (http://swissmodel.expasy.org/) [34] . human dihydropyrimidinase (pdb entry: 2vr2) was the first hit suggested as a template by the program. the amino acid sequences of human (with 519 aa) and p. aeruginosa dihydropyrimidinase (with 479 aa) shared 51% identity and 66% similarity (s2 fig). we also used (ps) 2 , another bioinformatic tool, for structural modeling [35] . (ps) 2 (http://140.113.239.111/~ps2v2/docs.php) is an automatic homology modeling server that combines both sequence and secondary structure information to detect the homologous proteins with remote similarity and target-template alignment. human dihydropyrimidinase was also the first hit suggested as a template by (ps) 2 because it had the highest score. results obtained from (ps) 2 analysis indicated that 99.58% of the secondary structure was aligned, which implied that human and p. aeruginosa dihydropyrimidinase shared a highly similar structure (s3 fig). mutational analysis of the residues within the active site according to the crystal structure of saccharomyces kluyveri (pdb entry: 2fvk) [41] and sinorhizobium meliloti dihydropyrimidinases (pdb entry: 3dc8) [42] , residues h59, h61, k150, h183, h239, and d316 of p. aeruginosa dihydropyrimidinase were crucial for the assembly of the binuclear metal center within the active site; meanwhile, residues y155, s289, and n337 were crucial for substrate binding (fig 4a) . the amino acid sequences of 497 sequenced dihydropyrimidinase homologs aligned using consurf [33] indicated that these residues were well conserved (fig 4b) . the importance of these residues was then probed by site-directed mutagenesis, in which alanine substitution was constructed and analyzed. as expected, the catalytic activities of these ala-substituted mutant proteins were severely impaired. h59a, h61a, k150a, h183a, h239a, d316a, y155a and s289a were inactive. only n337a mutant protein was found to be active, but its activity was about 20-fold less than that of the wildtype dihydropyrimidinase. to understand the inhibitory mechanism of dihydromyricetin and myricetin on dihydropyrimidinase, their structures found in the drugbank (http://www.drugbank.ca/) [36] were computationally docked into the 3d model of p. aeruginosa dihydropyrimidinase using patchdock (http://bioinfo3d.cs.tau.acil/patchdock/) [37] . docking was automatically performed after uploading the coordinates and topology files of the compound and dihydropyrimidinase. the docking models with the highest score in dihydropyrimidinase interacting with dihydromyricetin and myricetin are shown in fig 5. although dihydromyricetin and myricetin were docked inhibition of dihydropyrimidinase by flavonoids into the active site pocket of dihydropyrimidinase, their binding modes differed. on one hand, dihydromyricetin interacted with i95, s289, and d316 (fig 5a) , in which s289 and d316 were found to be crucial for the catalytic activity of dihydropyrimidinase. on the other hand, myricetin interacted with n157, q194, r212, and n337, in which only n337 was crucial. this docking study showed that myricetin only partially occupied the dihydropyrimidinase active site. the ring structure of dihydromyricetin was more flexible than that of myricetin ( fig 5) because of lacking the double bond on its ring (fig 3a) . we identified dihydromyricetin as a potent inhibitor with an ic 50 value of 48 μm on dihydropyrimidinase. to determine what type of inhibitor dihydromyricetin is, this compound (40 μm) was included in the standard assay for dihydropyrimidinase with different concentrations of dihydrouracil. as shown in fig 6, inhibition of dihydropyrimidinase by dihydromyricetin resulted in lineweaver−burk plots with lines that crossed the y-axis at a similar point, which indicated that dihydromyricetin was a competitive inhibitor of dihydropyrimidinase. the presence of 40 μm dihydromyricetin yielded v max and k m values, kinetic constants, of 5.4 ± 0.4 μmol/mg/min and 1.6 ± 0.2 mm, respectively. without dihydromyricetin, the kinetic constants v max and k m were 7.6 ± 0.4 μmol/mg/min and 0.7 ± 0.1 mm, respectively ( table 3 ). the k m value obviously increased by twofold, whereas v max was only slightly affected. these findings indicated the competitive inhibition of dihydropyrimidinase by dihydromyricetin; inhibition of dihydropyrimidinase by flavonoids specifically, dihydromyricetin could compete with dihydrouracil for the active site of dihydropyrimidinase [43] . to determine whether the inhibitory capabilities of myricetin and dihydromyricetin are correlated with their binding abilities, the dissociation constants (the k d values) of dihydropyrimidinase for myricetin and dihydromyricetin were determined through fluorescence quenching. quenching refers to the complex formation process that decreases the fluorescence intensity of the protein. the fluorescence emission spectra of dihydropyrimidinase were remarkably quenched with dihydromyricetin ( fig 7a) and myricetin (fig 7b) . upon adding 50 μm myricetin and dihydromyricetin, the intrinsic fluorescence of dihydropyrimidinase was quenched by 56.2% and 89.1%, respectively. as shown in fig 7b, adding different concentrations of myricetin resulted in a red shift (~7 nm; λ max = 333.5-340.5 nm) in the dihydropyrimidinase emission wavelength (λ em ). adding dihydromyricetin also caused a red shift in the dihydropyrimidinase emission wavelength (fig 7a) , which was much more significant (~25 nm; λ max = 333.5-358.5 nm). these observations indicated that myricetin and dihydromyricetin interacted with dihydropyrimidinase, thereby suggesting that myricetin and dihydromyricetin could form stable complexes with dihydropyrimidinase. the k d values of dihydropyrimidinase bound to myricetin and dihydromyricetin, as determined through their titration curves (fig 7c) , were 28.8 ± 2.2 and 22.6 ± 2.6 μm, respectively. our in silico experiments showed that myricetin and dihydromyricetin may use different modes to bind to dihydropyrimidinase (fig 5) . these different possible binding modes were also revealed by the distinct fluorescence quenching spectra, in which their λ max shifts differed significantly (fig 7) . the docking study showed that n337 interacted with the hydroxyl group on the ring of myricetin, but not dihydromyricetin. thus, fluorescence quenching of the n337a mutant was also carried out to test whether this residue is important for myricetin binding, but not for dihydromyricetin. myricetin and dihydromyricetin quenched the intrinsic fluorescence of dihydropyrimidinase by 58.4% and 86.3%, respectively (fig 8) . adding myricetin and dihydromyricetin resulted in red shifts in λ max of the n337a mutant from 333.5 nm to 341 nm (~7.5 nm) and 357 nm (~23.5 nm), respectively. the λ max shifts of the n337a mutant by myricetin and dihydromyricetin were similar, but still slightly different, to those of the wildtype dihydropyrimidinase (fig 7) . the k d value of the n337a mutant bound to myricetin ( fig 8c) , compared with that of the wild-type dihydropyrimidinase, was reduced to 59.3 ± 8.6 μm (twofold). however, the k d value of the n337a mutant for dihydromyricetin binding was 24.1 ± 1.7 μm, a value nearly identical to that of the wild-type dihydropyrimidinase, which suggested that n337 was important for myricetin binding but not for dihydromyricetin. these observations indicated that myricetin and dihydromyricetin could still form stable complexes with the n337a mutant, but the binding environment of the active site within the n337a mutant was somewhat different from that of the wild-type protein. substrate analogs for any enzyme are usually potential inhibitors, but this common rule is not applicable for dihydropyrimidinase (fig 2) . combating diseases caused by infections resistant to all antibacterial options requires the development of clinically useful small-molecule antibiotics and identification of novel targets. dna metabolism is one of the most basic biological functions that should be a prime target in antibiotic development. considering that dihydropyrimidinase is a component in the chain of pyrimidine catabolism required for metabolizing the dna base, blocking this enzyme's activity may be useful to limit bacterial growth and survival. although some chelators are known to inhibit dihydropyrimidinase, they are harmful to humans. in this study, we showed that dihydromyricetin, a flavonol, significantly inhibited the catalytic activities of dihydropyrimidinase toward both the natural substrate dihydrouracil and xenobiotic substrate 5-propyl-hydantoin (fig 3) . furthermore, the metabolic effects and safety of the flavonols are well established, making flavonols beneficial for humans. thus, some of these plant polyphenols may become the lead compounds for further antibiotic development and drug design. we found that the flavonols myricetin, quercetin, kaempferol, and galangin, which contain different numbers of hydroxyl substituents on their aromatic rings, exerted different inhibitory effects on dihydropyrimidinase (fig 3) . although flavonols are known to have several hydroxyl groups, thought to have remarkable potential for binding any protein, the strength of the flavonols' inhibitory effect (ic 50 ) on dihydropyrimidinase was not correlated with the number of hydroxyl substituents on the flavonols' aromatic rings. in addition, the inhibitory effect (ic 50 ) of flavonols on dihydropyrimidinase was substrate-dependent. for example, as shown in fig 3, the inhibitory effect of kaempferol on the activity of dihydropyrimidinase was significant only with dihydrouracil as a substrate (with ic 50 value of 50 ± 2 μm), but not with 5-propyl-hydantoin. although our docking results (fig 5) and the findings of the kinetic studies (fig 6) both showed a competitive inhibition of the flavonol dihydromyricetin on dihydropyrimidinase, further structure-inhibition relationship studies are still needed for deeper understanding of and myricetin with dihydropyrimidinase. an aliquot amount of dihydromyricetin and myricetin was individually added to the enzyme solution for each k d . the inhibition of dihydropyrimidinase by flavonoids the inhibition mechanism, particularly because we are still unsure whether each enzyme only contains a single dihydromyricetin within the active site of dihydropyrimidinase. flavonoids are the most common group of plant polyphenols with antioxidant, antiradical, anticancer, and antibacterial properties [19] . some flavonoids are atpase-inhibiting agents and competitors for atp-binding proteins [27, 32] . for example, several flavonoid derivatives have been developed as therapeutic agents for cancer [21] . myricetin and scutellarein strongly inhibit the atpase activity of nsp13 of the sars coronavirus helicase [44] . myricetin, luteolin, and morin inhibit the hexameric replicative helicases, and myricetin inhibits gram-negative bacteria growth [45] . myricetin also inhibits bacterial allantoinase and dihydroorotase [15] . given that myricetin is also a potent inhibitor of numerous dna and rna polymerases and telomerases [46] , these flavonoids, especially myricetin, may be a competent dirty drug or multi-target drug [43] . dihydropyrimidinase is a zinc enzyme. however, in this study, dihydropyrimidinase supplemented with co 2+ ions exhibited the highest activity ( table 2 ). due to the low bioavailability of cobalt on earth and in cells, we thought that the possibility of a biological role playing in dihydropyrimidinase may be ruled out. the higher activity of the co 2+ -substituted enzymes has been proposed. the higher activity followed by metal replacement in co(ii)-thermolysin than in native thermolysin may be due to the enhancement of stabilization of the transition state than those of the native thermolysin [47] . for imidase, the sizes of the metal are found as the important factors that affect the activity of imidase [4] . the position of nucleophilic attack on an imide substrate may fluctuate due to size of the coordinating metal ion. because the crystal structure of the co 2+ -dihydropyrimidinase is still lacking, both these possibilities cannot be ruled out. the chemical mechanism of the binuclear metal center-containing amidohydrolase likely involves three steps: (1) the hydrolytic water molecule must be activated for nucleophilic attack, (2) the amide bond of the substrate must be made more electrophilic by the polarization of the carbonyl-oxygen bond, and (3) the leaving group nitrogen must be protonated as the carbon-nitrogen bond is cleaved [38, 48] . this catalytic mechanism is applicable to each member in the cyclic amidohydrolase family [41, 48, 49] . however, a question remains as to why a common mechanism-based inhibitor for these imide-hydrolyzing enzymes, such as imidase, hydantoinase, dihydropyrimidinase, allantoinase, and dihydroorotase, is difficult to find. new and undetermined factors should be further studied and discovered in this protein family, such as the third metal ion recently found in dihydroorotase, which was found to be highly important for catalysis [50, 51] . further studies are also necessary to investigate the substrate specificity, selectivity, and catalytic mechanism of dihydropyrimidinase and hydantoinase, as well as other cyclic amidohydrolases. 2 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substitutions in the active site of thermolysin crystal structures of vertebrate dihydropyrimidinase and complexes from tetraodon nigroviridis with lysine carbamylation: metal and structural requirements for post-translational modification and function dihydropyrimidine amidohydrolases and dihydroorotases share the same origin and several enzymatic properties getting cad in shape: the atomic structure of human dihydroorotase domain structure, functional characterization, and evolution of the dihydroorotase domain of human cad conceived and designed the experiments: cyh. performed the experiments: cyh. analyzed the data: cyh. contributed reagents/materials/analysis tools: cyh. wrote the paper: cyh. key: cord-349029-zyfop43z authors: dobrovolny, hana m. title: modeling the role of asymptomatics in infection spread with application to sars-cov-2 date: 2020-08-10 journal: plos one doi: 10.1371/journal.pone.0236976 sha: doc_id: 349029 cord_uid: zyfop43z sars-cov-2 started causing infections in humans in late 2019 and has spread rapidly around the world. while the number of symptomatically infected and severely ill people is high and has overwhelmed the medical systems of many countries, there is mounting evidence that some of the rapid spread of this virus has been driven by asymptomatic infections. in this study, we use a compartmental mathematical model of a viral epidemic that includes asymptomatic infection to examine the role of asymptomatic individuals in the spread of the infection. we apply the model to epidemics in california, florida, new york, and texas, finding that asymptomatic infections far outnumber reported symptomatic infections at the peak of the epidemic in all four states. the model suggests that relaxing of social distancing measures too quickly could lead to a rapid rise in the number of cases, driven in part by asymptomatic infections. in late 2019, a novel coronavirus (sars-cov-2) began transmitting in humans in wuhan, china [1, 2] and has since spread widely around the world. the virus can cause a severe respiratory illness, known as covid-19, characterized by fever and cough that can lead to respiratory failure and death [3, 4] . the virus appears to spread easily from human to human [5, 6] , surviving well in aerosolized form and lasting for long periods of time on surfaces [7] . it has also become increasingly apparent that asymptomatic or unreported cases are playing a role in the rapid spread of the virus [8, 9] . a number of studies have investigated the possibility of asymptomatic carriers of sars-cov-2 and have tried to estimate their number using isolated clusters of cases. one study estimated that the asymptomatic proportion of sars-cov-2 infections on the diamond princess cruise ship docked outside tokyo while an outbreak spread onboard was *18% [10] . a study of japanese evacuees from wuhan, china estimated the proportion of asymptomatic infections at *30% [11] . a similar study of german nationals evacuated from wuhan found 2 of the 114 evacuees were asymptomatically infected with sars-cov-2 [12] . testing of every person in an isolated italian village revealed that 50-75% of people were asymptomatic [13] . even in high-risk populations, there appear to be some asymptomatic cases. a study of a nursing home in king county, washington found *30% of patients were asymptomatic on the day of testing for sars-cov-2, with *4% remaining asymptomatic upon followup a week later [14] . a study in hospitalized patients in beijing found that *5% of patients testing positive for sars-cov-2 had asymptomatic infections [15] . while the exact proportion of asymptomatic infections is still unclear, these studies indicate that asymptomatic infections with sars-cov-2 are not uncommon. however, it is not enough that asymptomatic infections exist. in order for asymptomatic individuals to spread the infection, they must be able to shed and transmit the virus to others. there are some studies that indicate this might in fact be the case for asymptomatic infections with sars-cov-2. zhang et al. [16] report on a familial cluster of covid patients initiated by an asymptomatic individual. there is also a second case report of a different asymptomatic individual infecting other members of his family [17] . a series of sars-cov-2 infections among business associates in germany was traced to a single pre-symptomatic individual [18] . in a more large-scale scenario, identification and rapid isolation of asymptomatic sars-cov-2 positive individuals led to quick decline in the number of new cases in an italian village [13] , suggesting that asymptomatic individuals were responsible for at least some of the spread of the virus. this is supported by a modeling study of transmission in china that suggests that *86% of infections were caused by asymptomatic or unreported cases [9] . these studies indicate that asymptomatic individuals could be a factor in the spread of sars-cov-2 and should be considered when predicting the scope of the epidemic and the effectiveness of mitigation strategies. mathematical modeling investigations of the role of asymptomatics during an epidemic have previously been done for other infectious diseases [19] [20] [21] [22] [23] [24] [25] [26] . these models have estimated the proportion of asymptomatic individuals [22, 23, 25, 26] and have shown how changes in the proportion of asymptomatic individuals might change the course of the epidemic [19, 23, 24] or affect mitigation strategies such as quarantine [23] and vaccination [20, 21] . in the current sars-cov-2 pandemic, isolation and social distancing are the primary weapons in stopping the spread of the infection. in order to estimate how effective these strategies will be, we will need a better understanding of the role of asymptomatic individuals in sars-cov-2 spread and the effect the proportion and relative infectiousness of asymptomatics have on the time course of the epidemic. in this paper, we study a compartmental epidemic model that includes asymptomatic infections to determine the role that asymptomatic individuals might play in the spread of sars-cov-2. we find that the relative infectiousness of asymptomatic individuals has more of an impact on the time course and size of the epidemic than the proportion of asymptomatic individuals, but that the proportion of asymptomatic individuals has a bigger impact on mortality. we apply our model to data from sars-cov-2 epidemics in california, florida, new york, and texas, finding that a large number of infections in these states are unreported and that relaxing social distancing measures too early will cause a rapid spike in infections driven in part by these hidden infections. we use a compartmental model that includes asymptomatic infections with assumptions geared towards modeling the current sars-cov-2 pandemic, where s is the pool of susceptible individuals, a are individuals infected asymptomatically, i are individuals infected symptomatically, r are recovered individuals, and d are those who have died. we assume that the course of the epidemic is short compared to the human lifespan and do not include births or deaths from other causes, instead assuming that the total population, n = s + a + i + r + d stays constant over the course of the epidemic. upon exposure to the virus, some fraction (given by p) of people become asymptomatically infected; the remaining people become symptomatically infected. we assume that the asymptomatics can infect susceptible individuals, but with a different infection rate, determined by the proportionality constant r. we assume that asymptomatic individuals will all recover (after an average time 1/ k), but that symptomatically infected individuals are removed either through recovery or hospitalization/isolation (at rate α) or die (at rate δ). to apply our model to the current pandemic, we use four us states as examples: california, florida, new york, and texas. we use data from the state health departments of california, florida, new york, and texas that tracks the cumulative number of infected individuals and the cumulative number who have died through april 17, 2020. data is included in s1 file. we assume that the epidemic starts with a single symptomatic individual, i(0) = 1 and that there are no asymptomatic, recovered or dead (a(0) = r(0) = d(0) = 0). since we do not know the time at which the infected individual arrived to start each epidemic, we introduce a free parameter, t d , to shift the data in time and set the appropriate start time of the infection. we fix the death rate of symptomatic individuals to δ = 0.056/d based on mean time from symptom onset to death [27] . since our data includes the cumulative number of infected individuals, we define a variable c, where dc dt ¼ ð1 à pþ b n si þ rsa ð þ tracks the cumulative number of symptomatically infected individuals and fit that to the cumulative infected individuals. early in the pandemic, testing was primarily limited to individuals who displayed symptoms, so the case count in this data set should primarily consist of symptomatic individuals. we simultaneously fit both the infected and dead to the model by minimizing the weighted sum of square residuals (ssr) using the nelder-mead algorithm implemented in octave. confidence intervals are determined through bootstrapping with 1000 replicates. we explored the effect of asymptomatics on the course of the infection using a base set of parameters given in table 1 . we used the population of the united states for the population size and used estimates for the remaining parameters from the literature. infections are initiated with a single symptomatic infected individual. the infection rate was taken from a model fit to data of coronavirus infections from china [9] ; they estimate infection rates before and after travel bans and social distancing measures were put in place. we use both values in our simulations. the mean time from symptom onset to death is 18 d [27] or δ = 0.056/d. we use a removal rate of α = k = 0.143/d based on data suggesting the mean time from symptom onset to hospitalization is 7 d [27] . although many people who are hospitalized eventually recover, once they are hospitalized they are not contributing much to widespread community transmission. to study the role of asymptomatic individuals in an epidemic, we varied both r (from 0 to 2) and p (from 0 to 1) and measured the case-fatality ratio, the size of the epidemic, and the time of peak of the epidemic. the case-fatality ratio is defined as the number of dead divided by the total number infected. the size of the epidemic is measured here as the proportion of the total population who become infected, either symptomatically or asymptomatically. results are shown as contour plots in fig 1 for both high (left column) and low (right column) infection rates. we see that the relative infectivity (r) of asymptomatic individuals has little effect on the case-fatality ratio, remaining essentially constant for all values of r except when p is near one. in the upper left corner, there is a pocket where the mortality is zero; this is particularly evident for the lower infection rate. this pocket is more clearly defined for the epidemic size and the time of peak. both the epidemic size and the time of peak are fairly constant until we approach the upper left hand corner where there is a rapid decrease in the epidemic size and a rapid increase in the time of peak. this boundary defines a threshold for the epidemicabove this boundary, the epidemic simply dies off. this boundary is determined by the basic reproduction number r 0 for the epidemic. r 0 for this model is given by r 0 here has two components: the first term, r a ¼ prb k , represents spread due to asymptomatic individuals, while the second term, r s ¼ ð1à pþb aþd represents spread due to symptomatic individuals. estimates of the basic reproduction number for sars-cov-2 outbreaks in various locations range from 0.5-4 [28] , however, fig 1 includes combinations of parameters leading to r 0 values much larger than 4, so are likely biologically unrealistic. it might be more interesting to understand the trade-off between the proportion of asymptomatic infections and the relative infectivity in contributing to a particular value of r 0 . we fixed r 0 to values within the known range for sars-cov-2 and plotted the values of p and r that will produce particular values of r 0 for both the high (fig 2 (left) ) and low (fig 2 (right) ) values of infection rate. solving for p in the r 0 equation gives the relationship between p and r, when the infection rate is high, a high proportion of asymptomatics and low relative infectivity is required for low values of r 0 . the range of possible p and r values expands as r 0 increases, with the relationship indicating that higher values of p require higher values of r for a particular r 0 . when the infection rate is low, we see a slightly different relationship between p and r for high r 0 -when r 0 = 3, increasing r values require lower values of p for a particular r 0 . this change in behavior occurs because we have crossed an asymptote at r = k/(α + δ) (*0.72 for these parameters). note that the r 0 = 4 curve does not appear in this plot because there are no biologically reasonable combinations of p and r that will result in r 0 values of 4. in the current covid pandemic, public health authorities have been attempting to lower the infection rate through social distancing measures, so we more closely examined the dependence of the epidemic threshold on infection rate. fig 3 shows the r 0 = 1 (the epidemic threshold value) curves for different values of infection rate (β). r 0 < 1 is to the left of each curve. we see that as the infection rate is lowered, there are more combinations of r and p that result in no epidemic. that is, the general infection rate is just not high enough to support spread of the infection no matter what the proportion of asymptomatic individuals or their relative infectivity. we fit the model to data from four states: california, florida, new york, and texas. data and model fits to the data are shown in fig 4. best fit parameter values with 95% confidence intervals are given in table 2 . parameter correlation and parameter distribution plots are included in s1 file. all four of these states have implemented some form of social distancing for a period of time, and the majority of the data is taken after these measures are in place. all four states have r 0 values near 2. estimates of the r 0 for sars-cov-2 before changes in social behavior in china run between 2 and 4.25 [29] [30] [31] , typically falling below 1 when complete lockdowns are imposed [28] . the r 0 values found here are on the low end for unmitigated sars-cov-2, suggesting that the social distancing measures are having some effect, but the measures taken in these states have not driven r 0 below the threshold value of 1. interestingly, the estimated r 0 https://doi.org/10.1371/journal.pone.0236976.g002 table 2 . https://doi.org/10.1371/journal.pone.0236976.g004 values are almost entirely determined by the basic reproduction number for asymptomatic spread in all four states, indicating that the spread of the infection is largely driven by asymptomatic individuals, at least in the early stages of the pandemic. the large r a value is almost entirely determined by the high proportion of asymptomatic individuals-about 99% for all four states. this is somewhat higher than the 86% unreported cases estimated in china [9] and might be indicative of the limited testing availability in the united states early in the pandemic. the undercounting of symptomatic infections is particularly problematic since each symptomatic person incorrectly classified as asymptomatic because they weren't tested might appear as several asymptomatic people in order to account for the difference in infectivity between the two groups. the high proportion could also be in part due to the lack of a pre-symptomatic phase in this model; recent studies have shown that symptomatic people can infect others during the pre-symptomatic phase [32] [33] [34] and within this model would be considered part of the asymptomatic population. the relative infectivity of asymptomatic patients ranges from *6-15%, so the high basic reproduction number for this group is driven by the large number of patients rather than their ability to easily infect others. the infectivity values for all four states are high, so based on the analysis of fig 2, we expect that there are few combinations of p and r that will result in r 0 values near 2 and that this can only happen if p is large and r is small. fig 5 shows the time course of asymptomatic individuals and symptomatic individuals for epidemics in all four states. in all cases, at the peak of the epidemic, there are far more asymptomatic individuals than symptomatic individuals. this is due not only to the high proportion of infections that are asymptomatic, but is also caused by a faster removal of symptomatic individuals as compared to asymptomatic individuals. while it might seem unusual that asymptomatic individuals are recovering slower than symptomatic individuals, given the heightened awareness of transmission of infectious diseases as well as the stay-at-home orders in these states, symptomatic individuals are likely to isolate themselves upon symptom onset, effectively removing themselves from participating in further transmission. while not true recovery, from the standpoint of the model, these symptomatic people are classified as recovered. therefore, asymptomatic individuals participate in transmitting the infection for a longer period of time and in greater numbers than asymptomatic individuals. it is also notable that the peak in asymptomatic infections occurs slightly after the peak in symptomatic infections. this could have repercussions for decisions on when to relax social distancing measures since we will observe a decline in infections while asymptomatic infections are still increasing. we also estimated the time at which the initial infected individual initiated the infection in each state. using our estimates of t d , we can calculate the estimated date of the start of the epidemic. for all four states, we find that the epidemics were initiated sometime in january 2020. for california, this is as early as january 15, while for florida the estimated infection start date is january 14. new york has the earliest estimated start date of january 11, while the latest is in texas on january 24. strict stay-at-home measures have severe economic implications and are difficult to maintain [35, 36] , so government officials want to relax social distancing measures as early as possible. given the large number of asymptomatic individuals predicted by our model, as well as the fact that the asymptomatic peak is slightly later than the symptomatic peak, we decided to investigate model predictions of what happens as social distancing measures are relaxed. based on a previous modeling study, we assume that stay-at-home orders cut the infection rate β in half [9] , so double the infection rate at two week intervals starting on the last day of our data (april 17, 2020). results are shown in fig 6 with the solid line indicating the original epidemic where social distancing was not relaxed, and dashed lines indicating predicted epidemics when social distancing is relaxed. we see that there is an immediate spike in both symptomatic and asymptomatic infections upon reopening and that the spike becomes smaller as reopening is delayed. interestingly, there is also a change in the decay rate of the epidemic as the re-opening date is changed. earlier re-openings have a larger spike in cases, but also exhibit a more cases, delaying re-opening lowers the size of the epidemic as well as the number of fatalities. even though immediate re-opening might result in a faster resolution to the epidemic, this comes at the cost of more illness and deaths. for california, florida, and texas, both the total epidemic size and the number of fatalities increase with any re-opening within the next 10 weeks, as compared to no relaxation of social distancing measures for the duration of the epidemic. we analyzed a compartmental model that includes asymptomatic infections finding that the relative infectiousness of asymptomatic individuals plays a larger role in changing the time course and size of the epidemic while the proportion of asymptomatic infections plays a larger role in determining mortality. yet testing in many countries is limited to people who are symptomatic, meaning that there is no data on the number of asymptomatic infections, so mathematical models cannot properly incorporate asymptomatic individuals. this not only hampers the ability of the model to accurately predict the time course of the epidemic, but could also lead to inaccurate predictions of the effect of isolation [23] and contact-tracing mitigation strategies. if a vaccine is eventually developed, previous studies have shown that the number of asymptomatic infections also alters the effectiveness of vaccination strategies [20, 21] . when we fit our model to data from the sars-cov epidemic in four states, we found that there were far more asymptomatic individuals than symptomatic individuals at the peak of the epidemic in all four states. this is consistent with recent studies of antibody seroprevalence that suggest a 50-85 fold undercount of covid-19 in santa clara county, california [37] and in los angeles [38] . our model has two different mechanisms by which the number of asymptomatic individuals can rise to high numbers. if the proportion of infections that become asymptomatic is high (p close to 1), then as the number of infections rise, more of these will be asymptomatic. it is also possible to accumulate large numbers of asymptomatic individuals if symptomatic infections are removed from the epidemic, either through death, recovery, or isolation, faster than asymptomatic infections. this might be the case if symptomatic individuals are quickly tested and then isolated thereby being removed from further participation in the infection as might be the case for these four states since testing for sars-cov-2 in all these states at the time this data was taken, was limited to symptomatic individuals. this also points to a limitation of this study. testing for sars-cov-2 started slowly in the united states [39] and remains inadequate to this day, so there is no tracking of the number of asymptomatic cases and likely an undercount of symptomatic cases. thus the asymptomatic compartment in this application of the model includes symptomatic patients who were unable to get tested. our model also does not include a latent or pre-symptomatic phase, which also lumps people who likely have different infection rates into one compartment. this mixing of individuals who should be in separate compartments as well as the inadequate counting of symptomatic infections leads to error in our parameter estimates and an inability to uniquely identify all parameters of the model [40] . correlation plots included in s1 file show that there are correlations between some parameter estimates, although there was no consistent pattern on which parameters were correlated between the four data sets used. adequate testing protocols that capture both symptomatic and asymptomatic infections will allow better parameterization of the model. additional data, such as tracking the number of recovered individuals would also help constrain parameters. while some state public health agencies are reporting the number of recovered, this number is really only reflective of symptomatic patients who have been hospitalized and released. symptomatic patients who remain at home are not being tracked, so their recovery is not recorded. accurately tracking the course of all aspects of the epidemic, particularly in its early phase [41] , is crucial for proper parameterization of mathematical models and will lead to more accurate model predictions. despite these limitations, fitting of our model to the data has resulted in some interesting parameter estimates. the basic reproduction number appears to be robust to limitations of parameter identifiability [42] . we found a basic reproduction numbers around 2 for all four states. this is at the lower end of sars-cov r 0 estimates in the absence of any behavioral changes [29] [30] [31] . these low values of r 0 likely reflect the effect of social distancing measures in the us, although these measures have not driven r 0 below 1 as a full lockdown would [28] . however, a recent study [43] indicates that sars-cov-2 oubreaks with r 0 < 1.5 can be contained with 50% contact tracing. even for r 0 up to 2.5, contact tracing only needs to capture 70% of infections to be effective. while the epidemiological model used here is not as complex as some others that have been suggested for sars-cov-2, it still provides insight into a key aspect of sars-cov-2 transmission. our model shows that asymptomatic infections can change the size and lethality of an epidemic even if they are not necessarily a large proportion of the infections. for the sars-cov epidemics examined here, the model predicts that there are far more asymptomatic or unreported cases at the peak of the infection, suggesting that there might be widespread community transmission if stay-at-home orders are relaxed too early. epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study a new coronavirus associated with human respiratory disease in china clinical characteristics of covid-19 in new york city review of the clinical characteristics of coronavirus disease 2019 (covid-19) first known person-toperson transmission of severe acute 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are tackling coronavirus worldwide studying the identifiability of epidemiology models using mcmc detecting presymptomatic infection is necessary to forecast major epidemics in the earliest stages of infectious disease outbreaks assessing parameter identifiability in compartmental dynamic models using a computational approach: application to infectious disease transmission models feasibility of controlling covid-19 outbreaks by isolation of cases and contacts key: cord-324410-be2ith3z authors: wang, qi; pang, yuan-ping title: accurate reproduction of 161 small-molecule complex crystal structures using the eudoc program: expanding the use of eudoc to supramolecular chemistry date: 2007-06-13 journal: plos one doi: 10.1371/journal.pone.0000531 sha: doc_id: 324410 cord_uid: be2ith3z eudoc is a docking program that has successfully predicted small-molecule-bound protein complexes and identified drug leads from chemical databases. to expand the application of the eudoc program to supramolecular chemistry, we tested its ability to reproduce crystal structures of small-molecule complexes. of 161 selected crystal structures of small-molecule guest-host complexes, eudoc reproduced all these crystal structures with guest structure mass-weighted root mean square deviations (mwrmsds) of <1.0 å relative to the corresponding crystal structures. in addition, the average interaction energy of these 161 guest-host complexes (−50.1 kcal/mol) was found to be nearly half of that of 153 previously tested small-molecule-bound protein complexes (−108.5 kcal/mol), according to the interaction energies calculated by eudoc. 31 of the 161 complexes could not be reproduced with mwrmsds of <1.0 å if neighboring hosts in the crystal structure of a guest-host complex were not included as part of the multimeric host system, whereas two of the 161 complexes could not be reproduced with mwrmsds of <1.0 å if water molecules were excluded from the host system. these results demonstrate the significant influence of crystal packing on small molecule complexation and suggest that eudoc is able to predict small-molecule complexes and that it is useful for the design of new materials, molecular sensors, and multimeric inhibitors of protein-protein interactions. in 1990, a computer was used to screen 10,000 chemicals in the cambridge structural database (csd) [1] , leading to the identification of a haloperidol analog capable of inhibiting hiv-1 and hiv-2 proteases with a k i of <100 mm [2] . the screening was accomplished using a computer docking program, dock, that docked each chemical of the database into the active sites of the enzymes and evaluated the shape complementarity of the docked compound relative to the active sites. inspired by this seminal work, the eudoc program was devised to search for the specific conformations, positions, and orientations of two threedimensional (3d) structures that permit the strongest nonbonded intermolecular interactions between the two. the eudoc program uses docking algorithms that differ from those of dock [3] . it addresses molecular flexibility by using conformation selection and conformation substitution mechanisms that enable massively parallel computing [3] . eudoc was devised to perform on a cluster of more than 300 loosely connected processors [3] and has recently been ported to the ibm blue gene/l supercomputer [4, 5] . this program has successfully predicted small-moleculebound protein complexes and identified drug leads from chemical databases [6] [7] [8] [9] [10] [11] [12] . the eudoc program is also efficient. in a computational screen of 23,426 chemicals (at a resolution of 1.0 å translation and 10u of arc rotation) for inhibitors of a chymotrypsin-like cysteine protease of the severe acute respiratory syndrome-associated coronavirus, the eudoc program is able to reduce the wall-clock time of the screen from 242 minutes using 396 xeon processors (2.2 ghz) on a beowulf cluster to 13 and 7 minutes using 2048 and 4096 powerpc-440 processors (700 mhz) on blue gene/l, respectively [4, 5] . because a large database can be divided into subsets, a sustained petaflops capability would be able to screen 23 million chemicals in about 10 minutes or to screen 20065000 billion chemicals for one drug target in a year [4] . this capability offers the possibility of identifying inhibitors that are effective enough for in vivo testing, eliminating the need of medicinal chemistry to improve the efficiency of inhibitor leads identified by terascale computers [4] . in the context of this promise, we seek to extend the application of the eudoc program to supramolecular chemistry. supramolecular chemistry deals with creation of a large molecule assembled with noncovalent bonding among small molecular units, in contrast to organic synthesis that involves breaking and making covalent bonds to create a new molecule [13] . such noncovalent bonding is reversible and comprises hydrogen bonding, metal coordination, hydrophobic force, van der waals force, p-p interaction, cation-p interaction, and/or long-range electrostatic interaction to assemble small molecules into a multimolecular complex. supramolecular chemistry principles have been used to develop new materials, molecular sensors, and multimolecular complexes designed to disrupt protein-protein interactions. to expand the application of the eudoc program to supramolecular chemistry, we tested its ability to reproduce the crystal structures of small-molecule guest-host complexes. previously we had tested the ability of the program to reproduce crystal structures of proteins in complex with small molecules and found that eudoc reproduced 97% of 154 crystal structures using the bound conformations of both proteins and their smallmolecule partners [3] . this success may not transfer to with smallmolecule guest-host complexes such as a crown ether in complex with 4-nitrobenzene-1,2-diamine, however, because the binding pocket or cavity in a small-molecule host is not as well formed as that in a protein. herein we report the results of our docking studies with 161 selected crystal structures of small-molecule guest-host complexes using the eudoc program. these results show that the program is able to reproduce all 161 crystal structures and that the average interaction energy of these small-molecule complexes (250.1 kcal/mol) is nearly half of that of the 153 small molecule-bound protein complexes we studied in previous tests (2108.5 kcal/mol). the results also demonstrate the significant influence of crystal packing on small-molecule complex crystal structures and suggest that the eudoc program is able to predict 3d structures of small-molecule guest-host complexes with reasonable reliability. the ability of eudoc to reproduce crystal structures of smallmolecule guest-host complexes was evaluated with the following procedure. the guest and host molecules in the complex crystal structure were separated, and the guest structure was then docked back into the host structure by the eudoc program. this docking process used translational and rotational increments of 1.0 å and 10u of arc, respectively, and a docking box that was defined to enclose the guest structure in the guest-host complex crystal structure. of many eudoc-generated guest-host complexes, only the complex with the strongest interaction energy was compared to the corresponding crystal structure of the complex. in this comparison, the host portion of the eudoc-generated complex was superimposed onto the host portion of the crystal structure, and the mass-weighted root mean square deviation (mwrmsd) of the guest portion between the two superimposed complexes was calculated. if the mwrmsd was ,2.0 or 1.0 å , the crystal structure of the complex was reproduced or accurately reproduced, respectively, by the eudoc program [3] . because the uncertainty in calculating the interaction energy using the eudoc program was estimated to be 0.7 kcal/mol [3] , occasionally, a few eudoc-generated complexes were considered to have the strongest interaction energy and compared to the crystal structure thus resulting in multiple mwrmsds, if their interaction energies differed from the strongest interaction energy by #0.7 kcal/mol. in that case, as long as one of the mwrmsds was ,2.0 or 1.0 å , the crystal structure of the binary complex was reproduced or accurately reproduced, respectively, by the eudoc program. a total of 161 crystal structures of small-molecule guest-host complexes were obtained from csd for this study [1] . the selection criteria included the followings: (1) no covalent bond between a host and a guest; (2) the r factor of ,15 to ensure good crystallographic quality; (3) a guest to host ratio of 1 in a unit cell; (4) no structures containing ni +2 , ag + , pd +2 , pt +2 , au + or ru +2 because force field parameters for these ions were unavailable in the eudoc program. the results of the docking studies with the 161 guest-host complexes using the procedure described above are listed in table 1 . as apparent from the mwrmsd distribution listed in table 2 , the eudoc program reproduced 93% and accurately reproduced 81% of the 161 complexes. the deviations between the eudoc-generated and crystal complexes at different mwrmsd values are depicted in figure 1 . docking with consideration of the influence of crystal packing figure 2 shows the difference (mwrmsd of 3.52 å ) between the eudoc-generated and crystal structures (csd code: xag-mat). complex xagmat is one of the 12 complexes that the eudoc program failed to reproduce. despite a favorable p-p interaction between the guest and host structures predicted by the eudoc program, in the corresponding crystal structure the guest structure surprisingly docks at a region at which it partly interacts with the host via a p-p interaction (see fig. 2 ). this discrepancy suggests that the guest might partly interact with host(s) and/or guest(s) in neighboring unit cells of the crystal structure. to confirm this, the docking study with complex xagmat was repeated with consideration of the influence of crystal packingnamely, the guest was docked into a multimeric host system that included neighboring host(s) and/or guest(s). these neighboring structures were generated by applying the symmetry of the space group of the crystal structure. the host(s) and/or guest(s) in neighboring unit cells were excluded if these structures were .4.0 å away from the guest to be docked. interestingly, when the influence of crystal packing was taken into account, the eudoc program accurately reproduced complex xagmat with an mwrmsd of 0.07 å , instead of the 3.52 å obtained without consideration of crystal packing. this result prompted a new docking study that considered the influence of crystal packing. the results of the docking studies with consideration of the influence of crystal packing are listed in table 3 . the 12 complexes (csd codes: ajuxuy, atukef, baxzab, bif-kik, cramcc10, fanjag, gugguk, kolmaz, lay-maz, nebqoa, qajkan, and ralqaw01) that were not reproduced previously by the eudoc program were accurately reproduced after the influence of crystal packing was taken into account. with consideration of the influence of crystal packing, the eudoc program reproduced all 161 complexes and accurately reproduced 99% of them (see table 2 ). two crystal structures (csd codes: xaqjaa and xaqjee) could not be accurately reproduced by the eudoc program even after consideration of the influence of crystal packing (xaqjaa: mwrmsd = 1.65 å ; xaqjee: mwrmsd = 1.89 å ). visual inspection of these structures revealed that the binding between the guest and host structures was mediated by crystallographically determined water molecules. this mediation suggested that, similar to the crystal packing, water molecules might also play an important role in guest-host complexation, and it might be necessary to include them in the multimeric host system for docking. accordingly, the docking studies with the 161 complexes were repeated with consideration of the influences of both crystal packing and structural waters. the results are listed in table 4 . indeed, the eudoc program accurately reproduced complexes xaqjaa and xaqjee with mwrmsds of 0.03 and 0.27 å , respectively. taking into account the influences of both crystal packing and structural waters, the eudoc program accurately reproduced all 161 complexes (see table 2 ). the influences of crystal packing and structural water on docking this study shows that 31 (19%) of the 161 complexes could not be accurately reproduced with mwrmsds of ,1.0 å by the eudoc program if neighboring host(s) and/or guest(s) in the crystal structure were not included as part of the multimeric host system, whereas only 2 (1%) of these complexes could not be accurately reproduced with mwrmsds of ,1.0 å if neighboring structures were included but water molecules were excluded from the host system. these results show that the influence of crystal packing or crystal environment on crystal structures of guest-host complexes is significant, which is consistent with the reported influence of crystal packing on protein structures [14] . these results also show that the influence of structural waters on guesthost complex crystal structures is insignificant, which is consistent with our reported finding that complexation between a small molecule and a protein is not commonly mediated by water molecules [3] . this study therefore suggests that crystal packing should be taken into account when reproducing crystal structures of small-molecule guest-host complexes through docking studies, whereas water molecules, counterions or other companying molecules such as ethanol can be excluded from the host system. this study also suggests that, ideally, to perform prospective and accurate docking of a small molecule into another small molecule, repetitive docking of a guest or host into a guest-host complex that is generated by the previous docking is preferred, because a host is sometimes too small to prevent a guest from interacting with nearby guest(s) and/or host(s). it is worth noting, however, that the success rate of docking a small molecule into another small molecule is about 93% if the influence of crystal packing is ignored. in the crystal structure of rebek's acridine diacid in complex with quinoxaline (csd code: yawjip) there are two crystallographically independent forms of the complex in the asymmetric unit [15] . this structure was used to benchmark the all-atom amber/opls force field [16] . in this study, complex yawjip was accurately reproduced by the eudoc program using the nonbonded force field parameters of the second-generation [3, 17] ; the mwrmsds of the guest position between the eudoc-generated and crystal complexes for forms a and b are both 0.16 å . the accurate reproduction of the remaining crystal structures (see table 2 ) further demonstrates the accuracy of the nonbonded force field parameters of the secondgeneration amber force field (parm99.dat) for reproducing crystal structures [3, 17] . the above results suggest that the eudoc program can predict small-molecule guest-host complexes with a reasonable success rate (93%), without consideration to the mechanism of small-molecule complex aggregation-namely, without being given the multimeric host system. to demonstrate this ability herein, the crystal structure of complex yawjip is used as a model system. based on the nmr spectroscopic data of complex yawjip, the guest structure quinoxaline was proposed to have face-to-face pstacking with the acridine portion of rebek's acridine diacids in an early report of the complex [18] . however, the face-to-face pstacking was found in neither the crystal structure of complex yawjip [15] nor the monte carlo statistical mechanics calculations of the complex [16] . to perform a prospective docking study, the two-dimensional structures of quinoxaline and rebek's acridine diacid were the van der waals component of the intermolecular interaction energy; 4 the electrostatic component of the intermolecular interaction energy. 5 eudoc identified one alternative binding mode that is energetically indistinguishable from the binding mode of the crystal structure. , in) , respectively. both 3d structures were refined with energy minimization monitored with a normalmode (nmode) analysis to ensure that the energy minimization stopped when the minimized conformation reached a local potential energy minimum. the energy minimization was performed by using the sander module of the amber5 program [19] with the second-generation amber force field [17] , and the nmode analysis was carried out using the nmode module of the amber8 program [19] . given the refined 3d structures of quinoxaline and rebek's acridine diacid, the eudoc program generated a complex nearly identical to the crystal structure of form a (mwrmsd: 0.7 å ) but not the proposed complex with nearly face-to-face p-stacking. this perspective docking result suggests that the eudoc program is a useful tool for predicting 3d models of guest-host complexes to aid the design of new molecular entities according to the principles of supramolecular chemistry. visual inspection of 154 reported crystal structures of proteins in complex with small molecules [3] and the 161 crystal structures of guest-host complexes reported herein suggested that the noncovalent interactions of the guest-host complexes are in general weaker than those of the protein complexes. the average of the interaction energies of the guest-host complexes listed in the van der waals component of the intermolecular interaction energy; 4 the electrostatic component of the intermolecular interaction energy; 5 structural water molecules were present in the multimeric host system. 6 eudoc identified one alternative binding mode that is energetically indistinguishable from the binding mode of the crystal structure. table vi (excluding 1pha) of reference 3 (2108.5 kcal/mol) quantitatively confirm the relatively weak noncovalent interactions of the guest-host complex. this confirmation suggests that to design high-affinity guest-host complexes it is of advantage to incorporate the entropic energy into the binding, because the number of functional groups that can be introduced onto the guest and host structures to confer the nonbonded interactions is limited by the size of the two partners. this ''saturation'' problem is more apparent for small-molecule complexes than for protein complexes. it is therefore conceivable that the eudoc program is also a useful tool for estimating the interaction energies of guest-host complexes to aid the design of new molecular entities according to the principles of supramolecular chemistry. the guest and host structures were taken from the crystal structures of their corresponding complexes obtained from csd [1] . water molecules, counterions, and solvent molecules such as ethanol were removed from the guest or host structure. hydrogen atoms were added by using the quanta97 program (accelrys software, inc, san diego, california) followed by energy minimization of the hydrogen atoms using the sander module of the amber5 program [19] with the second-generation amber force field (parm99.dat) [17] and a positional constraint on all non-hydrogen atoms. the protonation states of the guest and host structures shown in figures s1, s2 , s3, s4, s5, s6, s7, s8, s9, s10, s11, s12, s13, s14, s15 and s16 of supporting information were determined according to pka values of functional groups at ph of 7.0. the atomic charges of the guest and host structures listed in table s1 of supporting information were generated according to the resp procedure [20] with ab initio calculations at the hf/6-31g* level using the gaussian03 program [21] . the amber atom types of the guest and host structures listed in table s1 of supporting information were assigned by the antechamber module of amber7 [19] . the algorithm of the eudoc program has been reported elsewhere [3] . briefly, it uses a systematic search protocol, translating and rotating a guest in a putative binding pocket of a host to search for energetically favorable orientations and positions of the guest relative to the host. a docking box is defined within the binding pocket to confine the translation of the ligand. the intermolecular interaction energy is the potential energy of the guest-host complex relative to the potential energies of the two partners in their free state. this energy was calculated according to equations 1 and 2 using the second-generation amber force field [17] . in calculating the intermolecular interaction energy, the dielectric constant was set to 1.0, and the distance cutoffs for steric and electrostatic interactions were set to 10 9 å . a docking box was defined to enclose the guest structure in the crystal structure of the guest-host complex. the size of the docking box and the cutoff for the interaction energy used by the eudoc program are listed in table s2 of supporting information. the complex-prediction module of the eudoc program (version 41, executable available from ypp) was used to translate and rotate the guest around the host at increments of 1.0 å and 10u of arc, respectively, unless noted otherwise in table 1 . to consider the influence of crystal packing, the pymol program (delano scientific llc, south san francisco, california) was used to generate a multimeric host system by applying the symmetry of the space group of the crystal structure. the host or guest structure was excluded from the multimeric host system if the shortest distance between a heavy atom of the guest structure to be docked and the heavy atom of the host/guest structure in neighboring unit cells was .4.0 å . energy minimization was performed with the sander module of amber5 [19] using (1) maxcyc = 10 6 , (2) dielc = 80, (3) scnb = 2.0, (4) scee = 1.2, (5) ntr = 0, (6) ntmin = 1 or 2, (7) ncyc = 0, (8) cut = 12, and (9) drms = 10 27 . nmode analysis was performed with the nmode module of amber8 [19] using (1) ilevel = 0 or 1, (2) cut = 12, (3) scnb = 2.0, (4) scee = 1.2, (5) dielc = 80, and (6) idiel = 1. the mwrmsds were calculated by superimposing the host portion of the eudoc-generated complex over the corresponding host portion of the crystal structure followed by a calculation for the mwrmsd of all atoms of the guest portion in the two superimposed complexes using the ptraj module of amber8 [19] . the cambridge structural database: a quarter of a million crystal structures and rising structure-based design of nonpeptide inhibitors specific for the human immunodeficiency virus 1 protease eudoc: a computer program for identification of drug interaction sites in macromolecules and drug leads from chemical databases in silico drug discovery: solving the ''target-rich and lead-poor'' imbalance using the genome-to-drug-lead paradigm eudoc on blue gene: accelerating the transfer of drug discoveries from laboratory to patient successful virtual screening of a chemical database for farnesyltransferase inhibitor leads chemical database techniques in drug discovery structure of acetylcholinesterase complexed with the nootropic alkaloid discovery of a new inhibitor lead of adenovirus proteinase: steps toward selective, irreversible inhibitors of cysteine proteinases re-engineering butyrylcholinesterase as a cocaine hydrolase from genome to drug lead: identification of a small-molecule inhibitor of the sars virus serotype-selective, small-molecule inhibitors of the zinc endopeptidase of botulinum neurotoxin serotype a the limit of accuracy of protein modeling: influence of crystal packing on protein structure molecular-sructure of rebek diacid quinoxalineconfirmation of 2-point binding structure and binding for complexes of rebek's acridine diacid with pyrazine, quinoxaline, and pyridine from monte carlo simulations with an all-atom force field a second generation force field for the simulation of proteins, nucleic acids, and organic molecules molecular recognition: ionic and aromatic stacking interactions bind complementary functional groups in a molecular cleft amber, a package of computer programs for applying molecular mechanics, normal mode analysis, molecular dynamics and free energy calculations to simulate the structural and energetic properties of molecules application of the multimolecule and multiconformational resp methodology to biopolymers: charge derivation for dna, rna, and proteins gaussian 03, revision c.02 the authors acknowledge computing support from the university of minnesota supercomputing institute. key: cord-344357-ocyaqs1y authors: fu, yue-qiang; sun, yue-lin; lu, si-wei; yang, yang; wang, yi; xu, feng title: effect of blood analysis and immune function on the prognosis of patients with covid-19 date: 2020-10-30 journal: plos one doi: 10.1371/journal.pone.0240751 sha: doc_id: 344357 cord_uid: ocyaqs1y introduction: this retrospective study investigated the implications of changes in blood parameters and cellular immune function in patients with coronavirus disease 2019 (covid-19). methods: records were reviewed of 85 patients admitted with covid-19 between february 4 and 16, 2020. the primary outcome was in-hospital death. results: fourteen patients died. the baseline leukocyte count, neutrophil count and hemoglobin was significantly higher in non-survivors compared with survivors, while the reverse was true of lymphocyte count, platelet, pao(2)/fio(2), cd3+ count and cd4+ count. the percentage of neutrophil count > 6.3×10(9)/l in death group was significantly higher than that in survival group, and multivariate logistic regression showed neutrophil count > 6.3×10(9)/l was independently associated with mortality. however, there were not significant difference in igg, igm, iga, c3, c4 and the percentage of ige > 100 iu/ml between the death group and survival group. areas under the receiver operating characteristic curves of the following at baseline could significantly predict mortality: leukocyte, neutrophil, lymphocyte, cd3+ and cd4+ counts. conclusions: for hospitalized patients with covid-19, lymphocyte, cd3+ and cd4+ counts that marked decrease suggest a poor outcome. admission neutrophil count > 6.3 ×10(9)/l is independently associated with mortality. at admission, leukocyte, neutrophil, lymphocyte, cd3+ and cd4+ counts should receive added attention. fourteen patients died. the baseline leukocyte count, neutrophil count and hemoglobin was significantly higher in non-survivors compared with survivors, while the reverse was true of lymphocyte count, platelet, pao 2 /fio 2 , cd3+ count and cd4+ count. the percentage of neutrophil count > 6.3×10 9 /l in death group was significantly higher than that in survival group, and multivariate logistic regression showed neutrophil count > 6.3×10 9 /l was independently associated with mortality. however, there were not significant difference in igg, igm, iga, c3, c4 and the percentage of ige > 100 iu/ml between the death group and survival group. areas under the receiver operating characteristic curves of the following at baseline could significantly predict mortality: leukocyte, neutrophil, lymphocyte, cd3+ and cd4+ counts. for hospitalized patients with covid-19, lymphocyte, cd3+ and cd4+ counts that marked decrease suggest a poor outcome. admission neutrophil count > 6.3 ×10 9 /l is independently associated with mortality. at admission, leukocyte, neutrophil, lymphocyte, cd3+ and cd4+ counts should receive added attention. the first case of an unknown pneumonia was traced to the wuhan south china seafood market in december 2019 [1] . it was confirmed as an acute respiratory infectious disease caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2, formerly known as 2019-ncov) and disease has been subsequently named coronavirus disease 2019 (covid-19) by who on feb 11, 2020 [2] . at present, the covid-19 pandemic has wrought great damage in china, asia, europe, and north america, and globally all countries are under threat. however, medical research concerning this new virus is relatively limited, and the pathophysiological mechanisms underlying the cause of disease are still under study. pulmonary infection caused by virus is often accompanied by changes in hematologic and immune test parameters. sars-cov-2 and sars-cov belong to the family coronaviridae and the genus beta-coronavirus. previous studies on sars may give us some useful hints [3] [4] [5] [6] . in patients with severe acute respiratory syndrome (sars), an initial neutrophil count >7000/ml (odds ratio [or] 6.4) has been associated with fatality [3] . leong et al. [4] found that neutropenia was associated with poor prognosis in patients with sars, and li et al. [5] reported that the peripheral cd4+ and cd8+ t lymphocyte absolute counts were significantly lower in these patients. tang et al. [6] also showed that cd3+, cd4+, and cd8+ lymphocyte significantly decreased in the acute phase of sars, especially in patients who died. wang et al. [7] showed that most patients with covid-19 had marked lymphopenia, and non-survivors developed more severe lymphopenia over time during hospitalization. another study also indicated that covid-19 patients in the death group had significantly lower lymphocyte count on admission than the recovered group [8] . recently, several studies showed that compared to non-severe cases, the severe covid-19 cases had lower lymphocyte counts [9] [10] [11] [12] [13] [14] and higher leukocytes counts [9] [10] [11] . some studies mentioned that cd4+t and cd8+t cells decreased in the covid-19 patients [9, 14, 15] . however, whether there are changes in humoral immune function has not been studied yet. and the differences of lymphocyte subsets between the survival and the death patients with covid-19 are rarely studied. covid-19 may lead to hyperactivity of the inflammatory response, and suppression of the immune response. the objective of the present case series was to compare the clinical characteristics, blood analyses, and immune functions features between the survivors and non-survivors. the predictive value of neutrophil, lymphocyte, and lymphocyte subsets at admission for mortality was also studied. the ethics committee of children's hospital, chongqing medical university (institutional review board of children's hospital, chongqing medical university) approved this retrospective cohort study. the requirement for written informed consent was waived because of the retrospective design. the study population comprised patients with covid-19 pneumonia, who had been admitted to the ward of the third batch of chongqing medical aid team in wuhan city of hubei province in china, from 4 february 2020 to 16 february 2020. the data were analyzed anonymously. the patients in this study were confirmed to have sars-cov-2 infection by real-time reverse transcription polymerase chain reaction (rt-pcr) assay, from nose and throat swab samples. at least one ground glass change in the lung was indicated on chest computed tomography (ct) scan. a blood routine and immune function test was completed within 12 hours after admission. patients with any of the following were excluded from this study: death due to natural causes rather than viral infection; immunodeficiency; or with long-term use of glucocorticoids or immunosuppressants before admission. collected information included: age; gender; concomitant disease; symptoms; hemoglobin; platelet; leukocyte, neutrophil, and lymphocyte counts; crp, cd3+, cd4+, cd8+, cd4/8, cd19+, and cd16+56+;igg, igm, iga, ige, c3, and c4 chest ct; alanine aminotransferase, creatinine; arterial blood gas; procalcitonin; and the result of the rt-pcr assay of sars-cov-2 rna. the primary outcome was in-hospital death. in-hospital death was defined as death occurred in hospital directly related to covid-19, which was confirmed by rt-pcr assay. all survivors discharge before 1 april 2020. data were analyzed using spss 21.0 (spss inc. chicago, il, usa). continuous variables are shown as median (interquartile range or iqr), and categorical data as percentage. the comparison of two medians was performed using the mann-whitney u test; and the comparison of percentages with the chi-squared test. interaction term of age (years) × gender (male) was tested as candidate variable in the logistic regression model. all factors with p � 0.25 in the univariate logistic regression model were included in the multivariate logistic regression model. multivariate analysis was performed to determine whether the admission neutrophil count > 6.3 ×10 9 /l was a risk factor for mortality, after adjusting for other variables. receiver operating characteristic (roc) curves were used to determine blood parameters at admission as predictors of mortality. a p-value < 0.05 was considered statistically significant for all analyses. overall, 93 persons were treated during the study period, 85 (49 men) of whom met the inclusion criteria for this study (table 1) . six patients were excluded due to absence of specific blood test within 12 hours after admission or negative sars-cov-2 result of nasopharyngeal swab sample. one patient in his 90s died of old age, although the condition of covid-19 was mild during hospitalization. this old patient was also excluded. the age of the study population was 64.00 (54.50, 70.00) years (range 31-89 y). the interval between symptom onset and admission was 13.00 (10.00,15.00) days. of these 85 patients, 44 suffered from concomitant chronic diseases. there was at least one ground glass change in the chest ct scans of each of these patients. median hospitalization days of patients who died was 4.00 (2.75, 6.25) days. the most common clinical symptoms are fever and cough. for this analysis, the 85 patients were assigned either to the survival group or the death group. seventy-one patients survived to discharge, while 14 patients died in hospital ( table 1 ). all non-survivors died within the first week of hospitalization, and all of them were in the acute stage of covid-19. there was no significant difference in age between the survival and death groups [62.00(55.00, 70.00) y cf. 67.00(50.75, 74.25) y; table 1 ]. in the death group, the percentage of men (11/14, 78 .6%) and rate of accompanying disease (10/14, 71.4%) were higher compared with the survival group (38/71, 53.52%; and 37/71, 52.11%, respectively), but the differences were not significant. the incidence of dyspnea in the death group was higher than that in the survival group ( table 1 ). the survival and death groups were also comparable regarding the interval between onset and admission [13.00 (10.00,15.50) d cf. 12.00 (9.00,15.25) d, table 1 ]. admission serum procalcitonin was measured in 61 patients, 9 of whom died. admission procalcitonin of death group was significantly higher than that of survival group [0.054 (0.030, 0.084) ng/ml cf. 0.163 (0.079, 0.328) ng/ml, p = 0.002]. the following blood parameters in the death group were significantly higher than in the survival group (table 1) : total leukocyte count (p = 0.000); percentage of neutrophils (p = 0.000), neutrophil count (p = 0.000) and hemoglobin (p = 0.017). upon admission, the percentage of the death group with crp >60 mg/l was significantly higher than that of the survival group [(21/ 71 (29.58%) cf. 11/14 (78.6%)]. in addition, 12/14 (85.71%) patients in the death group had an effect of immune function on the prognosis of patients with covid-19 admission neutrophil count > 6.3 × 10 9 /l, while this was true of only 12/71 (16.90%) of the survival group. in the death group, the percentage of lymphocytes, lymphocyte count, platelet count, and pao 2 /fio 2 were significantly lower than that of the survival group (p = 0.000, 0.002, 0.004, 0.000, respectively). 12/14 (85.7%) patients in the death group had lymphocyte count at baseline < 1.1 × 10 9 /l, however this was true of 41/71 (57.75%) of the survival group. in the 85 patients, the baseline median of the cd3+, cd4+, and cd8+ counts were all lower than the lower limit of the normal range ( table 2 ). the cd3+, cd4+ and cd8+ counts in more than 60% of patients were lower than the lower limit of normal range, respectively; the incidence rates were higher in non-survivors than survivors ( table 2 ). the median cd3+%, cd3 + count, cd4+%, and cd4+ count in the death group were all significantly lower than that of the survival group (p = 0.020, 0.007, 0.006 and 0.001, respectively). the medians of the cd8 + count and cd16+56+ count, cd19+ count and cd4+/cd8+ ratio in the death group were lower than those of the survival group, but the differences were not statistically significant. these patients overall were tested for humoral immunity function. however, there was no significant difference in igg, igm, iga, c3, c4 and the percentage of ige > 100 iu/ml between the death group and the survival group (table 3) . univariate logistic regression analyses were performed (table 4) . on univariate analysis, bmi, hemoglobin, platelets, neutrophil count > 6.3 ×10 9 /l, cd3+ count, cd4+ count, crp > 60 mg/l, pao 2 /fio 2 and lactate were found to be significantly associated with mortality (table 4 ). in addition, the p value of male gender, age (years) × gender (male), any comorbidity, lymphocyte count < 1.1 ×10 9 /l was < 0.25, respectively. multivariate logistic regression analyses were performed to determine which admission factors were independently associated with mortality ( table 5 ). all factors with p � 0.25 in the univariate logistic regression model were included in the multivariate model. after controlling areas under the roc curves (aucs) as predictors of mortality, determined from the analyses of blood parameters at baseline, were calculated and compared ( table 6 ). the aucs showed that the following could predict death due to covid-19: leukocyte count; neutrophil percentage; neutrophil count; lymphocyte percentage; lymphocyte count; cd3+ %; cd3+ count; cd4+ %, cd4+ count and pao 2 /fio 2 . in the survival group (n = 71), after treatments of symptoms, cough, shortness of breath, and dyspnea were relieved to varying degrees, and the chest ct gradually improved. four patients were well enough for discharge within 2 weeks after admission. the remaining hospitalized survivors (n = 67) underwent blood tests, 2 weeks after treatment. lymphocyte count and neutrophil count return to normal range in most patients (table 7) . however, in 13 of the 67 surviving patients after 2 weeks of treatment, the lymphocyte count was still below the lower limit of normal (1.1 × 10 9 /l) ( table 7 ). in addition, in 4 of the 67 surviving patients after 2 weeks of treatment, the neutrophil count was above the higher limit of normal (6.3 × 10 9 /l) ( table 7) . in 53 patients who tested cellular immunity of the survival group after 2 weeks of hospitalization, the count of lymphocyte subsets returned to normal range in most patients (table 8) . however, in 13 of these 53 patients, the cd3+ count remained lower than the lower limit of the normal reference value (723/μl); in 10 of these 55 patients, the cd4+ count was still lower than the lower limit of the normal reference value (404/μl). since december 2019, covid-19 has been prevalent in wuhan, china. it is highly contagious through human-to-human transmission, and in severe cases can lead to death [16] . patients with severe disease may have respiratory manifestations such as fever, cough, shortness of breath, dyspnea, and hypoxemia. chest ct examination of the patients shows predominant ground glass opacities mixed with consolidations, mainly peripheral or combined peripheral and central distributions, bilateral and lower lung zones being mostly involved [17] . in addition, covid-19 can attack and lead to the collapse of the immune system in the critically ill. serious inhibition of the immune system may promote replication of the virus and its spread throughout the body, damaging the function of multiple organs. the present retrospective study of patients with covid-19 found no statistical association between age and mortality, and none between the interval from onset to hospitalization and mortality. the percentage of men and rate of concomitant disease were higher in the patients who died compared with survivors, but the difference was not significant, perhaps due to the smallness of the sample. however, the admission (baseline) leukocyte count, neutrophil percentage, and neutrophil count of the death group were all significantly higher than that of the survivors. chang et al. [3] reported that an initial neutrophil count >7000/ml was an independent risk factor for death in patients with sars. singapore scholars also concluded that neutropenia predicted poor prognosis in sars [4] . the leukocyte counts of severe patients with covid-19 were significantly higher than those of non-severe patients [9] [10] [11] . for the present patients with covid-19, we found that neutrophil count was above the higher limitation of normal range in 12 (85.71%) dead patients and an admission neutrophil count > 10 9 /l was an independent risk factor of mortality. this significant elevation of neutrophil count in the death group, may reflect a strong inflammatory response toward viral infection, or a possible combination with bacterial infection. the specific mechanism needs further study. the report of wang et al. [10] on patients with covid-19 showed that the lymphocyte count continued to decrease with progression, unto death. the latest researches showed that the severe covid-19 cases had significantly lower lymphocyte counts compared with nonsevere covid-19 cases [9] [10] [11] [12] [13] [14] . the present study also determined that more than half of the patients (53/85, 62.35%) had lower lymphocyte count than the lower limitation of normal, especially in those who died (12/14, 85.7%). and we found that the lymphocyte counts in dead group were significantly lower than those in survival group. it is not clear whether there are other mechanisms for lymphocyte decline in covid-19, other than the destruction of lymphoid organs by viral attack. lymphopenia in patients with sars before hormone therapy may be induced by a stress mechanism involving the hypothalamus-pituitary-adrenal axis, due to elevation of the cortisol level [18] . lymphocyte subsets have an important role in humoral and cellular immunity against viral infection. the prognosis of various viral diseases is closely linked to cellular immune function, especially t lymphocyte function. qin et al. [10] found that number of helper t cells, suppressor t cells and regulatory t cells decreased in patients with covid-19. wang et al. [14] reported that cd4+ t cells, cd8+ t cells, b cells, and natural killer (nk) cells decreased in patients with covid-19, and were significantly lower in severe cases than mild cases. chen et al. [9] showed that cd4+t and cd8+t cells reduced in nearly all patients with covid-19, and severe cases had a lower level than moderate cases. in the present study of patients with covid-19, the baseline median values of cd3+, cd4+, and cd8+ counts were lower than the lower limit of the normal range. what is more noteworthy is that the cd3+ and cd4 + count levels in the patients who died was significantly lower than that of the survivors. the baseline cd8+, cd19+, and cd16+56+ counts in those who died were also lower compared with the survivors, although the differences between the two groups were not statistically significant. a study reported the suppression of cellular immunity in patients who died of viral pneumonia [19] . one study found that the counts of peripheral cd4+ and cd8+ t cells were substantially lower than normal in a 50-year-old man who died from covid-19 infection [15] . we speculate that the decrease in number of lymphocyte subsets in covid-19 may be caused by an attack of the immune system by sars-cov-2. lam et al. [20] reported that cd3+, cd4+, cd8+, and natural killer cell counts were promising predictors for intensive care unit admission in patients with sars. in the present study, the roc analysis determined that admission leukocyte count, neutrophil percentage, neutrophil count, lymphocyte percentage, and lymphocyte count were able to predict the mortality of covid-19 patients. in terms of cellular immune function, cd3+%, cd3+ count, cd4+%, cd4+ count and pao 2 /fio 2 could also predict mortality. clinicians should pay attention to these parameters when caring for these patients. admission lymphocyte and its subsets count significantly decreased in the acute phase of covid-19, especially in non-survivors. in additional to physical examination and imaging examination, blood analysis, including lymphocyte count, neutrophil count and lymphocyte subsets count, is also an important method to assess the condition of patients with covid-19, guide treatment and predict outcome. at present, there is no study about humoral immunity function of patients with covid-19. we found that the difference of igg, igm, iga, c3, c4 and the percentage of ige > 100 iu/ ml was not significant between the survivors and non-survivors. the baseline platelet level in patients who died was lower than that of the survivors. reference to other virus researches, this may be due to platelet destruction caused by the virus, or inhibition of the production of platelets in bone marrow via viral infection [21] , or platelet consumption caused by the formation of thrombus at the injured site of lung [22, 23] . the hemoglobin level in the dead patients is higher than that in the surviving patients, which may be due to the severe lung injury in the dead patients and the irritant increase of hemoglobin caused by hypoxemia. compared with admission, after 2 weeks of hospitalization the blood routine examination of 67 patients in the group who survived showed neutrophil and lymphocyte count of most patients returned to the normal range. it is worth noting that the lymphocyte count levels in 13 of the 67 surviving patients did not rise to the lower limitation of normal levels (1.1 × 10 9 / l) after 2 weeks of treatment, suggesting that the immune function of these patients had not returned to normal. in addition, the lymphocyte subsets of some patients also did not return to the normal range, which suggested that some patients have a relatively long time of impaired cellular immune function. the positive rate of sars-cov-2 rna in nasopharyngeal swabs was not satisfactory. when considering the hospital discharge of patients with covid-19, improved symptoms, obvious absorption in the chest ct, and consecutive negative nucleic acid tests are important. however, we recommend in addition that lymphocyte count and cellular immune function tests should be conducted to ensure that the disease has been effectively controlled. a previous study of patients with sars showed that, after two years, the counts of peripheral blood lymphocytes and subsets, comprising lymphocytes, cd4+, cd8+, and nk cells (cd16+56+), had remained lower than that of the healthy controls [24] . in patients with covid-19, changes in lymphocytes and the lymphocyte subsets during recovery need to be studied. there are several limitations to the present work. firstly, it is a single-center retrospective with a small sample size. however, to the best of our knowledge, it is the first to analyze the dead and survival patients with covid-19 the routine blood and immune function indexes. secondly, in surviving patients the selected timepoints for examining blood indexes were at admission and 2 weeks after beginning hospitalized treatment, which may not accurately reflect the continuous dynamic changes in leukocytes, neutrophils, lymphocytes, and lymphocyte subsets. thirdly, medical work was carried out under emergency conditions in february 2020, and bacterial culture test was not performed in every patient. the situation of concomitant bacterial infection is not particularly clear. fourthly, the number of patients given immune function tests who later died is relatively small; and the only evaluations of immune function were for cellular immunity and humoral immunity. fifthly, this is an explorative study. sample size was small and formal sample size calculation for multivariate logistic regression analysis was not done in present study, which might affect the repeatability of the relevant results. it is expected to have a larger sample size study to further explore the effect of admission blood index on patients and valid the results. finally, a time to event analysis was not applied in present study, because of relatively small number of deaths and study population. of note, the mortality rate in this study was relatively high. one study of covid-19 patients reported a death rate of 6 patients (4.3%) among 138 patients [6] . in another study, the mortality rate of covid-19 was 11.0% [25] ; while a recent large-sample and multicenter study showed that only 1.36% of patients succumbed [26] . we should point out that all the patients in the present study who died did so before 11 february 2020. the higher mortality rate was likely the result of the heightened severity of disease of admitted patients at this early stage of the pandemic in china. in addition, the treatment area was a temporary structure, expediently built, without a well-equipped intensive care unit, and the medical conditions were insufficient for the need. in this population of covid-19 patients, the admission leukocyte count, neutrophil percentage and neutrophil count in those who died within 28 days were all higher than that of the survivors, and the lymphocyte count, cd3+ count and cd4+ count was lower. leukocyte, neutrophil, lymphocyte, cd3+ and cd4+ count at admission could predict the mortality due to covid-19. admission neutrophil count > 6.3×10 9 /l was independently associated with mortality. however, 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of 2019 novel coronavirus infection in china key: cord-310790-3ikgmiof authors: cherrak, sabri ahmed; merzouk, hafida; mokhtari-soulimane, nassima title: potential bioactive glycosylated flavonoids as sars-cov-2 main protease inhibitors: a molecular docking and simulation studies date: 2020-10-15 journal: plos one doi: 10.1371/journal.pone.0240653 sha: doc_id: 310790 cord_uid: 3ikgmiof a novel coronavirus responsible of acute respiratory infection closely related to sars-cov has recently emerged. so far there is no consensus for drug treatment to stop the spread of the virus. discovery of a drug that would limit the virus expansion is one of the biggest challenges faced by the humanity in the last decades. in this perspective, to test existing drugs as inhibitors of sars-cov-2 main protease is a good approach. among natural phenolic compounds found in plants, fruit, and vegetables; flavonoids are the most abundant. flavonoids, especially in their glycosylated forms, display a number of physiological activities, which makes them interesting to investigate as antiviral molecules. the flavonoids chemical structures were downloaded from pubchem and protease structure 6lu7 was from the protein data bank site. molecular docking study was performed using autodock vina. among the tested molecules quercetin-3-o-rhamnoside showed the highest binding affinity (-9,7 kcal/mol). docking studies showed that glycosylated flavonoids are good inhibitors for the sars-cov-2 protease and could be further investigated by in vitro and in vivo experiments for further validation. md simulations were further performed to evaluate the dynamic behavior and stability of the protein in complex with the three best hits of docking experiments. our results indicate that the rutin is a potential drug to inhibit the function of chymotrypsin-like protease (3cl pro) of coronavirus. the virus is responsible of an elevated number of deaths as its transfer speed is higher than other coronaviruses. sars-cov-2 infection has then become a threat to public health. this disease caused by sars-cov-2 is affecting patients of a mild disease in most of them. however, in few cases, patients develop severe acute respiratory distress syndrome [13] . until today, no specific vaccine has been developed to stop the spread of the virus and the need of a cure is now urgent. viruses possess the ability to adapt and mutate quickly, leading research to find new therapeutic approaches. the main approach is to block the life cycle of the virus or to interfere in its fusion with the membrane so that the life cycle can be interrupted [14] [15] [16] [17] . another approach is the development of new drugs based on computational research tools. these tools are now fundamental to gain time and accuracy for developing new pharmacophores [18] [19] [20] . the process of identifying novel antiviral drugs to face the disease can rely on the screening of existing natural compounds known for their antiviral activity [21] . as flavonoids (fig 1) are one of the most widely distributed plant compounds and play essential roles in plant physiology, they have been intensively investigated. in plants, flavonoids are mainly synthesized as aglycones, glycosides and methylated derivatives. they are biosynthesized through the phenylpropanoid pathway, transforming phenylalanine into 4-coumaroyl-coa, which then enters the flavonoid biosynthesis pathway [22] . they possess a wide range of biological activities such as antioxidant [23] [24] [25] [26] , anticancer [27, 28] , antibacterial [29, 30] , antifungal [31] and antiviral activity [32] [33] [34] . different classes of flavonoids display antiviral activities in vitro and in vivo specifically, apigenin, luteolin, quercetin [35, 36] . flavonols, the most widely spread flavonoids (fig 1) are characterized by a 3-hydroxy-2-phenylchromen-4-one backbone; and quercetin is the first representative compound and the most extensively investigated. recent investigations also pointed out the antiviral activity of quercetin against a wide spectrum of influenza virus strains. it interacts with influenza hemagglutinin protein, thereby inhibiting viral-cell fusion [37] . several other flavonols and derivatives have antivirals activities as a sulfated substituted rutin, showing significant activity against different hiv-1 isolates [38] . several flavonoids are known by inhibiting virus proliferation either by stopping virus multiplication or by blocking cell entry. indeed, several natural compounds including baicalin, scutellarin, hesperetin, nicotianamine and glycyrrhizin were predicted to have a capacity to binding ace2 receptor with potential anti-sars-cov-2effects [36] . moreover, quercetin, daidzein, puerarin, epigallocatechin, epigallocatechingallate, gallocatechingallate and kaempferol were reported to inhibit the proteolytic activity of sars-cov 3cl hydrolase [39] . proteases and spike proteins are targets of choice for inhibition of sars and mers, and constant work is made to find novel molecules which can be able to interfere with virus life cycle either by computational methods or by experimental investigation [17, [40] [41] [42] . the sars-cov-2 main protease (mpro), a chymotrypsine like hydrolase (3cl hydrolase) is an attractive target for anti-cov drug design due to its responsibility for the maturation of main functional enzymes such as replicase and helicase [43] [44] [45] . in this work, a screening of natural flavonoids has been made and their potential inhibitory effect against sars-cov-2 mpro by molecular docking and molecular dynamics has been tested. indeed, our computed data suggest that strong interaction occur between flavonoids with sugar moieties and the main protease of the sars-cov-2virus. these results can represent a promising starting point for antiviral therapies that are alternative to the vaccination strategy. the three-dimensional structure of flavonoids were obtained from pubchem, in.sdf format and the structures were optimized using the avogadro software and saved as mol2 files. the 3d structure of sars-cov2 main protease (mpro, 3clpro) (pdb id: 6lu7) protein was obtained from protein data bank (https://www.rcsb.org/). the resolution of the retrieved structure was 2.16 å. the mpro 3d structure was loaded into ucsf chimera for molecular docking preparation [46] . the inhibitory effect of thirty eight flavonoids on sars-cov-2 mpro was studied by docking experiments using autodock vina software. the protease crystal structure consists of a 306 residues length chain with an excellent resolution of 2.16 å co-crystallized with an engineered peptide inhibitor (n3) [47] . ucsf chimera was used to remove all water molecules, heteroatoms, and co-crystallized solvent. the inhibitor was also abstracted from the pdb file and saved as a pdb file and used as positive control during docking studies. finally, polar hydrogens and partial charges were added to the structure using ucsf chimera point for antiviral therapies that are alternative to the vaccination strategy [46] . molecular docking was finally performed by the autodock vina [48] program in a rectangular box. the retained flavonoids were those with conformations of lower binding energy which are matching the active site defined by the co-crystallized n3 inhibitor. molecular dynamics simulations were performed with the gromacs 2020 package using charmm36 force field installed on linux system [49, 50] . the protein topology was prepared with the pdb2gmx tool. the protein complexes were solvated in a triclinic box. energy minimization of the system was performed by using the steepest descent algorithm [51] . protein was solvated by tip3 solvent model and 4 na + ions were introduced into the system to make the whole system neutral. the complexes consisting of the flavonoids and the mpro were solvated in a water box with a minimal margin of 1.5 å from any edge to any protein atom. an overall pressure and a temperature equal to 1 bar and 300 k were used with a time gap of 2 fs to stabilize the system. temperature was kept constant inside the box, the vrescale, an optimized berendsen thermostat temperature coupling technique, was used. parrinello-rahman pressure coupling method was utilized in npt equilibration. energy minimization of the system was carried out by the steepest descent algorithm for 50,000 iteration steps and cut-off up to 1000 kj.mol -1 to reduce the steric clashes. particle mesh ewald (pme) was employed to treat long-range electrostatics interactions with a coulomb cut-off of 1.0 nm. the system was subjected to a final 50 ns molecular dynamic run each step of 2 fs. trajectory analysis was performed using various gromacs analysis tools. the gmx rms, and gmx rmsf tools were respectively used to calculate the root mean square deviation (rmsd) and the root mean square fluctuations (rmsf) of protein. hydrogen bonds were analyzed using gmx hbond tool, and the radius of gyration with the gyrate tool. finally, plots were prepared using grace software. the crystallographic structure of 3c like protease of sars-cov-2 (pdb id: 6lu7) in complex with a peptide-like inhibitor (n3) was made very recently available in the protein data bank. from the above mentioned structure, we can clearly see a closed binding pocket around the peptide-like inhibitor. docking of all flavonoid structures (fig 2) was done against the active site of mpro protein. docking analysis provided several configurations that were scored to determine favorable binding modes. the flavonoid structures with the highest docking scores are summarized in table 1 . the three compounds with highest affinity (fig 2) for the active site are quercetin 3-rhamonoside, myricetin 3-rutinoside and rutin with binding energies of -9.7, -9,3 and -9.2 kcal.mol -1 respectively. these values are higher than those obtained for the n3 inhibitor (-7.5 kcal.mol -1 ). thirty eight flavonoids have been tested in this study by molecular docking against the active site of the sars-cov-2mpro. the results which are summarized in table 1 show that natural aglycone flavonoids possess high docking scores; with a maximum score achieved by egcg with -7,9 kcal.mol -1 . this value is comparable to the score obtained for n3 inhibitor co-crystallized with the protease (6lu7), used here as a positive control (-7,7 kcal/mol). furthermore, we can clearly see that glycosylated flavonoids display the highest scores; with a maximum value of 9,7 kcal.mol -1 for quercetin 3-rhamnoside. as shown in fig 3, quercetin 3-rhamnoside is well installed in the active site and the position is matching with the n3 peptide inhibitor. the best compounds possess sugar moieties in their structure, and the position seems to be important. compounds substituted at the carbon 3 display higher scores than those substituted at the carbone 7. genistin, a flavonoid substituted at position 7 displays low score (-7,5 kcal. mol -1 ) compared to datiscin or hyperoside (-8,2; -8,4 kcal.mol -1 ) among others. glycosylated flavonoids as sars-cov-2 inhibitors: a molecular docking and simulation studies the nature of the sugar moiety is also important to the activity.as we can see, the top 5 compounds are flavonols and they possess either a rhamnose or a rutinoside (a dissaccharide: glucose-rhamnose) at position 3. quercetin 3-rhamnoside (fig 3) and myricetin 3-rutinoside bind with more strength than datiscin which is substituted at position 3 with glucose. however, low bioavailability of flavonoids is a major inconvenient to their use as natural drugs. the presence of sugar moieties usually leads to enhanced bioavailability of the respective flavonoid aglycone depending on the nature of the substituted sugar. flavonoids with glucose moieties seems to be absorbed more rapidly than flavonoids with rhamnosides, rhamnoglucosides, rutinosides and neohesperidosides substitutions [52] . it is assumed that bioavailability of glyco-flavonoids is achieved by aglycone part in blood plasma after the cleavage of the glycosidic part by hydrolyzing enzymes in the human gastrointestinal tract [53] . glucose moieties are easily hydrolyzed in the small intestine epithelial cells. however, the lack of α-l -rhamnosidase or rutinosidase in human cells makes the bioavailability of relative flavonoids largely dependent on their hydrolysis by intestinal bacteria [53] . furthermore, few intestinal bacterial strains can achieve cleavage of these types of bonds [54] . discovery studio visualizer was further used to determine hydrogen bonds occurring between amino acids of the active site and flavonoids (fig 5) . the n3 inhibitor was docked to the sars-cov-2mpro to be further compared to docked generated model with autodock vina. the superimposed structures of the co-crystallized n3 inhibitor (fig 4) are closely related indicating a good accuracy of the automated tool. results (table 2) show that compounds that undertake bonds with the highest strength usually bind to thr26, ty54, his41, asn142, gly143, cys145and glu166 in the substrate binding subsite s1 fig for the proteolytic activity [55, 56] , leading to strong binding due to hydrogen bonds. mpro consists of three domains and substrate binding site resides in a cleft between domain i and ii, while domain iii is involved in catalytic function. dimerization of the protein is required for its catalytic regulation [57] . glu166 residue is a key amino acid involved in the dimerization of mpro and creation of substrate binding pocket [45, 58] . cys141 and his41 residue forms a catalytic dyad on the active site of the protein essential for its catalytic function. similar results were recently obtained by molecular docking with different molecules against the mpro protein [57, 59, 60] . quercetin 3-rhamnose, a flavonol with a rhamnose at position 3 of the c ring, is involved in several hydrogen bonds contracted with thr26, phe140, leu141 and gly143 (fig 5a) . the second and third flavonoids with highest affinity, myrictin 3-rutinoside and rutin are also flavonols but with a rutinoside (glucose-rhamnose) at the 3 position. fig 5b and 5c show that both compounds display hydrogen bonds with mpro leading to high affinity binding to the active site. results show that glycosylated flavonols with a sugar moiety at position 3 with at least a rhamnose in their structure are the compounds that bind with higher affinity to the active site pocket of the mpro. molecular dynamics simulations are of proven in-silico methods for obtaining dynamic data at atomic spatial resolution [2, 51] . the docked mpro complexes with the three compounds with highest scores: quercetin -3 rhamnose, myricetin 3-rutinoside and rutin were subjected to md simulations for 50 ns to analyze the stability of the complexes. rmsd plot is usually used to understand the stability of the complex while rmsf is used to understand the structural flexibility (fig 6) the mpro in complex with rutin (green) showed the most stable rmsd during the 50 ns simulation around 0.2 nm. the m pro with compound myricetin 3-rutinoside (red) is also stable and constant range of rmsd between 0.15 nm and 0.25 nm. slight increase was observed after 40 ns simulation. the quercetin 3-rhamnose (black) showed stable rmsd between 5 and 40 ns with values close to 0.3 nm. after 40 ns rmsd value decreased to 0.25 nm (fig 6) . the differences of backbone rmsd in quercetin 3-rhamnoside and myricetin 3-rutinoside indicate that m pro undergoes conformational changes. rmsf trajectories provide important information regarding the stability of the complex. high fluctuations in the plot indicate more flexibility and unstable bonds. on the other side, low value or less fluctuation indicates well structured regions in the complex and less distortion. as depicted in fig 6, all the systems showed almost similar patterns. the overall value of rmsf for all the three complexes was around 0.1 nm as clearly seen in fig 6. in order to determine the compactness of the system, rg was plotted against time. higher rg values illustrate less compactness with conformational entropy, while low rg values are the simulation rg values of the three compounds reported as 2.2-2.3 nm. the fewer changes in the rg value showed the stability of the protein in the complex suggesting that the binding of these three molecules does not induce structural changes. the rg range of the three complexes (fig 7) support their condensed architecture as well as size. moreover; potential and kinetic energy plots confirm the stability of the complexes (s1 fig). hydrogen bonding plays an essential role in determining the binding strength between ligands and protein. rutin (green) id (black) has constant range of hydrogen bond between 2 to 4 in whole simulation while quercetin 3-rhamnose and myricetin 3-rutinoside showed changes in bonding. more hydrogen(>6) bonds between 10 to 25 ns for myricetin 3-rutinoside, suggesting a conformational change in the binding site during simulation (fig 7) . over all, observation suggested that mpro protein in complex with the three compounds is stable during simulation. however, it is clear that rutin complex is more stable and therefore a better candidate as potential drug for inhibiting the mpro protein. until now, the spread of sars-cov-2has stimulated research to find an existing drug for a rapid treatment. at that time, the confirmed cases are over 7 million and rising, and the deaths are approaching 400000, and governments fear a second infection wave. isolation strategy was mandatory to limit the spread of the virus, but solutions have to be found urgently. the main protease taken into account as molecular target for stopping the spread of the virus is an interesting strategy. results show that glycosylated flavonoids display a strong inhibitory activity on sars-cov-2 mpro. glycosylated flavonoids seems to bind with more strength than the n3 co-crystallized inhibitor. the binding of the flavonoids at the protease active site is clearly structure dependant. compounds binding with more strength are flavonols substituted with mono or disaccharides at position c3. the nature of sugar is also primordial to the activities, as flavonoids with a rhamnose possess the strongest binding affinity. as flavonoids with rhamnose moiety undergo no or little modification in the intestine, they might be a good candidate in the treatment of the sars-cov-2coronavirus. coronavirus disease covid-19: a new threat to public health identification of phytochemical inhibitors against main protease of covid-19 using molecular modeling approaches the sars-cov-2 outbreak from a one health perspective. one elucidating biophysical basis of binding of inhibitors to sars-cov-2 main protease by using molecular dynamics simulations and free energy calculations rg and hydrogen bonds plots for mpro complex with quercetin -3 rhamnose (black), myricetin 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biological significance? critical reviews in food science and nutrition isoquercitrin: pharmacology, toxicology, and metabolism hydrolysis of the rutinose-conjugates flavonoids rutin and hesperidin by the gut microbiota and bifidobacteria hispidin and lepidine e: two natural compounds and folic acid as potential inhibitors of 2019-novel coronavirus main protease (2019-ncovmpro), molecular docking and sar study. current computer-aided drug design combined drug repurposing and virtual screening strategies with molecular dynamics simulation identified potent inhibitors for sars-cov-2 main protease (3clpro) coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs identification of potential natural inhibitors of sars-cov2 main protease by molecular docking and simulation studies withanone and caffeic acid phenethyl ester are predicted to interact with main protease (mpro) of sars-cov-2 and inhibit its activity phylogenetic analysis and structural perspectives of rna-dependent rna-polymerase inhibition from sars-cov-2 with natural products. interdisciplinary sciences: computational life sciences key: cord-333853-p2kbjwpy authors: smee, donald f.; wong, min-hui; russell, andrew; ennis, jane; turner, jeffrey d. title: therapy and long-term prophylaxis of vaccinia virus respiratory infections in mice with an adenovirus-vectored interferon alpha (mdef201) date: 2011-10-13 journal: plos one doi: 10.1371/journal.pone.0026330 sha: doc_id: 333853 cord_uid: p2kbjwpy an adenovirus 5 vector encoding for mouse interferon alpha, subtype 5 (mdef201) was evaluated for efficacy against lethal vaccinia virus (wr strain) respiratory infections in mice. mdef201 was administered as a single intranasal treatment either prophylactically or therapeutically at doses of 10(6) to 10(8) plaque forming units/mouse. when the prophylactic treatment was given at 56 days prior to infection, it protected 90% of animals from death (100% protection for treatments given between 1–49 days pre-infection), with minimal weight loss occurring during infection. surviving animals re-challenged with virus 22 days after the primary infection were protected from death, indicating that mdef201 did not compromise the immune response against the initial infection. post-exposure therapy was given between 6–24 h after vaccinia virus exposure and protection was afforded by a 10(8) dose of mdef201 given at 24 h, whereas a 10(7) dose was effective up to 12 h. comparisons were made of the ability of mdef201, given either 28 or 1 day prior to infection, to inhibit tissue virus titers and lung infection parameters. lung, liver, and spleen virus titers were inhibited to nearly the same extent by either treatment, as were lung weights and lung hemorrhage scores (indicators of pneumonitis). lung virus titers were significantly (>100-fold) lower than in the placebo group, and the other infection parameters in mdef201 treated mice were nearly at baseline. in contrast, viral titers and lung infection parameters were high in the placebo group on day 5 of the infection. these results demonstrate the long-acting prophylactic and treatment capacity of mdef201 to combat vaccinia virus infections. the threat of using orthopoxviruses, variola and monkeypox, as bioterror weapons has led to increases in vaccination particularly in military personnel as well as the investigation of countermeasures (antiviral treatments) for such infections [1] . a number of compounds have been identified that exhibit direct antiviral activity against these and related poxviruses in animal models [2, 3] . from these investigations, three compounds stand out as being clinical candidates, cidofovir [4] , cmx001 (an orally active prodrug of cidofovir) [5] , and st-246 [6] . cidofovir, cmx-001, and st-246 have all been used in emergency settings to treat vaccination complications [7, 8] . immune system stimulation via exogenous recombinant interferon (rifn) is effective for treating vaccinia virus respiratory infections in mice [9] . single daily doses of rifna or rifnc rescued mice from lethality when administered 1-2 days after virus challenge and reduced lung virus titers. both vaccinia keratitis and vaccinia-induced skin lesions were treated with rifn (topically and injected respectively) in monkey models with success [10, 11] . vaccinia and other poxviruses have evolved comprehensive mechanisms to antagonize the interferon system [12] , that include blocking ifn gene induction, disrupting extracellular and intracellular signaling and slowing ifn induced gene (pkr and 2959 oas) activation. treatment with exogenous rifn only compensates for host ifn gene suppression and swamping the ifn binding protein so it must begin early after infection before the virus suppresses activation of the antiviral state. interferon has a short half-life of three hours [13] , which requires frequent treatment with large bolus doses resulting in commensurate adverse effects often leading to patient-initiated cessation of treatment [14, 15] . pegylated rifn has improved the in vivo half-life to allow for weekly injections, but results in a reduced activity of the protein and an increased cost. a needle-free, single dose drug capable of achieving steady-state method of delivering interferon would maximize the therapeutic benefits of ifn, while minimizing the bolus-induced toxicity. to this end, a replication-deficient adenovirus 5 (ad5) vector containing the human consensus interferon alpha gene, referred to as def201, has been developed. intranasal administration of def201 allows for the ad5 vector to infect respiratory cells and drives constitutive expression of the ifn transgene and secretion of fully glycosylated ifn. ad5-vectored mouse interferon (mdef201) resulted in sustained ifn levels [16] , that completely protected mice from a lethal western equine encephalitis virus infection when given intramuscularly at 10 7 plaque forming units (pfu)/ mouse up to 7 days prior to virus challenge [16] . against venezuelan equine encephalitis virus infection, mdef201 prevented death when administered intramuscularly at 10 7 pfu/mouse 24 h prior to infection but not when given 6 h after infection [17] . intranasally-administered mdef201 was also used to treat mice infected intranasally with sars virus (resulting in a lethal respiratory infection) [18] . it was 100% protective when administered prophylactically at 10 6 pfu/ mouse up to 14 days pre-virus exposure, with similar protection afforded by a 10 8 dose administered therapeutically at 12 h after infection [18] . adenoviral vectored human consensus ifn (def201), was recently used intranasally to treat hamsters infected intraperitoneally with yellow fever virus (resulting in a lethal hepatic infection) [19] . protection from death was observed for single administrations of def201 at 10 7.5 pfu/animal given 7 days before to 2 days after virus challenge, which demonstrated both prophylaxis and therapy in this model. def201 treatment reduced yellow fever virus titers in liver and spleen, and resulted in decreases in serum alanine transaminase levels. herein, mdef201 was evaluated for prophylaxis and therapy of vaccinia virus (wr strain) respiratory infections in mice. in this model the virus infection spreads systemically to include other tissues besides the lungs, particularly the liver and spleen [20] . increases in lung weight and lung hemorrhage (indicative of pneumonitis) are hallmarks of vaccinia infection, as is severe body weight loss. the results show that a single intranasal dose of mdef201 produced a remarkably prolonged 8-week prophylactic effect, and was also active therapeutically. the experiments were conducted in accordance with protocol 552 approved by the institutional animal care and use committee of utah state university. the work was done in the aaalac-accredited laboratory animal research center of utah state university in accordance with the national institutes of health guide for the care and use of laboratory animals (revision; 2010). female balb/c mice (charles river labs, wilmington, ma) weighing approximately 13-15 g at the time of first treatment were used. after a 48-hour quarantine period the animals were randomly assigned to cages. all work with these animals was performed in the biosafety level 2 area of the aaalacaccredited laboratory animal research center at utah state university. the adenovirus vectored mouse interferon alpha, subtype 5 construct mdef201 was prepared at the robert fitzhenry vector laboratory (mcmaster university, hamilton, canada). the methods of preparation have been described previously [16] . briefly, the mouse interferon alpha gene (subtype 5) was cloned into a replication deficient ad-5 vector (deletions of e1 and e3 gene deletions), amplified in 293 cells and purified by cesium chloride gradient centrifugation. stock solutions of mdef201 were stored at 280uc and were thawed on ice and diluted in saline to the appropriate dose immediately prior to a single administration. gilead sciences (foster city, ca) provided the positive control compound cidofovir. vaccinia virus (wr strain) was purchased from the american type culture collection (atcc, manassas, va). the virus was propagated in african green monkey kidney (ma-104) cells (from ma bioproducts, walkersville, md) for use in these studies. mice were anesthetized with ketamine/xylazine (50/5 mg/kg) by intraperitoneal (i.p.) injection for intranasal treatments and intranasal infection. the animals were initially infected intranasally with approximately 1-2610 5 pfu of vaccinia virus in a 50-ml volume. mdef201 was administered intranasally (50-ml) either prior to or after vaccinia virus exposure, according to the experimental schedule. placebo-treated mice were given saline. in order to maintain consistency since intranasal treatment alters the environment for infection, all mice were given intranasal saline when the mdef201 treatments were given 1 day before or after virus infection, or else additional placebo groups were used to accommodate delivery of intranasal liquid. all animals were weighed every other day. in some instances a normal control group (untreated, uninfected) of 10 mice was maintained throughout the study. for certain studies, the normal control group was infected with virus at the end of the initial 21-day antiviral study to serve as naïve, infected controls for the re-infection. treated mice surviving the primary infection were re-infected on day 22 with approximately 5610 5 pfu units of vaccinia virus in a 50-ml volume. this dose was larger than for the primary infection to account for the increased age of the mice, and older mice are less susceptible to vaccinia infection. the age-matched normal control (naïve) animals were infected for the first time with the same virus challenge. survival and body weights in these groups were determined over a 21-day period. separate animals were maintained for determination of tissue virus titers, lung hemorrhage scores, and lung weights. groups of 5 infected mice per day were sacrificed for removal of tissues. lungs were given a hemorrhage score (color change from pink to plum which occurs regionally in the lung rather than gradually over the entire lung) ranging from 0 to 4 (entire lung affected). lungs, spleens, livers, and snouts were weighed prior to homogenization which releases infectious virus for titration. homogenization of soft tissues was done in 1 ml of cell culture medium using a stomacher. snouts were ground in 1 ml of medium using sterilized mortars and pestles. samples were serially diluted in 10-fold increments and plaque titrated in 12-well microplates of vero cells. plaques were stained at three days with 0.2% crystal violet in 10% buffered formalin, then the plaques were counted with the aid of a light box. plaque numbers were converted to pfu per gram of infected tissue. initially, survivor numbers were compared by multiple group chi square analysis. when statistical significance was found, survivor numbers were evaluated using the two-tailed fisher exact test. differences in the mean day of death, tissue virus titers, lung weights, and lung hemorrhage scores were statistically analyzed using the two-tailed mann-whitney u-test. analyses were performed using the instath computer software program (graphpad software, san diego, ca), comparing treated and placebo groups. the short-term prophylactic activity of mdef201 administered at various intranasal doses one day prior to virus challenge is presented in table 1 . mdef201 was uniformly, 90-100% protective from a lethal vaccinia challenge at doses of 10 5 to 10 7 pfu/animal. this protective effect can be attributed to the mifn transgene because the empty adenovirus vector was not protective and produced similar complete mortality as the placebo control. cidofovir (positive control compound) was also 100% protective at 100 mg/kg/day when administered by intraperitoneal route starting 1 day post-infection. table 1 also shows the effects of treatment with mdef201 and cidofovir on lung infection parameters. a dose-responsive effect on lung virus titer was seen with mdef201 treatments, with the highest dose causing nearly an 800-fold reduction in lung virus titer. mdef201 doses of 10 5 and 10 6 pfu/mouse reduced virus titers 8-and 125-fold, respectively. all three mdef201 treatments significantly reduced lung weights and lung hemorrhage scores. in contrast, cidofovir treatment caused a only a 40-fold reduction in virus titer, and protected lungs from virus-induced pathology. lung virus titers and lung weights were also intermediate between the 10 5 and 10 6 mdef201 dosage groups. neither the control, nor adenovirus empty vector inhibited lung infection parameters. to measure the window of prophylactic activity, 10 7 pfu/mouse of mdef201 was administered at various times prior to virus infection ( table 2 ). in the first experiment a single dose of mdef201 was 100% protective when administered between 28 days and 1 day pre-virus exposure. mean body weights during the course of the primary infection are presented in figure 1a . all of the treatments with mdef201 largely protected the animals against weight loss. a slight drop in weight occurred on day 1 following intranasal exposure to virus, but the mice were back to the initial starting weight by day 3. seven placebo-treated animals survived the infection, differing from that of table 1 where no animals survived. this likely occurred because the mice at the time of infection were 28 days older, and susceptibility of the animals to infection diminishes with age. severe weight loss in the placebo group persisted until day 11 of the infection. after that time, the seven animals that survived the infection began to recover body weight. the surviving mice from experiment 1 were re-infected with a 5-fold higher challenge of vaccinia virus than was originally given, and all of these animals survived the re-challenge ( table 2) . infection of age-matched naïve mice resulted in expected 100% mortality. weight loss occurred in all mdef201 treated groups, but was much less severe than in naïve, infected mice ( figure 1b) . rebound from weight loss during the re-infection was fastest in the placebo group. this group contained the most seriously afflicted animals during the primary infection, thus would be expected to have a more robust immune response than treated animals that did not get as sick. based upon the successful extended prophylaxis up to 28 days pre-infection, a second experiment ( table 2 ) was initiated using starting times as early as 56 days before virus challenge. mdef201 was 90% protective when administered at -56 days, and 100% protective when given at all shorter time points (,49 days). because the virus challenge dose was higher than that given in experiment 1 (see footnote ''b'' in table 2 ), the result was that all placebo-treated animals in experiment 2 died from the primary infection. minimal weight loss occurred in groups treated prophylactically with mdef201 ( figure 1c ). decreases in body weight in mdef201 groups were primarily seen on days 5-9 of the infection, with the nadir being day 7. the surviving mice that were treated with mdef201 in the second experiment (table 2) were re-infected with a 2-fold higher challenge of vaccinia virus, and all of these animals survived. infection of age-matched naïve mice resulted in 100% mortality. weight loss occurred in all mdef201 treated groups, but was minimal and occurred on days 3-5 ( figure 1d ). this is in contrast to the rapid weight decline in naïve, infected mice. body weights during the re-infection were similar for most mdef201 groups. slightly less rapid recovery in body weight over 21 days was seen in the mdef201 group treated prophylactically at -56 days compared to the other treated groups. viral titers in various tissues were determined from mice treated with mdef201 either at 28 days or 1 day prior to infection. lung virus titers in mdef201 treated groups were at least 100-fold less than in placebo treated mice (figure 2a ). liver and spleen virus titers were below or near the level of detection in animals treated with mdef201, but increased over time in placebos ( figures 2b and 2d) . snout virus titers in mdef201 treated groups were significantly less than placebos on day 3 but not on day 5 ( figure 2f ). in comparing virus titers in the -28 day mdef201 group to that of the -1 day mdef201 group, the amounts of detectable virus were nearly equivalent in lung, liver and spleen. differences were observed in snout virus titers between these two groups, but due to variability the differences were not statistically significant. lung weights and lung hemorrhage scores were determined in infected mice in parallel with the determination of viral titers. lung weights in mdef201 treated animals were near normal, whereas on day 5 the placebos exhibited a large increase in lung weight ( figure 2c) . similarly, lung scores were near normal in the two groups treated with mdef201, but were severe on day 5 in the placebo group ( figure 2e ). the extent of inhibition of lung intranasal treatments with mdef201 (10 7 pfu/mouse) were given one time only on the indicated day prior to virus exposure. in experiment 1 the primary intranasal challenge was 1610 5 pfu of virus. since this challenge dose failed to cause 100% mortality (as mice age their susceptibility to infection wanes), the primary virus challenge dose was increased to 2.5610 5 pfu of virus for experiment 2. reinfection of both sets of mice was performed intranasally with 5610 5 pfu of virus. the data accompany those of table 2 . thus, the initial number of mice per group for each figure corresponds to the total number per group in the table. doi:10.1371/journal.pone.0026330.g001 disease in the -28 days mdef201 group was comparable to that seen in the -1 day mdef201 group. the results from these experiments indicate that mdef201 has an extremely long-acting prophylactic activity against vaccinia virus infections in mice. only in animals treated 56 days prior to infection was there a hint of waning activity, since one animal died from the infection in that group. mdef201 at two different doses was administered after infection to combat a vaccinia virus infection. preliminary studies with low dose mdef201 (10 5 or 10 6 pfu/mouse), given at 6, 12, or 24 h after infection provided no protection from the lethal infection (data not shown). however, higher doses of mdef201 (10 8 pfu/mouse) were 100% protective when administered either at 6, 12, or 24 h after infection (table 3) . a 10 7 pfu/mouse dose was 80-90% protective when given at 6 and 12 hours, but only 30% protective when administered at 24 hours. survival time in the lower dosage group receiving treatment at 24 hours was significantly increased relative to the placebo control. cidofovir was 100% protective when administered at 100 mg/kg/day for two days starting at 24 h. body weight changes for treatments with 100% survival during the course of the infection are shown in figure 3 (10 8 mdef201 and cidofovir). weight loss in mdef201 treated mice increased with longer delay to commencement of treatment, with all animals regaining their pre-challenge weight by day 18 of the experiment. given the weight loss in the +24h group, further delays in treatment would likely result in some mortality. mice treated daily with cidofovir starting 24h post-vaccinia challenge lost less weight. in these studies, a single intranasal dose of mdef201 was found to exhibit a potent prophylactic activity that endured for at least 56 days. an endpoint in the duration of the protection could not be projected because treatment at -56 days resulted in only one death intranasal treatments with mdef201 (10 7 pfu/mouse) were given one time only on the indicated day prior to intranasal virus exposure (2.5610 5 pfu). there were no survivors in the placebo group on day 7 for data determinations. this experiment was conducted concurrently with the second experiment in table 2 . each data point represents the mean for five animals per group. doi:10.1371/journal.pone.0026330.g002 and caused minimal weight loss during the infection. indeed, weight loss during infection for the -56 day treatment group was equivalent to the -1 day treatment group. investigation of other parameters of infection (tissue virus titers, lung weights, and lung hemorrhage scores) revealed that the inhibition of these parameters with a -28 day mdef201 treatment was quite comparable to that of a -1 day treatment. prophylaxis starting a day before infection could effectively be achieved with doses of 10 5 through 10 7 pfu/mouse. extended prophylaxis up to 56 days preinfection was only investigated at the 10 7 dose. other investigators have shown that def201 in both mouse and human constructs are active prophylactically [16, 17, 18, 19] . kumaki et al. showed 100% protection against sars infection in mice by a 10 6 dose given only at -14 days (other time points were not assessed) [17] . thus, it is possible that prophylaxis earlier than 14 days would be protective against sars infection. against yellow fever virus infections in hamsters, def201 was effective when given 7 days prior to infection but not at -21 days [19] . this breadth of viral efficacy indicates the potential of def201 to function as a truly broad-spectrum antiviral. remarkably, mdef201 was effective in mice against vaccinia virus infection when given nearly two months prior to virus exposure. this indicates that ad5-vectored ifn induced a longterm antiviral state that was completely protective with a single dose. this extended prophylaxis appears to exist in the absence of measurable serum ifn levels, since wu et al. [15] were only able to detect interferon alpha protein in mice treated intramuscularly with mdef201 at 24 and 48 h, but not at 7 days (times in between were not investigated). this study raises a number of important questions relative to the long acting protective effect of mdef201 against vaccinia virus infection in mice. questions such as how much interferon is produced, how long is it produced, and what cells are responsible for producing it are being asked and are currently under investigation. it has already been shown that interferon is successfully produced by the vector when administered intramuscularly to mice and the protein is detectable in the serum [16] . from that work we also know that the levels of interferon rise very quickly (within 3-5 h), are transiently high, and come down within several days. we can infer that the interferon protein will be delivered successfully to the lung and nostrils (and have since confirmed this in a separate study, unpublished). given that the protection lasts longer than the previously measured levels of interferon protein persist, we assume that the interferon is activating an immunological cascade that then protects the animal from infection by inducing an antiviral state. thorough investigation of induced genes, length of induction, and localization of induction are planned, but are beyond the scope of this study. the choice of intranasal route of administration is important, as this route has been demonstrated to bypass pre-existing immunity to the adenovirus vector [21] . intranasal delivery is also less invasive and easier to administer to large numbers of patients in the event of a mass infection due to a natural outbreak or intentional release. adenovirus was selected to be the vector for interferon due to its rapid infectivity, breadth of human safety data, and facility of intranasal administration. mdef201, administered by intranasal route, has already been shown to be effective in the treatment of sars coronavirus respiratory infections in mice [18] . this is the first description of adenoviral vectored interferon alpha being used, by any route, to prevent or treat vaccinia virus infections. a limited therapeutic window of time exists for treating vaccinia virus infections with interferon-based drugs like mdef201, and to achieve treatment higher doses of mdef201 are needed than for prophylaxis. indeed, low dose mdef201 (10 5 -10 6 pfu/mouse) that were effective prophylactically were not effective when administered even 6 h after infection. higher doses (10 8 pfu/ mouse) were required for full protection from lethality when given at +24 h, however considerable body weight loss resulted due to infection (figure 3 ). the single 10 8 dose administered at 24 h was not as effective as daily doses of 100 mg/kg of cidofovir that acts directly on vaccinia to inhibit replication. this may be due in part to the lag time between administration of mdef201 and the production of therapeutic levels of ifn within 6 h [15] . secondly, in situ produced ifn must overcome a high level of ifn system down regulation caused by the established vaccinia infection [12] . it has been demonstrated by other investigators that both mdef201 and def201 are effective as a treatment only when given within 1-2 days after virus exposure [16, 17, 18, 19] . how well ad-vectored ifn protects infected animals will depend upon each virus' virulence including the number and expression of different ifn system antagonists. however, the treatment efficacy of def201 in these unrelated viruses indicates real potential as a broad spectrum antiviral, and the extrapolation of these treatment windows into the clinical scenario may be significant. in these studies antiviral activity depended on the amount of mdef201 administered, which was the case in other published studies with unrelated viruses [16, 17, 18, 19] . the production of a steady-state level of interferon may bypass the need for bolus dosing associated with traditional interferon treatment, and thus reduce toxic side effects. nevertheless, safety of def201 is a consideration for eventual human use, and safety and toxicology studies are ongoing. it will need to be determined how long the ifn-induced antiviral state persists and its effects on treated individuals. this is the first study demonstrating that surviving mice treated with mdef201 are protected against re-infection with the same virus. this is not surprising, as mdef201 treatment did not completely prevent vaccinia virus production in the lungs and snouts of the animals (figures 2a and 2f) , thus driving an acquired immune response. the veracity of that immune response in mdef201 treated animals most likely was weaker than it would be in more severely infected mice, based upon weight comparisons to surviving placebos ( figure 1b) . assuming an adequate safety profile, one can envision the use of def201 as a means of quickly providing significant protection to first responders and medical chain personnel confronted with a deliberate release of variola or monkeypox virus into the environment. even if infected, def201 treated individuals could still acquire an immunity to the virus, as demonstrated by the present studies, thus rendering them 'vaccinated' against future exposure. the intranasal method of delivery of def201 facilitates rapid prophylaxis in people entering a suspected poxvirus environment. moreover, def201 has the potential to treat a large number of unrelated viruses simultaneously, for which there are no current treatments. pathogenesis and potential antiviral therapy of complications of smallpox vaccination a review of compounds exhibiting antiorthopoxvirus activity in animal models progress in the discovery of compounds inhibiting orthopoxviruses in animal models countermeasures to the bioterrorist threat of smallpox development of cmx001 for the treatment of poxvirus infections st-246 antiviral efficacy in a nonhuman primate monkeypox model: determination of the minimal effective dose and human dose justification severe eczema vaccinatum in a household contact of a smallpox vaccinee progressive vaccinia in a military smallpox vaccinee -united states prevention of lethal respiratory vaccinia infections in mice with interferon-alpha and interferon-gamma effect of human leukocyte interferon on vaccinia and herpes virusinfected cell cultures and monkey corneas prevention of vaccinia lesions in rhesus monkeys by human leucocyte and fibroblast interferon the interferon system and vaccinia virus evasion mechanisms pharmacokinetics of interferon alpha-2b in healthy volunteers pharmacokinetic properties of a 40 kda branched polyethylene glycol-modified form of consensus interferonalpha (peg-cifn) in rhesus monkeys phase i trial of consensus interferon in patients with metastatic renal cell carcinoma: toxicity and immunological effects pre-and post-exposure protection against western equine encephalitis virus after single inoculation with adenovirus vector expressing interferon alpha alpha interferon as an adenovirus-vectored vaccine adjuvant and antiviral in venezuelan equine encephalitis virus infection singledose intranasal administration with mdef201 (adenovirus vectored mouse interferon-alpha) confers protection from mortality in a lethal sars-cov balb/c mouse model treatment of yellow fever virus with an adenovirus-vectored interferon, def201, in a hamster model effects of cidofovir on the pathogenesis of a lethal vaccinia virus respiratory infection in mice nasal delivery of an adenovirus-based vaccine bypasses pre-existing immunity to the vaccine carrier and improves the immune response in mice key: cord-342730-b7y8mybg authors: dellagi, koussay; rollot, olivier; temmam, sarah; salez, nicolas; guernier, vanina; pascalis, hervé; gérardin, patrick; fianu, adrian; lapidus, nathanael; naty, nadège; tortosa, pablo; boussaïd, karim; jaffar-banjee, marie-christine; filleul, laurent; flahault, antoine; carrat, fabrice; favier, francois; de lamballerie, xavier title: pandemic influenza due to ph1n1/2009 virus: estimation of infection burden in reunion island through a prospective serosurvey, austral winter 2009 date: 2011-09-29 journal: plos one doi: 10.1371/journal.pone.0025738 sha: doc_id: 342730 cord_uid: b7y8mybg background: to date, there is little information that reflects the true extent of spread of the ph1n1/2009v influenza pandemic at the community level as infection often results in mild or no clinical symptoms. this study aimed at assessing through a prospective study, the attack rate of ph1n1/2009 virus in reunion island and risk factors of infection, during the 2009 season. methodology/principal findings: a serosurvey was conducted during the 2009 austral winter, in the frame of a prospective population study. pairs of sera were collected from 1687 individuals belonging to 772 households, during and after passage of the pandemic wave. antibodies to ph1n1/2009v were titered using the hemagglutination inhibition assay (hia) with titers ≥1/40 being considered positive. seroprevalence during the first two weeks of detection of ph1n1/2009v in reunion island was 29.8% in people under 20 years of age, 35.6% in adults (20–59 years) and 73.3% in the elderly (≥60 years) (p<0.0001). baseline corrected cumulative incidence rates, were 42.9%, 13.9% and 0% in these age groups respectively (p<0.0001). a significant decline in antibody titers occurred soon after the passage of the epidemic wave. seroconversion rates to ph1n1/2009 correlated negatively with age: 63.2%, 39.4% and 16.7%, in each age group respectively (p<0.0001). seroconversion occurred in 65.2% of individuals who were seronegative at inclusion compared to 6.8% in those who were initially seropositive. conclusions: seroincidence of ph1n1/2009v infection was three times that estimated from clinical surveillance, indicating that almost two thirds of infections occurring at the community level have escaped medical detection. people under 20 years of age were the most affected group. pre-epidemic titers ≥1/40 prevented seroconversion and are likely protective against infection. a concern was raised about the long term stability of the antibody responses. in april 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, ph1n1/ 2009v, occurred in mexico and the united states [1] . the rapid spread of infection to other continents led the world health organization (who) to declare on 11 june 2009 that a pandemic of ph1n1/2009v influenza was under way, which raised major international concern about the risk of high morbidity and lethality and the potential for severe socio-economic impact. actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated [2] . illness surveillance data do not allow to an accurate estimate of the true influenza infection rate, as a substantial proportion of infections are asymptomatic or mild [3] . serological surveys can overcome this limitation, but must take into account that a significant proportion of the population that exhibited crossprotective antibody titers before circulation of the ph1n1/2009v [4] . this so-called ''baseline immunity'' has to be subtracted from the seroprevalence observed after the pandemic wave, to determine seroincidence in serosurveys [5] [6] [7] [8] . however, except for few studies [9] [10] [11] , most of these serosurveys did not use serial measurements in the same person, which allows for a better understanding of antibody kinetics and the dynamics of infection within individuals and communities. reunion island (805,500 inhabitants) is a french overseas department located in the southwestern indian ocean, 700 km east of madagascar and 200 km southwest of mauritius. the first imported case of ph1n1/2009v was identified on 5 th july 2009 (week 29) in a traveller returning from australia. the first case indicating community transmission was detected on 21 st july (week 30). ph1n1/2009v became the predominant circulating influenza virus within four weeks of its first detection, its activity peaked during week 35 (24) (25) (26) (27) (28) (29) (30) and ended at week 38 [12] . contrary to initial fears, the health care system was not overwhelmed, as morbidity and mortality rates were lower than predicted [12] [13] [14] . in order to assess at the community level, the actual magnitude of the ph1n1/2009v pandemic and the extent of the herd immunity acquired after passage of the epidemic wave, a prospective population serosurvey was conducted in reunion island during the passage of the epidemic wave in the 2009 austral winter season (july-december 2009): prevalence of infection was assessed on a weekly basis and seroconversion rates were measured using paired sera. the copanflu-run was part of the copanflu international project, a consortium between the french national institute of health and medical research (inserm), the institute of research for development (ird) and the mérieux fondation under the promotion of the school of advanced studies in public health (ehesp). to enable the rapid implementation of the study in anticipation of the imminent spread of the pandemic wave, we used a pre-existing sample of 2442 households established in october 2006 for the investigation of the chikungunya outbreak (serochik) and updated in may 2008 throughout a follow-up telephone survey (telechik) on a basis of 1148 households [15, 16] . we took special attention to select households representing a wide range of geographic locations in order to minimize the repartition bias. the inclusion phase started on july 21 st (week 30) and was continued up to week 44, throughout the epidemic wave and beyond. a first serum sample (sample 1) was obtained from each household member. an active telephonic inquiry was then conducted twice a week to record symptoms compatible with influenza-like illness (ili) occurring in households. report of ili (fever $37.8uc associated with any respiratory or systemic symptom) led to three consecutive visits of a nurse to the incident case-dwelling (on day 0, +3 and +8 post-report) to record symptoms and collect nasal swabs from all family members (for qrt-pcr detection of ph1n1/2009v. at week 45, the active inquiry was discontinued and a second (post-epidemic) serum sample (sample 2) was obtained (weeks 45-52) to determine seroconversion rates. sera were aliquoted and stored at 280uc. the protocol was conducted in accordance with the declaration of helsinki and french law for biomedical research (nu id rcb afssaps: 2009-a00689-48) and was approved by the local ethics committee (comité de protection des personnes of bordeaux 2 university). every eligible person for participation was asked for giving their written informed consent. viral genome detection by rt-pcr. viral rna was extracted from 140 ml of nasal swab eluate using the qiaamp viral rna kit (qiagen) and processed for detection by taqman qrt-pcr targeting the heamagglutinin ha gene (superscript iii platinum one-step qrt-pcr system, invitrogen) according to the recommendations of the pasteur institute (van der werf s. & enouf v., sop/flua/130509). confirmed ph1n1/2009v infection was defined as a positive qrt-pcr detection of the ha gene in at least one nasal swab. hemagglutination inhibition assay (hia). a standard hemagglutination inhibition technique was adapted to detect and quantify ph1n1/2009v antibodies [17] . the antigen was prepared by diluting a non-inactivated cell culture supernatant producing a pdm h1n1v strain (strain opyflu-1 isolated from a young patient returning from mexico in early may 2009) [18] . briefly, the virus was propagated onto mdck cells under standard conditions. the last passage (used for antigen preparation) was performed in the absence of trypsin and ht-fbs. the supernatant was collected at day seven p.i. clarified by centrifugation at 8006 g for 10 min at room temperature, aliquoted and conserved at 280uc. the hemagglutinating titer of the non inactivated viral antigen was immediately determined under the hia format described below. the dilution providing 5.33 hemagglutinating units in a volume of 25 ml was used for subsequent hia. sera were heat-inactivated at 56uc for 30 min prior to use. sequential twofold dilutions in pbs (1/10 to 1/1280) in volumes of 25 ml were performed and distributed in v-bottom 96 well microplates. human red blood cells (rbc) were used for hemagglutination experiments. detection and quantification of antibody to ph1n1/2009v was performed as follows: 25 ml of virus suspension was added to the serum dilution (25 ml) and incubated for 1 hour at room temperature. each well was then filled with 25 ml of a 1% rbc suspension in pbs (v/v: 0.33%), followed by another 30 min incubation at room temperature. the hia titer was determined as the last dilution providing clear inhibition of hemagglutination. all experiments were performed in the presence of the same negative and positive controls, the latter including sera with 1/40, 1/80, 1/160 and 1/320 antibody titers. the results reported in this study were based only on serological analysis of paired sera. for the sake of analysis, four successive phases were identified throughout the pandemic wave: phase a (weeks 30-31) corresponded to early epidemic time, phase b (w32-39) to the epidemic unfolding, phase c (w40-44) to the immediate post-epidemic stage and phase d (w45-52) to the late post-epidemic stage. seropositivity was defined as a hia titer of 1/ 40 or more. the baseline-proxy seroprevalence rate was estimated on serum samples collected in phase a. the cumulative incidence rate of infection measured the raise between the raw seroprevalence rate at any given time during the epidemic phases (s2pi) and the age-specific baseline-proxy seroprevalence rate (s1pa) (s2 pi -s1 pa ). seroconversion was defined as a shift from seronegative at inclusion (sample 1: hia ,1/40) to seropositive on follow-up (sample 2: hia $1/40), or for sera tested seropositive on inclusion as a four-fold increase of hia titers between sample 1 and sample 2 paired sera. we also calculated the proportion of sera that tested seropositive in sample 1 for which the hia titer decreased fourfold and passed under the cut-off value of 1/40 in sample 2. we considered this proportion as a ''seronegation'' rate. the sample size was calculated for identifying risk factors in the prospective cohort study. considering on average three individuals per household, an intra-household correlation of 0.3, a power greater than 80% could be obtained with a sample size of 840 comprising 2500 individuals, assuming exposure levels ranging from 10% to 90% and a relative risk greater than 1.3. with 2,500 subjects, the study allowed 1-2% absolute precision around the estimated values for seroconversion rates. data entry used epidata version 3.1 (the epidata association, odense, denmark). sas version 9.1 (sas inc., cary, nc, usa) was used for statistical analysis. the characteristics of the study cohort were compared to those of the population of reunion island and a chi2 test (or fisher's exact test when non applicable) was used to analyse differences in age, sex and geographic location. cumulative incidence rates of infection (i.e. seroincidence) and seroconversion rates were standardized according to the age structure of the community (french national institute for statistics and economical studies (insee) source). baseline-proxy seroprevalence, cumulative incidence rates of infection, as well as seroconversion and seronegation rates, were expressed as percentages. cumulative reverse distribution curves were used to show the distribution of antibody titers. in all tests, a p value,0.05 was considered significant. we estimated 95% confidence intervals (cis) of proportions by using a cluster bootstrap technique with 1000 re-samples [19] . after bootstraping, we used an anova model to compare mean cumulative incidence proportions between pandemic phases, within each age group. we used an alternating logistic regression model (alr) with an exchangeable log odds ratio (or) to test the intra-household correlation-adjusted association between factors and the seroconversion outcome. data were analysed with respect to subject age. initially, four age groups were considered: the children and adolescents (,20 yrs), young adults (20-39 yrs), middle-age adults (40-59 yrs), and elderly adults ($60 yrs). as the cumulative incidence of infection of the second and third groups were very close, both groups were merged into one adults group (20-59 yrs). therefore we refer further in our study to three age groups: children and adolescents (,20 yrs), adults (20-59 yrs), elderly ($60 yrs). a total of 2,164 individuals from 772 households were enrolled between weeks 30 and 44 in the copanflu-run cohort, allowing the collection of 1,932 sera at inclusion (sample 1). during this period, 136 households (17.7% of households) containing 464 individuals (21.4% of individuals) reported at least one case of ili. sixty subjects among the 464 individuals (12.9%, belonging to 33 households [24.3%]) were qrt-pcr positive, which documented the ph1n1/2009v infection. no positive qrt-pcr could be detected after week 37 and no ili was reported after week 40, the end of the epidemic wave. the second follow up serum sample (sample 2) was obtained for 1,759 subjects at least five weeks after the end of the epidemic wave (weeks 45-52) which allowed the constitution of a serobank of 1,687 paired-sera. the profile of the cohort and the major outcomes are displayed in figure 1 . details on inclusions and serum sample timing with respect to the circulation of ph1n1/2009v over the island are provided in figure 2 . the socio-demographic and space-time characteristics of the cohort are detailed in table 1 . compared to the community of reunion island, the sample of 1,687 individuals for whom pairedsera were available, was older (,20 yrs: 27% vs 35%, and $60 yrs: 17,9% vs 11,3%) and composed of a slight excess of females (54.1% vs 51.5%). the imbalance was due to a deficit in subjects aged under 40 years, reflecting men at work and the fact that parents declined the second serum for children younger than five. baseline-proxy (,pre-epidemic) hia titers to the ph1n1/ 2009v were measured on sample 1 ( table 2) , obtained from 249 subjects (103 households) recruited at the very beginning of the investigation during weeks 30 and 31 (phase a, figure 2 ), when the epidemic activity in the cohort was still very low. age distribution in this group was similar to that of the whole cohort (data not shown). the overall, the baseline-proxy seroprevalence rate (hia $1/40), over all ages, was 43.4% (95%ci: 37.4%-49.6%). however the majority of positive sera had low antibody titers, at the cut off value for confirmation (i.e. = 1/40). the proportions of sera with hia titer .1/40 were 0%, 3.0% and 24.6% in the young, middle-aged and older age groups respectively. these results indicate that pre-epidemic baseline antibody cross reactivity was stronger in the elderly ($60 yrs) and weaker in children and adolescents (,20 yrs) and adults (20-59 yrs), with highly significant differences between age groups (p,0.0001). the reverse cumulative distribution curves of hia titers are displayed for each age group and for the whole cohort on figure 3 . the proportion of seropositive sera (hi $1/40) steadily increased during the epidemic unfolding (phase b, w32-39) and in immediate post epidemic period (phase c, w40-44) when it reached its maximum level, then declined in the late post epidemic period (phase d, w45-52). this decline was significant enough to return the reverse cumulative distribution curve to baseline levels in the elderly. the cumulative incidence rates, obtained after subtraction of the age-specific baseline-proxy seroprevalence from the raw seroprevalence at each phase of the epidemic are shown in table 2 (note that the cumulative incidence rates of infection represented for the group ''all ages'' were standardized according to age structure of the community). the cumulative incidence rates were much higher in children and adolescents (,20 yrs), indicating very active transmission of infection within this age group. as mentioned earlier, cumulative incidence rates peaked in phase c (w40-44), and then declined indicating some lability of the humoral immune response against the ph1n1/2009v. the age-related difference observed in the incidence rates was highly statistically significant (p,0.0001). to estimate more appropriately the decline of antibody titers occurring after the peak of the humoral response to the ph1n1/ 2009v, we considered paired-sera from the group of 264 subjects for whom the first serum sample (sample 1) was obtained just after the epidemic wave (phase c, w40-44), and the corresponding second sample was collected at the end of the survey (phase d, w45-52). seronegation rates were 27.0% (61/226) for all age groups, 17.4% (12/69) in children and adolescents (,20 yrs), 32.3% (41/127) in adults (20-59 yrs) and 26.7% (8/30) in the elderly ($60 yrs). differences between the seronegation rates according to age were statistically weakly significant (p = 0.0671). we then considered the 1687 individuals for whom paired sera were available and we measured the seroconversion rates according to age and to the time of first serum sample collection (phase a, b or c). criteria of seroconversion were defined in the method section. as shown in table 3, there was a sharp decline in seroconversion rates across all the age groups, depending on whether participants were enrolled during phase a, phase b, or phase c (p,0.0001). to interpret these data, one should remember that antibodies at seroprotective levels (hia $1/40), in serum samples 1 collected during the per epidemic phase b or early post epidemic phase c could represent either base line cross reactive antibodies or rising ph1n1/2009 specific antibodies due to a recent or ongoing infection. this ambiguity could lead to underestimation of the seroconversion rate for subjects enrolled in phases b and c. in order to solve this ambiguity, we specifically considered the group of 249 subjects in whom cross reactive antibodies were detected at the time of phase a (w30-31). the seroconversion rate of this group is the most indicative of the exposure of individuals to the whole epidemic wave. it was the highest (63,2%, p,0.0001) in children and adolescents (,20 yrs), and still significantly high in adults (39.4%, p,0.0001). we then tested in this particular group, the impact of (baseline) pre-epidemic cross reactive antibodies on the rate of seroconversion to ph1n1/2009 (table 4) . no subject with hia titer superior to 1/40 had evidence of seroconversion to ph1n1/2009. the seroconversion rate in individuals with a hia titer equal to 1/40 was linked with age, being more important in children and adolescents (,20 yrs). the highest seroconversion rate (.56%) was registered in subjects with hia titers inferior to 1/40, particularly for the under 20 years where it reached 85%. hence, the risk of seroconversion decreased when pre-epidemic hia titer was high after controlling for age (p,0.0001) (figure 4) . the multivariate adjusted odds ratio for seroconversion were 0.15 (95%ci: 0.06-0.37, p,0.0001) per two-fold increase in baseline titer, 1.79 (95%ci: 1.23-2.59, p,0.003) per other household members who seroconverted, 5.33 (95%ci: 1.56-19.27, p,0.008) figure 1 . the cohort profile and major outcomes. figure 1 details the three phases of the protocol: i) inclusion (weeks 30-44) and serum samples s1 collection; ii) follow up for detection of ili in households, qrt-pcr on nasal swabs and estimation of cumulative seroincidence rates; iii) end of the study (weeks 45-52) and samples s2 collection. hia on paired sera (s1+s2) allowed estimating seroconversion rates. doi:10.1371/journal.pone.0025738.g001 bp (baseline-proxy) seroprevalence rates were estimated on weeks 30-31 in each age group. b cumulative incidence rates measured the raise between raw seroprevalence rates and age-specific baseline-proxy seroprevalence rate. in the group ''all ages'', cumulative incidence rates were standardized according to age structure of the community. doi:10.1371/journal.pone.0025738.t002 data are numbers, percentages (95% confidence intervals) and alr parameter test p value for comparison of seroconversion proportions according to time of first sample (s1) collection at inclusion, in each age group, after controlling for household selection. in the group ''all ages'', rates of seroconversion were standardized according to age structure of the community. na: not assessed. seroconversion was defined as a shift from seronegative at inclusion (i.e. hia titer ,1/40) to seropositive on follow-up sample, or as a 4-fold increase of reciprocal hia titer between first and second paired samples for sera tested seropositive on inclusion (i.e. hia titer $1/40). for age ,20 years (vs age $60 years) and 11.35 (95%ci: 0.41-4.47, p = 0.62) for age 20-60 years (vs age $60 years). the observed and predicted seroconversion rates according to age and baseline hia titer are displayed figure 4 . finally, we considered the 46 subjects who had been infected by the pandemic virus over the course of the study, verified by a positive qrt-pcr nasal swab, and for whom paired sera were available. initial hia antibody titers in this group were ,1/40, the copanflu-run cohort was set up to conduct a prospective population-based study investigating the herd immunity induced by the 2009 pandemic influenza virus and identifying risk factors for ph1n1/2009v infection from paired sera collected in an entire community. most works published to date have used either extensive cross-sectional serosurveys on pre-and post-epidemic independent serum samples, the baseline immunity being assessed from stored frozen samples [5, 7, 8] , or non representative adult cohorts (military, health care workers, long-stay patients). antibody titers were measured by hia using a cut-off value set at 1/40 as classically recommended. this hia titer at 1/40 is considered protective, i.e. conferring 50% protection against a viral challenge [20] . our assay has introduced some changes in the experimental protocol compared to the classic one. the use of a non-inactivated viral antigen, i.e. a native virus, with nondenatured epitopes probably allows detection of antibodies to epitopes of the hemagglutinin not detected in the classic hia test. this can induce slight differences in the sensitivity of detection of cross-reacting antibodies, but this does not modify the kinetics of ab and the epidemiological evolution of seroprevalence and does not jeopardize the global comparability of serological results. this is confirmed by the fact that our hi assay detected seroprotective antibody titers in 93.5% and gave evidence seroconversion in 73.9% of qrt-pcr confirmed ph1n1/2009 influenza, all figures close to those reported in the literature [5, 21] . we considered that titers of .1/40, in sera collected from individuals enrolled during weeks 30 and 31 were cross reactive antibodies and not de novo antibodies triggered by the pandemic virus and hence used them as a proxy for baseline pre epidemic immunity. several arguments support this assumption: i) the first case indicating autochthonous transmission in reunion island was reported by the epidemiological surveillance department of la réunion on 21st july (week 30), i.e. the same day when inclusion started in our study cohort; ii) 7 to 15 days are required to develop an antibody response after viral infection; iii) on weeks 30 and 31, the epidemic activity due to the pandemic virus was very low in our study cohort and it became significant only after week 32. hence, during weeks 30-31, 103 households were recruited and only 2 households reported ili cases. nasal swabs collected from these 2 individuals were tested qrt-pcr negative to the pandemic virus whereas one had evidence of coronavirus and rhinovirus using a multiplex rt-pcr to respiratory viruses (h. pascalis, manuscript in preparation). in contrast, during weeks 32 to 39, 199 individuals belonging to 99 households reported ili, among whom 60 individuals had documented infection by the pandemic virus. our study shows that a substantial proportion of reunion island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly [5, 6, 8, [22] [23] [24] [25] [26] , though large variations do exist between countries [10, 11, 23, 27, 28] . these cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to h1n1 viruses circulating before 1957 [24, 25, 29, 30] . baseline seropositivity rates that we report in children and in younger adults (i.e. 30%-35%) were notably higher than those reported from other parts of the world [6, 8, 22, 23, [31] [32] [33] . however one should note that these baseline antibodies were of low titer, just at the level of the hia threshold (i.e. 1/40). several factors could have contributed to this comparatively high baseline rates found in our study: i) it may reflect the fact that the hi test used in our study was marginally more sensitive than the classic one [17] ; ii) some individuals may have already been infected with ph1n1/ 2009 virus at weeks 30 and 31 and may have triggered an antibody response to the virus. this hypothesis seems unlikely in view of the arguments presented above and of a similar high proportion of sera titering hia = 1/40 among 122 sera from adult patients sent for diagnostic purposes to the regional hospital microbiology laboratory, during the first half of 2009 (i.e. before the 2009 pandemic) (data not shown). however we cannot formally exclude this hypothesis in view of a recently reported study from taiwan [11] that showed evidence of subclinical community transmission with proved seroconversion several weeks before report of the first documented case in the island. a similar conclusion was also drawn from australia [34] ; iii) our serological test might detect cross-reactive antibodies triggered by recent vaccination with trivalent seasonal influenza vaccine as reported [4, [35] [36] [37] [38] [39] . however, seasonal influenza vaccines were of rather limited use in reunion island, especially in children and young adults; iv) finally the high baseline titers may reflect the infectious history of the individuals to seasonal influenza viruses cross antigenic with ph1n1/2009 virus as recently suggested for seasonal 2007 h1n1 infection [40] . this serosurvey indicates that a large fraction of the reunion island population was infected with the pandemic virus. younger people, have paid the main tribute to the epidemic as almost two thirds show evidence of seroconversion, confirming earlier clinical reports from the island [12] and accumulating reports from other countries [17, 32, 41, 42] and suggesting that school children have likely played the central role in the epidemic diffusion of the pandemic virus. lower infection rates were found in adults and the lowest rates were recorded in the elderly. based on clinical cases reported to the epidemiological surveillance services [12] , it was estimated that 66,915 persons in reunion island who consulted a physician were infected by the ph1n1/2009 virus during the 9 weeks of the epidemic, giving a cumulative attack rate of 8.26%. taking into account those who did not consult a physician, the number of symptomatic infected persons was estimated to 104,067 (attack rate: 12.85%). in fact, the attack rate of ph1n1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response (i.e., weeks 40-44), a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases the wide gap between the two estimates indicates that a large fraction (almost two thirds) of those who got infected by ph1n1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level. in england, baguelin et al. [43] estimated that the cumulative incidence rates of infection by the pandemic virus in children were 20 to 40 times higher than that estimated from clinical surveillance. our study, as others [6] , indicates that pre-existing cross reactive antibodies to ph1n1/2009 at titers $1/40 prevented from seroconversion in response to the pandemic virus. this level of pre-existing cross reactive immunity likely confers true protection against infection as about two thirds and one third of documented infection (qrt-pcr positive) in our series have occurred in individuals with baseline hia titers ,1/40 and = 1/ 40 respectively and less than 5% of documented infections occurred in individuals with base line titers .1/40. the protection was effective not only in older adults but also in younger persons. this indicates that protection was conferred not only by baseline cross reactive antibodies triggered by close ph1n1/2009 viruses that circulated before 1957 (as in the elderly), but also by antibodies likely resulting from recent exposure to seasonal influenza epidemics (as shown in younger persons) [40] . the observed seroconversion rates depend on age, after adjusting for baseline ph1n1/2009 titers. the protective role of increasing age might be explained by a stronger cross-immunity in adults and elderly or by a higher exposure of young subjects to the virus during the 2009 epidemic (due to social contacts and mixing patterns). it may also indicate that immune mechanisms other than cross reactive antibodies detected by hia (i.e. immunity to neuraminidase and conserved t cells epitopes [44] might develop throughout life, providing additional protection from infection or severe disease, especially in the elderly. interestingly, evidence is seen for a decline in antibody titers, which occurred soon after the passage of the epidemic wave. in paired sera, this decline was significant enough to bring, within a few weeks, almost 27% of sera that tested positive (i.e. hi titers $1/40) in the immediate post epidemic phase to levels under the cut-off value in the second serum sample. this decay accounts for the observation that older adults ($60 yrs) in the study cohort were apparently almost completely spared by the epidemic if one only considers cumulative incidence rates derived from iha titration on samples 2 (weeks 45-52). in fact, the cumulative incidence rate in older adults measured just after the epidemic peak (i.e. weeks 40-44) was 20.4%. similar results of early antibody decay were recently reported [10, 45] . more generally, these data show that serosurveys conducted months after passage of the epidemic, likely underestimate the real extent of ph1n1/2009 infection, compared to antibody titration performed earlier, when humoral responses are at their highest level. whether the decline in antibody titers has functional immunologic consequence to individuals or within the communities warrants further investigation. however, one should note that there was no second epidemic wave in reunion island during the subsequent austral winter seasons in 2010 and 2011. influenza during the 2010 winter was at a level not higher than the usual passages of seasonal flu, though almost two thirds of documented cases in 2010 were also due to ph1n1/2009v [46] . in addition many fewer pandemic virus isolates were noted during the ongoing 2011 austral winter, strongly suggesting that the first epidemic wave had conferred a solid herd immunity, at the community level. our study has some limitations. the fact that the epidemic progression coincided with the implementation of the prospective study, we were not able to collect, strictly speaking, pre-epidemic sera from the cohort members. therefore we used as proxy base line seroprevalence data from individuals recruited at the very beginning of the investigation when the epidemic activity in the cohort was very low. this may overestimate the base line immunity if subclinical community transmission 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of a/h1n1 2009 influenza infection in england from sequential antibody prevalence data using likelihood-based estimation preexisting immunity against swine-origin h1n1 influenza viruses in the general human population antibody dynamics of 2009 influenza a (h1n1) virus in infected patients and vaccinated people in china surveillance de la grippe à la réunion we acknowledge the nurses, investigators and technicians of the cic-ec de la réunion. key: cord-348243-e5tdb08v authors: schermer, bernhard; fabretti, francesca; damagnez, maximilian; di cristanziano, veronica; heger, eva; arjune, sita; tanner, nathan a.; imhof, thomas; koch, manuel; ladha, alim; joung, julia; gootenberg, jonathan s.; abudayyeh, omar o.; burst, volker; zhang, feng; klein, florian; benzing, thomas; müller, roman-ulrich title: rapid sars-cov-2 testing in primary material based on a novel multiplex rt-lamp assay date: 2020-11-02 journal: plos one doi: 10.1371/journal.pone.0238612 sha: doc_id: 348243 cord_uid: e5tdb08v background: rapid and extensive testing of large parts of the population and specific subgroups is crucial for proper management of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infections and decision-making in times of a pandemic outbreak. however, point-of-care (poc) testing in places such as emergency units, outpatient clinics, airport security points or the entrance of any public building is a major challenge. the need for thermal cycling and nucleic acid isolation hampers the use of standard pcr-based methods for this purpose. methods: to avoid these obstacles, we tested pcr-independent methods for the detection of sars-cov-2 rna from primary material (nasopharyngeal swabs) including reverse transcription loop-mediated isothermal amplification (rt-lamp) and specific high-sensitivity enzymatic reporter unlocking (sherlock). results: whilst specificity of standard rt-lamp assays appears to be satisfactory, sensitivity does not reach the current gold-standard quantitative real-time polymerase chain reaction (qpcr) assays yet. we describe a novel multiplexed rt-lamp approach and validate its sensitivity on primary samples. this approach allows for fast and reliable identification of infected individuals. primer optimization and multiplexing helps to increase sensitivity significantly. in addition, we directly compare and combine our novel rt-lamp assays with sherlock. conclusion: in summary, this approach reveals one-step multiplexed rt-lamp assays as a prime-option for the development of easy and cheap poc test kits. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the recent pandemic of sars-cov-2 is a major challenge for healthcare systems worldwide. lacking an effective and approved vaccine, reliable screening of samples from nasopharyngeal swabs for viral rna is a fundamental pillar for effective disease control. governmental measures (e.g. shutdown of social life) largely depend on such data to allow for a rapid adaptation to changes in epidemiologic indicators. furthermore, isolation and quarantine of infected and exposed individuals are key for effective outbreak control. here, the prompt establishment of a diagnostic real-time pcr based testing [1] has been instrumental already in the early phases of the pandemic. such qpcr-based tests are extremely powerful due to their high sensitivity and specificity. however, these assays require specific lab equipment and expertise which is typically not widely available directly at the point-of-care making transportation to a specialized facility necessary. furthermore, the material required for the different steps involved may become subject to shortages during a pandemic making alternative approaches an important goal. even though rna isolation and subsequent qpcr is performed in only a few hours, actual turnaround times from sample collection to diagnostic test results are often much longer. decision-making in both healthcare facilities and other spheres of public life would be facilitated enormously by direct testing on site with short turn-around times. such an approach could also limit the need for quarantine, allow healthcare workers after exposure to continue their work upon a daily negative swab and avoid shortages in systemically relevant personnel. recently, a number of pcr-independent methods have been proposed for this purpose including isothermal amplification (rpa, lamp [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] ) and their combination with genome editing tools such as cas12a [13] or cas13a [14, 15] for improved performance. the majority of these studies, however, have not been carried out on direct primary material but on rna isolated from patient samples or generated in vitro [3] [4] [5] [6] [7] [8] [9] 13 ]. in the study at hand, we use previously described rt-lamp and sherlock assays on both isolated rna and primary material from patients [9, 14] . in addition, we describe a newly designed multiplexed rt-lamp assay targeting orf3a and orf7a of sars-cov-2. orf3a and orf7a have been selected since these targets have not been used for any available diagnostic qpcr assay, to avoid the risk of amplicon cross-contamination between different types of assays. nasopharyngeal swabs were taken from symptomatic patients presenting at university hospital of cologne from march to april 2020. swabs were directly transferred in 1-3 ml of universal transfer medium (utm) or pbs. for diagnostic qpcr, rna was extracted from 500 μl utm / pbs of the swab samples using the automated magna pure 96 system (roche). diagnostic qpcr was performed using a realstar sars-cov-2 rt-pcr kit 1.0, with primers targeting e and s gene (altona diagnostics) on a lightcycler 480 (roche). positively and negatively tested utm/pbs samples were randomly selected for rt-lamp or sherlock assays. here, 0.5 to 1.9 μl isolated rna or 0.5 to 1.9 μl utm/pbs was used for subsequent rt-lamp/sherlock assays. this study was performed exclusively with surplus diagnostic material that was analyzed anonymously. no specific approval number from the irb (ethikkommission der medizinischen fakultaet der universitaet zu koeln (irb of university of cologne)) was required. for assays from direct material 10 μl of utm/pbs were incubated at 98˚c for 15 minutes in a pcr cycler with heated lid placed inside a safety cabinet. treatment of 15 min at 92˚c has been shown to inactivate sars-cov-2 [16] and 98˚c was found to be preferable for downstream rna-based applications. for orf1a and gene n we used previously described primer sets [9] . additional primer sets were designed for orf3a, orf7a and the m gene using the primerexplorer v5 tool (http:// primerexplorer.jp/e/). all primers were ordered from idt, purified with standard desalting, as page purification did not improve the performance of the assays. all primer sequences are listed in s1 table. all rt-lamp reactions were assembled on ice. in brief, each 20 μl reaction contained 10 μl warmstart colorimetric rt-lamp mix (neb), 2 μl primer mix (f3/b3 2 μm each; fip/bip 16 μm each; lf/lb 4 μm each), 40 mm guanidine hydrochloride (from a 4 m stock solution, ph8), dnase/rnase free water and 0,5 μl sample. for multiplexed rt-lamp the additional primer mix replaced 2 μl of dnase/rnase free water. initially, the assays were performed without guanidine hydrochloride at 65˚c for 40 minutes, while the multiplexed reaction was done at 60˚c for 40 to 50 minutes, as indicated in the figures. each assay was performed including several negative controls. positive reactions were identified due to a clear change in color from pink/red to orange/yellow. in two cases out of almost 200, the mere addition of 0.5 μl sample to the reaction resulted in a color change. these samples are not included in the data shown. the two step sherlock assay for orf1a and s -gene was performed according to the protocol established in feng zhang's lab at mit (https://zlab.bio/s/covid-19-detection-v20200321.pdf) [14, 15] . in brief, an rpa reaction was set up using 5.9 μl rpa mix (twist amp), 0.2 μl protoscript rt (neb), 1μl rpa primer mix (10 μm each), 1.9 μl sample (isolated rna or utm/pbs) and 0.5 μl magnesium acetate (280 mm stock solution). the reaction was mixed, spun down and incubated 42˚c for 25 min. the cas13a-based detection step was performed in a 20 μl reaction as follows: 2 μl tris-hcl buffer (400 mm, ph 7.4), 9.6 μl dnase/ rnase free water, 2 μl lwacas13a (corresponding to 120 ng of protein), 1 μl crrna (10 ng/μl), 1 μl lateral-flow-reporter (20 μm), 1 μl superase in rnase inhibitor (thermofisher scientific), 0.6 μl t7 polymerase (lucigen), 0.8 μl rntps (25 μm each; neb), 1 μl mgcl2 (120 mm stock solution) and 1 μl of previous rpa reaction. the reaction was mixed and spun down, then incubated at 37˚c for 30 min. next, the reaction was diluted with 80 μl of hybridetect buffer (milenia), mixed, and then a hybridetect dipstick (milenia) was placed in the reaction tube and incubated at rt. results were visible within 5 minutes. to combine the rt-lamp amplification with cas13a detection we added a t7 promoter in the loop region of the fip primers for the rt-lamp assay (s1 table) . after the rt-lamp reaction at 65˚c for 40 min, 0.5 μl were used in the cas13a detection assay assembled as described above, using a specific crrna targeting the rt-lamp gene n amplicon. to allow for the comparison of different nucleic acid detection methods for sars-cov-2 we collected redundant material from nasopharyngeal swabs obtained for qpcr testing in clinical routine due to suspected covid-19. 171 samples were selected randomly from samples collected in the period from march to april 2020. the cohort included individuals between the age of 1 month and 88 years with a close to equal distribution of men and women (s2 table) . we first tested two recently described assays for sars-cov-2 detection on isolated rna from patient samples. specifically, we performed colorimetric rt-lamp assays using primers targeting orf1a and gene -n [9] and two-step sherlock assays combined with lateral flow detection [14, 15] targeting orf1a and s gene (s1a-s1c fig) . even though both assays worked well on these samples, they failed to detect the virus in specimen that were positive in diagnostic qpcr at cycle threshold (ct) values > 30 for e and s gene (s1 fig). moreover, rt-lamp appeared to be slightly more sensitive and specific (s1c fig) . due to limited availability of the rpa reagents required for the first step of sherlock at that time paired with the results described above, we focused our efforts on validation of the rt-lamp assays. since detection worked on isolated rna, we went on to test this approach using primary material (i.e. transport medium from nasopharyngeal swabs without rna isolation) strikingly, both rt-lamp assays performed well on these specimens. using as little as 0.5 μl utm as input for each reaction to avoid inhibitory effects of the medium or of tissue contaminants on the reaction, detection worked in samples tested positive by qpcr (see ct as reference) (fig 1a) . in parallel, 5 additional sets of rt-lamp primers targeting additional genes of the sars--cov-2 genome (nc_045512) were designed, tested and validated. here, we preferred genes that are not target of any standard diagnostic qpcr assay, to minimize and avoid any possible interference of such point of care assays with other routine diagnostic pipelines. among these new primer sets, the oligos targeting orf7a showed the highest sensitivity and specificity in several tests on diluted isolated rna and was thus selected for further testing in primary material. in this second set of experiments we screened a total of 70 samples, 52 of which had been tested positive for sars-cov-2 by diagnostic qpcr. fig 1b displays representative results of the rt-lamp assays targeting gene n, orf1a and orf7a. assays targeting gene n and orf7a were more sensitive than the one targeting orf1a as indicated by the mean ct value of rt-lamppositive samples (fig 1c) , by the total number of samples that were correctly identified ( fig 1d and 1e ). 3 out of 70 specimens turned out positive for gene n and 1 out of 70 for orf1a and orf7 respectively, that were negative by qpcr. in order to increase the sensitivity of rt-lamp assays to a comparable level with qpcr we performed a "two-step" lamp reaction, either using a small amount (0.2 to 0.5 μl) of a first rt-lamp reaction as template for a second one or by refreshing the first reaction with new enzymes and dntps. all of these attempts resulted consistently in negative controls (water, empty utm or samples from negative patients) turning positive, either because of unspecific amplification or because of the necessary re-opening of the reaction tubes after the first amplification step. the use of mineral oil on top of every reaction-performed to avoid cross-contamination-did not improve these results. in an additional set of experiments, we combined the rt-lamp assays targeting gene n with lwacas13a mediated detection. to this end, we to increase accessibility of rna and efficiently inactivate the virus we incubated utm from swabs at 98˚c for 15 min. treatment at 92˚c for 15 minutes has been demonstrated to efficiently inactivate sars-cov-2 [16] , while 98˚c appeared a good choice for downstream rna applications in case of sars-cov-2 [17] . after incubation at 98˚c, some of the utm samples showed a gel-like consistency. this was observed in only a minor fraction of the samples and was most likely caused by protein and sugar supplements contained in one specific type of utm. this utm was easily recognizable since it was the only one containing phenol red as ph indicator. in these cases, pipetting about 10 times up and down with a p20 pipette allowed for complete homogenization and subsequently accurate pipetting. again, we only used 0.5 μl utm for each reaction. in addition, we added guanidine hydrochloride as a classical rnase inhibitor and lamp enhancer [18] to the multiplexed reaction at 60˚c. these modifications in sample preparation combined with our new multiplexed assay were used to analyze a set of 102 clinical samples consisting of 74 sars-cov-2 positive and 28 negative swabs (fig 2) . 54 sars-cov-2 positive samples with ct values up to 38 were positive in the rt-lamp assay (fig 2b) , while still 20 out of 74 qpcr positive samples were not detected in our assay on direct material. however, the vast majority of samples up to a ct of 30 were correctly identified (94%; 45 out of 48). meanwhile, rpa reagents arrived and we performed the two step sherlock assays on some of the very same direct samples. however, sensitivity was much lower with a cut-off threshold of 21 cycles (fig 2d; representative assays shown in s4 fig). in summary, our multiplex rt-lamp protocol is a simple and sensitive way to detect sars-cov-2 rna from clinical samples. based on our data we conclude, that multiplexing primers in rt-lamp reactions is a highly promising way to further increase sensitivity of these assays and to quickly develop a rapid poc test. currently, a test based on our multiplexed rt-lamp assay would-in contrast to a good specificity-most likely miss to identify those infected patients with very low amounts of viral rna in the nose or throat and would not yet reach the sensitivity of the gold-standard qpcr assays. however, the rt-lamp approach comes with several clear-cut advantages. in contrast to classical qpcr, it can be used in primary material without the need for rna isolation and yields results rapidly. furthermore, only little equipment is required (thermoblock) and could easily be made available in e.g. emergency rooms. the estimated costs for reagents are below 1.50 € per reaction with the advantage-compared to qpcr-that additional expenses for nucleic acid isolation are not required. regarding the target setting of primary screening, one could hypothesize, that identifying patients with high viral loads would detect those individuals that are highly infectious. consequently, in this setting, even a poc test with a lower sensitivity as compared to diagnostic qpcr would be extremely useful and could be backed up by a combination with qpcr the results of which get available later. currently, a relation between the level of viral rna in swabs and the infectivity of a patient has not yet been definitively established. detection of viral rna is not equivalent to detection of infectious virus. one retrospective study, however, suggested low infectivity of patients with ct-values >24 in e gene qpcr from swabs since the authors did not observe viral growth in exposed vero cells [19] . besides, a statement paper of the national centre for infectious diseases and the chapter of infectious disease physicians (singapore) refers to a study with 73 covid-19 patients where a ct >30 was found to be the threshold of infectivity [20] . additional studies report ct values between 31 and 34 as cut-off for infectivity in different set-up [21, 22] . a recently updated meta-analysis provides a very comprehensive overview on this important topic supporting the conclusion that infectivity is related to the cycle threshold level, but a clear cut-off can currently not yet be defined [23] . nonetheless, the ultimate goal would be a poc test that reaches the sensitivity of qpcr and can completely replace the current approach where favorable. studies in the near future will clarify, whether patients with high viral rna loads are indeed the most contagious individuals. besides, this knowledge will also be of greatest importance for the actual clinical interpretation of qpcr results in the future. by now, it has been shown that the viral load is already high before and maybe highest at onset of symptoms [24] and at the time point of presentation to the clinic followed by a steady decline [25] . consequently, when focusing the screening on pre-symptomatic individuals, sensitivity of the rt-lamp assay may actually be higher than in the current cohort. additionally, one thing to be kept in mind when directly comparing sensitivity between the different methods, is the fact that qpcr reaches this standard only in isolated rna whilst the multiplex rt-lamp assay attains an optimized detection rate already in primary material. of note, further addition of a step concentrating rna using bead-based pulldown to an rt-lamp-based protocol has also been successful [26] . remarkably, here the authors move from swabs, that require trained personnel and personal protection equipment, to a home-based gargle [26] and a very recent report using qpcr demonstrated, that even saliva could be used as a valid source for viral diagnostics [27] . optimizing also the collection of samples and including such specimens will be an important step towards broadly applicable poc testing. regarding specificity, only few specimens turned out to be positive in the multiplex rt-lamp assay that were negative in qpcr. since qpcr is the gold-standard this can be interpreted as a minor limitation in specificity. however, qpcr itself does not reach a sensitivity of 100% [28] . consequently, it is not clear yet whether these individuals were truly negative or missed by the qpcr assay. crispr/ cas12 [13] or cas13a [15] based assays are another promising way to detect rna in a pcr-independent manner. comparison of this approach with rt-lamp demonstrates a surprisingly high sensitivity even of the colorimetric one step rt-lamp assay. very recently, a novel protocol for cas13a -called stop ('sherlock testing in one pot')-has been described [29] . as this replaces the isothermal rpa reaction of the original sherlock protocol by a rt-lamp reaction, the authors use a thermo-stable cas13a enzyme to enable performing the entire reaction at the same temperature. whilst being a very interesting approach, nonetheless, this comes with the difficulty the reaction tube has to be opened for the final lateral flow assay used for detection. in the real-world poc testing setting, this would require the establishment of a 'pre-amp' and 'post-amp' area to avoid cross-contamination, which may limit its use. alternatively, lateral flow could be replaced by using a fluorescent probe together with an appropriate simple detection device. however, the highest ct value resulting in a positive stop assay is-at about 30 cycles-in a similar range compared to a recently described cas12a-based method (detectr) [29] . the rt-lamp assay described in our study works without re-opening the test tube after amplification and provides detection at higher ct values. on the other side, both sherlock and detectr add an additional level of specificity to the detection due to the crrna directing the cas12/13 enzyme. both sherlock and detectr require a considerable number of pipetting steps. in contrast, the multiplexed rt-lamp assay demonstrates a similar or even higher sensitivity and requires only two simple pipetting steps at the poc: (1) taking a aliquot of the utm for 'boiling' and (2) the addition of the 0.5 μl sample to the reaction tube, which could also be reached without a pipet by an inoculating loop. reaction tubes can be prepared in anticipation elsewhere (e.g. in any nearby central facility) and stored for hours at 4˚c. the preparation of these reaction tubes, is also done with only four simple pipetting steps (1. primer mix pre-diluted in water, 2. rt-lamp mix, 3. guanidine hydrochloride, ! aliquot in 200 μl tubes). this could even be further simplified by adding guanidine to the primer mix. after amplification, the tube and the according controls are photo-documented and discarded. in summary, we are convinced that systematically combining and testing different multiplex rt-lamp primer sets on primary swab material is one of the most promising approaches to develop a powerful poc test. transferring such assays to automated microfluidic formats [11] can become an important tool to support disease control strategies. detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr clinical assessment and validation of a rapid and sensitive sars-cov-2 test using reverse-transcription loop-mediated isothermal amplification development of a reverse transcription-loopmediated isothermal amplification as a rapid early-detection method for novel sars-cov-2 rt-lamp for rapid diagnosis of coronavirus sars-cov-2 a novel reverse transcription loop-mediated isothermal amplification method for rapid detection of sars-cov-2 development of a novel reverse transcription loop-mediated isothermal amplification method for rapid detection of sars-cov-2. virol sin. 2020 development of reverse transcription loop-mediated isothermal amplification assays targeting severe acute respiratory syndrome coronavirus 2 (sars-cov-2) rapid and visual detection of 2019 novel coronavirus (sars-cov-2) by a reverse transcription loop-mediated isothermal amplification assay rapid molecular detection of sars-cov-2 (covid-19) virus rna using colorimetric lamp sars-cov-2 on-the-spot virus detection directly from patients rapid isothermal amplification and portable detection system for sars-cov-2 journal of clinical virology: the official publication of the pan american society for clinical virology crispr-cas12-based detection of sars-cov-2 a protocol for detection of covid-19 using crispr diagnostics sherlock: nucleic acid detection with crispr nucleases evaluation of heating and chemical protocols for inactivating sars-cov-2 an alternative workflow for molecular detection of sars-cov-2-escape from the na extraction kit-shortage enhancing colorimetric loop-mediated isothermal amplification speed and sensitivity with guanidine chloride predicting infectious sars-cov-2 from diagnostic samples national centre for infectious diseases and the chapter of infectious disease physicians, academy of medicine presymptomatic sars-cov-2 infections and transmission in a skilled nursing facility viral rna load as determined by cell culture as a management tool for discharge of sars-cov-2 patients from infectious disease wards viral cultures for covid-19 infectivity assessment. systematic review temporal dynamics in viral shedding and transmissibility of covid-19 temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study rapid and highly sensitive isothermal detection of sars-cov-2 for laboratory and home testing saliva or nasopharyngeal swab specimens for detection of sars-cov-2. the new england journal of medicine correlation of chest ct and rt-pcr testing in coronavirus disease 2019 (covid-19) in china: a report of 1014 cases point-of-care testing for covid-19 using sherlock diagnostics we thank stefanie keller for her outstanding technical support during very special and challenging times. special thanks to the team of benchling for revolutionizing the work with dna/rna sequences. venn diagrams were generated using "venny 2.1" by j.c. oliveros (https://bioinfogp.cnb.csic.es/tools/venny/index.html, accessed in may 2020). key: cord-322827-h33su548 authors: guan, lili; liu, jin; wu, xia min; chen, dafang; wang, xun; ma, ning; wang, yan; good, byron; ma, hong; yu, xin; good, mary-jo title: unlocking patients with mental disorders who were in restraints at home: a national follow-up study of china’s new public mental health initiatives date: 2015-04-07 journal: plos one doi: 10.1371/journal.pone.0121425 sha: doc_id: 322827 cord_uid: h33su548 background: in 2005, china implemented a demonstration program known as “686” to scale-up nation-wide basic mental health services designed to improve access to evidence-based care and to promote human rights for people with severe mental disorders. as part of the 686 program, teams “unlocked” and provided continuous mental health care to people with severe mental disorders who were found in restraints and largely untreated in their family homes. we implemented a nation-wide two-stage follow-up study to measure the effectiveness and sustainability of the “unlocking and treatment” intervention and its impact on the well-being of patients’ families. methods: 266 patients unlocked from 2005 in “686” demonstration sites across china were recruited in stage one of the study in 2009. in 2012, 230 of the 266 cases were re-interviewed (the stage two study). outcome measures included the patient medication adherence and social functioning, family burden ratings, and relocking rate. we utilized pre-post tests to analyze the changes over time following the unlocking efforts. results: 96% of patients were diagnosed with schizophrenia. prior to unlocking, their total time locked ranged from two weeks to 28 years, with 32% having been locked multiple times. the number of persons regularly taking medicines increased from one person at the time of unlocking to 74% in 2009 and 76% in 2012. pre-post tests showed sustained improvement in patient social functioning and significant reductions in family burden. over 92% of patients remained free of restraints in 2012. conclusion: practice-based evidence from our study suggests an important model for protecting the human rights of people with mental disorders and keeping them free of restraints can be achieved by providing accessible, community based mental health services with continuity of care. china’s “686” program can inform similar efforts in low-resource settings where community locking of patients is practiced. 96% of patients were diagnosed with schizophrenia. prior to unlocking, their total time locked ranged from two weeks to 28 years, with 32% having been locked multiple times. the number of persons regularly taking medicines increased from one person at the time of unlocking to 74% in 2009 and 76% in 2012. pre-post tests showed sustained improvement in patient social functioning and significant reductions in family burden. over 92% of patients remained free of restraints in 2012. mental disorders constitute a huge global burden of disease, and there is a large treatment gap, particularly in low and middle-income countries (lmics). in china, mental and behavioral disorders contribute to 23.6% of years lived with disability (ylds) [1] . recognizing the gap between the population's needs for mental health care and the level of resources and services available [2] [3] [4] , the chinese government invested in an innovative reform of mental health services [5] and mental health legislation [6] [7] . in 2004, as part of an effort to rebuild the public health infrastructure following the severe acute respiratory syndrome (sars) outbreak, china officially placed mental health services within the public health system. in 2005, the government supported the launching of the national continuing management and intervention program for psychosis, known as "686" after initial funding of cny 6.86 million [5, [8] [9] [10] . this program has contributed to improving care for patients with severe mental disorders, including schizophrenia, psychosis, bipolar disorder and schizoaffective disorder, through increasing access to treatment and integrating hospital and community services designed to provide continuity of evidence-based care and to address patients' rights. the 686 program prioritized equal access to a basic package of community based mental health care for patients with severe mental illness, especially for those living in poverty. the program focused on promoting rehabilitation and recovery, family education and support, and making medications continuously available [11] . according to liu and colleagues [5] , implementing the 686 program has led to significant progress in scaling up mental health services in china. by 2005, 60 demonstration sites had been established, two in each of china's 30 provinces, covering 43 million people in "686" catchment areas. by the end of 2009, 96.88 million people from the general population in 112 cities were covered by this program, a total of 161,800 patients (0.17% of the general population) were registered, and 42,400 patients received regular follow-up [5] . as the 686 program was being initiated, teams found seriously mentally ill individuals who were physically restrained or "locked" by family members accounted for approximately 0.2% of the patients registered in the "686" demonstration sites. although these were rare events in the community, the patients indeed lived miserable lives, without care from medical personnel. many were secluded in separate rooms or outbuildings, such as huts; some were also restrained by ropes, belts or chains; a few were locked in iron cages. program staff used "free-medication" funds, which were developed by 686 program for providing a subsidy (cny 700 per case per year) for the expenses of essential antipsychotic medication to the patients who were socio-economically disadvantaged, to unlock and treat the patients in restraints between 2005 and 2008. from 2008, the 686 program provided additional funds (cny 5,000 per case per year) for the "unlocking and treatment" intervention to "free" the locked patients and to provide immediate medical care and enrollment in "686" follow-up services. widespread reports by journalists, human rights activists, and mental health specialists make it clear that in societies with limited mental health resources in asia and africa, many persons with severe mental illness are locked by family members and live in appalling conditions [12] [13] [14] [15] [16] . others are locked or chained in temples or compounds of traditional healers [17] . despite such reports, we find no report of empirical studies of the long-term effectiveness of programs designed to "unlock" the patients and provide mental health services in such settings. only one report published in english demonstrated that 23 restrained patients with schizophrenia-spectrum disorder exhibited clinical improvement within 19 months of psychiatric treatment after unlocking in bali, indonesia [18] . however, no specific outcome measures were clearly indicated. the present study aims to measure the long-term effects of an "unlocking and treatment" intervention undertaken from 2005 in the context of a larger mental health services reform program in china. patients with severe mental disorders were followed-up about their medication adherence, mental health status, social functioning and family burden in 2009 and 2012 to investigate the changes over time following the unlocking efforts. we hypothesized that the patients and families would benefit from the intervention and the improvements achieved could be largely sustained through 2012. this report concludes by suggesting the implications of this intervention for other settings with limited mental health resources. we designed a nation-wide two-stage follow-up study to investigate the initial phases of the 686 program "unlocking and treatment" intervention. individuals who fulfilled the following criteria were included in the study: 1) met diagnostic criteria for schizophrenia, schizoaffective disorder, bipolar disorder, delusional disorder, mental retardation with psychotic symptoms, or schizophrenia-like psychosis in epilepsy; 2) received the "unlocking and treatment" intervention provided by the 686 program between january 1, 2005 and july 31, 2009; and 3) were not inpatients at the time of investigation. by july 2009, the 686 program had enrolled and unlocked a total of 271 patients in the demonstration sites across 26 provinces of china. (no locked cases were reported by the demonstration sites in beijing, shanghai, jiangsu and zhejiang at that time.) these represented a complete sample of the locked individuals among the patients with severe mental disorders registered in "686" demonstration sites at that moment. among them, 266 were included in stage one of the study between august 22, 2009 and september 5, 2009 . information about patients and families were gathered for both the time when patients were unlocked (t0) and the time of stage one interviews (t1) to allow pre-post comparison. in june 2012, 230 of these patients and families were followed-up in stage two of the study (t2) (fig 1) . case report forms designed by the "686" implementation and evaluation research team from peking university institute of mental health (pkuimh) were utilized at both 2009 stage one data collection (concerning t0 and t1) and 2012 stage two data collection (t2) to collect the same data at each time point. t0 was defined as one month prior to the "686" unlocking action; t1 was defined as the month prior to participation in the stage one of the study in 2009; and t2 was defined as the month prior to the date of participation in the stage two study in 2012. one or two key investigators from each province were trained to collect patient clinical data concerning t0, t1 and t2 from existing medical records routinely recorded by clinical staff for each unlocked patient, dated from the time patients were first unlocked by the 686 program (i.e., t0). hospital psychiatrists and community case managers reviewed the accuracy of the case records from hospitalizations as well as records from the community clinics. in addition, the key investigators were trained to interview family members in their homes during stage one (concerning t0 and t1) and stage two (t2) studies to collect data about the locking and relocking history as well as family members' subjective experiences of family burden. the study was approved by the ethics review board at peking university institute of mental health (pkuimh). patients and/or their family members/guardians consented to participate in a written form. neighborhood/village committee staff, who were trained by 686 program to be mainly responsible for helping find the patients and leading community advocacy, provided information about the seriously mentally ill individuals who were locked in their family homes and assisted the unlocking team reach the patients. the unlocking process began with requesting permission from families of locked patients to allow a small group of trained professionals to free the patients. among the patients registered by 686 program, 271 were in restraints at home, and all of the families agreed to participate in the "unlocking and treatment" intervention program. the unlocking team consisted of three or more staff, including psychiatric health professionals (psychiatrists and psychiatric nurses), community persons who were often health workers, and neighborhood committee members. following unlocking, patients received physical exams and psychiatric diagnoses. patients assessed to be in need of hospitalization were admitted immediately to the provincial psychiatric hospitals. costs of medical and psychiatric treatment, including medications, were covered by the 686 program. upon discharge, patients were enrolled in "686" community services and followed monthly in their community by specially trained "686" case managers, who are mainly primary health care workers. persons unlocked who were assessed as not needing hospitalization were enrolled in "686" community services immediately after being freed; costs of antipsychotic and other prescribed medications, regular follow-ups, and community-based rehabilitation were covered by the 686 program. as an important component of the package of care provided by the 686 program, health education and psychosocial support were also provided to patients and their family members during follow-up visits at community health centers or village clinics. psychiatrists, psychiatric nurses, lay health care providers and non-health workers made up multidisciplinary teams to provide mental health services and non-professional services. changes in patients were assessed and recorded on a regular basis on follow-up by staff who were trained by the 686 program to conduct standardized clinical assessments. these procedures continue into the present. in case that there is failure of preventing "relocking", the "686" teams would take immediate action to restart the "unlocking and treatment" intervention as far as possible. mental health status, diagnoses, and violent behaviors. following unlocking, a psychiatrist conducted interviews with the patient and a family member who best knew the patient. overall psychiatric symptoms were assessed with the brief psychiatric rating scale (bprs) and global severity was assessed with clinical global impression (cgi) scales. a final diagnosis, based on icd-10 criteria, was made by a senior psychiatrist who reviewed the medical history, illness symptoms and associated impairments, and other available information. violent behaviors related to initiating restraint were evaluated on level 1 to level 5 by using the scale established by the national working group of 686 program [19] , defined as follows: level 1: threats other people verbally, or shouts at others, but doesn't damage any physical issue; level 2: damages only domestic property at home, and can be persuaded to stop; level 3: damages property at home or out of home, but cannot be persuaded to stop; level 4: repetitively damages property or hurt other people at home or out of home, but cannot be persuaded to stop; level 5: hurts other people with any kind of weapon, or sets fire, or explodes a bomb, etc. in the 686 program, level 3 and above is regarded as cases with high violence. medication adherence. adherence with antipsychotic medication over the previous month was rated by the clinician in closest contact with the patient and recorded on a three point measure as good adherence, uses antipsychotics intermittently, and does not use medications. "good adherence" was defined as patients actively take medications as prescribed, which includes following the prescribed dosing schedule, taking the full dose and refilling prescriptions. patients were rated as "uses antipsychotics intermittently" if they followed the prescription about half of the time during last month. "does not use medications" was defined as patients who did not receive antipsychotic medications for longer than two weeks. the rating was based on knowledge of the patient from routine clinical contact. social functioning. assessments of social functioning include five categories: daily life activities, housework, productive labor or work, learning ability, and interpersonal relationships. each category of social functioning was evaluated on a three point measure as good, average or poor [19] . the assessments were based on clinician's observation of the patient's performance as well as reports from patient and family members. family burden ratings. we explored the impact of patient mental illness on the well being of families, taking changes in caregivers' ratings of their family burden experiences as a subjective measure. family members were asked to rate their subjective experiences on analogue scales from 0 "no impact at all" to 10 "extremely negative impact" for the five categories of family burden-stigma, psychological pressures, economic burden, loss of personal energy, and interpersonal relationships. ratings concerning t0 were obtained by families' retrospective reflections at 2009 stage one study. relocking rate. patients remaining free of restraints after receiving "686" unlocking and treatment intervention was considered a sign of success of the program. the relocking rate was defined as the proportion of patients who were relocked after the initial "unlocking and treatment" intervention. the comparison of variables at t0 (before unlocking), t1 (after unlocking 2009) and t2 (after unlocking 2012) was performed using wilcoxon signed rank tests, as the data about medication adherence and social functioning were of ordinal categorical variables, and the data about family burden ratings were not normally distributed and did not meet the criteria of compound symmetry. chi-square tests were used to measure male/female and rural/urban differences in terms of psychiatric diagnosis and duration of illness. the level of statistical significance was defined a priori as a two-sided p-value of 0.05 or less. all calculations were performed with spss version 13.0. as 36 (14%) cases of the total sample dropped out of the study at t2, data from 230 (86%) cases were used in the statistical analysis and related comparisons at t2. based on our analysis of demographic features, restraint history, and measures of mental health status, violent behaviors, and social functioning at both t0 and t1, there was no evidence of systematic bias introduced by loss to follow-up of 36 patients. a total of 266 subjects aged 17-78 (mean age 38.6 [sd 9.9]) were included in the study at stage one. as presented in table 1 , the "locked" population suffered from grave mental illnesses of long duration, with 96% afflicted with schizophrenia. there were no male/female and rural/ urban differences in terms of psychiatric diagnosis or duration of illness (p>0.05). table 2 describes the restraint history of these patients before being unlocked (t0). total time of restraint varied from two weeks to 28 years, with 32% being locked multiple times. the vast majority of families reported that the top two reasons for "locking" a mentally ill family member were financial problems associated with caring for the patient (96%) and serious difficulties in finding capable caretakers (87%). families also reported that patient violence triggered the most recent restraint; 94% of patients exhibited serious violent behaviors, at level 3 or above on the 686 program's violent behavior scale [19] . after being unlocked by the 686 program, 233 patients (88%) were admitted to a psychiatric hospital for professional treatment; 33 patients (12%) were immediately enrolled in "686" community outpatient programs. upon discharge from psychiatric hospital, 91% of inpatients were regarded by medical staff as improved and 9% as significantly improved; two (<1%) showed no improvement. changes in patient medication adherence and social functioning table 3 illustrates changes in patients as measured in stage one (t0 and t1) and stage two (t2) studies. prior to unlocking, 82% of the patients were not receiving antipsychotic medication; 17% of the patients were receiving medications and used them intermittently; only one patient showed good adherence to medication. all 266 patients were assessed to require antipsychotic medications at the time of unlocking. significant changes in medication adherence occurred after patients were unlocked and entered into the "686" treatment program. three quarters of patients were assessed as having good adherence to medication treatment both at t1 and at t2. social functioning in each of the five categories also improved significantly, showing major changes from t0 to t1 (p<0.0001). although the proportion of patients with poor functioning increased slightly from t1 to t2 (p<0.05) except in "productive labor or work" category (p = 0.2452), most changes were sustained through 2012, as indicated by the t0-t2 comparisons (p<0.0001). table 3 . changes in patient medication adherence and social functioning (t0, t1 and t2 comparison). t0 t1 t2 t0-t1 test t0-t2 test t1-t2 as illustrated in table 4 , when comparing family members' evaluations on how much they were affected by the patients' mental illness, 2009 post-unlocking scores illustrated significant improvement in caregivers' experience with a reduction in rating scores on each of the measures of family burden from t0 to t1 (p<0.0001). although measures of family burden except stigma (p = 0.1089) increased slightly from t1 to t2 (p<0.0001), the improvement in five categories of family burden ratings were largely sustained as shown in the t0-t2 comparisons (p<0.0001). by the time of the stage two study, 21 (8%) of the initial 266 individuals had an episode of "relocking" following the "686" intervening efforts. poor adherence to treatment, including "uses antipsychotics intermittently" and "does not use medications", was found at t2 in 12 (57%) cases who were relocked. families noted the top two reasons for relocking to be similar to reasons for initial locking: no capable care-giver was available and financial difficulties. this study reports findings of an implementation research project designed to measure the effectiveness of an "unlocking and treatment" intervention that was developed in the context of china's 686 program [10] . our analysis of cases of patients who had been unlocked, entered into treatment, and returned to their homes-266 at baseline in 2009, and 230 in 2012-provides the first practice-based evidence for long-term benefits of an intervention approach that is linked to a major mental health services reform. the persons locked by family members in rural and urban china were primarily patients who had lived with severe mental illness for many years, as a result of a functional failure of the traditional hospital-based mental health model. less than 1% of the total sample were in regular treatment. unlocking patients and providing access to free hospital treatment and continuing mental health care in the community were shown to have powerful and sustained benefits for these patients. it is encouraging that the number of persons regularly taking medicines increased from one person at the time of unlocking to three quarters in 2009 and in 2012, table 4 . changes in family burden ratings (t0, t1 and t2 comparison). t0 a t1 t2 t0-t1 test t0-t2 test t1-t2 data are mean (sd). family members were asked to rate their subjective experiences on analogue scales from 0 "no impact at all" to 10 "extremely negative impact". the higher the score the higher the family members experience family burden that was associated with patients' mental illness. especially given that an epidemiological study suggests only 60% of persons with psychotic illness in china have ever sought specialized mental health care [4] . besides the improved clinical outcome achieved, improved social functioning was shown to hold not only through 2009 (t1) but through 2012 (t2), compared with the time the patients were freed by the 686 program (t0). improved access to care and social functioning of patients had direct benefits for the lives of families as well. family caregivers described significant reductions in feelings of stigma, psychological pressures, economic burden, loss of personal energy, and negative personal relationships at both t1 and t2. we observed some decline in patient social functioning and increased family burden from t1 to t2. this might be due to the habituation effect. over the years after the "unlocking", families became accustomed to the initial impact of the initiative on patients' clinical condition, and thereby their ratings were biased toward minimizing the initial improvement at t2. other possible explanations might be: 1) patients received more intensive services during t0-t1, especially immediately after being enrolled in the "unlocking and treatment" intervention, than during t1-t2; such services included inpatient care, assistance from the disabled federation and the civil affairs association, the "686" rehabilitation programs, etc. 2) those were the most vulnerable population in the community with the least societal resources. therefore the families had very limited ability to take care of the patients and cope with family burdens. it is suggested that further efforts are needed to continue to support the long-term recovery of patients from severe mental illness and to improve the well being of the families. our study suggests that successful eradication of restraint practices can be achieved and sustained by scaling up services to provide effective, accessible and affordable care for people living with mental disorders [20] [21] . the finding that more than 92% of those unlocked and entered into continuous treatment by the 686 program remained free of restraints by 2012 demonstrates the feasibility of improving the human rights of persons with severe mental illness by increasing access to mental health care in the community [22] , even with limited societal resources. nevertheless, the failure to prevent relocking for 21 individuals suggests that considerable room for improvement of our mental health care practice still exists. continuous and efficient treatment and strong social supports are warranted to prevent the recurrence of restraints. persons with severe mental illness worldwide live in "zones of abandonment" [23] . in north america, these include the streets, halfway houses, homeless shelters, and prisons. in countries with very limited mental health resources, these are often homes of family members, where a combination of lack of medical care, outbursts of violence inside the household or a tendency to wander outside the house, stigma and embarrassment, and lack of ability of families to provide care lead to seclusion and restraints [24] . this phenomenon, which occurs throughout the world [15] [16] [17] [18] [25] [26] [27] , represents human rights violations of individuals with mental and psychosocial disabilities [20] . to prevent patients with severe mental disorders from being pushed to the margins of society, continuous development of accessible services in the community should be guaranteed for the long run. policies and laws should be well formulated to stimulate advocacy and education campaigns, as well as establish and develop legal and oversight mechanisms to prevent human rights violations [7, 20] . limitations of this study grow out of the fact that it was designed after the program was underway; it is not a prospective study with an experimental design. family burden ratings at t0 were obtained by family care-givers' retrospective reflections, which may introduce bias; these findings should therefore be interpreted with caution given their subjective nature. however, family care-givers, usually parents and spouse of the patients, had very clear memories concerning the unique and painful experience of locking a mentally ill family member. we believe that with limitations, the rating of family burden based on retrospective recall thus has important validity. the sample of the study was 96% schizophrenia; this might make it difficult to generalize findings to locked individuals with other serious mental illnesses. at the time of this study, it was possible that one or more patients with severe mental disorders remained locked and were not reached by the 686 program during implementation in the demonstration sites. the 686 program was implemented according to the annual budget and tasks allocated by the central government, and patients who were the most mentally ill and lived in poverty were prioritized. the patients registered at the initial phase of the 686 program were 0.17% of the general population, and 0.2% of all those registered were found to be in restraints and therefore freed and treated by the program. in a few cases, although the exact number was not available, patients or families refused to be enrolled by 686 program for some concerns. further efforts are warranted for the program to cover the whole population of patients with severe mental disorders and provide effective mental health care for people in need. with such efforts, it is hoped that those individuals who remain in restraints at home can be fully identified, and "locking" of patients can be ultimately eliminated. there is obviously much to be done in china to scale up the 686 program for the whole nation [28] [29] and to improve quality of care. this will require substantially increased investment in mental health services. however, the success of the program in maintaining the severely ill individuals in treatment over three to seven years, and the benefits of the intervention for those who have lived for years with untreated psychosis and for their families, attests to the feasibility and social value of the "686" model. the chinese "686" model can serve to inform similar efforts in other lmics where restraint of the patients continues [15] [16] [17] [18] [25] [26] [27] . extending protection of human rights of persons with severe mental illness and providing patients equitable access to effective care in the community remain enormous challenges in the least resourced countries [30] [31] . findings of this study suggest that the chinese model may be one of the important models for low-resource settings. the 686 program demonstrates the feasibility of building model mental health programs to scale up services [21, 29] , by using limited investment of central government to mobilize administrative and societal resources, allocating funds from local government and lay organizations, as well as by involving lay health care providers and non-health workers in the multidisciplinary mental health service team [5, 32] . rapid health transition in china, 1990-2010: findings from the global burden of disease study prime: a programme to reduce the treatment gap for mental disorders in five low-and middle-income countries packages of care for mental, neurological, and substance use disorders in low-and middle-income countries prevalence, treatment, and associated disability of mental disorders in four provinces in china during 2001-05: an epidemiological survey mental health system in china: history, recent service reform and future challenges mental health law of the people's republic of china (english translation with annotations) china's national mental health law: a 26-year work in progress integrating mental health into primary care: the policy maker's perspective and experience in china integration of hospital and community services-the '686 project'-is a crucial component in the reform of china's mental health services significance of the 686 program for china and for global mental health packages of care for schizophrenia in lowand middle-income countries mentally iil man caged for decade in china lost in paradise: the chained-up mentally ill of bali another side of african psychiatry in the 21st century-chaining as containment breaking the chains local suffering and the global discourse of mental health and human rights: an ethnographic study of responses to mental illness in rural ghana treating the untreated: applying a community-based, culturally sensitive psychiatric intervention to confined and physically restrained mentally ill individuals in bali, indonesia. eur arch psychiatry china (2012) management and treatment guidelines for psychosis human rights violations of people with mental and psychosocial disabilities: an unresolved global crisis scale up services for mental disorders: a call for action the wpa action plan vita-life in a zone of social abandonment the zone of social abandonment in cultural geography: on the street in the united states, inside the family in india aceh free pasung: releasing the mentally ill from physical restraint global mental health: a failure of humanity a qualitative study of religious practices by chronic mentally ill and their caregivers in south india wpa guidance on steps, obstacles and mistakes to avoid in the implementation of community mental health care scale up of services for mental health in low-income and middle-income countries resources for mental health: scarcity, inequity, and inefficiency barriers to improvement of mental health services in low-income and middle-income countries treatment and prevention of mental disorders in low-income and middle-income countries we thank the patients and the family members who participated in the study. we acknowledge all "686" staff in china who contribute to implementing "unlocking and treatment" intervention and providing mental health care to the patients. key: cord-332922-2qjae0x7 authors: mbuvha, rendani; marwala, tshilidzi title: bayesian inference of covid-19 spreading rates in south africa date: 2020-08-05 journal: plos one doi: 10.1371/journal.pone.0237126 sha: doc_id: 332922 cord_uid: 2qjae0x7 the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) pandemic has highlighted the need for performing accurate inference with limited data. fundamental to the design of rapid state responses is the ability to perform epidemiological model parameter inference for localised trajectory predictions. in this work, we perform bayesian parameter inference using markov chain monte carlo (mcmc) methods on the susceptible-infected-recovered (sir) and susceptible-exposed-infected-recovered (seir) epidemiological models with time-varying spreading rates for south africa. the results find two change points in the spreading rate of covid-19 in south africa as inferred from the confirmed cases. the first change point coincides with state enactment of a travel ban and the resultant containment of imported infections. the second change point coincides with the start of a state-led mass screening and testing programme which has highlighted community-level disease spread that was not well represented in the initial largely traveller based and private laboratory dominated testing data. the results further suggest that due to the likely effect of the national lockdown, community level transmissions are slower than the original imported case driven spread of the disease. the first reported case of the novel coronavirus (sars-cov-2) in south africa was announced on 5 march 2020, following the initial manifestation of the virus in wuhan china in december 2019 [1] [2] [3] . due to its further spread and the severity of its associated clinical outcomes, the disease was subsequently declared a pandemic by the world health organisation (who) on 11 march 2020 [1, 2] . in south africa, by 26 april 2020, 4546 people had been confirmed to have been infected by the coronavirus with 87 fatalities [4] . numerous states have attempted to minimise the growth in number of covid-19 infections [1, 5, 6] . these attempts are largely based on non-pharmaceutical interventions (npis) aimed at separating the infectious population from the susceptible population [1] . these initiatives aim to strategically reduce the increase in infections to a level where their healthcare systems stand a chance of minimising the number of fatalities [1] . some of the critical indicators for policymaker response planning include projections of the infected population, estimates of health care service demand and whether current containment measures are effective [1] . as the pandemic develops in a rapid and varied manner in most countries, calibration of epidemiological models based on available data can prove to be [7] . this difficulty is further escalated by the high number of asymptomatic cases and the limited testing capacity [1, 2] . a fundamental issue when calibrating localised models is inferring parameters of compartmental models such as susceptible-infectious-recovered (sir) and the susceptible-exposedinfectious-recovered (seir) that are widely used in infectious disease projections. in the view of public health policymakers, a critical aspect of projecting infections is the inference of parameters that align with the underlying trajectories in their jurisdictions. the spreading rate is a parameter of particular interest which is subject to changes due to voluntary social distancing measures and government-imposed contact bans. the uncertainty in utilising these models is compounded by the limited data in the initial phases and the rapidly changing dynamics due to rapid public policy changes. to address these complexities, we utilise the bayesian framework for the inference of epidemiological model parameters in south africa. the bayesian framework allows for both incorporation of prior knowledge and principled embedding of uncertainty in parameter estimation. in this work we combine bayesian inference with the compartmental seir and sir models to infer time varying spreading rates that allow for quantification of the impact of government interventions in south africa. compartmental models are a class of models that is widely used in epidemiology to model transitions between various stages of disease [1, 8, 9] . we now introduce the susceptible-exposed-infectious-recovered (seir) and the related susceptible-infectious-recovered (sir) compartmental models that have been dominant in covid-19 modelling literature [1, 5, 6, 10] . the susceptible-exposed-infectious-recovered model. the seir is an established epidemiological model for the projection of infectious diseases. the seir models the transition of individuals between four stages of a condition, namely: • being susceptible to the condition, • being infected and in incubation • having the condition and being infectious to others and • having recovered and built immunity for the disease. the seir can be interpreted as a four-state markov chain which is illustrated diagrammatically in fig 1. the seir relies on solving the system of ordinary differential equations below representing the analytic trajectory of the infectious disease [1] . where s is the susceptible population, i is the infected population, r is the recovered population and n is the total population where n = s + e + i + r. λ is the transmission rate, σ is the rate at which individuals in incubation become infectious, and μ is the recovery rate. 1/σ and 1/μ therefore, become the incubation period and contagious period respectively. we also consider the susceptible-infectious-recovered (sir) model which is a subclass of the seir model that assumes direct transition from the susceptible compartment to the infected (and infectious) compartment. the sir is represented by three coupled ordinary differential equations rather than the four in the seir. fig 2 depicts the three states of the sir model. the basic reproductive number r 0 . the basic reproductive number (r 0 ) represents the mean number of additional infections created by one infectious individual in a susceptible population. according to the latest available literature, without accounting for any social distancing policies the r 0 for covid-19 is between 2 and 3.5 [2, 6, 10, 11] . r 0 can be expressed in terms of λ and μ as: extensions to the seir and sir models. we use an extended version of the seir and sir models of [6] that incorporates some of the observed phenomena relating to covid-19. first we include a delay d in becoming infected (i new ) and being reported in the confirmed case statistics, such that the confirmed reported cases cr t at some time t are in the form [6] : we further assume that the spreading rate λ is time-varying rather than constant with change points that are affected by government interventions and voluntary social distancing measures. we follow the framework of [6] to perform bayesian inference for model parameters on the south african covid-19 data. the bayesian framework allows for the posterior inference of parameters which updates prior beliefs based on a data-driven likelihood. the posterior inference is governed by bayes theorem as follows: where p(w|d, m) is the posterior distribution of a vector of model parameters (w) given the model(m) and observed data(d), p(d|w, m) is the data likelihood and p(d) is the evidence. the likelihood. the likelihood indicates the probability of observing the reported case data given the assumed model. in our study, we adopt the student-t distribution as the likelihood as suggested by [6] . similar to a gaussian likelihood, the student-t likelihood allows for parameter updates that minimise discrepancies between the predicted and observed reported cases. priors. parameter prior distributions encode some prior subject matter knowledge into parameter estimation. in the case of epidemiological model parameters, priors incorporate literature based expected values of parameters such as recovery rate(μ), spreading rate(λ), change points based on policy interventions etc. the prior settings for the model parameters are listed in table 1 . we follow [6] by selecting lognormal distributions for λ and σ such that the initial mean basic reproductive number is 3.2 which is consistent with literature [2, 5, 6, 10, 12] . we set a lognormal prior for the σ such that the mean incubation period is five days. we use the history of government interventions to set priors on change points in the spreading rate. the priors on change-points include 19/ 03/2020 when a travel ban and school closures were announced, and 28/03/2020 when a national lockdown was enforced. we keep the priors for the lognormal distributions of the spreading rates after the change points weakly-informative by setting the same mean as λ 0 and higher variances across all change points. this has the effect of placing greater weight on the data driven likelihood. similar to [6] we adopt weakly-informative half-cauchy priors for the initial conditions for the infected and exposed populations. markov chain monte carlo (mcmc). given that the closed-form inference of the posterior distributions on the parameters listed in table 1 is infeasible, we make use of markov chain monte carlo to sample from the posterior. monte carlo methods approximate solutions [6, 10] . in this work, we explore inference using metropolis-hastings (mh), slice sampling and no-u-turn sampler (nuts). metropolis hastings (mh). mh is one of the simplest algorithms for generating a markov chain which converges to the correct stationary distribution. the mh generates proposed samples using a proposal distribution. a new parameter state w t � is accepted or rejected probabilistically based on the posterior likelihood ratio: a common proposal distribution is a symmetric random walk obtained by adding gaussian noise to a previously accepted parameter state. random walk behaviour of such a proposal typically results in low sample acceptance rates. while sample acceptance is guaranteed with slice sampling, a large slice window can lead to computationally inefficient sampling while a small window can lead to poor mixing. hybrid monte carlo (hmc) and the no-u-turn sampler (nuts). metropolis-hastings (mh) and slice sampling tend to exhibit excessive random walk behaviour-where the next state of the markov chain is randomly proposed from a proposal distribution [13] [14] [15] . this results in low proposal acceptance rates and small effective sample sizes. hmc proposed by [16] reduces random walk behaviour by adding auxiliary momentum variables to the parameter space [15] . hmc creates a vector field around the current state using gradient information, which assigns the current state a trajectory towards a high probability next state [15] . the dynamical system formed by the model parameters w and the auxiliary momentum variables p is represented by the hamiltonian h(w, p) written as follows [15, 16] : where m(w) is the negative log-likelihood of the posterior distribution in eq 7, also referred to as the potential energy. k(p) is the kinetic energy defined by the kernel of a gaussian with a covariance matrix m [17] : the trajectory vector field is defined by considering the parameter space as a physical system that follows hamiltonian dynamics [15] . the dynamical equations governing the trajectory of the chain are then defined by hamiltonian equations at a fictitious time t as follows [16] : in practical terms, the dynamical trajectory is discretised using the leapfrog integrator. in the leapfrog integrator to reach the next point in the path, we take half a step in the momentum direction, followed by a full step in the direction of the model parameters-then ending with another half step in the momentum direction. due to the discretising errors arising from leapfrog integration a metropolis acceptance step is then performed in order to accept or reject the new sample proposed by the trajectory [15, 18] . in the metropolis step the parameters proposed by the hmc trajectory w � are accepted with the probability [16] : pðw � jd; a; b; hþ pðwjd; a; b; hþ algorithm 1 shows the pseudo-code for the hmc where � is a discretisation stepsize. the leapfrog steps are repeated until the maximum trajectory length l is reached. the hmc algorithm has multiple parameters that require tuning for efficient sampling, such as the step size and the trajectory length. in terms of trajectory length, a trajectory length that is too short leads to random walk behaviour similar to mh. while a trajectory length that is too long results in a trajectory that inefficiently traces back. the stepsize is also a critical parameter for sampling, small stepsizes are computationally inefficient leading to correlated samples and poor mixing while large stepsizes compound discretisation errors leading to low acceptance rates. tuning these parameters requires multiple time consuming trial runs. nuts automates the tuning of the leapfrog stepsize and trajectory length. in nuts the stepsize is tuned during an initial burn-in phase by targeting particular levels of sample acceptance. the trajectory length is tuned by iteratively adding steps until either the chain starts to trace back (u-turn) or the hamiltonian explodes (becomes infinite). we use the samplers described above to calibrate the seir and sir models on daily new cases and cumulative cases data for south africa up to and including 20 april 2020 provided by johns hopkins university's center for systems science and engineering(csse) [3] . sir and seir model parameter inference was performed using confirmed cases data up to and including 20 april 2020 and mcmc samplers described in the methodology section. each of the samplers are run such that 5000 samples are drawn with 1000 burn-in and tuning steps. we use leave-one-out(loo) cross-validation error of [19] to evaluate the goodness of fit of each model. table 2 shows the loo validation errors of the various models. it can be seen that the sir model with two change points as the best model fit with the lowest mean loo of 448.00. the seir model with two change points showed a mean loo of 459.94. we note that [6] similarly finds that the sir model displayed superior goodness of fit to the seir on german data. we now further present detailed results of the sir and seir models with inference using nuts, the trace plots from these models indicating stationarity in the sampling chains are provided in s2 and s5 figs. the trace plots for the sir and seir models using mh are provided in s3 and s6 figs, while similar trace plots for slice sampling are provided in s4 and s7 figs. the trace plots largely indicate that the nuts sampler displays greater agreement between parallel chains thus lower rhat values. time-varying spread rates allow for inference of the impact of various state and societal interventions on the spreading rate. fig 5 shows the fit and projections based on sir models with zero, one and two change points. as can be seen from the plot the two change point model best captures the trajectory in the development of new cases relative to the zero and one change point models. the superior goodness of fit of the two change point model is also illustrated in table 2 . the fit and projections showing similar behaviour on the seir model with various change points are shown in fig 6. the mean reporting delay time in days was found to be 6.848 (ci [5.178, 8.165 ]), literature suggests this delay includes both the incubation period and the test reporting lags. the posterior distribution incubation period from the seir model in fig 7 yields the inference of parameters is dependent on the underlying testing processes that generate the confirmed case data. the effect of the mass screening and testing campaign was to change the underlying confirmed case data generating process by widening the criteria of those eligible for testing. while initial testing focused on individuals that either had exposure to known cases or travelled to known covid-19 affected countries, mass screening and testing further introduced detection of community level transmissions which may contain undocumented contact and exposure to covid-19 positive individuals. we have performed bayesian parameter inference of the sir and seir models using mcmc and publicly available data as at 20 april 2020. the resulting parameter estimates fall in-line with the existing literature in-terms of mean baseline r 0 (before government action), mean incubation time and mean infectious period [2, 5, 6, 10] . we find that initial government action that mainly included a travel ban, school closures and stay-home orders resulted in a mean decline of 80% in the spreading rate. further government action through mass screening and testing campaigns resulted in a second trajectory change point. this latter change point is mainly driven by the widening of the population eligible for testing, from travellers (and their known contacts) to include the generalised community who would have probably not afforded private lab testing which dominated the initial data. this resulted in an increase of r 0 to 1.304. the effect of mass screening and testing can also be seen in fig 9 indicating a mean increase in daily tests preformed from 1639 to 4374. the second change point illustrates the possible existence of multiple pandemics, as suggested by [20] . thus testing after 28 march is more indicative of community-level transmissions that were possibly not as well documented in-terms of contact tracing and isolation relative to the initial imported infection driven pandemic. this is also supported by the documented increase in public laboratory testing (relative to private) past this change point, suggesting health care access might also play a role in the detection of community-level infections [21] . we have utilised a bayesian inference framework to infer time-varying spreading rates of covid-19 in south africa. the time-varying spreading rates allow us to estimate the effects of government actions on the dynamics of the pandemic. the results indicate a decrease in the mean spreading rate of 60%, which mainly coincides with the containment of imported infections, school closures and stay at home orders. the results also indicate the emergence of community-level infections which are increasingly being highlighted by the mass screening and testing campaign. the development of the community level transmissions (r 0 � 1.3041 (ci[0.887, 1.7748])) of the pandemic at the time of publication appears to be slower than that of the initial traveller based pandemic (r 0 � 3.278 (ci[2.715, 3.73])). a future improvement to this work could include extensions to regional and provincial studies as current data suggests varied spreading rates both regionally and provincially. as more government interventions come to play priors on more change points might also be necessary. on data-driven management of the covid-19 outbreak in south africa. medrxiv covid-19) an interactive web-based dashboard to track covid-19 in real time. the lancet infectious diseases coronavirus disease (covid-19) case data-south africa impact of nonpharmaceutical interventions (npis) to reduce covid19 mortality and healthcare demand inferring covid-19 spreading rates and potential change points for case number forecasts an epidemiological forecast model and software assessing interventions on covid-19 epidemic in china compartmental models in epidemiology an introduction to compartmental modeling for the budding infectious disease modeler fundamental principles of epidemic spread highlight the immediate need for large-scale serological surveys to assess the stage of the sars-cov-2 epidemic feasibility of controlling 2019-ncov outbreaks by isolation of cases and contacts. medrxiv detecting suspected epidemic cases using trajectory big data bayesian automatic relevance determination for feature selection in credit default modelling sampling techniques in bayesian finite element model updating automatic relevance determination bayesian neural networks for credit card default modelling bayesian learning via stochastic dynamics bayesian learning for neural networks mcmc using hamiltonian dynamics. handbook of markov chain monte carlo practical bayesian model evaluation using leave-one-out cross-validation and waic sa's covid-19 pandemic trends and next steps update on covid-19 key: cord-341097-c96hm610 authors: mayer, craig s.; williams, nick; huser, vojtech title: analysis of data dictionary formats of hiv clinical trials date: 2020-10-05 journal: plos one doi: 10.1371/journal.pone.0240047 sha: doc_id: 341097 cord_uid: c96hm610 background: efforts to define research common data elements try to harmonize data collection across clinical studies. objective: our goal was to analyze the quality and usability of data dictionaries of hiv studies. methods: for the clinical domain of hiv, we searched data sharing platforms and acquired a set of 18 hiv related studies from which we analyzed 26 328 data elements. we identified existing standards for creating a data dictionary and reviewed their use. to facilitate aggregation across studies, we defined three types of data dictionary (data element, forms, and permissible values) and created a simple information model for each type. results: an average study had 427 data elements (ranging from 46 elements to 9 945 elements). in terms of data type, 48.6% of data elements were string, 47.8% were numeric, 3.0% were date and 0.6% were date-time. no study in our sample explicitly declared a data element as a categorical variable and rather considered them either strings or numeric. only for 61% of studies were we able to obtain permissible values. the majority of studies used csv files to share a data dictionary while 22% of the studies used a non-computable, pdf format. all studies grouped their data elements. the average number of groups or forms per study was 24 (ranging between 2 and 124 groups/forms). an accurate and well formatted data dictionary facilitates error-free secondary analysis and can help with data de-identification. conclusion: we saw features of data dictionaries that made them difficult to use and understand. this included multiple data dictionary files or non-machine-readable documents, data elements included in data but not in the dictionary or missing data types or descriptions. building on experience with aggregating data elements across a large set of studies, we created a set of recommendations (called consider statement) that can guide optimal data sharing of future studies. in recent years, efforts to define research common data elements (cdes) attempt to harmonize data collection across clinical studies [1] . sheehan at al. defined a cde as 'a combination of a precisely defined question paired with a specified set of responses to the question that is common to multiple datasets or used across different studies' [2] . cdes have been defined on both a general level applicable to a broad range of diseases and studies, as well as on a disease specific level that focuses on data elements applicable to a narrow context. an example of general data elements are those defined by phenx initiative, such as employment status, education attainment, or health insurance coverage (all with appropriate permissible values for such elements). an example of disease specific cdes are those defined by the therapeutic area user guide for hiv [3] published by the clinical data interchange standard consortium (cdisc). cdes are expected to deliver the following benefits: faster and cheaper study start-up, improved comparability and aggregation of data across studies, improved study data collection and study data quality, and improved data organization for re-use. another phenomenon that highlights the importance of cdes is the requirement to share de-identified individual participant data (ipd) of a completed observational study or interventional trial [4] . when data is shared, a data dictionary is typically provided to describe individual data elements used in a study. in recent decades, tens of new data sharing platforms with the aim of facilitating secondary research have emerged [5] . identification and standardization of cdes is, however, an ongoing challenge in the field of clinical research informatics [6] . some leaders have argued strongly for adoption of policies that require much stronger sponsor-enforced standardization [2, 7] , while others point to possible restrictions and additional cde burden on principal investigators of future studies [8, 9] . increased pressure for cde adoption can be seen in research efforts triggered by the covid-19 pandemic [10, 11] . many cde initiatives use expert consensus to achieve standardization. we refer to this method as a top down approach. if the number of data elements to standardize is large, expert consensus can be very time consuming. with increased availability of de-identified ipd data from completed studies, it is possible to arrive at cdes using a data-driven approach. most common data elements will simply appear in a high number of shared study datasets if a simple usage frequency approach is used. we refer to this method as a bottom up approach. a data-driven approach can also handle a large volume of common data elements. however, this data-driven approach depends on studies sharing their data elements in an analyzable format. these two general clinical research trends (effort to arrive at standardized cdes and increased sharing of completed studies) have also impacted the clinical domain of infectious diseases and hiv. for example, at clinicaltrials.gov, the number of studies that provide a link to de-identified ipd increased from 4.4% in 2014 (3 hiv studies have link to ipd out of 68 total hiv studies that completed in 2014) to 12.6% in 2019 (8 hiv studies have link to ipd out of 63 total hiv studies that completed in 2019). such numbers demonstrate a gradual shift towards increased data sharing. despite improvements, there are still opportunities for further enhancements in terms of format and the extent of data sharing in the hiv domain as well in clinical research in general). we present a study to analyze the quality and usability of data dictionaries of hiv studies. we focused on data dictionaries because they are an important common metadata artifact for data sharing. we thus search for hiv/aids interventional trials or observational studies using several approaches. in our article, we use the term study to refer to both interventional trials and observational studies. our goal is to find hiv studies that provide a data dictionary with a list of data elements used in a study and relevant additional metadata. we analyze the format and content of those data dictionaries. the study contribution lies in the generating of recommendations that improve data sharing of future hiv studies. our study is the first informatics study of data sharing format that analyzes a large body of hiv studies shared to date via various data sharing mechanisms. the presented study is limited to data dictionary analysis, although the motivation is to later analyze a large body of past hiv data elements to inform data-driven consensus on cdes. this study is part of a larger research project titled 'identification of research common data elements in hiv/aids using data science methods' [12] . in this section, we outline how we identified a set of analyzed hiv studies and define important terms for the analysis of data dictionaries. we also describe relevant standards for study data dictionaries. we searched clinical study data sharing platforms [5] for hiv studies that shared ipd. we defined hiv studies as any study relating to hiv, which includes any study with hiv positive participants, or any study relating to hiv infection (i.e. hiv prevention or vaccine trials). a data sharing platform is a web-based repository of completed clinical studies with study ipd and other study artifacts (such as protocol, study publications or case report forms) for each included study. the platforms we searched included national institute of drug abuse (nida) data share [13] , the national heart, lung and blood institute (nhlbi) biolincc, the national institute of child health and human development data and specimen hub (nichd dash), vivli, clinicalstudydatarequest.com, project data sphere, the national institute of mental health data archive (nda) and the yale open data access project. we also searched for hiv clinical trial networks using a web search engine and requested ipd from completed studies conducted by those networks. we excluded studies for which a data dictionary could not be obtained or inferred from ipd or studies that were not registered at clinical-trials.gov. in order to aggregate this data across many hiv studies, we obtained institutional review board approval at our local institution (or exemption if the data were de-identified). we filed appropriate data requests at the platforms where we found relevant hiv studies. for each hiv study included in our analysis, we organized the available data artifacts into the following categories: ipd data files, data dictionary files, study protocol documents and study de-identification notes. within the data dictionary category, we observed several different formats. some studies used single or multiple comma separated value files (csv), a widely accessible and machine-readable file format. some studies used portable document format (pdf) or a proprietary sas data catalog format ( � .sas7bcat) [14] . because we used computerized methods for aggregation, we did not include studies whose data dictionaries were not in a machine-readable format or could not be readily converted into such a format. this includes pdfs with scanned content. in our analysis of data dictionaries and data elements, we adopt the definitions developed by nih cde task force of the nih data council [15] . they clearly defined terms such as data element, common data element, form and permissible value [16] . in addition to using terminology defined by the nih cde task force, we have introduced three types of data dictionaries. we use the term element data dictionary (or element dictionary in shorter form) to describe a spreadsheet that enumerates individual data elements used in a study with fields such as data element name and description. we combined all data element data dictionaries to create an aggregate data elements file that contains data elements from a set of studies. due to the large number of data elements and to limit the project scope, we did not perform any semantic mapping of identical des. this aggregated file targeted the following attributes about each data element (referred to as target de model): (1) data element description, (2) data element id (sometimes called de name, de short label, variable name, or variable id), (3) data type (such as character, numeric, date, enumerated, or boolean), (4) length (provides information about the length of the character string or maximum value or range for a numeric data element), and (5) group id (sometimes called form name or form id; provides information on which form the de is being collected). to demonstrate some examples of study data elements, table 1 shows the element data dictionary from study nct00099359: 'trial of three neonatal antiretroviral regimens for prevention of intrapartum hiv transmission.' for brevity, the table shows only 10 selected data elements out of all 577 data elements used in that study. for each element data dictionary, we analyzed the accuracy and completeness of the dictionary. this included an analysis of the previously specified features, such as the presence and clarity of the data element descriptions and the prevalence, common usage, and accurate representation of the data type for each listed data element. we use the term forms data dictionary (or forms dictionary in shorter form) to refer to a data dictionary that provides a full list of titles and descriptions of all case report forms (crfs) used in the study (or other relevant metadata for data element grouping). table 2 shows an example of a forms dictionary from study nct01751646: 'vitamin d absorption in hiv infected young adults being treated with tenofovir containing cart.' we followed the same approach as with the element data dictionary and combined all form dictionaries across all analyzed studies. the intention was to see whether some crf titles appear more frequently. to eliminate similarly named forms, we manually mapped synonymous form titles to their preferred title and identified common crf names that appeared in at least two studies. for example, forms by the names 'f89' for nct01751646 and 'hxw0100' for nct01418014 both map to a common form name of 'family history'. the semantic mapping was much more feasible for forms (compared to data elements, where it was out of project scope) due to the relatively small total number of forms across all studies. for harmonizing data collection across studies, the issue of copyright protection on case report forms must be considered [17, 18] . to measure the impact of copyright, we analyzed whether any of the form (across all studies in our sample) were marked as copyrighted. finally, we use the term permissible values data dictionary (or permissible values dictionary in shorter form) to list permissible values that are possible for categorical data elements. each permissible value (on separate rows) is linked to the data element it provides the value for (the link is via data element id). for example, in study nct01772823 'an open label demonstration project and phase ii safety study of pre-exposure prophylaxis' for data element offtxr, which describes the reason for discontinuation, its permissible value dictionary has 6 permissible values defined: 1 (viral breakthrough), 2 (adverse reaction), 3 (subject's decision), 4 (clinician's decision), 5 (course completed), and 99 (other). each row represents a single possible value (organized under a respective data element). the permissible value information model has the following columns defined: (1) permissible value id (or permissible value short label; this column is optional and can be missing), (2) permissible value, (3) permissible value description (if the previous field does not sufficiently define the permissible value), and (4) data element id (for proper linking of this permissible value dictionary to the element dictionary). for the permissible value dictionaries, we assessed commonly used formats for permissible values, as well as the most commonly used values. for all analyzed studies, we also looked at primary and secondary outcomes [1] as defined for each study at clinicaltrials.gov. the assumption was that every outcome on clinicaltrials. gov would be linked to at least one study data element. during our review of the study's clini-caltrials.gov record we also determined whether the clinicaltrials.gov study record reflects the availability of the study results data, ipd data [19] or data dictionary. finally, during aggregation of data dictionaries across studies, we recorded positive features of dictionaries as well as challenges of dictionary formats that complicated the analysis. our goal was to create a set of recommendations for optimal data sharing for future hiv studies (presented in the discussion section). in addition to analyzing a set of real studies, we also investigated relevant standards for clinical study data dictionaries. our reason why we looked into such standards was to inform our efforts to combine multiple data dictionaries into a single data structure. there are two relevant data dictionary standards: cdisc define-xml and redcap. cdisc define-xml standard is used by the food and drug administration in the us [20] and pharmaceuticals and medical devices agency in japan. it was first released in 2005 and it uses extensible markup language (xml) to describe a data dictionary of a clinical study. redcap data dictionary format is another standard defined by the redcap electronic data capture system used by more than 3200 institutions world-wide [21] . redcap stands for research data capture. the redcap software was first released in 2004 and it uses a zip compressed spreadsheet file to represent a data dictionary. while it is not widely used by individual studies, it is of note that, for example, the phenx initiative provides their cdes in the redcap data dictionary format [22] . we also acknowledge that iso 11179 specification aims at describing data element metadata, however it does not clearly define an exchange format [23] . our platform search identified relevant hiv studies on three data sharing platforms: nhlbi biolincc (3 studies), nichd data and specimen hub (dash) (11 studies) and nida data archive (5 studies). we searched 5 additional data sharing platforms which did not yield any more results due to several reasons, including: (1) we did not find any hiv studies on those platforms, (2) the study data request process was still pending at the start of our dictionary analysis, or (3) we could not obtain the data. our web search identified 5 hiv/aids clinical trial networks. they were the hiv vaccine trials network (hvtn), the hiv prevention trials network (hptn), the aids clinical trial group (actg), the international maternal, pediatrics adolescent aids and the microbicide trials network (mtn). after filing official or email requests, we obtained studies from two networks: (1) hptn (9 studies were obtained), and (2) hvtn (2 studies were obtained). one trial network, mtn, was inactive and for the two remaining networks, actg and impaact, our data request was pending at the time of our data dictionary analysis. our web search for individual hiv studies (outside any network) identified one additional hiv study (multicenter aids cohort study; macs). our searches were conducted between october 1, 2018 and march 31, 2019. after pooling all possible study acquisition channels, we acquired a total of 31 hiv studies from 5 distinct sources. nida data share was the only source with a widely used standard as it used cdisc. both hptn and hvtn stated that newer trials in their networks would be standardizing to cdisc as well. all other data sharing platforms and studies allowed for custom formats to be used by the studies present on their platform. of our set of studies, one from hptn and the 5 acquired from the nida data archive used a cdisc format. studies that used cdisc format were excluded from the main analysis presented here (we have a separate research project that focuses solely on cdisc-formatted studies). for another 7 studies in our input set, the data dictionary was either (1) not convertible into a machine-readable format, (2) the dictionary was not included in the shared data package, or (3) the dictionary could not be readily inferred from the provided ipd data. this resulted in a total of 18 studies in our final sample of studies that we analyzed. table 3 provides a clinicaltrials.gov study identifier and the study titles for this final set. seven studies (38.9%) in our sample were observational studies while the remaining 11 (61.1%) were interventional trials. we identified a total of 26 328 data elements across the analyzed 18 studies. see supplemental file s1 at the project repository, https://github.com/lhncbc/cde/tree/master/hiv/ datadictionary, to see the aggregated data element file (complete list of data elements). the median number of data elements for the studies in our set was 427 elements. the number of elements ranged between a minimum of 46 elements (study nct02404311) and a maximum of 9 945 elements (study nct00046280). a total of 14 studies used only csv files to provide their data dictionaries. in most cases, the studies provided a single csv dictionary file for the entire study. a single dictionary file is the most user-friendly format for data re-using researchers. a minority of studies split the dictionary into multiple files. the most extreme case of a split dictionary was a study that provided 55 dictionary files (one for each of the 55 study data files generated). four studies (nct00000590, nct00005273, nct00005274, and nct01418014) used pdf files to share the element dictionary. this pdf format required a manual conversion into a csv machine readable format. one study (nct01233531) had a mixture of formats with some data elements provided in a pdf format and some in csv format (spread across 8 files). for some of the studies where we also obtained ipd data (10 studies), we saw data elements present in data, but missing and not defined in the data dictionary, making the data dictionary incomplete. to quantify this level of completeness, we generated data element dictionaries for half of the trials we had ipd for and calculated the percentage of data elements included in the data dictionary. table 4 presents the results of this dictionary completeness analysis and shows that completeness ranges from 45.4% to 100%. we observed missing or incorrect information about data types within our sample of studies. explicit declaration of data type for each data element is important for proper data interpretation and correct data analysis. in one psychology study, incorrect results were published when a categorical variable (code for country of birth) was incorrectly used as a numerical variable in the model [24] . thanks to data sharing and secondary analysis by a researcher (external to the original study team) the error was discovered and revised study results were generated. in terms of missing data type, 12 studies provided data type for all des. one study (nct01492842) had missing data type for 49% of its data elements. data type was completely missing in four studies (22% out of 18 studies). on an aggregate level, across all studies, a total of 10 755 des had missing data type (40.9% out of all 26 328 des). however, this was mainly due to a single study (the multicenter aids cohort study [macs]; nct00046280) with 9 945 des without data type which accounted for 92.5% of all des with missing data type. the breakdown of the number of data elements for a given data type can be found in table 5 . for the 15 573 data elements where data type was declared the most common data type was string with 48.6%, followed by numeric with 47.8%. in terms of incorrect data type, we observed that no studies used a categorical data type. use of categorical variables is common in research. for example, all 13 studies in the national sleep research resource [25] , which is a sharing platform that we consider exemplary in terms of data dictionary format, have at least one categorical variable. in addition, a special case of categorical data type is a boolean data type and it was also not present in any of the element dictionaries. both cdisc define.xml and redcap standards clearly distinguish categorical data elements and support enumeration of permissible values. for the purpose of data analysis and when data is loaded into a database, a categorical variable may still be implemented as a string or a number; however, a flag that indicates that only a set of permissible strings or numbers are expected as values represent good analytical practice. by inspecting the data element title and description and study data, we found numerous categorical data elements; however, their formally listed data dictionary data type was not categorical and there was no flag marking them as categorical. we consider string data type (sometimes also called character) to be a free-text entry with no restrictions. in other words, no set of permissible values is defined for a string data element. an example of a data element of type string (from study nct01751646) is the element titled table 4 . proportion of data elements found in ipd data that are also present in the data dictionary. 'reason missed vitamins-specify' (data element id: vtmrsp). it asks about the specific reasons why the patient missed taking vitamins. the study does not provide any list of permissible values for this question, and there are 107 distinct responses in the ipd data). to demonstrate incorrect data type, we provide two examples. the first example shows the imperfect use of string data type. the study nct00099359 contains a data element 'severity grade of a medical event' (data element id: grade). this categorical data element has five permissible values: 'mild', 'moderate', 'severe', 'life threatening' and 'death'. the ipd data shows 5 distinct values that are all subsumed by this permissible value set. an accurate data dictionary should adopt and use categorical data type as one of the valid data types. if, for some reason, this semantically-rich modelling approach for data dictionary is not used, it should at least use a flag or other mechanism to distinguish data as 'string-categorical' versus 'string-proper'. the second example shows the other variant of this misclassification problem when a categorical data element is in the data dictionary marked as numeric data type (with numbers representing codes for a particular permissible value). as demonstrated in the previously cited retracted analysis [24] , this misclassification can be even more error-prone. in our sample, an example of a numeric-categorical data element (a data element that is not formally designated as categorical in the data dictionary and not distinguished from numerical-proper by any flag) is from study nct00005273 with title 'pain severity' that asks about severity of pain. it has the following permissible values: 0 for none, 1 for mild, 2 for moderate, 3 for severe, and 9 for unsure. not interpreting this element as numerical value is crucial for correct data analysis. if the data dictionary does not model correctly categorical variables, the provision of a complete permissible value dictionary can still fully compensate for the lack of distinction between numeric-categorical and numeric-proper or string-categorical and string-proper. however, we found that many studies in our sample did not provide the permissible value dictionary, and the problem can thus still occur. for data re-using researchers, the ability to properly interpret each data element is crucial. for that purpose, having an unambiguous description for each data element (in addition to a data element id) facilitates this proper interpretation. if the data element definition or meaning cannot be fully understood (description is missing or is vague), the resulting analysis can misinterpret these data elements (leading to incorrect results) or exclude them from the analysis (leading to incomplete results). we found missing data element descriptions in three studies. the element description field was missing for 4 447 des (16.9% out of all 26 328 des). one study (nct00005274) with 4 249 des omitted descriptions for all its data elements, while two studies (nct00865566 and nct01233531) had missing descriptions for some des. the remaining 15 studies (83.3% out of 18 studies) provided descriptions for 100% of their des. we also do, however, acknowledge the fact that a well formulated data element name can be in some cases sufficient to fully define an element. in the aggregate de file, we observed 327 des where the de description was identical to the de name (1.9% out of all 17 014 elements with descriptions). we also found that some de descriptions were vague or difficult to understand. des with confusing description can sometimes be disambiguated by inspecting the ipd data. although, such inspection may require the submission (and approval) of a formal data request that may not be necessary for the data dictionary alone. review of the clinicaltrials.gov records showed that 7 studies (38.9% out of 18 studies) provided study results. no study included a hyperlink (or actual file) for the data dictionary. two studies (11.1%) referenced ipd availability on clinical-trials.gov. although it is theoretically possible for a study to not organize its des into groups, all 18 studies in our sample did group their elements and utilized a group id mechanism. the majority of studies (11 out of 18 studies) used the case report form as the organizing principle (group id is the form name). the remaining 7 studies organized their elements by study data table and used the table name as the group id. the median number of forms in a study (or distinct groups) was 27 (ranging from a minimum of 2 (nct02404311) to a maximum of 124 (nct00005274). a review of form names (or group ids) can provide data re-using researchers a highly pragmatic and quick overview of what data was collected in a given study. if a standardized data collection instrument was used in a study, it may be easiest to discover it via the review of the forms dictionary. if a previously defined and standardized form addresses well the study's data collection needs, use of such a standardized form allows for a very straightforward method of meta-analysis across studies. for example, cdisc clinical data acquisition standards harmonization standard (cdash) defines such common forms for 'protocol deviations', 'demographics', 'adverse event', 'end of study', or 'concomitant medications' [26] . we identified a total of 28 common form names. some form names, such as 'off study' (in 11 of 18 studies, 61.1.6%), 'eligibility' (in 10 on 18 studies, 55.6%) and 'visit report' (in 10 of 18 studies, 55.6%) were very common in our set of studies. we defined very common as forms being present in 50% or more studies out of a set of studies that had a forms dictionary. table 6 shows the very common form names and quantitative measures of their use (count of studies and percentage). less common form names (present in two studies) were body measurements, family history, medical history, missed visit, behavior, and skin test. our results for common form names are affected by the fact that 10 (out of 18 studies) were executed by the same research network (adolescent medicine trials network for hiv/aids interventions). similarly, to data elements, we have observed form names data inaccuracies in 11 studies within our sample. we observed form names (or group names) that were ambiguous, and instances of identical names used to refer to two clearly different forms (see table 2 for an example: two forms both titled 'specimen tracking form'). for data recipients, unambiguous and good data descriptions are important for proper analysis. no study in our sample marked any of the forms as copyrighted. copyright protection typically does not impact the use of the collected data in a secondary research analysis. however, for researchers obtaining common forms and data elements with the intent to use the most prominent ones in a future study (especially those used frequently in past studies), the copyright status is important. use of categorical data elements in research is extremely common and, as stated earlier, most studies would be expected to provide a permissible value dictionary. table 6 . very common form names and the number and percentage of studies their used in. we were able to extract permissible values for 11 studies. the aggregate file contained a total of 7 669 permissible values for 1 815 des. the mean number of permissible values per data element is 4.23. use of numerical ids is most common: 1 511 categorical des (83.3%) used numbers as permissible values. the most frequent permissible value description was 'no' (for 1 334 des in 10 distinct studies). in some cases, permissible values represent administered drugs (e.g., values 3tc, nfv, nvp and zdv were permissible values for data element 'drugnm' which represents the drug name in trial nct00099359). permissible values can also represent laboratory tests. for example, 'cd4 t cell percent' is a permissible value for data element 'testnm' in study nct01772823. some permissible values can be further linked to established healthcare terminologies, such as rxnorm terminology for drugs or logical observation identifiers names and codes (loinc) terminology for laboratory tests. both data dictionary standards (define-xml and redcap) allow for such annotation of permissible values by relevant external terminology codes. no study in our sample made any such annotations. six hiv studies we obtained for our analysis used a cdisc format. cdisc specifications mandate ipd data to be represented in an sdtm (study data tabulation model) format and a corresponding data dictionary in the define-xml format. table 7 lists the studies that used cdisc format (which are excluded from the main analysis). the sdtm format accommodates some data elements directly as a column in a standardized spreadsheet (we refer to those as sdtm-model-hardcoded des) and for other data elements it uses an entity-attribute-value (eav) approach with concepts for those entities defined in cdisc controlled terminology (we refer to those as sdtm eav des). cdisc controlled terminology (ct) thus represents yet a third, tightly linked and relevant cdisc standard. in fact, cdisc ct concepts are also used for coded permissible values. all six studies that used some cdisc standard used sdtm format and also provided a define-xml dictionary. we obtained a large number of hiv trials and analyzed the format and content of their data dictionaries. to our knowledge, our study is the first to aggregate hiv data elements from a large set of completed hiv studies that were later shared via a platform or other mechanism. the majority of studies used a custom, non-standardized format that required significant processing to make the data machine-readable. the lack of consensus to use a single standard should be viewed in light of the fact that data sharing of clinical study data is still a developing and evolving scientific challenge. moreover, the studies in our sample may have been initiated at a time when the emphasis on proper data sharing (by research enterprise in general) was smaller. only in recent years has the importance of study metadata become more prominent. in the clinical domain of hiv, we did not find any prior study that analyzed hiv-specific research data elements. cdisc hiv therapeutic area user guide, published in january 2019, is the only relevant hiv-specific data element effort [3] (in addition to base sdtm elements, it highlights lab codes for cd4 count, loinc codes for hiv viral load testing and mother-infant data linking among many other things). a study focused on data elements and data sharing for hiv registries was published by our team in 2019 [27] . there are, however, prior studies that are not specific to hiv and cover data elements and dictionaries for medicine in general. sharma et al. analyzed three data dictionaries and used archetype modeling language developed by the clinical information modeling initiative [28] . they also developed a platform (called d2refine) for data element harmonization and standardization. in their report they discuss impediments to comparison and interoperability caused by the lack of standardization of data dictionaries from clinical studies. this lack of standardization we observed in our analysis as well. due to the evolution of some of the data dictionary standards and sharing policies, it is difficult to recommend a single data dictionary standard. as outlined in section 2.3, there are essentially two data dictionary standards (cdisc define-xml and redcap) to consider. although, the redcap format is not backed up by any formal standard developing organization (sdo). a recommendation to adopt a cdisc define-xml triggers the requirement to also adopt other related cdisc standards (including cdisc controlled term terminology and possibly cdisc sdtm) and such adoption requires significant training and technical expertise. we consider it too significant a hurdle to be universally recommending such an adoption. with regards to the redcap format, we view it to be very similar to simply formulating a good set of best practices to create a single spreadsheet, compliant with fair principles [31, 32] , that captures all significant parameters of all study data elements (either unique to the study or formal cdes adopted by the study). in other words, if redcap format is not formally utilized, we also find it acceptable to use an ad-hoc single spreadsheet-based data dictionary. based on transformation of data dictionaries and the creation of the aggregated de file, we have designed a set of general recommendations for the optimal sharing of data element metadata for future studies in the hiv domain. we, however, do believe that these recommendations also apply beyond hiv and to other medical domains. we enumerate some of the most important recommendations as a list below. we developed a more detailed set of recommendations as part of consider statement (consolidated recommendations for sharing individual participant data from human clinical studies) to improve the practice of data sharing and reuse. it is available at w3id.org/consider and includes details defining the desired best data sharing practices, positive examples of studies following the recommendations, challenging examples of studies where recommendations are not followed and notes on how to evaluate adherence of a study to a given recommendation (consider checklist). • provide data dictionary documentation separate from de-identified individual participant data. since it contains no participant level data and it does not require local ethical approval as a condition of releasing the data dictionary (avoid a request wall for the data dictionary). • share a data dictionary as soon as possible. do not wait until the data collection is complete. • provide the data dictionary in a single, machine-readable file. • for each data element, provide a data type (such as numeric, date, string, categorical). • for categorical data elements, provide a list of permissible values and distinguish when a numerical code or a string code is a code for a permissible value (versus when it is an actual number or string) • provide a complete data dictionary (all elements in the data are listed in a dictionary) and all types of applicable dictionaries (date elements, forms [or groupings], and permissible values). utilize a description field, in addition to title, to fully describe a data element or form. • at study design time and when resources allow, adopt previously defined applicable common data elements (including adoption of grouped data elements). within the analyzed data dictionaries, we saw instances of closely related des. for example, hiv 1 rna viral load and hiv 1 rna viral load date. this split into multiple self-standing des that are closely related data elements created higher counts of distinct data elements. in recent decades, several common data models (cdms) for electronic health record (ehr) data have emerged that group related data elements into more complex structures. similarly, the cdisc sdtm standard provides higher level structures (lb [= laboratory] domain) that group data elements together into one row of data in a higher level structure. this grouping has been referred to as the ehr data convention [20] and is increasingly becoming the method of choice for modelling closely related des. sharing of clinical study data is inherently linked to de-identification. if a study collected sensitive information (patient's exact date of birth or other identifying data enumerated in regulations or various policies), such information is removed or obfuscated prior to data sharing. nine studies in our sample provided de-identification notes. maintaining a data element dictionary can greatly facilitate ipd de-identification. for example, all elements with date data type may need a relative time obfuscation (shifting all events for a given participant by some fixed number of days). annotation of data elements with common terminologies can help in finding data elements that need to be de-identified and it can help identify an appropriate deidentification method based on a knowledge base organized by cde created from prior instances of study de-identification. our findings are time dependent and based on studies shared at the time of our review (october 2019; 18 studies). ongoing studies or studies currently being planned may use a more complete data dictionary and employ better dictionary formats. thanks to requirements to use cdes included in new funding opportunity announcements [2] , newly initiated studies are more likely to adopt cdes during the study design stage. because we searched specifically for hiv studies, our findings may not generalize fully to other clinical domains. we analyzed 26 328 des from 18 hiv studies. all analyzed studies organized their des into groups. an average study, represented by the median of our set, had 427 data elements. the majority of studies used csv files to share a data dictionary while 22% of the studies used a non-computable, pdf format. string and numeric data types were the most frequent data types with many studies incorrectly representing categorical data elements (100%) and not providing a full list of permissible values (39%). only a minority of studies reported study results or linked ipd within their clinicaltrials.gov record. we also identified two relevant data dictionary standards that have many features that encourage proper data sharing. using our analysis and review, we designed a set of recommendations that provide best practices for data sharing for future studies. analyzing real-world use of research common data elements improving the value of clinical research through the use of common data elements hiv therapeutic area user guide (taug) area user guide (taug) data sharing and embedded research data sharing platforms for de-identified data from human clinical trials clinical research informatics: supporting the research study lifecycle guidance to encourage the use of cdes data sharing celebrating parasites big data and collaboration seek to fight covid-19 [internet]. the scientist magazine® data sharing in the era of covid-19. lancet digit health website for research project: identification of research common data elements in hiv/aids using data science methods data share platform using sas catalogs to develop and manage sas data step programs nih scientific data council committee nih common data element task force: glossary sub-group: definitions of terms reflection paper on copyright, patient-reported outcome instruments and their translations. health qual life outcomes questions of copyright. health qual life outcomes sharing of individual participant data from clinical trials: general comparison and hiv use case study data for submission to cder and cber | fda the redcap consortium: building an international community of software platform partners redcap instruments zip files one step away from technology but one step towards domain experts-mdrbridge: a template-based iso 11179-compliant metadata processing pipeline retraction notice to: the negative association between religiousness and children's altruism across the world the national sleep research resource: towards a sleep data commons evaluation of research accessibility and data elements of hiv registries standardized representation of clinical study data dictionaries with cimi archetypes a concept for a data dictionary system supporting for clinical research the fair guiding principles for scientific data management and stewardship. sci data problems in fairifying medical datasets. stud health technol inform key: cord-339789-151d1j4n authors: hong, hyokyoung g.; li, yi title: estimation of time-varying reproduction numbers underlying epidemiological processes: a new statistical tool for the covid-19 pandemic date: 2020-07-21 journal: plos one doi: 10.1371/journal.pone.0236464 sha: doc_id: 339789 cord_uid: 151d1j4n the coronavirus pandemic has rapidly evolved into an unprecedented crisis. the susceptible-infectious-removed (sir) model and its variants have been used for modeling the pandemic. however, time-independent parameters in the classical models may not capture the dynamic transmission and removal processes, governed by virus containment strategies taken at various phases of the epidemic. moreover, few models account for possible inaccuracies of the reported cases. we propose a poisson model with time-dependent transmission and removal rates to account for possible random errors in reporting and estimate a time-dependent disease reproduction number, which may reflect the effectiveness of virus control strategies. we apply our method to study the pandemic in several severely impacted countries, and analyze and forecast the evolving spread of the coronavirus. we have developed an interactive web application to facilitate readers’ use of our method. a recent work [35] demonstrated that r 0 is likely to vary "due to the impact of the performed intervention strategies and behavioral changes in the population". the merits of our work are summarized as follows. first, unlike the deterministic odebased sir models, our method does not require transmission and removal rates to be known, but estimates them using the data. second, we allow these rates to be time-varying. some timevarying sir approaches [27] directly integrate into the model the information on when governments enforced, for example, quarantine, social-distancing, compulsory mask-wearing and city lockdowns. our method differs by computing a time-varying r 0 , which gauges the status of coronavirus containment and assesses the effectiveness of virus control strategies. third, our poisson model accounts for possible random errors in reporting, and quantifies the uncertainty of the predicted numbers of susceptible, infectious and removed. finally, we apply our method to analyze the data collected from the aforementioned github time-series data repository. we have created an interactive web application (https://younghhk.shinyapps.io/ tvsirforcovid19/) to facilitate users' application of the proposed method. we introduce a poisson model with time-varying transmission and removal rates, denoted by β(t) and γ(t). consider a population with n individuals, and denote by s(t), i(t), r(t) the true but unknown numbers of susceptible, infectious and removed, respectively, at time t, and by s (t) = s(t)/n, i(t) = i(t)/n, r(t) = r(t)/n the fractions of these compartments. the following ordinary differential equations (ode) describe the change rates of s(t), i(t) and r(t): with an initial condition: i(0) = i 0 and r(0) = r 0 , where i 0 > 0 in order to let the epidemic develop [36] . here, β(t) > 0 is the time-varying transmission rate of an infection at time t, which is the number of infectious contacts that result in infections per unit time, and γ(t) > 0 is the time-varying removal rate at t, at which infectious subjects are removed from being infectious due to death or recovery [33] . moreover, γ −1 (t) can be interpreted as the infectious duration of an infection caught at time t [37] . from (1)-(3), we derive an important quantity, which is the time-dependent reproduction number r 0 ðtþ ¼ bðtþ gðtþ : time-varying sir based poisson model for covid -19 to see this, dividing (2) by (3) leads to where (di/dr)(t) is the ratio of the change rate of i(t) to that of r(t). therefore, compared to its time-independent counterpart, r 0 ðtþ is an instantaneous reproduction number and provides a real-time picture of an outbreak. for example, at the onset of the outbreak and in the absence of any containment actions, we may see a rapid ramp-up of cases compared to those removed, leading to a large (di/dr)(t) in (4), and hence a large r 0 ðtþ. with the implemented policies for disease mitigation, we will see a drastically decreasing (di/dr)(t) and, therefore, declining of r 0 ðtþ over time. the turning point is t 0 such that r 0 ðt 0 þ ¼ 1; when the outbreak is controlled with (di/dr)(t 0 ) < 0. under the fixed population size assumption, i.e., s(t) + i(t)+ r(t) = 1, we only need to study i (t) and r(t), and re-express (1)-(3) as with the same initial condition. as the numbers of cases and removed are reported on a daily basis, t is measured in days, e.g. t = 1, . . ., t. replacing derivatives in (5) with finite differences, we can consider a discrete version of (5): iðt þ 1þ à iðtþ ¼ bðtþiðtþf1 à iðtþ à rðtþg à gðtþiðtþ; rðt þ 1þ à rðtþ ¼ gðtþiðtþ; where β(t) and γ(t) are positive functions of t. we set i(0) = i 0 and r(0) = r 0 with t = 0 being the starting date. model (6) admits a recursive way to compute i(t) and r(t): iðt þ 1þ ¼ f1 þ bðtþ à gðtþgiðtþ à bðtþiðtþfiðtþ þ rðtþg; for t = 0, . . ., t − 1. the first equation of (7) implies that β(t) < γ(t) or r 0 ðtþ ¼ bðtþg à 1 ðtþ < 1 leads to that i(t + 1) < i(t) or the number of infectious cases drops, meaning the spread of virus is controlled; otherwise, the number of infectious cases will keep increasing. to fit the model and estimate the time-dependent parameters, we can use nonparametric techniques, such as splines [38] [39] [40] [41] [42] [43] , local polynomial regression [44] and reproducible kernel hilbert space method [45] . in particular, we consider a cubic b-spline approximation [46] . denote by b(t) = {b 1 (t),. . .,b q (t)} t the q cubic b-spline basis functions over [0, t] associated with the knots 0 = w 0 < w 1 < . . . < w q−2 < w q−1 = t. for added flexibility, we allow the number of knots to differ between β(t) and γ(t) and specify log bðtþ ¼ when b 1 ¼ � � � ¼ b q 1 and g 1 ¼ � � � ¼ g q 2 , the model reduces to a constant sir model [46] . we use cross-validation to choose q 1 and q 2 in our numerical experiments. denote by β ¼ ðb 1 ; . . . ; b q 1 þ and γ ¼ ðg 1 ; . . . ; g q 2 þ the unknown parameters, by z i (t) and z r (t) the reported numbers of infectious and removed, respectively, and by z i (t) = z i (t)/n and z r (t) = z r (t)/n, the reported proportions. also, denote by i(t) and r(t) the true numbers of infectious and removed, respectively at time t. we propose a poisson model to link z i (t) and z r (t) to i(t) and r(t) as follows: we also assume that, given i(t) and r(t), the observed daily number {z i (t), z r (t)} are independent across t = 1, . . ., t, meaning the random reporting errors are "white" noise. we note that (9) is directly based on "true" numbers of infectious cases and removed cases derived from the discrete sir model (6) . this differs from the markov process approach, which is based on the past observations. with (6), (7) and (8), r(t) and i(t) are the functions of β and γ, since given the data (z i (t), z r (t)), t = 1, . . ., t, we obtain ðβ;ĝþ, the estimates of (β, γ), by maximizing the following likelihood or, equivalently, maximizing the log likelihood function where c is a constant free of β and γ. see the s1 appendix for additional details of optimization. we then estimate the variance-covariance matrix of ðβ;γþ by inverting the second derivative of −ℓ(β, γ) evaluated at ðβ;γþ. finally, for t = 1, . . ., t, we estimate i(t) and r(t) byîðtþ ¼ nîðtþ andrðtþ ¼ nrðtþ, whereîðtþ andrðtþ are obtained from (7) with all unknown quantities replaced by their estimates; estimate β(t) and γ(t) bybðtþ andĝðtþ, obtained by using (8) with (β, γ) replaced by ðβ;γþ; and estimate r 0 ðtþ byr 0 ðtþ ¼βðtþ=γðtþ. estimation: let n be the size of population of a given country. the date when the first case was reported is set to be the starting date with t = 1, i 0 = z i (1)/n and r 0 = z r (1)/n. the observed data are {z i (t), z r (t), t = 1, . . ., t}, obtained from the github data repository website mentioned in the introduction. we maximize (10) to obtainβ ¼ ðb 0 ;b 1 ; . . . ;b q 1 þ and γ ¼ ðĝ 0 ;ĝ 1 ; . . . ;ĝ q 2 þ. the optimal q 1 and q 2 are obtained via cross-validation. we denote by since the first case of covid-19 was detected in china, it quickly spread to nearly every part of the world [6] . covid-19, conjectured to be more contagious than the previous sars and h1n1 [48] , has put great strain on healthcare systems worldwide, especially among the severely affected countries [49] . we apply our method to assess the epidemiological processes of covid-19 in some severely impacted countries. the country-specific time-series data of confirmed, recovered, and death cases were obtained from a github data repository website (https://github.com/ulklc/covid19-timeseries). this site collects information from various sources listed below on a daily basis at gmt 0:00, converts the data to the csv format, and conducts data normalization and harmonization if inconsistencies are found. the data sources include in particular, the current population size of each country, n, came from the website of worldometer. our analyses covered the periods between the date of the first reported coronavirus case in each nation and june 30, 2020. in the beginning of the outbreak, assessment of i 0 and r 0 was problematic as infectious but asymptomatic cases tended to be undetected due to lack of awareness and testing. to investigate how our method depends on the correct specification of the initial values r 0 and i 0 , we conducted monte carlo simulations. as a comparison, we also studied the performance of the deterministic sir model in the same settings. fig 1 shows that, when the initial value i 0 was mis-specified to be 5 times of the truth, the curves of i (t) and r(t) obtained by the deterministic sir model (6) were considerably biased. on the other hand, our proposed model (9), by accounting for the randomness of the observed data, was robust toward the mis-specification of i 0 and r 0 : the estimates of r(t) and i(t) had negligible biases even with mis-specified initial values. in an omitted analysis, we mis-specified i 0 and r 0 to be only twice of the truth, and obtain the similar results. our numerical experiments also suggested that using the time series, starting from the date when both cases and removed were reported, may generate more reasonable estimates. using the cubic b-splines (8), we estimated the time-dependent transmission rate β(t) and removal rate γ(t), based on which we further estimated r 0 ðtþ, i(t) and r(t). to choose the optimal number of knots for each country when implementing the spline approach, we used 5-fold cross-validation by minimizing the combined mean squared error for the estimated infectious and removed cases. fig 2 shows sharp variations in transmission rates and removal rates across different time periods, indicating the time-varying nature of these rates. the estimated i(t) and r(t) overlapped well with the observed number of infectious and removed cases, indicating the reasonableness of the method. the pointwise 95% confidence intervals (in yellow) represent the uncertainty of the estimates, which may be due to error in reporting. fig 3 presents the estimated time-varying reproduction number,bðtþĝðtþ à 1 , for several countries. the curves capture the evolving trends of the epidemic for each country. in the us, though the first confirmed case was reported on january 20, 2020, lack of immediate actions in the early stage let the epidemic spread widely. as a result, the us had seen soaring infectious cases, and r 0 ðtþ reached its peak around mid-march. from mid-march to early april, the us tightened the virus control policy by suspending foreign travels and closing borders, and the federal government and most states issued mandatory or advisory stay-home orders, which seemed to have substantially contained the virus. the high reproduction numbers with china, italy, and sweden at the onset of the pandemic imply that the spread of the infectious disease was not well controlled in its early phases. with the extremely stringent mitigation policies such as city lockdown and mandatory mask-wearing implemented in the end of january, china was reported to bring its epidemic under control with a quickly dropping r 0 ðtþ in february. this indicates that china might have contained the epidemic, with more people removed from infectious status than those who became infectious. sweden is among the few countries that imposed more relaxed measures to control coronavirus and advocated herd immunity. the swedish approach has initiated much debate. while some criticized that this may endanger the general population in a reckless way, some felt this might terminate the pandemic more effectively in the absence of vaccines [50] . fig 3 demonstrates that sweden has a large reproduction number, which however keeps decreasing. the "big v" shape of the reproduction number around may 1 might be due to the reporting errors or lags. our investigation found that the reported number of infectious cases in that period suddenly dropped and then quickly rose back, which was unusual. around february 18, a surge in south korea was linked to a massive cluster of more than 5,000 cases [51] . the outbreak was clearly depicted in the time-varying r 0 ðtþ curve. since then, south korea appeared to have slowed its epidemic, likely due to expansive testing programs and extensive efforts to trace and isolate patients and their contacts [52] . . estimated i(t), r(t), β(t), γ(t) , and r 0 ðtþ. the us (left) and china (right) are shown based on the data up to june 30, 2020. the blue dots and the red dashed curves represent the observed data and the model-based predictions, respectively, with 95% confidence interval. more broadly, fig 3 categorizes countries into two groups. one group features the countries which have contained coronavirus. countries, such as china and south korea, took aggressive actions after the outbreak and presented sharper downward slopes. some european countries such as italy and spain and mideastern countries such as iran, which were hit later than the east asian countries, share a similar pattern, though with much flatter slopes. on the other hand, the us, brazil, and sweden are still struggling to contain the virus, with the r 0 ðtþ curves hovering over 1. we also caution that, among the countries whose r 0 ðtþ dropped below 1, the curves of the reproduction numbers are beginning to uptick, possibly due to the resumed economy activities. we have developed a web application (https://younghhk.shinyapps.io/tvsirforcovid19/) to facilitate users' application of the proposed method to compute the time-varying reproduction number, and estimated and predict the daily numbers of active cases and removed cases for the presented countries and other countries; see fig 4 for an illustration. our code was written in r [53] , using the bs function in the splines package for cubic b-spline approximation, the nlm function in the stats package for nonlinear minimization, and the jacobian function in the numderiv package for computation of gradients and hessian matrices. graphs were made by using the ggplot2 package. our code can be found on the aforementioned shiny website. the rampaging pandemic of covid-19 has called for developing proper computational and statistical tools to understand the trend of the spread of the disease and evaluate the efficacy of mitigation measures [54] [55] [56] [57] . we propose a poisson model with time-dependent transmission and removal rates. our model accommodates possible random errors and estimates a timedependent disease reproduction number, r 0 ðtþ, which can serve as a metric for timely evaluating the effects of health policies. there have been substantial issues, such as biases and lags, in reporting infectious cases, recovery, and deaths, especially at the early stage of the outbreak. as opposed to the deterministic sir models that heavily rely on accurate reporting of initial infectious and removed cases, our model is more robust towards mis-specifications of such initial conditions. applications of our method to study the epidemics in selected countries illustrate the results of the virus containment policies implemented in these countries, and may serve as the epidemiological benchmarks for the future preventive measures. several methodological questions need to be addressed. first, we analyzed each country separately, without considering the traffic flows among these countries. we will develop a joint model for the global epidemic, which accounts for the geographic locations of and the connectivity among the countries. second, incorporating timing of public health interventions such as the shelter-in-place order into the model might be interesting. however, we opted not to follow this approach as no such information exists for the majority countries. on the other hand, the impact of the interventions or the change point can be embedded into our nonparametric time-dependent estimates. third, the validity of the results of statistical models eventually hinges on the data transparency and accuracy. for example, the results of chinazzi et al. [58] suggested that in china only one of four cases were detected and confirmed. also, asymptomatic cases might have been undetected in many countries. all of these might have led to underestimation of the actual number of cases. moreover, the collected data could be biased toward patients with severe infection and with insurance, as these patients were more likely to seek care or get tested. more in-depth research is warranted to address the issue selection bias. finally, our present work is within the sir framework, where removed individuals include recovery and deaths, who hypothetically are unlikely to infect others. although this makes the model simpler and widely adopted, the interpretation of the γ parameter is not straightforward. our subsequent work is to develop a susceptible-infectious-recovered-deceased (sird) model, in which the number of deaths and the number of recovered are separately considered. we will report this elsewhere. containment of covid-19 requires the concerted effort of health care workers, health policy makers as well as citizens. measures, e.g. self-quarantine, social distancing, and shelter in place, have been executed at various phases by each country to prevent the community transmission. timely and effective assessment of these actions constitutes a critical component of the effort. sir models have been widely used to model this pandemic. however, constant transmission and removal rates may not capture the timely influences of these policies. we propose a time-varying sir poisson model to assess the dynamic transmission patterns of covid-19. with the virus containment measures taken at various time points, r 0 may vary substantially over time. our model provides a systematic and daily updatable tool to evaluate the immediate outcomes of these actions. it is likely that the pandemic is ending and many countries are now shifting gear to reopen the economy, while preparing to battle the second wave of virus attack [59, 60] . our 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cord-340937-6mpob1nx authors: varshney, mohit; parel, jithin thomas; raizada, neeraj; sarin, shiv kumar title: initial psychological impact of covid-19 and its correlates in indian community: an online (feel-covid) survey date: 2020-05-29 journal: plos one doi: 10.1371/journal.pone.0233874 sha: doc_id: 340937 cord_uid: 6mpob1nx background: the pandemic of corona virus (covid-19) hit india recently; and the associated uncertainty is increasingly testing psychological resilience of the masses. when the global focus has mostly been on testing, finding a cure and preventing transmission; people are going through a myriad of psychological problems in adjusting to the current lifestyles and fear of the disease. since there is a severe dearth of researches on this issue, we decided to conduct an online survey to evaluate its psychological impact. methods: from 26(th) to 29(th) march an online survey (feel-covid) was conducted using principles of snowballing, and by invitation through text messages to participate. the survey collected data on socio-demographic and clinical variables related to covid-19 (based on the current knowledge); along with measuring psychological impact with the help of impact of event–revised (ies-r) scale. results: there were a total of 1106 responses from around 64 cities in the country. out of these 453 responses had at least one item missing; and were excluded from the analysis. the mean age of the respondents was around 41 years with a male female ratio of 3:1 and around 22% respondents were health care professionals. overall approximately one third of respondents had significant psychological impact (ies-r score > 24). higher psychological impact was predicted with younger age, female gender and comorbid physical illness. presence of physical symptoms and contact history predicted higher psychological impact, but did not reach statistical significance. conclusion: during the initial stages of covid-19 in india, almost one-third respondents had a significant psychological impact. this indicates a need for more systematic and longitudinal assessment of psychological needs of the population, which can help the government in formulating holistic interventions for affected individuals. from 26 th to 29 th march an online survey (feel-covid) was conducted using principles of snowballing, and by invitation through text messages to participate. the survey collected data on socio-demographic and clinical variables related to covid-19 (based on the current knowledge); along with measuring psychological impact with the help of impact of eventrevised (ies-r) scale. there were a total of 1106 responses from around 64 cities in the country. out of these 453 responses had at least one item missing; and were excluded from the analysis. the mean age of the respondents was around 41 years with a male female ratio of 3:1 and around 22% respondents were health care professionals. overall approximately one third of respondents had significant psychological impact (ies-r score > 24). higher psychological impact was predicted with younger age, female gender and comorbid physical illness. presence of physical symptoms and contact history predicted higher psychological impact, but did not reach statistical significance. corona is a single stranded rna virus that had its roots into the world from almost 60 years since its discovery in late 1960s. corona viruses belong to the corona viridae family in the nidovirales order. the nomenclature of the corona virus is named after the crown-like spikes on the outer surface of the virus structure. [1] the virus has been infecting animals like chickens and pigs but there was no major human contraction to humans. [2] earlier, the allied viruses of the same family like the severe acute respiratory syndrome coronavirus sars-cov in 2003, human corona virus hcov nl63 in 2004 [3] , hku1 in 2005 [4] , middle east respiratory (mers) in 2012, have shown their outbreaks and now the novel version of this virus has presented a threat of unmatched severity. according to the classification of international taxonomy of viruses (ictv) has referred this novel pathogen as sars-cov-2 (formerly known as 2019-ncov) in 2019. [5, 6] the first case was identified in the city of wuhan, a chinese seafood market and since then it has been exponentially increasing with an evident human to human contact via respiratory droplets while sneezing and coughing. [7] the mode and transmission and other related details about the virus continue to be updated in every few weeks, leading to enhanced uncertainty. [8] during this period most of the research has been focused on understanding and preventing transmission; exploring treatment options and issues with global governance. however we think that the psychological impact of this pandemic like stress and anxiety among the general population is also a grave concern. [9] a study from china suggesting that more than half of the participants had a significant psychological impact of the covid-19 pandemic. another recent study from denmark reported psychological well-being as negatively affected. in the united states nearly half were found to be anxious as per the survey conducted by the american psychiatric association. [10] [11] [12] [13] the same has not been studied in indian population systematically; except anecdotal discussions and case reports. [14] in indian subcontinent, as of 30 march 2020, according to the ministry of health & family welfare (mohfw), a total of 1071 covid-19 positive cases (including 49 foreign nationals) were reported in 27 states/union territories. these include 99 cases that were cured / discharged, one person who has migrated and 29 deaths. [2] hospital isolation of all confirmed cases, tracing and home quarantine of the contacts is on-going. in india, spread of the initial disease could be traced mainly to the foreign nationals who visited the country as tourists from the disease affected countries and secondly due to the mass immigration of indian nationals from abroad; due to the fear of infection. as the pandemic outbreak in india was on-going, the government of india took stringent measures to limit the cases by far in that stage only, by initiating a major lockdown pan-india and also by shifting the immigrants to the special quarantine facilities prepared by the indian military directly from the airports and seaports for a minimum of 14 days. community health teams were also launched to spread awareness about the chances of spread and precautionary measures that one can use to protect themselves and others. [15] during the early stages of the pandemic in india, this study was focused mainly to assess its psychological impact. the lives of people were drastically affected with lock-down and fear related to the disease's potential effects and transmission [15] . the fear due to the contraction of covid -19 is on the rise because of the death tolls and global spread. [16, 17] . hence, this study attempted to find the initial psychological impact of covid-19 among general public; and understand its relationship with physical symptoms. this can potentially help policy makers in formulating comprehensive interventions. the study has been approved by the institutional ethics committee at institute of liver and biliary sciences, new delhi (letter no: iec/2020/73/ma04). a cross sectional survey design was decided to assess the initial psychological impact of covid-19, (fears worries and impairment in sleep). we collected data using an online (anonymous) survey platform (survey monkey) as per indian government's recommendations to minimise face-to-face or physical interaction as citizens continue to isolate themselves at home. potential respondents were invited through a text message, which lead them to a survey monkey page (designed by it team at ilbs, new delhi). all people who have registered at ilbs (2009 to present) since the inception were sent the sms for participation in the feel-covid survey. additionally, using the principles of snowballing, the link was circulated by the investigators through social media for capturing data from english speaking general population (who have some access to internet). an effort was made to capture healthcare workers who have handled patients / potential patients. additionally, family member of patients suffering from liver disease, being screened in institute of liver and biliary sciences, were requested to take the survey while waiting for their consultation. during offline requests all standard social distancing protocols were maintained as directed by indian government. we collected data anonymously, without collecting information that could identify the respondents. the period of data collection was between 26 th and 29 th march 2020. once the user clicked on the link they were given information about the nature and purpose of survey on the first page. subsequently, if they consented to participate, they were taken to the next page (first section) of the survey. the first part of the study questionnaire collected sociodemographic information (age, gender, occupational status, city of residence, type of family) and information regarding physical symptoms like presence of cough, cold, head ache breathing difficulty, fever and fatigue related to coronavirus disease. contact history variables included close contact with an individual with confirmed covid-19, indirect contact with an individual with confirmed covid-19, and contact with an individual with suspected covid-19 or infected material; and any foreign travel in the last 14 days. participants were also asked about being a healthcare worker and if they had a known pre-existing medical or psychiatric illness. the second part of the survey was adopted from impact of event scale-revised (ies-r). this tool comprised of 22-items questionnaire which measure the effect of routine life stress, everyday traumas and acute stress. for all questions, scores could range from 0 through 4. categorization of the score ranges from 24 to 32, 33 to 36 and more than 37 which signify mild, moderate and severe psychological impact respectively. [18, 19] . among this scale, the intrusion subscale is mean item response of items 1, 2, 3, 6, 9, 14, 16, 20 . the avoidance subscale is the mean item response of items 5, 7, 8, 11, 12, 13, 17, 22 . the hyperarousal subscale is the mean item response of items 4, 10, 15, 18, 19, 21. separately, the data on actual number of confirmed cases of covid-19 and deaths in the country was accessed through government of india website for general public which was available in the website url address "https://www.mygov.in/covid-19". for the purpose of this study we accessed the above website till 31 st march 2020. descriptive statistics were conducted for the socio-demographic variable and clinical parameters (like physical symptoms and contact history). normality of data was assessed using shapiro-wilk test. the scores of the ies-r and subscales were expressed as mean and standard deviation. we used linear regression to calculate the univariate associations between sociodemographic characteristics, physical symptom contact history variables, additional health information variables and ies-r score. all tests were two-tailed, with a significance level of p < 0.05. statistical analysis was performed using spss statistic 22.0 (ibm spss statistics, new york, united states). during the very early period, fig 1 depicts the progression of number of cases of covid-19 from 1 st february 2020 to 30 th march 2020 in india. the figure also has a timeline of events (first case, first recovered case, first death and curfew announced) to get a perspective of results during the initial period of covid-19 in india. the first case of covid-19 was reported on 1st february 2020 in india. thereafter there was a significant increase in the number of the confirmed, recovered and deceased individuals due to coronavirus outbreak up to 30th march 2020. ("https://www.mygov.in/covid-19). at the time of conducting the survey, the number of cases was building up. a total of 1106 responses were obtained in the study duration through the survey monkey platform. out of these 453 had at least one item missing in the psychological impact related responses and were excluded from analysis. the final analysis was done on rest of the 653 respondents. the mean age of the respondents were 41.82 years (sd = 13.85; range = 18-82) with a male preponderance [491(75.2%)]; among which 145 participants (22.2%) were health professionals. most of the respondents 400(61.3%) belong to nuclear families and 257(39.3%) respondents had reported a history of physical illness; including 125 (19.1%) with a history of known liver disease. the psychological impact of covid-19 outbreak, as measured by ies-r scale, revealed a mean score of mean of 19.79 ((sd) = 13.89) and median of 18.00. as it can be seen from the table 1 , most of the respondents 436 (66.8%) had minimal psychological impact 436 (66.8%) in reaction to covid-19 outbreak. around 98 (15.0%) had mild psychological impact (ies-r score of 24-32) and 36 (5.5%) had moderate psychological impact (ies-r score of 33-36) however, 83 (12.7%) reported severe psychological impact (ies-r score of >36). (table 1) (table 2) . linear regression showed that there was a statistically significant association found between male counter parts and minimal psychological impact which ranges from 0 to 23 on ies-r scale; and between age and psychological impact with higher age associated with lesser psychological impact. moreover, there was a significant association between history of any physical illness and psychological impact. however, there were no statistically significant association between any other demographic or clinical variables. as far as physical symptoms were concerned 62 (9.6%) respondents had reported the presence of cough. 34(5.2%) respondents reported presence of cold. however, diarrhoea was the least reported physical symptoms which accounts for merely in two (0.3%) respondents whereas headache was among 87 (12.3%) which was more frequently reported compared to other physical symptoms. interestingly, sore throat and myalgia were present in 51(7.7%) and 48(7.4%) respectively. only a few respondents had the symptoms of fever 17(2.6%) and breathing difficulty 11 (1.6%). univariate linear regression revealed that there was a statistically significant association with presence of diarrhoea and the impact on their psychological health (p = 0.006). there was no statistically significant association between the physical symptoms such as cough, cold, headache, sore throat, myalgia, fever and breathing difficulty. only nine (1.4%) respondents had travelled during past fortnight, 20 (3.1%) had visited covid-19 infected areas. 6(0.9%) had direct contact with the covid-positive persons. there was no statistically significant association between contact history of the respondents and their impact on psychological health. the current study investigated the initial psychological impact of covid-19 outbreak in indian population. as the disease progressed, concerns regarding health, economy, and livelihood increased day-to-day. the findings of the pandemic's impact on mental health could help inform health officials and the public to provide mental health interventions to those who are in need. this can guide researchers to plan prospective longitudinal studies for assessing treatment need. [20] there are mental health concerns like anxiety, worries and insomnia especially after the declaration of lockdown in india on 24 th march, 2020. government of india has launched helpline numbers to provide guidance and counselling, in collaboration with different institutes of national importance. [21] world health organization has urged to take the necessary precautions to tackle the negative impact of the spread of coronavirus on psychological health and well-being. [22] overall, among the 653 respondents 33.2% had significant (mild / moderate /severe) psychological impact regarding covid-19. this finding was different from the study conducted in china by wang et al which reported 53.8% of respondents suffered a psychological impact from the outbreak, ranging from moderate to severe among 1210 respondents. [10] since these findings were during the early phase of covid-19 outbreak in the country, chances are they could have changed over time and hence, should be interpreted accordingly. in the past, during outbreaks such as 'ebola virus', individual and community at national and international had a major and wide spectrum of psychosocial impacts due to the sudden outbreak of the disease. it is likely that people are relating contracting the virus with a fear of falling sick, helplessness, hopelessness, stigma and even death. [23] providing psychological first-aid & counselling are quintessential during an epidemic. it helps in reducing the psychological distress and promoting adaptive coping strategies to deal with the situation. [24] despite the efforts of who and other public health authorities to contain the covid-19 outbreak, this time of crisis is generating stress throughout the country [25] , much alike its impact on the global counterparts [26] . constant support for mental and psychosocial well-being in different groups during the outbreak should be of highest priority. [9, 16] demographic variables showcase that males had lesser psychological impact of covid-19 outbreak as compared to their female counterpart. the impact on females was found to be statistically significant. these findings were similar in the chinese community where females suffered a greater psychological impact of due to the coronavirus outbreak. [10, 27] this also corresponds to previously available extensive epidemiological literature which shows that women are at a higher risk. [28] in our survey, physical co-morbidities were a predictor for higher psychological impact in response to the outbreak, similar to the finds in existing research. [29] an unexpected finding was the non-statistically significance of impact of being a health care worker on psychological impact. this is contrary to existing literature [30] about them being more prone to unfavourable mental health outcomes. this could have been due to low sample size of healthcare professionals representation in the study; thus limiting generalizability of the findings. however, there are some more limitations to be considered while analysing the study results. first is the inherent design of the study like sampling technique being only restricted to people with internet access and having understanding of english; could also limit generalizability of the study. second are the concerns of social desirability while responding to questions on mental health issues. thirdly the study was conducted during a period of lockdown, which can have its own psychological impact and this confounder could not be addressed through the questionnaire used in the study. these issues could have caused under or over reporting in the rate of psychological impact found in the study. since approximately 20% of the study participants had history of some liver disease, there could be a sampling bias in the study. moreover, the questionnaire used has not been validated in indian population earlier. but we felt the timely need of conducting this survey in order to enhance the understanding of psychological concerns and hence a separate validation was not attempted before the study. despite the limitations, this study provides the first cross-sectional data on actual level of psychological impact among indian community; and how mental health of people is affected during a pandemic of this nature. online surveys (or self-administered questionnaires) have been found as an effective way of assessing problems related to mental health [31, 32] and this becomes a prudent method of conducting research in the period of lockdown. since these findings pertain to the initial period of pandemic in india, a larger longitudinal study should be conducted in the current time to guide policy makers in understanding the psychological impact. covid-19 pandemic has caused a lot of uncertainty in the lives of indian public, just like their global counterparts. our survey is one of the first mental health related data from india, during the initial phase of covid-19 pandemic and indicated that a significant proportion of them have had a psychological impact during the crisis. the factors that predicted higher impact were younger age, being female and having a known physical comorbidity. there is a need for considering mental health issues by the policy makers; while planning interventions to fight the pandemic. covid-19 infection: origin, transmission, and characteristics of human coronaviruses coronavirus disease (covid-19) coronavirus nl63-induced adult respiratory distress syndrome human coronavirus-hku1 infection among adults in cleveland, ohio. open forum infect dis evaluation and treatment coronavirus (covid-19) timely mental health care for the 2019 novel coronavirus outbreak is urgently needed clinical features of patients infected with 2019 novel coronavirus in wuhan centre for disease prevention and control. novel coronavirus disease 2019 (covid-19) pandemic: increased transmission in the eu/eea and the uk-sixth update-12 mental health and psychosocial considerations during covid-19 outbreak. world health organization immediate psychological responses and associated factors during the initial stage of the 2019 coronavirus disease (covid-19) epidemic among the general population in china covid-19 and mental health: a review of the existing literature the depressive state of denmark during the covid-19 pandemic especially for loved ones; older adults are less anxious how covid-19 is overwhelming our mental health focus on mental health during the coronavirus (covid-19) pandemic: applying learnings from the past outbreaks early release-public mental health crisis during covid-19 pandemic fear of covid 2019: first suicidal case in india! the impact of event scale-revised: psychometric properties in a sample of motor vehicle accident survivors the impact of event scale-revised: psychometric properties of the lithuanian version in a sample of employees exposed to workplace bullying the covid-19 outbreak: crucial role the psychiatrists can play coronavirus: government launches helpline for mental health issues during lockdown. deccan herald rethinking online mental health services in china during the covid-19 epidemic the 1995 kikwit ebola outbreak: lessons hospitals and physicians can apply to future viral epidemics initial public health response and interim clinical guidance for the 2019 novel coronavirus outbreak-united states covid-19, coronavirus and mental health rehabilitation at times of crisis patients with mental health disorders in the covid-19 epidemic depression after exposure to stressful events: lessons learned from the severe acute respiratory syndrome epidemic prevalence of depression in the community from 30 countries between comorbidity and its impact on 1590 patients with covid-19 in china: a nationwide analysis factors associated with mental health outcomes among health care workers exposed to coronavirus disease responses to mental health stigma questions: the importance of social desirability and data collection method measuring mental illness stigma with diminished social desirability effects we are grateful to the it team of ilbs-ms jyoti agarwal, lt. col. rajnish kishore (general manager, it); mr sandip kumar for all the support during launch of the survey and compiling results. we also acknowledge ms garema khurana (deputy editor at 9xmedia) for her valuable and free of cost english language editing. key: cord-318845-w7q5o8wc authors: pendell, dustin l.; marsh, thomas l.; coble, keith h.; lusk, jayson l.; szmania, sara c. title: economic assessment of fmdv releases from the national bio and agro defense facility date: 2015-06-26 journal: plos one doi: 10.1371/journal.pone.0129134 sha: doc_id: 318845 cord_uid: w7q5o8wc this study evaluates the economic consequences of hypothetical foot-and-mouth disease releases from the future national bio and agro defense facility in manhattan, kansas. using an economic framework that estimates the impacts to agricultural firms and consumers, quantifies costs to non-agricultural activities in the epidemiologically impacted region, and assesses costs of response to the government, we find the distribution of economic impacts to be very significant. furthermore, agricultural firms and consumers bear most of the impacts followed by the government and the regional non-agricultural firms. scientific laboratories designed to study diseases are not completely risk free, and the possibility exists that accidents of nature or deliberate acts of terror might cause the spread of a disease that the facility is trying to prevent. on the one hand animal and human health officials in the united states are interested in preventing the introduction and spread of diseases like ebola, rift valley fever or highly pathogenic avian influenza, while on the other hand scientists within the united states need small quantities of these materials for study and to develop response strategies should an outbreak occur. it is a tenuous balancing act with tradeoffs that can be staggering varying in outcomes and economic value. this study considers a timely and relevant case, food-and-mouth-disease (fmd), in one such facility, the national bio and agro defense facility (nbaf). we couple outcome from plume and epidemiological models with an economic model to calculate the potential economic consequences of several critical types of accidental releases from a research facility. the approaches used here are likely to have application for other diseases and research facilities, and in providing a monetary estimate of the risks posed by such facilities that can be compared against the benefits of better preparedness. in 2009, manhattan, kansas was selected as the site for the new national bio and agro defense facility. it is intended to replace the current research facility at plum island animal disease the united states were zoonotic, a disease that is transmissible between animals and humans, with 72% originating from wildlife [8] . trends suggest that the frequency of emerging infectious diseases will continue to increase, especially with the growing interface between humans, animals, and wildlife [9] . potential transmission and spread of foreign animal diseases (fad) and zoonotic diseases are driven by some of the same forces that propel the global economy. globalization has increased both international trade and human mobility. as the world human population and standard of living continues to grow, there is, and will be a continued increase in demand for protein from livestock. the united nations world tourism organization [10] forecasts a 3 to 4% increase over 2012s record one billion international tourist arrivals worldwide in 2013. the economic losses associated with emerging, highly contagious fads and zoonotic diseases can be significant. according to the world bank [11] , the direct costs (e.g., costs to public and animal health services and producer compensation for culled animals) and indirect costs (e.g., trade and tourism) of outbreaks during the 2000s surpassed $20 billion and $200 billion, respectively. hosono et al. [12] estimated that the 1998-1999 nipah virus outbreak in malaysia resulted in $90 million in losses from culled animals and $170 million in indirect effects. the 2003 severe acute respiratory syndrome (sars) outbreak in east asia and canada resulted in estimated losses of $40 to $50 billion [11] . in the 2001 fmd outbreak in the uk that lasted more than 220 days in duration, over six million animals were culled with estimated losses of $10 billion to $12 billion [13] . animal agriculture is important to the u.s. economy as it is a primary source of food and nutrition, a major contributor to exports, and it is valued at $165 billion [14] . with such a vital industry, the united states needs to position itself to defend against the threat of fads and zoonotic diseases. to do so, a modern biocontainment laboratory that is capable of conducting research and developing vaccines against such diseases is required, yet the united states does not currently have one. in 2004, in the homeland security presidential directive 9 (hspd-9): defense of united states agriculture and food, the administration showed the need for such a facility: "the secretaries of agriculture and homeland security will develop a plan to provide safe, secure, and state-of-the-art agriculture biocontainment laboratories that research and develop diagnostic capabilities for foreign animal and zoonotic diseases" [15] . there are 14 foot-and-mouth disease virus incidents known or believed to have been released over the past 50 years from biocontainment research laboratories worldwide, including czechoslovakia, demark, germany, spain, russia, united kingdom, and united states [2] . because of potential release and subsequent consequences of a contagious infectious disease, proper analyses and reviews must be conducted before constructing a new state-of-the-art biocontainment research facility. homeland security [16] has noted that piadc is at "the end of its lifecycle and is too small to meet the nation's research needs". it is the intent that nbaf will: (1) replace and enhance the current research at piadc; (2) enhance research capabilities diagnosing foreign animal, emerging and zoonotic diseases in large animal livestock; (2) develop new vaccines and other countermeasures for large animal livestock; (3) train veterinarians and animal agricultural specialists in preparing for and responding to animal diseases; and (4) give the united states its only bsl-4 research capacity of high-consequence diseases affecting large animal livestock. because of two government accountability office [2, 17] reports, the fy 2010 dhs appropriation act (p.l. 111-83) would not allow funds to be made available for construction of nbaf until it completed a site specific risk assessment to be reviewed by the national academies of sciences [1] . following the appropriations act of 2010, dhs commissioned the site-specific biosafety and biosecurity mitigation risk assessment in 2010, including a review by the national academy of sciences (nas). in the nas review, they noted several shortcomings in the site specific risk assessment (ssra), including an inadequate qualitative risk assessment, underestimation of the risk of a pathogen release and transmissions, and methodological flaws of the plume and epidemiological modeling [18] . to address these shortcomings, dhs conducted an updated site-specific biosafety and biosecurity mitigation risk assessment in 2012 [16] , which was also reviewed by the national academy of sciences [19] . the information from initial risk assessment completed in 2010 was used in altering the design of nbaf and changing the standard operating procedures, personnel training, and emergency response planning. for example, the initial design of nbaf would withstand wind speeds of 90 miles per hour [20] . following the updated risk assessment, the design of nbaf was modified to conform to nuclear regulatory standards and withstand with up to 228 miles per hour [16] . to assess the economic impacts of unintentional fmdv releases from nbaf, we follow [4, 7, [21] [22] to link supply shocks from an animal disease spread model with a multi-commodity, multi-market partial equilibrium model. this is supplemented with a regional input-output economic model to capture impacts on allied and associated businesses [4, 21] . additional economic shocks include domestic and international markets, which are discussed in detail below. government costs associated with controlling and eradicating and fmd outbreak are calculated as well. the epidemiological disease spread model used in this study is the north american animal disease spread model (naadsm). naadsm is a spatial, stochastic, state-transition simulation model that simulates highly contagious animal diseases [23] . naadsm has been used in numerous studies to evaluate the impacts of highly contagious animal diseases in various countries including several economic impact studies [3-4, 6, 21-22] . the naadsm framework requires extensive parameterization including information on animal population (e.g., location, production type, size of herd), disease manifestation (e.g., latently infected), disease transmission (e.g., direct, indirect, and aerosol), disease detection, surveillance, and control (e.g., animal movements, traceability, and vaccination). the parameters can take on an integer value (e.g., number of herds destroyed per day), probability (e.g., probability of infection given exposure to an infected herd), probability density function (e.g., length of time that an infected herd is subclinically infectious) or relational function (e.g., probability of detecting an infectious herd, which is a function of clinical signs being observed and being reported to authorities once clinical signs have been observed). the parameters are developed through literature searches, research, and expert opinions. given the epidemiological output is exogenous input into the economic model, and not the primary focus of this paper, the reader is referred to dhs [16, section 6.1.4] for complete documentation of the parameter values used in this study. naadsm is simulated 200 times for each scenario to generate a distribution of disease spread outcomes. in addition to the uncertainty resulting from the stochastic disease spread model, different starting locations for infection are also incorporated to better reflect uncertainty. unlike previous studies which pick a specific starting location (e.g., cow-calf operation or feedlot operation) to model the consequences of an fmd outbreak, this study includes all possible starting locations (e.g., cow-calf operations, feedlot operations, etc.) for each release scenario. in other words, for each starting location the fmd outbreak is modeled and the consequences are ranked. it is then possible to report the outbreak consequences at the probability levels (e.g., p5, p50, and p95) for the different starting locations. agricultural firms and consumers. a quarterly multi-market partial equilibrium simulation model is used to assess how unintentional releases of fmdv would impact u.s. agricultural producers and consumers. this study utilizes an updated version of the paarlberg et al. [22] partial equilibrium model. the partial equilibrium model includes major agricultural sectors along vertical and horizontal market chains beginning with livestock and grain production to meat processing and the final consumer, including both domestic and international. exogenous, production, domestic demand, and international trade, shocks resulting from an fmd outbreak are incorporated into the model as percentage changes from the baseline. the economic model then solves for the percent changes in the endogenous variables (prices and quantities) for each quarter. the percent changes in the endogenous variables are then applied to a baseline defined by the observed data for the first quarter of 2009 through the fourth quarter of 2018 of no-disease. this results in estimated changes in per capita consumer welfare and changes in quasi-profits and captured by returns to capital and management. parameters for the partial equilibrium model include livestock-feed balance information, revenue and factor shares, and elasticities. the livestock-feed balance information, revenue shares, and factor shares are retained as defined in [22] . the retail elasticity values for final meat demand for beef, pork, and poultry [24] , lamb [25] , and milk [26] are updated for this study. substitution elasticities for derived demand and trade elasticities remained unchanged. producer expectations regarding expected future returns are modeled as naïve expectations [27] . livestock supply, use, and price data, as well as forage prices are from the livestock marketing information center [28] . coarse grains, wheat, rice, and the soybean complex supply, use, and price data are from outlook reports and data prepared by the usda-economic research service [29] . total quarterly use was generated by feed balance equations from which data on animal numbers are combined with standard feeding practices to produce quarterly amounts of forage and pasture. hay, corn silage, and sorghum silage production is reported by the national agricultural statistics [30] . uncut grazed pasture is imputed for quarters 2 and 3. international trade data are derived from lmic [28] , usda-ers [31], and foreign agricultural service [32] . information concerning the crop policy mechanisms are from provisions of the federal agriculture improvement and reform act of 1996 [33] and the 2002 farm act [34] . regional non-agricultural sector. assessing the costs and disruptions to the non-agricultural activities in the epidemiologically impacted region can be very important [4, 21] . thompson et al. [13] estimates that the direct losses of tourism following the 2001 uk fmd outbreak were equal to the losses to the agricultural sector, excluding the producer compensation from the government. furthermore, the indirect effects to tourism were more than 20 times larger when compared to the indirect effects to agriculture. the regional impacts in this study are estimated using an input-output model which is a system of equations that describe the flow of income and product throughout an economy. specifically, the bureau of economic analysis's regional input-output modeling system (rimsii) is used because they provide a well-accepted and validated methodology to evaluate these impacts. moreover, the input-output industrial multipliers provide the flexibility to define the states defined by the disease spread model. rimsii integrates the input and output relationships of approximately 500 u.s. industries and regional economic accounts. the final-demand multipliers for output are used to estimate the indirect economic activity generated by a specific economic activity in a region. thus, the intent of using the rimsii data is to measure the effects of an fmd outbreak on the non-agricultural regional economy. calculations are structured to remove duplication or double counting of losses. the indirect effects evaluated include: (1) the effect of culling and destroying animals on the non-agricultural regional economy (e.g., retail trade); (2) the economic implication of a travel ban that would limit recreational and non-essential travel in and out of a region; and (3) the indirect effects from the stimulus to the region created by the expenditures during government eradication and clean-up efforts. travel bans, resulting in reduced tourism, are another important source of potential economic losses for the impacted region [35] . travel bans composed of transit and ground transportation; spectator sports; hotels and motels; and food and drink services are evaluated. the economic impact from the loss in travel expenditures can be measured using rimsii [36] . total domestic travel expenditures for overnight trips and day trips of over 50 miles in 2007 were obtained from the u.s. statistical abstract produced by the u.s. census bureau by state. however, the rimsii data separates the economic effects of various forms of travel (table 1) . thus, using data on the percentage allocations of travel expenditures from the bureau of labor statistics, expenditures are allocated by category for each state in the study region [37] . while not insignificant, travel and tourism is not a dominant sector in the region. kansas, nebraska, and oklahoma each constitute less than 1% of the u.s. domestic travel visits and expenditures (3.1% combined). travel and tourism are more important in texas, colorado and missouri which contribute 6.7%, 2.0% and 1.8% of domestic travel visits and expenditures, respectively. travel expenditures, by state, for trips of over 50 miles were obtained from the u. s. census bureau. additionally, the u.s. bureau of labor statistics reports the percentage of allocations of travel expenditures. thus, using data on the percentage allocations of travel expenditures, expenditures were allocated by category (e.g., air transportation) for each state in the study region [16] ). it appears likely that in major outbreaks travel restrictions to non-agricultural events will be lifted after two quarters so a maximum reduction of 4% of annual travel is realized. for outbreaks of less than two quarters, the travel reduction is computed from the number of days the outbreak lasts as a percentage of a full year's reduction. typical government costs associated with eradicating an fmd outbreak are calculated. these costs include appraisal, cleaning and disinfection, disposal, euthanasia, indemnification, quarantine, surveillance, and vaccination. indemnification payments reflect the value of culled animals at average market prices in the first quarter of 2009 prior to an fmdv release. table 2 reports the government costs used in this study which are based on published literature [38] [39] and the non-indemnification costs per animal are consistent in magnitude with those reported by abdalla et al. [40] . an fmd outbreak would result in shocks to production, domestic demand, and international trade [5, 7, 22] . these shocks are expressed as percentage changes for each quarter and incorporated into the economic model. production (supply) shock. output from the disease spread model is used to estimate the production or supply shocks. specifically, the number of animals culled by production type, by quarter is used to calculate the percentage reduction in supply. additionally, two emergency vaccinations scenarios are assumed: vaccinate-to-kill and vaccinate-to-live. no federal vaccination policy for fmd exists in the u.s. and no definitive precedent was uncovered in previous studies. after discussions with members of a u.s. government review panel, the vaccinationto-live and vaccination-to-kill policies were defined for this study as initial assessment. it is realized that different assumptions concerning a vaccination policy could be made (see [3] ). for outbreaks <180 days, the vaccinate-to-kill scenario is assumed where all vaccinated animals are assumed to be culled. for outbreaks >180 days, it is assumed that culling of the vaccinated cattle will occur until the 180 th day of the outbreak and then afterwards, any vaccinated cattle in the queue to be culled or newly vaccinated cattle will not be culled and remain in the cattle inventory. depending on the scenario (vaccinate-to-kill or vaccinate-to-live), the number of animals culled and/or animals vaccinated listed in table 3 and table 4 are used in calculating the production shock, respectively. domestic demand shock. there are anticipated decreases in consumer demand, even though fmd does not pose a human health concern. as such, reduction in u.s. consumer demand is incorporated to allow variations in the level of consumer perception of food quality [41] . these domestic demand shocks represent the share of the u.s. population decreasing consumption of a final good and provide a policy instrument by which to manage impacts on final demand. because there have been no fmd events on the u.s. mainland since 1929, it is [46] . given the above information, domestic demand shocks are specified for fmd across the scenarios. based on the epidemiological output (see table 4 ) smaller (larger) outbreaks coincided with outbreaks that lasted shorter (longer) than one quarter. consequently, following an fmd outbreak lasting less than one quarter, it was assumed that 5% of people would refrain from consuming beef, pork, and lamb while 2.5% would stop consuming milk and dairy products during the outbreak. this is consistent with consumer reactions to food safety events reported in [41, 44] . in the second quarter, domestic consumer demand for beef, pork and lamb declined by 2.5% and was fully recovered (i.e., 0% decline) for dairy and milk products. it is assumed that consumer demand for meat products would be fully recovered by the third quarter. in outbreaks lasting more one quarter, it was assumed that 10% of domestic consumers would refrain from consuming beef, pork and lamb while 5% would stop consuming milk and dairy products during the outbreak. following the end of the outbreak, it was assumed that domestic consumer demand would decrease by 5% for one quarter and 2.5% for another quarter for beef, pork, and lamb. consumer demand for dairy and milk products would decline by 2.5% for one quarter following the outbreak. international trade shock. the magnitude and duration of trade shocks assumed in this study are based on observations from previous events in throughout the world, including the united states. in 2003 and 2004, due to isolated incidences of bse, the u.s. and canada faced complete bans on beef in major overseas markets while beef and cattle imports and exports continued among the north america free trade agreement countries (canada, mexico, and the united states) under a variety of restrictions [47] . the united states has experienced a long recovery relative to pre-outbreak trade status as a result the isolated bse events. u.s. beef exports, as a percentage of beef production was 9.5% in 2003, dropped dramatically to 1.9% in 2004, and recovered to 7.4% in 2008 [28] . a review of previous literature is useful in identifying plausible time lengths defining trade bans for our fmd scenarios. the eu imposed a one year ban on the uk following its 2001 fmd outbreak. rich and winter-nelson [7] analyzed the 2000-2001 fmd outbreaks in the southern cone of south america and concluded short lived impacts on exports to argentina, brazil, and uruguay. randolph, morrison, and poulton [48] assumed a 12 month ban on exports during fmd outbreaks in zimbabwe. nogueira et al. [49] and tozer et al. [50] apply 1 to 2 year trade bans for hypothetical fmd outbreaks in mexico and australia, respectively. although the actual length of export restrictions will depend upon the actual product, disease, trade agreements, and countries involved, these observations provide valuable information on trade bans. given the information above, trade shocks are created in the following manner. first, 95% of all u.s. exports of beef, pork, lamb meat, cattle, swine, and sheep are halted during the full quarter of the outbreak and for one quarter after the last case appears. this assumes some processed/cooked beef is still exported after the outbreak. the interruption of exports for one quarter beyond the end of the outbreak (and for two quarters beyond the end of the outbreak when emergency vaccination without slaughter is practiced) is consistent with world organization for animal health (oie) guidelines and practices (chapter 8.5) during fmd outbreaks [51] . second, after the additional quarter ended with no fmd reported, it is assumed that gradual recovery of u.s. exports will occur until it reaches the baseline levels. full recovery is assumed to occur in approximately two years following one full quarter after the outbreak is eradicated. for fmd, the duration of the outbreak becomes a critical element in determining the economic effects from trade disruptions. the region of focus for this study includes seven states: colorado, iowa, kansas, missouri, nebraska, oklahoma and texas. in this region agriculture is economically important, especially for livestock. in 2009, cattle and calves are the most valuable agricultural commodity in four states in the study [52] . all seven states are in the top 10 cattle and calves inventory with 45.1 million head (47.7% of total u.s. inventory) located in those seven states. texas, nebraska, kansas, iowa, and colorado are the five largest states with cattle on feed; almost 10 million head on january 1, 2009 [52] . furthermore, hogs are recognized as one of the top five commodities those states. total hog and pig inventory on december 1, 2008 for the region was 30.8 million head (47.4% of total u.s. inventory). 31.2 percent of total u.s. sheep and lamb inventory (1.8 million head) occurred in those seven states on january 1, 2009 [52] . additionally, a significant percentage of state farm receipts in this region are derived from dairy. a unique feature of this study when compared with previous work is the size of the livestock population and number of herds. most hypothetical fmd studies in the united states focus on a small region at the state or county level [4, 6, 27, 38, [53] [54] . with nearly 83.76 million head and 0.45 million herds, this study contains one of the largest, if not the largest, number of susceptible animals/herds of any fmd economic modeling study ( table 5 ). the exact farm locations are not available in the united states. location data typically exists for medium and large sized operations. smaller operations were accounted for by incorporating locations from a dataset developed by lawrence livermore national laboratory (llnl) using the nass agricultural census data from 2007 [55] .the production types used in naadsm are adjusted to allow for use in the partial equilibrium economic model. the production types required by the partial equilibrium economic model are beef cattle, dairy, slaughter cattle, swine and sheep. as such, the production types used in naadsm (listed in table 5 ) are adjusted as follows: cow-calf + beef (backyard) = beef cattle; dairy = dairy; feedlot (small) + feedlot (large) = beef slaughter; swine = swine (small) + swine (large) + swine (backyard); goats + sheep + small ruminants (backyard) = sheep. as with any contagious disease research laboratory, there are multiple mechanisms and pathways in which a pathogen might be released from a containment laboratory. such pathways include: liquid (e.g., through a drain), solid waste (e.g., carcass disposal system), fomite and vectors (e.g., clothing, mosquito), and aerosol (e.g., air filtration system). although protocols to reduce the risk of infectious material leaving the laboratory exist, it is impossible to eliminate all of the risk associated when dealing with highly contagious pathogen research. thus, four plausible unintentional introduction events of the fmdv are evaluated. these events with examples of actual releases include: in august 2007, an fmd outbreak occurred near pribright, uk. according to defra [56] , it has been suggested that the probable cause of the outbreak was due to leaking drainage pipes at the institute for animal health, a zoonotic disease research laboratory. in september 1971, an air leak in a gasket around a research laboratory door was the cause of in an internal release of fmdv at piadc [2] . this scenario represents the virus accidentally released through an aerosol. in august 1980, several steers at piadc were found to contain a different strain of fmdv than the vaccine research being conducted. although the actual cause of this outbreak was never determined, it is likely a laboratory worker carried and transmitted the virus to steers [2] . although no know natural disaster has resulted in a release of fmdv, it is plausible that such an event could occur as 42 tornados, on average, are reported with 120 nautical miles of nbaf each year [16] . on june 11, 2008 , an enhanced fujita 4 tornado touched down in manhattan, kansas, including on the campus of kansas state university, which is where nbaf will be located. each of the four release scenarios have differing: (1) probabilities of an event occurring, (2) amount of fmdv released, and (3) the means by which the virus is transported in the environment. thus, this information is used to calculate the probability that any given premises becomes infected with fmdv. further details about the different release mechanisms can be found in [16] pages 405-412 and 480-507. additionally, the first three events represent unannounced events while the latter scenario represents an announced event. it is important to distinguish between these two types of unintentional releases as an unannounced release could continue to spread the fmdv until the disease is identified and confirmed by officials. in the event of an announced released, control and mitigation plans (e.g., animal movement bans, increased surveillance, etc.) could be immediately implemented, potentially reducing the impacts. the results from the disease spread model are summarized in tables 3 and 4 . results are reported for the distribution of outcomes at 5 th , 50 th , and 95 th percentiles of outbreaks based on animals culled. this provides a range of consequences that may arise from an fmd outbreak. to better reflect uncertainty inherent in the potential different outbreak starting locations (i.e., cow-calf operation, feedlot, etc.), additional consequences are reported for the 5 th , 50 th , and 95 th percentiles based on location of the index case. for example, p5/p50 implies the 5 th percentile of epidemiological output based on culled animals and the 50 th percentile of epidemiological output based on starting location. in all release events, there were several assumptions regarding vaccinations including: (1) an emergency vaccination program was implemented after the first animal was infected; (2) the fmd vaccination supply was not a limiting constraint; (3) a 10 km vaccination ring was employed; (4) all animals that were vaccinated were culled unless the outbreak lasted longer than 2 quarters, at which the culling of vaccinated animals ceased at the end of the 2 nd quarter. those animals that were vaccinated and not culled remained in the inventory. in the aerosol, liquid waste, and transference release events, there are 10 scenarios in which the outbreak lasted less than 2 quarters (i.e., vaccinate-to-kill policy); 7 of those scenarios were less than 49 days in duration (table 3) . additionally, the number of animals culled in those scenarios generally was less than 1.2 million head. the remaining scenarios were a vaccinate-tolive policy and ranged in duration from 266 to 533 days. the number of animals culled ranged between 6.0 and 27.4 million head for the longer duration scenarios. the tornado release event results have one noticeable difference: the upper bound of animals culled and duration of outbreaks was smaller when compared to the other three release events, but the lower bound was much higher. in other words, the 5 th percentile of epidemiological output resulted in 6.0 million head culled with a duration of 240 days, which is much larger than the other three release events ( table 3 ). the number of animals culled and length of the outbreak for the 95 th percentile of epidemiological output scenario was 20.6 million head and 533 days. although the duration of 533 days was the same as other three release event scenarios, the number of animals culled was much smaller in the 95 th percentile scenario. the impacts at the 5 th percentile were larger due to the initial spread of the virus by the tornado. because this release event is in effect a self-announcement, mitigation and control plans are put into place immediately and a higher probability of observing and reporting the disease by a producer, which can be seen at the 95 th percentile of epidemiological output based on culled animals. the numbers of livestock vaccinated follow a similar pattern as duration and number of animals culled. the p5/p5 and p95/p95 scenarios result in the smallest and largest number of vaccinations administered, respectively ( table 4 ). the fewest vaccines administered are 0 in the liquid waste release event for p5/p5 while the p95/p95 scenario for the transference release scenario results in over 65 million vaccinations. to determine the total economic impact for a scenario, the changes in producer returns to capital and management and consumer welfare, government indemnification and non-indemnification expenditures, and the costs to the non-agricultural regional sector were summed together. liquid waste release. losses for the liquid waste release scenario range from $0 to $138,889 million in damage ( table 6 ). the 5 th percentile epidemiological output and 5 th and 50 th location quartiles resulted in no detection and spread of fmd (table 4) . thus, no economic damages were incurred. in the p5/p95 scenario (5 th percentile in epidemiological output and 95 th percentile for starting location), changes in producer returns to capital and management were a decline of $19,920 million while changes in consumer welfare increased by $3,489 million. the positive effect on consumers was a result of a small production shock, small table 6 . agricultural and regional non-agricultural impacts and government costs of hypothetical fmd outbreak (millions $). producer returns to capital and management indemnification non-indemnification regional non-agriculture impacts liquid waste adverse reactions from consumers, and trade sanctions. in other words, it is possible for consumers of meat and dairy products to benefit from an fmd outbreak, if it is small in nature and consumer reactions are limited because of trade sanctions on agricultural exports leads to oversupply and reduce u.s. meat and dairy prices [3, 22, [49] [50] . producer and consumer impacts for p50/p5 are similar in size to p5/p95 while the remaining distribution of epidemiological and starting location impacts range from declines of $47,578 to $64,027 million and declines of $43,436 to $60,322 million for producers and consumers, respectively. in all release event scenarios, the negative impacts to consumers are smaller than that of the producers. the regional non-agricultural effects were negative and range from $0 to losses of $4,152 billion (table 6 ). although government indemnification payments replace the value of lost livestock, it is not enough to offset the full economic impact on the region. the government costs associated in eradicating an fmd outbreak range from $0 to $15,448 million with 50 to 85 percent of that due to indemnity payments (table 6) . table 6 summarizes the distributional cumulative economic impact across the entire study period for the liquid waste release (beginning in 2009 and ending in 2018). however, consequences of disease outbreaks are inherently dynamic in nature with benefits and costs accruing differently to producers and consumers across time, and this interplay has important policy implications [5] . fig 1 illustrates the changes in producer returns to capital and management by agricultural sector across time for the p50/p50 liquid waste scenario. after an outbreak is announced there is an immediate negative effect on the swine and beef cattle sectors of about $2,400 million. this is due to the loss of livestock, meat, and dairy export markets, reduced demand for red meat and dairy products by domestic consumers, and culled animals. when the outbreak is officially declared over (the 5 th quarter), the beef cattle sector's producers returns to capital and management has rebounded to the pre-outbreak levels while the swine sector's recovery to pre-disease outbreak levels occurs approximately four quarters later (the 9 th quarter). this result is mostly due to the amount of exports lost by both sectors; approximately 7% and 17% of u.s. beef and swine production in 2009 was exported, respectively. after the 5 th quarter, beef producers experience positive returns, and do so for the next 17 quarters, primarily a result of lower grain and forage prices, consumer demand fully recovering, and the gradual recovery of export markets. swine producers also have positive gains, but not as large of gains as the beef cattle producers. losses to the meat processing sector's returns to capital and management decline from the onset of the outbreak until the peak in the 8 th quarter, where the loss in that quarter is approximately $2,813 billion (fig 1) . these losses are a result of lower beef prices due to the oversupply of beef (i.e., loss of the export markets). similar to the meat processing, producer returns to management and capital in the crops sector decline. however, the losses in the crops sector are more severe and continue to decline until the 11 th quarter. these losses (which include grains, forage, and pasture) are a result of less demand from fewer cattle and swine. additionally, the 2009 grains prices were far above u.s. government support levels, thus, price support payments had little, if any, effect. both the meat processing and crops sectors producer returns do not fully recover to pre-disease levels by the 40 th quarter. although producer returns to capital and management for the remaining sectors (eggs and layers, dairy cattle and milk, lambs and sheep, and soybean processing) are positive or negative, they are very small. table 7 reports aggregate changes in producer returns to capital and management across the 40 quarters by sector. this perhaps gives the best overview of the impacts to the agriculture industry in this hypothetical fmd outbreak. additional liquid waste scenarios reported in dhs [16] remain qualitatively similar, but do vary according to the degree of the outbreak. aerosol release. in the event of an accidental release of the fmdv through an aerosol, the total economic impacts range from losses of $16,515 million to $139,464 million ( table 6 ). the range of losses to the agricultural producers was $19,733 million for the p5/p50 scenario to $62,610 million for the p50/p95 scenario. the small localized fmd outbreaks that lasted less than a quarter in duration, p5/p5, p5/p50, and p5/p50 scenarios, resulted in gains to consumers of about $3,500 million. the p5/p95 scenario resulted in a decline in consumer welfare of $26,128 million while the remaining scenarios saw much larger declines in consumer welfare ranging from $51,877 to $60,274 million because of the duration of the outbreak and number of animals culled and vaccinated. the government indemnification costs range from $30 million for the p5/p5 scenario to $9,850 million for the p95/p95 scenario. the non-indemnification costs are smaller with a range of $6 million to $5,600 million. finally, the impacts to the regional non-agricultural sectors are declines of $50 million to $4,645 million across the scenarios. the distribution of losses by production type across time is qualitatively similar to the liquid waste scenario described above. transference release. because the duration of the fmd outbreaks and number of animals culled and vaccinated are similar to the aerosol scenarios, the total economic impacts of the transference release scenarios are similar to impacts of an aerosol release, including the distribution of impacts by production types across time. losses to producers range from $19,660 million to $61,445 million while changes in consumer welfare are a positive $3,576 million to a negative $59,895. government indemnification and non-indemnification costs range from $163 million and $27 million to $9,850 million and $5,621 million, respectively. regional nonagricultural impacts range from $163 million to $4,087 million in the losses resulting from hypothetical fmd outbreaks. tornado release. tornado events are unlike the previous releases because of immediate, widespread dispersion and the events are effectively self-announced. the economic impacts were estimated in the event the fmdv is released because a tornado comprises the containment laboratories at nbaf. with a tornado release event, the duration of an outbreak under the p5/ p5 scenario is 240 days compared to 0, 28, and 35 days in the liquid waste, aerosol, and transference scenarios, respectively. additionally, the number of animals culled in the p5/p5 scenario is significantly higher when compared to the other three release scenarios (approximately 5.8 million more animals are culled). thus, the lower end of the range of total economic impacts for this scenario is much larger because more animals are culled and the duration is longer at the lower end of the distribution (p5/p5). although the impacts are larger at the lower end of the distribution, the impacts at the upper end of the distribution (p95/p95) are smaller because fewer animals are culled. the total economic impacts range from losses of $76,426 million to $137,565 million. similar to the aerosol and transference release events, producers lose along the entire distribution of outcomes. however, consumers do not gain in any at any point along the distribution. when compared to the other three release events, the range of government costs is smaller. however, the p5/p5 scenario has much larger government costs while the p95/p95 costs are smaller for both indemnification and non-indemnification. the lower and upper ends of the distribution for regional non-agricultural impacts are losses of $760 million and $3,828 million, respectively. united states animal agriculture is becoming a highly integrated and global system that is very important both domestically and internationally. this complex system combined with the increasing frequency of emerging infectious disease threatens the stability of the u.s. economy, food security, and livestock and public health. this economic analysis is part of a congressionally mandated site specific risk assessment, which links outcomes from plume and epidemiological models to risk outcomes. this study examines the economic consequences of foot-and-mouth disease virus (fmdv) releases from a national disease research facility. specifically, we investigate the economic impacts to consumers and firms, costs to the government, and disruptions to non-agricultural regional sectors. outcomes of the site-specific risk assessment have been and are currently being implemented in the form of feedback into the planning and construction process of the national bio-and agro defense facility (nbaf), along with improvements in scientific and economic modeling. given the projected investment costs of over $1 billion and the risks related to potential foreign animal and zoonotic disease releases, feedback into the planning and construction of the facility is critical to enhance future food security. unlike previous studies that focused on various alternate mitigation strategies, this study focuses on potential animal disease releases from nbaf. the release events modeled include aerosol, liquid waste, and transference (unannounced release events) and a tornado (announced release event). indeed, differences in the distribution of economic consequences arise between the unannounced and announced events. mitigation controls used includes stamping out, vaccinate-to-live and vaccinate-to-kill. although this is not a definitive study of fmd vaccination, the economic consequences from the selected scenarios are informative to government planners and policy makers. schroeder et al. [3] find that emergency vaccination can be a cost effective mitigation tool that can help reduce the spread of disease. they conclude that a high-capacity emergency vaccination program together with large vaccination zones results in significant savings to consumers, producers, and the government. total losses for the reported fmdv release events range from about $16 billion to $140 billion in damages. producer effects are always negative due to lost output and reduced prices and share the largest burden in losses. consumers realize negative or positive effects primarily contingent upon the size of the outbreak, export losses, and assumed demand shocks. regional non-agricultural losses and government indemnification (non-indemnification) costs are much smaller than the producer and consumer impacts, ranging from $39 million to over $4,500 million and from $18 million to nearly $10,000 million ($3 million to over $5,600 million) across the scenarios, respectively. these economic impacts across the four release events are similar, except for the tornado release event. because the tornado release event is effectively an announced event and the virus dispersion is greater, lower bound economic losses are much larger than the unannounced releases while the upper bound of the economic consequences is much smaller. several additional key insights are identified. first, it is important to integrate time into the economic analysis, as livestock are durable goods. costs and benefits evolve over time in response to producer decisions, consumer reactions, and international trade responses by trading partners. second, disaggregation among production types is vital to link epidemiological to economic models. furthermore, this allows for additional analysis on how the different production sectors are impacted. third, size of the outbreak and duration of trade sanctions are important. fourth, the timing of identifying an fmdv release (announced vs. unannounced) is important and has differing economic consequences. similar to all hypothetical foreign animal disease studies that report economic consequences, they are conditional in nature. not only are they conditional on the available information and modeling assumptions, but they are conditional on an outbreak occurring. while it is plausible that fmdv releases may arise from nbaf, estimated probabilities of the sequence of events leading to an outbreak are not large [16] . nevertheless, as actual events and empirical evidence demonstrate, fmd events are low probability high cost events that deserve continued vigilance and research to mitigate the consequences. this study is not without limitations. total costs could be overestimated if, for example, the impact of tourism is diverted to different areas or the purchase delayed/deferred. however, the estimated costs provide plausible estimates using standard economic techniques. the modeling approach assumes homogenous commodities and goods, including beef. fmd outbreaks are treated as shocks to economic system and are not endogenous to the model. trade is not differentiated among trading partners, but rather differentiated by domestic and international markets. capacity constraints need to be investigated for processing infected animals, as well as capital constraints. nevertheless, the modeling framework provides the appropriate fidelity to assess economic consequences. this study does raise questions for future research. first, additional research needs to be completed on vaccination strategies and capacity constraints. second, trade sanctions and trade agreements need to be studied further. third, government costs and the structure of government costs should be examined to better understand the nature of public expenditures. fourth, potential benefits resulting from research, diagnostic tests, and training of personnel would have positive impacts. the extent of those benefits needs to be investigated. fifth, sensitivity analysis on the economic parameters deserves further examination. finally, further implementation of an iterative risk assessment may provide interactive feedback and subsequent updating of information that could increase the efficiency and effectiveness of the risk assessment and facility construction process. department of homeland security appropriations act high-containment biosafety laboratories: dhs lacks evidence to conclude that foot-and-mouth disease research can be done safely on the u.s. mainland economic impact of alternative fmd emergency vaccination strategies in the united states the economic impacts of a foot-and-mouth disease outbreak: a regional analysis invasive species management: foot-and-mouth disease in the u.s. beef industry modeling alternative mitigation strategies for a hypothetical outbreak of foot-and-mouth disease in the united states an integrated epidemiological-economic analysis of foot and mouth disease: applications to the southern cone of south america global trends 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lessons learned pendell initiated this research while he was at colorado state university. the authors would like to thank philip paarlberg and national academies committee members for helpful comments. key: cord-314908-kp2jznwb authors: roczniewska, marta; rogala, anna; puchalska-kaminska, malwina; cieślak, roman; retowski, sylwiusz title: i believe i can craft! introducing job crafting self-efficacy scale (jcses) date: 2020-08-10 journal: plos one doi: 10.1371/journal.pone.0237250 sha: doc_id: 314908 cord_uid: kp2jznwb job crafting is beneficial for employees and organizations. to better predict these behaviors, we introduce the concept of job crafting self-efficacy (jcse) and define it as an individual’s beliefs about their capability to modify the demands and resources of their job to better fit their needs. this article describes the development and validation of a scale to measure jcse. we conducted a qualitative study to design and four quantitative studies to test the psychometric properties of this scale among polish and american employees in both paper-and-pencil and online versions. three independent (n(1) = 364; n(2) = 432; n(3) = 403) confirmatory factor analyses demonstrated a good fit to a 3-factor solution comprising jcse beliefs about increasing (a) structural job resources, (b) social job resources, and (c) challenging job demands. the 9-item jcse scale had good internal consistency, high time stability, and good validity. it correlated positively with general self-efficacy. jcse explained unique variance in job crafting behaviors over and above general self-efficacy, and was more important in predicting job crafting than contextual factors. we demonstrate the role of social cognitions in shaping job redesign behaviors and provide a useful tool to evaluate the effectiveness of interventions dedicated to empowering jcse. job design and redesign are usually seen as top-down processes in which people who manage an organization create jobs and then decide to introduce changes to them [1] . recently, a new perspective on this phenomenon has been suggested wherein job redesign is understood as a process depending on an individual's initiative. unbeknownst to their supervisors, employees may transform the characteristics of their jobs to match them to their needs and preferences. these proactive, bottom-up, and self-initiated actions have been labeled as job crafting [1, 2] . the current covid-19 pandemic crisis highlighted the importance of flexibility in how jobs are performed. thus, employee job crafting may form an especially important organizational asset. moreover, job crafting has numerous positive consequences for employees and organizations (for a meta-analysis, see: [3] ). therefore, scholars and practitioners are interested in promoting these acts in the workplace (e.g., [4] ). when are these behaviors most likely to occur? following social cognitive theory [5] , we argue that a key step for an employee to engage in job crafting is to build an expectancy that performance of this behavior is within this person's control. in this project we integrate social cognitive theory and the job demands-resources model [6, 7] to introduce the concept of job crafting self-efficacy (jcse). we define jcse as an individual's beliefs about his or her own capability to modify demands and resources present at their job to better fit their needs and preferences. we argue that the development of this construct is warranted because scholars and practitioners are interested in finding valid, precise, and useful predictors of job crafting to strengthen these behaviors in the workplace. while the literature points to a list of contextual and individual antecedents to job crafting [3] , not all of them are malleable (e.g., personality traits) or easily changed by the organizations (e.g., autonomy levels). this gap does not allow utilizing these factors as enhancers in introducing job crafting to the organizations. thus, a factor susceptible to interventions and change is practical. in the meta-analysis, general self-efficacy has been positively linked with job crafting behaviors [3] ; however, its predictive power was low to medium, and differed for distinct crafting behaviors. we propose that being a context-specific belief, jcse predicts jobcrafting behaviors more precisely than general measures of self-efficacy. by developing a more accurate predictor, we help to explain when job crafting occurs, which is relevant for both research and practice. we contribute to research and practice in three distinct ways. first, we introduce a malleable, context-specific construct to explain when employees engage in job crafting acts. by integrating social cognitive theory and the job demands-resources model, we explicate how such beliefs are formed and affect behaviors. this knowledge is vital for organizational practice, because it guides development of practices and interventions that may help with introducing job crafting in the workplace. next, we investigate the interplay between contextual (i.e., perceived opportunities to craft) and individual (i.e., jcse) factors predicting job self-efficacy to examine their relative importance. an answer to this question may help better guide organizational actions aimed at supporting proactivity in the workplace. finally, we provide an instrument to measure jcse beliefs, which was validated across different formats and two languages. this scale may be used to evaluate the effectiveness of interventions dedicated to empower jcse. the term job crafting was first coined by wrzesniewski and dutton [2] who described the bottom-up process of job re-design, wherein employees proactively introduce changes to certain aspects of their jobs. these authors proposed that individuals craft their jobs by changing their understanding of the role they perform (cognitive crafting), by altering the amount or type of tasks they pursue at work (task crafting), or by modifying their interactions with other people at work (relational crafting). the purpose of these behaviors is to increase the meaning of work and to transform the jobs people have into ones they want to perform [8] . tims and bakker [1] proposed an alternative to wrzesniewski and dutton's conceptualization by framing job crafting within the job demands-resources model (jd-r; [9, 10] ). these authors defined job crafting as a bottom-up process of shaping one's job characteristics, that is, demands and resources [1] . job demands are those aspects of the job that require physical or psychological effort, and are therefore associated with physiological and psychological costs [7] . some of the demands, like time pressure, are of a challenging character, that is, although they are appraised as stressful, they provide the potential for growth and may have a positive effect on an individual [11] . other types of job demands, like role conflict, serve as a hindrance to effective goal pursuit, and therefore negatively influence an individual [11] . job resources represent those aspects of the job that help buffer the potentially negative impact of job demands on individuals. they support goal pursuit and stimulate personal growth [7] . examples include work autonomy, feedback, and being provided with learning opportunities. jd-r theory poses that interactions between the levels of job demands and job resources determine employee well-being [7] . indeed, research demonstrates that high hindrance job demands combined with low amounts of job resources result in exhaustion, whereas high levels of both challenge demands and job resources create employee engagement [7] . given the assumptions of the jd-r theory, tims and bakker [1] argue that employees craft their jobs to achieve higher well-being by optimizing the levels of demands and job resources in their jobs. these authors proposed four crafting strategies. the first two relate to employees increasing their job resources. individuals can seek more structural job resources, for example, by creating opportunities for development at work or expanding their levels of job autonomy. employees may also seek more social job resources. for instance, they can look for help or advice from their colleagues to better deal with the demands of their job. individuals can also optimize the levels of their job demands. they do so by increasing challenging job demands (e.g., introducing new projects in the company), and by decreasing hindering job demands (e.g., reducing workload). individuals benefit from job crafting. those who more frequently initiate changes in their jobs experience higher job satisfaction and work engagement [12] , stronger person-job fit [13] , increased work meaning [14] , and better health [15] . although the primary aims of job crafting are self-serving, organizations benefit from individual job crafting acts as well. employees who engage in job-crafting are more productive and have more sustainable employment [16] . namely, job crafting predicts better in-role and extra-role performance, and weaker turnover intentions [3] . in addition, ghitulescu [17] found that job crafting is linked with reduced absenteeism. given the multiple positive consequences of job crafting, it is important to understand how to encourage it in the workplace. although job crafting concerns employees' self-initiated actions, these bottom-up re-design behaviors can be supported through organizational interventions (e.g., [15] ). employee intention to engage in these acts combined with managerial encouragement or workplace social norms may not be enough to pursue job crafting. we argue that yet another element has to be added to that equation: an expectancy that performance of the behavior is within a person's control, that is, they have the capability to implement it successfully. this element is called self-efficacy. self-efficacy represents people's beliefs in their capabilities to exert control over the environment and execute actions necessary to deal with future situations [18] . vough and parker [19] argue that employees who feel self-efficacious are more likely to act proactively. indeed, social cognitive theory postulates that people with high self-efficacy levels are more eager to perform challenging or risky tasks [5] . because job crafting is a demanding activity that requires moving beyond the job descriptions, we believe that the postulates of social cognitive theory apply to this phenomenon. when people with high self-efficacy realize that they are not completely satisfied with their jobs, they perceive that they are in control to change the situation. this realization may lead them to actually alter the characteristics of the job to fit them better [19] . indeed, a meta-analysis performed by rudolph and colleagues [3] revealed that general self-efficacy positively correlates with job crafting behaviors. although self-efficacy may be conceptualized and measured in a more global way as the general belief in one's competence to cope with a broader range of stressful or challenging demands [20] , it is usually defined and measured as a domain-specific construct. social cognitive theory argues that self-efficacy measures should be context-specific because self-efficacy itself is a context-specific belief [5, 21] . people usually hold distinct efficacy beliefs across disciplines, for example, one can feel very self-efficacious as an academic and, simultaneously, extremely inefficacious as a driver. indeed, previous studies suggest that applying context-specific self-efficacy measures allows outcomes to be predicted more successfully (e.g., [22] ). to better predict job crafting in the workplace we introduce a new, domain-specific selfefficacy construct: job crafting self-efficacy (jcse). we theoretically frame our understanding of jcse within the jd-r resources approach to job crafting. therefore, we define jcse as an individual's beliefs about his or her own capability to modify demands and resources present at their job to adjust them to their needs and preferences. in that, jcse is different from job crafting itself, because the latter describes employee acts of modifications, whereas the former encompasses employee beliefs about the ability to perform these modifications. further, jcse differs from general self-efficacy, because it is contextualized to focus on one's beliefs regarding the ability to alter their job characteristics rather than optimistic self-beliefs to cope with a variety of demands in life. overall, the proposed construct of jcse combines job crafting and selfefficacy, but is distinct from them. because job crafting consists of four conceptually different dimensions in the jd-r model, we propose four corresponding types of jcse beliefs about one's ability to: (a) increase structural job resources; (b) increase social job resources; (c) increase challenging job demands; and (d) decrease hindering job demands. we argue that these beliefs form separate dimensions because they describe independent behaviors that employees may engage in to align their jobs with their own preferences. therefore, we hypothesize that jcse consists of four subdimensions (hypothesis 1). based on the assumptions of social cognitive theory, we also expect that these specific beliefs predict matching job-crafting behaviors, for example, individuals who feel self-efficacious with respect to increasing challenging job demands are more likely to start new projects or learn about new developments at work and try them out. we hypothesize that there is a positive link between a specific jcse belief and a corresponding job crafting behavior (hypothesis 2). specific jcse beliefs may derive from more generalized self-efficacy beliefs. due to the fact that certain levels of hierarchy in self-efficacy have been detected, where general self-efficacy beliefs consist of domain-specific self-efficacy beliefs (e.g., [23] ), scholars posit that when individuals encounter recurrent successes in specific contexts, they develop a more generalized self-efficacy. then, this generalized belief may form the basis for an individual's assessment of prospective efficacy in new situations [24] . therefore, we expect a positive correlation between general self-efficacy and jcse (hypothesis 3). however, because jcse beliefs are context-specific, we argue that they predict job crafting behaviors more accurately than general self-efficacy (hypothesis 4). self-efficacy, next to organization-based self-esteem and optimism, has been included as a personal resource in an extension to the jd-r model [25] . xanthopoulou and colleagues [25] showed that personal resources mediated the relationship between job resources and engagement. this result indicates that the supply of job resources activates employees' self-efficacy, self-esteem, and optimism, and makes them feel more engaged, thus affecting the motivational process described by the jd-r model [7] . the study also showed that personal resources mediated the relationship between job resources and exhaustion. moreover, previous studies have supported the moderator role of personal resources in the relationship between job demands and well-being (e.g., [26] ). thus, personal job resources affect the health impairment process described in the jd-r model. overall, we expect that jcse is positively related to work engagement (hypothesis 5) and negatively to job burnout (hypothesis 6). finally, we examine the interplay between situational conditions and jcses in shaping job crafting. thus, we compare the predictive power of jcse and a newly-developed construct that measures perceived opportunities to craft (poc) in an organization [27] . this latter construct addresses an individual's perceptions regarding the possibilities his or her job provides to change certain aspects of it. these opportunities seem dependent on organizational-or jobrelated factors rather than individual self-efficacy beliefs. because self-efficacy has both direct and indirect (via intentions) links with actual behaviors [28] , we expect jcse to explain job crafting behaviors above and beyond poc (hypothesis 7). the aim of our project was to develop a four-dimensional measure for job crafting self-efficacy (jcse) that uses a limited number of items and can be applied regardless of cultural and occupational context. to achieve this goal, we conducted a series of studies with diverse procedures and samples. as the jcse scale (jcses) was constructed on the basis of the jd-r approach to job crafting [1, 29] , in line with guidelines [30] we started with conducting a confirmatory factor analysis to verify its four-dimensional factorial structure (hypothesis 1). then, in order to achieve intended parsimony of the scale, we reduced the number of jcses items, analyzing its internal and external consistency and judgmental qualities following the guidelines outlined by stanton, sinar, balzer, and smith [31] . next, we conducted a series of confirmatory factor analyses among distinct samples and formats, that is, paper-and-pencil (study 1) and online (study 2), as well as in an additional cultural context (study 3). subsequently, we analyzed jcses's convergent and criterion validity (study 1-4) to test hypotheses 2-7 regarding jcse relations with other constructs. moreover, we have assessed jcses's discriminant validity (study 2-3). the overview of jcses development procedure is presented in fig 1. three work and organizational psychologists, familiar with job crafting research and social cognitive theory, independently generated an initial pool of items. following the recommendation for self-efficacy scales [28] , self-efficacy items should be represented by confidencestatements with the following semantic structure: "i am confident that i can (perform something) despite (barrier)". this structure is warranted because human behavior is not only a function of intentions and cognitive control, but also is influenced by the perceived and the actual environment [32] . thus, introducing barriers provides an opportunity for more realistic assessments [32] . such structure was used in a variety of scales measuring a wide range of selfefficacy beliefs regarding health behaviors, e.g., physical activity [33] , regulation of eating [34] or resisting the urge to use drugs [35] . hence, following these recommendations, generated items comprised job crafting behaviors along with job crafting barriers. we defined barriers as aspects of the immediate work environment and the self that inhibit the translation of motivation and abilities into job crafting. typical barriers were selected on a basis of a semi-structured interviews, where participants were asked about constraints to their crafting behaviors (see: s1 appendix). examples included lack of energy, hostile relations among employees or a fear of being overwhelmed with additional tasks. we choose the most typical barriers and connected them with specific job crafting behaviors. specific job-crafting behaviors were chosen on the basis of the jd-r approach to job crafting [29] , e.g., asking one's supervisor for feedback or seeking opportunities for development. before we collected data on the jcses, all authors had discussions about the proposed definition of jcse and the constructed items. after eliminating clearly redundant items, the initial version of the jcse scale consisted of 27 items. importantly, the items of the jcse scale were worded in a simple way (avoiding complex terminology or professional jargon) making the instrument suitable for participants with different levels of education and professional backgrounds. example items for this initial version of the jcse scale include "how certain are you that you would be able to develop your knowledge and abilities despite a heavy workload?" (jcse in increasing structural resources), "how certain are you that you would be able to ask your supervisor for support, despite concerns about being judged?" (jcse in increasing social resources), "how certain are you that you would be able to set new job challenges, even though it leads to more responsibilities or a larger workload?" (jcse in increasing challenges) and "how certain are you that you would be able to reduce how work affects your emotions despite worries about how others will judge you?" (jcse in decreasing demands). analogical to other se scales [21] , response categories refer to the certainty of being capable of performing the indicated behaviors in one's job. we used a 10-point scale, ranging from 1 (definitely incapable) to 10 (definitely capable). this first version of the scale was assessed to test the factorial validity of the scale and choose items for the final version of the instrument. a summary of participants' characteristics across studies 1-4 is given in table 1 . all studies described in this manuscript were carried out in accordance with the recommendations of departmental ethics committee, who provided specific approval for this research project after the research was reviewed (wke/s16/v/18). we obtained written informed consent from all subjects in study 1 and 4, and online consent in studies 2 and 3, in accordance with the declaration of helsinki. all of our studies were based on convenience sampling. study 1. the first study had a cross-sectional design. participants were 364 employees recruited by seven research assistants who distributed paper-and-pencil questionnaires among working adults in poland. participants were not remunerated for participation. study 2. the second study was of a cross-sectional nature as well. participants were recruited by four research assistants who distributed invitations to the on-line study among polish working adults. participants took part in a prize draw to win one out of three vouchers for a bookstore worth about 20 eur each. five hundred fifty-four individuals agreed to participate in this research. missing data were deleted list-wise. due to participants quitting after completing the first scale in the procedure (i.e., jcse scale) and missing data in the next scales, the sample for the subsequent validity analysis was lower (n = 340) than for cfa (n = 432). study 3. the third study had a cross-sectional design and was conducted online on the u. s. sample. the scale was translated into english beforehand using a backward translation procedure [36] . participants were recruited through mechanical turk (mturk)-an online data collection website where individuals register to complete tasks for financial rewards. mturk was proven to be a reliable source of data collection in several studies (e.g., [37, 38] ). to be eligible to participate in our study, individuals had to be employed and reside in the united states. participants were instructed about requirements of the study and its purpose (i.e., research on work attitudes and behaviors). participants spent approximately 6 minutes on the study. after the completion of the study, each participant was rewarded with $0.75 in compensation. within the study survey, three validity check items were inserted to control participants' engagement in the study. there were 418 employees who agreed to participate. individuals who failed to pass the attention test (n = 9) were deleted from the database. due to missing data and participants quitting after filling in the first measure, that is, jcses, the final samples for cfa was n = 403 and n = 381 for the validity analysis. study 4. the fourth study was conducted by adopting a time-lagged design. the data were gathered using a paper-and-pencil procedure at two measurement points with a six-week time break between time 1 and time 2. at time 1 and 2 participants were asked to mark their questionnaire with a unique code. additionally, participants were informed at time 1 that there would be a second measurement point in 6 weeks, and they were kindly asked to attend the study if possible. there were 134 participants at time 1 and 119 at time 2. participants were not remunerated for participation. in each study we measured job crafting self-efficacy as well as actual job crafting behaviors. in study 2 we additionally tested general self-efficacy and work well-being indicators, i.e., work engagement and job burnout. in study 3 we also measured general self-efficacy. in study 4 we assessed perceived opportunity to craft in the organization. all used measures are described below. job crafting. this construct was assessed in study 1, 2 and 4 using four sub-dimensions of the polish version [39, 40] of the job crafting scale (jcs) originally developed by tims, bakker and derks [29] . in study 3 we used the english language version of the jcs. the dimensions are: increasing structural job resources (e.g., "i try to develop my capabilities"), increasing social job resources (e.g., "i ask colleagues for advice"), increasing challenging job demands (e.g., "when there is not much to do at work, i see it as a chance to start new projects") and decreasing hindrance job demands (e.g. "i make sure that my work is mentally less intense"). items were rated on a 5-point scale, ranging from 1 (never) to 5 (very often). general self-efficacy. the variable was measured in study 2 with the general self-efficacy scale (gses; [41] ) in adaptation by juczyński [42] . in study 3 the original english version of gses was used. the instrument consists of 10 items (e.g., "i can always manage to solve difficult problems if i try hard enough"). participants respond to these items using a 4-point scale ranging from 1 (not at all true) to 4 (exactly true). work engagement. this construct was measured in study 2 with the short version of the utrecht work engagement scale (uwes-9; [43] ) in adaptation by cieslak and colleagues [44] . the instrument consists of 9 items (e.g., "at work, i am bursting with energy", α = .90). participants respond to these items using a 7-point likert scale ranging from 0 (never) to 6 (always). job burnout. this variable was measured in study 2 with oldenburg burnout inventory (olbi) [45] in adaptation by baka and basińska [46] . the instrument consists of 16 items (e.g., "after my work, i usually feel worn and weary", α = .85). the scale ranged from 1 (totally disagree) to 4 (totally agree). perceived opportunity to craft in organization. the variable was measured in study 4 with perceived opportunity to craft scale (pocs; [27] ) translated into polish by the authors of this study using a back translation procedure. the scale consists of 5 items (e.g., "at work i have the opportunity to vary the type of task i carry out"). reliability of the scale was high and stable at time 1 (α = .86) and time 2 (α = .88). factorial structure of the jcses analytical strategy. to verify the factorial structure of jcses we performed a series of confirmatory factor analyses (cfa) using mplus 7.0 [47] . missing data were deleted list-wise. in step 1, we tested for the multivariate normal distribution. significant coefficients of skewness and kurtosis (p < .001) indicate that this assumption cannot be accepted in each reported sample. consequently, we used the maximum likelihood robust (mlr) estimator instead of maximum likelihood (ml) estimator to test the fit. according to a multifaceted approach to an assessment of the model fit, we considered the following fit indicates: comparative fit index (cfi; [48] ) and tucker and lewis index (tli; [49] ); root mean square error of approximation (rmsea; [50] ) along with 90% confidence interval limits. additionally, we report traditional chi-square statistics. the model is considered to fit the data when the following values are obtained: rmsea < .08 [50] , and tli and cfi > .90 [51] . we also tested χ2/df ratio across models, assuming that values below three generally indicate good model fit [52] . confirmatory factor analysis of the initial version of the jcses. the theoretically supported four-factor model was tested in study 1 against an alternative one-factor model. this comparison is derived from theory as self-efficacy may also be measured as a more general one-factor construct. the results of the cfa regarding the goodness-of-fit indices of the tested models are presented in table 2 (see: jcse-27). the results revealed that the one-factor model of 27-item jcses fits the data inadequately, with high rmsea (.108) and the chi-square/df ratio above 3 (5.26). the goodness-of-fit indices of the four-factor model are not highly satisfactory; however, they are better than a one-factor model with the results of rmsea (.09) and the chi-square/df ratio (3.67). the cfi and tli ratios are not satisfactory in both models: one-factor (cfi = .67; tli = .65) and four-factor (cfi = .80; tli = .78). we compared the models using satorra-bentler scaled chi-square [53, 54] . the results demonstrated that the 4-factor model had a significantly better fit than the 1-factor model, δχ 2 = 309.35, δdf = 6, p < .001. overall, none of the two models had good fit parameters. thus, we decided to modify the initial version of the jcses with the aim to reduce the number of items. scale reduction process. to achieve indented parsimony of the scale we selected three items per subscale. research suggests that scales consisting of three items may achieve adequate internal consistency reliability [55] . following the recommendations of stanton, sinar, balzer, and smith [31] regarding the reduction of the self-reported scales, we first analyzed the internal consistency and then the judgmental qualities of the jcses. see s1 table for jcses-27's reliability estimates and factor loadings. internal consistency refers to the overall degree that items of an instrument are intercorrelated and form a homogenous construct. since all cronbach's alpha reliability estimates were high and range from .80 (jcse in decreasing hindrance demands) to .90 (jcse in increasing structural resources), indicating good consistency of the scale [56] , we decided to study the factor loadings of items. according to stanton et al. [31] , factor analysis is a popular technique used in the scale reduction as retained items that fail to load strongly on the factor of interest may increase the internal consistency of the scale. since all of jcse-27 items had significant and satisfactory loadings (i.e., more than .30; see [31] ), we did not have any premises to exclude items upon this criterion. in the last step, an expert judge panel evaluate the qualities of the subscales such as clarity of expression, the semantic redundancy of an item's content with other items, and face validity [31] . this criterion of assessment is very important, since respondents might negatively view items that are highly redundant with each other. following this recommendation, three work and organizational psychology experts analyzed items and chose those that were clear and distinctive from each other. on this basis, we chose 3 items per dimension, resulting in a total of 12 items for future studies. confirmatory factor analysis of jcses-12. to verify the proposed four-factor model of the shortened version of jcses, we performed a confirmatory factor analysis (cfa), using mplus 7.0 [47] on a sample from study 2. as table 2 shows (see: jcse-12), in line with the results of study 1, the goodness-of-fit indices suggest that a one-factor model does not fit the data well, with rmsea higher than .08 (.092) and the chi-square/df ratio above 3 (4.63). the four-factor model is satisfactory as for rmsea (.066) and has an acceptable chi-square/df ratio (2.91). however, two out of three items from jcse in decreasing demands had low factor loadings (below .35) and one of them ("postpone making tough decisions, despite the pressure from your co-workers") did not load significantly on the latent factor (p = .26) (see table 3 ). this result may suggest that these items do not build a coherent latent factor of jcse in decreasing hindering job demands. therefore, we decided to reject the four-factor model and in the next step tested an alternative model of jcse without the dimension of decreasing hindering job demands. confirmatory factor analysis of jcses-9. in the next step we verified the hypothesis of the three-dimensional structure of jcse and contrasted it with the one-factor model. as table 2 demonstrates (jses-9 [pl]), the goodness-of-fit indices of the three-factor model regarding cfi and tli exceed the required .90 (cfi = .94; tli = .91). moreover, rmsea is low (.061) and the chi-square/df ratio is lower than 3 (2.63) indicating a good fit. the fit of the three-factor model is significantly better in comparison with the jcse as a one-factor model δχ 2 = 66.63, δdf = 3, p < .001. additionally, the one-factor model of jces-9 seems to fit the data rather poorly (rmsea = .099, the χ2/df = 5.22). we aimed to replicate the factor structure of jses-9 among a us sample (n = 403). as table 2 shows (jses-9 [us]), the fit of the three-factor model is significantly better in comparison with the jcse as a one-factor model δχ 2 = 75.33, δdf = 3, p < .001. the goodness-of-fit indices regarding cfi and tli are higher than 0.90 (cfi = .97; tli = .96), rmsea is low (.05) and the chi-square/df ratio is lower than 3, indicating a good fit. to sum up, our series of studies showed that the final version of the jcse scale consists of three dimensions-jcse in: (a) increasing structural resources, (b) increasing social resources, and (c) increasing challenging job demands. to achieve parsimony of the scale, each of jcse dimensions contains three items. this three-factor structure was confirmed among distinct samples and formats (see s2 appendix). the final version of the instrument can be found in s3 appendix. in the next step, we set out to test the convergent and criterion validity of the jcses. results regarding convergent and criterion validity are based on correlations and multivariable hierarchical regression analyses. hypotheses 2-4 regarding criterion validity were verified on samples from cross-sectional online studies: polish (study 2) and us (study 3), and hypotheses 5-6 about criterion validity with data from study 2. hypothesis 7 concerning jcse's relative role in comparison with perceived opportunity to craft as predictors of job crafting, and time stability of the scale were verified with data from study 4 with a time-lagged procedure (polish sample). results regarding discriminant validity are based on heterotrait-monotrait (htmt) ratio of correlations, using data from study 2 and study 3. the results of correlations from studies 2-3 are displayed in tables 4 and 5 . in line with hypothesis 2, there are positive and moderate correlations between each dimension of jcse and its parallel job crafting dimension, that is, increasing structural resources (study 2: r = .46, see table 4 , p < .001; study 3: r = .59, p < .01, see table 5 ), social resources (study 2: r = .48, see table 4 p < .001; study 3: r = .40, p < .01, see table 5 ), and challenging job demands (study 2: r = .56, p < .001, see table 4 ; study 3: r = .60, p < .01, see table 5 ). supporting hypothesis 3, we found significant and positive correlations between general self-efficacy and each dimension of jcse, i.e., increasing structural resources (study 2: r = .35, p < .001, see table 4 ; study 3: r = .45, p < .001, see table 5 ), social resources (study 2: r = .27, p < .001, see table 4 ; study 3: r = .35, p < .001, see table 5 ) and challenging job demands (study 2: r = .31, p < .001, see table 4 ; study 3: r = .33, p < .001, see table 5 ). to verify whether each jcses dimension has a stronger impact on specific job crafting behavior than general self-efficacy (hypothesis 4), we conducted multivariable hierarchical regression analyses among polish and us samples. in each model with job crafting behaviors as explained variables, general self-efficacy (gse) was entered in the first step (model 1), and in the second step we entered a relevant jcse dimension (model 2). the increment in rsquare was considered as a measure of the added value of the variable entered in the second step, and thus of its lack of redundancy with respect to what is explained in the first step [57] . table 6 presents results of regression analyses for job crafting dimensions in studies 2 (poland) and 3 (us). as table 6 indicates, adding jcse in step 2 contributed to additional unique explained variance in each type of job crafting behavior across both samples. this result means that a relevant jcse belief predicts additional variance in job crafting behaviors over and above gse. the results among polish and us samples are comparable. in both samples, gse predicted increasing structural resources (study 2: β = .37, p < .001; study 3: β = .53, p < .001) in model 1; however, after entering jcse in increasing structural job resources (model 2), gse's impact decreased (study 2: β = .25, p < .001; study 3: β = .33, p < .001). jcse in increasing structural job resources positively predicted increasing structural resources (study 2: β = .37, p < .001; study 3: β = .44, p < .001), and explained additional variance (study 2: δr 2 = .12, p < .001; study 3: δr 2 = .15, p < .001;) over and above gse. general self-efficacy also predicted increasing social resources (study 2: β = .16, p = .003; study 3: β = .15, p = .003); however, its impact stopped being significant with job crafting self-efficacy in increasing social resources (study 2: β = .03, p = .520; study 3: β = .01, p = .833) as a predictor in model 2. jcse in increasing social resources was a positive predictor of increasing social resources (study 2: β = .47, p < 001; study 3: β = .41, p < .001) and explained additional variance in increasing social resources (study 1: δr 2 = 21%; study 2: δr 2 = 14%). finally, general self-efficacy was a stronger predictor of increasing challenging demands in model 1 (study 2: β = .45, p < .001; study 3: β = .37, p < .001), than in model 2 (study 2: β = .30, p < .001; study 3: β = .20, p < .001). when jcse in increasing challenging demands was entered in model 2, both variables were a positive predictor of job crafting through challenges, but jcse was a stronger one (study 2: β = .47, p < .001; study 3: β = .54, p < .001). jcse in increasing challenging demands contributed significantly to the prediction of how often participants sought challenges (study 2: δr = .20, p < .001 study 3: δr = .26, p < .001). in hypothesis 5 we predicted that all types of jcse would be positively related to work engagement (we). in study 2 we found positive and low relations between we and jcse in table 6 . results of the multivariable hierarchical regression analysis in study 2 (n = 340) and study 3 (n = 381). increasing both structural (r = .27, p < .001, see table 4 ) and social (r = .22, p < .001, see table 4 ) job resources. the relation between we and jcse in increasing challenging job demands was positive and moderate (r = .40, p < .001, see table 4 ). in line with hypothesis 6, study 2 demonstrated that relations between jsce dimensions and burnout are significant and negative. these relations were similar in strength and low for each jcse dimension, that is, jcse in increasing structural resources (r = -.30, p < .001, see table 4 ), jcse in increasing social resources (r = -.23, p < .001, see table 4 ), and jcse in increasing challenging demands (r = -.25, p < .001, see table 4 ). to verify whether jcse predicts job crafting better than perceived opportunity to craft in an organization (hypothesis 7), we utilized a time-lagged design (study 4) with predictors measured at time 1, and explained variables-at time 2. we used a multivariable hierarchical regression analysis with pocs measured at time 1 entered in step 1 (model 1), and a relevant jcse dimension measured at time 1 and added as a second step variable (model 2). relevant job crafting behaviors measured at time 2 acted as an explained variable. table 7 presents study correlations and table 8 presents the results of the regression analysis. the results suggest that poc has a lower predictive power than jcse for job crafting behaviors. poc predicted increasing structural resources (β = .46, p < .001) in model 1; however, after entering jcse in increasing structural job resources (model 2), poc's impact decreased (β = .24, p < .001). jcse in increasing structural job resources positively predicted increasing structural resources (β = .46 p < .001), and explained additional variance (δr 2 = .16, p < .001) over and above poc. surprisingly, poc did not predict job crafting in increasing social resources (β = -.02, p = .84). as expected, jcse in increasing social job resources positively predicted increasing structural resources (β = .46 p < .001), model 2 indicated that when jcse in increasing social resources entered the model, the link between poc and job crafting in increasing social resources became significantly negative (β = -.24 p = .016). finally, poc is a [58] for both study 2 and 3. the htmt test 'requires the calculation of a ratio of the average correlations between constructs to the geometric mean of the average correlations within items of the same constructs' [59, p. 124 ]. the 0.85 threshold was used to indicate if the two constructs are distinguishable [58] . the results of both study 2 and study 3 suggest that all jcse dimensions show discriminant validity with respect to dimensions measuring job crafting behaviors, as well as to gse. all ratios resulting from the htmt analyses were lower than 0.85 cut-off point, and ranged from 0.12 to 0.78. cronbach's alpha values for all jcse dimensions in studies 2-4 ranged from .62 to .83, which is acceptable in preliminary research [60] . what is more, we examined test-rest reliability of time 1 and time 2 responses on the jcse scale by testing correlations between the two measurement points. as table 7 demonstrates, the results of the correlation analyses between job crafting self-efficacy dimensions measured both times are similar and high, that is, r = .77, p < .001, for jcse increasing structural resources; r = .76, p < .001, for jcse increasing social resources; r = .77, p < .001, for increasing challenging demands. in this paper we introduced the concept of job crafting self-efficacy and evaluated the psychometric characteristics of the jcse scale. job crafting self-efficacy is a person's beliefs about his or her own capability to modify demands and resources present at their job to better fit their needs and preferences. the results of our research suggest that jcse predicts actual job-crafting behaviors. these findings are consistent with social cognitive theory [5, 21] according to which self-efficacy beliefs 'play a key role in shaping the courses lives take by influencing the types of activities and environments people choose to get into' [61, p. 10] .the key strength of our studies is developing a more accurate predictor of job crafting behaviors, which has both theoretical and practical implications. below we expand on these contributions. the present series of studies showed that the final version of the jcses (jcses-9) consists of three dimensions-jcse in: (a) increasing structural resources, (b) increasing social resources, and (c) increasing challenging job demands. in the first two studies we tested a four-factor model of jcse, including the dimension of jcse in decreasing hindering job demands (jcses-12). in study 2, the four-factor model did not fit the data well; therefore, based on the results of confirmatory factor analysis, we proposed an alternative, three-factor model where we dropped "jcse in decreasing hindering job demands" as a factor. the three-factor model of jcse is supported not only by statistical means, but is also sound given the results of two recent meta-analyses on job crafting. rudolph and colleagues [3] found that there is low factor loading and a small amount of variance explained in overall job crafting by the dimension of decreasing hindering job demands. moreover, there is no link between this latter construct and general self-efficacy [3] . it is therefore possible that decreasing hindering job demands operates differently than other job crafting dimensions. similarly, lichtenthaler and fischbach [62] proposed a distinction between promotion-focused job crafting (increasing resources and challenging demands) and prevention-focused job crafting (decreasing hindering demands). these authors demonstrated that there is a positive relationship between promotion-focused job crafting and health, motivation and performance, while there is a negative relationship between prevention-focused job crafting and the aforementioned variables. it seems, therefore, that promotion-focused job crafting offers a more constructive way of optimizing the fit between one's job and their needs and preferences than a prevention-focused one. thus, we suggest that it is possible to differentiate between personal beliefs about one's abilities to perform promotion-versus prevention-focused job crafting behaviors, and that the jcses-9 developed here can be used to measure the first group of beliefs. recently, scholars have introduced the facet of job crafting which relates to optimizing (rather than avoiding) job demands [63] . initial research shows that optimizing demands is positively related to work engagement, which implies that-in contrast to decreasing hindering job demands-it may be a favorable behavior [63] . however, because optimizing job demands was not a part of the initial job crafting model [1] , as well as the corresponding scale to measure job crafting behaviors [29] , we have not included this in our theorizing. however, we encourage future researchers to develop jcse to capture self-efficacy in optimization of job demands. the results of our research support the validity of the jcse-9. across polish (study 2) and us samples (study 3) all jcse dimensions were positively associated with gse. moreover, jcse dimensions were predictive of their parallel job crafting dimensions (studies 2-4). we also showed that specific jcse dimensions predicted actual job crafting behaviors more accurately than general self-efficacy beliefs. further, jcse-9 dimensions were positively related to work engagement, and negatively-to job burnout. this result supports the positive role of personal job resources in motivational and health impairment processes as described by the jd-r model [6] . what is more, results of discriminant validity analysis showed that jcse dimensions are different from dimensions measuring job crafting behaviors, as well as from a measure of gse. thus, the results suggest that jcses, self-efficacy scale and job crafting scale do not overlap. finally, evidence from of our research showed that the distinguished dimensions can be reliably measured with our instrument as jcses-9 showed good internal consistency estimates (studies 1-4) and high stability over time (study 4). importantly, the results of our studies showed that the three-factor model had significantly better fit than the one-factor model. in this sense, it follows the results for job crafting instruments, where two recent metaanalyses [3, 62] revealed that specific job crafting dimensions may have distinct relationships with individual differences, job characteristics, and work outcomes. for example, only increasing structural resources and increasing challenging demands have negative relationships with turnover intentions. on the other hand, increasing social resources and increasing challenging demands are weakly, but positively associated with prevention focus [3] . these and our findings, combined with the results from cfas, discourage from using a total score when assessing job crafting behaviors. therefore, we highly recommend using the jcses-9 subscale scores rather than the total score. in our series of studies, we introduced the concept of jcse and consequently contributed to the theory and practice in three distinct ways. first, our research demonstrated that jcse, as a context-specific belief, is a more accurate predictor of job crafting than general self-efficacy. thus, we introduced a predictor that is not only more precise, but its advantage (as compared to, for example, personality traits) is the potential for malleability. self-efficacy is a modifiable belief [5] , which may be successfully enhanced using psychological interventions [44, 64] . techniques enhancing self-efficacy that are used in these interventions are usually based on sources of self-efficacy listed by bandura [18] , for example, vicarious or mastery experiences. therefore, we propose that it may be possible to promote job crafting behaviors through jcse interventions. for example, during workshops employees may be encouraged to share successful attempts in job crafting and plan how to overcome barriers that may interrupt the job crafting plans that they develop. moreover, since leaders have significant impact on employees [65] , we believe that they can enhance jcse among their subordinates through acting as role models (i.e., vicarious experience) by demonstrating how they craft their jobs. moreover, leaders can use verbal persuasion to encourage employees to craft. on a day-to-day basis, they may use feedback and appreciate employees' crafting attempts. further experimental investigations are needed to evaluate the effectiveness of this type of interventions. second, the evidence from study 4 also suggests that jcse is more relevant for job crafting than perceived opportunity to craft in an organization. an implication of this result is the possibility that in case of proactive behaviors, such as job crafting, the predictive power of personal resources may be stronger than factors that relate to environmental context. thus, our research demonstrates that is not enough to provide the opportunities or encouragement to craft for employees to start exhibiting these behaviors. our line of work suggests that organizations may benefit from introducing elements enhancing job crafting self-efficacy during job crafting interventions to increase the probability that employees implement job crafting practices in their jobs. moreover, the results of study 2 suggest that jcse, being a personal resource, is linked with enhanced work engagement and reduced job burnout. therefore, our research suggests that there is an opportunity to affect the level of the aforementioned variables through jcse psychological interventions. the final contribution of our research is that the jcses-9 may be helpful in monitoring employees' beliefs about their capability to modify demands and resources present at their job. this instrument may be used as one of the outcome evaluation tools of job crafting interventions. our research has some limitations. in all of our studies we applied a cross-sectional (study 1, study 2, and study 3) or time-lagged cross-sectional design (study 4), which precludes causal inferences. further studies using a longitudinal design need to be done to explore relationships between jcse and related constructs. however, the main goal of our series of studies was to validate a scale to measure jcse and a cross-sectional design is sufficient for these strictly psychometric purposes. both the strength and directions of the obtained relationships support the theoretical validity of the jcse scale. another limitation is that in all studies we used selfreport measures, which may lead to common method variance (monomethod bias; [66] ). to minimize this risk we avoided repeated use of the same anchor points in questionnaires, and in study 4 we spaced the predictors and explained variables in time [67] . to further reduce this method bias, future studies could also include various types of measures, such as peer-ratings or supervisor assessments of job crafting behaviors [29] . moreover, the results of our research suggest that jcse in increasing social resources may operate differently than other jcse dimensions. results of the regression analysis showed that the impact of gse on increasing social resources ceases to be significant when jcse in increasing social resources enters the model. with regard to environmental factors-when the dimension of jcse in increasing social resources enters the regression model as a predictor, the influence of perceived opportunity to craft in an organization on this parallel job crafting dimension starts to be negative. future studies need to examine how jcse in increasing social resources may operate in relationship with related constructs. job crafting is said to occur in every profession [68] , on every organizational level [69] , and on an everyday basis [70] . in our research we introduced the concept of jcse, which may constitute one of the key predictors of job crafting behaviors. we also developed and validated a measure to assess jcse, which 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engagement increase person-job fit? the role of job crafting and job insecurity work meaning in self and world perspective: a new outlook on the wami scale individual job redesign: job crafting interventions in healthcare how can i shape my job to suit me better? job crafting for sustainable employees and organizations shaping tasks and relationships at work: examining the antecedents and consequences of employee job crafting. 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job crafting crafting a job on a daily basis: contextual correlates and the link to work engagement we wish to thank research assistants who helped gather data for study 1 (sylwia dzikowska, andżelika krauze, patrycja łuszczyńska), study 2 (jolanta kwiecień, anna orzeszek), and study 4 (katarzyna jabłońska, wiktoria jarosz). key: cord-337630-ojhk5opy authors: tasic, velibor; hynes, ann marie; kitamura, kenichiro; cheong, hae il; lozanovski, vladimir j.; gucev, zoran; jutabha, promsuk; anzai, naohiko; sayer, john a. title: clinical and functional characterization of urat1 variants date: 2011-12-16 journal: plos one doi: 10.1371/journal.pone.0028641 sha: doc_id: 337630 cord_uid: ojhk5opy idiopathic renal hypouricaemia is an inherited form of hypouricaemia, associated with abnormal renal handling of uric acid. there is excessive urinary wasting of uric acid resulting in hypouricaemia. patients may be asymptomatic, but the persistent urinary abnormalities may manifest as renal stone disease, and hypouricaemia may manifest as exercise induced acute kidney injury. here we have identified macedonian and british patients with hypouricaemia, who presented with a variety of renal symptoms and signs including renal stone disease, hematuria, pyelonephritis and nephrocalcinosis. we have identified heterozygous missense mutations in slc22a12 encoding the urate transporter protein urat1 and correlate these genetic findings with functional characterization. urate handling was determined using uptake experiments in hek293 cells. this data highlights the importance of the urat1 renal urate transporter in determining serum urate concentrations and the clinical phenotypes, including nephrolithiasis, that should prompt the clinician to suspect an inherited form of renal hypouricaemia. in man, the level of serum uric acid is determined primarily by the production of urate, as an end product of purine metabolism (for which the liver enzyme xanthine oxidase is necessary) versus biliary and urinary tract elimination. in the majority of other mammals, uric acid is metabolized by uricase (urate oxidase) to allantoin, before urinary excretion. thus man (and other species lacking uricase, such as great apes), has comparably higher serum uric acid levels than most mammals. the renal handling of uric acid is a complex and incompletely understood process [1, 2] . uric acid is freely filtered at the glomerulus, the majority undergoes reabsorption via proximal tubular urate transporter proteins and a proportion (,10%) is secreted back into the filtrate in the late proximal tubule. molecular genetic and genome wide association studies have recently allowed the identification of several proximal tubule urate transporters including urat1 (alias slc22a12) [3] and glut9 (alias slc2a9) [4, 5, 6] . proposed models of urate transport in the proximal tubule [7] suggest an initial uptake of uric acid from the filtrate by urat1, coupled to organic acid transporters. glut9, in two different isoforms, allows for basolateral exit of urate from the proximal tubule (isoform i) and regulation of urate entry/exit at the apical membrane (glut9dn isoform). finally, in the late proximal tubule there are transporter proteins mediating uric acid secretion (including abcg2, npt1 and npt4) [7] . as uric acid excretion is mediated through molecular transporters, certain drugs such as fenofibrate, valproic acid, trimethoprim and losartan may be used to manipulate these processes [8, 9, 10] , thus allowing manipulation of serum uric acid levels. in humans, genetic defects in the activity of xanthine oxidase or an acquired defect in liver enzyme function or renal uric acid handling may result in hypouricaemia. acquired hypouricaemia may be seen in a number of clinical disorders, including fanconi syndrome [11] , type 1 and type 2 diabetes mellitus [12, 13] , thyrotoxicosis [14] , pseudohypoparathyroidism type 1b [15] , pseudoaldosteronism due to licorice ingestion [16] , distal renal tubular acidosis [17, 18] , obstructive jaundice [19] and severe acute respiratory syndrome [20] . idiopathic renal hypouricaemia is an inherited form of hypouricaemia that is characterized by excessive urinary wasting of uric acid leading to an increased clearance (and increased fractional excretion) of uric acid. the majority of patients are asymptomatic, but some may present with uric acid nephrolithiasis or acute kidney injury following severe exercise [21] . in 2002, enomoto et al. reported that mutations in gene slc22a12 encoding the urat1 transporter were responsible for most cases of idiopathic renal hypouricaemia [3] . recently anzai et al. found mutations in slc2a9, encoding glut9, in patients with severe renal hypouricaemia [22] . it is noteworthy that reports of idiopathic renal hypouricaemia secondary to mutations in uric acid transporters urat1 and glut9 were initially reported from japan, korea and china [23] . more recently, three jewish israeli families of iraqi origin have been reported to have renal hypouricaemia, with a common mutation in slc22a12 [24] . inactivating mutations in slc22a12 have not yet, to our knowledge, been reported in a caucasian population. the typical presentation of idiopathic renal hypouricaemia is that of exercise induced acute kidney injury with a preceding history of loin pain with nausea and vomiting for several hours after physical exercise. the exact mechanism of renal damage is unclear, but may relate to damage from oxygen free radicals [21] . in contrast to this dramatic presentation, most patients are well with no overt clinical symptoms, although renal stones and hematuria may be presenting symptoms and signs. here we present data from skopje (macedonia) and newcastle upon tyne (uk) where we have investigated the underlying genetic cause of hypouricaemia in patients of european descent. we present mutations in slc22a12 encoding urat1 alongside their clinical, biochemical and functional characterization. this data highlights the importance of renal urate transporters in determining serum urate concentrations, and the clinical phenotypes that should lead the renal clinician to suspect an inherited form of renal hypouricaemia. a total of thirty two patients with hypouricaemia were recruited (macedonia, n = 20 and united kingdom n = 12) for mutational analysis of the slc22a12 and slc2a9 genes. the basic demographic, clinical, laboratory and molecular genetic data from these patients are given in table 1 . we found changes in slc22a12 in five patients from macedonia and two patients from the united kingdom ( figure 1a ). no pathogenic mutations in slc2a9 were identified. an outline of the clinical, biochemical and molecular genetic features of each case is given below. patient sk-1: this 7 year old girl was referred to the nephrology unit with persistent vomiting, presumed to be secondary to a non-obstructive kidney stone. she had a history of similar episodes during the preceding 6 months. on admission she was alert, but pale and moderately dehydrated. clinical examination revealed that her abdomen was non-tender with no renal masses. a renal ultrasound scan (uss) revealed a single stone within the right kidney measuring 10 mm, without calyceal dilatation. laboratory investigations on admission revealed normal renal function (serum creatinine 26 mmol/l, estimated glomerular filtration rate 123 ml/min/1.73 m 2 (according to schwartz formula)). although she was moderately dehydrated, she had persistently low serum uric acid levels ranging from 0.72-1.18 mg/dl. fe urate was elevated at 20.8%. liver function tests, serum electrolytes, total protein and albumin were within reference values. urinary excretion of cystine, calcium, phosphate, oxalate, glucose, low molecular weight proteins, amino acids and organic acids were also within reference limits. in order to clarify the etiology of urolithiasis, urinary excretion of xanthine and hypoxanthine was measured, showing normal values. mutation analysis of slc22a12 revealed a heterozygous missense mutation, leading to amino acid change p.r434c. genetic screening of other family members revealed that her brother and father both carried the identical heterozygous change, but were asymptomatic in terms of renal stones, with normal renal ultrasound scan and no reported episodes of exercise induced renal failure. biochemical evaluation revealed that the father had normal values of serum uric acid while the brother had moderately decreased serum uric acid level (1.56 mg/dl). patient sk-2: this 18 year female was referred for management of hypertension. she had a past medical history of pyelonephritis and bilateral ureteric reflux with scarring of the left kidney, as determined by uss and tc 99 dmsa scan. laboratory investigations revealed serum creatinine and urea within normal limits. she was found to have low serum uric acid levels on two occasions (1.74 mg/dl and 1.78 mg/dl, respectively) with an increased fe urate . she had mild proteinuria (0.35 g/24 h) and a renal uss revealed a small left kidney, but no evidence of nephrolithiasis. mutational analysis of the slc22a12 gene was performed and a heterozygous missense mutation p.r434h was detected. patient sk-3: this 5 year old girl was admitted to the nephrology unit with severe dehydration, polyuria and vomiting. she was noted to have faltering growth and rickets. laboratory investigations revealed a hyperchloremic metabolic acidosis (ph 7.23, hco 3 13.6 mmol/l, be 212.6 mmol/l), hypokalemia (3.0 mmol/l), hypophosphatemia (0.84 mmol/l) and hypouricaemia (1.24 mg/dl). simultaneous measurement of urine ph with electrode revealed value of 6.77 which pointed to distal acidification defect (expected urine ph,5.5). there was evidence of proteinuria on urine dipstix testing (1+) which was characterized with sds-pag electrophoresis as complete tubular proteinuria. there was a generalized hyperaminoaciduria, but no glucosuria. there was also evidence of uricosuria (fe urate varied between 2431%) and hyperphosphaturia (fe po4 varied between 21-33%). ultrasound examination revealed bilateral nephrocalcinosis and a solitary cyst in the left kidney measuring 10 mm. following alkali therapy, metabolic compensation was achieved and her growth pattern improved. following correction of the metabolic acidosis, the proximal defects of aminoaciduria and low molecular proteinuria resolved. serum electrolytes also normalized, but there was persistent hypouricaemia during the two year observational period (1.24-1.36 mg/dl). since the hypouricaemia was associated with an elevated fe urate , we undertook mutational analysis of slc22a12 which revealed a heterozygous missense mutation, leading to amino acid change r434c. the mother of patient sk-3 (r434c) was also heterozygous for the r434c variant in slc22a12. biochemical evaluation confirmed she had a low normal serum uric acid level (2.3 mg/dl) with fractional excretion of urate of 19.3%. she had a normal renal ultrasound, but a past medical history of renal colic and passage of a single calculus 10 years ago. patient sk-4: this 8 year old boy presented with recurrent attacks of visible hematuria and sensorineural hearing loss. a percutaneous renal biopsy was performed which demonstrated minimal glomerular abnormalities on light microscopy. immunofluorescence studies did not reveal any immune deposits and electron microscopic analysis was not available. given a clinical suspicion of alport syndrome, genetic studies were performed, identifying a mutation in the col4a5 gene. following another attack of visible hematuria with very severe colicky pain, serum biochemistry revealed a low uric acid level. repeated examinations of the uric acid confirmed persistent hypouricaemia, with high fe urate . mutational analysis of slc22a12 revealed a heterozygous missense mutation, leading to the amino acid change r347s. genetic analysis confirmed that the mother of patient sk-4 was also heterozygous for the sequence variant r347s. patient sk-5: a 7 year old female presented with vitiligo, prompting a screen for autoimmune diseases. she was found to have compensated hypothyroidism, with typical ultrasound changes of the thyroid gland and increased antithyroid antibodies, suggesting hashimoto thyroiditis. she was also found to have hypouricaemia with significant hyperuricosuria. she underwent mutation analysis of slc22a12 which revealed a heterozygous missense mutation, p.r434h. patient nc-1: a 41 year old female presented with recurrent renal colic, with 2 episodes in less than 12 months. she received lithotripsy treatment for a left renal calculus (struvite stone). her past medical history was noteworthy for type 1 diabetes mellitus, since 10 years of age, complicated by peripheral neuropathy and urinary tract infections. she also had treated hypothyroidism. at presentation, serum electrolyte abnormalities revealed a borderline low uric acid level and a serum creatinine of 95 mmol/l. a fe urate was transiently raised at 16%. repeat serum biochemistry revealed a serum uric acid level of 3.86 mg/dl and a normalised fe urate . following successful lithotripsy she has remained stone free for 5 years, following advice regarding increased fluid intake. genetic analysis of slc22a12 revealed a heterozygous missense mutation, p.v388m. patient nc-2: a 45 year old lady presented to the regional lithotripsy unit for treatment. she had recurrent renal stone disease (calcium oxalate) for 7 years, requiring both lithotripsy and a right pyeloplasty. she was on no medications. serum electrolytes revealed a normal serum creatinine (75 mmol/l) with a low serum uric acid (2.01mg/dl). fe urate was not available for this patient. we performed genetic analysis of slc22a12, which revealed a heterozygous missense mutation, p.i75t. following the identification of variants within the slc22a12 gene encoding urat1 ( figure 1a ,b) we used online databases and mutation prediction software to attempt to score the pathogenicity of each variant. within the urat1 amino acid sequence, residues r347, v388 and r434 are highly conserved, whilst i75 is not well conserved (table 2) . sift and snps3d analysis predicted all 5 sequence variants to be either not tolerated (sift) or deleterious (snps3d). polyphen analysis predicted that 3 out of the 5 missense changes to be ''probably damaging'', with v388m and i75t predicted to be benign (table 2 ). using mammalian cells, we evaluated the urate transport function of urat1 sequence variants found in this study. urate uptake was measured in transiently transfected hek293 cells, comparing wild-type transporter activity to sequence variants i75t, r347s, r434c and r434h. following transient transfection of hek293 cells with flagtagged urat1 constructs, we demonstrate strong plasma membrane expression in wild-type urat1 (figure 2a) . plasma membrane expression levels of variants r434c and r434h were low whereas the intracellular localization was not strongly observed, possibly due to the stability of protein. for variant i75t the partial membrane expression was observed together with partial intracellular localization. for variants r347s and v388m strong plasma membrane expression, similar to wildtype levels was observed (figure 2a) . measurement of urate uptake in hek293 cells demonstrates a significant reduction of urate transport function by i75t, r347s, r434c and r434h variants of urat1 but not in v388m variant ( figure 2b ). idiopathic renal hypouricaemia is a disorder that has been characterized previously in patients from far eastern countries including japan, korea and china. the disease may be completely asymptomatic [23, 25, 26] , but there are many reports, particularly from japan, where patients present with acute kidney injury following strenuous physical activities [21, 27, 28, 29, 30, 31, 32, 33, 34] . the exact mechanism of acute kidney injury remains elusive, but it is believed that uric acid serves as an antioxidant, and in states of hypouricaemia this protective role is lost. kaneko et al. demonstrated in a 15 year old girl with idiopathic renal hypouricaemia an oxidative imbalance soon after exercise with a predisposition to exercise-induced acute renal failure [35] . in contrast, some patients may present with more minor renal features including nephrolithiasis or hematuria [23, 31, 36] . in 2002, enomoto et al. established that urat1 transporter was responsible for tubular reabsorption of urate [3] . slc22a12 encodes the protein urat1 and loss of function mutations are responsible for majority of patients with idiopathic renal hypouricaemia. the w258x variant of urat1 is a typical mutation found in japanese and korean populations [23, 31, 37, 38] . allele frequency of w258x in the general population in japan was found to be as high as 1.9% [39] . heterozygous carriers of urat1 mutations are usually asymptomatic but they may develop nephrolithiasis. prevalence of hypouricaemia in japan varies between 0.15% [40] and 0.23% from the analysis of serum urate levels in 1730 school children [41] . in korea, the prevalence of hypouricaemia in healthy adults is 3.3% [42] . therefore, school-age children who plan to performing competitive sporting activities are advised to have their serum uric acid level checked [43] . loss of function mutations affecting urat1 have not been previously reported in a caucasian population. however, single case reports from european patients presenting with clinical and biochemical features of hereditary renal hypouricaemia exist [44, 45, 46, 47] . tzovaras et al. tested nine greek subjects with primary renal hypouricaemia [48] . all had serum uric acid levels ,2.5 mg/dl, associated with a fe urate .10% and no other known cause of hypouricaemia. no definite pathogenic mutations were detected in this series and just one silent polymorphism (1246t.c) in exon 2 of the slc22a12 gene was noted. it is questionable why hereditary renal hypouricaemia is apparently so rare in caucasian populations. either the prevalence of urat1 mutations is indeed low in this population, or a decreased awareness of this disease and its presentation outside of the far east allows cases to go undetected. even in japan, there are patients who have clinical features of renal hypouricaemia but no slc22a12 mutations. very recently, a genome-wide association study was performed in 6890 african americans and 21708 european participants in order to try and identify risk alleles for elevated serum urate, associated with gout [49] . a novel urat1 variant (g65w) was identified (rs12800450) and was associated with a reduction in serum urate of around 1.2 mg/dl per copy of the minor allele. urate transport studies demonstrated a reduction in the urate transport for the g65w urat1 variant [49] . this study validates heterozygous changes within urat1 as a determinate of reduced serum urate levels. in this study the variant allele provided a protective affect against gout, but one may postulate that this may also be an at-risk allele for the hypouricaemia. both biochemically and clinically, a single heterozygous change in urat1 may be significant. cheong et al. reported a w258x homozygous mutation in a 7 year old child, whose mother and brother were also heterozygous for w258x and had mild hypouricaemia and abnormally high fe urate , whilst his father who was also heterozygous for w258x, had a normal serum uric acid level of 4.6 mg/dl [23] . in the same report, an 11-yearold girl with asymptomatic microscopic hematuria and low serum urate was a compound heterozygote for w258x/r477h. [23, 31, 36] . recently, the glut9 glucose transporter, encoded by slc2a9 gene has been shown to have an important functional role in transporting uric acid from the renal tubular cells through the basolateral membrane into interstitium [22] . two additional reports have confirmed this finding [51, 52] . matsuo et al. detected two heterozygous mutations in glut9 in two hypouricaemic subjects whom were negative for urat1 mutations (r380w in exon 10 and r198c in exon 6) and confirmed their reduced transport activity in xenopus oocyte expression system [51] whilst dinour et al. recently described two families with recessively inherited hypouricaemia who were negative for urat1 mutations [52] . among hypouricaemic subjects in both families, three subjects had nephrolithiasis and three subjects had a history of exercise induced acute kidney injury. with genome wide homozygosity screen and linkage analyses they established slc2a9 as a causative gene for renal hypouricaemia in these families. in these families, homozygous carriers of slc2a9 mutations had very low serum levels of uric acid and extremely high values of fe urate (at around 150%). in this report we present combined data from macedonian and english patients who are heterozygous carriers of slc22a12 sequence variants. although the presentations of the patients were varied, a systematic search for hypouricaemia identified patients with possible hereditary renal hypouricaemia and who are suitable for mutational analysis of slc22a12 and slc2a9 genes to try and identify abnormalities in the encoded urate transporters urat1 and glut9, respectively. we identified seven patients harboring five slc22a12 variants. interestingly, three macedonian patients carried mutations at amino acid position 434 of urat1. some of these variants in slc22a12 were discovered following significant clinical episodes including nephrolithiasis in patients sk-1, nc-1 and nc-2. there were however, no episodes of exercise induced acute kidney injury that we are aware of. the sequence variant r434h was identified in one of our patients with hypouricaemia. this variant has a reported heterozygosity index of 0.011 (table 2) , although this variant was not detected in our normal control subjects. additional studies are required to demonstrate whether this variant is a common cause of hypouricaemia. of note, in our cohort of patients, we found no novel sequence variants in the glut9 transporter (slc2a9), leaving a number of patients with unexplained hypouricaemia. however, given the complexity of proximal tubule urate handling, other urate transporter protein variants may account for hypouricaemia in the remaining patients. in patient sk-1 nephrolithiasis was likely to be due to multiple risk factors including renal wasting of urate, episodes of cyclic vomiting leading to concentrated urine and hypocitraturia. in patient sk-4, who also had a molecular genetic diagnosis of alport syndrome, attacks of gross hematuria were confusing due to presence of colicky pain and eumorphic red blood cells (100%), which are not usual features of alport syndrome. in this patient, we found the mutation p.r347s. cheong et al. reported a similar case to this, where a 14 year old girl who presented with acute post-streptococcal glomerulonephritis [23] . although her nephritis had a favorable course, the microhematuria persisted more than one year. on reevaluation this girl was found to have low serum uric acid (1.3 mg/dl) with increased fe urate (14.5%) in favor of idiopathic hypouricaemia. mutational analysis in this patient revealed heterozygous w258x mutation in slc22a12. patient sk-2 has hypertension and moderate proteinuria due to reflux nephropathy, presumably as a coincidental finding to the functionally significant p.r434h variant. as hypertension in the context of renal disease is often treated with ace inhibitors or angiotensin receptor antagonists, some caution is advisable. both losartan [8] and irbesartan [53] have an inhibitory action on urat1. thus treatment with these agents has potential to have a marked uricosuric effect in patients with homozygous urat1 mutations. patient sk-3 had a complex phenotype of distal renal tubular acidosis and renal hypouricaemia, associated with the p.r434c mutation within urat1. it is well known that hypouricaemia may be associated with distal renal tubular acidosis at diagnosis [17, 18] as a part of transitory proximal tubular dysfunction. in additional, pharmacological agents may also disrupt proximal tubular handing of urate. in our case, hypouricaemia persisted for more than 2 years despite a normalization of other proximal tubular functions. definitively, mutational analysis of slc22a12 with a functionally significant change (p.r434c) explained the persistent hypouricaemia in this patient. patient nc-1 was a recurrent calcium stone former, with a past medical history of type 1 diabetes. here, serum urate levels were only borderline low and the fe urate was also only transiently raised at 16%. the missense mutation p.v388m was associated with negative functional data, with no significant change in urate transport in hek293 cell experiments. inclusion of this case is helpful as the negative functional data and the transient hypouricaemia are consistent with this variant being benign. the dataset derived from the v388m variant allow a comparison to be made between it and the more functionally significant variants, acting as another negative control. given the hypouricaemia was transient in this case, we do not assume that this sequence variant is causative. from our sequence variants of urat1, mutations with an impact upon uric acid handling (when modeled in hek293 cells) are associated with a persistent hypouricaemia. patient nc-2 was also a recurrent stone former, with persistently low serum uric acid. the heterozygous missense mutation, p.i75t was confirmed to be functionally significant in hek293 urate uptake studies. in previously reported japanese and korean cases of idiopathic renal hypouricaemia secondary to slc22a12 mutations, the majority of patients have been shown to have either homozygous mutations or compound heterozygote mutations [23] . in our caucasian population, disease associations have been made with single heterozygous changes in urat1, suggesting that such a change is sufficient and that a dominant pattern of inheritance may be present. in conclusion, we have identified macedonian and british patients with hypouricaemia, who presented with symptoms including renal stone disease and haematuria. we have identified missense mutations in slc22a12 encoding urat1. this data highlights the importance of renal urate transporters in determining serum urate concentrations and the of clinical phenotypes that should lead the clinician to suspect an inherited form of renal hypouricaemia. for macedonia patients, the institutional review board and the ethics committee at the medical faculty and the ministry of education and science, skopje, republic of macedonia approved the study. informed written consent was obtained by participants' parents or legal guardians. for uk patients, full ethical approval was obtained by the newcastle and north tyneside research ethics committee and informed written consent were obtained from participants. macedonian patients: children attending in-patient or outpatient nephrology service at the university children's hospital, skopje were screened for hypouricaemia. serum uric acid level was determined in children with contraction or expansion of the extracellular volume, acute kidney injury, chronic kidney disease, nephrolithiasis, tubular disorders, those receiving diuretics or nephrotoxic drugs, liver diseases, diabetes mellitus and thyroid diseases. children less than two years of age were excluded from this study since they have physiologically lower levels of uric acid due to immaturity of the liver and renal tubular function. where serum uric acid levels were ,2 mg/dl and fractional excretion of urate (fe urate ) was .10% (normal range 2-8%) and these biochemical findings persisted despite clinical improvement and normalization of other biochemical indices, or if no explanation was found for lower serum levels of uric acid, than idiopathic renal hypouricaemia was considered to be a likely diagnosis [23] . patients with suspected renal hypouricaemia underwent additional studies, including ultrasound imaging of the urinary tract (if previously not performed) and molecular analysis of slc22a12 and slc2a9. united kingdom patients: adult kidney stone formers attending for lithotripsy at the newcastle upon tyne nhs foundation trust hospital, uk were recruited (following informed consent) and screened for serum and urinary biochemical abnormalities. twelve patients with hypouricaemia (,2.6 mg/dl) underwent molecular analysis of slc22a12 and slc2a9. mutational analysis of slc22a12 and slc2a9 genes was performed using exon pcr and direct sequencing. (oligonucleotide primer sequences are listed in table s1 ). for control patient dna screening, 92 samples were obtained from blood donor (healthy control) panels. online in silico analyses were performed when sequence variants were identified. these included sift (http://sift.jcvi. org/), polyphen (http://genetics.bwh.harvard.edu/cgi-bin/ggi/ ggi.cgi) and snps3d (www.snps3d.org/). full-length cdna of human urat1 was obtained as described previously [3] and tagged with 36 flag at the n-terminus. the quickchange site-directed mutagenesis kit (stratagene, la jolla, ca) was used to introduce point mutations into urat1 cdna in the expression vector according to the instructions. complementary oligonucleotides used for mutagenesis are described in table s2 . all the final cdna sequences were confirmed by dna sequencing. for the evaluation of transport function of slc22a12 mutations, we used a mammalian cell expression system as described previously [54] . briefly, hek293 cells [55] were maintained in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal bovine serum (fbs), 1 mm sodium pyruvate, 100 u/ml penicillin, and 100 mg/ml streptomycin (invitrogen, carlsbad, ca) at 37uc and 5% co 2 . transient transfection with lipofectamine 2000 (invitrogen) was performed according to the manufacturer's instructions. after transfection, the cells were grown 36-48 h before the experiments. cellular uptake of [ 14 c]ua were measured in hurat1 (or its mutants)-transfected hek293 cells [55] grown on poly-d-lysinecoated 24-well plates. the cells were incubated in chloride-free hanks' balanced salt solution (hbss) containing the following in mm: 125 na gluconate, 4.8 k gluconate, 1.2 kh 2 po 4 , 1.2 mgso 4 , 1.3 ca gluconate, 5.6 glucose and 25 hepes, ph 7.4) for 10 min. the uptake study was started by adding hbss containing [ 14 c]ua or to the plate. after 2 min, the cells were washed three times in ice-cold hbss, then lysed in 0.1 m naoh for 20 min followed by measurement of radioactivity by scintillation counting. the experiments were performed in quadruplicate per experiment and repeated twice. all the data are given as the mean 6 s.d. the student's t-test was used to determine significant differences. a value of p,0.05 was considered to be significant. immunocytochemical analyses were performed as previously described [56] . urat1 (or its mutants)-transfected hek293 cells were fixed with methanol and incubated with the anti-flag antibody (1:50) (sigma) followed by alexa fluorh 488-labelled goat anti-rabbit immunoglobulin (invitrogen; diluted 1:200). the staining was observed under a confocal laser scanning microscope (fluoview fv1000, olympus). alexa 488 fluorescence was excited by argon laser light of wavelength 488 nm. new insights into renal transport of urate renal solute transporters and their relevance to serum urate disorder molecular identification of a renal urate anion exchanger that regulates blood urate levels the glut9 gene is associated with serum uric acid levels in sardinia and chianti cohorts genomewide association study identifies genes for biomarkers of cardiovascular disease: serum urate and dyslipidemia slc2a9 is a newly identified urate transporter influencing serum urate concentration, urate excretion and gout a 'complexity' of urate transporters uricosuric action of losartan via the inhibition of urate transporter 1 (urat 1) in hypertensive patients hypouricemia in severely disabled children: influence of valproic acid and bed-ridden state the effect of trimethoprim on potassium and uric acid metabolism in normal human subjects hyperuricosuria in the fanconi syndrome renal handling of uric acid in patients with type 1 diabetes in relation to glycemic control hypouricemia and hyperuricemia in type 2 diabetes: two different phenotypes hypouricemia in a patient with thyrotoxicosis phenotypic and molecular genetic aspects of pseudohypoparathyroidism type ib in a greek kindred: evidence for enhanced uric acid excretion due to parathyroid hormone resistance pseudoaldosteronism with increased serum cortisol associated with pneumonia, hypouricemia, hypocalcemia, and hypophosphatemia proximal renal tubular dysfunction in primary distal renal tubular acidosis atypical presentation of distal renal tubular acidosis in two siblings hypouricemia in individuals admitted to an inpatient hospital-based facility renal hypouricemia is an ominous sign in patients with severe acute respiratory syndrome exercise-induced acute renal failure associated with renal hypouricaemia: results of a questionnairebased survey in japan plasma urate level is directly regulated by a voltage-driven urate efflux transporter uratv1 (slc2a9) in humans mutational analysis of idiopathic renal hypouricemia in korea urat1 mutations cause renal hypouricemia type 1 in iraqi jews hereditary renal hypouricemia a case of renal hypouricemia caused by urate transporter 1 gene mutations a case of exerciseinduced acute renal failure in a patient with idiopathic renal hypouricemia developed during antihypertensive therapy with losartan and trichlormethiazide exercise-induced acute renal failure in a patient with congenital renal hypouricaemia acute renal failure after exercise in a japanese sumo wrestler with renal hypouricemia the case: a young man with acute kidney injury after exercise. the diagnosis: exercise induced acute kidney injury in hereditary renal hypouricemia clinical and molecular analysis of patients with renal hypouricemia in japaninfluence of urat1 gene on urinary urate excretion two male siblings with hereditary renal hypouricemia and exercise-induced arf exercise-induced acute renal failure in a patient with renal hypouricemia a case of exercise-induced acute renal failure in a patient with enhanced renal hypouricaemia oxidative imbalance in idiopathic renal hypouricemia familial hypouricaemia associated with renal tubular uricosuria and uric acid calculi: case report analysis of mutations in the urate transporter 1 (urat1) gene of japanese patients with hypouricemia in northern japan and review of the literature the w258x mutation in slc22a12 is the predominant cause of japanese renal hypouricemia slc22a12 w258x frequency according to serum uric acid level among japanese health checkup examinees cause of persistent hypouricemia in outpatients recurrent urat1 gene mutations and prevalence of renal hypouricemia in japanese prevalence of hypouricaemia and slc22a12 mutations in healthy korean subjects renal hypouricemia in school-aged children: screening of serum uric acid level before physical training acute renal failure due to uric acid nephropathy in a patient with renal hypouricemia non-urate transporter 1-related renal hypouricemia and acute renal failure in an israeli-arab family absence of slc22a12 gene mutations in greek caucasian patients with primary renal hypouricaemia genome-wide association study for serum urate concentrations and gout among african americans identifies genomic risk loci and a novel urat1 loss-of-function allele molecular analysis of the slc22a12 (urat1) gene in patients with primary gout mutations in glucose transporter 9 gene slc2a9 cause renal hypouricemia homozygous slc2a9 mutations cause severe renal hypouricemia concentration-dependent inhibitory effect of irbesartan on renal uric acid transporters functional analysis of human organic cation transporter oct3 (slc22a3) polymorphisms the multivalent pdz domain-containing protein pdzk1 regulates transport activity of renal urate-anion exchanger urat1 via its c terminus human sodium phosphate transporter 4 (hnpt4/slc17a3) as a common renal secretory pathway for drugs and urate the authors thank a. yamanishi for technical assistance. key: cord-327257-doygrgrc authors: zhu, jocelyn; shen, beiyi; abbasi, almas; hoshmand-kochi, mahsa; li, haifang; duong, tim q. title: deep transfer learning artificial intelligence accurately stages covid-19 lung disease severity on portable chest radiographs date: 2020-07-28 journal: plos one doi: 10.1371/journal.pone.0236621 sha: doc_id: 327257 cord_uid: doygrgrc this study employed deep-learning convolutional neural networks to stage lung disease severity of coronavirus disease 2019 (covid-19) infection on portable chest x-ray (cxr) with radiologist score of disease severity as ground truth. this study consisted of 131 portable cxr from 84 covid-19 patients (51m 55.1±14.9yo; 29f 60.1±14.3yo; 4 missing information). three expert chest radiologists scored the left and right lung separately based on the degree of opacity (0–3) and geographic extent (0–4). deep-learning convolutional neural network (cnn) was used to predict lung disease severity scores. data were split into 80% training and 20% testing datasets. correlation analysis between ai-predicted versus radiologist scores were analyzed. comparison was made with traditional and transfer learning. the average opacity score was 2.52 (range: 0–6) with a standard deviation of 0.25 (9.9%) across three readers. the average geographic extent score was 3.42 (range: 0–8) with a standard deviation of 0.57 (16.7%) across three readers. the inter-rater agreement yielded a fleiss’ kappa of 0.45 for opacity score and 0.71 for extent score. ai-predicted scores strongly correlated with radiologist scores, with the top model yielding a correlation coefficient (r(2)) of 0.90 (range: 0.73–0.90 for traditional learning and 0.83–0.90 for transfer learning) and a mean absolute error of 8.5% (ranges: 17.2–21.0% and 8.5%-15.5, respectively). transfer learning generally performed better. in conclusion, deep-learning cnn accurately stages disease severity on portable chest x-ray of covid-19 lung infection. this approach may prove useful to stage lung disease severity, prognosticate, and predict treatment response and survival, thereby informing risk management and resource allocation. coronavirus disease 2019 (covid19) is an infectious disease that can cause severe respiratory illness [1, 2] . first reported in wuhan, china in december 2019 [3] , covid-19 was declared a pandemic on mar 11, 2020 (https://www.who.int). more than 2 million people have been infected and more than 120,000 have died of covid-19 (https://coronavirus.jhu.edu, a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 apr 14, 2020) . these numbers are predicted to continue to rise in the foreseeable future with a high likelihood of a second wave and recurrence. covid-19 has already overwhelmed many hospitals in many countries. portable chest x-rays (cxr) has become an indispensable imaging tool to imaging patients with contagious diseases because it is informative and the equipment can be readily disinfected. cxr provides both the extent and severity of lung infection, and is widely used to quantify disease severity in many lung diseases [4] . the hallmarks of covid-19 lung infection on cxr include bilateral, peripheral hazy opacity and airspace consolidation [5] . given the anticipated shortage of intensive care unit (icu) beds and mechanical ventilators in many hospitals, cxr has the potential to play a critical role in decision-making to determine which patients to put on a mechanical ventilator, monitor disease progression and treatment effects during mechanical ventilation, and determine when it is safe to extubate. similarly, computed tomography (ct), which offer better sensitivity than cxr, has been used in imaging suspected or confirmed covid-19 patients, albeit mostly in china early on in the pandemic [6] [7] [8] [9] . however, ct is less convenient, not practical in icu setting, and prone to cross contamination and, thus, it is not widely used in covid-19 infection or similar contagious disease in the united states. deep-learning artificial intelligence (ai) has become increasingly popular for analyzing diagnostic images [10, 11] . ai has the potential to facilitate disease diagnosis, diseases severity staging, and longitudinal monitoring of disease progression. one common machine-learning algorithm is the convolutional neural network (cnn) [12, 13] , which takes an input image, learns important features in the image such as size or intensity, and saves these parameters as weights and bias to differentiate types of images [14, 15] . cnn architecture is ideally suited for analyzing images. the majority of machine learning algorithms to date are trained to solve specific tasks, working in isolation. models have to be rebuilt from scratch if the feature-space distribution changes. transfer learning overcomes the isolated learning paradigm by utilizing knowledge acquired for one task to solve related ones. transfer learning in ai is particularly important for small sample size data because the pre-trained weights enables more efficient training and better performance [16, 17] . there are many ai algorithms that have already been developed for cxr applications (see review [18] ) and these ai algorithms can be repurposed to study covid-19 lung infection. a few studies have already reported ai applications to classify covid-19 versus non-covid-19 lung infection using cxr [19] [20] [21] [22] and ct [23] [24] [25] [26] . many of these referenced publications here are non-peer reviewed pre-prints. these studies aimed to classify covid-19 versus non-covid-19 images, not to stage lung disease severity. classification of covid-19 versus non-covid-19 images for the purpose of diagnosis made by ai unfortunately has poor specificity. the reverse-transcription polymerase chain reaction test of a nasopharyngeal or oropharyngeal swab is still needed for definitive diagnosis. by contrast, deep-learning ai is well suited to stage disease severity. to our knowledge, there have been no studies to date that use deeplearning ai to stage disease severity on cxr of covid-19 lung infection. the goal of this study was to employ deep-learning cnn to stage disease severity of covid-19 infection on cxr (as opposed to classify covid-19 versus non-covid-19 lung infection). the ground truths were the disease severity scores determined by expert chest radiologists. comparison was made with traditional and transfer learning. this approach has the potential to provide frontline physicians an efficient and accurate means to triage patients, assess risk, allocate resources, as well as to monitor disease progression and treatment response. this approach is timely and urgent because of the anticipated widespread shortage of icu beds and mechanical ventilators. the diagram of patient selection and experimental design is shown in fig 1. this is a retrospective study using publicly available de-identified data. images were downloaded from https:// github.com/ieee8023/covid-chestxray-dataset [27] on mar 30, 2020. the original download contained 250 images of covid-19 and sars. only anterior-posterior and posterior-anterior cxr from covid-19 were included in this study, resulting in final sample size of 131 cxrs from 84 covid-19 positive patients (51 males and 29 females; 4 with missing information). the mean and standard deviation of age was 55.1±14.9 years old for men and 60.1±14.3 years old for women. radiological scoring is widely used to stage disease severity in many lung diseases [4] . to establish the disease severity score, a group of 6 chest radiologists with at least 20 years of experience worked together to reach consensus by evaluating two dozen images of portable cxrs of covid-19 patients from stony brook university hospital (these testing cxrs were not used in this study). our scoring system was adapted from those by warren et al. [28] and wong et al [5] . two board-certified chest radiologists with 20+ years of experience and one radiology resident reviewed image quality and scored the cxr for disease severity using the following criteria based on degree of opacity and geographical extent. the degree of opacity score of 0-3 was assigned to each of the right and left lung as: 0 = no opacity; 1 = ground glass opacity; 2 = consolidation; 3 = white-out. the right and left lung were scored separately and were added together. the geographical extent score of 0-4 was assigned to each of the right and left lung depending on the extent of involvement with ground glass opacity or consolidation: 0 = no involvement; 1 = <25%; 2 = 25-50%; 3 = 50-75%; 4 = >75% involvement. the right and left lung were scored separately and were added together. sum of the two types of scores (0-14) and the product of the two types scores (0-48) were computed. the average of all radiologist scores were used for analysis. a convolutional neural network (fig 2a) [12, 13] with an additional regression layer was utilized to predict disease severity scores of cxr on a graded scale. the images were normalized, converted to rgb images, resized to 64x64 pixel images, and separated into 80% training and 20% testing datasets. this model architecture included convolutional, activation, batch normalization, max pooling, dense layers, and a final dense regression layer to fit the model to predict continuous values. specifically, for convolutional neural networks, deep learning models are well-suited to learn complex functions, and optimize performance of regularization methods. batch normalization layers served to stabilize the learning process by standardizing inputs, and standard rectified linear activation layers for cnns were used to optimize performance. in general, the number of layers and nodes depends heavily on the input data and prediction goals, therefore heuristics were used to generate a standard cnn model for image analysis, the model was then fine-tuned based on performance on the testing dataset. this resulted in three 3x3 kernel sized convolutional layers. l2 regularization and dropout layers, using a parametric probability of 0.3 as determined by analyzing performance results, were used to prevent overfitting. an optimal batch size of 8 was determined by five-fold cross validation, and the model was trained for 110 epochs. the loss function was measured by mean squared error as the model predicted continuous values, and the learning rate was set to 0.001, as it proved to perform more positively than the standard 0.01 rate in early training, and the adam optimizer proved the most successful in minimizing loss. given the limited dataset, a transfer learning method was also explored to optimize prediction. a vgg16 model was loaded with sample weights trained off the imagenet dataset [29] . to be compatible with vgg16, the data was normalized, converted to rgb images, and resized into 224x224 pixel images. the dataset was also split into 80% training and 20% testing. only the top layers were trained to prevent overfitting, and all convolutional layers were frozen. standard vgg16 architecture was utilized, with the exception of the fully connected layers ( fig 2b) . once out of the convoluted layers, the model output was flattened, then passed through three dense layers, including a regression dense layer. for this transfer learning method, cross validation revealed an optimal batch size of 8 and only 10 epochs were necessary to train the deep learning artificial intelligence staging of portable chest radiographs in covid-19 data, as opposed to the 110 needed for the traditional training method. adam optimizer and 0.001 learning rate were similarly employed in this model. the means and standard deviations of the radiologist scores, and the fleiss' kappa inter-rater agreement were calculated. correlation analysis between ai-predicted versus radiologist severity scores were analyzed with slopes, intercepts, correlation coefficients (r 2 ), p-values, and mean absolute errors (mae) reported. results using traditional and transfer learning were compared. all results are reported as means ± standard deviations. a p<0.05 was taken to be statistically significant unless otherwise specified. note that the prediction of graded values precluded receiver operating curve (roc) analysis. the final sample size consisted of 131 cxr from 84 covid-19 positive patients (51m and 29f; 4 missing information). the men were on average 55.1±14.9 years old and women were 60.1±14.3 years old. cxr examples demonstrating a range of severity scores which include extent and opacity score are illustrated in fig 3. cxr of covid-19 positive patients showed hazy opacities and/or airspace consolidation. it has a predominance of bilateral, peripheral deep learning artificial intelligence staging of portable chest radiographs in covid-19 and lower lung zone distribution. the results of three raters are summarized in table 1 . the mean opacity score was 2.52 (0-6), with a standard deviation of 0.25 (9.9%) across three readers. the mean geographic extent score was 3.42 (a range from 0 to 8), with a standard deviation of 0.57 (16.7%) across three readers. the low standard deviations across three raters suggest good agreement. fleiss' kappa for inter-rater agreement was 0.45 (z = 24.4) for opacity score and 0.71 (z = 29.5) for geographic extent score, indicating good to excellent agreement. the mean of sum of scores was 5.84 (0-14) and mean of product of scores was 11.86 (0-48). fig 4 shows the histograms of radiologist scores. the product score was slightly skewed toward lower values because numbers multiplied by a number close to zero tended to be of lower values. the data were separated into 80% training and 20% testing datasets. the loss function decreased and prediction accuracy improved with increasing epochs for both training and validation datasets. for the traditional training method, the loss function typically converged at training epochs of 30-50 epochs, and five-fold validation confirmed optimal hyperparameters of training on 110 epochs. for the transfer learning method employing a vgg16 model, 10 epochs were sufficient to train the model with high predictive ability; more epochs led to overfitting and increased computational time. fig 5 shows the ai-predicted scores versus radiologist severity scores of the "test" dataset, obtained for different types of scores using traditional and transfer learning. overall, there were strong correlations between the ai-predicted and radiologist scores. the slopes, intercepts, r 2 , p values, and mean absolute errors (mae) with traditional and transfer learning are summarized in table 2 . the slopes were generally (but not consistently across different scores) closer to unity, the intercepts were generally closer to zero, and r 2 are generally closer to unity for the transfer learning compared to traditional learning. the top model yielded a r 2 = 0.90 (range: 0.73-0.90 for traditional learning and 0.83-0.90 for transfer learning). the p-values for the transfer learning were consistently smaller than to those of the traditional learning. the maes for the transfer learning are consistently smaller than to those of the traditional learning. the top model yielded a mae of 8.5% (range: 17.2% to 21.3% for traditional learning, and 8.5% to 15.5% for transfer learning). transfer learning generally performed better than those traditional learning. this study tested the hypothesis that deep-learning convolutional neural networks accurately stage disease severity on portable chest x-rays using radiologists' severity scores as ground truths associated with covid-19 lung infection. disease severity scores by three radiologists yielded good inter-rater agreement. ai-predicted scores were highly correlated with radiologist scores. transfer learning improved efficiency and shorten computational time without compromising performance. although there are already over 5500 publications on covid-19 on pubmed (keyword "covid-19" apr 21, 2020), only a few studies have reported ai applications to classify covid-19 versus non-covid-19 lung infection on cxr [19] [20] [21] [22] and ct [23] [24] [25] [26] . all of these studies (including many non-peer-reviewed pre-prints cited here) aimed to classify covid-19 versus non-covid-19 images, not to stage disease severity. a major challenge for table 2 . summary of slope, intercepts, r 2 , and p values of the correlation analysis, and the mean absolute error (mae) of ai-predicted and radiologist scores with traditional and transfer learning. the % was obtained by dividing mae by the corresponding maximum score. table 2 ai in the application to disease diagnosis is that training datasets were limited to a few lung diseases, resulting in low generalizability. amongst these studies, most compare cxr of covid-19 infection with those of bacterial pneumonia and/or normal cxr. more importantly, ai diagnosis of disease in general, and covid-19 infection in particular, based on cxr has poor specificity because many pathologies have similar radiographic appearance as other infections and diseases on cxr. definitive diagnosis of covid-19 infection still requires a test by reverse transcription polymerase chain reaction of a nasopharyngeal or oropharyngeal swab. our study differs from these previous studies in that we used ai of cxr to stage disease severity on a graded scale in positive covid-19 patients. deep-learning ai, specifically a convolutional neural network, is well suited to extract information from cxr and stage disease severity by training using chest radiologist determination of disease severity scores. we evaluated individual and combination scores of severities as it is unknown which type of scores more accurately reflect lung infection disease severity. our findings showed that most scores yielded similar correlation coefficients. one possible explanation is that both the opacity score and geographic extent yielded similar information regarding disease severity. further studies are needed to determine which is superior. it is worth noting that performance by receiveroperating curve (roc) analysis (such as area under the curve (auc), accuracy, sensitivity and specificity) cannot be used for continuous variables. instead, correlation analysis and mean square error analysis were performed. there were strong correlations between the ai-predicted and radiologist scores, with the top model yielding a r 2 = 0.90 and a mean absolute error of 8.5%. this is remarkable performance. the ideal intercept should be zero and ideal slope should be unity. the intercepts were generally close to zero. however, all the slopes were consistently below the line of unity. a possible explanation is that the score distributions were skewed by low score values. as the models attempted to minimize loss, it tended to underestimate higher scores, resulting in slope less than unity. other explanations are possible. further studies are needed. another novelty of our study is that we compared traditional and transfer learning. transfer learning approach is expected to yield good performance with small datasets. most of the performance indices (such as r 2 , p values, and maes) were better with transfer learning compared to traditional learning. the training time with transferring learning was shorter without compromising performance. although radiology reports are informative, they are usually qualitative. a quantitative score of disease severity of portable cxr afforded by ai in an accurate and efficient manner should prove useful in clinical settings of covid-19 circumstance. with cxr being an indispensable imaging tool in the management of covid-19 lung infection, radiologists need an efficient and accurate means to stage disease severity and monitor disease progression and treatment response. furthermore, with the anticipated widespread shortage of icu beds and ventilators, frontline physicians need an efficient and accurate means to triage patients, assess risk and allocate resources. this ai approach to stage disease severity is ideally suited to tackle these challenges associated with the covid-19 pandemics or the like. this proof-of-concept pilot study has several limitations. first, this retrospective study was conducted on an open multi-institutional dataset with a small sample size. this topic is timely and only small dataset are currently available at the time of this writing. these findings need to be replicated on larger sample size with multi-institutional data. it may also be of important to investigate multiple longitudinal time points to evaluate disease progression. second, we did not study the correlation of radiographic score severity with clinical disease severity or clinical outcome which were unavailable. although radiographic score has been widely used as a surrogate marker of disease severity in a variety of lung diseases (see review [4] ), it is unknown at this time if radiographic scoring reflects functional or clinical outcome in the case of covid-19. third, a variety of radiographic scoring systems has been used in other studies [5, 28] , each has its advantages and disadvantages. a more sophisticated score system could be explored. future studies should also consider incorporating non-imaging data (such as demographics, co-morbidities, vitals, blood biomarkers). in conclusion, deep-learning convolutional neural networks accurately stage lung disease severity on portable chest x-rays associated with covid-19 lung infection. this approach has the potential to be used to prognosticate, stage disease severity, monitor disease progression and treatment response, which in turn could inform risk management and resource allocation associated with the covid-19 pandemic. conceptualization: jocelyn zhu, tim q. duong. the continuing 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convolutional neural networks improving neural networks by preventing co-adaptation of feature detectors very deep convolutional networks for large-scale image recognition deep machine learning-a new frontier in artificial intelligence research multi-task transfer learning deep convolutional neural network: application to computer-aided diagnosis of breast cancer on mammograms transfer learning with deep convolutional neural network for liver steatosis assessment in ultrasound images a systematic review of the diagnostic accuracy of artificial intelligence-based computer programs to analyze chest x-rays for pulmonary tuberculosis goldgof gm. finding covid-19 from chest x-rays using deep learning on a small datase a modified deep convolutional neural network for detecting covid-19 and pneumonia from chest x-ray images based on the concatenation of xception and resnet50v2. informatics in medicine unlocked covid-19: automatic detection from x-ray images utilizing transfer learning with convolutional neural networks automatic detection of coronavirus disease classification of covid-19 patients from chest ct images using multiobjective differential evolution-based convolutional neural networks artificial intelligence distinguishes covid-19 from community acquired pneumonia on chest ct covid-net: a tailored deep convolutional neural network design for detection of covid-19 cases from covid-19 image data collection severity scoring of lung oedema on the chest radiograph is associated with clinical outcomes in ards very deep convolutional networks for large-scale image recognition key: cord-315531-2gc2dc46 authors: mcgarvey, peter b.; huang, hongzhan; mazumder, raja; zhang, jian; chen, yongxing; zhang, chengdong; cammer, stephen; will, rebecca; odle, margie; sobral, bruno; moore, margaret; wu, cathy h. title: systems integration of biodefense omics data for analysis of pathogen-host interactions and identification of potential targets date: 2009-09-25 journal: plos one doi: 10.1371/journal.pone.0007162 sha: doc_id: 315531 cord_uid: 2gc2dc46 the niaid (national institute for allergy and infectious diseases) biodefense proteomics program aims to identify targets for potential vaccines, therapeutics, and diagnostics for agents of concern in bioterrorism, including bacterial, parasitic, and viral pathogens. the program includes seven proteomics research centers, generating diverse types of pathogen-host data, including mass spectrometry, microarray transcriptional profiles, protein interactions, protein structures and biological reagents. the biodefense resource center (www.proteomicsresource.org) has developed a bioinformatics framework, employing a protein-centric approach to integrate and support mining and analysis of the large and heterogeneous data. underlying this approach is a data warehouse with comprehensive protein + gene identifier and name mappings and annotations extracted from over 100 molecular databases. value-added annotations are provided for key proteins from experimental findings using controlled vocabulary. the availability of pathogen and host omics data in an integrated framework allows global analysis of the data and comparisons across different experiments and organisms, as illustrated in several case studies presented here. (1) the identification of a hypothetical protein with differential gene and protein expressions in two host systems (mouse macrophage and human hela cells) infected by different bacterial (bacillus anthracis and salmonella typhimurium) and viral (orthopox) pathogens suggesting that this protein can be prioritized for additional analysis and functional characterization. (2) the analysis of a vaccinia-human protein interaction network supplemented with protein accumulation levels led to the identification of human keratin, type ii cytoskeletal 4 protein as a potential therapeutic target. (3) comparison of complete genomes from pathogenic variants coupled with experimental information on complete proteomes allowed the identification and prioritization of ten potential diagnostic targets from bacillus anthracis. the integrative analysis across data sets from multiple centers can reveal potential functional significance and hidden relationships between pathogen and host proteins, thereby providing a systems approach to basic understanding of pathogenicity and target identification. the niaid (national institute of allergy and infectious diseases) biodefense proteomics program, established in 2004, aims to characterize the pathogen and host cell proteome by identifying proteins associated with the biology of microbes, mechanisms of microbial pathogenesis and host responses to infection, thereby facilitating the discovery of target genes or proteins as potential candidates for the next generation of vaccines, therapeutics, and diagnostics [1] . the program includes seven proteomics research centers (prcs) conducting state-of-the-art high-throughput research on pathogens of concern in biodefense and emerging/ reemerging infectious diseases, as well as a biodefense resource center for public dissemination of the pathogen and host data, biological reagents, protocols, and other project deliverables. the prcs work on many different organisms, covering bacterial pathogens (bacillus anthracis, brucella abortus, francisella tularensis, salmonella typhi, s. typhimurium, vibrio cholerae, yersinia pestis), eukaryotic parasites (cryptosporidium parvum, toxoplasma gondii), and viral pathogens (monkeypox, sars-cov, vaccinia). the centers have generated a heterogeneous set of experimental data using various technologies loosely defined as proteomic, but encompassing genomic, structural, immunology and protein interaction technologies, as well as more standard cell and molecular biology techniques used to validate potential targets identified via high-throughput methods. in addition to data, the prcs have provided biological reagents such as clones, antibodies and engineered bacterial strains, other deliverables include standard operating procedures (sops) and new technologies such as instrumental methods and software tools and finally publications related to all of these activities. consequently, there were a number of unique challenges facing the resource center: (i) how to coordinate with the seven prcs with various pathogens, technologies, processes, and data types; (ii) how to provide seamless integration of three institutions that make up the resource center; and (iii) how to provide timely and effective dissemination of newly discovered information to the user community. in particular, due to the breadth of the program, the potential user community is quite broad, from technology or informatics experts who may want to reanalyze the data or develop better algorithms, to a wide group of biomedical scientists who are interested in mining the data for their own studies or just finding new information on a protein or gene of interest quickly and easily. accordingly, we developed a set of functional requirements early in the biodefense resource center development: (i) to implement a center-specific submission protocol and data release plan for timely dissemination, (ii) to promote data interoperability, adopting common standards (such as hupo proteomic standards initiative [2, 3, 4] ), defining a core set of metadata with mapping to controlled vocabularies and ontologies, recommending preferred ids for gene/ protein mapping, and (iii) to provide value-added annotation to capture key findings and integration of the data with related resources for functional interpretation of the data. available online at http://proteomicsresource.org, the architecture, initial content and general features of the biodefense proteomics resource were briefly described elsewhere [5] . a breakdown of the resources content by organism, prc and other criteria can be seen at: http://www. proteomicsresource.org/resources/catalog.aspx. tutorials and help are provided on the website: http://www.proteomicsresource. org/resources/tutorials.aspx , http://proteininformationresource. org/pirwww/support/help.shtml#30 the objective of this study is to provide a systems approach to the study of pathogen-host interactions, connecting the various types of experimental data on genomics, proteomics and host-pathogen interactions with information on pathways, regulatory networks, literature, functional annotation and experimental methods. having most of this information accessible in one place can facilitate knowledge discovery and modeling of biological systems. like many problems in data integration, it is easy to know the general outline of what we want, but often much harder to implement and navigate the information, especially if the original data crosses disciplinary, laboratory and institutional boundaries. here we describe in detail a protein-centric approach for systems integration of such a large and heterogeneous set of data from the niaid biodefense proteomics program, and present scientific case studies to illustrate its application to facilitate the basic understanding of pathogen-host interactions and for the identification of potential candidates for therapeutic or diagnostic targets. several scientific use cases are presented that illustrate how one can search varied experimental data from different laboratories and even ones researching different infectious organism and their hosts to make potentially useful connections that could lead to new hypotheses and discoveries. based on the functional requirements of the resource center, we developed a bioinformatics infrastructure for integration of prc deliverables ( figure 1 ). in our workflow, multiple data types from prcs are submitted to the center using a data submission protocol and standard exchange format, with the metadata using controlled vocabulary whenever possible. for functional interpretation of the data, we then map the gene and protein data based on identifier (id) mapping or if necessary using peptide or sequence mapping to proteins in our data warehouse described below. all of the databases, along with information on the prcs and organisms under study are listed in the proteomics catalog accessible from the web portal. the key design principal in the resource center is protein-centric data integration. here the diverse experimental data are integrated and presented in a protein-centric manner where information is queried and presented via common proteins and connected to experimental data and the network of protein attributes, including information on the encoding genes, protein families, pathways, functions and more. in practice a protein-centric approach works well as proteins occupy a middle ground molecularly between gene and transcript information and higher levels of molecular and cellular structure and organization. proteins are often the functional molecules in biological processes described by pathways, molecular interactions and other networks. protein families, in turn, have proven to be invaluable in studying evolution and for inferring and transferring functional annotation across species. underlying the protein-centric data integration is a data warehouse called the master protein directory (mpd) where key information is extracted from the primary data and combined for rapid search, display and analysis capabilities. the mpd is built on the data and capabilities of iproclass [6] a warehouse of protein information, which in turn is built around uniprotkb [7] but supplemented with additional sequences from gene models in refseq [6] and ensembl [7] and additional annotation and literature from other curated data resources such as model organism databases [8, 9, 10, 11, 12, 13] and generif [14] . the biodefense data are essentially additional data fields added to a subset of iproclass entries to create the mpd. currently the mpd defines and supports information from the following types of data produced by the prcs: mass spectrometry, microarray, clones, protein interaction, and protein structure. more data types or attributes may be added in the future if needed. supplemental table s1 shows the common and unique fields used in the mpd for each data type. an advantage of the data warehouse design is that, if needed, additional fields can be extracted from the primary data and easily added as new attributes without greatly altering the existing database design or query mechanisms. the mpd data is stored in an oracle database along with iproclass data. the mpd including the website and ftp files is updated every 3 weeks in conjunction with iproclass or whenever new prc data is released. the various proteomics research centers all used different sources and identifiers for the nucleotide and protein sequences in their analysis pipelines and occasionally would change sources depending on the experiment. this is a common problem encountered when attempting to combine data across research laboratories unless identical sequence databases, processes, platforms and organism names are used. examples of database identifiers used include genbank/embl/ddbj accessions and locus tags, unigene accessions, refseq accessions, ipi accessions, ncbi gi numbers and ids unique to a sequencing center or organism-specific database. the first step was to map all experimental results to a common representation of a protein. this was achieved by mapping all protein and gene ids and names to iproclass proteins. the majority of the mapping using ids from public resources was done using mapping services and tables provide on the protein information resource (pir) web site (http:// proteininformationresource.org/pirwww/search/idmapping.shtml) and ftp site (ftp://ftp.pir.georgetown.edu/databases/iproclass/). however, some mapping problems needed to be addressed either by automated rules, direct sequence comparisons or manual analysis and annotation. mapping difficulties fell into 4 categories: 1) one-to-many mappings: a common problem, especially when eukaryotic host proteins derived from alternate splicing or viral polyproteins are involved. uniprotkb usually merges information on alternate splice forms or polyproteins, which helped minimize this problem for our purposes, but in cases where multiple mappings exist, we selected as most informative the entry in the manually reviewed uniprotkb/ swissprot section; if no swissprot entry was found, the longer sequence in uniprotkb/trembl section was selected. users could always find the alternate mappings via the iproclass related sequences link on the mpd entry page to precompiled blast results on all iproclass sequences. 2) retired sequences: genomic sequences from databases such as refseq, ipi, or unigene are not static and, as information changes, some gene predictions and translations are retired with each new build. retired sequences often required manual mapping by a curator to match original gene or peptide results to current protein sequences. primarily uniparc (uniprot sequence archive) [15] was used for this purpose. 3) protein sequences not available in iproclass or any public repository: this occurred most often with toxoplasma gondii whose genome sequencing was still in progress and stable builds were not yet available. however, the problem also occurred in some well-characterized organisms like vibrio cholera and bacillus anthracis. several data sets contained information on annotated but not translated pseudogenes. in the case of vibrio cholera, 21 of 48 pseudogenes cloned and sequenced by the harvard institute of proteomics did not contain the annotated point mutation or frame shift and appeared to produce full-length proteins [16] . in the case of bacillus anthracis, microarrays containing probes for 82 of 192 annotated pseudogenes also showed significant changes in rna expression in response to infection or other treatments [17, 18, 19] . in these cases, new database entries were created in the mpd to house the results. 4) alternate species or strain representations: several experimental data sets reported sequence identifiers for strains or variants other than the one used in the experimental sample. this is not an uncommon situation as the genetically most characterized variant is often an attenuated laboratory strain while the more virulent strains are either not yet sequenced or the sequence is of lower quality. often microarray chips or mass spectrometry databases are designed using the best available sequence from the research strain, yet then use rna and protein samples from another similar virulent strain. the question here was what organism strain to map to and represent on our website: the strain the rna or protein the sample came from, or alternatively, the strain that matched the identifier and sequence used in the research to detect the rna or protein. for the mpd we chose to map to the sequence identifier reported in the data files and related publications, with the additional virulent strain information noted in the results summary. data mining design goals. in consultation with niaid and prcs within the project's interoperability working group, we developed 3 goals for data mining. 1) all project data and other deliverables should be available via browsing and simple keyword searches. 2) the data and information provided by the resource should be sufficient to allow a skilled researcher to download and reanalyze or mine the data for additional information. 3) our target user was a biomedical scientist not expert in the technology used to produce the data or in bioinformatics, thus the data, procedures, publications and general results and conclusions of an analysis should be relatively easy to find on the project website for someone not familiar with the details of the particular technologies used to generate it. to allow both simple keyword searches and also boolean searches of the project data, we did the following: 1) we included in the mpd only ''validated'' results that were determined by the research centers to be significant using their methods. to do otherwise would confuse users not familiar with the technology and how results are filtered. results that fell below the significance threshold used by the research center were made available via download of data sets at the ftp site. 2) ''raw'' unprocessed machine specific data would be stored by the prc and available on request. 3) to facilitate and simplify searches across laboratories and data types, we omitted most data type or analysis specific numerical values and statistics from the general mpd search and display. these numerical values are usually platform, laboratory and method dependent and cannot easily be used to compare across datasets so including them might be confusing to users. instead, we focused on providing simple, yet powerful, queries of experimental summaries where a user can query if a gene/protein was presented in the results and, in some cases, if it showed a reported increase or decrease in expression or accumulation based on the prc's criteria. once a set of proteins of interest is identified, a user can then drill down to view the specific experimental values and methods employed to generate the particular dataset. links to details in the publications are provided and full data are available via ftp. since all protein attributes are included as search options, one can query beyond simply protein names, accessions or project data and search pathways, protein families, gene ontology (go) terms, database cross-references and many other attributes, providing many powerful options to the users. to provide a robust text search for the website, we used the pir text indexing system [20] in which over 100 text fields and unique identifiers from the mpd database are indexed using callable personal librarian (cpl) [21] which supports fast exact text search, substring & wildcard text search, range search and boolean searches. entry indexing and retrieval is supported by oracle. i: unstructured keyword search. a simple keyword search was implemented on every page of the resource's website. this searches all fields in the mpd. to further facilitate searches, the protein name field is supplemented by also searching the biothesaurus [10] containing all gene and protein name synonyms and textual variants for each protein from over 35 data sources. in addition, the default option searches all text from the pubmed abstract for all project publications, an abstract of each technology and all text in sops. the text indexed for publications, technologies and sops was annotated with additional standard keywords to facilitate searches. figure 2 shows the results of a simple keyword search with hits for proteins, reagents, publications, technologies and sops. ii: structured text search. the mpd database contains over 100 fields derived from iproclass and proteomics research center's data. currently 75 of these fields are available for individual searches and can be combined with boolean operators as seen in some of the use case examples in this paper. the protein-centric search results are presented in a customizable tabular format where users can add or delete columns. currently 62 fields can be customized. the tabular display has two modes: 1) a default mode which displays fields common to all the supported data types and 2) a data type specific mode which restricts the results to a particular data type and displays fields specific to that data type. see supplemental table s1 for a list of data type specific fields. additional filters for proteomic research center and organism are available as pull down menus to aid browsing and viewing the results of queries. examples of these functions are illustrated in the scientific use cases below and in help pages and tutorials available on the website. http:// www.proteomicsresource.org/resources/tutorials.aspx, http:// proteininformationresource.org/pirwww/support/help.shtml#30. the resource currently contains information on 35,112 proteins from 58 datasets, 35,819 reagents, 75 sops, 31 technologies and 88 manuscripts. table 1 shows statistics on proteins in the mpd. currently ,30% of the proteins are uncharacterized in that they are called either ''uncharacterized'' or ''hypothetical'' and have no other functional annotations or functional domains. of these uncharacterized proteins, ,85% have experimental data, such as mass spectrometry, microarray, or protein interactions, associated with them. the remaining 15% of uncharacterized proteins are available as full length clones for further research. though the program is focused on pathogen proteins, about 32% of the proteins are host proteins from mouse or human, as cell lines or tissue samples from both these organisms were used as infection models for multiple pathogens. simple keyword search. due to the popularity of internet searches, support of unstructured keyword queries, even for structured data, has become critical for any web site. to support this feature, the default protein-centric search returns results for all project deliverables, data, reagents, protocols, technologies and publications. an example is shown in figure 2 where a single keyword search ''bacillus anthracis'' finds 10,988 pathogen and host proteins with mass spec, microarray or protein interaction data and also 13,251 reagents in the master reagent directory (mostly orf and y2h clones and mutant bacterial strains of bacillus anthracis), 2 sops, 16 project publications and 2 technologies. the matched fields column allows users to refine their queries and construct simple boolean searches. example i: integrative analysis. structured fields allow boolean queries across organisms, data types and laboratories. figure 3 shows a query where we make use of the controlled vocabulary in the ''expression condition'' field and searched for common host proteins detected in studies of bacillus anthracis and salmonella typhimurium infection in mouse macrophage cell lines. currently 222 proteins meet these criteria, mostly from mass spectrometry studies by pnnl and the university of michigan; however, if we further restrict the search to include only those also detected in microarray experiments, we find 16 proteins with mass spec data from s. typhimurium infections at one research center, and mass spec and microarray studies done using b. anthracis at another research center. from the customizable results display shown in figure 3 we can view summary information on the proteins detected. full details on the protein and individual experimental results are available via links [5] . some benefits of the protein-centric mapping approach are visible in the default display. 1) minimizing redundancy, the column prc id shows the different identifiers from different databases used by the research centers. in the case of one protein q9wua3/k6pp_mouse 6-phosphofructokinase type c (ec 2.7.1.11), a total of 9 identifiers from 4 different databases (unigene, refseq, ipi, nr) were reported in the research results that all represent either the gene or protein sequence for this single mouse protein. 2) discovery of additional experimental information from other studies. fifteen of the sixteen proteins found in the query also have mass spec data from caprion proteomics, as indicated in the 'experiment' column by caprion_05, 06 and _10. caprion is studying brucella abortus and using a similar mouse macrophage model. currently, only the data for uninfected macrophages is available from caprion. additional data on brucella and mouse proteins from bacterial infected macrophages should be included in future releases. from the results display, one can follow links to view the iproclass protein report with executive summaries of the results from the prcs and information collected from over 100 public resources or drill down to view the specific peptides or expression values seen in these studies, read publications and methods about the experiments or download the data for additional analysis. with a comprehensive protein data warehouse, one can also broaden the search for relevant data and information from related organisms using protein cluster or family information. in figure 4 we illustrate this by selecting one protein, k1033_mouse, an uncharacterized protein seen in three datasets from infected mouse macrophages. using the uniref90 cluster id [22] to query for all proteins with at least 90% identity and no gaps, we find the human homolog k1033_human was detected in hela cells infected with vaccinia and monkypox virus. if we do a batch retrieval using uniref90 ids from all 16 original mouse proteins, we find 12 human homologs were also detected in studies of orthopox infection (not shown). the identification of a hypothetical protein with differential gene and protein expressions in two host systems (mouse macrophage and hela cells) infected by different bacterial (bacillus anthracis and salmonella typhimurium) and viral (orthopox) pathogens suggest that this protein should be prioritized for additional analysis and functional characterization. [23, 24, 25] . although different laboratories use different sample preparation, detection and analysis techniques making some direct comparisons difficult, having the data together in one place allows queries and comparisons between proteins and gene sets to be combined and additional analysis undertaken. in this example, data from different labs and data types are combined for further analysis. a query of the mpd on data type = ''interaction'' and organism name = ''virus'' finds 33 vaccinia virus proteins with interactions with human proteins determined by myriad genetics. browsing the results display shows that 25 of the proteins were also seen in mass spec work published by pnnl [26] . by combining the two experimental data sets experiment = ''myriad_05'' and ''pnnl_ms_09'', we find 83 virus and human proteins with both mass spec and interaction data from each laboratory. further investigation into the experimental details shows that a protein interaction network was determined by myriad genetics using a yeast-two hybrid assay to screen viral bait proteins against a library of human prey proteins cloned from different tissues. the mass spec data from pnnl was obtained from viral preparations isolated from infected human hela cells. the work from pnnl contained quantitative information in the form of spectral counts and accurate mass tag [27] intensities, downloadable from the project ftp site. we combined all the vaccinia plus human interaction data with peptide counts for each protein and visualized the results using cytoscape [28, 29] . the complete network of results is shown in figure 5a . various methods have been tried and compared for filtering large intra-species interaction networks to limit false positives and to select the biologically relevant interactions [30, 31, 32, 33, 34, 35] . relatively little has yet been done for inter-species pathogen-host networks. several common factors that have proved useful in other studies are 1) evaluating network hubs with many interactions and 2) using correlations between interacting pairs with similar gene expression patterns. for this small interactome, we looked for relatively abundant proteins associated with multiple interactions. we identified a single human protein interacting with three viral proteins, three being the largest number of viral interactions seen with a single host protein in this data set. the interactions are shown in figure 5b . the host protein keratin, type ii cytoskeletal 4 (p19013) interacts with three viral proteins (p11258 -14 kda fusion protein, a27l; q805h7 -chemokine-binding protein c23/b29; p17362 -protein c6l). the 14 kda fusion protein a27l is the most abundant protein seen in this data set and participates in virus penetration at during cell fusion [36, 37] . a27l facilitates initial attachment to cells by binding to glycosaminoglycans [38] . a27l is found in all orthopoxviruses and has no cellular or entomopoxvirus homologs. additional viral proteins involved in attachment (d8l, h3l) [39, 40] and fusion (f9l, i2l) [41, 42] were not observed. the chemokine-binding protein c23l belongs to a family of poxvirus chemokine-binding proteins that mimic the chemokine response and prevent activation and chemotaxis of leukocytes [43, 44] . protein c6l belongs to a family of poxvirus paralogs that may function as toll-like receptor inhibitors based on homology to a52r [45, 46, 47] . thus, this protein may modulate toll/il-1r signaling, resulting in a diminished host immune response and enhancing viral survival. p19013 -keratin, type ii cytoskeletal 4, the host protein, has tissue specificity in the suprabasal layer of the stratified epithelium of the esophagus, exocervix, vagina, mouth and lingual mucosa, and in cells and cell clusters in the mucosa and serous gland ducts of the esophageal submucosa [48] . transgenic knockout mice have shown keratin, type ii cytoskeletal 4 to play an important role in maintaining normal epithelial tissue structure [49] . keratin queries on cluster or family information can discover related information across laboratories and host pathogen systems. using the uniref90 cluster id for k1033_mouse, identified in figure 3 , to query for all proteins with at least 90% identity, we find the human homolog k1033_human was detected in hela cells infected with vaccinia and monkypox virus [12] . doi:10.1371/journal.pone.0007162.g004 4 in human saliva has been shown to interact with the protein srr-1 localized on the surface of streptococcus agalactiae and to play a critical role in colonization of this bacterial pathogen [50, 51] . little is known about the mechanisms by which poxviruses attach to and enter host cells. no receptor for virion attachment on the host cell surface has been found. poxvirus infection can occur through interaction with human as well as mice airway epithelia, [52, 53] we propose that the protein interactions outlined above may represent some of the initial interactions between host and pathogen. thus they represent potential therapeutic targets for further investigation. this is the first report describing the interaction of a poxvirus protein with a host keratin, type ii cytoskeletal 4 protein. proteins. unequivocal identification of pathogens is important so that adequate counter measures can be taken. currently over 700 pathogenic and non-pathogenic bacteria have been completely sequenced. the availability of sequence data allows identification of proteins that are unique at different taxonomic levels, thus providing a means to begin to distinguish pathogenic from non-pathogenic species. however, if the initial screening depends on sequence data alone, the list of potential targets for laboratory validation can be relatively long; by supplementing sequence results with experimental data one can prioritize the target list for validation in the laboratory. we used such an approach by computationally screening potential targets using cupid [54] , prc data and other computational means to produce a list of potential targets. identifying species-specific proteins can be done with confidence when multiple species and strains have been sequenced as is the case with bacillus anthracis. the approach relies on the fact that if a gene is conserved over time within multiple strains it gives confidence they will not be lost in the near future and hence are ideal for diagnostic targets. these ''core unique'' proteins have related sequences in all selected organisms (in this case all available strains of bacillus anthracis) but not in other related organisms. an initial total of 327 proteins unique to the bacillus anthracis proteome were identified using the cupid and uniprotkb version 13.0. (bacillus anthracis strain ames isolate porton, bacillus anthracis strain ames ancestor, bacillus anthracis strain sterne were compared to twelve other genomes in the bacillus genus). the two closest relatives of bacillus anthracis as determined by cupid are bacillus thuringiensis and bacillus cereus. the species most closely related to the selected organism is based on the best blast hits of its entire proteome [54] . one needs to be careful in choosing the diagnostic targets that these two non-pathogenic organisms are not being detected. the initial list of 327 was refined to identify ''core unique'' proteins that are 100 amino acids or more in length. the 100 residue cutoff was used to ensure that the target list consisted of proteins that are real (short proteins might not be real) and are unique, as identification of homologs for short proteins is not trivial [55] . this resulted in a list of 21 ''core unique'' proteins in the pathogenic strains. it is possible that the proteins found may have homologs in other organisms which were undetected by cupid because the genes were not annotated as open reading frames to confirm their uniqueness, the 21 proteins were screened for significant regions of similarity at the dna level (either pseudogenes or unannotated genes) using tblastn against the ncbi nr database. using ncbi's nr which is produced independently of similar, but not identical, sources as iproclass also helps assure no sequences were missing from our warehouse. this additional analysis resulted in a total of 10 bacillus anthracis specific proteins proposed as high-quality targets for development of diagnostic probes. we then supplemented this information with data from the prc projects and master protein directory to create a matrix of information ( figure 6 ). six of the ten targets have data from the university of michigan prc showing that they were differentially expressed in published microarray experiments. nine of the ten are available as clones produced by the harvard institute of proteomics. a search of all the microarray data from the university of michigan (using http://proteinbank.vbi.vt.edu/ proteinbank/p/search/searchproteins.dll) showed that the four proteins not differentially expressed (listed as clones only in figure 6 ) were still constitutively expressed well above background in all studies (not shown). either the proteins or the dna coding for these proteins can be used to develop and test pathogen detection systems. all the ''core unique'' proteins detected in this study lacked meaningful functional annotation (i.e., were annotated simply as ''uncharacterized protein''), which is not surprising as such unique proteins are not easy to characterize. one protein identified as a target is a remnant of a prophage protein. such proteins are well known to be related to virulence [56] . another protein is from the pxo2 plasmid. a similar approach was taken for salmonella species using cupid and public prc data and several candidate diagnostic proteins are currently being validated in the laboratory (data not shown). a systems approach to biology or medicine requires the sharing, integration and navigation of large and diverse experimental data sets to develop the models and hypotheses required to make new discoveries and to develop new treatments. to date this has most often been done with selected research data or within an institution or program where common instrumentation and methods make standardization of experimental practices and data management easier to achieve [57, 58, 59] . alternative approaches require a reanalysis of all the data by a common methodology as has been done in some data repositories [60, 61] or assigning some common statistical metric to all data of a certain type to allow functional coupling [62] . these approaches are all potentially useful, but practically difficult to achieve on a large scale with heterogeneous data. the protein-centric approach we employed is a relatively simple, yet powerful and practical, approach to integrate and figure 6 . ten potential diagnostic markers for pathogenic strains of bacillus anthracis. the prioritized protein list was obtained by computationally screening potential targets using cupid [54] , prc data and other computational means described in the text. doi:10.1371/journal.pone.0007162.g006 navigate diverse sets of omics data in a manner useful for systems biology. proteins are often the biologically functional elements in cellular networks; thus, many types of data can be mapped to and through proteins as a common biological object. the lightweight data warehouse approach used for the mpd proved useful in practice, especially with large datasets as its simple design and schema allows greater flexibility to add new data types and to modify search and analysis capabilities. similar lightweight approaches and schemas designed to optimize queries have been shown useful in integration of genomic data [63, 64] . the main drawback of this approach is that the warehouse does not contain all the data. however, this is rarely a problem if the data are available in some other data resource optimized for that particular data type and if some upfront analysis of the user's needs for query and analysis options is performed. for example, our use case analysis suggested that for microarray and mass spectrometry data, individual raw intensities, machine-specific parameters and most calculated numerical values were not required for general queries and analysis across the combined data as these values were only comparable between the particular analysis performed in one lab. as a result, most numerical values were not included in the mpd for the default search but are accessible for display via hyperlinks to our protein data center or ftp site. however, if a new attribute appear or users request searches on a particular value omitted from the warehouse, adding it is a relatively simple matter of adding new data columns. for instance, in example ii our combination and analysis of mass spectrometry and protein interaction data, we could include peptide counts directly in the mpd for immediate download instead of retrieving them from the ftp files. of course, no one approach can be perfect, as in biology and research there always seems to be exceptions and new data and multiple approaches need to be accommodated. efforts to standardize reporting requirements, vocabularies and develop common xml data formats for sharing data are welcome and can greatly ease the transfer and automated processing of a particular data type. however the current standards do not necessarily guarantee integration as the problems of reconciling gene and protein identifiers as well as differences in experimental methodology remain. we investigated and employed a few common data standards and ontologies in developing the biodefense proteomics resource. we provided some data using mzdata [65] and mage-ml [66] but also provided original dataspecific text files for download. we found that several ontologies to describe experimental methods were useful but incomplete and focused on higher eukaryotes and thus did not yet contain terms needed for microbial pathogens. most useful was the gene ontology [67] which has been widely adopted to annotate and classify large scale results and can be used for searching and classification in the mpd. here we have presented some unique examples to illustrate benefits, as well as the difficulties, associated with integration of a very diverse set of omics research data across different data types, laboratories and organisms. we illustrated with three examples how potential therapeutic and diagnostic targets can be identified from integrated data applying relatively simple and established tools and techniques. we continue to focus on data integration to allow biologists to find relevant data sets for further detailed analysis using the approaches and tools of their choice. in general the analysis of diverse omics data is an area of active research and a number of useful tools are under active development including cytoscape [28] , bioconductor [68] and galaxy [69] . in the future a more seamless integration between data repositories and analysis tools such as these would be the most useful approach to add additional analysis options for integrated data. author contributions building integrated approaches for the proteomics of complex, dynamic systems: nih programs in technology and infrastructure development the hupo proteomics standards initiativeeasing communication and minimizing data loss in a changing world the minimum information about a proteomics experiment (miape) the hupo proteomics standards initiative-overcoming the fragmentation of proteomics data an emerging cyberinfrastructure for biodefense pathogen and pathogen-host data the iproclass integrated database for protein functional analysis the universal protein resource (uniprot) ) dictybase, the model organism database for dictyostelium discoideum the arabidopsis information resource (tair): a model organism database providing a centralized, curated gateway to arabidopsis biology, research materials and community the mouse genome database (mgd): the model organism database for the laboratory mouse the yeast proteome database (ypd) and caenorhabditis elegans proteome database (wormpd): comprehensive resources for the organization and comparison of model organism protein information nucleic acids research, database issue the zebrafish information network: the zebrafish model organism database generif quality assurance as summary revision uniprot archive production and sequence validation of a complete full length orf collection for the pathogenic bacterium vibrio cholerae transcriptional profiling of the bacillus anthracis life cycle in vitro and an implied model for regulation of spore formation transcriptional profiling of bacillus anthracis during infection of host macrophages the global transcriptional responses of bacillus anthracis sterne (34f2) and a delta soda1 mutant to paraquat reveal metal ion homeostasis imbalances during endogenous superoxide stress the pir integrated protein databases and data retrieval system callable personal librarian (cpl) (version 6.5). callable personal librarian (cpl) (version 65) uniref: comprehensive and non-redundant uniprot reference clusters a data integration methodology for systems biology: experimental verification from bytes to bedside: data integration and computational biology for translational cancer research integration of metabolomic and proteomic phenotypes: analysis of data covariance dissects starch and rfo metabolism from low and high temperature compensation response in arabidopsis thaliana comparative proteomics of human monkeypox and vaccinia intracellular mature and extracellular enveloped virions the utility of accurate mass and lc elution time information in the analysis of complex proteomes cytoscape: a software environment for integrated models of biomolecular interaction networks exploring biological networks with cytoscape software comparative assessment of large-scale data sets of protein-protein interactions a direct comparison of protein interaction confidence assignment schemes a relationship between gene expression and protein interactions on the proteome scale: analysis of the bacteriophage t7 and the yeast saccharomyces cerevisiae protein interactions: two methods for assessment of the reliability of high throughput observations assessment of the reliability of protein-protein interactions and protein function prediction gaining confidence in high-throughput protein interaction networks vaccinia virus induces cell fusion at acid ph and this activity is mediated by the n-terminus of the 14-kda virus envelope protein a 14k envelope protein of vaccinia virus with an important role in virus-host cell interactions is altered during virus persistence and determines the plaque size phenotype of the virus the oligomeric structure of vaccinia viral envelope protein a27l is essential for binding to heparin and heparan sulfates on cell surfaces: a structural and functional approach using site-specific mutagenesis vaccinia virus envelope h3l protein binds to cell surface heparan sulfate and is important for intracellular mature virion morphogenesis and virus infection in vitro and in vivo vaccinia virus envelope d8l protein binds to cell surface chondroitin sulfate and mediates the adsorption of intracellular mature virions to cells vaccinia virus f9 virion membrane protein is required for entry but not virus assembly, in contrast to the related l1 protein the vaccinia virus gene i2l encodes a membrane protein with an essential role in virion entry blockade of chemokine activity by a soluble chemokine binding protein from vaccinia virus monkeypox virus viral chemokine inhibitor (mpv vcci), a potent inhibitor of rhesus macrophage inflammatory 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toll-like receptor 4 in pulmonary vaccinia infection computational identification of strain-, species-and genus-specific proteins a search method for homologs of small proteins. ubiquitin-like proteins in prokaryotic cells? the impact of prophages on bacterial chromosomes systems biology at the institute for systems biology a data integration methodology for systems biology integrated genomic and proteomic analyses of gene expression in mammalian cells open source system for analyzing, validating, and storing protein identification data peptideatlas: a resource for target selection for emerging targeted proteomics workflows global networks of functional coupling in eukaryotes from comprehensive data integration ensmart: a generic system for fast and flexible access to biological data biomart central portal-unified access to biological data further steps in standardisation design and implementation of microarray gene expression markup language gene ontology: tool for the unification of biology. the gene ontology consortium bioconductor: open software development for computational biology and bioinformatics galaxy: a platform for interactive large-scale genome analysis key: cord-348807-9xxc5hyl authors: cuomo, raphael e.; purushothaman, vidya; li, jiawei; cai, mingxiang; mackey, timothy k. title: sub-national longitudinal and geospatial analysis of covid-19 tweets date: 2020-10-28 journal: plos one doi: 10.1371/journal.pone.0241330 sha: doc_id: 348807 cord_uid: 9xxc5hyl objectives: according to current reporting, the number of active coronavirus disease 2019 (covid-19) infections is not evenly distributed, both spatially and temporally. reported covid-19 infections may not have properly conveyed the full extent of attention to the pandemic. furthermore, infection metrics are unlikely to illustrate the full scope of negative consequences of the pandemic and its associated risk to communities. methods: in an effort to better understand the impacts of covid-19, we concurrently assessed the geospatial and longitudinal distributions of twitter messages about covid-19 which were posted between march 3rd and april 13th and compared these results with the number of confirmed cases reported for sub-national levels of the united states. geospatial hot spot analysis was also conducted to detect geographic areas that might be at elevated risk of spread based on both volume of tweets and number of reported cases. results: statistically significant aberrations of high numbers of tweets were detected in approximately one-third of us states, most of which had relatively high proportions of rural inhabitants. geospatial trends toward becoming hotspots for tweets related to covid-19 were observed for specific rural states in the united states. discussion: population-adjusted results indicate that rural areas in the u.s. may not have engaged with the covid-19 topic until later stages of an outbreak. future studies should explore how this dynamic can inform future outbreak communication and health promotion. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 between the beginning of march 2020 and the end of april 2020, the number of recorded coronavirus disease 2019 (covid-19) cases grew from under 100,000 to over 3 million worldwide [1] . globally, growth has not been consistent, with recorded daily cases exhibiting noticeable but relatively muted spread in january, followed by low transmission in february, rapid daily increases in march, and high but sustained levels of new cases in april [2] . similarly, the spread of covid-19 has not had even geospatial distribution, with noticeably high levels of recorded cases in china, western europe, and new york; and relatively low levels of recorded cases in sub-saharan africa and central america [1] , though the exact global burden of covid-19 remains unknown. current evidence suggests that covid-19 has an appreciable case-fatality rate when compared to other diseases of similar epidemiological characteristics and infectiousness [3] . however, establishing even basic epidemiological data during a health emergency can be challenging, particularly in the context of lack of sufficient access to testing and diagnostic capacity, overburdened health systems, and variability in clinical protocols for testing, contact tracing, and other public health measures [4] . a commonly-employed strategy to reduce deaths from covid-19 when faced with unevenness in disease reporting is to prevent especially susceptible individuals, such as the elderly and immunocompromised [5] , from contracting the virus by implementing social distancing guidelines [6] . despite the rapid increase in number of deaths since the beginning of the covid-19 pandemic, there has existed wide variability in the seriousness with which governments and local communities have pursued, implemented and enforced social distancing guidelines [7] . concordantly, there may exist appreciable variability in the level of attention that local communities may have devoted to covid-19 and associated prevention measures. in addition, the covid-19 pandemic has numerous ramifications which extend beyond physical health, including those which are sociocultural, economic, and psychological [8] . individuals with certain mental health conditions or occupations may be adversely impacted by social distancing measures, though they may never contract covid-19. conversely, some individuals who become infected with covid-19 may be entirely asymptomatic [9, 10] . therefore, one additional reported case may not indicate any negative utility (if that person were asymptomatic), and case counts may not reflect all negative impacts of covid-19 (including psychological and economic impacts). this lack of comprehensive covid-19 burden is likely difficult to estimate, hence, necessitating alternative methodological approaches to assess interactions between outbreak trends from both a longitudinal and localized context. the use of non-traditional forms of epidemiological data, particularly those that originate from online interaction by users, can provide additional insights how much communities pay attention to emerging public health issues, particularly when such data has resolution to geospatial location data and longitudinal data [11] . in this study we leverage data from the popular microblogging social media platform twitter to rigorously describe the longitudinal and spatial distributions of covid-19 social media engagement at sub-national levels for the united states. analysis focused on longitudinal and geospatial assessments, both independently and concurrently, to better characterize community-level attention to the covid-19 pandemic and related opportunities to engage the public in outbreak communication. tweets related to covid-19 were collected prospectively from march 3rd to april 13th by using the twitter public streaming application programing interface (api) using a combination of cloud computing using virtual instances in amazon web services (aws) executed with scripts in the python programming language. tweets were included if they contained three pieces of information: (1) geo-identifiable characteristics (e.g. geocoded metadata); and (2) keywords related to covid-19, including: "corona outbreak," "corona," "anticorona," "coronavirus," "wuhan virus," "covid," "wuhan pneumonia," and "pneumonia of unknown cause." tweets were excluded if latitude and longitude coordinates were outside of land masses or if keywords were found outside the body of the tweet text. confirmed covid-19 cases, deaths, and recoveries were obtained from johns hopkins university github cssegisanddata/covid-19 repository [2] . these data were obtained for the united states at the secondary administrative level (i.e. counties). active covid-19 cases were based on diagnosed and reported cases of deaths and recoveries; specifically, active cases were calculated by subtracting a given day's deaths and recoveries from confirmed cases, following the conventional approach used to compute active covid-19 cases [12] . for normalization, population estimates for secondary administrative levels for the u.s. nationally were obtained from the american community survey [13] . in an effort to remove less relevant tweets, such as those relating to news media, machine learning was used to train a classifier to detect tweets exhibiting accounts of first-hand experience with covid-19. training data was taken from another study which used tweets that were derived using the same set of keywords as this study [14] . the classifier was computed using support vector machines (svm), utilizing a linear kernel algorithm via the e1071 package in r, applied to a document-term matrix computed from the training set. all longitudinal and geospatial analyses utilized tweets detected using this machine learning classifier. the c3 method of the early aberration reporting system (ears) was used to identify statistically significant aberrations in daily change in tweets related to covid-19 for each of the fifty states in the united states. the ears method is derived from a separate technique, the cumulative sum control chart (cusum) for sequential analysis. furthermore, ears is a syndromic surveillance approach used by the centers of disease control and prevention (cdc) and other public health agencies to detect statistically significant aberrations in infectious diseases [15] . all ears methods involve comparison with preceding records, and the c3 method is the most sensitive of the three ears methods. specifically, the first eleven days of twitter data was needed to establish a baseline for statistical comparison, and the earliest date it was possible to identify aberrations was march 14th. logistically, a dataframe was created in r for each state, with a factor of dates and a series of observed tweets per day, and each day following the baseline period was assessed for statistically significant longitudinal aberration via the surveillance package. geospatial analyses involved the use of arcgis to create choropleth maps with nine intervals to visualize the geospatial distribution of tweets and tweets per capita at the secondary administrative level for the united states. individual tweets were available as latitude/longitude point coordinates from the twitter api, and these point coordinates were aggregated within county spatial polygons in arcmap. for visualization of tweets per capita, the total number of tweets was divided by county-level population. equal intervals were selected as a blue-yellowred gradient in the "graduated colors" feature, thereby illustrating a consistent gradient of magnitudes. concurrent longitudinal and geospatial analysis was conducted by computing a space-time cube with 1-day time step intervals for 2,500 square-kilometer areas in the united states. the space-time cube is a construct whereby a record is produced for every combination of time step and space. after the space-time cube was created using built-in tools within arcmap, the emerging hot spot analysis feature was used to analyze the space-time cube to compute the getis ord gi � statistic for each area at each time point, thereby identifying of z-scores for hot spot and cold spot trends for each 2,500 square-kilometer area in the united states. specifically, this methodology utilizes the mann kendall trend test to compute z-scores for every square area, thereby relaying the degree to which the given area was becoming "hotter" or "colder." these z-scores were also visualized as a blue-yellow-red gradient so as to relay the distribution of longitudinal trends for tweets in all areas of the united states. the emerging hot spot analysis feature was also conducted on space-time cubes for active daily covid-19 cases per capita within the study time period. z-scores for both covid-19 cases per capita were then averaged for areas within each of the fifty states in the united states and the district of columbia, as were z-scores for tweets per capita in each state. ranking of average z-score was then computed for cases and tweets, and the largest discrepancies between rankings were reported. data management, statistical analysis, and geospatial visualization was conducted using r version 3.6.0 and arcgis version 10.6. from march 3rd through april 13th, 173,847,058 tweets related to covid-19 were collected. 1,244,478 of these tweets had geo-identifiable information which could be converted to point coordinates, of which 609,794 were from the united states. of these us tweets, machine learning classification selected 17,841 with evidence of first-hand experience with covid-19. in an analysis to detect aberrantly high levels of tweets within the in the 32-day period between march 14th and april 13th, inclusive, approximately two-thirds of statistically significant aberrations at the state level were detected in the seven-day period between march 28th and april 3rd, inclusive (table 1) . nearly all states with aberrantly high numbers of posts during this time period had relatively high proportions of rural populations. specifically, within this seven-day period, arkansas had four consecutive days of aberrantly high tweet volumes (march 28th through march 31st), with mississippi and missouri both having two consecutive days of high tweet volumes (march 29th and march 30th). sunday, march 29th was the only day to exhibit aberrantly high tweet volumes for four separate states. the number of tweets collected at the secondary administrative division in the united states were especially high in areas with high numbers of people (fig 1a) , with levels of tweets becoming much more evenly distributed after adjusting for population (fig 1b) . similarly, time-space cubes were computed for the united states at 2,500 square-kilometer units with 1-day time step intervals, for both tweet counts over time and population-normalized tweets per capita over time. the number of tweets over time significantly decreased (p = 0.0008), and this trend remained consistent after normalizing for county-level population (p = 0.0012). however, a visualization of z-scores across us spatial units revealed discrepancies in trends toward significant hot/cold spots for (fig 2) data, with appreciable discrepancies in the range of the distribution. a time-space cube was computed for population-normalized us daily active cases of covid-19, using county centroids with 1-day time step intervals. overall, a statistically significant increase in county-level population-normalized covid-19 cases was detected (p < 0.0001). z-scores for trends from the resultant emerging hot spot analysis for covid-19 cases per capita were compared to z-scores for trends from emerging hot spot analysis for covid-19 tweets per capita. average values were then computed for each state and the district of columbia. the range of state-level z-scores observed for the cases per capita analysis ) , and for the tweets per capita analysis was -10.78 (missouri) to 1.89 (alabama). furthermore, ranks were also computed for trend zscores of normalized cases and normalized tweets, with a rank of 1 for to the highest z-score value and a rank of 51 for the lowest z-score value. the largest discrepancies, in which a state had relatively high trend z-scores for increases in normalized cases while having relatively low trend z-scores for normalized tweets, were for new hampshire (tweet rank of 43, case rank of 32); vermont (tweet rank of 39, case rank of 8); and rhode island (tweet rank of 33, case rank of 4). our analyses uncovered a relative spike in tweets about covid-19 around march 29th for predominantly rural areas within the united states. this spike in tweets immediately follows a period during which aberrantly higher number of cases of covid-19 were reported in the united states. it may be that the rapid acceleration of reported cases caused increased concern about the pandemic, and that communities in these areas mostly ignored the public health threat until a relatively high threshold of cases were reported elsewhere. alternatively, it is possible that the increased attention to covid-19 in these communities was highly influenced by the social distancing guidelines and forced suspension of businesses that occurred during this same time period. this study suggests that, across subnational areas within the united states, there exists a highly variable threshold of perceived dangerousness and/or intrusiveness required to activate outbreak-related conversations on social media platforms such as twitter, a finding that can inform future outbreak communication and health promotion strategies. both of these causal scenarios may be greatly mediated by news media commonly more consumed in certain areas, and further study should be conducted to assess media-specific roles [11] . concurrent geospatial and longitudinal analyses also indicate that predominantly rural areas of the united states increased engagement in covid-19 social media conversations at later stages of the study timeframe. these analyses further suggest that there exists variability of engagement within states, as was observed for idaho, iowa, and south dakota; and that regions of increasing concern can also span across multiple states, as was the case for the montana-idaho and for the minnesota-wisconsin areas. interestingly, though several states exhibited a decline in social media engagement following relatively high points earlier in the study period, states eschewing this negative trend did not exhibit clear longitudinal patterns for recorded active covid-19 cases. these results suggest that increased communication in certain areas may be a reaction to conditions unrelated to reported covid-19 infection in their communities (such as discussion of social distancing guidelines or discussions of related government action). alternatively, increased social media messaging about covid-19 may result from higher numbers of people contracting covid-19 than have been reported in these areas, which itself may be related to insufficient testing capacity and healthcare access, as has been widely reported and remains an acute problem in rural communities [16] [17] [18] . this study is unique in that it uses twitter data as a proxy measure for assessing the concurrent longitudinal and geospatial distributions of attention to covid-19 across local and regional communities in the united states. however, a number of studies have utilized similar methods in covid-19 research, although usually for covid-19 infection cases and/or deaths. for example, time-space methods were used to differentiate the trajectories of covid-19 prevalence between rural and urban counties in the united states [19] , and sequential spatial cross-sections have been assessed to define the evolution of hot spots in subnational regions [20] . while twitter posts related to covid-19 have been assessed using spatial and longitudinal techniques [21, 22] , these studies have generally focused on country-level comparisons. as these analyses were ecological in nature, this study is primarily intended to generate hypotheses, and the findings in this study should be further corroborated with other sources of data that can better identify specific covid-19 hot and cold spots in relation to projected number of cases. other data includes the availability and volume of covid-19 testing kits, search engine query results (e.g. google trends data), covid-19 hospitalizations data, etc. tweets themselves were collected on the basis of containing certain keywords related to covid-19, though the actual content of these tweets may vary in their interpretability for estimating attention to covid-19 or disease burden. future work by co-authors is focused on developing machine learning approaches to identify users reporting covid-19-related symptoms, lack of testing access, and recovery, that could be used in a future study to better assess covid-19 hot spots and also used to compare against aberrations in reporting. while preliminary, this study suggests that there may exist an appreciable degree of variability in the patterns defining community attention to public health topics, particularly as pertains to online discussion of infectious disease outbreaks. further study should be conducted to characterize community-level moderators of these patterns and to understand the degree to which they may facilitate improved dissemination and impact of public health messaging. the use of ears and time-space cubes appear to have utility in identifying longitudinal trends and their discrepancies across space, and further academic research on the topic of community-level attention to public health issues may seek to leverage these analytical techniques. supporting information s1 data. 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misinformation sharing on twitter an exploratory study of covid-19 information on twitter in the greater region key: cord-337067-j8ebslif authors: mades, andreas; gotthardt, katherina; awe, karin; stieler, jens; döring, tatjana; füser, sabine; prange, reinhild title: role of human sec63 in modulating the steady-state levels of multi-spanning membrane proteins date: 2012-11-15 journal: plos one doi: 10.1371/journal.pone.0049243 sha: doc_id: 337067 cord_uid: j8ebslif the sec61 translocon of the endoplasmic reticulum (er) membrane forms an aqueous pore, allowing polypeptides to be transferred across or integrated into membranes. protein translocation into the er can occur coand posttranslationally. in yeast, posttranslational translocation involves the heptameric translocase complex including its sec62p and sec63p subunits. the mammalian er membrane contains orthologs of yeast sec62p and sec63p, but their function is poorly understood. here, we analyzed the effects of excess and deficit sec63 on various er cargoes using human cell culture systems. the overexpression of sec63 reduces the steady-state levels of viral and cellular multi-spanning membrane proteins in a cotranslational mode, while soluble and single-spanning er reporters are not affected. consistent with this, the knock-down of sec63 increases the steady-state pools of polytopic er proteins, suggesting a substrate-specific and regulatory function of sec63 in er import. overexpressed sec63 exerts its down-regulating activity on polytopic protein levels independent of its sec62-interacting motif, indicating that it may not act in conjunction with sec62 in human cells. the specific action of sec63 is further sustained by our observations that the up-regulation of either sec62 or two other er proteins with lumenal j domains, like erdj1 and erdj4, does not compromise the steady-state level of a multi-spanning membrane reporter. a j domain-specific mutation of sec63, proposed to weaken its interaction with the er resident bip chaperone, reduces the down-regulating capacity of excess sec63, suggesting an involvement of bip in this process. together, these results suggest that sec63 may perform a substrate-selective quantity control function during cotranslational er import. the er is a major site of synthesis for both secretory and membrane integrated proteins. generally, nascent polypeptides are translocated cotranslationally across or into the er membrane at sites termed translocons. the translocon minimally comprises one sec61a-b-c heterotrimer and associated proteins forming an aqueous conduit that aligns with the ribosome during translocation. in cotranslational translocation, signal sequences or transmembrane (tm) domains within a nascent polypeptide are recognized by the signal-recognition particle (srp). the srpbound ribosome nascent chain complex then binds to the membrane via the srp receptor, and after srp release the translating ribosome interacts with the translocon, thereby enabling the growing polypeptide to be inserted directly into the translocon channel. soluble proteins, such as those ultimately secreted from the cell or localized inside the er lumen, cross the membrane completely, while membrane proteins exit laterally into the lipid phase, a step referred to as membrane integration [1, 2, 3] . the events accompanying the gating of the lateral exit portal of the translocon are currently less resolved. numerous components near the mammalian sec61 core translocon complex have been described that could contribute to the overall efficiency of translocation. these include the spc (signal peptidase complex) [4] , ost (oligosaccharyl transferase complex) [5] , tram (translocation chain-associated membrane protein) [6, 7] , trap (translocon-associated protein) [8] , ramp4 (ribosome-associated membrane protein 4) [6] , er proteins with j domains inside the er lumen such as erdj1 and erdj2/sec63 [9, 10, 11] , the sec63-interacting sec62 protein [10] , and others. the functional roles of the translocon-associated sec62 and sec63 proteins in protein biogenesis at the er have been well established in the yeast saccaromyces cerevisiae, where nascent chains can be translocated either co-or posttranslationally [1, 2, 3] . both translocation pathways depend on the protein-conducting sec61 channel, but differ in their targeting mechanisms. in the cotranslational mode of translocation, nascent precursors are targeted in an srp-dependent manner. in the posttranslational mode of translocation, the sec61 channel partners with another membrane protein complex, consisting of the essential sec62p and sec63p proteins, and the non-essential sec71p and sec72p proteins, that together mediate targeting and translocation of completed polypeptide chains. the posttranslational pathway is used predominantly by precursors with low hydrophobic signal peptides [1, 2, 12] . in the mammalian er, the cotranslational mode of protein translocation dominates. even so, orthologs of the yeast sec62p and sec63p, referred to as sec62 and sec63, are found in stoichiometric amounts compared with sec61a and in association with the sec61 complex [10, 13] . sec62 is a double-spanning membrane protein with a cytosolic domain that associates with ribosomes, suggesting a cotranslational function of this protein in mammals [14] . sec62 also teams up with sec63, an integral membrane protein of the hsp40 co-chaperone family [10, 14] . similar to its yeast ortholog, mammalian sec63 contains three tm domains with a lumenal j domain that functionally interacts with the er resident bip chaperone (kar2p in yeast) [10, 12, 15, 16] . the conservation of structural and biological features of sec62 and sec63 from yeast to humans strongly suggests that these proteins are likewise involved in protein transport into the mammalian er, but their precise function is less known. loss of function mutations in the human sec63 are not lethal but are linked to polycystic liver disease, indicating that it may contribute to er import and/or folding of proteins involved in biliary cell growth [1, 17] . in the case of human sec62, its overexpression was found to be associated with sporadic colorectal cancer and prostate cancer [1] . the participation of sec62/sec63 in the biogenesis of particular er substrates is also suggested by the finding that both proteins can transiently associate with newly synthesized polypeptides during er membrane integration in cell-free systems [18] . our previous work has been focused on the biogenesis of three related envelope proteins of the hepatitis b virus (hbv) that are synthesized as multi-spanning proteins at the er membrane. while the small s and middle m envelope proteins are integrated into the er membrane in a typical cotranslational process, the large l envelope protein forms a dual topology via a process involving cotranslational membrane integration and subsequent posttranslational translocation of its pres subdomain into the er [19, 20, 21] . similar to posttranslational translocation processes in yeast, the posttranslational pres translocation of l requires bip [22, 23] , a known interaction partner of sec63 [10] . we took this as a rationale to study the function of human sec63 and its partner proteins in cell culture systems. against expectation, we find that sec63 does not play a role in the special topogenesis of the hbv l protein (k.a and r.p., unpublished observation), but is rather more generally involved in the control of the steady-state levels of polytopic membrane proteins. overexpressed sec63, but not sec62, reduces the steady-state level of the polytopic hbv.s envelope protein to study the role of the human sec62 and sec63 proteins, expression vectors encoding n-terminally myc-tagged versions of these proteins were constructed and transiently transfected into huh-7 human liver carcinoma cells. upon deconvolution immunofluorescence and phase contrast microscopy, the ectopically expressed sec62 and sec63 constructs yielded an er-like staining and partially colocalized with calnexin, a membraneanchored er chaperone (fig. 1a) . the constructs were next transfected together with a c-terminally hemagglutinin (ha)tagged version of the s envelope protein of hbv (hbv.s). hbv.s is cotranslationally integrated into the er membrane, directed by the action of an uncleaved signal-anchor and a stop-transfer sequence encoded within its first and second tm segments [24] . the hydrophobic c-terminal half of hbv.s is predicted to further span the membrane two times thereby resulting in a fourfold membrane-spanning protein. after transient coexpression of the constructs, cell lysates were prepared with the non-denaturing detergent np-40 and analyzed by myc-and ha-specific western blotting. as shown in fig. 1b , sec62 and sec63 were efficiently synthesized and appeared in about 46 and 87 kda forms, respectively, in accord with their calculated molecular masses. hbv.s was obtained in its characteristic doublet of a 24 kda nonglycosylated (p24) and a 27 kda glycosylated (gp27) form as a consequence of partial n-linked glycosylation [25] . intriguingly, the intracellular level of hbv.s was significantly lower when ectopically expressed sec63 was present. in contrast, excess sec62 reproducibly did not affect the level of hbv.s (fig. 1b) . similar results were obtained with cell lysates prepared with the denaturing detergent sds (fig. 1b) , indicating that the observed down-regulation of hbv.s by excess sec63 was not merely due to changes in the solubility profile. since the overexpression of sec63 might be harmful for the cells, cytotoxicity assays of cellular supernatants were performed which did not reveal significant indications of cell damage (fig. 1b) . because sec62 and sec63 have been shown to associate with each other [13, 14] , the upregulation of sec63 could evoke an imbalance of the complex partner. therefore, the fate of hbv.s was analyzed in cells overexpressing both sec62 and sec63 proteins. under these conditions, the levels of sec62 and sec63 were lower as compared to their individual expression. one explanation may be that sec63 also reduced the steady-state level of the double-spanning sec62 protein at the er. alternatively, this could be the consequence of a lower degree of transfection caused by the higher amount of plasmid dna used for triple transfection. nonetheless, even in consideration of a lower transfection rate, hbv.s was downregulated ( fig. 1b) , implicating that the steady-state level of sec62 is less important. in vitro-binding studies have identified the negatively charged c-terminus of sec63 as the interaction domain for basic oligopeptide motifs within the n-terminus of sec62 [14] . to examine whether the sec62/sec63 interaction might play a role in the function of sec63, we analyzed a c-terminally truncated sec63 mutant lacking large parts of the cytosolic domain including the sec62 binding site. this sec63dc mutant similarly down-regulated hbv.s (fig. 1b) , thus arguing against an essential role of sec62 in the observed action of sec63. following er translocation, the hbv.s protein is able to selfassemble into subviral envelope particles (svps) that bud into intralumenal cisternae of post-er/pre-medial-golgi compartments and exit the cell by the constitutive pathway of secretion [25, 26] . to investigate whether the intracellular reduction of hbv.s by overexpressed sec63 was simply due to an enhanced svp export, lysates and supernatants of cotransfected cells were examined by an hbv.s-specific elisa. this analysis, however, clearly revealed that excess sec63 reduced both the intra-and extracellular pool of hbv.s. in contrast, overexpressed sec62 did not interfere with the synthesis and secretion of hbv.s (fig. 1c ). overexpressed sec63 reduces the steady-state level of the polytopic hbv.s envelope protein in a dosedependent manner to ascertain the degree of sec63 overexpression, total sec63 levels were examined in control-versus sec63-transfected huh-7 cells by using a polyclonal sec63-specific antiserum for western blotting. in order to ensure the true identification of endogeneous sec63, cells were depleted for sec63 by treatment with specific sirna duplexes. as shown in fig. 2a , endogeneous sec63 was absent in lysates of sisec63-treated cells. due to the myc-tag, exogeneous sec63 displayed a slower electrophoretic mobility that allowed its distinction from the endogenous isoform ( fig. 2a) . upon serial dilution of the sec63-transfected lysate and quantification, we calculated that exogeneous sec63 was overexpressed about 3.2-fold. an accompanying titration experiment showed that sec63 down-regulated hbv.s in a dose-dependent manner (fig. 2b) . thereby, the most prominent decline in the hbv.s steady state level appeared when the concentration of the transfected sec63 dna was raised above 1.5 mg/10 6 cells (fig. 2b ). to rule out the possibility that up-regulated sec63 had effects on mrna production or stability, the hbv.s-specific transcript levels were measured by quantitative reverse transcription pcr. although we noticed a modest decline of the hbv.sspecific mrna in sec63-overexpressing cells (fig. 2c ), this drop is unlikely to account for the impact of sec63 on the steady-state hbv.s pool. to specify the role of excess sec63, we next analyzed the fate of an hbv.s mutant lacking the first tm segment (hbv.sdtm1). this mutant still enters the er pathway but fails to assemble into intralumenal secretory svps [27] . upon coexpression with sec63, the intracellular amount of hbv.sdtm1 was drastically decreased (fig. 3a) , similar to the situation of wild-type (wt) hbv.s. thus, excess sec63 depleted hbv.s proteins irrespective of their secretory phenotypes and therefore likely acts prior to svp assembly and secretion. to sustain this finding, the outcome of further hbv envelope proteins was studied. the l envelope protein of hbv (hbv.l) is unable to assemble into secretory svps due to its dual transmembrane topology [21, 25] . this deficit can be abrogated by the addition of a foreign signal sequence to the nterminus of hbv.l that blocks the formation of the dual topology and enables secretion [28] . the overexpression of sec63 decreased the non-glycosylated and glycosylated forms of both the wt hbv.l and hbv.ile9::l signal fusion proteins (fig. 3a ). this indicates that excess sec63 compromises the production of hbv envelope proteins independently of their particular secretory behavior. during er translocation, the hbv envelope proteins are modified by n-linked glycosylation conducted by the translocon-associated ost complex [5, 29] . because the overexpression of sec63 could impair the structural integrity of the ost complex concomitant with translocational constraints, we studied a glycosylation-defective hbv.l mutant (hbv.ldn309). due to the mutation of the n-glycan acceptor site, this mutant appeared only in the nonglycosylated p39 form (fig. 3a) . hbv.ldn309 was also downregulated by excess sec63, thus largely excluding a participation of the n-linked glycosylation machinery in this process. since hbv is a hepatotropic virus, we routinely used huh-7 cells for viral expression studies. some liver cells, however, have been implicated in sec63 abnormalities, as mutations in the sec63 gene contribute to the development of autosomal dominant polycystic liver disease [1, 17] . therefore, we examined the effect of excess sec63 in hek293t human embryonic kidney cells in parallel. as exemplified for the hbv.l protein, overexpressed sec63 reduced the intracellular hbv.l level to almost the same extent as compared to huh-7 cells (fig. 3b ), arguing against cell type-specific effects. in combining these data, we deduced that excess sec63 diminished the steady-state pools of the hbv envelope proteins synthesized at the er. however, there remained the possibility that the observed effects were simply due to an exceptionally high degree of cotransfection of the sec63 and hbv constructs, thereby provoking intracellular imbalances, unspecific protein interferences, and/or other effects. to address this issue, we used a bicistronic plasmid vector carrying expression units for ha-tagged hbv.l under the control of the human metallothionein iia (hmtiia) promoter and for ha-tagged hip (heat shock cognate protein 70 [hsc70] interacting protein) under the control of the cytomegalovirus (cmv) promoter [22] . human hip is a cytosolic cochaperone involved in the regulation of hsc70 chaperone activity. this construct offered the chance to monitor the effect of excess sec63 in the same cells and the same blot. upon cotransfection of hbv.l/hip with sec63, the level of hbv.l was again reduced, while the amount of cytosolic full-length hip was only marginally affected (fig. 3c ). for unknown reasons, we noted, however, that hip-specific degradation products were sensitive to excess sec63. to get insights into the underlying mechanism(s), we investigated the time frame of the action of up-regulated sec63. although the data presented so far favor a cotranslational mode of sec63 activity, posttranslational impacts of excess sec63, such as a disruption of the integrity of the er membrane, could not be ruled out. therefore, cells transiently expressing the hbv.s construct for two days were retransfected with sec63 and analyzed after an additional day. for comparison, cells were simultaneously cotransfected with hbv.s and sec63. as shown in fig. 4a , overexpressed sec63 did not affect a preexisting hbv.s pool. to exclude the possibility that the lack of sec63 action in the ''posttransfection'' experiment was due to constraints in the retransfection, we analyzed the rate of cotransfection by immunofluorescence. although the intensity of the hbv.s-specific signals was substantially lower as compared to cells expressing hbv.s alone, the amplification of the signals enabled the detection of transfected cells. upon quantification, 49614% or 4267% of transfected cells were determined to be co-or posttransfected with sec63, respectively (n = 5) (fig. 4a ). together, these data support a role for sec63 at an early stage of hbv.s protein synthesis, possibly occurring during translocation. nuclei and organelle structures in the cell periphery are shown in the right panels. bar, 10 mm. b. cells were transfected with ha-tagged hbv.s together with empty plasmid dna (control), myc-tagged sec62, sec63, or sec63dc at a 1:3 dna ratio (4 mg total dna), respectively. cotransfections of hbv.s with sec62 plus sec63 were done at a 1:3:3 dna ratio (7 mg total dna), respectively. three days after transfection, cellular lysates were prepared with np-40 (np-40) and analyzed by ha-or myc-specific western blotting (wb) to demonstrate expression of hbv.s (bottom) or sec62, sec63 and sec63dc (top). to confirm equal gel loading, the lysates were probed by anti-b-actin wb (middle top). numbers to the right refer to molecular weight standards in kd, while the non-glycosyated (p24) and glycosylated (gp27) forms of hbv.s (s) are indicated on the left (middle bottom). relative hbv.s expression values were determined by densitometric analysis and demonstrated below in the graph in percent amount relative to control cells. error bars indicate the standard deviation from three separate experiments. the analyses of sds lysates (sds) of correspondingly cotransfected cells are illustrated in the bottom panel. to probe for cell cytotoxicity, supernatants of cotransfected cells were assessed for ldh activity. c. cells were cotransfected with hbv.s and sec62 or sec63 exactly as in a. lysates (cell) and supernatants (medium) were examined by an s-specific elisa, and hbv.s levels are demonstrated in percent amount 6 sd relative to control cells (n = 3). doi:10.1371/journal.pone.0049243.g001 in yeast, sec63p and its kar2p partner protein have been indicated to be involved in the export of misfolded proteins to the cytosol during er-associated degradation (erad) by the proteasome [30, 31] . for the mammalian sec63, such a role is ill-defined. to examine whether the up-regulation of sec63 reduced the hbv.s level via an enforced export out of the er, transfected cells were treated with the proteasome inhibitor epoxomicin. the efficacy of the drug during the applied assay conditions was indicated by the appearance of c-terminally degraded sec63 derivatives that accumulated in epoxomicin-but not in mocktreated cells (fig. 4b) . importantly, the inhibition of the erad pathway did not abolish the ability of excess sec63 to downfigure 2 . overexpressing of sec63 reduces the steady-state hbv.s level in a dose-dependent manner without grossly affecting transcription. a. huh-7 cells were either treated with control sirna (sicon) or sirna duplexes targeting sec63 (sisec63) or were transfected with control dna (control) or myc-tagged sec63 (sec63; 3 mg dna). np-40 cell lysates were prepared after three days and probed by anti-sec63 wb. for calculation of the degree of sec63 overexpression, the sec63-transfected lysate was serially diluted prior to gel loading as indicated above the lanes (% load). b. cells were transfected with hbv.s (1 mg dna) together with increasing amounts of sec63 dna, as indicated above the lanes (in mg). total dna used for transfections was adjusted with empty plasmid dna (control). extracts were prepared with sds lysis buffer two days after transfection and assayed by myc-(top), b-actin-(middle), and ha-specific (bottom) wb. c. cells were cotransfected with hbv.s and control or sec63 plasmids as in regulate hbv.s (fig. 4b) , suggesting that the reduction of the hbv.s steady-state level is not mediated by proteasomal degradation. to account for this unexpected finding, different explanations are conceivable. first, non-proteasomal proteases, like amino-and carboxypeptidases, may contribute to the destruction of improperly translocated hbv.s chains. such proteases, acting in the cytosol and the er/secretory pathway, have been reported to participate in endogenous antigen processing [32] . second, excess sec63 may halt hbv.s translocation, thereby promoting cotranslational destruction prior to completion of protein synthesis. in doing so, full-length, cterminally tagged hbv.s chains would not be detectable. and third, autophagosomes/lysosomes may be involved in the disposal of hbv.s. this pathway has been described in cells permanently replicating hbv [33] . although being clients of sec63, the hbv envelope proteins do not directly interact with sec63 as measured in a split-ubiquitin yeast two-hybrid assay (data not shown). this prompted us to examine whether or not sec63 may play a more general role in the production of er proteins. to this aim, we employed a panel of different translocational reporter proteins. for soluble er marker proteins, hemopexin (hxp) and a yellow fluorescent er fusion protein (yfp.er) were used that are cotranslationally translocated across the er membrane by virtue of their n-terminal cleaved signal peptides [34] . while yfp.er is retained within the er lumen via its c-terminal kdel retention signal, hxp is secreted from the cells. as shown in fig. 5a , both proteins were efficiently synthesized in transfected huh-7 cells. because hxp is a substrate to n-and o-linked glycosylation [34] , it appeared in multiple forms with different electrophoretic mobilities. upon overexpression of sec63, the intracellular levels of both reporters as well as the glycosylation pattern of hxp were largely unaffected, indicating that excess sec63 did neither affect er translocation nor the steady-state levels of these soluble proteins. identical results were obtained when er membrane proteins with a single membrane-spanning segment were analyzed. here, we took use of either a golgi.yfp fusion construct encoding yfp with a nterminal membrane anchoring signal peptide of b1,4-galactosyltransferase or the g glycoprotein of vesicular stomatitis virus (vsv.g). vsv.g harbors a cleavable signal peptide and a single tm segment in its c-terminus and is a substrate to n-linked glycosylation [35, 36] . coexpression studies demonstrated that sec63 neither down-regulated golgi.yfp nor vsv.g (fig. 5b) . it also did not affect the glycosylation pattern of vsv.g. since these data are in seemingly conflict with the results obtained for the hbv envelope proteins, we next assessed the behavior of multispanning membrane proteins synthesized in the presence of upregulated sec63. the m envelope protein of the mouse hepatitis coronavirus (mhv.m) is a triple-spanning protein with its first and second tm segments serving as a signal-anchor and stop-transfer signal, respectively [37] . beside viral proteins, human cd63/ lamp-3 (lysosomal-associated membrane protein 3), a member of the tetraspanin family, was included as a cellular multi-pass reporter [38] . ha-tagged versions of mhv.m and cd63 were cotransfected with sec63 and cell extracts were probed by western blotting. consistent with published data [37, 38] , the mhv.m and cd63 proteins run as broad smear in sds-polyacrylamide gels likely due to glycosylation (fig. 5c) . importantly, overexpressed sec63 almost completely depleted each of the multi-pass reporters (fig. 5c) , as it is the case for the hbv envelope proteins. to probe whether the non-glycosylated yfp.er and golgi.yfp reporters were truly translocated into the er of sec63-overexpressing cells, trypsin protection experiments were performed. microsomes of cotransfected cells were prepared and either left untreated or treated with trypsin in the absence or presence of detergent. for both reporters, protection against trypsin was observed in intact microsomes (fig. 6a) . on disruption of microsomes with detergent, trypsin completely converted the proteins to tryptic fragments thereby leading to the disappearance of full-length yfp.er and golgi.yfp. we noted, however, that neither construct was fully protected from proteolysis in the absence of detergent (fig. 6a ), likely as a consequence of some leakage of the microsomes. nonetheless, the amounts of protected yfp.er and golgi.yfp chains were almost identical in controland sec63-overexpressing cells. thus, up-regulated sec63 does not interfere with the translocation of these substrates. as measured by quantitative reverse transcription pcr of representative reporter genes, up-regulated sec63 does also not impair the synthesis and stability of yfp.er-and vsv.g-specific mrnas (fig. 6b ). in the case of cd63, transcript levels were diminished in the presence of exogenous sec63 (fig. 6b ). this may in part, but not entirely contribute to the observed decline of the cd63 protein level (see fig. 5c ). in order to determine the step(s) at which sec63 is acting, we analyzed whether up-regulated sec63 affected the translation or cotranslational insertion of the multi-pass cd63 reporter. for this, we used two constructs encoding the cytosolic green fluorescent protein (gfp) or gfp with a c-terminal fusion of cd63 (gfp.cd63). both constructs were in identical vector backbones and under the control of identical transcriptional elements. upon cotransfection of either of the two constructs with control or sec63 dna, their common gfp-tag enabled immunodetection on the same gel. like the cd63 wt protein (see fig. 5c ), the gfp.cd63 fusion protein appeared in multiple bands likely as a result of glycosylation, implicating that it had entered the er/secretory pathway (fig. 7) . up-regulated sec63 diminished the yield of the gfp.cd63 membrane reporter, while it had little effects on the cytosolic gfp protein (fig. 7) . since the transcripts of the two reporters were generated from the same promoter/polyadenylation elements and hence should be similar, we inferred that excess sec63 appeared to affect the cotranslational insertion rather than the translation of polytopic proteins. collectively, these data suggest that excess sec63 interferes selectively with the formation of multi-spanning er proteins. therefore, we next analyzed er cargoes in sec63-deficient cells. for depletion of sec63, classic sirna duplexes (sisec63) as well as sirna molecules prepared by endoribonuclease digestion (esi-sec63) were transfected into huh-7 cells prior to transfection with the hbv.s construct. western blot analysis of cell lysates showed that both sirna species depleted sec63 by about 80% as compared to control sirna-treated cells (fig. 8a ). when lysates were probed for hbv.s, the knock-down of sec63 resulted in significant higher intracellular p24/gp27 levels as compared to control-treated cells. of note, the intracellular increase of hbv.s was not due to a block in svp secretion, since the extracellular amount of hbv.s was also elevated in sec63-depleted cells (data not shown). these results indicate that sec63 is per se not required for hbv.s production; rather, its limitation promotes hbv.s synthesis and/or stability. to corroborate these results, other er reporter proteins were studied under conditions of deficit sec63. as representative markers, the soluble hxp protein, the single membrane-spanning vsv.g protein, and the multiple-spanning proteins hbv.l and mhv.m were investigated. depletion efficacy of sec63 and intracellular steady-state levels of the marker proteins were monitored by specific immunoblotting. as shown in fig. 8b , the biogenesis of hxp and vsv.g were unaffected by the knock-down of sec63. conversely, the amounts of each of the multi-spanning reporters were increased in sec63-depleted cells. these observations suggest that human sec63 is not essential for cotranslational er translocation, but regulates the translocation efficiency of certain precursor polypeptides. beside sec63 (i.e., erdj2), six other resident er proteins with j domains inside the er lumen have been described in mammals [11] . erdj1 is a further translocon-associated erdj protein and constitutes a single-pass type i membrane protein. unlike sec63, it has the ability to associate with translating ribosomes, likely to modulate translation [9] . erdj4 is a soluble er protein and plays a role in the degradation of erad substrates, but has not been found in close contact with the sec61 translocon [39] . to analyze whether an up-regulation of these erdj proteins might also affect the level of a multi-spanning membrane protein, flag-tagged versions of erdj1 and erdj4 were cotransfected with hbv.s. flag-specific western blotting confirmed the ectopic expression of erdj1 and erdj4 in 63 and 25 kda forms, respectively, consistent with their theoretical molecular masses (fig. 9) . importantly, when lysates were probed for hbv.s, no significant change in the p24/gp27 level was noticeable (fig. 9) . thus, in contrast to sec63, the overexpression of neither erdj1 nor erdj4 compromises the production and/or life span of hbv.s. in the next set of experiments, we focused on the sec63/bip interplay. the yeast and mammalian sec63 proteins have been shown to interact with the er lumenal kar2p/bip chaperone in a productive manner [10, 12, 15] . these interactions require their lumenally exposed j domains containing the highly conserved his-pro-asp (hpd) motif [11, 40, 41] . because mutations of this sequence abolish the functional interaction between j domains figure 5 . overexpressing of sec63 reduces steady-state levels of polytopic proteins. er reporter constructs, including soluble (a), singlepass membrane (b), and multi-pass membrane (c) proteins were used for cotransfection studies as indicated above the panels. each reporter was transfected with control dna or myc-tagged sec63 at a 1:3 dna ratio, respectively, into huh-7 cells. three days later, cell lysates were subjected to specific immunoblotting. ectopic expression of sec63 was assessed with anti-myc antibodies, while anti-b-actin antibodies were used to demonstrate identical gel loading. hxp, mhv.m, and cd63 were probed with anti-ha antibodies, yfp.er and golgi.yfp were detected with anti-gfp antibodies, and vsv.g was verified with anti-g-specific antibodies. reporter expression values were determined by densitometry and demonstrated in percent amount 6 sd relative to control cells (n = 3). doi:10.1371/journal.pone.0049243.g005 and their chaperone partners [40, 41] , we reasoned to study a sec63 mutant devoid of the hpd motif (sec63dhpd). the wt and mutant sec63 proteins were coexpressed with hbv.s and cell extracts were examined by specific immunoblotting. the sec63dhpd mutant likewise reduced hbv.s, albeit its downregulating activity was reproducibly less pronounced as compared to wt sec63 (fig. 10a) . similar results were obtained with the polytopic cd63 reporter (fig. 10a) . these results suggest that the stimulation of the atpase activity of bip by the j domain of sec63 may not be mandatory for the role of sec63 in polytopic membrane homeostasis. however, they do not thoroughly exclude that sec63 may function in concert with bip. to dissect whether sec63 per se or the associated bip chaperone is responsible for hbv.s protein reduction, the reporter was studied in cells overexpressing sec63 and bip either individually or together. the up-regulation of bip neither reduced the level of hbv.s nor reverted the inhibitory action of excess sec63 on hbv.s (fig. 10b) . together, these data suggest that sec63 is primarily responsible for the control of membrane protein levels, possibly with aid of bip. this work provides support for a role of human sec63 in protein biogenesis at the er. by analyzing the effects of excess and deficit sec63 in human cell lines, we observed an inverse correlation between the cell content of sec63 and the level of particular er substrates. as reporters, we used soluble, single-and multispanning membrane proteins of viral, bacterial, and mammalian origin and observed that the overexpression of sec63 specifically reduces the steady-state levels of polytopic membrane proteins, while the limitation of sec63 has reciprocal effects. the data of the kinetic experiment support a role for sec63 at an early stage of multi-spanning membrane protein synthesis, and we favor a model where this occurs during translocation. overall, our data implicate that sec63 has a regulatory function during cotranslational insertion of multi-pass membrane proteins that is, however, not essential. its action as a translocational controller may explain why the loss of sec63 function associated with polycystic liver disease (pcld) is not lethal in humans [1, 17] . with regard to our finding that the depletion of sec63 augments the biogenesis of polytopic proteins, dysfunctional sec63 may evoke an unbalanced physiological situation in pcld patients, thereby eventually affecting proteins that are involved in the control of biliary cell growth and proliferation. previous studies have shown that the yeast and mammalian sec62 and sec63 proteins interact with each other [13, 14, 42] , suggesting a concerted action of these proteins. while this is true for posttranslational protein translocation into the yeast er [2, 39] , overlapping roles for the mammalian proteins have not been documented. rather, in vitro biochemical analyses of dog and bovine pancreatic microsomes revealed that only small fractions of sec62 and sec63 are associated with each other, while the majority of these proteins were not found in a complex [10, 13] . cross-linking experiments in cell-free systems also argue for figure 8 . silencing of sec63 increases the steady-state levels of polytopic proteins. a. huh-7 cells were treated with control sirna (sicon) or sirna duplexes (sisec63) and esirna molecules (esisec63) targeting sec63. two days later, cells were transfected with hbv.s and lysed after 48 h. lysates were subjected to sec63-specific wb to examine depletion. for hbv.s detection, anti-ha wb was used. to confirm equal loading of lysates, bactin levels were assessed. below the panels, the cell-associated hbv.s levels are demonstrated in percent amount 6 sd relative to control cells (n = 2). b. cells were transfected with control or sec63-specific sirnas as above, prior to transfection with the indicated er reporters. hxp was used as a soluble marker, vsv.g represented a single membrane-pass protein, while hbv.l and mhv.m served as multi-spanning membrane reporters. cell lysates were subjected to immunoblotting using anti-sec63 (top), anti-b-actin (middle), or anti-ha/vsv.g antibodies (bottom). reporter expression values were determined by densitometry and demonstrated in percent amount relative to control cells. doi:10.1371/journal.pone.0049243.g008 individual functions of mammalian sec62 and sec63 proteins, as sec63 was found in transient proximity to newly synthesized membrane proteins at early stages of membrane integration, while sec62 was detected at a later stage [18] . consistent with this, we show that the overexpression of sec62 did not limit hbv.s membrane protein expression. moreover, up-regulated sec63 exerts its inhibitory activity independent of the sec62 protein content of the cells and independent of its sec62-interacting motif, indicating that a stoichiometric complex formation between sec63 and sec62 may not be mandatory for function. a sec62independent action of sec63 is also used by the parasite trypanosoma brucei that lacks a sec62 ortholog, but engages sec63 for co-and posttranslational er protein import [43] . the substrate-specificity of the sec63 action is intriguing, as it selectively targets multi-spanning membrane proteins. a currently unresolved issue in membrane protein folding is how and when tms are released from sec61 once translocation is terminated. one view, supported by lipid cross-linking studies is that tms are dislocated from the translocon and move into the bilayer by passive thermodynamic partitioning through a lateral cleft in the sec61a subunit. hence, the driving force for the lateral exit of tms can be correlated to their intrinsic hydrophobicity. numerous studies have shown that such a model is likely to apply for the membrane integration of single-spanning membrane proteins [44, 45, 46] . the biogenesis of polytopic membrane proteins, however, presents distinct challenges to the er translocon and demands additional modulation. experimental data suggested that up to four tms of one polypeptide could be present in the translocation channel at the same time concomitant with widening of the pore [47, 48, 49] . moreover, tms appear to progress through different proteinaceous environments prior to their integration into the lipid bilayer either one by one, as pairs or even groups, suggesting the participation of additional factors beside the translocon core complex [50, 51, 52] . because mammalian sec63 is adjacent to sec61 [10] and because it selectively influences the synthesis of polytopic membrane proteins, it is tempting to speculate that sec63 may affect the structure and functionality of those translocons handling polytopic proteins. in this view, sec63 (i) may control expansion of the translocon to accommodate multiple tms, (ii) may act as a lateral gate keeper controlling the opening and closure of the exit portal towards the lipid layer, and/or (iii) may function as a chaperone or an escort for tms awaiting lipid integration. a regulatory function of sec63 may also explain why it is dispensable for cotranslational translocation into reconstituted proteoliposomes [6] . since excess sec63 inhibits the biogenesis of polytopic proteins, while its deficit has inverse effects, sec63 has features of a ''negative'' regulator. the concept of translocational regulation by substrate-selective trans-acting factors has been well documented [53] . for example, the translocon-associated tram and trap proteins can interact directly with some nascent chains and stimulate translocation in a signal sequence-dependent manner, but neither protein is absolutely required because at least some substrates can be translocated in their absence [7, 8, 54] . translocon-associated er lumenal factors, like the soluble bip chaperone or the membrane-embedded ost complex, appear to influence translocation of nascent chains by mechanisms including trapping, modification, and/or folding, thereby facilitating quality control of er substrate entry [5, 16] . for the translocon-associated sec63 protein, we suggest a quantity control function in such that it may regulate the amount of polytopic protein integration in order to prevent er membrane overload. at first glance, our rna interference data are in conflict with two recent works, studying the loss of sec63 function in vitro and in vivo. by using a mutant mouse model for pcld, fedeles et al. [55] demonstrated that sec63 differently affected the polycystic kidney disease gene products, polycystin-1 and polycystin-2. both proteins are synthesized as multi-spanning proteins at the er whereupon polycystin-1 uses a cleavable signal peptide, while polycystin-2 employs a non-cleavable signal [56, 57] . in sec63 knock-out cells, the biogenesis of polycystin-1 was strongly impaired, while polycystin-2 was only moderately affected [55] . by studying seven different lumenal, single-and multi-spanning cotranslational er reporters with and without cleavable signal peptides, lang et al. [58] showed that some proteins need the help of sec63, while others do not. the authors suggest that the sec63dependancy may be in part related to the properties of cleavable and non-cleavable signal peptides [58] . except for the hbv.i-le9::l reporter that contains an artificial, presumably uncleaved signal peptide (r. prange, unpublished observation), all polytopic cargos used in this work contain non-cleavable signal peptides. hence, apart from its quantity control function sec63 appears to play an additional ''active'' role in cotranslational protein import depending on the character of the signal peptide. a greater panel of proteins will have to be analyzed to deduce the exact function of sec63. the j domains of yeast and mammalian sec63 proteins have been demonstrated to functionally interact with the kar2p/bip chaperone which plays a role in many functions of the er, including lumenal gating of the translocon, assisting protein folding, and targeting misfolded proteins for proteasomal degradation [16] . thus, the up-regulation of sec63 could lead to trapping of bip concomitant with defects in er translocation and homeostasis. however, we do not favor this interpretation, because the overexpression of two other bip-interacting erdjs, erdj1 and erdj4, did not impair the hbv.s steady-state protein level. also, the simultaneous overexpression of sec63 and bip did not revert the inhibitory effect of excess sec63 on the hbv.s cargo. nonetheless, the question remains whether sec63 acts in conjunction with bip. in case of the yeast sec63p, mutations of the hallmark hpd motif of its j domain abolish the functional interaction with kar2p, i.e., lead to a failure to stimulate the atpase activity of the chaperone [15, 40] . the overexpression of a corresponding mutant of the human sec63 compromised the steady-state levels of polytopic reporters but half the amount as compared to the wt protein. one interpretation of this finding may be that bip is not absolutely required for the sec63 action. alternatively, the hpd mutation may weaken but may not completely prevent the sec63/bip interaction. in favor of the latter interpretation are observations that j domain proteins can form stable complexes with hsp70 chaperones in both the adpand atp-bound states [41, 59] . notably, a recent analysis of the multi-spanning cystic fibrosis transmembrane conductance regulator has shown that the release of tms from the sec61 translocon and their membrane integration is atp-dependent [60] , although -thus far -there are no known core translocon components that contain atpase activity. possibly, the sec63/bip participation may provide the missing link. human sec62, sec63, erdj1, erdj4, and bip were obtained as full-length cdna clones from imagenes. for n-terminal tagging with the myc epitope, sec62 and sec63 were cloned into pcmv-myc (bd biosciences, clontech). the sec63dhpd and sec63dc mutants were created by mutagenesis using the quikchangeh ii xl site-directed mutagenesis kit (stratagene). sec63dhpd carries alanine substitutions of the amino acids (aa) hpd at positions 132 to 134, while sec63dc contains a premature stop codon generated at aa position 444 thereby deleting the cterminal domain (aa 444-760) of sec63. erdj1, erdj4, and bip were inserted into p3xflag-cmv-14 (sigma-aldrich) thereby giving raise to c-terminally flag-tagged constructs. all constructs were verified by sequencing and cloning details are available on request. mammalian expression vectors carrying the wt or mutant hbv envelope genes have been described previously [22, 28] . briefly, pni2.s.ha (hbv.s) and pni2.l.ha (hbv.l) encode the s or the l gene, respectively, with a c-terminally fused ha epitope under the transcriptional control of the hmtiia promoter. the hbv.sdtm1 mutant lacks the first tm segment of the s protein, while the hbv.ile9::l mutant carries the l gene with a foreign n-terminal signal sequence derived from human interleukin-9 [27, 28] . the bicistronic plasmid pcdna/ha.hip-l (hbv.l/hip) encodes the ha-tagged l gene and the ha-tagged human hip gene under the control of the hmtiia or cmv promoter, respectively [22] . the m envelope gene of mhv strain a59, present in plasmid ptz19r-m [37] , was a gift from p. j. m. rottier (utrecht university, the netherlands) and was subcloned into pcmv.ha (bd biosciences, clontech) for c-terminal tagging with the ha epitope (mhv.m). plasmid pci-vsvg expresses the g glycoprotein of vsv under control of the cmv promoter [35] and was obtained from g. nolan (stanford university school of medicine, usa) as ''addgene plasmid 1733''. plasmid peyfp-er encodes yfp with an n-terminal er targeting sequence of calreticulin and a c-terminal er retrieval sequence (bd biosciences, clontech). plasmid peyfp-golgi carries a fusion gene consisting of yfp preceded by a n-terminal 81 aa sequence of human b1,4-galactosyltransferase (bd biosciences, clontech). the construct for the human ha-tagged hemopexin (hxp) has been described [61] . vectors encoding human cd63 with an nterminal ha-tag or n-terminal egfp fusion under control of the cmv promoter were gifts from g. spoden (university mainz, germany). for egfp tagging, the open reading frame of cd63 encoding aa 2-236 was fused in frame to aa 236 of egfp (cd63.gfp) present in plasmid pegfp-c1 (bd biosciences, clontech). to inhibit expression of sec63, sirna duplexes targeting the nucleotide positions 1942 to 1960 of sec63 (caa-gaatggtggtggcttt) or esirna were used (sigma-aldrich). as a control, a nonsense sirna with no known homology to mammalian genes was used (quiagen). commercially available antibodies were as follows: mouse antib-actin (sigma-aldrich), rabbit anti-calnexin (santa cruz biotechnology), mouse anti-flag (sigma-aldrich), mouse anti-gfp (bd biosciences, clontech), mouse anti-ha (babco), rat anti-ha (roche), mouse anti-myc (babco), rabbit antibody against human sec63 (sigma-aldrich), and mouse anti-vsv-g (sigma-aldrich). peroxidase-labeled, secondary antibodies were obtained from dianova, and fluorophor-labeled antibodies were purchased from molecular probes. to probe for protein expression, cells were lysed with either the non-denaturing detergent nonidet p-40 (np-40) or the denaturing reagent sodium dodecylsulfate (sds). np-40 lysates were prepared by incubating the cells with tris-buffered saline (50 mm tris-hcl ph 7.5/150 mm nacl) containing 0.5% np-40 for 20 min on ice. thereafter, lysates were centrifuged for 5 min at 13 000 g and 4uc. for lysis with sds, the cells were scraped from the plates using 1 6 laemmli buffer, and cell suspensions were boiled for 10 min prior to centrifugation. cell extracts were subjected to sds-page and western blotting analyses using standard procedures. immunoreactivity was determined by enhanced chemiluminescence (western lightningh plus-ecl, perkinelmer) and recorded on amersham hyperfilm ecl (ge healthcare). the densitometric analysis of western blot band intensities was based on linear, 8-bit, gray scale, transmitted light tiff scans of the exposed films and was performed by using imagequanth software (molecular dynamics). in parallel, cell culture media was harvested, cleared by centrifugation, and assayed for the secretion/release of proteins. the expression and secretion of the hbv.s envelope protein was measured using the murex hbsag version 3 elisa (abbott). to evaluate the presence of damage and toxicity of transfected cells, lactate dehydrogenase (ldh) activity was determined in culture media using a colorimetric quantification assay (roche). microsomes were prepared from homogenized cells and subjected to a trypsin protection assay as described [22] . briefly, microsomes were proteolyzed with trypsin in the presence or absence of 0.5% np-40 for 60 min on ice. after inactivation of trypsin with aprotinin, solubilized samples were subjected to sds-page and immunoblotting. for immunostaining, cells grown on cover-slips were fixed and permeabilized with ice-cold methanol containing 2 mm egta. cells were blocked in pbs containing 2% animal serum, incubated with the indicated primary antibodies for 1 h at 37uc, rinsed with pbs, and then incubated with alexafluor-tagged secondary antibodies for 1 h at 37uc. dna was stained with hoechst 33342 (sigma-aldrich). z-stack images were acquired separately for each channel using a zeiss axiovert 200 m microscope equipped with a plan-apochromat 1006 (1.4 na) and a zeiss axiocam digital camera and were optically deconvoluted using the software supplied by zeiss. phase contrast images were obtained with the same microscope using phase contrast optics. for quantitative immunofluorescence analyses, images of randomly selected cells (about 100 cells per coverslip; n = 5) were collected at 1006 magnification with identical settings. the mrna of cotransfected huh-7 cells was isolated using the dynabeadsh mrna direct tm kit (life technologies) accord-ing to the mini-scale guidelines. to eliminate trace amounts of genomic and plasmid dna, the mrna was treated with 5 u dnase i (roche applied science) at 30uc for 20 minutes. reverse transcription was performed with the transcriptor universal cdna master kit (roche applied science) and the cdna was diluted 1:3. the mrna levels were determined by a taqmanchemistry based quantitative real-time pcr as absolute quantification using the standard curve method. therefore, serial dilutions of plasmid dna containing the respective target dna (range 1610 6 to 1610 21 fg/ml) were amplified in parallel. the pcr was performed with a 7300 real-time pcr system and the sequence detection software 4.0 (life technologies). each pcr reaction contained 4 ml aqua bidest, 0.4 ml of each primer (100 mm), 0.2 ml of each taqman probe (100 mm), 12.5 ml taqmanh gene expression master mix (life technologies) and 7.5 ml cdna. primers and probes are listed in table 1 . the pcr reaction was initiated by a single step at 50uc for 2 minutes and 95uc for 10 minutes followed by 38 cycles at 95uc for 15 seconds and at 60uc for 60 seconds. amplifications of samples and standard curves were performed in duplicate. the calculated data were converted into percentages whereupon the mean value of the control samples was set to 100%. protein transport into the endoplasmic reticulum: mechanisms and pathologies protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes protein translocons: multifunctional mediators of protein translocation across membranes the beta subunit of the sec61 complex facilitates cotranslational protein transport and interacts with the signal peptidase during translocation an evolving view of the eukaryotic oligosaccharyltransferase protein translocation into proteoliposomes reconstituted from purified components of the endoplasmic reticulum membrane signal sequencedependent function of the tram protein during early phases of protein transport across the endoplasmic reticulum membrane substrate-specific function of the translocon-associated protein complex during translocation across the er membrane erj1p has a basic role in protein biogenesis at the endoplasmic reticulum homologs of the yeast sec complex subunits sec62p and sec63p are abundant proteins in dog pancreas microsomes life and death of a bip substrate bip and sec63p are required for both co-and posttranslational protein translocation into the yeast endoplasmic reticulum mammalian sec61 is associated with sec62 and sec63 evolutionary gain of function for the er membrane protein sec62 from yeast to humans interaction of bip with the j-domain of the sec63p component of the endoplasmic reticulum protein translocation complex the er function bip is a master regulator of er function mutations in sec63 cause autosomal dominant polycystic liver disease tailanchored and signal-anchored proteins utilize overlapping pathways during membrane insertion post-translational alterations in transmembrane topology of the hepatitis b virus large envelope protein a dramatic shift in the transmembrane topology of a viral envelope glycoprotein accompanies hepatitis b viral morphogenesis novel transmembrane topology of the hepatitis b virus envelope proteins chaperone action in the posttranslational topological reorientation of the hepatitis b virus large envelope protein: implications for translocational regulation mammalian bip controls posttranslational er translocation of the hepatitis b virus large envelope protein hepatitis b surface antigen: an unusual secreted protein initially synthesized as a transmembrane polypeptide hepatitis b virus morphogenesis hepatitis b surface antigen assembles in a post-er, pre-golgi compartment deletions in the hepatitis b virus small envelope protein: effect on assembly and secretion of surface antigen particles hepatitis b virus large envelope protein interacts with gamma2-adaptin, a clathrin adaptor-related protein posttranslational n-glycosylation of the hepatitis b virus large envelope protein mutant analysis links the translocon and bip to retrograde protein transport for er degradation one step at a time: endoplasmic reticulumassociated degradation multiple proteases process viral antigens for presentation by mhc class i molecules to cd8(+) t lymphocytes activation of erad pathway by human hepatitis b virus modulates viral and subviral particle production hemopexin: structure, function, and regulation rapid production of retroviruses for efficient gene delivery to mammalian cells using 293t cellbased systems a single n-linked oligosaccharide at either of the two normal sites is sufficient for transport of vesicular stomatitis virus g protein to the cell surface membrane assembly of the triple-spanning coronavirus m protein. individual transmembrane domains show preferred orientation the tetraspanin cd63 regulates escrt-independent and -dependent endosomal sorting during melanogenesis erdj4 protein is a soluble endoplasmic reticulum (er) dnaj family protein that interacts with erassociated degradation machinery the lumenal domain of sec63p stimulates the atpase activity of bip and mediates bip recruitment to the translocon in saccharomyces cerevisiae interaction of murine bip/ grp78 with the dnaj homologue mtj1 structural studies and the assembly of the heptameric post-translational translocon complex role of protein translocation pathways across the endoplasmic reticulum in trypanosoma brucei the proteinconducting channel in the membrane of the endoplasmic reticulum is open laterally toward the lipid bilayer the sec61p complex mediates the integration of a membrane protein by allowing lipid partitioning of the transmembrane domain x-ray structure of a protein-conducting channel sequential triage of transmembrane segments by sec61alpha during biogenesis of a native multispanning membrane protein two translocating hydrophilic segments of a nascent chain span the er membrane during multispanning protein topogenesis the hydrophobic core of the sec61 translocon defines the hydrophobicity threshold for membrane integration different transmembrane domains associate with distinct endoplasmic reticulum components during membrane integration of a polytopic protein cotranslational protein integration into the er membrane is mediated by the binding of nascent chains to translocon proteins forced transmembrane orientation of hydrophilic polypeptide segments in multispanning membrane proteins the concept of translocational regulation a protein of the endoplasmic reticulum involved early in polypeptide translocation a genetic interaction network of five genes for human polycystic kidney and liver diseases defines polycystin-1 as the central determinant of cyst formation pkd2, a gene for polycystic kidney disease that encodes an integral membrane protein epitope-tagged pkhd1 tracks the processing, secretion, and localization of fibrocystin differential effects of sec61alpha-, sec62-and sec63-depletion on transport of polypeptides into the endoplasmic reticulum of mammalian cells role of the j-domain in the cooperation of hsp40 with hsp70 sequencespecific retention and regulated integration of a nascent membrane protein by the endoplasmic reticulum sec61 translocon gammaadaptin, a novel ubiquitin-interacting adaptor, and nedd4 ubiquitin ligase control hepatitis b virus maturation we thank g. nolan, p. j. m. rottier, and g. spoden for generously providing plasmid dna constructs. key: cord-304616-k92fa15l authors: izes, aaron m.; kimble, benjamin; norris, jacqueline m.; govendir, merran title: assay validation and determination of in vitro binding of mefloquine to plasma proteins from clinically normal and fip-affected cats date: 2020-08-05 journal: plos one doi: 10.1371/journal.pone.0236754 sha: doc_id: 304616 cord_uid: k92fa15l the antimalarial agent mefloquine is currently being investigated for its potential to inhibit feline coronavirus and feline calicivirus infections. a simple, high pressure liquid chromatography assay was developed to detect mefloquine plasma concentrations in feline plasma. the assay’s lower limit of quantification was 250 ng/ml. the mean ± standard deviation intraand inter-day precision expressed as coefficients of variation were 6.83 ± 1.75 and 5.33 ± 1.37%, respectively, whereas intraand inter-day accuracy expressed as a percentage of the bias were 11.40 ± 3.73 and 10.59 ± 3.88%, respectively. accordingly, this validated assay should prove valuable for future in vivo clinical trials of mefloquine as an antiviral agent against feline coronavirus and feline calicivirus. however, the proportion of mefloquine binding to feline plasma proteins has not been reported. the proportion of drug bound to plasma protein binding is an important concept when developing drug dosing regimens. as cats with feline infectious peritonitis (fip) demonstrate altered concentrations of plasma proteins, the proportion of mefloquine binding to plasma proteins in both clinically normal cats and fip-affected cats was also investigated. an in vitro method using rapid equilibrium dialysis demonstrated that mefloquine was highly plasma protein bound in both populations (on average > 99%). successful treatment of feline infectious peritonitis (fip), a fatal, virulent coronavirus infection affecting predominantly younger cats, remains difficult [1] . reviews describing the virus responsible for fip as well as issues with diagnostics and therapeutics are available [1, 2] , providing an explanation as to why re-purposing drugs used in human medicine has been largely unsuccessful in treating fip infected cats [1] . recently, the use of antiviral therapies to alter the replication of virulent forms of feline coronavirus known as feline infectious peritonitis virus (fipv) has shown enormous hope for clinicians [3, 4] . however, veterinary access to these antiviral treatments is currently limited, leading to the global emergence of expensive, unregistered versions of these drugs without the necessary quality control assurances [4, 5] . accordingly, the need for inexpensive, safe, antiviral medications to treat fipv-infected cats remains. plos one | https://doi.org/10.1371/journal.pone.0236754 august 5, 2020 1 / 10 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 mefloquine is currently used for both prevention (as a monotherapy) and treatment (either alone, or in combination with artesunate) of chloroquine-resistant plasmodium falciparum malaria in humans [6] [7] [8] . it also has been shown to reduce the viral load of fipv and feline calicivirus (fcv) at low concentrations in infected crandell rees feline kidney cells without cytotoxic effects [9, 10] . using an in vitro assay, our team demonstrated that while mefloquine undergoes some phase i metabolism in cats, it does not undertake phase ii glucuronidative metabolism in this species [11] . in anticipation of conducting in vivo clinical trials of mefloquine to treat fipv-infected cats, mefloquine's plasma concentrations must be monitored to optimise the dosage regimen. knowledge of the amount of drug bound to plasma proteins is also important when optimising dosage regimens as only the unbound (or free) fraction is therapeutically active [12, 13] . although the plasma protein binding of mefloquine has been determined to be > 98% in humans [14] , its binding percentage in cats has not been reported. this information is particularly germane in any investigation of a potential fip antiviral as fip-affected cats have altered concentrations of the plasma proteins albumin (decreased) and globulins (elevated) compared to clinically normal cats [1, 15] . consequently, the aim of this study was two-fold: first, to develop and validate a high pressure liquid chromatography (hplc) method to detect mefloquine in feline plasma, and second, to determine the in vitro plasma protein binding of mefloquine in both clinically normal and fip-affected cats. mefloquine hydrochloride and verapamil hydrochloride (as the internal standard [is]) were purchased from sigma-aldrich (castle hill, nsw, australia). hplc grade methanol, hplc grade acetonitrile, phosphate buffered saline ph 7.4 (pbs) and triethylamine were purchased from thermo fisher scientific (macquarie park, nsw, australia). mefloquine and verapamil were quantified in feline plasma based on modified hplc method [16] . the hplc system consisted of a shimadzu lc-20at delivery unit, dgu-20a3 ht degassing solvent delivery unit, sil-20a auto injector, spd-20a uv detector and cto-20a column oven. shimadzu lc solution software version 1.24 (kyoto, japan) was used for chromatographic control, data collection and data processing. chromatographic separation was performed with a microsorb-mv c18 column (250 mm × 4.6 mm i.d., 5.0 μm; varian, mulgrave, vic, australia) with a 1.0 mm optic-guard c18 pre-column (choice analytical, thornleigh, nsw, australia) under a pressure of 1,900 psi at 25˚c. the isocratic mobile phase consisted of a mixture of 25 mm sodium phosphate buffer with 1.0% triethylamine adjusted to ph 2.5 and acetonitrile and methanol (50:25:25, v/v/v) at a flow rate of 1.0 ml/min. the injection volume was 10 μl per sample. the diode array detector was set at a wavelength of 220 nm. the retention times of verapamil and mefloquine were approximately 8.5 and 14.5 minutes, respectively. stock solutions of mefloquine and verapamil were prepared in 100% methanol and 100% acetonitrile, respectively, both at a concentration of 1.0 mg/ml. further working solutions of mefloquine were prepared in 100% methanol to achieve final concentrations of 500, 1,000, 2,500, 5,000, 10,000, 25,000 and 50,000 ng/ml. similarly, working solutions of verapamil were prepared in 100% acetonitrile to yield final concentrations of 12.5 μg/ml for the validation study and 5.0 μg/ml for the plasma protein binding study. stock solutions and working solutions were stored at 4.0˚c prior to use. blank, clinically normal, feline plasma was pooled (n > 5) and stored at -20˚c prior to use for preparation of standards and quality control (qc) samples. the origin of the plasma samples is described below. mefloquine standard samples (50, 100, 250, 500, 1,000, 2,500 and 5,000 ng/ml) and three quality control samples (qc) (250, 1,000 and 5,000 ng/ml) were prepared by spiking 10 μl of prepared working solutions of mefloquine into 90 μl of blank feline plasma. the proteins within the plasma samples were extracted through simple protein precipitation. specifically, 100 μl of acetonitrile, containing either 12.5 μg/ml of the is for the validation study or 5.0 μg/ml of the is for the plasma protein binding study, was added to the 100 μl feline plasma samples. the samples were then vortexed and centrifuged at 14,000 × g for ten minutes to remove any particulates. the supernatant was injected into the hplc system. assay selectivity was established by analysing pooled (n > 5) blank, clinically normal, feline plasma to ensure that there were no endogenous interference peaks around the retention times of mefloquine and verapamil. mefloquine concentrations were measured via standard curves performed on three replicates of each concentration of mefloquine (100, 250, 500, 1,000, 2,500 and 5,000 ng/ml) on three consecutive days. a weighting factor (1/x) was used to assign the relative importance of the observations in the regression; in this case, to ensure that larger observations were not over-fitted. the theoretical lower limit of detection (llod, the lowest analyte concentration that can be distinguished from the assay background noise) and the lower limit of quantification (lloq, the lowest concentration at which an analyte can be reliably detected at a specified level of accuracy and precision) were estimated as follows [17] : llod = 3.3 x σ /s, where σ is the standard deviation of the y-intercepts from the regression lines and s is the mean slope of the calibration curves. thereafter, the lloq was calculated using the formula lloq = 3 x llod. acceptance criteria for the lloq was defined as precision with a cv � 15% and accuracy within ± 20% of nominal concentration with repeated analyses [18] . intra-and inter-day precision, expressed as cv (%), were analysed from triplicates of qc samples (250, 1,000 and 5,000 ng/ml), both within a single day and on three consecutive days, respectively. intra-and inter-day accuracy, expressed as bias, were determined by a percentage difference between the estimated value and the nominal value of mefloquine as follows: bias (%) = 100 -(estimated value / nominal value × 100). absolute recovery of mefloquine and is were determined by comparing the peak area of pre-spiked plasma samples (n = 9) at concentrations of 250, 1,000 and 5,000 ng/ml with corresponding concentrations of mefloquine and is in mobile phase. each assay was conducted in triplicate. the linearity of the data was assessed through linear regression analysis with graphpad prism software version 8.1.1 (graphpad software, inc., ca, usa). with owner verbal or written consent, blood was collected from both clinically normal and fip-affected cats. the use of the residual plasma from this patient for this study was approved by the university of sydney animal ethics committee (protocol: 2016/1027). the animals were considered clinically normal based on inclusion criteria modified from that of norris et al. (2007) [19] . specifically, the cats designated for inclusion as clinically normal subjects were systemically well and had their blood collected for purposes other than investigating a current illness. examples of clinically normal cats included those presenting for annual examinations, routine vaccinations or for routine screening tests prior to sedation or general anaesthesia for de-sexing, grooming, routine dental scaling or radiography post-acute trauma [19] . in contrast, a cat's fip status was confirmed by direct immunofluorescence of effusion samples and immunohistochemistry on tissue samples (n = 5), or simply immunohistochemistry (n = 5) using methods outlined by worthing et al. (2012) [20] . additionally, other haematological tests were performed including quantification of albumin: globulin which was consistent with the hyperglobulinaemia observed with cats infected with fipv [15] . all plasma samples were stored at -20˚c prior to analysis. plasma samples from both clinically normal and fip-affected cats were provided by the paddington cat hospital (sydney, nsw) and the veterinary pathology and diagnostic service, sydney school of veterinary science. the in vitro plasma protein binding (ppb) of mefloquine was determined by the rapid equilibrium dialysis (red) method. the red assay was performed according to manufacturer's (thermo fisher scientific, macquarie park) instructions. pooled plasma samples (n > 5) were assigned to one of two experimental groups, depending on health status, i.e., either clinically normal cat plasma or fip-affected plasma. prior to experimentation, the ph of the pooled plasma (approximately ph 8) was adjusted to ph 7.3 [21] , to mimic the physiologic ph of cats. likewise, the total protein (tp), albumin and globulin concentrations of the pooled plasma samples were quantified prior to the assay by the vpds using a thermo fisher scientific konelab prime 30i analyzer (scoresby, vic, australia) employing conventional protocols. for each experimental group, two final concentrations of mefloquine (either 5,000 or 10,000 ng/ml) were tested using three replicates for each concentration. these concentrations were selected as they are comparable to the 10 μm concentration (10 μm = 3.78 μg/ml or 3,780 ng/ml) tested by mcdonagh et al. (2014) [10] in fipv-infected crandell rees feline kidney cells. accordingly, 300 μl of plasma pre-spiked with mefloquine was added to the redringed chamber of the red device whilst 550 μl of pbs was added to the white-ringed chamber. the unit was covered with sealing tape (thermo fisher scientific, scoresby, vic, australia, product no. 15036) and incubated on an orbital shaker at 250 rpm for four hours. the temperature was set at 38˚c to mimic the normal body temperature of cats. following incubation, 100 μl of post-dialysis samples were removed from the red-ringed chambers. these samples were then placed in separate microcentrifuge tubes and precipitated using 100 μl of chilled acetonitrile pre-spiked with the is (5.0 μg/ml verapamil). after vortexing, the mixtures were centrifuged at 14,000 × g for ten minutes. the supernatant was injected into the hplc system. similarly, 200 μl volumes of post-dialysis samples were removed from the whiteringed chambers. these samples also underwent hplc analysis. all samples were prepared and analysed in triplicate. the ppb of mefloquine was determined by the following equation: %free ¼ ðconcentration buffer chamber=concentration plasma chamberþ � 100% %bound ¼ 100 à %free statistical data analysis was performed using graphpad prism software version 8.1.1 (graph pad software, inc., ca, usa). prior to parametric testing, a shapiro-wilk normality test performed on the mefloquine ppb data indicated a normal distribution. likewise, an inspection of a normal quantile-quantile (qq) plot of this data further corroborated its normality. a twoway anova evaluating the effects of health status and drug concentration on mefloquine ppb was performed. results were considered statistically significant at p < 0.05. based on the uv spectra, the greatest area under the mefloquine peak was at a wavelength of 220 nm. the retention times of the is and mefloquine were approximately 8.5 and 14.5 minutes, respectively. no endogenous interference was observed at the retention times of mefloquine and the is. chromatograms of feline plasma pre-spiked with mefloquine and the is as well as blank feline plasma are shown in fig 1. selectivity. pooled blank feline plasma and feline plasma pre-spiked with mefloquine (250, 1,000 and 5,000 ng/ml) and the is (12.5 μg/ml) were used to check the selectivity of this method. due to the limited volume of fip-affected feline plasma available, only blank plasma of clinically normal cats was used. as demonstrated in fig 1, no endogenous plasma components interfered with elution of analytes. linearity, llod, lloq, accuracy and precision. the plasma peak ratio (area of mefloquine divided by the is area) versus the concentration was plotted and determined to be linear for the concentration range used (100 to 5,000 ng/ml). the mean regression standard curves (n = 3) for mefloquine were described as y = 9.374 e-005 x + 0.001417, with a weighting factor of 1/x. the correlation coefficient value (r 2 ) for each curve � 0.97. estimated from standard curves, the llod for mefloquine was 20.5 ng/ml and the lloq was 61.5 ng/ml. however, as mefloquine concentrations < 250 ng/ml were not accurate (>20% accuracy), the lloq was set as 250 ng/ml. intra-and inter-day precision expressed as cvs ranged from 5.30 to 8.74% and 3.77 to 6.32%, respectively. intra-and inter-day accuracy expressed as a percentage of the bias ranged from -12.67 to 13.96% and -15.01 to 9.04%, respectively. these values satisfied the guidelines regarding assay reliability [18] . intra-and inter-day precision and accuracy are summarised in table 1 . drug recovery from plasma. the absolute recovery rates of mefloquine expressed as percentages ± standard deviation (s.d.) from the 250, 1,000, and 5,000 ng/ml qc samples (n = 3) were 107.82 ± 5.93; 99.91 ± 5.58; and 105.21 ± 6.32, respectively. the absolute recovery rate of the is expressed as a percentage ± s.d. was 123.74 ± 3.77 (n = 9). in vitro plasma protein binding of mefloquine in healthy and fip-affected cats. the measured concentrations of total protein, albumin and globulin in the pooled plasma for the respective experimental groups are provided in table 2 . although reference intervals (ri) vary depending on the laboratory and the methodology used for quantification, reference intervals from cornell university's animal health diagnostic center [22] were used as none were provided by the testing laboratory. the results of in vitro plasma protein binding of mefloquine in clinically normal and fipaffected cats are provided in table 3 . a significant difference was found between the plasma protein binding of mefloquine in clinically normal and fip-affected cats (p = 0.0004), even though mefloquine was determined to be highly plasma protein bound (on average > 99%) in both groups. moreover, a significant interaction effect between health status and mefloquine concentration was identified (p = 0.0007). in contrast, no significant difference was discovered between the plasma protein binding of the two concentrations of mefloquine (p = 0.11). there has been recent interest in the suitability of mefloquine as an antiviral treatment for fip [9, 10, 23] . the majority of publications that detect and quantify mefloquine use liquid chromatography. liquid chromatography demonstrates good sensitivity (i.e., it can detect low concentrations of mefloquine), is highly specific and the machinery is affordable for veterinary diagnostic laboratories. although an hplc assay to detect mefloquine in an in vitro matrix has been reported [23] , in contrast, this study describes an assay to quantify mefloquine table 1 . intra-and inter-day precision and accuracy for the qc samples (250, 1000 and 5000 ng/ml of mefloquine) tested in triplicate for each concentration. to the authors' knowledge, this is the first report of mefloquine ppb in a non-human species. this study provides the novel finding that mefloquine is highly plasma protein bound in cats. in humans, mefloquine is also highly plasma protein bound (> 98%) [14] , which may account, in part, for its long half-life of approximately three weeks in healthy human subjects [24] . high plasma protein binding can result in the drug-protein complex acting as a reservoir for the physiologically active free drug concentration and consequently prolonging its duration of action [12, 25] . likewise, it should not be assumed that a drugs' ppb is constant across species [25] . in a comparative species study investigating the plasma protein binding of 574 compounds, drugs tend to be slightly more bound in human plasma proteins in comparison to the plasma proteins of rats, mice and dogs [26] . some of the compounds that showed significant interspecies differences in plasma protein binding included diazepam (98% ppb in humans versus 84.8% in rats), prazosin (94.7% ppb in humans versus 65% in rats versus 63% in dogs) and sildenafil (93.6% ppb in humans versus 74% in dogs) [26] . diseases, such as fip, can alter the concentrations of plasma proteins and in turn impact upon a drug's plasma protein binding and ultimately its therapeutic efficacy [27] . for example, in humans, the proteins albumin and α 1 -acid glycoprotein (aag) provide the largest contribution to the protein binding of drugs [27, 28] . yet, with acute falciparum malaria, serum concentrations of aag in non-immune human patients increase two-fold within 24 hours whereas plasma levels of albumin decrease by 30% [29] . likewise, in fip-affected cats, common serum protein abnormalities may include elevated aag [30] and globulin levels [1, 15] as well as decreased albumin levels and a low a: g ratio [15] . here, as described in the literature, the confirmed fip plasma samples demonstrated elevated globulin and decreased albumin levels as well as a low a: g ratio. mefloquine has a high affinity for aag binding, preferentially binding to aag over albumin [27] . thus, if more aag is present in fip-affected cats, it may be preferentially bound by mefloquine. yet, small changes in the unbound drug fraction of highly protein bound drugs can have a significant therapeutic impact [28] . for example, the reduction of ppb from 99.9% to 99.8% can lead to a doubling of therapeutically active unbound drug concentration in plasma [28] . here, although a significant difference was found between the plasma protein binding of mefloquine in clinically normal and fip-affected cats, due to the unknown biological variability of the assay, it is likely that this difference is equivocal. fundamentally, it is difficult to generalise from two pooled plasma samples to two populations of individuals since the biological variability in each population is unknown. the variability (standard deviation) used to evaluate differences in ppb were derived from the assay method and do not represent biological differences. yet, it is also unlikely that further in vitro mefloquine feline plasma protein binding studies will resolve this issue. on the contrary, observing the clinical response of mefloquine in fip-affected animals ultimately may decide mefloquine's therapeutic efficacy in this patient population. in vitro studies to quantify plasma protein binding have some limitations. whilst the major advantage of the red method include its speed, simplicity, reliability and cost-effectiveness [31] , its drawbacks, which also serve as limitations to this study, involve nonspecific membrane binding of the drug [31] as well as changes in oncotic pressure leading to an overestimation of unbound drug concentration [32] . moreover, in vitro ppb may not mirror in vivo ppb in the live animal as the structure of the plasma proteins and their affinity to bind substrates can vary with age, disease and /or presence of competing endogenous and exogenous compounds such as dietary constituents or other therapeutic drugs [33, 34] . furthermore, the drug-protein dissociation rate, which can also greatly affect highly ppb drugs [35] , was not determined in this study. likewise, an additional limitation to this project was the scarcity of confirmed fipaffected plasma as this impacted upon its availability for use in both the hplc validation and ppb protocols. finally, given mefloquine's purported mechanism of action as a schizonticide, another potential limitation concerns the effects of the intraerythrocytic accumulation of mefloquine on plasma protein binding. however, previous studies have demonstrated that red blood cells do not serve as a significant depot for mefloquine [36, 37] . recently, the broad spectrum coronavirus protease inhibitor, gc376 [38] , and the adenosine nucleoside analogue, gs-441524 [3, 4] have been shown to be safe and efficacious antiviral agents for use against fipv. yet, as neither of these agents has obtained registration for veterinary use, investigations into more readily available drugs with anti-fipv activity, such as mefloquine, are urgently required for this invariably fatal disease. this study has validated an accurate and reliable assay to detect mefloquine in feline plasma and demonstrated that mefloquine is highly plasma protein bound in both clinically normal and fip-affected cats. further studies describing mefloquine's pharmacokinetic profile in the cat should be progressed. an update on feline infectious peritonitis: diagnostics and therapeutics a review of feline infectious peritonitis virus infection: 1963-2008 the nucleoside analog gs-441524 strongly inhibits feline infectious peritonitis (fip) virus in tissue culture and experimental cat infection studies efficacy and safety of the nucleoside analog gs-441524 for treatment of cats with naturally occurring feline infectious peritonitis fifty years' fascination with fip culminates in a promising new antiviral mefloquine for preventing malaria in pregnant women the position of mefloquine as a 21 st century malaria chemoprophylaxis patient age does not affect mefloquine concentrations in erythrocytes and plasma during the acute phase of falciparum malaria antiviral effect of mefloquine on feline calicivirus in vitro identification and characterisation of small molecule inhibitors of feline coronavirus replication in vitro hepatic metabolism of mefloquine using microsomes from cats, dogs and the common brush-tailed possum (trichosurus vulpecula) predicting plasma protein binding of drugs: a new approach drug-protein binding: a critical review of analytical tools clinical pharmacokinetics of mefloquine positive predictive value of albumin: globulin ratio for feline infectious peritonitis in a mid-western referral hospital population a high performance liquid chromatographic assay of mefloquine in saliva after a single oral dose in healthy adult africans defining limit of detection and limit of quantitation as applied to drug of abuse testing: striving for a consensus international conference on harmonisation of technical requirements for registration of pharmaceuticals for human use prevalence of feline immunodeficiency virus infection in domesticated and feral cats in eastern australia risk factors for feline infectious peritonitis in australian cats ph adjustment of human blood plasma prior to bioanalytical sample preparation chemistry (cobas) reference intervals in vitro hepatic metabolism of mefloquine using microsomes from cats, dogs and the common brush-tailed possum clinical application of mefloquine pharmacokinetics in the treatment of p. falciparum malaria in vitro binding of cefovecin to plasma proteins in australian marsupials and plasma concentrations of cefovecin following single subcutaneous administration to koalas (phascolarctos cinereus) species differences in drug plasma protein binding selective plasma protein binding of antimalarial drugs to α1-acid glycoprotein plasma protein binding and blood-free concentrations: which studies are needed to develop a drug? serum protein concentrations in plasmodium falciparum malaria critical assessment of the diagnostic value of feline α1-acid glycoprotein for feline infectious peritonitis using the likelihood ratios approach protein binding of antimicrobials: methods for quantification and for investigation of its impact on bacterial killing errors in estimating the unbound fraction of drugs due to the volume shift in equilibrium dialysis clinical pharmacology: plasma protein binding of drugs what is the true clinical significance of plasma protein binding displacement interactions? drug safety characterization of plasma protein binding dissociation with online spe-hplc characterization of in vivo metabolites of wr319691, a novel compound with activity against plasmodium falciparum studies of the disposition and metabolism of mefloquine hcl (wr 142,490), a quinolinemethanol antimalarial, in the rat reversal of the progression of fatal coronavirus infection in cats by a broad-spectrum coronavirus protease inhibitor professor michael court (washington state university, college of veterinary medicine) provided invaluable insight into the interpretation of the mefloquine plasma protein binding results. we also thank the veterinary pathology and diagnostics services, sydney school of veterinary science and dr. randolph baral (the paddington cat hospital, sydney, nsw), for providing the feline plasma samples. key: cord-349781-l93978vq authors: cong, yu; hart, brit j.; gross, robin; zhou, huanying; frieman, matthew; bollinger, laura; wada, jiro; hensley, lisa e.; jahrling, peter b.; dyall, julie; holbrook, michael r. title: mers-cov pathogenesis and antiviral efficacy of licensed drugs in human monocyte-derived antigen-presenting cells date: 2018-03-22 journal: plos one doi: 10.1371/journal.pone.0194868 sha: doc_id: 349781 cord_uid: l93978vq middle east respiratory syndrome coronavirus (mers-cov) presents an emerging threat to public health worldwide by causing severe respiratory disease in humans with high virulence and case fatality rate (about 35%) since 2012. little is known about the pathogenesis and innate antiviral response in primary human monocyte-derived macrophages (mdms) and dendritic cells (mddcs) upon mers-cov infection. in this study, we assessed mers-cov replication as well as induction of inflammatory cytokines and chemokines in mdms and immature and mature mddcs. immature mddcs and mdms were permissive for mers-cov infection, while mature mddcs were not, with stimulation of proinflammatory cytokine and chemokine upregulation in mdms, but not in mddcs. to further evaluate the antiviral activity of well-defined drugs in primary antigen presenting cells (apcs), three compounds (chloroquine, chlorpromazine and toremifine), each with broad-spectrum antiviral activity in immortalized cell lines, were evaluated in mdms and mddcs to determine their antiviral effect on mers-cov infection. while chloroquine was not active in these primary cells, chlorpromazine showed strong anti-mers-cov activity, but it was associated with high cytotoxicity narrowing the potential window for drug utilization. unlike in established cells, toremifene had marginal activity when tested in antigen presenting cells, with high apparent cytotoxicity, also limiting its potential as a therapeutic option. these results demonstrate the value of testing drugs in primary cells, in addition to established cell lines, before initiating preclinical or clinical studies for mers treatment and the importance of carefully assessing cytotoxicity in drug screen assays. furthermore, these studies also highlight the role of apcs in stimulating a robust protective immune response to mers-cov infection. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 middle east respiratory syndrome coronavirus (mers-cov) was first isolated in saudi arabia in 2012 from a patient with severe acute respiratory disease complicated by renal failure [1, 2] . since that time, the virus has caused sporadic outbreaks of mild-to-severe respiratory disease. approximately 80% of human cases have been reported in saudi arabia with 211 cases occurring in the first 9 months of 2017 [3] . beginning in may 2015, a large hospital-associated outbreak of mers occurred in the republic of korea. the outbreak in korea resulted in a total of 186 mers-cov cases, including 36 deaths, and was the largest outbreak of mers occurring outside of the arabian peninsula [4] . this outbreak highlighted the risk of international dissemination of mers-cov and the continued risk of nosocomial infection. as of september 6, 2017, the number of confirmed global cases of mers-cov infection reported to world health organization was 2079 cases in 27 countries with 722 fatalities, resulting in a case fatality rate around 35% [3] . mers-cov is a zoonotic virus that is transmitted from animals to humans with camels likely serving as the principal host for mers-cov [5] . while nosocomial infections are common, barrier nursing practices can limit spread of the virus as the virus does not seem to pass easily from person-to-person unless close contact occurs [6] . in humans, mers-cov infection typically causes a lower respiratory tract disease such as pneumonia, and common symptoms include fever, cough, sore throat, myalgia, and shortness of breath [7] . symptoms such as gastrointestinal complications and renal failure have also been reported in patients, especially those with severe chronic illness such as diabetes [6, 8] . systemic dissemination has been documented in locations such as the circulatory system and respiratory tract [9] . in the studies presented here, we had two principal objectives. the first was to determine whether human antigen presenting cells (apcs) were permissive to mers-cov infection. the second objective was to determine if these cells were suitable or appropriate for secondary screens for drugs that have been identified as effective in continuous culture cell lines. macrophages and dendritic cells (dcs) are professional apcs linking innate and adaptive immunity. these and other apcs act as a first defense against viral infection by stimulating immune surveillance, priming, and tolerance [10, 11] . appropriately functioning apcs are critical for the ability to mitigate infection and limit the development of disease. apcs are abundant in the respiratory tract where they provide immune surveillance and respond to local tissue inflammation in the airways and the distal lung. an important role of apcs is mitigating infection by producing cytokines that stimulate an inflammatory response and recruiting memory and effector cells to the site of infection [12] . professional apcs are also an important source of type i interferons (ifn-α/β). type i ifns have a significant bystander effect on uninfected neighboring cells by inducing an antiviral state, activating innate immune cells, and priming adaptive immunity. currently, no prophylactic or therapeutic options are proven as effective interventions for infection with mers-cov, severe acute respiratory syndrome coronavirus (sars-cov), or any other coronaviruses. to rapidly identify potential therapeutic options against emerging viral infections, investigators have adopted the approach of screening existing licensed drugs for efficacy against novel viral pathogens. screening licensed drugs could expedite the implementation of new medical countermeasures by providing an avenue for off-label use of compounds shown to be useful for the treatment of specific viral diseases. a number of pharmaceutical agents have potential for the treatment of coronaviruses, including neurotransmitter inhibitors, estrogen receptor antagonists, kinase signaling inhibitors, protein-processing inhibitors, and antiparasitic agents [13, 14] . results from previous studies found toremifene citrate (tomf), chlorpromazine (cpz) and chloroquine (cq) to be [13, 14] . we established a cell differentiation method to generate large amounts of monocyte-derived macrophages (mdms) and dendritic cells (mddcs) of high quality (>95% purity) for evaluating compounds against other viruses such as yellow fever and ebola viruses [15] . to address pharmaceutical efficacy against mers-cov infection in primary cells critical for blocking infection, we tested several candidate mers-antivirals in human mdms and immature dendritic cells. the tested compounds included tomf, an estrogen receptor antagonist, cpz, a neurotransmitter inhibitor, and cq, an antimalarial agent. all viral infection-related work was performed in the biocontainment laboratory at the integrated research facility (irf) of national institute of allergy and infectious disease (niaid)/ national institute of health (nih) using procedures and processes appropriate for the facility and the agent under study. all staff performing this work were trained to work in the biocontainment space and with mers-cov. human mdms and mddcs were differentiated at the irf as described previously [15] . briefly, cd14 + monocytes were isolated from human peripheral blood mononuclear cells for differentiation into mdms and mddcs. mdms were differentiated by culturing the purified cd14+ monocytes with human macrophage-colony stimulating factor (m-csf) (r&d systems, minneapolis, mn) and kpb-m15 conditioned medium (scgf research laboratory, ukyo-ku, jp) for 7 days. to generate mddcs, cd14+ monocytes were supplied with 20 ng/ml of human granulocyte macrophage-colony stimulating factor (gm-csf) and 10 ng/ml of interleukin (il)-4 in r-10 medium [rpmi-1640 (lonza, allendale, nj)] containing 10% heat inactivated fbs (millipore sigma). the cells were then incubated for 6 days at 37˚c and 5% co 2 with a change of medium every second day. immature mddcs were harvested at this point or stimulated to induce maturation. to induce maturation, the medium was replaced with a mddc maturation cocktail (r-10 medium containing 10 ng/ml of tumor necrosis factor (tnf)-α, 10 ng/ml of il-1β, 15 ng/ml of il-6, 1 μg/ml of prostaglandin e2 (peg2), 20 ng/ ml of gm-csf, 10 ng/ml of il-4) on day 6 post-plating and incubated overnight at 37˚c and 5% co 2 . the cells were then collected for phenotyping and further assays. mdms and mddcs were stained and analyzed as mentioned previously [15] . mdms and mddcs (immature and mature) were seeded at 5 × 10 5 cells per well in 48-well plates and incubated overnight at 37˚c and 5% co 2 . the next day, the cells were infected at a moi of 2 for 1 (hour) h at 37˚c and 5% co 2 . after the inoculum was removed, cells were washed twice with phosphate-buffered saline and cultured with r-10 medium. cell-free supernatants were collected, aliquoted to multiple microtubes at the indicated time-points, and frozen at -80˚c until used. the titer of virus stocks and cell-free supernatants collected from kinetics studies were determined by plaque assay on vero e6 cells. test samples were diluted serially 1:10, and 100 μl of the sample was added to each well of 6-well plates containing an 80-90% confluent monolayer of vero e6 cells. the plates were then incubated for 1 h at 37˚c and 5% co 2 with rocking every 15 mins for virus adsorption and infection. following incubation, a pre-warmed semisolid overlay (0.8% tragacanth [f/c] (millipore sigma) mixed in 1x eagle's minimum essential medium containing 2% fbs) was gently added to the cells. the plates were then incubated for 5 days at 37˚c and 5% co 2 without being disturbed. to fix the plates, the overlay was aspirated, and the cells were fixed and stained with neutral buffered formalin (nbf) (thermo fisher scientific) containing 0.2% crystal violet (f/c) (millipore sigma) for 30 min at room temperature. the plates were then washed with running water and air-dried. the plaques were then enumerated, and the virus titer was calculated. inflammatory mediator expression was measured using a custom human 10-plex cytokine bead arrays (cat# hcytomag-60k, millipore sigma, billerica, ma) on a luminex flex-map 3d tm system equipped with xponent 4.2 software. (luminex, austin, tx). cytokines and chemokines included in the panel were: ifn-α2, ifn-γ, tnf-α, il-6, il-8, il-12p40, monocyte chemotactic protein (mcp)-1, macrophage inflammatory protein (mip)-1α, interferon-inducible protein (ip)-10, and regulated upon activation normal t-cell expressed and secreted (rantes). the assays were performed according to the manufacturers' instructions in 96-well mylar plates (millipore) as described previously [15] . cell-free supernatants harvested from kinetics studies were mixed with beads coated with capture antibodies to various cytokines and chemokines. the mixtures were incubated with biotinylated detection antibodies. finally, pe-conjugated streptavidin was added, and the fluorescent signals were measured using a flexmap equipped with xponent 4.2 software (luminex corp). the raw data were measured as mean fluorescence intensity (mfi), and the concentration of each analyte was calculated using a 4-or 5-parameter logistic fit curve generated for each analyte from the 6-point concentration standard curve. the concentrations of all the analytes in the quality control reagents were found to be within the expected ranges. the lower limit of quantification (lloq) was determined using the lowest point on the standard fit curve that was at least 3 times above background. the calculation of the lloq was performed by subtracting the mfi of the background (diluent) from the mfi of the lowest standard concentration and back-calculating the concentration from the standard curve. data were further analyzed using prism 6.0 software (graphpad software, la jolla, ca). mdms or mddcs (100 μl) were plated in black opaque (cytotoxicity assay) or clear bottom (testing viral inhibition) 96-well greiner microplates (greiner bio-one, monroe, nc, 655948) at 1 × 10 5 cells/well and incubated at 37˚c and 5% co 2 overnight. drugs in powder formulations were dissolved in (dmso) (millipore sigma). final dmso concentrations were lower than 0.05%. drug solutions diluted in rpmi-1640 with 10% fbs (r-10 medium) to a 4x concentration were added into a 96-well, cell-free, drug dilution plate. drugs (50 μl/well) were transferred from the drug dilution plates to cell plates using the 96-well liquidator (rainin instrument, oakland, ca) 1 h prior to infection. duplicate cell plates for each cell type were infected with mers-cov at a moi of 0.1 by adding 50 μl of virus diluted in r-10 medium directly to the drug mixture in the cell plate and the plates incubated at 37˚c and 5% co 2 . after 48 h, cell culture supernatants were collected, and the cells were fixed with 10% nbf. the collected supernatants were clarified by centrifugation and stored at -80˚c for future use. to quantify the antiviral activity of the candidate compounds, a cell based immunofluorescence enzyme-linked immunosorbent assay was used as described previously [13] . the fixed plates, as described above, were stained with a rabbit polyclonal antibody to the mers-cov--emc/2012 s protein (sino biological) at 1:1000 followed by staining with alexa fluor 594-conjugated goat anti-rabbit igg (h+l) antibody (thermo fisher scientific) at 1:2500. fluorescence was quantified on a plate reader (infinite m1000 pro; tecan us, morrisville, nc) with an excitation wavelength of 590 nm and an emission wavelength of 617 nm. cytotoxicity assays were performed in parallel to measure drug toxicity. one black opaque cell plate for each cell type was mock infected using r-10 medium under the same conditions cd14 + monocytes were isolated from human peripheral blood mononuclear cells (pbmcs). cells were then differentiated to monocyte-derived macrophages (mdms) by adding macrophage-colony stimulating factor (m-csf) or to immature monocyte-derived dendritic cells (mddcs) by adding granulocyte macrophage-colony stimulating factor (gm-csf) and interleukin-4 (il-4). immature mddcs were exposed to mddc maturation cocktail (r-10 medium containing 10 ng/ml of tumor necrosis factor (tnf)-α, 10 ng/ml of il-1β, 15 ng/ml of il-6, 1 μg/ml of prostaglandin e2 (peg2), 20 ng/ml of gm-csf, 10 ng/ml of il-4) overnight for maturation. mdms and mddcs were characterized by flow cytometry for expression of major cell-surface markers. using these protocols, the mdms are predominantly cd14+, cd11b+, hla-dr+, cd169+, cd163+, and cd86+. the mddcs are predominantly cd14-, cd11c+, hla-dr+, cd80+, and cd86+. mature mddcs are cd83+, while immature mddcs are cd83-. https://doi.org/10.1371/journal.pone.0194868.g001 as the infected cells, and cell viability was measured using the celltiter glo luminescent cell viability assay kit (promega, madison, wi) according to the manufacturer's instructions. luminescence was read on the infinite1 m1000 pro plate reader (tecan). antiviral activity was evaluated by means of a 50% tissue culture infectious dose (tcid 50 ) assay using the spearman/kaerber method [16, 17] . briefly, a 10-fold serial dilution, 10 −1 to 10 −7 , was made from cell-free supernatant samples collected from the drug efficacy evaluation studies for cell-based enzyme-linked immunosorbent assay. these dilutions were used to infect vero e6 cells on 96-well plates with seven infected wells for each dilution. samples containing only cell culture media were used as a negative control, and virus stock was used as a positive control. test samples were added to each well at a volume of 100 μl. the plates were then incubated for 6 days at 37˚c and 5% co 2 to observe the mers-cov-induced cytopathology. following the incubation, the media were removed, and the cells were fixed and stained with 10% nbf containing 0.2% crystal violet. the plates were incubated for 30 min at room temperature and then washed with running water. the tcid 50 titer was determined by identifying the 50% endpoint in cytopathic effects in the cell monolayer. the results were calculated by applying the spearman/kaerber method and presented as log 10 tcid 50 /ml. non-linear regression analysis was performed to calculate 50% effective concentration (ec 50 ) of compounds and their specific toxicity (50% cytotoxicity concentration, cc 50 s) using prism 6.0 software (graphpad software) from the dose-response curves of the drugs. selectivity index (si) is defined as the ratio of the concentration of 50% cellular cytotoxicity to 50% effective concentration (si = = cc 50 äec 50 ), indicating the relative efficacy of a compound in viral replication inhibition as opposed to causing cytopathic effect. error bars represent the standard deviation of three replicates unless indicated otherwise. statistical significance determinations were performed using the results of at least two individual experiments or duplicate or triplicate replicates from a single experiment. for these studies, mdms and mddcs were carefully characterized to ensure uniformity and reproducibility between experiments. differentiated mdms and mddcs were characterized by flow cytometry as performed previously [15] . the mdms generated were predominantly cd14 + , cd11b + , hla-dr + , cd169 + , cd163 + , and cd86 + , confirming that the mdm population was highly purified, and over 95% of the cells generated were positive for macrophages (fig 1) . phenotyping of human mddcs showed they were cd14 -, cd11c + , hla-dr + , cd40 + , cd80 + , and cd86 + . the dc maturation marker, cd83, was present on 95.1% of mature mddcs (fig 1) compared to 8.13% of the immature mddcs and 0.98% of the unstained control cells (fig 1) . the isolation protocol used in these studies consistently generated highly purified phenotypically homogeneous apcs with minimal inter-experimental or inter-operator variability. to determine if primary apcs are permissive to mers-cov infection, kinetics studies were performed to measure virus titer following infection using cells isolated from four different donors. a 2-to 4-log 10 increase in viral titers was observed in mdms and immature mddcs despite variation between donors (fig 2a and 2b) . no significant increase in viral titer was observed in mature mddcs up to 8 days pi (fig 2c) indicating a restriction in virus infection or virus replication. virus clearance in mdms began at day 3 pi, and no virus could be detected by 6 to 8 days pi. however, mers-cov continued to propagate in immature mddcs up to 8 days pi, demonstrating differential infection and replication capabilities in mdms and immature mddcs. to compare the ability of mers-cov to induce innate immune responses in three types of apcs, the release of cytokines and chemokines was measured from virus-or mock-infected cells. the 10-plex human panel included the antiviral cytokines (ifn-α2 and ifn-γ), proinflammatory cytokines (tnf-α, il-6, and il-12p40), and chemokines (ip-10, mcp-1, mip-1α, rantes and il-8). in mdms, mers-cov infection resulted in increased concentrations of almost all antiviral or proinflammatory cytokines and most chemokines (except mcp-1), many of these cytokines or chemokines were released as soon as 1 h pi ( fig 3a) and may have been the result of direct cell stimulation rather than active infection. the high induction of ifn-γ, and ip-10 over the course of the infection in mdms was dramatic, indicating a very strong antiviral response was induced upon mers-cov infection. mers-cov infection repressed il-6 expression from mdm and this repression decreased over the course of several days ( fig 3a) with a similar response profile seen with chemokines mip-1α and mcp-1. increased mip-1α and ip-10 expression was observed in immature mddcs (fig 3b) . however, other than a bolus of ifn-γ released immediately after the infection of mddc, the release of most of the cytokines or chemokines from immature and mature mddcs were negligible over the course of the mers-cov infection relative to mock-infected cells (fig 3b and 3c) . as expected in a human population, considerable donor-to-donor variation in the cytokine response was observed following infection with mers-cov. a cell-based immunofluorescence assay was applied to evaluate the antiviral effectiveness of cq, cpz, and tomf in mdms, which adhere to 96-well plates and could be fixed and stained. cytotoxicity assays were performed in parallel using mock-infected cells after drug addition. tomf performs well inhibiting mers-cov replication in vero e6 cells (ec 50 = 12. 9 μm) [13] . in mdms, the activity of tomf at the dose range tested was too low to determine an ec 50 and the cytotoxicity was relative high (fig 4a) . unlike in vero e6 cells (ec 50 = 6.3 μm) [13] , cq did not show any detectable antiviral activity in mdms, although no cytotoxicity was associated with this compound (fig 4b) . cpz inhibited mers-cov in vero e6 cells (ec 50 = 9.5 μm) with low cytotoxicity [13] . however, in mdms, the cytotoxicity of cpz was relatively high (fig 4c, cc 50 = 25.64 μm. antiviral activity of cpz (ec 50 = 13.58 μm) did not separate well from cytotoxicity and led to a low selectivity index (si = 1.9). the cell-based immunofluorescence assay could not be applied to immature mddcs due to their poor adherence to the 96-well plate; cells could not be fixed for further antibody staining. therefore, the efficacy of the candidate compounds on mers-cov-infected apcs was evaluated by a median tcid 50 assay. mature mddcs did not produce detectable infectious virus after infection and were therefore excluded (fig 2c) in these drug activity studies. cell culture supernatants saved from the cell-based assay were titrated by tcid 50 to detect mers--cov to evaluate the efficacy of the compounds. there was no virus yield reduction in cqtreated mdms or immature mddcs, though the cytotoxicity was relativity low (fig 5a and 5b ). in cpz treated cells, around a 2 log 10 virus reduction was observed in both cell types. however, high cytotoxicity narrows the therapeutic window in both mdms and mddcs ( fig 5a and 5b) . a 1-1.5 log 10 virus reduction was measured in tomf-treated cells, but only when the dose is higher than 20 μm, with a corresponding increase in cytotoxicity (fig 5a and 5b) . these observations were similar when compared to the cell-based assay mentioned above. virus yield reduction data for tomf, cq and cpz in vero e6 cells showed that cpz was active against mers-cov infection with a 3.1 log 10 drop at the drug dosage of 15 μm, but tomf and cq were not active (fig 5c) . these data suggest a strong correlation between veroe6 cells and mdms when virus titers are determined by tcid 50 . as sporadic outbreaks of mers continue to occur, our current options for treatment of lifethreatening zoonotic coronavirus infections in humans are still lacking. the lack of treatment is due to the poorly understood pathogenesis of mers and other coronavirus infections, despite the extensive research efforts triggered by the 2003 sars outbreak [18] . human apc cells, mdms and mddcs, play important roles in bridging innate and adaptive immunity during viral infections by presenting viral antigens to t cells and by secreting appropriate cytokines and chemokines [19] . mdms and immature mddcs reside at the sites where most infections occur, such as the mucosal surfaces within the respiratory tract. in their role in the immune response, apcs are also a potential initial site for viral replication. additionally, apcs can act as viral reservoirs for further dissemination to organs as already shown for other human infectious diseases like measles and human immunodeficiency virus (hiv) infection [20, 21] . in this study, we assessed the characteristics of mers-cov growth kinetics in mdms and mddcs. we found that mers-cov can readily infect and robustly replicate in human mdms and immature mddcs, while the virus does not establish a notable infection in mature mddcs. high viral infectivity in mdms and immature mddcs indicates that the virus can overcome the initial antiviral response, but that the maturation of mddc imparts a restriction that prevents virus replication. the capacity of mddc to stimulate t cells is closely related to their maturation stage [22] . immature dcs are highly specialized at antigen uptake and processing, but are poor stimulators of primary immune responses [23] [24] [25] . productive infection of immature dcs may delay the activation of t cells, therefore allowing more time for mers-cov replication or dissemination. an additional reason that immature mddcs may be more susceptible to mers-cov infection than mature mddcs to mers-cov could be their ability to internalize and process viral antigens. after uptake of antigens or pathogens, immature mddcs may undergo maturation, migrate to lymphatic tissues, and trigger adaptive immune responses. in theory, the ability of sampling and presenting antigen should be enhanced through mddcs maturation. for example, immature mddcs may promote viral dissemination of sars-cov within the host by recruiting the virus to enter and damage the lymphatic tissues [26]. a recent study has shown that abortive herpes herpesvirus (hhv)-1 (herpes simplex virus-1) infection of mature mddc is able to modulate their inhibition of t-cell stimulatory function [27] . endocytosis in mature dcs is slowed somewhat relative to immature dcs, potentially the result of changes to their endocytic mechanism or receptor expression [28] . similarly, virus was disseminated systemically in a patient with mers [29]. following mers-cov infection, the induced immune response in apcs was consistent with their susceptibility to infection and the kinetics of virus propagation. the production of proinflammatory cytokines is essential for host resistance against mers-cov infection. infection of mdms with mers-cov appreciably induced the secretion of antiviral cytokines (ifn-α2, ifn-γ, and il-12p40), moderately or appreciably up-regulated proinflammatory cytokines (tnf-α, il-6), and variably upregulated inflammatory chemokines (mip-1α, ip-10, rantes, il-8). clinical studies have shown that expression of proinflammatory cytokines/chemokines, such as il-6, il-8, ifn-γ, or ip-10, is highly correlated with disease severity and mortality of sars patients [30] . nicholls et al reported that macrophages in lung tissues of sars patients were identified as the location where the "cytokine storm" and virus pathogenesis originated [31] . mdms secrete proinflammatory cytokines and chemokines to activate antiviral mechanisms, which may cause the dysregulated production of these inflammatory mediators leading to damage to neighboring tissues [32] . expression of il-6 and tnf-α are a potential indicators of severe respiratory viral infection, such as sars and human infection by avian influenza viruses [33] . our data indicate the possibility of an ifnγ th1-mediated inflammatory response that may cause severe mers disease before an adaptive host immune response is mounted. however, restriction of virus growth in mature mddcs results in no antiviral activation by ifn-α2. nevertheless, only moderate expression of ip-10 and minimal induction of mip-1α, mcp-1, and rantes were observed in immature mddcs. significant upregulation of most cytokines and chemokines was not observed in either mature or immature mddcs. overall, the limited antiviral cytokine response in mddcs suggests a possible mechanism of immune escape during mers-cov infection as seen in sars-cov infection [26] . a review of the literature suggests that there is a significant gap in knowledge regarding the role on intracellular regulatory mechanisms such as pathogen recognition receptors (prr) in the control of coronavirus infection. in this study there was essentially no change in the concentration of cytokines/chemokines measured in mature (and non-permissive) mddcs. in immature mddcs, while the measured concentration was less than the control cells in many cases, the concentrations of a number of cytokines/chemokines (il-6, ip-10, mip-1α, mcp-1) increased over the course of the infection, specifically on days 6 and 8 when titers were highest. these data suggest that prr may have been activated, as would be anticipated in a productive viral infection, but the scale and scope was not evaluated. in addition, data obtained for cytokine release very early after infection cannot be properly evaluated due to the potential presence of cellular components in the virus preparations. in several reports of immune responses triggered by apc, findings are similar, or conflicting compared to our results. [19, 32] . these discrepancies may relate to differences in experimental conditions, the protocols for generating and phenotyping mdms and mddcs in different laboratories, or variability of individual blood donors [36] . appreciable efforts have been made to identify novel antiviral therapeutics for mers-cov since the initial outbreak occurred. compounds that target host effectors could be beneficial during the course of infection [37] . mers-cov drug screens have been performed mostly in established cell lines such as vero and huh 7 cells. very little drug evaluation has been performed in primary cell lines. here we picked three fda-approved broad-spectrum inhibitors (cpz, cq, tomf) that were shown to be effective against mers-cov infection in immortalized cell lines [13, 14] and evaluated their antiviral activities in mdms and mddcs. we excluded mature mddcs for drug screening due to the lack of productive mers-cov infection. surprisingly, our findings for tomf activity in mers-cov-infected mdms and immature mddcs differed from results of previous studies in vero e6 cells. tomf showed minimal activity against mers-cov infection in mdms. tomf also had little antiviral activity in immature mddcs until reaching a high concentration (>20 μm) that was associated with high cytotoxicity. tomf is an estrogen receptor modulator that has been shown to inhibit filoviruses [38] and block both mers-cov and sars-cov with ec 50 s12.9 and 11.97μm, respectively, in vero e6 cells [13] . this drug inhibits late phrase of virus entry by interfering the fusion of filoviruses [38] . however, in primary cells, the ec 50 increased to 38 μm though lower cytotoxicity was seen (cc 5 0 = 22.54 μm). thus, the therapeutic window for effective treatment with tomf in primary apcs is narrow. previously, cq, a well-established antiparasitic agent, showed strong inhibition on mers-cov and sars-cov with low ec 50 s (range from 5.76 to 12.9 μm) and low toxicity [13] . cq likely accumulates in lysosomes where it sequesters protons and increases the ph. the drug interacts with a variety of host proteins and cellular processes, resulting in modulation of immune responses [39] . cq has also been reported to inhibit replication of multiple viruses such as flaviviruses [40] , influenza viruses [41, 42] , hiv [40] , ebola [43] and nipah viruses [44] in vitro. in our study, cq had minimal anti-mers activity in either mers-cov-infected mdms or immature mddcs, though appreciable cytotoxicity was not observed. cpz is a neurotransmitter inhibitor, which was the first antipsychotic drug developed for treatment of schizophrenia treatment [45] . cpz inhibits clathrin-mediated endocytosis through preventing the formulation of clathrin-coated pits at the plasma membrane [46] . previous work has reported that cpz inhibits mers-cov infection of huh 7 cells with an ec 50 of 4.9, vs cc 50 of 21.3 μm [14] . in our primary cells, some activity against mers-cov was observed in cpz-pretreated mdms and immature mddcs (ec 50 = 13.58 μm). however, the high cytotoxicity (25.64 μm) observed with cpz on both types of primary cells precludes further development. these studies demonstrate the tested compounds, each of which were shown to be efficacious in continuous cell lines, were not effective at inhibiting mers-cov infection in primary apcs. in conclusion, mers-cov infection in primary mdms induces overexpression of inflammatory cytokines/chemokines potentially leading to tissue damage and systemic virus dissemination. on the other hand, the mechanism of immune escape through mddc results in inefficient viral clearance, which may explain the high pathogenicity and clinical manifestations seen in mers. this study also provides the first demonstration of medical countermeasure evaluation for mers-cov in primary apcs and illustrates the importance of using nonimmortalized primary cells for the evaluation of drug efficacy. these data also suggest that, given the established mechanisms of action of tomf, cq and cpz, the mechanisms of virus internalization and exocytosis may be different from those used in established cell lines. before performing in vivo studies, potential compounds should be evaluated in primary cells as major differences may occur between immortalized cell lines and primary cells. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group middle east respiratory syndrome coronavirus (mers-cov)-saudi arabia. disease outbreak news. geneva, ch: world health organization computer-generated vs. physiciandocumented history of present illness (hpi): results of a blinded comparison middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus (mers-cov) severe acute respiratory syndrome vs. the middle east respiratory syndrome. current opinion in pulmonary 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pharmacology and clinical effects of current and future therapeutic agents infectious entry of west nile virus occurs through a clathrin-mediated endocytic pathway we thank irf cell culture staff in preparing the established cells used in this study. we thank irf immunology staff in characterizing the primary cells used in this study. we thank dr. key: cord-342476-0rupk21u authors: van rijn, anneloes l.; van boheemen, sander; sidorov, igor; carbo, ellen c.; pappas, nikos; mei, hailiang; feltkamp, mariet; aanerud, marianne; bakke, per; claas, eric c. j.; eagan, tomas m.; hiemstra, pieter s.; kroes, aloys c. m.; de vries, jutte j. c. title: the respiratory virome and exacerbations in patients with chronic obstructive pulmonary disease date: 2019-10-24 journal: plos one doi: 10.1371/journal.pone.0223952 sha: doc_id: 342476 cord_uid: 0rupk21u introduction: exacerbations are major contributors to morbidity and mortality in patients with chronic obstructive pulmonary disease (copd), and respiratory bacterial and viral infections are an important trigger. however, using conventional diagnostic techniques, a causative agent is not always found. metagenomic next-generation sequencing (mngs) allows analysis of the complete virome, but has not yet been applied in copd exacerbations. objectives: to study the respiratory virome in nasopharyngeal samples during copd exacerbations using mngs. study design: 88 nasopharyngeal swabs from 63 patients from the bergen copd exacerbation study (2006–2010) were analysed by mngs and in-house qpcr for respiratory viruses. both dna and rna were sequenced simultaneously using an illumina library preparation protocol with in-house adaptations. results: by mngs, 24/88 samples tested positive. sensitivity and specificity, as compared with pcr, were 96% and 98% for diagnostic targets (23/24 and 1093/1120, respectively). additional viral pathogens detected by mngs were herpes simplex virus type 1 and coronavirus oc43. a positive correlation was found between cq value and mngs viral normalized species reads (log value) (p = 0.002). patients with viral pathogens had lower percentages of bacteriophages (p<0.001). no correlation was found between viral reads and clinical markers. conclusions: the mngs protocol used was highly sensitive and specific for semi-quantitative detection of respiratory viruses. excellent negative predictive value implicates the power of mngs to exclude any pathogenic respiratory viral infectious cause in one test, with consequences for clinical decision making. reduced abundance of bacteriophages in copd patients with viral pathogens implicates skewing of the virome during infection, with potential consequences for the bacterial populations, during infection. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 chronic obstructive pulmonary disease (copd) is characterized by exacerbations with high morbidity and mortality, and over 65 million patients suffer from this disease worldwide [1] . a copd exacerbation is an acute event leading to worsening of the respiratory symptoms and is associated with a deterioration of lung function [2] . exacerbations are mainly associated with infections, of which a large part is caused by viruses (22-64%) [3] [4] [5] [6] . however, in part of the exacerbations an etiologic agent is not detected. current routine virus diagnostics is based on polymerase chain reactions (pcr) and inherently the number of detectable pathogens is restricted to the ones included in the assay. rare, mutated and pathogens with an uncommon clinical presentation will be missed, along with new and currently unknown ones. over the last decades, several previously unidentified viruses have been discovered as respiratory pathogens, including metapneumovirus [7] , middle-east respiratory syndrome coronavirus [8] and human bocavirus [9] . metagenomic next generation sequencing (mngs) is an innovative method, which enables the detection of all genomes in a given sample. proof of principle studies have shown that mngs on respiratory samples can confirm and extend pcr results and deliver typing and resistance data at the same time [10] [11] [12] [13] [14] . the performance of mngs in the clinical diagnostic setting, especially the positive and negative predictive value, has not yet been elucidated and is likely to differ per clinical syndrome and sample. previous data from reports on 16s rrna analysis from the respiratory tract have led to increased insight in the microbiome in patients with copd [15] . changes in bacterial populations have been associated with exacerbation events and clinical phenotypes [15] . however, these studies are intrinsically limited to analysis of the bacterial part of the microbiome. so far only a few studies using shotgun metagenomics have focussed on the respiratory virome in children with acute respiratory infections [16, 17] . in this study, we analyse the composition of the virome in adult patients with exacerbations of copd. the aim of this study was to correlate the respiratory virome in copd patients as found by mngs with qpcr and clinical data. patients with copd were included in the bergen copd exacerbation study (bces) between 2006 and 2010 in bergen, norway [18] . all patients lived in the haukeland university hospital district. baseline data taken during the first visit while in the stable state included amongst others exacerbation history, medications, comorbidities, spirometry and global initiative for chronic obstructive lung disease (gold 2007) categorisation. patients were given a telephone number to a study nurse, whom they would contact in case of an exacerbation. patients with an exacerbation according to a predefined set of symptoms were scheduled for an appointment with a study physician the next working day. during exacerbations, nasopharynx swabs were sampled and two different markers for the severity of the exacerbation were scored. after an exacerbation a control visit was scheduled. during the study period 154 patients had at least one exacerbation and in total 325 exacerbations were included in bces, of which 88 exacerbation samples were tested in the current study. nasopharyngeal samples were frozen and stored at -80˚c. in total 88 nasopharyngeal samples of patients at the time of exacerbation were selected based on the availability of other samples (outside the current focus) and sent to the leiden university medical center (the netherlands) for further testing. the viral respiratory panel covered by the multiplex real-time pcr (qpcr) developed in our laboratory consists of coronavirus 229e, coronavirus hku1, coronavirus nl63, coronavirus oc43, influenza a, influenza b, human metapneumovirus, parainfluenza 1-4 (differentiation with probes), respiratory syncytial virus, and rhinovirus [19] . total nucleic acids (na) were extracted directly from 200 μl clinical sample, using the total nucleic acid extraction kit on the magnapure lc system (roche diagnostics, almere, the netherlands) with 100 μl output eluate. nucleic acid amplification and detection by real-time pcr was performed on a biorad cfx96 thermocycler, using primers, probes and conditions as described previously [19] . cq values were normalized using a fixed baseline fluorescence threshold. the metagenomics protocol used has been described and optimized for simultaneously rna and dna detection previously [14] . in short, internal controls, equine arteritis virus (eav) for rna and phocid herpesvirus-1 (phhv) for dna (kindly provided by prof. dr. h.g.m. niesters, the netherlands), were spiked in 200 μl of the virus transport medium in which the nasopharyngeal swab was stored. nucleic acids were extracted directly from 200 μl clinical sample using the magnapure 96 dna and viral na small volume extraction kit on the mag-napure 96 system (roche diagnostics, almere, the netherlands) with 100 μl output eluate (an updated version of the isolation method used for qpcr, tested previously [20] ). extraction buffer was used as negative control (for extraction, library preparation, and sequencing). for library preparation, 7 μl of nucleic acids were used, using the nebnext 1 ultra™ directional rna library prep kit for illumina 1 , with several in-house adaptations to the manufacturers protocol in order to enable simultaneous detection of both dna and rna. the following steps were omitted: poly a mrna capture isolation, rrna depletion and dnase treatment step. this resulted in a single tube per sample throughout library preparation containing both dna and rna. metagenomic sequencing was performed on an illumina nextseq 500 sequencing system (illumina, san diego, ca, usa), and approximately 10 million 150 bp paired-end reads per sample were obtained. after quality pre-processing, sequencing reads were taxonomically classified with centrifuge [21] using an index constructed from ncbi's refseq and taxonomy databases (accessed february 2019) with reference nucleotide sequences for the viruses, bacteria, archaea, fungi, parasites, and protozoa. reads with multiple best matches were uniquely assigned to the lowest common ancestor (k = 1 centrifuge setting; previously validated [14] ). both negative and positive results were confirmed using genomedetective website [22] version 1.111 (accessed december 2018-january 2019) and horizontal coverage (%) was determined using genomedetective. read counts were normalized, dividing the rawread count by the total number of reads in the sample and by the (average) genome size, and multiplied by 10^11 (to achieve comprehensible read counts in the same order of magnitude as the raw read counts). for samples with dubious or inconclusive classification results a de novo assembly was performed. pre-processed short reads assigned to a higher taxonomic level of a suspected viral target were extracted and de novo assembled with spades version 3.11.1 [23] into longer stretches of contiguous sequences (contigs). the resulting contigs were then run against the blast ncbi's nucleotide (nt) database (accessed 2017) using blastn 2.7.1 [24] . after identification of a putative target sequence, all the reads from the original sample were mapped against the identified best blast hit for further confirmation using bwa 0.7.17 software package [25] . sensitivity, specificity, positive and negative predictive values were calculated based on 24 pcr positive and 1120 pcr negative target results of 88 samples. correlation between qpcr cq value and logarithm of normalized numbers of mngs viral reads was tested with population pearson correlation coefficient. potential correlations of mngs data with clinical variables were tested as follows. cq value/ viral reads and clinical parameters (exacerbation severity, duration of exacerbation or decrease/increase in forced expiratory volume in 1 second (fev 1 , control visit compared to baseline) were tested with one-way anova and kruskal-wallis test when appropriate (depending on distribution). comparison of the percentage of phages of all viral reads (after subtraction of the internal control eav and phhv reads) between mngs virus positive samples and negative samples was tested with mann-whitney u test, comparison with clinical parameters with kruskal-wallis test. diversity of the virome in different patient groups was characterized by shannon diversity index (h) and tested with welch two sample t-test. statistical analyses were performed using ibm spss statistics version 25 software for windows <0.05 were considered statistically significant. prior to inclusion all subjects received written and oral information and signed informed consent. the bces study was approved by the regional ethical committee in western norway (rek-vest, case-number 165.08). the performance of this study, including mngs, was approved by the medical ethics review committee of the leiden university medical center (cme number b16.004); no additional consent was necessary. in total 63 patients with 88 exacerbations were included with a median of one exacerbation per patient (range 1-5). baseline patient characteristics and exacerbation characteristics are shown in tables 1 and 2 respectively. of the 88 samples, 23 (26%) tested positive with in-house pcr: 14 (61%) were rhinovirus positive, three influenza a, two coronavirus nl63, one coronavirus oc43, two parainfluenza 3 and one parainfluenza 4. cq values ranged from 19-38 (table 3 ). a median of 11 million (7,522,643-20,906,019) sequence reads per sample were obtained. of the 11 million reads, approximately 93% were homo sapiens reads, 3% were bacterial and 0.1% viral (table 4) . a median of 3% of the reads could not be assigned to sequences in the centrifuge index database (ncbi refseq). of the 23 qpcr positive samples, 22 tested positive with mngs, resulting in a sensitivity of mngs of 96%. only one sample, that was rhinovirus positive by qpcr (cq 38), could not be detected by mngs ( table 3) . coverage of reference genomes was high (93-100%) with the exception of three samples: 30% coverage of rhinovirus c (1,401,120 mapped reads, accession ay391777.1). a coverage plot of all reads against this reference strain (fig 1) showed good horizontal and vertical coverage (read coverage depth 428). the original oc43 qpcr amplification appeared to have been inhibited, and repeated oc43 qpcr confirmed the positive mngs result (cq 25). sensitivity, specificity and predictive value. the sensitivity, specificity and predictive values of mngs were calculated based on 24 pcr positive and 1120 pcr negative target results of 88 samples and the normalized read counts (table 5 ). calculations were made using different cut-off values of respectively �0, �15 and �50 normalized read counts. with a cutoff of �15, the sensitivity was 92% and specificity 100% and the positive predictive value (ppv) increased to 92%. the negative predictive value (npv) was 100% for all cut-off levels. a roc curve (fig 2) , using youden's index [26] demonstrated that the optimal sensitivity and specificity were achieved using a cut-off of 5 reads (96% (23/24) and 100% (1115/1120) respectively). typing mngs provides additional typing data, as compared to qpcr. of the 13 rhinoviruses detected with mngs, 6 (46.2%) were rhinovirus a, 2 (15.4%) rhinovirus b and 5 (38.5%) rhinovirus c. the three influenza viruses were assigned to be h3n2 strains by mngs. in order to analyse the semi-quantitative quality of the mngs assay, the number of the normalized sequence reads (log) mapping to qpcr target viruses (species level) as obtained with mngs were compared to the cq values of qpcr. a significant negative correlation was found (fig 3; pearson correlation coefficient ρ = -0.6, p = 0.002). the following markers were tested for potential associations with clinical severity of exacerbation (exacerbation severity, self-reported exacerbation severity), length of exacerbation and a decrease/increase in fev 1 (control visit compared to baseline): mngs pathogen positive versus negative exacerbation (qpcr targets), the number of normalized reads (log, cutoff of �5normalized reads) for the different target viruses (species level). no correlation was found between these markers and the different disease severity parameters (results not shown). overall proportions of normalized read counts of viral families (excluding eav and phhv control reads, cut-off of �5normalized reads) detected by mngs per patient are shown in fig 4. patients with viral pathogens (pcr target viruses) had significantly reduced proportions of bacteriophages when compared to patients without viral pathogen:0% and 79% bacteriophages respectively (p<0.001) bacteriophage reads vs. all viral reads, normalized reads excluding eav and phhv control reads. the shannon diversity scores for bacteriophages (normalized reads, cut-off of �5normalized reads) were comparable for copd exacerbations of viral aetiology in pcr positive versus negative patients (fig 5) . shannon diversity (normalized reads, excluding internal controls) was significant lower for all viral reads (p<0.001) and eukaryotic viruses (p = 0.028) in patients with viral pathogens (pcr target viruses positive). no significant association was found between the diversity scores, nor the percentage of bacteriophages, and the following parameters: disease severity, length of exacerbation, number of exacerbations during the study period, difference in fev 1 , gold stage, smoking, crp level, and the virus species (results not shown). the most prevalent phyla were proteobacteria, firmicutes actinobacteria and bacteroidetes, see fig 6. the normalized bacterial read count of the most prevalent phyla was not significantly different between patients with a pcr-target virus positive and pcr-target negative patients. pathogenic bacterial species detected with an abundance of >10% of the bacterial reads were: h. influenza (five samples); m. catarrhalis (20 samples); s. pneumoniae (one sample); and the respiratory virome in copd patients s. aureus (one sample). no apparent association with bacteriophages was found, or was a high abundance of bacteriophages associated with copd exacerbations of viral cause. the raw sequence data of the samples, after removal of human reads have been deposited to sequence read archive database (http://www.ncbi.nlm.nih.gov; accession number srx6713943-srx6714030). in this study, the respiratory virome in patients with copd exacerbations was analysed with both mngs and qpcr, and combined with clinical data. the incidence of viral pathogens was 26% with both mngs and qpcr. mngs failed to detect one rhinovirus with low load (cq 38) and pcr failed to detect one betacoronavirus oc43 (72644 reads), due to one of the limitations of pcr, i.e. inhibition of amplification. one additional viral pathogen, not present in the respiratory pcr panel, was detected: herpes simplex virus 1, found by others to be associated with copd [27] . the incidence of viral pathogens was comparable to that in previous publications (22-64%) [3, 5, 6] . the viral pathogen with the highest incidence was rhinovirus, followed by influenza, coronaviruses and para-influenza viruses. interestingly, subtyping data was readily available by mngs, accentuating the high resolution of mngs, with rhinovirus (rv) species a and c being most frequent, followed by rv-b. rv-c was first identified in 2006 and associated with high symptom burdens in children and asthmatics [28, 29] . recently, an asthma-related cadherin-related family member 3 (cdhr3) gene variant [30] was associated with greater rv-c receptor display on pulmonary cell surfaces of children and adults, and associated with higher susceptibility to severe virus-triggered asthma episodes [31, 32] . in line, romero-espinoza et al detected predominantly rv-c in children with acute asthma exacerbations by mngs [33] . the significance of rv-c infection in the adult population is less well studied. although rv-c has been previously associated with exacerbations of copd [34, 35] , to our knowledge, to date, cdhr3 polymorphisms have not yet found to be associated with copd. the sensitivity, specificity and positive and negative predictive values of mngs were high: 96%, 100%, 82% and 100%, respectively, when encountering a cut-off of �5normalized reads, with a detection limit of approximately cq 38. the high negative predictive value implicates the potential of mngs to exclude the most prevalent viral respiratory infections in one test. the potential to exclude any infectious cause, both viral and bacterial, would have significant consequences for starting and/or continuation of antimicrobial or, at the other end of the spectrum, immune-modulating treatment. the normalized viral species sequence read count might give an indication of the viral burden and the clinical relevancy of the detected virus. although in our dataset we could not find any correlation with disease severity, several paediatric studies demonstrated a correlation between virus load and severity in respiratory infections [36] [37] [38] [39] . further analysis with a larger number of infected patients and/or a different spectrum of exacerbation severity will be needed to demonstrate or exclude such an association in copd patients. furthermore, the complete respiratory virome showed a high bacteriophage abundance that could be linked to the absence of viral pathogens. lower bacteriophage abundance may be the result of viral expansion. hypothetically, a healthy virome size and diversity fits a certain size and diversity of bacteriophages, while during viral infection, pathogens predominate the virome. alternatively, others have hypothesized that viral and microbial diversity may play a role in infection susceptibility and the development of acute and chronic respiratory diseases [33] . our results indicate that virome dysbiosis may be accompanied by bacteriome dysbiosis, though no significant differences were detected in line with other reports [40, 41] . however, these studies don't compare between copd exacerbation with and without viral infections. others have found a higher phage abundance in a patient with severe copd when compared with one patients with moderate copd and healthy controls, dna sequencing, in line with the hypothesis of a state of dysbiosis that increases with disease progression [27] . in copd patients, viral infections have been suggested to trigger bacterial overgrowth and infections [42, 43] , demonstrating the significance of viral-bacterial interactions. moreover, hypothetically, bacteriophages play a role in the horizontal gene transfer of bacterial virulence factors. the most abundant bacterial phyla detected in this study were comparable with other reports. although the percentage of proteobacteria was relatively high when compared to other studies of the nasopharyngeal microbiome, our swabs are sampled during copd exacerbations [44, 45] . study of the lower airways by means of e.g. protected brushes during bronchoscopy is needed for further analysis of bacterial and viral (sub)populations including comparison with pcr and culture results. studies comparing the respiratory virome during stable disease and exacerbations are needed to determine a potential correlation between the virome/bacteriome during stable state and disease progression or exacerbation frequency. in the current study, most respiratory pathogens detected were rna viruses. this is in line with previous literature [3, 5, 6] . however, it must be noted that, despite the fact that a wide range of dna viruses have been detected with the current protocol (dna bacteriophages with high abundance, herpes simplex virus, bocavirus, anelloviruses, cmv, ki polyomavirus [14] ), we cannot exclude suboptimal detection of some dna viruses. furthermore, highly divergent viruses with sequences deviating from their representative ncbi refseq sequences may have been missed, as has been described by others [46] . however, bioinformatic classification using alternative databases (both genomedetective and local databases) did not result in additional findings. though mngs renders the possibility to detect all viruses in direct respiratory material, this revolutionary method is not yet used as routine accredited diagnostic procedure for pathogen detection. before mngs can be implemented as a routine diagnostics, the optimal protocol must be defined and analysis and interpretation of the metagenomic data must be standardized, followed by external quality assessment. this study demonstrates good performance of our mngs protocol, in line with other studies [37, 38, 47, 48] and seems to overcome some of the current thresholds for implementation in clinical diagnostics. the mngs protocol used was highly sensitive and specific for semi-quantitative detection of respiratory pathogenic viruses. excellent negative predictive value implicates the potential of mngs to exclude a known viral infectious cause in one test, with consequences for clinical decision making. reduced abundance of bacteriophages in copd patients with viral pathogens implicates skewing of the virome, and speculatively the bacterial population, during infection. management and prevention of exacerbations of copd copd exacerbations: defining their cause and prevention copd exacerbations. 2: aetiology the role of viral infections in exacerbations of chronic obstructive pulmonary disease and asthma. therapeutic advances in respiratory disease a newly discovered human pneumovirus isolated from young children with respiratory tract disease isolation of a novel coronavirus from a man with pneumonia in saudi arabia cloning of a human parvovirus by molecular screening of respiratory tract samples exploring the potential of next-generation sequencing in detection of respiratory viruses genomic characterization of a newly discovered coronavirus associated with acute respiratory 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with severe exacerbations cadherin-related family member 3, a childhood asthma susceptibility gene product, mediates rhinovirus c binding and replication genetic association of the functional cdhr3 genotype with early-onset adult asthma in japanese populations unbiased detection of respiratory viruses by use of rna sequencing-based metagenomics: a systematic comparison to a commercial pcr panel we thank our generade project partners floyd wittink, wouter suring (hogeschool leiden), danny duijsings (baseclear) and christiaan henkel (leiden university). we also like to thank tom vreeswijk, lopje höcker and mario van bussel (kml, lumc) for help with the pre-sequencing experiments and jeroen laros (human genetics, lumc) for help with the bioinformatics. we thank caroline brouwer for technical assistance (kml, lumc). key: cord-339578-eg19rfvi authors: garcia-garcia, maria luz; calvo, cristina; ruiz, sara; pozo, francisco; del pozo, victoria; remedios, laura; exposito, nadia; tellez, ana; casas, inmaculada title: role of viral coinfections in asthma development date: 2017-12-05 journal: plos one doi: 10.1371/journal.pone.0189083 sha: doc_id: 339578 cord_uid: eg19rfvi background: viral respiratory infections, especially acute bronchiolitis, play a key role in the development of asthma in childhood. however, most studies have focused on respiratory syncytial virus or rhinovirus infections and none of them have compared the long-term evolution of single versus double or multiple viral infections. objective: our aim was to compare the frequency of asthma development at 6–8 years in children with previous admission for bronchiolitis associated with single versus double or multiple viral infection. patients & methods: a cross-sectional study was performed in 244 children currently aged 6–8 years, previously admitted due to bronchiolitis between september 2008 and december 2011. a structured clinical interview and the isaac questionnaire for asthma symptoms for 6-7-year-old children, were answered by parents by telephone. specimens of nasopharyngeal aspirate for virological study (polymerase chain reaction) and clinical data were prospectively taken during admission for bronchiolitis. results: median current age at follow-up was 7.3 years (iqr: 6.7–8.1). the rate of recurrent wheezing was 82.7% in the coinfection group and 69.7% in the single-infection group, p = 0.06. the number of wheezing-related admissions was twice as high in coinfections than in single infections, p = 0.004. regarding the isaac questionnaire, 30.8% of coinfections versus 15% of single infections, p = 0.01, presented “wheezing in the last 12 months”, data that strongly correlate with current prevalence of asthma. “dry cough at night” was also reported more frequently in coinfections than in single infections, p = 0.02. the strongest independent risk factors for asthma at 6–8 years of age were: age > 9 months at admission for bronchiolitis (or: 3.484; ci95%: 1.459–8.317, p:0.005), allergic rhinitis (or: 5.910; 95%ci: 2.622–13.318, p<0.001), and viral coinfection-bronchiolitis (or: 3.374; ci95%: 1.542–7.386, p:0.01). conclusions: asthma at 6–8 years is more frequent and severe in those children previously hospitalized with viral coinfection-bronchiolitis compared with those with single infection. allergic rhinitis and older age at admission seem also to be strong independent risk factors for asthma development in children previously hospitalised because of bronchiolitis. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 more frequently in coinfections than in single infections, p = 0.02. the strongest independent risk factors for asthma at 6-8 years of age were: age > 9 months at admission for bronchiolitis (or: 3.484; ci95%: 1.459-8.317, p:0.005), allergic rhinitis (or: 5.910; 95%ci: 2.622-13.318, p<0.001), and viral coinfection-bronchiolitis (or: 3.374; ci95%: 1.542-7.386, p:0.01). there is strong evidence that respiratory viruses play a key role in the development of asthma in children. classic studies by sigurs et al. showed that respiratory syncytial virus (rsv) bronchiolitis, severe enough to require hospitalization, is a risk factor for asthma at the age of 7, 13 and 18 [1, 2, 3] . ng asthma at 6 years being more than four-fold higher compared with hrvnegative cases [4, 5] . other studies have reported that early human metapneumovirus (hmpv) infection during infancy is also an independent risk factor for the development of preschool asthma [6] ]. however, most studies have focused on rsv or hrv infections and only one of thm has evaluated the long-term evolution of viral coinfections, although focused exclusively on human bocavirus (hbov) and hrv coinfections in 3 to 35 month-old children with their first or second wheezing episode [7] . our main objective was to compare the frequency of asthma and other respiratory symptoms, at 6-8 years of age, in children with previous admission with bronchiolitis associated with viral co-infection versus simple viral infection. this is a substudy of an ongoing prospective investigation of respiratory tract infections in children, approved by the medical ethics committee. inclusion criteria were children 6-8 years of current age, with a previous hospitalization with bronchiolitis at 0-24 months of age, between september 2008 and december 2011. during the hospital stay a physician filled out a study-questionnaire with the following variables: age, sex, month of admission, clinical diagnosis, history of prematurity, need for oxygen therapy-evaluated via transcutaneous oxygen saturation -, axillary temperature (! 38˚c), presence of infiltrates and/or atelectasis in chest x-rays, administration of antibiotic therapy, length of hospital stay, total white blood cell (wbc) count, c-reactive protein (crp) serum levels and blood culture results (for those cases where such tests were performed) and results of the virological study. parents were contacted by phone between october 2016 and january 2017, and were invited to take part in the study. a telephone interview based on a structured questionnaire was performed, to obtain information on wheezing episodes, related hospital admissions, physician-diagnosed atopic dermatitis, allergic rhinitis, food allergy, use of bronchodilators and maintenance medication for asthma. information on pet contacts, parental smoking habits, presence of allergy, eczema and asthma in first order family members (mother, father or siblings) that had been diagnosed by a medical doctor was also recorded. furthermore, the present investigation used the isaac questionnaire for asthma symptoms for 6-7-year-old children, previously validated and translated to spanish [8] , which was answered by parents over the phone. the researchers were blinded to the status of the child when the interviews were performed. informed consent was obtained from parents or legal guardians. exclusion criteria were refusal to participate. specimens consisted of nasopharyngeal aspirates (npa) that were taken from each patient at admission. each specimen was sent for virological investigation to the respiratory virus and influenza unit at the national microbiology center (isciii, madrid, spain). npas were processed within 24 hours after collection. upon receipt, three aliquots were prepared and stored at -70˚c. both the reception and the npa sample processing areas were separate from those defined as working areas. polymerase chain reactions (pcr) methods for detection of sixteen respiratory viruses. three reverse transcription (rt)-nested pcr assays were performed to detect a total of sixteen respiratory viruses. in these assays, the reverse transcription and first amplification round were carried out in a single tube using the qiagen onestep rt-pcr kit (qiagen). influenza a, b and c viruses were detected by using primer sets only to amplify influenza viruses in a multiplex pcr assay as previously described [9] . a second multiplex pcr was used to detect parainfluenza viruses 1 to 4 (piv), human coronaviruses (cov) 229e and oc43, enteroviruses (ev) and hrv [10] . presence of rsv a and b types, hmpv, hbov and adenoviruses (adv) were investigated by a third multiplex rt-nested pcr-brq method [11] (s1 table) . bronchiolitis. all the classic criteria, present in an initial episode of acute onset expiratory dyspnea with previous signs of viral respiratory infection-whether or not this was associated to respiratory distress or pneumonia-were applied in diagnosing bronchiolitis [12] . the term recurrent wheezing was used to imply more than one episode of wheezing verified by a physician. the children who answered affirmatively to question 2 of the isaac questionnaire, "wheezing in the last 12 months", being the one that in the validation studies has shown a better correlation with the current prevalence of asthma, were considered to have asthma [13] . allergic rhinitis. was defined as rhinitis appearing at least twice after exposure to a particular allergen and unrelated to infection. viral coinfection. was defined as the detection of more than one viral pathogen in the same sample. continuous variables were described using mean and standard deviation. categorical variables were described using absolute and relative frequencies. clinical characteristics of patients with simple infections were compared with those associated with coinfections. to compare qualitative variables, chi 2 test or fisher's exact test was used if there were 5 items of data in a cell. for quantitative variables, as all of them followed a normal distribution, the means were compared using the students´s t to compare two groups. a two-sided value of p < 0.05 was considered statistically significant. multivariate stepwise logistic regression analysis was used to calculate the adjusted odds ratios (or) with 95% confidence intervals for estimating the association between different factors and asthma. all analyses were performed using the statistical package for the social sciences (spss), version 21.0. of the 351 children previously admitted with bronchiolitis, with positive viral detection and current age between 6 and 8 years, 244 (52 coinfections and 192 single infections) could be located and agreed to participate in the study. clinical characteristics during admission for bronchiolitis are presented in table 1 . no significant clinical differences could be detected between both groups, coinfections and single infections, in terms of age at admission, gender, prematurity, fever, hypoxia or length of hospital stay. the most frequently identified viruses in single infections were: rsv (100, 52%), hrv (29, 15%), hmpv (15, 8%), adv (5, 2.6%) and hbov (3, 1.6%). the most frequent coinfections were rsv + hrv (15, 29%) and rsv + hbov (12, 23%). with respect to comparability between both groups, no differences were found between coinfections and single infections regarding allergic rhinitis, atopic dermatitis or food allergy ( table 2) . several possible hereditary and environmental factors for the development of recurrent wheezing or asthma were also evaluated. no differences were observed in the family history of asthma or atopy. nearly one-third of children were exposed to tobacco smoke, with similar percentages in both groups ( table 2) . median current age at follow-up contact was 7.3 years (iqr: 6.7-8.1), with a slight predominance of females (52.5%). the rate of recurrent wheezing was 82.7% in the coinfection group and 69.7% in the single-infection group, p = 0.06, needing rehospitalization for wheezing 21% and 29% respectively, p = 0.251. the number of wheezing-related admissions was twice as high in coinfections than in single infections, p = 0.004. a similar percentage of children were prescribed inhaled corticosteroids in both groups. however, montelukast was used more frequently in coinfections than in simple infections, p = 0.01 (table 3) . regarding the isaac questionnaire answers, nearly 70% had "ever wheezed", without significant differences between both groups. however, 30.8% of coinfections versus 15% of single infections, p = 0.01, presented "wheezing in the last 12 months", data that strongly correlate with current prevalence of asthma. "dry cough at night" was also reported more frequently in coinfections than in single infections, p = 0.02. the questions: "wheezing during exercise?" and "ever had asthma?" were affirmatively answered with similar frequency in both groups. (table 4 ). in the combined coinfection and single infection group, several possible hereditary and environmental risk factors for asthma at the age of 6-8 were evaluated using a univariate analysis ( table 5 ). the risk of asthma was increased in children previously hospitalized for bronchiolitis with viral coinfection (or = 2.498; 95% ci: 1.229-5.077), in those who were older at the table 5 with a p-value < 0.10 were entered in a stepwise logistic regression analysis to estimate which factors were independently related to the development of asthma at 6-8 years. age > 12 months at admission for bronchiolitis (or: 7.389; 95%ci: 2.683-20.352, p<0.001), age > 9 months at admission for bronchiolitis (or: 3.484; ci95%: 1.459-8.317, p:0.005), allergic rhinitis (or: 5.910; 95%ci: 2.622-13.318, p<0.001), and viral coinfection-bronchiolitis (or: 3.374; ci95%: 1.542-7.386, p:0.01) were the strongest independent risk factors for asthma at 6-8 years of age. our current study shows for the first time to our knowledge, that there is a strong and independent association between severe bronchiolitis associated with viral coinfection and the development of asthma at 6-8 years old. viral coinfections are usually detected in up to 30% of children with an acute respiratory tract infection [14] . our group, in previous studies, found viral coinfections in 22.8% of 318 infants less than 2 years old admitted with bronchiolitis [11] and in 35% of 2,993 children less than 14 years old admitted with lower respiratory tract infection [15] . our current results confirm that the most frequently identified viral coinfections are dual rsv-hrv and rsv-hbov infections. results from cohort studies evaluating the severity of acute respiratory viral coinfections are conflicting. our group previously found that clinical severity was associated mainly with rsv infections, either alone or in combination with other viruses [15] . however, asner et al. [16] in a recent systematic review and meta-analysis found no differences in clinical disease severity between viral coinfections and simple infections, although an increased risk of mortality was observed amongst preschool children with coinfections. they conclude that large, prospective, well designed studies with objective outcomes are needed to better understand the clinical significance of viral respiratory coinfections. regarding the role of early viral respiratory coinfections in the development of wheezing or asthma, so far, no study evaluating this possible association has been published. our results confirm that recurrent wheezing and asthma are very common in the first years of life after a severe bronchiolitis. but the likelihood of developing asthma was 2.5 times higher if bronchiolitis was associated with viral coinfection compared to single infections. in addition, the number of rehospitalizations for asthma was also significantly higher in coinfections than in single infections, suggesting that coinfections are associated not only with more frequency of asthma but also with greater severity. the mechanism by which respiratory infections are associated with subsequent asthma is not fully understood but the different inflammatory responses released after viral infections seem to play a relevant role. the induction of a prevalent th2-type response, with the production of il-4, il-5, and il-13, results in a less robust and less efficient reaction to the infection, clinically presenting as increased disease severity and risk for future recurrent wheezing [17, 18] . several reports showed that thymic stromal lymphopoietin (tslp) is a key initiator of allergic airway responses [19] and significant negative correlation has been described between tslp levels in induced sputum and fev-1 in asthmatic children [20] . tslp is secreted by rsv-infected airway epithelial cells (aecs) to promote the activation of dendritic cells (dcs) [21, 22] . the activated dcs can then induce a th2 cell polarization response in the lung, which could contribute the development of asthma in rsv-infected individuals, as well as induction of exacerbations in asthmatic rsv-infected patients. it has been suggested that the ability of rsv to induce tslp expression by aecs is responsible for the subsequent th2 aspects of the immune response [23] . other recent studies performed in young children with hrv infection, have also found higher levels of nasal tslp [24, 25] . on the other hand, various genetic studies in humans have identified both il-33 and its receptor st2, as being key regulators in the development of asthma [26] and to have a strong th2-promoting ability in animal models of asthma [27] . il-33 is responsible for the immunopathophysiological response observed following neonatal rsv infection in mice. its presence in nasal aspirates of human infants with severe rsv, together with the finding that by blocking the receptor of interleukin 33 in vivo [28] , th2 responses are greatly reduced, suggest it has a role in disease severity and asthma [29] . our findings, in a recent study conducted in hospitalized infants with bronchiolitis and in healthy controls, showed that nasal tslp and il-33 are detected more frequently in viral coinfections than in single infections. in addition, infants with dual rsv+hrv infection were 9 times more likely to have detectable nasal tslp and this association was independent of other factors such as age or illness severity [30] . it is worth noting that no patient with bronchiolitis but with negative viral detection had detectable levels of nasal tslp or il-33, suggesting that the release of these proteins may be mediated by respiratory viruses. it can be hypothesized that this stronger tslp response after viral coinfection bronchiolitis, could stimulate a vigorous production of th2-associated effector cytokines, such as il-4, il-5, il-13, as was reported in asthmatic adults by ying et al, that could be associated with higher frequency of wheezing and asthma development later on [31] . as far as we know, only lukkarinen et al [7] have compared the systemic th1-type, th2-type and proinflammatory cytokine profiles in young children with simple versus viral coinfection, being hrv and hbov1 the viruses involved. they included hospitalized children less than 35 months of age with their first or second wheezing episode. they observed that wheezing children with hrv had higher proinflammatory responses than did those with hbov1 or those with coinfection hrv+hbov1, suggesting that hbov1 may interfere with hrv-induced immune responses. furthermore, this immunological response was accompanied by the clinical finding that children with hrv+hbov1 wheeze tended to develop recurrent wheezing later and less often than did those with hrv wheeze. our previous and current results suggest that an interaction may also occur in rsv and hrv coinfections, but in an opposite way, towards a predominant th2 response that could increase the development of recurrent wheezing/asthma. the risk of asthma in our study, like in other cohort studies on hospitalized children with lower respiratory symptoms, was directly dependent on age [4, 32] : the adjusted or was 3.4 for asthma in children with bronchiolitis aged > 9 months and 7.3 for asthma in school age in bronchiolitis children aged > 12 months. however, lukkarinen et al [33] , in a 7-year followup found asthma inversely dependent on age, probably because they studied infants with wheezing only, vs. bronchiolitis, with or without wheezing, in other studies as in ours. this different inclusion criteria may bias the kind of patients included in the studies. rhinitis is a known risk factor for asthma in children [34] . our results suggest that rhinitis is an independent risk factor for asthma persistence in school-age children with previous severe bronchiolitis. lauhkonen et al [35] in a prospective follow-up study, in 102 children hospitalised for bronchiolitis, also found that current asthma was associated with prolonged rhinitis and a positive skin prick test at five to seven years. on the other hand, recent reports have demonstrated that the severity of rhinitis and asthma are closely related in children [36] . thus, as other authors have stated [37] , all these results highlight the convenience to assess nasal symptoms in infants and children with recurrent wheezing and asthma. in conclusion, asthma at the age of 6-8 is more frequent and severe in those children previously hospitalized with viral coinfection bronchiolitis compared with those with single infection. moreover, viral coinfection, allergic rhinitis and older age at admission seem also to be strong independent risk factors for asthma development in children previously hospitalised because of bronchiolitis. however, given the complexity of the immunological mechanisms involved, more studies are needed to confirm this association and better understand its physiopathological mechanism. supporting information s1 respiratory syncytial virus bronchiolitis in infancy is an important risk factor for asthma and allergy at age 7 severe respiratory syncytial virus bronchiolitis in infancy and asthma and allergy at age 13 asthma and allergy patterns over 18 years after severe rsv bronchiolitis in the first year of life rhinovirusinduced wheezing in infancy-the first sign of childhood asthma? infantile respiratory syncytial virus and human rinovirus infections: respective role in inception and persistence of wheezing human metapneumovirus bronchiolitis in infancy is an important risk factor for asthma at age 5 human bocavirus 1 may suppress rhinovirus-associated immune response in wheezing children validation of the spanish versión of the phase iii isaac questionnaire on asthma simultaneous detection of influenza a, b, and c viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-pcr assay simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays detection of new respiratory virus in bronchiolitis: 3-year study what's in the name? validation of questionnaire and bronchial hyperresponsiveness against respiratory physician assessment in the diagnosis of asthma two-year prospective study of single infections and co-infections by respiratory syncytial virus and viruses identified recently in infants with acute respiratory disease respiratory syncytial virus coinfections with rhinovirus and human bocavirus in hospitalized children clinical disease severity of respiratory viral co-infection versus single viral infection: a systematic review and meta-analysis host response to respiratory syncytial virus infection respiratory syncytial virus-host interaction in the pathogenesis of bronchiolitis and its impact on respiratory. morbidity in later life stromal lymphopoietin as a key initiator of allergic airway inflammation in mice increased expression of thymic stromal lymphopoietin in induced sputum from asthmatic children tslp-activated dendritic cells induce an inflammatory t helper type 2 cell response through ox40 ligand tslp-activated dendritic cells induce human t follicular helper cell differentiation through ox40-ligand thymic stromal lymphopoietin is induced by respiratory syncytial virus-infected airway epithelial cells and promotes a type 2 response to infection rhinovirus infection in young children is associated with elevated airway tslp levels rhinovirus-induced airway cytokines and respiratory morbidity in severely premature children defining the contribution of snps identified in asthma gwas to clinical variables in asthmatic children il-33 is more potent than il-25 in provoking il-13-producing nuocytes (type 2 innate lymphoid cells) and airway contraction role of interleukin 33 in respiratory allergy and asthma respiratory syncytial virus disease is mediated by age-variable il-33 thymic stromal lymphopoietin, il-33, and periostin in hospitalized infants with viral bronchiolitis thymic stromal lymphopoietin expression is increased in asthmatic airways and correlates with expression of th2-attracting chemokines and disease severity wheezing rhinovirus illnesses in early life predict asthma development in high-risk children prednisolone reduces recurrent wheezing after first rhinovirus wheeze: a 7-year follow-up allergic rhinitis as a predictor for wheezing onset in school-aged children following up infant bronchiolitis patients provided new evidence for and against the united airway disease hypothesis severity of rhinitis and wheezing is strongly associated in preschoolers: a population-based study preschool-age wheezing phenotypes and asthma persistence in adolescents key: cord-340713-v5sdowb7 authors: bird, jordan j.; barnes, chloe m.; premebida, cristiano; ekárt, anikó; faria, diego r. title: country-level pandemic risk and preparedness classification based on covid-19 data: a machine learning approach date: 2020-10-28 journal: plos one doi: 10.1371/journal.pone.0241332 sha: doc_id: 340713 cord_uid: v5sdowb7 in this work we present a three-stage machine learning strategy to country-level risk classification based on countries that are reporting covid-19 information. a k% binning discretisation (k = 25) is used to create four risk groups of countries based on the risk of transmission (coronavirus cases per million population), risk of mortality (coronavirus deaths per million population), and risk of inability to test (coronavirus tests per million population). the four risk groups produced by k% binning are labelled as ‘low’, ‘medium-low’, ‘medium-high’, and ‘high’. coronavirus-related data are then removed and the attributes for prediction of the three types of risk are given as the geopolitical and demographic data describing each country. thus, the calculation of class label is based on coronavirus data but the input attributes are country-level information regardless of coronavirus data. the three four-class classification problems are then explored and benchmarked through leave-one-country-out cross validation to find the strongest model, producing a stack of gradient boosting and decision tree algorithms for risk of transmission, a stack of support vector machine and extra trees for risk of mortality, and a gradient boosting algorithm for the risk of inability to test. it is noted that high risk for inability to test is often coupled with low risks for transmission and mortality, therefore the risk of inability to test should be interpreted first, before consideration is given to the predicted transmission and mortality risks. finally, the approach is applied to more recent risk levels to data from september 2020 and weaker results are noted due to the growth of international collaboration detracting useful knowledge from country-level attributes which suggests that similar machine learning approaches are more useful prior to situations later unfolding. according to the future of humanity institute there is a 2.05% chance that mankind will go extinct by the year 2100, through either a natural or engineered pandemic [1] . if there is one lesson to learn from the ongoing covid-19 coronavirus (sars-cov-2) pandemic, it is that a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 we were not prepared. the virus initially spread rapidly across the globe, mortality began to rise, and countries desperately struggled to test their citizens for the virus once it became known that many infectious carriers of it show no noticeable symptoms [2] [3] [4] . this suggests three main risk factors to be observant of: the initial risk of transmission due to varying factors such as, for example, population density [5] and international travel [6] ; the risk of mortality due to ageing populations [7] and underlying health issues [8, 9] ; and finally the risk of a country not being able to test citizens aptly and thus producing possibly under-reported measures of the previous two [10] . machine learning has shown success in contributing to research during the covid-19 pandemic. health service data trend models have shown to aid in classification of the virus [11, 12] , vaccine design [13] , estimation of cases, deaths, and recoveries [14, 15] , simulating what could have happened if 'lockdown' was not instituted [16] , and also simulating behaviour of the spread of the disease by prior knowledge from other locations [17] . in this work, we devise a machine learning based strategy to predict three-fold risk at the country-level: (i) risk of transmission, (ii) risk of mortality, and (iii) risk of inability to test. through these three quantifiable measures, preparedness and risk can be assessed, providing some quantitative reasoning behind global decisions, should another deadly disease grip our species again. our main contribution is the exploration of the idea that country-level demographic and geopolitical attributes can aid in the classification of pandemic risk and preparedness in terms of transmission, mortality, and an inability to test (which the previous two depend on, since testing allows for accurate measurements of transmission and mortality). in order to do this, various supervised learning classifiers are explored in order to discern how much useful information these country-level attributes carry for the classification of these three risks. we note that the classification problems are difficult, where many powerful techniques achieve unsatisfactory scores on the dataset, scoring around 10-20% higher than an approximate 25% random guess on the dataset, showing that learning useful rules from the data is not an easy task. this is not unexpected, since the classes have not been directly derived from the data used to predict them, rather, they have been derived from covid-19 statistics and then given as classes for country-level demographic and geopolitical information. due to this, strategies of linear searching and genetic optimisation are also followed in order to achieve more accurate results. although results are varied, the fact that all final models achieve much higher than 25% accuracy we formulate the problem as a 4-class problem. (which would be achieved via a random guess), shows that the geopolitical and demographic attributes at the country-level do carry predictive ability when it comes to pandemic risk and preparedness. the final models chosen are characterised by high classification accuracy for the risks of transmission, mortality, and inability to test, and are trained with no prior knowledge of the new coronavirus pandemic (other than the class). this may allow for generalisation to classify a nation's risk in the early days of a future pandemic. the remainder of this work is organised as follows: section 2 details the method followed with subsection 2.1 describing machine learning approaches in particular. section 3 presents the results for the risk of transmission (3.1), the risk of mortality (3.2) and the risk of inability to test (3.3) . finally, the limitations of the study are described, future work is suggested, and the study is concluded in section 4. collected from [18] formalised on the 12th may 2020, updated experiments for newer data can be found in section 3.7, with the relative ordering based on the three metrics with regards to population (cases, deaths, and tests per million). the risk classes are low, medium-low, medium-high and high for each type of risk. as defined in other works [19] [20] [21] , discretisation of the continuous features into bins is performed by the k% method in which k = 25 (equal frequency binning), resulting in four close-to-equal classes, with the difference being that the highest risk class is a minor 1.2% larger than the other three classes. future work aims to explore other methods of discretisation, whereas this work initially focuses on the machine learning pipeline on the basis of equal class error weighting. covid-19 data are then removed, and the attributes to complement the country-level classes are the following: un region [22] , 2020 population estimate [18] , median age [18] , population density per km 2 [18] , urban population % [18] , urban population total [18] , nursing and midwifery personnel per 10,000 (most recently recorded) [23] , medical doctors per 10,000 (most recently recorded) [23] , tobacco prevalence 2016 [24] , obesity prevalence 2016 [25] , gross domestic product 2019 [22] , land area km 2 [22] , net migration [22] , infant mortality per 1,000 births [22] , literacy rate % [22] , arable land % [22] , crop land % [22] , other land % [22] , climate classification type [22] , birth rate per 1,000 [22] , death rate per 1,000 [22] , gdp expenditure on agriculture [22] , gdp expenditure on industry [22] and gdp expenditure on services [22] . since some countries are not recorded by the world health organisation, figures for nursing, midwifery and medical doctors personnel per 10,000 people from hong kong are collected from an alternative source [26] . missing data which occurred mostly for tobacco prevalence, was given as '-1', which flags as an attribute that the data have not been collected (which could in itself provide useful information). the classification problem of risk is therefore formulated based on prior knowledge of the pandemic in terms of class only, but the attributes to attempt to classify them are purely country-level information regardless of number of cases, deaths and other coronavirus specific data. thus the problem becomes a pandemic risk and preparedness classification problem based on demographic and geopolitical attributes only. we aim for a generalisable model, which can be applied to the future state of countries, should another potential pandemic begin prior to any meaningful measurements being available. the method is illustrated in fig 1. following this, a set of machine learning models are tasked with predicting a country's risk class by learning from all other countries in a process of leave one out cross-validation [27], which is performed for all three types: finally, the best models for each risk factor are organised into a predictive framework, which produces an output for the three risks. since testing is taken into account, countries that have not reported testing data cannot be considered, but are later classified by the model generalised on those countries that do. a three-fold machine learning approach is proposed following observing the maps for the three separate risk quarters in figs 2, 3 and 4 which show the discretised inability to test risk, transmission risk, and mortality risk respectively. we note that the countries with seemingly fewer cases have performed far fewer tests as can be observed in that the growth of cases and testing tend to increase alongside one another. that is, a country with more cases will test more, and as such will have more confirmed cases, since the larger number of tests have identified more cases. the data for the two experiments were accessed on 12 th may 2020 and 16 th september 2020. trained with the strategy of leave one out cross-validation (loo cv) where every country's risk is predicted based on learning from all other countries, a set of supervised classification models are benchmarked. this section details the models focused upon, and the methods used to search for others. the metrics reported following the models described in this section are mean classification accuracy due to close-to-equal class balance [28] (low, med-low, med-high are equal and high is minimally larger by a factor of 1.2%) and high variance often observed due to the nature of loo cv [29, 30] . decision trees are tree structures, where each internal node represents a condition based on attributes that allows splitting the data and leaf nodes represent class labels [31] . a random decision forest (rdf) [32], used in this study, creates multiple random decision trees, where note that many countries at "low risk" by number of cases are at "high risk" for inability to test. https://doi.org/10.1371/journal.pone.0241332.g003 note that many countries at "low risk" by number of deaths are at "high risk" for inability to test. https://doi.org/10.1371/journal.pone.0241332.g004 each decision tree votes on the class of the input data object, and the predicted class is that, which receives the majority vote. splitting of the trees is based on information gain: where ig is the observed difference in information entropy, which is expressed in eq (2), that is, the nodes split data based on reducing the randomness of object class distribution. k-nearest neighbours (knn) is similar to an rdf in that the prediction is derived by a majority vote. the voters, rather than decision trees, are the data objects within the observations that are closest in terms of n-dimensional euclidean space where n is the number of attributes. gradient boosting [33] forms an ensemble of weak learners (decision trees) and aims to minimise a loss function via a forward stage-wise additive method. in these classification problems, deviance is minimised. at each stage, four trees (n = classes) are fit on the negative gradient of the multinomial deviance loss function, or cross-entropy loss [34, 35]: where, for k classes, i is a binary indicator of whether the prediction that class y is the class of observed data x is correct, and finally p is the probability that aforementioned data x belongs to the class label y. xgboost [36] differs slightly in that it penalises trees, leaves are shrunk proportionally, and extra randomisation is implemented. naïve bayes is a probabilistic classifier that aims to find the posterior probability for a number of different hypotheses and selecting the most likely case. bayes' theorem is given as: where p(h|d) is the posterior probability of hypothesis h given the data d, p(d|h) is the conditional probability of data d given that the hypothesis h is true. p(h) i.e., the prior, is the probability of hypothesis h being true and p(d) = p(d|h)p(h) is the probability of the data. naïvety in the algorithm is due to the assumption that each probability value is conditionally independent for a given target, calculated as pðdjhþ ¼ q n i¼1 pðd i jhþ where n is the number of attributes/ features. linear discriminant anaylsis (lda), based on fisher's linear discriminant [37] , is a statistical method that aims to find a linear combination of input features that separate classes of data objects, and then use those separations as feature selection (opting for the linear combination) or classification (placing prediction objects within a separation). classes k 2 {1, . . ., k} are assigned priorsp k ( (3) in mind, maximum-a-posteriori probability is thus calculated as: where f k (x) is the density of x conditioned on k: s k is the covariance matrix for samples of class k and class covariance matrices are assumed to be equal. the class discriminant function δ k (x) is given as: wherem k is the class mean, and finally classification is performed via quadratic discriminant analysis (qda) is an algorithm that uses a quadratic plane to separate classes of data objects. following the example of lda, qda estimates the covariance matrices of each class rather than operating on the assumption that they are the same. qda follows lda with the exception that: support vector machines (svm) optimise a high dimensional hyperplane to best separate a set of data point by class by maximising the margin and minimising the empirical risk, and then predict new data points based on the distance vector measured from the hyperplane [38]. the optimisation of the hyperplane is to achieve the goal of maximising the average margins between the points and separator. generation of a multi-class svm is performed through sequential minimal optimisation (smo) [39] by breaking down the optimisation into smaller linearly-solvable sub-problems. for multipliers a, reduced constraints are given as: where there are data classes y and k are the negative of the sum over the remaining terms of the equality constraint. stacked generalisation (stacking) [40] is the process of training a machine learning algorithm to interpret the predictions of an ensemble of algorithms trained upon the dataset in a process of meta-learning. generally, a stack can represent any kind of ensemble, but the interpretation algorithm is often logistic regression. it has been noted in multiple domains that stacking often outperforms the individual models in the ensemble [41-43]. it was observed during experimentation that the classification problems were difficult, leading to many models achieving relatively bad results, i.e., the results outperformed an approximate 25% chance random guess by around 10-20% classification accuracy, with many stateof-the-art models predicting the wrong value more than half of the time (< 50%). the solutions explored to solve this are the following: a linear search is performed for random decision forests (rdf) and k-nearest neighbours (knn) from 10, 20, . . ., 1000 decision trees and 1, 2, . . ., 50 neighbours, respectively. random forests are often found to be powerful ml algorithms, and so an in-depth search is performed in order to maximise their ability. this is also followed for knn since it is of low complexity and can thus be quickly benchmarked. a genetic search is also performed via the tree-based pipeline optimization tool (tpot) algorithm detailed in [44] with consideration to the whole scikit-learn toolkit [45] where not detailed in the previous section, more information is available on the models in [46] . tpot is an algorithm that treats each machine learning operator as a genetic programming (gp) primitive which include, modified features, feature combinations, feature selections and dimensionality reductions, learning algorithms as well as their predictions (for exploration of ensembles). gp trees were chosen since they best represented a machine learning pipeline and are implemented with the deap framework [47] , and best solutions are selected by the multi-objective nsga-ii algorithm [48] by aiming to increase classification accuracy while reducing minimising the number of machine learning operators as previously described. 5% of offspring produced by the best models cross-over with another through a process of onepoint crossover, and the remaining offspring randomly mutate at a 33% chance of point, insertion, or shrinkage. thus, the algorithm introduces and tunes ml operators with promising effect and removes operators that cause the results to degrade. finally, the best machine learning pipeline is presented from the search. to conclude, the method described in this section follows the process of manual exploration, linear search, and genetic programming in order to explore the best classification models for these problems in terms of classification accuracy. as previously described, accuracy is chosen as the metric of comparison since the datasets are closely balanced, and the drawback of loo is high variance (large standard deviation due to binary per-fold results) while enabling classification model validation of a small dataset. all of the experiments in this paper were performed using the scikit-learn toolkit [45] implemented in python. the algorithms were executed on an intel core i7 processor (3.7ghz). due to the large computational complexity when searching a problem space with loo, the algorithm was executed three times with a population size of 10 for 10 generations, if a model scored lower than the manually or linearly explored models then it was discarded, and otherwise presented if it achieved a higher score. this decision was based on the fact that results for the three problems attained were only 49.67%, 43.79%, and 56.21%, and more robust models were required in order to provide accurate predictions. in this section, the three sets of results are presented. for readability purposes, linear searches of rdf and knn are presented as the same figs (6) and (7). the linear searches for rdf and knn are shown in figs 6 and 7, respectively. the best rdf was a forest of 230 trees which scored 43.8%, and the best knn had a value of k = 30 which scored 36.6%. fig 9 shows the model comparison for risk of mortality. the difficulty of the problem can be seen with the low results achieved, with the exception of two models discovered by the genetic model search algorithm. the second best model, which utilised extra trees via recursive feature elimination scored 61.97% and the best model found was a process of stacking svm and extra trees which had a classification ability of 71.24%. country-level pandemic risk and preparedness classification based on covid-19 data fig 10 shows a comparison of other models that were explored. many solutions were quite weak, but achieving higher results in comparison to the other two problems, suggesting that the problem is a slightly less difficult one. the best algorithms, as was the case for the other problems, were also discovered by the genetic search algorithm. unlike the previous two problems, the best models found were singular rather than either an ensemble or feature elimination pipeline, where extra trees scored 71.21% and gradient boosting scored 77.12%. following the original outline of the experiment in figs 1 and 11 builds upon this by including the best findings from the three benchmarking experiments. the best model for risk of transmission was a stacking algorithm combining gradient boosting and a decision tree for 74.51% accuracy, the best model for risk of mortality was a stacking algorithm combining support vector machine and extra trees for 71.24% accuracy, and the best model for risk of inability to test was a gradient boosting algorithm for 77.12% accuracy. all of the best models were found by the genetic model search algorithm. as previously discussed, the classification of risks must be interpreted relative to one another. for example, if the maps in figs 2, 3 and 4 are observed, note that countries that do not test much also seemingly, on the surface, report fewer cases and deaths per million. on one hand, this could simply be due to the fact that there are fewer cases and thus fewer tests are country-level pandemic risk and preparedness classification based on covid-19 data required, but on the other hand, could imply that fewer tests performed have themselves led to unreported figures of the other two [49] [50] [51] [52] . with this in mind, it is important to consider the output for risk of inability to test in order to interpret the other two risks. in the case where the risk of inability to test is towards the lower end of the spectrum, then risks for transmission and mortality are more likely to be an accurate representation of the situation. vice versa, though, where there is a high risk of inability to test, this in itself should be considered the most descriptive risk factor for the country since there is less prior knowledge to base risks of transmission and mortality upon. country-level pandemic risk and preparedness classification based on covid-19 data table 1 shows the predicted class values for the best models applied to each of the respective risk classification problems. please note the discussion of interpretation in section 3.4, where high inability to test is often coupled with lower risks of the prior two, as can be seen in fig 5, for as of yet unknown reasons i.e. they could either be actually true to the pattern observed, or on the other hand, very low testing leads to naturally fewer reported cases and deaths than the actual values. many countries bare similarity to others and so have been generalised, further outliers still such as china may not have accurately predicted labels since the population is much larger than those observed in the training data, likewise this may be the case with other geopolitical information within the outlier set. in this section, we perform a preliminary exploration of how useful country-level attributes are in addition to lag-window features (seven days prior, with mean and standard deviation for days 1 − n via a growing lag-window). the process is implemented via a 10-fold temporal validation process (predicting future fold k from growing training data 1 to k − 1). this approach is explored for the forecasting of cases and deaths. appendix a in s1 appendix shows the pearson correlation coefficient of each attribute in relation to the total cases for each day. as can be expected, the most correlative feature are the cases recorded for the previous day. interestingly, mean values of the previous two and three days have more correlation to the total cases on the current day compared to the previous day lag value alone. gross domestic product and urban population have a weak but useful correlation for regression of the total cases. as can be expected, the singular pearson's correlation coefficient of each of the isolated attributes tend to be low with exception to the lag windows due to the nature of increasing growth in infections. the tables within appendices b, c, and d in s1 appendix detail the scores given to the attributes by linear regression, m5p and svr respectively. it is observed that the rankings achieved by the lag window attributes are the same for each algorithm, and the order otherwise is relatively similar. all algorithms then rank the country at the same place above other features, which actually had a negligible correlation of 0.03. another interesting observation is that the m5p algorithm ranks medical doctors per 1,000 population as relatively high in the ranking, second only to country when lag windows are not considered. urban population totals are considered important by all of the algorithms, likely since this is an indication of both spread as well as a rule of thumb for total number of infected. table 2 shows the results for total case prediction by all of the chosen algorithms. the best algorithm achieved a rmse of 325.66 when considering 41 features chosen by linear regression ranking, which were the 19 time-window attributes and 22 geopolitical or demographic attributes. this provides a decrease in rmse of 17.08 when this algorithm only considers lags of the series, and many instances can be observed in which this metric was reduced by considering additional attributes explored within this study. the best results achieved by all of the seven algorithms considered at least two of the additional attributes, it is worth noting that the best of the best models is also the model which chose the most of the additional attributes (as well as the best svr, which also chose 41 attributes in total). appendix e in s1 appendix shows the correlation of each singular attribute towards the prediction of deaths. as can be observed, the rankings of the lag windows are the same as those for total confirmed infections described in the previous section. otherwise, rankings are similar and differ only slightly, as well as their observed correlation. appendices f, g, and h in s1 appendix detail the scores given to each attribute by the linear regression, m5p and support vector regression algorithms respectively. as can be expected, the rankings match those of the highest correlation coefficient. interestingly, a small change is noted within the attributes for svr whereas quite a disparity can be seen when observing the scores given by the other two algorithms. table 3 shows the 189 models trained for forecasting total deaths. similarly to the total case predictions, the best model found was within a voting ensemble of linear regression and svr. unlike total case predictions, introducing geopolitical and demographic attributes had negative effect on the result, with the best model taking only the temporal lag window features as input. once 40 attributes were introduced, the linear regression model had an absurdly high rmse of 2.93e+05, which since average values were taking during voting regression, also affected the ensembles that included it. given the nature of research and peer review, the approach in this work was formalised on the 12th of may 2020 and as such the data is over three months out of date at the time of writing (16th of september, 2020). given this, the experiments devised in this work are re-applied to the new data. it was noted that all manual models failed to generalise with the new data. that is, a range of scores between 24.95% to 28.63% for all models, for all three risk classification problems. this is most likely due to international collaboration towards the three risk factors, and as such, country-level attributes lose classification prediction ability towards the risk factors. with the previous successful experiments in mind, this argues that risk classification would be more useful when performed prior to the situation unfolding, given that country-level information is seemingly more important at this stage when compared to the current postpeak climate. though much weaker results are now observed, this could in fact be viewed as a positive situation, given that country-level data i.e. who you are and where you are from no longer impacts risk as it was observed to in the initial experiments performed in may 2020. it has been noted during mid-2020 that organisations such as the united nations and world health organisation have implemented and released humanitarian packages to lower economically developed countries (ledcs) [53] [54] [55] . it has also been noted that many healthcare professionals returned to their native countries (often also ledcs) in order to aid in tackling the virus [56] . the positive effects of these factors likely contribute towards the reason why country-level information was useful for risk classification earlier in the pandemic, but are less-so later on post-peak. with the nature of the data streaming from the ongoing pandemic with regards to the time taken to run model benchmarks, the largest and most obvious limitation to this study is that the models are constantly going out of date by the day, since more up to date data is constantly becoming available. it is for this reason that the models should be updated at a later date, and the statistical differences that occur, if any, noted. secondly, though relatively good results were found through a complex process of genetic optimisation, further models could be explored in order to possibly reach better results than the final models in this study. finally, the interpretation that is required as aforementioned, i.e. that the risk of inability to test is the most important metric and possibly enables the other two for interpretation, suggests that the ternary approach followed could be better optimised through a unified approach. that is, one singular "metric of risk" that is calculated via the three metrics explored in this work as separate problems. prior to this study, a metric of (c + d)/t was explored (where c, d, and t denote cases, deaths, and tests respectively, all with regards to per million population), but this metric is, at this point, impossible to classify. the k% method was used to divide the continuous features into four bins where k = 25. other methods of binning such as mdl [57] , caim, cacc, and ameva [58] could also be explored and benchmarked in future experiments. to conclude, the main hypothesis that this work has argued in favour of is that geopolitical and demographic attributes at the country-level hold value in terms of classifying risk produced by the covid-19 dataset. this was shown when the four class distribution which was close to equal ('high' was 1.2% larger than the other classes) could be classified far above the approximate 25% class distribution through loo cv. though this is observably possible from the results presented in this study, it is worth noting that the classification problem proved extremely difficult for many powerful machine learning techniques, which often scored around only 40%, and a genetic search had to be followed in order to devise complex strategies of ensemble and hyperparameter optimisation in order to achieve better results at 74.51%, 71.24%, and 77.12% for the three problems. future work aims to keep the data up to date to the point at which the pandemic is over, and also to explore other methods of solving the issue of risk and preparedness classification through a more unified approach as well as through stronger machine learning models, if possible. global catastrophic risks survey presumed asymptomatic carrier transmission of covid-19 estimation of the asymptomatic ratio of novel coronavirus infections (covid-19) covid-19: identifying and isolating asymptomatic people helped eliminate virus in italian village sjö din h. high population densities catalyse the spread of covid-19 the effect of travel restrictions on the spread of the 2019 novel coronavirus (covid-19) outbreak case-fatality rate and characteristics of patients dying in relation to covid-19 in italy obesity in patients younger than 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covid-19 in various countries: preliminary retrospective results based on wavelets and deterministic modeling estimating the fraction of unreported infections in epidemics with a known epicenter: an application to covid-19 will covid-19 be a litmus test for post-ebola sub-saharan africa? the coronavirus knows no borders. tidsskrift for den norske legeforening health prevention and response policies against infectious diseases: is the world ready for a novel coronavirus pandemic? proceedings book the world health organisation. how is who responding to covid-19? azerbaijani doctors return home to help their country face covid-19 multi-interval discretization of continuous-valued attributes for classification learning supervised and unsupervised discretization of continuous features the authors would like to show their gratitude to all of the medical professionals working to treat and cure covid-19 across the world, as well as the researchers working vigorously on vaccines and antibody tests for the disease. we would also like to thank all of the key workers for their effort to make life as normal as possible during these difficult times. key: cord-325113-sou8xyld authors: kuiper, johannes w. p.; baade, timo; kremer, marcel; kranaster, ramon; irmisch, linda; schuchmann, marcus; zander, johannes; marx, andreas; hauck, christof r. title: detection of sars-cov-2 from raw patient samples by coupled high temperature reverse transcription and amplification date: 2020-11-02 journal: plos one doi: 10.1371/journal.pone.0241740 sha: doc_id: 325113 cord_uid: sou8xyld sars-cov-2 is spreading globally with unprecedented consequences for modern societies. the early detection of infected individuals is a pre-requisite to contain the virus. currently, purification of rna from patient samples followed by rt-pcr is the gold standard to assess the presence of this single-strand rna virus. however, these procedures are time consuming, require continuous supply of specialized reagents, and are prohibitively expensive in resource-poor settings. here, we report an improved nucleic-acid-based approach to detect sars-cov-2 with the ability to detect as little as five viral genome equivalents. the approach delivers results without the need to purify rna, reduces handling steps, minimizes costs, and allows evaluation by non-specialized equipment. the use of unprocessed swap samples is enabled by employing a heat-stable rnaand dna-dependent dna polymerase, which performs the double task of stringent reverse transcription of rna at elevated temperatures as well as pcr amplification of a sars-cov-2 specific target gene. as results are obtained within 2 hours and can be read-out by a hand-held led-screen, this novel protocol will be of particular importance for large-scale virus surveillance in economically constrained settings. as of september 2020, severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is responsible for more than 32.7 million covid-19 cases and associated with more than 991 000 fatalities (who; covid-19 weekly epidemiological update [accessed 30. september 2020]. available from: https://www.who.int/emergencies/diseases/novel-coronavirus-2019/ situation-reports). within a few months of its emergence, this infectious agent has spread globally and is derailing societies worldwide. sars-cov-2 is an enveloped plus-strand rna virus of the beta-coronavirus genus with closely related strains circulating in bats and some other mammals indicating a zoonotic origin [1, 2] . upon host cell infection, beta-coronaviruses generate a minus-strand rna copy of their genome by a virus-encoded rna-dependent rna-polymerase [3] . furthermore, the virus directs the synthesis of several subgenomic minus strand rnas, which are complementary to the 3' end of the viral genome and which encode several non-structural proteins including the n gene [3] . therefore, infected cells harbor plus and minus strand rnas of the coronavirus, with an overabundance of transcripts derived from the 3' end of its genome [4, 5] . as with other rna viruses, confirmation of sars-cov-2 infection is based on the molecular biological detection of the viral genome and its transcripts in patient samples by nucleic acid amplification techniques (naats) [6] . to allow sensitive and accurate detection of viral ribonucleic acids, primary patient samples such as nasopharyngeal swabs, sputum or stool are further processed to isolate the total rna. using reverse transcriptase, the rna is then reverse transcribed into dna. next, the dna is pcramplified by a thermo-stable dna-dependent dna polymerase using specific primers and probes to detect the presence of sars-cov-2 sequences. facilitated by genome sequencing and rapid information sharing, such a naat-based diagnostic surveillance of sars-cov-2 has been in place right from the beginning of the covid-19 pandemic [7, 8] . however, the current methodology is laborious, requires multiple handling steps and expensive consumables, resulting in a costly diagnostic procedure, which can constrain covid-19 testing in economically tight settings. furthermore, the overwhelming worldwide demand for some of the same reagents (such as those needed for rna isolation) and diagnostic kits has produced shortages and unnecessarily delayed or restricted testing. from a clinical point of view, rapid testing and early decision making on further isolation measures for patients and health care workers remains a critical issue. we have developed a thermostable dna polymerase, which can mediate dna synthesis from both rna as well as dna templates [9] . by targeted modifications, we have further improved the accuracy and processivity of this enzyme [10] , which lays the foundation of the commercialized volcano3g formulations. we reasoned that such a bi-functional enzyme might allow us to improve and simplify the detection of rna viruses. here we show that vol-cano3g can be used in a coupled high-temperature reverse transcription and amplification reaction to detect sars-cov-2 with high sensitivity and specificity. moreover, our findings demonstrate the usefulness of such a thermo-stable rna-and dna-reading dna polymerase to simplify covid-19 diagnostics. most importantly, we can show that this robust enzyme allows detection of sars-cov-2 directly from unprocessed patient material. accordingly, this streamlined procedure, which does not depend on limited reagents, nor requires expensive equipment, is poised to enable large scale sars-cov-2 surveillance in a multitude of resourceor time-constrained settings. a rna-and dna-reading heat-stable polymerase reverse transcribes and amplifies viral rna evidence of an acute sars-cov-2 infection depends on the detection of viral rna species in patient samples, which necessitates reverse transcription of rna followed by pcr amplification of the resulting dna. to confirm that the volcano polymerase is able to perform both of these steps, we employed an in vitro transcribed synthetic sars-cov-2 rna template covering a~750 nt stretch within the 3'-end of the viral genome ( fig 1a) . this target region is also included in the cdc panel of primers (division of viral diseases, national center for immunization and respiratory diseases, centers for disease control and prevention, atlanta, ga, usa; https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html; the target sequences for the n1 primers and probe are marked in red and green, respectively. the r2 primer binding sequence is underlined. sequences divergence between sars-cov-2 and sars-cov-1 genomes are highlighted in blue. b) performance of volcano3g polymerase was compared to taq polymerase using plasmid dna or in vitro transcribed rna as template (5000 viral genome equivalents). c) determination of the linear dynamic range for the volcano3g protocol with or without an additional primer (r2) for optimized reverse transcription at a final concentration of 250 nm. in vitro transcribed rna containing the sars-cov-2 n amplicon was serially diluted in the range from 1x10 6 copies to 10 copies. d) limit of detection (lod) was assessed with serial dilutions ranging from 20 to 1 copy per reaction (n = 6 for each dilution). the fraction of positive reactions (y-axis) were plotted against the log-transformed number of rna copies per reaction. addition of r2 primer enhances the performance at lower copy-numbers. e) amplification curves showing the performance of volcano3g on isolated rna from two covid-19 patients in presence or absence of r2. https://doi.org/10.1371/journal.pone.0241740.g001 accessed on 5.5.2020). when 5000 genome equivalents of the purified, in vitro transcribed viral rna was used as a pcr template for a generic, heat-stable dna-dependent dna polymerase (taq dna polymerase) no amplification occurred, demonstrating the absence of a contaminating dna template ( fig 1b) . in contrast, when volcano3g was employed, the expected amplification product was obtained, confirming that the volcano3g polymerase can read the rna template to produce and amplify a specific dna sequence ( fig 1b) . clearly, the taq dna polymerase was able to yield an amplicon, when dna instead of rna was used as a template (fig 1b) . not surprisingly, the volcano3g polymerase was able to operate with primers and probes supplied by other manufacturers targeting the same region of the sars-cov-2 n gene (s1a and s1b fig) . due to the thermostability of the volcano3g polymerase, this rna-reading enzyme could perform, in contrast to other widely used enzymes, the reverse transcription under stringent, high-temperature conditions. reverse transcription at elevated temperatures could help to overcome stable rna-secondary structures present in beta-coronavirus genomes [11, 12] . to take advantage of this particular feature of the volcano3g polymerase, we added an extended, high-melting temperature virus-specific primer (r2) to the reaction, and adjusted the pcr protocol to include a high-temperature reverse transcription step. as the 3'-end of the viral genomic plus-strand rna and its minus-strand complementary sequences are the most abundant nucleic acids in coronavirus-infected cells, we focussed on the coronavirus n gene, which is located at the very 3'-end of the sars-cov-2 genome [13] . furthermore, the amino terminus of the n protein varies greatly between different humanpathogenic beta-coronavirus isolates (s1c fig) , making this region an ideal target for sars-cov-2-specific primers. accordingly, the high-temperature rt primer r2 was designed to be complementary to sequences at the 5' end of the n gene ( fig 1a) . the addition of the r2 primer consistently increased the performance of the volcano3g reaction resulting in lower cq-values over a wide range of template concentrations ( fig 1c) . the high-temperature reverse transcription afforded by volcano3g polymerase and the r2 primer worked at temperatures >70˚c (s1d fig) . while the optimal elongation temperature for the purified rna template was at the lower end of these temperatures (s1d fig) , a slightly elevated temperature (75˚c) was chosen with more complex rna samples to achieve a balance between stringency and polymerase acitivity. under these experimental conditions, addition of r2 lowered the limit of detection (lod) to five copies of the viral genome equivalent ( fig 1d) . to assess, if this adapted procedure allows the sensitive detection of sars-cov-2 in patient material, we used rna isolated from nasopharyngeal swabs of two confirmed covid-19 cases. in both samples, amplification of the human rnasep transcript demonstrated the integrity of the samples and resulted in similar cq-values in the presence or absence of the additional r2 primer, while the non-template control (ntc) gave no signal ( fig 1e) . most importantly, the volcano3g polymerase detected viral rna in both samples, but produced exceptionally low cq-values upon addition of the r2 primer, suggesting that the high-temperature reverse transcription by the thermo-stable rna-reading dna polymerase opens an additional window of opportunity for hypersensitive detection of viral rna. together, these initial results encouraged the further exploration of the volcano3g polymerase for facilitating the detection of sars-cov-2 rna. to evaluate the potential of the high-temperature rt-pcr protocol using volvano3g for the detection of viral rnas in patient material, we assessed the presence of sars-cov-2 in rna isolated from a small cohort of covid-19 suspected cases. rna was isolated from nasopharyngeal swabs and the isolated nucleic acid was then evaluated in parallel by i) a commercial in vitro diagnostic kit (allplex, seegene) and ii) high-temperature rt-pcr with volcano3g. of the 43 samples, the allplex-assay detected the sars-cov-2 n gene in 35 samples, while 8 samples remained negative (fig 2a) . the eight negative samples also did not yield amplicons in the allplex-assay for the rdrp or the e gene (data not shown). when the 43 rna samples were employed in high-temperature rt-pcr with volcano3g polymerase, the identical results were obtained, showing complete consistency with regard to positive and negative outcomes (fig 2a and 2b ). pairwise comparison of the cq-values obtained for the isolated rna samples in detecting the n gene revealed that the high-temperature rt-pcr with volcano3g resulted in lower cq-values throughout all samples suggesting a slightly increased sensitivity (fig 2c) . in addition to the resolution of rna secondary structures, the increased sensitivity might be due to the fact that the high temperature reverse transcription step involves several cycles, which allow initial highly stringent amplification of viral target genes. though the high temperature rt-pcr with volcano3g was slightly more sensitive, the results correlated extremely well over a wide range of cq-values with the results obtained by the commercial assay (r 2 = 0.980, p<0.0001, fig 2d) . together, these findings suggest that high temperature rt-pcr with volcano3g could be an additional option to rapidly detect rna-virus genomes with high specificity and sensitivity. besides enhanced stringency and possible resolution of rna secondary structures, we speculated that high-temperature rt-pcr might promote viral lysis and allow the detection of sars-cov-2 rna directly from unprocessed patient samples. to this end, we employed a second cohort of samples, where each nasopharyngeal swab was initially resolved in 300 μl of distilled water. while 150 μl of these diluted samples were used for rna extraction and standard rt-pcr, 12 μl of the diluted sample were directly employed in high-temperature rt-pcr with the volcano3g polymerase. importantly, even with this raw patient material, the hightemperature rt-pcr yielded clear-cut results without increasing the background (fig 3a) . each sample was analysed repetitively in three to four separate high-temperature rt-pcr runs with the volcano3g polymerase and the results obtained were compared to the standard rt-pcr from purified rna of the identical samples. importantly, all negative samples were consistent between the two approaches suggesting that there is no increased risk of producing false positive results, when using the unprocessed patient samples (fig 3b) . moreover, 100% of the samples showing cq-values of � 24 for the sars-cov-2 n gene in the standard rt-pcr assay with purified rna were correctly identified by the volcano3g high-temperature rt-pcr employing raw patient samples (fig 3b and 3d) . also, 10 out of 12 samples showing cq-values >24 and � 30 for the sars-cov-2 n gene in standard rt-pcr from purified rna were detected by direct high-temperature rt-pcr from unprocessed samples in all replicates (fig 3b) . the remaining 2 samples of this set were positive in 1 or 3 out of four replicates, respectively (fig 3b) , samples yielding cq-values above 30 in the standard rt-pcr following rna extraction, which would represent a low viral load, were only rarely detected as positive, when patient samples were used without further processing (fig 3b) . none of those patients, from which samples with low viral loads (cq-value >30) were obtained, was hospitalized and all of them showed only mild symptoms including sore throat and rhinitis. it is important to stress that clinical studies of viral dynamics in patients have shown that sars-cov-2 titers in swab samples are most often high during the initial phase of the infection, even before onset of symptoms [14] [15] [16] [17] . in contrast, low titers are typically seen at the later phase of the infection and can even be detected after the resolution of symptoms [18] . observations from laboratoryconfirmed sars-cov-2 transmission pairs indicate that transmissability is highest around the onset of symptoms, when viral titers in nasopharyngeal swabs are high [19] , and that infectiousness of clinical samples towards cultured cells is negligible, if cq-values are above 24 [20] . indeed, virological assessment of patient-derived swap material has shown, that samples yielding cq-values > 30 in regular rt-pcr from purified rna (corresponding to~10 6 viral copies/ ml of sample) do not allow productive infection of cell cultures [21] . based on these findings, the simple and rapid detection of people sustaining high sars-cov-2 titers as afforded by high-temperature rt-pcr of unprocessed patient material might be sufficient to break chains of viral transmission. interestingly, for most positive samples detected by the high-temperature rt-pcr with volcano3g, the cq-values were lower compared to the standard rt-pcr (fig 3c and 3d) , indicating that the detection of sars-cov-2 from unprocessed patient material is not limited by the sensitivity of this direct approach. as unprocessed swab material might contain numerous patient-derived proteins, mucus, or membrane fragments, we speculated that substance(s) within these raw samples could interfere and inhibit the detection of sars-cov-2 when only high-temperature rt-pcr using volcano3g polymerase allows sars-cov-2 detection from unprocessed patient samples. a) nasopharyngeal-and throat swab samples (prepared in water) were added directly as template for rt-qpcr using the volcano3g protocol. representative amplification curves of patients with high (dark blue), medium (medium blue) and low cq as well as negative patients (light blue) are shown. b) rna was isolated from the remaining patient material and analysed in an accredited diagnostic lab using the allplex 2019-ncov assay from seegene. the samples were divided in 3 groups based on cq (<24, 24-30 and >30) and each sample was analysed repeatedly (3-4 times) by high-temperature rt-pcr. the bar diagram depicts the average frequency of detection (± sem) for each group (patient samples in each group: cq < 24 n = 12; cq 24-29 n = 12; cq > 30 n = 9 and negative samples n = 12). c) the direct and rapid sars-cov-2 detection a low viral load is present. therefore, we tested the inhibitory potential of the eluted swab material. using the in vitro transcribed viral rna as a template, we spiked the volcano3g reaction mix with increasing amounts of swab-derived material (fig 3e) . importantly, there was a clear dose-dependent inhibition of the amplification reaction due to the material eluted from the swabs (fig 3e and s2 fig). the extent of inhibition showed variability between individual samples (s2 fig), with some samples leading to complete inhibition of viral detection at 10% added swab material ( fig 3e) . especially, for samples containing low viral titers this could significantly elevate the lod. therefore, we supplemented the distilled water used for eluting the nasopharyngeal swabs with betaine, bsa, carrier rna, dtt or treated them with proteinase k or combinations of these reagents, with the idea that these substances might be able to alleviate the inhibition due to inhibitory proteins or rna degrading or oxidizing agents. indeed, several of the treatments enhanced the detection of sars-cov-2 from an unprocessed patient sample demonstrating that it is possible to partially overcome the inhibitory effect of the raw material (s3a and s3b fig) . as patient samples are expected to contain molecules that sequester magnesium ions (e.g. dna, proteins) and calcium ions and therefore compete with magnesium for binding to polymerases, we hypothesized that increasing the concentration of mgcl 2 might augment the pcr reaction. indeed, increasing concentrations of mgcl 2 lowered the threshold of detection of rna template in presence and absence of spiked-in (sars-cov-2 negative) patient material proteinase k treatment is normally performed at relatively high temperatures (50˚c-70˚c) to promote protein unfolding and enhance enzyme activity. afterwards, inactivation of proteinase k is required to protect the polymerase from proteolysis. though inactivation of proteinase k is commonly achieved by heat treatment (5-10 min at 95˚c) these high temperatures also promote rna degradation, especially in the presence of divalent cations (s3e fig). to circumvent the thermal inactivation of proteinase k we included instead the serine protease inhibitor phenylmethylsulfonyl fluoride (pmsf) in our final optimized protocol (s3f fig). to re-evaluate 10 nasopharyngeal swabs, which had shown low viral titers on initial testing (conventional rt-pcr cq > 30), we eluted these swabs again in 50 μl rnase-free water supplemented with 10 ng/μl carrier rna and stored for 1 month at -20˚c. this small volume (compared to 350 μl for the cohort in fig 3a and 3b ) was chosen to increase the concentration of eluted virus from these low-titer samples. these re-eluted low-titer samples were subjected to our optimized protocol (s3f fig). again, we did not observe any false-positives, but were able to detect 4 out of 10 positive low-titer samples (cq > 30, fig 3f) . even though the quality of this low-titer cohort was suboptimal due to re-using of swabs, we were able to decrease the false-negative rate for samples with cq > 30 from 96% (fig 3b) to 60% (fig 3f) . it is important to note that the number of tested samples is very low and therefore it will be necessary to test cq values of each patient sample are compared between the reference protocol and the volcano3g direct approach. dotted red line indicates the cut-off, were the direct assay looses sensitivity. d) for each positive patient sample, the cq values obtained in 3-4 repetitions with the high-temperature rt-pcr (volcano3g direct input; mean +/-sd) were plotted against the cq-values obtained with the standard rt-pcr on isolated rna for linear regression analysis (r 2 = 0.779, p<0.0001). e) rt-pcr analysis of 100 copies of in vitro transcribed rna spiked with varying amounts of pooled patient material from 5 confirmed negative patients. f) 10 swabs were eluted a second time according to our optimized protocol (low elution volume, protease k treatment and additional mgcl 2 ). in addition, 10 confirmed negative swab samples were included. g) 5 volcano3g reactions from the cohort of unprocessed samples presented in fig 3a ( in low-translucent white tubes) were photographed on a blue light transilluminator. reference cq values (seegene allplex on purified rna) and cq values obtained with volcano3g using unprocessed samples (measured cq) are depicted above each tube. https://doi.org/10.1371/journal.pone.0241740.g003 larger cohorts to obtain robust numbers on false-negative rates. moreover, additional parameters might require optimization such as determining the optimal sampling location (nose swabs, throat swabs, sputum, or saliva) and the influence of swab type [22] . though further improvements will be necessary to consistently detect sars-cov-2 rna in unprocessed lowtiter samples, bypassing the need for rna purification dramatically accelerates and simplifies the evaluation of patient material. as an additional asset of the simplified approach, we have observed that the endpoint of the high temperature rt-pcr with volcano3g polymerase can also be easily evaluated on a bluelight led screen, yielding strong green fluorescence for positive samples and allowing clearcut differentiation by the naked eye (fig 3g) . together, the use of a heat-stable, rna-and dna-reading polymerase is poised to simplify detection of viral rnas. by reducing handling steps, the chances of sample cross-contamination are minimized. moreover, screening of unprocessed samples might be an economical way to identify highly contagious, asymptomatic individuals in population-wide screening campaigns, as high viral loads are present at the initial stage of infection [17] . the overwhelming dynamic spread of sars-cov-2 and the apparent bottle-necks in virus testing highlight the need for additional methodology that can be deployed in resource poor settings and that is operative in situations, where particular reagents such as rna isolation kits might become scarce. therefore, the direct detection of virus rna from unprocessed patient samples and the ability to perform and read-out complex naat procedures using standard field pcr machines and blue-light screens could dramatically expand the covid-19 testing capabilities and might also afford an important economic relief for health care providers in large parts of the world. nasopharyngeal swabs were collected at the covid-19 test center hosted at the klinikum konstanz. samples used in this study were procured with sterile dry swabs (copan 160c rayon, www.copaninnovation.com; eurotubo collection swab 300253, www.deltalab.es; culture swab without transport medium 09-516-5009, www.nerbe-plus.de) and samples were processed within 24h for rna isolation. material eluted in rnase/dnase free deionized water not directly used for rna isolation was stored at -20˚c for further analysis. the study was approved by the local ethics committee of university of konstanz (05/2020). the authors confirm that all research was performed in accordance with relevant guidelines/regulations and that informed consent was obtained from all participants and/or their legal guardians. nasopharyngeal swabs were eluted in 350 μl rnase/dnase free deionized water and 150 μl of the eluate was used for rna isolation using the nucleospin 96 rna kit (macherey-nagel, düren, germany), where the ra1 lysis buffer included in the kit was supplemented with 10 μg/ ml of carrier rna (qiagen, hilden, germany) and 15 mm dtt. automated handling of the samples was accomplished using the integra assist plus pipetting robot (integra, biebertal, germany). finally, purified rna was eluted into 60 μl rnase/dnase free deionized water. position 27923-28648 of the sars-cov2 genome (nc_045512.2) was amplified with pfu dna polymerase (primers clone_n1_f and clone_n1_r, see table 1 ). the resulting 726 bp amplicon was purified, digested (kpni/bamhi) and ligated in pbluescript-ks(+). the resulting plasmid was linearized with bamhi, purified and transcribed in vitro (t7 dna polymerase, neb) for 4 hours at 37˚c to yield a 753 nt transcript. the purified transcript was treated with rnase-free dnase (roche) for 4 hours at 37˚c and purified again (viral rna isolation kit, qiagen). reference cq values were obtained using the allplex 2019-ncov assay (seegene, seoul, south korea) according to the manufacturer's instructions. 8 μl of purified rna were used to run 25 μl reactions on a bio-rad cfx96 and analysed with seegene viewer software version 3.18. throughout this study high-temperature rt-pcr on rna purified from either the in vitro transcribed viral genome fragment or the swab material was performed with the volcano3g rt-pcr probe 2x master mix (in short: volcano3g) (mypols biotec, konstanz, germany) using the cdc-approved n1 primer/probe mix from integrated dna technologies (idt, san diego, ca, usa). in addition, sequence-identical primers and probe were obtained from microsynth ag (balgach, switzerland) to produce data presented in s1 fig. typically, 10 μl reactions were set up containing 1 μl rna, 1x volcano3g mastermix, cdc_n1 or rnasep primer/probe mix (see table 1 ) and 250 nm r2 primer. a three-stage thermal cycling program was performed on a roche lightcycler 96, consisting of 1) 150 seconds at 75˚c, 2) 10 cycles of 95˚c for 5 seconds, 150 seconds at 75˚c, 3) 45 cycles of 95˚c for 10 seconds, 57˚c for 35 seconds. all assays were performed in white low profile tubes with ultra-clear caps (thermo-fisher, cat no: ab1771) as singleplex with fam/bhq1-labelled probes. cq values were determined with roche lightcycler software 1.1.0. nasopharyngeal swabs were eluted in 350 μl rnase/dnase free deionized water. these unprocessed samples were added directly to the pcr reaction. reaction conditions were identical to those described for purified rna as template, with the exception that the reaction volume was increased to 50 μl and 12 μl unprocessed sample was used (unless otherwise stated in the results section). in addition, successful amplification could be visualised by detection of the dequenched fluorescein signal (fam) on a blue light transilluminator (safe imager 2.0, invitrogen). in an effort to improve the efficiency of our protocol on unprocessed patient samples, several treatment regimens were tested. nasopharyngeal swab samples in distilled water were supplemented with 1 ng/μl carrier rna (jena analytik), 100 mm betaine (molecular grade, sigma aldrich), 0.05% bovine serum albumin (bsa) or a combination of all three, before adding the samples directly to the volcano3g master mix. in another approach, swab samples were treated with 130 μg/μl nuclease-free proteinase k (analytik jena) for 10 minutes at 70˚c in 5 mm hepes (ph 7.4) followed by inactivation for 10 minutes at 95˚c. alternatively, samples were treated with 2.5 mm nuclease-free dtt (thermo fisher scientific) for 10 minutes at 70˚c or a combination of proteinase k and dtt. in the final optimized protocol ( serial dilutions of the in vitro transcribed amplicon were made in water containing 5 ng/μl rna carrier mix (innuprep virus dna/rna kit, analytik jena) and real-time pcr reactions (10 μl reaction volume) were set up as described above. three to eight replicate reactions were conducted containing 1x10 6 , 1x 10 5 , 1x 10 4 , 1x10 3 , 100, 10, 5, 3 or 1 copies per reaction. to estimate the lod, the fraction of positive reactions (cq < 40) was plotted against the logtransformed number of amplicon copies. linear regression analysis was performed with prism (graphpad, la jolla, ca, usa). genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding a pneumonia outbreak associated with a new coronavirus of probable bat origin the nonstructural proteins directing coronavirus rna synthesis and processing equine arteritis virus subgenomic rna transcription: uv inactivation and translation inhibition studies arterivirus nsp1 modulates the accumulation of minusstrand templates to control the relative abundance of viral mrnas rapid point of care diagnostic tests for viral and bacterial respiratory tract infections-needs, advances, and future prospects. the lancet infectious diseases a new coronavirus associated with human respiratory disease in china detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr. euro surveillance: bulletin europeen sur les maladies transmissibles = european communicable disease bulletin evolving thermostable reverse transcriptase activity in a dna polymerase scaffold structure and function of an rnareading thermostable dna polymerase characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot a three-stemmed mrna pseudoknot in the sars coronavirus frameshift signal high-resolution analysis of coronavirus gene expression by rna sequencing and ribosome profiling evaluation of sars-cov-2 rna shedding in clinical specimens and clinical characteristics of 10 patients with covid-19 in macau viral load of sars-cov-2 in clinical samples. the lancet infectious diseases sars-cov-2 viral load in upper respiratory specimens of infected patients quantitative detection and viral load analysis of sars-cov-2 in infected patients clinical and virological data of the first cases of covid-19 in europe: a case series. the lancet infectious diseases temporal dynamics in viral shedding and transmissibility of covid-19 predicting infectious sars-cov-2 from diagnostic samples virological assessment of hospitalized patients with covid-2019 impact of nasopharyngeal swab types on detection of bordetella pertussis by pcr and culture we thank the members of the ag hauck and ag groettrup for their help in processing patient samples, thomas meyer and silke müller from the screening center of university of konstanz for automated rna isolation, and susanne feindler-boeckh for expert technical assistance. key: cord-346089-u31n0qxa authors: mcdade, thomas w.; mcnally, elizabeth m.; zelikovich, aaron s.; d’aquila, richard; mustanski, brian; miller, aaron; vaught, lauren a.; reiser, nina l.; bogdanovic, elena; fallon, katherine s.; demonbreun, alexis r. title: high seroprevalence for sars-cov-2 among household members of essential workers detected using a dried blood spot assay date: 2020-08-14 journal: plos one doi: 10.1371/journal.pone.0237833 sha: doc_id: 346089 cord_uid: u31n0qxa objective: serological testing is needed to investigate the extent of transmission of sars-cov-2 from front-line essential workers to their household members. however, the requirement for serum/plasma limits serological testing to clinical settings where it is feasible to collect and process venous blood. to address this problem we developed a serological test for sars-cov-2 igg antibodies that requires only a single drop of finger stick capillary whole blood, collected in the home and dried on filter paper (dried blood spot, dbs). we describe assay performance and demonstrate its utility for remote sampling with results from a community-based study. methods: an elisa to the receptor binding domain of the sars-cov-2 spike protein was optimized to quantify igg antibodies in dbs. samples were self-collected from a community sample of 232 participants enriched with health care workers, including 30 known covid-19 cases and their household members. results: among 30 individuals sharing a household with a virus-confirmed case of covid-19, 80% were seropositive. of 202 community individuals without prior confirmed acute covid-19 diagnoses, 36% were seropositive. of documented convalescent covid-19 cases from the community, 29 of 30 (97%) were seropositive for igg antibodies to the receptor binding domain. conclusion: dbs elisa provides a minimally-invasive alternative to venous blood collection. early analysis suggests a high rate of transmission among household members. high rates of seroconversion were also noted following recovery from infection. serological testing for sars-cov-2 igg antibodies in dbs samples can facilitate seroprevalence assessment in community settings to address epidemiological questions, monitor duration of antibody responses, and assess if antibodies against the spike protein correlate with protection from reinfection. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 serological testing for sars-cov-2 igg antibodies identifies prior viral exposure and, potentially, immunity. recent surveys suggest a range of seroprevalence rates, with relatively low rates in much of the us [1, 2] . optimal surveillance of seroprevalence ideally avoids contact between community members and health care providers and surveyors, since such contact may carry risk of exposure and discourage survey participation. an alternative to venipuncture blood collection is finger stick dried blood spot (dbs) sampling [3, 4] . dbs relies on a finger prick with blood drops captured on filter paper, and dbs can be performed in the home with return of sample by mail. dbs sampling has served as the foundation for nationwide newborn screening programs since the 1960s and is increasingly applied as a minimally-invasive alternative for community health research. the centers for disease control and us postal service consider dbs specimens nonregulated, exempt materials for return of samples to laboratories [5] . a robust and quantitative elisa was granted emergency use authorization from the fda; this elisa measures sars-cov-2 antibodies in serum and has been determined to not cross-react with other common coronavirus strains [6] . this same elisa was recently used to detect a higher than expected seroprevalence for antibodies to sars-cov-2 among health care workers and patients in a pediatric dialysis unit [7] . we adapted this elisa to measure igg antibodies to the receptor binding domain (rbd) of the sars-cov-2 spike protein in dbs samples. the rbd is often the target of neutralizing antibodies, although data on the frequency of rbd-binding antibodies with neutralizing activity is still limited [8] . the primary objective of this paper is to describe our protocol and present data on assay performance and validity. in addition, we demonstrate the feasibility and utility of quantifying sars-cov-2 antibodies in self-collected dbs with results from a community-based sample enriched with health care workers. a detailed assay protocol is supplied in the s1 file. all research activities were implemented with written informed consent under protocols approved by the institutional review board at northwestern university (#stu00212457 and #stu00212472). a community-based sample of 232 adults was recruited through electronic communication initiated by the investigators. participants completed a brief survey of covid-19 symptoms and diagnosis, and were provided with materials for dbs collection. dbs sample collection occurred between april 18 and may 20, 2020. all participants returned a blood sample of sufficient quantity and quality for analysis. a set of matched serum and dbs samples was collected from 17 individuals in the study. in addition, dbs samples from 23 individuals presumed to be negative based on the pre-pandemic date of sample collection (2018) were used as negative controls for assay validation. samples were analyzed in duplicate, with the average result reported. statistical analyses were performed with prism (graphpad, la jolla, ca). an unpaired twotailed t-test was used to compare negative and confirmed positive samples. error bars represent ± standard error of the mean (sem). passing-bablok regression and bivariate correlation were used to evaluate patterns of statistical association across matched dbs and serum samples. cr3022, an antibody with defined affinity to the rbd, was used to validate dbs samples in an elisa. we observed a strong dose-response between cr3022 concentration and optical density (od) in wells coated with 2.0 μg/ml rbd ( fig 1a) , with no response in the absence of rbd antigen. responses were approximately linear up to 6.25 μg/ml, with a flattening of response at higher concentrations of cr3022. based on a calibration curve generated from the cr3022 dilution series, the lower limit of detection (lld) was determined to be 0.032 μg/ml. the cut-off for low seropositivity was set at the od value produced by the 0.39 μg/ml cr3022 calibrator; this low seropositive cut-off is well above lld and greater than 3 standard deviations above od values for known negative samples. the cut-off for full seropositivity was set at the od value corresponding to the 0.60 μg/ml cr3022 calibrator. the low seropositive cut-off range contained 2 of 30 (6.6%) known pcr+ samples. 90% of the known pcr confirmed positive cases were above the 0.60 μg/ml seropositive threshold. there was clear distinction between positive and negative samples (fig 1b) . with one exception, all pcr confirmed positive samples were more than 3 sd from the mean od for the negative samples. intra-assay variability (percent coefficient of variance (%cv), based on analysis of 10 replicates) at the seropositivity threshold of 0.60 μg/ml was 4.23, and 6.56 and 6.20%cv at 1.56 and 6.25 μg/ml, respectively. results from matched dbs and serum samples showed tight agreement (fig 1c) . we evaluated igg levels to rbd protein in 232 dbs samples acquired from the community ( table 1) . thirty cases had previous positive viral pcr testing for covid-19, with 60-85% reporting chills, fatigue, cough, or headache as the most common symptoms. twenty-seven of 30 were seropositive with igg concentrations from 0.6 μg/ml to 10 μg/ml (fig 2) . the median time between serological testing and viral pcr testing was 28 days (range 16-43 days). two participants had low seropositivity (igg<0.6 μg/ml but >0.39 μg/ml) and both had asymptomatic infections. the single seronegative participant was pregnant, and 52 days after the first viral pcr test she tested positive for virus again. among the remaining 202 participants, none of whom tested positive for sars-cov-2 with pcr, 60% reported some covid-19-like symptoms with headache, fatigue, or rhinorrhea being most common. fifteen of these participants were pcr tested and determined viral negative; of these, 9 were determined to be seronegative, 4 were low seropositive, and 2 were seropositive in dbs. only one participant required hospitalization for dyspnea (0.8%), and this individual tested viral negative and was low seropositive. follow-up dbs samples were acquired from 28 seronegative and 19 low seropositive participants, approximately 2-3 weeks (range from 13-23 days) after the original dbs sampling date from may 5 through june 4, 2020. igg to rbd increased in 10 (35%) of the seronegative participants, shifting 1 to seropositive and 4 to low seropositive (fig 3) . fifteen of 19 (79%) low seropositive samples increased igg, with 11 of 19 (58%) becoming seropositive. thirteen of 13 (100%) known viral positive sars-cov-2 participants remained low seropositive or seropositive upon resampling (mean 41 days post positive viral swab test). for the 202 unconfirmed/negative individuals, igg concentrations ranged from 0-10 μg/ ml, with 33 (16%) seropositive, 40 (20%) low seropositive, and 129 (64%) seronegative ( fig 4a) . in the sample 30 participants shared households with individuals confirmed to have sars-cov-2 through a viral pcr test. the virus positive individuals in these households were disproportionately healthcare workers or first responders (19/20). among the 30 household members, 21 (70%) were seropositive, 3 (10%) were low seropositive, and 6 (20%) were seronegative ( fig 4b) . a number of participants were from a single healthcare facility in which a number of healthcare workers tested positive for virus while others were found to be viral negative between march 24 to april 3, 2020. those healthcare workers that remained asymptomatic were not tested for active virus. we evaluated the seroconversion of 48 household members from 33 essential healthcare worker households, 40-50 days post the initial exposure date ( fig 4c) . participants sharing a household with a known covid-19 positive healthcare worker had a high seroconversion rate with 9/17 (53%) seropositive, 3/17 (18%) low seropositive, and 5/17 (29%) seronegative. this was in contrast to 10/10 (100%) of household members remaining seronegative when sharing a household with a virus-negative health care worker. household members that resided with healthcare workers that were not tested for virus were 2/21 (10%) seropositive, 3/21 (14%) low seropositive, and 16/21 (76%) seronegative. these data show a high transmission rate among households of front-line essential workers and the dynamic nature of seroconversion in the community. of 30 covid-19 exposed household members, 70% were seropositive and 10% low seropositive. c) increased seroconversion in household members of known viral pcr positive healthcare workers (53% seropositive and 18% low positive) compared to 100% seronegative in known viral pcr negative healthcare worker households. household members of healthcare workers with no viral testing had exposure rates more similar to the community acquired rates. https://doi.org/10.1371/journal.pone.0237833.g004 covid-19 is now widely acknowledged to have high rates of community spread. strategic testing in community-based settings is critical for tracking spread of sars-cov-2 and for identifying the factors that mitigate transmission. we have validated a dbs assay to facilitate large-scale serological testing of sars-cov-2 igg antibodies, and results from our feasibility study document a high rate of household transmission. in addition, based on hains et al. [7] and the data here, indeterminate levels of igg merit retesting and current estimates of seroprevalence may underestimate community spread. because of their potential risks to patients, health care workers and first responders generally had priority for viral pcr testing to ascertain acute infection. however, limited supplies of viral testing materials often restricted access to viral pcr testing during march and april 2020, which likely resulted in under-testing and under-reporting of covid-19 cases. serological testing is positioned better to inform viral exposure especially during the interval when viral pcr testing was less available or weeks after symptom onset. furthermore, improved and reliable serological surveys, combined with close clinical follow up, can elucidate whether serological status predicts immune protection from repeat infection or disease. dbs sampling can aid in these surveys. advantages of dbs include a low cost and non-invasive approach to blood collection. dbs samples do not require special handling prior to transport and analysis, and igg antibodies remain stable in dbs for at least 8 weeks at room temperature [3, 9] . while point-of-care lateral flow immunoassay tests share some of the advantages of dbs sampling, they are difficult to implement in the home and they do not provide the same level of accuracy and quantification as lab-based tests for sars-cov-2 antibodies [10] . limitations of dbs include small sample volume, and the possibility for errors in self-collection (i.e., blotting, smearing of blood on filter paper) that can interfere with quantification. widespread serological testing is needed to more accurately assess viral spread. while our sample is not representative of the general population, the observation of high household seroconversion, using an assay restricted to a small component of the viral spike protein, suggests there may be higher exposure to covid-19 among some members of the community than previously appreciated. seroprevalence of sars-cov-2-specific antibodies among adults in performance characteristics of the abbott architect sars-cov-2 igg assay and seroprevalence in what a drop can do: dried blood spots as a minimally invasive method for integrating biomarkers into population-based research state of the science in dried blood spots shipping guidelines for dried blood spot specimens a serological assay to detect sars-cov-2 seroconversion in humans asymptomatic seroconversion of immunoglobulins to sars-cov-2 in a pediatric dialysis unit cross-neutralization of sars-cov-2 by a human monoclonal sars-cov antibody epstein-barr virus antibodies in whole blood spots: a minimally invasive method for assessing an aspect of cell-mediated immunity evaluation of antibody testing for sars-cov-2 using elisa and lateral flow immunoassays. medrxiv key: cord-337879-liqhbqxl authors: kriesel, john d.; hobbs, maurine r.; jones, brandt b.; milash, brett; nagra, rashed m.; fischer, kael f. title: deep sequencing for the detection of virus-like sequences in the brains of patients with multiple sclerosis: detection of gbv-c in human brain date: 2012-03-08 journal: plos one doi: 10.1371/journal.pone.0031886 sha: doc_id: 337879 cord_uid: liqhbqxl multiple sclerosis (ms) is a demyelinating disease of unknown origin that affects the central nervous system of an estimated 400,000 americans. gbv-c or hepatitis g is a flavivirus that is found in the serum of 1–2% of blood donors. it was originally associated with hepatitis, but is now believed to be a relatively non-pathogenic lymphotropic virus. fifty frozen specimens from the brains of deceased persons affected by ms were obtained along with 15 normal control brain specimens. rna was extracted and ribosomal rnas were depleted before sequencing on the illumina gaii. these 36 bp reads were compared with a non-redundant database derived from the 600,000+ viral sequences in genbank organized into 4080 taxa. an individual read successfully aligned to the viral database was considered to be a “hit”. normalized ms specimen hit rates for each viral taxon were compared to the distribution of hits in the normal controls. seventeen ms and 11 control brain extracts were sequenced, yielding 4–10 million sequences (“reads”) each. over-representation of sequence from at least one of 12 viral taxa was observed in 7 of the 17 ms samples. sequences resembling other viruses previously implicated in the pathogenesis of ms were not significantly enriched in any of the diseased brain specimens. sequences from gb virus c (gbv-c), a flavivirus not previously isolated from brain, were enriched in one of the ms samples. gbv-c in this brain specimen was confirmed by specific amplification in this single ms brain specimen, but not in the 30 other ms brain samples available. the entire 9.4 kb sequence of this gbv-c isolate is reported here. this study shows the feasibility of deep sequencing for the detection of occult viral infections in the brains of deceased persons with ms. the first isolation of gbv-c from human brain is reported here. multiple sclerosis (ms) is a chronic demyelinating disease of unknown cause, which affects the brain and spinal cord of about 400,000 individuals in the u.s. a number of viral infections of the cns can lead to demyelination, including distemper (dogs), measles (sspe, humans), and influenza (humans). [1] viruses have long been suspected as causative agents in ms based on the epidemiology of the disease including geographic patterns, isolated outbreaks, and migration studies. [2, 3, 4, 5] novel viruses that cause human disease continue to be discovered using molecular techniques including hepatitis c (1989), corona virus nl63 (2004), bocavirus (2005) , and rhinovirus c group (2007) . [6, 7, 8, 9] new human polyoma and arenaviruses, identified by high-throughput or ''deep'' sequencing of pathologic specimens, were recently discovered as causes of serious human diseases (2008) . [10, 11] to date most studies into the possible viral etiology of ms have focused on human dna viruses. unfortunately, because dna viruses can become latent or quiescent within the cns, it is difficult to discern whether latent, commensal viruses associated with ms lesions (e.g. hhv-6 and ebv) are causative or just opportunistic (i.e. ''along for the ride''). to avoid the identification of latent viruses that are not transcriptionally active, the present investigation focused on viral rna -both genomic (from rna viruses) and message (from either rna or dna viruses). deep sequencing offers a new approach for the discovery of pathogens. in contrast to pcr, this approach allows a relatively unbiased survey of nucleic acid sequences found within a sample. deep sequencing provides millions of short sequences less than 100 bp in length. the completion of the human genome project allows subtraction of human sequences from the dataset resulting in a large number of plausibly-nonhuman sequences that can then be compared to existing databases such as genbank. this technique was recently employed to discover novel picornaviruses linked to respiratory illness and diarrhea in children. [12, 13] gb virus-c (gbv-c or hepatitis g virus) is a human flavivirus originally isolated from a surgeon (with the initials g.b.) who was experiencing acute hepatitis. [14] the gb viruses are approximately 10 kb in size and are most closely related to hepatitis c virus. other flaviviruses include dengue viruses, west nile virus, yellow fever virus, and the tick-borne encephalitis viruses. in addition to humans, gbv-a and gbv-b infect monkeys, gbv-c infects chimpanzees. the recently described gbv-d infects only bats. [15, 16] animal inoculations suggested that gbv-c might be a cause of hepatitis, but subsequent human observations did not confirm this. gbv-c is known to be lymphotropic and has been found in human serum, muscle, lymphoid tissue, spleen, kidney and liver, but not brain (0 of 8 specimens) or spinal fluid (0 of 17 specimens). [17, 18, 19] surveys show that active viral replication occurs in the blood in 1-2% of human blood donors while about 5% of the general population is seropositive. [20] viral replication can persist for years at high titer (up to 50 million copies per ml blood). this virus is believed to be nonpathogenic, but has been associated with non-hodgkin's lymphoma. [21, 22, 23] resolution of gbv-c infection is often accompanied by antibody production against the envelope (e2) protein. gbv-c infection appears to induce antibodies that cross-neutralize hiv, probably explaining the observed survival benefit of persons infected with both viruses over those with hiv alone. [24] we report the results of deep sequencing of diseased brain tissue (plaque) taken from deceased patients with multiple sclerosis. this method identified one occult viral infection with gbv-c in a patient who died with primary progressive ms. one hundred mg pieces of cryopreserved white matter from ms plaques or control specimens were obtained from the human brain and spinal fluid resource center, west los angeles va medical center and from the rocky mountain ms center, university of colorado, denver. these specimens were collected after death and were either fresh frozen or snap frozen in liquid nitrogen. this research was submitted to the university of utah health sciences irb and, since it was performed on deidentified pathologic material, was found to be exempt from review and oversight. dnase treated rna was extracted from the tissue sections using qiagen rna-lipid kits (valencia, ca) on dry ice, under laminar airflow. purified rna was quantified on a thermo scientific nanodrop spectrophotometer (waltham, ma). samples with absorbance (a 260 /a 280 ) ratio ,1.8 were rejected and re-extracted. fifty ng of rna from each sample was submitted the huntsman cancer institute (hci) microarray core facility lab for analysis on an agilent 2100 bioanalyzer nanochip (agilent technologies, usa). the extracted rna samples were evaluated for their distribution of rna sizes, their relative abundances, the height and relative ratio of the 28s and 18s rrna peaks, and for rna integrity (rin) as determined by the agilent software. each sample was evaluated by an experienced technician and then independently by one of the investigators. samples with an unfavorable electrophoresis trace or rin were not sequenced. to enhance the detection of non-human sequences, rna samples that passed the quality control step above were subjected to rrna removal using the ribominus kit (invitrogen inc., carlsbad, ca). recovered rna (post rrna removal) was again quantified and subjected to bioanalyzer analysis. rna samples that passed the rna quality control were submitted to the huntsman cancer institute microarray core facility for reverse transcription, cdna library preparation, and high-throughput sequencing. datasets of 36 bp sequences, or reads, were obtained from each brain sample. low complexity (lzw compressibility .0.4) reads, reads with homopolymeric stretches longer that 18 bases and those with bustard quality scores #5 in more than 13 positions were excluded from the analysis. the remaining reads are defined as ''high quality reads.'' reads which perfectly matched ucsc build 18 of the human genome, as judged by bowtie alignments, were excluded from the analysis. weaker matches to mammalian rrna transcripts were detected and removed from the data set using megablast (word size = 12) with a database of 45s pre-rrna and 28s sequences (gis: 1261918 and 225637499). the remaining high quality, nonhuman, nonribosomal reads were defined as ''screened reads.'' comparative enrichment of viral sequence was then determined using these filtered sequences (screened reads). a non-redundant database of viral sequences was built to reduce noise from highly oversampled taxa in the public databases. this nonredundant database is 98 megabases in size organized into 4080 viral taxa. all viral sequence in genbank was obtained (more than 600,000 records). the longest sequence from each viral taxa was extracted from the input sequence set and placed in the non-redundant output set. all subsequences longer than 23 bases in the input that were compared to the sequence that was just extracted. identities were removed from the input by replacing the identical subsequence with x's. the longest remaining sequence, thus screened, was then extracted from the input, placed in the non-redundant output and used to screen the remaining input sequences. the process was repeated until no unscreened subsequences longer than 49 bases remained in the input. the resulting database, nr_virobank is available online (http://fischer-lab.path.utah.edu/data/gbv-c). megablast was used to align the brain sequences to nr_virobank using a low-stringency protocol. a word size of 16 was used, sequence filtering was disabled, and sequence-virus pairs with expect values #1.0 were considered in further analysis. the hit-rate (hr t-s ) of a sample to a viral taxon was calculated using equation 1 where the number of alignments to a given taxon (a t-s ) is normalized by the number of filtered nonribosomal sequences obtained for the specimen (r s ): the mean and standard deviation of the normalized hit rates in the control specimens were determined for each viral taxon. the distribution of control hits was examined for normality. taxa with non-normally distributed control hit rates were excluded from further analysis. the hit rates of each individual ms sample were then compared to the control hit-rate distribution for each taxon. using the python programming language and scipy, the bonferroni corrected student's t-test was used to quantify the statistical significance of any overrepresentations within each ms sample. cluster and java treeview were used to generate an unsupervised cluster of significant sample-taxa p-value pairs. [25, 26] detection of gbv-c pcr primers were designed from 4 regions of the gbv-c genome based on the deep sequencing results. reverse transcription was performed using independent forward and reverse gene specific priming. primers were selected from the 59-ntr, e2, and ns3 gene regions based on souza et. al. with modifications as suggested by the deep sequencing results. [27] primer sequences (59-39) employed were: three anti-gbv-c antibody preparations and controls were kindly provided by the jack stapleton laboratory at the university of iowa. these included pre-and post-immune rabbit polyclonal antiserum against gbv-c e2 protein [24] , mouse monoclonal anti-e2 #7067 [24] , and mouse monoclonal 1c4 [28] , plus control mouse igg1 (vector laboratories, burlingame, ca). frozen ms and control brain tissues were thawed and fixed in 4% paraformaldehyde. five micron paraffinized sections were used. antigen retrieval was performed (vector product h-3300). the tissues were penetrated with triton-x and endogenous peroxidase was quenched. sections were blocked with 5% normal goat serum (vector product s-1000). appropriate goat anti-rabbit or goatanti-mouse igg secondary antibodies (santa cruz biotechnology, products sc-2004 and sc-2060) were diluted 1:500-1:000 as recommended by the manufacturer. signal was developed with novared (vector product sk-4800). thirty-two specific primers to the ms-6 gbv-c isolate were designed using the gaii sequences that were highly homologous to known gbv-c isolates. overlapping amplicons were obtained using rt-pcr with freshly extracted total rna from sample 3840. amplicons were topo-cloned and sequenced using an abi 3730 sequencer. the sanger sequences were processed and assembled using the phred, phrap and consed programs using a known gbv-c scaffold (gi: 1666805). [29, 30] the gaii reads were added to this intermediate assembly using consed. fifty frozen ms plaque and 15 control, undiseased brain specimens were extracted. among these, 17 ms and 11 control brain specimens were selected for sequencing based on favorable rna quality profiles. these brain specimens were collected between the years 1983 and 2004. they were kept frozen at 270 degrees until being subjected to rna extraction in our laboratory. characteristics of the sequenced brain specimens are shown in table s1 . the post-mortem intervals between the ms and control specimens were similar: 12.962.2 vs. 11.561.6 hours (p = 0.64 by unpaired t-testing). the ms subjects were younger than the controls at the time of specimen collection: a median of 55 vs. 69 years, (p = 0.01 by unpaired t-testing). the proportion of specimens obtained from each of the two brain banks was similar between the ms and control groups (p = 0.7 by fisher's exact test). the 17 sequenced ms specimens came from deceased patients that carried the diagnoses of primary progressive ms (3), chronic progressive multiple sclerosis (5), secondary progressive ms (5), relapsing-remitting ms (2), or multiple sclerosis, subtype not specified (2). high-throughput sequencing on the illumina gaii yielded 4-24 million 36 bp sequences (''reads'') from each of the 28 brain specimens (17 ms, 11 normal controls). these reads are designated as ''high quality reads'' in table s1 . these sequences represent cellular rna, not dna, and therefore correspond either to viral genomes or to actively transcribed genes, both human and nonhuman, present in the specimen. human and ribosomal reads were subtracted from the dataset, providing 0.9-7.4 million screened reads for analysis. there were no significant differences in the number of high quality reads or screened reads between the ms and control specimens (p.0.5 by mann-whitney testing). viral alignments were performed on the sequences derived from each of the ms and control brain specimens. hit rates were calculated for each taxon represented in the nonredundant viral database (n = 4080). the mean and standard deviation of the hit rates in the control specimens were determined for each taxon. only 109 out of 7947 (1.4%) viral taxa showed a non-normal distribution of control hit rates. these were removed from further consideration. bacteriophage and plant virus taxa were excluded from further analysis due to less biologic significance compared with human and animal viruses. retroviruses were reserved for later analysis due to the ubiquitous expression of human endogenous retroviruses (hervs) in human specimens. hits were observed to 2153 remaining taxa. the hit rates to each taxon for each ms sample were then compared to the control hit rate distribution for that taxon. student's t-test corrected for multiple comparisons using the bonferroni method was used to quantify the statistical significance of any apparent over-representation observed in the ms samples. the 12 taxa with a least one ms sample with a over-representation more significant than p = 0.05 were placed into a hierarchical cluster (figure 1 ). over-representation in at least one taxon was observed in 7 of the 17 ms samples. the 36 bp reads from the illumina sequencing are not likely to identify any viruses unambiguously. overrepresented viral taxa (shown in figure 1 ) had all the deep sequencing reads from the implicated sample aligned to a database containing all genbank sequences for the taxon of interest. the reads were then joined into contiguous regions (contigs) using ssake, a short read assembler. [31] contigs generated by ssake were then aligned back to the taxon specific database. these newly assembled contigs were then aligned to both host and taxon specific viral databases again. contigs that had improved alignment quality (compared to single reads) to the candidate viral sequences and worse alignment to human sequences were used to design primers for virus specific rt-pcr. the results of this analysis are shown in table 1 . this strategy identified gbv-c as a promising candidate for pcr follow up in a single ms brain sample. table 2 shows the analysis of deep sequencing results for viral taxa previously implicated in the pathogenesis of ms. overall, the ms specimen hit rate was relatively high for the human herpesviruses compared to other viral taxa, probably due to the detection of rna expressed during latency. it is also possible that these higher hit rates are driven by short sequences derived from abundant human transcripts that align to the herpesvirus taxa. however, none of the human herpesvirus nor measles virus hit rates differed significantly between the ms and control distributions. one subject who died with primary-progressive ms had .1000 36 bp sequences detected that mapped to gbv-c virus (hepatitis g), a human flavivirus not known to cause any persistent disease and never before detected in human brain. [14, 17, 18] this brain specimen was investigated further. an initial attempt to amplify gbv-c from this specimen using published primers was unsuccessful, later determined to be due to sequence variation of the isolate. [27] pcr primers for 3 viral regions (59-ntr, e2, and ns3) were designed from the contigs assembled from the illumina sequencing data on this particular subject. rt-pcr amplification of these regions revealed amplicons of the expected sizes, approximately 300 bp (figure 2) . sequencing of the amplicons showed 96-99% identity to .100 known gbv-c sequences. quantitative rtpcr of this brain sample showed that there were between 1.7610 6 and 2.4610 6 viral genome copies per gram of tissue. interrogation of csf from this subject showed 1.1610 6 viral genome copies/ml. blood from this subject was not available for analysis. anti-gbv-c antibodies were not detected in csf from this subject (personal communication from dr. jack stapleton). specific rt-pcr amplification of 31 affected (ms) and 23 unaffected (control) brain samples was performed with each of the 3 gbv-c primer sets testing for both positive and negative strand viral rna, using the existing gbv-c positive sample as a positive control. both strands of gbv-c rna were detected only in subject ms-6, both before and after ribosomal removal, and not in any of the other 53 brain rna specimens studied. twenty ms and seven control csf samples were interrogated with 3 separate gbv-c specific primers (ntr-1, e2, and ns3). the ms-6 csf sample was used as a positive control. gbv-c was amplified from none of these other 27 csf samples. the recovered sequence of gbv-c uu1 contained a open reading frame of 2842 codons, containing the structural and to gbv-c uu1 was the east african isolate gbv-c (ea) (gi:1666805). [32] at the amino acid level the viral polyprotein was 97.7% identical to gbv-c (ea) (64 coding differences). the sequence of gbv-c uu1 has been deposited with genbank (gb jn127373). immunohistochemistry with controls was attempted on gbv-c infected, ms afflicted brain (ms-6); other ms brain specimens where gbv-c was not detected by pcr; and several normal brain controls. some anti-gbv-c dependent staining of neuronal cytoplasm was observed in both infected and uninfected brain sections with the polyclonal rabbit anti-gbv-c preparation. the monoclonal ab preparations did not specifically detect gbv-c in any of the human brain tissues examined, including that from ms-6 (shown by molecular methods to contain gbv-c rna). this work demonstrates the feasibility of deep sequencing for the detection of occult viral infections in brain tissue. this method revealed several viral candidates. the presence of one of these candidates, gbv-c, was confirmed in the brain of one of the subjects who suffered and died with progressive ms. this virus has never been detected before in human brain tissue. it's relationship with the underlying disease, primary progressive multiple sclerosis, in this single person is not clear. rna sequences from other viruses previously implicated in the pathogenesis of ms including number of ms brain specimens that had reads with significant homology (megablast expect #0.1) to the indicated viral sequences. 4 the assembly of short reads did not improve alignment with the specified viral sequences. 5 assembly revealed homology to human mitochondrial and host integration sites. doi:10.1371/journal.pone.0031886.t001 viral taxa are defined as sequences in genbank below the level of family. this includes sequences identified as belonging to genus or species or subspecies (strain). it also includes sequences not assigned to a specific species or genus. 2 the ms specimen hit rate is defined as the number of hits for a given taxon or species divided by the number of total non-ribosomal reads. this method is used to normalize these values between specimens. the range of hit rates among the 17 sequenced ms brain specimens is displayed. 3 the control specimen hit rates for each viral taxon are expressed as the mean 6 sem among the 8 control specimens. 4 null hypothesis = the tested ms specimen falls within the expected distribution of control samples. values provided are two-tailed. ebv, cmv, hhv-6, and measles, were not significantly overrepresented in the diseased brain specimens compared with controls. gbv-c or hepatitis g virus is currently believed to be nonpathogenic. however, this virus was originally isolated from a human who was suffering from acute hepatitis. [33, 34] prior attempts to isolate gbv-c from the brain in infected subjects were unsuccessful. [18] other flaviviruses certainly do infect the human central nervous system including west nile virus, dengue [35, 36] , tickborne encephalitis viruses, and japanese encephalitis virus. even hepatitis c virus has been isolated from diseased brain in a patient with fatal demyelinating disease. [37] the epidemiology of gbv-c infection is similar to that of ms itself where infection/ disease is rare in children and much more common in women of childbearing age. [38] gbv-c is, in part, sexually transmitted. [39, 40, 41] the sex predominance of gbv-c infection is about 2:1 women:men, again similar to ms. [42] the presence of both positive (rna genome) and negative (replication intermediate) strand flaviviral rna in ms-6 suggests that the virus was actively replicating in the brain of this subject. active replication is also supported by this subject's pathology report showing ''active plaques'' with a perivascular lymphocytic infiltrate (data not shown). the clinical history revealed onset of primary progressive ms beginning at age 44 in an otherwise healthy caucasian woman. the disease was relentless causing the subject to use a wheelchair by age 55 and to become bed bound at age 60. active replication gbv-c in the brain of ms-6 is also supported by the relatively high titer of virus observed in both tissue and csf (1-2 million copies per ml) and the absence of anti-gbv-c antibodies in the csf. actively replicating gbv-c in this subject's brain may or may not be causal. gbv-c may be found in the blood of humans for years after acute infection and it may be present at high titer -in the range of 10 8 copies per milliliter of blood. [14, 20] resolution of gbv-c viremia usually coincides with the development of antibodies to the virus. gbv-c replicates primarily in peripheral blood mononuclear cells, including lymphocytes, so its presence in the brain of subject ms-6 may simply reflect penetration of the figure 2 . amplification of gbv-c rna from the brain of ms-afflicted subject ms-6. following the failure of published primers to amplify sequences in this specimen, specific primers were constructed based on the sequences of specific reads obtained from the illumina gaii sequencing of this specimen. [27] a. four regions of the gbv-c genome were selected for amplification including 2 sites in the 59 non-translated region, the e2 (envelope protein) gene, and ns3 (non-structural). b. amplicons were derived from each of these primer sets, including + (genome) and 2 (replication intermediate) strands. sequencing of 3 of these amplicons spanning 906 bp revealed identity with .100 published gbv-c isolates, indicating that this subject had a novel strain of gbv-c in her brain tissue which appeared to be replicating at the time of her death. doi:10.1371/journal.pone.0031886.g002 damaged blood-brain barrier by serum or virus-infected lymphocytes. [18] on the other hand, persistent gbv-c infection of the brain might have manifestations not previously described, such as progressive demyelination. animal studies demonstrate that viral infections of the cns can result in a demyelinating disease that is similar to ms. [43, 44, 45, 46, 47] the failure to specifically detect gbv-c by immunohistochemistry in brain sections from subject ms-6 was disappointing. this may be due to a relative absence of viral antigen in these tissues or the lack of suitable reagents for the detection of gbv-c antigen in fixed, paraffinized tissue. in any case, the lack of detectable gbv-c rna from 30 other ms brain specimens suggests that this virus will not be isolated frequently from the brains of ms patients. deep sequencing is a powerful tool that has revealed new pathogens. this report shows the feasibility of this technique for the study of brain tissues affected by ms. a novel isolate of gbv-c was identified here in this manner. the presence of gbv-c in this brain specimen was confirmed by designing pcr primers from the sequencing results. the deep sequencing analysis in this study was based on the early illumina gaii technology and limited by read length (36-base pairs) and sequencing from only a single end of the library inserts. the investigators believe that further improvements in sequencing technologies, such as longer reads and perhaps paired-end strategies, will significantly simplify the bioinformatics required for future clinical viral metagenomic studies. table s1 demographic, sequencing, and disease characteristics of the sequenced ms and control brain specimens. the source, age, sex, year of collection, post-mortem interval, and diseases associated with each specimen used for this study is shown here, along with the number of reads obtained. 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recombinant hsv-1 expressing il-2 induces t-cell auto-reactivity and cns demyelination theiler's virus infection: a model for multiple sclerosis two models for ms: experimental allergic encephalomyelitis and theiler's murine encephalomyelitis virus the authors would like to acknowledge drs. jack stapleton and james mclinden from the university of iowa for gbv-c related advice, reagents, and csf serology. brian dalley from huntsman cancer institute microarray core facility supervised the preparation of cdna libraries and the illumina sequencing. theodore wilson helped with the genome assembly. key: cord-323133-gdg50omp authors: buzatto, g. p.; tamashiro, e.; proenca-modena, j. l.; saturno, t. h.; prates, m. c.; gagliardi, t. b.; carenzi, l. r.; massuda, e. t.; hyppolito, m. a.; valera, f. c. p.; arruda, e.; anselmo-lima, w. t. title: the pathogens profile in children with otitis media with effusion and adenoid hypertrophy date: 2017-02-23 journal: plos one doi: 10.1371/journal.pone.0171049 sha: doc_id: 323133 cord_uid: gdg50omp objectives: to evaluate the presence of viruses and bacteria in middle ear and adenoids of patients with and without otitis media with effusion (ome). methods: adenoid samples and middle ear washes (mew) were obtained from children with ome associated with adenoid hypertrophy undergoing adenoidectomy and tympanostomy, and compared to those obtained from patients undergoing cochlear implant surgery, as a control group. specific dna or rna of 9 respiratory viruses (rhinovirus, influenza virus, picornavirus, syncytial respiratory virus, metapneumovirus, coronavirus, enterovirus, adenovirus and bocavirus) and 5 bacteria (s. pneumoniae, h. influenzae, m. catarrhalis, p. aeruginosa and s. aureus) were extracted and quantified by real-time pcr. results: 37 ome and 14 cochlear implant children were included in the study. at the adenoid, virus and bacteria were similarly detected in both ome and control patients. at the middle ear washes, however, a higher prevalence of bacteria was observed in patients with ome (p = 0.01). s. pneumoniae (p = 0.01) and m. catarrhalis (p = 0.022) were the bacteria responsible for this difference. although total virus detection was not statistically different from controls at the middle ear washes (p = 0.065), adenovirus was detected in higher proportions in adenoid samples of ome patients than controls (p = 0.019). conclusions: despite both ome and control patients presented similar rates of viruses and bacteria at the adenoid, children with ome presented higher prevalence of s. pneumonia, m. catarrhalis in middle ear and adenovirus in adenoids when compared to controls. these findings could suggest that these pathogens could contribute to the fluid persistence in the middle ear. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 otitis media with effusion (ome) is a common childhood disease characterized by the presence of fluid in the middle ear, with no symptoms and/or signs of acute inflammation [1, 2] . in the united states, approximately 90% of all children develop an episode of ome before they reach school age, mainly between the ages of 4 months and 6 years [2] . the presence of ome is associated with severe negative impact on child development, including hearing loss with long-term consequences for speech and language acquisition, poor school performance, and imbalance issues [1] [2] [3] . moreover, children with ome are five times more susceptible to develop acute otitis media than controls [4, 5] . the pathogenesis of ome is still not fully understood. epidemiological data suggest that the pathogenesis of ome is multifactorial, involving anatomical, immunological, genetic, microbial, and environmental factors [6] [7] [8] [9] . however, it is widely accepted that the dysfunction of the eustachian tube play a key role in the development of ome in all ages. mechanical obstruction of the eustachian tube is one of the main identifiable causes of eustachian tube dysfunction, especially due to adenoid hypertrophy (ah) in the pediatric age. the presence of ah can obstruct the nasopharyngeal ostium of the eustachian tube, leading to negative pressure in the middle ear cavity, and eventually mucosal transudation [10] . indeed, ah is a frequent condition observed in patients with ome [7] [8] [9] 11] . in children with ah and ome, the surgical removal of the adenoid (associated with ventilation tube insertion) accelerates the recovery of the middle ear mucosa, decrease the risk of recurrence and need of repetitive surgical procedures, and reduce treatment failure rate. however, new evidence point out that the benefits of adenoidectomy in children with ome older than 4 years-old is independently of the adenoid size [12] , suggesting that the removal of a local reservoir of pathogenic microbiota (viruses, planktonic bacteria, and biofilms) is the key for such benefits [13] [14] [15] . despite some studies have evaluated the microbial colonization of adenoid and middle ear, no studies have thoroughly studied the association of respiratory pathogenic microbiota between these two niches. therefore, the present study was carried out to compare the detection of common respiratory viruses and bacteria in adenoids and middle ear fluid in children with ome and in controls. moreover, adenoid can be reservoirs of pathogens that may reach the middle ear [13] . adenoid samples obtained from patients with chronic adenoiditis are highly colonized by multiple species of viruses and bacteria. the rate of detection in hypertrophic adenoids is higher than 85% for viruses [14] and almost 100% for bacteria [15] . bacteria and viruses have been detected in the middle ear fluid (mef) from children with ome. using an rt-pcr-based assay only for three respiratory viruses (rhinovirus, respiratory syncytial virus and human coronavirus), positivity was detected in 30% of mef from children with ome in finland [16] . in a study conducted in pittsburgh, 75 of 97 (77.3%) samples of mef from children with ome were positive by pcr for one or more of three bacteria (m catarrhalis, h influenza and s pneumoniae) [17] . the study was conducted following the principles expressed in the declaration of helsinki, after approval by the ethics review committee of the clinical hospital of the university of sao paulo school of medicine in ribeirão preto, brazil (#10466/2008). written informed consent was obtained from all parents/guardians. the study enrolled patients seen at the division of otorhinolaryngology from may 2010 to august 2012. two groups of patients were studied: a) children with ome and adenoid hypertrophy (ah) who underwent adenoidectomy and tympanostomy with ventilation tube insertion, and b) children with no middle ear disease, who underwent cochlear implantation due to severe hearing loss. all patients were evaluated by otoscopy, audiometry, tympanometry, and nasal endoscopy. ome was diagnosed by medical history suggesting persistence of middle ear fluid for more than 3 months without acute signs of inflammation, confirmed by the presence of tympanic membrane opacity, retraction, or air-liquid interface, and by a type b tympanogram and/or conductive hearing loss. ah was defined at nasal endoscopy by the presence of adenoid in contact with the torus tubaris (grade 3) and/or vomer (grade 4) [18, 19] . exclusion criteria were: prior surgical procedures in the upper airways, including tympanostomy for tube placement; use of antibiotics or symptoms of respiratory infection within the last month; history of recurrent acute otitis media or recurrent upper airways infection; tympanic membrane perforation, or the presence of genetic syndromes, such as down syndrome. all patients with ome and ah were under treatment with nasal corticosteroids for at least one month during the preoperative period. the control group consisted of children undergoing cochlear implant due to severe hearing loss with no medical history of otitis media, no respiratory infection within the last month, absence of tympanometric alterations, nor adenotonsillar hypertrophy. all patients followed the vaccination protocols of the brazilian ministry of health [20] , which includes some pathogens targeted in the present study, such as pneumococcal conjugated. additionally, patients in the control group were immunized with pneumococcus 23-valent vaccine and meningococcus c, according to the cochlear implant surgery protocol. fragments of adenoid (surface and stroma) and middle ear washes (mew) were obtained from all patients under general anesthesia. briefly, adenoids fragments were collected using beckmann adenoid curette (from ome-ah patients) or punch biopsy forceps, under direct visualization after soft palate retraction, in order to avoid contamination (controls). adenoids samples were placed in eagle's minimal essential medium (mem) with 15% of a solution containing 20,000 u/ml of penicillin-streptomycin and 200 μg/ml of amphotericin b, 10% of fetal bovine serum (gibco 1 , life technologies, carlsbad, ca, usa), and kept on ice until further processing. adenoids were washed twice in mem to remove blood and tissue debris and the fragment was macerated in trizol 1 (invitrogen, life technologies, carlsbad, ca, usa) for later nucleic acid extraction. in children with ome, mew was performed with 0.5 ml of sterile saline through a small tympanostomy using a sterile needle coupled to a 5 ml syringe, after mechanical cleaning of the external ear canal. after that, the mew was conditioned into a polypropylene microtube and treated with antimycotic and antibiotic solution (gibco 1 , grand island, ny, usa) for one hour at 4˚c. aliquots were made in thrice the volume of trizol, and frozen at −80˚c until further processing. from each patient, samples were collected from only one side, which was randomly chosen when bilateral disease was present. in control patients, mew was obtained after partial mastoidectomy was performed, when direct access to the middle ear was possible through the attic, with the same sterile technique utilized to collect samples in ome patients. nucleic acid extraction and pathogen detection rna was extracted from 250 μl of mew or from approximately 30 mg of adenoid tissue using 750 μl of trizol 1 [21] , according to the manufacturer's protocol. dna was further extracted using dna purification kit (promega 1 , fitchburg, wi, usa) starting with the dna-enriched fraction obtained with trizol 1 . samples were tested by real-time pcr for rhinovirus (hrv), influenza virus (flu), parainfluenza virus (hpiv), syncytial respiratory virus (hrsv), metapneumovirus (hmpv), coronavirus (hcov), enterovirus (hev), adenovirus (hadv) and human bocavirus (hbov) following an in house real-time pcr protocol, based on primers listed on s1 table and taqman probes, with the same procedures published elsewhere (14] . real-time pcr was also used to detect the presence of s. pneumoniae, h. influenzae, m. catarrhalis, p. aeruginosa, and s. aureus, using the primers listed on s2 table. the fisher's exact test was used to compare rates of virus and bacteria detection in both groups of patients. wilcoxon test was done to assess correlation between bacteria and virus detected in each patient. the analysis was performed using graphpad prism 5 (la jolla, ca, usa). 14 control patients (2-12 years-old; mean 4 years 6 months; sd 2.64; 9 male / 5 female) and 37 children with ome and ah (2-12 years-old; mean 6 years 1 month; sd 1,97; 19 male / 18 female) were included in this study. no severe complications related to the sampling procedure, such as nasal or oral bleeding, dysphagia and otorrhea, were observed in the control group as well as in the ome-ah group within the first 48 hours after surgery or after 3 weeks postoperative reassessment. at least one virus species was detected, considering adenoid samples, in 32 of 37 (86.5%) patients with ome-ah and in 11 of 14 (78.6%) control patients. differently, at least one virus was detected in mews from 19 of 37 (51.3%) patients with ome, and in 3 of 14 (21.4%) control patients. the overall rates of virus detection were significantly higher in adenoids as compared to mew in both groups (p = 0.002 for the ome group and p = 0.007 for the control group), but the virus detection rates in mew and adenoids were not significantly different between patients with ome and control children (respectively p = 0.06 and p = 0.66). despite this fact, hadv was more often found in adenoids of ome-ah group as compared to controls (p = 0.01) ( table 1) . the rates of virus co-detection in positive samples were 59.3% (19/32) in adenoids and 21% (4/19) in mews. two or 3 viruses were simultaneously detected in adenoid tissues from 15 (40.5%) and 4 (10.8%) patients with ome. in contrast, only 3 (8.1%) control patients had 2 viruses and only 1 (2.7%) had 3 different viruses detected in mews (fig 1) . there was no significant concordance between detection of viruses in adenoid and the middle ear (p = 0.65). only 6 (16.2%) patients with ome (5 hev and 1 hbov) and 1 control patient (hev) had the same virus detected in mews and adenoid tissue, suggesting that the viral colonization of the middle ear is independent from viral infection of adenoids. notably, the rates of bacteria detection in mews was significantly higher in patients with ome than in control patients (p = 0.01), especially due to s. pneumococcus and m. catarrhalis. overall bacterial detection rates in adenoids were similar between ome and control patients (p = 0.29). twelve of 14 control patients (85.7%) had at least one species of respiratory pathogenic bacteria detected in adenoid tissues, reinforcing previous data that indicate that potentially pathogenic bacteria colonize adenoids of healthy children [22, 23] (table 2) . with regard to bacteria co-detection in adenoids and mews from ome patients, 18 patients (48.6%) with ome had more than one bacterium detected in adenoid, and two, three, or four bacteria were simultaneously detected respectively in 16.2%, 18.9%, and 13.5% of patients. six patients had 2 (16.2%), four had 3 (10.8%) and two patients had 4 bacteria (5.4%) simultaneously detected in mews for an overall frequency co-detection of bacteria in mews of 32.4% (12 of 37 patients). bacteria co-detection was also frequent in the control group, in which five patients had 2, three patients had 3 and one patient had 4 different bacteria simultaneously detected in adenoid tissues. bacteria co-detection was not observed in mews from control patients (fig 2) . similar to the findings regarding viruses, there was no concordance between bacteria detected in adenoids and mews, both in controls and ome patients (p = 0.18). the same bacteria were detected in the mews and adenoids was observed in 13 (35.1%) patients with ome, and in 2 (14.2%) controls (fig 2) . multiple associations between viruses and bacteria in the adenoid and middle ear were investigated. the only microbes that presented positive association was s. pneumoniae and hadv in hypertrophic adenoids of patients with ome (p = 0.02) (tables 3 and 4) . we did not find any significant association between virus and bacteria in the adenoid or middle ear in patients of the control group. although the pathogenesis of ome is not fully understood, there is evidence that adenoid may play an important role in this disease, either by mechanically impairing the eustachian tube function, or by acting as a microbial reservoir for ascending infection to the middle ear [13, 14, 24] . children are often exposed to pathogens that may chronically persist in tissues in the upper airways, especially in the adenoid and tonsils [13, 14, 25, 26] . thus, as the adenoid has an intimate anatomical relation with the eustachian tube and ultimately to the middle ear, it is important to understand how the presence of microbes in the adenoid could lead to the development or persistence of ome in children. to the best of our knowledge, this is the first case-control study that compared adenoid and middle ear microbiota of ome patients with healthy children. in our study, we used a sensitive method to detect nucleic acid of a comprehensive panel of respiratory viruses and bacteria to compare the microbial colonization of adenoid and its correspondence in the middle ear in both ome children and controls. the overall detection rate of bacteria in the middle ear was significantly higher in patients with ome than in controls, but was similar for viruses. the higher overall frequency of bacteria detection in mews from ome patients was expected, since the accumulation of middle ear effusion favors growing of bacterial elements that may have been suctioned through the tube from the nasopharyngeal microbiota. moreover, these bacteria may play roles as chronic inflammatory stimuli to the mucosa of the middle ear and contribute to the dysfunction of eustachian tube. there was little or no concordance of the microbe detected in the adenoid and the middle ear, both in patients with ome as well as in controls. this finding is in agreement with a previous report that performed 16s rdna pyrosequencing of both niches, showing that the adenoid microbiota was more diverse and complex than of the middle ear in a child with ome [27] . as the adenoid is exposed to the nasal and oral microbial contents, differently from the enclosed cavity of the middle ear, this makes the adenoid more susceptible to be colonized by a higher load and more variety of microorganisms. it is generally accepted that the development of negative pressure within the middle ear cavity suctions microbial components of the nasopharyngeal microbiota but, at present, there is no enough data to rule out the establishment of a selected microbiome especial to the middle ear cavity. interestingly, adenovirus detection was more frequent in hypertrophic adenoid tissues from patients with ome than in normal adenoids from healthy controls. this is in agreement with a paper previously published by our group reporting high rates of adenovirus detection by pcr in hypertrophic adenoids [14] . it has not been clear whether adenovirus has any role in pathogenesis of adenoid hypertrophy, especially because this virus is also frequent in nasopharyngeal secretions from healthy individuals [28] . since some hadv species are more prone than others to have more prolonged shedding [29] , prospective longitudinal studies of viruses in patients with hypertrophic tonsils should include determination of adenovirus species to evaluate whether they are associated with chronic tonsillar hypertrophy. the observed association of hadv with s. pneumoniae in adenoids could lead to speculate that the epithelial damage induced by hadv could somehow favor the secondary local proliferation of s. pneumoniae [30] . some viruses, for instance influenza, are known to favor pneumococcal colonization of the upper airways by multiple mechanisms, including exposure of receptors for pneumococcal adherence and by providing nutrients sources for bacterial growth [31] . however, the simultaneous presence of hadv and s. pneumoniae in adenoid tissue could be independent observations, due to the high frequencies of both microorganisms in the adenoid. s. pneumoniae and m. catarrhalis were detected more often in middle ear washes from ome patients than controls. considering that s. pneumoniae and m. catarrhalis are the most frequent pathogens detected in acute otitis media [6] , and are able to ascend through the eustachian tube [32] , causing ciliary damage to the airway epithelium and disrupting mucociliary flow, this may result in conditions for persistence in the middle ear compartment [33] [34] [35] [36] . in some cases, especially when ome is not clearly related to a mechanical obstruction of the eustachian tube, the toynbee effect of negative pressure within the middle ear may help ascendance of microbes through the tube, leading to colonization of the middle ear, which may be pivotal in ome pathogenesis. m. catarrhalis has been widely studied because of its importance in middle ear diseases, and this interest has even led to vaccines for m. catarrhalis being proposed [37] . it's mechanisms of adherence and triggering of the immune response have been well studied and even when not related to clinically relevant infection processes, appears with a high level of colonization during childhood [38] . once regarded as nonpathogenic, m. catarrhalis could be related to several chronic affections of the upper airways [39] . microorganisms in sessile biofilms are frequently resistant to the innate and adaptive immune responses and to antimicrobial agents. in ome, there has been evidence that the presence of middle ear fluid is associated to formation of biofilm in the middle ear, and that these biofilms are related to the local inflammation [9, 24] . in conventional, culture-based microbiology, detection of these microorganisms is not always possible, and the use of sensitive molecular methods, such as real-time pcr, enables detection of multiple microbes in complex niches like the middle ear. although pcr cannot distinguish viable from dead microbes, the specificity of the primers coupled with careful sampling technique in order to minimize crosscontamination between sampling sites, ensure that the microorganisms were indeed present in the middle ear. viruses and bacteria interact in many different ways on different mucosal surfaces [40] , including the upper respiratory tract mucosa and tonsils [41] . persistence of viral infections includes a complete reprogramming of the local immune response, attenuation of production of type 1 interferon, and local alteration of the cd4+/cd8+ t cell balance [41, 42] . the airway mucosa affected by viral infection becomes more susceptible to bacterial adherence and mucosal inflammation [30, 43, 44] . therefore, associations of viruses and bacteria could be acting in concert to modify the course of the ome. in the present study, there was an association of detection of hadv and s. pneumoniae in adenoid tissues from patients with ome, suggesting that microbial correlations already described in acute middle ear disease [16, 45] could play a role in such chronic processes. clinical studies based on microbial detection in patients with adenoid hypertrophy and ome have rarely included a healthy control group. a notable example was a study of biofilms in middle ear samples, including adults and children [25] . importantly, biofilms were not observed in ome patients, but not in control specimens of middle ear mucosa obtained from patients undergoing cochlear implantation, strongly suggesting that biofilm production is an important factor in development of ome. through a minimally invasive method (mew) and a very sensitive procedure (real time pcr), we were able to compare samples of healthy children with those of children with ome. in the light of that result, the findings of some potentially pathogenic bacteria in the middle ear of patients undergoing cochlear implant suggests that their presence in the middle ear is independent of biofilm formation. unfortunately, in the present study no attempt was made to verify the presence of biofilm. concluding, in children with ome and adenoid hypertrophy we observed higher detection rates of potentially pathogenic bacteria, but not respiratory viruses, by real-time pcr in middle ear samples, as compared to control patients without adenoid hypertrophy. we did not observe correlation between the microbiota of adenoid and middle ear, neither in ome children nor in controls. this adds evidence that microbial community present in the middle ear may be associated with persistence of fluid and pathogenesis of ome. supporting information s1 conceptualization: gpb ea et wta-l fcpv. formal analysis: gpb. clinical practice 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proteins from cell and tissue samples differences in nasopharyngeal bacterial carriage in preschool children from different socio-economic origins differences in nasopharyngeal bacterial flora in children with nonsevere recurrent acute otitis media and chronic otitis media with effusion: implications for management role of adenoid biofilm in chronic otitis media with effusion in children direct detection of bacterial biofilms on the middle-ear mucosa of children with chronic otitis media respiratory viruses are continuously detected in children with chronic tonsillitis throughout the year the otologic microbiome: a study of the bacterial microbiota in a pediatric patient with chronic serous otitis media using 16srrna gene-based pyrosequencing otitis media with effusion a comparison of viral fitness and virulence between emergent adenovirus 14p1 and prototype adenovirus 14p strains the airway epithelium: soldier in the fight against respiratory viruses influenza promotes pneumococcal growth during coinfection by providing host sialylated substrates as a nutrient source genetic similarity between adenoid tissue and middle ear fluid isolates of streptococcus pneumoniae, haemophilus influenzae and moraxella catarrhalis from iranian children with otitis media with effusion secretory otitis media and the nature of the mucociliaryy system otitis media with effusion: components which contribute to the viscous properties viral rna in middle ear mucosa and exudates in patients with chronic otitis media with effusion mucociliary clearance defects in a murine in vitro model of pneumococcal airway infection moraxella catarrhalis: from emerging to established pathogen moraxella catarrhalis, a human respiratory tract pathogen moraxella catarrhalis: pathogenic significance in respiratory tract infections treated by community practitioners viruses and the microbiota distinct regulation of tonsillar immune response in virus infection innate and adaptive immune regulation during chronic viral infections mucosal exudation of fibrinogen in coronavirus-induced common colds respiratory viruses augment the adhesion of bacterial pathogens to respiratory epithelium in a viral species-and cell typedependent manner natural history of untreated otitis media key: cord-349217-vpih1wvs authors: petropoulos, fotios; makridakis, spyros title: forecasting the novel coronavirus covid-19 date: 2020-03-31 journal: plos one doi: 10.1371/journal.pone.0231236 sha: doc_id: 349217 cord_uid: vpih1wvs what will be the global impact of the novel coronavirus (covid-19)? answering this question requires accurate forecasting the spread of confirmed cases as well as analysis of the number of deaths and recoveries. forecasting, however, requires ample historical data. at the same time, no prediction is certain as the future rarely repeats itself in the same way as the past. moreover, forecasts are influenced by the reliability of the data, vested interests, and what variables are being predicted. also, psychological factors play a significant role in how people perceive and react to the danger from the disease and the fear that it may affect them personally. this paper introduces an objective approach to predicting the continuation of the covid-19 using a simple, but powerful method to do so. assuming that the data used is reliable and that the future will continue to follow the past pattern of the disease, our forecasts suggest a continuing increase in the confirmed covid-19 cases with sizable associated uncertainty. the risks are far from symmetric as underestimating its spread like a pandemic and not doing enough to contain it is much more severe than overspending and being over careful when it will not be needed. this paper describes the timeline of a live forecasting exercise with massive potential implications for planning and decision making and provides objective forecasts for the confirmed cases of covid-19. the accuracy of traditional forecasting largely depends on the availability of data to base its predictions and estimates of uncertainty. in outbreaks of epidemics there is no data at all in the beginning and then limited as time passes, making predictions widely uncertain. on february 18, 2020, a new york times article [1] cautioned against excessive optimism about the crisis peaking, even though there were close to 50 days since the virus had been identified. besides, there are concerns that the data may not be reliable, as was the case of bird flu and sars when the number of affected people and deaths were misreported to hide the extent of the epidemic. similarly, in the case of covid-19, the reporting did not reflect the correct numbers as well when on the february 13 a new category of "clinically diagnosed" was added to "lab-confirmed" ones [2] . such problems decrease forecasting accuracy and increase uncertainty, making the drawing of definite conclusions more difficult. related to forecasting accuracy and uncertainty, there is a more severe problem that has to do the perception of epidemics and pandemics. politicians are concerned with regards to the a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 measures to be taken while the general population fears about the impact on the epidemic on their health/lives. furthermore, the pharmaceutical firms are working on vaccinations for the new virus with considerable commercial interest. this was the case with sars when governments persuaded on the severity of the virus bought large numbers of vaccines that were never used as its spread stopped without the need to vaccinate people. of course, the big problem is the asymmetry of risks and the irrational fear of a pandemic with its possible catastrophic consequences, as happened with the 1918 spanish flu that killed an estimated 50 million worldwide. in contrast, the sars killed a total of 774 in 2003 and the bird flu around 100 in 1997. covid-19 has resulted in an estimated 5.8 thousand deaths until now (15/03/2020). at the same time, there is much less concern over the seasonal flu that kills about 646,000 people worldwide each year [3] . medical predictions are often not accurate while their uncertainty is seriously underestimated [4] . predicting the future of epidemics and pandemics is much more difficult as the number of cases to be studied can be measured in one hand. at one end of the scale is the case of sars where the fear of becoming a pandemic was overblown, resulting in overspending and the application of restrictive measures to be contained that it turned out to be unnecessary. at the other end is the spanish flu that turned out to be a serious pandemic with catastrophic consequences, arguably in a different era when communication and the ability to raise public awareness (and possibly exaggerated fear) were limited. despite the inaccuracies associated with medical predictions, still forecasting is invaluable in allowing us to better understand the current situation and plan for the future. in this paper, we provide statistical forecasts for the confirmed cases of covid-19 using robust time series models, and we analyse the trajectory of recovered cases. we focus on the cumulative daily figures aggregated globally of the three main variables of interest: confirmed cases, deaths and recoveries. these were retrieved by the center for systems science and engineering (csse) at johns hopkins university (https://github.com/ cssegisanddata/covid-19 accessed on 12/03/2020) and are presented in fig 1. the data refer to daily cumulative cases and cover the period from january 22, 2020 until march 11, 2020. we include both "lab-confirmed" and "clinically diagnosed" cases. we emphasise the importance of the recovered cases, which is not covered in media as widely as the confirmed cases or the deaths. while all three data patterns show an exponential increase, the trends of both the confirmed cases and the deaths were reduced in the mid of february; a second exponential increase is observed in late february and march as a result of the increased number of cases in south korea, iran, and europe. at the same time, the number of recovered cases is steadily increasing. to forecast confirmed cases of covid-19, we adopt simple time series forecasting approaches. we produce forecasts using models from the exponential smoothing family [5, 6] . this family has shown good forecast accuracy over several forecasting competitions [7] [8] [9] and is especially suitable for short series. exponential smoothing models can capture a variety of trend and seasonal forecasting patterns (such as additive or multiplicative) and combinations of those. we limit our attention to trended and non-seasonal models, given the patterns observed in fig 1. note that we follow a pragmatic approach in that we assume that the trend will continue indefinitely in the future. this approach contradicts other modelling approaches to covid-19 using an s-curve model (logistics curve) that assumes convergence. while statistical approaches to model selection (such as information criteria, which measure the maximum likelihood of a model while penalising for its complexity) could be used, we judgmentally select a model [10] to better reflect the nature of the data. we opt for an exponential smoothing model with multiplicative error and multiplicative trend components. even if in some cases an additive trend model gave lower information criteria values, we opted for the multiplicative trend model given the asymmetric risks involved as we believe that it is better to err to the positive direction. we produce ten-days-ahead point forecasts and prediction intervals and update our forecasts every ten days. please note that this is not an ex-post analysis, but a real, live forecasting exercise. we have been posting and evaluating our forecasts publicly in social media (please, refer to the twitter accounts of the authors, @fotpetr and @spyrosmakrid). we first started at the end of january 31, 2020 and only had ten actual data points in hand. we decided to use a multiplicative trend exponential smoothing model. the forecasts (and 90% prediction intervals) produced at the end of 31/01/2020 are presented in fig 2 with red (and pink) colour. note that the vertical axis is log-scaled. the mean estimate (point forecast) for the confirmed cases ten-days-ahead was 209 thousand with the 90% prediction intervals ranging from about 38 to 534 thousand cases. the actual confirmed cases on 10/02/2020 were just under 43 thousand. we observe a considerable forecast error from the mean estimate equal to 166 thousand cases (an absolute percentage error of 388%), with the forecasts being extremely positively biased. still, the actual cases lie within the prediction intervals. then, we increased the historical number of our data to include cases up to the end of february 10, 2020 (20 data points). we once again produced ten-days-ahead predictions. the forecasts and prediction intervals are depicted in fig 2 with blue colour. we observe that the actual values for the period 11/02/2020 until 20/02/2020 closely follow the mean estimate. the forecast error on 20/02/2020 was 5.8 thousand cases (an absolute percentage error of 7.7%). this was despite the change that was made on 13/02/2020 with regards to how confirmed cases are recorded to now include "clinically diagnosed" instances as opposed to exclusively lab-confirmed. one crucial observation is that this more accurate forecast came with a significant decrease in the steepness of the slope compared to the forecast for the previous ten-days period. another observation is that at the end of 20/02/2020, we were still over-forecasting the number of confirmed cases. finally, all actual values lied well inside the prediction intervals range. we produced a third set of forecasts and prediction intervals using the data up until 20/02/ 2020. the forecasts are presented in fig 2 with green colour. the mean estimate for ten-daysahead (01/03/2020) was 83 thousand cases. the slope of the forecasts was, once again, lower compared to the previous two sets of forecasts, confirming the fact that the observed confirmed cases (up until 20/02/2020) show a steady decrease. we also observed a significant decrease in the associated forecast uncertainty, with the prediction intervals being much tighter compared to our past forecasts. the 90% prediction intervals worst-case scenario was about 600 thousand cases, which is halved compared to that of the last round of forecasts (1.2 million cases). the actual confirmed cases at the end of 01/03/2020 were 88 thousand. at the end of this third round of forecasts, we recorded an error of 5.5 thousand cases (6.2%). while this error was lower than the previous round (in both absolute and percentage terms), it was the first time that our 10-days ahead forecast were below the actual values (under-forecasting). this was because the virus had been spreading in three countries outside mainland china (south korea, iran, and italy). our fourth round of forecasts is shown in fig 2 with purple colour. the mean estimate for 11/ 03/2020 was 112 thousand confirmed cases, with the uncertainty levels being similar to the previous round: there was a 5% chance that they would exceed 613 thousand by the end of 11/ 03/2020. the observed actual confirmed cases at the end of this period were almost 127 thousand. the absolute forecast error at the end of the last period (11/03/2020) was 15.4k (12.1%), higher compared to the previous set of forecasts but still well within the prediction intervals. for the second round in a row, we were consistently under-forecasting the actual cases. this was due to the exponential increase of the confirmed cases mostly in europe, iran and the us, with south korea managing to decrease the number of new daily cases significantly. we produced a final set of forecasts and prediction intervals using the most recent data, up until 11/03/2020. these are presented in fig 2 with yellow/gold colour. note that we estimated three levels of uncertainty (50, 70 and 90%). the trend of our forecasts is much increased compared to the last two rounds: we predict 83 thousand new cases for this round (a total of 210 thousand cases). the associated levels of uncertainty are also increased: there is a 25% chance that the total confirmed cases will exceed 413 thousand by the end of 21/03/2020; and a 5% chance that they will exceed 1.19m. we also attempted to produce forecasts by splitting the recorded confirmed cases into two groups: cases within mainland china and cases anywhere else, as the trends into these two groups are different. we fitted independent exponential smoothing models, and then we summed up the forecasts (bottom-up hierarchical forecasting). we notice that using this approach, the mean estimate is close to that if all data are considered together (207 versus 210 thousand cases). however, the estimated uncertainty by splitting the data is considerably lower, possibly since the confirmed cases outside mainland china have significantly increased only recently. we next turn our attention to the recovered cases that have received little attention until now. we focus on the number of the recovered cases as a percentage of the total confirmed cases as well as the ratio of recovered cases versus deaths. we are particularly interested in the trajectory of these two ratios. fig 3 presents this analysis. first, we observe the solid relationship between the two curves. second, we notice that despite the very small percentages of recovered cases until the end of january (less than 2%), currently, about 1 out of 2 confirmed cases have recovered (52.8% of the total confirmed cases). moreover, the ratio of recovered cases versus deaths is currently above 14:1. despite this, we observe a reverse of both curves since 08/03/ 2020, which is associated with the increasing number of cases outside mainland china. the uncertainty surrounding an unknown, novel coronavirus can spark a global alarm, leading a harvard professor stating that 40-70% of the global population might be infected in the coming year [11] which matches chancellor angela merkel's warning regarding the effects of the novel coronavirus in germany [12] . norman, bar-yam and taleb [13] discuss the systemic risk of pandemics, the existence of fat-tailed processes due to global interconnectivity and the negatively biased estimates of spread, reproduction and mortality rates. on the opposite side, others are arguing about people overly panicking [14] and neglecting the probabilities [15, 16] with the new virus being the first "infodemic" as a result of the hyper-connectivity offered by today's social media [17, 18] . the polarisation of the opinions globally can be summarised by the quotes of three renowned personalities: • elon musk: "the coronavirus panic is dumb" [19] . • nassim nicholas taleb: "saying the coronavirus panic is dumb is dumb" [20] . • bill gates: "i hope it's not that bad, but we should assume it will be until we know otherwise." [21] . regardless of what one's beliefs are, we believe that forecasts and their associated uncertainty can and should be an integral part of the decision-making process, especially in highrisk cases. apart from the significant public health concerns, the dangers imposed on global supply chains and the economy as a whole are also considerable [22, 23] . risk-averse people can focus on the worst-case-scenarios and act accordingly. deciding to discard any formal, statistical forecasts and acting conservatively, still implies an underlying forecasting process, even if this process is not formalised (personal judgment/belief). in this exercise, we used univariate time series models, which assume that the data is accurate and past patterns (including precautionary measures) will continue to apply. significant, consistent forecast errors (potentially spanning outside the prediction intervals) should be associated with changes in the observed patterns and the need for additional actions and measures in the case of negatively biased forecasts. we believe that the significant forecast error at the end of the first forecast period (from 01/ 02/2020 to 10/20/2020) as depicted in fig 2 could be the result of two factors: • while the forecasts that we produced using the data up until 31/01/2020 would be a good estimate in the scenario of "business-as-usual" (nothing changes), they disregard the fact that the world will act to get the virus under control. the chinese authorities managed to rapidly construct two new hospitals, in huoshenshan and leishenshan areas in wuhan, that opened on 03/02/2020 and 08/02/2020 respectively. multiple commuting restrictions were applied both within china and internationally. the world health organisation helped in creating awareness of the novel virus. so, the decline in the spread of the covid-19 during this first round could well be linked with these attempts from local and global authorities. • there may be a "garbage-in, garbage-out" situation. as mentioned above, our analysis and forecasts assumed that the data are accurate. it could be the case that the positive bias of the first-period forecasts is not as significant as it seems dues to potential inaccuracies in the actual data and the under-accounting of confirmed cases. this is especially true given the delay effects in diagnosing covid-19 cases [24] . our second and third sets of forecasts that cover the period 11/02/2020 to 01/03/2020 were very close to the recorded confirmed cases (the forecast error was lower than 6 thousand cases at the end of each 10-day period). the slowing down of the trend during this period suggested that covid-19 would not cause any serious problems, particularly outside of mainland china. unfortunately, that was not the case. the last two sets of forecasts that cover the period 02/03/2020 to 21/03/2020 show a significant increase in the trend of cases globally coupled with an increase in the associated uncertainty. we hope that our forecasts will be a useful tool for governments and individuals towards making decisions and taking the appropriate actions to contain the spreading of the virus to the degree possible. conceptualization: fotios petropoulos, spyros makridakis. formal analysis: fotios petropoulos. coronavirus epidemic keeps growing, but spread in china slows sharp increase in deaths and cases in hubei flu kills 646,000 people worldwide each year: study finds predicting medical risks and appreciating uncertainty a state space framework for automatic forecasting using exponential smoothing methods exponential smoothing with a damped multiplicative trend the accuracy of extrapolation (time series) methods: results of a forecasting competition the m3-competition: results, conclusions and implications the m4 competition: 100,000 time series and 61 forecasting methods judgmental selection of forecasting models harvard professor sounds alarm on 'likely' coronavirus pandemic: 40% to 70% of world could be infected this year up to 70% of germany could become infected-merkel systemic risk of pandemic via novel pathogens-coronavirus: a note coronavirus: there are better things to do than panic the cognitive bias that makes us panic about coronavirus coronavirus and its global sweep stokes fear over facts. experts say it's unlikely to produce 'apocalyptic scenario coronavirus in europe: epidemic or 'infodemic'? mit technology review. the coronavirus is the first true social-media "infodemic bill gates and elon musk just issued very different responses to the coronavirus. it's a lesson in emotional intelligence the black swan' author to elon musk:'saying the coronavirus panic is dumb is dumb responding to covid-19-a once-in-a-century pandemic? how coronavirus could impact the global supply chain by mid-march jpmorgan officially forecasts a coronavirus-driven recession will rock the us and europe by july characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china visualization: fotios petropoulos. writing -review & editing: fotios petropoulos, spyros makridakis. key: cord-330079-pdaowkop authors: xu, lin; he, xia; zhang, ding-mei; feng, fa-shen; wang, zhu; guan, lin-lin; wu, jue-heng; zhou, rong; zheng, bo-jian; yuen, kwok-yung; li, meng-feng; cao, kai-yuan title: surveillance and genome analysis of human bocavirus in patients with respiratory infection in guangzhou, china date: 2012-09-11 journal: plos one doi: 10.1371/journal.pone.0044876 sha: doc_id: 330079 cord_uid: pdaowkop human bocavirus (hbov) is a novel parvovirus associated with respiratory tract diseases and gastrointestinal illness in adult and pediatric patients throughout the world. to investigate the epidemiological and genetic variation of hbov in guangzhou, south china, we screened 3460 throat swab samples from 1686 children and 1774 adults with acute respiratory infection symptoms for hbov between march 2010 and february 2011, and analyzed the complete genome sequence of 2 hbov strains. specimens were screened for hbov by real-time pcr and other 6 common respiratory viruses by rt-pcr or pcr. hbov was detected in 58 (1.68%) out of 3460 samples, mostly from pediatric patients (52/58) and inpatient children (47/58). six adult patients were detected as hbov positive and 5 were emergency cases. of these hbov positive cases, 19 (32.76%) had co-pathogens including influenza virus (n = 5), rsv (n = 5), parainfluenza (n = 4), adenovirus (n = 1), coronavirus (n = 7). the complete genome sequences of 2 hbovs strains (genbank no. jn794565 and jn794566) were analyzed. phylogenetic analysis showed that the 2 hbov strains were hbov1, and were most genetically close to st2 (genbank accession number dq0000496). recombination analysis confirmed that hbov strain gz9081 was an intra–genotype recombinant strain among hbov1 variants. human bocavirus (hbov), recently identified as a new member of the parvoviridae family from the respiratory secretions of children suffering from lower respiratory tract infection [1] , has a single-stranded dna genome of ,5.2kb, which contains three open reading frames (orfs), encoding two non-structural proteins (ns1 and np1) and two viral capsid proteins (vps) [2] . the nucleotide sequences are highly conserved among hbovs circulating in different geographic regions [3] , with the vp1/ vp2 gene displaying relatively commonly found nucleotide polymorphisms [2] . since its first identification, hbov has been detected in 1.5%-19% of respiratory tract secretions [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] and 0.8%-9.1% of fecal samples [13, [16] [17] [18] [19] , respectively, from patients with acute respiratory tract illnesses or gastroenteritis worldwide, it is of note that most of these reports were mainly derived from young children and infants, with only a few exceptions testing adult patients. more recently, three more genotypes of hbov (hbov2-4) were found, [2, 20, 21] , and reports have shown that inter-genotype and intra-genotype recombinations are present among bocavirus [22] . all 4 genotypes of hbov have been identified in children with acute gastroenteritis (age), whereas only hbov1 and hbov2 were reported in respiratory tract samples. due to the higher rates of co-infections with other pathogens, it remains to be clarified whether in these diseases hbovs are the key etiologic agent or just a concomitant virus bystander. to better understand the epidemiology of the hbov infection, in conjunction of a viral surveillance program, we investigated the presence of hbov in patients with acute respiratory infection in guangzhou, a city located in south china. geographically, the city is characteristics of a tropical-subtropical climate, with the average annual temperature of 20-22uc and average relative humidity of 77%. the city is also highly populated, with a resident population of 12.70 million, plus a non-residential population of 4.76 million. these socio-natural factors make the region generally vulnerable to air-borne as well as food-borne viral infection. the epidemiological status and genomic characteristics of hbov prevailing in pediatric and adult patients with respiratory infection in the region, however, remains unknown. in our current study, we screened throat swab specimens from patients with acute respiratory tract infection symptoms for hbov and other common respiratory viruses over a 12-month period using polymerase chain reaction (pcr) methods, and in addition, the molecular phylogeny and complete genome sequences of 2 hbov strains were also analyzed. all research involving human participants was approved by the medical ethics review board of zhongshan school of medicine, sun yat-sen university, in accordance with the guidelines for the protection of human subjects. written informed consent was obtained from each participant/guardian. from march 2010 to february 2011, 3460 throat swabs were obtained from 1686 children and 1774 adult patients who had been admitted to five hospitals in guangzhou, china. they were only taken from individuals with # 3 days of fever (temperature $37.5uc), and with cough, sputum, throat sore or other respiratory tract infection symptoms. there were 2009 male and 1451 female patients with age ranging from 1 day to 95 years. demographic, epidemiology and clinical information including case history, symptoms, physical signs and examination results etc. were collected using a standardized questionnaire. all specimens were added to 2 ml vtm (consists of earle's balanced salt solution (biosource international, usa), 4.4% bicarbonate, 5% bovine serum albumin, 100 mg/ml vancomycin, 30 mg/ml amikacin, and 40 u/ml nystatin) according to a standard protocol and transported within 8 hr at 4uc to biosafety laboratory of sun yat-sen university, where they were divided into aliquots, and stored at 280uc until processing further. all specimens were tested for 7 common respiratory viruses, including influenza virus types a, b and c (inf-a, inf-b and inf-c), parainfluenza (piv) types 1-4, respiratory syncytial virus (rsv), human metapneumovirus (hmpv), human coronavirus (hcov), adenovirus (adv) and hbov using pcr, rt-pcr or real-time pcr methods as described below. information of patients whose throat swabs were found positive for hbov was analyzed retrospectively. dna and rna were simultaneously extracted from 200 ml of throat swab specimen using qiaamp minielute virus spin (qiagen, germany). reverse transcription of virus rna was conducted by superscript iii transcriptase and random hexamer primers (invitrogen, life technology, usa), both kits were used according to the manufacturer's instructions. inf-a, -b and -c, piv -1, -2, -3 and -4, rsv-a and -b, hmpv, hcov, and adv were detected by a standard reverse transcription-pcr (rt-pcr) or pcr techniques as previously described using specific primers listed in table s1 [11, [23] [24] [25] , and amplified products were detected by agarose gel electrophoresis. screening of hbov used real-time pcr. the full sequence of hbov was referred from the st2 strain (genbank accession number dq000496, or nc_007455). taqman real-time pcr primers (np1-f and np1-r) and probe (synthesized by invitrogen, life technology, usa) were designed to bind the np1 highly conserved region of different hbov strains and analyzed by primer express software (version 3.0, applied biosystems, usa) (for primer and probe sequences, see table 1 ), with regard to optimal g-c content (55%, 44% and 60% for np1-f, np1-r and probe, respectively), melting temperature (58.4uc, 59.3uc and 70.0uc for np1-f, np1-r and probe, respectively), and amplicon length (88bp). each reaction mixture consisted of 10 ml 26 iq supermix reaction mixture (bio-rad, usa), 2 ml of viral dna, 0.5 mm each of the forward and reverse primers, and 0.3 mm of the probe, and nuclease-free water to a final volume of 20 ml. real-time pcr was conducted for 95uc for 15 min, followed by 45 cycles of 95uc for 15 s, 60uc for 1 min on the abi7500 realtime pcr system. the complete genomes of hbov strains were amplified using primers designed for complete genome by the primer premier 5.0 software to bind relatively conserved regions of hbov as available in the genbank database (primer sequences shown in table 1 ). the pcr was carried out using the platinum pfx taq polymerase (invitrogen, usa) in a prepared reaction mix according to the following condition: 95uc for 5 min, followed by 40 cycles of 94uc for 15 s, 53uc to 58uc (see table 1 for melting temperature of different primers) for 45 s, and 68uc for 2 min, and a final extension at 72uc for 7 min. pcr products for genome analysis was purified by agarose gel dna purification kit (takara, china), and the pcr products of terminal sequences were cloned into pcr-blunt 4-topo vector (zero blunt topo pcr cloning kit for sequencing, invitrogen, usa). all pcr products used for cloning and sequencing were from three independent pcr reactions. sequencing was performed by a commercial service of invitrogen co. according to the method described in ref. [26] (guangzhou, china) and submitted to the genbank database. the genomic sequences and orfs of hbov were comparatively analyzed with complete genome sequences of other hbov strains in the genbank (including hbov reference strains st1, st2, parvovirus b19, bovine parvovirus, canine minute virus, and virus strains obtained from several countries). these sequences were aligned by the clustal x program, and a neighbor-joining tree was constructed using the mega 4.0 software. potential recombinant sequences and parental sequences were analyzed using the recombination detection program (rdp) [27] . rdp scanning was performed by geneconv, bootscan, max-chi, chimaera, and siscan methods. a multiple comparison corrected p-value cutoff of 0.001 was used throughout. simplot checking was used to confirm and evaluate localization of possible recombination break points by bootscan program [28] . recombinant validation was done by checking the bilateral gene sequences of the recombinant site using phylogenetic trees. of the 3460 samples collected from patients with respiratory tract infection symptoms and signs enrolled in the study during the period between march 2010 and february 2011, detection for 7 viruses, namely, influenza, piv, rsv, hmpv, hcov, adv and hbov, showed that 1275 (36.8%) were found positive for one single virus and 112 (3.2%) were infected by more than one virus. as shown in table 2 , 21.1% of patients tested were found positive for inf-a, -b, or -c (median age 25 years), 7.7% for rsv (median age 0.91 years), 4.2% for adv (median age 5 years), 3.5% for piv (median age 1.2 years), 2.7% for hmpv (median age 2 years), and 2.6% for hcov (median age 3 years). hbov dna was detected in 58 samples (1.68%) (median age 1.5 years) by real-time pcr, including 52 pediatric (47 inpatient, 4 outpatient and 1 emergency patient) and 6 adult (1 inpatient and 5 emergency patients) cases. the monthly distribution of 7 respiratory viruses tested in patients with indications for respiratory infection from march, 2010 to february, 2011 showed biannual peaks. the highest peak of total positive rate of hbov and other 6 common respiratory viruses appeared in august (56.2%, chi-square test, p,0.05 compared with other months), and another lower peak appeared in winter to spring (january to march). interestingly, influenza virus was prevalent throughout the year, and peaked in august (fig. s1 ). it is also of note that hbov was detected in nearly all months except january during the study year, and the peak was present in may and june (5.4% and 6.3% respectively, fig. 1 ). patients enrolled in this study aged from 1 day to 95 years, including 1686 children (#15 years old) and 1774 adults (.15 years old) with a median age of 16 years. the total infection rate of common respiratory virus in children is 41.6% (701 positive out of 1686 pediatric subjects), as compared to that of 38.7% (686 positive out of 1774 adult subjects) in adults, for most of the screened respiratory viruses, the infection rate of pediatric patients was higher than adult patients (p,0.05) except influenza virus, which tended to infect adults (see fig. s2 for the age distribution of common respiratory viruses). in contrast to influenza virus, which displayed a higher infection rate (540 out of 731 influenza-infected individuals) in adult patients aged from 15 to 64 years old (fig. s2) , hbov tended to mostly infect infants younger than 2 years of age (41 out of 58 all hbov-positive subjects) with a few adult infection (6 in age .15 years old group amongst 58 all hbov-positive subjects). (fig. 2 ). in this study, the common symptoms of patients detected as hbov positive included cough (91.4%), fever (100%),rhinorrhea (36.2%), sputum (36.2%). it is noteworthy that while 43 (74.1%) out of the 58 hbov-positive patients were clinically diagnosed as lower respiratory tract infection including bronchopneumonia, acute asthmatic bronchopneumonia, and severe pneumonia, only 15 (25.9%) patients met the criteria of severe respiratory infection as featured by dyspnoea. in addition, 9 (15.5%) of the hbovpositive patients were clinically presented as acute upper respiratory tract infection, and 3 were diagnosed, respectively, as bronchial asthma, herpangina and infectious mononucleosis. of note, a 77-year-old patient with acute exacerbation of copd was found infected by hbov without the presence of any of other 6 respiratory viruses tested in this study. this patient displayed a normal hemogram but a chest radiograph of coarse lung marking. to better understand the hbov pathogenicity, clinical characteristics of hbov-positive outpatients/emergency cases were compared with those of hbov-positive inpatients ( table 3 ). the hbov positive rate in outpatients/emergency was 0.56% (10 out of 1800 outpatients/emergency cases), significantly lower than that of 2.89% in inpatients (48 out of 1660 inpatients) (chi-square test, p,0.01). the odds of infection with hbov resulting in severe disease (or admission) were 5.21 (95% ci 2.64-10.25). hbov positive pediatric patients were more diagnosed as lower respiratory tract illness than adults in both outpatients and inpatients (3 out of 5 pediatric vs 0 out of 5 adults in outpatient/emergency hbov-positive patients and 40 out of 47 pediatric vs 0 out of 1 adult in inpatients), especially in infants under 2 years old (35 out of 52 hbov-positive pediatric patients) ( table 3 ). in contrast, hbov positive adults were mainly diagnosed as acute upper respiratory tract infection (auri) ( table 3) . hbov-positive pediatric inpatients were more likely to be co-infected with other viruses than adult inpatients. however, hbov-positive adult outpatients were more frequently co-infected with influenza than pediatric outpatients, and the reason may lie in the high influenza infection rate in adults ( table 2 and table 3 ). the surveillance data of 7 common respiratory viruses showed that among the 3460 samples, 112 were tested as more than one virus positive. although influenza was the most common coinfecting virus, the highest co-infection rate occurred in hcov and hbov (table 4 ). in 19 of 58 hbov positive specimens (32.76%), other virus can be found, and hcov was the most commonly co-detected virus with hbov, accounting for 7 out of 19 (36.84%) hbov co-infection cases. there were totally 8 triple virus co-infection cases (table 4 ). it is noteworthy that despite of the high co-infection rate, in as high as 67.24% hbov-positive patients, no other screened common respiratory virus was found, especially in pediatric patients (36/52, 69.2%), and most of them the complete genomes of two hbov strains gz4785 (genbank no. jn794565) and gz9081 (genbank no. jn794566) obtained in this study were highly conserved with 98.8% identity to each other, and showed more than 99% nucleotide identity to st2 strain of hbov1 (genbank accession no. dq0000496). hbov strains used in the phylogenetic analysis included the strains obtained in this study in guangzhou (gz4785 and gz9081), representative strains of hbov1-4, human parvovirus b19, bovine, and canine minute virus. based on complete genome, the phylogenetic analysis results showed that the two guangzhou strains gz4785 and gz9081 were genetically close to hbov1 (fig. 3) , consistent with the sequence comparison analysis results. from the phylogenetic tree based on complete genome, different strains of hbov1 were clearly divided into three groups (fig. 4a) , and the representative strain of group i and group ii was the prototype strains st1 and st2, respectively. group iii included 4 strains which came from taiwan, thailand, and guangzhou. most chinese strains obtained from respiratory specimens belonged to group i, but it is noteworthy that gz9081 strain obtained in this study belonged to group iii. no apparently genotypic differences existed between the phylogenetic trees based on the 3 hbov orfs (ns1, np1 and vp1/vp2) and the complete genome of 23 hbov1 strains (fig. 4b-d) . similar to previous studies [2, 24] , ns1 appeared to be the most conserved gene, whereas vp1/vp2 had the most nucleotide polymorphisms. the phylogenetic trees were almost identical between vp1/vp2 gene and complete genome, which indicated that vp1/vp2 can be used instead of complete genome to analyze the genetical relationship of hbovs. in phylogenetic analysis, we found that hbov strain gz9081 belonged to a group different from most of chinese strains obtained from respiratory specimens. interestingly, full-length genome analysis showed that gz9081 strain contained an ns1 gene closely homologous to that of the st1 group, and that the rest of its genome (np1 and vp1/vp2 genes) resembled the cu74 group, suggesting that gz9081 might represent a hybrid virus, prompting us to conduct the recombinant analysis on this strain. therefore, rdp was further carried out. the bootscan plot of the recombination event was showed in fig. 5 , which confirmed the daughter linage gz9081 was a recombinant of parental strains st1 and cu74. two trees of the relevant strain were constructed respectively on the recombinant region (position: 1-1272+4385end) and non-recombinant region (position: 1272-4385), which further confirmed that gz9081 was closely related with st1 on the recombinant region (fig. 5) . it was very probable that gz9081 was an hbov1 intra-genotypic recombinant. human bocavirus (hbov) was a newly discovered parvoviridae virus. by now, little is known about its epidemiology and genetic characteristics in guangzhou, china. the pathogenicity of hbov is still in uncertain because of its high co-infection rate with other pathogens, and it remains unclear whether hbovs are sole etiologic agent or just a concomitant virus bystander. therefore, to understand the prevailing status and pathogenicity of hbov, other pathogens needed to be simultaneously examined. so in this study, a surveillance of 12 months period during 2010-2011 in guangzhou was established to understand the prevalence and pathogenicity of hbov in patients with acute respiratory infection symptoms. other 6 common respiratory viruses (influenza virus, parainfluenza virus, adenovirus, coronavirus, rsv, metapneumovirus) were screened at the same time to understand their coinfection status with hbov. the results revealed that the overall monthly distribution of hbov and other 6 common respiratory viruses were typical, in accordance with the epidemics of respiratory viruses in tropical/subtropical areas like guangzhou, which demonstrated that the surveillance data in this study were highly reliable. it is notable that hbov, like rsv, tended to mainly infect #2 year old infants, with only a few adult infections, and the majority of the hbov positive patients (67.24% of all hbov positive patients and 69.23% of hbov positive children) were hbov single positive, indicating it may have potential pathogenicity, especially in infants. therefore, to better elucidate its pathogenic roles, the clinical characteristics of hbov-positive outpatients/emergency cases were analyzed for comparison with hbov-positive inpatients ( table 3 ). the results showed that the hbov positive rate in outpatients/emergency was statistically lower than inpatients, and the odds of infection with hbov resulting in severe disease (or admission) were as high as 5.21. hbov positive pediatric patients were more diagnosed as lower respiratory tract illness than adults in both inpatients and outpatients, and pediatric inpatients were more likely coinfected with other viruses. although there was possibility that other copathogens not be screened in this study may exist, these results showed that hbov may have pathogenic role in causing severe disease/admission or lower respiratory tract illness of children. in addition, our results showed that hbov was related to asthma or its exacerbation, which occurred in 10 pediatric patients. previously, only limited data in adults were available for the study of hbov, especially in large samples. many reports of adult hbov infection enrolled no more than 100 samples [2, 8, 12, 29, 30] . the prevalence and associated illness of hbov in adults have not been well characterized in guangzhou. in our study, we found 6 hbov-positive adult cases in 1774 adult patients with upper respiratory tract infection, and the 3.4% prevalence rate was in accordance with those previously reported for adults [2, 8, 12, 29, 30] . although hbov infection was rare in adults with respiratory infection in our study population, it was still notable that hbov may cause exacerbation in adults with basic or primary pulmonary disease like copd. we found 52 hbov-positive pediatric patients, which represented 3.1% positive rate in pediatric patients with respiratory infection. this is similar to other reports of 1.5%-19%. consistent with other studies [2, 8, 30] , the prevalence rates were higher in children under 2 years of age, but generally decrease with the increase of age (fig. 2) , which implied that antibodies against hbov acquired during early life may provide protection. in our study, up to 32.76% co-infection rate of hbov was observed from throat samples, and the rate may be higher if more viruses were screened. the most frequently detected co-pathogen was hcov, different from previous reports in china [12, 15] , and the reason may lie in different climate/geography conditions and virus distribution. there was no obvious evidence that co-infection of hbov and other common respiratory viruses can increase the disease severity, since no correlation was found between coinfection and clinical symptoms, and the rate of lower respiratory tract illness did not increase in co-infection cases. in order to improve the diagnostic sensitivity, our present study employed real-time pcr to screen hbov with the primers and probes binding the np1 conserved region. to exclude pcr contamination and prevent false positive and false negative results in hbov and other 6 common respiratory viruses screening, the following strategies were used. firstly, all pcr was strictly carried out in 4 separate rooms, namely, reagent preparation, sample preparation, pcr and pcr-product rooms. secondly, each pcr/ real-time pcr assay was performed in duplicates and repeated three times. thirdly, positive samples were further confirmed by pcr using another set of primers, and for some of the 6 common respiratory viruses positive samples, viral isolation and determination was performed to further confirm the pcr positive results. complete genome sequence of 2 hbov strains were obtained in our study. gene analysis showed high identity (98.8%) between each other, and phylogenetic analysis demonstrated that they belonged to hbov1, which was more frequently detected in respiratory tract illness than other genotypes. phylogenetic tree of complete genome showed that different strains of hbov1 can be divided into three groups. it is noteworthy that the two strains identified in this study (gz4785 and gz9081), which were circulating at the same time during the 1 year study period, belonged to different groups. furthermore, our phylogenetic analysis results showed that most chinese hbov1 strains obtained from respiratory samples belonged to group i which was genetically closely to st2, but gz9081 was different, which belonged to group iii. phylogenetic trees based on different hbov orfs showed that its ns1 gene was closely homologous to the st1 group, whereas its np1 and vp1/vp2 genes resembled the cu74 group, suggesting it may be a recombinant. although generally hbov1 sequences were highly conserved, there were still evidences that the recombination existed among hbovs [22] , and co-infection of hbovs [20] might increase the chance of recombination between hbovs. therefore, we suspect that gz9081 may be a hybrid virus. so a recombinant detection program was performed to analyze gz9081. the result confirmed that gz9081 was an intra-genotype of hbov1 recombinant, originated from the parental strains st1 and cu74 (fig. 5) . as far as we know, this is the first time that recombination between hbov1 is reported and this is the first recombinant strain of hbov1 reported in china. since recombination could change virulence or antigenicity of viruses, we believe that the finding of hbov recombination might have significance on the epidemiology, seroprotection and pathogenicity study of hbov. further studies are needed to examine the virulence and antigenic changes of gz9081 strain. continuous surveillance and genome sequence analysis are needed to obtain more information on the genotypic variation and molecular evolution of hbov in china. human bocavirus and acute wheezing in children cloning of a human parvovirus by molecular screening of respiratory tract samples human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus realtime pcr for diagnosis of human bocavirus infections and phylogenetic analysis realtime pcr assays for detection of bocavirus in human specimens human bocavirus: prevalence and clinical spectrum at a children's hospital human bocavirus in patients with respiratory tract infection human bocavirus in italian patients with respiratory diseases human bocavirus in french children high prevalence of human bocavirus detected in young children with severe acute lower respiratory tract disease by use of a standard pcr protocol and a novel real-time pcr protocol frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections clinical and molecular epidemiology of human bocavirus in respiratory and fecal samples from children in hong kong clinical and genetic analysis of human bocavirus in children with lower respiratory tract infection in taiwan bocavirus infection in hospitalized children human bocavirus infection in young children with acute respiratory tract infection in lanzhou human bocavirus infection in children with acute gastroenteritis in japan and thailand human bocavirus infection in children with acute gastroenteritis and healthy controls human bocavirus infection in children hospitalized with acute gastroenteritis in china human bocavirus in children hospitalized for acute gastroenteritis: a case-control study human bocaviruses are highly diverse, dispersed, recombination prone, and prevalent in enteric infections human bocavirus species 2 and 3 in brazil recombination analysis based on the complete genome of bocavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia children with respiratory disease associated with metapneumovirus in hong kong simultaneous detection of influenza a, b, and c viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-pcr assay dna sequencing with chainterminating inhibitors rdp2: recombination detection and analysis from sequence alignments a modified bootscan algorithm for automated identification of recombinant sequences and recombination breakpoints human bocavirus infections in hospitalized children and adults human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand all enrollees participating in the surveillance project are appreciated. we owe our special thanks to the participated doctors and nurses of the 5 hospitals conceived and designed the experiments: k-yc lx. performed the experiments: lx xh d-mz f-sf zw l-lg. analyzed the data: lx xh k-yy m-fl. contributed reagents/materials/analysis tools: rz b-jz j-hw m-fl. wrote the paper: lx xh m-fl k-yy. key: cord-352222-zq9o66i4 authors: rajatonirina, soatiana; razanajatovo, norosoa harline; ratsima, elisoa hariniana; orelle, arnaud; ratovoson, rila; andrianirina, zo zafitsara; andriatahina, todisoa; ramparany, lovasoa; herindrainy, perlinot; randrianirina, frédérique; heraud, jean-michel; richard, vincent title: outcome risk factors during respiratory infections in a paediatric ward in antananarivo, madagascar 2010–2012 date: 2013-09-12 journal: plos one doi: 10.1371/journal.pone.0072839 sha: doc_id: 352222 cord_uid: zq9o66i4 background: acute respiratory infections are a leading cause of infectious disease-related morbidity, hospitalisation and mortality among children worldwide, and particularly in developing countries. in these low-income countries, most patients with acute respiratory infection (ari), whether it is mild or severe, are still treated empirically. the aim of the study was to evaluate the risk factors associated with the evolution and outcome of respiratory illnesses in patients aged under 5 years old. materials and methods: we conducted a prospective study in a paediatric ward in antananarivo from november 2010 to july 2012 including patients under 5 years old suffering from respiratory infections. we collected demographic, socio-economic, clinical and epidemiological data, and samples for laboratory analysis. deaths, rapid progression to respiratory distress during hospitalisation, and hospitalisation for more than 10 days were considered as severe outcomes. we used multivariate analysis to study the effects of co-infections. results: from november 2010 to july 2012, a total of 290 patients were enrolled. co-infection was found in 192 patients (70%). co-infections were more frequent in children under 36 months, with a significant difference for the 19–24 month-old group (or: 8.0). sixty-nine percent (230/290) of the patients recovered fully and without any severe outcome during hospitalisation; the outcome was scored as severe for 60 children and nine patients (3%) died. risk factors significantly associated with worsening evolution during hospitalisation (severe outcome) were admission at age under 6 months (or = 5.3), comorbidity (or = 4.6) and low household income (or = 4.1). conclusion: co-mordidity, low-income and age under 6 months increase the risk of severe outcome for children infected by numerous respiratory pathogens. these results highlight the need for implementation of targeted public health policy to reduce the contribution of respiratory diseases to childhood morbidity and mortality in low income countries. respiratory infections are a major cause of infectious diseaserelated morbidity, hospitalisation, and mortality among children under 5 years old worldwide, and particularly in developing countries [1] . these diseases are responsible for 4 million deaths worldwide every year [4] : they are thus among the leading causes of death and the most common infectious cause of death [2] . although many episodes of respiratory infection are mild, 7 to 13 percent are severe enough to warrant hospitalisation [3] . these diseases are responsible for at least 6% of the world's disability and death [1] . it is estimate that there are 33.7 million cases of respiratory infection annually among african children and longitudinal studies suggest that the overall mortality rate among children in developing countries is between 6.6% and 14.1% [5] . despite respiratory infections being a public health priority, data on their epidemiology in africa are sparse. diagnostic methods to identify the pathogen rapidly are not available in lowincomes settings, so most patient with respiratory infections are treated empirically with antibiotics based on local epidemiology [6] . administering appropriate therapy at the appropriate time would improve outcomes. these diseases also have an economic impact, as they lead to substantial consumption of antimicrobial agents in both community and hospital settings. furthermore, the high frequency of respiratory infections and the excessive use of antimicrobial agents are major contributors to the development of antibiotic-resistant pathogens. knowing the local epidemiology of respiratory infections is essential if appropriate empiric therapy is to be prescribed. evaluation of mixed infections and the relative importance of each potential pathogen may also contribute to improving the understanding of the pathogenesis of these infections [7, 8] . our study aimed to evaluate the risk factors associated with the evolution and outcome of respiratory illnesses in patients aged under 5 years old hospitalised in one of the four main public hospitals in antananarivo. the study was conducted at the ''centre hospitalier de soavinandriana'' (cenhosoa) in antananarivo, the capital city of madagascar, which is located in the central highlands. antananarivo consists of administrative, commercial, industrial and residential areas, with patches of agricultural land, mostly rice fields. the city is divided into six administrative districts. in madagascar, hib vaccine was introduced in 2008, and pneumococcia vaccine in 2012 cenhosoa is a national referral hospital in the third district of the urban municipality of antananarivo; hospitalization and care at this hospital is paid for by the patients. it is located near the institut pasteur of madagascar (ipm). the paediatric and neonatology ward of cenhosoa has 54 beds, and the annual number of children hospitalized for respiratory infection was estimated to be 150. a prospective, hospital-based, study was conducted at cen-hosoa from november 2010 to july 2012. the case definition and eligibility criteria used during this study are described in figure 1 . demographic, socio-economic, clinical and epidemiological data were recorded in case report forms (crfs). paediatricians completed the crfs which were then validated by a clinical research officer. instruction sheets were provided to clinical research officers to ensure accurate data collection. the data collected was managed by a senior medical epidemiologist. on admission, trained personnel using standard operating procedures had collected nasal and throat swabs, and sputum sample by aspirating airway secretion from the hypopharynx through the nose or tracheal aspirates. nasopharyngeal secretions, tracheal aspirates and sputum were obtained before start of antibiotic therapy. specimens were transported within 30 minutes of sampling to the ipm for tests of respiratory pathogens. hiv serostatus was not determined; the prevalence of hiv in madagascar is low, estimated to be around 1 per 1000 [7] . the study was approved by the national ethics committee of the ministry of health of madagascar. written informed consent was obtained from parents or legal guardians of children enrolled in the study. consenting to be enrolled in the study required consent to provide samples for diagnosis of influenza and other respiratory pathogens and to fill out a case report forms (crfs). refused consent and hospitalization within the two weeks before consultation were reasons for exclusion from the study. virological analyses were performed by national influenza centre (nic). a panel of multiplex real-time rt-pcr was used for the detection of viral pathogens in nasopharyngeal swabs. primers and probes were obtained from the authors of reported studies or were developed at the nic [8] . viral rna and dna were extracted from 140 ml of each specimen using the qiaamp viral rna mini kit and qiaampminelute virus kit (qiagen, courtaboeuf, france) according to the manufacturer's protocols. an abi 7500 fast real-time pcr system (applied biosystems) was used for amplifications. each sample was subjected to five different amplification reactions, each containing primers and probes specific for two or three viruses such that the following 12 respiratory viruses were targeted as previously described: parainfluenza virus 1-3 (piv), human coronaviruses (hcov) -oc43, -229e, -nl63 and hku1, respiratory syncytial virus (rsv), human metapneumovirus (hmpv), human rhinovirus (hrv), adenovirus (adv) and bocavirus (bov). as part of our routine influenza surveillance, screening for influenza viruses is performed by mdck cell inoculation and realtime rt-pcr according to the cdc protocols [9] . bacteriological analyses were performed by the medical analysis laboratory of ipm gram-stained sputum preparations were examined for polynuclear neutrophils (pn) and epithelial cells. the areas of maximal purulence were examined and graded as follows: class 1: more than 25 cells and fewer than 10 pn, class 2: more than 25 cells and 10-25 pn, class 3: more than 25 cells and more than 25 pn, class 4: 10-25 cells and more than 25 pn and class 5: fewer than 10 cells and more than 25 pn. only the classes 4 and 5 present a high positive predictive value. if the laboratory determined that the sample was of oral origin it had been rejected. so, classes 1 to 3 had been considered as rejection criteria. quantitative sputum cultures were performed on each specimen by a previously described method [10] : sputum specimens were homogenized with an equal volume of digest-eurh on a vortex mixer; 10 ml aliquots of the homogenate were serially diluted in saline and 10 ml aliquots of the dilutions were plated with a sterile inoculator on blood columbia agar with nalidixic acid and colistin (anc), chocolate polyvitex (pvx) agar, and drigalski agar. plates were incubated under 5% co 2 at 35uc for 24 to 48 hours. colonies growing on plates inoculated with suitable dilutions were counted to enumerate each bacterial species present. the threshold of significance was set at $10 7 /ml (for one or two species only). isolates were identified by standard laboratory methods. mono-infection was defined as the presence of only one pathogen as determined by viral and bacterial analysis. patients for which more than one pathogen was detected were considered to have co-infections. the outcome was scored as severe if the patient died, or presented a wrong clinical evolution during hospitalisation (complication) or the hospitalization was longer than 10 days (3 rd quartile). analyses were performed with r software [11] . the nonparametric wilcoxon test was used to compare means. qualitative variables were compared with the fisher exact test. statistical differences were considered significant for p-values,0.05. multivariate backward step-by-step logistic regressions were performed to take into account confounders, bias and interactions involving pathogens linked to the dependent variable. all variables with a pvalue,0.20 were included in the initial model. five age groups were defined for the analysis: [0-5] months, [6] [7] [8] [9] [10] [11] [12] months, [13] [14] [15] [16] [17] [18] months, [19] [20] [21] [22] [23] [24] months, [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] months, .36 months. from november 2010 to july 2012, 290 children were recruited from the 301 hospitalized patients eligible. eleven eligible patients refused to participate in the study, and they did not differ from included patients for age, sex or diagnosis on admission. the baseline demographic and clinical characteristics of the patients are shown in table 1 . the sex ratio (male/female) was 1.3. the mean of age was 1.1 years [95% ci: 1.0 to 1.2], the age range was from 1 day to 5 years, and 250 (86%) patients were 2 years old or younger. the diagnoses on admission (table s1 ) were significantly different between the age groups: bronchiolitis was found more frequently among patients under 6 months old (55%, p,0.01); ltri and pneumonia were more frequent among patients aged over 12 months (respectively, 68% and 85%,p,0.01). forty-nine (17%) patients had at least one underlying disease, with asthma being the most frequent (5%). some of the children had underlying conditions, particularly atopy (52; 18%) and passive exposure to tobacco smoke (113; 39%). seventy-one (24%) had been hospitalized during the previous 12 months. patients aged under 18 months had significantly more prior hospitalisations (p = 0.01). the difference between sexes was not statistically significant. around 45% (131/290) of patients had been treated with antibiotics before hospitalisation (table s2 ). significantly more patients under than over 12 months old had received antibiotic treatment before hospitalisation (p,0.01). the difference between sex was not statistically significant. the frequency of bacterial detection was lower in the group with prior antibiotic treatment (49% versus 65%, p = 0.01). no such relationship was found for viral results. accurate information about immunisation was available on vaccination cards for 82 (26%) children. eighty-seven percent of children had completed vaccination against bcg, 77% had received one haemophilus influenzae type b vaccination. no child was vaccinated against streptococcus pneumoniae and only two were vaccinated against influenza. the average of duration of hospitalisation was 7.9 days [95% ci: 7.1 to 8.6], range 1 to 79 days. the median duration of hospitalisation was 7 days, and 61% of patients were hospitalised for more than 7 days. the duration of hospitalisation for living patients was 8.0 [7.2-8.7 ] days, and for fatal cases was 3.9 [1.4-9.2]. there were no significant differences between sex and age groups. discharge information was available for all cases: 79% (230/ 290) of patients fully recovered without any complication during their hospital stay; the condition of 60 children worsened during hospitalisation, and nine patients (3%) died. all deaths were of children under 18 months of age, and six (67%) were of children less than 6 months old. for the patients who died, streptococcus pneumoniae (56%, 5/9) was the pathogen most frequently detected, followed rsv (33%, 3/9) and influenza type a virus (22% 2/9). for multivariate analysis of factors possibly associated with coinfection, sex, age group, number of rooms in the home and antibiotic treatment before hospitalisation were included in the initial model. the multivariate analysis indicated that children under 36 months of age were more often infected by more than one pathogen; the difference was statistically significant for the 19-24 months old group (adjusted or = 8.0) ( table 1) . previous antibiotic treatment before hospitalization was a significantly protective factor against co-infection (or = 0.5). in univariate analysis, influenza a, rhinovirus, streptococcus pneumoniae, and haemophilus influenza type b were significantly associated with co-infections. haemophilus influenzae and s. pneumoniae were most often associated with co-infection (adjusted or = 20.5 and 11.1, respectively) ( table 1) . univariate analysis showed that a severe outcome was significantly more frequent for patients with comorbidity (or = 3.9), including prematurity or congenital disease (such as heart disease or cerebral malformation). also, two of the three patients presenting with malnutrition died. hospitalization during the 12 months prior to admission was also associated with a severe outcome (or = 2.1). a low household income, of less than 455 us$ (less than 182 us$ or = 4.4 and between 182 and 455 us$ or = 3.5) was associated with severe outcomes. for multivariate analysis, age group, monthly household income, admission diagnosis, comorbidities, previous hospitalisation, and infection with influenza virus type a, rsv and rhinovirus were included in the initial model. multivariate analysis (table 3 ) adjusted for these variables showed that age below 6 months (or = 5.3), living in a household with low income (or = 4.1), and the presence of comorbidities (or = 4.6) were significantly aggravating factors. infection with an influenza type a virus was a significantly protective factor against a severe outcome (or = 0.4 [95% ci = 0.2-0.9]). in the paediatric ward of cenhosoa, most children under 36 months, hospitalised for respiratory infections, were co-infected, and the most frequently encountered pathogens were h. influenza, s. pneumonia, influenza a and rhinoviruses. during hospitalisation, the evolution of respiratory infections was worse for the youngest children and for those presenting comorbidities and living in lowincome household. over all, viral infections made a large contribution to the burden of infectious respiratory disease. viral pathogens were found in 85% of the patients and bacterial pathogens in 57%, although for 45% of the patients had been treated with antibiotics before hospitalisation, and this presumably affected the observed prevalence of bacterial infections. our findings for viruses were similar to those of other studies. for example, the detection rate for viral pathogens was 72% in a study in vietnam [12] , 50% in a study in mozambique [13] , both focusing on hospitalised children. these results are consistent with those for the paediatric ward in cenhosoa. our findings are also consistent with another report from madagascar indicating that rsv (30.5%) and by influenza a (22.6%) are the most frequent viral causes of community respiratory infection [8] . few studies in developing countries have addressed the role of bacterial co-infection in severe respiratory illness. most focus on the viral aetiology because molecular diagnosis tools are available for these pathogens allowing to identify the infection in 80%-95% of cases [14, 15] . in developing countries, laboratory methods to identify bacterial pathogens are more difficult to implement, because of the lack of appropriate facilities and material in hospital laboratories and then, long transit times are prejudicial for stability of specimens. in our study the transit time was reduced because of the proximity of the cenhosoa and the ipm. unfortunately, our findings for the rate of bacterial infection may be artificially low due to prior antibiotic use. in our study, streptococcus pneumoniae was the most common bacterial pathogen as in other studies of hospitalised patients with acute respiratory illness, and haemophilus influenzae type b was the next most frequent [16] [17] [18] [19] . primary infection by a respiratory virus increases the risk of secondary bacterial pneumonia, and viral and bacterial coinfection is common in young children with pneumonia in developing countries (about 20-30% of episodes) [20, 21] . evidence of probable mixed viral-bacterial infection has been recorded in up to 45% of cases of community acquired pneumonia in children [22] [23] [24] [25] . a large proportion of our patients had coinfection (70%; 192/273) and the [0-6] months age group had the highest risk. these results are consistent with a study of patients with community respiratory infection in madagascar [8] . viral and bacterial co-infection was found in 57% of our patients; the most frequent combination was streptococcus pneumoniae with one of several respiratory viruses (88%). data from gambia and nigeria indicate that mixed bacterial and viral infections may occur in 8 -40% of cases of childhood pneumonia [26, 27] . recent data from south africa indicate that in at least 31% of cases, viral-associated pneumonia may be due to concurrent infection with streptococcus pneumoniae in the absence of vaccination with pneumococcal conjugate vaccines [28] . appropriate management of respiratory infections can have a significant impact on child mortality and it is important to target priority groups of children most at risk of dying: these groups include very young infants, children infected with hiv and malnourished children [29] . in our study, the outcome was severe for all of the children who presented malnutrition and 67% (2/3) died. chisti et al show that the combination of pneumonia with severe or moderate malnutrition is associated with a high risk of death: the rr ranges from 2.9 to 121.2 for severe malnutrition, and from 2.5 to 15.1 for moderate malnutrition. for moderate malnutrition, rr = 1.2 to 36.5. several other studies also lead to the same results [30] [31] [32] [33] . ballard et al. report that malnutrition and level of parental education both have an impact on the incidence of respiratory infections [34] . pneumonia is the leading causes of childhood mortality, and is responsible for approximately 21% of deaths of african children under five years of age each year. however, little information is available about the causes of childhood pneumonia in developing countries. in our study, only 3% of the patients died; this value is low. note that the study population consists of patients from families with a socioeconomic level that is above the average for the general population in madagascar. the patients who died were more frequently co-infected and under 6 months old. these results are consistent with other studies on this topic in developing countries [35, 36] . however, study design may have an impact on the mortality rate observed: indeed all fees for hospitalisation were paid by the study to allow follow-up to be optimised. the study identified living in a low-income household as a risk factor for severe outcome. this is important for patient management during hospitalisation and also highlights the consequences of financial barriers to access to vaccination against influenza and pneumococci. these vaccinations were not included in the panel of vaccinations in expanded programme of immunisation (epi) during this period in madagascar. they were only available in private centres and were expensive, and therefore inaccessible for much of the population. only two of the patients included in this study had received vaccination against influenza and none had received vaccination against pneumococci according to their vaccination cards. our statistical analysis identified influenza a infection as a protective factor against a fatal outcome; however, it is well-known that influenza a may have an impact on high mortality during major epidemics [37] [38] [39] . the co-circulation of influenza a virus, rsv and the high frequency of streptococcus pneumoniae in co-infections in children aged less than 5 years raised issues about the indirect impact of influenza vaccination on respiratory infections. in particular, the benefits of establishing a system of vaccination against infectious agents for which a vaccine is available should be considered to provide access to a larger proportion of the population. in conclusion, we advocate to further research in developing countries to document in more detail the impact of viral and bacterial co-infections on the prognosis of severe respiratory illness. as numerous infections found in our study could be avoided by immunisation, we recommend that appropriate vaccination must be rapidly available for all the malagasy children. this would reduce mortality among the youngest children, as this age group currently suffers the greatest burden of morbidity and mortality associated with respiratory infections. table s1 diagnosis at admission according with age groups. (docx) estimates of world-wide distribution of child deaths from acute respiratory infections community-acquired pneumonia epidemiology and etiology of childhood pneumonia study of community acquired pneumonia aetiology (scapa) in adults admitted to hospital: implications for management guidelines community-acquired pneumonia in children british thoracic society guidelines for the management of community acquired pneumonia in childhood bayesian mapping of pulmonary tuberculosis in antananarivo viral etiology of influenza-like illnesses in antananarivo who | cdc protocol of realtime rtpcr for influenza a (h1n1) (n.d.). who. available remic référentiel en microbiologie médicale r: a language and environment for statistical computing. r foundation for statistical computing viral etiologies of acute respiratory infections among hospitalized vietnamese children in ho chi minh city viral acute respiratory infections among infants visited in a rural hospital of southern mozambique intranasal fluticasone propionate does not prevent acute otitis media during viral upper respiratory infection in children human bocavirus and acute wheezing in children epidemiology and etiology of childhood pneumonia etiology of community-acquired pneumonia in hospitalized patients in northern israel etiology of severe pneumonia in children in developing countries the bacterial etiology of community-acquired pneumonia in korea: a nationwide prospective multicenter study etiology of acute lower respiratory tract infections in gambian children: ii. acute lower respiratory tract infection in children ages one to nine years presenting at the hospital diagnoses of acute lower respiratory tract infections in children in rawalpindi and islamabad induced sputum in the diagnosis of childhood community-acquired pneumonia etiology of community-acquired pneumonia in 254 hospitalized children etiology of community-acquired pneumonia in hospitalized school-age children: evidence for high prevalence of viral infections malnutrition as an underlying cause of childhood deaths associated with infectious diseases in developing countries etiology of acute lower respiratory tract infections in gambian children: ii. acute lower respiratory tract infection in children ages one to nine years presenting at the hospital the etiology of pneumonia in malnourished and well-nourished gambian children a trial of a 9-valent pneumococcal conjugate vaccine in children with and those without hiv infection challenges to improving case management of childhood pneumonia at health facilities in resource-limited settings multihospital surveillance of pneumonia burden among children aged ,5 years hospitalized for pneumonia in bangladesh bacterial aetiology and outcome in children with severe pneumonia in uganda etiologic agents and outcome determinants of community-acquired pneumonia in urban children: a hospital-based study improvements in nutritional management as a determinant of reduced mortality from community-acquired lower respiratory tract infection in hospitalized children from rural central africa the effects of malnutrition, parental literacy and household crowding on acute lower respiratory infections in young kenyan children bacterial etiology of serious infections in young infants in developing countries: results of a multicenter study. the who young infants study group neonatal pneumonia in developing countries excess mortality associated with the 2009 a(h1n1)v influenza pandemic in antananarivo this study would not have been possible without the excellent support from all the clinicians (dr emma rasoanirina, dr roland randriamirado, dr harimboahangy rakotoarison, dr lucien radozahana, dr rani razafindrakoto and dr domohina rakotovao), and all the nurses of the paediatric ward of the cenhosoa, antananarivo. we are deeply indebted to all staff who contribute, every day, to the hospital surveillance programme. we also acknowledge the tremendous work of all technicians of the virology unit and cbc.disclaimer: the views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the ministry of health. conceived and designed the experiments: sr. performed the experiments: sr jmh vr. analyzed the data: sr vr nhr. contributed reagents/ materials/analysis tools: nhr ao ehr lr rr ph fr zza ta. wrote the paper: sr nhr ehr vr. key: cord-308764-9z4qcoqz authors: wei, lin; li, shenghua; liu, shenggui; he, anna; wang, dan; wang, jie; tang, yulian; wu, xianjin title: transcriptome analysis of houttuynia cordata thunb. by illumina paired-end rna sequencing and ssr marker discovery date: 2014-01-02 journal: plos one doi: 10.1371/journal.pone.0084105 sha: doc_id: 308764 cord_uid: 9z4qcoqz background: houttuynia cordata thunb. is an important traditional medical herb in china and other asian countries, with high medicinal and economic value. however, a lack of available genomic information has become a limitation for research on this species. thus, we carried out high-throughput transcriptomic sequencing of h. cordata to generate an enormous transcriptome sequence dataset for gene discovery and molecular marker development. principal findings: illumina paired-end sequencing technology produced over 56 million sequencing reads from h. cordata mrna. subsequent de novo assembly yielded 63,954 unigenes, 39,982 (62.52%) and 26,122 (40.84%) of which had significant similarity to proteins in the ncbi nonredundant protein and swiss-prot databases (e-value <10(−5)), respectively. of these annotated unigenes, 30,131 and 15,363 unigenes were assigned to gene ontology categories and clusters of orthologous groups, respectively. in addition, 24,434 (38.21%) unigenes were mapped onto 128 pathways using the kegg pathway database and 17,964 (44.93%) unigenes showed homology to vitis vinifera (vitaceae) genes in blastx analysis. furthermore, 4,800 cdna ssrs were identified as potential molecular markers. fifty primer pairs were randomly selected to detect polymorphism among 30 samples of h. cordata; 43 (86%) produced fragments of expected size, suggesting that the unigenes were suitable for specific primer design and of high quality, and the ssr marker could be widely used in marker-assisted selection and molecular breeding of h. cordata in the future. conclusions: this is the first application of illumina paired-end sequencing technology to investigate the whole transcriptome of h. cordata and to assemble rna-seq reads without a reference genome. these data should help researchers investigating the evolution and biological processes of this species. the ssr markers developed can be used for construction of high-resolution genetic linkage maps and for gene-based association analyses in h. cordata. this work will enable future functional genomic research and research into the distinctive active constituents of this genus. saururaceae, a member of the paleoherbs, is an ancient family with six species in four genera, anemopsis, gymnotheca, houttuynia and saururus [1] . houttuynia cordata thunb. (yuxingcao in chinese) is the only species in the genus houttuynia [2, 3] . it is distributed mainly in the central, southeastern and southwestern regions of china, and extends to japan, korea and southeast asia, where it grows in moist, shady places [4] . h. cordata is an important traditional medical herb native to china and other asian countries [5, 6] . it plays a unique role in improving the immune system of patients with severe acute respiratory syndrome (sars) [7, 8] . extracts of h. cordata have diverse pharmacological effects including anticestodal [9] , antibacterial [10, 11] , antiviral [12] [13] [14] [15] , anticancer [16, 17] , antioxidant [18, 19] , antiallergenic [20, 21] , anti-inflammatory [22] [23] [24] , antimutagenic [18] and anti-obesity [25] activities. h. cordata is also consumed as a vegetable in china for its special aroma. although h. cordata is of high medicinal and nutritional value, there are no genomic resources for this non-model genus. this lack of genomic information has become a limitation for extensive and intensive research on this important traditional medical herb. previous studies on this plant have mainly focused on cultivation techniques [26, 27] , its physiological and biochemical properties [28, 29] , its genetic relationships and the diversity among h. cordata germplasm collections from different places [4, 30, 31] , and its pharmacological effects [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] . to date, few gene sequences or novel genes have been reported on this species, although much effort has been devoted to cloning key genes. rna-seq, which is based on next generation sequencing, is a high throughput technology that has great advantages in examining the fine structure of a transcriptome [32] . when no genome sequence is available, transcriptome sequencing provides an effective way to obtain large amounts of sequence data [33] . rna-seq has been widely used in many organisms to obtain mass sequence data for transcriptional analysis, gene discovery and molecular marker development [34] [35] [36] . the genetic relationships and diversity among h. cordata germplasm collections have been investigated mostly using aflp [37] , issr [4] , pcr-rflp [30] and rapd markers [31] . no simple sequence repeat (ssr) markers have been reported in h. cordata. compared with other types of molecular markers, ssr markers have many advantages, such as simplicity, effectiveness, abundance, hypervariability, reproducibility, codominant inheritance, and extensive genomic coverage [38] . because of the lack of effective molecular markers, marker-assisted selection and molecular breeding of h. cordata has lagged behind other medicinal plants such as panax notoginseng (burkill) f.h.chen [39] , gastrodieae elata blume [40] , and glycyrrhiza uralensis fisch. [41] . thus, a rapid, low-cost and effective approach is required to develop ssrs molecular markers for h. cordata. in this study, we applied the next-generation massively parallel sequencing technique (illumina hiseq 2000) to the sequencing and analysis of the complete h. cordata transcriptome for the first time. we sampled the pooled transcriptomes of flower, leaf, stem and rhizome tissues of h. cordata and used illumina paired-end sequencing technology to generate a large-scale est database and develop a set of ssr markers. these results provide a very useful genomic resource for research on h. cordata in the future. to obtain a global overview of the h. cordata transcriptome and gene activity at the nucleotide resolution, rna was extracted from four different h. cordata tissues including the rhizome, stem, leaf and flower, and mixed at equivalent concentrations. sequencing was performed on an illumina hiseq2000 genome analyzer. each sequencing pass can yield two 90-bp independent reads from either end of a dna fragment. in all, 56,668,324 sequence reads were generated, of which 51,973,070 were of acceptable quality after cleaning the low-quality reads (table 1) , then we used the trinity short reads assembly program to assemble the reads for non-redundant consensus [42] several complementary methods were used to annotate the assembled sequences. first, the assembled sequences were queried against the national center for biotechnology information (ncbi) nonredundant protein (nr) and swiss-prot protein databases using blastx to search for similar sequences (e-value ,10 25 ). of the 63,954 assembled sequences, 39,982 (62.52%) showed homology to sequences in the nr database (table s1 ), while 26,122 (40.84%) unigenes had homology to proteins in the swiss-prot database. in addition, 99.06% of the unigenes over 1,000 bp in length showed homologous matches, whereas only 29.28% of the unigenes shorter than 300 bp showed matches (fig. 2) . the 2,624 unigenes that had no matches in either the nr or swiss-prot databases were subjected to gene prediction analysis using estscan (ver-sion3.0.2) [43] . in total, 42,785 unigenes were detected by homology analysis using the nr and swiss-prot databases or estscan prediction. the e-value distribution of the top hits in the nr database showed that 62.2% of the mapped sequences had strong homology (e-value ,10 230 ) and 50.8% had very strong homology (e-value ,10 245 ) to available plant sequences, whereas 37.8% of the homologous sequences had e-values in the range 10 25 to 10 230 (fig. 3a) . the similarity distribution of sequences database showed that 3,880 (9.70%), 12,513 (31.30%), 16 biaceae), populus trichocarpa (salicaceae), glycine max, medicago truncatula (leguminosae), oryza sativa japonica group, and sorghum bicolor (gramineae), respectively. and 18.35% of the distinct sequences had matches to sequences from 'other' species. (fig. 3c ). based on nr annotation, 30,131 unigenes were assigned gene ontology (go) terms. the sequences that belonged to the biological process, cellular component, and molecular function clusters were categorized into 46 functional groups (fig. 4) . 'cellular processes' and 'metabolic processes', 'cell' and 'cell part', 'antioxidant activity' and 'binding' were the dominant groups among the three main categories (biological process, cellular component and molecular function), respectively. however, we did not find any genes in the clusters 'carbon utilization', 'locomotion', 'nitrogen utilization', 'sulfur utilization', 'viral reproduction', 'extracellular matrix', 'extracellular matrix part', 'extracellular region part', 'channel regulator activity', 'metallochaperone activity', 'protein tag' or 'translation regulator activity' (fig. 4) . in addition, all unigenes were subjected to a search against the cluster of orthologous groups (cog) database for functional prediction and classification. overall, 15,363 of the 39,982 sequences showing nr hits were assigned cog classifications. the cog-annotated putative proteins were functionally classified into at least 25 molecular families including cellular structure, biochemistry metabolism, molecular processing, and signal transduction (fig. 5) . the cluster for 'general function prediction only' (5,362, 13.58%) represented the largest group, followed by transcription (4,124, 10.44%), replication, recombination and repair (3, 242, 8 .21%), posttranslational modification, protein turnover and chaperones (3,014, 7.63%), function unknown (2,787, 7.06%), signal transduction mechanisms (2,614, 6.62%), translation, ribosomal structure and biogenesis (2,603, 6.59%), cell cycle control, cell division, chromosome partitioning (2,446, 6.19%), carbohydrate transport and metabolism (2,340, 5.93%), cell wall/ membrane/envelope biogenesis (2,263, 5.73%), intracellular trafficking, secretion, and vesicular transport (1,244,3.15%), amino acid transport and metabolism (1,177,2.98%), secondary metabolites biosynthesis, transport and catabolism (1,109, 2.81%), inorganic ion transport and metabolism (909, 2.30%), lipid transport and metabolism (887, 2.25%), energy production and conversion (823, 2.08%), coenzyme transport and metabolism (501, 1.27%), defense mechanisms (447, 1.13%), cytoskeleton cell (398, 1.00%), motility (385, 0.98%), nucleotide transport and metabolism (298, 0.75%), chromatin structure and dynamics (270, 0.68%) and rna processing and modification (222, 0.56%), whereas only a few unigenes were assigned to nuclear structure and extracellular structure (13 and 11 unigenes, respectively). the cog function classification of h. cordata unigenes is shown in a total of 24,434 assembled sequences were associated with 128 predicted kegg metabolic pathways. the number of sequences ranged from 3 to 6,718. the top 20 pathways with the greatest number of sequences are shown in table 2 . the greatest number of transcripts was found in the metabolic pathways. the top 10 metabolic pathways were: glycerophospholipid metabolism (1,974), ether lipid metabolism (1,853), starch and sucrose metabolism (869), purine metabolism (697), pyrimidine metabolism (637), phenylpropanoid biosynthesis (381), oxidative phosphorylation (298), amino sugar and nucleotide sugar metabolism (table s2 ). for further assessment of the assembly quality and development of new molecular markers, all 63,954 unigenes generated in this study were used to mine potential microsatellites, which were defined as di-to hexa-nucleotide ssrs with a minimum of four repetitions for all motifs. the ssrs that were only located in one single read had been eliminated. using the microsatellite (misa, http://pgrc.ipk-gatersleben.de/misa/) tool, a total of 4,800 potential ssrs were identified in 4,413 unigenes, 357 of which contained more than one ssr; 164 ssrs were present in compound form ( table 3) . the frequency, type and distribution of the 4,800 potential ssrs were also analyzed in this study. the compilation of all ssrs revealed that, on average, one ssr could be found every 9.04 kb in the unigenes. among the 4,800 ssrs, tri-nucleotide repeat motifs were the most abundant type (1,994, 41.54%), followed by mono-(1,313, 27.35%), di-(1,278, 26.63%), hexa-(110, 2.29%), penta-(66, 1.38%) and tetra-nucleotide (39, 0.81%) repeat motifs. the mono-to hexa-nucleotide motifs were further analyzed for ssr repeat numbers (table 4) . ssr length was mostly distributed in the 12 to 20 bp range, accounting for 87.04% (4035 ssrs) of the total ssrs, followed by 21-99 bp (562 ssrs, 12.12%). there were 39 ssrs longer than 100 bp. within the ssrs, 119 motif sequence types were identified, of which mono-, di-, tri-, tetra-, penta-and hexa-nucleotide repeats had 2, 4, 10, 11, 31 and 58 types, respectively. the most abundant motif detected in the ssrs was the a/t mono-nucleotide repeat (1,296, 27. fifty primer pairs (designated hm_1-hm_50) were randomly selected from the microsatellites, excluding mono-nucleotide repeats motif, to evaluate their applicability and the polymorphism across 30 individuals of h. cordata (table s3) table 5 ). next generation sequencing technologies provide a low cost, labor saving and rapid means of transcriptome sequencing and characterization [44] , which enables various functional genomic studies on an organism. although the 454 life sciences (roche) technology is often used for transcriptome analysis of non-model organisms, it is more expensive than the illumina technology [45] . de novo assembly of short reads without a known reference is considered difficult [46] , but de novo assembly of transcriptomes using short reads has received attention [47] . in this study, we demonstrated a strategy for de novo assembly of a transcriptome using short reads for a non-model medicinal plant, h. cordata, for which sequence data is very limited in the public databases at present. we showed that assembly program parameters and sequence quality have a significant effect on the assembly output. although the length of contigs were often less than 500 bp, the illumina sequencing solution was reliable. such as the average contig size of sesame was less than 200 bp [48] , whitefly was only 40 bp [49] , sweetpotato was 202 bp [50] . compared with these reports, the assembled contigs in this study was quite long (253 bp). this suggested that the coverage was relatively high. greater n50 and average lengths are considered indicative of a better assembly. here, the n50 length of the unigenes was 1,051 bp and the average length was 679 bp, which suggests that the relatively short reads from illumina paired-end sequencing for this non-model organism have been effectively and accurately assembled. illumina sequencing yielded 56.67 million paired-end reads for h. cordata. the 63,954 unigenes produced here may be useful for further research into h. cordata functional genomics. of the h. cordata unigenes, 39,982 (62.52%) showed homology to sequences in the nr database. comparatively, in epimedium sagittatum [51] , whitefly [49] , sweet potato [50] and sesame [48] , only 38.50%, 16.20%, 46.21% and 54.03% of the unigenes, respectively, had homologs in the nr database. the average unigene length in our database was 679 bp, compared with 246, 266, 581 and 629 bp, respectively, in the four studies mentioned above. the higher percentage of hits found in this study was partially a result of the increased number of long sequences in our unigene database; the results for whitefly [49] and sesame [48] support this conclusion. homologs in other species were not found for 18.3% of the unique sequences. specifically, only 29.28% of the unigenes shorter than 300 bp showed matches, meaning that 70.72% produced no hits (fig. 3) . these shorter sequences may lack a characterized protein domain, or they may contain a known protein domain but the query sequence is too short to show sequence matches, resulting in false-negative results. additionally, little genomic and transcriptomic information is currently available for h. cordata, and consequently, many h. cordata lineage-specific genes might not be included in current databases. both gene annotation and kegg pathway analyses are useful for predicting potential genes and their functions at a wholetranscriptome level. in the h. cordata transcriptome, the predominant gene clusters are involved in the cellular process and metabolic process categories of the biological process go domain, the cell and cell part categories of the cellular component domain, and antioxidant activity and binding categories of the molecular function domain. similar results were found in sesame [51] and whitefly [49] . however, in the chickpea transcriptome, the sequences were found to be mainly involved in protein metabolism (biological process) and transferase activity (molecular function) [52] . this suggests remarkable differences among different species of plants. kegg analysis showed that 24,434 sequences were involved in 128 known metabolic or signaling pathways, including endocytosis, plant hormone signal transduction and plant-pathogen interaction. h. cordata is one of the most important medicinal plants and is rich in secondary metabolites, which makes it a very important target for genomic studies. in this study, 2,448 (10.02%) sequences of h. cordata were associated with biosynthesis of secondary metabolites (table 2) . these results may be useful for further investigation of gene function in the future. the large number of sequences generated for h. cordata in this study for the first time provides valuable sequence information at the transcriptomic level for screening of novel functional genes, or for investigation of molecular mechanisms. ssr markers play an important role in genetic diversity research, population genetics, linkage mapping, comparative genomics, and association analysis [38, 53] . previously, genetic diversity analysis of h. cordata germplasm was restricted to aflp [37] , issr [4] , pcr-rflp [30] and rapd markers [31] . one of the main reasons for this was the lack of a genome sequence or transcriptome information for h. cordata. our results have resolved this problem and enabled development of ssr markers for this species. in the present study, 4,800 perfect microsatellites exceeding 12 bp were identified from the h. cordata dataset, and 119 motif sequence types were identified. if mono-nucleotide repeats were excluded, di-nucleotide repeats were the most abundant type, followed by tri-nucleotide repeats, which is consistent with previous reports [54] [55] [56] [57] . the fact that the most abundant di-and tri-nucleotide motifs were ag/tc and aag/ ttc, respectively, was also coincident with previous reports on other species of plants [54] [55] [56] . in this study, 48 (96%) of the primer pairs designed from the unigenes successfully yielded high-quality amplicons. these results suggested that the unigenes were suitable for specific primer design, that the assembled unigenes were of high quality, and that the ssrs identified from our dataset could be useful in the future. five primer pairs produced products that deviated from the expected size, which might have been caused by the presence of introns [51, 58, 59] , large insertions or repeat number variations, or a lack of specificity [51] . the failure of two primer pairs to produce amplicons might have been because of the primer(s) being located across splice sites, large introns, chimeric primer(s), poorquality sequences, or assembly errors [51, 59] . the 43 primer pairs in our dataset were used to investigate polymorphisms in 30 individuals of h. cordata from 15 populations located across the natural distribution of the species in 13 provinces of china. the results indicated that the level of polymorphism was relatively high, which was also coincident with previous reports using issr [4] , rapd [31] and ramp markers [60] . since we identified other ssrs in our dataset, more pcr primers could be developed that would be very useful in germplasm polymorphism assessment, quantitative trait loci mapping [61] , comparative genomics [62] , functional genomics and proteomics studies. in this study, we have analyzed the transcriptome of h. cordata using high-throughput illumina paired-end sequencing. we obtained 39,982 sequences and demonstrated some important features of the h. cordata transcriptome through gene annotation and kegg pathway analysis. in addition, we identified reliable genetic markers in the form of 4,800 ssrs. fifty primer pairs were randomly selected to detect polymorphism among 30 h. cordata accessions, and 43 (86%) of these primer pairs successfully amplified fragments, revealing abundant polymorphisms. the ssr markers developed in this study can be used for construction of high-resolution genetic linkage maps and to perform gene-based association analyses in h. cordata. this is the first application of illumina paired-end sequencing technology to investigate the whole transcriptome of h. cordata and to assemble rna-seq reads without a reference genome. this study will provide useful resources and markers for functional genomics and proteomics research on h. cordata in future. h. cordata, whose seeds came from national forest park of zhong po mountain in huaihua city of hunan province, was grown in the experimental station of the department of life sciences, huaihua university, huaihua, china. the individuals have 18 chromosomes and diploid karyotype (fig.7) . flower, leaf, stem and rhizome tissues were harvested 14 weeks post planting,because h. cordata was planted in spring, and their flowers began to open after 14 weeks. all of the samples were immediately frozen in liquid nitrogen and stored at 280uc until use. total rna was isolated using the trizol reagent according to the manufacturer's instructions (invitrogen, carlsbad, ca, usa). table 5 . cont. forward primer reverse primer no. of alleles it was treated with rnase-free dnase i (worthington, lakewood, co, usa) for 30 min at 37uc to remove any residual dna. the quality and quantity of each rna sample was determined by biophotometer (eppendorf, germany). only those rna samples with an a 260 :a 280 ratio from 1.9 to 2.1 and an a 260 :a 230 ratio from 2.0 to 2.5 were used for the analysis. a total of 20 mg of rna was equally pooled from the four different tissues for cdna library preparation. beads with oligo(dt) were used to isolate poly(a) mrna after total rna was collected from the samples. fragmentation buffer was added to disrupt the mrna into short fragments. taking these short fragments as templates, random hexamer primers were used to synthesize first-strand cdna. second-strand cdna was then synthesized using buffer, dntps, rnase h and dna polymerase i. the short fragments were purified with a qiaquick pcr extraction kit and resolved with eb buffer for end reparation and a tailing. the short fragments were then connected with sequencing adapters. after agarose gel electrophoresis, suitable fragments were selected for pcr amplification as templates. the cdna library was sequenced on an illumina hiseq2000 sequencing platform. the raw reads were cleaned by removing adapter and lowquality sequences, because sequencing errors can create difficulties for the short-read assembly algorithm. we therefore carried out a stringent filtering process. firstly, we discarded all reads with adaptor contamination. secondly, we ruled out low-quality reads with ambiguous sequences ''n''. thirdly, the reads with more than 10% q,20 bases were also removed. de novo transcriptome assembly was carried out with the short reads assembly program in the trinity software(release-20120608) [42] . contigs were created by combining reads that had a certain length of overlap. the reads were then mapped back to the contigs; with paired-end reads we were able to detect contigs from the same transcript as well as the distances between the contigs. the contigs were connected using the trinity software to get sequences that could not be extended at either end. such sequences were defined as unigenes. these unigenes were further processed by sequence splicing and redundancy removal using the tgicl software(version 2.1) [63] to acquire non-redundant unigenes that were as long as possible. after gene family clustering, the unigenes could be divided to two classes. one was clusters, including unigenes that were .70% similar to each other; the other was singletons. in the final step, the sequence direction of the unigenes was determined. the unigenes were first aligned to sequences in the ncbi nr and swiss-prot protein databases with an e-value ,10 25 using blastx. unigenes that did not have homologs in the databases were scanned using estscan (version3.0.2) [43] . blast2go(version 2.5.0) [64] was used to obtain go annotations for the unigenes based on blastx hits against the ncbi nr database with an e-value threshold of ,10 25 . wego [65] was used for go functional classification of all unigenes and to plot the distribution of the h. cordata gene functions. the unigene sequences were also aligned to the cog database to predict and classify their functions. pathway assignments were carried out based on the kegg database [66] , which contains a systematic analysis of inner-cell metabolic pathways and the functions of gene products. a microsatellite program (misa; http://pgrc.ipkgatersleben. de/misa/) was used to identify and localize microsatellite motifs. we searched for all types of simple sequence repeats (ssrs) from mononucleotide to hexanucleotide using the following parameters: at least 12 repeats for mono-, six repeats for di-, five repeats for triand tetra-, and four repeats for penta-and hexa-nucleotide simple repeats. primer pairs were designed using the software primer 3-2.2.2. the major parameters for primer design were set as follows: primer length of 18-25 bases (optimal 21 bases), pcr product size of 80-200 bp (optimal 100-150 bp), gc content of 40-60% (optimal 50%), and dna melting temperature of 57-64uc (optimal annealing temperature 55-59uc). thirty individuals of h. cordata from 15 populations located across the natural distribution of the species in 13 provinces of china (table s3) were selected for polymorphism investigation with the ssrs. leaf samples were collected, dried and preserved in silica gel until dna extraction. genomic dna was extracted from the leaves of each individual using the ctab protocol [67] , dissolved in double distilled water, and quantified using agarose gel electrophoresis. the dna concentration was calculated according to dna standards. pcr amplification was performed in 16 ml reaction mixtures. each reaction contained 0.2 ml taq dna polymerase (0.5 u/ml), 2.5 ml pcr buffer, 1.5 ml mgcl 2 (25 mmol/l), 0.5 ml dntps (2.5 mmol/l), 0.4 ml each primer (10 pmol/l), 2.0 ml template dna (50 ng/ml), and 8.5 ml sterilized h 2 o. the temperature profiles were: initial denaturation at 94uc for 3 min, 35 cycles of denaturation at 94uc for 30 s, annealing at the optimal temperature of each primer pair for 30 s, and extension at 72uc for 45 s. final extension was at 72uc for 5 min, and then samples were held at 4uc. after pcr amplification, 6 ml aliquots of the amplified pcr products were loaded onto an 8% polyacrylamide gel. after 3-4 h of electrophoresis (250 v), the gels were stained with silver nitrate (silver staining). perfectly amplified loci were tested for polymorphism by genotyping 30 individuals of h. cordata. the genetic diversity and mean allele number were calculated using popgene version 1.32 [68] . polymorphism information content (pic) was derived according to the following formula: where n is the number of alleles at one locus; pi and pj are the frequencies of the ith and jth alleles at one locus; j = i+1. table s1 the statistics and annotations of unigenes. 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(glycine max l. merr.) genetic map and their association with est markers the genome sequence of caenorhabditis briggsae: a platform for comparative genomics tigr gene indices clustering tools (tgicl): a software system for fast clustering of large est datasets blast2go: a universal tool for annotation, visualization and analysis in functional genomics research wego: a web tool for plotting go annotations kegg: kyoto encyclopedia of genes and genomes dna protocols for plants ctab total dna isolation population genetic analysis of co-dominant and dominant markers and quantitative traits we thank the anonymous referees and the editor for their comments and suggestions that helped improve the manuscript. key: cord-316047-d9cpe9yl authors: gonzalez, t.; de la rubia, m. a.; hincz, k. p.; comas-lopez, m.; subirats, laia; fort, santi; sacha, g. m. title: influence of covid-19 confinement on students’ performance in higher education date: 2020-10-09 journal: plos one doi: 10.1371/journal.pone.0239490 sha: doc_id: 316047 cord_uid: d9cpe9yl this study analyzes the effects of covid-19 confinement on the autonomous learning performance of students in higher education. using a field experiment with 458 students from three different subjects at universidad autónoma de madrid (spain), we study the differences in assessments by dividing students into two groups. the first group (control) corresponds to academic years 2017/2018 and 2018/2019. the second group (experimental) corresponds to students from 2019/2020, which is the group of students that had their face-to-face activities interrupted because of the confinement. the results show that there is a significant positive effect of the covid-19 confinement on students’ performance. this effect is also significant in activities that did not change their format when performed after the confinement. we find that this effect is significant both in subjects that increased the number of assessment activities and subjects that did not change the student workload. additionally, an analysis of students’ learning strategies before confinement shows that students did not study on a continuous basis. based on these results, we conclude that covid-19 confinement changed students’ learning strategies to a more continuous habit, improving their efficiency. for these reasons, better scores in students’ assessment are expected due to covid-19 confinement that can be explained by an improvement in their learning performance. the coronavirus covid-19 outbreak disrupted life around the globe in 2020. as in any other sector, the covid-19 pandemic affected education in many ways. government actions have followed a common goal of reducing the spread of coronavirus by introducing measures limiting social contact. many countries suspended face-to-face teaching and exams as well as placing restrictions on immigration affecting erasmus students [1] . where possible, traditional classes are being replaced with books and materials taken from school. various e-learning platforms enable interaction between teachers and students, and, in some cases, national television shows or social media platforms are being used for education. some education systems announced exceptional holidays to better prepare for this distance-learning scenario. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 in terms of the impact of the covid-19 pandemic on different countries' education systems many differences exist. this lack of homogeneity is caused by such factors as the start and end dates of academic years and the timing of school holidays. while some countries suspended in-person classes from march/april until further notice, others were less restrictive, and universities were only advised to reduce face-to-face teaching and replace it with online solutions wherever practicable. in other cases, depending on the academic calendar, it was possible to postpone the start of the summer semester [2] . fortunately, there is a range of modern tools available to face the challenge of distance learning imposed by the covid-19 pandemic [3] . using these tools, the modification of contents that were previously taught face-to-face is easily conceivable. there are however other important tasks in the learning process, such as assessment or autonomous learning, that can still be challenging without the direct supervision of teachers. all these arguments end in a common topic: how to ensure the assessment's adequacy to correctly measure students' progress. thus, how can teachers compare students' results if they differ from previous years? on one hand, if students achieve higher scores than in previous years, this could be linked with cheating in online exams or with changes in the format of the evaluation tools. on the other hand, lower grades could also be caused by the evaluation format change or be attributable to autonomous learning as a less effective teaching method. the objective of this article is to reduce the uncertainty in the assessment process in higher education during the covid-19 pandemic. to achieve this goal, we analyze students' learning strategies before and after confinement. altogether, our data indicates that autonomous learning in this scenario has increased students' performance and higher scores should be expected. we also discuss the reasons underneath this effect. we present a study that involves more than 450 students enrolled in 3 subjects from different degrees from the universidad autónoma de madrid (spain) during three academic years, including data obtained in the 2019/2020 academic year, when the restrictions due to the covid-19 pandemic have been in force. e-learning has experienced significant change due to the exponential growth of the internet and information technology [4] . new e-learning platforms are being developed for tutors to facilitate assessments and for learners to participate in lectures [4, 5] . both assessment processes and self-evaluation have been proven to benefit from technological advancement. even courses that solely offer online contents such as massive open online courses (moocs) [6, 7] have also become popular. the inclusion of e-learning tools in higher education implies that a greater amount of information can be analyzed, improving teaching quality [8] [9] [10] . in recent years, many studies have been performed analyzing the advantages and challenges of massive data analysis in higher education [11] . for example, a study of gasevic et al. [12] indicates that time management tactics had significant correlations with academic performance. jovanovic et al also demonstrated that assisting students in their management of learning resources is critical for a correct management of their learning strategies in terms of regularity [13] . within few days, the covid-19 pandemic enhanced the role of remote working, e-learning, video streaming, etc. on a broad scale [14] . in [15] , we can see that the most popular remote collaboration tools are private chat messages, followed by two-participant-calls, multiperson-meetings, and team chat messages. in addition, several recommendations to help teachers in the process of online instruction have appeared [16] . furthermore, mobile learning has become an alternative suitable for some students with fewer technological resources. regarding the feedback of e-classes given by students, some studies [17] point out that students were satisfied with the teacher's way of delivering the lecture and that the main problem was poor internet connection. related to autonomous learning, many studies have been performed regarding the concept of self-regulated learning (srl), in which students are active and responsible for their own learning process [18, 19] as well as being knowledgeable, self-aware and able to select their own approach to learning [20, 21] . some studies indicated that srl significantly affected students' academic achievement and learning performance [22] [23] [24] . researchers indicated that students with strongly developed srl skills were more likely to be successful both in classrooms [25] and online learning [26] . these studies and the development of adequate tools for evaluation and self-evaluation of learners have become especially necessary in the covid-19 pandemic in order to guarantee good performance in e-learning environments [27] . linear tests, which require all students to take the same assessment in terms of the number and order of items during a test session, are among the most common tools used in computerbased testing. computer adaptive test (cat), based on item response theory, was formally proposed by lord in 1980 [28] [29] [30] , as is the case with linear testing. some platforms couple the advantages of cat-specific feedback with multistage adaptive testing [38] . the use of cat is also increasingly being promoted in clinical practice to improve patient quality of life. over the decades, different systems and approaches based on cat have been used in the educational space to enhance the learning process [39, 40] . considering the usage of cat as a learning tool, establishing the knowledge of the learner is crucial for personalizing subsequent question difficulty. cat does have some negative aspects such as continued test item exposure, which allows learners to memorize the test answers and share them with their peers [41, 42]. as a solution to limit test item exposure, a large question bank has been suggested. this solution is unfeasible in most cases, since most of the cat models already require more items than comparable linear testing [43]. the aim of this study is to identify the effect of covid-19 confinement on students' performance. this main objective leads to the first hypothesis of this study which can be formulated as h1: covid-19 confinement has a significant effect on students' performance. the confirmation of this hypothesis should be done discarding any potential side effects such as students cheating in their assessment process related to remote learning. moreover, a further analysis should be done to investigate which factors of covid-19 confinement are responsible for the change. a second hypothesis is h2: covid-19 confinement has a significant effect on the assessment process. the aim of the project was therefore to investigate the following questions: 1. is there any effect (positive or negative) of the covid-19 confinement on students' performance? 2. is it possible to be sure that the covid-19 confinement is the origin of the different performance (if any)? 3. what are the reasons for the differences (if any) in students' performance? 4. what are the expected effects of the differences in students' performance (if any) in the assessment process? we have used two online platforms. the first one is e-valuam [44] , an online platform that aims to increase the quality of tests by improving the objectivity, robustness, security and relevance of assessment content. e-valuam implements all the cat tests described in the following sections. the second online platform used in this study is the moodle platform provided by the biochemistry department from universidad autónoma de madrid, where all the tests that do not use adaptive questions are implemented. adaptive tests have been used in the subjects "applied computing" and "design of water treatment facilities". traditional tests have been used in the subject "metabolism". 2.1.1 cat theoretical model. let us consider a test composed by n q items. in the most general form, the normalized grade s j obtained by a student in the j-attempt will be a function of the weights of all the questions α and the normalized scores ψ (s j = s j (α, φ)), and can be defined as: where the φ i is defined as where δ is the kronecker delta, a i the correct answer and r i the student's answer to the i-question. by using this definition, we limit φ i to only two possible values: 1 and 0; φ i = 1 when the student's answer is correct and φ i = 0 when the student gives a wrong value. this definition is valid for both open answer and multiple-choice tests. in the case of multiple-choice test with n r possible answers, φ i can be reduced to consider the random effect. in this case: independently of using eqs 2 or 3, to be sure that s j (α, φ) is normalized (i.e. 0< = s j (α, φ)< = 1), we must impose the following additional condition on α: in the context of needing a final grade (fg) between 0 and a certain value m, which typically takes values such as 10 or 100, we just need to rescale the s j (α, φ) value obtained in our model by a factor k, i.e. fg j = k s j (α, φ). we will now include the option of having questions with an additional parameter l, which will be related to the level of relevance of the question. l is a number that we will assign to all the questions included in the repository of the test (i.e. the pool of questions from where the questions of a j-test will be selected). the concept of relevance can take different significances depending on the context and the opinion of the teachers. in our model, the questions with lower l values will be shown initially to the students, when the students answer correctly a certain number of questions with the lower l value, the system starts proposing questions from the next l value. by defining n l as the number of possible l values, the l value that must be obtained in the k-question of the j-test can be defined as: where trunc means the truncation of the value between brackets. it is worth noting that l k is proportional to the sum of the student's answers to all the previous questions in the test. this fact means that, in our model, the l k depends on the full history of answers given by the student. l k is inversely proportional to n q , which means that it takes a higher number of correct answers to increase l k . once l k is defined, a randomly selected question is shown to the student. another important fact that implies the use of eq 5 in the adaptive test is that we will never have l k <l k-1 . in other words, once a student starts answering questions from a certain l value, they will never go back to the previous one. in fig 1 we show a simple example of the multiple possible paths that a student can follow when facing a test that uses our model. in the example shown in fig 1 we have used n q = 6 and n l = 3. in this very straightforward example, the l value changes every time a student gives 2 correct answers (following diagonal arrows on the diagram). wrong answers imply a vertical drop on the diagram. in this case, the l value will not change (l k = l k-1 ). the final student's grade g is shown on the lower part of the diagram. the lowest grade (g = 0) corresponds to a test where the student has failed all the n q questions. in this case, l = 1 for all the possible j values. the highest grade (g = 6) implies that the student has faced questions from all the possible l values (l = {1,2,3} in fig 1) . ma-ts are used for evaluation and self-evaluation of theoretical contents. in this case, we use a standard format where a single correct answer must be chosen from a pool of possible answers shown by the system. since students can sometimes answer correctly by a random selection, eq (3) must be used when evaluating the score of any item. the format of the ma-t in our cat method requires the following elements: in addition, the e-valuam platform, where the method proposed in this article has been implemented, allows optional use of images or sounds for both the statement and the answers. e-valuam shows the statement and the possible answers. all other information relating to the cat method such as the level of the item is hidden from the student. the interface also shows optional feedback information about the performance of the learner in the previous item. finally, it shows the time remaining to finish the test. to train the contents of numerical problems, an oa-t was developed in which the statements include at least one parameter that will change its value with each execution of the application. this kind of question requires the following elements to be created: • statement with explicit indication of the modifiable parameter(s). • minimum and maximum values of each modifiable parameter. • programming code (matlab in e-valuam) that calculates the solution to the problem. • level of the item. as with ma-t, there is also a possibility of including multimedia files in the item. however, in this type of question, the multimedia option is only available in the question as the answer is numerical and included by the user in all cases. in this case the interface has a space for entering the numerical answer. in this figure, we also show the feedback of the application when an incorrect previous answer has been introduced. traditional tests have been used in the subject "metabolism" from the human nutrition and dietetics degree. the course contents are divided into 6 parts corresponding to different aspects of human metabolism. each part is taught with face-to-face lectures followed by different on-line activities that the students perform in the moodle platform and that are later discussed between them and with the professor in face-to-face workshops. all 6 parts of the course have a similar format, being a lecture followed by a selection of online activities in this order: "exercises workshop", "discussion workshop", "self-assessment" and "test". except for the discussion workshops outlined below, all activities are on-line questionnaires (closed, multiple-choice questions) that the students must perform in moodle, achieving a score that is automatically calculated and showed to them as part of their continuous assessment. exercises workshops and self-assessments are completed by the students without tutor supervision, whereas the tests are done on-line but under controlled conditions, just in the same way as a regular examination. each student should perform a total of 15 online activities: 5 exercises workshops, 6 self-assessment activities and 4 tests. discussion workshops are practical cases that must be debated by the students and sent to the tutor on moodle. as these activities do not obtain an automatic score but require a correction by the professor, they have not been considered in this study. the control group of our study is formed by the students of "applied computing" and "metabolism" from academic years 2017/2018 and 2018/2019 and by students of "design of water treatment facilities" from academic year 2017/2018. in the case of "design of water treatment facilities", a longitudinal study has been performed in academic year 2017/2018 to analyse the effect of rewards in the students' learning strategies, especially those related to time management. the experimental group of our study are students of "applied computing" and "metabolism" from academic year 2019/2020. in the longitudinal study of "design of water treatment facilities", experimental group corresponds to the third stage of the study. to answer our research questions, we first set up an experiment that obtains accurate measurements of the autonomous learning activities both in the control and the experimental groups. high accuracy in the measurements of the autonomous learning activities is achieved by using the previously described adaptive tools both in learning and assessment. in our experiment, the evaluation format and the e-learning tools are known to the students from the start, which motivates them to use the tools and perform the tests more consistently. in fig 2 we show the procedure used in our experiments to measure autonomous learning. there are two kinds of contents in the subjects included in the experiment: theory and numerical problems. in the case of theory, students can use the e-valuam platform through ma-t. the final evaluation is performed by oa-t. in the case of numerical problems, both learning and evaluation use oa-t. in this case, student motivation for using the platform is even higher because their final scores will be obtained by the same tool they use for their autonomous learning. this system is applied in all the experiments related to analysing the autonomous learning activity. in the case of "design of water treatment facilities", an additional longitudinal study has been performed and is shown in fig 2. this study has been performed in three stages. in the first stage, the self-evaluation material was presented to the students right before the evaluation test. in the second stage, the self-evaluation material was available 3 weeks before. to manage the theory contents, a ma-t with three possible answers to each question and only one correct has been created. in the first stage, with the test made available only one day before the exam, students had at their disposal a ma-t composed of 16 questions and with a time limit of 15 minutes. in the second stage, the time available was increased to 30 minutes and the question number to 18. these changes are related to the nature of the subject contents and do not have a significant impact on the study. the oa-t tests had 6 and 9 questions in the first and second stage, respectively. in order to maintain consistency in the subsequent study, an additional effort has been made to keep the difficulty of the questions as similar as possible. stage 3 had no new material added, instead, it used all the material created in stages 1 and 2. stage 3 corresponded to the time window between the end of classes and the last exam. in this stage, all the material was available from the beginning. the protocol used in this stage only varied from stage 2 in the inclusion of a reward for students who used the application on 3 or more different days. the reward was related to a bonus in the final grades. once the correct measure of the autonomous learning is ensured, as explained in the previous section, we must develop an experiment to describe the effect of covid-19 confinement. this experiment compares results of control and experimental groups in the whole assessment process. there are two stages that must be considered to determine the effect of the covid-19 confinement. the first one corresponds to the period without confinement in the year 2019/2020 (before march 11), when all the measurable activities are performed in similar conditions for experimental and control groups. the second stage corresponds to the period of covid-19 confinement (after march 11), where some measurable activities were performed in a different format and statistical differences can be found by comparing experimental and control groups. in the case of "applied computing", students use the e-valuam application on a continuous basis as it is available from the beginning of the course. they use the same oa-t for training and for their final exam, ensuring often and consistent use of the platform throughout the course. for this reason, in "applied computing", data analysis is performed over a continuous self-evaluation process over a single test along the whole academic year. the test performed by the students did not change its format or available questions in the whole three years under study. in the case of "metabolism", the confinement started just before the beginning of part iv of the subject. therefore, in this second stage, the scheduled in-person lectures from part iv were recorded on video by the tutor and provided to the students. afterwards, the students had an opportunity to perform the corresponding on-line continuous assessment activities, just in the same way as they would have done under normal conditions (as these activities were always not face-to-face). as this subject has already had an important number of on-line activities programmed prior to the suspension of face-to-face teaching, it is an extremely valuable tool for analysing the effect of the confinement on students' performance. in fig 3, we show the full experiment described in this section. first, the two studies performed in years prior to covid-19 confinement. in these first studies, we focus on the learning strategies of the students and their response to reward scenarios. information from these first studies will be useful for later discussion of the reasons for the changes caused by the covid-19 confinement. the second part of our experiment is related to the effect of covid-19 confinement, where we compare results of similar assessment activities between control and experimental groups. in fig 3 we include information about all control and experimental groups in the different interventions. this work involves data that has been collected from 458 students of the universidad autónoma de madrid. this data has been collected in the following manner: 1. it does not involve minors. 2. it has been collected anonymously. students have been identified by a numerical code, avoiding gathering of any personal information. 3. students have been informed by the lecturers that some information about their activity could be anonymously collected for statistical purposes. authors of this study did not receive any objections. 4. the tasks related to this study were completely voluntary and they did not in any form alter students' activities, classes, or the assessment process. considering these circumstances, we have received confirmation from the research ethics committee of universidad autónoma de madrid that we do not need to apply for ethics approval from our university since no personal data, minors or potentially hazardous activities were involved in the study. teachers involved in the study (coauthors of the present manuscript) who were responsible for the subjects taught also gave consent to carry out the study. we obtained verbal consent from the participants in the study. experiments have been performed in the subjects "applied computing", "metabolism" and "design of water treatment facilities". 2.3.1 applied computing. this study with real students took place during the 2017/2018, 2018/2019 and 2019/2020 academic years in the first-year subject "applied computing". 97, 73 and 91 students were involved each academic year, respectively. this subject was taught through theory lessons and practical classes in the computer laboratory. this course corresponds to 6 ects and belongs to the chemical engineering degree in the faculty of sciences from universidad autónoma de madrid, spain. due to covid-19 pandemic, the face-to-face teaching was cancelled on march 11, having a strong impact on the 2019/2020 course since it started in the first week of february and lasted until mid-may. the data for this study has been obtained from real students enrolled on the "metabolism" course in the human nutrition and dietetics degree from universidad autónoma de madrid, spain. this is a 6 ects compulsory subject taught during the second semester of the first year of the degree. the study comprises data from 2017/2018 and 2018/ 2019 academic years, with 64 and 63 enrolled students, respectively, and from the present 2019/2020 academic year (47 students), which has been strongly affected by the restrictions caused by the covid-19 pandemic. this year, the course started on january 28 and finished on april 30. face-to-face teaching cancellation and beginning of confinement occurred approximately halfway through the semester. the experiment with real students took place during the 2017/2018 academic year. 23 students were involved in the optional fourth-year subject "design of water treatment facilities". this subject was taught through theoretical and practical classes in the classroom and laboratory, respectively. our study focused on the theoretical part of the subject. this course corresponds to 6 ects and belongs to the chemical engineering degree in the faculty of sciences of the universidad autónoma de madrid, spain. 2.4.1 applied computing. in this subject we have expressed student performance as a 0-10 score and analyzed it across all tests performed in self-evaluation. those oa-t format tests were available from the beginning of the academic year and students used them on a continuous basis. data from this subject imply results from an adaptive test. this imply that there are some points in the test where students get stacked and some scores are repeated more often than others. we tested if data followed a gaussian distribution using a d'agostino and pearson omnibus normality test [45] . as the data did not pass the normality test, we used nonparametric statistical tests to compare the data from all 3 academic years. means were compared by a kruskal-wallis test [46] and, when differences were found, by a mann-whitney post hoc test [47] to determine which pairs of means were different. all statistical analysis was performed using graphpad prism 6 (graphpad software, la jolla, california, usa). data is presented as mean±sd and statistical significance was set at p<0.05. here, we have analysed 2 variables, the score (0-10) obtained by the students in the different activities and the proportion of students that passed the activity (score�5), from 2019/2020 and the 2 previous academic years. firstly, we checked if the results from the previous 2 years (2017/2018 and 2018/2019) were similar, thus allowing them to be compared with results from the present year. to perform the analysis, we tested the score data for normality using a d'agostino and pearson omnibus normality test [41], later comparing them with an unpaired 2-tailed t-test, if they were normal, or with a non-parametric mann-whitney test, if they were not normal. afterwards, we proceeded similarly to compare the results from the present year with those from the previous 2 years. when data from each activity followed a normal distribution, we compared the 3 means by 1-way anova followed by an unpaired 2-tailed t-test. when the data did not follow a normal distribution, we compared the 3 means with a kruskal-wallis test followed by a mann-whitney post hoc test. in order to compare the proportion of students that passed the activities (score�5) in different years, we performed a z-test (after confirming that our data met the central limit theorem). all statistical analysis was performed using graphpad prism 6. data is presented as mean±sd and statistical significance was set at p<0.05. in order to substantiate related samples of results in our experiment, we performed a wilcoxon signed rank test which compares two related samples that cannot be assumed to be normally distributed. in the experiments related to this subject, the same students were involved in both samples. data is presented as mean ±sd and statistical significance was set at p<0.05. in this section, we analyze students' self-learning strategies in the subject "applied computing" in the years that were not influenced by covid-19 (2017/2018 and 2018/2019). however, since the data is very similar in both courses, we will focus con 2017/2018 for clarity. all statistical results can be also applied to 2018/2019. in both years, theoretical lessons were taught in the classroom and practical lessons in computer laboratories with the teacher physically present. practical lessons always used the e-valuam platform and the adaptive test described previously. in fig 4a, we show the scores obtained by all 2017/2018 students in all the attempts made with adaptive tests. attempts are ordered chronologically. a score of -1 in the figure implies that the student did not finish the test and did not obtain a numerical score. those tests have been included in the figure as a part of student's autonomous work and must be considered as learning time; however, we will exclude them from score data analysis. there are some patterns in the figure that must be considered. first, certain scores repeat more than others (2.5, 5 and 7.5), which corresponds to a jump in question level (l) (level 1: 0-2.5, level 2: 2.5-5, level 3: 5-7.5 and, level 4: 7.5-10), meaning that students were not yet able to answer more difficult questions. another pattern that can be easily seen in the figure is a region where a huge number of tests had a score of -1 (between 600 and 700 approximately). on those days teachers asked the students to find the most difficult questions in the test. for that reason, students interrupted the tests when they reached those questions. however, the most interesting pattern, which can be observed in fig 4a, is the distribution of the tests in time, with a much bigger number of tests done in may, being the last month of the academic year for this subject. students used the platform 688 times in total in may and only 486 times in the period between february and april. it is worth noting that the academic year for this subject ended with the final exam on may 21. in fig 4b we show the distribution of attempts between february and may. we found 62 in february, which is a low but reasonable number since students did not yet have enough knowledge to face tests properly. they generally started using the platform in the middle of the month. in march, we noted an increase in the number of attempts followed by a slight decrease in april. this difference can be justified by the easter holidays (one week) in april. the number of attempts strongly increased in may. even when counting only the first two weeks of the month, we have more attempts than in previous months (292 from may 1-15). in the period of may 16-21, we have recorded 396 attempts (469 if we count the attempts in the final exam). in the inset of fig 4b we show the attempt distribution in the last period (may 16-21). as we can see, students used the platform 127 times the day before the exam and 173 times the day of the final exam (95 for studying and 78 in the exam itself). more than 33% of tests were performed in the last 6 days before the final exam (and more that 50% of those tests were performed the day before the exam). to study the effects of a rewarded scenario, we have developed an experiment in the academic year 2017/2018 for students from the subject "design of water treatment facilities". we have used 3 stages (fully described in the materials and methods section): stage 1, where the self-evaluation tests have been made available for 1 day; stage 2, where the self-evaluation tests have been made available for 3 weeks; and stage 3, where self-evaluation tests were available for a longer period with a reward related to their use. fig 5 shows the attempts distribution in all the stages. focusing on the ma-t, 13/20 students made more attempts in the first stage than in the second, which is striking considering that in the second stage application was available for longer. three students have not made any attempts in either of the 2 stages. as for the group of students who have used the application for more than one day, we noted only 3 individuals working for 3 days or more. considering that the application was available for 3 weeks in this second stage, these results show the very low willingness of the students to work continuously. focusing on the oa-t, this effect is even more significant, as only two cases in which students used the application for more than one day were recorded. increasing student motivation and analyzing the results of a more persistent use of the tool was the objective of stage 3. in fig 5 we can see the distribution of attempts made by the students in the whole study (3 stages) . there is a fourth stage in the figure that corresponds to an additional exam taken by the students that did not pass the previous ones. since only a few of them were involved in this fourth stage, it was excluded from the analysis. as we can see in the figure, attempts from stage 1 were limited to a couple of days, which corresponds to the short period when the test was available. some attempts can be found in a wider window in stage 2. however, a much higher number of attempts extended in time can be found in stage 3, which corresponds to the rewarded stage. in order to measure the use of the tool over time, we summarized the use of the system by students in stage 2 and 3 by comparing the number of days the students attempted the stage 2 ma-t and oa-t tests during the 3 weeks leading up to the exams given after each stage. it is interesting to note that, during stage 3, six more students utilized the system than in stage 2; however, the total attempts reduced between the 2 stages from 123 to 102. this translates to a 23% increase in the average use of the application over time from 1.9 days in stage 2 to 2.4 days in stage 3 combined with a reduction of attempts per student from 9.5 in stage 2, to 5.4 in stage 3. in the earlier stage, fewer students used the application intensively by making multiple attempts a day. students who logged in at the later stage were more likely to use the application once or twice a day over several days because of the reward related to this behavior. out of a total sample of 19 students who used the tool in stage 3, 5 were eliminated by the test as they accessed the tests the same number of days in both stages. then, for the use of the application by the remaining 14 students the w-value was of 15.5, with the critical value of w = 25 at p.0.05. therefore, the result is significant. we have studied two cases related to two different and common scenarios in covid-19 distance learning. the first one is related to subjects where teachers increased the number of tasks that students should perform autonomously. the second one is related to subjects where all the autonomous tasks were the same as in previous courses. in this case, only face-to-face sessions were replaced by distance learning activities such as online sessions or recorded classes. 3.2.1 additional autonomous activities during confinement. in fig 6 we show the scores obtained by all the students in the 3 courses analysed in all the e-valuam tests performed in the subject "applied computing". in the figure we indicate the period of the covid-19 confinement during the 2019/2020 course (from march 11 to march 30). for clarity, we also indicate the equivalent period in the two previous years. as we can see in the figure, the number of tests performed before confinement was similar (225 and 246) in the two previous years (control group), but there is a remarkable increment in the number of tests (328) performed in the experimental group. these differences can be easily explained normalizing these numbers to the number of students each year (97, 73 and 91 respectively). by doing that, we obtain 2.31, 3.37 and 3.6 test/student, respectively. thus, differences between courses 2018/ 2019 and 2019/2020 are reduced, although there is still some difference with respect students from course 2017/2018, that can be explained by the fact that some of these students were retaking the subject and, therefore, did not use the e-valuam application often until the contents were more difficult. if we count the number of tests finished until the end of the academic year in courses 2017/2018 and 2018/2019, we find 1345 and 1175 tests respectively. normalizing with the number of students, we find 13.87 and 16.1 test/student respectively. this is a difference of 2.23 test/student in a period of 3.5 months, which means a difference of less than 1 test/student each month. the conditions after the covid-19 confinement radically changed in the course 2019/ 2020 because students were required to perform a higher number of tests each week. as we can see in fig 6, there are still small differences between courses 2017/2018 and 2018/2019. however, the number of tests taken in 2019/2020 is almost 5 times higher. in the inset of fig 6 we show a histogram with the data for course 2019/2020. adaptive tests used in this experiment induce the effect of higher number of attempts at scores 2.5, 5 and 7.5, which are the points where the level of the questions increases. for that reason, the curve is not normal. this fact is statistically tested in the following analysis. when comparing the mean scores from the period before confinement (fig 7) , we found that they were statistically different between the 3 academic years. differences between the mean score from the 2019/2020 (experimental group) and both the other 2 years (control group) were . the mean scores are significantly different both in the control and in the experimental groups between both periods (before and after confinement). we have also seen an increase in the mean score differences after the confinement. this study has been performed with students from the subject "metabolism", which is a subject where no additional tasks have been imposed to students because of the covid-19 confinement. we have analysed students' scores and the proportion of students that passed the activity (score�5). as it can be observed in fig 8a, the scores obtained by students in the different activities during the course were consistently similar in both years of the control group, with the only exception in the second activity where there was a slightly decrease in year 2018/2019 (p<0.05). similarly, the proportion of students with a score�5 was very similar in the control group (p>0.05) ( fig 8b) . therefore, students' performance in the previous 2 years (control group) was not significantly different, allowing us to compare these results to the results from 2019/2020. scores obtained by the students in the 2019/2020 academic year before confinement were similar to those obtained by students from the control group, although some differences were found at the beginning of the course (activities number 2, 3 and 4, p<0.05). we think that this could be due to the adaptation of the students to the course and to the new assessment methodology, being afterwards similar again. however, after the end of face-to-face teaching and at the beginning of the confinement, students' scores were significantly higher than in the previous academic years (fig 8a) . the most remarkable differences are evident in activities 10 and 11, that have always been on-line activities. thus, in activity 10, mean score in the present year (2019/2020) was 8.1±0.2 vs. 6.5±0.2 (p<0.0001) and vs. 6.7±0.3 (p = 0.0005) in 2017/2018 and https://doi.org/10.1371/journal.pone.0239490.g008 significant differences when comparing students' performance in confinement with the performance in previous periods where activities were not limited to distant learning. at this point, it is clear that the variable that correlates with the change in students' performance is the beginning of the confinement. however, we cannot yet establish if the difference is due to either: • the new learning methodology, or • the new assessment process. for those reasons, we should now answer research question 2 and establish whether the differences found when answering research question 1 are only due to the covid-19 confinement. the problem with confinement is that not only the learning, and teaching strategies should be modified, but also the assessment process as it cannot be done face-to-face. there are some concerns related to these new assessment methods such as the opportunity for cheating by the students. this is the reason why we have chosen only subjects that include several tests that have not been modified because of the confinement. the results of our study show that students also achieved significant improvements in their scores even in tests that were performed in the on-line format in previous years. moreover, this improvement is only significant when comparing data after the confinement (i.e. there are no significant differences in on-line tests that were performed before the confinement). these findings reveal that the new assessment process cannot be the reason for the improvement in students' performance because the learners also achieved better scores when the format of the assessment did not change. for these reasons, we establish that the new learning methodology is the main reason for the change in students' performance during the confinement. now, we shall discuss research question 3 (what are the reasons of the differences (if any) in students' performance?). we have proven that something has changed in students' learning methodology. the question is: what is the common element in those methodologies that caused the improvement in the learning process? we have analyzed data from two different subjects that used two very different learning strategies during the confinement (section 3.2). in one of them (section 3.2.1), additional elearning tasks were imposed on the students. theoretical lessons were replaced by written documents. in the other one (section 3.2.2), no additional e-learning tasks were given, but theoretical lessons were replaced by multimedia classes. in both cases, we have found a significant increment in students' performance in the evaluation tests after the confinement. it seems that students' performance is increased independently of the learning strategies followed by teachers. since we have established that the assessment process cannot be responsible for the differences, and we cannot find any common elements in the learning methodologies, we must conclude on a general change in the autonomous learning process. we have also analyzed data from the previous years in two subjects that demonstrate that students do not work on a continuous basis (section 3.1). in both subjects, students work hard only in the last days before the final exams. in one subject, we have found that more than 33% of the autonomous work was done in the 5 days immediately preceding the final exam (section 3.1.1). in the other subject, students only used the e-learning material in the last 2 days before the final exam, even when it had been provided three weeks in advance (section 3.1.2). in this study, we have also found that students easily change their learning behavior and study continuously when a reward is offered. this extra motivation dramatically changed their learning strategy and students worked in a much wider time window, increasing their performance. an increase in the students' performance due to adequate time management in the learning process is well-established in the literature [12, 13] . in the present covid-19 confinement, students can find by themselves many different motivations (rewards) to work on a continuous basis. first, the confinement is a new scenario that has never been faced by the students. for this reason, students do not have any previous experience to use as a reference in their learning process. without any previous reference, students need to be confident that they are following the course correctly and therefore, work continuously in order not to miss any important content. another interpretation is that they are afraid of missing the academic year because of the covid-19 confinement and they work harder to overcome any difficulty. finally, students might be motivated by their intrinsic responsibility in a very confused situation and work hard to contribute as much as they can to solve the problems that higher education is facing. most probably, different students will find different motivations in this new scenario (probably a combination of many). we conclude that there is a real and measurable improvement in the students' learning performance that we believe can guarantee good progress this academic year despite the covid-19 confinement. answering question 4, we have demonstrated that students get better grades in activities that did not change their format after the covid-19 confinement. moreover, we have demonstrated that there is an improvement in their learning performance. in conclusion, higher scores are expected due to the covid-19 confinement that can be directly related to a real improvement in students' learning. supporting information s1 data. (xlsx) better performance of students was strongly evident when analysing the proportion of students that passed these 2 activities (fig 8b). thus, 95.6% of students passed activity 10 (p<0.0047 vs tracking e-learning through published papers: a systematic review a comparative analysis of forums and wikis as tools for online collaborative learning designing videogames to improve students' motivation investigating variation in learning processes in a future-learn mooc the mooc model for digital practice. sshrc application, knowledge synthesis for the digital economy teamwork assessment in collaborative projects through process mining techniques assessment of collaborative learning experiences by graphical analysis of wiki contributions. interactive learning environments mining theorybased patterns from big data: identifying self-regulated learning strategies in massive open online courses complexity leadership in learning analytics: drivers, challenges and opportunities analytics of time management strategies in a flipped classroom predictive power of regularity of pre-class activities in a flipped classroom covid-19 epidemic as e-learning boost? chronological development and effects at an austrian university against the background of the concept of "e-learning readiness campus traffic and e-learning during covid-19 pandemic online assessment in higher education in the time of covid-19. education in the knowledge society e-learning during covid 19 pandemic comparing students' self-discipline and self-regulation measures and their prediction of academic achievement investigating self-regulation and motivation: historical background, methodological developments, and future prospects the metacognitive control components of self-regulated learning student differences in self-regulated learning: relating grade, sex, and giftedness to self-efficacy and strategy use using formative assessment to influence self-and coregulated learning: the role of evaluative judgement theorizing and researching levels of processing in self-regulated learning developing web-based assessment strategies for facilitating junior high school students to perform self-regulated learning in an e-learning environment applying a web-based training to foster self-regulated learning-effects of an intervention for large numbers of participants. the internet and higher education comparing online and blended learner's self-regulated learning strategies and academic performance. the internet and higher education the authors want to thank dr. gema perez-chacón for her valuable comments on statistical analysis. conceptualization: santi fort. the aim of this study was to identify the effect of covid-19 confinement on students' performance. therefore, we conducted an experiment among 450 students from three subjects in different degrees of higher education at universidad autónoma de madrid, spain. the results of this study answer our 4 research questions:1. is there any effect (positive or negative) of covid-19 confinement on students' performance?2. is it possible to be sure that covid-19 confinement is the origin of the different performance (if any)?3. what are the reasons of the differences (if any) in students' performance?4. what are the expected effects of the differences in students' performance (if any) in the assessment process?with respect to research question 1, the results analyzed in section 3.2 show that there is a significant positive effect of covid-19 confinement on students' performance. the results indicate that students obtained better scores in all kinds of tests that were performed after the beginning of confinement. different sources of error have been removed from our study by including only subjects that fit the following requirements for the last three academic years:• same teaching methodology and teachers in all the years.• same assessment process in all years, including both distance and on-site activities.• tests performed both before and after the covid-19 confinement in the present academicyear.• tests that only imply an objective assessment process in order to avoid any possible subjective interpretation by the lecturers.we have compared results of students from the present 2019/2020 academic year (experimental group) with results of students from the last two academic years 2018/2019 and 2017/ 2018 (control group). students from the two previous academic years had similar circumstances from the beginning to the end of their course. when compared to students of the 2019/2020 academic year, we have two periods with different conditions. the first period is taught in the same conditions as in previous years (before confinement). in the second period, those conditions dramatically changed, and all the teaching and learning activities were limited to distance learning. the results of our studies (section 3.2) clearly indicate that there are key: cord-335245-1eksm537 authors: pattyn, els; verhee, annick; uyttendaele, isabel; piessevaux, julie; timmerman, evy; gevaert, kris; vandekerckhove, joël; peelman, frank; tavernier, jan title: hyperisgylation of old world monkey isg15 in human cells date: 2008-06-18 journal: plos one doi: 10.1371/journal.pone.0002427 sha: doc_id: 335245 cord_uid: 1eksm537 background: isg15 is an ubiquitin-like protein, highly induced by type i interferons. upon the cooperative activity of specific ubiquitinating enzymes, isg15 can be conjugated to its substrates. increasing evidence points to a role for protein isgylation in anti-viral and anti-tumoral defense. principal findings: we identified isg15 from old world monkeys (owm) as a hyper-efficient protein modifier. western blot analysis visualized more efficient conjugation of owmisg15 relative to huisg15 in human (hu), monkey and mouse (mo) cell-lines. moreover, the substrates of owmisg15 identified upon tandem affinity purification followed by lc-ms/ms identification largely outnumbered these of huisg15 itself. several ubiquitin-conjugating enzymes were identified as novel isgylated substrates. introduction of a n89d mutation in huisg15 improved its isgylation capacity, and additional q31k/t33a/d133n mutations yielded a huisg15 variant with an isgylation efficiency comparable to owmisg15. homology modeling and structural superposition situate n89 in the interaction interface with the activating enzyme. analysis of the ube1l residues in this interface revealed a striking homology between owmube1l and huube1, the activating enzyme of ubiquitin. in line with this observation, we found efficient activation of agmisg15, but not huisg15 or moisg15, by huube1, thus providing a likely explanation for owm hyperisgylation. conclusions: this study discloses the poor conjugation competence of huisg15 compared to owmisg15 and maps the critical determinants for efficient conjugation. hyperisgylation may greatly assist isgylation studies and may enhance its function as positive regulator of interferon-related immune responses or as anti-tumoral modulator. type i interferons (ifn)s are involved in host defense mechanisms, particularly against viral infections. they induce a so-called ''antiviral state'' by inducing both cytosolic and nuclear events. ifn stimulated gene 15 (isg15) is an ubiquitin (ub)-like molecule (ubl), highly induced upon both type i and type ii ifn treatment [1] . it is expressed as a 17 kda protein, containing 2 ub domains and a c-terminal oligopeptide (eg. octapeptide in human, hexapeptide in mice). maturation of isg15 includes n-terminal met excision [2] and removal of the c-terminal peptide giving a 15 kda protein [3] . maturated isg15 can then be conjugated via an isopeptide binding to the e-amino group of a lys residue in the target protein [4] . alternatively, the processed 15 kda isg15 molecule can be secreted and exerts immunoregulatory functions on peripheral blood lymphocytes [5] . conjugation of ub(l) requires the cooperative activity of at least 3 enzymes. the ubiquitination cascade is initiated by an ub-activating enzyme (termed uba, ube or e1) adenylating the cterminus of ub(l), thereby forming an acyl-phosphate linkage with amp. the catalytic cys residue in ube1 subsequently attacks this high energy bond, forming a thiolester bond to the c-terminal gly of ub(l). in humans, ub molecules are found to be activated by ube1 (also known as a9s1) [6] or ube1l2 (also named uba6) [7, 8] , isg15 by ube1l [9] , sumo by aos-uba1 [10] and nedd8 by appbp1-uba3 [11] . ub(l) molecules thiolester-linked to its ub-activating enzyme are transferred to a ub-conjugating enzyme (termed ubc or e2), also by a thiolester linkage on a cys residue. ubch8 has been identified as a major ub-conjugating enzyme effecting isg15 conjugation [12, 13] . around 400 proteins are recognized as ub-ligases or e3s. roughly, they can be discerned as ring (really interesting new gene)-finger proteins, acting as a molecular scaffold, and hect (homologous to e6-ap c-terminus)-domain proteins, which also exert a catalytic contribution. ub-ligases confer specificity, and place the ub or ubl molecule in close proximity to the lys residue of the substrate. the formation of polyubiquitin chains is a process mediated by the ub-conjugating enzyme together with the ub-ligase. recently, the ifn-induced herc5 has been identified as an isg15 e3-ligase in human cells [14] . the estrogen-response finger protein (efp), also an ifn-induced protein, functions as an e3-ligase for isgylation of 14-3-3d [15] . definitely, these recently discovered isg15-ligases are only the onset of a more extensive list. as ub, most ubls are synthesized as inactive precursors, being processed by de-ubiquitinating enzymes (dubs) exposing the mature protein with a c-terminal gly residue. dubs not only exert their function by protein processing, they also have a function in removal of the ub(l) from their substrate. ub specific protease 2 (usp2), usp5 (also named isopeptidase t), usp13 (isot3) and usp14 have been identified as proteases with a dual specificity for ub and isg15 [16, 17] . usp18 (also named ubp43) specifically cleaves isg15 [18] . of note, usp18 also competes with jak1 for binding to the type i ifn receptor ifnar2 [19] . isg15 is upregulated upon viral infection. its role in antiviral defense is underscored by viral mechanisms to counteract isg15 function. for example, the influenza b non-structural ns1b protein binds isg15, thereby preventing its association to ube1l [9] . also the papain-like protease of the severe acute respiratory syndrome (sars) coronavirus, counteracts isg15 functioning by removing it from its substrate [20] . the hepatitis c virus ns3/4a protease cleaves ifn promoter-stimulator-1 (ips-1) leading to reduced isg15 expression and conjugation [21] . in a more direct way, ectopic expression of isg15 in cells lacking a functional ifn response reduces newcastle disease virus and influenza virus replication [22] , and also overcomes fatal intracerebral infection in ifnar 2/2 mice by sindbis virus [23] . isg15 knock-out mice proved to be more sensitive to influenza, herpes and sindbis viral infection [24] . an inverse dose-response correlation of exogenously expressed isg15 and release of human immunodeficiency virus (hiv) virions is also found and suppression of isg15 expression by sirna counteracts ifn-mediated inhibition of hiv virion release [25] . the precise mode of action of isg15 is not yet established. isgylation is believed to counteract ubiquitination by competing for the same lys in substrates [26] . studies with ubch6 and ubch13 showed that isgylation of these e2s hampered their ability to form a thiolester intermediate with ub [27, 28] . this competition may protect substrate proteins from proteasomal degradation. we here report the surprising finding that isg15 from old world monkeys is a far more efficient protein modifier than its human counterpart in human, monkey and mouse cells. we identified novel isgylation substrates and mapped the critical residues in this isgylation process. this hyperisgylation competence of agmisg15 was validated by an alternative activation mechanism. these findings will be useful for a better understanding of isg15 biology. viral resistance strongly varies between species. in this respect, old world monkeys (owm)s receive much attention by their remarkable hiv resistance. they are distinguished from apes (here represented by the chimpanzee) in that most have tails and are distinguished from new world monkeys (not included in this study) in that their tails are never prehensile. we compared isg15 sequences from hominid origin with those of owm, which diverged some 25 million years ago, and of murine origin, which diverged from primates approximately 75 million years ago [29] . isg15 from different origins were aligned to two ub molecules ( figure 1 ). the nand c-terminal part of isg15 share respectively 32 and 37% sequence identity with ub. the isg15 variability largely exceeds what can be expected by genetic drift alone [2] . we isolated isg15 cdnas of humans (allelic variant with n at position 83 and s at position 94) (homo sapiens, hereafter huisg15), chimpanzee (pan troglodytes, cpzisg15), african green monkey (cercopithecus aethiops, agmisg15), rhesus monkey (macaca mulatta, rhmisg15) and mouse (mus musculus, moisg15). all were cterminally cloned after a flag recognition tag. huisg15 and cpzisg15 only differ by one amino acid (respectively a ser and asn residue at position 21) and represent hominid isg15 (homisg15). agmisg15 and rhmisg15 differ from each other by two amino acids (respectively a ser and asn residue at position 21 and arg and lys at position 77), and are both part of the old world monkeys (hereafter referred to as owmisg15) (see table 1 ). total isgylation patterns were visualized by western blot analysis on total cell lysates. as can be seen in figure 2a , agmisg15 and rhmisg15 outperform homisg15 and moisg15 in its isgylation capacity in human hek293t cells. moisg15 also conjugates more favorably to human proteins compared to huisg15, albeit not to the same extent as owmisg15. to rule out the possibility that conjugation of ectopic huisg15 could be hindered by competition with endogenous isg15, the same blot was stripped and reprobed with an antibody recognizing hu and owmisg15, showing essentially the same results (data not shown). similar findings were obtained in monkey cos cells and mouse n38 cells (resp. figure 2b and figure 2c ). note that all these experiments were performed without extra stimuli (e.g. ifn treatment) or co-transfection of any ubiquitinating enzyme meaning that in all tested cell-lines, target isgylation by agmisg15 could be achieved by endogenously available ubiquitinating enzymes. figure 1 . isg15 orthologues show a low degree of amino acid conservation. isg15 from different species were aligned with two molecules of ubiquitin using clustalx [61] . sequence conservation is visualized using the amas server [62] with a conservation threshold of 7. residues with a conservation above this threshold are shown on a grey background, residues that are identical in all sequences have a black background. doi:10.1371/journal.pone.0002427.g001 we wanted to address whether the observed differences in isgylation capacity was only quantitative or also qualitative. therefore, tandem affinity purification (tap) experiments were performed to identify isgylated substrates by hu and agmisg15. to this end, a tandem proteina binding domain and flag-tag, separated by a tobacco etch virus protease cleavage domain was fused n-terminally to hu or agmisg15. human fibroblast 2ftgh cells were stably transfected with these constructs, and 2 cell clones expressing tap-tagged huisg15 or tap-tagged agmisg15 with comparable expression levels were selected (see figure s1 ). to rule out experimental variation, this experiment was also performed in transiently transformed hekt cells with either tap-tagged huisg15 or agmisg15. as shown in table 2 , the tap experiments identified substantially more isgylation substrates of agmisg15 compared to huisg15 in human celllines. details of the identified peptides can be found in table s1 . in addition to an overlap with the already described isg15 substrates [30] [31] [32] [33] [34] , we here report the ub-conjugating enzymes ubch10, ubch16 are ubch17 as novel isgylation substrates of agmisg15. the cdnas of the ub-conjugating enzymes ubch6, 7, 8, 10, 13, 16 and 17 were isolated by rt-pcr from 2ftgh cells and linked to a c-terminal v5-tag. these constructs were transfected in hekt cells together with either a mock construct, huisg15 or agmisg15. ubch7 was included as a control, as it was neither in our experiments nor in others identified as a substrate for isgylation. western blot analysis on total cell lysates containing b-me confirmed isgylation of ubch10, h13 and h17 with agmisg15 but not with huisg15, as seen by a 15 kda shift upon staining with anti-v5 ab detecting the ectopic expressed ubch proteins (figure 3a ,b and figure s2a ). in addition, co-immunoprecipitation experiments were performed by binding isgylated proteins through isg15's flag-tag to an anti-flag affinity gel. also here, the isgylation of ubch10, h13 and h17 by agmisg15 could be established ( figure 3c ). worth mentioning, when the co-immunoprecipitated samples were obtained in a more concentrated way, a faint band of co-immunoprecipitated ubch13 and ubch17 could be seen with huisg15 ( figure s2b ), hinting at a qualitative rather than quantitative difference in isgylation between hu and agmisg15. of note, all the experiments were performed without cotransfection of ube1l and/or ubch8 or ifn stimulus. previous studies using western blot analysis in hekt cells revealed hu or moisg15 conjugation to substrates such as ubch13 only upon co-transfection of at least ube1l -and generally also ubch/m8or upon ifn stimulation. moreover, in most cases an additional immunoprecipitation or pull-down purification step was required [27, 28, [30] [31] [32] [33] . alternatively, experiments were performed in usp18-deficient murine embryonic fibroblasts [30, 34] . we next replaced the differing amino acids in huisg15 by their agmisg15 counterparts (see figure 1 and 4). four of these residues (i.e. residues 89, 113, 114 and 133) are situated near the interaction interface between isg15 and its activating enzyme, ube1l, predicted by narasimhan et al. [35] (red highlighted in figure 4 ). the n83s and s94n allelic variants of huisg15 were also included. both conjugation to various ubchs and total isgylation patterns of different huisg15 mutants were tested. the effect of mutating huisg15 residues situated near the predicted ube1l interface and the different allelic variants on conjugation to ubch proteins is shown in figure 5a and s3a. strikingly, the single huisg15 n89d variant displayed a greatly enhanced isgylation efficiency. no effect was seen for all other mutants, including the n83s or s94n human allelic variants. note that also in this experiment the cell lysates were boiled in sds loading buffer containing b-me. the slower migrating band differs 15 kda from the unconjugated form of the ubchs, and is thus the result of isg15 bound by an isopeptide linkage, not by a thiolester bond. as with agmisg15, no co-transfection of any activating or conjugating enzyme was required to visualize its conjugation by western blot analysis on total cell lysates in hekt cells. a more detailed study including all differing residues (and also the n89e variant as it occurs in mice), demonstrated that only mutation at position 89 affected the total isgylation pattern. however, the effect of this mutation is only intermediary as compared with agmisg15 (see figure 5b ). we next combined the huisg15 n89d variant with additional mutations. as shown in figure 5c , mutation of d133n and qit31-33kia in the huisg15 n89d variant further enhanced its isgylation in human hekt cells. finally, the triple huisg15 mutant n89d, d133n and qit31-33kia displayed an isgylation efficiency comparable to the monkey orthologues (see figure 5d and s3b). the interaction interfaces of nedd8 bound to its activating enzyme, appbp1-uba3, [36] and sumo bound to sae1-sae2 [37] have recently been mapped and show a remarkable high degree of similarity. we built a similar homology model for the ube1l-isg15 complex (figure 6a ), based on the crystal structure of the appbp1-uba3-nedd8-atp complex [38] . this superposition brings the critical residue 89 in isg15 in the interaction interface with ube1l. therefore, we compared the sequences of hu and owmube1l in their predicted interfaces with isg15 and found an interaction region displaying significant substitutions (i.e. the sequence 563-gtsgtwg-569 in huube1l corresponding to 560-gtlgtrg-566 in owmube1l). residue w568 in huube1l juxtaposes to isg15 n89 (figure 6a ). this residue is substituted with an arg residue in owmube1l. strikingly, the corresponding sequence in owmube1l shows high similarity with huube1 ( figure 6b ). this observation prompted us to test whether agmisg15 could be activated by huube1. we performed an in vitro assay analyzing the loading of either huisg15 or agmisg15 with huube1 or huube1l. thiolester formation of both isg15 orthologues with huube1l was observed, as expected. importantly, as anticipated from the sequence similarity, huube1 was also found to be able to form thiolester bonds with agmisg15, whilst no loading of huisg15 could be seen (figure 7a ). the thiolester nature of the bond was verified by addition of b-me. western blot analysis evaluating the total isgylation pattern further confirmed this alternative activation pathway for agmisg15 ( figure 7b for the experiment in hekt cells, figure 7c in cos cells). the cells were transfected with combinations of an empty vector or vectors encoding huube1l, huube1 and ubch8 together with flagtagged huisg15, agmisg15 or moisg15. visualising the isgylation pattern on the different samples only observed significantly enhanced isgylation by hu and moisg15 upon ectopic expression of ube1l. a beneficial effect on hu/ moisgylation efficiency upon ectopic expression of ube1 was minimal. by contrast, even with the higher basal isgylation level of agmisg15, overexpression of both ube1l and ube1 were able to further markedly enhance its conjugation capacity. the isg15 gene emerged upon the duplication of an ub dimer and insertion in an ifn-controlled area [39] . in sharp contrast with ub itself, of which 72 of the 76 amino acid are invariant between animals, plants and fungi [40] , isg15 is not well conserved between species [2] . this variability in isg15 sequence may indicate a redundant function of isg15. alternatively, it could point towards a role for isg15 in the immune system, since high interspecies sequence divergence is very often associated with genes involved in host defense. organism-specific niche occupation may lead to distinct repertories of invading pathogens causing species-specific pressures for adaptation of the host immune system [41, 42] . this increased evolutionary rate in immune genes may be further enhanced by the high mutation rates of the pathogens [43] . in line with the assignment of a specific role for isg15 in host defense, naturally occurring loss-of-function isg15 mutants have not yet been observed sofar, whereas a duplication of the isg15 gene has been seen in crucian carp [44] . although the precise function of isg15 remains enigmatic, several lines of evidence point to its role as an antiviral agent. isg15 is one of the most prominently induced genes upon type i ifn treatment, and several viruses such as influenza b and sars have developed mechanisms to counteract isg15 function [9, 20] . isg15 was also found to be critical for the ifnmediated inhibition of hiv replication and release [25] . moreover, although isg15 knock-out mice were initially found to be as vulnerable to viral infections as their wild type countermates [45] , a subsequent study discovered isg15 knock-out mice to be more vulnerable to influenza, herpes and sindbis viral infection [24] . a key finding of this report is the significant difference in isgylation capacity between isg15 orthologues. in human cells, protein modification by isg15 of owm greatly outperforms that of hu and moisg15. mutation of the residues in huisg15 to the corresponding residues in the owm orthologue mapped amino acid 89 as a critical residue for isgylation. the occurrence of an acidic residue (preferably an asp in human cells and glu in mouse cells) at this position greatly enhances its isgylation capacity. the asp residue at position 89 in owmisg15 also occurs in sheep and cows. in rodents and fish, this residue is a glu residue. the incidence of the unfavorable asn residue at position 89 for isg15 modification in huisg15 is also found in chimpanzee and dog isg15. however, notwithstanding the greatly enhanced isgylation capacity by mutation of the single asn 89 residue in huisg15 to an asp residue, the overall isgylation pattern in human hekt cells was still intermediary compared to agmisg15. additional mutations were created in the huisg15 n89d mutant. mutation of d133n and qit31-33kia further increased its isgylation capacity. the triple huisg15 mutant n89d d133n qit31-33kia proved to be as effective as agmisg15 in conjugating target proteins in hekt cells. the use of this hyperefficient isg15 variant may help to unravel the physiological function of isg15. the ub(l) modification process is initiated by its dedicated activating enzyme, a crucial step which is also described to dictate selectivity. ub-activating enzymes are composed of three modular domains: an n-terminal domain which targets atp and ub(l)binding (residues 1-595 in ube1l), a second domain harboring the catalytic cys residue (embedded in residues 596-880) and an c-terminal ubl-fold domain (residues 914-1013), which selects for and binds to the cognate ub-conjugating enzymes [8, 46] . based on interaction interfaces of nedd8 bound to appbp1-uba3 [36] , narasimhan and colleagues predicted 7 hot spot residues on isg15 constituting the interface with ube1l. three of these residues show a high degree of conservation among the different ub(l)s (i.e. r92, e132 & r153 in isg15), 4 other residues are presumed to confine specificity for ube1l (r87, k90, w123 & f149) [35] . we built a similar homology model for the ube1l, using the crystal structures of the appbp1-uba3-nedd8-atp complex. structure superposition shows that residue 89 corresponds to l8 in nedd8 (figure 6a) , a well-conserved residue that is also found in ub (figure 1 ). mutation of l8 in ub significantly reduced its target conjugation ability [47] , and in yeast this residue proved to be essential for viability [48] . this suggests that residue 89 in isg15 and l8 in ub have a similar important role in conjugate formation. l8 in nedd8 is part of a hydrophobic patch (l8, i44, v70) that interacts with v323 and y331 of uba3, embedded in the two b-sheets preceding its cterminal domain. mutation of these residues greatly reduced nedd8 adenylation and consequently conjugation [36] . however, residues l8, i44 and v70 in nedd8 correspond with the nonhydrophobic residues n89, t125 and n151 in isg15, suggesting different types of interaction in this region. the two uba3 bstrands correspond to the region 880-895 in the ube1l model, but the alignment in this region is too poor to allow reliable modeling of the actual interactions. moreover, residues 152-lrl-154, which are part of the c-terminal region of isg15 juxtapose to n89 (figure 4 ). this region aligns with 71-lrl-73 in ub which was described to be of high importance for ube1 binding affinity and for substrate specificity [49] . our model suggests that residue 89 makes a contact with r565 in owmube1l, which lies in the n-terminal domain, known to select for a specific ubl [36, 37] . in huube1l, this arg is replaced by a trp. strikingly, the sequence 560-gtlgtrg-566 in owmube1l is very similar to the corresponding region 602-gtlgtkg-608 in huube1 (see also figure 6b ). based upon this observation, we experimentally demonstrated that huube1 could efficiently activate agmisg15, in contrast to hu and moisg15. this non-redundant role for ube1l in activating moisg15 is also in line with the loss of moisg15 conjugation in ube1l knock-out mice [50] . unlike ube1l, which is ifn-inducible and has a tissue and cell-line specific expression profile [51] , ube1 is ubiquitously expressed. taken together, our findings support a role for residue 89 in isg15 as a hot spot residue in the interface with its activating enzyme, thus providing an explanation for the hyperisgylation seen for owmisg15. recently, with the discovery of ube1l2 (uba6) as the activating enzyme for fat10 and as a second activating enzyme for ub [7, 8] , the theory of an activating enzyme harboring unilateral assignment for a specific ub(l) was overthrown. here, we describe promiscuity of ube1 for owmisg15 activation. this is the first report of isg15 being activated by another activating enzyme than ube1l. moreover, to the best of our knowledge, this is also the first report of an ublother than ubiquitin-being activated by ube1. in contrast, homology modeling and structural superpositions do not predict a direct role for residue d133 or residues qit31-33 of isg15 in the activation step (figure 4 and 6a) . the d133 residue is part of a ridge of negatively charged residues along the isg15 molecule [35] . the function of this ridge is unclear. d133 in huisg15 corresponds to a negatively charged residue in ub, sumo, nedd8, rub1, apg12 (all asp residues) and urm1 (occupied by a glu residue). models based on the appbp1-uba3-nedd8-atp complex or the sae1-sae2-sumo-mg-atp complex, do not predict direct contact of residue 133 in isg15 with ube1l. the same hold true for residues 31-33. this suggests that mutations figure 6 . residue 89 in isg15 is situated in the predicted contact area with its activating enzyme. (a) isg15 (red ribbons) overlaid to nedd8 (green ribbons) bound to its activating enzyme appbp1-uba3 (grey ribbons). residue 89 (dark blue) of isg15, makes contact with residues in the two b-strands (shown in brown) of the activating enzyme that precedes its c-terminal domain. k193 in uba3 (orange) is located very close to n89, and corresponds to w568 in human ube1l. the other residues of isg15 with an important effect on total isgylation, situated at positions 133 (yellow) and 31-33 (cyan blue), do not make contact with ube1l and are likely to be involved in another feature of the isgylation process. (b) huube1l was aligned with huube1 and rhmube1l (ensembl peptide sequence en-smmup00000027609). the region 563-569 in huube1l shows two substitutions (s565l and w568r, red coloured) in the corresponding region of rhmube1l, which better resembles the corresponding region in huube1 (k607). doi:10.1371/journal.pone.0002427.g006 at position 31-33 and 133 influence total isgylation by a different mechanism than mutation at position 89. this must occur later in the isgylation process, since no effect of these mutations can be observed in the absence of the n89d mutation. of note, notwithstanding their position on different ub domains in isg15, residues 31, 33 and 133 are situated at the same face of the molecule (see figure 4 ) close to the electronegative ridge. as the total protein isgylation results from the balance between conjugation and deconjugation, mutation of qit31-33 to kia and d133 to n in huisg15 n89d conceivably could affect recognition or activity by (a) discriminate dub(s), retaining the targeted protein in the isgylated state. in line with this hypothesis, a role has been described for the n-terminal ub fold of isg15 in the efficiency of dub recognition/activity of the sars coronavirus papain-like protease [52] . in this study we also present the ub-conjugating enzymes ubch10, h16 and h17 as new isgylation targets. ubch10 (ube2c) acts as an ubch for the anaphase promoting complex (apc), promoting cyclina degradation and mitotic exit. during the g1 phase, ubch10 is auto-ubiquitinated allowing reaccumulation of cyclina and entry in the s phase [53] . ubch10 is often referred to as the cancer-related ubch as it is markedly overexpressed in the majority of cancerous cell lines [54] . recently, also ubch16 (e2-25k or hip2) has been identified as an apc-dependent ubch promoting ub k48-linked chain extensions on pre-attached ubs [55] . ubch17 (ube2t) has recently been identified as the ubch essential to ubiquinate fancd2, which needs to be ubiquitinated in order to bind brca2 and take part in the dna repair process. ubch17depleted cells have abnormal chromosomes, characteristic for in vitro assay revealing the capability of agmisg15 to form thiolester bonds with ube1, contrary to huisg15. 2.5 mm ubch8 was incubated with 200 nm of either ube1 or ube1l, and 7.5 mm of either mature hu-or agmisg15 under conditions as described in materials and methods. the samples were divided in two aliquots; and were subjected to sds-page under non-reducing or reducing conditions. the proteins were stained with irdye blue protein stain and visualized using the odyssey infrared imaging system. the extra band representing thiolester formation between ube1 and agmisg15 is flanked by two asterisks. these thiolester bonds are disrupted by the addition of reducing agent (here b-me). (b) ube1 can efficiently activate agmisg15, but not hu or moisg15. hekt cells were transfected with an empty vector, or combinations of vectors encoding huube1l, huube1 and ubch8 together with huisg15, agmisg15 or moisg15 as indicated. the total isgylation patterns were evaluated by western blot analysis using the flag tag of the isg15 proteins. fanconi anemia [56, 57] . these findings may help explain the role for isg15 in anti-tumor defense. in conclusion, we report that owmisg15 more efficiently isgylates proteins compared to human isg15. it remains to be determined whether this reflects merely quantitative or also qualitative differences in the role of isg15 among species. as isg15 is implicated in antiviral protection against various viruses including sars, influenza, hepatitis c and hiv, these differences in isg15 conjugation efficiency may help explain human sensitivity to various viruses. human isg15 (allelic variant with n at position 83 and s at position 94) was isolated from cdna of hek293t cells, african green monkey isg15 from cdna of vero cells and mouse isg15 from cdna of l929sa cells. all huisg15 mutants as well as the other human allelic variants (n83s and s94n) were created through site-directed mutagenesis (stratagene). chimpanzee isg15 was obtained by site-directed mutagenesis of huisg15 (s21n mutation), rhesus monkey isg15 was obtained by 2 consecutive site-directed mutations on agmisg15 (s21n and k77r mutation). all cell-lines were cultured in a 8% co2 humidified atmosphere at 37uc and grown in dulbecco's modified eagle's medium (invitrogen) with 10% fetal calf serum (cambrex corp.). for standard isgylation experiments in hek239t cells, 3,5610 5 cells were seeded the day before transfection in 6-well plates and transfected for 6 h using a standard calcium phosphate precipitation procedure. typically 0.75 mg of isg15variant, if indicated supplemented with 0.25 mg e1 and 0.5 mg e2 or, applicable supplemented with 0.75 mg substrate was used in a total volume of 250 ml dna/capo4-transfection mixture. fugenehd (roche) was used for transfection in cos cells and lipofecta-mine2000 (invitrogen) for transfection in n38 cells. since fugenehd induces a minimal ifn response, this reagent was exceptionally used for the hek293t transfections in figure 7b . however, no differences could be seen between this transfection reagent and the calcium phosphate precipitation method regarding the isgylation difference between hu and agmisg15. tap purification experiments were performed in 2ftgh cells stably expressing either hu or agmisg15 and hekt cells transiently expressing either hu or agmisg15 with an n-terminal tap-tag. single colonies of the 2ftgh cells were selected upon co-transfection of the isg15 constructs with a puromycin resistance marker and selection on 3 mg/ml puromycin. in order to check the expression levels of the tap-isg15 proteins compared to the endogenous isg15 levels, the different stable cell-lines were stimulated for 26 h with ifnb (peprotech) prior to western blot analysis. two days after transfection, cells were lysed with 150 ml 26 sds loading buffer (62 mm tris hcl ph 6.8, 2% sds, 8% glycerol, 5% b-me, 0.01% brome phenol blue) and homogenized on a qiashredder mini spin column (qiagen). 35 ml of the boiled lysate was loaded on a sds-polyacrylamide gel. blotting efficiency was checked using ponceau s staining (sigma). the precision plus protein standard all blue (biorad) was used as molecular weight marker. flag-tagged proteins were revealed using a 1/8000 dilution of anti-flag m2 mouse monoclonal ab (sigma), v5-tagged proteins by 1/5000 dilution of anti-v5 mouse monoclonal ab (invitrogen). hu and owmisg15 could be detected by an anti-human isg15 rabbit polyclonal ab (abcam). human isg15 was also detected with the anti-human isg15 mouse monoclonal ab, a generous gift from dr. e. borden. anti-human actin rabbit polyclonal ab (sigma) 1/3000 diluted was applied as control loading. either goat anti-mouse irdyeh 800cw or anti-rabbit irdyeh 680 (li-corh biosciences) was used as secondary ab. targeted proteins on the blots were visualized using the odysseyh infrared imaging system (li-corh biosciences). the 2ftgh or hek293t cells expressing either tap-tagged agmisg15 or huisg15 were lysed in cell lysis buffer (50 mm tris-hcl ph 8, 10% glycerol, 1% np40, 150 mm nacl, 5 mm naf, 5 mm zncl 2 , 1 mm na 3 vo 4 , 10 mm egta, complete tm protease inhibitor cocktail (roche)). the insoluble fraction was spun down and the supernatant was incubated with igg sepharose (amersham biosciences) overnight. the beads were washed three times with washing buffer (2 mm tris-hcl ph 7.5, 5% glycerol, 0.1% np40, 150 mm nacl) and twice with tev (tobacco etch virus) protease cleavage buffer 1 (10 mm tris-hcl ph 8, 150 mm nacl, 0.1% np40, 0.5 mm edta) and were then incubated with tev protease in tev protease cleavage buffer 1 for 2 hours. the beads were then spun down and the supernatant was incubated with anti-flag agarose (sigma) in tev protease cleavage buffer 2 (10 mm tris-hcl ph 8, 150 mm nacl, 0.1% np40) for 2 to 4 hours. the anti-flag agarose beads were washed three times with washing buffer and incubated with 250 mg/ml flag peptide in flag elution buffer (2 mm tris-hcl ph 7.5, 5% glycerol, 150 mm nacl) for 10 min to elute for the flag-tagged proteins. the resulting peptide mixture was precipitated by addition of tca to a final concentration of 10%. it was incubated overnight, centrifuged and washed with ice-cold acetone containing 0.05n hcl and dried. pellets were re-dissolved in 3 ml 50 mm nh 4 hco 3 (ph 7.9) containing 8m ureum for 20min periodic vortexing, 21 ml 50 mm nh 4 hco 3 (ph 7.9) was added in aliquots to lower the final concentration of ureum to 1 m. the resulting peptide mix was digested in solution by trypsin and applied for nano-lc-ms/ms analysis as described before using a waters q-tof mass spectrometer [58] or a bruker esquire hct ion trap mass spectrometer [59] . the generated peptide fragmentation spectra were searched using the mascot database search engine (http://www. matrixscience.com) in the swissprot database (taxonomy was set to human). the following mascot parameters were set. the enzyme setting was trypsin with a maximum of 1 missed cleavage allowed. variable amino acid modifications that were allowed are acetylation (n-term), carbamylation (lys and n-term), deamidation (asn and gln), formation of pyroglutamate (n-terminal gln), oxidation of met to its sulfoxides and propionamide modification of cys. allowed peptide and fragment ion mass tolerance were 0.3 da (q-tof) or 0.5 da (ion trap). mascot's instrument setting was ''esi-quad-tof'' (q-tof) or ''esi-trap'' (ion trap) for calculating theoretical peptide fragmentation spectra. following database searching, only spectra that exceeded the corresponding mascot's threshold score for identify (set at the 95% confidence level) are here reported. the matching estimated false positive discovery rate is well acceptable and is only between 2 to 4% at the individual spectrum level as previously assessed [60] . co-immunoprecipitation procedure 2610 6 hek293t cells were transfected with the indicated expression vectors. cleared lysates (modified ripa lysis buffer: 200 mm nacl, 50 mm tris-hcl ph 8, 0,05% sds, 2 mm edta, 1% np40, 0,5% doc, complete tm protease inhibitor cocktail (roche)) were prepared two days after transfection. the samples were incubated with anti-flag m2 affinity gel (sigma). after immunoprecipitation, sds-page and western blotting, interactions were detected using an anti-v5 antibody (invitrogen) production and purification of hu and agmisg15 proteins mature isg15 (with removed c-terminal peptide) was cloned in the ptyb1 vector and purified with the impact tm procedure (intein mediated purification with an affinity chitin-binding tag) (new england biolabs) when the cultures reached an od600 of 0.6, isopropyl b-d-thiogalactopyranoside (iptg) was added to a final concentration of 0.3 mm, and were grown at 15uc overnight. the next day, cells were removed from the medium by centrifugation. the cell pellets were resuspended in ice-cold cell lysis buffer (20 mmtris ph 7.5, 500 mm nacl) and disrupted mechanically by sonication. after removal of cell debris by centrifugation, the clarified lysate was loaded onto a chitin column (equilibrated with 10 volumes of the column buffer (20 mm tris, 500 mm nacl, 1 mm edta, ph 9.0). three bed volumes of the cleavage buffer (20 mm tris, 1 mm edta, 50 mm dtt, ph 9.0) were loaded onto the column, and incubated overnight. the isg15 protein was eluted from the column using twice 4ml column buffer and dialysed against 3 times 5 l pbs overnight. the concentration of the protein was determined by the bradford assay. 30 ml was loaded on a 12% sds page gel and silver stained to check the quality and purity of the isg15 proteins. purified recombinant ube1, ube1l, ubch8, ubch10 and ubch13 were purchased (boston biochem). the in vitro thiolester reactions were performed based on the isg15 conjugation initiation kit (boston biochem). all samples were prepared in 20 ml containing 50 mm hepes ph 8.0 and 100 mm nacl. the concentration of the recombinant proteins is indicated in the figure legend figure s1 the selected stable 2ftgh cell clones express comparable levels of the tap-tagged isg15 orthologues. 2ftgh cells were stably transfected with the indicated tap-tagged constructs as described in materials and methods. four clones were selected for the tap experiments, 2 clones expressing taptagged huisg15 and 2 clones expressing tap-tagged ag-misg15. one cell-line stably expressing tap-tagged prmt (protein arginine n-methyltransferase) was used as a control (lane 2). the different cell-lines were seeded at the same density in 12well plates. 16 h after seeding, the cell-lines were stimulated with ifnb(1 ng/ml). one non-stimulated (ns) control was included (lane 1). 26 h after ifnbtreatment, cell lysates were prepared and separated by sds page. (a) western blot using anti-flag ab, revealing the tap-tagged constructs. open arrow indicates the ectopic expressed tap-tagged isg15 constructs. the prmt control construct is a bigger protein. (b) western blot using anti-huisg15 ab (gift of dr. e borden). open arrow indicates the ectopic expressed tap-tagged isg15 constructs (note the weaker cross-species recognition of agmisg15 by the antibody). closed arrow indicates the induced endogenous isg15 as a result of the ifn stimulation. interferon induces a 15-kilodalton protein exhibiting marked homology to ubiquitin isg15: a ubiquitin-like enigma precursor processing of pro-isg15/ucrp, an interferon-beta-induced ubiquitin-like protein the interferon-inducible 15-kda ubiquitin homolog conjugates to intracellular proteins immunoregulatory properties of isg15, an interferon-induced cytokine ubiquitin-activating enzyme. mechanism and role in protein-ubiquitin conjugation ube1l2, a novel e1 enzyme specific for ubiquitin dual e1 activation systems for ubiquitin differentially regulate e2 enzyme charging influenza b virus ns1 protein inhibits conjugation of the interferon (ifn)-induced ubiquitin-like isg15 protein the ubiquitin-like protein smt3p is activated for conjugation to other proteins by an aos1p/uba2p heterodimer identification of the activating and conjugating enzymes of the nedd8 conjugation pathway the ubch8 ubiquitin e2 enzyme is also the e2 enzyme for isg15, an ifn-alpha/ beta-induced ubiquitin-like protein interferoninducible ubiquitin e2, ubc8, is a conjugating enzyme for protein isgylation herc5, an interferon-induced hect e3 enzyme, is required for conjugation of isg15 in human cells the interferon-inducible ubiquitin-protein isopeptide ligase (e3) efp also functions as an isg15 e3 ligase specific and covalent targeting of conjugating and deconjugating enzymes of ubiquitin-like proteins screen for isg15-crossreactive deubiquitinases ubp43 (usp18) specifically removes isg15 from conjugated proteins ubp43 is a novel regulator of interferon signaling independent of its isg15 isopeptidase activity the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme viral and therapeutic control of ifn-beta promoter stimulator 1 during hepatitis c virus infection mx genes show weaker primary response to virus than other interferon-regulated genes identification of interferon-stimulated gene 15 as an antiviral molecule during sindbis virus infection in vivo ifn-stimulated gene 15 functions as a critical antiviral molecule against influenza, herpes, and sindbis viruses innate antiviral response targets hiv-1 release by the induction of ubiquitin-like protein isg15 isg15-dependent regulation link between the ubiquitin conjugation system and the isg15 conjugation system: isg15 conjugation to the ubch6 ubiquitin e2 enzyme isg15 modification of ubiquitin e2 ubc13 disrupts its ability to form thioester bond with ubiquitin evidence for higher rates of nucleotide substitution in rodents than in man proteomic identification of proteins conjugated to isg15 in mouse and human cells identification and herc5-mediated isgylation of novel target proteins human isg15 conjugation targets both ifn-induced and constitutively expressed proteins functioning in diverse cellular pathways herc5 is an ifn-induced hecttype e3 protein ligase that mediates type i ifn-induced isgylation of protein targets high-throughput immunoblotting. ubiquitiin-like protein isg15 modifies key regulators of signal transduction crystal structure of the interferon-induced ubiquitin-like protein isg15 the structure of the appbp1-uba3-nedd8-atp complex reveals the basis for selective ubiquitin-like protein activation by an e1 structures of the sumo e1 provide mechanistic insights into sumo activation and e2 recruitment to e1 insights into the ubiquitin transfer cascade from the structure of the activating enzyme for nedd8 ubiquitin-conserved protein or selfish gene? ubia, the major polyubiquitin locus in caenorhabditis elegans, has unusual structural features and is constitutively expressed of mice and not men: differences between mouse and human immunology divergent and convergent evolution of nk-cell receptors evolutionary process and the ecology of human immune function the innate immune response to grass carp hemorrhagic virus (gchv) in cultured carassius auratus blastulae (cab) cells isg15, an interferon-stimulated ubiquitin-like protein, is not essential for stat1 signaling and responses against vesicular stomatitis and lymphocytic choriomeningitis virus structural basis for recruitment of ubc12 by an e2 binding domain in nedd8's e1 surface hydrophobic residues of multiubiquitin chains essential for proteolytic targeting distinct functional surface regions on ubiquitin site-directed mutagenesis of ubiquitin. differential roles for arginine in the interaction with ubiquitin-activating enzyme ube1l and protein isgylation are not essential for alpha/beta interferon signaling involvement of ube1l in isg15 conjugation during retinoid-induced differentiation of acute promyelocytic leukemia selectivity in isg15 and ubiquitin recognition by the sars coronavirus papain-like protease autonomous regulation of the anaphasepromoting complex couples mitosis to s-phase entry ubch10 is the cancer-related e2 ubiquitin-conjugating enzyme sequential e2s drive polyubiquitin chain assembly on apc targets ube2t is the e2 in the fanconi anemia pathway and undergoes negative autoregulation fanconi anemia and ubiquitination chromatographic isolation of methionine-containing peptides for gel-free proteome analysis: identification of more than 800 escherichia coli proteins a complex interaction pattern of cis and socs2 with the leptin receptor improved recovery of proteome-informative, protein n-terminal peptides by combined fractional diagonal chromatography (cofradic) the clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools protein sequence alignments: a strategy for the hierarchical analysis of residue conservation we are greatly indebted to dr. ernest borden for the very generous gift of anti-huisg15 antibody. key: cord-331885-8zmuhebu authors: xu, xiuyan title: risk factor analysis combined with deep learning in the risk assessment of overseas investment of enterprises date: 2020-10-02 journal: plos one doi: 10.1371/journal.pone.0239635 sha: doc_id: 331885 cord_uid: 8zmuhebu to evaluate the overseas investment risks of enterprises and expand the application and development of deep learning methods in risk assessment, 15 national clusters are utilized as samples to analyze and discuss the overseas investment risk indicators of enterprises. first, based on the indicator system of overseas investment risks, five major types of investment risks are identified. second, the deep neural network (dnn) is introduced; a risk evaluation model is constructed for enterprise overseas investment. finally, the investment attractiveness index in the fraser risk assessment learning label is adopted as the evaluation results of the model. according to the classification of risks, the model is trained and its performance is tested. the results show that the major source of overseas investment risks includes basic resources, political systems, economic and financial development, and environmental protection. the corresponding risk score is high. north american country clusters and oceanian country clusters have lower investment risks, while the investment risks in africa, latin america, and asia are affected by multiple factors of the specific cities. this is closely related to the resources and legal systems possessed by the country clusters. this is of great significance for enterprises to conduct risk assessment in overseas investment. in the overseas investment activities of enterprises, risk is the most critical problem and challenge [1, 2] . especially, for mining enterprises, their overseas investment projects are affected by multiple factors such as long periods and various uncertainties. the impact on the ecological environment is large. under the action of multiple factors, there is a greater degree of uncertainty between the investment expenditure of mining enterprises and the expected benefits. therefore, the application of scientific and effective solutions to assess the investment risks has great significance in reducing losses of enterprises and ensuring the utilization of overseas resources and energy. the assessment of investment risks has been extensively discussed by researchers. researched the risk management strategy of chemical industry clusters and found that when unexpected losses increased, enterprises were more inclined to adopt high-level investment strategies [3] . for the difficulty in calculating the a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 weight of risk assessment indicators in traditional investment risk assessment models, ren and du (2018) constructed an industrial investment risk assessment indicator system from the levels of systemic risks and non-systematic risks; on this basis, they proposed a support vector machine (svm)-based industrial investment risk assessment model for the marine foreign trade and economic zone; the results suggested that the model could assess risks through empirical analysis [4] . liu and zeng (2017) adopted the system dynamics method to analyze and discuss the investment risks of renewable energy projects,' the results revealed that policy risks, technical risks, and market risks had greater impacts [5] . yao et al. (2017) proposed a tactical exploration framework that applied grammar-based genetic planning to the generation and evolution of tactics in combat-level simulations, which could effectively reduce investment risks [6] . due to its unique advantages, deep learning has been widely used in many fields [7] . the applications of deep learning methods in the field of risk or investment have been researched extensively. xie et al. (2020) analyzed and discussed the application of deep learning in financial data processing, investment model, and investment strategy establishment, which had reference value for improving investors' investment strategy and rate of return [8] . sohangir et al. (2018) explored the application of convolutional neural network (cnn) in stock market investment forecasting; they found that deep learning models could be effectively applied to financial analysis, and cnn had the best effect [9] . liu (2019) believed that the prediction accuracy and portfolio optimization of financial risks were very important and compared the performance of support vector machines (svm) and long short term memory recurrent neural networks (lstm-rnns) in classification; the results suggested that the latter had better performance, which was of great significance for maximizing investment profits [10] . kim and won (2018) proposed a new hybrid lstm model and found that the model could be applied to the prediction of stock market volatility [11] . the above research indicates that investment assessment and investment risks have been discussed a lot. at the same time, deep learning methods have been widely adopted in market forecasting and investment. meanwhile, the assessment of overseas investment risks is less researched, let alone the applications of deep learning methods in overseas investment risk assessment. the effective assessment of risks is influential. to build a risk assessment model for overseas investment of enterprises and evaluate investment risks scientifically and effectively, the deep neural network (dnn) based on deep learning is introduced, which is an innovation. the built risk assessment model is built to provide a reference for enterprises to assess investment risks and reduce risks. from the perspective of enterprises, overseas investment risks are affected by multiple factors, and the function of each influencing factor is very unclear. it is widely recognized that the overseas investment risks of enterprises are a huge and complex system. therefore, it is necessary to design and construct an investment risk assessment system. during the entire process of constructing the assessment system, determining the construction principles and the composition of the investment risk influencing factors are the key links, which helps refining the influencing factors and indicators. on this basis, the review of literature, on-site inspections, and expert consultation are the means of model construction; in the meantime, the construction principles of scientificity, reliability, and comparability are persisted. the assessment system is divided into risk categories, project components, and risk factors. for the evaluation of venture capital, common methods include variance method [12] , coefficient and capital asset pricing model [13] , "a" scoring method [14] , value-at-risk evaluation model [15] , and comprehensive risk index model [16] . due to the complexity of different country clusters, the comprehensive risk index evaluation method is adopted. the risks in the overseas investment assessment system are divided into basic resources, economics and finance, political systems, environmental protection, and resources and energy. then, these five categories are refined. under the category of basic resources, the investment goal is to improve the infrastructure equipment in the process of overseas investment and resource development of enterprises; specifically, it includes health care, basic communications, power energy, and land resources [17] . the category of economic and finance is correlated to the economic development and financial system in the destinations of overseas investment. the investment goal of this category is to promote the optimization of enterprise profits. the risk can be evaluated by the inflation rate and gdp. here, this risk category is refined into economic growth, financing, changes in interest rates and exchange rates, inflation, credit systems, and total tax rates [18] . the risk category of political systems mainly characterizes the degree of political influences, as well as the stability of a particular country. this is a key factor in assessing the diplomatic situation, which also includes the level of rule of law and policy support. in particular, the major risk factors of the political system include regime alternation, contradictions and conflicts among surrounding powers, law system, social credit, foreign shareholding, and partners [19] . the risk factor category of environmental protection is essential for investment enterprises. when enterprises make overseas investments, they will always consider environmental protection factors, such as the legal requirements of environmental protection, the environmental law enforcement level of the investment destinations, and the impact of natural factors on environmental protection. specifically, this category, a major risk factor, is refined into environmental law enforcement and environmental expenditures [20] . the risk factor category of resources and energy includes the development and utilization of minerals and other resources. specifically, this category is refined into resource application potential and development conditions [21] . an overseas investment risk assessment system of enterprises including five major categories is thereby built. the details of categories are shown in table 1 below. of late years, deep learning technology has developed rapidly. dnn has been applied in image recognition, text detection, and other fields. compared with previous machine learning methods, deep learning technology has better performance in data fitting of complex factors [22, 23] . therefore, the deep learning method is adopted to build the overseas investment risk assessment model of enterprises. the overall construction idea includes three steps: preliminary model construction, model training, and analysis and characterization of model performance. deep learning technology includes a variety of models, for dnn, strong feature extraction ability, easy training, fast convergence speed, and simple structure composition are its characteristics [24] . considering the characteristics of investment risk assessment, dnn is applied to the construction of the enterprise overseas investment risk assessment model. in deep learning neural network models, the structure of dnn is different from traditional neural network models. it consists of the input layer, the hidden layer, and the output layer [25] . a basic model y k corresponding to dnn can be expressed as: in (1), x i represents the neural unit node of the dnn, w ik b k represents the value of the weight, b k represents the bias, and y k represents the activation function. the training process of dnn is shown in fig 1 below . on this basis, the input and output of dnn in overseas investment risk assessment are defined. x is utilized as the input value of the evaluation model, and the above five table 1 . the composition of the risk assessment indicator system for overseas investment of enterprises. basic resources health care a1 -health care (+), risk ( categories of investment risks are converted into corresponding risk indicators. first, the evaluation model is input into the sample feature dataset, which is represented by the following equation: in (2), f represents the number of features corresponding to each data, which corresponds to the risk factor indicator in the assessment model; l represents the number of labeled data, which corresponds to the investment destination in the assessment model and the training sample; u represents the number of unlabeled data, which also corresponds to the test sample. if y is the output value of the evaluation model, it will also correspond to the sample risk label dataset. in (3), c represents the number of categories corresponding to the sample label. refined into the investment risk assessment, the investment risks are divided into five different levels; level i indicates that no investment can be made, level ii indicates that the investment risk is serious, level iii indicates that the investment risk is high, level iv indicates that the investment risk is average, and level v indicates that the investment risk is low. these five different risk levels correspond to five label categories and the realization of the labeling rules, which can be expressed as: for the sample dataset in the input investment risk assessment model, feedforward calculation should be utilized to judge and express the input data features. after successive propagation throughout the layers, the data dimensionality is finally reduced. in this process, the corresponding data are constantly becoming abstract, and finally, the result can be predicted. the risk assessment model of enterprise overseas investment built here is actually a process of reducing multidimensional data to five dimensions. to solve this problem, the feedforward propagation of information in dnn can be expressed as: in (5), z (p) represents the specific state of the eigenvalues corresponding to the p-tier investment risk assessment, w (p) represents the weight matrix, f p represents the activation function corresponding to the eigenvalues of the p-tier investment risk assessment, and b (p) is the offset. the information is passed successively throughout the layers in dnn, and finally, the output of the network can be obtained. considering that data can have limitations in the target countries or regions for enterprises investment overseas, the application of labeled data can play an important role in the training of the network. if there is a set of data samples (x (i) , y (i) ): the corresponding output of the neural network after the feedforward propagation is f(x|w, b): then, the corresponding objective function o(•) can be expressed as: in (8), w represents the weight matrix corresponding to each layer, and b represents the bias vector corresponding to each layer in the neural network. based on the goal of minimizing investment risk output, the gradient descent method is applied to realize the iteration and update of parameters. the corresponding calculation is: in (9), δ represents the update rate corresponding to the parameter, and λ represents the parameter. furthermore, the error term is defined as: in (10), � represents the dot product operator of vectors. after obtaining the error term corresponding to each layer, the gradient value corresponding to each layer parameter can be obtained. according to above analysis, the entire training process of dnn is achieved through the calculation of the state and activation value of each layer, as well as the calculation of the error corresponding to each layer in the neural network, which helps update the relevant parameters. the expression level of dnn also plays an extremely important role, which can be achieved by the introduction of nonlinear activation functions. the logistic function, tanh function, and relu function are the most commonly used activation functions in traditional neural network models. however, the first two activation functions have the problem of gradient disappearance during neural network training [26] . therefore, in the proposed model, relu is used as the activation function, and its corresponding equation is: compared with the other two activation functions, the gradient calculation corresponding to the relu function can be expressed as: ( this makes the problem of gradient disappearance in the entire backpropagation process alleviated. to test the effectiveness of the dnn-based enterprise overseas investment risk assessment model, the fraser risk assessment is chosen as the learning label. this risk assessment includes multiple countries and regions [27, 28] . here, countries and regions that have continuity in the fraser risk assessment learning label from 2018 to 2019 are selected as the research samples. specifically, a total of 15 countries are included as the target countries. besides, president xi jinping of china has visited all the included countries, sharing close cooperative relations with china. in addition, the selected countries have rich resources such as minerals. the five risk assessment levels proposed above are utilized to analyze and characterize the proposed dnn-based enterprise overseas investment risk assessment model. the selected research samples are shown in table 2 table 1 above. furthermore, for output y of the risk assessment model, the investment attractiveness index of the target country or region in the fraser risk evaluation learning label is adopted as the evaluation result of the model. in addition, the corresponding label of the model output is divided into five risk levels according to the corresponding scores. therefore, the model is trained and its performance is tested. during 2018-2019, the selected overseas investment risk assessment indicators for north american country clusters and latin american country clusters are shown in fig 2a-2d below. data analysis shows that the risk assessment indicators of the two countries in north america, the united states and canada, show different indicators among the five risk levels. however, because the united states and canada have richer reserves of resources and energy, coupled with a higher level of economic development, better political systems, and sound infrastructure, the overall investment environment is better, and the overall investment risks are also lower. in contrast, however, several countries in latin america have different levels of investment risks. this is mainly due to differences in the level of economic development between countries, as well as the differences in the supply of basic resources and the rule of law system. during 2018-2019, the selected overseas investment risk assessment indicators for asian country clusters are shown in fig 3a and 3b below. data analysis shows that asian countries have richer resources and energy reserves. however, due to multiple factors, such as the development level of industries, professional skills, and capital supply, generally, these countries show higher investment risks. this is inseparable from a country's political development trends and geographic location. during 2018-2019, the selected overseas investment risk assessment indicators for african country clusters are shown in fig 4a and 4b below. analyzing the risk assessment indicators of each risk level shows that african country clusters also have abundant resources and energy reserves. however, because the overall level of economic development is poor, these countries are highly dependent on mineral resources and energy exports. in addition, the political system are often traditional so that the degree of implementation is also poor. the political situation in africa is generally stable but partially turbulent, leading to a higher level of investment risk. during 2018-2019, the selected overseas investment risk assessment indicators for european and oceanian country clusters are shown in fig 5a and 5b below. changes in the risk indicators show that the overall resource and energy reserves of the european country clusters are general; however, the basic resources, such as infrastructure, are sound and complete. the overall macroeconomic development level is lower. the overall risks at all levels are lower in several cities. australia, a representative country of oceania, has abundant minerals, coal, and other natural resources, with independent legislation among states, stable political trends, and sound infrastructure; therefore, the overall investment environment is good. the above analysis suggests that a country's infrastructure, health care conditions, economic development level, environmental protection conditions, and political system are the key components that affect its risks of enterprise overseas investment [29, 30] . in contrast, north american countries are more favorable target countries for overseas investment, which are inseparable from their resources and energy, health care conditions, economic development, and political systems. among the investment risks, the biggest disadvantage of the north american clusters is that if the investment is associated with mining, due to the high requirements for environmental protection, the labor factors and other issues must be considered. meanwhile, in latin american clusters, the main problems are the level of economic development and the imperfections of infrastructure and legal construction. in the meantime, considering the actual situation during the current covid-19 (corona virus disease 2019) pandemic, the situations in the united states and other countries are severe, which also needs to be noted in investment. asian countries have abundant resources and energy reserves; besides, the overall political development situation is stable [31] . however, the policies for investment are not perfect and stable enough. changes in leadership will trigger adjustments and changes in policies; therefore, problems in the efficiency and concepts of the government have led to an increase in investment risks. in the meantime, the presentation of the final result of investment risks is inseparable from a country's geographic location. in terms of development stage, africa is still in the initial period of industrialization. although the momentum of development in recent years has become stronger, the overall facilities and systems need to be improved. in addition, the health care conditions in africa are backward. natural disasters and infectious diseases occur frequently. this is a concrete manifestation of the risks of african country clusters in overseas investment, which is also an aspect that needs attention. the enterprise overseas investment risks of european and oceanian clusters also illustrate the important role of several risk factors in investment. a total of 15 country clusters with different geographical locations are chosen as the research objects. based on risk factor analysis, the dnn-based enterprise overseas investment risk assessment model is tested. the results suggest that the investment risk indicators can clearly show the level of investment risks. the investment risk level of each country is comprehensively affected by basic resources and energy, political systems, and economic and financial development levels. the above results have provided basic data support for the risk assessment of enterprises overseas investment. however, the assessment of enterprise investment risks is still in the exploratory stage. affected by multiple objective factors, the sample data only include the risk indicators during 2018-2019. as the research continues to be expanded, the sample data will be increased in the future to obtain more valuable results. supporting information s1 data. 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based natural language processing deepsurv: personalized treatment recommender system using a cox proportional hazards deep neural network hierarchical modeling of molecular energies using a deep neural network deep binary reconstruction for cross-modal hashing health care professionals' attitudes towards population-based genetic testing and risk-stratification for ovarian cancer: a survey study anticipated health behaviour changes and perceived control in response to disclosure of genetic risk of breast and ovarian cancer: a quantitative survey study among women in the uk overseas direct investment and exports in korea: a time series approach the influence of investor-centered values in the operation of political risk insurance risk and opportunity assessment for water cooperation in transboundary river basins in south asia resources: xiuyan xu. writing -review & editing: xiuyan xu. key: cord-346858-18q8rxzg authors: hossain, md. tanvir; ahammed, benojir; chanda, sanjoy kumar; jahan, nusrat; ela, mahfuza zaman; islam, md. nazrul title: social and electronic media exposure and generalized anxiety disorder among people during covid-19 outbreak in bangladesh: a preliminary observation date: 2020-09-11 journal: plos one doi: 10.1371/journal.pone.0238974 sha: doc_id: 346858 cord_uid: 18q8rxzg classified as a pandemic by the world health organization, the novel coronavirus disease (covid-19) has spread to bangladesh since early march of 2020, and people are getting daily updates from the social and electronic media. we aimed at assessing the prevalence of anxiety among bangladeshi people during the pandemic in connection with social media exposure (sme) and electronic media exposure (eme). for this cross-sectional study, data were collected from 880 participants by a self-administered online-based questionnaire relating personal characteristics, self-rate health (srh), sme, and eme with anxiety. findings show that around half of the surveyed population experienced a spike of anxiety (49.1%) during the pandemic, ten times higher than the national anxiety rate in 2019. the participants with an increased sme of over four hours per day experienced a higher level of anxiety than individuals with < = 2 hours exposure to social media. similarly, the anxiety was higher among people with fair/bad srh compared to individuals with excellent srh. it is highly recommended to develop active surveillance and effective monitoring systems to reduce the spread of misinformation from both social and electronic media to improve the state of mental health conditions during the pandemic. the spread of novel coronavirus disease , also known as severe acute respiratory syndrome coronavirus 2 (sars-cov-2), among 114 countries and territories across the globe has made the world health organization (who) declare a global pandemic [1] . originated in wuhan, china, from december 2019 [2] , the covid-19 infected 8.7 million human beings across 210 countries and territories with confirmed cases of death around 0.47 million by 22 june 2020 [3] . the outbreak of made the governments and health practitioners across the world to promote psychological crisis interventions along with other necessary preventive social safety protocols for the citizens as well as for the healthcare workers during the pandemic [4] [5] [6] [7] [8] [9] [10] . despite the preventive measures of international organizations and governments of different countries to minimize the spread of the covid-19 [11, 12] , the news of an increasing number of infected as well as deceased in different countries and regions have steered panic among the mass people [13] [14] [15] [16] [17] . the situation has been elevated further with the exposure to exaggerated 'viral' news in social media, such as facebook, messenger, twitter, and so on, as well as 'misinformation' by electronic sources, like news reports and online blogs [5, 7, [18] [19] [20] . the rampant misinformation and false reports, together with the negative attitude of people towards the infected, have fueled a wide range of psychopathological consequences, including fears, depression, and anxiety [13, 18, 21] . the mental health burden of the covid-19 infected patients and the healthcare professionals, fearing the persisting social prejudice and stigma generated from 'overexposure' to media 'misinformation,' forced some people to commit suicide [22] [23] [24] . a study in south korea during middle east respiratory syndrome coronavirus (mers) found a positive relationship between media exposure and risk perceptions [25] . however, some others are suggesting that the exposure to media during pandemic and epidemic increased severe mental health outcomes, including suicidal behavior [26] [27] [28] . apart from the social exclusion and mental health issues, employment and financial issues also led to suicide [21, 28, 29] . bangladesh, following the first confirmed covid-19 case in early march of 2020 [30] , initiated all possible preventive measures, such as nationwide lockdown, closing government and private offices as well as educational institutions, and deploying military forces to curb human transmissions. yet, there are 112,306 confirmed covid-19 cases, with 1,464 fatalities as of 22 june 2020 [31] . moreover, bangladesh has been experiencing a flood of disinformation both in mainstream social as well as electronic media [32] spreading hatred and social stigma against the healthcare providers, security forces, and people with mild symptoms, but not covid-19 positive [33] . bangladesh has witnessed its first covid-19 related suicide on 25 march 2020, though the victim was not diagnosed with covid-19 [34] . the suicide marks the strong presence of fears and stresses among people, as evident in various countries of the world [28, 29, 35] . at present, the level of anxiety generated from the exposure to social and electronic media during covid-19 pandemic is not known in bangladesh, while some other countries have addressed the issue vigorously [13, 18, 36] . therefore, the purpose of the current study was to fill the void in exploring the presence of anxiety among people in bangladesh during the covid-19 pandemic and to identify its determinants to devise preventive measures to curb the symptom of anxiety associated with the pandemic. this was a cross-sectional study. after the identification of the first case of covid-19 patient on 8 march 2020, the government of bangladesh declared a countrywide lockdown. thus, this study was undertaken online to comply with the who recommended 'social distancing' to avoid face-to-face contact with the potential participants. data were collected in the third week of march, started from 19 april to 25 april, and the participants responded to the e-questionnaire anonymously. a good many quantitative studies have been undertaken in different countries following this technique [13, 18] , making it popular as well as proving its effectiveness. the target population was the bangladeshi people, staying within the country during the data collection period, aged 16 and older, able to understand english. the e-questionnaire, based on the widely used google form, was forwarded to the participants with valid facebook account. of the initial 937 responses, a total of 880 responses was deemed suitable to retain in the study after careful and rigorous scrutiny. this study was carried out in accordance with the declaration of helsinki, and it was approved by the ethical clearance committee of khulna university, bangladesh. all the participants responded to the online survey by filling up a written informed consent letter in the first section of the e-questionnaire. the participants were free to decline from the survey at any moment without prior justification. socio-demographics. a range of factors were considered as explanatory variables, based on the previous studies, to assess the impact of sme and eme on anxiety. factors comprised of age, sex and place of residence [18, 37, 38] , educational level [18, 38] , occupation, marital status and srh [18] , and division [18, 39, 40] . social and electronic media exposure. the social media exposure (sme) and electronic media exposure (eme) were assessed by asking the participants about how often they were exposed to the social media, (e.g., facebook, messenger, whatsapp, instagram, twitter, skype, viber), and electronic media, (i.e., television, radio, internet) during the past two weeks of lockdown to get news and information regarding the covid-19. their responses, for both sme and eme, were measured by five-point likert-scale items, including 'never,' 'occasionally,' 'sometimes,' 'often' and 'always.' the five-point scale was later transformed into four-levels for both sme and eme, where 'never' and 'occasionally' were merged into 'less,' while the rest three remained the same, i.e., 'sometimes,' 'often,' and 'always' in the final analysis. the participants were also asked to report their favorite source information (recoded 'facebook and others' for sme, and 'internet and others' for eme), the time spent to get news and information (< = 2 hours, 2-4 hours and more than 4 hours), and changes in sme and eme compared to the pre-covid-19 situations. however, the changes of time spent in sme and eme to compare the pre-covid-19 and during the pandemic situations were categorized into 'increased more,' 'about the same' and 'decreased more'. self-rate health. we adopted the 'short-form survey instrument' (sf-36) developed by the rand corporation [41] to measure the self-rate general health conditions. unlike the sf-36, containing 36-items to assess the health conditions, we used seven-items, and the participants were asked to rate their health conditions as well as their ability to get involved in various activities by a five-point likert-scale, including 'excellent,' 'very good,' 'good,' 'fair,' and 'bad,' and it was recoded as 'excellent,' 'very good,' 'good' and 'fair/bad.' the cronbach's α in this study was 0.713. the other question was directed to address the existing 'chronic' health conditions of the participants, and the response was recoded as 'yes' and 'no.' anxiety. the anxiety of the participants, as applied by previous studies [18, 19, 36, 37, 42] , was measured by the generalized anxiety disorder scale (gad-7) [43]. the self-reported gad-7 consisted of seven symptoms, and the participants were asked how often they were bothered by each of these symptoms in the last two weeks. the responses were categorized into fourpoint scale, including 'not at all' (score = 0), 'several days' (score = 1), 'more than half of the days' (score = 2) and 'nearly every day' (score = 3). a score of 10 or greater signifies the case of anxiety [43] . the cronbach's α in the study was 0.873. data were analyzed in two consecutive stages using the statistical package for social sciences (spss), v20. firstly, the pearson's chi-square (χ 2 ) test of independence was executed to measure the association of the explanatory variables with sme and eme at 5% level of significance. finally, the multivariable logistic regression model was performed considering the sme and eme related variables found statistically significant in the pearson's chi-square (χ 2 ) test. findings were shown using the adjusted odds ratio (aor) with 95% confidence intervals (ci). table 1 presents the personal characteristic of the participants. the mean (±standard deviation) age of 880 participants was 26.3 (±7.2) years, and the highest 42.0% were from the age cohort of 21-25 years. most of the participants in this study were male and unmarried, and their proportion counted for 70% each. more than half (56.0%) were students, and lived in khulna division (55.9%), whereas two-thirds (66.5%) were from urban areas. only 10.8% of them had different diseases, including diabetes, heart, and lung-related, while more than half (50.5%) of the participants reported having a good health condition. table 1 also shows the association between exposure to social media and different characteristics of the participants. of the total responses, the proportion of 'less,' 'sometimes,' 'often' and 'always' of sme was 7.4%, 21.7%, 27.0%, and 43.9%, respectively. findings indicate that age (p<0.001), education (p = 0.001), marital status (p = 0.004), occupation (p = 0.001), place of residence (p<0.001) and srh (p = 0.003) were significantly associated with the sme. the exposure to social media for both male (44.2%) and female (43.2%) was almost the same; however, the sme was highest among the age group of 26-30 years than the younger ones (aged < = 20 years). likewise, the sme was higher among the highly educated (master or above) and married (50.0%) compared to those having lower educational qualifications (37.2%) and unmarried (41.2%). the proportion of sme was also higher for urban (46.7%) areas in comparison with rural settings (38.3%). no statistically significant differences were observed between the administrative divisions of bangladesh and sme. but the participants reporting bad/fair health conditions or health-related problems had greater exposure to social media. the association of eme and personal attributes of the participants were presented in table 2 . findings indicate that of the total responses, the proportion of 'less,' 'sometimes,' 'often,' and 'always' of eme was 9.3%, 26.5%, 28.5%, and 35.7%, respectively. factors, such as age (p = 0.012), education (p = 0.006), marital status (p = 0.016), occupation (p = 0.007), place of residence (p = 0.007) and srh (p = 0.001) were significantly associated with the eme. the use of electronic media was higher among male (36.5%) than female (33.7%), older (above 30 years) than younger (< = 20 years) and private employees (39.3%) over other occupational groups, including students (35.5%) and government-funded jobs (34.8%). there were no differences observed between married and unmarried as well as between khulna and other divisions with eme. in contrast, the participants from urban areas (37.9%) and having excellent health had greater exposure to electronic media compared to their respective counterparts. moreover, the participants reporting health-related problems had proportionately greater eme than the people with no complex health issues. table 3 shows the prevalence (95% ci) of anxiety in relation to a range of personal characteristics of the participants. the overall prevalence of anxiety was 49.1% (95% ci: 45.8-52.4%). findings indicate that the participants with different characteristics, such as belonging to the age group of 21-25 years, being married, living in urban areas, having greater exposure to social (facebook) and electronic media (internet), and spending more than 4 hours in sme and eme were more likely to show anxiety symptoms. significant factors from the pearson's chi-square (χ2) test of independence for both sme and eme were retained in the multivariate analysis to investigate the impact of these factors on anxiety in bangladesh (table 3) . findings suggest that after adjusting the sme and eme related factors, several factors, such as time and changes in sme as well as srh, were the most significant determinants of anxiety. results revealed that the participants with an sme of over four hours a day had 1.52 times (95% ci: 1.01-2.31, p = 0.049) higher anxiety compared to those with < = 2 hours exposure to social media. likewise, the adjusted odds ratio (aor) of [26, 51, 52] , as well as the recent covid-19 pandemic [18, 39, 53, 54] . the presence of severe anxiety among bangladeshi people can be related to their exposure to both social and electronic media as the current study found that more than one-third of the participants were always using the social and electronic media to get updated information regarding the covid-19 situation. the reliance of the mass people on social and electronic media to get informed on current events, however, is not uncommon as the who and concerned governments usually provide updates about surveillance and active cases on social and electronic media during different crisis moments [5, 26, 55, 56] . however, the overexposure to media misinformation might lead to anxiety symptoms [18, 54] , as it increased the fear of the findings of the current study complement the previous studies as the odds of anxiety were highly associated with the time spent in the social media (�4 hours) as well as the increased tendency of social media use. a study on university students in bangladesh suggested that isolation with minimum physical activities and poor sleep quality were the major risk factors of over 'exposure' or 'addiction' to social media [59] . in addition to the continuous exposure to and utilization of social media, the poor health condition was found to have an inverse relation with anxiety. the odds of anxiety were the highest among individuals with bad srh compared to those with excellent srh, and such results correspond with previous studies [18, 51] . being home bounded, the critically ill individuals, with an exposure to the flood of negative media projections, often experience an aggravation of mental health problems [7] as well as low life satisfaction [60] . with the imminent threat, both physical and mental, the physically ill individuals, without family and social support, are more susceptible to suicidal behavior [22, 34] , as was the case during the sars endemic in hong kong [26] . some limitations should be accounted for the generalization of findings of the study. the survey was online based, a popular technique for a quick situation analysis. however, the selection biasness, by age groups or use of social media, might have influenced the results. moreover, it does not cover a national representative sample as most of the participants were from the khulna division, the southwestern region of bangladesh. the study, cross-sectional in nature, might not accurately explain the causal relationship between anxiety and sme as well as eme. the study assessed the presence of anxiety among people under a sudden emergency without considering their mental health in pre-lockdown conditions. despite the efforts of selecting all possible factors influencing the anxiety among people in an emergency, there may have some other confounding issues that remained unattended. this study provides a preliminary idea of the pretext of the mental health conditions of bangladeshi people during the covid-19 pandemic. our findings suggest that sme is the key factor responsible for increasing anxiety among the population of bangladesh. hence, the government must develop active surveillance and effective monitoring system to minimize the spread of misinformation in both social and electronic media without sacrificing the democratic spirit. the authority also needs to broadcast positive and supportive information through both 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bangladesh: mahfuz anam covid-19 status bangladesh dhaka, bangladesh: ministry of health and family welfare psychosocial, and socio-economic crisis in bangladesh due to covid-19 pandemic: a perception-based assessment matiur rahman first covid-19 suicide case in bangladesh due to fear of covid-19 and xenophobia: possible suicide prevention strategies hospital suicide due to non-treatment by healthcare staff fearing covid-19 infection in bangladesh? study of knowledge, attitude, anxiety & perceived mental healthcare need in indian population during covid-19 pandemic. asian journal of psychiatry prevalence of mental disorders in china: a crosssectional epidemiological study. the lancet psychiatry study on the public psychological states and its related factors during the outbreak of coronavirus disease 2019 (covid-19) in some regions of china prevalence and predictors of ptss during covid-19 outbreak in china hardest-hit areas: gender differences matter the association between facebook addiction and depression: a pilot survey study among bangladeshi students unprecedented disruption of lives and work: health, distress and life satisfaction of working adults in china one month into the covid-19 outbreak the authors are grateful to all the participants and express their deepest respect and gratitude to the medical professionals, social workers and security forces for fighting the covid-19 pandemic, and for inspiring the nation by setting examples of patriotism. conceptualization: md. tanvir hossain, benojir ahammed. key: cord-320938-f526k9q1 authors: chen, hongjun; ye, jianqiang; xu, kemin; angel, matthew; shao, hongxia; ferrero, andrea; sutton, troy; perez, daniel r. title: partial and full pcr-based reverse genetics strategy for influenza viruses date: 2012-09-28 journal: plos one doi: 10.1371/journal.pone.0046378 sha: doc_id: 320938 cord_uid: f526k9q1 since 1999, plasmid-based reverse genetics (rg) systems have revolutionized the way influenza viruses are studied. however, it is not unusual to encounter cloning difficulties for one or more influenza genes while attempting to recover virus de novo. to overcome some of these shortcomings we sought to develop partial or full plasmid-free rg systems. the influenza gene of choice is assembled into a rg competent unit by virtue of overlapping pcr reactions containing a cdna copy of the viral gene segment under the control of rna polymerase i promoter (pol1) and termination (t1) signals – herein referred to as flu pcr amplicons. transfection of tissue culture cells with either ha or na flu pcr amplicons and 7 plasmids encoding the remaining influenza rg units, resulted in efficient virus rescue. likewise, transfections including both ha and na flu pcr amplicons and 6 rg plasmids also resulted in efficient virus rescue. in addition, influenza viruses were recovered from a full set of flu pcr amplicons without the use of plasmids. type a influenza (flu) viruses belong to the family orthomyxoviridae and their genome consist of eight segments of single strand rna of negative polarity [1] [2] [3] . the viral rna (vrna) is found in the virion and infected cells in the form of viral ribonucleoprotein particles (vrnps) associated with three polymerase subunits (pb1, pb2, and pa) and the nucleoprotein (np) [4, 5] . the virus encodes for two surface glycoproteins, hemagglutinin (ha) and neuraminidase (na), the proton pump transmembrane protein (m2), the matrix protein (m1), the nuclear export protein (ns2/nep), the nonstructural protein (ns1) and, in some influenza viruses (from an alternative translation start site in segment 1) the pb1-f2, an apoptosis modulatory protein [6] [7] [8] . additional viral protein products include pb1-n40, derived from an alternative start site within the pb1 orf, resulting in a protein product that lacks the first 39 aa of pb1, and pa-x, derived from the pa mrna and consists of the n-terminal 191 aa of pa fused to 61 aa that result from +1 frameshifting [9, 10] . de novo synthesis of influenza viruses by reverse genetics (rg) requires not only the viral rna but also the viral protein components [11] [12] [13] [14] . thus, rg systems for influenza rely invariably on a dual promoter concept: one for the synthesis of vrna segments and another for the synthesis of viral mrnas [11] . since the termini of influenza vrnas are crucial for virus replication, plasmids carrying a rna polymerase i (pol1) or t7 rna polymerase promoters have been used to generate vrnas with the exact 39 end, whereas a pol1 terminator sequence (t1) or a hepatitis h ribozyme have been used to generate the exact 59 end. plasmids carrying typical rna polymerase ii (pol2) promoters (cmv and/or chicken b-actin promoters) have been utilized for the synthesis of influenza mrnas [15] [16] [17] [18] [19] . despite the great advantages of this technology, and although variations to the plasmid-based approach have been developed, they inevitably rely on a cloning step [20, 21] . a system that does not rely on cloning could speed up studies on the significance of mutations in the viral genome for replication and/or modulation of virulence. in this report, flu pcr amplicons, instead of plasmids, are an efficient and viable alternative to the plasmid-based rg system. flu gfp pcr amplicons were derived from phw72-egfp [21] . in order to determine whether a flu pcr amplicon could be transfected into cells and be amplified by the influenza polymerase complex, a pcr product was produced encoding the gfp reporter gene in negative orientation flanked by the influenza segment 7 untranslated regions (utrs) and further flanked by the human pol1 promoter and the mouse t1 termination signal, pol1egfpt1 (fig. 1a, fig. s1a , table s1 ). co-transfection of the pol1egfpt1 amplicon along with 4 protein expression plasmids encoding the influenza virus polymerase complex (3p) and np into 293t cells resulted in efficient amplification of the reporter replicon and detection of green fluorescence signal ( fig. 2a) . the proportion of green cells observed was comparable to those observed in the positive control cells co-transfected with phw72-egfp and the 3p and np expression plasmids (fig. 2b) . the fluorescence signal of another amplicon, pol1egfputr, which lacks the t1 signal, was present in fewer cells compared to the pol1egfpt1 amplicon indicating that run off transcription by the rna pol1 complex may result in vrna fragments with incorrect and/or incomplete influenza sequences (fig. 2c ). as expected, no fluorescence signal appeared when cells were transfected with a flu gfp pcr amplicon lacking the pol1 and t1 elements (utregfputr) (fig. 2d ) or by removing the pb1 plasmid in cotransfected cells with either pcr amplicons or phw72-egfp plasmid ( fig. 2e and data not shown) . these initial studies were expanded in order to test whether pcr amplicons containing rna polymerase ii (pol2) and polyadenylation sequences flanking an appropriate orf would result in gene expression (fig. s1b) . thus, pcr amplicons of the 3p and np genes were obtained using a set of primers spanning the cytomegalovirus promoter (cmv) and bovine growth hormone (bgh) polya elements. co-transfection of the pol2pb2bgh, pol2pb1bgh, pol2pabgh, and pol2npbgh, along with phw72-egfp, resulted in efficient reporter replicon expression indicating that pcr amplicons with either pol1 or pol2 transcription elements are appropriately transcribed by the corresponding transcription complexes (fig. 2f ). in order to demonstrate whether flu pcr amplicons could be used to replace plasmids in the rg system, the strains mouse-adapted a/california/04/2009 (h1n1) [22] and a/chicken/ north sumatra/072/2010 (h5n1) -herein referred to as h1n1pdm and 072, respectively -were used as donors for the ha and na genes ( fig. 1b and fig. 3) . a specific set of internal primers designed within conserved regions of these gene segments were then developed in order to maximize gene amplification from viral cdna preparations and to assemble the appropriate ha and na pcr amplicons (fig. 3, fig. s 2a and b) . the pol1ha pdm t1 pcr amplicon carried a full-length copy of the ha gene from the h1n1pdm strain flanked by the pol1 and t1 signals ( fig. 2a) . with respect to the 072 ha gene, the internal primers were designed to delete (d) the polybasic amino acid signal sequence (rerrkrrr) and replace it with one carrying a monobasic cleavage site (tetr) (fig. 3b, fig. s 2) . similar strategies were followed to create the na amplicons pol1na pdm t1 and polna 072 t1 from viral cdnas ( fig. 3c and d) . full-length pol1ha pdm t1 and pol1na pdm t1 pcr amplicons were obtained and confirmed by sequencing (fig. 2e ). in addition, full-length ha and na pcr amplicons were generated lacking either the t1 sequence or both the pol1 and t1 sequences, which serve as controls for efficiency of virus rescue as described below. sequencing results confirmed the amplification of an overlapping dh5 ha amplicon, pol1ha d072 t1, with a deleted polybasic cleavage site and the full-length amplification of the polna 072 t1 (fig. 3f, fig. s 2) . . pcr-based reverse genetics. a) pcr-based flu reporter replicon encoding gfp: pcr amplification was performed using primers spanning the pol1 to t1 sequences and phw72egfp. after agarose gel purification and testing to show that the pcr product is devoid of plasmid dna contamination, the flu gfp amplicon is transfected into 293t cells along with four expression plasmids encoding the polymerase complex of influenza virus. expression of gfp reflects influenza polymerase activity on a vrna flu gfp replicon generated from pol1 transcription of the flu gfp amplicon. variations to this these are described in the main text and shown in fig. 2 . b) starting with a influenza virus candidate, vrna, cdna and reconstitution of a full-length flu pcr amplicon (in this case, the ha and na pcr amplicons are depicted) is performed. transfection of flu pcr amplicons along with appropriate complementary rg plasmids into susceptible cells leads to the generation of recombinant influenza viruses with the desired gene constellation. the strategy speeds up the reverse genetics process by obviating a classical cloning step, which is currently part of the plasmid-based rg system. doi:10.1371/journal.pone.0046378.g001 efficient influenza virus rescue using flu pcr amplicons in either ''1+7'' or ''2+6'' modes the pol1ha pdm t1 or pol1ha d072 t1 ha pcr amplicons (table 1) were co-transfected into co-cultured 293t/mdck cells in a ''1+7'' mode along with 7 rg plasmids encoding the corresponding additional gene segments from the influenza a/puerto rico/ 8/1934 (h1n1) strain (pr8). at 48 h and 72 h post-transfection (hpt) cells co-transfected with the pol1ha pdm t1 pcr amplicon plus 7 rg pr8 plasmids (h1 pdm :7pr8) showed typical virus-induced cytopathic effect (cpe). h1 pdm :7pr8 virus titers at 72 hpt reached 3.16610 4 tcid 50 /ml, which was 5 times lower than the one obtained using the corresponding ph1pdm rg plasmid (ph1 pdm :7pr8, 1.58610 5 tcid 50 /ml) ( table 1) . after a subsequent blind passage in mdck cells or 9 day-old embryonated chicken eggs, virus titers increased .10 7 tcid 50 /ml with either the pol1ha pdm t1 pcr amplicon or the whole plasmid-based rg system. likewise, the dh5n1 virus could be rescued using the pol1had 072 t1 ha pcr amplicon and 7 rg pr8 plasmids (h5d 072 :7pr8, table 1 ). at 72 hpt, h5d 072 :7pr8 virus titer in transfected cells was 1.58610 4 tcid 50 /ml, and increased to 2.32610 8 tcid 50 /ml when passaged in eggs (table 1) . at 72 hpt, the ticd 50 titer of the pcr-based h5d 072 :7pr8 virus was 10, 000 times less than that of ph5d 072 :7pr8 virus, which was obtained with the bidirectional prf1437 plasmid ( table 1) . the identity of the 1+7 reassortants was further confirmed by sequencing the ha gene, and by immunofluorescence assay (ifa) (fig. 4a ) and plaque assay (fig. 4b ). as expected, no cpe and no virus was detected after transfection of cells with 7 rg pr8 plasmids (and in which the plasmid encoding the ha segment was omitted, not shown). by plaque assay, the h5d 072 :7pr8 virus was found to form plaques in agar plates in the presence, but not in the absence, of 1 mg/ml tpck(fig. 4c) , consistent with the reconstitution of a monobasic cleavage site in the ha gene during pcr amplification. to further confirm that these viruses carry a dh5 gene and/or do not present additional mutations introduced during pcr amplification, each of these viruses was plaquepurified and the ha gene was sequenced and analyzed for 48 plaque-purified viruses for each of three rg viruses, h5d 072 :7pr8, ph5d 072 :7pr8 , and control rg-derived pr8. every single virus isolate from the h5d 072 :7pr8 virus group contained a monobasic cleavage site identical to the one designed in the pcr primers. mutations were identified elsewhere but in only 4 out of the 48 sequences analyzed for the h5d 072 :7pr8 virus and in each case corresponded to single nucleotide changes (table s2 ). the mutations caused amino acids changes in 3 of them; however, these changes did not alter the antigenic make up of these viruses by hi assay (table s2) . no mutations were identified in ha sequences derived from plaques obtained from the rgderived pr8 or ph5 d072 :7pr8 viruses (data not shown). these results highlight the high fidelity of the pcr approach. because the pr8 virus is a fully laboratory adapted strain and can be recovered very efficiently by rg, studies were performed to test whether the pol1ha pdm t1 or pol1had 072 t1 pcr amplicons could be recovered in the background of other rg systems including the a/guinea fowl/hong kong/wf10/1999 (h9n2) [23] and the cold-adapted a/ann arbor/6/1960 (h2n2) [24] strains, herein referred to as wf10 and aa60 ca , respectively (table 1 ). in addition, virus rescue was performed in the context of the h1n1pdm background ( table 1) . regardless of the rg virus background used, virus rescued was possible. this was particularly the case using the wf10 background in which high virus titers (1.58610 6 tcid 50 /ml) in the initial co-transfected cells were obtained with either pol1ha pdm t1 or pol1had 072 t1 ha pcr amplicons (h1 pdm pcr:7wf10 and h5d 072 :7wf10, respectively, table 1 ). when passaged into eggs, virus titers increased significantly, about 1,000 fold for the h5d 072 :7wf10 virus. virus titers below or just at the limit of detection were observed in cells co-transfected in the context of 7 rg plasmids from the aa60 ca strain carrying either the pol1ha pdm t1 (h1 pdm :7aa60 ca ) or pol1ha d072 t1 (h5d 072 :aa60 ca ) ha pcr amplicons or the control plasmid ph1pdm (ph1 pdm :7aa60 ca ). however, a .1,000 fold increase in virus titers were observed after blind passage in eggs of supernatants of the aa60 ca -based co-transfected cells from these reassortant groups (table 1 ). in the 2+6 mode, efficient virus rescue was also obtained. no statistical differences were observed in rescue efficiency between the flu ha pcr amplicon plus 7 pr8 rg plasmids pol1egfputr and 3p and np plasmids, d) utregfputr (without hpol1 and t1 sequences) and 3p and np plasmids, e) phw72-egfp plasmid and 2p (minus pb1) and np plasmids, and f) pol2pb2bgh, pol2pb1bgh, pol2pabgh, and pol2npbgh amplicons and phw72-egfp plasmid. pictures obtained at 24 hpt (206 magnification using the carl zeiss axiophot photomicroscope and uv filter). the amplicons were amplified by the method described in the supplemental information. doi:10.1371/journal.pone.0046378.g002 (h1 pdm :7pr8) compared to the ha and na amplicons and 6 pr8 rg plasmids (h1n1 pdm :6pr8). if the wf10 background was used, co-transfection of the ha and na amplicons resulted in approximately 10 fold less virus (h1n1 pdm :6wf10) in the supernatant of transfected cells compared to using the ha amplicon alone (ha pdm :7wf10). a similar trend was observed when the aa60 ca background was used (compare reassortants h1n1pdm:6aa60 ca with ha pdm :7aa60 ca ). dh5n1 viruses were rescued from the 072 strain using the amplicons pol1ha d072 t1 and pol1na 072 t1 co-transfected with either the pr8, wf10, or aa60 ca backgrounds with efficiencies similar to those obtained using the ha and na amplicons from the h1n1pdm strain. it must be noted that reassortant viruses carrying 7 gene segments from either wf10 or aa60 ca encode a n2 na subtype, which may have affected the rescue efficiency of the h1 pdm or dh5 ha gene segments. nevertheless, the results showed that either the 1+7 or 2+6 strategies using flu pcr amplicons is a suitable method to speed up the recovery of influenza viruses by rg. since the pcr strategy with pol1 and t1 signals was initially used, we wanted to determine whether amplicons lacking the t1 signal could be better substrates for the generation and subsequent amplification of vrna segments. pcr amplicons were prepared and designated as pol1ha pdm utr or pol1na pdm utr using the overlapping pcr method mentioned above, and in which the t1 signal was omitted. similarly, pcr amplicons for ha and na lacking both the pol1 and t1 signals were prepared and used as controls. using either the pr8 or h1n1pdm virus backgrounds, virus rescue was possible with ha and na pcr amplicons lacking the t1 signal, although the rescue efficiency was 100,200 fold lower overlapping pcr strategy and reconstitution of ha and na pcr amplicons. the strategy to produce full-length ha and na pcr amplicons was based on amplification of the pol1 promoter from the pdp2002 vector, subtype specific internal primers for ha and na and, depending on the product, primers containing a t1 termination signal. a) the ha pdm was amplified with the following overlapping fragments: 1, fragment spanning the primer set pt1fragfwd and swha-931r (which incorporates the t1 signal), 2, fragment spanning from the swha-752f and polfragrev, and 3, poli promoter fragment amplified using polf and hpol1rev primers. the three pcr products above were purified by agarose gel electrophoresis and combined in equal proportions (10 ng each) to create the full length pol1ha pdm t1 pcr amplicon using the primer pair pt1fragfwd and hpol1rev. b) an overlapping pcr strategy to produce an h5 ha segment in which the polybasic cleavage site from the chicken/north sumatra strain was removed and replaced by the sequence of low pathogenic virus following the strategy described above. c) and d) depict the strategies used for generation of full length n1 pcr products from the h1n1 pdm and 072 h5n1 strains. e) alternative pcr products were generated to serve as controls for pcr-based reverse genetics. agarose gel shows the expected size for lane 1, hpol1 pcr amplicon; lane 2, generuler tm 1 kb plus dna ladder; lane 3, pol1ha pdm t1; lane 4, pol1ha pdm utr; lane 5, pol1na pdm t1; lane 6, pol1na pdm utr; lane 7, utrha pdm utr; and lane 8, utrna pdm utr. f) polha d072 t1 (lane 1) and polna d072 t1 (lane 3) amplicons were prepared following the strategy described above and depicted in b) and d), respectively. lane 2 corresponds to generuler tm 1 kb plus dna ladder (75-20,000 bp) from fermentas inc (lane 2). all the pcr products were amplified here and in the supplemental information. doi:10.1371/journal.pone.0046378.g003 than using the same amplicon with the t1 signal. after passage in mdck cells, virus titers of these reassortants (ha pdm utr:7pr8, ha pdm utr:7pdm, h1n1 pdm utr:6pr8, and h1n1 pdm utr:7pdm) were increased although they were 10,30 fold lower than those obtained with the full-length t1 signal-containing amplicons (ha pmd :7pr8, ha pdm :7pdm, h1n1 pdm :6pr8, and h1n1 pdm :7pdm, table 1 ). these results are consistent with the previous observation using the flu gfp amplicon lacking the t1 signal, which suggests that the presence of the t1 signal helps generate optimal full-length flu pcr amplicons. to expand the potential use of pcr amplicons as a suitable surrogate system to recover influenza viruses without the use of plasmids, each virus segment was amplified to generate a full set of flu pcr amplicons encoding each one of the viral segments ( fig. s 3) . the optimal cocktail of eight pcr amplicons, based on the ha and na genes of the h1n1 pdm and 6 other amplicons from the pr8 strain, consisted of polpb1 pr8 t1 (1 mg), polpb2 pr8 t1 (1 mg), polpa pr8 t1 (1 mg), polnp pr8 t1 (1 mg), polha pdm t1 (0.5 mg), polna pdm t1 (0.5 mg), polm pr8 t1 (0.5 mg), and polns pr8 t1 (0.3 mg), along with the 3p (1 mg each) and np (1 mg) expression helper plasmids. the mixture was transfected into co-cultured 293t/mdck cells with a ratio of 500/1 and at a density of 5610 5 cells. it resulted in low efficiency virus rescue with a titer of 1.58610 2 tcid 50 after 72 hpt with no detectable ha titer (table 1 , 8pcr:3p/np (pr8) virus). blind passage in mdck cells, resulted in virus titers in the order of 1610 6 tcid 50 /ml with an ha titer of 128. these studies were further expanded to include a full set of pcr products in which the 3p and np expression plasmids were replaced by the corresponding pol2pb2bgh, pol2pb1bgh, pol2pabgh, and pol2npbgh amplicons in a reaction including 12 pcr amplicons and no plasmids ( because vero and mdck cells have been approved for influenza vaccine production, we investigated whether flu pcr amplicon rescue, either in 1+7 or 2+6 modes, was possible in these cells. vero cells co-transfected with the flu ha (alone or in combination with the na) pcr amplicon from the h1n1pdm strains and 7 (or 6) pr8 rg plasmids resulted in virus rescue that was observed at 120 hpt (,10 2 tcid 50 /ml) with about 500 fold lower efficiency compared to the whole plasmid-based system ( table 2 ). blind passage of supernatants of vero cells at 72 hpt into mdck cells resulted in virus titers similar to those obtained using the whole plasmid rg system (around 10 7 tcid 50 /ml). using the 1+7 approach, virus rescue was also possible in mdck cells with ha pcr amplicons from two h5n1 strains, 072 and a/viet nam/1203/2004 (vn1203) ( table 3 ). in this case, ha pcr amplicons were prepared carrying the canine pol1 promoter (k9pol1) and termination signals (k9t1). both ha genes were amplified using overlapping pcrs that removed the gene's polybasic cleavage site sequences (fig. s 4) . co-transfections of the ha pcr amplicons and 7 k9pol1-driven rg plasmids from the vn1203 strain in mdck cells resulted in virus rescue with titers of ,10 5 tcid 50 /ml at 120 hpt and 10 8 tcid 50 /ml after blind passage in mdck cells (table 3 , reassortant viruses had 072 :7vn1203 and had vn1203 :7vn1203). like in the previ-ous transfection studies, removing the k9t1 signal from the pcr products resulted in impaired virus rescue (reassortant viruses had 072 utr:7vn1203 and had vn1203 utr:7vn1203), and removing both the k9pol1 and k9t1 sequences resulted in no virus rescue. in this report, a significant modification was introduced to the plasmid-based reverse genetics system [25, 26] for influenza based on pcr amplicons. in order to optimize and maximize amplification of the genes of interest, a strategy involving overlapping pcr fragments for each segment was designed and used in conjunction with a high fidelity polymerase and corresponding buffer, phusion high-fidelity pcr master mix with gc buffer (new england biolabs). this enzyme performed the best in our hands, compared to seven other commercially available dna polymerases (supplementary materials and methods s1). the synthesis of full length flu pcr amplicons implies producing overlapping pcr fragments with distinct differences in gc-versus at-rich regions. the human and canine pol1 promoters are approximately 75% gc-rich whereas the ha segment is approximately 60% at-rich (data not shown). the approach succeeded in producing overlapping pcr amplicons for the ha and na segments from different subtypes, including h1n1pdm, h5n1, or h9n2 (not shown) and from the 6 internal gene segments of the pr8 strain by designing overlapping primers and optimizing the pcr conditions (fig. s 2) . the amount obtained in each reaction for full length flu pcr amplicons was in the order of 1,5 mg (fig. 2) , which is sufficient for transfection and virus rescue and comparable to the amount of plasmid dna used for transfection in the conventional plasmid-based rg system (table 1) . for the h5n1 vaccine candidates, the polybasic cleavage site (rerrrkkr) in highly pathogenic strains was easily removed and replaced by a low pathogenic sequence (tetr) by virtue of adequate set of primers and overlapping pcr (table s1, fig. 3 and fig. s 2) . plaque assays revealed that the rescued h5d 072 pcr:7pr8 virus was strictly dependent on the presence of tpck-trypsin, which strongly suggests that the virus viruses recovered by reverse genetics were used to infect mdck cells at a moi of 0.001. at 36 hpi, cells were fixed with 4% paraformaldehyde in pbs solution (santa cruz) and viral antigen detected by ifa using subtype specific monoclonal antibodies and fitc-labeled antimouse antibodies (green). counterstaining was performed with propidium iodide (red). a) mab 3b2 specific for the ha h1n1 pdm virus has no reaction with the ha of pr8 strain (only cell nuclei can be seen in red reacting with propidium iodide) (x10). b) and c) positive viral antigen green signal detected using mab 3b2 in cells infected with either the h1n1 pdm utr:6pr8 or h1n1 pdm :6pr8 viruses, respectively (x10). d) mab dpjy01 specific to h5n1 virus has no reaction with pr8-infected cells (x20). e) positive reaction of mab dpjy01 cells infected with h5d 072 n1:6pr8 (x20). f) sequence of ha cleavage site of h5d 072 n1:6pr8 virus indicates the presence of a low pathogenic pattern (tetr), which was created by overlapping pcr as indicated in the materials and methods and fig. 3b . g) plaque assay was performed as described in materials and methods. the pr8 and h5d 072 :7pr8 viruses form plaques in agarose plates in the presence of 1 mg/ml tpck-trypsin but not in its absence. doi:10.1371/journal.pone.0046378.g004 preparation was devoid of any wild type virus contaminant (fig. 4c . this is not surprising since each overlapping pcr fragment is produced and purified independently prior to assembly the reaction to generate the full length pcr product. in addition, a low mutation rate was observed with approximately 8% of the plaque-purified viruses having a single nucleotide mutation. it is not fully understood at this stage whether those mutations are naturally present in the wild type 072 virus. notably, the ha gene of clone 43 (table s2 ) contained a mutation at aa position 151, which was shown related to a switch of receptor specificity from a 2,3 to a 2, 6 linked sialic acids [27] . since the 072 virus used in this report has undergone more than one passage in eggs, it is possible that the homogeneity of its population had contributed to the finding of the small mutation rate among the h5d 072 pcr:7pr8 clones. nevertheless, the results clearly show that the pcr-based rg strategy does not create a heterogeneous virus population. in this regard, it could be argued that the pcr-based approach will be most likely better represent the virus population present in the original virus isolated compared to the plasmid-based system. since it was not clear whether the presence of the t1 signal would have either a positive or negative effect on the production of full-length flu pcr amplicons, it was important to determine whether run off pol1 transcription would make a difference in virus rescue efficiency using pcr amplicons. the results show that the presence of the t1 signal at the 39 end of the flu pcr amplicon greatly improved virus rescue efficiency, approximately 100-200 fold (tables 1 and 3 ). this observation was consistent throughout the studies since omitting the t1 signal resulted in less efficient amplification of the flu gfp replicon (fig. 2c ) and significantly less virus rescue in both human and canine cells (tables 1 and 3) . the ha and na pcr amplicons contributed to produce virus efficiently in the background of not only the laboratory adapted strain pr8 but also in other strains like the aa60 ca live attenuated vaccine strain or the wf10 and h1n1pdm wild type strains (table 1) . furthermore, the approach is not limited to production of virus from transfected 293t or co-cultured 293t/mdck cells. transfection of vero cells in a 1+7 and 2+6 mode also resulted in virus rescue (table 2 ). virus rescue of the dh5n1 1+7 virus in mdck cells, with the attenuated ha pcr amplicons carrying the k9pol1 and k9t1 signals, was also successful ( table 3) . the boundaries of the system were extended by showing that a complete set of 8 flu pcr amplicons can be effectively recovered by rg in the context of 4 expression plasmids encoding the influenza polymerase complex (table 1) . more importantly, an expression competent pcr version of the 3p and np was also effective in rescuing the virus in a transfection reaction that contained no plasmids (table 1 ). our studies imply that protein expression studies could be performed without preparation of clones, although, it must be emphasize that systematic studies to evaluate the differences in protein expression of pol2-based pcr amplicons and their corresponding plasmids were not performed. although it can be argued that rescue efficiency was lower than using a plasmid-based approach, further optimization of the pcrbased system is likely possible by manipulating the amount and proportion of each amplicon in the transfection. such analysis is beyond the scope of the present report. it could also be argued that the pcr-based system may produce a more variable virus population than the one that is obtained using the plasmid-based system. although there is such possibility, it is an inherent nature of influenza viruses to evolve through point mutations and therefore no reverse genetics system is error free. however, the sequencing of reassortants produced in this study does not show mutations that would alter the antigenicity of the ha surface proteins. in this regard, for vaccine development, as long as the vaccine seed stock is antigenically identical to the vaccine candidate, other mutations would be irrelevant. in fact, inactivated influenza vaccines prepared by classical reassortment have only two pre-requisites: 1) ha surface gene derived from the vaccine candidate and 2) high growth in eggs. full genome sequencing of vaccine viruses is not a pre-requisite for approval of the vaccine by the fda. overall, the implications of the pcr-based approach for rg development are highly significant. using a combination of pcr amplicons and plasmids, it would be possible to streamline the study of gene variants for one or more gene segments and determine fitness, pathogenesis or any other biological aspect of the virus. several mutant viruses with mutations in one or more genes could be produced without having to prepare individual clones. recovery of viruses entirely from pcr products implies that other viral systems could be amenable to a similar strategy. this could be particularly important for viruses with genomes larger than the influenza virus that are occasionally associated with cloning difficulties or plasmid instability. in fact, pcr amplification for the purpose of virus generation has already been done for coronaviruses, although the pcr product was used as template for in vitro rna transcription and the resulting rna was later transfected into cells [28] . in summary, a rg system for influenza was developed that does not require a cloning step for recovery of viruses and has profound implications for vaccine development, pandemic preparedness, and for the study of influenza viruses. the mouse-adapted h1n1pdm virus and wf10 viruses have been previously described [22, 23] . the highly pathogenic h5n1 virus strain a/chicken/northsumatra/mdn/072/ 2010 (strain 072) was a gift from teguh prajitno (vaksindo, indonesia) and sent to us by ron fouchier (erasmus medical center, the netherlands). the vn1203 virus (h5n1 clade 1) was obtained from the centers for disease control and prevention, atlanta, ga (cdc). the influenza pr8 strain was grown from a reverse genetics clone obtained from peter palese, mount sinai school of medicine, new york, ny (h1n1) (pr8). virus stocks were prepared in specific pathogen free 9-day old embryonated chickens eggs following standard techniques for growth of influenza viruses. mdck and vero cells were maintained in modified eagle's medium (mem) (sigma-aldrich, st. louis, mo) containing 5% fetal bovine serum (fbs) (sigma-aldrich). human embryonic kidney cell-line 293t (hek293t) were cultured in opti-mem i (gibco, grand island, ny) containing 5% fbs. the rg 8-plasmid system for the pr8 virus was a gift from dr. peter palese, mount sinai school of medicine, new york, ny. the rg system for the ann arbor cold adapted (ca) virus was produced from the homologous strain provided to us by ruben donis, cdc, and cloned into the pdp2002 vector (sutton et al, unpublished). the rg 8-plasmid systems for the h1n1pdm and wf10 have been previously described [22, 29] . the bidirectional pgd2007 rg vector containing the canine pol1 and t1 sequences was a gift from ruben donis. the 8 gene segments of vn1203 were subcloned into the pgd2007 vector from clones originally prepared in the pdp2002 vector (data not shown). the plasmids prf1428 and prf1437 were provided by ron fouchier and encode the wt h5 ha and dh5 ha genes, respectively, from the 072 virus. the plasmids pcdna774pb1, pcdna762pb2, pcdna787pa and pcdna693np have been previously described [30] . the phw72-egfp plasmid encoding the influenza egfp reporter replicon was a gift from robert webster, st jude children's research hospital. memphis, tn [21] . the vrnas and cdnas from wild type and blind passage reassortant viruses were prepared as previously described [22, 31] . briefly, total rnas were extracted by using the rneasy kit (qiagen, valencia, ca) following manufacturer's instructions. reverse transcription was carried out with the uni12 primer (59-agcaaaagcaagg-39) and avian myeloblastosis virus (amv) reverse transcriptase (promega, madison, wi). the cdnas were stored at 280uc until use. the flu egfp replicon was amplified with the primers pt1fragfwd and hpol1rev using phw-egfp plasmid dna as the template [30] . pcr amplicons from pcdna762 (pb2), pcdna774 (pb1), pcdna787 (pa) or pcdna693 (np) were generated with the primers pcmvf and pbghr, and were designated as pol2pb2bgh, pol2pb1bgh, pol2pabgh and pol2npbgh, respectively [21, 30, 32] . the pol2-based flu pcr amplicons were flanked by sequences corresponding to the immediate-early human cytomegalovirus (cmv) promoter and bovine growth hormone (bgh) polyadenylation signal. pcr conditions were similar to the overlapping pcr of pol1ha pdm t1 and pol1na pdm t1 amplicons except for the use of 10 pg of the corresponding plasmid dna template (see below). after pcr amplification, two methods were used to demonstrate that pcr products were devoid of spurious plasmid dna contamination. pcr products were digested with dpn i (new england biolabs, ipswich, ma) for 1 h, and then separated and purified by agarose gel electrophoresis, and subsequently 100 ng of the purified pcr product were used to transform e. coli top10 cells (invitrogen, carlsbad, ca). alternatively, the primer pairs, pcmvf and pt1fragrev, pdp2066-2090f and pdp2392-2416r, and pt1-2f and utr-h1rev were used to demonstrate lack of plasmid dna contamination in purified pcr reactions (table 1) . this strategy was also used to demonstrate no plasmid dna contamination after pcr amplification of the pol1 promoter from the rg vector pdp2002. in all instances, no plasmid dna contamination was observed by either method (data not shown). pcr strategy of overlapping ha and na gene segments with human pol1 promoter a schematic representation of the overlapping pcr approach is shown in fig. 2 using the set of primers described in table s1 . two overlapping pcr fragments were generated for the ha pdm gene: the first fragment spans from the primer set pt1fragfwd, which incorporates the t1 signal, and swha-931r. the second fragment was amplified with the primer pair swha-752f and polfragrev. the human pol1 promoter was pcr amplified using primers polf and hpol1rev and the pdp2002 plasmid vector as template. details of amplification conditions for each pcr fragment are provided with the supplementary information (fig. s. 2a and b) . the 3 pcr products above were purified by agarose gel electrophoresis and combined in equal proportions to generate a full-length ha pcr amplicon using the primer pair pt1fragfwd and hpol1rev. the 50 ml pcr reaction mixture contained 10 ng of each pcr product, 25 ml of master pcr mix, 1.5 ml 100% dmso, and 50 pmol/ml of each primer. pcr reaction conditions were 98uc for 30 sec, and then 30 cycles at 98uc for 8 s, 56uc for 1 min and 72uc for 3 min, ending with 72uc for 10 min. pcr products were amplified using the phusion high-fidelity pcr master mix with gc buffer (new england biolabs, ipswich, ma). alternative ha pcr products without t1 signal sequence or lacking both pol1 and t1 elements were generated to serve as controls for pcr-based reverse genetics (fig. 2 and table 2 ). pcr products were purified by agarose gel electrophoresis and quantitated after gel purification using nanodrop 1000 (nanodrop, wilmington, de). overlapping pcr products were produced for the na pdm gene using the primer pair ptifragfwd and swna-763r and n1-562f and polfragrev, whereas the full length na pcr amplicon was generated with the primer pair pt1fragfwd and hpol1rev. alternative full-length na amplicons were generated using specific primers as noted in table 1 . similar strategies were used to amplify the had 072 and had vn1203 pcr products in which the polybasic cleavage site sequence were removed using overlapping pcr products spanning sequences from the primer pairs ptifragfwd and indoh5-clvr and indoh5-clvf and polfragrev whereas the full length ha pcr amplicon as generated with the primer set pt1fragfwd and hpol1rev. the full length na gene segment from indo072 strain was amplified without the generation of internal overlapping fragments using the primer set htin1fwd and poln1rev rather and then subsequently introduced in a pcr reaction to generate the full-length pol1na 072 t1 pcr amplicon carrying the pol1 promoter (table s1 ). to compare the rescue efficiency of pcr amplicons, prf1437 plasmid was also used as control. to set up a reverse genetics system using a full set of pcr amplicons, the h1n1 pdm surface gene segments and the pr8 virus 6 internal gene segments were selected. the internal genes were amplified from the total cdnas prepared from a wildtype pr8 strain, with an initial titer of 1.58610 9 tcid 50 /ml in mdck cells. for the overlapping pr8 pb2 pcr amplicon with pol1 and t1 signal sequences, the ptifragfwd and pb2-1811r primers were used to produce the n terminal fragment of pb2 (pb2-n), and then the pb2-1643f and polfragrev primers were used to produce the c terminal fragment (pb2-c). pb2-n, pb2-c, and pol1 fragments were mixed (10 ng each), and the overlapping pcr reaction was performed using primers pt1fragfwd and hpol1rev. the pcr parameters were similar to those of pol1ha pdm t1 and pol1na pdm t1 amplicons described above. the final product was labeled pol1pb2 pr8 t1. a similar strategy was followed for other gene segments fused to pol1 and t1 signal sequences, using the primer pairs: pb1-1240f/pb1-1531r, pa-892f/pa-1314r, ha-760f/ha-1274r, np-1116f/np-1441r, na-743f/ na-905r, m-741f/m-915r, or ns-469f/ns-887r primers respectively. final pcr amplicons were designated as pol1pb1 pr8 t1, pol1pa pr8 t1, pol1ha pr8 t1, pol1np pr8 t1, pol1na pr8 t1, po1m pr8 t1, and pol1ns pr8 t1, respectively. additional details are provided with the supplementary information (fig. s. 3 ). overlapping pcr with canine pol1 promoter the generation of the ha pcr amplicon from h5n1 containing the k9pol1 promoter was performed as follows: the k9pol1 promoter was amplified from the pgd2007 vector using the primer pair k9pol1f and k9pol1r under these conditions: the initial denaturation at 98uc for 30 sec, 30 cycles of 98uc for 8 sec, 60uc for 30 sec, and 72uc for 1 min and the final extension at 72uc for 10 min (table s1 ). overlapping ha fragments, one containing the k9t1 signal was produced with the primer pair ktiuni12f and indoh5-clvr (h5n1) and the second one with the primer pair indoh5-clvf (h5n1) and kpolutrr under the following conditions: pre-pcr treatment at 98uc for 30 sec, 30 cycles of 98uc for 8 sec, 56uc for 30 sec, and 72uc for 1 min and the final extension at 72uc for 10 min (table s1 ). the amount of overlapping ha pcr products and the k9pol1 amplicons were calculated and mixed together at a concentration of 10 ng each. a full length ha pcr amplicon was generated with the primer pair k9tiuni12f and k9pol1r. the thermal profile was: denaturation at 98uc for 30 sec, 30 cycles of 98uc for 8 sec, 56uc for 1 min, and 72uc for 4 min, and then extension at 72uc for 10 min. all the pcr products were amplified using the phusion high-fidelity pcr master mix with gc buffer. for partial plasmid-free rescue, the plasmid of choice was replaced with the corresponding flu pcr amplicon and virus rescue performed essentially as described [26] with minor modifications. briefly, co-cultured 293t/mdck cells at a ratio of 500:1 (5610 5 cells per well) was seeded into each well of a 6-well tissue culture plate. the plates were incubated at 37uc overnight. the following day, 1 mg of each plasmid or flu pcr amplicons was incubated for 45 min with 16 ml of transit-l1 transfection reagent (mirus bio llc, madison, wi ) and then the transfection allowed to occur overnight before the media was replaced with fresh serum-free opti-mem. at 24 h post-transfection (hpt), l-(tosylamido-2-phenyl) ethyl chloromethyl ketone (tpck)-treated trypsin (1 mg/ml) was added to the cell supernatants. mdck and vero cells were grown to 70% confluency in 75cm 2 flasks and then trypsinized with trypsin-edta (invitrogen) and resuspended in opti-mem i containing 5% fbs. cell suspensions were seeded into 6-well tissue culture plates and incubated at 37uc overnight before transfection. transfections and post-transfection steps proceeded as described for the 293-t/ mdck co-cultured cells, except that vero cells were incubated with 2 mg/ml of tpck-trypsin. supernatant of transfected cells were collected at the times indicated in tables 1, 2 and 3 and blind passage in either or both mdck cells or 10-day old embryonated chicken eggs to monitor for the presence of rescued viruses. tcid 50 titers were determined in mdck cells by the reed and muench method as described [33] . virus stocks were prepared and frozen at 280uc until use. the rescued viruses were examined by plaque assay in mdck cells (30) . briefly, confluent cell monolayers in 6-well plates were infected with 10-fold dilutions of virus in a total volume of 0.4 ml pbs for 1 h at 37uc. cells were washed twice with pbs and covered with an overlay of modified eagle's medium containing 0.9% agar, 0.02% bsa, 1% glutamine, and, when noted, 1 ug/ml tpck treated-trypsin. the plates were then incubated at 37uc under 5% co 2 . after 3 days of incubation the overlays were removed and the cells were stained with 0.1% crystal violet. for sequencing the ha gene from plaque-purified viruses, the agarose was plucked from areas of the plate were plaques were observed. viruses were eluted from the agarose pluck in tissue culture media and later expanded in mdck cells for 72 h prior to rna extraction, cdna synthesis, pcr, and sequencing. antisera against the dh5n1 indo072 virus were raised in chickens by infecting them with a live attenuated version of the dh5n1 indo072 virus in the background of the wf10att virus similar to as previously described [34] . collected sera were treated with receptor-destroying enzyme (accurate chemical and scientific corp., westbury, ny) prior to hi assays as described in the who animal influenza training manual (who/cds/csr/ ncs/ 2002.5) (30). sequencing of overlapping pcr products, viral cdnas and ha sequences from the purified clones of pr8 or rescued reassortants was performed using a combination of universal primers [31] and custom made primers (available upon request) and the big dye terminator v3.1 cycle sequencing kit (applied biosystems, foster city, ca) on a 3500 genetic analyzer (applied biosystems, foster city, ca) according to the manufacturer's instructions. sequence analysis was performed using software available through the lasergene package (dnastar inc., madison, wi). cells grown in 96-well plates were infected with rescued influenza viruses at a dose of 1 tcid 50 /well. at 36 hpi, the cells were washed in precooled 0.01 m phosphate buffered saline (pbs) buffer and fixed in neutral formaldehyde for 20 min at room temperature. the cells were then incubated with blocking solution (10% normal goat serum in pbs) for 1 h and probed with a primary antibody for 30 min. two monoclonal antibodies were used to identify the recombinant influenza viruses: mab 3b2 is specific for the ha protein of h1n1pdm viruses and which does not react with the ha of pr8 and other subtypes of influenza a viruses [35] . mab dpjy01 is specific for the ha of h5 subtype influenza viruses [36] . the antibody-antigen complexes were further incubated with fluorescein isothiocyanate (fitc)-conjugated goat anti-mouse ig (h+l) (southernwest biotech associates inc, birmingham, al) for 30 min at room temperature. the cells were washed three times with pbs after incubation and then counterstained with propidium iodide (pi) and examined under an axiophot photomicroscope produced by carl zeiss (lex of 488/ 543 nm, lem of 522/590 nm for 100 ms). materials and methods s1 enzymes and kits used to generate full-length flu pcr amplicons. figure s2 generation of ha and na amplicons. cdnas from the h1n1pdm and h5n1 072 viruses were prepared as described in the main text. m, generuler tm 1 kb plus dna ladder. lane 1, unspecific pcr products obtained using one-step rt-pcr to generate the full length of ha pdm gene (1,840 bp) with the primers pt1hf and polhr. lane 2, the n terminus of ha pdm specific pcr product (998 bp) obtained using the primer pair pt1fragfwd, which incorporates the t1 signal, and swha-931r. lane 3, the c terminus of overlapping ha pdm specific pcr product (1,022 bp) using the primer pair swha-752f and polfragrev. lane 4, the n terminus of na pdm specific pcr product (799 bp) obtained using the primer pair ptifragfwd and swna-763r. lane 5, the c terminus of na pdm specific pcr product (924 bp) from n1-562f and polfragrev primer set. lane 6, the first had 072 specific pcr fragment (1,090 bp) obtained with the primers ptifragfwd and indoh5-clvr. lane 7, the second ha d072 specific pcr fragment (762 bp) obtained with the primers pair indoh5-clvf and pol1fragrev. lane 8, the full-length na d072 amplicon (1,460 bp) generated with the primer set htin1fwd and poln1rev. the 25 ml pcr reaction mixture contained 10 ng of cdnas, 12.5 ml of master pcr mix, 0.6 ml 100% dmso, and 10 pmol/ml of each primer. the pcr reaction conditions were 98uc for 30 sec, and then 30 cycles at 98uc for 8 s, 56uc for 1 sec and 72uc for 2 min, ending with 72uc for 10 min. pcr products were amplified using the phusion high-fidelity pcr master mix with gc buffer. (468 bp) with primer pair ns-469f and polfragrev. pcr conditions were similar to those described in sfig 2. b) full-length pr8 pcr amplicons. overlapping pcr products generated in a) were mixed at a concentration of 10 ng (each product) and amplified with the forward primer pt1fragfwd and the reverse primer hpol1rev as described in the main text. the thermal profile was: denaturation at 98uc for 30 sec, 30 cycles of 98uc for 8 sec, 56uc for 2 min, and 72uc for 4 min, and then extension at 72uc for 10 min. all the pcr products were amplified using the phusion high-fidelity pcr master mix with gc buffer. the final overlapping pcr amplicons were designated as pol1pb2 pr8 t1 figure s4 ha pcr amplicons flanked with k9pol1 promoter. two produce overlapping pcr products for ha d072 and ha dvn1203 gene segments. pcr conditions used were similar to those described in sfig 2 and in the main text. lane 1, the k9pol1 promoter (351 bp) was amplified from the pgd2007 vector using the primer pair k9pol1f and k9pol1r. lane 2, pcr fragment containing the n-terminus of ha d072 and the k9t1 signal (36 bp) was produced with the primer pair ktiuni12f and indoh5-clvr with a size of 1,091 bp. lane 3, pcr fragment containing the c-terminus of ha d072 (760 bp) produced with the primer pair indoh5-clvf and kpolutrr. lane 4, pcr fragment containing the n-terminus of ha dvn1203 (1,091 bp) amplified as in lane 2. lane 5 pcr fragment containing the c-terminus of ha dvn1203 (760 bp) produced as in lane 3. lane 6, the two overlapping ha d072 pcr products and the k9pol1 pcr fragment were mixed at a concentration of 10 ng (each product) to generate the full length of k9pol1ha d072 t1 pcr amplicon (2,144 bp) using the primer pair ktiuni12f and k9pol1r. lane 7, the two overlapping ha dvn1203 pcr products and the k9pol1 pcr fragment were mixed at a concentration of 10 ng each and amplified to generate the full length k9pol1ha dvn1203 t1 amplicon (2,144 bp) using the primer pair ktiuni12f and k9pol1r. lane 8, same as in lane 6, except that k9pol1ha d072 utr (2, 109 bp) lacks the k9t1 signal after amplification with the primer pair bm-ha-1f and k9pol1r. lane 9, same as in lane 7, except that k9pol1ha dvn1203 utr (2,109 bp) lacks the k9t1 signal after amplification. m, generuler tm 1 kb plus dna ladder. (jpg) influenza viruses in animal wildlife populations in vivo analysis of the promoter structure of the influenza virus rna genome using a transfection system with an engineered rna the synthesis of influenza virus negative-strand rna takes place in insoluble complexes present in the nuclear matrix fraction the influenza a virus pb2 polymerase subunit is required for the replication of viral rna the molecular anatomy of influenza virus rna polymerase molecular anatomy of 2009 influenza virus a (h1n1) influenza a virus pb1-f2 gene a novel influenza a virus mitochondrial protein that induces cell death an overlapping protein-coding region in influenza a virus segment 3 modulates the host response de novo replication of the influenza virus rna genome is regulated by dna replicative helicase influenza virion-derived viral ribonucleoproteins synthesize both mrna and crna in vitro different de novo initiation strategies are used by influenza virus rna polymerase on its crna and viral rna 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potential for strains with altered virulence phenotype? role of quail in the interspecies transmission of h9 influenza a viruses: molecular changes on ha that correspond to adaptation from ducks to chickens multiple amino acid residues confer temperature sensitivity to human influenza virus vaccine strains (flumist) derived from cold-adapted a/ann arbor/6/60 rescue of influenza a virus from recombinant dna a dna transfection system for generation of influenza a virus from eight plasmids acquisition of human-type receptor binding specificity by new h5n1 influenza virus sublineages during their emergence in birds in egypt reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus replication and transmission of h9n2 influenza viruses in ferrets: evaluation of pandemic potential the matrix 1 protein of influenza a virus inhibits the transcriptase activity of a model influenza reporter genome in vivo universal primer set for the full-length amplification of all influenza a viruses functional analysis of pa binding by influenza a virus pb1: effects on polymerase activity and viral infectivity a simple method for estimating fifty percent endpoints a new generation of modified liveattenuated avian influenza viruses using a two-strategy combination as potential vaccine candidates a novel monoclonal antibody effective against lethal challenge with swine-lineage and 2009 pandemic h1n1 influenza viruses in mice intranasal delivery of an iga monoclonal antibody effective against sublethal h5n1 influenza virus infection in mice we would like to express our gratitude to qiong chen and johanna lavigne for their technical assistance. key: cord-321705-6a7avlro authors: hou, tianya; zhang, taiquan; cai, wenpeng; song, xiangrui; chen, aibin; deng, guanghui; ni, chunyan title: social support and mental health among health care workers during coronavirus disease 2019 outbreak: a moderated mediation model date: 2020-05-29 journal: plos one doi: 10.1371/journal.pone.0233831 sha: doc_id: 321705 cord_uid: 6a7avlro purposes: during the outbreak of coronavirus disease 2019 (covid-19) all over the world, the mental health conditions of health care workers are of great importance to ensure the efficiency of rescue operations. the current study examined the effect of social support on mental health of health care workers and its underlying mechanisms regarding the mediating role of resilience and moderating role of age during the epidemic. methods: social support rating scale (ssrs), connor-davidson resilience scale (cd-risc) and symptom checklist 90 (scl-90) were administrated among 1472 health care workers from jiangsu province, china during the peak period of covid-19 outbreak. structural equation modeling (sem) was used to examine the mediation effect of resilience on the relation between social support and mental health, whereas moderated mediation analysis was performed by hayes process macro. results: the findings showed that resilience could partially mediate the effect of social support on mental health among health care workers. age group moderated the indirect relationship between social support and mental health via resilience. specifically, compared with younger health care workers, the association between resilience and mental health would be attenuated in the middle-aged workers. conclusions: the results add knowledge to previous literature by uncovering the underlying mechanisms between social support and mental health. the present study has profound implications for mental health services for health care workers during the peak period of covid-19. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the coronavirus disease 2019 (covid19) as an unprecedented threat has infected more than 2,800,000 people by 27 april, 2020 [1] , which has attracted international attention as a public health emergency of international concern [2] . considerable countries are confronting escalating pandemics and tremendous burden. the availability of skilled health care workers is the decisive factor in overcoming a viral epidemic [3] . in the most affected areas such as hubei province, china where the confirmed cases were first reported, nearly all health care workers work directly with the infectious patients, whereas in the less affected areas, some health care workers directly fight against covid-19 and others are prepared to fight. however, the bravery of health care workers cannot protect them away from mental health problems during covid-19. according to the research during the 2003 severe acute respiratory syndrome (sars) outbreak, health care workers have shown mental health problems with 10% hospital workers reporting higher levels of stress [4, 5] . another study during ebola outbreak presented that health care workers of sierra leone had significant psychological symptoms including depression, interpersonal sensitivity and even paranoid ideation [6] . more importantly, evidence from previous literature showed that the stress levels for high-and low-risk health care workers were equivalent during the outbreak of epidemic [3] . psychological intervention and mental health services were in need to prevent health care workers from being traumatized as they were emotionally affected during the epidemic [6, 7] . mental health is fundamental to an individual's overall well-being and absolutely essential to a productive and efficient life. in workplace, mental health problems are found to be associated with plenty of negative influences, such as reduction of efficiency, loss of productivity, disability and absenteeism [8, 9] . given the adverse impacts, it is of great importance to investigate the potential factors and mechanisms that could enlighten the improvement of the mental health and maintenance of productivity of health care workers in the mist of the epidemic. among all the influential factors, social support has been recognized as one of the protective factors for mental health [10, 11] . thus, the aim of the present study was to replicate the relationship between social support and mental health based on the health care workers during covid-19 outbreak, and further extend the previous studies by exploring the potential mechanisms in the relationship. social support is individuals' perception or experience in terms of being involved in a social group where people mutually support each other [12] . previous research has repeatedly emphasized the role of social support in the promotion of mental health [13] . not only crosssectional studies [14, 15] , but also a large body of longitudinal studies emerging recently [13, 16] have confirmed the positive association between social support and mental health outcome robustly. although a substantial number of research found this strong relation can hold across a broad range of samples, such as cancer patients, patients with multiple sclerosis, nurse students and so on [17, 13, 11] , a handful of studies have presented different results. a meta-analysis conducted by ge, zhao, liu, zhou, guo & zhang [18] has concluded that for the aged people, mental health was weakly or extremely weakly associated with social support. fiori et al. [10] has claimed that the emotional support was only related to mental health in females only, not in males. therefore, it is vital to note the previous studies have found the relation between social support and mental health based on certain samples, however, whether the conclusion could be duplicated to health care workers during the outbreak of covid-2019 still remains unexplored. moreover, the mediating mechanisms (i.e., how social support correlates with mental health?) and moderating mechanisms (i.e., when this relationship is most potent?) underlying the association between social support and mental health also stay largely unknown. answering these questions could be of vital importance to further understand the mental health of medical workers and advance the more effective interventions to ensure the productivity of health care worker during covid-19. thus, the present research employed a sample of chinese health care workers during covid-19 outbreak to explore a conceptual model in which, on the one hand, resilience mediated the association between social support and mental health; on the other hand, the indirect relationships between social support and mental health via resilience were moderated by age group. social support and resilience. resilience is an individual's capacity to deal with significant adversity and quick recover [19] . a great many of studies based on various methodologies and samples have provided robust evidence with respect to the association between social support and resilience. numerous cross-sectional studies have revealed a positive association between social support and resilience [20] [21] [22] . in addition, a longitudinal study conducted by liu, he, jiang, & zhou [23] utilized a sample of adolescents from 5.12 earthquake-hit region and confirmed social support as the protective factor of resilience after a one-year followup. furthermore, a meta-analysis including 14 studies also has concluded that social support, particularly the utilization of the support, could enhance children's resilience [24] . thus, it is possible that social supports could enhance the resilience of health care workers. resilience and mental health. mental health is the critical component of personal development and growth, which is more than the absence of mental illness [25] . plenty of empirical studies reach the consensus that resilience exerts an positive effect on mental health [25] [26] [27] and the association is presented to be consistent across different samples with diverse background [28] [29] [30] . also, psychological resilience can help protect individuals against mental illness and thrive from the adversity [31] . in fact, a few researchers have investigated the relation between social support, resilience and wellbeing [14, 13, 15, 32] . it is worth noting although the previous studies explored the relation, some treated resilience as a covariate or moderator, while others did analyze resilience as a mediator. however, as far as we know, no research has examined whether the relationship between social support and mental health via social support could be applied to the health care workers who are prepared and fight during the epidemic. although social support may have an impact on mental health indirectly via resilience, not all people with lower resilience suffer from lower level of mental health. therefore, it is essential to explore the influential factors that could strengthen or attenuate the link between social support, resilience and mental health. this research examined a hypothesis that the resiliencemental health link in the indirect association between social support and mental health would be moderated by age group. several studies have concluded the possibility of the receipt of depression treatment diminished as getting older [33, 34] . robb, haley, becker, polivka & chwa [35] have compared the similarity and difference between younger and older adults in the attitudes towards mental health service, and found younger adults showed more willing to seek mental health services compared with the elder. in addition, dinapoli, cully, wayde, sansgiry, yu, & kunik [36] have examined the role of age in the prediction of mental health service use. the results have presented that younger adults with depression or anxiety disorders were more likely to utilize mental health service compared with the middle-aged adults. the mental health services include mediation, yoga and stress management, which are mainly focusing on the enhancement of resilience [37, 38] . in sum, younger adults might be more likely to receive resilience training to improve the mental health than middle-aged adults. thus, the mental health of younger adults might rely more on resilience than other factors. furthermore, health care workers during the pandemic are in highly psychological stressing condition when fighting against covid-2019 outbreak [39] . the middle-aged workers usually have longer length of employment and more working experience than the younger workers. meanwhile, the odds of participation in the fight against other epidemics before might be higher for the middle-aged in comparison to the younger. all these past experiences would make them less stressful, less anxious, less fearful and better mental health state when facing the epidemic [40] . taken all together, the mental health of the middle-aged workers would be less dependent on resilience, which indicates the link between resilience and mental health would be attenuated in middleaged adults than younger adults. in fact, a meta-analysis including 60 studies has specifically investigated the moderating role of age in the relation between trait resilience and mental health [25] . however, the study just concluded the relation was stronger for adults than children and adolescence, without the comparison between younger adults and middle-aged adults. unlike the previous research, the current study examined the potential difference between the younger and middle-aged medical workers in the link between resilience and mental health. taken all together, the aims of this research were twofold: (a) to examine whether the mediating role of resilience in social support and mental health could be duplicated to the health care workers from a less affected area during the covid-19 epidemic, and (b) to test whether the relationship between social support and mental health via resilience is moderated by age group. the current study constructed a conceptual model to address both mediation and moderation effects (see fig 1) . based on the literature review, the following hypotheses were proposed: hypothesis 1: resilience would mediated the relationship between social support and mental health of health care workers during covid-19 pandemic. hypothesis 2: age group would moderate the indirect association between social support and mental health via resilience such that the resilience-mental health pathway would be stronger in younger age group in comparison with the middle-age group. given that we suppose age would only moderate the second stage of the mediation path, the present study would call it "a second stage moderation model". the cross-sectional study was conducted from 1 st to 7 th february, 2020, which was the peak period of covid-2019 outbreak in china. the participants were health care workers from local hospitals, community health service centers and government department in jiangsu province who participated in the fight against covid-19. the questionnaires were distributed through internet. all subjects were given informed written consent before completing the online survey concerning demographic information, social support, resilience and mental health. all the subjects were free to withdraw from the research at any time. the research was approved by the ethics committees of the second military medical university. a total of 1521 health care workers completed the survey in the present study. considering the present study was to compare the indirect effect of social support on mental health via resilience between the young and middle-aged heath care workers, participants aged 50 or over were excluded. finally, 1472 subjects were included in the analysis. social support. the social support rating scale (ssrs) developed by xiao was utilized to measure social support [41] . the 10-item scale consists of 3 dimensions including objective support, subjective support and availability. a representative item was "how many close friends do you have to get support and help?". higher scores indicate higher levels of social support. the scale has presented impressive validity and reliability in chinese population [42] . the cronbach's alpha for the present study was 0.949. resilience. the connor-davidson resilience scale (cd-risc) was used to assess resilience [43] . the 25-item likert scale consists of five dimensions: (a) personal competence, high standards, and tenacity; (b) trust in one's instincts, tolerance of negative affect, and strengthening effects of stress; (c) positive acceptance of change and secure relationships; (d) control; (e) spiritual influence [44, 45] . participants rated each item from 0 (not true at all) to 4 (true all the time). the range of total scores is from 0 to 100, with higher scores representing higher levels of resilience. the scale has presented good psychometric properties [43] . in this study, the cronbach's alpha was 0.955. mental health. symptom checklist 90 (scl-90) developed by derogatis and cleary [46] was administrated to evaluate mental health [47, 48] . the 90-item scale is widely applied to measure clinical psychiatric symptoms and differentiate individuals with mental illness from healthy people [49, 50] . each item is rated from 1 (no symptom) to 5 (severe symptom). the higher total scores the participants got, the worse mental health condition they were in. in this research, scl-90 � 160 was defined as psychological abnormality [51] . the scale has shown good validity and reliability in chinese population [52] . in this research, the cronbach's alpha was 0.983. firstly, the present study calculated the descriptive statistics and bivariate correlations among variables of interest by statistical package for social science (spss) 21.0 for windows. a twotailed p-value smaller than 0.05 indicated the presence of statistical significance. secondly, structural equation modeling (sem) conducted by amos 23.0 through maximum likelihood method was performed to examine the mediating role of resilience in the relation between social support and mental health. the model fit index included root mean square error of approximation (rmsea), standardized root mean square residual (srmr), goodness of fit index (gfi) and comparative fit index (cfi). as recommended by previous literature, the values of rmsea and srmr smaller than 0.08 and the values of gfi and cfi more than 0.9 indicate an acceptable fit [53] . bias-corrected bootstrap method was used to examine the significance of mediation effect. specifically, we used 2000 bootstrap samples and determined the bias-corrected 95% confidence interval. if the confidence does not contain zero, it means the significance of the effects [54] . finally, the moderated mediation model was tested by using hayes [55] process macro (model 14). the 95% bias-corrected confidence interval from 5000 resamples was generated by bias-corrected bootstrapping method to examine the significance of moderated mediation effect. the sociodemographic characteristics of the participants and the distribution of scl-90 scores were presented in table 1 . most health care workers were females (76.5%), middle-aged (55.0%), married (73.0), and reported 12-16 years of schooling (64.9) and less than 10 years of working (61.6). the prevalence of psychological abnormality was 7% among health care workers. the mean ± sd total score of scl-90 was 110.28 ± 28.89. there were no significant differences in scl-90 scores associated with gender, age, marital status, years of schooling and years of working. social support was positively correlated with resilience and age group, and negatively correlated with scl-90 scores (all p < 0.001). resilience was positively associated with age groups and negatively associated with scl-90 scores (all p < 0.001). structural equation model was employed to examine the mediating role of resilience. firstly, the direct path coefficient from social support to scl-90 scores in the absence of resilience was significant, γ = -0.39, p < 0.001. secondly, the structural equation model regarding the 2) . a bootstrap procedure conducted to examine the mediation effects. 2000 bootstrapping samples was generated from the original dataset (n = 1472) via random sampling. the indirect effect of social support on scl-90 scores through resilience was -0.120 (se = 0.019, 95%ci = [-0.156, -0.084], p = 0.001). the 95% biased-corrected confidence interval did not contain zero, which verified the indirect relationship between social support and scl-90 scores via resilience. it has been expected that age group would moderate the second stage of the mediation process. as shown in table 2 , in model 1, social support positively predicted resilience, β = 0.408, p < 0.001. model 2 revealed that the effects of resilience on scl-90 scores was moderated by age group, β = 0.126, p < 0.01. for descriptive purpose, the present study plotted the relationship between resilience and scl-90 scores, separately for younger and middle-aged groups (see fig 3) . simple slope test presented that for subjects from younger group, resilience was significantly and negatively associated with scl-90 scores, β simple = -0.343, p < 0.001. for subjects from middle-aged group, resilience was still negatively correlated with scl-90 scores, but much weaker, β simple = -0.217, p < 0.001. the biased-corrected 95% confidence interval for index of moderated mediation was from 0.0065 to 0.0981, which did not contain zero. this further presented that the indirect effects of social support on scl-90 scores via resilience significantly differed between groups. evidence from previous literature has already found health care workers with higher levels of social support are more likely to show higher levels of mental health [56, 57] . nevertheless, issues regarding the underlying mediating and moderating mechanisms and whether this could be applied to health care workers who are fighting with the outbreak of covid-2019 stay largely unanswered. to our knowledge, the present study is the first to report the effect of social support on mental health based on health care workers from a less affected area during covid-19 outbreak. this research built a moderated mediation model to test whether resilience mediated the association between social support and mental health of health care workers and whether this indirect relationship was moderated by age groups. the results showed that (1) the mediating role of resilience in the association between social support and mental health could be replicated to the health care workers during the epidemic; (2) age moderated the indirect link between social support and mental health (resilience-mental health path), with younger workers showing stronger than middle-aged workers. the prevalence of psychological abnormality was 7% among health care workers in our study, lower than that (17.3%) of chinese health care workers during the sars epidemic [58] . a recent study presented the rate of mental abnormality among nurses in guangdong province was 11.9% during the non-epidemic phase [51] . the difference might be attributed to the fact that jiangsu province was less affected during the covid-19 pandemic. additionally, different instruments and regional differences might also contribute to the discrepancy. generally speaking, the prevalence of psychological abnormality cannot be neglected and more attention should be paid to address this issue. in line with our hypothesis, resilience mediates the relationship between social support and mental health of health care workers during the epidemic. this is aligned with previous research based on different samples [13, 16] . this study is the first to explore the relation with the focus on the population who are facing the emergency events of the public health. this finding can be explained by the "buffer" hypothesis developed by cohen and wills [59] , which revealed the buffering effect of social support on the impact of stress upon mental health. the previous research has highlighted that finding effective approaches to deal with stress is of great importance in positive health outcomes [12] . social support could protect individual from stressful conditions and poor health state [60] . meanwhile, individuals with higher levels of social support might be more inclined to believe that they could get the help needed when facing the stressful event regarding the outbreak of the epidemic. this notion would enhance their beliefs to deal with the adversity and difficulty in the battle with covid-19, which further leads to the higher levels of resilience [61] . in addition, the reports of previous literature have demonstrated that resilience, as one kind of personal resources, also buffered the impact of stress on mental health [62] [63] [64] [65] . taken all together, resilience plays a mediating role in the association between social support and mental health for the health care workers fighting with the epidemic. the findings also revealed the moderating role of age groups in the association between resilience and mental health of the health care workers. in the past research, a small handful of previous studies have found that age moderated the link between resilience and mental health by the comparison between younger adults and older adults (usually age 50 and over) [66, 67] , whereas some other researchers focused on the contrast between the adults and minors [25] . however, these studies neglected the potential differences between the younger adults and middle-aged adults. unlike the past research, this study took the potential difference between younger and middle-aged health care workers into consideration innovatively. consistent with our hypothesis aforementioned, the increase in age (from young adults to middle-aged adults) attenuated the relationship between resilience and mental health. specifically, the younger health care workers showed stronger association between resilience and mental health compared to the middle-aged ones. this result might be interpreted by erikson's theory of life cycle development [68] , which postulates eight stages of human life. younger adults belong to stage vi with the emphasis on intimacy, while middle-aged adults are attributed to stage vii focusing on generativity. erikson's generativity stage is similar to self-actualization in maslow's hierarchy of needs [69, 70] . hence, compared with younger adults, the mental health of middle-aged adults relies less on resilience but other factors, such as the feeling of self-fulfillment, personal growth and so on. the findings of this research have profound implications since the data were collected during the peak period of covid-19 in china, which is the stage other countries are currently experiencing. first, the results stress the crucial role of social support in mental health. during the outbreak, the maintenance of mental health of health care workers is crucial for the productivity and work efficiency. therefore, it is of great importance to provide a full range of social support including instrumental support, emotional support and so on. second, the findings also highlight the potential value of resilience-focused intervention in health care workers. the resilience-focused mental health promotion program usually contains coping skills, stress management, positive attitude and so on [71] . finally, the indirect link between social support and mental health via resilience is stronger in younger adults, which implies we should give priority to younger health care workers with respect to resilience-boosting intervention. meanwhile, we also need to find other influential factors of mental health for middle-aged health care workers. several study limitations must be noted. first, the current study employed a cross-sectional design since health care workers who have consecutively worked for several days would be replaced by others, which made it hard to revisit them after a certain period. however, the cross-sectional design is insufficient to infer the causality in terms of the relationship analyzed. also, as existing literature showed, social support and resilience might influence each other reciprocally [23, 72] , the reverse causality cannot be ruled out. future research could conduct longitudinal study to further explore the moderated mediation model. second, the study used convenient sampling and all data were collected through self-report, which undermined the generalization of the results. the participants in our study were all from jiangsu province, china, which was geographically limited for a wider generalization. future research could test the relation based on more typical samples from multiple regions and manage to collect data from multiple sources (e.g., colleagues). finally, mental health is a complex concept, which could be influenced by numerous factors. social support and resilience could just explain a limited part of mental health. sleep quality and fatigue might be two important factors of mental health, but we failed to investigate. during the covid-19 outbreak, health care workers had to work excessive hours and suffered from disrupted circadian rhythms, which would contribute to fatigue and undermine sleep quality. hence, future study might focus on a more integrated model of mental health with diverse influential factors. in spite of the limitations, the current study contributes to the previous literature theoretically and practically. theoretically, this study adds knowledge to the previous research by exploring the moderated mediation model, which would help further understand the relationship between social support and mental health. practically, the findings are essential for the maintenance of mental health of health care workers during the outbreak of covid-2019. in conclusion, this study presented the protective role of social support in mental health among health care workers. moreover, resilience could be one of the pathways through which social support contributes to mental health. furthermore, the effect of social support on mental health via resilience is attenuated in middle-aged health care worker compared with the younger ones. supporting information s1 file. (sav) s1 data. strobe statement-checklist of items that should be included in reports of cross-sectional studies. 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veterans study childhood and society motivation and personality generativity versus stagnation: an elaboration of erikson's adult stage of human development systematic review of resilience-enhancing, universal, primary school-based mental health promotion programs the influence of the resilience on the organizational commitment of kindergarten and childcare teachers: social support as a mediating variable the authors would like to acknowledge all participants and the research assistants from naval medical university. key: cord-344839-r05p9h0e authors: majmundar, monil; kansara, tikal; lenik, joanna marta; park, hansang; ghosh, kuldeep; doshi, rajkumar; shah, palak; kumar, ashish; amin, hossam; chaudhari, shobhana; habtes, imnett title: efficacy of corticosteroids in non-intensive care unit patients with covid-19 pneumonia from the new york metropolitan region date: 2020-09-09 journal: plos one doi: 10.1371/journal.pone.0238827 sha: doc_id: 344839 cord_uid: r05p9h0e introduction: the role of systemic corticosteroid as a therapeutic agent for patients with covid-19 pneumonia is controversial. objective: the purpose of this study was to evaluate the effect of corticosteroids in non-intensive care unit (icu) patients with covid-19 pneumonia complicated by acute hypoxemic respiratory failure (ahrf). methods: this was a single-center retrospective cohort study, from 16(th) march, 2020 to 30(th) april, 2020; final follow-up on 10(th) may, 2020. 265 patients consecutively admitted to the non-icu wards with laboratory-confirmed covid-19 pneumonia were screened for inclusion. 205 patients who developed ahrf (spo(2)/fio(2) ≤ 440 or pao(2)/fio(2) ≤ 300) were only included in the final study. direct admission to the intensive care unit (icu), patients developing composite primary outcome within 24 hours of admission, and patients who never became hypoxic during their stay in the hospital were excluded. patients were divided into two cohorts based on corticosteroid. the primary outcome was a composite of icu transfer, intubation, or in-hospital mortality. secondary outcomes were icu transfer, intubation, in-hospital mortality, discharge, length of stay, and daily trend of spo(2)/fio(2) (sf) ratio from the index date. cox-proportional hazard regression was implemented to analyze the time to event outcomes. result: among 205 patients, 60 (29.27%) were treated with corticosteroid. the mean age was ~57 years, and ~75% were men. thirteen patients (22.41%) developed a primary composite outcome in the corticosteroid cohort vs. 54 (37.5%) patients in the non-corticosteroid cohort (p = 0.039). the adjusted hazard ratio (hr) for the development of the composite primary outcome was 0.15 (95% ci, 0.07–0.33; p <0.001). the adjusted hazard ratio for icu transfer was 0.16 (95% ci, 0.07 to 0.34; p < 0.001), intubation was 0.31 (95% ci, 0.14 to 0.70; p– 0.005), death was 0.53 (95% ci, 0.22 to 1.31; p– 0.172), composite of death or intubation was 0.31 (95% ci, 0.15 to 0.66; p– 0.002) and discharge was 3.65 (95% ci, 2.20 to 6.06; p<0.001). the corticosteroid cohort had increasing spo(2)/fio(2) over time compared to the non-corticosteroid cohort who experience decreasing spo(2)/fio(2) over time. conclusion: among non-icu patients hospitalized with covid-19 pneumonia complicated by ahrf, treatment with corticosteroid was associated with a significantly lower risk of the primary composite outcome of icu transfer, intubation, or in-hospital death, composite of intubation or death and individual components of the primary outcome. the role of systemic corticosteroid as a therapeutic agent for patients with covid-19 pneumonia is controversial. the purpose of this study was to evaluate the effect of corticosteroids in non-intensive care unit (icu) patients with covid-19 pneumonia complicated by acute hypoxemic respiratory failure (ahrf). this was a single-center retrospective cohort study, from 16 th march, 2020 to 30 th april, 2020; final follow-up on 10 th may, 2020. 265 patients consecutively admitted to the non-icu wards with laboratory-confirmed covid-19 pneumonia were screened for inclusion. 205 patients who developed ahrf (spo 2 /fio 2 � 440 or pao 2 /fio 2 � 300) were only included in the final study. direct admission to the intensive care unit (icu), patients developing composite primary outcome within 24 hours of admission, and patients who never became hypoxic during their stay in the hospital were excluded. patients were divided into two cohorts based on corticosteroid. the primary outcome was a composite of icu transfer, intubation, or in-hospital mortality. secondary outcomes were icu transfer, intubation, in-hospital mortality, discharge, length of stay, and daily trend of spo 2 /fio 2 (sf) ratio from the index date. cox-proportional hazard regression was implemented to analyze the time to event outcomes. plos one | https://doi.org/10.1371/journal.pone.0238827 september 9, 2020 1 / 14 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is the rna virus that causes coronavirus disease 2019 . this virus is responsible for a spectrum of disease presentation, which ranges from asymptomatic infection to severe pneumonia, respiratory failure, and death [1] . to date, sars-cov-2 has caused a significant degree of morbidity and mortality within the united states, with a large proportion of these cases concentrated in new york city [1] . given the novelty of this virus, there is limited data on treating these patients. as a result, management protocols vary and are rapidly evolving with emerging data and clinical experiences. the use of systemic corticosteroids in the management of covid-19 infection is widely debated. the use of corticosteroids with influenza pneumonia has previously been associated with a higher risk of death [2, 3] and delayed viral clearance during severe acute respiratory syndrome-related coronavirus (sars) and middle east respiratory syndrome (mers) outbreaks [4] [5] [6] . alternatively, few studies supported the use of corticosteroids at a low-to-moderate dose in patients with coronavirus and influenza a (h1n1) and sars pneumonia [7, 8] . on the other hand, the world health organization has recommended against routine use of systemic corticosteroids to patients with covid-19 [9] ; nevertheless, a consensus statement by the chinese thoracic society recommends judicious use of corticosteroids in these patients [10] . recently, recovery trial demonstrated the beneficial clinical effect of dexamethasone among patients with covid-19 who required oxygen with and without invasive mechanical ventilation [11] . although recovery trial demonstrated a beneficial effect of dexamethasone on mortality, there are no other analogous studies that have reproduced similar results, and the current study is unique in terms of dose, duration, and type of corticosteroids used. we performed a retrospective cohort study on patients admitted to the general inpatient wards with a diagnosis of covid-19 pneumonia complicated by acute hypoxemic respiratory failure. the purpose of this study was to determine the clinical efficacy of corticosteroids on outcomes of intensive care unit (icu) transfer, intubation, in-hospital death, discharge, and length of stay. we hypothesized that systemic corticosteroid use would be associated with a lower risk of a composite endpoint of icu transfer, intubation, or death. this is a retrospective cohort study of confirmed cases of covid-19 pneumonia hospitalized at metropolitan hospital center serving the east harlem community in new york city. all patients were diagnosed with covid-19 pneumonia as per the world health organization's interim guidance document [12] . we collected information on consecutive patients admitted to the general wards in metropolitan hospital from march 15, 2020, to april 30, 2020, as per our inclusion and exclusion criteria. the ethics committee of new york city health and hospital (star) and brany institution review board approved this study and permitted a waiver of informed consent from the study participant. we accessed the hospital databases from may 1st to 10th, 2020. patients were eligible for the study if they met the following inclusion criteria 1) age � 18 years old, 2) confirmed cases of sars-cov-2 by pcr method, 3) admitted in general wards, 4) pao2/fio2 (pf) ratio <300 if arterial blood gas was available or spo 2 /fio 2 (sf) ratio <440, 5) bilateral infiltrate on chest imaging validated by radiology staff. the exclusion criteria were 1) patients with severe immunosuppression (hiv infection, long term use of immunosuppressive agents), 2) pregnant woman or lactating women, 3) oral glucocorticoids were needed for other diseases, 4) direct admission to intensive care unit (icu), 5) if had any of primary composite outcome within first 24 hours of admission, 6) patient who never required oxygen during the hospital course, 7) patients who left against medical advice. a team of resident physicians reviewed and collected demographic, laboratory, clinical, and outcomes data from electronic medical records between march 15 and april 30, 2020. the inclusion criteria, exclusion criteria, individual components of all definitions of clinical outcomes were recorded separately and checked by two authors (m.m. and i.h.) (s1 table) . two independent residents adjudicated all the outcome data, and any disparity was resolved by consulting the primary investigator. patient confidentiality was protected by allocating a deidentified patient identification, and the electronic data was stored in a locked, password-protected computer. nasopharyngeal swab samples were obtained from all patients at admission and tested using real-time reverse transcriptase-polymerase chain reaction assays at labcorp laboratory to identify sars-cov-2 infected patients. the decision to give corticosteroids was at the discretion of the treating physician. all corticosteroid dosages were converted to the equivalent dose of methylprednisolone [13, 14] . the calculation of the sf ratio and pf ratio has been elaborated in the s1 file. in the study, the index date was not taken as the date of admission. in the non-corticosteroid cohort, the index date was taken as the date when the patient's sf ratio went below 440, or the pf ratio went below 300. for the corticosteroid cohort, the date when corticosteroid was started was taken as the index date. our primary outcome was the composite outcome of intensive care unit (icu) transfer, intubation, or death. secondary outcomes were discharge, intensive care unit transfer, intubation, death, composite of intubation or death, length of stay, and a daily trend of sf ratio since the index date. baseline characteristics of both cohorts were expressed using descriptive statistics. the continuous variables were exhibited as a mean ± standard deviation or a median with interquartile range (iqr) for normal and non-normal distribution, respectively. categorical variables were extrapolated in frequency and proportions. the t-test and mann-whitney-wilcoxon tests were applied for normal and non-normal distribution, respectively, to compare continuous variables between two cohorts. fisher's exact test or pearson's chi 2 tests were implemented to compare categorical variables. time to event (composite primary outcome and secondary outcomes) was defined as the time from the index date of the study to the specified events. we used the cox-proportional hazard model to determine univariable and multivariable hazard ratio (hr) and 95% confidence interval (ci) for the corticosteroid group compared with non-corticosteroid group on the development of composite primary outcome, icu transfer, intubation, death, composite of intubation or death, and discharge. the multivariable cox-proportional model included sf ratio, age, gender, chronic lung disease, white blood cell count, platelet count, tocilizumab, and therapeutic dose of enoxaparin (for the construction of multivariate model-s2 file). we applied the test for proportionality assumption based on the schoenfeld residuals. for the primary outcome and other secondary outcomes, we constructed kaplan-meier curves and used the log-rank test to compare. censoring was applied on the day of discharge. linear regression was used to show the difference between the length of stay between two cohorts. mixed-effects linear regression with restricted maximum likelihood was used to perform a longitudinal analysis to assess the trend of sf ratio over time between two cohorts. the fixed-effect constant was suppressed, and unstructured residual errors were generated. a likelihood ratio test vs. linear model chi-square test was used to assess the appropriateness of using a multilevel model. margins were calculated to display predicted probabilities. missing data were not imputed. all tests were 2-sided, and a p-value less than 0.05 was considered statistically significant. all analyses were performed with stata software, version 16.0 (statacorp llc). we screened 265 consecutive patients admitted to the metropolitan hospital, new york medical college, from march 15 to april 30, 2020, and included 205 patients in the final analysis of the study. the date of the final follow-up was may 10, 2020. the mean age of the entire cohort was 57.61±15.86 years, 153 (74.63%) patients were male, and 149 (73.04%) patients were of hispanic ethnicity/race. the common comorbidities were hypertension (103, 50.24%), obesity (84, 42%), and diabetes (83, 40.49%) ( table 1 ). at the time of analysis, a total of 14 patients (out of 205) were still admitted to the hospital. of 205 patients, 60 (29.27%) patients received systemic corticosteroids, and 145 (70.73%) patients did not receive it. patients in the corticosteroid cohort received systemic corticosteroids in the form of methylprednisolone (n = 29, 48.33%), prednisone (n = 10, 16.67%), hydrocortisone (n = 1, 1.67%), and dexamethasone (n = 20, 33.33%). corticosteroid was commenced at a median of 2 days (iqr, 1-5) following admission, on a median or equivalent dose of 80 mg per day (iqr, 60 a comparison of demographic, clinical, and laboratory parameters of patients with and without the composite primary outcome is demonstrated in out of 202 eligible patients, 13 (22.41%) patients developed primary outcome in the corticosteroid cohort compared with 54 (37.5%) patients in the non-corticosteroid cohort (p = 0.039). in both unadjusted and adjusted analysis, patients who received corticosteroids were less likely to have had a primary outcome [(unadjusted hazard ratio, 0.45; 95% ci, 0.24 to 0.82; p-0.009), (table 3, fig 1, panel a) , (adjusted hazard ratio, 0.15; 95% ci, 0.07 to 0.33; p < 0.001) ( table 3) ]. proportionality assumption was not violated (global test p = 0.153). in the corticosteroid cohort, 12 (20.69%) patients were transferred to icu as compared with 47 (32.64%) patients in the non-corticosteroid cohort (p = 0.09). in the unadjusted and adjusted cox-analysis, patients who received corticosteroid were less likely to require icu transfer [(unadjusted hazard ratio, 0.49; 95% ci, 0.26 to 0.93; p-0.029), (adjusted hazard ratio, 0.16; 95% ci, 0.07 to 0.34; p < 0.001)] (table 3, s1 fig). in the corticosteroid cohort, 11 (18.97%) patients were intubated compared with 36 (25.35%) patients in the nonfig 1, panel b) . in the corticosteroid cohort, 13 (22.41%) patients intubated or died compared with 46 (31.94%) patients in the non-corticosteroid cohort (p = 0.178). in the unadjusted and adjusted cox-analysis, patients who received corticosteroid were less likely to have a composite of intubation or death events [(unadjusted hazard ratio, 0.60; 95% ci, 0.33 to 1.12; p-0.108), (adjusted hazard ratio, 0.31; 95% ci, 0.15 to 0.66; p-0.002)] ( table 3, fig 1, panel c) . in the corticosteroid cohort, 47 (85.45%) patients were discharged compared with 102 (75%) patients in non-corticosteroid cohort (p = 0.11). in the unadjusted and adjusted cox-analysis, patients who had received corticosteroid had more likely to be discharged [(unadjusted hazard ratio, 1.17; 95% ci, 0.83 to 1.65; p-0.380), (adjusted hazard ratio, 3.65; 95% ci, 2.20 to 6.06; p < 0.001)] ( table 3, s3 fig) . proportionality assumptions were not violated for any of the secondary outcomes tested by cox-regression. the median length of stay was higher in the corticosteroid cohort (9 days, iqr (6-17)) compared to the non-corticosteroid cohort (7 days, iqr (5-13.5); p = 0.025) (tables 1 and 3) . using multivariable linear regression, length of stay was lower in corticosteroid cohort but statistically non-significant (coefficient -1.06; 95% ci, -4.26 to 2.14; p = 0.515). furthermore, significant interactions between treatment and time demonstrated that the noncorticosteroid cohort experienced a decrease in sf ratio over time compared to the corticosteroid cohort, who were found to experience an increase in sf ratio over time. the likelihood ratio test vs. linear model chi-square test indicated that the use of a multilevel model was appropriate for the data. the results of our study showed that the administration of corticosteroids in patients admitted to the general medical ward with ahrf and a diagnosis of covid-19 pneumonia was associated with a lower risk of developing the primary outcome composite of icu transfer, intubation or death. regarding secondary outcomes, patients who received corticosteroids were found to have a lower risk of icu transfer, intubation, in-hospital death, composite of intubation, or death and were more likely to be discharged. in the corticosteroid cohort, however, a lower hazard of mortality did not show statistical significance likely due to a smaller sample size. as discussed in the introduction, the findings from earlier studies have indicated the controversial role of corticosteroids in covid-19. a preliminary report from the recovery trial [11] demonstrated a beneficial role of low dose (6 mg) dexamethasone in reducing the risk of death among patients who required oxygen with or without invasive mechanical ventilation. the study by wu et al. demonstrated a beneficial role of methylprednisolone in reducing the risk of death in a subgroup analysis of 84 patients with ards, from an observational study of 201 patients with covid-19 [15] . a meta-analysis of randomized clinical trials suggested corticosteroid could reduce mortality and need for mechanical ventilation in patients with severe community-acquired pneumonia, irrespective of bacterial or viral etiology [16, 17] . on the contrary, another meta-analysis showed higher mortality among patients receiving corticosteroids [18] . however, this meta-analysis included patients with sars, mers, covid-19, and had high heterogeneity. a subgroup analysis from the same study reported no significant difference in mortality between two cohorts when included only covid-19 patients [18] , suggesting corticosteroids may not be harmful in these patients. we assume studies that showed higher or no difference in mortality with corticosteroid were critically ill with ards, on mechanical ventilation, and they might have passed the point where adverse outcomes could be modified by corticosteroid. in covid-19 pneumonia, lung injury is associated with a direct virus-induced injury. however, the severity of illness is associated with the virus triggered immune hyper-response that is characterized by activation of various immune cells and release of numerous pro-and anti-inflammatory cytokines, including tumor necrosis factor (tnf), interleukin-6 (il-6), and many more. overwhelming secretion of cytokines leads to severe lung damage manifested as the destruction of the small airway, alveolar epithelium, and vascular endothelium that progresses to pulmonary edema and hyaline membrane formation [19, 20] . in severe covid-19 pneumonia, patients' symptoms worsen and become more hypoxic during the 4-7 days after onset of symptoms [21] . hence, it is vital to suppress the cytokine storm before that period. we, therefore, believe a majority of patients survive and recover if they overcome the period of the cytokine storm. corticosteroid is a classical immunosuppressive drug that helps in delaying or halting the progress of pneumonia and has been effective for the treatment of ards [22, 23] . in addition to immunosuppressive properties, corticosteroid possesses anti-inflammatory activity that reduces systemic inflammation, decreases exudation into the lung tissue, promotes the absorption of inflammation, and prevents alveolar damage [24] . these effects of corticosteroid help in relieving hypoxemia earlier, preventing further progression of respiratory insufficiency, and hence associated with improved primary, secondary outcomes, and sf ratio in the study. this study was also unique and different from the recovery trial in the following ways. compared with the recovery trial, the average duration of corticosteroids administration was half, the median equivalent dose of dexamethasone was two times, and various types (methylprednisolone, dexamethasone, and prednisone) of corticosteroids were used. the present study cohort only included non-intubated patients admitted to general wards. in the current study, a hazard ratio of a composite of intubation or mortality was 0.31 (0.15-0.66), intubation was 0.31 (0.14-0.70), and death was 0.53 (0.22-1.31). in the recovery trial, the subgroup of patients on oxygen, a hazard ratio of the composite of invasive mechanical ventilation or mortality was 0.87 (0.79-0.96), and of mortality was 0.82 (0.72-0.94) when they excluded patients who were receiving invasive mechanical ventilation at randomization. the difference in the dose and duration of corticosteroid between two studies prompts the possibility of greater beneficial effect with a higher dose and shorter duration of the corticosteroid on clinical endpoints. traditionally corticosteroids are not used early in viral pneumonia due to concerns of delayed viral clearance. however, delayed treatment may predispose to worsening and progressive inflammatory response and multiorgan failure. hence, the authors presume that careful monitoring of inflammatory markers might help guide the judicious use of corticosteroid. this study highlights that early administration of moderate-dose of any systemic corticosteroid (oral or intravenous) for a shorter duration in covid-19 viral pneumonia may not be as harmful as initially suspected, and even more beneficial than shown by the recov-ery trial. the lower hazard of icu transfer, intubation, and a higher rate of discharge might be linked to a better quality of life of the patient if corticosteroids are given during the early period of illness. however, data on readmission to hospital or ed visits post-discharge would be required to confirm this presumptive role. this study has several limitations, given the observational nature of the study. it is a single-center study, and most of the patients were hispanic that limits the generalizability of the study. however, this study could be taken as a role of corticosteroid in the predominantly hispanic population. there were missing data for inflammatory markers and potential inaccuracies in the documentation of variables from the electronic health records; however, as mentioned above, two authors made sure the accuracy of the collected database. due to the retrospective nature of the study, it was not possible to collect data on side effects such as secondary superimposed infection and hyperglycemia. hence, one should be vigilant about these adverse effects while using it. authors believe that the physicians are familiar with the side effects of corticosteroid and ways to prevent and treat its complications as it is a long-known drug compared with newer medications on which we have very limited data. due to missing data for inflammatory markers, adjustment with those variables was not possible. despite the extensive adjustments, it is still possible that unmeasured confounding prevails. we did not have longitudinal data with follow-up. a randomized clinical trial is the best approach to determine whether the benefit can be ascribed to any given therapeutic intervention as the trial design diminishes the two major hurdles of observational studies, namely unmeasured confounding and bias. a randomized clinical trial from china has been registered in which patients were randomly assigned to receive 1 mg/kg methylprednisolone (nct04273321) or placebo. the authors tried to minimize confounding by choosing the best possible model in multivariable regression in this retrospective cohort study. in our analysis of hospitalized patients in the general ward with covid-19 pneumonia complicated by acute hypoxic respiratory failure, early use of moderate dose systemic corticosteroid for the shorter duration was associated with a significantly lower rate of the primary outcomes of icu transfer, intubation, or in-hospital death. given the observational design, the study should be interpreted with caution due to potential bias and residual confounders. presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new york city area the effect of corticosteroids on mortality of patients with influenza pneumonia: a systematic review and meta-analysis corticosteroid therapy for critically ill patients with middle east respiratory syndrome systemic corticosteroid therapy may delay viral clearance in patients with middle east respiratory syndrome coronavirus infection the use of corticosteroid as treatment in sars was associated with adverse outcomes: a retrospective cohort study treatment of severe acute respiratory syndrome with glucosteroids effect of low-to-moderate-dose corticosteroids on mortality of hospitalized adolescents and adults with influenza a(h1n1)pdm09 viral pneumonia clinical management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected expert consensus on the use of corticosteroid in patients with 2019-ncov pneumonia dexamethasone in hospitalized patients with covid-19-preliminary report world health organization. global surveillance for human infection with coronavirus disease (covid-19) hä ussler u. pharmacokinetics and pharmacodynamics of systemically administered glucocorticoids steroid conversion calculator-mdcalc risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease corticosteroid therapy for patients hospitalized with community-acquired pneumonia: a systematic review and metaanalysis. annals of internal medicine. american college of physicians corticosteroids for pneumonia. cochrane database of systematic reviews the role of corticosteroids in the management of critically ill patients with coronavirus disease 2019 (covid-19): a meta-analysis pathological findings of covid-19 associated with acute respiratory distress syndrome pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology clinical characteristics of coronavirus disease 2019 in china glucocorticoids and acute lung injury. critical care medicine immune regulation by glucocorticoids antiinflammatory action of glucocorticoids-new mechanisms for old drugs we acknowledge the dedication, commitment, and sacrifice of the staff, providers, and personnel in our institution through the covid-19 crisis and the suffering and loss of our patients as well as their families and our community. we acknowledge ms. ejiro c. gbaje mph for her help in the analysis of data. key: cord-303034-w72oeoxq authors: haischer, michael h.; beilfuss, rachel; hart, meggie rose; opielinski, lauren; wrucke, david; zirgaitis, gretchen; uhrich, toni d.; hunter, sandra k. title: who is wearing a mask? gender-, age-, and location-related differences during the covid-19 pandemic date: 2020-10-15 journal: plos one doi: 10.1371/journal.pone.0240785 sha: doc_id: 303034 cord_uid: w72oeoxq masks are an effective tool in combatting the spread of covid-19, but some people still resist wearing them and mask-wearing behavior has not been experimentally studied in the united states. to understand the demographics of mask wearers and resistors, and the impact of mandates on mask-wearing behavior, we observed shoppers (n = 9935) entering retail stores during periods of june, july, and august 2020. approximately 41% of the june sample wore a mask. at that time, the odds of an individual wearing a mask increased significantly with age and was also 1.5x greater for females than males. additionally, the odds of observing a mask on an urban or suburban shopper were ~4x that for rural areas. mask mandates enacted in late july and august increased mask-wearing compliance to over 90% in all groups, but a small percentage of resistors remained. thus, gender, age, and location factor into whether shoppers in the united states wear a mask or face covering voluntarily. additionally, mask mandates are necessary to increase mask wearing among the public to a level required to mitigate the spread of covid-19. wearing a mask in public is currently a controversial and politicized issue in the united states, even with case evidence from other countries that face coverings help to control the spread of coronavirus disease 2019 (covid-19) [1] . as taiwan, hong kong, south korea, and other countries with almost universal masking have been some of the most successful at reducing daily case and death rates [2, 3] , the united states has largely trended in the other direction, setting national and state records for daily new cases of covid-19 at the end of june 2020. as of september 2020, the united states had recorded more than seven million confirmed cases and over 200,000 deaths [4] . despite the controversy around masking, transmission of covid-19 (caused by the virus sars-cov-2) occurs primarily through respiratory particles from infected individuals and even asymptomatic cases can be contagious [5] . experts in aerosol science, virology, infectious diseases, and epidemiology acknowledge the potential for a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 airborne transmission of the virus [6] . thus, without a vaccine in distribution, masks are one of the few control measures available for protection against the virus because they serve as a physical barrier between people [7, 8] . not only do masks protect the wearer from droplets and some smaller airborne particles, but they also provide source control, stopping particles coming from a wearer. study of the filtration efficiency of different fabrics has shown that even cotton weaves and blends can block viral transmission [9] , so masks can be made virtually cost-free with household materials. while up to 50% of person-to-person transmission of the virus may occur through asymptomatic individuals [10] , many people who have been infected with covid-19 experience significant functional complications within their organ systems. in children and young adults, the disease appears to be mild, but even among previously infected collegiate athletes, potentially life-altering inflammation within the cardiovascular system was detected [11] . thus, even fit young adults are not immune to the harm that the virus can cause within the human body. systemically, cardiovascular [12] , neurological [13] , hepatic [14] , renal [15] , and respiratory [16] complications of the virus have been reported across most age groups. older adults however are the most likely to experience severe outcomes, with one study showing that 86% of hospitalized individuals were at least 50 years old [17] . furthermore, 60% of hospitalized cases were men in this study, and consistent with a meta-analysis showing that males are at greater risk for severe outcomes from covid-19 [18] . overall, death rate increases drastically with age and comorbid conditions, and mortality among hospitalized cases may be as high as 20% [19] . new data is also emerging that suggests previously asymptomatic or "mild" cases are experiencing debilitating negative effects months after infection [20] [21] [22] [23] . given the easy transmission of this debilitating virus from person-to-person and the lifechanging complications across populations, mask wearing is demonstrated to be a very effective and proactive public health tool that will save lives and lessen future public health burden. though the evidence of the efficacy and importance of masks is clear, store policies and public mandates requiring masks in america have been met with protests [24] and, in rare cases, violence [25, 26] . public health research shows these measures may already have reduced cases in the united states by 450,000 through may 22 nd [27] , but the messaging from top-level government officials has been inconsistent [28] and polling suggests that a sizeable portion of the general population are still going out in public without masks [29, 30] , though self-reported behavior is not always reliable. a previous meta-analysis concluded that women were 50% more likely than men to engage in nonpharmaceutical protective behaviors (e.g. mask wearing) during epidemics and pandemics [31] , and in the covid-19 pandemic women report wearing masks more often than men [30] , despite the fact that male sex may be a risk factor for more severe outcomes of the disease [18] . older adults are at higher risk for more severe cases of covid-19 [17, 32] , so it is reasonable to expect that this demographic may wear masks more than younger individuals. mask wearing by retail shoppers may also vary by location, in that groups that report greater resistance to mask wearing may be more concentrated outside of urban areas [33] . specifically, rural areas tend to have higher concentrations of conservative-leaning voters [34] who are reported to be more resistant to wearing a mask [33] . while the initial outbreak in the united states was primarily in cities, rural counties have also some of the highest case rates in the united states per capita [35] . this geographical spread is particularly concerning given that populations that live in non-metropolitan areas are more vulnerable to covid-19 [36] and highlights the need to study pandemic-related health behaviors like masking in rural locations. learning what demographic groups are wearing masks and how mask mandates impact behavior among these groups will help officials to better target public health messaging that promotes the practice among groups with lower usage. to facilitate greater understanding and reliable experimental data on whether gender, age, location, and the presence of mask mandates influence mask wearing in the united states, we conducted a direct observational study at retail stores in wisconsin. observations occurred between june and august 2020. from june 9 th to august 3 rd , 2020, the 7-day average case rate in wisconsin more than doubled (341 to 844 cases) and the percentage of positive cases had increased from 3.5% to 7.4% [37] . thus, during this period, many major retail chain [38] and wisconsin state [39] mask mandates were enacted, allowing for study of mask wearing before and after these regulations went into effect. the primary aim of our study was to determine mask use by gender expression, estimated age, and location. we hypothesized that, without a mandate, mask use among females would be greater than males across all age groups and locations. additionally, that mask use would be greater among older adults (>65 years old) than middle-age (30-65 years old) and young individuals (2-30 years old) and would be less in rural than in urban or suburban areas. a secondary aim of this project was to revisit stores on dates near the implementation of store and state mask mandates to characterize how mask-wearing behavior changes in response to these requirements. we addressed our primary aim by visiting 36 different retail locations across five different counties in southeastern wisconsin. retail stores selected for observation were based largely on geographical spread across milwaukee and the surrounding areas. in the interest of exploring mask wearing among urban, suburban, and rural stores, we designated a center point of the city (united states postal service main office; 345 w st. paul ave, milwaukee, wi, usa) and recorded the linear distance of each store to the city center. based on distance, stores were then placed into urban (<6.1 km from city center; n = 15, 3.2 ± 1.8 km to city center), suburban (11.5-32.1 km; n = 13, 20.5 ± 7.2 km), and rural (>36.9 km; n = 10, 55.8 ± 21.4 km) store groups. visits to stores (n = 38) occurred at various times between 9am and 8pm (june 3 rd -9 th , 2020) and lasted at least 15 minutes (average = 43 min), with observers recording data with writing utensil and paper or mobile device. to address our secondary aim, stores were revisited and data was collected in the same manner on dates shortly before store or state mandates were implemented (n = 7; july 24 th -31 st , 2020), when there was a store mandate but not a state mandate (n = 20; july 22 nd -july 31 st , 2020), and after the wisconsin state mandate began (n = 10; august 1 st -3 rd , 2020). note that the overlap in the secondary visit dates is related to corporate differences in the dates mask mandates began at various stores. stores consisted of grocery and other large big-box retail stores and shoppers were not aware they were being observed. children under the estimated age of two were not recorded and estimated age categories were defined as young = 2-30 years old, middle age = 30-65 years old, older: >65 years old. these ranges reflect the substantial increase in stratified death rates for individuals 30-39 and 65-74 years old when compared to prior age groups (19) . for all observations that were collected, summary sheets were crosschecked by other observers. all procedures involved public observation or accessed public information and did not require review by an institutional review board. to determine the impact of gender, age, location, and their interactions on mask wearing during the initial visit period (june 3 rd -9 th , 2020), multiple logistic regression analysis was performed. sixty individuals who were wearing their mask or face covering improperly (not over nose and mouth) were recorded but excluded from this analysis as they could not be grouped into a dichotomous outcome (mask/no mask). after excluding these individuals, 5517 observations remained for the analysis, and gender expression, age, and location independent variables were dummy coded and entered into a backward elimination procedure with their associated interactions (gender-age, gender-location, age-location, and gender-age-location). limit for variable removal and test classification cutoff were set at 0.025 and 0.5, respectively. adjusted odds ratios (aor) are expressed with respect to reference groups (gender: male, age: young, location: rural) with 95% confidence intervals and significance was determined at p < 0.05. percentage data including incorrect mask wearers from the initial visit (june 3 rd -9 th , 2020) was used to compare to later data (july 24 th -aug 3 rd , 2020) and evaluate the changes in maskwearing behavior in response to mask mandates. all analyses were performed with either microsoft excel (microsoft, redmond, wa, usa) or ibm statistical package for social sciences version 26 (ibm, armonk, ny, usa). over the course of 75 visits, 9935 individuals were observed entering retail stores. of the 5517 individuals we observed during the first round of data collection that could be grouped into a dichotomous outcome (mask/no mask) (fig 1) , 41.5% were wearing a mask or face covering with the general trend across gender, age, and location largely aligning with our original hypotheses (table 1) . females wore masks 7.6% more than males (fig 2a) . additionally, masks were seen at a higher percentage in older than middle-age (+16.1%) and young (+19.8%) individuals (fig 2b) . urban and suburban mask wearing was similar but was much lower at rural stores (suburban: -28.7%; urban: -26.4%; 55.81 ± 21.37 km) (fig 2c) . regression analysis revealed that gender, age, and location all significantly impact the odds of an individual being observed to wear a mask (p < 0.001; fig 3) . results indicate the odds of a female wearing a mask are significantly greater than males (aor = 1.470, 95% ci = 1.313-1.646). odds are also greater for middle age (aor = 1.597, 95% ci = 1.359-1.877) and older adults (aor = 3.434, 95% ci = 2.811-4.195) than younger individuals. the odds of observing someone in urban (aor = 3.847, 95% ci = 3.157-4.689) or suburban (aor = 4.124, 95% ci = 3.418-4.975) areas wearing a mask is also much higher than in rural locations, reflecting the much lower prevalence of masks seen at rural stores. the significant age-location interaction effect (p < 0.001) is largely driven by differential changes in mask-wearing behavior between older adults and the other age groups (fig 2d) . while estimating age and gender is an obvious limitation of this study, the sample was large (~10,000 observations) increasing the study power to offset the effect of variability from the estimations. currently, no peer-reviewed and direct-observational studies of mask wearing are available for comparison to our study findings. preprint study akin to ours also reflects alarming and similarly low percentages of mask wearing in wisconsin [40] . additionally, our data can be discussed in relation to national polls and surveys that addressed mask wearing at about the same time. a may-to-june survey of adults in wisconsin and four other surrounding states indicated 45.1% always wear a mask when they leave home [29] , and data in our study lends credibility to this number. notably, other regions of the united states surveyed reported different habits (33.5-64% mask usage) so direct observational studies in other parts of the country would be beneficial to confirm national variability when mask mandates are not in place. another poll conducted in mid-april indicated that only 36% of adults always wear a mask or face covering when outside the home [30] , so perhaps usage nation-wide has gradually improved even when masks are not required. women also reported wearing a mask more often than men. so why are men wearing masks in public places less than women? perhaps masks are viewed as a sign of fragility or weakness among some men in the usa, as suggested in previous preprint work [41] . in this case, public health messaging that focuses on aligning masks with masculinity would likely be beneficial to improve usage among males in the united states. alternatively, women may be more likely to protect themselves and others by wearing a mask because they handle the majority of caregiving within families [42] , or because of awareness of the preexisting gender inequalities in social, political, and economic systems that have been further amplified due to the pandemic [43] . it is not surprising that our june data showed that older individuals wear masks more than middle-age and young people because older adults are at higher risk for more severe cases of covid-19. however, the low percentage of young individuals wearing a mask combined with their potential to be asymptomatic [44] creates problems for case containment. a key and underreported benefit of masks is source control, so mask wearing is important across all age groups. lower-risk individuals put older adults and those with preexisting conditions of all ages at risk of severe illness by not wearing a mask due to the potential for asymptomatic viral transmission. one of the most interesting findings of the current study is the evidence of drastically different mask-wearing behavior in rural areas compared with urban and suburban shoppers when mask requirements are not in place. the odds of urban and suburban shoppers wearing a mask was about 4x greater than for rural store-goers during our june data collection period, possibly reflecting the fact that individuals shopping in rural areas perceive lower risk. however, it has been previously reported that mask-wearing habits do not appreciably differ between counties with low and high numbers of covid-related deaths [30] . further, while living population density may be lower in rural regions, the size of retail stores is relatively uniform. thus, differences in the density of shoppers in stores across regions is likely far less than the actual population density differences reflect. understanding why rural shoppers may resist the use of masks more than shoppers in other areas is an important issue because the health care system in these areas of the united states may be less equipped to handle large spikes in cases. rural residents may also need to travel further for emergency care, putting them at greater risk for severe consequences of infection. for these reasons, public health messaging that promotes masks in rural communities is critically important. by the last week of july 2020, for stores with no mask mandate, wearing increased to about 80% overall (fig 4) . this may reflect some improvement in voluntary compliance, as many stores had announced mandates to be enforced at later dates. thus, some individuals may have been moved to comply due to the prospect of needing to wear a mask for future shopping trips. within these data there were no discernable age-related differences, but it was still evident that more females (82.2%) were wearing masks than males (77.8%). data after store and the wisconsin state mask mandates began shows mask wearing increased to over 90% overall (fig 4) . modeling studies suggest mask usage needs to be to nearly universal to have a significant effect on the epidemiological curve [45, 46] , and our results emphasize that mask mandates are necessary to approach this goal. if public officials do not enact these mandates the welfare of shoppers will continue to be left to retail corporations. while retail store mandates obviously improve mask-wearing compliance among shoppers, our data may indicate that that store regulations could be slightly less effective than mandates enacted by government as a slightly higher percentage of individuals were observed correctly wearing masks after a state mandate began (96% vs 93% with store mandate only). mask mandates obviously improve usage, but it deserves mention that a small percentage of the population still resist masks even with retail store and government regulations in place. though it may be argued that some shoppers might have a health condition that precludes wearing a mask, the centers for disease control and prevention only recommend against mask wearing if an individual has trouble breathing or is unable to remove a face covering without assistance [47] so it is improbable that this is a valid explanation for all of the mask resistors observed with a state mandate (2%; fig 4) . mask-wearing mandates are necessary to reduce person-to-person transmission of covid-19 and save lives as a portion of the general public will resist wearing masks in retail stores without them. even with mandates in place, some individuals still resist or wear them incorrectly, putting employees, other shoppers, and themselves at risk due to the potential for viral transmission from asymptomatic carriers [5, 10] . encouraging voluntary compliance to mask wearing is critical so that retail employees do not have to play the role of "mask police", risking verbal or physical abuse from resistant shoppers. close encounters (within six feet) are a great risk to adjusted odds ratios (aor) and 95% confidence interval plots of mask usage for gender expression, age, and location during the initial data collection period from june 3 rd -9 th , 2020. odds ratios are expressed in relation to reference groups (gender: male; age: young; location: rural). https://doi.org/10.1371/journal.pone.0240785.g003 who is wearing a mask? employees if the resistor without a mask is infected. though some stores provide masks to shoppers after entering, there is still a short period that these individuals are inside of the store without masks on. as the possibility of aerosolized transmission of covid-19 was recently confirmed [48] , every breath and word spoken without a mask on (or without one covering mouth and nose) and especially indoors, increases risk of aerosolized virus spread. thus, it is critical to wear a mask from entrance to exit of any public indoor space. to further reduce the number of individuals entering without a mask, stores may benefit from providing masks to shoppers that need them outside of the entrance, where possible. averting cases of covid-19 will save lives so wearing a mask is a public health service and should not be viewed as a symbol of fragility of the individual. wearing a mask is not about stopping transmission of all cases but minimizing case rates and lessening the public health burden. importantly, the long-term effects of covid-19 are still unknown so mask wearing is also vital to reduce burden from future comorbidities that may result within individuals who were infected. masks are an essential public health intervention for combatting the pandemic, and only 41% of individuals observed in our june sample were wearing a mask when entering a retail store. this is less than half the percentage needed to have a significant effect on the reducing the mask-wearing compliance was poor in june but had improved immediately before mandates began. compliance rose to > 90% with a mandate in place across all demographics, but a small percentage of the population still resisted. the overlap in "before" and "with store mandates" dates is related to corporate differences in the dates mask mandates began. https://doi.org/10.1371/journal.pone.0240785.g004 spread of covid-19 [45, 46] . males, younger individuals, and shoppers in rural communities were observed wearing masks less than other groups. however, with store mandates and a state-wide mandate to wear a mask, we showed that mask-wearing compliance rose to over 90% in all groups, including those that resisted mask wearing in june. as officials around the united states continue to debate mask mandates at local and state levels, our data indicated that mandates are necessary to ensure mask-wearing compliance among the public meets the minimum threshold to take control of the covid-19 pandemic. our data also highlights that a portion of the general population does not follow public health recommendations without them. furthermore, even with mandates in place a portion of shoppers (~4%) still resist or wear masks ineffectively. individual shoppers may consider this information to evaluate the potential risks of in-person shopping based on store and local mask policies, location, and the age and gender of the store's clientele. our findings are also important to inform policy makers that mask mandates are necessary to improve compliance to a level that can help flatten the epidemiological curve, saving lives and decreasing burden on the united states health care system. supporting information s1 file. june observations raw data. its contains raw data for the initial data collection period in june. (xlsx) s2 file. july and august observations raw data. its contains raw data for the second data collection period in july and august. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 reducing transmission of sars-cov-2 a modelling framework to assess the likely effectiveness of facemasks in combination with 'lock-down' in managing the covid-19 pandemic united states coronavirus info; c2020 temporal dynamics in viral shedding and transmissibility of covid-19 enviromental health matters initiative face coverings for the public: laying straw men to rest face masks for the public during the covid-19 crisis physical distancing, face masks, and eye protection to prevent person-to-person transmission of sars-cov-2 and covid-19: a systematic review and meta-analysis. the lancet estimating the generation interval for coronavirus disease (covid-19) based on symptom onset data cardiovascular magnetic resonance findings in 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about "long covid the lasting misery of coronavirus long-haulers. nature anti-vaccine activists, mask opponents target public health officials-at their homes. los angeles times black mayor of kansas city says he was called n-word, received death threat over mask rule. nbc news mcdonald's customer attacks drive-through worker over safety rule community use of face masks and covid-19: evidence from a natural experiment of state mandates in the us mixed messaging on masks set u.s. public health response back. npr the northeast leads the country in mask-wearing. cnn new april guidelines boost perceived efficacy of face masks a meta-analysis of the association between gender and protective behaviors in response to respiratory epidemics and pandemics older adults and covid-19 republicans, democrats move even further apart in coronavirus concerns how the rural-urban divide became america's political fault line latest map and case count rural america is more vulnerable to covid-19 than cities are, and it's starting to show. the conversation wisconsin department of health services covid-19: wisconsin summary data mcdonald's joins walmart and dozens of other chains with mask mandates emergency order #1: relating to preventing the spread of covid-19 by requiring face coverings in certain situations. the state of wisconsin office of the governor use of face coverings by the public during the covid-19 pandemic: an observational study the effect of messaging and gender on intentions to wear a face covering to slow down covid-19 transmission geographies of care and responsibility united nations secretariat. the impact of covid-19 on women age-dependent effects in the transmission and control of covid-19 epidemics bidirectional impact of imperfect mask use on reproduction number of covid-19: a next generation matrix approach. infectious disease modeling this simple model shows the importance of wearing masks and social distancing. the conversation considerations for wearing masks viable sars-cov-2 in the air of a hospital room with covid-19 patients key: cord-335859-k37jivp6 authors: wu, daphne c.; jha, prabhat; lam, teresa; brown, patrick; gelband, hellen; nagelkerke, nico; birnboim, h. chaim; reid, angus title: predictors of self-reported symptoms and testing for covid-19 in canada using a nationally representative survey date: 2020-10-21 journal: plos one doi: 10.1371/journal.pone.0240778 sha: doc_id: 335859 cord_uid: k37jivp6 random population-based surveys to estimate prevalence of sars-cov2 infection causing coronavirus disease (covid-19) are useful to understand distributions and predictors of the infection. in april 2020, the first-ever nationally representative survey in canada polled 4,240 adults age 18 years and older about self-reported covid experience in march, early in the epidemic. we examined the levels and predictors of covid symptoms, defined as fever plus difficulty breathing/shortness of breath, dry cough so severe that it disrupts sleep, and/or loss of sense of smell; and testing for sars-cov-2 by respondents and/or household members. about 8% of canadians reported that they and/or one or more household members experienced covid symptoms. symptoms were more common in younger than in older adults, and among visible minorities. overall, only 3% of respondents and/or household members reported testing for sars-cov-2. being tested was associated with having covid symptoms, indigenous identity, and living in quebec. periodic nationally representative surveys of symptoms, as well as sars-cov-2 antibodies, are required in many countries to understand the pandemic and prepare for the future. the pandemic of sars-cov-2 infection causing coronavirus disease-2019 (covid-19, hereafter "covid") has affected almost every country globally [1] . understanding the sociodemographic characteristics of covid patients can inform efforts to reduce transmission [2] . in canada and other high-income country settings where reliable data can be gathered, a combination of population-based surveys (including surveys and testing), hospitalizations, and mortality data can produce an accurate profile of the impact of the pandemic. here, we report on the results of the first nationally-representative poll in canada of selfreported covid symptoms conducted by the angus reid forum in early april 2020 covering symptoms reported mostly in march 2020, prior to the peak month of test-reported cases in april. specifically, we seek to understand the distribution and predictors of canadians reporting possible covid symptoms and testing for sars-cov-2 using the current standard (polymerase chain reaction or pcr-based) test. we discuss these findings in the context of the age distribution of covid hospitalizations and deaths, and a planned survey of antibodies to sars-cov-2 in a random sample of canadians. the angus reid institute (ari) conducted an online survey from april 1-5, 2020, among a nationally representative randomized sample of 4,240 canadian adults who are members of angus reid forum, drawing upon 70,000 adults in a distributed online panel used for policy research by public sector, not-for-profit, media and commercial organizations (with sampling units approximately corresponding to each federal riding) [3] . a probability sample of this size carries a margin of error of +/-2 percentage points, 19 times out of 20. the sample frame was established to match the canadian census data from statistics canada [4] . the survey was commissioned and paid for by ari. respondents were required to be 18 years or older and to speak english or french. overall, the survey respondents were broadly representative of canadian society in terms of gender, age, regional distribution, and numbers of household members (table 1) . survey respondents were less representative of canada in terms of ethnicities other than indigenous. the survey had fewer single-member households than in the canadian census, and had slightly higher education levels than did the 2019 canadian population. the survey questionnaire included questions related to covid and socio-demographic characteristics among both the respondents and members of their households. questions related to covid symptoms included whether the respondent experienced any of a list of eight symptoms: i) difficulty breathing or shortness of breath, ii) a fever, iii) a mild dry cough, iv) a severe dry cough so severe that it disrupts sleep, v) sore throat, vi) frequent sneezing, vii) loss of sense of smell, viii) fever with hallucinations over the past month. questions related to testing for covid included whether the respondent have been tested for covid, scheduled to be tested, trying to get tested but have not been able to, have done a self-assessment through government website/app, or have not been tested nor planning to be tested or self-assessed. respondents living in households with more than one person were also asked if anyone else in the household had experienced any of the covid symptoms and have been tested for covid. the questionnaire used for this study was developed based on expert opinion, and the choice of covid symptoms were based on expert opinion and physicians from unity health toronto. the full survey questionnaire is provided in the (s1 appendix). the survey instrument was pre-tested in 60 individuals prior to the main survey. the data were collected by angus reid institute and were analysed at angus reid institute and centre for global health research, unity health toronto. all personal identifiers were removed from the collected data and each participant was then assigned a randomly-generated code identifier before analysis. research) provided support in the form of salaries for authors (dcw, hg), but did not have any additional role in the study design, data collection and analysis, decision to public, or preparation of the manuscript. the specific roles of these authors are articulated in the 'author contributions' section. the authors of this manuscript have read the journal's policy and have the following competing interests: hcb is a paid employee of deltadna biosciences, inc., but the company did not fund the study. there are no patents, products in development or marketed products associated with this research to declare. this does not alter our adherence to plos one policies on sharing data and materials. to understand the socio-demographic predictors of covid symptoms, we conducted a logistic regression analysis where the outcome was self-reported symptoms suggestive of covid infection which we defined in this study as the respondent reporting himself/herself and/or at least one member of the household having had a combination of fever (with or without hallucinations) and any of i) difficulty breathing/shortness of breath or ii) dry cough so severe that it disrupts sleep or iii) a loss of a sense of smell in the past month; and the explanatory variables were gender (male, female, or other), education level (high school and under, or some college/ university and higher), province, age, ethnicity (indigenous, english and other european, or others), visible minority (defined as persons, other than aboriginal peoples, who are nonwhite in race or colour) [6] , and number of household members. we defined indigenous ethnicity as whether the person reported identifying with the aboriginal peoples of canada which includes first nations, métis or inuit and/or those who reported registered or treaty indian status [7] . we also used logistic regression analysis to identify predictors of testing, which included the above explanatory variables and also covid symptoms in the respondent or a household member. respondents were considered "tested" if he/she had been tested or were scheduled to be tested. respondents who did not report on any of the explanatory variables were excluded from these analyses. results of the regression analyses were presented after adjusting for possible confounding effect of other explanatory variables as adjusted odds ratios (or) with 95% confidence intervals (ci). discrepancies in or between totals are due to rounding. since the sample frame matched the canadian census data from statistics canada, no additional survey weights were applied. we used rstudio version 1.1.453 for analyses. the angus reid forum obtained consent from all participants. the data without any personal identifiers are made available openly to bona-fide researchers. irb approval for this study was obtained from unity health toronto research ethics board (reb# 20-107). of the 4,240 respondents, 334 (or 7.9%) reported either themselves or at least one of the household members having covid symptoms. of these, 210 (5.0%) reported covid symptoms only in themselves. the adjusted or of the respondent having symptoms when at least one of the household members reported symptoms was 1.45 (95% ci 1.41-1.49); the or was similar for at least one household member having symptoms if the respondent reported symptoms. in terms of testing for sars-cov-2, 126 (or 3.0%) reported some household testing or being scheduled for testing, and only 68 (or 1.6%) reported that they have been or are scheduled for testing. details of the variation in covid symptoms and sars-cov-2 testing in this sample across provinces have already been published [3] . table 2 shows the adjusted or of respondents or a member of the household, and respondents only having covid symptoms after adjustment for other variables examined. we excluded 99 (or 2.3%) respondents who did not report on at least one of the variables. the proportion of respondents reporting covid symptoms within the household decreased with age: 11.2% of those aged 18-45 years, 5.6% among those aged 46-65 years, and 3.1% among those aged 66 years and older. the lower prevalence at higher ages was similar among those reporting covid symptoms only themselves. after controlling for gender, province, age, ethnicity, visible minority, and number of household members, older adults were significantly less likely to report having covid symptoms themselves or within the household (age 46-65, or = 0.55, 0.42-0.71; age 66+, or = 0.30, 0.18-0.47). those who reported themselves as a visible minority were significantly more likely to have covid symptoms (12.9%) compared to those who did not (7.1%; adjusted or = 1.55; 1.08-2.20). similar results were found when we examined covid symptoms only among respondents. the associations changed substantially between unadjusted and adjusted analyses, most notably for ethnicity, and somewhat for being a visible minority, suggesting that some residual confounding factors were present. table 3 shows the adjusted or of respondents or a member of the household being tested or scheduled for sars-cov-2 testing. the strongest predictor of testing was having covid symptoms among members of the household, of whom about 16.5% were tested, compared to 2.1% among those without covid symptoms among household members (or = 6.63, 4.46 a nationally representative survey of canadians finds that about 8% of adults report that they or someone in their household reported symptoms suggestive of covid in march 2020. being a visible minority was associated with higher self-reported covid symptoms. selfreported symptoms were notably less common at older ages than in younger adults. only 3% of canadian adults reported that they or someone in the household had been tested for sars--cov-2. the main predictors of being tested were the presence of covid symptoms, being of indigenous identity, and living in quebec. testing rates were somewhat lower in older adults. there are surprisingly few national studies, and despite some limitations, this study represents the first to document self-reported symptoms in a reasonably representative sample. covid symptoms overlap with some other infections, notably seasonal influenza, which could have inflated the coronavirus rates in this survey. however, a study comparing the covid symptom syndrome with other infections in the united states suggests that most are actually due to covid [8] . we found possible covid symptoms to be less prevalent and the levels of testing marginally less prevalent in older adults than other age groups, despite the certainty that the vast majority of covid hospitalizations and deaths occur at older ages. however, a weakness of our sample is the lack of representation from nursing and long-term care residents, in whom more than three-quarters of all covid deaths occur [1] . approximately 0.3 million (out a total population of 38 million) canadians are estimated to live in nursing homes/long-term care institutions [9] . this would represent less than 1% of the expected population in the survey. such populations were under-represented in the survey. there may be additional reasons for the age-specific findings, however. anecdotal reports suggest that older adults do not experience the symptoms used to define covid infection in the poll, but may report vaguer symptoms such as dizziness and confusion [10] . further surveys focused on syndromes that might occur in older adults are warranted. the testing results are broadly consistent with reports of the general levels of access to sars-cov-2 testing during the survey time period, including a higher level of testing in quebec than in ontario. however, testing results tend not to be representative of the population [3] . this syndromic survey provides some insights into the prevalence of actual sars-cov-2 in canada. this time period of symptoms in march is consistent with testing data showing a peak exposure and incidence of covid occurred in mid-to late april, presumably about 10-14 days in those adults who were tested because of their symptoms [1] . a forum research and mainstreet research reported an estimated prevalence of 5-8% in early-to-mid april in ontario, which is consistent with our overall results for that province. however, they used a narrow range of symptoms from those we defined in our study [11] . in the uk, pilot results from the covid-19 infection survey being carried out by the office for national statistics found that 0.25% of the community population in england above the age of 2, tested positive for the sars-cov-2 antigen in early-to-mid may 2020. testing involved home self-tests of nasal swabs, and excluded those in hospitals, care homes, or other institutional settings [12] . the prevalence was lower than expected in the pilot, and larger studies are planned, along with antibody surveys. a nationally representative sero-epidemiological study is needed to establish the population distribution of cumulative sars-cov-2 infection, which would capture both symptomatic and asymptomatic cases. such studies have now been conducted in england [13] , iceland [14] , brazil [15] , and spain [16] . we have begun such a study in canada called action to beat coronavirus/action pour battre le coronavirus (ab-c). the study protocol is provided in the (s2 appendix). this study will determine the cumulative prevalence of sars-cov-2 infection during march-may 2020 in canada through testing for igg antibodies (using dual assays), paired with household questionnaire data on covid experience. a second round of questionnaires and antibody testing four to six months later in the same individuals will provide information on ongoing transmission, changes in the immune status of the population, and the durability of the immune response. the angus reid forum is partnering with the centre for global health research at unity health toronto and the university of toronto on this study. the expected sample frame is 14,000 individuals surveyed, of which we expect about 8,000 to agree to provide a blood sample. the present analysis informs the design of antibody surveys. for example, since the strongest predictor of covid testing was the presence of symptoms, a similar higher prevalence of antibodies might be expected among those reporting symptoms (who may be more likely to enroll in the survey). thus, ensuring that the sample size is sufficiently large to examine the seroprevalence in those without symptoms is key. similarly, while the current study did not examine geographic clustering of covid symptoms, this might well be the case with actual sars-cov-2 infection. there have been calls for use of self-reported syndromic data to monitor the epidemic [17] . we believe, that these strategies should include also large and as diversely representative survey as possible. older adults bear the brunt of covid hospitalization and deaths. however, this population reported a lower prevalence of symptoms than younger adults. thus, the ab-c study will oversample the population age 60 or older, particularly to understand the possible role of asymptomatic infections at older ages. as the ab-c study is not likely to capture the prevalence of infection among nursing home residents, ancillary studies are needed to quantify hazards among this important group. self-reported symptoms are, by their nature, subject to limitations and misclassification. however, the trends over time, even with misclassification, are useful for understanding trends in the actual underlying prevalence of infection. this is because the "noise" of covid symptoms should change little from one survey to the next, and provided there is large "signal" due to the covid pandemic-certainly the case with this virus-reliable estimation of the trends of infection are possible. similar methodological insights have arisen from hiv-1 testing in pregnant populations in various countries [18] . the biases in enrollment are also inherent in any polling, but the angus reid polling showed reasonably good consistency with the canadian 2016 census. moreover, the testing results did not appear to be biased greatly, and the more common testing reported in quebec is consistent with public health reports. another limitation of the study is that the survey questionnaire was not validated against serological results as none were available at the time of study design. however, subsequent studies comparing antibody with symptoms which we defined as suggestive of covid in our study confirmed that our choice of symptoms was appropriate [19] . in conclusion, covid surveillance using nationally representative surveys is essential, and ideally needs to be accompanied by antibody determination of infection. particular attention must be paid to symptoms and testing levels in older adults. covid: recent updates on the coronavirus pandemic characteristics of adult outpatients and inpatients with covid-19-11 academic medical centers morbidity and mortality weekly report; v. 69, early release the incidence of covid infection in canada? new survey points to over 100,000 households census of population table 17-10-0005-01 population estimates on july 1st, by age and sex visible minority of person aboriginal identity of person covidview: a weekly surveillance summary of u.s. covid activity clhia report on long-term care policy: improving the accessibility, quality and sustainability of long-term care in canada seniors with covid show unusual symptoms, doctors say. cnn health 2020 -for immediate release covid-19 infection survey (cis) antibody prevalence for sars-cov-2 in england following first peak of the pandemic: react2 study in 100,000 adults humoral immune response to sars-cov-2 in iceland remarkable variability in sars-cov-2 antibodies across brazilian regions: nationwide serological household survey in 27 states prevalence of sars-cov-2 in spain (ene-covid): a nationwide, population-based seroepidemiological study putting the public back in public health-surveying symptoms of covid-19 trends in hiv-1 in young adults in south india from 2000 to 2004: a prevalence study coronavirus (covid-19) infection survey: characteristics of people testing positive for covid-19 in england the action to beat coronavirus in canada/action pour battre le coronavirus (ab-c) study group key: cord-340766-aic570x8 authors: kim, se jin; kim, kang; park, sung bum; hong, duck jin; jhun, byung woo title: outcomes of early administration of cidofovir in non-immunocompromised patients with severe adenovirus pneumonia date: 2015-04-15 journal: plos one doi: 10.1371/journal.pone.0122642 sha: doc_id: 340766 cord_uid: aic570x8 the benefits of treatment with antiviral therapy for severe adenovirus (adv) pneumonia are not well established. we described the clinical characteristics and treatment outcomes of early cidofovir treatment of severe adv pneumonia in non-immunocompromised patients. we retrospectively reviewed the medical records of all patients diagnosed with severe adv pneumonia between 2012 and 2014. a total of seven non-immunocompromised patients with severe adv pneumonia were identified, and all isolates typed (n = 6) were human adv-b55. all patients had progressive respiratory failure with lobar consolidation with or without patchy ground glass opacity. three patients required vasopressors and mechanical ventilation. all patients had abnormal laboratory findings including: leukopenia, thrombocytopenia, or elevated liver enzymes. after admission, all patients received antiviral therapy with cidofovir, and the median time from admission to cidofovir administration was 48 h and median the time from onset of symptoms to cidofovir administration was 7.1 days. after cidofovir administration, complete symptomatic improvement occurred after a median of 12 days and radiographic resolution occurred after a median of 21 days. consequently, all patients completely improved without complications. our data suggest that early administration of cidofovir in the course of treatment for respiratory failure as a result of adv pneumonia in non-immunocompromised patients could be a treatment strategy worth considering, especially in cases of hadv-55 infection. adenoviruses (adv) are non-enveloped, double-stranded dna viruses that can cause diseases of the upper and lower respiratory, gastrointestinal, and ocular systems [1] . respiratory infection caused by adv in non-immunocompromised patients is usually mild and self-limited. in contrast, in immunocompromised patients, adv infection can be disseminated and result in a considerable mortality rate [2] [3] [4] . despite the low incidence of serious illness in non-immunocompromised patients, levin et al. reported fatal adv pneumonia in a previously healthy adult in 1967 [5] , and sporadic cases of severe respiratory disease in non-immunocompromised adults have been reported [6] [7] [8] [9] [10] [11] since. these reports include several outbreaks of adv respiratory infections in previously healthy adults in vulnerable populations, such as residents of mental health care centers and military recruits [12] [13] [14] [15] [16] [17] . previous investigations described the clinical characteristics of severe adv pneumonia as being distinct from those of other causative agents [8, 18, 19] , and revealed that several adv serotypes were associated with severe disease states [6, [19] [20] [21] . however, these efforts have not influenced the overall clinical outcomes in cases of severe adv pneumonia. currently, there are no approved antiviral agents for the treatment of severe adv pneumonia, and limited data on the clinical response to antiviral therapy in immunocompromised patients are available [6, 8] . our institution, a military hospital with the highest referral rate in south korea, encounters several cases of adv pneumonia annually, some of which are severe cases [17, 22] , thus we have aimed to produce favorable outcomes by applying antiviral therapy upon confirmation of the diagnosis of severe adv pneumonia. the present study describes in detail the clinical characteristics and favorable treatment outcomes of non-immunocompromised adults who had experienced severe adv pneumonia and received early cidofovir administration. we retrospectively reviewed the medical records of all consecutive patients diagnosed with adv pneumonia between december 1, 2012 and june 1, 2014 at the armed forces capital hospital (874 bed referral military hospital) in gyeonggi province, south korea. the diagnosis of adv pneumonia was considered certain when it was associated with the following: 1) lower respiratory and/or systemic symptoms, 2) lung infiltration on chest radiography or computed tomography (ct) scan, and 3) evidence of adv infection identified by positive multiplex polymerase chain reaction (pcr) for adv from lower respiratory tract samples, such as sputum, or bronchoalveolar lavage (bal) fluid. only non-immunocompromised adult patients who fulfilled the criteria for severe community-acquired pneumonia, set out in the infectious diseases society of america/american thoracic society consensus guidelines [23] , and admitted to the intensive care unit with progressive respiratory failure, defined as a partial pressure of arterial oxygen (pao 2 )/fraction of inspired oxygen (fio 2 ) ratio of < 300 mmhg and/or tachypnea (respiration rate >30 breaths/min) [24] , were included in the analysis. criteria for severe community-acquired pneumonia requiring admission to the intensive care unit are one or more major criteria or three or more minor criteria [23] . major criteria include: 1) invasive mechanical ventilation and 2) requiring vasopressors due to septic shock. minor criteria include: 1) respiratory rate 30/min, 2) pao 2 /fio 2 ratio 250 mmhg, 3) multilobar pneumonia in chest radiographs, 4) decreased level of consciousness, 5) blood urea nitrogen 20 mg/dl, 6) white blood cells < 4,000/mm 3 , 7) platelets < 100,000/mm 3 , 8) core temperature < 36°c, and 9) hypotension requiring aggressive fluid therapy. the institutional review board of the armed forces capital hospital approved this study and permitted review and publication of patient records. all data on the study patients were analyzed anonymously. the requirement for informed consent by individual patients was waived given the retrospective nature of the study. findings including leukopenia or extrapulmonary symptoms were suspected of having atypical pneumonia [25] , and underwent thorough examinations. infectious etiologies were investigated using peripheral blood, sputum, and bal fluid utilizing techniques such as staining and microbiological culture for bacteria and mycobacterium tuberculosis; multiplex real time pcr testing for respiratory bacterial agents including streptococcus pneumoniae, haemophilus influenza, mycoplasma pneumoniae, chlamydia pneumoniae, legionella, and bordetella; and multiplex real time pcr testing for respiratory viruses. nucleic acid extraction was performed on the magna pure lc 2.0 (roche diagnostics, mannheim, germany) using a magna pure lc nucleic acid isolation kit i (roche diagnostics) according to the manufacturer's instructions. more than fifty microliters of clinical samples were used for nucleic acid extraction and eluted in 50 μl of elution buffer. multiplex real-time pcr was performed to screen for 15 commonly isolated respiratory viral pathogens, including adenovirus, rhinovirus, influenza virus a/b, respiratory syncytial virus a/b, bocavirus, coronavirus 229e/oc43/nl63/hku1, parainfluenza virus 1/2/3, and metapneumovirus, using a real-q rv detection kit (biosewoom, seoul, korea) on a roche light cycler 480 ii instrument (roche diagnostics, mannheim, germany) according to the manufacturer's instructions. the presence of specific viral sequences in the reaction was detected by an increase in the fam, vic and cy5 fluorescence from the relevant dual-labeled probe. five primer/probe sets were used to detect respiratory viruses. the serotype of human-adv-positive samples was evaluated by sequence analysis using the partial hexon genomic region. cidofovir (mylan institutional) was initiated upon confirmation of adv infection. cidofovir, at a dosage of 5mg/kg weekly, was administrated in combination with oral probenecid. hydration consisted of 1-2 l of normal saline before cidofovir infusion and 1-2 l immediately thereafter to prevent renal toxicity. a total dose of 2-g probenecid was given orally 3 h before infusion, and 1 g at 2 and 8 h after the cidofovir infusion. the number of cidofovir courses administered was determined by the attending physician. eleven patients were newly diagnosed with adv pneumonia and admitted during the study period; all were non-immunocompromised adults. of these, four patients did not fulfill the criteria for severe community-acquired pneumonia [23] , improved clinically with no antiviral therapy, and were excluded from the analysis. consequently, seven patients with severe adv pneumonia were analyzed in this study; their baseline characteristics are shown in table 1 . all patients were previously healthy young males without underlying conditions. three (43%) patients were non-smokers. body mass index (kg/m 2 ) of patients varied from 21.7-32.4. the illnesses of most of the patients (n = 6) occurred in early spring, while that of one occurred in the winter. three (43%) patients were from a military training facility and all patients were from different operational areas. all patients presented with acute respiratory or systemic symptoms, such as fever (n = 7), cough (n = 6), purulent sputum (n = 6), blood-tinged sputum (n = 3), and dyspnea (n = 7). one patient had concurrent upper respiratory tract symptoms, and three patients had extrapulmonary symptoms such as diarrhea. all patients had respiratory distress, with hypoxemia (pao 2 /fio 2 ratio <300 mmhg). six (86%) patients had tachycardia (>100 beats/min) or tachypnea (>30 breaths/min). three (43%) patients needed vasopressors and mechanical ventilation. the median duration from onset of symptoms to admission was 5 days. five (71%) patients received empirical antibiotics including quinolone (n = 3) and third-generation cephalosporins (n = 2) before admission. the two remaining patients received quinolone (n = 1; identified as patient 3) and piperacillin (n = 1; identified as patient 6) on admission. initial chest ct findings in patients with severe adv pneumonia are shown in table 2 . most patients (n = 5, 71%) had bilateral lobar consolidation with ground glass opacity in the upper and lower lobes (fig 1) ; only two patients (29%) had unilateral lobar consolidation with or without ground glass opacity. the most commonly involved lobe was the left lower lobe (n = 3), followed by the right lower lobe (n = 2), left lower lobe (n = 1), and both lower lobes (n = 1). pleural effusion was observed in all patients. the results of initial laboratory tests are shown in table 3 . all patients had thrombocytopenia (<150,000/μl), most (n = 6, 86%) patients had leukopenia (<4,000/μl), and none had leukocytosis (>10,000/μl). c-reactive protein levels were elevated in all patients, with a median of 8.31 mg/dl. procalcitonin levels were elevated in five (71%) patients, with a maximum of 24.23 ng/ml. hyponatremia was observed in two (29%) patients, and all patients had elevated liver enzymes. pleural fluid analysis was performed in four (57%) patients; all results were lymphocyte-dominant exudates without evidence of empyema. adv was identified in all patients by pcr in sputum (n = 7) and bal fluid (n = 2). serotyping by sequence analysis was available for six patients; all isolates typed (n = 6) were human adv-b55 (s1 fig). bacterial co-infection was identified in the sputum of two (29%) patients by pcr; s. pneumoniae (patient 3) and s. pneumoniae + h. influenzae (patient 5) were identified. no other causative agent was identified. clinical responses to antiviral therapy in each study patient the clinical responses to treatment in each patient are shown in figs 2 and 3. fig 2 shows the clinical responses of patients 1-3, who needed vasopressors and mechanical ventilation due to septic shock and severe respiratory failure. in patient 1, after cidofovir administration, blood pressure stabilized after approximately 4 days, and tachypnea, tachycardia, and fever improved within 5 days. oxygenation completely improved within 14 days (fig 2a) . in patient 2, blood pressure stabilized after approximately 2 days, and tachypnea, tachycardia, and fever improved within 4 days. oxygenation completely improved within 8 days of cidofovir administration (fig 2b) . in patient 3, blood pressure stabilized after approximately 4 days, and tachypnea, tachycardia, and fever within 7 days. oxygenation completely improved within 9 days of cidofovir administration (fig 2c) . in patient 4, tachypnea, tachycardia, and fever improved within 3 days, and oxygenation completely improved within 5 days, of cidofovir administration (fig 3a) . in patient 5, tachypnea, tachycardia, and fever improved in 3 days, and oxygenation completely improved within 12 days, of cidofovir administration (fig 3b) . in doi:10.1371/journal.pone.0122642.g001 patient 6, tachypnea, tachycardia, and fever improved in 2 days, and oxygenation completely improved within 2 days, of cidofovir administration (fig 3c) . in patient 7, tachypnea, tachycardia, and fever improved in 2 days, and oxygenation completely improved within 6 days, of cidofovir administration (fig 3d) . overall treatment outcomes of study patients are summarized in table 4 . median time from admission to identification of adv infection was 2 (range, 1-6) days. all patients received cidofovir, and most patients (n = 6, 86%) received cidofovir within 72 h (minimum, 25 h and median, 48 h); only one patient (14%) received cidofovir 156 h after admission. the median time from onset of symptoms to cidofovir administration was 7.1 days. three patients (patients 1, 2, and 3) who needed vasopressors and mechanical ventilation received two doses of cidofovir. the other four patients (patients 4, 5, 6, and 7) received single doses of cidofovir. six (86%) patients received adjuvant intravenous immunoglobulin for 3 days. after cidofovir administration, clinical, radiological, and laboratory improvement occurred in all patients. complete symptomatic improvement occurred after a median of 12 (range, 7-25) days, and complete radiographic resolution occurred after a median of 21 (range, 13-40) days (fig 4) . normalization of white blood cell count and c-reactive protein level after a median of 6 (range, 3-11) days and 11 (range, 4-13) days, respectively. adverse reactions (skin rash) to cidofovir occurred in one patient, and resolved completely with only conservative management. all patients recovered completely without complications. in the present study, we described favorable outcomes to antiviral therapy with cidofovir in non-immunocompromised adult patients with severe adv pneumonia. our data suggest that early administration of cidofovir in the course of respiratory failure could be a treatment strategy worth considering in severe adv pneumonia. in the seven cases discussed, cidofovir was administered more promptly than in previous fatal cases. in previous studies, most patients did not receive antiviral therapy with cidofovir; indeed, even in the few patients who did receive cidofovir, the administration time was not early in the course of respiratory failure [6, 8, 12] and they had poor outcomes. for example, in a recent study that evaluated outcomes in five patients with severe adv pneumonia caused by adv-55 infection in non-immunocompromised adults [26] , similar to our study group, four (80%) of the five patients died despite receiving appropriate respiratory support and antiviral therapy, including acyclovir, ganciclovir, and ribavirin, without using cidofovir. in a study of the etiology and epidemiology of acute viral lower respiratory tract infections of south korean soldiers in our institution, six cases of severe adv pneumonia were identified. all six patients required mechanical ventilation, most (5/6) did not receive cidofovir, and half (3/6), including one patient who was treated with cidofovir 7 days after admission, died [17] . moreover, in a case report of fatal adv pneumonia in immunocompetent adult identical twins, one patient did not recover despite administration of cidofovir antiviral therapy no sooner than 8 days after the onset of symptoms [6] . in the present study, in contrast, time from admission to identification of adv was short (median, 2 days), and all patients received antiviral therapy on the day of diagnosis of adv infection. given this background, our findings may indicate that administration of cidofovir early in the course of respiratory failure may be a beneficial treatment strategy in severe cases of adv respiratory infection, and this may assist physicians in making decisions regarding diagnosis and treatment. in our study, all isolates typed (n = 6) were human adv-b55 [27, 28] . to date, more than 60 human adv types have been identified [29] [30] [31] [32] , and serotypes 1-5, 7, 11, 14, 21 , and 41 are most commonly associated with human disease. among these, human adv-b55 has been identified as an emergent acute respiratory disease pathogen, fully characterized by wholegenome sequencing, and has caused two recent outbreaks; one in china in 2006 [33] and one in singapore in 2005 [14] . in one study that evaluated 48 cases of adv pneumonia [19] , patients infected with human adv-55 had higher pneumonia severity index scores compared with those infected with other serotypes. thus, human adv-55 could be a major pneumonia pathogen in the general population, and further surveillance and monitoring of this agent as a cause of pneumonia are warranted. however, to date, limited data on the correlation between human adv-55 and clinical severity or clinical courses are available; and no data are available on the treatment responses to cidofovir in cases of human adv-55 infection. in these contexts, our data may have clinical significance and further studies on the associations of adv serotypes with severe respiratory infection are necessary. the pathophysiology of adv pneumonia remains unclear; few studies have been performed. for example, prince et al. [34] and gregory et al. [35] indicated that much of the acute damage in adenovirus infection appears to be due to dysregulation of the immune system. these features may, in part, explain the self-limiting nature of cases of even severe adv infection without antiviral therapy, and suggest that the host's immunological reaction to the infection is a major factor in the pathogenesis of the lung injury. moreover, this implies that antiviral therapy administered late in the course of respiratory failure may not be effective. however, because there are no accurate data on the pathophysiology of adv, further studies are needed. this study had several limitations. firstly, the study was not controlled with respect to the treatment administered because of its retrospective design. although we obtained favorable outcomes, other factors may have influenced the outcomes in the study patients, such as the supportive treatment strategies, including ventilator settings and the prone position, or host factors, including young age and previous good health status, and moreover, self-limiting cases of severe adv infection have been reported previously [9] [10] [11] 16] . to date, no controlled study has been reported on the benefits of antiviral therapy in severe adv pneumonia. this may be mainly because of the rarity of severe adv pneumonia in non-immunocompromised adults and ethical problems in using antiviral agents that have not obviously been demonstrated to have clinical effect. in this context, our data, despite the limitations, may suggest possible benefits of administering cidofovir as early as possible in severe adv pneumonia caused mainly by human adv-55. based on our clinical data, further controlled studies are warranted to confirm the benefits of cidofovir. second, despite extensive microbiological testing, the possible influences of bacterial or other co-infection on treatment outcomes cannot be excluded. furthermore, this study included only a small number of patients. additionally, the study population was not representative of the general population in terms of age or gender because our study was conducted at a military hospital; thus, large prospective studies are needed. we described favorable clinical outcomes to antiviral therapy with cidofovir in non-immunocompromised adult patients with severe adv pneumonia. our data suggest that early administration of cidofovir in the course of treatment for respiratory failure as a result of adv pneumonia in non-immunocompromised patients could be a treatment strategy worth considering, especially in cases of hadv-55 infection. late fatal adenovirus pneumonitis in a lung transplant recipient treatment of adenovirus pneumonia with cidofovir in pediatric lung transplant recipients lethal hemorrhagic alveolitis after adenovirus pneumonia in a lung transplant recipient fatal nonbacterial pneumonia associated with adenovirus type 4. occurrence in an adult fatal type 3 adenoviral pneumonia in immunocompetent adult identical twins 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community-acquired respiratory viral infections in adult inpatients with lower respiratory tract infections emergence of community-acquired adenovirus type 55 as a cause of community-onset pneumonia adenovirus type 7 associated with severe and fatal acute lower respiratory infections in argentine children adenovirus serotype 14 pneumonia at a basic military training site in the united states, spring 2007: a case series high isolation rate of adenovirus serotype 7 from south korean military recruits with mild acute respiratory disease infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults acute respiratory distress syndrome: the berlin definition laboratory diagnosis of atypical pneumonia emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study internally controlled real-time pcr monitoring of adenovirus dna load in serum or plasma of transplant recipients real-time pcr with an internal control for detection of all known human adenovirus serotypes genetic analysis of a novel human adenovirus with a serologically unique hexon and a recombinant fiber gene novel human adenovirus strain computational analysis of human adenovirus type 22 provides evidence for recombination among species d human adenoviruses in the penton base gene computational analysis identifies human adenovirus type 55 as a re-emergent acute respiratory disease pathogen genomic analyses of recombinant adenovirus type 11a in china pathogenesis of adenovirus type 5 pneumonia in cotton rats (sigmodon hispidus) implications of the innate immune response to adenovirus and adenoviral vectors we thank all the patients and physicians involved in the current study. conceived and designed the experiments: bwj sjk kk sbp djh. analyzed the data: bwj sjk. contributed reagents/materials/analysis tools: bwj sjk kk sbp djh. wrote the paper: bwj sjk kk sbp djh. key: cord-319538-bawzonq1 authors: krause, martin; douin, david j.; tran, timothy t.; fernandez-bustamante, ana; aftab, muhammad; bartels, karsten title: association between procalcitonin levels and duration of mechanical ventilation in covid-19 patients date: 2020-09-18 journal: plos one doi: 10.1371/journal.pone.0239174 sha: doc_id: 319538 cord_uid: bawzonq1 background: patients diagnosed with covid-19 frequently require mechanical ventilation. knowledge of laboratory tests associated with the prolonged need for mechanical ventilation may guide resource allocation. we hypothesized that an elevated plasma procalcitonin level (>0.1 ng/ml) would be associated with the duration of invasive mechanical ventilation. methods: patients diagnosed with covid-19, who were admitted to any of our health system’s hospitals between march 9(th)-april 20(th), 2020 and required invasive mechanical ventilation, were eligible for this observational cohort study. demographics, comorbidities, components of the sequential organ failure assessment score, and procalcitonin levels on admission were obtained from the electronic health record. the primary outcome was the duration of mechanical ventilation; secondary outcomes included 28-day mortality and time to intubation. outcomes were assessed within the first 28 days of admission. baseline demographics and comorbidities were summarized by descriptive statistics. univariate comparisons were made using pearson’s chi-square test for binary outcomes and mann-whitney u test for continuous outcomes. a multiple linear regression was fitted to assess the association between procalcitonin levels and the duration of mechanical ventilation. results: patients with an initial procalcitonin level >0.1 ng/ml required a significantly longer duration of mechanical ventilation than patients with a level of ≤0.1 ng/ml (p = 0.021) in the univariate analysis. there was no significant difference in 28-day mortality or time to intubation between the two groups. after adjusted analysis using multivariable linear regression, the duration of mechanical ventilation was, on average, 5.6 (p = 0.016) days longer in patients with an initial procalcitonin level >0.1 ng/ml. conclusion: in this cohort of 93 mechanically ventilated covid-19 patients, we found an association between an initial plasma procalcitonin level >0.1 ng/ml and the duration of mechanical ventilation. these findings may help to identify patients at risk for prolonged mechanical ventilation upon admission. since the initial outbreak in the hubei province of china in november 2019, severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has spread around the world and was declared a pandemic by the world health organization [1, 2] . based on observational studies from the epicenters of the pandemic in wuhan, china, the lombardy region in italy, and the new york city area in the united states, a significant portion of patients diagnosed with coronavirus disease 2019 (covid19) were admitted to the intensive care unit (icu) for ventilatory support: between 17%-24% of hospitalized patients and up to 72% of patients admitted to the icu have required invasive mechanical ventilation [2] [3] [4] [5] . while most ventilated patients fulfill the berlin definition for acute hypoxemic respiratory distress syndrome, the unique and variable clinical course of covid-19 has made it difficult to predict the disease's progression [6, 7] . laboratory values, including plasma c-reactive protein, d-dimer, lactate dehydrogenase, and procalcitonin, are often elevated in patients with covid-19 who required icu-admission, mechanical ventilation, or died [8] [9] [10] [11] . knowledge of laboratory tests associated with prolonged mechanical ventilation and mortality in a health care environment constrained by high demand and limited resources, especially regarding ventilatory support, is critical. in a critically ill covid-19 cohort from a large united states healthcare system, we hypothesized that elevated plasma procalcitonin levels would be associated with a longer duration of mechanical ventilation. ethical approval was obtained from the institutional review board (colorado multiple institutional review board #20-0677) on april 3 rd , 2020, and the requirement for informed consent was waived. data were collected retrospectively for any events before april 3 rd , and prospectively going forward. we followed the strengthening the reporting of observational studies in epidemiology (strobe) guidelines for reporting observational studies [12] . we aimed to identify if plasma procalcitonin levels on admission are associated with the duration of mechanical ventilation (primary outcome), 28-day mortality, and time to intubation (secondary outcomes) in a cohort of covid-19 patients requiring mechanical ventilation. we designed an observational cohort study using automated data collection and manual chart review from the electronic health record (ehr) from 12 hospitals within a large united states health care system. patients with covid-19 that were admitted between 03/09/2020 and 04/20/2020 to one of the health system's icus requiring mechanical ventilation were eligible for inclusion in this study [13] . exclusion criteria were age <18 years, patients' or their proxies' objection to observational data collection for research, or no procalcitonin level drawn during the admission (fig 1) . patient characteristics including relevant baseline demographics, chronic comorbidities, components of the sequential organ failure assessment (sofa) score, and procalcitonin levels were obtained from the ehr. demographics included: age in years, self-reported gender, body mass index (bmi) in kg/m 2 , race, ethnicity, and insurance status. we chose chronic comorbidities according to previously reported findings from our group [13] and others [10, 14] based on relevant pre-existing international classification of diseases codes (icd-10) information from the ehr. comorbidities were comprised of any history of cardiac disease, pulmonary disease, hypertension, diabetes mellitus, renal disease, liver disease, and solid malignancies. to calculate the sofa score at hospital admission, we used the first drawn platelet count, creatinine, and bilirubin, the first recorded glasgow coma scale score, and the lowest mean arterial pressure or the highest dose of vasoactive agents within the first eight hours of admission [15] . furthermore, we obtained the first drawn procalcitonin level during the index admission. in-hospital mortality within 28 days was assessed based on the date of death as recorded in the ehr. information on the duration of mechanical ventilation during the first 28 days of admission was obtained using manual chart review. following published literature [16, 17] , days were counted towards invasive mechanical ventilation, if the patient was mechanically ventilated within the first 28 days of admission, if a patient was reintubated within 48 hours (failure to extubate), or if a patient with a tracheostomy was on noninvasive ventilation or not requiring invasive/noninvasive ventilation for less than 48 hours (failure to wean). furthermore, a patient who died within the first 28 days of admission was accounted for 28 days of invasive mechanical ventilation [16, 17] . the day of admission accounted for day 1 to assess time from admission to intubation and time from admission to death. results were summarized using descriptive statistics, and continuous data were assessed for normality of distribution. univariate comparisons were performed using mann-whitney u tests for continuous outcomes, and pearson's chi-square test for the binary mortality outcome. we then assessed if there was an association between an initial procalcitonin level of >0.1 ng/ ml [18] vs. less and the duration of mechanical ventilation by fitting a linear regression model. age, bmi, hypertension, diabetes mellitus, chronic cardiac disease, sofa score on admission, and the first available procalcitonin level during the hospitalization were chosen as covariates in the model. statistical analysis was performed using the software program spss, version 26 (ibm corporation, armonk, ny). a basic power analysis for a linear multiple regression model was performed using g � power, version 3.1.9.2 [19] . imputing an effect size f 2 of 0.15, the number of tested predictors as 1, and the total number of predictors as 4, an alpha = 0.05 and a desired power of 90%, the sample size required would have been n = 73. a total of 93 mechanically ventilated patients who tested positive for covid-19 and had a procalcitonin level on file were included. the median duration of mechanical ventilation was 14 days. patients' demographics and comorbidities, sofa scores on admission, initial procalcitonin levels, and outcomes are summarized in table 1 . in the univariate analysis, patients with an initial procalcitonin level >0.1 ng/ml had a median duration of 17 days mechanical ventilation compared to patients with an initial procalcitonin level �0.1 ng/ml, who had a median duration of 10 days (p = 0.021) ( table 2 ). there was no significant difference in duration of mechanical ventilation for patients with a procalcitonin level >0.25 ng/ml vs. lower or >0.5 ng/ml vs. lower. there was also no significant difference in 28-day mortality or time from admission to intubation for patients with a age-years 59 (15) (continued ) procalcitonin level >0.1 ng/ml vs. lower (table 2 ). in deceased patients with an initial procalcitonin level >0.1 ng/ml, death occurred at a median duration of 11 days. the results from a fitted multivariable linear regression model with the duration of mechanical ventilation in days as the dependent variable are presented in table 3 . after adjustment for age, bmi, chronic cardiac disease, diabetes mellitus, hypertension, and sofa score on admission, the multivariable linear regression model demonstrated that the duration of mechanical ventilation was on average 5.6 days (95% confidence interval 1.09-10.17, p = 0.016) longer in patients with an initial procalcitonin level >0.1 ng/ml compared to those with a lower procalcitonin level. in addition, older age (b = 0.22, 95% confidence interval 0.10-0.35, p = 0.001) and higher bmi (b = 0.23, 95% confidence interval 0.02-0.43, p = 0.033) were associated with a longer duration of mechanical ventilation. the other independent variables included in the regression model were not significantly associated with the duration of mechanical ventilation. in the univariate analysis of our observational cohort study, procalcitonin levels >0.1 ng/ml on admission were associated with prolonged mechanical ventilation in critically ill covid-19 patients. the multiple linear regression model revealed that the duration of mechanical ventilation was, on average, 5.6 days longer in patients with an initial procalcitonin level >0.1 ng/ ml. our findings demonstrate an association between elevated procalcitonin levels on admission and duration of mechanical ventilation in patients with covid-19, thereby affirming our hypothesis. given a shortage of ventilators in epicenters of this pandemic and the association between limited resources and covid-19 mortality, we view our findings as highly relevant [20, 21] . in addition, prolonged mechanical ventilation is associated with icu-acquired weakness and neurocognitive decline, which both contribute to short-and long-term-mortality [22, 23] . early studies from china identified an elevated procalcitonin level as a predictor of the severity of covid-19 infections but did not specifically explore an association between procalcitonin and duration of mechanical ventilation [3, 9, 11] . procalcitonin is currently used to predict treatment failure and mortality in lower respiratory infections [24] . however, its plasma level often remains within the normal range in non-complicated cases of covid-19. it has, therefore, been suggested as a helpful early marker of bacterial superinfections and disease progression [25] . indeed, mechanically ventilated covid-19 patients are more likely to suffer from bacteremia [26] . identifying patients with covid-19 who are at risk for prolonged mechanical ventilation may assist clinicians in allocating scarce resources and aid in patients' risk stratification. our multivariate regression analysis was adjusted for severity of disease by including the sofa score, for predisposing characteristics by including age, bmi, diabetes mellitus, hypertension, and chronic cardiac disease, and for procalcitonin levels on admission based on our own preliminary findings and other literature [8-11, 13, 27-29] . besides a procalcitonin level of >0.1 ng/ml, duration of mechanical ventilation was associated with age and bmi in our multivariate regression model. indeed, early literature from china and europe identified age and obesity as risk factors for invasive mechanical ventilation [2, 7, 14, 30] . we did not find a correlation between elevated procalcitonin levels and 28-day mortality. however, 17 of 18 patients who died (94.4%) showed an elevated procalcitonin level suggesting that appropriately powered retro-or prospective studies might be able to identify such an association. our study has multiple weaknesses: first, an observational cohort study is not designed as a randomized controlled trial, and residual confounding remains possible [31] . for example, exclusion of patients without a documented procalcitonin level may have induced a selection bias. however, only three patients were excluded due to the lack of laboratory testing. furthermore, our findings may not be representative of other areas of the united states or other countries. lastly, prolonged mechanical ventilation and in-hospital mortality were determined within 28 days of the patient's admission. a later follow-up could potentially show significant associations between demographics, comorbidities, and laboratory values in either of our outcomes, which were not identified at 28 days. given the need for additional timely information during this pandemic as well as previous work by others [16, 17] , we chose 28 days as the cutoff time-point for this study. our observational study including 93 critically ill covid-19 patients requiring mechanical ventilation found an association between a procalcitonin value of >0.1 ng/ml on admission and the duration of mechanical ventilation. procalcitonin could be used to risk-stratify mechanically ventilated covid-19 patients. a novel coronavirus from patients with pneumonia in china baseline characteristics and outcomes of 1591 patients infected with sars-cov-2 admitted to icus of the lombardy region cardiovascular implications of fatal outcomes of patients with coronavirus disease 2019 (covid-19) clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a 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requiring invasive mechanical ventilation the sofa score-development, utility and challenges of accurate assessment in clinical trials reappraisal of ventilator-free days in critical care research statistical evaluation of ventilator-free days as an efficacy measure in clinical trials of treatments for acute respiratory distress syndrome procalcitonin algorithm in critically ill adults with undifferentiated infection or suspected sepsis. a randomized controlled trial statistical power analyses using g*power 3.1: tests for correlation and regression analyses. behavior research methods potential association between covid-19 mortality and healthcare resource availability critical supply shortages-the need for ventilators and personal protective equipment during the covid-19 pandemic clinical practice guidelines for the management of pain, agitation, and delirium in adult patients in the intensive care unit clinical review: intensive care unit acquired weakness prognostic value of procalcitonin in respiratory tract infections across clinical settings procalcitonin in patients with severe coronavirus disease 2019 (covid-19): a meta-analysis clinical characteristics of covid-19 in new york city high prevalence of obesity in severe acute respiratory syndrome coronavirus-2 (sars-cov-2) requiring invasive mechanical ventilation covid-19 and african americans risk factors for the development of acute respiratory distress syndrome in mechanically ventilated adults in peru: a multicenter observational study association of public health interventions with the epidemiology of the covid-19 outbreak in wuhan, china bias in observational study designs: case-control studies key: cord-308249-es948mux authors: dokuka, sofia; valeeva, diliara; yudkevich, maria title: how academic achievement spreads: the role of distinct social networks in academic performance diffusion date: 2020-07-27 journal: plos one doi: 10.1371/journal.pone.0236737 sha: doc_id: 308249 cord_uid: es948mux behavior diffusion through social networks is a key social process. it may be guided by various factors such as network topology, type of propagated behavior, and the strength of network connections. in this paper, we claim that the type of social interactions is also an important ingredient of behavioral diffusion. we examine the spread of academic achievements of first-year undergraduate students through friendship and study assistance networks, applying stochastic actor-oriented modeling. we show that informal social connections transmit performance while instrumental connections do not. the results highlight the importance of friendship in educational environments and contribute to debates on the behavior spread in social networks. social environment has a significant impact on individual decisions and behavior [1] [2] [3] . people tend to assimilate the behavior, social norms, and habits of their friends and peers. it is empirically shown that social interactions play a key role in the spread of innovations [4] , health-related behavior [5, 6] , alcohol consumption and smoking [7, 8] , delinquent behavior [9, 10] , happiness [11] , political views [12, 13] , cultural tastes [14] , academic performance [15] [16] [17] [18] [19] . although there is an extensive body of research showing that a large proportion of social practices disseminates across social networks [3] , the question of what types of social contacts cause the spread of specific behavior remains open [1, 20] . in this paper, we analyze the diffusion of academic performance across different types of student social networks. while these social networks are extensively studied in the literature [15] [16] [17] [18] 21, 22] , there is a lack of agreement on whether social networks are effective channels for the academic performance spread [16, 18, 23] . and if they are, what types of networks serve the best for the propagation of the academic-related behavior? we analyze the spread of academic achievements within two different social networks of first-year undergraduate students. we test two mechanisms of academic performance diffusion in the student social networks. first, we analyze the academic performance spread through the friendship network, which can be considered as a network of informal social a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 interactions. second, we study the academic performance spread in the study assistance network which is aimed at study-related information and knowledge transmission, and serves for problem-solving [24] . we apply stochastic actor-oriented model (saom) for joint modeling of networks and behavior dynamics [25] . we model the evolution of two social systems. in the first model, we analyze the coevolution of friendship network and academic achievements. in the second model, we study the coevolution of study assistance network and academic achievements. both models are controlled with a variety of structural and behavioral properties such as a tendency to form mutual ties and befriend similar others. results show that academic performance spreads through friendship connections, while the study assistance ties do not cause the performance transfer. social networks are the pathways for the behavior transmission. this process may be guided by various factors, including the network topology [26] , type of propagated behavior, nature of social contacts, and other features of the social environment. the majority of recent studies on this topic are concentrated on the structural properties of the networks that drive the behavior diffusion processes. for example, it was experimentally shown that short average path length and high clustering cause a faster behavior spread [27] that can be explained by the formation of the dense network communities with the fast information and behavior exchange within these cohesive groups. in [28] it was demonstrated that there are differences in spreading processes initiated by well-connected actors, or hubs, and by actors with a few social connections. hubs are effective in information propagation due to the high number of connections, while actors with a few ties are more efficient in spreading messages that are controversial or costly. the probability of behavior adoption by an individual is also highly correlated with the number of social contacts that directly influence this individual [29] . the influence by many peers, or so-called "complex contagion", results in faster and easier behavior adoption, rather than the influence by one person, or "simple contagion" [26] . the efficacy of social contagion is often associated with the type of propagated behavior. centola and macy outline the danger of considering the social contagion studies in the 'whatever to be diffused' way [30] . for example, the adoption of information is much less risky, costly, and time-consuming, rather than the adoption of health-related behavior, sports habits, and academic achievements. the nature of social ties is also a significant factor for behavior transmission. social connections are traditionally divided into "weak" and "strong" ties, and they exhibit completely different spreading patterns [31] . strong ties are formed within the dense network communities such as family or friends, while weak ties, according to granovetter's definition, emerge during the whole life and represent people who are marginally included in the network of contacts, such as old college friends or colleagues [31] . empirical literature shows that both types of relationships can serve as channels for the diffusion of behavior or information [1, 20] . but weak ties are important instruments for information propagation, while strong ties are more successful in costly behavior transmission. although the vast array of theoretical and empirical studies improved our understanding of the behavior transmission processes, there is still an open question regarding the differences of the behavior spread in networks of different natures. social ties can vary both in the level of their strength and intensity, as we outlined above, and in their origins. networks can be based on friendship, romantics, advice seeking, social support, and many other relationships. despite the huge variance in the social network types, the majority of the research on social diffusion is concentrated on the networks of friendship ties. however, the relationships of distinct nature can result in completely different behavior transmission processes. in this paper, we consider the transmission of academic performance within student social networks. this process attracts the attention of researchers since the publication of the famous "coleman report" [21] . this report showed that students tend to obtain similar grades as their peers, classmates, and friends, and this effect remains strong after controlling for a variety of socio-economic and cognitive variables. further empirical studies demonstrated the presence of this effect in various case studies. for example, it was shown that student grade point average (gpa) increases if her dormmate is in the highest 25th gpa percentile [32] . in [15] , mba students tend to assimilate the grades of their friends and advisers. it was also demonstrated that this social influence is associated with the personal characteristics of students and the nature of their social connections. for instance, lower-achieving students are more influenced by their peers [33, 34] , the diffusion of academic performance is stronger among women than men [35] , can be related to the race of a peer [36, 37] , and is stronger from close peers such as friends [38] . at the same time, online communication networks do not serve as effective channels for performance transmission. students tend to segregate in online networks based on their performance and this prevents the diffusion of achievements through online ties [18, 19] . summarizing, the majority of studies demonstrate that social networks are effective channels for the performance diffusion. it was shown that achievements spread well within friendship networks, while other types of ties (e.g. online relationships) do not serve as channels for the performance transmission. in this paper, we examine the diffusion of academic achievements in two distinct social networks: friendship and study assistance. we demonstrate that, despite the significant overlap in these networks, they exhibit different patterns of behavior transmission. we analyze the longitudinal data on friendship and study assistance networks and gpa of a first-year student cohort of the economics department in one of the leading russian universities in 2013-2014 academic year. in this university, students are randomly assigned by the administration to different study groups of up to 30 students. lectures are usually delivered to all the cohort simultaneously while seminar classes are delivered to each study group separately. in the first year, most of the courses are obligatory. therefore, students have a limited possibility to form networks with students from other groups, programs, or year cohorts. the academic year consists of four modules of two or three months. at the end of each module, students take final tests and exams. the grading system is at a 10-point scale where a higher score indicates a higher level of academic achievement. the course grade is the weighted average of midterm and final exams, homework, essays, and other academic activities during the course. the sample consists of 31% males and 69% females. the data for this study was gathered from two sources: the longitudinal student questionnaire survey (3 waves during the first academic year: october 2013, february 2014, and june 2014) and the university administrative database. in total, our dataset consists of 117 students that took part in at least two surveys with up to 700 connections between them in total. the detailed over-time aspect of the networks gives us a rich dataset of links of diverse nature. the sample can be considered representative to student cohorts in selective universities. in the questionnaire survey, we ask students about their connections within their cohort. the questions were formulated in the following way: 1. please indicate the classmates with whom you spend most of your time together; 2. please indicate the classmates whom you ask for help with your studies. the role of distinct social networks in academic performance diffusion. there were no limitations in the number of nominations. additionally, students were asked to indicate those classmates whom they knew before the admission to the university. we also gather information about students' study-group affiliation from the administrative database. in total, we have four different network types: friendship, study assistance, knowing each other before studies, and being in the same study group. from an administrative database of the university, we gather data about student performance (grade point average, or gpa at the end of the first year) that is measured on a scale from 0 to 10. we transform the data on performance from continuous to categorical scale and distinguish four performance groups based on the grading system of the university: high performing students (their gpa is equal or higher than 8), medium high performing students (with gpa from 6 to 8), medium low performing students (with gpa from 4 to 6) and low performing students (with gpa lower than 4). it is important to mention that the information about individual student grades is publicly available in this university. this is common in some russian universities but very different from educational systems in the european union and the us. in russian universities, grades are often publicly announced by teachers to the class. in the studied university, final grades are additionally published online on the university website. this creates a specific case when students know about the grades of each other and can coordinate their social connections depending on this information. individuals who did not participate in the questionnaire survey were excluded from the analysis (14 individuals, 10.7% of the sample). these missing data were not treated in a special way. we followed the recommendations (40) , suggesting that 'up to 10% missing data will usually not give many difficulties or distortions, provided missingness is indeed non-informative'. data collection procedures are described in the "data collection" section in s1 file. the descriptive statistics of the sample are presented in the si ("case description" section and tables 1-3 in s1 file). the network visualizations are presented in figs 1-6. standard statistical techniques such as regression models are not applicable for the analysis of social networks due to the interdependence of network observations [39] . therefore, we apply a stochastic actor-oriented model (saom) that allows to reveal the coevolution of network properties and behavior of actors [25, 40] . this dynamic model is widely used for studying the joint evolution of social networks, actor attributes, and separating the processes of social selection and social influence. in total, we estimate two models: the first model estimates the coevolution of friendship network and academic performance, the second one estimates the coevolution of study assistance network and academic performance. the saom's underlying principles are the following. firstly, network and behavior changes are modeled as markov processes which means that the network state at time t depends only on the (t-1) network state. secondly, saom is grounded on the methodological approach of structural individualism. it is assumed that all actors are fully informed about the network structure and attributes of all other network participants. thirdly, time moves continuously and all the macro-changes of the network structure are modeled as a result of the sequence of the corresponding micro-changes. this means that an actor, at each point in time, can either change one of the outgoing ties or modify his or her behavior. the last principle is crucial for the separation of the social selection and social influence processes. there are four sub-components of the coevolution of network and behavior: network rate function, network objective function, behavior rate function, and behavior objective function [25, 40] . the rate functions represent the expected frequencies per unit of time with which actors get an opportunity to make network and/or behavioral micro-changes (40) . the objective functions are the primary determinants of the probabilities of changes. the probabilities of the network and/or behavior change are higher if the values of the objective functions for the network/behavior are higher [25, 40] . the objective functions for the network (eq 1) and behavior change (eq 2) are calculated as a linear combination of a set of components called effects: where s ki (x) are the analytical functions (also called effects) that describe the network tendencies (40) ; β kz s ki z (x, z) are functions that depend on the behavior of the focal actor i, but also on the behavior of his or her network partners and a network position [22] ; β k and β k z are statistical parameters that show the significance of the effects. saom coefficients are interpreted as logistic regression coefficients. parameters are unstandardized, therefore, the estimates for different parameters are not directly comparable. during the modeling, saom allows the inclusion of endogenous and exogenous covariates. as endogenous variables, we include in our models network density, reciprocity, popularity, activity, transitivity, 3-cycles, transitive reciprocated triplets, and betweenness [40] . density and reciprocity show the tendency of students to form any ties and to form mutual ties. transitivity, 3-cycles, transitive reciprocated triplets, and betweenness measure a propensity of students to form triadic connections with their peers. popularity and activity are included to control the tendency of actors to receive many ties from others and to nominate a large number of actors. to control for social selection, we include the selection effect based on academic achievement. it shows whether students with similar levels of academic achievements tend to form connections with each other. we also controlled for the tendency of students with high grades to increase their popularity and activity over time. to test the presence of social influence, we include the effect of performance assimilation. it shows whether students tend to assimilate the academic achievement levels of their peers. in addition, we controlled for the propensity of students with high levels of popularity and activity to change their academic performance. in the model construction we follow the general network modeling requirements necessary for saom [40] . the role of distinct social networks in academic performance diffusion. all research protocols were approved by the hse (higher school of economics) committee on interuniversity surveys and ethical assess of empirical research. all human subjects gave their informed verbal consent prior to their participation in this research, and adequate steps were taken to protect participants' confidentiality. table 1 presents the modeling results of two separate models. in the first, we model the coevolution of friendship network and academic performance. in the second one, we model the coevolution of study assistance network and academic performance. social influence [effect 24] is positive and significant in the friendship social network. this means that the academic performance of students tends to become similar to the performance of their friends. in other words, academic achievements diffuse through friendship ties. in the study assistance network, however, social influence is not present. this indicates that students the role of distinct social networks in academic performance diffusion. do not assimilate the performance of their study assistants; this network channel does not propagate the spread of academic achievements. positive indegree effect [effect 25] suggests that students who are often asked for help increase their performance over time. the non-significant estimates of the linear and quadratic shape parameters [effects 22 and 23] for friendship indicate that the influence of peers sufficiently explains the performance dynamics [15] . the negative effect of the quadratic shape parameter [effects 23] for the study assistance network shows the convergence of the academic performance to unimodal distribution [15] . the effect of performance selection [effect 19] is positive for the study assistance network. it suggests that students with similar levels of academic achievements tend to ask each other for help. the effect of social selection in the friendship network is not significant. this means that students do not have a preference to befriend students with similar academic achievements. positive estimates for the performance of alter [effect 17] in both social networks suggest that individuals with high performance are popular in friendship and study assistance networks. positive effect for the performance of ego [effect 18] for friendship network shows that high performing students tend to create friendship connections. the role of distinct social networks in academic performance diffusion. we find the presence of gender homophily [effect 16] in both friendship and study assistance social networks. students tend to create friendship and study assistance connections with individuals with the same gender. positive effect of ego for males [effect 15] in the friendship network suggests that males tend to nominate more friends. the network control effects [effects 3, 4, 7, 8, 9, and 10] that were included in the models show expected signs and significance scores, as in most student social networks [25] shows that transitivity is less important for friendship ties when reciprocity is present (and vice versa) [41] . the combination of negative betweenness [effect 10] and positive transitivity [effect 7] in both networks demonstrate that individuals do not seek for brokerage positions and do not want to connect peers from different network communities and study groups. positive activity effect [effect 6] in friendship network indicates that students with many ties tend to create new friendship relationships. positive effect of popularity [effect 5] in study assistance network suggests that individuals ask for help those students, who are often asked for help by others. in friendship network this effect is negative, which means that students do not tend to befriend popular individuals, i.e. those who already have a lot of friends. in both networks rate parameters are larger in the first period rather than in the second, indicating that the tie formation stabilizes over time. the modeling results also show that students tend to create friendship and study assistance ties with individuals they knew before the enrollment [effect 11] and individuals from the same study group [effect 12]. also, students tend to create friendship connections with their study assistants [effect 13.1] and they seek for study assistance from their friends [effect 13.2]. we conducted the time heterogeneity test for both network models [40] . this test is used to examine whether the parameter values β k of the objective function are constant over the the role of distinct social networks in academic performance diffusion. periods of observation. we find the time heterogeneity in models. in both networks, parameters such as betweenness, acquaintance before enrollment, popularity and activity of the high performing individuals are heterogeneous. in the friendship network, there is also time heterogeneity for gender of alter and ego, performance social selection and influence. in the study significance codes ��� p < 0.001 �� p < 0.01 � p < 0.05. the models converged according to the t-ratios for convergence and the overall maximum convergence ratio criteria suggested in (40) . goodness of fit is adequate for all models. https://doi.org/10.1371/journal.pone.0236737.t001 the role of distinct social networks in academic performance diffusion. assistance network, we find time heterogeneity for studying in the same group, gender of ego, and performance of ego. the cases of previous acquaintance or being in the same study group can be explained by the nature of these types of ties. for instance, the acquaintance before enrollment can play a significant role at the beginning of studies, while after several months' students will tend to expand their networks and will not seek connections with individuals they knew before studies. the same explanation can be used for the case of being in the same study group. at the beginning of studies, students will form ties within their study groups but later they will tend to expand their network and form ties with other group members. differences in time heterogeneity of academic achievements may be related to the decreased statistical power of these effects between different models. the effects of academic performance on network evolution processes may be understood in details by considering all the performance-related effects simultaneously [40] . in table 2 , we present log-odds for the performance selection within different achievement groups. the higher the estimate, the higher the probability of a study assistance tie formation between students from different performance groups. table 2 shows that there is a significant tendency toward selection of high-performing individuals as study assistants, and this tendency is present among all groups of students. similarly, in table 3 we present precise estimates for the social influence process for all achievement groups. each row of the table corresponds to a given average behavior of the friends of an ego. values in the row show the relative 'attractiveness' of the different potential values of ego's behavior. maximum diagonal value indicates that for each value of the average friends' behavior the actor 'prefers' to have the same behavior as all these friends (40) . this shows that individuals tend to assimilate their friends' performance. in this paper we explore the academic performance diffusion through two social networks of different natures: friendship and study assistance. we empirically confirm that educational the role of distinct social networks in academic performance diffusion. outcomes of students are diffused in different ways within friendship and study assistance networks. ties in the friendship network transmit academic achievements, while ties in the study assistance network do not. the absence of the social influence process along the presence of social selection in the study assistance network may suggest the presence of social segregation based on performance [19] . this can be related to the high competitiveness of the university environment under the study. we expect that some students are highly motivated to receive higher grades and prefer to invest time and effort in their high academic results rather than help their less academically successful peers. our findings demonstrate that the efficacy of academic achievements diffusion is determined by the nature of the social network. it was established that social integration in the classroom is positively associated with the higher academic performance of students [17, 19] . here we claim that it is extremely important to integrate individuals specifically in the network of informal friendship interactions and motivate them to create connections with higher-performing students. these findings support the idea that the nature of social relationships is crucial for the transmission of specific types of information and behavior in social networks. close friendship relationships serve as effective channels for the spread of various complex behaviors, including very costly behavior types such as health behavior [1] . academic performance is one of the examples of these behavior types that are not easily transmittable. in contrast, the instrumental study assistance ties do not produce the propagation of academic achievements from successful students to their lower-performing peers. to sum up, we show that costly and complex behavior (such as academic achievement) diffuses more effectively in the network of strong close connections such as friendship. these findings contribute to the current debates on behavior propagation in social networks and propose new insights on factors that impact the success of behavior transmission. this study has several limitations. first, we analyze social networks of first-year students. this time frame, when students start their educational path at an undergraduate level, receives a lot of attention in the literature [17, 19, 42] due to the fast speed of social tie formation. at the same time, it would be beneficial to investigate the diffusion of academic achievements through social networks along the full period of studies. second, we examine only two types of social relations, however, the spectrum of social ties that can serve as channels of the performance diffusion is much wider. it is a potential avenue for future studies to estimate the efficacy of other types of social networks such as cooperation, competition, romantic relationships, and negative ties on the process of academic achievement diffusion. the data on some of these networks is difficult to collect (e.g., negative relationships) due to the high sensitivity of studied relationships but these types of ties can be nevertheless significant for behavior transmission. in the time of the covid-19 pandemic and after it, it is also extremely important to examine the effect of online networks on academic performance transmission because online interaction remains the only communication channel for students. our empirical findings have several policy implications. academic achievements are one of the key components of financial success and individual well-being [43, 44] , that makes the performance increase is one of the main goals of the educational system. however individual achievements are quite stable and largely driven by heritable factors [45] which make interventions aimed at the academic performance growth highly complex and difficult to implement. one of the possible mechanisms of performance increase is social influence, as we show in this paper. teachers can pay additional attention to the development of informal friendship relationships between students with various performance levels during classes. it can be reached by group work assignments, in which group membership is defined by the teachers and is not based on the personal preferences of students. long-term group assignments such as working on a research project together can stimulate students from different achievement groups to develop friendship ties with each other. the creation of recreation and open spaces within the university building can also give additional options for students with distinct performance to meet, interact, and form friendship ties. the combination of these actions would help students to build and sustain their informal networks, which, in turn, serve as key channels of the academic performance diffusion and lead to a positive behavior change. supporting information s1 file. 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[1] . within the 50 states, there is an apparent disparity in the number and causes of infections and their spread within each state. however, what is common to all cases reported thus far, is the rates of mortality and hospitalizations that appear to be highest among relatively older populations and populations with underlying medical conditions that facilitate morbidity due to covid-19 [2] [3] [4] [5] [6] [7] [8] . much research has focused on clinical outcomes, epidemiological modeling, and transmission dynamics of the novel coronavirus (see for example, [9] [10] [11] [12] ), but less focus has been placed on risk and vulnerability to contracting the disease. emerging studies have begun to report on the impacts of social vulnerability on covid-19 from an incidence and outcome standpoint [2] [3] [4] [5] [6] [7] 13] . however, the spatial resolution of most studies to date has been at the global or country level, and less attention has been paid to finer spatial resolutions such as the census tract scale within a county. a finer spatial resolution is important from a vulnerability and risk standpoint as demonstrated in a recent study that showed that the poorest neighborhoods in houston, texas, might be at a higher risk of hospitalization from covid-19 [14] based on an analysis of the centers for disease control (cdc) underlying risk factors for severe covid-19 cases [4] that include: asthma, chronic obstructive pulmonary disease (copd), heart disease, hypertension, diabetes, and a history of heart attacks or strokes. while the aforementioned underlying medical conditions are important risk factors, they weigh in on the risk of hospitalization but not necessarily on the risk of contracting the disease. as such, underlying medical conditions and sociodemographic variables may not fully represent the magnitude of the risk and the challenge in managing and mitigating disease in affected populations from pandemics such as covid19 . environmental pollutants such as air quality [15] , co 2 emissions [13] , and ambient conditions such as temperature and humidity [5, 16] showed correlations with covid-19 morbidity. furthermore, environmental exposures due to proximity to contaminated areas such as superfund sites, hazardous waste sites, landfills, and leaky petroleum tanks has long-term adverse effects on public health, immune systems, and vulnerability to certain diseases [17] [18] [19] [20] [21] . public health is further exacerbated by natural disasters, such as hurricanes and severe storms [22] [23] [24] that expose populations to pathogens and pollutants in floodwater and their flooded homes and potentially contribute to weakened immune systems. behavioral and lifestyle factors could also affect the vulnerability of a population to an infectious disease such as covid-19. obesity, in recent covid-19 data, has been shown to be prevalent in hospitalized patients [7] , and smoking has been associated with disease progression [25] . finally, it should be noted that because the risk is unevenly distributed, shortages in hospital beds, personal protective equipment (ppe), and medications have emerged in some but not all communities [26] [27] [28] , thereby widening disparities and exposing systemic shortcomings [29] . limited access to medical services, especially with less than fullyfunctional transportation systems combined with lack of insurance coverage, could worsen the impact of covid-19 for people with less favorable sociodemographic metrics and people in rural regions. thus, a more holistic view of the vulnerability of communities to covid-19 that considers all of the aforementioned variables is needed to guide decision-makers in identifying the areas and populations in their jurisdictions that require specific resources, response, and mitigation actions. while there are numerous indices with different applications, none, by themselves, can provide a comprehensive description of the influential factors in the covid-19 pandemic. among the existing indices, the social vulnerability index (svi) developed by the geospatial research, analysis, and services program (grasp) at the centers for disease control and prevention (cdc) and agency for toxic substances and disease registry [30] is the most commonly used one and has been used to explain the variability in covid-19 spread [6] . while svi considers socioeconomic status, household composition/disability, minority status/ language, and housing/transportation, it does not take environmental exposure, underlying medical conditions, behavioral, and lifestyle factors, and vulnerability to natural hazards into account. moreover, access to medical services, which is a key factor in a pandemic, is not included in svi. the same comments are valid for other similar indices such as concentrated disadvantage measures [31] , and the social deprivation index [32] ; these indices have not been developed with a pandemic such as covid-19 in mind, but rather for identifying disparities in population with regards to specific determinants. in this study, we develop a rigorous planning tool at the census tract level that examines influential determinants of vulnerability to covid-19 in 5 broad categories (with 46 variables) that include: 1) access to medical services, 2) underlying medical conditions, 3) environmental exposures, 4) vulnerability to natural disasters and 5) sociodemographic, behavioral, and lifestyle factors. to the best of the knowledge of the authors, none of the existing studies provide a holistic perspective on covid-19 vulnerability. the goals for developing the planning tool are to better understand medical access gaps and identify parts of the county where more protective measures and response actions need to be put in place. such a planning tool is critical in order to mitigate the impact of covid-19 and prepare for future pandemics. using this tool, policymakers can identify neighborhoods with a higher potential for becoming the next hot spots, efficiently match community resources with community needs, and ensure that equipment, personnel, medications, and support are available to everyone, particularly the most vulnerable and those in greatest need. this strategy is essential to address historical trends that have preferentially delivered resources to those with means resulting in gaps in quality [33] [34] [35] . the planning framework developed in the study is readily transferable to other counties in the us and can be expanded to the state level for decisionmaking on a short-term or long-term basis towards improving the overall health of communities in each state. harris county, located in the southeastern part of texas (fig 1) , is the third-most populous county in the u.s., with more than 4.7 million residents [36] . while ranked number 2 in the nation in terms of gross domestic product (gdp) growth, the county exhibits geospatial socioeconomic disparities among its population. while harris county was experiencing fewer cases, and lower rates of transmission relative to the rest of the u.s., starting around mid-june 2020, the pandemic resurged, and the number of covid-19 cases has increased substantially. fig 2 shows the number of confirmed cases of covid-19 in harris county compared to new york county, for example. as shown in fig 2, both the total number of confirmed cases [1] and the slope of the spread during the initial phase of the spread are significantly higher in new york compared to harris county. however, over time, the spread curve was flattened in new york while a second surge wave of the pandemic occurred in harris county. this is important to note because it potentially offers the county the opportunity for using the developed tool for improved long-term planning to respond to community health needs and disparities in response to covid-19 and other pandemics or natural disasters. the total number of confirmed covid-19 cases at the county level was downloaded from the johns hopkins center for civic impact's coronavirus resource center [1] . for harris county and nueces county, the total number of confirmed covid-19 cases at the zip code levels were compiled from the harris county public health database [37] and the city of corpus christi covid-19 dashboard [38] . the zip code-level cases were converted to tract-level using the "spatial join" tool in arcmap and then were normalized by dividing the number of cases to the population of each tract. all census data (2018) at the census tract level were compiled from the national historical geographic information system (nhgis) database [39] . total population, the number of households, median income and income per capita (adjusted to 2018 u.s. dollars), percent of the population below the poverty line, with cash public assistance or food stamps/snap (supplemental nutrition assistance program), living alone, with health insurance coverage, with a disability, education, and age distribution for each tract in harris county were accessed. using the detailed variables in the census data, education in this study was defined as the percent of the population with high school diplomas or higher degrees. due to the importance of age in the vulnerability to covid-19, both median age and the percent of the population in decadal age intervals were calculated. the percent of the population below the poverty line was chosen as the main economic variable, and the household density was calculated by dividing the total number of households by the area of each census tract. two measures of vulnerability to flooding were defined, using data from hurricane harvey that had severe impacts on harris county in 2017: i) the ratio of the number of households that filed damage claims based on federal emergency management agency (fema) data [40] to the total number of houses in each tract, and ii) the ratio of the wetted areas (with water depth greater than zero) during hurricane harvey in a census tract to the total area of the tract (the specific methodology for this approach is described in [41] ). locations and types of medical facilities including nursing facilities, federally qualified health centers, hospitals, rural health clinics, urgent care centers, and harris county health system facilities were obtained from the health resources and services administration (hrsa) query data explorer tool [42] , harris county health system [43] , and the homeland infrastructure foundation-level data (hifld) database [44] . the microsoft bing maps platform apis [45] was used to estimate the drive time from the centroid of each tract to all of the available medical facilities nearby. arcmap was used to extract the coordinates of both origins (centroids) and destinations (medical facilities), and the minimum travel time in minutes was then recorded for each tract in microsoft excel. the underlying conditions that might affect the vulnerability to covid-19 (arthritis, asthma, high blood pressure (hbp), cancer (except skin cancer), high cholesterol, chronic kidney and heart diseases, copd, diabetes, poor physical and mental health, and stroke); as well as increased-risk behaviors/conditions (binge drinking, smoking, no leisure time physical activity, obesity, sleep less than 7 hours), and preventive indicators (annual doctor and dentist checkups, medication for high blood pressure (hbp), cholesterol screening, and routine physical exams) were all acquired from the 500 cities mapper database [46] (table 1) , which draws from the centers for disease control and prevention's (cdc) behavioral risk factor surveillance system. it should be noted that data from [46] were only available at 584 out of 786 (73.8%) census tracts in harris county. census tracts without data are clearly identified in all figures. as noted before, ambient conditions such as temperature and humidity could affect the spread of covid-19; however, in this study, an ambient gradient in harris county was neglected as the spatial change over the county is expected to be minimal. three indicators of air quality: ozone, nitrogen dioxide (no 2 ), and particulate matter smaller than 2.5 micrometers (pm 2.5 ), were downloaded from the texas air monitoring information system (tamis) database [47] . for ozone, the 8-hour average concentrations were calculated using ibm spss (version 26) for all of the available monitoring stations (40) and compared with the 70 ppb standard established by the united states environmental protection agency (epa). the number of exceedances of the epa standard for each monitoring station over the period of 2000-2019 was then calculated. "interpolation" tools in arcmap were used to convert the median measured concentration for each station to a continuous raster to overcome the spatial sparsity in measurements. the generated raster was then used to calculate the concentration of ozone for each census tract using the "zonal statistics" tool in arcmap. one particular station (695: uh moody tower) was removed from the ozone calculation due to its extremely low temporal resolution compared to the other stations. similar approaches were taken for no 2 (hourly measurements for 21 stations with 100 ppm standard) and pm 2.5 (averaged daily data for 12 stations with a 35 μg/m 3 standard). environmental releases from various sources to air, water, soils in harris county were obtained from the united states coast guard national response center database [48] . the total number of emissions, pollution spills, or contaminant discharge events that occurred during the period between 2000 and 2020 for each zip code was extracted from the database by combining different years, filtering the actual events for harris county, and removing redundant data points. the "spatial join" tool in arcmap was used to convert the total number of events in each zip code to a summed total number of events for each census tract. the resulting value was added to the number of leaking petroleum storage tanks (underground and aboveground tanks) reported by the texas commission on environmental quality (tceq) [49] in the tract. a hazardous sites shapefile was developed by merging two databases: the epa superfund enterprise management system database [50] , and the texas commission on environmental quality (tceq) gis database [49] . from the latter source, the locations of municipal solid waste sites/landfills were acquired. the "near" tool in arcmap was used to calculate the distance between the centroid of each census tract to the nearest aforementioned hazardous sites. a second environmental variable was defined as the sum of the total number of dry cleaners, petroleum storage tanks (all underground and aboveground tanks), and sites that are part of an industrial and hazardous waste corrective action (ihwca) program located within each census tract; data for those was obtained from [49] . both shapiro-wilk and kolmogorov-smirnov tests conducted in ibm spss showed that none of the datasets were normally distributed. a principal component analysis (pca) with orthogonal rotation (varimax with kaiser normalization) was conducted in ibm spss as the first step to reduce the dimensions. due to the limitation in data availability, as noted before, the pca was performed for data from 584 tracts with all available data. eigenvalues from random values were generated and compared with the values in this study using a parallel analysis engine [51] . this comparison was made to determine the number of components that should be retained in the analysis; components with eigenvalues greater than the randomized method were kept. the first five components that could explain~80% of the variability in the 46 variables showed eigenvalues larger than the ones generated by the engine. s1 table in s1 file shows the most dominant variables in each component (category). the choice of variables for the study (table 1 ) was based on the results of the pca in addition to findings reported in previous studies, and data availability. category 1 includes access to medical services, including medical facilities, medications, and insurance coverage, routine checkups, and physical exams, as well as household density as a surrogate for interaction among individuals within each tract (e.g., how crowded grocery stores could be in the tract). category 2 includes chronic diseases, medical conditions, disability that could potentially affect the vulnerability to covid-19, and age distribution. for environmental exposure, pollution events from various sources, the 3-air quality indicators, and the presence of hazardous sites were included. flooding from hurricane harvey was the only metric in category 4, although this could be expanded in future work to include heat, drought, wildfires, and other natural disasters. finally and for category 5, a combination of social, economic, behavioral, and lifestyle factors that could potentially threaten the health of individuals during the covid-19 pandemic was considered. vulnerability analysis. the step-by-step methodology applied in this study is depicted in fig 3. two classification approaches were used in the study with the goal of identifying the most vulnerable populations to covid-19; a rank-based exceedance method developed in microsoft excel, and a standard k-means cluster analysis (k = 3) using ibm spss. validation of any developed models for the vulnerability was not possible due to lack of data at the desired spatial resolution and the fact that the pandemic is still developing. thus, the second model (k-means) was used as a benchmark for the first model for comparison purposes. in the rank-based exceedance method, for each variable, sorting the data in microsoft excel developed the rank of each census tract relative to other tracts within harris county. the exceedance rate (percentile) was calculated as follows: where m is the rank, and n is the total number of tracts (786 in harris county). the calculated exceedance for a given tract represents the percent of tracts that have a better condition than the selected one. to ensure that the direction of exceedance is the same among all variables, (1 -exceedance) was used for variables with positive nature such as insurance coverage, education, access to medication, and preventive tests. for each category, the average value of exceedance for all of the variables within that category was calculated and reported. in addition to classifying the tracts for each of the aforementioned categories, an overall vulnerability was defined by averaging the exceedance rates of the five defined categories. the percentile associated with each averaged value (for each category and for the overall vulnerability) was calculated and exported to arcmap to generate decision-support level maps. in the k-means cluster analysis (k-means is an unsupervised machine learning algorithm), three classes were defined for each category. as a result, the output classes were ordered as high (severe), average, and low depending on the order of the final cluster centers. the anova test was conducted on the clusters to ensure that the values of the different variables were significantly different between clusters. similar to the exceedance method, an overall vulnerability for each census tract was determined by averaging the five output class numbers (i.e., 1, 2, and 3) associated with the five categories. for illustration purposes, the percentile rank for each of the tracts was calculated and exported to arcmap. although it is possible to assign weights to the categories and calculate a weighted average, equal importance for the categories was assumed in this study. assigning weights is beyond the scope of this paper as there is not enough evidence to support such assignments as of this writing. spearsman's correlation analysis was performed to find correlations, if any, among the exceedance rates of vulnerabilities and normalized number of covid-19 cases at the census tract level. box plots were used to demonstrate the distribution of covid-19 cases among the defined quantiles of overall vulnerability using both k-means cluster analysis and exceedance methods. geospatial distribution of determinants in the 5 categories s2 table in s1 file provides a summary of statistics for all of the 46 variables used in the study. among the 46 variables, maps are only presented for those that were not based on publicly available data. fig 4 shows the locations of medical facilities (all types as described in methods) within and around harris county as well as the drive time to the nearest facility for each census tract. the drive time varies from seconds to 25.23 minutes in the no traffic condition, with a median of 4.74 minutes (s2 table in s1 file). as can be seen from fig 4, people who live in areas located farther away from the center of the county, especially in the western and northeastern parts, have a longer drive time to a medical care facility. this longer drive time becomes even more critical if an individual does not have a personal car and needs to use the less than a fully functional transportation system. the travel time is even longer to facilities managed by the harris health system (typically used by individuals with no insurance or documentation). from a planning standpoint , fig 4 below , when combined with vulnerabilities, can be used to drive decisions related to the establishment of field hospitals during periods of widespread transmission. importantly, the data can be used to develop a more holistic response plan directing persons with various severity or symptoms of the disease to different types of medical intervention facilities. the distance to and location of hazardous sites (superfund sites, landfills, and industrial hazards) are shown in fig 5. the distance ranges from 79 to 9,386 m with a median of 2,105 m. the hazardous sites are spread over the entire county but are more concentrated closer to the industrial areas (see fig 1) , water bodies (houston ship channel (hsc), and galveston bay (gb)). the tracts in less developed areas have the longest distance to a hazardous site indicating a higher potential vulnerability to pollution in the more developed parts of harris county. s1-s3 figs in s1 file show the median concentration of ozone, no 2 , and pm 2.5 for each tract in addition to the number of times during 2000-2019 that a monitoring station exceeded the epa standard. it should be noted that the number of stations and, consequently, the geospatial coverage was significantly lower for no 2 and pm 2.5 when compared to ozone. stations with the highest number of exceedances of epa standards for all three measures are located near industrial areas (see fig 1) . while the central parts of the county showed the highest concentration of no 2 and pm 2.5 , ozone concentrations were highest closer to the industrial areas. the higher levels of no 2 in central parts of harris county could be attributed to emissions from mobile sources that are more abundant in downtown houston [52] . the observed pattern for ozone is a result of industrial activities, the ozone-no 2 relationship, and the wind pattern in houston [53, 54] . in the case of pm 2.5 , the higher concentrations in harris county have been associated with regional aerosols, biomass burning, and gasoline combustion [55] that are higher in the central part of the county. the median concentrations for ozone, no 2 , and pm 2.5 were 21.24 ppb, 8.32 ppm, and 9.98 μg/m 3 , respectively, for the period of 2000-2019. what is interesting to note is the fact that the three variables have different spatial distributions thereby indicating potentially more important involvement in covid-19 based on recent research showing increased vulnerability due to pm 2.5 pollution in covid-19 patients [56] and cdc's indication that "people with moderate to severe asthma may be at higher risk of getting very sick from covid-19." contaminant discharge events (s4 fig in s1 file) and the second environmental variable representing dry cleaners, petroleum storage tanks, and ihwca sites (s5 fig in s1 file) were substantially higher in industrial areas close to the hsc and gb: la porte, baytown, deer park, and channelview, with the number of events as high as 1,449 (2000-present) . the median was 12 events across all census tracts. fig 6 shows the percentile of flooding across harris county based on the filed claims to fema. areas closer to the bayous/streams and flood control dams showed a higher vulnerability. a similar distribution was observed using the geospatial inundation modeling approach (s6 fig in s1 file) . finally, s7 fig in s1 file shows the distribution of educated persons in harris county, defined as the ratio of age 25 years and over with a high school diploma or higher degree to the total population for each census tract. the developed illustrative maps, data and tabulated findings in this work can be directly used by decision-makers to make quantitative comparisons that fit their needs. the results are illustrated here using generalized descriptive language based on the geospatial visualization in order to identify the overall areas of the county with the most vulnerable population. it is noted that using the same methodology and set of variables, except for category 4 that could be customized based on location, similar tables and maps could be created for any location of interest. for instance, category 4 could be defined as being vulnerable to drought, wildfire, tornados, earthquakes, and so on. such vulnerability originates from the fact that i) in the aftermath of each of these natural hazards, the individuals who were impacted may have severe socio-economic-health issues, and ii) a compound event where any of these hazards overlap with the pandemic could substantially amplify the effect of each of the hazards. table in s1 file. s8-s12 figs in s1 file show the class of each tract (i.e., high/severe, average, and low) for category 1 through category 5, respectively. the results in all categories were similar to the exceedance methods, validating the choice of methodology. the overall vulnerability generated by the k-means methods led to a very similar map (s13 fig in s1 file) to the exceedance approach (fig 8) . fig 9a shows the normalized total number of confirmed covid-19 cases as of august 16, 2020, in harris county at the census tract level. it should be noted that the original dataset at the zip code level was converted to census tract level using arcmap. since the conversion was completed using an area-based weighted average, the maximum number of normalized cases is different from the original dataset; 453 and 212 confirmed covid-19 cases per 10,000 persons for the converted and original datasets, respectively. by comparing figs 9 and 7, a lot of similarities in the geospatial distribution of vulnerability (fig 7) and morbidity (fig 9) can be observed. spearman's correlation analysis showed a significant and relatively strong correlation (p-value = 2.6e-51, ρ = 0.570) between the normalized number of cases and the overall vulnerability exceedance rate. while all categories showed significant correlations with the normalized cases, cat 5 followed by cat 1 showed the most strong relationships with a correlation coefficient of 0.616 (p-value = 7.2e-62) and 0.566 (p-value = 2.3e-50), respectively. the correlation coefficients for cat 2, cat 3, and cat 4 were 0.39 (p-value = 1.6e-22), 0.344 (pvalue = 3.4e-23), and 0.203 (p-value = 9.3e-09), respectively. fig 9b depicts the distribution of normalized cases among tracts with different levels (quantiles) of overall vulnerabilities. the general increasing trend in the box plots shown in fig 9b supported by visual similarities and, most importantly, by statistical tests validates the performance and reliability of the developed tool. to further validate the developed tool, the vulnerability to natural disasters was estimated for nueces county, tx, during hurricane harvey using the fema data. a significant and relatively strong correlation (p-value = 0.017, ρ = 0.471) was observed between the number of covid-19 confirmed cases (as of august 16, 2020, s14a fig in s1 file) , and the total number of harvey claims at the zip code level (s14b fig in s1 file) . overall, the vulnerabilities associated with each category exhibited varying geospatial distributions with some commonality (i.e., some census tracts had elevated vulnerabilities in each category). category 1, 2, and 5 vulnerabilities shown in fig 7 ( access to medical services, underlying medical conditions, and sociodemographic, respectively) indicated a similar finding; the most severe vulnerability can be observed in areas with the least favorable conditions represented by the three categories (lowest income, lower education levels, less insurance coverage, unhealthy diet and lifestyles, and more underlying medical conditions). category 3 (environmental exposures) showed a spatially declining gradient from east to west with some hotspots around downtown houston. this gradient could be explained by the presence of the majority of industrial activities in the eastern part of harris county, and worse air quality near downtown houston. category 4 (vulnerability to natural disasters) showed the highest risk in the vicinity of major bayous/streams in the county, as discussed before. looking at fig 8, it could be concluded that the most vulnerable persons to covid-19 in harris county, are living in the eastern part of the county, specifically areas next to the hsc and gb, and areas identified as opportunity zones [57] . the residents in these neighborhoods are individuals belonging to disadvantaged or historically marginalized groups, are exposed to several chemicals (with industrial sources), and subject to flooding both from rainfall and storm surge (such as what was experienced during hurricane ike in 2008). the relationship between race and ethnicity and physiological vulnerability to covid-19 is beyond the scope of this paper since the medical data are insufficient at this time to complete such an analysis. however, due to disparities in the distribution of wealth, welfare, and services in the us, african-american and hispanic populations are more likely to have lower levels of health, education, and income. the relative ratios of african-american and hispanic populations across harris county are shown in s15, s16 figs in s1 file, respectively. by looking at figs 8, 9a, and s15, s16 figs in s1 file, it could be concluded that the majority of tracts with the highest ratio of african-american and hispanic population are located in areas with the highest overall vulnerability and normalized covid-19 cases. individuals living in the western and southeastern fringe of the county are least vulnerable. however, it is noted that the underlying medical condition data were not available for those tracts. it is also noted that individuals in those areas, if infected, especially in the western fringe, will have significantly limited access to medical services compared to the other parts of the county. for each category, the total population and the distribution of population in two age intervals, 45-65 (the age group with the highest number of reported covid-19 cases), and +65 (the age group with the highest mortality rate), over different percentiles (from low to high with regards to the severity of conditions within each category) is shown in table 2 . using the vulnerability findings presented above for harris county (fig 7, and yellow highlighted values in table 2 ); a total of 59,307; 98,702; 78,723; 105,431; and 59,624 seniors (+65 years), who are at most risk of covid-19 mortality, are living in areas with the highest vulnerability in category 1 through 5, respectively. considering the fact that harris county is prone to flooding and the hurricane season is in progress from may through the end of november, a potential hurricane combined with the covid-19 pandemic could lead to a compound natural disaster event affecting significant numbers of senior citizens as shown in table 2 . decision-makers, to prepare for the worst-case pandemic scenario and occurrence of a hurricane, in particular, could use the numbers in category 1 and 4 for planning response and recovery measures that take into account flooding and increased vulnerability to covid-19. for overall vulnerability (fig 8 and cyan highlights in table 2 ), a total of 722,357 persons (~17% of the population of the county) including 171,403 with ages between 45-65 (~4% of the total population of harris county), and 76,719 seniors (~2% of the population of the county and 10.6% of total identified vulnerable population), are at a higher overall risk. as of august 16, 2020, 92,944 confirmed covid-19 cases were reported in harris county [37] , which is 12.7% of the estimated vulnerable people identified in this study. among those,~14,600 (15.7% of total confirmed cases) are seniors compared to the 10.6% estimated by the model. the disagreement between the model estimates and reported numbers by harris county [37] could be due to the testing rates. according to [58] , texas is among the states with the lowest testing rates (15.46%), with a higher testing rate among the seniors. it is expected that as time passes, the number of cases increases in the county both because of expanding the testing rates and due to further spread of the pandemic. the limitation in data availability should be considered when interpreting these results. first, data was lacking for some of the variables for a number of census tracts in harris county; the cdc's 500 cities dataset [46] for example is limited to the city of houston and does not include the rest of the county. the analysis findings might vary if a houston-specific dataset is used. however, the downside is that the approach would not incorporate important countylevel considerations. second, the use of the covid-19 dataset up to current dates in terms of manuscript preparation (august 16, 2020 in this study) and not having the full benefit of the comprehensive infection dataset prior to the availability of vaccinations or effective treatments could affect the results and, consequently, interpretations of the findings. future work could include new variables such as occupational exposure to covid-19, and taking into account populations that have the ability to work from home and those that do not. this may be a key component of vulnerability, especially in the context of stay home orders issued by various states. in this novel project, we develop a planning tool that can help identify populations at higher risk of infection, morbidity, and mortality from covid-19 at the census tract level. these findings can guide the allocation of scarce resources, and thus, are relevant to policymakers at all levels of government. effectively using the results from the planning tool to inform actions could mean the difference between suppressing the virus and allowing it to re-emerge. in comparison, it is noted that studies that map the geospatial spread of coronavirus from wuhan to neighboring communities are starting to emerge [59, 60] , and similar efforts need to be launched in the u.s. the application of geospatial methods to case data enables significantly more rigor in understanding the confluence of various factors that jointly increase vulnerabilities and reduce resilience to covid-19 spread, impact, re-emergence in new hot spots or on a seasonal basis. while geospatial indices exist [30, 32] , they are not tailored to the unique features of this virus. lastly, the findings from this study enable public health departments to efficiently and equitably allocate resources, including preventive, therapeutic, and rehabilitative measures. for example, our results can guide where testing and vaccine distribution sites should be located, which communities would benefit most from policies that mandate physical distancing and masks, which hospitals need additional support because they are at risk for exceeding their capacities, and where contact tracers should target their efforts. reports of long wait times for testing and uncertainty about where to invest resources [61] suggest that policy makers lack the data they need to make these decisions. supporting information s1 file. 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disadvantage and homicide measures of social deprivation that predict health care access and need within a rational area of primary care service delivery unequal treatment: confronting racial and ethnic disparities in health care crossing the quality chasm: a new health system for the 21st century. washington, dc: the national academies press the association between income and life expectancy in the united states new census bureau estimates show counties in south and west lead nation in population growth county/houston covid-19 cases city of corpus christi covid-19 dashboard ipums national historical geographic information system: version federal emergency management administration (fema) characterization of vulnerability of road networks to fluvial flooding using sis network diffusion model health resources and services administration (hrsa) query data explorer tool harris county health system. harris health system locations homeland infrastructure foundation-level data (hifld)-urgent care facilities microsoft bing maps platform apis the 500 cities mapper united states coast guard. national response center superfund: national priorities list (npl) parallel analysis engine to aid determining number of factors to retain constraining nox emissions using satellite no2 measurements during 2013 discover-aq texas campaign a 15-year climatology of wind pattern impacts on surface the relation between ozone, nox and hydrocarbons in urban and polluted rural environments the characterization of fine particulate matter downwind of houston: using integrated factor analysis to identify anthropogenic and natural sources exposure to air pollution and covid-19 mortality in the united states opportunity zones spatio-temporal patterns of the 2019-ncov epidemic at the county level in hubei province the changing patter of covid-19 in china: a tempo-geographic analysis of the sars-cov-2 epidemic texas lawmakers warn white house of "catastrophic cascading consequences key: cord-342519-tjr6dvtt authors: souza, thiago moreno l.; salluh, jorge i. f.; bozza, fernando a.; mesquita, milene; soares, márcio; motta, fernando c.; pitrowsky, melissa tassano; de lourdes oliveira, maria; mishin, vasiliy p.; gubareva, larissa v.; whitney, anne; rocco, sandra amaral; gonçalves, vânia maria c.; marques, venceslaine prado; velasco, eduardo; siqueira, marilda m. title: h1n1pdm influenza infection in hospitalized cancer patients: clinical evolution and viral analysis date: 2010-11-30 journal: plos one doi: 10.1371/journal.pone.0014158 sha: doc_id: 342519 cord_uid: tjr6dvtt background: the novel influenza a pandemic virus (h1n1pdm) caused considerable morbidity and mortality worldwide in 2009. the aim of the present study was to evaluate the clinical course, duration of viral shedding, h1n1pdm evolution and emergence of antiviral resistance in hospitalized cancer patients with severe h1n1pdm infections during the winter of 2009 in brazil. methods: we performed a prospective single-center cohort study in a cancer center in rio de janeiro, brazil. hospitalized patients with cancer and a confirmed diagnosis of influenza a h1n1pdm were evaluated. the main outcome measures in this study were in-hospital mortality, duration of viral shedding, viral persistence and both functional and molecular analyses of h1n1pdm susceptibility to oseltamivir. results: a total of 44 hospitalized patients with suspected influenza-like illness were screened. a total of 24 had diagnosed h1n1pdm infections. the overall hospital mortality in our cohort was 21%. thirteen (54%) patients required intensive care. the median age of the studied cohort was 14.5 years (3–69 years). eighteen (75%) patients had received chemotherapy in the previous month, and 14 were neutropenic at the onset of influenza. a total of 10 patients were evaluated for their duration of viral shedding, and 5 (50%) displayed prolonged viral shedding (median 23, range = 11–63 days); however, this was not associated with the emergence of a resistant h1n1pdm virus. viral evolution was observed in sequentially collected samples. conclusions: prolonged influenza a h1n1pdm shedding was observed in cancer patients. however, oseltamivir resistance was not detected. taken together, our data suggest that severely ill cancer patients may constitute a pandemic virus reservoir with major implications for viral propagation. the emergence of the novel influenza a/h1n1 pandemic virus (h1n1pdm) significantly affected the utilization of healthcare resources and increased morbidity and mortality in children and young adults [1, 2] . from april through september 2009, during the fall/winter in the southern hemisphere, brazil experienced the first wave of the h1n1pdm virus, and by the end of december 2009, over 1600 h1n1pdm-related deaths had been reported in brazil [3] . emerging data on the clinical course of severe h1n1pdm infection have allowed the identification of high-risk groups, which include pregnant women and patients with morbid obesity [4, 5] . however, an analysis of the impact of this novel virus in a highly susceptible population, such as cancer patients, through clinical and virological perspectives, needs to be highlighted [6, 7, 8, 9, 10, 11] . the atypical clinical presentation of influenza infections in cancer patients, which delays clinical suspicion, antiviral treatment and adequate prevention of viral transmission, is a major challenge for clinical management in this population [12] . cancer patients are more likely to suffer from severe seasonal influenza infections [12, 13, 14] and prolonged viral shedding, as has been reported for an h3n2 seasonal virus [15] . prolonged shedding and the development of oseltamivir resistance in cancer patients infected with the h1n1pdm virus have not been thoroughly evaluated. data on these aspects could have major implications for the clinical management and infection control practices for h1n1pdm-infected cancer patients [16] . because the analysis of this novel viral infection in cancer patients is an important component of the 2009 pandemics, we conducted a prospective cohort study aimed at evaluating the clinical course of influenza infection, the duration of viral shedding, h1n1pdm evolution and the emergence of antiviral resistance in hospitalized cancer patients with a severe h1n1pdm infection in a reference cancer center during the winter of 2009 in brazil. during the study period, 44 hospitalized cancer patients with a suspected influenza infection were screened, and 24 had a confirmed influenza a diagnosis using a rapid indirect immunofluorescence (ifi) test or world health organization (who)recommended real-time rt-pcr (rrt-pcr) ( figure 1 and table s1 ). among these, 20 patients were confirmed to be positive for the h1n1pdm virus using rrt-pcr ( figure 1 and table s1 ). the remaining four patients were positive for influenza a using ifi only. considering the pandemic case definitions with reference to international guidelines [17] , these last four cases were categorized as h1n1pdm-confirmed cases. altogether, these 24 cases constituted the study population. all of the respiratory samples collected from the 20 rrt-pcr-confirmed patients were inoculated in cell cultures. we recovered the virus from 13 individuals after at least two passages in mdcks, constituting 15 isolated samples. these isolates were also analyzed for oseltamivir resistance using a functional assay. patients diagnosed with h1n1pdm were young (median age = 14.5, range 3-69 years). in total, 14 (58.3%) were under 18 years old, and 17 (70.8%) were less than 50 years old. hematologic cancer occurred in 75% (18) of the patients, whereas solid tumors occurred in 25% (6) patients (tables s2 and s3) . a total of 22 (,92%) patients had received immunosuppressive therapy in the previous 30 days. among these individuals, 18 patients (75%) were on chemotherapy, 14 (58.3%) received systemic corticosteroids and 1 (4%) received radiation therapy (table 1 and s4) . no patient received erythropoietin (epo) or immunomodulatory agents. a total of 14 patients (58.3%) presented febrile neutropenia (,500 neutrophils/mm 3 ) at the time h1n1pdm was diagnosed. the median duration of neutropenia after the onset of viral disease was two days (ranging from one to six days; table s5 ). according to the brazilian national cancer institute's protocol, all patients that presented neutropenia received g-csf until normalization of neutrophil counts. the clinical characteristics and comparisons among groups are shown in table 1 and tables s1, s2, s3, s4, s5, s6, s7, s8, s9, s10, and s11. the overall mortality in our cohort was around 21% (n = 5), and four patients (n = 16.6%) had at least one comorbidity besides cancer. of these patients with comorbidities, one died. no pregnant or morbidly obese patients were identified. a total of 23 (95.8%) patients were treated with oseltamivir, and the median time from the initial symptoms to the initiation of therapy was three days (0-15 days; tables 1, s2 and s6). one patient that died due to severe acute respiratory failure 24 h after clinical suspicion of h1n1pdm infection never received antiviral treatment. oseltamivir was used for a median of seven days (0-19 days), and double doses (150 mg bid for adults and twice the recommended dose per kg for children) were administered for 11 (47.8%) patients (table s6) . a total of 11 (47.8%) patients received oseltamivir for more than seven days. six patients (25%) received this antiviral within 48 h of clinical suspicion. all patients that died received oseltamivir more than 48 h after the onset of the illness. however, when we compared the mortality of patients that received oseltamivir either within or after 48 h of the onset illness, no significant difference was observed (0/6 vs. 5/18, p = 0.28). no differences in prolonged viral shedding were observed between these two groups. at presentation, all patients were treated with broad-spectrum intravenous antimicrobial agents to combat community-acquired pneumonia and/or febrile neutropenia [18] , and five (20.8%) had concomitant positive cultures (tables s7 and s8) . hypoxemia was frequent, and the median pao2/fio2 on the first arterial blood gas evaluation was 192 mmhg (range: 64-367 mmhg). overall, 13 patients (five adults and eight children) were admitted to the icu. six patients were directly admitted from the emergency department, and the other seven patients were transferred from other hospital wards (table s2) . ventilatory support was given to 12 patients (table 1 and s9). invasive mechanical ventilation was performed in 10 patients (76.9%), and non invasive ventilation (niv) was performed in 3 patients (23.1%; table 1 ). among the niv patients, one required subsequent endotracheal intubation and mechanical ventilation, and all three patients were discharged from the hospital. extra-pulmonary organ failure occurred in eight patients (33.3%; table 1 and s9). of the 13 critically ill patients, 12 were treated with oseltamivir, and treatment was initiated 48 h after the first signs/symptoms of viral infection in 5 of them. adjunct or non-conventional supportive therapies for ards were performed for 12 of the 13 patients that entered the icu (92.3%). a total of 10 patients (76.9%) received systemic corticosteroids (eight due to previous use and two for shock and persistent ards); five (38.5%) were ventilated in a prone position, and four (30.8%) required recruitment maneuvers. no patient received extra-corporeal membrane oxygenation. although prolonged influenza a shedding has been observed for a cancer patient infected with the h3n2 seasonal virus [15] , more detailed data on h1n1pdm secretion in severely ill cancer patients are required. we evaluated viral shedding in 10 mechanically ventilated patients by collecting sequential respiratory samples at different time-points after the onset of illness. the duration of viral shedding was considered to be the time frame from the initial symptoms to the last h1n1pdm-confirmed sample. five (50%) patients in this group showed viral shedding for at least 11 days during oseltamivir treatment ( figure 2 and table s12 ). the median duration of h1n1pdm shedding was 23 days (ranging from 11 to 63 days; figure 2 and table s12 ). most importantly, the maximum duration of h1n1pdm shedding in our investigation was 63 days, followed by 44 days for another patient. to our knowledge, these periods constitute the longest registered cases of h1n1pdm shedding described to date. all rrt-pcr-positive samples from these patients with the longest viral shedding durations (5645 and 5899) were culturable, meaning that these were infectious viruses ( figure 2 and table s12 ). in addition, the last h1n1pdm-confirmed samples from these patients were only detected using cell culture assays, suggesting the presence of low viral loads in these specimens ( figure 2 and table s12 ). these patients still shed the virus for an additional 25 to 40 days after cessation of the antiviral treatment ( figure 2 ). to date, no significant variation has been detected at the amino acid level in the hemagglutinin (ha) of the 2009 pandemic virus [19] . therefore, we examined the genetic diversity of the h1n1pdm virus recovered from these severely ill patients by performing sequence analysis of the viral ha gene. no significant divergence was found in the h1n1pdm isolates collected during the onset of illness from the four patients with prolonged shedding ( figure s1 ). to evaluate the genetic characteristics of the isolates from patients with prolonged viral shedding, we sequenced two consecutive samples from a single individual (5645s2/09 and 5645s3/09) and compared their ha sequences to other h1n1pdm viruses from mild, severe and fatal cases from different countries. we observed that samples collected a month apart (5645s2/09 and 5645s3/09) clustered together and displayed a relatively large branch length from other h1n1pdm viruses ( figure 3 ). this result may have occurred because strain 5645s2/09 diverged from ca/ 04 in the amino acid residues l52s, l70p, p100s, c153l, t214a, q293r and i321v (h1 numbering). additionally, isolate 5645s3/ 09, which was sequenced from amino acid residues 167 to 413, also had the mutations t214a, q293r and i321v fixed in the viral population a month after the initial sampling. in the isolate 5645s3/09, another mutation was also acquired, d238p, suggesting continuous viral evolution. although we cannot determine the role of these mutations in viral pathogenesis by this result alone, the retention of the changed residues over time strongly suggests viral persistence rather than re-infection. considering that influenza resistance to antivirals is likely to emerge in immunocompromised individuals and that, with respect to the h1n1pdm virus, only a few studies have detected oseltamivir resistance [20] , we investigated the emergence of antiviral resistance in h1n1pdm samples isolated in cell culture. thus, oseltamivir carboxylate ic 50 values were measured for 15 h1n1pdm samples isolated from 13 patients. we found that 10 isolates were sensitive (0.8560.27 nm), and 5 displayed high ic 50 values for the antiviral used (165.13642.26 nm). these samples were sent to the who collaborating center at the cdc in atlanta for resistance confirmation. we found that the isolates with high ic 50 values were endowed with a neuraminidase (na) activity that was cross-resistant to oseltamivir, zanamivir and peramivir, suggesting the presence of a co-pathogen endowed with na activity within these isolates. co-infections with other respiratory viruses (coronavirus (229, 43 and 63), parainfluenza (1, 2, 3 and 4), human metapneumovirus, parechovirus, rhinovirus, rsv a/b, adenovirus and enterovirus) or atypical bacteria (mycobacterium pneumonia) were not identified in these samples, suggesting that no other viral source of na activity was responsible for these high ic 50 values. however, we co-isolated a streptococcus sp. from the samples with high ic 50 values. the na activity of this bacterial strain displayed a phenotype resistant to nais. in further testing with bacteria-free h1n1pdm isolates, the virus' ic 50 were consistent with a sensitive isolate. pyrosequencing analyses were also performed and revealed that the other high-ic 50 samples had the wt h275 residue and thus did not contain this oseltamivir resistance marker. viruses isolated from the initial onset of illness were a/california/07-like viruses ( figure s2 ). prolonged influenza shedding in cancer patients has been observed for seasonal strains [15] . regarding the h1n1pdm virus, it has been shown that prolonged virus shedding in cancer patients may occur, although such a phenomenon has only been documented through cases involving a single studied patient [21, 22, 23, 24, 25] . here, we prospectively and systematically collected information from a cohort of hospitalized cancer patients with severe h1n1pdm infections. these patients presented high mortality, prolonged viral shedding and h1n1pdm evolution without the emergence of oseltamivir resistance. this is the first study to address viral shedding and resistance in cancer patients with h1n1pdm infections; thus, it may provide insight into the role of cancer patients as potential human reservoirs for this pandemic virus. unlike previous reports, our population was composed of hospitalized, severely immunocompromised cancer patients [26] . most of them were young, had hematologic malignancies and received chemotherapy and systemic steroids in the weeks that preceded the h1n1pdm infection. the patients were treated with oseltamivir in the early course of the infection (the median time to antiviral initiation was three days). a total of 13 patients (54%) required intensive care and presented severe respiratory distress. in these patients, the mortality rates were higher (38%) than those observed for general icu patients suffering from h1n1pdm infections [2, 27] as well as for non-critically ill cancer patients [26] . however, the outcomes were not different from those reported for cancer patients requiring mechanical ventilation [28, 29] . interestingly, during the influenza season, 14 patients (58.3%) with febrile neutropenia were identified as h1n1pdm cases, a condition that is not usually investigated in this scenario. however, febrile neutropenic cancer patients have an increased risk of developing respiratory distress and multi-organ failure. therefore, screening for respiratory viruses and prompt initiation of oseltamivir treatment should be considered in these patients. febrile neutropenia indicates a poor prognostic with respect to a patient's outcome, but neutropenia duration in our cohort of patients was less than seven days. thus, prolonged viral shedding might not have a correlation with neutropenia. we observed the persistence of h1n1pdm in 5 out of 10 patients studied for this purpose. in these individuals, viral shedding continued for at least 11 days, despite the use of oseltamivir. the median duration of viral shedding in our population was 23 days, and two pediatric patients with acute lymphoblastic leukemia showed even longer virus secretions (44 and 63 days; figure 2 and table s10), although it is difficult to determine whether viral persistence was due to cancer per se or to acute lung injury and mechanical ventilation. influenza shedding is not considered to last long, and it disappears seven days after the onset (2.4 and 4.5 days for oseltamivir-and placebo-treated groups, respectively) [30] . studies aimed at monitoring 2009 h1n1pdm virus shedding using randomized trials with appropriate controls, such as outpatients and hospitalized or immunocompromised individuals, have not yet been conducted. because we were also unable to establish agematched controls with or without immunosuppression, since this study was conducted during the peak of the first wave of the 2009 pandemics in brazil, we compared our work to other studies on h1n1pdm shedding in general, hospitalized or immunocompromised populations [21, 22, 23, 24, 25, 31, 32, 33, 34, 35, 36] . in table 2 , we summarize the cohort used in each study, whether or not they were immunocompromised, their underlying diseases and the number of patients analyzed for viral shedding in each of these studies. we compared the maximum periods of shedding among these different populations and the number of patients that secreted the virus for more than seven days. these data would be more informative and relevant from a public health point of view because the duration of the quarantine for h1n1pdm was approximately that long [21] . we found ( table 2 ) that in households in hong kong [31] and canada [34] , the maximum periods of h1n1pdm shedding ranged from 8 to 11 days. these periods were not different from what was observed with military cadets [22] and during the containment phase of the pandemics in vietnam [23] (table 2) . regarding h1n1pdm shedding among infants, hien et al. showed that children five to nine years old could secrete the h1n1pdm virus for five to six days, which is markedly lower than what is observed for the seasonal influenza virus [30, 33] . compared to our results, we observed higher periods of viral shedding in two seven-year-old patients with acute lymphoblastic leukemia (table 2 and figure 2 ). however, a single report on two travelers in france showed that these apparently immunocompetent individuals secreted the h1n1pdm virus for 14 and 28 days [25] (table 2 ). although these periods of time are comparable to the time frame of virus secretion in hospitalized patients in china [23] and our work, the study from felury et al. might be as biased as ours by the small size of the cohort ( table 2) . influenza-infected immunocompromised individuals may have prolonged influenza shedding [15, 21, 32, 36] ; however, more insights are necessary to better comprehend the dynamics of the h1n1pdm virus in these individuals. mora et al. showed that in hiv-1-infected individuals, co-infection with the h1n1pdm virus might lead to an outcome not different from the one expected for immunocompetent subjects, although no systematic analysis of viral shedding was performed [36] . a similar conclusion was also drawn for transplant recipient individuals, whose longest periods of viral shedding did not exceed 11 days [32] (table 2 ). in our study, we found periods of h1n1pdm shedding similar to what the cdc observed for leukemia patients [21] (table 2) . although the small size of these cohorts of immunocompromised individuals [21, 32] , including ours, may require a more conclusive and mechanistic analysis, these observations may stimulate further systematic studies to understand or gain insight into factors associated with prolonged h1n1pdm shedding. in addition, it might give insights on basic studies on influenza pathogenesis. our results highlight the need for closer surveillance of cancer patients with h1n1pdm infections until the detection of the first negative sample. we hypothesize that follow-up protocols aimed at monitoring the persistence of viral shedding in cancer patients may be relevant if patients are submitted to immunosuppressive therapies in the days or weeks prior to or following an h1n1pdm infection. next, we sought to determine viral evolution during prolonged shedding. we found that some amino acid changes persisted from the initial symptoms until 30 days thereafter, suggesting that these patients had viral persistence rather than re-infection. in addition, an extra amino acid change (d238p) was found in the viral ha sequenced a month after the onset of illness, suggesting continuing viral evolution. although some of the mutations that we found (l52s, l70p, p100s, c153l, t214a, d238p, q293r and i321v) in strains 5645s2/09 and 5645s3/09 have not been previously described, other amino acid residue changes that were detected in our study (p100s and t214a) have been found in h1n1pdm viruses throughout the world without a significant link to viral pathogenesis or antigenic variation [37, 38, 39] . influenza viruses resistant to antiviral drugs have been reported in immunocompromised patients, [21, 40] and this resistance might be associated with prolonged viral shedding [41, 42] . notably, five isolates from two patients had high ic 50 values to neuraminidase inhibitors (nais). an na activity that was multiresistant to nais was identified and could be due to the presence of a streptococcus strain found in throat swabs and tracheal aspirates. pyrosequencing analyses of samples with high ic 50 values revealed that these specimens were h275 wild-type sensitive viruses. these results reinforce the need for additional genotyping assays to confirm the identification of putative resistant strains identified using functional assays. in addition, our findings show the need for investigating other sources of na activity in virus isolates with odd ic 50 values. the apparent paradox of prolonged viral shedding without antiviral resistance could be explained by either the inability of the immunocompromised host to effectively clear the h1n1pdm virus [12, 43] or inefficient absorption of the drug [44] . because we combined both clinical and molecular virology data, our results might contribute to the discussion on the adequate duration and type of anti-h1n1pdm treatment in immunocompromised patients with a protracted course. in these patients, the use of parenteral systemic or inhaled antivirals should also be investigated. although our work further investigates the unique dynamics of h1n1pdm virus infection in immunocompromised hosts, some caveats must be noted. because our investigation started during a new pandemic, the clinical evaluation and management protocols changed during the course of the study as new data emerged from the literature and from updated recommendations [45] . as the pandemic reached its peak in south america, the establishment of a larger and more diverse cohort with age-matched controls with or without immunosuppression became complex. thus, more indepth multivariate and mechanistic clinical analyses were limited. moreover, no recommendations for monitoring viral persistence were available; therefore, only a subset of severely ill patients admitted to the icu was evaluated. despite that, an important connection between clinical and laboratory information was studied, revealing the continuous evolution of h1n1pdm ha sequences and the stability of the na gene in severely ill patients [14] . in conclusion, this study provides evidence that severe h1n1pdm infection is associated with significant morbidity and mortality in cancer patients. in these patients, viral persistence without the emergence of antiviral resistance may occur during the clinical course of the disease. this result has major implications for the clinical management of h1n1pdm infections and infection control strategies. our study may provide insights into h1n1pdm shedding and might contribute to the development of new guidelines to manage cancer patients with h1n1pdm infection. the ethics committee (comitê de é tica em pesquisa; cep; http://www.inca.gov.br/conteudo_view.asp?id = 2380) at the instituto nacional de câncer (inca), rio de janeiro, brazil, headed by dr. adriana scheliga approved the study under protocol #18/ 2010 and waived the need for informed consent. this was a prospective cohort study conducted in the hospital do câncer-i, instituto nacional de câncer (hc-i-inca), rio de janeiro, brazil, from july 8 th to october 1 st , 2009. the hci-inca is a 160-bed comprehensive cancer center primarily for the population of rio de janeiro and neighboring states. the present study was strictly observational, and every clinical decision was at the discretion of the attending physician. all patients with a definite diagnosis of cancer requiring hospital admission for any reason and who displayed influenza-like illness were evaluated. patients in complete remission from cancer for more than five years were not considered. data were collected using a standardized case report form that included demographic data, clinical presentation, comorbidities, cancer status, use of immunosuppressive therapies, time course of acute illness, need for intensive care, use of antivirals, adjunctive therapies, advanced life support and in-hospital mortality (supporting information; si). patients were included if they had a fever (.37.8uc) and/or respiratory influenza-like illness and confirmed influenza a h1n1pdm diagnosis (by at least one of three assays, ifi, rrt-pcr or cell culture, and according to case definitions from the who [46] ). patients were treated according to the brazilian public health guidelines [47] . nasopharyngeal dacron-swab specimens were collected from all patients and placed onto transport medium (hanks solution with 100 u/ml penicillin and 100 mg/ml streptomycin) at the initial evaluation. tracheal aspirates were also obtained if the patient required tracheal intubation. patients' clinical samples were directly tested for a panel of respiratory viruses using an ifi assay for influenza a (respiratory virus panel; biotrin, mount merrion, co. dublin, ireland.). specimens were also sent to the brazilian national influenza center (ioc/fiocruz) for h1n1pdm confirmation using rrt-pcr, which was performed in accordance with the current guidelines from the who/cdc [46] . viral shedding was evaluated in the subset of patients that remained under mechanical ventilation for longer than seven days and in those patients with persistent hypoxemia and pulmonary infiltrate. these patients received a specific number to which their sample number was appended. that is, the first sample was ''s1'', and subsequent specimens were numbered consecutively (table s12 ). viral secretion was evaluated using both cell culture and rrt-pcr assays until it was negative. virus isolation was performed in madin-darby canine kidney (mdck) cells and/or embryonated eggs (see text s1 and table s12 ). the functional antiviral assay was performed using the na-star kit (applied biosystems, ca), according to the manufacturer's instructions. the rt-pcr protocol for sequencing using the sanger method and pyrosequencing [48] are presented in the si, as well as the phylogenetic analysis. blood samples were routinely sent for bacterial culturing, as were tracheal aspirates if the patient was intubated. standard descriptive statistics were used to describe the study population. continuous variables were reported as the mean 6 standard deviation or median (range) as appropriate. univariate analysis was used to identify factors associated with hospital mortality. two-sample t-tests and a chi-square or fisher's exact test were also used. two-tailed p values ,0.05 were considered statistically significant. text s1 clinical investigation and influenza virus assays. figure s1 phylogenetic tree of ha gene from the classical swine, eurasian swine, american avian and human seasonal lineages. the bootstrap probability is indicated for each interior branch, all values below 80% are hidden. the scale bar indicates the number of amino acid changes per site. colored circles indicate the samples from our study. this tree is unrooted. each influenza ha lineage is displayed beside their respective clade. found at: doi:10.1371/journal.pone.0014158.s014 (0.12 mb tif) figure s2 phylogenetic tree of na gene from the followed-up cohort. the bootstrap probability is not indicated for each interior branch since it is below 85%. the scale bar indicates the number of amino acid changes per site. the tree is rooted by california/ 07/2009 na sequence. found at: doi:10.1371/journal.pone.0014158.s015 (0.18 mb tif) critically ill patients with 2009 influenza a(h1n1) in mexico critically ill patients 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markers of drug resistance in 2009 pandemic influenza a (h1n1) viruses by pyrosequencing we are indebted to the instituto nacional de câncer, fiocruz, faperj and cnpq for their support. the authors thank marc-alain widdowson (cdc/ccid/ncird) for critical review of the manuscript. alicia fry (cdc atlanta epidemiology intelligence officer) was also extremely helpful in discussions on virus shedding. key: cord-335404-s48psqth authors: mukandavire, zindoga; nyabadza, farai; malunguza, noble j.; cuadros, diego f.; shiri, tinevimbo; musuka, godfrey title: quantifying early covid-19 outbreak transmission in south africa and exploring vaccine efficacy scenarios date: 2020-07-24 journal: plos one doi: 10.1371/journal.pone.0236003 sha: doc_id: 335404 cord_uid: s48psqth the emergence and fast global spread of covid-19 has presented one of the greatest public health challenges in modern times with no proven cure or vaccine. africa is still early in this epidemic, therefore the extent of disease severity is not yet clear. we used a mathematical model to fit to the observed cases of covid-19 in south africa to estimate the basic reproductive number and critical vaccination coverage to control the disease for different hypothetical vaccine efficacy scenarios. we also estimated the percentage reduction in effective contacts due to the social distancing measures implemented. early model estimates show that covid-19 outbreak in south africa had a basic reproductive number of 2.95 (95% credible interval [cri] 2.83–3.33). a vaccine with 70% efficacy had the capacity to contain covid-19 outbreak but at very higher vaccination coverage 94.44% (95% crl 92.44–99.92%) with a vaccine of 100% efficacy requiring 66.10% (95% crl 64.72–69.95%) coverage. social distancing measures put in place have so far reduced the number of social contacts by 80.31% (95% crl 79.76–80.85%). these findings suggest that a highly efficacious vaccine would have been required to contain covid-19 in south africa. therefore, the current social distancing measures to reduce contacts will remain key in controlling the infection in the absence of vaccines and other therapeutics. the coronavirus disease 2019 (covid-19) originated in wuhan, china, in december 2019 and has rapidly spread around the world [1] . as this is a new and novel virus, there is a huge scientific evidence gap and therefore limited understanding of the epidemiology of sars--cov-2, the pathogen that causes the disease covid-19. currently, the epicentre of the virus is a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 in europe and new york in the united states of america [2] . in africa, the virus is just starting to set its foothold, with south africa now reporting the majority of cases in the continent. the first case of the covid-19 in south africa was reported on the 5 th of march 2020 [3] . measures to contain the epidemic culminated in the declaration of the state of disaster leading to a national lockdown on the 26 th of march 2020 with gauteng, western cape, kwazulu-natal and the free state provinces reporting most of the covid-19 cases. the map in fig 1 shows the distribution of covid-19 confirmed cases in south africa before the government mandated a lockdown. gauteng province appeared to be the "epicentre" of covid-19 in south africa for a number of reasons. first, the province has the largest population density [4, 5] and the urban population is poor with 20% of its population being food insecure [4] . second, gauteng province has two international airports including or tambo international airport handling over 21 million passengers annually [6] . third, the volume of people that use public transport runs into millions daily creating social networks and patterns that are key in accelerating the spread of the disease. finally, the province is the country's economic hub, and many people (including international visitors) travel in and out of the province daily [4] . with the majority of confirmed cases early in the outbreak having been linked to international travel [7] , it is not surprising that the most affected provinces (gauteng, western cape and kwa-zulu-natal) have international airports with direct flights to affected global regions (fig 1) . the cases in the free state province have mainly been attributed to a cluster transmission resulting from a mega church gathering [8] . with covid-19 having been declared a global pandemic [9] and the urgent need to have an effective vaccine to control the pandemic [10] , there is a need to understand the utility of mass vaccination campaigns for this pandemic. critical in the early stages of the disease is the need to clearly understand the spectrum of disease severity and transmission characteristics of the disease in order to identify optimal control measures. many of the control measures suggested for this pandemic have been attributed to the lessons learnt in wuhan, china [11] . the challenges associated with real-time analysis of an evolving epidemic are well articulated in [12] . these include testing capacity and delayed appearance of symptoms and asymptomatic carriage. the impact of covid-19 on south africa may differ from that on china and other regions such as europe and north america. south africa has unique circumstances, for example, it has the highest numbers of people living with hiv, with a significant proportion not on treatment, and one of the largest tuberculosis (tb) burdens in the world [13, 14] . moreover, underlining disease conditions such as diabetes, hypertension and chronic obstructive pulmonary disease are prevalent in south african and these are known to be risk factors for covid-19 infection and mortality [15] . the age distribution for south africa is also different from china and europe as its young population accounts for the majority of the population [16] . data from china and other settings have shown that sars-cov-2 is more infectious than influenza, and has an incubation period of about 5 days (median time) and a doubling time of 3 days [17, 18] . however, we have a limited understanding of the infectiousness of the virus in settings with different populations and a huge burden of other chronic conditions such as africa. mathematical models provide important insights in the understanding of emerging infectious diseases and informing public health policies. several mathematical models have been used to understand covid-19 transmission dynamics and inform public health policy [12, 19, 20, 21, 22] . the reproductive numbers of the covid-19 epidemic in china have been determined in several modelling studies (table 1) . here, we adapt a susceptible-exposed-infected-removed (seir) compartmental model to quantify early transmissibility of covid-19 in south africa and explore the potential utility of a vaccine in containing the disease. the seir model has been used to model respiratory infections including middle east respiratory syndrome (mers) [23, 24] , covid-19 in wuhan china [11] , influenza [25, 26] and global tracking of covid-19 [27] . in addition, we estimate the reduction in effective contacts after the implementation of the severe and extreme shutdown of the society, and this is critical in determining the impact of social distancing in the south african context. we use a standard deterministic compartmental seir model to simulate covid-19 in south africa. the model classifies the human population into four epidemiological compartments at any time t, the susceptible s(t), exposed e(t), infected i(t) and the recovered r(t). the total population is thus given by susceptible individuals are infected upon interaction with infectious covid-19 individuals and the rate of daily generation of newly infected cases is given by λ(t) = βs(t)i(t)/n, where the parameter β is the effective contact rate, i.e. the contact that will result in an infection. the lockdown effect is modelled with parameter, �2(0,1) where �'0 implies an ineffective lockdown and �'0 implies a completely effective lockdown. the effective contact reduction term multiplies the effective contact rate in the model to give (1−�)β. exposed individuals in the e(t) compartment become infectious at a constant rate σ and move to the i(t) class. infected individuals i(t), recover at a constant rate γ to the removed class r(t). the schematic model flow diagram is presented in s1 fig. the model assumptions result in the following system of differential equations. following a similar approach in [37] , we use a markov chain monte carlo (mcmc) within a bayesian framework (in r fme package [38] ) to fit the model to the cumulative data of confirmed covid-19 cases in south africa and estimate the magnitude of the epidemic using the basic reproductive number and quantify required vaccines' attributes to stem similar outbreaks. we used data on covid-19 cases published by the south african department of health from the 5 th of march 2020 to 28 march 2020 prior to the lockdown to estimate the basic reproductive number [39] . we set the model lockdown effect parameter � = 0 when estimating the basic reproductive number. the percentage reduction in effective contacts after the lockdown � was estimated by fitting the model to cumulative covid-19 cases reported a week after the lockdown (from 29 th march to 11 th april 2020). cumulative data for covid-19 cases reported in south africa from the 5 th march to 11 th april 2020 is shown in s1 table. in the fitting, we set the lockdown effect parameter � = 0 and varied β, σ, γ (within parameter ranges in s2 table) and initial infected population in order to estimate the basic reproductive number. in estimating the lockdown effect, we varied � and kept parameters used to estimate the basic reproductive number constant. gaussian likelihood was used to draw model parameter posteriors assuming uniform non-informative priors while the variances were regarded as nuisance parameters. the mcmc chain was generated with at least 100 000 runs for the final fitting excluding the burn-in period. chain convergence was examined visually and using the coda r package [40] . uncertainty of each estimated parameter was evaluated by analysing the mcmc chains and calculating the 2.5% and 97.5% quantiles to give the 95% credible interval (cri). the basic reproductive number (r 0 ), is as a measure of the average number of secondary cases generated by a primary case and is an important statistic for quantifying intervention programmes [41, 42] . using an intuitive mathematical approach, the reproductive number of model system (1) is given by r 0 = β/γ. the corresponding minimum vaccination coverage (c) for covid-19 vaccine for different vaccine efficacy scenarios was estimated using the mathematical expression c � ð1 à r à 1 0 þ=s where s the proportional reduction of the susceptibility for individuals partially immunized. estimates of effective contact rate (β), the incubation period (1/σ), infectious period (1/γ), the percentage reduction in effective contacts (�) and the basic reproductive number, r 0 for south africa are shown in table 2 . the mathematical model (of the seir type) was fitted to the cumulative covid-19 cases for south africa at the national level (fig 2(a) ). we estimated an effective contact rate 1.30 (95% crl 1.21-1.39) per day, incubation period of 3.21 days (95% crl 3.04-3.44 days), infectious period of 2.27 days (95% crl 2.04-2.74 days) and r 0 of 2.95 (95% crl 2.83-3.33) before the lockdown. the result r 0 >1 clearly shows disease sustainability in the country. estimates of r 0 were used to conduct sensitivity analysis based on different covid-19 vaccines' efficacy assumptions to explore possible scenarios that may arise from mass vaccination campaigns, as scientists attempt to develop effective vaccines for covid-19 [43, 44] . the vaccine efficacy scenarios were assumed to vary in the range of 50-100% (fig 2(b) ). the results suggest that a vaccine with more than 70% efficacy could have the potential to contain the covid-19 outbreak in south africa but at extremely high vaccination coverage rates of 94.44% (95% crl 92.44-99.92%). as expected, vaccination coverage for epidemic control decreases with an increase in vaccine efficacy, with a vaccine of 100% efficacy requiring table 2 before lockdown [45] . we also quantified the percentage reduction in effective contacts as a result of the lockdown mandated by the government of south africa. fig 3 shows that the epidemic is slowing down after the implementation of a lockdown. the results showed that the lockdown resulted in 80.31% (95% crl 79.76-80.85%) reduction in effective contacts ( table 2 ) and consequently resulted in a reduction in the number of covid-19 cases reported in the first two weeks of implementation. this confirms results in china that demonstrated the importance of quarantine, social distancing, and isolation in containing the pandemic [46] . covid-19 has spread rapidly globally assisted by air travel in an increasingly connected world [47, 48] . globally most countries, including south africa, have adopted one form or another of the lockdown approaches in an attempt to curb disease transmission within their borders [11, 49, 50] . our model estimate of r 0 >1 confirms covid-19 persistence in south africa and indicate that the outbreak has the momentum to rapidly spread and spill over to other geographic regions of the country, in particular if the coming winter season (may to july) presents ideal environmental conditions for persistence of the virus. the estimate of r 0 for south africa is in a similar range published for covid-19 in other modelling studies (table 1) . hypothetical scenarios on vaccine efficacy demonstrated that, a vaccine of at least 70% efficacy would have been sufficient to contain the spread of covid-19 in south africa although at high vaccination coverage. however, it is important to note that expectations that the development of a highly effective vaccine for the novel-coronavirus will be achieved in the coming months are extremely optimistic, especially when considering that a vaccine has still not been successfully created for viruses like hiv, severe acute respiratory syndrome (sars) and mers, with the hiv vaccine being in development for many years [44, 51] . nevertheless, the huge global interest in quickly identifying an effective vaccine could increase the possibility that a successful vaccine candidate can be developed in the coming months [43] . even when a safe and effective vaccine becomes available, there are several logistical and operational challenges that need to be addressed for successful deployment and for the vaccine to achieve the desired coverage [52, 53] . the modelled lockdown demonstrated 80.31% reduction in effective contacts, showing that it is an effective measure to bring the disease under control. however, the reduction in the number of daily reported cases should be interpreted with caution as this could also have been a result of many other factors such as reduced international travel to high-risk regions and behaviour change. as the epidemic continues to unfold, it remains to be seen what trend the epidemic will follow if local transmissions are sustained within south africa. the implementation of this society shut down is not sustainable in the long run as it is unlikely to be tolerated for too long by the population. a vaccine would be an ideal preventative strategy for covid-19 but it appears that it should be complemented with prevention approaches such as isolation, quarantine, personal hygiene and limitations of public gatherings in order to achieve optimal protection of the population in south africa. the economic and social burden of the disease continues to be felt and this likely to be enhanced by an extension of the current lockdown of 21 days by a further 2 weeks [54] . however it is unclear whether a stringent lockdown could be maintained for a longer period given the socio-economic challenges of the country where a significant percent of adults are involved in informal employment and others have jobs that do not allow them to work from home. this could affect the effectiveness of the lockdown in many of the townships as individuals will have to ease lockdown conditions in order to be economically active and prevent financial woes on individuals in urban communities. while the epidemic seems to have slowed down as a results of the lockdown (fig 3) , there is need for continued scientific investigation including explorations through mathematical models to monitor the trend with the aim of informing public health policy in the short-term. the study has some limitations. the estimate of the reproductive number is based on available data and this estimate could possibly change depending on the quality of the data from the start of the epidemic (with possible under-reporting of cases in the initial phases of the epidemic). we note that spatial modelling mainly in the affected provinces would have been ideal but we did not have good data on a finer resolution to effectively parameterise a spatial model but as the epidemic evolves, nascent data on local covid-19 transmission in south africa is becoming available. we used a simple mathematical model without other population demographics as these were not important for short-term prediction [55] and such models are also important when epidemiological and clinical disease characteristics of the disease are not well established as is the case for covid-19 [56] . the simple model is only intended to give preliminary estimates for an epidemic that is evolving and whose trend has the potential to change dramatically overtime. however, it would be interesting to see how our results will change when a more complicated model is used. despite these shortfalls, findings in this study are important in understanding the transmissibility of the virus and informing the development of robust covid-19 prevention and control programmes in south africa and outlining mass vaccination expectations. the covid-19 pandemic has continued to spread and causing many deaths than any infectious disease we have seen in recent years and this calls for an urgent and well-coordinated timely and effective public health response. currently there is no proven treatment or vaccines for covid-19 and countries have embraced quarantine, social distancing, and isolation of infected individuals to contain the pandemic. thus, as more setting-specific data about the transmission dynamics of the virus become available, the building of suitable mathematical models to weight out the impact of current public health control measures and explore the potential utility of anticipated biomedical interventions such as vaccines is paramount. supporting information s1 fig. schematic covid-19 model diagram outlining infection progression. the arrows connecting compartments denote covid-19 infection at rate βs(t)i(t)/n, progression to infectiousness σe and recovery rate γi respectively. (docx) s1 coronavirus disease 2019 (covid-19) situation report-62 an interactive web-based dashboard to track covid-19 in real time first case of covid-19 coronavirus reported in sa battleground gauteng: epicentre of the pandemic statistics south africa national institute for communicable diseases, covid-19 update: 274 confirmed cases-76% have history of travel free state races to curb covid-19 outbreak as angus buchan tests positive and country cases rise to 927 who director-general's opening remarks at the media briefing on covid-19 ensuring global access to access to covid-19 vaccines a conceptual model for the coronavirus disease 2019 (covid-19) outbreak in wuhan, china with individual reaction and governmental action early dynamics of transmission and control of covid-19: a mathematical modelling study world health organization targeting the hiv epidemic in south africa: the need for testing and linkage to care in emergency departments with most coronavirus cases in africa, south 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analysis in r using package fme covid-19 / novel coronavirus coda: convergence diagnosis and output analysis for mcmc estimating the reproductive numbers for the 2008-2009 cholera outbreaks in zimbabwe cholera in haiti: reproductive numbers and vaccination coverage estimates preliminary identification of potential vaccine targets for the covid-19 coronavirus (sars-cov-2) based on sars-cov immunological studies sars-cov-2 and covid-19: the most important research questions global routine vaccination coverage world health organisation preparedness and vulnerability of african countries against importations of covid-19: a modelling study passengers' destinations from china: low risk of novel coronavirus (2019-ncov) transmission into africa and south america the positive impact of lockdown in wuhan on containing the covid-19 outbreak in china distribution of the covid-19 epidemic and correlation with population emigration from wuhan, china coronavirus vaccine in 18 months? experts urge reality check logistical and structural challenges are the major obstacles for family medicine physicians' ability to administer adult vaccines operational challenges of vaccination, 10th annual african vaccinology course (aavc) nationwide lockdown extended by two weeks is modelling complexity always needed? insights from modelling prep introduction in south africa epidemiological and clinical aspects of covid-19; a narrative review key: cord-328206-iylw1bvw authors: yu, daojun; chen, yu; wu, shenghai; wang, baohong; tang, yi-wei; li, lanjuan title: simultaneous detection and differentiation of human papillomavirus genotypes 6, 11, 16 and 18 by allglo quadruplex quantitative pcr date: 2012-11-09 journal: plos one doi: 10.1371/journal.pone.0048972 sha: doc_id: 328206 cord_uid: iylw1bvw background: human papillomaviruses (hpv) are classified into high-risk hpv and low-risk hpv. the most common high-risk hpv types in cervical cancer are hpv 16 and 18, and the most common low-risk types causing genital warts are hpv 6 and hpv 11. in this study, applying novel allglo fluorescent probes, we established a quadruplex quantitative pcr method to simultaneously detect and differentiate hpv 6, 11, 16 and 18 in a single tube. methods: the specificity, the sensitivity, the detection limit, the reproducibility and the standard curve of this method were examined. finally, clinical samples that had been tested previously by taqman pcr and hpv genoarray (ga) test were used to verify the accuracy and sensitivity of the method. results: the assay has a sensitivity of 10(1) to 10(2) copies/test and a linear detection range from 10(1) to 10(8) copies/test. the mean amplification efficiencies for hpv 6, 11, 16, and 18 were 0.97, 1.10, 0.93 and 1.20, respectively, and the mean correlation coefficient (r(2)) of each standard curve was above 0.99 for plasmid templates ranging from 10(3) to 10(7) copies/test. there was 100% agreement between the allglo quadruplex quantitative pcr, hpv ga test and taqman uniplex qpcr methods. conclusions: allglo quadruplex quantitative pcr in a single tube has the advantages of relatively high throughput, good reproducibility, high sensitivity, high specificity, and a wide linear range of detection. the convenient single tube format makes this assay a powerful tool for the studies of mixed infections by multiple pathogens, viral typing and viral load quantification. more than 120 human papillomaviruses (hpv) genotypes have been found throughout the world. the most common high-risk hpv types in cervical cancer are hpv 16 and 18, and the most common low-risk types causing genital warts are hpv 6 and hpv 11 [1] . some studies suggest that about 70% of cervical epithelial cell dysplasia and cervical cancer is closely related to hpv 16 and 18 infection, while more than 90% of external genital warts is caused by hpv 6 and 11 infection [2, 3] . hpv 16 and 18 are regarded as high-risk hpv, while hpv 6 and 11 are thought as low-risk hpv [4] [5] [6] . a persistent infection may generate a particularly high hpv dna load through viral replication [7] [8] [9] . because hpv 6 or 11 can cause genital warts and hpv 16 or 18 may lead cervical cancer, preventing infections from these viruses can be an effective way to control the incidence of cervical cancer and cauliflower excrescence. a natural hpv-infection gives little or none protection against new infections due to low immune response, but the hpv-vaccine does.several hpv vaccines are commercially available [10] [11] [12] [13] [14] [15] [16] and have been shown to be effective in preventing infections caused by hpv 6, 11, 16 and 18, there are already several commercial tests for hpv 6, 11, 16 and 18 available. it is obvious a marked progression for these tests. this assay will facilitate molecular epidemiological studies and drug development. hpv genotyping is mainly determined by molecular methods of detecting viral dna [17] [18] [19] [20] [21] . the multiplex real-time fluorescence quantitative pcr (qpcr) technique is an effective method which can simultaneously type many viruses [22] [23] [24] . however, existing qpcr has some disadvantages such as low throughput [25, 26] , complicated techniques [27, 28] , and expensive reagents [27, 29] . moreover, it was not able to type four hpv viruses simultaneously in a single tube in a quantitative manner [26] in order to meet the requirement for detecting multiple subtypes of hpv in a clinical setting. the allglo probe is the latest generation of fluorescent quantitative probes developed by allelogic biosciences corporation (hayward, california). it replaces the quencher with a fluorophore that is identical the first fluorophore. both of the fluorophores are in a quenched state when the probe is intact, but become dequenched when the probe is cleaved. an allglo probe generates two light-emitting fluorophores per cleavage event, leading to greater fluorescence gain in fluorescence pcr and lower ct value ( figure 1 ). in addition, the optimal tm of the oligonucleotide of the allglo probe is the same temperature for annealing/extension, not ten degrees higher as is required for taqman. as a result, allglo probes tend to be shorter, usually 15-16 nucleotides long. this short oligonucleotide is advantageous in single mismatch discrimination. so allglo quadruplex quantitative pcr has the advantages of relatively high throughput, good reproducibility, high sensitivity, high specificity, it is easy for designing the probes and primers of multiplex qpcr and can increase the detection throughput. in this study, we used allglo probe and developed a method to simultaneously detect four types of hpv (hpv 6, 11, 16, 18) in a single-tube format. this quadruplex assay was validated with an existing uniplex taqman assay and a hpv ga test. we carried out a series of control experiments and demonstrated the high specificity, sensitivity, and repeatability of this approach. clinical specimens which included vaginal secretions (220 specimens), leucorrhoea (30 specimens), cauliflower excrescence warts (200 specimens), and other tissues (10 specimens)were collected from hangzhou first people's hospital, china. the patients(210 m, 250f) aged 20 to 50 years.informed written consent to utilize their specimens for this study was obtained from the patients (approved by the ethics review committee of hangzhou first people's hospital). after pre-treatment, all of the specimens were subject to dna extraction, to detection of hpv genotype by hpv ga test (hybribio ltd., hong kong), and then to quantitative detection of viral loads (only hpv 6, 11, 16 and 18) by fluorescence quantitative pcr (da'an reagent, guangzhou da'an diagnostic co., ltd., china). the positive samples with known concentrations were conserved at -80 uc for future use. we used pcr to gain anticipated size of dna fragments for each hpv target gene (table 1) and then ligated to pmd-19t plasmids at 16uc overnight. the ligated products were transformed into e. coli dh5a and plated on lb plates containing l00 mg/l ampicillin. plasmid dna was extracted from bacterial culture using an extraction kit (takara reagent). the plasmid insert dna sequences (supporting information s1) were confirmed by sanger sequencing (invitrogen biotechnology shanghai, china). the plasmid dna samples from positive clones were quantified by uv spectrophotometry. plasmids were diluted to 10 10 copies/ml in te (ph 8.0) buffer for storage at -20uc. secretion swab specimens were dissolved in normal saline. tissues and cauliflower excrescence warts were infiltrated in 4% sodium hydroxide solution and comminuted with metal bar. the dissolved samples were oscillated and resuspended for 1 min, followed by centrifuging for 5 min (4 uc, 12,0006g/min). the supernatant was discarded, and the sediment was resuspended in 50 ml of dna extraction solution (guangzhou da'an diagnostic co., ltd., china). after heating at 100 uc for 10 min, the sample was centrifuged for 5 min (4 uc, 12,0006g/min). the supernatant was saved for qpcr analyses. primer and probe design. conserved regions sequences of the four hpv type-specific genes were obtained from gene bank. after sequence comparison by clustralw software, the most conservative regions were used to design the primers and probes with primer premiers 5.0. by blast sequence comparison, the primers and probes with the best specificity were selected. the primers and allglo probes used in the multiplex qpcr were synthesized by chaoshi bio-company (shanghai, china). to ensure the specificity and sensitivity of the multiplex qpcr, all primers and probes were designed to have similar tm (table 1) . to enhance the hybridization specificity of each probe to its cognate target sequence, each probe was designed to have at least nine mismatched base pairs with its noncognate targets sequence. determination of the specificity and the sensitivity of the primer and the probe. to determine the specificity and the sensitivity of the primer and the probe, the uniplex fluorescence quantitative pcr experiments were first carried out on a qiagen rotor gene 6000 fluorescence quantitative pcr instrument, which can simultaneously detect five wavelengths. parameter variables such as cycle temperatures, concentration of primers and probe, the detection limit and replicating steps were all optimized. for every reactions, each 25-ml pcr reaction contained 11 ml of deionized water,10 ml of 2 6 qpcr mix (chaoshi bio-company, shanghai, china), 1 ml individual hpv plasmid dna template(from 10 2 copies/ml to10 5 copies/ml), 2 ml primer (1 ml forward primer, 1 ml reverse primer)and 1 ml probe (four primer/probe combinations and concentrations: 400 nm/400 nm, 400 nm/ 200 nm, 200 nm/200 nm, or 200 nm/100 nm). each pcr reaction was performed in triplicate. a blank plasmid without an hpv dna insert and other hpv types dna (genotypes identified through hpv ga test and sanger sequencing,except hpv genotypes 6, 11, 16, 18, such as hpv genotypes 32, 39, 52, 58 etc.) was used as a negative control, and deionized water was used as the no-dna blank control. the cycling conditions were a 5-min incubation at 95uc followed by 45 temperature cycles moving between 95uc for 10 s and 52uc for 20 s (during the cycling phase, the optimization annealing temperatures were varied from 52 to 66uc in single-degree increments). the specificity and sensitivity of primers and probes were determined according to the lowest ct value, the highest gain in fluorescence and detection limit of the uniplex fluorescence qpcr, and the quadruplex qpcr described in the following sections referred to the parameters of the uniplex fluorescence qpcr. optimization of quadruplex quantitative pcr. thanks to the specificity of the primer and the probe we selected here, we found we could take conditions such as the primer/probe combinations form uniplex assay to multiplex directly. then we optimized annealing temperature (from 52uc260uc) and determined the compromise annealing temperature used for multiplex detection was 58?. quadruplex quantitative pcr was performed in the same manner as the uniplex qpcr assay described above, pcr mixtures contained 10 ml of 2 6 qpcr mix, 1 ml individual standard curve for quadruplex quantitative pcr. each quantitative pcr reaction, which was used to construct a standard curve, contained mixture of the four types of hpv plasmids (the concentration of each hpv types was equal) at final concentrations (copies/ml) of 10 3 , 10 4 , 10 5 , 10 6 , and 10 7 , as well as primers and probe at optimized concentrations and combinations. the qpcr assay were performed in duplicate, and a standard curve was constructed by the instrument's software. the replicability test of quadruplex quantitative pcr. five aliquots plasmids dna were made from the mixed stock of four types hpv plasmids and stored at -20 uc, the selected concentrations of each hpv type were 10 2 , 10 4 , 10 6 ,and 10 8 (copies/test). five side-by-side quadruplex quantitative pcr assay were carried out using the aliquots plasmids dna in triplicate to determine the mean ct value. the cv% between each aliquot was calculated to determine the replicability of our method. detection of clinical specimens. 270 clinical samples conserved at -80 uc whose hpv genotype and viral loads (only hpv6, 11, 16 and 18) had been previously determined were divided into nine groups (table 2) , each group contained 30 samples. dna was extracted from these samples with a da'an extraction kit (guangzhou da'an diagnostic co. ltd., guangzhou, china.). the extracted dna was divided into five aliquots. two aliquots were used for the detection of hpv6-11 and hpv16-18 mixed types by taqman uniplex probe fluorescence quantitative pcr (guangzhou da'an diagnostic co., ltd., china). the third aliquot was used for the detection of hpv 6, 11, 16 and 18 with allglo probe quadruplex fluorescence qpcr. the fourth aliquot was used for the hpv genotype detection with hpv ga test again. the remaining aliquot was conserved at -80 uc to further verify the specificity of the quadruplex fluorescence qpcr method, and the same amount of dna template was used in each experiment. the result was determined only when all parallel detection results were consistent. finally, the results obtained by the quadruplex qpcr were compared with the results obtained with the uniplex taqman qpcr and hpv ga test. table 3 . comparison of sensitivity and detection limit between taqman uniplex qpcr and allglo quadruplex qpcr. titrations of primer/probe combinations gave a positive result in uniplex qpcr and quadruplex qpcr. the 400/200 combination had the highest amplification efficiency and the lowest ct value ( table 3 ). the optimum annealing temperatures (uc) for hpv 6, 11, 16 and 18 amplification were: 60, 58, 58 and 58, respectively. in quadruplex fluorescence quantitative pcr, the compromise annealing temperature used for multiplex detection was 58uc, the primer/probe combinations was 400/200 (nm). all of the four hpv types in quadruplex qpcr were detected positively, while the other type of hpv were negative in the same detection system (figure 2 ). the sensitivities (copies/test) for hpv 6, 11, 16, and 18 were 10 1 , 10 2 , 10 1 , and 10 2 respectively ( table 4 ).the detection result of quadruplex qpcr is consistent with the uniplex qpcr(p.0.05). the coefficients of variation (cvs) of the five parallel tests for the four types of hpv plasmids were all below 5% (table 4 ). five side-by-side allglo quadruplex quantitative pcr assay were carried out using the dna aliquots in triplicate to determine the mean ct value, and the mean ct value was used for statistics of coefficient of variation (%) for four hpv types. *copies/test. # intra-assay coefficient variation. inter-assay coefficient variation. doi:10.1371/journal.pone.0048972.t004 five concentrations of plasmids of each type (10 3 , 10 4 , 10 5 , 10 6 , and 10 7 copies/test) were used to construct a standard curve with the allglo quadruplex fluorescence quantitative pcr system. the mean amplification efficiencies for hpv 6, 11, 16, and 18 were 0.97, 1.10, 0.93 and 1.20, respectively, and the mean correlation coefficient (r 2 ) of each standard curve was above 0.99. the results obtained with allglo quadruplex fluorescence qpcr, hpv ga test and taqman uniplex qpcr method were summarized in table 2 .the agreement rate between allglo quadruplex fluorescence qpcr, hpv ga test and taqman uniplex qpcr was 100%. single-tube allglo probe quadruplex fluorescence qpcr could simultaneously type hpv 6, 11, 16, and 18 and quantitatively detect the viral load of each hpv at the same time. the detection sensitivity of allglo probe quadruplex fluorescence qpcr was higher than that of hpv ga test and taqman uniplex qpcr. the fluorescence pcr technique is becoming increasingly important in the diagnosis of human diseases, such as influenza virus, avian flu virus [30, 31] , cytomegalovirus (cmv) [32] , and severe acute respiratory syndrome (sars) virus [33] . fluorescence pcr has advantages over conventional pcr [34, 35] .the former generates an amplification kinetics curve, which directly reflects the dynamic changes of the pcr process in real time. with the development of molecular biology and molecular marker techniques, multiplex fluorescence quantitative pcr technology is becoming more widely used. multiplex pcr not only significantly reduces the workload but also increases the accuracy and comparability of the experiment. methodologically, the multiplex fluorescence quantitative pcr technique is also suitable for hpv viral load and genotyping detection, especially for detecting a large number of samples simultaneously. quantitative pcr has been developed based on conventional pcr techniques. the quantitation of dna or rna templates is achieved by determining how many cycles are needed to reach a certain level of fluorescence. currently used probes in fluorescence pcr include taqman probes, fret probes, molecular signal probes, new fluorescent double-stranded replacement probes [36, 37] and allglo probes. fret probes are not commonly used in real-time monitoring but are used in melting-point analysis and genotyping after the completion of pcr amplification [38] . it is difficult to achieve the specificity in genotyping with conventional taqman probes; mgb was reported to enhance the specificity. however, mgb increases the cost and the difficulties in probe design and synthesis [39] . molecular signal probes have complicated designs when used in genotyping, and they are also inefficient in single-base discrimination [40] . currently, the most widely used probe in the clinical laboratory is taqman. however, it is difficult to design taqman experiments to detect multiple targets simultaneously, and this seriously limits the application of real-time pcr in genotyping [41, 42] . the allglo probe is the latest generation of quantitative fluorescent probes invented by allelogic biosciences corporation. allglo probes use two identical fluorophores that are specially selected in such a way that the two fluorophores are mutually quenched when both are conjugated to the probe oligonucleotide. both fluorophores become dequenched when the probe is cleaved. this study, the allglo probe quadruplex quantitative pcr was performed to simultaneously detect and differentiate human papillomavirus genotypes 6, 11, 16 and 18. samples of four types of hpv (hpv 6, 11, 16, and 18) were pcr-amplified and sequence-confirmed in order to ensure the reliability of the fluorescence qpcr. plasmids containing the target gene were cloned by t-a cloning for the preparation of standard dna stock. by optimizing the concentrations and combinations of primers and probes in allglo quadruplex quantitative pcr, we finally resolved the issue of mutual interference among primers in multiplex pcr and established a single-tube quadruplex fluorescence quantitative pcr technique based on the allglo probe. this new approach allows simultaneous detection and typing of four hpv types in a single tube. the r 2 values of all of the standard curves were larger than 0.99, and the amplification efficiency was between 0.91 and 1.23, which meets the requirement for fluorescence quantitative pcr standard curves [43, 44] . the agreement rate between allglo quadruplex fluorescence qpcr, hpv ga test and taqman uniplex qpcr was 100%. single-tube allglo probe quadruplex fluorescence qpcr could simultaneously type hpv 6, 11, 16, and 18 and quantitatively detect the viral load of each hpv at the same time. compared with the hpv ga test and taqman uniplex qpcr method, the allglo quadruplex qpcr method enjoys a high sensitivity and a wide linear range. in our experiment, the sensitivity of quadruplex fluorescence quantitative pcr can reach 10 to100 copies/test, while the sensitivity of hpv ga test and taqman uniplex qpcr method was above 100 copies/test. for the same sample, the positive rate and accuracy of the allglo quadruplex qpcr method was higher than those of the hpv ga test and taqman uniplex qpcr method. in addition, allglo probe quadruplex fluorescence quantitative pcr also has the advantages of relatively high throughput, time savings, simple operation, and lower cost, which are key factors that are needed in order to be qualified for clinical applications. the repeatability, sensitivity and specificity were high enough to be qualified for both qualitative and quantitative detection in clinical settings. allglo quadruplex quantitative pcr has the advantages of relatively high throughput, good reproducibility, high sensitivity, high specificity, and a wide linear range of detection. the convenient single-tube format makes this assay a powerful tool for the study of mixed infections caused by multiple pathogens, viral typing and viral load quantification,meanwhile, this method could be used in single nucleotide polymorphism (snp) and mutation analysis, prenatal diagnosis and genetic disease screening, drug efficacy analysis, tumor gene expression detection, and so on. the assay will facilitate molecular epidemiological studies and drug development. supporting information s1 the dna target sequences of hpv (hpv 6, 11, 16, 18) inserted in plasmid was confirmed by sanger sequencing. hpv prevalence and type distribution in women with or without cervical lesions in the northeast region of romania external genital warts: diagnosis, treatment, and prevention comparison of the immunogenicity of the human papillomavirus (hpv)-16/18 vaccine and the hpv-6/11/16/18 vaccine for oncogenic non-vaccine types hpv-31 and hpv-45 in healthy women aged 18-45 years comparison of the novel human papillomavirus 4 auto-capillary electrophoresis test with the hybrid capture 2 assay and with the pcr hpv typing set test in the detection of highrisk hpv including hpv 16 and 18 genotypes in cervical specimens comparative analysis of 19 genital human papillomavirus types with regard to p53 degradation, immortalization, phylogeny, and epidemiologic risk classification human papillomavirus and cervical cancer: biomarkers for improved prevention efforts associations of human leukocyte antigen class ii genotypes with human papillomavirus 18 infection and cervical intraepithelial neoplasia risk effect of hiv viral load, cd4 cell count and antiretroviral therapy on human papillomavirus prevalence in urine samples of hiv-infected men human papillomavirus type 16 (hpv-16) viral load and persistence of hpv-16 infection in women infected or at risk for hiv hpv vaccine against anal hpv infection and anal intraepithelial neoplasia human papillomavirus (hpv) 6, 11, 16, and 18 prevalence among females in the united states-national health and nutrition examination survey end-of-study safety, immunogenicity, and efficacy of quadrivalent hpv (types 6, 11, 16, 18) recombinant vaccine in adult women 24-45 years of age immunogenicity and reactogenicity of alternative schedules of hpv vaccine in vietnam: a cluster randomized noninferiority trial efficacy of quadrivalent hpv vaccine against hpv infection and disease in males impact of human papillomavirus (hpv)-6/11/16/18 vaccine on all hpv-associated genital diseases in young women sexual behaviour and hpv infections in 18 to 29 year old women in the pre-vaccine era in the netherlands hpv molecular assays: defining analytical and clinical performance characteristics for cervical cytology specimens molecular methods for a correct diagnosis of multiple hpv infections and clinical implications for vaccine diagnostic methods and techniques in cervical cancer prevention part ii: molecular diagnostics of hpv infection molecular detection and genotyping of human papillomavirus in cervical carcinoma biopsies in an area of high incidence of cancer from moroccan women simultaneous amplification and identification of 25 human papillomavirus types with templex technology development and evaluation of a new detection tool-visual dna microarray for simultaneous and specific detection of human immunodeficiency virus type-1 and hepatitis c virus multiplex fluorescent quantitative pcr for detecting deep fungal infection in patients with systemic lupus erythematosus a novel multiplex real-time rt-pcr assay with fret hybridization probes for the detection and quantitation of 13 respiratory viruses multiplex quantitative competitive polymerase chain reaction based on a multianalyte hybridization assay performed on spectrally encoded microspheres quantitative multiplex pcr assay for the detection of the seven clinically most relevant high-risk hpv types multiplex fluorescent rt-pcr to quantify leukemic fusion transcripts molecular beacon-based real-time pcr method for detection of 15 high-risk and 5 low-risk hpv types development of a real-time multiplex pcr assay for the detection of multiple salmonella serotypes in chicken samples a fret based melting curve analysis to detect nucleotide variations in ha receptor-binding site of h5n1 virus design of multiplexed detection assays for identification of avian influenza a virus subtypes pathogenic to humans by smartcycler real-time reverse transcription-pcr quan-quantitation of cytomegalovirus (hcmv) dna and beta-actin dna by duplex real-time fluorescence pcr in solid organ (liver) transplant recipients a novel coronavirus associated with severe acute respiratory syndrome ante-and post-mortem diagnosis of rabies using nucleic acid-amplification tests development of a real-time multiplex pcr assay for detection of viral pathogens of penaeid shrimp new approach to real-time nucleic acids detection: folding polymerase chain reaction amplicons into a secondary structure to improve cleavage of forster resonance energy transfer probes in 5'-nuclease assays templated chemistry for sequence-specific fluorogenic detection of duplex dna a miniaturised integrated biophotonic point-of-care genotyping system quantitative detection method for roundup ready soybean in food using duplex real-time pcr mgb chemistry silicon-based biosensors for rapid detection of protein or nucleic acid targets novel application of locked nucleic acid chemistry for a taqman assay for measuring diverse human immunodeficiency virus type 1 subtypes application of semi-quantitative m gene-based hydrolysis probe (taq-man) real-time rt-pcr assay for the detection of peste des petits ruminants virus in the clinical samples for investigation into clinical prevalence of disease revisiting the sigmoidal curve fitting applied to quantitative real-time pcr data unreliable real-time pcr analysis of human endogenous retrovirus-w (herv-w) rna expression and dna copy number in multiple sclerosis key: cord-334955-gnu92up6 authors: sutton, jeannette; renshaw, scott l.; butts, carter t. title: covid-19: retransmission of official communications in an emerging pandemic date: 2020-09-16 journal: plos one doi: 10.1371/journal.pone.0238491 sha: doc_id: 334955 cord_uid: gnu92up6 as the most visible face of health expertise to the general public, health agencies have played a central role in alerting the public to the emerging covid-19 threat, providing guidance for protective action, motivating compliance with health directives, and combating misinformation. social media platforms such as twitter have been a critical tool in this process, providing a communication channel that allows both rapid dissemination of messages to the public at large and individual-level engagement. message dissemination and amplification is a necessary precursor to reaching audiences, both online and off, as well as inspiring action. therefore, it is valuable for organizational risk communication to identify strategies and practices that may lead to increased message passing among online users. in this research, we examine message features shown in prior disasters to increase or decrease message retransmission under imminent threat conditions to develop models of official risk communicators’ messages shared online from february 1, 2020-april 30, 2020. we develop a lexicon of keywords associated with risk communication about the pandemic response, then use automated coding to identify message content and message structural features. we conduct chi-square analyses and negative binomial regression modeling to identify the strategies used by official risk communicators that respectively increase and decrease message retransmission. findings show systematic changes in message strategies over time and identify key features that affect message passing, both positively and negatively. these results have the potential to aid in message design strategies as the pandemic continues, or in similar future events. as the most visible face of health expertise to the general public, health agencies have played a central role in alerting the public to the emerging covid-19 threat, providing guidance for protective action, motivating compliance with health directives, and combating misinformation. social media platforms such as twitter have been a critical tool in this process, providing a communication channel that allows both rapid dissemination of messages to the public at large and individual-level engagement [1] . for all their virtues, however, these platforms also pose their own challenges. prominent among these is the problem of successfully reaching members of the public beyond their relatively small circle of immediate followers, a difficulty compounded by the highly crowded online media environment [1] . to reach a large audience, a message must typically be retransmitted by the sender's followers to others on the platform, a process that depends critically on the salience of the message to the user base and users' own decisions to retransmit it or not [2] . while each retransmission event is inevitably idiosyncratic, past research has shown that message style, structure, and content, as well as the sending context, can affect the overall rate at which messages are passed on, providing "levers" that can be exploited to maximize message amplification [3] . characterizing the specific factors shaping message retransmission in the unfolding covid-19 pandemic is thus important for evidencebased messaging strategies to be used in the months ahead. in this paper, we examine message retransmission on one of the most widely used social messaging systems (twitter), focusing on original messages posted by public agencies responding to covid-19. using a census of messages posted by approximately 700 accounts during the first three months of the pandemic's presence in the united states, we evaluate style, content, and contextual factors enhancing or inhibiting message retransmission. as we show, many factors affecting message retransmission rates are similar to those observed for other hazard events; however, the unique circumstances of the pandemic lead to some distinctive patterns of message passing that may inform communication strategies. public health and emergency management agencies are on the front lines of informing and educating the public about the science of virus transmission and prevention [4] . in response to a threat such as covid-19, their mission requires them to communicate accurate and credible information to local populations using all means of information delivery. currently, one of the most effective means of directly reaching the public is through the use of digital and social media. social media enables delivery of timely and actionable risk information to the public while also supporting ongoing dialog, potentially increasing trust and reducing fear and erroneous rumors fueled by misunderstanding [5] . this is increasingly important in the case of the coronavirus pandemic, which has now affected all 50 states across the country and requires nearly hourly updates about changing conditions and policies affecting at risk individuals. during this uncertain time period, online information seeking and sharing is likely to see a dramatic increase, as seen in research on past events [6, 7] requiring effective use of online communication infrastructure to alert and inform a public at risk. informal online communication channels have the potential to serve as a platform for risk communication amplification and to facilitate engagement through dialog. communication of timely risk information is absolutely vital for behavioral change to protect public health and safety; however, prior work on messaging in response to emerging health threats such as zika [8] and ebola [9] has shown that effective messaging strategies can depend upon details of the threat itself, while work on more conventional infrastructure-disrupting hazards such as fires and floods has likewise suggested that providing critical information regarding "hazard-adjacent" events such as closures, outages, and transportation interruptions can be vital for effective communication with the public [3] in contrast, covid-19 has affected every state and u.s. territory, resulting in a nationwide state of emergency where multiple levels of response are actively communicating about the risk. in addition to the pervasive threat posed by infection with the sars-cov-2 virus itself, mitigation measures intended to slow the spread of the infection have been complex and disruptive, requiring both individuals and organizations to radically alter or curtail their normal patterns of activity (often at severe personal or economic cost). in a very real sense, every member of the population is both a responder and a victim of the pandemic, whatever their age, occupation, or place of residence. this is a marked departure from more commonly experienced hazards (including public health hazards) in which there is a clear separation between the impacted population, those responding to it, and the remainder of the country, and in which mitigation measures place few demands on those not directly involved in the incident itself. adding to the difficulty is the fact that social distancing associated with covid-19 mitigation attenuates informal social contacts that are normally an important source of information and support during crises. as such, the need for official messaging during the covid-19 pandemic is arguably far greater than in more commonly encountered hazard settings, and the demands on communicators to inform and motivate are higher. with no immediate end to the event in sight, these needs are likely to continue to grow. in this study we examine two aspects of official communication on twitter during the first three months of the response (february-april). first, we investigate the messaging strategies used by public agencies in the evolving covid-19 response. in particular, we investigate how official tweets differ before and after march 13, the day on which the federal government announced a nationwide emergency. second, we investigate the predictors of message retransmission. we include message features, that is, the design choices and the message content that is shared by our sample of official risk communicators. we also investigate the effect of message senders, that is, the type of account (public health, emergency management, or elected official) and the level of government involved (local, state, or federal), as well as the size of the follower networks associated with each account. finally, we include a measure of salience, pre and post announcement of a nationwide emergency, to investigate the effects of time period on message retransmission. as we show, a wide range of practical and technical content types have been important promoters of information passing during the period, rather than any one content type proving to be of critical interest; salience-enhancing tactics such as the use of interrogative and exclamatory language have not been effective in promoting message retransmission, although inclusion of videos has a fairly large effect. these findings may be useful for informing communication strategy as the pandemic continues to unfold. covid-19 emerged as an international threat in the early part of 2020, as it spread from china into other regions of the world. by early january, the u.s. centers for disease control began to issue public alerts about a novel coronavirus and by the end of january, the world health organization announced that the outbreak was a public health emergency of international concern. on february 11, the who announced covid-19 as the name of the disease that results from the virus (ultimately dubbed sars-cov-2). on march 11, the who recognized the spread of covid-19 as a pandemic and on march 13, the white house proclaimed a national emergency concerning the covid-19 outbreak. epidemic management traditionally has an "urgent but narrow focus: containing the spread of disease and caring for the sick and dying" [10] . such a focus suggests communication priorities that are limited to alerting populations to effective protective actions that can be taken individually and collectively while official attention is devoted to surveillance and the development and deployment of treatments. outbreaks, however, while initially acute, may result in multiple waves of transmission as the virus passes from community to community, requiring a longitudinal communication and a sustained effort to reach persons. moreover, in comparison to prior pandemics, the covid-19 pandemic has occurred during a period of substantially greater epidemiological sophistication and public health preparedness. the disease itself-and its infectious agent-were identified relatively quickly following the initial outbreak in wuhan, with the viral genome being sequenced by late january of 2020 and models for disease propagation spun up almost immediately. this information, combined with early field reports, made it possible to quickly recommend guidance for effective containment and control (successful in e.g. south korea), or, barring that, mitigation in the form of social distancing measures intended to slow diffusion and reduce the risk of catastrophic failure of healthcare delivery systems. these measures have been effective: covid-19 has been contained in some locales, and in most others its spread has been sufficiently inhibited to allow hospitals to adapt to the patient load (aka "flattening the curve"). however, a side effect of this strategy has been a considerable prolongation of the public response, accompanied by a need for widespread and ongoing collective action to maintain social distancing measures. both factors have resulted in an enhanced requirement for communication with the public on an ongoing basis, not only to pass on warnings and alerts, but also to inform, educate, and continuously motivate individuals in their roles as de facto responders in the ongoing disaster. at this time, it is uncertain how long the covid-19 pandemic will last, and whether additional infection waves will occur, and whether it will end with natural elimination by herd immunity, suppression via immunization, or conversion to endemicity. with future outbreak events potentially looming on the horizon, and the need to sustain intrusive social distancing measures over a long period, public communication will play an every greater role in the public health response. while all channels are vital, reaching the public directly in the current era increasingly necessitates the use of social media. prior work on social media use and message retransmission in the context of hazard response has revealed a number of factors that are frequently involved in determining messaging effectiveness. here we briefly review some of the key issues pertinent to the covid-19 case. engagement has long been identified as a goal of organizational communication on social media [11] and was originally operationalized as two-way dialogic communication [12] . more recently, social media engagement has been conceptualized as a continuum of communication and includes all communicative acts made by an organization such message content choices to build relationships as well as using hashtags (#), including a hyperlink (url), and direct replies to exchange messages with other users [5] . research characterizing online engagement among governmental agencies (local weather forecast offices-wfo-for the national weather service) revealed various content-oriented strategies, e.g. to build community and inspire action, as well as message-structure strategies, e.g. replies, hashtags, and urls, both facilitate interaction and dialogue [5] . however, the use of these strategies by wfos were found to differ relative to the occurrence of a threat, such as being in a period of an active weather warning. for example during nonthreat periods, engagement content included community building and action-oriented messages encouraging followers to get involved in local education or other efforts. furthermore, in nonthreat periods, messages were more likely to include urls and images. in contrast, during threat periods, wfos tended to share messages about current conditions, "nowcasts," and weather warnings; they also increased their number of direct replies [5] . in the context of the first several months of the covid-19 event in the u.s. context, the most obvious transition point corresponding to an identified "threat period" was the declaration of a national state of emergency in march; this not only validated the emerging consensus regarding the seriousness of the virus threat in the u.s., but also marked a direct change from earlier federal communications downplaying the risk of a major outbreak. this hence motivates our first research question: rq: how do risk communication messaging strategies change relative to the declaration of a national state of emergency? message diffusion, in the form of passing messages from one account to another or retransmission by retweeting, is key to amplifying messages in the social media environment [13] . the more that a message is retweeted, the further the message is spread across online networks, allowing more persons to attend to, and possibly act upon, the information being shared. for risk communicators who send messages during an acute threat period, amplification is a primary objective for reaching those who are vulnerable. based upon the findings of seven case studies, vos et al. [9] proposed the risk communication on social media (rcsm) model, which identifies key factors that influence message passing under conditions of acute threat. these factors include the design of message itself (including the contents and structural features), the characteristics of the account sending the message (such as organization type), the number of followers associated with the account, and the time during which the message was sent. results from previous studies [2, 14] found that messages containing information about the severity of the threat and actionable information were shared at a higher rate than those that did not contain those contents. additionally, messages containing media, such as an attached image, were also shared more often [9] . some features that facilitate engagement, such as the inclusion of a url or direct replies, were found to decrease message passing [3] . similar results were identified when investigating longitudinal engagement on social media [15] . returning to the wfo accounts described above, sutton et al. [3] modeled message retransmission relative to the timing of threat and nonthreat events. they found that actionable and instructive messages and those that included a visual image were highly shared regardless of the time period; however, daily updates in the form of forecasts or current weather conditions involving little uncertainty, as well as message features that increase interaction, such as direct replies and urls, decreased message passing. importantly, in threat periods, they also found that warning messages, in particular those that offered no instruction, decreased message passing. longitudinal communication by public safety agencies are likely to follow patterns similar to nws wfos, where threats emerge and fade, requiring continuous monitoring of the environment and shifting communication tactics to meet public needs. engagement strategies designed to further relationship building during nonthreat periods are likely to shift during threat periods in order to privilege communication about the severity of a hazard, its potential impact, and recommendations for protective actions. such strategies are also likely also to affect message retransmission and the ability to amplify key messages. this motivates our second research question: we identified 690 accounts representing public health, emergency management, and elected officials (see table 1 ). public health accounts were identified by drawing on publicly available lists [16] and subsequent projects on social media risk messaging [9, 17] . the accounts for state governors (plus the district of columbia and puerto rico) (n = 52) and state emergency management organizations (n = 52) local mayors (n = 100) and local emergency management agencies (n = 100) for the 100 largest cities in the u.s. were collected by manually searching organization websites for associated social media accounts. between 1 february 2020 and 30 april 2020, we collected 149,335 tweets produced by the twitter accounts associated with 690 accounts offices using the twitter representational state transfer (rest) api, which allows data to be collected on specific accounts including account and tweet metadata, prior messages posted by the account, and follower count at time of query, and other features (fig 1) . each targeted account was queried at least once per 24 hours during data collection, allowing retrieval of the complete text of all messages posted during the period, as well as the exact time at which they were posted. on average, each account produced 216.43 messages during the study period, with 12 accounts producing only a single tweet and @who producing the most (n = 2624). lexicons-specialized keyword lists that identify topics in the context of a specific corpushave been developed in previous research by identifying crisis-specific keywords that can be used for monitoring twitter to detect information relevant to a disaster as it is emerging [18, 19] . such systems have generally focused on extracting features from the message, and classifying each tweet based upon user-defined categories. because we selected targeted accounts belonging to state and local agencies, we were most interested in identifying keywords within our existing corpus. our goal was not to develop a lexicon that could be utilized on a different set of accounts or applied to a different set of hazards. instead, we wanted to identify keywords that aligned with 1) theoretical concepts, 2) drew from previous research on twitter messages in disaster, and 3) observed content and language use during the covid-19 event. keywords (see table 1 ) were identified manually by reviewing a random sample of 100 tweets per day over the 90-day data collection period (n = 9,000) to identify words or phrases commonly used by our targeted accounts to communicate about the pandemic and associated topics. we began by reviewing past work on message retransmission on twitter in the context of acute threat and longitudinal risk communication to identify relevant thematic areas [3, 9] . this work also includes fear appeals [20, 21] with content themes around individual susceptibility and self efficacy, and the severity of the threat. because of the collective nature of the pandemic, we also include the keywords for content representing collective efficacy [21] . additionally, we identified keywords associated with the secondary threats that emerged over time as the pandemic continued. and finally, because there were a number of tweets within out dataset that were not about the pandemic, we also identified keywords associated with those off-topic tweets. susceptibility is a state of being likely to be influenced or harmed by a particular thing [9, 22] . keywords describe individuals or groups at risk of covid-19 infection including older adults, persons with pre-existing health conditions, or institutionalized populations such as persons in nursing homes or those who are incarcerated as well as homeless persons. additional keywords relevant to susceptibility include expressions of uncertainty such as low risk, high risk, and increased risk. severity represents the magnitude, or impact, of a threat [9, 22] . here, we divide severity keywords into two categories: surveillance strategies to identify the impact on the population such as test result, presumptive positive, contract trace, and person under investigation and symptoms of covid19, such as shortness of breath, cough, and fever. efficacy information [9, 22] instructs individuals about how they can protect themselves against a threat. efficacy keywords express actions that an individual can take to protect themselves from being exposed to coronavirus or exposing others to coronavirus such as staying home, social distancing, washing hands, or wearing a mask. collective efficacy refers to the capacity to achieve an intended effect [23] such as limiting population exposure and reducing virus transmission in order to protect vulnerable populations and critical resources. collective efficacy includes phrases such as slow the spread, good neighbors, united, solidarity, flatten the curve, and stay home save lives. technical information includes keywords describing the mechanisms of how the virus spreads [9] . example words and phrases include droplet, transmission, incubation, person-to-person, and large gatherings. official response includes keywords about governmental responses and how to access information [9] . example words and phrases include policy, declaration, proclamation, restrictions, and surveillance as well as briefing, resource guide, and press conference. resilience keywords express thanks and appreciation to the work of community members who provide essential services and supplies during the response [3] . these words include hero, salute, champion, honor, grateful, and thank. closures and openings keywords indicate the suspension of services, activities, and facilities [3] . keywords include visitation, lock down, mandatory, recreation, gatherings, parade, ceremony, as well as salon, spa, nightclub, bar, nba, and ncaa. thematic categories that emerged included keywords about the primary threat, including covid19, coronavirus, and ncov-sars and secondary threats that developed over the course of the event. this includes topics such as mental health, substance abuse, child abuse, domestic violence, food and housing insecurity, blood shortages, scams and misinformation, unemployment, stigma and discrimination, schools, price gouging, medical supply and equipment shortages, essential services, and field hospitals. we also include keywords for information sharing, such as webinar, resource guide, hotline, and briefing, identifying places, times, or strategies to obtain additional information about the ongoing pandemic. and finally, because our data include tweets from the month of february, when there was considerably less attention on the pandemic, we found a number of keywords that helped to identify clearly off topic tweets. these keywords were primarily associated with current events, such as super bowl, puppy bowl, valentines day, black history month, groundhog day, state of the union, and census. we also found a number of campaigns, frequently associated with hashtags, such as #goredforwomenday and #cancerawareness. we used regular expressions to code messages for content, structure, and style (see table 1 ). codes were distinctive but not mutually exclusive. messages could exhibit characteristics for each stylistic and structural feature as well as multiple content keywords. messages were coded for the thirteen content codes described previously, and nine structural features. structural features are coded for presence or absence in a given message, which included a video, an image, a hashtag, a hyperlink (url), a mention (@username), or the reply function. we also include "quote tweets" as a type of message structure. quote tweets are tweets that repost the original tweet with additional comment. to help understand the sentence style of a given message we also coded for punctuation, demonstrated by the presence of a question mark or an exclamation point. engagement. first, to assess whether message content and structure varied between the two time periods of interest (before and after the national emergency declaration), we used chi-square tests to examine the association for message content and message structural features and whether the message was published before and after march 13, 2020. since chi-square tests can only inform us whether an association between two variables exists, to understand the magnitude and direction of their relationship we calculated the log odds ratios and odds ratios. odds ratios are more naturally interpreted on log scale due to their being multiplicative: null effects are mapped 0 and effect sizes can be compared symmetrically to one another. message features that exhibit positive log odds ratios indicate that there is an increase use during the post-declaration period, while a negative log odds-ratio indicates a lower use during the post-declaration period. each message feature's chi-square statistic, log odds ratio, and raw odds ratios were calculated separately using the r statistical programming language. message retransmission. the other outcome of interest for this study is the propensity for messages to be retransmitted. retransmission was operationalized as the total number of times that a given message was retweeted or passed on-with the core question for our analyses being how do risk communication strategies affect message retransmission relative to the declaration of a national emergency? to estimate the contribution of multiple mechanisms to information retransmission, we perform a negative binomial regression of the retweet count on predictors related to message style, content, and posting context. we parameterize our model as. where y i is the retweet count for the ith message (with expectation ey i and covariate vector x i ), β is a vector of regression coefficients, and φ is the dispersion parameter for the negative binomial distribution. as this implies, a one-unit change in x i under this model is associated with a change of β in the log expected retweet count, or equivalently a multiplicative change in the expected retweet count by a factor of exp(β). use of the negative binomial likelihood allows us to account for the high level of overdispersion in the data, arising from the well-known tendency of some messages to "go viral" while other, similarly situated messages receive less attention. as has been found in prior work on retransmission in the domain of twitter [3] , the majority of messages published by official agencies do not get retweeted, with 65% of messages in our sample being passed on less than eight times. however, there are some messages that are retweeted at much higher rates, which results in a highly skewed distribution (mean = 59, median = 3, mode = 0 [26% of all messages], standard deviation = 685.27, skewness = 44.63, kurtosis = 2954.36). the marginal distribution of retweet frequency is shown in fig 2. as noted above, we account for this highly skewed distribution by employing a negative binomial model [24]; models were fit using the mass and glmmadmb packages in the r statistical programming language [25, 26] . we also control for various contextual factors that are unrelated to the content and structural features of a given message using fixed effects. these factors include seasonal effects including the month, the day of the week, and the time of the day a message was sent. (the large volume of available messages allows us to reliably estimate daily cycles at hourly resolution, as can be seen from the standard errors in table 3 .) we also control for the type of account sending the message: public health organizations, governors, mayors, state emergency management agencies, and local emergency management agency accounts. messages most often contained content about official response (n = 28,677; 19%), individual efficacy (n = 28, 324; 19%), and surveillance (n = 27,465, 18%). in regard to message structure presented in tweets, just over 49% (n = 73,091) of tweets included urls, 45% included a hashtag (n = 67,876) and 45% included a photo (n = 66,830). the chi square analyses indicate significant differences in message content and message structure in relation to the pre-and post-emergency declaration periods. (odd ratios reported in table 2 ). first, the inclusion of some content categories and structural features decrease following the emergency declaration. for example, off-topic content decreased by 63.5%, while content about technical information decreased by 18.6%, content about susceptibility decreased by 17.5%, and content about closures and openings decreased by 17% (fig 3) . we also find that messages containing an exclamation point or a question mark decrease by 48.6% and 27% respectively following the emergency declaration. the use of various message structural features also decrease post-emergency declaration. for example, we find a 26% decrease of the inclusion of images, a 17% decrease in mentions, a 12% decrease in the use of hashtags, and a 10% decrease in the inclusion of urls following the emergency declaration. in contrast, the inclusion of some message contents and structures increase post-emergency declaration. for example, content about collective efficacy increases by 78%, while content about information sharing increases by 61%, content about secondary impacts increases by 50%, content about surveillance increases by 50%, and content about individual efficacy increases by 45%. additional messaging content changes post-declaration include increases in content about official response activities (3.7%), the primary threat (25%), and symptoms (27%). there are also several message structural features that increase in frequency. for example, quote tweets increase by 10% and direct replies to other users increase by 32.5%. we also find that there are a few message features that remain fairly consistent regardless of the period during which they were used. for example, video attachments and content about resilience are constant in messages. and finally, we find that organizations varied their frequency of tweeting pre-and postemergency declaration. following the declaration, messages posted by local emergency management accounts decreased by 22% while messages by state emergency management accounts decreased by 6%. in contrast, mayors accounts increased their messaging by 3.6% and governors accounts increased the rate of their messaging by 23.6%. there were no significant differences in frequency of messages posted by public health organizations before or after the emergency declaration. the reported model (see table 2 ) indicates that the retweeting of risk communication messages during the first three months of the covid-19 pandemic is influenced by message content, message features, organizational type of the account, the number of followers for an account, and the time/day at which a message is sent. message contents, structure, and style jointly influence message retransmission. content features that most strongly influenced message retransmission pertained to information characterizing the impacts of the virus, its spread, and actions individuals can take to protect themselves (see fig 4) . messages that include surveillance content were passed on 40% more often than messages that did not include that content. messages that include technical information were passed on 30% more often; those containing content about efficacy were passed on 28% more often; and those containing content about symptoms were passed on 27% more often than messages that did not include those contents. messages including content naming the primary threat, were passed on 21.5% more, while secondary threats from the virus were passed on 20% more. messages containing content about official responses to the pandemic were shared 19% more often, while messages with content about collective efficacy were passed on 12.5% more and closures and openings were passed on 12% more than those that did not contain that content. messages containing content about resilience and susceptibility had the smallest effect on message retransmission with increases of 6.8% and 4.6% respectively. while most message contents have a positive effect on message retransmission, we find that messages containing content about information sharing decrease the likelihood of being shared by 11%. we also find that some structural features increase message retransmission (see fig 5) . the inclusion of media, both video and images increase message passing. messages that include videos are retransmitted 63% more often and messages containing photos or other images are passed on 27% more often than those that do not, holding all other features constant. we also find that the use of hashtags increase message passing by about 12% more than those that do not include a hashtag. importantly, there are several structural features that decreased message retransmission. for example, 'quote tweets,' that is, posting original content while quoting another user's message leads to a 7% decrease in message passing. we also find that mentioning another account or directly replying to another user decrease message passing by 23% and 82% respectively. table 2 ); whiskers indicate 95% confidence intervals. a wide range of covid-19 related message content enhances retransmission, with technical information, information related to disease surveillance and symptoms, and actions that can be taken to prevent infection being among the most important predictors. by contrast, content identifying sources of follow-up information (information sharing) tends to suppress retransmission. and finally, we find that messages containing weblinks (url) decrease the likelihood of retransmission by 30% holding all other features constant. we also find that sentence style affect message passing. messages that include exclamation marks were 23% less likely to be shared; messages including question marks were 7% less likely to be shared. this is distinct from some other settings, in which such saliency-enhancing features increase retransmission rates. the date at which the message was sent also makes a difference in message retransmission. for example we find that messages published after the national emergency declaration on march 13 th were passed on 44% more frequently than those sent before the declaration. we also find that, when compared to the reference category of february, messages posted in the month of march were retransmitted 153% more often; those posted in april had an increase of 58%. the account organizational type also had an impact on message passing. when compared to the reference category of the public health accounts, we find that messages posted from governors accounts were retransmitted 172% more frequently, while those from mayors accounts were shared 14% more often. in contrast, we find that messages posted by emergency management accounts decreased message retransmission. those posted by state emergency table 2 ); whiskers indicate 95% confidence intervals. videos substantially enhance retransmission, while photos and hashtags have much more modest effects. informal language (e.g., exclamatory and interrogative text), audience-narrowing features (e.g., mentions and replies), and urls tend to suppress retransmission. finally, we find that the number of followers has a significant effect on message retransmission. we find that a one unit increase in the log count of an account's followers coincides with a 113% increase in message passing. period, organization and follower effects are summarized in fig 6. this analysis has investigated two aspects of messaging by official accounts from february 2020, before widespread acknowledgment or impact of covid-19 across the u.s. to the end of april 2020, when health officials began to emphasize the use of masks and face coverings to limit the spread of the virus and more than 60,000 persons had died. we first examined the volume of messages by account type and the inclusion of message features, including content, structure, and style, and how those features differed pre-and post-emergency declaration. next we modeled the use of those message features to determine how their inclusion positively or negatively affected message retransmission. here, we discuss how message content and features employed by official accounts interact with message retransmission. one of the strongest contributors to message retransmission is the message structures that are technological affordances of the twitter platform. in the case of the pandemic, we find that table 2 ); whiskers indicate 95% confidence intervals. relative to the pre-declaration period, retransmission rates are higher in the post-declaration period; base retransmission rates increased further in march, 2020, while falling back somewhat in april. relative to public health agencies, elected officials are frequently retweeted, while state and local emergency management accounts see less retransmission. as in other settings, follower counts are also an important predictor of retransmission. https://doi.org/10.1371/journal.pone.0238491.g006 the inclusion of media, both videos and photos, lead to significantly greater message sharing. prior research has shown that media is an important feature that draws attention to messages, helping them to stand out in a sea of text [9] . in this research, while there is little change in the frequency of including videos with tweets, there was a decrease in the use of images postdeclaration. the inclusion of hashtags also increase message passing. prior research has shown that hashtags serve as strategy for organizing informational topics [27] and helping groups to form and exchange information [28] , and they become especially important for coordinating localized information [15] . here we find that while hashtag use decreases post-declaration, it remains an important predictor of message passing. it is possible that organizations were actively using hashtags to communicate about campaigns or other planned events in the predeclaration period in a coordinated fashion among organizations, which then declined when attention was turned to the pandemic [29] . prior research has also shown that structural features also lead to a decrease in message retransmission. in particular, the inclusion of directed messages, in the form of replies, consistently and negatively affects message retransmission under conditions of imminent threat [3] and during emerging infectious disease. directed messages serve as a strategy for engagement between an organization and a single message sender; therefore it is unsurprising that these messages would not be retransmitted broadly. in this case, we find that the use of directed messages significantly increased post-emergency declaration. this is similar to the finding from prior research on the use of twitter during periods of increased threat by national weather service weather forecast offices (wfos). under conditions of greater uncertainty, wfos increased their direct replies to users; in this research we find that public agencies also increase their replies, suggesting an effort to engage with persons in an ongoing manner [15] . while directed messages do not lead to increased message retransmission, they may serve to increase trust in the organization due to its responsiveness [5] . another structural feature that has been found to negatively affect message passing is the inclusion of a url [3] . here we find a decline in the use of this messaging strategy in the postdeclaration period. however, this consistently negative effect on message passing suggests that it may be more effective to deliver additional information by attaching content as an image if the goal is to increase message passing among online individuals. we also find that quote tweets also negatively affect message retransmission. while this structural feature increased in use post-declaration, its use leads to a decrease in message passing. such a finding suggests that attaching commentary to a previously constructed message doesn't aid in the diffusion of that content. if the goal of the sending organization is to increase the reach of an original message, it may be better to simply retweet it. message content also affects retransmission. prior research found that messages that contain information about the hazard and its impact [3] or its severity [9] will increase message passing. here we find that tweets containing content about surveillance, referencing the extent of those infected, hospitalized, dead, or recovered, and tweets containing technical information, that is how the hazard is spread among populations, both positively and significantly affect message retransmission. such attention to the hazard and its ongoing impact is not unexpected given the initial uncertainty about how it spread and the severity of the impacts on different populations. as multiple versions of epidemiological models were developed and shared, attempts to predict the infection rate with and without mitigation could only be verified by the posting of daily surveillance numbers that report deaths and any evidence that the curve was flattening. messages containing surveillance content increased by more than 50% post-declaration. additionally, protective action guidance, in the form of a warning or advice [3] or efficacy [9] , has previously been shown to increase message retransmission as individuals pass along information about what can be done to limit exposure to a threat and to protect themselves from harm. here we find that messages with content about individual efficacy were passed on 28% more often than those without efficacy content. however, in the case of covid-19, we also find that officials posted content about collective efficacy, that is content about social actions that taken together as a broad population are necessary to reduce the spread of the disease to the most vulnerable. messages exhorting people to "flatten the curve," "stop the spread," and "stay home save lives", increased by 78% post-emergency declaration and had a positive effect on message retransmission. in contrast, we find that information sharing has a negative effect of retransmission. messages that include information sharing decreases retransmission; however, this content increased post emergency declaration by 61%. previous research also showed that the style of a message, in the form of exclamatory or interrogative sentences, has an effect on message retransmission. in particular, messages with exclamation points frequently draw attention to the text and suggest urgency on the part of the sender [3] . here we see not only a decrease in the use of exclamatory sentences post-declaration, we also find the use of exclamation marks decreased retransmission. it is possible that in this case of a slow moving virus where orders were released and widespread measures were implemented gradually, perhaps very little was so urgent to warrant an exclamation point. equally plausible, however, is the observation that exclamatory and interrogative language is relatively informal, and potentially viewed as less authoritative than declarative language. in an environment in which members of the public are seeking authoritative guidance regarding a threat that they already perceive, trading authority for salience may be a poor bargain. it is possible that this balance may shift in the future, if gaining and maintaining attention becomes a challenge due to response fatigue. we also find that timing of the message has a strong effect on message retransmission. previous research has found an increase in messages posted leading up to and during imminent threat events by both members of the public [6] as well as response organizations [2] serves as a measure of salience about the event (how many people are paying attention) and also demonstrating the usefulness of social media as a platform for collective communication. we also find that the type of organization posting messages affects retransmission. perhaps it is expected that accounts representing individuals who are responsible for decision making, including monitoring the health of its local citizenry, declaring shelter in place orders, and placing restrictions on businesses and other organizations, would also receive a great deal of attention during a widespread pandemic. governors and mayors not only posted messages more often post-declaration, their messages were also retweeted more frequently than other organization types. in contrast, emergency management accounts at both the state and local level decreased message frequency post-declaration. and finally, follower numbers matter. due to the effects of network diffusion, messages posted by accounts with more followers will not only receive greater immediate exposure but are likely to receive more retweets overall (thus exposing them to yet more users). this effect is a consistent finding in work on organizational communication on twitter, emphasizing the importance of building a following over the long term. this research provides insight into the content and style features employed by official accounts pre-and post-emergency declaration and models what features increase or decrease message retransmission. from this, we are able to draw conclusions about how to improve message design to increase diffusion of official accounts during an emerging public health crisis. we do not know how these results apply to other types of accounts, such as media or other entities. additionally, we note that all social media efforts will be hampered by the algorithms deployed by the platforms themselves, which prioritize some accounts and messages to the detriment of others. we also cannot know the effects these messages actually have on behaviors offline. while retransmission demonstrates some form of interaction with content leading to a decision to share the message more broadly, we don't know what occurs beyond choosing to amplify the message. and, finally, while the findings on message retransmission remain fairly consistent across hazards and across account types, changing circumstances may lead to different patterns in the future. usage of these findings to guide practice should bear in mind the potential for retransmission predictors to evolve as the pandemic unfolds. a central feature of the covid-19 pandemic is the need to respond to rapidly changing circumstances, due both to the changes in the state of public health knowledge (e.g. on the efficacy of masks for personal protection) and the evolving political and economic situation (e.g. distancing regulations and resistance thereto). pandemics are, ultimately, disasters, and the critical role of improvisation that is central to effective disaster response is also inescapable here: it is unlikely in the case of covid-19 that conditions can be expected to fully stabilize in the near future, and risk communicators within official agencies need to establish processes to continuously re-assess and re-evaluate messaging practices in light of changing events (where possible, anticipating messaging that could be used if particular future conditions were to obtain). our work has shown that, in this uncertain environment, the public has been attentive to-and likely to retransmit-a wide range of practical information regarding the health impacts of covid-19, protective action measures, and the progress of the pandemic itself. at the same time, we have also found that some saliency-enhancing tactics useful in other disasters (such as sentence styles that use exclamatory and interrogative punctuation) have been counterproductive in the covid-19 pandemic. employing these factors when crafting messages may help public agencies in reaching the public in later stages of the covid-19 pandemic, or in the next public health emergency to arise. public health in the era of social media warning tweets: serial transmission of messages during the warning phase of a disaster event a cross-hazard analysis of terse message retransmission on 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retransmission in the boston marathon bombing response. effective communication during disasters getting the word out, rain or shine: the impact of message features and hazard context on message passing online. weather, climate, and society social media adoption in local health departments nationwide. american journal of public health social media messages in an emerging health crisis: tweeting bird flu crisislex: a lexicon for collecting and filtering microblogged communications in crises practical extraction of disaster-relevant information from social media putting the fear back into fear appeals: the extended parallel process model key: cord-315343-ywgoqlxj authors: ribeiro, haroldo v.; sunahara, andre s.; sutton, jack; perc, matjaž; hanley, quentin s. title: city size and the spreading of covid-19 in brazil date: 2020-09-23 journal: plos one doi: 10.1371/journal.pone.0239699 sha: doc_id: 315343 cord_uid: ywgoqlxj the current outbreak of the coronavirus disease 2019 (covid-19) is an unprecedented example of how fast an infectious disease can spread around the globe (especially in urban areas) and the enormous impact it causes on public health and socio-economic activities. despite the recent surge of investigations about different aspects of the covid-19 pandemic, we still know little about the effects of city size on the propagation of this disease in urban areas. here we investigate how the number of cases and deaths by covid-19 scale with the population of brazilian cities. our results indicate small towns are proportionally more affected by covid-19 during the initial spread of the disease, such that the cumulative numbers of cases and deaths per capita initially decrease with population size. however, during the long-term course of the pandemic, this urban advantage vanishes and large cities start to exhibit higher incidence of cases and deaths, such that every 1% rise in population is associated with a 0.14% increase in the number of fatalities per capita after about four months since the first two daily deaths. we argue that these patterns may be related to the existence of proportionally more health infrastructure in the largest cities and a lower proportion of older adults in large urban areas. we also find the initial growth rate of cases and deaths to be higher in large cities; however, these growth rates tend to decrease in large cities and to increase in small ones over time. human activities have become increasingly concentrated in urban areas. a direct consequence of this worldwide urbanization process is that more people are living in cities than in rural regions since 2007 [1] , and projections indicate that the world urban population could reach more than 90% by the end of this century [2] . besides being increasingly urbanized, we live in an unprecedentedly connected, and highly mobile world with air passengers exceeding 4 billion in 2018 [3] . on the one hand, a highly connected and highly urbanized society brought us innovation, economic growth, more access to education and healthcare; on the other, it has also lead to pollution, environmental degradation, privacy concerns, more people living in substandard conditions, and suitable conditions for dissemination of infectious diseases over the globe. in particular, the emergence of infectious disease outbreaks has significantly increased over time, and the majority of these events are caused by pathogens originating in wildlife [4] , which in turn has been associated with changes in environmental conditions and land use, agricultural practices, and the rise of large human population settlements [5] . the ongoing outbreak of the novel coronavirus (sars-cov-2) seems to fit well the previous context as it was first identified in wuhan in december 2019, an influential chinese city exceeding 11 million inhabitants, and apparently originated from the recombination of bat and malayan pangolin coronaviruses [6] . the coronavirus disease 2019 (covid-19) initially spread in mainland china but rapidly caused outbreaks in other countries, making the world health organization first declare a "public health emergency of international concern" in january 2020, and in mid-march, the outbreak was reclassified as a pandemic. as of 16 august 2020, over 21.2 million cases of covid-19 have been confirmed in almost all countries, and the worldwide death toll exceeds 761 thousand people [7] . the covid-19 pandemic poses unprecedented health and economic threats to our society, and understanding its spreading patterns may find important factors for mitigating or controlling the outbreak. recent works have focused on modeling the initial spreading of covid19 [8] or the fatality curves [9], projecting the outbreak peak and hospital utilization [10] , understanding the effects of mobility [11] , demography [12] , travel restrictions [13] , behavior change on the virus transmission [14] , mitigation strategies [15] , non-pharmaceutical interventions [16] , networkbased strategies for social distancing [17] , among many others. despite the increasing surge of scientific investigations on the subject, little attention has been paid to understanding the effects of city size on spreading patterns of cases and deaths by covid-19 in urban areas. the idea that size (as measured by population) affects different city indicators has been extensively studied and can be summarized by the urban scaling hypothesis [18] [19] [20] [21] . this theory states that urban indicators are non-linearly associated with city population such that socio-economic indicators tend to present increasing returns to scale [18, 22, 23] , infrastructure indicators often display economy of scale [18, 19] , and quantities related to individual needs usually scale linearly with city population [18, 19] . urban scaling studies of health-related quantities have shown that the incidence and mortality of diseases are non-linearly related to the city population [24] [25] [26] [27] . despite the existence of several exceptions [27] , noninfectious diseases (such as diabetes) are usually less prevalent in large cities, while infectious diseases (such as aids) are relatively more common in large urban areas. this different behavior is likely to reflect the fact that people living in large cities tend to have proportionally more contacts and a higher degree of social interactions than those living in small towns [19, 28] . within this context, the recent work of stier, berman, and bettencourt [29] has indicated that large cities in the united states experienced more pronounced growth rates of covid-19 cases during the first weeks after the introduction of the disease. similarly, cardoso and gonçalves [30] found that the per capita contact rate of covid-19 increases with the size and density of cities in united states, brazil and germany. these findings have serious consequences for the evolution of covid-19 and suggest that large metropolises may become infection hubs with potentially higher and earlier peaks of infected people. investigating whether this behavior generalizes to other places and how different quantities such as the number of cases and deaths scale with city size are thus important elements for a better understanding of the spreading of covid-19 in urban areas. here we investigate how population size is associated with cases and deaths by covid-19 in brazilian cities. brazil is the sixth most populous country in the world, with over 211 million people, of which more than 85% live in urban areas. while it is likely that the novel coronavirus was already circulating in brazil in early february 2020 [31] , the first confirmed case in the country dates back to 26 february 2020, in the city of são paulo. between the first case and 12 august 2020, brazil has confirmed 3,088,670 cases of covid-19 (second-largest number) spread out over 98.9% of the 5,570 brazilian cities. this disease caused 102,817 deaths (second-largest number) with 3,892 cities reporting at least one casualty as of 12 august 2020. we start by briefly presenting our data set (see methods for details). our investigations rely on the daily reports published by the health offices of each of the 27 brazilian federative units. these daily reports update the number of confirmed cases (y c ) and the number of deaths (y d ) caused by covid-19 in every brazilian city from 25 february 2020 (date of the first case in brazil) to 12 august 2020 (date of our last update). from these data, we create time series of the number of cases y c (t c ) for each city, where t c refers to the number of days since the first two daily cases reported in each city. similarly, we create time series of the number of deaths y d (t d ), where t d refers to the number of days since the first two daily deaths reported in each city. by doing so, we group all cities according to their stage of disease propagation (as measured by t c or t d ) to investigate the evolution of allometric relationships between total cases or deaths and city population. we have also considered different number of daily cases or deaths as the reference point, and our results are robust against different choices (from one to seven daily cases or daily deaths, see the number of cases is well described by a power-law function of the city population where β c is the so-called urban scaling exponent [18] . similarly, fig 1b shows the association between the number of casualties and the city population on logarithmic scale (logy d versus logp) for different numbers of days since two daily deaths first reported (t d = 15, 50, 85 and 120 days). again, the results indicate that the number of deaths is approximated by a powerfunction of the city population where β d represents the urban scaling exponent for the number of deaths. the results of fig 1 also show the adjusted allometric relationships (dashed lines) and the best fitting scaling exponents β c and β d (see methods for datails). these exponents exhibit an increasing trend with time so that β c and β d exceed one after some number of days after the first two daily cases or deaths. this dynamic behavior is better visualized in fig 2, where we depict β c and β d as a function of the number of days since the first two daily cases (t c ) or deaths (t d ). the scaling exponent for the number of cases β c is sub-linear (β c < 1) during the first four months and appears to approach a super-linear plateau (β c > 1) as the number of days t c further increases. the dynamic behavior of the scaling exponent for deaths β d is similar to β c ; however, β d appears to be approaching a plateau larger than the one observed for β c . the evolution of the scaling exponents for cases and deaths indicates that small cities are proportionally more affected by covid-19 during the first four months. however, this initial apparent advantage of living in large cities vanishes with time, and become a disadvantage after about four months. this is more evident by estimating the number of cases per capita from eq (1), that is, y c /p � p β c −1. similarly, we can estimate the number of deaths per capita from eq (2), yielding y d /p � p β d −1. thus, we expect the number of covid-19 cases or deaths per capita to decrease with the city population if β c < 1 and β d < 1; conversely, these per capita numbers are expected to increase with the city population if β c > 1 and β d > 1. for instance, because β c � 0.77 and β d � 0.85 after 75 days since the first two daily cases or deaths, the number of cases and deaths per capita decreases with population as y c /p � p −0.23 and y c /p � p −0.15 . at those particular values of t c and t d , an 1% rise in the population is associated with a �0.23% decrease in the incidence of covid-19 cases and �0.15% reduction in the incidence of deaths. in a concrete example for t c = t d = 75 days, we expect a metropolis such as são paulo (with �12 million people) to have �54% less cases and �39% less deaths per capita than a medium-sized city such as maringá/pr (with �420 thousand people, �1/30 of são paulo), which in turn is expected to have �41% less cases and �29% less deaths per capita than a small-sized city such as paranaíba/ms (with �42 thousand people, �1/10 of maringá). however, both scaling exponents increase with time, such that this urban advantage vanishes and become a disadvantage during the long course of the pandemic. by considering our latest estimates for the scaling exponents, we find β c � 1.04 (t c = 144 days) and β d � 1.12 (t d = 120 days). thus, at these particular values of t c and t d , we expect the number of cases per capita to slightly increase with population (y c /p � p 0.04 ) and the number of fatalities per capita to increase with population as y d /p � p 0.12 . thus, for β d � 1.12 at t d = 120 days, we expect a metropolis such as são paulo (�12 million people) to have �50% more deaths per capita than maringá/pr (�420 thousand people), which in turn is expected to have �32% more deaths per capita than paranaíba/ms (�42 thousand people). figs 8-14 in s1 appendix show that the scaling relations for number of cases and deaths per capita support the previous discussions. the latest estimates of β c found for cases of covid-19 are smaller than those reported for the 2009 h1n1 pandemic in brazil (β c � 1.2) and hiv in brazil and united states (β c � 1.4) [27] . similarly to what we observe for the cases of covid-19, the allometric exponent for hiv cases in brazil was initially sub-linear during the 1980s, became super-linear after the 1990s, and started to approach a super-linear plateau after the 2000s [27] . however, the evolution of the allometry for hiv has been much slower than what we have observed for the covid-19. another interesting point reported by rocha, thorson, and lambiotte [27] is that the number of h1n1 cases in brazil started to scale linearly with city population in 2010 (one year after the first outbreak). these authors also argue that this reduction in the scaling exponent possibly reflects a better response for the spread of h1n1 after the pandemic outbreak. if the behavior observed in the 2009 h1n1 pandemic generalizes (at least in part) for the current covid-19 pandemic, we would expect a decrease in values of β c in the future. the lastest estimates of β d for covid-19 deaths are larger than those reported for diabetes (β d � 0.8), heart attack (β d � 1) and cerebrovascular accident (β d � 1) in brazil after the 2000s [27] . conversely, scaling exponents related to disease mortality in brazil displayed a decreasing trend with time, and values as high as 1.25 were observed for diabetes in 1996 (β d � 1.22) and heart attack in 1981 (β d � 1.25) [27] . the convergence of these exponents to linear or sub-linear regimes may reflect the increasing access to medical facilities in urban areas [27] . based on currently available data (fig 2) , it is hard to confidently assert whether the values of β c and β d will remain larger than one during the long-term course of the pandemic. however, the persistence of this behavior indicates large cities are likely to be more affected at the end of the covid-19 outbreak. part of this behavior may be due to large cities testing for covid-19 proportionally more than small ones. results for the united states indicate that more rural states have lower testing rates and detect disproportionately fewer cases of covid-19 [32] . as brazilian cities are likely to suffer from this bias, we would expect a decrease in the scaling exponent β c after the observed increasing trend depending on the magnitude of this effect (that is, as small cities increase their testing capabilities, their number of cases tend to increase and bend the scaling law downwards). on the other hand, it is clearer that large cities were proportionately less affected during the initial months (since the first two daily cases or deaths) of the pandemic. we believe there are at least two possible explanations for this behavior. first, it may reflect an "increasing urban advantage" where the larger the city, the more access to medical facilities and so the chance of receiving more appropriate treatment against the coronavirus disease. a second cause can be associated with age demographic changes with the city population; specifically, a smaller proportion of older adults at high risk for severe illness and death from covid-19 leads to a reduced number of deaths per capita. another possibility is that the strategies and policy responses of large and small cities to covid-19 are different, which in turn may lead to different efficiency in containing the pandemic. these responses are highly heterogeneous at the national level [33, 34] as well as among counties in the united states [35] . among these three possibilities, we did not explore the possible effects of different city strategies against the covid-19, but in light of the findings for the united states [35] , this effect is likely to play an important role in the brazilian case and may deserve further investigation. to test for an increasing urban advantage for the treatment of covid-19 during the initial spread of the disease, we investigate the scaling relation between the number of hospital intensive care unit (icu) beds and city population. because critically ill patients frequently require mechanical ventilation [36, 37] , the number of icu beds has proved to be crucial for the treatment of covid-19. fig 3a shows the allometric relationship between the number of icu beds from private and public health systems (y icu , as of april 2020) and the population, where a super-linear relationship emerges with scaling exponent β icu � 1.16. the super-linear scaling of icu beds indicates that large brazilian cities are better structured to deal with critically ill patients, which in turn may partially explain the reduction of deaths per capita with the city size during the initial three-four months since the first two daily deaths. it is worth noting that the brazilian public unified health system (sistema ú nico de saúde-sus) is decentralized and composed of "health regions", contiguous groups of cities usually formed by a large city and its neighboring cities [38] . cities within the same health region may share medical services, which may in turn partially explain the reduction of the structural advantages of large urban areas during the long-term course of the pandemic. we have also investigated how age demographic distribution changes with city population. estimates have shown that the case fatality rate of covid-19 is substantially higher in people aged more than 60 years (0.32% for those younger than 60 years versus 6.5% for those older than 60 years [39] ). thus, the age demographic of cities represents an important factor for the number of deaths caused by covid-19. fig 3b and 3c show how the number of people older (p hr , the high-risk population) and younger (p lr , the low-risk population) than 60 years change with the total population (p). we note that the high-risk population increases sub-linearly with city size with an exponent β hr � 0.91, while the low-risk population scales linearly (β lr � 1) with city size. this result shows that large cities have a lower prevalence of adults older than 60 years, such that a 1% increase in city population is associated with a 0.91% rise in the high-risk population. in a more concrete example, we expect a city with one million people to have proportionally �19% fewer adults older than 60 years when compared with a city of 100 thousand inhabitants. thus, a low prevalence of elderly in large urban areas may also partially explain the initial reduction of the number of deaths per capita with the increase of city population. in addition to addressing the urban scaling of cases and deaths of covid-19, we have investigated associations between the growth rates of cases and deaths and the city population (figs 16-22 in s1 appendix). as mentioned, the work of stier, berman, and bettencourt [29] shows that the initial growth rates of covid-19 cases in metropolitan areas of the united states scale as a power-law function of the population with an exponent between 0.11 and 0.20. by using our data and as detailed in methods, we have estimated the growth rates of cases (r c ) and deaths (r d ) for brazilian cities. in agreement with the united states case, our results also indicate that covid-19 cases initially grow faster in large cities (fig 23 in s1 appendix) , such that r c � p b r c with b r c between �0.1 and �0.3 during the first three months (t c ≲ 90, fig 23 in s1 appendix). we also found similar behavior for the growth rate in the number of deaths r d , where a power-law relation r d � p b r d is a reasonable description for the empirical data with a scaling exponent b r d between �0.1 and �0.5 during the first three months (t d ≲ 90, fig 23 in s1 appendix). the growth rate depicts a more instantaneous picture of the covid-19 spreading process, and its association with size may change during the long-term evolution of the pandemic. these changes may reflect the different actions taken by each city to face the covid-19 pandemic and other particularities affecting the covid-19 spreading. for the spreading of covid-19 in the united states, heroy [40] has reported that large cities appear to enter in an exponential spreading regime earlier than small ones. to better investigate these possibilities in our data, we have estimated the average relationship between the growth rate of cases (r c ) and deaths (r d ) and the city rank s (s = 1 represents the largest city in data, s = 2 the second-largest, and so on) at different periods. fig 4a shows the results for the growth rates in the number of cases (r c ). in agreement with the power-law association between r c and the city population (figs 16-22 in s1 appendix), we note that lower values of the city rank s are associated with higher growth rates r c in the initial days since the first two daily cases. however, as time goes by, the growth rate of cases starts to decrease in large cities (low-rank values) and to increase in small ones (high-rank values). this result appears to agree with the findings of heroy [40] in the sense that there is a delay in the emergence of high growth rates of cases between large and small cities. fig 4b shows the same analysis for growth rate in the number of deaths r d . while we also observe a decrease in r d for large cities and increase for small ones, the differences in r d are less pronounced than in r c . these findings also emerge when investigating the scaling exponents associated with the growth rates of cases (b r c ) and deaths (b r d ). the results of fig 23 in s1 appendix show that these exponents start to decrease around t c � t d � 100 days and become negative in our latest estimates. it is worth remembering that the time t c (or t d ) is measured in days since the first two daily cases (or first two daily deaths) for each city; thus, the results of fig 4 do not reflect delays in the emergence of the first case in each city. we have studied scaling relations for the number of covid-19 cases and deaths in brazilian cities. similarly to what happens for other diseases, we found the number of cases and deaths to be power-law related to the city population. during the initial three-four months since the first two daily cases or deaths, we found a sub-linear association between cases and deaths by covid-19, meaning that the per capita numbers of cases and deaths tend to decrease with population in this initial stage of the pandemic. we believe this behavior can be partially explained by an "increasing urban advantage" where large cities have proportionally more icu beds than small ones. in addition, changes in age demography with city size show that large cities have proportionally less elderly people who are at high risk of developing severe illness and dying from covid-19. this may also partially explain the initial reduction of fatalities per capita with the city population. in addition, we have argued that the strategies and policy responses of large and small cities to covid-19 may also be different and lead to different efficiency in containing the pandemic. however, we found that this "urban advantage" vanishes in the long-term course of the pandemic, such that the association between cases and deaths by covid-19 with population becomes super-linear in our latest estimates since the first two daily cases or deaths. thus, the persistence of this pattern indicates that large cities are expected to be proportionally more affected at the end of the covid-19 pandemic. this result is in line with the findings for other infectious diseases [25, 27] and probably reflects the existence of a higher degree of interaction between people in large cities [19, 28] . because social distancing is currently the only available measure to mitigate the impact of covid-19, our results suggest that large cities may require more severe degrees of social distancing policies. in agreement with the results for metropolitan areas in the united states [29] , we have found that large cities usually display higher growth rates in the number of cases during the initial spread of the covid-19. however, our results also show that these growth rates tend to decrease in large cities and to increase in small ones in the long-term course of the pandemic. this behavior suggests the existence of a delay in the emergence of high growth rates between large and small cities. similar behavior was also found in the united states [40] , where large cities appear to enter an exponential growth regime earlier than small towns. the existence of this delay suggests that the initial slow-spreading pace of the covid-19 in small cities is likely to be a transient behavior. together with the recent findings of stier-berman-bettencourt [29] and heroy [40] for the united states, as well as those of cardoso and gonçalves [30] for united states, brazil and germany, our results suggest that social distancing policies and other actions against the pandemic should take into account the non-linear effects of city size on the spreading of the covid-19. the primary data set used in this work was collected from the brasil.io api [41] . this api retrieves information from covid-19 daily reports published by the health offices of each of the 27 brazilian federations (26 states and one federation district) and makes it freely available. this data set comprises information about the cumulative number of cases and deaths of covid-19 from 25 february 2020 (date of the first case in brazil) until 12 august 2020 (date of our last update) for all brazilian cities reporting at least one case of covid-19. the brasil.io api also provides population data of brazilian cities, which in turn relies on population estimates for the year 2019 released by the brazilian institute of geography and statistics (ibge). there is a total of 5,507 brazilian cities with at least one reported case of covid-19 on 12 august 2020, corresponding to 98.9% of the country's total number of cities. in addition, 3,892 cities suffered casualties from this disease, representing 69.9% of the total. to ensure that our estimates rely on at least 50 cities, we consider a suitable upper threshold for the time series length (fig 24 in s1 appendix) . the data about age demographics refer to the latest brazilian census that took place in 2010, while the data about the number of icu beds are from april 2020. these two data sets are maintained and made freely available by the department of informatics of the brazilian public health system (datasus) [42] . urban scaling [18] usually refers to a power-law association between a city property y and the city population p, and it is expressed by where y 0 is a constant and β is the urban scaling exponent. eq (3) can be linearized by taking the logarithmic on both sides, that is, where log y and log p are the dependent and independent variables of the corresponding linear relationship between log y and log p. we have estimated the power-law exponents in eq (3) by using the probabilistic approach of leitão et al. [43] . specifically, we have found the probabilistic model with lognormal fluctuations and where the fluctuations in log y are independent of p to be the best description of our data in the majority of scaling laws. thus, we assume these lognomal fluctuations in all adjusting procedures in order to estimate the values of β. it is worth mentioning that this maximum-likelihood estimate for scaling exponents is analogous to the one obtained via usual least-squares with the log-transformed variables (log y versus log p). let us consider that x t (t = 1, . . ., n) represents the cumulative number of cases (y c ) or the cumulative number of deaths (y d ) for covid-19 in a given city at time t (number of days since first case t c or death t d ). the logarithmic growth rate r t at time t is defined as where τ is a time delay. if we assume the numbers of cases or deaths to initially increase exponentially (x t � e rt , where r is the exponential growth rate), r t represents an estimate for the growth rate of this initial exponential behavior (r). we have estimated r t for the number of cases (r c ) and deaths (r d ) up to values of t c and t d ensuring a sample size of at least 50 cities for the allometric relations between these growth rates and the city population (fig 24 in s1 appendix). all results in the main text were obtained for τ = 14 but our discussion is robust for τ between 9 and 21 days (figs 25-38 in s1 appendix). supporting information s1 appendix. supplementary figs (1-38) world urbanization prospects: urban population (% of total) global urbanization projections for the shared socioeconomic pathways international civil aviation organization, civil aviation statistics of the world and icao staff estimates global trends in emerging infectious diseases origins of major human infectious diseases isolation of sars-cov-2-related coronavirus from malayan pangolins projecting hospital utilization during the covid-19 outbreaks in the united states spread and dynamics 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large cities in the united states in years surrounding the 1918 pandemic growth patterns and scaling laws governing aids epidemic in brazilian cities statistical signs of social influence on suicides the non-linear health consequences of living in larger cities the scaling of human interactions with city size covid-19 attack rate increases with city size urban scaling of covid-19 epidemics tracking the onset date of the community spread of sars-cov-2 in western countries a commentary on rural-urban disparities in covid-19 testing rates per 100,000 and risk factors. the journal of rural health the effect of large-scale anti-contagion policies on the covid-19 pandemic quantifying policy responses to a global emergency: insights from the covid-19 pandemic creatures of the state? metropolitan counties compensated for state inaction in initial us response to covid-19 pandemic. mansueto institute for urban innovation research paper critical care utilization for the covid-19 outbreak in lombardy, italy: early experience and forecast during an emergency response covid-19: a novel coronavirus and a novel challenge for critical care brazil's unified health system: the first 30 years and prospects for the future estimates of the severity of coronavirus disease 2019: a model-based analysis. the lancet infectious diseases metropolitan-scale covid-19 outbreaks: how similar are they? boletins informativos e casos do coronavírus por município por dia public healthcare system (sus), department of data processing (datasus) is this scaling nonlinear the authors have declared that no competing interests exist. key: cord-327199-ggomuomb authors: moerdyk-schauwecker, megan; hwang, sun-il; grdzelishvili, valery z. title: cellular proteins associated with the interior and exterior of vesicular stomatitis virus virions date: 2014-08-08 journal: plos one doi: 10.1371/journal.pone.0104688 sha: doc_id: 327199 cord_uid: ggomuomb virus particles (virions) often contain not only virus-encoded but also host-encoded proteins. some of these host proteins are enclosed within the virion structure, while others, in the case of enveloped viruses, are embedded in the host-derived membrane. while many of these host protein incorporations are likely accidental, some may play a role in virus infectivity, replication and/or immunoreactivity in the next host. host protein incorporations may be especially important in therapeutic applications where large numbers of virus particles are administered. vesicular stomatitis virus (vsv) is the prototypic rhabdovirus and a candidate vaccine, gene therapy and oncolytic vector. using mass spectrometry, we previously examined cell type dependent host protein content of vsv virions using intact (“whole”) virions purified from three cell lines originating from different species. here we aimed to determine the localization of host proteins within the vsv virions by analyzing: i) whole vsv virions; and ii) whole vsv virions treated with proteinase k to remove all proteins outside the viral envelope. a total of 257 proteins were identified, with 181 identified in whole virions and 183 identified in proteinase k treated virions. most of these proteins have not been previously shown to be associated with vsv. functional enrichment analysis indicated the most overrepresented categories were proteins associated with vesicles, vesicle-mediated transport and protein localization. using western blotting, the presence of several host proteins, including some not previously shown in association with vsv (such as yes1, prl1 and ddx3y), was confirmed and their relative quantities in various virion fractions determined. our study provides a valuable inventory of virion-associated host proteins for further investigation of their roles in the replication cycle, pathogenesis and immunoreactivity of vsv. vesicular stomatitis virus (vsv, family rhabdoviridae) is a prototypic nonsegmented negative-strand rna virus (order mononegavirales) serving as a model for other human and animal pathogens including other rhabdoviruses such as rabies virus. vsv is also being widely investigated as a vector for vaccines (reviewed in [1] ), gene therapy (reviewed in [2] ) and oncolytic (anticancer) virotherapy (reviewed in [3, 4] ). as a result, currently two phase i human clinical trials evaluating the safety of the vsv-based hiv vaccines (clinicaltrials.gov, trials nct01438606 and nct01578889) are currently in progress. in addition, a phase i human clinical trial using replication-competent oncolytic vsv against hepatocellular carcinoma is in progress (trial nct01628640). vsv replicates in the cytoplasm and contains a nonsegmented, negative-strand rna genome of approximately 11 kb encoding five viral proteins, all of which are included in the mature virion. the large polymerase protein (l) and phosphoprotein (p) together form the viral rna dependent rna polymerase (rdrp). in mature virions, the rdrp is associated with the nucleocaspid (n) protein encapsidated viral genome, together forming the ribonucleoprotein (rnp) complex. the matrix (m) protein is responsible for nucleocapsid condensation and is the primary driving force behind viral budding through the host cell plasma membrane, whereby the virion obtains its lipid bilayer envelope. embedded in that envelope is the transmembrane glycoprotein (g), which is essential for receptor binding and cell entry (reviewed in [5] ). vsv virions, like those of many viruses, contain not only virus encoded-proteins, but also host (cellular) proteins [45] . some of these proteins are enclosed within the virion structure, while others, in the case of enveloped viruses like vsv, are embedded in the host-derived membrane [6] . while many of these incorporations could be ''accidental'', others may reflect the mechanism of virus assembly, be necessary for viral function, impact host range and infection efficiency, or influence the outcome of subsequent infections. for example, a recent study showed that depletion of a number of host proteins normally incorporated into herpes simplex virus type-1 virions not only reduced virion production in those cells, but that new infections with the resulting virions also yielded fewer progeny virions even in cells expressing normal levels of the host protein [7] . the host-derived proteins of a virus may also affect the host immune response. this is an especially important consideration for viruses, including vsv, used in therapeutic applications where large numbers of virus particles are administered, as it may influence efficacy as well as the potential for adverse side effects. incorporation of icam-i into the envelope of human immunodeficiency virus type-1 (hiv-1) not only increased infection efficiency [8, 9, 10, 11] but also interfered with virus neutralization by host antibodies [12, 13, 14, 15] . in another example, the presence of host complement control proteins such as cd46, cd55 and cd59 in the viral envelope has been shown to protect against antibody dependent complement mediated virus lysis in several viruses including human t cell leukemia/ lymphoma virus type i [16] , human cytomegalovirus [16] , hepatitis c virus [17] , hiv-1 [18, 19] , extracellular enveloped vaccinia virus [20] , simian virus 5 [21] and mumps virus [21] . to comprehensively look at host protein incorporation, a number of purified viruses have been analyzed by mass spectrometry including poxviruses [22, 23, 24] , herpesviruses [25, 26, 27, 28, 29, 30, 31, 32] , orthomyxoviruses [33] , coronaviruses [34, 35] , retroviruses [36, 37, 38, 39] , paramyxoviruses [40, 41] , baculoviruses [42] , hytroviruses [43] and arteriviruses [44] . our previous study examined vsv virions grown in three different cell lines (bhk-21, 4t-1 and a549) originating from different species, and found a number of similarities and differences in the host protein content [45] . here we conduct an analysis not only of intact (''whole'') virions, but also virions treated with proteinase k (prok) to remove surface proteins to look at the localization of host protein incorporation into the virion. syrian golden hamster kidney fibroblast cells (bhk-21; atcc# ccl-10) were grown in monolayer cultures maintained in minimum essential medium (eagle's mem, cellgro) supplemented with 9% fetal bovine serum (fbs, gibco), 0.3% glucose (w/v), 3.4 mm l-glutamine, 90 u/ml penicillin and 90 mg/ml streptomycin, and kept in a 5% co 2 atmosphere at 37uc. infectivity [plaque forming units (pfu) per ml] of virus stocks was determined by standard plaque assay on bhk-21 cells. recombinant wild-type (wt) vsv (indiana serotype) was generated previously [46] using pbs-l, pbs-p, pbs-n, and pvsvfl(+) plasmids, and was kindly provided by john k. rose (yale university) [47] . to grow and purify virus vsv, bhk-21 cells were infected with at a multiplicity of infection (moi) of 0.001 and incubated at 37uc in media containing 5% fbs. virus containing media was collected around 24 hours (h) post infection (p.i.) when most cells were infected but significant cell detachment had not yet occurred in order to maximize exclusion of cellular debris. the media was centrifuged at 3,0006 g for 10 minutes (min) to remove large cellular debris. the virus was then purified as described in [48] , with slight modifications. in brief, clarified supernatants were underlayed with 5 ml 20% (w/v) sucrose in hen buffer (10 mm hepes ph 7.4, 1 mm edta, 100 mm nacl) and centrifuged at 28,000 rpm and 4uc for 3.5 h in a beckman sw32 ti rotor. the resulting virus-containing pellet was resuspended overnight in hepes buffered saline, ph 7.5 [hbs; 21 mm hepes, 140 mm nacl, 45 mm kcl, 0.75 mm na 2 hpo 4 , 0.1% (w/v) dextrose] and then centrifuged in a 7.5-27.5% continuous gradient of optiprep (axis shield) in hbs at 26,500 rpm and 4uc for 30 min using a beckman sw40 ti rotor. the virus-containing band was removed from the gradient, diluted with et buffer (1 mm tris-hcl ph 7.5, 1 mm edta), pelleted by centrifugation at 27,000 rpm and 4uc for 1.5 h using a beckman sw40 ti rotor and resuspended in et buffer. for protease treatment, purified virions were treated with 0.08 mg prok per 1 mg total protein, re-purified by centrifugation through a sucrose cushion as previously described [45] and resuspended in et buffer. for isolation of viral rnp complexes, purified virions not treated with prok were disrupted as previously described [49] . in brief, virions were disrupted in a buffer containing a final concentration of 10 mm tris-hcl (ph 8.0), 5% glycerol (v/v), 0.4 m nacl, 1.85% triton x-100, and 0.6 mm dtt, and pelleted through a 30% glycerol cushion onto a 100% glycerol cushion by centrifugation at 38,000 rpm and 4uc for 2 h using a tla 100.2 rotor. under these conditions, proteins with moderate to high affinity for the viral nucleocapsid, including the viral l/p polymerase complex, remain associated with the nucleocapsid [49] . the recovered rnp complexes were diluted 1:1 (v/v) with a buffer containing 50 mm tris-hcl (ph 8.0), 10 mm nacl, 5 mm mgcl 2 and 2 mm dtt. virions were absorbed to carbon-formvar coated grids (electron microscopy sciences) by floating grids on 5 ml drops of sample for 30 seconds (s). grids were blotted dry, stained with 2% uranyl acetate in water for 30 s, blotted to remove excess stain and airdried. samples were visualized using a jeol jem 2100 lab6 transmission electron microscope. total protein equaling 50 mg for purified virion samples and 60 mg for prok treated virions was separated on a 10% tris-glycine sds-page gel under reducing conditions and stained with coomassie brilliant blue r250. these quantities were chosen so that the total amount of viral n plus p protein was approximately equivalent for both sample types. each gel lane was cut into 13 or 14 gel bands for analysis. gel pieces were subjected to in-gel trypsin digestion and the resulting peptides were extracted from the gel matrix, separated using waters acquity ultra performance liquid chromatography (uplc) with nano-split and analyzed using a ltq-xl tandem mass spectrometer (thermofisher scientific, waltham, ma) as described previously [50] . briefly, samples were separated by a 65 min linear gradient from 95% solvent i (0.1% formic acid in water)/solvent ii (0.1% formic acid in acetonitrile) to 50% solvent i/ii at a flow rate of 500 nl/min on reversed phase chromatography using a trap/elute method with a in-house c 18 sample trap in line with a c 18 analytical column. each full ms scan was followed by eight ms/ ms scans of the most intense ions with data-dependent mode using the dynamic exclusion option (top 8 method). the spectra were searched using the sequest algorithm of the bioworks software (thermofisher, san jose, ca; version 3.3.1 sp1) against the ipi.human.v.3.18 and ipi.mouse.v.3.18 databases concatenated with a vsv and sendai virus protein database. a parent ion mass tolerance of 2.0 da, fragment ion mass tolerance of 1.0 da, and a 16 da differential modification for methionine oxidation were used for search parameters. protein identifications were accepted when the peptide probability was greater than 95.0% [51] , the protein probability was greater than 99.0%, and contained at least two unique peptides. scaffold software was used for data compiling of each group and calculating spectral counts, unique peptides, percent coverage and empai [52, 53, 54] . while a single set of gel bands was isolated for each sample, three replicate uplc-ms/ms runs were conducted for the whole and prok virion samples, although one set of runs could not be used for the whole virions due to poor data quality. protein enrichment was analyzed using the database for annotation, visualization and integrated discovery (david) v.6.7 [55, 56] . using the mus musculus genome as the background data set, protein sets were analyzed for enrichment using the terms from the gene ontology (go) biology process, cellular component or molecular functions fat databases. the go fat databases contain the more specific go terms while excluding the more general terms. the enriched terms were then subjected to cluster analysis using the default settings, to identify groups of related enriched terms, with overall enrichment scores based on the ease scores of the member terms. the broadest term, representing most if not all the proteins in the cluster, is used here to describe the cluster. cellular lysates were prepared by mock infecting bhk-21 cells or by infecting them with vsv at a moi of 0.05. cells were harvested at 18 h p.i. and lysed in ripa buffer (25 mm tris-hcl ph 7.6, 150 mm nacl, 1% np-40, 1% sodium deoxycholate and 0.1% sds). protein concentrations of cellular lysates, purified virions, prok treated virions and rnp complexes were determined by bradford assay. 50 mg of purified virions, 60 mg prok treated virions, 15 mg of rnp complexes and 10 mg of cellular lysates were separated on tris-glycine 10% sds-page gels, transferred to pvdf membranes and rapidly stained with the reversible dye ponceau s prior to the use of antibodies to confirm viral protein loading and the quality of protein transfer from gel to membrane. membranes were blocked in tbs (0.5 m nacl, 20 mm tris ph 7.5) with 0.1% tween 20 and 5% non-fat milk powder and then probed with antibodies against cc2d1a (a300-285a; bethyl laboratories), yes1 (3201; cell signaling), itch (32/itch; bd transduction laboratories), hsc70 (k-19; santa cruz biotechnology), prl2 (05-1583; millipore), ck1 (2655; cell signaling), or ddx3y (pa5-22050; thermo scientific). detection was with species-specific horseradish peroxidase-conjugated secondary antibodies using the enhanced chemiluminescence plus (ecl+) protein detection system (ge healthcare) and captured using a chemidoc-it imaging system (uvp imaging, upland ca). recombinant wt vsv was grown in bhk-21 cells and purified using gradient centrifugation. the purity of the resulting material was then examined by electron microscopy (em). as seen in figure 1 , nearly all of the material present was clearly identifiable as vsv virions, although many of them were bent, a previously observed form that is generally believed to be infectious [57] and may represent an em processing artifact [58, 59] . the titer of these purified virions on bhk-21 cells was 1.4610 11 pfu/ml, demonstrating this preparation was highly infectious. a portion of these whole virions was used directly for proteomic analysis and additional confirmation assays, while the other portion was treated with prok and re-purified (fig. 2) . prok treatment cleaves all proteins on the exterior of the virions as well as the extracellular domain of all transmembrane proteins. however, it cannot penetrate the viral envelope, leaving all vsv proteins except for g intact, as well as all host proteins contained within the virion and the transmembrane and cytoplasmic domains of the host membrane proteins located in the viral envelope. this treatment also tends to alter the density of any remaining cell derived vesicles relative to the treated virions, facilitating their removal during the subsequent repurification step [60] . however, proteins on the interior of any residual cellular vesicles would still be detectable. for mass spectrometry (ms) analysis, total protein from 50 mg purified virions or 60 mg prok treated virions was separated by 1d-sds-page. these quantities were chosen so that the amount of viral n and p protein was approximately equal in both sample types as determined by coomassie staining (fig. 3a) to facilitate comparisons between samples. importantly, while n, p and l bands were similar in both virion preparations, no full length g protein was visible for prok treated virions, indicating the prok treatment was highly successful. after separation by sds-page, the resolved proteins were cut out in a series of bands as indicated in figure 3a . these bands were then subjected to in-gel trypsin digest and the resulting peptides were extracted, separated by uplc and analyzed by tandem ms (ms/ms). in this study, virions were grown on bhk-21 cells as it is the standard cell line for growth of vsv and the high virion yields aid in purification. however, a complete database of syrian hamster proteins is not available. instead, peptides were identified by matching them to either a human or a mouse database. the results from both searches were highly similar; therefore only those from the search of the mouse database are presented here. in addition to all five viral proteins, 257 different host proteins were identified in total (table s1) , with 181 proteins identified in whole virions and 183 proteins identified in prok treated virions (fig. 3b ). this is a considerably larger number of host protein than identified in our previous study where 30 proteins were identified in a different preparation of bhk-21 derived virions [45] . this is likely primarily due to dividing the samples into a larger number of bands following 1-d sds-page. in addition, in the present study we used enhanced sample separation through use of uplc, which should also allow for more sensitive detection. of the 30 proteins detected in bhk-21 derived whole virions in our previous study, 27 were detected in at least one sample type here, showing consistency in the proteins detected in independent virion preparations purified using two different methodologies (continuous iodixanol gradient versus discontinuous sucrose gradient). differences in the purity of the virions prepared using these two methods could also contribute to differences in the number of host proteins identified. however, em images, staining of total protein on sds-pages gels and infectivity (4.2610 7 pfu/mg total protein for the present method versus 2.4610 7 pfu/mg total protein for our previously published method [45] ) all indicate virions prepared using the method presented here are of equal or greater purity to those used previously. surprisingly, more than 40% of proteins found in prok treated virions were not found in whole virions even though the prok treated virions were derived from the whole virion preparation (fig. 2) . one possibility is that some of these proteins may have been masked by more abundant host or viral proteins that were removed by treatment. for example, 25% (19/76) of the proteins detected in prok treated virions but not whole virions were from the position correlating to that of the viral g protein in the whole virion sample (table s1 and fig. 3a) . another possible explanation is that these proteins are in low abundance or possess other properties making them difficult to detect consistently by ms. of the proteins found only in prok treated virions, 50% of proteins were identified based on detection of only two unique spectra (the minimum number allowed under our criteria), as opposed to 15% for proteins also identified in whole virions (table s1 ). unsurprisingly, ease of detection also seemed to be a major determinate of consistency of detection between technical replicates. proteins found in all technical replicates were identified based on only two unique spectra in 19% and 17% of cases for whole virions and prok treated virions respectively, while proteins found in only one technical replicate were identified based on only two spectra in 71% and 77% of cases (fig. 3c-d and table s1 ). in general, the position of the identified proteins on the gel was consistent with the predicted molecular mass of the proteins (table s1 and fig. 3a) although some appeared at higher molecular masses, likely due to post-translational modifications affecting protein mobility. exceptions to this general pattern were keratins, common environmental contaminants, which were found across a wide range of molecular weight and therefore excluded from this analysis. following prok treatment, 74 of the 181 proteins identified in whole virions were no longer detectable, likely due to their removal by the treatment. however, other proteins were still detected in the prok treated sample but appeared at a lower molecular mass versus the predicted molecular mass and/or that observed for whole virions, likely due to the removal of the extracellular domain. for example, hepatocyte growth factor receptor was found in a slice spanning approximately 140-225 kda in the whole virion while it was found in slices spanning approximately 50-80 kda in prok treated virions. this demonstrates the value of information about the approximate size of the proteins in the sample (as determined by the position of the gel band) in interpreting data from proteinase treated samples. although the primary focus of this study was determining host protein incorporation and localization in vsv virions, our search database also included vsv protein sequences allowing for their detection. in untreated samples, a mean of 1231-182 spectra per technical replicate were detected for each of the five vsv encoded proteins (fig. 4a) . the number of spectra went up upon prok treatment for all vsv proteins except g where there was a sharp reduction. this is consistent with our other data (fig. 3a) demonstrating that prok treatment is effectively removing the extracellular domain of the vsv g protein. removal of the extracellular domain was confirmed by determining the distribution of the spectra and peptides derived from the g protein (fig. 4b) . the mean number of spectra associated with the transmembrane domain and cytoplasmic tail of the g protein were unchanged between whole and prok treated virions, while prok treatment decreased the number of spectra associated with the extracellular domain of g. in looking at the distribution of the detected peptides in the g protein, prok treatment completely eliminated peptides associated with the n-terminal portion of the extracellular domain while a few were still detected in the more c-terminal portion of the domain, suggesting this region has a slight degree of resistance to proteinase cleavage, although the decrease in spectral counts suggests the majority the g proteins were cleaved. while ms analysis of unlabeled proteins cannot be used to make quantitative comparisons of protein abundance, spectral counts or measures derived from spectral counts (including empai values shown in table s1 ), can be used to make semi-quantitative comparisons [61] . based on spectral counts, few if any of the host proteins come close to matching the viral proteins in abundance within the virion. only one host protein, low-density lipoprotein receptor-related protein 1 (lrp1), had a mean of more than 100 total spectra per technical replicate in untreated whole virions, while only 17 (9.4%) had a mean of 20 or more (table s1 ). no proteins found only in prok treated virions had a mean spectral count of more than 20, indicating that these proteins are present in the virions in low abundance. as discussed in the previous section, proteins not associated with the interior of the virion, including proteins embedded in the host derived viral envelope, can be identified by their absence in prok treated samples or by a size shift upon prok treatment. here we wanted to examine the localization within the vsv virion of host proteins that did not display any of these patterns (i.e. proteins that do not appear to be associated with the viral envelope). five of the chosen proteins were found in both whole and prok treated virion samples and had similar spectral counts in both sample types (table s1 ). we also chose two proteins, proto-oncogene tyrosineprotein kinase yes (yes1) and protein tyrosine phosphatase type iva 2 (ptp4a2; also called prl2), detected in prok treated virions but not whole virions, as proteins found in the treated samples would also be expected to be found in the whole virions from which they were derived (fig. 2) . to identify proteins associated with vsv rnp complexes, we also isolated and analyzed viral rnp complexes from a separate preparation of purified vsv virions by treating them with detergent and salt to release the nucleocapsid from the envelope and m protein while maintaining its association with the l/p polymerase complex. because protein quantities were chosen so that the amount of n and p protein was approximately equal for all virion samples (fig. 5) , the host protein associated with whole virions that was retained in the treated samples could be estimated. based on this, proteins were determined to be associated with the viral rnp complex, associated primarily with the interior of the virion but not the rnp or associated primarily with the exterior of the virion. most proteins were barely detectable in the rnp complexes, suggesting they do not associate with the viral rnp or that the association was disrupted under the conditions used for rnp isolation. only for e3 ubiquitin-protein ligase itchy (itch) were substantial amounts of protein detected in rnp. the m protein of vsv, as well as other rhabdoviruses, contains a late domain (ldomain) including a proline rich ppxy motif known to bind ww domains found in a number of cellular proteins [62] . vsv m protein has been shown to bind the ww domain of nedd4 (also detected in this analysis) [63] , and can presumably also bind other hect domain-containing e3 ubiquitin ligases, including itch, as has been reported for other viruses with l-domains including ppxy motifs [64] . however, given that itch is present in rnp complexes at levels that nearly equal the other two sample types, while m protein is sharply reduced, it seems likely that itch may also interact with one of the rnp proteins (n, p or l). three of the tested proteins, yes1, casein kinase i isoform alpha (csnk1a1) and heat shock cognate 71 kda protein (hspa8; also known as hsc70), while barely detectable in rnp, were found at similar levels in whole and prok treated virions, indicated they are primarily associated with the interior of the virions. in contrast, levels of ptp4a2 and putative atp-dependent rna helicase pl10 (d1pas1; also known as ddx3y), dropped sharply in the prok and rnp samples indicating they are not primarily located in the interior of the virion. one protein, coiled-coil and c2 domain-containing protein 1a (cc2d1a), identified by ms could not be detected by wb, indicating either protein levels below the threshold of detection, a misidentification of the protein based on homology, or a false-positive identification. the latter seems the least likely as the protein was identified in both sample types, with at least 6 consecutive amino acids matched in manual inspection. of the 6 proteins that could be detected by western blot, only 4 were present at similar levels in whole and prok treated virions, despite similar spectral counts in both sample types and the absence of a size shift. therefore, while these characteristics may suggest a protein associated with the interior of the virion, localization should be independently confirmed. to obtain an overview of the types of proteins most commonly associated with purified vsv virions, an enrichment analysis was conducted using gene ontology terms. this analysis was done for all three go databases (biological process, cellular component and molecular function), first using all the host proteins found in our analysis then looking specifically at proteins associated with prok treated virions (fig. 6) , as this is the highest purity sample and excludes proteins found exclusively on the exterior of the virion. related enriched terms were then clustered together to give an overall enrichment score. the most highly enriched clusters, when looking at all the proteins, were vesicles and vesicle mediated transport, protein localization and nucleotide binding (fig. 6 ). these were also the most highly enriched clusters when looking specifically at proteins identified in prok treated virions. the cluster cell adhesion was also relatively highly enriched when looking at all proteins, but less so in prok treated virions as would be expected. enrichment of proteins involved in vesicles and vesicle mediated transport, protein localization and cytoskeletal organization is consistent with known features of vsv assembly and budding. more than one third (88/257) of the proteins identified are represented in at least one of these 4 clusters, indicating many virion-incorporated host proteins are likely involved in these functions. transport of the vsv nucleocapsid to the site of budding has been shown to be dependent on microtubules [65] and is an important step in vsv assembly. furthermore, as seen for many enveloped viruses (reviewed in [66] ), vsv budding appears to use the host proteins involved in multivesicular body (mvb) formation which are relocated from endosomal membranes to the plasma membrane in an m protein dependent manner (reviewed in [67] ). however, the budding of vsv and related viruses may still have some unique features. while the specific members of the mvb pathway utilized by vsv for its budding are not clear, it is known that, unlike what has been observed for many other viruses, tsg101 is not essential for the budding of vsv and rabies virus [68] , while contradictory reports exist regarding the importance of vps4a [68, 69] . in contrast, the host ubiquitinproteasome system does appear to be essential [63, 69] . the lipid composition of the vsv envelope is consistent with that of its host cell, although levels of cholesterol and sphingomyelin are elevated [48] , indicating that unlike some other viruses such as influenza virus and hiv-1 [70, 71, 72] , vsv does not bud through host membrane regions enriched in lipid rafts. instead, vsv g and m proteins appear to initially localize to separate microdomains of the host plasma membrane but then either merge or cluster together with each other and host protein containing microdomains at the site of virus budding [73] . therefore, vsv readily incorporates the membrane proteins of its host. in pseudotyping experiments using both mixed virus infections and expression of viral or host proteins from vsv recombinants, non-vsv proteins can make up a significant portion of the protein in the vsv envelope, with incorporation levels up to 31% of that seen for vsv g protein reported [74, 75] . consistent with these observations, 46% (118/257) of proteins identified in our study are associated with the cellular component go term ''plasma membrane'', suggesting they were acquired along with the envelope. the tendency of vsv to indiscriminately acquire relatively large numbers of host membrane proteins has the potential to be exploited to help fine tune therapeutic vectors through the choice of cell line used to generate the viruses. for example, growing oncolytic vsvs in a cell line naturally expressing or engineered to express high levels of complement control protein may improve efficacy by slowing the rate of clearance by the host immune system following administration. while many of the proteins identified in vsv virions appear to be associated with viral assembly, budding or the host-derived viral envelope, they may also have additional functions that affect virus replication. furthermore, proteins were also identified that do not figure 5 . confirmation of host protein incorporation in virion preparations. protein lysates from uninfected (bhk) and vsv infected (bhk+ vsv) bhk-21 cells were separated by sds-page along with total protein from whole virions, proteinase k (prok) treated virions, and viral ribonucleoprotein complexes (rnp). loading of the virion samples was confirmed by ponceau s staining of the membrane. the position of the viral glycoprotein (g), nucleocapsid protein (n), phosphoprotein (p), and matrix protein (m) is indicated. western blots using primary antibodies against coiled-coil and c2 domain-containing protein 1a (cc2d1a), protein tyrosine phosphatase type iva 2 (ptp4a2), putative atp-dependent rna helicase pl10 (d1pas1), proto-oncogene tyrosine-protein kinase yes (yes1), casein kinase i isoform alpha (csnk1a1), heat shock cognate 71 kda protein (hspa8) and e3 ubiquitin-protein ligase itchy (itch), were conducted as indicated to determine the presence of the host proteins. approximate molecular mass of the proteins is given on the left. doi:10.1371/journal.pone.0104688.g005 have known associations with these functions. our study provides a valuable inventory of virion-associated host proteins for further investigation into their potential roles in vsv replication cycle, pathogenesis, and immunoreactivity. table s1 cellular proteins identified in whole and prok treated vsv virions following 1-d sds-page and uplc-ms/ms. (xlsx) figure 6 . functional enrichment analysis. enrichment analysis and clustering based on gene ontology terms was conducted for (a) all proteins identified and (b) proteins identified in the proteinase k (prok) treated virions, as described in the materials in methods. the five clusters in each database with the highest enrichment score are depicted here. numbers above the bars indicate the number of identified proteins associated with each cluster. doi:10.1371/journal.pone.0104688.g006 nonsegmented negative-strand viruses as vaccine vectors recombinant rhabdoviruses: vectors for vaccine development and gene therapy vesicular stomatitis virus as a flexible platform for oncolytic virotherapy against cancer vesicular stomatitis virus as an oncolytic vector rhabdoviridae. in: in: knipe dm and plunder and stowaways: incorporation of cellular proteins by enveloped viruses analysis of virion-incorporated host proteins required for herpes simplex virus type 1 infection through a rna 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raft-derived microdomains: a quantitative analysis of the surface distribution of ha, na and m2 proteins evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid-enriched membrane lipid rafts retroviruses human immunodeficiency virus and murine leukemia virus are enriched in phosphoinositides plasma membrane microdomains containing vesicular stomatitis virus m protein are separate from microdomains containing g protein and nucleocapsids foreign glycoproteins expressed from recombinant vesicular stomatitis viruses are incorporated efficiently into virus particles role of heterologous and homologous glycoproteins in phenotypic mixing between sendai virus and vesicular stomatitis virus the authors thank dr. sue moyer (university of florida college of medicine) for providing vsv reagents for this project and eric hastie for critical comments on the manuscript. key: cord-340027-6l55rcfm authors: mamode khan, naushad; soobhug, ashwinee devi; heenaye-mamode khan, maleika title: studying the trend of the novel coronavirus series in mauritius and its implications date: 2020-07-10 journal: plos one doi: 10.1371/journal.pone.0235730 sha: doc_id: 340027 cord_uid: 6l55rcfm mauritius stands as one of the few countries in the world to have controlled the current pandemic, the novel coronavirus 2019 (covid-19) to a significant extent in a relatively short lapse of time. owing to uncertainties and crisis amid the pandemic, as an emergency announcement, the world health organization (who) solicits the help of health authorities, especially, researchers to conduct in-depth research on the evolution and treatment of covid-19. this paper proposes an integer-valued time series model to analyze the series of covid-19 cases in mauritius wherein the corresponding innovation term accommodates for covariate specification. in this set-up, sanitary curfew followed by sanitization and sensitization campaigns, time factor and safe shopping guidelines have been tested as the most significant variables, unlike climatic conditions. the over-dispersion estimates and the serial auto-correlation parameter are also statistically significant. this study also confirms the presence of some unobservable effects like the pathological genesis of the novel coronavirus and environmental factors which contribute to rapid propagation of the zoonotic virus in the community. based on the proposed com-poisson mixture models, we could predict the number of covid-19 cases in mauritius. the forecasting results provide satisfactory mean squared errors. such findings will subsequently encourage the policymakers to implement strict precautionary measures in terms of constant upgrading of the current health care and wellness system and re-enforcement of sanitary obligations. four months since the start of the covid-19 outbreak, most affected states have imposed strict confinement measures, largely disrupting the daily sequence of works in various spheres. most people are working from home with limited resources, online teaching instead of traditional classroom teaching are being conducted and strict sanitization measures in public places, especially in super, and hypermarkets have been imposed. overall, the world is in an unexpected turmoil. however, there is still a ray of hope, especially, when developing sub a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 saharan african countries with limited economic resources, like mauritius and seychelles, are combating the pandemic with full vigor. setting the focus on mauritius, this is one of the countries whose economy has made great strides and as of the end of april 2020, the health authorities officially reported that more than 90 percent of covid-19 patients have recovered (312 recovered cases out of 332 confirmed cases, with 7 active cases). indeed the situation was alarming when the first cases of covid-19 was reported on the 18th march 2020 and within four weeks, the total number of confirmed cases climbed to 325, an exponential increase, and the total number of deaths was hovering at 9. considering the severity of the situation, policy makers announced a six-week national complete lockdown, effective from the 20th march 2020, high-tech medical diagnostic apparatus, key protective hygiene and sanitation equipment and life saving medical drugs like hydroxychloroquine tablets, worth a colossal amount, were purchased and even obtained free of charge from sister countries like india, to upgrade the quality of health services and fight the pandemic. thorough covid-19 screening is being conducted on a daily basis. within one month, by the end of april 2020, around 17,196 polymerase chain reaction (pcr) tests in addition to 19 ,393 rapid swab tests have been conducted among "frontliners" who are hospital health care workers and essential service workers, representing around 3 per cent of the whole population. this measure is indeed applaudable as by early detection of the virus, some patients can be cured quickly. as pro-active measures, mauritius is better equipped with an average of 3.4 hospital beds per 1000 population, as compared to developed countries like the united kingdom (uk) and other sub saharan countries [1] and with the collaboration with private stakeholders, an adequate number of quarantine centers have been instituted. all these measures, along with the positive changes in the population behaviour in regard to confinement, have altogether helped to contain the pandemic in mauritius. the measures adopted by mauritius have highly been praised by international organizations such as world health organization (who) and united nations (un). as a retrospective, it is important to retrieve, via inferential statistical methods, the factors that have contributed significantly in controlling the propagation of the novel coronavirus in mauritius. besides, since the covid-19 data is in the form of an integer-valued time series [2] layout, it is of research importance to develop an integer-valued regression time series process that can provide insights on the significance of explanatory variables as well set a platform to forecast the possible number of covid-19 infected cases which is quite tedious as illustrated in [3, 4, 5] . admittedly, research findings in [6] indicate that the covid-19 data exhibits lots of dynamic and latent features and hence, to cater for such volatilities, we introduce random effects in our model specification. in this context, we propose gamma [7] and normal [8] random effects that, in addition to the covariate specifications, would render the model estimators less biased and improve the forecasts. in the same vein, such forecasts will aid policymakers in articulating post pandemic measures, since mauritius, which is highly dependent on the hospitality sector, is expected to face a drastic contraction in its economic growth. the research findings in this paper will evidently guide policy makers and add value to the limited research undertaken so far in the case of covid-19 in mauritius. it is important to understand the evolution of covid-19 in mauritius otherwise as an import-dependent country, mauritius may find itself engulfed in the darkness of covid-19 in terms of loss in economic wealth and deterioration in population health state. the paper therefore proposes an integer-valued auto-regressive model (inar(1)) with conway-maxwell poisson (com-poisson) mixed innovation terms that can accommodate for covariate, random effect and serial auto-correlation specifications. the organization of the paper is as follows: in section 2, material in terms of time series of covid-19 in mauritius and proposed methods principally the inar(1) process and the com-poisson mixture innovations have been provided. estimation of parameters are in section 2 as well. in section 3, the results have been interpreted followed by some discussions in section 4. finally, section 5 concludes the study. the local data on covid-19 is publicly available in european union open data portal at s1. as aforementioned, mauritius registered its first three cases on the 18th march 2020 and as at 11th april 2020 or after 23 days, the health authorities of mauritius officially reported 318 active cases of covid-19 and 7 deaths. this signifies an overall average of 13.8 covid-19 cases per day with a variance of 102.2. detailed statistics for the 23 days are given at s2. currently, three important peaks have been noted on day 8 (25th march 2020), day 18 (5th april 2020) and day 22 (10th april 2020) where 33, 31 and 41 patients were tested positive for covid-19 respectively. based on these statistics, it is obvious that the data are exhibiting severe over-dispersion. this is confirmed by the qcc.overdispersion.test in r statistical software via the qcc package that yields a chi-squared p-value of 8.4477e − 13, showing a high level of significance. moreover, the rcorr and the adftest illustrate that the data exhibit some serial correlation and are non-stationary. this indicates that the number of infected cases in mauritius shows some fluctuations as well. the varselect routine in the library vars showed that the akaike information criterion (aic) yielded a lag order of 1, and hence we can conclude that the model proposed in section 3 is suitable to model the above series. let y t indicates the number of infected or covid-19 positive case at the t th time point, t = 1, 2, 3, . . ., t and e y t ¼ y t jg, where γ describes the aforementioned latent effect. in the subsequent parts of this section, we show the different probability models for γ. thus, the vector of time series conditional count observations is represented by e y ¼ ½ e y 1 ; e y 2 ; . . . ; e y t ; . . . ; e y t � 0 . it is important to assume that the observations e y t and g y ðtþhþ are likely to be serially correlated, where the lag h 2 z þ ; 1 � h � t à 1. due to the latent effect γ, the covariance between the unconditional observation y t and y t + h is computed as: we further assume that the observation y t is subject to a p × 1 vector of covariates, denoted by x t ¼ ½x t;1 ; x t;2 ; x t;3 ; . . . ; x t;k ; x t;ðkþ1þ ; . . . ; x t;p � 0 that comprises of both time-variant and timeinvariant exogeneous variates. the regression vector β is denoted by β = [β 1 , β 2 , . . ., β k , β k+1 , . . ., β p ]. as stated earlier, we assume that the conditional observation e y t follows the inar(1) process that satisfies the first-order auto-regressive equation [9] e where r t is the random innovation component and ρ 2 (0, 1) independent of the observation e y t , covðg y tà k ; e r t þ ¼ 0, for k 2 z þ and � indicates the binomial thinning operator where r � g y tà 1 jg y tà 1 � binomialðg y tà 1 ; rþ [9] . hence, the above auto-regressive equation can be re-written in terms of the error components r t [10] as: and hence the moments of e y t are derived from the distributional properties of e r t and using the binomial thinning properties. we now introduce the com-poisson mixture model, wherein the innovation term e r t in eqn(1) follows the com-poisson mixture as: . hence, the com-poisson generalizes some important discrete models and for some substitutions, the com-poisson acts a bridge between the binomial, geometric and negative binomial. however, in practice, the exact evaluation of zð e l t ; nþ is nearly impossible for a general ν > 0. the following approximation was suggested in [11] zð e l t ; nþ ¼ expðn and this proves to yield satisfactory results especially for very large λ, as illustrated in [12, 13] . in fact, under this approximation, the moments of e r t can be expressed suitably in closed forms as follows: n ; whilst still, the above approximations satisfy the over-(under) dispersion assumptions for ν < (>)1. in fact, e l t ¼ expðx 0 t b þ gþ. in the same vein, an alternative re-parameterization of the com-poisson was proposed in [14] , by allowing e m t ¼ expðx 0 t b þ gþ. as for the model specification for γ, we let w = w(κ 2 ) = exp(γ) follows the exponential of the respective probability function, assuming κ 2 = σ 2 + ν. the moments of the unconditional response, y t are given as below and the detailed proof is in s1 appendix: it is clear, from the above marginal moments that the ratio vðy t þ eðy t þ > 1 and hence the above model is suitable to model over-dispersion, in particular, severe over-dispersion. we assume the following single-parameter models-gamma and normal distributions for the random effect w = exp(γ), given as: with where κ 2 = σ 2 + ν. by assuming e y t ¼ r � g y tà 1 þ e r t , the conditional log-likelihood function for the set of observations (y 1 , y 2 , . . ., y t , . . ., y t ) is written as: where f ðe y t j g y ðtà 1þ ; gþ ¼ x min ðe y t ;g y ðtà 1þ þ s¼0 g y ðtà 1þ s � � r s ð1 à rþ g y ðtà 1þ à s � f e r t ðe y t à sþ ð6þ from the above, the evaluation of the integrand in eq (5) is performed using the numerical quadrature techniques using the integral, fastghquad packages in r and likelihood is thereon optimized using the optim function. further to the model construction and estimation procedures, in this section, some time-independent and time-variant covariates that could explain the fluctuations in the number of confirmed covid-19 active cases in mauritius have been considered. the following covariates are measured as from 18th march 2020 until 11th april 2020. many songs of hope in local and international languages, with the aim to communicate the seriousness of the situation and encourage the population to stay home, have been playing on social media. slogans like "stayhomestaysafe" and "besafe-moris" have been trending on social media to draw attention on the importance of confinement. in some strategic areas, the police force even addressed the public on important confinement-related matters using loudspeakers. the results illustrate that the climatic conditions do not contribute quite significantly to the evolution of the covid-19 in mauritius, while the remaining variables do impact significantly on the number of covid-19 cases. it can be noticed that the sanitary curfew/lockdown, sanitization and sensitization campaigns and safe shopping guidelines have helped to curb down, by a large extent, the number of covid-19 cases in mauritius. hence, such preventive and pro-active measures, with the main focus on sanitization measures at grocery stores and in busy public places, should be maintained even after lifting lockdown orders. we can also note that the number of contravention fines and traffic offences tackled by the officers of the mauritius police force have also helped in containing the virus as due to road blockages, the mobility of people are minimised leading to no public gatherings. the treatment variable confirms availability of adequate hydroxychloroquine tablets to treat the number of covid-19 cases in mauritius. the number of quarantine centers have significantly helped in timely containment of the novel coronavirus, eliminating to a great extent the risk of spreading in the local community. as per the protocols set by the ministry of health and wellness, as soon as people were repatriated to mauritius, they were immediately self-isolated. based on the numerical evidence, the age composition, in the local context, shows that people below 60 years of age has been more affected by covid-19. the value of σ 2 confirms the presence of some unobservable effects that are possibly contributing to the propagation of the virus. this could be linked to research findings in [17, 18] which showed that the sars-cov-2 is an aerosolized virus, particularly found in patients' bathrooms, whilst others concluded that the small airborne particles could remain suspended in air and even flow up to six feet with the air current [19] . the results in table 1 were obtained assuming the training dataset from 18th march 2020 to 11th april 2020. we use the above regression estimates or weights to forecast the number of infected covid cases in mauritius from 12th april 2020 to 23rd april 2020. from table 2 , the mean squared error (mse) based on the proposed com-poisson gamma mixture is computed as 3.8598 with relatively lower corresponding aic, which can be viewed as satisfactorily adequate. in fact, the model slightly overstates the number of cases which is surely more beneficial and prudent to the policymakers. local authorities, through provision of sufficient ppes to "frontliners", re-enforcement of the sanitary obligations and by upgrading the sanitization campaigns, can help to contain the virus more rapidly. also, using these forecasts, the local health authorities can make reliable action plans, especially, regarding bed capacity in each covid-19 hospitals in case of sudden increase in the number of covid-19 cases in the future. note that a similar prediction exercise was reported in italy [20, 21] using an alternative forecasting method, but since not all its regions were under confinement and, most importantly, due to delay in policy making, the scenario went uncontrollable. in line with confinement measure, new feasible approaches including broadcasting of educational content on television for primary and secondary grade levels, work from home schemes, setting up of online purchasing platforms, provision of urgent court services via technological communication and a more safe and hygienic lifestyle have been adopted to limit socio-economical shocks. however, many individuals, especially working people and children, are exposed to this unprecedented stressful situation of unknown duration. it is proven that changes in work schedule and requirements and home schooling affect the psychological behaviours of most individuals [22, 23] . here, right parenting skills and sustainable programmes by local stakeholders become crucial. for the tertiary sector, academics used intensively interactive online teaching platforms for virtual lecturing, as well as open source learning platforms for assignment practices. for adequate food supply and a reasonable price mechanism on the local market, a controlled mark-up of 20 percent on some basic commodities and restrictions on quantity of basic groceries purchased, have been implemented. local crop production is being encouraged through provision of diverse rebate schemes and reduction in the price of vegetable seeds. vulnerable families, where the head of the household are self-employed, or are informal sector workers or lead a small and medium enterprise, are financially being assisted based on the new social protection schemes. the aforementioned economic activities have recently faced a drastic fall in performance due to the prolonged movement restrictions. it is compulsory for any employee to be in possession of a work access permit, when travelling to work. misuse of the permit will lead to serious legal convictions. recently, the covid-19 (miscellaneous provisions) bill and the quarantine bill were implemented with foremost aim to drive the mauritian economy and to prevent any resurgence of the disease. through setting up of a comprehensive set of strict sanitary measures, improvement in surveillance control and in the level of preparedness in the health care system, mauritius remain on alert to stave off a second wave of sars-cov-2 in the country. the covid-19 (miscellaneous provisions) bill brought in amendments to safeguard the fundamental rights of mauritians and most importantly the public health. in this context, it is believed that the findings from this study will act as a yardstick for policy makers when planning for preparedness measures and response strategies. every stakeholders, be it the individuals, the governmental or non-governmental organisations and the principal actors of the private sector, should bring in their collective capacities to work towards a "covid-19-free" mauritius. as of now, physical distancing is the new "norm" of the society. the results of this research revealed that several factors, the most significant being confinement measure, and least one being climatic conditions, affect the number of covid-19 cases in mauritius. it is worth mentioning that the proposed model adapts to the local data since the number of covid-19 cases in mauritius is mostly low integer-valued, hence, its usage is not generic as it depends on the nature and pattern of the covid-19 data. besides, some countries like, seychelles, faces a zero-inflated pattern which implies the present model needs to be modified to accommodate for zero-inflated cases. however, as for mauritius, this shall guide the policy makers to implement stricter preventive measures associated with legal obligations and economical tools to curb the pandemic on time and capitalise the mauritian economy. the state of mauritius should ensure that other jurisdictions like rodrigues, agalega and chagos archipelago, which lack high quality health care services are not affected by covid-19. research on covid-19 should be ongoing due to pathological complexities of the novel coronavirus and unexplored area of research like seasonal effect on the spread of the virus [24] . mauritius heads into coronavirus storm with strong social welfare buffers. the conversation autoregressive moving-average processes with negative binomial and geometric marginal distrbutions composite monte carlo decision making under high uncertainty of novel coronavirus epidemic using hybridized deep learning and fuzzy rule induction finding an accurate early forecasting model from small dataset: a case of 2019-ncov novel coronavirus outbreak forecasting models for coronavirus disease (covid-19): a survey of the state-of-the-art investigating the cases of novel coronavirus disease (covid-19) in china using dynamic statistical techniques on familial longitudinal poisson mixed models with gamma random effects random effects models for longitudinal data discrete operator self-decomposability and queing networks. stochastic models first-order integer-valued autoregressive process a useful distribution for fitting discrete data the com-poisson model for count data: a survey of methods and applications approximating the conway-maxwell-poisson distribution normalizing constant reparametrization of com-poisson regression models with applications in the analysis of experimental data. statistical modelling eco-epidemiological assessment of the covid-19 epidemic in china temperature, humidity, and latitude analysis to predict potential spread and seasonality for covid-19. ssrn preprint the coronavirus pandemic and aerosols: does covid-19 transmit via expiratory particles aerodynamic analysis of sars-cov-2 in two wuhan hospitals consideration of the aerosol transmission for covid-19 and public health modelling and predicting the spatio-temporal spread of coronavirus disease 2019 (covid-19) in italy. statistical modelling covid-19 and italy: what next dealing with sleep problems during home confinement due to the covid-19 outbreak: practical recommendations from a task force of the european cbt-i academy mitigate the effects of home confinement on children during the covid-19 outbreak seasonality and immunity to laboratory-confirmed seasonal coronaviruses (hcov-nl63, hcov-oc43, and hcov-229e): results from the flu watch cohort study special thanks to ministry of health and wellness in mauritius and to world health organization (who) for disseminating covid-19 related information on a daily basis. conceptualization: maleika heenaye-mamode khan. key: cord-322446-ddv86eoy authors: sharma, kulbhushan; åkerström, sara; sharma, anuj kumar; chow, vincent t. k.; teow, shumein; abrenica, bernard; booth, stephanie a.; booth, timothy f.; mirazimi, ali; lal, sunil k. title: sars-cov 9b protein diffuses into nucleus, undergoes active crm1 mediated nucleocytoplasmic export and triggers apoptosis when retained in the nucleus date: 2011-05-27 journal: plos one doi: 10.1371/journal.pone.0019436 sha: doc_id: 322446 cord_uid: ddv86eoy background: 9b is an accessory protein of the sars-cov. it is a small protein of 98 amino acids and its structure has been solved recently. 9b is known to localize in the extra-nuclear region and has been postulated to possess a nuclear export signal (nes), however the role of nes in 9b functioning is not well understood. principal findings/methodology: in this report, we demonstrate that 9b in the absence of any nuclear localization signal (nls) enters the nucleus by passive transport. using various cell cycle inhibitors, we have shown that the nuclear entry of 9b is independent of the cell cycle. further, we found that 9b interacts with the cellular protein crm1 and gets exported out of the nucleus using an active nes. we have also revealed that this nes activity influences the half-life of 9b and affects host cell death. we found that an export signal deficient sars-cov 9b protein induces apoptosis in transiently transfected cells and showed elevated caspase-3 activity. conclusion/significance: here, we showed that nuclear shuttling of 9b and its interaction with crm1 are essential for the proper degradation of 9b and blocking the nuclear export of this protein induces apoptosis. this phenomenon may be critical in providing a novel role to the 9b accessory protein of sars-cov. severe acute respiratory syndrome (sars) was a new respiratory illness that emerged in china in 2003 and spread globally [1, 2] . the causative agent was identified as a new coronavirus and was named sars coronavirus (sars-cov) [3] [4] [5] . the sars-cov genome consists of approximately 29,700 nucleotides encoding 28 putative proteins [6, 7] . just like other coronaviruses, the sars-cov genome also contains several small open reading frames (orfs) in addition to those encoding for structural proteins [6] [7] [8] [9] . these small orfs are presumed to encode 8 group specific, accessory proteins viz. orf3a, 3b, 6, 7a, 7b, 8a, 8b and 9b [8] . one of these accessory proteins, the 9b protein is encoded by orf-9b of the sars-cov genome. just like the internal (i) gene of other group ii coronaviruses, the orf-9b of sars-cov overlaps with its nucleocapsid orf [8, [10] [11] [12] . however, there is no homology between the sars-cov 9b and i protein of other coronaviruses. the 9b protein has been shown to get expressed in sars-cov-infected cells and antibodies against it have been found in the sera of sars infected patients, demonstrating that the protein is produced during infection [13] [14] [15] [16] , but its actual function is not yet determined. studies on 9b-structure by meier et al., (2006) revealed a 2-fold symmetric dimer having a lipid binding cavity and proposed its role in virus assembly [17] . cellular localization of 9b has been previously reported to be predominantly cytoplasmic and membranous. also, a nuclear export signal (nes) present in its 46-lrlgsqlsl-54 amino acid region has been suggested to be responsible for its nucleocytoplasmic export [18] . keeping this in mind, we studied the cellular localization pattern of 9b and found that in addition to the cytoplasm, some of the 9b protein was also present in the nucleus. this entry of 9b into the nucleus was independent of cell cycle progression. further, we showed that 9b which lacks the nuclear localization signal (nls) continued to enter the nucleus passively and was able to exit the nucleus due to its functional nes. also, nuclear export was found to be crm-1 dependent and blocking nes based export resulted in an increased half-life of 9b, which accumulated in the nucleus. finally, our studies revealed that when 9b remained within the nucleus, it triggered caspase 3 mediated apoptosis in transiently transfected mammalian cells. the requirement for caspase 3 in apoptosis induction was further confirmed using the cell permeant caspase inhibitors, z-vad-fmk (general caspase inhibitor) and z-devd-fmk (caspase 3 inhibitor). to the best of our knowledge, this is the first report showing the nuclear localization of 9b, its passive diffusion into and active crm-1 dependent transport out of the nucleus. also, this is the first report associating 9b with nucleocytoplasmic export linked apoptosis. the sars-cov (genbank accession number nc_004718) 9b gene was pcr amplified and cloned into pcdna3.1/v5-his topo vector (invitrogen) using gene specific primers; f1 (59 gtaatggaccccaatcaaaccaac 39) and r1 (59 tttt-gccgtcaccaccacgaa 39) . in order to clone 9b into peyfpn1, it was pcr amplified using f2 (59 cgggaattcc-tgatggaccccaatcaaacc 39) and r2 primers (59 gta-tggatcccgttttgccgtcaccaccac 39) . a mutant of 9b was made using commercially available, pcr based site directed mutagenesis services (banglore genei, banglore) where all leucine present in the nes region of 9b were replaced by alanine (mu-nes-9begfpn1: 46-lrlgsqlsl-54 to 46-aragsqasa-54) and the mutant was further cloned into egfpn1 vector (clontech). creatine phosphate, creatine phosphokinase, leptomycin b (lmb), digitonin and cell-cycle inhibitors were purchased from sigma. wheat germ agglutinin (wga) was purchased from invitrogen. caspase inhibitors were purchased from bd pharmingen. cleaved caspase-3 antibody was purchased from cell signaling. the sars-cov 9b specific antibody, alexa fluorh488 goat-anti-rabbit igg and crm 1 antibodies were purchased from abgent, invitrogen and abcam, respectively. vero cells were maintained in dmem supplemented with penicillin, streptomycin and 10% fbs. approximately 0.6 million cells were plated in a 60 mm dish. the total amount of transfected dna was 3 mg per 60 mm dish. the plasmid was mixed with lipofectamine 2000 (invitrogen) in serum-free dmem media (invitrogen) and the cells were transfected as per manufacturer's protocol. for metabolic labeling, 36 hrs post-transfection, cells were starved for 1 h in cysteine/methionine-deficient medium (invitrogen), and were then labeled with 100 mci of [ 35 s] cysteine/ [ 35 s] methionine promix for a specific time period. after labeling, cells were washed once in pbs and lysed in radioimmunoprecipitation assay (ripa) buffer (150 mm nacl, 1% np40, 0.5% deoxycholate, 0.1% sds and 50 mm tris/hcl, ph 8.0 with protease inhibitor cocktail). all transfection experiments were performed in triplicates for at least three times. standard deviation was calculated wherever needed. vero e6 (ve6) cells (atcc, global bioresource center) were maintained with dulbecco's modified eagle medium (dmem) supplemented with 10% fetal bovine serum, streptomycin, penicillin and l-glutamine at 37uc and 5% co 2 . chamber-culture slides (bd falcon) were seeded with vero e6 cells to form confluent monolayers. chambers were inoculated with sars-cov (tor-3 strain) at an moi of 5 tcid50 per cell. infected cells were fixed in 2% paraformaldehyde in pbs, ph = 7, for 15 minutes at room temperature, at 12, 18, 24, 36 and 48 hours post infection. prior to use, culture slides were rinsed twice in pbs and subjected to a 10 min permeabilization step with pbs containing 0.25% triton x-100. after washing 365 min in pbs and blocking with 1% (v/v) bsa in pbs containing 0.01% tween-20 (pbst) for 1 hour at rt, slides were incubated overnight at 4uc with 1:100 dilution of primary antibody in 1% bsa pbst (sars virus pup6 polyclonal, n-terminal) (abgent). slides were rinsed 365 min in pbs, and then incubated with secondary antibody, alexa fluorh488 goat-anti-rabbit igg (invitrogen) for 1 h at rt in 1% bsa pbst. after additional rinsing 365 min in pbs, slides were air dried and mounted in 49,6-diamidino-2-phenylindole (dapi) antifade reagent (jackson immunoresearch), mounted with coverslips, and stored in darkness at 4uc until examination. slides were imaged using a zeiss lsm700 laser scanning microscope with zen2009 version 5.5 software. three-dimensional volume projections were rendered in ''volume viewer'' using ''imagej'' version 1.43 g. controls included mock-infected cells. the 9b transfected cells were fixed 36 hrs post-transfection in 2% paraformaldehyde (in pbs, ph = 7) for 15 min at room temperature. paraformaldehyde was removed and cells were washed once with 1xpbs and then rehydrated in 1xpbs for 20 min. next, the cells were mounted using antifade reagent having dapi (invitrogen). for localization studies, cells were imaged using lsm 510 confocal microscope (carl-zeiss, ag, germany) fitted with axiovert 200 m inverted microscope (carl-zeiss). images were captured with either 63x oil objective or 40x objective using the 488 nm for gfp and 514 nm for yfp. filter used at the detector channel for the respective proteins were bp 505-530 nm for gfp, and lp 530 nm for yfp. for (dapi) stained nuclei, 405 laser line was used for excitation and signals were captured using lp 420 detector filter. the captured images were processed using both 'image j' as well as 'lsm image browser' software. approximately 200 cells were counted from each sample and protein localization was scored. data represents analysis from 3 independent sets of experiments. vero cells were synchronized for 18 hrs by serum starvation. further, these cells were transfected in duplicates with the plasmid of interest and were kept in complete media for 12 hrs. the cells were treated for 16 hrs with either nocodazole or aphidicolin (1 mg/ml and 1.5 mg/ml, respectively) and were placed in complete media for the next 24 hrs. subsequently, one set of drug treated cells were processed for confocal microscopy and another set were used for flow cytometry experiments. for leptomycin b (lmb) treatment, cells were synchronized and were treated with 25 nm drug for 16 hrs and further incubated in complete media for another 24 hrs. for cyclohexamide treatment, transfected cells were treated with 15 mg/ml of the drug 34 hrs post-transfection. two hours post-treatment, cells were processed for microscopy. z-fafmk, z-vad-fmk and z-devd-fmk were dissolved in dmso to give a final concentration of 50 mm and stored at -80uc until use. immediately prior to addition to cells, the stock solutions were diluted into the culture medium. for propidium iodide (pi) staining, cell pellets from vero cells were fixed in 70% ethanol at 4uc for 45 min. after being washed twice with ice-cold pbs, the cell pellet was resuspended in 500 ml pisolution in pbs (40 mg/ml pi from 50x stock solution (2.5 mg/ml), 0.1 mg/ml rnase a and 0.05% triton x-100) and incubated at 37uc for 30 min. for each sample, about 10,000 events were acquired using a cyan-adp flow cytometer (dako, glostrup, denmark) at 488 nm excitation and the results were analyzed using 'summit (version 4.3)' and 'flowjo (version 6.4)' software. all values were expressed as mean 6 sd and a paired t-test was performed. fluorescent protein for in-vitro transport assay vero cells were transfected with the appropriate plasmid and were processed 36 hrs post-transfection. the cells were washed at least two times with cold phosphate buffer saline (pbs), ph 7.4, by resuspension and centrifugation, 5000 rpm, 4uc. the cells were then washed with 10 mm hepes, ph 7.3, 110 mm potassium acetate, 2 mm magnesium acetate, 2 mm dtt and then pelleted. the cell pellet was lysed in 1.5 vol of lysis buffer (5 mm hepes, ph 7.3, 10 mm potassium acetate, 2 mm magnesium acetate, 2 mm dtt, 1 mm pmsf, and 1 mm na 3 vo 4 with a protease inhibitor cocktail) (amersham biosciences, nj, usa) and was processed for in-vitro transport assay as described by adam et al., 1990 [19] . in-vitro transport assay was performed as explained by adam et al., 1990 with minor modifications [19] . briefly, vero cells were grown on coverslips and were permeabilized by immersion in ice cold transport buffer containing 40 mg/ml digitonin for 5 min. after 5 min, cells were washed and kept in cold transport buffer. the coverslips were then blotted to remove excess buffer and inverted over a drop of complete transport mixture on a sheet of parafilm in a humidified box. the complete transport mixture contained 50-75% fluorescent protein (freshly made as explained above) diluted with transport buffer to give the following final conditions: approx. 25-35 mg/ml fluorescent-tagged fusion protein + transport buffer (20 mm hepes, ph 7.3, 110 mm potassium acetate, 5 mm sodium acetate, 2 mm dtt, 1.0 mm egta, 1 mm atp, 5 mm creatine phosphate (sigma), 20 u/ml creatine phosphokinase (sigma), and protease inhibitor cocktail (amersham biosciences, nj, usa)). the fluorescent protein was prepared as explained above and the entire box was then floated in a water bath at 30uc. as a positive control, we have used the np protein of h5n1 which is known to have two nls [20] . the 3a protein of sars-cov has been used as a negative control as it has been shown to localize in the extranuclear region [21] . at the end of the assay, each coverslip was rinsed and mounted in a small amount of transport buffer (without dapi) and was observed using the confocal microscope. coverslips containing transfected vero cells were incubated with transport buffer containing 50 mg/ml wheat germ agglutinin (wga) for 15 min at room temperature. the coverslips were then blotted to remove excess buffer, inverted on a drop of complete transport mix and processed for in-vitro transport assay. at the end of the assay, each coverslip was rinsed and mounted in a small amount of transport buffer (without dapi). samples were observed by confocal microscope (carl-zeiss) and processed using 'image j' software. vero cells were resuspended in 400 ml buffer a (10 mm hepes ph 7.9, 10 mm kcl, 1 mm edta, 1 mm egta, 1 mm dtt, 1 mm pmsf and protease inhibitor mix). further, the mixture was placed on ice for 15 min. after 15 min, 25 ml of 10% np-40 was added to the cells and vortexed for 10 seconds. cells were centrifuged at 1000 rpm for 10 min at 4uc. the supernatant was labeled as cytoplasmic fraction. the pellet was resuspended in 200 ml of buffer c (10 mm hepes ph 7.9, 400 mm nacl, 1 mm edta, 1 mm egta, 1 mm dtt, 1 mm pmsf and protease inhibitor mix). the samples were kept on ice for 20 min. subsequently, the samples were centrifuged, 14000 rpm, 10 min, at 4uc and the supernatant was labeled as nuclear fraction. immunoprecipitation was performed as described previously [22] . for sars-cov infected cells, nuclear and cytoplasmic fractions were prepared using cellytictm nucle-artm extraction kit (sigma, mi, usa) according to manufacturer's protocol. vero e6 cells infected by sars-cov (frankfurt i strain) at moi = 1, were treated with or without 50 nm lmb for 24 or 48 hrs in 6 well plates. western blot was then performed on nuclear and cytoplasmic fractions. asynchronously growing vero cells were transfected with appropriate constructs and incubated for 36 hrs. subsequently, transfected or untransfected cells were pulse-labeled for 30 minutes and chased for 0, 30 and 60 minute time-points. whole cell lysate was then prepared by cell lysis as described above in fluorescent protein preparation method. immunoprecipitation was performed using a polyclonal anti-9b antibody and band intensities were quantified by image j software. cell death was detected using cell-death-detection tunel assay kit (roche biochemicals), according to the manufacturer's instructions. apoptotic cells were quantified by counting tunel-positive cells in each set of experiments. at least 200 cells from three separate images were inspected, and percentages were calculated. cell extracts from transfected cells were assayed for cleaved caspase-3 by western blotting. briefly, cell lysates were analyzed on sds-page, transferred onto nitrocellulose membrane and a western was performed using cleaved caspase-3, caspase-7 and caspase-9 specific antibodies separately (cell signaling, usa). band intensities were quantified using image j software. vero e6 cells were infected with sars cov (moi = 1) for 1 hr, then washed and treated with or without 50 nm lmb for 24 or 48 hrs. cell viability was then determined using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) colorometric detection assay from cell biolabscytoselect tm cell viability and cytotoxic assay kit (cell biolabs, inc) according to manufacturer's protocol. experiments were performed in triplicates and were repeated at least three times. statistical analysis was done using the graphpad software. unpaired t-test was performed and two-tailed p-value was calculated. statistical significance of variations was considered. cells infected with sars-cov showed faint cytoplasmic staining with antibodies directed at the 9b protein at 12 h post-infection (fig. 1a) . by 18 h post-infection, many discrete cytoplasmic foci were observed, and by 24 h post-infection, small foci of nuclear staining appeared, while control uninfected cells showed no staining (fig. 1b) . typical infected cells showed numerous nuclear foci of 9b protein by 36 h post-infection, as well as extensive, larger areas of cytoplasmic inclusions ( fig. 1a and 1b) . by 48 h post-infection, many nuclei were seen to be changing their appearance and undergoing apoptosis, and majority of the cells at this time point contained fewer foci of the 9b protein (fig. 1b, 48 h time point, second panel). stacks of confocal images were used to generate three-dimensional projections, with merged data from the alexa fluor and dapi channels (fig. 1b) . when viewed from the side and from the top, as well as sections through the volume projections, it was observed that the antibody staining (in green) was within the nucleus as well as the cytoplasm. these foci of fluorescence first appeared in the nucleus at 24 h post infection to reach a maximum at about 36 hr. p.i., and are somewhat reduced in numbers at 48 h p.i. (fig. 1b) . after the observation that some proportion of the 9b protein enters into the nucleus, we planned to check its localization in 9b transfected cells. vero cells were transfected with the plasmid constructs peyfpn1-9b (having 9b sequence fused to a yellow fluorescent protein coding sequence at the c-terminus region). analysis of 9b-yfp localization showed that in addition to the extranuclear region, some amount of 9b was also present within the nucleus similar to the sars-cov infected cells (fig. s1 , panel (i), (ii) and (iii)). in a parallel experiment, only peyfpn1 vector was transfected in order to rule out any effect due to the vector itself (fig. s1 , panel (iv), (v) and (vi)). only cells showing clear nuclear localization of 9b were scored positive. similar results were also obtained with hek-293t and cos-7 cells (data not shown). in order to biochemically confirm the nuclear localization of the 9b protein, we transfected vero cells with pcdna3.1-v5 his-9b construct. after 36 hrs, cells were processed for nuclear and cytoplasmic extract preparation. as shown in figure 2 , 9b was present both in cytoplasm as well as nucleus. the purity of the extracts was checked using anti-actin and anti-polymerase ii antibodies for the cytoplasmic and nuclear extracts, respectively ( the cellular localization of many proteins is cell-cycle specific [23] [24] [25] . in order to examine the effects of cell-cycle on 9b localization, we blocked cells either in the g1 or g2 stage, treating them with different cell-cycle inhibitors. for g1 phase, the cells were treated with aphidicolin (g1-s phase inhibitor) at a concentration of 1.5 mg/ml and processed for microscopy and flow cytometry analysis. the microscopy results clearly showed that the cellular localization pattern of 9b remained unchanged in the presence of aphidicolin (fig. 3a) . flow cytometer analysis was done to confirm the efficacy of the drug. the percentage of cells arrested at g1-s phase of the cell-cycle after aphidicolin treatment were found to be 57.9% [60.794%] as compared to untreated cells (44.8%60.361%) confirming a significant arrest at this phase (p = 0.0015) (fig. 3b) . blocking cell-cycle at the g2 phase (using nocodazole) did not affect the cellular localization pattern of the 9b protein, indicating that there was no effect of cell-cycle progression on the cellular localization and nuclear transport of the 9b protein (fig. 3c) . flow cytometry analysis showed that the percentage of cells arrested at the g2-m phase of the cell-cycle after nocodazole treatment was 71.4% [62.689%] as compared to untreated cells (35.8%61.453%) showing significant cell-cycle arrest at this phase (p = 0.0027) (fig. 3d) . sars-9b protein lacks an active nls and enters the nucleus by passive mode of transport cellular localization of some well known proteins like p27kip1, transcription factor lymphoid enhancer factor 1 (lef-1), t-cell factor 1 (tcf-1), cystinosin and some viral proteins have been confirmed by in-vitro transport assays using digitonin-permeabilized cells [26] [27] [28] [29] . to investigate the mode of nuclear transport of 9b, an in-vitro transport assay was performed. initially, the 9b-yfp fusion protein was expressed in vero cells separately and then their lysate was added to digitonin permeabilized cells. the cells were supplied with all necessary components needed for in-vitro transport as detailed in materials and methods. results showed that both h5n1-np (a positive control in the assay) and 9b-yfp were able to enter the nucleus in digitonin permeabilized cells (fig. 4 , panel (i) and (ii), respectively). on the contrary, the 3a-gfp protein (negative control) was not able to get into the nucleus (fig. 4, panel (iii) ). a protein sorting signal analysis of the 9b sequence (using psort program, http://psort.org/) predicted absence of nls in it and suggested its nuclear import to be passive [30] . further, in order to rule out the possibility that the 9b protein uses an active transport mode to enter the nucleus, we used wheat germ agglutinin (wga), a lectin known to inhibit nls dependent active nuclear transport [31] . when wga was added to the cells, h5n1-np (known to undergo nls dependent nuclear import) was found to get restricted to cytoplasm indicating that wga was able to inhibit the active transport of h5n1-np protein through the nuclear membrane (fig. 4, panel (iv) ). however, sars-cov 9b showed similar nuclear localization even in the presence of wga (fig. 4, panel (v) ). thus, our data clearly shows that although the sars-cov 9b protein lacks a nls, it enters the nucleus by passive transport. as expected, the negative control, sars-3a remained cytoplasmic (fig. 4, panel (vi) ). the nuclear export signal (nes) has been reported to be present in the c-terminal region of the 9b protein [18] . to confirm the involvement of the nes in 9b export, we mutated the essential amino-acids of the nes region (46-lrlgsqlsl-54 to 46-aragsqasa-54) and cloned it into the pegfpn1 vector (now onwards called as mu-nes-9b). cells were transfected with this mutant and were analyzed 36 hrs after transfection. microscopic analysis clearly revealed that most of the fluorescence accumulated in the nucleus (fig. 5a, panel i-iii) . further, in order to check the role of nes in nuclear export of 9b, we treated the 9b-eyfpn1 transfected cells with leptomycin-b (lmb); a drug known to inhibit active nes based export [32, 33] . we found that most of the 9b protein accumulated within the nucleus upon lmb treatment, confirming the role of nes in its nuclear export (fig. 5b, panel iv-vi) . in order to reduce the cytoplasmic staining resulting from the newly synthesized 9b protein, transfected cells were treated with cyclohexamide and processed for microscopy. the reduction in cytoplasmic fluorescence in lower panels of figure 5a and 5b confirms the effect of cyclohexamide treatment (fig. 5a , panel iv-vi and fig. 5b, panel vii-ix) . the data obtained was confirmed by performing biochemical analysis of the fractionated lysates of transfected cells. nuclear and cytoplasmic extracts were prepared 36 hrs post-transfection and were analyzed on 15% sds-page gel followed by a western blot using anti-9b polyclonal antibody. purity of the extracts was checked as described before. results confirmed our previous observations (fig. 5c) . taken together, all these results proved that 9b follows a nes dependent, active nuclear export. in order to confirm the localization pattern of 9b in the presence or absence of lmb, sars-cov infected cells were processed for western blotting at two different time points, 24 hrs and 48 hrs. calnexin was used as purity control for nuclear and cytoplasmic extract preparation. the 9b protein was seen in both nucleus as well as cytoplasm at both 24 hrs and 48 hrs (lane 2 and 5, fig. 5d, upper panel) . this shows that 9b localizes both in the nucleus as well as in the cytoplasm in both transfection and infection conditions. further, the effect of lmb was checked and the proportion of 9b in nuclear and cytoplasmic extracts was compared in reference to total protein using densitometric analysis (fig. 5d, lower panel) . as shown in figure 5d , total 9b in lbm treated cells seem slightly lower compared to untreated cells. this is most probably due to more cells in apoptotic phase. however, densitometry graphs clearly show that the percentage of nuclear 9b gets increased on lmb treatment which further supports our transfection data. the effect seems more pronounced at the 48 hrs time point. the only difference we observed between transfection and infection conditions was that even without lmb, a lot of 9b was seen in the nucleus 48 hrs post infection. this may be attributed to the impact of other sars-cov proteins (expressed during infection) on 9b localization. cells were synchronized in duplicates by starvation and were transfected with peyfpn1-9b. drug treatment was given. one set was processed for microscopy and the other set was used for flow cytometric analysis. a. the effect of aphidicolin treatment on 9b protein localization pattern. synchronized peyfpn1-9b transfected cells were treated with 1.5 mg/ml aphidicolin (sigma). yellow color of 9b was converted into green. panel (i), (ii) and (iii) shows 9b, dapi and a merge image respectively. arrow shows nuclear entry of 9b. b. the efficacy of aphidicolin was confirmed by flow cytometeric analysis. left histogram shows control cells and right histogram shows drug treated cells. aphidicolin inhibited the s-phase entry of cells. c. the effect of nocodazole treatment on 9b protein localization pattern. synchronised cells were treated with 1 mg/ml nocodazole (sigma). yellow color of 9b was converted into green. panel shows the gfp tagged sars-cov 3a protein that was used as a negative control for the in-vitro transport assay. arrows clearly show that the 3a protein is not able to enter the nucleus. panel (ii) shows that even in in-vitro transport assay, sars-cov 9b protein localizes in both cytoplasm as well as nucleus. cytoplasmic 9b protein was somewhat diffused which may be explained as the effect of digitonin permeablization. arrows shows 9b protein localization in digitonin permeablized cells. lower panels: an in-vitro transport assay for 9b protein performed in the presence of wga. the in-vitro transport assay performed in presence of 50 mg/ml wga shows a passive mode of import for sars-cov 9b protein. panel (iv) shows that the nucleocapsid protein of influenza was unable to enter the nucleus in presence of wga showing that wga was able to block the nls dependent active transport into the nucleus. as shown in panel (vi), the sars-3a protein was unable to get into the nucleus in wga treated, digitonin permeablized cells. as shown in panel (v), the sars-cov 9b protein was able to enter the nucleus even in the presence of wga showing that its entry is independent of active transport pathway. arrows in various panels show the localization of protein. nuclear and cytoplasmic extracts were prepared 36 hrs post 9b transfection and were ran on a 15% sds-page gel followed by a western blot using active nuclear export takes place by interaction of the nuclear export signal (nes) with a protein, known as crm-1 and is critical for rna and protein export from the nucleus [32, 33] . synchronized cells were transfected with the appropriate constructs and the lysate was immunoprecipitated using the mentioned antibodies. the complex was subjected to sds-page followed by western blotting using either anti-9b or anti-crm1 antibodies. as a control, the levels of crm-1 were measured by performing immunoprecipitation using anti-crm-1 antibody followed by western blotting with the same antibody. results show an interaction of 9b with the crm-1 protein, clearly supporting our previous observations (fig. 5e, lane 2) . further, mu-nes-9b was unable to bind crm-1, confirming the presence of nes activity in the 46 to 54 amino-acid region (fig. 5e, lane 3) . many cellular proteins are known to follow the ubiquitin/ proteasome pathway for their degradation [34] . for shuttle proteins, nuclear export is known to promote degradation [35] . our data has already indicated that 9b behaves like a shuttle protein, going into the nucleus passively and coming out using the nes. keeping these facts in mind, we designed experiments to investigate whether nuclear export influences the intracellular stability of 9b protein. vero cells transiently expressing 9b-eyfpn1 or mu-nes-9b were metabolically pulse-labeled followed by a chase period for 0, 30 and 60 min. compared to 9b, we observed a significantly prolonged half-life for mu-nes-9b, indicating that nuclear export is continuously supplying substrate for the degradation machinery. similar results were observed for 9b-eyfpn1 transfected cells treated with a nuclear export inhibitor, lmb (fig. 6) . the experiment was performed in triplicates and the significance of the data was calculated using ''graphpad'' software. the differences between 9b, mu-nes-9b and 9b+lmb for time point of 30 min and 60 min were significant as the p value ranged from p = 0.0004 to p = 0.0013. during our pulse-chase analysis explained above, it was discovered that a number of 9b-eyfpn1 transfected cells died on treatment with lmb. for quantification of the apoptotic cells, tunel assay was performed (fig. 7a, left panel) . the number was significantly higher for lmb treated 9b transfected cells as compared to untreated cells. similar results were observed for mu-nes-9b (fig. 7a, right panel) . these results indicated that blocking 9b nuclear export may induce cell death by apoptosis. similar results were obtained when we checked the cell viability in sars-cov infected cells by mtt assay. maximum viability of sars-cov infected cells was lost on lmb treatment suggesting that 9b nuclear export may be important for cell survival (fig. 7b) . however, the role of other viral proteins in this process can not be ignored. saponin was used as control. to check the pathway thus involved in induction of apoptosis, we determined the levels of various cleaved caspases (caspase 3, 7 and 9) in transiently transfected cells. western blot results show that 9b-eyfp induce apoptosis (only if its nuclear export is blocked) by increasing the cellular levels of cleaved caspase-3 (fig. 7c, lanes1-4) . as expected, mu-nes-9b induced apoptosis even in the absence of lmb. to confirm that the apoptosis is caspase dependant, z-fa-fmk, z-vad-fmk and z-devd-fmk (200 mm each) were added to cells 16 hrs post-transfection, and then every 4 hrs thereafter, for a total of four doses. the lysates were checked for the levels of cleaved caspase-3. results confirmed that apoptosis induced by 9b (blocked in the nucleus) was caspase dependent (fig. 7c, lanes 5-8) . all these results collectively showed that the export deficient 9b protein lead to caspase-3 dependent apoptosis of the host cells. therefore, an nes based nuclear export of 9b appears to be very significant for host survival and may play a vital role in supporting the sars-cov life cycle. anti-9b polyclonal antibody. left panel shows purity of extracts. results in right panel show that both mu-nes-9b as well as lmb treated 9b have increased nuclear localization of 9b. c and n represent cytoplasmic and nuclear extracts respectively. d. biochemical analysis of the localization pattern of 9b protein in sars-cov infected cells. synchronized cells were infected with the virus. nuclear and cytoplasmic extracts were prepared 24 and 48 hrs post infection and were analyzed on 15% sds-page gel followed by western blotting using appropriate antibody. the upper panel shows that 9b is present in both nuclear as well as cytoplasmic extracts. calnexin was used to check the purity of extracts. lower panel shows the densitometric comparison of nuclear and cytoplasmic 9b using image j program. results show that lmb treatment (50 nm) leads to an increased nuclear localization of 9b as compared to untreated cells. e. the sars-cov 9b protein interacts with crm-1. synchronized cells were transfected with the appropriate constructs and the lysate was immunoprecipitated using the mentioned antibodies. next, western blotting was performed using either anti-9b or anti-crm1 antibodies as per the case. lane 2 in the lowermost panel clearly shows that 9b is able to pull out the endogenous crm-1 protein. however, mutated nes clearly shows inhibition of binding of 9b with crm-1 further confirming the presence of nes in 46-54 amino acid region. lane 1 represents peyfp-n1 vector which has been used as a negative control. uppermost and central panel shows the expression level of crm-1 and 9b respectively in the transfected cells. ip represents immunoprecipitation; w represents western blotting. doi:10.1371/journal.pone.0019436.g005 figure 6 . nuclear export of sars-cov 9b protein promotes its degradation. pulse chase analysis of 9b protein shows that the half life of 9b-eyfpn1 was approximately 20 minutes in cell culture. exportdeficient 9b mutant showed a significant increase in half-life of 9b. similar results were observed upon inhibition of 9b export by lmb. band intensities from three independent experiments were quantified using a phosphoimager and graphed with standard errors for 9b-eyfpn1, lmb treatment as well as mu-9b nes. the p value for differences between 9b, mu-nes-9b and 9b+lmb was calculated using ''graphpad'' software and ranged from p = 0.0004 to p = 0.0013. doi:10.1371/journal.pone.0019436.g006 the sars-cov 9b protein passively diffuses into the nucleus and undergoes active crm-1 mediated nucleocytoplasmic export the present study uncovers an important step in understanding the roles of accessory proteins in the sars-cov life-cycle. in this study, we characterized the nucleocytoplasmic shuttling of the 9b protein and found that 9b enters the nucleus by a passive mode of transport whereas its export is active and nes-crm1 interaction dependent. further, in transfection experiments, we showed that this localization pattern of 9b was independent of cell-cycle stages. a time course study of 9b revealed that the pattern of its cellular localization remained unchanged irrespective of post-transfection time (data not shown). disruption of 9b-crm1 binding after mutating the critical residues within the nes, and nuclear accumulation of 9b on treatment with lmb, a crm1 antagonist, reconfirmed our hypothesis. earlier studies on 9b show that it localizes in the cytoplasm [17, 18] . we also found the cytoplasmic localization of 9b, however our biochemical as well as confocal analysis clearly showed that 9b, in addition to cytoplasm, also localizes in the nucleus. this import is not active and is nls independent as 9b is able to enter into the nucleus even in the presence of wga. wga is known to bind to the nuclear pore complex and inhibit the nuclear localization signal (nls)-dependent intracellular transport [8, 36] . proteins below 50 kda have been reported to get into the nucleus passively [37, 38] . sars-cov 9b is a small protein of about 13 kda, thus there is a fair possibility of passive diffusion of 9b even as a dimer. however, the possibilities of exposure of a hidden nls after conformational changes cannot be ignored. our preliminary data suggests that 9b interacts with many nuclear proteins ( fig. s2a-c) . these interactions may also provide a possible reason for the presence of 9b in the nucleus. cell-cycle inhibitors are known to affect the cellular localization of some proteins [24, 25, 39] . to confirm the effect for 9b, we used various cell-cycle inhibitors. aphidicolin, arrests g1-s phase progression of the cell-cycle by specially inhibiting dna polymerase a, responsible for dna replication [25, 40, 41] . nocodazole is an anticancer drug that interferes with the structure and function of microtubules during interphase and blocks cellcycle progression at the g2-m phase [41] . our results show that the localization of 9b is independent of cell-cycle stages and the pattern of distribution of 9b remains unaltered in the presence of the drugs used. a low level of apoptosis observed for nocodazole treated cells is a well established effect of this drug [42] . lmb inhibits nes dependent protein export [32, 33] . the suggested mechanism involves direct binding of lmb to crm1 (exportin 1), which blocks the binding of crm1 to proteins containing the nes. deletion of the nes present in 9b as well as lmb treatment of cells has been reported to retain 9b in the nucleus [18] . we observed similar results when we repeated these experiments. further when we mutated the essential amino-acids within the nes by site-directed mutagenesis or used cells treated with lmb; we found accumulation of 9b in the nucleus. we performed a blast analysis of the nes region of sars-cov 9b protein which revealed that this region was well conserved (except the replacement of q by n at amino-acid position 51 in a few cases) irrespective of the host of the virus. interestingly; this phenomenon of nuclear transport exhibited by 9b is similar, but not identical to some other known proteins. proteins like mapk and survivin are reported to localize in both cytoplasm as well as the nucleus [43, 44] . similar to 9b protein, survivin protein does not have nls but contains nes. survivin also enters passively into the nucleus. however, blocking nuclear export of survivin enhances its half-life [44] . our results show that the 9b behaves similar to the survivin protein. we found that the half-life of 9b protein in cells is about 20 minutes and an active nuclear export is mandatory for proper degradation of the 9b protein. our pulse-chase analysis clearly shows an approximate two-fold increase in the half-life of 9b protein, when its export from nucleus is blocked. the dependence of 9b degradation on nes is also supported by increased half-life of both; mutant (lacking essential amino acids of its nes region) as well as 9b treated with lmb. the nes based nuclear export has been shown to promote degradation of shuttle proteins like nuclear factor kappa b or inhibitory kappa b by the proteasome or ubiquitin pathway [35] . similar to 9b, the degradation of many shuttle proteins has been reported to get delayed if their export is inhibited [44] . the sars-cov 9b protein triggers caspase 3 mediated apoptosis when retained in the nucleus of mammalian cells while performing pulse-chase assays, we found that a significant number of vero e6 cells, in which nuclear export of 9b has been inhibited (either by treating with lmb or using nes deficient 9b), were showing caspase 3 dependent apoptosis. the absence of significant cell death in case of eyfp-n1 transfected, lmb treated cells, ruled out the possibility of drug induced cell death. all these results indicate that 9b if blocked in the nucleus, activates apoptotic signaling pathway/s. however, the possibilities for this observation to be cell-type specific can not be ignored. summing up, we propose a hypothesis describing the possible role of the sars-cov 9b protein in maintaining the cell survival figure 7 . inhibition of nuclear export of sars-cov 9b protein induces apoptosis. vero cells were transfected with appropriate plasmid. in order to chemically block active export, cells were treated with lmb for 16 hrs. a. the sars-cov 9b protein induces apoptosis, if blocked in the nucleus. a tunel assay was conducted in order to quantify the apoptotic cells. tunel-positive cells were counted from a total sample size of 200 cells in each set of experiments. standard deviation was calculated from three independent experiments. the graph shows percentage of apoptotic cells. arrows in left panel shows tunel positive cells. b. mtt assay was performed to check the effect of lmb (50 nm) on cell viability in sars-cov infected cells. saponin was used as control. maximum viability was lost when sars cov infected cells were treated with lmb. c. apoptosis induced by the nuclear blocked sars-cov 9b protein is caspase 3 dependent. cell lysate was used to measure cleaved caspases by western blotting. among all, only cleaved caspase 3 was picked up demonstrating the role of caspase 3 mediated apoptosis in cells having export deficient 9b protein. lane 2 and 3 clearly shows that the cleaved caspase 3 is present only in lmb treated 9b protein or nes mutant of 9b respectively. lane 1 and lane 4 shows the controls used in the assay. a faint band for cleaved caspase 3 was also seen in lane 4 which is attributed to the effect of drug treatment. to confirm that the apoptosis is caspase dependant, caspase inhibitors like z-vad-fmk and z-devd-fmk (200 mm each) were added to cells 16 hr post transfection, and then every 4 h thereafter for a total of four doses. the cells were lyzed 36 hrs post transfection and the lysates were checked for the levels of cleaved caspase-3. both z-devd-fmk and z-vad-fmk were able to inhibit apoptosis (lane 5 and lane 6). the negative control inhibitor (z-fafmk) had no inhibitory effect on apoptosis (lane 7). results confirm that apoptosis induced by 9b (if blocked in the nucleus) is caspase dependent. doi:10.1371/journal.pone.0019436.g007 after viral infection (fig. 8 ). there are certain proteins which function as transcription factor and on activation (by stress or infection) move into the nucleus [45, 46] . nuclear localization of such proteins further regulates the genes that play a role in apoptosis and cell-cycle progression. we propose that the nes of 9b may remain unexposed until it binds to the nuclear proteins. our preliminary results show that 9b binds with the nuclear proteins (fig. s2) . these interactions may be exposing the nes of 9b leading to the export of 'x' proteins along with 9b using the crm1 dependent pathway. this event may stop host cell response against stress caused by the viral infection. recent studies on accessory proteins of coronaviruses including sars-cov reveal that these proteins are not essential for virus replication and pathogenecity [47] [48] [49] , but studies are there to support the fact that accessory proteins do affect virus release, stability, pathogenesis, and finally contribute towards virulence [50] . in mhv, i protein (an accessory viral structural protein) can contribute to plaque morphology [10] . also, sars-cov (9b protein) has been shown to be a structural component of the virions which again indicates the importance of 9b protein for the virus [51] . however, more studies need to be performed on the functional analysis of the 9b protein. thus, besides other mechanisms like dimerization, lipid binding or interaction with other proteins, export of 9b seems to be a very important phenomenon for both functional activities of 9b as well as the sars-cov life-cycle. in-vivo studies as well as identification of the nuclear protein/s specifically binding to 9b will help in further understanding the role of 9b in nucleocytoplasmic shuttling. while it is not sure whether sars-cov will again emerge in the human population, it has spurred on the awareness against it. hopefully, this study will add a step towards the better understanding of the behavior and function of the 9b accessory protein in the life-cycle of the sars-cov. figure s1 microscopy results show that the sars-9b protein localizes in both cytoplasm as well as nucleus when expressed in transfected mammalian cells. vero cells were transfected with either peyfpn1-9b or peyfpn1 alone. after 36 hrs, cells were fixed and mounted. arrow in panel (i) indicates that some protein also enters into the nucleus. panel (iv) shows the localization pattern of peyfpn1 vector as a control. panel (ii) and (v) corresponds to the dapi staining of panel (i) and (iv) respectively. panel there are certain proteins which function as transcription factor and on activation (by stress or infection) move into the nucleus. nuclear localization of such proteins further regulates the genes that play a role in apoptosis and cell cycle progression. we propose that nes of 9b may remain unexposed until it binds to the nuclear proteins (represented as 'nuclear protein' = 'x'). our preliminary data shows that 9b binds with the nuclear proteins (fig. s2 ). this interaction may be exposing nes of 9b leading to the export of 'x' proteins along with 9b using crm1 dependent pathway. this event may stop host cell response against stress caused by the viral infection. doi:10.1371/journal.pone.0019436.g008 and processed by autoradiography, a band of approx. 17 kda was seen on the autoradiogram (lane 2). lane 1 shows the mock lysate. b. the tnt expressed 9b protein was immunoprecipitated using anti-9b specific antibody (abgent). the antibody was able to recognize the 9b protein (lane 2). m represents mock lysate. c. vero cells were processed and the nuclear proteins were extracted as explained in material and methods. the tnt expressed 9b protein was added to the nuclear extract and a pull down assay was performed using 9b specific antibody (abgent). in parallel, one control reaction having nuclear extract incubated with the tnt product of an empty pcdna 3.1 vector (labeled as mock lysate) was also assembled. the pulled-out proteins were run on a 15% sds page followed by coomassie blue staining. lane 1 shows the proteins pulled out with 9b protein. lane 2 shows the proteins pulled out with mock lysate. lane 3 shows a pull-down using a non-specific antibody. the protein ladder is shown in lane 4. n.e. represents nuclear extract. n.s. represents non-specific. arrow indicates the 9b protein on the gel. (tif) who recommended measures for persons undertaking international travel from areas affected by severe acute respiratory syndrome (sars) a major outbreak of severe acute respiratory syndrome in hong kong identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage mechanisms and enzymes involved in sars coronavirus genome expression the internal open reading frame within the nucleocapsid gene of mouse hepatitis virus encodes a structural protein that is not essential for viral replication bovine coronavirus i protein synthesis follows ribosomal 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in cell cultures and mice coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus severe acute respiratory syndrome coronavirus accessory protein 9b is a virion-associated protein we thank ravinder kumar for lab support; md. atif bin towheed, manjusha and shailja for technical assistance and swapnil sharma for assistance in manuscript preparation. k.s. is a research associate of the department of biotechnology (dbt), india. key: cord-340195-425rd7ul authors: smith, kristine m.; anthony, simon j.; switzer, william m.; epstein, jonathan h.; seimon, tracie; jia, hongwei; sanchez, maria d.; huynh, thanh thao; galland, g. gale; shapiro, sheryl e.; sleeman, jonathan m.; mcaloose, denise; stuchin, margot; amato, george; kolokotronis, sergios-orestis; lipkin, w. ian; karesh, william b.; daszak, peter; marano, nina title: zoonotic viruses associated with illegally imported wildlife products date: 2012-01-10 journal: plos one doi: 10.1371/journal.pone.0029505 sha: doc_id: 340195 cord_uid: 425rd7ul the global trade in wildlife has historically contributed to the emergence and spread of infectious diseases. the united states is the world's largest importer of wildlife and wildlife products, yet minimal pathogen surveillance has precluded assessment of the health risks posed by this practice. this report details the findings of a pilot project to establish surveillance methodology for zoonotic agents in confiscated wildlife products. initial findings from samples collected at several international airports identified parts originating from nonhuman primate (nhp) and rodent species, including baboon, chimpanzee, mangabey, guenon, green monkey, cane rat and rat. pathogen screening identified retroviruses (simian foamy virus) and/or herpesviruses (cytomegalovirus and lymphocryptovirus) in the nhp samples. these results are the first demonstration that illegal bushmeat importation into the united states could act as a conduit for pathogen spread, and suggest that implementation of disease surveillance of the wildlife trade will help facilitate prevention of disease emergence. no adequate estimate of numbers of wildlife traded throughout the world exists given the large size and covert nature of the business. beyond the threats to conservation, the intermingling of wildlife, domestic animals and humans during the process of wildlife extraction, consumption, and trade can serve as a vessel for pathogen exchange [1] . nearly 75% of emerging infectious diseases in humans are of zoonotic origin, the majority of which originate in wildlife [2, 3] . therefore infectious diseases acquired from contact with wildlife, such as occurs via the wildlife trade, are increasingly of concern to global public health. trade in live animals and animal products has led to the emergence of several zoonotic pathogens, of which rna viruses are the most common. sars emerged as a respiratory and gastrointestinal disease in southwest china and within months had spread to 29 other countries, eventually leading to 8,098 cases and 774 deaths. masked palm civets (paguma larvata) traded in the markets of guangdong were found to be infected and a large proportion of the early cases were restaurant workers who bought and butchered wildlife from these markets [4] . the united states is one of the world's largest consumers of imported wildlife and wildlife products [5] . between 2000 and 2006, approximately 1.5 billion live wild animals (around 120,000,000 per year) were legally imported into the united states nearly 90% of which were destined for the pet industry [6] , and an average of over 25 million kilograms of non-live wildlife enter the united states each year [5] . new york is the most frequently used port of entry into the united states, and in combination with los angeles and miami accounts for more than half of all known wildlife imports. imports most often refused entry (i.e., deemed to be illegal) into the united states included those from china, philippines, hong kong, thailand, and nigeria [5] -countries with endemic pathogens such as highly pathogenic h5n1 influenza virus, nipah virus, and simian retroviruses. health risks to the us public, agricultural industry, and native wildlife posed by the wildlife trade have generally not been quantified due to minimal surveillance of live animal imports and the absence of surveillance of wildlife product imports. despite this, known examples of disease introductions to the united states via the wildlife trade have included pathogens of risk to wildlife, livestock and public health such as amphibian chytridiomycosis, exotic newcastle's disease, and monkeypox, respectively. the monkeypox outbreak showed that a single shipment of infected animals can result in serious impact on public health, highlighting the challenges faced by agencies attempting to regulate both legal and illegal wildlife trade. the usda regulates certain exotic ruminant species, some birds, some fish, a few species of tortoise, hedgehogs, tenrecs, and brushtail possums for specific foreign animal diseases to protect agricultural health. in general, there is no current remit for usda to regulate species as potential threats to wildlife or public health. species restricted by centers for disease control and prevention (cdc) include certain turtles, nhps, bats, civets, and african rodents. hunting and butchering of bushmeat (for the purpose of this paper to be defined according to oxford dictionary as the meat of african wild animals) has been increasingly recognized as a source of disease emergence. harvest of nhp bushmeat and exposure to nhps in captivity have resulted in cross-species transmission of several retroviruses to humans including simian immunodeficiency virus (siv), simian t-lymphotropic virus (stlv), and simian foamy virus (sfv) [7, 8] . while siv and stlv adapted to humans and spread to become the global pathogens human immunodeficiency virus (hiv) and human t-lymphotropic virus (htlv), less is known about the distribution and public health consequences of sfv infection [7] . much of the bushmeat smuggled into the united states from africa by air passes through europe en route, although amount and characteristics of bushmeat reaching us borders is not well described. one study estimated that 273 tons of bushmeat was imported every year into paris roissy-charles de gaulle airport in france on air france carriers alone [9] . under the authority of the public health service act, the us department of health and human service (dhhs), cdc is responsible for preventing the introduction, transmission, and spread of communicable diseases, including those from animals or animal products to humans. cdc recognizes the potential public health risk posed by illegal trade in wildlife and regulations are in place that prohibit the importation of bushmeat products derived from cdc-regulated animals. to better understand and educate the public about risks to public health from smuggled bushmeat, beginning in 2008 cdc and inter-agency and non-governmental partners initiated a cooperative effort to assess those risks. this effort includes a pilot study to screen for evidence of zoonotic pathogens in cdc-regulated wild animal products. here we report finding sequences of simian retroviruses and herpesviruses in bushmeat confiscated at five us airports. this pilot study was initiated at john f. kennedy airport (jfk) in queens, ny, where cdc-regulated wildlife products were seized by us customs and border protection (cbp) between october 2008 to september 2010. beginning in april 2010, additional seizures from another four airports that receive international flights (philadelphia, washington dulles, george bush intercontinental-houston, and atlanta hartsfield-jackson international) were included in the study. illegally imported shipments were confiscated opportunistically and thus the pilot study established only the presence and not the prevalence of zoonotic agents in the specimens. site of origin and destination, flight data, mail shipment or carrying passenger identification, date of arrival, and date of sample collection were recorded for each confiscation. items were photographed and identified to genus and species if possible. biological samples were processed for aliquoting and storage at the cdc quarantine laboratory at jfk airport, and any remaining tissues were incinerated according to standard protocols. all items were sampled while wearing full personal protective equipment and sterile instruments were used to avoid cross-contamination. the freshest part of each item was located (muscle appearing red or raw, joint fluid, bone marrow, etc.) and several samples were taken from each item, placed in cryotubes, and preserved immediately in liquid nitrogen. an additional collection of bushmeat items was seized by us fish and wildlife service (usfws) at jfk airport in 2006, and provided for this study by usfws and the united states geological survey national wildlife health center (nwhc). specimens included those central to a 2006 federal case against a person caught smuggling bushmeat into new york for resale [10] . these samples had been stored at usfws forensic laboratories at 220uc from 2006 until 2010, when they were shipped to the nwhc for processing as part of this study. all specimens were then stored at 280uc, and thawed at 220uc before processing at the nwhc. tissue dissection was performed as described above with some minor differences; 0.5 cm 2 samples were preserved in 1 ml nuclisens lysis buffer (biomerieux inc, cat# 284135) prior to immediate storage at 280uc. permission was obtained from the new york department of agriculture and markets to transfer the frozen specimens from jfk airport to cdc national center for hiv/aids, viral hepatitis, std, and tb prevention (nchhstp), and/or columbia university's center for infection and immunity (cii) for testing. when an assured gross identification of species could not be made, samples were genetically identified by phylogenetic analysis of mtdna genes, including cytochrome c oxidase subunits i and ii (cox1/2), and/or cytochrome b (cytb) [11] [12] [13] [14] [15] . nucleic acids were extracted from 10-30 mg of tissue using mechanical disruption (qiagen tissue lyser ii or next advance inc bullet blender), followed by proteinase k treatment until complete digestion of the tissue was achieved. purification of subsequent homogenates was performed using the qiagen all-prep dna and rna extraction kit or dneasy blood and tissue kits according to the manufacturer's instructions. nucleic acid quality was determined using the agilent bioanalyser (agilent rna nano 6000) or ß-actin pcr as previously described [16] . samples were screened for multiple pathogens as described in detail elsewhere, including: leptospira and anthrax [17] , herpesviruses [18] , filoviruses [19] , paramyxoviruses [20] , coronaviruses [21] , flaviviruses [22] , orthopoxviruses [23] and simian retroviruses (siv, stlv, sfv) [24] [25] [26] [27] [28] [29] . all pcr-amplified bands approximately the expected size were confirmed by sequencing. raw sequences were analyzed and edited in geneious pro v5.1.7 and mega 5.03. multiple sequence alignments were constructed using clustalw and phylogenetic comparisons made using neighbor-joining (nj) and maximum likelihood (ml) algorithms. modeltest was used to select the most appropriate nucleotide substitution model. support for branching order was evaluated using 1,000 nonparametric bootstrap support. sequence identity was calculated using uncorrected p-distances in paup* and blast. from october 2008 to september 2010, 8 postal shipments confiscated at jfk airport were included in this study. from june 2010 to september 2010, an additional 20 passenger-carried packages confiscated at the four other international airports were sampled for this study. additional confiscations were made but were not included in this study due to poor condition of sample (e.g., severely degraded or chemically treated). in many cases multiple separate packages were included in a single shipment or carried by a single passenger. specimens varied in condition, including items that were fresh, raw transported in a cooler, lightly smoked, or well dried (fig. 1a-d) . most items contained moist inner tissue. rna quality was low with a predominance of degraded, low molecular weight fragments in the samples, while bactin sequences were detected in the nhp specimens suggesting the presence of amplifiable dna (data not shown). samples from approximately 44 animals were included in this study, including 9 nhps comprising 2 chimpanzees (pan troglodytes), 2 mangabeys (cercocebus spp.), and 5 guenons (cercopithecus spp.; one of which was further analyzed and identified as cercopithecus nictitans, white-nosed guenon) all confirmed by phylogenetic analysis; and 35 rodents comprised of 14 cane rats (thryonomys sp.) confirmed by gross or phylogenetic analysis, 18 suspected cane rats (based on gross identification), and 3 rats (unknown species) confirmed by gross identification. the usfws specimens from 2006 included an additional 20 nhp tissues from 16 individual animals including 10 baboons (papio sp.) and 6 african green monkeys (agms; chlorocebus sp.) all confirmed by phylogenetic analysis. both sfv and herpesviruses were detected in the nonhuman primate bushmeat samples. all positive nhp samples are presented in table 1 . all nhp samples were negative for siv and stlv sequences. all rodent samples were negative for leptospira, anthrax, herpesviruses, filoviruses, paramyxoviruses, coronaviruses, flaviviruses, and orthopoxviruses. sfv polymerase (pol, 465-bp) and long terminal repeat (ltr, ,357-bp) sequences were detected at cdc in tissues from one chimpanzee (bm013) and one mangabey (bm008). sfv ltr sequences were also identified in a second mangabey (bm010). blast analysis of the 425-bp pol sequences from bm013 and bm008 showed maximum nucleotide identity to sfvs from p. t ellioti and mangabey (cercocebus atys and cercocebus agilis), respectively. phylogenetic analysis of the two pol sequences with those available on genbank confirmed that the chimpanzee sfv was highly related to sfv from p. t. ellioti whereas the mangabey sfv clustered tightly with sfv from sooty mangabeys (cercocebus atys) ( figure 2) . p. t. ellioti are endemic to west-central africa in nigeria and cameroon while cercocebus atys are found in west africa from senegal to ghana. phylogenetic analysis was not performed on ltr sequences since only limited sfv sequences in this region are available at genbank. blast analysis was similarly limited and gave the highest nucleotide identity to chimpanzee and mandrill (m. sphinx) sfv ltr sequences, respectively. the two ltr sequences from mangabeys (bm008 and bm010) were 94% identical to each other due to an 8-bp deletion in the ltr of bm008 and 8 nucleotide substitutions. in the usfws samples sfv pol sequences were present in 3/10 baboons, and in 1/6 agms. the baboon sfvs shared .97% nucleotide identity, and had 88-90% nucleotide identity with the agm sfv. phylogenetic analysis of the short (156 bp) pol sequences shows that the three baboon sfvs clustered together, yet separately from the agm sfv -suggesting some genetic relatedness that reflects host specificity as previously demonstrated [13] (figure 3 ). however, while the short baboon sfv pol sequences detected in this study clustered together, they did not cluster with other published sequences from baboons (80.1-84.2% nucleotide identity). similarly the agm sequences did not cluster with published agm sequences (85.8-86.5% nucleotide identity). these results may reflect poor phylogenetic signal from limited sequence data in this region. all simian dna samples from usfws were also screened for larger sfv pol sequences (465-bp) as done at the cdc but were found in only one baboon sample (cii-163). phylogenetic analysis of the larger pol sequence inferred a significant relationship to sfv from guinea baboons (p. papio) (figure 3 ), which correlated with the origin of the shipment (guinea). our inability to detect larger pol sequences in other sfv-positive baboon and agm samples may be due to highly degraded nucleic acids in those specimens (confiscated in 2006) which limits detection of longer sequences. two genera of herpesvirus were detected in nhp specimens, including cytomegaloviruses (cmv; betaherpesvirus) and lymphocryptoviruses (lcv; gammaherpesvirus) ( table 1) . cmv sequences from baboons cii-028 and cii-163 shared .99.5% nucleotide identity indicating they are likely to be the same virus. comparison of this virus with the cmv sequence from whitenosed guenon bm002 showed these two cmvs are 91% identical. overall, nucleotide sequence identity within the cmvs (for sequences included here) was shown to be 68.4-100% (m = 85.0%). lcvs were detected in four agms, two baboons, and one mangabey. lcv sequences in agms cii-044 and cii-144 were .99% identical and likely represent the same virus. a comparison of this virus with the other lcvs detected showed 88.2-95.5% sequence identity. sequence identity for the entire lcv group was calculated to be 81.0-100% (m = 87.5). phylogenetic analysis confirmed the presence and phylogenetic relatedness of cmv and lcv in these nhp specimens (figure 4 ). multiple viruses were detected within some samples. both lcv and sfv were detected in the bone marrow of agm cii-051 and muscle of mangabey bm008 (table 1) . cmv, lcv, and sfv were detected in baboon cii-163 (table 1) . new sfv, herpesvirus, and mtdna sequences identified in the current study have been deposited at genbank with the following accession numbers: jf810903-jf810914 and jf828317-jf828329. sequences less than 200 bp are available upon request. our study is the first to establish surveillance for zoonotic viruses in wild animal products illegally imported into the united states in an effort to prevent the transmission of infectious agents from these shipments. the restricted number of samples included in this study were tested for a limited range of pathogens only and thus presence of additional pathogens not included in this study cannot be ruled out. we identified four sfv strains and two different herpesviruses (in some cases in the same tissues) in smuggled nhp bushmeat. using phylogenetic analysis and gross examination, we were able to determine that bushmeat from nine nhp species and at least two rodent species were attempted to be smuggled into the united states. these results are consistent with the origin of the shipments from west africa and included species of conservation importance (p. papio, cercocebus atys, and p. t. ellioti are classified as ''near threatened'', ''vulnerable'', and ''endangered'', respectively by the international union for conservation of nature), suggesting more education efforts or harsher penalties are needed regarding the handling, consumption, and illegal transportation of products from wildlife of conservation concern. in addition, the finding of mangabey, guenon, and cane rat bushmeat in our study is consistent with that reported by chaber et al who found these and bushmeat from nine other species entering paris-charles de gaulle airport [9] . our finding of sfv dna in smuggled nhp specimens comprising of four species (baboon, chimp, mangabey, and agm) is significant because sfv is a known zoonotic infection of humans exposed to nhps. however, the mode of transmission to humans is poorly understood and while most infected people reported sustaining a nhp exposure (mostly bites) others did not, suggesting a less invasive mode of infection is possible [7] . these viruses are probably not easily spread from human-to-human, although persistent infection has been documented [7] . several sfv-positive people reported donating blood while infected and because blood banks do not screen for sfv, secondary transmission via contaminated blood donations may be possible [7] . further research into the possibility of secondary transmission of sfv is required. the finding of sfv dna in the bushmeat samples highlights a potential public health risk of exposure to these tissues along the hunting, transportation, and consumption continuum with multiple opportunities for primary transmissions. unlike most retroviruses whose rna genome is packaged in the viral particles, foamy viruses are unusual in that dna and/or rna can be present in the infectious virus particles. thus, finding of only dna does not exclude that sfv in these tissues is not infectious, especially in the more recently cdc confiscated items which contained fresher tissue compared to the usfws items confiscated in 2006 that were partially degraded at the time of analysis in 2010. human infection with sfv is of further concern because increases in the pathogenicity of simian retroviruses following cross-species transmission have been documented (e.g., hiv-1 and hiv-2) [30, 31] . however, the limited number of cases, short follow-up duration, and selection biases in the enrolling of healthy workers or hunters to identify cases all limit the identification of potential disease associations [7] . although we did not find siv or stlv in the limited number of specimens in this study, these viruses have been found in high prevalences in nhp specimens at bushmeat markets and in hunted nhps [8, 32, 33] . hiv-1 and hiv-2 emerged as a result of several spillover events of siv from chimpanzees and mangabeys, respectively, that were likely hunted for bushmeat in central and western africa [30] . serosurveillance studies have shown thirtyfive different species of african nhps harbor lentivirus infections, with a prevalence of siv in up to 35% of free-ranging chimpanzees, and 30-60% of free-ranging sooty mangabeys and green monkeys [30, 31, 33, 34] . to date, four groups of htlv viruses found in humans are believed to have originated from corresponding stlv strains in nhp species (including mangabeys, baboons, and chimpanzees) via multiple transmission events [35] . htlv-1, closely related to stlv-1 group viruses, infects 15 to 20 million people worldwide and is spread from person to person via bodily fluids [35] . these viruses are capable of causing leukemia, lymphoma and neurologic disease in humans [35] . discoveries of htlv-3 and htlv-4, and a novel stlv-1 strain were recently made in nhp hunters in cameroon [7] , and 89% of hunted bushmeat in cameroon has been shown to be infected with stlv strains [8, 32] . although imported wildlife products are often not in a freshly-killed state, many are not smoked or processed in any manner, thus screening of larger sample collections of smuggled bushmeat may reveal evidence of these viruses. like retroviruses, herpesviruses can cause long-term latent infections in their host. most herpesviruses are host-specific, yet particular strains are capable of causing severe disease in the nonhost, examples of which include agents of malignant catarrhal fever and herpes b virus [36, 37] . cmvs are in the betaherpesvirus subfamily. human cmv is typically asymptomatic in humans, with the exception of immunocompromised persons. similarly, many nhps are asymptomatic hosts of cmv that do not typically infect other species, including humans. however, baboon cmv (bcmv), like that identified in our study, has been shown to replicate in human tissues in vitro as well as infect and replicate in humans following a bcmv-positive liver xenotransplant [38] . lymphocryptoviruses (lcv) are in the gammaherpesvirus subfamily, and include human lcv, and epstein-barr virus (ebv), the agent of infectious mononucleosis. nearly 90% of adults in the united states have antibodies indicating exposure at some point to ebv. lcvs are typically asymptomatic in their host, with the exception of immunocompromised individuals who may develop b-cell tumors. although much less efficient, baboon lcv can infect human b cells in immunocompromised persons or in persons co-infected with ebv and replicate in ebv-immortalized b cells with the theoretical potential for viral recombination [39] . however, it is unknown if the novel herpesviruses found in bushmeat specimens in our study can easily infect humans handling these tissues. systematic studies examining herpesvirus transmission risks associated with handling or consumption of infected animal tissues have not been reported. in addition, virus isolation was not performed in our study to determine the infectiousness of the specimens at the time of confiscation. in summary, our study establishes initial surveillance methodology to detect and identify zoonotic pathogens and species of origin of wildlife products entering the united states. while we were successful in demonstrating the presence of sfv and herpesviruses in bushmeat specimens, our pilot study was limited by the range, number, and variable condition of products available to us and was not intended to be a comprehensive review of presence or to measure prevalence of all pathogens imported in wildlife products. because our study only included a small number of cdcregulated species and excluded products of ungulate, carnivore, reptile, avian and other origin, as well as any live animal imports, all of which may carry zoonotic pathogens or diseases that threaten domestic livestock or native wildlife, in addition to the fact that virus isolation was not performed in our study to determine the infectiousness of the specimens at the time of confiscation, there is a large component of zoonotic disease risk assessment not included in this study. a further understanding of pathogen movements through the trade will only be recognized through broader surveillance efforts and pathogen identification and discovery techniques in wildlife and wildlife products arriving at us ports of entry so that appropriate measures can be taken to further mitigate potential risks. wildlife trade and global disease emergence host range and emerging and reemerging pathogens overviews of pathogen emergence: which pathogens emerge, when and why epidemiologic clues to sars origin in china united states fish and wildlife service office of law enforcement intelligence unit. us wildlife trade: an overview for reducing the risks of the wildlife trade molecular detection of viral pathogens simian t-cell leukemia virus (stlv) infection in wild primate populations in cameroon evidence for dual stlv type 1 and type 3 infection in agile mangabeys (cercocebus agilis) the scale of illegal meat importation from africa to europe via paris the problems and promise of dna barcodes for species diagnosis of primate biomaterials identification of mosquito blood meals using mitochondrial cytochrome oxidase subunit i and cytochrome b gene sequences ancient co-speciation of simian foamy viruses and primates universal primer cocktails for fish dna barcoding dna primers for amplification of mitochondrial cytochrome c oxidase subunit i from diverse metazoan invertebrates naturally acquired simian retrovirus infections in central african hunters molecular detection of anthrax spores on animal fibres detection and analysis of diverse herpesviral species by consensus primer pcr rapid molecular strategy for filovirus detection and characterization sensitive and broadly reactive reverse transcription-pcr assays to detect novel paramyxoviruses identification of a severe acute respiratory syndrome coronavirus-like virus in a leaf-nosed bat in nigeria identification of a kunjin/west nile-like flavivirus in brains of patients with new york encephalitis detection of orthopoxvirus dna by realtime pcr and identification of variola virus dna by melting analysis retroviral antibodies in indians the simian foamy virus type 1 transcriptional transactivator (tas) binds and activates an enhancer element in the gag gene frequent simian foamy virus infection in persons occupationally exposed to nonhuman primates coinfection of ugandan red colobus (procolobus [piliocolobus] rufomitratus tephrosceles) with novel, divergent delta-, lenti-, and spumaretroviruses human foamy virus: further characterization, seroepidemiology, and relationship to chimpanzee foamy viruses myasthenia gravis accompanied by thymomas not related to foamy virus genome in belarusian's patients aids as a zoonosis: scientific and public health implications origins of hiv and the evolution of resistance to aids simian t-lymphotropic virus diversity among nonhuman primates in cameroon risk to human health from a plethora of simian immunodeficiency viruses in primate bushmeat chimpanzee reservoirs of pandemic and nonpandemic hiv-1 primate parasite ecology: the dynamics and study of host-parasite relationships new hosts for equine herpesvirus 9 b-virus (cercopithecine herpesvirus 1) infection in humans and macaques: potential for zoonotic disease detection of infectious baboon cytomegalovirus after baboon-to-human liver xenotransplantation infection of human b lymphocytes with lymphocryptoviruses related to epstein-barr virus we thank the usfws forensics laboratory, ashland, oregon, and nathan ramsay, usgs national wildlife health center for assistance with this study. we thank the contributions of ecohealth alliance's consortium for conservation medicine. use of trade names for identification only and does not imply endorsement by the us geological survey national wildlife health center, the department of health and human services, the public health service, or the centers for disease control and prevention. the findings and conclusions in this report are those of the authors and do not necessarily represent the views of the national wildlife health center or the centers for disease control and prevention. key: cord-341914-l2bomgji authors: flies, andrew s.; mansfield, linda s.; grant, chris k.; weldele, mary l.; holekamp, kay e. title: markedly elevated antibody responses in wild versus captive spotted hyenas show that environmental and ecological factors are important modulators of immunity date: 2015-10-07 journal: plos one doi: 10.1371/journal.pone.0137679 sha: doc_id: 341914 cord_uid: l2bomgji evolutionary processes have shaped the vertebrate immune system over time, but proximal mechanisms control the onset, duration, and intensity of immune responses. based on testing of the hygiene hypothesis, it is now well known that microbial exposure is important for proper development and regulation of the immune system. however, few studies have examined the differences between wild animals in their natural environments, in which they are typically exposed to a wide array of potential pathogens, and their conspecifics living in captivity. wild spotted hyenas (crocuta crocuta) are regularly exposed to myriad pathogens, but there is little evidence of disease-induced mortality in wild hyena populations, suggesting that immune defenses are robust in this species. here we assessed differences in immune defenses between wild spotted hyenas that inhabit their natural savanna environment and captive hyenas that inhabit a captive environment where pathogen control programs are implemented. importantly, the captive population of spotted hyenas was derived directly from the wild population and has been in captivity for less than four generations. our results show that wild hyenas have significantly higher serum antibody concentrations, including total igg and igm, natural antibodies, and autoantibodies than do captive hyenas; there was no difference in the bacterial killing capacity of sera collected from captive and wild hyenas. the striking differences in serum antibody concentrations observed here suggest that complementing traditional immunology studies, with comparative studies of wild animals in their natural environment may help to uncover links between environment and immune function, and facilitate progress towards answering immunological questions associated with the hygiene hypothesis. evolutionary processes have shaped the vertebrate immune system over time, but proximal socio-ecological factors mediate the activation, duration, and intensity of immune responses [1, 2] . for example, an animal's sex, diet, sociality, life-history stage, and pathogen pressure can influence its immune function [2] [3] [4] [5] [6] [7] . one proximal factor widely believed to shape immune function during ontogenetic development is exposure to potential pathogens, including both microbes and macroparasites. in fact, reduced parasitic helminth exposure has been correlated with the increased prevalence of allergy and autoimmune disease in some human populations (reviewed in [8] ). the hygiene hypothesis was originally proposed as an explanation for the commonly observed pattern of increased occurrence of allergic disease in relatively hygienic environments [9] and in human populations with access to modern medicine. this hypothesis has been modified many times, but one of its basic tenets remains the idea that reduced exposure to potential pathogens leads to dysregulated development of immune defense pathways, and consequently to increased risk of allergy and autoimmune disease. although debate about the implications and breadth of the hygiene hypothesis continues, it is clear that environmental exposure to potential pathogens can play a critical role in the development of the immune system [10] [11] [12] . wild-type animals, which are those that putatively represent the normal phenotype for a given species as it occurs in nature, represent a critical component of many laboratory studies, yet few studies have actually examined immune function in wild animals living in their natural environment, and fewer still have compared immune function between wild and captive conspecifics [12] [13] [14] [15] [16] [17] . differences between captive and wild animals with respect to immune activation, duration and intensity can be expected due to regular cleaning of captive facilities, implementation of pathogen control programs, reduced energy expenditure in captive animals, and a more stable environment in captivity than in the wild [6, 18, 19] . a more robust understanding of the effects of ecological variables such as pathogen exposure on immune function might be gained by studying immune function in non-traditional species, and assessing how basic immune defenses differ between wild and captive animals with similar genetic backgrounds. carnivores are particularly interesting with respect to immune function because they are regularly exposed to pathogens that are capable of infecting both prey and predator. for example, particular salmonella spp. and shiga toxin-producing escherichia coli are commonly found in wildlife carcasses [20, 21] on which carnivores feed. furthermore, several carnivores in africa, including spotted hyenas (crocuta crocuta), regularly test seropositive for anthrax antibodies, yet they show no signs of disease [22] . wild hyenas are also known to harbor an abundance of parasitic worms of several genera [23] , and survive exposure to a wide array of viral pathogens including but not limited to: rabies virus, canine distemper virus, corona virus, calicivirus, and parvovirus [24, 25] . in the wild, spotted hyenas live in large social groups, which increases the probability of acquiring socially transmitted pathogens [26] . furthermore spotted hyenas can live more than 20 years, which likely makes their acquired immune system, specifically recall responses, more important than in shorter-lived species, such as mice, that are not re-exposed to the same pathogens over the course of many years [27] . high pathogen exposure rates coupled with low mortality rates from infectious disease suggest that immune function may be particularly robust in wild spotted hyenas. our primary objective in this study was to assess differences in immune function between wild spotted hyenas inhabiting a pathogen-rich and highly variable environment in their natural habitat, and captive hyenas inhabiting a more stable captive environment in which efforts were made to minimize the hyenas' exposure to pathogens. to accomplish this, we used an immune defense component model (fig 1) , modified from schmid-hemp and ebert [28] , to compare immune defenses in wild and captive hyenas along two continua: 1) constitutive to induced defenses, and 2) non-specific to specific defenses. constitutive defenses are innate defenses that are present in the absence of infection and are functional without prior exposure [28] . patterns shown here are hypothesized based on existing literature and the locations within the plot were adjusted based on the results of this study. bacterial killing capacity (bkc) is the least specific defense and also falls at the constitutive end of the x-axis. specific igg falls near the induced end of the x-axis, and is the most specific defense represented here. antinuclear antibodies (anas) are specific to nuclear proteins and nucleic acids and can be induced following ligation of endosomal pattern recognition receptors. natural igg and igm are non-specific and can be produced independent of cd4 t cell help. the dashed lines represent primary exposure (left) and secondary (right) exposure. to a pathogen [28] . induced defenses can be innate or adaptive and are initiated in response to the recognition of specific molecular patterns that are often associated with threats to physiological homeostasis [28] . specific defenses are effective against only a limited range of antigens and specificity can be modified during a host's lifetime. specific defenses are most effective upon repeated exposure to the same pathogen, because they are maintained after initial infection, allowing a much faster response to subsequent infections. non-specific defenses target a broader range of antigens but are not modified over time. natural antibodies (nabs) are germline encoded, undergo little or no somatic hypermutation, and are thus largely non-specific, but can be rapidly induced by the microbiota or infection by pathogens [29] [30] [31] [32] . by contrast, specific antibodies produced by b-2 cells, are primarily of the igg isotype in non-mucosal regions, are produced in response to a strong stimulus, such as an infection, and undergo somatic mutation and affinity maturation [31] . nabs are primarily of the igm isotype, but can also be of the igg and iga isotypes [33, 34] . natural igm makes up the majority of total igm in mice, and functions in concert with complement to provide first line defenses to inhibit pathogen invasion and replication [30] . anti-nuclear antibodies (ana) represent a subset of autoantibodies (aabs) that bind to intracellular self-antigens, and are commonly derived from nabs independently of help from t cells, but can also be induced via t cell dependent mechanisms [35] . complement, the primary mediator of serum bacterial killing capacity (bkc), is a first-line immune defense that is continually produced at low levels in the absence of pathogenic challenge, and functions to prevent infection [36, 37] . complement levels can increase quickly following infection, but the duration of the complement pulse is generally short compared to the rise in antibody levels following infection (fig 1) [38] . we hypothesized here that higher levels of induced immune defenses (fig 1: quadrants i and iv) would be observed in a pathogen-rich natural environment than in a relatively clean captive environment. by contrast, we expected that constitutive immune defenses (fig 1: quadrants ii and iii), which are less dependent on pathogen exposure and maintained at baseline levels in the absence of such exposure, should be similar in pathogen-rich wild and pathogenpoor captive environments [14, 28] . we measured relative concentrations of total igg and igm antibodies, autoreactive igg (aigg) and autoreactive igm (aigm) that bind nuclear antigens, natural igg (nigg) and natural igm (nigm) against keyhole limpet hemocyanin (klh), and complement-mediated bacterial killing capacity (bkc) of serum. based on these studies we found strikingly different serum antibody concentrations in captive and wild hyenas. captive spotted hyenas used in this study were born and housed at the field station for behavioral research at the university of california, berkeley (ucb) (37.882843°n, -122.240330°e). the colony was founded by importing 20 infant hyenas from the talek region of kenya in 1984-1985 [39] . all hyenas were treated with anti-helminthic drugs prior to importation from kenya and no parasitic worms have been detected in the colony since its founding, whereas a high diversity and abundance of parasitic worms have been detected in the feces of the wild hyenas [23] . the captive hyena enclosures have cement floors that are hosed down each day with water, and steam cleaned annually. fecal material is removed from medium-sized external enclosures on a daily basis, and fecal material is removed from large outdoor enclosures on a monthly basis. the captive hyenas used in our study have been in captivity for less than four generations, and were derived directly from the same wild population under study here, located in the talek region of the masai mara national reserve, kenya. no disease outbreaks have ever occurred in the captive population, but many virulent viral, bacterial, and protozoan pathogens have been documented in wild spotted hyenas in east africa [22, 24, 25, [40] [41] [42] . ticks, fleas, tsetse flies, and other biting insects, any of which are capable of transmitting pathogens to hyenas, are commonly observed on wild hyenas, but not on captive hyenas. additionally, the captive hyenas are fed only usda approved meat and standard carnivore chow (nebraska brand, usa), whereas the wild hyenas feed on fresh ungulate carcasses and rotting carrion [43] . the captive hyenas exhibit most of the same behaviors as wild hyenas, including scent marking, establishment of dominance hierarchies, greeting ceremonies, and mating [39] , but social group size is much smaller in the captive group than in the wild group. pathogen transmission is often positively correlated with host density, and the effect becomes more pronounced in species that live in large aggregations [44] . wild hyenas live in social groups that can include more than 100 individuals [45] . by contrast, captive hyenas are housed in groups of two or three. the low number of hyenas in each enclosure reduces the possibility of direct transmission of pathogens among group members via social interactions. interspecific pathogen transmission between captive hyenas and wild local animals is probably rare because wild animals seldom stray into the hyena enclosures. wild hyenas are regularly observed feeding on carcasses from which lions, jackals, and vultures, have also fed, and wild hyenas often engage in fierce battles with other carnivores. the institutional animal care and use committees at both the university of california, berkeley and michigan state university approved this study, under approval numbers # r091-0609r and # 07/08-099-00, respectively. captive hyenas were immobilized with blow dartdelivered intramuscular injections of ketamine (4-6 mg/kg) and xylazine (1 mg/kg). blood was drawn from the jugular vein and serum was aliquoted, and frozen at -80°c until use. sera from wild spotted hyenas in the maasai mara, kenya (-1.44290°n, 35.20731°e), were collected as part of the long-term michigan state university hyena research project, which has continuously monitored and recorded demographic information about the wild hyena clan since 1988. research on wild hyenas was approved by the kenya wildlife service and carried out under research permit # ncst/rri/12/1/bs-011/24 from the kenyan national council for science, technology & innovation. hyena tissue samples were exported under the genetic materials access permit #0004 from the national environment management authority of kenya. wild spotted hyenas were immobilized using dart-delivered intramuscular injections of telazol (6.5 mg/kg; zoetis, usa) in a plastic dart fired from an air rifle (telinject inc., usa) [46] . serum was collected as described above, but was snap frozen in liquid nitrogen in the field, then transported to michigan on dry ice, and stored at -80°c until use. total igg and igm were quantified using sandwich elisas. checkerboard 2-fold dilutions of sera and detection antibodies were used to determine optimal dilutions for assays. first, capture antibodies (igg: bethyl labs # a20-117a; igm: custom monoclonals international # cm7) were diluted into coating buffer (0.05 m carbonate-bicarbonate, ph 9.6) at 10 μg/ml, aliquoted into 96-well plates. the plates were then washed twice with tbst and non-specific binding was then blocked 1% bsa in tbs. all standards, sera, and detection antibodies were diluted in 1% bsa in tbst. we used pooled sera from both captive and wild hyenas to create a standard curve on each plate. standard curves were run in duplicate on each plate. the starting dilutions of the pooled sera standard curve were 1:10,000 and 1:100 for total igg and igm, respectively, and 12 doubling dilutions were performed. serum samples were diluted 1:100,000 for igg and 1:1,000 for igm and 100 μl were aliquoted into each well and incubated for 60 minutes. all samples were tested in triplicate. the plates were washed three times with tbst before adding 100 μl of horseradish peroxidase (hrp)-conjugated anti-igg (kirkegaard & perry laboratories # 04-20-02) or anti-igm (kirkegaard & perry laboratories # 04-20-03) detection antibodies to the plates at concentrations of 0.2 μg/ml and 0.25 μg/ml, respectively. the plates were then incubated 60 minutes and washed four times. 100 μl/well of 3,3',5,5'-tetramethylbenzidine (tmb) was used as the substrate for the hrp, and the color change reaction was stopped using 0.2 m h 2 so 4 . absorbance was read at 450 nm on a standard plate reader. relative serum concentrations were calculated using the "calibfit" package in r [47] . we used naturally-occurring anti-klh antibodies as our measure of nabs [48] . klh is a standard immunogen in laboratory studies; it is unlikely that either captive or wild hyenas could ever have been exposed to klh, which is derived from marine gastropods [14] . natural anti-klh is detectable in pre-immune human serum, and concentrations are relatively stable over time [49, 50] . nabs were quantified using the same protocol as above, but with the following changes. instead of coating capture antibodies to the plates, we coated klh (emd millipore # 374805) at 10 μg/ml. the pooled sera again underwent 12 doubling dilutions, with starting dilutions of 1:50 and 1:12.5 for igg and igm, respectively. serum samples were diluted 1:250 for igg and 1:100 for igm, and 100 μl were aliquoted into each well and incubated for 60 minutes. the horseradish peroxidase (hrp)-conjugated anti-igg or anti-igm detection antibodies were added to the plates at concentrations of 0.2 μg/ml and 0.25 μg/ml, respectively. we used commercially available in vitro diagnostic ana kits (hemagen # 6659 96, columbia, md, usa) to assess aigg and aigm concentrations [51] . sera were diluted 1:1,600 and 1:400 for igg and igm, respectively. we again used hrp-conjugated anti-igg or anti-igm detection antibodies added to the plates at concentrations of 0.2 μg/ml and 0.25 μg/ml, respectively. see the manufacturer's product data sheet for a full description of the test kit. two strains of bacteria were tested: escherichia coli (atcc# 8739) was chosen because it has been used as a standard bacterial strain in several other studies of bactericidal capacity [52] ; proteus mirabilis (atcc# 35659) was chosen because, like e. coli, it is a gram negative bacterium that is commonly found in the gastrointestinal tract of many animals and was more susceptible to complement-mediated killing than most other strains in preliminary testing. all bacterial killing assays were conducted in 96-well round bottom plates. all serum samples from both wild and captive hyenas were passed through a 0.22 μm filter prior to adding to the 96-well plate to remove residual red blood cells and other cellular debris. sera were then diluted in phosphate buffered saline (pbs), added to row 1 and serially diluted in pbs. 50 μl of bacteria in cation-adjusted mueller-hinton broth ii (mhbii) was then added to each well [53, 54] to reach a total volume of 100 μl, with the final serum dilutions ranging from 1:8 to 1:1,024 for e. coli 1:4 to 1:512 for p. mirabilis control wells were loaded with bacteria, but no serum, and blank wells were loaded with pbs and mhbii without bacteria. one day prior to beginning the bacterial killing assay, e. coli or p. mirabilis were inoculated into mhbii and incubated overnight at 37°c while shaking at 120 rpm. after overnight incubation, bacterial cell concentration was adjusted to approximately 10 8 cfu/ml by diluting bacteria in sterile pbs until optical density at 600 nm matched the corresponding turbidity of the mcfarland turbidity standard (bd biosciences # 287298). bacteria were then serially diluted in pbs to a concentration of 10 4 cfu/ml. control wells, serum wells and antibiotic wells were next quickly loaded with 50 μl stock bacteria-containing broth, resulting in a final concentration of 500 cfu/well and a total volume of 100 μl/well. 50 μl of sterile mhb were added to blank wells to bring the total volume of each well to 100 μl. plates were then placed in an incubator at 37°c and turbidity was measured after 24 hours on a bio-tek plate reader at 600 nm. percent inhibition was calculated by dividing the optical density of each sample well by the mean optical density of the control bacteria wells. because the assay allows the bacteria to grow to saturation, there was a clear distinction among wells exhibiting bacterial growth and those without growth. the minimum inhibitory concentration (mic) was defined as the negative log 2 of the lowest dilution that exhibited over 90 percent inhibition compared to the mean of the control wells [55, 56] . for example, if 1:40 was the lowest dilution exhibiting complete growth inhibition, the response variable value would be:-log 2 (1/40) = 5.32. additionally, the plates were visually inspected to ensure that there was no bacterial growth at the calculated mic. the mean of triplicates for each sample was used as the final mic, and we used the combined mean mic for both e. coli and p. mirabilis as a general measure of serum bkc. we used version 3.0.3 of the software package r [47] to create linear regression models to assess the effects of captivity on immune function in spotted hyenas. age and sex are known to affect aspects of immune function in some species, so we included age in months and sex as covariates in all of the models. because no single model is a perfect representation of nature [57] , we used an information theoretic multimodel inference approach rather than choosing a single best model [58] . the full models included captivity status, sex, and age as input variables, as well as all twoway interactions between input variables. we explored all possible subsets of the full model, which yielded 18 candidate models for each dependent variable [58] . all subset models were ranked according to akaike's information criteria corrected for small sample size (aicc) [59] . models with a difference in aicc of less than two (δ aicc < 2) are considered to be equally good [59] . the top models, those with δ aicc < 2, were then averaged using the 'mumin' package in r to produce weighted averages of regression parameter coefficients, 95% confidence intervals, and p-values for each input variable in the top models [47] . in cases where only a single model had δ aicc < 2, we report the results directly from the linear model, rather than weighted averages. the response variables total igg, total igm, anti-ana igg, anti-ana igm, anti-klh igg anti-klh igm, and bkc were normalized by log transformation; the input variable age was normalized using a square root transformation. all continuous response and predictor variables were then centered and standardized by subtracting the mean and dividing by the standard deviation; standardization allows for more interpretable comparisons of effect sizes by measuring all effects on a common scale, and centering makes the main effects more biologically interpretable when interactions are present [60, 61] . prior to the beginning of data analysis, we excluded four captive hyenas from the analysis of anti-klh natural antibodies because they were immunized with klh one year prior to the collection of the serum samples used in this study; anti-klh igg and igm levels in these individuals had already returned to preimmunization levels [62] , but, nevertheless, we excluded these animals because their anti-klh antibodies no longer satisfied the criteria for natural antibodies. one wild hyena was excluded from the study prior to beginning data analysis because its serum contained a large lipid globule and could not be filtered in a manner consistent with the other serum samples. additionally, we ran out of sera from two wild hyenas prior to performing the anti-ana igm assay so their values are not represented in that assay. we also performed univariate non-parametric tests of the effects of captivity on immune function and the results were similar to the aicc model selection approach. correlations among total igg, total igm, anti-ana igg, anti-ana igm, anti-klh igg, anti-klh igm, and bkc were assessed using pearson product-moment correlation tests. differences in variability between immune measures in captive and wild hyenas were assessed using levene's test for homogeneity of variance. we assessed the relative concentrations of total igg and igm in 15 captive and 14 wild hyenas. for total igg, only a single model had δ aicc < 2; captivity status was the only input variable in this model, indicating the wild hyenas had significantly higher serum concentrations of total igg than did captive hyenas (p < 0.001) (fig 2a) . wild hyenas also had significantly more total igm than captive hyenas (p < 0.001) (fig 2b) ; here age was non-significant (p = 0.100), s1 table for full list of effect sizes, confidence intervals, and p-values for the analysis of total igg and igm). we measured natural antibodies to klh in sera from 11 captive hyenas and 14 wild hyenas. as with total igg, there was again only a single model with δ aicc < 2, however, this time the model included captivity status, sex, and an interaction between captivity status and sex. wild hyenas had significantly higher concentrations of anti-klh nigg than captive hyenas (p < 0.001) (fig 3a) . there was also a significant interaction between captivity status and sex (p = 0.003), with all wild female hyenas having higher concentrations of anti-klh nigg than wild male hyenas (fig 3a) . the weighted averages from the four anti-klh nigm models with δ aicc < 2 suggest that there was no statistically significant difference (p = 0.116) between anti-klh nigm concentrations in wild hyenas than captive hyenas (fig 3b) . a marginally nons2 table for full list of effect sizes, we assessed the relative concentrations of aigg in 15 captive and 14 wild hyenas, and aigm in 15 captive and 12 wild hyenas. wild hyenas had significantly higher levels of both aigg (p < 0.001) and aigm (p < 0.001) (fig 4) . aigm decreased with age (p = 0.039), particularly in females. (see s3 table for full list of effect sizes, confidence intervals, and p-values for the analysis of aigg and aigm) we assessed bkc for 15 captive and 14 wild hyenas and found that none of the input variables or interactions significantly affected the bkc in the hyenas we tested. captivity status and sex were included in the best fitting model set (δ aicc < 2), but neither captivity status (p = 0.336) nor sex (p = 0.433) significantly affected bkc (fig 5) . the null model, which included only the intercept, had the lowest aicc value. (see s4 table for full list of effect sizes, confidence intervals, and p-values for the analysis of bkc) levene's tests revealed no difference with respect to homogeneity of variance between captive and wild hyenas for any immune measure (p > 0.45 in all cases), indicating that variance in immune measurements was homogenous across all samples. additionally, see table 1 for a full list of correlations among immune measures. here we show that wild hyenas inhabiting a pathogen-rich environment have higher levels of induced immune defenses than captive hyenas, whereas we observed no significant difference between wild and captive hyenas with respect to the constitutive immune defenses we tested. our study is one of the first to use a non-rodent species to make comparisons of antibody concentrations using wild and wild-derived animals that are genetically similar. importantly, our captive population was derived directly from the wild population, and has been maintained in captivity for less than four generations. previous research that compared immunoglobulin levels among human populations reported that total igm concentrations were greater in populations where infectious disease prevalence was higher [63, 64] , suggesting the environmental factors can influence total igm concentrations. however, several studies comparing laboratory mice inhabiting antigen-free, germ-free, or specific pathogen-free environments detected no difference in total igm concentrations in the three different environments, suggesting that in mice igm is primarily a constitutive defense that is not affected by environment [65] [66] [67] . similarly, devalapalli, lesher, et al. [15] found no difference in total igm levels between laboratory mice and wild mice. however, devalapalli, lesher, et al. [15] reported significantly greater concentrations of total igm in wild-caught rats than in laboratory rat strains, and these studies have recently been extended to demonstrate that biome enrichment are important for the development of the humoral immune system [12] . here we found that total igm concentrations are significantly greater in wild hyenas than in a captive population. collectively, these studies indicate that total igm concentrations in some species may be affected by their environment, and highlight the importance of using multiple model systems, including some natural models, when attempting to understand the role of the immune system in health and disease. here we observed significantly greater nigg against klh in wild than in captive hyenas. previous research in mice showed that approximately 20% of the expanded nab-producing b-1 cell population showed an activation-induced cytidine deaminase igm to igg isotype switch [68] . repeated stimulation of b-1 cells could lead to a higher proportion of nigg-producing b-1 cells compared to nigm-producing b-1 cells, and competition for antigen could further augment production of nigg and decrease production of nigm. this might account for the lack of a significant difference in nigm against klh in the captive and wild hyenas, and suggests that over the long-term nigm is the least inducible antibody type described in fig 1. however, quantification of nabs against targets other than klh and a larger sample size would provide greater statistical power to detect differences in nigm and would help to further clarify this issue. interestingly, wild females had higher concentrations of nigg against klh than did the wild males, but this difference was not apparent in captive hyenas. previous work has shown that nabs, particularly natural autoantibodies, are important for the establishment of self-tolerance [34, 69, 70] . very little is known about self-tolerance and autoimmunity in wild animals, but in humans, autoimmune diseases are more common in women than men, and are often associated with hormonal changes associated with pregnancy and tolerance to fetal antigens [71] . despite the positive correlation between autoantibody levels and some autoimmune diseases such as systemic lupus erythematosus, high levels of aigg are associated with a reduced risk of alzheimer's disease, parkinson's disease, and multiple sclerosis [72] . in a well-studied wild population of soay sheep (ovis aries) females had higher levels of aigg than did males, and aigg-positive females were more likely to survive harsh winters, had higher offspring survival, but reduced fecundity than did autoantibody-negative females [51] , again demonstrating a context-dependent positive role for aigg. interestingly, in the study of wild of the soay sheep the concentration of serum anas was positively correlated with the concentration of antibodies that bind antigens derived from an important parasitic trematode [51] . no parasitic helminths have been detected in the captive hyena colony in nearly 30 years by either fecal flotation or during postmortem assessment of the gi tract, whereas every fecal sample ever tested from wild hyenas harbored at least one genus of parasitic helminth egg, and many samples contained thousands of eggs [23] . here we show that both aigg and aigm levels are significantly higher in wild hyenas than in captive hyenas. low-affinity aabs, which may be derived from b-1 or marginal zone b cells, might be critically important for maintenance of physiological homeostasis in wild hyenas that are faced with a relentless assault of viral pathogens, because aabs are critical for efficient clearance of virally-infected apoptotic and necrotic cells. further research is needed to differentiate between high-and low-affinity anas in hyenas, and this could provide much needed insight into the relationship between autoimmunity and pregnancy in female mammals. surprisingly, nigm and aigm were negatively correlated with age in females, and there was no association between any measure of igg and age. several studies have found that nigg and aigg increase with age [48, 73, 74] . it is possible that isotype switching from igm to igg may account for the decreased nigm and aigm observed in older hyenas. an alternative to the hypothesis that differences in pathogen exposure lead to the observed differences in induced immune function observed in wild and captive hyenas is that resource availability varies between the two populations and could drive the observed differences. the captive hyenas used in this study were fed a standard diet of 0.5-1 kg of carnivore chow and approximately 0.5 kg of meat and bone daily, which is adequate for maintaining stable body mass. several days may pass between meals for wild hyenas, and their body masses are highly variable, depending on the size and timing of the last meal. if resources are the primary mediator of the immune defenses tested here, then we would expect to see more variable immune defense levels in wild hyenas. however, our results indicate that variance in immune defenses was similar among captive and wild hyenas. nevertheless we would need serum samples collected at multiple time points from the same individuals to rule out this hypothesis altogether. currently there is a dearth of data in regard to allergy and autoimmunity in animals other than humans and rodents, but collaborative studies between wildlife biologists, immunologists, and managers of captive animals could greatly expand the range of comparative work in this area. importantly, the groundwork for this type of comparative research is already in place in the form of zoos and wildlife research studies that routinely collect tissue samples from captive animals and keep accurate records of the medical history for each animal. the feasibility and impact of comparative immunology research should be augmented by the rapidly increasing availability of published genomes and decreasing costs of monoclonal antibody production, which should allow reagents, such as isotype subclass specific antibodies, to be developed in a cost-effective manner for non-traditional study species. the recent discovery of the important role of tlr10, which is expressed in human and hyenas but not in mice [75] , in the innate immune response to influenza virus in humans [76] suggests that some infectious disease research might progress more rapidly by incorporating comparative studies of non-traditional species. however, in future studies, we do caution that the genetic distance between the captive and wild populations should be minimal, and meticulous monitoring and record keeping are needed to establish unambiguous differences between the captive and wild populations. this work shows that wild hyenas from the masai mara in kenya had significantly higher serum antibody concentrations than hyenas born and raised in a captive facility. importantly, the captive hyena population was derived directly from the wild population and has been in captivity for less than four generations. the striking differences observed here suggest that environmental and ecological factors are important modulators of the immune response, and that comparative analysis of captive and wild animals may help to uncover patterns that are not apparent in traditional laboratory studies of immune defenses. supporting information s1 dataset. spreadsheet containing the data used in this study. 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responses in b-1a and their relationship to natural immunity beyond clonal selection and network definition of human autoimmunity-autoantibodies versus autoimmune disease sex differences in autoimmune disease natural igg autoantibodies are abundant and ubiquitous in human sera, and their number is influenced by age, gender, and disease age-related autoantibody production in a nonhuman primate model do natural antibodies compensate for humoral immunosenescence in tropical pythons? characterization of toll-like receptors 1-10 in spotted hyenas toll-like receptor 10 is involved in induction of innate immune responses to influenza virus infection this study was possible only with the help of dr. stephen e. glickman and the staff of the field station for behavioral research at uc berkeley. we would like to thank the kenya wildlife service and narok county council for allowing us to perform the field research in masai mara. we also thank dr. julia bell for her advice on laboratory procedures, emily johnston flies for her help with the research and writing, and dr. john hayball and dr. dallas flies for their comments on the manuscript. key: cord-352403-4591ewsa authors: hartwig, stacey m.; holman, kaitlyn m.; varga, steven m. title: depletion of alveolar macrophages ameliorates virus-induced disease following a pulmonary coronavirus infection date: 2014-03-07 journal: plos one doi: 10.1371/journal.pone.0090720 sha: doc_id: 352403 cord_uid: 4591ewsa coronaviruses cause respiratory disease in humans that can range from mild to severe. however, the pathogenesis of pulmonary coronavirus infections is poorly understood. mouse hepatitis virus type 1 (mhv-1) is a group 2 coronavirus capable of causing severe morbidity and mortality in highly susceptible c3h/hej mice. we have previously shown that both cd4 and cd8 t cells play a critical role in mediating mhv-1-induced disease. here we evaluated the role of alveolar macrophages (am) in modulating the adaptive immune response and subsequent disease. depletion of am using clodronate liposomes administered prior to mhv-1 infection was associated with a significant amelioration of mhv-1-induced morbidity and mortality. am depletion resulted in a decreased number of virus-specific cd4 t cells in the lung airways. in addition, a significant increase in the frequency and total number of tregs in the lung tissue and lung airways was observed following mhv-1 infection in mice depleted of am. our results indicate that am play a critical role in modulating mhv-1-induced morbidity and mortality. coronaviruses (cov) such as 229e, oc43, nl63 and hku1 have been shown to cause mild acute respiratory disease in humans [1] [2] [3] [4] [5] . severe acute respiratory syndrome (sars) is caused by a group 2 cov termed sars-cov that emerged from china in late 2002 [6] [7] [8] [9] [10] [11] [12] [13] . although most cov do not induce respiratory disease in mice, intranasal infection with the group 2 cov mouse hepatitis virus type 1 (mhv-1), results in a pulmonary infection in mice [14] . we have previously shown that intranasal mhv-1 infection of c3h/hej mice, which harbor a natural mutation in the gene that encodes toll-like receptor 4 (tlr4) [15, 16] , results in increased morbidity and mortality along with severe pulmonary disease as compared to the wild-type c3h/hen mice [17] . both cd4 and cd8 t cells contribute to the mhv-1-induced disease, as depletion of either subset ameliorates morbidity and mortality [18] . here we assessed the role of alveolar macrophages (am) in the induction of the pathogenic t cell response following intranasal mhv-1 infection in susceptible c3h/hej mice. administration of clodronate liposomes (cl) prior to mhv-1 infection resulted in the depletion of am within the lung. we demonstrate that mice treated with cl exhibit a significant decrease in morbidity and mortality following mhv-1 infection as compared to control mice. in addition, amelioration of mhv-1-induced disease correlated with a significant increase in the frequency and total number of foxp3 + tregs in the lung airways and the lung tissue. our data indicate that am play a critical role in controlling mhv-1induced disease severity. all experimental procedures utilizing mice were approved by the university of iowa animal care and use committee. the experiments were performed under strict accordance to the office of laboratory animal welfare guidelines and the phs policy on humane care and use of laboratory animals. female c3h/hejcr and c3h/hencr mice were purchased from the national cancer institute (nci, fredrick, md) and were used at 5 to 8 weeks of age. all animals were maintained in accredited facilities at the university of iowa (iowa city, ia). mhv-1 was obtained from the american type culture collection (atcc manassas, va) and propagated in dbt cells as previously described [18] . mice were infected intranasally (i.n.) with 5610 3 plaque forming units (pfu) of mhv-1 in a total volume of 50 ml while under light anesthesia with isoflurane. each mouse was monitored daily for weight loss and severity of illness, using a previously described scoring system [19] . airway function was measured using a whole-body plethysmograph (buxco electronics, wilmington, nc) as previously described [18] . conscious, unrestrained mice were monitored daily. baseline measurements of enhanced-pause (penh) were recorded over 5 min and reported as an average. lung function was measured daily prior to and following infection. whole lung and spleen homogenates were collected from mhv-1-infected c3h/hejcr mice and viral titers were determined by plaque assay as previously described [17] . cl were obtained from nico van rooijen (vrije university, amsterdam, the netherlands). mice were depleted of am by administering 75 ml of cl i.n. 6 hours (hr) prior to mhv-1 infection. control mice were administered 75 ml of dulbecco's phosphate buffered saline (pbs; gibco, grand island, ny). bronchoalveolar lavage (bal) cells were harvested by inserting a cannula into the trachea and performing 3 consecutive 1 ml washes with rpmi 1640 supplemented with 10 u/ml penicillin g, 10 mg/ml streptomycin sulfate, 2 mm l-glutamine, 0.1 mm nonessential amino acids, 1 mm sodium pyruvate, 10 mm hepes (all from gibco), 5610 25 m 2-mercaptoethanol (sigma-aldrich, st. louis, mo) and 10% fetal calf serum (fcs; atlanta biologicals, lawrenceville, ga) into the lung airspace. lungs were subsequently perfused by slowly injecting 5 ml of pbs into the right ventricle of the heart. the tissue was dissected and cut into small pieces and placed in 4 ml of hbss w/o cacl 2 , mgcl 2 and mgso 4 (gibco) supplemented with 125 u/ml collagenase type ii (invitrogen, grand island, ny) and 60 u/ml dnase i (sigma-aldrich). lungs were incubated on a rotator at 37uc for 30 min. lymph nodes were similarly digested in 1 ml of hbss containing collagenase and dnase i as described above. lungs were pressed through a wire mesh screen (bellco glass, vineland, nj) to create a single-cell suspension. lymph nodes and spleens were pressed between the frosted ends of glass slides to create single-cell suspensions (surgipath, richmond, il). single-cell suspensions of the various tissues were plated into 96well round bottom plates at 1.5610 6 to 2610 6 cells/well. cells were washed with staining buffer (pbs containing, 2% fcs and 0.02% sodium azide), blocked with anti-fccrii/iii monoclonal antibody (mab; clone: 24g.2) and stained with fluorochromeconjugated antibodies. the following antibodies were used: cd4peridinin-chlorophyll proteins (percp; clone: rm4-5, biolegend), cd49d-alexa fluor 647 (clone: r1-2, biolegend), cd8-percp-cy5.5 (clone: 53-6.7, biolegend), cd11a-phycoerythrin (pe; clone: m17/4, ebioscience) and cd90.2-fluorescein isothiocyanate (fitc; clone: 53-2.1, ebioscience). samples were acquired on a facscanto flow cytometer (becton dickinson, san jose, ca) and analyzed using flowjo software (tree star, inc., ashland, or). cells were stimulated for 5 hr at 37uc in rpmi 1640 with 10 mg/ml of brefeldin a (bfa) alone or bfa with mhv-1 derived peptides. mhv-1-specific cd4 t cell responses were assessed using a pool of known cd4 t cell epitopes at a final concentration of 10 mg/ml: m196-210, n346-360, n376-390, s171-185, s881-895 and s921-935. mhv-1-specific cd8 t cell responses were assessed following stimulation with 1 mm of the n421-428 peptide. cells were washed with staining buffer and surface stained with either thy1.2 fitc and cd8 percp-cy5.5 or thy1.2 and cd4 for 30 min at 4uc. cells were washed twice with staining buffer and subsequently fixed and red blood cells simultaneously lysed with facs lysing solution (bd bioscience) for 10 min at room temperature. cells were washed with permeabilization buffer and stained with ifn-c allophycocyanin (apc; clone: xmg1.2, biolegend) and tnf-a pe (clone: mp6-xt22, ebioscience) mab for 30 min. cells were washed again with permeabilization buffer followed by a wash with staining buffer. in some cases cells were also stained with foxp3 (clone: fjk-16s, ebioscience) using the foxp3 staining kit (ebioscience) per the manufacturer's instructions. samples were run on a facscanto flow cytometer (bd bioscience) and data analyzed using flowjo software. all statistical analyses were performed using prism software (graphpad software, inc., san diego, ca). statistical significance was determined by using an unpaired t test. a value of p,0.05 was considered significant. asterisks indicating significance were defined as follows: *, p,0.05; **, p,0.01; ***, p,0.001. to verify that cl treatment would result in the selective depletion of am within the lung, we examined the frequency and total number of am in the lung at 6, 24 and 48 hr following i.n. cl administration. depletion of am defined as, cd11b lo / cd11c hi /mhcii lo /siglec f + , was observed at 6 and 24 hr (data not shown) and was maintained for at least 48 hr ( figure 1a , b). the total number of am in the lung at 48 hr was significantly (p,0.05) reduced as compared to pbs controls. other pulmonary cell populations were unaffected at all time points examined (data not shown). in order to evaluate the role of am during respiratory mhv-1 infection, am were depleted from c3h/hej mice via i.n. administration of 75 ml of cl 6 hr prior to infection. c3h/hej mice administered cl exhibited significantly less weight loss between days 6-7 post-infection (p.i.) as compared to pbs-treated control mice (figure 2a) . similarly, cl-treated mice showed significantly reduced airway resistance (penh) between days 4-7 p.i. as compared to the pbs control mice following mhv-1 infection ( figure 2b ). consistent with the reduced weight loss and improved airway function, the cl-treated mice also exhibited significantly increased survival ( figure 2c ). the timing of am depletion was critical as mice administered cl 48 hr after mhv-1 infection exhibited no difference in weight loss, penh, or survival as compared to pbs control mice (data not shown). thus, depletion of am prior to mhv-1 infection results in significantly reduced morbidity and mortality. to determine the impact of cl administration on virus replication, viral titers were determined in the spleen and lung at days 4 and 14 p.i.. at day 4 p.i., the cl-treated mice exhibited significantly (p,0.01) reduced viral titers in the spleen and lung as compared to the pbs controls ( figure 3a , c). however, by day 7 p.i., viral titers were not significantly different between the groups (data not shown). at day 14 p.i., surviving mice from both groups had either cleared the virus from the spleen ( figure 3b ) or exhibited similarly low levels of virus in the lung ( figure 3d ). thus, depletion of am was associated with a decreased viral load early after infection. we have previously reported that both cd4 and cd8 t cells contribute to mhv-1-induced morbidity and mortality [18] . therefore we examined the impact of am depletion on the mhv-1-induced t cell response. at day 6 p.i., prior to the onset of mhv-1-induced mortality, there was no significant difference between the cl-treated mice and the pbs control mice in total cd4 t cell numbers in the bal ( figure 4a ). however, we observed a significant (p,0.01) increase in the total number of cd4 t cells in the lung tissue of cl-treated mice as compared to the pbs control mice ( figure 4a ). using cd11a and cd49d expression to identify antigen-specific cd4 t cells [20] , we observed no significant difference in the total number of cd11a hi / cd49d + cd4 t cells in either the bal or lungs of cl-treated mice as compared to pbs control mice ( figure 4b ). we have previously identified six mhv-1-derived cd4 t cell epitopes in c3h mice [21] . because the frequency of the responding cd4 t cells to any one of these epitopes is relatively low, we used a pool of all six epitopes to stimulate the cd4 t cells ex vivo. we observed a significant (p,0.05) decrease in the total number of mhv-1-specific ifn-c-producing cd4 t cells in the bal of cl-treated mice as compared to pbs controls ( figure 4c ). in addition, there was a slight decrease in the total number of ifnc-producing cd4 t cells in the lungs of cl-treated mice as compared to the pbs controls, though it did not reach significance ( figure 4c ). we also evaluated the cd8 t cell response. at day 6 p.i., we observed a significant (p,0.05) decrease in the total number of cd8 t cells in the bal of cl-treated mice as compared to the pbs control mice ( figure 5a ). in contrast, the total number of cd8 t cells in the lung tissue was similar between the groups. using surrogate markers to identify antigen-specific cd8 t cells [22, 23] , we observed a significant (p,0.05) decrease in the total number of cd8 lo /cd11a hi cd8 t cells in the bal of cl-treated mice as compared to the pbs control mice ( figure 5b ). however, we observed no difference in the total number of cd8 lo /cd11a hi cd8 t cells in the lungs of cl-treated mice compared to the pbs control mice ( figure 5b ). in order to evaluate the virus-specific cd8 t cell response, we stimulated cells isolated from the lung with the d k -restricted n421-428 epitope [21] . the total number of mhv-1-specific ifncproducing cells in the bal and the lungs was slightly reduced in mice administered cl as compared to the pbs control mice, however this reduction did not reach significance ( figure 5c ). regulatory t cells (tregs) are known to suppress t cell responses [24] . because our data indicated a general trend of reduced cd4 and cd8 t cell responses in the lung airways, we hypothesized that am depletion may have altered the treg response. we had noted earlier that the total number of cd4 t cells in the lungs of cl-treated mice is increased, but there was not a similar increase in the number of the virus-specific cd4 t cells. therefore, we questioned if an increase in foxp3 + tregs would account for this apparent discrepancy. we observed significant increases in the both frequency and total number of foxp3 + cd4 t cells in the bal (p,0.01) and the lungs (p,0.05) of the cltreated mice at day 6 p.i. as compared to that of the pbs control mice ( figure 6a, b) . we also observed that the ratio of foxp3 + cd4 t cells to foxp3 2 cd4 t cells was significantly increased in the bal (p,0.01) and although it did not reach significance, the lung exhibited a similar trend ( figure 6c) . thus, depletion of am was associated with a significant increase in the number of tregs in the lung airways and lung tissue following mhv-1 infection. here we examined the impact of am depletion via cl treatment on mhv-1-induced morbidity and mortality in susceptible c3h/hej mice. we have previously shown that c3h/hej mice are more susceptible than either balb/c or c57bl/6 mice to an intranasal mhv-1 infection [17] . our results indicate that mhv-1-induced morbidity and mortality is reduced in mice administered cl prior to infection as compared to pbs control mice. it is known that some strains of mhv can replicate within macrophages [25] . therefore, it is possible that the protection we observe following am depletion is due to the elimination of a population of potential viral target cells. this may explain why we observe a significant reduction in peak viral titers in cl-treated mice as compared to pbs controls. in contrast to our results, work by luker et al. has shown that during a vaccinia virus infection, depletion of am results in increased weight loss and viral replication [26] . although vaccinia virus may also infect am [26] , in the study by luker et al. cl were administered intratracheally (i.t.) 2 days prior to infection [26] as compared to i.n. administration 6 hr prior to infection as we have done here, which may account for the differences in outcomes. am are known to suppress the immune response [27] [28] [29] . depletion of am protects balb/c mice from a lethal infection with a mouse-adapted strain of sars-cov [29] . similar to our findings, sars-cov-infected mice quickly regain body weight. we demonstrate that depletion of am prior to infection also results in reduced total numbers of cd4 and cd8 t cells in the bal of cl-treated mice as compared to pbs control mice. we also observe reduction in the total number of antigen-specific cd4 and cd8 t cells in cl-treated mice as compared to pbs control mice. however, in contrast to our results, am depletion resulted in an increase in the total and sars-cov-specific t cell response in the lung tissue [29] . the differences between our results and those detailed above could be explained by the timing of cl administration. in the sars-cov studies, cl was administered 3 days prior to sars-cov infection, which may have allowed for am numbers to recover earlier during the course of infection. openshaw et al. demonstrated that balb/c mice treated with cl 3 days prior to an acute respiratory syncytial virus (rsv) infection results in no significant changes in either cd4 or cd8 t cell recruitment or t cell activation [30] . in addition, morbidity and mortality of these mice were also unaffected. an increase in viral titers at the peak of infection was noted, but no significant differences were observed in viral clearance. thus, it is clear that the timing of am depletion and the viral system examined both contribute to the impact of am depletion on a pulmonary viral infection. a recent study by soroosh et al. explored the relationship between am and the development of foxp3 + inducible tregs (itregs) in the lung. cd11c + f4/80 + macrophages were found to promote the generation of foxp3 + itregs through the expression of tgf-b and retinoic acid under steady state conditions [31] . following allergen exposure, the am lost their capacity to promote itreg generation and converted to an inflammatory phenotype [31] . work by savage et al. also showed that treg induction could be regulated by the activation state of am. cd4 t cells exhibit a more functional suppressor phenotype after encounter with anti-inflammatory type 2 macrophages whereas pro-inflammatory type 1 macrophages promote proliferation in the presence of antigen [32] . these studies demonstrate the importance of am and their role in controlling lung inflammation and influencing the generation of tregs. tregs have been shown to play an important roll in modulating the adaptive immune response, as well as play a critical roll limiting immunopathology during an acute pulmonary virus infection, as seen with rsv [33] and influenza a virus (iav) [34] . the frequency of tregs in the lung has been reported to be increased following an infection with either rsv [33] or iav [34] . we observed a significant increase in the total number of cd4 t cells in the lung tissue of cl-treated mice. we also observed a significant increase in the total number and frequency of tregs following mhv-1 infection in both the airways and lung tissue of the cl-treated mice. the increased treg response we observe may help modulate the pathogenic cd4 and cd8 t cell response resulting in reduced morbidity and mortality. in this report we show that the depletion of am by cl administration prior to a mhv-1 infection is associated with a significant amelioration of disease. depletion of am prior to mhv-1 infection significantly reduces weight loss and airway function and increases survival in susceptible c3h/hej mice. we have previously shown that both cd4 and cd8 t cells contribute to morbidity and mortality in the model [18] . our results suggest that depletion of am prior to mhv-1 infection modulates the development of a pathogenic t cell response. the morphology of three previously uncharacterized human respiratory viruses that grow in organ culture a new virus isolated from the human respiratory tract cultivation of a novel type of common-cold virus in organ cultures identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia aetiology: koch's postulates fulfilled for sars virus sars-associated coronavirus a novel coronavirus associated with severe acute respiratory syndrome a major outbreak of severe acute respiratory syndrome in hong kong the genome sequence of the sars-associated coronavirus identification of severe acute respiratory syndrome in canada description and clinical treatment of an early outbreak of severe acute respiratory syndrome (sars) in guangzhou, pr china epidemiology and cause of severe acute respiratory syndrome (sars) in guangdong, people's republic of china murine hepatitis virus strain 1 produces a clinically relevant model of severe acute respiratory syndrome in a/j mice defective lps signaling in c3h/hej and c57bl/10sccr mice: mutations in tlr4 gene characterization of a congenitally lps-resistant, athymic mouse strain toll-like receptor 4 deficiency increases disease and mortality after mouse hepatitis virus type 1 infection of susceptible c3h mice protective and pathologic roles of the immune response to mouse hepatitis virus type 1: implications for severe acute respiratory syndrome differential role of gamma interferon in inhibiting pulmonary eosinophilia and exacerbating systemic disease in fusion protein-immunized mice undergoing challenge infection with respiratory syncytial virus quantifying antigen-specific cd4 t cells during a viral infection: cd4 t cell responses are larger than we think t cell epitope specificity and pathogenesis of mouse hepatitis virus-1-induced disease in susceptible and resistant hosts quantitating the magnitude of the lymphocytic choriomeningitis virus-specific cd8 t-cell response: it is even bigger than we thought tracking the total cd8 t cell response to infection reveals substantial discordance in magnitude and kinetics between inbred and outbred hosts regulatory t cells: friend or foe in immunity to infection? correlation between growth potential of mouse hepatitis viruses in macrophages and their virulence for mice murine alveolar macrophages limit replication of vaccinia virus downregulation of the antigen presenting cell function(s) of pulmonary dendritic cells in vivo by resident alveolar macrophages regulation of tcell function in lung tissue by pulmonary alveolar macrophages evasion by stealth: inefficient immune activation underlies poor t cell response and severe disease in sars-cov-infected mice alveolar macrophages are a major determinant of early responses to viral lung infection but do not influence subsequent disease development lungresident tissue macrophages generate foxp3 + regulatory t cells and promote airway tolerance human anti-inflammatory macrophages induce foxp3 + gitr + cd25 + regulatory t cells, which suppress via membrane-bound tgfbeta-1 foxp3 + cd4 regulatory t cells limit pulmonary immunopathology by modulating the cd8 t cell response during respiratory syncytial virus infection influenza a virus infection results in a robust, antigen-responsive, and widely disseminated foxp3 + regulatory t cell response the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health. conceived and designed the experiments: smh kmh smv. performed the experiments: smh kmh. analyzed the data: smh kmh smv. wrote the paper: smh kmh smv. key: cord-322414-dpx191xh authors: harke, nina n.; radtke, jan p.; hadaschik, boris a.; bach, christian; berger, frank p.; blana, andreas; borgmann, hendrik; distler, florian a.; edeling, sebastian; egner, tobias; engels, christina l.; farzat, mahmoud; haese, alexander; hein, rainer; kuczyk, markus a.; manseck, andreas; moritz, rudolf; musch, michael; peters, inga; pokupic, sasa; rocco, bernardo; schneider, andreas; schumann, andré; schwentner, christian; sighinolfi, chiara m.; buse, stephan; stolzenburg, jens-uwe; truß, michael c.; waldner, michael; wülfing, christian; zimmermanns, volker; witt, jörn h.; wagner, christian title: to defer or not to defer? a german longitudinal multicentric assessment of clinical practice in urology during the covid-19 pandemic date: 2020-09-15 journal: plos one doi: 10.1371/journal.pone.0239027 sha: doc_id: 322414 cord_uid: dpx191xh introduction: after the outbreak of covid-19 unprecedented changes in the healthcare systems worldwide were necessary resulting in a reduction of urological capacities with postponements of consultations and surgeries. material and methods: an email was sent to 66 urological hospitals with focus on robotic surgery (rs) including a link to a questionnaire (e.g. bed/staff capacity, surgical caseload, protection measures during rs) that covered three time points: a representative baseline week prior to covid-19, the week of march 16(th)-22(nd) and april 20(th)-26(th) 2020. the results were evaluated using descriptive analyses. results: 27 out of 66 questionnaires were analyzed (response rate: 41%). we found a decrease of 11% in hospital beds and 25% in or capacity with equal reductions for endourological, open and robotic procedures. primary surgical treatment of urolithiasis and benign prostate syndrome (bps) but also of testicular and penile cancer dropped by at least 50% while the decrease of surgeries for prostate, renal and urothelial cancer (tur-b and cystectomies) ranged from 15 to 37%. the use of personal protection equipment (ppe), screening of staff and patients and protection during rs was unevenly distributed in the different centers–however, the number of covid-19 patients and urologists did not reach double digits. conclusion: the german urological landscape has changed since the outbreak of covid-19 with a significant shift of high priority surgeries but also continuation of elective surgical treatments. while screening and staff protection is employed heterogeneously, the number of infected german urologists stays low. during rs) that covered three time points: a representative baseline week prior to covid-19, the week of march 16 th -22 nd and april 20 th -26 th 2020. the results were evaluated using descriptive analyses. 27 out of 66 questionnaires were analyzed (response rate: 41%). we found a decrease of 11% in hospital beds and 25% in or capacity with equal reductions for endourological, open and robotic procedures. primary surgical treatment of urolithiasis and benign prostate syndrome (bps) but also of testicular and penile cancer dropped by at least 50% while the decrease of surgeries for prostate, renal and urothelial cancer (tur-b and cystectomies) ranged from 15 to 37%. the use of personal protection equipment (ppe), screening of staff and patients and protection during rs was unevenly distributed in the different centershowever, the number of covid-19 patients and urologists did not reach double digits. on march 11 th 2020 the who declared the coronavirus disease 2019 (covid-19) caused by sars-cov-2 (severe acute respiratory syndrome coronavirus 2) a pandemic. at that time, 1,567 cases were confirmed in germany with three deaths due to covid-19 after the first german patient was identified on january 27 th 2020 [1] . germany is a federal state, and handling of the lockdown stayed in the responsibility of the individual states. therefore, different steps were taken at different time points: the first states declared the lockdown in the week of march 16 th 2020. measures included social distancing to "flatten the curve" based on simulation scenarios as well as drastic changes in the health care system [2] . in an efficient manner, triage systems and recommendations were developed by several international societies/associations to meet these challenges also in urology [3] [4] [5] . based on those recommendations, both the healthcare system and the individual hospital had to respect different groups: infected patients, diagnosed and future urological patients as well as healthcare workers. this study aims to give an insight to the urological situation and changes in surgical capacities, therapeutic and deferral strategies and management of staff and patients since the covid-19 outbreak in germany. on may 8 th 2020, 66 urological departments in germany with a focus on laparoscopic/robotic surgery were contacted following an initiative of the german association of urology working group "laparoscopy and robot-assisted surgery". an email was send to the heads of the urological departments which included a link to an online questionnaire using the google docs open-source survey tool [6] . the survey was conducted in accordance with the checklist of reporting results of internet-e-surveys (cherries) [7] . for successful completion of the survey, answering of all questions was not mandatory. a meticulous completion of the questionnaire was estimated to take 90 to 120 minutes. all members of our working group piloted the survey and no technical problems occurred. a similar survey was conducted by an italian covid-19 group and published by rocco et al. [8] . the complete survey (s1 file) included detailed queries on numbers of available hospital beds and operating room (or) capacity, staff members, surgical caseloads with subcategorization of surgeries at three different time points: week 1) baseline week that portrays the numbers of a regular/representative week before the outbreak of covid-19, week 2) march 16 th to 22 nd 2020 which represent the first week after the lockdown in germany (confirmed cases in germany on march 16 th 2020: 6,012 with 13 deaths) and week 3) april 20 th to 26 th (confirmed cases in germany on april 20 th 2020: 141,672; 4,404 deaths and approximately 91,500 recovered) [1] . for weeks 2 and 3, participants were asked about the rates of infected urologists and patients as well as protective measures during daily routine and robotic surgery (rs). preliminary results were presented during a webinar focusing on the development of rs after the outbreak of covid-19 organized by the dgru on may 14 th 2020, and participants were reminded to complete the questionnaire before the data collection was closed on may 24 th 2020. the daily situation report of the pandemic in germany and constantly updated case numbers of infected/recovered patients and death rates, including analysis of the 16 german federal states can be found on the homepage of the german government´s agency for disease control and prevention, "robert koch institute" (rki) [1] . spss 25 was used for statistical analysis. categorial data are shown as frequencies and proportions; continuous variables are given as median and range. for improved visualization in the graphs, continuous data (e.g. number of staff members, cases) were summed to a total number with subsequent division to percentages. chi-square and fisher's exact test were used to identify differences in the the varying practice settings with a statistical significance p<0.05. 27 completed questionnaires were analyzed (41%) and included 14 responses from teaching hospitals, eight university hospitals and five non-academic centers. each center (19 public and eight private) documented shifts in their numbers and capacities after the beginning of the pandemic compared to the representative baseline week before the outbreak. while only a moderate cut-down of available beds within the different urological departments was found (median beds at baseline: 49 vs. 45 in march and 43 in april), the or capacity was reduced distinctly by more than 25%, however this was handled heterogeneously and varied from a decrease to 22% of the initial capacity to no reduction. in total, 4571 surgeries were documented in the three requested weeks with 1195 emergency cases and 3376 scheduled cases. the number of scheduled patients per center declined from a median of 58 scheduled surgeries in the baseline week compared to 31 and 29 cases in march and april. in the same period, an increase of emergency cases could be observed with 363 in a regular week vs. 448 after the covid-19 outbreak in march and 384 emergency surgeries in april (fig 1) . no significant differences in management of or capacities could be observed between university, teaching and non-academic hospitals in march (p = 0.53) and april (p = 0.29). the majority of scheduled cases in the baseline week represented oncological surgeries (44% of total scheduled cases) followed by urolithiasis (24%), surgical therapy of bps (15%) and other surgeries (e.g. reconstructive, transgender, pediatric) with 18%. in the following weeks, declines could be observed in each of these subgroups (fig 2) . compared to the average numbers in the preceding weeks with a total of 366 cases, stone therapy (47% kidney and 53% ureteral stones) declined by 50%. oncological surgeries were subclassified according to their priority and less urgent cancer therapies for low risk prostate cancer (baseline week: a total of 43 cases compared to 24 in march and 17 in april) and small renal masses (srm) (reduction to 49% in march and april) were postponed, resulting in an increased or stable portion of surgeries for more advanced tumor constellations; the total number of prostatectomies for advanced prostate cancer rose from a total of 22 in the baseline week to 28 cases in april. urothelial cancer treatment dropped from 297 cases to 211 in march and 195 in april with a comparable decrease for tur-b and radical cystectomies. remarkably, the rates of rare tumor entities including upper tract urothelial cancer as well as penile and testicular cancer decreased by 48-85% in comparison to the pre-covid-19 case numbers (fig 3) . in the pre-covid-19 era, a median capacity of 4 robotic operating rooms/week was reported by the respondents ranging from two up to 20 robotic days/week with a maximum of 45 robotic cases in one high-volume center. comparable to the developments for endourological (25%) and open/laparoscopic or capacities (27%), robotic or capacity decreased by 26% to a median of three days/week and a maximum of 12 robotic or days/week in march but slowly increased to 83% of the initial capacity in april. three centers suspended their urological robotic program in the weeks of march or april¸one department paused rs completely since covid-19 screening at the time of admission of patients comprised questionnaires in 78% of the institutions and physical examination for fever in 44%. in 52%, patients undergoing surgery were tested for covid-19 prior to the operation with a comparable percentage in public and private hospitals (p = 0.21). a chest ct for screening was routinely employed in only one hospital. in seven departments patients were identified as covid-19 positive either after screening (n = 3) or presentation of symptoms (n = 4). to minimize the risk of virus transmission to the hospital staff, wearing of masks was required in all hospitals. this included regular surgical masks but also hand-sewn masks (19%). regular screening of the staff with testing for covid-19 was reported by 7% of the respondents and in only two urological departments the staff members' temperature/fever was checked daily. to reduce the exposition to potentially infected patients, home office or (un)paid leave was enabled in 59% resulting in a reduction of the median staff members per center from 17 in the baseline week to 16 in march and april. eight out of a total of 505 urologists (1.6%) were tested positive for sars-cov-2 infection. when covid-19 hit europe, the impressive rates of infected patients and deaths in italy as the first affected european country indicated that immediate and extraordinary steps were needed to face this threat. this resulted in unprecedented changes in the healthcare systems across the world. in hospitals, surgical capacity decreased significantly to reserve ventilators and icu beds for covid-19 patients. a global survey by teoh et al. summarized a drop of 81-100% of outpatient clinic appointments and 48% of urological surgeries after the beginning of the pandemic [9] . to manage these reductions, new triage systems were implemented to identify those at high risk of covid-19 and those at risk from being affected by delayed treatment of their urological disease. in april 2020 the european association of urology published recommendations in a remarkable short time frame based on the available information [3] . surgeries should be scheduled only after risk stratification into one of four categories: low, intermediate, high priority and emergency. however, elective surgeries within the lowest risk category (including treatment of urolithiasis and bps) were reported in march and in april. on the other hand, a 25% decrease of intermediate and high priority surgeries was also observed. this might be contributed to the differing policies of healthcare systems in the german federal states as well as hospital policies which may also explain the heterogeneous handling of or capacities: while some respondents reported a reduction of more than 50%, others continued with a mostly unchanged program. unsurprisingly, these findings for germany are in line with a global survey that also revealed an inconsistent implementation of urological covid-19 guidelines on procedure prioritization across the different practice settings and regions worldwide [10] . major concern of the elective treatment of patients potentially harboring covid-19 without clinical symptoms or initial negative testing have been raised after lei et al. published results of 34 patients undergoing surgery while unknowingly being infected in january 2020 in wuhan [11, 12] . all of these patients developed covid-19 pneumonia with need for icu care in 44% and a death rate of 21%. this disadvantageous postoperative course for covid-19 positive patients was also supported by nepogodiev [13] . the diagnosis related group system in germany determines a minimum hospital stay depending on the type of procedure. major urological surgeries require a postoperative stay of at least 4-10 days, a time span that largely covers the incubation period of covid-19 [14] . in a german study of 337 patients after radical prostatectomy (february to april 2020), no clinically evident covid-19 infection occurred in the early postoperative time [15] . accordingly, only a minority of the respondents reported covid-19 positive patients in their departments in march and april. the deferral of procedures and the fear of virus transmission in a urological department (outpatient or hospital) in combination with social distancing resulted in a further worrisome phenomenon: novara et al. described a 55% decrease of urological consultations in emergency departments after the outbreak of the pandemic in italy [16] . in contrast, our study confirmed an increase in urological emergency cases, like ureteral stent-placement, but we noticed a distinct reduction of tur-bs and primary surgical treatment of testicular cancer. especially for the latter, this was surprising, as the reduced or capacity was a rather negligible aspect; an orchiectomy is a short operation which can be done in a same-day surgery setting [17] . more likely, patients postponed an urological consultation for macrohematuria or a testicular nodule due to fear of a sars-cov-2 infection [18] . this is corroborated by further studies showing that the number of cardiac catherization for st-elevation myocardial infarction decreased by 40 and 38% during the first weeks of covid-19 [19, 20] . this observation in the context of a drop in prostate biopsy rates across europe to 38% may indicate that a new wave of patients with possibly even more advanced disease might occur in the next months when outpatient care normalizes [21] . 40% of the european participants of a global survey reported about voluntary or mandatory deployment to covid-19 patient care and/or staff shortage [9] . hence, in addition to continued high-quality patient care, protection of the healthcare workers is of utmost importance and the risk of virus transmission due to (unknown) contact with infected patients should be minimized. our study shows a wide range of screening measures that was not always in line with the eau recommendations: while some hospitals implemented triage admission wards with routine swabs on every patient, others relied solely on questionnaires. porter et al. suggested in a recent review that every patient should be managed as covid-19 positive and one of the first recommendations in personal protection equipment (ppe) in the daily routine in and outside of the hospital is the use of a mask [22] . according to a german survey by paffenholz et al. only a minority of respondents experienced a regular or continuous shortage of masks or gowns with no significant differences between university and regional hospitals [23] . in each of the 27 participating centers of the present study masks were included in the regular ppe. to adapt to a potential shortage of masks especially in the early days of the pandemic, some hospitals also used hand-sewn masks for patient care outside of the operating room. surgical teams are exposed to a high risk of contagion [24] . especially in the initial phase with intubation and the preceding manual ventilation a high risk of contagion was described during the sars ("bird flu") outbreak in 2002 [25] . in the later period, surgical plume may endanger the operating staff as the generated aerosols may theoretically contain viable particles as shown for several viruses [26] [27] [28] . hence, several surgical societies recommended a restrained use of laparoscopic or robotic procedures due to a possibly higher risk of aerosol generation [29, 30] . in our study we found an overall reduction in or capacity by one fourth, but we did not observe a higher cut-back in robotic or capacity compared to endourology/ open procedures. notably, less than 50% of the respondents reported taking special safety measures during robotic surgery, and only a minority routinely used ffp2/3 mask or a closed filtration system to protect the staff. this rather restricted intraoperative use of ppe can also be observed in other surgical specialties: routine ppe was reported by only 9.1% of the respondents of a global survey conducted by the international society of university colon and rectal surgeons [31] and solely 35% of minimally invasive emergency general surgeries in italy were performed using measures for reduced aerosol dispersion [32] . however, with a rate of 1.6% sars-cov-2 positive (tested) urologists in our study this rather deviant inconsistent implementation of the "eau robotic urology section (erus) guidelines during covid-19 emergency" [5] did not translate into a higher infection rate. several limitations have to be discussed. the response rate was below 50% possibly leading to a selection bias and the distribution of the online questionnaire focused on departments (sub)specialized on rs. therefore, these findings cannot be generalized for institutions focusing on urolithiasis or reconstructive surgery as well as outpatient care. furthermore, our results also reflect the differences in the german federal healthcare system and further analyses should elucidate the potential influence of varying states and hospital policies. yet, in the present preliminary analysis, we observed no significant differences between varying practice settings (university, teaching and non-academic hospitals and private vs. public), a fact that was also confirmed globally [10] . the survey included detailed questions on capacities and case numbers but intentionally precluded further information on patient outcomes for ethical reasons. this initial study wanted to describe the situation in germany during the first weeks of the pandemic and further studies are needed to elucidate the individual patient results before, during and hopefully after the covid-19 era. however, in case of a continued pandemic, the evaluation of the changes in practice patterns and patient management during the first wave of covid-19 and the subsequent impact on sars-cov-2 infection and death rates might be a foundation for future particularized and evidence-based guidelines to protect our patients and staff. the outbreak of covid-19 has changed healthcare systems worldwide including the daily routine in urological departments. while several urological societies recommended postponement of elective therapies, we found a heterogeneous implementation in germany with continuation of low priority cases as well as deferrals or shifts even of urgent surgeries. screening strategies and staff protection is varying widely between the responding institutions, however, the numbers of infected german urologists and patients stayed low in our survey. covid-19) daily situation report of the robert koch institute feasibility of controlling covid-19 outbreaks by isolation of cases and contacts european association of urology guidelines office rapid reaction group: an organisation-wide collaborative effort to adapt the european association of urology guidelines recommendations to the coronavirus disease sages and eaes recommendations for minimally invasive surgery during covid-19 pandemic robotic urology section) guidelines during covid-19 emergency administer and collect medical questionnaires with google documents: a simple, safe, and free system improving the quality of web surveys: the checklist for reporting results of internet e-surveys (cherries) the dramatic covid-19 outbreak in italy is responsible of a huge drop in urological surgical activity: a multicenter observational study a global survey on the impact of covid-19 on urological services prioritising urological surgery in the covid-19 era: a global reflection on guidelines clinical characteristics and outcomes of patients undergoing surgeries during the incubation period of covid-19 infection author's reply-clinical characteristics and outcomes of patients undergoing surgeries during the incubation period of covid-19 infection elective surgery cancellations due to the covid-19 pandemic: global predictive modelling to inform surgical recovery plans clinical characteristics of coronavirus disease 2019 in china martini-klinik experience on prostate cancer surgery during the early phase of covid-19 impact of covid-19 pandemic on the urologic practice in the emergency departments in italy risks from deferring treatment for genitourinary cancers: a collaborative review to aid triage and management during the covid-19 pandemic non-covid-19 visits to emergency departments during the pandemic: the impact of fear reduction in st-segment elevation cardiac catheterization laboratory activations in the united states during epub 2020/04/14 stemi care during covid-19: losing sight of the forest for the trees. jacc case rep the impact of covid-19 outbreak on uro-oncological practice across europe: which burden of activity are we facing ahead? eur urol society of robotic surgery review: recommendations regarding the risk of covid-19 transmission during minimally invasive surgery impact of the covid-19 pandemic on urologists in germany personal protective equipment for common urologic procedures before and during the united states covid-19 pandemic: a single institution study aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review human immunodeficiency virus-1 (hiv-1) in the vapors of surgical power instruments human papillomavirus dna in co2 laser-generated plume of smoke and its consequences to the surgeon detecting hepatitis b virus in surgical smoke emitted during laparoscopic surgery minimally invasive surgery and the novel coronavirus outbreak: lessons learned in china and italy. ann surg. 2020. epub 2020/03/30 covid-19: considerations for optimum surgeon protection before, during, and after operation covid-19 and the global impact on colorectal practice and surgery emergency general surgery in italy during the covid-19 outbreak: first survey from the real life key: cord-329468-vjsurl60 authors: okino, cintia hiromi; mores, marcos antônio zanella; trevisol, iara maria; coldebella, arlei; montassier, hélio josé; brentano, liana title: early immune responses and development of pathogenesis of avian infectious bronchitis viruses with different virulence profiles date: 2017-02-15 journal: plos one doi: 10.1371/journal.pone.0172275 sha: doc_id: 329468 cord_uid: vjsurl60 avian infectious bronchitis virus (ibv) primarily replicates in epithelial cells of the upper respiratory tract of chickens, inducing both morphological and immune modulatory changes. however, the association between the local immune responses induced by ibv and the mechanisms of pathogenesis has not yet been completely elucidated. this study compared the expression profile of genes related to immune responses in tracheal samples after challenge with two brazilian field isolates (a and b) of ibv from the same genotype, associating these responses with viral replication and with pathological changes in trachea and kidney. we detected a suppressive effect on the early activation of tlr7 pathway, followed by lower expression levels of inflammatory related genes induced by challenge with the ibv b isolate when compared to the challenge with to the ibv a isolate. cell-mediated immune (cmi) related genes presented also lower levels of expression in tracheal samples from birds challenged with b isolate at 1dpi. increased viral load and a higher percentage of birds with relevant lesions were observed in both tracheal and renal samples from chickens exposed to challenge with ibv b isolate. this differential pattern of early immune responses developed after challenge with ibv b isolate, related to the downregulation of tlr7, leading to insufficient pro-inflammatory response and lower cmi responses, seem to have an association with a most severe renal lesion and an enhanced capability of replication of this isolate in chicken. avian infectious bronchitis virus (ibv) is a highly infectious causative agent of avian infectious bronchitis (ib), a disease of high economic impact, which affects poultry worldwide. ibv replicates primarily in tracheal epithelial cells, inducing several mucosal pathological changes, including ciliary loss, degeneration and necrosis of epithelial cells, glandular degeneration, inflammatory cell infiltration and epithelial hyperplasia [1, 2] . after replication at primary site in tracheal mucosa, viraemia and ibv secondary replication are also found in other respiratory a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 tissues (nose, lungs and air sacs) and in many non-respiratory epithelial tissues (kidneys, testes, oviduct, and gastrointestinal tract). nephritis is commonly observed, mainly in broilers, and depending on the pathotype of the ibv strain and on the bird age, it may cause high mortality, and microscopic lesions of degeneration and necrosis of the renal tubular cells and interstitial inflammatory cell infiltrate [3, 4, 5, 6] . prevention of ibv infection is currently achieved through vaccination, especially by attenuated viral vaccines, suggesting that local mucosal immunity is essential for induction of effective protection against disease [7, 8, 9] . in a previous study, we demonstrated that the dose of attenuated vaccine administered by local route is closely related to humoral and cellular immune responses at tracheal mucosal sites and their ability to confer effective protection against disease [8] . the continuous emergence of new ibv variants in several countries [3, 10, 11, 12, 13] are routinely pointed out as a cause of outbreaks in vaccinated flocks, leading to significant economic losses to the poultry industry [14] . despite the great number of ibv strains and variants described over the last years [14] , most of the studies have been limited to the classification and differentiation of ibv strains in genotypes, pathotypes, protectotypes, and/or serotypes, while, the specific immune mechanisms involved in pathogenesis of this virus remains poorly elucidated. a recent study has observed differential early immune response after infection with ibv when comparing ibv-susceptible and ibv-resistant chicken lines. even before infection, specific genes, including genes related to innate immune response, were found to be differentially expressed in each chicken line. despite these differences, viral loads were similar in tracheal samples of both chicken lines, indicating that ibv resistance might be associated with how the birds respond to the virus and not how they can prevent an initial infection [15] . the innate immune response is the first line of host defense against infections, and several immune and non-immune cells on mucosal surfaces are involved on recognition of pathogen associated molecular patterns (pamp) of microorganisms. pamps are recognized through pattern recognition receptors (prr), among which toll-like receptors (tlr) are an important group. during a rna-virus infection, activation of innate immune responses depends on the interaction of endosome-associated (tlr3 and tlr7) and/or cytosol-located (rig-i like receptors) prrs. tlr3 and tlr7 interact with double-stranded (ds) rna or single-strand (ss) rna, respectively, and the subsequent activation pathways for tlr3 and tlr7 are mediated by trif (tir-domain-containing adapter-inducing interferon-β) and myd88 (myeloid differentiation primary response gene 88), respectively, which lead to the production of anti-viral type i interferons (ifnα and ifnβ) and pro-inflammatory cytokines (il1β and il6) [16] . expression of genes related to the tlr pathway activation and type i ifn were already found to be upregulated in tracheal samples from birds challenged with reference strains of ibv [7, 16, 17] . the inducible nitric oxide synthase (inos or nos2), responsible for the production of nitric oxide (no) by macrophages in response against microbial infections, is often associated with synergic effects combined with acute phase proteins and cytokines, as ifns and tnfs, leading to enhanced phagocytosis. alternatively, an excessive production of inos and consequently no may have negative effects by promoting excessive inflammation or apoptosis [18, 19] . although a previous report did not identify a change in inos transcripts in both tracheal and lung samples during the early phase of ibv infection [16] , increased levels of this enzyme was associated with high degree of disease severity in chickens infected with avian influenza h5n1 [18] , and virulent newcastle disease [20] . the important role of pro-inflammatory cytokines on ibv pathogenicity was demonstrated in a previous evaluation of the kinetics of the inflammatory and cell-mediated immune responses in tracheal mucosa of birds infected with the m41 strain of ibv. tnfsf15 and tgfβ transcripts were found to peak at 1 day post infection (dpi), followed by higher upregulation of il6, il1β and ifnγ. these increased levels of pro-inflammatory cytokines were associated to highest scores of lesions and viral load at 3dpi, whereas later, at 7dpi, highest increases in cd8 and granzyme homolog a mrna expression were detected and associated to a significant decrease in lesions and viral load [2] . it has been recognized that il6 is associated with development of tissue damage in chickens challenged with ibv, including nephritis [21] . levels 20 times higher of il6 mrna after ibv challenge were observed in an ibv-susceptible chicken line compared to the disease-resistant chicken line [22] . high level of il6 gene expression were detected in renal samples of chickens challenged with a nephropathogenic ibv strain and it has been well correlated with viral load and influx of inflammatory cells in this organ [6] . during the development of adaptive immunity against ibv infection, the local cell-mediated immune (cmi) response was associated with cd3+, cd8+ and cd4+ cells influx in the trachea from 3 to 7 dpi with ibv [1, 9] . furthermore, cd8 and other cmi related genes were markedly increased during secondary response related to memory protection induced by the first antigen ibv exposition at 1dpi (8) . conversely, the cmi genes were found most upregulated later, at 5dpi, in the primary adaptive immune response of non-vaccinated chickens and correlated with high virus load in trachea, indicating this response might be more involved in the ibv pathogenesis in this post-infection period of non-immune chickens [8] . mechanisms underlying immune-pathogenesis caused by ibv and those relating to viral clearance have begun to be elucidated [2, 4, 7, 8, 16, 17, 22, 23] . however, there are no studies regarding the existence of differential pathological and immunological profiles induced by ibv strains or isolates differing in virulence for chickens. the aim of this study was to investigate and compare the pathogenesis and the innate and adaptive immune responses induced by the infection with two brazilian field isolates of ibv classified in the br-i genotype. two brazilian field isolates bi/br/embrapa/331/2000 (accession number: ku727196, identified as ibv a isolate) and bi/br/embrapa/127/2006 (accession number: ku727200, identified as ibv b isolate), were isolated from clinical cases of avian infectious bronchitis in poultry flocks located in southern brazil. virus isolation and titration were carried out by inoculation of five 10-day-old specific pathogen-free (spf) embryonated chicken eggs/sample via the allantoic sac route [24] and virus titers were expressed as 50% embryo infectious doses (eid 50 ) according to the reed & muench method [25] . ibv field strains were genotyped by automated dna sequencing (abi3130xl, applied biosystems) of a 1059 bp portion of the s1 gene amplified as described [26] . the deduced amino acid sequences of s1 protein of ibv a and b isolates have 93.16% of identity, and with regard h120 strain (massachusetts serotype) of ibv they have 69.4% and 70.0% identity, respectively. three groups of specific pathogen free (spf) chicks (white leghorn lineage) obtained from valo biomedia (uberlândia, sp, brazil) were housed in separate positive pressure isolators. at 28 days of age, groups a and b were respectively challenged with 10 4.0 eid 50 /bird of a strain of ibv and 10 4.0 eid 50 /bird of b strain of ibv by intra-ocular and intra-nasal routes. one group remained mock infected (negative control group, referred as nc). during whole experimentation, chickens were monitored twice a day. if animals have presented severe depression and lethargy, they were separated to be euthanized, though, there were no animals in these conditions during experiment. the birds from groups a, b and nc were randomly euthanized by cervical dislocation, at 1, 5 and 8 day post-infection (dpi). tracheal and renal samples were collected from each group; a portion of the proximal third from each tracheal sample and a fragment of distal left kidney were immediately frozen in liquid nitrogen and kept at -70˚c until processing for rna extraction. another portion of tracheal and renal samples were subjected to histopathological analysis. throughout the experimental period, birds received water and feed "ad libitum", the room temperature was adapted according with the bird´s age, and the monitored ammonia concentration levels remained below 2 parts per million (ppm). tracheal samples were divided into three fragments (proximal, medial and distal), and a portion of each fragment was prepared for histopathology. the severity of observed lesions scores were determined as previously described [8] . absence of injury was classified as 0, while mild, moderate and severe were scored as grades 1 to 3. morphological characteristics observed by histopathology included loss of cilia and epithelial cells, degeneration or necrosis of epithelial cells, degeneration of mucous glands, mucosal inflammatory infiltrate and epithelial hyperplasia. a sum of all scores of microscopic lesions from all evaluated fragments was amounted to final score per bird, ranging from 0 to 45. birds presenting score values ! 2 in two or more evaluated parameters or presenting marked heterophilic infiltration were considered as relevant lesions induced by ibv. the scores of microscopic lesions in the kidney samples were determined as previously described [6] . the parameters evaluated included tubular degeneration and necrosis, and presence of inflammatory infiltrate, and the scores ranged from 0 to 3 according to the severity of lesions. rna extractions from the proximal third of tracheal and renal samples of experimentally infected chickens were performed using trizol reagent (invitrogen) followed by rna purification using rneasy mini kit (qiagen). the extracted rna purity and quantification were estimated by 260nm ultraviolet absorbance and readings at 260/280nm, respectively. rna quality was verified with agilent rna 6000 nano kit (agilent technologies) in an agilent 2100 bioanalyzer instrument (agilent technologies) for determination of rin (rna integrity number) or by rna analysis in 1% gel electrophoresis. all tracheal and renal samples were tested for ibv viral load by rt-qpcr (hydrolysis probe system) using agpath-id one step rt-pcr kit (ambion), primers and lna-probe (5´fam-3 bhq1, idt) for amplification of the 3´utr genome region of ibv, as described [27] . cq (cycle quantification) results were used to calculate the log of rna copies (log10) using the linear equation from a standard curve. samples presenting cq 36 were classified as positive for ibv. two-step rt-qpcr was used for the relative quantification of gene expression in the tracheal samples. cdnas were synthesised according to instructions provided with high capacity cdna reverse transcription kit (applied biosystems) and 1μl (500 ng/μl) of oligo(dt) primers (idt). the pcr reactions contained 20 ng of cdna, 7.5 μl of 2x quantifast sybr green master mix (qiagen), and 3 μm of each primer (table 1 ) in a final volume of 15 μl. amplification included a pre-incubation step at 95˚c for 5 min, followed by 40 cycles of 95˚c for 15 seconds and 60.0˚c for 35 seconds. after amplification, a melting curve analysis was performed by raising the incubation temperature from 65˚c to 95˚c in 0.2˚c increments with a hold step of 1 sec at each increment. all oligonucleotides used were designed using primer3 [http://frodo.wi.mit.edu] software, spanning exons according to gene sequences from ensembl [http://ensembl.org] and mrna sequences deposited in genbank. except for ifnα and ifnβ which are intron-less mrnas, then, residual genomic dna was digested with 2 μl dnase i (promega) before cdna synthesis. efficiency of each specific real-time pcr was calculated table 1 using two-fold serial dilutions of cdna pooled from all tested animals, and cq values plotted into the linear equation. the relative expression of all tested genes (table 1) in tracheal samples of ibv-infected chickens was quantified as the fold change relative to the non-infected group (negative control), and the gene expression from each sample were standardised using the cq value of the top2b/hprt1 constitutive reference genes for the same sample [28] . the stability of the reference genes was tested using four candidates (top2b, hprt1, gapdh and histone h3) and the best genes were selected using bestkeeper and normfinder softwares. the comparisons of relative changes in gene expression, viral load and microscopic lesions between the experimental groups were performed using the kruskal-wallis test followed by wilcoxon test. all analyses were conducted using the sas 9.4 software (2012), and the probability level for significance was set as p 0.05. the tracheal lesions observed in groups challenged with a or b ibv isolates at 1 dpi were characterized by mild acute tracheitis, consisting of the presence of heterophils and mucus exudation in the tracheal lumen with congestion and heterophilic infiltration in the lamina propria and epithelial cell desquamation (fig 1a) . the most prominent lesions were observed at 5 dpi, with lesion scores ranging from 20 to 30, consisting of tracheitis. the microscopic pathological [1] , congestion [2] , heterophilic cell infiltrate [3] . (b) proximal third of trachea from the group challenged with ibv a strain, at 5dpi, showing ciliary loss [1] , degeneration and necrosis of some epithelial cells [2] , lymphoid cell infiltrate [3] and epithelial hyperplasia [4] . (c) proximal third of trachea from nc group. changes consisted mainly of lymphoplasmacytic inflammatory infiltrate in the mucosa, ciliary loss and epithelial hyperplasia (7 1b). degeneration of the mucous glands and of epithelial cells was also observed at lower frequency and intensity. at 8 dpi, lesions were mild, tending to tissue recovery. scores between 7 and 15 were found mainly in the challenged groups. the main histopathological changes were degeneration of the mucous glands, lymphocytic infiltration in the mucosa and epithelial hyperplasia. no relevant microscopic tracheal alterations were observed in the nc group at all post-infection intervals analyzed (fig 1c) . marked lymphoplasmacytic interstitial nephritis (lesion score 3) was only observed in the challenged groups (a and b), at 8dpi (fig 2a) . tubular necrosis was not observed in any renal sample from all experimental groups. a higher percentage of birds challenged with ibv b isolate showed relevant tracheal (at 1dpi and 5dpi) and renal (at 8dpi) microscopic lesions ( table 2 ) compared to the birds challenge with ibv a isolate. no positive samples were detected for the presence of ibv genome in tracheal and renal samples from the negative control (nc) group in any of the post-infection intervals analysed (fig 3 and table 3 ). high percentages (87.5% and 100%) of tracheal samples were positive at 1 dpi for the presence of ibv genome from chickens challenged with ibv a and b strains, and differed significantly from tracheal samples from nc group (fig 3a and table 3 ). at 5dpi, all chickens from the challenged groups were ibv positive, with similar virus loads to those detected at 1dpi, differing also significantly different from nc group. the renal samples from chickens of a group showed 88.89% of positivity for ibv genome in kidney samples while all the renal samples from chickens of b group were positive for ibv genome, (fig 3 and table 3 ). the presence and quantity of ibv genome in renal samples differed significantly from those of nc group. the viral load decreased at 8dpi in tracheal samples from all ibv challenged groups (a and b) (fig 4b) . despite of lower virus levels, all challenged groups maintained 100% of positive birds (table 3) . additionally, all the chickens from group challenged with ibv b isolate were positive for the presence of ibv genome in renal samples and only 16.67% were positive for the a challenged group, a significant higher viral load was found in b group when compared to other experimental groups (fig 3b and table 3 ). significantly higher means of ibv genome load (10 to 1000 times higher) were detected in group challenged with ibv b isolate compared to the group challenged with ibv a isolate, at 5dpi interval in tracheal samples (p = 0.0041) and at 8dpi in renal samples (p = 0.0041). (fig 3) . the mrna expression data for tlr3, tlr7, myd88, inos, ifnα and ifnβ in tracheal samples are illustrated in fig 4. tlr3 was significantly upregulated and peaked at 1dpi in ibv ibv positive birds at 1dpi ibv positive birds at 5dpi ibv positive birds at 8dpi trachea trachea kidney trachea kidney nc 0/8 (0%)* 0/9 (0%) 0/9 (0%) 0/5 (0%) 0/5 (0%) a 7/8 (87.5%)* 9/9 (100%) 8/9 (88.89%) 6/6 (100%) 1/6 (16.67%) b 9/9 (100%) 9/9 (100%) 9/9 (100%) 5/5 (100%) 5/5 (100%) nc = negative control group; a = group challenged with ibv a isolate; b = group challenged with ibv b isolate. * one sample was lost during processing. challenged groups. at 5dpi, tlr3 transcripts also remained upregulated in challenged groups, although the levels were lower compared to values found at 1dpi. at 8dpi, tlr3 mrna dropped markedly in challenged groups, but still remained significantly upregulated (fig 4a) . no significant differences were detected in the expression of tlr3 gene between the tracheal samples of chickens infected with a and b ibv isolates, and significant differences were seen by comparing the birds from groups a and b with those from control mock-infected birds. tlr7 transcripts were downregulated (5 times) at 1 dpi only in the tracheal samples from b-challenged group, but their levels of expression rose at 5dpi and maintained at high levels at 8dpi in all challenged groups (a and b) (fig 4b) . myd88 mrna transcripts were upregulated in tracheal samples from all challenged groups (a and b) and at all post-infection intervals. at 8dpi, the samples from a group presented higher levels than those from b group (p = 0.0176) (fig 4f) . ifnα was upregulated at 5dpi and 8dpi in tracheal samples from chickens of b group, and only at 8dpi in the samples from a group chickens. ifnβ transcripts were upregulated in chickens from b group at 1dpi and 5dpi, while in chickens from the a group, only at 5dpi (fig 4c and 4d) . the inos gene showed a significant increase in mrna expression at 1dpi in challenged groups. at 5dpi and 8dpi, these transcripts dropped to basal levels ( fig 4e) . il1β transcripts were significantly upregulated in tracheal samples from all challenged groups at 1, 5 and 8 dpi and the highest levels of il1β expression were reached at 8dpi. (fig 5a) . il6 mrna transcripts were significantly upregulated in tracheal samples from challenged groups at 1 and 5 dpi. at 8 dpi, only the samples from group a showed upregulation in the expression of this cytokine gene (fig 5b) . the tnfsf15 mrna was only significantly upregulated at 8dpi, in the group challenged with ibv a isolate (fig 5c) . the cd3 and cd4 mrna transcripts were downregulated at 1dpi only in the tracheal samples from the b group. at 5dpi and 8dpi, all tracheal samples from challenged groups showed upregulation of cd3 and cd4 expression, and the highest levels of expression of these genes were observed at 5dpi (fig 6a and 6b) . at 5dpi and 8dpi, all challenged groups presented upregulation of cd8β transcripts, and the highest levels of expression of this gene were detected at 5dpi (fig 6c) . the levels of ifnγ gene expression started to increase at 1dpi in group challenged with ibv a isolate. all challenged groups, presented upregulation of ifnγ transcripts, at 5dpi and 8dpi and the highest levels were found 5dpi (fig 6d) . the transcripts of granzyme homolog a mrna were upregulated in all challenged groups at the three post-infection intervals evaluated, except for the group challenged with ibv b isolate, at 1dpi (fig 6e) . the host-virus interactions that result in more severe pathogenesis induced by different pathotypes of ibv are not fully elucidate, but the ibv pathogenesis has been associated with high virus replication rates, induction of exacerbated inflammatory responses and delayed activation of anti-viral effector mechanism of innate and adaptive immune responses [2, 16, 31] . in this study, we found a suppressive effect on expression of some early innate and adaptive cell-mediated immune genes in the primary site of virus replication (trachea) from chickens infected with one of the tested ibv isolates (b). this possibly contributed to an exacerbated pathogenicity of this ibv isolate, especially for kidney tissues, but not for a isolate. ibv b isolate demonstrated an enhanced ability for viral replication in chicken host, as we observed significantly higher ibv genome loads in tracheal samples (at 5dpi), and in kidneys (at 8dpi) of birds challenged with this isolate, compared to the group infected with ibv a isolate. furthermore, a higher percentage of positive birds for ibv genome and the presence of pathological alterations were observed in the ibv b infected group, especially in renal samples, that were ibv positive in birds infected with this isolate at 8dpi, while only 17% were positive in the group infected with ibv a isolate. this enhanced viral replication of ibv b isolate compared to a isolate, may have played a role as one of the determinants for the higher percentage of chickens with relevant microscopic tracheal and renal lesions in birds from the ibv b challenged group. this could result in increased pathology of the ibv b isolate. however, the reason for the differences observed in tissue tropism and pathogenicity of different ibv strains remains an open question and cannot be associated in the present study to differences in s1 protein sequences, as a and b ibv isolates have an identity of xx % for this protein. similarly, a recent study demonstrated that despite the same efficiency of s1 protein binding to the epithelial cells of oviduct, in a sialicacid dependent manner, observed for qx ibv strain (associated with nephropathogenicity and reproductive tract disorders) and b1648 ibv strain (associated with nephropathogenicity), a high susceptibility to infection was observed for qx strain in contrast to the resistance to infection with b1648 strain [29] . the authors concluded that other cellular receptors and post-virus binding activation steps could be involved and play a role in the development of ib disease, although the attachment of ibv to host cells is considered as the first important step in virus infection and for determining the tissue tropism for this virus [29] . in view of the differences observed here for these two brazilian ibv isolates (a and b) in terms of viral replication and pathological alterations induced, the current study investigated how innate and adaptive immune responses are developed after challenge with each one of these viruses, aiming to better understand the virus-host interactions and the putative immune-related mechanisms that might determine the course of pathogenicity induced by ibv isolates with different virulence profiles. several studies on innate immune responses elicited by viruses and mediated by prrs have demonstrated that the levels of expression of tlr genes are usually upregulated, in order to activate intra-cellular pathways involved in the production of type i anti-viral ifns and proinflammatory cytokines [30, 7, 16, 17] . tlr7 was reported to be upregulated at 1dpi in trachea from chickens experimentally infected with a connecticut strain of ibv, but then the expression of this gene decayed to basal levels and remained unaltered at 2dpi and 3dpi [16] . in contrast, in the current study an unexpected suppressive effect in the tlr7 gene expression was observed only during the early phase of ibv b isolate infection (1dpi), as the transcripts of this gene were found 5 times downregulated. moreover, at 8dpi, other genes related to activation of tlr7 pathway were also affected, including the adapter myd88 and the pro-inflammatory cytokine gene (tnfsf15), which showed lower levels of expression in the birds from the group challenged with b isolate compared to the group challenged with isolate a. thus, these results demonstrate a differential profile of early type of innate immune responses induced by ibv b isolate compared to the ibv-induced innate immune responses of another study [16] . in the later study, an upregulation in the transcription of tlr7 and myd88 genes was observed at 1dpi in tracheal samples from chickens infected with a connecticut strain of ibv, that has a typical respiratory and pathogenicity for chicken respiratory tract. although other genes related to innate immune responses were also found to be differentially expressed in response to infection with the brazilian ibv isolates, including tlr3, type i ifns (ifnα and ifnβ) and inos, no significant differences were observed between the group infected with ibv b isolate and that infected with ibv a isolate. these results suggested that the identified differences between the two ibv isolates are not related to the biological activities associated with these innate response genes. however, tlr3 and inos genes were significantly upregulated and peaked at 1dpi, while type i ifns were significantly upregulated and peaked at 5dpi. these results contradict other findings for tlr3, ifnα, ifnβ and inos gene expression in the respiratory tract of chickens challenged with a connecticut strain of ibv [16] , since no significant differences were observed for inos and ifnα gene expression, and tlr3 was unaltered at 1dpi and ifnβ was upregulated at 1dpi. this indicates the likely existence of others factors and pathways in the innate immune responses that are triggered by the interaction of host and different pathotypes of ibv as well possible differences in structural and non-structural proteins of ibv strains involved in virus evasion from the immune responses [31] . as memory immune responses induced by ibv vaccination could have some delay until reach effective anti-viral activities, the innate immune responses might be crucial to reduce the viral replication and pathological alterations induced by a virus pathogen as well exert a relevant role in the activation and shaping of the anti-viral adaptive immune responses. thus, we hypothesize that the suppressive effect on tlr7 pathway could be associated with an enhanced ability for viral replication and induction of lesions for both tracheal and renal tissues. however, further analyses of these assumptions are necessary. in addition, a reduction of expression of cmi-genes for adaptive immune responses was also observed only in the group challenged with ibv b isolate, as the transcript levels of cd3 and cd4 were significantly downregulated at 1dpi only in this group. granzyme homolog a and ifnγ transcripts were also differentially expressed between the challenged groups in this interval, as significant upregulation was observed only in a group. therefore the downregulation of cell-mediated immune related genes could be a consequence of a primary virus challenge effect and could have negative consequences for the chicken host. in a previous study, we were able to detect an increase in the expression of cell-mediated immune related genes at 1 dpi in tracheal samples from birds vaccinated with massachusetts attenuated ibv strain and challenged with a virulent homologous strain. this early cmi response, was associated with the level of cellular memory immune responses conferred by immunization, and this effect was dependent on the vaccine dose administered and was negative correlated with the tracheal pathological changes induced by m41 strain of ibv [8] . although we have not evaluated in this study the gene expression and/or the biological activity of mhc i and ii pathways, or other genes related to antigen presentation by dendritic cells, we can speculate that ibv b isolate might have some differentiated properties that confer to this virus a greater ability to decrease antigen presentation and to evade the host adaptive immune responses against this virus. this assumption is supported by the findings that all cellmediated immune related genes analyzed here have unaltered expression or were significantly downregulated in the group infected by b isolate of ibv at 1dpi, as shown the significantly downregulated levels of expression observed for cd4 and cd3 transcripts, and unaltered expression for ifnγ and granzyme homolog a genes in birds of this group. in contrast, at 1dpi, the ugroup challenged with ibv a isolate showed unaltered levels of cd3 and cd4 mrna, while ifnγ and granzyme homolog a transcripts were significantly upregulated. natural killer (nk) cells have important roles in the initial mechanisms of innate immunity against ibv, and were also important source of granzyme homolog a and ifnγ. although some authors have associated ifnγ production with the activity of nk cells after challenge with ibv [32] , in our experiment, the profile of expression of ifnγ and granzyme homolog a may as well be associated with t cd8+ activation, since cd8β and cd3 were also found to be upregulated in coincident time-points post-challenge in which ifnγ and granzyme homolog a genes were upregulated, especially when considering that nk cells are unable to express cd8β and cd3 genes. in summary, this study has contributed to the better understanding part of host-ibv interactions, and to our knowledge, it is the first study that demonstrate that expression of distinct innate and cell-mediated adaptive immune genes are induced by two ibv isolates that were classified in the same genotype but differing in their virulence activities. overall, the results point out to the relevance of the involvement of the tlr7 pathway and the associate factors related to the suppression of early innate immune responses as well the early cell-mediated adaptive immune responses and the implications on the pathogenesis of infection by a more virulent ibv isolate. however, further studies are required to confirm this association, and to verify other cellular response pathways that may also be affected by this virus. it remains also to be further investigated the differences in the immune responses triggered by the ibv infection with more distinct genotypes of ibv strains that can differ in pathogenicity for the respiratory, urinary or other tissue targets for ibv infection in chickens. moreover, it is important for poultry industry and indirectly for human health to better understand how coronaviruses infect the chicken host and which genes and/or pathways are effectively involved in increasing the severity of this disease, especially considering the diseases caused by other coronaviruses such as severe acute respiratory syndrome-associated coronavirus and middle east respiratory syndrome coronaviruses. kinetics of lymphocytic subsets in chicken tracheal lesions infected with infectious bronchitis virus inflammatory and cellmediated immune responses in the respiratory tract of chickens to infection with avian infectious bronchitis virus nephritis associated with infectious bronchitis virus cal99 variant in game chickens transcriptome analysis of chicken kidney tissues following coronavirus avian infectious bronchitis infection characterization of nephropatogenic infectious bronchitis virus dmv/1639/11 recovered from delmarva broiler chickens in 2011 increased expression of interleukin-6 related to nephritis in chickens challenged with an avian infectious bronchitis virus variant. 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susceptible and resistant birds induction of innate immune response following infectious bronchitis corona virus infection in the respiratory tract of chickens transcriptome of local innate and adaptive immunity during early phase of infectious bronchitis viral infection increased inducible nitric oxide synthase expression in organs is associated with a higher severity of h5n1 influenza virus infection nitric oxide and macrophage function virulent newcastle disease virus elicits a strong innate immune response in chickens immune responses to virulent and vaccine strains of infectious bronchitis viruses in chickens interleukin-6 expression after infectious bronchitis virus infection in chickens effects of avian infectious bronchitis virus antigen on eggshell formation and immunoreaction in hen oviduct detection of viral antigen following exposure of oneday-old chickens to the holland-52 strain of ibv a simple method of estimating fifty per cent end points sequence analysis of nephropathogenic infectious bronchitis virus strains of the massachusetts genotype in beijing lna probe-based real-time rt-pcr for the detection of infectious bronchitis virus from the oviduct of unvaccinated and vaccinated laying hens analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method differences in the tissue tropism to chicken oviduct epithelial cells between avian coronavirus ibv strains qx and b1648 are not related to the sialic acid binding properties of their spike proteins pathogenicity and tissue tropism of infectious bronchitis virus is associated with elevated apoptosis and innate immune responses recent progress in studies of arterivirus-and coronavirus-host interactions rapid nk-cell activation in chicken after infection with infectious bronchitis virus m41 the authors thank tania alvina potter klein, dejalmo alexandre da silva, altair althaus and gláucio mata mattos (in memorian) for support provided during experimental infection in chickens. the authors also thank to francielle ianiski for processing of tissue samples for histology.validation: cho lb. writing -review & editing: cho hjm. key: cord-346067-zly8p0y7 authors: ruiz-irastorza, guillermo; pijoan, jose-ignacio; bereciartua, elena; dunder, susanna; dominguez, jokin; garcia-escudero, paula; rodrigo, alejandro; gomez-carballo, carlota; varona, jimena; guio, laura; ibarrola, marta; ugarte, amaia; martinez-berriotxoa, agustin title: second week methyl-prednisolone pulses improve prognosis in patients with severe coronavirus disease 2019 pneumonia: an observational comparative study using routine care data date: 2020-09-22 journal: plos one doi: 10.1371/journal.pone.0239401 sha: doc_id: 346067 cord_uid: zly8p0y7 objective: to analyze the effects of a short course of methyl-prednisolone pulses (mp) during the second week of disease (week-2) in patients with severe coronavirus disease 2019 (covid-19) pneumonia. methods: comparative observational study using data collected from routine care at hospital universitario cruces, barakaldo, bizkaia, spain in patients with covid-19 pneumonia. we compared patients who received week-2-mp (125–250 mg/d x3) with those who did not, with the end-points time to death and time to death or endotracheal intubation. results: we included 242 patients with covid-19 pneumonia and elevated inflammatory markers at admission. sixty-one patients (25%) received week-2-mp. twenty-two patients (9%) died and 31 (12.8%) suffered death or intubation. the adjusted hrs for death and death or intubation for patients in the week-2-mp group were 0.35 (95%ci 0.11 to 1.06, p = 0.064) and 0.33 (95%ci 0.13 to 0.84, p = 0.020), respectively. these differences were specifically seen in the subcohort of patients with a spo2/fio2 at day 7 lower than 353 (adjusted hr 0.31, 95% ci 0.08 to 1.12, p = 0.073 and hr 0.34, 95%ci 0.12 to 0.94, p = 0.038, respectively) but not in patients with higher spo2/fio2. patients receiving out-of-week-2-mp, non-pulse glucocorticoids or no glucocorticoids had an increased adjusted risk for both outcomes compared with week-2-mp group: hr 5.04 (95% ci 0.91–27.86), hr 10.09 (95% ci 2.14–47.50), hr 4.14 (95% ci 0.81–21.23), respectively, for death; hr 7.38 (95% ci 1.86–29.29), hr 13.71 (95% ci 3.76–50.07), hr 3.58 (95% ci 0.89–14.32), respectively, for death or intubation. these differences were significant only in the subgroup with low spo2/fio2. conclusions: week-2-mp are effective in improving the prognosis of patients with covid-19 pneumonia with features of inflammatory activity and respiratory deterioration entering the second week of disease. the recognition of this high-risk population should prompt early use of mp at this point. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 95% ci 0.08 to 1.12, p = 0.073 and hr 0.34, 95%ci 0.12 to 0.94, p = 0.038, respectively) but not in patients with higher spo2/fio2. patients receiving out-of-week-2-mp, non-pulse glucocorticoids or no glucocorticoids had an increased adjusted risk for both outcomes compared with week-2-mp group: hr 5.04 (95% ci 0.91-27.86), hr 10.09 (95% ci 2.14-47.50), hr 4.14 (95% ci 0.81-21.23), respectively, for death; hr 7.38 (95% ci 1.86-29.29), hr 13.71 (95% ci 3.76-50.07), hr 3.58 (95% ci 0.89-14.32), respectively, for death or intubation. these differences were significant only in the subgroup with low spo2/ fio2. beginning in december 2019, a novel coronavirus, designated sars-cov-2, has caused an international outbreak of respiratory illness termed covid-19 [1, 2] . glucocorticoids were banned in the initial recommendations for treating the disease [3] . however, severe lung and systemic inflammation may take place usually during the second week of disease, being the main cause of admission to intensive care units, need for mechanical respiratory support and death [4] . admission of patients with covid-19 started at hospital universitario cruces in march 2020, reaching its peak between the 27 th of march and early april. an initial period of no use of glucocorticoids [3] was followed by the administration of non-pulse glucocorticoids and later methyl-prednisolone pulses (mp) to selected patients. on april 3 rd 2020, a joined protocol by the services of infectious diseases and internal medicine included mp in certain scenarios (see methods). the aim of this study is to analyse the effects of mp on the clinical course of patients with covid-19 pneumonia admitted to the services of infectious diseases and internal medicine of hospital universitario cruces. our working hypothesis was that a short course of mp administered within the second week after the onset of symptoms (week-2-mp) would improve the outcome of patients with covid-19 pneumonia with markers of inflammation and signs of progressive respiratory deterioration. we conducted an observational study using data collected from routine care to assess the efficacy of week-2-mp to treat patients with covid-19 pneumonia. the protocol was approved by the basque country research ethics committee (code epa2020032) in accordance with the declaration of helsinki's guidelines for research in humans. this study has been registered in the eu pas register with the number eupas36287. all patients admitted between march 1 st and april 30 th to the services of infectious diseases and internal medicine, hospital universitario cruces, with a diagnosis of covid-19 pneumonia, supported by chest x-ray and/or ct scan informed by skilled radiologists. all cases were confirmed by positive reverse-transcriptase-polymerase-chain-reaction (rt-pcr) assay for sars-cov-2 in nasopharyngeal swabs, were initially selected for the study. patients were excluded if they had no inflammatory markers at admission (see therapeutic protocols), died within the first week of symptoms, were admitted to hospital after the end of the second week, died of causes non-related to covid-19 infection or were initially admitted to the intensive care unit. initial management included supportive therapy with fluids and oxygenation with the goal of an o2 saturation �92%. the antiviral protocol was developed based on the recommendations by the spanish ministry of health and the spanish agency for medicines and health products (aemps) in the weeks prior to april 3, 2020 [5, 6] . hydroxychloroquine for 5 days associated with lopinavir/ritonavir for 7-10 days were recommended for all patients with pneumonia. selected patients with severe pneumonia -curb65 �2 [7] and/or spo2 in ambient air o2 <90%-could be treated with remdesivir for 5 to 10 days, at the discretion of the clinician, after enrolment in a clinical trial [8] or as an offlabel drug. following the recommendations of the world health organization (who) [3] , glucocorticoids were not used during the initial weeks of the pandemic, unless needed for comorbid conditions. after the recognition of the inflammatory phase of covid-19 pneumonia [4] , glucocorticoids were empirically given to selected patients with severe disease and positive inflammatory markers, defined as any of the following: lymphocyte count <800/mm3 (normal 1300-2900), platelet count <150000/mm3 (normal 135000-150000), ferritin >1000 ng/ml (normal 15-300), c-reactive protein >100 mg/l (normal 0-11), d-dimer >1000 ng/ml (normal 0-500). this was an empiric definition, in which the levels of ferritin, crp and d-dimers well above the upper normal limit were set taking into account the marked elevation in such parameters seen in many patients with covid-19 pneumonia. glucocorticoids were initially given at doses around 1 mg/kg/d during several days and later as mp, 125 to 250 mg/d for 3 consecutive days, similar to the scheme used in our patients with systemic autoimmune diseases [9] . our therapeutic protocol was updated on april 3 rd 2020, including the recommendation of mp for patients with covid-19 pneumonia with altered/worsening inflammatory parameters (lymphopenia, thrombocytopenia, rising ferritin, d-dimers and or c-reactive protein) and clinical deterioration, particularly those showing impending respiratory failure with decreasing spo2/fio2 values. mp were encouraged to be given during the second week after the onset of symptoms, always according to the attending physician best judgment. this therapeutic protocol remained unchanged until the end of the study period. all the clinical data were extracted from hospital universitario cruces electronic medical records by the study investigators. databases were accessed to extract the study data between 11 th and 30 th may 2020. all data were fully anonymized before the analysis and the basque country research ethics committee waived the requirement for informed consent. baseline variables included age, gender, previous diagnosis of diabetes mellitus, obesity (body mass index �30), arterial hypertension, chronic pulmonary disease, active neoplastic, neurodegenerative disease or systemic autoimmune disease and immunosuppressive therapy. the curb65 scale [7] was calculated at admission and divided into three categories (low, medium and high risk). in order to assess severity within the second week of disease, the spo2/fio2 at that point was included in the database. the definition of high inflammatory state at admission described in the therapeutic protocol in use (see above) was assumed for the purposes of this study. therapeutic variables included the following: lopinavir/ritonavir, hydroxychloroquine, non-pulse glucocorticoids (including the average daily dose and the number of days of treatment), low molecular weight heparin and mp (including the week of administration, counting after the onset of symptoms). we used two primary outcomes: time to death (attributed to covid-19) and time to death or endotracheal intubation. mean and standard deviation or median and interquartile range were used to describe continuous variables, according to their distributional characteristics. counts and relative frequencies describe categorical variables. the time of onset of symptoms (fever and/or persistent cough), as reported by the patient, was the time origin for calculation of time to events. life tables were used to describe risk of events over the follow-up period, which ended either at the occurrence of the event of interest, at discharge or at the end of the study follow-up on 20 th may 2020. kaplan-meier failure curves with log-rank test were calculated for each of the two primary outcomes, using the dichotomous variable week-2-mp, yes/no, for group comparisons. adjusted cox proportional risk models were fitted to assess the effect of the dichotomous variable week-2-mp as the predictor of main interest. the aforementioned variables were included in the full model. likelihood ratio tests were used in a sequential fashion in order to find a reduced adjusted model containing statistically significant covariates. hazard ratios (hr) with 95% confidence intervals were used to estimate the magnitude of the association between risk predictors and outcomes. proportionality of hazards was tested through the use of comparison of adjusted and predicted survival curves and schoenfeld residuals. all the univariate and multivariate analyses were repeated after stratification by the median spo2/fio2 at week 2 (equal to 353), in two groups: spo2/fio2 �353 and >353 (designated as low spo2/fio2 and high spo2/fio2), as this variable showed a significant departure from the proportionality of hazards assumption. finally, the cox analysis was repeated using a four-category predictor in order to better illustrate the effects of glucocorticoids according to the dose, duration and time of administration. patients were divided into 4 groups: a) no-glucocorticoids, i.e. patients not receiving glucocorticoids in any form (n = 122); b) non-pulse glucocorticoids, i.e. patients receiving glucocorticoids at doses lower than 100 mg/d for periods longer than 3 days (n = 36, with 10 of them also receiving pulses); c) out-of-week-2-mp, i.e. mp at week 1 or 3, with no additional glucocorticoids at lower doses (n = 30); d) week-2-mp, i.e. patients receiving mp during week 2, with no additional glucocorticoids at lower doses (n = 54). as our proposed schedule consists of mp with no following tapering scheme, we decided to group all patients receiving nonpulse courses of glucocorticoids for longer than 3 days into the same category and keeping in the mp groups those patients receiving only pulses. neither patients nor the public were involved in the conception or conduct of the study. three hundred and forty-three patients with covid-19 pneumonia were initially identified. of these, 252 had an inflammatory state at admission; after excluding 2 patients with early death within the first week of disease course, 2 patients dying with covid-19 but with death attributable to terminal cancer and 6 patients admitted after the end of the second week of disease, 242 patients were selected for the analysis of the primary and secondary outcomes. sixty-one patients (25%) received week-2-mp. in addition, 33 patients (14%) received outof-week-2-mp (week 1 or 3). the remaining 148 patients (61%) did not receive mp. table 1 shows the clinical characteristics of the whole cohort and according to whether or not patients received week-2-mp. twenty-two patients (9.1%) died during the study period. the proportion of deceased patients was lower in the week-2-mp group: 4/61 (6.6%) vs. 18/181 (9.9%). the kaplan-meier failure curves ( fig 1a) showed a non-significant decreased risk of death of patients in the week-2-mp group (log-rank test, p = 0.102). the final cox model showed an adjusted hr for death of 0.35 (95%ci 0.11 to 1.06, p = 0.064) for patients in the week-2-mp group. other independent predictors of death included a previous diagnosis of arterial hypertension, the use of non-pulse glucocorticoids, a high-risk curb65 category and spo2/fio2 at week 2 ( table 2 ). in the subgroup with low spo2/fio2, 3/42 ( the final cox model in this subcohort showed a protective effect of week-2-mp (hr 0.31, 95% ci 0.08 to 1.12, p = 0.073), with the rest of independent predictors being unchanged from the whole cohort model. no differences were seen in the univariate analysis between patients with or without week-2-mp in the subpopulation with high spo2/fio2. only 5/120 (4%) patients died in this subgroup. the multivariate cox model could not identify any significant predictor of the outcome. thirty-one patients (12.8%) suffered death or intubation. week-2-mp patients had a lower incidence of the combined outcome: 6/61 (9.8%) vs. 25/181 (13.8%). the kaplan-meier failure curves (fig 2a) showed a non-significant largest time free of events in patients receiving week 2 mp, (log-rank test p = 0.125). in the final cox model, the adjusted hr for week-2-mp was 0.33 (95%ci 0.13 to 0.84, p = 0.020). the same independent predictors than in the model with death as the outcome variable, with the exception of arterial hypertension, were retained (table 3 ). in the subcohort with low spo2/fio2, the combined outcome was met by 5/42 (12%) vs. 20/80 (25%) patients receiving and not receiving, respectively, week-2-mp. the failure curves on fig 2b showed a significantly better outcome in the former group (log-rank test, p = 0.032). in the final cox model, week-2-mp were beneficial (hr 0.34, 95%ci 0.12 to 0.94, p = 0.038). the model retained the same final variables than the whole cohort analysis. six patients suffered the combined outcome in the subcohort of patients with high spo2/ fio2. again, no differences were seen between patients with or without week-2-mp in the univariate or multivariate analysis. however, the use of non-pulse glucocorticoids was associated with an increased risk for the combined outcome (hr 10.5, 95%ci 0.94 to 116.84, p = 0.056). table 4 depicts the clinical characteristics of the four subgroups. in the model with mortality as single outcome, week-2-mp patients showed lower mortality (hr 0.24, 95%ci 0.05 to 1.24, p = 0.088), whilst non-pulse glucocorticoid patients had an increased risk of death (hr 2.44, 95%ci 0.88 to 6.76, p = 0.087), both compared with patients with no glucocorticoids. the rest of the model included the same variables than the previous analysis (table 5 ). in the subcohort with low spo2/fio2, week-2-mp decreased mortality compared with no glucocorticoids, hr 0.14, 95%ci 0.02 to 1.28, p = 0.082. the remaining subgroups showed non significant differences. in the model with the combined death or intubation outcome, 2-week-mp were protective (hr 0.28, 95%ci 0.07 to 1.12, p = 0.072), whilst non-pulse glucocorticoids increased the risk table 6) . none of the analysis in the subcohort with high spo2/fio2 at week 2 showed significant differences between the four therapeutic groups. the results of the four-level glucocorticoid cox analysis with 2-week-mp as the main comparator are presented in table 7 . week-2-mp was superior to all the other therapies in order to prevent both main outcomes. the effects were more marked among patients with low sao2/fio2. this study supports the utility of glucocorticoids to improve the outcome of patients with covid-19 pneumonia. glucocorticoid use, however, should not be indiscriminate, but rather restricted to patients with laboratory evidence of inflammation and progressing respiratory compromise, and best used as short-course pulse therapy (125-250 mg/d of methyl-prednisolone during 3 days) administered during the second week after the onset of symptoms, where the hyperinflammatory reaction takes usually place. by the time the outbreak reached our region in late february 2020, the use of glucocorticoids was discouraged by the who [3] . this recommendation was based on a review of the clinical evidence of steroid therapy in other similar viral diseases, such as sars-cov-1, middle east respiratory syndrome (mers), influenza and respiratory syncytial virus, and on the idea that glucocorticoids could actually delay viral clearance [10] . however, an observational study of 201 patients with covid-19 pneumonia admitted to wuhan jinyintan hospital published in early march showed a reduced mortality among patients receiving methyl-prednisolone, although the dose, the duration and the time of administration were not specified [11] . indeed, new insights into the pathogenesis of the disease seem to support immunoregulation of some kind in patients affected by sars-cov-2, with the recognition of an inflammatory phase of the disease that can lead to extensive lung and multisystemic injury [4] . this second phase usually takes place during the second week after the onset of symptoms. recent data also support the utility of glucocorticoids in covid-19 disease [12] [13] [14] . in an observational study of 213 patients from detroit [12] , investigators compared patients of two groups, the standard of care (soc) and early corticosteroid protocol groups (cp). patients a second observational study from madrid compared 396 patients admitted for covid-19 treated with glucocorticoids with 67 not treated [13] . in the glucocorticoid-treated group, methyl-prednisolone was given either at 1 mg/kg/d (78.3%) or as 2 to 4-day pulses, up to 500 mg/d (21.7%) with subsequent tapering in 25% patients. the duration of therapy with 1 mg/ kg/d was not specified. the median time from disease onset to initiation of glucocorticoid was 10 days. this study showed a significant reduction in mortality among glucocorticoid users (13.9% vs. 23.9%, hr 0.51, 95%ci0.27-0.96), with differences being significant only in the subgroup classified as moderate-severe disease. there was no difference in mortality between patients receiving 1 mg/kg/d or pulses, however, 70 patients received mp a mean of 5 days after the failure of the 1 mg/kg/d scheme. the preliminary results from the recovery trial have just been released [15] . patients with suspected or confirmed covid-19 infection were randomised to receive dexamethasone (n = 2104) or soc (n = 4321). the dexamethasone protocol consisted of 6 mg/d for up to 10 days, although the actual median number of days was 6, and 7% of soc patients also received dexamethasone. the primary outcome, all-cause mortality within 28 days of randomization, was met by 21.6% patients allocated to dexamethasone vs. 24.6% allocated to usual care (rr 0.83; 95% ci 0.74 to 0.92). the reduction of mortality was only significant in patients receiving respiratory support. following the press release of these results, the who has already demanded an increase in dexamethasone production [15] . in contrast, a more recent brazilian double-blind, randomized, placebo-controlled trial in patients with clinical or radiological suspicion of covid-19 pneumonia, with spo2 � 94% at table 4 . clinical characteristics of the cohort according to type of glucocorticoid therapy. non-pulse-glucocorticoids (n = 36) week-2-mp (n = 54) room air or need for non-invasive or invasive ventilation reported no benefit from the use of methyl-prednisolone [16] . they were treated with methyl-prednisolone, 0.5 mg/kg/12h, or placebo for five days. the 28-day mortality rates in the 393 patients who completed the followup was 37.1% vs. 38.2% in the intervention and placebo groups, respectively. of note, the median number of days between disease onset and randomization was 13 days in both groups. authors reported, in the subgroup of patients older than 60 years who also had higher levels of c-reactive protein, a lower mortality among patients treated with methyl-prednisolone. thus, a beneficial role of glucocorticoids in patients with severe covid-19 disease has been generally found [12] [13] [14] 16] . whether they should be used in short-term pulses (i.e. doses of methylprednisolone between 100-500 mg/d for 3-4 days) or at lower doses for more prolonged periods of time is still an open question. the advantages of dexamethasone over methyl-prednisolone are not well established; besides the large sample size and the randomised controlled trial design of the recovery study [14] , the difference between the treated and untreated groups was quantitatively smaller than in the studies using methyl-prednisolone [12, 13] . the time of administration of glucocorticoids has been variable, although in the brazilian study, which concluded with mostly negative results, they were administered within the third week of disease in 50% of patients [16] . thus, specific recommendations about which corticosteroid should be administered, at what dose, for how long and when in the clinical course, could not be yet made. our study confirms that patients with covid-19 disease with a high inflammatory profile and respiratory compromise are most likely to benefit from glucocorticoid therapy. in addition, our study found that only in patients treated in the second week after the onset of symptoms seemed mp to be effective. we also observed a clear difference between the use of a short course of mp and more prolonged reduced doses of glucocorticoids, the former preventing and the second being associated to an increased risk of both unfavourable outcomes in all developed models. the presence of unidentified sources of bias cannot be excluded even after statistical adjustment for other prognostic factors and thus we cannot assure this deleterious effect on non-pulse methyl-prednisolone, moreover in the light of the results of previous studies. however, the superiority of week-2-mp over all the remaining options was clearly identified in our cohort. our study does not support the use of non-pulse glucocorticoids in the setting of severe covid-19 pneumonia, either alone or combined with mp. these findings are actually in agreement with the results seen in patients with systemic lupus erythematosus and other autoimmune diseases. the activation of the non-genomic way by mp, given during a short period of time, maximises the anti-inflammatory activity of glucocorticoids without relevant side effects [17] [18] [19] . on the other hand, doses in the range of 40-90 mg/d during a period of one week or more are less effective and can increase the risk of infections. the reason for this is the full activation of the genomic way, which is responsible for most of the serious secondary effects of glucocorticoids [17] [18] [19] . the most important limitation of this study is the observational design with a high variability in the therapeutic schemes, a consequence of the rapidly changing situation during this highly stressing pandemic. indeed, the actual number of patients treated with mp was lower than expected after the approval of the revised protocol. patients receiving mp also had a more severe disease. thus, although the analysis included a high number of potentially predictive variables in the initial multivariate cox models, it is possible that some unidentified confounder may have influenced our results. we also had a low number of patients meeting the final endpoints compared with other studies [12] [13] [14] 16] . a possible explanation for this is that, despite the high pressure attained, our hospital did not collapse as many others in spain. despite the consequent limited statistical power, the risk reduction among patients receiving week-2-mp was consistently between 60-70% for both endpoints in all the models, which supports a clinically relevant effect. a critical point in the clinical application of our results is the correct identification of the point of disease onset, in order to make the correct evaluation and starting mp, if needed, early in the second week. this is not always easy, since non-specific symptoms may precede the fully symptomatic phase of the disease by several days and because some patients even with radiological features of severe pneumonia can be oligosymptomatic [20] . thus, a thorough anamnesis must be made at admission with specific questions regarding the starting date of fever, persistent cough and/or dyspnea. this study confirms that mp, 125-250 mg/d for 3 consecutive days given during the second week of disease without subsequent tapering, improve the prognosis of patients with covid-19 pneumonia, features of inflammatory activity and respiratory deterioration. our results open the door to a more rational and planned management of patients with covid-19. those with negative inflammatory markers and normal spo2 seem to have a good prognosis, thus clinical observation monitoring oxygen saturation (even at home), could be appropriate. on the other hand, those approaching the second week of disease with worsening inflammatory markers and respiratory failure would greatly benefit from a short course of mp, which could not only be life-saving, but also help avoid the overload of critical care units. clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study coronavirus disease 2019-covid-19 clinical management of severe acute respiratory infection when novel coronavirus (no) infection is suspected (who/2019-ncov/clinical/2020.4) covid-19 illness in native and immunosuppressed states: a clinical-therapeutic staging proposal manejo clínico del covid-19: tratamiento mé dico tratamientos disponibles para el manejo de la infecció n respiratoria por sars-cov-2.18 de marzo de 2020 defining community acquired pneumonia severity on presentation to hospital: an international derivation and validation study study to evaluate the safety and antiviral activity of remdesivir (gs-5734™) in participants with severe coronavirus disease (covid-19) mobile app. version 1.05. osakidetza clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in wuhan, china early short course corticosteroids in hospitalized patients with covid-19 a retrospective controlled cohort study of the impact of glucocorticoid treatment in sars-cov-2 infection mortality dexamethasone in hospitalized patients with covid-19-preliminary report covid-19: demand for dexamethasone surges as recovery trial publishes preprint for the metcovid team. methylprednisolone as adjunctive therapy for patients hospitalized with covid-19 (met-covid): a randomised, double-blind, phase iib, placebo-controlled trial seventy years after hench's nobel prize: revisiting the use of glucocorticoids in systemic lupus erythematosus unraveling the functions of the membrane-bound glucocorticoid receptors: first clues on origin and functional activity glucocorticoids in the treatment of rheumatic diseases: an update on the mechanisms of action ct features of sars-cov-2 pneumonia according to clinical presentation: a retrospective analysis of 120 consecutive patients from wuhan city we thank all the health professionals from hospital universitario cruces involved in the care of patients with covid19 formal analysis: guillermo ruiz-irastorza, jose-ignacio pijoan.methodology: guillermo ruiz-irastorza.supervision: guillermo ruiz-irastorza. key: cord-345019-i7zm9bt1 authors: al-waleedi, ali ahmed; naiene, jeremias d.; thabet, ahmed a. k.; dandarawe, adham; salem, hanan; mohammed, nagat; al noban, maysa; bin-azoon, nasreen salem; shawqi, ammar; rajamanar, mohammed; al-jariri, riyadh; al hyubaishi, mansoor; khanbari, lina; thabit, najib; obaid, basel; baaees, manal; assaf, denise; senga, mikiko; bashir, ismail mahat; mahmoud, nuha; cosico, roy; smith, philip; musani, altaf title: the first 2 months of the sars-cov-2 epidemic in yemen: analysis of the surveillance data date: 2020-10-29 journal: plos one doi: 10.1371/journal.pone.0241260 sha: doc_id: 345019 cord_uid: i7zm9bt1 introduction: yemen was one of the last countries in the world to declare the first case of the pandemic, on 10 april 2020. fear and concerns of catastrophic outcomes of the epidemic in yemen were immediately raised, as the country is facing a complex humanitarian crisis. the purpose of this report is to describe the epidemiological situation in yemen during the first 2 months of the sars-cov-2 epidemic. methods: we analyzed the epidemiological data from 18 february to 05 june 2020, including the 2 months before the confirmation of the first case. we included in our analysis the data from 10 out of 23 governorates of yemen, located in southern and eastern part of the country. results: a total of 469 laboratory confirmed, 552 probable and 55 suspected cases with onset of symptoms between 18 february and 5 june 2020 were reported through the surveillance system. the median age among confirmed cases was 46 years (range: 1–90 years), and 75% of the confirmed cases were male. a total of 111 deaths were reported among those with confirmed infection. the mean age among those who died was 53 years (range: 14–88 years), with 63% of deaths (n = 70) occurring in individuals under the age 60 years. a total of 268 individuals with confirmed sars-cov-2 infection were hospitalized (57%), among whom there were 95 in-hospital deaths, conclusions: the surveillance strategy implemented in the first 2 months of the sars cov 2 in the southern and eastern governorates of yemen, captured mainly severe cases. the mild and moderate cases were not self-reported to the health facilities and surveillance system due to limited resources, stigma, and other barriers. the mortality appeared to be higher in individuals aged under 60 years, and most fatalities occurred in individuals who were in critical condition when they reached the health facilities. it is unclear whether the presence of other acute comorbidities contributed to the high death rate among sars-cov-2 cases. the findings only include the southern and eastern part of the country, which is home to 31% of the total population of yemen, as the data from the northern part of the country was inaccessible for analysis. this makes our results not generalizable to the rest of the country. although pneumonia caused by a novel coronavirus was first described in china in december 2019, it was officially named as coronavirus disease 2019 (covid-19) on 11 february 2020 [1] . the virus that causes the disease was named severe acute respiratory syndrome coronavirus-2 (sars-cov-2) due to its genetic similarities to the coronavirus responsible for the severe acute respiratory syndrome (sars) epidemic in 2003 [2] . sars-cov-2 is transmitted through respiratory droplets and the case fatality rate ranges from 0.3% to 15% among the confirmed cases, primarily due to pulmonary complications [1, 3] . the incubation period is estimated to be from 2 to 14 days, with majority of the cases developing symptoms 5 days after exposure to the virus. the signs and symptoms may include fever, cough, difficult breathing, fatigue, headache and others [4] . the virus can also be transmitted by individuals who are asymptomatic carriers of the virus [5] . the development of symptoms and fatality varies by age group, with older people being most at risk of becoming symptomatic and die [6] . yemen was one of the last countries in the world to declare the first case of the pandemic, on 10 april 2020 [7] . fear and concerns of catastrophic outcomes of the epidemic in yemen were immediately raised, as the country is facing a complex humanitarian crisis [7] . suppression measures were already being implemented by the government before the notification of the first case, including curfew, closure of schools, airports, markets, mosques, and prohibiting public gatherings [8] . about 50% of the population of yemen is estimated to be in acute need of health care, with high rates of malnutrition, child and maternal mortality [7] . in addition, the limited availability of safe drinking water, people living crowded houses, inadequate sanitation, and stigma constitute barriers for effective response and control of the epidemic in yemen [9] . due to the ongoing armed conflict, less than 50% of the health facilities in yemen are fully functional [7] and more that 2 million children are malnourished [7] . yemen has a population of 30 million people, and the armed conflict which started in 2015 led to a fragmentation of the healthcare system [7, 8] . for early detection of sars-cov-2 in yemen, as in other countries, a case definition, active surveillance, and contact tracing were required [10, 11] . the purpose of this report is to describe the epidemiological situation in yemen during the first 2 months of the sars-cov-2 epidemic, from 5 april to 5 june 2020. the report also includes the 2 months before the notification of the first confirmed case from 18 february to 10 april 2020, as well as the challenges and information gaps. we analyzed the epidemiological data from 18 february to 05 june 2020. we included in our analysis the data from 10 out of 23 governorates of yemen, namely abyan, aden, al-dhale'e, al-mahrah, hadramout, lahj, marib, shabwa, taizz and the island of socotra, located in southern and eastern part of the country controlled by the internationally recognized government. yemen has a total population of 30 million according to projections from the 2004 census, with 31% of the population located in the southern and eastern governorates. the data from the northern governorates were not available for analysis. the cases and contacts were investigated by 5-member multidisciplinary rapid response teams (rrts) in each district, comprising a clinician, laboratory technician, surveillance officer, risk communication officer and an environmental health officer. a line list of cases and contacts was compiled daily in a microsoft excel spreadsheet using the data received from all the governorates. all the variables in the line list were extracted from the case-based form used in the country. these included demographic information, signs and symptoms, history of contact with other cases, history of travel, comorbidities, and hospitalization data. the comorbidities only included some specific non-communicable diseases, although information regarding communicable diseases and other health conditions was sometimes recorded in the comments section of the form. contact tracing data were available only from hadramout governorate. we constructed the chains of transmission of the other governorates using the information available in the line list. the contact tracing activities started with the reporting of the first confirmed case, who had onset of illness on 5 april 2020. we decided to limit the analysis of the chains of transmission to the first 250 cases, with onset of illness from 5 april to 25 may 2020, when the collection of the information was more consistent. the contacts were followed up daily by the rrts. each team was responsible to follow up a maximum of 10 contacts to ensure quality and consistence of contact tracing data. there was no specialized software available for contact tracing and the daily analysis was done through microsoft excel. a temporary interruption followed by inconsistency of contact tracing data reporting was observed after 25 may 2020. when the number of contacts exceeded the recommended 10 contacts per contact tracing team the daily information and analysis regarding the status of the contacts became irregular. we used the microsoft excel to perform univariate analysis of the cases and contacts data. yemen adopted the who case definition of suspected, probable, and confirmed cases in march 2020 [12] (fig 1) . the suspected cases had to present acute respiratory illness and were subdivided in the following components: a component "a" with history of travel to affected countries, a component "b" with history of contact with confirmed or probable cases and a the first 2 months of the sars-cov-2 epidemic in yemen component "c" requiring hospitalization, without a clear diagnosis, regardless to history of travel of contact with sick people. due to limited laboratory supplies, component "c" of definition of a suspected case was given low priority for several weeks. probable cases were suspected cases either not tested or with inconclusive laboratory results. cases with positive laboratory results for covid-19 were classified as confirmed. real-time reverse transcriptase polymerase chain reaction (rt-pcr) performed to nasopharyngeal swabs specimens was used for laboratory confirmation of sars-cov-2 infection. the rrts transported the specimens to the laboratories in viral transport medium. only cases that were positive for sars-cov-2 using rt-pcr were classified as confirmed. a total of 5 central public health laboratories (cphls) were available in different geographical areas of the country to perform rt-pcr, namely in aden, mukalla, sayoun, taizz, and sana'a. data for the cphl in sana'a were not provided by the local authorities from the north of yemen, and therefore were not included in our analysis. the aden cphl started sars-cov-2 testing on 21 march 2020, and confirmed the first case on 29 april 2020. mukalla cphl started sars-cov-2 testing on 8 april 2020, and confirmed the first case confirmed on 10 april 2020. this was the first confirmed case in yemen. the taizz cphl starting testing for sars-cov-2 on 27 april, and confirmed the first case on 1 may 2020. sayoun was the last cphl to start testing for sars-cov-2 on 6 may 2020, and confirmed the first case on 9 may 2020. the analysis and publication of the data was authorized by the scientific committee of the ministry of public health and population in aden. no additional ethical approval was required because the data collection and analysis were part of an outbreak response and not a research. the requirement for informed consent was waived because the study is based on a retrospective analysis of routine surveillance data collected in an emergency. the patients and health workers details were kept confidential. a total of 469 laboratory confirmed, 552 probable and 55 suspected cases with onset of symptoms between 18 february and 5 june 2020 were reported through the surveillance system (table 1) . of the confirmed cases, 3 had history of travel abroad within 14 days before the the first 2 months of the sars-cov-2 epidemic in yemen onset of symptoms, namely to saudi arabia (n = 2) and egypt (n = 1). the median age among confirmed cases was 46 years (range: 1-90 years), and 75% of the confirmed cases were male ( table 2) . three confirmed cases were reported in children under 5 years old. a total of 111 deaths were reported among those with confirmed infection, 71% of them (n = 79) occurring in males. the mean age among those who died was 53 years (range: 14-88 years), with 63% of deaths (n = 70) occurring in individuals under the age 60 years. hadramout governorate reported the highest number of deaths (n = 40). among those with confirmed infection, diabetes (11%) was the most common comorbidity among those with confirmed infection, and the first 2 months of the sars-cov-2 epidemic in yemen hypertension (17%), diabetes (16%), and chronic lung diseases (13%) were the most common comorbidities among those who died. one of the fatal cases from hadramout governorate was in an individual with hiv infection. a pregnant woman of approximately 20 weeks gestation was reported in hadramout. her pregnancy ended in a spontaneous miscarriage after the onset of her covid-19 symptoms. no underlying conditions were reported in 61% of the cases. the most common symptoms were fever, cough, and difficulty breathing and sore throat ( table 3 ). in addition to the symptoms shown in the table, 26 individuals reported a headache and 6 reported a loss of smell and taste. housewives were the most commonly affected occupational category, followed by healthcare workers and soldiers (table 4 ). among the healthcare workers, medical doctors and laboratory technicians were the most frequently affected, with 3 deaths of medical doctors and 2 deaths of laboratory technicians reported. a marked increase occurred in number of cases reported according to the date of onset of symptoms from 24 april 2020, with a peak on 15 may 2020 (fig 2) . aden and hadramout were the governorates that reported the highest numbers of confirmed cases, followed by taizz. although the first case that was confirmed in aden tested on 29 april 2020, 20 other cases in the governorate were subsequently confirmed that had earlier dates of onset of symptoms than the first case. a total of 268 individuals with confirmed sars-cov-2 infection were hospitalized (57%), among whom there were 95 in-hospital deaths, and 27 were admitted to an intensive care unit (icu), including 14 of those who died subsequently. of the patients admitted to an icu, 11 were provided with mechanical ventilation, including 8 of those who died (table 5) . among the 95 patients who died in hospital, 44 had both the date of admission and the date of death the first 2 months of the sars-cov-2 epidemic in yemen recorded in the line list. of these, 73% (n = 32) died within 24 hours after admission. the mean time from admission to death was 1.1 days. the mean time from admission to death and from onset of symptoms to death per district is described in the s1 table. the date of death was missing in 51 cases (s1 table) . of all the fatal cases reported, 6 individuals were reported to be dead on arrival (3 from mukalla city in hadramout, 2 from qairah district in taizz, and 1 from salh district in taizz). al-mukalla city in hadramout governorate reported 28% (n = 31) of all the deaths in the country (s2 table) (fig 3) . (fig 4) . all the chains of transmission had 1 generation of cases, except one which had 2 generations. the average number of cases generated by each index case was 2 (range: 1-5). the average time between the onset of symptoms of the index case to onset of symptoms of the contacts was 8.8 days (range: 2-22 days). the source of infection and the cases generated by 180 cases were not identified. the contact tracing activities were interrupted in aden governorate in the first week of the epidemic due to limited human resource capacity, community resistance, and security issues. the first 2 months after confirmation of the sars-cov-2 epidemic in yemen was characterized by a 57% hospitalization rate in the southern and eastern parts of the country included in the first 2 months of the sars-cov-2 epidemic in yemen our study, 63% of deaths occurring in individuals aged <60 years, confirmatory testing of <50% of the suspected cases, and majority of cases were not related to a defined chain of transmission. therefore, the country satisfied the criteria to be classified as a community transmission setting [12] . the percentage of severe cases requiring admission in yemen was more than the double that reported in several other countries including iran, where the percentage was reported to be approximately 20% [1, 5, 13, 14] . however, the mean time from onset of symptoms to admission to the health facility was 5.4 days, which is comparable to the average of 5-10 days reported in other countries [1, [15] [16] [17] [18] . closure of some hospitals, and other hospitals refusing to receive and admit patients with acute respiratory illness may had led to notification of more serious cases, as cases of mild and moderate severity were not self-reported [19] . the closure of health facilities was mainly due to a shortage of personal protective equipment and other supplies to manage sars-cov-2 [7] . in addition, there was a shortage of trained rrts, who were responsible for investigation and collection of laboratory samples of each case reported in the community and in health facilities [19] . this may also have led to underreporting, especially of mild cases that were not self the first 2 months of the sars-cov-2 epidemic in yemen reported due to limited resources, stigma, and other barriers. more than 50% of the cases were classified as probable due to a shortage of laboratory supplies to investigate all the cases reported [19] . the case fatality rate was also overestimated in china (>20%) at the beginning of the epidemic, when testing capacity was limited, but dramatically decreased once tests became widely available in the country [5] . as yemen was one of the last countries in the world to notify the first case, the sensitivity of the case definition remained high for long time, capturing mainly individuals with history of travel, and those with severe disease. therefore, it can be inferred that most of the mild and moderate cases were not reported. the fact that the index case had no clear source of infection and was identified on the day after the laboratory start testing for sars-cov-2 suggests that the infection was probably circulating in the country for a period prior to confirmation of the first case. the low prioritization of the component "c" of the case definition may have led to misdiagnosis of severe cases admitted to the hospitals before the confirmation of the first case. the epidemiological curve shows 20 confirmed cases reported in aden with date of onset of symptoms in the days before the confirmation of the first case and with no clear source of infection. however, it is possible that those who died of the infection may have had other acute and chronic comorbidities besides those captured in the line lists which may have contributed to their deaths [20] . the mean age at death was 54 years, which is much younger than that reported in iran, china and other countries where the mean age varies from 65 to 73 years [14, 21, 22] . in addition, 63% of all deaths were in individuals aged under 60 years, while other countries have reported less than 20% of deaths in this age group [5] . considering that the older people are at high risk for severe illness [6] , in yemen many of them likely died at home without reaching the health facilities. among the individuals who died in the hospitals, the mean time from admission to death was 1.1 days and 73% of deaths occurred within 24 hours of hospital admission. in other countries, in-hospital deaths due to sars-cov-2 have generally been reported to occur several days after admission [15, 16, 23, 24] . this indicates that individuals tended to reach health facilities late, with less chance of survival, especially among those who required mechanical ventilation [25] . as in other countries, there was no deaths reported in children aged under 15 years in yemen [24] . yemen is experiencing outbreaks of other infectious diseases such as dengue fever, measles, diphtheria, cholera [7, 19] , and the h1n1 influenza virus has also been identified in yemen [26] . therefore, co-existence of these diseases among sars-cov-2 infections should be considered. khat is a plant consumed in few countries, including yemen as stimulant of central nervous system with effects similar to amphetamines [27] . the majority of adults in yemen consume khat, but the effect that this has among individuals with acute and chronic illnesses remains unclear [8, 28] . it has been described in studies conducted in yemen that khat reduces the appetite, increases blood pressure and induces respiratory diseases [29] [30] [31] . however, no study has been conducted to assess the magnitude of these respiratory diseases, and so the possible effect of khat among individuals with sars-cov-2 infection is unknown. malnutrition, especially among adults should also be considered among the comorbidities contributing to sars-cov-2 deaths in yemen [7] . a higher number deaths was also observed in the areas with more limited resources iran and china in the early stages of sars-cov-2 epidemic [23, 32] . the mean age among confirmed cases was 47 years, with higher percentage of cases among males. this is similar that the demographics described in china in the early stage of the epidemic [18, 20] . however, considering that population pyramid in poor countries is usually younger that in developed countries, we also consider underreporting of cases among young people with mild symptoms in yemen. the signs and symptoms of the cases in yemen were also similar to those described in china [1, 33, 34] . in yemen, some patient reported a loss of the sense of taste and smell as described in other countries [35] . the contact tracing was successful in the beginning of the epidemic, especially in hadramout governorate, where the index case was detected. however, it was more complex in aden, where the first cases detected had no clear source of infection, human resources were limited, there was community resistance, and limited supplies, especially ppes for the contact tracers. in addition, contact tracing in conflict settings may be also a challenge due to security and accessibility issues [36] . however, household contacts, and healthcare workers were among the most affected people, which is similar to what was described in china [17] . our study has several potential limitations. the findings only include the southern and eastern part of the country, which is home to 31% of the total population of yemen, as the data from the northern part of the country was inaccessible for analysis. this makes our results not generalizable to the rest of the country, because besides the higher population, the northern part of yemen has different characteristics including lower temperature and higher altitude. some studies suggest that different climatic conditions, including temperature and altitude may affect the transmission and mortality due to sars-cov-2 infections [37, 38] . the case fatality rate is unclear, as most of the cases reported through the surveillance system were severe. therefore, we excluded this indicator from our analysis to avoid misinterpretations. the nutrition status of the cases among both adults and children was not reported in the line list, making it impossible to determine the prevalence of malnutrition among sars-cov-2 cases. the majority of sars-cov-2 articles published with detailed epidemiological analysis are from china and other developed countries. this makes comparisons challenging as yemen is a country with very different characteristics. the line list used for the analysis had a lot missing information, including key dates and key variables. however, this description of the findings and challenges may be useful for documentation and as a basis for assessing the improvement of the surveillance system in the later stages of the sars-cov-2 epidemic in yemen. the surveillance strategy implemented in the first 2 months of the sars-cov-2 in yemen, where 5 rrt members were responsible of investigation of each case in the communities and health facilities was shown to have limited effectiveness, especially in the areas when the available resources were the most limited. the surveillance system in the southern and eastern governorates included in our study captured mainly severe cases, making it difficult to interpret the mortality data. the mortality appeared to be higher in individuals aged under 60 years, and most fatalities occurred in individuals who were in critical condition when they reached the health facilities. it is unclear whether the presence of other acute comorbidities contributed to the high death rate among sars-cov-2 cases in yemen. we recommend a revision of the surveillance strategy, to reduce the burden on the rrts and the investigation of additional acute comorbidities among sars-cov-2 patients in yemen, including h1n1, dengue fever, malnutrition, and the effects of khat. supporting information s1 table. time novel coronavirus covid-19: current evidence and evolving strategies perspective sars-cov-2 vaccines: status report the many estimates of the covid-19 case fatality rate covid-19 and medical education the epidemiology and pathogenesis of coronavirus disease (covid-19) outbreak sars-cov-2 and coronavirus disease 2019: what we know so far estimating clinical severity of covid-19 from the transmission dynamics in wuhan, china covid-19 and conflict: the devastating impact of withdrawing humanitarian support on universal health coverage in yemen yemen is free of covid-19 responding to the covid-19 pandemic in complex humanitarian crises the implementation of integrated disease surveillance and response in liberia after ebola virus disease outbreak 2015-2017 enhancing ebola virus disease surveillance and prevention in counties without confirmed cases in rural liberia: experiences from sinoe county during the flare-up in monrovia global surveillance for covid-19 caused by human infection with covid-19 virus: interim guidance characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention epidemiological characteristics of coronavirus disease 2019 (covid-19) patients in iran: a single center study case report: pathological findings of covid-19 associated with acute respiratory distress syndrome clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study. lancet risk factors for severity and mortality in adult covid-19 inpatients in wuhan clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china world report fears of "highly catastrophic" covid-19 spread in yemen comorbidity and its impact on 1590 patients with covid-19 in china: a nationwide analysis understanding of covid-19 based on current evidence clinical characteristics of 25 death cases with covid-19: a retrospective review of medical records in a single medical potential association between covid-19 mortality and healthcare resource availability presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new york city area predictors of 24-h mortality after inter-hospital transfer to a tertiary medical intensive care unit epidemiology of fatal cases associated with pandemic influenza reported in yemen qat habit in yemen society: a causative factor for oral periodontal diseases the subjective effects of chewing qat leaves in human volunteers medical and social aspects of qat in yemen: a review a medical evaluation of the use of qat in north yemen coronavirus disease 2019 (covid-19) outbreak in iran: actions and problems review of the clinical characteristics of coronavirus disease 2019 (covid-19) epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study smell and taste dysfunction in patients corona virus infection in syria, libya and yemen; an alarming devastating threat significance of geographical factors to the covid -19 outbreak in india high-altitude populations need special considerations for covid-19 we thank all the rapid response teams' members across the country who conducted the investigations of the cases under difficult circumstances. we also thank the health workers and the partners who support the response activities in the country. key: cord-318696-jheb2fnn authors: kesic, matthew j.; meyer, megan; bauer, rebecca; jaspers, ilona title: exposure to ozone modulates human airway protease/antiprotease balance contributing to increased influenza a infection date: 2012-04-09 journal: plos one doi: 10.1371/journal.pone.0035108 sha: doc_id: 318696 cord_uid: jheb2fnn exposure to oxidant air pollution is associated with increased respiratory morbidities and susceptibility to infections. ozone is a commonly encountered oxidant air pollutant, yet its effects on influenza infections in humans are not known. the greater mexico city area was the primary site for the spring 2009 influenza a h1n1 pandemic, which also coincided with high levels of environmental ozone. proteolytic cleavage of the viral membrane protein hemagglutinin (ha) is essential for influenza virus infectivity. recent studies suggest that ha cleavage might be cell-associated and facilitated by the type ii transmembrane serine proteases (ttsps) human airway trypsin-like protease (hat) and transmembrane protease, serine 2 (tmprss2), whose activities are regulated by antiproteases, such as secretory leukocyte protease inhibitor (slpi). based on these observations, we sought to determine how acute exposure to ozone may modulate cellular protease/antiprotease expression and function, and to define their roles in a viral infection. we utilized our in vitro model of differentiated human nasal epithelial cells (necs) to determine the effects of ozone on influenza cleavage, entry, and replication. we show that ozone exposure disrupts the protease/antiprotease balance within the airway liquid. we also determined that functional forms of hat, tmprss2, and slpi are secreted from human airway epithelium, and acute exposure to ozone inversely alters their expression levels. we also show that addition of antioxidants significantly reduces virus replication through the induction of slpi. in addition, we determined that ozone-induced cleavage of the viral ha protein is not cell-associated and that secreted endogenous proteases are sufficient to activate ha leading to a significant increase in viral replication. our data indicate that pre-exposure to ozone disrupts the protease/antiprotease balance found in the human airway, leading to increased influenza susceptibility. influenza a virus is responsible for the seasonal epidemics and reoccurring pandemics, which represents a worldwide threat to global public health [1] . it is a major cause of morbidity and mortality worldwide, as during the recent h1n1 pandemic. in the u.s. alone, over 100,000 individuals are hospitalized and over 20,000 people die every year due to influenza virus infection and related diseases [2, 3] . despite large-scale efforts in vaccination and antiviral therapies, the morbidity and mortality rates associated with influenza infections have not significantly changed in recent years [4, 5] . in the context of potentially pandemic respiratory viral infections, it is important to identify molecular targets/pathways for therapeutic intervention to protect susceptible sub-populations. epidemiologic studies show that exposure to inhaled oxidants such as cigarette smoke, diesel exhaust, and ozone can modulate immune function and increase susceptibility to respiratory viral infections [6, 7, 8, 9, 10, 11] . despite the extensive study into different factors influencing susceptibility to infection, the mechanism(s) by which inhaled oxidants modify viral pathogenesis are very complex and have yet to be fully elucidated. the ability of oxidants to cause lung injury/dysfunction is dependent, in part, on the delicate equilibrium that exists between oxidants and antioxidants. oxidative stress is caused by an imbalance between the production of reactive oxygen species (ros) and the body's ability to readily detoxify reactive intermediates. ozone is one of the most abundant components of air pollution in urban areas, and has been shown to be a potent inducer of oxidative stress causing airway inflammation and increased respiratory morbidities [8, 9, 12] . the mexico city metropolitan area (mcma), one of the most densely populated areas in the world, experiences high levels of air pollutants such as environmental ozone and particulate matter (pm) [13, 14, 15] . the mcma is located 2240 m above sea level and is surrounded by mountains. due to this geographical location, there is less available oxygen, making combustion less efficient, which produces more polycyclic aromatic hydrocarbon (pah) pollutants. for these reasons, mexico city receives higher levels of environmental ozone and various other types of photochemical smog [13] . it was here that the first influenza pandemic of the 21 st century emerged in march 2009. it was responsible for an estimated ,258,698 laboratory confirmed cases and roughly ,1,370 deaths by december 2009 [1, 16, 17] . the normal ''flu season'' occurs during the colder half of the year in each hemisphere [18] . interestingly, this outbreak emerged during the dry and warmer months when ozone levels were significantly higher [19, 20] . possible mechanisms by which oxidative stress alters the airway environment leading to broadened cellular tropism and/or susceptibility to viral infections have been proposed. [21, 22, 23] the relationship between oxidative stress and the cellular protease/antiprotease balance has been considered to be a major contributor in the development of numerous airway pathologies [24] . early studies demonstrated that the cleavage of influenza ha is essential for viral penetration into host cells and that this mechanism was mediated by cellular trypsin-like serine proteases. these proteases, in turn, are regulated by mucus antiproteases, such as slpi and a 1 -antitrypsin (a1at) [25, 26, 27, 28, 29] . recent studies have identified specific cellular proteases and antiproteases that may be involved in influenza infection, which include transmembrane protease serine 2 (tmprss2), human airway trypsin-like protease (hat), and secretory leukocyte proteinase inhibitor (slpi) [30, 31, 32, 33, 34] . the expression of these proteases in the lung are necessary for cleavage of the viral ha surface protein, thus allowing viral fusion and entry into the host cell. studies have shown a correlation between inflammation and oxidative stress which alters expression of these proteases and antiproteases [29, 35] . specifically, hat has been shown to be released into the airway fluids under inflammatory conditions, particularly in asthmatics [31, 36] . in addition, a murine study showed slpi expression was suppressed and protease expression was increased in nrf2-deficient mice which led to increased inflammation, further demonstrating a relationship between oxidative stress and protease expression [37] . oxidative stress derived from cigarette smoke or addition of reactive oxygen intermediates has been shown to decrease antiprotease activity [35, 38] and a murine study demonstrated that decreased antiprotease expression increased influenza infectivity [39] . the imbalance of protease/antiprotease expression caused by oxidative stress has been attributed to both an increase in inflammatory cells, which release proteolytic enzymes capable of destroying lung tissue, and a functional deficit of antiproteases due to oxidation of their active site [35, 40] . in addition to influenza, studies have shown that regulated proteolysis is required for the spread/ propagation of many human viruses, including human immunodeficiency virus (hiv), nipah, ebola, severe acute respiratory syndrome coronavirus (sars-cov), and metapneumoviruses [41, 42, 43, 44, 45] . although it has been shown that oxidative stress increases severity of viral infections, the exact mechanism as to how and why this happens and the role of ozone exposure in these responses are not fully understood. while the deleterious health effects of both ozone and influenza have been well documented, few studies have looked at the effects of ozone in the context of an influenza infection. herein, we used our established cell culture model of differentiated human nasal epithelial cells (necs) [46] , exposed them to 0.4ppm of ozone, and determined the effects on susceptibility to an influenza a infection. we show that there is a delicate balance of proteases and antiproteases present in the human airway surface liquid. additionally, we found exposure to ozone disrupts this equilibrium by enhancing protease secretion while inhibiting antiprotease expression leading to increased susceptibility to viral infection. we directly show that ozone increases soluble protease expression and that they are functional for cleaving the viral ha protein and activation of the intact influenza virion. primary human nasal epithelial cells (necs) were obtained from healthy adult volunteers. this protocol was approved by the university of north carolina school of medicine institutional review board for biomedical research. in addition, written informed consent was provided by each study participants and/or their legal guardians. primary human nasal epithelial cells (necs) were obtained as previously described [6] . briefly, necs were obtained from healthy adult volunteers by gently stroking the inferior surface of the turbinate several times with a rhino-probe curette (arlington scientific, arlington, tx), which was inserted through a nasoscope. this protocol was approved by the university of north carolina school of medicine institutional review board for biomedical research. nec were expanded to passage 2 in bronchial epithelial growth medium (begm, cambrex bioscience walkersville, inc., walkersville, md) and then plated on collagencoated filter supports with a 0.4 mm pore size (trans-clr; costar, cambridge, ma) and cultured in a 1:1 mixture of bronchial epithelial cell basic medium (bebm) and dmem-h with singlequot supplements (cambrex), bovine pituitary extracts (13 mg/ml), bovine serum albumin (bsa, 1.5 mg/ml), and nystatin (20 units) . upon confluency, all-trans retinoic acid was added to the basolateral medium to establish air liquid interface (ali) culture conditions (removal of the apical medium) to promote differentiation. mucociliary differentiation was achieved after 18-21 days post-ali. we obtained the madin darby canine kidney (mdck) cell line from the american type culture collection (atcc, manassas, va). the beas-2b cell line was derived by transforming human bronchial cells with an adenovirus 12-simian virus 40 construct [47] . we obtained our beas-2b cells from the american type culture collection (atcc, manassas, va). beas-2b cells were grown in keratinocyte basal medium (kbm) supplemented with 30 mg/ml bovine pituitary extract, 5 ng/ml human epidermal growth factor, 500 ng/ml hydrocortisone, 0.1 mm ethanolamine, 0.1 mm phosphoethanolamine, and 5 ng/ml insulin. the primary human necs were exposed under ali conditions to filtered air or 0.4 ppm ozone for 4 h in the exposure chambers operated by the u.s. environmental protection agency, environmental public health division. in noted experiments, 10 mm of a cell-permeable form of reduced glutathione, glutathione-ethylester (gsh-et) (sigma-aldrich st. louis. mo) or 1 mm of egcg (sigma-aldrich st. louis. mo) was added to the basolateral medium 30 min prior to ozone exposure, similar to our previous studies [6, 46] . construction of vlp expression plasmids. to generate the b-lactamase-m1 fusion expression plasmid (pcaggs-b-lacm1 pr8) the influenza a/puerto rico/8/34/mount sinai (h1n1) m1 sequence was pcr amplified from the m1 expression vector pdz-m (which was kindly provided by dr. adolfo garcia-sastre, mount sinai school of medicine) and inserted into the pcaggs vector [48, 49, 50] . the b-lactamase gene was pcr amplified from pcdna3.1 and fused n-terminally to m1 within pcaggs to create a modified b-lactamase-m1 fusion. in the modified b-lactamase, the first 24 amino acids were excluded to remove a secretion signal and his 24 was substituted with asp to create an optimal kozak consensus sequence. the pcdna3.1-blactamase construct has been described previously [51] . the influenza a/puerto rico/8/34/mount sinai (h1n1) ha (pcaggs-ha) and na (pdz-na) over-expression vectors were generously provided by dr. aldolfo garcia-sastre and have been described previously [48, 49, 50, 52] . production of vlps. to generate influenza a/pr/8/h1n1 b-lactamasem1vlps (pr8 b-lacm1 vlps), 293t cells were cotransfected with 3 mg each of pcaggs-ha, pdz-na, and pcaggs-b-lacm1 pr8 using fugeneh hd (roche applied science, indianapolis, in) according to manufacturer's instructions. the supernatant containing the vlps were collect 48 h post-transfection and clarified of floating cell debris by centrifugation at 3,000 rpm for 10 min. the vlps were concentrated once by low-speed centrifugation through an amicon ultra 100 kd centrifuge filter unit (millipore; billerica, ma), and the retentates were aliquoted and stored at 280uc. vlp entry assay. the vlp entry assay was performed as previously described [6] with modifications. briefly, target cells were exposed to filtered air or 0.4 ppm ozone for 4 h. 24 h postexposure, vlps were added to the apical surface in a total volume of 100 ml and incubated at 37uc for 3 h. the cells were washed twice with hbss to remove unbound virus and infected cells were detected by using geneblazer tm fret in vivo cell-based assay system substrate ccf2-am according to manufacturer's recommendations (invitrogen). samples were lysed in hbss by freeze-thaw method repeatedly treated for 3 cycles (frozen in liquid nitrogen for 3 min and thawed in a 65uc water bath for 3 min). viral entry was quantified by using the polarstar optima plate reader (bmg labtech, inc.). all experiments were performed in triplicate in three independent experiments. we used influenza a/bangkok/1/79 h3n2 serotype (which was kindly provided by dr. melinda beck, university of north carolina). we also obtained influenza a/malaya/302/1954 h1n1 serotype from the american type culture collection (atcc, manassas, va) for western blot analysis of the cleavage products of the viral ha protein. virus was propagated in 10-dayold embryonated hen's eggs. the virus was collected in the allantoic fluid and titered by 50% tissue culture infectious dose in madin-darby canine kidney cells and hemagglutination as described before [53] . stock virus was aliquoted and stored at 280uc until use. unless otherwise indicated, for infection about 5610 5 cells were infected with approximately 128 hemagglutination units (hau) of influenza a, which resulted in approximately 10% of the cells being infected with influenza 24 h post-infection. total rna, total protein, basolateral supernatants, and apical washes were collected 24 h post-infection. apical surface liquid was collected at 24 h post-exposure by washing the apical surface of the cells with hbss and total protein concentrations were determined by bradford protein assay (bio-rad). cell lysates were prepared at 24 h post-infection in passive lysis buffer (promega, madison, wi) with a protease inhibitor mixture (cocktail set iii; calbiochem, san diego, ca) as well as phosphatase inhibitors (0.5 mm navo4, 1 mm b-glyceropho-phate) on ice for 30 min. after centrifugation, total protein concentrations were determined by bradford protein assay (bio-rad). both the apical supernatants and cellular lysates were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and transferred to nitrocellulose (schleicher & schuell biosciences, keene, nh). proteins were detected using specific antibodies to hat, tmprss2, and spli (1:1,000; santa cruz biotechnology, santa cruz, ca) or influenza a virus hemagglutinin h1 antibody (1:1,000; abcam, cambridge, ma). b-actin (1:2,000; us biological, swampscott, ma), which was used as a loading control. antigen-antibody complexes were stained with anti-rabbit or anti-mouse, horseradish peroxidase (hrp)-conjugated antibody (1:2000, santa cruz biotechnology) and detected with supersignal west pico chemiluminescent substrate (pierce, rockford, il). densitometry was performed using a fujifilm las-3000 imager (fuji film global tokyo, japan). apical surface liquid was collected by washing the apical surface of the cells with hbss and total protein concentrations were determined by bradford protein assay (bio-rad). 50 mg of total protein were immunoprecipated overnight at 4uc with 200 ng of anti-tmprss2, anti-hat, or igg isotype as a control, followed by 1 h incubation with 50 ml of protein g-agrose beads (pierce, rockford, il). cleared supernatants were incubated with 50 ul of influenza a/malaya/302/1954 h1n1 for 30 min at 32uc. virus was subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and transferred to nitrocellulose (schleicher & schuell biosciences, keene, nh). proteins were detected using specific antibodies to hat, tmprss2, and spli (1:1,000; santa cruz biotechnology, santa cruz, ca) or influenza a virus hemagglutinin h1 antibody (1:1,000; abcam, cambridge, ma). influenza virus titers in apical washes were determined by 50% tissue-culture infectious dose (tcid50) in madin darby canine kidney (mdck) cells and evaluated using agglutination of red blood cells as an indicator according to a modified protocol described before [54] . briefly, mdck cells grown in roundbottom 96-well plates were inoculated with virus-containing samples diluted in serum-free dmem containing 20 mg/ml trypsin using log10 dilutions. after 3 days incubation, a suspension of human erythrocytes (0.5%) was added to each well. wells exhibiting hemagglutination were considered positive and virus titers were expressed as log tcid 50 . a modification to the above protocol was performed to test secreted protease activity. in these experiments, necs were exposed to either 0.4ppm of ozone or air for 4 h. 24 h postexposure, apical supernatants were collected and protein concentrations were determined by bradford protein assay. 100 mg of protein within the apical supernatant, which contained the endogenous secreted proteases, was incubated with 80 ml of virus for 30 min at room temperature. mdck cells grown in roundbottom 96-well plates were inoculated with the above samples diluted in serum-free dmem or serum-free dmem containing 20 mg/ml trypsin, as a positive control, using 10-fold dilutions. after 3 days incubation, a suspension of human erythrocytes (0.5%) was added to each well. wells exhibiting hemagglutination were considered positive and virus titers were expressed as log tcid 50 . 10 mm phenylmethyl sulfonyl floride (pmsf) (sigma-aldrich st. louis mo) was used to inhibit trypsin protease activity in control samples. recombinant human slpi (rhslpi) (r&d systems minneapolis mn) was added to serum-free dmem containing 20 mg/ml trypsin and incubated for 60 min at room temperature prior to addition of 80 ml of virus. mdck cells grown in round-bottom 96well plates were inoculated with the above samples diluted in serum-free dmem using 10-fold dilutions. after 3 days incubation, a suspension of human erythrocytes (0.5%) was added to each well. wells exhibiting hemagglutination were considered positive and virus titers were expressed as log tcid 50 . all experiments were performed in triplicate in three independent experiments. total rna was extracted using trizol (invitrogen) according to the supplier's instruction. first-strand cdna synthesis and realtime rt-pcr was performed as described previously [46, 53] using commercially available primers and probes for ha (inventoried taqman gene expression assays) purchased from applied biosystems (foster city, ca). interleukin-6 (il-6) levels necs were exposed to filtered air or 0.4 ppm ozone for 4 h. 24 h post exposure, basolateral supernatants were collected for the measurement of ldh concentration according to the manufacturer's recommendations (takara bio inc. madison, wi). released ldh concentration was expressed as optical density. measurements of il-6 concentration were determined according to the manufacturer's recommendations (bd biosciences san diego, ca). released il-6 concentration was expressed as pg/ml. to measure trans-activation of the slpi promoter, 1.2610 5 beas-2b were cotransfected with either 0.1 mg of slpi-luciferase reporter plasmid (which was kindly provided by dr. rosalia simmen, arkansas children's hospital research institute) along with 0.02 mg of thymidine kinase-renilla luciferase by using fugene 6 (roche) according to the manufacturer's recommendations. after 24 h, cells were treated with 1 mm egcg or dmso. 8 h post-treatment cells were pelleted and lysed in passive lysis buffer (promega, madison, wi). the trans-activation activity was measured as luciferase light units as described previously [55] . all transfection experiments were performed in triplicate and normalized for transfection efficiency by using renilla luciferase. unpaired student's t-test and one-way anova were used for determination of statistically significant differences. the use of the term significant in text refers to a comparison of values for which p,0.05. our group has recently demonstrated that oxidative stress increases susceptibility to influenza virus and that these responses may be mediated via increased viral entry [6, 46] . therefore, we wanted to evaluate how exposure to ozone affects viral entry and replication. we chose to examine influenza infection in human necs exposed to 0.4ppm ozone for 4 h, based on our group's recent in vivo [12, 56] and in vitro [9] studies. to assess the effects of ozone on necs, we determined ldh and il-6 levels in basolateral supernatants as markers of cytotoxicity and inflammation, respectively ( figure 1a and 1b) . as expected, exposure to 0.4ppm of ozone for 4 h causes a significant increase in both inflammation and cytotoxicity [57] . subsequently, 24 h postexposure, cells were infected with influenza a/bangkok/1/79 or a mock control. 24 h post-infection total rna was subjected to real time rt-pcr to quantitate the influenza viral hemagglutinin transcript (ha) ( figure 1c ). as depicted, we saw a significant increase in the amount of viral ha mrna produced in the cells previously exposed to ozone as compared to the filtered air control. similarly, by analyzing the apical washes for influenza viral titers 24 h post-infection, we saw a significant increase in viral titers in ozone exposed cells as compared to the control ( figure 1d ). these results demonstrate that pre-exposure to ozone significantly increased viral replication in necs. to determine the potential mechanism(s) mediating the enhancement of viral replication following acute ozone exposure, we assessed whether innate antiviral immune response mediators were modulated. specifically, we analyzed the expression of interferon-b (ifn-b), interferon-a (ifn-a), retinoic acid inducible gene i (rig-i), and toll-like recptor-3 (tlr-3). human necs were exposed to ozone and 24 h post-exposure were infected with influenza a. 24 h post-infection total rna was isolated and rt-pcr was performed to quantitate the amount of each cellular transcript. ozone alone had no significant effect on mrna expression in any of the four antiviral genes as compared to the air control (figure 2a-d) . as expected, we found substantial inductions of these four antiviral mediators 24 h post-infection with influenza a, but without significant differences in ozone exposed compared to control cells. these data demonstrate that exposure to ozone does not alter baseline expression of host antiviral immune response genes, and does not interfere with their induction in response to influenza infection. since we have shown that ozone exposure did not abrogate antiviral responses, we wanted to determine if the increase in influenza virus infection is due to the oxidative stress pathway/ mechanism induced by ozone exposure. to test this hypothesis, we treated our necs with either a potent phase-ii antioxidant inducer or a direct antioxidant. we have previously shown that oxidative stress increases susceptibility to influenza infection [6] . previous reports have shown that nrf2 gene expression and protein expression can be induced via antioxidant supplementation [58, 59, 60] , more specifically by the addition of the polyphenolic catechin, epigallocatechin-3-gallate (egcg). this compound has the ability to induce nrf2 activation which in turn up-regulates the expression of multiple phase-ii antioxidants [6, 61, 62] . based on our previous published data [6, 46] , we determined that 1 mm of egcg or 10 mm of gsh-et is sufficient to detect an increase in nrf2 expression or counteract oxidative stress induced by diesel exhaust exposure in necs. these concentrations also correlate with levels of egcg that are achievable in vivo [63, 64, 65] . necs were treated with either 1 mm egcg, 10 mm gsh-et, or dmso as a vehicle control for 30 min prior to a 4 h 0.4ppm ozone exposure. 24 h post-ozone exposure cells were infected with influenza a virus and total rna was isolated 24 h post-infection to determine the level of influenza ha transcripts by rt-pcr. as shown in figure 3a , exposure to ozone significantly increases viral ha expression. interestingly, the addition of either egcg or gsh-et significantly inhibited viral ha transcription as compared to the vehicle control. we again collected apical washes to perform viral titer assays. our results, shown on a log scale, indicate that both egcg and gsh-et significantly reduced viral replication ( figure 3b ). taken together, these results demonstrate that antioxidants, either through nrf2 activation (egcg) or by direct addition of an antioxidant (gsh-et), counteract the oxidative stress induced by ozone exposure and can suppress influenza virus replication in necs. ozone not only triggers intracellular oxidative stress, but has also been shown to directly affect and modify the cellular lipid membrane [66, 67] and transmembrane molecules [68] . since the influenza virion must first bind and enter the target cell prior to replication, we hypothesize that the increased susceptibility to influenza after ozone exposure most likely happens early in the virus lifecycle, upstream of viral replication. to determine which step(s) in the virus life cycle are affected by ozone exposure, we employed an enzymatic virus-like particle (vlp) assay. this assay quantitatively measures the amount of virus that enters the cells. similar to our previous work [6] , cells were infected with a vlp that only express the hemagglutinin (ha), neuraminidase (na), and matrix (m) proteins along with a functional b-lactamase reporter fusion (pr8 b-lacm1 vlp) prior to loading with the fluorogenic substrate ccf2-am . in cells in which the pr8 b-lacm1 vlp has entered, the ccf2-am substrate will be cleaved, disrupting fret of the substrate, and resulting in increased ccf2 emission at 447 nm. to determine the effects of ozone exposure on virus entry, necs and the cell line mdck as a control were exposed to air or ozone and 24 h post-exposure, cells were infected with pr8 b-lacm1 vlp. in this experiment, mdck cells are used as a control cell line which does not produce proteases required to activate the viral ha protein. figure 4 shows that exposure to ozone results in a significant increase in viral entry in the necs alone. in summary, these data demonstrate that acute exposure to ozone enhances viral entry, consistent with the increased viral replication shown in figure 1c . ozone exposure disrupts host protease/antiproteases balance as stated earlier, recent reports have identified two endogenous human trypsin-like serine proteases (tmprss2 and hat) that possess the ability to cleave influenza virus in vitro [30, 32, 33, 69] . viral ha proteolytic cleavage is required for viral fusion and entry into the host cell. proteases are regulated by antiproteases, such as slpi and to a lesser extent a1at. studies have shown that a disruption of the protease/antiprotease balance is a hallmark of numerous lung diseases and pathologies including copd, emphysema, asthma, and cancer [40, 70, 71, 72] . hennet et al, demonstrated a link between oxidative stress and protease expression which led to increased influenza infection in mice [39] . linking this work with our current hypothesis, we investigated the protease/antiprotease balance on the epithelial surface in the context of an acute ozone exposure. for these experiments we exposed our necs to either air or ozone for 4 h. 24 h post-exposure, both apical surface liquid and cell lysates were collected to characterize secreted/soluble and membrane-bound intracellular levels of hat, tmprss2, and slpi. western blots revealed that intracellular levels of slpi decreased slightly with ozone exposure, while hat and tmprss2 expression remained relatively unchanged ( figure 5a ). although it is well known that slpi is secreted and is present in normal human airway surface liquid, only recently was the transmembrane protease, hat, found to be secreted in an in vitro overexpression system [33] . we found the levels of the secreted forms of hat, tmprss2, and slpi to be significantly changed by ozone exposure ( figure 5b ). under normal conditions, slpi is in greater abundance than the proteases, yet 24 h post ozone exposure, we found a decrease in slpi but a significant increase in both hat and tmprss2. it is worth noting that the time at which these samples were taken corresponds to the exact time post ozone exposure when the cells were infected with influenza in the data shown in figures 1, 2 and 3, thus representing the levels of protease and antiprotease present at the time of infection. to determine if disruption in the protease/ antiprotease equilibrium is due to oxidative stress imposed by the ozone exposure, we treated our necs with the potent nrf2 inducer egcg prior to ozone challenge. figure 5c shows that egcg did increase levels of slpi which corresponds with a decrease in protease expression. finally, figure 5d demonstrate that the addition of 1 mm egcg significantly increases the transactivation of the slpi promoter which correlates with the increase in slpi protein expression displayed in figure 5c . taken together, these results indicate that ozone-induced oxidative stress disrupts the protease/antiprotease balance, in favor of protease activity. furthermore, we demonstrate that the equilibrium can be restored with the induction of nrf2 via antioxidant supplementation resulting in an increase in the antiprotease slpi. we characterized the cleavage products of the viral ha protein to determine if the proteases produced by acute ozone exposure can cleave an intact influenza virion. we first investigated where ha cleavage takes place. necs were exposed to air or ozone and 24 h post-exposure, cells were infected with influenza a/malaya/ 302/1954 h1n1. the cell lysates were analyzed by western blotting for detection of the ha cleavage products. figure 6a shows that the majority of the viral ha protein within the cell is cleaved and there is not a significant change with ozone exposure. in addition, western blot analysis of apical washings incubated with virus showed that ha0 was cleaved and that there was an increase in the cleavage of ha from the ozone exposed cells as measured by densitometry. quantification of the h2 cleavage products demonstrated that ozone exposure leads to enhanced proteolytic activation of the virion. this data demonstrate that ha cleavage can take place in both the intra-and extracellular environment and that ozone exposure increases the proteolytic activation of the virus in the airway ( figure 6b ). to characterize the function of tmprss2 and hat in ha cleavage, we depleted the proteases via immunoprecipitation prior to addition of the virus. figure 6c shows the apical washes from necs that were immunoprecitipated with anti-hat, anti-tmprss2, and igg as a control. the cleared supernatants were incubated with influenza a/malaya/302/1954 h1n1. as shown in figure 6d , there is little difference in the cleavage of the ha protein when the protease are depleted separately, but we demonstrate a significant decrease in the cleavage of ha when both proteases were removed. this data show that the protease(s) found in the apical surface liquid are able to cleave an intact influenza a virion. in addition we show that there are additional proteases secreted into the airway liquid space that are able to activate the virus. although we show that slpi, hat, and tmprss2 are secreted by necs, the question remains as to whether they are functional. to test functionality and the role they play in a viral infection, we employed a modified infectivity viral titer assay. these experiments are very similar to the viral titer assays mentioned earlier, but with a few modifications. similar to the cultures of differentiated human epithelial cells were exposed to either 0.4ppm of ozone or air for 4 h. 24 h post-exposure, samples were collected and subjected to western blot analysis to detect slpi, hat, tmprss2, and b-actin. a) total cellular lysates were analyzed for intracellular slpi, tmprss2, and hat protein expression by western blot. membrane was stripped and analyzed for b-actin as a loading control. b) 24 h post-exposure, apical supernatants were analyzed for secreted slpi, tmprss2, and hat protein levels. c) cultures of differentiated human epithelial cells were treated with egcg or dmso as a vehicle control for 30 min before exposure to either 0.4ppm of ozone or air for 4 h. 24 h post-exposure, apical supernatants were analyzed for secreted slpi, tmprss2, and hat protein levels. d) function activity of the slpi promoter was determined using a dual-luciferase reporter assay. beas-2b cells were co-transfected with slpi-luc and tk-renilla. cells were treated with egcg or dmso as a vehicle control. cells were harvested 8 h post-treatment and analyzed for luciferase expression. the values represent luciferase production from a representative experiment performed in triplicate. necs were obtained from four healthy volunteers (n = 4). densitometry was used to quantitate the amounts of protein, and the numbers below the gel indicate the ozone/air or egcg/dmso sample ratio. doi:10.1371/journal.pone.0035108.g005 figure 6 . secreted proteases are functional for hemagglutinin cleavage. cultures of differentiated human epithelial cells were exposed to either 0.4ppm of ozone or air for 4 h. a) 24 h post-exposure, cells were infected with influenza a/malaya/302/1954. 24 h post-infection, total cellular lysates were analyzed for intracellular cleavage of the viral ha protein by western blot. b) 24 h post-exposure, apical supernatants were collected and incubated with influenza a/malaya/302/1954. cleavage of the viral ha protein was analyzed by western blot. c) cultures of differentiated human epithelial cells were exposed to either 0.4ppm of ozone or air for 4 h. 24 h post-exposure, apical supernatants were immunoprecipitated (ip) with anti-hat, anti-tmprss2, or an isotype control (igg). protein levels were analyzed by western blot. d) apical supernatants of differentiated human epithelial cells, were immunoprecipitated (ip) with anti-hat, anti-tmprss2, or an isotype control (igg) followed by incubation with influenza a/ malaya/302/1954. cleavage of the viral ha protein was analyzed by western blot. necs were obtained from four healthy volunteers (n = 4). densitometry was used to quantitate the amounts of cleaved h2 protein, and the numbers below the gel indicate the ozone/air ratio. doi:10.1371/journal.pone.0035108.g006 assays before, we utilized the well-characterized mdck cell line which does not produce the proper proteases to activate the influenza virion, which can be overcome by the addition of exogenous trypsin. in contrast to the previous titer experiments above, we did not add exogenous trypsin to enable multicycle replication of influenza virus in these cells. these experiments allowed us to test if secreted proteases present in the apical surface liquid from necs exposed to air or ozone were able to facilitate multiple rounds of viral replication in mdcks. we show that the secreted proteases present in the apical supernatants from necs are functional and can propagate viral entry and replication. we also demonstrate that the ozone-exposed nec supernatants displayed significantly greater infection kinetics than the air control ( figure 7a ). this correlates with the increase in protease expression post ozone exposure ( figure 5b ), viral entry (figure 4) , and viral replication ( figure 1c and 1d ). in addition we show that this is a protease-mediated event, since the addition of a known serine protease inhibitor, pmsf, significantly inhibits viral replication. to demonstrate that slpi has the ability to protect airway epithelium from influenza infection by inhibiting proteolytic cleavage, we incubated various amounts of recombinant human slpi (rhslpi) to our traditional viral titer assay. briefly, we incubated our viral titer media, serum-free dmem containing 20 mg/ml of exogenous trypsin, with varying amounts of rhslpi prior to addition of influenza. as shown in figure 7b , addition of rhslpi significantly inhibits multicycle viral replication. we conclude that the addition of rhslpi directly inhibits trypsinmediated cleavage of viral ha leading to decreased infection and replication. taken together, these data demonstrate that exposure to ozone disrupts the protease/antiprotease balance resulting in greater secretion of endogenous proteases leading to increased viral cleavage/activation culminating in enhanced viral entry and replication. numerous epidemiological studies link increased ambient ozone levels with respiratory viral infections [1, 73, 74] . however, the role of ozone exposure in viral replication and pathogenesis remains to be fully defined. because ambient ozone is a major source of oxidative stress in the airway epithelium and this is the primary site for influenza infections, it is important to determine the role ozone exposure plays in viral infections. although it has been shown that ozone exposure can modulate many aspects of the immune response [8] , how these alterations influence a viral infection have yet to be elucidated. recent studies have demonstrated the importance of the protease/antiprotease balance in the context of normal lung homeostasis, and have shown that oxidative stress can disrupt this delicate equilibrium [40, 75] . it has been well documented that many lung pathologies are dependent on the regulatory interplay between oxidative stress and protease expression [40, 70, 71] . although there is speculation that mexico city's increased influenza-related morbidity and mortality rates were due to exposures to higher levels of ozone, little research has been done looking at the effects of pre-exposure to ozone in the context of an influenza infection in humans. herein, we demonstrate that acute ozone exposure disrupts the protease/ antiprotease balance resulting in increased secreted protease expression in primary human epithelial cells, resulting in increased ha cleavage and activation of the influenza virion, which correlated with enhanced viral entry and replication. numerous host cell-dependent factors can affect and control influenza virus attachment and uptake by: (i) proteolytic cleavage of viral ha by host cell-derived serine proteases [30, 33] , (ii) host cell derived innate immune defense molecules aimed at inhibiting the infectious virions [25, 76] , and (iii) antiviral mediators limiting viral replication and shedding of virus particles [6, 52] . we first focused on potential effects of ozone on host antiviral defense responses. our data showed that exposure to ozone elicited the typical pro-inflammatory and cellular damage responses as seen with increased release of il-6 and ldh. we found that the same exposure regimen also resulted in significant increase in viral replication. we initially hypothesized that this could be due to a disruption in one of the cellular antiviral response mechanism/ pathways, and assessed the transcription levels of 4 classical antiviral mediators; rig-i, ifn-a, ifn-b, and tlr-3. similar to our previous studies using exposure to diesel exhaust [46] , we found that ozone did not decrease expression of these mediators or their response when challenged with influenza. in our previous study [46] , we found that exposure to an oxidant pollutant increased influenza virus attachment and/or entry, but did not distinguish between these two steps in the viral infection cycle. therefore, we focused our attention on steps upstream of viral replication to identify mechanisms by which oxidants, such as ozone, affect influenza infectivity. to dissect specific points in the virus life cycle upstream of viral replication that could determine the role ozone exposure plays in viral susceptibility, which ultimately dictates viral pathogenesis and outcome, we utilized our previously described enzymatic virus-like particle (vlp) assay [6] . our data demonstrated that ozone exposure significantly increased influenza virus entry. this is consistent with our previous work showing that exposure to other oxidant pollutants and suppression of nrf2 increases influenza virus entry [6, 46] . similar to these previous studies, data shown here demonstrated that the mechanism(s) through which ozone exposure alters influenza virus entry is mediated by oxidative stress. since ozone directly interacts with the apical surface of the respiratory epithelium we hypothesize that ozone either directly, or via the induction of biologically active ozone reaction products, modifies either the expression or function of surface proteins, as previously been demonstrated for human surfactant protein a (sp-a) [77] . among the most prominent proteins on the apical surface potentially regulating infection are the proteases. to date, at least five different proteases have been identified in the airways of animals and humans [78] . despite many years of effort, the exact protease(s) that activates influenza has yet to be identified. numerous groups have reported that the family of type ii transmembrane serine proteases and trypsin-like proteases are responsible for viral cleavage [26, 30, 33, 79] . previous studies have examined these proteases in the context of influenza infections, specifically tmprss2 and hat. although these studies demonstrated that these proteases are capable of cleavage, the experiments were performed in cell lines such as mdcks [33] or the colorectal adenocarcinoma cell line caco-2 [32] , neither of which are natural target cells for influenza. since the cellular protease determines the tropism as well as efficiency of viral replication, we utilized fully differentiated human primary nasal epithelial cells (necs) in this study, a natural host cell for influenza virus. we show that hat and tmprss2 are not only present, but secreted from necs ( figure 5b ), and that ozone exposure enhances the release of the proteases into the apical compartment. this is consistent with reports showing that oxidative stress increases cellular protease activity [38] . since proteases in the upper airway are inhibited by slpi [78] , we focused on this antiprotease and its expression in relation to hat and tmprss2. similar to previous reports, we report that slpi is detected in the secreted airway and oxidative stress reduces the expression and function of the protein ( figure 5b ) [35, 80] . these data are consistent with our hypothesis that influenza virions can become activated in the airway surface liquid prior to binding to the epithelial surface. virion activation in the airway prior to binding would eliminate the need for a membrane-bound protease thus broadening viral tropism from airway epithelium, that contain the necessary proteases, to any mammalian cell, since 2,6-linked sialic acid is expressed by most mammalian cells ranging from lymphoid to glial cells [81, 82] .this could explain the increased morbidity and mortality associated with the mexico city 2009 h1n1 pandemic which resulted in increased infection of multiple cell types including distal airway epithelium and immune cells including alveolar macrophages [83, 84] as compared to previous seasonal outbreaks. egcg has been shown to have potent antioxidant capabilities; in part by inducing the expression of a number of antioxidant enzymes [85] . in vitro and in vivo studies have shown that this supplement induces the expression of phase ii antioxidant genes such as gstm1, which was associated with nrf2-are signaling [58, 86] . whether and how activation of antioxidants expression is involved in the potential antiviral effects of egcg is not known. previous studies have indicated that it directly binds influenza virus and therefore prevents attachment and entry into host cells [63, 87] . however, these studies were conducted in mdck cells, which are not a natural host cell for influenza and require addition of exogenous proteases to achieve viral entry [63] . iizuka et al, demonstrated that nrf2-knockout mice display protease/antiprotease imbalance leading to increased susceptibility to cigarette smoke-induced emphysema [37] . in addition, the same group showed that nrf2 expression exerts a protective effect through the transcriptional activation of antiproteases [88] . our data demonstrate that supplementation of differentiated nasal epithelial cells with egcg from the basolateral side (to eliminate direct interaction with the virus during infection) significantly increases slpi production ( figure 5c ). it is worthy to note that we show a direct inverse relationship between the levels of secreted slpi and secreted protease expression, suggesting a potential novel antiviral mechanism by which egcg could be protective against influenza infections. we next determined that the secreted proteases are functional. we demonstrate that proteases present in the apical surface liquid were able to activate the virus ( figure 6b ) and produce multiple rounds of viral replication ( figure 7a ). in addition, apical supernatants from ozone-exposed epithelial cells had significantly higher viral titers, which correlate with the increased protease expression displayed in figure 5b . in the final experiment we utilized rhslpi and show a dose dependent protective response ( figure 7b ). this demonstrates that that the antiprotease slpi has a protective affect against viral activation and replication. in conclusion, this is the first study to demonstrate that secreted proteases from primary human respiratory nasal epithelium proteolytically activate influenza virions and that exposure to a common ambient air oxidant pollutant increases these effects. we speculate that individuals with increased airway oxidative stress, whether due to an underlying medical condition or exposure to high levels of air pollutants, would develop disruption of the epithelial protease/antiprotease balance and become more susceptible to infections with viruses like influenza, sars-cov, and metapneumovirus. disruption in the protease/antiprotease balance has been described in several respiratory diseases including copd, emphysema, and asthma [70, 71, 89] . this mechanism could be exploited as a novel anti-viral therapy in combination with conventional antiviral treatments. antiproteases, such as slpi appear to protect the respiratory epithelium from influenza virus infection and nutritional supplements may increase the antiprotease:protease ratio. taken together, the data shown here provide further support to the concept that targeting haactivating proteases and decreasing oxidative stress to arrest viral infection and spread in conjunction with antiviral agents is a promising potential therapeutic intervention approach against influenza. characterizing the epidemiology of the 2009 influenza a/h1n1 pandemic in mexico occurrence of 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oxidative injury oral absorption and bioavailability of tea catechins inhibition of the infectivity of influenza virus by tea polyphenols transcription factor nrf2 plays a pivotal role in protection against elastaseinduced pulmonary inflammation and emphysema protease inhibitors in respiratory disease: focus on asthma and chronic obstructive pulmonary disease we thank ms. luisa e. brighton for her expert technical assistance and dr. philip bromberg, m.d., for his critical review of the manuscript. key: cord-350842-4m82l5t8 authors: xing, jun; sun, ning; xu, jun; geng, shuling; li, yuqian title: study of the mental health status of medical personnel dealing with new coronavirus pneumonia date: 2020-05-19 journal: plos one doi: 10.1371/journal.pone.0233145 sha: doc_id: 350842 cord_uid: 4m82l5t8 this paper studied the relationship between personality traits and mental health conditions of medical personnel to provide a basis and reference for the implementation of targeted education on mental health. a self-report inventory, the symptom checklist-90 (scl-90), was used to investigate the mental health status of 548 medical personnel dealing with the new coronavirus pneumonia in eight provinces and cities of china. the overall mean scl-90 score and mean values of factors (somatization, obsessive-compulsive, anxiety, phobic anxiety, and psychoticism) of the medical personnel were significantly higher than in the norm group (p < 0.05), while their average interpersonal sensitivity score was significantly lower (p < 0.01). in addition, personal factors affecting the mental health status of medical personnel were identified (all p < 0.05). the overall mental health status of medical personnel responding to new coronavirus pneumonia is generally higher than that of the norm group in china. the results of this study should contribute to measures to alleviate the psychological pressures on medical personnel dealing with the new coronavirus epidemic in china. novel coronavirus pneumonia (ncp) is a pathogenic coronavirus often referred to as the novel coronavirus. on january 12, 2020, who officially named the disease coronavirus disease 2019 (covid-19). the first case of covid-19 was reported in wuhan in china on december 12, 2019 [1] [2] as causing severe acute respiratory infection (sari). coronaviruses are a large family of viruses known to cause illnesses such as cold and more serious diseases such as middle east respiratory syndrome (mers) and severe acute respiratory syndrome (sars). covid-19 is a new strain of coronavirus that has never been found in the human body before [3] [4] . covid-19 patients typically show symptoms such as fever, coughing, shortness of breath, and difficulty in breathing. in more severe cases, the disease can lead to pneumonia, severe acute respiratory failure, kidney failure, and even death [5] . there is as yet no specific treatment for covid-19. patients mainly receive symptomatic treatment and care for the prevention of complications, while supplementary medical services also appear to be very effective for infected people. the rate of covid-19 infections has increased rapidly in a short period of time. as of february 17, 2020, the number of confirmed cases exceeded 70,000, with 1,770 deaths [6] . in addition, the disease can cause secondary infections, which has created a huge burden and pressure for the prevention and treatment of the disease in various places. at present, more than 30,000 medical personnel from various medical teams across the country have provided support in wuhan [7] . though the help from these medical personnel has relieved the local pressure for medical care to save critically ill covid-19 patients, there have been serious infections among the medical staff in wuhan and other places in hubei province. at present, over 3000 medical personnel have been infected, which has greatly increased the psychological pressure they experience. in the face of the catastrophic health emergency of covid-19, medical personnel have been affected by different kinds of subjective and objective factors and confront several mental health problems. mental illness is a form of human stress response, an explanatory, emotional, and defensive response within the human body, and a physiological response of the human body to the impact of needs or injuries. therefore, this study aims to analyze the psychological state of medical personnel dealing with covid-19 and its influencing factors in order to provide an objective basis for the prevention of further transmission, interventions, and countermeasures for covid-19. this study adopted convenience sampling to recruit research subjects. from january 25 to february 16, 2020, 560 medical personnel from 12 hospitals in eight provinces and cities across the country were enrolled as research subjects. ① survey of demographic characteristics of medical personnel, including 18 questions related to the following aspects: province, hospital, department, occupation, gender, age, highest education level, work experience, level of expertise, marital status, children, living status, whether you have participated in training for handling of public health emergencies, whether family members support your working on the front line against coronavirus, whether you have supported in affected areas in hubei, designated hospitals, department of infectious diseases, fever clinics or emergency department, level of concern whether you and your family have been infected, degree of suspicion that you were infected when coronavirus-related symptoms occurred, and whether you have received medical observation recently. ② the scl-90 self-report inventory: the scl-90 inventory, compiled by derogatis in 1975, includes 10 factors and a total of 90 items. it had been translated into chinese version and used in the study [8] . each factor reflects the symptoms and pain of a patient in a certain aspect, and the distribution of symptoms can be understood through the factor scores. the 10 factors include somatization, obsessive-compulsive, depression, anxiety, hostility, phobic anxiety, paranoid ideation, psychoticism, sleep, and diet. each item was scored using a 5-point likert scale, ranging none, mild, moderate, moderate to severe, to severe. the total score is the sum of the scores of the 90 items. in previous studies, the homogeneity reliability of the total scl-90 scale was 0.97, and the homogeneity reliability of each sub-scale was also greater than 0.68. the test-retest reliability was greater than 0.7, and the content validity was above 0.85, which suggest sound reliability and validity [8] . 2.2.2 survey methods and medical ethics. the study used online questionnaires for data collection. researchers conducted surveys upon the completion of general training. all procedures were approved by the ethics committee of harbin medical university (hrbykd-a26). the research purpose and methods were explained to subjects to seek their cooperation. online informed consent forms were signed by participants. they were informed that their participation was completely voluntary, and they could withdraw from the study at any time. the link of the online questionnaire was then sent to a total of 560 medical personnel via the internet. 1. in this study, data analysis was performed after logical checks using the statistical software spss 22.0. a p-value less than 0.05 (p < 0.05) was considered statistically significant. 2. statistical description: the mean value, standard deviation, and frequency were used to describe the demographic data of medical personnel, while the mean value and standard deviation were used to describe the scores for the mental health status of medical personnel responding to covid-19. 3. statistical inference: multivariate linear regression was adopted to analyze the impact of the demographic data of medical personnel on their mental health status. a total of 560 questionnaires were distributed, and 548 valid questionnaires were recovered, for an effective recovery rate of 97.90%. these 548 questionnaires were completed by medical personnel from eight provinces and cities in china, namely heilongjiang, liaoning, jilin, inner mongolia, tianjin, sichuan, shanxi, and shandong. details of the respondents' personal information are given in table 1 . the overall average of scl-90 and mean values of factors (somatization, obsessive-compulsive, anxiety, phobic anxiety, and psychoticism) of medical personnel were significantly higher than that of the norm group (p < 0.05 or p < 0.01), while the average score of the interpersonal sensitivity factor of medical personnel was significantly lower than that of the norm group (p < 0.01). details are provided in table 2 . stepwise linear regression was performed using the total score of mental health status as the dependent variable and 17 items of personal information as independent variables. the 17 items include: hospital, department, occupation, gender, age, highest education level, work experience, level of expertise, marital status, any children, living status, whether you have participated in training for handling of public health emergencies, whether family members support your working on the front line against coronavirus, whether you have supported in affected areas in hubei, designated hospitals, department of infectious diseases, fever clinics or emergency department, level of concern whether you and your family have been infected, degree of suspicion that you were infected when coronavirus-related symptoms occurred, and whether you have received medical observation recently. the α-values for importing and exporting a variable in the regression equation were set to 0.10 and 0.15, respectively. factors affecting the mental health and status of medical personnel based on their significance from high to low are: the degree of suspicion that they were infected when the novel coronavirusrelated symptoms occurred, the level of concern whether they and their family members have been infected, age, whether they have supported in affected areas in hubei province, designated hospitals, and other places for the novel coronavirus, and whether family members support them working on the front line (p < 0.05). details of the regression results are listed in table 3 . covid-19 is a fulminant infectious disease. as it is highly contagious, many people are frightened by it and even talk fearfully about coronavirus, which can also be observed in front-line medical staff. li et al. reported how much people and medical staff suffer from vicarious traumatization and how this vicarious traumatization of non-front-line medical staff is more serious than that of front-line medical staff [10] . as in south and southeast asia countries, also in italy, there are similar problems in medical staff due to high workload and intermittent lack of protective devices. in addition, some slight form of racism is demonstrated against health care professionals who potentially have a higher risk of being infected and between non-front-line medical staff towards front-line medical staff [11] . the results of the study have shown that the overall mean of the scl-90 and the mean values of the factors (somatization, obsessivecompulsive, anxiety, phobic anxiety, and psychoticism) of medical personnel were significantly higher than that of the norm group (p < 0.05), while the average score of the interpersonal sensitivity factor of medical personnel was significantly lower than that of the norm group (p < 0.01). the results were partly similar with some research in wuhan city [12] . more specifically, medical personnel are most of the people closest to covid-19 patients, so they are at high risk of exposure to the disease. moreover, they have a deep understanding of the dangers of covid-19, so they are prone to anxiety and fear. the infection protection procedures for covid-19 are highly complex and medical staff need to constantly change clothes and replace protective equipment, so they are more likely to establish obsessive-compulsive behaviors. medical personnel, especially young medical staff, have less experience in the field and in dealing with difficulties and hardships in life. therefore, when they suddenly encounter such sudden public health events, they tend to suffer anxiety and phobic anxiety, leading to physical and mental problems. that is why scores of the factors somatization, obsessive-compulsive, anxiety, phobic anxiety, and psychoticism were significantly higher than in the norm group. this result suggests that psychologists and team leaders should pay more attention to the anxiety, phobic anxiety, and psychoticism issues of the medical personnel in a team. the average score for the interpersonal sensitivity factor of medical personnel was significantly lower than that of the norm group. this shows that in the event of an infectious disease epidemic, the majority of medical personnel are united and have good professional strengths and qualities for self-regulation and self-protection. the results of this study show partial consistency with the studies of the mental health status of front-line medical personnel for sars in 2003 [13] . the results of this study have shown that the factors affecting the mental health status of medical personnel based on the significance from high to low are: the degree of suspicion that they were infected when the novel coronavirus-related symptoms occurred, the level of concern whether they and their family members have been infected, age, whether they have supported in affected areas in hubei province, designated hospitals, and other places for the novel coronavirus, and whether their family members support them working on the front line. specific reasons are given in the following sections. degree of suspicion that they were infected when the novel coronavirus-related symptoms occurred, the level of concern whether they and their family members have been infected, whether they have supported in affected areas in hubei province, designated hospitals, and other places for the novel coronavirus, and whether family members support them working on the front line. covid-19 patients are the main source of transmission of the disease. patients with latent infection (i.e., asymptomatic infection) may also constitute a source of infection [14] . medical personnel are in frequent close contact with patients during their treatment and care, hence the high risk of infection [15] . among 138 patients admitted consecutively from january 1 to 28, 2020, to zhongnan hospital of wuhan university, the proportion of medical personnel was as high as 29% [16] . a retrospective analysis of 1099 confirmed covid-19 patients from 552 hospitals in 31 provinces (diagnosis as of january 29) found that the proportion of medical staff was 2.09% [17] . therefore, medical staff are at high risk of infection and are under great psychological pressure. suicidal cases were reported in india but also in other countries, italy included, where two infected italian nurses committed suicide in a period of a few days probably due to fear of spreading covid-19 to patients. it is possible that fear and anxiety of falling sick or dying, helplessness will drive an increase in the 2020 suicide rates [18] . if they become infected as a result of supporting affected areas in hubei and covid-19 designated hospitals, it will not only affect their physical and mental health but also that of their families. therefore, with the emergence of symptoms and the increase in the level of concern, the mental health status of clinical medical staff may deteriorate. furthermore, if their families do not support them working on the front line against the disease, then the psychological burden of the medical staff will also increase due to the resulting sense of conflict with professional ethics, resulting in further impact on their physical and mental health. we found that the higher the age, the higher the mental health score and the more psychological problems. based on the age distribution of patients across the country, all ages have no resistance to the novel coronavirus, and a person of any age can be infected as long as virus transmission conditions are met [19] . an analysis of 4021 confirmed patients nationwide (diagnosis date as of january 26) also shows that people of all ages are generally vulnerable to the disease, of whom 71.45% are aged 30 to 65 years [19] . as people get older, the risk of exposure to the disease may increase in people with underlying illness such as asthma, diabetes, and heart diseases [20] . therefore, older medical personnel have more psychological stress when dealing with covid-19 patients. it is advised that older medical personnel receive psychological counseling before and during work to help them adjust their status as soon as possible. despite all the above, this study believes that after such a "smokeless" war against the novel coronavirus, the psychological quality of medical personnel can be improved to a certain extent. in this study, the applicability of the results is limited by the nature of cross-sectional studies, and because of its use of convenience sampling from 12 hospitals in eight provinces and cities of china. in subsequent research in this project, a longitudinal study should be conducted that uses a wider sample and measures the mental health status of medical personnel from multiple dimensions, which can help better identify the mutual influence between demographic data and mental health status. in the face of the catastrophic health emergency caused by covid-19, medical staff have been affected by different kinds of subjective and objective factors. their mental health problems are a form of human stress response, an explanatory, emotional, and defensive response within the human body, and a physiological response of the human body to the invasion of needs or injuries. in this special environment, their work, life, and emotions tend to be regularly abnormal. due to the requirements for isolation and disinfection, medical personnel need to wear several layers of protection clothing. this increases the intensity of their work and requires great physical energy, causing severe hypoxia and physical symptoms such as headache and muscle soreness. other symptoms such as obsessive-compulsive symptoms, interpersonal sensitivity, depression, anxiety, phobic anxiety, hostility, and paranoid ideation are all normal psychological reactions in the handling of emergencies and environmental stimuli. in face of a disaster, persons with good mental health will tend to actively take measures such as catharsis, transference, compensation, relaxation, humor, self-consolation, and rational response. the results of this study show that the overall mental health of medical staff is generally poor when dealing with covid-19. psychological tests show that people have a process of adaptation to catastrophic emergencies, from initial rejection, shock, and fear, to habituation, acceptance, and calm, to co-existence and living together, which is a regular process. in the face of such a sudden disaster as covid-19, these psychological symptoms have manifested in both doctors and patients. for medical personnel, it is particularly important to pay attention to mental health conditions while fulfilling their responsibilities. in future research, it is worth exploring how to strengthen the monitoring of mental health conditions of medical personnel and establish an active, systematic, and scientific psychological defense system under such special circumstances. report of clustering pneumonia of unknown etiology in wuhan city chinese center for disease control and prevention. wuhan municipal health committee's report on the status of viral pneumonia of unknown cause world health organization. novel coronavirus-china 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origins and receptor binding a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china the continuing 2019-ncov epidemic threat of novel coronaviruses to global health: the latest 2019 novel coronavirus outbreak in wuhan fear of covid 2019: first suicidal case in india national health commission of the people's republic of china novel coronavirus (2019-ncov) advice for the public: myth busters the authors would like to thank the medical personnel who participated in the study. conceptualization: ning sun. key: cord-320091-2lrqubdl authors: badawi, alaa; velummailum, russanthy; ryoo, seung gwan; senthinathan, arrani; yaghoubi, sahar; vasileva, denitsa; ostermeier, emma; plishka, mikayla; soosaipillai, marcel; arora, paul title: prevalence of chronic comorbidities in dengue fever and west nile virus: a systematic review and meta-analysis date: 2018-07-10 journal: plos one doi: 10.1371/journal.pone.0200200 sha: doc_id: 320091 cord_uid: 2lrqubdl background: flavivirus diseases such as dengue fever (denv), west nile virus (wnv), zika and yellow fever represent a substantial global public health concern. preexisting chronic conditions such as cardiovascular diseases, diabetes, obesity, and asthma were thought to predict risk of progression to severe infections. objective: we aimed to quantify the frequency of chronic comorbidities in flavivirus diseases to provide an estimate for their prevalence in severe and non-severe infections and examine whether chronic diseases contribute to the increased risk of severe viral expression. methods: we conducted a comprehensive search in pubmed, ovid medline(r), embase and embase classic and grey literature databases to identify studies reporting prevalence estimates of comorbidities in flavivirus diseases. study quality was assessed with the risk of bias tool. age-adjusted odds ratios (ors) were estimated for severe infection in the presence of chronic comorbidities. results: we identified 65 studies as eligible for inclusion for denv (47 studies) and wnv (18 studies). obesity and overweight (i.e., bmi> 25 kg/m(2), prevalence: 24.5%, 95% ci: 18.6–31.6%), hypertension (17.1%, 13.3–21.8%) and diabetes (13.3%, 9.3–18.8%) were the most prevalent comorbidities in denv. however, hypertension (45.0%, 39.1–51.0%), diabetes (24.7%, 20.2–29.8%) and heart diseases (25.6%, 19.5–32.7%) were the most prevalent in wnv. ors of severe flavivirus diseases were about 2 to 4 in infected patients with comorbidities such as diabetes, hypertension and heart diseases. the small number of studies in jev, yfv and zika did not permit estimating the prevalence of comorbidities in these infections. conclusion: higher prevalence of chronic comorbidities was found in severe cases of flavivirus diseases compared to non-severe cases. findings of the present study may guide public health practitioners and clinicians to evaluate infection severity based on the presence of comorbidity, a critical public health measure that may avert severe disease outcome given the current dearth of clear prevention practices for some flavivirus diseases. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the flaviviridae is a large family of positive-strand rna viruses, that comprises four genera: flavivirus, pegivirus, pestivirus, and hepacivirus [1] . the flavivirus genus consists of more than 70 viruses, many of which are arthropod-borne human pathogens that cause a variety of clinical diseases, ranging from asymptomatic to mild fever to more severe diseases including encephalitis and hemorrhagic fever [1, 2] most flaviviruses are transmitted through the bite of an infected arthropod vector, mainly aedes genus (aedes aegypti and to a lesser extent, aedes albopictus) and cluex mosquitos, and most were once maintained by animal reservoirs in sylvatic transmission cycles [2] . many flaviviruses, however, such as dengue virus, yellow fever and zika virus, are now principally maintained by mosquito-borne transmission with a possible human-to-human transmission through transfusion of infected blood or transplantation of infected tissue [3] . some flaviviruses can cause globally significant vector-borne diseases with a substantial public health impact such as dengue virus (denv), japanese encephalitis virus (jev), west nile virus (wnv), zika virus (zikv) and yellow fever virus (yfv) [4] . other members of the flaviviridae family that have a more regional impact include murray valley encephalitis virus (mvev) in oceania, st. louis encephalitis virus (slev) in north america, and tick-borne encephalitis virus (tbev) in europe [1] . over the past few decades, many of these flaviviruses have re-emerged for a range of reasons including decreases in mosquito control efforts, rapid changes in climate and vector's demography, dense urbanization, population growth and globalization with increased transportation and trade activities [5] . examples include the geographic spread of denv throughout the tropical world; jev throughout south asia, australasia and the pacific; zikv into south and central america; yfv into the americas and the invasion of wnv into much of north america [1, 5] . it is estimated that there are over 390 million denv infections per year, of which 96 million manifests clinically with varying degrees of severity [6] and 3.9 billion people in 128 countries are at risk of infection [7] . similarly, high incidence rates for symptomatic cases of jev were reported over the past three decades to reach 2.4 per year per 100,000 population [8] . epidemic waves of yfv are projected to result in 30,000 to 200,000 clinical cases per year with case-fatality rates ranging from 2 to 15% [9] [10] [11] [12] . wnv, first appeared in the northeastern usa in 1999, are spread presently across much of the usa and southern canada. for example, in 2015, the cdc reported 2,175 cases of wnv, of which 1,616 (74%) were hospitalized and 146 (7%) died [13] . in the developing world, wnv incidence is likely to be underestimated due to political, psychological, and economic barriers to reporting [12, 14] . although most human flavivirus infections are asymptomatic or have an undifferentiated febrile illness, a small percentage of affected individuals develop acute fever that can progress to severe clinical manifestations such as hemorrhage, vascular leakage and encephalitis [15] . currently, our knowledge of the host-related factors that influence the pathogenesis of severe disease is inadequate to allow prediction of who will develop severe clinical illness. however, some mechanisms and etiological factors underlying inter-individual variations in response to flavivirus infections have been identified. interactions of virus-encoded proteins with human innate immune pathways [15] ; the effect of host-cell surface molecules in virus binding and entry [16] ; the role of viral protein nuclear localization in the host cell response [17] ; and the flavivirus replication dynamics within multiple immune systems [18] have all been considered as host-pathogen interaction events that may regulate viral virulence or attenuation and the subsequent disease severity. over the past few years, however, host-related factors such as preexisting chronic conditions, e.g., cardiovascular diseases, diabetes, obesity, and asthma have received attention as predictors for increased risk of progression to severe flavivirus infection [19] [20] [21] . recent studies have raised the proposition that cardiovascular disease, stroke, diabetes, respiratory diseases and renal disorders may contribute, together with old age, to severe clinical manifestations of dengue [19, 20] . a few studies of wnv [14] and jev [22] infections, and responses to yfv vaccination [23] , have also explored the role of chronic comorbidities in the prognosis of infections. given the lack of specific medical treatment for flavivirus diseases, effective public health surveillance for vector-borne infections together with continuing vector control efforts will be critical to preventing infection. however, elucidating the impact of comorbidities to the severity of disease when infection occurs will be critical to identifying vulnerable populations, to whom effective interventions protocols and individually-tailored clinical monitoring practices should be particularly targeted. the objective of this study is to systematically review the existing literature on the prevalence of the most common non-communicable comorbidities related to the cluster of metabolic syndromes-associated diseases, such as diabetes mellitus, heart diseases, hypertension, asthma, stroke and obesity in flavivirus infections and to evaluate the difference of their prevalence in severe vs. non-severe clinical outcomes to infection. identifying and characterizing associations between comorbidities and severity of flavivirus infections will be significant factor in designing public health measures that aim to prevent the severe outcomes of infection. we carried out a systematic literature search that conforms to the preferred reporting items for systematic reviews and meta-analysis (prisma) guidelines [24] (see s1 table) , in pubmed, ovid medline(r), embase and embase classic databases from inception to the last week of november 2016 (november 25, 2016). the date for searching database is the same date for the upper limit of the period considered. a grey literature search was also conducted in the american society of tropical medicine and hygiene and open forum infectious diseases-infectious diseases society of america for the two most recent years. using the pico format (acronym for "population or problem", intervention or exposure of interest", "comparison" and "outcome") [25], the research questions were: "what is the frequency of chronic comorbidities in flavivirus infections?" and "is the severity of flavivirus infection associated with higher prevalence of comorbidities?" (s2 table) . the search related terms (mesh), inclusion and exclusion criteria and synonyms are shown in table 1 . non-english language reports were excluded from this study. to identify relevant studies, we used the comprehensive fourstep search strategy of prisma (fig 1) , i.e., (i) identification, (ii) screening, (iii) eligibility and (iv) inclusion of studies. we evaluated 47 studies in denv , 18 in wnv [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] [86] [87] [88] [89] [90] and two in jev [22, 91] for inclusion (see fig 1) . given the small number of studies in jev, the two articles were excluded from further quantitative analysis. no studies met the inclusion criteria for yfv and zikv infections. the titles and abstracts of the identified studies were reviewed independently by two reviewers using covidence systematic review software, (veritas health innovation, melbourne, australia. available at www.covidence.org). differences and conflicts were resolved by a third reviewer and through discussions for a consensus to be reached. percentage agreement and cohen's kappa (κ) statistic [92] were calculated and interpreted in accordance with landis and koch's benchmarks for assessing the agreement between reviewers [93] as poor (<0), slight (0.0-0.20), fair (0.21-0.40), moderate (0.41-0.60), substantial (0.61-0.80), and excellent (>0.81). the methodological quality of each study-from the standpoint of evaluating the prevalence of chronic comorbidities in flavivirus infections-was assessed as part of the data extraction. with some modification, we used the standards tool for evaluating and reporting epidemiologic studies on chronic disease incidence or prevalence, designed to assess population-based prevalence studies [94] [95] [96] . to assess the risk of bias, each study was rated against each of the ten following criteria: 1) clearly stating the research objective, 2) distinctly defining the study population, 3) homogeneity of the study subjects (e.g., age, ethnicity, gender ratio and origin of the studies population), 4) sample size and power justification, 5) assessing the infection prior to identifying the comorbidity, 6) evaluating levels of infectious disease severity and the related frequency of comorbidity, 7) standardizing the infectious disease assessment across all study subjects, 8) standardizing the assessment of table 1 . keywords for the search related terms and synonyms. population individuals with a flavivirus infections (both severe and non-severe cases), all age groups. dengue fever, yellow fever, west nile virus, zika, japanese encephalitis. diabetes, hypertension, heart disease (including: cardiovascular disease, coronary artery disease, coronary vascular disease, atrial fibrillation, chronic ischemic heart disease, acute coronary syndrome for duration less than 6 months, cardiac disorder, cardiac heart failure, congestive cardiac failure), stroke, obesity, asthma (does not include chronic obstructive pulmonary disease; copd). proportion/rate frequency of at least one of the comorbidities, percent comorbidity, death/fatality, incidence, hospitalization, mortality, mortality rate. comorbidities across all study subjects, 9) assessment of comorbidities blindly of the infectious disease status, and 10) considering the potential confounders with clear and adjusted statistical analyses. each criterion was rated dichotomously (yes: low risk = 1 point; no: high risk = 0 point). an overall score was calculated by adding all the items rated as low risk. thus, higher scores indicated lower risk of bias and stronger method quality. grey literature (mainly conference abstracts) was not assessed in this way as the required information was unavailable. data extracted from the selected studies in duplicate by two reviewers and included the first author's name, publication date, country, dates of recruitment, total sample size (divided to males and females), age estimates (from reported mean, median or the mid-point for age range of the highest subject frequency), procedures for case identification, type of flavivirus infection, severity of infection, prevalence of clinical manifestations (mild symptoms such as fever, headache, muscle pain, rash, and malaise together with severe symptoms as described below) and percentage of comorbidities including diabetes (both type i and type ii, if mentioned), hypertension, heart diseases (due to the small sample size of individual conditions, we (table 1) . all denv and wnv were confirmed cases in the selected studies and infection was identified either by rt-pcr, elisa, report reviews or from national surveillances. chronic comorbidities were either self-reported or record-based and did not allow for differentiating between those diagnosed before, after or during the infectious episodes. weighted average was used to calculate the overall age. severe cases of denv were defined as those with any form of the disease that develops major complications such as dengue hemorrhagic fever (dhf, grades i and ii), dengue shock syndrome (dss) (dhf grades iii and iv), infections developing organ failures (e.g., acute renal failure and acute respiratory failure), clinically significant bleeding, cases requiring icu hospitalization and/or fatal cases . severe cases of wnv were defined as those developed wnv neuroinvasive diseases, such as, cases of wn encephalitis (wne), wn meningitis (wnm), poliomyelitis or acute flaccid paralysis, cases requiring icu hospitalization, need for rehabilitation, wnv-associated retinopathy (wnvr), chorioretinitis or fatal cases [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] [86] [87] [88] [89] [90] . to measure the prevalence rates of comorbidities, we extracted the proportions or percentages reported in the selected studies from the total number of flavivirus cases and from non-severe and severe cases. publication bias was assessed by the visual inspection of funnel plot (s1 fig). egger's test [97] was also used to assess publication bias and the tendency for the effects estimated in small sample size studies to differ from those estimated in larger studies. the results of egger's test were presented as t-value and p for publication bias for studies reporting the comorbidity of interest (s4 table) . significant egger's test for publication bias was based on p<0.1. the primary outcome measure was to evaluate the overall prevalence of comorbidities in the flavivirus infection and the estimates stratified by severity. meta-analyses were carried out to assess the pooled prevalence (and 95% ci) of clinical symptoms and the proportions of each comorbidity in the total, severe and non-severe cases of each infection. meta-analysis tests were conducted using comprehensive meta-analysis software, cma version 3.9 (englewood, nj, usa) [98] . variances of raw proportions or percentages were pooled based on a binary random-effects model [99] , given the population heterogeneity and assuming the relationships between comorbidities and flavivirus infections vary across populations. forest plots were used to illustrate the prevalence of comorbidities in flavivirus infections from the selected studies prior to stratifying the proportions by severity. comparisons of proportions for the prevalence of clinical symptoms between diseases and the pooled prevalence estimates of comorbidities in relation to severity were carried out using the chi-squared test as previously recommended [100, 101] . ors adjusted for age (and 95% ci) for severe infection outcome in patients with comorbidities were calculated using the cochran-mantel-haenszel test as previously described [102] [103] [104] . all statistical tests were two-sided and conducted using spss statistics, version 21.0 (spss inc., chicago). assessing the heterogeneity among the selected studies was carried out using the q test [105] that informs about the presence versus the absence of heterogeneity. the q test, however, does not report the extent of heterogeneity and has inadequate power to detect heterogeneity among the small number of studies identified for some comorbidities. therefore, we calculated the i 2 index to complement the q test to describe the degree of between-study heterogeneity [106] . i 2 index values were categorized as low (0-30%), moderate (30-60%), substantial (60-90%), and considerable (>90%) as recommended [107] . we also quantified the true heterogeneity by estimating the within-study variance in the random-effects model (τ 2 ), as previously described [108] . all data generated or analyzed during this study are included in this published article (and its supporting information files). the original search resulted in 1,628 articles selected for title review as they satisfied our selection criteria (s3 table) . additional 11 articles were identified through bibliography search from previously identified systematic reviews (fig 1) . after de-duplication, a total of 1,373 original titles were selected for abstract review. abstract review resulted in the exclusion of 1,031 reports. full text assessment was conducted on the remaining 342 articles by at least two reviewers and resulted in the selection of 47 studies in denv [26-72], 18 in wnv [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] [86] [87] [88] [89] [90] and two in jev [22, 91] (those were further excluded) for inclusion (see fig 1 for exclusion criteria and breakdown). the agreement on the inclusion between two reviewers was 86.5% with weighted κ = 0.62 (95% ci: 0.52-0.72). this substantial agreement (0.61-0.80) may relate to the extent of clarity in the assessed abstracts when reporting the rates or the presence of comorbidities at the initial stage of the selection process. no studies were identified, however, for yfv and zikv infections. after excluding the 2 studies on jev, the quality of each of the remaining 65 studies, involving 61 sets of patients, was assessed. with a maximum quality score of 10, a good score (!7 points) was achieved in 35% of the studies (16 studies on denv and 7 on wnv); 30% of the studies were scored as being of fair quality (5-7 points including13 studies on denv and 6 on wnv) and 20% of the studies were scored as low quality ( 5 points including 11 studies on denv and 14 on wnv) ( table 2) . no score was assessed for the remaining 9 reports from the grey literature (abstracts; 7 on denv and 2 on wnv). more than 70% of the reports (48 studies) were retrospective in nature with the rest being age-and sex-matched or nested case-control studies, surveillance reports, or prospective studies. in these studies, there was a wide variation in the sample size ranging from five [81] to 6,070 [46] patients. same cohort was likely to be reported for denv in two occasions, i.e., by chen et al., 2015 [39] and lee et al., 2006 [70] and also by lee et al., 2009 [63] and lee et al., 2008 [88] . to avoid repeated counting of the cases from the same study cohort, we only included the study with the larger sample size both in evaluating the total number of subjects and in the analysis of average age of the studied cases. furthermore, one study [30] reported that out of the 1,734 studied denv cases, about 10% were classified as highly suggestive whereas the remaining 90% were confirmed by the diagnostic algorithm. this report did not separate or identify this small number of cases (~170 cases) that represents only 0.53% of the total number evaluated here and were included into our analysis. moreover, one study [28] included fatal cases of denv and chikungunya virus coinfection. although these cases may represent an unusual clinical population, they were included here for our report to be inclusive as the number of subjects was extremely small (n = 7) and they only had 3 cases of hypertension (representing <0.08% of the entire hypertensive cases). to examine if the study estimates are related to the size of the study, publication bias was assessed by visual inspection of funnel plots (s1 fig) and by egger's test (s4 table) . funnel plot inspection demonstrated a seemingly non-symmetrical distribution of the effect size on either side of the pooled estimate, suggesting some evidence of publication bias. results of egger's test, however, for most of the associations between denv or wnv and the comorbidities showed p>0.1 (except for the prevalence of hypertension in denv and heart diseases in wnv, p = 0.03), given the assumption for evidence of smallstudy effects is based on p<0.1 as previously reported [109] . the studies selected for denv were geographically diverse and included 18 countries. the reports were from southeast asia, south america, eastern mediterranean, and western pacific regions. most of the wnv reports were, however, from the united states with few studies (table 4 ). in wnv, however, meningitis and encephalitis were present in >35% of the severe cases, representing the highest prevalent severe condition. obesity/overweight was the most prevalent comorbidity in denv patients (24.5%, 95% ci: 18.6-31.6%), followed by hypertension (17.1%, 95% ci: 13.3-21.8%) and diabetes (13.3%, 95% ci: 9.3-18.8%) (fig 2) . heart disease, asthma and stroke were present in about 5.0% of the denv cases. on the other hand, in wnv cases, hypertension was the most frequent comorbidity (45.0%, 95% ci: 39.1-51.0%), followed by heart diseases (~25%), diabetes (~25%) and stroke (10.1%, 95% ci: 7.1-14.3%) (fig 3) . no study in wnv has reported the incidence of obesity or asthma. overall, there was low (i 2 = 0-40%) to moderate (i 2 = 30-60%) heterogeneity among the identified studies. when cases of denv and wnv were stratified by severity (table 5) , there was 3-to 4-fold higher prevalence (p<0.0001) of diabetes and heart diseases in severe denv and wnv cases, respectively compared to their rates in the non-severe disease. hypertension, however, was about 2-fold more prevalent in severe cases of both flavivirus infections (p<0.0001) than nonsevere cases. asthma and obesity were only examined in denv as none of the selected studies reported their prevalence in wnv. asthma and obesity were~1.5-fold more frequent (p<0.001) in patients with severe denv than in non-severe cases. the frequency of stroke was assessed only in one study in non-severe denv [69] and wnv [74] , which did not permit comparison between severe and non-severe cases. based on the differences in prevalence of comorbidities between severe and non-severe cases of denv and wnv, we estimated the age-adjusted or of severe infection outcome in patients with chronic comorbidities to be 3.41 (95%ci: 2. 54-4.59) in denv patients with obesity/overweight, 2.76 (95%ci: 2.54-299) in patients with diabetes, 2.4 in those with heart diseases (95% ci: 1.65-1.70) and 1.61 (95% ci: 1.52-1.70) in patients with hypertension. the or of severe wnv was 4.21 in patients with diabetes (95% ci: 2.22-7.92) and 2.72 in those with hypertension (95% ci: 1.78-4.14) (table 5 ). the present study evaluates the frequency of comorbidities in denv and wnv as examples to flavivirus infections that represent a major global health problem. datasets were few or unavailable for jev, zikv and yfv. only two studies were found to satisfy our selection criteria for jev [22, 91] and most of the reports on zikv were related to co-existing infectious diseases such as chikungunya or hiv [110] [111] [112] or to gestational diabetes in a single case [113] . studies in yfv, however, primarily evaluated the safety and effectiveness of vaccination (17d-yf) in people with pre-existing chronic illnesses [114] [115] [116] . a few systematic reviews on the frequency of comorbidities in flavivirus infection exist and none have used meta-analysis to synthesize global prevalence estimates. we observed a large difference in the volume of literature and total number of patients evaluated for each of the examined condition where 47 studies were identified for denv [26-72] with 31,969 subjects and 18 for wnv [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] [86] [87] [88] [89] [90] with 3,050 patients. almost 40% of the selected studies were assessed to have good quality score for evaluating and reporting chronic disease incidence or prevalence [94] [95] [96] . however, chronic diseases were not clearly defined in the vast majority of the studies as they were either selfreported or record-based and did not allow for differentiating between those diagnosed before, after or during the infectious episodes. very few of the selected reports provided sample size justification and all had infectious disease status assessed prior to the chronic disease scoring. the development of chronic diseases following infection was not reported in any of the selected studies. furthermore, none of the selected studies reported either denv or wnv as a coinfection for one another. moreover, a few number of studies provided information on the status of more than one chronic disease comorbidity in the same patient. additionally, assessment of the chronic diseases was not blindly evaluated with respect to the infectious diseases status. these factors meant that 60% of the selected studies had good-to-fair quality scores. this made us choose to include studies that were assessed to be of "fair" or "low" quality in our meta-analysis to attain more comprehensive estimates with larger sample size and to avoid the bias when excluding studies with small or no effect. the geographical diversity of denv reports compared to wnv studies likely reflects the fact that dengue now is the most common flavivirus infection globally with transmission occurring in at least 128 countries and almost 4 billion people around the world [7] . wnv, on the other hand, despite being an important cause of human disease worldwide with continual increase in transmission over the past 77 years [5, 14] , is spreading in a notably lesser rate than denv [5]. only one study of the 18 selected reports on wnv were from regions other than europe or the usa [89] . given the repeated wnv outbreaks between 1996 and 2015 in israel and europe and its movement through north america over the last decade that resulted in sustained presence in the community [5, 13, 14] , it was predictable to identify a limited set of wnv studies from regions other than those primarily affected. most clinically apparent acute flavivirus infections progress from a classic presentation of mild fever, headache, muscle pain, rash, and malaise to a more disease-specific syndrome of acute febrile illness to further severe states of hemorrhagic fever and encephalitis. pooled prevalence analysis of these clinical symptoms indicated their comparatively lower frequencies in , heart diseases (c), asthma (d), stroke (e) and obesity/overweight (f) in dengue fever patients and 95% ci. heterogeneity analysis was carried out using q test, the among studies variation (i 2 index) and within-study variance in the random-effects model (τ 2 ). wnv than in denv. it is known that wnv infection is commonly asymptomatic, with~20% of infected persons having clinically apparent disease [117] [118] [119] . symptomatic patients mostly present with fever, headache and malaise and less commonly with myalgia, rash, neck pain, and arthralgia [76] . on the other hand, patients infected with denv are asymptomatic in the majority of cases but a small proportion may develop an array of clinical symptoms ranging from mild flu-like syndrome, such as fever, skin rash, headache, myalgia, and arthralgia, to severe forms of the disease. only rash was 2-fold more frequent in denv than wnv cases. according to the 2009 who classification system [120] , denv patients can be clinically classified as with 'probable dengue', 'dengue with warning signs' or 'severe dengue'. this deviates from the 1997 who classification system [121] , categorizing patients as with df, dhf or dss. over 90% of the denv patients in the selected set of studies presented with df. the progression of df to severe clinical manifestations is mostly unpredictable. case fatality rate may exceed 20% if timely and appropriate differential therapy is not introduced to reduce the impact of disease complications [120] . recent estimates for the global burden of dengue suggest the epidemics mainly affect children and young adults between the ages of 5 and 30 years old in terms of death, years lived with disability (ylds), years of life lost (ylls) and disability-adjusted life-years (dalys) [122] . however, an age shift to more adult cases with severe denv (and more comorbidities) is presently demonstrated in several reports from around the world (see below) [120, 123, 124] . in addition to this age-shift in severe denv cases, the incursion of denv into new world regions may have influenced the age-related increased incidence of the disease. advanced age, on the other hand, has long been known as a frequent risk factors for severe disease outcome of wnv [14] . average annual incidence of severe wnv cases reported to cdc between 1999-2015 for cases over 70 years old was~1.3 per 100,000 population per year compared to only~0.5 for patients <10-39 years old [125, 126] . introduction of the competent vectors into natural environments and urban areas, together with the changing societal factors (migration, industrialization, trade, urbanization and population growth), may all have facilitated the geographical expansion of flavivirus diseases into different regions of the world. examples include the expansion of denv from the caribbean islands to brazil and from the pacific islands to other regions in south asia [127, 128] and wnv from africa and the middle east to europe and north america [5] . many of the regions where these vector-borne diseases spread are also experiencing an epidemiological shift from communicable diseases to non-communicable disorders as the primary causes of the morbidity and mortality [129] . with the ageing population, non-communicable diseases now account for nearly half of the disease burden in low-and middle-income countries [130] . therefore, flavivirus diseases, particularly denv, may have been shown now to affect older adults, an age group with inherently more comorbidities [130, 131] . the present study demonstrates that hypertension and diabetes are the most prevalent comorbidity in both denv and wnv with obesity/overweight and heart diseases being present, respectively, in about 20% of the denv and wnv cases. although the prevalence of diabetes, hypertension and heart diseases varied widely among the denv selected studies (ranging from 60-to 100-fold), the vast majority of the reports showed values clustering around the pooled estimated averages for each comorbidity, as evidenced by the low i 2 index values. obesity, stroke and asthma proportions varied only by 5-to 30-fold among the studies with moderate i 2 index values. however, in wnv studies, the among-studies prevalence of comorbidities varied by 3-to 12-fold for diabetes, hypertension, heart diseases and stroke with low to moderate i 2 indexes. these variations in the prevalence of comorbidities in wnv cases may relate to the higher average patients' age (compared to denv) since older age is a risk factor for a number of non-communicable diseases, e.g., diabetes and heart conditions [132] [133] [134] [135] [136] . on the other hand, the wider variation in prevalence of comorbidities in denv than wnv can be linked to the different patterns of geographical distribution between the two diseases. diabetes, hypertension and heart diseases were, respectively, 2-to 4-fold significantly more prevalent in severe denv and wnv than in non-severe cases. this observation is supported by a number of case-control studies implicating comorbidities in severe outcome of flavivirus infections. over 2-fold higher frequency of diabetes were found in severe denv cases than in cases with df [45, 47, 57, 59] . furthermore, severe clinical presentation of denv was likely to develop in patients with diabetes than in non-diabetic subjects with the odds ratio (or) ranging from 1.26 (95% ci 0.8-2.0) [50] to 26 (95% ci 2.5-273.7) [45] . this is line with the or of about 2 to 4 for severe denv and wnv observed here among patients with diabetes or heart diseases. diabetes risk factors such as glucose intolerance [137] and hyperlipidemia [53] were prevalent in 54% and 17% of severe denv cases, respectively and more frequent in elder patients [61, 66] . similarly, diabetes (19-42%), hyperlipidemia (17-19%) and coronary artery disease (5-17%) were prevalent in wnv cases than controls [76, 78] with or of neuroinvasive outcome varying between 2.4 (95% ci 0.57-10.4) and 4.5 (95% ci 0.88-23.1) [76] . diabetes was 23% more frequent in cases with wne and wnm than in wnv fever [74] . developing encephalitis was more likely to occur in wnv cases who also had diabetes (or = 2.0; 95% ci: 1.1-3.7) or cardiovascular diseases (or = 28.3; 95% ci: 5.9-134.9) [87] . furthermore, hypertension was shown to be present in 50% of the severe or fatal denv cases [1] , the manifestation of acute respiratory failure [6], and in higher proportion of elder patients [1, 3] with or varying from 1.6 (95% ci 1.1-2.1) [4] to 44.3 (95% ci 6.2-315.5) [5]. in contrast, some studies did not show such a relationship in severe denv [8, 9, 138] . in wnv, patients with hypertension had higher odds of developing neuroinvasive outcome (or 1.88, 95% ci 0.63-5.58) [78] and encephalitis (or = 5.1, 95% ci 2.5-10.4) [87] than non-hypertensive cases. high prevalence of hypertension (46%) was also noted in patients with wne, wnm and fatal outcome compared to those in wnv fever [74] . in line with these findings, the results of the present study strongly suggest that diabetes, heart diseases and hypertension are more prevalent in severe denv and wnv cases than in the non-severe disease and may present significant risk factors-together with advanced age-in complication of flavivirus infection. diabetes, hypertension, cardiac diseases and obesity are interrelated as they share similar cardiometabolic risk factors that result in the development of metabolic syndrome and the subsequent manifestation of this range of chronic diseases. these metabolic syndrome related diseases may impair the immune system to increase the level and duration of viremia [109, 139] and facilitate the passage of neurotropic flavivirus across the blood-brain barrier to predispose patients to neurologic complications [88, 118, 140] . impairment of the innate immune system-that mediates the host defense to infection-render individuals more susceptible to a range of infectious diseases and severe illnesses [141] [142] [143] [144] . in fact, the metabolic syndrome related chronic conditions are linked to endothelial dysfunction, attenuation of anti-inflammatory responses and a generation of a pro-inflammatory state; features that are also common in many infectious disorders [21, 141, 145] . for overproduction of pro-inflammatory cytokines such as ils, tnf-α, ifn-γ and tgf-β is known to occur in severe denv [146] , wnv [147] and yfv [148] leading to cytokine storm and vasculopathy, hemorrhage, tissue damage and septic shock characteristic of severe flavivirus infections. cytokine synthesis shift to the th1 (microbicidal action of pro-inflammatory ifn-γ) from th2 (anti-inflammatory il-4, -10 and -13) in severe infection, when accompanied by the increased pro-inflammatory cytokine levels arising from chronic diseases, both can lead to endothelial dysfunction and a subsequent range of complications, including allergy, vascular leakage, ascites and pericardial effusion as observed in denv [19, 141, [149] [150] [151] . in support, mononuclear cells from diabetic patients, when infected with denv, produced significantly higher levels of il-4, il-10 and granulocyte-macrophage colony-stimulating factor compared to their healthy counterparts [152] . conditions such hyperglycemia and cellular insulinopenia may also impair macrophage and lymphocyte functions leading to a status of reduced acquired immune response [143] that was linked to about 60% increased risk of pneumonia-related complications and hospitalization [153] . further evidence for a possible role of altered innate immunity in mediating the association between metabolic syndrome-related comorbidities and severe clinical presentation of flavivirus infections can be also substantiated from the relatively high prevalence of asthma in severe denv cases. patients with asthma normally exhibit altered th1 and th2 responses [50, 59] . in general, although the etiological relationship between chronic comorbidities and severity of flavivirus diseases is yet to be fully elucidated, it may be reasonable to suggest that in infected patients, chronic conditions may synergistically attenuate both the innate and adaptive immune systems [154] . this may further impair critical components of immunity such as chemotaxis, phagocytosis, and the bactericidal activity of neutrophils and macrophages as well as downregulate the functions of t cells and neutrophils [142, 154] to exacerbate the complications of the infectious diseases. although the present study is the first to systematically report and quantify the prevalence of comorbidities in flavivirus infections, it has several limitations. it does not address the effect of flavivirus infection on the development of chronic comorbidities or attempting to substantiate a causality between the two conditions. also, the study is not addressing the effect of integrated vector management practices on the infectious or chronic disease incidence or causal associations. we are simply reporting the prevalence of comorbidities in flavivirus severe and non-severe cases to warrant further studies investigating the effect of chronic comorbidities on the infectious disease outcome. the selected studies were mostly retrospective in nature with variable clinical and laboratory diagnostic criteria and control groups and were heterogeneous for exposures and outcomes. in the denv studies, although the majority were conducted after the who adopted the new case classification of 2009 [120] to improve clinical management, many of the reports still used the who 1997 classification [121] . this absence of similar endpoint measures hindered the results comparability and may limit the generalizability of conclusions to other geographical regions or older age groups since the who 1997 criteria were based on disease patterns of children in thailand [21, 121] . in addition to the retrospective nature of many of the identified studies, incomplete clinical datasets, relatively small sample size, inappropriate definition or selection of control population were noted as shortcomings in various reports leading to further limitations in interpreting the findings. in fact, most of the studies were hospital-based with a high potential for selection bias of appropriate control groups. additionally, the studies retrieved in this review measured different outcomes for denv (dhf and dss-with different grades and definitions of severity) and wnv (wne, wnm, poliomyelitis or acute flaccid paralysis) making results difficult to compare, broadening the scope of the outcome and rendering the findings challenging to extrapolate. furthermore, the identified reports have shown several-fold of among-studies variance in the proportion of comorbidities which may have contributed to the significant heterogeneity observed in our report. additional sources of heterogeneity may relate to the large amongstudies variation in sample size and surveillance approaches. the heterogeneity of the selected studies was evident from the publication bias that may have been driven from the small-study effect, i.e., the possibility of including small studies with spuriously overstated estimates while discounting those without statistically significant effects that may have a lower possibility of being published. this was addressed, however, by stratifying the analysis by the flavivirus infection and including all studies related to the research question rather than excluding reports based on lack of quality. this may levy some limitations on the estimated contribution of comorbidities to severe flavivirus infections and render our results as a guide to generate more accurate estimates for national or international intervention strategies in subjects with comorbidities. when assessing comorbidities, the studies showed that the various co-existing conditions have been either self-reported or record-based and did not allow for differentiating between those diagnosed before, after or during the infectious episodes. furthermore, most of the retrieved reports did not provide clearly characterized, valid and reliably-defined comorbidities that were implemented consistently across studies. for example, the majority of the reports did not distinguish between the prevalence of type 1 and type 2 diabetes where both were combined despite their different characteristics, etiological factors and clinical features. given this paucity of information, it was challenging to distinguish between the two types of diabetes for their role in viral diseases severity and complication. furthermore, the lack of consistent reporting for the statuses of heart diseases made it difficult to evaluate the frequency of each heart condition. therefore, we combined the different heart conditions under a single comorbidity. the utility of pooling the clinically different and heterogeneous pathologies of heart diseases may lead to losing significance of this observation and result in misinterpretation. this observation warrants developing more rigorous studies exploring the role of various heart condition in the severity of flaviviral infections. given the possible extent of under-diagnosis for many of the assessed comorbidities, particularly in the developing world, misclassification of many co-existing medical conditions is likely substantial. together with the possible underestimation for the frequency of infectious disease where not all asymptomatic cases will be detected, these factors may have led to lower estimates of prevalence for many of the assessed comorbidities. lastly, since we identified only two studies for jev and no studies met the inclusion criteria for yfv and zikv infections, the present report may be viewed as focusing primarily on denv and wnv. this lack of available information on the prevalence of comorbidities in flavivirus diseases other than denv and wnv, calls for developing large-scale studies to cover this knowledge gap. in conclusion, the study of comorbidities in flavivirus infection is important for reducing the burden of the disease via guiding approaches for improved patient outcome or differential case management. we provided evidence for a higher prevalence of diabetes, hypertension, and heart diseases in severe cases of flavivirus infections such as denv and wnv than in nonsevere cases. these findings do not implicate causality between the chronic comorbidities and severe denv or wnv. it simply demonstrates different profiles of comorbidities at different stages of the infectious diseases. our results warrant further assessments to identify the nature and extent of the co-existence between comorbidities and infection. for example, standardized prospective case-control studies in regions of high infection prevalence would contribute to a better understanding for the etiological role of comorbidities in severe disease outcome when conducted with an agreed protocols of comorbidity assessments, infection classification, disease biomarker analysis and appropriate control groups. however, even in the absence of causal inference between the non-communicable and infectious diseases, 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nicholas ogden for constructive discussions and comments. key: cord-309471-lr68epyb authors: xia, jingya; veselenak, ronald l.; gorder, summer r.; bourne, nigel; milligan, gregg n. title: virus-specific immune memory at peripheral sites of herpes simplex virus type 2 (hsv-2) infection in guinea pigs date: 2014-12-08 journal: plos one doi: 10.1371/journal.pone.0114652 sha: doc_id: 309471 cord_uid: lr68epyb despite its importance in modulating hsv-2 pathogenesis, the nature of tissue-resident immune memory to hsv-2 is not completely understood. we used genital hsv-2 infection of guinea pigs to assess the type and location of hsv-specific memory cells at peripheral sites of hsv-2 infection. hsv-specific antibody-secreting cells were readily detected in the spleen, bone marrow, vagina/cervix, lumbosacral sensory ganglia, and spinal cord of previously-infected animals. memory b cells were detected primarily in the spleen and to a lesser extent in bone marrow but not in the genital tract or neural tissues suggesting that the hsv-specific antibody-secreting cells present at peripheral sites of hsv-2 infection represented persisting populations of plasma cells. the antibody produced by these cells isolated from neural tissues of infected animals was functionally relevant and included antibodies specific for hsv-2 glycoproteins and hsv-2 neutralizing antibodies. a vigorous ifn-γ-secreting t cell response developed in the spleen as well as the sites of hsv-2 infection in the genital tract, lumbosacral ganglia and spinal cord following acute hsv-2 infection. additionally, populations of hsv-specific tissue-resident memory t cells were maintained at these sites and were readily detected up to 150 days post hsv-2 infection. unlike the persisting plasma cells, hsv-specific memory t cells were also detected in uterine tissue and cervicothoracic region of the spinal cord and at low levels in the cervicothoracic ganglia. both hsv-specific cd4(+) and cd8(+) resident memory cell subsets were maintained long-term in the genital tract and sensory ganglia/spinal cord following hsv-2 infection. together these data demonstrate the long-term maintenance of both humoral and cellular arms of the adaptive immune response at the sites of hsv-2 latency and virus shedding and highlight the utility of the guinea pig infection model to investigate tissue-resident memory in the setting of hsv-2 latency and spontaneous reactivation. hsv-2 infection is widespread globally with an estimated 23.6 million new infections occurring each year [1] . although disease associated with hsv-2 infection is often limited in severity, more serious manifestations may also occur. hsv-2 present in the birth canal of infected mothers may be passed to neonates during vaginal delivery resulting in serious morbidity and mortality [2, 3] . vigorous cell-mediated responses are normally responsible for diminishing the severity and duration of hsv-2 disease with immune compromised individuals being more likely to experience severe complications resulting from hsv-2 infection [4, 5] . hsv-2 infection also has been shown to increase the risk of hiv infection and increased hiv shedding is often observed during an active hsv-2 infection in co-infected individuals [6, 7] . hsv-2 has co-evolved with humans and is an extremely successful pathogen, capable of residing long-term in its host and of effective transmission to uninfected individuals. genital hsv-2 infection of the epithelia spreads to sensory neurons and ultimately results in lifelong latent infection of the innervating sensory ganglia and spinal cord [8, 9, 10] . once thought to reactivate only occasionally from latency, it is now generally held that reactivation events for most individuals are quite frequent [11] and result in virus shedding often in the absence of overt clinical symptoms. further, clinical evidence suggests that the period of virus shedding following reactivation is most often of relatively short duration [12, 13] due perhaps to the clearance of virus by hsv-specific t cells residing at the site of previously infected skin [14, 15] . similar populations of tissue-resident hsv-specific cd4 + and cd8 + t cells have been found in latently infected trigeminal ganglia of humans [16, 17, 18] and in mice following ocular hsv-1 infection [19, 20] . however, the long term presence and immune function of virus-specific t cells in neural tissues following genital hsv-2 infection has received much less study and less information is available. sacral ganglia-resident memory cell populations are not currently amenable to study in humans. infection of mice with fully virulent hsv-2 commonly results in encephalitis and death, precluding easy analysis of the magnitude, phenotype and function of virus specific ganglia-and spinal cord-resident memory t cells in this animal model. the guinea pig model of genital hsv-2 infection represents a unique system to address the nature of both genital-resident and neural tissue-resident immune memory. genital infection of guinea pigs results in a self-limiting vulvovaginitis with neurologic manifestations mirroring those found in human disease. virus is transported by retrograde transport to cell bodies in the sensory ganglia and autonomic neurons in spinal cords [10] . during this phase of infection, the virus establishes a latent infection and, similar to humans, the animals undergo spontaneous, intermittent reactivation of virus. hsv-2 recurrences may manifest as clinically apparent disease with erythematous and/or vesicular lesions on the perineum or as asymptomatic recurrences characterized by shedding of virus from the genital tract. candidate prophylactic vaccines against hsv-2 have been capable of inducing high levels of systemic, hsv-specific immune responses but have failed to prevent hsv-2 infection or hsv disease in clinical trials [21, 22] . a different vaccine approach is needed to impact hsv-2 disease, perhaps involving development of strong immune responses to hsv-2 at the sites of virus infection. tissue resident memory immune cells are strategically located to protect against re-infection or interfere with hsv-2 shedding following release of virus after reactivation from latency. the potential for protection conferred by these tissue-resident cell populations has profound implications for hsv-2 vaccine strategies, but the nature of tissue-resident memory immune cells in neural tissues following genital hsv-2 infection, the distribution of immune b and t cells within the reproductive tract, the nature of tissue-resident hsv-specific antibody secreting cells (ascs) and the functional activity of locally-produced antibody is not well understood. we utilized a genital hsv-2 infection of guinea pigs to address these issues and demonstrate the utility of this approach in assessing the nature of tissue-resident immune cell populations in an animal model that most effectively recapitulates human hsv-2 infection. virus hsv-2 strain ms stocks were prepared on vero cell monolayers and stored at -80˚c as described previously [23] . both the replication-defective hsv-2 strain, hsv-2 dl5-29, deleted of the hsv dna replication protein genes ul5 and ul29, and the complementary cell line v529 expressing the ul5 and ul29 proteins [24] were a kind gift of dr. david knipe (harvard medical school, boston, ma). virus stocks were prepared by infection of v529 cells with hsv-2 dl5-29 at an moi of 10. following development of cytopathic effect (typically 24 hours), cells were subjected to three cycles of freeze-thaw, cell debris pelleted by centrifugation, and the supernatant was aliquoted and frozen at -80˚c. female hartley guinea pigs were purchased from charles river (burlington, ma) and strain 13 guinea pigs were obtained from dr. marisa st. claire, national institutes of allergy and infectious disease, nih. female guinea pigs weighing 275 to 300 g were infected by intravaginal (ivag) inoculation with 200 ml of a suspension containing 10 6 pfu of hsv-2 strain ms as described previously [23] . animals were observed daily and primary disease severity and frequency of spontaneous recurrent disease were scored daily as described previously [25] . all animals survived the hsv-2 infection and all animals exhibiting either primary or recurrent disease symptoms were included in the studies. this study was carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health. guinea pigs were maintained under specific pathogen free conditions and were supplied with food and water ad libitum at the association for assessment and accreditation of laboratory animal care-approved animal research center of the university of texas medical branch. all animal research was humanely conducted and approved by the institutional animal care and use committee of the university of texas medical branch with oversight of staff veterinarians (protocol number 0204025b). genital tract and neural tissues (spinal cord and sensory ganglia) were collected from guinea pigs and finely minced. genital tract tissue was successively digested with dnase i and dispase ii (roche diagnostics, mannheim, german) for 45 min followed by collagenase (roche diagnostics), hyaluronidase (sigma-aldrich, inc. st. louis, mo) and dnase i digestion for 60 min at 37˚c. neural tissues were digested with liberase (roche diagnostics) and dnase i for 45 min at 37˚c. lymphocytes from genital tract and neural tissues were isolated by centrifugation over optiprep density gradients and percoll gradients, respectively. assays to quantify hsv-specific ascs were performed as described previously [26] on hsv-glycoprotein-coated plates. to quantify hsv-specific t cells, lymphocytes isolated from spleen, genital tract, or neural tissues of uninfected or hsv-2 infected guinea pigs were stimulated by 48 h culture with splenocyte-or mesenteric lymph node cell-antigen presenting cells (stimulator cells) infected with hsv-2 dl5-59 and pulsed with uv-killed hsv-2 to ensure that both mhc class i and class ii antigen presenting pathways were engaged to stimulate both virus-specific cd8 + and cd4 + t cells. lymphocytes were treated with medium as a control. cultures were incubated in the presence of recombinant human il-2 (ebioscience, san diego, ca) on anti-ifn-c antibody-coated plates. hsv-specific ifn-c secreting t cells were detected using monoclonal antibody v-e4 as the capture antibody and biotinylated anti-guinea pig ifn-c antibody n-g3 as the detection antibody [27] (both antibodies a kind gift of dr. hubert schäfer, robert koch institute, berlin, germany). spots were visualized by incubation with streptavidin peroxidase and aec (3-amino-9-ethylcabazole) substrate (sigma-aldrich, st. louis, mo). hsv-specific ascs and ifn-c secreting cells were quantified using an immunospot reader and analyzed with immunospot software (cellular technology ltd, cleveland, oh). enriched lymphocyte populations from the spleen, bone marrow, lower genital tract (vagina and cervix), and neural tissues (spinal cord, sensory ganglia) were cultured at 4610 6 cells/ml in t cell medium alone or with 4.0 mg/ml lps and 1.0 mg/ml cpg for 72 hrs. preliminary studies indicated maximum recovery of antibody secreting cells at 72 h of culture. hsv-specific ascs from stimulation cultures were quantified on hsv-glycoprotein-coated elispot plates. hsv-specific iga, igg, igg1 and igg2 titers from immune serum or asc culture supernatants were obtained by elisa as performed previously [28] . plate-bound immunoglobulins were detected by addition of polyclonal goat anti-guinea pig igg (#a60-110a) or polyclonal rabbit anti-guinea pig iga (#a60-105) (bethyl laboratories, inc., montgomery, tx) followed by polyclonal hrp-rabbit antigoat igg (#a50-100p) or polyclonal hrp-goat anti-rabbit igg (#a120-101p; bethyl laboratories, inc., montgomery, tx), respectively. igg1 and igg2 titers were detected by incubation of plate bound hsv-specific antibodies with polyclonal biotinylated goat anti-guinea pig igg1 (#abin457757) or polyclonal biotinylated goat anti-guinea pig igg2 (#abin457760; antibodies-online inc., atlanta, ga) followed by streptavidin peroxidase (sigma-aldrich, st. louis, mo). normalized optical density readings at 490 nm (od 490 ) obtained from serial dilution of serum or culture supernatants were analyzed by non-linear regression. the end point titer was defined as the serum dilution resulting in an od 490 value equivalent to three standard deviations above od 490 values from naive sera or medium only. for determination of glycoprotein specificity, elisa plates were coated with hsv-2 recombinant glycoprotein d (rgd2) and recombinant glycoprotein g (rgg2) purchased from meridian life science, inc., memphis, tn. neutralizing antibody titers from immune serum or asc culture supernatants were determined by a modification of the technique described previously [29] . neutralizing antibody in supernatants was also demonstrated by adding a series of two-fold dilutions of virus to undiluted asc supernatant containing low tox m rabbit c (accurate chemical and scientific, westbury, ny) at a final dilution of 1/ 15. following incubation at 37˚c for one hour, hsv-2 in each sample was quantified by plaque assay on vero cell monolayers. vero cells (american type culture collection number ccl-81) were obtained originally from the laboratory of dr. lawrence stanberry, columbia university school of medicine, new york, ny). single cell suspensions of genital tract lymphocytes and neural tissue lymphocytes were blocked with anti-fc rii/iii mab (#553142; bd biosciences, san jose, ca) and surface stained with monoclonal mouse anti-guinea pig cd8-fluorescein isothiocyanate (fitc; #mca752f; clone ct6) or monoclonal mouse anti-guinea pig cd4-phycoerythrin (pe; #mca749pe; clone ct7; abd serotec, oxford, uk), labeled with anti-fitc or anti-pe microbeads, respectively, (miltenyi biotec, bergisch gladbach, germany) and applied to a magnetic column (miltenyi biotec) according to the manufacturer's protocol. the phenotype of columnbound and column-flow through populations was assessed by flow cytometry. data were acquired on a bd facscanto ii (bd biosciences) at the utmb flow cytometry core facility and analyzed using flowjo software (tree star, ashland, or). column enrichment of cd4 + t cells commonly resulted in greater than 90% cd4 + cells (0.64% cd8 + cells). column enrichment of fitc-labeled cd8 + t cells was less efficient (typically 12-29% of collected cells were cd8 + ) although less than 0.5% of obtained cells were cd4 + . statistical differences for b lymphocyte assays, t lymphocyte assays, and antibody titers were determined using student's t test. differences in cell frequency were determined using the chi square test. values for p,0.05 were considered significant. all calculations were performed using graphpad prism software version 5.0 (graphpad software, san diego, ca). we previously demonstrated that hsv-specific asc were detected long-term at the site of hsv-2 latency in the sensory ganglia and at the site of virus shedding in the female genital tract in guinea pigs infected ivag with hsv-2 [28] . we hypothesized that these asc represented long-lived plasma cells existing in survival niches at these sites of chronic inflammation although an alternative explanation is that these asc derived from tissue-resident memory b cells as a result of periodic exposure to hsv antigens released during reactivation events. hsv-specific memory b cells were detected functionally because antibodies for guinea pig memory b cell markers are not currently available. memory b cells, but not plasma cells, become activated and differentiate into asc upon stimulation with polyclonal activating agents such as tlr-agonists [30, 31] . the presence of memory b cells in the culture is therefore detected as a tlr-agonist-induced increase in the number of antigen-specific, ascs. to test for the presence of memory b cells at peripheral tissues, we stimulated lymphocyte populations isolated from bone marrow, spleen, vagina/cervix, or spinal cord/sensory ganglia of previously infected guinea pigs with a combination of lps and cpg oligonucleotides and quantified hsv-specific asc by elispot. hartley guinea pigs were infected with hsv-2 strain ms. fourteen of the 19 animals used for assessment of hsv-specific asc were scored for both primary and recurrent hsv-2 disease to document hsv disease. primary hsv-2 disease was observed in 12 of 14 animals with a mean disease score of 5.4¡1.1 and at least one recurrent lesion was detected in 12 of 14 infected animals between days 15 pi to the day of euthanasia resulting in a mean recurrent disease score of 1.2¡0.26. tissues were harvested from individual animals between days 49-70 after infection. less than five hsv-specific ascs were detected in all tissues from uninfected control animals. however, as shown in fig. 1 , hsv-specific ascs were detected in all tissues from hsv-2 infected animals following control treatment. significantly greater numbers of hsv-specific asc were detected in lps/cpg-stimulated splenocyte cultures compared to medium-stimulated cultures (p,0.0001, student's t test) indicating the presence of hsv-specific memory b cells. additionally, a smaller but still significant increase in asc number was also detected in lps/cpg-stimulated bone marrow cells compared to stimulation with medium alone (p,0.05, student's t test). in contrast, there was no difference in hsv-specific asc number following medium-or lps/cpg-stimulation of lymphocytes isolated from the vagina/cervix or spinal cord/sensory ganglia. therefore, hsv-specific memory b cells were readily detectable in the spleen and to a lesser extent in the bone marrow of hsv-2 infected guinea pigs but not at the peripheral sites of hsv-2 infection. these results suggest that the vast majority of hsv-specific asc present in the lower genital tract, lumbosacral ganglia and adjacent spinal cord on days 49-70 post infection represented a persisting population of plasma cells. to more precisely define the tissue distribution of hsv-specific asc, lymphocytes from uterus, vagina/cervix, cervicothoracic sensory ganglia, cervicothoracic spinal cord, lumbosacral sensory ganglia and the adjacent spinal cord were isolated from individual animals between 49-70 days post infection and hsv-specific asc quantified by elispot. as shown in fig. 2 , hsv-specific ascs were detected in lymphocyte populations isolated from tissues of infected-but not uninfected-guinea pigs. the presence of genital tract-resident, hsv-specific asc was limited mainly to the vagina and cervix as only a very low number of hsvspecific ascs were isolated from the uterus of a single infected animal ( fig. 2a) . hsv-2 genomes were detected at this time period in the vagina/cervix tissue from two of four infected animals which most likely represented hsv-2 shedding; however, no virus genomes were detected in the uterus. similarly, hsv-specific ascs were detected in the lumbosacral ganglia innervating the genital tract of five of seven animals, but not in the cervicothoracic ganglia of infected guinea pigs (fig. 2b) . hsv-specific ascs were detected in spinal cord tissue isolated from the cervicothoracic region in five of ten infected animals whereas significantly higher numbers of hsv-specific asc were detected in the lumbosacral region of the spinal cord in nine of ten infected animals (p,0.05, student's t test, fig. 2c ). to characterize the antibody produced by tissue-resident asc and to determine if the isotype and igg subclass profile of antibody from hsv-specific asc isolated from peripheral sites of hsv-2 infection were similar to hsv-2-specific antibodies in immune serum, asc were isolated from selected tissues and the antibody-containing culture supernatant was harvested after three days of culture peripheral tissue-resident immune memory to hsv-2 for analysis by elisa (fig. 3) . in sera from immune animals, titers of hsvspecific igg were significantly higher than iga titers (p,0.001, student's t test) and the igg2 subclass predominated the response. a similar isotype profile was apparent for hsv-specific antibody produced by bone marrow-, vagina/cervix-, and spinal cord/ganglia asc. antibody was also tested for binding to recombinant hsv-2 glycoproteins representing known targets of antibodies in convalescent sera. as shown in fig. 4 , hsv-specific serum igg bound at a nearly equivalent level to full-length or truncated recombinant hsv-2 glycoprotein d (rgd2) while binding to recombinant hsv-2 glycoprotein g (rgg2) was detected at a significantly lower level. the same binding pattern was observed for igg produced by bone marrow-and spinal cord/ganglia-asc (fig. 4b, c) . the level of igg produced by asc from the vagina/cervix was more variable and therefore binding to the two forms of rgd2 and rgg2 was not significantly different (fig. 4b) . hsv-2 neutralizing antibody was present at a titer of 1:1023¡196 in the serum of hsv-2 infected guinea pigs. functional hsv-2-neutralizing antibody was also produced by ascs cultured from the lumbosacral ganglia and spinal cords of hsv-2 infected animals (fig. 4e, f) . incubation of hsv-2 with supernatants of spinal cord/ganglia-resident asc decreased viral titers over a range virus inoculums compared to the medium control (fig. 4e) . additionally, asc supernatant could be diluted approximately 5-fold and retain the ability to neutralize 50% of input virus (fig. 4f) . neutralizing antibody was not detected in supernatants of asc from genital tracts perhaps due to an approximately 10-fold lower number of hsv-specific asc isolated from genital tract asc compared to lumbosacral spinal cord asc ( fig. 2a compared to fig. 2c ). to our knowledge, these studies represent the first characterization of hsv-specific antibodies produced by neural tissue-resident asc during hsv latency. the activation and infiltration of hsv-specific t cells producing ifn-c at the peripheral sites of acute hsv-2 infection in the genital tract and sensory ganglia has been observed in murine models of genital hsv-2 infection [29, 32, 33] and from hsv lesion-derived human t cells [34, 35] . to further characterize t cell immunity in the genital tract, lumbosacral ganglia and spinal cord, strain 13 guinea pigs were inoculated ivag with hsv-2 strain ms. all animals experienced primary hsv disease resulting in a mean disease score of 5.7¡1.1. on day 7 post infection, hsv-specific, ifn-c-secreting t cells from the spleen, vagina/cervix, and lumbosacral ganglia and the adjacent spinal cord were quantified using a pair peripheral tissue-resident immune memory to hsv-2 of previously described anti-guinea pig ifn-c -specific monoclonal antibodies [27] in an ifn-c elispot assay. lymphocytes from the indicated tissues were stimulated by culture with stimulator cells infected with hsv-2 dl5-29 and pulsed with uv-inactivated hsv-2 to ensure that both mhc class i and class ii antigen presenting pathways were engaged to stimulate both cd4 + and cd8 + t cells. as shown in fig. 5a-b , ifn-c secreting cells (sc) were readily detected in tissues from hsv-infected, but not uninfected guinea pigs. hsv-specific ifn-c scs detected in the spleen, vagina/cervix, and spinal cord/lumbosacral ganglia of hsvinfected guinea pigs were over four logs greater than in uninfected animals ( fig. 5b) . although the total number of hsv-specific, ifn-c scs was similar in all three tissues, the frequency of these cells isolated from the spleen was somewhat variable among individual animals and significantly lower overall than the frequencies of hsv-specific, ifn-c sc isolated from the vagina/cervix or spinal cord/ganglia (p,0.0001, chi square). the ifn-c elispot assay was modified for use in outbred animals and used to detect and quantify hsv-specific memory t cells from tissues of hsv-2-infected hartley guinea pigs. animals were inoculated ivag with hsv-2 strain ms and the primary and recurrent disease were scored. ten of the 11 animals utilized for memory t cell analysis experienced primary hsv disease (mean score 6.7¡1.0) and 10 of 11 animals experienced at least one recurrent lesion (mean score 5.2¡0.7) between day 15 and the time of euthanasia (day 99-150). tissues were harvested from individual animals between days 99-150 post infection. cells from spleen, vagina/cervix, uterus, lumbosacral ganglia, cervicothoracic ganglia and the corresponding adjacent region of spinal cord were stimulated with stimulator cells infected with hsv-2 dl5-29 and pulsed with uv-inactivated hsv-2 to stimulate both cd4 + and cd8 + memory t cells. as shown in fig. 6a-b , hsv-specific memory t cells were readily detected in the spleen of hsv-2 infected animals (mean 291,426¡72,324 ifn-c sc/spleen). hsv-specific memory t cells were also detected in both the upper and lower genital tract of all animals tested (fig. 6c, d) . although present at significantly higher frequency in the vagina/cervix than in the uterus (p,0.0001, chi square; fig. 6c) , overall, the total number of hsvspecific memory t cells was not different between these tissues (fig. 6d) reflecting differences in the yield of lymphocytes isolated from these tissues. hsv-specific memory t cells were detected over the entire length of the spinal cord. however, the frequencies of tissue-resident, memory t cells detected in lumbosacral region spinal cord and cervicothoracic region spinal cord were significantly different (p,0.0001, chi square; fig. 6e ) and the total number of hsv-specific memory t fig. 4 . igg antibody from tissue-resident asc isolated from hsv-2-infected guinea pigs is reactive with hsv-2 glycoproteins and neutralizes hsv-2. hartley guinea pigs were infected ivag with hsv-2 strain ms, serum was collected and lymphocytes were isolated from the indicated tissues between days 48-57 post infection. supernatants from asc cultures were collected on day three after isolation and the endpoint titer of antibodies reactive with fulllength recombinant hsv-2 gd (rgd2), truncated rgd2, or truncated recombinant hsv-2 rgg (rgg2) was determined by elisa. (*p,0.05; ** p,0.01; *** p,0.001 compared to hsv-2 gg2 titer). virus neutralization was detected by incubating a constant virus titer in serial dilutions of asc supernatant to determine a 50% neutralizing titer (e) or by incubating serial dilutions of hsv-2 virions in undiluted asc supernatant (f). results shown are from a representative experiment of three performed. doi:10.1371/journal.pone.0114652.g004 cells was significantly greater in spinal cord tissue from the lumbosacral region (p,0.05, student's t test) . similarly, hsv-specific memory t cells were detected in both the lumbosacral and cervicothoracic ganglia. importantly, virus-specific memory t cells were also detected at significantly higher frequency in the lumbosacral compared to cervicothoracic ganglia (p,0.0001, chi square; fig. 6g ) and the total number of hsv-specific memory t cells was significantly greater in lumbosacral ganglia compared to cervicothoracic ganglia (p,0.01, student's t test, fig. 6h ). lymphocytes were harvested from peripheral tissues of individual infected animals between days 99-150 post infection and passed over selection columns to enrich for cd4 + or cd8 + subsets to further characterize the t cell memory response. cd4 + enriched populations were stimulated with stimulator cells pulsed with uv-inactivated hsv-2 while cd8 + enriched populations were stimulated with stimulator cells infected with hsv-2 dl5-29. hsv-specific memory t cells representing both cd4 + and cd8 + subsets were readily detected in the vagina/ cervix of hsv-2-infected guinea pigs at 2-6 months post infection (fig. 7) . hsvspecific cd4 + t cells were detected in the vagina/cervix at significantly lower total cell number compared to hsv-specific cd8 + t cells (p,0.05, student's t test). both cd4 + and cd8 + hsv-specific memory t cells were maintained in the sensory ganglia/spinal cords of hsv-2 infected guinea pigs (fig. 7) . the total number of ganglia/spinal cord-resident, hsv-specific cd4 + and cd8 + t cells was not different demonstrating the maintenance of both subsets of virus specific memory t cells at the sites of hsv-2 latency during the initial months after hsv-2 infection. hsv-specific, tissue-resident cd4 + and cd8 + memory t cells have been detected in genital skin at the site of previous hsv-2 infection [15, 36, 37] and at the site of hsv latency in the trigeminal ganglia of hsv-1-infected humans and mice [16] [17] [18] [19] [20] . using a guinea pig model of hsv-2 genital infection, we extend these results and show here that a full complement of adaptive immune cells including hsvspecific b cells, cd4 + t cells, and cd8 + t cells resides in the genital tract site of hsv-2 infection and at the site of latency in the sensory ganglia. additionally, consistent with the recent observation that genital hsv-2 infection results in virus infection of autonomic neurons in the spinal cord [10] , we show here that the development and maintenance of all these hsv-specific memory immune cells extends also to hsv-2 infected spinal cords. these tissue-resident populations are appropriately positioned to play a rapid and critical role in modulation of hsv-2 reactivation and to control hsv-2 shedding. the induction of these tissue-resident memory cells by immunization would most likely be an important contributor to successful immunization strategies against hsv-2. the cellular components comprising b cell memory are quiescent memory b cells and antibody-secreting, long lived plasma cells (llpc). memory b cells normally reside in secondary lymphoid tissue although they have also been detected at peripheral sites of previous virus infection [38] . because immune reagents that distinguish memory b cells are not yet available for guinea pigs, we utilized the ability of memory b cells to become activated and secrete antibody in response to polyclonal stimulation to phenotypically identify these cells. tlr agonist-stimulated memory b cells were readily detected in the spleen and to a lesser extent in the bone marrow of infected animals but not in lymphocytes isolated from genital or neural tissues. while the assay may have lacked the sensitivity to absolutely exclude the presence of memory b cells in these tissues, peripheral tissue-resident immune memory to hsv-2 the results strongly suggest that the majority of the hsv-specific asc detected in these tissues represent persistent plasma cell populations. llpc reside primarily in lymphoid tissues such as the spleen or bone marrow and are responsible for maintaining serum antibody titers. however, populations of llpcs have also been detected at sites of chronic inflammation including the nervous systems of humans infected with polio [39] or animals persistently infected with coronaviruses [40, 41] . hsv-specific ascs were predominantly detected at peripheral tissue sites that had experienced active hsv-2 infection suggesting that antigen and/or active inflammation are required for the establishment of these populations. the influence of intermittent exposure to hsv antigen following spontaneous hsv-2 reactivation on the population dynamics of these asc is not yet known. however, this population is most likely maintained in an antigenindependent fashion in survival niches at sites of chronic hsv infection in which survival stimuli such as il-6 or cxcl12 are consistently present [42] . both igg and iga have been detected in serum and vaginal secretions of guinea pigs following ivag infection with hsv-2 [43] or chlamydia [44] . although hsvspecific iga was consistently detected, igg antibody represented the predominant isotype produced by genital tract resident asc as reported in both mice and humans [45, 46] . additionally, the predominance of igg2 expression by antibodies derived from peripheral tissue asc mirrored that of serum antibody and bone marrow-derived asc. antibody produced by genital-and neural tissueresident asc recognized rgd2 and rgg2 and hsv-specific antibody produced by neural tissue-resident asc was capable of neutralizing hsv-2. together these results demonstrate the ability of these cells to produce functionally relevant antibody at sites of chronic hsv infection. igg may enter vaginal secretions by transudation from the serum or by receptor-mediated transport across the vaginal epithelium by fcrn [47] and does not require the presence of genital-resident asc. however, igg production by genital-resident, virus-specific asc would increase the local concentration of virus-specific igg resulting in increased movement of this antibody into vaginal secretions via simple diffusion or fcrn-mediated transport. the role for local antibody in protection of humans against hsv-2 infection or hsv disease is uncertain. the presence of hsv-specific maternal antibody in vaginal secretions decreases the risk of hsv-2 acquisition by neonates [2] . by analogy, locallyproduced hsv-specific antibodies in mucosal secretions might prevent or limit virus infection. it has been shown that re-infection of ganglia is extremely difficult to achieve in previously infected animals due in part to the presence of preexisting hsv-specific antibody [48, 49] . locally-produced antibody in neural tissues may limit virus access to neurons, limit spread and acute replication of virus within the ganglia, and, upon reactivation from latency, interfere with virus spread from infected nerve ending to epithelial cells [50] [51] [52] . plasma cells are also a source of cytokine production in inflamed tissues and b cell-derived cytokines such as il-6, il-12, tgf-b, and il-10 that may play a role in regulating the inflammatory response in sites chronically infected with hsv-2. given these functions of hsv-specific b cells, the populations of hsv-specific asc we detected long term in genital tract, latently-infected sensory ganglia and spinal cords seem strategically located to play an active role in modulation of recurrent disease and shedding or in protection of genital and neural tissue against reinfection upon subsequent hsv-2 exposure. in humans, hsv-specific cd8 + t lymphocytes have been isolated from hsv lesion material and from genital skin at the dermal-epidermal junction after hsv-2 infection [14, 15, 36] . hsv-specific cd4 + t cells have also been detected in the vaginal epithelium long-term after hsv-2 genital infection of mice [29, 32, 53] and have also been isolated from hsv lesion material from humans [34, 35] . the development of genital tract-resident memory t cells in guinea pigs as a result of genital hsv-2 infection is apparently very similar and it is of note that a strong ifn-c response is detected upon stimulation of these cells as has been detected in both mice and humans [29, 36, 37] . prophylactic vaccination to induce genital tract-resident memory t cells would theoretically provide protection against the initial infection whereas therapeutic vaccination to enhance a genital tractresident memory t cell population might aid in modulating virus shedding from the genital tract of those individuals already infected. hsv-specific, ifn-c secreting effector t cells were detected in the lumbosacral ganglia and spinal cord during acute hsv-2 infection. importantly, large populations of hsv-specific, cd4 + and cd8 + memory t cells were detected at these sites up to 150 days after hsv-2 infection. the maintenance of memory t cell populations at the sites of hsv-2 latency following genital hsv-2 infection has not been extensively studied previously. sacral ganglia-resident t cell populations are not readily accessible in humans and infection of mice with hsv-2 frequently results in mortality complicating long-term study of memory t cells. we know from ocular hsv-1 infection of mice that hsv-specific cd8 + t cells surround hsv-1 infected neurons in the trigeminal ganglia of mice [20, 54] and may be involved in the maintenance of hsv latency via non-lytic mechanisms involving ifn-c and release of granzyme b [54, 55] . similarly, hsv-2 -specific cd4 + t cells have been shown to play a role in resolution of an acute virus infection of the lumbosacral ganglia neurons following genital hsv-1 infection [33] . the role of these neural tissue-resident memory t cells in modulating hsv-2 reactivation and how the population dynamics of these populations is influenced by chronic exposure to hsv-2 is currently unclear. utilization of this animal model in which spontaneous reactivation of hsv-2 occurs should help resolve these issues. in contrast to the tissue location of hsv-specific asc, the hsv-specific memory t cells were more broadly dispersed in both the genital tract and neural tissues. these results suggest differences in either the inflammatory milieu or antigen load between the t and b cell experiments or different sensitivities of our assays to detect hsv-specific memory t and asc populations. our results also demonstrate that beyond the ganglia site of hsv-2 latency, hsv-specific resident memory t cells become established in the spinal cord following hsv-2 genital infection. ohashi et al. [10] detected the greatest quantities of latent hsv-2 genomes in the sacral region of the spinal cord. consistent with this finding, although hsv-specific memory t cells were detected throughout the entire spinal column, the highest frequency and the greatest total number of hsv-specific memory t cells were detected in the lumbosacral region. the current study therefore extends previous results regarding the nature of immune memory in the sacral ganglia and spinal cord sites of latency following genital hsv-2 infection. guinea pigs infected ivag with hsv-2 experience intermittent, spontaneous reactivation events resulting in recurrent virus shedding and development of clinical symptoms remarkably similar to humans. a roadblock to studying immune control of hsv-2 in this model has been the lack of reagents for immune response proteins. we previously isolated genital tract resident lymphocytes to detect hsv-specific asc in the genital tracts and neural tissue of hsv-2-infected guinea pigs for time periods up to 8 months after genital infection [28] . our current studies extend these previous results and demonstrate that asc releasing functionally protective, hsv-specific antibody are present at the sites of hsv-2 latency and sites of virus shedding. the nature of immune memory at sites of hsv-2 infection and latency was further clarified by development of an ifn-c elispot to detect and quantify hsv-specific effector and memory t cells and demonstrate vigorous hsv-specific memory t cell responses at these epithelial and neural sites. future studies utilizing the guinea pig hsv-2 infection model will allow characterization of important properties of these cells and test immunization regimens to induce genital tract resident t cell populations in the context of virus reactivation and shedding that most effectively models human hsv-2 infection. an estimate of the global prevalence and incidence of herpes simplex virus type 2 infection effects on infants of a first 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protective immunity to vaginal infection host defenses in herpes simplex infection of the nervous system: effect of antibody on disease and viral spread genital reinfection after recovery from initial genital infection with herpes simplex virus type 2 in guinea pigs effect of immune serum on the establishment of herpes simplex virus infection in trigeminal ganglia of hairless mice role of antibody in primary and recurrent herpes simplex virus infection neutralizing antibodies inhibit axonal spread of herpes simplex virus type 1 to epidermal cells in vitro analysis of herpes simplex virus-specific t cells in the murine female genital tract following genital infection with herpes simplex virus type 2 gamma interferon can prevent herpes simplex virus type 1 reactivation from latency in sensory neurons noncytotoxic lytic granule-mediated cd8+ t cell inhibition of hsv-1 reactivation from neuronal latency we thank dr. hubert schäfer for the anti-ifn-c hybridomas and dr. richard pyles for critical reading of the manuscript. key: cord-324091-nljd2ok1 authors: gordon, jennifer l.; balsom, ashley a. title: the psychological impact of fertility treatment suspensions during the covid-19 pandemic date: 2020-09-18 journal: plos one doi: 10.1371/journal.pone.0239253 sha: doc_id: 324091 cord_uid: nljd2ok1 purpose: to examine the psychological impact of fertility treatment suspensions resulting from the covid-19 pandemic and to clarify psychosocial predictors of better or worse mental health. methods: 92 women from canada and the united states (ages 20–45 years) whose fertility treatments had been cancelled were recruited via social media. participants completed a battery of questionnaires assessing depressive symptoms, perceived mental health impact, and change in quality of life related to treatment suspensions. potential predictors of psychological outcomes were also examined, including several personality traits, aspects of social support, illness cognitions, and coping strategies. results: 52% of respondents endorsed clinical levels of depressive symptoms. on a 7-point scale, participants endorsed a significant decline in overall quality of life (m(sd) = -1.3(1.3), p < .0001) as well as a significant decline in mental health related to treatment suspensions on a scale from -5 to +5 (m(sd) = -2.1(2.1), p < .001). several psychosocial variables were found to positively influence these outcomes: lower levels of defensive pessimism (r = -.25, p < .05), greater infertility acceptance (r = .51, p < .0001), better quality social support (r = .31, p < .01), more social support seeking (r = .35, p < .001) and less avoidance of infertility reminders (r = -.23, p = .029). conclusion: fertility treatment suspensions have had a considerable negative impact on women’s mental health and quality of life. however, these findings point to several protective psychosocial factors that can be fostered in the future to help women cope. one in six reproductive-aged couples experience infertility, defined as being unable to achieve pregnancy despite �12 months of focused attempts to conceive [1] . although male and female-factor infertility are equally prevalent, women generally bear the brunt of infertilityrelated burden: even in cases of male-factor infertility, treatments such as intrauterine insemination (iui) or in vitro fertilization (ivf) require that women attend near-daily ultrasounds, self-inject gonadotropins, and undergo invasive and painful procedures. women also carry a disproportionate share of the psychological burden associated with infertility, with infertile women consistently reporting lower self-esteem, more depression and anxiety, and lower life satisfaction, than their male partners [2] . studies of women presenting for the evaluation of infertility in a tertiary care setting suggest that approximately 30-40% of these women experience clinically significant depression or anxiety [3] [4] [5] [6] . in fact, quality of life and levels of depression and anxiety among women undergoing fertility treatments are indistinguishable from those of individuals undergoing cancer treatments and cardiac rehab following a heart attack [7] . on march 17 th , 2020, the american society of reproductive medicine and the canadian fertility and andrology society announced their recommendations to immediately suspend all in-person fertility treatments throughout canada and the u.s. indefinitely due to the covid-19 pandemic [8, 9] . these recommendations included delaying the start of new treatment cycles but also, in many cases, abandoning treatment cycles that had already begun. while perhaps the right decision given the circumstances, this was devastating news for thousands of couples: unsurprisingly, one early survey of patients at a new york city fertility clinic found that 55% of patients reported being "very" to "extremely" upset over the cancellation of fertility cycles [10] . however, to our knowledge, there have been no studies examining the mental health impacts of fertility treatment suspensions. this was therefore the purpose of the current investigation. furthermore, the current study aimed to clarify predictors of better or worse mental health during this challenging time. women from across canada and the united states were recruited to participate in the current online study via advertising on social media. to qualify, women had to report having had their fertility treatments suspended due to the covid-19 pandemic and that their treatments had not yet resumed. individuals were compensated with a $15.00 amazon e-gift card for their participation. the consent form for this study consisted of the first page of the survey-all participants provided consent by clicking on the "accept" button and continuing to the survey itself. the study was reviewed and approved by the university of regina research ethics board. the study was advertised on facebook between april 26 th and june 13 th , 2020. the study ad instructed prospective participants to message the research team if they were interested. a member of the team then verified their eligibility to participate. if deemed eligible, they were provided with a link and password to access an online survey using qualtrics survey software. any diagnoses known to contribute to their infertility and to report which treatments had been suspended due to the pandemic. patient health questionnaire-9 (phq-9). the phq-9 [11] consists of 9 items based on dsm-iv criteria for diagnosing depressive disorders and is capable of determining both disorder presence and severity. items are scored on a 4-point likert scale ranging from 0 (not at all) to 3 (nearly every day), which indicates the degree participants have been bothered by the listed problems in the past 2 weeks (total scores ranging from 0-27). in the current study, internal consistency was found to be α = .84. an internal consistency above 0.7 suggests that the items of a scale are sufficiently correlated as to suggest that they measure the same general construct. intolerance of uncertainty scale short form-12 (ius-12). the ius-12 [12] includes 12 items on a 5-point scale ranging from 1 (not at all characteristic of me) to 5 (entirely characteristic of me). the questionnaire features three subscales: inhibitory anxiety, prospective anxiety, and a total score. in the current study, internal consistency was found to be α = .87. revised life orientation test (lot-r). the lot-r [13] is a 10-item questionnaire focusing on pessimism and optimism. the lot-r is on a 5-point scale ranging from 0 (strongly disagree) to 4 (strongly agree). of the 10-items, 6 items contribute to an overall optimism score and 4 are filler questions. in the current study, internal consistency was found to be α = .80. revised defensive pessimism questionnaire (dpq-r). the dpq-r [14] is a 17-item questionnaire focusing on defensive pessimism. the dpq-r is on a 7-point likert scale for a maximum total score of 119. in the current study, internal consistency was found to be α = .71. illness cognition questionnaire (icq). the icq [15] is an 18-item questionnaire with responses on a 4-point scale from "not at all" to "completely". for the purposes of this study, the words "my illness" were replaced with "my infertility. this measure has a three-factor structure, these factors being "helplessness" (e.g. my infertility controls my life"), "acceptance" (e.g. "i have learned to live with my infertility"), and "perceived benefits" (e.g. "i have learned a great deal from my infertility". infertility coping questionnaire. this questionnaire (s1 file) was developed by our research team because a comprehensive infertility-specific coping questionnaire was not deemed to exist. its creation was based on a careful review of all existing infertility-specific coping questionnaires. it began as a list of 117 items, which was reduced to 39 items after redundant items were removed, in collaboration with our patient-advisors. these 39 items were retained to represent each of the following subscales: 1) avoidance, 2) active coping, 3) find meaning, 4) defensive pessimism, 5) optimism, 6) seek social support, 5) behavioral engagement. responses are on a 5-point scale from "not at all" to "always". its internal consistency in the current study was found to be α = .81. indicators of social support. participants were asked to indicate the number of people, apart from their romantic partner, with whom they speak openly about their infertility. they were also asked to rate, on a 5-point scale from 'very unhelpful' to 'very helpful', the degree to which their partner and the degree to which others have helped them cope with the suspension of fertility treatments. psychosocial impact of treatment suspensions. participants were asked to rate on a scale from -5 (very negative impact) to +5 (very positive impact), the extent to which their mental health had been impacted by treatment suspensions. they were also asked to rate the extent to which their mental health had been impacted by the other components of the pandemic (e.g. worries about the virus, living with restrictions on daily activities, unemployment, financial difficulties). quality of life before and after the pandemic. a 1-item quality of life measure [16] asked participants to answer the following question: "taking everything in your life into account, please rate your overall quality of life on the following 7-point scale. one (1) means life is very distressing; it's hard to imagine how it could get much worse. seven (7) means life is great; it's hard to imagine how it could get much better. four (4) means life is so-so, neither good nor bad." they were asked to answer this question twice: first, with the present in mind and second, thinking about their state in the month leading up to treatment suspensions. all analyses were conducted using sas 9.4. the two primary outcomes for this study were change in quality of life rating (from before treatment suspensions) and mental health impact ratings. the wilcoxon signed rank test, a non-parametric test that does not require data to be normally distributed, was used to assess whether change in quality of life and mental health impact were significantly different from 0. spearman correlations, also a non-parametric test that does not require normally-distributed data, were used to examine the relationship between indicators of social support, personality traits, aspects of illness cognition, and coping strategies, on these two outcomes. for both illness cognitions and coping strategies, an additional linear regression analysis using proc glm was conducted including all cognitions or coping factors as predictors within the same model to clarify their independent effects. these were repeated after log-transforming both change in quality of life rating and mental health impact ratings to improve normality of these dependent variables, both of which were leftskewed (towards the negative end of the scale). power calculations were carried out in g � power. setting alpha at 0.05, this study had 80% power to detect a correlation coefficient of 0.3, which we judged to be the smallest clinically significant effect that we would want to be powered to detect. a total of 187 women contacted us through facebook with interest in the study. thirty-eight participants did not pursue the conversation past an initial explanation of the study and 43 were deemed ineligible to participate. finally, 14 participants were removed from all analyses because the survey took less than 5 minutes to complete, raising questions about the validity of responses. thus, 92 participants were remaining for the current analysis. table 1 reports the demographic and reproductive health characteristics of these 92 women. participant ages ranged from 20 to 45 years and time spent trying to conceive ranged from 5 to 180 months. more than half of the participants had had an ivf cycle cancelled and approximately one third were in the midst of iui. 36% of the participants were ages 35-39 and 12% were aged 40 or older. table 1 summarizes the median psychosocial variables assessed in the study. median depressive symptoms were quite high based on the patient history questionnaire, with over half of the sample scoring above the clinical cut-off of 10. there was a statistically significant decline in self-reported quality of life of 1.3/7 following the suspension of fertility treatments (s(88) = -1221, p < .0001). when rating the mental health impact of treatment suspensions on a scale other 6% patient history questionnaire-9 score (/27; 10 = clinical cut-off) 10 . median mental health impact of other pandemic components (-5 to +5) -1.9 (1.9) mean # of confidants about infertility 2.6 (3.0) 0 18% from -5 (very negative impact) to +5 (very positive impact), respondents endorsed a median score of -3.0, which is significantly different from 0 (s(86) = -1481, p < .0001) (fig 1) . overall, 86% of respondents reported that treatment suspensions had had a negative impact on mental health. baseline characteristics. neither age, years of education, annual income, nor the number of children a woman had were correlated with either change in quality of life or perceived mental health impact (p >.05). however, the length of time a woman had been trying to conceive was associated with a greater negative perceived mental health impact (r(77) = -.30, p = .008) of treatment suspensions. personality traits. table 2 depicts the correlation between three personality traits (trait optimism, defensive pessimism, and intolerance of uncertainty) that were considered potentially relevant under the current circumstances, in relation to the overall change in quality of life and the mental health impact attributed to fertility treatment suspensions. with regards to the psychological impact of fertility treatment suspensions during the covid-19 pandemic personality traits, defensive pessimism was related to a greater negative change in overall quality of life as well as a greater negative mental health impact of treatment suspensions. neither trait optimism nor intolerance of uncertainty were significantly related to any outcomes (p >.05). infertility-related cognitions. greater acceptance was associated with a more positive change in overall quality of life and lesser mental health impact attributed to treatment suspensions (table 2) . furthermore, greater benefit-finding was associated with a smaller mental health impact of treatment suspensions while helplessness was associated with a greater negative mental health impact of treatment suspensions. however, when all three illness cognition factors were included in the same regression model as three simultaneous predictors of quality of life change, only acceptance was a significant predictor of treatment suspensions (β(se) = 0.8(0.3), p < .01) while helplessness (β(se) = 0.3(0.2), p = .163) and benefit-finding (β(se) = 0.1(0.2), p = .831) were not. the same pattern was seen when examining the impact of illness cognitions on perceived mental health impact: acceptance was a significant predictor (β(se) = 0.3(0.1), p < .001) while helplessness (β(se) = 0.0(0.1), p = .592) and benefit-finding (β(se) = 0.1(0.1), p = .229) were not. these results remained unchanged when quality of life change and mental health impact were log-transformed to improve normality of the dependent variable. coping strategies. the internal consistency of each of the seven subscales was examined. in instances where it was below 0.7, items were removed to improve it. this resulted in 7 items being removed. the remaining 32 items are included in table 3 . the internal consistency of the psychological impact of fertility treatment suspensions during the covid-19 pandemic the final subscales are as follows: 1) avoidance, α = 0.82, 2) active coping, α = 0.71, 3) finding meaning, α = 0.72, 4) defensive pessimism, α = 0.66, 5) optimism, α = 0.80, 6) seek social support, α = 0.77, and 7) behavioural engagement, α = 0.67. as shown in table 2 , spearman correlations revealed that seeking social support was correlated with a more favourable change in quality of life and that engaging in less avoidance was associated with a more favourable change in mental health. when all seven coping factors were included in the same regression model, seeking social support remained the only factor predicting change in quality of life: specifically, a 1-point increase in mean item endorsement was associated with a 0.6-point increase in quality of life (β(se) = 0.6(0.2), p = .004). avoidance remained predictive of a greater negative mental health impact (β(sem) = -0.7(3), p = .033). however, optimism emerged as predictive of a more favourable mental health impact of treatment suspensions (β(sem) = 0.8(0.3), p = .015). the other coping factors were not significant predictors (p > .05). these results remained unchanged when quality of life change and mental health impact were log-transformed. sensitivity analyses: predictors of the mental health impact of other pandemic aspects. on a scale from -5 to +5, participants were asked to reflect on the extent to which their mental health had been impacted by aspects of the covid-19 pandemic that were unrelated to their fertility treatments being cancelled (e.g. activity restrictions, social isolation, financial hardship, job loss). of the variables listed in table 2 , defensive pessimism (r(83) = -.29, p = .007), infertility acceptance (r(83) = .24, p = .024), and infertility benefit-finding (r (83) = .22, p = .049) were weakly correlated with this outcome. the current study sought to examine the mental health impact of fertility treatment suspensions resulting from the covid-19 pandemic. furthermore, it sought to explore the psychosocial predictors of this psychological impact. overall, results suggest that the mental health impact of treatment suspensions is substantial: indeed, over 50% of respondents reported clinically significant depressive symptoms, a rate that is considerably higher than typical rates observed in this population, which hover closer to 30% (nelson, shindel, naughton, ohebshalom, & mulhall, 2008; volgsten et al., 2008) . similarly, over 50% of respondents endorsed a "-3" or worse on a scale from -5 to +5 to reflect the mental health impact that treatment suspensions have had. these findings mirror a survey of patients at a new york city fertility clinic finding that 55% reported being "very" to "extremely" upset over treatment cancellations [10] . however, the current study extends these findings by conducting a more detailed assessment seek information or advice that can help me achieve pregnancy 40 take time to understand, identify, or express my feelings 19 try to find meaning in my experience 26 try to grow as a person as a result of this experience 28 accept the situation as it is 16 decide that i don't care 8 prepare myself for the worst 42 tell myself that it would be for the best if i didn't get pregnant 17 tell myself that having biological children is not important 29 believe that everything will work out 33 stay optimistic that my efforts will be successful 33 fantasize about how things might turn out 55 believe that i will feel better in time 32 try to find humor where i can 32 seek social support seek emotional support professionals (e.g., counsellor, doctor) 15 seek emotional support from friends or loved ones 22 seek emotional support on the internet (e.g., blogs, chatrooms) 33 seek emotional support from others with similar experience 24 practice self-care (e.g., meditation, watch movie) 26 of the mental health impact of treatment suspensions, as well as factors moderating this impact. several variables were found to moderate the psychological impact of treatment suspensions. perhaps unsurprisingly, women who had been trying to conceive for a longer time reported a greater negative mental health effect of treatment suspensions. furthermore, while the number of confidants a person had was unrelated to the impact of treatment suspensions, women who reported better emotional support from their partner and others also reported a smaller negative mental health impact. this is consistent with prior research finding that better quality social support is associated with lower infertility-related distress [17] . it is noteworthy that the mean number of reported confidants was very small and that only 42% of respondents reported that their confidants were at least "somewhat" helpful in their efforts to cope with fertility treatment suspensions. women found their partners to be somewhat more helpful, however, as 58% were rated as at least "somewhat" helpful. furthermore, only 22% of women endorsed seeking social support from friends and loved ones at least "often". overall, these findings suggest that quality social support is beneficial for women who are struggling with the stresses of infertility but that this is not available to most women. this is consistent with research finding that unsupportive social interactions are commonly reported by women struggling with infertility and that these interactions predict greater distress [18, 19] . thus, interventions aimed at improving the quality of the social support that they receive from others may be beneficial for this population. for example, a recent pilot study [20] found that couples experiencing infertility benefited greatly from 12 sessions of interpersonal therapy, a therapeutic approach that is primarily focused on improving the quality of one's interpersonal relationships. furthermore, in this study, interpersonal therapy was found to result in significantly better outcomes when compared to brief supportive therapy. with regards to personality traits, defensive pessimism was associated with a greater decline in quality of life, as well as a greater negative mental health impact of fertility treatment suspensions. defensive pessimism is the tendency to, in times of uncertainty, focus on potential negative outcomes and have low expectations in an effort to avoid disappointment [14, 21] . although this approach may prove useful in motivating an individual to take steps toward avoiding an unwanted outcome under certain circumstances (spencer & norem, 1996) , our findings suggest that it is maladaptive in the context of the largely uncontrollable condition of infertility. spearman correlations revealed that greater acceptance and benefit-finding were associated with a lesser mental health impact of fertility treatment suspensions while greater helplessness was associated with a greater negative impact. however, in a regression model including all three infertility-related cognitions as predictors simultaneously, only acceptance was found to be a significant predictor. the importance of illness acceptance-that is, the extent to which an individual changes their focus from the elimination of their illness to living as well as possible with their illness-has been shown to be a strong predictor of wellbeing among those living with chronic pain [22] [23] [24] . consistent with this, meta-analytic evidence suggests that acceptance and commitment therapy (act) [25] , which aims to foster greater illness acceptance and reduce illness-related avoidance, is an effective intervention for chronic pain [26] . to our knowledge, only one case study has documented the use of act for infertility-related distress [27] , with promising effects. with regards to coping strategies, seeking social support and endorsing greater optimism were associated with better outcomes. in contrast, avoidance of infertility reminders was associated with worse outcomes. this latter finding is consistent with prior research finding that the endorsement of an avoidant coping style has been associated with worse psychological outcomes in the context of infertility [28] [29] [30] [31] . furthermore, in one of the few existing longitudinal studies of coping and psychological outcomes in infertility, the avoidance of reminders and thoughts concerning one's infertility was found to be a mediator between baseline vulnerability for psychopathology and 12-month distress levels [32] . interventions targeting avoidance as a coping strategy, such as acceptance and commitment therapy, may be well-suited for infertility-related distress. although avoidance is often a clinical target in cognitive behavioural therapy, most studies that have applied this treatment approach to infertility have not directly targeted infertility-related avoidance [33, 34] . the current study findings should be interpreted in light of some limitations. first, it is a cross-sectional investigation, providing only a snapshot of the interrelationships between psychosocial factors and infertility-related distress in the context of the covid-19 pandemic. the sample size was somewhat limited and is also not guaranteed to be representative of all women under similar circumstances-potentially, more distressed women were compelled to complete the survey as it appeared on their facebook feed. nonetheless, it provides some valuable insights into the emotional challenges that the covid-19 pandemic has introduced in the lives of women struggling with infertility. furthermore, it points to several targets for future interventions specifically targeting infertility-related distress. in summary, the current study suggests that the suspension of fertility treatments have had a significant negative impact on women's mental health and quality of life. low defensive pessimism, high-quality social support, greater infertility acceptance, and less use of avoidance, were all found to be protective factors against the negative effects of treatment suspensions on wellbeing. these findings suggest that additional mental health resources are likely to be needed in this population; they also suggest that infertility-specific psychological interventions should target social support, infertility acceptance, and infertility-related avoidance. supporting information s1 file. the infertility coping questionnaire, developed by the research team for the purposes of this study. (pdf) estimating the prevalence of infertility in canada psychometric evaluation of infertile couples a study on psychological strain in ivf patients sundstrom poromaa i. prevalence of psychiatric disorders in infertile women and men undergoing in vitro fertilization treatment the psychological impact of infertility: a comparison with patients with other medical conditions patient management and clinical recommendations during the coronavirus (covid-19) pandemic: american society of reproductive medicine the emotional impact of the asrm guidelines on fertility patients during the covid-19 pandemic. medrxiv monitoring depression treatment outcomes with the patient health questionnaire-9 fearing the unknown: a short version of the intolerance of uncertainty scale distinguishing optimism from neuroticism (and trait anxiety, selfmastery, and self-esteem): a reevaluation of the life orientation test defensive pessimism: harnessing anxiety as motivation the construct validity of the illness cognition questionnaire: the robustness of the three-factor structure across patients with chronic pain and chronic fatigue quality of life while living and aging with a spinal cord injury and other impairments dyadic dynamics of perceived social support in couples facing infertility the impact of social relations on the incidence of severe depressive symptoms among infertile women and men longitudinal analyses of the relationship between unsupportive social interactions and psychological adjustment among women with fertility problems. social science & medicine interpersonal psychotherapy versus brief supportive therapy for depressed infertile women: first pilot randomized controlled trial. archives of women's mental health reflection and distraction defensive pessimism, strategic optimism, and performance adjustment to chronic pain: the role of pain acceptance, coping strategies, and pain-related cognitions acceptance of pain is an independent predictor of mental well-being in patients with chronic pain: empirical evidence and reappraisal a prospective analysis of acceptance of pain and values-based action in patients with chronic pain. health psychology acceptance and commitment therapy: altering the verbal support for experiential avoidance acceptance and commitment therapy versus traditional cognitive behavioral therapy: a systematic review and meta-analysis of current empirical evidence. international journal of psychology and psychological therapy using acceptance and commitment therapy to treat infertility stress the psychological well-being of infertile women after a failed ivf attempt: the effects of coping counselling in infertility: individual, couple and group interventions. patient education and counseling gender differences in how men and women who are referred for ivf cope with infertility stress resilience and social support promote posttraumatic growth of women with infertility: the mediating role of positive coping psychosocial vulnerability, resilience resources, and coping with infertility: a longitudinal model of adjustment to primary ovarian insufficiency treatment of depression and anxiety in infertile women: cognitive behavioral therapy versus fluoxetine psychotherapy for infertility: a cognitive-behavioral approach for couples we thank tianna sauer for her help with data collection. conceptualization: jennifer l. gordon, ashley a. balsom. key: cord-351098-x729wpp7 authors: long, rachel b.; krumlauf, kristi; young, anna m. title: characterizing trends in human-wildlife conflicts in the american midwest using wildlife rehabilitation records date: 2020-09-11 journal: plos one doi: 10.1371/journal.pone.0238805 sha: doc_id: 351098 cord_uid: x729wpp7 human-wildlife conflict is difficult to measure, but the analysis of records from wildlife rehabilitation facilities has shown potential as a technique for characterizing human impacts on wildlife. to examine the value of wildlife rehabilitation records for characterizing local human-wildlife conflicts and prevalence of select wildlife diseases, we reviewed 45,668 records representing over 280 species admitted to a wildlife rehabilitation facility over a 10-year period (2005–2014). we identified the most frequently recorded causes of admission for commonly admitted species, and evaluated how causes of admission may vary across taxa throughout the year. our analyses support the value of wildlife rehabilitation facility data for characterizing some pressures from human-wildlife conflict and select disease trends for certain taxa, as well as utility for informing topics to emphasize in local conservation education efforts. for example, orphaned neonatal wildlife accounted for the largest proportion of admissions to this facility, and highlights a opportunity for conservation education regarding when wildlife is truly orphaned and requires professional intervention. additionally, domestic dog attack cases accounted for a proportion of admissions comparable to that of domestic cat attacks, demonstrating a need for the conversation surrounding the impact of domestic pets on local wildlife to expand to include dogs in addition to cats. interactions between humans and wildlife have become more frequent as a consequence of encroachment, resulting in an increase in the likelihood of human-wildlife conflict events and zoonotic disease transmission [1, 2] . human-wildlife conflicts-negative interactions between humans and wildlife that pose a real or perceived threat to either party [3] -are substantial causes of wildlife mortality and population decline, and manifest differently across taxa and anthropogenic contexts [4, 5] . examples of conflicts demonstrated to significantly impact wildlife populations include vehicle strikes [6, 7] , mid-flight collisions with windows [8] , domestic cat predation [9] , and anthropogenic sources of ecological contamination [10, 11] . quantifying causes of morbidity and mortality in wildlife populations is often difficult, as there are significant logistical challenges associated with in situ studies of wildlife health [12, 13] . it can also be challenging to navigate the behavioral biology of wild populations in a minimally invasive manner; high stress events and capture myopathy can be significant welfare risks to surveys of live individuals [14] . continued innovation of methods that can be used to identify threats to wildlife health with fewer logistical and welfare challenges is necessary [13] . the analysis of records from admissions to wildlife rehabilitation facilities has potential to be a useful technique for characterizing human-wildlife conflicts and disease trends that may be impacting local wildlife, as this method would not be as subject to the challenges of assessing wildlife health in the field as described by ryser-degiorgis [13] and spalding and forrester [12] . additionally, there is an impressive number of wildlife rehabilitation facilities located around the world-for example, there are currently over 60 wildlife rehabilitation organizations located in the state of ohio [15] , over 330 wildlife rehabilitation carers in australia as of 2000 [16] , and over 65 wildlife rehabilitation facilities in the united kingdom [17] . these facilities each frequently admit hundreds to thousands of animals annually-uniquely positioning them to assess pressures faced by their respective local species [16] . the studies to date from wildlife rehabilitation institutions have focused primarily on identifying common causes of morbidity and mortality for specific taxa, including raptors [18] [19] [20] , reptiles [21] , and black cockatoos [22] , as well as comparing rehabilitation outcomes [20, 23] , causes for admission across taxa, and causes for admission across ecological niches [24] . a broader analysis of wildlife rehabilitation records could be ideal for identifying human-wildlife conflicts of particular significance for conservation, such as the recent study that used these records to demonstrate the impact of domestic cat predation on a variety of native species [25] . analyses that span longer time periods could also provide insight regarding how causes of morbidity and mortality may fluctuate seasonally-valuable information for epidemiological studies of disease and human-wildlife conflicts [26, 27] , as well as conservation education efforts. one such study on australian wildlife found that admissions peaked in the spring and summer during breeding seasons [28] . the purpose of this study was to examine the value of wildlife rehabilitation facility admissions records for informing understanding of local human-wildlife conflicts and wildlife disease trends by 1) identifying the top reasons for admission to this facility, 2) examining how occurrences as measured by admitted cases may fluctuate monthly and across taxa, and 3) analyzing trends in commonly admitted disease cases. this information could have direct implications for wildlife rehabilitators and conservation educators, as well as offer information relevant to human and domestic animal health [12, 16, 29] . this study utilized records from admissions to a wildlife rehabilitation facility, but did not involve direct interaction with or experimental manipulation of live animals. as such, iacuc approval was not required. however, the permission of the wildlife rehabilitation facility to utilize these records for the purposes of this study was sought and granted. we utilized records from admissions to a wildlife rehabilitation facility veterinary hospital in central ohio, united states of america. this facility was located in a suburban context, with urban and rural areas in near proximity. animals were regularly admitted from all three contexts, though most frequently from urban and suburban areas. most admissions (>70%) originated from the county in which this facility was located and the six immediately surrounding counties (franklin, licking, delaware, union, madison, fairfield, pickaway). animals were also admitted from more distant regions of ohio; at least one animal was admitted to this facility from 74 of the 88 counties in ohio during the span of this study. we reviewed records spanning a 10-year period (2005-2014), which included 45,668 individuals from over 280 species, including rabies vector species. each individual admitted to this facility had been assigned a unique record entry, regardless of life stage or being admitted with conspecifics at the same intake event. the records included both animals that were alive on presentation to the hospital as well as animals that were dead on arrival. admissions records had been recorded in a microsoft access database from 2005-2012, and in the wildlife incident log/ database and online network (http://www.wild-one.org) database from 2013-2014. the two databases differed in how information was recorded, so consolidation of all records in the time span of this study necessitated standardization to facilitate analysis across the 10-year period. we assigned each case to one of eight taxonomic groups: mammals, reptiles, amphibians, waterfowl, water/shorebirds, raptors, gallinaceous birds, and songbirds+. we assigned all mammalian, reptilian, and amphibian species to their respective taxonomic categories. we assigned orders anseriformes, podicipediformes, and gaviiformes to the waterfowl taxonomic group, orders pelecaniformes, gruiformes, ciconiiformes (excluding vultures), and charadriiformes to the taxonomic group of water/shorebirds, the orders falconiformes, accipitriformes, strigiformes, and vulturine ciconiiformes to raptors, and the order galliformes to gallinaceous birds. we categorized all passerines and other small bodied terrestrial birds, namely the orders piciformes, caprimulgiformes, apodiformes, and columbiformes, into the songbirds+ taxonomic group. we classified records where species was not identified as unknown. information in the records regarding the circumstances that led to an animal being admitted to this wildlife rehabilitation facility was documented according to what was stated by the individuals admitting the animal to the facility and/or derived from physical examinations by a veterinarian upon admission. causes for admission were numerous and there was variation in how the causes were documented in the records, so to facilitate analysis we created a categorization system that involved assigning each case a broad cause of admission category and a specific subcategory. the broad cause of admission categories we created for the purposes of this study were modelled after the "circumstance" categories utilized by the wild-one database system. we divided some of these categories into multiple categories (ex: we divided wild--one's "collision" category into the broad categories of "collision with non-moving object" and "collision with moving object") to enable us to analyse groups of similar specific causes for admission with more precision. our list of specific cause of admission categories was formulated based on the more detailed information contained in records related to the circumstances that led to an animal being admitted. these lists of broad cause of admission categories and specific cause of admission subcategories were then applied to all records across both the microsoft access and wild-one databases resulting in 83 broad categories and 20 subcategories (s1 table) . if an animal was admitted with multiple injuries, we assigned its cause of admission based on the primary reason the animal was brought to the wildlife hospital. if an animal was admitted with both a disease and an injury-for example, a raccoon (procyon lotor) that had been struck by a vehicle but also exhibited signs of canine distemper virus-we categorized the animal with the cause of admission categories corresponding to both the source of its injury and its disease to ensure that disease occurrences were represented appropriately. this dual categorization was only necessary for 14 cases. while most records explicitly stated the reasons that individuals were admitted to the hospital and cause for admission categories were assigned accordingly, there were records where the circumstances that resulted in an animal's injury were either unknown by the individual who admitted the animal, or were not recorded. in unknown cases, it was possible in many instances to reasonably assign a cause for admission based on corroborating information and/ or findings from physical examinations-an approach also utilized by other facilities [30] . a frequently encountered example of this involved animals that had been found by the side of a road, but had not been observed or recorded being hit by a vehicle. if a record in this instance contained at least two other items of information (fractures, abrasions, contextual information, etc.) that would implicate a car strike, we assigned the individual the broad category of "collision with moving object" and specific subcategory of "hit by vehicle." if a record stated an individual was "found by road" but there was not sufficient information to implicate a car strike, we assigned the broad category of either "unknown" if no other relevant information was included, or "injury unspecified cause" and the appropriate subcategory if at least one injury was noted. for the purposes of this study, causes of admission considered to be human-wildlife conflicts involved animals being admitted due to an adverse direct interaction with a human (ex: vehicle strikes, lawn mowing equipment strikes, or intentionally inflicted trauma) or an adverse indirect interaction with a human or a human-associated phenomenon (ex: window strikes or domestic pet attacks). there are many reasons that wildlife may become orphaned, ranging from misinformed human interferences, the caregiving parent(s) becoming deceased due to a human-wildlife conflict, predation, etc.; the reason neonates become orphaned is rarely known unless the event was directly observed. we have chosen to include animals with the "orphaned" cause of admission designation to be part of the conversation regarding local human-wildlife conflict as it is postulated by wildlife rehabilitators that a noteworthy portion of neonate and juvenile animals admitted as orphans were likely not truly orphaned. while the prevalence of some diseases can be exacerbated by human influence, such as how close contact with infected individuals at bird feeders or contact with contaminated bird feeders contributes to the spread of mycoplasmal conjunctivis in finches [31] , diseases are considered separately from human-wildlife conflicts. as this wildlife rehabilitation facility is a non-profit organization that relies largely on donations/grants to operate, and as such has limited resources to dedicate to diagnostic testing, some disease cases were identified based on a syndromic approach, where a presumed diagnosis is made based on clinical signs, relevant history, and/or response to treatment. mycoplasmal conjunctivitis and avian botulism are two examples of diseases in this study where cases were typically identified using this approach, as they have very characteristic clinical signs. suspected west nile virus cases were at times presumed based on a syndromic approach, but were typically also supported via serum antibody blood tests as resources allowed. however, it was not indicated in this data set which cases were confirmed and which were not. while this approach is certainly not without limitation and possibility for error, it is common in wildlife rehabilitation facilities across the united states due to similar resource constraints, and does have some merit; syndromic surveillance for wnv was recently described as still potentially conferring value to wnv surveillance efforts [32] . suspected canine distemper virus cases in mesopredator species such as raccoons were typically confirmed via serum antibody tests. rabies testing was pursued in mentally inappropriate mesopredators when there was a known situation where another animal or a human may have been exposed. to examine how human-wildlife conflict as measured by admissions to this facility may vary seasonally and across taxonomic groups, we assessed changes in mean cases admitted per month via chi-squared tests. we also described the total number of each taxonomic group admitted, the top 20 species admitted, the top five broad causes for admission, the top five specific causes for admission, and the top three specific causes for admission for each taxonomic group. to characterize the most common causes for admission to this wildlife rehabilitation facility, we first identified the top four specific causes for admission. for each of the top four specific causes, we assessed the number of cases admitted, the percentage that those cases constituted of all cases admitted to the facility, the taxonomic groups affected, and how mean cases per month fluctuated with chi-squared tests. for one of the top four specific causes for admission, vehicle strikes, we utilized average daily traffic volume data [33] for the county where this facility was located to evaluate the contribution of monthly and annual changes in traffic volume to trends in vehicle strike cases with linear regression tests. to examine the potential of wildlife rehabilitation records to contribute to disease monitoring, we assessed the number of cases admitted for five diseases of particular importance to the wildlife rehabilitation facility-canine distemper virus, west nile virus, avian botulism, mycoplasmal conjunctivitis, and rabies. we identified the species that were affected by each of these five diseases, as well as the percentage of all cases admitted that these diseases constituted. for the most represented of the five diseases, canine distemper virus, we also analyzed fluctuations in mean cases per month and in total cases per year via chi-squared tests as it was the only disease with a degree of representation in the records that facilitated meaningful analysis (n>100 cases across 10 years). a total of 45,668 cases were admitted to this wildlife rehabilitation facility from 2005-2014, with a mean of 4,562.7 ± 226.51 cases admitted annually. of these records, 98% contained sufficient information to be included in some level of analysis and 92% contained sufficient information to be assigned a broad and specific cause of admission category. total admissions varied across all 10 years (w 2 9 ¼ 112:44, p < 0.001), ranging from 4,105 to 4,848. the mean cases admitted each month fluctuated throughout the year (w 2 11 ¼ 3467:74, p < 0.001). more cases were admitted from may through august than october through march, with may being the peak month for total cases admitted (1,083.5 ± 89.29). the month with the fewest mean cases admitted was december (65.9 ± 13.29); (fig 1) . of the eight defined taxonomic categories, mammals were the most frequently admitted, representing over half of all cases (table 1 ). of the 20 species most frequently admitted, seven were mammals, with the eastern cottontail rabbit (sylvilagus floridanus) accounting for nearly one-fourth of all admitted cases. songbirds+ also accounted for a notable percentage of cases admitted, followed by waterfowl, raptors, and reptiles. water/shorebirds, amphibians, and gallinaceous birds accounted for only a small portion of all cases admitted, 0.4% collectively. the top five most frequently assigned broad causes for admission categories account for 83.5% of all cases admitted, and the top five most frequently assigned specific causes for admission subcategories account for 81.5% of all admitted cases ( table 2 ). the most assigned broad category and specific subcategory was "orphaned", encompassing just over half of all cases admitted. domestic animal interactions were the second most frequent cause of admission at nearly one in five of all cases admitted; within this broad category, both cat and dog attacks each accounted for close to a tenth of all cases admitted. collisions with moving objects constituted nearly a tenth of all cases admitted, with vehicle strikes accounting for most of these incidents. the most frequently observed specific causes for admission varied across taxonomic groups (table 3) . being orphaned was the most frequent reason for admission for mammals, song-birds+, waterfowl, water/shorebirds, and gallinaceous birds, and the second most common reason for raptors and reptiles. vehicle strikes were the top cause of admission for raptors, reptiles, and amphibians. domestic animal attacks were either the second or third top cause for admission of mammals, songbirds+, reptiles, and gallinaceous birds. collisions with buildings/ windows were the third most recorded cause of admission for songbirds+ and raptors. amphibians were most frequently admitted due to lawn mower strikes. we further examined the top four identified specific causes of admission to the wildlife rehabilitation facility: orphaned, dog attacks, cat attacks, and vehicle strikes. orphaned a total of 23,201 neonate and juvenile individuals were admitted as having been orphaned, constituting 50.8% of all cases admitted during the study period, a large proportion of admissions during the spring and summer months (figs 1 and 2) , and one of the top three specific causes for admission for seven of the eight taxonomic groups ( table 3 ). the mean orphan cases varied significantly across months (w 2 11 ¼ 2727:59, p < 0.001), with the peak month being may and the month with the fewest cases being january (fig 2) . the total orphan cases also varied annually (w 2 9 ¼ 337:05, p < 0.001), with the most admitted in 2010 and the fewest admitted in 2007. a total of 4,491 individuals were admitted due to being attacked by a cat, and 3,755 individuals were admitted due to a dog attack. cat attacks and dog attacks account for comparable percentages of all cases admitted to the wildlife rehabilitation facility (9.8% and 8.2% respectively). the three taxonomic groups most impacted by cat attacks were mammals (2,593 cases), song-birds+ (1,805 cases), and waterfowl (64 cases), collectively representing 69 species. mammals (3,262 cases), songbirds+ (403 cases), and reptiles (29 cases) were most impacted by dog attacks. cat attacks impacted 1,413 more songbirds+ individuals than dog attacks, and dog attacks impacted 682 more mammals than cat attacks. the mean cases of cat attacks (w 2 11 ¼ 361:99, p < 0.001) and dog attacks (w 2 11 ¼ 323:10, p < 0.001) varied across months. the peak month for mammal cases admitted due to either cat or dog attacks was may, and the peak month for songbirds+ cases admitted due to either cat or dog attacks was june. the months with the fewest dog attack or cat attack cases admitted were december and january (fig 3) . the total cases of dog attacks (w 2 9 ¼ 45:5, p < 0.001) and cat attacks (w 2 9 ¼ 91:63, p < 0.001) also differed annually, with peak admissions for dog attacks occurring in 2013, peak admissions for cat attacks occurring in 2009, and the fewest cases of either cat or dog attacks being admitted in 2005. vehicle strikes represented 8% of all cases admitted to the wildlife hospital. diverse taxa were admitted due to vehicle strike cases, with canada geese (branta canadensis), virginia opossums (didelphis virginiana), eastern cottontail rabbits, mallards (anas platyrhynchos), and eastern gray squirrels (sciurus carolinensis) being the five most admitted species. the mean vehicle strike case admissions varied significantly across months (w 2 11 ¼ 115:48, p < 0.001), and the total cases differed annually (w 2 9 ¼ 34:38, p < 0.001). the peak month for cases admitted due to vehicle strikes occurred in june, and the month with the fewest vehicle strike cases was january (fig 4) . the peak month for mean daily traffic volume in franklin county, ohio, usa was june, and the month with the lowest mean daily traffic volume was january (fig 4) . a strong linear relationship was found between mean daily traffic volume per month and mean monthly vehicle strike cases admitted (f 1,10 = 13.63, p = 0.004, r 2 = 0.577), but not between mean annual traffic volume and total annual vehicle strike cases admitted (f 1,10 = 0.416, p = 0.539, r 2 = 0.056). raccoons (procyon lotor) and 30 (5.5%) cases observed in striped skunks (mephitis mephitis). the mean cases of canine distemper per month exhibited some fluctuation (fig 5) , but not to a statistically significant degree (w 2 11 ¼ 3:63, df = 11, p = 0.979). however, across all 10 years there was a fluctuation in total cases admitted annually for both raccoons (w 2 9 ¼ 182:77, df = 9, p < 0.001) and skunks (w 2 9 ¼ 34, df = 9, p = 0.009) with peak years in 2008 for raccoons and 2009 for skunks. mycoplasmal conjunctivitis (mycoplasma spp.) accounted for 8% of all disease cases admitted, and largely impacted house finches and goldfinches (89% collectively). west nile virus (family flaviviridae, genus flavivirus) was recorded for 6% of all disease cases, avian botulism (clostridium spp.) accounted for 4%, and other miscellaneous disease states accounted for 28%. a total of three individuals, all big brown bats (eptesicus fuscustested), tested positive for rabies (family rhabdoviridae, genus lyssavirus), accounting for 0.3% of disease cases admitted, and 0.007% of all cases admitted to the wildlife rehabilitation facility during the examined time period. our study demonstrates the value of wildlife rehabilitation records for characterizing local human-wildlife conflicts and potentially select disease trends, as well as how occurrences may fluctuate seasonally and impact taxa differently. while our analysis is most directly applicable to the region where this wildlife rehabilitation facility is located, we believe this technique can be applied in other regions as well. a similar study in australia found that car strikes, dog and cat attacks, and orphans were also top reasons for admissions, although they additionally found entanglement and disease to be highly prevalent [28] . using the records, we were able to identify what are likely some of the predominant types of human-wildlife conflicts in the central ohio region, including domestic animal attacks, vehicle strikes, lawn mower strikes, and collisions with buildings, and how they impact taxa differently. it is likely that wildlife rehabilitation facilities have a greater monitoring capacity for some taxa more than others; mammals, songbirds+, and waterfowl constituted the majority of admissions to this facility. it should be noted that distinctive species or subgroups within the taxonomic categories utilized in this study may experience human-wildlife conflict differently; here we focused on broad trends, future studies are needed to tease apart which threats may apply more to specific species or taxonomic subgroupings. additionally, this facility primarily depends on the public to bring in animals they find, which results in the predominantly admitted species being those that are more frequently seen by people, and those that non-professional individuals are willing to attempt to handle. however, meaningful trends were still able to be discerned for some of the taxa that were admitted, such as mammals being more likely to be admitted due to domestic dog attacks than cat attacks. these findings also suggest utility for future studies that examine how human-wildlife conflicts may vary or be experienced differently by taxonomic groups more specific than those examined in this study. it was also found that other forms of human-wildlife conflict, such as vehicle strikes, impacted a larger assortment of taxonomic groups. vehicle strikes were one of the top three specific causes of admission for five of the eight taxonomic groups utilized in this study (table 3) . this suggests that ongoing study and education is needed regarding vehicle strike deterrents such as wildlife-friendly driving practices. orphaned. orphaned neonates and juveniles were a primary cause of admission for seven of the eight taxonomic groups admitted, accounting for the largest source of admissions to this facility ( table 2 ) and highlighting the ongoing need for public education regarding legitimately orphaned wildlife. orphaned wildlife admissions have constituted a predominant cause for admission in other wildlife rehabilitation facilities as well [19, 34] . this facility has protocols to help screen for neonatal wildlife that may not actually be orphaned, but they mostly entail conversational questioning, and are dependent on the honesty and existing knowledge of the members of the public presenting the animals to the hospital. thus, increasing the rigor and/or consistency of these screenings may be useful to some degree, but increasing public understanding of topics such as the biology of local neonatal wildlife, how to discern whether found neonates are legitimately orphaned, and local resources that can be contacted for assistance would likely be the more ultimate solution. domestic animal attacks. domestic animal attacks demonstrated significant month-tomonth fluctuations, likely corresponding with seasonal changes in the biology of local wildlife related to habitat use [35, 36] , breeding seasons [37] , migration [37, 38] , and range size [39, 40] . domestic animal attack cases may also exhibit this seasonality as during the months of april-august in the northern hemisphere, domestic animals maintained as household pets may be more likely to spend more time outside and thus have a greater likelihood of encountering wildlife. the seasonal influx and thus increased density of migratory songbird species may also be a contributing factor. a similar trend in seasonal rates of domestic cat attacks on wildlife was also reported for another u.s. wildlife rehabilitation facility's records [25] . this highlights a time of year where it is particularly important to emphasize supervising pets outdoors to decrease the frequency of domestic animal attacks on wildlife. a similar study that was broader in region, including all north america wild-one data but focusing on cat-wildlife conflicts over a shorter period of time, also determined domestic pet attacks to be a top reason for admission at 14%, comparable to our finding of 18% for this ohio facility. unlike our study, they found that birds presented to the facility due to a cat attack more than other taxa, but similarly the species most commonly admitted for cat attacks were the eastern cottontail rabbit, american robin, and eastern gray squirrel [41] . this suggests that our findings are representative of national trends for at least domestic felid attacks. our study also suggests that the current conversation regarding the impact of domestic animal attacks on wildlife needs to expand. we found evidence for the previously demonstrated magnitude with which domestic cat predation impacts songbird and mammalian taxa [9, 25, 42] , but our findings also illuminate that in terms of total cases admitted, dog attacks exert pressure comparable to cats. it should be noted that cats are primarily predating taxa that are typically considered of higher conservation concern, namely songbirds. although none of the species in this study were threatened or endangered, many admitted songbird species are protected under the migratory bird treaty act. however, dogs are impacting small mammals to an extent that is of notable concern from an animal welfare standpoint, and they could pose a conservation threat in regions where endangered small mammals are more prevalent than in central ohio. while it has been previously suggested that dog attacks may have a noteworthy impact on wildlife populations [43, 44] , we provide evidence that dog attacks are another source of anthropogenic pressure on wildlife that should be addressed in wildlife education efforts and accounted for in conservation decision-making for songbird and mammalian taxa. vehicle strikes. our findings suggest that changes in traffic volume throughout the year are not the primary driver of the fluctuations observed in vehicle strike cases. while a relationship was found between traffic volume and vehicle strikes across months within a mean year, this was not the case across years. it is likely that seasonal changes in wildlife dispersal, migration, and behavioral biology contribute more significantly to vehicle strikes than the fluctuations in traffic volume recorded in this area. these findings support the relevance of vehicle strikes as a significant source of morbidity and/or mortality in wildlife [45, 46] , as well as the need for additional study regarding effective wildlife/vehicle strike prevention measures such as wildlife crossing structures or warning signage for motorists [47] . additionally, these findings lend support to the value of incorporating mitigation techniques such as wildlife crossing structures into roadway preconstruction planning [47] , as well as heightened education efforts during times of the year where changes in wildlife behavioral biology may contribute to increased likelihood of vehicle strikes. disease. while the mean canine distemper virus cases admitted per month were not found to fluctuate to a statistically significant degree, there was fluctuation in total raccoon and skunk canine distemper cases admitted annually from 2005-2014. these species are known reservoirs of canine distemper virus in this region [48] [49] [50] . an increase in admissions of both species due to canine distemper may suggest an increase in overall prevalence of canine distemper cases during those years. this facility also admitted a noteworthy number of cases of mycoplasma conjunctivitis, an ocular infectious disease primarily affecting finch species that was first observed in ohio in 1994 [31, 51] , throughout the examined time period, these findings, in conjunction with other recent work that has observed annual fluctuations in west nile virus cases admitted to a minnesota wildlife rehabilitation facility [32] and characterized european hedgehogs as a natural reservoir for some coronaviruses via surveillance testing of admissions to a facility in northern italy [52] , may implicate value for wildlife rehabilitation admissions for disease monitoring. however, additional research is needed which compares wildlife records and monitored wildlife population disease prevalence before conclusions can be drawn. it should be noted that resources available for diagnostic testing and relatively low rates of diseased individuals being admitted may limit the ability of wildlife rehabilitators to discern small fluctuations in disease occurrences locally [27] , and diseases that do not show obvious clinical signs will be underrepresented in this dataset. however, in the event of significant spikes in disease prevalence in local populations, wildlife rehabilitation facilities would be among the first to detect it via abnormal increases in cases admitted, potentially such as those observed in canine distemper cases admitted in 2008 and 2009. this would corroborate speculation in other literature regarding the potential value of wildlife rehabilitation records for wildlife disease monitoring [19, 27, 53] . while rabies is one of the diseases of greatest public health concern, it was only confirmed in three cases out of over 45,000 admitted across 10 years. this could be due to the public being less likely to approach animals exhibiting signs that can be associated with rabies, but is also likely a reflection of the low prevalence observed in this region [54]. while wildlife management challenges are multifaceted, wildlife rehabilitation facilities could feasibly serve as one piece of the puzzle by contributing information regarding human-wildlife conflict and some disease trends in urban and suburban areas broadly, or more specifically by taxon. a standardized system of record keeping would optimize the potential effectiveness of rehabilitators as a resource in this way. progress has been made with the creation of standardized national databases such as wild-one (http://www.wild-one.org/), wildlife rehabilitation md (http://wrmd.org/), and state-specific online databases, but there is a need for cohesiveness between organizations and public health agencies regarding data collection [53] . some of the most significant challenges for wildlife health surveillance are related to the lack of confirmed cases, underreporting of confirmed cases, and lack of infrastructure to facilitate assembling records for surveillance [55] . an increase in federal financial support of wildlife rehabilitation facilities could enable increased staff power, resources for disease-related diagnostics, and a cohesive nationwide database. this infrastructure would facilitate more uniform, cohesive monitoring of human-wildlife conflicts and disease occurrences nationally and internationally, and potentially enable wildlife rehabilitation facilities to contribute to monitoring of these trends on both local and global scales. additionally, our findings highlighted several needs for public education outreach efforts-namely, the impacts of both domestic cat and dog predation on wildlife, how to recognize when neonatal wildlife is truly orphaned and resources to contact for professional intervention, time periods throughout the year when different human-wildlife conflicts are most likely to occur, and affected wildlife taxa. the identification of such trends and subsequent development of relevant public outreach campaigns has been shown to contribute to wildlife population recovery and maintenance [43] , and our findings suggest that wildlife rehabilitation facilities are uniquely positioned to contribute to these efforts. supporting information s1 table. all broad and specific causes for admission. a complete list of the broad specific causes for admission and the corresponding specific causes for admission that were used to categorize the admissions records from a wildlife rehabilitation facility in the midwest, usa. 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in microbiology and immunology we would like to thank dusty lombardi and the other staff and volunteers of ohio wildlife center for their enthusiastic partnership in this study and remarkable work on behalf of wildlife. we would also like to specifically acknowledge the late dr. donald burton, founder of ohio wildlife center and revered wildlife veterinarian. thank you also to barbara ray for support in the early stages of this project, the young lab at otterbein university for their feedback on multiple manuscript drafts, and dr. michelle willette for her feedback on a later draft of this manuscript. key: cord-330749-xt4aa2ur authors: schilling, stefan; fusco, francesco maria; de iaco, giuseppina; bannister, barbara; maltezou, helena c.; carson, gail; gottschalk, rene; brodt, hans-reinhard; brouqui, philippe; puro, vincenzo; ippolito, giuseppe title: isolation facilities for highly infectious diseases in europe – a cross-sectional analysis in 16 countries date: 2014-10-28 journal: plos one doi: 10.1371/journal.pone.0100401 sha: doc_id: 330749 cord_uid: xt4aa2ur background: highly infectious diseases (hids) are (i) easily transmissible form person to person; (ii) cause a life-threatening illness with no or few treatment options; and (iii) pose a threat for both personnel and the public. hence, even suspected hid cases should be managed in specialised facilities minimizing infection risks but allowing state-of-the-art critical care. consensus statements on the operational management of isolation facilities have been published recently. the study presented was set up to compare the operational management, resources, and technical equipment among european isolation facilities. due to differences in geography, population density, and national response plans it was hypothesized that adherence to recommendations will vary. methods and findings: until mid of 2010 the european network for highly infectious diseases conducted a cross-sectional analysis of isolation facilities in europe, recruiting 48 isolation facilities in 16 countries. three checklists were disseminated, assessing 44 items and 148 specific questions. the median feedback rate for specific questions was 97.9% (n = 47/48) (range: n = 7/48 (14.6%) to n = 48/48 (100%). although all facilities enrolled were nominated specialised facilities' serving countries or regions, their design, equipment and personnel management varied. eighteen facilities fulfilled the definition of a high level isolation unit'. in contrast, 24 facilities could not operate independently from their co-located hospital, and five could not ensure access to equipment essential for infection control. data presented are not representative for the eu in general, as only 16/27 (59.3%) of all member states agreed to participate. another limitation of this study is the time elapsed between data collection and publication; e.g. in germany one additional facility opened in the meantime. conclusion: there are disparities both within and between european countries regarding the design and equipment of isolation facilities. with regard to the international health regulations, terminology, capacities and equipment should be standardised. the term highly infectious diseases (hid) defines mostly viral and bacterial infections that (i) are easily transmissible from person to person; (ii) cause a life-threatening clinical illness with no or few treatment options; and (iii) pose a threat for both health care workers and the public, thus requiring specific infection control measures and public health planning [1] . included in this definition are viral haemorrhagic fevers (e.g. filo-, arena-or bunyavirus infections); some respiratory syndromes (e.g. severe acute respiratory syndrome (sars)-coronavirus, or pneumonic plague); as well as any (re2) emerging infectious agent transmissible from person-to-person [2] . in europe, hids have caused several events within the last decade: sars affected eight european nations in 2003; lassa virus has repeatedly been imported to europe; and crimean congo haemorrhagic fever virus infections are increasing in several mediterranean regions, and have also been imported to central europe and the united kingdom (uk) [3] [4] [5] [6] [7] . recently detected agents like the new human middle east respiratory syndrome coronavirus underline the continuous challenge faced [8] . patients with suspected or proven hids should be cared for in a clinical environment that provides safe, secure, high-quality, and appropriate care with optimal infection containment, prevention and control procedures [1] . consensus statements on the operational management and design of isolation facilities have been published in europe and the united states of america [1;9] . in addition, the european commission has funded projects to enhance early recognition of cases by training front-line health care workers (hcws) and standardizing diagnostic methodology [10] [11] [12] . despite such efforts, no pooled data on isolation facilities resources, such as infrastructure design, technical equipment, capacity and access to intensive care, do exist. the study presented was performed by the european network for highly infectious diseases, euronhid, and set up to compare the operational management, resources, and technical equipment among isolation facilities with recommendations published. due to differences in geography, population density, national response plans, and experience with hids in participating countries, it was hypothesized that the level of adherence to recommendations may vary. the objective of this article is to present data about infrastructure design and resources, technical equipment, available personnel, and access to intensive care. euronhid consists of infectious disease clinicians and public health officers with expertise in the management of hids identified via their national health authorities. administrative and scientific aspects are managed by an italian coordination team, and a steering committee, including partners from france, germany, greece, and the uk. both committees started their work in 2007, and reported all organizational and scientific developments to the project members. names and countries of all project members are listed above.\ three checklists were developed and based on projectmember's experience, available literature, national preparedness plans, and guidelines of international authorities for the management of hids [13] . each checklist contained questions addressing specific items of interest, and answers were structured as openended, closed, semi-open or free text options. overall, the checklists included 44 items and 148 specific questions. checklist one contained three major sections: infrastructure; technical equipment; and personnel management. a fourth section on emergency departments was not obligatory [14] . all checklists can be downloaded after free registration from: www.eunid.eu. the checklists underwent a pilot application to identify structural gaps and sources of misinterpretation [13] . the facilities of the steering committee members and one external facility applied the checklists and cross-checked all itmes for their applicability, but no external validation process was conducted. following minor changes, the checklists were disseminated to all eligible facilities until december 2009 and expected to be resubmitted within three months. data provided were cross-checked by personal on-site visits by a coordination team member in all but four centres enrolled. investigator (the national project representative) and observer (the coordination team member) bias may be ruled out, as all data recorded were cross-checked on-site and agreed with the facilities operational manager or director. until november 2009, the coordination team collected data on eligible isolation facilities to be included into the survey. national health authorities of all european union (eu) member states were contacted to identify their will to participate. participating national health authorities were requested to identify clinical facilities responsible for the admission, assessment, and care of hid patients in their country, and to nominate a national project representative. euronhid was co-funded by the european commission/ director general for consumers and health (dg sanco) for an initial period of 36 months and extended to 42 months without additional funding. a technical and scientific report on all data collected during the project was delivered to the commission in december 2010. for details please follow: http://ec.europa.eu/ research/health/infectious-diseases/emerging-epidemics/projects/ 143_en.html. until mid of 2010, 48 isolation facilities in 16 european countries were recruited into this survey (table 1 ). recruitment was closed by the end of 2009 with the exception of norway, who received questionnaires by end of 2009 and submitted data by spring of 2010. data collection and on-site visits were finalised in spring 2010. except for norway, all participating countries were eu member states. data were collected from all existing facilities in participating countries except for spain, where data were only collected in the catalonia region. overall, the median feedback rate for checklist 1 was 97.9% (n = 47/48), with a range from n = 7/48 (14.6%) to n = 48/48 (100%) of centres providing valid answers to specific questions. all data provided in this article represent a minimum feedback from 44 facilities (.90%). the majority of facilities were constructed or underwent major re-construction in the year 2001 or later (n = 32/47; 66.7%) and are located on the same campus or in direct connection with a general hospital (n = 41/48; 85.4%). most facilities have designated rooms (n = 23/48; 47.9%) or wards (n = 18/48; 37.5%) located within other hospital structures, and seven facilities are located within a separate building (n = 7/48; 14.6%). national guidelines for the construction of isolation facilities were available for 17 facilities whereas 29 were (re2) constructed in the absence of specific requirements (37% and 63%, respectively). three facilities accept paediatric (6.3%), and 14 (29.2%) accept adult patients, only, whereas most facilities provide care for both (n = 31/48; 64.6%). the number of isolation beds per facility ranges from one to 56, with a median of 9.3 beds for adult and 7.2 for paediatric patients. with respect to the overall population, the number of beds ranges from 0.05 to 17.9 per one million inhabitants (median: 1.7) ( table 1 ). the majority of all facilities uses their beds on a daily basis for routine patients (n = 37/48; 77,1%), while the remainder provide bed capacities reserved for hid cases, only (n = 11/48, 22.9%). except for the united kingdom, all countries (n = 46/48; 95.4%) use barrier nursing techniques with high level personal protective equipment (hl-ppe) (21) . 37 facilities (n = 37/46; 80.4%) have a response time of less than five hours after a case is notified, the remainder demand up to 10 hours to be fully operational (n = 9/46; 19.6%). only eight (16.7%) of all facilities evaluated lacked any experience in the management of suspected or proven hid cases. among the remainder, most had experience with infections due to sars (n = 13/48; 27.8%), and viral haemorrhagic fevers (n = 9/48; 18.8%). essential technical requirements for isolation facilities includes: negative pressure in the isolation room(s); an anteroom; aerosoltight doors and windows; high efficacy particulate air (hepa) filtration of exhausted air; and a surface of walls, floors and ceiling withstanding disinfection procedures [1] . all but five facilities enrolled (n = 43/48; 89.6%) provide negative pressure in the isolation area, and the majority (n = 34/48; 70.8%) is also equipped with all other essential equipment investigated. in facilities with two or less additional items, most often hepa filtration of exhausted air and anterooms were missing (n = 7 and 5/14 facilities, respectively) ( figure 1 ). additional technical requirements not considered essential, but benefitting infection control and hcw safety, includes: self-closing doors; audio and/or visual negative pressure indicators; an exclusive evacuation pathway; and an internal communication system (for hcw-hcw and hcw-patient communication) [1] . at least 3 additional technical requirements are found in the majority of facilities (n = 34/48; 70.8%). most often, an internal communication system (n = 43/48; 89.6%) and negative pressure indicators (n = 40/48; 83.3%) were present, while an exclusive evacuation pathway was missing in half of all facilities enrolled (n = 27/48, 56.3%). thirty-three facilities (68.8%) provide intensive care, available for patients within the isolation facility. twelve facilities (25%) rely on a designated isolation room within standard intensive care units, three (6.3%) have no access to intensive care capacities ( figure 2 ). equipment for the monitoring of vital parameters and advanced life support is available in all 48 facilities. in contrast, mechanical ventilators are available either permanently in twelve (25%) or on call in thirty-two facilities (66.7%), but not accessible at all in four (8.3%). forty-two facilities (87.5%) have either permanent or on-call access to blood gas analyzers, whereas six have not (12.5%). hence, out of forty-five facilities reporting intensive care provision, one (2.2%) lacks a mechanical ventilator, and three (6.7%) have no ability to monitor their ventilation therapy. almost all facilities (93.6%; n = 44/47) report permanent access to specifically trained infectious diseases doctors. 45/48 (93.75%) facilities report providing intensive care, but only 34 of those (75.5%) have access to specifically trained intensive care specialists. in addition, out of 34 facilities indicating to provide care for pediatric hid patients, only 11 (32.4%) report either permanent or on-call access to pediatricians. compared with available doctors, even fewer facilities report access to specifically trained infectious diseases nurses (n = 41/48; 85.4%), but intensive care nurses are available in a comparable number of facilities (n = 32/ 45; 71.1%) (figure 3 ). all but two facilities indicate 24-hour access to specifically trained medical and non-medical staff (n = 45/47; 95.7%), although 72.9% (n = 35/48) only have specific protocols to contact staff responsible for the operation of the facility. a shift plan to limit the number of hcws exposed to patients as well as the duration of working under hl-ppe exists in 70.8% of facilities (n = 34/48). permanent access to technicians was lacking in eleven facilities (n = 11/48; 22.9%). all participating facilities were evaluated for their adherence to recommendations for the operational management of isolation facilities published [1] . as hypothesized, the level of adherence to recommendations varied, both within and between participating countries. hence, three categories of isolation facilities representing different levels of adherence were defined: (i) high level isolation units (hliu), defined as operating independently from other hospital resources and specifically equipped; (ii) isolation rooms, defined as specifically equipped but only partially independent from other hospital resources; and (iii) referral centers, neither specifically equipped nor functionally independent. according to this classification, all facilities enrolled were categorized as shown in figure 4 . few facilities completely fulfilled recommendations for hlius (n = 18/48; 37.5%). six of those are located in germany, five in france, two in italy, and one each in the uk, finland, greece, ireland, and norway. such hlius are constructed and equipped for the admission, assessment and longterm critical care of hid patients. most facilities assessed did partially meet the recommendations, but lacked infrastructural components, or preconditions for personnel management (n = 25/48; 52%). such were labeled 'isolation rooms' considered effective to assess hid patients and provide short-term medical care until a patient can be transferred to a hliu. of these, five each are located in france, greece, and spain; two in germany; and one each in denmark, finland, ireland, luxembourg, malta, poland and slovenia. the remaining facilities (n = 5/48; 10.4%) are considered to function as 'referral centers' where suspected hid cases can be assessed within daily routine, but even short-term critical care cannot be provided without an increased risk of nosocomial infections. two of such facilities are located in bulgaria and france, each, and one in austria. within the last decades, autochthonous outbreaks or imported cases of hids have affected europe with significant impact, and new pathogens are emerging. most european countries have established national response plans including specialised clinical care facilities for the management of such scenarios [3] [4] [5] [6] [7] [15] [16] [17] . this article presents the first standardised analysis on the operational management, infrastructure, and technical equipment of 48 isolation facilities in 16 european countries. data provided may support national authorities to assess their level of preparedness but have to be adapted to single member states' needs and resources. with regard to the implementation of the international health regulations [18] , terminology, capacities and equipment accessible in isolation facility should be documented and standardised on a european level. although all eu member states were invited to join the project by dgsanco, only 16 agreed to participate, thus data presented are not representative for the eu in general. a major limitation of this study is the time elapsed between data collection and publication: e.g. in germany one another facility has opened in the meantime. hence, a periodical re-assessment of facilities and countries should be achieved, aiming for a permanently updated record of national preparedness. in addition, the checklists applied did not undergo an external validation process (e.g. a delphi cycle) due to limitations of funds and project duration, and present published guidelines, only. hence, within the data collection process, misinterpretations occurred and items needed to be excluded from the data analysis presented here. half of all countries assessed have at least one hliu according to the recommendations [1] . compared to the definition, 18 facilities fulfilled the criteria applied, but the majority of all facilities enrolled could not operate independently from other facilities. the high rate of incompliance with the recommendations depicted here can be explained by the absence of legally binding documents by the time most facilities were constructed. after sars and the us anthrax attacks in 2001, the political will was to enhance public preparedness, but no standard was agreed on. only few countries (e.g. the uk) had specific guidelines at hand, but the vast majority of facilities were constructed using laboratory-or operational theatre guidelines adapted to local needs and infrastructure prerequisites. an earlier inventory of isolation rooms in europe by our group faced this problem, when eu countries were asked to report on negative pressure rooms for hid patients [19] : the overall number of rooms exceeded most expectations, but both technical and infrastructural conditions were not well defined. hence, an overreporting of any isolation room' with available led to the impression of a well prepared health system, although other aspects of equipment and personnel were not addressed. given the definitions for different levels of isolation facilities proposed now, a reassessment of the eu's capacities could lead to more accurate data. furthermore, the allocation of funds for hospital's biological event preparedness has decreased since the sars pandemic, and is not harmonised within the eu. hence, as our data show, the level of adherence to the recommendations mirrors the economical situation in the eu, with the highest level of adherence in the economical most stable countries. the situation in the united states of america is comparable: a consensus on the operational management of isolation units consistent with the european approach exists, but only three facilities fulfill those criteria, although hospital preparedness for biological events is mandatory [9, 20] . the contrary can be found in israel, where the capability to manage hid patients is mandatory for every public hospital, and dedicated hlius are absent [21] . as hlius are designed for single imported cases or small clusters in the beginning of an outbreak, only, they do not compensate for surge capacity planning in major biological events [1] . besides infrastructure and equipment, additional features which support effective functioning of such specialised centres were addressed in this survey and recently published [22] [23] [24] [25] . access to a transportation system for hid patients plays an important role in distribution of isolation facilities and is reflected by the overall amount isolation facilities per country. balancing the necessary access to a focus of expertise with available transport logistics leads to either a centralised or a de-centralised approach to the location of specific facilities, and ambulance dispatch recording can be of use in early warning systems for outbreak detection [26] . most documented hid cases demanded supportive intensive care [16, 17, 27] , and access to such is considered essential for hlius [1] . in order to allocate funds most effectively, a balance between infection control excellence and operational feasibility should be sought. basic specifications for hliu provision, including access to intensive care equipment and trained personnel, should be developed in order to facilitate a common standard of best clinical practice. key issues are the composition of a permanently available multidisciplinary medical and nonmedical team, the ability and physical fitness to work with ppe, and reliable timelines and shifts to prevent accidents or mistakes due to exhaustion [1, 28] . data depicted in this article do highlight available personnel as the most crucial pitfall in operating isolation facilities: compared to the complicane with technical equipment recommendations, the lack of specifically trained staff is surprising, especially for hcws trained in intensive and paediatric care. it should be mentioned that all facilities with experience in proven hids provide more sophisticated human resources management such as shift-and surge capacity planning as an outcome of their experience. the overall investment for an isolation facility, and hlius in particular, is high, in terms of costs for construction and maintenance, medical and infection control equipment, human resources and training activities. even in time of economical constraints, employers ''are obliged to ensure the health and safety of workers'', as defined by the european commission [29] . without adequate protection, hcws may acquire and transmit infections, with potential dramatic impact on the health system, and the economic solidity of a country, as documented during the sars epidemic in canada. for these reasons, we believe that specialized isolation facilities, and hlius in particular, still represent a critical infrastructure to which each eu member state should have access, within the country or through predefined agreements with neighbouring countries where such facilities are available. whether such capacities should be reserved for hid cases, only, or also used on routine basis, is part of an ongoing discussion. financial constraints within hospitals and the host country may benefit from a routine-use concept, although such facilities demand more frequent maintenence due to damage on technical equipment. in addition, access to routine use beds for training of hcws is reduced, and patients need to be relocated to other rooms once the facility receives an hid case. in contrast, reserved bed capacities demand additional personnel and material for routine functional checks, thus considered cost-ineffective. the major benefit from reserved capacities is a full access for training and no delay in patients admission since no evacuation of others is needed. the who ihrs define the need of preparedness for infectious diseases outbreaks, but a european, if not international, consensus on the funding and minimum number of isolation capacities is not in sight. although hids are rare events, they cause dynamic and often rapidly evolving issues in need of comprehensive solutions and may challenge the capacity of healthcare systems. leadership and funding at both national and european level are required to harmonize preparedness plans, terminology and communication to weaken the impact of future infectious disease outbreaks with cross-border potential. in order to achieve a balance between saving lives and protecting hcws in hazardous environments, national and international collaboration should continue to share experiences, and provide standardized training and equipment. the 'european network for highly infectious diseases -euronhid' is co-funded by the european commission/dg sanco under the eu contract number 2006205. neither the commission nor dg sanco had an influence on the study design, data collection, or interpretation of results. other members of the euronhid have supported the authors heli siikamaki (finland), christian perronne (france), all members of the ''stä ndige arbeitsgemeinschaft der kompetenz-und behandlungszentren -stakob framework for the design and operation of high level isolation units: consensus of the european network of infectious diseases infection control in the management of highly pathogenic infectious diseases: consensus of the european network of infectious disease severe acute respiratory syndrome: identification of the etiological agent a fatal case of lassa fever in london the first case of lassa fever imported from mali to the united kingdom first confirmed case of crimean-congo haemorrhagic fever in the uk crimean-congo haemorrhagic fever isolation of a novel coronavirus from a man with pneumonia in saudi arabia designing a biocontainment unit to care for patients with serious communicable diseases: a consensus statement a curriculum for training healthcare workers in the management of highly infectious diseases the european network of biosafety-level-4 laboratories: enhancing european preparedness for new health threats community research and development service european training and research centre for imported and highly contagious diseases euronhid checklists for the assessment of highlevel isolation units and referral centres for highly infectious diseases: results from the pilot phase of a european survey infection control management of patients with suspected highly infectious diseases in emergency departments: data from a survey in 41 facilities in 14 european countries acute liver failure, multiorgan failure, cerebral oedema, and activation of proangiogenic and antiangiogenic factors in a case of marburg haemorrhagic fever novel coronavirus associated with severe respiratory disease: case definition and public health measures implementing the international health regulations (2005) in europe isolation rooms for highly infectious diseases: an inventory of capabilities in european countries department of health and human services, health resources and services administration world health organization, regional office for europe (2012) assessment of heath-system crisis preparedness: israel. world health organisation personal protective equipment management and policies: european network for highly infectious diseases data from 48 isolation facilities in 16 european countries infection control practices in facilities for highly infectious diseases across transportation capacity for patients with highly infectious diseases in europe: a survey in 16 nations diagnostic issues and capabilities in 48 isolation facilities in 16 european countries: data from the euronhid surveys surveillance of ambulance dispatch data as a tool for early warning on behalf of the ehec-hus consortium (2012) validation of treatment strategies for enterohaemorrhagic escherichia coli o104:h4 induced haemolytic uraemic syndrome: case-control study on the introduction of measures to encourage improvements in the safety and health of workers at work key: cord-310396-jitao9k0 authors: lei, yu; moore, chris b.; liesman, rachael m.; o'connor, brian p.; bergstralh, daniel t.; chen, zhijian j.; pickles, raymond j.; ting, jenny p.-y. title: mavs-mediated apoptosis and its inhibition by viral proteins date: 2009-03-07 journal: plos one doi: 10.1371/journal.pone.0005466 sha: doc_id: 310396 cord_uid: jitao9k0 background: host responses to viral infection include both immune activation and programmed cell death. the mitochondrial antiviral signaling adaptor, mavs (ips-1, visa or cardif) is critical for host defenses to viral infection by inducing type-1 interferons (ifn-i), however its role in virus-induced apoptotic responses has not been elucidated. principal findings: we show that mavs causes apoptosis independent of its function in initiating ifn-i production. mavs-induced cell death requires mitochondrial localization, is caspase dependent, and displays hallmarks of apoptosis. furthermore, mavs(−/−) fibroblasts are resistant to sendai virus-induced apoptosis. a functional screen identifies the hepatitis c virus ns3/4a and the severe acute respiratory syndrome coronavirus (sars-cov) nonstructural protein (nsp15) as inhibitors of mavs-induced apoptosis, possibly as a method of immune evasion. significance: this study describes a novel role for mavs in controlling viral infections through the induction of apoptosis, and identifies viral proteins which inhibit this host response. in recent years, knowledge of host cell signaling responses to viral infection has progressed rapidly. it is known that cells of the immune system contain toll-like receptors (tlrs) capable of detecting extracellular or endosomal viral nucleic acid and activating appropriate signal transduction pathways leading to the up-regulation of immune and inflammatory cytokines. besides detecting extracellular viral products, somatic cells can also respond to intracellular viral rna by activating the recently identified mitochondrial antiviral signaling pathway. following cytoplasmic detection of viral nucleic acid by the rig-i-like helicases (rlh) family of receptors, these and other signaling proteins are recruited to the mitochondria where they interact with the mitochondrial antiviral signaling adaptor protein mavs (ips-1, visa and cardif) [1, 2, 3, 4] . in vitro and in vivo experiments have revealed a critical role for mavs and its mitochondrial localization in the activation of host antiviral responses [1, 5] . although the role of mavs in type-1 interferon (ifn-i) responses is known, the localization of mavs to the mitochondria suggests other putative mitochondrial functions for mavs, prominent among these is apoptosis. however, to date, there are no comprehensive studies focused on testing this hypothesis. notably, host cell apoptosis is a successful strategy to impede viral replication and restrict virus spreading during a productive infection [6] . multicellular organisms are equipped with at least two evolutionarily conserved defensive arms to eradicate viral infections: programmed cell death and innate immune responses. many proteins which function in both apoptotic and inflammatory signaling cascades contain a caspase recruitment domain (card), which functions as a homotypic interaction motif. in fact, the biological function of the card domain was initially described in a subset of caspases which activate mitochondria-dependent apoptotic signaling [7] . for example, the card containing apaf-1 (apoptosis protease-activating factor-1) protein binds to cytochrome c and forms a ternary multimeric protein structure called the apoptosome which functions to activate caspase-9 via a proximity-induced mechanism [8] . other card-containing proteins including some members of the nlr (nucleotide-binding domain and leucine-rich repeat containing) protein family have been linked with both apoptotic and inflammatory signaling [9] . for example, the card-containing nlr, nod1, has been shown to activate a caspase-9 dependent apoptosis and play a positive regulatory role in pathogen-induced nf-kb activation [10] . similarly, nod2, a protein linked with the etiology of the autoinflammatory crohn's disease, has been reported to augment caspase-9-induced apoptosis when overexpressed [11] . a third card-containing nlr, nlrc4 (ipaf), mediates cell death through a caspase-1 dependent fashion [12, 13, 14] . similar to the aforementioned proteins, mavs contains an nterminal card-like domain, in addition to a central proline-rich region and a c-terminal transmembrane (tm) domain, which targets mavs to the mitochondrial outer membrane [1] . recent crystal structure analysis reveals that the card-like domain of mavs is indeed a classical card fold with surface charge profiles of a typical card domain involved in homotypic associations [15] . consequently, the presence of a card-like domain coupled with its mitochondrial localization suggests a putative role for mavs in both immune and cell death responses. in fact, both the n-terminal card-like and tm domains are indispensable for mavs-mediated activation of interferon regulatory factor-3 (irf-3) and subsequent transcription of the antiviral ifn-i, suggesting that these domains are critical to mavs function [1] . as a survival mechanism, it is known that some viruses have evolved strategies to inhibit mavs function through selective targeting of these functional domains. for example, the genome of hepatitis c virus (hcv) has evolved to include a serine protease, ns3/4a, which cleaves the mavs tm domain and dislodges mavs from the mitochondria, thereby abrogating mavs mediated ifn-i production [4, 16] . similar to hcv, hepatitis a virus (hav) encodes for the 3abc protein, which localizes to the mitochondria and inhibits mavs signaling via proteolytic cleavage [17, 18] . currently, there are no reports of viral proteins targeting mavs for inhibition of virus-induced cell death responses. host cell apoptosis has been reported to suppress viral replication and the subsequent production of infectious progeny viruses [19] . for example, adenoviruses and baculoviruses which are defective in anti-apoptotic genes are compromised in producing progeny viruses [19] . in addition, several viruses infectious to humans, including the coronaviruses, are known to modulate host cell apoptotic responses [20, 21] . in 2003, researchers from several labs identified a unique coronavirus linked with the pathogenesis of severe acute respiratory syndrome (sars) in humans [22, 23, 24] . the spread of sars-cov, reaching near pandemic levels, resulted in the death of over 900 individuals with a case fatality rate of 11% [25] . since that time, the sequencing of the complete sars-cov genome has allowed scientists to study the function of each sars-cov encoded protein in greater details. phylogenetic analyses revealed that sars-cov was not closely related to any other characterized coronaviruses [26, 27] . the distinct nature of the sars-cov genome suggests possible unique strategies employed by this virus to subvert host defense mechanisms. notably, unlike other common human respiratory viral infections, such as influenza a virus, the viral loads in the upper airway of sars patients progressively increase, reaching a peak around 10 days after the initial onset of symptoms [28] . this indicates that at least during the initial stages of sars-cov infection, this virus might suppress host defensive responses. in fact, several sars-cov proteins have been shown to inhibit host ifn-i responses [20, 21, 29] . although the fact that certain clinical manifestations of sars such as lymphopenia and cell death in the lung or liver are thought to be related to the ability of sars-cov to induce apoptosis in specific cell types and that several pro-apoptotic proteins have been found in sars-cov genome [30, 31, 32] , the early pathogenesis of sars is poorly understood and how apoptosis contributes to the initial pathological changes is largely unknown. in addition, the replication of sars-cov seems to be restrained to the first two weeks upon symptom onset with little evidence of continued replication after this time window [33] . clearly, the temporal and cell-type specific expression of sars-cov proteins could account for the dynamics of sars-cov infection and given that host cell fate decisions are a part of overall host responses to any pathogen, it is conceivable that like sars-cov inhibitory effects on ifn-i responses, this virus could employ proteins which function as inhibitors of host cell death. in this report, we describe a novel function of mavs in mediating virus-induced apoptosis, and identify viral proteins as inhibitors of this response. transient expression of mavs protein causes hek293t cells to crenate and lose adherence, suggesting that mavs is cytotoxic (fig. 1a) . although some cell death was observed at 24 hours posttransfection, it was most evident at 48 hours post-transfection, which was quantitated by the trypan blue exclusion test of cell viability. in this assay, we observed a dose-dependent increase in the percentage of trypan blue positive cells at 48 hours posttransfection with mavs plasmid (fig. 1b) . this result was confirmed by measurements of cell viability via xtt assay. mavs-induced cell death is potent as 10 ng of mavs plasmid was sufficient to decrease cell viability by 40% (fig. 1c ). mavs contains a n-terminal card-like domain, which is well conserved from human to pufferfish [1] ; and the card domain of other proteins has been shown to mediate the activation of caspases, facilitating apoptosis [34] . therefore, we next sought to test the hypothesis that mavs induces apoptosis through a caspase-dependent mechanism. it is well known that blockade of caspase activity will inhibit the intrinsic apoptotic pathway [35] . thus, we treated hek293t cells with a pan-caspase inhibitor (z-vad-fmk) followed by transfection of increasing amounts of mavs plasmid. as expected, transient mavs expression in untreated cells resulted in a significant loss of cell viability as measured by xtt assay ( fig. 2a , black bars); and application of the pan-caspase inhibitor resulted in a complete reversal of mavs-induced cell death ( fig. 2a , grey bars). it is known that caspase-dependent apoptosis can cause poly (adp-ribose) polymerase (parp) cleavage, thus we investigated the effect of mavs expression on the triggering of parp cleavage. hek293t cells were transfected with two different doses of mavs plasmid and both adherent and floating cells were harvested and lysed 24 and 48 hours post-transfection. immunoblotting followed by densitometry measurements revealed a dose-dependent increase in parp cleavage (fig. 2b, 2c ). in congruence with the parp cleavage pattern, caspases-3 and caspase-9 protein levels also showed dose-dependent increases following mavs expression (fig. 2b, 2c ). transmission electron microscopy examination of hek293t cells transfected with mavs or empty vector was performed at 48 hours post-transfection (fig. 2d ). unlike the otherwise healthy cells transfected with empty vector (fig. 2d , top panel), mavs-expressing cells exhibit the morphological hallmarks of apoptosis including an intact plasma membrane (fig. 2d , bottom panel arrow i), crenation, condensed and marginated chromatin (arrow ii), large vacuoles (arrow iii), cytoplasm shrinkage, membrane blebbing, an intact nuclear envelope, and swollen mitochondria (arrow iv) [36] . a kinetic analysis shows that mavs expression reached a peak at 24 h post-transfection (fig. s1a) , while mavs-associated apoptosis lagged behind reaching a peak at 48 h post-transfection (fig. s1b ). thus the late onset of apoptosis does not appear to be due to the lack of mavs expression. it is possible that there are several molecular steps that have to occur before mavs could cause caspase-3 and caspase-9 activation, leading to apoptosis. the aforementioned studies analyzed the effect of transient expression of mavs on cell death. to explore the physiological role of endogenous mavs in mediating virus-induced apoptosis, we extended these findings to investigations of recombinant sendai virus expressing gfp (rsev-gfp) infected mouse embryonic fibroblasts (mefs) isolated from mavs knockout or wild type littermate control mice. mavs deficiency was confirmed by western blot (fig. s2 ). at 48 hours post-infection, we infected mavs 2/2 and wildtype littermate control mefs with rsev-gfp, which is a known inducer of apoptosis [37] . infection efficiency in each cell line was monitored by gfp positivity, which was similar between mavs wildtype and knockout mefs (fig. 3a , top graph). in contrast, the percentage of annexin v positive cells was .30% among infected wildtype mefs compared to ,8% for the mavs 2/2 mefs (fig. 3a, bottom graph) . similarly, mavsdeficient mefs maintained a spindle-like fibroblastic morphology (fig. 3b , gfp bottom panel), while wildtype mefs crenated and became detached from the plate. in addition, rsev-gfp infected wildtype mefs have higher propidium iodide/hoechst staining ratio as compared to mavs 2/2 , further indicating an increase in cell death (fig. 3b , hoechst and pi panels). transmission electron micrographs taken for both cell types, with or without sev, demonstrated that sev infection resulted in the presence of typical apoptotic features in the wildtype mefs (fig. 3c , left panels). however, sev infected mavs 2/2 showed little signs of apoptosis and are indistinguishable from the uninfected controls (fig. 3c , right panels). previous reports have shown that the mavs c-terminal transmembrane domain (tm) is essential to mavs function as an adaptor in ifn-i signaling [1] . therefore we sought to determine the essential domain that mediates mavs-induced apoptosis. we expressed full length mavs protein or three truncation mutants (fig. 4a ) in hek293t cells and measured annexin v and 7-aad staining. the mavsdtm mutant, which lacks the mitochondrial transmembrane sequence, was completely incapable of inducing apoptosis, suggesting that similar to the role of mavs in interferon signaling, mitochondrial localization is essential to mavs activation of apoptosis ( (fig. s3a, s3b ), thus inability of these constructs to induce apoptosis is not due to ineffective protein expression. to solidify the mitochondrial dependency of mavs-induced cell death, we co-expressed mavs with the hepatitis c virus (hcv) protein ns3/4a. ns3/4a is a serine protease which has been shown to target the mavs tm domain for cleavage and subsequent inhibition of mavs antiviral signaling [4, 17] . consistent with its role in inhibiting mavs mediated ifn-i signaling, ns3/4a inhibited mavs induced apoptosis (fig. 4b , bottom right panel). the inhibitory effects of mavsdtm mutant and ns3/4a were also verified by a measurement of cell viability via xtt assay (fig. 4c ). mitochondrial membrane potential collapse marks a point-of-no-return during apoptosis and occurs earlier than dna fragmentation [38, 39] . only wildtype mavs resulted in compromised mitochondrial membrane potential as measured by tmre staining while the addition of ns3/4a abrogated this effect (fig. 4d ). it has been shown that exogenous mavs expression triggers ifn-i production [1, 5] . type-1 ifns mediate their effects by binding to cell surface receptors, activating downstream interferon-stimulated genes or isgs, among which more than 15 genes have pro-apoptotic functions [40] . we sought to determine if mavs-induced apoptosis was the result of activation of a distinctive signaling pathway or as a consequence of ifn production. first, we induced ifn-i production in hek293t cells by ectopic expression of upstream signaling molecules that activate mavs-mediated ifn production. as previously reported, a helicase domain truncation mutant of rig-i [41] , full-length mavs [1] , or mda-5 in conjunction with its ligand poly(i:c) [42] zare all potent ifn-i inducers (fig. s4) . neither drig-i nor mda-5 plus poly(i:c) induced any observable cell death or annexin vpositive cells (fig. 5a, bottom panels) . only full-length mavs triggered apoptosis as quantified by annexin v and 7-aad staining (fig. 5a, top right panel) . to explore the role of ifn-b in mavs-induced apoptosis, hek293t cells were treated with 200 neutralization iu/ml ifn-b antibody prior to transient expression of mavs followed by xtt measurements. we found that blocking ifn-b from binding to its cell surface receptor had no effect on mavs-induced apoptosis ( fig. 5b ) even though the antibody efficiently blocked the function of secreted ifn-b (fig. s5 ). in congruence with these findings, rsev-gfp infections of the interferon-a/b receptor (ifnar) knockout and wildtype mefs demonstrated that this receptor is not required for sev-induced cell death as visualized by pi staining (fig. 5c ). mavs is also capable of inducing the transcription factor nf-kb, which is known to activate a wide array of genes linked to immune, inflammatory, and cell-fate processes [43] . to test the involvement of nf-kb in mavs-induced apoptosis, we co-expressed mavs with the non-degradable superrepressor form of ikba. this super-repressor is a known potent inhibitor of nf-kb activation and as expected inhibited mavsinduced nf-kb activation (fig. s6 ) [44] . however, transient expression of mavs in hek293t cells induced apoptosis even in the presence of the nf-kb super-repressor (fig. 5d ). there are 14 subtypes of type-1 ifns, and a plethora of other secreted soluble factors can be induced by mavs expression. therefore, we performed a transwell assay designed to definitively answer if mavs-induced cell death was intrinsic to the host cell or caused by some unknown secretory factor. hek293t cells were plated in two different chambers (top and bottom) separated by an insert filter membrane, permissive to all secretory cytokines but impermeable to fugene6:dna complexes. in this experiment, only the lower chamber was transfected with mavs. cells in the upper chamber were not transfected with mavs, but were exposed to the same cytokine milieu. as expected, mavs induced cell death in the lower chamber (transfected cells); however the cells in the upper chamber (untransfected cells) remained healthy and unchanged from controls (fig. 5e ). this indicates that the mavs-induced death is not caused by an undefined secretory product(s) which includes the interferon family members. irf3 is a critical transcription factor for ifn-i production but it has been associated with apoptosis, and furthermore can be activated by mavs [37, 45] . we assessed whether mavs-induced apoptotic signaling depends on irf3. we made numerous attempts to introduce mavs into irf3 2/2 fibroblasts, however these cells were much more difficult to transfect than wildtype fibroblasts, hence the different efficiencies of transfection made the interpretation of data difficult. instead we used sirna to reduce irf3 expression. briefly, hek293t cells were plated in 96-well plate and endogenous irf3 expression was reduced by transfecting a pool of four sirna into the cells. mavs was introduced 24 hours after sirna transfection. cell viability was measured by xtt assay 48 hours after cells were transfected with an expression plasmid containing mavs or a control empty vector. the reduction of irf3 did not prevent mavs-induced loss of cell viability ( figure 6a ). the same sirna was introduced into 5610 5 cells plated in a 6-well plate, and an immunoblot was used to verify the efficiency of sirna 48 h post-transfection ( figure 6b ). as an alternate approach to measure cell death, the experiment was repeated in 6 well plates and cells were harvested 48 hours after plasmids transfection. half of the cells from each well were stained for annexin v and analyzed by flow cytometry, and the other half of the cells were lysed in ripa buffer for western blotting analyses of irf3. as expected we were able to greatly reduce the endogenous irf3 expression, yet mavs-induced apoptosis was not abrogated (fig. 6c, 6d) . while host response that elicits apoptosis may function as an antiviral strategy, viruses have also evolved diverse mechanisms to evade these host antiviral responses. therefore we performed a functional screen of 12 sars-cov-encoded proteins to identify any potential modulators of mavs-induced apoptosis. each of these sars-cov genes were cloned into an expression vector and co-expressed with mavs plasmid followed by xtt cell viability measurements. protein expression was verified by an immunoblot (fig. s7 ). of the 12 tested proteins, only one sars-cov protein, nsp15, significantly altered mavs-induced apoptosis of hek293t cells (fig. 7a) . further examination showed that nsp15 significantly inhibited mavs-induced apoptosis in a dose-dependent manner (fig. 7b) . the anti-apoptotic function of nsp15 displayed specificity since it did not inhibit staurosporine-induced apoptosis (fig. 7c) . we sought to investigate if the anti-apoptotic function of nsp15 was specific for sars-cov or shared by other coronaviruses. when the nsp15 protein encoded by the sars-cov, hku1 or nl63 genome was coexpressed with mavs, only sars-cov nsp15 inhibited mavs-mediated apoptosis as measured by annexin v and 7-aad staining, while the others showed little effect on cell viability (fig. 7d ). host cellular response to virus infection involves the concomitant activation of parallel signaling pathways leading to the transcription of a plethora of cytokine genes, prominent among these are the genes encoding type-1 interferons (ifn-i). it is the autocrine and paracrine action of these and other cytokines which encompasses the comprehensive host immune response designed to defend against viral infection. this is accompanied by a reciprocal activation of programmed cell death in infected host cells, which is also known to reduce viral load. apoptosis has been suggested to play a protective role at the organismal level in preventing the virus from completing its replication and producing infectious progeny viruses [19] . consequently, the exact mechanisms underlying both virus-induced apoptotic signaling in addition to viral strategies to subvert these responses is currently a topic of intensive research. while pathways that govern ifn-i have been extensively investigated, the molecular mediators that activate host cell apoptosis during viral infection are less known. in this study we have identified a role for mavs in the initiation of virus-induced apoptosis (fig. 8) . furthermore, we have identified hepatitis c virus ns3/4a and sars-cov nsp15 proteins as inhibitors of mavs-mediated apoptosis. it is known that mavs is a potent inducer of ifn-i responses and that ifn-i can activate host apoptotic responses, therefore it was important to determine whether the cell death responses observed in the current study were a consequence of ifn-i secretion. in fact, ifn-i consist of several species including ifn-a, ifn-b, ifn-v and ifn-k and these cytokines bind to surface receptors leading to the activation of the jak-stat signal transduction pathway resulting in the transcriptional activation of virtually hundreds of ifn-stumulated genes (isgs), whose protein products play pivotal roles in a variety of biological events, including but not limited to immunomodulation, cell differentiation, anti-angiogenesis and programmed cell death. for example, type-1 ifn induction of apoptotic responses can be mediated by a multitude of isgs, illustrated by the finding that more than 15 isgs have pro-apoptotic functions [40] . in addition, the involvement of proteins on ifn axis in virusinduced host cell apoptosis has been implicated in another previous report, in which mavs has been shown to be critical for reovirus-triggered caspase-3/7 activation in hek293t cells [46] , however, the study did not evaluate whether mavs mediates virus-induced apoptosis and what roles type 1 ifns play in mavs-mediated apoptosis. using a multi-pronged approach we demonstrate that mavs-mediated apoptosis is not a consequence of ifn-i induction by mavs. we show that (a) the over-expression of the truncation mutant of rig-i that induce ifn-i does not lead to apoptosis; (b) anti-ifn-b antibody does not ablate mavs-induced apoptosis; (c) the targeted deletion of ifn receptor does not alter mavs-induced apoptosis; (d) the co-culture of mavs-transfected cells with nontransfected cells separated by an insert filter membrane does not induce apoptosis in the latter, indicating that a soluble secretory factor is not likely responsible for the apoptosisinducing activity. in the ifn-i induction pathway mediated by endogenous rig-i like helicases, irf3 lies downstream of mavs, and exogenous expression of mavs can lead to the phosphorylation and nuclear translocation of irf3 [1] . in addition, others have shown that the expression of constitutively active form of irf3 mutant is toxic to cells and the transfection of wild type irf3 expression can augment sev-induced apoptosis [37, 45] . hence it was important to evaluate if mavs-induced apoptosis depends on the presence of irf3. our data shows that depletion of endogenous irf3 by means of rnai did not reverse mavs-induced cell viability loss. together with our findings that mavs-induced apoptosis is ifni-independent, we speculate that mavs-induced apoptotic signaling pathway is different from the classical mavs-mediated ifn-i response. furthermore, it is possible that the reported proapoptotic effects of the constitutively active form of irf3 might be independent of its ifn-i-inducing function. in fact our data suggests other ifn-i signaling molecules such as the constitutively active form of rig-i and mda5 do not lead to apoptosis despite of their roles in inducing ifn-i. the dual functions of mavs in virus-induced ifn-i and apoptosis highlight this molecule as a putative target for viral evasion strategies designed to escape host immunity. for example, it is already known that hepatitis c virus (hcv) produces a serine protease, ns3/4a, which disrupts ifn-i production through the targeted cleavage of the mavs transmembrane region and the subsequent dislodging of mavs from the mitochondria [4, 16] . in fact, we found that loss of mavs mitochondrial localization through mutation of the transmembrane domain, also completely abolished its pro-apoptotic effects. consistent with these results, when hcv ns3/4a protein was co-expressed with mavs, this viral protein completely inhibited mavs-induced cell death. this result would suggest that the host immune evasion strategies of the hcv ns3/4a protein may be extended to inhibition of mavsmediated apoptosis. similarly hepatitis a virus (hav), a picornavirus, employs a cysteine protease 3abc to abrogate ifn-i production by targeting mavs [17] . therefore, it is likely that other uncharacterized viral mechanisms target mavs for modulation of host cell death responses. one novel strategy described in this study is employed by the sars-cov. similar to sars-cov abilities to inhibit ifn-i responses, we have found that this virus is also capable of interfering with host cell death responses by targeting mavs. we showed that among the 12 sars-cov proteins tested, nsp15 alone can completely abrogate mavs-induced apoptosis. the underlying mechanism is currently unclear since the function of nsp15 is not yet fully defined. however, an earlier report indicates that this protein is indeed important for sars pathogenesis in that it is essential for viral replication [47] . however, until we understand the exact temporal expression patterns of each of the sars-cov proteins during the course of an infection, the relative contributions of each to the evasion of host immunity and apoptosis will not be fully understood. further studies are needed to determine the exact mechanism of action for nsp15 on inhibiting mavs-induced cell death. since sars-cov replication is limited to the first two weeks after symptom onset and nsp15 is critical for its replication [33, 47] , this would support the theory that nsp15 may function as an inhibitor of host cell death during this time, which would possibly benefit viral replication. further studies are ongoing to determine how nsp15 and other sars-cov proteins contribute to overall viral evasion strategies. the finding that mavs mediates virus-induced apoptosis posits a new mechanism by which mitochondria serves as a bona fide intracellular sentinel for antiviral and apoptotic responses. it is well established that the permeabilization of the mitochondria outer membrane by the pro-apoptotic bcl-2 family member bak results in the activation of caspase-9 in a classical apaf-1-dependent or an alternate apaf-1-independent pattern [48] . furthermore, it is known that proteins localized on the mitochondria are targeted by viruses to modulate host responses. for example, the cytomegalovirus rna can interact with the mitochondrial enzyme complex i (reduced nicotinamide adenine dinucleotide-ubiquinone oxido-reductase) to modulate the classical mitochondria-mediated apoptosis [49] . recent discoveries also underscore the mitochondria as a platform orchestrating host antiviral type-1 ifns through the mitochondrial mavs protein. this study describes, for the first time, a duality of function for the mavs protein in regulating both ifn-i and apoptotic antiviral responses from within the mitochondria and suggests that mavs is a pivotal molecule in the bifurcation of host responses following viral challenge. furthermore, the identification of hcv ns3/4a and sars-cov nsp15 as inhibitors of mavs-mediated apoptotic responses reveals both novel host defense mechanisms as well as viral immune-evasion mechanisms that might serve as useful templates for the development of anti-viral drug strategies. hek293t cells, mavs +/+ , mavs 2/2 , ifnar 2/2 mouse embryonic fibroblasts were maintained in dmem media supplemented with 10% fbs, 1% penicillin and 100 mg/ml streptomycin. cells were passed every three days and grown at 37uc in 5% co 2 . the mammalian expression plasmids of ns3/4a, wild type mavs and truncation mutants were kindly provided by dr. zhijian chen at the university of texas southwestern medical center. ha-tagged sars-cov proteins expression plasmids, hku1 nsp15 and nl63 nsp15 expression plasmids were provided by drs. ralph baric and matthew frieman at the university of north carolina. transfections and viral infections hek293t cells were seeded in 96-well or 6-well plates, and the total dna transfected into these cells was 400 ng/well and 1 mg/ well respectively. standard transfection protocol was performed using fugene6 (roche applied science) according to the commercial protocol. cells were incubated for the indicated times prior to assay. rsev-gfp is a recombinant sendai virus expressing gfp and was originally made by dr. daniel kolakofsky [50] . for viral infections, 1.6610 4 mefs were plated into 12-well plate one day prior to sendai virus (sev) infection. viral infections were performed when cells reached 60% confluence. mefs were incubated with sev for 1 h in serum-free dmem supplemented with 0.5% tryple select at the moi of 0.5. serum-free dmem supplemented with 0.5% tryple select was used for mock infection. virus inoculum was removed and cells were replenished with complete media supplemented with 0.5% tryple select following incubation with virus. cells were harvested and washed in cold facs buffer (5% fbs in 16 pbs) twice and in 16 annexin v binding buffer (bd bioscience, san diego, ca) once. cells were transferred into 96well plate and resuspended in 45 ml annexin v binding buffer. cells were stained according to standard cell staining protocol [51] using annexin v conjugated to fitc or apc (bd biosciences, san diego, ca). cells were washed in facs buffer and stained with 7-aad (invitrogen, carlsbad, ca) for 10 min at room temperature. cells were washed twice and resuspended in facs buffer containing 1 mg/ml actinomycin d. after 5 min incubation, all samples were fixed in 500 ml 1% em grade formaldehyde (polysciences, warrington, pa) for immediate flow cytometry analyses. for the rsev-gfp infected samples, gfp positive cells were gated; annexin v and 7-aad positive populations were assessed thereafter. mitochondrial membrane potential dy was assessed by flow cytometry analysis on a dysensitive fluorophore tetramethylrhodamine ethylester (tmre) (molecular probes, eugene, or). after treatment, hek293t cells were incubated with tmre at the concentration of 10 nm per 1610 6 cells for 40 min at 37uc. immediate flow cytometry analysis was performed after staining. all flow cytometry data were collected on either a facscalibur (bd biosciences, san jose, ca) or cyan flow cytometer (dako north america, carpinteria, ca) and then analyzed by flowjo software (tree star, ashland, or). transmission electron microscopy examination was performed as described [52] . mefs or hek293t cells were harvested and fixed in glutaraldehyde and all samples were post-fixed in 1% oso 4 for transmission electron microscopy examination. cells were embedded in london resin white and sections were analyzed using a leo em-910 transmission electron microscope (leo electron microscopy inc., thornwood, ny). hoechst and propidium iodide (pi) dual staining was used to evaluate sevinduced cell death. samples were photographed using a leica dmirb inverted fluorescence microscope (leica microsystems inc., bannockburn, il) with a digital camera (micropublisher, q-imaging, burnaby, bc, canada). cells were lysed in ripa lysis buffer (1% triton x-100, 0.25% doc, 0.05% sds, 50 mm tris ph8.0, 150 mm nacl and 50 mm naf) containing proteinase inhibitor cocktails (roche) for 30 min at 4uc. whole cell lysates were suspended in laemmli's sample buffer and loaded onto nupage bis-tris 4-12% gradient pre-cast gels (invitrogen, carlsbad, ca) for sds-page and subsequent immunoblotting for parp, caspase-3 and caspase-9 (abcam, cambridge, ma), irf3 (santa cruz biotechnology, santa cruz, ca), rodent specific mavs (cell signaling, danvers, ma). densitometry was performed using imagej analysis software. identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction visa is an adapter protein required for virus-triggered ifn-beta signaling cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus the specific and essential role of mavs in antiviral innate immune responses viral homologs of bcl-2: role of apoptosis in the regulation of virus infection card games in apoptosis and immunity apaf-1, a human protein homologous to c. elegans ced-4, participates in cytochrome c-dependent activation of caspase-3 the nlr gene family: a standard nomenclature nod1, an apaf-1-like activator of caspase-9 and nuclear factor-kappab nod2, a nod1/apaf-1 family member that is restricted to monocytes and activates nf-kappab caspase-1 activator ipaf is a p53-inducible gene involved in apoptosis differential activation of the inflammasome by caspase-1 adaptors asc and ipaf identification of ipaf, a human caspase-1-activating protein related to apaf-1 crystal structure of human ips-1/ mavs/visa/cardif caspase activation recruitment domain hepatitis c virus protease ns3/4a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity disruption of innate immunity due to mitochondrial targeting of a picornaviral protease precursor regulation of mitochondrial antiviral signaling pathways regulators of apoptosis on the road to persistent alphavirus infection open reading frame 8a of the human severe acute respiratory syndrome coronavirus not only promotes viral replication but also induces apoptosis induction of apoptosis by the severe acute respiratory syndrome coronavirus 7a protein is dependent on its interaction with the bcl-xl protein aetiology: koch's postulates fulfilled for sars virus identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study sars coronavirus and innate immunity severe acute respiratory syndrome coronavirus gene 7 products contribute to virusinduced apoptosis a sars-cov protein, orf-6, induces caspase-3 mediated, er stress and jnk-dependent apoptosis pathogenesis of severe acute respiratory syndrome time course and cellular localization of sars-cov nucleoprotein and rna in lungs from fatal cases of sars the mitochondrial apoptosome: a killer unleashed by the cytochrome seas caspases in cell survival, proliferation and differentiation the morphology of apoptosis the irf-3 transcription factor mediates sendai virus-induced apoptosis sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo apoptosis and interferons: role of interferon-stimulated genes as mediators of apoptosis the rna helicase rig-i has an essential function in double-stranded rnainduced innate antiviral responses shared and unique functions of the dexd/h-box helicases rig-i, mda5, and lgp2 in antiviral innate immunity nf-kappab signaling pathways in mammalian and insect innate immunity nf-kappab activation provides the potential link between inflammation and hyperplasia in the arthritic joint irf-3 activation by sendai virus infection is required for cellular apoptosis and avoidance of persistence retinoic acid-inducible gene-i and interferon-beta promoter stimulator-1 augment proapoptotic responses following mammalian reovirus infection via interferon regulatory factor-3 major genetic marker of nidoviruses encodes a replicative endoribonuclease apoptotic pathways: ten minutes to dead complex i binding by a virally encoded rna regulates mitochondria-induced cell death sendai virus trailer rna binds tiar, a cellular protein involved in virus-induced apoptosis c-c chemokine receptor 5 on pulmonary fibrocytes facilitates migration and promotes metastasis via matrix metalloproteinase 9 nlrx1 is a regulator of mitochondrial antiviral immunity we thank victoria madden for technical support with electron microscopy. we thank dr. mark heise and catherine cruz for the interferon protection assay. sars-cov proteins expression plasmids, hku1 and nl63 nsp15 expression plasmids were provided by drs. ralph baric and matthew frieman, university of north carolina at chapel hill. nf-kb super-repressor plasmid is a kind gift of dr. albert baldwin, university of north carolina at chapel hill. rsev-gfp was generously provided by dr.daniel kolakofsky, university of geneva. we also acknowledge visiscience inc. for scienceslides 2008 application in cartoon preparation. key: cord-339327-4422s317 authors: norris, susan l.; sawin, veronica ivey; ferri, mauricio; raques sastre, laura; porgo, teegwendé v. title: an evaluation of emergency guidelines issued by the world health organization in response to four infectious disease outbreaks date: 2018-05-30 journal: plos one doi: 10.1371/journal.pone.0198125 sha: doc_id: 339327 cord_uid: 4422s317 background: the production of high-quality guidelines in response to public health emergencies poses challenges for the world health organization (who). the urgent need for guidance and the paucity of structured scientific data on emerging diseases hinder the formulation of evidence-informed recommendations using standard methods and procedures. objectives: in the context of the response to recent public health emergencies, this project aimed to describe the information products produced by who and assess the quality and trustworthiness of a subset of these products classified as guidelines. methods: we selected four recent infectious disease emergencies: outbreaks of avian influenza a—h1n1 virus (2009) and h7n9 virus (2013), middle east respiratory syndrome coronavirus (mers-cov) (2013), and ebola virus disease (evd) (2014 to 2016). we analyzed the development and publication processes and evaluated the quality of emergency guidelines using agree-ii. results: we included 175 information products of which 87 were guidelines. these products demonstrated variable adherence to who publication requirements including the listing of external contributors, management of declarations of interest, and entry into who’s public database of publications. for guidelines, the methods for development were incompletely reported; who’s quality assurance process was rarely used; systematic or other evidence reviews were infrequently referenced; external peer review was not performed; and they scored poorly with agree ii, particularly for rigour of development and editorial independence. conclusions: our study suggests that who guidelines produced in the context of a public health emergency can be improved upon, helping to assure the trustworthiness and utility of who information products in future emergencies. in the context of the response to recent public health emergencies, this project aimed to describe the information products produced by who and assess the quality and trustworthiness of a subset of these products classified as guidelines. we selected four recent infectious disease emergencies: outbreaks of avian influenza a-h1n1 virus (2009) and h7n9 virus (2013), middle east respiratory syndrome coronavirus (mers-cov) (2013), and ebola virus disease (evd) (2014 to 2016). we analyzed the development and publication processes and evaluated the quality of emergency guidelines using agree-ii. we included 175 information products of which 87 were guidelines. these products demonstrated variable adherence to who publication requirements including the listing of external contributors, management of declarations of interest, and entry into who's public database of publications. for guidelines, the methods for development were incompletely reported; who's quality assurance process was rarely used; systematic or other evidence reviews were infrequently referenced; external peer review was not performed; and they scored poorly with agree ii, particularly for rigour of development and editorial independence. the world health organization (who) shapes public health policy for united nations member states through the development of technical guidance. more than a decade ago, in response to harsh external criticism of its guidelines, who instituted the guidelines review committee (grc) with the mandate to develop rigorous methods and procedures ensuring that each who guideline is evidence-based and meets the highest international standards so that guidelines are credible, trustworthy and relevant to end-users [1] . who quality standards dictate that guidelines must address a critical public health problem, use transparent and explicit processes minimizing potential sources of bias such as conflicts of interest, include diverse perspectives in the guideline development group, reflect the current state of the evidence, and provide a clear link between the evidence and recommendations taking into consideration the balance of benefits and harms of interventions and other important considerations [2] . who's scope of work includes producing guidelines and other information products in the context of public health emergency response such as natural and technological disasters, disease outbreaks, armed conflicts and other humanitarian crises. when relevant guidelines do not exist, either because the situation is new or because current guidelines are not applicable, it is extremely challenging to produce new guidelines. significant uncertainty in the field leads to urgent need, imposing short development timelines; scientific data are scarce and the collection of new data is hindered; health systems may be poorly functioning or nonexistent; and resources (both money and expertise) for guideline development may be scant. despite this challenging context, adherence to the principles and standards for high-quality, trustworthy and usable guidelines is essential such that end-users adopting and implementing recommendations in the field can optimally impact population health outcomes. the best way to apply traditional quality standards for guidelines to the context of emergencies remains unclear. who does not currently have an established approach with defined processes and tools to develop emergency guidelines. thus, guideline development groups at who and in other organizations must improvise and adapt standard methods on an ad hoc basis, potentially compromising the quality of the final product. to date there are no comprehensive evaluations of guidelines produced in the context of a public health emergency. the aim of this study is to describe the information products produced by who in recent emergencies and to assess the quality and trustworthiness of a subset of these products classified as guidelines. the overarching goal of this evaluation was to inform future processes and methods for producing these types of guidelines at who and by other organizations globally. we examined information products issued by who for four recent public health emergencies: outbreaks of avian influenza a-h1n1 virus (2009), avian influenza a-h7n9 virus (2013), middle east respiratory syndrome coronavirus (mers-cov) (2013) and ebola virus disease (evd) (2014 to 2016). there were other outbreaks, humanitarian emergencies, and natural disasters between 2009 and 2016 that were graded by the global emergency management team [3] , however we selected these four emergencies as purposeful sample as they all focused on infectious diseases outbreaks, and involved a significant response by who including the production of a large number of information products. all occurred after the institution of the grc so that a standard approach to develop guidelines in the non-emergency context was in place at who. we included all information products published in any language that were specifically developed or adapted by who for the four selected emergencies. we excluded testimonials, media releases, situation reports and meeting reports. two independent reviewers assessed each document against study inclusion criteria. as per who's definition [2] , we classified an information product as a guideline based on the content of that document (i.e., whether it contained a specific recommendation for the end-user), rather than by the process for development. guidelines included products that contained recommendations and did not reference an underlying, source guideline. these covered a wide range of topics including clinical care [4] , water and sanitation [5] , and occupational health and safety [6] , among many other topics. all other information products were classified as non-guidelines, which included, for example, publications derived from existing guidelines [7] , assessments of preparedness in countries [8] , and tools for assessment or response [9] . non-guidelines are not expected to contain detailed methods and other types of information that is expected in a high-quality guideline. we identified eligible information products available on 24 april 2016, from lists of technical documents related to each emergency assembled by the who department of communicable disease surveillance and response. we had difficulty identifying relevant documents and therefore searched multiple potential sources such as who's global digital library (institutional repository for information sharing (iris)), hand-searched who websites, consulted key who staff in technical units and reviewed who resource toolkits. two independent reviewers extracted information product characteristics and publication format, and identified translations. for guidelines, we extracted data on standard who guideline development processes (e.g., based on a systematic review of the evidence, involved external experts in the formulation of recommendations) and had two independent reviewers appraise each guideline using the appraisal of guidelines for research and evaluation (agree) ii instrument [10] . this instrument, the most widely-used guideline quality appraisal tool, is comprised of 23 items, six quality domains and a 7-point likert-like response scale. we report the prevalence of key characteristics for information products in each emergency. for continuous data, we report the mean and standard deviation (sd). we summarize agree ii scores for each domain (scaled to a percentage of the maximum score) across guidelines using the median value and the interquartile range, as scores were not normally distributed and sample sizes were small for some emergencies. all statistical analyses were conducted using stata 12.1 (statacorp lp, college station, texas, usa). we identified 175 information products of which 87 were classified as guidelines. table 1 summarizes the characteristics of all information products. table 2 outlines the methods and processes used in the development of guidelines specifically. for the h1n1 emergency response, 54 information products met our inclusion criteria: 15 guidelines and 39 other information products [11] . systematic reviews were cited in only two guidelines (13%) and four other guidelines (27%) referred to a literature search. external experts were involved in 53% of guidelines, but only four named these contributors of which three reported that declaration of interests (doi) had been obtained. peer review rarely occurred. dates of expiration or planned update for the guidelines were provided in eight title contains: "guideline" or "guidance" "interim" documents (53%). h1n1-related guidelines received the highest scores in the agree ii domains of clarity of presentation (median 78%; interquartile range (iqr) 74%-83%) and scope and purpose (median 69%; iqr 63%-85%) (fig 1) . the lowest scores were assessed in editorial independence (median 0%; iqr 0%-0%), rigour of development (median 13%; iqr 4%-20%), and applicability (median 15%; iqr 9%-19%). for h7n9, we identified 14 emergency information products, five emergency guidelines and nine other information products. all but one document was available only in english. one guideline referenced a systematic review; another cited evidence, but did not indicate whether the evidence review was systematic. of the three guidelines that reported involving external experts, only one listed those individuals and provided their doi. none of the guidelines provided a date of expiration or planned update, though one guideline indicated that recommendations would be updated as new information emerged. based on the agree ii instrument, h7n9 emergency guidelines scored well in clarity of presentation (median 89%; iqr 86%-89%) and scope and purpose (median 72%; iqr 69%-78%). the lowest-scoring agree ii domains were rigor of development (median 7%; iqr 6%-25%), applicability (median 19%; iqr 15%-27%) and editorial independence (median 0%; iqr 0%-0%) (fig 1) . twenty-three information products, including 16 emergency guidelines and seven other information products, met our inclusion criteria for mers-cov documents. eleven guidelines (73%) were available in at least two languages (english and arabic); we identified only an english version of the remaining information products. none of the mers-cov emergency guidelines referenced a systematic review. although five guidelines (31%) reported that external experts contributed to the recommendations, only four of these listed the names of individual experts and only one reported that doi had been collected. three guidelines (19%) provided a specific date for the expiration or update of the guideline. the agree ii domains that scored the highest across these mers-cov-related emergency guidelines were clarity of presentation (median 83%; iqr 72%-92%) and scope and purpose (median 76%; iqr 53%-78%). the lowest scoring domains were editorial independence (median 0%; iqr 0%-0%) and rigour of development (median 5%; iqr 1%-9%) (fig 1) . we included 84 evd information products, including 52 emergency guidelines and 32 other information products. overall, 42 (50%) information products were available in english and french, including 32 (62%) guidelines and 10 (31%) non-guidelines; the remainder were available only in english. only two evd guidelines reported or referenced a systematic review, although a third guideline reported that a rapid review of the evidence had been performed. an additional five guidelines (10%) referenced evidence or indicated that a literature search had been performed, but it was unclear if the review methods were systematic. four guidelines (8%) reported that recommendations were based entirely on expert opinion. most guidelines (81%) cited other publications, including other who information products. four guidelines (8%) reported that doi had been collected from external experts. four guidelines provided a date for the expiration or update of the guideline; eight additional guidelines indicated that regular updates would be provided as new information became available. evd guidelines scored the highest in the agree ii domains of clarity of presentation (median 81%; iqr 69%-86%) and scope and purpose (median 69%; iqr 60%-81%). the lowest scores were assessed in editorial independence (median 0%; iqr 0%-0%) and rigour of development (median 7%; iqr 2%-18%) (fig 1) . approximately half (51%) were published in 2014 (fig 2) . guidelines were published an average of 178 days (sd 200) following the initial emergency grading of evd in sierra leone on 25 july 2014; two documents (4%) were published prior to the assignment of the emergency grade. information products issued by who during four recent infectious disease epidemics were difficult to find, were often published at long intervals after outbreaks were graded, and demonstrated variable adherence to who publication and reporting requirements. few guidelines met international standards for the development and presentation of trustworthy and usable guidelines, as assessed with tools designed for standard (non-emergency) guidelines. the principles for trustworthy guidelines were developed in the context of standard (nonemergency) clinical guidelines where there is a much longer time-period for development, end-users' needs may be much clearer, there may be much more (high-quality) evidence, and the implementation setting and health systems may be much less fragile and chaotic [1] . universally agreed-upon standards and methods for the optimal development of public health guidelines in the context of emergencies have not yet been established and thus our assessments using these standards must be interpreted with caution. nonetheless, the principles of transparency, minimization of risk of bias in the formulation of recommendations, and the presentation of implementable, impactful recommendations inarguably apply to all types of guidelines and recommendations. the difficulty we faced to identify and retrieve relevant documents suggests that end-users may have similar or greater difficulties during the emergency response. in addition, incomplete reporting of key information such as the responsible technical unit and publication date may prevent end-users from determining the relevance of a product to their specific context as the epidemic trajectory changes or new information emerges. there was no consistent format of the information products that we examined, including within specific emergencies. information products should vary in their content and appearance to meet the needs of the intended end-users, however who products should be readily identifiable with consistent appearance and branding, including the who logo. the labelling of information products varied across products of the same or similar types, even within emergencies. in particular, guidelines were referred to by a vast array of terms that did not appear to have a specific meaning. for example, "interim guidance" was relatively frequently used in evd-related documents, appropriately suggesting a limited shelf-life for these documents, however specific plans for updating were rarely presented. we noted incomplete implementation of who's mandatory policy on conflicts of interest of external contributors and the reporting of funding sources for guidelines. this is an important finding as the interests of both author and funder are correlated with the conclusions of systematic reviews and the recommendations in clinical practice guidelines [12] . although doi does not in itself minimize the risk of bias associated with those interests, disclosure is widely regarded as the first and essential step in addressing competing interests and their potential effects on the conclusions of an evidence review or the recommendations in a guideline. despite the who grc's responsibilities, only three guidelines were approved by the quality assurance agency. the specific reasons were unclear, although the pressure to produce guidelines quickly could lead to ad hoc and variable processes and procedures. nonetheless, quality assurance and control processes must still be used for all who information products. although incomplete reporting likely impeded our assessment, the rigour of development of the guidelines examined in our study appears to be low, as assessed by agree ii and infrequent reference to systematic reviews. compressed timelines in response to urgent needs necessitate abbreviated methods, however an explicit description of the approaches used to develop a guideline is always possible. the information and considerations used to inform each recommendation must be clearly described and when direct evidence is scarce, it is even more important to be transparent about the basis for recommendations. the applicability of indirect evidence must be carefully assessed and the rationale for its inclusion provided (for example, the use of data from related viruses or other infectious agents with similar vectors). draft guidelines rarely underwent peer review by individuals and organizations distinct from the primary contributors to the guideline. this is an important gap in the development process, as peer review is a standard step in guideline development [1, 2] , and has important benefits including stakeholder engagement, identification of errors and attention to key implementation issues. peer review can be executed within days and comments can be quickly evaluated with careful planning and ready access to a cohort of experts and stakeholders. a clear indication of plans for updating recommendations is a critical issue in the emergency context, as the evidence base and other considerations such as those related to implementation are continually evolving. all information products developed in response to an emergency, particularly those labelled "interim", should have a "review by" date and sufficient resources must be available to evaluate and update information products as required. high scores in the agree ii domains of clarity of presentation and scope and purpose may be attributable to the operational focus of most emergency guidelines, which typically requires a concise, clear format. other agree ii domain scores suggest significant room for improvement. for example, guidelines scored inconsistently in the domain of applicability, which includes criteria such as descriptions of barriers and facilitators to implementation of the recommendations, and provision of or reference to other tools to support implementation. although this evaluation examines a number of important measures, there are many additional questions that were not addressed. we focused on emergency information products for infectious disease outbreaks and it is important to examine information products developed in response to other types of emergencies such as natural disasters and humanitarian crises caused by displacement of populations secondary to conflict or climate change. we did not evaluate dissemination, utilization, perceived usefulness by end-users, implementation and uptake of recommendations, or most importantly, the impact on health outcomes and wellbeing. such impact evaluations are needed. nor did we try to identify gaps in the guidance covered by the available information products. despite our efforts to compile comprehensive lists of information products developed by who in response to h1n1, h7n9, mers-cov and evd, we may not have captured all relevant documents. we examined only the latest version available: some information products may have been updates which may have different characteristics than previous versions that were developed in shorter timelines or earlier in the emergency response. for practical reasons, we evaluated only information reported in the documents. we may have misclassified derivative products as guidelines if they failed to reference the underlying guideline. although widely used to assess the quality of reporting and conduct of guidelines, the agree ii instrument has limitations [13] , and it was developed for clinical guidelines and not for public health or emergency guidelines. thus, the domain scores that we report should be interpreted with caution. the agree ii tool is also limited in its scope. for example, the domain of applicability examines the inclusion of tools for implementation and discussion of barriers, cost implications, and monitoring of the guideline's recommendations, but it does not address actual implementation or impact on health. in addition, we had only two assessors for the agree ii appraisals; although acceptable according to agree ii guidance, four reviewers would have enhanced the reliability of these assessments [10] . this study did not intend to compare the quality of guidelines for the four responses with each other or with standard who guidelines developed in stable settings. it is challenging to compare guidelines across the four epidemics as each event differed with respect to incidence, mortality rate, rate of change in the burden of disease, geographic spread, prior experience with the disease, and the availability of resources. who guidelines produced in the context of a public health emergency can be improved upon, helping to assure the trustworthiness and utility of who information products in future emergencies. the principles of transparency, minimization of the risk of bias, and reliance on research and other evidence over expert opinion, apply to guidelines developed in any setting or context. with careful self-evaluation, thoughtful reflection on lessons learned in these emergencies, and a firm commitment to continuous improvement, who will continue to provide timely and high-quality guidance in the context of public health emergencies. supporting information s1 clinical practice guidelines we can trust world health organizaton. who handbook for guideline development-2nd edition. geneva: world health organization emergency response framework (erf) world health organization. facilitation manual: psychological first aid during ebola virus disease outbreaks ebola virus disease: key questions and answers concerning water, sanitation and hygiene world health organization. ebola virus disease (evd): occupational safety and health: joint who/ilo briefing note for workers and employers infection prevention and control (ipc) guidance summary: ebola guidance package world health organization. ebola virus disease preparedness strengthening team: mali country visit 20-24 world health organization. facility questionnaire for ebola virus disease/viral haemorrhagic fever diagnosis capacity agree ii: advancing guideline development, reporting and evaluation in health care pandemic (h1n1) 2009 guidance documents conflict of interest in clinical practice guideline development: a systematic review tools for assessing the content of guidelines are needed to enable their effective use-a systematic comparison the authors gratefully acknowledge the administrative support of ms myriam felber and dr martin mendelson. key: cord-330537-xz0wt1sz authors: biermann, olivia; tran, phuong bich; viney, kerri; caws, maxine; lönnroth, knut; sidney annerstedt, kristi title: active case-finding policy development, implementation and scale-up in high-burden countries: a mixed-methods survey with national tuberculosis programme managers and document review date: 2020-10-28 journal: plos one doi: 10.1371/journal.pone.0240696 sha: doc_id: 330537 cord_uid: xz0wt1sz background: the world health organization (who) stresses the importance of active case-finding (acf) for early detection of tuberculosis (tb), especially in the 30 high-burden countries that account for almost 90% of cases globally. objective: to describe the attitudes of national tb programme (ntp) managers related to acf policy development, implementation and scale-up in the 30 high-burden countries, and to review national tb strategic plans. methods: this was a mixed-methods study with an embedded design: a cross-sectional survey with ntp managers yielded quantitative and qualitative data. a review of national tb strategic plans complemented the results. all data were analyzed in parallel and merged in the interpretation of the findings. results: 23 of the 30 ntp managers (77%) participated in the survey and 22 (73%) national tb strategic plans were reviewed. ntp managers considered managers in districts and regions key stakeholders for both acf policy development and implementation. different types of evidence were used to inform acf policy, while there was a particular demand for local evidence. the nsps reflected the ntp managers’ unanimous agreement on the need for acf scale-up, but not all included explicit aims and targets related to acf. the ntp managers recognized that acf may decrease health systems costs in the long-term, while acknowledging the risk for increased health system costs in the short-term. about 90% of the ntp managers declared that financial and human resources were currently lacking, while they also elaborated on strategies to overcome resource constraints. conclusion: ntp managers stated that acf should be scaled up but reported resource constraints. strategies to increase resources exist but may not yet have been fully implemented, e.g. generating local evidence including from operational research for advocacy. managers in districts and regions were identified as key stakeholders whose involvement could help improve acf policy development, implementation and scale-up. tuberculosis (tb) remains one of the top 10 causes of death worldwide [1] . the world health organization (who) end tb strategy [2] and the united nations (un) sustainable development goals [3] aim at ending tb by 2030. international attention has also been drawn to tb with the 2017 global ministerial conference on ending tb [4] and the un's 2018 general assembly high-level meeting on tb [5] . due to a combination of underreporting of detected cases and underdiagnosis, there is still a gap of three million between estimated incident tb cases and those notified worldwide [6] . ending tb will require increased and early case detection [2] by applying strategies such as systematic screening, i.e. the "systematic identification of people with suspected active tb in a predetermined target group, using tests, examinations or other procedures that can be applied rapidly" [7] . active case-finding (acf) is tantamount to systematic screening for active tb, though it implies screening outside of health facilities [7] . the potential benefits and risks of acf need to be carefully balanced when developing and implementing acf policies [7] [8] [9] [10] [11] . this study focuses on the 30 high tb burden countries, as they account for 85-89% of the global tb burden [12] . moreover, this study focuses on national tb programme (ntp) managers, key stakeholders in developing and implementing acf policies [13] . ntps are usually housed within national ministries of health, though the activities of the programme are implemented at different levels of the health system [14] . depending on the context, ntps are responsible for delivering high-quality and effective diagnostic, treatment and preventive services [13, 14] . the aim of this study was to describe attitudes of ntp managers related to acf policy development, implementation and scale-up in the 30 high-burden countries, which potentially impact the development and implementation of national acf policies. this was a mixed-methods study with an embedded design (fig 1) [15] , complemented by a document review. the study comprised a cross-sectional survey with ntp managers from 30 high tb burden countries, which included closed and open-ended questions designed to elicit quantitative and qualitative information, enhancing each other [15] . the document review included a sample of national tb strategic plans (nsps) from the high tb burden countries and was designed to complement the findings from the interviews. we implemented all surveys through structured interviews and therefore refer to them as "interviews" in the subsequent text. the policy analysis triangle [16] considers how the content of a policy, actors, context and processes shape policymaking. we used the triangle as a conceptual framework, adapting it by putting "context", "processes" and "actors" as the corner stones of the triangle (instead of having "actors" in the middle of the triangle, as in the original version) and including example topics we covered (fig 2) . the framework shapes the presentation of the study findings. questionnaire. the development of the questionnaire was informed by a scoping review and an expert interview study on factors influencing the development and implementation of acf policies [11, 17] . the preceding work considered acf globally, while this survey focuses on the 30 high tb burden countries. two independent researchers with expertise in survey design provided feedback on the survey to minimize biases [18] . the questionnaire (s2 appendix) included sections on the general views on acf, national acf policies, evidence use, contextual factors, scale-up, monitoring and evaluation, and lessons learned. question formats included likert scales, lists, yes/no questions and open-ended questions (without probing questions). five-point likert scales investigated 1) agreement with 23 national tuberculosis (tb) programme managers participated in the cross-sectional survey, which was analyzed using descriptive statistics and manifest content analysis. 22 national tb strategic plans were reviewed, and data extracted to complement the survey findings. all data were analyzed in parallel and merged in the interpretation of the findings. statements about the potential benefits and risks of acf, 2) degree of influence of contextual factors on acf policies, and 3) frequency of use of different types of evidence in acf policy. the ntp managers also confirmed which stakeholders were involved in acf policy development and implementation, using a list of 14 types of stakeholders. participants. ntp managers from all 30 high-burden countries were contacted via email. in total, 23 ntp managers agreed to be interviewed (participation rate: 77%). 17 ntp managers took part in the interview themselves, whereas six appointed one of their team members to participate on their behalf. in this article, we refer to "the ntp managers", meaning all 23 respondents. the ntp managers did not receive an incentive for participation. ntp managers from the following 23 countries are represented: bangladesh, brazil, cambodia, china, congo, democratic republic of the congo, ethiopia, indonesia, kenya, lesotho, mozambique, myanmar, namibia, nigeria, pakistan, papua new guinea, philippines, south africa, tanzania, thailand, vietnam, zambia and zimbabwe. ntp managers from seven countries did not participate in the study: angola, central african republic, democratic people's republic of korea, india, liberia, russia and sierra leone. five were not interested in or did not have time for being interviewed and one did not reply. one ntp manager started but discontinued the interview due to an unstable telephone connection; several attempts were made to complete the survey but remained unsuccessful due to limited interest and/or time. data collection and management. the interviews were conducted in english between march and october 2018. as the pilot interview took 54 minutes, we condensed the survey. we included the pilot results in our dataset, apart from the questions that we deleted in the revised questionnaire. the remaining interviews took on average 48 minutes (28-68 minutes). the primary investigator (ob) conducted ten interviews in person during international conferences, five at a who meeting, and eight on the phone/skype, depending on the participants' preferences. all ntp managers were provided with information about the study and the questionnaire when accepting to participate. after providing informed consent, the interviews were voice-recorded. ob or pt transcribed the responses verbatim and shared the transcriptions with the ntp managers (and appointed team members) for their information and review. five ntp managers (22%) replied after having received the filled-in survey; one included additional information. if the ntp managers did not respond within four weeks, the survey was considered complete. one interview was terminated half-way due to limited time; we still included the data. pt entered all responses into redcap (research electronic data capture), a secure, web-based platform [19] . the anonymity and confidentiality of the ntp managers were ensured by removing all identifiers in the presentation of the results. data analysis. using stata 15 (statacorp llc), we computed descriptive statistics (frequencies, mean, median and proportions). we used cronbach's alpha to assess the internal consistency of the likert scale items as background analysis when developing the instrument [20] . we added additional indicators to investigate any patterns in the responses: 1) country income level and 2) region [21] , and 3) proportion of ntp budget consisting of domestic funding, 4) international funding and 5) being unfunded [6] . we analyzed open-ended answers with nvivo 11 (qsr international) using content analysis [22] , focusing on the manifest content and generating meaning units, codes and categories (table 1 ). in the results section, we first describe quantitative then qualitative findings and add information from the document review where applicable. the checklist for reporting results for internet e-surveys (cherries) was applied for reporting [23] . we identified 18 nsps through an online search, whilst an additional four nsps were shared with us by staff from the stop tb partnership or who. nsps from the following 22 countries are included: angola, bangladesh, brazil, cambodia, ethiopia, india, indonesia, kenya, liberia, mozambique, myanmar, namibia, nigeria, pakistan, papua new guinea, philippines, sierra leone, south africa, tanzania, vietnam, zambia and zimbabwe. the nsps from angola, brazil and mozambique were available in portuguese. eighteen of these countries overlap with the countries that are represented in the survey (the ones that do not overlap are angola, india, liberia and sierra leone). we were not able to obtain nsps from the following eight countries: china, congo, central african republic, democratic people's republic of korea, democratic republic of congo, lesotho, russia and thailand. congo and lesotho do not have an nsp according to the interviews performed. china, democratic republic of the congo and thailand have nsps based on the interviews, but we were unable to gain access. whether the democratic people's republic of korea, the democratic republic of congo and russia have nsps or whether the nsps are not publicly available remained unclear. active tuberculosis case-finding in high-burden countries data extraction from the national strategic plans. we developed a data extraction table, which we filled with data on the development of the nsp, background and aims, targets, operational and ethical considerations, budget estimates and funding sources (s1 appendix). a native portuguese speaker extracted and translated the data from the nsps from angola, brazil and mozambique. ethics approval and consent to participate and for publication. this study has been approved by the swedish ethical review authority in stockholm (2017/2281-31/2). of the 23 ntp managers who responded to the survey, 39% were female (n = 9). seventy-four percent (n = 17) of the ntp managers identified themselves as policymakers, whereas 13% (n = 3) identified themselves as researchers or "other". among the 20 participants who identified themselves either as policymakers or "other", 90% (n = 18) reported having research experience. in each of the following sections, we state the total number of participants who answered a particular question, as not all questions were answered by all 23 participants. the perceived benefits of acf. all ntp managers (n = 23) asserted that acf has some benefits (fig 3) . they agreed with the sentiments that acf leads to early detection, diagnosis and treatment, and reduced tb transmission and incidence. one ntp manager (4%) from a lower middle-income country disagreed that acf leads to improved treatment outcomes, while another ntp manager (4%) from a low-income country disagreed that acf leads to reduced future health system cost, and positive social and economic consequences for a tb patient. other potential benefits of acf included better health system performance (e.g. increased access and care-seeking, increased human resource capacity and synergies), epidemiological impact (e.g. reduced tb mortality) and health educational benefits through increased knowledge and awareness. "improving knowledge about tb among patients and the community i think is the most important component of the programme." (ntp manager 22) the perceived risks of acf. the ntp managers' views on potential risks of acf were more heterogenous compared to their views on the potential benefits (fig 4) . more than half of the ntp managers (55%, n = 12/22) neither agreed nor disagreed that acf leads to increased false-positive diagnoses of tb; they emphasized that the risk of a false-positive diagnosis depends on the diagnostic test used. four of the five upper middle-income countries (80%) included in this study were among those who neither agreed nor disagreed. moreover, while 83% (n = 19/23) of the ntp managers affirmed that acf leads to increased health system costs in the short term, 83% disagreed that acf would lead to increased health system costs in the long term (over 10 years); both estimates included ntp managers from low-, lower middle-and upper middle-income countries. the ntp managers brought up two examples of how acf could impair health system performance; by overburdening health care workers and increasing waiting times for non-tb patients. acf policies in high-burden countries. according to the ntp managers, a written acf policy exists in 91% (n = 21/23) of the countries, typically as part of an nsp. in line with that, the majority (87%, n = 20/23) said that acf contributes to the achievement of the goals outlined in their nsp. in 43% (n = 9/21) of the countries, the national acf policy has been evaluated or formally assessed. the document review supported this and showed that 86% (n = 19/22) of the nsps stated aims directly related to acf. another 86% reported a case detection gap. all nsps explicitly mentioned the need to undertake contact tracing and described one or more additional target groups for acf. yet, only six (27%) nsps outlined concrete targets for the number of people screened and three (14%) (two lower middle-income countries and one low-income country) specified targets for the number of people tested. a wide variety of factors play a role in both policy development and implementation according to the ntp managers. some factors are important across many settings, e.g. the country and health system context, and funders' priorities. fifty-five percent (n = 12/22) of the ntp managers articulated that factors at the level of the overall country context influenced the development of acf policies to a high degree. they highlighted the influence of politics, including the political system, commitment, agendas, political change and turnover. in terms of acf policy implementation, country-level factors also influenced to a high degree according to 45% (n = 10/22) of the ntp managers. such factors included local geography, climate and culture. the majority of the ntp managers said that health system factors influenced acf policy development and implementation to high degrees (77%, n = 17/22; and 68%, n = 15/22, respectively). most ntp managers emphasized the importance of human and financial resources for acf policy implementation and buy-in at sub-national level. focusing on human resources, many nsps documented the need to not only expand human resources, but to increase their capacity for acf, and to improve their coordination, engagement and supervision. many ntp managers expressed that factors at the level of organizations and the community moderately influenced the development and implementation of acf policies; (55%, n = 12/22; and 45%, n = 10/22). they stressed the importance of community engagement to develop good, implementable acf policies. ninety-one percent (n = 21/22) and 86% (n = 20/22) of the ntp managers specified that the priorities of funding organizations influenced acf policy development and implementation, respectively. according to who, the ntp budget in five of the included countries does not include any international funding [6] . the ntp managers elaborated that funders demanded seeing impact on case detection and imposed timelines for acf activities. overall, funders were described as influential, but not as powerful as the government in policy development. "you have to have your policy first and then you prioritize. so, the funders will have a choice. the policy will influence the funders, but not the other way around." (ntp manager 22) the views of ntp managers on the influence of individual level factors (e.g. knowledge about tb) on acf policy development were mixed; 18% (n = 4/22) of the respondents considered such factors as highly influential, another 18% saw no influence at all. the ntp managers described the need for tb patients to be at the center of the discussion when developing acf policies, whilst they would seldom directly influence policy development processes. "what we are trying to do is providing them a service they haven't demanded or didn't know they will get, what we need to think about is their expectation, their fear, and take those onboard when we design the policy." (ntp manager 13) when it comes to acf policy implementation, all ntp managers declared that the individual level context would influence it to at least some degree. they elaborated on the importance of willingness and trust from the side of individuals participating in acf, and their knowledge and awareness of tb. types of evidence and frequency of their use for acf policymaking. the ntp managers were asked about their use of who guidelines, international scientific evidence, national scientific evidence, expert knowledge and personal experience. the findings are summarized in fig 5, showcasing that all different types of evidence are used in making acf policies, many of them frequently. all ntp managers used who guidelines. they described using them as a reference document, but also underscored the need for contextualization. conversely, international scientific evidence was utilized less; 21% (n = 5/23) rarely or never used it. the ntp managers described applying international scientific evidence when local data and evidence were limited, to learn from other countries experiences or when in doubt about who guidelines. "if we have an argument about what who says, then we look for more evidence." (ntp manager 2) national scientific evidence was also frequently used; 8% (n = 2) of the ntp managers stated they rarely or never utilized it. they stressed the importance of national scientific evidence (e.g. publications in national journals), especially for implementation. we investigated if upper middle-income countries used national evidence more compared to other countries, given that many have a better national research infrastructure, but replies were mixed. finally, most ntp managers frequently used expert knowledge and personal experience in acf policymaking; one ntp manager (4%) asserted not relying on those types of evidence. according to the qualitative data, ntp managers often drew on expert knowledge through institutionalized processes, e.g. regularly convening working groups and committees. experts could be from a neighboring country with a similar context or from international organizations. nsps furthermore described the use of data in nsp development, including from burden of disease analyses, prevalence and drug resistance surveys, and surveys on knowledge, attitudes and practice in acf policymaking. besides, the nsps affirmed the use of country assessments, external program reviews, monitoring reports and lessons learnt for nsp development. stakeholders influencing acf policy development and implementation. the 'top three' stakeholders perceived to be influencing acf policy development were international organizations, policymakers in the national government and managers in a district or region. in terms of acf policy implementation, the 'top three' stakeholders reported to influence implementation were managers in a healthcare institution (e.g. a hospital), managers in nongovernmental organizations and, again, managers in a district or region. many ntp managers pointed out policymakers in the national government as most powerful stakeholders in acf policy development and policymakers at sub-national level for implementation. "i think the sub-national level, especially the implementers, [are the most powerful] since they have the experience on how to do the activity and we can incorporate the lessons learned based from their experience in the policy to be formulated." (ntp manager 23) moreover, the document review underlined implementing partners such as community health workers, volunteers, private providers, pharmacies, religious leaders and setting-specific implementing partners, e.g. prison health workers and wardens. considerations for scaling up acf. all ntp managers articulated that acf should be scaled up in their countries (100%, n = 22). according to them, scaling up acf would necessitate political commitment, appropriate diagnostics, data and evidence, community engagement, knowledge and awareness about tb, and human and financial resources. ninety-one percent (n = 20/22) of the ntp managers proclaimed that financial resources for acf were insufficient. nineteen of 20 countries (95%) received support for tb from the global fund to fight aids, tuberculosis and malaria. when asked about strategies for increasing financial resources, the ntp managers underlined the generation of evidence to show impact, advocacy for domestic funding, diversification of funding sources, and the development and/or exploration of new funding mechanisms. eighty-nine percent (n = 17/19) of the ntp managers furthermore said that human resources for acf were inadequate. strategies for fighting human resource constraints included advocacy for more (and domestically funded) human resources and human resource training. this study provided insights into acf policy development, implementation and scale-up in high tb burden countries, based on a mixed-methods survey with ntp managers and a document review of nsps. ntp managers considered the national governments the most powerful stakeholder for acf policy development, while sub-national actors were perceived most powerful for acf policy implementation. the only 'top' stakeholders named for both acf policy development and implementation were managers in districts and regions. different types of evidence were used for developing and implementing acf policies, while there was a particular demand for local evidence to inform local decisions. many potential benefits of acf were highlighted and the need for scale-up was unanimously agreed upon. the nsps reflected the ntp managers' support of acf, but not all included explicit aims, targets and target groups related to acf. the ntp managers acknowledged the risk that acf may cause increased health system costs in the short-term, but also recognized the possibility of decreasing costs in the long-term. about 90% of the ntp managers declared that financial and human resources were currently lacking, while they also mentioned strategies to overcome resource constraints. many of the described factors influencing acf policy implementation align with the available evidence in the field. firstly, acf policy implementation has been widely studied, as documented by a scoping review [17] and more recent studies [24, 25] . in line with our study, a major barrier for acf implementation is the lack of human and financial resources [26] [27] [28] [29] [30] . the lack of resources is also reflected in the world tb report 2019 [6] , which showed that only five of the high tb burden countries had a national tb budget consisting of more than 50% domestic resources. another five countries had tb budgets largely based on international funding, while the budget of 11 countries remained mostly unfunded [6] . secondly, the perceived benefits and risks of acf have been documented in the literature [12, [31] [32] [33] [34] [35] , while this study provides unique insights into ntp managers' value judgements. the fact that all ntp managers agreed on the need for scaling up acf seems important given that their leadership and commitment for acf is considered paramount for acf policy [11] . the ntp managers commitment to scaling up acf may be particularly important when tb patients' access to care is impeded, such as during the covid-19 pandemic. this study provides new insights regarding key stakeholders and evidence use in acf policy development and implementation. firstly, the only 'top' stakeholder named in both acf policy development and implementation were managers in districts and regions. their involvement in acf policy processes may increase ownership and relevance of policies [36] , and thus increase the likelihood for implementation. secondly, besides describing the types of evidence and frequency of evidence use, this study showed that most ntp managers had research experience and many countries seemed to have institutionalized processes for acf policy development in place, such as regularly convening working groups and committees. both are indicators conducive to evidence-informed policymaking [37, 38] . ntp managers mentioned strategies to overcome resource constraints for acf, which appear necessary to implement and scale up acf. strategies include a) the diversification of funding sources and b) the generation of local evidence to inform resource allocation for acf. the importance to invest in local evidence has been highlighted [39] , while acf-related projects offer opportunities for generating context-specific evidence [11] . meanwhile, local research infrastructure and research capacity may be limited, especially in low-and lower middle-income countries [40, 41] . in terms of diversified funding, our study showed that some countries already have a diverse funding base, including the government, national health insurances, the private sector, etc. even out-of-pocket payments were explained as a funding source for acf implementation, while acf should rather be seen as a strategy to decrease patient cost [42] [43] [44] . demonstrating the effectiveness of acf investments in relation to other underfunded interventions is important to limit the opportunity cost of scale-up. adding to the strategies that ntp managers described, acf could be anchored more strongly in the countries' nsps, e.g. with concrete targets. implementation, policy and programmatic research, as well as cost-effectiveness studies on human and financial resource strategies for acf may be useful to explore the "how to" questions which this survey started to assess. in line with who guidance concerning the monitoring and evaluation for acf, operational research that builds on available local data may help further build the local evidence base to inform local decision-making around acf. similar types of surveys and mixed-methods studies targeting sub-national stakeholders may provide relevant local evidence for acf policy development, implementation and scale-up, complementing the findings of this study. for this study, we developed a new questionnaire which allowed us to quantify and qualify the attitudes of ntp managers. we backed up our findings by including information from nsps. the survey had a high participation rate of 77%. though innovative, the questionnaire had not been validated and included likert scales with limited reliability based on cronbach's alpha [20] . we aimed to increase the reliability of the ntp managers' responses by implementing the survey as structured interviews. despite the geographic diversity, we were able to conduct some interviews in person. those interviews were generally longer, and the quality of the responses may be higher compared to those interviews conducted via phone/skype. however, the validation process (i.e. sharing the filled-in surveys with the participants) helped us to ensure the quality and depth of the data from all interviews. the survey was conducted in english which was not the native language for many of the participants. this may have led to poorer quality responses. however, english is the working language for most ntp managers, especially in international collaboration that all of them participate in. in general, survey questionnaires are prone to various types of biases [18] . we attempted to minimize biases by involving two independent researchers from the evaluation unit at karolinska institutet to critically assess our survey questionnaire. yet, item social desirability may still have occurred, i.e. in terms of questions being written in such a way as to reflect more socially desirable attitudes [45] . likewise, mood state bias [45] may be prevalent, i.e. the predisposition of ntp managers to view the topic in certain terms, e.g. generally positive. this may have been influenced by the timing of the survey interview, e.g. ntp managers who participated in the survey while at a who meeting might have been in a certain state of mind and away from their day-to-day work. it is possible that questions about the use of evidence may have been affected by this bias, as ntp managers who participated in the survey while at a who meeting may have been exposed to who guidelines more recently and thus valued them more in their responses. we tried to mitigate these biases by giving ntp managers opportunities to reflect, take breaks and elaborate on their responses. the ntp managers' unanimous agreement on the need for acf scale-up was reflected in the nsps, but not all nsps included explicit aims and targets related to acf. most ntp managers identified human and financial resource constraints as the main barriers to acf implementation and scale-up. strategies to increase resources exist but may not yet have been fully implemented, e.g. generating local evidence for advocacy. managers in districts and regions were identified as a key stakeholder whose involvement could help improve acf policy development, implementation and scale-up. global burden of disease tuberculosis collaborators. the global burden of tuberculosis: results from the global burden of disease study world health organization. the end tb strategy. global strategy and targets for tuberculosis prevention, care and control after 2015. geneva: world health organization transforming our world: the 2030 agenda for sustainable development ministerial conference on tb political declaration of the high-level meeting of the general assembly on the fight against tuberculosis geneva: world health organization world health organization. systematic screening for active tuberculosis: principles and recommendations. geneva: world health organization systematic screening for active tuberculosis: rationale, definitions and key considerations world health organization regional office for europe. screening programmes: a short guide. increase effectiveness, maximize benefits and minimize harm. copenhagen: who regional office for europe power plays plus push": a qualitative study of experts on factors influencing the development and implementation of active tuberculosis case-finding policies a double-edged sword': perceived benefits and harms of active case-finding for people with presumptive tuberculosis and communities-a qualitative study based on expert interviews use of high burden country lists for tb by who in the post-2015 era. discussion paper. geneva: world health organization what can national tb control programmes in low-and middle-income countries do to end tuberculosis by 2030? implementing the who stop tb strategy: a handbook for national tuberculosis control programmes. geneva: world health organization mixed methods in biomedical and health services research reforming the health sector in developing countries: the central role of policy analysis factors influencing active tuberculosis case-finding policy development and implementation: a scoping review a catalog of biases in questionnaires research electronic data capture (redcap)-a metadata-driven methodology and workflow process for providing translational research informatics support best practices for developing and validating scales for health, social, and behavioral research: a primer. front. public health classifying countries by income qualitative content analysis in nursing research: concepts, procedures and measures to achieve trustworthiness improving the quality of web surveys: the checklist for reporting results of internet e-surveys (cherries) enablers and challenges in the implementation of active case findings in a selected district of karnataka, south india: a qualitative study active case finding for tuberculosis through touch agents in selected high tb burden wards of kolkata, india: a mixed methods study on outcomes and implementation challenges community-based active tuberculosis case finding in poor urban settlements of phnom penh, cambodia: a feasible and effective strategy active case finding of tuberculosis: historical perspective and future prospects improving tuberculosis case detection in underdeveloped multi-ethnic regions with high disease burden: a case study of integrated control program in china challenges from tuberculosis diagnosis to care in communitybased active case finding among the urban poor in cambodia: a mixed-methods study tuberculosis case finding in remote mountainous areas-are microscopy camps of any value? experience from nepal the benefits to communities and individuals of screening for active tuberculosis disease: a systematic review programmatic approaches to screening for active tuberculosis benefits of community-based tb screening vs. passive case finding patient characteristics, health seeking and delays among new sputum smear positive tb patients identified through active case finding when compared to passive case finding in india community-wide screening for tuberculosis in a high-prevalence setting research, evidence and policymaking: the perspectives of policy actors on improving uptake of evidence in health policy development and implementation in uganda creating a knowledge translation platform: nine lessons from the zambia forum for health research capturing lessons learned from evidence-to-policy initiatives through structured reflection ending tuberculosis by 2030-pipe dream or reality? assessing capacity for health policy and systems research in low-and middle-income countries world health organization. world report on knowledge for better health: strengthening health systems. geneva: world health organization the role of active case finding in reducing patient incurred catastrophic costs for tuberculosis in nepal mitigating financial burden of tuberculosis through active case finding targeting household and neighbourhood contacts in cambodia active case finding among marginalized and vulnerable populations reduces catastrophic costs due to tuberculosis diagnosis common method biases in behavioral research: a critical review of the literature and recommended remedies first and foremost, we wish to thank the ntp managers and ntp staff who participated in the survey. we would also like to acknowledge the contributions of terese stenfors and per j palmgren from karolinska institutet in developing the survey questionnaire; of noemia teixeira de siqueira-filha from the liverpool school of tropical medicine in extracting information in portuguese language for the document review; of the stop tb partnership and who for their support in identifying nsps for the document review; and of who for inviting ob to join their meetings to conduct survey interviews. key: cord-350443-ca5avyjf authors: zhang, lei; wilson, david p. title: trends in notifiable infectious diseases in china: implications for surveillance and population health policy date: 2012-02-16 journal: plos one doi: 10.1371/journal.pone.0031076 sha: doc_id: 350443 cord_uid: ca5avyjf this study aimed to analyse trends in notifiable infectious diseases in china, in their historical context. both english and chinese literature was searched and diseases were categorised according to the type of disease or transmission route. temporal trends of morbidity and mortality rates were calculated for eight major infectious diseases types. strong government commitment to public health responses and improvements in quality of life has led to the eradication or containment of a wide range of infectious diseases in china. the overall infectious diseases burden experienced a dramatic drop during 1975–1995, but since then, it reverted and maintained a gradual upward trend to date. most notifiable diseases are contained at a low endemic level; however, local small-scale outbreaks remain common. tuberculosis, as a bacterial infection, has re-emerged since the 1990s and has become prevalent in the country. sexually transmitted infections are in a rapid, exponential growth phase, spreading from core groups to the general population. together human immunodeficiency virus (hiv), they account for 39% of all death cases due to infectious diseases in china in 2008. zoonotic infections, such as severe acute respiratory syndrome (sars), rabies and influenza, pose constant threats to chinese residents and remain the most deadly disease type among the infected individuals. therefore, second-generation surveillance of behavioural risks or vectors associated with pathogen transmission should be scaled up. it is necessary to implement public health interventions that target hiv and relevant coinfections, address transmission associated with highly mobile populations, and reduce the risk of cross-species transmission of zoonotic pathogens. china has experienced a large decline in the spread and burden of infectious diseases since the early 1960s, associated with effective and large-scale public health interventions and large populationbased vaccination programmes. china successfully eliminated 11 infectious diseases -including smallpox from the general chinese population in early 1960s [1] , 19 years before its global eradication -and another 10 infectious diseases, including poliomyelitis, have more recently been eliminated [2] . a further 13 diseases, including measles, are thought to be contained well at low endemic levels [3] . surveillance systems for infectious diseases in china are mainly hospital based. the latest available statistics (from 2006) indicate that china has 18,703 county hospitals, 40,907 township hospitals and 201,562 medical clinics [4, 5] . hospitals at the prefecture level or above are usually equipped with reference laboratories that are capable of carrying out molecular surveillance for cases. together with laboratories from academic institutions, they form the front line of surveillance for outbreak detection and notification of infectious diseases in china. all hospitals and clinics are obliged to report both suspected and confirmed cases of notifiable infectious disease to their nominated county centre for disease control (cdc). after recording the details of the reported cases (including their geographical location, demographicl information and infection status), county cdcs send the information to the country's central cdc, through the national infectious diseases monitoring information system database, which was established in 2004 [6] . this reporting mechanism bypasses the previous stepwise hierarchical reporting framework, thus allowing information to flow directly from grassroots cdcs to china's central disease database. however, regional, provincial and national cdcs do not have equal access to all of the data in the database: their access rights are limited to their own administrative regions, and only national cdcs has the full access to all data [7] . in addition to their responsibility for verifying the disease information from their administrative regions, municipal and provincial cdcs are required to report to the appropriate level of the bureaus of health or department of health and to form networks with local research bodies, universities and other health organisations. at the top of the hierarchy, china's central cdc is the overseeing organisation with responsibility for assembling and analysing diagnostic data for all diseases and then presenting a final report to the ministry of health, which is the main public health policymaker in china. the central cdc is also the only legitimate office for disseminating infectious disease information to the public, but provincial and municipal cdcs are also authorised to publicise the information to people in their jurisdiction, under the supervision of the ministry of health [8] . in contrast to disease surveillance systems in europe, the chinese surveillance system uses a multilayer administrative mechanism that enables rapid and efficient upward flow of epidemic information. china is a populous country of 1.3 billion people [9] . over the last 30 years of economic reform in the country, there have been environmental, demographic, social and behavioural changes in the population. as china's ties with the rest of world become increasingly strong, infectious diseases in china no longer remain a domestic issue. a thorough review of infectious disease surveillance in china is timely and important for the country's disease prevention and control strategies. currently, 39 infectious diseases are classified as notifiable in china. this study reviews trends in notifiable infectious diseases in china, in their historical context, discusses the current epidemiological state of these infections and their implications for disease surveillance and public health interventions. the review is structured by category of disease or transmission route. currently, 39 infectious diseases are notifiable in china, classified as a, b or c according to their epidemic levels and potential population threats. groups a and b (total 28 diseases) represent categories of diseases with high risk of outbreaks or that are likely to result in rapid spread once an outbreak occurs. mortality and morbidity related to group a and b diseases are reported and published by the chinese ministry of health on a monthly basis. group c diseases are less infectious and, when outbreaks occur, are epidemiologically less severe. they are required to be reported only when outbreaks occur. in this review, we searched published peer-reviewed research articles as well as online reports and grey literature from 1985 to 2010 relevant to disease surveillance in china in the following databases: pubmed, chinese scientific journals fulltext database (cqvip), china national knowledge infrastructure (cnki) and wanfang data. keywords used in the database search included ['chinese' or 'china'] and ['infectious diseases surveillance' or 'surveillance system' or 'infectious diseases monitoring' or 'infectious diseases information' or the type of infectious disease or the name of individual infectious diseases]. we also searched governmental reports, reports of non-governmental organisations and other grey literature from online sources. we then collated data on notified cases and mortality related to these diseases, using the latest available information from the ministry of health [10] . notifiable infectious disease data, including morbidity and mortality rates, was summarised by chinese ministry of health and published annually in chinese health yearbook and online accessible through cnki database. notably, this dataset does not contain data on sars and influenza a(h5n1) virus infection. hence we did not include them in our statistical analysis, but describe them in the text. the selected diseases were categorised into eight major types according to their diseases characteristics and origins. the morbidity and mortality for each disease type were calculated as the sum of the corresponding rates of individual diseases. the total number of diagnosed and death cases were estimated by multiplying morbidity and mortality rates by the overall chinese population in the study years. case fatality rate was defined as the percentage of persons diagnosed with the disease who die as a result of the illness during the calendar year, and was estimated by dividing mortality rate by morbidity rate of the diseases. further, for each individual disease, we calculate the disease-specific mortality rates among the chinese population in five-year intervals (1999-2003 and 2004-2008) . for each disease, the mean annual increase or decrease during 1999-2008, with 95% confidence intervals, is estimated, based on linear regression of logarithmic values of the number of annual death cases. trends in infectious diseases in china, 1999 china, -2008 morbidity of notifiable infectious diseases in china, represented by estimated numbers of new cases, declined substantially (.90%) from 22,000 cases per million in 1975 to a nadir level of 1,800 cases per million in 1995 (figure 1a ). since then, the rate of new infectious disease cases gradually reverted and maintained an upward trend. in 2008, the estimated rate of infectious diseases among the general chinese population reached over 3000 cases per million population. the composition of diagnosed diseases cases also changed substantially, in 1975, the three most reported diseases were gastrointestinal diseases (41.9%), vector-borne diseases (30.8%) and vaccine-preventable diseases (21.1%), corresponding to a total of 93.8% of all diagnosed cases ( figure 1a ). additionally, these three types of diseases account for 35.5, 21.7 and 18.4 million reported cases respectively during the period 1975-1979 (table 1 ). in contrast, in 1995, although gastrointestinal diseases remained the dominating disease type (41.6%), the proportion of all cases that were due to viral hepatitis quickly rose to 35.7%. sexually transmitted diseases re-emerged, and together with hiv, consisted of a substantial 6.3% of all reported cases. in 2008, the three most frequently reported disease types included viral hepatitis (38.3%), bacterial infections (33.3%) and stis and hiv (9.8%), which account for 5.4, 4.8 and 1.4 million diagnosed cases respectively during the period 2005-2008 (table 1) . rapid declines in infectious diseases mortality and its similar saddle pattern were also observed in the past 35 years. the overall mortality rate in china decreased from 66 cases per million in 1975 to 5 cases per million in 1995, then it gradually reverted to 10 cases per million in 2008 ( figure 1b ). vaccine-preventable diseases, bacterial infections and gastrointestinal diseases were the greatest causes of death, accounting for 30.0%, 24.0% and 19.5% of reported infectious diseases death cases among chinese population in 1975 ( figure 1b) . however, the rank of composition shifted to zoonoses (22%), viral hepatitis (17.3%) and quarantinable diseases (15.3%) in 1995. since then, the proportion of deaths caused by stis and hiv, and bacterial infections rapidly increased. by 2008, stis and hiv (39.5%) has become the mostly deadly infectious disease, followed by bacterial infections (24.7%) and zoonoses (20.0%). during the period 2005-2008, these three types of diseases have led to approximately 12,500, 13,300 and 11,400 deaths in china, respectively (table 1) . case fatality rate measures the percentage of deaths among people who contracted a disease. during 1975-2008, the disease type with the highest fatality rate is zoonoses, consistently causing 5-15% of deaths among the infected population. following that, quarantinable diseases killed 1.4-5.6% of its infected population during the same period. in comparison, fatality rates in vaccinepreventable (0. (table 1) . plague, cholera and epidemic haemorrhagic fever (which is caused by hantaviruses and mainly transmitted by rodents) have had diverse impacts on the chinese population at different stages of recent history. during 1944 to 1949, there were 179 notified outbreaks of plague across china, which resulted in an estimated 2.4 million deaths. from 1950 to 1954, the mean number of annual notifications of plague infection was 1,373 cases per year [11, 12] . this was then substantially reduced to 20 cases per year over the following 30 to 40 years. since 1990, the number of annual reported plague cases has increased to approximately 52, which is equivalent to 0.004 per 100,000 population per year [11, 12] . there is no observed trend in the table 1 . estimated number of diagnosed cases, death cases and case fatality rate of eight major infectious disease types in china in 5-year intervals. period 1975 period -1979 period 1980 period -1984 period 1985 period -1989 period 1990 period -1994 period 1995 period -1999 period 2000 period -2004 period 2005 period -2008 quarantinable diseases mean annual mortality due to plague during 1999-2008 (table 2) . cholera has caused two major outbreaks in china in recent decades: the first started in 1973, leading to a large peak in the number of cases in 1980, with an annual notification rate of four cases per 100,000 population ( figure 2a ). the rate then gradually subsided but there was a second major outbreak in the early 1990s. during 2004 to 2008, cholera cases were reported at relatively low incidence levels: the disease is well controlled, with a mean annual decline in mortality due to the disease of 48% (95% ci, 12-70%, table 2 ). despite a 13-fold decrease in the epidemic haemorrhagic fever notification rate since 1987 and continuous decline in the diseasespecific mortality rate, china is still the country most severely affected by this disease, with 90% of the global cases of haemorrhagic fever with renal syndrome (hfrs) occurring in china [13, 14] . hfrs remains an important public health issue in china, as an estimated 20,000-50,000 new cases are reported each year [15] . it is important to establish effective strategies to reduce the incidence of hfrs. the mean annual decline in mortality due to this disease is 12% (95% ci, 3-20%, table 2 ). persistent public health campaigns have been effective in reducing the number of rodents, which has been largely responsible for the decreasing trend in plague and epidemic haemorrhagic fever notifications. improved hygiene and sanitary conditions have also led to the decline in incidence and mortality due to cholera. overall, these quarantinable diseases persist at low endemic levels, but still pose potential threats. in the early 1960s, a nationwide vaccination programme was implemented to eradicate smallpox, measles, poliomyelitis, tuberculosis (tb), pertussis, diphtheria and tetanus. measles vaccine (one-dose schedule) was introduced in the country in 1965, and from 1978 it has been administered routinely to all infants through the country's expanded programme on immunisation [16] . measles mortality and morbidity has declined continuously and substantially since 1978. during 1995 to 2007, the incidence was less than one per 100,000 population (figure 2b), with fewer than 250 deaths due to measles reported annually [17] . however, limited small-scale outbreaks continue to occur among susceptible children in rural areas with low routine immunisation coverage and among susceptible children of 'floating immigrants' (the relatively large population of internal chinese immigrants who leave their rural hometown and move to urban areas to seek better employment opportunities) [18] . in addition, although a two-dose measles vaccination schedule has been recommended in china since 1986, the second dose is widely perceived as a nonmandatory booster. coverage of the second dose is not reported, but is thought to be low [19] . a global effort to eradicate polio began in 1988 [20] ), wild-type poliovirus was eradicated in china in 1994 [21, 22] ; extensive surveillance for acute flaccid paralysis is thought to have strongly contributed to polio eradication in the country [23] . vaccination against diphtheria, pertussis and tetanus was also made mandatory for all infants under the country's expanded immunisation programme since 1960s. in 2006, vaccination coverage of the vaccine against the three diseases reached 99.0%, comparable with polio vaccination (99.0%) and measles vaccination (98.6%) [24] . since 2006, the total annual incidence of reported cases of diphtheria, pertussis and tetanus decreased to below 0.5 cases per 100,000 population ( figure 2b ). widespread vaccination against these diseases is one of the important contributors to improvements in child health in china: infant mortality rate dropped from 20% in 1950 to 1.7% in 2006 [24] . mandatory vaccination of children throughout the past decades has been demonstrated to be successful in containing the spread of these diseases in china. it is estimated that polio vaccination alone has saved a total of 1,128,000 children in china from disabling disease and suffering [24] . mortality due to pertussis and tetanus experienced significant annual decays at rates of 44% (95% ci, 23-59%) and 12% (95 ci%, 30-65%) ( table 2) . trends in tb are discussed in the section on bacterial infections. the number of reported cases of bacillary and amoebic dysentery has declined rapidly since 1975 in china (figure 2c ). an initial large decline occurred in the mid-1970s, largely due to substantial improvements in sanitary conditions and quality of drinking water. however, approximately 84 million diarrhoeal episodes were still reported in china each year at the end of the 1990s: 25% of the episodes were in children under five years of age [25] . shigella bacilli and entamoeba histolytica have been found to be the main aetiological organisms [25] . a live oral shigella vaccine was then developed in 1997 in china, which provided 60-70% protection against s. flexneri serotype 2a and s. sonnei infection [26] . between 1999 and 2008, reported deaths due to dysentery decreased by a mean of 10% (95 ci%, 1-18%) per year ( table 2 ). the number of notified cases of typhoid and paratyphoid fevers is very low in china, with an annual rate of 1.2 notifications per 100,000 population in 2008 (figure 2c ). the number of reported deaths due to these infections has decreased at a mean rate of 22% (95% ci, 2-39%) per year (table 2) . however, these infections remain endemic in some rural areas of southern china, where there is a lack of adequate sewage disposal and safe water supplies [27] . a large, localised outbreak occurred during 1996 to 1998 in xing-an county in the southern province of guangxi, which resulted in an annual incidence rate of 39 to 103 cases per 100,000 population [28] . subsequently 61,030 doses of vi polysaccharide vaccine were administered in 1999 and the epidemic quickly subsided. no further typhoid fever outbreaks were reported in china. national public health campaigns since 1949 in china effectively improved hygiene and sanitary conditions, which substantially reduced most of vector-borne infectious diseases. in 1955, a total of 5,970,000 malaria cases were reported nationally, accounting for 68% of the total number of reported infectious disease cases and an incidence of 1,028 per 100,000 population. malaria disproportionately affected some regions, with incidence rates as high as 10,360 per 100,000 population in hainan province [29] . in 1960 and 1970, there were major outbreaks of the disease, leading to a national incidence of 1,554 and 2,961, respectively, per 100,000 population [29] . it took considerable time for the incidence to fall: the number of cases did not decrease to 1955 levels until 1982, but it has since continued to decrease. by 1998, the rate of notifications decreased to 31.3 per 100,000 population, corresponding to more than a 99% reduction and accounting for only 1.3% of the total number of reported cases of notifiable conditions [29] . data for 2004 to 2008 indicate that the mean annual mortality rate due to malaria was reduced to 0.004 per 100,000 population (table 2) , corresponding to only 52 reported deaths per year countrywide. malaria no longer represents a major threat to the chinese population. the ongoing governmentmobilised mass movement for vector control is probably an effective method for eliminating mosquitoes and their habitats and consequently reducing the disease burden due to malaria in the country. the universal healthcare system provided an effective base for the implementation of countrywide interventions [6] . as a result of the national public health campaigns to eliminate infectious disease vectors, forest encephalitis (also named russian spring-summer encephalitis), tick-borne relapsing fever, tsutsuga-mushi disease (also known as scrub typhus) and epidemic typhus were reduced to very low levels by the late 1980s and were then no longer notifiable. the number of visceral leishmaniasis (also known as kala-azar), japanese encephalitis, malaria and dengue fever epidemics has also decreased, with few cases per year (figure 2d) . however, there are signs that climate change may have led to local outbreaks of some these vector-borne diseases in the past decade in china [30] . rabies is one of the zoonotic diseases on the rise in china. during a 58-year period between 1950 and 2007, a total of 117,530 human rabies cases were reported, corresponding to an incidence rate of 9.1 cases per 100,000 population. three major epidemics were observed [31, 32] : the first occurred in the mid-1950s when the annual number of reported cases reached a peak of about 2,000. the epidemic subsided in the 1960s and then started to surge again in the early 1970s. after reaching a sustained peak from 1982 until the end of the decade, at a level of 5,000 to 6,000 cases per year [33] , in 1996 the number of cases was at its lowest, with 159 reported cases [33] . as the disease in humans is closely associated with transmission of rabies virus by infected dogs, in the past epidemics, strict policies for vaccination of owned dogs and elimination of stray dogs were put in place, resulting in effective control of the epidemic. however, since 2000, due to a new wave of rapid expansion of the pet dog population in urban china, there has been a third outbreak, in which the number of cases has increased rapidly: in 2007, the number of notified cases climbed to 3,250 [34, 35] . the rabies-related mortality rate increased during 1999 to 2008 by a mean of 26% (95% ci, 14-37%) per year ( table 2 ). this should serve as a call for action to prevent the further spread of rabies in china. severe acute respiratory syndrome (sars), caused by a coronavirus that was transmitted to humans through close contact with civet cats, spread from guangdong, china, quickly leading to a worldwide epidemic in 2003. china reported 5,327 (66%) of the 8,071 sars cases globally and 349 sars-related deaths during the epidemic, which lasted for approximately a year [36] . the first human case of highly pathogenic avian influenza a (h5n1) virus infection was reported in hong kong in 1997. by 2009, mainland china had 88 avian influenza outbreaks among birds in 23 provinces and a total of 38 human cases and 25 deaths had been reported [37] . in 2010, the epidemic had spread to 15 countries and resulted in 296 deaths, out of 500 cases [38] . in comparison, the 2009 influenza a(h1n1) pandemic caused more than 154,000 human cases and 842 deaths in china alone [39] , despite the virus being much less virulent than influenza a(h5n1) virus. china, with concentrated human activity in its fast-growing major cities that may lead to effective transmission of human-tohuman transmission of h1n1, is very vulnerable to the transmission of respiratory infectious diseases of zoonotic origin. during the mid-to-late stages of the sars outbreak and, more apparently throughout the course of the 2009 influenza pandemic, the chinese government responded swiftly with openness and transparency to control both diseases and prevent further episodes. in both cases, the epidemics caused widespread social panic. instead of the ministry of health, the state councilsrepresenting the highest administrative authority of the chinese government -assumed direct leadership in combating the epidemics. case notifications for leptospirosis, anthrax and brucellosis have remained at low levels since 2000. there was an increase in the number of notified case of brucellosis from 0.17 per 100,000 population in 2000 to 1.50 per 100,000 population in 2007, but this was probably due to improved surveillance and is believed not to reflect an increase in incidence [40] . the annual number of notified cases of leptospriosis and anthrax dropped below 0.01 per 100,000 population in 2008. the mortality rate due to brucellosis remains very low and stable, whereas the rates due to leptospriosis and anthrax have dropped significantly, by 20% and 31%, respectively, during 1999 to 2008 (table 2) . china has the second largest tb epidemic in the world (after india) [41] . in 2008, some 4,500,000 patients were living with pulmonary tb in china, corresponding to an incidence of 111 per 100,000 population, and resulting in 130,000 deaths from tb in the same year [42] . since 2005, an estimated 20,000 additional tb reactivation cases have arisen per annum in china due to human immunodeficiency (hiv) coinfection [42] . the tb epidemic has spread unevenly in china: in 2005, china reported a national tb prevalence of 0.2% [41] , whereas the southern province of guangxi, which borders vietnam, reported a much higher prevalence of 0.65% [43] . in 1991, in adopting dots -the internationally recommended tb control strategy -china launched a 10-year infectious and endemic disease control project in an effort to curb its expanding tb epidemic in 13 of its 31 provinces [44, 45] . it is estimated that the number of chinese people infected with tb between 1990 and 2000 decreased by 30% and that 300,000 tb deaths were averted. with a success rate greater than 90% [45] , dots has led to the avoidance of an estimated 1.5 million tb cases [44, 45] . however, since 2000, multidrug-resistant (mdr) tb and extensively drug-resistant (xdr) tb have emerged as a severe public health issue in the country. in 2004, china reported approximately 140,000 mdr tb cases, accounting for about one third of the estimated global burden of mdr tb [46] . in some provinces, the proportion of mdr tb among newly notified tb cases and previously treated cases was found to exceed 10% and 30%, respectively [47] . the latest study indicates that the prevalence of mdr tb among tb patients is as high as 19.4%, and 14.9% of mdr tb cases were xdr during 2007 to 2009 [48] . the mortality due to tb increased at a rate of 28% (95% ci, 14-43%) per year. hydroelectric projects that led to the formation of a large number of dams in china, as well as climate change, have substantially increased the risk of schistosomiasis occurring and its spread to non-endemic areas of the country, leading to small-scale outbreaks and increases in rates of case notifications near the affected areas [49, 50] . the incidence of schistosomiasis has increased from 0.10 cases per 100,000 population in 2004 to 0.26 per 100,000 population in 2008 (figure 2f ). since the founding of china in 1949, laws have been enacted to make commercial sex illegal. brothels were closed down and sex workers were sent to camps to be 're-educated'. special institutions for treatment of sexually transmitted infections (stis), at the time mostly syphilis, gonorrhoea and chlamydia, were established for the training of medical personnel, who were then sent to areas previously associated with large sex industries to treat stis at beginning of 1950s. during 1950s, a universal healthcare system was established that provides treatment for sti patients free of charge. designated laboratories for stis were built for diagnosis and research [51] . more importantly, population-based health education campaigns about stis were carried out, and detection and treatment of stis were portrayed as patriotic actions [52] . as a result, the number of cases of notifiable stis decreased from 10 million in 1950 to reportedly eliminated from 1964 [51, 53, 54] . however, notifiable stis have re-emerged, leading to a fastspreading epidemic in the country. in 1987, the incidence for stis was 6.64 per 100,000 population and in 1996, the rate increased to 34.6 per 100,000 population, which corresponded to 1.8 million new sti cases [52] . the annual growth rate of the number of sti cases in china exceeds 20% [52] . the rise of sti incidence is mainly due to unprotected sexual acts and other high-risk behaviours [55, 56] . since early 1980s, underground prostitution has again become rampant in major cities in china [57] . the number of chinese men, increased purchasing power of urban residents and trends away from traditional chinese family values of fidelity all led to an increased number of male clients for commercial sexual services [58] . in addition, lack of sexual health knowledge among commercial sex workers and their inability to effectively negotiate condom use during sexual acts substantially increased the risk of sti transmission [57, 59] . the previously hidden population of men who have sex men (msm) is becoming more overt due to increasing acceptance in society. high-risk sexual behaviours, including a low percentage of condom use, in this population is also contributing to the fast transmission of stis [60] . for example, in 1998, the incidence of syphilis in china was 0.17 per 100,000 population: it increased 20-fold to 4.31 cases per 100,000 population in 2002 and further to 15.88 cases per 100,000 population in 2007 [10, 51] (figure 2g ). in particular, msm, injecting drug users (idus) and female sex workers have the highest syphilis prevalence in china, of 10-20% [61, 62] , 5.4% [63] and 7-12% [64] , respectively. the first case of acquired immunodeficiency syndrome (aids) in china was reported in 1985, but a local hiv epidemic was not detected until 1989 when a cluster of infections was diagnosed among idus in yunnan province [65] . before 2000, china had fewer than 20,000 reported hiv/aids cases, but in 2007, the number rose to 200,000. the latest estimates are that 700,000 people were living with hiv/aids in china at the end of 2007, with 70,000 new infections added every year since then [66] and 20,000-30,000 annual aids-related deaths [67] . although the hiv/aids epidemic first began and predominated among idus, it rapidly spread to other population groups through both heterosexual and homosexual transmission. the latest figures demonstrate that heterosexual transmission has accounted for approximately 38-50% of new hiv infections since 2005 [68] , which may level at or exceed the percentage (30-49%) caused by transmission due to the sharing of injection equipment among idus [69, 70] . notably, hiv infection has become more prevalent among msm, as male homosexual activities account for approximately 10% of new infections [71] . aids mortality rate increased from 0.10 in 1999 to 1.01 cases per million in 2005, then to 4.58 cases per million in 2008, corresponding to an annual growth rate of 44% (95% ci, 30-58%) the rate is expected to increase sharply if the epidemic remains uncontrolled and there is no large procurement of antiretroviral drugs. cumulatively, over 9000 infants were infected through mother-to-child transmission by 2005 and it was estimated that about 500 million chinese were carriers of mycobacterium tuberculosis in 2003 [72] : coinfection of hiv and m. tuberculosis could therefore have a large epidemiological impact, in the absence of appropriate public health interventions. viral hepatitis is prevalent in china, but the prevalence differs according to the type of virus. before the 1990s, hepatitis a virus (hav) was the most prevalent. in the early 1990s, the annual number of reported hav diagnoses was more than 50 per 100,000 population. this dropped by 90% to 5 per 100,000 population in 2005 to 2006 [73] (figure 2h ). the mean annual death rate due to hav decreased at a rate of 21% (95% ci, 8-32%) during 1999 to 2008 ( table 2 ). the decrease is probably a result of mass vaccination efforts in introducing the hav vaccine (h2 strain) in the 1990s and improvement of hygiene and sanitary conditions that broke the cycle of food and water contamination [73] . according to 2008 data [74] , hav persists at low, endemic levels ( figure 2h ). in comparison, infections of hepatitis b virus (hbv), which is transmitted mainly through body fluid from mother to child, and hepatitis c virus (hcv), transmitted predominantly by intravenous injections or blood transfusion, are becoming alarmingly widespread in china. the annual number of new hbv cases increased rapidly from 20 per 100,000 population in 1990 and reached 100 per 100,000 population in 2008, despite an increasing vaccination coverage of newborns from 30% in 1992 to 93.4% in 2005 [75] . hcv infections followed a similar trend and in 2007, the annual notification rate was about 8.8 per 100,000 population. the 2007 hcv and hbv prevalence among the general population was 0.5% and 6%, respectively [74] . mortality due to hbv and hcv increased at annual rates 9% (95% ci, 1-20%) and 30% (16-46%) respectively. in contrast to hav infection, which always causes acute hepatitis but never develops into a chronic condition, hcv and hbv infections often cause chronic hepatitis and may develop into cirrhosis and hepatocellular carcinoma [76] . coinfection of hbv or hcv with other pathogens causing chronic infections, such as hiv, results in substantial increases in the disease burden on individuals, accelerates disease progression and complicates treatment [77, 78] . since hbv and hcv can both be transmitted through exchange of body fluids, coinfection of these hepatitis viruses with hiv is common in at-risk groups in china. approximately 67-71% of the 700,000 registered idus in china are infected with hcv [79, 80, 81] and 17-26% of them are infected with hbv [79, 82] . the prevalence of hiv/hcv coinfection is 6.45% in idus [80] . among blood donors, the prevalence of hiv/hcv coinfection is 5.8-6.5% [80, 83] , whereas the figure for hiv/hbv coinfection is 3.7% [84] . in comparison, coinfection of hiv and hcv among the general population remains unknown as there is currently no reports on its prevalence. however, coinfection is expected to be increasingly noticeable as the hiv epidemic transforms into a generalised epidemic in china [6] . overall, this study investigated the temporal trends of major types of notifiable infectious diseases in china. our analysis indicated that while the morbidity and mortality of most infectious diseases reduced substantially during 1975-1995, there is an increasing trend of re-emergence of previously prevalent diseases and emergence of new infectious diseases since 1995, in particular, stis and hiv, viral hepatitis and zoonoses diseases. these diseases accounted for the majority of deaths associated with infectious diseases in china since 2005. consistent with this, analysis of case fatality rates indicated a higher percentage of deaths among zoonotically infected people (5-15%) and an elevated proportion (0.894%) among stis and hiv-infected individuals. our study has important implications for the surveillance and control of infectious diseases in china. first, the persistent small outbreaks of particular infectious diseases across the country indicate that any decrease in prevention efforts will probably trigger re-emergence of the diseases. surveillance activities are therefore essential to inform and prioritise public health responses. although efficient and well-developed disease surveillance systems have been implemented in many urban areas, hygiene conditions, health services and monitoring of patterns of spread and disease burden are largely lacking in rural china [6] . second, the rapid rise in the number of notified cases of stis, especially hiv infection, and viral hepatitis in china is associated with growth of the sex industry, increasingly frequent risky sexual behaviours and an increasing number of sexual partners in the general chinese population. the newly implemented internetbased national infectious diseases monitoring information system database not only provides a platform for integrating epidemiological data on hiv infection and other stis, but also initiated second-generation behavioural surveillance of the at-risk populations [70] . our analysis indicates that both epidemiological and behavioural surveillance of hiv infection and other stis is essential to understand and forecast the trends in these epidemics. extending surveillance efforts into population groups that were previously considered to be less at risk may further improve the quality and reliability of surveillance data. third, coinfections, especially those involving hiv, are likely to become major public health issues in the future. we demonstrate that tb/hiv, hbv/hiv and hcv/hiv coinfections have become increasingly prevalent in china and the trend is likely to continue in the absence of strong public health interventions. thus, inclusion of testing for tb and viral hepatitis coinfections as part of hiv voluntary counselling and testing could be useful among at-risk populations. scaling up of free-of-charge antiretroviral therapy should also include treatment for these coinfections. fourth, surveillance of zoonotic infections becomes increasingly important due to the interactions between humans and animals in china. china has established an efficient surveillance system for human infectious diseases [6, 85] : a parallel national centralised system for surveillance of disease in animals may be useful for reducing the risk of animal-to-human transmission. one of the potential limitations in our investigation may be poor data quality in the passive notification-based surveillance system. it is widely perceived that underreporting exists in the chinese infectious diseases surveillance system, but little is known about the extent of underreporting as systematic evaluations have never been conducted [6] . this issue requires further consideration in the future. an overview of prevalence studies would be useful for comparison with numbers of notified cases in order to estimate numbers of undiagnosed cases. a comprehensive evaluation would also involve assessments of the efficiency and effectiveness of the surveillance system, both quantitative and qualitative. understanding changes in testing rates in various population groups will also be useful for interpreting trends in notification data. but such investigations are beyond the scope of this review. infectious disease surveillance and interventions in china face several major challenges. the country's major economic reform in 1979 has created a large economic disparity between rural and urban areas, resulting in a large population of floating immigrants (also known as 'peasant workers'). this accounts for 10% of the total chinese population in 2005 [86] . the vast majority of these immigrants are from rural environments but work in urban areas, away from their families, and hence regularly travel between the two sites. in addition to the natural population growth of major cities, the inflow of internal immigrants substantially increases the population size and density in urban areas. these immigrants often live in overcrowded residential areas with compromised hygiene conditions. female immigrants often become targets of those seeking to recruit them for commercial sex work [57, 87] . these floating immigrants are often more disadvantageous than the local residents. they tend to be less educated compared with others in urban areas who have permanent resident status and less likely to seek medical treatment when sick due to financial constraints or lack of health insurance [6] . not surprisingly, the population of floating immigrants has been found to be more vulnerable to infectious diseases, especially stis, hiv and viral hepatitis [6] . additionally, their high-risk behaviours and mobility promotes transmission of infectious disease agents and creates a major challenge for disease detection and control. given the characteristics, behaviours and the increasing population size of floating immigrants, it becomes apparent that the capacity of current chinese healthcare system cannot meet the needs of both the surveillance activities and health problems of these migrant workers. the infectious disease surveillance system needs to be tailored for its high mobility, and a dramatic expansion of the private health sector is required to address the health needs of this population. transmission of zoonotic infectious agents relies on close interaction between infected animals and humans. as a result of the increasing social trend of keeping house pet dogs, it is estimated that the number of pet dogs in china reached 80-200 million in 2004 [88] . however, only 30% of dog owners register their animals with government authorities and only 2-8% of dogs are vaccinated against rabies [31, 89] -coverage of greater than 70% is needed to sufficiently control rabies, as determined by the world health organization (who) [90] . the high rabies prevalence (6.4%) among dogs and the expanding number of pet dogs pose a continual and increasing threat of transmission of rabies virus from dogs to humans in china [89] . in addition, the consumption of game meat, especially in southern china, is one of the important sources of zoonotic diseases [91] . markets in guangdong provinces sell live poultry, fish, reptiles and mammals, including dogs and civet cats, for food [92] : the housing of these live animals, often in packed conditions, together with human activities, makes such markets the ideal hub for cross-species transmission of zoonotic agents [91] . since there is no systematic surveillance of these live markets, it is difficult to estimate the number of such markets and the amount of game meat consumed by residents in southern china. the sars epidemic in 2003 demonstrates that transmission of pathogens originally associated with animals can cause worldwide outbreaks in human populations. the current absence of both epidemiological and behavioural data, including their frequency of contacts and means of animal handling, on animal infectious diseases is a strong barrier to effective surveillance and control. environmental changes facilitate the emergence and transmission of 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infectious diseases in floating population: prevalent features and control migration and health in china: problems, obstacles and solutions. singapore: asian metacentre for population and substainable development analysis pivotal role of dogs in rabies transmission inferior rabies vaccine quality and low immunization coverage in dogs (canis familiaris) in china world health organization (1996) world health organization expert consultation on rabies the role of the legal and illegal trade of live birds and avian products in the spread of avian influenza severe acute respiratory syndrome analysis of factors affecting the epidemiology of tuberculosis in china key: cord-337585-kpghvb6u authors: moustaqim-barrette, amina; papamihali, kristi; mamdani, zahra; williams, sierra; buxton, jane a. title: accessing take-home naloxone in british columbia and the role of community pharmacies: results from the analysis of administrative data date: 2020-09-11 journal: plos one doi: 10.1371/journal.pone.0238618 sha: doc_id: 337585 cord_uid: kpghvb6u introduction: british columbia’s (bc) take-home naloxone (thn) program provides naloxone to bystanders for use in cases of suspected opioid overdose. this study seeks to provide trends and analysis from the provincial bc thn program since inception in 2012 to the end of 2018. materials and methods: bc thn shipment and distribution records from 2012–2018 were retrieved. frequency distributions were used to describe characteristics of individuals accessing the program. to evaluate correlates of distribution after the addition of hundreds of pharmacy distribution sites, an analytic sample was limited to records from 2018, and multivariate logistic regression was used to evaluate correlates of collecting naloxone at a pharmacy site. results: since program inception to the end of 2018, there were 398,167 naloxone kits shipped to distribution sites, 149,999 kits reported distributed, and 40,903 kits reported used to reverse an overdose in bc. there was a significant increasing trend in the number of naloxone kits used to reverse an overdose over time (p<0.01), and more than 90% of kits that were reported used were distributed to persons at risk of an overdose. individuals not personally at risk of overdose had higher odds of collecting naloxone at a pharmacy site, compared to other community sites (including harm reduction supply distribution sites, peer led organizations, drop-in centers, and supportive housing sites) (adjusted odds ratio (aor): 2.69; 95% ci: 2.50–2.90). conclusions: this study documents thousands of opioid overdose reversals facilitated through the bc thn program. while those at highest risk of overdose may preferentially access naloxone through community sites, naloxone distribution through pharmacies has allowed the bc thn program to expand dramatically, increasing naloxone availability through longer opening hours on evenings and weekends. and in rural and remote regions. a diversity of naloxone distribution sites and strategies is crucial to prevent rising opioid overdose deaths. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 college of pharmacists of bc made it available as a schedule ii drug (behind the counter) [12] . in september 2016, the college of pharmacists of bc changed the status of naloxone for emergency use to 'unscheduled', making it available outside of pharmacies [14] . furthermore, the bc thn program removed the requirement to report individual naloxone kit ids and/or patient names in october 2016 [15] . in december 2017, the bc thn program officially expanded to select community pharmacies to improve bc thn access across the province. pharmacies are available widely, including in remote and hard-to-reach communities, with longer hours (including weekends and evenings), and they may be more acceptable to clients less likely to frequent harm reduction supply distribution sites [16] . the program partnered with pharmacy wholesalers, banners and chains to onboard pharmacies through centralized distribution models initially, later reaching stand-alone pharmacies in remote areas with limited access to harm reduction supply distribution sites. by the end of 2018, there were 1,448 active take-home naloxone distribution sites across bc [17] , including 562 community pharmacy sites [18] -representing 40.5% of all pharmacies [19] . understanding correlates of take-home naloxone distribution and access is crucial for planning and targeting services aimed at curbing opioid overdoses. while analyses from the bccdc shows that the bc thn program, combined with other harm reduction interventions, has averted thousands of opioid overdose deaths [20, 21] , there is still relatively little data in the published literature examining community reach and barriers to access of naloxone distribution programs in canada. furthermore, preliminary evidence from the bc coroners service suggests a spike in drug toxicity deaths corresponding with covid-19 physical distancing measures and changes to the illicit drug supply; the coroners service reported a 130% increase in drug toxicity deaths in june 2020 compared to june 2019 [22] . as overdose deaths continue to rise and organizations critical in the training and distribution of naloxone face reduced hours and temporary closures due to covid-19 [23] , there is a need to understand barriers and access associated with different avenues of distribution. the current paper seeks to first provide an overview of uptake of the bc thn program since inception in august 2012 to the end of 2018, as well as correlates of naloxone distribution through pharmacy sites after the bc thn's pharmacy expansion in late 2017. findings may help inform optimization of take-home naloxone programs across jurisdictions. this study analysed anonymous administrative data. ethics approval was obtained from the university of british columbia behavioural ethics board (h12-02557). the bc thn program draws on two key sources of data to monitor and evaluate naloxone distribution and use across bc. firstly, 'supply order forms' were used internally at the bccdc to track the number of kits shipped, to participating distribution sites [9] . second, 'distribution records' were used by naloxone distribution sites to track characteristics of individuals who receive a naloxone kit [10] . no personal identifiers were collected, and sites were asked to return completed distribution records to the bccdc on a monthly basis. since program inception, distribution records have included site-specific information (site's unique id, city, and the date the kit was distributed to an individual) completed by on-site staff, as well as a 'site type' identifier (corrections facilities, pharmacy, post-secondary, and other (housing sites, treatment centres, non-governmental and peer-led organizations). individuals collecting a kit were asked to self-report their gender, their age range, and whether they are collecting a first or replacement kit (due to the previous kit being used, lost, stolen, expired or confiscated). following the unscheduling of naloxone and thn program expansion in 2017 to individuals not personally at risk of an overdose, distribution record forms were amended to include self-reported risk of overdose i.e. personally at risk of overdose or at risk of witnessing an overdose (i.e. friends and family of someone at risk of overdose). before this date, all kit recipients were assumed to be personally at risk of overdose. distribution records often experience a delay in return and entry due to two reasons: 1) a delay and general decrease in submission of records from distribution sites and, 2) a lag in manual entry of records at the bccdc. together, we estimate the return window to be on average between three to six months. supply order forms were geographically coded by health authority (vancouver coastal health, fraser health, island health, interior health, and northern health) and used to provide the number of naloxone kits shipped to distribution sites over time. distribution records were the primary data source used in this study. from these records, site-specific geographical information was used to classify observations by health authority (vancouver coastal health, fraser health, island health, interior health, and northern health). records were also used to classify records by age group (<19, 19-30, 31-60, >60, unknown), gender (male, female, other (trans, gender non-conforming), unknown), reason for collecting a kit (1 st kit, replacement kit-used, replacement kit-other (previous kit lost, stolen, confiscated or expired), site type (corrections facility, pharmacy, post-secondary institution, other (housing sites, treatment centres, non-governmental and peer-led organization), and 'eligibility for collecting a naloxone kit (not personally at risk of overdose, peronlly at risk of overdose, unknown). this study includes findings from distribution records and supply order forms across bc from program inception on august 31 st , 2012 to december 31 st , 2018. to evaluate the effect of the thn program's expansion into community pharmacies, a separate analytic sample was created which limits distribution records to those returned between january 1 st , 2018 and december 31 st , 2018. data for this study were retrieved and analyses conducted in january 2020. all analyses were conducted using r version 3.5.3 [24] . frequency distributions were used to describe characteristics of individuals accessing the bc thn program from 2012 to the end of 2018, as well as to compare shipment and distribution data. a chi-squared test for trend (cochran-armitage test) was used to evaluate differences in the proportion of naloxone kits used to respond to an opioid overdose over time. frequency distributions were subsequently used to examine the separate analytic sample, limited to 2018 distribution records. in this sample, all non-pharmacy sites were grouped as 'other' sites to compare with pharmacy distribution. multivariate logistic regression was used to examine factors associated with distribution site type. the model building strategy described by hosmer and lemeshow [25] , and further operationalized by zhang [26] , was employed. all available covariates were included for bivariable analysis to explore trends and examine distributions by the outcome variable. covariates were cross-tabulated with the outcome variable and those with a p-value < 0.25 and were deemed to be conceptually relevant as indicated in the literature, were included for further multivariate analysis. as per the purposeful selection model building strategy, variables which did not meet the p-value cut-off but were known to be conceptually relevant were included in further analyses. adjusted odds ratios (aor), 95% confidence intervals (ci), and p-values are reported. pvalues less than 0.05 were considered statistically significant. table 1 provide a summary of naloxone kits shipped, reported distributed, and reported used to reverse an overdose from the bc thn program, from august 31 st , 2012 to december 31 st , 2018. during this time period, a total of 398,167 kits were shipped and 149,999 naloxone kits were reported distributed, according to records returned. data show a consistent increase in kits shipped, however the number of kits reported distributed increased through 2017 but dropped in 2018 (fig 1) . a total of 41,197 kits (27.5% of those distributed) were reported to have been obtained to replace a kit used to respond to an opioid overdose. as a proportion of naloxone kits reported distributed, naloxone kits reported used to reverse an overdose increased steadily over time, from 8.7% in the pre-2015 period (168/1921), to 12 table 2 presents summary characteristics of bc thn distribution records, from program inception to the end of 2018. for all variables, missing information was categorized as 'unknown'. overall, 24,890 kits (16.6%) were distributed to replace a lost, stolen, confiscated or expired kit. the majority of thn distribution records were received in 2017 (42.2%) and 2018 (40.1%). the large majority (94.2%) of thn kits were distributed at 'other' sites (including housing sites, health and treatment centres, non-governmental and peer-led organizations). the overall kit distribution rate 2012-2018 was 31.4 kits per 1000 bc population but varied between health authorities (based on average population estimates 2012-2018). the rate of kit distribution varied between health regions, from 22.4 to 50.4 kits/1,000 population, while the proportions distributed were similar between the four most populous health regions (20.8% -26.7%), with 5.2% of kits distributed in the least populous region-see the table in s1 appendix for details. overall, most thn kits were distributed to individuals who identified as male (43.4%) compared to female (36.2%), other genders (1.2%), and unknown (19.3%), and to those between the ages of 19-30 (27.9%) or 31-60 (45.4%). eligibility criteria was introduced in 2017, when the thn program was expanded to individuals not personally at risk of overdose. all individuals who received a kit before 2017 were considered personally at risk of overdose, and account for the majority of the 'unknown' category. for the age and gender variables, a high proportion of 'unknown' is likely due to the nature of ensuring a low-barrier thn program-some information is not always reported by thn distribution sites or by recipients. table 3 presents the analytic sample characteristics after limiting naloxone distribution records to 2018, stratified by eligibility. by the end of 2018, there were 562 community pharmacy distribution sites. a breakdown of thn distributing pharmacy sites by health authority is provided in the table of s2 appendix. a total of 57,524 records were included, of which 39,208 (68.2%) self-reported being personally at risk of overdose, compared to 18,316 (31.8%) of individuals who reported not being personally at risk of overdose. of naloxone kits reported used to respond to an opioid overdose in 2018, 90.2% were kits obtained by individuals who were personally at risk of overdose. even after the introduction of pharmacy sites across bc, a large majority (93.2%) of individuals continued to access naloxone at 'other' community site types, compared to pharmacy sites (6.8%). furthermore, the majority of those who self-reported not being personally at risk of overdose accessed naloxone at a pharmacy (60.1%), while the majority of individuals personally at risk of overdose accessed naloxone at other sites (70.2%). table 4 presents the analytic sample characteristics after limiting naloxone distribution records to 2018, as well as multivariate logistic regression results (adjusted odds ratios and 95% confidence intervals) for correlates of thn kit collection at a pharmacy site, compared to other sites. in bivariate analyses, all covariates were statistically significant and conceptually relevant, and were included in the analysis. after adjustment, and consistent with descriptive statistics, individuals who were not personally at risk of overdose had more than twice the odds of collecting a thn kit at a pharmacy site, compared to other sites (aor = 2.69 [95% ci 2.50-2.90]). individuals collecting a replacement kit were less likely to collect those kits at a pharmacy site compared to first time kit the current study provides an overview of uptake of the bc thn program. while all canadian provinces and territories have publicly funded thn programs, research and evaluation of these programs in the scholarly literature is limited. a recent study from ontario describes uptake of the province's pharmacy-dispensed naloxone kits in ontario [27] , and preliminary analyses of provincial thn programs in ontario [28] , alberta [29] , manitoba [30] , and british columbia [15] have been published. data from other jurisdictions are not directly comparable, due to differences in program structure, eligibility criteria, and scope [9] . nevertheless, analyses across provincial thn programs are valuable for examining program uptake across jurisdictions. an environmental scan of provincial programs and distribution has also been conducted [9] , and our group is involved in efforts to combine aggregate jurisdictional level data nationally. the current study finds that the bc thn program expanded to a total of approximately 1,500 sites across bc by the end of december 2018, including 562 pharmacy sites (40.5% of all community pharmacies in bc) since december 2017. overall, almost 400,000 naloxone kits were shipped and 150,000 reported distributed since the program was launched in 2012. our findings show that there is a significant increasing trend in the proportion of kits reported used to reverse an overdose over time. most individuals collecting a naloxone kit selfreported being male and between the ages of 31-60. this corresponds with data from the bc coroners service, which has found that the group at highest risk of overdose death in bc are men between the ages of 40-59 years old, as well as those 19-39 years [31] . importantly, our findings also indicate that the vast majority of kits reported used to reverse an overdose were distributed to persons themselves at risk of an overdose. while it is possible that individuals collect naloxone kits for others to use on them in the event they overdose, most individuals who use drugs do so around other people who use drugs. we believe our findings suggest then that people who use drugs are most likely to respond to overdoses in their communities, though further research to help elucidate this question is warranted. after evaluating data from 2018, our analysis also finds that individuals at risk of opioid overdose were significantly less likely to collect a naloxone kit at a pharmacy site compared to community-based sites. in developing the bc thn program, the priority was to ensure a low barrier environment for individuals receiving overdose response training, collecting naloxone kits, and seeking other supports. consistent with our findings, evidence from the literature suggests that people with lived and/or living experience of substance use may be reluctant to access pharmacies and other medical services due to past experiences of stigma or discrimination [32, 33] . nevertheless, data from this study suggests that first time naloxone kit recipients may be more likely to collect kits from pharmacy sites, suggesting that these sites are important in reaching different cohort of clients who may not access traditional distribution sites aimed at higher-risk communities. the authors believe that these findings offer support for increased access to naloxone through pharmacy sites in bc, and highlights the importance of having a variety of distribution site types. a previous evaluation from our group suggests pharmacists are willing to be involved in the bc thn initiative, despite some initial concerns about lack of remuneration for training and dispensing. there are mixed views regarding further program growth; some pharmacy managers feel that naloxone is now fairly accessible and that there is no need for further expansion, while others feel that expansion to more pharmacy banners and chains would be beneficial [16] . due to the covid-19 crisis, some harm reduction services and supply distribution sites have temporarily closed or have reduced hours [23] , while pharmacies remain an essential service that continue to be widely available, open, and hence accessible to distribute thn. online training is available for those who need refresher training or to be newly trained, allowing for physical distancing during the training process. the thn kits contain non-latex gloves which responders are advised to put on before responding to an overdose and to remove carefully after responding, as well as to practice hand hygiene. with recent changes of temporary exemptions [34] and clinical opioid prescribing guidelines [35] in relation to covid-19, individuals may be receiving new opioid agonist therapy (oat) prescriptions, longer prescriptions, and/or delivery of medications by pharmacies. in many cases, pharmacists have been advised to co-dispense naloxone. future research is needed to explore changes in distribution and the role of pharmacies in providing naloxone to individuals at risk of overdose during the dual public health emergency period. while data suggests a decline in naloxone kits reported distributed compared to kits shipped in 2018, it is important to note that distribution numbers may not accurately reflect naloxone kits in public circulation. uneven response rates across sites, delays in returning distribution forms, and delays in entering data means that the true count of naloxone kits distributed to the public likely exceeds the reported kits distributed. the authors note that the observed drop in reported kit distribution in 2018 is likely due to a lower return rate, and does not represent a true decrease in naloxone kits distributed to the public. relatedly, the higher proportion of pharmacy naloxone kit distribution in the vancouver coastal region in the 2018 analytic sample is likely due to low reporting in high volume, low-capacity community sites in the vancouver region compared to other regions. operationally, the program has confirmed with high volume sites, which have low reported distribution forms, that kits ordered are being distributed and not held in inventory or being thrown away due to naloxone expiry. the large majority of sites reorder kits as needed on a weekly or monthly basis. thus, we believe that true distribution is likely much higher than the number of distribution records returned to the bccdc. as previously mentioned, careful interpretation of this data is required; the program is low barrier and to ensure anonymity identifiers are not collected from naloxone kit recipients. therefore, we cannot identify how many unique individuals are accessing or using naloxone kits, or how many kits are distributed and used per individual in bc. self-reported data is also susceptible to response bias. stigma around drug use may discourage high-risk individuals from providing information or self-identifying as being at risk of experiencing an overdose, which may in turn bias results. between 2012 and 2018, more than 40,000 take-home naloxone kits have been reported used to reverse an opioid overdose in bc. community distribution sites are crucial for providing naloxone to people at high risk of overdose, who may be most likely to respond to overdoses in their communities. pharmacy sites appear to reach a cohort of first-time kit recipients, sustain longer opening and weekend hours, and support access in rural and hard-to-reach areas. findings from this study highlight the importance of making naloxone available through a variety of distribution sites that are both widely accessible and responsive to the needs of people who require naloxone. current health topics-province of british columbia canadian broadcasting corporation (cbc). b.c. declares public health emergency with 186 cases of covid-19 and 7 deaths | cbc news world health organization overview of national data on opioid-related harms and deaths [internet]. government of canada illicit drug overdose deaths in bc bc: coroners service of bc, ministry of public safety & solicitor genera are take-home naloxone programmes effective? systematic review utilizing application of the bradford hill criteria reducing the harm of opioid overdose with the safe use of naloxone: a pharmacologic review management of opioid analgesic overdose environmental scan naloxone access and distribution in canada bc centre for disease control. training & resources [internet]. toward the heart about toward the heart timeline of community naloxone in british columbia british columbia centre for disease control an evaluation of take home naloxone program implementation in british columbian correctional facilities college of pharmacists of british columbia. naloxone [internet]. college of pharmacists of british columbia lessons learned from ramping up a canadian take home naloxone programme during a public health emergency: a mixed-methods study evaluation of british columbia's take home naloxone program in community pharmacies take home naloxone report: review of data to centre for disease control (bccdc) bc centre for disease control. find a site college of pharmacists of british columbia distribution of takehome opioid antagonist kits during a synthetic opioid epidemic in british columbia, canada: a modelling study modelling the combined impact of interventions in averting deaths during a synthetic-opioid overdose epidemic virus measures may be hurting overdose prevention in vancouver, official says the r foundation. r: the r project for statistical computing model-building strategies and methods for logistic regression model building strategy for logistic regression: purposeful selection the uptake of the pharmacydispensed naloxone kit program in ontario: a population-based study development and implementation of an opioid overdose prevention and response program in toronto, ontario. can j public health rev can sante publique alberta's provincial take-home naloxone program: a multi-sectoral and multi-jurisdictional response to overdose at-a-glance-lessons learned from launching the manitoba take-home naloxone program. apercu-lecons tirees lancement programme naloxone emporter domic manit government of british columbia perpetuating stigma or reducing risk? perspectives from naloxone consumers and pharmacists on pharmacy-based naloxone in 2 states the feasibility of pharmacy-based naloxone distribution interventions: a qualitative study with injection drug users and pharmacy staff in rhode island exemptions for practitioners and pharmacists prescribing and providing controlled substances, and for patients, during the coronavirus pandemic covid-19: information for opioid agonist treatment prescribers and pharmacists british columbia: bc centre on substance use (bccsu) the authors would like to thank the take-home naloxone (thn) team at the british columbia centre for disease control (bccdc) (sierra williams, emily ogborne-hill, laura moore, amrit parmar, olivia oldesloe, and sara young) for their tireless work on the thn program. we are equally indebted to the harm reduction coordinators and distribution sites across british columbia (bc) dedicated to getting naloxone into the hands of those who need it, and to the many communities and individuals who have received overdose response training and saved lives using naloxone. all authors would like to acknowledge that they work and live on the unceded traditional territories of the skwxwú7mesh (squamish), selílwitulh (tsleil-waututh), and xwməθkwəýəm (musqueam) nations and that the take-home naloxone program operates across the unceded traditional territories of 198 first nations. key: cord-339869-euikj8fv authors: cebey-lópez, miriam; herberg, jethro; pardo-seco, jacobo; gómez-carballa, alberto; martinón-torres, nazareth; salas, antonio; martinón-sánchez, josé maría; justicia, antonio; rivero-calle, irene; sumner, edward; fink, colin; martinón-torres, federico title: does viral co-infection influence the severity of acute respiratory infection in children? date: 2016-04-20 journal: plos one doi: 10.1371/journal.pone.0152481 sha: doc_id: 339869 cord_uid: euikj8fv background: multiple viruses are often detected in children with respiratory infection but the significance of co-infection in pathogenesis, severity and outcome is unclear. objectives: to correlate the presence of viral co-infection with clinical phenotype in children admitted with acute respiratory infections (ari). methods: we collected detailed clinical information on severity for children admitted with ari as part of a spanish prospective multicenter study (gendres network) between 2011–2013. a nested polymerase chain reaction (pcr) approach was used to detect respiratory viruses in respiratory secretions. findings were compared to an independent cohort collected in the uk. results: 204 children were recruited in the main cohort and 97 in the replication cohort. the number of detected viruses did not correlate with any markers of severity. however, bacterial superinfection was associated with increased severity (or: 4.356; p-value = 0.005), picu admission (or: 3.342; p-value = 0.006), higher clinical score (1.988; p-value = 0.002) respiratory support requirement (or: 7.484; p-value < 0.001) and longer hospital length of stay (or: 1.468; p-value < 0.001). in addition, pneumococcal vaccination was found to be a protective factor in terms of degree of respiratory distress (or: 2.917; p-value = 0.035), picu admission (or: 0.301; p-value = 0.011), lower clinical score (-1.499; p-value = 0.021) respiratory support requirement (or: 0.324; p-value = 0.016) and oxygen necessity (or: 0.328; p-value = 0.001). all these findings were replicated in the uk cohort. conclusion: the presence of more than one virus in hospitalized children with ari is very frequent but it does not seem to have a major clinical impact in terms of severity. however bacterial superinfection increases the severity of the disease course. on the contrary, pneumococcal vaccination plays a protective role. to correlate the presence of viral co-infection with clinical phenotype in children admitted with acute respiratory infections (ari). we collected detailed clinical information on severity for children admitted with ari as part of a spanish prospective multicenter study (gendres network) between 2011-2013. a nested polymerase chain reaction (pcr) approach was used to detect respiratory viruses in respiratory secretions. findings were compared to an independent cohort collected in the uk. results 204 children were recruited in the main cohort and 97 in the replication cohort. the number of detected viruses did not correlate with any markers of severity. however, bacterial superinfection was associated with increased severity (or: 4.356; p-value = 0.005), picu admission (or: 3.342; p-value = 0.006), higher clinical score (1.988; p-value = 0.002) respiratory introduction molecular techniques including polymerase chain reaction (pcr) have increased the sensitivity of detection for common and emerging respiratory viruses, and often reveal the presence of more than one pathogen in respiratory patients. [1] , [2] the importance of viral co-infections in the pathogenesis, severity or course of respiratory infections is not well established. the high sensitivity of molecular techniques raises questions about the clinical relevance of positive test results. the presence of a virus does not necessarily indicate causation of clinical symptoms or disease [3] . on the contrary, bacterial co-infection is usually associated with a more severe diseases course and worse prognosis, despite the precise interaction between bacteria and viruses is not always clear. our study aims to analyze the relationship between viral or bacterial co-infection detected by molecular methods, and the clinical phenotype of children admitted to hospital with lower tract acute respiratory infections (lt-ari). two independent observational prospective patient groups were enrolled in spain (main group) and in the united kingdom (replication group). spanish children were recruited between january 2011 and january 2013 through a national hospital based research network: gendres (genetic, vitamin d and respiratory infections research network-www.gendres. org), which includes 13 spanish tertiary hospitals. uk children were recruited between october 2009 and may 2010 at st mary's hospital (uk). in both cohorts, eligible study participants were children under 14 years of age with a lower respiratory tract illness of sufficient severity to warrant admission to hospital, and a virus identified on nasopharyngeal swab or aspirate sample. all types of lt-ari were included, from bronchiolitis to pneumonia, with or without wheezing, fever, rhinorrhea and respiratory distress. a nasopharyngeal sample (aspirate or swab) was obtained during admission for viral pathogen detection. besides the diagnostic procedures performed at origin in the referring hospital, viral detection was performed in all recruited subjects using a panel of 19 viruses, by nested pcr: respiratory syncytial virus; influenza virus (a, b); parainfluenza virus types (1-4); adenovirus (a-f); rhinovirus; metapneumovirus; coronavirus (nl63, 229e, oc43) and bocavirus (methods previous described in cebey-lópez et al. [4] ). no difference was found on the molecular diagnosis yield in our series when using aspirate or swab as sample source. micropathology ltd. provided support in the form of salaries for authors es and cf, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. the specific roles of these authors are articulated in the 'author contributions' section. competing interests: the authors have the following interests. edward sumner and colin fink are employed by micropathology ltd. there are no patents, products in development or marketed products to declare. this does not alter the authors' adherence to all the plos one policies on sharing data and materials, as detailed online in the guide for authors. all investigators were trained in the study protocol for patient recruitment, sample processing and sample storage. the study was performed according to good clinical practice. written informed consent was obtained from a parent or legal guardian for each subject before study inclusion. detailed clinical data on each patient were collected using a secured web-based platform. this included risk factors for lt-ari (prematurity, immunization status, obesity, diabetes, asthma and previous admissions to hospital), current medications, and family history of asthma or other respiratory diseases. the severity of each respiratory case was ranked as follows: 1) physician criteria (mild, moderate or severe); 2) wood-downes scale (0 to 10 points; mild < 3, moderate 4-7, severe > 8); and 3) a newly developed scale -named genvip score-(0 to 20 points) that assesses food tolerance, degree of medical intervention needed, respiratory distress, respiratory frequency, apnea, malaise and fever (see s1 table for further details on this scale). supplemental oxygen and / or mechanical ventilation requirement during admission were also recorded. bacterial superinfection diagnosis was established according to the referring physician criteria, based on clinical data, inflammatory markers, and radiological findings, and/or appropriate cultures in sterile sites (e.g blood or cerebrospinal fluid). for the study purposes, cases classified as bacterial co-infections included both cases with and without microbiological confirmation as in our series, cultures were not carried out systematically, but only when a bacterial superinfection was suspected by the referring physician. in this analysis, we compared clinical data to the results of pathogen identification in respiratory samples. we performed all the analysis using the r software, version 3.0.2 (www.rproject.org). general data were presented as means with 95% confidence intervals (ci). different statistical models were used to assess the bivariate association between the variables depending on the dependent variable. the relationship between demographic and clinical variables with mono-infection and co-infection was analysed using simple logistic regression. a binary logistic model was used for the binary variables (co-infection status, oxygen requirements, respiratory support needed and picu admission), linear model for continuous variables (wood-downes score and genvip score), negative binomial regression model for counted data (hospital stay length) and logistic multinomial model for the multinomial variable (respiratory distress status). multiple regression models were considered using the significant risk factors obtained in the bivariate analysis and sex and age variables. in order to reduce the likelihood of false significant results due to too many statistical comparisons, the bonferroni multiple test correction were considered. a χ2 test was performed to evaluate the correlation between bacterial superinfection and pneumococcal vaccine. level of statistical significance was set at 0.05. the gendres cohort included nasopharyngeal samples from 204 patients with a median age of 16.7 (95% ci: 12.7-20.6) months and a male-to-female sex ratio of 1.7. one patient was excluded due to incomplete clinical data. in the uk a cohort of 97 patients' data was analyzed, with a median of age of 36.6 (95% ci: 27.6-45.6) months and a male-to-female ratio of 0.94 (s1 fig) . comparison between both cohorts is shown in table 1 . the pcr results were preciously described in cebey-lópez et al. [4] . clinical data, family or past medical history, need of picu admission or hospital length of stay, and oxygen or respiratory support need, were equivalent in the gendres and the uk cohort (table 2 ). in the gendres cohort the presence of rhinovirus as co-pathogen was associated with a significantly increased wood-downes score by 1.289 points (95% ci: 0.387, 2.192); pvalue = 0.006. rsv infection was associated with increased oxygen requirements [or (95% ci): 3.154 (1.302, 7.966); p-value = 0.012] ( table 3) . these isolated findings were not replicated in the uk cohort (table 3) . these findings were not replicated in the uk cohort (fig 1; s1 -s7 tables). our study revealed that even though multiple viral detection is frequent in hospitalized children with lt-ari, this association is not related to either disease severity or to any other clinical features studied. picu admission, disease severity according to different scales, need for respiratory support, and length of hospital stay followed a similar pattern in viral mono-versus co-infected children. contrariwise, bacterial superinfection increased the severity of the disease course, while pneumococcal vaccination played a protective role. the detection of multiple coincident viruses in clinical settings is becoming more common since the introduction of molecular based multiplex tests, but the clinical significance of these findings remains unclear and seems to have no impact in disease severity [5] . both an increase in disease severity in relation to dual infections [6] [7] [8] [9] and the absence of this association [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] have been reported. richard et al. [8] found that co-infected children were almost three times more likely to be admitted to the picu than those with single viral infections. compared to our study richard et al. developed a retrospective and monocentric study in which they only considered dual infections, infants and bronchiolitis. there is contradictory evidence linking disease severity with specific respiratory viruses. a shorter hospital stay has been reported in children with rhinovirus bronchiolitis than with rsv [20] . rhinovirus and rsv co-infection is reported to increase the risk of severe disease [8] or the bronchiolitis relapse [21, 22] . other studies did not find significant differences in severity between co-infection and single infection [12, 18, 23, 24] . in our study we did not find increased severity of illness in children with rsv-rhinovirus dual infection. in our series, only rsv as mono-infection increased oxygen requirements, and rhinovirus as a co-infecting pathogen increased the wood-downes score in the spanish cohort, but these isolated findings arising from the multivariate analysis could not be replicated in the uk cohort. several studies have reported increased severity with bocavirus (hbov) co-infections [13, [25] [26] [27] ; this was not the case in our series (also in agreement with pen et al. [15] ). hbov was commonly detected in our patients, with no impact in the severity of the illness. as hbov was detected in alongside other respiratory viruses with an established pathogenic potential, it is possible that hbov detection reflects asymptomatic persistence or prolonged viral shedding [28] . bacterial superinfection was the only factor consistently linked to greater severity. studies of the pandemic influenza indicate that respiratory viruses predispose to bacterial complication and interaction between viruses and bacteria in respiratory infections has been extensively reported in the literature [29] , but the underlying mechanisms between viral and bacterial synergism are complex and remain unclear [30] . common respiratory viral infections, such as influenza or respiratory syncytial virus have been linked to seasonal increases in streptococcus pneumoniae disease [31] . the relationship between bacterial and viral infection is clouded by the low sensitivity of bacterial detection in sterile-site samples by traditional culture methods, and the reliance on non-specific clinical data for the for diagnosis of bacterial co-infection, including inflammatory markers, radiological findings and / or appropriate cultures, resulting in 30% of the cases in the gendres cohort and 55% in the uk cohort. bacterial superinfection increased most measures of severity in both cohorts (picu admission, respiratory support requirement, genvip score, hospital stay length and respiratory distress). interestingly, pneumococcal vaccination was revealed as an independent protective factor of disease severity in our patients. pneumococcal vaccine reduced the severity of viral lt-aris through a reduction in oxygen requirement, invasive and non-invasive ventilation, admission to picu, respiratory distress, and genvip score. a reduced incidence of viral alveolar pneumonia has been previously reported after pneumococcal vaccination [31, 32] , although there was no demonstrable reduction in the number of confirmed pneumococcal infections. this is likely to reflect the limited sensitivity of culture-proven pneumococcal disease in pneumonia. the protective effect of pneumococcal vaccines found in our study might reinforce the importance of the paradigm of viral-penumococci interaction at nasopharynx level in the pathogenesis and clinical course of the disease [33] current pneumococcal conjugate vaccines significantly decrease nasopharyngeal carriage of pneumococci [34] and thus reduces the possibility of this viral-pneumococci direct interaction. one of the limitations of the present study is that our samples were not tested for viral load by quantitative pcr and the viral load of certain viruses-like rsv-has been associated with the co-infection status and the severity [3] . also, the study did not consider milder or asymptomatic children. in addition, bacterial superinfection rate in our series might be overestimated as diagnosis was accepted as true even without microbiological confirmation, just based on referring physicians' criteria. several studies had shown that viruses can be found in children with no respiratory infections [6, 35] , and further research is needed to understand the natural history of respiratory viral carriage and infection. however, our findings were consistent in both independent cohorts very different between them, so this makes the outcomes more robust. in summary, the severity and course of an acute respiratory episode requiring hospitalization in children did not correlate with the presence of one or more viruses. in contrast, bacterial co-infection was associated with more severe disease, whereas pneumococcal vaccination decreased severity. future studies are needed to investigate whether particular viruses, or combinations of viruses, influence the risk of bacterial co-infection. supporting information s1 fig. flow chart of study population of the main cohort (gendres cohort) and replication cohort (uk-cohort). (jpg) s1 table. genvip score. (docx) s2 table. demographic characteristics, family and patient medical history, clinical course and principal virus in children with ari and disease severity, considering respiratory support and oxygen requirement the characteristics that described the severity of the illness of the main cohort. (docx) s3 table. variables analyzed in children with ari and disease severity, considering the clinical scales the characteristics that described the severity of the illness of the main cohort. (docx) s4 table. demographic characteristics, family and patient medical history, clinical course and main virus in children with ari and disease severity in gendres cohort. (docx) s5 table. children's with ari characteristics and moderate and severe respiratory distress. (docx) s6 table. comparison of virus and disease severity of the replication cohort (uk cohort) considering the virus as single pathogen or as co-infection in the sample. (docx) s7 table. demographic characteristics, clinical course and principal virus in children with ari and disease severity, considering respiratory support and oxygen requirement the characteristics that described the severity of the illness of the uk-cohort are presented. (docx) s8 table. variables analyzed in the uk-cohort children and disease severity according to hospital stay length and picu admission are shown. (docx) hospital materno infantil virgen del camino francisco giménez sánchez, miguel sánchez forte eider oñate vergara (hospital de donostia we thank stuart gormley and the clinical team at st mary's hospital for assistance in recruiting the uk cohort. we also extend our gratitude to the nursery and laboratory service from the hospital clín newly discovered respiratory viruses: significance and implications. current opinion in pharmacology comparison of multiplex pcr assays and conventional techniques for the diagnostic of respiratory virus infections in children admitted to hospital with an acute respiratory illness clinical utility of pcr for common viruses in acute respiratory illness viral co-infections in pediatric patients hospitalized with lower tract acute respiratory infections clinical disease severity of respiratory viral co-infection versus single viral infection: a systematic review and meta-analysis respiratory pathogens in children with and without respiratory symptoms frequent detection of viral coinfection in children hospitalized with acute respiratory tract infection using a real-time polymerase chain reaction the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis viral etiology of acute febrile respiratory illnesses in hospitalized children younger than 24 months diagnostic value of respiratory virus detection in symptomatic children using real-time pcr infection with multiple viruses is not associated with increased disease severity in children with bronchiolitis. pediatr pulmonol the association of newly identified respiratory viruses with lower respiratory tract infections in korean children comparison of human metapneumovirus, respiratory syncytial virus and influenza a virus lower respiratory tract infections in hospitalized young children multipathogen infections in hospitalized children with acute respiratory infections polymicrobial acute respiratory infections in a hospital-based pediatric population prevalence and clinical characteristics of human metapneumovirus infections in hospitalized infants in spain. pediatric pulmonology single versus dual respiratory virus infections in hospitalized infants: impact on clinical course of disease and interferon-gamma response respiratory syncytial virus, its coinfection and paediatric lower respiratory infections. the european respiratory journal: official journal of the european society for clinical respiratory physiology hospital length-of-stay is associated with rhinovirus etiology of bronchiolitis multicenter study of viral etiology and relapse in hospitalized children with bronchiolitis association of rhinovirus infection with increased disease severity in acute bronchiolitis. american journal of respiratory and critical care medicine prospective multicenter study of the viral etiology of bronchiolitis in the emergency department prospective multicenter study of viral etiology and hospital length of stay in children with severe bronchiolitis. archives of pediatrics & adolescent medicine cloning of a human parvovirus by molecular screening of respiratory tract samples the incidence of human bocavirus infection among children admitted to hospital in singapore bocavirus infection in hospitalized children human bocavirus: passenger or pathogen in acute respiratory tract infections? clinical microbiology reviews viral and bacterial interactions in the upper respiratory tract influenza and rsv make a modest contribution to invasive pneumococcal disease incidence in the uk. the journal of infection influence of pneumococcal vaccines and respiratory syncytial virus on alveolar pneumonia, israel. emerging infectious diseases vaccine trialist g. a role for streptococcus pneumoniae in virus-associated pneumonia respiratory syncytial virus increases the virulence of streptococcus pneumoniae by binding to penicillin binding protein 1a. a new paradigm in respiratory infection. american journal of respiratory and critical care medicine evolving role of 13-valent pneumococcal conjugate vaccine in clinical practice detecting respiratory viruses in asymptomatic children we greatly thank all the patients that kindly contributed to this study. the authors thank the study investigators of gendres network (www. key: cord-336441-m6pur6td authors: wang, changjian; wu, kangmin; zhang, xinlin; wang, fei; zhang, hongou; ye, yuyao; wu, qitao; huang, gengzhi; wang, yang; wen, bin title: features and drivers for energy-related carbon emissions in mega city: the case of guangzhou, china based on an extended lmdi model date: 2019-02-11 journal: plos one doi: 10.1371/journal.pone.0210430 sha: doc_id: 336441 cord_uid: m6pur6td based on the apparent energy consumption data, a systematic and comprehensive city-level total carbon accounting approach was established and applied in guangzhou, china. a newly extended lmdi method based on the kaya identity was adopted to examine the main drivers for the carbon emissions increments both at the industrial sector and the residential sector. research results are listed as follow: (1) carbon emissions embodied in the imported electricity played a significant important role in emissions mitigation in guangzhou. (2) the influences and impacts of various driving factors on industrial and residential carbon emissions are different in the three different development periods, namely, the 10(th) five-year plan period (2003–2005), the 11(th) five-year plan period (2005–2010), and the 12(th) five-year plan period (2010–2013). the main reasons underlying these influencing mechanisms were different policy measures announced by the central and local government during the different five-year plan periods. (3) the affluence effect (g-effect) was the dominant positive effect in driving emissions increase, while the energy intensity effect of production (e-effect-production), the economic structure effect (s-effect) and the carbon intensity effect of production (f-effect-production) were the main contributing factors suppressing emissions growth at the industrial sector. (4) the affluence effect of urban (g-effect-aui) was the most dominant positive driving factor on emissions increment, while the energy intensity effect of urban (e-effect-urban) played the most important role in curbing emissions growth at the residential sector. with anthropogenic releases of carbon emissions contributing toward climate change primarily characterized by global warming, many policymakers and scientific-researchers are seeking scales. therefore, comprehensive inventory and specific boundary are the basis for city-level carbon emissions accounting, as well as local emission mitigation strategies [28] . the second strand of research focuses on the city-level driving factors and influencing mechanisms. decomposition analysis (e. g. index decomposition analysis (ida) and structural decomposition analysis (sda)) and regression method (e. g. stochastic impacts by regression on population, affluence, and technology (stirpat) model) are the most commonly applied methods for the scientific evaluation and quantitative analysis of factors influencing city-level carbon emissions, especially the logarithmic mean divisia index (lmdi) method based on the ida framework. as shown in table 1 , the sda method based on the io technique applied in the city-level driving factors and influencing mechanisms research in china were mainly focused on the four municipalities (i. e. beijing, shanghai, tianjin and chongqing), because of the available io data base compiled by the municipality bureau of wang et al. [41] 1997-2010 stirpat beijing wang et al. [42] 2000-2010 lmdi beijing mi et al. [43] 2010 io beijing wang et al. [44] 1997-2010 sda beijing tian et al. [45] 1995-2007 sda beijing wang et al. [46] 1996-2010 lmdi and tapio index beijing, tianjin wang et al. [47] 1998-2009 stirpat shanghai shao et al. [48] 1994-2009 stirpat shanghai zhao et al. [49] 1996-2007 lmdi shanghai shao et al. [50] 1994-2011 lmdi shanghai kang et al. [51] 2001-2009 lmdi tianjin li et al. [52] 1996-2012 stirpat tianjin tan et al. [53] 2000-2012 lmdi chongqing wang et al. [54] 2005-2010 lmdi suzhou chen et al. [55] 2000-2013 decoupling index macao liu et al. [56] 1995-2009 lmdi beijing, shanghai, tianjin, and chongqing statistics. in other words, the io data sources were lacking in other provincial capital cities and medium-sized cities in china. consequently, the ida methods were used more frequently than the sda methods based on io technique [59] [60] [61] [62] , owing to its applicability in which each model can be applied to any available data at any aggregation level in a period-wise or timeseries manner [63] [64] [65] , where the preferred one in the lmdi [66] [67] [68] [69] [70] [71] [72] [73] . by means of lmdi method, wang et al. [46] investigated the main driver for industrial carbon emissions in beijing-tianjin-hebei economic band from 1996 to 2010, and the results showed that economic output and energy intensity were both the most important influencing factors. wang et al. [42] decomposed the influencing factors of residential carbon emissions in beijing and found that the rising per capita gdp and the declining energy intensity contributed the most to emissions changes during 2000 to 2010. similar results, conducted by wang et al. [54] , can also be found in suzhou city. zhao et al. [49] decomposed the main drivers for industrial carbon emissions in shanghai and found that industrial output, energy intensity, energy and industrial structure were major determinants for emissions changes. shao et al. [50] adopted an extended lmdi method and investigated the techno-economic drivers for industrial carbon emissions in shanghai, and the results showed that industrial output scale was mainly responsible for emission increments while industrial structure adjustment, r&d intensity, and energy intensity were most significant factors in mitigating emissions. kang et al. [51] performed a multi-sectoral decomposition analysis of city-level greenhouse gas emissions in tianjin from 2001 to 2009, including the agricultural, industrial, transportation, commercial and other sectors, and the results showed that economic growth was the most important driver for emissions increments while energy efficiency was primarily responsible for emissions reductions. hopefully, previous studies about driving factors and influencing mechanisms of city-level carbon emissions performed on china's megacities such as beijing, shanghai etc., have made great contributions to the realization of regional low-carbon road map. but, the previous lmdi studies mainly focused on the economic growth effect, the energy intensity effect, the population size effect, and the technology progress effect influencing the change of carbon emissions. it fails to distinguish the effects of economic structure, population structure, and energy structure on the change of carbon emissions. in fact, the strongest factors that offset china's energy consumption and carbon emissions have shifted from efficiency gains to structural changes [74, 75] . structural indicators combined with other traditional and emerging factors should also be deeply considered and investigated in china's regional energy and carbon researches, which may perform different influencing mechanism in different regions during different development stages. compared with four municipalities, there were relatively few studies conducting on guangzhou city, one of china's first-tier cities, with the considerable gdp scale and total energy consumption amount (table 2) . meanwhile, guangzhou has the highest level of per capita gdp and per capita energy consumption than the four municipalities. guangzhou is the provincial capital of guangdong province, the most developed coastal region in the southeast of china (fig 1) . in addition, guangzhou city is an important manufacturing base in china as well as a pilot zone for low-carbon city announced by china's national development and reform commission (ndrc) in 2012. therefore, guangzhou city may serve as a demonstration of how to realize the targets of low-carbon city development, thereby highlighting the importance and representativeness of studying its features and drivers for carbon emissions in detail. the innovation and contribution of this study compared with other references mainly lies in the following two aspects. firstly, city-level total carbon accounting approach based on the apparent energy consumption data from the comprehensive energy balance table was established. secondly, driving factors including structural indicators, e. g. economic structure, energy structure, and population structure, were investigated systematically by adopting the newly extended lmdi method based on the kaya identity and the ida theory. it was aimed to provide theoretical references for making more targeted policies on energy saving and emission reduction in china's mega cities. this study is organized as follows: after the introduction, section 2 presents the methodology of city-level carbon accounting in guangzhou and the method to decompose the changes in carbon emissions during the whole research period. section 3 addresses the case analysis and the main results of guangzhou city. finally, we conclude our study and provide some policy implications for low-carbon city development. city-level total carbon accounting as mentioned in table 1 , urban emissions inventory is the key for city-level total carbon accounting. however, there are accounting discrepancies between the different types of methods [21] . based on china's energy statistic and the reference approach of ipcc guideline for national greenhouse gas emission inventories, there are mainly three approach to account, i. e. primary energy consumption (e. g. coal, oil, natural gas, and renewables) [63] , final energy consumption (e. g. coal, oil, natural gas, renewables, and electricity/heating) [61, 62] , and total energy consumption from the "energy balance table" including the aggregate summary of energy production, energy transformation and final consumption [76, 77] . compared with the primary energy consumption accounting and the final energy consumption accounting, apparent energy consumption data [78, 79] from the comprehensive energy balance table was recommended for this study. as shown in the table 3 , the comprehensive energy balance table is mainly based on three modules, namely, 1) total energy available for local consumption as a result of interprovincial and international energy trades, 2) input-output of energy conversion transformation for thermal power, heating, coking, petroleum refining, and gas production, 3) final consumption due to primary industry, secondary industry, tertiary industry, and residential consumption. primary industry, namely, agriculture, mainly includes farming, forestry, animal husbandry, and fishery. secondary industry includes manufacturing industries and construction industry. tertiary industry includes transportation, storage, post and telecommunication services, wholesale, retail trade and catering services, and other service sectors. then, we calculated energy-related carbon emissions by energy types according to the ipcc guidelines for national greenhouse gas inventories based on the apparent energy consumption data from the comprehensive energy balance table, as follows the equation: greenhouse gas (ghg) in our case study is referred in particular to carbon emissions-that directly affect climate change, which didn't include other ghgs (i. e. ch 4 , n 2 o, etc.). where the subscript i is the various fuels in this case study, t means the time in years. c t represents total carbon emissions in year t (in million tons, mt). e i t represents the total energy consumption of fuel type i in year t (in million tons, mt), and ncv i is the net caloric value of fuel i. cc i t is the carbon content of the fuel type i, and o i is the oxidation rate of fuel i. the conversion factors, net caloric value, oxygenation efficiency and carbon content of the various fuels are listed in table 4 . data sources, consisting of the gross domestic product including agriculture, production, construction, and service, the population including rural population and urban population, the total energy consumption based on industrial and residential perspective, covering the period from 2003 to 2013, were all available. economic, population and energy data were collected from the guangdong province statistical yearbook (2003-2014) and guangzhou city statistical yearbook (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) . economic data was measured by gdp in chinese yuan in time series. energy data includes physical quantity of total energy consumption by fuel types measured by tons of standard coal equivalent (tce), which were compiled by the guangdong province statistical bureau and guangzhou city statistical bureau. as mentioned and reviewed in table 1 , the lmdi methods based on the ida theory, were used frequently to quantitatively analyze the drivers of carbon emissions at the city-level. the lmdi technique based on the ida theory and an extended kaya identity was conducted to uncover the main driving forces for energy-related carbon emissions in guangzhou city. the kaya identity [18, 64, 80, 81] expresses carbon emissions as a product of four underlying driving factors: where p represents the population, g represents the gdp, e represents the total energy consumption; g p represents the per-capita gdp, e g represents the energy consumption intensity, and c e represents the carbon intensity of energy. then, the kaya identity was extended as follow: where x n k¼1 c i (i = 1, 2, 3, 4, 5, 6) represents carbon emissions from agriculture (i = 1), production (i = 2), construction (i = 3), service (i = 4), urban resident (i = 5), and rural resident (i = 6). k (k = 1, 2, 3, . . .n) represents the various energy types in this case study, mainly including coal, coke, crude oil, gasoline, diesel oil, kerosene, fuel oil, lpg, natural gas, and electricity. e i (i = 1, 2, 3, 4, 5, 6) represents total energy consumption by different sectors. gdp i (i = 1, 2, 3, 4) represents gross domestic product by different industries. p urban and p rural represent the total population of urban and rural residents, respectively. ti urban and ti rural represent the total income of urban and rural residents, respectively. ai urban and ai rural represent the average income of urban and rural residents, respectively. then, eq (3) can be rewritten as follow: where p = p, represents the total population size, ur represents the level of urbanization. g ¼ gdp p , represents the per capita gdp. e i ¼ e i gdp i (i = 1, 2, 3, 4, 5, 6), represents the energy consumption intensity of agriculture, production, construction, service, urban resident, and rural resident, respectively. , represents the energy carbon intensity of agriculture, production, construction, service, urban resident, and rural resident, respectively. s i ¼ gdp i gdp (i = 1, 2, 3, 4), represents the economic structure of agriculture, production, construction, and service, respectively. then, the changes of regional carbon emissions between two years can be decomposed as: following the preferred lmdi method proposed by ang et al. [66, 82, 83] , the various effects of each factor can be quantificationally calculated as: in the newly established lmdi method, the population effect (δc p-effect ), the affluence effect (δc g-effect ), the energy intensity effect (δc e-effect ), the economic structure effect (δc s-effect ), and the carbon intensity effect (δc f-effect ). in addition, social and economic factors such as urbanization (δc ur-effect ), family income in urban and rural households (δc ai-effect ) were analyzed to further explain the changes of carbon emissions at the city level. economic development in guangzhou was described with gdp, which was converted into the 2003 constant prices (fig 2) . according to the urban emissions inventory in guangzhou and eq (1), we calculated the energy-related carbon emissions from 2003 to 2013. total energy-related carbon emissions mainly include two parts, namely, carbon emissions generated in the boundary from fossil fuel combustion and out of the boundary embodied in the imported electricity (fig 3) . carbon emissions embodied in the imported electricity need to be calculated by considering the total amount of imported electricity and the corresponding emissions factor of power generation the case of guangzhou based on extended lmdi model [56] . as for china's power supply system, electricity is supplied by the main six regional grids five-year plan period, respectively.), which were issued by the central government and performed by the local government. but in fact, carbon emissions mitigation in the boundary mainly benefited from contributions of the imported electricity from the neighbors guizhou and yunnan provinces via the cross-province electricity trades. during the same period, carbon emissions embodied in the imported electricity dramatically increased 3.236 million tons in 2010 to 4.379 million tons in 2013. rapidly increasing carbon emissions embodied in imported electricity indicated that there were large amounts of fossil energy were burned for power. final products-thermal power was consumed in guangzhou, while carbon emissions were discharged in its neighbor producers. if these carbon emissions embodied in the imported electricity were allocated to the final consumer, guangzhou would have paid more efforts and implemented more strict mitigation measures to achieve its energy saving and emission reduction targets. aiming to have a better understanding of the complex various influencing factors for carbon emissions in guangzhou city, we decomposed the total carbon emissions into two parts including the industrial sector and the residential sector. driving factors of carbon emissions in industrial sector in guangzhou. driving factors of carbon emissions in industrial sector were decomposed yearly first (table 5 ). in order to have a better understanding of influencing factors in long time series, we divided the carbon emissions process into 3 periods (fig 4) , according to the regional five-year plans for socioeconomic development, combined with a certain historical background. during the 10 th five-year plan period (2003) (2004) (2005) , guangzhou announced the overall objective in the 10 th five-year plan that it would take the lead in realizing socialist modernization and building the modernization central city over china. guangzhou's industrial carbon emissions increased by 6.29 million tons mainly because the rapid economic development. the affluence effect (g-effect) was the dominant positive effect in driving carbon emissions increase, bringing in a 5.43 million tons increase, accounting for 86.34% of the total industrial carbon emissions changes, which was followed by the energy intensity effect of service (e-effect-service), the carbon intensity effect of service (f-effect-service), and the economic structure effect (s-effect). in order to achieve the basic standards of modernization (i. e. per capita gdp is more than 5000 u.s. dollar, and the third industry added value accounted for more than 50% of gdp etc.), guangzhou paid more efforts to foster the three pillar industries (i. e. electronic information manufacturing industry, automobile industry, and petrochemical table 5 period the case of guangzhou based on extended lmdi model economic structure effect (s-effect). the adjustment of industrial structure in guangzhou was mainly focused on upgrading the traditional advantage industries (i. e. iron and steel, shipbuilding, machinery and equipment, electricity, paper, and textile etc.) and fostering the new and high technology industries (i. e. software, new material, new energy, and digital industry etc.), announced in the 11 th five-year plan for socioeconomic development in guangzhou driving factors of carbon emissions in residential sector in guangzhou. driving factors of carbon emissions in residential sector were decomposed yearly first (table 6 ) and divided into 3 periods (fig 5) . although the residential carbon emissions in guangzhou accounted for a relatively small proportion of the total carbon emissions, it exhibited a rapid growth trend during the whole research period along with the improvement of urbanization level. the total residential carbon emissions increased from 0.55 million tons in 2003 accounting for 3.61% of the total carbon emissions to 2.01 million tons accounting for 6.59% in 2013. furthermore, the residential carbon emission increments were mainly generated by the urban residents, which increased from 0. the case of guangzhou based on extended lmdi model was always two times of the average income of rural residents during the same period. the energy intensity effect of urban (e-effect-urban) played the most important role in curbing carbon emissions growth, resulting in a 0.26 million tons decrease, accounting for 34.87% of the total residential carbon emissions changes in absolute value. during the 12 th five-year plan period (2010-2013), guangzhou's residential carbon emissions increased by 0.51 million tons. the affluence effect of urban (g-effect-aui) and the carbon intensity effect of urban (f-effect-urban) were the most two dominant positive driving factors on the carbon emissions increment, bringing in 0.49 million tons and 0.29 million tons increase, accounting for 95.88% and 57.43% of the total residential carbon emissions changes, respectively. the energy intensity effect of urban (e-effect-urban) played the most important role in curbing carbon emissions growth, resulting in a 0.33 million tons decrease, accounting for 64.95% of the total residential carbon emissions changes in absolute value. based on the apparent energy consumption data from the comprehensive energy balance table, a systematic and comprehensive city-level total carbon accounting approach was established and applied in one of the china's mega city-guangzhou. total carbon emissions including the fossil fuel combustion in the city's boundary and embodied in the imported electricity out of the boundary were still performing a rising tendency. carbon emissions embodied in the imported electricity played a significant important role in emissions mitigation in guangzhou, especially after 2009. examining the effects of different boundaries on carbon accounting at city level is crucial for the emissions mitigation in urban china. then, the newly extended lmdi method based on the kaya identity was adopted to examine the main drivers for the carbon emissions increments both at the industrial sector and the residential sector in guangzhou city. quantitative analyses and time-series analysis were performed on the influencing mechanism for various influencing factors both at the industrial sector and the residential sector for the three different development periods, namely, the 10 th five-year plan period (2003) (2004) (2005) , the 11 th five-year plan period (2005-2010), and the 12 th five-year plan period (2010-2013). the influences and impacts of various driving factors on industrial and residential carbon emissions are different in the three different development periods. overall, the affluence effect (g-effect) was the dominant positive effect in driving carbon emissions increase, while the energy intensity effect of production (e-effect-production), the economic structure effect (s-effect) and the carbon intensity effect of production (f-effect-production) were the main contributing factors suppressing the carbon emission growth at the industrial sector. considering the high urbanization level in guangzhou, the affluence effect of urban (g-effect-aui) was the most dominant positive driving factor on the carbon emissions increment, while the energy intensity effect of urban (e-effect-urban) played the most important role in curbing carbon emissions growth at the residential sector. in conclusion, affluence effect-economic growth, was still the most important contributor to the increases in carbon emissions in urban china. however, structural indicators, such as economic structure and energy intensity of different industrial sector, were playing significant negative effects on carbon emissions. in the future, economic structure adjustment and energy intensity improvement at the detailed production industries should be deeply investigated, in order to make these indicators play more negative effects on carbon emissions. considering the significant important role played by the emissions embodied in the imported electricity via the cross-province electricity trades, guangzhou should adjust its current emissions mitigation policies which only consider its emissions occurring within the city's boundary. guangzhou should make more joint efforts to help its neighbors improve the efficiency of thermal power generation. guangzhou might make efforts to buy more clean power such as hydropower from its neighbors yunnan and guangxi provinces to replace a certain amount of thermal power. in addition, carbon trading scheme [87] about the cross-province electricity trades should be introduced and conducted on the imported electricity in guangzhou. in order to further promote the negative effects of the economic structure effect (s-effect), the energy intensity effect of production (e-effect-production), and the carbon intensity effect of production (f-effect-production), guangzhou should pay more efforts to the optimization of energy consumption structure and industrial structure at the industrial sector. (1) coal consumption in energy mix should be further decreased, while relatively low carbon energy such as natural gas should be accelerated both in the industrial and residential sectors. renewable energy such as solar pv and hydropower should also be encouraged. (2) efficiency of thermal power generation should be further improved in guangzhou, especially the major power plants such as the pearl river power plant and the huarun power plant in nansha district, the guangzhou power plant in liwan district, the hengyun power plant, the huangpu power plant, and the ruiming power plant in huangpu district. (3) energy consumption structure and energy utilization technique should be also effectively optimized and improved, especially in the pillar industries (i. e. electronic information manufacturing industry, automobile industry, and petrochemical industry), the traditional advantage industries (i. e. iron and steel, shipbuilding, machinery and equipment, paper, and textile etc.) and the new and high technology industries (i. e. software, new material, new energy, and digital industry etc.) in guangzhou in the current and near future. urbanization level of guangzhou is relatively high than other cities in guangdong province and even in china. the rapid increase in the average income of urban residents along with the high level of urbanization has made the affluence effect of urban (g-effect-aui) become the most dominant positive driving factor on the residential carbon emissions increment during the whole research period. the residential energy consumption structure was mainly based on 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combustion reduced carbon emission estimates from fossil fuel combustion and cement production in china examining the driving factors of energy related carbon emissions using the extended stirpat model based on ipat identity in xinjiang global and regional drivers of accelerating co2 emissions 82. ang key: cord-333650-4towah1t authors: malmo, jostein; moe, nina; krokstad, sidsel; ryan, liv; loevenich, simon; johnsen, ingvild b.; espevik, terje; nordbø, svein arne; døllner, henrik; anthonsen, marit w. title: cytokine profiles in human metapneumovirus infected children: identification of genes involved in the antiviral response and pathogenesis date: 2016-05-12 journal: plos one doi: 10.1371/journal.pone.0155484 sha: doc_id: 333650 cord_uid: 4towah1t human metapneumovirus (hmpv) causes severe airway infection in children that may be caused by an unfavorable immune response. the nature of the innate immune response to hmpv in naturally occurring infections in children is largely undescribed, and it is unknown if inflammasome activation is implicated in disease pathogenesis. we examined nasopharynx aspirates and blood samples from hmpv-infected children without detectable co-infections. the expression of inflammatory and antiviral genes were measured in nasal airway secretions by relative mrna quantification while blood plasma proteins were determined by a multiplex immunoassay. several genes were significantly up-regulated at mrna and protein level in the hmpv infected children. most apparent was the expression of the chemokine ip-10, the pro-inflammatory cytokine il-18 in addition to the interferon inducible gene isg54. interestingly, children experiencing more severe disease, as indicated by a severity index, had significantly more often up-regulation of the inflammasome-associated genes il-1β and nlrp3. overall, our data point to cytokines, particularly inflammasome-associated, that might be important in hmpv mediated lung disease and the antiviral response in children with severe infection. our study is the first to demonstrate that inflammasome components are associated with increased illness severity in hmpv-infected children. the negative sense single strand rna virus human metapneumovirus (hmpv) has since its discovery in 2001 emerged as a commonly detected respiratory tract pathogen involved in the npas were tested using pcr for hmpv (a1, a2a/b, b1, b2), adenovirus, human bocavirus-1, coronavirus (oc43, 229e and nl63), enterovirus, influenza a and b virus, parainfluenza virus type 1-4, parechovirus, rsv, rhinovirus, bordetella pertussis, chlamydophila pneumoniae and mycoplasma pneumoniae. nucleic acid was extracted using nuclisens easy-mag according to the manufacturer's protocol (biomerieux). all pcrs were in-house realtime assays based on taqman probes [16] . for hmpv detection, primers targeting the n-gene allowing detection of all four hmpv genotypes were used as described elsewhere [17] . genotyping was performed by sequencing an amplified f-gene pcr product [18] using a 3130 genetic analyzer (applied biosystems) and comparing sequenced data with the nucleotide blast database (www.ncbi.nlm.nih.gov/blast/). a total of 162 children tested positive for hmpv (6.1%) in npa. for the purpose of the present study, 30 children satisfying all of these criteria were recruited: an npa was sampled less than one week after onset of symptoms and within one day after admittance, either hmpv genotypes a2 or b2 were detected in npa, no viral co-infections detected in npa, and serum levels of crp <100 mg/l. clinical data were recorded prospectively or retracted from medical records. upper respiratory tract infection (urti) was diagnosed when rhinitis, pharyngitis, tonsillitis and/or otitis media (serous, simplex or purulent) was found without signs of lower respiratory tract infection (lrti). lrti was diagnosed in children with signs and symptoms of respiratory difficulty such as tachypnea, retractions and nasal flaring, signs of lower airway obstruction (wheezing, prolonged expiration, rhonchi), focal findings (crepitations) and/or a positive chest x-ray (infiltrates, atelectasis, air trapping). lrti was divided in bronchiolitis (age <2 years, tachypnea, retractions, wheezing, crepitations), obstructive bronchitis (age >2 years, signs of lower airway obstruction), asthma exacerbation with signs of lower airway obstruction, and pneumonia (cough, tachypnea, localized crepitations, x-ray infiltrates). a clinical severity score previously used to determine hmpv/rsv disease severity in children [7, 8] was calculated for each infected child as the sum of these variables 1) length of hospital stay 5 days (1 point), 2) oxygen demand (1 point), 3) ventilatory support by continuous positive airway pressure ventilation (1 point), and 4) ventilatory support by endotracheal intubation and overpressure ventilation (2 points). ten children admitted for elective surgery with no symptoms of rti in the last two weeks were randomly selected and included as controls. the clinical and virological findings are summarized in table 1 , and a detailed overview for the individual patients is presented in the s1 table. mrna quantification cdna was synthesized from rna isolated from the collected npas using the qscript kit according to the manufacturer's protocol (quanta). quantitative pcr (qpcr) was performed using perfecta sybr green reaction mix (quanta) and a steponeplus instrument (life technologies) with the temperature profile 95°c for 20 s, 40 cycles at 95°c for 3s and 60°c for 30s. fold-change in ifn-β, ifn-γ, il-28, iκbα, il-1β, il-18, nlrp3, ip-10, tnf-α, il-6 and isg54 gene expression relative to the control with highest levels of target gene mrna was calculated using the ddc t -method with gapdh as housekeeping gene. data was plotted as boxplots where the box represents 50% of the values, the horizontal line indicates the median, whiskers show the maximum and minimum values, and the closed circles indicate outliers. primer sequences used in this study are shown in the s2 table. protein quantification blood samples were collected in vacuette edta tubes (greiner bio-one) and the fractions separated according to the manufacturer's protocol. the concentrations of proteins in the plasma fraction were measured using a bio-plex multiplex system (bio-rad) and cytokine assays for ifn-β, ifn-γ, il-28a, il-1β, il-18, ip-10, tnf-α, il-6, gro-α and gro-β (bio-rad). categorical variables were analyzed with the pearson chi-square test or the 2-tailed fisher's exact test. continuous and nearly normal distributed variables were analyzed with the student's t-test or anova-test, and non-parametric variables were compared by mann-whitney u tests. a two-sided p-value less than 0.05 was considered statistically significant. normally distributed and continuous variables are presented as average±standard deviation and nonnormally distributed variables as median (range). analyses were performed using the statistical package of social science (spss, version 19.0) and sigmaplot (version 12.0). the study was approved by the regional committee for medical and health research ethics (rek, mid-norway). caregivers to all patients and controls received written information about the study. written consent was obtained at the hospital from the caregivers of all controls and the majority of the patients included in the cohort. due to practical challenges by enrolling patients 24 hours a day, 7 days a week, some patients were enrolled after discharge from the hospital. their caregivers received written information after the hospital stay and children were included if the caregivers did not resist enrollment by taking contact to the hospital within 4 weeks. this study was based on a cohort involving 2656 children hospitalized with respiratory tract infection (rti) where 162 tested positive for hmpv. from the hmpv positive children we selected samples without detectable co-infections (n = 30) as described in the methods section. further, children admitted for elective surgery without recent symptoms of rti were randomly selected and included as controls (n = 10). table 1 shows that the majority of the children were diagnosed with lower respiratory tract infection (bronchiolitis or pneumonia), and the disease severity ranged from 0-4. one half of the patients had more severe disease with a score higher than zero (s1 table) . during the period of this study, a2 (43% of the samples) and b2 (42%) were the dominant genotypes found in the cohort and therefore our focus. the length of hospitalization was average 4.1±4.2 days for a2 and 3.1±2.0 days for b2 infected children (p>0.05, student's t-test). the maximum c-reactive protein (crp) values in the patients ranged from approximately 5 to 100 mg/l with the majority below 50 mg/l, indicating that severe bacterial infections were unlikely. to determine the presence of antiviral cytokines in children infected with hmpv and controls, we initially investigated the expression of type i, ii and iii ifns. fig 1a shows that only a2 infected children had slightly elevated mrna levels of the type i ifn-β compared to the controls. as shown in fig 1b, the mrna levels of the type ii ifn-γ was not elevated in any of the patient groups relative to the controls. in correspondence with this, ifn-γ protein was not detected in the blood samples from any of the investigated children (data not shown). finally we evaluated the mrna expression of the type iii ifn il-28. fig 1c shows that both the a2 and b2 positive patients had significantly increased levels of il-28 expression. next, we wanted to investigate the possible involvement of nf-κb and inflammasome-associated genes in response to hmpv infection. fig 2 shows the mrna expression of a) iκbα, a repressor gene induced by nf-κb activation [19] , b) il-1β, c) il-18 and d) nlrp3 in hmpv infected children and controls. the expression of il-18 was significantly increased for both a2 and b2 infected children. however, for the other genes investigated their expression was not significantly increased compared to the controls. it should be noted that the standard deviations for the iκbα, il-1β and nlrp3 expression were high. this was caused by a considerable fraction of the hospitalized children having undetectable expression while others had high mrna levels relative to control subjects. in contrast, children from the control group exhibited similar levels of iκbα, il-1β and nlrp3 mrna levels. to illustrate this individual variation, differences in gene expression for individual a2 and b2 positive patients are outlined in fig 3. from this figure, it can be seen that a) iκbα, b) il-1β and c) nlrp3 were significantly upregulated in several patients. interestingly, most of the patients with upregulation of iκbα, il-1β and nlrp3 had increased expression of all three genes. to evaluate the involvement of other relevant immunomodulator genes, we measured the mrna expression of ip-10, tnf-α, il-6 and isg54. as shown in fig 4a, the expression of ip10 was increased for both the a2 and b2 infected patients. the tnf-α expression (fig 4b) was significantly higher in patients infected with b2 compared to the controls. further, the expression of il-6 ( fig 4c) was not increased at mrna level in any of the groups with hmpv infected children. finally, the expression of isg54 (fig 4d) was markedly increased for both a2 and b2 infected patients. as mentioned earlier, the expression of inflammatory genes can possibly promote pathogenic events during rti. consequently, we wanted to investigate if the gene expression profiles differed in children with a severity index of zero compared to those with more serious disease and a higher severity score. as summarized in table 2 , the mrna expression of il-1β and nlrp3 were more frequently upregulated in the children with a severity score of 1-4. finally, the concentration of several secreted cytokines in blood plasma was measured for a random subset of the patients (n = 10) and the controls (n = 10) as shown in fig 5. herein, we also included the chemokines gro-α and gro-β to evaluate implications of neutrophil recruitment to the lungs. fig 5 shows that all of the cytokines included in the analysis, except for il-28, were detected at significantly higher concentrations in patients compared to the controls. the percentage with up-regulated genes is given in the brackets. statistical significance is indicated with the p-value from a fisher's exact test comparing the two groups. further, as mentioned earlier, ifn-γ was not detected above the threshold value in the controls or patient samples. this study aimed to identify inflammatory genes associated with hmpv mediated disease. we performed an analysis of npas and blood samples collected from children hospitalized with rti. herein, we identify specific cytokines, such as ip-10, il-18, tnf-α, and isg54, that are elevated in hmpv mediated lung disease. of noteworthy interest is the up-regulation of il-1β and nlrp3 in samples from children with the most severe respiratory tract infection. this suggests that the inflammasome is involved in the innate immune response to hmpv in these patients, either as a protective mechanism or as a response to lung damage. type i and iii ifns are responsible for the induction of an antiviral state in cells immediately after infection [20] . while type i ifns are produced by many different cell types, type iii ifns seems to be mostly produced by plasmacytoid dendritic and epithelial cells where the epithelial cells are the main targets for activity [21] . due to their powerful and potentially harmful effects, the production of these cytokines is tightly regulated [22] . our results showed that the type i ifn-β mrna in npa was marginally up-regulated for the a2 infected patients, but the protein concentrations in plasma were markedly increased for both a2 and b2 positive patients. this inconsistency could be explained by the protein being produced by tissue resident cells in e.g. the lung or lymph nodes. another explanation could be a lag in the induction of protein synthesis in the epithelial cells after the elevation of mrna transcription, since it is assumed that cytokines are able to enter the bloodstream from lung tissue during inflammation. for type iii ifn il-28 we observed increased mrna expression both for a2 and b2 infected children. however, il-28 protein was not significantly elevated in blood plasma. il-28 is prominently expressed in lung epithelial cells, and suggested to prevent viral invasion through skin and mucosal surfaces [23] . hence, the lack of increased il-28 protein in blood could, similar as suggested for ifn-β, be explained by a lag in the induction of protein synthesis. the expression of il-28 has been shown to be triggered by viral infection, and it displays immunological properties similar to the type i ifn-α/β [23] . recently, it has been shown that hmpv induces expression of il-28 in mice and the cytokine was suggested to have an important protective role [24] . for rsv, which is phylogenetically and clinically closely related to hmpv, an in vitro study has shown that il-28, in contrast to ifn-α/β, is induced during infection of primary respiratory tract cells [25] . further, a study on infants hospitalized with rsv infection and bronchiolitis showed induced expression of il-28 in the airways [26] . overall, our results adds to existing data suggesting that ifn-β and il-28 are induced to some extent during hmpv infection. chemoattractants are induced during acute inflammation events to recruit effector cells of the immune system. in our study, elevated mrna expression of the chemoattractant ip-10 was observed for a2 and b2 infected patients. further, elevated levels of ip-10 protein, and also of the chemokines gro-α and gro-β were found in blood plasma. ip-10 has previously been shown to be significantly elevated seven days post infection with hmpv a2 in mice [27] . the expression of ip-10 may be induced by ifn-γ [28] , ifn-α/β [29] or tnf-α [30] secreted by different immune-cells. in our study, despite elevated ip-10, we did not measure any increased expression of the type ii interferon ifn-γ at mrna or plasma protein levels. the lack of ifn-γ detection in npas from hmpv infected children has also recently been reported elsewhere [31] . our data suggests that ifn-β and tnf-α, which was significantly detectable in plasma, might be responsible for induction of ip-10. in addition, ifn-γ-independent induction of ip-10 has been demonstrated in human peripheral blood mononuclear cells [32] . in this setting the type iii interferon il-29, which is closely related to il-28 that was shown to be upregulated herein, was correlated to ip-10 induction. of note, when infecting human epithelial cells with hmpv in vitro, we observed that il-28 mrna was induced and that il-29 showed a similar trend (data not shown), as also suggested elsewhere [24] . hence, il-28/29 could mediate ifn-γ independent ip-10 induction in hmpv infection. ifn-γ-independent ip-10 expression has also previously been reported in bal samples from patients during respiratory tract virus infection [33] . we found that mrna expression of the pro-inflammatory cytokine tnf-α was elevated only in the b2-infected children. however, the level of tnf-α protein in plasma was significantly increased both for a2 and b2 infected patients. an explanation for this could be different expression kinetics for mrna and protein. tnf-α induces nf-κb [34] and, among other functions, it is involved in the recruitment of neutrophilic cells to the lungs [35] . tnf-α production has previously been shown to be at high levels in nasopharyngeal secretions from rsor influenza-virus infected children [36] . when comparing the patients with a severity index of 0 and those with an index of 1-4, tnf-α had significantly higher levels of expression (p<0.001, student's t-test). this could indicate that the presence of tnf-α in the respiratory tract directly or indirectly affects the severity of disease in hmpv infected children. it is worth mentioning that the hmpv c t value was similar for the patients with severity index 0 and higher (s1 table) , suggesting similar extent of viral replication in these patients. we did not detect any increase in mrna levels of the pro-inflammatory cytokine il-6. on the other hand, the levels of il-6 in blood plasma were significantly increased for the hmpv infected patients. similar as for ifn-β, this observation could be explained by il-6 protein being produced by tissue-resident cells or, alternatively, downregulation of gene expression after a peaked response. a previous study comparing the expression of several inflammatory cytokines in hmpv, rsv and influenza virus, detected elevated levels of tnf-α, il-6 and il-1β protein in nasal washes from infants with rti [9] . together with our data showing increased detection of these proteins in blood from patients with rti compared to the controls, this could suggest that at least a part of the protein detected in blood might have been secreted from cells in the respiratory tract. the antiviral cytokine isg54 was expressed at the highest relative levels of any of the genes included in this study. the mrna expression was up-regulated for both hmpv a2-and b2-positive npa samples. isg54 is known to be induced by viruses downstream of pattern recognition receptors and ifns [37] . the expression of isgs is initiated by type i and type iii interferons such as ifn-β and il-28. we did indeed find that these ifns were up-regulated at mrna and/or protein level in hmpv infected children. the functional effects and clinical involvement of isg54 are largely unknown. nevertheless, isg54 has been shown to inhibit viral replication in vitro and in vivo [38] . interestingly, mice lacking isg54 have recently been shown to be subject to high mortality when infected with the mouse respiratory tract pathogen sendai virus-a negative sense, single-stranded rna paramyxovirus [39] . this suggests that isg54 has a protective function and limits pathogenesis in mice. however, in our study, we were not able to identify any differences with respect to length of hospitalization or severity index when comparing the groups of patients expressing isg54 or not (p>0.05, student's ttest), suggesting a general protective function of this cytokine in children. our study reveal that most of the hmpv-infected children exhibited low levels of mrna coding for inflammasome-associated proteins, except for il-18 which was induced in both a2 and b2 infected patients, both at mrna and protein levels. interestingly, some patients showed distinct expression profiles with elevated levels of several genes involved in the inflammasome mediated immune-response. more specific, approximately 1/4 of the patients showed increased expression of iκbα, il-1β and nlrp3. as mentioned earlier, the repressor gene iκbα expression is induced by nf-κb activation [19] . nf-κb induces mrna expression of the potent proinflammatory cytokines pro-il-1β and pro-il-18 [40] . further, increased level of nlrp3 mrna expression is the initial essential step in activating the nlrp3-inflammasome. taken together, the increased levels of nlrp3 mrna in this group of children along with enhanced il-1β and il-18 mrna expression and protein secretion could suggest that the nlrp3-inflammasome is activated to produce il-1β and il-18 in the respiratory tract of these patients. previously, it has been shown that influenza virus is able to activate the nlrp3-inflammasome in mice [14] . this was found to be critical for survival and reduction of viral titers in mice lung. further, rsv was recently shown to activate the nlrp3-inflammasome in vitro using primary human lung epithelial cells [15] . our results show that the hmpv infected children with a severity score exceeding zero more frequently exhibited elevated levels of il-1β and nlrp3 than children with severity score zero. this suggests a possible involvement of these genes in hmpv infected children with severe disease. as mentioned earlier, previous studies on rsv have shown a variation in disease severity for different genotypes, but similar studies on hmpv have been inconclusive [5] [6] [7] [8] . consequently, we wanted to see if our data suggested any differences in gene expression or disease severity for the hmpv a2 and b2 genotypes. however, we did not detect any differences between a2 and b2 with respect to expression profiles except for ifn-β and tnf-α which was significantly higher expressed in a2 and b2 positive patients, respectively, at mrna level. further, we did not detect any differences in the length of hospital stay or disease severity in the a2 and b2 infected children. our study population includes infants and up to 8 years old children. consequently there is a risk that children with both primary and secondary hmpv infections are included. these might experience different immune responses. to address this we compared children less than one year of age with older children. we found no differences in gene expression profiles (data not shown). in addition, the average age of children with severity score 1-4 was not significantly different from those with severity score zero (p = 0.28, student's t-test), indicating that the youngest children where not more disposed to severe disease compared to the older. of note, there was a difference in age distribution between hmpv-infected and control children. the controls were symptom-free and screened for the same airway pathogens as the patient samples, and no pathogens were detected. hence, we would not anticipate that the inflammatory genes are expressed above a basal level in the controls. further, to our knowledge, no studies so far have shown that basal expression is significantly different in children less than one year and older. our use of clinical samples from selected, well-classified patients is a strength of the study. for studies aimed to examine immune responses to a particular viral infection in vivo it is a challenge, especially in children, to obtain sufficient number of samples collected at similar conditions. in particular, interfering viral and bacterial co-infections are common. in our study population extensive viral analyses of each sample revealed that nearly half of the population had viral co-infections and were excluded from further analysis. in addition, bacterial infections were unlikely because most children had received one or more conjugated pneumococcal vaccinations, and only patients with maximal crp levels less than 100 mg/l were included. furthermore, we only included samples that were collected within the first week after onset of symptoms. finally, separating the samples based on the two most frequent hmpv genotypes enabled us to see if there was a genotype-specific cytokine gene expression profile or differences in disease severity. this allowed us to study the initial host immune response towards particular hmpv genotypes, which extends current literature. however, our selection criteria limited the total number of patients that were eligible for our study. consequently, it will be necessary to confirm our findings in other cohorts, in particular with respect to inflammasome-associated genes in children with severe rti. in summary, our study has identified inflammasome components that may be key players in the innate immune response against hmpv in children. the recent discovery of nlrp3 inhibitors [41] that may prevent or even treat rti-mediated inflammation, emphasizes the need to establish the role of inflammasomes in the response to rti in children. our study provide an important contribution in this regard. supporting information s1 a newly discovered human pneumovirus isolated from young children with respiratory tract disease outbreak of human metapneumovirus infection in norwegian children human metapneumovirus: lessons learned over the first decade novel human metapneumovirus sublineage correlation between respiratory syncytial virus genotype and severity of illness genetic variability of human metapneumovirus infection: evidence of a shift in viral genotype without a change in illness differences in clinical severity between genotype a and genotype b human metapneumovirus infection in children comparison of risk factors for human metapneumovirus and respiratory syncytial virus disease severity in young children differential production of inflammatory cytokines in primary infection with human metapneumovirus and with other common respiratory viruses of infancy cytokines and chemokines in respiratory secretion and severity of disease in infants with respiratory syncytial virus (rsv) infection regulation of inflammasome signaling the role of the nlrp3 inflammasome in the pathogenesis of airway disease lethal influenza virus infection in macaques is associated with early dysregulation of inflammatory related genes the nlrp3 inflammasome mediates in vivo innate immunity to influenza a virus through recognition of viral rna human respiratory syncytial virus viroporin sh: a viral recognition pathway used by the host to signal inflammasome activation coronavirus causes lower respiratory tract infections less frequently than rsv in hospitalized norwegian children real-time reverse transcriptase pcr assay for detection of human metapneumoviruses from all known genetic lineages molecular epidemiology of human metapneumovirus in ireland i kappa b: a specific inhibitor of the nf-kappa b transcription factor induction and function of type i and iii interferon in response to viral infection interferon-[lambda] in the context of viral infections: production, response and therapeutic implications posttranscriptional control of type i interferon genes by ksrp in the innate immune response against viral infection ifn-lambda (ifn-λ) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo impact and regulation of lambda interferon response in human metapneumovirus infection type-iii interferon, not type-i, is the predominant interferon induced by respiratory viruses in nasal epithelial cells interferon lambda 1-3 expression in infants hospitalized for rsv or hrv associated bronchiolitis pulmonary infection of mice with human metapneumovirus induces local cytotoxic t-cell and immunoregulatory cytokine responses similar to those seen with human respiratory syncytial virus biochemical characterization of a gamma interferon-inducible cytokine (ip-10) interferon-independent, human immunodeficiency virus type 1 gp120-mediated induction of cxcl10/ip-10 gene expression by astrocytes in vivo and in vitro essential involvement of cross-talk between ifn-γ and tnf-α in cxcl10 production in human thp-1 monocytes premature infants have impaired airway antiviral ifn[gamma] responses to human metapneumovirus compared to respiratory syncytial virus interferon lambda-1 (ifn-[lambda]1//il-29) induces elr-cxc chemokine mrna in human peripheral blood mononuclear cells, in an ifn-[gamma]-independent manner detection of respiratory viruses and the associated chemokine responses in serious acute respiratory illness tnf activates nf-κb by phosphatidylcholine-specific phospholipase c-induced "acidic" sphingomyelin breakdown role of p38 mitogen-activated protein kinase in a murine model of pulmonary inflammation severe human lower respiratory tract illness caused by respiratory syncytial virus and influenza virus is characterized by the absence of pulmonary cytotoxic lymphocyte responses the broad-spectrum antiviral functions of ifit and ifitm proteins ifit1 is an antiviral protein that recognizes 5[prime]-triphosphate rna sendai virus pathogenesis in mice is prevented by ifit2 and exacerbated by interferon tlrs, nlrs and rlrs: a trinity of pathogen sensors that co-operate in innate immunity inflammasome inhibition: putting out the fire we would like to thank the research nurses at the children's clinic (st. olav's hospital) and the technicians at the department of medical microbiology (st. olav's hospital). key: cord-336420-1a2u9p4t authors: söderman, martina; rhedin, samuel; tolfvenstam, thomas; rotzén-östlund, maria; albert, jan; broliden, kristina; lindblom, anna title: frequent respiratory viral infections in children with febrile neutropenia a prospective follow-up study date: 2016-06-16 journal: plos one doi: 10.1371/journal.pone.0157398 sha: doc_id: 336420 cord_uid: 1a2u9p4t objective: febrile neutropenia is common in children undergoing chemotherapy for the treatment of malignancies. in the majority of cases, the cause of the fever is unknown. although respiratory viruses are commonly associated with this condition, the etiologic significance of this finding remains unclear and is therefore the subject of this study. study design: nasopharyngeal aspirates were collected during 87 episodes of febrile neutropenia in children age 0–18 years, being treated at a children’s oncology unit between january 2013 and june 2014. real-time polymerase chain reaction was used to determine the presence of 16 respiratory viruses. follow-up samples were collected from children who tested positive for one or more respiratory viruses. rhinoviruses were genotyped by vp4/vp2 sequencing. fisher’s exact test and mann-whitney u test were used for group comparisons. results: at least one respiratory virus was detected in samples from 39 of 87 episodes of febrile neutropenia (45%), with rhinoviruses the most frequently detected. follow-up samples were collected after a median of 28 days (range, 9–74 days) in 32 of the 39 virus-positive episodes. the respiratory viral infection had resolved in 25 episodes (78%). the same virus was detected at follow-up in one coronavirus and six rhinovirus episodes. genotyping revealed a different rhinovirus species in two of the six rhinovirus infections. conclusion: the frequency of respiratory viral infections in this group of patients suggests an etiologic role in febrile neutropenia. however, these findings must be confirmed in larger patient cohorts. febrile neutropenia is a common complication in children undergoing chemotherapy for the treatment of malignancies. because septicemia (which is potentially lethal) is difficult to rule out at the onset of fever, empiric treatment with broad-spectrum antibiotics is promptly initiated based on wide indications [1] . however, in most cases, no underlying cause of the fever can be identified [2] . febrile neutropenia is associated with long hospitalization [3] [4] [5] which has negative social effects for the child and its family [6] . in addition, hospitalization and the use of broad-spectrum antibiotics increase the patient's risk of subsequent infection with antibiotic-resistant bacteria [7, 8] and fungal infections [9, 10] . a better understanding of the etiology of febrile neutropenia is thus needed in order to decrease unnecessary hospitalization and excessive antibiotic use. as infections with respiratory viruses are a common morbidity in children [11] respiratory viruses likely play a significant role in childhood febrile neutropenia. advances in molecular methods have increased the sensitivity of viral diagnostics tests, with recent studies reporting the detection of respiratory viruses in the nasopharynx in 44-57% of childhood febrile neutropenia episodes using real-time polymerase chain reaction (pcr) [3] [4] [5] 12] . however, the clinical significance of positive pcr findings is unclear, as respiratory viruses have also been detected in asymptomatic immunocompetent children [13] [14] [15] [16] . in addition, some respiratory viruses can be detected weeks after the first infection, which is suggestive of prolonged viral shedding [17, 18] . here, we describe the results of a longitudinal study involving repeated sampling and the assessment of a broad panel of respiratory viruses by pcr to clarify whether respiratory viruses play a causal role in childhood febrile neutropenia. children aged 0-18 years, who were treated for a malignancy at the childhood cancer unit at astrid lindgren children's hospital in stockholm, sweden, between january 2013 and june 2014, were eligible for enrollment in this study. all the patients who met the criteria for febrile neutropenia were asked to participate. patients could be enrolled multiple times if recurrent episodes of febrile neutropenia occurred during the study period. to be enrolled for a new episode, the patient needed to have been afebrile for more than 72 hours and have completed the antibiotic treatment for the previous episode of febrile neutropenia. febrile neutropenia was defined as a body temperature of 38.5°c on one occasion or 38.0°c on two occasions at least 60 minutes apart, combined with an absolute neutrophil count of either 0.5×10 9 /l on one occasion or 1.0×10 9 /l with a decline to less than 0.5×10 9 /l over a subsequent 48-hour period. oral and written information regarding the study were provided to each patient prior to enrollment, and signed consents were obtained from their caretakers. the study was approved by the regional ethical review board in stockholm. university laboratory (iso: 15189:2012) for microbiological analysis. viral nucleic acids were extracted from the npa with a magattract virus mini m48 kit (qiagen, sollentuna, sweden) and analyzed with in-house real-time pcrs for the following 16 viruses: adenovirus (hadv); bocavirus (hbov); coronaviruses nl63/oc43/229e/hku1 (hcov); enterovirus (ev); influenza virus a, including a(h1n1)pdm09 and b (flu); metapneumovirus (hmpv); parainfluenza viruses 1-3 (piv); respiratory syncytial virus (rsv) and rhinovirus (rv) [19] . a semiquantitative assay was used and the actual cycle thresholds-values (ct-values) were provided, however, the npa has not been validated for quantitative data. in all study subjects who initially tested positive for one or more respiratory viruses, a follow-up sample was collected at the time of their next visit to the hospital. this follow-up sample was collected regardless of the patient's absolute neutrophil count or symptoms and was analyzed in the same manner as the first sample. however, if the patient had a new episode of febrile neutropenia at the time of the follow-up, this sample was considered both a follow-up sample and a new episode sample, which required an additional follow-up sample if the new episode tested positive for a respiratory virus. rv/ev genotyping was performed by vp4/vp2 sequencing for samples that were pcr-positive for rv and/or ev using a recently published method [20] based on a method and primers originally published by wisdom et al. [21] . briefly, viral rna was extracted as described above for most samples, but the rneasy lipid tissue mini kit (qiagen, sollentuna, sweden) was used for samples that were negative in the vp4/vp2 pcr after magattract extraction. the rna was used for the nested reverse transcriptase (rt)-pcr with one-step superscript-platinum taq (life technologies, stockholm, sweden) for the first (outer) pcr, and platinum taq for the second (nested) pcr. sequencing was done on an abi 3730 instrument and the rv/ev species and type was determined by maximum likelihood phylogenetic trees constructed using phyml [22] and the sequences have been deposited in genbank under accession numbers kx15472-kx154585 for rv-a and kx290514-kx290520 for rv-c. two of the rv-c samples were not submitted to the genbank because the sequences were of suboptimal quality due to a probable infection with more than one rv genotype. blood cultures were collected from all patients for the detection of bacterial infections and analyzed at the karolinska university laboratory, as per routine clinical procedures. clinical data, including the results of biochemical and microbiological analyses, patient's characteristics, treatment during the febrile episode, fever characteristics, and the duration of hospitalization, were collected from the medical records. data were analyzed using graphpad prism 6.0 software (graphpad prism, san diego, ca). fisher's exact test and the mann-whitney u test were used for group comparisons of categorical and continuous data, respectively. a p-value less than 0.05 was considered statistically significant. a total of 56 patients representing 92 episodes of febrile neutropenia were enrolled in the study. five episodes were excluded due to incomplete sampling. therefore, the analyses included 54 patients with 87 episodes of febrile neutropenia (ranges, 1-5 episodes per patient and 8-214 days between episodes). the characteristics of the study participants are listed in table 1 . the median age across all episodes was 7 years (range, 0.5-17.7 years), and 59% of the episodes occurred in females. the patient was undergoing treatment for a hematologic malignancy in 51% of the episodes and for a solid tumor in 49% of the episodes. respiratory symptoms were noted in 70% of the episodes of febrile neutropenia ( table 1) . at least one respiratory virus was identified in 39 (45%) of the 87 episodes. a single respiratory virus was detected in 34 (39%) of the episodes (table 1) . multiple viruses (range, 2-4 viruses) were detected in two episodes (2%), whereas co-presence with a respiratory virus and septicemia was detected in three episodes (3%) ( table 1) . of the 87 episodes, rv was the most frequently detected respiratory virus (n = 21, 24%), followed by hcov (n = 7, 8%), flu (n = 4, 5%), rsv (n = 3, 3%), piv (n = 3, 3%), hmpv (n = 2, 2%), hbov (n = 2, 2%), and hadv tumor type 0.14 0.04 hematologic d malignancy days with antibiotics (n = 1, 1%) ( table 2 ). in 12 of the virus-positive episodes, the patient was <4 years of age. the most common respiratory virus identified in the group <4 years of age was rv (n = 9), followed by piv (n = 2), hcov (n = 2), hadv (n = 1), hbov (n = 1) and rsv (n = 1). in addition, one episode with co-presence with a respiratory virus and septicemia was identified in the group <4 years of age. of the 36 episodes involving only respiratory viral infection (i.e., infection with either a single respiratory virus or multiple respiratory viruses) respiratory symptoms were detected in 31 (86%) ( table 1) . no symptoms were apparent in four episodes involving rv and one episode involving hcov (table 2) . of the 31 episodes involving respiratory symptoms, 26 reported the appearance of respiratory symptoms at time of fever onset ( table 2 ). in the remaining five episodes, the respiratory symptoms appeared over 6 days before the onset of fever. these five episodes represented one flu b and four rv ( table 2 ). bacterial blood cultures were positive in 13 (15%) of the episodes of febrile neutropenia. five cultures were excluded from further analysis and not defined as septicemia because they were determined to be either contaminants or of no clinical relevance by the treating clinician and/ or the laboratory: micrococcus species (n = 1), coagulase-negative staphylococcus (n = 1), staphylococcus epidermidis (n = 2), and unspecified gram-positive bacteria (n = 1). eight episodes were therefore considered true septicemia. of these, five episodes involved only septicemia (6%) ( table 3 ) (i.e. they tested negative for respiratory viruses by pcr). three episodes involved co-presence with a respiratory virus and septicemia; gram-positive bacteria (staphylococcus epidermidis (n = 2) and alpha streptococcus (n = 1)) were detected in all three episodes and one episode was also positive for gram-negative bacteria (escherichia coli). in all three episodes involving co-presence of respiratory virus and septicemia the patients were being treated for a hematologic malignancy, and all had respiratory symptoms ( table 1 ). the viruses found in this group were rv (n = 2) and hbov (n = 1) ( table 2 ). in 43 episodes (49%), no respiratory virus or septicemia were detected and the febrile episode was therefore defined as a fever of unknown origin (table 1) . when comparing episodes involving only respiratory viral infection (i.e., infection with either a single respiratory virus or multiple respiratory viruses) with episodes involving only septicemia or fever of unknown origin, no statistically significant differences were observed with respect to age, gender, days with fever, or maximum temperature (table 1) . however, the presence of respiratory symptoms was significantly higher in the episodes involving only respiratory viral infection (86%) compared with both the episodes involving only septicemia (40%) and fever of unknown origin (58%) (p = 0.043 and p = 0.007, respectively). the episodes with only respiratory viral infection were also more often treated for a hematological malignancy (58%) compared to the fever of unknown origin episodes (35%) (p = 0.04 (table 4) . a new respiratory virus was detected in six follow-up samples: flu a (n = 2), rv (n = 2), hcov (n = 1), and rsv (n = 1). of these six, the episodes for all except one involving rv exhibited respiratory symptoms, and one (flu) represented a new episode of febrile neutropenia. rv genotyping was performed to determine whether the episodes exhibiting repeated pcr positivity involved a new or persistent infection. of samples from the 21 rv-positive episodes, 20 were successfully sequenced, resulting in the identification of 11 rv-a and 9 rv-c species ( table 2 ). before sequencing, four patients, representing six episodes, remained rv-positive at follow-up (table 4 ). one patient was rv-positive in three follow-up samples; however sequencing revealed that two of these follow-up samples contained a new rv specie (fig 1) . sequencing also revealed that the three remaining patients all had the same genotype (fig 1) . all of the rv-c had cleared by the time of follow-up (table 2) , and the four persistent episodes involving rv were associated with rv-a infections ( table 2 ). to the best of our knowledge, this is the first longitudinal study to assess respiratory viral persistence in children presenting with febrile neutropenia. we found that respiratory viral infections are common in children with febrile neutropenia, in the majority of episodes accompanied by respiratory tract symptoms and that the infection had resolved at follow-up in the majority of the episodes. these results support the theory holding that there is a causal relationship between respiratory viral infections and episodes of febrile neutropenia, but proving this theory will require more longitudinal studies with asymptomatic neutropenic control cohorts. in addition, prolonged viral persistence with regard to these respiratory tract infections seems to be uncommon in children undergoing chemotherapy for the treatment for malignancies. in a previous study we detected respiratory viruses in 46% of episodes of febrile neutropenia [3] . subsequent studies have confirmed this finding, with detection rates ranging between 52-57% [4, 5] . the detection rate of 45% in our current study is consistent with earlier studies. it is crucial to correctly diagnose febrile neutropenia in immunosuppressed children, as infections with viruses [23, 24] , bacteria [25, 26] or fungi can be fatal [26] in this group of patients. the clinical significance of a single time-point pcr finding of certain pathogens has been debated. studies on immunocompetent children have reported that viruses such as hbov, hcov, and rv are detected in asymptomatic children at rates ranging between 27-40% [13] [14] [15] [16] , which could be suggestive of prolonged viral shedding from an earlier infection or the incubation period prior to a symptomatic[17] , or asymptomatic infection [13, 14, 17] . to clarify this issue, more longitudinal studies with appropriate control groups are needed. rhedin et al. conducted a case-control study to investigate the etiologic role of respiratory viruses in a group of children with respiratory tract symptoms seeking medical care at a pediatric emergency center [13] . the control group consisted of children on routine visits to child welfare centers for vaccination within the childhood immunization program. the detection rate of respiratory viruses in the control group was high (35.4%) with rv and hcov the most commonly detected viruses in asymptomatic children and hmpv, piv and rsv detected only rarely. the authors concluded that hmpv, piv and rsv play an etiologic role and suggested that findings of rv and hcov must be carefully interpreted [13] .we chose a longitudinal design for the present study, and found that 78% of the infections had resolved by the median follow-up time of 28 days. in addition, two of the rv infections identified at follow-up represented new species of rv from the previous infection, which increases the proportion of resolved infections to 84%. one major limitation of the current study was the lack of a control cohort of neutropenic patients without fever to address the question as to whether respiratory viruses are frequently found in asymptomatic immunosuppressed children. however, the use of a longitudinal design allowed us to collect follow-up samples from patients both with and without respiratory symptoms. only five patients at the first sampling and two patients at follow-up sampling were positive for a respiratory virus without the presence of respiratory symptoms, which suggests that the frequency of asymptomatic infections is low in this group of patients. interestingly, rv was detected in five of these seven episodes and hcov was detected in the other two, which are in line with other studies [13] . the presence of respiratory symptoms was significantly higher in the episodes involving only respiratory viral infection, compared with both episodes involving only septicemia and fever of unknown origin, with symptoms appearing with the onset of fever in the majority of the episodes. peck et al. investigated the incidence of respiratory virus in patients receiving a hematopoietic stem cell transplant using longitudinally collected respiratory tract samples regardless of symptoms [27] . in line with our results, a majority of the respiratory viruses detected could be correlated with respiratory tract symptoms, with the exception of piv, which was detected in asymptomatic patients. infection with rv or hcov was not investigated in that study. in addition, high viral loads were correlated to more symptoms. our real-time-pcr technique was not validated for use in reporting clinical viral loads; thus the presented ct-values must be interpreted with extreme caution. nevertheless, the ct-values <30 observed in 27 of the 39 episodes of febrile neutropenia are suggestive of high viral loads ( table 2) . considered together, our data suggests that respiratory viruses play an etiologic role in febrile neutropenia, but our results should be interpreted carefully, especially those regarding rv, which is commonly detected in asymptomatic patients [13] . despite the above-mentioned limitations, we believe that our findings of a respiratory virus together with other clinical parameters such as respiratory symptoms, negative blood cultures after 48 hours, and a history of short duration of myelosuppression, may help to reduce the treatment time with broad-spectrum antibiotics and the need for prolonged hospitalizations. we were particularly interested in rv in our study because it was the most frequently detected virus, was still detected at follow-up, and has been reported in asymptomatic immunocompetent children [13] [14] [15] [16] . the clinical impact of rv has been correlated with severe respiratory diseases in children under 5 years of age [28] . in our study, only the a and c species of rv were identified. these species are believed to be more pathogenic and more strongly associated with hospital treatment than rv-b [28] [29] [30] . rv-a and rv-c causing both upper and lower respiratory tract infections were the most frequently detected species in another study that investigated rv infection in immunosuppressed children [31] . in the only case of rv-b reported in that study, the patient had an upper respiratory tract infection. in our study cohort, all episodes with a detected rv infection were hospitalized as a result of their febrile neutropenia, and therefore the severity of the disease in relation to the rv specie was difficult to evaluate. respiratory viruses and their shedding times have not been thoroughly investigated in immunosuppressed children. earlier studies on rsv have shown prolonged viral shedding in immunosuppressed children compared with healthy children [32] . martin et al. conducted a longitudinal study of healthy immunocompetent children attending a daycare center [33] and found prolonged shedding (i.e., persistent virus >7 days) of all viruses examined except flu a and b. jartti et al. reported shedding times of 2-3 and 5-6 weeks for ev and rv, respectively, in immunocompetent children [17] . however, rv was not sequenced in that study; therefore, it is unclear whether the sample at 5 weeks represented the same or a new genotype of rv. another study reported that prolonged persistence (>30 days) of the same rv strain is uncommon (<5%) [34] . shedding times were of special interest in our cohort of immunosuppressed children because viral shedding necessitates isolation from other immunosuppressed children. the design of our present study did not allow us to determine the exact shedding time, which is a major limitation. however, the clearance of all viruses except rv-a and hcov at a median follow-up time of 28 days, together with respiratory symptoms appearing at the time of fever onset in a majority of the patients, suggests that the shedding time for viruses such as flu, hmpv, rsv and piv is limited. the same rv genotypes were detected from follow-up samples in four episodes, with a follow-up time of 12-51 days. furthermore, five patients positive for rv reported symptom appearance 6 days or longer before fever onset. that could indicate longer shedding times for rv, which is in line with results from studies on immunocompetent children [17] . however, this needs to be addressed in additional studies with repeated followup sampling, preferable on a weekly basis. hbov has been associated with acute wheezing in immunocompetent children [35] . in immunosuppressed children, hbov has been detected both together with other viruses and also detected repeatedly, suggesting prolonged shedding or reactivation [36, 37] . in this study, hbov was detected twice, with hadv, rsv and rv in one episode and together with septicemia in another episode; both episodes involved respiratory symptoms. at follow-up sampling, hbov was no longer detectable. in our previous study [3] , we detected hbov in three episodes: together with one other virus (rv) with septicemia and as the only agent, making it difficult to ascertain the clinical relevance. our data strengthen the evidence suggesting that respiratory viruses play an etiologic role in febrile neutropenia in children receiving treatment for a malignancy. if confirmed in future studies, the findings reported here will have implications for the clinical management of these patients. our finding could lead to a decrease in the duration of both hospitalization and treatment with broad-spectrum antibiotics, leading to positive social effects for the child and his or her family as well as decreasing the risk of subsequent infection with antibiotic-resistant bacteria. clinical practice guideline for the use of antimicrobial agents in neutropenic patients with cancer: 2010 update by the infectious diseases society of america a prospective study on the epidemiology of febrile episodes during chemotherapy-induced neutropenia in children with cancer or after hemopoietic stem cell transplantation respiratory viruses, a common microbiological finding in neutropenic children with fever frequency and clinical outcome of respiratory viral infections and mixed viral-bacterial infections in children with cancer, fever and neutropenia nasopharyngeal detection of respiratory viruses in febrile neutropenic children health-related quality of life anticipated with different management strategies for paediatric febrile neutropaenia european guidelines for empirical antibacterial therapy for febrile neutropenic patients in the era of growing resistance: summary of the antibiotic resistance is associated with longer bacteremic episodes and worse outcome in febrile neutropenic children with cancer invasive fungal infections in paediatric acute myeloid leukaemia the 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respiratory samples for detection of human rhinoviruses (hrvs) and enteroviruses: comprehensive vp4-vp2 typing reveals high incidence and genetic diversity of hrv species c new algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of phyml 3.0 management of respiratory viral infections in hematopoietic cell transplant recipients and patients with hematologic malignancies influenza a/h1n1 in pediatric oncology patients pathogenesis of bloodstream infection in children with blood cancer a prospective study of septicaemia on a paediatric oncology unit: a three-year experience at the royal liverpool children's hospital, alder hey, uk respiratory virus infection among hematopoietic cell transplant recipients: evidence for asymptomatic parainfluenza virus infection human rhinovirus species associated with hospitalizations for acute respiratory illness in young us children human rhinovirus c infections mirror those of human rhinovirus a in children with community-acquired pneumonia high prevalence of human rhinovirus c infection in thai children with acute lower respiratory tract disease human rhinovirus c infections in pediatric hematology and oncology patients respiratory syncytial viral infection in children with compromised immune function epidemiology of multiple respiratory viruses in childcare attendees duration of rhinovirus shedding in the upper respiratory tract in the first year of life clinical assessment and improved diagnosis of bocavirus-induced wheezing in children human bocavirus in children with acute lymphoblastic leukemia persistence of human bocavirus dna in immunocompromised children key: cord-344870-tbgqulpu authors: lloyd-smith, james o. title: maximum likelihood estimation of the negative binomial dispersion parameter for highly overdispersed data, with applications to infectious diseases date: 2007-02-14 journal: plos one doi: 10.1371/journal.pone.0000180 sha: doc_id: 344870 cord_uid: tbgqulpu background: the negative binomial distribution is used commonly throughout biology as a model for overdispersed count data, with attention focused on the negative binomial dispersion parameter, k. a substantial literature exists on the estimation of k, but most attention has focused on datasets that are not highly overdispersed (i.e., those with k≥1), and the accuracy of confidence intervals estimated for k is typically not explored. methodology: this article presents a simulation study exploring the bias, precision, and confidence interval coverage of maximum-likelihood estimates of k from highly overdispersed distributions. in addition to exploring small-sample bias on negative binomial estimates, the study addresses estimation from datasets influenced by two types of event under-counting, and from disease transmission data subject to selection bias for successful outbreaks. conclusions: results show that maximum likelihood estimates of k can be biased upward by small sample size or under-reporting of zero-class events, but are not biased downward by any of the factors considered. confidence intervals estimated from the asymptotic sampling variance tend to exhibit coverage below the nominal level, with overestimates of k comprising the great majority of coverage errors. estimation from outbreak datasets does not increase the bias of k estimates, but can add significant upward bias to estimates of the mean. because k varies inversely with the degree of overdispersion, these findings show that overestimation of the degree of overdispersion is very rare for these datasets. the negative binomial (nb) distribution has broad applications as a model for count data, particularly for data exhibiting overdispersion (i.e. with sample variance exceeding the mean). in the biological literature, classical uses of the nb distribution include analysis of parasite loads, species occurrence, parasitoid attacks, abundance samples and spatial clustering of populations [1] [2] [3] [4] [5] [6] [7] . the range of applications of the nb distribution was extended recently to include the epidemiology of directly-transmitted infections, as the nb distribution was shown to be a suitable model for the 'offspring distribution' for a number of disease transmission datasets [8] . the offspring distribution, a concept arising in the theory of branching processes [9] , is the probability distribution for the number of individuals (termed 'secondary cases') infected directly by each infectious individual in a disease outbreak. estimation of nb parameters for empirical offspring distributions revealed a high degree of overdispersion-particularly for severe acute respiratory syndrome (sars), measles, and smallpox-signalling an unexpectedly large influence of individual variation and 'superspreading' on the dynamics of disease emergence [8] . however, the authors emphasized the challenges inherent in estimating nb parameters and the confidence intervals (cis) associated with those estimates, and noted that previous work on nb parameter estimation had not explored the parameter ranges of interest for epidemiological studies. a particular concern is whether the results were influenced by small sample size in the datasets analyzed, or biases peculiar to disease transmission data. this study uses simulated data to assess the bias and precision of nb parameter estimates and the coverage accuracy of cis for highly overdispersed datasets, addressing the challenges of small datasets as well as potential biases arising in the data collection process. the popularity of the nb distribution is due largely to its ability to model count data with varying degrees of overdispersion. the distribution is commonly expressed in terms of the mean m and dispersion parameter k such that the probability of observing a non-negative integer x is the variance of the nb distribution is m (1+m/k), and hence decreasing values of k correspond to increasing levels of dispersion. the poisson distribution is obtained as kr', and the logarithmic series distribution is obtained as kr0 [1, 10] . when k = 1, the nb distribution reduces to the geometric distribution. note that recent work in the statistical literature uses the quantity a = 1/k due to its preferable properties for inference (discussed below), but studies applying the nb distribution in ecology and epidemiology are overwhelmingly posed in terms of k. accordingly, all calculations in this study were conducted using a, but all results and discussion are posed in terms of k. (confusingly, the term 'dispersion parameter' can refer to either k or a; other terms for k include 'shape parameter' and 'clustering coefficient'.) the dispersion parameter k is commonly used as an inverse measure of aggregation in biological count data [1] [2] [3] [4] [5] 8, 11, 12] , yet its estimation from finite datasets is a recognized challenge. many simulation studies have examined the efficacy of different estimators of nb parameters for finite datasets [11, [13] [14] [15] [16] 17 ; also see review in 14] , but owing to precedent most of these have focused on k$1 and hence do not apply to highly overdispersed data. one biologically motivated study did explore values of k,1 [16] , but it did not test the maximum-likelihood (ml) methods of estimation that have become standard owing to their asymptotic efficiency and low bias [12, 13, 17] . the small-sample accuracy of ml estimates of k has not been tested for nb distributions with moderate to high degrees of overdispersion. moreover, little attention has been paid to the accuracy of cis of such nb parameter estimates. the first aim of this study, therefore, is to investigate the bias, precision and ci coverage accuracy of ml estimates of k for small samples. the investigation focuses on datasets with k,1, to address the gap in existing studies, but results for k$1 are included to establish continuity with earlier work. the second aim is to investigate how estimates of k are affected by potential biases of the data collection process, in particular systematic under-counting of events and the selection bias inherent in disease outbreak data. the disease transmission datasets analyzed by lloyd-smith et al. [8] fell into two broad categories, surveillance and outbreak datasets, each of which presents challenges due to the processes by which data are generated and collected. surveillance datasets combine information about many separate introductions of a disease into a population of hosts. empirical offspring distributions can be constructed by counting the number of secondary cases infected by the first infectious individual in each outbreak, but ignoring all subsequent generations of transmission (which often are not reported in detail, or may be influenced by outbreak control measures). the resulting datasets are analogous to many other datasets in biology, compiling many independent records of unrelated events. datasets of this type can be affected by two broad classes of under-counting error. first, data points may be underestimated, due to the possibility that some of the secondary cases will be overlooked, misdiagnosed, or not traced to the individual that infected them. second, individuals who do not transmit the disease may be more likely to be missed by surveillance programs, because they do not initiate a cluster of cases and thus are less likely to attract the attention of health authorities. therefore instances of a particular value (i.e. x = 0, for no secondary cases) may be systematically under-counted in the surveillance samples. these two classes of under-counting error are common to many types of biological data [e.g. 18, 19, 20] . outbreak datasets, comprising the second category of disease transmission data, are more unique to epidemiology and disease ecology. offspring distributions drawn from outbreak data include the number of secondary cases caused by many individuals within a single disease outbreak. these datasets arise when several generations of epidemic spread (typically early in an outbreak, before control measures are imposed) are fully reconstructed by contact tracing, so the number of secondary cases caused by each infectious case can be determined. lloyd-smith et al. [8] showed that when the degree of infectiousness is highly overdispersed (e.g. when the offspring distribution is nb with k,1), many outbreaks will die out stochastically in their first few generations of spread. in such situations, the outbreaks that survive tend to be those where a highly infectious individual (i.e. an individual whose number of secondary cases is drawn from the right-hand tail of the offspring distribution) appears in the early generations [8] . because outbreak datasets necessarily are drawn from successful outbreaks, there is the possibility of selection bias for an increased proportion of exceptionally infectious individuals, or 'superspreaders' [21] . intuitively, this risk appears to be particularly acute for offspring distributions with lower mean values, for which the epidemic's growth is more dependent on chance. (note that the mean of the offspring distribution corresponds to the basic reproduction number r 0 of the disease [8, 22] ). four types of simulated datasets were examined. in all cases, the datasets comprised n values, x i (i = 1, 2, …, n), generated as described below. in the epidemiological context that motivated this study, these values x i correspond to the numbers of secondary cases that were infected by n different infectious individuals, but similar data could arise from many other processes. all simulations were conducted using matlab v6.1 (mathworks, cambridge ma). 2.1.1 negative binomial data because the nb random number generator in matlab v6.1 (nbinrnd) does not allow noninteger values of k, nb random variates were simulated using the fact that the nb distribution can be derived as a poisson distribution with gamma-distributed intensity, i.e. a poisson-gamma mixture [23, 24] . first, n values g i were drawn from a gamma distribution with mean m and dispersion parameter k. second, each of these values was used as the intensity parameter for a poisson random variate to yield a nb-distributed value x i , i.e. x i = poisson(g i ). random variates were generated using the matlab functions gamrnd and poissrnd. counting to simulate surveillance datasets with uniform under-counting of data, it was assumed that each secondary case can be missed by surveillance with a fixed probability p u . raw data were drawn from a nb distribution with parameters m and k, as described in section 2.1.1 above. each value x i was then decreased by an amount d i ,binomial(x i , p u ), generated using the matlab function binornd, to represent under-counting. zeroes to simulate the possible under-reporting of individuals who cause no secondary infections, it was assumed that all individuals who caused x i = 0 cases can be overlooked with some fixed probability p z , while all other individuals have their full case-count recorded. nb samples were generated as in section 2.1.1, then any value x i = 0 was deleted with probability p z and replaced by another nb random variate. if the new value was also 0, then it was again replaced with probability p z . this process was repeated until a sample of n values was generated, in which each remaining value x i = 0 had avoided replacement exactly once. 2.1.4 outbreak data to generate outbreak datasets, stochastic disease outbreaks were simulated as discrete-time branching processes with nb offspring distributions, using the method described by lloyd-smith et al. [8] . each outbreak was assumed to begin with a single infected individual, who transmits the disease to x 1 other individuals, where x 1 is drawn from a nb distribution with parameters m and k. each of these second-generation cases infects x i other individuals, where the x i are independent and identically distributed draws from the same nb offspring distribution; the number of cases in the third generation is then x x1z1 i~2 x i . this process was repeated until the cumulative number of cases exceeded n, and the x i values corresponding to the first n infectious cases were used as the simulated outbreak dataset. to mirror the selection bias in using real outbreak datasets of a given size, outbreaks were simulated repeatedly until the cumulative number of cases was n or higher. outbreaks that died out with fewer than n total cases were not used. large outbreaks were less likely when m,1, particularly for k.1 where extremely infectious individuals (who cause large superspreading events) were very rare. no results were reported for parameter sets for which fewer than 1 in 10 5 simulated outbreaks had n cases or more. otherwise, simulations were repeated until the desired number of datasets was obtained. for each of the above classes of simulated data, 10,000 simulated datasets were generated for each combination of the mean m = {0.5, 1.0, 3.0}, the dispersion parameter k = {0.1, 0.3, 0.7, 1.0, 3.0, 10.0}, and the sample size n = {10, 30, 100, 300}, in a full factorial design. datasets with no non-zero values of x i were rejected, as k cannot be estimated from all-zero data. for each simulated dataset, the ml estimate k was determined as described below. the 90% ci was calculated, and it was recorded whether the true value k fell within the ci, above its upper bound (termed a ci underestimate), or below its lower bound (a ci overestimate). the 90% ci was studied instead of the 95% interval because the more extreme values of k are most difficult to estimate accurately, and to match results presented in lloyd-smith et al. [8] . an extensive statistical literature exists on ml estimation of nb parameters [1, 10, 11, 13, 15, 17] . this work shows that it is better to make inferences about k indirectly via its reciprocal a = 1/k, for two reasons. first, use of the reciprocal avoids discontinuities for homogeneous datasets, because increasing homogeneity yields ar0 instead of kr'. indeed, there is a continuous transition to values a,0 corresponding to underdispersion (when sample variance is less than the mean), for which direct estimation of k is problematic [14, 25] . second, the sampling distribution for a tends to be more symmetric than that for k [13] (an example using outbreak data is shown in fig. si-1 of lloyd-smith et al. [8] ). in this study ml estimation was conducted for the parameter a, but results are reported in terms of k = 1/â because k is more familiar to epidemiologists and ecologists. estimates of â were restricted to positive values, because the allowed range for k was (0,'). underdispersed datasets were assigned the minimum value of â, corresponding to kr'. this approximation is reasonable because the study focuses on highly overdispersed nb distributions (with k,1); estimation of â for underdispersed data is discussed indepth elsewhere [14, 15, 17, 25] . the ml estimate of m is the sample mean, x [10] . the ml estimate of a was determined by unidimensional numerical maximization of the log-likelihood function [15] , conducted using the fminbnd function of matlab 6.1 over the interval (0.001,1000). the termination tolerance was set sufficiently small that negligible accuracy was lost in inverting the estimates, and direct ml estimates of k (obtained by maximizing the log-likelihood function derived from equation (1)) matched k = 1/â to beyond the fourth decimal place. reported estimates of k thus are drawn from the range (0.001,1000), which is much broader than the range of k commonly estimated from epidemiological data (e.g. the range of k was [0.032,5.1] in 11 uncontrolled outbreak datasets [8] , or [0.038,6.014] in 49 macroparasite burden datasets [4] ). nb distributions with k = 1000 and kr' (the poisson distribution) are indistinguishable in practice. confidence intervals for k were estimated from the asymptotic variance of the sampling distribution, given by the inverse of the information matrix [24] . for 11 outbreak datasets, intervals estimated in this way were very similar to those estimated using bias-corrected bootstrap methods (both parametric and nonparametric) and asymptotic variance for the zero-class estimator of k [8] . for ml estimates of k or â, the asymptotic sampling variances (s 2 k or s 2 a ) cannot be expressed in closed form but are easily calculated numerically [10, 17] . these variances are related by s 2 a~s 2 k .k 4 [13] . in this study s 2 a a was calculated for each simulated dataset, and the 90% ci for â was estimated as [â2z 0.95 s â , â+z 0.95 s â ], where z 0.95 is the 95 th percentile of the standard normal distribution [24] . the ci for k was generated by inverting and reversing the endpoints of the interval for â. when â2z 0.95 s â ,0, the upper bound of the interval for k was assumed to be kr'. the results for unaltered nb datasets are shown in figure 1 . boxplots show the median, interquartile range (iqr) and [5 th , 95 th ] percentile interval of 10,000 ml estimates k for each parameter set, while vertical lines show the true value of k. in general, the estimates are biased upward (i.e. favoring values k.k) but converge on the true value k as sample size n increases. for a given n, estimation tends to be less biased (the median value of k is closer to k) and more precise (the iqrs of k are smaller) for larger values of m and smaller values of k. numbers to the right of each subplot in figure 1 show the coverage accuracy of the cis estimated for k. the two numbers y/ z show, respectively, the percentage of simulations for which the true value of k fell below and above the estimated ci. for the 90% intervals estimated here, perfect coverage would yield values 5.0/5.0. for almost all parameter sets the proportion of ci overestimates (when the lower bound of the ci exceeds the true k) is greater than 5%, sometimes substantially so. this pattern is broken only for small n and large k. for all parameter sets the proportion of ci underestimates (when the upper bound of the ci is below the true k) is less than 5%. when the proportion of ci overestimates is very high (.10%, say), ci underestimates tend to be almost non-existent. the true coverage of the estimated 90% cis (calculated as (1002y2z)%) is generally less than 90%, although it often approaches this value for n = 300. again, there is an exception for small n and large k, when realized coverage exceeds 90% and reaches 100% in some instances (when the ci is extremely broad). the results for nb surveillance datasets subject to uniform undercounting are shown in figure 2 . results are shown for two values of the probability p u that any given secondary case is missed by surveillance. when p u = 0.2 (fig. 2a) , estimates of k from these datasets differed only slightly from estimates from raw nb data (fig. 1) , exhibiting all the same qualitative patterns and slightly worse bias and precision. when p u = 0.5 (fig. 2b) , results exhibited similar, but more extreme, differences from the raw nb results. results of estimation from nb surveillance datasets with underreporting of the zero class, in which individuals who caused x i = 0 cases were omitted from simulated datasets with probability p z , are shown in figure 3 . for both p z = 0.2 (fig. 3a) and p z = 0.5 (fig. 3b) , estimates of k are biased upward significantly. notably, this effect does not diminish as sample size increases. indeed, for most parameter sets the proportion of ci overestimates increases with higher n, as the sampling distribution narrows around the biased value. estimates from simulated outbreak datasets are shown in figure 4 . for m = 0.5 and k.0.1, no results are presented for n$100 because fewer than 1 in 10 5 simulated outbreaks reached 100 cases. for other values of m and k, estimates of k are quite robust (fig. 4a ). comparing these results to estimates from figure 1 , it is evident that estimates from outbreak datasets have similar biases (slightly positive for small n, but diminishing as n increases) and precisions that are as good and sometimes better than those from unaltered nb data. the outbreak datasets yield slightly more ci overestimates for m = 3, even though the iqr and [5 th , 95 th ] percentile interval of the sampling distribution is often smaller. outbreak datasets yield fewer ci overestimates for k = 0.1, m = 0.5 or 1.0, and n = 10 or 30. ml estimates of the mean are shown for these datasets as well (fig. 4b ). there is a striking positive bias evident in the estimates of m for m = 0.5; in all cases shown, the distribution of m estimates has median value .1 and 5 th percentile value $1. for m = 1, there is an upward bias in the m estimates that decreases as sample size rises. for m = 3, the upward bias persists but is very slight for k$0.3 or n$30. this study makes three novel contributions to the established literature on estimation of the nb dispersion parameter k. it provides the first comprehensive evaluation of ml estimation of k for highly overdispersed datasets (i.e. those with k,1); it reports the coverage accuracy of cis derived from those estimates; and it examines potential biases in estimation due to methods and errors of data collection, with application to epidemiological datasets in particular and biological datasets in general. the major qualitative results are summarized in table 1 . the results for unaltered nb datasets confirm and extend the findings of earlier studies. small-sample estimates of k were biased toward overestimating k-and hence underestimating the degree of overdispersion in the data-as reported in previous studies using ml and related methods of estimation for k$1 [14, 15, 17] . the positive bias in k arises because smaller samples are less likely to include values from the right-hand tail of the nb distribution, without which the dataset appears more homogeneous. estimates of k were less biased and more precise for larger values of m, possibly because such datasets had higher total numbers of nonzero events. estimates were more biased and less precise for higher values of k (particularly in the previously-studied range of k$1), corresponding to the known instability of ml estimates when data are closer to being fitted by a poisson distribution [13] . intuitively, this effect arises because a nb distribution with k = 10 is qualitatively similar to one with k = 50 or kr', and quite dissimilar to one with k = 1, so the range of k estimates for small samples tends to be large and skewed upwards. one previous simulation study [16] presented in-depth results for estimation of k,1 (specifically, for k = 0.4), employing methodof-moments estimates k mom rather than the ml estimates assessed here. that study reported that smaller sample sizes from nb datasets led to systematic underestimation of the mean and variance and overestimation of k; the variance/mean ratio was also biased downward by small n. there is one interesting difference between the method-of-moments estimates results of gregory and woolhouse [16] and the present results for ml estimation: the positive bias of k mom was fairly constant as m increased (though the range of k mom values was greatest for lower m), while the bias of ml estimates k decreased for higher m (fig. 1) . it is notable that their values of m ranged from 1.25 to 160 (for k = 0.4), while the values used here ranged from 0.5 to 3 (for k between 0.1 and 10). several salient patterns emerged regarding the realized coverage of 90% cis, as estimated using the asymptotic variance of ml estimates. the true coverage of the nominal 90% intervals was typically less than 90%, and ci overestimates were much more numerous than ci underestimates. for all parameter sets considered, ,5% of cis had upper bounds below the true value of k. the realized coverage of the cis is driven by the interplay of two factors: the value of the estimates, k, and the breadth of the intervals (determined by the sampling variance, s 2 k k ). the upward bias of k increases for lower values of n and m and higher values of figure 1 . estimated values of k and confidence interval coverage for nb datasets. 10,000 datasets were simulated as described in section 2.1.1 of the text, using mean m, dispersion parameter k, and sample size n as shown. boxes show the median and interquartile range (iqr) of 10,000 resulting ml estimates of k, and whiskers show the 5 th and 95 th percentile values. numbers to the right of each subplot show the percentage of simulations for which the true value of k was outside (below (ci overestimate)/above (ci underestimate) for the numbers y/z, respectively) the 90% confidence interval estimated for k the vertical line in each subplot shows the true value of k. to facilitate comparison among parameter sets, the horizontal axis of all subplots is scaled from 0 to 10 times the true value of k. doi:10.1371/journal.pone.0000180.g001 k; lower values of n, m, or k lead to increases in s 2 k and hence broader intervals. overestimates of k favor ci overestimates by setting a high mid-point for the estimated intervals, and by reducing the estimated sampling variance (because s 2 k is calculated with an inflated value of k) and thus leading to narrower intervals. the gross patterns in the frequency of ci overestimates thus are driven primarily by patterns of bias in k. to understand the finer patterns in ci coverage accuracy, particularly for ci underestimates and for ci overestimates for higher values of k, it is necessary to consider how the cis are calculated. recall that intervals were estimated for a = 1/k as [â2z 0.95 s â , â+z 0.95 s â ], then converted into intervals for k. ci underestimates for k occur when a,â2z 0.95 s â . the complete absence of ci underestimates in many small-n parameter sets arises because â,z 0.95 s â such that the lower bound of the ci for â is ,0. in these instances, the upper bound of the ci for k is set to the maximum value for k and cannot be exceeded. as n, m, or k increases, s â decreases and the cis narrow such that some ci underestimates occur. similarly, ci overestimates occur when a.â+z 0.95 s â . as k increases, ci overestimates become less frequent (despite the high frequency of k overestimates) because a = 1/k is often smaller than z 0.95 s â . because â is constrained to positive values in these simulations, ci overestimates are impossible when a,z 0.95 s â . accordingly, for given values of k.1, ci overestimates are more frequent for higher values of n and m (corresponding to lower values of s â ). this study's focus on overdispersed datasets, and hence on the positive values of k familiar to biologists, has thus influenced the determination of ci coverage in some regions of parameter space. estimation procedures allowing for underdispersed data (â,0) may show different results. investigators requiring cis guaranteed to reach nominal levels of coverage should consult the literature on exact cis for discrete distributions [e.g. 26] . the simulation results from surveillance and outbreak datasets (figs. 2-4) can be interpreted readily in light of the raw nb results discussed above. for datasets where individual values correspond to completely unconnected events (e.g. epidemiological surveillance of multiple independent introductions of a disease, or many other biological observations), the effects of two forms of underreporting were assessed. in uniform under-counting, each instance of the quantity being counted (e.g. secondary cases, in the epidemiological context) can be overlooked with equal probability p u . the expected value of each datum x i in the raw dataset (drawn from an nb distribution with parameters m and k) is reduced to (12p u ) x i , and the resulting distribution is nb with parameters (12p u ) m and k (as argued under the topic of 'population-wide control measures' by lloyd-smith et al. [8] ). thus uniform undercounting does not introduce systematic bias to ml estimates of k, but does cause a slight increase in the small-sample bias and decrease in precision (fig. 2) corresponding to the effect of a lower mean, as characterized for raw nb data (fig. 1) . in contrast, the second class of under-reporting bias, in which x i = 0 events are omitted from datasets with probability p z , leads to systematic overestimation of k that does not vanish as n increases (fig. 3) . nb distributions with low k are characterized by large zero classes and long tails (giving rise to the large variance-to-mean ratios that define overdispersion). decreasing the proportion of zeroes (hence replacing x i = 0 events by x i .0 events) leads to higher sample mean m and lower sample variance ŝ 2 . as is readily seen from the method-of moments estimator k mom = m 2 /(ŝ 2 2m ) [10] , this will bias estimates of k to higher values. investigators should be vigilant for this class of under-reporting bias, and conduct estimation using a zero-modified nb distribution [27] if zero under-counting is suspected. outbreak datasets involve a mechanism of data generation that is particular to epidemiological (or demographic) processes. earlier analyses have shown that when offspring distributions are highly overdispersed (e.g. nb with k,1), the outbreaks that succeed tend to be those with early superspreading events [8] . the present results show that this does not cause underestimation of k as had been feared; estimates of k from outbreak data (fig. 4a ) exhibited similar properties to those from raw nb data (fig. 1) . indeed, outbreak estimates had slightly smaller bias and greater precision for smaller n, probably because the use of outbreak data (biased toward including high-x i events) counteracts the usual smallsample bias (which arises because small datasets often lack high-x i events). therefore the selection bias inherent in outbreak datasets acts to offset somewhat the usual upward bias in estimates of k. in sharp contrast, estimation of m from outbreak datasets (assessed by simulation because, unlike the surveillance cases, the potential bias cannot be computed directly) is strongly biased upward when m is below or near 1 (fig. 4b) . this is unsurprising because the minimum value of m for an outbreak with n cases is (n21)/n (for an outbreak that dies out immediately following the n th case), while higher values are quite feasible. (recall that m is estimated as the mean number of secondary cases generated by the first n cases in an outbreak, regardless of whether the outbreak continues beyond n cases. if the cumulative number of cases after the r th generation of transmission is j, then the mean value of x i for i = 1 to j is (j21)/j. if the n th case then occurs in the (r+1) th generation of transmission, then all infections caused by the final n2j individuals in the dataset (i.e. x i for i = j+1 to n) serve to inflate m above its minimum value of (n21)/n.) the greatest bias in m occurs for low k and n, when large superspreading events in the final generation can have disproportionate effect on the sample mean. for m = 1.0, the bias decreases as n increases, probably because higher-n datasets involve more generations of disease transmission, so the 'left-over' cases of the final generation (i.e. the final n2j individuals in the example above) make a smaller proportional contribution. for m = 3.0, there is no substantial bias for any parameters (with a minor exception for k = 0.1 and n = 10). the results presented here suggest several avenues for future work. this study has focused on ml estimation only, and it would . estimated values of (a) k and (b) m for outbreak datasets generated by branching process simulations with nb offspring distributions. 10,000 datasets were simulated as described in section 2.1.4 of the text. circles indicate parameter sets for which fewer than 1 in 10 5 simulated outbreaks had n cases or more. other plotting details are described in figure 1 be fruitful to extend the conclusions to other methods of estimating k, such as maximum quasi-likelihood [14] , method-of-moments with small-sample correction [16] , or bias-corrected ml [17] . further studies on estimation of m will be interesting, particularly in the epidemiological context where the mean of the offspring distribution is equivalent to the crucial quantity r 0 [8, 22] . in particular, it will be important to learn how the overdispersion observed in disease transmission data [8] influences estimation of r 0 from continuous-time outbreak data such as daily case reports [28, 29] , as opposed to estimation directly from known chains of transmission as assessed here. overdispersed offspring distributions cause outbreaks to either die out stochastically or grow explosively [8] , so estimation of r 0 from daily case reports (of successful outbreaks only, necessarily) may exhibit bias beyond that shown in figure 4b . in summary, this study showed that there is minimal risk of underestimating k-and hence of overestimating the degree of overdispersion in the data-due to small sample size or any of the three process biases considered here. there is substantial risk of overestimating k, particularly when sample sizes are small or the zero-class is systematically under-counted. all of the systematic biases identified in this study favored higher values of k, and instances when confidence intervals excluded the true value k were predominantly overestimates. note that an independent risk of underestimating k can arise from pooling data from heterogeneous groups: the dispersion parameter estimated from pooled data is nearly always less than the average of values estimated for the individual groups [11, 16] . regarding sample sizes for nb datasets with k#1, n = 100 or more allows accurate and precise ml estimation of k, while for n = 30 the median estimates showed minimal bias but the sampling distribution skewed to high values. a sample size of 10 yields unreliable estimates, particularly for m#1. these findings will help guide prospective design of sampling regimens, or, when sample size cannot be increased, will aid investigators in understanding the limitations of ml estimates of k and associated cis. fitting the negative binomial distribution to biological data -note on the efficient fitting of the negative binomial analysis of frequency count data using the negative binomial distribution patterns of macroparasite aggregation in wildlife host populations comparative performance of species richness estimation methods linear model analysis of net catch data using the negative binomial distribution spatial modelling of individual-level parasite counts using the negative binomial distribution superspreading and the effect of individual variation on disease emergence the theory of branching processes sampling theory of the negative binomial and logarithmic series distributions small sample comparison of different estimators of negative binomial parameters heterogeneities in macroparasite infections: patterns and processes the negative binomial distribution estimation of the negative binomial parameter kappa by maximum quasi-likelihood maximum-likelihood estimation for the negative binomial dispersion parameter quantification of parasite aggregationa simulation study bias-corrected maximum likelihood estimator of the negative binomial dispersion parameter towards reliable bird surveys: accounting for individuals present but not detected different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures selecting a distributional assumption for modelling relative densities of benthic macroinvertebrates epidemiological and genetic analysis of severe acute respiratory syndrome mathematical epidemiology of infectious diseases: model building, analysis, and interpretation chance mechanisms generating the negative binomial distribution mathematical statistics and data analysis multistage estimation compared with fixed-sample-size estimation of the negative binomial parameter k confidence curves and improved exact confidence intervals for discrete distributions models for counts data with many zeros infectious diseases of humans: dynamics and control estimation and inference of r 0 of an infectious pathogen by a removal method i am grateful to leo polansky, sadie ryan and maria sanchez for helpful comments on the manuscript. key: cord-342639-vf9n2vf9 authors: chang, chung-ke; chen, chia-min michael; chiang, ming-hui; hsu, yen-lan; huang, tai-huang title: transient oligomerization of the sars-cov n protein – implication for virus ribonucleoprotein packaging date: 2013-05-23 journal: plos one doi: 10.1371/journal.pone.0065045 sha: doc_id: 342639 cord_uid: vf9n2vf9 the nucleocapsid (n) phosphoprotein of the severe acute respiratory syndrome coronavirus (sars-cov) packages the viral genome into a helical ribonucleocapsid and plays a fundamental role during viral self-assembly. the n protein consists of two structural domains interspersed between intrinsically disordered regions and dimerizes through the c-terminal structural domain (ctd). a key activity of the protein is the ability to oligomerize during capsid formation by utilizing the dimer as a building block, but the structural and mechanistic bases of this activity are not well understood. by disulfide trapping technique we measured the amount of transient oligomers of n protein mutants with strategically located cysteine residues and showed that ctd acts as a primary transient oligomerization domain in solution. the data is consistent with the helical oligomer packing model of n protein observed in crystal. a systematic study of the oligomerization behavior revealed that altering the intermolecular electrostatic repulsion through changes in solution salt concentration or phosphorylation-mimicking mutations affects oligomerization propensity. we propose a biophysical mechanism where electrostatic repulsion acts as a switch to regulate n protein oligomerization. introduction a fundamental part of viral self-assembly is the packaging of the viral genome into ribonucleoprotein (rnp) complexes called capsids. capsid formation requires two key activities: the interaction between protein and nucleic acid and the ability of the complex to oligomerize [1] . the severe acute respiratory syndrome coronavirus (sars-cov) nucleocapsid (n) protein is a phosphoprotein that performs both functions [2] . while the interaction between sars-cov n protein and nucleic acids has been examined at the genetic, biochemical and biophysical levels, understanding of sars-cov n protein oligomerization is relatively limited. although it is generally accepted that the n protein dimer serves as the basic building block of the nucleocapsid in coronaviruses, the structural and mechanistic bases of how n protein dimers oligomerize to form larger entities remain a mystery [3, 4, 5, 6, 7, 8, 9] . sars-cov n protein consists of two structural domains interspersed between intrinsically disordered regions (idr), a unique arrangement shared among coronaviruses [10] (fig. 1a) . the n-terminal structural domain (ntd, res. 45-181) adopts an ob (oligonucleotide binding)-like fold capable of binding to nucleic acids, whereas the c-terminal structural domain (ctd, res. 248-365) is responsible for dimer formation through a domain-swapping mechanism [3, 8, 9, 11] . we have shown that the ctd dimer is also capable of binding directly to nucleic acids [9, 12] . intriguingly, idrs also seem to modulate nucleic acid binding of sars-cov n protein, with at least one idr, the flexible linker region (lkr, res. 182-247), capable of direct interaction with rna under in vitro conditions [13] . like many capsid proteins, sars-cov n protein has a high isoelectric point (pi), with a large excess of positively charged residues distributed throughout the protein (+6 charges between residues 176-204, the sr-rich region; +7 charges between residues 249-267 in ctd region; +8 charges between residues 370-389 in the c-terminal idr region) that are important for the nucleic acid binding activity [11, 12, 13] . in the absence of nucleic acids, full-length sars-cov n protein predominantly exists as a dimer in solution [14] . however, a truncated mutant that includes part of the c-terminal structural domain and the c-terminal idr (res. 283-422) was shown capable of forming high-order oligomers in a concentrationdependent manner [15] . x-ray crystallography results of the ntd and ctd imply that the two structural domains also have oligomerization potential, even though they showed no indication of forming oligomers in solution [16] . in particular, we have shown that the ctd packed in a helical conformation with dimension and charge distribution that conforms to that observed in em structures ( figure 1b , 1c) [9, 17] . these studies suggest that sars-cov n protein could potentially form high-order oligomers by itself if protein concentrations were high enough, and small populations of oligomers could exist even at low concentrations, but could not be detected through conventional methods. closer inspection revealed that the interface area between the dimers in the helical packed crystal structure of ctd is small, consistent with the inability to detect higher order ctd oligomer in solution. nonetheless, such an oligomerization behavior may be relevant to rnp packaging. sars-cov n protein is highly phosphorylated in cells, and tentative phosphorylation sites have been mapped to the ser/argrich portion of the lkr [18, 19, 20] . notably, it has been suggested that the lkr is directly involved in n protein oligomerization [21] . a conflicting report, however, indicates that the lkr interfered with n protein oligomerization when the c-terminal domain is present [22] . peng et al demonstrated that phosphorylation of the lkr by the sr protein kinase-1 (srpk1) partially impaired the self-association of the full-length protein [19] . although there is a connection between phosphorylation and n protein oligomerization, whether this is due to a simple charge effect, conformational changes or involves interactions with other components of the virus-host system remains unclear. in this study, we used in vitro disulfide trapping and nmr spectroscopy to detect oligomeric species of the sars-cov n protein ctd in solution. we found that interactions between ctd dimers conform to that predicted from the helical crystal packing. extending our findings to a di-domain construct (dd, res. 45-365) which includes the ntd, ctd and lkr, we conclude that the dd is also able to form oligomers through the ctd in solution. introduction of negative charges to the lkr through phosphorylation-mimicking mutations in the dd construct resulted in enhanced oligomerization. we discuss the implications of our findings in the context of viral capsid assembly and propose a model where coronavirus n protein oligomerization is regulated by the modulation of charges on the protein. various protein domains were cloned and expressed as described previously [8, 10] . for disulfide trapping experiments, we chose mutation sites that would form disulfide linkages based on the crystal packing structures of the sars-cov n protein ctd ( figure 1 ) [9] . sites for the phosphomimicking mutations were chosen based on recent literature [18, 19, 20] . standard pcr techniques were employed to obtain mutant clones, which were then confirmed through dna sequencing. mutant proteins were expressed in escherichia coli bl21(de3) cells, followed by affinity and size-exclusion chromatography purification as previously described [8, 10] . isotope-labeled proteins for nmr studies were prepared using established protocols [8, 10] . purification of u-15 n, 2 h-ctd q290c tetramer was performed by collecting the tetramer fraction from a superdex 75/60 size exclusion column after treating the protein with 10 mm b-mercaptoethanol at 4uc for one hour and extensive washing with fplc buffer (50 mm sodium phosphate, 150 mm nacl, 1 mm edta, ph 7.4) in an amicon centrifugal concentrator (millipore, ma). nmr spectroscopy. samples contained 0.1-0.4 mm protein in nmr buffer containing 10 mm sodium phosphate, ph 6.0, 50 mm nacl, 1 mm edta, 0.01% nan 3 , 10% d 2 o and complete protease inhibitor cocktail (roche, germany). samples of cys mutants also contained 10 mm dithiothreitol (dtt) to avoid formation of disulfide bonds. all experiments were performed on bruker 600-mhz spectrometers equipped with cryoprobes at 30uc unless stated otherwise. the acquired data were processed with the topspin suite (bruker biospin, germany) or inmr (nucleomatica, italy). fixed amounts of purified proteins containing cysteine mutations (3 mg/ml for the ctd and 1.5 mg/ml for the dd) under various buffer conditions were allowed to react with 10 mm of bmercaptoethanol for 1 hour at 4uc, followed by extensive washing on an amicon centrifugal concentrator with fplc buffer to remove the b-mercaptoethanol. molecules in close contact and correct orientation spontaneously form disulfide linkages, which can be assayed through size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page). controls in the form of dd q290c were performed for each experiment to ensure b-mercaptoethanol efficacy. experiments were conducted on an akta fast-performance liquid chromatography (fplc) system (ge healthcare, ca) equipped with a superdex 75/60 column at an elution rate of 1.0 ml/min. apparent molecular weights of the proteins were estimated from the elution profile calibrated with the lmw gel filtration calibration kit (ge healthcare, ca). elution volume and molecular weight have the following relationship: where mw is the molecular weight in daltons and e.v. is the elution volume in ml. peak quantification was achieved through analysis with igor pro software (wavemetrics, or), where each peak in the chromatogram was fit with a gaussian curve and the underlying area integrated in the program. area integration errors were estimated from the residual between fitted and experimental curves. disulfide trapping samples were boiled for 2 min at 94uc in the presence and absence of b-mercaptoethanol. the boiling time was kept short (2 min) to avoid protein aggregation. the samples were then loaded on a nupage 4-12% bis-tris gel (invitrogen, ca) and run at room temperature according to manufacturer's instructions. the gel was digitized on a uvp biodoc-it equipped with a fluorescent plate (uvp, ca). quantification of the disulfide-linked and unlinked bands was achieved through the imagej software (national institutes of health, ma). the solution structure (pdb id: 2jw8) and crystal structure (pdb id: 2cjb) of the ctd spanning residues 248-365 were obtained from the protein data bank (www.rcsb.org). all structure images were prepared with pymol (www.pymol.org). within the crystal asymmetric unit, the sars-cov n protein ctd packs as an octamer which stacks to form a helical arrangement with a continuous positively charged surface that could potentially allow the rna to bind to it through electrostatic interactions ( fig. 1 ) [9] . such organization suggests a rnabinding facilitated mechanism for the formation of a helical rnp particle. it is unclear whether the oligomer structure is biologically relevant, since there have been no reports of oligomer species being detected in solution. to test the possibility that the oligomer structure reflects the existence of transient interactions that have been trapped during the crystallization process, we applied an in vitro disulfide trapping technique in an attempt to capture these transient interactions in solution. since wild-type sars-cov n protein contains no cysteine residues, we engineered single-site cysteine mutations at various locations (t264c, q290c, r294c, r320c, and s328c) on the ctd dimer. q290 and r294 are located at helix 2 and s328 is located at the loop region connecting the two b-strands. based on the crystal packing of the ctd structure as shown in figures 1b-1e , both q290 residues (colored magenta) in a ctd-dimer are located at the interface of the adjacent dimers in the oligomer. the distance between the bcarbons of the q290-q290 pair is within 8 å , a suitable distance for disulfide trapping reactions ( figure 1c ) [23] . similarly, the bcarbons of the r294-r294 pair (colored green) are also within a distance suitable for disulfide bond formation. on the other hand, s328 residues (colored cyan) of the ctd dimer are located in the central cavity of the ctd super-helix and are in a favorable position to form inter-dimer disulfide bonds with another s328 residue. the rest of the mutations are not located at the interface and were engineered as negative controls. the mutants did not perturb the structure of the ctd when monitored through 15 nedited heteronuclear single-quantum coherence (hsqc) nmr spectroscopy as judged by the absence of chemical shift perturbation greater than 0.03 ppm other than that of the mutated residues. we then tested for the ability of these mutants to spontaneously form disulfide linkages at 150 mm salt concentration through sizeexclusion chromatography. none of the mutants are located close enough to form intra-dimer disulfide linkages, thus any disulfide linkage must be due to inter-dimer protein disulfide bond formation. the elution profile from a superdex-75/60 sizeexclusion chromatography (sec) column (molecular weight cutoff 75 kda) of the ctd mutants, shown in figure 2 , fall in two groups: ctd q290c , ctd r294c and ctd s328c had high oligomerization potential; whereas ctd t264c and ctd r320c had little to no oligomerization potential. we observed three major peaks with molecular weight of 32 kda, 64 kda and higher molecular weights for peaks d, t and h, respectively, in the chromatograms of ctd q290c , ctd r294c and ctd s328c . the first peak eluted out from the column, peak h, has a molecular weight outside of the resolution limit of the resin. these molecular weights correspond to dimer and tetramer for the d and t peaks. the populations of the oligomers (t + h) are plotted on figure 2b . our results are in general agreement with the proposed crystal packing structure of the ctd with gln290, arg294 and ser328 having higher populations of oligomers. these results qualitatively suggest that the ctd of sars-cov n protein is capable of transient self-association through the oligomer interface identified in the crystal structure. we also tested the oligomerization potential of the ntd since its crystal packing hinted at the possibility of ntd oligomerization [16] . we introduced cys mutations at arg69, asn127, glu137 and thr167; sites that have the potential to form spontaneous disulfide linkages based on the trimeric crystal packing structure of the ntd [16] . according to the structure, arg69-glu137 and asn127-thr167 pairs are close in space and should have high probability of forming spontaneous disulfide linkages. however, we did not observe significant oligomer formation from our chromatograms ( figure 2c) , suggesting that the ntd either does not form oligomers or forms oligomers through an unidentified intermolecular interface. electrostatic screening drives self-association of sars-cov n protein ctd sars-cov n protein is highly electropositive due to an abundance of basic residues. these charges are considered important for rna binding, but they are also potentially deterring the self-association of the protein through electrostatic repulsion [11, 12] . the inter-protein charge-charge interaction can be affected by salt concentration, thus we first studied the effect of buffer salt concentration on trapped self-associated species of ctd q290c and the results are shown in figure 3 . the systematic shift of the chromatograms toward higher retention volume (lower molecular weight) with increasing salt concentration ( figure 3a ) are due to non-specific adsorption of the protein to the superdex matrix [24] . the relative amount of tetramer and larger oligomers in solution increases with increasing salt concentration ( figure 3b ), suggesting that reducing charge repulsion by increasing salt concentration enhances self-association of ctd. salt-induced conformational changes, electrostatic screening or a combination of both potentially could promote self-association of the protein [25] . however, one would expect the resonance positions of the 15 n-edited hsqc of the ctd q290c in low-salt (50 mm nacl) and high-salt (500 mm nacl) conditions to be different if there were changes in the conformation. since the two spectra are virtually identical ( figure 3c ), one can rule out the possibility of conformational change-induced oligomerization. we further tested the oligomerization behavior of a q290c mutant of the di-domain construct consisting of the ntd, the linker region and the ctd (dd q290c , a.a. 45-365). we utilized a non-reducing denaturing polyacrylamide gel electrophoresis (page) assay due to the large molecular weight of the dd dimer (, 70 kd), which is close to the void volume of the sec column used for the ctd studies. because the positions of the two gln290 in the dimer are far apart, formation of disulfide bonds in the q290c mutant would require at least two dimers to draw close together in space, such as formation of tetramers of higher order oligomers. denaturation of the dimer under non-reducing conditions would form monomers, whereas under the same conditions, high-order oligomers would form a mixture of dimers (those that formed a disulfide bond) and monomers. so presence of dimer bands on a non-reducing denaturing gel would reflect the presence of tetramers or high-order oligomers. consistent with our previous results, we also detected oligomers of the dd q290c as shown in figures 4a and 4b . interestingly, we saw little effect on the oligomerization of dd q290c when changing solution salt concentration. although the reason for the discrepancy between dd q290c and ctd q290c is unknown, one possible explanation is that the additional positive charges of dd q290c require much stronger electrostatic screening (e.g. even higher salt concentrations) to have an observable effect on the oligomerization of the protein, akin to the electrostatic screening threshold observed in crystallization studies [26] . we further examined whether changing the electrostatic properties of the protein itself would affect transient selfassociation. we chose the putative phosphorylation sites on the flexible linker as our prime target, and assayed the effect of negative charges on n protein self-association by changing these sites from ser to glu in the dd q290c mutant [18, 19, 20] . in figure 5 , we observed that gradual introduction of negative charges on the unstructured linker had a positive effect on the oligomerization of the dd when compared to the dd q290c control, with maximum effect achieved when 3 negative charges were introduced per each chain (see figure s1 ). further increases in negative charges (s185/189/195/202/203/207e) were less effective in enhancing dd q290c oligomerization. we checked the overall structure of mutants mimicking putative phosphorylation with known kinases [19, 20] , i.e. s189e, s207e, s202/203e and s189/202/203/207e with 15 n-hsqc and found that the extra negative charges did not affect the overall structure of the dd (data not shown), suggesting that electrostatic interactions rather than conformational changes are responsible for the increased tendency to self-associate. overall, our results suggest that hyperphosphorylation of the lkr, which reduces the total positive charge of the n protein, can enhance and regulate oligomerization of dd through electrostatic effects. understanding the self-association of capsid proteins is a fundamental step towards unraveling the mechanism of viral self-assembly. the study of self-association of coronavirus n proteins poses a particular challenge due to the intricate arrangement of structured and disordered domains that have been implicated in protein self-interaction. the literature on how sars-cov n protein oligomerize is rather confusing and the mechanism by which n protein associate with rna to form the helical rnp is also far from understood. it has been previously established that the sars-cov n proteins exist as dimers in solution with little indication of forming higher order oligomers in the absence of nucleic acids [8, 10, 14] . through a combination of disulfide trapping and structure-based mutagenesis, we successfully trapped transiently interacting oligomers of the ctd, suggesting that transient self-association between dimers do occur at specific site(s). the q290c mutation proved to be particularly useful in this study because it had little effect on the structure of the ctd and was electrostatically neutral, yet allowed for the selective detection of direct physical interaction between dimers. it enabled us to explore the effect of various variables (e.g. salt concentration and phosphorylation) on the oligomerization of the sars-cov n protein in a controlled environment. this study provides biochemical evidence that the ctd, but not ntd, is a transient intermolecular self-association site of sars-cov n protein. our results further suggest that transient self-association can be regulated by lowering intermolecular electrostatic repulsion, either through environmental charge screening or charge modifications on the protein itself. interestingly, our data is contrary to what was found in a number of previous studies. luo et al. reported that oligomerization of a sars-cov n protein construct spanning residues 283-422 was not affected by salt [15] . peng et al. found that sars-cov n protein phosphorylated by srpk1 induced less oligomerization than its unphosphorylated counterpart [19] . the disagreement most likely lies in the differences between the cross-linking techniques employed by the different groups. our experimental design requires that two dimers must physically interact with each other close to the mutation site before crosslinking can take place, whereas the use of a chemical cross-linker in the previous studies did not distinguish between dimers that arise from intra-dimer or inter-dimer associations. in luo et al. 's construct, the loss of residues 248-282 not only strips away most of the positive charges on the surface of the dimer, but also results in the loss of three n-terminal helices, which could cause adverse effects on the structural integrity of the protein [12] . over all, we believe that our method provides a more direct measure of the selfassociation of the basic building blocks involved in capsid assembly by taking into account the structural modularity of sars-cov n protein. it should also be noted that although the free-form nterminal domain did not oligomerize in our system, under in vivo conditions it still could form oligomers in the presence of rna as proposed by fan et al for the n protein of the infectious bronchitis virus [5] . other parts of the coronavirus n protein, e.g. so-called c-terminal tail, have also been implicated in protein oligomerization [15, 27] . in fact, the multitude of regions found to contribute towards oligomerization of coronavirus n proteins suggests that n protein self-association is an intricate affair involving intermolecular interactions among multiple domains of the protein. our results show that it is possible to change the oligomerization potential of sars-cov n protein by tinkering with the electrostatic characteristics of the system (figures 3 and 4) . normally, the positively charged n protein exerts a repulsive force that prevents self-association. in the case of the ctd, we enhance the charge screening effect and reduce the intermolecular repulsion when increasing the salt concentration, thus resulting in enhanced oligomerization. however, this salt dependency was lost when we changed the system to the dd construct ( figure 4 ). this could arise from the large number of charges in the dd (+36 charges/dimer) versus in the ctd (+14 charges/dimer), which would make it more difficult to screen the charges using the salt concentrations in this current study. interestingly, our observations could be related to those by verheije et al [28] . they observed in situ that self-interaction of ctd was independent of rna, whereas homotypic interaction of full-length n protein required the presence of rna. one could speculate that the cellular environment provided enough charge screening for ctd selfassociation, whereas rna was required to counterbalance the excessive charges of the full-length protein. n proteins of coronaviruses are highly phosphorylated in infected cells [29, 30] . sars-cov has also been shown to be phosphorylated in cells by several kinases, mainly at the serine residues in the sr region [18, 19, 20] . however, the in vivo phosphorylation sites of sars-cov n protein in actual infection have not been mapped. furthermore, peng et al. reported that phosphorylation of sars-cov n at the sr-rich motif could modulate its translation inhibitory activity and also its multimerization activity [19] , thus phosphorylation may play a role in regulating the viral life cycle. our findings that electrostatic repulsion acts as a means of modulating nucleocapsid assembly and that an optimal number of charges is favored for multimer formation suggest a possible mechanistic roles for phosphorylation in the regulation of sars-cov nucleocapsid assembly. in a fully formed capsid, each n protein would be stabilized by a network of weak protein-protein interactions with adjacent n proteins and protein-rna interactions with the viral genome. the protein-rna interactions neutralize the excessive charges on the n protein, thus negating the effect of electrostatic repulsion. under these conditions, extensive phosphorylation of sars-cov n protein is not necessary and the protein can maintain a hypophosphorylated state within the virion as proposed in a past study [20] . this hypophosphorylated state does not necessarily imply complete dephosphorylation. in fact, it would be advantageous to ''tune'' the n protein such that it retained a small number of phosphorylated sites even in the assembled form (e.g. the ''diphosphorylated'' mimics in fig. 5b ). one could then envision that further dephosphorylation events would enhance uncoating of the viral rna due to electrostatic repulsion between n protein dimers (e.g. ''mono-phosphorylated'' mimics in fig. 5b ). such a possibility has been proposed for jhmv, a neurotropic strain of mouse hepatitis coronavirus (mhv), where dephosphorylation of the n protein by an endosomal-associated cellular protein phosphatase was found to facilitate viral infections [31] . newly produced n protein, on the other hand, would be able to bind to rna with high affinity, but capsid formation could be impaired due to lack of protein-protein interactions caused by residual electrostatic repulsion. in this case, hyperphosphorylation of n protein could become a critical assisting factor, i.e. a ''last straw'' in neutralizing excessive electrostatic forces, in nucleocapsid assembly (e.g. ''triphosphorylated'' mimics in fig. 5b ). electrostatic repulsions have been shown to play similar roles in other viruses, where n proteins often have excessive positive charges. hepatitis b virus (hbv) n protein, for example, has an isoelectric point of 9.93, and forms icosahedral capsids rather than helical ones found in coronaviruses. an increase in the solution salt concentration has been shown to induce the formation of empty hbv capsids, and theoretical studies have attributed the phenomenon to charge screening effects [32, 33] . recently, a ''charge balance hypothesis'' was proposed to explain hepatitis b virus (hbv) capsid stability, assembly, rna encapsidation, and dna replication [34, 35, 36] . this hypothesis emphasized the importance of a balanced electrostatic interaction between the positive charge from the arginine-rich domain (ard) of the core protein (hbc) and the negative charge from the encapsidated nucleic acid. our results reinforce the common theme that electrostatic repulsion is a universal mechanism of capsid assembly modulation in viruses. the observation of trapped transient oligomer shown in the present study does not mean that ctd acts as the nucleation center for the capsid formation. in fact, we had previously shown that n protein interacts with nucleic acid at multiple sites and with high affinity, thus n-rna interaction presumed to be the driving force for the ribonucleoprotein (rnp) formation [13] . in this rna binding-coupled rnp packing scenario, the multitude of weak protein-protein interactions reinforces the protein-rna interaction and contributes towards the assembly of a stable rnp, which guarantees the formation of nucleocapsids containing genetic material. the modular structure and multiple sites of moderate rna binding affinity of the n protein not only allow the packaging of a stable rnp but also offer an energetically favorable condition for the expression of the viral genomic information. one can envision an unzipping mechanism for unwinding of the viral rna molecule and dissociation of the rna molecule from the n protein in a stepwise manner, one module at a time, without the need to overcome a high-energy barrier. this is consistent with the concept for virus assembly that capsid proteins associate through locally weak interactions to form globally stable structures [25] . weak interactions between capsid proteins minimize formation of kinetic traps, allow a greater degree of regulation of assembly, and may be essential for viruses where dissociation is part of the virus life cycle. on the other hand, newly synthesized n protein would also be involved in many rna-protein or protein-protein interactions, most likely modulated by n protein phosphorylation state, affecting viral and host processes other than nucleocapsid assembly [2] . more works need to be performed to understand the effect of n protein phosphorylation on viral and host processes other than nucleocapsid assembly. by disulfide trapping technique we measured the amount of transient oligomers of n protein mutants with strategically located cysteine residues and showed that sars-cov n protein is capable of transient oligomerization in solution through the ctd in the absence of nucleic acids. the data is compatible with the helical oligomer packing model of n protein observed in crystal. this transient oligomerization process is driven by the neutralization of excessive charges on the protein, which can be accomplished through changes in environmental salt concentration and protein phosphorylation. the data is consistent with a general mechanism whereby the electrostatic repulsion between n protein molecules acts as an oligomerization switch. it also reinforces the concept of rna-binding coupled rnp packaging in sars-cov. figure s1 representative strips from sds-page of selected dd mutants after disulfide trapping. the arrow denotes the trapped species originating from tetramers or higher order oligomers. 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review e, statistical, nonlinear, and soft matter physics exposure of rna templates and encapsidation of spliced viral rna are influenced by the arginine-rich domain of human hepatitis b virus core antigen (hbcag 165-173) testing the balanced electrostatic interaction hypothesis of hepatitis b virus dna synthesis by using an in vivo charge rebalance approach testing an electrostatic interaction hypothesis of hepatitis b virus capsid stability by using an in vitro capsid disassembly/reassembly system the nmr experiments were carried out with nmr spectrometers of the high-field nuclear magnetic resonance center (hfnmrc) supported by the national research program for genomic medicine, the national science council of the republic of china. key: cord-346314-o9fjpqaj authors: jarboui, mohamed ali; bidoia, carlo; woods, elena; roe, barbara; wynne, kieran; elia, giuliano; hall, william w.; gautier, virginie w. title: nucleolar protein trafficking in response to hiv-1 tat: rewiring the nucleolus date: 2012-11-15 journal: plos one doi: 10.1371/journal.pone.0048702 sha: doc_id: 346314 cord_uid: o9fjpqaj the trans-activator tat protein is a viral regulatory protein essential for hiv-1 replication. tat trafficks to the nucleoplasm and the nucleolus. the nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rrna and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. importantly, transient nucleolar trafficking of both tat and hiv-1 viral transcripts are critical in hiv-1 replication, however, the role(s) of the nucleolus in hiv-1 replication remains unclear. to better understand how the interaction of tat with the nucleolar machinery contributes to hiv-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of jurkat t-cells stably expressing hiv-1 tat fused to a tap tag. using an organellar proteomic approach based on mass spectrometry, coupled with stable isotope labelling in cell culture (silac), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in jurkat t-cell nucleolus upon tat expression. numerous proteins exhibiting a fold change were well characterised tat interactors and/or known to be critical for hiv-1 replication. this suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by tat provide an additional layer of control for regulating cellular machinery involved in hiv-1 pathogenesis. pathway analysis and network reconstruction revealed that tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, t-cell signaling pathways and genome integrity. we present here the first differential profiling of the nucleolar proteome of t-cells expressing hiv-1 tat. we discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust hiv-1 production. the nucleolus is a highly ordered subnuclear compartment organised around genetic loci called nucleolar-organising regions (nors) formed by clusters of hundreds of rdna gene repeats organised in tandem head-to-tail repeat [1, 2] . a membrane-less organelle originally described as the ''ribosome factory'', the nucleolus is dedicated to rna-polymerase-i-directed rdna transcription, rrna processing mediated by small nucleolar ribonucleoproteins (sornps) and ribosome assembly. ribosome biogenesis is essential for protein synthesis and cell viability [2] and ultimately results in the separate large (60s) and small (40s) ribosomal subunits, which are subsequently exported to the cytoplasm. this fundamental cellular process, to which the cell dedicates most of its energy resources, is tightly regulated to match dynamic changes in cell proliferation, growth rate and metabolic activities [3] . the nucleolus is the site of additional rna processing, including mrna export and degradation, the maturation of uridine-rich small nuclear rnps (u snrnps), which form the core of the spliceosome, biogenesis of t-rna and micrornas (mirnas) [4] . the nucleolus is also involved in other cellular processes including cell cycle control, oncogenic processes, cellular stress responses and translation [4] . the concept of a multifunctional and highly dynamic nucleolus has been substantiated by several studies combining organellar proteomic approaches and quantitative mass spectrometry, and describing thousands of proteins transiting through the nucleolus in response to various metabolic conditions, stress and cellular environments [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16] . collectively, the aforementioned studies represent landmarks in understanding the functional complexity of the nucleolus, and demonstrated that nucleolar proteins are in continuous exchange with other nuclear and cellular compartments in response to specific cellular conditions. of importance, the nucleolus is also the target of viruses including hiv-1, hcmv, hsv and kshv, as part of their replication strategy [2, 17] . proteomics studies analysing the nucleoli of cells infected with human respiratory syncytial virus (hrsv), influenza a virus, avian coronavirus infectious bronchitis virus (ibv) or adenovirus highlighted how viruses can distinctively disrupt the distribution of nucleolar proteins [2, 17, 18, 19, 20, 21, 22, 23, 24] . interestingly, both hiv-1 regulatory proteins tat and rev localise to the nucleoplasm and nucleolus. both their sequences encompass a nucleolar localisation signal (nols) overlapping with their nuclear localisation signal (nls), which governs their nucleolar localisation [25, 26, 27, 28, 29, 30, 31] . furthermore, tat and rev interact with the nucleolar antigen b23, which is essential for their nucleolar localisation [25, 26, 27, 28, 29, 30] . nevertheless, a recent study described that in contrast to jurkat t-cells and other transformed cell lines where tat is associated with the nucleus and nucleolus, in primary t-cells tat primarily accumulates at the plasma membrane, while trafficking via the nucleus where it functions [32] . while the regulation of their active nuclear import and/or export, as mediated by the karyopherin/importin family have been well described, the mechanisms distributing tat and rev between the cytoplasm, nucleoplasm and the nucleolus remains elusive [33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48] . importantly, two major studies by machienzi et al. have revealed important functional links between hiv-1 replication and the nucleolus [49, 50] . first, they could inhibit hiv-1 replication and tat transactivation function employing a tar decoy specifically directed to the nucleolus. furthermore, using a similar approach, with an anti-hiv-1 hammerhead ribozyme fused to the u16 small nucleolar rna and therefore targeted to the nucleolus, they could dramatically suppress hiv-1 replication. collectively, these findings strongly suggest that hiv-1 transcripts and tat nucleolar trafficking are critical for hiv-1 replication. however the nature of these contributions remains to be elucidated. in this report, we systematically analysed the nucleolar proteome perturbations occurring in jurkat t-cells constitutively expressing hiv-1 tat, using a quantitative mass spectrometry approach. following the detailed annotation of the quantitative abundance changes in the nucleolar protein composition upon tat expression, we focussed on the tat-affected cellular complexes and signalling pathways associated with ribosome biogenesis, spliceosome, molecular chaperones, dna replication and repair and metabolism and discuss their potential involvement in hiv-1 pathogenesis. in this study, we investigated the quantitative changes in the nucleolar proteome of jurkat t cells constitutively expressing hiv-1 tat (86aa) versus their tat-negative counterpart, using stable isotope labelling with amino acids in cell culture (silac) technology, followed by esi tandem mass spectrometry and implemented the experimental approach described in figure 1a . first, using retroviral gene delivery, we transduced hiv-1 tat fused to a tandem affinity purification (tap) tag (consisting of two protein g and a streptavidin binding peptide) or tap tag alone (control vector) in jurkat leukemia t cell clone e6-1 and sorted the transduced cells (gfp positive) by facs. this resulted in a highly enriched population of polyclonal transduced cells presenting different expression levels of the transgene ( figure 1b) . the functionality of tap-tat was confirmed by transfecting jurkat tap-tat and tap cells with a luciferase reporter gene vector under the control of the hiv-1 ltr (pgl3-ltr) [36] . tap-tat up regulated gene expression from the hiv-1 ltr by up to 28 fold compared to control ( figure 1c ). to further address the functionality of tat fused to tap, we compared jurkat tap-tat with jurkat-tat, a cell line stably expressing untagged tat [51] . both cell line exhibited comparable hiv-1 ltr activity following transfection with pgl3-ltr ( figure s1 ). next, tat expression and subcellular localization was verified by subcellular fractionation followed by wb analysis ( figure 1e ). tap-tat displayed a prominent nuclear/nucleolar localization but could also be detected in the cytoplasm. these observations were further validated by immunofluorescence microscopy ( figure 1e ). of note, jurkat-tat presented similar patterns for tat subcellular distribution as shown by immunofluorescence microscopy and subcellular fractionation followed by wb analysis (figure s2 and s3). we next compared the growth rate and proliferation of the jurkat tap and tap-tat cell lines (materials and methods s1), which were equivalent ( figure s4a ). similarly, facs analysis confirmed that the relative populations in g1, s, and g2/m were similar for jurkat tap-tat and tap cells ( figure s4b ). we labeled jurkat tap-tat and jurkat tap cells with light (r0k0) and heavy (r6k6) isotope containing arginine and lysine, respectively. following five passages in their respective silac medium, 85 million cells from each culture were harvested, pooled and their nucleoli were isolated as previously described ( figure 1a ) [52] . each step of the procedure was closely monitored by microscopic examination. to assess the quality of our fractionation procedure, specific enrichment of known nucleolar antigens was investigated by western blot analysis ( figure 1d ). nucleolin (110 kda) and fibrillarin (fbl) (34 kda), two major nucleolar proteins known to localise to the granular component of the nucleolus, were found to be highly enriched in the mixed nucleolar fraction. of note, nucleolin was equally distributed between the nuclear and cytoplasmic fractions. this distribution pattern for nucleolin appears to be specific for jurkat t-cells as show previously [52, 53] . the nuclear protein parp-1 (poly adpribose polymerase 1) (113 kda) was present in the nuclear and nucleoplasmic fraction but was depleted in the nucleolar fraction. alpha-tubulin (50 kda) was highly abundant in the cytoplasmic fraction and weakly detected in the nuclear fractions. collectively, these results confirmed that our methods produced a highly enriched nucleolar fraction without significant cross contamination. subsequently, the nucleolar protein mixture was trypsindigested and the resulting peptides were analysed by mass spectrometry. comparative quantitative proteomic analysis was performed using maxquant to analyse the ratios in isotopes for each peptide identified. a total of 2427 peptides were quantified, representing 520 quantified nucleolar proteins. the fully annotated list of the quantified nucleolar proteins is available in table s1 and the raw data from the mass spectrometry analysis was deposited in the tranche repository database (https:// proteomecommons.org/tranche/), which can be accessed using the hash keys described in materials and methods. we annotated the quantified proteins using the toppgene suite tools [54] and extracted gene ontology (go) and interpro annotations [55] . the analysis of go biological processes ( figure 1f ) revealed that the best-represented biological processes included transcription (24%), rna processing (23%), cell cycle process (13%) and chromosome organisation (15%), which reflects nucleolar associated functions and is comparable to our previous characterisation of jurkat t-cell nucleolar proteome [52] . subcellular distribution analysis ( figure 1f ) revealed that our dataset contained proteins known to localise in the nucleolus (49%), in the nucleus (24%) while 15% of proteins were previously described to reside exclusively in the cytoplasm. the subcellular distribution was similar to our previous analysis of the jurkat t-cell nucleolar proteome [52] . table s1 . the distribution of protein ratios are represented in figure 1g as log 2 (abundance change). the silac ratios indicate changes in protein abundance in the nucleolar fraction of jurkat tap-tat cells in comparison with jurkat tap cells. the distribution of the quantified proteins followed a gaussian distribution ( figure 1g ). a total of 49 nucleolar proteins exhibited a 1.5 fold or greater significant change (p,0.05) upon tat expression (table 1) . of these, 30 proteins were enriched, whereas 19 proteins were depleted. cells displayed no changes in the steady state content of some of the major and abundant constituents of the nucleolus, including nucleophosmin (npm1/ b23), c23, fbl, nucleolar protein p120 (nol1), and nucleolar protein 5a (nol5a). the distinct ratios of protein changes upon tat expression could reflect specific nucleolar reorganization and altered activities of the nucleolus. we performed wb analysis to validate the silac-based results obtained by our quantitative proteomic approach ( figure 2 ). 15 selected proteins displayed differential intensity in the nucleolar fractions upon tat expression, including 9 enriched (hsp90b, stat3, prb, ck2a, ck2a', hsp90a, transportin, zap70, ddx3), and 3 depleted (ilf3, bop1, and ssrp1) proteins. in addition, we also tested by wb analysis, protein abundance not affected by tat expression (importin beta, fbl, b23, c23). these results highlight the concordance in the trend of the corresponding silac ratios, despite some differences in the quantitative ranges. of note, using wb, we could observe a change of intensity for protein with a silac fold change as low as 1.25-fold. of note, the question remains as to which fold change magnitude might constitute a biologically relevant consequence. on the one hand, the threshold of protein abundance changes can be determined statistically and would then highlight the larger abundance changes as illustrated in table 1 . alternatively, the coordinated enrichment or depletion of a majority of proteins belonging to a distinct cellular complex or pathway would allow the definition of a group of proteins of interest and potential significance. therefore, we next focused on both enriched or depleted individual proteins with activities associated with hiv-1 or tat molecular pathogenesis, and on clustered modifications affecting entire cellular signaling pathways and macromolecular complexes. we initially focused on signaling proteins interacting with tat and/or associated hiv-1 molecular pathogenesis and whose abundance in the nucleolus was modulated by tat expression. phospho-protein phosphatases. phospho-protein phosphatase pp1 and pp2a are essential serine/threonine phosphatases [56, 57] . importantly, pp1 accounts for 80% of the ser/thr phosphatase activity within the nucleolus. in our study, pp1 was found to be potentially enriched by 1.52-fold in the nucleolus of jurkat cells expressing tat, which supports previous studies describing the nuclear and nucleolar targeting of pp1a by hiv-1 tat and how pp1 upregulates hiv-1 transcription [58, 59, 60, 61, 62] . pp1 c was also identified as part of the in vitro nuclear interactome [63] . similarly, ppp2ca, the pp2a catalytic subunit (1.29-fold) and its regulatory subunit pp2r1a (1.27-fold) were similarly enriched upon tat expression. interestingly, tat association with the pp2a subunit promoters results in the overexpression and up regulation of pp2a activity in lymphocytes [64, 65] . furthermore, pp2a contributes to the regulation of hiv-1 transcription and replication [61, 66] . retinoblastoma protein. the tumour suppressor gene prb protein displayed a 1.4-fold change in the nucleolus upon tat expression [67] . furthermore, wb analysis confirmed the distinct translocation of prb from the nucleoplasm to the nucleolus by tat ( figure 2 ). depending on the cell type, prb can be hyperphosphorylated or hypophosphorylated upon tat expression and can negatively or positively regulate tat-mediated transcription respectively [68, 69, 70] . interestingly, the hyperphosphorylation of prb triggers in its translocation into the nucleolus [71] . phosphorylation of prb is also associated with an increase in ribosomal biogenesis and cell growth [72] . stat3. the transcription factor signal transducer and activator of transcription 3 (stat3) was significantly enriched (1.86-fold) in the nucleolar fraction by tat constitutive expression. furthermore, wb analysis indicated that tat expression could promote the relocalisation of stat3 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( figure 2) . interestingly, previous studies have demonstrated tat-mediated activation of stat3 signaling, as shown by its phosphorylation status [73] . interestingly, stat3 phosphorylation induced dimerisation of the protein followed its translocation to the nucleus [74] . ybx1. ybx1, the dna/rna binding multifunctional protein was enriched by 1.38-fold in the nucleolus of jurkat cells upon tat expression. interestingly, ybx1 interacts with tat and tar and modulates hiv-1 gene expression [63, 75] . zap70. the protein tyrosine kinase zap70 (zeta-chainassociated protein kinase 70) was enriched by 1.24-fold in the nucleolus of jurkat cells expressing tat [76] . furthermore, wb analysis revealed that tat expression could promote the relocalisation of zap70 from the cytoplasm to the nucleus, with a distinct enrichment in the nucleolus ( figure 2 ). of note, zap70 is part of the in vitro nuclear tat interactome [63] . matrin 3. the inner nuclear matrix protein, matrin 3 (matr3), presented a 1.39-fold change in the nucleolus of jurkat cells expressing tat. it localizes in the nucleolasm with a diffuse pattern excluded from the nucleoli [77] . matrin 3 has been identified as part of the in vitro hiv-1 tat nuclear interactome [63] . two recent studies have described matrin 3 as part of ribonucleoprotein complexes also including hiv-1 rev and (rev response element) rre-containing hiv-1 rna, and promoting hiv-1 post-transcriptional regulation [78, 79, 80] . casp10. the pro-apototic signaling molecule, caspase 10 (casp10), was significantly depleted from the nucleolus of jurkat-tat cells (0.82-fold) [81] . importantly, tat expression downregulates casp10 expression and activity in jurkat cells [82] . adar1. adenosine deaminase acting on rna (adar1), which converts adenosines to inosines in double-stranded rna, was significantly depleted from the nucleolus of jurkat-tat cells (0.78-fold). interestingly, adar1 over-expression up-regulates hiv-1 replication via an rna editing mechanism [83, 84, 85, 86, 87, 88] . furthermore, adar1 belongs to the in vitro hiv-1 tat nuclear interactome [63] . to underline the structural and functional relationships of the nucleolar proteins affected by hiv-1 tat, we constructed a network representation of our dataset. we employed cytoscape version 2.6.3 [89] and using the mimi plugin [90] to map previously characterised interactions, extracted from protein interaction databases (bind, dip, hprd, ccsb, reactome, intact and mint). this resulted in a highly dense and connected network comprising 416 proteins (nodes) out of the 536 proteins, linked by 5060 undirected interactions (edges) ( figure 3a ). centrality analysis revealed a threshold of 23.7 interactions per protein. topology analysis using the centiscape plugin [91] showed that the node degree distribution follows a power law ( figure s5 ), characteristic of a scale-free network. importantly, when we analysed the clustering coefficient distribution ( figure s6 ) we found that the network is organised in a hierarchical architecture [92] , where connected nodes are part of highly clustered areas maintained by few hubs organised around hiv-1 tat. furthermore, node degree connection analysis of our network identified hiv-1 tat as the most connected protein ( figure s6 ). specifically, the topology analysis indicated that the values for tat centralities were the highest (node degree, stress, radiality, closeness, betweeness and centroid), characterising tat as the main hub protein of the nucleolar network. indeed, a total of 146 proteins have been previously described to interact with tat ( figure 3b , table s2 ). these proteins are involved in a wide range of cellular processes including chromosomal organization, dna and rna processing and cell cycle control. importantly, aver the third of these proteins exhibit an increase in fold ratio change (59 proteins with a ratio .1.2 fold). in parallel, we characterised the magnitude of the related protein abundance changes observed in distinct cellular pathways ( figure 4) . ribosomal biogenesis. we initially focused on ribosome biogenesis, the primary function of the nucleolus. we could observe a general and coordinated increase in the abundance of ribosomal proteins in the nucleolus by tat expression (figure 4 ). while some ribosomal proteins remained unaffected, tat caused the nucleolar accumulation of several distinct large and small ribosomal proteins, except rpl35a, for which tat expression caused a marked decrease at the nucleolar level (0.29-fold). similarly, several proteins involved in rrna processing exhibited an overall increase in nucleolar accumulation upon tat expression. these include human canonical members of the l7ae family together with members participating in box c/d, h/aca and u3 snornps ( figure 4) . conversely, bop1, a component of the pebow (pescadillo bop1 wdr12) complex essential for maturation of the large ribosomal subunit, was significantly depleted from the nucleolus of jurkat tap-tat cells (0.81-fold) and this was confirmed by wb analysis (figure 2 ) [93] . nevertheless, the other pebow complex components, pes1 (0.94-fold) and wdr12 (1.1fold), were not affected by tat expression. of note, we did not detect change in the abundance of protein participating in rdna transcription such as rnapoli, ubf. spliceosome. we identified and quantified in our dataset 55 proteins out of the 108 known spliceosomal proteins [94] . these proteins include the small nuclear ribonucleoproteins u1, u2 and u5, sm d1, d2, d3, f and b, and the heterogeneous nuclear ribonucleoproteins. our data suggested a distinct increase in the abundance of specific spliceosome complex proteins upon expression of hiv-1 tat in jurkat t-cells (figure 3 and 4) . the only three proteins that were significantly depleted from the nucleolus upon expression of hiv-1 tat were rbmx (0.89-fold), hnrnpa2b1 (0.84-fold) and snrpa (0.81-fold). several investigations showed expression alteration in cellular splicing factors in hiv-1 infected cells [95, 96] . molecular chaperones. we have identified several molecular chaperones, co-chaperones and other factors involved into proteostasis to be highly enriched in the nucleolus of t-cells upon tat expression (figure 3 and 4) , many of which were previously characterised as part of the tat nuclear interactome [63] . several heat-shock proteins including dnajs, specific hsp90, hsp70 and hsp40 isoforms and their co-factors were distinctively enriched in the nucleolar fraction of jurkat cells expressing tat ( figure 4 ). as shown by wb, while hsp90a and b are mostly cytoplasmic, tat expression triggers their relocalisation to the nucleus and nucleolus, corroborating our proteomic quantitative approach (figure 2) . similarly, heat-shock can cause the hsp90 and hsp70 to relocalise to the nucleolus [97, 98, 99, 100, 101] . in a recent study, fassati's group has shown that hsp90 is present at the hiv-1 promoter and may directly regulate viral gene expression [102] . we also observed the coordinated increased abundance of class i (groel and groes) and class ii (chaperonin containing tcp-1 (ctt)) chaperonin molecules (figure 3 and 4) upon tat expression. ubiquitin-proteasome pathway. the ubiquitin-proteasome pathway is the major proteolytic system of eukaryotic cells [103] . importantly, the nuclear ubiquitin-proteasome pathway controls the supply of ribosomal proteins and is important to ribosome biogenesis [104, 105] . the 26s proteasome is composed of the 20s core particle (cp) and the 19s regulatory particle (rp). alternatively, cp can associate with the 11s rp to form the immunoproteasome. all the quantified proteins in our study are part of the 19s regulatory complex and include psmd2 (1.5-fold), psmd3 (1.32-fold), psmd11 (1.25-fold) and psmd13 (0.72-fold), the only proteasome component significantly depleted from the nucleolus in the presence of tat (figure 4) . interestingly, tat interacts with distinct subunits of the proteasome system, including the 19s, 20s and 11s subunits. the consequences of these interactions include the competition of tat with 11s rp or 19s rp for binding to the 20s cp, which resulted in the inhibition of the 20s peptidase activity [106, 107, 108, 109, 110, 111] . furthermore, tat was shown to modify the proteasome composition and activity, which affects the generation of peptide antigens recognized by cytotoxic t-lymphocytes [112] . importantly, a recent study demonstrated that in the absence of tat, proteasome components are associated to the hiv-1 promoter and proteasome activity limits transcription [113] . addition of tat promoted the dissociation of the 19s subunit from the 20s proteasome, followed by the distinct enrichment of the 19s-like complex in nuclear extracts together with the tat-mediated recruitment of the 19s subunits to the hiv-1 promoter, which facilitated its transcriptional elongation [113] . we also quantified uba1 (1.36-fold), the e3 ubiquitin-protein ligase uhrf1 (1.13-fold), ubc (1-fold) and two ubiquitinspecific-peptidases, usp30 (1.28-fold) and usp20 (0.06-fold) (figure 4) . dna replication and repair. upon hiv-1 tat expression, we observed the coordinated nucleolar enrichment of several cellular factors associated with dna replication and repairs pathways (figure 4) . tat induced the coordinated enrichment of the miniature chromosome maintenance mcm2-7 complex (from 1.23-to 3.30fold, respectively) [114] . mcm7, 6 and 3 were identified as part of the in vitro nuclear interactome of hiv-1 tat [63] . the structural maintenance of chromosomes 2, smc2, was enriched (1.35-fold) in the nucleolar fraction by tat expression. smc2 was identified as part of the in vitro nuclear interactome of hiv-1 tat [63] . while replication factor c1 (rfc1) and rfc2 (1.31-and 1.28-fold respectively) displayed an increased fold change and rfc5/3 were not affected, rfc4 was severely depleted (0.69-fold) from the nucleolar fraction upon tat expression [115] . rfc1 and rfc2 were identified as part of the in vitro nuclear interactome of hiv-1 tat [63] . tat induced the enrichment of xrcc6 (1.27-fold) and xrcc5 (1.36-fold) in the nucleolus, which are involved in the repair of non-homologous dna end joining (nhej) [116] . xrcc6 associates with viral preintegration complexes containing hiv-1 integrase and also interact with tat and tar [117, 118, 119] . furthermore, in a ribozyme-based screen, xrcc5 (ku80) knockdown decreased both retroviral integration and tatmediated transcription [120] . as part of the base excision repair (ber), we have identified a major apurinic/apyrimidinic endonuclease 1 (apex1) (1.29-fold) . importantly, in a sirna screen targeting dna repair factors, apex1 knockdown was found to inhibit hiv-1 infection by more 60% [121] . the high mobility group (hmg) protein, hmga1 (1.30-fold), was enriched in the nucleolus following tat expression [122] . hmga1 interact with hiv-1 integrase and is part of the hiv-1 pre-integration complex [123, 124] . importantly, hmga1 has been identified in a proteomic screen, as a cellular cofactor interacting with the hiv-1 59leader [125] . metabolism. our proteomic data suggest that tat induces perturbations in glycolysis, the pentose phosphate pathway, and nucleotide and amino acid biosynthesis (figure 4 and figure s7 ). notably, in t cells expressing tat, we detected co-ordinated changes in the abundance of proteins not previously known to be associated with tat pathogenesis, which revealed unexpected connections with with glycolysis and the pentose phosphate pathway, including the following glycolitic enzymes, lactate dehydrogenase b (ldhb) (1.37-fold), glyceraldehyde 3-phosphate dehydrogenase (gapdh) (1.17-fold) and phosphoglyceric acid mutase (pgam1) (0.89-fold) ( figure 4 and figure s7 ). briefly, gpi catalyzes the reversible isomerization of glucose-6-phosphate in fructose-6-phosphate. subsequently, pfkp catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis. at the end of the glycolytic pathway, pkm2, in its tetrameric form, is known to generate atp and pyruvate, while ldhb diverts the majority of the pyruvate to lactate production and regeneration of nad+ in support to continued glycolysis, a phenomenon described for proliferative tcells [126] . of note, in highly proliferating cells, pkm2 can be found in its dimeric form and its activity is altered. this upregulates the availibility of glucose intermediates, which are rerouted to the pentose phosphate and serine biosynthesis pathways for the production of biosynthetic precursors of nucleotides, phospholipids and amino acids. as part of the pentose phosphate pathway, we have characterised the significant enrichment of glucose-6-phosphate dehydrogenase (g6pd) (2.11-fold), which branches of the glycolysis pathway to generate nadph, ribose-5phosphate an important precursor for the synthesis of nucleotides. consistent with this, we detected the coordinated increase in the abundance of enzymes which plays a central role in the synthesis of purines and pyrimidines. more specifically, impdh2 (1.66fold), a rate-limiting enzyme at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides, phosphoribosyl pyrophosphate synthetase 2 (prps2) (1.41-fold), cytidine-5-prime-triphosphate synthetase (ctps) (1.74-fold) which catalyses the conversion of utp to ctp and the ribonucleotide reductase large subunit (rrm1) (1.56-fold). in parralel, we noted the increased abundance of the phosphoserine aminotransferase psat1 (1.90-fold), an enzyme implicated in serine biosynthesis, which has been linked with cell proliferation in vitro. the host-virus interface is a fundamental aspect in defining the molecular pathogenesis of hiv-1 [127, 128, 129, 130, 131, 132, 133] . indeed, with its limited repertoire of viral proteins, hiv-1 relies extensively on the host cell machinery for its replication. several recent studies have capitalized on the recent advances in the ''omics'' technologies, and have revealed important insights into this finely tuned molecular dialogue [132, 134] . hiv-1 tat is essential for viral replication and orchestrates hiv-1 gene expression. the viral regulatory protein is known to interact with an extensive array of cellular proteins and to modulate cellular gene expression and signaling pathway [135, 136] . we and others have employed system-level approaches to investigate tat interplay with the host cell machinery, which have characterised hiv-1 tat as a critical mediator of the host-viral interface [137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149] . here, we have investigated the nucleolar proteins trafficking in response to hiv-1 tat expression in t-cells, with the view to provide unique and novel insights on the role of proteins compartimentalisation by tat in the fine-tuning of protein availability and function. we have developed for this study, a cellular model using jurkat t-cells stably expressing tat fused in its n-ternminal to tap-tag. jurkat t-cells are robust and present the advantage to grow without stimulations and are easely transduced using retroviral gene delivery. importantly, they have been widely employed to evaluate tat-mediated pathogenesis using system-wide approaches and to analyse t-cell key cellular signaling pathways and functions [144, 150, 151, 152] . indeed, we have found them particularly suited for prolongued in vitro culture in silac medium and subsequent isolation of their nucleolus followed by ms analysis, which requires up to 85 millions of cells. we fused tat to the tap tag to enable future downstream applications such as tandem affinity purification or chromatin ip analysis. importantly, we have confirm that n-terminal tap-tag did not interfere with tat function nor its localisation in jurkat cells, when compared to untagged-tat. of note, tat subcellular distribution can vary according to the cell type employed. while tat is known to accumulate in the nucleus and nucleolus in jurkat cells and other transformed cell lines, in primary t-cells, tat was described to primarily accumulate at the plasma membrane, while trafficking via the nucleus where it functions [32] . these differences remain to be characterised but could be related to different expression levels of transport factors in transformed cell lines versus primary cells, as recently described by kuusisto et al. [39] . furthermore, stauber and pavlakis have suggested that tat nucleolar localisation could be the results of tat overexpression [31] . here, we have selected and employed a polyclonal population of jurkat t-cells expressing tat at different levels. we propose that this heterogeneity in tat expression levels might reflect tat stochastic expression described during viral replication [153] . using a quantitative proteomic strategy based on an organellar approach, we quantified over 520 nucleolar proteins, including 49 proteins exhibiting a significant fold change. the extent to which the induced variations in the abundance of nucleolar proteins are biologically relevant and can affect cellular and/or viral processes remains to be determined. nevertheless, the biological nature of the pathways and macromolecular complexes affected enable us to discuss their potential associations with hiv-1 pathogenesis. hiv-1 tat is expressed early following hiv-1 genome integration and mediates the shift to the viral production phase, associated with robust proviral gene expression, viral proteins assembly and ultimately, virions budding and release. in this context and based on our results, we propose that tat could participate in shaping the intracellular environment and metabolic profile of t cells to favor host biosynthetic activities supporting robust virions production. indeed, we observed the distinct nucleolar enrichment of ribosomal proteins and enzymes associated with ribosomal biogenesis, which could be indicative of an increase in protein synthesis. with the notable exeption of rpl35a nucleolar depletion, ribosomal proteins and enzymes associated with ribosomal biogenesis were in the top 20 most enriched nucleolar proteins (nhp2l1, rlp14, rpl17, rpl27, rps2, rpl13). furthermore, this effect appears to be specific to hiv-1 tat since transcription inhibition by actinomycin d resulted in the overall depletion of ribosomal proteins in the nucleolus [9] . moreover, quantitative proteomics analysis of the nucleous in adenovirus-infected cells showed a mild decrease in ribosomal proteins [24] . whether this reflect a shift in ribosome biogenesis and/or a change in the composition of the ribosomal subunits remains to be determined. nevertheless, the adapted need for elevated ribosome production is intuitive for a system that needs to support the increased demand for new viral proteins synthesis. in parralel, we observed the concordant modulation of pathways regulating protein homeostasis. we noted the significant nucleolar accumulation of multiple molecular chaperones including the hsps, the tcp-1 complex, and canx/calr molecules and the disrupted nucleolar abundance of proteins belonging to the ubiquitin-proteasome pathway, which controls the supply of ribosomal proteins [104, 105] . these observations further support previous studies describibing the modulation of the proteasomal activity by tat, which affect the expression, assembly, and localization of specific subunits of the proteasomal complexes [106, 107, 108, 109, 110, 111, 113] . we also observed the concomitant depletion of casp10 in the nucleolus of jurkat tap-tat. it has been suggested that casp10 could be targeted to the nucleolus to inhibit protein synthesis [154] . interestingly, the presence and potential roles of molecular chaperones in the nucleolus have been highlighted by banski et al, who elaborate on how the chaperone network could regulate ribosome biogenesis, cell signaling, and stress response [97, 155] . as viral production progresses into the late phase and cellular stress increases, nucleolar enrichment of molecular chaperones by tat could not only enable adequat folding of newly synthetised viral proteins but could also promote tolerance of infected cells to stress and maintain cell viability. coincidentally, we observed the marked nucleolar enrichment of enzymes belonging to metabolic pathways including glycolysis, pentose phosphate, nucleotide and amino acid biosynthetic pathways. similarly, these pathways are elevated in proliferative t-cells or in cancer cells following a metabolic shift to aerobic glycolysis, also known as the warburg effect [156, 157, 158, 159] . there, glucose intermediates from the glycolysis pathway are not only commited to energy production and broke-down into pyruvate for the tca cycle, but are redirected to alternative pathways, including the pentose phosphate pathway, and used as metabolic precursors to produce nucleotides, amino acids, acetyl coa and nadph for redox homeostasis. consistently, we also noted the concomittant nucleolar enrichment of enzymes belonging to the nucleotide synthesis pathway, including imph2, a rate limiting enzyme known to control the pool of gtp. similarly, we noted the nucleolar enrichment of psat1, an enzyme involved in serine and threonin metabolism, which is associated with cellular proliferation [160] . collectively, we propose that by controlling protein homeostasis and metabolic pathways, tat could meet both the energetic and biosynthetic demand of hiv-1 productive infection. of note, while nucleotide metabolism enzymes are associated with the nucleus, glycolysis takes place in the cytoplasm. nevertheless, glycolytic enzymes have been detected in both the nuclear and nucleolar fractions by proteomic analyses [8, 161] . furthermore glycolytic enzymes, such as pkm2, ldh, phosphoglycerate kinase, gapdh, and aldolase, also have been reported to display nuclear localization and bind to dna [162] . more specifically, pkm2 is known to associate with promoter and participate in the regulation of gene expression as a transcriptional coactivator [163] . hiv-1 tat has previously been described as an immunoregulator and more specifically, has been reported both to inhibit or to promote tcr signaling [164] . we have observed the nucleolar enrichment by tat of key proximal or downstream components of t-cell signaling pathways, including zap70, ilf3 and stat3, which play crucial roles in t-cell development and activation. we had previously identified them as t-cell specific components of the nucleolus, and if studies suggested that their association with the nucleolus could be regulated by specific conditions [165] . our results further support that tat could contribute to the dysregulation of tcr-derived signals and that the nucleolus could represent an important spatial link for tcr signaling molecules. we observed the coordinated nucleolar enrichment of key components of the dna replication, recombination and repair pathways by tat. these include xrcc5 and xrcc6, hmga1, apex1, mcm2-7, smc2, rfc1 and rfc2, while rfc4 was found to be significantly depleted. interestingly, these cofactors have been associated with the efficiency of retroviral dna integration into the host dna or the integrity of integrated provirus [166] . whether the increased abundance of these factors within the nucleolus could be associated with their potential participation in the integration and maintenance of provirus gene integrity, remains to be determined. the mechanisms of tat-mediated segregation and compartimentalisation of proteins in or out of the nucleolus may depend on factor(s) inherent for each protein and the nature of their relationship with tat, since subcellular fractionation combined with wb analysis showed that the pattern and extent of subcellular redistribution between proteins varied. we could observe cases where tat upregulated the expression of proteins which resulted in a general increase of theses proteins throughout the cellular compartments including the nucleolus (ddx3, tnpo1). alternatively, tat could trigger the nucleolar translocation of proteins directly from the cytoplasm or the nucleoplasm (prb). additionally, we observed cytoplasmic proteins redistributed to both the nucleoplasm and nucleolus upon tat expression (stat3, zap70 and hsp90). finally, we also noted protein depletion in the nucleolar fraction accompanied by an increase in the nucleoplasm (ssrp1). it remains difficult at this stage, to appreciate whether the accumulation of specific proteins would result in their activation or inhibition by sequestering them away from their site of action. conversely, the depletion of a protein from the nucleolus could either result in the down-regulation of its activity in this location or could be the result of its mobilization from its storage site, the nucleolus, to the nucleoplasm or cytoplasm where it can perform its function. remarkably, we identified several known hiv-1 tat partners involved in hiv-1 pathogenesis, which suggests that tat could physically modulate their nucleolar targeting or their recruitment to specific site in the nucleoplasm or cytoplasm. tat could also promote post-translational modifications, which could mediate the targeting of specific proteins to the nucleolus. this is exemplified by the following enriched proteins, prb, pp1 and stat3, for which phosphorylation is induced by tat. importantly, their phosphorylation status determines their subcellular distribution, thus providing a potential mechanism for their redistribution by tat. moreover, our data indicates that serine/threonine kinases (ck2 a') and phosphatases (pp1) were significantly enriched in the nucleolar fractions of jurkat tap-tat. these enzymes account for the majority of the phosphorylation/ dephosphorylation activity in the nucleolus and can act as regulators of nucleolar protein trafficking. in addition, tat significantly decreased the levels of sumo-2 in the nucleolus. similarly, sumo-mediated post-translational modifications are known to modulate nucleolar protein localization [104] . given the potential importance of post-translational modifications, including phosphorylation in the tat-mediated change of abundance of nucleolar proteins, a more targeted proteomic approach such as the enrichment for phosphopetides, would extend the resolution of our screening approach. the control of protein turnover is also an important mean to modulate the abundance of nucleolar proteins. ribosomal proteins are degraded by the ubiquitin-proteasome pathway to ensure their abundance matches up with rrna transcription levels. conversely, heat shock proteins hsp90s protect them from degradation. interestingly, our data showing that tat modulation the abundance proteins associated with the ubiquitin-proteasome and heat-shock pathway. this could contribute to the observed enrichment of ribosomal proteins by tat. nevertheless, we cannot exclude that the increased abundance of ribosomal proteins in the nucleolus could be the result of tat-mediated prevention of their export to the cytoplasm. interestingly, using a different cellular system, a drosophila melanogaster tat transgenic strain, ponti et al, analysed the effects of tat on ribosome biogenesis, following 3 days heat shock treatment to induce tat expression under the control of the hsp70 promoter [167] . following tat expression, they observed a defect in pre-rrna processing associated with a decrease in the level of 80s ribosomes [167] . nevertheless, the different cellular system employed combined with the 3 days heatshock induction make their results difficult to compare with ours. while previous system-level studies have monitored the effects of hiv-1 tat expression on t cells, to our knowledge, we have presented here the first proteomic analysis of dynamic composition of the nucleolus in response to hiv-1 tat expression. using quantitative proteomics, we have underlined the changes in abundance of specific nucleolar proteins and have highlighted the extensive and coordinated nucleolar reorganization in response to tat constitutive expression. our findings underscore that tat expressing t-cells exhibit a unique nucleolar proteomic profile, which may reflect a viral strategy to facilitate the progression to robust viral production. importantly, we noted the functional relationship of nucleolar proteins of our dataset with hiv-1 pathogenesis and hiv-1 tat in particular. this further increases our confidence in our experimental strategy and suggests a role for tat in the spatial control and subcellular compartimentaliation of these cellular cofactors. ultimatly, our study provides new insights on the importance of tat in the cross talk between nucleolar functions and viral pathogenesis. importantly, we have also identified changes in nucleolar protein abundance that were not previously associated with hiv-1 pathogenesis, including proteins associated with metabolic pathways, which provide new potential targets and cellular pathways for therapeutic intervention. jurkat t-cells, clone e6.1 (atcc), jurkat ntap-tat and jurkat ntap were maintained in rpmi-1640 medium supplemented with 10% (v/v) foetal bovine serum (gibco, eu approved), and antibiotics. phoenix-gp cells (g.p. nolan; www.stanford.edu/ group/nolan/), were maintained in dmem medium supplemented with 10% (v/v) foetal bovine serum (gibco, eu approved). cells were counted using scepter tm 2.0 cell counter (millipore). the sequence of hiv-1 tat (hiv-1 hxb2, 86 amino acids) was sub-cloned into pentr 2b vector (invitrogen, a10463). using the gateway technology (invitrogen), we introduced the hiv-1 tat sequence into the plasmid pcemm-ntap(gs)-gw [168] . phoenix cells (g.p. nolan; www.stanford.edu/group/ nolan/), were transfected using fugene 6 (roche) with 5 mg of the plasmid ntap-tat or ntap and 3 mg of the pmdg-vsvg. viral supernatants were collected after 48 h, filtered and used to transduce the jurkat cell lines. the construct is termed ntap-tat, the empty vector was termed ntap. using retroviral gene delivery, we stably transduced jurkat cells (clone e6.1 (atcc)). the positive clones named jurkat ntap-tat and jurkat ntap were sorted to enrich the population of cells expressing gfp using the bc moflo xdp cell sorter (beckman coulter). sub-cellular fractions (10 mg) were resolved by sds-page and transferred onto biotrace pvdf membranes (pall corporation). the following primary antibodies were used: a-tubulin (sc 5286), c23 (sc 6013), and fibrillarin (sc 25397) were from santa cruz biotechnology, and parp (am30) from calbiochem, mouse anti-zap 70 (05-253, millipore), rabbit anti-stat3 (06-596, millipore), rabbit anti-ilf3 (ab92355, abcam), rabbit anti-hsp90 beta (ab32568, abcam), mouse anti-adar1 (ab88574, abcam), rabbit anti-hdac1 (ab19845, abcam), rabbit anti-ssrp1 (ab21584, abcam) rabbit anti-bop1 (ab86982, abcam), mouse anti-kpnb1 (ab10303, abcam), rabbit anti-hiv-1 tat (ab43014, abcam), rabbit anti-ck2a (ab10466, abcam), rabbit anti-ddx3x (ab37160, abcam), mouse anti-tnpo1 (ab2811, abcam), mouse anti-hsp90a (ca1023, merck), and rabbit-anti rb1 (sc-102, santa cruz).the following secondary antibodies were used ecl: anti-mouse igg and ecl anti-rabbit igg (ge healthcare), and donkey anti-goat igg (sc 2020) (santa cruz biotechnology). for silac analysis silac-rpmi r0k0 and silac-rpmi r6k6 (dundee cells) media supplemented with 10% dialyzed fbs (gibco, 26400-036) were used. the jurkat cells expressing ntap-tat and ntap were serially passaged and grown for five doublings to ensure full incorporation of the labelled amino acids. cells viability was checked with trypan blue (0.4% solution, sigma) and further confirmed using pi staining and facs analysis. cells were mixed to the ratio 1:1 to obtain 140610 6 cells. nucleoli were isolated from the mixed cell population as previously described in jarboui et al., [165] . nucleolar extracts (100 mg) were resuspended in 50 mm ammonium bicarbonate and in solution trypsin digested as previously described in jarboui et al. [165] . sample was run on a thermo scientific ltq orbitrap xl mass spectrometer connected to an eksigent nano lc.1dplus chromatography system incorporating an auto-sampler. sample was loaded onto a biobasic c18 picofrittm column (100 mm length, 75 mm id) and was separated by an increasing acetonitrile gradient, using a 142 min reverse phase gradient (0-40% acetonitrile for 110 min) at a flow rate of 300 nl min-1. the mass spectrometer was operated in positive ion mode with a capillary temperature of 200uc, a capillary voltage of 46v, a tube lens voltage of 140v and with a potential of 1800 v applied to the frit. all data was acquired with the mass spectrometer operating in automatic data dependent switching mode. a high resolution ms scan was performed using the orbitrap to select the 5 most intense ions prior to ms/ms analysis using the ion trap. the incorporation efficiency of labelled amino-acids was determined by analysing the peptides identified in isolated nucleoli from cell population maintained in ''heavy'' medium as described in [169] . our analysis showed that we had an incorporation efficiency .95% (data not shown). the ms/ms spectra were searched for peptides identification and quantification using the maxquant software [170] (version 1.1.1.36), the human ipi database (version 3.83) and the andromeda search engine associated to maxquant [171] . standard settings were used for maxquant with the acetyl (protein n-term) as variable modification and carbamidomethyl (cys) as fixed modification, 2 missed cleavage were allowed, except that the filtering of labelled amino acids was prohibited. initial mass deviation of precursor ion and fragment ions were 7 ppm and 0.5 da, respectively. each protein ratio was calculated as the intensity-weighted average of the individual peptides ratios. proteins were identified with the minimum of one peptide with a false discovery rate less than 1%. gene ontology, kegg pathway and pfam terms were extracted from uniprot entries using perseus, a software from the maxquant data analysis package (http://www.maxquant.org ), and the toppgene suite tools [54] . the jurkat ntap-tat and jurkat ntap were transfected using the amaxa electroporation system (amaxa biosystem) with the pgl3 (pgl3-ltr) (promega) as recommended by amaxa biosystem. dual-luciferase assays (promega) were performed according to the manufacturer's instructions. luciferase activity was measured and normalized against the total amount of proteins as quantified by the bca protein quantification kit (pierce, thermo scientific). to preserve their original shape, we performed immunostaining of jurkat cells in suspension. cells were fixed in 2% pfa for 10 min at rt, permeabilised in 0.5% triton x-100 for 15 min at rt and blocked with 5% fcs. cells were incubated with the rabbit hiv-1 tat antibody (ab43014, abcam) followed by the secondary antibody anti-rabbit alexa fluor 647 (a-21246, invitrogen). cells were allowed to attach to cell-tak (bd) coated silanised slides (daocytomation), and stained with dapi. images were captured with a carl zeiss confocal microscope equipped with a plan-apochromat 63x/1.4 oil dic objective. the proteomics raw data file from the mass spectrometry analysis was deposited to the tranche repository(https:// proteomecommons.org/tranche/) [172] . the file can be accessed and downloaded using the following hash key: (r3o5sv5z6hvwqrbndhp21txfetludwyxvwmifu-h6e1kmgaraucsq4dlncxeuvfohdezledcg4x5y8resb6-mua6wm1kiaaaaaaaab/w = = ). materials and methods s1 description of the methods employed to examine cell cycle, cell viability and cell proliferation analysis. 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in adenovirus-infected cells viral nucleolar localisation signals determine dynamic trafficking within the nucleolus protein b23 is an important human factor for the nucleolar localization of the human immunodeficiency virus protein tat spatial association of hiv-1 tat protein and the nucleolar transport protein b23 in stably transfected jurkat t-cells specific complex of human immunodeficiency virus type 1 rev and nucleolar b23 proteins: dissociation by the rev response element effects of a highly basic region of human immunodeficiency virus tat protein on nucleolar localization a region of basic amino-acid cluster in hiv-1 tat protein is essential for trans-acting activity and nucleolar localization intracellular trafficking and interactions of the hiv-1 tat protein phosphatidylinositol-(4,5)-bisphosphate enables efficient secretion of hiv-1 tat by infected t-cells the ins and outs of hiv-1 tat tuning the transport properties of hiv-1 tat arginine-rich motif in living cells in vivo study of hiv-1 tat arginine-rich motif unveils its transport properties direct interaction of the human i-mfa domain-containing protein tat results in cytoplasmic sequestration and control of tat activity the arginine-rich domains present in human immunodeficiency virus type 1 tat and rev function as direct importin betadependent nuclear localization signals the hiv-1 tat nuclear localization sequence confers novel nuclear import properties global enhancement of nuclear localization-dependent nuclear transport in transformed cells intermolecular masking of the hiv-1 rev nls by the cellular protein hic: novel insights into the regulation of rev nuclear import nuclear factor 90, a cellular dsrna binding protein inhibits the hiv rev-export function functional analysis of the interaction of the human immunodeficiency virus type 1 rev nuclear export signal with its cofactors the ins and outs of hiv rev the hiv-1 rev protein the specificity of the crm1-rev nuclear export signal interaction is mediated by rangtp requirement of ddx3 dead box rna helicase for hiv-1 rev-rre export function mechanisms of receptor-mediated nuclear import and nuclear export the nuclear pore component nup358 promotes transportin-dependent nuclear import a nucleolar tar decoy inhibitor of hiv-1 replication ribozyme-mediated inhibition of hiv 1 suggests nucleolar trafficking of hiv-1 rna constitutive expression of hiv-1 tat protein in human jurkat t cells using a bk virus vector gautier vw proteomic profiling of the human t-cell nucleolus nucleolin undergoes partial n-and o-glycosylations in the extranuclear cell compartment toppgene suite for gene list enrichment analysis and candidate gene prioritization interpro: the integrative protein signature database mitotic phosphatases: no longer silent partners emerging roles of nuclear protein phosphatases nuclear targeting of protein phosphatase-1 by hiv-1 tat protein expression of a protein phosphatase 1 inhibitor, cdnipp1, increases cdk9 threonine 186 phosphorylation and inhibits hiv-1 transcription regulation of hiv-1 transcription by protein phosphatase 1 dephosphorylation of cdk9 by protein phosphatase 2a and protein phosphatase-1 in tat-activated hiv-1 transcription nuclear protein phosphatase-1 regulates hiv-1 transcription vitro nuclear interactome of the hiv-1 tat protein association of tat with promoters of pten and pp2a subunits is key to transcriptional activation of apoptotic pathways in hiv-infected cd4+ t cells pp2a targeting by viral proteins: a widespread biological strategy from dna/rna tumor viruses to hiv-1 protein phosphatase 2a enhances activation of human immunodeficiency virus type 1 by phorbol myristate acetate rb1, development, and cancer human immunodeficiency virus type 1 tat protein modulates cell cycle and apoptosis in epstein-barr virus-immortalized b cells hiv-1 tat elongates the g1 phase and indirectly promotes hiv-1 gene expression in cells of glial origin retinoblastoma gene inhibits transactivation of hiv-ltr linked gene expression upon co-transfection in he la cells nucleolar size and activity are related to prb and p53 status in human breast cancer nucleolus, ribosomes, and cancer intracellular tat of human immunodeficiency virus type 1 activates lytic cycle replication of kaposi's sarcoma-associated herpesvirus: role of jak/stat signaling getting the message across, stat! design principles of a molecular signaling circuit interaction of yb-1 with human immunodeficiency virus type 1 tat and tar rna modulates viral promoter activity the structure, regulation, and function of zap-70 bipartite nuclear localization signal of matrin 3 is essential for vertebrate cells matrin 3 and hiv rev regulation of mrna characterization of the hiv-1 rna associated proteome identifies matrin 3 as a nuclear cofactor of rev function matrin 3 is a co-factor for hiv-1 rev in regulating post-transcriptional viral gene expression playing the disc: turning on trail death receptor-mediated apoptosis in cancer hiv-1 tat protein concomitantly down-regulates apical caspase-10 and up-regulates c-flip in lymphoid t cells: a potential molecular mechanism to escape trail cytotoxicity enhancement of replication of rna viruses by adar1 via rna editing and inhibition of rna-activated protein kinase multiple levels of pkr inhibition during hiv-1 replication adar2 editing enzyme is a novel human immunodeficiency virus-1 proviral factor alternate rrna secondary structures as regulators of translation adar1 interacts with pkr during human immunodeficiency virus infection of lymphocytes and contributes to viral replication double-stranded rna adenosine deaminases enhance expression of human immunodeficiency virus type 1 proteins cytoscape: a software environment for integrated models of biomolecular interaction networks michigan molecular interactions (mimi): putting the jigsaw puzzle together analyzing biological network parameters with centiscape network biology: understanding the cell's functional organization interdependence of pes1, bop1, and wdr12 controls nucleolar localization and assembly of the pebow complex required for maturation of the 60s ribosomal subunit comprehensive proteomic analysis of the human spliceosome the expression of the essential nuclear splicing factor sc35 is altered by human immunodeficiency virus infection hiv-1 infection induces changes in expression of cellular splicing factors that regulate alternative viral splicing and virus production in macrophages nucleolar targeting of the chaperone hsc70 is regulated by stress, cell signaling, and a composite targeting signal which is controlled by autoinhibition stress inhibits nucleocytoplasmic shuttling of heat shock protein hsc70 heat-induced nuclear accumulation of hsc70s is regulated by phosphorylation and inhibited in confluent cells intracellular localization of the 90 kda heat shock protein (hsp90alpha) determined by expression of a egfp-hsp90alpha-fusion protein in unstressed and heat stressed 3t3 cells localization of heat shock proteins in mouse male germ cells: an immunoelectron microscopical study gyrase b inhibitor impairs hiv-1 replication by targeting hsp90 and the capsid protein ubiquitin-like and ubiquitin-associated domain proteins: significance in proteasomal degradation ubiquitin and ubiquitin-like proteins in the nucleolus: multitasking tools for a ribosome factory analysis of nucleolar protein dynamics reveals the nuclear degradation of ribosomal proteins the rtp site shared by the hiv-1 tat protein and the 11s regulator subunit alpha is crucial for their effects on proteasome function including antigen processing hiv-1 tat inhibits the 20 s proteasome and its 11 s regulator-mediated activation peptide sequencing identifies mss1, a modulator of hiv tat-mediated transactivation, as subunit 7 of the 26 s protease new human gene encoding a positive modulator of hiv tat-mediated transactivation a cdna for a protein that interacts with the human immunodeficiency virus tat transactivator human immunodeficiency virus-1 tat protein interacts with distinct proteasomal alpha and beta subunits hiv-1 tat protein modulates the generation of cytotoxic t cell epitopes by modifying proteasome composition and enzymatic activity the proteasome regulates hiv-1 transcription by both proteolytic and nonproteolytic mechanisms oncogenic activity of mcm7 transforming cluster unzipped and loaded: the role of dna helicases and rfc clamp-loading complexes in sister chromatid cohesion the xrcc genes: expanding roles in dna double-strand break repair role of the non-homologous dna end joining pathway in the early steps of retroviral infection lupus autoantigen ku protein binds hiv-1 tar rna in vitro effect of ku80 depletion on the preintegrative steps of hiv-1 replication in human cells identification of cellular cofactors for human immunodeficiency virus replication via a ribozyme-based genomics approach sirna screening of a targeted library of dna repair factors in hiv infection reveals a role for base excision repair in hiv integration the dynamics of hmg proteinchromatin interactions in living cells retroviral cdna integration: stimulation by hmg i family proteins hiv-1 cdna integration: requirement of hmg i(y) protein for function of preintegration complexes in vitro activity of the human immunodeficiency virus type 1 cell cycle-dependent internal ribosomal entry site is modulated by ires trans-acting factors fuel feeds function: energy metabolism and the t-cell response human immunodeficiency virus type 1, human protein interaction database at ncbi patterns of hiv-1 protein interaction identify perturbed host-cellular subsystems the biological context of hiv-1 host interactions reveals subtle insights into a system hijack hivhost interactions: a map of viral perturbation of the host system cataloguing the hiv type 1 human protein interaction network retroviral proteomics and interactomes: intricate balances of cell survival and viral replication dual role of host cell factors in hiv-1 replication: restriction and enhancement of the viral cycle decoding the multifaceted hiv-1 virus-host interactome functions of tat: the versatile protein of human immunodeficiency virus type 1 chromatin dynamics associated with hiv-1 tat-activated transcription modifications in host cell cytoskeleton structure and function mediated by intracellular hiv-1 tat protein are greatly dependent on the second coding exon pathway analysis in hek 293t cells overexpressing hiv-1 tat and nucleocapsid expression profiles and pathway analysis in hek 293 t cells overexpressing hiv-1 tat and nucleocapsid using cdna microarray proliferative activity of extracellular hiv-1 tat protein in human epithelial cells: expression profile of pathogenetically relevant genes proteomics analysis of human astrocytes expressing the hiv protein tat hiv-1 tat reprograms immature dendritic cells to express chemoattractants for activated t cells and macrophages gene expression profile of hiv-1 tat expressing cells: a close interplay between proliferative and differentiation signals modifications in the human t cell proteome induced by intracellular hiv-1 tat protein expression cell-type-specific proteome and interactome: using hiv-1 tat as a test case differentially expressed genes in hiv-1 tat-expressing cd4(+) t-cell line hiv-1 tat assembles a multifunctional transcription elongation complex and stably associates with the 7sk snrnp the histone chaperone protein nucleosome assembly protein-1 (hnap-1) binds hiv-1 tat and promotes viral transcription genome-wide binding map of the hiv-1 tat protein to the human genome jurkat t cells and development of the t-cell receptor signalling paradigm global survey of human t leukemic cells by integrating proteomics and transcriptomics profiling a genome-wide short hairpin rna screening of jurkat t-cells for human proteins contributing to productive hiv-1 replication stochastic gene expression in a lentiviral positive-feedback loop: hiv-1 tat fluctuations drive phenotypic diversity dedd and dedd2 associate with caspase-8/10 and signal cell death chaperones and multitasking proteins in the nucleolus: networking together for survival? evidence for an alternative glycolytic pathway in rapidly proliferating cells glutamine uptake and metabolism are coordinately regulated by erk/mapk during t lymphocyte activation on the origin of cancer cells understanding the warburg effect: the metabolic requirements of cell proliferation characterization of human phosphoserine aminotransferase involved in the phosphorylated pathway of lserine biosynthesis proteomics profiling of nuclear proteins for kidney fibroblasts suggests hypoxia, meiosis, and cancer may meet in the nucleus glycolytic enzymes as dna binding proteins emerging roles of pkm2 in cell metabolism and cancer progression viral modulation of t-cell receptor signaling proteomic profiling of the human t-cell nucleolus the role of unintegrated dna in hiv infection the hiv tat protein affects processing of ribosomal rna precursor an efficient tandem affinity purification procedure for interaction proteomics in mammalian cells use of stable isotope labeling by amino acids in cell culture as a spike-in standard in quantitative proteomics maxquant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification andromeda: a peptide search engine integrated into the maxquant environment proteomecommons.org collaborative annotation and project management resource integrated with the tranche repository the authors wish to acknowledge access to and use of the ucd conway mass spectrometry resource instrumentation at the mass spectrometry resource (msr), conway institute for biomolecular and biomedical research, university college dublin is gratefully acknowledged. the authors wish to acknowledge prof. dimitri scholz director of biological imaging, conway institute, ucd, for his assistance with image acquisition and analysis. key: cord-352720-z1cvjc2y authors: díaz-corvillón, pilar; mönckeberg, max; barros, antonia; illanes, sebastián e.; soldati, arturo; nien, jyh-kae; schepeler, manuel; caradeux, javier title: routine screening for sars cov-2 in unselected pregnant women at delivery date: 2020-09-29 journal: plos one doi: 10.1371/journal.pone.0239887 sha: doc_id: 352720 cord_uid: z1cvjc2y background: south america has become the epicenter of coronavirus pandemic. it seems that asymptomatic population may contribute importantly to the spread of the disease. transmission from asymptomatic pregnant patients’ needs to be characterized in larger population cohorts and symptom assessment needs to be standardized. objective: to assess the prevalence of sars cov-2 infection in an unselected obstetrical population and to describe their presentation and clinical evolution. methods: a cross-sectional study was designed. medical records of pregnant women admitted at the obstetrics & gynecology department of clínica dávila for labor & delivery, between april 27(th) and june 7(th), 2020 were reviewed. all patients were screened with rt-pcr for sars cov-2 at admission. after delivery, positive cases were inquired by the researchers for clinical symptoms presented before admission and clinical evolution. all neonates born from mothers with confirmed sars cov-2 were isolated and tested for sars cov-2 infection. results: a total of 586 patients were tested for sars cov-2 during the study period. outcomes were obtained from 583 patients which were included in the study. thirty-seven pregnant women had a positive test for sars cov-2 at admission. cumulative prevalence of confirmed sars cov-2 infection was 6.35% (37/583) [ci 95%: 4.63–8.65]. from confirmed cases, 43.2% (16/37) were asymptomatic. from symptomatic patients 85.7% (18/21) had mild symptoms and evolved without complications and 14.3% (3/21) presented severe symptoms requiring admission to intensive care unit. only 5.4% (2/37) of the neonates born to mothers with a positive test at admission had a positive rt-pcr for sars cov-2. conclusion: in our study nearly half of pregnant patients with sars cov-2 were asymptomatic at the time of delivery. universal screening, in endemic areas, is necessary for adequate patient isolation, prompt neonatal testing and targeted follow-up. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 coronavirus disease 2019 (covid-19), caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), has been defined as a global public health emergency [1] . six months after the emergence of this novel virus, south america has become the epicenter of covid-19 pandemic. it has been proposed that pregnant women should be considered a high-risk population, since gestation itself could be related with several pregnancy-related complications, higher susceptibility to respiratory pathogens and also can generate problems in terms of the spread of the infection due to the multiple interactions with the health-care system [2] . while initial evidence suggests that pregnant women were not at increased risk for covid-19, neither developed a more severe disease compared to non-pregnant adults [3, 4] , recent reports suggest increased rates of preterm birth [5] , pneumonia and intensive care unit admission [6] , and maternal mortality [6, 7] . currently, it has become evident that asymptomatic-people dissemination may play an important role in the spread of the virus [8] . the reported rates of asymptomatic pregnant women ranges from 43% to 89%, with estimates from 4 to 9 undetected cases per each symptomatic patient, supporting universal screening as a possible strategy [9] [10] [11] [12] [13] [14] [15] . it is also well established that pregnant women keep their pregnancy supervised by healthcare professionals, allowing close follow-up of their clinical conditions. therefore, it has been proposed that women admitted for delivery could provide a potential study group with useful estimates of virus circulation among general population [12, 13, 16] . given the possibility there is a higher prevalence of sars cov-2 infection than reported just by symptoms, screening of unselected population may give a more accurate estimate. the former, becomes clinically relevant due to administration of personnel protection measures, proper patient isolation, prompt neonatal testing and targeted follow-up. the main objective of this study was to assess point-prevalence of sars cov-2 infection in unselected obstetrical population at the time of delivery and to describe the presentation and clinical evolution of confirmed cases. the study was conducted at the obstetrics & gynecology department of clínica dávila, santiago, chile. our institution is a private healthcare center that provides obstetrical care to nearly 5000 pregnant women per year. it is currently one of the largest obstetrics facilities in our country. women were screened for covid-19 clinical symptoms including fever, cough and shortness of breath by trained personnel, and rt-pcr for sars cov-2 (allplex tm 2019-ncov assay [17] ) was performed by nasopharyngeal swab, unless a prior test with no more than 48 hours to admission was reported. clinical management was carried out with personal protective equipment levels c or d following recommendations [18] , until rt-pcr for sars cov-2 report was provided. after delivery, patients with a positive rt-pcr for sars cov-2 were inquired by researchers for clinical symptoms presented before the diagnosis (fever � 37.8, cough, headache, shortness of breath, myalgia, odynophagia, nasal congestion, digestive symptoms (diarrhea / vomiting), anosmia, dysgeusia, anorexy) and followed-up for clinical evolution. (s1 appendix) following institutional guidelines, neonates born from mothers with the diagnosis of covid-19, regardless of symptoms, were isolated and sars cov-2 rt-pcr was performed at 6 hours and 48-72 hours after delivery. patients with history of covid-19 confirmed by rt-pcr during pregnancy, or with less than 24 weeks of gestational age at admission were excluded. the main objective was to establish the point-prevalence of sars cov-2 infection in our obstetrical population at delivery. secondary objectives were: i) describe the rate of newborns confirmed with positive rt-pcr for sars cov-2; ii) evolution of confirmed cases; iii) frequency of adverse maternal outcomes (maternal intensive care unit admission, need of invasive ventilatory support, maternal death); iv) frequency of adverse perinatal outcomes (preterm birth, small for gestational age, 5 minute apgar < 7, admission to neonatal intensive care unit, perinatal death). this study was approved by the institutional review board of clínica dávila, and a waiver of consent was granted. to estimate the point-prevalence of sars cov-2 infection at the time of delivery, a sample size estimation was performed based on the following statistical assumptions: i) a target population of unknown size; ii) 95% confidence intervals; iii) precision of the prevalence estimator of 2.5%; iv) expected point-prevalence of 10% or less. previous reports in literature have reported point-prevalence's that ranged from 15.4% to 19.9% in obstetric population [7, 8, 11] . at the time this study was conceived, the national sars cov-2 incidence in chile was in its initial stages; therefore a prevalence of 10% or less was considered plausible for our target population. the estimated sample size required to assess the prevalence of disease was 553 pregnancies. assuming a maximum loss to follow-up of 5% throughout the study, a final sample of 583 patients was expected to be included. based on the number of deliveries in our facility, we estimated that the entire sample required would be successfully obtained in a six-week period. in quantitative variables, normality of distribution was assessed using shapiro-wilk normality test, and homogeneity of variances between groups was tested using levene´s test. in variables fitting a gaussian distribution, comparisons between groups were made using student´s t-test (with adjustment for unequal variances if necessary). in variables not fitting a gaussian distribution, mann-whitney u-test was used for comparisons. comparison of categorical variables between groups was performed using chi-square test or fisher´s exact test as appropriate. the overall prevalence of confirmed sars cov-2 infection at delivery was described using 95% confidence intervals. estimates of prevalence were also obtained for each of the six weeks of the present study. the daily screening positivity rate observed in the study, and the dailyincidence rate in the city of santiago (reported by the ministry of health) [19] were modeled using 5-period moving averages time series. correlation between the observed screening positivity rate and the daily-incidence rate reported in the city of santiago was estimated using spearman's rho correlation coefficient. maternal and perinatal outcomes were described using absolute frequencies (percentages) and means (standard deviations). odds ratios and mean differences were used to compare outcomes between groups. in categorical variables, risk estimations were calculated using simple or multivariate logistic regression analysis accounting for potential covariables if appropriate. in numerical variables, mean differences between groups were estimated using simple or multiple linear regression models, accounting for potential covariables if considered necessary. two-sided p-values of less than 0.05 were considered statistically significant. the statistical package used for analysis was stata v.14. a total of 586 patients were admitted and tested for sars cov-2 during the study period. three cases were excluded: one was less than 24 weeks at the time of admission and the other two cases were term pregnancies, who had a previous diagnosis of covid-19, with complete quarantine for 14 days, and no longer considered as active cases. finally, a total of 583 patients who delivered 586 newborns were included. among them, 37 had a positive result for sars cov-2 at admission. mean maternal age was 30.3 years and 48.9% of patients were nulliparous. nearly 16% of our population presented at least one described risk factor for severe disease [20] . overall, there were no significant differences between confirmed cases and controls in any of the maternal characteristics (table 1) . during the 6 weeks study period, the cumulative prevalence of confirmed sars cov-2 infection was 6.35% [ci 95%: 4.63-8.65]. interestingly, we were able to observe a progressive increase in the rate of positive tests, starting with a point prevalence of 3.03% (3/96) during the first week and reaching an 8.89% (8/82) during the last week of the study. when we compared the daily positivity rate observed in our study group with the daily-incidence rate reported in santiago de chile, there was a statistical significant positive correlation between them (rho: 0.559, p-value < 0.001) (fig 1) , meaning that during the same period of time, regional incidence rate showed similar trends. from the 37 confirmed cases, 43.2% (16/37) were asymptomatic and 56.8% (21/37) were symptomatic at admission. table 2 summarizes the characteristics of these patients. among symptomatic cases, 71.4% (15/21) mentioned no symptoms at admission. however, after a structured interview was applied, they referred at least one symptom present during the previous days and were classified as symptomatic cases. s1 fig shows symptom distribution according to patient survey. based on covid-19 disease severity characteristics by wu et al. [21] of symptomatic cases, 85.7% (18/21) had mild symptoms and evolved positively during hospitalization. the other 14.3% (3/21) presented severe symptoms and required admission to intensive care unit. of them, 2 required invasive ventilatory support, representing 9.5% (2/21) of symptomatic cases and 5.4% (2/37) of all confirmed cases. there were no maternal deaths during the study period. overall, the mean gestational age at delivery was 38.8 weeks of gestational age, and the rate of preterm birth (<37 weeks of gestational age) was 5.29% (31/586). mean birthweight was 3337.1 grams and the rate of small for gestational age newborns (<10 th centile) was 5.12% (30/ 586). there were 49.5% (290/586) cesarean sections, and 10.1% (30/296) instrumental deliveries. mean apgar score at 1 and 5 minutes was 9, and the rate of low apgar (<7 at 5 minutes) was 0.8% (5/585). overall, there were no differences between confirmed cases and controls for each outcome evaluated table 3 summarize main perinatal outcomes and s1 table compares results among symptomatic and asymptomatic cases. during the study period, there were 33/586 (5.6%) newborns who required neonatal admission (either neonatal intensive care unit or neonatal intermediate care unit). among them, 5 cases were deliveries from rt-pcr positive patients; newborn from case #10 was admitted due to cyanosis; newborn from case #19 was admitted due to transient tachypnea; newborn from case #22 was admitted due to septic shock; newborn from case #23 was admitted due to 30 weeks preterm birth; newborn from case #25 was admitted due to prenatal diagnosis of early fetal growth restriction. all of them presented a negative rt-pcr for sars cov-2. among 37 confirmed maternal cases, all newborns were initially isolated. of them 94.6% (35/37) had a negative rt-pcr for sars cov-2 analysis at 6 and 72 hours after delivery. there were 2 (5.4%) newborns, both from asymptomatic mothers, who presented a positive rt-pcr for sars cov-2 at 6 and 72 hours, both were kept in isolation during maternal admission, evolved without symptoms, and were discharged to complete domiciliary quarantine with their mothers. during our study four perinatal deaths were registered, 2 stillbirth and 2 neonatal death. of the stillbirths, both mothers had a negative rt-pcr for sars cov-2. the first one occurred at 38 weeks and was attributable to a prenatal diagnosis of trisomy 18. the second, was a perinatal death at 40 weeks of gestational age, without any referable cause at the moment of this report. of the neonatal deaths, the first one was a spontaneous preterm birth of 27 weeks who died after 6 hours of delivery, with negative maternal rt-pcr for sars cov-2 but findings consistent with severe connatal infection. the second one was a newborn delivered at 37 weeks of gestational age, from a patient with a positive rt-pcr for sars cov-2 at admission (case #22), who rapidly evolved with a septic shock and died after 26 hours. the neonatal rt-pcr for sars cov-2 taken at 6 hours of life was negative. finally, based on a positive blood culture, neonatal death was attributed to a severe sepsis caused by streptococcus agalactiae. our study on universal screening among unselected obstetrical population reveals an overall prevalence of 6.35% of sars-cov-2 infections at delivery. interestingly, nearly half of these cases were asymptomatic at the time of delivery, and of the symptomatic cases nearly 70% referred symptoms only after a targeted interrogation. the later, demonstrates a not negligible reporting bias among patients with very mild symptoms. it could be argued that previous reports on universal screening in obstetrics population do not provide information on the local situation of the pandemic, so it is difficult to estimate the real implications and external validity of their findings. moreover, as it has been stated, the different time points of patient recruitment along with the rising and falling phase of the pandemic curve, explain the different rates of positive rt-pcr results [22] . therefore, in areas with high prevalence of infection, it could be expected that more women may be positive but asymptomatic [23] . this could be seen in reports by sutton et al. [10] , vintzileos et al. [9] , bianco et al. [13] and dória et al. [16] all of them were conducted in areas and timepoints with reported high prevalence of infection [24] , and showed higher observed sars-cov-2 infection prevalence, ranging between 11 and 19%, with up to 15% of asymptomatic confirmed cases among screened population. on the other side, reports by naqvi et al. [25] and gagliardi et al. [12] , reported a low performance of universal screening based either on an overall lower disease burden in their region or due to a referred "steady state" of virus circulation, with less than 1% of asymptomatic confirmed cases. in an intermediate epidemiological situation [24] reports by khalil et al. [11] , miller et al. [14] and ochiai et al. [26] presented observed sars-cov-2 infection prevalence ranging between 3 and 7%, with up to 6% of asymptomatic confirmed cases among screened population, which are similar to our findings. in our study, at the moment of the initial recruitment, according to the official data reported by the national ministry of health, there were about 7858 confirmed cases of sars cov-2 in the city of santiago, with a cumulative incidence of 96.7 per 100000 habitants. in the following weeks, there was a progressive increase in daily incidence, reaching at the end of our study a total of 112136 confirmed cases and a cumulative incidence of 1380 per 100000 habitants. the above explains the increase in cases in our obstetrical population registered during the study period. according to our results, it could be argued that current rates of sars cov-2 infection among general population are underestimated, and this is only partially explained by asymptomatic cases. regarding asymptomatic population, almost all previous studies report higher rates of asymptomatic patients [9-13, 16, 26] . this could be explained by how case definition changed as the pandemic evolved, leading to a non-standardized definition in literature, and by differences among studied populations. also, the extent of symptom evaluation at admission is a key factor in the reported prevalence of asymptomatic patients. in our study there was a high rate of reporting bias (patients not referring symptoms spontaneously but with positive ones when a targeted anamnesis was applied) of nearly 70%. this could be due to the fact that a significant number of symptoms of covid-19 disease overlap with those related to physiological changes during pregnancy. the above highlights the importance of targeted symptom assessment, the need of proper patient education on signs & symptoms and the potential limitation of a diagnostic strategy based only in patients' symptoms report. regarding perinatal outcomes, we found no significative differences between groups. it's important to highlight that our study was not designed to assess the differences in perinatal outcomes, so careful interpretation should be taken. however, we did notice a trend to a higher rate of preterm birth among our study population, which is in line with recent reports [5, [27] [28] [29] [30] . we also had an unexpected high rate of perinatal death, but after analyzing each case individually, causal association with sars cov-2 infection seems unlikely. taking into account our findings and the available literature, in endemic areas, it seems reasonable to perform universal screening for sars cov-2 infection to all patients admitted in labor as it detects an important percentage of patients that would not be detected by conventional clinical screening based only in clinical manifestations of the disease. moreover, when compared against data reported by the national ministry of health, trends among our obstetric population may be a reflection of what was happening in the city of santiago at the same time. as it has been suggested that there is a strong possibility that community infection prevalence may far exceed what is currently being reported [14] . so, universal screening to obstetric population may provide insight to estimates general population prevalence of sars cov-2 infection. real implications of covid-19 disease in the obstetrics population are still largely unknown. so far, conclusions drawn from available literature are reasonably hindered by the context of an evolving pandemic and different approaches across nations. until larger nation-based reports are available, definitive conclusions cannot be made. also, given the consistently high rates of asymptomatic infection, implications and longterm outcomes among this non-identified subset of "recovered" ongoing pregnancies (and their newborns) are somehow alarming. serological assessment at third trimester as a way of screening for this population could be considered, yet several issues such as cross reactivity, antibody kinetics and cost effectiveness remain to be resolved [31] . the main strength of our study is that our institution is one of the largest obstetric centers in our country allowing to gather a large sample of patients' representative of local population in a short period of time. this is important due to the unpredictable behavior of the pandemic. also, to the best of our knowledge, this is one of the largest reports on universal screening in unselected obstetrics population. we also acknowledge several limitations: first, we were not able to assess for maternal serologic status, therefore we cannot rule-out that some of our patients may already have recovered from an asymptomatic infection. second, we did not perform an active follow-up, including targeted anamnesis, of non-infected cases, so we are not able to assess if some proportion of our patients were at early stages of incubation and developed the disease after being discharged from our institution. third, several sources of variability have been reported from rt-pcr obtained from nasopharyngeal swab [32] , so the possibility of false negative results in our patients, especially those who were asymptomatic at admission could not be discarded, and point prevalence could be underestimated. bronchoalveolar lavage has been reported to present higher sensibilities than nasopharyngeal swab [33] , but the use of invasive (high-risk aerosolizing) diagnostic measure seems disproportionate in the context of asymptomatic population. also, it has been reported that non-enhanced chest ct presents higher sensibilities than rt-pcr [34] . however, according with current recommendations of the american college of radiology (acr) [35] , we did not perform on systematic basis imagenologic studies to all patients. finally, regarding newborns with confirmed infection, we did not perform amniotic fluid nor placental analysis, therefore we are not able to establish any conclusion regarding potential vertical transmission. the point prevalence found in our study is 6.35%, with nearly 50% of them being asymptomatic. universal screening in unselected population at delivery, should be considered in endemic areas as provide good estimates of population-level prevalence of sars cov-2 infection, allowing adequate with protection of health team, proper patient isolation, prompt neonatal testing and targeted follow-up. who director-general's opening remarks at the media briefing on covid-19-11 coronavirus disease 2019 (covid-19) pandemic and pregnancy coronavirus disease 2019 (covid-19) in pregnant women: a report based on 116 cases care of the pregnant woman with coronavirus disease 2019 in labor and delivery: anesthesia, emergency cesarean delivery, differential diagnosis in the acutely ill parturient, care of the newborn, and protection of the healthcare personnel rates of maternal and perinatal mortality and vertical transmission in pregnancies 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weeks of confirmed presentations to an affiliated pair of new york city hospitals covid-19 during pregnancy: a case series from an universally tested population from the north of portugal the allplex 2019-ncov (seegene) assay: which performances are for sars-cov-2 infection diagnosis? who | personal protective equipment for covid-19 world health organization risk factors for 2019 novel coronavirus disease (covid-19) patients progressing to critical illness: a systematic review and meta-analysis characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention the "scar" of a pandemic: cumulative incidence of covid-19 during the first trimester of pregnancy executive management summary and short report of outcome covid-19)-world health organization severe acute respiratory syndrome coronavirus 2 (sars-cov-2) universal testing experience on a los angeles labor and delivery unit universal screening for sars-cov-2 in asymptomatic obstetric patients in clinical presentation and outcomes of pregnant women with covid-19: a systematic review and meta-analysis effects of coronavirus disease 2019 (covid-19) on maternal, perinatal and neonatal outcomes: a systematic review outcome of coronavirus spectrum infections (sars, mers, covid 1-19) during pregnancy: a systematic review and meta-analysis characteristics and outcomes of pregnant women admitted to hospital with confirmed sars-cov-2 infection in uk: national population based cohort study the important role of serology for covid-19 control inappropriate nasopharyngeal sampling for sars-cov-2 detection is a relevant cause of false-negative reports detection of sars-cov-2 in different types of clinical specimens sensitivity of chest ct for covid-19: comparison to rt-pcr acr recommendations for the use of chest radiography and computed tomography (ct) for suspected covid-19 infection to the midwives and physicians of our institution. key: cord-340703-vtuy806l authors: cascio, antonio; colomba, claudia; di carlo, paola; serra, nicola; lo re, giuseppe; gambino, angelo; lo casto, antonio; guglielmi, giuseppe; veronese, nicola; lagalla, roberto; sergi, consolato title: low bone mineral density in hiv-positive young italians and migrants date: 2020-09-03 journal: plos one doi: 10.1371/journal.pone.0237984 sha: doc_id: 340703 cord_uid: vtuy806l background: human immunodeficiency virus (hiv) infected individuals may have osteoporosis. we aimed to evaluate the bone mineral density (bmd) in naïve antiretroviral (arv) treated hiv positive patients comparing native italian group (itg) to a migrants group (mig) upon arrival in italy. methods: we conducted a cross-sectional study on 83 hiv patients less than 50 years old. we used the dual-energy x-ray absorptiometry (dxa) within six months from the hiv diagnosis. participants were categorized as having low bmd if the femoral neck or total lumbar spine z-score was– 2 or less. results: mig showed low bmd more often than itg (37.5% vs.13.6%), especially for the female gender (16.7% vs. 0.0%). a low cd4 rate (<200 cells/μl) was most often detected in mig than itg. in particular, we found most often male italians with abnormal cd4 than male migrants (67.8% vs. 33.3%) and vice versa for females (30.5% vs. 66.7%). we found an abnormal bone mineral density at the lumbar site. low bmd at the lumbar site was more frequently observed in female migrants than female italians. both male and female migrants had a z-score value significantly lower than male and female italians, respectively. by logistic regression low vitamin-d level was positively correlated to low bmd in itg only. all data were verified and validated using a triple code identifier. conclusions: both dxa and vitamin-d evaluation should be offered after the diagnosis of hiv infection. lumbar site low bmd is an initial condition of bone loss in hiv young patients, especially in female migrants. vitamin d levels and supplementation may be considered after hiv diagnosis independently of age to improve bone health. highlights: this study evaluates the frequency of bone mineral density in hiv positive patients naive to antiretroviral therapy. it compares the density of the native italian population with that of hiv migrants upon arrival in italy. the results show that hiv positive migrants, even if younger than 50 years of age, are at risk for osteoporosis, especially if they are female. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 osteoporosis is a process characterized by increased bone resorption without reciprocal bone apposition. the decreased bone mass leads to an increased risk of bone fractures, which are particularly as the subject ages [1, 2] . currently, lifestyle factors, including alcohol, diet, hormones, physical activity, and smoking, are influencing the bone mass, and osteoporosis is affecting younger people than before [2] [3] [4] . the incidence of osteoporosis and osteoporosisrelated fractures varies across the european union/european economic area (eu/eea), and especially in the mediterranean area, it is increasing, as it has been evidenced in the literature with a clear link to nutrition [1, 2] . among human immunodeficiency virus (hiv)-related comorbidities, the bone disease has emerged as a significant question in this chronically infected population [3] [4] [5] [6] [7] [8] . several studies suggest that fracture rates are higher in communities with hiv than among matched uninfected controls despite anti-retroviral therapy (art) and gender [9] [10] [11] . the causes of a low bone mineral density (bmd) could be different in hiv-infected patients by considering the time of hiv infection [12] . previous studies conducted in our geographical area established that the prevalence of osteoporosis was significantly higher in hiv-infected than in uninfected subjects, which mirrors a result similar to previous meta-analyses [12] [13] [14] [15] . the prevalence of osteopenia and osteoporosis in hiv mono-infected patients in our geographical area was about 44.9%, and 20.9% in comparison with 18% reported in a healthy italian population [16] . a recent analysis of bone-healthy in the immigrant population conducted in sweden showed that women had low bmd for age according to the american and african-american referents [17] [18] [19] . moreover, a study conducted in german-turkish immigrants osteopenia was diagnosed in 32% and osteoporosis in 8% of young migrants [20] . in the migrant setting, it has been hypothesized that bmd is intriguingly associated with lifestyle, body mass index (bmi), and vitamin d levels. recently, low level of vitamin d were also reported in somali migrant women in sweden and refugees in canada using the calgary refugee health program, an urban family practice that serves newly arrived refugees in calgary, alberta [21] [22] [23] . although the number of migrants and refugees crossing the mediterranean sea has decreased in 2018-19 and 2020, during the covid-19 pandemic, this number has been unprecedented in the last ten years compared with the past twenty or thirty years [5] [6] [7] . topical italian data showed that the new diagnosis of hiv infection in 72% of migrants was late and less than six months before developing aids with half of the subjects coming from sub-saharan africa (ssa, 59.4%) and with an increasing percentage of fertile females [22] [23] [24] [25] [26] . further, the incidence of bmd loss and related fractures attributed to a specific eating pattern has also been targeted for guidelines and suggestions within the eu/eea [8] . our study aims to emphasize the burden of bone health in naïve arv hiv positive patients and compare the bone density of the native italian population group (itg) with that of hiv migrants (mig) upon arrival in italy. this investigation is a retrospective cross-sectional study. we gathered data from 83 arv naïve subjects consecutively admitted between january 2010 and may 2015 at the sicilian aids center of the university of palermo, italy. we retrospectively analyzed all patients who underwent dual-energy x-ray absorptiometry (dxa) within six months from hiv diagnosis independent of their bmi. in this study, we excluded all patients with the treatment of steroidinduced bone loss and subjects with active tuberculosis. dxa measures bmd utilizing two x-ray beams with different energy levels, which are aimed at the patient's bones (see below). of the 83 subjects, 59 were italian, and 24 were migrant patients living in italy for less than twelve months. table 1 shows the characteristics of enrolled patients. we collected the medical records and entered into an anonymous database, as previously reported [8, 9] . cd4+ t-cell count and plasma hiv-rna levels were assessed as previously reported [9] . 25-hydroxy-vitamin d (25(oh)d) was assayed as previously reported [21, 22, 25] . we considered a serum 25(oh)d concentration of 30 ng/ml as a threshold value for identifying low levels of vitamin d, as previously reported (standard value of our laboratory: 30-80 ng/ml) [13] . all subjects gave their informed consent for inclusion before they started participating in the study, which was conducted by the declaration of helsinki. the ethics committee of the university of palermo approved the study protocol. each participant was free to decline or withdraw from the study at any time, and this was explained in their original language or a language that the migrant knew. bmd was assessed by dxa, using a qdr discovery hologic dxa in the femoral neck and dxa in the lumbar spine by total body dxa. for each scan, bmd, z-scores were recorded as previously reported [9] [10] [11] . dxa measurements were performed in the femur (femoral neck and/or total hip) and lumbar spine in each patient. since the age of our patients ranged from 30 to 50 years, the use of z-scores (defined as an individuals' bmd in comparison to agematched normal individuals) was used for all the analyses, according to world health organisation (who) recommendation [27] . participants were categorized as having low bmd if the femoral neck or total lumbar spine z-score was-2 or less. to individualize sample sizes statistically significant in this study, we considered a bernoulli sampling for both italian and migrant groups [28] . for itg, the minimum sample size for this study was estimated equal to 41 patients affected by low bmd. it was obtained considering a statistical z-score at 95%, an error ε = 15% and hypothesizing a prevalence π, about 60% according to the studies of cavalli et al. [16] and tomažič et al. [29] . in this case, we estimated a prevalence range equal to 45%-75%. for mig, the minimum sample size for this study was estimated equal to 22 patients affected by low bmd. it was obtained considering a z-score at 95%, an error ε = 20%, and hypothesizing a prevalence π about 35%, according to varenna et al. [30] . in this way we estimated a prevalence range equal to 15%-55%. in this case, we considered an error ε greater to an error in itg because of the amount of information that could be gathered in migrants in italy. also, the sample size for itg and mig were increased to 59 and 24 patients, respectively considering the possibility of unexpected events and, consequently, the likelihood of patients' data loss. the statistical analysis was performed by matlab statistical toolbox version 2008 (math-works, natick, ma, usa). data are presented as number and percentage for categorical variables. numerical data are expressed as the mean ± standard deviation (sd) or median and confidence interval at 95% (ci). the χ 2 test and fisher's exact tests were performed to evaluate significant differences of proportions or percentages between two groups. the fisher's exact test was used where the χ 2 test was not appropriate. the mann-whitney test was used to test the difference between two independent samples (itg and mig group). it was the alternative for a t-test dealing with independent samples when the distribution of the samples is not normal. normal distributed independent samples were studied using the d'agostino-pearson test. linear correlation analysis was also performed, and the test on pearson's linear correlation coefficient r was performed with the t-student test under the null hypothesis of pearson's linear correlation coefficient r = 0. the logistic regression was performed to analyze the relationship between low bmd (dichotomous variable) and the independent variables: gender (dichotomous-m = 1, f = 0), previous fractures (dichotomous-yes = 1, no = 0), bmi (continuous), cd4 cells (continuous), and 25-hydroxy-vitamin d (continuous). finally, all tests with p-value < 0.05 were considered significant. the 83 patients, composed by 59.4% males and 40.6% females, with ages into range 30-50, mean 44.2 years old and standard deviation (s.d.) equal to 4.9 years old, was subdivided into two groups (table 1) in table 2 , we report the differences between itg and mig group and statistical tests. low bmd was significantly more frequent in mig group in comparison to itg group (37.5% > 13.6%, p = 0.0324), particularly it was significantly more frequently found in migrant females than in italian females (16.7% > 0.0%, p = 0.0058). significant differences were seen in low-bmd values. in fact, both female and male italians had a measure of bmd significantly greater than migrant females and males respectively (median: -1.0 > -2.3, p = 0.0272; 0.05 > -0.8, p = 0.0291). in addition, we found major presence of migrant females with abnormal lumbar value in comparison to italian females (16.7% > 0.0%, p = 0.0058). conversely, no significant differences between itg and mig were found considering the femoral bmd values. in examining the gender in mono-infected hiv patients, the percentages of hiv patients were significantly greater in italian males in comparison to migrant males (57.6% > 25%, p = 0.007). on the other hand, hiv was most frequently observed in female migrants in comparison to female italians (58.3% > 23.7%, p < 0.003). for co-infected patients, there were no significant differences in gender between itg and mig. also, for aids patients there was a significant difference between itg and mig (45.8% < 83.3%, p = 0.0017). in particular, aids was most frequent in female migrants in comparison to female italians (8.3%>8.5%, p<0.0001). we found a higher percentage of male italians with abnormal values of cd4 in comparison to male migrants (67.8% > 33.3%, p = 0.0039), and vice versa for the female gender ( in migrant patients, 25-oh-vitamin d mean values were significantly lower than in italian patients (14.8 > 11.8 ng/ml, p = 0.0466). also, male italians had mean value significantly figs 1 and 2 , the heat maps of lbd and fbd values for itg and mig groups are shown, respectively. every line represents the fbd and lbd values for each patient, and the graduation of the colors are representative of the z-score. in fig 3, we showed the scatter plots for itg and mig group between cd4 values with lbd and fbd (a-d). for every scatters plot, the red points on graphs are individuated by cd4 values and correspondent lumbar bmd and femoral bmd z-scores. the blue line is the best linear fit, and the red lines define the 95% prediction interval for the regression curve. for any given value of the independent variable (cd4), this interval represents the 95% probability for the values of the dependent variable (lbd or fbd). by linear correlation analysis, it results that for the itg group, there was a significant positive linear correlation between cd4 and femoral bmd (r = 0.34, p = 0.009) and between cd4 and lumbar bmd (r = 0.30, p = 0.020). in contrast, the mig group showed no significant correlation between cd4 and femoral bmd (r = 0.0, p = 1.0) and between cd4 and lumbar bmd (r = 0.23, p = 0.27). in other words, in italian patients, an increase/decrease of cd4 values implicate an increase/decrease of lumbar bmd or femoral bmd scores. we did not observe these correlations in the mig group. finally, in table 3 , we report the logistic regression analysis between low bmd variable (dichotomous) and the independent variables: gender (dichotomous), bmi (continuous), hydroxy-vitamin d (continuous), cd4 (continuous), and previous fractures (dichotomous) for the total sample, itg, and mig. for this scope, two models were considered. the null model: -2ln(l 0 ), where l 0 was the likelihood of obtaining the observations if the independent variables did not affect the outcome, and the full model: -2ln(l 0 ), where l 0 was the likelihood of obtaining the observations with all independent variables incorporated in the model. the difference between these two yields was estimated with the chi-square test to define how well the independent variables affect the outcome or dependent variable. if the chi-square test was positive (p < 0.05), then there was evidence that at least one of the independent variables contributes to the prediction of the outcome. by logistic regression, it resulted that only hydroxy-vitamin d was negatively correlated to low bmd both in total sample (odds ratio, or = 0.85 and p = 0.0063), and in itg (or = 0.84 and p = 0.0309). in other words, an increase (decrease) of hydroxy-vitamin d contribute to decreasing (increasing) of low bmd. in other words, a decrease of hydroxy-vitamin d contributes to increasing the probability of osteoporosis or osteopenia in the total sample, and particularly, in the italian patients. in the migrant group, we did not observe significant correlations by regression analysis, and the small sample size of migrants considered in our study may be the reason (see below). our study shows that "low bmd" was significantly more frequent in the mig group in comparison to the itg group (migrants 37.5% vs. 13.6%). in particular, it was significantly more frequently found in migrant females than in italian females. our previous reports [13, 14] on the prevalence of low-bmd in hiv mono-infected patients who underwent arv therapy showed higher percentage rates of osteopenia (44.9%) and osteoporosis (20.9%) than an agerelated healthy italian population (18%) [16] . our current study showed that the percentage of abnormal bmd was mostly restricted to the lumbar site. the low bmd of the lumbar site in female migrants was more evident than in female italians, and both males and female migrants had a z-score value significantly lower than males and females italians. studies conducted on hiv negative patients have, indeed, shown how osteophyte formation, bone sclerosis, disk space narrowing, and spondylolisthesis are positively correlated with lumbar spine bmd. at the same time, there was no association with femoral neck bmd [31] . in general, anatomical studies suggest that sex-differences in the lumbar spine appear to be supported by postural differences in sacral bone-orientation and morphological differences in the vertebral bodies of females [32] . moreover, emerging data show that pregnant and breastfeeding women are associated with early osteoporosis, especially in the thoracolumbar spine [12] . the involvement of the lumbar site is probably justified by the fact that our sample is younger than in other studies conducted on older populations of hiv negative subjects [13-15, 33, 34] . hcv is a risk factor for osteoporosis and fractures in the hiv population. in our studied population, hcv co-infection was found in 18.1% of the enrolled sample. recently, one of the authors identified a more lumbar than femoral "low bmd" in hiv/hcv coinfected patients [16] . the comparison of aids diagnosis between italian and migrants showed that aids was more frequent in migrants, especially in females. in general, we found more frequently low cd4 values (<200 cells/μl) in both male and female migrants in comparison with italian patients. these data are in agreement with the emerging late diagnosis of hiv infection in young italians comparing with age-matched female migrants [24, 25] . by linear correlation analysis, an increase/decrease of cd4 values implicates an increase/decrease of lumbar or femoral bmd scores only in italian patients. in contrast, we did not observe similar correlations in the mig group. this aspect is probably due to the element that all migrants had lower cd4 values than age-matched italians. indeed, no migrant had values of cd4 � 350. although the mean of bmi in migrants was lower than italian patients, the statistical analysis of this variable evidenced that low bmd was significant only in female migrants. on the other hand, the comparison of the percentage of patients with abnormal bmi was significant in male italians. the logistic regression showed a relationship between low hydroxy-vitamin d and low bmd only in the italian group. this data should be validated on a larger sample size. low consumption of dairy products, high consumption of soft drinks, moderate sun exposure, and a high prevalence of vitamin d deficiency have been identified among youth as risk factors for early osteoporosis. the nutrition factor is probably intrinsically associated with the geographical region and climate extremes (droughts and storms) may aggravate the bone mass loss of hiv-infected youth. as reported in other studies, we observed early natural menopause status both in italian than in migrant women. this finding was analogous to both groups. apart from hiv-related immunologic status (cd4 count and viral load) other socio-demographic variables (marital status, parity, education, or income) and religious clothing of the female migrant sample may have influenced the low bmd observed in our study. to the best of our knowledge, this study is the first dxa-based investigation to study and assess the bmd among the flow of migrants and refugees from countries with a high incidence of hiv infection across the mediterranean area. in this respect, the fact that our study includes only migrants, who were resident in italy for no more than one year, could offer the opportunity to carefully assess the risk factors, which stress the bone composition in the hiv infected population. a recent paper on osteoporosis among hiv positive patients highlighted how an assessment of baseline bone mass loss within the first two years from the start of arv is often lacking in longitudinal studies and claimed that there is some evidence concerning bone loss within the first year of hiv infection and art initiation [13, 17] . the findings of a high prevalence of low bmd in hiv infected migrants aged less than 50 years of age could suggest that the first screening for osteoporosis should be carried out as soon as possible and before the start of art. this aspect should help the clinician in arv management. planning hiv therapy to prevent bone loss is crucial. we suggest the use of tenofovir alafenamide (taf) molecule instead of tenofovir to reduce the risk of bone toxicity [35, 36] . a recent study enrolled young hiv positive with low bmd showed tenofovir as a key molecule in arv therapy [11] . taf is a hepatitis b virus (hbv) nucleotide reverse transcriptase inhibitor for the treatment of chronic hbv infection in adults with compensated liver disease and has been approved by the food and drug administration (fda) for the treatment of hiv-1 in november 2015. the altered bone remodeling in young hiv positive women may suggest some alertness about our current preventive health care services. most of the immigrants diagnosed with hiv are pregnant women who have undergone tests for sexually transmitted diseases. although this study is original and discloses essential steps to improve current management strategies because of new migrations from underdeveloped countries, there are some limitations. our research involves a small size of the groups, but the cross-sectional study design still maintains its validity. this study does not permit us to establish a cause-effect relationship from our results or to evaluate the impact of osteoporosis risk factors and/or lifestyle risk factors on the management of bone disease in the hiv population. thus, a multicentric and prospective study is warranted. in conclusion, our study raises the question of the need to plan preventive strategies in hiv migrants and refugees, also in the light of the great movement of people from southern mediterranean areas to northern european countries with low sun exposure [21, 22] . the female migrants must be protected from precocious low bmd with an additional layer of safety [17-19, 21, 22, 33] . the approach requires different and specific intervention strategies in the future. our data confirm that early screening for low bmd and other risk factors associated with bone loss in hiv patients is useful [2, 7, 15, [18] [19] [20] . in agreement with the osteoporosis foundation (http://share.iofbonehealth.org/wod/ compendium/2019-iof-compendium-of-osteoporosis-press.pdf) the authors stress the strategies for preventing osteoporosis. the role of nutrition in maintaining bone health associated with a personalized anti-hiv treatment and daily vitamin supplementation to reduce early bone loss as well as a baseline dxa are critical steps for hiv migrants and refugees after arrival in the host countries [13, 17, 20, 23, 36] . high-risk groups should be first identified, and osteoporosis investigations should be carried out earlier than at 50 years of age [37] [38] [39] . as indicated above, our results should be evaluated with caution due to the retrospective nature of the study. also, despite the estimated minimum sample sizes of itg and mig were correctly applied and the conditions for the sample size according to bernoulli are met, prospective and more extensive studies are needed to confirm our results. assessment of fracture risk and 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fractures in a migrant population from southern to northern italy: a cross-sectional, comparative study insulin/igf-1r, sirt1, and foxos pathways-an intriguing interaction platform for bone and osteosarcoma morphological and postural sexual dimorphism of the lumbar spine facilitates greater lordosis in females calcium and vitamin d in human health: hype or real? phalangeal quantitative ultrasound: cheaper methods for screening and follow-up of bone pathologies in hiv-infected women? optimizing antiretroviral therapy for women living with hiv planning hiv therapy to prevent future comorbidities: patient years for tenofovir alafenamide osteoporosis risk factors in hiv positive women with osteoporosis: a retrospective analysis vitamin d status and the relationship with bone fragility fractures in hiv-infected patients: a case control study subsequent fracture risk of women with pregnancy and lactation-associated osteoporosis after a median of 6 years of follow-up the authors ac, pdc, and ns, contributed equally to this paper. we are deeply grateful to the patients and their families for participating in the study. preliminary results of this study were presented at the hiv glasgow conference, oct. 23-26, 2016, glasgow, united kingdom. key: cord-356132-1nisyl5r authors: wang, huiyao; xia, qian; xiong, zhenzhen; li, zhixiong; xiang, weiyi; yuan, yiwen; liu, yaya; li, zhe title: the psychological distress and coping styles in the early stages of the 2019 coronavirus disease (covid-19) epidemic in the general mainland chinese population: a web-based survey date: 2020-05-14 journal: plos one doi: 10.1371/journal.pone.0233410 sha: doc_id: 356132 cord_uid: 1nisyl5r as the epidemic outbreak of 2019 coronavirus disease (covid-19), general population may experience psychological distress. evidence has suggested that negative coping styles may be related to subsequent mental illness. therefore, we investigate the general population’s psychological distress and coping styles in the early stages of the covid-19 outbreak. a cross-sectional battery of surveys was conducted from february 1–4, 2020. the kessler 6 psychological distress scale, the simplified coping style questionnaire and a general information questionnaire were administered on-line to a convenience sample of 1599 in china. a multiple linear regression analysis was performed to identify the influence factors of psychological distress. general population’s psychological distress were significant differences based on age, marriage, epidemic contact characteristics, concern with media reports, and perceived impacts of the epidemic outbreak (all p <0.001) except gender (p = 0.316). the population with younger age (f = 102.04), unmarried (t = 15.28), with history of visiting wuhan in the past month (t = -40.86), with history of epidemics occurring in the community (t = -10.25), more concern with media reports (f = 21.84), perceived more impacts of the epidemic outbreak (changes over living situations, f = 331.71; emotional control, f = 1863.07; epidemic-related dreams, f = 1642.78) and negative coping style (t = 37.41) had higher level of psychological distress. multivariate analysis found that marriage, epidemic contact characteristics, perceived impacts of the epidemic and coping style were the influence factors of psychological distress (all p <0.001). epidemic of covid-19 caused high level of psychological distress. the general mainland chinese population with unmarried, history of visiting wuhan in the past month, perceived more impacts of the epidemic and negative coping style had higher level of psychological distress in the early stages of covid-19 epidemic. psychological interventions should be implemented early, especially for those general population with such characteristics. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the epidemic of the 2019 coronavirus disease has aroused widespread concern throughout society in china. because the virus can be transmitted through droplets, contact, etc. [1] , cities in many regions of china have closed non-essential public places, restricted mass gathering activities, and enacted other control measures to effectively control the spread of the virus [2] . the epidemic has had a strong impact on general population's daily life. at the same time, as the epidemic continues, general population gradually experience different levels of psychological distress, such as nervousness, fear of infection, anxiety, depression, sleep problems, and inattention [3, 4] . previous studies have reported that some psychological problems often occur during similar epidemic [5, 6] or other traumatic stress events, such as natural disasters [7, 8] , disease [9] , or long-term employment in high stress occupations [10] [11] [12] , and may last for a long time [13, 14] . when faced with stress or traumatic experiences, general population often responds differently, with some responding positively and others responding negatively. evidence has suggested that coping styles in the face of stress have an impact on the quality of general population's life [15, 16] , and negative coping styles may be related to psychological distress or mental illness such as post-traumatic stress disorder (ptsd), anxiety, and depression [7, 8, 12] . for this reason, we conducted this study in the early stages of this epidemic to investigate the general population's psychological distress and coping style related to the epidemic of covid-19 so that those who have high levels of psychological distress and/or respond negatively can be detected early and undergo timely intervention. this study was conducted through an online survey, starting at 16:00 on february 1, 2020 and ending at 24:00 on february 4, and the survey was approved by the ethical review board of the west china hospital of sichuan university. the snowball sampling method was used to invite subjects. all invitees completed the questionnaire online via questionnaire star (https://www. wjx.cn). an initial set of invitees (10 participants) was chosen to ensure broad representation of age, gender, occupation, education level, and city. this set of invitees then forwarded the questionnaire to 10 companions whom they considered suitable for the survey, and this second set forwarded the questionnaire in the same way. the study included a general population aged 18 years or older who volunteered to participate in the study. the participants received a complete description of this survey and were asked to sign an online informed consent prior to data collection. respondents were excluded if they reported a history of mental illness and/ or could not complete the online survey independently. a self-made questionnaire was used to collect demographic and epidemiological information of participants, including gender, age, marriage, epidemic contact and concern characteristics, and perceived epidemic impacts of the epidemic of covid-19. the kessler 6 psychological distress scale (k6) was used to assess the psychological distress of participants; this scale has been proven to have cross-cultural reliability and validity [17] . it contains six questions that ask participants to rate how often they have felt 'nervous', 'hopeless', 'restless or fidgety', 'so depressed that nothing could cheer you up', 'that everything was an effort', and 'worthless' during the past 30 days. the simplified coping style questionnaire (scsq) was used to assess the participants' coping styles during the covid-19 epidemic; this questionnaire has been proven to have good reliability and validity in chinese [18] . the scsq contains twenty items, with each item using a four-point score (0 = never, 1 = seldom, 2 = often, 3 = always), and two subscales: positive coping (12 items) and negative coping (8 items) . according to the average and standard deviation of the positive coping style and the negative coping style scores, a z conversion is used to calculate their respective standard scores, and then, the negative coping standard scores are subtracted from the positive coping style standard scores to calculate the tendency value of coping style. a result greater than 0 was defined as a participant adopting a positive coping style when faced with stress, and a result less than 0 was defined as a participant adopting a negative coping style [19] . differences in psychological distress (k6 score) among categorical variables were tested by ttests or one-way analysis of variance. a stepwise multiple linear regression analysis was performed to identify the influence factors of psychological distress. all statistical analyses were conducted in spss version 22.0 (ibm, chicago, il, usa), and p<0.05 was considered to be statistically significant. the same ip address could be used only once to complete the questionnaire, which did not collect any personal information such as names, thereby ensuring anonymity and honest responses. the time spent on each questionnaire was monitored automatically, and the whole questionnaires completed in fewer than 120 seconds were rejected as invalid. there were 1607 individuals from 26 regions of china who completed this survey, and 1599 (99.5%) were included in the analysis participants. among all participants, 1068 (66.8%) were female, 531 (33.2%) were male; ages ranged from 18 to 84 years old (mean 33.9±12.3 years); 914 (57.2%) were married, 685 were unmarried (42.8%); 326 (20.4%) had a history of visiting wuhan; and 333 (20.8%) had a history of epidemics occurring in their community; 1583 (99.0%) concern with media reports related to the epidemic; 911 (56.9%) feel nervous, 767 (48.0%) feel difficult to control emotion and 612 (38.3%) have epidemic-related dreams in perceived impacts of the epidemic; 547(34.2%) respond with negative coping styles (table 1) . the results revealed significant differences in the participants' psychological distress based on age (f = 102.04), marriage (t = 15.28), history of visiting wuhan or not (t = -40.86), history of epidemics occurring in the community or not (t = -10.25), concern with media reports related to the epidemic (f = 21.84), and changes over living situations (f = 331.71), emotional control (f = 1863.07), and epidemic-related dreams (f = 1642.78) in perceived impacts of the epidemic (all p <0.001); there were no significant differences based on gender (t = -1.00, p = 0.316). as age increases and marital status changes, k6 scores have a downward trend. those with a history of visiting wuhan and a history of epidemics occurring in the community have higher level of psychological distress than those without such experiences. the psychological distress tended to increase with concern with media reports related to the epidemic and perceived impacts of the epidemic. at the same time, the results also show that those with negative coping style have higher level of psychological distress than those with positive coping style (t = 37.41, p <0.001) ( table 2 ). age, marriage, epidemic contact characteristics, perceived impacts of the epidemic and coping style were included in the multivariate analysis. factors' values were listed in (table 4 ). unmarried, history of visiting wuhan, more serious changes over living situations, more difficult of emotional control, higher frequency of epidemic-related dreams, and negative coping style in the general population showed higher level of psychological distress. the results of the present study suggest that the general population in china mainland reported higher level of psychological distress in the early stages of covid-19 than those in non-epidemic period [20] . the present study was conducted during the first two weeks of the covid-19 outbreak, so it indicated that the general population have already presented psychological distress in the early stages of epidemic. this finding is consistent with the previous studies. the traumatic stress experiences during the occurrence of emergency events, such as major public events or natural disasters, were often related to the general population's early psychological distress and subsequent mental illness [5, 6, 8] . the psychological distress could cause the impairment of individual's normal daily activities and was associated with worse social functioning [21] . so, our result suggested that the psychological intervention for the general population should be provided urgently after the outbreak of covid-19 epidemic. the multivariate analysis showed that marriage was the influence factor of psychological distress in general population. the population with unmarried showed higher level of psychological distress. unmarried status, lack of a major social support system, was found to be related to psychological distress [22] . the other study also found single mothers without additional personal support showed higher values of psychological distress [23] . these might imply the importance of the basic social support of marriage when an individual faced the covid-19 epidemic and suggest that mental health workers should pay increased attention to unmarried population. our results also found that the history of visiting wuhan in the past month was the influence factor of psychological distress. the population with history of visiting wuhan showed higher level of psychological distress. mandatory contact tracing and 14 days quarantine, which form part of the public health responses to the covid-19 outbreak when having the history of visiting wuhan in the past month. the quarantine may perpetuate the sense of danger and uncertainty and increase individuals' psychological distress about the effects of contagion, infection, and stigma on their families and friends [3] . it indicated that the general population who have the history of visiting wuhan should be paid more attention in the early stages of covid-19 epidemic. perceived impacts on changes over living situations, emotional control, and epidemicrelated dreams were found to be the influence factors of psychological distress. the population with more serious changes over living situations, more difficult of emotional control, and higher frequency of epidemic-related dreams showed higher level of psychological distress. previous investigations on ebola virus and severe acute respiratory syndrome (sars) have found the similar phenomena that individuals might perceive the infectious diseases as a type of life-threatening life event which could cause various negative emotions, such as anxiety, depression, fear, and a series of sleep problems [3, [24] [25] [26] [27] . in addition, these symptoms may be risk factors for individuals suffering from mental illness in the future and affect individual's attitudes and behaviors towards the epidemic prevention [25] . as of jan 25, 2020, 30 chinese provinces, municipalities, and autonomous regions have initiated first-level responses to major public health emergencies [3] . a range of measures has been urgently adopted, including closed non-essential public places, restricted mass gathering activities, delineating control areas, contact tracing and monitoring to effectively control the spread of the virus, which may bring public uncertainty and a sense of crisis [28, 29] . so, the sudden changes over living situations may increase individual's anxiety and nervous. the sleep problems, especial recurrent distressing dreams in which the content are related to the traumatic event are the core symptoms in patients with ptsd [30] . so, this indicated that the general population may present the symptoms of ptsd in the early stages of covid-19 epidemic. difficult to control the emotion and emotional instability could lead to worsening psychological problems which have been reported in the outbreak of sars [31] . taken these results together, suggested that the psychological intervention should be implemented urgently for the general population who perceived more impacts on changes over living situations, emotional control, and epidemicrelated dreams to prevent suffering from ptsd in the later time. the multivariate analysis also showed that the coping style was the influence factor of psychological distress. the population with negative coping style showed higher level of psychological distress. the previous study related to traumatic stress events has reported that those in the general population with traumatic stress experiences were more likely to adopt a negative coping style [9] . many previous studies have shown that different coping styles, especially negative coping styles, for trauma stress events are also related to subsequent mental illness [7, 10, 32] . in contrast, a positive coping style may promote emotional well-being [33] . therefore, the general population with negative coping styles should be given attention and the appropriate psychological interventions should be considered urgently. there are several limitations in our study. firstly, the survey method is based on network invitation rather than face-to-face random sampling, and participants need to be able to use network tools. as a result, the status of the general population who cannot use network tools is unclear. secondly, we did not assess whether and how respondents were engaging in prevention. finally, our study design is cross-sectional and thus cannot capture changes in psychological distress and its predictors over the course of the covid-19. our study revealed that the epidemic of covid-19 caused high level of psychological distress in the general population. the general mainland chinese population with unmarried, history of visiting wuhan in the past month, perceived more impacts of the epidemic and negative coping style showed higher level of psychological distress in the early stages of covid-19 epidemic. psychological interventions should be implemented urgently, especially for those in the general population with such 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beijing: people's military medical press comparative study of the kessler 10 and kessler 6 scales on chinese university students' mental health. sciencepaper online psychological distress and social functioning in elderly spanish people: a gender analysis associated risk factors for psychological distress in patients with gastric epithelial neoplasm undergoing endoscopic submucosal dissection psychological distress and socioeconomic status in single mothers and their children in a german city an evaluation of psychological distress and social support of survivors and contacts of ebola virus disease infection and their relatives in lagos, nigeria: a cross sectional study-2014 the impact of community psychological responses on outbreak control for severe acute respiratory syndrome in hong kong a novel coronavirus outbreak of global health concern the continuing 2019-ncov epidemic threat of novel coronaviruses to global health-the latest 2019 novel coronavirus outbreak in wuhan, china diagnostic and statistical manual of mental disorders, 5th edition, and clinical utility the immediate psychological and occupational impact of the 2003 sars outbreak in a teaching hospita the mediating effects of coping strategies in the relationship between automatic negative thoughts and depression in a clinical sample of diabetes patients positive emotions trigger upward spirals toward emotional well-being we thank the general population who participated in this survey and bravely resisted the covid-19 epidemic, and thank the questionnaire star (https://www.wjx.cn) for providing us with a data survey platform. key: cord-318614-518giv0m authors: tsai, jih-jin; liu, wei-liang; lin, ping-chang; huang, bo-yi; tsai, ching-yi; lee, pei-yu alison; tsai, yun-long; chou, pin-hsing; chung, simon; liu, li-teh; chen, chun-hong title: a fully automated sample-to-answer pcr system for easy and sensitive detection of dengue virus in human serum and mosquitos date: 2019-07-10 journal: plos one doi: 10.1371/journal.pone.0218139 sha: doc_id: 318614 cord_uid: 518giv0m background: the insulated isothermal pcr (iipcr) technology enables consistent pcr amplification and detection in a simple heating device. a pan-dengue virus (denv) rt-iipcr, targeting the 5’ untranslated region, was validated previously on the semi-automated pockit combo system (involving separate devices for nucleic acid extraction and pcr amplification/detection) to offer performance comparable to a laboratory real-time pcr. working on the same technologies, a compact automated sample-in-answer-out system (pockit central nucleic acid analyser) has been available commercially for iipcr, minimizing human error risks and allowing easy molecular bio-detection near points of need. here, we evaluated the analytical and clinical performance of the pan-denv rt-iipcr on the fully automated system by comparison to those on the semi-automated system. methodology/principal findings: testing sera containing serial diluted denv-1, -2, -3, or -4 cell culture stock, the pan-denv rt-iipcr system had similar 100% detection endpoints on the two systems; i.e. at 1, 10, 1 and 10 pfu/ml, respectively, on the fully automated system, and at 10, 1, 10 and 10 pfu/ml, respectively, on the semi-automated system. furthermore, both fully automated and semi-automated pcr system can detect all four denv serotypes in mosquitos. clinical performance of the reagent on the two systems was evaluated by testing 60 human serum samples. both systems detected the same 40 samples (ten denv-1, -2, -3, and -4 positive each) and did not detect the other 20; 100% agreement (κ = 1) was found between the two systems. conclusions/significance: with performance comparable to a previously validated system, the fully-automated pcr system allows applications of the pan-denv reagent as a useful tool near points of need to facilitate easy, fast and effective detection of dengue virus and help mitigate versatile public health challenges in the control and management of dengue disease. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 dengue virus (denv), a member of the genus flavivirus in the family flaviviridae, causes dengue in humans. dengue virus infection, one of the most important and mosquito-borne viral diseases, is considered a major public health problem in developing tropical and subtropical countries mainly in asia, south america, and the caribbean with major disease outbreaks and endemic every year [1] . four serotypes of dengue virus (denv-1, -2, -3, and -4) have been identified, with distinct genotypes within each serotype. multiple virus serotypes have been found co-circulating in the hyperendemic regions in southeast asia and pacific [1, 2] . its single-stranded, positive-sense genomic rna contains 5'-and 3'-untranslated regions (utr) and a single open reading frame encoding for a single polyprotein that could be cleaved into three structural proteins (capsid [c] , premembrane/membrane [prm/m], and envelope [e] ) and seven nonstructural proteins (ns1, ns2a, ns2b, ns3, ns4a, ns4b, and ns5). denv is primarily transmitted by the mosquito species aedes aegypti present in tropical and sub-tropical regions, and less efficiently by a. albopictus [3] , particularly during the viremic phase of the disease in which high levels of denv viremia are present in the blood stream. positive association between denv infection in mosquitoes and human has been found; human cases were reported at about one week after appearance of denv-positive a. aegypti [4, 5] . a. aegypti can also pick up denv from people showing no symptoms or oligo symptom, resulting in silent transmission [6] . therefore, for dengue disease control, it is important to extend denv surveillance in the human and mosquito populations to near points of care (poc)/point of needs (pon), generating early alert signals to prevent and/or control denv epidemics and reducing the spread of denv infection [7] . denv causes different clinical signs in humans, from mild febrile illness (dengue fever, df) to life-threatening condition such as hemorrhagic fever/dengue shock syndrome (dhf/ dss) [8, 9] . early, accurate and rapid diagnosis of dengue is critical for confirmation of clinically suspected cases to ensure timely management of severe dengue disease [10] . a number of ns1 rapid diagnostic tests are commercially available to easily detect ns1 antigen, but they are not sensitive or specific enough [11] [12] [13] . during the acute phase of the illness when viremia levels are high, the viral rna or soluble antigens can be easily detected [14] . for rapid and accurate diagnosis of dengue virus in early phases of acute infections, detection of denv rna is recommended as the most sensitive and specific method to diagnose dengue in the acute phase of the illness by who [9] . after the first developed nucleic acid detection test for dengue diagnosis over 20 years ago [15] , there have been a number of rt-pcr assays for denv detection and serotype identification with high sensitivity and specificity [16] [17] [18] [19] [20] . combined with automated rna extraction protocols, rt-pcr can potentially facilitate fast and accurate diagnosis of dengue suspected cases with a single sample obtained during the patient's first visit to the clinician. however, the requirement of a pre-pcr nucleic acids extraction step, an expensive and not easy to use thermal cycler, and a highly trained technician make pcr technology not accessible in resource-limited regions where dengue is endemic [10, 21] . molecular diagnosis tools for dengue is still much needed at or near poc/pon, particularly for developing countries with limited public health resources. for poc/pon dengue diagnosis, several isothermal amplification methods such as nucleic acid sequence-based amplification (nasba), thermophilic helicase-dependent amplification (thda), loop-mediated isothermal amplification (lamp), recombinase polymerase amplification (rpa), and insulated isothermal pcr (iipcr) have been developed and reported previously as potential field-deployable tools for the detection of different microorganisms [7, 10, [22] [23] [24] [25] . based on these technologies, various all-in-one molecular diagnostics have been made available to help minimizing risks of human errors and allowing easy molecular bio-detection at or near poc/pon in the last few years, but, to our knowledge, no reagents are available for denv testing on these platforms. the pan-denv reverse transcription-iipcr (rt-iipcr), previously validated to work with the filed-deployable semi-automated pcr system, pockit combo (genereach biotech, taichung, taiwan), is a potential poc/pon tool for the detection of denv. it offered great analytical and clinical performances for rapid detection of denv in human serum; its clinical performance was comparable to that of the reference multiplex rt-pcr assay in studies testing samples collected in sri lanka and taiwan [7, 26] . the field-deployable system includes one automated na extraction device (taco mini automatic nucleic acid extraction system, taco mini; genereach) and one compact pcr device (pockit nucleic acid analyzer, pockit; genereach) in a durable suitcase for easy field deployment. the iipcr system is unique in its employment of a single heating source in a thermally baffled device to drive rayleigh-bernard convection in a capillary tube, allowing the three steps of pcr (denaturation, annealing, and extension) to occur sequentially and continuously [27, 28] . since the first report of the assay in 2012, many pcr/rt-iipcrs on the pockit system have been validated for sensitive and specific detection of various parasitic, bacterial, and viral pathogens in animals and humans, including plasmodium spp, salmonella spp, influenza a virus, middle east respiratory syndrome virus, zika virus and dengue virus [7, 26, [29] [30] [31] [32] . requiring several manual liquid transfer steps to assemble the pcr tests, the complexity of the semi-automated pcr system is still relatively high. a compact fully automated sample-toanswer system (pockit central nucleic acid analyser, pockit central) is commercially available recently, in which modules in the existing semi-automated system (automated na extraction and compact pockit pcr modules) were integrated with a liquid handling module. this device can be set up and run with minimal set-up time and steps to allow quick and easy detection of denv in human at settings such as a local hospital laboratory or airport and in mosquitos for surveillance and monitoring of denv. therefore, the new assay has potential to enable timely early diagnosis of denv infection, facilitating effective disease management and control particularly in regions of low medical resources. in addition, the system can facilitate monitoring of the mosquitos carrying denv in the field to provide timely information for local government to take immediate measures to facilitate effective local management and control of dengue. in this study, we evaluated the performance of the pan-denv rt-iipcr reagent on the pockit central for sensitive and specific detection of denv1-4 serotypes in human serums and mosquitos. serum samples were collected from clinically suspected dengue patients for routine diagnosis using rt-pcr methods [33] at the tropical medicine center, kaohsiung medical university hospital, kaohsiung, taiwan in 2012. the use of retrospective clinical specimens in this study was approved by the kaohsiung medical university hospital institutional review board (kmuhirb-f(i)-20180032); waiver of informed consent was obtained. all collected samples were anonymized. stocks of four denv isolates, denv-1 (hawaii strain; 1.8 x 10 3 pfu/ml), denv-2 (ngc strain; 2.075 x 10 6 pfu/ml), denv-3 (dn8700829a strain; 1 x 10 6 pfu/ml), and denv-4 (dn9000475a strain; 1.9 x 10 5 pfu/ml), were prepared in the mosquito c6/36 cell line (a. albopictus). the virus was diluted in rpmi1640 medium (gibco-life technologies, grand island, ny, usa) containing 1% fcs (gibco-life technologies) and added to the cell at a multiplicity of infection of 0.01. after incubation at 28˚c, 5% co 2 for 4-7 days. viruses were harvested when cytopathic effect was observed. titers of denv stocks and human serum of dengue confirmed cases were determined by plaque assay. briefly, 10-fold serial dilutions of the denv stock were made in mem medium (gibco-life technologies) and added in duplicate to bhk-21 cells in 6-well plates (about 1× 10 6 cells per well). mem medium was used in mock infection. adhesion was allowed at 37˚c under 5% co 2 for 2 h before addition of 3 ml overlay medium containing 1.2% methylcellulose. cells were incubated further for 5-10 days until plaques became visually apparent by microscopy, fixed, and stained with crystal violet. plaques were counted manually and plaque forming units (pfu) per ml were determined with the plaque quantification program [34, 35] . two viruses known to also cause febrile illness or skin rash illness were tested for analytical specificity. zika virus (mr766, prvabc59 strain) were from the american type culture collection, manassas, va, usa. chikungunya virus (ck9500004 strain) was from the taiwan center of disease control, taipei, taiwan. a total of 60 archived serum specimens (30 denv positive, 10 of denv-1, -2 and -3 each; 30 denv negative) were collected from clinically suspected dengue patients with informed consents at the tropical medicine center, kaohsiung medical university hospital, kaohsiung, taiwan, for routine diagnosis in 2012. all samples were stored at -80˚c until nucleic acid extraction. due to the lack of denv-4 271 positive clinical samples in the region, denv-4 samples were prepared by spiking 10 of the denv-negative human serum specimens with different concentrations of the denv-4 dn9000475a stock (1.9 x 10 5 pfu/ml). adult female mosquitoes, aged 7-8 days, were cold anesthetized and inoculated using a microcapillary needle that had been pulled to a point with needle puller. the 4 serotype of dengue virus stocks were standardized to 2 x 10 6 pfu/ml, and 0.2 μl was injected into each mosquito (approximately 400 pfu/mosquito). infected mosquitoes were maintained in cages at 28 ± 1˚c and 70% ± 5% relative humidity with a 12 h/12 h light-dark cycle and fed with 10% sucrose solution. a. aegypti mosquitoes (ugal strain) were injected individually with denv-1 (hawaii strain, 400 pfu), denv-2 (ngc strain, 400 pfu), denv-3 (dn8700829a strain, 400 pfu), or denv-4 (dn9000475a strain, 400 pfu) by micro-injection (nanoinjector) into the thoracic cavity. whole mosquitoes were homogenized for plaque forming assay after 0, 1, 3, 5, 7, and 14 days post-infection [36] . denv was found to be detectable (10 3 -10 4 pfu/ml) in mosquitoes, and viral titers (infectivity) were maintained in the mosquitoes that had been cultured for 2 weeks without significant attenuation. three infected mosquitoes were collected on day 7 post infection, and frozen at −80˚c until further use. for nucleic acid extraction before pcr analysis, each mosquito was homogenized in 250 μl pbs with a disposable grinder and centrifuged briefly. subsequently, 200 μl of the upper aqueous sample were transferred into the first well of the preloaded extraction plate or cartridge. the pan-denv rt-iipcr reagent, targeting the 5' utr sequence, was performed as described in the user manual (pockit dengue virus reagent set, genereach biotech). the semi-automated pockit combo system includes an automated taco mini for nucleic acid extraction and a pockit device for pcr detection. nucleic acid extraction on the taco mini was done using the taco preloaded dna/rna extraction kit (genereach biotech) according to the manufacturer's instructions. briefly, 200 μl of the sample were added into the first well of the extraction plate before the automatic extraction steps. all nucleic acids were collected individually and placed at -80˚c until further use. subsequent pcr detection was done manually on a pockit device. first, 50 μl of premix buffer was added to reconstitute each lyophilized dengue virus premix, followed by the addition of 5 μl of test nucleic acid extract. a 50 μl volume of the premix/sample mixture was transferred into an r-tube, which was sealed subsequently with a cap, spun briefly in a microcentrifuge (cubee, genereach biotech), and placed into a pockit device. the default program, including an rt step at 50˚c for 10 min and an iipcr step at 95˚c for about 30 min; qualitative results were shown on the display screen in less than one hour. to run the pan-denv rt-iipcr reagent (pockit dengue virus reagent set) on the fully automated sample-to-answer pockit central system, the premix tube of the reagent was placed into the designated well in the transfer cartridge of the pockit cartridge set (gen-ereach biotech). after 200 μl of the clinical sample were added into well one of the extraction cartridge of the pockit cartridge set, and sample and reagent information were logged into the device, the cartridges were placed into the pockit central device accordingly, and the program was started. the nucleic acid extraction, iipcr amplification and detection steps were completed automatically in 80 minutes. qualitative results were displayed on the screen at the end of the program. to compare the performance of two methods, inter-rater agreement was assessed with a 2 x 2 contingency table and determined by cohen's kappa statistic using commercial software spss 14.0 (spss inc., chicago, il). the values � 0.20, 0.21-0.39, 0.40-0.59, 0.60-0.79, 0.80-0.90, and > 0.90 were interpreted as no, minimal, weak, moderate, strong, and almost perfect agreement, respectively [37] . to test analytical sensitivity, 10-fold serial dilutions (1,000, 100, 10, 1, 0.1, and 0.01 pfu/ml) of the denv-1, 2, 3, or 4 isolates were made separately in pooled denv-negative human serum and subjected in triplicate to the pan-denv rt-iipcr test with the semi-automated pockit combo and automated pockit central systems in parallel. the 100% detection end points were at 1, 10, 1, and 10 pfu/ml for denv-1, -2, -3 and -4 with the fully automated pcr system, respectively; while the end points were at 10, 1, 10, and 10 pfu/ml for denv-1, -2, -3 and -4 with the semi-automated pcr system, respectively (table 1, s1 table) . the results indicated that performances of the pan-denv rt-iipcr on the two pcr systems were comparable. similar to the observation with the semi-automated pcr system, the pan-denv rt-iipcr with the automated pcr system detected the four denv serotypes (denv-1, -2, -3, and -4) and did not react with the two zika virus and one chikungunya virus strains, indicating that the assay also had great specificity for denv rna on the fully automated system ( table 2) . performance of the pan-denv rt-iipcr on the semi-automated pockit combo system (see materials and methods for detail) was previously validated to be equivalent to that of the cdc denv-1-4 real-time rt-pcr [7] . the clinical performance of the pan-denv rt-iipcr reagent on the fully automated pockit central system was evaluated by comparison with the performance on the semi-automated pcr system. for this purpose, clinical serum samples were tested from sample to results using the pan-denv rt-iipcr reagent on the fully automated and semi-automated pcr systems in parallel. a total of 60 archived serum specimens (30 denv positive, 10 of denv-1, -2 and -3 each; 30 denv negative) collected from fully automated sample-to-answer pcr system for dengue virus detection clinically suspected dengue patients were tested. there were no severe dengue patients in the 30 denv-positive group. the 30 control cases were selected from age-and sex-matched dengue-suspected cases; dengue virus infections were later ruled out clinically and by laboratory tests. all clinical samples were collected within 6 days from the day of illness onset. based on analysis from a previous study, the median age was 45 years old (range 20-65) in the case group and 46 years old (range 21-67) in the control group. the percentage of male subjects was 53.3% in the case group and 50% in the control group. the median viral titers in different serotypes were 8.2 x 10 2 pfu/ml (denv-1), 3.5 x 10 3 pfu/ml (denv-2) and 2.7 x 10 5 pfu/ ml (denv-3) (s2 table) . denv-4 samples were prepared by spiking 10 of the denv-negative human serum specimens with different concentrations of the denv-4 dn9000475a stock (1.9 x 10 5 pfu/ml). the results showed that the pan-denv rt-iipcr detected 40 positive samples and 20 negative samples by both pcr systems (table 3, s2 table) . the overall agreement between the two systems was 100% (ci 95% , 95.7-100%; κ = 1.0), indicating the reagent can work with the fully automated pcr system to provide performance equivalent to that with the semi-automated pcr system for the detection of denv rna in human serum. denv titers can reach 10 2.67 ± 0.33 to 10 4.09 ± 0.71 pfu equivalents/ml in infected female a. aegypti after oral infection with denv [36] . the titers are higher than the sensitivity of the pan-denv rt-iipcr/pockit central system (table 1) . preliminary evaluation of the performance of the fully automated and semi-automated pcr systems was done using mosquitos collected on day 7 post intra-thoracic infection with one of the four serotypes. the results shows that both pcr systems could detect denv serotype 1-, 2-, 3-, and 4 rna in denvtable 2 . analytical specificity of pan-denv rt-iipcr on the fully automated pockit central system comparison with the semi-automated pockit combo system. infected mosquitos (table 4 ; each serotype tested in triplicate, s3 table) , suggesting both systems can be a useful tool for screening denv-infected mosquitos. testing with the pan-denv rt-iipcr, the analytical and clinical performance of the fully automatic pockit central system was comparable to those of the semi-automatic pocki combo system, which was validated previously to offer performances equivalent to the cdc denv1-4 real-time rt-pcr for the detection of denv in human serum [7, 24, 26] . validation with 60 serum samples prepared from dengue-suspected patients demonstrated that the pan-denv rt-iipcr performed equally well on both the semi-automated and fully automated equipment (100% agreement; ci 95% , 98.81~100%; κ = 1.0; table 3 ). moreover, the pan-denv rt-iipcr can work on both systems to detect the four denv serotypes in denvinfected mosquitos (table 4) . timely on-site detection of denv in human and mosquito can potentially alert front-line health professionals to allow timely implementation of intervention strategies and help focusing efforts in targeted areas to help mitigate disease outbreaks [1, 38] . performance of such tests near poc/pon could help shift health service more to local levels, achieve disease diagnosis at early stages, and reduce costs and turn-around time, improving health management quality in underreached communities. currently, commercially available ns1 immunological test products are rapid and do not require trained personnel to detect denv in both human and mosquitos [39] . however, they are reported as less sensitive than elisa and may cross react with other flaviviruses [11] [12] [13] . moreover, their sensitivities were relatively low on days 1 and 2 and after day 5 postsymptomatic onset in human, compared to that seen with the rt-pcr methods [40] . pcr testing after an ns1 positive results has been recommended for confirmation of denv infection, but the samples have to be shipped to a central laboratory currently. there are needs for sensitive and specific molecular diagnostic tests for dengue at poc/pon to allow early intervention for clinical management, surveillance, and outbreak investigations [26] . the previously available pan-denv rt-iipcr/semi-automated pcr system enabled sensitive and specific molecular testing near poc/pon. in this study, we showed that the reagent also worked well on the fully automated pcr system, which can serve as an advanced simple tool at settings with limited human resources and infrastructure to set up a pcr laboratory. one major advantages of the fully automated pcr system is its simple protocol (fig 1) ; users simply have to put 200 ul serum into a preloaded extraction cartridge, place both the extraction and reagent cartridges into the device, and start the reaction through the user interface on the screen; the results are ready in 85 min. compared to conventional pcr methods and to the semi-automated pcr system (see fig 1 for procedures involved) , the fully automated pcr system offers much shorter set up time; its capacity to process 8 reactions in one test run makes it suitable for applications at near-patient settings where the sample numbers are relatively small and a sophisticated testing procedure is not possible. moreover, the pan-denv rt-iipcr reagent is available in a lyophilized format to facilitate shipment at ambient temperatures and storage at 2-8˚c for at least 2 years, greatly reducing shipping and storage fully automated sample-to-answer pcr system for dengue virus detection costs. hence, this system can facilitate relatively inexpensive, rapid, and simple poc/pon detection of denv in routine dengue diagnosis. moreover, the fully automated and semi-automated pcr systems allows applications of the pan-denv rt-iipcr to detect denv in the local mosquito populations even at remote regions; the combined results can help decision making to effectively allocate control and prevention efforts into areas most in need of dengue control. therefore, the fully automated pockit central device and field-deployable semi-automated pockit combo system has potential to serve as a flexible mobile pon tool for rapid denv detection in both human and mosquito. studies to verify and validate further the performance of the pan-denv rt-iipcr reagents on both pcr systems for denv detection in mosquitos are underway. in summary, the pan-denv rt-iipcr coupled with the fully automated and semi-automated pockit platforms can serve as a rapid, accurate, and effective tool for use as a poc/ pon test system for early differential diagnosis of denv infection in human especially in local clinics, laboratories and hospitals, or for surveillance of denv in mosquitos. in the semi-automatic system, users add sample (serum, homogenized mosquitos) into the preloaded extraction plate (a) and place the plate into the nucleic acid extraction device for automatic nucleic acid extraction (b). the resulted nucleic acid extracts are added manually to the lyophilized reagent reconstituted first with provided buffer; the final pcr mixture is transferred manually to the reaction vessel (c). the reaction vessels are placed into the pcd device, which performs pcr amplification, detection and data interpretation automatically to provide qualitative results on the monitor (d). in the fully automatic system, users add serum sample to the preloaded extraction cartridge, place the extraction and reagent/consumable cartridges into the device, key in reagent and sample information, and start the assay (e). the device automates nucleic acid extraction, pcr reagent preparation, and pcr amplification, detection, and data interpretation to provide qualitative results on the monitor (f). table. sample info and test results of mosquito samples with pan-denv rt-iipcr on fully automated pockit central system and semi-automated pockit combo system. (docx) world health organization-geneva.global strategy for dengue prevention and control 2012-2020. who library cataloguing-in-publication data dengue hemorrhagic fever epidemic in taiwan. emerging infectious diseases consequences of the expanding global distribution of aedes albopictus for dengue virus transmission characteristics of the spatial pattern of the dengue vector, aedes aegypti fine scale spatiotemporal clustering of dengue virus transmission in children and aedes aegypti in rural thai villages asymptomatic humans transmit dengue virus to mosquitoes validation of the pockit dengue virus reagent set for rapid detection of dengue virus in human serum on a field-deployable pcr system dengue fever and dengue haemorrhagic fever: challenges of controlling an enemy still at large. reviews in medical virology diseases tit, diseases whodocont, epidemic who, alert p. dengue: guidelines for diagnosis, treatment, prevention and control: world health organization potential 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wolbachia infection on vector competence of potential dengue vectors aedes aegypti and aedes albopictus in the transmission of dengue virus serotype 1 in southern taiwan interrater reliability: the kappa statistic. biochemia medica: biochemia medica a new paradigm for aedes spp. surveillance using gravid ovipositing sticky trap and ns1 antigen test kit detection of dengue virus ns1 antigen in infected aedes aegypti using a commercially available kit. the american journal of tropical medicine and hygiene comparison of nonstructural protein-1 antigen detection by rapid and enzyme-linked immunosorbent assay test and its correlation with polymerase chain reaction for early diagnosis of dengue key: cord-311531-wezrs7gc authors: parčina, marijo; schneider, uffe vest; visseaux, benoit; jozić, robert; hannet, irene; lisby, jan gorm title: multicenter evaluation of the qiastat respiratory panel—a new rapid highly multiplexed pcr based assay for diagnosis of acute respiratory tract infections date: 2020-03-12 journal: plos one doi: 10.1371/journal.pone.0230183 sha: doc_id: 311531 cord_uid: wezrs7gc acute respiratory tract infections (arti), including the common cold, pharyngitis, sinusitis, otitis media, bronchiolitis and pneumonia are the most common diagnoses among patients seeking medical care in western countries, and account for most antibiotic prescriptions. while a confirmed and fast arti diagnosis is key for antibiotic prescribing, empiric antimicrobial treatment remains common, because viral symptoms are often clinically similar and difficult to distinguish from those caused by bacteria. as a result, inappropriate antibiotic prescriptions are high and in certain settings likely higher than the commonly estimated 30%. the qiastat respiratory panel(®) assay (qiastat rp) is a multiplexed in vitro diagnostics test for the rapid simultaneous detection of 21 pathogens directly from respiratory samples, including human mastadenovirus a-g, primate bocaparvovirus 1+2, human coronavirus (hku1, nl63, oc43, 229e), human metapneumovirus a/b, rhinovirus/enterovirus, influenza a virus (no subtype, subtype h1, h1n1/2009, h3), influenza b virus, human respirovirus 1+3, human orthorubulavirus 2+4, human orthopneumovirus, bordetella pertussis, chlamydia pneumoniae, mycoplasma pneumoniae and legionella pneumophila. we describe the first multicenter study of 445 respiratory samples, collected through the 2016–2017 and 2018 respiratory seasons, with performance compared against biofire filmarray rp v1.7 and discrepancy testing by seegene allplex rp. the qiastat rp demonstrated a positive percentage of agreement of 98.0% (95% ci: 96.0–99.1%) and a negative percentage agreement of 99.8% (95% ci: 99.6–99.9%). with use of this comprehensive and rapid test, improved patient outcomes and antimicrobial stewardship may potentially be achieved. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 discrepancies between the two test methods for both clinical sites were resolved by allplex rp (seegene, seoul, south korea) at the clinical site in bonn. testing at both clinical sites was performed by trained laboratory personnel, who had shown proficiency with both methods. residual respiratory samples as nasopharyngeal flocked swabs in utm (copan, brescia, italy) received for routine bacterial and viral testing from 445 patients were included in the study. samples were enrolled consecutively according to instrument capacity. clinical samples were included from two study sites after meeting the following inclusion criteria: respiratory samples were collected utilizing flocked swab (floqswabs™,copan, brescia, italy) placed in utm and were sent for viral and/or bacterial testing. the residual volume of all included samples was above 600 μl. all samples were tested by the qiastat rp and the filmarray rp within 4 days of collection when stored at 2-8˚c, and within 90 minutes after thawing when stored at -80 c. the samples were anonymized and assigned a study number linked to patient demographic information, including age, sex and hospitalization status (general practice, hospitalized, pediatric hospital/ward, intensive care unit). the samples had been collected under an institutional review board (irb)-approved protocol, which included a waiver of informed consent for the use of residual anonymized samples. the protocol was approved by ethikkommission an der medizinischen fakultät der rheinischen friedrich-wilhelms-universität bonn under the approval 052/18 and by de videnskabsetiske komiteer center for sundhed, region hovedstaden under the approval h-18001793. the qiastat dx system is a new highly multiplexed platform for integrated nucleic acid extraction and multiplex, rt-real-time pcr detection. the qiastat rp (catalogue number 691211) is designed to run on the qiastat diagcore analyzer testing respiratory samples from individuals with signs and symptoms of an acute respiratory tract infection. each qiastat rp cartridge contains a full process internal control (ic), which is a ms2 bacteriophage that is included in dried format and gets automatically rehydrated upon sample loading. the ic verifies all steps of the analysis process during testing, including sample resuspension/homogenization, lysis, nucleic acid purification, reverse transcription, and pcr. the cartridge has two distinct loading ports and can be inoculated directly with a dry swab or with transport medium. in brief, samples were homogenized by vigorous inversion of the vial and 300 μl utm was transferred into the assay cartridge using the provided transfer pipette. the inoculated cartridge was placed on the instrument. the test started automatically and ran for 69 minutes. upon completing the test and ejecting the cartridge, the analyzer interpreted results and displayed a test summary. the analyzer will also report errors that may occur during processing. amplification curves and cycle threshold (ct) values can be viewed for detected pathogens and for the ic. a report can be printed or exported to an external usb storage device. the analyzer can be bidirectionally connected to laboratory information systems. the the filmarray rp is a multiplex sample-to-answer pcr panel that tests for 20 viral and bacterial pathogens on nasopharyngeal swabs in utm at a time. a sample volume of 300 μl is required for the testing with approximately two minutes of hands-on-time in the set-up of the test. the system consists of one to eight modules (biofire flimarray torch, salt lake city, utah, usa) that that allows for time independent parallel testing of one sample per module. the test runs for approximately 65 minutes. a report can be printed or exported to an external usb storage device. the analyzer can be bidirectionally connected to laboratory information systems. the for human coronavirus hku1 the filmarray rp assay was accepted as correct as the pathogen is not included in the panel on the allplex rp. samples that yielded discordant results between the qiastat rp and the filmarray rp were discrepancy tested by the allplex rp assay. the allplex rp is a rt-pcr assay that tests for 19 viral and seven bacterial pathogens on nasopharyngeal swabs, nasopharyngeal aspirate or bronchoalveolar lavage at a time. the allplex rp runs on the seegene workflow that consists of a module for nucleic acid extraction and pcr setup, a 96-well pcr thermocycler and a computer for interpretation and reporting with a total sample-to-answer time in less than one day. the the allplex rp assay was used as the comparator method for primate bocaparvovirus 1+2, as this analyte is not included in the filmarray rp panel and the allplex rp results were accepted as correct. sensitivity and specificity are reported as the qiastat rp could be compared to a combined result from the filmarray rp and the allplex rp. results are reported as true positive (tp), false positive (fp), false negative (fn) and true negative (tn) results. binomial two-sided 95% confidence intervals were calculated using the wilson score method. for human coronavirus hku1 and primate bocaparvovius 1+2 results were compared by calculation of positive percent agreement (ppa) and negative percent agreement (npa) as only results from the qiastat rp and either the filmarray rp or allplex rp were available. primate bocaparvovirus 1+2 is not included as a target in the filmarray rp and the human coronavirus hku1 is not included in the allplex rp making it impossible to determine the sensitivity and specificity for these two targets. accordingly, overall agreement is also reported as ppa and npa as sensitivity and specificity was not determined for all targets. the ppa represent how often a new test (nt) agrees with a non-reference standard (nrs) and is calculated as ppa = "nt and nrs positive" / ("nt and nrs positive" + "nt negative and nrs positive"), whereas the npa is calculated as npa = "nt and nrs negative" / ("nt and nrs negative" + "nt positive and nrs negative"). 445 patients were included in the study. gender distribution was almost equal with 223 female patients and 222 male patients. 122 patients (27.4%) were younger than six years, 56 patients (12.6%) were between six and 21 years, 124 patients (27.9%) were between 22 and 49 years and 137 patients (30.8%) were 50 years or older. no age information was available for six patients (1.3%). the majority of samples, 307 (69.0%) were referred for testing by general practitioners. 106 samples (23.8%) were from hospitalized patients, 30 (6.8%) were from a pediatrics hospital/ward and two samples (0.4%) came from an intensive care unit. all patients had presented with signs and symptoms of an acute respiratory tract infection. the qiastat rp detected one or more potential pathogens in 333 (74.8%) of the 445 tested samples (table 1 ) for a total of 415 pathogens ( table 2) . multiple pathogens were detected in 19.5% of the positive samples (65/333), and the highest number of pathogens detected in a single sample was five (human respirovirus 3, human orthorubulavirus 4, human coronavirus oc43, rhino-/enterovirus and primate bocaparvovirus 1+2). the majority of multiple infection samples contained two pathogens, which was the case for 53/65 samples (81.5%). the qiastat rp detected a bacterial pathogen in 7.6% of all samples (34/445). in 25 out of 34 samples only bacteria were detected and in the remaining nine samples, additional viral pathogens were detected (four samples with two pathogens, four samples with three pathogens and one sample with four pathogens). the number of each potential pathogen detected by the qiastat rp in co-infections, by hospitalization and by age group is presented in table 2 . the most frequently detected pathogen was human orthopneumovirus, which was detected in 98 samples, 71 of which (72.4%) came from patients less than 6 years of age. the second most common pathogen was influenza a virus, which was detected in 75 samples, equally distributed between the h1 pdm2009 and h3n2 strains. rhino-/enterovirus was detected in 63 samples and influenza b virus was detected in 51 samples, highlighting the very active influenza b virus season in early 2018. for 440 of 445 specimens (98.9%), a valid result was obtained by the initial qiastat rp testing. five samples (1.1%) were invalid after the first qiastat rp testing, comprising one run aborted by the analyzer (0.22%), three runs with software errors (0.67%), and one run in which the internal control failed to amplify (0.22%). all five samples were retested with the qiastat rp and yielded a valid result after the second qiastat rp testing. the performance characteristics for individual qiastat rp targets before discrepancy resolution are presented in table 3 and the performance characteristics after discrepancy resolution are presented in table 4 . before resolution by discrepancy testing, qiastat rp and filmarray rp agreed on the detection of 376 pathogens in the 445 samples (table 3) . for all pathogens except human mastadenovirus a-g, rhino-/enterovirus, hmpv, influenza a virus no subtype and human respirovirus 3, the agreement was excellent (table 3 ). for human respirovirus 3 and influenza a virus no subtype the total number of positive samples by at least one method was only nine and five respectively. for human mastadenovirus a-g the qiastat rp and filmarray rp agreed on 27 positive samples out of 41 initial positive samples (table 3 ). after discrepancy testing the sensitivity for the qiastat rp was 97.1% (33/34, table 4 ). for rhino-/enterovirus the qiastat rp and filmarray rp agreed on 49 positive samples out of the 71 initial positive samples. after discrepancy testing the sensitivity for the qiastat rp was 94.4% (52/55). for hmpv the qiastat rp and filmarray rp agreed on 19 positive samples out of 21 initial positive samples. after discrepancy testing the sensitivity was 95.0% (19/20). after resolution by discrepancy testing on the allplex rp, a total of 402 pathogen results were considered as true positive (table 4) , of which qiastat rp detected 394, for an overall ppa of 98.0% (95%ci 96.0%-99.1%). after discrepancy testing, the overall npa of the qiastat rp was 99.8% (95%ci 99.6%-99.9). twenty-two pathogens detected by filmarray rp were not detected by qiastat rp ( table 3) . fourteen of these were also not detected by allplex rp and were considered as true negatives, leaving 8 pathogen detections as false negatives (table 4 ). thirty-nine pathogens detected by qiastat rp were not detected by filmarray rp (table 3) . twenty-one of these were also not detected by allplex rp and were considered as false positives ( table 4) . three of the 22 pathogens detected by filmarray rp but not detected by qiastat rp were detected in single infection samples and additional two of the pathogens not detected by qiastat rp were both detected in the same double infection sample. the five pathogens involved were rhino-/enterovirus (three), human mastadenovirus a-g (one) and human orthopneumovirus (one). all off these five pathogens were detected by the discrepancy method (allplex rp). the remaining 17 pathogens not detected by qiastat rp but detected by filmarray rp were from 17 samples with two or more pathogens. three of these 17 pathogens (one hmpv, one influenza a virus, no subtype and one human orthopneumovirus) were detected by the discrepancy method adding up to a total of 8 false negative pathogens. qiastat rp detected 39 pathogens not detected by filmarray rp. 10 of these pathogens were in single infection samples and the detected pathogens were rhino-/enterovirus (n = 1, ct value 27.6), influenza b virus (n = 1, ct value 20.7), influenza a virus, no subtype (n = 1, ct value 34.2), influenza a virus subtype h3n2 (n = 2, ct values 32.7 and 33.7), human orthopneumovirus (n = 2, ct values 28.8 and 32.9) and human mastadenovirus a-g (n = 3, ct values 21.3, 22.4 and 22.7) . the influenza a virus with no detected subtype sample could not be discrepancy tested due to insufficient residual volume and was accordingly considered false positive by the qiastat rp. one additional single-infection pathogen (the rhino-/enterovirus) was considered false positive, as this result could not be confirmed positive by discrepancy testing. the remaining eight single-infection pathogens were confirmed positive by the discrepancy method and thus considered true positives ( it was not possible to assess the sensitivity of the qiastat rp for legionella pneumophila or influenza a virus subtype h1n1 as these organisms were not detected by any of the methods. primate bocaparvovirus 1+2 was detected by the qiastat rp in five specimens, all containing at least one other potential pathogen. the allplex rp confirmed the detected primate bocaparvovirus 1+2 in three of five specimens. because primate bocaparvovirus 1+2 is not included in the panel of the reference method, the results are listed separately and are expressed as ppa/npa. this study demonstrates for the first time the clinical performance of the new qiastat rp assay for detection of 21 respiratory pathogens. by comparison to the filmarray rp, the overall ppa and npa after discrepancy testing were 98.0% (95% ci: 96.0%-99.1%) and 99.8% (95% ci: 99.6%-99.8) respectively. in general, the qiastat rp assay showed excellent agreement with filmarray rp. for all pathogens included on the qiastat rp test menu, except rhino-/ enterovirus (52 tp and 379 tn samples), influenza a virus, no subtype (3 tp and 440 tn samples) and hmpv (19 tp and 425 tn samples), the qiastat rp showed sensitivity/ppa >97% and specificity/npa >99% (table 4 ). the reason for the discordant results for rhino-/enterovirus is not known but may potentially be explained by different diagnostic tests targeting different genetic regions of the rhino/enterovirus genome. the ct values for the pathogen targets present in the discrepant qiastat positive rp and filmarray rp negative samples were detected by qiastat with ct values in the range 30-37, which may be considered as weakly positive. only three targets were detected as qiastat rp positive and filmarray rp negative target with ct values below 30, one human orthopneumovirus (ct 16.2) and two rhino-/enterovirus (ct 26.9 and 29.7) and thus as strong positive samples. it would have been beneficial to resolve discrepancy results by sequencing as this potentially would have clarified the discordant results for rhino-/enterovirus. this was not possible as both the qiastat rp and filmarray rp as assays are performed in sealed samples-to-answer cartridges that are constructed to prevent access to sample material remaining in the cartridge. the qiastat rp assay requires one manual pipetting step to load the patient sample into test cartridge. the test cartridge id and patient/sample id is entered by on-board scanner and the total assay time is approximately 69 minutes. the qiastat dx diagcore analyzer offers a small footprint, traceable internal inhibition controls (by amplification curve as well as ct value), low maintenance requirements and low failure rate as well as seamless connectivity and integration with hospital and laboratory information systems. in addition, the qiastat dx diagcore analyzer offers access to amplification curves and ct values of all detected pathogen targets. the qiastat rp assay cartridge has on-board wet and dry reagents, build-in amplification inhibition controls, detects 21 pathogens in a total of 8 separate reaction chambers, offers a direct dry swab testing option via a separate cartridge input port and stores at room temperature. the qiastat rp is ce ivd marked and fda approval is pending. the syndromic diagnostic approach for arti may provide multiple benefits for the institution (e.g. prudent use of side room isolation facilities, reduced prescription of antimicrobials, targeted use of antivirals) the benefits have been partly described in previous publications. trabattoni et al reported a significant reduction in emergency department (ed) length of stay as well as a significant reduction in hospitalization rate for patients tested by the alere i influenza assay in the ed [22] , and hansen et al reported a positive impact on admittance rate as well as total cost by roche liat influenza testing in the ed setting [14] . brendish et al reported on encouraging findings of poc testing of respiratory viruses, as their studies showed that this testing was associated with a reduced length of hospital stay, resulted in more single doses or brief courses of antibiotics as well as in an improved influenza detection and antiviral use [15] . however, understanding of the full impact on patient management by the syndromic testing approach for arti as well as the cost-benefit will require more prospective outcome studies. such studies would also provide information regarding which patient population(s) will benefit the most from respiratory tract syndromic testing and what the added benefits of syndromic testing compared to selected target testing (e.g. influenza a/b virus, human orthopneumovirus, hmpv, bordetella pertussis or combinations hereof). this study has several limitations. first, as this study was not designed as an epidemiological study, the included clinical samples were not collected consecutively and the detected coinfection rates and the frequency of different pathogens in the co-infected samples may not reflect the actual co-infection rates in the patient population at the time of sample collection. second, as several pathogens, e.g. coronaviruses, b. pertussis, c. pneumoniae and parainfluenzavirus 1-4, were only detected in few clinical samples, the performance of the qiastat rp assay for detection of these pathogens is uncertain. in conclusion, in this first clinical study of the new qiastat rp assay performed in two diagnostic laboratories providing 445 clinical samples for analysis, we 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retrospective study prevalence of respiratory viruses among adults, by season, age, respiratory tract region and type of medical unit viral-bacterial coinfection affects the presentation and alters the prognosis of severe community-acquired pneumonia prevention strategies for seasonal influenza in healthcare settings systematic review of antibiotic prescription associated with upper respiratory tract infections in china prevalence of inappropriate antibiotic prescriptions among us ambulatory care visits point-counterpoint: large multiplex pcr panels should be first-line tests for detection of respiratory and intestinal pathogens clinical decision making in the emergency department setting using rapid pcr: results of the clade study group routine molecular point-of-care testing for respiratory viruses in adults presenting to hospital with acute respiratory illness (respoc): a pragmatic, open-label, randomised controlled trial impact of early detection of respiratory viruses by multiplex pcr assay on clinical outcomes in adult patients impact of a rapid respiratory panel test on patient outcomes impact of turnaround time on outcome with point-of-care testing for respiratory viruses: a post hoc analysis from a randomised controlled trial the frequency and seasonality of influenza and other respiratory viruses in tennessee: two influenza seasons of surveillance data quantifying reporting timeliness to improve outbreak control clinical practice guidelines by the infectious diseases society of america: 2018 update on diagnosis, treatment, chemoprophylaxis, and institutional outbreak management of seasonal influenza implementation of alere i influenza a & b point of care test for the diagnosis of influenza in an ed the authors would like to thank laboratory technician danah knudsen, who participated in the testing at copenhagen university hospital hvidovre. conceptualization: marijo parčina, benoit visseaux, irene hannet, jan gorm lisby. key: cord-321855-7b1c2xdh authors: alshami, alanoud; alattas, rabab; anan, hadeel; alhalimi, abdulbary; alfaraj, ahmed; al qahtani, hadi title: silent disease and loss of taste and smell are common manifestations of sars-cov-2 infection in a quarantine facility: saudi arabia date: 2020-10-30 journal: plos one doi: 10.1371/journal.pone.0241258 sha: doc_id: 321855 cord_uid: 7b1c2xdh objectives: in this study, we aimed to study the clinical presentations, and viral clearance of sars-cov-2 positive quarantined individuals. design: cross-sectional study. setting: governmentaldesignated facility in the eastern province, saudi arabia. participants: 128 laboratory-confirmed covid-19 quarantined individuals who had a history of travel abroad in the last 14 days before the quarantine or were in direct contact with laboratory-confirmed cases. the study was from march 18th-till april 16th. primary and secondary measures: the clinical presentation, prevalence of asymptomatic carriers among sars-cov-2 positive quarantined subjects, and the difference between virus clearance among symptomatic and asymptomatic individuals. results: sixty-nine of the 128 residents (54%) were completely asymptomatic until the end of the study. the remaining 59 residents (46%) had only mild symptoms. the most common symptom was a sudden loss of smell and taste, accounting for 47.5%. the median time to virus clearance was significantly different between the two groups. symptomatic residents cleared the virus at a median of 17 days (95% ci, 12.4–21.6) from the first positive pcr vs. 11days (95% ci, 8.7–13.3) in the asymptomatic group (p = 0.011). false-negative test results occurred in 18.8% of the total residents and false-positive results in 3%. conclusion: the prevalence of asymptomatic carriers among quarantined travelers and those identified by contact tracing is high in our study. therefore, testing, tracing, and isolating travelers and contacts of laboratory-confirmed cases, regardless of symptoms, were very effective measures for early disease identification and containment. loss of taste and smell were the most common presentations in our mild symptomatic residents and should be used as a screening tool for covid-19. the persistent positive pcr beyond 14 days observed in the mild symptomatic residents despite being symptoms free, warrant further studies to determine its implications on disease spread and control. introduction a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), was first discovered on december 31st, 2019, in wuhan city, china, after a cluster of atypical pneumonia was observed. this infection was labeled as corona virus disease 2019 (covid 19) [1] . this has led to an outbreak of infection in china that spread globally over a few months and was declared a pandemic by the who on march 11th, 2020. currently, most reported cases are from outside china. as of july 19th, 2020, there are 14 270 805 confirmed cases worldwide, and 250,920 of these cases are in saudi arabia, with 2486 reported deaths. although we have gradually gained some insight into the clinical presentation spectrum of covid-19 from other countries' experiences and published data, we still do not have enough information about the real spectrum of the disease and the prevalence and clinical characteristics of asymptomatic carriers. goa et al. have conducted the first and largest covid-19 epidemiological study in china. their study included 72, 314 subjects, and they described a mild disease course in about 80% of their cohort, severe disease in 14%, critical in 5%, and asymptomatic in only 1.2% [2] . the possibility of developing asymptomatic infection has further been reported in larger percentages in multiple small reports [3, 4] however large data is still lacking. hu et al. have examined 24 asymptomatic infected individuals with a history of close contact with sars-cov-2 confirmed cases and found that only 20% of them developed symptoms. the importance of detecting asymptomatic carriers relies on their ability to spread the disease. this assumption has been illustrated by different reports [4] [5] [6] . however, it has not been validated yet in large scale studies. a recent study has shown that the viral load detected in asymptomatic patients was similar to the viral load of symptomatic patients suggesting that asymptomatic patients can also transmit the disease [7] . the fact that there is a group of asymptomatic carriers who are roaming around unrecognized and might be able to transmit the disease imposes a serious public health threat if not early identified and contained. identifying the asymptomatic carriers early on will help us better understand the dynamics of the disease spread and guide us to find better measures of disease containment and mitigations. in this study, we aimed to study the clinical characteristics among quarantined individuals with positive sars-cov-2 pcr and viral clearance in quarantined individuals with covid-19. this study was approved by the institutional review board at king fahad specialist hospital-dammam. as this study does not carry more than minimal risk, we only obtained a verbal consent that was taken in the beginning of the phone call conversation. the verbal approval to participate was documented by the research team using a web-based data collection tool (red-cap). for all minors involved in the research, verbal consent and clinical information were taken from their parents. this is a cross-sectional study conducted in a quarantine facility in al-khobar city in the eastern province of saudi arabia. the facility was designated only for sars-cov-2 positive cases confirmed by real-time polymerase chain reaction (rt-pcr) using combined nasopharyngeal and oropharyngeal samples. residents were travelers who tested positive, confirmed cases with mild disease transferred from hospitals, or confirmed cases admitted directly from the community after being traced by the regional public health authority. all residents admitted from march 16th until april 6th, whether adults or children, were included. admission to the quarantine was restricted to low-risk stable patients with mild symptoms only. elderly patients >65 years of age with more than one comorbidity were not allowed in this quarantine. all residents were followed up daily by nurses with symptoms screening checklist. following up, residents on daily bases have allowed us to differentiate between 3 categories of patients: true asymptomatic carriers (no symptoms at all), pre-symptomatic (developed symptoms in the quarantine), and symptomatic patients who had symptoms during or before admission. all data was collected upon presentation to the quarantine. data were collected prospectively by nurses using the standardized case report forms, generated by modified who/ international severe acute respiratory and emerging infection consortium case record form for severe acute respiratory infections [8] . all data collected was verified by the research team through a short phone survey to ascertain the accuracy of the data, especially the symptoms presented within the last 14 days before admission to the quarantine. the collected data included symptoms-based screening checklists, such as a new onset of cough, sore throat, runny nose, fever, and myalgia-atypical symptoms like headache, nausea and vomiting, diarrhea, and loss of taste and smell. history of comorbidities was taken by self-reported medical history, and it included: hypertension, diabetes mellitus, chronic lung disease, renal disease, and cardiovascular disease. history of pregnancy and current smoking status were also recorded. pcr samples were taken for each resident upon admission and were repeated every 72 hours as long they were positive. these samples were sent to the regional laboratory for processing, and the results were uploaded on the hesn database (saudi public health electronic database). patients were deemed infection-free if they had two negative pcrs 24 hours apart (as per saudi cdc guideline). we also calculated the percentage of false-negative and falsepositive tests. a false-negative test was defined as a negative test that was preceded and followed by a positive test within 24-72 hours' time frame. a false-positive result was defined as a positive test that was preceded and followed by a negative test within 24-72 hours' time frame. we mainly used descriptive statistics. quantitative/continuous variables were presented as mean ± standard deviation (sd) or median with inter-quartile range (iqr). frequencies, proportions were used to describe qualitative data. bivariate analysis was performed using an independent sample t-test or mann-whitney u test, which is appropriate. survival analysis was used to determine the time from the first positive pcr until the first truly negative one. rt-pcr test and sampling. detection of sars-co-v-2 in respiratory samples was performed by rt-pcr using the altona kit (real star sars-cov-2 rt-pcr kit) (usa). the kits include detection of e gene sequence specific for all b-beta-corona virus and sars-cov-2 specific sequence of s gene. samples for pcr were collected simultaneously from the nasopharynx and oropharynx following the who instructions for sample taking. each set of the test was reported as screening and confirmatory. rna was extracted and reverse-transcribed to cdna. then real-time pcr was performed as instructed by the manufacture using specific primers and probes targeting the sars-cov-2 genomes. internal control is included in the assay to rule exclude reaction inhibition. no patients or public were involved in this study during the design, conduct, or analysis phases. among the 128 patients included in the study, 69 (53.9%) were female table 1. the mean age was 39.6 years, with a range of (8-76 years). among the 128 residents, four children <16 years were included. three out of the four children were asymptomatic, and one child presented with mild fever and cough. sixty-five residents (50.8%) had a history of travel outside the kingdom within the last 14 days before admission to the quarantine. iran and iraq being the most visited countries accounting for (68.2%) followed by the uk (10.61%) and the rest of countries accounting for the remaining 21.2%. sixty-two residents (49.2%) had a history of direct exposure to a lab-confirmed case, and one resident was screened based on symptoms and tested positive without a clear history of travel or contact with a suspected or lab-confirmed case. sixty-nine patients (54.3%) did not exhibit any symptoms (asymptomatic carriers/ silent disease), while the other 59 patients (45.7%) had only mild symptoms. the most common reported symptom among our residents was a complete and sudden loss of smell and taste, 28 (47.5%). these symptoms appeared at a median of 6 days (iqr, 4-9 days) after the onset of fever or upper respiratory tract symptoms. however, it was the only clinical symptom in 18.65% of patients. residents also reported a history of cough in (40.7%), myalgia in (39%), and headache in (37.3%). the median time for symptoms resolution was five days (iqr 3-11days) . out of the 59 symptomatic patients, 11(18.64%) were pre-symptomatic, who subsequently developed symptoms at a mean of 7.78 days (+/-5.7 sd) after the first positive pcr while in quarantine. the median incubation period in symptomatic residents was seven days (iqr, 3.75-12 days). a total of 664 pcr tests have been done for the 128 residents. the median number of tests per person was 5 (iqr, 4-6 tests). twenty-four residents had at least one false negative test (18.8%) in (table 2) . symptomatic and asymptomatic residents were significantly different in the timing of clearing the virus. the median time to develop a real negative pcr (2 negative pcrs 24 hours apart) in the symptomatic group was 17 days (95% ci, 12.38-21.6) vs. 11 days (95% ci, 8.66-13.34) in the asymptomatic group (p = 0.01)) (fig 1) . the first confirmed case of sars-cov-2 in saudi arabia was on march 2nd, 2020, for a saudi traveler who came back from iran. in response to the covid19 pandemic, saudi arabia was one of the first countries that applied early and stringent mitigation measures while the number of confirmed cases was still low (300 cases) [9] . on march 15th, 2020, the saudi cdc (weqaya) imposed new regulations on all travelers coming from outside the kingdom to prevent importing infection from these countries. all travelers and close contacts of laboratoryconfirmed cases were tested and quarantined for 14 days in a designated facility regardless of the presence or absence of symptoms. this regulation has helped us to better understand the prevalence of sars-cov-2 infection in asymptomatic patients. in our cohort, the majority of residents were either truly asymptomatic (54%) or had mild symptoms (46%). the proportion of asymptomatic carriers in our cohort is considered to be one of the highest reported so far. however, these findings don't reflect the true prevalence of asymptomatic carriers in the general population as particularly vulnerable groups who were expected to have a more severe course were excluded. having said that, people > 65 years of age constitute only 3.41% of the general saudi population, while the majority (71.72%) are between 15-64 years of age. our proportion of asymptomatic carriers was somehow close to the reported proportion of the diamond princess cruise ship passengers in japan (50%) [10] . however, a lower prevalence has been reported in other studies. a report from germany on 116 travelers coming back from wuhan city to frankfort has shown a prevalence of 1.7% [11] . the most common symptoms were sudden and complete loss of taste and smell occurring in almost half of them (47.5%). more interestingly, these symptoms were found to be the only presenting symptoms in 18.7% of our residents, and this in itself should be considered as a red flag and should be reported as one of the common symptoms of mild disease. a similar prevalence of isolated loss of smell was demonstrated by varia et al. in a survey of 2428; he found a 74% prevalence of loss of smell, of which 17.3% were isolated [12] . furthermore, isolated olfactory dysfunction was also reported as the only clinical manifestation of covid-19 by simon b et al. [13] . the majority of our symptomatic patients report that they could not smell even strong odors like perfumes and could not differentiate between sweet and sour food. loss of taste and smell was reported as a rare clinical manifestation of covid-19 in a chinese study, accounting only for 5% of the symptoms [14] . however, subsequent studies have shown that these symptoms were more prevalent than previously reported. our findings are in light with a recent study that reported a 59% prevalence of loss of taste and smell in a cohort of covid-19 patients [15] . these symptoms were also detected in 70% of covid-19 patients in the early disease phase in a study from italy [16] . a multi-center european cohort of 417 covid-19 patients with mild to moderate disease has also reported a very high prevalence of olfactory (85.6%) and gustatory dysfunction (88%) [17] . although these symptoms were reported more commonly in mild disease, these findings might be subjected to selection and reporting bias. our study only included patients with mild disease and excluded those with severe disease who might not be able to report these symptoms. most of the residents who were quarantined because of contact with lab-confirmed cases were identified by contact tracing programs. although this information might be subjected to recall bias, none of our patients with a history of direct contact with lab-confirmed sars-cov-2 infection (including household contacts) reported exposure to symptomatic individuals. therefore, it indicates that they probably contracted the infection from asymptomatic or pre-symptomatic individuals. however, our study cannot confirm these findings. our study is unique in terms of the numbers of pcr performed per patient and also unique in terms of frequently testing the asymptomatic residents. a median of 5 tests per resident was done for both groups. this has allowed us to assess the virus clearance time accurately and to shed light on some of the pcr tests short comes. because we have enrolled asymptomatic residents in our study, we elected to use the first available positive pcr as the starting point for virus clearance time calculation. we are keeping in mind that those asymptomatic residents might have contracted the virus a few days or weeks before the quarantine. although our symptomatic group had mild symptoms, they have shed the virus for a longer period of time when compared with the asymptomatic group (17 vs. 11 days). nevertheless, we had a few outliers. we had a 32 years old asymptomatic lady who cleared the virus after 36 days of admission. the clinical implications for persistently positive pcr in true asymptomatic and mildly symptomatic patients need to be further studied. as a pcr test will not differentiate between a person who is contagious from a person who is not, further studies utilizing virus cultures and neutralization assays to help dictate the best duration of the quarantine. our study result is different than what was reported by lui et al. in which he found that majority of patients with mild symptoms cleared the virus at 10 days, while those with severe symptoms lasted for more than 10 days [18] . a false-negative test in a previously positive patient was not an uncommon finding in our cohort. this is most likely related to sampling techniques and specimen source. so, if the negative test was not repeated, we would have 18.8% of the time mistakenly reported these results as negative. so, we probably need to always do a repeat confirmatory test before deeming that the patient is infection-free. we also had about 3% of false-positive test results, which might be secondary to the detection of dead virus rna particles rather than re-infection, as described recently by the south korea group. this study is one of the few studies that assessed the prevalence of asymptomatic disease among quarantined individuals. it is unique in terms of the numbers of pcr performed per patient and the frequent tests performed in the asymptomatic group. this study is a cross-sectional study, so symptoms ascertainment before admission was subjected to recall bias. however, we find that majority of patients were well aware of their symptoms, and they were able to give us the date of onset of symptoms precisely. this study results cannot be generalized on all covid-19 patients as we have excluded some high-risk groups and people with moderate to severe disease. in conclusion, the prevalence of true asymptomatic carriers among covid-19 quarantined individuals in our study was high. however, this high prevalence might not represent the true prevalence in the general population. these findings re-enforce the importance of using testbased strategies and contact tracing rather than symptoms-based screening checklist when dealing with travelers and subjects with direct contact with a lab-confirmed case. a testingbased screening strategy perhaps should be extended to first-line healthcare workers and atrisk groups. also, countries planning for mitigation measures release should enhance their testing abilities to prevent new outbreaks. sudden onset of loss of smell and taste were prevalent in our study and were key symptoms of mild disease. these symptoms alone or in combination with other symptoms might as well be utilized as a marker for testing patients who may be positive for the virus. the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19)-china the epidemiological characteristics of 2019 novel coronavirus diseases (covid-19 covid-19 in south korea-challenges of subclinical manifestations transmission of 2019-ncov infection from an asymptomatic contact in germany clinical characteristics of 24 asymptomatic infections with covid-19 screened among close contacts in nanjing asymptomatic cases in a family cluster with sars-cov-2 infection sars-cov-2 viral load in upper respiratory specimens of infected patients laboratory testing for coronavirus disease 2019 (covid-19) in suspected human cases preparedness and response to covid-19 in saudi arabia: lessons learned from mers-cov estimating the asymptomatic proportion of coronavirus disease 2019 (covid-19) cases on board the diamond princess cruise ship sars-cov-2 infection among travelers returning from wuhan olfactory and gustatory function impairment in covid-19 patients: italian objective multicenter-study isolated sudden onset anosmia in covid-19 infection. a novel syndrome? neurologic manifestations of hospitalized patients with coronavirus disease 2019 in wuhan loss of smell and taste in combination with other symptoms is a strong predictor of covid-19 infection remote psychophysical evaluation of olfactory and gustatory functions in early-stage coronavirus disease 2019 patients: the bologna experience of 300 cases olfactory and gustatory dysfunctions as a clinical presentation of mild-to-moderate forms of the coronavirus disease (covid-19): a multi-center european study viral dynamics in mild and severe cases of covid-19 key: cord-323185-n0rubc72 authors: varshney, bhavna; agnihotram, sudhakar; tan, yee-joo; baric, ralph; lal, sunil k. title: sars coronavirus 3b accessory protein modulates transcriptional activity of runx1b date: 2012-01-12 journal: plos one doi: 10.1371/journal.pone.0029542 sha: doc_id: 323185 cord_uid: n0rubc72 background: the causative agent of severe acute respiratory syndrome, sars coronavirus (sars-cov) genome encodes several unique group specific accessory proteins with unknown functions. among them, accessory protein 3b (also known as orf4) was lately identified as one of the viral interferon antagonist. recently our lab uncovered a new role for 3b in upregulation of ap-1 transcriptional activity and its downstream genes. thus, we believe that 3b might play an important role in sars-cov pathogenesis and therefore is of considerable interest. the current study aims at identifying novel host cellular interactors of the 3b protein. methodology/principal findings: in this study, using yeast two-hybrid and co-immunoprecipitation techniques, we have identified a host transcription factor runx1b (runt related transcription factor, isoform b) as a novel interacting partner for sars-cov 3b protein. chromatin immunoprecipitaion (chip) and reporter gene assays in 3b expressing jurkat cells showed recruitment of 3b on the runx1 binding element that led to an increase in runx1b transactivation potential on the il2 promoter. kinase assay and pharmacological inhibitor treatment implied that 3b also affect runx1b transcriptional activity by regulating its erk dependent phosphorylation levels. additionally, mrna levels of mip-1α, a runx1b target gene upregulated in sars-cov infected monocyte-derived dendritic cells, were found to be elevated in 3b expressing u937 monocyte cells. conclusions/significance: these results unveil a novel interaction of sars-cov 3b with the host factor, runx1b, and speculate its physiological relevance in upregulating cytokines and chemokine levels in state of sars virus infection. severe acute respiratory syndrome (sars) emerged in the guangdong province of china in november 2002 and swept through more than 29 countries. its spread infected more than 8000 people with a high mortality rate of 10%. it was found to be associated with a novel coronavirus named sars-cov [1, 2] . sars-cov, like other coronaviruses, is a positive sense, singlestranded enveloped rna virus with a huge 29.7 kb genome [3] . its genome comprises of 14 orfs which encode non-structural genes, structural genes and several unique group specific accessory proteins namely 3a, 3b, 6, 7a, 7b, 8a, 8b and 9b. [4] . recognition of peptides derived from accessory proteins by convalescent sera of sars-cov infected patients [5] as well as their immunohistochemical detection in infected veroe6 cells and in clinical specimens [6] corroborates their expression during viral infection. however, these accessory proteins have been found dispensable for viral replication [7] . sars-cov accessory protein 3b is a 154 amino acid (aa) protein and has been characterized as one of the interferon antagonist encoded by sars-cov genome [8] . gfp tagged 3b has been reported to localize in the nucleus, nucleolus and mitochondria in cells [9, 10, 11] . a recent report delineated a unique nucleo-mitochondrial shuttling behaviour of 3b-gfp wherein 3b was found to inhibit rig-i and mavs induced type i interferon induction in the mitochondria [9] . recently, we published a role of 3b in induction of ap-1 transcriptional activity that was mediated by the activation of erk and jnk pathways [12] . being an interferon antagonist that is dispensable for viral replication and observing its effect on the activity of crucial host transcription factors, 3b probably plays a role in disease progression by mediating viral-host interactions, which are poorly understood. to uncover host interacting partners, of sars-cov 3b, we conducted a yeast two-hybrid screen of human lung cdna library using 3b as bait. the screen identified runx1b (runt related transcription factor 1, isoform b) as one of the host interacting partners of 3b. runx1 belongs to the runx family of genes which includes runx2 and runx3 additionally [13] . runx genes encode the a subunit, which heterodimerizes with the b subunit, cbfb to form transcription factor cbf (core binding factor) [14] . runx1 has a 128 aa runt domain through which it binds cbfb as well as the consensus dna element, tgt/cggt [14, 15] . runx1 has three isoforms: runx1a, runx1b and runx1c. runx1a is a 250 aa protein with a runt domain. runx1b is a 453 aa protein and possess additional pst (proline, serine and threonine rich) region downstream to runt domain. runx1c differs from runx1b by 32 aa at n-terminus and is presumed to have similar functions in cells as runx1b [16] . runx1 is crucially required for definitive hematopoiesis and tlymphocyte differentiation [17, 18] . at the molecular level, runx1 isoforms have been shown to regulate transcription of a number of genes including cytokines (il2, il3, gm-csf etc.) and chemokines (mip-1a, csfr etc.). based on the yeast two-hybrid screening results, we conducted our current study with the runx1b isoform. in this study, we confirmed the putative interaction of 3b and runx1b and observed in vivo recruitment of 3b on the runx1 binding element on the il2 promoter in transiently transfected human t, jurkat cells. further, 3b was found to increase the transactivation potential of runx1b on the il2 promoter, which may partly be attributed to the enhanced erk dependent phosphorylation of runx1b in 3b expressing cells. we next determined the positive effect of 3b-runx1b interaction on the expression of runx1b regulated chemokine mip-1a, reported to be upregulated in sars-cov infected monocyte derived dendritic cells. thus, we report a novel interaction of sars-cov 3b with runx1b and discussed its plausible significance in sars virus pathogenesis. to identify cellular interacting partners of sars-cov accessory protein 3b, yeast two-hybrid screening of human lung cdna library with full-length 3b as bait was conducted. the bait plasmid was constructed by cloning 3b coding sequence in-frame with the lex a dna binding domain (fig. 1a) . the human lung cdna library, cloned in-frame with the b42 activation domain vector figure 1 . sars-cov 3b interacts with runx1b. a. a schematic representation of full-length 3b, phyblexa/zeo-3b bait plasmid, full-length runx1b and pyestrp2-runx1b (51-421 aa) prey plasmid. the 3b protein has a nucleolar localization signal (nols) at the c-terminus. the runx1b protein has a runt homology domain (rhd, 50-177 aa), an activation domain (ad, 291-171 aa), and an inhibitory domain (id, 346-411 aa). b. the 3b-runx1b interaction was assessed on a selective growth media (supplemented with 3-at) and by filter lift b-galactosidase activity assay in a yeast two-hybrid experiment. phyblexa/zeo, pyestrp2, phyblexa/zeo-3b, pyestrp2-runx1b, phyblexa/zeo-fos and pyestrp2-jun constructs were cotransformed in l40 in combinations tabulated above. phyblexa/zeo-fos and pyestrp2-jun were used as positive control. c, d. in vitro analysis of 3b and runx1b interaction. c. in vitro translated s 35labelled 3b and runx1b lysates (input) were subjected to co-immunoprecipitation alone or together, using a-runx1 antibody. d. total cell lysates of huh7 cells expressing indicated proteins were immunoprecipitated with a-flag antibody and western blotted with a-myc antibody to probe 3b protein (panel 1). lysates were probed for the expression of runx1b and 3b with a-flag and amyc antibodies, respectively. doi:10.1371/journal.pone.0029542.g001 was purchased commercially. from the screening, a clone encoding 51-421 aa of runt related transcription factor 1, isoform b (runx1b, accession no. np_001001890) (fig. 1a) was obtained. yeast co-transformants containing pyestrp2-runx1b (51-421 aa) and phyblexa/zeo-3b withstood stringent growth conditions of media (supplemented with 5 mm 3-aminotrizol) and scored positive for b-galactosidase filter lift assay (fig. 1b) whereas co-transformants of phyblexa/zeo and pyestrp2 (empty bait and prey plasmids); phyblexa/zeo-3b and pyestrp2 (bait plasmid with empty prey plasmid); and pyestrp2-runx1b and phyblexa/zeo (prey plasmid with empty bait plasmid) did not. pyestrp2-fos and phyblexa/zeo-jun co-transformed yeast colonies were used as positive control. runx1b and 3b physical interaction was confirmed in vitro by co-immunoprecipitation in two separate experiments. firstly, coimmunoprecipitation using in vitro transcribed and translated fulllength [ 35 s]-3b and [ 35 s]-runx1b showed pull-down of 3b by anti-runx1 from 3b-runx1b lysate mixture whereas no corresponding band for 3b was visible from the 3b and runx1b lysates alone (fig. 1c) . the interaction was further confirmed in huh7 cells. cells expressing flag tagged runx1b (pcmv-tag2b flag runx1b or flag-runx1b) and myc tagged 3b (pcdna 3.1 myc/his-3b or myc-3b), alone or together, were subjected to co-immunoprecipitation assay. immunoprecipitation by anti-flag detected 3b from cells co-expressing runx1b and 3b (lane 3, fig. 1d ) whereas no corresponding band for 3b was seen from cells expressing runx1b or 3b alone (lane 1 and 2, fig. 1d ). reciprocal co-immunoprecipitation using anti-gfp antibody in cells expressing egfp, 3b-egfp, 9b-egfp (negative control) and runx1b expression constructs showed pull-down of runx1b with 3b specifically, confirmed physical interaction of 3b with runx1b. 3b partially co-localizes with runx1b in the nucleus cellular distribution of full-length 3b and runx1b was visualized in hek293 cells using immunofluorescence assay. hek293 cells transfected with flag-runx1b and ha-3b (pxj40-ha-3b) expression plasmids were subjected to immunofluorescence assay using anti-ha and anti-runx1 antibodies. confocal microscopy revealed localization of runx1b in the nucleus, as reported earlier [19] and 3b in the nucleus with majority inside the nucleolus, as seen by yuan et al [11] . cotransfected cells expressing 3b and runx1b showed significant partial co-localization of the two proteins in the extra-nucleolar nucleus area with pearson's correlation coefficient = 0.633 and mander's coefficient = 0.9 (fig. 2 ). similar levels of partial colocalization were also seen in cos7 cells (data not shown). 3b gets recruited on the runx1 binding element on the il2 promoter runx1b together with cbfb forms cbf, which further interacts with other transcription factors, co-activators or corepressors on runx1 binding elements and regulates transcription of target genes. the il2 gene promoter harbours three runx1 binding sites and is reported to be regulated by runx1b in t cells [19] . to investigate whether 3b-runx1b interaction leads to the recruitment of 3b on runx1 binding elements on the endogenous il2 promoter, chip assays were performed in runx1b/cbfb endogenously expressing jurkat cells that are abortively infected by sars-cov. jurkat cells transfected with empty vector or ha-3b were subjected to the assay after 48 h of transfection. chip results from 3b transfected cells using anti-ha depicted co-immunoprecipitation of the il2 promoter region containing runx1 binding site but not from mock transfected cells (fig. 3) . however, the 39-distal il2 promoter region, which does not contain runx1 binding site, was not immunoprecipitated by anti-ha in either of the samples, suggesting that 3b recruitment on the il2 promoter region is specific to the runx1 binding. control chip assays using anti-runx1 (positive control) and no antibody (negative control) were conducted simultaneously. these results clearly demonstrate an in vivo recruitment of 3b on the runx1 binding element on the il2 promoter. viruses employ various strategies to manipulate activities of host transcription factors in their favour. to investigate the effect of sars-cov 3b protein on the runx1b transcriptional activity, reporter gene assays were performed using the mouse il2 promoter. hek293 cells were co-transfected with wild-type (wt) il2 promoter luciferase plasmid, flag-runx1b, flag-cbfb and myc-3b in combinations mentioned. renilla luciferase plasmid (prl-tk) was co-transfected as an internal control. 3b alone showed no significant increase in the luciferase activity whereas 3b along with runx1b and cbfb showed increase in the luciferase activity in a dose dependent manner (fig. 4a ). transfection in jurkat cells also showed 1.5-5.0 fold increase in the luciferase activity with increasing doses of myc-3b (fig. 4b) , suggesting that the increase in the luciferase activity in hek293 and jurkat cells was specific to the 3b expression. to confirm whether 3b induced increase in the il2 promoter activity was due to runx1b binding on the promoter, the il2 promoter with all three runx1 binding sites mutated (mutant il2-luc) was used. myc-3b transfected jurkat cells showed merely 0.3 fold increase in the luciferase activity with mutant il2-luc as against 2.0 fold with wt il2-luc (fig. 4c) ; confirming that 3b dependent increase in figure 2 . 3b partially co-localizes with runx1b in the nucleus. cellular distribution of 3b and runx1b proteins were visualized by subjecting flag-runx1b and ha-3b transfected hek293 cells to immunofluorescence assay. 3b was visualized using primary a-ha and alexa-488 conjugated secondary antibody. runx1b was visualized using primary a-runx1 and alexa-594 conjugated secondary antibody. nuclei were visualized by dapi (496-diamidino-2-phenylindole) staining. arrows indicate extra nucleolar nucleus area of partial co-localization. doi:10.1371/journal.pone.0029542.g002 the il2 promoter activity relies on the runx1b transcription factor complex binding. hence, we conclude that 3b potentiates runx1b transcriptional activity on the il2 promoter. runx1b transcriptional activity is regulated by various posttranslational modifications like phosphorylation, acetylation, ubiquitination etc [20] . phosphorylation of runx1b by ser-thr kinases/erk on serine 249/266 has been reported to potentiate its transactivation potential [21] . sars-cov 3b has been reported to activate erk pathway [12] . therefore, determination of erk dependent phosphorylation levels of runx1b in 3b expressing cells was of our interest. to investigate this, an in vitro kinase assay with 3b and vector transfected hek293 cells was performed. runx1b immunocomplex from transfected cells served as a substrate for the kinase assay. results depicted a 1.5 fold increase in phosphorylated runx1b levels in 3b transfected cells compared to control transfected cells (fig. 5a ). this suggests that 3b activated erk may partly be responsible for the stimulated runx1b transcriptional activity in 3b transfected jurkat cells. to test this hypothesis, the reporter gene assay was performed in the presence of erk inhibitor, u0126. 3b transfected jurkat cells were treated with dmso or u0126 for 24 hrs prior to measurement of luciferase activity. treatment of cells with u0126 led to the inhibition of luciferase activity which was otherwise observed in 3b expressing dmso treated cells (fig. 5b) . this experiment points out that 3b mediated increase in runx1b transactivation potential may also be contributed by stimulated erk activity. 3b and runx1b cooperatively enhance mip-1a mrna levels promoter of macrophage inflammatory protein (mip-1a), a well characterised pro-inflammatory cytokine [22] have been documented to be regulated by runx1b through its proximal and distal runx1 binding elements [23] . sars-cov infection has been found to stimulate monocyte derived-dendritic cells to express mip-1a [24] . in order to study the effect of 3b expression on endogenous mip-1a mrna levels, real-time pcr was performed in human monocytic u937 cells. cells overexpressing runx1b showed 3.0 fold increase in mip-1a mrna levels; whereas those expressing 3b showed 5.0 fold increase as compared to vector. interestingly, cells overexpressing 3b along with runx1b showed 13 fold increase in mip-1a mrna levels ( fig. 6) , suggesting that 3b significantly enhances runx1b transactivation potential on the endogenous mip-1a promoter. sars pathogenesis, caused by evasion of the host innate immunity, is characterized by a remarkable cytokine storm and lymphopenia. sars coronavirus antagonizes host interferon action by encoding a few interferon antagonists like 3b, sars 6, nucleocapsid, nsp1, nsp3 and recently reported, m protein [8, 25, 26] . among them, 3b, like sars6, belongs to a group of accessory proteins that are unique to sars coronavirus. a recent study brought out plausible contribution of 3b in sars pathogenesis, by affecting levels of chemokines that are found upregulated in sars. we believed that the accessory protein 3b has more roles to play in virus pathogenesis which are still unknown. to unveil new cellular interactors of 3b, we employed a yeast two-hybrid screen of human lung cdna library and identified transcription factor runx1b as a putative interactor of 3b. the interaction was validated in vitro by co-immunoprecipitation. runx1b plays a crucial role in development of myeloid and lymphoid lineage cells. it was originally identified at a break point on human chromosome 21 in the t(8;21) translocation. it is a most common target of chromosomal translocation in human leukaemias [16, 27] including acute myeloid leukaemia, b-cell acute lymphoblastic leukaemia and t-cell lymphoblastic leukaemia [28, 29] . importantly, runx1b is involved in transcriptional regulation of several genes including cytokines and chemokines like il2, il3, gm-csf, mip-1a, csfr etc. [19, 23, 30, 31, 32, 33, 34, 35] . it synergises with other transcription factors to activate transcription [36, 37, 38] and acts as a transcriptional activator or repressor depending upon its association with co-activators such as cbp, p300, moz [39, 40] and co-repressors, msin3a [41] , tle1 [42, 43] and ncor [28] . several reports have described the involvement of runx1 isoforms in transcription and replication of viruses like murine leukaemia virus [14, 44, 45] , maedi visna virus [46] , polyomavirus [47] and human papilloma virus [48, 49] . b-cell proliferation and immortalization by epstein-barr virus is mediated by downregulation of runx1, a consequence of the binding of runx3 on the runx1 promoter [50] . runx1 has been found to be upregulated in latently infected gm-ps by human cytomegalovirus (cmv), a species specific herpesvirus [51] . in adenovirus infected cells, runx1 leads to significant mislocalization of e4orf6, thus interfering with viral replication [52] . a comparative study by microarray analysis of host gene transcription in the huh7 cell line infected with sars-cov and human coronavirus 229e found significantly increased runx1 expression with sars-cov infection as compared to human cov22e [53] . this observation prompted us to study this novel interaction of sars-cov accessory protein 3b with runx1. we observed significant partial co-localization of 3b and runx1b in the extra-nucleolar nucleus region. next, 3b was found to get recruited on the runx1 binding elements which led to increased transactivation of runx1b on the il2 promoter, as depicted by reporter gene assays. additionally, 3b also affected its transactivation on the il2 promoter by modulating phosphorylation levels of runx1b through erk. a similar mode of regulation of immediate early gene x-1 through erk-induced runx1 phosphorylation in response to thrombopoetin was reported by hamelin and colleagues [54] . in order to explore the effect of 3b on runx1b target gene other than il2, we measured mrna levels of a mip-1a, the promoter of which is reported to be regulated by runx1b [23] . mip-1a has been observed to get upregulated in monocyte derived dendritic cells infected with sars-cov [24] . in our study, overexpression of 3b in monocytic cells u937 resulted in 5 fold increase in mip-1 mrna levels which rose to 13 folds, when overexpressed with runx1b. interestingly, a recent report by chen et al. figure 5 . 3b expression increases phosphorylated runx1b levels through erk activation. a. hek293 cells were transfected with vector, 3b and runx1b expression plasmids. erk immunoprecipitated from vector and 3b lysates were subjected to kinase assay with runx1b beads. phosphorylated runx1b was visualized by autoradiography. input levels of immunoprecipitated erk and phospho erk levels in lysates were probed by western blotting. graph depicts fold increase in the levels of phosphorylated runx1b procured after three independent experiments. bar represents mean6sd of values obtained by densitometry. #, p,0.05. b. jurkat cells were transfected with wt il2-luc in the presence or absence of myc-3b and treated with dmso or u0126. relative luciferase activity was measured and is shown as the mean6sd of three independent experiments performed in triplicates. *, p,0.005. phospho erk levels in lysates were probed by western blotting. doi:10.1371/journal.pone.0029542.g005 characterizing cellular immune response to sars-cov infection in senescent mouse models showed increase in mip-1a and il2 levels at early and late stages of infection [55] , a result that supports our observations. to the best of our knowledge, this is the first report unveiling the effect of sars-cov protein 3b on the transcriptional activity of a host transcription factor, runx1b, and its downstream target genes il2 and mip-1a. jurkat and monocyte cells are abortively infected by sars-cov. upon entry in these cells, rna genome of the sars-cov will get transcribed and translated and expectedly, would lead to the synthesis of 3b along with other proteins. however, until now there is no direct report on the expression levels of 3b in these virus infected cells. therefore, based on our study we have speculated the role of 3b in virus infected monocytic and t cells and put forth a plausible role of 3b in sars-cov pathogenesis which gives new directions to the understanding of sars. 3-aminotrizole was purchased from sigma. luciferase assay kit (e1500) and in vitro transcription and translation kit (l4610.) were purchased from promega. trizol was purchased from invitrogen. antibodies against runx1, ha and c-myc were purchased from santa cruz. anti-flag was purchased from roche. alexa 488 conjugated anti-mouse and alexa 594 conjugated anti-rabbit were purchased from molecular probes. the sars-cov 3b gene (25689-26153 bp), was pcr amplified from the sars-cov genome (nc_004718) and cloned in pxj40-ha vector as described earlier [56] . the 3b insert was further cloned into phyblexa/zeo and pcdna3.1(-) myc/his vector. pcmvflag tag 2b-runx1b, mouse wt il2-luc and mutant il2-luc (all three runx1 binding sites mutated) constructs were generously provided by dr. shimon sakaguchi (institute for frontier medical sciences, kyoto university, japan), migri-cbfb was gifted by dr. nancy a speck (university of pennsylvania school of medicine, philadelphia). lex a based screening system (hybrid hunter, version f), comprised of saccharomyces cerevisiae strain l40 [mata his3d200 trp1-901 leu2-3112 ade2 lys2::(4lexaop-his3)ura3::(8lexaop-lacz) gal4], phyblexa/zeo and pyestrp2 as binding domain and activation domain vectors, respectively and human lung cdna library cloned in pyestrp2, was purchased from invitrogen. screening was performed as per manufacturer's protocol. the bait plasmid was constructed by cloning 3b coding sequence in-frame with the lexa dna binding domain in phyblexa/zeo. phyblexa/zeo-3b was co-transformed with cdna library in l40 and co-transformants were selected for the activation of two reporter genes, his3 and lacz. strength of the interaction in selected co-transformants were assessed by their ability to grow on his 2 trp 2 and zeo + yc media supplemented with 5 mm at (3amino-1,2,3-trizole, competitive inhibitor of his3) and for positivity of filter b-galctosidase activity assay. filter-lift assay was performed as described before [57] . plasmids were isolated from positive co-transformants and shuttled into e.coli dh5a and sequenced. l40 co-transformed with phyblexa/zeo-fos and pyestrp2-jun was used as the positive control and l40 cotransformed with phyblexa/zeo and pyestrp2 was used as the negative control for the library screening. cell culture and transfection hek293, human embryonic kidney cells and huh7, human hepatoma cells were maintained in dmem (dulbecco's modified eagle's medium). human leukemic t cells, jurkat and human monocytic cells, u937, were maintained in rpmi1640. all cell lines were maintained in media supplemented with 10% fcs (fetal calf serum, v/v) and antibiotics (penicillin and streptomycin). transient transfections in huh7 and hek293 cells were carried out using fugene 6 (roche) and lipofectamine 2000 (invitrogen) as per manufacturer's protocol and in jurkat and u937 cells were carried out by electroporation at 260 v and 975 mf in 4 mm cuvettes, using biorad gene pulser. for immunofluorescence assay, hek 293 cells were seeded at 50% confluency on coverslips in a 12 well plate. cells were singly or co-transfected with runx1b (pcmv-tag2b flag runx1b) and 3b (pxj40 ha-3b) expression vectors and the assay was performed 24 h post-transfection as described by korkaya et al. [58] . 3b and runx1b was stained using mouse anti-ha and rabbit anti-runx1 antibodies, respectively. alexa 594 conjugated goat anti-rabbit and alexa 488 conjugated goat anti-mouse were used as secondary antibodies. coverslips were mounted on to the glass slides in anti-fade (with dapi) medium. confocal images were collected using a 606 objective on a nikon a-1rconfocal microscope and analysed by imaging software, nis-elements. for in vitro co-immunoprecipitation, [ 35 s] radiolabelled full length runx1b and 3b were expressed using a coupled in vitro transcriptional/translation system as per manufacturer's protocol. 3b lysate, runx1b lysate and 3b-runx1b lysate mix were incubated with anti-runx1 in 500 ml lysis buffer (20 mm tris ph 7.4, 150 mm nacl, 0.05% np40) at 4uc for 4-6 h. immunocomplexes were precipitated by adding 50 ml sepharose-a (50% v/v) beads at 4uc for 1 h. beads were washed thrice with lysis buffer and resuspended in sds-page loading buffer. samples were run on sds-page and bands were detected by autoradiography. for in vivo co-immunoprecipitaion, huh7 cells were transiently transfected with expression plasmids of runx1b (pcmv-tag2b flag runx1b) and 3b (pcdna3.1 myc-3b) in combinations mentioned. total dna amount was normalized by adding pcdna3.1. 48 h post transfection, cells were lysed and immunoprecipitation and western blotting was performed as described earlier [59] . for chip, 10-15610 6 jurkat cells were transfected with 10 mg vector or 3b (pxj40 ha-3b) plasmid. 48 h post transfection, chip assay was performed as described earlier [60] . dna fragments were analysed for the recruitment of runx1b and 3b on the runx1 binding site on the il2 promoter as well as on 59 distal region of the human il2 gene (non-runx1 binding site). sequences of the primers used for pcr: forward primer for human il2 promoter: 59-ctctagctgacatgtaagaagc-39; reverse primer for human il2 promoter: 59-ctacactgaa-catgtgaatagc-39; forward primer for 39 distal region of the human il-2 gene: 59-aaatgttgcaggatccttgc-39; reverse primer for 39 distal region of the human il-2 gene: 59-tgagctctgacatgatgctc-39. luciferase assay was performed as per manufacturer's protocol (promega). for the assay, cells were transfected with reporter plasmid (wt il2-luc or mut il2-luc), prl-tk and indicated expression plasmids. pegfp-n1 plasmid was transfected as a negative control in cells not expressing 3b. drug treatment (u0126, 10 mm) was performed for 24 h prior to harvesting. all transfections were normalized for the amount of total dna by adding pcdna3.1. luciferase activity was measured 48 h post transfection and normalized against renilla luciferase activity. relative luciferase activity was expressed as mean6standard deviation (sd) of three independent experiments. statistical significance was calculated using student's t test. p values are given in the figure legends. error bars represent the sd. in vitro kinase assay erk activity in 3b transfected cells was assayed using immunoprecipitated runx1b as the substrate. hek293 cells were transfected with expression plasmids of vector (control), 3b, and runx1b. 48 h after transfection, cells were lysed in lysis buffer (20 mm tris-hcl ph 7.5, 150 mm nacl, 1 mm edta, 1 mm egta, 1% triton, 2.5 mm sodium pyrophosphate, 1 mm b-glycerophosphate, 1 mm sodium orthovandate) and equal amounts of vector and 3b lysates were subjected to immunoprecipitaion using anti-erk antibody and runx1b lysate, using anti-runx1 antibody. sepharose a beads were washed thrice with the lysis buffer and twice with the kinase buffer (20 mm tris-cl ph 7.5, 10 mm mgcl 2 , 1 mm dtt, 5 mm sodium orthovandate). for the assay, erk beads were incubated with runx1b beads in 20 mm tris-cl (ph 7.5), 10 mm mgcl 2 , 1 mm dtt, 2 mm b-glycerophosphate and 50 mm atp, 5 mci [c-32 p]-atp at 37uc for 30 min. reaction mixtures were run on sds-page, and analyzed by autoradiography. rna was isolated from transfected u937 cells using trizol, as per manufacturer's protocol. 5 mg rna was reverse transcribed using oligo-dt primer and mulv reverse transcriptase as per manufacturer's protocol (promega). primers used for pcr were: mip-1a forward: 59 acttgctgctgacacgccga 39 and reverse: 59 cacagacctgccggctt cgc 39; actin forward: 59 tgacggggtcacccacactgtgcccatcta39 and reverse: 59 ctagaagcatttgcggtggacgatggaggg39. data is represented as bar graph and is mean6sd of three independent observations. identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus sars corona virus peptides recognized by antibodies in the sera of convalescent cases coronaviral hypothetical and structural proteins were found in the intestinal surface enterocytes and pneumocytes of severe acute respiratory syndrome (sars) severe acute respiratory syndrome coronavirus group-specific open reading frames encode nonessential functions for replication in cell cultures and mice severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists molecular determinants for subcellular localization of the severe acute respiratory syndrome coronavirus open reading frame 3b protein mitochondrial location of severe acute respiratory syndrome coronavirus 3b protein nucleolar localization of non-structural protein 3b, a protein specifically encoded by the severe acute respiratory syndrome coronavirus lal sk sars-cov accessory protein 3b induces ap-1 transcriptional activity through activation of jnk and erk structure and regulated expression of mammalian runx genes molecular cloning and characterization of pebp2 beta, the heterodimeric partner of a novel drosophila runt-related dna binding protein pebp2 alpha identification of aml-1 and the (8;21) translocation protein (aml-1/eto) as sequence-specific dna-binding proteins: the runt homology domain is required for dna binding and proteinprotein interactions alternative splicing and genomic structure of the aml1 gene involved in acute myeloid leukemia aml-1 is required for megakaryocytic maturation and lymphocytic differentiation, but not for maintenance of hematopoietic stem cells in adult hematopoiesis differential requirements for runx proteins in cd4 repression and epigenetic silencing during t lymphocyte development foxp3 controls regulatory t-cell function by interacting with aml1/runx1 post-translational modifications of runx1 regulate its activity in the cell the extracellular signal-regulated kinase pathway phosphorylates aml1, an acute myeloid leukemia gene product, and potentially regulates its transactivation ability macrophage inflammatory protein-1 transcriptional regulation of the human mip-1alpha promoter by runx1 and moz chemokine upregulation in sars-coronavirus-infected, monocyte-derived human dendritic cells severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type i interferon, in infected cells severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf3.tank.tbk1/ikkepsilon complex fusion of the tel gene on 12p13 to the aml1 gene on 21q22 in acute lymphoblastic leukemia a mechanism of repression by acute myeloid leukemia-1, the target of multiple chromosomal translocations in acute leukemia a new translocation that rearranges the aml1 gene in a patient with t-cell acute lymphoblastic leukemia the identification of (etv6)/runx1-regulated genes in lymphopoiesis using histone deacetylase inhibitors in etv6/runx1-positive lymphoid leukemic cells cooperation between core binding factor and adjacent promoter elements contributes to the tissue-specific expression of interleukin-3 aml1a and aml1b can transactivate the human il-3 promoter regulation of gm-csf gene transcription by core-binding factor identification of a region which directs the monocytic activity of the colonystimulating factor 1 (macrophage colony-stimulating factor) receptor promoter and binds pebp2/cbf (aml1) pebp2/ cbf, the murine homolog of the human myeloid aml1 and pebp2 beta/cbf beta proto-oncoproteins, regulates the murine myeloperoxidase and neutrophil elastase genes in immature myeloid cells ccaat enhancer-binding protein (c/ebp) and aml1 (cbf alpha2) synergistically activate the macrophage colony-stimulating factor receptor promoter cooperative binding of ets-1 and core binding factor to dna runx1 and gata-1 coexpression and cooperation in megakaryocytic differentiation interaction and functional cooperation of the leukemia-associated factors aml1 and p300 in myeloid cell differentiation activation of aml1-mediated transcription by moz and inhibition by the moz-cbp fusion protein groucho/tle/r-esp proteins associate with the nuclear matrix and repress runx aml/pebp2(alpha)) dependent activation of tissue-specific gene transcription tle, the human homolog of groucho, interacts with aml1 and acts as a repressor of aml1-induced transactivation transcriptional repression by aml1 and lef-1 is mediated by the tle/ groucho corepressors transcriptional activity of core binding factor-alpha (aml1) and beta subunits on murine leukemia virus enhancer cores transcriptional activation of a retrovirus enhancer by cbf (aml1) requires a second factor: evidence for cooperativity with c-myb regulation of the long terminal repeat in visna virus by a transcription factor related to the aml/pebp2/cbf superfamily a new transcription factor family associated with human leukemias competitive binding of viral e2 protein and mammalian core-binding factor to transcriptional control sequences of human papillomavirus type 8 and bovine papillomavirus type 1 a new cellular factor recognizes e2 binding sites of papillomaviruses which mediate transcriptional repression by e2 downregulation of runx1 by runx3 requires the runx3 vwrpy sequence and is essential for epstein-barr virus-driven b-cell proliferation impact of human cytomegalovirus latent infection on myeloid progenitor cell gene expression runx1 permits e4orf6-directed nuclear localization of the adenovirus e1b-55k protein and associates with centers of viral dna and rna synthesis comparative host gene transcription by microarray analysis early after infection of the huh7 cell line by severe acute respiratory syndrome coronavirus and human coronavirus 229e thrombopoietin regulates iex-1 gene expression through erk-induced aml1 phosphorylation cellular immune responses to severe acute respiratory syndrome coronavirus (sars-cov) infection in senescent balb/c mice: cd4+ t cells are important in control of sars-cov infection over-expression of severe acute respiratory syndrome coronavirus 3b protein induces both apoptosis and necrosis in vero e6 cells the orf3 protein of hepatitis e virus interacts with liver-specific alpha1-microglobulin and its precursor alpha1-microglobulin/bikunin precursor (ambp) and expedites their export from the hepatocyte the orf3 protein of hepatitis e virus binds to src homology 3 domains and activates mapk the nucleocapsid protein of severe acute respiratory syndrome-coronavirus inhibits the activity of cyclin-cyclindependent kinase complex and blocks s phase progression in mammalian cells rb interacts with histone deacetylase to repress transcription we are grateful to the following scientists for generously providing us with constructs: dr. shimon sakaguchi for providing pcmvtag2bflag-runx1b, wt il2-luc and mutant il2-luc constructs; dr. nancy a. speck for providing cbfb-migri construct. we thank dr. majula kalia for her guidance in confocal experiments. we also acknowledge the technical help from mr. r. kumar in cell culture maintenance and propagation and ekta khattar for help with the manuscript. key: cord-341880-wxliz485 authors: mottaleb, khondoker abdul; mainuddin, mohammed; sonobe, tetsushi title: covid-19 induced economic loss and ensuring food security for vulnerable groups: policy implications from bangladesh date: 2020-10-16 journal: plos one doi: 10.1371/journal.pone.0240709 sha: doc_id: 341880 cord_uid: wxliz485 at present nearly half of the world’s population is under some form of government restriction to curb the spread of covid-19, an extremely contagious disease. in bangladesh, in the wake of five deaths and 48 infections from covid-19, between march 24 and may 30, 2020, the government imposed a nationwide lockdown. while this lockdown restricted the spread of covid-19, in the absence of effective support, it can generate severe food and nutrition insecurity for daily wage-based workers. of the 61 million employed labor force in bangladesh, nearly 35% of them are paid on a daily basis. this study examines the food security and welfare impacts of the covid-19 induced lockdown on daily wage workers both in the farm and nonfarm sectors in bangladesh. using information from more than 50,000 respondents complied with the 2016–17 household income and expenditure survey (hies) in bangladesh, this study estimates daily wage rates as bangladesh taka (bdt) 272.2 in the farm sector and bdt 361.5 in the nonfarm sector. using the estimated daily wage earnings, this study estimates that a one-day complete lockdown generates a us$64.2 million equivalent economic loss only considering the wage loss of the daily wage workers. after estimating the daily per capita food expenditure separately for farm and nonfarm households, this study estimates a minimum compensation package for the daily wage-based farm and nonfarm households around the us $ 1 per day per household to ensure minimum food security for the daily wage-based worker households. by september 14, 2020, nearly 29 million people in 216 countries and territories have been sickened by the severe acute respiratory syndrome coronavirus-2 (sars-cov-2) or covid-19 [1] . by that time, the covid-19 induced death toll had reached more than 925 thousand globally [1] . the virus was first detected on december 31 st 2019 in wuhan province, china, and on january 11, 2020, china confirmed the first covid-19 induced death followed by the first death in the united states on january 21, 2020 [2] . on january 30, 2020, the world health organization (who) declared covid-19 a global public health emergency of international concern [3] . covid-19 is an extremely contagious disease, spreading rapidly through human to human contact [4] [5] [6] . until the time of writing this article, there is no effective medicine to cure or vaccine to protect from this virus. to curb the spread of this disease, national governments have imposed varying levels of movement restrictions. for example, by april 7, 2020, national governments of 32 countries and territories in asia, 43 in europe, 38 in the americas and the caribbean, and 44 in africa imposed varying levels of movement restrictions [7] . studies and opinions warned that the covid-19 lockdown and restricted labor movement could generate havoc across the world [8, 9] , and could cause severe global food shortages by disrupting the supply chain [10] [11] [12] [13] [14] [15] [16] [17] [18] . particularly, the one-size-fits-all approach of lockdown can aggravate the plight of daily wage workers in low-income countries. considering this, one suggestion is to expand social safety net programs in developing countries [14] . however, to our knowledge, there is no solid study based on microdata that examines the economic loss due to the covid-19 induced lockdown and the minimum compensation required to ensure the basic nutrition of the poor households. the present study uses information from 50,000 economically active workers in bangladesh, collected by the bangladesh bureau of statistics (bbs), to quantify the economic loss due to the covid-19 lockdown based on the lost wage earnings of the daily wage workers in the farm and nonfarm sectors of bangladesh. then, applying simple econometric estimation processes, this study estimates the minimum compensation packages for the daily wage-based farm and nonfarm households of bangladesh that ensure their minimum food security during the lockdown. the rest of the study is organized as follows. the next section describes the current poverty situation and the covid-19 induced restricted movement and lockdown in the world and bangladesh. section 3 explains materials and methods, and section 4 presents major findings. section 5 provides conclusions and policy implications. to control the spread of covid-19, at least 157 countries and territories in the world imposed varying levels of movement restrictions [7] . in asia, 12 countries implemented localized lockdowns, 10 countries implemented national lockdowns, and the rests implemented localized or suggested national recommendations, such as maintaining social distance [7] . these covid-19 induced lockdowns and movement restrictions can exacerbate the already worsening global poverty situation. despite the extraordinary success in alleviating abject hunger and extreme poverty since the 1990s [19] , the share of hungry people in the globe has been increasing in recent years [20, 21] . in 1950, more than 63% of the total population of the world was extremely poor, living on less than us$ 1.90/day [19] . in 1990, globally, the extremely poor reduced to 35 .9% (1,895 million), which further reduced to 20.8% (1,352 million) in 2005 [22] . however, since 2015, the absolute number of hungry people has started to increase. in 2015, out of a global population of 7.3 billion, 784 million (10.7%) were hungry, which increased to 821 million (10.9%) in 2017 [23] . it is projected that covid-19 will further exacerbate the poverty and abject hunger in the world in a number of ways. it is argued that the overburden on the health sector causes the reallocation of resources to the health sector to save lives, which may reduce resource allocation to the agriculture and industrial sectors, thereby hampering the food production and input supply chains [16] . more importantly, covid-19 induced lockdowns and movement restrictions have already increased unemployment and under-employment, which has severely reduced the purchasing power of consumers [10] . the international labor organization (ilo) estimates that the participation of the global working force in the first quarter of 2020 declined by 4.5% which is equivalent to 130 million full-time jobs [8] . in a recent report, asian development bank (adb) projected that the covid-19 induced global loss will range between $6.1 to $9.1 trillion compared to a no-covid-19 baseline, which is equivalent to 7.1% to 10.5% of the global gdp [24] . the international monetary fund (imf), warned that due to the covid-19 induced lockdown and resource reallocation to the health sector to save lives, the global gdp could be reduced by 4.9% in 2020 compared to the previous year [25] . the economic commission for latin america and the caribbean (eclac) and ilo [26] projected that due to the covid-19 pandemic, the economy of the latin american and caribbean region will shrink by 5.3% and the unemployment rate will increase from 3.5% to 11.5%, equivalent to more than 11.5 million new unemployed persons. furthermore, the poverty rate in the region will increase by 4.4% and the extreme poverty rate will increase by 2.6% [26] . studies also warned about a possible severe food crisis, particularly in countries currently experiencing economic shocks, weather extremes, or conflicts [10, 27] . it is observed that during epidemics like hiv/aids and ebola, food prices in affected countries increased significantly, with severe impacts on food security, especially for vulnerable populations including women, children, and marginal people [28] . for example, in sierra leone, the triennium average ending in 2013 for rice (paddy) and cassava prices were us $614.3/ton and us$303.7/ton, respectively [29] . however, when the ebola epidemic hit the country in 2014, the price of rice (paddy) rose to us$707.3/ton (+15%) and the cassava price to us$ 521.1/ton (+72%) [29] . increased food prices can disproportionately affect vulnerable groups, such as marginal households and women [30, 31] . in bangladesh, for example, the food price hikes of 2007-08 pushed an additional 13 million people below the poverty line [32] . given the covid-19 induced chaos, if major food and cereal exporting countries restrict their exports, a severe crisis may be generated in the food and cereal import-dependent countries [28, 33] . in 2008, droughts in australia and argentina and rising oil prices prompted a number of major cereal exporting countries such as india to ban cereal and food exports, aggravating the food price crisis [28] . it is reported that kazakhstan has already banned wheat flour exports and restricted exports of buckwheat, vegetables, onions, carrots, and potatoes; while vietnam has temporarily stopped rice exports [11] . in fact, in its latest report, the united nations warned that world trade could shrink by 15%, the global economic output could be slashed by us$ 8.5 trillion over the next two years, and the economic growth of developed countries could reduce to -5%. consequently, the global economy is projected to shrink by 3.2% and some 34.3 million people are projected to fall below the poverty line due to covid-19 [34] . unu-wider warned that in the most extreme case, covid-19 induced responses could reduce 20% of the income or consumption expenditure at the global level, and poverty levels could increase by an additional 420-580 million people compared to 2018 levels [35] . covid-19 could create a severe setback to attaining the zero hunger goal of the united nations by 2030 [23] . in 2019, a total of 135 million people in the world were severely food insecure [10] . food insecurity triggered by conflicts affected 77 million people in 22 countries; economic shockrelated food insecurity affected 22 million people in eight countries, and weather extremes caused food insecurity for 34 million people in 25 countries [10] . the world food program (wfp) cautioned that in the absence of swift and effective actions, the number of severely food insecure people could reach 265 million in 2020 [27] . in addition, at present, nearly 2 billion people in the world are moderate to severely food insecure [36] . these food vulnerable people are mostly concentrated in africa, west asia, and latin america [36] . although has been affecting high-and low-income countries, the aftermath of covid-19 could have severe negative impacts on low-income developing countries. in bangladesh, on march 23, 2020, in the wake of three deaths and 25 infections of covid-19 [37] , the bangladesh government imposed a nationwide holiday and lockdown initially from march 26 to april 4, 2020 [38] , and later extending the general holiday to april 11, and then to april 30, 2020 [39] . finally, the government extended the general holiday and airport and road lockdown until may 30, 2020 [39] . until april 17, 2020, out of 64 districts of bangladesh, 38 districts were completely under lockdown and 17 were under partial lockdown [40] (fig 1) . as of september 14, 2020, bangladesh had a total of 341,056 confirmed covid-19 cases, with 245,594 recovered and 4,802 deaths [37] . since 2000, bangladesh has continued to achieve stunning economic progress. after independence in 1971, the per capita gdp of bangladesh was $133.6, which had increased to $418.1 in 2000, and $1688 in 2018 [41] . due to the enormous population pressure, the average farm size in bangladesh is as small as 0.68 ha [42] . despite the fact, bangladesh became the fourth-largest paddy-rice-producing country (49.5 mmt) in the world after china (214 mmt), india (169 mmt), and indonesia (81.4 mmt) [43] . today, bangladesh is self-sufficient in rice, the major staple of the country. the 1971-73 triennium average daily per capita dietary energy intake was 1963 kcal, which has increased to 2574 kcal/daily in 2015-17 [44] . in 2019, bangladesh ranked 83 rd out of 113 countries on the global food security index [45] , whereas it ranked 102 nd out of 119 countries in 2006 [46] . the covid-19 induced turmoil, however, could substantially undermine the economic achievement of bangladesh by affecting trade and the garment industry of bangladesh. bangladesh is the second largest garment exporting country in the world after china, where, in more than 4,000 garment factories, more than 4.4 million workers are employed, the majority of whom are female [47] . more than 80% of the total export earnings of the country solely come from the garment industry [48] . this industry is thus dependent on foreign export demand, especially from developed economies and also on imported materials from abroad and particularly from china. in 2015, the total merchandise trade value (export + import) of commodities and goods (excluding services) of bangladesh was us $79.8 billion, of which total trade value with china was $11.1 billion [49] , which was nearly 14% of the total commodity trade value of bangladesh. in march 2020, organization for economic co-operation and development (oecd) projected that in 2020 global gdp will drop by 2.4% compared to 2019, and china's gdp growth rate would be below 5% [50] . thus, negative impacts of covid-19 in china and in other high-income economies could be transmitted to bangladesh through the commodity trade channel. alarmingly, it is already reported that the unemployment rates in china and the united states have increased to unprecedentedly high levels [51] . in bangladesh, reportedly, until august 2020, export order worth of us $ 3.18 billion has been cancelled, which affected the employment of 2.28 million garment workers [52] . the nationwide lockdown and movement restriction can also severely undermine the economic achievement of bangladesh by directly curbing the wage earning opportunity of the economically employed labor force. in 2016-17, the economically active employed labor force (15 years and above) of the country was 60.8 million, of which 85.1% were informal workers ( table 1 ). the share of informal workers is the highest in the farm sector, in which out of total 24.7 million employed workers, 95.4% of them are informally employed [53] . the international labor organization (ilo) stressed that the income opportunity of the informal workers and casual labor in both farm and nonfarm sectors are mostly affected by the covid-19 induced contraction of employment and the restricted movement measures [8] . the severe labor shortages problem in harvesting boro rice (dry-season irrigate rice crop) in 2020 has not only spoiled farmers' harvest, but also curbed income opportunity of the casual labor, who in 2000, 48.9% of the total population of 127.7 million, were classified as poor, which reduced to 31.5% in 2010 [41] . however, in 2016, out of 158 million people, 24.3% were classified as poor [41] . in 2015, children under 5 years of age, every 2 children in 5, were stunned, and 14% were wasted [54] . a recent report warns that if lockdown continues for a long time, the current poverty rate and undernutrition could deteriorate significantly [55] . for example, it is stressed that in bangladesh, at least 13 million extra people have fallen below the poverty line due to covid-19 [56] . a recent survey asserts that 75% of the sampled respondents reported not having enough food, while 91% reported of not having enough money to purchase food [38] . what is missing in the existing literature is an estimate of the magnitude of the economic loss associated directly with the lockdown measure taken by the government to curb the spread of the coronavirus, as opposed to the estimate of overall negative impacts. using bangladesh as a case, this study quantifies the daily economic loss due to wage earning forgone of daily wagebased workers both in farm and nonfarm sectors. as to food security, it has already been suggested to expand the public support system [14] . the present study, in addition, suggests that the minimum compensation package should be provided to ensure the minimum food security of the daily wage-based workers. this study relies on bangladesh's 2016-17 household income and expenditure survey (hies) data collected by the bangladesh bureau of statistics (bbs). since 1971, bbs has conducted 15 rounds of surveys, with the hies 2016-17 survey being the 16 th series. the monetary poverty of the country is measured mainly based on the hies, which is a nationally representative comprehensive survey [57] . to enhance data precision by improving the data collection process, the world bank provides technical support. bbs uses a two-stage stratified random sampling process. in the first stage, primary sampling units (psus), consisting of specific geographic areas, are selected. in the second stage, from each psu, 20 households are randomly selected for a detailed interview. the 2016-17 dataset comprises 2,304 psus covering all 64 districts of bangladesh and 46,080 households. the 2016-17 hies survey is a multipurpose survey, with nine sections detailing information on household level demographics, food consumption, farm and nonfarm economic activities, and labor allocation. this study uses data on labor allocation, daily wage earnings, and food consumption. as each household was comprised of 4.0 persons on average, information on age, education, marital status, rural-urban affiliation, and job status in the sampled seven days were given for all 184,320 members of the sampled 46,080 households. in this study, however, we have only considered the economically employed respondents, who were more than 15 or less than 60 years old. thus, this study uses the sub-sample consisting of 50,671 individuals, of which 34,152 (67.4%) were from rural areas, 16,519 (32.6%) from urban areas, 6,522 (12.9%) female and 44,149 (87.1%) male. to calculate the economic loss associated with the covid-19 induced lockdown, we estimate the daily wage earnings (w is ) of a respondent i, working in sector s (= farm and nonfarm separately. the wage function is specified as follows: where lnw is is the natural log of daily wage earned by a sampled respondent i in sector s (= farm f or nonfarm nf), and cereal price is the price of cereals per kg in bangladesh taka (bdt/ kg). this variable is included in the regression to control for a possible association between cereal price and wage payment of the daily wage workers. the ilc is is a vector of variables, which includes respondent-level characteristics, such as: • years of schooling, • female dummy (fm) (female = 1, 0 otherwise); • marital status (married = 1, 0 otherwise); • age in years; the vector of variables rlc is includes a rural dummy that assumes a value of 1, if a respondent is from a rural area, and 0 otherwise, and 63 dummies for 64 districts of bangladesh. in addition, in estimating daily wage income, we include inverse mill's ratio calculated after estimating eq (7)-the occupation choice model, to take into account a possible self-selection bias in selecting wage receiving methods of the respondents as will be explained below. by multiplying the estimated per head average daily wage earnings (in bdt) in the farm sector w f and that in the nonfarm sector w nf by the absolute number of daily waged workers in those in bangladesh, the expected loss of daily wage earnings due to the covid-19 induced lockdown is calculated as follows: to estimate the minimum compensation package for the daily wage-based workers to ensure the minimum food security, we specify and estimate a function that explains household level daily food expenditure for the household of daily wage worker i (that is, survey respondent i) in sector s, as follows: where lnedfx is the natural log of the household level daily total expenditure on all food (in terms of bdt); wage earnings, lndweh is is the natural log of the total daily wage earning of the household of respondent i calculated as: the definitions of the vectors of variables ilc is and rlc is are the same as explained in eq (1). the vector of variable hlc is includes: • household level income from rent of land and other properties, insurance income, profit and dividend income, lottery and prize money, charity, gift, royalty and assistance both in cash and kind, remittance income, gratuity and retirement benefits, interest received and other income in cash or kind. to make it daily, we divided by 365; • household level receipt from social safety nets, including any positive income from any social safety net programs by any member of the household. to make it daily, we divided it by 365; and • land owned (acres). in estimating eq (3), we have estimated, firstly without including division dummies; secondly including seven division dummies for eight divisions, and finally including 63 district dummies for 64 districts to capture the regional heterogeneity on the daily household level food expenditure. as both the dependent variable (lnedfx is ), and the estimated household level total daily wage earnings ðlndweh is þ are in log form, the coefficient g 2ŝ in eq (3) is simply the elasticity. it provides the share of total wage earnings are spent on household level daily food expenditure of a sampled day-labor household in farm and nonfarm sectors. assuming, zero income of the daily wage-based households in a very strict lockdown situation, the minimum daily support can be calculated as: where edfx is is the estimated daily household level total food expenditure in sector s. in reality, however even under a stringent lockdown measure, daily wage earners may desperately search for any miscellaneous work, and thus can earn some positive income (>0). the earnings of a daily wage worker during lockdown time, however, can be lower by a fraction by θ than the normal time, where 0 < θ < 1. it means, in the lockdown time, the total wage earnings of a daily wage-based household will be ydweh is , which is lower than the normal time household level daily total earnings dweh is in eq (3), where θ = 1. consequently, the logarithm of the household level daily total food expenditure with this lost income (ydweh is ) would be smaller than that without income loss by: by rearranging eq (5), we obtain where (ydwehþ is is the lockdown induced reduced daily total wage earnings of a daily wagebased household, θ is the lockdown induced wage earning fraction (0< θ < 1), and g 2ŝ is the estimated coefficient of wage earning in explaining the daily household level total food expenditure lnedfx is specified in eq (3) a reasonable amount of minimum daily food expenditure support would be the average of the difference between food expenditures with and without income loss due to lockdown measures. using the estimated coefficient of the daily wage earning g 2ŝ from eq (3) and assuming different rates of income share, modifying eq (5) we recalculate the minimum required daily support to be provided to each household in sector s as follows: where ms s is the minimum required daily support to a household, edfxðdwehþ s is the estimated household level daily food expenditure that is obtained by estimating eq (3), and θ is the estimated coefficient. eq (8) indicates that the calculated daily minimum support is now contingent upon the fraction of daily wage earning θ, the value of g 2ŝ and the household level daily total food expenditure in the normal time. under the assumption that a daily wage-based household earns only 10% of their wage income (θ = 0.1) in the covid-19 induced lockdown time compared to the daily wage earning in the normal time, then the household level daily total food expenditure will become fraction 0.1^(g 2ŝ ) of the initial per capita food expenditure, which is the estimated value of the dependent variable of eq (3). in that case, the minimum support will be 1-0.1^(g 2ŝ ) estimated value of the hosehold level daily food expenditure in suggesting daily minimum support to ensure minimum food security of the daily wagebased households, we assume a range of income loss. in estimating the wage function (eq (1)), we have incorporated the inverse mill's ratio for controlling the possible self-selection bias due to occupation choice (daily wage-based workers in the farm, or nonfarm sectors. the multinomial logistic regression estimation procedure was employed to characterize the daily wage-based workers in the farm and nonfarm sectors (i), setting the non-daily wage workers as the base (= 0) and assigning a value of 1 for daily wagebased farm workers (n f ), and a value of 2 for daily wage nonfarm workers (n nf ) as follows: pðdaily; farmþ pðnon dailyþ pðdaily; nonfarmþ pðnon dailyþ where hlc is , ilc is , and rlc is are the same as in eq (2), and homestead is is the size of the homestead land owned (acres). some basic background information of the sampled respondents is presented in table 2 . the sampled respondents are divided into three groups based on the wage payment methods: daily wage workers in farm and nonfarm sectors and other workers. out of 50,671 sampled respondents, 34,301 (67.7%) were paid other than daily basis mode, 7,552 (14.9%) worked in the farm sector and were paid daily, and 8,818 (17.4%) worked in the nonfarm sector and were paid daily. on average, a sampled respondent was more than 36 years old, with nearly five years of schooling, more than 80% were married, and more than 67% were from rural areas ( table 2 ). in addition, a sampled household comprised of 4.4 family members, with 1.6 earners ( table 2) . female workers were less likely to be paid on the daily basis. in general, the daily wage workers were mostly engaged in the open field related physical works. in bangladesh, it is not socially acceptable that females and males perform physical work together in an open field. a closer scrutiny of table 2 reveals that, on average, the daily wage-based workers in the farm sector are statistically significantly older (40.1 years) with significantly fewer years of schooling (2.2 years) and was more likely to be from rural areas compared to others. in addition, the number of family members and earners were lower for households of daily wage workers in both farm and nonfarm sectors compared to the other group. thus, daily wagebased workers in general, are relatively more resource poor compared to other workers. more than one-fifth of the sampled respondents were from dhaka division, followed by chattogram (18.3%) and khulna divisions (15.2%) ( table 2) . dhaka and chattogram are the largest garment industry clusters in bangladesh [58] , where more than 4.4 million workers are employed, the majority of which are female [47] . thus, as there are more nonfarm work opportunities in dhaka and chattogram than other divisions of bangladesh, the largest share of sampled respondents was drawn from these two divisions. nearly 20% of the daily wage-based farmworkers were from rangpur division ( table 2) . of the total population of bangladesh, 23.4% live below the national poverty line [57] . rangpur division has the highest incidence of income poverty (30.5%) [57] , which is reflected in the concentration of daily waged-based farmworkers. as per table 3 , more than 13% of respondents did not have their own homestead land, and more than 11% were landless. on average, the daily wage earning in the farm sector was bdt 314, and bdt 421 in the nonfarm sector ( table 3 ). the yearly average income from land rent, rent of other properties, insurance, profits and dividends, lottery and prize money, charity, gifts, royalties and other assistance both in cash and kind, remittances, gratuities and retirement benefits, interest received and other income in cash or kind, was bdt 11,427. however, this income was significantly lower for daily wage workers in the farm sector (bdt 4,661.6) and the nonfarm sector (bdt 5,343.5). more than 5% of respondents reported inclusion in the social safety net programs, with the yearly average receipt from them being nearly bdt 10,414. it shows that daily wage workers were more likely to be included in a social safety net program than others ( table 3 ). the average daily household level total food expenditure was bdt 213.5, of which the expenditure only on cereals accounted for 30% (bdt 64.7). the daily per household level consumption of cereals was 1.8kg (table 3) . this indicates the importance of cereals as a cheap source of dietary energy for the poor in developing countries. importantly, as expected, the daily household level total food expenditure was significantly lower for daily wage farmworkers, which confirms that the daily wage-based workers are more distressed than others. tables 4-5 present estimated functions specified in eqs (1) and (3). table 4 presents the estimated functions explaining the (ln) daily earning per earner for farm and nonfarm workers. the estimates were obtained by means of the generalized linear model (glm) estimator. there is a positive relationship between years of schooling and the daily wage earnings in the nonfarm sector, however, such relation is insignificant in the case of the farm sector (table 4) . on average, the female daily wage workers were paid less than their male counterparts in both farm and nonfarm sectors ( table 4 ). the coefficients on the district dummies can be seen in s1 table. the estimated daily wage earnings for farm workers is bdt 272.2, and bdt 361.5 for nonfarm workers (table 4 ). in 2016-17, there were 24.7 million workers in the farm sector and 36.1 million in the nonfarm sector (table 1) . there is no straightforward information on the share of workers who were paid on daily basis. in 2016-17, however, 44.4% workers in the rural area, and 18.5% workers in the urban area were paid on daily basis [53] . as most of the workers in the rural area of bangladesh are mainly engaged in farming, we assume that out of 24.7 million workers in the farm sector, 44.7% of them, or 10.9 million were paid on daily basis. similarly, as in the urban area, most of the workers are mostly engaged in the nonfarm sector, we assume that out of 36.1 million workers in the nonfarm sector, 18.5% of them, or 6.7 million, were paid on daily basis. following eq (2), multiplying the estimated daily wage earnings for farmworkers as bdt 272.2/day and for the nonfarm worker as bdt 361.5/day, under the assumption of a complete lockdown with no-one allowed to work, the economic loss in one day is estimated at bdt 5389.03 million or approximately us$ 64.2 million. assuming 50% of the daily wage workers are not allowed to work and the rest are, the economic loss/day will be bdt 2694.5 million or us$ 32.1 million. table 5 presents the estimated functions applying the ordinary least square (ols) estimation approach, explaining the (ln) daily household level total food expenditure of the sampled farm and nonfarm daily wage-based households. we have presented the results of three different models. in model (1), we did not include the division or district dummies, whereas in model (2) we included seven division dummies for eight divisions; and in model (3) we included 63 district dummies for 64 districts. in the estimated functions, we have included the natural log (ln) of the estimated daily total wage earnings of a sampled household in explaining the daily household level total food expenditure of the sampled daily wage-based farm and nonfarm households ( table 5 ). the estimated (ln) daily household level total wage earnings are highly statistically significant and positive in explaining the (ln) daily household level total expenditure on food (table 5) . it shows that a 1% increase in total household level daily wage earnings leads to an increase in the daily districts effects are controlled by including 63 district dummies for 64 districts (s1 table) inverse household level food expenditure by 0.2% (model 2) at the minimum to 0.34% at the maximum for the daily wage-based farm households in the farm sector, and a 1% increase in wage earnings leads to an increase in the daily per capita food expenditure by 0.29% (model 6) to table) districts effects are controlled by including 63 district dummies for 64 districts (s2 table) 0.41% (model 4) at the maximum for the daily wage-based nonfarm households ( table 5 ). the income from other sources, years of schooling and age also positively and significantly affect the daily per capita food expenditure of the sampled farm and nonfarm workers. this shows that, on average, both farm and nonfarm daily wage-based households in khulna, mymensingh, rangpur and rajshahi divisions spend statistically significantly less on food per capita compared to the households in barishal division, which is the base division. the coefficients on the district dummies can be seen in s2 table. in table 5 , in the case of the sampled daily wage-based household in the farm sector, the estimated coefficient of the daily total wage earnings in the household level daily total food expenditure ðg 2f þ are 0.34, 0.20 and 0.23, respectively (models 1-3) . the estimated daily household level food expenditures ðedfx f þ are bdt149.5, bdt150.6 and bdt152.5, respectively (models 1-3) . similarly, the estimated share of the daily total wage earnings in the household level daily total food expenditure ðg 2f þ are 0.41, 0.30 and 0.29, respectively (models 4-6), and the estimated daily household level food expenditures ðedfx nf þ are bdt167.2, bdt168.8, and bdt170.8 respectively (models 4-6). assuming 10%, 20% and 30% of the daily earnings of the daily wage-based households in the farm and nonfarm sectors (θ = 0.1, 0.2 and 0.3) compared to the normal time daily wage earnings, the estimated daily minimum supports are presented in table 6 . in estimating the daily minimum support, we have set γ 2f = 0.34 and edfx f ¼ bdt 152.5 for the daily wage-based farm household, and γ 2nf = 0.41 and edfx nf ¼ bdt 178.5, for the daily wage-based nonfarm households. our estimation shows that the estimated daily minimum support is ranged from bdt 51.2-83 depending on the share of the daily wage earnings (θ) for the daily wage-based farm households, and it ranged between bdt63-104 in the case of the daily wage-based household in the nonfarm sector under the assumptions of 70%, 80% and 90% income loss (θ = 0.1, 0.2 and 0.3) due to covid-19 induced lockdown time ( table 6 ). the estimation suggests a common minimum support at us $ 1 per daily wage-based household in bangladesh to ensure minimum food security during covid-19 induced lockdown time. however, our estimation process is flexible which contingent upon the share of income loss due to the severity of the lockdown. this flexibility in estimating the daily minimum support will allow policymakers to set the daily minimum support based on the severity of a lockdown situation in a specific region. table 7 presents the estimated functions that characterize the sampled daily wage workers (eq 9), applying the multinomial logit estimation procedure setting the workers who receive their remuneration, not on a daily basis as the base (= 0). compared to the base workers, respondents with higher income, with more family members and more years of school and female respondents were less likely to be daily wage-based workers (table 7 ). in contrast, married and rural respondents; and relatively older respondents, were more likely to be daily table 6 . calculation of minimum daily support using the estimated daily household level food expenditure and the estimated coefficient ðg 2 þ reported in table 5 wage-based workers both in farm and nonfarm sectors. female respondents were in general less likely to be daily wage-based workers, and specifically, female respondents in age groups 15-24, 25-34, and 35-44 years old were less likely to work as daily wage-based workers in both farm and nonfarm sectors. in characterizing the daily wage-based workers, the district level effects are also included by including 63 dummies for 64 districts setting bagerhat district as the base (= 0). the coefficients of the district dummies can be seen in s3 table. after estimating the functions that characterize the sampled daily wage-based workers reported in table 7 , the generalized inverse mill's ratios are calculated separately for daily wage-based workers in farm and nonfarm sectors following vella's [59] procedure, and plugged in into eq (1) in estimating daily wage earnings (table 4 ). it is noted that the methodology of this study is simple and relatively easily replicable for other countries. for example, in india, 34.5% of employed workers are casual labor, of which 21% are engaged in agriculture [60] . the daily average wage earnings per day by a casual labor ranged between india rupee (rs.) 253-282 [60]. a recent report stressed that in india, in april 2020, 12 million people became jobless, and the unemployment rate had increased from 8.8% in march 2020 to 23.5% in april 2020, due to covid-19 induced turmoil [61] . by examining the share of expenditure of the daily income on food, it is possible to suggest a minimum support package to ensure the food security of casual labor-based households in india during the covid-19 induced lockdown period. using information of more than 50,000 respondents from the hies 2016-17 dataset, this study, firstly quantified the economic loss due to the covid-19 induced lockdown and suggested the minimum support package to ensure food security of the daily wage-based workers in bangladesh. nearly half of the world's population is now under some form of restrictions imposed by national governments to curb the spread of covid-19. while this lockdown may restrict the spread of covid-19, in the absence of effective support, it may also generate severe food and nutrition insecurity for daily wage-based workers, particularly in the developing countries [10, 27] . the ilo warned that due to covid-19 induced movement restrictions and lockdown, the participation of the global working force in the first quarter of 2020 has declined by 4.5% which is equivalent to 130 million full-time jobs [8] . in bangladesh, out of a 60.8 million employed labor force, 24.7 million (40.6%) are directly engaged in the farm sector, of which 44.4% are paid on daily basis, and 36.1 million are engaged in the nonfarm sector, of which 18.4% are paid on daily basis. this study stressed that, compared to others, the daily wage-based workers in both farm and nonfarm sectors are comparatively more resource poor in terms of land ownership and education. based on the estimated daily wage earnings, this study found that under the assumption of complete lockdown, the daily economic loss due to prohibiting daily wage-based workers to work would be us$ 64.2 million. this study also demonstrated that the average wage elasticity in explaining the daily food expenditure at the household level ranged from 0.20 to 0.34, and the daily household food expenditure ranged from bdt 149.5-152.5 for the daily wage-based farm households. for the daily wage-based nonfarm households, the average wage elasticity in explaining the daily food expenditure at the household level ranged from 0.29 to 0.41, and the daily household food expenditure ranged from bdt 167-171. assuming 10%, 20% and 30% of the daily earnings of the daily wage-based households in the farm and nonfarm sectors (θ = 0.1, 0.2 and 0.3) compared to the normal time income, we have estimated that on average it is necessary to provide daily bdt 51-104 or around us $ 1 per daily wage-based households during the covid-19 induced lockdown time. it is important to mention here is that, the suggested minimum support us$ 1/day/household is calculated based on considering only food expenditure. the suggested minimum support package is thus only suitable for the short-term. in the case of a long-term lockdown situation, it is necessary to include the costs of health, education, clothing and housing. recently, the government of bangladesh announced the provision of approximately us$ 24/month to two million to households [62] . in addition, 20kg cereals and food will be provided to 1 million households in bangladesh [63] . while the amount of support is in line with our findings, in the case of a lengthy lockdown period, it is necessary to consider other household costs, such as clothing, medicine and education in the support package. moreover, as the labor market of bangladesh is comprised of 60.8 million active workers, the coverage of the safety net should be expanded in the case of a lengthier lockdown. as food and nutrition insecurity can have long-run impacts on human capital formation, the government must expand the emergency safety network program to include almost all marginal households or consider loosening restrictions in the agriculture sector. without effective support programs, an implementation of a strict lockdown for a long time may be very difficult, if the poor households are forced to come out to search for works, money and food. in such case, to support smallholder agriculture, wage workers and agricultural value chains, the government should consider issuing movement passes to persons and carriers of agricultural input and output in the case of a very strict lockdown scenario. finally, it is also imperative to take necessary steps against government failure in the form of leakage in distributing compensation packages. otherwise, the benefits of government effort may not reach to the vulnerable groups. supporting information s1 table. district dummies included in explaining daily wage earnings (reported in table 4 ) of the daily wage workers in the farm and nonfarm sectors (bagerhat district is the base = 0). (docx) s2 table. district dummies included in explaining the daily household level total food expenditure (reported in table 5 ). base district: bagerhat = 0. (docx) table 7 ). base district: bagerhat. covid-19) pandemic: public advice. in: world health organization (who) timeline: how coronavirus got started: the outbreak spanning the globe began in december covid-19) events as they happen early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study temporal dynamics in viral shedding and transmissibility of covid-19 the world in lockdown in maps and charts how the world shut down third edition updated estimates and analysis. geneva the great lockdown. int monet fund global report on food crises: joint analysis for better decisions. rome; 2020 coronavirus measures could cause global food shortage, un warns. the guardian covid-19 and food security in vulnerable countries food security and covid-19 ensuring food security during the covid-19 pandemic covid-19: channels of transmission to food and agriculture. rome how covid-19 may disrupt food supply chains in developing countries addressing covid-19 impacts on agriculture, food security, and livelihoods in india. washington d.c.: international food policy research institute (ifpri); 2020 impacts of covid-19 on the agri-food sector: food security policies of asian productivity organization members the short history of global living conditions and why it matters that we know it global hunger continues to rise, un report says. in: world health organization (who) the state of food security and nutrition in the world 2017: building resilience for peace and food security. rome: food and agriculture organization of the united nations poverty and shared prosperity 2018: piecing together the poverty and puzzle. washington d.c.: world bank sustainable development goals 2: zero hunger the impact of covid-19 on developing asian economies: the role of outbreak severity, containment stringency, and mobility declines a crisis like no other, an uncertain recovery. int monet fund covid-19): employment situation in latin america and the caribbean covid-19 will double number of people facing food crises unless swift action is taken preventing global food security crisis under covid-19 emergency. washington d.c.: international food policy research institute (ifpri); 2020 do market shocks generate gender-differentiated impacts? policy implications from a quasi-natural experiment in bangladesh are female-headed households more food insecure? evidence from bangladesh did the commodity price spike increase rural poverty? evidence from a long-run panel in long-run panel in bangladesh tracking government responses affecting global food markets during the covid-19 crisis world economic situation and prospects. covid-19 disrupting lives estimates of the impact of covid-19 on global poverty safeguarding against economic slowdowns and downturns. rome: food and agriculture organization of the united nations (fao) covid-19: bangladesh multi-sectoral anticipatory impact and needs analysis. dhaka; 2020 the daily prothom alo. the holiday has increased to covid-19 in bangladesh: 55 districts under partial or complete lockdown. covid-19 in bangladesh world development indicators conversion of agricultural land to non-agricultural uses in bangladesh: extent and determinants data: production. in: online database on crop production,yield and harvested area online database on food balance sheets strengthening food systems and the environment through innovation and investment, the economist and intelligence unit the challenge of hunger global hunger index: facts, determinants, and trends. food policy bangladesh garment industry rebounds un comtrade database the world economy at risk. oecd interim econ assess united nations dev program china impact of covid-19 on bangladesh rmg industry dhaka: ministry of health and family welfare, government of bangladesh the political economy of the stimulus package for covid-19 induced economic crisis in bangladesh opinion on fighting on covid-19 induced increasing poverty. the daily ittefaq bbs. preliminary report on households income and expenditure survey an inquiry into the rapid growth of the garment industry in bangladesh estimating models with sample selection bias: a survey job losses may have narrowed the daily prothom alo. governmet will provide monthly bdt 2000 to 2000000 hosueholds of bangladesh. stuff report 00000 households will get 20kg food support per month. the daily prothom alo key: cord-352511-gkm7i62s authors: yamada, yoshiyuki; liu, xiao bo; fang, shou guo; tay, felicia p. l.; liu, ding xiang title: acquisition of cell–cell fusion activity by amino acid substitutions in spike protein determines the infectivity of a coronavirus in cultured cells date: 2009-07-02 journal: plos one doi: 10.1371/journal.pone.0006130 sha: doc_id: 352511 cord_uid: gkm7i62s coronavirus host and cell specificities are determined by specific interactions between the viral spike (s) protein and host cell receptor(s). avian coronavirus infectious bronchitis (ibv) has been adapted to embryonated chicken eggs, primary chicken kidney (ck) cells, monkey kidney cell line vero, and other human and animal cells. here we report that acquisition of the cell–cell fusion activity by amino acid mutations in the s protein determines the infectivity of ibv in cultured cells. expression of s protein derived from veroand ck-adapted strains showed efficient induction of membrane fusion. however, expression of s protein cloned from the third passage of ibv in chicken embryo (ep3) did not show apparent syncytia formation. by construction of chimeric s constructs and site-directed mutagenesis, a point mutation (l857-f) at amino acid position 857 in the heptad repeat 1 region of s protein was shown to be responsible for its acquisition of the cell–cell fusion activity. furthermore, a g405-d point mutation in the s1 domain, which was acquired during further propagation of vero-adapted ibv in vero cells, could enhance the cell–cell fusion activity of the protein. re-introduction of l857 back to the s gene of vero-adapted ibv allowed recovery of variants that contain the introduced l857. however, compensatory mutations in s1 and some distant regions of s2 were required for restoration of the cell–cell fusion activity of s protein carrying l857 and for the infectivity of the recovered variants in cultured cells. this study demonstrates that acquisition of the cell–cell fusion activity in s protein determines the selection and/or adaptation of a coronavirus from chicken embryo to cultured cells of human and animal origins. interspecies adaptation, replication and transmission in cells are essential steps for an animal virus to emerge successfully in a human population. virus-cell and cell-cell membrane fusion, mediated by fusion proteins associated with viral envelope, is crucial for the entry of enveloped viruses into cells and for rapid spread of infection to the neighboring cells. this membrane fusion process may, therefore, be a limiting point for efficient adaptation and infection of an animal virus in cells from a different host species. in this study, we report that acquisition of the cell-cell fusion activity by point mutations in the spike (s) protein of avian coronavirus infectious bronchitis virus (ibv) plays a critical role in adaptation and/or selection of a variant that infects cultured cells. coronavirus is a large family of enveloped, positive-stranded rna viruses that cause respiratory and intestinal infections in avian and mammalian species [1] . ibv, the prototype member of coronavirus, causes highly contagious diseases in chicken and is a constant threat to the poultry industry. coronavirus was traditionally considered to have narrow host specificities [2] . however, the outbreaks of severe acute respiratory syndrome (sars), a serious zoonotic transmission event caused by a novel coronavirus, demonstrate that a certain coronvirus species may exhibit wider host specificities and suggests the possibility of crossspecies transmission of animal coronaviruses to human [3, 4] . cross-species transmission was also observed in coronavirus transmissible gastroenteritis virus (tgev) and human coronavirus oc43 [5] [6] [7] . these events highlight the importance of understanding the mechanisms of interspecies adaptation and transmission of coronavirus. the beaudette strain of ibv was previously adapted to embryonated chicken eggs. this embryo-adapted ibv strain was subsequently adapted to cultured cells originated from chicken and monkey. for example, the virus was adapted by serial passages to primary chicken kidney (ck) cells [8, 9] and the african green monkey kidney cell line vero cell [8] [9] [10] [11] [12] [13] . furthermore, the veroadapted ibv is able to infect cultured human and animal cell lines [14, 15] . in a previous report, a total of 49 amino acid substitutions was found during adaptation of ibv from chicken embryo (ep3) to vero cells (p65) [10] [11] [12] . among them, 26 amino acid substitutions are in the s protein [10] . in this study, expression of s protein cloned from ibv strains ep3, ck, passage 7 (p7) and p65 of vero-adapted ibv showed induction of cell-cell fusion by s(ck), s(p7) and s(p65) constructs. however, no formation of syncytial cells was observed in cells expressing s(ep3). construction of chimeric s constructs and site-directed mutagenesis studies identified a leucine to phenylalanine substitution at the amino acid position 857 in the heptad repeat 1 region (l857-f) that confers the non-fusogenic s protein to fusogenic. re-introduction of the f857-l mutation back to the genome of vero-adapted ibv (p65) showed rescue of virus containing the f857-l mutation. however, compensatory mutations occurred in the s1 region that could rescue the cell-cell fusion activity of s constructs carrying the f857-l mutation. cells were maintained in dmem supplemented with 10% newborn calf serum. the vero-adapted ibv and recombinant vaccinia/t7 virus was propagated and titrated on vero cells. virus stocks were kept at 280uc until use. cell monolayers grown on 4 well slide chambers were infected with vaccinia/t7 virus for 1 hour followed by transfection of indicated plasmids using the effectene transfection kit (qiagen). at 12 hours post-transfection, cells were washed with phosphate buffered saline (pbs) supplemented with 10% normal goat serum, fixed with 4% paraformaldehyde in pbs for 15 minutes, and permeabilized with 0.2% triton x-100. immunofluorescent staining was performed by incubation of cells with rabbit anti-ibv s polyclonal antibodies and subsequently with fitcconjugated anti-rabbit igg. cells were examined by fluorescent microscopy and digital images were collected. protein samples were prepared from cells harvested at 12 hours post-transfection, separated by sds-page and transferred to pvdf membranes. the membranes were incubated with rabbit anti-ibv s polyclonal antibodies or mouse anti-b-tubulin monoclonal antibody (sigma aldrich), and subsequently with hrp-conjugated anti-rabbit or -mouse igg (dako). polypeptides were detected using the enhanced chemiluminescence (ecl) detection reagents (amersham). vero cells were transfected as described above, and harvested at 12 hours post transfection. cells were washed once with pbs, resuspended in blocking buffer containing 20% fbs and 1% bsa in pbs, and incubated on ice for 30 minutes. subsequently, cells were incubated with 0.1% saponin in facs washing buffer containing 2.5% fbs and 0.05% sodium azide in pbs for 10 minutes at room temperature when required. immunofluorescent staining was carried out with 1:100 diluted rabbit anti-ibv s polyclonal antibodies, and 1:20 diluted fitc-conjugated swine anti-rabbit antibody (dako). after washing two times with the facs washing buffer, cells were fixed with 1% ice cold paraformaldehyde and analyzed by flow cytometry. viral rna was extracted from the culture supernatants or infected cells using the rneasy mini kit (qiagen) according to the manufacturer's instructions. rt-pcr was performed using the expand reverse transcription and high fidelity pcr kits (roche). the pcr products were cloned into pcrh-xl-topoh vector (invitrogen) and sequenced by automated sequencing. construction of the full-length ibv cdna clones from p65 of vero-adapted ibv was previously reported [16, 17] . the f857-l point mutation was introduced into the corresponding fragment using quickchange site-directed mutagenesis kit (stratagene), and subsequently ligated into the full-length cdna clone. the full-length transcripts were generated in vitro using the mmessage mmachine t7 kit (ambion) according to the manufacturer's instructions with certain modifications. briefly, 30 ml of transcription reaction with a 1:1 ratio of gtp to cap analog were sequentially incubated at 40.5uc for 25 minutes, 37.5uc for 50 minutes; 40.5uc for 25 minutes, and 37.5uc for 20 minutes. the transcripts were extracted with phenol/chloroform. vero cells were grown to 90% confluence, trypsinized, washed twice with cold pbs, and resuspended in pbs. rna transcripts were added to 400 ml of vero cell suspension in an electroporation cuvette, and one electrical pulse at 450 v, 50 mf was given using a bio-rad gene pulser ii electroporator. the transfected vero cells were cultured overnight in 1% fbs-containing mem in a 60 mm dish or a six-well plate and further cultured in mem without fbs. the s genes from different passages of the vero-adapted ibv strain and ck-adapted ibv were amplified and cloned into pkt0 vector [18] . chimeric s constructs were made by overlapping pcr [19] . point mutations were introduced by site-directed mutagenesis using the quikchange tm kit (stratagene). all constructs were confirmed by automated nucleotide sequencing. cell-cell fusion activity of s proteins cloned from ep3 and ck-adapted beaudette strain of ibv acquisition of the cell-cell fusion activity is essential for selection and adaptation of coronavirus ibv from chicken embryo to cultured cells [10] . sequence comparison of two s protein constructs, s(ep3) and s(ck), cloned from ep3 and ck-adapted ibv strains, respectively, showed amino acid substitutions at 31 positions (fig. 1a) . the cell-cell fusion activity of these two s constructs was analyzed by transfection into vero cells using the vaccinia/t7 recombinant virus system. western blot analysis showed the presence of major forms of s protein, including the 180-kda glycosylated (s*) and 130-kda unglycosylated full-length s (s) and the cleaved s1 and s2 species (s1/s2) in cells expressing the two constructs (fig. 2a, lanes 2 and 3) . it was noted that the expression level of s(ck) was higher than s(ep3) (fig. 2a) . as a negative control, cells transfected with ibv n protein were included, and the expressed n protein was detected by western blot with anti-n antibodies (fig. 2a, lane 1) . immunofluorescent staining of vero cells expressing s(ck) clearly showed syncytia formation at 12 hours post-transfection (fig. 2b , panel s(ck)). however, in vero cells expressing s(ep3), no obvious syncytia was observed (fig. 2b , panel s(ep3)). in the negative control cells, no fusion of the transfected cells was detected (fig. 2b, panel n) . to investigate the possibility that intrinsic differences in cell surface translocation of the two s constructs may affect their cellcell fusion activity, cell surface expression of the two proteins was analysed by flow cytometry after immunofluorescent staining with anti-s antiserum. as shown in fig. 2c acquisition of the cell-cell fusion activity by mutation of a conserved leucine residue to phenylalanine (l857-f) in the heptad repeat 1 region of s protein to map the amino acid mutation(s) responsible for acquisition of the cell-cell fusion activity of s(ck), three chimeric constructs were first made. construct ep3-ck(1) was made by replacing the c-terminal 412 amino acid region of s(ep3) with the corresponding region from s(ck), ep3-ck(2) was made by replacing the cterminal 280 amino acid region of s(ep3) with the corresponding region from s(ck), and ck-ep3 was made by replacing the nterminal 882 amino acid region of s(ep3) with the corresponding region from s(ck) (fig. 3a) . western blot analysis of cells expressing these constructs detected the s1 and s2 species as well as the glycosylated and unglycosylated forms of the full-length s protein (fig. 3b , lanes [3] [4] [5] . immunofluorescent staining showed cell-cell fusion and syncytia formation in cells expressing both ep3-ck(1) and ck-ep3 (fig. 3c , panels ep3-ck(1) and ck-ep3), but not ep3-ck(2) (fig. 3c , panel ep3-ck (2)). the relative cell-cell fusion activities of these s constructs were semiquantitatively defined by comparing the average size of syncytia induced by different s constructs with the average size (considered as 1) of cells expressing s(ep3), and are listed in the order from high to low as follows: ck-ep3.ck = ep3-ck(1)&ep3 = ep3-ck(2) (.indicates the relative activity is within 1 fold higher, and &indicates more than 1 fold higher). these results demonstrate that the region between amino acids 750 and 882 may determine the fusogenic difference between s(ep3) and s(ck). examination of this region showed two amino acid substitutions from s(ep3) to s(ck), i.e. n826 to s and l857 to f (fig. 1b) . to determine which amino acid substitution dictates the fusogenic change, three mutant constructs were made. constructs ck3(s826-n) and ck(f857-l) were made by mutation of the s826 and f857 residues in s(ck) to n and l, respectively (fig. 3a) . construct ep3(l857-f) was made by mutation of the l857 residue in s(ep3) to f (fig. 3a) . western blot analysis of cells expressing these constructs detected the s1 and s2 species as well as the fulllength forms (fig. 3b , lanes 6-8). immunofluorescent staining showed formation of syncytia in cells expressing ck(s826-n) (fig. 3c , panel ck(s826-n), suggesting that mutation of s836 to n did not affect the cell-cell activity of s(ck). cell-cell fusion and syncytia formation were also observed in cells expressing ep3(l857-f) but not ck(f857-l) (fig. 3c , panels ep3(l857-f) and ck(f857-l)), demonstrating that the l857-f mutation introduced into s(ep3) renders the protein fusogenic in cultured cells. on the other hand, mutation of the f857 residue to l totally abolishes the fusion activity of s(ck) (fig. 3c , panel ck(f867-l). the relative cell-cell fusion activities of these s constructs are ck(s826-n).ep3(l857-f)&ep3 = ck(f857-l). these results confirm that s(ck) gains the cell-cell fusion activity by l857-f mutation in the heptad repeat 1 region of the protein. the f857-l substitution was then introduced into the s constructs cloned from vero-adapted ibv (p7) and (p65), respectively, generating s(p7) and s(p65) (fig. 3a) . expression of these constructs showed cell-cell fusion and syncytia formation in cell expressing wild type s(p7) and s(p65), but not the mutant s proteins (fig. 3c , panel p7, p7(f857-l), p65 and p65(f857-l)). the expression levels of both f857-l mutants were lower than the wild type constructs, but no significant difference in the s1/s2 cleavage was observed (fig. 3b, lane 9-12 ). the relative cell-cell fusion activities of these s constructs are p65.p7&ep3 = p7(f857-l) = p65(f857-l). these data indicate that s protein acquires its cell-cell fusion activity by the l857-f mutation during adaptation to both ck and vero cells. further mutations of the l857 residue to other amino acids based on s(ep3) were made. as shown in fig. 3a , the l857 was mutated to y, s, e, i and k, respectively. expression of these mutant constructs showed that mutations of l857 to y and s exhibited similar effect on cell-cell fusion as the l857-f mutant. cell-cell fusion and syncytia formation were observed in cells expressing these two mutants (fig. 3c) . however, much less cell-cell fusion and smallersized syncytia were observed in cells expressing l857-e, l857-k and l857-i mutant constructs (fig. 3c) . the relative cell-cell fusion activities of these s constructs are ep3(l857-y).ep3(l857-s).ep3(l857-e).ep3(l857-i) = ep3(l857-k).ep3. introduction of the f857-l substitution back to the genome of vero-adapted ibv and analysis of its effect on viral infectivity in cultured cells the f857-l mutation was then introduced back to the genome of vero-adapted ibv by using an infectious clone system based on p65 [16, 17] to test its influence on viral recovery and infectivity. in vitro synthesized full-length transcripts derived from wild type (ribv) and mutant (fl) clones were introduced into vero cells by electroporation. at 3 days post-electroporation, syncytia formation the recombinant wild type and mutant viruses (p0) were recovered from the culture media at 3 and 6 days post-electroporation, respectively, and further propagated on vero cells for 5 passages. total rna was extracted from the culture media of cells infected with each passage of the mutant virus and rt-pcr was carried out to amplify the s gene. the rt-pcr products were cloned, 10 bacterial clones were randomly chosen from p0, and the complete nucleotide sequence of the s gene was determined to confirm if the recovered virus maintains the f857-l substitution. as shown in table 1, l857 was found in all 10 clones. however, only five clones had an identical sequence with the original mutant s gene (type fl), and additional mutations at other positions were found in the other five clones (table 1) . among them, two clones contain a t773-s substitution (flv1), one contains an i769-v substitution (flv2), and two contain q523-l and i769-v substitutions (flv3) ( table 1 ). these results demonstrate that the recovered fl mutant virus from p0 contains a mixed population of quasispecies. to investigate which quasispecies would become dominant in the subsequent passages, sequencing analysis of bacterial clones containing the pcr fragments from p1, p3 and p5 was performed. in the four clones chosen from p1, a homogenous s gene with both q523-l and i769-v (flv3) mutations was found (table 1) . subsequent sequencing of clones derived from p3 and p5 each showed that six out of 10 clones from p3 and two out of 10 clones from p5 are flv3 (table 1 ). the dominant clones contain an additional proline to serine substitution at amino acid position 327 (flv4) ( table 1) . the recovered viruses were then plaque-purified. compared to wild type ibv, ribv showed similar growth kinetics in vero cells (fig. 4b) , but formed slighlty smaller plaques (fig. 4a) with lower expression level of s protein (fig. 4c) . a total of 20 mutant viruses was plaque-purified from passages 3 and 5, and the s gene of all purified viruses was shown to share the same sequence as flv4 ( table 1 ). the flv4 mutant virus formed similar-sized plaques as ribv (fig. 4a) with slightly lower expression of s protein (fig. 4c) . interestingly, the mutant virus produced up to 10-fold higher titers of virus, compared to ribv (fig. 4b) . restoration of the cell-cell fusion activity of s(p65) protein carrying the f875-l mutation by compensatory mutations in the s1 region the cell-cell fusion activity of s proteins cloned from the mutant ibv construct fl and the four variants (flv1, flv2, flv3 and flv4) was analyzed by expression in vero cells. once again, expression of these constructs led to the detection of s1 and s2 species as well as the full-length forms (fig. 5a) . higher levels of s protein were detected in cells expressing s(flv3) and s(flv4), comparing to cells expressing the other two s constructs (fig. 5a) . immunofluorescent staining showed the formation of giant syncytia in cells expressing s(flv3) and s(flv4) (fig. 5b , panels s(flv3) and s(flv4)), but much smaller syncytia were observed in cells expressing s(flv2) (fig. 5b, panel s(flv2) ). no obvious cell-cell fusion was observed in cells expressing s(fl) and s(flv1) (fig. 5b , panels s(fl) and s(flv1)). the relative cell-cell fusion activities of these s constructs are flv4.flv3.flv2&ep3 = fl = flv1. these results confirm that acquisition of the cell-cell fusion activity is an important step for adaptation of ibv in cultured cells. since amino acid difference between s(flv2) and s(flv3) was only at the 523 th residue, the s(fl(q523-l)) construct was also created and expressed. the results showed that it displayed a similar cell-cell fusion activity as flv2 ( = fl(i769-v)) ( fig. 5a further enhancement of the cell-cell fusion activity of s protein and adaptation of ibv to cell culture by g405-d substitution vero-adapted ibv gradually increased its infectivity in vero cells by serial passages and a significant difference between p7 and p65 was observed [10] . comparison of amino acid sequences between s(p7) and s(p65) revealed a single mutation at the amino acid position 405 (g405-d) in s(p65) (fig. 1a) . to analyze the possibility that the enhanced infectivity of p65 virus is due to the enhanced cell-cell fusion activity of the corresponding s protein, s(p7) and s(p65) constructs were created and expressed in vero cells. efficient induction of cell-cell fusion was observed in cells transfected with both constructs (fig. 6a, panels s(p7) and s(p65) and 6b, lanes 3 and 4) . comparatively, significantly larger syncytia was observed in cells expressing s(p65) construct than in cells expressing s(p7) (fig. 6a) , demonstrating that the additional g405-d mutation in s(p65) may enhance its cell-cell fusion activity. the g405-d mutation was then introduced into s(ep3) and s(ck) and expressed (fig. 6a) , showing that introduction of g405-d mutation into s(ck) drastically enhanced its cell-cell fusion activity (fig. 6b) . interestingly, introduction of the mutation into s(ep3) and expression of the construct in vero cells showed formation of small syncytial cells (fig. 6b) . the relative cell-cell fusion activities of these s constructs are ck(g405-d).p65.ck&p7.ep3(g405-d).ep3. avian coronaviruses have been isolated from chicken, turkey and pheasant and may exist in many other avian species [20] . ibv is usually associated with respiratory disease in domestic fowl, and was believed to have a limited host range. chicken is the only natural host. similarly, coronaviruses originated from human and other animal species were considered to have narrow host specificities until the identification of sars-cov as the causative agent of sars outbreaks in 2003. the current model of animal origin of sars-cov highlights the importance of cross-species adaptation and transmission of animal coronaviruses to human. cross-species transmission of virus infection has long been recognized as a way for the emergence of many zoonotic diseases. the molecular basis for this phenomenon would lie on the rapid adaptation of certain viruses to a changing environment through selection of minor variants from quasispecies, accumulation of mutations, recombination between minor variants, and reassortment of their genomes. a better understanding of the underlying mechanisms that control these events would be essential for providing safeguards to limit the impact of these devastating diseases. in this study, we show that acquisition and enhancement of the cell-cell fusion activity by amino acid substitutions in the s protein are critical for interspecies adaptation and infectivity of ibv to cultured cells. data present clearly show that the l857-f mutation in the heptad repeat 1 region of s proteins derived from cell-culture-adapted ibv is important for adaptation of the virus to cell culture systems, and an additional mutation in the s1 region (g405-d) could enhance this process. as s protein carrying the l857-f mutation is able to induce cell-cell fusion, but losses the activity when f857 was mutated back to l, it suggests that induction of cell-cell fusion is an essential step in adaptation/ selection of ibv to cultured mammalian cells. coronavirus s protein is the major determinant for viral entry and host specificity. it is a class i fusion protein and mediates viral entry by specific binding of the s1 domain to a host cell receptor [21] [22] [23] [24] [25] . the cellular receptors for several coronaviruses have been identified, including members of the cacinoembryonic antigen family of cell adhesion molecules as the receptor for mhv, angiotensin converting enzyme ii for sars-cov and human coronavirus nl63, and aminopeptidase n for human coronavirus 229e and tgev [26] [27] [28] [29] [30] . to date, the receptor(s) for ibv has not been identified in its native or adapted host cells. it is assumed that a mammalian counterpart on vero cells could be used as a receptor for ibv at low affinity, and might have structural and functional similarities to the native receptor on chicken cells. at the initial stages of the adaptation process, a certain proportion of ep3 would weakly bind to this molecule and gains entry into the cells by endocytosis. in addition, binding of ibv to sialic acid was reported to be important for adaptation of the virus to human cells [31, 32] . the beaudette strain of ibv was also reported to have an additional binding activity to heparin-like structures [33] . these additional binding activities may help to initiate infection and thus allow the virus to adapt to the new host receptor by mutation. to uncoat the engulfed virion and to establish subsequent infection cycles as well as to spread infection to neighboring cells, acquisition of virus-cell/cell-cell membrane fusion and enhancement of the cell-cell fusion activity would be an essential step for successful selection/adaptation of virus to the new host cells. membrane fusion mediated by coronavirus, similar to other viruses, is a multistep process. it includes binding of the s protein to one or more receptors, conformational changes of the protein to a fusionactive form and the actual fusion process. the membrane-fusion activity of coronavirus s protein is mainly associated with domains in the s2 region of the protein [34] [35] [36] [37] [38] . in this study, we demonstrate that l857-f substitution in the s2 region of the ibv s protein confers the s protein from non-fusogenic to fusogenic. this mutation may affect one of these fusion steps and thus modify the fusion activity of s protein and syncytia formation. at the same time, the virus was successfully adapted to the cultured cells with enhanced infectivity, confirming that acquisition of membrane fusion is an important step in selection/adaptation of ibv to cell culture and may also play a crucial role in cross-species adaptation and transmission of ibv in cultured cells. further enhancement of the cell-cell fusion activity of ibv s protein was achieved by a single amino acid substitution (g405-d) in p65 virus. this mutation, meanwhile, enhances the infectivity of the virus in cultured cells. the enhancement effect by mutations in the s1 region and its significance on viral infectivity was further demonstrated by cloning and expression of s gene derived from the ibv variants rescued from the full-length transcripts containing the f857-l mutation. in variants flv3 and flv4, additional amino acid substitutions (q523-l and i769-v) greatly enhanced the cell-cell fusion activity of the l857-containig s protein and the infectivity of the recovered virus. the involvement of residues in the s1 region in the cell-cell fusion activity of s protein was also demonstrated in other coronaviruses [25] . these results illustrate the complexity of the fusion process and the involvement of multiple domains in the induction of membrane fusion. it is worth mentioning that the cell-cell fusion activity of various s constructs was approximated by the degree of cell-cell fusion induced in cells overexpressing individual constructs. in a previous report, we showed nice correlation between the cell-cell fusion activity of two s constructs and their expression levels in the cells [39] . this correlation was also observed in this study with more wild type and mutant s constructs. based on data generated from adaptation [10] and cell-cell fusion studies presented here, a model of two-step adaptation process is proposed (fig. 7) . in this model, the adaptation was divided into primary and secondary adaptation (fig. 7) . early passages of vero-adapted ibv, including p7, p12, p14 and p20, belong to the primarily adapted group (fig. 7) . other cell cultureadapted strains, including the ck-adapted and beaudette-us strains cac39114 and cac39300 [8, 9] , and the vero-adapted strain aav98206 described by youn et al. [13] , also belong to this group (fig. 7) . late passages of vero-adapted ibv, including p36, p50 and p65, contain the additional g405-d amino acid substitution and belong to the secondarily adapted group (fig. 7) . except in the cell culture-adapted ibv strains, the l857 residue was found to be absolutely conserved in all coronaviruses sequenced so far. as structural information for this ibv s protein is currently lacking [40, 41] , we are unclear the overall role of this residue on the formation and stability of the six-bundle structure of the protein. further structural and functional studies are required to delineate the precise roles of this mutation in the fusion process. mutation of this residue to either a ser or a tyr showed similar effect on the cell-cell fusion activity of the s protein as a phe. on the other hand, when the residue was mutated to ile, glu or lys, a much lower cell-cell fusion activity of the s protein was observed. interestingly, mutations in the s1 and some distant s2 regions could compensate the effect of f857-l mutation. this may explain why s protein from several other coronaviruses, such as mhv and human coronavirus, could induce efficient virus-cell/cell-cell fusion although a conserved leu residue was found at the equivalent position [42, 43] . it is worth mentioning that the cell-cell fusion activities of different s constructs were qualitatively and semi-quantitatively determined in cells overexpressing individual s constructs using the vaccinia/t7 expression system. attempts to obtain more rigorous quantitative data were unsuccessful. as shown in this figure 7 . diagram showing a two-step adaptation process of chicken embryo-adapted ibv to vero cells. also shown are the numbers of amino acid changes during each adaptation process. a the accession no. for s genes from ep3 is dq001338, p7 is dq001337, p65 is dq001339. b the accession no. for this vero-adapted strain is aav98206. c the accession no. for these two strains are cac39114 and cac39300. doi:10.1371/journal.pone.0006130.g007 study, s protein is inefficiently translocated to the cell surface, probably due to the presence of an er retention signal [44] . since cell surface expression of s protein is a prerequisite for the induction of cell-cell fusion, disruption of the er-retention signal may facilitate cell surface expression as well as quantitative analysis of the cell-cell fusion activity of the protein. as virus-cell/cell-cell fusion is essential for efficient propagation of viral infection, attempts to interfere this process with fusion inhibitors, such as peptides or small molecules, are being made for several viruses, including hiv, sars-cov and influenza virus. the involvement of multiple domains in the induction of cell-cell fusion demonstrated here would complicate the design of such inhibitors. furthermore, mutations in regions beyond the target sequence, in the case of coronaviruses the s1 and some distant s2 regions, may lead to the emergence of drug-resistant strains. understanding of the fusion mechanisms in more detail would, therefore, help design more efficient inhibitors. conceived and designed the experiments: dxl. performed the experiments: yy xbl sf fpt. analyzed the data: yy xbl sf dxl. wrote the paper: xbl dxl. the molecular biology of coronaviruses the biology and pathogenesis of coronavirus isolation and characterization of viruses related to the sars coronavirus from animals in southern china bats are natural reservoirs of sars-like coronaviruses murine encephalitis caused by hcov-oc43, a human coronavirus with broad species specificity, is partly immune-mediated genetic evolution and tropism of transmissible gastroenteritis coronaviruses circulation of genetically distinct contemporary human coronavirus oc43 strains replication and morphogenesis of avian coronavirus in vero cells and their inhibition by momensin coronavirus ibv: partial amino terminal sequencing of spike polypeptide s2 identifies the sequence arg-arg-phe-arg-arg at the cleavage site of the spike precursor propolypeptide of ibv strains beaudette and m41 selection of and recombination between minor variants lead to the adaptation of an avian coronavirus to primate cells emergence of an avian coronavirus infectious bronchitis virus (ibv) mutant with a truncated 3b gene: functional characterization of the 3b gene in pathogenesis and replication single amino acid mutation in the spike protein of coronavirus infectious bronchitis virus hampers its maturation and incorporation into virions at the nonpermissive temperature in vitro assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication induction of p53-independent cell cycle arrest at s-and g 2 /m-phase in cells infected with the coronavirus infectious bronchitis virus promotes viral replication identification of two new polypeptides encoded by mrna5 of the coronavirus infectious bronchitis virus an arginine-to-proline mutation in a domain with undefined functions within the helicase protein (nsp13) is lethal to the coronavirus infectious bronchitis virus in cultured cells amino acid residues critical for rna-binding in the n-terminal domain of the nucleocapsid protein are essential determinants for the replication and infectivity of coronavirus in cultured cells further characterization of the coronavirus infectious bronchitis virus 3c-like proteinase and determination of a new cleavage site membrane association and dimerization of a cysteinerich, 16-kda polypeptide released from the c-terminal region of the coronavirus infectious bronchitis virus 1a polyprotein coronaviruses from pheasants (phasianus colchicus) are genetically closely related to coronaviruses of domestic fowl (infectious bronchitis virus) and turkeys the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex murine coronavirus with an extended host range uses heparan sulfate as an entry receptor cloning of the mouse hepatitis virus (mhv) receptor: expression in human and hamster cell lines confers susceptibility to mhv feline aminopeptidase n serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup i a 12-amino acid stretch in the hypervariable region of the spike protein s1 subunit is critical for cell fusion activity of mouse hepatitis virus aminopeptidase n is a major receptor for the enteropathogenic coronavirus tgev human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry angiotensinconverting enzyme 2 is a functional receptor for the sars coronavirus receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins human aminopeptidase n is a receptor for human coronavirus 229e sialic acid is a receptor determinant for infection of cells by avian infectious bronchitis virus infection of hela cells by avian infectious bronchitis virus is dependent on cell status heparan sulfate is a selective attachment factor for the avian coronavirus infectious bronchitis virus beaudette receptor-induced conformational changes of murine coronavirus spike protein n-terminal domain of the murine coronavirus receptor ceacam1 is responsible for fusogenic activation and conformational changes of the spike protein fusogenic properties of uncleaved spike protein of murine coronavirus jhmv the n-terminal domain of the murine coronavirus spike glycoprotein determines the ceacam1 receptor specificity of the virus strain conformational changes in the spike glycoprotein of murine coronavirus are induced at 37uc either by soluble murine ceacam1 receptors or by ph 8 coronavirus spike protein inhibits host cell translation by interaction with eif3f structural basis for coronavirusmediated membrane fusion. crystal structure of mouse hepatitis virus spike protein fusion core crystal structure of severe acute respiratory syndrome coronavirus spike protein fusion core amino acid substitutions and an insertion in the spike glycoprotein extend the host range of the murine coronavirus mhv-a59 acquired fusion activity of a murine coronavirus mhv-2 variant with mutations in the proteolytic cleavage site and the signal sequence of the s protein infection of the tracheal epithelium by infectious bronchitis virus is sialic acid dependent key: cord-354000-jxqskt4k authors: warren, cody j.; griffin, laura m.; little, alexander s.; huang, i-chueh; farzan, michael; pyeon, dohun title: the antiviral restriction factors ifitm1, 2 and 3 do not inhibit infection of human papillomavirus, cytomegalovirus and adenovirus date: 2014-05-14 journal: plos one doi: 10.1371/journal.pone.0096579 sha: doc_id: 354000 cord_uid: jxqskt4k type i interferons (ifn-α and β) induce dynamic host defense mechanisms to inhibit viral infections. it has been recently recognized that the interferon-inducible transmembrane proteins (ifitm) 1, 2 and 3 can block entry of a broad spectrum of rna viruses. however, no study to date has focused on the role of ifitm proteins in dna virus restriction. here, we demonstrate that ifn-α or -β treatment of keratinocytes substantially decreases human papillomavirus 16 (hpv16) infection while robustly inducing ifitm1, 2 and 3 expression. however, ifitm1, 2 and 3 overexpression did not inhibit hpv16 infection; rather, ifitm1 and ifitm3 modestly enhanced hpv16 infection in various cell types including primary keratinocytes. moreover, ifitm1, 2 and 3 did not inhibit infection by two other dna viruses, human cytomegalovirus (hcmv) and adenovirus type 5 (ad5). taken together, we reveal that the entry of several dna viruses, including hpv, hcmv, and ad5 is not affected by ifitm1, 2 and 3 expression. these results imply that hpv, and other dna viruses, may bypass ifitm restriction during intracellular trafficking. human papillomaviruses (hpvs) are small, non-enveloped double-stranded dna viruses causally associated with multiple human cancers. over 170 different genotypes have been identified and collectively categorized into high-risk or low-risk genotypes depending on their oncogenic capacity [1, 2] . the high-risk types are most commonly associated with cervical cancer [3, 4] and increasing evidence points to a contributing role in other cancers including head-neck [5, 6] and anogenital cancers [7] . hpv16 is the most prevalent high-risk genotype and serves as the main vaccine target along with hpv18 [8, 9] . hpv is the most common sexually transmitted pathogen in the united states [10] . despite high exposure rates, most people clear their infections naturally within 1-2 years [11] . however, longterm persistent infections are established in approximately 10% of women [12] . since persistence is required for cancer progression [13, 14] , it is critical to understand host immune features that are responsible for viral clearance so that new approaches targeting persistent hpv infections can be developed. in order to establish long-term infections, hpvs must actively avoid both adaptive and innate immune responses. hpvs prevent adaptive immune detection by several mechanisms in their unique life cycle. first, viral antigen production is limited to terminally differentiated keratinocytes of the mucosal and cutaneous epithelia. these cells are programmed to die of terminal differentiation, thus virus release coincides with limited inflammation and release of danger signals [15] . additionally, there is no viremic stage of the hpv life cycle, which minimizes the activation of systemic immune responses [16] . despite eliciting weak adaptive immune responses, the majority of primary hpv infections are cleared, thus suggesting the involvement of additional immune-mediated control mechanisms. keratinocytes intrinsically express low-levels of interferons (ifns) a, b, and k which induce interferonstimulated gene (isg) expression [17, 18] . however, the hpv oncoproteins e6 and e7 actively target the ifn regulatory transcription factors irf-3 and irf-1, respectively, resulting in an overall dampening of isg responses during infection [19] [20] [21] . active subversion of the ifn pathway suggests that the innate immune response, specifically ifn-regulated genes, may interfere with hpv persistence. the ifn-inducible transmembrane (ifitm) proteins are a family of ubiquitously expressed restriction factors that mediate ifn-induced antiviral activity [22, 23] . the antiviral effects of ifitms were first discovered in a genetic screen for host factors that restrict influenza a virus replication [24] . follow-up studies revealed ifitm type-specific restriction of an array of rna viruses including marburg and ebola filoviruses (marv, ebov), dengue and west nile (wnv) flaviviruses, sars coronavirus (sars-cov), hepatitis c virus (hcv), human immunodeficiency virus (hiv), rift valley fever virus (rvfv), respiratory syncytial virus (rsv), and reovirus [25] [26] [27] [28] [29] [30] . one of the common entry mechanisms shared by all these viruses, except hiv, is the requirement for low ph in late endosomes or lysosomes to facilitate genome release into the cytosol [22, 23] . hpvs encapsidate their 8 kb, double-stranded dna genome, in a desiccant-resistant icosahedral capsid composed of the major and minor capsid proteins l1 and l2, respectively [31] . devoid of an envelope, initial cell contact is mediated at the basement membrane of epithelial cells by direct binding of the l1 capsid protein to heparan sulfate proteoglycans [32] . while downstream events in entry are not yet fully elucidated, it is well known that uncoating and genome translocation to the nucleus is dependent on the low ph encountered in acidified late endosomes and lysosomes [33] [34] [35] . as the antiviral activity of ifitms is likely mediated by preventing endosome fusion and viral entry into the cytosol [22, 23, 36] , we hypothesized that one or more types of ifitm proteins inhibit hpv entry by preventing escape from the endocytic pathway. our study demonstrates that type i ifns inhibit hpv infection in keratinocytes, thus suggesting that isg induction may be critical for hpv restriction. surprisingly, overexpression of ifitm1 and ifitm3 consistently enhanced hpv infectivity in various epithelial cell lines and keratinocytes, while knockdown of endogenous ifitms yielded no effect. additionally, two other dna viruses, hcmv and ad5, were unaffected by ifitm overexpression. analysis of the rate of hpv capsid protein degradation in ifitm1 overexpressing primary keratinocytes revealed a delay in proteolytic degradation of virus capsids. collectively, our results suggest that endosome trafficking, altered by ifitm overexpression, preferentially routes hpv to a more productive infectious pathway. based on these results, we present the first study detailing the role of ifitms in the entry of dna viruses, showing that ifitm1, 2 and 3 proteins do not restrict were calculated by student's two-tailed t-test using prism version 6.0 for mac, graphpad software (san diego california usa, www.graphpad.com). significant differences (*p , 0.05, **p , 0.01, ***p , 0.001 and ****p , 0.0001) compared to pbs treated cells are marked with asterisks. (c) ifitm1, 2 and 3 expression levels were determined by rt-qpcr using ifitm specific primers (table s1 ). data are presented as fold change compared to untreated cells. (d) hacat cells were treated with 100 u/ml of ifn-b or medium alone and endogenous ifitm1 or ifitm3 protein expression in cell lysate was analyzed by western blotting using specific antibodies. pageruler plus ladder (thermo) was used to approximate molecular weights in kilodaltons (kd): ifitm1, 15 kd; ifitm3, 17 kd; b-actin, 46 kd. doi:10.1371/journal.pone.0096579.g001 hpv, hcmv, and ad5 infections. our results suggest an evolutionarily conserved entry mechanism by these dna viruses that bypasses the antiviral function of ifitms that restrict many rna viruses. hpv16 virions and pseudovirions were prepared as described previously [37, 38] . plucf, used in generating hpv16-lucf pseudovirions, was a kind gift from chris buck. adenovirus type 5 (ad5-cmv-gfp), human cytomegalovirus (hcmv tb40e mcherry) and sars-cov pseudotyped lentivirus [39] were generous gifts from drs. jerome schaack, caroline kulesza, and kathryn holmes, respectively (university of colorado school of medicine). human leukocyte ifn-a (nr-3078) and human recombinant ifn-b (nr-3085) were obtained from bei resources (manassas, va). 293ft cells purchased from invitrogen (grand island, ny) and hela cells obtained from dr. paul lambert (university of wisconsin-madison) were maintained in dulbecco's modified eagle's medium (dmem) (thermo scientific/hyclone, logan, ut) supplemented with 10% fetal bovine serum (fbs) (hyclone). hacat cells [40] were also obtained from dr. paul lambert and were cultured in e-medium (3 parts dmem, 1 part ham's f-12 nutrient mixture) supplemented with 5% fbs. human foreskin keratinocytes (hfks), derived from 3 neonatal foreskin donors, were purchased from invitrogen (cascade biologics, portland, or) and cultured in epilife medium with 60 mm calcium supplemented with human keratinocyte growth supplement (invitrogen/cascade biologics) according to the manufacturer's protocol [41] . human lung epithelial a549 cells transduced with vector alone (pqcxip) or stably expressing c-myc tagged ifitm1, 2, or 3 were generated previously [25] and maintained in roswell park memorial institute (rpmi) 1640 (hyclone). hela cell lines stably expressing prs vector-based control shrna and shrna targeting ifitm1 or 3 [25] were maintained as described above. hela and hacat cell lines transfected with vector alone or stably expressing ifitm proteins were selected with 3 mg/ml of puromycin (invitrogen). to produce transducing retroviruses, 293ft cells were transfected using the pei method with a 1:3:4 dna ratio of pcmv-vsv-g, pmlv gag-pol (dr. jerome schaack) and transfer vector, respectively. after 48 and 72 h, culture supernatants were combined, passed through a 0.45 mm syringe filter and concentrated 100 times by centrifugation at 25,000 rpm for 3 h at 4uc. hpv16-lucf and sars-cov pseudovirion infectivity were measured by luciferase assay as described previously [39, 41] . infections with hpv16-lucf pesudovirions were performed at 10 to 10,000 viral genome equivalents (vge)/cell, which is equivalent to approximately 0.3 to 300 multiplicity of infection (moi). gfp and phycoerythrin (pe) signals from cells infected with ad5-cmv-gfp and hcmv tb40e mcherry, respectively, were collected from at least 20,000 cells using a facscalibur flow cytometer (bd bioscience). data were analyzed using flowjo software (tree star). cells were lysed in ripa buffer containing protease inhibitor cocktail (roche) and 20 mg of total protein was separated by sds-page. antibodies: mouse monoclonal anti-l1 (1:1000, cam-vir-1, abcam), mouse monoclonal anti-c-myc (1:1000, 9e10, santa cruz biotechnology), mouse monoclonal anti-b-actin (1:5000, 8h10d10, cell signaling technology), rabbit polyclonal anti-ifitm1 (1:1000, 11727-3-ap, proteintech), rabbit polyclonal anti-ifitm3 (1:500, h00010410-d03p, novus biologicals) and hrp-conjugated secondary antibodies (1:10,000, jackson immu-noresearch laboratories). proteins were visualized using clarity western ecl substrate (bio-rad) and the chemidoc xrs system (bio-rad). total rna was extracted using rneasy kit (qiagen) and cdna was synthesized using oligo (dt) and superscript ii reverse transcriptase (invitrogen). cdna was analyzed by quantitative pcr (qpcr) using fast start universal sybr green master mix (roche). primers specific for selected isgs (table s1 ) were designed using primer-blast (http://www. ncbi.nlm.nih.gov/tools/primer-blast/) and b-actin sequences have been described previously [42] . qpcr was performed using cfx connect real-time pcr detection system (bio-rad). using the 2 2ddct method, relative quantities were calculated and normalized to b-actin. to determine whether type i ifns interfere with hpv16 infection, hacat keratinocytes were pre-treated with ifn-a or ifn-b for 24 h and then infected with hpv16 luciferase reporter pseudovirions (hpv16-lucf) in the presence of ifn-a or ifn-b. at 48 h post infection (hpi), infectivity was measured by relative luciferase activity [43] . although both ifn-a and -b treatments significantly inhibited hpv16-lucf infection, the reductions seen with ifn-b treatment were more robust at lower doses (fig. 1a) . rt-qpcr of selected isgs (mx1, ifi44, ifit1, ifitm1, 2, and 3) revealed enhanced expression of mrnas following ifn-a treatment (fig. s1 ). this indicates that, although keratinocytes are responsive to ifn-a stimulation, ifn-b treatment is more effective at reducing infectivity despite sharing a common surface receptor [44] . to exclude the possibility that the observed effect of ifn-b on hpv infection might reflect cytotoxicity rather than inhibiting infection, we measured cell viability using a luminescence-based atp quantification assay in parallel (promega). no significant effect on keratinocyte viability was observed up to 300 u/ml of ifn-b (fig. 1b) . ifitm protein expression induced by type i ifn inhibits infection of many rna viruses [22] [23] [24] [25] [26] [27] [28] [29] [30] . therefore, we analyzed induction of ifitm1, 2 and 3 expression by ifn-b treatment in human keratinocytes, the natural host cells for hpv. hacat cells were treated with increasing doses of ifn-b for 24 h, followed by rna extraction and rt-qpcr to assess ifitm mrna expression levels. ifitm1, 2 and 3 mrna expression was increased by ifn-b in a dose dependent manner (fig. 1c) . it appeared that the magnitude of ifitm1 induction by ifn-b treatment was higher than the levels of ifitm2 and ifitm3 induction. however, this may be due to the low basal level of endogenous ifitm1 in human keratinocytes (fig. 1d) . while differing in endogenous expression levels, ifitm1 and ifitm3 proteins were dramatically increased by ifn-b treatment (fig. 1d) . to determine the effect of ifitms on hpv16 entry, hela cells stably expressing c-myc-tagged ifitm1, 2 or 3 or with vector alone ( fig. 2a-b) were infected with hpv16-lucf. at 48 hpi, infectivity was measured by luciferase assay and percent infectivity was calculated after normalization to cells transduced with the empty vector control. despite high expression (fig. 2b) , ifitm1, 2 or 3 did not inhibit virus infection ( fig. 2a) . comparable results were obtained in a549 cells and hacat keratinocytes (fig. 2c-f) . surprisingly, overexpression of ifitm1 and ifitm3 consistently enhanced hpv16 infection by 30-60% while the effects of ifitm2 were negligible ( fig. 2a, 2c and 2e) . to examine the effect of ifitm1 in primary human keratinocytes, human foreskin keratinocytes (hfks) were transduced with c-myc-tagged ifitm1 using a retroviral delivery system [25, 41] . consistently, ifitm1 overexpression enhanced hpv16 infection even higher at .3-fold ( fig. 2g and 2h) . these results suggest that ifitm1 overexpression may facilitate hpv infection in human keratinocytes. we further investigated if ifitms restrict infection of other dna viruses that also require acidic compartments for entry into host cells. in epithelial cells, ad5 and hcmv entry and egress from endosomal compartments depends on low ph [45, 46] . given that ifitm restriction is mediated along this pathway [22, 23] , we hypothesized that ifitm overexpression would affect the entry processes of ad5 and hcmv. recombinant ad5 and hcmv (strain tb40e) expressing gfp and mcherry, respectively, were used to infect hela cells stably expressing ifitm1, 2, or 3 or with vector alone. at 48 hpi, the percentage of infected cells was determined by flow cytometry. our results showed that ad5 and hcmv infections were unaffected by ifitm protein expression ( fig. 3a and b) . it has been previously reported that s-protein mediated entry of sars-cov is broadly restricted by the ifitm1, 2 and 3 proteins [25] . using a sars-cov s-protein pseudotyped lentivirus [39] as a positive control, we demonstrate a similar restriction of sars-cov infection mediated by ifitm proteins in hela cell lines (fig. 3c) . mean % infectivity from quadruplicate samples representative of at least two independent experiments with standard error. p-values were calculated as described in fig. 1 to determine whether endogenous ifitm expression affects hpv entry, ifitm1 or ifitm3 was knocked down in hela cells by stable expression of ifitm1, ifitm3 or control scrambled shrna [25] . since ifitm2 expression appeared to have no effect on hpv16 infectivity, we focused only on ifitm1 and ifitm3. the levels of target gene knockdown were determined by rt-qpcr (fig. 4a) . although ifitm1 and ifitm3 show some sequence homology, no off target effects were noted by shrna knockdown (fig. s2) . interestingly, knockdown of ifitm1 or ifitm3 expression had no effect on hpv16-lucf infection (fig. 4b) . taken together, our results suggest that, compared to rna viruses, the dna viruses hpv, hcmv, and ad5 might rely on different entry mechanisms to avoid ifitm restriction. previous studies show that overexpression of ifitm3 disrupts the composition of endosomal compartments [36, 47] and influ-ences the rate of reovirus capsid protein degradation [29] . given the consistent effects of ifitm1 overexpression on hpv16 infection in primary keratinocytes, we determined whether ifitm1 altered the kinetics of hpv16 l1 major capsid protein degradation. hfks were inoculated with hpv16 virions containing full-length viral genomes for 1 h at 4uc to allow for virus attachment but not internalization. internalization was initiated by shifting the cells to 37uc for 0, 2, 4, 6 and 12 h. at the indicated time points, the cells were trypsinized to remove non-internalized virus, then harvested for hpv16 l1 western blotting. intriguingly, hfks overexpressing ifitm1 exhibited delayed l1 degradation compared to the empty vector control, showing reduced accumulation of the 22 and 12 kd degradation products as early as 2 hpi (fig. 5) . interestingly, we previously reported that the autophagy inhibitor, 3-methyladenine (3-ma), delays the degradation of hpv16 l1 capsid proteins during entry in a strikingly similar manner as ifitm1 overexpression [41] . further, 3-ma also significantly enhanced hpv16 infection in hfks [41] . as feeley et al. demonstrated a role for ifitm3 in autophagosome-lysosome fusion during influenza a virus infection [36] , our results imply that ifitm1 overexpression may alter cellular compartments in a similar manner that partially protects the hpv capsid from degradation in lysosomes and autolysosomes. it is well known that innate immune responses are critical for protecting host cells from early establishment of virus infection. following recognition of viral components by pattern recognition receptors, type i ifn production is induced which triggers localized antiviral activities through various isgs [48] . here we report the role of type i ifns, ifn-a and ifn-b, in restricting hpv entry into human keratinocytes. as shown in figure 1 , ifn-b significantly inhibited hpv infection at much lower concentrations than ifn-a, while ifn-a efficiently induced isgs in human keratinocytes. one explanation for these differential effects is that ifn-a and -b induce different groups of isgs. microarray analysis of cells treated with either ifn-a or ifn-b identified more than 20 candidate genes whose mrna levels are selectively induced by ifn-b but not ifn-a. these most notably include pkr, isg-56 (ifit1), isg-58 (ifit5), and hif-1a, while the remaining genes lack annotated antiviral activity [49] . an unbiased analysis of genes preferentially induced by ifn-b in keratinocytes may be useful in identifying candidate host factors that restrict hpv entry and replication. alternatively, the differences in hpv infectivity may be due to differences in interruption of cell proliferation triggered after ifn exposure. ifn-b exhibits strong anti-proliferative effects on human cancer cells at concentrations far lower than those for ifn-a [50] . as we previously revealed that hpv requires host cell mitosis for virus entry [43] , disrupting cell proliferation by ifn-b may interfere with hpv infection. ifitm proteins inhibit several rna viruses that rely on low-ph in late endosomes or lysosomes for virus entry [22, 23] . it is well established that hpv requires these acidified compartments for uncoating and egress from the endosomal pathway [33] [34] [35] , thus ifitms would serve as an attractive candidate for hpv restriction. using various epithelial cell lines and primary keratinocytes expressing ifitms, we show that hpv infection is surprisingly enhanced by ifitm1 and ifitm3 overexpression (fig. 2) . a similar enhancement has been observed with pseudovirions expressing the entry proteins of several arenaviruses and a retrovirus, moloney murine leukemia virus (mmlv) [24] . thus, our and previous results [24] suggest that resistant viruses may take advantage of endocytic trafficking altered by overexpression of ifitm. additionally, we demonstrate that ifitm1, 2 and 3 did not inhibit infection of two other dna viruses, ad5 and hcmv, whereas infection of sars-cov, a positive control, was broadly restricted (fig. 3) . the low ph of the endocytic pathway is required for adenovirus infection [51, 52] . however, it has been reported that adenovirus egress from endosomes is rapid and likely mediated by penetration in early endosomes but not late endosomes [45, 53] . entry mechanisms of hcmv vary depending on different virus strains and the host cell types [54] . while hcmv entry into fibroblasts is mediated by ph-independent fusion at the host cell plasma membrane [55] , virus entry into epithelial cells requires endocytosis and ph dependent fusion within endosomes [46] . because we utilized an epithelial cell model (hela), hcmv entry might occur along the endocytic pathway, although the exact point of egress has still yet to be determined. nevertheless, the tested dna viruses, hpv, hcmv, and ad5, similarly require endocytosis and low ph for entry into host cells, and are not inhibited by ifitms. further comparative studies of larger panels of dna viruses, with various entry mechanisms, might aid in defining the role of ifitms during dna virus infection and clarify the differential routes of endosome trafficking in rna and dna virus entry into host cells. our results provide the first evidence that ifitms do not restrict and potentially enhance infection of dna viruses (fig. 2) . overexpression of ifitm1 delayed l1 capsid protein degradation in primary keratinocytes (fig. 5) , which may explain an enhancement of virus infection by ifitm1. further investigations into how viruses bypass ifitm restriction may provide more detailed mechanistic insights into virus trafficking and help clarify productive routes of infectious entry. taken together, our results suggest that hpv and other dna viruses may have evolved to avoid ifitm1, 2 and 3 restriction during entry into host cells. figure s1 ifn-a treatment stimulates the induction of isgs in keratinocytes in a dose dependent manner. hacat cells were treated with 70 or 350 u/ml ifn-a for 24 h. total rna was extracted and expression levels of the indicated isgs were measured by rt-qpcr. data is presented as fold change relative to b-actin mrna, followed by normalization to pbs-treated cells. error bars represent sem. (tif) figure s2 ifitm knockdown in hela cells is specific. total rna was isolated from hela cells stably expressing scrambled shrna or shrna targeting ifitm1 or ifitm3. expression levels of ifitm1 and ifitm3 mrna were measured by rt-qpcr and normalized by b-actin mrna levels. data is presented as % change in mrna expression of knockdown cells compared to cells with scrambled shrna. (tif) table s1 list of primers used for rt-qpcr. (pdf) chapter 3: hpv type-distribution in women with and without cervical neoplastic diseases classification of weakly carcinogenic human papillomavirus types: addressing the limits of epidemiology at the borderline the causal relation between human papillomavirus and cervical cancer human papillomavirus is a necessary cause of invasive cervical cancer worldwide a causal role for human papillomavirus in head and neck cancer human papillomavirus-associated head and neck squamous cell carcinoma: mounting evidence for an etiologic role for human papillomavirus in a subset of head and neck cancers human papillomaviruses in the pathogenesis of anogenital cancer prevalence of human papillomavirus in cervical cancer: a worldwide perspective american cancer society guideline for human papillomavirus (hpv) vaccine use to prevent cervical cancer and its precursors sexually transmitted diseases among american youth: incidence and prevalence estimates the natural history of type-specific human papillomavirus infections in female university students persistence of human papillomavirus infection: keys to malignant progression natural history of cervical human papillomavirus infection in young women: a longitudinal cohort study baseline cytology, human papillomavirus testing, and risk for cervical neoplasia: a 10-year cohort analysis immune responses to human papillomavirus immune responses to human papilloma viruses epigenetic silencing of interferon-kappa in human papillomavirus type 16-positive cells human papillomaviruses and the interferon response human papillomavirus 16 e6 oncoprotein binds to interferon regulatory factor-3 and inhibits its transcriptional activity inactivation of interferon regulatory factor-1 tumor suppressor protein by hpv e7 oncoprotein. implication for the e7-mediated immune evasion mechanism in cervical carcinogenesis the human papillomavirus e7 oncoprotein abrogates signaling mediated by interferon-alpha the broad-spectrum antiviral functions of ifit and ifitm proteins ifitms restrict the replication of multiple pathogenic viruses the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus isg56 and ifitm1 proteins inhibit hepatitis c virus replication the ifitm proteins inhibit hiv-1 infection ifitm-2 and ifitm-3 but not ifitm-1 restrict rift valley fever virus interferoninducible transmembrane protein 3 (ifitm3) restricts reovirus cell entry defining the range of pathogens susceptible to ifitm3 restriction using a knockout mouse model atomic model of the papillomavirus capsid current understanding of the mechanism of hpv infection a membrane-destabilizing peptide in capsid protein l2 is required for egress of papillomavirus genomes from endosomes entry of human papillomavirus type 16 by actin-dependent, clathrin-and lipid raft-independent endocytosis human papillomavirus types 16, 18, and 31 share similar endocytic requirements for entry ifitm3 inhibits influenza a virus infection by preventing cytosolic entry production of infectious human papillomavirus independently of viral replication and epithelial cell differentiation efficient intracellular assembly of papillomaviral vectors role of the spike glycoprotein of human middle east respiratory syndrome coronavirus (mers-cov) in virus entry and syncytia formation normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line human papillomavirus infection is inhibited by host autophagy in primary human keratinocytes fundamental differences in cell cycle deregulation in human papillomaviruspositive and human papillomavirus-negative head/neck and cervical cancers establishment of human papillomavirus infection requires cell cycle progression evidence that types i and ii interferons have different receptors adenovirus endocytosis human cytomegalovirus entry into epithelial and endothelial cells depends on genes ul128 to ul150 and occurs by endocytosis and low-ph fusion the antiviral effector ifitm3 disrupts intracellular cholesterol homeostasis to block viral entry the host type i interferon response to viral and bacterial infections identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays antitumor activities of interferon alpha, beta, and gamma and their combinations on human melanoma cells in vitro: changes of proliferation, melanin synthesis, and immunophenotype stepwise dismantling of adenovirus 2 during entry into cells adenovirus protein vi mediates membrane disruption following capsid disassembly infectious adenovirus type 2 transport through early but not late endosomes entry route of hcmv into endothelial cells human cytomegalovirus penetrates host cells by ph-independent fusion at the cell surface we thank jerry schaack for providing pvsv-g, mlv-gag-pol, ad5-cmv-gfp and useful suggestions for ad5 infectivity assays, katherine holmes and zhaohui qian for providing sars-cov pseudotyped lentivirus, caroline kulesza and chris abraham for hcmv tb40e mcherry and advice on flow cytometry assays, chris buck and john schiller for providing plucf and p16shell, ken tyler and scott seitz for the polyclonal anti-ifitm3 antibody, and paul lambert for hela and hacat cell lines. we also thank the members of our lab for useful comments and suggestions. the following reagents were obtained through bei resources, niaid, nih: human leukocyte ifn-a (nr-3078) and human recombinant ifn-b (nr-3085). conceived and designed the experiments: cjw mf dp. performed the experiments: cjw lmg asl dp. analyzed the data: cjw lmg dp. contributed reagents/materials/analysis tools: cjw lmg ih mf dp. wrote the paper: cjw lmg dp. key: cord-326568-twv2i3fb authors: bruminhent, jackrapong; ruangsubvilai, nattanon; nabhindhakara, jeff; ingsathit, atiporn; kiertiburanakul, sasisopin title: clinical characteristics and risk factors for coronavirus disease 2019 (covid-19) among patients under investigation in thailand date: 2020-09-15 journal: plos one doi: 10.1371/journal.pone.0239250 sha: doc_id: 326568 cord_uid: twv2i3fb to manage coronavirus disease 2019 (covid-19), a national health authority has implemented a case definition of patients under investigation (puis) to guide clinicians’ diagnoses. we aimed to determine characteristics among all puis and those with and without covid-19. we retrospectively reviewed clinical characteristics and risk factors for laboratory-confirmed covid-19 cases among puis at a tertiary care center in bangkok, thailand, between march 23 and april 7, 2020. reverse transcription-polymerase chain reaction for sars-cov-2 rna was performed. there were 405 evaluable puis; 157 (38.8%) were men, with a mean age ± sd of 36.2 ± 12.6 years. the majority (68.9%) reported no comorbidities. there were 53 (13.1%) confirmed covid-19 cases. the most common symptoms among those were cough (73.6%), fever (58.5%), sore throat (39.6%), and muscle pain (37.4%). among these patients, diagnoses were upper respiratory tract infection (69.8%), viral syndrome (15.1%), pneumonia (11.3%), and asymptomatic infection (3.8%). multivariate analysis identified close contact with an index case (or, 3.49; 95%ci, 1.49–8.15; p = 0.004), visiting high-risk places (or, 1.92; 95%ci, 1.03–3.56; p = 0.039), productive cough (or, 2.03; 95%ci, 1.05–3.92; p = 0.034), and no medical coverage (or, 3.91; 95%ci, 1.35–11.32; p = 0.012) as independent risk factors for covid-19 among the puis. the majority had favorable outcomes, though one (1.9%) died from severe pneumonia. covid-19 was identified in 13% of puis defined per a national health authority’s case definition. history of contact with a covid-19 patient, visiting a high-risk place, having no medical coverage, and productive cough may identify individuals at risk of covid-19 in thailand. coronavirus disease 2019 (covid19) is an emerging respiratory tract infection caused by severe acute respiratory syndrome coronavirus-2 (sars-cov-2), a novel coronavirus initially reported in china and later spreading worldwide [1] . in january 2020, the world health organization (who) declared covid-19 a public health emergency of international concern [2] . covid-19 patients can present as asymptomatic, mild upper respiratory tract disease or potentially severe pneumonia. consequentially, those with severe infection are at potential risk for acute hypoxemic respiratory failure requiring mechanical ventilation, a condition with substantially high morbidity and mortality. the approximate mortality rate has ranged from 1% to 10% depending on patients' clinical presentations and the allocation of medical resources, varying among resource-adequate and constrained settings [3, 4] . on january 12, 2020, the ministry of public health (moph), thailand, reported the first imported patient who tested positive for covid-19 outside china [5] . as of april 25, 2020, a total of 2,907 people in thailand were diagnosed with covid-19, with a mortality rate of approximately 1.8% [6] . among the cases, the greatest risk factors of contracting covid-19 were found to be close contact with an index case or a history of travel to a high-risk area. based on a number of recent case reports, these seem to be crucial clues for a diagnosis of covid-19 in thailand [7, 8] . the moph has therefore set out a definition of patients under investigation (puis) to identify patients at risk of contracting covid-19; this is based on the united states centers for disease control and prevention (cdc) guidelines. a stratified investigation was attempted to better identify patients at risk and who needed investigation based on capacity and accessibility to nucleic acid amplification testing, which was considered unaffordable for some areas of thailand. specific risk factors could assist clinicians in predicting which puis were infected with covid-19. although there was a previous case series of covid-19 patients who were hospitalized [9] , there has been no study focused on this population. moreover, the rate of cases testing positive for covid-19 based on the case definition had not been assessed and clinical characteristics and risk factors for non-infected versus infected puis has not been explained. we therefore aimed at a large-scale investigation of this all entities and expected to define better criteria for identifying cases. this would lead to improved diagnoses, prompt therapy, and infection control. the present study was conducted at ramathibodi hospital, a 1,200-bed, university hospital in the center of the bangkok metropolitan area in thailand. we conducted a retrospective review examining for covid-19 in puis aged �15 years covering march 23 to april 7, 2020, when the highest rate of cases was reported in thailand. a list of puis was retrieved from a database of the infection prevention and control services at our hospital. the department of disease control, moph, on march 2, 2020, defined puis as follows [10] . first, these patients have a history of fever or fever �37.5˚c (99.5˚f) and one respiratory tract symptom (e.g., cough, runny nose, sore throat, tachypnea, dyspnea, difficulty breathing), and during the 14 days before developing symptoms they: (a) traveled to or from thailand or lived in an area with a report of an ongoing outbreak of covid-19; (b) worked and had close contact with tourists, worked in a crowded place, or had contact with many people; (c) had contact with confirmed patients or with respiratory droplets of patients suspected of or confirmed with having covid-19, and without appropriate protective equipment; or (d) had a history of going to a community location or a place with groups of people (e.g., market, department store, hospital) as announced by the provincial communicable disease committee. second, they are pneumonia patients with a history of one of the following: (a) had close contact with a covid-19 patient; (b) had unexplained pneumonia and the clinical condition did not improve within 48-72 hours; or (c) had pneumonia with a profile consistent with that of covid-19. third, the patients are medical personnel with a history of fever or fever �37.5˚c (99.5˚f) and one respiratory tract symptom, and the physician in charge or the communicable disease control officer advised an examination for sars-cov-2. last, there was detection of a group of cases in the same place, within the same week, and with an epidemiologic connection. we also included those who were not entirely matching the case definition but were deemed to be at risk based on their substantial exposure or typical symptoms (or both). the latter was determined by a certified infectious diseases specialist designated to decide who should be investigated as a pui. per practical flow, each pui was initially determined as a non-severe or severe case based on our institution criteria developed by members of the infectious diseases division in conjunction with the pulmonary and critical care division. non-severe puis underwent an interview and physical examination at a designated acute respiratory infection clinic at the faculty of medicine ramathibodi hospital. severe puis were defined as one of the following: peripheral capillary oxygen saturation (spo 2 ) <92% with room air, spo 2 92%-95% (room air) with respiratory rate (rr) >30 breaths/min, or rr <30 breaths/min with signs of impending respiratory failure. those classified as severe were directly admitted to an airborne infection isolation room (aiir) in an intensive care unit. all confirmed covid-19 patients were mandatorily admitted to the hospital. demographic data including sex, age, home address, occupation, health insurance scheme enrollment, underlying disease, and presenting symptoms were obtained by reviewing medical records. those with risk of sars-cov-2 infection in accordance with the definitions of puis as per the moph (as described above) were also retrieved and reviewed. we also collected complete blood count, chemistry laboratory testing results, chest x-ray findings, and the results of sars-cov-2 rna detection. we divided patients' final diagnoses into two groupspatients with positive and negative results of sars-cov-2 rna-to determine clinical characteristics and risk factors for covid-19. members of the infectious diseases division developed the treatment regimen at our institution in conjunction with the pulmonary and critical care division; this was guided by the department of disease control, moph [11] . (hydroxy)chloroquine and boosted protease inhibitors, either boosted lopinavir or darunavir, were given to those with mild (with comorbidities) and moderate symptoms. favipiravir, an anti-viral agent, was added for those diagnosed with pneumonia. supportive treatment was offered for all patients. nasopharyngeal and throat swabs or endotracheal aspirates from those who were intubated were collected from puis using copan floqswabs, and a sterile tube containing copan's universal transport medium (copan diagnostics inc.). viral rna was extracted from the samples using magdea dx reagents (precision system science co., ltd.) with a fully nucleic acid extraction system. the detection of sars-cov-2 was performed using reverse transcription-pcr (rt-pcr); this was performed using a cfx96 real-time pcr detection system (bio-rad laboratories, inc.). amplification of sars-cov-2 orf1ab and n gene fragments, using a sars-cov-2 nucleic acid diagnostic kit (sansure biotech inc.), was approved by the national medical products administration and certified by the china food and drug administration [12] . physicians were allowed to investigate for other respiratory viruses, such as influenza or other pathogens, for patients deemed to be at risk. the study protocol was approved by the institutional review board (irb) of the faculty of medicine ramathibodi hospital, mahidol university, bangkok, thailand, with the provisions of the good clinical practice guidelines and the declaration of helsinki (approval number: coa. mura2020/557). all data were fully anonymized before accessed and the irb waived the requirement for informed consent. median values (with interquartile range, iqr) or mean (with standard deviation, sd) were used to describe the patients' baseline characteristics, and laboratory investigations for continuous data and frequency were used for categorical data. a chi-square test or fisher's exact test and wilcoxon rank-sum test were used to compare categorical variables and continuous variables between the two groups, respectively. univariate and multivariate logistic regression analyses were performed to determine the factors associated with positive results for sars--cov-2 rna. variables that presented p <0.05 in the univariate analysis were considered in a multivariate logistic regression model after assessment of multicollinearity of variance inflation factors. variables were selected into a multiple logistic regression model with forward stepwise selection, and those that attained significance were retained in the model. the odds ratio (or) and its 95% confidence interval (ci) were estimated. p <0.05 was considered statistically significant. all statistical analyses were performed using stata statistical software version 15 a total of 414 patients during the study period were investigated for covid-19. table 1 shows the baseline characteristics of all puis. nine were excluded because of incomplete data (n = 5) and because pcr for sars-cov-2 was not tested (n = 4). among the remaining 405 evaluable patients, 157 (38.7%) were men, with a mean age ± sd of 36.2 ± 12.6 years. the majority (96.8%) were of thai ethnicity and (85.2%) lived in the bangkok metropolitan area. a total of 149 (36.8%) patients were unemployed and 297 (73.3%) had to self-pay their full medical expenses. only around one-third (31.1%) had underlying diseases, including allergic rhinitis (6.4%), diabetes mellitus (5.7%), hypertension (4.2%), and dyslipidemia (1.7%). few patients (2.5%) had immunocompromised conditions. there were 347 (85.7%) and 58 (14.3%) patients who were fulfilled the criteria and designated as a pui, respectively. twenty-six (6.4%) severe puis and 379 (93.6%) non-severe puis were classified as the aforementioned criteria. among 400 (98.8%) patients underwent nasopharyngeal and throat swabs and 5 (1.2%) patients provided endotracheal aspirates for sars-cov-2 pcr, a total of 53 (13%) patients were confirmed as having covid-19; 18 (34%) patients were men with mean age ± sd of 36.3 ± 10.2 years. the majority were of thai ethnicity (98.1%), lived in the bangkok metropolitan area (84.9%), were unemployed (39.6%), and worked at a restaurant (34%). most (92.4%) did not have medical coverage. most (81.1%) had no underlying diseases and none were immunocompromised. confirmed covid-19 patients were classified as having upper respiratory tract infection (69.8%), viral syndrome (15.1%), pneumonia (11.3%), or asymptomatic infection (3.8%). the patients were admitted into an aiir located in the general ward (98.1%) or intensive care unit (icu) (1.9%). among the latter, 5 (1.2%) patients underwent endotracheally intubation on arrival due to acute respiratory failure and therefore endotracheal aspirates were collected accordingly. compared with non-covid-19 patients (table 2) , there were no differences in the proportion of male sex, median age, nationality, home address, or underlying disease between groups (p >0.05 for all). those with covid-19 lacked medical coverage at a significant rate compared with those without covid-19 (92.4% vs. 7.04%; p = 0.001). compared with non-covid-19, those with covid-19 reported a history of close contact with an index case (86.8% vs. 63.1%; p = 0.001), visiting a crowded place, or attending an activity where people gathered, in the 14 days before symptom onset (41.5% vs. 26.7%; p = 0.026). however, there was no significant difference regarding history of traveling abroad in the 14 days before symptom onset (1.9% vs. 8.2%; p = 0.100). both groups reported having symptoms on arrival at a rate as high as 94%. those with covid-19 complained significantly more frequently of productive cough (34% vs. 21.6%; p = 0.047). they also experienced slightly more subjective fever (58.5% vs. 44.3%; p = 0.054) and anosmia (5.7% vs. 1.1%; p = 0.051), though slightly less diarrhea (3.8% vs. 12.5%; p = 0.062). other respiratory symptoms showed no significant difference, including dry (table 3) , those with covid-19 presented with significantly lower median white blood cell counts, at 6,100 cells/mm 3 , compared with 9,600 cells/mm 3 in non-covid-19 patients (p = 0.002). the median percentages of lymphocytes (25.5% vs. 14%; p = 0.056) and monocytes (10.5% vs. 7%; p = 0.030) were slightly more prominent in covid-19 patients. prevalence of lymphopenia and thrombocytopenia did not significantly differ. levels of liver function tests, lactate dehydrogenase, c-reactive protein, and procalcitonin were indistinguishable between the groups (p >0.05 for all). abnormal chest x-ray was more frequently observed in the covid-19 group, including patchy opacity old infiltration 0 (0.0) 4 (11.4) others 0 (0.0) 1 (2.9) spo 2 : peripheral capillary oxygen saturation; heent: head, ear, eye, nose, and throat; iqr: interquartile range https://doi.org/10.1371/journal.pone.0239250.t002 table 3 . laboratory results of patients under investigation with and without covid-19. white blood cells, cells/mm 3 6,100 (4,500-6,800) 9,600 (7,500-12,900) 0 (28%) and reticular/interstitial opacity (57%). those with covid-19 were more likely to be diagnosed with pneumonia compared with upper respiratory tract infection (11.3% vs 3.1%, p = 0.029). among 352 (86.9%) puis who did not have a positive result for covid-19, 40 (11.4%) patients underwent further investigations. there were seven patients in the non-covid-19 group diagnosed with infections with non-covid pathogens: influenza virus (n = 2), pneumocystis jirovecii (n = 1), haemophilus influenzae (n = 2), klebsiella pneumoniae (n = 1), and staphylococcus aureus (n = 1). the majority of diagnosed covid-19 patients had favorable outcomes, though one (1.9%) died from severe pneumonia. univariate analysis (tables 4 and 5 the present study appears to be one of the first and largest to evaluate incidence of and predictors for covid-19 in a setting in which patients were investigated under the impetus of a national health authority's criteria. we found approximately one in eight puis had covid-19 confirmed by molecular testing. most patients had no comorbidities, presented with upper respiratory tract infection, and had a favorable outcome. apart from close contact with an infected case and visiting high-risk places, we found that having no medical coverage and presenting with productive cough were predictors of being diagnosed with covid-19 among puis. sars-cov-2 is an emerging respiratory virus that commonly causes no or mild respiratory tract infection and is occasionally complicated by severe pneumonia [1] . the strategy for identifying index case has varied among different settings depending on risk and exposure. a targeted approach rather than universal testing may be more practical in areas where resources are limited. in thailand, case definition-driven cases were previously used for diagnosis of middle east respiratory syndrome coronavirus (mers-cov); however, the spread and impact of mers-cov were considered less than with sars-cov-2 [13] . we were able to use our national authority's case definition to identify covid-19 cases. additionally, we validated histories of close contact with an index case and of visiting high-risk places, which were already included in the criteria. chen et al. reported that half of patients with a history of exposure to the seafood market suspected to be the sources of the virus, and close contact with a covid-19-infected individual, were major risk factors among people who lived in wuhan, hubei, china during the initial outbreak [14] . in thailand, people who had been to bars and boxing events were the first two clusters of cases reported in the heart of the bangkok metropolitan area in early march 2020 [15] . we also discovered that having no medical coverage and having productive cough, which were not included in the case definition, were novel risk factors. lack of medical coverage in thailand would likely reflect challenging socioeconomic status, such as living in crowded households and certain types of workplaces. our study supported possible human-to-human transmission in a closed environment and among family members [7, 16, 17] . a national policy is also implemented to monitor those who were closely contacted with an index patient (covid-19 patient) and complimentarily investigated for covid-19 should new symptoms occur. individuals who pay for their own medical care out-of-pocket may also have superior access to medical services. furthermore, productive cough remained associated with covid-19 after adjusting for other covariables. sputum production was previously reported in roughly one-third of a cohort roughly one-third apart from dry cough [18] . an unexpected caveat is although the male gender predominance was observed among several cohorts regarding vulnerability to covid-19, our result instead revealed female gender is more frequently diagnosed. a reason to explain this disparity has been proposed but not entirely clear, however, an outcome seemed indifferent [19, 20] . furthermore, a greater proportion of female puis in our cohort likely from a coincidence or possibly more attention in their health conditions was more prominent among the female population. among confirmed covid-19 patients, our patient data regarding clinical symptoms and signs were both comparable and somewhat different from those in previous reports from china [14, 21] . measured body temperatures were similar to those in previous studies that were found more likely to be observed at admission [18] . another study found subjective fever was more likely to be reported among covid-19 patients [21] . respiratory symptoms were indistinguishable between those with and without covid-19 in our cohort, except for productive cough, which was contrary to the previous cohort. patients diagnosed with covid-19 in china reported having cough in up to 80% of cases; and more specifically, dry cough in nearly 60% [14, 21] . we also found anosmia was more likely to be reported among confirmed covid-19 cases; it was significantly more frequent among covid-19 patients than among influenza patients in a pilot case-control study [22] . to the contrary, gastrointestinal symptoms such as diarrhea, nausea, and vomiting were more likely to present in non-covid-19 patients. chen et al reported gastrointestinal symptoms in only 1%-2% of covid-19 pneumonia patients [14] . a case series of thai covid-19 hospitalized patients showed fever and respiratory symptoms were not present at significant levels even when all patients were diagnosed with radiologically confirmed pneumonia [9] . this agreed with our finding that respiratory symptoms were not prominent. wang et al. reported anosmia, dyspnea, sore throat, dizziness, and abdominal pain were more common among covid-19 patients who were admitted to an intensive care unit (icu) in china [21] . a case definition focused on fever and respiratory signs and symptoms may inadequately detect some cases, especially those later in the disease course. we found faster heart rate, lower oxygen saturation, and borderline low blood pressure were associated with covid-19. there was no such association, however, in a logistic analysis. this was likely owing to the small number of patients. our study confirmed that most of the infected patients were asymptomatic or had mild symptoms. they therefore could pose a greater risk of transmitting the disease within a community. asymptomatic patients with upper respiratory specimens testing positive for sars--cov-2 transmission were previously reported [23, 24] . co-infection with other pathogens coinciding with sars-cov-2 is plausible; however, the patients in the present study did not receive further testing after covid-19 because they all had mild symptoms and further workups did not show evident impact on disease management [9] . regarding laboratory investigations, covid-19 patients presented with significantly lower wbc count compared with non-covid-19 patients. lymphocytosis was commonly seen in viral infections in general. however, we observed the percentages of lymphocytes and monocytes were slightly more prominent in the covid-19 patients. our data did not reveal lymphopenia or thrombocytopenia because the patients in our study had slightly less severe cases than those in another cohort [18] . lymphocyte count was lower in icu compared with non-icu covid-19 patients [21] . among thai covid-19 patients who were hospitalized, those with severe covid-19 tended to have leukopenia, lymphopenia, and slight thrombocytopenia [9] . other tests, including liver function, lactate dehydrogenase, inflammatory markers such as c-reactive protein (crp), and procalcitonin, were unable to distinguish covid-19 and non-covid-19 cases in our pui cohort. the nucleic acid amplification testing used in our hospital detects sars-cov-2 by targeting the specific conserved sequence approved by the who [12, 25] . the primers and probes target the orf1ab and n genes of sars-cov-2. covid-19 serological tests were not performed because the test was unavailable during the study period. inability to access medical resources and high costs of pcr testing are potential barriers to implementing universal screening in a general population. this study had several limitations. first, recall bias was inevitable based on the nature of the retrospective study. a record-keeping form implemented at acute respiratory infection clinics would better assist physicians in gathering important information, performing a more thorough review, and collecting as much data as possible. second, risk factors identified in our cohort are more specific for the local population investigated and may not generalizable. we do, however, encourage clinicians to investigate those variables for their relevant populations because lifestyle and exposure may vary among regions. third, some covid-19 patients may have been overlooked, especially those presenting with atypical or non-respiratory symptoms as have been reported in the literature [26, 27] . fourth, our study was covered for only 2-week period due to an epidemiological characteristic of covid-19 in thailand is relatively brief. therefore, we tentatively selected this specific period when the majority of puis attended healthcare facilities for an investigation when the information regarding some specific risk factors was completely collectable and deem interpretable. finally, diagnostic criteria and identification of puis have evolved as more data have emerged. the case definition should be revised based on the current situation in thailand. a previous version of the definition was more specific for imported cases while <7% of puis were detected at airport screening [15] . we also acknowledge incomplete data gathering and examination among those with mild cases, due to an attributable effect of the contagious disease. however, the present study is one of the first conducted in thailand involving an intervention on puis using a screening test to include those at risk of sars-cov-2 infection acquisition. our hospital also, by far, conducts the most virology studies of any institution in thailand. we believe the aforementioned variables could better stratify those at risk in addition to a criterion. more importantly, we encourage a case definition combined with thorough history-taking to improve sensitivity of this definition. a national health authority criterion could potentially predict covid-19 diagnosis during an outbreak setting in thailand. clinicians should be aware of those who have no medical care coverage and present with productive cough as an initial manifestation, and these factors should be included in the criteria to increase sensitivity of diagnosis among suspected cases of covid-19. supporting information s1 a novel coronavirus from patients with pneumonia in china covid-19: who declares pandemic because of "alarming levels" of spread, severity, and inaction fair allocation of scarce medical resources in the time of covid-19 critical supply shortages-the need for ventilators and personal protective equipment during the covid-19 pandemic world health organization. novel coronavirus-thailand (ex-china) 2020. available from world health organization. who thailand situation report photo quiz: a 50-year-old male taxi driver with acute hypoxemic respiratory failure journey of a thai taxi driver and novel coronavirus clinical characteristics of patients hospitalized with coronavirus disease, thailand. emerging infectious diseases case definition for patients under investigation (puis) with covid-19 guidelines for clinical practice, diagnosis, treatment and prevention of healthcare-associated infection in response to patients with covid-19 infection laboratory diagnosis of emerging human coronavirus infections-the state of the art. emerging microbes & infections epidemiology and clinical characteristics of patients under investigation for middle east respiratory syndrome coronavirus infection in thailand epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study novel coronavirus 2019 pneumonia situation: thailand situation importation and human-to-human transmission of a novel coronavirus in vietnam a familial cluster of infection associated with the 2019 novel coronavirus indicating possible person-to-person transmission during the incubation period. the journal of infectious diseases clinical characteristics of coronavirus disease 2019 in china impact of sex and gender on covid-19 outcomes in europe gender differences in patients with covid-19: focus on severity and mortality. front public health clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china acute-onset smell and taste disorders in the context of covid-19: a pilot multicenter pcrbased case-control study sars-cov-2 viral load in upper respiratory specimens of infected patients presumed asymptomatic carrier transmission of covid-19 update on covid-19 in vitro diagnostics listed by national regulatory authoritiesin imdrf jurisdiction 2020 neurological complications of coronavirus disease (covid-19): encephalopathy. cureus cardiac and arrhythmic complications in patients with covid-19 we thank all infection prevention and control nurses and all staffs at acute respiratory infection clinic at the faculty of medicine ramathibodi hospital, mahidol university, bangkok, thailand. conceptualization: jackrapong bruminhent, sasisopin kiertiburanakul.data curation: jackrapong bruminhent, nattanon ruangsubvilai, jeff nabhindhakara. key: cord-344782-ond1ziu5 authors: zhang, jing; finlaison, deborah s.; frost, melinda j.; gestier, sarah; gu, xingnian; hall, jane; jenkins, cheryl; parrish, kate; read, andrew j.; srivastava, mukesh; rose, karrie; kirkland, peter d. title: identification of a novel nidovirus as a potential cause of large scale mortalities in the endangered bellinger river snapping turtle (myuchelys georgesi) date: 2018-10-24 journal: plos one doi: 10.1371/journal.pone.0205209 sha: doc_id: 344782 cord_uid: ond1ziu5 in mid-february 2015, a large number of deaths were observed in the sole extant population of an endangered species of freshwater snapping turtle, myuchelys georgesi, in a coastal river in new south wales, australia. mortalities continued for approximately 7 weeks and affected mostly adult animals. more than 400 dead or dying animals were observed and population surveys conducted after the outbreak had ceased indicated that only a very small proportion of the population had survived, severely threatening the viability of the wild population. at necropsy, animals were in poor body condition, had bilateral swollen eyelids and some animals had tan foci on the skin of the ventral thighs. histological examination revealed peri-orbital, splenic and nephric inflammation and necrosis. a virus was isolated in cell culture from a range of tissues. nucleic acid sequencing of the virus isolate has identified the entire genome and indicates that this is a novel nidovirus that has a low level of nucleotide similarity to recognised nidoviruses. its closest relatives are nidoviruses that have recently been described in pythons and lizards, usually in association with respiratory disease. in contrast, in the affected turtles, the most significant pathological changes were in the kidneys. real time pcr assays developed to detect this virus demonstrated very high virus loads in affected tissues. in situ hybridisation studies confirmed the presence of viral nucleic acid in tissues in association with pathological changes. collectively these data suggest that this virus is the likely cause of the mortalities that now threaten the survival of this species. bellinger river virus is the name proposed for this new virus. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the bellinger river snapping turtle, myuchelys georgesi, is a species of freshwater turtle that, prior to this outbreak, was rare and has a very restricted habitat. it is confined solely to a 60 kilometre section of the bellinger river, and a short section of the adjacent kalang river in northern coastal new south wales (nsw), australia. while the turtle had been described as "locally abundant" it was also described as "meets the criteria for being listed as a vulnerable species under the threatened species conservation act of nsw" [1] . in 2014 it was estimated that there were approximately 2500 of this species in the wild [2] . commencing in mid-february 2015, a number of people using and managing the environment surrounding the bellinger river observed a large number of deaths in m. georgesi. a multi-agency investigation was undertaken to establish the cause and extent of the outbreak. mortalities continued for 7 weeks and involved mostly adult animals. more than 400 dead or dying animals were observed and population surveys conducted after the outbreak had ceased indicated that only a small proportion of the total population had survived with very few adults remaining. no other species, including the sympatric murray river turtle (emydura macquarii), appeared to be affected. full details of the prevailing environmental conditions and the extent of the investigation have been described by others [2] . a number of moribund and dead m. georgesi were collected for post mortem examination at the australian registry of wildlife health, taronga conservation society australia mosman, nsw. tissue samples were referred to a number of different laboratories to test for a wide range of potential pathogens, toxins and water quality assessment. no consistent results were obtained from bacterial cultures and tests for mycoplasma, trichomonas, chlamydia and toxins gave negative results. on the basis of the histological changes and a failure to identify any other causative agent, a viral aetiology was suspected. testing was undertaken at several other laboratories and ranaviruses, adenoviruses, paramyxoviruses (ferlavirus) and herpesviruses were excluded [2] . samples were also referred to the elizabeth macarthur agriculture institute, menangle nsw. here we report the isolation and characterisation of a novel nidovirus and provide evidence for its involvement as the principal pathogen in this disease outbreak. the nsw office of the environment and heritage (oeh) was consulted during the early stages of the investigation and confirmed that, as this was a diagnostic investigation, no animal care and ethics (aec) or other approvals were necessary. when sample collection was undertaken as part of the follow-up epidemiology investigations, all procedures were approved by the aec of the taronga conservation society of australia (aec approval number 3e/10/ 15). twenty one turtles that were either dead or moribund and then euthanased were submitted to the australian registry of wildlife health, taronga conservation society of australia where post mortem examinations were conducted. various samples from each of the 21 m. georgesi were submitted to the virology laboratory at the elizabeth macarthur agriculture institute. swabs had been collected from the buccal cavity (n = 5), conjunctiva (n = 5) and cloaca (n = 5) and placed in 3ml of phosphate buffered gelatin saline (pbgs, ph 7.3) and held at 4˚c. samples of brain (n = 4), conjunctiva (n = 5), kidney (n = 10), liver (n = 10), lung (n = 9), myocardium (n = 5), ovary (n = 2), spleen (n = 10), urinary bladder (n = 5) and serum or plasma (n = 11) (either fresh or after holding frozen at -80˚c) were sent on frozen blocks for 'same day' delivery to the elizabeth macarthur agriculture institute. samples of frozen kidney (n = 5) or liver (n = 3) were also submitted from an archival collection of m. georgesi tissues that had been held at the australian museum, sydney since collection in 1991. suspensions (approximately 20%, w/v) of fresh tissues from affected animals were prepared by homogenising in serum free minimal essential medium (mem), containing penicillin (230μg/ml), streptomycin (250μg/ml) and amphotericin-b (5μg/ml), using a bead beater. the supernatant was collected after centrifugation at approximately 3000g for 20 min at 4˚c and filtered using a 0.45μm syringe filter. a wide range of tissues was also collected and fixed for 48 hours in 10% neutral buffered formalin solution. sections of formalin fixed tissue were cut and stained with haematoxylin and eosin, giemsa and periodic acid-schiff stains using standard methods and examined by light microscopy. sub-confluent monolayer cultures of buffalo african green monkey kidney (bgm) cells [3] initially grown at 37˚c in 10ml cell culture tubes containing mem supplemented with 10% (v/v) foetal bovine serum (fbs), penicillin (115μ/ml), streptomycin (125μg/ml) and amphotericin-b (2μg/ml) were used for the first virus isolation attempts. immediately before use the culture medium was replaced with fresh maintenance medium (mem, 2% fbs and antibiotics) and 200μl of filtered supernatant from tissue homogenate was added to the medium. based on experience with other aquatic pathogens, all cultures were maintained at 25˚c. cells were observed frequently and passaged after 7 days by scraping the cells off the tube surface and adding 200μl of suspension to new sub-confluent monolayers. after changes were observed in the morphology of bgm cell monolayers, tissue culture fluids were also passaged onto sub-confluent monolayers of other mammalian, avian, fish, reptile or mosquito cell cultures. full details of the cell cultures used and the relevant culture conditions are summarised in table 1 . cell culture supernatants were examined by electron microscopy by placing 10μl of sample on parlodion/carbon coated 400 mesh copper grids. after washing briefly with de-ionised water, negative staining was achieved by the addition of 2% aqueous uranyl acetate. the stained specimens were examined in a philips 208 transmission electron microscope. culture supernatant from bgm cell cultures showing advanced cytopathology was clarified by centrifugation at 3,000g for 30 min and passed through a 0.45μm filter. the virus was then pelleted at 25,000g for 2 hours at 4˚c and resuspended in 500μl of nuclease free water. host cell nucleic acids were removed by incubating with dnase i (250u; stratagene) and an rnase cocktail (0.5u rnase a and 20u rnase t1; ambion) at 37˚c for 2 hours. total ribonucleic acids were then extracted and eluted in 20μl of rnase free water using an rneasy minikit (qiagen). a dna library was prepared using a truseq stranded mrna sample prep kit, omitting the poly (a) mrna purification step and sequencing with 150bp paired end reads was completed on an illumina miseq platform at the australian genome research facility (brisbane, australia). raw ngs data has been submitted to the sequence read archive (sra) (accession srp158959) [6] .trimmomatic software [7] was used to determine and remove low quality bases and adapter sequences using a minimum quality score of 20. scaffolds (nodes) were then assembled in velvet optimiser [8] and run in the megablast 2.2.26 software [9] to identify homology with sequences in the genbank database [10] . some scaffolds were identified as having homology with ball python nidovirus sequences. to remove extraneous sequence data only the trimmed sequences that aligned to ball python nidovirus genome (kj541759), using burrows-wheeler aligner [10] , were assembled into scaffolds using velve-toptimiser [8] . the scaffolds were then aligned in sequencher [11] to form a single contig. additional sanger sequencing was completed after further amplification using the primers listed in s1 table. open reading frames (orfs) were determined from the full sequence using open reading frame viewer software [12] . similarity to other viruses for each of the orfs and their predicted amino acid sequences were determined by searches using blastn and blastp [13] algorithms through the ncbi server (http://blast.ncbi.nlm.nih.gov/blast.cgi). a region of the orf1b which was predicted to code for an rna-dependant rna polymerase (rdrp) was selected for comparative studies because this region was found to be the most highly conserved across the torovirinae subfamily. the amino acids of this conserved region of the orf1b, were aligned against other members of the torovirinae and representatives from the other families within the order nidovirales. a phylogenetic tree was produced using the maximum likelihood method based on the jtt matrix-based model [14] with 1000 bootstrap replicates. initial trees for the heuristic search were obtained automatically by applying the neighbor-joining method to a matrix of pairwise distances estimated using a jtt model. the analysis involved amino acid sequences from 44 virus strains. all positions containing gaps and missing data were excluded. there was a total of 268 amino acid positions in the final dataset. phylogenetic analyses and percentage similarities were calculated using clustal w alignment in mega6 [15] . using the methods described above, similar comparisons were made for the conserved putative helicase (342 positions) and m pro c-like (302 positions) domains of polyprotein 1ab, as well as the whole length polyprotein 1a and spike protein. after the detection of a novel rna viral sequence by ngs, qrt-pcr taqman assays were designed to detect nucleic acid sequences that were specific for the novel virus and directed at the sequence encoding the presumptive polyprotein 1a (replicase 1a). the resulting assay was used to test serum and a range of tissue homogenates from affected animals. later, a selection of samples was also tested in an assay directed at the region encoding the 'spike' protein. for testing of samples in these assays, total nucleic acid was extracted using a magnetic bead based kit and a magnetic particle handling system [16] . the details of primers and probe are as follows: the qrt-pcr assays, both of which produced a 77bp product, used agpath mastermix (life technologies) and was run on an abi 7500 thermocycler for 45 cycles under standard conditions as specified by the mastermix manufacturer. on each occasion the assay was run, included on the plate were two positive control samples, representing approximately 10000 (pc1) and 1000 (pc2) copies of viral rna, a negative control (nc, a trna solution) and a 'no template' control (ntc), the latter consisting of nuclease free water. the positive and negative controls were included throughout the procedure from extraction through to the completion of the pcr reactions while the ntc was only added during the pcr setup. an exogenous rna control [16] was also included to monitor the efficiency of the nucleic acid extraction and pcr reaction and to detect the presence of inhibitors. any evidence of rna amplification was recorded and results were expressed as cycle threshold (ct) values as described previously [16] . a conventional rt-pcr was run to produce template for in situ hybridisation (ish) probe production. pcr template was created using conventional pcr primers targeting the gene encoding a putative viral membrane protein (m). primer details were as follows: brvmp forward: 5' atggagtccacctcga 3', brvmp reverse: 5' ttatggtaggatg gctgtt 3'. rt-pcr reactions were prepared using the mytaq one-step rt-pcr kit (bioline) according to the manufacturer's instructions and pfuultra ii fusion dna polymerase (agilent) was added to the reaction at a final dilution of 1/100. the template used (5μl) was purified from the cell culture isolate as described previously. cycling parameters for the pcr assay were a 20 min initial reverse transcription step at 45˚c and a 1 min denaturation step at 95˚c, followed by 40 cycles of denaturation at 95˚c for 10s, annealing at 56˚c for 30s, extension at 72˚c for 30s and a final extension step at 72˚c for 7 min. the resulting template (of approximately 650 base pairs) was visualised using electrophoresis in a 1.5% agarose gel stained with gel red nucleic acid gel stain (biotium). approximately 1ng of pcr product was used in the pcr digoxigenin (dig) ish probe synthesis kit (roche) with thermocycling parameters as described in the kit instructions. the size of the dig-labelled probe was determined using electrophoresis (as described above) and was quantified using a nanodrop spectrophotometer (thermo fisher). a hybaid omnislide thermal cycling system (thermo scientific) was used to maintain humidity and temperature control during all incubation steps. hybrislip coverslips (sigma) were used at every step. sections of tissue 4μm thick were cut onto superfrost plus slides (menzel gläser) and dried at 65˚c for 30 min. slides were dewaxed in xylene and rehydrated in an ethanol series. slides were treated with 100 μl proteinase k (dako) for 15 min at 37˚c, followed by a 3 min tris buffer wash (0.1 m, ph 8.0) and pre-hybridisation for 1h at 37˚c in 100 μl prehybridisation buffer (50% formamide, 1 x denhardt's solution, 4 x ssc, 0.25 mg yeast trna/ml, 10% dextrans sulfate). an exchange was made with 100 μl hybridization buffer containing prehybridisation solution with 5 ng/μl ish probe and slides were heated at 95˚c for 5 min. slides were cooled on ice for 5 min then incubated overnight at 42˚c. the next day slides were washed in wash buffer (0.1 m maleic acid, 0.15 m nacl and 0.3% tween20, ph 7.5) (roche) at 40˚c for 10 min. sections were then blocked with 500 μl blocking buffer (1% roche blocking reagent in wash buffer) at room temperature (rt) for 30 min. blocking buffer was exchanged with a 1/200 dilution of anti-dig antibody (roche) in blocking buffer and incubated for 1 hr. sections were washed for 30 min at rt in wash buffer and equilibrated for 5 min at rt in detection buffer (roche) and incubated with 500 μl bcip/nbt in the dark at rt for 5 h. slides were then rinsed in tap water and counterstained with 0.2% fast green (australian biostain) for 20 sec and mounted in dpx mounting medium (sigma-aldrich). negative control slides included additional tissue sections processed without the ish probe and sections with ish probe applied that were taken from an eastern long neck turtle (chelodina longicollis) that had given negative results by pcr. the latter samples were included in the absence of fixed tissues from presumptively uninfected m. georgesi. following the detection of the novel virus, in november 2015 (about 6 months after the cessation of the outbreak) an intensive survey of the parts of the river where affected turtles had been detected [2] was undertaken by groups of biologists and ecologists and samples collected from a wide range of aquatic species and some terrestrial animals (n = 360) to establish the size of the remaining population and whether any other animals were carrying this virus. a total of 502 samples were collected, consisting of various amphibians, arthropods, fish and reptiles. for animals of sufficient size, swabs were collected separately from mucosal surfaces (usually conjunctiva, oral and cloaca), placed in pbgs and held at approximately 4˚c for up to 10 days before being sent to the laboratory. blood samples were also collected from larger animals (n = 83) and serum was submitted for testing. small invertebrates and small vertebrates were preserved in absolute ethanol. pooled tissues or whole bodies from ethanol fixed animals were prepared for nucleic acid extraction by first digesting in proteinase k solution as described previously [17] . full details of the species, the samples collected and the numbers examined are listed in table 2 . when examined at necropsy, animals were usually in poor body condition, had bilateral swollen eyelids and conjunctivitis. some animals had tan foci on the skin of the ventral thighs. consistent histological findings included severe peri-orbital, splenic and nephric inflammation and necrosis. a proportion of affected animals also had evidence of a fibrinoid vasculopathy. full details of the gross and histopathology will be described elsewhere. when bgm cells were inoculated with pooled homogenates of spleen and lung tissues from 5 animals, after 2 passages cytopathology consisting of lytic destruction of cells in small foci was in subsequent studies, this virus replicated in mdbk cells but without cytopathology. replication could only be detected by pcr and virus loads were lower than those obtained in cv-1 cells. examination of the culture supernatant from cv-1 cells by electron microscopy identified bacilliform particles approximately 170 nm long and 25 nm wide (fig 1) . the name "bellinger river virus" (brv) is proposed for this virus. subsequent to the successful detection of virus in the pool of tissues, further virus isolation attempts were undertaken on a number of individual tissue samples. viruses were isolated in cell culture from heart (10/10 samples), kidney (11/14) , plasma (2/4) and spleen (4/5). the nucleic acid sequencing analysis identified a sequence of 30742 nucleotides of a virus with a genome organisation most closely related to viruses from the family coronaviridae and subfamily torovirinae (fig 2) . this sequence was subsequently confirmed by primer walking and further sanger sequencing to establish what is considered to be the full length sequence (genbank reference mf685025) of a nidovirus. the pattern of orfs of this virus closely matches the pattern observed for other nidoviruses. however, this appears to be a novel nidovirus with less than 50% nucleotide similarity to recognised nidoviruses. phylogenetic analysis using aligned amino acid sequences from the conserved rdrp site of polyprotein 1b (pp1b) confirmed the classification of the virus and places it in the proposed barnivirus (bacilliform reptile nidovirus) genus [18] (fig 3) . very similar results were obtained from comparisons involving the conserved putative helicase and m pro c-like domains of polyprotein 1ab, as well as the whole length polyprotein 1a and spike protein (s1-s4 figs). this new nidovirus appears most closely related to ball python nidovirus 1 [18, 19] , python nidovirus [20] and morelia viridus nidovirus [21] , however these viruses have yet to be assigned to a genus. the highest amino acid sequence similarity was found in the pp1b region (68-69%), while all other proteins had lower levels of similarity (table 3) . blast search also identified lower levels of homology with shingleback nidovirus (sbnv) [22] , a virus of australian shingleback lizards, two virus sequences that were identified from intestinal nematodes in asian snake species [23] , and four virus sequences identified in asian snake species [24] . these viruses are likely to also be classified within the proposed genus barnivirus. in common with most other members of the coronaviridae [25-27] a ribosomal frameshift sequence was identified within the first orf of the turtle nidovirus sequence. an alignment of this region across the possible members of the proposed barnivirus genus is shown in fig 4. watson-crick base pairing of the rna sequence shows a common pseudo-knot motif downstream to the ribosomal frameshift sequence. this pseudoknot secondary structure is postulated to play an important role in the discontinuous synthesis of subgenomic rnas [28] . the structure of this pseudo-knot differs from most other similar structures in coronaviruses by the presence of an additional stem in the region between the first stem and the "kissing stem loop". this predicted structure is common to all of the putative members of the proposed barnivirus genus [18, 29] (fig 5) . the qrt-pcr assay that was developed to detect the presumptive replicase 1a protein coding region of this virus allowed the detection of viral rna in tissues from affected animals. samples of kidney, liver, lung and spleen from the 5 animals that had contributed to the virus isolation pool were tested. very high virus loads were detected in kidneys and spleen (ct range 15.9-16.4) of 2 of the 5 animals but variable and moderately high levels of viral rna were also detected in other tissues of these and the other 3 animals (table 4 , animals 1-5). testing of a wider range of tissues from another 5 dead animals from the outbreak demonstrated high virus loads in heart, kidney, lung and spleen while virus was present in all other tissues at moderate levels ( table 4 , animals 6-10). relatively high levels of viral rna were also detected in oral, conjunctival and cloacal swabs of these animals (table 5 ). serum samples from another 11 animals gave strong to moderate reactivity (ct range 19.4-35.6) ( table 6 ). very similar results were obtained for all samples when tested in the spike protein assay. as the reactivity in both assays was extremely similar, the extracts of the archival samples were only tested in the replicase 1a qrt-pcr and gave negative results. ish was conducted on a selection of affected tissues and confirmed the presence of viral nucleic acid in association with severely necrotic histological lesions. within a severely affected lacrimal gland there was staining of residual glandular epithelial cells and staining within areas of necrotising inflammation. the kidney lesions from two affected turtles showed similar staining in areas containing intense inflammatory infiltrates and necrotic cellular debris, within degenerate or necrotic renal tubule epithelial cells (fig 6a and 6b ) and within foci of vasculitis. staining was also detected within dense foci of necrotising cystitis, as well as in scattered granulocytes in the multifocally oedematous urothelium (fig 6c and 6d) . occasional granulocytes stained within the myocardial interstitium. no staining was observed in the negative control preparations which included tissue sections processed without the probe and tissues from an eastern long neck turtle (chelodina longicollis) that had given negative results by pcr). samples of all animals collected (table 2) were tested in the replicase 1a qrt-pcr assay. of the myuchelys georgesi turtles tested (n = 31), viral rna was detected in conjunctival swabs from eight animals and also in oral swabs from 2 of these animals. ct values were consistently high (ct >31), with most ct >35. low levels of viral rna were also detected in conjunctival swabs from two of the 49 emydura macquarii turtles tested and in 2 of 13 samples of egg casings of unknown species (ct >37 in each instance) ( table 2) . these casings had been adherent to the plastron of 2 m. georgesi. the reactivity detected in each of these samples collected during this field survey was confirmed with similar results obtained when tested in the replicase 1a qrt-pcr. the virus described in this study, for which the name "bellinger river virus" (brv) is proposed, is believed to have had a profound impact on the wild myuchelys georgesi population. the data presented provide strong, indirect evidence that this virus is the principal aetiological agent involved in the deaths of these m. georgesi. unfortunately, as this is a now a critically endangered species of turtle [30], it is not possible to undertake experimental transmission studies to fulfil koch's postulates. nevertheless, the criteria for disease causation defined by fredericks and relman [31] have been met. brv, as a novel nidovirus, was isolated from tissues of diseased animals, very high levels of viral rna were detected in tissues with marked pathological changes and in situ hybridisation assays demonstrated the presence of specific viral rna in lesions in kidneys and eye tissue-two of the main affected organs. no or very low levels of viral rna were detected in normal animals tested at the time of the outbreak. collectively these data suggest that this virus is the likely cause of these mortalities. the high levels of viral rna in several different organ systems would suggest that this virus is actively replicating in these organs and these detections are not an incidental finding or due to contamination as a result of either ingestion or inhalation of virus from the environment. nevertheless, although we believe that brv is the principal pathogen, it is inevitable that other factors are likely to have contributed to the onset and severity of disease. the turtles were probably already in a stressed and potentially immunosuppressed state as they had lost considerable body condition [2] . the higher water temperatures may have supported and perhaps enhanced virus replication, a phenomenon that is well known for a number of aquatic viruses [32, 33] and, although other pathogens have not been identified, it is likely that other microbes, even commensals, may have contributed to disease severity and ultimately the death of these turtles. the m. georgesi population has been reduced to such an extent that the species has now been classified as "critically endangered" [30], with perhaps less than 100-200 animals present in the wild. the survival of the species may be dependent on a captive breeding population [2] due to the very small number of mature adults (possibly 10-15) that have survived in the wild (chessman and jones, pers comm [s1 file]. the impact of bellinger river virus, the constrained geographical location of m. georgesi, the potential for hybridisation with emydura macquarii and perhaps a range of environmental factors [2] have combined to seriously threaten the survival of this species of turtle. there are few documented instances where a pathogen has been directly incriminated in the potential extinction of an animal species [2] and to have such an impact in a very short time period. for example, while the chytrid fungus of batrachochytrium dendrobatidis has resulted in significant declines in amphibian populations in a number of countries and on different continents [34, 35] , its impact on amphibian populations has taken place over almost two decades, admittedly on a broad geographical scale. table 5 . qrt-pcr results for viral transport medium containing swabs collected from mucosal surfaces of affected m. georgesi turtles using an assay detecting a segment of the putative replicase 1a gene. results for samples with very high rna loads (ct<20) shown in bold. these animals correspond to the st 5 animals from which tissues were collected (table 4) . at the time of its identification, this nidovirus was the first of the members of the proposed barnivirus genus (family coronaviridae, subfamily torovirinae) to be associated with disease in an aquatic reptile. other barniviruses have been linked to infection and often disease in terrestrial reptiles. these include infections in ball pythons (python regius) [18, 19, 36] , indian rock python, (python molurus) [20, 36] , burmese python (python bivittatus) [36] , green tree python (morelia viridis) [21] , carpet python (morelia spilota) [36] , boa constrictor (boa constrictor) [36] and shingleback lizards (tiliqua rugose) [22] . in the terrestrial reptiles, the predominant clinical presentation has been respiratory disease [18] [19] [20] [21] [22] 37] . experimental infections undertaken in ball pythons [37] have also demonstrated a tropism for the respiratory tract. in this respect, brv differs in that, while there are pathological changes in several organ systems, the most severe changes are in the kidneys, corresponding to the high viral loads detected. it is interesting to note that there was only evidence of brv replication in vitro in kidney cell cultures. while there was evidence of visible cytopathology in primate kidney cell cultures, virus replication was also detected in mdbk cells when culture supernatants were tested by qrt-pcr. the tropism of this virus for kidney cells would suggest that renal disease was a key factor contributing to the death of these turtles. chelonian species have been reported to be infected by a wide range of viral taxa including adenoviruses, bunyaviruses, flaviviruses, herpesviruses, paramyxoviruses, picornaviruses, ranaviruses, retroviruses and togaviruses [38, 39] . a virus in the order nidovirales, family arteriviridae, has been identified in the chinese softshell turtle, pelodiscus sinensis, during an outbreak of severe haemorrhagic disease affecting multiple organs including gonads, intestine, kidney, liver, lung, and spleen [40] and, recently, partial sequence of another virus from the arteriviridae was detected by nucleic acid sequencing of the gut, liver and lungs of an apparently normal chinese broad headed pond turtle (mauremys megalocephala) [24] . based on the extent of the epizootic in the bellinger river, it would appear that brv was introduced into a naive population. the origin of brv is yet to be determined as there was no evidence of virus in other species sampled from the affected area, except for the detections of low levels of brv rna in emydura macquarii, a closely related species that can interbreed with m. georgesi and in 2 clusters of egg casings. in each instance the viral rna levels were close to the limit of detection and are of uncertain significance. the possibility that these are the result of superficial contamination from the environment cannot be excluded, and is likely for the egg casings as they were removed from the carapace of m. georgesi turtles in the bellinger river. brv is the first nidovirus in the proposed barnivirus genus that has been isolated from a non-squamatid reptile and is phylogenetically placed between the recognised python nidoviruses and the shingleback lizard nidovirus. this may indicate that the barniviruses are quite widespread among both aquatic and terrestrial reptile species. the apparent abundance of reptilian nidoviruses would suggest that this turtle virus may have originated in a snake, lizard or other reptile and that m. georgesi could be an incidental host. however, as detections of viruses from the order nidovirales have also been reported in fish [41] [42] [43] [44] [45] and more distantly related viruses even in insects [46] [47] [48] , there are many potential reservoirs for this virus. finally, from a practical perspective, the detection of viral rna in cloacal and ocular swabs, serum and plasma indicates that these are samples that could be collected from live endangered animals for future screening or surveillance. these sample types have already been used to select a group of presumptively virus-free animals to establish a captive breeding colony [2] that remains healthy and apparently free of virus after 2 years in isolation. serum, conjunctival and cloacal swabs from the animals in this captive colony have given negative results in the brv qrt-pcr assays on several occasions over this 2 year period. however, the development of serological assays to detect 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nidovirus infects spotfin shiner cyprinella spiloptera and golden shiner notemigonus crysoleucas identification and characterization of genetically divergent members of the newly established family mesoniviridae discovery of the first insect nidovirus, a missing evolutionary link in the emergence of the largest rna virus genomes an insect nidovirus emerging from a primary tropical rainforest we acknowledge staff of the nsw office of environment and heritage, particularly gerry mcgilvray and shane ruming for their coordination of the disease response. the nsw office of environment and heritage, nsw department of primary industries, nsw local lands services and taronga conservation society australia provided considerable financial and logistical support. we also thank bellingen shire council, dr mark crane and his staff at bellingen veterinary hospital, adam skidmore, and paul thompson and herpetology staff from taronga, and ecologist bruce chessman for their considerable contributions to the disease response. the willingness of scott ginn and colleagues from the australian museum, sydney to provide valuable tissues from archived m. georgesi is greatly appreciated. we are indebted to the staff of virology laboratory who assisted with sample preparation, cell culture and virus isolation studies and for assistance with the pcr assays. key: cord-310466-0lbbiq7u authors: fu, yang-chih; wang, da-wei; chuang, jen-hsiang title: representative contact diaries for modeling the spread of infectious diseases in taiwan date: 2012-10-03 journal: plos one doi: 10.1371/journal.pone.0045113 sha: doc_id: 310466 cord_uid: 0lbbiq7u recent studies of infectious diseases have attempted to construct more realistic parameters of interpersonal contact patterns from diary-approach surveys. to ensure that such diary-based contact patterns provide accurate baseline data for policy implementation in densely populated taiwan, we collected contact diaries from a national sample, using 3-stage systematic probability sampling and rigorous in-person interviews. a representative sample of 1,943 contact diaries recorded a total of 24,265 wide-range, face-to-face interpersonal contacts during a 24-hour period. nearly 70% of the contacts occurred outside of respondents' households. the most active age group was schoolchildren (ages 5–14), who averaged around 16–18 daily contacts, about 2–3 times as many as the least active age groups. we show how such parameters of contact patterns help modify a sophisticated national simulation system that has been used for years to model the spread of pandemic diseases in taiwan. based on such actual and representative data that enable researchers to infer findings to the whole population, our analyses aim to facilitate implementing more appropriate and effective strategies for controlling an emerging or pandemic disease infection. many policy implementations for disease control rely on modeling how infectious diseases spread. to increase the effectiveness of such modeling, one of the most comprehensive efforts seeks to collect empirical data by means of large-scale probability surveys and infers the findings to the population upon which a policy is to be implemented. such a task becomes more urgent for densely populated societies that may be more susceptible to a pandemic outbreak. in particular, recent studies of infectious diseases have constructed more accurate parameters of interpersonal contact, based on diary-approach surveys from both europe [1] [2] [3] [4] and asia [5] . other studies have used web-based surveys [6] or timeuse methods [7] . to ensure that such diary-based contact patterns provide accurate baseline data for policy implementation, we collected contact diaries from a representative national sample in taiwan and extracted realistic parameters for our simulation modeling of disease infections. using empirical contact data to model how diseases spread has two major policy implications. first, unlike earlier studies, largescale surveys enable researchers to derive and incorporate more accurate parameters into their models [8] [9] . earlier modeling of disease infections, for example, estimated that an average person had contact with either 10 or 20 people at work in a typical day. the estimated number of contacts at school ranged widely from 20, 79, 128 to 155 [10] [11] [12] . whether based on simulations or small samples of interviews, such estimates may not reflect overall contact patterns in the whole population. more recent attempts, however, reveal how people actually interact under various circumstances, which helps predict possible trends of disease infections. second, diary-based large surveys help distinguish contact patterns not only among various social groups within a population, but also across different countries. comparative european surveys, for example, have found that residents in southern europe have more interpersonal contacts than those in northern europe [1] . another survey revealed fewer contacts among vietnamese [5] . such different contact patterns serve to inform researchers and policy makers by helping them model how diseases may spread and rethink how certain intervention strategies may work. the latest attempts have helped approach the ultimate goal of clarifying how infectious illnesses actually spread among people. a critical point that determines the success of such attempts lies in how rigorously they follow probability sampling and conduct the interviews, both necessary conditions for collecting a sample that can represent the whole population. recent diary-based large surveys have relied mostly on quota sampling [1] [2] , partly because any systematic sampling of a national population has been inherently sophisticated and the data collection tedious and costly. when a survey's respondents all live in rural areas [5] , furthermore, the findings will be less compatible with those of other studies. our diary-based survey in taiwan emphasizes the rigorous requirements on both systematic probability sampling and in-person (face-to-face) household interviews to ensure proper inference to the population as a whole. only when the modeling of disease infections relies on surveys that target the right populations and apply rigorous procedures in sampling schemes and data collection can policy makers implement effective measures for disease control. our data were drawn from a survey conducted in 2010 in taiwan. at the time, no internal review board or research ethics committee was established in taiwan to review ethics of studies in social and behavioral sciences (pilot research ethics committees for such a purpose are being tested by the national science council as of spring 2012). being fully aware of possible ethics issues involved in our study, however, we took the following steps to protect our survey respondents and anyone mentioned in the survey. first, we used the personal information from our sample list, as regulated by the law, only to locate and identify our targeted respondents and then destroyed the name list after the interviews. second, all interviews required respondents' prior written informed consent (or that by the parents of respondents who were under 18 years old). when a targeted respondent was younger than age 9, a parent or a guardian who knew the child well answered the questions on the child's behalf. third, we kept all completed questionnaires under strict anonymity and confidentiality, left no identifiable personal information (such as names of respondents or anyone listed in the contact diaries) in our electronic data set, and destroyed the completed questionnaires after data cleaning. as one of asia's developed economies and emerging democracies, taiwan presents an interesting case for studies in disease infection. first of all, taiwan is densely populated (about 634 persons per square kilometer in 2010), yet the infrastructure of public health has been well established [13] [14] . with its long and extensive experiences with large-scale social surveys, partly facilitated by the comprehensive household and population registration system, taiwan has conducted the most interviews of any general social survey series in the world [15] . this study follows that tradition in sampling and field interviews, while adding questions about disease infections into the survey designed mainly for 24-hour contact diaries. under the influence of the chinese culture that overly stresses guanxi (or relationship) [16] [17] , taiwan's residents tend to make contact with others often in their everyday lives. asking questions about interpersonal contacts thus turns out to be significant not only for research on disease infections but also for more general social issues [18] . under such circumstances, taiwan offers an opportunity for exploring how rigorous diary-approach surveys may further facilitate researchers' and policy makers' understanding of how infectious diseases spread. fielded in spring 2010, our survey aimed to gain insight into the patterns of interpersonal contact among taiwan's 23 million residents. the timing of the survey was crucial, given the pandemic h1n1 influenza that caused world-wide concerns in 2009. taiwan's residents were particularly alert, in the shadow of having endured the most confirmed cases of severe acute respiratory syndrome (sars) outside of hong kong and mainland china during the 2003 outbreak [19] [20] . the core survey instrument was a 24-hour contact diary log that recorded every face-to-face interpersonal contact the respondent had made during the past 24 hours. to capture the possible channel of air-borne transmission of influenza, we asked respondents to record physical contacts and those nonphysical contacts with verbal communication made within 2 meters. part of the diary log followed those used in recent studies elsewhere [1] [2] [3] [4] [5] . this instrument went through a series of modifications after cognitive interviews and a pretest. to ensure that our survey results are representative, we targeted the whole registered population in taiwan without any age limit. the survey sampling and in-person household interviews were both conducted by the academia sinica, taiwan. to obtain a representative sample of taiwanese residents, we followed the sampling procedures used in the well-established taiwan social change survey (tscs) [21] [22] . we first categorized all of taiwan's 358 towns and cities into 7 strata based on factor analyses of 6 demographic and socioeconomic indices at the town/city level (number of persons per square kilometer, % of college graduates among those over 15 years old, % of those over age 65, % of those aged 15-64, % of those working in the manufacturing sector, and % of those working in the service sector, all taken from census and household registration data). with a goal of collecting 1,900 successful cases (which is more than sufficient for statistical inference and analyses), we followed the rule of ''probability proportionate to size'' (pps) to set the number of our targeted respondents in each of these 7 strata, making sure that these numbers were in proportion to the subpopulations. according to this sampling framework, we used systematic sampling in each of the following three stages to determine our targeted respondents: (1) we first selected 34 out of the 358 towns and cities grouped by strata, (2) from each sampled town/city we then picked 2 villages/precincts, and (3) in each of the 68 villages/ precincts, we randomly selected 28 to 86 residents directly (skipping the household sampling), depending on the numbers of targets we set earlier. to achieve our goal in the third stage, the aforementioned institutes asked the ministry of the interior to provide a computer file that contained all individuals listed in the household and population register (with names, gender, birth dates, and addresses) in each of the 68 targeted villages/precincts. following strict survey standards, no substitute respondents were allowed. as a result, we sampled 4,207 residents as our targeted respondents. these targets ranged from 1.4 to 4.0 times more than the expected successful sample size, varying among different towns and cities according to the average response rates from various nationwide surveys over the past 5 years. a total of 41 trained interviewers visited respondents' residences to conduct face-to-face interviews to collect both individual background information and details about all interpersonal contacts during the past 24 hours. when the interviewers could not reach targeted respondents on the sample list, they were instructed to go back to each address at least three more times (in different time slots and on different days of the week) before giving up on the interview. to examine how the empirical data of interpersonal contact may contribute to modeling, we formulate the findings from our survey and apply them to a pandemic flu simulation system developed at academia sinica, taiwan. the detailed implementation of the simulation system has been described elsewhere [23] . we construct contact patterns and social-mixing groups based on the average number of contacts, duration of each contact, and frequency of contact for all age groups. to facilitate easier modeling, we first convert each category of contact duration into mean contact duration. for example, we use 2.5 minutes for ''less than 5 minutes,'' 12 minutes for ''5-19 minutes,'' 37 minutes for ''15-59 minutes,'' 150 minutes for ''1 to 4 hours,'' and 240 minutes for ''more than 4 hours.'' for the same reason, we create mean contact frequency, a specific fraction for each category of contact frequency, such as 1 for ''daily,'' 0.5 for ''almost daily,'' 1.5/7 for ''once or twice weekly,'' 1.5/30 for ''once or twice monthly,'' 0.5/30 for ''less than once per month,'' and 1/365 for ''never.'' we then calculate weighted contact duration by multiplying mean contact duration by mean contact frequency. next, by dividing weighted contact duration by 1,440 (the number of minutes in a day), we obtain weighted contact probabilities. based on the same principle, we are also able to calculate interpersonal weighted contact probabilities in various social-mixing group settings. finally, we reconstruct contact probabilities between age groups under various ''contexts'' of contact (household, household cluster, etc.) [11, [24] [25] , combining the aforementioned age groups into a simplified classification of age groups. the simplified classification divides all respondents into only 2 groups: child (ages 0-18) and adult (age 19 and over). to reveal more details, we also compute contact probabilities for child 0-4, child 5-19, and adult 19-64, respectively. to perform a comparative analysis of the simulation results with findings from previous studies, we further introduce a scale factor to adjust each of the contact probabilities in the various groups to a reasonable range. the case with which we compare is a simulation model for pandemic influenza in the united states (as reported in proceedings of the national academy of sciences in [11] , hereafter pnas). based on these realistic parameters, we perform 100 baseline simulations with a basic reproduction number (r 0 ) of 1.6 for each of the 2 sets of contact probabilities, and calculate the average daily new clinical cases of all 100 simulated results. after two-and-a-half months of fieldwork, we completed 1,954 successful interviews. the respondents (or their parents or guardians) provided 1,943 contact diaries that recorded every person with whom they had contacted during the past 24 hours (up to a maximum of 40 persons, a cutoff set to limit time and space). the ''crude'' (or ''minimum'') response rate was about 46.4% (or 51.3% after excluding ineligible cases such as wrong addresses, etc.)(the response rates are calculated by international standards, cf. [21] [22] . using the 2010 household and population register as the criterion, a test of goodness of fit shows that our targeted sample and taiwan's total population did not differ in any of the distributions for gender, age groups, or the interaction between gender and age groups (p = 0.831, 0.715, and 0.862, respectively). our completed sample (n = 1,954) was also evenly distributed between males (50.9%) and females (49.1%), although the age distribution was a little skewed towards both ends. the median age was 37, ranging from 0 to 97, with an interquartile range (iqr) from 19 to 54. to modify for the underrepresented subsamples (for ages 21-40), we used a weighted sample in further analyses. respondents were well distributed over all geographic regions and across different urbanization levels (about 13.6% rural and 86.4% urban) that matched the distributions of the total population. the household size averaged 4.5. for those over 18 years old (adults), 56.4% were married, and 37.6% received at least college education. about 37.9% of adults were service-sector workers, and 23.8% were manual workers; others were either students (5.5%) or not working (32.8%). our 1,943 contact diaries recorded a total of 24,265 contacted persons during the past 24 hours. the median age of these contacted persons was 33, with an iqr range from 17 to 48. as among the respondents, young adults (ages 20-29) were also underrepresented among the contacted persons. furthermore, subsamples at both ends of the age structure were even smaller, indicating that our respondents rarely made contact with the very old and the very young. much content of the contact diaries indicates that interpersonal contact often occurs with those in one's immediate social circles. for example, about 25.8% of contacted persons were family or household members, and 36.9% were related to one's school or workplace (table 1 ). about two thirds of these persons had contact with the respondents on a daily basis. nearly one third of the contacts involved a physical contact or lasted over 4 hours. a little more than half of the contacts also took place within 1 km from home. despite such an indication of contact with strong ties, the contact diaries also reveal extensive contacts with those beyond one's core social networks. in particular, nearly 70% of contacts occurred away from respondents' homes (such as at school, in the workplace, on public transportation, during leisure time, etc.). about 17.4% of contacted persons had maintained infrequent contact with respondents (less often than weekly). the most common and fundamental indicator used in modeling disease infections has been the average number of persons contacted by an individual or a group. with an upper cut off at 40, the average number of persons in our 24-hour diaries reaches 12.5 (s.d. = 9.3), which is close to that in european countries (13.4) yet much higher than in vietnam (7.7) [2, 5] . within taiwan, most regional differences are not significant (for example, the average number of contacts is 12.4 in the north and 12.3 in the south), except for those living in the more remote east (only 9.5). as shown in figure 1 , an average male respondent contacts 12.7 persons, only slightly more than the average female respondent (12.2) . age groups, however, vary extensively on how many people are contacted each day. for example, infants, toddlers, and preschoolers (under age 5) have contact with only about 10 persons per day. those over age 60 also contact fewer than 10. in contrast, middle-aged adults (particularly those in their 30 s and 40 s) seem to be well connected with others through face-to-face contact, although the males in their 20 s are not as active. the most active age group of all is schoolchildren (ages 5-14), who average around 16-18 contacts. these super spreaders of influenza viruses make 2-3 times as many contacts as the least active age groups [3, 8, 12] . furthermore, about half of these children's contacts involve a touch or other kind of physical contact. a comparison of the findings of our study with those of a previous pnas study in the united states is summarized in table 2 . after implementing the simulations, we can clearly determine that the adjusted contact probabilities would result in a slightly faster and more pronounced epidemic outbreak in taiwan ( figure 2) . a similar pattern can also be observed from the distribution of group infection cases, as shown in figure 3 . diary-based surveys help generate realistic parameters for simulating how influenza-like diseases may spread. being constructed from 24-hour diaries, patterns of interpersonal contact have been particularly helpful in revealing the underlying factors that influence the scope, direction, and speed of disease infections. because interactions vary by social norms, which often differ from culture to culture, it is essential to design policies based on countryspecific empirical data. in pursuit of such a goal, recent attempts at collecting comparative survey data represent a major step towards policy implementations that target the right populations. to extend these latest efforts, this paper focuses on how to collect and analyze contact diaries from a probability sample that is representative of the national population. we then use the representative contact diaries to construct simulation models. in particular, we model disease infections with matching age groups between infectors (diary keepers) and infectees (contacted persons), and with various occasions of contact. our findings suggest that, compared to other studies, the representative contact diaries in taiwan yielded a wider range of interpersonal contacts. for example, a recent study in rural vietnam collected 865 contact diaries showing that 85% of all contacts occurred at home, and 93% were with those who kept in touch on a daily basis [5] . with only 30.9% of contacts taking place at home and 66.5% interacting daily in our case, our systematic sampling helped obtain a sample that displays more diverse contact patterns in a highly urbanized society. compared with findings from european surveys, however, our study reveals less physical contact even in the private realm, a pattern similar to vietnam. for example, about 53% of home contacts in taiwan (45% in vietnam) were physical, which is significantly lower than in european societies (75%). of the contacts that took place daily, only 41% involved physical touch, which was nearly identical to the finding in vietnam (40%) but again much lower than in europe (60%) [2, 5] . physical contacts among the taiwanese were also less common in public realms: about 43% of school contacts and 27% of leisure contacts were physical in taiwan, compared to 50% for both occasions in europe [2] . although contact patterns in various settings imply different risks of disease infection, cross-national variations further help explain how societies differ in influenza infections. as documented in recent studies that used diversified contact parameters for social groups [1] [2] [3] [4] [5] [6] , any efforts to model realistic disease infections should take into account how various groups differ in contact patterns in everyday life. such variations also play a critical role in simulation modeling. our attempt helps modify a sophisticated national simulation system that has been used for years to model the spread of pandemic diseases in taiwan. the results brought by the new parameters also show noticeable differences from similar modeling based on data from the united states. given the nature of disease infections, the simple diary-based approach shall continue to be the most comprehensive method to collect details about face-toface interpersonal contacts in everyday life. the difficulties of data collection may be eased by the use of more technically advanced methods, such as online surveys. nonetheless, because such surveys tend to undersample technically disadvantaged groups (who may also be more or less vulnerable to disease infections), one should pay special attention to the validity of statistical inferences based on data collected from the use of such methods. more country-specific findings are expected as an increasing amount of comparable diary-based probability survey data becomes available from other countries. for example, preliminary analyses show that the taiwanese have a different cross-generational pattern of interpersonal contact. while european diary-based studies reveal frequent interactions between children and adults about 30-40 years apart [1] [2] [3] [4] , taiwan diaries show additional and noticeable interactions between age cohorts that are about 60 years apart. this latter pattern suggests that the living or parenting arrangements in asia may differ from those in the west. it is not uncommon in taiwan for people from three generations to live under the same roof, or for grandparents to help take care of their young grandchildren on a daily basis. in such a circumstance, both care givers (seniors) and care takers (toddlers or young children) may well belong to the age groups that are also most susceptible to influenza infections. does this special feature of age composition within the household contribute to any divergent cross-cultural patterns of disease infections? when contact patterns in everyday life are subject to such cultural norms, policy makers should make the best use of diary-generated empirical data and design intervention strategies accordingly. in the wave of facing the global threat by new influenza (such as pandemic h1n1 2009) and other infectious diseases, taiwan and other countries have implemented more robust infrastructure and nationwide surveillance systems for disease control [26] [27] [28] . to further strengthen intervention strategies and other policy implementations, it is crucial to apply sociological tools about probability sampling and direct recordings of actual human behaviors via contact diaries. a critical value of these tools lies in their capability to make statistical inferences from a representative sample to the target population. although probability sampling has been widely used in large social surveys, our study combines rigorous procedures in both systematic sampling and in-person household interviews with 24-hour diary recording. unlike small scale studies of contact diaries that aim to build long-term actual and comprehensive archives of interpersonal contacts [29] [30] [31] , our large-scale probability survey helps generate contact diaries that are short-term yet representative. with actual data and a better understanding of contact patterns, such studies should help efforts to implement more appropriate and effective strategies in controlling an emerging or pandemic disease infection. contact profiles in eight european countries and implications for modeling the spread of airborne infectious diseases 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agents advances in health care in taiwan: lessons for developing countries does universal health insurance make health care unaffordable? lessons from taiwan social science research and the general social surveys social connections in china: institutions, culture and the changing nature of guanxi gifts, favors, and banquets: the art of social relationships in china measuring personal networks with daily contacts: a single-item survey question and contact diary the impact of the sars epidemic on the utilization of medical services: sars and the fear of sars the evolution of the taiwan social change surveys different survey modes and international comparisons efficient simulation of the spatial transmission dynamics of influenza estimating infectious disease parameters from data on social contacts and serological status matrix models for childhood infections: a bayesian approach with applications to rubella and mumps design of a robust infrastructure to monitor the safety of the pandemic a(h1n1)2009 vaccination program in taiwan historical review of pandemic influenza a in taiwan nationwide surveillance of influenza during the pandemic (2009-10) and postpandemic (2010-11) periods in taiwan contact diaries: building archives of actual and comprehensive personal networks social networks in post-soviet russia: continuity and change in the everyday life of st we thank team members of the social mixing for influenza like illness (smili), an international joint project led by john edmunds and jonathan read, for sharing research ideas and instruments. part of the questionnaire was based on methods used in an earlier polymod social contact study. we also thank szu-ying lee and max chern for helping with data analyses and manuscript preparation. conceived and designed the experiments: ycf dww jhc. performed the experiments: ycf. analyzed the data: ycf dww. contributed reagents/ materials/analysis tools: ycf dww. wrote the paper: ycf dww. restructure paper contents and formats: jhc. key: cord-333248-5342lyeu authors: elenius, varpu; palomares, oscar; waris, matti; turunen, riitta; puhakka, tuomo; rückert, beate; vuorinen, tytti; allander, tobias; vahlberg, tero; akdis, mübeccel; camargo, carlos a.; akdis, cezmi a.; jartti, tuomas title: the relationship of serum vitamins a, d, e and ll-37 levels with allergic status, tonsillar virus detection and immune response date: 2017-02-24 journal: plos one doi: 10.1371/journal.pone.0172350 sha: doc_id: 333248 cord_uid: 5342lyeu background: tonsils have an active role in immune defence and inducing and maintaining tolerance to allergens. vitamins a, d, and e, and antimicrobial peptide ll-37 may have immunomodulatory effects. we studied how their serum levels were associated with allergy status, intratonsillar/nasopharyngeal virus detection and intratonsillar expression of t celland innate immune response-specific cytokines, transcription factors and type i/ii/iii interferons in patients undergoing tonsillectomy. methods: 110 elective tonsillectomy patients participated. serum levels of vitamins a, 25(oh)d, and e, ll-37 and allergen-specific ige as well as nasopharyngeal/intratonsillar respiratory viruses were analyzed. the mrna expression of ifn-α, ifn-β, ifn-γ, il-10, il-13, il-17, il-28, il-29, il-37, tgf-β, foxp3, gata3, rorc2 and tbet in tonsils were analyzed by quantitative rt-pcr. results: the median age of the patients was 16 years (range 3–60), 28% of subjects had atopy, and 57% carried ≥1 respiratory virus in nasopharynx. detection of viruses decreased by age. higher vitamin a levels showed borderline significance with less viral detection (p = 0.056). higher 25(oh)d was associated with less allergic rhinitis and atopy (p < 0.05) and higher vitamin e with less self-reported allergy (p < 0.05). in gene expression analyses, 25(oh)d was associated with higher il-37, vitamin a with higher ifn-γ and vitamin e with less il-28 (p < 0.05). ll-37 was associated with less foxp3, rorc2 and il-17 in tonsils (p < 0.05). conclusions: vitamin d and e levels were associated with less allergic disorders. vitamin a was linked to antiviral and vitamin d with anti-inflammatory activity. ll-37 and was linked to t regulatory cell effects. epidemiologic and multiple observational studies suggest that deficiencies of vitamins a, d and e may be associated with development of asthma and allergic disorders [1] [2] [3] [4] . it was found in several studies that vitamin a deficiency is associated with a higher risk of asthma [5] [6] [7] , but randomized trials with vitamin a supplementation were less supportive [8, 9] . prospective studies have shown that vitamin d supplementation reduces the risk of recurrent respiratory infections, virus-induced wheezing and asthma exacerbations although some of the studies have shown conflicting results [10] [11] [12] [13] [14] . vitamin d is known to induce antimicrobial peptide ll-37, which has anti-viral, -bacterial and -fungal effects [15] . maternal vitamin e intake during pregnancy has been negatively associated with wheezing and eczema in children of atopic mothers [16, 17] . we determined serum levels of vitamins a, d, and e and antimicrobial peptide ll-37 in patients undergoing tonsillectomy. tonsils are the first contact point of the immune system to various infectious agents, food and aeroallergens [18] and they have an active role in inducing and maintaining tolerance to various allergens [19] . however, it is not known how they regulate these functions. we studied how serum vitamins and antimicrobial peptide ll-37 levels and allergic and tonsillar diseases were associated with direct in vivo detection of respiratory viruses and t cell subset-related transcription factors, cytokines as well as type i, ii and iii interferons in tonsils. human tonsil samples were obtained from 110 elective tonsillectomy patients (table 1) from satakunta central hospital, pori, finland, from april 2008 to march 2009 and biobanked. tonsillectomy was done according to clinical indications. written informed consent was obtained from the study patients and/or their guardians. the ethics committee of turku university hospital approved the study. all patients filled a standard questionnaire to obtain information of their allergic diseases and respiratory symptoms. atopy was defined as positive immunoglobulin e (ige) antibody (>0.35 ku/l) to any of the following allergens: codfish, cow's milk, egg, peanut, soybean, wheat, cat, dog, horse, birch, mugwort, timothy, cladosporium herbarum or dermatophagoides pteronyssinus (phadiatop combi 1 , phadia, uppsala, sweden). animal sensitization was defined as positive ige antibodies to cat, dog, horse or dermatophagoides pteronyssinus. birch, mugwort, timothy and cladosporium herbarum were considered as pollen aeroallergens. the eczema was defined as atopic eczema, if a child was atopic and had typical symptoms that included pruritus, typical morphology and chronicity of atopic eczema (hanifin and rajka diagnostic criteria for atopic dermatitis). vitamins and ll-37 vs. allergy, virus infection and tonsillar immune response serum samples were taken before surgery. tonsillectomy was performed according to clinical routine. tonsil tissue was immediately cut in 3-4 mm cubes in sterile conditions, stored in rnalater rna stabilization reagent (qiagen, hilden, germany), incubated at 2-8˚c until the next working day and stored in -80˚c after removal of the non-absorbed reagent [20] . the nasopharyngeal aspirate samples were obtained during the operation using a standardized procedure as previously described [21] . both nasopharyngeal aspirate and sera were stored in -80˚c before analyses. retinoic acid (vitamin a) and alpha tocopherol (vitamin e) levels were determined by highperformance liquid chromatography (hplc) in the vita laboratory, helsinki, finland. serum total 25(oh)d measurement was done using an immunoassay (abbott architect, chicago, usa) and ll-37 was measured using elisa (hycult biotech, uden, the netherlands), both in massachusetts general hospital, boston, usa. bioavailable levels of 25(oh)d were estimated using additional serum measurements (d-binding protein and albumin) and published formulae [22] . serum specific ige levels against common airborne and food allergens were determined by using a fluoroenzyme immunoassay (cut-off for specific allergens 0.35 ku/l; immunocap, phadia, uppsala, sweden) in turku university hospital, turku, finland. [20] . a nasopharyngeal aspirate sample was suspended into 1 ml of pbs, and nucleic acid was isolated from 550 μl of the suspension using nuclisense easymag automated nucleic acid extractor (biomerieux, boxtel, the netherlands) with on-board lysis. intratonsillar samples (approximately 300 μg each) were homogenized and the total rna was isolated from tonsil tissues as previously described [20] . reverse transcription was performed with the revert aid m-mulv reverse transcriptase (fermentas, st. leon-rot, germany) using random hexamer primers according to the manufacturers protocol. we analyzed intratonsillar mrna expression levels of the cytokines and transcription factors related to t subsets cells relevant to allergic responses as well as type i/iii interferons related to antiviral responses ( table 2) . gene expressions of ifn-α, ifn-β, ifn-γ, il-10, il-13, il-17, il-28, il-29, il-37, tgf-β, foxp3, gata3, rorc2 and tbet were analyzed by quantitative real-time as previously described [20] . elongation factor 1α (ef1α) was used as a housekeeping gene. data are shown as relative expressions, which show 2 -(δct) values multiplied by 10 4 , where δct corresponds to the difference between the ct value for the gene of interest and ef1α. statistical analysis continuous variables were described as means (sds) or medians (interquartile ranges) when appropriate, and categorical variables as frequencies and percentages. the subjects with and without serum samples were compared using mann-whitney u-test and chi-square test. correlations were calculated using spearman rank-order correlations coefficients due to mainly skewed distributions. the associations of serum levels of vitamins and ll-37, allergy status and virus detection with intratonsillar cytokine and transcription factor expressions were analyzed using univariable and age-adjusted linear regression. the modifying effects of age (<16 vs. !16 years) and indication of tonsillectomy (recurrent tonsillitis vs. hypertrophic tonsils) on the associations were also examined. analyses were also adjusted for smoking. before analyses, vitamin d and ll-37 levels and gene expression values were log transformed because of positively skewed distributions. statistical significance was established at the level of p < 0.05. statistical analyses were done using sas system for windows (version 9.4, sas institute inc. cary, nc, usa). initially, tonsil samples were available from 143 patients and analysed for clinical data, nasopharyngeal/intratonsillar virology and intratonsillar gene expression. serum samples were available from 110 subjects of these, who were included in the study. the subjects without serum samples did not differ from the analytic cohort in regard to age, sex, allergy or nasopharyngeal/intratonsillar virus detection (all p > 0.1). the median age of the study subjects was 16 years (range 3-60) and 45% were males. main indications for tonsillectomy were hypertrophic tonsils (43%), recurrent tonsillitis (38%), other indications (5%) or a combination of these indications (14%) ( table 1 ). altogether, 51% of patients had self-reported allergy and 28% had atopy, 27% had physician-diagnosed allergic rhinitis, 14% physician-diagnosed atopic eczema and 12% physician-diagnosed asthma (table 1) . seventeen % of patients had respiratory symptoms on the operation day (table 1) . the median level for serum vitamin a was 1. both vitamin a and e levels increased by age (p <0.0001) (fig 1a and 1b) , but serum bioavailable 25(oh)d levels slightly decreased by age (p = 0.02) (fig 1c) . total 25(oh)d and antimicrobial peptide ll-37 levels did not vary by age (p = 0.57) (s1 fig) . in the nasopharyngeal aspirates, 57% of the patients had at least one virus and 23% had 2 or more viruses (fig 2a) . rhinovirus (47%) was the most prevalent virus, followed by bocavirus-1 (14%), adenovirus (9%), enteroviruses (8%), coronavirus (6%) and other viruses (<3% each) (fig 2a) . in tonsils, 25% of patients had at least one virus and 6% had 2 or more viruses (fig 2a) . bocavirus-1 was detected in 7%, adenovirus and enteroviruses in 8%, and parainfluenza and rhinovirus in 4% of the tonsils (fig 2a) . virus detection rates strongly decreased by age (both p < 0.0001) (fig 2b) . overall virus prevalence was 100% in children under age 5 years, but only 13% after 40 years of age ( fig 2b) . in age-adjusted analyses, higher vitamin a tended to associate with less nasopharyngeal virus detection (p = 0.056) (fig 3a) . lower bioavailable 25(oh)d levels were associated with allergic rhinitis (p = 0.046) ( fig 3b) and lower vitamin e levels with self-reported allergy (p = 0.0086) (fig 3c) . lower total (p = 0.036) and bioavailable 25(oh)d (p = 0.0031) levels were associated with atopy (fig 3d and 3e, respectively) . no other significant associations were found. age or indication for tonsillectomy did not effect on these clinical associations. in age-adjusted analysis, we observed that higher bioavailable 25(oh)d levels were associated with higher expression levels of newly discovered anti-inflammatory cytokine il-37 (p = 0.024) ( fig 4a) and higher vitamin a levels were associated with higher expression of ifn-γ (p = 0.043) (fig 4b) . higher vitamin e levels were associated with lower il-28 expression (p = 0.016) (fig 4c) . in addition, higher serum antimicrobial peptide ll-37 levels were associated with lower expression of intratonsillar foxp3 (p = 0.011) (fig 4d) , rorc2 (p = 0.015) ( fig 4e) and il-17 (p = 0.044) (fig 4f) . no other significant associations were found between serum levels of vitamins and ll-37 and the "immune activation/regulatory" cluster of cytokines and their transcription factors in tonsils. age, smoking or indication for tonsillectomy did not have modifying effects on the mrna expression associations. this study provides new insights into connections between serum levels of vitamins a, d, and e and antimicrobial peptide ll-37 and several important outcomes: allergy, respiratory virus detection and tonsillar immune responses. we found that higher bioavailable 25(oh)d levels were associated with lower prevalence of allergic rhinitis and atopy, higher vitamin e levels with lower prevalence of self-reported allergy, and higher vitamin a showed borderline significance for an association with less respiratory virus detections. in line with this finding, we found that higher vitamin a levels were associated with higher intratonsillar expression of ifn-γ. also, higher serum bioavailable 25(oh)d levels were associated with higher intratonsillar expression of novel anti-inflammatory cytokine il-37, which is known to suppress immune responses, regulate t-reg development and induce tolerance [23] . exacerbations of childhood and adult asthma are often caused by viral infection [24] . it is generally accepted that, low or deficient innate and adaptive immune responses may contribute to the morbidity of viral infections [25] . we show that multiple viruses exist in nasopharynx and tonsils in relatively asymptomatic patients. less vitamin a was associated with less ifn-γ and tendency for more viral detection, which may partly explain the association previously seen with vitamin a deficiency and asthma exacerbations [1] . the inverse association between vitamin a levels and virus detection in our study is interesting since multiple observational studies have shown that vitamin a deficiency is associated with a higher risk of asthma and wheezing [5-7, 26, 27] . vitamin a has been shown to enhance treg activity via foxp3 and inhibit th17 development via retinoid orphan receptor γt (rorγt) [28] [29] [30] [31] , but we did not find any significant association between serum vitamin a levels and intratonsillar antiviral/immunoregulatory gene expression, except for ifn-γ. ifn-γ is a critical molecule in immune system with multiple functions, mostly related to th1 response against bacterial, viral and fungal infections [32] . vitamin d has beneficial pleiotropic effects on both the innate and adaptive immune system [33] . high vitamin d levels during maternity are associated with less childhood wheezing [34] [35] [36] [37] and vitamin d deficiency appears to contribute to increased susceptibility to infections and wheezing [12, 38, 39] . prospective studies have shown that vitamin d supplementation reduces the risk of recurrent respiratory infections, virus-induced wheezing and asthma exacerbations [12] . even though multiple studies suggest that vitamin d has beneficial effects on the immune defense and on allergic disease, the exact mechanisms are not well defined. our finding of the positive association with higher vitamin d status and higher intratonsillar expression of anti-inflammatory cytokine il-37 might partly explain some of these results. il-37 is an anti-inflammatory cytokine which suppresses immune responses and inflammation [23] . vitamin d is also known to enhance the expression of antimicrobial peptide cathelicidin, often referred in its active form as ll-37 [15] . ll-37 is not only an endogenous antibiotic peptide that destroy bacteria, virus and fungi, but can also act as an immune modulator [40] . we found that higher serum ll-37 levels were associated with lower intratonsillar expression of il-17 and its transcription factor rorc2, both needed to th17 cell development [41] , as well as lower expression of foxp3, a transcription factor known to induce treg cells [41, 42] . in our study cohort, serum ll-37 levels tended to increase with vitamin d levels, but this correlation did not reached statistical significance (s2 fig). our patients did not have acute infection at the time of operation and collection of blood samples. it might be that without acute infection, serum ll-37 levels are not elevated. this data may suggest that a critical balance appear to lie between ll-37 expression due to infections and th17 and treg cell development. vitamin e levels have been shown to associate with th1 and th17 development [43] . our data shows that serum higher vitamin e levels were associated with less self-reported allergy. in agreement with this finding, maternal vitamin e intake during pregnancy has been associated with less wheezing and eczema in children [16, 17] . we found weak or no associations between serum vitamin e levels and the expression of cytokines or transcription factors in tonsils. strengths of our study are simultaneous measurement of multiple vitamins and a novel antimicrobial peptide ll-37, comprehensive viral and atopy characterization and complete clinical data of over 100 patients. statistical analyses were conducted carefully and adjusted for age, smoking and indication for tonsillectomy. according to a previous report from our group, indication for operation, mainly tonsil hypertrophy or recurrently infected tonsils, do not play a role in expression of studied genes [20] . however, we do not have yet any mechanistic data to understand how these regulatory networks crosstalk. in summary, our study provides new evidence suggesting that vitamin a may have antiviral effects. also, our study suggests potentially important roles for vitamin d and antimicrobial peptide ll-37 in th17 and treg cell regulation and development of allergic disease. clinically, our study suggests that vitamin d may promote anti-inflammatory mechanisms. further studies are needed to understand the crosstalk between regulatory networks in allergy and viral infections. nutrients and foods for the primary prevention of asthma and allergy: systematic review and meta-analysis prenatal intake of vitamins and allergic outcomes in the offspring: a systematic review and meta-analysis vitamin d insufficiency and asthma in a us nationwide study prenatal, perinatal, and childhood vitamin d exposure and their association with childhood allergic rhinitis and allergic sensitization vitamin a status in children with asthma serum vitamin a concentrations in asthmatic children in japan serum retinol and airway obstruction maternal vitamin a supplementation and lung function in offspring supplementation with vitamin a early in life and subsequent risk of asthma vitamin d supplementation for childhood asthma: a systematic review and meta-analysis effect of vitamin d3 supplementation during pregnancy on risk of persistent wheeze in the offspring: a randomized clinical trial vitamin d supplementation in children may prevent asthma exacerbation triggered by acute respiratory infection effect of prenatal supplementation with vitamin d on asthma or recurrent wheezing in offspring by age 3 years: the vdaart randomized clinical trial in utero exposure to 25-hydroxyvitamin d and risk of childhood asthma, wheeze, and respiratory tract infections: a meta-analysis of birth cohort studies cutting edge: 1,25-dihydroxyvitamin d3 is a direct inducer of antimicrobial peptide gene expression antenatal determinants of neonatal immune responses to allergens antioxidant intake in pregnancy in relation to wheeze and eczema in the first two years of life mucosal immune response in the ear, nose and throat induction and maintenance of allergen-specific foxp3+ treg cells in human tonsils as potential first-line organs of oral tolerance distinct regulation of tonsillar immune response in virus infection respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children bioavailable vitamin d is more tightly linked to mineral metabolism than total vitamin d in incident hemodialysis patients from il-2 to il-37: the expanding spectrum of anti-inflammatory cytokines epidemiology of respiratory viruses in patients hospitalized with near-fatal asthma, acute exacerbations of asthma, or chronic obstructive pulmonary disease interactions between innate and adaptive immunity in asthma pathogenesis: new perspectives from studies on acute exacerbations plasma lycopene and antioxidant vitamins in asthma: the plava study vitamin a deficiency alters airway resistance in children with acute upper respiratory infection all-trans retinoic acid mediates enhanced t reg cell growth, differentiation, and gut homing in the face of high levels of co-stimulation retinoic acid increases foxp3+ regulatory t cells and inhibits development of th17 cells by enhancing tgf-beta-driven smad3 signaling and inhibiting il-6 and il-23 receptor expression stat6 inhibits tgf-beta1-mediated foxp3 induction through direct binding to the foxp3 promoter, which is reverted by retinoic acid receptor th17 responses in chronic allergic airway inflammation abrogate regulatory t-cell-mediated tolerance and contribute to airway remodeling clinical implications of interferon-γ genetic and epigenetic variants what is new in vitamin d maternal vitamin d intake during pregnancy and early childhood wheezing maternal intake of vitamin d during pregnancy and risk of recurrent wheeze in children at 3 y of age maternal vitamin d status and childhood asthma, wheeze, and eczema: a systematic review and meta-analysis infant respiratory tract infections or wheeze and maternal vitamin d in pregnancy: a systematic review low serum 25-hydroxyvitamin d levels are associated with increased risk of viral coinfections in wheezing children is vitamin d deficiency a marker of severity of wheezing in children? a cross-sectional study antimicrobial peptides and the skin immune defense system mechanisms of peripheral tolerance to allergens vitamin a and retinoic acid in t cell-related immunity the role of retinoic acid in tolerance and immunity key: cord-355259-779czzzx authors: yang, xiaoyun; steukers, lennert; forier, katrien; xiong, ranhua; braeckmans, kevin; van reeth, kristien; nauwynck, hans title: a beneficiary role for neuraminidase in influenza virus penetration through the respiratory mucus date: 2014-10-15 journal: plos one doi: 10.1371/journal.pone.0110026 sha: doc_id: 355259 cord_uid: 779czzzx swine influenza virus (siv) has a strong tropism for pig respiratory mucosa, which consists of a mucus layer, epithelium, basement membrane and lamina propria. sialic acids present on the epithelial surface have long been considered to be determinants of influenza virus tropism. however, mucus which is also rich in sialic acids may serve as the first barrier of selection. it was investigated how influenza virus interacts with the mucus to infect epithelial cells. two techniques were applied to track siv h1n1 in porcine mucus. the microscopic diffusion of siv particles in the mucus was analyzed by single particle tracking (spt), and the macroscopic penetration of siv through mucus was studied by a virus in-capsule-mucus penetration system, followed by visualizing the translocation of the virions with time by immunofluorescence staining. furthermore, the effects of neuraminidase on siv getting through or binding to the mucus were studied by using zanamivir, a neuraminidase inhibitor (nai), and arthrobacter ureafaciens neuraminidase. the distribution of the diffusion coefficient shows that 70% of siv particles were entrapped, while the rest diffused freely in the mucus. additionally, siv penetrated the porcine mucus with time, reaching a depth of 65 µm at 30 min post virus addition, 2 fold of that at 2 min. both the microscopic diffusion and macroscopic penetration were largely diminished by nai, while were clearly increased by the effect of exogenous neuraminidase. moreover, the exogenous neuraminidase sufficiently prevented the binding of siv to mucus which was reversely enhanced by effect of nai. these findings clearly show that the neuraminidase helps siv move through the mucus, which is important for the virus to reach and infect epithelial cells and eventually become shed into the lumen of the respiratory tract. pigs are naturally susceptible to three subtypes of influenza a viruses: h1n1, h3n2 and h1n2, all of which have a strong tropism for the pig respiratory tract mucosa [1] [2] [3] . swine influenza virus particles are transmitted by direct contact and through the air in large droplets or as aerosols [1, 4, 5] . during the transmission from pig to pig, the virus first encounters mucus, the first barrier of the respiratory tract. after overcoming this barrier, the virus reaches the target cells in the mucosal epithelium. influenza virus infects host cells by binding to cellular receptors via one of the major viral glycoproteins, hemagglutinin (ha). ha binds to sialic acids (sa) on the cell surface and mediates the subsequent membrane fusion leading to virus entry [6] . neuraminidase (na) catalyzes the removal of terminal sialic acids on the cellular surface to release the progeny virus [7] . it is well documented that the na functions at the releasing stage of the virus replication [8] [9] [10] , while little is known if na plays a role during the virus entry into host cells and even less on if it helps the virus overcome the mucus layer. mucus is a complex mixture of mucous glycoproteins (mucins), proteins, proteases and protease inhibitors, lipids and water [11, 12] . mucins, the major component of mucus, are highly oglycosylated with glycans covalently linked via n-acetylgalactosamine (ganac) to the hydroxyl groups of serine and threonine residues of the mucin backbone [12, 13] . most of the sugar chains of mucin monomers are terminated with sialic acid, which is also known to be the cellular receptor of influenza viruses. it is hypothesized that influenza viruses bind to these extracellular receptors, get entrapped in the mucus and then are removed by ciliary clearance [14] [15] [16] . several studies have shown that interaction of influenza virus with mucus results in competitive inhibition of the virus. roberts et al. [17] showed that preincubation of human h3n2 virus strain a/victoria/3/75 with ferret nasal washes containing mucus clearly reduced the virus infectivity, and this inhibition was correlated to competitive binding of the virus with alpha 2,3 and 2,6 linked sialic acids (a2,3-and a2,6-sa) present in the mucus secretions. the protective effect of the mucus barrier was confirmed by a recent study using a transgenic mouse model that overexpressed sa a2-3 gal rich muc5ac. transgenic mice challenged with a/pr8/34 h1n1, which preferentially binds a2,3-sa showed significant less infection than the normal mice [18] . these studies suggest that mucus or mucins block the influenza virus infection by competitively inhibiting ha-mediated cell adsorption. despite this inhibitory function of the mucus, the virus is ultimately able to reach the susceptible epithelial cells. it has long been assumed that na promotes virus access to target cells in the airway by mucus degradation. however, this concept is scarcely supported by experimental data. cohen et al. [19] incubated a/ pr/8/34 h1n1 and a/aichi/2/68 h3n2 virus with human salivary mucins which were previously coated on magnetic beads, and after extensive washings, detected the remained neu5ac on the mucins. they showed that these human influenza viruses had cleaved away 40-60% of neu5ac content of the mucins by their viral neuraminidase. the effective cleavage may allow the efficient release of virus from the mucus. this contrasts with the findings of ehre et al. [18] who demonstrated a strong protection of muc5ac up-regulated mice against a/pr/8/34 h1n1 virus infection. hence, the purified human salivary mucins may not fully reflect the natural mucus as these mucins had been highly modified after attaching to magnetic beads. unraveling the mechanism behind the penetration of viruses across the mucosal barriers has potentially significant implications for the development of novel antiviral strategies. therefore, an in vitro model resembling the in vivo situation is needed and the interactions of influenza virus with natural mucus should be studied in depth. in the present study, we aimed to address the following questions: (1) is the virus entrapped or able to penetrate through the native respiratory mucus? (2) can viral neuraminidase use mucin sialic acids as substrates and catalyze the cleavage of them away? (3) is this cleavage sufficient to liberate the virions and allow them to penetrate through the mucus layer? to this purpose, we applied swine influenza virus to a model we previously set up using porcine respiratory mucus, pseudorabies virus (prv) and single particle tracking (spt) [20] . in addition, the penetration of siv was studied by the use of a mucus layer on which an appropriate amount of virus particles was added. the microscopic diffusion and macroscopic translocation were evaluated. next, the effects of neuraminidase on the virus mobility in mucus were examined. tracheas were collected from 6-month-old pigs which were negative for swine influenza a viruses as shown by a hemagglutination inhibition (hi) test. using of tracheas from euthanized animals was approved by the ethical and animal welfare committee of the faculty of veterinary medicine of ghent university. two days before euthanization, the pigs were treated intramuscularly with ceftiofur (naxcel, pfizer-1 ml/20 kg body weight) to clear the respiratory tract from possible bacterial infections. the tracheas were dissected and the mucus was gently scraped with a spoon, collected with a syringe, and the mucus samples were stored separately at 270uc until use. these samples were negative for neuraminidase determined by na assay using fluorescent na substrate (4-methylumbelliferyl-n-acetylneuraminic acid [mu-nana]). fluorescence lectin staining of a2,3and a2,6-sa in mucus freshly collected mucus was filled in a gelatin capsule (2.3 cm 6 0.8 cm), snap frozen in methocel (fluka). cryosections of 12 mm were made with a trimming interval of 400 mm between each section. the mucus sections were blocked in 1% (w/v) bovine serum albumin for 1 h, followed by incubation with biotin conjugated sambucus nigra lectin (sna-i) (ey laboratory, ca, usa; 1:100) for 1 h, at room temperature. after 2 washings in phosphate buffered saline (pbs), the sections were incubated with streptavidin-texas red (invitrogen, 1:200) and fitc labeled maackia amurensis lectin (maa) (ey laboratory, ca, usa; 1:25) for 1 h, at room temperature. afterwards, the sections were washed, and mounted in 90% glycerin containing 2.5% 1,4diazobicyclo-(2,2,2)-octane (dabco). images of the fluorescence staining were acquired using a confocal microscope (leica tcs sp2 laser scanning spectral confocal system, leica microsystems gmbh) and the fluorescence signal was analyzed with imagej. the coverage of either sa was calculated by ratio of fluorescence signal to the region of interest (roi). madin darby canine kidney (mdck) cells were maintained in minimum essential medium (mem, gibco) supplemented with 10% fetal calf serum (gibco), 100 mg/ml of streptomycin and 100 units/ml of penicillin (invitrogen). the avian-like h1n1 swine influenza virus sw/belgium/1/98 was used at the third passage on mdck cells. the virus was propagated in mdck cells in mem supplemented with 5 mg/ml trypsin (gibco), 100 mg/ml of streptomycin and 100 units/ml of penicillin (gibco). confluent mdck cells were inoculated with siv at a multiplicity of infection (m.o.i) of 0.01 in mem. twenty hours post inoculation, the supernatant was harvested. the cellular debris was removed by ultracentrifugation at 7 000 6g for 20 min at 4uc in a type 35 rotor (beckman, fullerton, ca, usa) and the suspension was clarified by filtration with a 0.45 mm filter (millipore). afterwards, the virus was pelleted at 75 000 6g for 2 h at 4uc in a type 35 rotor. following resuspension in pbs (1/ 100 of the original volume), the virus suspension was brought on a discontinuous optiprep (sigma) gradient containing 10-30% (w/ v) of iodixanol and centrifuged at 100 000 6g for 3 h at 4uc in an sw41ti rotor (beckman, fullerton, ca, usa). the visible opalescent virus bands at the interfaces were harvested separately. the buffer was exchanged with hne (5 mm hepes, 150 mm nacl, 0.1 mm edta, ph 7.4) buffer by the use of a 50 kda filter device (millipore). to verify the purity of the virus from each band, double staining was performed by using 3,39-dioctadecyloxacarbocyanine perchlorate (dio) and hyperimmune swine serum directed to influenza sw/belgium/1/98 virus, followed by texas redconjugated goat anti-swine igg antibody. hyperimmune swine serum was diluted (1:50) and mixed with the virus suspension (1:1, v/v). after 2 h incubation on ice, texas red-conjugated goat antiswine igg secondary antibody (1:50, invitrogen) was added and incubated further on ice for 2 h. afterwards, the resulting mixture containing virus, hyperimmune serum, and secondary antibody was equilibrated to room temperature. dio solution (1 mm in dmso) was mixed with virus suspension (1:100, v/v) by fierce vortexing, followed by incubation at room temperature for 20 min. the resulting suspension was ultracentrifuged at 100 000 6g for 1.5 h in a type 35 rotor to remove the free antibodies and dyes. after resuspension in pbs, the virions were clarified by a further ultracentrifugation at 100 000 6g for 1.5 h. the staining was analyzed with confocal microscope by randomly selecting 10 regions. the ratio of viral antigen positive particles (virions) versus dio positive particles was calculated, which is referred to as the degree of viral purity. the band containing the most purified virus was obtained and the buffer was exchanged with hne buffer. after incubation with dio solution as previously described, the virus suspension was filtered by the use of a sephadex g-50 column (ge healthcare, belgium) to remove unbound dye. the unlabeled virus that was eluted through the sephadex g-50 column was used as negative control. dio is a lipophilic dye which integrates into the lipid components of the viral envelope. to determine that the integration of the dio dye into the viral envelope does not change the biophysical properties of the virus, the size and surface charge (zeta potential) of the labeled and unlabeled virions were measured by dynamic light scattering and laser doppler anemometry as previously described [20] . infectivity and hemagglutination activity of the dio-labeled viral particles were tested by virus titration and ha assay as previously described [21] . the na enzymatic activity was determined according to the protocol adapted from adamo et al. [22] . briefly, 25 ml of fluorescent na substrate (4 the trajectories of fluorescent viral particles in porcine tracheal respiratory mucus were recorded by a fast and sensitive electronmultiplying charge-coupled device (emccd) camera (cascade ii: 512; roper scientific, tucson, az, usa) mounted on an inverted epifluorescence microscope (nikon te2000e, nikon belux, brussels, belgium) equipped with a 1006 oil-immersion objective (plan apochromat, nikon). tracking experiments were performed in press-to-seal silicone isolators (20 mm diameter, 0.5 mm deep, invitrogen, merelbeke, belgium). our previous study shows that prv was highly immobilized while 100 nm pgeylated beads were diffusive in porcine respiratory mucus, hence gfp-prv was used as a negative control and the latter was used as a positive control [20] . gfp-prv was semi-purified according to the previous protocol [20] . three microliters of siv (10 8.5 tcid 50 / ml) or prv (10 8.5 tcid 50 /ml) suspension or 100 nm pegylated beads (0.004%, w/v) were mixed with 100 ml of porcine tracheal respiratory mucus by gentle stirring. to determine if na would affect siv diffusion, the virus was added to the mucus with or without the presence of 0.02 mm zanamivir (sigma) or 10 mu/ml arthrobacter ureafaciens neuraminidase (roche applied science). the mixture was placed in a custom made glass chamber. the samples were incubated at 37uc for 10 min on the microscope using a stage-top incubator (tokai hit, fujinomiya, japan) before spt measurement. movies were captured with the nis elements ar software (nikon) at a temporal resolution of 33 ms for 5 s. the illumination time was 30 ms per frame. trajectories of n $ 500 particles were analyzed for each experiment and three independent experiments were performed for each condition. movies were analyzed with the image processing software (ips, in-house developed software) [23] to extract x, y positional data over time. the apparent diffusion coefficient (da) was calculated as a function of the time scale (t) for each particle. analysis of the movies was performed with ips. the centroids of individual particles were identified in each frame of a movie. based on the positions of the centroids, the trajectories of the particles can be determined by a nearest neighbor algorithm. the apparent diffusion coefficient da corresponding to the first time lag dt was calculated according to the classical formula: da = msd/4dt [24] . afterwards, the distribution of diffusion coefficient of the particles was obtained by maximum entropy method (mem) analysis [25] . freshly collected mucus sample (150 ml) was added to a gelatin capsule (2.3 cm 60.8 cm) to create a mucus ''layer'' at the bottom (referred to as virus in-capsule-mucus penetration system, fig. 1 ). afterwards, eight microliter of virus suspension containing approximately 10 6.5 tcid 50 purified siv were brought in the form of 5 droplets onto the surface of the mucus. immediately, 10 min and 30 min after virus addition, the capsules were snap frozen in methocel. due to a delay of freezing process, the time point ''immediately after addition'' was designated as 2 min. to determine if the na would influence the siv penetration, the virus was added with or without the presence of 0.1 mm zanamivir (sigma) or 50 mu/ml arthrobacter ureafaciens neuraminidase onto the mucus, followed by snap-freezing at 30 min post virus addition. cryosections of 12 mm were made with a trimming interval of 400 mm between each section. double immunofluorescence staining was performed using mouse anti-muc5ac igg1 (45m1, lifespan biosciences, 1:100) and mouse anti-np igg2a (hb-65, atcc, 1:50) monoclonal antibodies, followed by texas red conjugated goat anti-mouse igg1 and alexa fluor 488 conjugated goat anti-mouse igg2a secondary antibodies (invitrogen), respectively. ten sections were made for each condition, then 10 images were taken, and finally translocations of the virions was measured, as shown in fig. 1 , from the top of the mucus layer until the deepest point of the viral signal. mucus sections (12 mm) were made, and incubated with 30 ml suspension containing 10 6 tcid 50 siv in the presence or absence of 0.1 mm zanamivir or 50 mu/ml arthrobacter ureafaciens neuraminidase at 37uc, for 1 h. the sections were then fixed with 4% paraformaldehyde for 20 min, followed permeabilization in 0.1% (v/v) triton x-100 for 10 min. immunofluorescence staining was performed using a mouse igg antibody to siv np, followed by goat anti-mouse igg antibody conjugated with fitc to visualize the virions. fluorescence images were acquired on randomly selected regions with a confocal microscope, and the bound virions on the mucus were calculated with imagej. five sections were examined for each mucus sample for the semi-quantification of a2,3and a2,6-sa coverage in the mucus. for each section, 2 images were taken and thus 10 images were obtained for each sample. as shown in fig. 2 , the mucus consisted of mixed and heterogeneous a2,3and a2,6-sa. the sa coverage was calculated by the ratio of the pixels of positive signal to the total pixels measured. the a2,6-sa covered over 50% the region of interest (roi), while merely 11% of the region was constituted by a2,3-sa. after ultracentrifugation over a discontinuous optiprep gradient containing 10% to 30% of iodixanol, three visible opalescent bands were collected, named band 1, band 2 and band 3, respectively, from top to bottom (fig. 3b) . the purity of virus from each band was assessed by confocal microscopy following dio lipophilic dye labeling and siv immunofluorescence staining. as dio integrates into the lipophilic components of virus and cellular debris, dio staining was used as a total-particle assessment. the red color visualized the viral particles, and the green color represented dio-labeled particles (fig. 3a) . the percentage of double positive particles versus dio positive particles represents the virus purity in a ratio extent. consequently, the highest viral purity (over 0.9 for the ratio of double positive particles/dio positive particles) was found in band 2 (fig. 3c) . therefore, the virus preparation from band 2 was used for further analysis. after incubation with 10 mm dio dye at room temperature, followed by elution in a sepharose g-50 column, the labeled and unlabeled siv were analyzed for different characteristics. the results show that the hemagglutination activity and infectivity were not altered by labeling. the neuraminidase activity of dio-labeled siv was 91% of that of unlabeled siv. measured by dynamic light scattering and laser doppler anemometry, the size and surface charge of the labeled virions were not significantly altered (table 1) . the motion of siv in porcine respiratory mucus was investigated and compared with the diffusion of prv and 100 nm pegylated beads. trajectories of 8 steps were analyzed, from which a distribution of the apparent diffusion coefficients was obtained. similar to our previous data, prv was highly hindered in the porcine respiratory mucus, while the 100 nm pegylated beads diffused freely. the distribution of diffusion coefficient clearly demonstrated that, compared to one immobile fraction for prv or a mobile fraction for the 100 nm beads, siv experienced two diffusion patterns in porcine respiratory mucus, with 70% of viral particles being trapped while the rest of particles moving rapidly (fig. 4a) . the average diffusion coefficient of siv in mucus was 11-fold higher than that of prv (fig. 4b) . the similar size 100 nm pegylated beads are muco-innert which indicates that these particles did not interact with any type of the mucus moieties. thus to the opposite, the viral particles were immobilized probably due to binding interactions with the mucus. these data suggest that binding and releasing effects were present in the interactions of siv with porcine respiratory mucus. the depth of siv penetration could be visualized by double immunofluorescence staining of the muc5ac and siv nucleoprotein (np). the distance from the surface down to the deepest point of virus translocation was measured and designated as the depth of virus penetration (fig. 1) . three independent experiments were performed and in total 120 measurements were conducted. distribution of the penetration depth for each condition was eventually obtained. immediately after virus addition, the virions rapidly entered the mucus layer and reached a depth of 31 mm within 2 min, due to a passive diffusion effect (fig. 5a) . incubated at 37uc, the virions spread further in the mucus with time. the distribution of penetration depth shows that the majority of siv particles travelled 10 mm further in the mucus from 2 until 10 min after virus addition and reached a depth of up to 180 mm at 30 min after addition (fig. 5a) . similarly to the microscopic diffusion, the distribution of siv penetration clearly shows two fractions at 30 min after virus addition (fig. 5b) . about 65% of the viral particles penetrated at 30 min more than 2-fold further than 2 min post virus addition (fig. 5b) . the average depth of virus penetration at 30 min was significantly higher than that of earlier time points (fig. 5c) , suggesting that the siv virions were able to actively penetrate the mucus layer. movies were captured with spt software, and the siv microscopic diffusion in mucus in the presence or absence of zanamivir or exogenous neuraminidase was analyzed with ips. as shown in fig. 6a , the mobile fraction of siv diffusion was severely diminished by nai treatment, while was elevated by the addition of arthrobacter ureafaciens neuraminidase. approximately 55% of the mobile viral particles (with da larger than 0.2 mm 2 /s) became stuck by the effect of zanamivir whereas the exogenous neuraminidase increased the mobile particles by approximately 15% (fig. 6b) . consistently, the presence of zanamivir in virus suspension almost completely inhibited the siv macroscopic penetration which contrasts the further penetration by the effect of exogenous neuraminidase (fig. 6c) . the average penetration of mock treated siv was significantly higher than that of nai treated virus, while the rise of average penetration from mock to neuraminidase treated virus was also significant (fig. 6d) . these data imply that neuraminidase helped siv penetrate through the porcine respiratory mucus. virus attaching to the mucus sections was visualized by immunofluorescence staining to the siv np. the virus binding to 5 mucus sections was analyzed, 2 images were taken for each section and in total 10 images were obtained for virions quantification. the virions that attached to a mucus region of 10 5 mm 2 were calculated. three independent experiments were performed. the representative confocal photomicrographs show that zanamivir clearly enhanced the attachment of siv to the mucus. in contrast, the exogenous neuraminidase depleted the virus binding to the mucus by 2-fold (fig. 7) . these data clearly demonstrated that na was able to release the siv particles which may have been bound by interaction of ha with mucins, moving the virions through the mucus. influenza viruses are highly contagious and readily spread by aerosol transmission. the mucus is the first barrier for the small aerosol droplets to settle and overcome. in the present study, we applied spt technique and a custom made virus in-capsule-mucus penetration system to visualize the microscopic diffusion and macroscopic penetration of siv in porcine respiratory mucus. spt is a unique model for rigorous analysis of virus-mucus interactions from the mobility point of view. the virus in-capsulemucus penetration system allows the visualization of virus penetration in mucus layer thereby mimicking the natural conditions. by the use of these models, we were able to track the diffusion of siv in natural respiratory mucus. in the spt assay, there were two fractions based on the virus diffusion coefficient, a mobile and an immobile fraction. the ability of siv to detach from mucus was attributed to the na activities, as inhibiting na by the use of zanamivir significantly suppressed the liberation of the virus from the mucus network (fig. 6a, 6b ). this is also in line with a previous report by matrosovich et al [26] , which describes that blocking of the na activities by oseltamivir efficiently inhibited influenza a viruses from infecting the differentiated human airway epithelium cultures which were probably covered by mucin secretions. furthermore, exogenous neuraminidase was shown to promote both the microscopic diffusion and macroscopic penetration detected by the spt and virus in-capsule-mucus penetration system (fig. 6) . this does not only confirm the beneficial effect of neuraminidase on releasing siv from respiratory mucus, but also highlights bidirectional synergistic interactions between influenza virus and bacterial infections. the influenza virus predisposition to secondary bacterial infections has been thoroughly studied [27, 28] , however, little information exists regarding the impact of bacterial neuraminidase on influenza virus entry and transmission, and further research is needed. neuraminidase was indicated to play a role in the siv releasing from mucus, however, two fractions of viral motion in the mucus lead to a discussion of the way that the virus binds to the mucus. mucins are the major constitute of mucus and are highly decorated by glycans terminated by sialic acids, thus they are likely to be attributed to the immobilization of siv in porcine respiratory mucus. mucins may play direct and indirect roles in host defense distinct from their ability to form adhesion decoys. in addition to mucins, the aqueous mucus layer consists of a great number of host defense agents including lysozyme, lactoferrin, secretory iga (siga), collectins, defensins, cathelicidins, histatins and surfactant proteins [29] [30] [31] . these molecules may function by binding the virions in a receptor-independent pattern. it has been shown that surfactant protein d (sp-d) binds via a carbohydrate recognition domain in a ca 2+ -dependent manner to n-linked high-mannose carbohydrates present on the ha and na of the influenza viruses [32] . in addition, siga is retained at high concentrations in mucus where it can efficiently trap the pathogens. last but not least, defensins are of great interest with respect to respiratory viral infection. human defensins have been shown to bind several types of viruses and inhibit the entry of the viruses to target cells [33, 34] . these components may be retained in mucus by direct binding with mucins or by the biophysical properties of mucus and thus become part of the gel network and provide an immobilized reservoir of protective effectors. alternatively, it is possible that the viral particles that were immobilized in the mucus were actually incomplete or defective virions or became inactive while being prepared. the production of defective particles has been reported for influenza a viruses [35, 36] . however, the approach of distinguishing active and inactive virions in an entity has not been readily achieved, and will be further examined. while vaccination remains the primary option for the prevention and control of influenza, anti-influenza virus drugs are considered as a complementary approach, as vaccine production may not be rapidly achieved. our findings provide experimental evidence for the essential role of na in influenza virus penetration through the respiratory mucus. blocking the na activities clearly suppressed the movement of virus in mucus (fig. 6) , illustrating that na played a role in removing the sialic acids on mucins, which may enable the virus to gain access to the cellular receptors. this suggests the usefulness of neuraminidase inhibitors as prophylactic treatment for influenza. on the other hand, preventative treatment with oseltamivir (tamiflu) failed to protect the lung from virus replication or inflammation in an in vivo influenza infection study in pigs despite reduced clinical symptoms and virus shedding [37] . this highlights the complexity of the in vivo situation and the minimal benefits neuraminidase inhibitors may have. the ability of an influenza virus passing through the mucus may serve as a determinant for influenza virus transmission in addition to efficient virus attachment, high potential of replication and low infectious dose required [5, 38, 39] . combining the study of cohen et al. [19] , it can be noticed that human influenza viruses could bind and be released from human salivary mucins but not from porcine submaxillary mucins, whereas, swine influenza virus was able to escape from porcine airway mucus, suggesting there may be different interactions between different influenza viruses and the mucus of different species. a balance of binding to and releasing from the mucin sialic acids, which is determined by the functional balance of ha and na, may influence how efficiently the virus avoids sticking to mucus. fluorescence lectin staining on mucus cryosection showed that both a2,3and a2,6-sa were present in the porcine respiratory mucus, with distinct predominance for the latter (fig. 2) . the binding profile of the siv strain was not investigated in this study, however, it has been well documented that swine influenza virus isolates, especially those with the avian-like h1 and h3 hemagglutinins showed receptor specificity for both a2,3and a2,6-sialylated glycans [40] [41] [42] . probably the mucus provides sufficient amount of receptors for siv binding. the binding of siv via ha to the porcine respiratory mucus was proved in the present study, and the amount of viral or exogenous na indeed modulated the extent of viral binding to and releasing from the porcine mucus (fig. 7) . concerning the releasing effect, na which mediates the process also has a substrate preference. it was demonstrated that na of human and swine influenza viruses have a preferential specificity for a2,3-sa although they cleave both linked sialylated glycans [43, 44] . therefore, we assume that the sialic acids in respiratory mucus secretions may exert an effect on influenza virus transmission. since the majority of viral particles were incapable of penetrating through the mucus layer, why do influenza viruses invade the respiratory tract of the animals after all [1, 3, 45] ? based on our experimental findings and present literature, we propose several strategies the influenza viruses may use to overcome the mucus barrier and find their way to establish infection: (1) production of enzymes that aid the virus movement through the mucus. influenza virus binds to and uses sialic acid-containing molecules as receptors. it is because of this capability that influenza virus has evolved a second viral surface protein, neuraminidase, as a receptordestroying enzyme that cleaves sialic acid, allowing the virus to be released after binding to sialic acid-containing molecules that do not lead to viral infection. this strategy is utilized as well by many other microbes, such as e. histolytica [46, 47] , vibrio cholerae [48] , helicobacter pylori [49] , reovirus [50] and coronavirus [51] , to subvert or avoid the mucus barrier. the production of enzymes, including mucinase, sialidase, glycosidase, elastase, and hydrolase, which are capable of degrading mucin core proteins and mucin carbohydrates facilitates microbes to swim through the mucus layer. furthermore, the enzymes that the microbes produce may also facilitate the invasion of other pathogens. in women with bacterial vaginosis, the overgrowth of anaerobic gram-negative bacteria that produce sialidase, glycosidases and other mucin-degrading enzymes causes a breakdown in the barrier properties of cervicovaginal mucus, thereby destroying the mucus gel and helping other sexually transmitted pathogens such as human immunodeficiency virus (hiv) to invade [52] . (2) the use of abundant and ubiquitous molecules as receptors. although there may be a risk of binding to decoy receptors, the use of abundant and ubiquitous molecules as receptors provides the apparent advantage to the virus for allowing infection of multiple cell types and species. this can result in a low minimal infectious dose for initial infection. based on the data of diffusion and penetration, the effects of the mucus network that virions encounter are so extreme that only a part of the particles can escape and reach susceptible target cells ultimately. thus the viruses which require lower minimal infectious doses for the same tissues may gain higher chance to establish an infection. (3) spreads via aerosol. the slow settle of aerosols in the air can cause prolonged contact of the virus with the respiratory tract which benefits the virus penetration through the mucus layer. furthermore, aerosol droplets can travel much more efficiently to the lower respiratory tract and the mucociliary apparatus may need a longer time to transport and exclude the virions out of the respiratory tract, which increases the chance of these viruses to penetrate through the mucus layer and reach the target cells eventually. the issues on if siv would be able to penetrate through the porcine respiratory mucus and if the neuraminidase would contribute to move the virus through the mucus layer have been addressed. however, the ability of the viral neuraminidase to cleave sialic acid from mucus has not been investigated due to technical limitation. the viscous property impedes the separation of the free sialic acids from the mucus even if they would have been cleaved by the viral neuraminidase. investigating the role of influenza virus neuraminidase in the cleavage of sialic acid from mucus may shed some light on unravelling the mechanism of influenza pneumonia. hence the effect of influenza virus neuraminidase on mucus needs to be studied. review: influenza virus in pigs influenza a viruses: new research developments evolution and ecology of influenza a viruses transmission of influenza a virus in pigs aerosol transmission of influenza a virus: a review of new studies cell receptors for influenza a viruses and the innate immune response influenza virus neuraminidase: structure, antibodies, and inhibitors functional significance of sialidase during influenza virus multiplication: an electron microscope study influenza virus neuraminidase: structure and function airway mucus: its components and function structure and function of the polymeric mucins in airways mucus barrier properties of mucus nasal mucus and influenza viruses. i. the haemagglutinin inhibitor in nasal secretions mucins in the mucosal barrier to infection natural and synthetic sialic acid-containing inhibitors of influenza virus receptor binding lack of transmission of a human influenza virus with avian receptor specificity between ferrets is not due to decreased virus shedding but rather a lower infectivity in vivo overexpressing mouse model demonstrates the protective role of muc5ac in the lungs influenza a penetrates host mucus by cleaving sialic acids with neuraminidase immobilization of pseudorabies virus in porcine tracheal respiratory mucus revealed by single particle tracking comparative pathogenesis of an avian h5n2 and a swine h1n1 influenza virus in pigs optimizing viral protein yield of influenza virus strain a/vietnam/1203/2004 by modification of the neuraminidase gene single particle tracking single-particle tracking: applications to membrane dynamics sizing nanomatter in biological fluids by fluorescence single particle tracking neuraminidase is important for the initiation of influenza virus infection in human airway epithelium influenza virus neuraminidase contributes to secondary bacterial pneumonia influenza virus infection decreases tracheal mucociliary velocity and clearance of streptococcus pneumoniae detection of surfactant proteins a, b, c, and d in human nasal mucosa and their regulation in chronic rhinosinusitis with polyps host defense effector molecules in mucosal secretions the airway epithelium: soldier in the fight against respiratory viruses evidence for a protective role of pulmonary surfactant protein d (sp-d) against influenza a viruses direct inactivation of viruses by human granulocyte defensins interactions of alpha-, beta-, and theta-defensins with influenza a virus and surfactant protein d most influenza a virions fail to express at least one essential viral protein influenza virus morphogenesis and budding efficacy of influenza vaccination and tamiflu(r) treatment-comparative studies with eurasian swine influenza viruses in pigs predicting 'airborne' influenza viruses: (trans-) mission impossible? interspecies and intraspecies transmission of influenza a viruses: viral, host and environmental factors comparison of the receptor binding properties of contemporary swine isolates and early human pandemic h1n1 isolates (novel 2009 h1n1) receptor specificity of subtype h1 influenza a viruses isolated from swine and humans in the united states receptor-binding properties of swine influenza viruses isolated and propagated in mdck cells characterization of the hemagglutinin receptor specificity and neuraminidase substrate specificity of clinical isolates of human influenza a viruses amino acid residues contributing to the substrate specificity of the influenza a virus neuraminidase swine influenza virus: zoonotic potential and vaccination strategies for the control of avian and swine influenzas roles for the galactose-/n-acetylgalactosamine-binding lectin of entamoeba in parasite virulence and differentiation movement of entamoeba histolytica trophozoites in rat cecum and colon intact mucus blankets and harvested mucus gels haemagglutinin/protease expression and mucin gel penetration in el tor biotype vibrio cholerae helicobacter pylori moves through mucus by reducing mucin viscoelasticity a glycosyl hydrolase activity of mammalian reovirus sigma1 protein can contribute to viral infection through a mucus layer sialic acids as receptor determinants for coronaviruses glycosidase and proteinase activity of anaerobic gram-negative bacteria isolated from women with bacterial vaginosis the authors would like to express their gratitude to lieve sys for the technical guidance and discussions on influenza viruses. thanks go to zeger vandenabeele and loes geypen for their help in preparing the trachea samples, and nele dennequin for preparing cell samples. conceived and designed the experiments: xy ls kf kvr hn. performed the experiments: xy. analyzed the data: xy ls kf rx hn. contributed reagents/materials/analysis tools: kf rx kb kvr. wrote the paper: xy. key: cord-333955-bnzbppof authors: biesold, susanne e.; ritz, daniel; gloza-rausch, florian; wollny, robert; drexler, jan felix; corman, victor m.; kalko, elisabeth k. v.; oppong, samuel; drosten, christian; müller, marcel a. title: type i interferon reaction to viral infection in interferon-competent, immortalized cell lines from the african fruit bat eidolon helvum date: 2011-11-30 journal: plos one doi: 10.1371/journal.pone.0028131 sha: doc_id: 333955 cord_uid: bnzbppof bats harbor several highly pathogenic zoonotic viruses including rabies, marburg, and henipaviruses, without overt clinical symptoms in the animals. it has been suspected that bats might have evolved particularly effective mechanisms to suppress viral replication. here, we investigated interferon (ifn) response, -induction, -secretion and -signaling in epithelial-like cells of the relevant and abundant african fruit bat species, eidolon helvum (e. helvum). immortalized cell lines were generated; their potential to induce and react on ifn was confirmed, and biological assays were adapted to application in bat cell cultures, enabling comparison of landmark ifn properties with that of common mammalian cell lines. e. helvum cells were fully capable of reacting to viral and artificial ifn stimuli. e. helvum cells showed highest ifn mrna induction, highly productive ifn protein secretion, and evidence of efficient ifn stimulated gene induction. in an alphavirus infection model, o'nyong-nyong virus exhibited strong ifn induction but evaded the ifn response by translational rather than transcriptional shutoff, similar to other alphavirus infections. these novel ifn-competent cell lines will allow comparative research on zoonotic, bat-borne viruses in order to model mechanisms of viral maintenance and emergence in bat reservoirs. the order chiroptera (bats) is one of the most diverse and geographically wide-spread orders within the mammals constituting 20% of all mammalian species [1] . chiroptera are subdivided into two suborders yango-and yinpterochiroptera. the latter includes frugivorous/nectarivorous bats (flying foxes) with species like rousettus aegyptiacus (r. aegyptiacus), pteropus alecto (p. alecto) and eidolon helvum (e. helvum) as well as the insectivorous bat rhinolophus cf. landeri (r. cf. landeri, rhinolophidae). yangochiroptera comprise bats from the three superfamilies emballonuroidea, noctilionoidea, vespertilionoidea including the insectivorous species myotis daubentonii (m. daubentonii), pipistrellus spec. (both vespertilionidae), tadarida brasiliensis (t. brasiliensis, molossidae), hipposideros cf. caffer/ ruber (h. cf. caffer/ruber, hipposideridae) [2, 3] . bats have been shown to host relevant human pathogens like rabies-, ebola-, marburg-, henipaviruses (hendra-, nipah-) and severe acute respiratory syndrome (sars)-like coronaviruses [4] . the ability to control such highly pathogenic viruses raises the question whether bats might have evolved particularly effective mechanisms of immune control [5] [6] [7] . little is known about the immune system of bats. it has been shown that bats develop immunoglobulins after infection and have lymphoid development similar to that in other mammals [8] [9] [10] . however, information on the innate immune response of bats is particularly scarce. toll-like receptor genes have been identified in r. leschenaulti and p. alecto [11, 12] . cells from pteropus species have been shown to produce high amounts of interferon (ifn)-l after stimulation with the double-strand (ds)rna analogue poly ic, and after infection with the bat-associated paramyxovirus, tioman [13] . conversely, infection with the highly pathogenic paramyxovirus hendra virus resulted in no induction of ifn expression and concomitant inhibition of ifn signaling, suggesting the presence of specific viral ifn antagonists [14] . a conserved functionality of ifn signaling in different mammalian cell cultures including epithelial lung cells from tadarida brasiliensis (tb1-lu) was already described earlier [15, 16] . however, there remains a fundamental lack of knowledge on the ways type i ifns are induced and ifn signals are processed in bat cells. because type i ifn is a major barrier towards virus infection, quantitative comparisons between different mammalian systems are of particular interest. currently there are hardly any bat cell lines available whose fundamental properties in ifn induction and -response have been characterized in a comparative manner. here we present a set of essential tools to characterize ifn induction and -response in bat cells, and introduce a novel group of highly ifn-competent, immortalized bat cell lines from the species e. helvum that hosts relevant zoonotic viruses including henipa-and lyssaviruses [17, 18] . we compare paramount patterns of ifn induction and response in these e. helvum cells with that in prototype murine and primate cell lines. for all capturing and sampling, permission was obtained from the wildlife division, forestry commission, accra, ghana. samples were exported under a state contract between the republic of ghana and the federal republic of germany, and under an additional export permission from the veterinary services of the ghana ministry of food and agriculture (permit no. chrpe49/09; a04957). all cells were cultivated in dmem (dulbecco's modified eagles medium) (paa, cölbe, germany) with 4.5 g/l glucose (paa), supplemented with 10% fetal bovine serum (paa), 1% penicillin/streptomycin 1006 concentrate (penicillin 10000 units/ml, streptomycin 10 mg/ml) (life technology), 1% l-glutamine 200 mm, 1% sodium pyruvate 100 mm (paa), 1% mem nonessential amino acids (neaa) 1006 concentrate (paa). cells were generally incubated at 37uc and 5% co 2 . as prototype mammalian cells we applied simian virus (sv) 40 large t antigen immortalized mouse embryonic fibroblasts (mef) generated inhouse from 129/svj mice [19] , african green monkey kidney cells (ma104, kindly provided by friedemann weber, university of marburg) and human lung adenocarcinoma epithelial cell line (a549, ccl-185). for titration of o'nyong nyong virus (onnv) vero e6 cells (atcc crl-1586) were used. under the auspices of ghana authorities bats were caught with mist nets, anaesthetized with a ketamine/xylazine mixture and euthanized to perform organ preparations (permit no. chrpe49/09; a04957). organs from e. helvum (embryo kidney and lung), r. aegyptiacus (kidney), m. daubentonii (lung), pipistrellus spec. (kidney), h. cf. caffer/ruber (embryo) and r. cf. landeri (kidney) were minced, trypsinized, and cultured in dmem medium by titration and seeding at 1:100 dilution in cell culture flasks as described previously [20] . imipenem (zienam, msd, haar, germany) and amphotericin b (paa) were added to minimize contamination risks. immortalization was done by lentiviral transduction of the large t antigen of sv40. immortalized cells were expanded and stock frozen or processed further for subcloning. all cell cultures were genotyped by amplification of mitochondrial cytochrome b as previously described using primers l14724 and h15149 (table s1 ) [21, 22] and were controlled for mycoplasma [23] , sv 5 (in-house assay, table s1), lyssaviruses [24] and filoviruses [25] by rt-pcr. nucleic acid extraction and real-time rt-pcr viral rna was extracted from cell culture supernatant with the qiaamp viral rna mini kit (qiagen, hilden, germany). total rna from 90% confluent cells was isolated using the rneasy mini kit (qiagen) and reverse-transcribed with random hexamer primers (life technologies, karlsruhe, germany). fragments of target genes were amplified from cdna by lowstringency pcr. after initial denaturation for 2 min at 94uc, touchdown pcr was done for 10 cycles (94uc/20 s, 66-56uc, delta 1uc/10 s, 72uc/60 s). the remaining 30 pcr cycles included 94uc/20 s, 56uc/20 s, 72uc/60 s. fragments were sequenced and used to design real-time rt-pcr assays targeting genes relevant for the ifn system as well as reference genes, in domains conserved among bat species (table s1) . probes were tagged with 59-carboxyfluorescein and 39-black hole quencher (biomers, ulm, germany). real-time rt-pcr was processed using the lightcyclerh 480 real-time pcr system (roche, basel, switzerland). for quantification of onnv genome equivalents (ge) the dilution end-point was defined as one pcr unit. log pcr units per ml for each experimental sample were calculated from the linear equations of the dilution series [26] . to determine the foldinduction of the different target genes (ifn, mxa, isg56) the 2 2ddct method was applied with tata-box binding protein (tbp) as housekeeping gene [27] . onnv infections were performed at multiplicity of infection (moi) of 2.5 and 0.0025 respectively. virus was diluted in serumfree medium optipro (life technologies). cells were inoculated for 1 h at 37uc and washed twice with pbs after infection. samples were taken at time points 0, 8 and 24 h post infection (hpi). titration of onnv was performed by plaque assay as previously described [26, 28] . vero e6 cells were seeded in 24-well plates at a density of 4610 5 cells per ml. virus supernatant was added and after 1 h incubation the inoculum was removed and cells were washed with pbs (paa). 26 mem (biochrom, berlin, germany), supplemented with 0.44% (w/v) nahco 3 (roth, karlsruhe, germany), 20% fcs (paa) and 2% penicillin/ streptomycin, was mixed in a 1:1 ratio with 2.4% avicel (fmc, biopolymers, brussels, belgium) and 0.5 ml per well was added. after 2 days the overlay was discarded and cells were stained with a 0.2% crystal violet and 20% ethanol (roth) containing solution. rift valley fever virus clone 13 (rvfv 13) infection for ifn induction was done as described above by infecting cells at an moi of 1. after 24 h, all supernatants were harvested and virus was inactivated by b-propiolactone (b-pl, ferak berlin, germany) treatment as described below. ifn stimulation by poly ic was performed by transfection of 8610 5 cells per 6-well with 5 mg poly ic using nanofectin (paa) according to the manual instructions. rvfv 13-and onnv-containing supernatants were inactivated with b-pl as previously described [29] [30] [31] . briefly, the supernatants were collected and rvfv 13 was inactivated by incubating with 0.05% of b-pl at 4uc for 16 h. the hydrolysis of b-pl was achieved by incubation at 37uc for 2 h. for onnv a final concentration of 0.1% of b-pl was needed for total inactivation. all b-pl treated samples were subjected to a virus plaque assay to control complete inactivation. to ensure equal treatment of the samples, supernatants of negative controls and cells stimulated with poly ic were also treated with b-pl. a classical vsv bioassay was performed as described previously [32] . eidni/41.3, mef and ma104 cells were seeded in 12-well plates at a density of 4610 5 cells per ml. after 24 h the inactivated supernatants or a pan-species ifn-a (pan-ifn, a universal recombinant type i human ifn-a a/d hybrid, pbl biomedical laboratories/axxora, lörrach, germany) standard dilution series were added to the respective cell lines. an internal standard curve comprising five concentrations (0.5, 1, 10, 20 and 40 units/ml) of pan-ifn diluted in medium of mock treated cells was applied in every experiment. the external pan-ifn standard curves for the ec 50 calculation were performed in quadruplicates and comprised pan-ifn concentrations between 0.25 and 150 units/ml diluted in dmem. 24 h posttreatment, ifn containing supernatants were removed and cells were washed with pbs. the cells were then infected with vsv at an moi of 0.025. after 1 h of virus adsorption cells were washed again and overlayed with avicel as described above. after 2 days the overlay was discarded, the cells were fixed and stained as before. the amount of plaques of the applied pan-species ifn standard curves or of the test samples was calculated in percentage (maximal plaque count from the negative control was set to 100%). the equation of the internal standard was used to calculate ifn amounts in the samples. ec 50 values were defined as ifn concentrations which reduced the number of plaques to 50%. afterwards the values were multiplied by the dilution factors and figures were normalized to the amount of ifn per ml. to define comparable, species-independent ifn concentrations the calculated amount of ifn was divided by the ec 50 of each internal standard curve. thus, the normalized amount of ifn could directly be compared. protein analysis was essentially done as described elsewhere [33] . generally, cells were lysed in ripa lysis buffer (150 mm nacl, 1% igepal ca-630, 0.5% sodium deoxycholat, 0.1% sds, 50 mm tris (ph 8.0), protease inhibitor cocktail iii, benzonase 25 units/ml, 5 mm dithiothreitol (merck, darmstadt, germany)) and separated on a 12% sds-page gel. western blotting was performed by using mouse-anti-mx1/2/3 (c-1, santa cruz, heidelberg, germany), goat-anti-ifit/isg56 (l-16, santa cruz), mouse-anti-actin (sigma) immunoglobulins at dilutions 1:1000. secondary detection was done with the help of horseradish peroxidase labeled goat-anti-mouse and rabbit-anti-goat antibody (1:20000, dianova, hamburg, germany) and supersignalh west femto chemiluminescence substrate (thermo fisher scientific, bonn, germany). old world frugivorous/nectarivorous bats or flying foxes (pteropodidae; formerly classified as ''megachiroptera'') comprise species like r. aegyptiacus and e. helvum. whereas r. aegyptiacus is a known reservoir for marburg virus [34] [35] [36] , e. helvum was shown to carry henipa-like viruses [18] and lagos bat virus [17] . only for r. aegyptiacus [37] and tadarida brasiliensis (atcc: ccl-88) cell cultures are commercially available. e. helvum bats roosting in kumasi, ghana, were investigated shortly before the parturition season in 2009. one pregnant female was euthanized by injection of ketamine/xylazine according to approved protocols, under a license from the veterinary services and the ministry of food and agriculture, accra, ghana. primary cell cultures from embryonic kidney tissue of e. helvum were generated at the kumasi collaborative centre for research in tropical medicine, kumasi ( figure 1a) . after expansion of primary cells to confluent monolayers, cells were immortalized by lentiviral transduction of the sv40 large t antigen gene [19] . primary cells could be passaged only twice, while transduced cells could be passaged continuously (.30 passages). out of the second passage, clonal cell lines were prepared by end point dilution plating. a clonal cell line named eidni/41.3 ( figure 1a ) was chosen for in-depth characterization. for comparison, two clonal cell lines from the same preparation (eidni/41.1 and eidni/41.2), as well as the 21st passage of a mixed culture (eidni/41), were also preserved ( figure 1a) . additionally, a kidney cell culture (4 th passage) from an adult r. aegyptiacus bat was prepared (data not shown) to investigate putative differences between the two related frugivorous bats. all bat cell cultures were genotyped by amplifying mitochondrial cytochrome b fragments [22] followed by a blast analysis. eidni and roni cells showed 100% identity with cytochrome b (genbank accession no. ab359172.1 (e. helvum) and jf728760.1 (r. aegyptiacus), data not shown). as already shown for primary cells of the pteropus species, bat cells can readily secrete ifn upon virus infection or poly ic transfection [13, 14, 38, 39] . however, immortalization of cells can damage cytokine genes and signaling pathways. [29, 40] . second, cells were transfected with poly ic [41] . to measure the induction of ifn transcription, a real-time rt-pcr assay for the ifn-b gene was designed. to this end the ifn-b gene was amplified by nested rt-pcr from cultures of a phylogenetically representative range of bats differing in main diet and hunting strategies including the insectivorous m. daubentonii, pipistrellus spec. (vespertilionidae), h. cf. caffer/ruber (hipposideridae),and r. cf. landeri (rhinolophidae) as well as the frugivorous/nectarivorous r. aegyptiacus and e. helvum (pteropodidae) ( figure 1b) , using primers designed upon alignments of available ifn-b genes from horses, swine, cattle, humans, mice and rats. real-time rt-pcr primers (see table s1 for genbank no. and primer sequences) were placed in sites conserved among bats ( figure 1c) . as shown in figure 1d , eidni/41.3 cells were readily infected by rvfv 13, and luciferase expression representing virus replication levels was comparable with that in several reference cell cultures including mef, ma104 and vero e6 cells. nevertheless, the induction of the ifn-b gene in a single cycle infection experiment, at an moi = 1, was approximately 30-fold stronger in eidni/41.3 cells than in mef and ma104 cells ( figure 1e ). the induction in vero e6 cells was not examined because of their intrinsic ifn-b gene defect [42, 43] . interestingly, excessive ifn induction as compared to reference cells was only observed upon virus infection, but not upon poly ic transfection. it was concluded that the capability of eidni/41.3 cells to react on natural and artificial ifn stimuli had not been affected by immortalization. since cellular mrna levels are prone to differential expression over time ifn protein secretion was analyzed next. it was tested whether eidni/41.3 cells were able to produce and secrete effective doses of type i ifn. to this end it was necessary to compare and calibrate species-specific ifn secretion between primate, rodent, and bat cells, using an appropriate experimental model. to achieve this, a vsv-based ifn bioassay was established. vsv growth and plaque morphology was compared in all cell lines (figure 2a) . countable plaques were visible after 2 days post infection (dpi). to obtain a calibration of ifn secretion across species, all cell lines were incubated with gradients of equivalent concentrations of recombinant pan-species ifn and subjected to vsv plaque reduction assays (exemplified for eidni/41.3 in figure 2b ). the antiviral responses in eidni/41.3, mef, and ma104 cells upon equivalent doses of ifn are compared in figure 2c . ec 50 values (amount of pan-species ifn to achieve a 50% vsv plaque reduction) were calculated for all cell lines ( figure 2c ). using this calibration, it was possible to compare the equivalent activity levels of species-specific ifn secreted from each cell line upon stimulation. in accordance with the ifn mrna induction, the highest equivalent amount of bioactive secreted ifn upon rvfv 13 virus infection and poly ic transfection was measured in eidni/41.3 cells, followed by mef and ma104 (figure 3 ). the observed difference in ifn mrna induction between rvfv 13 virus and poly ic transfection ( figure 1e) was not seen in this case favoring the idea of differential mrna regulation in e. helvum cells upon poly ic treatment. in conclusion, eidni/41.3 cells were capable of inducing ifn upon natural and artificial stimuli, to secrete active type i ifn, and to induce an antiviral state upon ifn stimulation. since e. helvum cells produced considerable amounts of ifn upon infection with rvfv 13 we investigated in addition the effects of infection with a non-attenuated wild-type rna virus. the old world alphavirus onnv was chosen because alphaviruses have been detected previously in bats [44, 45] and exhibit strong induction of ifn [46] . interestingly, alphaviruses utilize a rather general mechanism to evade ifn, by causing a translational shutoff that affects cellular translation more than viral translation [46] [47] [48] . to establish a synchronized infection of onnv, a high moi = 2.5 was used. virus growth was measured by an onnv-specific real-time rt-pcr and titration of virus in supernatants 24 hpi. virus rna and infectious virus were detected in all cell lines (figures 4a and b) . increases of infectious virus formation were about 1000-fold within 24 hpi, and specific infectivities, expressed as pfu per genome equivalent (pcr units), were highly comparable between cell cultures ( figure 4c) . to investigate if onnv infection caused ifn induction, the amount of ifn mrna (y-axis; for absolute values refer to figure s1 ) was directly compared to viral rna (x-axis) from supernatant at equivalent time points in each cell line ( figure 5a ). whereas no induction of ifn mrna was detectable at 8 hpi, onnv replication clearly induced ifn mrna expression by 24 hpi. comparison of secreted ifn with infectious virus production at 24 hpi revealed that increased viral titers corresponded to lower amounts of secreted ifn protein ( figure 5b and s1). eidni/ 41.3 cells showed highest levels of secreted ifn but the lowest level of infectious particles, suggesting that virus replication might be limited in response to ifn. to determine whether virus control correlated with levels of secreted ifn or merely with the induction of genes under control of the ifn promoter, the ratios of secreted ifn to ifn mrna were determined in all cell lines. in all cells stimulated by rvfv 13 or poly ic, the ratios of secreted ifn versus ifn mrna induction was comparable. in contrast, in all cells infected with onnv the relative levels of secreted ifn were clearly reduced, while overall ifn mrna induction was less affected ( figure 5c ). this effect was independent of the cell species. in order to exclude that the onnv-mediated reduction of ifn protein production was cell-clone specific, another cell line named eidni/41.2 as well as the original mixed cell culture eidni/41 was analyzed, essentially showing the same reduction of relative ifn secretion upon onnv infection ( figure 5d ). similar effects were also seen in a mixed cell culture generated from the related flying fox r. aegyptiacus (roni/7). a human lung adenocarcinoma epithelial cell line (a549) showed an even higher decrease of ifn secretion than ma104 and mef ( figure 5d) . alphaviral induction of a transcriptional and/or translational shutoff affecting mainly the cellular but not the viral protein production has been described in other cells [46] . while our results suggested similar effects to be in place in e. helvum and r. aegyptiacus cells, control of onnv replication seemed to be relatively more efficient in both applied bat cells. one reason might be differences in the capability of bat cells to react on ifn signaling. to investigate this, the mrna abundance of two different isgs (mxa and isg56) as well as two reference genes (actin-b and tbp) was analyzed and compared to the amounts of secreted ifn at 0 and 24 hpi. whereas the expression of mxa is strictly ifn signalingdependent, isg56 can also be directly activated in the absence of secreted ifn and without involvement of the jak/stat pathway [49, 50] . as shown in figure 5e , the ratios of mxa and isg56 mrna to secreted ifn showed an approximately 10fold increase 24 hpi. thus, transcription did not seem to be generally blocked after onnv infection in all cells. in bat cells (eidni/41.3) but not in murine or primate cells, the level of mxa mrna induction in relation to ifn in supernatant was more than 1000-fold higher compared to the same ratio for isg56. this was also the case in different clonal and mixed e. helvum kidney cells (eidni/41.2 and eidni/41), as well as in r. aegyptiacus kidney (roni/7) cells ( figure 5f ), suggesting that ifn signaling-dependent induction of isgs may be highly efficient in e. helvum and r. aegyptiacus bat cells. in our experiments this explained an apparently superior control of onnv replication in both applied bat cells. to confirm alphaviral translational shutoff in bat cells, protein expression of mxa, p56 and actin was determined by western blot analysis 24 hpi. as depicted in figure 6 , rvfv 13 infection clearly caused higher or similar (ma104 cells) expression of mxa in cells from all species while expression of p56 was hardly increased over levels seen in uninfected cells. in contrast, onnv infection resulted in a reduced expression of mxa and p56 proteins. this effect was generally more pronounced for mxa than for p56, suggesting that ifndependent signaling rather than jak/stat-independent induction were affected ( figure 6 ). interestingly, this effect was observed in all applied bat cell lines but not in mef ( figure 6a ). in summary, while e. helvum and r. aegyptiacus bat cells showed particularly efficient induction of mxa mrna, the production of this highly active antiviral protein was antagonized by onnv in those bat cells to a larger extent than in cells from rodents or primates. finally, it had to be excluded that the observed reduction of ifn and isg protein levels might be due to attenuation of virus replication. alphaviruses have been shown to acquire mutations in the 59utr [51, 52] , nsp3 [53] , or nsp2 genes [54] , which resulted in low level replication and enabled virus persistence without ifndependent elimination. the observed increase of ifn mrna upon onnv infection ( figure 5a ) and viral titers of up to 10 5 pfu per ml ( figure 4b ) would in fact speak against this hypothesis. however, to exclude attenuated replication in bat cells we analyzed virus growth at a very low moi of 0.0025. virus replication was monitored by real-time rt-pcr at time point 0, 8 and 24 hpi ( figure 7a ) and viral supernatants were titrated after 24 h ( figure 7b) . additionally, the specific infectivity of virus supernatants was compared 24 hpi ( figure 7c ). all cell cultures supported virus growth efficiently, despite 1000-fold lower mois applied in this experiment. the specific infectivity was higher compared to the infection at moi = 2.5, suggesting an efficient infectious particle formation in all cells at low moi. whereas ma104 primate cells yielded a 10 to 100-fold higher specific infectivity compared to the high moi experiment, eidni/41.3 and mef had similar ratios at both doses. taken together the highly productive virus formation at a low moi suggested that decreased ifn and isg protein levels were generally not due to attenuation of replication. in conclusion, this study is the first to directly compare ifn induction, secretion and signaling in african fruit bat cell lines with that in other mammalian cell lines. we show that these bat cells were fully capable of reacting to viral and artificial ifn stimuli. our bat cells showed strikingly high ifn mrna induction, efficient ifn protein secretion, and evidence of highly efficient isg induction. onnv exhibited typical strong ifn induction but evaded the ifn response by translational rather than transcriptional shutoff, as typical in alphavirus infection of mammalian cells. these well-characterized, highly interferoncompetent cell lines will enable comparative research on zoonotic, bat-borne viruses in order to model mechanisms of viral maintenance and emergence in bat reservoirs. we thank ute winke and stephan kallies for excellent technical assistance, antje seebens (noctalis) and peter vallo (academy of sciences of the czech republic) for field work in ghana, and beate kümmerer for critical review of the data. we are grateful to alexander pfeifer, university of bonn, for providing large t antigen lentiviruses, to winfried barchet, evolution. an eocene big bang for bats a molecular phylogeny for bats illuminates biogeography and the fossil record the evolution of echolocation in bats bats: important reservoir hosts of emerging viruses experimental inoculation of plants and animals with ebola virus animal models of henipavirus infection: a review nipah virus infection in bats (order chiroptera) in peninsular malaysia role of immunoglobulin classes in experimental histoplasmosis in bats immunofluorescence analysis of immunoglobulin bearing lymphocytes in the indian fruit bat: pteropus giganteus analysis of immunocompetent cells in the bat, pteropus giganteus: isolation and scanning 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strong effect on rna replication and subgenomic rna synthesis, but not on translation of the encoded proteins iresdependent replication of venezuelan equine encephalitis virus makes it highly attenuated and incapable of replicating in mosquito cells high-resolution functional mapping of the venezuelan equine encephalitis virus genome by insertional mutagenesis and massively parallel sequencing roles of nonstructural protein nsp2 and alpha/beta interferons in determining the outcome of sindbis virus infection university of bonn, for mouse embryonic kidney cells and to friedemann weber, university of marburg, for the donation of rvfv 13. key: cord-347317-qcghtkk0 authors: russo, lucia; anastassopoulou, cleo; tsakris, athanasios; bifulco, gennaro nicola; campana, emilio fortunato; toraldo, gerardo; siettos, constantinos title: tracing day-zero and forecasting the covid-19 outbreak in lombardy, italy: a compartmental modelling and numerical optimization approach date: 2020-10-30 journal: plos one doi: 10.1371/journal.pone.0240649 sha: doc_id: 347317 cord_uid: qcghtkk0 introduction: italy became the second epicenter of the novel coronavirus disease 2019 (covid-19) pandemic after china, surpassing by far china’s death toll. the disease swept through lombardy, which remained in lockdown for about two months, starting from the 8th of march. as of that day, the isolation measures taken in lombardy were extended to the entire country. here, assuming that effectively there was one case “zero” that introduced the virus to the region, we provide estimates for: (a) the day-zero of the outbreak in lombardy, italy; (b) the actual number of asymptomatic infected cases in the total population until march 8; (c) the basic (r(0))and the effective reproduction number (r(e)) based on the estimation of the actual number of infected cases. to demonstrate the efficiency of the model and approach, we also provide a tentative forecast two months ahead of time, i.e. until may 4, the date on which relaxation of the measures commenced, on the basis of the covid-19 community mobility reports released by google on march 29. methods: to deal with the uncertainty in the number of the actual asymptomatic infected cases in the total population volpert et al. (2020), we address a modified compartmental susceptible/ exposed/ infectious asymptomatic/ infected symptomatic/ recovered/ dead (seiird) model with two compartments of infectious persons: one modelling the cases in the population that are asymptomatic or experience very mild symptoms and another modelling the infected cases with mild to severe symptoms. the parameters of the model corresponding to the recovery period, the time from the onset of symptoms to death and the time from exposure to the time that an individual starts to be infectious, have been set as reported from clinical studies on covid-19. for the estimation of the day-zero of the outbreak in lombardy, as well as of the “effective” per-day transmission rate for which no clinical data are available, we have used the proposed seiird simulator to fit the numbers of new daily cases from february 21 to the 8th of march. this was accomplished by solving a mixed-integer optimization problem. based on the computed parameters, we also provide an estimation of the basic reproduction number r(0) and the evolution of the effective reproduction number r(e). to examine the efficiency of the model and approach, we ran the simulator to “forecast” the epidemic two months ahead of time, i.e. from march 8 to may 4. for this purpose, we considered the reduction in mobility in lombardy as released on march 29 by google covid-19 community mobility reports, and the effects of social distancing and of the very strict measures taken by the government on march 20 and march 21, 2020. results: based on the proposed methodological procedure, we estimated that the expected day-zero was january 14 (min-max rage: january 5 to january 23, interquartile range: january 11 to january 18). the actual cumulative number of asymptomatic infected cases in the total population in lombardy on march 8 was of the order of 15 times the confirmed cumulative number of infected cases, while the expected value of the basic reproduction number r(0) was found to be 4.53 (min-max range: 4.404.65). on may 4, the date on which relaxation of the measures commenced the effective reproduction number was found to be 0.987 (interquartiles: 0.857, 1.133). the model approximated adequately two months ahead of time the evolution of reported cases of infected until may 4, the day on which the phase i of the relaxation of measures was implemented over all of italy. furthermore the model predicted that until may 4, around 20% of the population in lombardy has recovered (interquartile range: ∼10% to ∼30%). italy became the second epicenter of the novel coronavirus disease 2019 (covid-19) pandemic after china, surpassing by far china's death toll. the disease swept through lombardy, which remained in lockdown for about two months, starting from the 8th of march. as of that day, the isolation measures taken in lombardy were extended to the entire country. here, assuming that effectively there was one case "zero" that introduced the virus to the region, we provide estimates for: (a) the day-zero of the outbreak in lombardy, italy; (b) the actual number of asymptomatic infected cases in the total population until march 8; (c) the basic (r 0 )and the effective reproduction number (r e ) based on the estimation of the actual number of infected cases. to demonstrate the efficiency of the model and approach, we also provide a tentative forecast two months ahead of time, i.e. until may 4, the date on which relaxation of the measures commenced, on the basis of the covid-19 community mobility reports released by google on march 29. to deal with the uncertainty in the number of the actual asymptomatic infected cases in the total population volpert et al. (2020), we address a modified compartmental susceptible/ exposed/ infectious asymptomatic/ infected symptomatic/ recovered/ dead (seiird) model with two compartments of infectious persons: one modelling the cases in the population that are asymptomatic or experience very mild symptoms and another modelling the infected cases with mild to severe symptoms. the parameters of the model corresponding to the recovery period, the time from the onset of symptoms to death and the time from a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 exposure to the time that an individual starts to be infectious, have been set as reported from clinical studies on covid-19. for the estimation of the day-zero of the outbreak in lombardy, as well as of the "effective" per-day transmission rate for which no clinical data are available, we have used the proposed seiird simulator to fit the numbers of new daily cases from february 21 to the 8th of march. this was accomplished by solving a mixed-integer optimization problem. based on the computed parameters, we also provide an estimation of the basic reproduction number r 0 and the evolution of the effective reproduction number r e . to examine the efficiency of the model and approach, we ran the simulator to "forecast" the epidemic two months ahead of time, i.e. from march 8 to may 4. for this purpose, we considered the reduction in mobility in lombardy as released on march 29 by google covid-19 community mobility reports, and the effects of social distancing and of the very strict measures taken by the government on march 20 and march 21, 2020. based on the proposed methodological procedure, we estimated that the expected dayzero was january 14 (min-max rage: january 5 to january 23, interquartile range: january 11 to january 18). the actual cumulative number of asymptomatic infected cases in the total population in lombardy on march 8 was of the order of 15 times the confirmed cumulative number of infected cases, while the expected value of the basic reproduction number r 0 was found to be 4.53 (min-max range: 4.40-4.65). on may 4, the date on which relaxation of the measures commenced the effective reproduction number was found to be 0.987 (interquartiles: 0.857, 1.133). the model approximated adequately two months ahead of time the evolution of reported cases of infected until may 4, the day on which the phase i of the relaxation of measures was implemented over all of italy. furthermore the model predicted that until may 4, around 20% of the population in lombardy has recovered (interquartile range: *10% to *30%). the butterfly effect in chaos theory underscores the sensitive dependence on initial conditions, highlighting the importance of even a small change in the initial state of a nonlinear system. the emergence of a novel coronavirus, sars-cov-2, that caused a viral pneumonia outbreak in wuhan, hubei province, china in early december 2019 has evolved into the covid-19 acute respiratory disease pandemic due to its alarming levels of spread and severity, with more than 3.5 million cases and 250,000 deaths globally, as of may 7, 2020 ( [1, 2] ). the seemingly far from the epicenter, old continent became the second-most impacted region after asia pacific, mostly as a result of a dramatic divergence of the epidemic trajectory in italy first, where there have been 214,457 total confirmed infected cases and 29,684 deaths, and then in spain where there have been 220,325 total confirmed infected cases and 25,857 deaths, as of may 7, 2020 ( [1, 2] ). the second largest outbreak outside of mainland china officially started on january 31, 2020, after two chinese visitors staying at a central hotel in rome tested positive for sars-cov-2; the couple remained in isolation and was declared recovered on february 26 [3] . a 38-year-old man repatriated back to italy from wuhan who was admitted to the hospital in codogno, lombardy on february 21 was the first secondary infection case ("patient 1"). "patient 0" was never identified by tracing the first italian citizen's movements and contacts. in less than a week, the explosive increase in the number of cases in several bordering regions and in the two autonomous provinces of trento and bolzano (the northerner in italy) placed enormous strain on the decentralized health system. following a dramatic spike in deaths from covid-19, italy transformed into a "red zone", and the movement restrictions were expanded to the entire country on the 8th of march. all public gatherings were cancelled and school and university closures were extended through at least the next month. in an attempt to assess the dynamics of the outbreak for forecasting purposes, it is important to estimate epidemiological parameters that cannot be computed directly based on clinical data, such as the transmission rate (or as otherwise called "effective contact rate" of the disease and the basic reproduction number, r 0 . the transmission rate is defined as the product of the probability of transmitting the virus given a contact between a susceptible and an infected individual and the average rate of contacts between susceptibles and infected. r 0 is defined as the expected number of exposed cases generated by one infected case in a population where all individuals are susceptible [4] . since the first confirmed covid-19 case many mathematical modelling studies have already appeared. the first models mainly focused on the estimation of the basic reproduction number r 0 using dynamic mechanistic mathematical models ( [5] [6] [7] [8] ), but also simple exponential growth models (see e.g. [9, 10] ). compartmental epidemiological models like sir, sird, seir and seird have been proposed to estimate other important epidemiological parameters, such as the transmission rate and for forecasting purposes (see e.g. [8, 11] ). other studies have used metapopulation models, which include data of human mobility between cities and/or regions to forecast the evolution of the outbreak in other regions/countries far from the original epicenter in china [5, 7, 12, 13] , including the modelling of the influence of travel restrictions and other control measures in reducing the spread [14] . among the perplexing problems that mathematical models face when they are used to estimate epidemiological parameters and to forecast the evolution of the outbreak, two stand out: (a) the uncertainty regarding the day-zero of the outbreak, the knowledge of which is crucial to assess the stage and dynamics of the epidemic, especially during the first growth period, and (b) the uncertainty that characterizes the actual number of the asymptomatic infected cases in the total population (see e.g. [15, 16] ). at this point we should note that what is done until now with dynamical epidemiological models is the investigation of several scenarios including different "days-zero" or just fixing the day-zero and run different levels of asymptomatic cases e.t.c. to cope with the above problems, we herein address a methodological framework that provides estimates for the day-zero of the outbreak and the number of the asymptomatic cases in the total population in a systematic way. towards this goal, and for our demonstrations, we address a conceptually simple seird model with a total of five compartments, with one of them modelling the asymptomatic infected cases in the population and another one modelling the part of the infected cases that will experience mild to severe symptoms, a significant share of which will be hospitalized, admitted to intensive care units (icus) or die from the disease. the proposed approach is applied to lombardy, the epicenter of the outbreak in italy. furthermore, we provide a twomonths ahead of time forecast from march 8 (the day of lockdown of all italy) to may 4 (the first day of the relaxation of the strict isolation measures). the above tasks were accomplished by the numerical solution of a mixed-integer optimization problem using the publicly available data of daily new cases for the period february 21-march 8, and the covid-19 community mobility reports released by google on march 29. we address a compartmental seiird model that includes two categories of infected cases, namely the asymptomatic (unknown) cases in the total population and the cases that develop mild to more severe symptoms, a significant share of which are hospitalized, admitted to icus and a part of them dies. in agreement with other studies and observations, our modelling hypothesis is that the confirmed cases of infected are only a (small) subset of the actual number of asymptomatic infected cases in the total population [7, 8, 16] . regarding the confirmed cases of infected as of february 21, a study conducted by the chinese cdc which was based on a total of 72,314 cases in china, found that about 80.9% of the cases were mild and could recover at home, 13.8% severe and 4.7% critical [17] . on the basis of the above findings, in our modelling approach, it is assumed that the asymptomatic or very mildly symptomatic cases recover from the disease relatively soon and without medical care, while for the other category of infected, on average their recovery lasts longer than the non-confirmed, they may also be hospitalized, admitted to icus or die from the disease. based on the above, let us consider a well-mixed population of size n. the state of the system at time t, is described by (see also fig 1 for a schematic) s(t) representing the number of susceptible persons, e(t) the number of exposed, i(t) the number of asymptomatic infected persons in the total population who experience very mild or no symptoms and recover relatively soon without any other complications, i c (t) the number of infected cases who may develop mild to more severe symptoms and a significant part of them is hospitalized, admitted to icus or dies, r(t) the number of asymptomatic cases in the total population that recover, r c (t) the number of the recovered cases that come from the compartment of i c and d(t) the reported number of deaths. for our analysis, and for such a short period, we assume that the total number of the population remains constant. based on demographic data, the total population of lombardy is n = 10m; its surface area is 23,863.09 kmq and the population density is *422 (inhabitants/kmq). the rate at which a susceptible (s) becomes exposed (e) to the virus is proportional to the density of infectious persons i. the proportionality constant is the "effective" disease transmission rate, say b ¼ � cp, where � c is the average number of contacts per day and p is the probability of infection upon a contact between a susceptible and an infected. our main assumption here is that only a fraction, say � of the actual number of exposed cases e will experience mild to tracing day-zero and forecasting the covid-19 outbreak in lombardy more severe symptoms denoted as i c (t) and a significant part of them will be hospitalized, admitted to icus or die. thus, we assume that the infected persons that belong to the compartment i c go into quarantine at home or they are hospitalized, and, thus, it is assumed that for any practical means they don't transmit further the disease. here, it should be noted that a wide testing policy may also result in the identification of asymptomatic cases belonging to the compartment i that would then be assigned to compartment i c . however, as a generally reported rule in italy, tests were conducted only for those who presented for treatment with symptoms like fever and coughing. thus, people who did not seek medical attention were tested very scarcely [18] [19] [20] . thus, for any practical means the compartment i c reflects the reported confirmed infected cases. a fraction of the i c cases that is given by the fatality ratio f = d(t)/(i c (t) + r c (t) + d(t)) dies with a mortality rate δ the inverse of which is the average time from the onset of symptoms to death, while the remaining part ((1 − f)) of the i c compartment recovers with a rate γ c , the inverse of which corresponds to the average time from the onset of symptoms to full recovery. we note that while more compartments could conceptually be included, we aimed at keeping a low level of complexity in order to avoid the introduction of more parameters, and thus a model that would suffer from the "curse of dimensionality". at this point we should note that on march 8, the date of the general lockdown, the number of confirmed infected cases was 3,372, the number of cases in icus was 399 and the number of hospitalized persons was 2,616 [21] . that is, until march 8, the number of confirmed cases was approximately equal to the number of hospitalized cases and the cases that were admitted to icus. therefore, until march 8, any difference between the asymptomatic cases as represented by our model by the compartment i and the compartment i c would approximately reflect a level of under-reporting of the actual asymptomatic cases in the total population. we should also note that in the available data of reported cases [21] there is no distinction between cases that recovered at home and those that recovered at and were dismissed from hospitals. thus, in the absence of such information, if one were to consider as a separate category the cases that are hospitalized, an extra parameter would have to be introduced (the fraction of recovered cases dismissed from hospitals). on one hand, such a piece of information is not available, and, on the other, such an attempt would add an extra degree of freedom that would need calibration or to be fixed at a certain value; however, due to the small size of the data and the "curse of dimensionality", this would also introduce unnecessary computational burden and further modelling uncertainty. thus, our discrete mean field compartmental seiird model reads: sðt à 1þiðt à 1þ à seðt à 1þ ð2þ the above system is defined in discrete time points t = 1, 2, . . ., with the corresponding initial condition at the very start of the outbreak (day-zero): the the term fδi c (t − 1) in eq 4 represents the fraction f of the i c cases that dies with a mortality rate γ and the term (1 − f)γ c i c (t − 1) represents the complementary part ((1 − f)) of the i c cases that recovers with a rate γ c . the parameters of the model are: is the average per-day "effective" rate at which an exposed person becomes infectious, is the average per-day "effective" recovery rate within the group of asymptomatic cases in the total population, is the average per-day "effective" recovery rate within the subset of the i c infected cases that finally recover, • δ(d −1 ) is the average per-day "effective" mortality rate within the subset of i c infected cases that finally die, • f is the probability that a i c case will die. here, this, is given by the "emergent" case fatality ratio, computed as f is the fraction of the actual (all) cases of exposed in the total population that enter to the compartment i c . here, we should note the following: as new cases of recovered and dead at each time t appear with a time delay (which is generally unknown but an estimate can be obtained by clinical studies) with respect to the corresponding infected cases, the above per-day rates are not the actual ones; thus, they are denoted as "effective/apparent" rates. the values of the epidemiological parameters σ, γ, γ c , δ that were fixed in the proposed model were chosen based on clinical studies. in particular, in many studies that use seird models, the parameter σ is set equal to the inverse of the mean incubation period (time from exposure to the development of symptoms) of a virus. however, the incubation period does not generally coincide with the time from exposure to the time that someone starts to be infectious. regarding covid-19, it has been suggested that an exposed person can be infectious well before the development of symptoms [22] . with respect to the incubation period for sars-cov-2, a study in china [23] suggests that it may range from 2-14 days, with a median of 5.2 days. another study in china, using data from 1,099 patients with laboratory-confirmed 2019-ncov ard from 552 hospitals in 31 provinces/provincial municipalities suggested that the median incubation period is 4 days (interquartile range: 2 to 7). in our model, as explained above, 1/σ represents the period from exposure to the onset of the contagious period. thus, based on the above clinical studies, for our simulations, we have set 1/σ = 3. regarding the recovery period, in a study that is based on 55,924 laboratory-confirmed cases, the who-china joint mission has reported a median time of 2 weeks from onset to clinical recovery for mild cases, and 3-6 weeks for severe or critical cases [24] . based on the above, and on the fact that within the subset of confirmed cases the mild cases are the 81% [17] , we have set the recovery period for the confirmed cases' compartment to be δ c = 1/21 in order to balance the recovery period with the corresponding characterization of the cases (mild, severe/critical). the average recovery period of the unreported/non-confirmed part of the infected population, which in our assumptions experiences the disease like the flu or a common cold, is set equal to one week [25], i.e. we have set δ = 1/7. this choice is based also on reports on the serial interval of covid-19. the serial interval of covid-19 is defined as the time duration between a primary case-patient (infector) having symptoms and a secondary case-patient having again symptoms. for example, it has been reported that the serial interval for covid is estimated at 4.4-7.5 days [26] ; for the case of lombardy, the average serial number has been estimated to be 6.6 days [27] . in our model, the 1/σ = 3 period refers to the period from exposure to the onset of the contagiousness. in this period, obviously there are no symptoms. thus, the serial interval in our model is 7 days (this is the average number of days in which an infectious becomes recovered and no longer transmits the disease). importantly, there are studies (see e.g. nushiura et al. [28] ) suggesting that a substantial proportion of secondary transmission may occur prior to illness onset. thus, the 7 days period that we have taken as the average period that an infectious person can transmit the disease before he/she recovers, reflects exactly this period; it refers to the serial interval for the cases that are asymptomatic and for cases with mild symptoms. finally, the median time from the onset of symptoms until death for italy has been reported to be eight days [29] , thus in our model we have set γ = 1/8. we have set f = 11% for the optimization. for the forecasting (i.e. for the period march 9 to may 4), the value of f was not fixed but it was computed dynamically each day t through the model simulations as the transmission rate β, as it cannot be obtained in general by clinical studies, but only by mathematical models, was estimated through the optimization process. regarding day-zero in lombardy, that is also unknown and estimated by the optimization process, what has been officially reported is just the date on which the first infected person was confirmed to be positive for sars-cov-2. that day was february 21, 2020, which is the starting date of public data release of confirmed cases. the day-zero of the outbreak, the per-day "effective" transmission rate β, and the ratio � were computed by the numerical solution of a mixed-integer optimization problem with the aid of genetic algorithms to fit the reported data of daily new cases (see the discussion in [30] ) from february 21 to march 8, the day of the lockdown of lombardy. as already mentioned, on march 8, the number of confirmed infected cases in the population was 3,372, the number of cases in icus was 399 and the number of hospitalized persons was 2,616 [21] . that is, until march 8 the number of mild and severe cases that were hospitalized and admitted to icus was approximately equal to the number of confirmed infected cases. thus, for the period of calibration, it is reasonable to assume that for any practical means the number of confirmed cases was approximately the same as for those that experienced mild to more severe symptoms and were admitted for medical care. thus, until march 8, the parameter � reflects also the level of under-reporting of the asymptomatic cases in the total population. here, for our computations, we have used the genetic algorithm "ga" provided by the global optimization toolbox of matlab [6] to minimize the following objective function: where, δx seird (t), (x = i, r, d) are the new cases resulting from the seird simulator at time t. the weights w 1 , w 2 , w 3 correspond to scalars serving in the general case as weights to the relevant functions for balancing the different scales between the number of infected, recovered cases and deaths. the convergence tolerance was set to 1.e −06 , the population size (distributed between the lower and upper bounds) was selected as 40 resulting from min(max (10 � nvars,40),100) for mixed-integer problems (here nvars = 3), ceil(0.05 � populationsize) individuals are guaranteed to survive to the next generation, the migration fraction was set to 0.2, while the number of generations that take place between migrations of individuals between subpopulations was set to 20 [6] . at this point we should note that the above optimization problem may in principle have multiple nearby optimal solutions (mnos). finding and assessing the information contained from mnos (known also as niching) is a particular challenging problem [31] . here, we created a grid of initial guesses within the intervals in which the optimal estimates were sought: for the day-zero (t 0 ) we used a step of 2 days within the interval december 27, 2019 until the 5th of february, 2020 i.e. ±20 days around the 16th of january, for β we used a step of 0.05 within the interval (0.3, 0.9) and for � we used a step of 0.02 within the interval (0.01, 0.29). the numerical optimization procedure was repeated 48 times for each combination of initial guesses. for our computations, we kept the best fitting outcome for each combination of initial guesses. next, in order to reveal structured patterns of distributions vs. uniformly random distributions, we fitted the resulting probability distributions of the optimal values using several functions, including the normal, log-normal, weibull, beta, gamma, burr [32] , exponential and birnbaum-saunders [33] distributions and kept the one resulting in the maximum log-likelihood (see in the supporting information for more details). for the computed parameters of the corresponding best distributions, we also provide the corresponding 95% confidence intervals. such fitting can demonstrate that the obtained values are not uniformly distributed. finally, we have run simulations based on all obtained values to assess the efficiency of the model and obtained results and the forecasting uncertainty until may 4. for our computations, we used the parallel computing toolbox of matlab 2020a [34] utilizing 6 intel xeon cpu x5650 cores at 2.66ghz. estimation of the basic reproduction number. here, we note that we provide an estimation of the basic reproduction number r 0 based on the estimation of the total number of (asymptomatic) infected cases in the population. thus, it is expected that the estimated r 0 will be larger than the ones reported using just the confirmed number of cases; the latter may underestimate the actual r0. initially, when the spread of the epidemic starts, all the population is considered to be susceptible, i.e. s � n. on the basis of this assumption, we computed the basic reproduction number based on the estimates of the epidemiological parameters computed using the data from the 21st of february to the 8th of march with the aid of the seiird model given by eqs (1)-(7) as follows. note that there are three infected compartments, namely e, i, i c and two of them (e,i) determine the outbreak. thus, considering the corresponding equations given by eqs (2), (3) and (4) , and that at the very first days of the epidemic s � n and d � 0, the jacobian of the system as evaluated at the disease-free state reads: the eigenvalues (that is the roots of the characteristic polynomial of the jacobian matrix) dictate if the disease-free equilibrium is stable or not, that is if an emerging infectious disease can spread in the population. in particular, the disease-free state is stable, meaning that an infectious disease will not result in an outbreak, if and only if all the norms of the eigenvalues of the jacobian j of the discrete time system are bounded by one. jury's stability criterion [35] (the analogue of routh-hurwitz criterion for discrete-time systems) can be used to determine the stability of the linearized discrete time system by analysis of the coefficients of its characteristic polynomial. the characteristic polynomial of the jacobian matrix reads: where a 2 ¼ 1 the necessary conditions for stability read: the sufficient conditions for stability are given by the following two inequalities: the first inequality (13) results in the necessary condition bð1 à �þ g < 1: it can be shown that the second necessary condition (14) and the sufficient condition (15) are always satisfied for the range of values of the epidemiological parameters considered here. thus, the necessary condition (16) is also a sufficient condition for stability. hence, the disease-free state is stable, if and only if, condition (16) is satisfied. note that in this necessary and sufficient condition (16) , the fraction (1 − �)/δ is the average infection time of the compartment i. thus, the above expression reflects the basic reproduction number r 0 which is qualitatively defined by r 0 = β � 1/infection time. hence, our model results in the following expression for the basic reproduction number: note that for � = 0, the above expression simplifies to r 0 for the simple sir model. estimation of effective reproduction number. for the estimation of the effective reproduction number r e , representing the average number of secondary infections from an infectious individual when in the population exist already non-susceptible individuals. for the calculation of r e , we now use the next generation matrix approach [36] . the next generation matrix g with elements = g ij is formed by the average number of secondary infections of type i from an infected individual of type j. formally, it is constructed by: where f is the vector containing the transmission rates from the model, and v is the vector containing the transition rates between the infected compartments. in our model: the effective reproduction number r e is the spectral radius, i.e. the dominant eigenvalue of g. thus, r e ðtþ ¼ bð1 à �þ g sðt à 1þ n à dðt à 1þ à r c ðt à 1þ à i c ðt à 1þ : ð20þ as discussed, we used the proposed approach to forecast the evolution of the pandemic in lombardy from march 8 to may 4, i.e. from the first day of lockdown to the first day of the relaxation of the social isolation. our estimation regarding the as of march 8 reduction of the "effective" transmission rate was based on the combined effects of prevention efforts and behavioral changes. in particular, our estimation was based on (a) the covid-19 community mobility reports released by google on march 29 [37] , and, (b) an assessment of the synergistic effects of such control measures as the implementation of preventive containment in workplaces, stringent "social distancing", and the ban on social gatherings, as well as the public awareness campaign prompting people to adopt cautious behaviors to reduce the risk of disease transmission (see also [38] [39] [40] [41] ). the effect of the distribution of contacts at home, work, when travelling, and during leisure activities can be also assessed. for example, based on an analysis for the social contacts and mixing patterns relevant to the spread of infectious diseases that was conducted in various countries, it has been found that for italy, around 20-23% of all physical social contacts during a day are attributed to workplaces, around 17-18% to schools, 2-3% to transportation, 20% to leisure activities, 15-18% to home, 15% to other activities (contacts made at locations other than home, work, school, travel, or leisure) and a 7-8% to contacts made at multiple other locations during the day, not just at a single location [42] . on the basis of the google covid-19 community mobility report released on march 29 [37] , the average reduction in the mobility in lombardy during the period february 16-march 8, compared to the period before february 16, was *15% in retail & recreation activities, *20% in transit stations, *12% in workplaces, while it was increased in parks by * 11% and was almost the same in groceries and pharmacies. in the period march 9 to march 20, the mobility was reduced by an average of *73% in retail & recreation activities, by *75% in transit stations, by *55% in workplaces, by *58% in parks and by * 32% in groceries and pharmacies. thus, taking into account the coarse effect of different activities in the physical contact [42] , the average reduction was of the order of *40% in mobility when compared to the period february 21-march 8. in fact, on march 17, based on the release of mobile phone data, the vice-president of lombardy, announced that the average mobility in the region (for distances more than 500 meters) had been reduced by a *50% with respect to the period before february 20 [43] . on march 20, the government announced the implementation of even stricter measures that included the closure of all public and private offices, closing all parks, walking only around the residency and not even in pairs, and the prohibition of mobility to second houses [44, 45] . according to the google covid-19 community mobility reports [37] , from march 20-21 until april 30, activities were reduced by an average of *87% in retail & recreation activities, by *84% in transit stations, by *70% in workplaces, by *80% in parks and by * 50% in groceries and pharmacies. thus, taking into account the coarse effect of different activities in the physical contact [42] , the average reduction was of the order of *65% in the mobility when compared to the period of february 21-march 8. a further reduction may be attributed to behavioral changes [46] . for example, it has been shown that social distancing and cautiousness reduce the disease transmission rate by about 20% [40] . thus, based on the above, it is reasonable to consider a (1-0. as discussed, for our computations we ran 48 times the numerical optimization procedure for each combination of initial guesses based on the daily reported new cases from february 21 to for all the near-optimal points obtained using the genetic algorithm optimization, the residuals were of the order of * 4,750,000. regarding the values of the optimal parameters, we fitted their cumulative probability distributions using several functions, including the normal, log-normal, weibull, beta, gamma, burr, exponential and birnbaum-saunders functions, and kept the one yielding the maximum log-likelihood (see in the s1 file). table 1 summarizes the mean values of the optimal parameters and their interquartiles, for the day-zero, the transmission rate (β) and the fraction (�) of the actual cases of exposed in the total population that enter to the compartment i c , and also the information on the values of the parameters of the best fitting distributions. note that the optimal values of day-zero were between january 5-january 23 (interquartile range: january 11 to january 18) (see s1 fig in s1 file) , the optimal values of β were between 0.636 and 0.86 (interquartile range: 0.665 to 0.719) (see s2 fig in s1 file) , and the optimal values of � were between 0.01 and 0.249 (interquartile range: 0.023 to 0.1) (see s3 fig in s1 file) . the best fit to the distribution of optimal values of the day-zero was obtained using a normal cdf with mean 1.67 (i.e. sim 2 days before the 16th of january) (95% ci:1.53, 1.80) and variance 4.43 (95% ci: 4.33, 4.52); thus taking the round value at 2 days, the expected day-zero corresponds to january 14 (interquartile range: january 11 to january 18). the best fit to the distribution of the optimal values of β, was given by fitting a burr cdf with α = 0.654 (95% ci: 0.653, 0.655), c = 276.061 (95% ci: 248.911, 306.172), k = 0.0537 (95% ci: 0.048, 0.060) having a mean value of 0.7 (interquartile range: 0.665 to 0.719). finally, the best fit to the distribution of the optimal values of � was given by fitting a birnbaum-saunders cdf with parameters μ = 0.0492 (95% ci: 0.048, 0.0505) (scale parameter) and α = 0.934 (95% ci: 0.914, 0.954) (shape parameter), resulting in an expected value 0.07 (interquartile range: 0.023 to 0.1) (see in s1 file). thus, based on the derived values of the "effective" per-day disease transmission rate, the basic reproduction number r 0 is 4.53 (min-max range: 4.40-4.65). finally, we ran the simulator for all values of the optimal triplets from the corresponding distributions as found by the solution of the optimization problem using the data from february 21 until march 8, then from march 9 to march 20 for validation purposes and from march 29 to may 4 for forecasting purposes. figs (2)-(4) depict the simulation results based on the optimal estimates, until the 19th of march. to validate the model with respect to the reported data of confirmed cases from march 9 to march 19, we have considered an (1-0.4)(1-0.2) reduction in the "effective" transmission rate and as initial conditions the values resulting from the simulation on march 8 as described table 1 . optimal parameter values and interquartiles for day-zero, transmission rate (β) and fraction (�) of the cases of exposed individuals in the total population that enter to the compartment i c . the resulting value of r 0 along with the minimum and maximum value is also given. tracing day-zero and forecasting the covid-19 outbreak in lombardy in the methodology. thus, the model approximated fairly well the dynamics of the pandemic in the period from february 21 to march 19. as discussed in the methodology, we also attempted based on our methodology and modelling approach to forecast the evolution of the outbreak until may 4, the first day of the relaxation of the measures. to do so, as described in the methodology, we have considered a (1-0.65) (1-0.2) reduction in the "effective" transmission rate starting on march 20 (compared to the period february 21-march 8), the day of announcement of even stricter measures in the region of lombardy (see in methodology). the result of our forecast is depicted in fig 5. as shown, the model predicts fairly the evolution of the epidemic two months ahead of march 8 (the model parameters and the day-zero were estimated using the reported data from february 21 to march 8). note that, regarding the confirmed cases, the mean values of i c (t) from over all simulations almost coincide with the reported values of the confirmed cases (see top panel of fig 5) . the reported recovered cases are in the lower part of the model predictions (r c (t)) (see medium panel of fig 5) . also, the model predicts fairly the total number of deaths until may 4 (see bottom panel of fig 5. however, at may 4 there is a significant difference between the mean value of deaths as obtained from simulations and the actual number of deaths. this is due to the following reasons. first, the difference that is observed with respect to the reported number of deaths and model forecasts in the period from march 18 to april 15 can be attributed to facts that the model did not take into account, such as the saturation of icus in that period, which could potentially lead to a larger number of deaths. indeed, on march 8, 400 new deaths were reported. however, we note that the model was calibrated with the data reported until march 8. until march 8, the case fatality ratio was of the order of *9-10% and the number of cases admitted to icus was relatively small. after march 20 when there were many cases admitted to icus, the death toll increased significantly, it almost doubled, thus reaching the *17-18% of the confirmed cases. second, regarding the difference that is observed between the model predictions and actual number of deaths at may 4, this is due to the fact that we did not calibrate the model parameters to take into account the changes in the death and recovery rates. it is expected that when the evolution of the epidemic is abrupt, as it was in lombardy in the period march 12 until the early of april, due to the saturation of the tracing day-zero and forecasting the covid-19 outbreak in lombardy health system and icus the mortality rates would be higher and the recovery rates longer. indeed, during this period, it has been reported, that the median time from the onset of symptoms until death in lombardy was eight days [29] which a very short period. for example, in other studies it has been reported that the time between symptom onset and death ranged from about 2 weeks to 8 weeks which from two to seven times longer than the one reported in lombardy [24, 47] . so, this difference explains the difference between simulations and reported number of deaths at may 4. here, for our demonstrations we have used the information about the epidemiological parameters, that was available at the early phase of the epidemic. thus, we must underline the very good agreement of the model predictions with the actual number of confirmed infected cases for the entire period march 8-may 4. this number, in contrast to the number of recovered and number of deaths is not shaped/biased by the capacity of icus and this is the number on which policy makers should focus in order to decide ahead of time of the necessary resources (e.g. necessary number of beds and icus) needed to keep the fatality ratio as low as possible. the model predicts that until may 4, an average of 20% of the population in lombardy has already recovered (interquartile range: *10% to *30%) (see fig 6) . finally, in fig 7, we report the evolution of the effective reproduction number r e until may 4. on may 4 the estimated mean value of r e was 0.987 (interquartile range: 0.857 to 1.133), thus marking the onset of a critical date for the post-lockdown period. tracing day-zero and forecasting the covid-19 outbreak in lombardy the crucial questions about an outbreak is how, when (day-zero), why it started, and if and when it will end. answers to these important questions would add critical knowledge to our arsenal to combat the pandemic. the tracing of day-zero, in particular, is of outmost importance. it is well known, that minor perturbations in the initial conditions of a complex system, such as the ones of an outbreak, may result in major changes in the observed dynamics. undoubtedly, a high level of uncertainty for day-zero, as well as the uncertainty in the actual numbers of exposed people in the total population, raise several barriers to our ability to correctly assess the state and dynamics of the outbreak and to forecast its evolution and its end. such pieces of information would lower the barriers and help public health authorities respond fast and efficiently to the emergency. for example, an over-or under-estimation of day-zero would result in an under-or over-estimation of the the transmission rate β and therefore of the basic reproduction number r 0 and consequently the effective reproduction number r e . furthermore, the correct estimation of day-zero is important for the assessment of the number of asymptomatic and actual recovered cases in the total population. this is turn will bias the assessment and ultimately the design of efficient control policies in real time. this study aimed exactly at shedding more light into this problem. to achieve this goal, we addressed a conceptually simple compartmental seiird model with two infectious compartments in order to bridge the gap between the number of asymptomatic cases in the total population and the cases that will experience mild to more severe symptoms. what is done until now with mathematical epidemiological models is the investigation of several scenarios, by changing e.g. the (or assuming a fixed) initial day (day-zero), the level of asymptomatic cases etc. our work, is the first that introduces a methodological framework, to estimate the day-zero as well as the level of asymptomatic cases in the total population in a tracing day-zero and forecasting the covid-19 outbreak in lombardy systematic way. following the proposed methodological framework, we found that the dayzero in lombardy was around the middle of january, a period that precedes by one month the fate of the first confirmed case in the hardest-hit northern italian region of lombardy. interestingly enough, when we submitted a preprint of the work at medrxiv on march 20 2020 ( [48] , another study that was also submitted at the same day at medrxiv, that was based on genomic and phylogenetic data analysis, reports the same time period, between the second half of january and early february, 2020, as the time when the novel coronavirus sars-cov-2 entered northern italy [49] . our analysis further revealed that the actual number of asymptomatic infected cases in the total population in the period until march 8 was around 15 times the number of confirmed infected cases, which until march 8 was also approximately equal to the number of cases that were hospitalized and admitted to icus. our model and methodological approach assume that there was one effective "zero" infected case that introduced the virus to the region; one could certainly argue that there were more than one cases that introduced the virus to the region on the same day; such scenarios can be investigated in a straightforward manner based on our proposed methodological approach. furthermore, the proposed approach could be used for the quantification of the uncertainty of the evolving dynamics, taking into account the reported, from clinical studies, distributions of the epidemiological parameters rather than their expected values. a critical point that is connected with the above is that with such a small number of infectious at the initial stage of the simulations, a stochastic model or a hybrid stochastic model could be more realistic in which uncertainty could be modelled in the form of realistic perturbations (see for example [50] ). furthermore, we did not consider the effect of an ongoing sampling strategy in the total population for the estimation of the level of under-reporting (represented in our model with the variable �). as mentioned in the methodology, within the first period of the outbreak the number of confirmed cases was approximately equal to the number of hospitalized cases, i.e. there was no sampling strategy. furthermore, as also reported for the later period, tests were conducted only for those who seeked medical care and had symptoms like fever and coughing. thus, people who did not seek for medical attention were tested very scarcely [18] [19] [20] . we will consider such type of modelling and analysis in a future work. regarding the forecasting in lombardy from march 8 until may 4 (the first day of relaxation of the measures), we have taken into account the very latest facts on the drop of human mobility, as released by google [37] until april 30 for the region of lombardy; these were shaped by the very strict measures announced on march 20-21 that included the closure of all parks, public and private offices and the prohibition of any pedestrian activity, even individually [45] . our modelling approach approximated fairly well the reported number of infected cases in lombardy two months ahead of time. the mean value of the evolution of the compartment that in our model reflects the confirmed cases, almost coincides with the reported cases for the entire period from march 8 to may 4. the differences that are observed between the reported number of deaths and simulations as discussed in the results should be attributed to the very short times from the onset of symptoms to death that have been reported in lombardy at the early phases of the pandemic in the region (8 days instead of 2 to 8 weeks that have been reported in other studies for china that is linked with the saturation of the icus [47, 51] . furthermore another important factor that is missing from the data used is that the number of deaths is largely affected by the criteria of death notification. indeed as it has been reported, the global coronavirus death toll could be 60% higher than the confirmed [52] . however, this fact did not affect the model predictions with respect to the confirmed cases, but the rate with which the confirmed cases die; the later is also dependent on the rate of the outbreak and the relevant capacity of the icus. to this end, we would like to make a final comment with respect to the basic reproduction number r 0 , the significance and meaning of which are very often misinterpreted and misused, thereby leading to erroneous conclusions. here, we found a r 0 * 4.5, which is higher compared to the values reported by many studies in china, and also in italy, and in lombardy in particular. for example, zhao et al. estimated r 0 to range between 2.24 (95% ci: 1.96, 2.55) and 3.58 (95% ci: 2.89, 4.39) in the early phase of the outbreak [9] . similar estimates were obtained for r 0 by imai et al., 2.6 (95% ci: 1.5, 3.5) [ [8] . regarding italy, d'arienzo and coniglio [54] used a sir model to fit the reported data in nine italian cities and found that r 0 ranged from 2.43 to 3.10. in another study, the authors provided an estimate of the basic reproduction number by analyzing the first 5,830 laboratoryconfirmed cases. by doing so they estimated the basic reproduction number at 3.1 [27] . first, we would like to stress that r 0 is not a biological constant for a disease as it is affected not only by the pathogen, but also by many other factors, such as environmental conditions, demographics, as well as, importantly, by the social behavior of the population (see for example the discussion in [4] ). thus, a value for r 0 that is found in one part of the world (e.g. in china), and even in a region of the same country, e.g. in tuscany, italy, cannot be generalized as a global biological constant for other parts of the world, or even for other regions of the same country. obviously, the environmental factors and social behavior of the population in lombardy are different from the ones, for example, prevailing in hubei. second, most of the studies that provide estimates of the basic reproduction number are based solely on the reported cases, thus the actual number of infected cases in the total population, that may be asymptomatic but transmit the disease, is not considered; this fact may lead to an underestimation of the basic reproduction number. moreover, in our approach as compared to clinical studies, the computation of r 0 comes out as the necessary and sufficient condition of the stability as derived from the proposed model whose parameters are computed based on the available reported data, thus with a delay between the first actual case (see also the discussion in [55] . our relatively simple conceptually model and approach do not aspire to accurately describe the complexity of the emergent dynamics, which in any case is an overwhelmingly difficult, if not impossible, task in the long run, even with the use of detailed agent-based models. we tried to keep the structure of the model as simple as possible in order to be able to model (in a coarse way) the uncertainty in both the "day-zero" and the number of asymptomatic actual cases in the total population using as few parameters as possible. the results of our analysis have indeed proved that the modelling approach succeeded in providing fair predictions of the evolution of the epidemic two months ahead of time. such an early assessment would help authorities to evaluate the required measures to control the epidemic, such as the scale of diagnostic tests that have to be performed and the number of icu beds required. while more complicated models can, in principle, be constructed to take into account more detailed information, such as the number of hospitalized patients and patients in icus, for any practical means such an approach would suffer from the "curse of dimensionality" as it would introduce many more parameters that would need calibration based on a relatively small size of data especially at the beginning of an outbreak. an attempt to compute the values of some of these additional parameters, which can only be roughly estimated by clinical studies at the early stages of an emerging novel infectious disease, would introduce additional uncertainty, thereby further complicating matters rather than solving the problem. to this end, we hope that our conceptually simple, but pragmatic, modelling approach and methodological framework help to provide improved insights into the currently uncontrolled pandemic and to contribute to the mitigation of some of its severe consequences. author contributions coronavirus disease 2019 (covid-19). situation report coronavirus covid-19 global cases by covid-19: preparedness, decentralisation, and the hunt for patient zero complexity of the basic reproduction number (r0) nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study transmissibility of 2019-ncov estimating the scale of covid-19 epidemic in the united states: simulations based on air traffic directly from wuhan, china data-based analysis, modelling and forecasting of the covid-19 outbreak preliminary estimation of the basic reproduction number of novel coronavirus (2019-ncov) in china, from 2019 to 2020: a data-driven analysis in the early phase of the outbreak covid-19 and italy: what next? breaking down of the healthcare system: mathematical modelling for controlling the novel coronavirus (2019-ncov) outbreak in wuhan estimating the risk on outbreak spreading of 2019-ncov in china using transportation data using predicted imports of 2019-ncov cases to determine locations that may not be identifying all imported cases the effect of travel restrictions on the spread of the 2019 novel coronavirus (covid-19) outbreak coronavirus-scientific insights and societal aspects current us coronavirus cases are "just the tip of the iceberg,-former usaid director says the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) in china in one italian town, we showed mass testing could eradicate the coronavirus perch& x00e9; non stiamo facendo pi& x00f9; tamponi? covid-19-situazione in italia prevention, how covid-19 spreads early transmission dynamics in wuhan, china, of novel coronavirus infected pneumonia report of the who-china joint mission on coronavirus disease how will country-based mitigation measures influence the course of the covid-19 epidemic? the early phase of the covid-19 outbreak in lombardy, italy (2020) serial interval of novel coronavirus (covid-19) infections superiore di sanit characteristics of covid-19 patients dying in italy report based on available data on avoidable errors in the modelling of outbreaks of emerging pathogens, with special reference to ebola niching methods and multimodal optimization performance cumulative frequency functions a new family of life distributions inners and stability of dynamic systems the construction of next-generation matrices for compartmental epidemic models advancing the right to health: the vital role of law, world health organization case investigations of infectious diseases occurring in workplaces, united states quantifying social distancing arising from pandemic influenza nonpharmaceutical measures for pandemic influenza in nonhealthcare settings-social distancing measures, emerging infectious diseases social contacts and mixing patterns relevant to the spread of infectious diseases il 40% ancora si sposta a milano militari per le strade. posti di blocco a roma coronavirus, ordinanza lombardia con nuove limitazioni modeling the interplay between human behavior and the spread of infectious diseases real estimates of mortality following covid-19 infection tracing day-zero and forecasting the covid-19 outbreak in lombardy, italy: a compartmental modelling and numerical optimization approach genomic characterisation and phylogenetic analysis of sars-cov-2 in italy bounded noises in physics, biology, and engineering who statement regarding cluster of pneumonia cases in wuhan global coronavirus death toll could be 60% higher than reported | free to read early transmission dynamics in wuhan, china, of novel coronavirus infected pneumonia assessment of the sars-cov-2 basic reproduction number, r0, based on the early phase of covid-19 outbreak in italy a new framework and software to estimate timevarying reproduction numbers during epidemics key: cord-349476-iac9fak3 authors: mao, liang title: evaluating the combined effectiveness of influenza control strategies and human preventive behavior date: 2011-10-17 journal: plos one doi: 10.1371/journal.pone.0024706 sha: doc_id: 349476 cord_uid: iac9fak3 control strategies enforced by health agencies are a major type of practice to contain influenza outbreaks. another type of practice is the voluntary preventive behavior of individuals, such as receiving vaccination, taking antiviral drugs, and wearing face masks. these two types of practices take effects concurrently in influenza containment, but little attention has been paid to their combined effectiveness. this article estimates this combined effectiveness using established simulation models in the urbanized area of buffalo, ny, usa. three control strategies are investigated, including: targeted antiviral prophylaxis (tap), workplace/school closure, community travel restriction, as well as the combination of the three. all control strategies are simulated with and without regard to individual preventive behavior, and the resulting effectiveness are compared. the simulation outcomes suggest that weaker control strategies could suffice to contain influenza epidemics, because individuals voluntarily adopt preventive behavior, rendering these weaker strategies more effective than would otherwise have been expected. the preventive behavior of individuals could save medical resources for control strategies and avoid unnecessary socio-economic interruptions. this research adds a human behavioral dimension into the simulation of control strategies and offers new insights into disease containment. health policy makers are recommended to review current control strategies and comprehend preventive behavior patterns of local populations before making decisions on influenza containment. during the past decade, influenza has obtained unprecedented attention due to widespread occurrence of novel viruses, such as the bird flu in 2003 and the swine flu in 2009 [1, 2] . recent estimates by the center of disease control and prevention (cdc) indicated that the 2009 swine flu is responsible for 274,000 hospitalizations and 12,470 deaths in the united states [3] . these staggering health burdens call for effective measures to control and prevent future outbreaks. the control of influenza primarily involves applying health resources to affected people, known as control strategies, for example, medical treatment for infected individuals, closure of affected workplaces/schools, and travel restriction to affected communities [4] . the prevention of influenza emphasizes healthy people and depends on their voluntary behavior against the disease, referred to as the preventive behavior. as recommended by cdc, the preventive behavior against influenza include receiving vaccination, wearing facemasks, washing hands frequently, taking antiviral drugs, and others [5] . while devising various control strategies and evaluating their effectiveness, few studies have incorporated the preventive behavior of individuals [6, 7] . in most cases, individuals are often assumed to passively comply with control strategies, but their active prevention against the disease has been overlooked. in reality, the preventive behavior of individuals also reduces infections and takes effect concurrently with typical control strategies. for instance, individuals may voluntarily protect themselves from infection, once they realize some control strategies being applied to their family members, colleagues, or communities [8, 9, 10] . by far, the combined effectiveness of control strategies and individuals' preventive behavior remains unclear, and little attention has been paid to this issue. lack of such knowledge may bias estimation of health resources needed to suppress an outbreak, and mislead the real practice of influenza containment. the purpose of this article is to evaluate the combined effectiveness of control strategies and individual preventive behavior. agent-based stochastic simulations are used to investigate three control strategies, including the targeted antiviral prophylaxis (tap), workplace/school closure, and travel restriction, as well as combinations of all three. the urbanized area of buffalo, new york, usa, is taken as a study area. the control effectiveness with and without considering individual preventive behavior is compared to indicate if there exists a significant difference. cost-effective strategies are suggested based on the comparison analysis. the remainder of this article is organized as follows. the method section that follows reviews two established influenza models for simulation and describes the design of control strategies being simulated. the result section presents and compares the simulation results. the discussion section concludes this article with implications. epidemic models, including mathematical and computer models, have been extensively used to investigate disease control strategies, because of their ease and flexibility to deal with different scenarios. the classic mathematical model, the sir model, and its variants employ differential equations to describe continuous variations between three subpopulations, i.e., the susceptible, infectious and recovered [11, 12] . various control strategies are often expressed as different initial conditions (e.g., the size of susceptible population) or parameter settings (e.g., the infection rate) of differential equations. the computer-based simulation models have recently gained their impetus in epidemiology [13, 14, 15, 16] . these models study population-level health outcomes through the simulation of individuals and their microinteractions. control strategies can be represented by altering individuals' health status and their behavior, such as endowing them immunity against infection and prohibiting their out-ofhome activities. all of these epidemic models provide solid platforms to evaluate and compare alternative strategies, thus informing health policy making [17, 18] . epidemic models without considering individual preventive behavior are widely seen in the literature and hereinafter referred to as 'influenza-only' models, because they primarily focus on influenza transmission. in this research, an influenza-only model is implemented in the study area, which includes a total number of 985,001 individuals. these individuals live in 967 census block groups and 400,870 households according to us census 2000 [19] , and carry out daily activities in 36,839 business locations [20] . the model involves an agent-based stochastic simulation, discrete time steps, and spatially explicit representation of individuals. each individual is a modeling unit with a set of characteristics (e.g., age, occupation, infection status, location and time of daily activities) and behaviors (e.g., traveling between locations for activities and having contact with other individuals) [21, 22] . individuals and households are simulated under the constraints of census data so that the modeled population matches the age and household structure of the real study area. individuals are also assigned to business locations to represent their daily activities, such as working, shopping, eating out, etc. ( figure s1 ). the contacts between individuals take place when individuals meet at the same time and location, such as homes, workplaces, shops, and restaurants. because individuals travel over time and location, their mobility weaves a spatio-temporally varying contact network (see text s1 section 1.1). through such a network, influenza viruses diffuse from one individual to another. each individual is allowed to take one of four infection status during a time period, i.e. susceptible, latent, infectious, and recovered. the progress of infection status follows the natural history of influenza, including the latent, incubation, and infectious periods (table s1 ). during the infectious period, individuals may manifest symptoms and become symptomatic. to initiate the disease transmission, five infectious individuals are randomly seeded into the study area at the first day of simulation, which then lasts for 150 days. in each day, the model traces susceptible contacts of infectious individuals, and stochastically identifies the next generation of infections using the monte-carlo method (see text s1 section 1.2). in order to further consider individual preventive behavior, this research employs an agent-based 'dual-diffusion' stochastic model that simulates the concurrent diffusion of both influenza and individual preventive behavior [23] . the preventive behavior is considered as a practice or information that also diffuses over contact networks through inter-personal influence. these two diffusion processes interact with one another, i.e., the diffusion of influenza motivates the propagation of preventive behavior, which in turn limits the influenza diffusion [24, 25, 26 ]. in the model, the diffusion of influenza is simulated similarly to the influenza-only model aforementioned. the diffusion of individual preventive behavior is propelled by two types of inter-personal influence through the contact network: one is the perceived infection risk and the other is the perceived social standard. the former is represented as the proportion of influenza cases among an individual's contacts, while the latter is expressed as the proportion of behavioral adopters among the contacts [23] . individuals are simulated to evaluate these two proportions every day through the contact network. once either proportion exceeds a corresponding threshold, an individual will be convinced to adopt and practice preventive behavior [27, 28] . the estimation of individualized thresholds toward adoption is based on a health behavioral survey approved by the social and behavioral sciences institutional review board, university at buffalo, state university of new york. the waiver of informed consent was obtained from the university review board for this research (see text s1 section 2 and figures s2-s3 ). compared to the influenza-only model, individuals in the dual-diffusion model have additional characteristics, such as their adoption status of preventive behavior and thresholds toward adoption. individuals also have more behaviors, for example, evaluating infection risks and social standards from their contacts, making decision to adopt, and carrying out preventive behavior against influenza. for illustrative purposes, the use of flu antiviral drugs (e.g., tamiflu) is taken as an example of preventive behavior in the simulation, because its clinical efficacy is more conclusive than other behaviors, for instance, washing hand and wearing facemasks. specifically, if an individual uses antiviral drugs, the chance of being infected and infecting others can be reduced by 70% and 40%, respectively [14, 29] . implementation details of these two models are not the focus of this article, and readers could refer to text s1 section 1.3 and table s2 . influenza control strategies are mostly applied at three levels: the individual level, group level, and community level. for each level, one strategy is selected for subsequent investigation, namely, a targeted antiviral prophylaxis (tap) strategy at the individual level, a workplace closure strategy at the group level, and a travel restriction strategy at the community level, as shown in table 1 . detailed descriptions of the three strategies are provided below. first, the tap strategy identifies symptomatic individuals (influenza cases), searches their household members, and then targets antiviral drugs to all these individuals [13, 30] . this strategy has been recommended to be quite effective if stockpiles of antiviral drugs are sufficient and infections can be quickly detected [4] . to account for limited health personnel, this research assumes that only a proportion of influenza cases, 60% (60%tap) and 80% (80%tap), can be identified during a day, following the design by germann et al. [13] . second, the workplace closure strategy shuts down a proportion of workplaces/schools where influenza cases are identified [31] . this strategy has been suggested to be useful to socially distance individuals, delay the disease spread, and win time for developing vaccines and antiviral drugs [32] . following the work by ferguson et al. [33] , a low-level scenario (10%wc) closes 10% affected workplaces and 100% affected schools during a day, while a high-level scenario (33%wc) closes 33% affected workplaces and 100% affected schools. third, the travel restriction strategy aims to reduce the trips into and out of affected communities [33, 34] . each of the 967 census block groups in the study area is treated as a community. following the project by germann et al. [13] , a low-level scenario (10% tr) restricts 10% trips into and out of all affected communities, while a high-level scenario prohibits 50% trips (50% tr). in addition to testing the three control strategies individually, the combinations of all three are also evaluated. a low-level combination scenario (referred to as the combined-low) includes all three strategies at their respective low levels. likewise, a high-level combination (referred to as combined-high) contains all three single strategies at high levels. the three control strategies and their combinations in table 1 are simulated by the influenza-only model and the dual-diffusion model, respectively. results from the influenza-only model indicate the effectiveness of control strategies without individual preventive behavior. meanwhile, outcomes from the dual-diffusion model show the combined effectiveness of both control strategies and individual preventive behavior. these two modeled effectiveness are compared to a baseline epidemic scenario, which represents a worst situation of no control strategies and no preventive behavior. all strategies are assumed to be implemented at the time when the cumulative number of influenza cases exceeds 1,000 (1% of the population), and last until the end of the epidemic. individuals having or having not adopted preventive behavior are treated the same by all control strategies, so that the control effectiveness from two models are comparable. for each model and each strategy scenario in table 1 , the simulation is performed 50 realizations to reduce randomness, resulting in a total of 1,000 realizations (5 strategies62 scenarios62 models650 realizations). each simulation records the time and location of every infection event during a 150-day period. for each strategy scenario, the control effectiveness is measured by an epidemic curve that depicts the number of daily new influenza cases from day 1 to day 150. the number of daily new cases is averaged from 50 model realizations, and then plotted against time to form an averaged epidemic curve (figure 1 ). associated characteristics of this epidemic curve are also derived, including an overall attack rate (the percentage of influenza cases in the population) and epidemic peak time (table 2 ). for the ease of comparison, a relative effectiveness of a control strategy is also calculated as an index ranging from 0 to 1. the relative effectiveness is defined as a ratio of the attack rate reduced by a strategy from the baseline to the baseline attack rate, i.e., (baseline attack rate2attack rate under a strategy)/baseline attack rate. a zero value represents the baseline scenario without any control strategy (the attack rate under non-strategy = the baseline rate), while a higher value close to 1 indicates that a control strategy produces a smaller attack rate. an effective strategy is expected to produce a low epidemic curve, small attack rate, and high relative effectiveness. in this research, an epidemic is assumed to be successfully contained, if the overall attack rate is below 5%. this is because reported influenza epidemics often have a 5% or higher attack rate [35, 36] . the spatial effectiveness of control strategies is also of interest, and thus a series of infection intensity maps are displayed in figure 2 . the infection intensity represents the density of total infections as points occurring within every geographic unit (50 m650 m) during the entire 150-day epidemic. the intensity value at each cell location is also the average from 50 model realizations and is converted to a unit of infections per sq km 2 for the ease of comparison. an effective strategy is expected to reduce infection intensity at every location, and meanwhile confine the spatial extent of affected areas. on average, the baseline epidemic scenario (red curves in figure 1 ) causes an 18.6% of the population developing influenza symptoms ( table 2 ). the epidemic peaks at day 77 with approximately 6,000 new cases occurring at the peak time. the application of 60% tap and 80% tap scenario (blue curves in figure 1a -b) significantly reduces the overall attack rate to 6.87% and 4.74%, respectively. these two tap scenarios also postpone the peak time by 5-13 days. without considering preventive behavior, the 80% tap scenario seems effective to contain the epidemic, because it manages to lessen the overall attack rate under the 5% epidemic criterion. by further adding the preventive behavior (hereinafter abbreviated as pb), both 60%tap+pb and 80%tap+pb scenarios (green curves in figure 1a -b) result in even lower attack rates around 4.3% ( table 2 ). the epidemic peaks can be limited around 1,000 daily new cases, while the peak time remains similar to the baseline scenario. this is because the diffusion of preventive behavior quickly exhausts the pool of susceptible individuals, and need to prepare a smaller stockpile of antiviral drugs than would otherwise being expected. turning to the workplace closure strategies (blue curve in figure 1c ), the 10% wc scenario slightly reduces the overall attack rates to 11.87%, and delays the peak time only a little ( table 2 ). in contrast, the 33% wc scenario ((blue curve in figure 1d ) lessens the attack rate to a much lower level of 4.86%, and advances the peak time by approximately 1 week. for the purpose of containing the epidemic, the 33% wc scenario, i.e., the closure of 33% affected workplaces, is needed to achieve an attack rate under 5%. by further including the individual preventive behavior (green curves in figure 1c -d), the 10% wc+pb and 33%wc+pb scenarios produce a much smaller attack rate of 3.99% and 1.83%, respectively ( table 2) . the time to reach epidemic peaks is shortened to 61-66 days, roughly 2 weeks earlier than the baseline scenario. the relative effectiveness of 10% wc scenario is doubled by considering preventive behavior. a primary reason is that a number of susceptible individuals voluntary protect themselves from infection. these individuals, therefore, cannot be infected or infect others at workplaces and schools, largely limiting the disease transmission. the comparison suggests that given the preventive behavior of individuals is counted, a 10% workplace closure strategy, instead of the 33% one, would be adequate to contain an influenza epidemic. surprisingly, the 10% tr scenario ( figure 1e ) alone causes an even worse situation than the baseline scenario. the overall attack rate reaches 20%, and is 1.4% higher than the baseline rate, leading to a negative effectiveness ( = 20.07 in table 2 ). a possible reason is that the travel restriction strategy extends the time of individuals spent at home, thereby intensifying the within-home transmission. since only 10% of trips into and out of affected communities are restricted, the disease can still be easily transported from one affected community to another through the 90% unrestricted trips. the epidemic thus develops faster and affects more individuals. as the travel restriction level elevates to 50% (the 50% tr in figure 1f ), much more trips into and out of affected communities are restricted. although the infections at homes are intensified, most infections can only take place within communities, instead of between communities. as a result, the overall attack rate drops to 5.91% and the epidemic peak is greatly mitigated. nevertheless, the 50% tr does not suffice to contain the epidemic, because the attack rate remains above 5%. the simulation results are distinctly different if adding individual preventive behavior (green curves in figure 1e -f). the 10%tr+pb scenario produces a much better outcome than that from the 10% tr alone, because the relative effectiveness jumps from 20.07 to 0.62. the overall attack rate and epidemic peak size are remarkably reduced, although the attack rate remains above 5% ( table 2 ). the 50%tr+pb scenario turns out to be effective for influenza containment, because the overall attack rate can be lowered to 1.65%, much less than the 5% epidemic criterion. the combined strategies (the blue curves in figure 1g -h) outperform each of the three single strategies. the total infections can be contained far below 5% of the population, with a small peak size under 1,000 cases. particularly, the combined-high scenario is capable of preventing the epidemic, given only 0.68% of the population being infected (table 2) . among the three single strategies, the tap strategy reduces infections within households, the workplace closure strategy tends to prevent infections at workplaces, and the travel restriction limits the disease transmission between communities. these three single strategies work together as complements, leading to a significant improvement in control effectiveness (relative effectiveness.0.9). without considering preventive behavior, the combined-high scenario seems necessary to contain the epidemic, while the combined-low scenario is insufficient. this argument, however, may be changed by incorporating individual preventive behavior (green curves in figure 1g -h). the combined-low+pb scenario now is adequate to reduce the overall attack rate below 5% and thus contain the epidemic, while the high-level scenario is no longer a necessity. based on the comparison analysis above, the tap 60%+pb, 10% wc+pb, 50% tr+pb, and the combined-low+pb scenarios are suggested to be cost-effective in controlling influenza epidemics. therefore, their spatial effectiveness is further examined and compared through infection intensity maps (figure 2 ). for description purposes, the mapped infection intensity is further categorized into 6 levels, i.e., very low (0-50 infections/km 2 ), low (50-100), moderate (100-200), high (200-500), very high (500-1,000), and extremely high (.1,000) . the baseline scenario (figure 2a ) induces an extremely high intensity of infections in the central business district of the study area. the infection intensity decreases in an outward direction to suburbs. this is because the central business district has the densest residential population and highly concentrated business locations. compared to the baseline map, the 60% tap+pb scenario ( figure 2b ) greatly reduces the infection intensities all around the study area, although the central business district retains a high-level intensity. the spatial effectiveness of 10%wc+pb ( figure 2c ) is similar to the 60% tap+pb, but moderate infections are more scattered in the suburban areas. the 50% tr+pb is capable of confining the wide spread of influenza over the study area ( figure 2d ), leaving only a small number of separated areas with high infection intensity. these hotspots are most located within cbd, university campuses, and large industrial plants, where a large number of people work and live. this is probably because the city-wide travels of individuals are partially prohibited, and hence disease can only develop locally. finally, the combined low+pb scenario not only reduces the intensity of the infections at all locations, but also confines spatial extent of disease spread ( figure 2e ). the infections in the central business district are reduced to a moderate level, while a vast proportion of the study area has only a small number of infections. in summary, previous studies on influenza containment have only considered the effectiveness of applying control strategies, while overlooking the effectiveness from individuals' preventive behavior. this research estimates the combined effectiveness of both control strategies and individual preventive behavior. the results imply that previous studies on control strategies are incomplete, and the control effectiveness might be under-estimated. the comparison between two model results indicates that preventive behavior of individuals has an extra effectiveness, in addition to the effectiveness from typical control strategies alone. this extra effectiveness produces an even smaller attack rate of influenza, lower epidemic peak, and earlier peak time. by considering the combined effectiveness, the control of influenza epidemics may not require as much health resources as estimated in previous studies. for example, the 80% tap strategy could be replaced by the 60% one, reducing the burden of local agencies to prepare health resources. likewise, the 10% workplace closure strategy, rather than the 33% strategy, would be sufficient to control the seasonal influenza epidemic in the study area. enormous socio-economic disruptions could be possibly avoided. a low-level combination of the three strategies is recommended to suppress influenza epidemic in the study area, while a high-level combination is no longer a must. particularly, with the help of individual preventive behavior, the 50% travel restriction strategy and the lowlevel combined strategy can successfully confines the spatial dispersion of influenza in the study area. similar to any modeling analysis, this research has a number of limitations. first, the simulation models focus on one us metropolitan area, one influenza virus strain, and one preventive behavior. it is possible that the model outcomes vary in different cities and different disease parameters, such as a pandemic influenza virus. the interpretation of model outcomes should be limited to seasonal influenza and in the study area. although the use of antiviral drugs is taken as an example in this research, the methodology can be easily extended to other preventive behavior, such as washing hands and wearing facemasks, once their preventive efficacy is conclusively quantified. second, the mass media also influences people's decision to adopt preventive behavior, especially for diseases that are highly infectious or pose severe health risks, such as the severe acute respiratory syndrome (sars). this research has not modeled the mass media because its effects on flu-related preventive behavior remain inconclusive. in addition, the seasonal influenza simulated in this research has a relatively mild infectivity and limited risks, thus is usually not a focus of mass media attention. third, the model assumes that individuals adopt preventive behavior immediately after the threshold effects happen. in reality, individuals' adoption of a behavior may take a relatively longer period as it may involve a number of psychological steps [37] . a more sophisticated behavioral approach may improve the modeling reality, but also increase the complexity of model structure. a trade-off between model performance and detail levels is always a challenge for modelers [38] . ongoing research is intended to address these limitations and challenges. control strategies enforced by health agencies and preventive behavior voluntarily practiced by the public are two intertwined components of disease containment. ignoring either component may prevent us from effectively mitigating burdens of influenza on public health. it is hard to resist citing and rephrasing the argument by funk et al. [26] that ''individual self-initiated behavior can change the fate of an outbreak, and its combined effectiveness with control strategies requires proper understanding if we are to fully comprehend how these control measures work''. this research attempts to fuse the human behavioral dimension into the study of control strategies, and thus offers more comprehensive understandings on disease containment. health agencies are recommended to gain prior knowledge about behavioral patterns of local people before choosing influenza control strategies. the findings of this research call for a review of current control strategies and re-estimate the health resources that are necessary to contain epidemics. it is believed that such a review would shed new insights on improving control effectiveness for looming influenza pandemics. figure s1 the simulation of contact network. the assignment of individuals to households, workplaces, service places and neighbor households based on the attribute and spatial information of individuals. text s1 (doc) conceived and designed the experiments: lm. performed the experiments: lm. analyzed the data: lm. wrote the paper: lm. emergence and pandemic potential of swine-origin h1n1 influenza virus public health risk from the avian h5n1 influenza epidemic updated cdc estimates of 2009 h1n1 influenza cases, hospitalizations and deaths in the united states planning for smallpox outbreaks preventing seasonal flu capturing human behaviour can influenza epidemics be prevented by voluntary vaccination the spread of awareness and its impact on epidemic outbreaks the health belief model and personal health behavior endemic disease, awareness, and local behavioural response a contribution to the mathematical theory of epidemics a structured epidemic model incorporating geographic mobility among regions mitigation strategies for pandemic influenza in the united states containing pandemic influenza with antiviral agents modeling targeted layered containment of an influenza pandemic in the united states flute, a publicly available stochastic influenza epidemic simulation model modeling influenza epidemics and pandemics: insights into the future of swine flu (h1n1) the effectiveness of policies to control a human influenza pandemic: a literature review data releases spatial-temporal transmission of influenza and its health risks in an urbanized area a conceptual framework for an individual-based spatially explicit epidemiological model agent-based simulation for a dual-diffusion process of influenza and human preventive behavior the impact of information transmission on epidemic outbreaks multiple sources and routes of information transmission: implications for epidemic dynamics modelling the influence of human behaviour on the spread of infectious diseases: a review threshold models of collective behavior social network thresholds in the diffusion of innovations perspectives on antiviral use during pandemic influenza containing pandemic influenza at the source individual-based computational modeling of smallpox epidemic control strategies simulation suggests that rapid activation of social distancing can arrest epidemic development due to a novel strain of influenza strategies for mitigating an influenza pandemic the effect of travel restrictions on the spread of a moderately contagious disease key facts about seasonal influenza (flu) global epidemiology of influenza: past and present a model of the precaution adoption process: evidence from home radon testing choosing the best model: level of detail, complexity, and model performance key: cord-316287-4i1grvlr authors: yim, sung sun; bang, hyun bae; kim, young hwan; lee, yong jae; jeong, gu min; jeong, ki jun title: rapid isolation of antibody from a synthetic human antibody library by repeated fluorescence-activated cell sorting (facs) date: 2014-10-10 journal: plos one doi: 10.1371/journal.pone.0108225 sha: doc_id: 316287 cord_uid: 4i1grvlr antibodies and their derivatives are the most important agents in therapeutics and diagnostics. even after the significant progress in the technology for antibody screening from huge libraries, it takes a long time to isolate an antibody, which prevents a prompt action against the spread of a disease. here, we report a new strategy for isolating desired antibodies from a combinatorial library in one day by repeated fluorescence-activated cell sorting (facs). first, we constructed a library of synthetic human antibody in which single-chain variable fragment (scfv) was expressed in the periplasm of escherichia coli. after labeling the cells with fluorescent antigen probes, the highly fluorescent cells were sorted by using a high-speed cell sorter, and these cells were reused without regeneration in the next round of sorting. after repeating this sorting, the positive clones were completely enriched in several hours. thus, we screened the library against three viral antigens, including the h1n1 influenza virus, hepatitis b virus, and foot-and-mouth disease virus. finally, the potential antibody candidates, which show k(d) values between 10 and 100 nm against the target antigens, could be successfully isolated even though the library was relatively small (∼10(6)). these results show that repeated facs screening without regeneration of the sorted cells can be a powerful method when a rapid response to a spreading disease is required. for the last two decades, monoclonal antibodies and antibody fragments have been proven to be effective as therapeutic and diagnostic agents, and have long been invaluable tools in various fields of biological research [1, 2] . for the development of antigenspecific antibodies, hybridoma technology that relies on animal immunization has been traditionally employed [3] . the recent progress in combinatorial technologies because of in vitro antibody repertoires and high-throughput screening methodologies has allowed the development of target-specific antibodies without animal immunization [4, 5] . in these technologies, various protein display systems including phage display, ribosome display, and cell-surface display, have been widely used for the initial isolation of antibodies specific to antigens from huge libraries, as well as for engineering the antibodies towards desired functions, e.g., enhanced affinity and higher thermostability. [6, 7, 8] . however, the most recent tools require repeated screenings in order to isolate potential candidates from the library, and consequently, they require relatively long time periods (several days to weeks) to complete the screening. the recent emergence and rapid dissemination of new viruses that cause serious human and animal diseases, such as sars coronavirus, swine flu h1n1 virus, and avian influenza h5n1 virus, has raised world concerns. the development of new tools to quickly isolate antibodies against rapidly spreading infectious viruses for treatment as well as early diagnosis is urgently required. currently, fluorescence-activated cell sorting (facs) has been used in high-throughput screening of huge libraries (generally bigger than 10 6 cells) that are constructed in various display systems in bacteria or yeast as the host [6, [8] [9] [10] . the following strategy is usually used for screening a recombinant antibody library: (i) cultivation of library cells; (ii) fluorescent-antigenpeptide or protein labeling of the library cells; (iii) facs sorting of the highly fluorescent population; (iv) regeneration of the sorted cells by regrowth or re-cloning of the sorted target genes; (v) repetition of steps i-iv until a highly fluorescent population is separated from the negative control population; and (vi) analysis of the individual clones. among these steps, the step determining the screening time is the regeneration of the sorted cells (step iv). in all of the current screening strategies, the sorted cells need to be regenerated for the next round of sorting, which can be done by cultivating the cells for at least one day [6, 9, 10] or by re-cloning the genes, which takes several days [11] . in addition to the regeneration time, contamination of the sorted cells by nonspecific clones also needs to be considered. during the cultivation for regeneration of sorted cells, differential growth rates among various clones (particularly non-specific clones) due to unregulated protein expression and differing cell viability can decrease the library screening efficiency, resulting in more rounds of sorting (longer duration) to isolate the potential antibody candidate [12] . herein, we report the development of a new high-throughput screening strategy based on escherichia coli protein display and facs sorting, which allows the simple and rapid isolation of potential candidates from a huge library in one day. first, we constructed the fully synthetic human antibody library in which antibody fragments (single-chain variable fragment, scfv) were produced in the periplasm of e. coli. after library cultivation and permeabilization, the cells were labeled with fluorescent antigen probes, and the highly fluorescent cells were sorted by using a high-speed cell sorter. immediately after the first-round sorting, the sorted cells were reused in the next round of sorting, without regeneration of the sorted cells. this resorting was repeated until a highly fluorescent population became enriched as the major population and, using the high-speed facs sorter, the best candidates could be isolated in one day. the overall strategy of this rapid screening is illustrated in the figure 1 . the proof of this concept was successfully demonstrated by the isolation of specific antibody fragments against three model viral antigens: h1n1 influenza virus, hepatitis b virus (hbv), and foot-and-mouth disease virus (fmdv) serotype o. the whole facs screening rounds of the synthetic human antibody library against each viral antigen could be done in one day, and these results show that repeated facs screening without regeneration of the sorted cells can be a rapid and efficient method to isolate potential antibody candidates in case of urgent requirements. the bacterial strains and plasmids used in this study are listed in the table s1 . e. coli jude-1 was used as the main host for gene cloning and library screening. e. coli hm130 was used for the production and purification of the isolated antibodies (scfv). the e. coli cells were inoculated into luria-bertani (lb) medium (10 g/l of tryptone, 5 g/l of yeast extract, and 10 g/l of nacl) containing 2% (w/v) glucose. after an overnight cultivation at 37uc and 200 rpm, the cells were transferred into 100 ml of fresh lb medium without glucose in a 500-ml flask and incubated at 37uc and 200 rpm. when the cell density (od 600 ) reached 0.6, the cells were induced with 1 mm isopropyl-b-d-thiogalactopyranoside (iptg) and were further cultivated at 25uc at 200 rpm for 4 h. the cells were harvested by centrifugation at 6,000 rpm for 10 min at 4uc for further analysis. in all the cultivations, ampicillin (50 mg/ml) was used as the sole antibiotic. for the facs screening of a human synthetic antibody (scfv) library, three fluorescent antigen probes were chemically synthesized: (i) fitc-crdnwhgsnrpw as an n1 epitope of h1n1 influenza virus [13] ; (ii) fitc-nsttfhqalldprvrglyf-pagg as a pres2 epitope of hbv [14] ; and (iii) fitc-pvtnvrgdlqvlaqk as a vp1 epitope of fmdv [15] . one aliquot (100 ml) of the library stocks frozen at 280uc was thawed and inoculated into 100 ml of lb medium containing 2% (w/v) glucose and ampicillin. after cultivation under the condition described above, the cells were harvested by centrifuging for 10 min at 6,000 rpm and 4uc. for efficient labeling with fluorescent probes, cell pellets were resuspended in 56 tris-kcl buffer (150 mm tris-hcl at ph 7.4 and 750 mm kcl), which dramatically increased the permeability of the e. coli outer membrane and allowed the fluorescent antigen probes to permeate into the periplasm [9, 16] . the resuspended cells were incubated with 5 mm of antigen peptides (n1, pres2, or vp1 epitope) conjugated with fitc for 1 h at 4uc. the cells were then washed twice with the same buffer (56 tris-kcl), and the fluorescent probe-labeled cells were sorted using a high-speed flow cytometer (moflo xdp, beckman coulter, miami, fl). in facs sorting, the cells were selected on the basis of high fluorescence intensity detection through a 530/40 band-pass filter for obtaining the fitc emission spectrum. ''purify mode'' was used as the sorting mode, which sorts only those drops that contain positive cells. all the e. coli cells sorted in each round of screening were immediately reused for the next round of facs sorting without regeneration. the sorting was repeated until the highly fluorescent population was fully enriched. to obtain the required sample volume, 500 ml of sheath buffer was added to the sorted samples during each round. after the final sorting, the scfv genes were amplified from the sorted cells by performing pcr with primers, assembly-f and assembly-r. after digestion with sfii, the amplified scfv genes were cloned into pmopac16 and transformed into e. coli jude-1 for the further analysis of the single clones. for enrichment of cells producing flash tag (flnccpgccmep) from non-specific cells, cells harboring pmopac16-mbp were diluted with non-specific cells harboring pmopac16 at 1:10000 ratio. after labeling with with 25 mm of flash-edt 2 (invitrogen), the mixed cells were applied to facs and the fluorescent cells were sorted by the repeated facs screening as described above. for the production of the isolated antibody fragments, the plasmids recovered from the isolated clones were introduced into e. coli strain hm130. after cultivation in 500 ml of lb medium, the cells were harvested by centrifugation for 10 min at 6,000 rpm and 4uc. the cells were then washed twice with pbs and resuspended in the same buffer. crude extracts of the cells were prepared by sonication (20 min at 50% pulse and 20% amplitude), and the extracts were centrifuged for 10 min at 10,000 rpm and 4uc to yield soluble lysates. the soluble lysates were filtered through a 0.45-mm syringe filter, and the soluble lysates were poured into poly-prep chromatography columns (bio-rad, hercules, ca) filled with talon metal-affinity resin (clontech, mountain view, ca). the resin was washed twice with 10 ml of washing buffer, and the 6-his tag fused to scfv was eluted using 2 ml of the elution buffer. the purified scfvs were used for elisa analysis. for the preparation of gst-fused antigens (n1 of h1n1, pres2 of hbv, and vp1 of fmdv), three vectors were constructed. the primers used in the construction of the gst-fused antigens are listed in the table s2 . the plasmid pgex-4t-1 containing the gst gene was used as the template, and the n1 epitope sequence (crdnwhgsnrpw) of the h1n1 influenza virus [13] was fused to the c-terminus of gst by performing pcr with the primer sets gst-f and gstn1-r. for the synthesis of the pres2 epitope sequence (nsttfhqalldprvrglyfpagg) of hbv [14] and the vp1 epitope sequence (pvtnvrgdlqvlaqk) of fmdv [15] , the same pcr strategy was used, except that the reverse primers-gstpres2-r and gstvp1-r-were used for synthesizing the pres2 and vp1 epitope genes, respectively. the pcr products containing the gst-fused n1 sequence, pres2 sequence, or vp1 epitope sequence were digested using ndei and hindiii, and then were cloned into pmopac1 to yield pmopac1-gst-n1, pmopac1-gst-pres2, or pmopac1-gst-vp1, respectively. e. coli jude-1 harboring pmopac1-gst-n1, pmopac1-gst-pres2, or pmopac1-gst-vp1 were cultivated in 500 ml of lb medium in a 2-l shaking flask at 37uc at 200 rpm. when the cell density (od 600 ) reached 0.6, the cells were induced with 1 mm of iptg and were further cultivated at 37uc at 200 rpm for 6 h. the cells were harvested by centrifugation at 6,000 rpm for 10 min at 4uc. the cells were then resuspended in 50 ml of pbs and were lysed by sonication (20 min at 50% pulse and 20% amplitude). after filtration with a 0.45-mm syringe filter (sartorius stedim biotech, goettingen, germany) to remove the residual insoluble debris, the gst-fused antigens were purified by using glutathione sepharose resin (ge healthcare biosciences ab, uppsala, sweden) as per the method specified by the manufacturer. the gst-fused antigens were mixed with 0.05 m carbonatebicarbonate coating buffer (ph 9.6) to a final concentration of 2 mm. the antigen solution (100 ml) was loaded onto 96-well elisa plate, which was then incubated for 2 h at 37uc. subsequently, each well was washed four times with pbs-t (135 mm nacl, 2.7 mm kcl, 4.3 mm na 2 hpo 4 , 1.4 mm kh 2 po 4 , and 0.5% tween-20 at ph 7.2) and filled with 200 ml of 5% bsa solution, and the plate was incubated for 1 h at 37uc. after washing with pbs-t four times, each scfv sample (soluble lysate or purified sample) was loaded on to the plate, and the plate was incubated overnight at 4uc. each well was washed four times with pbs-t, after which 1:5,000-diluted monoclonal anti-his antibody conjugated to horseradish peroxidase (hrp) (sigma-aldrich, st. louis, mo) was added, and the plates were incubated for 1 h at 37uc. finally, the wells were washed with pbs-t, and tetramethylbenzidien (tmb) was added for the colorimetric detection of the bound scfv clones. the reaction was arrested by adding 2 m of h 2 so 4 stop solution. the absorbance was measured at 450 nm by using a tecan infinite m200 pro elisa plate reader (tecan group ltd., mä nnedorf, switzerland). to confirm binding activity of scfv against inactivated whole fmdv, an fmdv serotype o kit (priocheck fmdv type o, prionics, switzerland) was used; elisa was performed in the same manner with the exception of the antigencoating step. protein samples were analyzed by performing electrophoresis on a 12% (w/v) sds-polyacrylamide gel electrophoresis (sds-page) gel. for the immunodetection of the his-tag-fused scfv proteins, a monoclonal anti-his antibody conjugated to horseradish peroxidase (hrp) (sigma-aldrich) was used. an ecl kit (amersham ecl prime western blotting detection reagent, ge healthcare) was used for signal detection. to prove the concept of our strategy (repeated facs screening without regeneration) (fig. 1) , we first conducted the enrichment of probe-specific cells from mixture with non-specific cells. in this enrichment experiment, flash-edt 2 which can interact with flash tag (flnccpgccmep) and give fluorescent signal [17] was used as probe for cell labeling. cells producing flash-tag fused maltose binding protein (mbp) were mixed with cells producing no-tag protein at 1:10000 dilution ratio, and the enrichment of positive cells were performed by repeated facs screening strategy. from the mixture, the fluorescent cells (top ,1.5% of the total population) were sorted and, immediately after the first round sorting, the sorted cells were applied to the next round of sorting by facs without cell regeneration. as shown in fig. 2 , the fluorescent cells began to be enriched after second round and fully enriched after fourth round sorting. this successful enrichment of probe-specific clones clearly indicate the repeated facs screening strategy can be used for the rapid screening of antigen-specific antibody from library. a fully synthetic human antibody (scfv) library with six diversified complementarity-determining regions (cdrs) was constructed. the detailed procedures for synthetic library construction are described in the figure s1 . briefly, by following the kabat definition [18] and using the database of human germline genes (http://www.bioinf.org.uk/abysis), the single chain variable fragment (scfv) was designed to have eight framework regions (frs) and six cdrs. regarding the frequency of usage and expression in e. coli, the dp-47 and dpk 22 human germline genes were chosen for the framework region of vh and vl, respectively. for the diversity of each cdr loop, except cdr h3, degenerate oligonucleotides were designed on the basis of the frequency of amino acids for each site in the human germline gene database (table s3 ). in the case of cdr h3, much greater diversity in amino acid composition and length was introduced, unlike that in the case of the other cdrs, by using 13 sets of degenerate oligonucleotides with an nnk random sequence and 13 different lengths (10-22 amino acids) (fig. s1 ). each vh and vl gene was assembled by pcr and, to eliminate the clones containing stop codons or frameshifts in the vh or vl gene, each pcr product of vh and vl was cloned into a b-lactamase fusion system. in the selection system, the e. coli clones containing inframe genes of the each chain produced the functional b-lactamase fusion proteins in the periplasm and were selected using ampicillin-containing agar plates [19, 20] . after selection, the inframe vh and vl genes were rescued, and the gene sequences were determined by sequencing. through sequencing experiments, we found that all the clones contained in-frame codons, and no stop codons were observed within the coding genes (data not shown). the rescued vh genes and vl genes were linked for a full antibody fragment (scfv) format and the in-frame scfv genes were also selected in the b-lactamase fusion system. from the selected clones, all in-frame scfv genes were rescued and cloned into pmopac16 for constructing a synthetic human antibody (scfv) library. from the library, 20 clones were randomly chosen, and their sequences were analyzed, and it was clearly confirmed that all 20 clones contained various sequences in the cdrs (fig. s2) . also the randomly selected clones showed good levels of scfv gene expression in e. coli (fig. s3) . the size of the final constructed library was about 3.3610 6 , which is sufficient for the initial isolation of antibody candidates against the target antigens. with this synthetic library, we conducted the isolation of antibody candidates against three antigens: (i) n1 antigenic epitope of h1n1 influenza virus; (ii) pres2 antigenic epitope of hbv; and (iii) vp1 antigenic epitope of fmdv. in order to isolate the n1 epitope-specific antibody (scfv), the synthetic library was screened as shown in the figure 1 . after cultivation of library, cells were harvested and treated with 56 tris-kcl buffer to improve the permeability of the outer membrane and to increase the labeling efficiency of the cells for fluorescent probes [9, 17] . then, cells were mixed with 5 mm of n1 antigenic peptide conjugated with fitc, and cell fluorescence was analyzed by facs. compared to the negative control (e. coli harboring pmopac16 vector only), the cells from the original library showed a slightly higher, but mostly similar fluorescence intensity (fig. 3a) . from the original library, the most fluorescent cells (top ,0.28% of the total population) were sorted until the number of sorted events reached 510,434. immediately after the first round of sorting, the sorted cells were applied to a second round of sorting by facs, and the most fluorescent population of the sorted cells (top ,3.06% of total population) were sorted again. after repeating this sorting three more times, the highly fluorescent population became the major population in the final (5 th ) round of sorting. the fluorescence of the sorted cells was clearly observed to be fully separated from that of the original black bar indicate signals from bsa coated wells, and grey bars indicate signals from gst-fused n1 antigen coated wells. (c) elisa analysis with the purified scfvs. symbols: circle, square and triangle represent the purified antibody from clones s1, s5 and s16, respectively. the closed and open symbols represent the coating of gst-fused n1 antigen and bsa on 96-well plates, respectively. doi:10.1371/journal.pone.0108225.g003 library (fig. 3a) . finally, 1,561 fluorescent cells were collected and used for analysis of individual clones. the sorting results are summarized in the table 1 . after the final screening, the scfv genes of the 1,561 sorted cells were rescued by pcr, and the pcr product was cloned into pmopac16 for the periplasmic expression of scfvs. after transformation into e. coli jude-1, a total of 16 colonies were randomly picked from the agar plate, and the binding activities of the isolated antibodies were analyzed by performing elisa. most clones, except for one (s10), exhibited higher binding activity against the n1 peptide than the non-specific bsa control (fig. 3b) . from the sequencing experiments, we found that among the 16 clones, six clones including s2, s4, s6, s9, s11, and s13, had the same dna sequences. two other clones, s8 and s12, were also same clones, while another two clones, s3 and s10, were same. the same clones with the same sequences showed similar levels of binding activity, except for s3 and s10 (fig. 3b) . among the 16 clones, four, including s1, s5, s7, and s16, showed relatively higher binding activity than the other clones; therefore, we tried purification of scfvs to analyze their specific activities. the four scfv genes were transformed into the e. coli strain hm130, which is protease-deficient and suitable for higher scfv production [21] . after cultivation, scfvs of three clones s1, s5, and s7 could be purified with high purity, but we failed to purify scfv of clone s16 (data not shown). the specific activities of three purified scfvs (s1, s5, and s7) against the n1 epitope were analyzed by elisa and we observed that the binding activities of s1 and s5 were higher than that of s7 and a non-specific bsa control (fig. 3c) . the sequence information of three isolated scfv (s1, s5 and s7) was provided in the figure s4 . to demonstrate the general use of our strategy for antibody screening, we also isolated a potential antibody against another target, pres2 of hbv. after cultivation and permeabilization of the library, cells were labeled with pres2 antigenic peptide conjugated with fitc, and the labeled library cells were sorted by facs. from the original library, the most fluorescent cells (top ,5.53% of total population) were sorted until the number of sorted events reached 1,531,233. then, through repeated sorting without regeneration (three more times), highly fluorescent cells were enriched as a major population, and finally 1,412 cells were collected ( fig. 4a and table 1 ). the scfv genes from the final 1,412 sorted cells were regenerated by pcr, cloned into pmopac16, and then transformed into e. coli strain jude-1. among the regenerated clones, 20 total colonies from the plate were randomly picked, and their binding activities were analyzed by elisa. most clones exhibited good binding activity against antigen pres2 of hbv, but among the 20 clones, four (sp1, sp4, sp14, and sp19) exhibited relatively higher binding activity than the other clones (fig. 4b) . from the sequencing experiment, it was confirmed that two clones (sp4 and sp14) were identical, and so three clones (sp1, sp4, and sp19) were chosen for further analysis. after cultivation, each antibody was successfully purified (data not shown) and the specific binding activities of purified antibodies were evaluated by elisa. we observed that the scfvs purified from clones sp1 and sp19 had much higher binding activity than that from the sp4 clones (fig. 4c) . the sequence information of three isolated scfv (sp1, sp4, and sp19) was provided in the figure s4 . foot-and-mouth disease (fmd) is a highly contagious viral disease infecting cloven-hoofed animals, such as cattle and swine, and it has resulted in massive livestock losses around the world. the diagnosis of fmdv at an early-stage of contamination is crucial to the prevention of the contagion. for the development of an immunodiagnostic system, a highly specific antibody is necessary; therefore, we chose the vp1 antigenic epitope of fmdv as another target antigen for the application of the repeated facs screening system. the synthetic antibody library cells were cultivated and labeled with fitc-conjugated vp1 antigenic peptide, and the labeled library cells were sorted by facs. from the original library, the most fluorescent cells (top ,1.87% of total population) were sorted until the number of sorting events reached 246,139. after two more rounds of facs screening, the highly fluorescent cells were enriched as a major population, and finally 5,262 cells were collected (fig. 5a and table 1 ). the scfv genes from the final 5,262 sorted cells were regenerated by pcr, cloned into pmopac16, and then transformed into e. coli strain jude-1. from the regenerated cells, we randomly picked 20 clones and obtained four clones (sv7, sv9, sv19, sv20) expressing active scfvs against the vp1 antigenic epitope (fig. 5b) . the four scfvs (sv7, sv9, sv19, sv20) that showed high binding were purified for further analyses. in elisa, we observed that one scfv (sv7) has much higher binding activity than the others (fig. 5c) . finally, we also examined the binding activity of the isolated scfv (sv7) against whole (inactivated) fmdv. it was clearly observed that the isolated sv7 antibody fragment exhibited high binding activity against fmdv, while negative control m18 scfv, which can specifically bind to anthrax toxin pa [11] , showed a negligible signal (fig. 6) . the sequence information of four isolated scfv (sv7, sv9, sv19 and sv20) was provided in the figure s4 . binding affinity of the isolated antibody the binding activity (k d ) of isolated antibody against each antigen were also determined by spr analysis. each antigen was immobilized on cm5 chip and the purified antibodies were loaded and binding affinities were analyzed. the binding affinities (k d s) of isolated antibodies, s5 scfv against n1 epitope, sp1 scfv against pres2, and sv7 scfv against were 11 nm, 11.5 nm and 54.9 nm, respectively (fig. s5) , which were similar to those typically obtained using hybridoma technology and other antibody screening strategies including phage display etc. in spr analysis, the affinity can be overestimated due to the avidity effect of dimerized or multimerized antibodies. to check the presence of dimeric antibodies in purified samples, all purified antibodies were analyzed by size exclusion column chromatography and by sds-page analysis in non-reducing and reducing conditions. in all experiments, we clearly confirmed the purified samples were present mainly as a monomeric form (fig. s6 and fig. s7 ) and so the possible avidity effect in spr analysis could be excluded. and the specificity of the isolated scfvs were also confirmed further by western blot on complex protein mixtures containing wildtype gst or antigen-fused gst. as shown in the figure s8 , each scfv could bind specifically to its own viral antigen-fused gst in the soluble lysate of e. coli host. taken all these data, we concluded that, in our screening, each antibody could be isolated not by nonspecific binding of antibody on the cell surface (stickiness) but by the histograms of original library, 1 st round, and 2 nd round sorted cells are represented by red, orange, and green curves, respectively. the 3 rd round sorted cells were used for regeneration of scfv genes by pcr, and its histogram is not shown here. (b) elisa with the soluble lysates of selected clones. black bar indicate signals from bsa coated wells, and grey bars indicate signals from gst-fused vp1 antigen coated wells. (c) elisa analysis with the purified scfvs. symbols: circle, square, triangle and diamond represent the purified antibody from clones sv7, sv9, sv19, and sv20, respectively. the signals were detected from the wells coated with gst-fused vp1 antigen on 96-well plates. doi:10.1371/journal.pone.0108225.g005 specific binding of antibody to antigen probe, and this means that our strategy is valid for the rapid isolation of antibody from library. screening libraries constructed by animal immunization (mainly mouse) with antigens has several benefits, but mouse antibodies give rise to a human anti-murine antibody response (hama) [22] . to reduce the undesired immunogenicity of the isolated mouse antibodies, ''humanizing'' procedures are commonly required, in which the non-human cdrs are conjugated into a given human sequence [23] . however, the exchange of the mouse framework with the human framework does not always guarantee similar affinity, and often leads to less immunogenicity; therefore, it is not suitable for the rapid isolation of effective antibodies. instead of an immune antibody library, non-immune synthetic antibody libraries have been developed that are generally constructed in human antibody frameworks [24, 25] . in the synthetic library, the quality and diversity are of particular importance in the isolation of the antibody; therefore, the six complementarity-determining regions are fully randomized and inserted into a single human antibody framework. for the efficient and soluble production of antibodies in a bacterial host, as well as for less immunogenicity in the human body, the choice of framework in library construction also needs to be considered seriously, and therefore, the isolated clones from the synthetic library are likely to have favorable properties for production and further therapeutic applications. within the human genome, it is known that 51 v h , 30 v l , and 40 v k gene segments exist. although all gene segments can produce a functional antibody, the level of gene expression and the stability of the antibody are very different [24] . in our synthetic library, the dp-47 and dpk 22 human germline genes, which are highly prevalent in human antibodies and are known to be well expressed in e. coli [26] [27] [28] [29] , were chosen for the framework region of vh and vl, respectively. the resulting antibody library showed diversity size of 3.3610 6 . we know that this library size is relatively small compared to diversity of natural human antibody repertoire and, the construction of much bigger library (.10 9 ) is needed to increase the probability for the generation of antibodies against various antigens. however, although the size of library is not large, the quality of our library was enough to be used for the isolation of antigen-specific antibody. to reduce the incidence of frame shifts and stop codons during library construction, the libraries were subjected to in-frame selection by fusion with b-lactamase and ampicillin resistance selection. the quality of the library was validated by sequencing clones that were randomly selected from the library and by analysis of antibody production in e. coli (fig. s2 ). most clones selected randomly from the constructed library and isolated clones by screening exhibited good expression levels with high solubility in e. coli; therefore, we concluded that the high-quality human antibody library with high diversity in six cdrs was successfully synthesized and was suitable for the rapid isolation of antigen-specific antibodies. for the isolation of highly potential clones from these huge libraries, large microbial libraries need to be screened through multiple rounds of sorting due to the contamination of non-specific false-positive clones; however, in most strategies, this regeneration step takes a long time, which makes it difficult to rapidly isolate potential antibodies from a combinatorial library [30] . our strategy, however, does not include time-limiting regeneration steps for the sorted population. the sorted population is used immediately for the next round of sorting, and this re-sorting process is repeated until the highly fluorescent population becomes separated from the negative population. without this regeneration step, highly fluorescent cells that produce potential antibody candidates could be collected in four hours through repeated sorting. for this sorting, we used high-speed facs sorter which can screen up to 70,000 cells per sec and, with this sorting rate, the first round to sort 0.5 to 1.5 million cells from library took approximately 2 h and all repeated sorting (4-5 times) could be completed in 4 hours. in the initial round of sorting, false-positive clones, where fluorescent antigen probes are non-specifically bound to antibodies on cells, can be found; however, those clones can be removed during the repeated sorting due to the fact that the binding of these antigen probes is not strong or cannot be maintained over repeated sorting for four hours. with this strategy, the overall procedure from cell cultivation to final round sorting could be completed in one day, with much time saved, unlike that in the previous strategies. in addition, we need to consider the effect of differing cell growth on the sorted cell population in the next round of sorting. stressful conditions during the facs screening process and antibody expression can severely affect cell viability [12] . therefore, during overnight cultivation for regeneration, the more viable cells tend to overgrow and become the major population simply because of their viability, and not because of their binding activity. the use of this unfavorable population in the next round of screening makes it difficult to isolate the positive clones. however, by eliminating the need for regeneration, our strategy can minimize the risk of overgrowth of false-positive clones, as well as losing positive candidates during the sorting process. consequently, antibody candidates can be rapidly isolated in one day, as we successfully demonstrated with three antigen models (n1 antigenic epitope of h1n1 influenza virus, pres2 antigenic epitope of hbv, and vp1 antigenic epitope of fmdv), where the isolated antibodies exhibited highly specific and strong activity against their antigen targets. although we could get successful screening results for three examined antigens, it does not mean that the use of our screening strategy guarantee the successful screening of antibody against various antigens. as one limitation, we can consider the variability of the performance of facs screening in respect of individual antigen. due to the different properties (stickiness, size and permeability) of individual antigen probes, cells can be nonspecifically labeled with probes and, unwanted screening results (isolation of non-specific and low-affinity antibody) can be obtained. to minimize the non-specific labeling of probe, we may need to modify the labeling conditions and display system according to the properties of probes. for example, in current display system, big size (.10 kda) probe cannot be used for screening because it cannot enter to periplasm of cell, and so our strategy is not suitable for antibody screening against big size antigens including whole virus. to accommodate big size probes, we can suggest the use of apex system [11, 31] instead of current periplasmic expression system. in apex system, outer membrane can be partially removed by spheroplasting treatment which allows the entrance of big size antigens to periplasm. with apex system, it was already demonstrated the antibody can interact with antigen as large as 250 kda in periplasm of e. coli [11] . our strategy can be easily combined with apex system. using apex system, cells can be labeled with big size antigens, and the positively labeled cells can be isolated by our repeated sorting. like this, the display system and labeling conditions can be modified if necessary, and the isolation of positive clones can be done by repeated sorting strategy. in conclusion, we developed a new strategy for isolating antigenspecific antibodies in e. coli by simply repeating facs using a highspeed cell sorter. a fully synthetic human antibody library with six diversified cdrs was constructed in e. coli [32] , and with repeated sorting, potential antibody candidates against three viral antigens (n1 of h1n1 influenza virus, pres2 of hbv, and vp1 of fmdv) could be successfully isolated in one day. the isolated antibody candidates were then easily purified, and their high activities against each antigen were confirmed by performing elisa. in the case of fmdv vp1, the final isolated antibody candidate (sv7) was also revealed to have high affinity against not only the vp1 antigenic peptide probe, but also the entire fmdv. therefore, this study has shown that repeated facs screening without regeneration of sorted cells can be an alternative strategy to isolate potential antibody candidates for therapeutic or diagnostic use in emergencies, and our screening strategy is expected to be used in situations requiring a rapid response to spreading disease [33] . figure s4 amino acid sequence of the isolated antibody against three antigens. s1, s5 and s16 scfvs are against n1 epitope of h1n1; sp1, sp4, and sp19 scfv are against pres2 epitope of hbv; sv7, sv9, sv19 and sv20 scfvs are against vp1 of fmdv. strategies and challenges for the next generation of therapeutic antibodies high level production of a kringle domain variant by high-cell-density cultivation of escherichia coli advances in the production of human monoclonal antibodies selecting and screening recombinant antibody libraries synthetic therapeutic antibodies yeast surface display for screening combinatorial polypeptide libraries applications of display technology in protein analysis targeting recombinant antibodies to the surface of e. coli: fusion to a peptidoglycan associated lipoprotein isolation of high-affinity ligand-binding proteins by periplasmic expression with cytometric screening (pecs) separation of e. coli expressing functional cell-wall bound antibody fragments by facs anchored periplasmic expression, a versatile technology for the isolation of highaffinity antibodies from escherichia coli-expressed libraries development of an optimized expression system for the screening of antibody libraries displayed on the e. coli surface nanogap field-effect transistor biosensors for electrical detection of avian influenza label-free optical diagnosis of hepatitis b virus with genetically engineered fusion proteins high-yield production of the vp1 structural protein epitope from serotype o foot-andmouth disease virus in escherichia coli directed evolution of a g protein-coupled receptor for expression, stability, and binding selectivity universal genetic assay for engineering extracellular protein expression kabat database and its applications: 30 years after the first variability plot simultaneous mutagenesis of antibody cdr regions by overlap extension and pcr a vector for the removal of deletion mutants from antibody libraries secretory production of recombinant protein by a high cell density culture of a protease negative mutant escherichia coli strain development of human anti-murine antibody (hama) response in patients the immunogenicity of humanized and fully human antibodies: residual immunogenicity resides in the cdr regions fully synthetic human combinatorial antibody libraries (hucal) based on modular consensus frameworks and cdrs randomized with trinucleotides construction of a large synthetic human scfv library with six diversified cdrs and high functional diversity biophysical properties of human antibody variable domains rapid construction and characterization of synthetic antibody libraries without dna amplification recombining germline-derived cdr sequences for creating diverse singleframework antibody libraries a novel synthetic naïve human antibody library allows the isolation of antibodies against a new epitope of oncofetal fibronectin flow cytometric screening of cellbased libraries apex 2-hybrid, a quantitative protein-protein interaction assay for antibody discovery and engineering circular polymerase extension cloning for highthroughput cloning of complex and combinatorial dna libraries isolation and expression of recombinant antibody fragments to the biological warfare pathogen brucella melitensis key: cord-316319-m6uha1qn authors: daleno, cristina; piralla, antonio; scala, alessia; senatore, laura; principi, nicola; esposito, susanna title: phylogenetic analysis of human rhinovirus isolates collected from otherwise healthy children with community-acquired pneumonia during five successive years date: 2013-11-19 journal: plos one doi: 10.1371/journal.pone.0080614 sha: doc_id: 316319 cord_uid: m6uha1qn in order to evaluate the circulation of the different human rhinovirus (hrv) species and genotypes in italian children with radiographically confirmed community-acquired pneumonia (cap), a nasopharyngeal swab was obtained from 643 children admitted to hospital because of cap during five consecutive winter and early spring seasons (2007-2012). real-time reverse transcriptase polymerase chain reaction (rt-pcr) was used to identify hrv, and the hrv-positive samples were used for sequencing analysis and to reconstruct the phylogenetic tree. hrv was identified in 198 samples (42.2%), and the vp4/vp2 region was successfully amplified in 151 (76.3%). hrv-a was identified in 78 samples (51.6%), hrv-b in 14 (9.3%) and hrv-c in 59 (39.1%). forty-seven (31.1%) of the children with hrv infection were aged <1 year, 71 (47.0%) were aged 1-3 years, and 33 (21.9%) were aged ≥4 years. blast and phylogenetic analyses showed that the hrv strains were closely related to a total of 66 reference genotypes, corresponding to 29 hrv-a, 9 hrv-b and 28 hrv-c strains. nucleotide variability was 37% between hrv-a and hrv-b, 37.3% between hrv-a and hrv-c, and 39.9% between hrv-b and hrv-c. a number of sequences clustered with known serotypes and, within these clusters, there were strains circulating during several seasons. the most frequently detected genotypes were hrv-a78 (n=17), hrv-a12 (n=9) and hrv-c2 (n=5). this study shows that, although it is mainly associated with hrv-a, pediatric cap can also be diagnosed in subjects infected by hrv-c and, more rarely, by hrv-b. moreover, a large number of genotypes may be involved in causing pediatric cap and can be different from year to year. although the prolonged circulation of the same genotypes can sometimes be associated with a number of cap episodes in different years. the use of molecular methods of respiratory viral screening has recently made it possible to establish that human rhinoviruses (hrvs) are not only the main cause of the common cold (as thought since they were first identified several decades ago), but also common etiologic agents of lower respiratory tract infections (lrtis) such as bronchiolitis and community-acquired pneumonia (cap) [1] [2] [3] [4] [5] [6] . it has also been reported that they are the pathogens most frequently associated with asthma exacerbations [7, 8] . these findings have significantly increased interest in better defining hrv epidemiology mainly in order to clarify the possible association between specific strains and the development of more severe respiratory problems. a number of studies have reported epidemiological data showing hrv infection rates in children and adults [4] [5] [6] [9] [10] [11] [12] . it has been generally found that various hrv genotypes simultaneously circulated in a given period of time in a specific geographic area, most of which belonged to the a and c species; furthermore, the genotypes belonging to species a were mainly associated with cap and those belonging to species c were mainly associated with wheezing. however, most of these studies were carried out in a single year and involved a relatively small number of patients, and only a few analysed specific lrtis. consequently, there are few data concerning the circulation of hrvs over a long period of time or the real role of the different species and genotypes in causing lrtis. the aim of this study was to evaluate the circulation of the different hrv species and genotypes in italian children with radiographically confirmed cap during the winter and early spring of five consecutive years as this information could help to develop tailored strategies for the prevention and treatment of pediatric hrv infections. the study was carried out in pediatric clinic 1 of the department of pathophysiology and transplantation of the university of milan during five consecutive years. the enrolment occurred between november 1 and april 30 in the years 2007-2008, 2008-2009, 2009-2010 and 2011-2011 and between november 1 and june 30 in 2011-2012. it was approved by the institutional review board of the fondazione irccs ca' granda, ospedale maggiore policlinico, milan, italy. the written informed consent of a parent or legal guardian was required, and the older children were asked to give their assent. all of the children aged between one month and 14 years seen in the emergency room (er) of the department of pathophysiology and transplantation who had fever (defined as an axillary temperature of >38°c), signs and symptoms of lower respiratory tract infection (i.e. cough, tachypnea, dyspnea or respiratory distress, and breathing with grunting or wheezing sounds with rales) and a chest radiograph consistent with cap (in accordance with the world health organization criteria for the standardized interpretation of pediatric chest radiographs for a diagnosis of pneumonia [13] ) were considered eligible for the study. the exclusion criteria were chronic diseases increasing the risk of respiratory infections, including premature birth; chronic disorders of the pulmonary or cardiovascular systems, including asthma; chronic metabolic diseases, including diabetes mellitus; neoplasia; kidney or liver dysfunction; hemoglobinopathies; immunosuppression; diseases requiring long-term aspirin therapy; and genetic or neurological disorders. the children with presumed nosocomial cap (i.e. appearing more than 48 hours after admission or within two weeks of hospital discharge) were also excluded. a sample of nasopharyngeal secretions was collected from all of the children with radiographically confirmed cap using a flexible pernasal flocked swab that was immediately placed in a mini-tube containing 1 ml of universal trasport medium (utm-rt kit cat. no. 360c, copan italia, brescia, italy). viral nucleic acids were extracted from the nasopharyngeal swabs using a nuclisens easymag automated extraction system (biomeriéux, craponne, france), and the extracts were tested for respiratory viruses using the rvp fast assay (luminex molecular diagnostics inc., toronto, canada) in accordance with the manufacturer's instructions. the rvp fast assay consists of a single multiplex polymerase chain reaction (pcr) with labelled primers, followed by the single-step hybridization of the pcr products with the fluorescent bead array and incubation with reporter reagents. the plate was analysed using a bio-plex 200 system (bio-rad laboratories, milan, italy) and its associated software luminex x ponent version 3.1 (luminex molecular diagnostics inc., provided by abbott), and the median fluorescent intensity (mfi) was determined. an mfi above the threshold level determined by the manufacturer for a particular target indicated a positive result for that target. the mean positive fluorescence intensities were established using tag-it data analysis software (tdas, luminex). the rvp fast assay simultaneously detects influenza a virus (subtyped h1 or h3), influenza b virus, respiratory syncytial virus (rsv)-a and -b, parainflunzavirus-1, -2, -3 and −4, adenovirus, human metapneumovirus (hmpv), coronaviruses 229e, nl63, oc43 and hku1, enterovirus/ rhinovirus, and human bocavirus. the samples that were positive for enterovirus/rhinovirus were retested by means of one-step real-time rt-pcr in order to identify the rhinovirus [6] . the hypervariable part of the 5' ncr (non-coding region), the entire vp4 gene and the 5' terminus of the vp2 gene in the hrv-positive samples were amplified by means of a rt-pcr as previously described [6, 14] . the pcr products were purified using the wizard sv gel and pcr clean-up system (promega, milan, italy), and then sequenced in both directions using the same forward and reverse primers as those used in the pcr. the nucleotide sequences were obtained by means of automated dna sequencing using an abi prism 3730 genetic analyser (applied biosystems, foster city, ca). the newly determined sequences were edited using sequencher software, and aligned using the clustalw program integrated in the mega version 5.0 package [15] . the analysed fragment was 393 nt in the vp4/vp2 region. the hrv in each sample was determined on the basis of the phylogenetic tree and by comparing it with all of the available rhinovirus reference prototypes encoding vp4/vp2 protein retrieved from genbank (http://www.ncbi.nlm.nih.gov). the phylogenetic tree was reconstructed using the neighbour-joining method and parameters selected by the mega model test program. branch support was assessed by means of bootstrap analysis with 1000 replicates. genotypes were assigned on the basis of their clustering with known prototype reference strains present in genbank. the genetic distances between and within hrv-a, hrv-b, hrv-c were tested using the mega program and compared by means of student's t-test. the graphs were made using graphpad prism version 5.01 for windows (graphpad software, san diego, ca). the 151 vp4/vp2 sequences described in this study have been deposited in genbank under accession numbers kf427709 -kf427859. during the five years of the study, 643 children with radiographically confirmed cap were enrolled. of these, 469 (72.9%) were positive for at least one virus, and 174 (27.1%) were negative. hrv was identified in 198 samples (42.2%), in 60% of the cases as single infectious agent. the vp4/vp2 region was successfully amplified in 151 (76.3%); the remaining 47 samples (23.7%) were excluded from the phylogenetic analysis because of poor sequence quality. all three hrv types were circulating in the study population: type hrv-a was identified in 78 samples (51.6%), hrv-b in 14 (9.3%), and hrv-c in 59 (39.1%). as shown in table 1 , 47 (31.1%) of the children with hrv infection were aged <1 year, 71 (47.0%) were aged 1-3 years, and 33 (21.9%) were aged ≥4 years. hrv-a and hrv-c were equally frequent in the patients aged <1 year (44.7% vs 42.6%), whereas their incidence was different in the children aged ≥4 years (57.6% vs 30.3%). hrv-b was the less frequently species in all three age categories. figure 1 shows the monthly distribution of the hrv species over the 5-year study period. hrv-a and hrv-c was circulating in almost all of the months of surveillance without any clear seasonal pattern: hrv-a cases were frequently observed in february (20/26; 76.9%) and march (17/29; 58.6%), whereas hrv-c cases were frequently observed in december (25/52; 48.1%). hrv-b occurred only sporadically throughout the study period. on the basis of blast and phylogenetic analyses, the italian hrv strains were closely related to a total of 66 reference genotypes corresponding to 29 hrv-a, 9 hrv-b and 28 hrv-c strains. table 2 shows the frequencies of the hrv genotypes detected in each season during the study period. the hrv sequences showed marked genetic diversity. the hrv-c sequences were the most heterogenous, with an intraspecies nucleotide p-distance of 0.25, which was greater than the p-distance within hrv-a (0.20) or hrv-b (0.21). nucleotide variability was 37% between hrv-a and hrv-b, 37.3% between hrv-a and hrv-c, and 39.9% between hrv-b and hrv-c. figure 2 shows the phylogenetic tree constructed on the basis of the vp4/vp2 region of the 151 sequences from this study and the references sequences. a number of sequences clustered with known genotypes and, within these clusters, there were strains circulating during hrv-a78-like strains were observed during three alternate seasons (2007-08, 2009-10, 2011-12) . the italian hrv-a78 sequences seem to cluster into two different groups (a and b, figure 3a ): in comparison with the reference strain, mean nucleotide divergence in cluster b was significantly greater than in cluster a (10.2% vs 6.9%; p<0.001). the hrv-a12 strain circulated in four consecutive seasons (2007-2011). six of the nine hrv-a12-like sequences (66.6%) showed high degree of identity (mean 99%) in comparison with that between them and the reference strain (mean 92%); the remaining three strains were dispersed with hrv-a12-like strains circulated worldwide (fig. 3b) . it is worth noting that two identical hrv-a12 strains were observed in two different seasons (mi10262-jan08 and mi532-dec08), and the same strains were also found in the usa in 2007, and italy and switzerland in 2009. hrv-a22-like strains clustered in two different groups with respectively 8.2% and 11.6% nucleotide divergence from the reference strain. these sequences circulated in two nonconsecutive seasons (2009-10 and 2011-12); however, one strain found in 2011-2012 (mi3543) showed a greater degree of nucleotide similarity with two hrv-a22-like sequences circulating in 2009-2010 than with another strain circulating in the same year. this could mean that the hrv-a22-like strains remained in the studied population for a long time, probably more than two years ( figure 3c ). an identical hrv-a68-like strain was identified in samples taken during two non-consecutive seasons (mi124-nov08 and mi124-feb11). in addition, a series of hrv-a-like sequences showed a nucleotide divergence of >10.5% from their reference strains: mi19 and mi1089 vs hrv-a20, and mi24 and mi91 vs hrva-80 (figure 2a ). all of the sequences defined as being hrv-b86-like and hrv-b48-like strains showed a nucleotide divergence of >9.5% from the reference strains (mean 9.9% and 11.6%) ( figures 3d and 3e ). hrv-b86-like strains were the most frequent b genotype, and circulated in three different seasons. hrv-b35like strains were found in two different seasons with the presence of the same strain in both years (mi10018-dec07 and mi2009-apr09), and a nucleotide divergence of 8.4% from the reference strain. two strains (mi1717-mar09 and mi3507-dec11) clustered alone without any reference strains ( figure 2b ). the nucleotide identity of the two sequences with hrvc pat10 (eu590054, which is not currently assigned to genotypes) was >90% (figure 3 f) . the four hrvc-23-like strains were all observed in december of three different seasons. two of them (mibr19-dec11 and mibr5-dec11) showed significantly greater nucleotide divergence from the reference strain than the other table 2 . hrv genotypes within each species detected among study subjects. species and genotypetotal 07-08 08-09 09-10 10-11 11-12 two strains (mi829-dec09 and mi575-dec08) (mean 11.7% and 4.2%; p<0.001), which suggests that they may belong to a new hrv-c genotype ( figure 3g ). however, the sequencing of vp1 for these two strains is required for type assignment. the mi8-dec08 and mi875-dec09 sequences showed more than 10.5% nucleotide divergence (14.2% and 12.9%, respectively) from their most similar reference strains (hrv-c29 and hrv-c35). these two sequences respectively grouped with the unassigned serotype sequences hrvc pat17 (eu752398) and hrvc pat19 (fj598096) ( figure 2b ). this study is based on five years' surveillance of hrv infection in italian children with radiographically confirmed cap and confirms the high frequency of the association between this virus and pediatric cap. the presence of a virus in the nasopharynx of a child with cap does not necessarily mean that it is the etiological agent of the disease because it may only indicate a coincidental upper airways infection, or be due to a carrier state or the prolonged shedding of a pathogen that caused a previous infection. this may be particularly important in the case of rvs because a number of epidemiological studies have shown that they can be found in the respiratory secretions of 12-22% of asymptomatic subjects [16, 17] . however, in our study population, we found hrv as a single agent in about 60% of the children in which this pathogen was identified. this strongly suggests that it was the cause of the cap diagnosed in many of our study patients, particularly in those in whom very low values of wbc counts and crp were found. the most frequently detected hrv species was hrv-a (51.6%), followed by hrv-c (39.1%) and hrv-b (9.3%). species and genotypetotal 07-08 08-09 09-10 10-11 11-12 these detection rates are similar to those recently reported in various countries [18] [19] [20] , but different from those of calvo et al. [21] and linsuwanon et al. [22] , who studied patients with lrtis and found that hrv-b was predominant when cap was diagnosed. however, the diagnosis of cap in the last two studies was not always radiographically confirmed and this could have affected the results. we detected hrvs more frequently in children aged <1 year (31.1%) and in those aged 1-3 years (47%), than in those aged ≥4 years (21.9%), as has been reported in other studies [18, 23] . in terms of species, hrv-a was more frequently detected than hrv-c in children aged 1-3 and ≥4 years, although a recent study in thailand found that hrv-c was the most frequent species in children with lrtis in the same age groups [24] . in our study the circulation of hrv was evaluated only during the winter and spring months. this means that data do not indicate the real circulation of hrv species during the whole year. however, ours is the first study defining the circulation of hrv species and genotypes in children with cap that is based on such a large number of samples collected over a 5-year observation period, which is significantly longer than in other studies [19, [25] [26] [27] [28] and can give a precise information on the circulation in time of the different hrv species. . the hrv-positive samples contained a total of 66 genotypes (29 hrv-a, nine hrv-b and 28 hrv-c), which is quite similar to the number reported in a recent 4-year study of children and adults with lrtis in sweden [29] , but higher than the 45 genotypes found in a study carried out in central italy [27] . however, the observation period of this study was limited to one year and only a small number of cap patients were enrolled. we classified the hrv genotypes using vp4/vp2-based phylogenetics, and distinguished the inter-and intra-serotypes using the nucleotide divergence threshold very recently proposed by mcintyre et al.: 10.5% for hrv-a, 9.5% for hrv-b, and 10.5% for hrv-c [30] . however, in the absence of a vp1 sequence, the putative new types are considered provisionally assigned. our data show that a large number of genotypes can circulate during each observation period, and that some can circulate in more than one year and simultaneously in different geographic areas. this is clearly shown by the similarity between the italian hrv-a strains identified in the present study and those found in spain [31] and jordan [32] during 2008, and the fact that we also found the genotypes most frequently detected in sweden by sansone et al.: a78, a12, a56, a10, c9 and c43 [29] . considering only the 2009-2010 season, genotypes a12, a40, a89 and c2 found in this study were also found in a study carried out in france and involving a population similar to ours [33] . furthermore, hrv-a12 was the most common genotype found in children with a primary diagnosis of viral cap in beijing, china, during the 2007-2008 season [34] . there was a wide range of genotypes with different degrees of nucleotide variations from their reference strain. increased nucleotide variability with respect to prototype strains has been observed in studies carried out all over the world [20, 26, 29, [34] [35] [36] . the considerable attention given to sequencing over the last few years and the development of efficient molecular methods capable of characterising hrv species and genotypes will allow the discovery of new variants in the near future. our findings show that, although cap is mainly associated with hrv-a, it can also be diagnosed in subjects infected by hrv-c and, more rarely, hrv-b. our analysis of the genotypes associated with pediatric cap suggests that a large number of genotypes may be involved in causing the disease. these may vary from year to year although the prolonged circulation of the same genotypes can sometimes be associated with a number of cap episodes in different years. this could be the case of hrv-a78-like and hrv-a12-like strains, which were the most frequently found in this study. however, the importance of these types 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patients phylogenetic patterns of human respiratory picornavirus species, including the newly identified group c rhinoviruses, during a 1-year surveillance of a hospitalized patient population in italy genetics, recombination and clinical features of human rhinovirus species c (hrv-c) infections; interactions of hrv-c with other respiratory viruses rhinovirus infections in western sweden: a four-year molecular epidemiology study comparing local and globally appearing types proposals for the classification of human rhinovirus species a, b and c into genotypically assigned types human rhinovirus a isolate so6316 polyprotein gene genbank fj615683.1 human rhinovirus sp. isolate 202692 polyprotein gene human rhinovirus c infections mirror those of human rhinovirus a in children with community acquired pneumonia evidence of recombination and genetic diversity in human rhinoviruses in children with acute respiratory infection screening respiratory samples for detection of human rhinoviruses (hrvs) and enteroviruses: comprehensive vp4-vp2 typing reveals high incidence and genetic diversity of hrv species c phylogenetic analysis of rhinovirus isolates collected during successive epidemic seasons impact of rhinovirus nasopharyngeal viral load and viremia on severity of respiratory infections in children key: cord-351990-aham72b9 authors: radin, jennifer m.; hawksworth, anthony w.; kammerer, peter e.; balansay, melinda; raman, rema; lindsay, suzanne p.; brice, gary t. title: epidemiology of pathogen-specific respiratory infections among three us populations date: 2014-12-30 journal: plos one doi: 10.1371/journal.pone.0114871 sha: doc_id: 351990 cord_uid: aham72b9 background: diagnostic tests for respiratory infections can be costly and time-consuming. improved characterization of specific respiratory pathogens by identifying frequent signs, symptoms and demographic characteristics, along with improving our understanding of coinfection rates and seasonality, may improve treatment and prevention measures. methods: febrile respiratory illness (fri) and severe acute respiratory infection (sari) surveillance was conducted from october 2011 through march 2013 among three us populations: civilians near the us–mexico border, department of defense (dod) beneficiaries, and military recruits. clinical and demographic questionnaire data and respiratory swabs were collected from participants, tested by pcr for nine different respiratory pathogens and summarized. age stratified characteristics of civilians positive for influenza and recruits positive for rhinovirus were compared to other and no/unknown pathogen. seasonality and coinfection rates were also described. results: a total of 1444 patients met the fri or sari case definition and were enrolled in this study. influenza signs and symptoms varied across age groups of civilians. recruits with rhinovirus had higher percentages of pneumonia, cough, shortness of breath, congestion, cough, less fever and longer time to seeking care and were more likely to be male compared to those in the no/unknown pathogen group. coinfections were found in 6% of all fri/sari cases tested and were most frequently seen among children and with rhinovirus infections. clear seasonal trends were identified for influenza, rhinovirus, and respiratory syncytial virus. conclusions: the age-stratified clinical characteristics associated with influenza suggest that age-specific case definitions may improve influenza surveillance and identification. improving identification of rhinoviruses, the most frequent respiratory infection among recruits, may be useful for separating out contagious individuals, especially when larger outbreaks occur. overall, describing the epidemiology of pathogen specific respiratory diseases can help improve clinical diagnoses, establish baselines of infection, identify outbreaks, and help prioritize the development of new vaccines and treatments. acute respiratory infections make up a huge proportion of disease burden in the united states and globally, with an estimated 94 037 000 disability adjusted life years and 3.9 million deaths worldwide each year [1] . respiratory infections are often difficult to diagnose clinically due to nonspecific and overlapping symptoms. additionally, diagnostic tests can be time-consuming and costly and often require trained and well-equipped laboratories, making laboratory confirmation of each case impractical. however, laboratory results from various surveillance populations can be paired with clinical, demographic, and seasonality variables to create models that can give timely predictions of disease outcomes. preventive measures and treatments to reduce respiratory disease burden can also be improved through routine surveillance by gaining a better understanding of the percent positivity of pathogens among acute respiratory cases, seasonality, and coinfection occurrence. currently, limited respiratory disease etiology studies have been done in the united states [2, 3, 4] , despite many being done in other countries [5, 6, 7, 8, 9, 10, 11, 12, 13, 14] . additionally, few viral etiology studies have collected clinical signs and symptoms and assessed their association with a broad range of respiratory pathogens [9, 13, 14] . most descriptive studies and predictive models for respiratory diseases have focused on identifying influenza using clinical signs and symptoms [15, 16, 17, 18, 19, 20] , however few have been age-stratified and therefore may have missed some important differences in clinical presentation by age. understanding us-specific disease burden and seasonality is important, since disease incidence, distribution, and seasonality may vary between populations, regions, and climates. this study aimed to describe characteristics associated with specific respiratory pathogens, as well as the etiology, seasonality, and coinfection rates among three us populations: military recruits, department of defense (dod) beneficiaries, and civilians living near the us-mexico border. the results of this study can help improve timely and more accurate diagnosis, inform treatment plans, establish baselines of infection, identify outbreaks, and help prioritize the development of new vaccines and future treatments. participants fri and sari surveillance was conducted between october 2011 and march 2013 among three surveillance groups in the united states: civilians near the us-mexico border, dod dependents, and military recruits. separate seasonality data from the same populations was available for january 2012 through december 2013. recruits are typically young and healthy adults who enter into an 8-12 week ''boot camp'' training program, which involves strenuous, and physically demanding activities and living in high-density barracks. during the first week of training, recruits receive a series of vaccinations, including influenza (seasonally) and adenovirus. most training centers also administer at least one dose of bicillin to incoming trainees as prophylaxis against streptococcus bacteria. the dod dependent population is made up of the families of active duty and retired military personnel. this population consists of all ages and has good access to health care through the tricare health care program. this research was conducted in compliance with all applicable federal and international regulations governing the protection of human subjects in research. the research conducted in the recruit and dod beneficiary populations underwent nhrc irb approval (nhrc protocols 31230 and nhrc.1999.0002) and written consent or parental guardian consent for minors was obtained for all participants. the data collected from the border population was part of a surveillance program run by the us centers for disease control and prevention (cdc) and was considered non-research by the nhrc irb. nhrc staff members only provided diagnostic support and only received non-personally identifiable data. case definitions were slightly different for each of the three populations. in the recruit population, an fri case was defined as a person who sought medical care and had an oral temperature $38.0uc (100.5uf) and either cough or sore throat. for the beneficiary population and border populations, the same fri case definition was used; however, a fever $37.8uc (100.0uf) or subjective fever was used. additionally, inpatients who met sari case definition at select border sites were also sampled. the sari case definition included people who presented with either fever $37.8uc (100.0uf) or feeling feverish/chills, in addition to cough, and hospital admission, with onset in the last 10 days. additionally, children under age five were included if they were admitted to the hospital with clinical suspicion of pneumonia. nasal or combination nasal/pharyngeal swabs and questionnaire data were collected from a convenience sample of up to 20 patients per week per site who sought medical attention, met the fri or sari case definition, and provided written informed consent. specimens were placed in universal transport medium [21] , preserved at 280uc, and later transported on dry ice to the reference laboratory at the nhrc every one to two weeks for testing. additionally, the following demographic and clinical signs and symptoms were collected from each fri and sari case: sex, age, study population, month of illness, pneumonia, sore throat, cough, nausea, shortness of breath, congestion, pink eye, body ache, headache, temperature, number of days of symptoms before seeking care, and date of seeking care. samples were extracted using the roche magna pure lc extraction system following manufacturer's instructions. samples were then tested for a broadspectrum panel consisting of standard pcr gel tests: hmpv (single plex), covnl63 (single plex), and cov229e and covoc43 (multiplex). real-time polymerase chain reaction (pcr) assays were run on the applied biosystems 7500 fast real-time pcr system testing for influenza a, influenza b, adenovirus, rsv, rhinovirus, and a bacterial multiplex consisting of m. pneumoniae, c. pneumoniae, and b. pertussis. the rhinovirus primers used in our study have been found to correctly identify 87 of the 100 distinct rhinovirus subtypes and to accurately distinguish rhinoviruses from enteroviruses [22] . all pcr tests were done using in-house primers, except for the influenza a and influenza b which were done using cdc primers. additionally, viral culture testing for parainfluenza was done for a systematic sample of specimens not positive for influenza or adenovirus. the frequencies and means of clinical and demographic characteristics across participants positive for each of the six viruses, bacterial infections, co-infections and no/unknown pathogen were compared. (table 1) . additionally, characteristics of influenza, other pathogen, or no/unknown pathogen were compared among age stratified groups of dod beneficiaries and us-mexico border populations ( table 2) and characteristics of rhinovirus, other pathogen, and no/ (24) 14 (15) 4 (5) 6 (10) 4 (3) 13 (27) 19 (43) unknown pathogen were compared among military recruits (table 3) . chi-square tests for categorical variables and analysis of variance (anova) tests for continuous variables were used to identify signs, symptoms, associated with each pathogen group from all populations. variables that were univariately associated with the pathogens (p,0.15) were investigated further in a multinomial logistic regression model. variables with a p,0.05 were considered in the final adjusted model (tables s1-s2 in s1 file). coinfections were coded as ''other pathogen'' even if one pathogen was influenza or rhinovirus. statistical analysis was conducted using sas software (version 9.3, sas institute, inc., cary, north carolina). proc logistic with link5glogit was used for the multinomial modeling and proc anova and proc freq were used for anova and chi-squared analyses, respectively. during the 18 months of the study, october 2011 through march 2013, 1444 patients met the fri or sari case definitions and were enrolled in this study, consisting 406 (28%) from the fri/sari border, 423 (29%) from the dod beneficiary, and 615 (43%) from the fri recruit populations. the percent positivity for each pathogen out of all specimens tested was rhinoviruses (16%), influenza (14%), rsv (6%), adenovirus (2%), coronaviruses (5%), bacterial (2%), hmpv (1%), and coinfections (6%). influenza a (h3n2) was the most common influenza subtype, making up 54% of influenza specimens, followed by influenza b (31%), and ph1n1 (13%). among coronaviruses, covoc43 was the most commonly identified strain, making up 67% of coronavirus specimens, seasonal patterns were apparent for influenza, rhinovirus, and rsv, whereas more consistent low levels of infection were seen for adenovirus and bacterial infections. the recruit population had a more constant number of fri cases sampled than the other two populations throughout the study period. overall, they also had a greater percent positivity for rhinovirus compared with the other two populations, but lower percent positivity for influenza and rsv. rhinovirus appeared to have two peaks: one in spring and one in summer/fall among the border and beneficiary populations and consistently higher levels among the recruit population. in all populations influenza and rsv peaked in the winter (fig. 1) . influenza a typically peaked before influenza b. rhinovirus and bacterial infections were more frequently isolated from recruits and men who make up the majority of the recruit population; whereas influenza and rsv were less frequent among these groups compared with the dod beneficiary and border populations. fewer rhinovirus and more rsv pathogens were identified from sari cases compared to fri. pneumonia and shortness of breath were more frequent among rhinovirus cases and less common among influenza cases. however, time to seeking care was shorter for influenza and hmpv and longest for bacterial infections. influenza, rsv, adenovirus, and hmpv cases all had higher temperatures compared with no/unknown pathogens, and rhinovirus has significantly lower temperature (table 1) . descriptive statistics for influenza, other, and no/unknown pathogen among dod beneficiaries and us-mexico border populations showed different clinical presentation across three age groups. among 0-4 year olds, influenza was more frequent among us-mexico border populations compared to dod beneficiaries. in the youngest age group, sore throat and fever were also more frequent among those positive for influenza compared to other or no/unknown pathogen and among 5-24 year olds, cough, fever, and short time to seeking care were more frequent. finally, among the 25 and older age group, sore throat, nausea, congestion, pink eye, body ache, and head ache were more frequently seen in the influenza group compared to the other groups. interestingly, fever was the least frequent among influenza positive participants in the oldest age group (table 2) . clinical presentation among recruits also differed by those with laboratory confirmed rhinovirus, other pathogen, or no/unknown pathogen. a higher percentage of males were diagnosed with rhinovirus or other pathogen compared to the other and no/unknown pathogen. additionally, participants with rhinovirus had higher percentages of pneumonia, shortness of breath, congestion, and less fever and longer time to seeking care than the other groups and participants with either rhinovirus or other pathogen had higher cough than the no/unknown pathogen group (table 3) . building on the descriptive statistics, we found that fever was predicative of influenza compared to no/unknown pathogen among 0-4 year olds. among 5-24 year olds, fever, cough, and short time to seeking care were predicative of influenza compared to no/unknown pathogen. finally, among those 25 years and older, sore throat and nausea were predictive of influenza compared to no/ unknown pathogen. (table s1 in s1 file). among the recruit population, cough epidemiology of respiratory infection and less fever were predictive of rhinovirus, and cough, fever, and less shortness of breath were predictive of other pathogen compared to no/unknown pathogen. (table s2 in s1 file). at least one pathogen was identified in 51% of all fri and sari cases. coinfections were found in 81 (6%) of all fri/sari cases tested, among which were 76 double, four triple, and one quadruple coinfections. the most frequent pathogen associated with a coinfection was rhinovirus, followed by rsv and covoc43. the top three most common coinfections were rhinovirus and rsv (15% of all coinfections, 11 cases), rhinovirus and adenovirus (13% of all coinfections, 10 cases), and rhinovirus and covoc43 (12% of all coinfections, 9 cases). interestingly, our study found an overall coinfection percent positivity of 6% which declined with age: 11% of 0-4 year olds, 5% of 5-24 year olds, 3% of 25-49 year olds and 2% of 50+ year olds. (table 4 ). in order to reduce respiratory disease burden, it is necessary to gain a better understanding of the percent positivity, coinfection rates, and seasonality of specific respiratory pathogens. additionally, a predictive clinical model that uses symptoms, seasonality, and patient demographics can also help improve prevention efforts and patient treatment. timeliness is especially important for influenza antivirals, which work best if given within the first 48 hours of symptoms. however, rapid diagnostic tests have poor sensitivity [23] and multiplex pcr tests are impractical for most clinical settings. additionally, treatment with antibiotics can often be incorrectly prescribed for viral infections, leading to increased antibiotic resistance. therefore, creating models to better predict the type of pathogen using symptoms and characteristics easily collected by a clinician at the time of visit could improve treatment accuracy and help protect the effectiveness of existing antibiotics and antivirals. many respiratory etiology studies have been done outside the united states among patients with both severe, lower respiratory illness [24, 25, 26] , as well as more mild, upper respiratory disease [7, 8, 9, 10, 11, 12] . these studies are important because viral etiologies vary across populations and regions, depending on factors such as population susceptibility, age, circulating strains, climate, comorbidities, and vaccination coverage. our study adds to the very limited number of etiology studies done in the united states [2, 3, 4] by examining viral etiology among three different us populations: military recruits, dod dependents, and a us-mexico border civilians and including all ages. our study is also unique in that it collected clinical signs, symptoms, and demographics for each case tested with the broad-spectrum respiratory panel. similar studies have been limited and have used smaller sample sizes, focused on one age group, and did not test for as many respiratory pathogens. identifying population-specific baselines of infection enables us to identify elevated rates, which may indicate an outbreak or the start of a pandemic. recognizing associated symptoms can help determine the most likely pathogen, as was seen in 2009 with the pandemic influenza (h1n1) strain first identified in brawley and san diego, california, in two of the populations in this study [27] . there were several key differences of infection among the three populations. the recruit population had consistently higher levels of rhinovirus and bacterial infections than the other groups, which may be reflective of close living conditions and a younger age group, mostly 18-24 years old. this population is also highly vaccinated for influenza and showed the smallest amount of influenza infection compared with the other two populations. the higher frequency of influenza among 0-4 year olds in the us-mexico border population (61%) compared to beneficiaries (39%) may be reflective of different exposures or differences in vaccination uptake. however, the overall frequency of influenza found between the two groups was similar. adenovirus, which historically had a large impact on recruits, was also low as a result of resumption of the adenovirus vaccines in october 2011 [28] . rsv, which usually infects young children, was more commonly found in the dod dependent (48%) and border populations (48%) compared with the recruits (4%). overall, rhinoviruses were the most common respiratory pathogen identified (236 cases, 16%), followed by influenza (197 cases, 14%), rsv (85 cases, 6%), and coronaviruses (66 cases, 5%), which coincides with other etiology studies [13] . the results of the age-stratified influenza, other pathogen, and no/unknown results showed some interesting differences across age groups. most interesting was the relatively low percentage of influenza positive participants with fever (42%) in the 25 and older year age group, compared to the 0-4 (74%), and 5-25 year olds (76%). fever may sometimes be masked by the consumption of antipyretics, although this group also complained of high headache (81%) and body ache (90%). the presentation differences of influenza by age could have some important implications for influenza surveillance, as the standard influenzalike illness and sari case definitions require [29] . consequently, an age-stratified case definition may be more appropriate as the standard case definitions may be underestimating the burden of influenza in older age groups. circulating strains may also influence clinical presentation across age groups. (table 2, table s1 in s1 file). in our 5-24 year olds we found fever, cough, and short time to seeking care to be predictive of influenza infection. unfortunately, previous studies assessing the diagnostic accuracy of fever, cough, and acute onset to predict flu have found them to have low sensitivity (20%), and high specificity (96%) [18] . another study, found that sore throat and fever in participants less than 5 years old had a sensitivity of 51% and specificity of 54% and cough and fever among those greater than 5 years had a sensitivity of 80% and specificity of 42% [30] . although identifying predictive symptoms can be useful, it is important to recognize that diagnostic accuracy may still be low due to overlapping symptoms of many respiratory infections and may change due to fluctuations in circulating strains, age of the population and comorbidities. ( table 2, table s1 in s1 file). rhinovirus was the most frequently identified pathogen among recruits, although it typically does not present with severe illness or acute onset, which is reflected in the long time to seeking care among rhinovirus positive participants. interestingly, we also found the lowest percentage of fever among participants positive for rhinovirus compared to any other respiratory pathogen (table 1) . these findings are similar to other studies [13, 14, 31] and could have important implications for designing respiratory disease surveillance systems to capture outbreaks of viruses in the same family. for example, the recent outbreak of enterovirus 68 has similar symptoms of low fever [32] . (table 3, table s2 in s1 file). understanding coinfections can also be useful for preventing respiratory disease. our study found around 30% to 40% of coronavirus and adenovirus infections occurred as coinfections and they most frequently occurred with rhinovirus. similarly, other studies have also found the highest ratio of coinfections among adenovirus and coronaviruses [33, 34] , and have found rhinovirus to be part of the most frequently occurring coinfections [26, 33] . these results suggest that infection with some viruses, such as rhinoviruses, could create opportunistic environments for colonization with other viruses and bacteria. interestingly, coinfections were most common among the youngest age group, newborn to four years, which did not have the highest rhinovirus rate. targeting rhinovirus infection through creation of new vaccines or treatment could have more far-reaching benefit in protecting a person from other infections. respiratory coinfection rates have varied across studies and are likely influenced by age, type of case definition used to enroll participants, and pathogens tested. previous studies with a median age of 2-3 years old have found coinfections among sari and acute respiratory tract infections of 17% and 19%, respectively [26, 33] . a third study among childcare attendees in the us found even higher coinfection rates of 47% [4] . finally, a study from scotland that tested respiratory specimens from all age groups found a coinfection rate of 5% [34] . these studies coincide with ours and show high coinfection rates among children. declining coinfection rates with age may be a result of overall increased immunity to a broad range of respiratory pathogens as people get older. one limitation of this study is that it only captured people with fri/sari who sought medical care. military recruits may be less likely to seek care than other groups due to concern over losing training time or having to restart the program. therefore, the etiology of more mild infections may be underrepresented for these two groups. additionally, the case definitions were slightly different for the three populations, which may have influenced which pathogens were identified in each group. although this study involved three different us populations, the results of this study may be less generalizable to the general us public who are not associated with the military or living on the us-mexico border. despite this, signs and symptoms from these pathogens should be similar across other populations in similar age groups and with similar vaccination coverage. additionally, we found that seasonality of infection for recruits was similar to that of the border and beneficiary populations for several pathogens, but with different intensity. consequently, illness surveillance in recruits, which made up the largest proportion of our study population, can be beneficial in informing disease trends in the general public. although we tested for many pathogens, there are likely still circulating viruses and bacteria for which we did not test, such as bocavirus, covhku1, or potentially unrecognized viruses; therefore, these cases were likely incorrectly classified as part of the ''no pathogen'' group. additionally, the timing of sample collection in the course of illness could impact whether or not viruses were identified by pcr. symptom collection may also be biased in the youngest age group of 0-4 year olds, who may not be able to articulate how they are feeling. despite this, our study was part of a well-established existing surveillance program that consistently collected and tested a substantial number of specimens at many different sites across the united states. in the future, additional years of surveillance data will continue to improve our understanding of seasonality. although nothing will replace the accuracy of laboratory diagnostics, a broader understanding of seasonality, clinical presentation, demographics, and coinfection rates of pathogen specific respiratory diseases can improve diagnosis and treatment, by informing clinicians on appropriate antiviral and antibiotic treatment during the patient's visit. this can ultimately reduce the number of lost work days and transmission. additionally, describing baseline of disease and seasonality for specific pathogens can improve our ability to detect outbreaks. identifying the percent positivity of each pathogen, coinfection rates, and risk factors for disease will help inform vaccination programs, and possible investment in the development of future vaccines or treatments. supporting information s1 file. tables s1 and s2. of defense, or the us government. approved for public release; distribution is unlimited. u.s. government work (17 usc 105) . not copyrighted in the u.s. this research was conducted in compliance with all applicable federal and international regulations governing the protection of human subjects in research (protocols nhrc 31230 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detection of human rhinovirus low sensitivity of rapid diagnostic test for influenza community-acquired pneumonia distinguished from influenza infection based on clinical signs and symptoms during a novel (swine) influenza a/h1n1 pandemic multicentered study of viral acute lower respiratory infections in children from four cities of argentina, 1993-1994 respiratory viral coinfections identified by a 10-plex real-time reverse-transcription polymerase chain reaction assay in patients hospitalized with severe acute respiratory illness-south africa initial identification and characterization of an emerging zoonotic influenza virus prior to pandemic spread history of the restoration of adenovirus type 4 and type 7 vaccine, live oral (adenovirus vaccine) in the context of the department of defense acquisition system world health organization. who surveillance case definitions for ili performance of case definitions used for influenza surveillance among hospitalized patients in a rural area of india rhino/enteroviruses in hospitalized children: a comparison to influenza viruses severe respiratory illness associated with enterovirus d68 -missouri and illinois epidemiology of respiratory viral infection using multiplex rt-pcr in epidemiology and clinical presentations of four human coronaviruses 229e, hku1, and oc43 detected over 3 years using a novel multiplex real-time pcr method we thank maria rosario araneta, patrick blair, daisy cabrera, johnnie conolly, jennifer cortinas, larivhie delacruz, daniel edgeworth, maria fierro, james key: cord-339724-roj8ksvc authors: lan, jiaming; deng, yao; chen, hong; lu, guangwen; wang, wen; guo, xiaojuan; lu, zhuozhuang; gao, george f.; tan, wenjie title: tailoring subunit vaccine immunity with adjuvant combinations and delivery routes using the middle east respiratory coronavirus (mers-cov) receptor-binding domain as an antigen date: 2014-11-18 journal: plos one doi: 10.1371/journal.pone.0112602 sha: doc_id: 339724 cord_uid: roj8ksvc the development of an effective vaccine is critical for prevention of a middle east respiratory syndrome coronavirus (mers-cov) pandemic. some studies have indicated the receptor-binding domain (rbd) protein of mers-cov spike (s) is a good candidate antigen for a mers-cov subunit vaccine. however, highly purified proteins are typically not inherently immunogenic. we hypothesised that humoral and cell-mediated immunity would be improved with a modification of the vaccination regimen. therefore, the immunogenicity of a novel mers-cov rbd-based subunit vaccine was tested in mice using different adjuvant formulations and delivery routes. different vaccination regimens were compared in balb/c mice immunized 3 times intramuscularly (i.m.) with a vaccine containing 10 µg of recombinant mers-cov rbd in combination with either aluminium hydroxide (alum) alone, alum and polyriboinosinic acid (poly i:c) or alum and cysteine-phosphate-guanine (cpg) oligodeoxynucleotides (odn). the immune responses of mice vaccinated with rbd, incomplete freund’s adjuvant (ifa) and cpg odn by a subcutaneous (s.c.) route were also investigated. we evaluated the induction of rbd-specific humoral immunity (total igg and neutralizing antibodies) and cellular immunity (elispot assay for ifn-γ spot-forming cells and splenocyte cytokine production). our findings indicated that the combination of alum and cpg odn optimized the development of rbd-specific humoral and cellular immunity following subunit vaccination. interestingly, robust rbd-specific antibody and t-cell responses were induced in mice immunized with the rrbd protein in combination with ifa and cpg odn, but low level of neutralizing antibodies were elicited. our data suggest that murine immunity following subunit vaccination can be tailored using adjuvant combinations and delivery routes. the vaccination regimen used in this study is promising and could improve the protection offered by the mers-cov subunit vaccine by eliciting effective humoral and cellular immune responses. in 2012 a novel human coronavirus, middle east respiratory syndrome coronavirus (mers-cov), caused outbreaks of a sars-like illness in the middle east, and is now considered a threat to global public health [1, 2] . as of july 23, 2014, the world health organization (who) reported 837 confirmed cases of mers-cov infection, including 291 deaths (a case fatality rate of 34.8%) [3] . now, studies show that camels are a likely primary source of the mers-cov that is infecting humans [4, 5, 6] . but the routes of transmission between camels and people which is the key point to stop transmission of the virus, is far from clearly understood. the continued threat of mers-cov necessitates the development of an effective vaccine. some studies have indicated that recombinant receptor-binding domain (rrbd) protein of mers-cov spike (s) is a good candidate antigen for a mers-cov subunit vaccine [7, 8, 9, 10] . however, highly purified proteins are typically not inherently immunogenic, as they usually lack the means to directly stimulate the innate immune system [11] . besides, they are often prone to degradation. hence, they call for efficient delivery systems and potent immunostimulants, jointly denoted as adjuvant(s) to evoke the desired antigen-specific immune response phenotype enabling successful vaccination [12] . aluminium is one of the most common adjuvant in non-living vaccines, has a record of successful use in human vaccination where it promotes antibody-mediated protective immunity [13] . another classic adjuvant is that based on a water-in-oil-emulsion formulation, such as incomplete freund's adjuvant (ifa). recently, researches have focused on adjuvants that signal through pattern recognition receptors (prrs), such as toll-like receptors (tlrs) [14] . cysteine-phosphate-guanine (cpg) oligodeoxynucleotides (odns), which activate b cells and plasmacytoid dendritic cells via tlr9 and induce both innate and adaptive immunity, are currently being developed as a vaccine adjuvant [15] . another frequently used adjuvant is polyriboinosinic acid (poly(i:c)), a synthetic dsrna that mimics the effects of naturally occurring dsrna, a tlr3 agonist [16, 17] . beside of enhancing the immune response, adjuvant(s) can tailor-make the polarization immune response. for example, ppolarized th1-type immunity can be achieved by the addition of freund's adjuvant or cpg dna to an antigen. on the other hand, th2 antibody responses can be induced by the alum, as indicated by increased igg1 relative to igg2a [18, 19] . however, in situations where both th1 and th2 responses are required for protection, the choice of one regimen over another might be counter effective. this has led to additional research for alternative adjuvants or adjuvant combinations that promote balanced mixed th1/th2 responses [18] . in recent years, the combination of antigens with more than one adjuvant, called the adjuvant system approach has produced vaccines with the ability to generate effective immune responses adapted to both the pathogen and the target population [20] . by using multiple adjuvants in combination, antigen presenting cell (apc) activation is influenced at more than one level, guiding the subsequent adaptive pathways and ultimately inducing a more robust immune response [20] . the induction of a robust humoral, including potent neutralizing antibodies, and cellular immune response is likely essential for immediate and sustained protective immunity in a mers-cov vaccine design. in this study, different adjuvants combination regimens including alum, ifa, cpg and poly(i:c) were compared in an effort to promote balance between th1 and th2 immune response to bystander rrbd antigen spanning residues 367-606 of mers-cov s in a murine model to develop an effective vaccine against mers-cov infection. animal studies were carried out in strict compliance with the guide for the care and use of laboratory animals of the people's republic of china. the study protocol was approved by the committee on the ethics of animal experiments of the chinese centre for diseases control and prevention. all procedures were performed under ethylether anesthesia and all efforts were made to minimize suffering. mers-cov rrbd protein, containing a 240-amino-acid fragment spanning residues 367-606 ( figure 1a ) of genbank number jx869059, was prepared using a bac-to-bac baculovirus expression system as described in detail previously [21] . the required rrbd was measured by sds-page ( figure 1b) and western blot ( figure 1c ) with a mice polyclonal antibody against spike of mers-cov ( figure 1c ). before vaccination, the rrbd protein was quantified by bradford method. the rrbd protein was combined with different adjuvants immediately prior to immunisation. aluminium hydroxide was kindly provided by the north china pharmaceutical group corporation genetech biotechnology development company. the odn motif containing unmethylated cpg (59-tccat-gacgttcctgacgtt-39) was synthesised by takara bio inc. poly(i:c) and ifa were purchased from sigma (st. louis, mo). a single dose (10 mg) of rrbd protein (100 ml) was combined with either 100 mg of alum alone (rbd/a), alum plus 10 mg of cpg (rbd/a+c), alum plus 50 mg of poly(i:c) (rbd/ a+p) or 10 mg of cpg and 100 ml of ifa (rbd/i+c). six-to-eight-week-old female balb/c mice (animal care centre, chinese academy of medical science, beijing, china) were randomly distributed into eight groups. eight mice of each group were vaccinated three times with rrbd proteins at 3-week intervals by either an intramuscular (i.m.) or a subcutaneous (s.c.) route (table 1 ). sera were collected 2 weeks after each vaccination and heat-inactivated at 56uc for 30 min before detection of rbdspecific and neutralizing antibodies. mice were scarified 2 weeks after the last immunisation, and their lungs and spleens were harvested for detection. a schematic of the vaccination and analysis timeline is shown in figure 2 . elisa was used to detect the mers-cov rbd-specific antibody response in immunised mice. briefly, 96-well elisa plates were pre-coated with rrbd protein (100 ng/well) overnight at 4uc and blocked with 2% non-fat milk for 2 h at 37uc. serially diluted sera of eight mice in each group were added to the plates and incubated at 37uc for 1 h, followed by four washes with phosphate-buffered saline (pbs) containing 0.1% tween 80 (pbst). bound antibodies were incubated with hrp-conjugated anti-mouse igg, igg1, igg2a or igg2b (1:5,000, sigma) for 1 h at 37uc. the reaction was visualised by using 3, 39, 5, 59tetramethylbenzidine (tmb) peroxidase substrate solution (invitrogen) and stopped by addition of 2 m h 2 so 4 . absorbance at 450 nm was measured using an elisa plate reader (wellscan mk 3). the cut-off value was set 2.1-fold above that of the negative control. antibody avidity was determined using the elisa method described by vermont et al [22] . briefly, sera were diluted to a titre of 1:100, and an ascending concentration of the chaotropic agent nascn (0-7 m) was added to the plate. plates were incubated for 15 min at room temperature (rt) before washing and development to determine total igg. as a control for antibody specificity, elisa was used to measure the total anti-mers-cov igg titres of pre-and post-vaccination sera samples. the conventional neutralization assay using live mers-cov is cumbersome and has to be performed in biosafety level-3 facilities. therefore, we adapted a mers-cov pseudovirus system which is sensitive and quantitative, and can be conducted in biosafety level-2 facilities as reported by zhao et al [23] . in brief, 293t cells were co-transfected with a plasmid encoding codon-optimized mers-cov s protein and a plasmid encoding env-defective, luciferaseexpressing, hiv-1 genome (pnl4-3r-e-luc) using fugene hd reagents (roche, basel, switzerland). supernatants containing mers-cov pseudovirus were harvested 48 h post-transfection and used for single-cycle infection. huh7.5 cells were plated at 10 4 cells/well in 96-well tissue-culture plates and grown overnight. the supernatants containing pseudovirus were pre-incubated with 2-fold serially diluted mouse sera at 37uc for 1 h before addition to cells. the culture was refed with fresh medium 24 h later and incubated for an additional 48 h. cells were washed with pbs and lysed using lysis reagent included in a luciferase kit (promega). aliquots of cell lysates were transferred to 96-well costar flatbottom luminometer plates (corning costar), followed by addition of luciferase substrate (promega). relative light units were determined immediately in the gaomax luminometer (promega). all experiments were carried out in triplicate. pseudovirus inhibition (pi) rate was calculated as: (relative luciferase units of mock sera -relative luciferase units of immune serum for a given dilution)/relative luciferase units of mock sera. to evaluate the antigen-specific t-cell response induced by the vaccination regimes, an ifn-c elispot assay was performed as described previously [17] . briefly, 96-well plates were coated with 100 ml per well of 5 mg/ml anti-mouse ifn-c antibody (bd pharmingen) overnight at 4uc and then blocked for 2 h at rt. freshly harvested splenocytes (5610 5 per well) or lung lymphocytes of eight mice in each group were isolated as described previously [22] . then, 4 mg/ml of a synthesised 18-mer peptide library, which overlapped the mers-cov s rbd by 10 amino acids, was added to the wells in triplicate. next, a biotinylated detection antibody (bd pharmingen) and streptavidin-horseradish peroxidase were added. blots were developed by the addition of an aec (3-amino-9-ethylcarbazole) substrate solution, which produced a coloured spot after 5-min rt incubation in the dark. finally, ifn-c spot-forming cells (sfcs) were counted. phorbol 12-myristate 13-acetate (pma) and ionomycin were added to the positive-control group, whereas the negative-control group received no stimuli. the number of peptide-specific ifn-c secreting t cells was calculated by subtracting the negativecontrol value from the sfc count. a cba analysis was conducted to investigate the levels of th1and th2-type cytokine secretion [18] in mice after three times' immunization. in brief, splenocytes (5610 5 per well) of eight mice in each group were distributed in 96-well plates and stimulated with 4 mg/ml of pooled rbd peptide. plates were incubated for 24 h at 37uc and supernatants were harvested. the concentrations of cytokines, including il-2, il-4, il-6, il-10, tnf-a, il-17a and ifn-c, were measured using a mouse th1/th2/th17 cytokine kit (bd biosciences) and a facs calibur flow cytometer (becton dickinson). data were analysed using the fcap array software (becton dickinson). statistical analysees were conducted using the one-way anova function in the spss 17.0 software package. a p-value less than 0.05 were considered to indicate statistical significance. the vaccination regime affects the rbd-specific igg response in mice to assess the humoral immune response to different immunisation regimens, mice were immunised with rrbd protein combined with different adjuvants three times at 3-week intervals. serum samples were collected 2 weeks after each vaccination and total anti-mers-cov rbd igg antibody titres were determined by elisa. the results indicated that rrbd protein combined with any adjuvant, including alum, ifa, cpg or poly(i:c), could induce a rbd-specific igg antibody response in the majority of mice after the second immunisation. in a few vaccinated mice, rbd-specific igg antibodies could be detected even after the first immunisation. the seroconversion rates of the different groups following the first and the second immunisations are shown in table 1 . as shown in figure 3a , there was no discernible increase in igg titres after the third immunisation compared to the second immunisation. among the vaccination regimes, rbd/a+c and rbd/i+c elicited the highest total igg titres (p,0.05, figure 3a) . besides, the difference of igg titer in these two groups was not significant (p$0.05, figure 3a ). similarly shown in figure 3a , the difference of igg titer in rbd/a and rbd/a+p groups had no significance. the rbd-specific antibodies were lower than 1:10 in the adjuvant control groups at each of the three vaccinations. the responses to the various vaccination regimes were investigated using nascn antibody-displacement elisa to measure antibody avidity ( figure 3b ). mice received the rbd/ a+c or rbd/i+c regimes had higher antibody avidity 2 weeks after the final vaccination than those received the rbd/a or rbd/a+p regimes. it was also noteworthy that high antibody avidity correlated with a high igg titre in mice. to further characterise the immune response to the different vaccination regimes, igg isotype analyses were performed 2 weeks after the final vaccination using secondary antibodies against igg1, igg2a and igg2b. as shown in figures 3c, d , e and f, mice immunised with rbd/a+c or rbd/i+c produced higher igg1 and igg2a titres than mice immunised with rbd/a or rbd/a+p. also, the igg1 to igg2a ratio revealed a th1 skewed response in mice that received the rbd/a+c or rbd/i+c regimes. in contrast, the rbd/a and rbd/a+p regimes produced a higher igg1/igg2 ratio, indicating a th2 response. the titres of rbd-specific igg2b antibodies, however, were not significantly different among the vaccination groups (p$0.05). nneutralizing antibodies in the sera of mice immunised with different vaccination regimes were evaluated with a pseudovirusbased neutralization assay. a low level of neutralizing antibodies were detected 2 weeks after the first or second vaccination in aall of the sera tested, although the total igg antibody levels had almost peaked after the second vaccination. the highest level of neutralizing antibodies was induced after the last vaccination ( figure 4 ). the pseudovirus inhibition (pi) rates are shown in figure 4 . as shown, the rbd/a+c regime had the highest neutralizing antibody activity (p,0.01). surprisingly, there was low level of detectable neutralizing antibody in the sera of mice immunised s.c. with the rbd/i+c regime, although sera from this to characterise the cellular immune responses elicited by the vaccination regimes, single ifn-c-producing cells were quantified by elispot. both systemic and local cellular immune responses were assessed using lymphocytes from the spleen and lungs of immunised mice. the peptide library used to stimulate the lymphocytes was described in the materials and methods section. results are expressed as the number of sfcs per 10 6 input cells. adjuvants without rrbd did not elicit a clear cellular response in the spleen 2 weeks after the third immunisation ( figure 5a ). neither rbd/a nor rbd/a+p induced a significant cellular immune response. in contrast, rbd/a+c and rbd/i+c regimen enhanced a detectable systemic cellular immune response. furthermore, the rbd/i+c regimen induced the greatest cellular immune response with the greatest number of ifn-c-producing cells in the spleen (p,0.05). a significant cellular immune response in the lung was induced only by the rbd/i+c regime, although a few ifn-c producing cells were detected in all immunised mice ( figure 5b ). we therefore concluded that while both the rbd/a+ c and rbd/i+c regimes could induce a systematic cellular immune response in mice, only the rbd/i+c regime could elicit a significant local cellular immune response in the lung. a higher frequency of rbd-specific, tnf-a-and il-4producing t cells were induced with alum and cpg via an i.m. route the cytokine profiles of spleen cells from immunised mice were analysed after stimulation with rbd-specific peptides. during cba, splenocytes from mice immunised with rbd/a+c or rbd/i+c produced ifn-c ( figure 6a ). in contrast, il-2 was produced by splenocytes following immunisation with rrbd combination of any adjuvants ( figure 6b ). but the differences in ifn-c and il-2 production among the groups were not significant (p$0.05). compared with other groups, splenocytes from mice immunised with rbd/a+c induced significantly higher levels of tnf-a ( figure 6c ) and il-4 ( figure 6d ) (p,0.01). all these indicated that the adjuvants of alum and cpg combination could induce a th1 and th2 mixed immune responses in the rrbd antigen model of mers-cov, though the responses revealed a th1 polarization in the isotype elisa and elispot detection. similarly, the high levels of ifn-c, il-2 and il-6 also indicated a th1 and th2 mixed immune responses could be induced by the rbd/i+c regimes. (figure 6a, 6b, 6e) . different from all of these, as shown in figure 6f , the rbd/a+p regimes induce the highest level of il-10 (p,0.01), which indicated a th2 response inclination consistent with the results of igg isotype. however, il-17a was not detected in any of the vaccination groups (data not shown). coronaviruses can adapt rapidly to new hosts, and an adaptation of mers-cov that allowed the virus to efficiently replicate in humans would be a major public health concern, since such an adaptation could trigger a pandemic [24] . the development of an effective vaccine is critical to prevent a potential mers-cov pandemic. previous studies have shown that vaccination with the sars-cov rbd induces highly potent neutralizing antibodies and significantly inhibits sars-cov infection [25, 26] . therefore it was proposed that vaccination with the rbd of mers-cov, which belongs to the same betacoronavirus genus as sars-cov [27, 28] , might also inhibit mers-cov infection and induce a neutralizing antibody response against mers-cov. du et al [8] identified a recombinant protein containing a 212-amino acid fragment (residues 377-588) in the truncated rbd of mers-cov spike protein fused with human igg fc fragment (s377-588-fc) was able to induce in the vaccinated mice strong mers-cov sspecific antibodies, which blocks the binding of rbd to dipeptidyl peptidase 4 (dpp4), the human mers-cov receptor [29] and effectively neutralizes mers-cov infection. besides, they [9] showed that residues 377 to 662 in the s protein of mers-cov induced significant neutralizing antibody responses, suggesting that this region had a potential to be developed as a mers-cov vaccine. mou et al [10] showed the polyclonal antibodies in rabbits against the rbd in the s protein to a 231-amino-acid fragment (residues 358 to 588) efficiently neutralized virus infectivity. however, none of the studies evaluated the immunogenicity of rrbd protein systematically in an animal model. recently, ma et al [7] ssuggested the possibility of developing a recombinant rbd protein containing residues 377-662 into an effective and safe mucosal mers vaccine through the intranasal route in the presence of the only poly(i:c) adjuvant in a mouse model. while the need for vaccines with the ability to generate an effective immune response has led to the combination of antigens with more than one adjuvant, the 'adjuvant system' approaches. the adjuvant system approach aids in the development of vaccines that generate effective immune responses [11, 30] . in this study, the roles of three adjuvants-alum, ifa, cpg and poly (i:c)-in rrbd subunit vaccination were investigated aimed at inducing an effective immune response through use of tailored adjuvant combinations and delivery routes. consistent with above studies, all vaccination regimes containing rrbd induced an rbd-specific cellular and humoral immune response. however, a more robust immune response was elicited when mice were immunised with the rbd/a+c and rbd/i+c regimes. an unexpected result was the absence of neutralizing antibodies in the sera of rbd/i+c immunised mice, despite anti-rbd specific igg titres being similar for the rbd/i+c and rbd/ a+c regimes. to further understand the riddle, we detected the aantibody avidity of different vaccination regimes by aavidity elisa. however, the results showed the high antibody avidity correlated with a high igg titre in mice of rbd/i+c and rbd/ a+c groups. so, we speculated maybe the adjuvants of destroyed the conformation of rrbd and covered the antigen binding sites. another probable cause of the low titer of neutralizing antibodies in the sera of rbd/i+c immunised mice was the delivery route of subcutaneous. as known, the subcutaneous may be associated with degradation at injection site, which leads to decreased bioavailability [31] . whatever, further studies are in process. compared with other studies, the regimes in this study induced lower titres of neutralization antibodies. for example, the pi 50 of alum plus cpg, the group showing the highest titer of neutralizing antibody in all immunization groups was 1:500. while the rrbd protein in the above studies acquired a 1:1000 in mice neutralization antibody titre. the differences may be caused by the detection methods of neutralization antibody. as showed in the materials and methods parts, the neutralization antibodies in this study were detected by a pseudovirus system which can be conducted in biosafety level-2 facilities. while the differences of induced neutralization antibodies among different groups can be shown clearly. the subclass of immunoglobulin induced after immunization is an indirect measure of the relative contribution of th1-type cytokines vs. th2-type cytokines [18] . to characterise the immune response of the different vaccination regimes, igg isotype including igg1, igg2a and igg2b analyses were performed. as expected, the rbd/a regimes produced a th2 response with high igg1/igg2 ratio. in contrast, mice received the rbd/a+c or rbd/i+c regimes revealed a th1 skewed response. consistently, the rbd/a+c or rbd/i+c regimes induced a systematic cellular immune response in mice by elispot analysis. the high level of ifn-c and il-2 in the cba was also a proof of the cellular immune response in mice. besides, the mice in the rbd/a+c group had a high level of il-4 and il-10, which were an index of th2 skewed response. taken together, the rbd/a+c induced a th1 and th2 mixed immune responses, though the responses had a th1 inclination. it was our original intention of mixed th1/th2 responses for better protection. similarly, the rbd/i+c regimes induced a mixed th1 and th2 responses. however, it was a pity that the rbd/i+c regimes could not induce an effective neutralization antibody, which was the most important factor of a prophylactic vaccine. above all, in this study, mers-cov s rrbd combined with the adjuvants alum and cpg produced the most robust immune response. it indicates that the combination of alum and cpg was the optimal strategy for i.m. rrbd antigen delivery in a murine model. this result will facilitate future mers-cov vaccine design. the results of the present study also support the importance of the adjuvant system approach, although adjuvant combinations do not always produce the desired response, as seen with rbd/i+c. consistent with the results of the present study, cpg plus alum was found to induce protective humoral, as well as cellular immunity, in mice immunised with a recombinant haemagglutinin vaccine that protected against influenza virus challenge [32] . the ideal immunity of the cpg and alum combination may be the result of mutual complementation of these two adjuvants. it is well known that alum can promote antibody-mediated protective immunity. however, alum is a poor inducer of cellular immune responses [12] . recently, adjuvants including oil-in-water emulsions have shown improved efficacy for avian influenza protection suggesting that even for diseases where humoral immunity can confer protection, cellular immune responses may be necessary in vaccine design [33] . the key features of cpg-odn used as a vaccine adjuvant, include the ability to elicit th1 cell, but only under certain conditions, cd8+ cytotoxic t cell responses and an additional ability to divert the pre-existing th2 response in neonates and elderly mice toward a th1 phenotype [34] . thus, we expect that the combination of alum and cpg will prove applicable in a range of infectious diseases that have defeated current immunisation strategies. except for a choice of adjuvants in combination with optimal protective antigen, practical items such as the antigen: adjuvant ratio, dose, vaccination regimen and often route of administration will strongly impact on both the effectiveness and safety of the vaccine formulation. in most cases, an experimental vaccine will be initially tested in an animal model [29] . to evaluate the immunogenicity of rrbd protein thoroughly, it is necessary to test the protective effects of rrbd subunit immunisation in an animal model of mers-cov infection. to date, rhesus macaques have been reported to generate pneumonia-like symptoms within 24 h of mers-cov infection [35] , and we are testing the effects of rrbd immunisation in rhesus macaques. considerable efforts are being made to establish a small animal model of mers-cov infection. though the lung cells of the syrian hamster express the receptor for mers-cov, they are not susceptible to mers-cov infection [35, 36] . recently, a mouse model of mers-cov infection was reportedly generated by transduction of mice with adenoviral vectors expressing dpp4 [24] . in the future, we expect the protective effect of the rbd/a+c vaccination should be investigated in this murine model of mers-cov infection. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov): challenges in identifying its source and controlling its spread middle east respiratory syndrome coronavirus (mers-cov) -update evidence for camel-to-human transmission of mers coronavirus concerns about misinterpretation of recent scientific data implicating dromedary camels in epidemiology of middle east respiratory syndrome (mers) middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels intranasal vaccination with recombinant receptor-binding domain of mers-cov spike protein induces much stronger local mucosal immune responses than subcutaneous immunization: implication for designing novel mucosal mers vaccines a truncated receptor-binding domain of mers-cov spike protein potently inhibits mers-cov infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines identification of a receptorbinding domain in the s protein of 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syrian hamsters key: cord-344152-pb1e2w7s authors: kolatkar, anand; kennedy, kevin; halabuk, dan; kunken, josh; marrinucci, dena; bethel, kelly; guzman, rodney; huckaby, tim; kuhn, peter title: c-me: a 3d community-based, real-time collaboration tool for scientific research and training date: 2008-02-20 journal: plos one doi: 10.1371/journal.pone.0001621 sha: doc_id: 344152 cord_uid: pb1e2w7s the need for effective collaboration tools is growing as multidisciplinary proteome-wide projects and distributed research teams become more common. the resulting data is often quite disparate, stored in separate locations, and not contextually related. collaborative molecular modeling environment (c-me) is an interactive community-based collaboration system that allows researchers to organize information, visualize data on a two-dimensional (2-d) or three-dimensional (3-d) basis, and share and manage that information with collaborators in real time. c-me stores the information in industry-standard databases that are immediately accessible by appropriate permission within the computer network directory service or anonymously across the internet through the c-me application or through a web browser. the system addresses two important aspects of collaboration: context and information management. c-me allows a researcher to use a 3-d atomic structure model or a 2-d image as a contextual basis on which to attach and share annotations to specific atoms or molecules or to specific regions of a 2-d image. these annotations provide additional information about the atomic structure or image data that can then be evaluated, amended or added to by other project members. the laboratory environment has changed considerably over the last decade as larger projects and broader scientific challenges have required scientists to cooperate and collaborate with peers around the globe. thus, few research labs remain isolated, independent units. the internet has enabled these research teams to work in trans-disciplinary environments, with researchers from many disciplines, nations, time zones, and languages working together on large-scale research projects. while this new development enables researchers to address these new challenges, a new approach to conducting daily research is required. despite changes to the typical laboratory environment, data often continues to be kept in disparate forms of media. for example, protein structure/activity data annotations and images may be kept in paper lab notebooks, manuscripts might be stored electronically in portable document format (pdf), and molecular structure coordinate files may be stored on a hard disk to be viewed and analyzed in graphical molecular viewers, to name a few. however, most of these storage methods are static, one-or two-dimensional, and not connected in real time and not contextually related. in addition, there is a need to manage the changes in these data over the lifetime of a project and avoid differing versions of documents amongst the project collaborators. geographic separation of laboratory units can also present challenges, especially for large-scale research projects. the time and cost for traveling, the efficient distribution of information among collaborators, and the streamlining of workflow processes can be key concerns for laboratory heads and organizational directors. to address these concerns, ''collaboratory systems'' have been put into operation. collaboratory systems are often computerbased systems that manage research workflow processes and allow different lab units to communicate and share data. wulf defined a collaboratory as a ''center without walls, in which the nation's researchers can perform their research without regard to physical location, interacting with colleagues, accessing instrumentation, sharing data and computational resources, and accessing information in digital libraries'' (wulf w (1989) ''the national collaboratory.'' towards a national collaboratory. unpublished report of a national science foundation invitational workshop, new york: rockefeller university). bly refines the definition to ''a system which combines the interests of the scientific community at large with those of the computer science and engineering community to create integrated, tool-oriented computing and communication systems to support scientific collaborations'' [1] . chin and lansing state that the research and development of scientific collaboratories has, thus far, been a tool-centric approach [2] . the main goal was to provide tools for shared access and manipulation of specific software systems or scientific instruments. such an emphasis on tools was necessary in these early development years because of the lack of basic collaboration tools-text chat, synchronous audio, videoconferencing-to support rudimentary levels of communication and interaction. today, however, such tools are available in off-the-shelf software packages, such as microsoft livemeeting, and ibm lotus sametime. the design of collaboratories can move beyond developing general communication mechanisms to evaluating and supporting data sharing in the scientific context. new communication channels can be added to facilitate collaborative exchange of ideas and data. social network services are an example of a novel communication channel that has attracted large numbers of participants. services like myspace and facebook provide a web site for members with similar interests to interact and share information through a variety of formats, such as, discussion groups, voice and video, file sharing, and email. the success of these services has to be considered within a scientific context to provide an effective on-line environment for scientific collaboration. a key challenge is to represent and visualize molecular and cellular data as a function of time, space, and ontological state, and then to efficiently and productively manage and share that data with collaborators and the scientific community. effective proteome-wide prediction of cell signaling interactions require that data on biological relevance, structural accessibility, and molecular sequence be combined to allow for the accurate modeling of cell signaling pathways, for example [3] . when structural genomics intersect with functional proteomics, real-time three-dimensional (3-d) annotation is needed to integrate the two by providing the structural contextual basis upon which to attach the functional information. another challenge is to efficiently communicate and discuss these models and their interpretation with collaborators. research teams are increasingly interdisciplinary and collaborative among laboratories in different departments and institutions located around the world, so it is important for them to have the tools to bridge the gaps of specialization, geography, and time. researchers must employ data organization and workflow management tools to share the context of their data. the ability to quickly search and retrieve complex 3-d data in real time is critical to the efficiency and productivity of large-scale research work. currently, the insufficient level of detail made available by published literature, both in print and online, makes it challenging for researchers to share their knowledge. for instance, data from large-scale structural genomics initiatives , such as, psi i & ii (us), sgc (europe), spine (europe), riken (japan), will require centralized and automated facilities across continents, new methods to divide labor, and novel tools to disseminate and annotate the data to put it into the larger scientific context of systems biology. a number of approaches to combine the power of computerized data storage with advanced visualization technology have been developed in the past. some of the systems below are specific to molecular structures and others are more broad-based client/ server collaboration systems offering additional communication and data sharing functionality. kinemage was the first desktop software tool used to visualize macromolecules, making it possible to enhance the communication of 3-d concepts in journal articles with electronic mass distribution of interactive graphics images. developed at duke university medical center, kinemage illustrated a particular idea about a 3-d object, rather than neutrally displaying that object [4] . kinemages, at the time, were a new type of published illustration, providing 3-d views in addition to standard figures, stereo figures, and color plates. operations on the displayed kinemage could be rotated in real time, parts of the display could be turned on or off, points could be identified and annotated, and changes between different forms could be animated. this was the first widely available molecular viewing and communication tool that allowed researchers to better communicate ideas that depended on 3-d information. the molecular interactive collaborative environment (mice) is an application that provides collaborative, interactive visualization of complex scientific data in 3-d environments within which multiple users can examine complex data sets in real time [5] . mice is portable and enables users to not only view molecular scenes on their own computer, but to distribute these scenes and interact with other users anywhere on the internet. components of mice are already in use in other san diego supercomputer center projects that require platform-independent collaborative software. the biocore system developed at the university of illinois is a large, complex collaboration environment that provides many features, such as data sharing, electronic laboratory notebooks, collaborative molecular viewing and message boards [6] . the biocore system is being used for research and data management as well as for training and uses a combination of a web browser and specialized molecular viewer software to interact with the system and other users. the emsl collaboratory provides both a scientific annotation middleware (sam) server and an electronic laboratory notebook (eln) client as tools for developing collaboration systems [7] . in particular, these tools have been used to develop an nmr virtual facility which enables remote nmr operation as well as sharing documents, data, and images and interacting with personnel at the nmr facility. isee is a software tool that allows users to leverage structural biology data by integrating data from many sources into a single data file and browser [8] . structures can be annotated with methods, key points of interests, and alignments. users can then browse the annotated structures, add new annotations, and evaluate existing ones. the files are small enough to be sent as an e-mail message, and the browser runs under windows, mac, and linux operating systems. though isee offers saved view states, it is not quite a scientific wiki-where users can make annotations without restrictions-because of its prepackaged annotation system. most recently we have developed the collaborative molecular modeling environment (c-me), a new collaboratory system that integrates many of the key features available on kinemage, mice, isee, and biocore systems into one thin-client windows application. the key features that c-me provides include 2-d image and 3-d molecular structure annotation support in realtime, as well as centralized data storage and organization using hierarchical layers (projectsrentitiesrannotations); the annotation system is not limited to predefined ontological categories ( figure 1 ). collaborative molecular modeling environment (c-me) is a collaboratory system developed for the purpose of supporting research projects involving the molecular systems biology and structural proteomics of the sars corona virus as well as a cancer diagnostic research project. these projects require a simple but secure process to store all relevant data in a variety of formats in a single location that are then available either through the c-me client or through a web interface for those collaborators who do not have the c-me client. the c-me backend architecture enables these features and is built on windows server 2003, microsoft sql server 2005, and microsoft office sharepoint server 2007 (moss 2007). c-me includes a public domain client application for the windows xp and vista operating systems that can be downloaded from http://c-me.scripps.edu. c-me is built using commercial off-the-shelf (cots) software as an open-ended content management and sharing system. the primary advantage for using cots components to build c-me is that it reduces overall system development costs and requires less development time-only the components required to perform a specific function need to be developed. developing a custom system from scratch could involve creating basic functionality, retrieving data from a database, or rendering or moving a threedimensional image on the screen, all of which might already be available commercially. c-me uses cots components for most of its back-end functions. windows server 2003, sql server 2005 and moss 2007 provide the central authentication, database, and web infrastructure that enable an application like c-me to be developed quickly. windows server 2003 provides the active directory authentication functionality. users can be added and removed from the directory and permissions assigned according to project or affiliation. sql server 2005 is the relational database management system that provides the data storage, indexing, and retrieval functionality for all data stored on moss 2007. thus, any information exposed by c-me is actually stored in a sql server 2005 database and made available through moss 2007 either directly using a browser or through c-me, using web services to access the data. moss 2007 is the portal system that organizes and makes available the data that is used by c-me including the root project image, protein data bank (pdb) coordinate files, and 2-d images, as well as all annotations. the user can access this information with c-me which uses web services to read and write data to and from the moss 2007 server. the user also has the option to access that data through a standard web browser ( figure 2 ). c-me is being developed using a rapid prototyping methodology and new programming tools from microsoft, including the c# programming language, .net 3.0 framework, and the windows presentation foundation (wpf). other systems, such as java, eclipse and netbeans, also provide built-in features for rapid application development. however, since we are leveraging moss 2007 for the data organization and storage functionality, we are using the .net 3.0 framework and wpf because of their tight integration with the microsoft windows and server systems figure 1 . c-me application window displaying a project. the project, entity and annotations list boxes are displayed along the left hand side and the graphical window for displaying the 3d molecule representations or 2d images to the right. a project can have an associated 2d image to provide additional context as shown in the graphical window for the selected cancer therapeutics project. for this project, a protein structure entity is displayed on the project image as a thumbnail image of the cartoon representation of its 3d structure which can then be opened for viewing and annotation by double-clicking it. doi:10.1371/journal.pone.0001621.g001 [9] . wpf is the graphical subsystem of the .net 3.0 framework and provides a programming model for building applications with a separation between the user interface and the underlying logic. the built-in wpf features for rendering 2d and 3d graphic objects were particularly helpful in more quickly assembling the functionality that supports c-me. the rapid prototyping has enabled us to review iterations of the prototypes in two-week cycles providing bug and usability feedback to the developers for the next cycle of development. figure 3 illustrates the relationships among the image rendering components and the back-end components, which communicate through industry-standard web services. the c-me graphical client application communicates via web service calls to the moss 2007 application programming interface (api) to read or write data to a specific moss 2007 entity or project site or to create new project and entity sites. figure 4 shows the flow of data as it is entered into c-me and directed to the appropriate site on the moss 2007 server which uses sql server 2005 as the relational database system for data storage. allowing multiple participants to jointly create and edit web pages is known as a ''wiki,'' first developed and implemented by ward cunningham in 1995 (http://en.wikipedia.org/wiki/wiki). the wikipedia is perhaps the most widely known and used wiki and is essentially an encyclopedia collaboratively written by volunteers [10, 11] . by allowing researchers to publish their data sets to a larger community through a publicly or privately available data portal, c-me creates the possibility of a ''wiki-like'' research community, where any number of authorized participants can view and annotate research results interactively in a specific 2-d or 3-d context. importantly, c-me is a collaboratory system that enables the sharing and analysis of biological data with project level security in addition to the large wiki features through: controlled access-users are authenticated against an active directory to determine their level of access to projects and entities. access can also be provided to accounts residing on external ldap repositories via moss. anonymous read-only access is also available. simple graphical interface-the graphical interface provides a standard set of mouse and keyboard controls to manipulate and annotate images and structures. data organization-data entered into c-me is organized in a hierarchical system that allows creation of projects containing multiple sub-projects. data consistency-all data is stored in one location so that all users access the same data from the c-me application. all changes are immediately available to all users. robust data backend-all c-me data is stored in an industrystandard sql server 2005 database accessed through the moss 2007 application programming interface. context-specific annotation-annotations are related graphically to the feature in the image or molecular structure that is pertinent. the system supports several data formats, has data translation capabilities, and is able to interact and exchange data with other sources, such as external databases. by leveraging moss 2007 functionality, c-me offers subscription capabilities, performs user authentication, establishes and manages permissions and privileges, and has data encryption capabilities to ensure secure data transmission as part of its security package. c-me organizes data into a hierarchical structure that displays the organization of a data set at three abstraction levels: the project (multiple) level, the entity level, and the annotation level. each layer of the hierarchy contains one or more instances of the layer below it. the user may select a particular level from one the three list boxes along the left side of the c-me window. a project is a collection of entities created around a particular research goal, organization, or any other meaningful category that a research team might create. each project can have an associated image to provide context for any data to be added to the project. projects, in turn, contain entities, which can be either a 3-d atomic coordinate file created in protein data bank format, a 2-d image file created in png, gif, or jpg format, or a set of microscope images from a clinical blood sample in the case of specimen entities. 3-d entities can be rotated, zoomed and translated to orient the view as required. entities can then be annotated, by adding notes, documents, images, e-mail threads, or other forms of information. annotations are anchored to specific locations in the 2-d or 3-d entities, so an annotation is always placed in a specific context and in a precise relationship to related annotations. the c-me application does not provide advanced molecular viewing features, such as surface display, geometry analysis, and electron density viewing. it is not intended to replace existing advanced or specialized molecular viewers, such as pymol or coot [12, 13] . instead, other viewers can be launched from c-me to provide more detailed molecular analysis. c-me provides basic 2-d image analysis functionality which includes the following image manipulation functions: rgb color filtering, zooming, and basic image enhancements such as contrast and brightness control. advanced image analysis operations should be performed with an appropriate external image manipulation application (e.g. adobe photoshop or a purposespecific image analysis application). c-me presents a user with an intuitive interface that reflects the three levels of information within the system. individual windows on the left side of the screen reflect the selections made by the user at each level of the c-me system ( figure 5 ). the top left window reflects the projects that are currently established on the system. the middle left window reflects the entities that have been chosen for examination by the user. the lower left window reflects the list of annotations that have already been entered by other users and allows users to navigate through them as well as to open the annotations for viewing. since moss 2007 creates a central repository for data in almost any format, users are not limited in the ways they organize their data. being able to link multiple kinds of data to an image or a protein structure, for example, makes it easier to organize experiments, collect and distribute results, and speed up the process of uncovering deeper knowledge from various experiments. each entity, sample, or laboratory report can become its own site. all information about that entity can be collected and retrieved from one point either using c-me or a web browser. a movie demonstrating major c-me features and usage is available (video s1, supporting information). once an entity is created, a user can begin annotating the molecule, image or specimen. annotations to molecule entities can be attached to the entire molecule, a set of user-defined amino acid residues, or a set of user-defined atoms. for an image entity, annotations are attached to the entire image or to a user-selected region of the image. specimen entities are different in that the annotations are actually the set of images (called cell hit annotations) associated with a specific sample added to the entity when the specimen entity was created. additional images can be added to a specimen entity by right-clicking on the entity name in the entities window at the left. annotations provide additional information and points of discussion for a molecular structure. text notes, urls, files, screen captures and discussions are all types of annotations that can be added to a structure or image. a user can then either double-click the annotation from the list in the lower left window or select the annotation flag from the graphical window to open that annotation. office documents and pdf files are opened for reading in a new tab in the c-me graphical window ( figure 6 ). the user may then decide to open that document for editing. any file can be attached as an annotation. commonly used file annotations include generic text files, images, audio or video media files, and pdf files. c-me relies on the user's installed software to open these files with the appropriate application. in addition, microsoft word, excel and powerpoint documents can be attached as annotations in c-me and are more tightly integrated with c-me and open in a separate tab in the graphical window. for example, excel spreadsheets can be attached as annotations and accessed through c-me, where data or formulas in the cells can be modified by users with appropriate permissions from local or remote locations. multiple users can open a given document simultaneously. a check-in/check-out feature is available. when a user edits an annotation, it is checked out to the first person who edits it. another user who wants to edit the same file at the same time can only open it as a ''read-only'' version. general text descriptions can be added as a note annotation and can be attached to the entire entity or to a specific region of the entity. a note annotation flag appears as a small pad-and-pen icon. a uniform resource locator (url) annotation is an html link that can be attached to an entity. this allows any web page to be added as additional information without having to actually extract information from the web site. a screen capture annotation allows an arbitrary rectangle of the user's screen to be captured as an image and attached to an entity. when selecting a screen capture annotation, c-me is minimized and the rest of the desktop is shown with a light grey color. the mouse cursor becomes a cross-hair cursor which can be used to drag out a rectangle to save as the screen captured image. a window then appears with the captured image in the bottom pane and a place to enter a title for the screen capture annotation, and optionally, an associated url and additional notes. users can also discuss aspects of an entity by creating a discussion thread. when creating a new discussion annotation, a window appears to enter the subject of the discussion and the actual text of the discussion. another user can then double-click this discussion and select a particular subject and click on the reply button to continue the discussion. other users with access to that entity can view all the entries in the discussion and contribute if desired. microsoft infopath forms, which are xml-based and are used to enter data about experiments and samples, can be published directly into a custom list or automatically sent via an e-mail message to a predefined e-mail account. infopath forms are currently being used to gather pathologist reviews of microscope images of stained cells. these forms are then processed to determine an overall score for each image from each pathologist's review. these scores can then be used to classify a cell image. by relying on industry-standard commercial off-the-shelf software products, c-me addresses three areas of concern related to security: authentication and information access, data backup, and data transport. c-me is built on the windows server 2003 operating system, and relies on the active directory for authentication and access control. system administrators or lab directors can control access and grant varying levels of privilegefrom ''read only,'' to ''contribute,'' to ''design pages''-or deny access entirely. the data backup function is built into the moss 2007 data portal and data contributed to c-me is automatically backed up. furthermore, data is encrypted and transferred using the ssl standard. generally, c-me is being used during regular laboratory operations to discuss and share current progress and problems in a particular research project. rather than having to create new powerpoint presentations, the presenter can start the c-me application and browse to the appropriate project to share the current thoughts and results. the annotations placed there collaboratively by other researchers working on that project can be viewed and edited, and new annotations can be added on the spot. similarly, c-me is being used to provide a tour of a completed crystal structure using the published research paper as a basis. now, the reader can step through the c-me annotations extracted from the paper and watch as the relevant portions of the structure are highlighted to place additional data, such as activity assays, sequence comparisons, and protein purification gels, in the context of the structural features. in addition, this guided display feature of c-me will also be used to share ideas, concepts and data with external collaborators allowing them to review, amend or add annotations. from the bench-top perspective, c-me is being used in both structure-based and cell-based research projects. there are currently two primary projects which represent similar but different challenges for the collaborative environment. the functional and structural proteomics analysis of sars-cov related proteins (fsps, http://visp.scripps.edu/sars/default. aspx) is an nih-funded project that requires the production of over 30 sars virus proteome proteins, their crystallization, and eventually, determination of their crystal structures. c-me is being used to collect, organize and share the known fsps structures as well as to annotate them with functional study information. the second project is the cancer bioengineering research project (cbrp, http://cancer.scripps.edu). this project is focused on detecting rare circulating tumor cells in blood specimens from cancer patients. these cells have similarities that allow for their automated detection, and once detected, a 2d image of each cell is produced and is analyzed in multiple ways, requiring annotation of various types. these image analysis results need to be associated with the patient sample they came from, and each specimen might result in 200+ different images, each of which requires evaluation and annotation by a pathologist. c-me is being used to store and organize the scanned microscope cell images, with the image analysis annotations and scoring attached to each stored image. in addition, infopath forms are used to allow pathologists to input the image analysis data to determine whether circulating tumor cells exist. c-me is being used in clinical classrooms. for example, the viewing and annotation features make it suitable for advancedlevel molecular biology courses. using c-me, instructors can present molecular-based material to students at a level of detail not available in print textbooks. it also organizes course materials in hierarchical trees and makes it possible for students to experience the process of research collaboration with their instructors. an instructor in a graduate level pathology course, for example, uses c-me to present students with examples of pathology reports in 2-d image formats. the instructor prepares appropriate annotations ahead of class time, and students reviewing the material after class can add comments or leave questions for the instructor. additionally, image-based medical specialties can use the features of c-me for education. in a pathology fellowship training program, where visual microscopic skills are paramount, learning modules for various pathologic diseases are created. this gives trainees the opportunity to practice making diagnoses by looking at microscopic images, as they will in their future daily practice. but unlike in real life, these microscopic images come with embedded annotations, files, powerpoint presentations and on-line literature reviews, highlighting the corresponding disease information the trainees need to master in association with each image of abnormal cells. trainees can also add modules to c-me as they pursue special interests in one disease or another, eventually creating a training 'wiki' for future year trainees. while c-me contains significant capability for use by researchers today, the application will be enhanced to increase its utility. currently, c-me requires an internet connection to access the project and entity data and annotations. we are planning enhancements that would allow offline use of c-me with data synchronization when c-me is re-connected to the internet. c-me will also be extended to leverage the search and indexing functionality already present in moss 2007 to provide searching functionality from the c-me client application. a c-me user would be able to perform searches within and across projects or entities to find relevant documents, notes, and other annotations related to the search criteria. in addition, c-me will detect user presence to signal a user that a collaborator is currently on-line. this would allow users to send an email or initiate an instant messenger session to discuss annotations and results if the other users or collaborators are currently using c-me. currently, infopath forms are used to capture pathologists' scores from surveys designed to evaluate cell images. the use of infopath forms will be extended to include tests and quizzes for use by instructors to evaluate student performance and understanding of material presented in a c-me project or entity. collaborative molecular modeling environment (c-me) enhances existing laboratory resources with interactive, real-time annotation and visualization capabilities. with this technology, researchers can create wiki-style collaborations for internal or distributed laboratory environments. as a content management and collaboration tool, collaborative molecular modeling environment (c-me) has the potential to improve research efficiency by providing collaborators real-time access to data organized at the molecular and project levels. its platform consolidates data from disparate sources, allowing users to easily compare, contrast, and merge information from different file systems. it complements existing visualization and collaboration software tools, such as isee, kinemage, mice, and biocore by allowing for real-time two-dimensional and three-dimensional annotation, hierarchical project and data organization, and dynamic collaboration. the c-me installation is available for download for computers running the windows xp or vista operating system (http://c-me. scripps.edu). this web site also contains additional information about the c-me application. video s1 video demonstration of the c-me application in use. the major c-me features are shown using a 3d protein structure entity and its associated annotations as an example. found at: doi:10.1371/journal.pone.0001621.s001 (9.82 mb swf) capturing and supporting contexts for scientific data sharing via the biological sciences collaboratory structures in systems biology the kinemage: a tool for scientific communication a prototype molecular interactive collaborative environment (mice) biocore: a collaboratory for structural biology electronic notebooks: an interface component for semantic records systems disseminating structural genomics data to the public: from a data dump to an animated story applications = code+markup: a guide to the microsofth windowsh presentation foundation reference revolution, news@nature wikinomics: how mass collaboration changes everything the pymol molecular graphics system coot: model-building tools for molecular graphics we would like to thank the members of the kuhn and stevens laboratories at the scripps research institute for discussions, testing the c-me application, and providing feedback. this is tsri manuscript no.19247. conceived and designed the experiments: pk ak th rg. analyzed the data: ak jk dm kk dh kb. wrote the paper: pk ak. other: implemented the application: ak kk dh jk. key: cord-352200-i05h8csb authors: xu, yi; zhou, wenwu; zhou, yijun; wu, jianxiang; zhou, xueping title: transcriptome and comparative gene expression analysis of sogatella furcifera (horváth) in response to southern rice black-streaked dwarf virus date: 2012-04-27 journal: plos one doi: 10.1371/journal.pone.0036238 sha: doc_id: 352200 cord_uid: i05h8csb background: the white backed planthopper (wbph), sogatella furcifera (horváth), causes great damage to many crops by direct feeding or transmitting plant viruses. southern rice black-streaked dwarf virus (srbsdv), transmitted by wbph, has become a great threat to rice production in east asia. methodology/principal findings: by de novo transcriptome assembling and massive parallel pyrosequencing, we constructed two transcriptomes of wbph and profiled the alternation of gene expression in response to srbsdv infection in transcriptional level. over 25 million reads of high-quality dna sequences and 81388 different unigenes were generated using illumina technology from both viruliferous and non-viruliferous wbph. wbph has a very similar gene ontological distribution to other two closely related rice planthoppers, nilaparvata lugens and laodelphax striatellus. 7291 microsatellite loci were also predicted which could be useful for further evolutionary analysis. furthermore, comparative analysis of the two transcriptomes generated from viruliferous and non-viruliferous wbph provided a list of candidate transcripts that potentially were elicited as a response to viral infection. pathway analyses of a subset of these transcripts indicated that srbsdv infection may perturb primary metabolism and the ubiquitin-proteasome pathways. in addition, 5.5% (181 out of 3315) of the genes in cell cytoskeleton organization pathway showed obvious changes. our data also demonstrated that srbsdv infection activated the immunity regulatory systems of wbph, such as rna interference, autophagy and antimicrobial peptide production. conclusions/significance: we employed massively parallel pyrosequencing to collect ests from viruliferous and non-viruliferous samples of wbph. 81388 different unigenes have been obtained. we for the first time described the direct effects of a reoviridae family plant virus on global gene expression profiles of its insect vector using high-throughput sequencing. our study will provide a road map for future investigations of the fascinating interactions between reoviridae viruses and their insect vectors, and provide new strategies for crop protection. rice viral diseases are major threats to rice production and have been distributed worldwide across regions depending on rice cultivation [1] . the most prevalent rice viruses are plant-infecting reoviruses in the genera phytoreovirus, fijivirus and oryzavirus of the family reoviridae. southern rice black streak dwarf virus (srbsdv), commonly known as rice black-streaked dwarf virus 2 (rbsdv 2), is a novel member of the fijivirus [2] . since its first observation in 2001 in guangdong, china, srbsdv has spread rapidly and now causes large yield losses throughout southern and central china, northern vietnam, and recently has been identified in northern china [3, 4] and japan [5] . when infected with srbsdv, rice often develops stunted stems, dark green, twisted leaves, and white waxy swellings along veins on the abaxial surface of the leaves. when infected in seedling stage, rice shows more severe stunting and occasionally dried necrotic leaves. with infections after tillering, stunting is usually not so obvious, but disrupted head development and shrunken grains are apparent in many fields. srbsdv is non-enveloped and has an icosahedral capsid (t = 13) composed of an outer and inner protein shells, similar to other known fijiviruses. virions contain ten linear genomic segments (named s1, s2, s3, s4, s5, s6, s7, s8, s9, s10) of double-stranded rna (dsrna), which range from approximately 4.5 to 1.4 kb in size [2, 6] . sequence comparisons and phylogenetic analyses show that srbsdv has a high sequence similarity with rbsdv, and that segments s1 and s10 have the highest relatedness [7] . although the p7-1 protein of srbsdv was recently reported to induce the formation of tubular structures in insect cells and may be a virus movement protein [8] , the molecular functions of the translated proteins have not been documented. srbsdv is transmitted by the delphacid member white-backed planthopper (wbph), sogatella furcifera (hemiptera: delphacidae), in a persistent-propagative manner [2] . wbph is one of the most economically important insect pests in asian countries [9] and as an oligophagous plant-feeder, wbph can cause great harm by direct feeding or vectoring srbsdv to crops, such as rice, wheat and maize [2, 10] . wbph is known for its long-distance migratory habits, the areas affected migration have been regarded to be the central and southeastern part of china, and vietnam, and these areas are consistent with spread and emergence of srbsdv during the past years [11, 12] . the ecological and physiological perspectives of wbph and other hemipteran insect pests have been extensively studied [13, 14] , but molecular mechanisms whereby the insect causes crop damage and yield losses are poorly understood. recently, transcriptomes of the planthoppers nilaparvata lugens and laodelphax striatellus were reported using next-generation dna sequencing techniques [15, 16] . transcriptional response of whitefly to a geminivirus was also reported [17] . these papers provide considerable information relevant to the genomics of planthoppers and whiteflies, and also generate some insight into the molecular mechanisms of insect defense against virus infection. however, the genomic resources available for wbph are still scarce and searches in genbank identified only about 156 wbph ests. hence, this limited amount of data provides little information for transcriptional, proteomic, and gene functional analysis of wbph and almost no information is available to dissect the complicated interactions between the newly emerged srbsdv and its vector wbph. however, next-generation high-throughput dna sequencing technique has provided unprecedented fascinating opportunities for gene discovery of wbph and detection of the global transcription responses to srbsdv infection. here, we constructed two transcriptomes of wbph and profiled the alternation of gene expression in response to srbsdv infection at the transcriptional level. as a whole, 81388 distinct unigenes have been identified and the results indicated that srbsdv infection can potentially perturb primary metabolism and the ubiquitin-proteasome pathway of wbph and activate immune regulatory systems, such as rna interfering, autophagy and antimicrobial peptide production. to our knowledge, this is the first report to define the wbph transcriptome. illumina sequencing and reads assembly as described in the methods, viruliferous and non-viruliferous wbph whole body cdna libraries were subjected to illumina sequencing platform, resulting in 40,665,360 and 39,135,954 reads, respectively. after cleaning and quality checks, short sequences were assembled, resulting in 136031 and 144604 contigs and data are archived at the ncbi sequence read archive (sra) under accession number srp009194 (http://www. ncbi.nlm.nih.gov/sra). using paired end-joining and gap-filling methods, these contigs were further assembled into 241,380 scaffolds with a mean length of 295 base pair (bp). after clustering the scaffolds with the nucleotide sequences available at ncbi, sequence data from the two libraries were combined, and 81388 unigenes were finally obtained with a mean length of 555 bp ( table 1 ). the length distribution of total unigenes had similar patterns between viruliferous and non-viruliferous wbph samples, suggesting there was no bias in the construction of the cdna libraries ( figure 1 ). however, some unigenes were obtained only from viruliferous or non-viruliferous samples (data not shown) and we believe these differences may be caused by distinctions that arise from long-term ecological adaptation to virus infection. all files of the assembled contigs and scaffolds from viruliferous, nonviruliferous wbph, and the combined est libraries are available upon request. to annotate the unigenes, we searched reference sequences using blastx against the non-redundant (nr) ncbi protein database with a cut-off e-value of 10 25 . a total of 28909 (35.52% of all distinct sequences) unigenes provided a blast result (table s1 ). the species distributions of the best match result for each sequence were shown in figure 2 and table s2 . the sequences of wbph had a 16.17% matches with the red flour beetle (tribolium castaneum) sequences, followed by 14.04% and 12.49% with honey bee (apis mellifera) and the pea aphid (acyrthosiphon pisum), respectively. it was surprising that wbph shared the highest similarity with the red flour beetle in the blast annotation. a similar pattern has also been reported in the brown planthopper (nilaparvata lugens) with the transcriptome of n. lugens having a similarity of 18.89% matches with the red flour beetle sequences, 14.80% with the body louse (pediculus humanus corporis) and 13.19% with the pea aphids, respectively [15] . we used the go assignment to classify the functions of predicted wbph unigenes. go is a gene functional classification system which offers a dynamic-updated controlled vocabulary and a strictly defined concept to comprehensively describe properties of genes and their products in any organism [18] . go has three ontologies: molecular function, cellular component and biological process. among 30,987 annotated unigenes, approximately 18045 (58.23%) of the unigenes could be annotated in go based on sequence homology (table s3) . when compared with the other two rice planthoppers (n. lugens and l. striatellus), wbph had a very similar go distribution ( figure 3 ). this distribution is quite reasonable given that the three sap-feeding planthopper species not only belong to the same family delphacidae in the evolutionary level, but also have similar ecological niches in the rice eco-system. in addition, go analysis also showed a similar distribution of gene functions for non-viruliferous and viruliferous wbph (figure 4 ), indicating that the number of genes expressed in each go category was not significantly affected by srbsdv infection. molecular markers are widely applied in the study of insect evolution and differentiation. as mentioned before, wbph is well known for its yearly migration across asian countries, resulting in the spread of srbsdv in east asia. a critical problem in the study of wbph migration is lack of an effective molecular marker, which would be helpful for define of migration pathways. by using de novo assembly of transcriptome sequences, we have identified 7291 simple sequence repeats (ssrs) or microsatellites in wbph. according to predictions, about 7.26% of protein-coding sequences possessed such repeats, of which 62.43% were trinucleotide repeats, with (aag) n being the most frequent motif (39.8%), followed by 16.62% dinucleotide and 0.7% tetranucleotide repeats ( figure 5 , table s4 ). the results are consistent with the recent reports indicating that trinucleotide repeats are the most abundant microsatellites in coding ests [19, 20] . previous work revealed that (aac) n is the most frequent motif in l. striatellus [16] . the different predicted trinucleotide repeats in the two transcriptomes defined for the two available rice planthopper genomes may reflect distinct adaptive behaviors of these related insects. the large numbers of potential molecular markers found in our study will be particularly useful for future genetic mapping, parentage analysis, genotyping and gene flow studies of these species. to identify differentially expressed genes in response to srbsdv infection, the numbers of clean tags for each gene were calculated, and then individual sets of reads were mapped back to the previously assembled transcript and counted as a proxy for gene expression. the differentially expressed transcripts between the two samples were identified using an algorithm developed by audic et al [21] . the expressed transcripts were assigned into groups according to their functions, such as biological process (3315 sequences, figure 6a , table s5 ), cellular component (2965 sequences, figure 6b , table s5 ) and molecular function (3721 sequences, figure 6c , table s5 ), and the distribution of every ontology was shown in figure 6 . totally, 58% of the differentially expressed genes were up-regulated in the viruliferous wbph ( figure 7 and figure 8a ). the detected fold changes (log 2 ratio) of gene expression ranged from 215 to +16, and more than 80% of the genes were up-or down-regulated between 1.0 to 5.0 fold ( figure 8b ). among the molecular function assignments, a high percentage of genes were assigned to cellular and metabolism process genes (35.6%), and 11.8% were related to biotic stimulus genes ( figure 6 ). to validate the digital gene expression (dge) result, we compared the expression profiles of the non-viruliferous and viruliferous wbph using qrt-pcr. we selected 42 unigenes randomly, 33 of which demonstrated a concordant direction of change for both dge and qrt-pcr (table s6) . of the selected unigenes, nine were inconsistent between dge and qrt-pcr. this difference might be caused by a lower sensitivity of qrt-pcr than dge, and read coverage may be uneven across the transcript length, owing to sequencing biases [22, 23] . nevertheless, qrt-pcr analysis confirmed the direction of change detected by dge analysis, indicating that our results are reliable. as mentioned above, viral infections cause dramatic changes in cellular and metabolic processes. for the primary metabolism analyses, 225 out of 3315 genes (6.8%) were down-regulated in viruliferous wbph, most of which were involved in translation, amino acid metabolism, and biosynthesis of ribosomes, spliceosomes and aminoacyl-trnas (table 2 and figure 9 ). these results suggest that protein synthesis and amino acid metabolism of viruliferous wbph were inhibited by srbsdv infection, which are consistent with previous reports in wasps (campoletis sonorensis) and whiteflies [17, 24] . in wasps, when infected by a polydnavirus, translation of specific growth-associated host proteins was inhibited [24] ; and in whiteflies, the primary metabolism genes were dramatically down-regulated by tomato yellow leaf curl china virus (tylccnv) infection [17] . in addition, our results indicated that a large percentage of the genes involved in lipid metabolism and lipogeneic compound metabolism were upregulated (table 2 and figure 9 ). in contrast to whiteflies, tylccnv infection causes the down-regulation of lipid metabolism. a possible explanation for this phenomenon is the differences in replication styles of the two viruses. srbsdv, a typical dsrna virus, replicates their genomes strictly in the cytoplasm, and the lipid biosynthesis and related pathways are necessary for membrane proliferation that occurs in infected cells. tylccnv is a dna virus that enters nucleus directly for its genome replication, membrane proliferation is less extensive. effects on lipid metabolism have also been reported in other virus infections, for instance, human cytomegalovirus (cmv) infection resulted in increases in the flux of the fatty acid biosynthesis pathway genes that were essential for optimal viral growth in fibroblasts [25] . furthermore, hepatitis c virus has been shown to co-opt the prenylation pathway to promote the efficient replication of its genome and possibly encapsidation [26, 27] . all of these observations together with our data suggest that perturbation of lipid metabolism in a range of virally infected cells is a hallmark of cellular changes associated with viral infection. viruses depend on host's machineries to replicate and express their genomes, and replicating cells have large pools of deoxynucleotides and high levels of key enzyme activities that viruses exploit during replication [28] [29] [30] . so viruses have developed strategies to regulate the host cell cycle to facilitate their replication. our results indicated that 231 out 3315 genes (7.0%) related to cell cycle were altered (table 3) . compared with the mitotic (m) phase, there were more up-regulated genes during inter-phase according to our dge results (data not shown). meanwhile, pathway enrichment analyses also showed that gene expression patterns in the ubiquitin-proteasome pathway were significantly altered. the proteasome is the major non-lysosomal proteolytic machine in eukaryotes [31] . in animals and plants, perturbation of the ubiquitin-proteasome pathway has already been shown to be caused by many viruses [32, 33] . for example, adenovirus e1a protein can directly interact with the 19 s proteasome regulatory components (s4 and s8), that are involved in regulation of the activities of 26s proteasome [34] . inhibition of the proteasome by different chemical compounds not only impairs entry but also affects rna synthesis and subsequent protein expression in coronavirus infections [35] . ubiquitination and deubiquitination of the nucleoprotein (np) were also reported to regulate the replication of influenza a virus rna [36] . in the plant phyla, the geminivirus bsctv c2 protein was reported to attenuate the degradation of samdc1 and suppresses dna methylation-mediated gene silencing by inhibiting 26 s proteasome pathway in arabidopsis [37] . also in arabidopsis, perturbation of ubiquitin-proteasome system affects accumulation of turnip yellow mosaic virus (tymv) rna-dependent rna polymerase during viral infection [38] . in our work, 72 out of 3315 differentially expressed genes were related to the ubiquitinproteasome pathway, and a majority of these were down-regulated (table 3 and figure 9 ). the ubiquitin-proteasome pathway is involved in regulation of metabolic adaptation and immune responses and may function by degrading numerous short-lived proteins, such as regulatory proteins [39] [40] [41] . indeed, all of these pathways were significantly affected in viruliferous wbph. among the differentially expressed genes in the viruliferous wbph, 5.5% (181 out of 3315) genes related to cytoskeleton organization were altered, including 70 microtubule cytoskeleton organization genes and 74 actin cytoskeleton organization genes. cytoskeleton-dependent intracellular transport is a common strategy for virus transport to intracellular destinations [42] [43] [44] [45] [46] [47] . for example, association with microtubules is necessary for the release of rice gall dwarf virus (rgdv) from cultured insect vector cells [48] ; the pns10 of rice dwarf virus (rdv) induces tubular structures that facilitate virus spread in the vector nephotettix cincticeps [49] . rgdv and rdv, together with srbsdv that we studied in this work, all belong to the reoviridae family, so a potential similar cytoskeleton regulatory pathway may exist among these viruses. meanwhile, according to a recent work using a recombinant insect baculovirus expression system, the srbsdv p7-1 protein formed tubular structures in insect cells [8] . by integrating these data with our transcriptome results, it is reasonable to believe that the dynamics of cytoskeleton has a major role in virus infection. cellular and humoral responses are major response systems used by insects against microbial infection [50] [51] [52] [53] . among the differentially expressed genes in viruliferous wbph, many genes related to cellular and humoral immune response were upregulated (table 4 and figure 9 ). for example, 65% (52 out 80) of the unigenes were up-regulated in the lysosome pathway. b-cell lymphoma 6 protein (bcl-6), an important regulation factor in humoral immune responses, is believed to be involved in germinal center b-cell functions and the immune response [54, 55] . this protein acts as a sequence-specific repressor of transcription, and has been shown to modulate the transcription of startdependent il-4 responses of b cells and altered expression of bcl-6 leads to modulated il-4 levels and increases in the immune response [56] . also, among the differentially expressed genes, antimicrobial peptide and melanization-related product synthesizing genes were also up-regulated in the wbph. phospholipase a2 products are considered to interfere with viral infection and it has been found that several phospholipases a2 can efficiently protect host cells from hiv-1 replication [57, 58] . down-regulation of the phospholipase a2 inhibitor subunit b (table 3 ) may be helpful for the releasing of active phospholipases a2, and thus improve host defenses against virus attacks. rna interference (rnai) silences gene expression through small interfering rnas (sirnas) and micrornas (mirnas) [59, 60] . the rnai system was initially described in plants, and in recent years, some work has revealed that rnai is a major mechanism to target viruses in some insects [60, 61] . in d. melanogaster, dicer-2 produces sirnas, whereas dicer-1 recognizes precursors of mirnas. in these cases, the small rnas are assembled with an argonaute (ago) protein into related effector complexes, such as the rna-induced silencing complex (risc), to guide specific rna silencing [59, 61, 62] . in our transcriptome data, we found 31 records of genes (10 agos, 16 dicers, 4 dsrna binding proteins) involved in the rna interference pathway. in the dge results (table 4) , some involved genes were up-regulated in viruliferous wbph, such as dicer 1 and dicer 2, but no substantial difference in the expression levels of putative rnai related genes was found in the l. striatellus after infection with rice table 3 . expression profiles of genes in the ubiquitin-proteasome and cell cycle pathways. stripe virus (rsv), a tenuivirus [16] . these differences may be caused by the diversity among the replication cycles of the two viruses or the host defense systems. the latter hypothesis is supported by recent findings that rsv and rdv (both plant reoviruses) caused different effects in the rice rna interference pathway [63] . we employed massively parallel pyrosequencing to collect ests from viruliferous and non-viruliferous samples of wbph. in total, we obtained 81388 different unigenes, and have provided a major genomic resource that will be useful for subsequent investigations of the biology of wbph and generation of new insecticide targets. we for the first time described the direct effects of a reoviridae family plant virus on global gene expression profiles of its insect vector using high-throughput sequencing. the genes involved in primary metabolism, ubiquitin-proteasome, cytoskeleton dynamics and immune responses were up regulated in viruliferous wbph. in addition, we found that an rnai pathway exists in wbph, and this pathway may play an important role in virus defenses. our study will provide a road map for future investigations of the fascinating interactions between reoviridae viruses and their insect vectors, and provide new strategies for crop protection. the wbph culture used in this study originated from jiangsu province, china. srbsdv-infected and un-infected rice (oryza sativa cv. nipponbare, k818) plants were used to feed wbph in all experiments. for viruliferous wbph, srbsdv-infected rice seedlings were collected from fields and then moved to a beaker after discarding the old leaves. then 200 wbph second instar nymphs were swept into the beaker, and the beaker was enclosed with nylon mesh. after two days of feeding, the rice seedlings were gently turned upside-down, and the juvenile planthoppers were swept onto healthy rice seedlings. all plants were grown in soil at 2561uc, 80% relative humidity in a growth incubator, under a long day photoperiod of 14 h in light and 10 h in darkness. sample preparation and rna isolation after two days of feeding on srbdv infected rice, wbph were maintained on uninfected rice seedlings for more than 12 days (viral circulative period). pcr was then used to ensure that viral rnas were present in the individual wbph. during this time, non-viruliferous wbph, as the control group, were treated identically. approximately 100 non-viruliferous and viruliferous wbph nymphs and adults were collected for rna extraction. total rna was isolated using the trizol reagent (invitrogen, carlsbad, ca, usa) according to the manufacturer's protocol. the concentration and quality of total rna were determined by a nano-drop spectrophotometer (thermo fisher scientific, wilmington, de, usa). cdna library preparation and illumina sequencing for transcriptome analysis rna was purified from 20 mg of pooled total rna using oligo (dt) magnetic beads and fragmented into 200-700 nucleotides length sequences in the fragmentation buffer. the cleaved poly-(a) rna was transcribed using oligo (dt) (takara, japan), and then second-strand cdna synthesis was performed. after end repair and ligation of adaptors, the products were amplified by pcr and purified using qiaquick pcr purification kit to create a cdna library. the cdna library was sequenced on the illumina sequencing platform (hiseq2000). the raw reads from the images were generated by solexa ga pipeline 1.6. after removal of low quality reads, processed reads were assembled using short oligonucleotide analysis package (soap) de novo software and clustered with tigr gene indices (tgi) clustering tools [64] . all raw transcriptome data have been deposited in the sra database (ncbi). the generated unigenes larger than 350 bp were analyzed by searching the genbank and swissprot databases with the blastx algorithm. (http://www.ncbi.nlm.nih.gov/). gene ontology annotations and cog classfication of the unigenes were determined with the blast2go (http://www.blast2go.org/) and inter-proscan software (http://www.ebi.ac.uk/tools/pfa/ iprscan/). ssrs were identified with the microsatellite identification tool (misa) (http://pgrc.ipk-gatersleben.de/misa/). misa is based on perl script designed to allow the identification and characterization of microsatellites in a comparative genomic context. detection criteria were constrained to perfect repeat motifs of 1-6 bp and a minimum repeat number of 12, 8, 5, 5, 5 and 5, for mono-, di-, tri-, tetra-, penta-and hexa-nucleotide microsatellites, respectively. a rigorous algorithm was developed to identify differentially expressed genes (degs) between the non-viruliferous and viruliferous wbph as described [21] . short-read sequence data from non-viruliferous and viruliferous wbph were mapped separately against the reference set of assembled transcripts with burrows-wheeler aligner (bwa) 0.5.8a [65] , relative transcript levels were output as rpkm (reads per kilobase of exon model per million mapped reads) values, which were taken into account the transcript abundance. the false discovery rate (fdr) was used to determine the threshold p-value in multiple tests. a fdr,0.001 and an absolute value of the log 2 ratio .1 provided thresholds to determine significant differences in gene expression. the differentially expressed genes were used for go enrichment analyses as described [18] . the correlation between two libraries was statistically assessed by calculation of the pearson correlation coefficient (data not shown). to confirm the results of pyrosequencing analysis, the expression levels of 42 randomly selected genes were measured in non-viruliferous and viruliferous insects by qrt-pcr. total rnas from each sample were extracted using trizol reagent (invitrogen) and treated with dnase i (invitrogen) according to the manufacturer's protocol. the concentration of each rna sample was adjusted to 1 mg/ml with nuclease-free water and total rna was reverse transcribed in a 20 ml reaction system using the amv rna pcr kit (takara). qpr-pcr was carried out on the lightcycler 480@ ii with lightcycler 480@ sybr i master (roche applied science, basel, switzerland) under the following conditions: 95uc for 5 min; and 40 cycles of 95uc for 10 s, 60uc for 15 s, and 72uc for 20 s, followed by melting curve generation (68uc to 95uc). primers used in qrt-pcr for validation of differentially expressed genes were shown in table s7 . each gene was analyzed in triplicate, after which the average threshold cycle (ct) was calculated per sample, and an endogenous 18 s rrna gene of wbph was used for normalization. a no template control sample (nuclease free water) was included in the experiment to detect contamination and determine the degree of dimer formation (data not shown). biology and epidemiology of rice virus southern rice 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antiviral immunity in drosophila melanogaster viral infection induces expression of novel phased micrornas from conserved cellular microrna precursors de novo assembly of human genomes with massively parallel short read sequencing fast and accurate short read alignment with burrows-wheeler transform we thank tong zhou, fenfen wang, yanyuan bao for assistance in feeding and collecting insects, jian xue for advice on data analysis, and xiaoying chen and chuanxi zhang for supplying the gene ontology data of l. striatellus and n. lugens. conceived and designed the experiments: xz jw. performed the experiments: yx. analyzed the data: yx wz. contributed reagents/ materials/analysis tools: yx yz jw. wrote the paper: yx xz jw. key: cord-352564-2j4pjjwk authors: dominguez, samuel r.; travanty, emily a.; qian, zhaohui; mason, robert j. title: human coronavirus hku1 infection of primary human type ii alveolar epithelial cells: cytopathic effects and innate immune response date: 2013-07-24 journal: plos one doi: 10.1371/journal.pone.0070129 sha: doc_id: 352564 cord_uid: 2j4pjjwk because they are the natural target for respiratory pathogens, primary human respiratory epithelial cells provide the ideal in vitro system for isolation and study of human respiratory viruses, which display a high degree of cell, tissue, and host specificity. human coronavirus hku1, first discovered in 2005, has a worldwide prevalence and is associated with both upper and lower respiratory tract disease in both children and adults. research on hcov-hku1 has been difficult because of its inability to be cultured on continuous cell lines and only recently it was isolated from clinical specimens using primary human, ciliated airway epithelial cells. here we demonstrate that hcov-hku1 can infect and be serially propagated in primary human alveolar type ii cells at the air-liquid interface. we were not able to infect alveolar type i-like cells or alveolar macrophages. type ii alveolar cells infected with hcov-hku1 demonstrated formation of large syncytium. at 72 hours post inoculation, hcov-hku1 infection of type ii cells induced increased levels of mrnas encoding il29,cxcl10, ccl5, and il-6 with no significant increases in the levels of ifnβ. these studies demonstrate that type ii cells are a target cell for hcov-hku1 infection in the lower respiratory tract, that type ii alveolar cells are immune-competent in response to infection exhibiting a type iii interferon and proinflammatory chemokine response, and that cell to cell spread may be a major factor for spread of infection. furthermore, these studies demonstrate that human alveolar cells can be used to isolate and study novel human respiratory viruses that cause lower respiratory tract disease. respiratory tract cells are structurally and functionally different in the upper respiratory tract (nasal and sinusoidal epithelia), conducting airways (tracheal and bronchial epithelia), and alveoli (alveolar epithelia). because they are the natural target cells for respiratory virus infection, primary human respiratory epithelial cell cultures provide the ideal in vitro systems for investigation of cell factors required for growth of respiratory human viruses, for analysis of their interactions with viruses and their innate immune responses to infection, and for isolation and propagation of novel respiratory pathogens. the alveolar epithelium consists of both alveolar type i cells (ati), which make up 95% of the surface area of the lung, are terminally differentiated, non-dividing, and function in gas exchange and fluid homeostasis; and alveolar type ii cells (atii), which produce surfactant proteins and lipids, divide and differentiate to replace damaged ati cells, participate in fluid homeostasis, and contribute to the innate defenses of the lung [1, 2, 3, 4] . our laboratory has developed a system to isolate and culture primary atii cells from human lungs and under specialized culture conditions transdifferentiate the atii cells into an ati-like phenotype [5] . in 2005 the fifth human coronavirus, hcov-hku1, was discovered by rt-pcr screening with conserved coronavirus primers on respiratory samples from adult patients with pneumonia that were negative for the severe acute respiratory syndrome coronavirus (sars-cov) [6] . to date, hcov-hku1 has been associated with both upper and lower respiratory tract illness in children and adults [7, 8, 9, 10, 11, 12] . until recently, research on hcov-hku1 has been limited because it could not be isolated from clinical specimens in continuous cell lines in vitro, and there are no reports of hcov-hku1 infecting animals. using primary human ciliated airway epithelial cell cultures, pyrc et al isolated and propagated hcov-hku1, and more recently, dijkman et al extended these observations to additional primary isolates of hcov-hku1 [13, 14] . similarly, we developed a primary human bronchial-tracheal epithelial cell (htbec) culture system at the air-liquid interface (al/i) and have successfully propagated hcov-hku1 from patient specimens. in parallel to these advances, and as an alternative approach to study hcov-hku1, we reasoned that hcov-hku1, like sars-cov [15] , may be able to infect and be serially propagated in human alveolar cells since this virus is known to cause pneumonia in a subset of patients. furthermore, we reasoned that infecting primary human alveolar cells would allow us to determine which subset of cells is susceptible to infection, and to characterize initial innate immune responses of alveolar epithelial cells to viruses that cause lower respiratory tract diseases. here we demonstrate that hcov-hku1 can infect and be serially propagated in primary human alveolar type ii cells but not in alveolar type i-like cells or alveolar macrophages at the air-liquid interface. human alveolar macrophages and alveolar cells were isolated as previously described [5, 16, 17] . briefly, de-identified donor lungs that were not suitable for transplant were obtained through the national disease research interchange (philadelphia, pa) and the international institute for the advancement of medicine (edison, nj). the isolation of cells was conducted as previously described with the exception that the type ii cells were positively selected using epcam (cd326) magnetic beads (miltenyi, auburn, ca). cells were plated in dmem with 10% fbs on millicell inserts (millipore, bedford, ma) coated with a mixture of 80% rat tail collagen and 20% matrigel (bd biosciences, san jose, ca). cells were allowed to adhere to inserts for 2 days submerged in dmem with 10% fbs, then cultured at an air-liquid interface in dmem with 1% charcoal-stripped serum (cs-fbs) supplemented with keratinocyte growth factor (k) for 2 days, and then switched to media that additionally had isobutylmethylxanthine (i), 8-bromo-camp (a), and dexamethasone (d) for 2 days to achieve the atii differentiated phenotype prior to infection (day 6 after plating). the alveolar macrophages and alveolar type i-like cells were cultured and characterized as reported previously [5, 16, 17] . briefly, to transdifferentiate type ii cells to type i-like cells, type ii cells were plated on rat tail collagen-coated dishes at a density of 0.5-1.0610 5 /cm 2 in dmem with 10% fbs (15) . after 24-48 h the medium was changed to 5% fbs without additives. type ilike cell phenotype was documented by positive staining for receptor for advanced glycation end products (rage, r & d systems, minneapolis, mn) and epithelial membrane protein 2 (emp2, sigma-aldrich, inc, st.louis, mo). for alveolar macrophages, the middle lobe of the lungs was perfused, and then lavaged with hepes-buffered saline containing 2 mm edta and then hepes-buffered saline alone. the macrophage purity of the cells were inoculated with primary clinical isolates of hcov-hku1 or isolates of hcov-hku1 which had been passaged one time on primary, differentiated, human bronchial-tracheal epithelial cells (htbec) (p1 virus) (lifeline cell technology, walkersville, md), at 1:10 dilution in dmem with 1% bovine serum albumin (bsa). after 4 hour incubation, the inoculum was removed and cells were incubated at 34 o c. for cells kept at an air-liquid interface (al/i), the apical surface of the cells were washed every 24 hours with dmem +1% bsa and collected for pcr analysis. for cells kept immersed, the media was collected and replaced every 24 hours. cell morphology was monitored daily to observe for any cytopathic effects. three days after inoculation, cells were fixed with 100% methanol and viral antigens and cell markers were detected using immunostaining. hcov-hku1 was detected in infected cells using a rabbit polyclonal antibody or a mouse-monoclonal antibody directed against the hcov-hku1 viral spike glycoprotein developed in our laboratory and visualized using a goat anti-rabbit or goat antimouse antibody conjugated to alexa 488 fluorophore (life technologies, grand island, ny). cells were also stained for cell type specific markers [atii cells: sp-a (pe-10, a gift from yoshio kuroki, university of sapporo, japan), prosp-b (millipore, billerica, ma), ttf-1 (leica biosystems, buffalo grove, il), and mouse igm anti-dobbs (hu-280, a gift from leland dobbs, university of california -san francisco); basal cells: keratin-5 (thermo scientific, pittsburgh, pa); alveolar macrophages: cd68, (clone kp1, dako, inc., carpenteria, ca)]. immunostained cells were imaged using a zeiss axioskop 2 fluorescent microscope with axiovision software and a zeiss lsm 700 laser scanning confocal microscope with zen software (national jewish cytometry core). viral rna from pooled apical washes was extracted using qiagen ez1 virus mini kits (valencia, ca) on a biorobot ez1 extractor (qiagen, valencia, ca) following the manufacturer's instructions. virus yield was determined by a quantitative real time rt-pcr (qrt-pcr) which targeted the hcov-hku1 polymerase 1b gene with comparison to copy number from a plasmid encoding the region recognized by this assay. the rt-pcrs were performed using a 1-step reverse-transcription pcr master mix (rna ultrasense one-step quantitative rt-pcr system, invitrogen life technologies, carlsbad, ca). reaction mixtures were prepared using a 500-nm final concentration of the forward primer (59-tggtggctgggacgatatgt-39), 250 nm of the reverse primer (59-ggcatagcacgatca-cacttagg-39), and 100 nm of the probe (59-6fam-ataatcccaacccatrag-minor groove binder nonfluorescent quencher-39). 10 ul of rna was added to 10 ul of master mix containing an additional 1.3 mm mgcl. rt-pcr was performed using the following cycling conditions: 50uc for 15 minutes, 95uc for 2 minutes, and 40 cycles of 95uc for 15 seconds, 60uc for 30 seconds [12] . at 72 hours post-infection, cells were harvested for rna analysis using the rneasy rna extraction kit (qiagen, valencia, ca), cdna synthesis using qscript cdna supermix (quanta biosciences, gaithersburg, md). to quantitate mrnas encoding interferons, cytokines and chemokines, we used taqman the committee for the protection of human subjects at national jewish health has deemed the use of human lung donor cells as nonhuman subject research, since there is no risk to donors and all donors are de-identified. use of the banked virus specimens and clinical data for this research was approved by the colorado multiple institutional review board. the 5 clinical isolates of hcov-hku1 from denver, co, utilized in the study were part of a larger set of hcov-hku1 samples collected as part of a study on the epidemiology of cov infections in pediatric patients detailed elsewhere [12, 18] . the clinical details of the patients from whom the isolates were obtained are summarized in table 1 . all of these hcov-hku1 isolates were hcov-hku1 genotype a. to determine which subset of primary human alveolar cells are susceptible to hcov-hku1 infection, human alveolar type i-like cells, alveolar type ii cells, and alveolar macrophages were tested. in addition, two different conditions for cell growth were compared, submerged cultures versus cultures at the air-liquid interface (al/i). cultures were inoculated with primary clinical isolates of hcov-hku1 or passage 1 (p1) virus. titers of viral rna in the wash from the apical surface of the cells were determined by qrt-pcr at the indicated time points, and hcov-hku1 infection of type ii alveolar cells plos one | www.plosone.org cultures were fixed and immunolabeled with antibodies to the hcov-hku1 spike glycoprotein to identify infected cells. neither the submerged nor air-liquid interface type 1-like cells nor alveolar macrophages were susceptible to infection by hcov-hku1 ( figure 2 ). in the submerged culture of type ii cells only a few cells were infected with hcov-hku1. in contrast, type ii cells maintained at the air-liquid interface supported infection, although the level of infection varied with individual subjects. type ii cells infected with hcov-hku1 showed formation of large syncytia indicating cell to cell spread may be a major factor for virus spread within the lung as it is for respiratory syncytial virus and parainfluenza viruses (figures 2 and 3 ). this pattern of susceptibility of atii cells at the al/i to hcov-hku1 infection was reproducible in most human donors, since cultures from 17 of 20 different donors tested were susceptible to infection with hcov-hku1, although cells from different donors demonstrated significant variation in the percent of hcov-hku1 infected cells. similarly, all 5 of our clinical hcov-hku1 specimens were able to infect type ii cells at the air-liquid interface. the atii cells had a morphological appearance of a cobblestone epithelial monolayer, typical for type ii cells. these cells were fluorescently labeled with antibodies to surfactant protein a (sp-a), pro-surfactant protein b (prosp-b), thyroid transcription factor-1 (ttf-1), and a type ii specific transmembrane protein (hu-280) using an antibody obtained from dr. leland dobbs, university of california, san francisco [19] ( figure 1 ). there were a few clumps of airway basal cells in these cultures. these cells stained for keratin 5 but were not infected by hcov-hku1. other than the formation of large syncytia, cytopathic effects (cpe) were not detectable in any of the type ii cell culture at the al/i. to examine the kinetics of hcov-hku1 replication in type ii alveolar cells, primary clinical specimens containing hcov-hku1 rna were used to inoculate the apical surface of type ii alveolar cells for 4 hrs and then returned to the al/i again. apical surfaces were washed 24, 48, 72, 96, and 120 hrs post inoculation and assayed for hcov-hku1 rna by qrt-pcr. viral genome copies increased approximately 1000 fold during the course of the infection indicating productive infection and replication (figure 4a) . to determine if human alveolar cells could be utilized to propagate hcov-hku1, apical surfaces were inoculated and incubated for 4 hr at 34 o c with a clinical sample of hcov-hku1 and then removed. the apical surfaces were then washed every 24 hr for the next 5 days. serial passage of hcov-hku1 infection was performed by inoculating naïve cultures with 100 ml of the day 5 (120 hr post inoculation) apical wash from the previous passage. viral genome copy numbers were maintained over the course of three passages (figure 4b ). immunofluorescence assays (ifa) with antibodies to the hcov-hku1 spike glycoprotein performed at 120 hours confirmed the presence of infected cells at the end of each passage. these experiments demonstrated that genome copies detected in the apical wash were infectious and did not represent defective particles, confirming that hcov-hku1 was truly replicating in type ii alveolar cells at the al/i. because alveolar type ii cells at the air-liquid interface were highly susceptible to hcov-hku1 infection, we could measure changes in the level of expression of select genes of the innate immune system in these cells in response to hcov-hku1 infection. at 72 hours post inoculation, hcov-hku1 infection induced markedly increased levels of mrnas encoding type iii interferons (il29), interferon-responsive genes (cxcl10), proinflammatory chemokines (ccl5 and cxcl10), and il-6. there was no significant increase in ifnb, the only type i interferon reported to be expressed by alveolar type ii cells [20] (figure 5a ). the levels of relative cellular gene expression varied significantly between human donors tested (figure 5b) . thus, human alveolar type ii cells induce significant innate, anti-viral responses to infection with hcov-hku1, but the extent of these responses varied in different humans with different levels of infection. this is the first study to demonstrate infection of primary human alveolar type ii cells at the al/i with primary clinical specimens containing hcov-hku1 rna. human alveolar type ii cells maintained at an air-liquid interface were highly susceptible to hcov-hku1 infection, but alveolar macrophages and type i-like cells were not susceptible to hcov-hku1. this supports our hypothesis that primary human alveolar cells can be a suitable culture system for isolation and propagation of novel human respiratory viruses that cause lower respiratory tract disease and which display a stringent degree of cell, tissue, and host specificity. utilization of this culture system is appealing because it most closely represents the in vivo physiological conditions of the lung [2, 21] . the type ii culture system allows identification of which terminal human respiratory epithelial cells are susceptible to infection and might provide insight into the spread of infections from the conducting airways required for the development of pneumonia and the acute respiratory distress syndrome (ards). these results are agreement with what is known about the sars-cov, another beta-coronavirus (lineage b) that causes more severe lower respiratory tract disease than hcov-hku1. autopsy studies of patients with sars-cov demonstrated expression of viral antigen in type ii pneumocytes [22, 23, 24] , and previous studies in our laboratory showed that in vitro cultures of human alveolar type ii cells at the al/i are susceptible to infection with sars-cov [15, 25] . similarly, a recent study using human lung organ cultures demonstrated that various subtypes of human influenza a viruses, which varied in their pathogenic potential, all preferentially infected type ii alveolar cells, with minimal infection of alveolar macrophages and no infection of alveolar type i cells hcov-hku1 infection of type ii alveolar cells plos one | www.plosone.org [21] . their study also highlighted that the different pathogenic potential of influenza a viruses was not attributed to differences in cellular tropism, as they all infected the same cells, but rather to the capacity of the viruses to productively replicate in type ii alveolar cells. overall, these studies emphasize the importance of atii cells in the pathogenesis of viral induced lower respiratory tract disease. in contrast to these viruses, human coronavirus 229e, an alphacoronavirus, can readily infect human alveolar macrophages, but not type i-like or type ii alveolar cells [17] . interestingly, in contrast to sars-cov, hcov-229e is a common cause of upper respiratory tract infection and rarely causes severe lower respiratory tract disease [26, 27] . the differences in clinical presentation between these coronavirus may be partially explained by whether that can infect type ii alveolar cells. several observations regarding infection of the type ii cells with hcov-hku1 are worth noting. growing and infecting the cultures at an air-liquid interface, as opposed to keeping them submerged, greatly increased the susceptibility of type ii cells to hcov-hku1. these results are identical to what we previously described for sars-cov [25] . interestingly, there is very little fluid at the apical surface of the alveolar epithelium [28, 29] , except in diseased states, and therefore the al/i conditions more accurately approximate in vivo conditions for type ii cells. over the course of our experiments we infected cells from 20 different donors. type ii cells from 17 of these donors were susceptible to hcov-hku1 infection, although there was significant variability in the degree of susceptibility between donors. one possible explanation for these differences might be differential levels of receptor expression. as the receptor for hcov-hku1 is currently unknown we were not able to explore this hypothesis. elucidating the mechanism(s) for the increased susceptibility in the al/i conditions and the variability exhibited between donors might unravel important cellular determinants of infection. induction of syncytia formation by hcov-hku1 most likely indicates direct cell to cell spread of virus and undoubtedly plays hcov-hku1 infection of type ii alveolar cells plos one | www.plosone.org an important role in the pathogenesis of hcov-hku1 in lung disease and escape from immune surveillance. many infections with enveloped viruses can directly move from cell to cell by fusion of adjacent plasma membranes resulting in large, multinucleated giant cells. this ability confers multiple potential pathogenic advantages over cell free spread. direct cell to cell spread is more efficient by eliminating the intrinsic barriers of fluid-phase diffusion required by cell free spread to encounter another target cells and engage the appropriate receptors. movement of infection between cells also allows the virus to evade innate humoral and cellular defenses [30, 31] . viral fusion with cell membranes of their target cells (virus-cell fusion) is an essential first step in infection for all coronaviruses. syncytia formation is also commonly seen in other coronaviruses and plays an important step in viral pathogenesis [32, 33, 34] . syncytia formation of dendritic cells resulting in cell death was recently shown to be important in an in vitro infection model of another human respiratory coronavirus, 229e [35] . syncytia forming activity has been shown to greatly enhance the infectivity of avian coronavirus infectious bronchitis [36] . modulation of syncytial formation helps to establish viral persistence in in vitro culture models of infection with the murine coronavirus, mouse hepatitis virus (mhv) [37] . both virus-cell entry and syncytia formation are mediated by the coronavirus spike protein, a class 1 viral fusion protein that is a major determinant of cell and host specificity. these two important processes in the pathogenesis of hcov-hku1, however, may occur through different mechanisms as has been shown for sars-cov and mhv [38, 39, 40, 41] . the innate immune system serves as the first line of defense in protection of the host against viral infections and helps to shape the resultant adaptive immune response. the characteristics of the initial immune response can greatly impact the resulting clinical outcome [42] . expression of pro-inflammatory cytokines in response to viral infection argues that type ii alveolar cells are not only target cells for infection but are also immune-competent and may directly influence the immune response to viral infection. many of the cytokine responses of the human type ii alveolar cells at the al/i to hcov-hku1 (induction of cxcl-10, il29, and ccl5) were similar to those we previously described for influenza and sars-cov [20] [25] . one significant difference was a lack of expression of the type i interferon, ifnb, which may reflect that our current assays were conducted at a later time point during the course of infection. alternatively, it could be due to the presence of unique viral proteins which can suppress the ifn response. another difference was that the degree of induction of these cytokines was overall approximately 10 fold less for hcov-hku1 compared with influenza and sars-cov. this may have important implications as recently it was shown that the level of cytokine responses in type ii alveolar cells induced by different strains of influenza a viruses were correlated with degree of pathogenicity [21] . similarly, the differences in cytokine responses exhibited by different human donors might partially explain the variability in clinical outcomes to hcov-hku1 infections. in summary, hcov-hku1 can infect, be serially propagated, and induce an anti-viral response in human alveolar type ii cells maintained at an air-liquid interface. type ii cells infected with hcov-hku1 demonstrated syncytia formation indicating cell to cell spread may be a major factor for virus spreading in vivo, suggesting a potential mechanism for evasion of host immune surveillance. these experiments demonstrate that hcov-hku1 has strong tropism for type ii alveolar cells and demonstrate the ability to use human alveolar cells to isolate and study novel human 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cleavage and cell fusion proteolytic activation of the spike protein at a novel rrrr/s motif is implicated in furin-dependent entry, syncytium formation, and infectivity of coronavirus infectious bronchitis virus in cultured cells receptor-independent spread of a highly neurotropic murine coronavirus jhmv strain from initially infected microglial cells in mixed neural cultures a human coronavirus responsible for the common cold massively kills dendritic cells but not monocytes acquisition of cell-cell fusion activity by amino acid substitutions in spike protein determines the infectivity of a coronavirus in cultured cells fusion resistance and decreased infectability as major host cell determinants of coronavirus persistence different host cell proteases activate the sars-coronavirus spike-protein for cell-cell and virus-cell fusion ready, set, fuse! the coronavirus spike protein and acquisition of fusion competence furin cleavage of the sars coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry cleavage inhibition of the murine coronavirus spike protein by a furin-like enzyme affects cell-cell but not virus-cell fusion immunity and immunopathology to viruses: what decides the outcome? the authors would like to thank yoko ito, elise messier, and karen edeen for assistance with isolation and culture of human alveolar cells and andrew berglund and philip weston for technical assistance with infection assays. we thank kathryn holmes for critical review of the manuscript. key: cord-339920-dr5bvpm0 authors: soberman, roy j.; mackay, christopher r.; vaine, christine a.; ryan, glennice bowen; cerny, anna m.; thompson, mikayla r.; nikolic, boris; primo, valeria; christmas, peter; sheiffele, paul; aronov, lisa; knipe, david m.; kurt-jones, evelyn a. title: cd200r1 supports hsv-1 viral replication and licenses pro-inflammatory signaling functions of tlr2 date: 2012-10-17 journal: plos one doi: 10.1371/journal.pone.0047740 sha: doc_id: 339920 cord_uid: dr5bvpm0 the cd200r1:cd200 axis is traditionally considered to limit tissue inflammation by down-regulating pro-inflammatory signaling in myeloid cells bearing the receptor. we generated cd200r1(−/−) mice and employed them to explore both the role of cd200r1 in regulating macrophage signaling via tlr2 as well as the host response to an in vivo, tlr2-dependent model, herpes simplex virus 1 (hsv-1) infection. cd200r1(−/−) peritoneal macrophages demonstrated a 70–75% decrease in the generation of il-6 and ccl5 (rantes) in response to the tlr2 agonist pam(2)csk(4) and to hsv-1. cd200r1(−/−) macrophages could neither up-regulate the expression of tlr2, nor assemble a functional inflammasome in response to hsv-1. cd200r1(−/−) mice were protected from hsv-1 infection and exhibited dysfunctional tlr2 signaling. finally, both cd200r1(−/−) mice and cd200r1(−/−) fibroblasts and macrophages showed a markedly reduced ability to support hsv-1 replication. in summary, our data demonstrate an unanticipated and novel requirement for cd200r1 in “licensing” pro-inflammatory functions of tlr2 and in limiting viral replication that are supported by ex vivo and in vivo evidence. the host response to viral pathogens is initiated by the interaction of viruses, viral genomes, viral proteins, and viral replication intermediates with host pathogen recognition receptors (prrs) to trigger the release of cytokines, chemokines, and interferons (ifn) [1, 2, 3, 4, 5, 6] . ultimately, cells and organisms must integrate input from different signaling pathways in the correct temporal and spatial manner to achieve an appropriate functional state and biological response. in the case of herpes simplex virus 1 (hsv-1) the interaction of the virus with toll-like receptor (tlr) 2 is critical. the interaction of tlr2 with hsv-1 triggers nf-kb dependent cytokine production [6, 7, 8, 9] leading to the recruitment and maturation of effector cells for pathogen clearance. excessive signaling through tlr2 can cause a ''cytokine storm'', leading to excessive inflammation and tissue damage resulting in a potentially lethal outcome [7, 9, 10, 11] . in response to hsv-1 infection, tlr2 2/2 mice show both dramatically enhanced survival and attenuated intracranial generation of ccl5 (rantes), ccl2 (mcp-1) and il-6; levels of these cytokines are directly correlated with the severity of hsv-1 infection and the development of immunopathology [9, 12, 13, 14, 15] . therefore, survival from hsv-1 encephalitis is determined not only by lytic viral infection but also by the balance between pro-inflammatory and downregulatory anti-inflammatory mechanisms (as well as anti-viral signaling pathways) that limit the pro-inflammatory tlr2 response to infection. however, down-regulatory pathways that interface with tlr2 signaling are poorly described. the hsv-1 model of encephalitis is an ideal model to probe the role of how down-regulatory pathways impact tlr2 biology. mouse cd200r1 is a 326 amino acid glycoprotein expressed on the surface of myeloid and glial cells [16] . cd200r1 interacts with cd200, which is expressed on the surface of neurons, epithelial cells, endothelial cells, and lymphocytes [17, 18] . cd200r1 has a 67 amino acid intracellular domain with a unique inhibitory tyrosine motif. stimulation of cd200r1 by cd200 leads to the phosphorylation of these tyrosines, which recruit the adapter proteins docking proteins (doks) 2 and 1 and sh2 domain-containing phosphatase-1 (shp-1) to initiate inhibitory signaling and suppress cytokine and chemokine generation [19, 20, 21] . in contrast to cd200r1, the small cytoplasmic domain of cd200 lacks a signaling motif [16, 17] . the interaction of cd200 with cd200r1 down-regulates innate and adaptive immune responses in models of experimental autoimmune encephalitis (eae), arthritis, transplantation, and acute pulmonary inflammation [16, 17, 18, 22] . however, the specific pro-inflammatory signal transduction pathways in macrophages, dendritic cells, and mast cells that are down-regulated by cd200r1 [19, 20, 21] , are poorly described. specifically, how cd200r1 impacts tlr2 signaling, particularly in macrophages, is not known. studies of the role of cd200r1:cd200 interactions in determining the host response to pathogens are complex. to date, they have employed cd200 2/2 mice, limiting their scope to probing the entire cd200:cd200r1 axis and not capable of detecting other roles for cd200r1 in myeloid cells, i.e., intrinsic functions of cd200r1 independent of cd200 ligand interaction. in the case of influenza and meningococcus, cd200 2/2 mice are prone to the effects of inflammation/sepsis [18, 23] . this is also the case for toxoplasma encephalitis [24] . in regards to leishmania, redox defense mechanisms used by the host are suppressed by the cd200r1:cd200 axis in l. amazonensis, which induces cd200 expression in host cells, so that cd200 2/2 mice are protected from the pathogen. however l. major grows more slowly in macrophages and does not induce cd200 expression in host tissue [25] . more recently studies of mouse hepatitis corona virus [26] have shown that the release of inhibition by disrupting the cd200:cd200r1 axis leads to an augmentation of pro-inflammatory signaling and increased recruitment of leukocytes leading to the rapid clearance of virus. recently, the concept that inhibitory receptors can be multifunctional and may be required for pro-inflammatory signaling has emerged. for example, cd150 contains a txyxxv/i motif in its cytoplasmic tail that can bind sh2containing molecules, ranging from tyrosine and inositol phosphatases and src family kinases to adaptor molecules. the function of cd150 can vary depending on the molecule with which it interacts [27] . cd200r1 has neither a conventional itim nor an itsm domain, and the range of its intracellular functions is not known. to understand how cd200r1 signaling impacts tlr2 function, we generated cd200r1 2/2 mice. in vitro, we compared the ability of cd200r1 +/+ and cd200r1 2/2 macrophages to generate cytokines/chemokines in response to the tlr2 agonist pam 2 csk 4 lipopeptide and to hsv-1. cd200r1 2/2 macrophages showed a marked decrease in the generation of both il-6 and ccl5 (rantes) in response to stimulation by both pam 2 csk 4 and hsv-1, whereas no difference was observed in the generation of any cytokine/chemokine in response to the tlr4 ligand bacterial lipopolysaccharide (lps). cd200r1 2/2 macrophages also lacked the ability to up-regulate tlr2 expression in response to hsv-1 infection. cd200r1 2/2 embryonic fibroblasts and macrophages exhibited a defect in the replication of hsv-1. in vivo, we found that cd200r1 2/2 mice showed both markedly decreased morbidity and increased survival. cd200r1 2/2 and cd200r1 +/+ mice did not show a statistically significant difference in the brain inflammatory response. however, cd200r1 2/2 mice showed a marked decrease in intracranial hsv-1 titers and expression of hsv-1 envelope glycoproteins. cd200r1 2/2 showed a decrease in brain levels of ifn-b consistent with the lower viral titers. these results are distinct from those previously observed with tlr2 2/2 mice, and support a defect in the responses to hsv-1 that include an inability to support viral replication in the absence of cd200r1 in addition to a defect in tlr2 responses. this study was approved and performed in strict accordance with the guidelines set forth by both the university of massachusetts medical school department of animal medicine institutional animal care and use committee (iacuc) (assurance number a3306-01) and by the massachusetts general hospital (mgh) center for comparative medicine, subcommittee on research animal care (srac), which serves as the institutional animal care and use committee (iacuc) at mgh (assurance number a3596-01). mice were bred and maintained under specific-pathogen-free conditions at the animal facilities at both the university of massachusetts medical school and the massachusetts general hospital charlestown facility, and all efforts were made to minimize suffering. anti-cd200r1 and anti-cd200 for cell surface staining were purchased from abd serotec. goat anti-cd200r1 intracellular domain-specific antibody was from santa cruz. anti-il-1b was goat polyclonal anti-mouse il-1b (af-401-na; r&d systems). the primary antibody used in immunohistochemical studies was rabbit polyclonal anti-hsv2 (b0116; dako) and the secondary antibody used was biotin-conjugated goat anti-rabbit (h+l) igg (656140; invitrogen). the secondary anti-rabbit antibody used in blotting studies was rabbit anti-goat (h+l) igg hrp conjugate (172-1034; bio-rad). mice used were backcrossed to generation n9 or n10. to identify the knockout allele in genomic dna, tail vein dna was purified using the dneasy kit (qiagen). pcr was then performed using the forward primer neo1, located in the 59promoter region of the neo gene cassette (59-tgcgaggc-caggccacttgtgtagc-39) combined with the reverse primer cd200r1-rev, located outside the short arm of the knockout construct (59-gggatgcagaacataggaggcag-39) corresponding to a sequence within the first third of intron 1 of the cd200r1 gene. the pcr product yields a 1.5 kbp product. to identify the wild-type allele primer wt1 (59-cagtagttttggagaatgtgacag-39) was combined with wt-rev (59-gatagcccttgctcctatgactgag-39) to yield a 1.4 kbp product corresponding to a region of intron 1 that is present in wt dna but is deleted by insertion of the targeting construct. the pcr conditions for both sets of primers were: (95uc, 15 min; 94uc, 30 sec; 62uc, 1 min; 72uc 2 min; 35 cycles using thermo start reddymix pcr master mix (thermo scientific). exon-specific screening. the following forward and reverse primers were combined to probe the expression of cd200r1 exons in peritoneal macrophages: exon 1f (59-atgttttgcttttggagaact-39) and exon 7r (59-cta-gattccaatggccgacaa-39) to amplify the full length cdna; exon 1f (above) and exon 5r (59-cacctctact-cagttctatgg-39) to amplify exons 1-5; exon 5f (59-tgaagtaacctactttccaga-39) and exon 7r (above) to amplify exons 5-7. rna was prepared using rneasy (qiagen) and rt-pcr was performed using 1 unit taq polymerase, and q solution (qiagen). the pcr conditions for all pairs of primers were: (94uc, 20 sec; 62uc, 20 sec; 72uc 2 min; 35 cycles). mice were injected with 4% thioglycollate and peritoneal exudate cells were routinely harvested 3-4 days later [6] . to generate macrophages, peritoneal exudate cells were plated at 10 6 cells per well in 24-well plates in dmem containing 10% fcs. lipopolysaccharide (lps) was obtained from sigma and phenol extracted as previously described [28] . pam 2 csk 4 was obtained from emc microcollections (tubingen, germany). tissues were recovered from mice at necropsy, fixed in bouin's solution (sigma-aldrich), and embedded in paraffin. for routine histology, 5 mm sections were stained with hematoxylin and eosin. the sections were evaluated by a pathologist without knowledge of the experimental design. for immunohistochemistry, fixed sections from cd200r1 +/+ and cd200r1 2/2 mice were deparaffinized, antigen-unmasked using citric acid, probed with antibody to hsv antigens [6] , stained with biotinconjugated secondary antibody, and detected using avidin:biotin-complexed hrp. the localization of hrp-labeled ab was detected using 3,39-diaminobenzidine; hematoxylin was used as a counterstain. histological scoring of brains 48 hours post-infection, mice were anesthetized with isoflurane and euthanized by exsanguination. brains were dissected and stored in bouin's fixative formula (fisher scientific, pittsburgh, pa) for 24 h. each brain was cut into 4 coronal sections, paraffin embedded, sectioned, mounted and stained with hematoxylin and eosin (h&e) by the histology core at university of massachusetts medical school, worcester, ma. to quantify the severity and the extent of pathologic inflammation in the brains of hsv-1 infected mice, a histologic inflammation scoring system was used. each of seven regions of the brain was assigned an inflammation score of 0 (no apparent inflammation), 1 (minimal inflammation), or 2 (above minimal inflammation). inflammation was judged on the presence and extent of cellular infiltrate and reactive gliosis. the regions of the brain that were scored were: frontal cerebral cortex, posterior cerebral cortex, hippocampus, diencephalon/mesencephalon (which includes the thalamus, hypothalamus and brainstem), cerebellum, the caudal paraventricular structures (defined as those areas adjacent to the lateral ventricles), and the rostral paraventricular structures (defined as those areas adjacent to the 3 rd ventricle, the cerebral aqueduct, and the 4 th ventricle). each brain was scored blindly by two observers. the scores for each of the seven regions were summed to arrive at a total brain inflammation score for each mouse with a maximum possible score of 14. to analyze the generation of viral-specific cd8+ t cell generation, mice were infected intraperitoneally with 10 4 pfu of hsv-1. on days 5-9, 50 ml of peripheral blood was withdrawn from tail veins and cells were stained for cd8 antigen using an anti-cd8 apc-conjugated monoclonal antibody, and probed for binding of the ssiefarl-h2-k d -pe gb peptide-(aa 498-505) (gb 498-505 ) mhc pentamer [29] to identify hsv-1 specific cd8+ positive t cells [30] . the percentage of cd8 positive cells that were also positive for gb pentamer binding was determined by flow cytometric analysis. (all antibodies were from ebioscience and bd biosciences, san diego, ca, while hsv-1 gb + pentamer was from proimmune inc., bradenton, fl). cells were incubated with cd3/cd11b/cd8/gb + 498-505 pentamer and monoclonal antibodies following the manufacturer's protocol. stained cells were washed twice with facs buffer and fixed with bd cytofix/cytoperm solution for 20 min at 4uc. following fixation, the cells were washed twice in bd perm/wash buffer, resuspended in 4% paraformaldehyde, and analyzed using a multicolor five-laser lsr ii instrument (bd biosciences, san diego, ca). hsv-1 strains including viruses expressing icp8-gfp [31] were generated in the laboratory of dr. david knipe. viruses were added to mefs and peritoneal macrophages at an m.o.i. of 10:1. an unpaired, two-tailed student's t-test or a kruskal wallis test was used to determine statistical significance where indicated. statistics were performed using graphpad (prism v5.0d) software. values of p,0.05 were considered significant. we developed a gene targeting strategy to delete the expression of the entire cd200r1 protein including the cytoplasmic tail and transmembrane region ( figure s1 ) to prevent the formation of a truncation mutant that could function as a dominant negative, potentially disrupting the interaction of other signaling molecules. this strategy was therefore distinct from that of a previously generated cd200r1 2/2 mouse in which only exons 2-4 (the extracellular domain) were targeted [32] . the targeting vector ( figure s1a ) was constructed by using a 1.3 kbp dna fragment as the short arm. the long arm was a 10 kbp genomic fragment, which starts from ecorv to spei. in this strategy, 1.2 kbp upstream of the atg start codon, exon 1, and 2.9 kbp of intron 1 were replaced by the neo gene cassette, resulting in a frame shift mutation and the generation of stop codons in each of the 3 reading frames. to confirm successful gene targeting, dna was prepared from tail veins and analyzed by both pcr and southern blot. for southern blotting, tail vein genomic dna was digested with nsi1, resolved by 0.7% agarose gels, transferred to nitrocellulose membranes, and then probed with a 550 bp genomic fragment localized within intron 1 to distinguish targeted and cd200r1 +/+ dna. as shown in figure s1b , dna from cd200r1 +/+ mice gave a predicted single band of 7.8 kbp. cd200r1 2/2 dna showed a single band of the predicted size of 2.7 kbp. both bands were detected in dna from heterozygote mice. when analyzed by pcr, tail vein dna of cd200r1 2/2 mice showed a characteristic 1.5 kbp band that was absent in cd200r1 +/+ dna ( figure s1c ), whereas cd200r1 +/+ tail vein dna showed only a 1.4 kbp band. dna from heterozygous mice showed both bands. finally, when analyzed by flow cytometry, cd200r1 +/+ , but not cd200r1 2/2 elicited peritoneal macrophages expressed cd200r1 ( figure s1d ). hsv-1 initiates cytokine and chemokine synthesis in the brain via stimulation of tlr2 expressed on macrophages, which are derived from peripheral blood monocytes [6, 7, 8, 9] , as well as on glial and microglial cells. we therefore examined the interaction of hsv-1 with cd200r1 +/+ and cd200r1 2/2 macrophages. elicited macrophages from both cd200r1 +/+ and cd200r1 2/ 2 mice were infected with hsv-1 (multiplicity of infection; moi = 10), or challenged with pam 2 csk 4 (100 ng/ml), or lps (100 ng/ml), and levels of il-6, ccl5 (rantes), or ccl2 (mcp-1) were measured at 24, 48, and 72 h. hsv-1 infected cd200r1 +/+ macrophages produced 35,000 pg/ml il-6 24 h after stimulation, which rose to 60,000 pg/ml by 48 h and 72 h ( figure 1a ). in contrast, cd200r1 2/2 macrophages only generated between 20,000 to 30,000 pg/ml il-6 in response to hsv-1 stimulation. pam 2 csk 4 stimulated il-6 levels averaged 70,000 to 80,000 pg/ ml over the 3 day experiment in cd200r1 +/+ cells, whereas cd200r1 2/2 macrophages generated only 10,000 to 20,000 pg/ ml. similar to the il-6 response, the generation of ccl5 (rantes) by cd200r1 2/2 macrophages stimulated with hsv-1 was blunted when compared to cd200r1 +/+ macrophages. to address whether the decrease in ccl5 (rantes) was a property of signaling by tlr2, or secondary to low levels of ifn, we determined whether cd200r1 2/2 peritoneal macrophages exhibit a blunted ccl5 (rantes) response to stimulation with the tlr4 ligand lps. in contrast to the attenuated response to tlr2 stimulation, there was only a modest decrease in the levels of il-6 at 24 h and 48 h and no difference in the levels of ccl5 (rantes) generated in response to tlr4 stimulation via lps in cd200r1 +/+ or cd200r1 2/2 cells at any time point ( figure 1a ). in the case of ccl2 (mcp-1) generation, no difference was found between cd200r1 2/2 and cd200r1 +/+ with any stimulant; though lps induced a marked increase in ccl2 (mcp-1) expression in cd200r1 2/2 cells. the formation of mature il-1b requires two distinct steps [33, 34] . the first signal induces the generation of pro-il-1b. expression of pro-il-1b is regulated at the transcriptional level by nf-kb, which, for hsv-1, is activated downstream of tlr2 signaling. the second signal activates assembly of the inflammasome complex and cleavage of pro-il-1b into its mature secreted form, il-1b. this can be triggered by a variety of mechanisms, including extracellular atp acting on the p2x receptor, reactive oxygen species, or potassium efflux triggered by the antibiotic nigericin [35] . the interaction of double stranded dna and viral replication intermediates with intracellular dna sensors can also induce inflammasome assembly [3, 36, 37, 38, 39, 40] . the assembly and function of the inflammasome is therefore dependent on the quantity of viral replication intermediates within cells. we therefore hypothesized that if cd200r1 was required for efficient viral replication, then cd200r1 2/2 macrophages should show lower levels of inflammasome formation and impaired conversion of pro-il-1b to il-1b. stimulation of cd200r1 2/2 peritoneal or bone marrowderived macrophages with hsv-1 led to the production of approximately 20% of the mature il-1b levels seen in cd200r1 +/+ cells ( figure 1b) . in contrast, no difference was found in mature il-1b levels between cd200r1 +/+ or cd200r1 2/2 macrophages stimulated with lps (100 ng/ml) for 3 h followed by the addition of atp (1 mm) for 1 h. these results suggested that that there was no intrinsic defect in inflammasome formation or in the capacity of cd200r1 2/2 macrophages to generate pro-il-1b mrna while virus-induced inflammasome formation was significantly attenuated in cd200r1 2/2 macrophages. the difference in the production of mature il-1b between cd200r1 +/+ and cd200r1 2/2 macrophages following hsv-1 infection were confirmed by western blot ( figure 1c ). because of the key role of tlr2 in mediating the response of macrophages to hsv-1 [6, 9] , we examined the relationship between tlr2 expression on the surface of peritoneal macrophages, cd200r1 expression, and hsv-1 infection. the expression of cd200r1 on the surface of wt and tlr2 2/2 elicited macrophages was determined by flow cytometry 24 h after infection with hsv-1 ( figure 2a) . as an internal control, cd200r1 2/2 cells were also included. there was no difference in cd200r1 expression levels between tlr2 +/+ and tlr2 2/2 macrophages. interestingly, the surface expression of cd200r1 decreased 24 h after hsv-1 infection in both wt and tlr2 2/2 macrophages (figure 2a) . this was contrasted with the effect of cd200r1 on tlr2 expression. following hsv-1 infection, tlr2 expression levels increased 12-fold in wt cells, but tlr2 expression was not increased in cd200r1 2/2 macrophages ( figure 2b ). these results indicated that cd200r1 directly controls the inducible surface expression of tlr2 either directly or secondary to decreased viral replication and that it may play a role in the amplification of hsv-1 infection. cd200r1 2/2 mice are protected from intracranial hsv-1 infection the cd200r1:cd200 axis is known to down-regulate intracranial inflammation, typified by studies of the eae model [17, 18] . because morbidity and mortality to hsv-1 infection is secondary to increased brain inflammation (as well as lytic virus replication), we predicted that cd200r1 2/2 mice would have a marked increase in both brain inflammation and mortality when compared to cd200r1 +/+ mice. to examine the effect of the cd200r1:cd200 axis in the intracranial inflammation associated with hsv-1 infection, cd200r1 +/+ and cd200r1 2/2 mice were infected with 10 5 pfu of hsv-1 by intracranial injection, and the survival in each group was followed daily for 15 days. cd200r1 +/ + mice showed a characteristic survival curve (figure 3) , with 40% of the mice surviving by day 6 and 26% by day 9. no further mortality after day 9 was observed. in contrast, cd200r1 2/2 mice exhibited significantly higher survival rates, with 75% of the mice surviving on day 6, and no further deaths within the course of the experiment. cytokine/chemokine levels in the brains of cd200r1 +/+ and cd200r1 2/2 mice is indistinguishable after intracranial hsv-1 infection to explain these apparently paradoxical results, we examined a series of parameters that define intracranial inflammation associated with hsv-1 infection. cd200r1 +/+ and cd200r1 2/ 2 mice were infected with hsv-1 and their brains were harvested and analyzed to assess inflammation 1 and 4 days post-infection. brains were homogenized and intracranial levels of three cytokines known to be associated with and/or causative for hsv-1 mediated brain inflammation and that are also linked with the outcome of hsv-1 encephalitis, il-6, ccl5 (rantes) and ccl2 (mcp-1) [6, 9, 12, 13, 14, 15] were measured. levels of il-6 are known to correlate with the level of viral infection [12, 15] , and il-6 2/2 mice have more severe hsv-1 encephalitis, with increased morbidity and death than il-6 +/+ mice [12] . levels of il-6 were statistically indistinguishable between cd200r1 +/+ and cd200r1 2/2 brains on day 1, and were essentially at baseline levels by day 4 ( figure 4a ). similarly, ccl5 (rantes) and ccl2 (mcp-1) levels were not statistically different between cd200r1 +/ + and cd200r1 2/2 brains on day 1 or day 4 ( figures 4b and 4c) , though the differences in rantes (ccl5) and mcp-1 (ccl2) on day 4 were trending towards significance with p-values of p = 0.09 and p = 0.07, respectively. during the initial phase of hsv-1 disease, viral replication is controlled to a large extent by the production of type i ifn and the expression of ifn-response genes [9, 41, 42, 43, 44, 45, 46] . therefore, we determined levels of ifn-b in the brains of cd200r1 2/2 and cd200r1 +/+ mice following intracranial hsv-1 infection. on day 1, cd200r1 2/2 brains had significantly lower levels of ifn-b than cd200r1 +/+ mice ( figure 4d ). on day 4, the levels ifn-b in cd200r1 2/2 remained statistically lower than cd200r1 +/+ mice, though both exhibited a decrease from day 1 levels. in addition, serum ifn-b levels in cd200r 2/2 mice were 76% lower compared to the levels found in cd200r1 +/+ mice. cd200r1 2/2 mice have lower brain viral titers than cd200r1 +/+ mice no difference in gross pathology between cd200r1 +/+ and cd200r1 2/2 brains was observed on day 3 post hsv-1 infection ( figure 5a ). leukocyte infiltration was assessed in 7 regions of the brain and each region received a score of 0 (no leukocytes), 1 (rare or occasional leukocyte infiltration) or 2 (marked leukocyte infiltration). total scores were determined in a blinded manner by 2 observers. both cd200r1 +/+ and cd200r1 2/2 brains showed indistinguishable degrees of leukocyte infiltration in both regional and compiled pathologic scores ( figure 5b ). the lack of difference in brain leukocyte scores, and in ccl2 (mcp-1), ccl5 (rantes), and il-6 levels indicated that cd200r1 2/2 mice had equivalent or modestly reduced brain inflammation, rather than an enhanced, inflammatory response to hsv-1 infection compared to cd200r1 +/+ mice (see above). when combined with the observation that cd200r1 2/2 mice had lower levels of ifn-b in brain and serum, the results suggest that there is decreased replication of hsv-1 in cd200r1 2/2 brains. to test this conclusion, cd200r1 +/+ and cd200r1 2/2 mice were infected intracranially with 10 5 pfu hsv-1, and virus titers in whole brain homogenates were determined by plaque assay using vero cells on days 1, 3, and 4 post-infection ( figure 5c ). on day 1, brain titers were comparable between cd200r1 +/+ and cd200r1 2/2 mice. though viral titers figure 1 . cd200r1 licenses pro-inflammatory signaling in peritoneal macrophages. (a) cd200r1 +/+ (wt) or cd200r1 2/2 (ko) elicited peritoneal macrophages were stimulated with either hsv-1 (moi 10:1, left column), pam 2 csk 4 (100 ng/ml, center column), lps (100 ng/ml, right column), or medium for 24, 48, or 72 h. il-6 (upper row, pg/ml), ccl5/rantes (middle row, pg/ml), and ccl2/mcp1 (lower row, pg/ml) levels were measured by elisa. data shown are mean and sd of representative experiment (total of 3 experiments, n = 4, wt and ko). an unpaired, two tailed student's t-test was used to determine statistical significance of independent experiments, p values; * p,0.05, { p,0.01. (b) supernatant il-1b levels from wt and ko elicited peritoneal macrophages (left panels) or bone marrow macrophages (right panels) 16 h after addition of hsv-1 (moi 10:1) or after a 3 h stimulation with lps (100 ng/ml) followed by atp (1 mm increased in both mice on day 2, cd200r1 2/2 mice exhibited significantly lower titers than cd200r1 +/+ mice (not shown). hsv-1 titers were negligible in cd200r1 2/2 brains by day 3, with 60% of cd200r1 2/2 mice having brain titers that were below the limit of detection. in contrast, hsv-1 titers from cd200r1 +/+ brains were unchanged from day 1 to day 3. on day 4, viral titers from cd200r1 +/+ brains had dropped slightly compared to their titers on day 3 but this was not statistically different. cd200r1 2/2 mice, however, continued to have a high percentage of animals (42%) that had completely cleared the infection. overall, the hsv-1 titers in cd200r1 2/2 mice were lower compared to cd200r1 +/+ mice on day 4 but the difference in titers did not reach statistical significance. importantly, approximately 50% of the cd200r1 2/2 mice had cleared the infection by days 3 and 4, while hsv-1 continued to replicate at high levels in 100% of cd200r1 +/+ mice. to assess the extent and distribution of hsv-1 infection in cd200r1 2/2 and cd200r1 +/+ brains, we determined the expression of hsv-1 viral antigens by immunohistochemical staining 3 days post-intracranial infection. hsv-1 antigens were detected in a large percentage of neurons in and around the hippocampus of cd200r1 +/+ mice ( figure 5d ). in contrast, cd200r1 2/2 mice had much less extensive neuron involvement with fewer hsv-1 positive neurons in the hippocampal region. in response to hsv-1 infection, the host generates cd8 + cells that recognize the hsv-gb protein. the hsv-1-specific cd8+ response is dependent on the viral load [29, 30] . a majority of the cd8+ cells are directed to a single dominant gb498-505 peptide and will specifically bind a mhc-gb-peptide-pentamer that can be monitored by flow cytometry [29, 30, 47, 48, 49] . cd200r1 +/+ and cd200r1 2/2 mice were infected with 10 4 pfu hsv-1 by intraperitoneal injection. the development of hsv-gb pentamerspecific cd8 + peripheral blood t cells was monitored up to 9 days post-infection ( figure 5e ). in cd200r1 +/+ mice, the percentage of gb-pentamer positive cells peaked 7 days after infection at approximately 14.4%, which decreased by half by day 9 ( figure 5e ). in contrast, cd200r1 2/2 mice showed a blunted and delayed response that gradually rose to approximately 6.5% by day 9. this blunted cd8+ response is consistent with reduced hsv-1 replication in cd200r1 2/2 mice and provide additional evidence that cd200r1 2/2 mice either more efficiently eliminate hsv-1 or lack the ability to support its sustained replication. to explore the role of cd200r1 in supporting viral replication, we generated mouse embryonic fibroblasts (mefs) from cd200r1 +/+ and cd200r1 2/2 mice and infected them with an hsv-1 strain that expressed a green fluorescent protein (gfp) fusion protein of the delayed early gene icp8 (icp8-gfp). we monitored the expression of gfp by flow cytometry for up to 20 h after infection. the expression of icp8-gfp peaked in cd200r1 +/+ cells at 12 h after infection, with over 40% of the cells expressing gfp ( figure 6a ). in contrast, cd200r1 2/2 mefs expressed less than half this amount. we then tested the ability of cd200r1 2/2 and cd200r1 +/+ peritoneal macrophages to be infected with hsv. macrophages were infected with icp8-gfp expressing hsv-1 and gfp expression was monitored for up to 144 h. icp8-gfp expression peaked at 24 h, at which time 10.8% of cd200r1 +/+ macrophages expressed icp-gfp. in contrast, only 4.4% of cd200r1 2/2 macrophages expressed icp8-gfp ( figure 6b ). this was consistent with the results seen in mefs. thus hsv-1 infection was attenuated both in cd200r1 2/2 macrophages and fibroblasts. we confirmed that both elicited peritoneal macrophages and mefs expressed cd200r1 by immunofluorescence ( figure 6c ). furthermore, neither macrophages nor mefs express cd200 message or protein (not shown); therefore the impact of cd200r1 on hsv-1 infection was independent of cd200r1:cd200 interaction but was dependent on cd200r1 expression itself. overall, our findings indicate that cd200r1 plays an important intrinsic role in regulating tlr2 expression and signaling. in a tlr2-dependent viral model like such as hsv-1 infection, lack of cd200r1 expression leads to decreased inflammatory cytokine/chemokine production as well as a failure to upregulate tlr2, thus suggesting a link between tlr2 signaling and decreased viral titers. the cd200r1:cd200 axis has traditionally been considered to be down-regulatory in inflammatory settings. in models of eae, toxoplasma encephalitis, experimental autoimmune retinitis, collagen-induced arthritis and influenza-induced pneumonitis, cd200 2/2 mice show enhanced inflammatory responses [17, 18, 24] . in the case of mouse hepatitis corona virus, the increased survival was attributed to release of inhibition, increased inflammation, and increased viral clearance. therefore, the dramatic increase in survival of cd200r1 2/2 mice compared to cd200r1 +/+ mice in response to intracranial hsv-1 appears to be paradoxical, or potentially explained by the same process. though a large component of the mortality seen in hsv-1 encephalitis in c57bl/6 wt mice (with an intact type i ifn response) is secondary to the inflammatory response [6, 8, 9] , our results emphasize that viral replication, as opposed to inflammation, can also be a key component of the lethal effects of hsv-1 encephalitis in mice. this was supported by the observation that no difference was found between brain chemokine/cytokines levels (with the exception of interferon-b, a marker for viral load) or in leukocyte recruitment into the brains of cd200r1 +/+ and cd200r1 2/2 mice. in contrast, our data strongly support a requisite role for cd200r1 in supporting the replication of hsv-1, and implicate decreased viral replication as the reason for increased viability of cd200r1 2/2 mice. thus, although brain titers of hsv-1 in cd200r1 +/+ and cd200r1 2/2 mice were similar on day 1 post-infection, sustained virus infection was found only in wild type and not in knockout mice. a large proportion of cd200r1 2/2 mice were essentially free of replicating virus by day 3-4 post-infection in contrast to cd200r1 +/+ mice which continued to produce infectious hsv-1 virions in their brains over the entire 4 day course of the study. our results demonstrate decreased hsv-1 viral titers (in many cases below the limit of detection) and reduced immunohistochemical detection of hsv-1 in the brains of cd200r1 2/2 mice. consistent with the reduced levels of brain infection in cd200r1 2/2 mice we found decreased interferon levels in the brains of cd200r1 2/2 compared to cd200r1 +/+ mice. the decreased ability of hsv-1 to infect and/or replicate in tissues outside the brain in cd200r1 2/2 mice was supported by the blunted hsv-1-specific cd8+ t cell response to intraperitoneal hsv-1 infection. finally, a role for cd200r1 in supporting viral replication was found by comparing elicited peritoneal macrophages and mefs from cd200r1 +/+ and cd200r1 2/2 mice, in which viral infection was decreased in cd200r1 2/2 cells. a unique role for cd200r1 in supporting (licensing) proinflammatory signaling by tlr2 was also found. this role was revealed by studies with elicited peritoneal macrophages in which the generation of il-6 and ccl5 (rantes) in cd200r1 2/2 macrophages was blunted by ,80% in response to tlr2 ligands and hsv-1; this was not observed in response to lps suggesting a specific defect in tlr2-driven responses. furthermore, cd200r1 2/2 macrophages lacked the ability to assemble a functional inflammasome. it is unclear why il-6 and ccl5 (rantes) but not mcp-1 levels were reduced in cd200r1 2/2 macrophages. it may be that there is differential regulation of il-6 and ccl2 (mcp-1) generation via tlr2 signaling. although these cytokines are both nf-bb driven, there are distinct transcription factors that drive expression of il-6 or mcp-1. in previous studies we have noted that hsv-induced il-6 is strictly dependent on tlr2 expression while some mcp-1 secretion still occurs in tlr2 2/2 cells, albeit at reduced levels compared to tlr2 +/+ macrophages [9] . currently, the mechanism(s) are not known. it is also possible that cd200r1 regulates il-6 and ccl2 (mcp-1) differently. it is also unclear why the expression of ccl5 (rantes) but not ccl2 (mcp-1) is decreased in cd200r1 2/2 mice. it is possible that ccl5 (rantes) secretion is secondary to decreased ifn, though ultimately this also is a consequence of the control of tlr2 signaling by cd200r1. an additional defect in tlr2 function, the inability to up-regulate the expression of tlr2 in response to hsv-1 infection, may contribute to the inability to perpetuate and amplify hsv-1 infection, though this may be secondary to the role of cd200r1 in regulating viral replication. the role of cd200r1:cd200 interaction in determining the host response to pathogens is complex, and, when considered in the context of our current studies, is likely to be dependent on the mechanisms that the host uses to kill the specific pathogen. in the case of influenza and meningococcus, cd200 2/2 mice are prone to the effects of inflammation/sepsis [18, 23] , indicating that cd200r1:cd200 interaction is not likely to be involved in the regulation of cellular anti-viral mechanisms. as described above, this is also the case for toxoplasma encephalitis [24] . in regards to leishmania, redox defense mechanisms used by the host are suppressed by the cd200r1:cd200 axis in l. amazonensis, which induces cd200 expression in host cells, so that cd200 2/2 mice are protected from the pathogen. however l. major grows more slowly in macrophages and does not induce cd200 expression in host tissue [25] . in the case of hsv-1 and other members of the herpesviridae family, the situation is somewhat analogous, but is more complex and involves a clearly distinct mechanism. in hsv-1, cd200r1 supports infection at or before the stage of early gene expression, which in turn regulates the surface expression of tlr2. whether additional cd200r1-independent or cd200r1dependent (intrinsic) intracellular mechanisms controlling viral replication are involved are not yet known, but would not be surprising. the decreased infection of hsv-1 at all time points in cd200r1 2/2 mefs as well as macrophages indicates that the situation is more complex than simply the suppression of a virustoxic metabolite such as no, which can be driven or amplified by tlr2. the effect of cd200r1 on hsv-1 infection in mefs and macrophages occurred in the absence cd200 expression suggesting that cd200r1 intrinsically regulates virus replication. because hsv-1 is a neurotropic virus and neurons express cd200 but not cd200r1 on their surface [50, 51] , how the macrophage-specific mechanisms elucidated by our work function to exert their profound impact on hsv-1 replication and host survival in vivo are not straightforward. even though macrophages and dendritic cells are relatively resistant to hsv-1 infection [52] , our data indicate that they have the potential to be infected, and therefore could be important in mediating intraneuronal transmission or to serve as a reservoir of virus. as cd200r1 2/2 macrophages would be deficient in these processes, the lack of cd200r1 could potentially slow the spread of virus within the brain. alternatively or concomitantly, cd200r1 +/+ macrophages may generate a factor that is required to sustain viral replication in neurons, or cd200r1 2/2 macrophages may contribute to increased viral killing. furthermore, how the impact of cd200r1 on tlr2 signaling affects viral replication in vivo is not fully elucidated. hsv-1 is known to target components of the tlr2 signaling pathway via expression of the viral protein icp0, suggesting that tlr2 biology and hsv-1 biology are linked [53] . the selective requirement for the induction of il-6 and ccl5 (rantes) expression by tlr2 ligands and hsv-1 is consistent with the emerging concept that inhibitory receptors can be multifunctional and be required for pro-inflammatory signaling. cd150 contains a txyxxv/i motif in its cytoplasmic tail that can bind sh2-containing molecules, ranging from tyrosine and inositol phosphatases and src family kinases to adaptor molecules. the function of cd150 can vary depending on the molecule with which it interacts [27] . cd200r1 has neither a conventional itim nor an itsm domain, and the range of potential interactions with its cytoplasmic tail is not known. another example of a dual role for inhibitory receptors is the fact that for their activation receptors to become activated, nk cell inhibitory receptors must first interact with their cognate self-mhc ligands [54] . in fact, the role of cd200r1 in supporting the synthesis of il-6 and ccl5 (rantes) may not be direct. the blunted generation of il-6 and ccl5 (rantes) was determined after 24 h, so signaling by cd200r1 could impact the synthesis of other cytokines, which in turn regulate the generation of il-6 and ccl5 (rantes). however, the role of cd200r1 in regulating the surface expression of tlr2 expression suggests a direct link between these pathways. figure s1 gene targeting of cd200r1. (a) development of the targeting construct. (b) southern blot of tail vein dna from cd200r1 2/2 (2/2), cd200r1 +/+ (+/+), and cd200r1 +/2 (+/ 2) mice. the 7.8 kbp band indicates the wt allele, whereas the 2.8 kbp band is diagnostic of a gene-targeted allele. (c) pcr analysis of tail vein dna from cd200r1 2/2 (2/2), cd200r1 +/ + (+/+), and cd200r1 +/2 (+/2) mice. the gene-targeted allele is represented by a 1.5 kbp band and the wt allele is represented by a 1.4 kbp band. (d) flow cytometric analysis of elicited peritoneal macrophages from cd200r1 +/+ (wt) and cd200r1 2/2 (ko) macrophages probed for the expression of cd200r1. 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immune inhibitory molecules cd200 and cd200r in the normal central nervous system and multiple sclerosis lesions suggests neuron-glia and glia-glia interactions the immune inhibitory complex cd200/cd200r is developmentally regulated in the mouse brain a role for the jak-stat1 pathway in blocking replication of hsv-1 in dendritic cells and macrophages herpes simplex virus immediate-early icp0 protein inhibits toll-like receptor 2-dependent inflammatory responses and nf-kappab signaling alternative cross-priming through ccl17-ccr4-mediated attraction of ctls toward nkt cell-licensed dcs the authors would like to thank c. stark for technical assistance with screening cd200r1 2/2 mice and animal husbandry. key: cord-324359-88vy3dre authors: kamara, foday mamoud; mokuwa, esther yei; richards, paul title: how villagers in central sierra leone understand infection risks under threat of covid-19 date: 2020-06-24 journal: plos one doi: 10.1371/journal.pone.0235108 sha: doc_id: 324359 cord_uid: 88vy3dre background: concern has been expressed over how well africa is prepared to cope with the pandemic of covid-19. will rural populations with low levels of education know how to apply community-based infection control? we undertook fieldwork in two villages in central sierra leone to gain insight into how rural people faced with covid-19 assess epidemic infection risks. methods: two communities were selected based on prior contrasted exposure to ebola virus disease–one with substantial number of cases and the other having resisted infection through strong community sequestration measures. we assessed understanding of infection risks via an experimental game. this asked players to express a preference for one of two diseases, one resembling ebola with lower risk of infection and the other resembling covid-19 with lower risk of death. players were not told the identity of the diseases. results: in total 107 adult villagers played the game (58% women). half (52%) preferred the disease model with lower risk of infection, 29% preferred the model with lower risk of death, while 21% saw the combined risk of infection and death as being equivalent. differences in reactions between the two locations were small despite different experiences of ebola. asked to explain their choices 48% of players cited information on infection risks modelled by the game and 31% stated that their choices reflected awareness of the need for personal action and respect for local regulations. we concluded that villagers thoughtfully assess disease risks and that some are good intuitive statisticians. conclusions: results suggest rural people in sierra leone retain the lessons of experience from the ebola outbreak of 2014–15 and will be able to apply these lessons to a new infectious disease for which have no prior practical experience. our expectation is that rural populations will understand covid-19 control measures, thus reducing need for draconian enforcement. concern has been expressed in media debate about the potential damage that could be caused by the spread of covid-19 in africa on account of weak health systems and lower levels of education. in particular, there has been some speculation about capacity to apply epidemiological concepts. social distancing, for example, demands not only physical separation but also an appreciation of the need to reduce chances of social contacts. to contribute to a better appreciation of these concerns this paper addresses the question of how well rural african populations understand and apply information relating to risks associated with infectious diseases. the data relate to rural populations in sierra leone exposed both to ebola virus disease (evd) in 2014-15 and now to covid-19 in 2020. numerical ability among people without formal education has been the subject of some research enquiry by anthropologists and others. by and large these studies have demonstrated that alleged computational handicaps among non-literate populations were more apparent than real. gay and cole found that non-literate rice farmers were better able to estimate volumes than us college graduates deployed by the peace corps to teach mathematics in liberian schools [1] . lave documented the truly astonishing capacities of tailors in monrovia to cut garments to fit varying body sizes and shapes without use of patterns [2] . the emergence and species-wide distribution of basic statistical capacities is a topic addressed by evolutionary anthropologists. in an influential contribution, cosmides and tooby conclude that the view most common in the literature on judgment under uncertaintythat our inductive reasoning mechanisms do not embody a calculus of probability-will have to be re-examined. from an ecological and evolutionary perspective, humans may turn out to be good intuitive statisticians after all [3] . broadly speaking, it has been assumed that popular decision-making regarding hazards is undertaken under uncertainty, guided by common-sense heuristics [4, 5] . here, we attempt to assess whether or not african villagers conceptualise decisions regarding infection in terms of risks. our paper offers provisional findings on this topic, based on data gathered in two ebolaaffected rural communities in central sierra leone immediately prior to the 3-day lockdown for covid-19 in the first week of april 2020. we collected data using a rapid action research approach. two communities were chosen because our research team was well-known in these locations through work on impact of evd [6] , and because we had ethical approval for ethnographic and survey work on pandemic preparedness provided by the institutional review board of njala university. this meant we could implement fieldwork quickly to provide information on how well covid-19 prevention strategies were likely to work in these settings. for data collection we used a simple game or test devised to encourage villagers to talk comparatively about infection risks. each iteration of the game took about 15 minutes to complete, including time taken to explain the game, and for the person tested briefly to explain their choices. in each village we first held a public consultation with the chief, explaining that the purpose of our visit was to run a small game, and seeking permission to proceed. we also explained that we would return to explain the results of the game when results were processed (see below). sampling was opportunist. research assistants (ras) approached all adult male or female villagers present, explained the game and asked who would like to play. willingness to play constituted informed consent. this was witnessed by the field supervisor and the community chiefs, since a majority of participants do not read or write. for the larger of the two villages (both are here identified by pseudonyms) we have a baseline census undertaken in mid-2019 as part of a larger research project on pandemic preparedness, so were able to check the biases inherent in this mode of sampling. from this, we know the sample under-represents adult males (= 41%). many of the absentee males were busy clearing rice farms at this time. the two communities were contrasted in terms of their exposure to evd in 2014-15. one village (taninihun, popn. c. 250) had a substantial number of cases early in the 2014-15 epidemic [6] . the other community (tawoveihun, popn. c. 850) was located in a chiefdom (lowest level of the hierarchy of local government administration) which prevented evd infection throughout the 2014-15 epidemic through rigorously enforced community lockdown. both communities were exposed to information on covid-19, mainly by radio broadcasting. at the date of writing (early may 2020) no cases had been reported as testing positive for covid-19 in the two districts concerned (bo and moyamba). altogether 72 iterations of the game were carried out in tawoveihun and 35 iterations in taninihun (labelled "village 2a" in [6] ). this represents approximately one fifth of the total adult population of each village. in every run of the game the researcher (a trained mende-speaking research assistant) lays out two sets of twenty stones or pebbles in two ranks of ten stones. each set of stones, represented by the leaf of an orange and a mango tree, two common fruit trees in every mende village, represents one of two epidemic disease. these diseases are referred to throughout as "orange" and "mango". neither pattern of stones was identified explicitly as a proxy for covid-19 or ebola, though judging by the comments (see s1 data) some players may have guessed the implied identities. for each disease players are told the first rank of stones represented the chance of being infected and of not being infected. for "orange" this is 5 stones (infected) and 5 stones (not infected). for "mango" this is 1 stone (infected) and 9 stones (not infected). the second rank of ten stones represents the risk of dying from or surviving the disease. for "orange" this is 1 stone (death) and 9 stones (recovery), and for "mango" this is 5 stones (death) and 5 stones (recovery). these values only roughly match the infection and death risks associated with covid-19 and ebola. more stones would have achieved a better match, but we judged that this might make the game more difficult for the players. nevertheless, the two patterns capture a major contrast between the two diseases, that with evd there are few infections but more of those infections prove deadly, and that with covid-19 there are many infections, but fewer are deadly. it will also be realised, from the set-up of the game, that we deliberately chose to set the combined probability of infection and death as the same in both cases (p = 0.05). again, this was not pointed out to villagers. instead, ras were instructed carefully to note down any player who reported this equivalence, but without commenting further, to ensure that if other players arrived at the same conclusion it was not because it had been endorsed by our field team within their hearing. our aim was to find out if there was any intuitive grasp of the multiplication rule for independent probabilities. players were invited to explain which disease they thought was preferable, and why. iterations of the game were documented in field notebooks against the name and gender of the player, together with each player's brief comment on the game or their choices. field notebooks were then photographed and sent for data analysis to the principal investigator in the netherlands via whatsapp (an encrypted connection). the anonymised spreadsheet version is made available in the s1 data. overall (table 1) , there was a higher preference (52% of all responses) for "mango" (modelling evd). disease "orange" (modelling covid-19) attracted just over a quarter (27%) of all responses. players finding no difference between the two disease models accounted for 21% of all responses. gender differences in preferences ( table 2) were not statistically significant. in tawoveihun, "orange" is chosen more often by women than by men (table 3 ), but the gender difference is not statistically significant. in taninihun "mango" is chosen more often by women than men (table 4) . again, the difference is not statistically significant. three kinds of responses (table 5 , 89% of all remarks) dominated comments after people made choices in the game-need for rules governing e.g. quarantine, comments on personal capacities to prevent or avoid infection (often made in the context of justifying a choice for disease "mango"), and comments about the relative risks to be inferred from "reading the stones" (i.e. recognition of a pattern in the way the stones were cast, as in divination practices). the largest group of comments (48 per cent) related to what can be learnt from the game itself (table 5) . within this set, 25 comments specifically mentioned the distribution of the stones in the game (s1 data). the second largest group of comments (31 per cent) related to steps that can be taken to avoid or prevent infection (table 5 ). ebola virus disease is a terrifying disease. thus, it seems at first sight paradoxical that villagers should consistently express a preference for "mango", the disease with a risk profile in the game similar to evd. this preference is consistent across the two sites and genders. our suggested explanation relates to recent experience. ebola had a low risk of spread once infection control measures were stringently applied. this was well understood in both villages. in tawoveihun the chiefs led a community-based quarantine initiative and no cases occurred. in taninihun there was a devastating outbreak caused by a funeral for a respected islamic teacher as a result of which many of his pupils died. enforced quarantine was a very painful experience, but people later realised this action had ended the outbreak. it seems clear from comments given to explain preferences that prospects for infection control had considerable weight. choices were related to preventive measures or local byelaws in 42 per cent of comments (table 5 and s1 data). one player from tawoveihun made the link between community action and avoidance of infection explicit by commenting that with covid-19 "we will survive through rules and regulations like we did during the ebola crisis" (female, tawoveihun, our emphasis). perhaps disease preferences also reflected lack of confidence in the possibility of cure. neither local nor hospital treatments were effective for evd. at the outset, only 30 per cent or so patients recovered, and even in the later stages, survival was hardly better than 60 per cent. whether someone sick with the disease lived or died was more a matter for god. for this reason, it may have seemed better to pick the option with the lower risk of infection in the first place and stick to the rules. nevertheless, a considerable minority (27 per cent) preferred disease "orange" (with a covid-19-like profile). these players mention the lower death rate. some appear to have had the evident awfulness of death from ebola in mind when rejecting the "mango" option. one man in tawoveihun rationalised his choice for disease "orange" with the comment that "some sicknesses are worse than death" (s1 data). just over one fifth of players (21 per cent) considered that there was no difference in ultimate outcome from the two disease profiles. comments included that "the diseases are the same" (female, taninihun), "[both] seem the same and both are bad, except god helps you" (female, tawoveihun) and "i fear both because of the poverty of africa" (male, tawoveihun). a large number of comments suggested that players were taking specific note of information conveyed in the distribution of the stones. some players were explicit in relating their preferences to numerical weights-for example "nine chances of not being infected" (s1 data). if there were intuitive probabilists among the players of the game we expect these to be among the 21 per cent who decided (correctly) that the combined probabilities of infection and death were the same in both models. this suggests a grasp of the multiplication rule for probability, even though players lacked much (or any) formal schooling. we did not collect data on educational levels for players in this game, but census work for a larger project of which this study is a part tells us that older male farmers and village women it has been suggested that situated learning often does not transfer outside its immediate context-that judgments cannot be shifted between contexts. our data, especially the comments of those who saw the two variants of the game having the same risk of eventual death, suggest the contrary, and that the two diseases-one known from experience, the other (as yet) only from reports-were handled according to a single calculus of risk. response to the game is an important part of what we set out to discover. players of the game adapted to it with apparent ease, perhaps because talking about problems in this way reflects experience in village divination sessions, often conducted with pebbles, cowrie shells or nuts. several informants reported their decisions in terms of information conveyed by the stones, an idiom familiar from the process of village divination [7] . in these cases, explanation of choices was phrased in language such as "the stones have already told me how i need to protect myself". one woman noted that the stones "are talking about death" in both versions of the game. these results suggest that games modelled on divinatory practices could provide public health practitioners with a generally useful means of conveying information about infection risks. the game also provides a specific context to engage further in comparative discussion of infection risks associated with covid-19. there has been widespread initial fear in rural sierra leone that covid-19 is another ebola. for effective response, the differences between evd and covid-19 need to be more fully explored. specifically, we are in the process of returning to the two communities to discuss the implications of the results reported above. we then plan to use the game to set up discussion of variations in risks of covid-19 by age. we intend also to follow up on comments by players that require further contextualization. one of the most interesting (made by two people) is that "no one will swear on sickness". village disputes often are resolved by asking a person to swear publicly to the truth of their evidence. no one can be sure whether or not the swearer is a brazen liar. but sickness, it is suggested, is not something that can be evaded by bluster; risks of infection are real. overall, our evidence suggests that rural people in sierra leone respond to infection risks in rational and calibrated ways. the benefits of this response were experienced in the ebola epidemic of 2014-15 [8] . we see no evidence that the same will not also be the case with covid-19. communities should be trusted to play a fuller part in infection control. supporting information s1 data. (xlsx) the new mathematics and an old culture. a study of learning among the kpelle of liberia are humans good intuitive statisticians after all? rethinking some conclusions from the literature on judgment under uncertainty judgment under uncertainty: heuristics and biases gigerenzer g gut feelings: the intelligence of the unconscious re-analysing ebola spread in sierra leone: the importance of local social dynamics shaw r memories of the slave trade: ritual and the historical imagination in sierra leone how a people's science helped end an epidemic we wish to thank our research assistants esther bowie, francis baigeh johnson, and theresa koroma for their excellent work. we also offer our thanks to two anonymous referees, and to professor melissa leach and dr hayley macgregor, for helpful comments on the draft. conceptualization: foday mamoud kamara, esther yei mokuwa, paul richards. key: cord-346347-r4ork18p authors: borrion, hervé; kurland, justin; tilley, nick; chen, peng title: measuring the resilience of criminogenic ecosystems to global disruption: a case-study of covid-19 in china date: 2020-10-14 journal: plos one doi: 10.1371/journal.pone.0240077 sha: doc_id: 346347 cord_uid: r4ork18p this paper uses resilience as a lens through which to analyse disasters and other major threats to patterns of criminal behaviour. a set of indicators and mathematical models are introduced that aim to quantitatively describe changes in crime levels in comparison to what could otherwise be expected, and what might be expected by way of adaptation and subsequent resumption of those patterns. the validity of the proposed resilience assessment tool is demonstrated using commercial theft data from the covid-19 pandemic period. a 64 per cent reduction in crime was found in the studied city (china) during an 83-day period, before daily crime levels bounced back to higher than expected values. the proposed resilience indicators are recommended as benchmarking instruments for evaluating and comparing the global impact of covid-19 policies on crime and public safety. crime patterns vary by place and can fluctuate over short time periods. studies on the temporal patterns of crime have focused on seasonal patterns such as time of year, day of week, holidays, and hours of darkness [1] [2] [3] . event generated perturbations in crime patterns have been less studied. these can be brought about by large-scale disruption to the public safety of communities and to the infrastructure upon which they rely. while exceptional in scale, such events are not rare. across the globe, natural disasters (epidemics, hurricanes, volcanic eruptions, earthquakes, cyclones, etc.), man-made catastrophes (terrorist attacks, industrial accidents, fires, etc.), and even mega sporting events (summer and winter olympics, football world cup, etc.) that radically disrupt everyday life are not uncommon [4] . covid-19 comprises a major source of global disruption, whose scale is unprecedented. reports both in the media and by police already suggest that covid-19 is having a substantial and immediate impact on criminal behaviour across diverse jurisdictions [5, 6] . with the important exception of domestic violence, most types of offence appear to have fallen steeply. the longer-term consequences for crime patterns are clearly not yet known at the time of writing but we anticipate crime rates for offences that dropped will likely increase once lockdown measures are lifted. this paper provides a methodological framework and a set of metrics for analysing the effect of covid-19 and associated (individual and formal) responses on crime, within and across cities. the framework used is grounded in 'resilience' theory and the metrics draw upon what might be expected on the basis of analogous disruptive events of different kinds, and their immediate and longer-term impacts on institutions and behaviours as they normalise. we begin by explaining our rationale for using resilience as a lens through which to analyse the immediate disruption of criminal activity and what might be expected in the longer term assuming the criminal behaviour has resilient qualities. next, we describe indicators and mathematical models devised to measure the characteristics of the disruption to, and resilience of the criminogenic ecosystem. finally, we briefly illustrate the application of these metrics using empirical data from an anonymous city in china (m1-city) that unlike much of the developed world, has gone through a complete wave of the covid-19 pandemic and the majority of routine activities have now resumed. resilience in the social-ecological context relates to the ability of human agents to learn and innovate, to manage shocks, and to also find new trajectories for communities and social-technical systems. more specifically, it focuses on (a) the amount of change a system can undergo (and, therefore, the amount of disturbance it can sustain) while still retaining the same controls on structure and function); (b) the degree to which the system is capable of self-organization and (c) the degree to which the system can build the capacity to learn, adapt, and ultimately offer a 'soft landing' for the organisation or system [7, 8] . systems are resilient to the extent to which they can adapt to and survive an event that threatens to disrupt them. such threats can be internal or external. they can arise as a result of human actions (such as war) or natural disasters (such as hurricanes and disease outbreak). the threats can apply, for example, to commercial organisations, families, economies, religious groups, or political systems. resilience studies explore ways in which (eco)systems are initially thrown off course, then adjust to disruptive events and ultimately revert to their previous state, fail gracefully, or some new normal [9] [10] [11] . much national planning has been concerned with resilience in the face of expected threats including those that spring from pandemics of the kind being experienced in the case of covid-19 [12] . most discussions of what is and can be done relate to the public interest in maintaining organisations and behaviours in the face of potentially disruptive threats with the majority of this work focusing on natural hazards and human error. similarly there has been a focus on security and the ways in which threats can be absorbed if encountered or anticipated in advance and therefore geared up for when they occur [13] [14] [15] . the latter is sometimes referred to as 'antifragility', following taleb [16] . strong organisations are those that have increased capacity to withstand successive threats by building on lessons learned from each and preparing accordingly [17] . studying the impact of the 7/7 terrorist attack, cox et al. [18] , for example, attempted to measure resilience as a function of the speed and extent to which the london underground was able to adapt and resume its core functions. they considered the number of daily journeys as a measure of performance and found that resilience was a function of not just the public transport system, but also individual users. in contrast to the literature on disruptive threats to public interest and how they might be mitigated, here we are concerned specifically with changes in criminal behaviour patterns generated as a consequence of the disruption created by exceptional events. there is a small literature on what has been observed in the short term in relation to patterns of criminal behaviour following disruptive natural and man-made disasters. in what follows previously established disaster categories and peril terminology for operational purposes are utilised to classify studies conducted in the criminological and sociological literature [19] . summarised in tables 1 and 2 previous studies found no one-to-one relationship between major types of disaster and their crime patterns consequences. however, they do show that effects are produced. a more nuanced discussion is required to fully unpack what happens within individual disasters and responses to them to understand the short and longer term impacts on crime and public safety. taking covid-19 as an example, the following discussion sketches a theoretical model, rooted in notions of resilience, of what would be expected in relation to crime pattern changes following a pandemic. there is good reason to believe that many patterns of criminal behaviour have been disrupted by the covid-19 pandemic and/or subsequent public-health measures. indeed, between 1 january 2020 and 5 may 2020, police data shows there has been a significant and unusual drop in rape, theft, and assault to name a few in many cities including new york (-29%; -10%; -5%), chicago (-15%; -7%; -22%) and los angeles (-25%; -25%; -4%) when compared to counts from the previous year [6, 41, 42] . these changes are not altogether surprising as crime is a function of patterned opportunity that arises from the routine activity of everyday human life [43] . moreover, the fundamental organising principles of society revolve around tasks that require people to act jointly in collective efforts related, for example, to work, school, and various forms of leisure. as restrictions inhibiting the movement potential of individuals to engage in the activities that underpin community structure are altered, so too will be the rhythm associated with them. indeed, even precautionary behaviours in anticipation of a pending disruptive event can disturb this periodicity. in turn, the ecological conditions required for many different crime categories shaped by the coordination of these rhythms will be thrown into disarray. take residential burglary, for example. this requires a motivated offender to identify a suitable target in the absence of a guardian that can intervene. the scarcity of guardian-free residences during the lockdowns introduced in response to covid-19 coupled with offenders' inability to 'forage', as doing so may draw unnecessary attention thus undermining the anonymity desired, combine to increase the risk of committing a burglary. however, this pattern will likely be temporary and levels of residential burglary and other crimes will return to previous (or higher) levels for several reasons. for instance, some offenders may adapt to these new conditions, finding new and innovative ways to take advantage of drastically reduced footfall and the natural surveillance it provides along with other elements of the criminogenic ecosystem. alternatively, a return to ecological equilibrium, brought about by lifting stringencies on behaviour, may lead back to the previous rhythms that naturally afforded offenders opportunities they could exploit. fine-grained data that can be used to capture empirical nuances of resilience across crime types and sub-types during and following covid-19 will soon become available. at that point, a common set of indicators will be necessary to perform a global analysis of crime during the pandemic period. numerous analytic methods and indicators have been used in different sectors to evaluate and measure resilience [44] . shin et al. [45] reviewed quantitative approaches for measuring the resilience of water infrastructure systems and evaluated 21 indicators for assessing major features of resilience. however, continuous variables (quantity and flow of water) cannot be used in the analysis of crime. furthermore, concepts found in supply and demand problems (such as reserve capacity and availability) are not directly applicable to public safety problems because offenders and victims have competing goals. a useful review from the transportation sector by sun et al. [46] pointed to metrics that are more relatable to criminogenic ecosystems by making a distinction between functionality and resilience metrics: functionality metrics (or performance measures) provide information about the (static or dynamic) state of a system, whereas resilience metrics are used to evaluate the continued functionality of a system during a disturbance. the former include metrics related to the topological features of a system as well as metrics related to traffic flow and system capacity such as travel time, throughput, and congestion. in the public safety domain, crime incidence (number of crimes) or the amount of harm (e.g., financial loss, environmental damage) caused by these events can serve as functionality metrics [47] . along the same public safety vein, resilience metrics can use the above-mentioned metric values to quantify a system's functional resilience. in other disciplines a common approach has been to compare the performance measure before and after a disruptive event, or to quantify the social and economic impact. the resilience triangle is a tool proposed by bruneau et al. (2003) for the purpose of modelling the loss of resilience after the occurrence of disruption to a system (fig 1) . the results are normalised, enabling the loss in performance, δy(t) = y 1-y(t), to be more readily interpreted in relation to loss in the worst case scenario, max(δy(t)) = 1. more specifically, the resilience triangle provides a measure of both the loss of functionality in a system in the wake of disaster (t de ) and the amount of time required for a system to recover. several metrics have been proposed that include the recovery time, the slope of the recovery speed (α), the length of the recovery path, which in combination quantify the area of the 'resilience triangle' (eq 1) along with the 'resilience index' that captures the average functionality level until the end of the study period (t h ) (eq 2): where, y(t) is the system functionality at time t, t de is the time when the disruptive event occurs, t rp is the recovery point, and t h is the time horizon of the analysis. other metrics have been created to follow the evolution of resilience over time. for example, ouyang and wang [49] proposed a metric that compares actual functionality (y a ) with a target functionality (y t ) between t 0 (the start of the prevention stage) and t h . (eq 3): socioeconomic resilience metrics have also been used to capture the economic impact of disruption. focusing on short term impact, rose [50] presented a metric called direct static economic resilience (dser) that represents 'the extent to which the estimated direct output reduction deviates from the likely maximum potential reduction given an external shock' (eq 4): where, %δy m is the maximum percent change in direct output and %δy is the (estimated) actual percent change in direct output. the selection of an appropriate functionality metric or performance measure depends on the system under analysis. for a commercial organisation, the y(t) function may be based on sales, profit, return on investment, and market share [51] . for criminals, different variables are required as metrics. a lone offender may be regarded as analogous to a sole tradesman; a small group of offenders to a small organisation; and a large criminal network (e.g., the mafia) to a multinational firm. in the case of common crimes such as burglary, most offenses are committed by single offenders or small groups of co-offenders [52] . naturally, daily crime counts for a given area may be considered an appropriate performance metric in this case. an alternative, and closely linked to the economic models of resilience, would be the daily revenue generated from offenses [53] . there are reasons to believe that crime-related performance measures during a pandemic period, or other disasters, may not match the idealised resilience triangle, but rather resemble the dipper-shaped function depicted in fig 2. this point is developed further in what follows: 1. resilience curves often assume that functionality and performance start to decrease immediately after the disruptive event occurs. this may not always be the case with crime. as an example, the predicted increase in domestic abuse may only begin several days after households began to be confined to their homes. consequently, it is reasonable to consider that there may be a lag (τ^) between the disruptive event (t de ) and departure point (t dp ) from the normal state of a system. 2. resilience curves assume that the loss of functionality/performance happens after the disruptive event. while true for most systems, humans are often able to anticipate and preempt disrupting events [54] . covid-19 prompted many people to begin stocking up on essential products. in turn, this anticipation by individual actors and the associated response to stock up on essential products appeared to generate, at least according to media reports, a series of physical and/or verbal assaults prior to a single observable case of covid-19 [55] . for this reason, the analysis window also includes an initial phase, starting at t i , considered the earliest point when performance could have been affected by the disruptive event. the time between t i and t dp is referred to as τ s. 3. resilience curves often represent loss of functionality as a quasi-instantaneous effect, such as when a light goes off during a blackout. this is, however, not always the case (c.f., [49, 50, 56] ). while many systems might experience a sharp drop in functionality or performance, the criminogenic factors that influence crime frequency during exceptional times are unlikely to change instantaneously. in the case of pandemics, for example, it is known that public-health measures affect biological and socio-technical systems (i.e., virus, individuals, communities, infrastructures) gradually. therefore, the damping period (τ -) starting with the departure point (t dp ) and leading to the landing point (t lp ) before the trough may last several days or even weeks. 4. resilience curves commonly found in the literature generally assume that disruptive events are punctual instances (such as an earthquake) and that a recovery phase starts almost immediately thereafter. however, some disruptive events can transpire over a longer interval causing sustained disruption to the environment as noted in other recent resilience studies on this pandemic [57] [58] [59] . in the case of covid-19, various different contagion prevention measures were introduced and enforced across several weeks in a staggered pattern as opposed to a singular instance (see [51] ). for this reason, t i should be regarded as the potential start of a disruptive process, with the maximum performance loss occurring over a sustained period (τ min ) between what we refer to as the landing point (t lp ) and ascent point (t ap ). 5. the duration of the recovery phase (τ r ) is also an interesting feature to observe as it provides information about the ability of criminogenic ecosystems to 'bounce back'. unlike the resilience triangle in fig 1, it is possible that the system finds an alternative equilibrium point with a lower or higher crime level. in the case of the latter, a restoration point (t rp ) demarcates the time when performance returns to its normal level and further delineates two sub-phases: the pre-restoration phase (τ + ) and the post-restoration phases (τ ++ ). the duration of these phases provide additional information for understanding the trajectory of a new equilibrium. fluctuating rates are an additional motivation for adapting the resilience triangle to crime phenomena. more specifically, the assumption that underpins this particular indicator is the stationarity of performance under normal times, a condition violated by the inconstant patterning of crime events across time. to overcome this violation of assumption we propose indicators that take account of the difference between crime levels under two scenarios: with and without the disrupting event (eq 5): where, d(n) is the difference between y f (n) and y a (n), the forecasted crime (under normal conditions) and actual crime time-series (daily crime counts), and n is the (temporal) index of the time-series. the relative difference in crime levels, rd(n), represents the number of crimes that were not committed relative to the number of crimes expected to occur on any given day (eq 6): measuring the resilience of criminogenic ecosystems to global disruption: a case-study of covid-19 in china rd(n) provides a useful measure to monitor the operating level (%) of offenders. however, it cannot be used directly to calculate cumulative losses in performance, that is the total number of offenses that did not occur. with varying forecasted crime levels (y f ), the difference, d(n), in crime levels is a more appropriate measure for this. another measure more closely aligned with the concept developed by bruneau et al. [48] is the normalised difference in crime levels. nd(n) describes the difference in crime levels relative to the mean of the forecasted crime level (eq 7): represented in figs 3 and 4, this non-performance measure (see [52] ) can be used to quantitatively describe the resilience of different criminogenic ecosystems. in these two models, t i, t de , t dp , t lp , t ap , t rp , and t rp correspond to the onset of the different phases described above. the principal resilience indicator, the normalised difference resilience indicator (ndri), is an estimate of crime reduction over the period of interest as a proportion of the total number of crimes expected in that period (eq 8): where, n i is the index of the initial point (the earliest reasonable opportunity for crime to change as a result of the disruptive event) and n h is the index of the time horizon of the analysis. six associated indicators described in eqs 9-14 can be used to characterise ecosystem resilience during the damping, trough, recovery and post-recovery phases, separately. similar to the ndri, the damping phase resilience indicator (dpri) focuses on the second phase of figs 2 and 3. this indicator is concerned with the area of the lightest coloured orange triangle in the damping phase and can be calculated using eq 9: the trough phase resilience indicator (tpri) is used to assess an ecosystem's resilience. represented in eq 10, it quantifies the relative loss in performance sustained by the ecosystem the recovery phase resilience indicator (rpri) in eq 11 can be used to measure the ecosystem's resilience during the recovery phase and corresponds to the brown coloured triangular area in fig 3: rpri ¼ in the case where the crime level in this phase exceeds the pre-disruptive event (t de ) level (μ 7 �μ 1 ), two sub-indicators can be used to measure the ecosystem's resilience during the recovery phase. they are, the pre-and post-restoration phase resilience indicators (pre-rpri and post-rpri) in eqs 12 and 13, and correspond to the coloured triangular areas in phases 5 and 6 in fig 3: pre à rpri ¼ post à rpri ¼ the posterior phase resilience indicator (ppri) estimates the relative loss in the new equilibrium phase when t h � t rp (eq 14). it corresponds to the brown coloured rectangular area in this section empirically illustrates the above indicators by using anonymised and aggregated crime data from during the covid-19 pandemic period. the people's republic of china is considered the first country to experience covid-19 [60] . it also happens to be one of the first places in the world where authorities not only introduced public-policy measures aimed at slowing or stopping contagion, but has also lifted them [61] [62] [63] . the exact beginning of the pandemic is difficult to trace. according to reports, the earliest indication of a pending epidemic available to members of the public (at the time) was a post on a chinese social media platform (wechat) issued from a hospital in wuhan on 1 january 2020 indicating that there was a pneumonia-like disease of unknown origins. the same day, chinese authorities shut down huanan seafood wholesale market, from which a number of cases were found to have emanated from (bbc news, 2020). the people's daily, a chinese newspaper, referred to covid-19 for the first time on 21 january and measures being introduced by the government in an effort to curb its spread. lockdown measures (expected to be the greatest source of societal disruption) followed shortly after (late january, early february) for most chinese cities. the measure experienced a high degree of compliance owing to strict enforcement supported by multiple means of detection and deterrence [64] . china began easing restrictions in a staggered approach with, for example, the majority of schools reopening on 16 march, public transport returning on 28 march, and the restriction on internal travel being lifted on 8 april 2020. thus routine activity has largely resumed across china with few localised exceptions [61] . although life has not completely returned to normal, many of the criminogenic conditions that were disrupted appear to have recovered to pre-covid-19 patterns. consequently, various cities in china provide a suitable setting to draw data from in an effort to illustrate the proposed concepts and metrics. for our illustrative example, victimisation data for retail theft was compiled from one of the largest cities (11 m. pop., 8000 km 2 , urbanization rate: 80%) in china. before the pandemic, the city had one of the highest gdp in the country and an unemployment rate around 3%. for anonymity, it is referred to as 'm1-city' throughout. according to chinese criminal law retail theft is a form of theft that involves entering and stealing from business premises [65] . (not to be confused with the western category of the same name, this crime type is referred to as 'commercial burglary' in china.) it was selected for analysis specifically because the number of potential theft opportunities is known to have greatly reduced during the covid-19 outbreak period. , as well as knn-multiple input multiple output, and knn-recursive) were tested to identify the approach with the greatest predictive accuracy for this particular time-series. the time-series data was partitioned into training (26 september 2017-2 august 2019, 707 days) and test (3 august 2019-31 december 2019, 120 days) datasets and the forecasting model that produced the least error over this 120-day test time horizon was identified for use. a novel ranking procedure for forecasting approaches using data envelope analysis [66] identified tbats [67] as the model which generated the least error (rmse = 6.7, mae = 5.4, mase = 0.31) and was then applied to a subset of the data (the first 827 days) to generate the counterfactual (y f ) between 1 january 2020 and 29 april 2020 (120 days). the theoretical model presented in fig 3 was then fitted to nd(n), the difference between y f and y a . no smoothing filter was applied to the data as this would have affected the pattern of crime. without definitive knowledge of what caused crime to drop in this period, it was not possible to specify the date of the disruptive event (t de ). however, 1 january 2020 was selected as the initial point (t i ) because of the potential coincidence between the public's awareness of an outbreak in a hospital in wuhan as indicated above. the time horizon for the analysis, t h , was selected based on the availability of data at the time of writing and also because it yielded a four-month long study period. all other dates for the theoretical model (t dp, t lp, t ap, t rp and t rp ) were estimated by applying a multiple change points detection method [68] and fitting the seven-phase model described above to the crime difference, nd(n). the result was then used to estimate the six indicators that were also introduced in the previous section. but it also predicted a drop in crime at roughly the same time as the 2020 spring festival holiday (25) (26) (27) (28) (29) (30) . the normalised difference in crime levels (eq 7) was then examined more closely to determine the different inflection points in the time-series. the stochastic models generated from it by the multiple change points detection method provided both a range of possible dates for each discontinuity and the shape of the resilience curve. the shape of the resilience curve generated from the stochastic method is provided in s1 fig. a consequence of the large variance during both the first and third phases meant that finding the precise time that difference levels departed from their previous state with absolute certainty was not achievable. the algorithm provided a range of dates from as early as 16 and as late as 22 for the departure point (t dp ). sensitivity analysis led us to adopt the 20 january 2020 for model fitting (τ s = 19 days). the same strategy was adopted for identifying t lp, t ap, and , t rp from the stochastic models. however, the overall shape of the resilience curve was less sensitive to small variations of these points. of particular interest is the ascent point (31 march 2020) when crime levels started to increase again. fig 6 is a graphical display of the resilience curve generated from the data derived from the stochastic model. the period between the departure point (t dp ) and recovery point (t rp ) lasted from day 20 to 103 (τ dr = 83 days). within this period, the damping phase was the shortest one (τ − = 3 days), suggesting the criminogenic ecosystem was poorly resilient. however, this result should be interpreted with caution because, as noted above, there were multiple alternatives identified as a potential departure point. for example, the classification of t dp to an earlier date (e.g., 18 january) would imply a slightly more gradual effect on crime. the trough was the longest phase of the model (τ min = 66 days), with the recovery phase only starting more than two months (τ − +τ min = 69 days) after the departure point. the recovery phase (τ r = 14 days) was split between a pre-restoration phase (τ + = 7 days) where crime levels returned to the pret dp level, and a post-restoration phase (τ ++ = 7 days) prior to a new equilibrium being found. the resilience indicators (summarised in s1 table) calculated based on the resilience curve generated from the stochastic method, hereafter referred to as the linear nd model, are very close to those directly estimated from the data, and easier to visually interpret. comparison measuring the resilience of criminogenic ecosystems to global disruption: a case-study of covid-19 in china between τ − and τ + shows it took significantly longer for crime to drop than to return to its expected level. the large difference between dpri (0.33) and pre-rpri (0.4) further suggests that the cumulative loss in crime performance was higher during the restoration phase than in the damping phase. the high value of tpri (0.78) shows the criminogenic ecosystem had limited resiliency during the trough phase. while some thefts were recorded during this period, the numbers fell to 30 per cent of the expected level (μ 4 = 0.7). although the number of thefts is expected to stabilise in the near future (if they have not already), it is worth noting that the average post-recovery level is 47 per cent higher than pre-covid-19 levels corresponding to a negative change δμ 1-7 = -0.60. this phenomenon may be due to several factors discussed in the section that follows. despite this, the cumulative counts suggest there was an overall reduction in theft, with an estimated 883 incidents that did not occur (36 per cent fewer than the expected total) during this period. assuming retail theft rates stay at the level seen in the post-recovery (posterior) phase, the cumulative loss in crime would be null by the 177 th day (26 june). however, this is not certain as it may return to its pre-t dp level before that date. several criminogenic factors influencing retail theft risk were likely affected by communications regarding the covid-19 outbreak and/or the lockdown measures imposed to tackle it. while it is not possible to draw any definitive conclusions regarding these from this data, a number of plausible explanations exist. it is conceivable, for example, that some store managers decided to fit stores with additional, or more effective security, during this period. increased natural surveillance inside stores that remained open, primarily grocery and other essentials, might have had a suppressing effect on levels of theft as customers were afforded fewer opportunities for deception. reduced in-store footfall and greater inter-customer distance might have facilitated detection of shoplifters. formal surveillance by the authorities is likely to have increased too, as part of their general public order activities. this is especially likely as many chinese municipalities have recently deployed dense cctv networks that can be used for public safety and security [69, 70] . with few people in the streets, those who attempted to steal may have been more easily identified and arrested (or deterred) by the police. similarly, the number of individuals commuting from outside the city able to commit theft would be diminished with restrictions on travel. the crime drop observed after the point of departure supports the general hypothesis that the covid-19 crisis had a significant impact on retail theft and potentially public safety more generally. the fact that covid-19 cases in china are traced back to late 2019 suggests the main source of disruption to offenders was not the virus itself, but rather awareness of the outbreak (unofficial announcement of a respiratory disease of unknown origins occurred on 1 january while an official announcement of human-to-human transmission was made on the 20 january 2020) and/or contagion measures imposed to control the spread of the virus. the overall shape of the resilience curve suggests it may take time for offenders to adapt their behaviour, and provides further empirical support for the notion that offenders are (relatively) rational agents who weigh the associated costs and benefits of committing an offense. routine activity and the perception of risk, reward and effort associated with offending are therefore likely to have played an important role in the damping and recovery phases. police agency and media reports suggest that similar drops have occurred in other countries. however, scientific inquiry is necessary to compare patterns in different places. while restrictions on travel and various other prevention measures meant to curb the spread of covid-19 remain in place in most countries around the world, it is too early to compare the restoration phase found herein with developments elsewhere. while on the one hand the limited research conducted in relation to criminality in the face of disaster may be positive as it is an indicator of how infrequently as a species we have had to face potential existential crises, on the other, it has limited the efforts made by the security and safety science community of researchers to quantify and better understand the crime and disorder patterns that emerge during such disasters so that measures can be put in place to prevent particular suboptimal outcomes. this article, including the resilience indicators contained within it, provides one of the first efforts (of which the authors are aware) to describe, quantitatively, how a given criminogenic ecosystem functions throughout such a disaster. of course, describing what has occurred creates a set of expectations grounded in empirical evidence. the indicators provide a way to capture a return to optimal functionality, as illustrated in the case of retail theft in m1-city. further, they can inform policy-makers responsible for the allocation of resources during disaster periods, police and private security professionals, and business owners, that they may face a bounce-back in criminal activity, which they may be able to pre-empt with security measures before restrictions on public life are eased. retail theft in m1-city in china, is one of any number crime categories that can be examined using the indicators developed in this paper. indeed, these non-performance metrics are applicable to any category of crime and disorder during a disaster and can be used to analyse the same category of crime within cities, and across other geographic scales. thus, they enable comparisons to be made between different urban environments such as m1-city, but also compare urban patterns with rural ones. the metrics developed in this paper are crime-, disaster-, and resolution-agnostic enabling any category of crime, for any disaster, at any geographic scale to be described. the approach opens up the opportunity for cross-national comparisons of the impact of covid-19 and the associated stringency measures, as well as at the state-, province-, or county-level, and even at the city-and neighbourhood-levels. interestingly, the indicators also offer a unique opportunity to analyse the resilience of offenders and the other constellation of actors that exist within the criminogenic ecosystem (e.g., human victims, property targets, guardians, and other types of informal and formal controllers). these metrics may give rise to more complex questions related to what has ultimately driven particular patterns to emerge throughout the disaster period. for example, if the rate of crime receded over a longer period of time in a1-city than in b1-city, was it a function of victims in a1-city not modifying their behaviour in relation to the stringencies that impacted the ecosystem; or, conversely, was it that offenders in b1-city adapted to the new ecological equilibrium more rapidly. similarly, and in line with what was found in m1-city, is the bounce-back that was described found elsewhere? while the indicator cannot provide a clear indication of whether the bounceback was the result of offender adaptation or a lack of preparedness on the part of business owner to modify or adopt new public safety and security measures, the latter seems the more likely of the two. the coincidence of stringency orders being lifted that opened up internal mobility, coupled with the reopening of schools and public transport, suggests that it was a lack of preparedness on the part of commercial businesses in relation to their susceptibility to retail theft in this new normal that emerged from the disaster period. an alternative explanation may be that with the lifting of stringencies comes a period of hyper-criminality among hardened offenders who perhaps felt pressure to make up for lost opportunities to offend emanating from the disaster. either scenario suggests that this is something that will have to be followed closely as disaster periods end across the globe and a return to normalcy (or a new normal) sets in. it is important to recognise the limitations of the findings reported here. it is possible that the pattern observed in m1-city is unique, describing a trend that is not found anywhere else in china, or other cities around the world. we believe this is unlikely given the magnitude, spread, and nature of the covid-19 stringency measures that have been adopted throughout the world and the associated anecdotal evidence that has repeatedly trickled into media reports about the general decline of many crimes during this period. this being said, it may be that some of the metrics are misleading. let us consider the recovery of retail theft in m1-city as an example. it may be that what we describe is not a function of offender adaptation, hyper-criminality after a period of criminogenic deprivation, or even a lack of preparedness by commercial businesses to retail theft, but rather a reporting error generated as a function of a return to normalcy. more specifically, much has been written about the temporal nature of crime reporting and the challenge of identifying the period in which a criminal event occurs. in fact, methods have emerged in the criminological literature in an effort to overcome this limitation (see, for example, [71] ). hence, the apparent bounce-back may be an artefact of a large number of businesses reporting incidents they learned of when stringencies were lifted. however, we believe that the large disparity in the cumulative count of expected versus actual retail theft events that occurred during the disaster period makes this unlikely. future studies of the same crime category across different scales and geographies should be conducted to more formally test this potentiality. further possible limitations relate to recording practices and the confluence of stringency orders. like the crime reporting bounce-back artefact noted above it may be that a large, disproportionate number of arrests were made during the 'lockdown' period when individual movement was largely prohibited. future studies examining changing patterns of arrest to explore the extent to which these individuals have existing criminal records, may throw some light on this. the phenomenon of offender self-selection [72] , whereby those committing more serious offences also commit more minor ones, may mean that those liable to commit crimes such as retail theft receive enhanced police attention as breaches in restrictions in everyday activity are vigorously enforced during lockdowns. again research on police activity and arrest patterns may be able to examine whether this is the case. finally, as noted earlier in this paper, domestic violence appears to have increased rather than decreased during lockdown, due to the enforced confinement of families in contrast to of types of offence. this will require its own analysis. the resilience indicators introduced in this paper provided a set of tools to quantify the (in) ability of an ecosystem to maintain a certain level of criminal activity. demonstrated through a case-study, it constitutes an important benchmark for an area of public safety research that requires significant attention as recently called for in the resilience literature as it relates the current pandemic (see [73] ). indeed, it fills a gap as no unifying theoretical framework existed prior to this work for describing the impact of disasters on criminogenic ecosystems and public safety (see [74] ). what is gleaned from the approach can ultimately aid in the development of a greater understanding of what can be expected during disasters and consequently will enable those involved in the practice and policy of public safety to better anticipate needs. the extant public safety literature that has focused on criminality during 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decis the case for a criminology of disaster key: cord-353253-kk2q71vg authors: itokawa, kentaro; sekizuka, tsuyoshi; hashino, masanori; tanaka, rina; kuroda, makoto title: disentangling primer interactions improves sars-cov-2 genome sequencing by multiplex tiling pcr date: 2020-09-18 journal: plos one doi: 10.1371/journal.pone.0239403 sha: doc_id: 353253 cord_uid: kk2q71vg since december 2019, the coronavirus disease 2019 (covid-19) caused by a novel coronavirus sars-cov-2 has rapidly spread to almost every nation in the world. soon after the pandemic was recognized by epidemiologists, a group of biologists comprising the artic network, has devised a multiplexed polymerase chain reaction (pcr) protocol and primer set for targeted whole-genome amplification of sars-cov-2. the artic primer set amplifies 98 amplicons, which are separated only in two pcrs, across a nearly entire viral genome. the original primer set and protocol showed a fairly small amplification bias when clinical samples with relatively high viral loads were used. however, as sample’s viral load become low, rapid decrease in abundances of several amplicons were seen. in this report, we will show that dimer formations between some primers are the major cause of coverage bias in the multiplex pcr. based on this, we propose 12 alternative primers in total in the artic primer set that were predicted to be involved in 14 primer interactions. the resulting primer set, version n1 (niid-1), exhibits improved overall coverage compared to the artic network’s original (v1) and modified (v3) primer set. the realtime surveillance of pathogen genome sequences during an outbreak enables monitoring of numerous epidemical factors such as pathogen adaptation and transmission chains in local to even global scale [1, 2] . since it was first identified in hubei, china in december 2019 [3] , the novel coronavirus, sars-cov-2, responsible for the atypical respiratory illness covid-19, has become a major concern for the medical community around the world. the relatively large genome size of corona viruses (approx. 30 kb) and varying levels of viral load in clinical specimens have made it challenging to reconstruct the entire viral genome in a simple and cost-effective manner. in january 2020, a group of biologists comprising the artic network (https://artic.network/), designed 196 primers (98 pairs) (https://github.com/articnetwork/artic-ncov2019/tree/master/primer_schemes/ncov-2019/v1) for targeted amplification of the sars-cov-2 genome by multiplexing pcr. these primers and method were based on a primer design tool primal scheme and a laboratory protocol primalseq that had been previously developed for sequencing outbreaking rna virus genomes directly from clinical samples using portable nanopore sequencer or other ngs platforms [4, 5] . the artic primer set for sars-cov-2 (hereafter, artic primer set v1) is designed to tile amplicons across nearly entire sequence of the published reference sars-cov-2 genome mn908947.3 [6] . the 98 primer pairs are divided into two separate subsets (pools 1 and 2), such that no overlap between pcr fragments occurs in the same reaction. the artic primer set v1 and the published protocol [7] worked well for samples with a relatively high viral load (ct < 25 in clinical qpcr tests). for these samples, all designated amplicons are amplified with an acceptable level of coverage bias for subsequent ngs analysis. however, a gradual increase in the overall coverage bias was observed as a sample's viral load decreased. although this phenomenon is generally expected in such highly multiplexed pcr, the coverage for the two particular pcr amplicons, 18 and 76, decays far more rapidly than other targets (fig 1a) . in our experience, the low to zero depth for those two amplicons was the most frequent bottleneck for using the artic primer set v1 to sequence all targeted genomic regions from samples with middle to low viral load (ct > 27). because the amplicon 18 overlaps the gene for nonstructural protein 3 (nsp3) in orf1a, which is essential for assembly of the replicase-transcriptase complex, and the amplicon 18 overlaps the gene of spike (s) protein, which mediates receptor biding and virus-cell fusion [8] , successful sequencing of those regions are potentially important for studies to understand evolution of the virus pathogenicity and infectivity. in situation with a high coverage bias in genome sequencing such as seen in fig 1a, however, excessive sequencing efforts are required to obtain viral genome sequences with no or few gaps. thus, minimizing the overall coverage bias will benefit the research community by both enabling more multiplexing in a given sequencing capacity and lowering the sequencing cost per sample. in this report, we first show that the acute dropout of amplicons 18 and 76 was due to the formation of a single dimer between the forward primer for amplicon 18 and the reverse primer for amplicon 76. the replacement of one of the two interacting primers resolved the dropout of both amplicons. we further detected an additional 13 other potential primer interactions that may be responsible for low coverage in other regions covered by the affected amplicons. our modified primer set, version n1 (niid-1), which includes 12 primer replacements from the artic primer set v1, yielded improved overall genome coverage in clinical samples compared to v1 and another modified primer set, v3. the results indicated that preventing primer dimerformation is an effective measure to improve coverage bias in the artic network's sars-cov-2 genome sequencing protocol, and may be applicable to other primalseq methods in general. in the original artic primer set v1, pcr amplicons 18 and 76 were amplified by the primer pairs 18_left & 18_right and 76_left & 76_right, respectively. those primers were included in the same multiplexed reaction, "pool 2." we noticed that two of those primers, 18_left and 76_right, were perfectly complementary to one another by 10-nt at their 3 0 ends (fig 1) . indeed, we observed ngs reads derived from the predicted dimer in raw fastq data. from this observation, we reasoned that the acute dropouts of those amplicons were due to an interaction between 18_left and 76_right, which could compete for amplification of the designated targets. next, we replaced one of the two interacting primers, 76_right, in the pool 2 reaction with a newly designed primer 76_rightv2 (5 0 -tctctgccaaattgt tggaaaggca-3 0 ), which is located 48-nt downstream from 76_right. fig 1a and 1b show the coverage obtained with the v1 set and the v1 set with 76_right replaced with 76_rightv2 for cdna isolated from a clinical sample obtained during the covid-19 cruise ship outbreak, which was previously analyzed (epi_isl_416596) [9] . the replacement of the primer drastically improved the read depth in the regions covered by amplicons 18 and 76 without any notable adverse effects. the replacement of the primer 76_right improved coverage not only for amplicon 76, but also for 18 as well, supporting the hypothesis that the single primer interaction caused dropout of both amplicons. given this observation, we identified an additional 13 primer interactions using in silico analysis (fig 2a and 2b ). those primer interactions predicted by primerroc algorithm [10] , which gave the highest score for the interaction between 18_left and 76_right among all 4,743 possible interactions, were likely involved in producing the low coverage frequently seen in our routine experiments. we, then, designed an additional 11 alternative primers, which resulted in a new primer set [artic primer set ver. niid-1 (n1)] including 12 primer replacements from the original v1 primer set (s1 table) . the n1 primer set eliminated all interactions shown in fig 2a, and was expected to improve amplification of up to 22 amplicons (1, 7, 9, 13, 15, 18, 21, 29 , 31, 32, 36, 38, 45, 48, 54, 59, 66, 70, 73, 76, 85, and 89). alongside with this modification, the artic network itself released another modified version of primer set known as v3 in 24 th march 2020 [11] after we reported our result on the replacement of primer 76_right in a preprint [12] . the v3 primer set included 22 spike-in primers, which were directly added into the v1 primer set to aid amplification of 11 amplicons (7, 9, 14, 15, 18, 21, 44, 45, 46, 76, and 89) . we compared the performance of the original primer set (v1) and the two modified primer sets (v3 and n1) by observing their responses to different annealing/extension temperatures (ta) in the thermal program (98˚c for 30 sec followed by 30 cycles of 98˚c for 15 sec and ta table) are shown in green. (b) violin plots showing the distributions of dimer-scores (the nearest-neighbor δg adjusted ˚c for 5 min) using the gradient function of a thermal cycler. we surmised that this gradient temperature experiment would enable us to examine the dynamics of amplification efficiencies for each amplicon over varying annealing conditions. in general, amplicons suffering from primer interactions were expected to drop rapidly as ta decreases. fig 3 indicates the abundances of the 98 amplicons at eight different ta, ranging from 63.1-68.6˚c, using same dilution from a cdna sample with high viral load (ct = 16.0), which has previously been obtained from patients during the cruise ship outbreak sequenced (epi_isl_416584). with the v1 primer set, amplicons 18 and 76 exhibited extremely low coverage for all ta values, with only a slight improvement above 67˚c. in addition to those two amplicons, many other amplicons exhibited reduced coverage in these lower ta range. most of those amplicons were related to the predicted primer interactions depicted in fig 2a. although the dropout for amplicons 18 and 76 resolved with the v3 primer set, many amplicons still suffered low coverage in the low ta region. compared to the v1 and v3 primer sets, the modifications in the n1 primer set resulted in improved robustness of coverage over a broader ta range for relevant amplicon. the improvement, however, made potentially weak amplicons 74 and 98 more apparent (fig 3) . the abundance of amplicons 74 gradually decreased with decreasing ta, in contrast, the abundance of amplicon decreased with increasing ta. these amplicons seemed equally weak in all three primer sets rather than specific in n1 primer set. so far, we have not yet identified interactions involving the primers for those amplicons. the gradient experiment also revealed relatively narrow range of optimal temperature for ta for the v1 and v3 primer set, around 65˚c, which was broaden for the n1 primer set. nevertheless, while ta = 65˚c is a good starting point, a fine tuning of this value may help improving sequencing quality since an even slight difference between thermal cyclers, such as systematic and/or well-to-well accuracy differences and under-or overshooting, may affect the results of multiplex pcr [13] . finally, we further compared the v1, v3 and n1 primer sets for three other clinical samples with relatively low viral loads using a standard temperature program (ta = 65˚c). in all three clinical samples ( fig 4a and s1 fig) , the n1 primer set showed the most even coverage distribution. the amplicons whose primer was prevented dimer formation (fig 2a) by the primer modification in this study showed significantly improved coverage compared from v1 to n1 (fig 4b) . the results of this study implicate importance of primer design which avoid dimer formation for primalseq approach. we consider this insight will be helpful especially when revision of primer set (either for v3 or n1) become necessary in future to cope with the ever-increasing diversity of sars-cov-2 genomes (https://nextstrain.org/ncov). the formation of primer-dimers is a major cause of coverage bias in the artic network's multiplex pcr protocol for sars-cov-2 genome sequencing. eliminating these problematic primer interactions improves sequence coverage and will likely increase the quality of genome sequencing for sars-cov-2 and other viruses based on the primalseq protocol. the ncov-2019_76_rightv2 primer was redesigned using primer3 software [14] set 65˚c as optimal tm and 20 nucleotides as optimal length. other alternative primers were basically by empirically determined penalty and bonus scores [10] ) at all heterodimers in pools1 and 2 reported by the primerroc algorithm (n = 4,743 for each pool). the scores of interactions depicted in fig 2a are re-designed just by shifting their position several nucleotides toward the 5 0 ends. for primer 36_leftv2 and 38_rightv2, extension on the 5 0 end were applied to set the medium dissociation temperature (tm) predicted by the neb website tool (https://tmcalculator.neb.com/) more than 63˚c. primer 66_leftv2 (tm = 69˚c) was designed by removing three nucleotides on the 3 0 ends from 66_left (tm = 68˚c) without position shift because this operation did . for all data, reads were down-sampled to normalize average coverage to 500x before analysis. the green lines and points indicate the abundances of amplicons whose primers in v1 primer set were subjected to modification in the n1 primer set. the orange lines and points indicate the abundances of amplicons whose primers were not modified but predicted to be eliminated the adverse primer interactions in the n1 primer set. other amplicons which were not subjected to the modification are indicated by black lines and points. the plots in the left column shows results of all 98 amplicons while only amplicons targeted by modification are shown in the plots in the right column. horizontal dotted line indicates fragment abundance = 30. red vertical lines indicate normal annealing/extension temperature, 65˚c. all those experiments were conducted with the same pcr master mix (except primers) and in the same pcr run in the same thermal cycler. and two modified artic primer sets (v3 and n1) on a same clinical sample (previously deposited to gisaid with id epi_isl_416596, ct = 28.5, 1/25 input per reaction). regions covered by amplicons with modified primers and with not modified but interacting primers are highlighted by green and orange colors, respectively. for all data, the reads were downsampled to normalize average coverage to 250x. horizontal dotted line indicates depth = 30. these two experiments were conducted with the same pcr master mix (except primers) and in the same pcr run in the same thermal cycler. (b) volcano plot for coverages of all 98 amplicons in pcr using the primer set n1 compared with pcr using the primer set v1 among three clinical samples (shown in fig 4a and s1 fig) . points with green color indicate amplicons whose primer(s) was subject to replacement. points with orange color indicate amplicons whose primers were not replaced but had been predicted to interact with either of the replaced primers as shown in fig 2a. https://doi.org/10.1371/journal.pone.0239403.g004 not lower the tm. see details of modifications on primers indicated in s1 table. all new primers were assessed by primerroc [10] (http://www.primer-dimer.com/) to ensure no significant interactions with the remaining primers were predicted. all primer sequences included in the primer set n1 and information for their genomic positions were deposited to https:// github.com/itokawak/alt_ncov2019_primers. all primers used in this study were synthesized as opc purification grade by eurofins genomics in japan. four cdna samples obtained from clinical specimens during the covid-19 outbreak on a cruise ship february 2020 [9] were reused in this study. the use of human specimens was approved by the research ethics committee of the national institute of infectious diseases (approval no. 1091). it was conducted according to the principles of the declaration of helsinki, in compliance with the law concerning the prevention of infections and medical care for patients of infections of japan. the ethical committee waived the need for written consent regarding the research into the viral genome sequence. the personal data related to the clinical information were anonymized, and our procedure is not to request written consent for all patients suffering from covid-19. the cdna had been synthesized from rna of sars-cov-2 positive pharyngeal swabs detected by the real time rt-pcr assay [15] . reverse transcription was conducted using protocol published by the artic network [7] , and then diluted the purified pcr product was subjected to illumina library prep using qiaseq fx library kit (qiagen) in 1/4 scale and using 6 min fragmentation time [16] . after the ligation of barcoded adaptor, libraries were heated to 65˚c for 20 min to inactivate the ligase, and then all libraries were pooled in a 1.5 ml tube. the pooled library was first purified by ampurexp at 0.8x concentration, and then again at 1.2x concentration. the purified library was sequenced for 151 cycles at both paired-ends in illumina iseq100. the obtained reads were mapped to the reference genome of sars-cov-2 mn908947.3 [6] using bwa mem [17] . the depth function in samtools [18] with 'aa' option was used to determine coverage at each base position. then, the 's' option of samtools view function was used for subsampling reads from each mapping data for normalization. to calculate coverage (fragment abundance) per pcr amplicon, 98 representative nucleotide (s1 appendix) each unique to the 98 amplicons amplified by either the v1 or n1 primer set were defined to avoid duplicated count of same amplicon molecules which were fragmented in library preparation. those representative nucleotides also reside within overlapped regions where original and corresponding alternative primers in the v3 primer set amplify (e.g. the nucleotide for the product 7 overlap with both amplicons amplified by 7_left & 7_right and 7_left_alt0 & 7_left_alt5). the start and end mapping positions of whole insert sequences of paired-end reads were determined by the bamtobed function in bedtools [19] with 'bedpe' option. the number of insert fragments overlapping each defined representative nucleotide was counted by the coverage function in the bedtools. fragment inserts of unexpectedly long length (>500 bp from start to end positions) were filtered out from the analysis. the depth counts were summarized and visualized using the python3. 6 supporting information s1 table. changes in the n1 primer set from v1 primer set. (xlsx) s1 fig. performance comparison of the three multiplex pcr primer sets. depth plots of the original (v1) and two modified artic primer sets (v3 and n1) for two clinical samples (newly deposited to gisaid with id epi_isl_416749, ct = 27.3 for a and previously deposited with id epi_isl_416596, ct = 26.5 for b, each 1/25 input per reaction). regions covered by amplicons with modified primers and with not modified but interacting primers are highlighted by green and orange colors, respectively. for all data, reads were down-sampled to normalize average coverage to 250x. horizontal dotted line indicates depth = 30. these two experiments were conducted with the same pcr master mix (except primers) and in the same pcr run in the same thermal cycler. (pdf) s1 appendix. bed format file for representative nucleotides used for calculating amplicon abundances. towards a genomics-informed, real-time, global pathogen surveillance system nextstrain: real-time tracking of pathogen evolution a novel coronavirus from patients with pneumonia in china an amplicon-based sequencing framework for accurately measuring intrahost virus diversity using primalseq and ivar multiplex pcr method for minion and illumina sequencing of zika and other virus genomes directly from clinical samples a new coronavirus associated with human respiratory disease in china ncov-2019 sequencing protocol an overview of their replication and pathogenesis. coronaviruses: methods and protocols haplotype networks of sars-cov-2 infections in the diamond princess cruise ship outbreak primerroc: accurate condition-independent dimer prediction using roc analysis. sci rep hcov-2019/ncov-2019 version 3 amplicon set a proposal of an alternative primer for the artic network's multiplex pcr to improve coverage of sars-cov-2 genome sequencing (manuscript version 1) performance evaluation of thermal cyclers for pcr in a rapid cycling condition primer3-new capabilities and interfaces development of genetic diagnostic methods for novel coronavirus 2019 (ncov-2019) in japan ncov-2019 sequencing protocol for illumina protocol v1 fast and accurate short read alignment with burrows-wheeler transform the sequence alignment/map format and samtools bedtools: a flexible suite of utilities for comparing genomic features first, we must note the huge contribution of the artic network, who developed the original primer set v1 and immediately opened it to the community. we deeply thank the staff of the quarantine station for the collection and transport of clinical specimens and the staff of the influenza virus research center and department of virology iii, national institute of infectious diseases for providing clinical samples used in this study. visualization: kentaro itokawa. writing -review & editing: tsuyoshi sekizuka, makoto kuroda. key: cord-343135-m0pdixw5 authors: marguet, christophe; lubrano, marc; gueudin, marie; le roux, pascal; deschildre, antoine; forget, chantal; couderc, laure; siret, daniel; donnou, marie-dominique; bubenheim, michael; vabret, astrid; freymuth, françois title: in very young infants severity of acute bronchiolitis depends on carried viruses date: 2009-02-25 journal: plos one doi: 10.1371/journal.pone.0004596 sha: doc_id: 343135 cord_uid: m0pdixw5 background: rt amplification reaction has revealed that various single viruses or viral co-infections caused acute bronchiolitis in infants, and rv appeared to have a growing involvement in early respiratory diseases. because remaining controversial, the objective was to determine prospectively the respective role of rsv, rv, hmpv and co-infections on the severity of acute bronchiolitis in very young infants. methods and principal findings: 209 infants (median age: 2.4 months) were enrolled in a prospective study of infants <1 year old, hospitalized for a first episode of bronchiolitis during the winter epidemic season and with no high risk for severe disease. the severity was assessed by recording sao(2)% at admission, a daily clinical score (scale 0–18), the duration of oxygen supplementation and the length of hospitalization. viruses were identified in 94.7% by rt amplification reaction: rsv only (45.8%), rv only (7.2%), hmpv only (3.8%), dual rsv/rv (14.3%), and other virus only (2%) or coinfections (9%). rv compared respectively with rsv and dual rsv/rv infection caused a significant less severe disease with a lower clinical score (5[3.2–6] vs. 6[4–8], p = 0.01 and 5.5[5–7], p = 0.04), a shorter time in oxygen supplementation (0[0–1] days vs. 2[0–3] days, p = 0.02 and 2[0–3] days, p = 0.03) and a shorter hospital stay (3[3–4.7] days vs.6 [5–8] days, p = 0.001 and 5[4–6] days, p = 0.04). conversely, rsv infants had also longer duration of hospitalization in comparison with rsv/rv (p = 0.01) and hmpv (p = 0.04). the multivariate analyses showed that the type of virus carried was independently associated with the duration of hospitalization. conclusion: this study underlined the role of rv in early respiratory diseases, as frequently carried by young infants with a first acute bronchiolitis. rsv caused the more severe disease and conversely rv the lesser severity. no additional effect of dual rsv/rv infection was observed on the severity. acute bronchiolitis is the most common respiratory disease in infants under 2 years of age. although most infants recover within five days, annual bronchiolitis hospitalization among infants younger than 1 year has been assessed at 31.2/1000 [1] . during the epidemic season, up to 90% of this acute wheezing disease has been attributed to respiratory syncytial virus (rsv). however, the recent use of rt amplification reaction has modified our knowledge as regards the epidemiology during the winter epidemic season, revealing the role of other viruses such as rhinovirus (rv) and human metapneumovirus (hmpv), and at a less frequency enterovirus, coronavirus or bocavirus [2] . moreover, dual viral infection appeared more frequently, estimated from 20% to 30% [3] [4] [5] . accompanying the change in epidemiology, the question was raised concerning severity as regards these various carried viruses. the results have remained controversial. for some authors, the severity was similar between rsv and rv [6] , rsv and hmpv bronchiolitis [4] . however, korppi et al. [6] defended the fact that rsv might be responsible for a more severe disease, as this virus concerned younger infants [7] . in comparison with rsv, rv has also been associated with either a more severe bronchiolitis [3] , or a shorter duration of the disease [8] . in one study, hmpv caused more moderate bronchiolitis than those related to rsv [9] . the involvement of viral co-infections in the expression of the severity of bronchiolitis was also debated. in fact, dual rsv/hmpv or rsv/rv was shown to worsen the clinical severity and increase the duration of hospitalization [10] [11] [12] . conversely, xepapadaki et al [4] and papadopoulos et al. [3] did not confirm the aggravation effect of these dual infections. recently, the fact that carrying at least any two viruses augmented the risk to be transferred to picu has been reported [5] . the aim of this prospective study was to assess the clinical severity and viral etiology in infants hospitalized with a first episode of acute bronchiolitis during the epidemic season. within this very young population, rsv caused more marked severe bronchiolitis than did rv, and rv co-infection but neither aggravated the clinical features nor the length of the hospitalization. hmpv related bronchiolitis were more frequently associated with hypoxemia at admission. ethics statement. this study was conducted according to the principles expressed in the declaration of helsinki. the french north west i hospital ethics committee (comité de protection des personnes ''nord-ouest i'', university hospital, rouen, france) approved the research study. all patients provided written informed consent for the collection of samples and subsequent analysis. a prospective multicentre study was conducted in infants hospitalized for a first episode of acute bronchiolitis during three epidemic seasons (november to march 2002 march -2004 . four pediatric centers in a same geographical location (north west of france) participated in the study. inclusion criteria were as follows: infants aged from 1 month to 1 year, requiring hospitalization for a first episode of acute bronchiolitis. the diagnosis was based on dyspnea and wheezing or crackles associated with lung hyperinflation on chest x-ray. exclusion criteria were any administration of steroids, history of wheezing, or underlying disease susceptible to modify the natural outcome (immunodeficiency, chronic lung disease, cystic fibrosis, congenital heart disease etc.). medical history and demographic data were collected. atopy was defined by asthma, allergic rhinitis or eczema in first degree relatives, or by eczema in infants. diagnosis and severity of bronchiolitis were first established in the pediatric emergency department, where the decision to be hospitalized was made. the severity of the disease was assessed with sao 2 %, a clinical score, the duration of oxygen requirement, and the length of hospitalization. the clinical score associated standard criteria previously described [13, 14] (table 1) . each item was scaled from 0 to 1 and the score varied from 0 to 18. because the disease could worsen after admission, the infant was further scored daily. the highest score observed during the hospitalization was retained for determining the severity of the disease. the discharge of the patient was based on the following criteria: sao 2 %$95%, normal respiratory rate (rr#47/min for infants aged ,2 mo, and #42/min when older) and no alteration in feeding during the past 24 h nor other complications directly related to the bronchiolitis. the length of the hospitalization was calculated as the time between the admission and the validation of the discharge criteria. any prolonged hospitalization for other reasons was therefore not taken into account. the rouen hospital ethics committee approved the research study and informed consent was obtained from the parents. antigens for rsv, influenza virus a and b, parainfluenza type 1, 2, and 3, and adenovirus were detected by direct immunofluorescence assay (dfa) from nasopharyngeal aspiration (npa) samples, employing commercial monoclonal antibodies conjugated with fluorescein isothiocyanate (imagen, dako, denmark) [15] . a positive result was indicated by dfa, and to the finding of at least one cell showing a typical fluorescence pattern, provided that at least 20 respiratory cells were available in the sample. all the samples were further analyzed by using molecular methods including four multiplex rt-pcr, as previously described [2] , and an adenovirus pcr (adenovirus consensush, argene). the multiplex pcr targeted 14 respiratory viruses: rsv, influenza viruses a, b and c, hmpv, parainfluenza viruses (piv) types 1, 2, 3, 4, rv, enterovirus (ev), human coronavirus oc43, 229e and nl63. the molecular methods allowed us to confirm the presence of the viruses which were detected by the dfa, and to find viruses that were not included in the dfa. pairwise correlation among the sao 2 %, clinical score, length of oxygen supply, demographic data and hospitalization were assessed using spearman's rank test (rho). for nominal variables, namely virus groups, a kruskall-wallis or a mann-whitney's test were also used. univariate analyses were performed to assess the association between these variables and severity criteria. severity was then analyzed as nominal variables using for clinical score, length of oxygen supplementation and hospitalization the 50 th percentile, and for sao2% the significant hypoxemia threshold of 92%. multivariate analyses were performed by using stepwise logistic regression to select variables independently associated with the virus-related severity. a chi-2 test, and fischer's test if required, was applied for the qualitative analyses. in all the analyses, a value of p,0.05 was considered statistically significant. the data were displayed as median and 25 th -75 th quartiles. the statistical analyses were performed using statview 5.0 (sas institute inc., cary-nc ca). two hundred nine infants were included. the median age was 2.4[1.6-3.9] months, range (1-10.5), of which 91%,6 months, and the sex ratio was 1.3. a total of 94.7% infants carried at least one virus (table 2) , 93.2% of rsv-associated bronchiolitis were detected by if and the remaining 6.7% by rt-pcr. twenty-one (10%) infants had a severe bronchiolitis with a clinical score .7, 96 (46.9%) required oxygen supplementation, seven were transferred to picu and four intubated. the clinical score, length of oxygen supplementation and duration of hospitalization were significantly correlated to each other: score and duration of oxygen supplementation (rho = 0.482, p,0.0001); score and length of hospitalization (rho = 0.393, p,0.0001); duration of oxygen supplementation and hospitalization (rho = 0.527, p,0.0001). the age was inversely correlated with the length of hospitalization (rho = 20.296, p,0.0001). the girls had an higher clinical score than the boys: 6 [4] [5] [6] [7] [8] vs. 5 [4] [5] [6] [7] p,0.036; the infants with a history of family atopy had a lower clinical score (5 [4] [5] [6] [7] vs. 7 [5] [6] [7] [8] [9] , p = 0.002) and required a shorter time of oxygen supplementation (2[0-4]d vs. 1[0-3]d, p = 0.009). passive tobacco exposure was associated with a longer duration of hospitalization: 5 [4] [5] [6] [7] [8] vs. 5 [3] [4] [5] [6] , p = 0.004. the dichotomous analysis by identified virus is displayed in the large variety of viruses and viral co-infections found restricted the analyses to rsv alone, rv alone, dual rsv/rv infection, and hmpv alone, the other groups being too small ( table 2) . for this subpopulation, the clinical score was 6 [4] [5] [6] [7] , sao 2 % was 95[92.5-97]% length of o 2 supplementation and hospitalization were 1[0-3] day and 5 [4] [5] [6] [7] days, respectively. there was a significant difference in the severity between rv, rsv, rv/rsv and hmpv as shown in table 4 and figure 1 . compared to rsv, infants with rv had a lower risk to present a severe disease as regards being supplied with oxygen (or = the availability of multiplex rt amplification reactions has modified our knowledge as regards the viral epidemiology of the winter bronchiolitis, where rsv has for a long time been considered the unique etiologic agent. it is now well established that rhinoviruses and human metapneumoviruses have also been involved in infantile bronchiolitis. however, the phenotypes as regards these new viruses should to be clarified, as the available data still remain controversial. in the present study, by analyzing various severity criteria, we showed that rsv remained the most aggressive agent in young infants, and conversely rv caused less severe bronchiolitis. furthermore, dual rsv/rv infection did not aggravate the severity of illness, tending to be more moderate than rsv related bronchiolitis. in addition, hmpv bronchiolitis has been distinguished by a more marked hypoxemia at the admission. the use of molecular amplification reaction was expected to achieve a better viral detection, which ranged from 70.5% to 96.1% in comparable studies [2, 3, 5, 16, 17] . in agreement with our previous reported experience [2] , the high rate of virus detected (94.7%) in this study underlined the various viral etiologies during the rsv epidemic season in very young infants with acute bronchiolitis. dual rsv/rv infection was shown as the second cause of bronchiolitis, while rsv remained the first and, rv and hmpv the third and fourth respectively. the prevalence of rsv, rv and hmpv was indeed comparable to other studies [3, 5, 8, 16, 17] , but we conversely found no difference in the age of the infants as regards the viruses involved. on the other hand, the seldom detection of parainfluenza virus or influenza a virus could be explained respectively by the recruitment restricted to the winter season and the very young age of the infants. the other viruses that were encountered were frequently found among various combinations of co-infection. lastly, bocaviruses, prevalent viruses in acute bronchiolitis, were not researched in this series. however, its pathogenicity was to date uncertain, as it is mainly detected in coinfection [18] . we chose to enroll only hospitalized infants with a first acute bronchiolitis, with no underlying disease and no previous treatment in the way to respect the natural history. in this selected very young population, the distribution of the viruses allowed comparing infants with four different viral carriages: rsv alone, rv alone, hmpv alone and dual rsv/rv infection. interestingly, the infants with rv had a less severe disease than those who carried either rsv alone or dual rsv/rv infection. furthermore, they recovered quickly with a shorter stay in hospital. this result was contrary to the previously reported series from papadopoulos et al [3] . these authors established that rv caused a 5.6 fold risk to be included in a high risk group defined by a clinical score above the 50 th percentile, and an earlier admission in the emergency unit. in our study, 56% of rsv infants and only 33% of rv had a score above this threshold and the infants with rv bronchiolitis presented at a later stage of the disease than the rsv infants. in addition, these authors found a difference in age and weight as regards the virus carried. therefore, the discrepancies between both studies may likely be explained by the different population enrolled, and the clinical assessment of severity. more recently, richard et al. [5] reported no difference in the distribution of rv or rsv among young infants hospitalized either in short term unit or picu. mansbach et al. [19] observed a better outcome for infants with rv bronchiolitis in emergency departments, and rsv was in other series identified as the more causative aggressive agent [12, 20] . lastly, hmpv bronchiolitis featured a more initial hypoxemic disease without worsening the further outcome, as they differed in a shorter hospital stay from rsv but not from rv or rsv/rv illness. this associated hypoxemia was also observed by mullins et al. [21] but not reported in other studies [4, 22] . the statement of viral co-infections in acute bronchiolitis raised the question of a cumulative pathogenic effect and consequently a more severe disease, although other hypotheses as successive infections or healthy carriage might be also supported [23] . in this study, the clinical severity of dual rsv/rv infection was comparable to that observed with rsv, but differed in a shorter duration of hospitalization in the coinfected infants. this underlined that rsv remains the most aggressive virus on the bronchi, and masked a clinical relevant additional effect of rv, in agreement with previous reports [3, 12, 20] . the other co-infections were not analyzed, because of the small number of infants in each category. among the coinfections, the comparison of rsv and dual hmpv/rsv lower respiratory tract infection has been the most studied, and was associated with either no greater severity [16, 24] or an increased risk to undergo mechanical ventilation [25] . richard et al. [5] also reported that any dual viral infection conferred a 2.7-fold increase in the risk to be admitted in picu for ventilator assistance. this studied population was however characterized by a high proportion of premature newborns or history of underlying chronic illness and the assessment of the severity differed completely. the young age has been established as one of the most relevant risk factors in rsv bronchiolitis. commonly detected in infants younger than those who carried rv [3, 6] or hmpv [22, 26] , rsvassociated severity was suggested therefore to be related to a younger age [6] . although the age was strongly correlated with the duration of hospitalization, this hypothesis was not supported in our study, as the infants forming each viral subgroup were aged in a similar range. in addition, we found a slight increase of clinical severity in the girls, a longer time of hospitalization due to tobacco exposure and a protective effect of family history of atopy, which was also observed by bradley et al. [27] . the multivariate analysis confirmed that the type of virus involved in the infection influenced independently the length of hospitalization. other known virus-related risk factors of severity were not studied in this series, as well as the viral load [28] , the host dependent [29] or virus-related inflammatory response [14] . in conclusion, the severity of a first acute episode of bronchiolitis in very young infants depends on the causative agents. although rsv remains the main concern in this frequent respiratory illness, the prevalence of dual rsv/rv and rv infection is high and permitted to reconsider the use of corticosteroids [30] . however, proposing new management implies the routine use of rt molecular amplification, which is currently the only tool to easily target the detection of rhinoviruses, but would include an increasing cost in the investigations. bronchiolitis-associated hospitalizations among us children comparison of multiplex pcr assays and conventional techniques for the diagnostic of respiratory virus infections in children admitted to hospital with an acute respiratory illness association of rhinovirus infection with increased disease severity in acute bronchiolitis human metapneumovirus as a causative agent of acute bronchiolitis in infants the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis rhinovirus-associated wheezing in infancy: comparison with respiratory syncytial virus bronchiolitis respiratory syncytial virus infection prospective multicenter study of bronchiolitis: predicting safe discharges from the emergency department clinical impact and diagnosis of human metapneumovirus infection impact of human metapneumovirus and respiratory syncytial virus co-infection in severe bronchiolitis preliminary evaluation of a multiplex reverse transcription-pcr assay combined with a new dna chip hybridization assay for detecting respiratory syncytial virus 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infection among children hospitalized with acute respiratory illness epidemiological and clinical features of hmpv, rsv and rvs infections in young children role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life. a birth cohort study human metapneumovirus infections cause similar symptoms and clinical severity as respiratory syncytial virus infections dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis role of metapneumovirus in viral respiratory infections in young children severity of respiratory syncytial virus bronchiolitis is affected by cigarette smoke exposure and atopy respiratory syncytial virus infections in hospitalized infants: association between viral load, virus subgroup, and disease severity il-10 gene polymorphism at -1082 a/g is associated with severe rhinovirus bronchiolitis in infants are responses to treatment virus-specific in wheezing children? key: cord-339157-wj47xeqj authors: zhang, chao; chen, shuaiyin; zhou, guangyuan; jin, yuefei; zhang, rongguang; yang, haiyan; xi, yuanlin; ren, jingchao; duan, guangcai title: involvement of the renin-angiotensin system in the progression of severe hand-foot-and-mouth disease date: 2018-05-23 journal: plos one doi: 10.1371/journal.pone.0197861 sha: doc_id: 339157 cord_uid: wj47xeqj background: hand-foot-and-mouth disease (hfmd) is generally considered as a mild exanthematous disease to infants and young children worldwide. hfmd cases are usually mild and self-limiting but for few cases leads to complicated severe clinical outcomes, and even death. previous studies have indicated that serum ang ii levels in patients with h7n9 infection were related to the severity of infection. however, the mechanisms underlying the pathogenesis of severe hfmd remain unclear. this study was undertaken to clarify the role of the renin-angiotensin system (ras) in the progression of severe hfmd. methods: in the present study, 162 children including hfmd patients and healthy controls were recruited. the data was analyzed by time-series fashion. concentrations of angiotensin ii (ang ii) and noradrenaline (na) in serum of patients were measured with elisa. we established a mouse model for enterovirus 71 (ev71) infection and determined concentrations of ang ii, na in tissue lysates at 3, 5 and 7 days post infection (dpi). results: the concentrations of ang ii and na in serum of the hfmd patients with mild or severe symptoms were significantly higher than that in healthy controls. additionally, the concentrations of ang ii and na in serum of severe cases were significantly higher than those mild cases and the increased concentrations of ang ii and na showed the same time trend during the progression of hfmd in the severe cases. furthermore, the concentrations of ang ii and na in target organs of ev71-infected mice including brains, skeletal muscle, and lungs were increased with the progression of ev71 infection in mice. histopathological alterations were observed in the brains, skeletal muscle and lungs of ev71-infected mice. conclusion: our study suggested that activation of the ras is implicated in the pathogenesis of severe hfmd. in the present study, 162 children including hfmd patients and healthy controls were recruited. the data was analyzed by time-series fashion. concentrations of angiotensin ii (ang ii) and noradrenaline (na) in serum of patients were measured with elisa. we established a mouse model for enterovirus 71 (ev71) infection and determined concentrations of ang ii, na in tissue lysates at 3, 5 and 7 days post infection (dpi). the concentrations of ang ii and na in serum of the hfmd patients with mild or severe symptoms were significantly higher than that in healthy controls. additionally, the concentrations of ang ii and na in serum of severe cases were significantly higher than those mild cases and the increased concentrations of ang ii and na showed the same time trend during the progression of hfmd in the severe cases. furthermore, the concentrations of ang ii and na in target organs of ev71-infected mice including brains, skeletal muscle, and lungs were increased with the progression of ev71 infection in mice. histopathological alterations were observed in the brains, skeletal muscle and lungs of ev71-infected mice. plos in recent years, several hand-foot-and-mouth disease (hfmd) outbreaks have occurred in asia-pacific region and europe [1] [2] [3] [4] . among them, enterovirus 71 (ev71) and coxsackie a16 (ca16) infection is main cause of hfmd outbreaks, and patients with ev71 infection are inclined to develop into severe symptoms [5, 6] . hfmd is generally considered as a mild exanthematous disease. generally, most of hfmd cases are usually mild and self-limiting but for few cases viral infection leads to complicated clinical outcomes including brainstem encephalitis, aseptic meningitis, encephalitis, and acute flaccid paralysis (afp), and even fatal cardiopulmonary failure [7] [8] [9] .numerous studies have shed light on the etiology and epidemiology of hfmd, and have helped medical professionals and public health officials worldwide understand this condition. previous studies have found that ev71 enters into the digestive tract first and then enters the lymphatic system, and ultimately into the central nervous system (cns) [10, 11] . cns injury can lead to a rapid increase in intracranial pressure, increased sympathetic tone, and excessive activation of local renin-angiotensin system (ras), further results in over-secretion of many cytokines into circulation, leading to peripheral vasoconstriction. peripheral vasoconstriction will cause vascular endothelium injury and increased permeability, further inducing blood accumulation in the lower pulmonary region [12] [13] [14] [15] . the ras plays a critical action regulating the circulation in the human body in response to low blood pressure or decrease in serum sodium levels [16] .a component of this system is angiotensinogen (agt) that is synthesized and released by the liver into the general circulation. agt is converted through another reaction into angiotensin i (ang i) by the protein renin from the renal juxtaglomerular apparatus. subsequently, ang i is converted to angiotensin ii (ang ii) by the pulmonary angiotensin-converting enzyme (ace). angii is an active octapeptide that acts primarily on ang ii receptor type 1 (at1r) [16] . angiotensin ii (ang ii) is a key factor to activate at1r, further inducing water and sodium storage by promoting the release of aldosterone and excessive activation of other neurohormonal system components by promoting the secretion of endothelin and noradrenaline (na) [17, 18] . high concentration of na in circulation is thought to associate with pulmonary edema [19] , which may involve with fatal pulmonary edema in severe hfmd. the ras can also activate related cells and regulates the expression of many mediators concerning cell growth and inflammatory responses [20, 21] . however, the role of ras in the process of hfmd has not been well characterized yet. in the present study, we assumed that ras participated in the progression of hfmd and tried to uncover the ras-related potential mechanism in the progression of severe hfmd. the study was reviewed and approved by the life sciences and ethics committee of zhengzhou university and the ethics committee of the zhengzhou children's hospital. written informed consent was obtained from each case's guardian before enrollment. total 162 subjects (10-60 months age) including 132 hfmd cases with mild (n = 79) or severe symptoms (n = 53) and healthy controls (n = 30) were recruited from zhengzhou children's hospital during april 2013 through june 2013. all hfmd cases were divided into different subsets of 1, 2, 3, 4, 5 days post infection (dpi) based on the hospitalization and initial onset time. according to the "diagnosis and treatment guideline on hand-foot-and-mouth disease (2010)", patients younger than 60 months with severe symptoms including meningitis, pulmonary edema, and mild cases without any nervous system lesions or pulmonary edema were included in this study. the children without any disease were classified as control. the patients with congenital disease, acute or chronic hepatitis, cardiovascular disease, intestinal diseases, and other infectious diseases were excluded from this study. balb/c mice (spf degree) were purchased from the medical animal center in zhengzhou university, henan, china, and raised in individual ventilation cage (ivc) system. as described in our previous study [14, 15] , 3-day-old balb/c mice were i.p. inoculated with ev71 strain (2×10 6 pfu/mouse) and sacrificed with isoflurane anesthesia on 3, 5 and 7 days post infection (dpi). the 3-day-old mice injected with the same volume of rd cell culture supernatants were used as controls and sacrificed with isoflurane on 3, 5 and 7 dpi. the brains, skeletal muscle and lungs of mice were ground into tissue homogenate with cold phosphate-buffered saline (pbs) at 4˚c. tissues were quickly removed and stored at -80˚c. tissues were grounded at 4˚c and repeated freezing and thawing for three times, followed by centrifugation at 3,000×g for 10 min at 4˚c. mice were sacrificed on 3, 5, 7 dpi, and the brains, skeletal muscle and lungs of mice were immediately fixed in 4% paraformaldehyde at 4˚c overnight. after fixation, paraffin-embedded tissues of 5 μm in thickness were stained with h&e. the concentrations of ang ii and na in serum or tissue lysates were measured by enzymelinked immunosorbent assay (elisa) kits (tsz, boston, usa). spss 17 .0 (ibm, nc, usa) was used for the statistical analysis. one-way anovas or student's t test depending on the validity of the normality assumption and the homogeneity of variance and a pearson correlation analysis were performed. a significance level <0.05 was used for this study. one hundred thirty-two patients with hfmd were identified, including 79 mild hfmd patients, 53 severe hfmd patients, and 30 healthy children were used as controls. there was no significant difference in sex ratio (male ratio, controls: 1.31/1; mild: 1.47/1; severe: 1.35/1, p>0.05) and age (mouths, controls: 24.11±8.62; mild: 22.45±10.71; severe: 26.18±10.53, p>0.05) among these three groups. the concentrations of ang ii and na are presented in fig 1. the concentrations of ang ii and na in serum of the hfmd patients with mild or severe symptoms were significantly higher than that in healthy controls (p<0.001). additionally, the concentrations of ang ii and na in serum of severe cases were significantly higher than those mild cases, p<0.05. pearson correlation analysis (fig 2) indicated that the concentration of ang ii was positively correlated to the concentration of na during the progression of hfmd in both mild and severe (r = 0.492, p<0.001 for the mild; r = 0.645, p<0.001 for the severe). together, our results suggested that the concentrations of ang ii and na in serum were increased in hfmd cases. as shown in fig 3, the concentration of ang ii in serum of the severe cases was significantly higher than that in the mild cases from 1 dpi to 5dpiduring the progression of hfmd. the concentrations of ang ii among the 5 subgroups of the severe were significantly different and increased from the 2 dpi to 4 dpi (p<0.001). the highest level of ang ii in serum of the severe cases occurred at 3 dpi. the concentrations of ang ii among the 5 subgroups in mild cases were not significantly different. the concentration of na in serum of severe cases was significantly higher than those in mild cases during the progression of hfmd from 1 dpi to 5 dpi. the concentrations of na among the 5 subgroups in severe cases were significantly different and increased from 1 dpi to 4 dpi (p<0.001). the highest level of na in severe cases occurred as shown in fig 4, expression of ang ii and na presented similar temporal trends in the corresponding tissues of ev71-infected mice. the concentrations of ang ii and na in ev71-infected mice were significant increased in brains from 3 dpi to 7 dpi, skeletal muscle at 5 dpi and 7 dpi, lungs at 7 dpi compared to controls. the results demonstrated that ev71 infection increased ang ii and na levels in target organs. histopathological alterations of mice at 3, 5, 7 dpi were examined with haematoxylin and eosin (h&e) staining. as shown in fig 5 , the brain tissues from ev71-infected mice exhibited pathological changes including perivascular cuffing compared with controls at 3, 5 and 7 dpi. mice were sacrificed on 3, 5, 7 dpi. the removed organ or tissue were sliced and stained with h&e. brain from ev71-infected mice exhibited perivascular cuffing (indicated by red solid arrows). skeletal muscle from ev71-infected mice appeared necrotizing myositis with muscle fibers rupture (indicated by orange solid arrows) at 5 dpi and 7 dpi. erythrocyte-filled fluid in the alveolar spaces was found in lungs (indicated by black solid arrows) from ev71-infected mice at 7 dpi. bar = 100μm. https://doi.org/10.1371/journal.pone.0197861.g005 skeletal muscle from ev71-infected mice demonstrated necrotizing myositis with muscle fibers rupture and inflammatory cells infiltration at 5 dpi and 7 dpi. lung lesions such as swollen alveoli and erythrocyte-filled fluid in the alveolar spaces were detected in lungs of ev71infected mice at 7 dpi. the above results indicated that ev71 infection induced obvious histopathological alterations in target organs. hfmd is a common viral infectious disease mainly caused by enterovirus 71(ev71) and coxsackievirus (cv) a16, which is mostly prevalent in infants and young children [9] . from 2008 to 2012, the mortality of severe hfmd is 1.9%, which is extremely higher than total mortality of hfmd [6, 22] . to date, no effective treatment for severe cases has been found. thus, to explore early biomarkers to identify cases with potential to develop into severe symptoms will be beneficial to direct early clinical intervention and reduce mortality of hfmd. in the present study, we found that the concentrations of ang ii and na were increased in serum of hfmd cases with mild or severe symptoms. increasing concentrations of ang ii and na in tissue lysates of mice were also determined during ev71 infection. the current understanding of ras is far more complex than its classic point, and two significant concepts have been added. first, the system is not only expressed at a systemic level but also works locally in a paracrine function in the vasculature, kidney, heart, lungs and cns et al. [16] . in our study, we found that serum concentrations of ang ii and na were elevated in hfmd cases, especially in severe cases, suggesting ras activation. the concentration of ang ii was positively related to the concentration of na during the progression of hfmd in both the mild and severe cases. the highest levels of ang ii and na occurred at 3 dpi in hfmd cases. as shown in our previous study, the highest levels of some inflammatory cytokines occurred at 3 dpi in hfmd cases. in terms of above results, ras activation may induce large amounts of pro-inflammatory cytokines [23] . numerous studies on the pathogenesis of other viral diseases have also shown that pathogens can affect the stability of the ras [24] [25] [26] [27] . thus evidence support our results, and activation of ras may involve with the development of hfmd. to further confirm the involvement of ras in the pathogenesis of hfmd, we establish an ev71-infected mouse model [14, 15] . in our study, we found that ev71 infection induced ang ii and na expression in the brains, skeletal muscle, and lungs of ev71-infected mice. we also observed histopathological alterations in these target organs, implying ras activation may participate in the process of ev71 infection. furthermore, pulmonary edema was also determined in ev71-infected mice. previous studies have indicated that serum ang ii levels in patients withh7n9 infection were higher than controls and were related to the severity of infection [24, 26] . the s protein of sars-cov virus can down regulate the expression of ace2 in the lungs and cause an accumulation of ang ii and acute lung injury (ali). after the intervention of the ace2 protein, symptoms of ali were alleviated [25] . zou, z et al. found that with h5n1 infection, the ras was involved and final concentration of ang ii were able to be used as prognostic indicators [27] .these studies suggested that with viral infection, the pathogens could damage the homeostasis of the lung ras, alter the local blood pressure and vascular permeability, and aggravate the imbalance of pro-inflammatory and anti-inflammatory mediators, thus affecting the occurrence and development of lung injury. yumiko i et al. found that an excessive expression of ang ii can enhance the permeability of the lung through at1r, resulting in pulmonary edema [28] . together, above evidence suggest that ev71 infection-induced pulmonary edema may be an outcome of ras activation. in summary, our study for the first time finds that activation of the ras may involve in the progression of severe hfmd. an epidemic of enterovirus 71 infection in taiwan. taiwan enterovirus epidemic working group. the new england journal of medicine the enterovirus 71 epidemic in 2008-public health implications for hong kong epidemiology and control of hand cyclical patterns of hand, foot and mouth disease caused by enterovirus a71 in malaysia effects of relative humidity on childhood hand, foot, and mouth disease reinfection in hefei, china. the science of the total environment spatiotemporal cluster patterns of hand, foot, and mouth disease at the county level in mainland china severe hand, foot and mouth disease in shenzhen, south china: what matters most? epidemiology and infection update on hand-foot-and-mouth disease in children; an epidemic associated with coxsakie virus a-16 foot and mouth disease in singapore: a comparison of fatal and non-fatal cases immunodeficient mouse models with different disease profiles by in vivo infection with the same clinical isolate of enterovirus 71 role of the renin-angiotensin system in ventilator-induced lung injury: an in vivo study in a rat model pathologic characterization of a murine model of human enterovirus 71 encephalomyelitis involvement of inducible nitric oxide synthase and mitochondrial dysfunction in the pathogenesis of enterovirus 71 infection pulmonary edema following central nervous system lesions induced by a non-mouse-adapted ev71 strain in neonatal balb/c mice renin angiotensin system and its role in biomarkers and treatment in gliomas intensity of hydration changes the role of reninangiotensin-aldosterone system blockers in contrast-induced nephropathy risk after coronary catheterisation in patients with chronic kidney disease changes in angiotensin ii and angiotensin-converting enzyme of different tissues after prolonged hyperoxia exposure. undersea & hyperbaric medicine : journal of the undersea and hyperbaric medical society effect of norepinephrine dosage on mortality in patients with septic shock role of renin-angiotensin aldosterone system on short-term blood pressure variability in hypertensive patients effects of calcium channel blockers comparing to angiotensin-converting enzyme inhibitors and angiotensin receptor blockers in patients with hypertension and chronic kidney disease stage 3 to 5 and dialysis: a systematic review and meta-analysis. plos one hand, foot, and mouth disease in china, 2008-12: an epidemiological study. the lancet infectious diseases serum inflammatory cytokine levels correlate with hand-foot-mouth disease severity: a nested serial case-control study angiotensin ii plasma levels are linked to disease severity and predict fatal outcomes in h7n9-infected patients a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury angiotensin-converting enzyme 2 (ace2) mediates influenza h7n9 virus-induced acute lung injury angiotensin-converting enzyme 2 protects from lethal avian influenza a h5n1 infections angiotensin-converting enzyme 2 protects from severe acute lung failure the authors thank doctor yuanfang shen and doctor yunping qiao, zhengzhou children's hospital, for allowing testing of the patients. key: cord-317779-j67vb7f3 authors: irizarry, kristopher j. l.; downs, eileen; bryden, randall; clark, jory; griggs, lisa; kopulos, renee; boettger, cynthia m.; carr, thomas j.; keeler, calvin l.; collisson, ellen; drechsler, yvonne title: rna sequencing demonstrates large-scale temporal dysregulation of gene expression in stimulated macrophages derived from mhc-defined chicken haplotypes date: 2017-08-28 journal: plos one doi: 10.1371/journal.pone.0179391 sha: doc_id: 317779 cord_uid: j67vb7f3 discovering genetic biomarkers associated with disease resistance and enhanced immunity is critical to developing advanced strategies for controlling viral and bacterial infections in different species. macrophages, important cells of innate immunity, are directly involved in cellular interactions with pathogens, the release of cytokines activating other immune cells and antigen presentation to cells of the adaptive immune response. ifnγ is a potent activator of macrophages and increased production has been associated with disease resistance in several species. this study characterizes the molecular basis for dramatically different nitric oxide production and immune function between the b2 and the b19 haplotype chicken macrophages.a large-scale rna sequencing approach was employed to sequence the rna of purified macrophages from each haplotype group (b2 vs. b19) during differentiation and after stimulation. our results demonstrate that a large number of genes exhibit divergent expression between b2 and b19 haplotype cells both prior and after stimulation. these differences in gene expression appear to be regulated by complex epigenetic mechanisms that need further investigation. discovering genetic biomarkers associated with disease resistance and enhanced immunity is critical to developing advanced strategies for controlling viral and bacterial infections in various species. plos disease resistance and susceptibility depends on a variety of factors including genetics. in numerous species, disease resistance has been associated with major histocompatibility complex (mhc) haplotype, as well as polymorphisms in several immune genes such as tgfβ and tnfα [1, 2] . cytokine production, specifically secretion of pro-inflammatory molecules, has also been associated with increased resistance against disease [3, 4] . studies have demonstrated association of mhc-b haplotype in chickens and resistance to a variety of viral pathogens, including aiv, marek's disease virus (mdv), avian leukosis virus, newcastle disease virus and rous sarcoma virus [5] [6] [7] [8] [9] [10] as well as other pathogens [11, 12] . b2 haplotype chickens are more resistant to avian coronavirus infection than b19 haplotypes and these differences in disease resistance were observed early after infection in our previous studies [10] . this suggests that innate immunity plays a major role with the macrophage being a key player in this enhanced immune response as evidenced by the b2 haplotype birds' greater capability to produce nitric oxide (no) in response to ifnγ and poly i:c [13] . in addition, b2 macrophages activated t cells more efficiently than macrophages derived from b19 haplotypes [14] . macrophages are directly involved in cellular interactions with pathogens and demonstrate distinct immune responses from more disease resistant animals in response to infection [15] [16] [17] [18] [19] [20] . in addition, macrophages release cytokines activating other immune cells and antigen presentation to cells of the adaptive immune response [21] [22] [23] . it has become increasingly clear that dysregulation of macrophage function is involved in inflammatory disease processes such as rheumatoid arthritis, inflammatory bowel disease and cancer [24] [25] [26] . involved in these interactions are crucial molecules such as toll-like receptors (tlrs) that recognize invading microorganisms, resulting in communication with the adaptive immune system such as increased expression of mhc surface molecules, t cell receptors and secreted cytokines [21, 23] . genetic differences in any of those molecules can potentially account for differences in immune competence and thus provide potential immunogenetic markers for disease resistance to various pathogens. ifnγ is a potent activator of macrophages and increased production has been associated with disease resistance in multiple species [27] [28] [29] [30] [31] . these findings indicate that chickens with enhanced ifnγ production are more resistant to certain infections. ifnγ enhances macrophage activation, expression of mhc and nitric oxide release which aides in killing of pathogens and also increases activity of cytotoxic t cells and secretion of th1 cytokines [31, 13] , underscoring how crucial this process is for innate immune competence. macrophage tlrs appear to be primed by ifnγ, reprogramming cellular responses to other cytokines, such as type i interferons and il-10 and activating the jak-stat pathway (janus kinase and signal transduction and activator of transcription) [24, 32, 33] . ifnγ, which increases tlr receptor availability for interaction with its ligands, has been shown to induce tlr2, 4, 6 and 9 [34] [35] [36] [37] . the response of macrophages to an immune stimulus is not just dependent on cell surface receptor and cytokine expression. other factors include the differentiation of monocytes into functional macrophages, a tightly regulated process that influences immune competence [38] . recent studies demonstrated a critical role for molecules such as a2b adenosine receptor for differentiation and proliferation of monocytes and macrophage function in immunity and inflammation [39, 40] . a2b expression is induced by ifnγ and leads to increase of anti-inflammatory signaling counteracting the inflammatory response activated within the ifnγ pathway. taken together, these studies emphasize the genetic basis of the activation of macrophages by ifnγ playing an important role in the innate immune response signaling and providing resistance to disease. in addition to inflammatory signaling, a number of transcription factor pathways and epigenetic mechanisms all contribute to immune function. dysregulation of any of these events will lead to an impaired innate immune response and consequently, increased susceptibility to disease. using an ex vivo model, we investigated the gene expression in macrophages from haplotypes b2 and b19 during differentiation and after stimulation with ifnγ. our experimental design leveraged an initial 6 day window for monocytes to differentiate into macrophages, which was followed by ifnγ stimulation between 1 and 24 h to further characterize subsequent rna gene expression and the molecular basis for dramatically different nitric oxide production and immune function between the b2 and the b19 haplotype chicken macrophages animal protocols were performed under the approval of the institutional animal care and use committee at western university of health sciences, pomona, california (westernu). fertilized eggs, descended from modified wisconsin line 3, were obtained from dr. w. elwood briles, northern illinois university, and incubated and hatched under standard conditions at (38˚c/50-65% humidity) [10, 13] at westernu. in addition to daily health monitoring, fresh food and water were provided ad libitum. experimental animals were euthanized by insufflation of isoflurane gas (butler, dublin, oh). whole blood samples were collected via jugular venipuncture in edta tubes from age matched chicks at 14-18 weeks old. at no time did the amount of blood harvested from each animal exceed 1% of body weight. and stored at -80˚c. the morphology of adherent cells was observed daily under bright field microscopy (20x objective). purity of monocyte cultures using this culture method was confirmed by ifa and facs using monoclonal antibody kul01 as previously described as part of a different aspect of this study [13] . a 50 ρg/ml ch-ifnγ solution (invitrogen, carlsbad, ca) was prepared in rpmi w/o phenol red culture medium (invitrogen, carlsbad, ca). after washing the cells twice with warm pbs, macrophage cultures were stimulated with 1 ml of rpmi-ch-ifnγ mixture [13] . nitric oxide production was measured [10, 43, 44] to confirm macrophage stimulation in assays by interferon (data not shown). stimulation was evaluated as yes/no based on previously published results from b2 and b19 ifnγ stimulated macrophages (10) sample collection and rna sequencing a total of 145 gigabytes of rna sequence data was obtained from b19 and b2 haplotypes of chickens. two birds from each haplotype were selected for inclusion in the sequencing. each bird provided blood for extraction and isolation of peripheral blood mononuclear cells. purified monocytes were cultured for differentiation and cell samples were collected from nine time points for each bird. samples were collected for sequencing on the day they were cultured (day t-6), as well as on day -3 (t-3), day 0 (called 0 hours), and then six additional times over a 24 hour period corresponding to 1 hour, 2 hours, 4 hours, 8 hours, 16 hours and 24 hours after interferon stimulation. cells were lysed in wells with rlt buffer containing beta-mercaptoethanol (qiagen, valencia, ca) and stored at -80˚c. rna was processed with the qiashredder and rnaeasy kit from qiagen (valencia, ca) according to manufacturer's instructions and sent on dry ice to dr. calvin keeler at the university of delaware for generation of libraries and sequencing with an illumina hiseq 2000. an rna sequence library was constructed from purified rna. the library was fragmented in order to generate appropriately sized rna fragments suitable for templates in random primed first-strand cdna synthesis. second strand synthesis was completed in accordance with specifications for sequencing with illumina's hiseq2000 platform. the samples corresponding to each time point from each bird were sequenced and the data was stored in a unique file for each sequenced sample and time point. forty fasq files were generated from the data totaling 145 gigabytes. the average file size was 3.65 gigabytes and the standard deviation was 2.25 gigabytes. the sequencing data provided 933,107,885 reads across the biological samples and time points (table 1 ). across all time points for the two b2 samples, one produced 298,903,517 reads and the other produced 165,589,594 reads. similarly, across all time points, the b19 samples produced 285,392,384 reads and 183,222,390. for each time point (across all four birds) sequencing reads ranged from a low of approximately 78 million reads to a high of just over 171 million reads, with most time points producing over 88.4 million reads each and a few producing over 100 million reads each. the chicken reference genome washuc2, corresponding to ensembl release 70, was downloaded from ensembl.org (http://www.ensembl.org/info/data/ftp/index.html). annotation files included the small rna annotation files obtained from mibase release 19 (http://www. mirbase.org/). sequenced reads were filtered to remove low quality sequences from the data. filtered sequences were aligned to the reference genome using bowtie and tophat, available along with the software package cufflinks, from john hopkins university center for computational biology (https://ccb.jhu.edu/software.shtml). the aligned reads generated by bowtie produced gapped alignments on the reference genome which tophat used to identify splice junctions flanking exons. the resulting aligned reads were analyzed by cufflinks to construct transcripts corresponding to mrna sequences. next, cufflinks was employed to estimate transcript specific expression levels across the transcripts and genes within the reference genome based on the number of sequence reads for each mrna. the sequence read data was normalized using the fragments per kilobase of transcript per million mapped reads (fpkm) method to more accurately determine expression levels. the resulting transcriptome data was loaded into the mysql relational database to more effectively manage, explore, mine and annotate the data. gene expression data was hierarchically clustered using 1-pearson correlation on the rows and keeping the column order conserved. the resulting clustered data set was visualized as a heat map with black representing lack of gene expression, and darker shades of blue indicating lower expression values. dark purple represents higher expression values than any shade of blue while bright pink represents the highest expression values. for visualization purposes, the heat maps were generated with maximum heat map color assigned to expression set lower than the absolute maximum expression value contained in the entire data set, subsequently all values of expression greater than or equal to the assigned expression threshold (for example, 1000) shared the same color on the heat map (regardless of whether the actual expression level was 1000, 2000, 20,000 or 90,000) . this setting provided the optimal visualization of both high and low expressed genes in the heat maps. gene enrichment calculations were performed using the david bioinformatics database tool version 6.8 (https://david.ncifcrf.gov/). the analysis was performed using comparisons of successive time points within the b2 haplotype data to identify sets of genes that were enriched (p-value < 0.05). the b2 haplotype represents the robust macrophage phenotype as characterized by nitric oxide production compared to the b19 haplotype. subsequently, the gene enrichment was performed on the b2 data. gene enrichment was determined using three distinct databases: gene ontology biological process, kegg pathways, and reactome pathways corresponding to s2, s3 and s4 tables respectively. because a large number of enrichment annotation terms were produced, a subset of representative highlights from each of these three enrichment analyses was chosen for inclusion in the results. highlights were selected to provide examples of the biological process annotations, kegg pathways annotations and reactome annotations. realtime pcr was performed on a selected number of target genes to validate rna sequencing results. rna was taken from macrophages stimulated with ifnγ as described above for 2 and 4 hours, unstimulated samples (0h) served as control. for realtime rt-pcr, cdna synthesis was performed using superscript iii first strand synthesis kit (invitrogen, waltham, ma), according to manufacturer's instructions. pcr conditions were as follows: 95˚c for 10 minhot start, 40 cycles of 95˚c for 15 sec, 60 or 63˚c depending on gene (see primers) for 30 sec according to manufacturer instructions for the biotool 2x sybr green qpcr mix (biotool, houston, tx). primer sequences were designed using primer 3 (atp6voc, litaf, il18r, tln-1.) primer sequences previously published were used for tlr2, tlr4, tlr5, tlr6 and tlr7 [45] . atp6voc (annealing 60˚c) forward tgttgtcatggcgggtatta, reverse acaaataacctgggctgctg; litaf(annealing 60˚c) forward atcctcacccctaccctgtc, reverse gacgtgtcacgatcatctgg; il18r (annealing 63˚c) forward ctcttcgtgcctcca ttgat, reverse accaagttcaactggccaaa; tln-1(annealing 60˚c) forward tcaagcaga agttgcacacc, reverse gggagccattaaggatgtca. pcr analysis was done using the δδ method with 18s serving as housekeeping gene control. statistics were done using graphpad software (prism version 7), paired t-test, two-tailed. a total of 179 gene expression measurements were extracted from a published paper describing the fold change in expression levels of genes induced after 4 h exposure to cytomegalovirus [46] . the data was converted to a tab-delimited text file containing the official gene symbol and the reported expression level. the file was loaded into a mysql relational database and joined to the expression data produced from the b2 and b19 cells. the data was joined on the gene symbol and a set of 54 genes were identified. the fold change for the b2 and b19 expression data was calculated by taking the log-2 (4 h expression / 0 h expression). b2 and b19 genes having expression = 0 for the initial time point were converted to 0.1 to prevent division by zero. additionally, the fold-change reported for il6, 280.8, was changed to 35, in order to preserve the scale of the graphs and legibility of the resulting data represented in the histograms. the fold-change in expression for the b-haplotype birds and the published data was plotted using microsoft excel. a set of 13,618 unique genes from among all mapped sequencing reads was generated from the 4 birds across all 9 time points. next, we analyzed the expression data to determine the number of genes expressed in each haplotype within each time point. within the minus 6-day (t-6) time point, representing the time point after plating and adherence of monocytes and the start of differentiation into mature macrophages, 11,785 genes were expressed in the b2 birds while 12,089 were observed in the b19 birds, with 11,216 genes expressed in both. interestingly, 4770 genes were off in both b19 and b2 haplotypes while just 569 genes exhibited expression in only the b2 chickens and 873 genes were expressed only in the b19 birds. similar relationships were detected in each of the remaining eight time points. the t-3 day time point, representing 3 days of differentiation in cell culture, exhibited the greatest expression of genes with a total of 11,429 expressed in both b19 and b2 birds while just 4068 genes lacked evidence of expression in both haplotypes. also, during the t-3 day time point the greatest number of genes (1118) exhibit evidence of expression in the b2 birds while lacking evidence of expression in the b19 birds. at the t0 time point, after 6 days of differentiation and immediately before stimulation with interferon, 10,975 genes were expressed in both haplotypes while 4547 genes were not expressed in macrophages of either haplotype. likewise, the 1 h and 2 h time points exhibited 11,349 genes and 10,789, on in both haplotypes, respectively. it is worth noting that the time point with the most genes off in both haplotypes is 16 h with 5238 genes. overall the data indicates that approximately 10,000 to 11,000 genes are on in both haplotypes at each time point while roughly 4000 to 5300 genes are off in both haplotypes at each time point. the number of genes on in one haplotype, while off in the other haplotype, ranges from about 400 to 1140 depending upon the haplotype and time point (fig 1) in order to better understand the cell biology underlying differences in macrophage differentiation and activation between b19 and b2 birds, we searched for genes exhibiting statistically significant differences between different time points within a single b-haplotype haplotype as well within the same time point between haplotypes. when comparing the expression profiles between the b2 and b19 haplotypes, we identified 210 genes exhibiting differential expression at the t-6 day time point. these genes represent 198 genes with higher expression in the b2 birds and just 12 genes for which expression was greater in the b19. after three days, at the t-3 day time point, thousands of genes exhibited altered expression patterns between the two groups. surprisingly, 7000 genes showed higher expression in the b19 birds while only 14 genes were expressed at higher levels in the b2 birds. by t0 hrs, which corresponds to 6 days of monocyte differentiation into macrophages, we observed 955 genes with significant expression patterns between the haplotypes. of these genes, 544 exhibited greater expression in the b2 haplotype while 411 exhibited higher expression in the b19 haplotype. cells were stimulated with ifnγ immediately following rna collection at the t0 hr time point. at 1 h (t1) post-stimulation 665 genes show evidence of significant patterns of expression between the haplotypes where b19 birds had 109 genes expressed to higher levels while the b19 haplotype was associated with 556 genes having greater expression compared to t0. this pattern of increased expression in the b19 group is reversed by the 2 h time point. at 2 h after ifnγ treatment, the b2 cells show a global increase in expression for 5989 genes while the b19 cells have just 18 genes on at higher levels than the b2 birds. by 4 hours after stimulation, the b2 birds still exhibit greater expression for 1029 genes while the b19 birds exhibit higher expression for 12 genes. this trend changes by 8 hours after treatment, at which time the slower responding b19 group begin showing increased expression in 797 genes while the b2 cells have greater expression for just 15 genes. by 16 hours after stimulation, only 66 genes are differentially expressed between the two haplotype groups. and, at the 24 hour mark, 406 genes show evidence of statistically significant differences in expression between them with the b2 cells exhibiting greater expression for 339 genes while the b19 cells have higher expression for 67 genes ( table 2) . the b2 and b19 haplotype birds represent distinct genetic variation within the b-locus on chromosome 16. subsequently, patterns of gene expression variation of the genes located within this region were investigated. among the seventeen genes exhibiting statistically significant differences in expression between the b2 and b19 birds, many displayed divergent gene expression patterns prior to ifnγ stimulation. in the b2 cells, gene expression peaks on day t-6 and expression is effectively inhibited by day t-3. this is not the case in the b19 cells. rather than reach maximum expression levels in a single day, the b19 cells don't achieve maximum expression until day t-3 ( fig 2) . for example trim7, trim27.1, bf2, tpn, and trim41 exhibit strong expression on day t-6 in the b2 cells while the same genes exhibited prolonged expression over day t-6 and day t-3 in the b19 cells. members of the trim (tripartite motif) family have been implicated in antiviral immune defense and several are ubiquitin ligases [47, 48] . tpn (tapasin) is a co-factor for mhc i critical for antigen presentation to cytotoxic t-cells and chickens express the single mhci locus termed bf-2 which is working with tpn in antigen presentation and it has been shown that there are differences in the selection of high affinity peptides in b19 vs b15 haplotypes [49] highlighting their critical role in immune competence. additional genes within the b-locus display a similar pattern of pre-stimulatory differences in gene expression between the two different haplotypes, including genes involved in differentiation, cell growth and apoptosis such as ptpn2 (tyrosine protein phosphatase non-receptor2) and nfkb. gene expression decreases to approximately baseline levels by time point t0 hours. a second distinction in the gene expression patterns between b19 and b2 cells is that b2 cells exhibited a fairly robust expression at 2 and 4 hours after interferon stimulation. unlike the b2 haplotype, the b19 haplotype appears incapable of generating such a rapid, robust and coherent gene expression profile. in contrast, the b19 cells generate a delayed, weak and uncoordinated lower level of expression that extends up to 8 hours, and in some cases even 16 distinct temporal gene expression patterns in b2 versus b19 monocytes/macrophages. b-locus haplotypes in chickens provide a mechanism for genetically perturbing the cluster of immunologically important genes on chromosome 16 and producing phenotypic variation affecting infectious disease susceptibility and resistance. the heat map allows visualization of gene expression between the two genetically distinct haplotypes. each row represents a gene within the b-locus (listed on the right) and each column corresponds to a particular time point when cells were collected for rna sequencing. black pixels indicate zero gene expression for a particular gene at a specific point in time, and dark blue corresponds to very low expression, while brighter blue indicates the next higher levels. dark purple represents higher expression levels than blue colors, and pink represents the highest levels of gene expression. monocytes were obtained from each haplotype of chicken and allowed to differentiate into macrophages in vitro for seven, days beginning on day minus 6 (t-6). rna was sampled on day t-6, day t-3, and again three days later which is denoted as 0 hours (t0), when ifnγ was initially added to the cultures. on t0, rna was sampled immediately before stimulation with ifnγ. subsequent time points correspond to the time following interferon stimulation, in hours (1 hour, 2 hours, 4 hours, 8 hours, 16 hours and 24 hours). as visible on the heat map, there are distinct differences in gene expression between the b2 and b19 cells. the most dramatic difference occurs on day t-6. b2 cells exhibit a rapid burst of gene expression, indicated as a single column of pink on the left most edge of the heat map. in contrast, the b19 cells appear to undergo a much slower and prolonged gene expression program that was not as rapidly down regulated as in genes in the b2 cells. additional gene expression data for a number of proteins involved in cell growth and apoptosis, is shown in the bottom half of the figure to highlight a similar pattern in gene expression and kinetics. the green border indicates the b2 haplotype expression pattern and the red border corresponds to the b19 expression pattern. hours. overall, this global pattern of temporally dysregulated gene expression represents a reoccurring theme with the b19 monocytes and macrophages. the divergent timing of gene expression observed in the b-locus genes is mirrored in many other genes as well, including members of the tlr signaling pathway, cellular mediators of apoptosis and cell survival, and components of cytokine signaling. the global dysregulation of gene expression among 700 genes at the t-3 day time point, as well as the expression pattern of 6000 genes exhibiting altered expression led us to explore the pattern of gene expression changes within each haplotype group over all of the time points. at the onset of the study, the b2 cells were actively expressing a diverse set of genes, however by the day t-3, most of those genes displayed reduced expression in the b2 group. even so, the b19 haplotype cells continue to express these 7000 genes at higher levels than the b2 birds. after stimulation, b2 macrophages again show different patterns of expression compared to b19 cells in regards to timing of peak expression and coherence of expression. four distinct patterns of divergent gene expression were identified between the b2 haplotype birds and the b19 haplotype birds (fig 3) . the first interesting divergent pattern shows strong gene expression on day t-6 in the b2 birds while relatively low levels of expression are observed in the b19 birds on the same day. this pattern is of interest because it represents a group of genes that are differentially regulated at the onset of the experimental time course. specifically, these genes include the macrophage m1 marker ptgs2, as well as the b-locus gene cyp21. other genes exhibiting this pattern include secreted interleukin ligands il-1β, il4i1, and il6, along with genes associated with inhibition of cellular processes including irg1 and mip-3α. interestingly, the adenosine receptor also displays this pattern of expression. these genes may represent initial modulators of divergent monocyte to macrophage differentiation between the b2 and b19 cells. the second example of divergent expression patterns is the single peak of day t-6 expression in the b2 haplotype cells compared to the prolonged multiple day expression in the b19 haplotype cells. some of these genes are macrophage differentiation mediators, like gata2 [50] , and fadd, while others are macrophage podosome (primary matrix structure) markers, including vcl and gsn. other genes exhibiting this divergent expression pattern include chemokine receptors, like cxcr4, fatty acid transport, such as slc25a17, and ubiquitin related factors, like dd5, which is associated with proteasomal degradation of gene products. additional interesting divergent gene expression patterns were observed between the two haplotypes occurring after stimulation by ifnγ (fig 3) . a notable difference in post-stimulatory induction of gene expression is a four-hour difference in peak expression timing for a large number of induced genes. in the b2 haplotype macrophages, the peak expression occurs between 2 and 4 hours, while in the b19 macrophages, the peak expression occurs between 4 and 8 hours. some of the most noticeable genes exhibiting this divergent gene expression pattern include litaf, il-1β, il12, and ifih1, genes involved in macrophage signaling and m1 macrophage polarization [26] . additionally, a number of genes implicated in invadosome assembly and function also exhibit this temporally displaced pattern of induction such as cd44, rac1, and src. another discernable difference in post-stimulatory induction of genes between the b2 macrophages compared to the b19 macrophages is one of coherence (fig 3) . specifically, there are a number of genes for which the b2 macrophages are able to rapidly turn on and reach relatively high levels of expression within 2 to 4 hours of ifnγ stimulation. in contrast, these same genes fail to exhibit a coherent peak of expression, even after 4 to 8 hours, in the b19 cells. instead, they exhibit a dispersed "smear" of gene expression extending from approximately 1 hour after stimulation to 16 hours post-stimulation. some of the most represented genes exhibiting this divergent pattern of expression include molecules involved in lysosome function and phagocytosis. cttn and actr3, genes implicated in fcr mediated phagocytosis, four distinct patterns were identified as representative of the types of divergent gene expression that re-occur across many genes involved in macrophage differentiation, activation and function in b2 versus b19 macrophages. 1. day t-6: b2 high vs b19 low. this divergent pattern exhibits strong expression of genes on day -6 in the b2 birds while relatively low levels of expression are observed in the b19 birds at the same time point. genes of interest include an adenosine receptor (p2ry12) 2. day t-6: b2 = 1 day vs. b19 = 3 days. this example of divergent patterns is the single peak of day t-6 gene expression in the b2 haplotype cells compared to the prolonged multiple day expression until day t-3 in the b19 haplotype cells. genes of interest include macrophage differentiation gene gata, adenosine receptor a2a and macrophage podosome markers vcl and gsn. macrophages. another interesting divergent gene expression pattern observed between the two haplotypes occurs after stimulation by ifnγ. there is a four-hour difference in peak expression timing for a large number of induced genes. in the b2 haplotype macrophages, the peak expression occurs between 2 and 4 hours, while in the b19 macrophages, the peak expression occurs between 4 and 8 hours. 4. maximum ifnγ stimulation: b2 = coherent vs. b19 = non-coherent another discernable difference in post-stimulatory induction of genes between the b2 macrophages compared to the b19 macrophages is one of coherence. specifically, there are a number of genes for which the b2 macrophages are able to rapidly turn on and reach relatively high levels of expression within 2 to 4 hours of ifnγ stimulation. in contrast, these same genes fail to exhibit a coherent peak of expression, even after 4 to 8 hours, in the b19 cells. instead, they exhibit a dispersed "smear" of gene expression extending from approximately 1 hour after stimulation to 16 hours post-stimulation. https://doi.org/10.1371/journal.pone.0179391.g003 along with lysosomal-associated molecules, like laptm5 and lamp1, as well as the lysosomal transporter molecules atp6ap1, atp6v1g1 and atp6v0c, exhibit this non-coherent pattern of expression in the b19 macrophages. in contrast, immediately following stimulation, the b2 cells rapidly induce expression of roughly 6000 genes by the 2 h following stimulation; while, at the same time, the cells derived from the b19 birds show no signs of induction among these genes until after 4 h. it is interesting to note that while the b2 birds show a statistically significant increase in expression for 6100 genes between 1 h and 2 h, the b19 cells exhibit increased expression for just 66 genes at this time point. the largest wave of increased gene expression occurs in the b19 cells during the transition from 2 h to 4 h post stimulation, when 1164 genes increase significantly over this time period. at the transition between 8 h and 16 h, the b2 haplotype group only exhibits differences in expression for 83 genes, with 44 having higher expression at the 16 h time point. yet, the b19 cells show differences in 386 genes during this same period, but interestingly, 356 of these genes exhibit decreased expression during this same time interval. taken together, these results suggest that a global disruption of temporal gene expression underlies the observed differences in differentiation, activation and nitric oxide production from macrophages derived from the two different mhc haplotypes. gene expression was measured in separate samples of b2 and b19 cells following stimulation with ifnγ. change in expression was assessed at 2 hours and 4 hours post stimulation. atp6v0c exhibited the greatest induction of all genes assayed, showing an increased expression in the b2 cells at 4 hours that was 20 times the initial expression at 0-hours. expression of atp6voc was dramatically less in the b19 birds. similarly, il18r exhibited greater than 9 times the initial expression in the b2 cells at 4 hours compared to the b19 cells which exhibited less than 2 times the initial expression at 0-hours. litaf and tlr2 exhibited more than 7 times the expression at 4 hours in the b2 macrophages, while tln-1, tlr-5, tlr-6 and tlr-7 exhibited greater than 4 times the initial expression in the b2 macrophages. in contrast, the b19 macrophages failed to exhibit comparable induction of these genes (fig 4) . in addition to the rt-pcr validation of gene expression, 54 genes, for which gene expression changes were described following cytomegalovirus stimulation were used as comparisons for the corresponding genes in the b2 and b19 haplotype birds (fig 5) . a total of 25 published genes exhibited decreases in expression following cytomegalovirus stimulation while 29 genes exhibited increased expression following stimulation. interestingly, all but one gene (fez1) in the b2 cells exhibited increased expression following ifnγ stimulation. in contrast, ten genes displayed decreased expression in the b19 cells. of the ten exhibiting fold-change < 0 in the b19 cells, 70% also exhibited decreased expression in the cytomegalovirus stimulated cells. in total, 28 genes (52%) expressed in the b2 cells matched the direction of the fold change reported in the published data while 33 genes (61%) corresponded between the b19 cells and the published data. of the ten published genes reported as having greater than 5-fold increased expression, 90% of the b2 genes exhibited fold-change in the same direction. overall, this data, in conjunction with the rt-pcr data, provides a comprehensive set of validation data providing evidence that the b2 and b19 gene expression data is reproducible and similar to expression patterns observed in cells stimulated towards macrophage activation pathway. visualization of gene expression via heat maps facilitated the identification of distinct expression patterns between the b2 and b19 haplotypes. because any initial differences in gene expression existing 6 days before ifnγ stimulation represent candidates responsible for the observed phenotypic differences between the two haplotypes. genes exhibiting divergent gene expression patterns between b2 and b19 birds on day -6 were identified (fig 6) . the genes cluster into four major clades (clade1, clade2, clade3, and clade4 with a singleton labelled clade 5). among these genes, represented in clade1 and clade2, are a number of mirnas exhibiting strong expression in b2 cells (mir-147, mir-146b, mir-1618, mir-200a, mir-1649, and mir-1648a) compared to the b19 samples. likewise, mirnas contained in clade3 and clade4 exhibit greater expression in b19 cells (mir-1627, mir-222b, mir-1633, and mir-19a). a number of small nucleolar rnas (snornas) exhibit similarly dichotomous gene expression patterns (clade4). for example, snord24, snoz40, snord74, snora17, and snord12 exhibit substantially higher levels of expression in b19 cells on day -6 compared to b2 cells (fig 6) . however, b2 cells also express snornas exhibiting divergent expression patterns between the two haplotypes, such as snou2_19 (clade2). in addition to non-coding rnas, divergent expression patterns are also observed with protein-coding rnas (fig 6) . for example, clade1 contains il6, il18, il-1β, ccl1, ptpn2 and mmp10, which exhibit higher initial expression on day -6 in the b2 birds. in contrast, the protein-coding genes lamp2, ubxn7, ube4b, pk3ca, ube2w, and cx3cr1, in clade3, exhibit higher initial expression patterns in b19 birds. clade5 contains the single gene ifnγ, which exhibits relatively low expression early in both b2 and b19 cells, but following stimulation rises to a higher level at 8 hours in the b19 birds. 54 genes, for which gene expression changes were previously described following cytomegalovirus stimulation were used as comparisons for the corresponding genes in the b2 and b19 haplotype birds. a total of 25 published genes exhibited decreases in expression following cytomegalovirus stimulation while 29 genes exhibited increased expression following stimulation. all but one gene (fez1) in the b2 cells exhibited increased expression following ifnγ stimulation. in contrast, ten genes displayed decreased expression in the b19 cells. of the ten exhibiting fold-change < 0 in the b19 cells, seven exhibited decreased expression in the cytomegalovirus stimulated cells. twenty-eight genes (52%) expressed in the b2 cells matched the direction of the fold change reported in the published data while 33 genes (61%) corresponded between the b19 cells and the published data. of the ten published genes reported as having greater than at least 5-fold increased expression, 90% of the b2 genes exhibited fold-change in the same direction. considering the diverse expression patterns discovered and the results indicating the involvement of non-coding rnas, further results including divergent non-coding rna expression will be described in more detail in further publications. enrichment analysis of genes exhibiting statistically significant differences in expression between time points and/or haplotypes (table 3 , s2, s3 and s4 tables) was performed using gene ontology and both kegg and reactome pathways. the results of the gene enrichment identification of divergent gene expression patterns between b2 and b19 macrophages. visualization of divergent gene expression patterns between the b2 and b19 haplotypes. a subset of genes exhibiting divergent gene expression were identified and visualized in heat following hierarchical clustering of the genes (rows), but not the time points (columns). the genes cluster into four major clades (clade1, clade2, clade3, and clade4) with a singleton gene (labelled clade 5). among these genes, represented in clade1 and clade2, are a number of mirnas exhibiting strong expression in b2 cells (mir-147, mir-146b, mir-1618, mir-200a, mir-1649, and mir-1648a) compared to the b19 samples. likewise, mirnas contained in clade3 and clade4 exhibit greater expression in b19 cells (mir-1627, mir-222b, mir-1633, and mir-19a). additionally, a number of small nucleolar rnas (snornas) exhibit similarly dichotomous gene expression patterns (clade4) such that snord24, snoz40, snord74, snora17, and snord12 exhibit substantially higher levels of expression in b19 cells on day -6 compared to b2 cells while b2 cells express such as snou2_19 (clade2). https://doi.org/10.1371/journal.pone.0179391.g006 provided a high-resolution perspective of the functional role of mrna sequenced within the b2 cells across the experimental time points. between the -6 day and -3 day time points, a large number of genes exhibit reduced expression. these genes are enriched for biological processes such as transcription, mrna splicing, the transition from day -3 to t0 (just prior to ifnγ stimulation) correlates with down regulation of genes associated with chromosome segregation, mitotic nuclear division and dna repair, cell cycle pathways, acetylation and some immunological functions of interleukin 3 and 5 signaling and interleukin receptor signaling. genes exhibiting increases in expression during this interval were enriched in processes and pathways related to interleukin 4, biosynthesis of amino acids, and metabolic pathways. within an hour of ifnγ stimulation genes exhibiting increased expression were associated with inflammatory and defense responses, toll-like receptor signaling pathways, cell chemotaxis, myd88 signaling, jak-stat signaling, cell adhesion, foxo signaling, and raf activation. genes exhibiting an increase in expression within two hours of ifnγ stimulation are enriched for biological processes of intracellular protein transport, er-to-golgi vesical mediated transport, endosome to lysosomal transport, chromatin remodeling, histone h3 acetylation, regulation of vesicle fusion and protein import into the nucleus. among the kegg pathways that exhibit enrichment for these genes are rna transport, protein export, lysosome, mrna surveillance pathway, endocytosis and mtor signaling. similarly, the enriched reactome pathways mirror these processes and include cytosolic sensors of pathogen-associated dna, rna polymerase ii initiation and promoter clearance, mtorc1-mediated signaling, map2k and mapk activation, downstream tcr signaling, fcε receptor 1 mediated mapk activation, and clec7a signaling. genes exhibiting decreased expression between 2 hours and 4 hours following ifnγ stimulation correspond to reduced expression of cell-cycle pathways and mediators, as well as genes implicated in g1/s transcription, dna replication, and separation of sister chromatids. biological processes identified within these genes include mitotic spindle assembly, chromosome segregation, and mitotic spindle checkpoint assembly. conversely, genes exhibiting increased expression during this same time are enriched for biological processes of inflammatory response, defense response to virus, positive regulation of interleukin 12 production, negative replication of viral genome replication, bacterial defense processes and positive regulation of il1α secretion. kegg pathways associated with these genes include cytokine-cytokine receptor interaction and genes implicated in influenza a signaling. reactome processes identified included stem cell population maintenance and tor signaling. over the remaining time points, from 4 hours to 8 hours, from 8 hours to 16 hours and from 16 hours to 24 hours the b2 cells exhibit a systemic down regulation of the genes that were initially activated during the ifnγ stimulation. overall, the gene enrichment analysis of the rna sequence data provides a cellular-level picture of the specific biological processes that occur over time following activation of monocyte-derived macrophages. previous work in our laboratories investigated the association between chicken haplotype and disease resistance, specifically the enhanced resistance of b2 haplotypes to avian coronavirus ibv [10] and the influence of innate immunity leading to decreased clinical signs of illness. we showed that macrophages play an important role in this enhanced immunity, demonstrating much better activation in response to stimulation [13] . to analyze the gene expression involved in this process leading to increased macrophage nitric oxide release in b2 haplotypes, we stimulated macrophages from b2 and b19 chicks for rna sequencing. in addition, we had observed different cell morphology when isolated monocytes from b2 and b19 haplotypes were differentiating into macrophages and therefore time points before stimulation, during differentiation of the macrophages, were included in this study. the rationale for investigating the gene expression differences between the b2 and b19 haplotype birds was to address underlying questions that were raised at the end of our previous studies: 1. why do the ifnγ stimulated b19 derived macrophages exhibit decreased nitric oxide production compared to the ifnγ stimulated b2 derived macrophages? 2. how do the two lineages of macrophages differ at the gene expression level? 3. what specific patterns of gene expression correlate with divergent macrophage differentiation, activation and function? 4. what is the underlying cause of the divergent gene expression patterns observed between b2 and b19 macrophages? the data collection, analysis and interpretation of results described herein provide plausible answers to these questions based on bioinformatics and functional genomics approaches. although these answers are more realistically new hypotheses for further investigation, they do represent significant advances in the understanding of b2 and b19 monocyte differentiation into macrophages and the resulting divergent patterns of b2 and b19 macrophage activation and function. ultimately, the findings and interpretations we report must be functionally and experimentally validated. even so, the use of computational methods to answer these questions represents a valuable first step in deciphering the cellular phenotypes underlying mhc haplotype variation in macrophage cells. our results demonstrate that there are large numbers of genes differentially expressed in the two haplotypes, both during differentiation of peripheral monocytes into mature macrophages, as well as after stimulation of differentiated macrophages with interferon. the answer to the question of why the ifnγ stimulated b2 haplotype cells produce more nitric oxide than the ifnγ stimulated b19 cells lies in the timing of macrophage differentiation and the phenotypic variation that is set up early prior to ifnγ stimulation, such as divergent expression of genes involved in differentiation and immune competence. at day t-6, after plating of monocytes without interferon stimulation, several genes relating to inflammation, interferon responses and differentiation are upregulated in the b2 haplotype. this is to be expected as adherence of the monocytes is actually an activation signal, but it is notable that this signal is not resulting in the same gene expression pattern in the b19 haplotype. this pattern can be observed for genes such as il1β, ptsg2, il6, which are mainly associated with the inflammatory m1 phenotype. on the other hand, adenosine receptor a2b is also showing increased gene expression at this time in b2 cells, and this receptor plays an important role in differentiation, as well as in the inflammatory response. expressions of these genes are consequently initially increased at the time of adherence, and then again after stimulation with interferon, in the b2 haplotype. some, but not all of these genes are expressed after stimulation with interferon in the b19 haplotype, but not to the same extent as the b2 macrophages, which appears to relate to the initial lack of expression at t-6 days, the beginning of differentiation. another interesting observation was the differential expression of genes at day t-6 versus day t-3 in the two haplotypes, as a large number of genes is highly expressed in both haplotypes at day t-6, but then is completely shut down in b2 haplotypes while showing delayed expression until day t-3 in the b19 birds. this seems to indicate a lack of appropriate regulation in the b19 birds, consequently leading to less coherent initiation of gene expression when stimulated. some of the genes showing this pattern are macrophage differentiation associated gata2 and fadd, as well as macrophage podosome markers vcl and gsn. taken together, these results appear to suggest that the regulation of b2 differentiation from monocyte to macrophage is very tightly regulated with many genes increasing in expression, quickly followed by shutting down this increased gene expression. in contrast, the regulation of b19 gene expression is not well regulated, appearing to "linger" with either delayed or extended gene expression. consequently, we observed differences in expression of genes after stimulation with interferon. specifically, genes that were strongly expressed at day t-6 and not expressed (or only weakly expressed) at day t-3 in the b2 birds, were robustly increased at 2 and 4 hours of stimulation. in contrast, the same genes showed weak and delayed expression in b19 birds after stimulation, emphasizing the importance of the regulation of gene expression during differentiation. this relates very well to the differences we previously reported in morphology of b2 and b19 macrophages during differentiation and after stimulation. our results provide insight into the complexity associated with macrophage differentiation, activation and function. thousands of genes are up-regulated and then down-regulated in a 24-hour period following ifnγ stimulation. the coordinated activity of multiple regulatory and gene expression control mechanisms is required to effectively achieve the dramatic changes in internal cellular programing that occur. although the b2 and b19 birds' haplotypes differ within the mhc locus, the functional consequences of this genetic difference extend well beyond the genes encoded within the b-locus, including macrophage differentiation, m1 and m2 macrophage markers, lysosomal factors involved in phagocytosis, podosome development, invadosome capabilities, chemotaxis potential and matrix degradation ability. additionally, during the process of differentiation and activation, thousands of genes associated with basic cellular biology undergo rapid changes in expression in coordination with the expression of factors associated with cell renewal and proliferation such as cell cycle regulators, mitotic spindle components, factors involved in chromatin remodeling, molecules required for chromosome segregation and nuclear division. taken together, our data and interpretations provide a framework of possible mechanisms of b2 and b19 macrophage biology in differentiation and activation. as such, our findings offer a number of hypotheses about macrophage cell biology that can be used for subsequent studies aimed at validating our findings. although we performed rt-pcr on a set of differentially expressed genes between the b2 and b19 macrophages, it is not feasible, or possible, to systematically verify, via rt-pcr, each and every transcript, observed at each time point, in the experiment. even so, our pcr validation provides independent evidence that the pattern of gene expression we observed in the rna sequence data, was consistent and reproducible which is also in line with our previous research detailing differences in macrophage activation and function [13] conclusions we have tried to elucidate possible mechanisms involved in enhanced disease resistance and macrophage functions displayed by b2 haplotype chickens compared to b19. this study highlights the complex gene expression patterns involved in macrophage differentiation and activation. one of the main conclusions from the large number of differences seen in the gene expression of the two haplotypes is the fact that there are not just a few genes or genetic markers that can be readily identified as being the ultimate cause of enhanced macrophage function in b2 chickens. rather, it appears that events during differentiation of monocytes into macrophages have a significant impact on the subsequent ability for stimulation of immune genes after ifnγ treatment. the differences in gene expression correlate with the previously observed differences in morphology of the two haplotypes, with b2 macrophages having a more typical macrophage appearance [13] . considering the global temporal dysregulation of many genes in b19 haplotypes compared to the more resistant b2 chicks, it seems likely that a variety of genomic regulatory mechanisms (such as transcription factors, mirnas, snornas, ubiquitin mediated proteasomal degradation, and epigenetic regulation) might play a major role in this process which will be further detailed in a future publication. it will be of great interest to further elucidate these mechanisms and their connection to enhanced immunity. ultimately, our detailed model of macrophage differentiation, activation and function following ifnγ stimulation provides a high resolution molecular map of cellular biology which can be leveraged by other investigators to further explore the role of these genes in immunology. supporting information s1 table. significant differences in gene expression with p-values. pairwise gene expression differences between samples (b2 and b19 haplotype) and timepoints (-6 days, -3 days, 0 days, 1 hr, 2 hrs, 4 hrs, 8 hrs, 16 hrs, 24 hrs) are provided with p-values in excel format. the analysis described within this manuscript focused on differences between [1] matched time points between b2 and b19 haplotype chickens (such as b2 1 hr versus b19 1 hr) as well as [2] progressive timepoints within the same haplotype group (such as b2 1hr versus b2 2hr and b19 4 hr versus b19 8 hr). subsequently the data contained in this supplemental file also focuses predominantly on those comparisons. this file contains a total of 163,043 rows including the header line containing field names (genename-identifier for each gene either as gene symbol or ensemble geneid; locus-chromosome number and the start-end base pair location of the gene; sample1 and sample2-the "paired" samples compared for significant gene expression, note 'ab' corresponds to b2 haplotype and 'ec' corresponds to b19 haplotype; teststatusindication that the analysis method performed by cuffdiff program within the cufflinks package was 'ok'; fpkm1 and fpkm2-the fpkm values for sample 1 and sample 2 respectively; log2fpkm-the log of the ratio of fpkm1 and fpkm2; teststat-the test statistic generated during the statistical analysis; pvalue-the p-value corresponding to the difference in expression between sample1 and sample2; qvalue-a multiple testing corrected p-value; signif-'yes ' indicates that the pairwise difference in expression is in fact statistically significant). (xlsx) s2 table. significant gene ontology biological process enrichment analysis results. enriched gene ontology biological process terms identified within up and down regulated genes within the b2 haplotype chicken samples across progressive time points (-6 days, -3 days, 0 days, 1 hr, 2 hrs, 4 hrs, 8 hrs, 16 hrs, 24 hrs) are provided in excel file format. since the b2 chickens exhibited the most robust macrophage phenotype these samples were used for the analysis as a means of characterizing the biological processes that were associated with the altered gene expression across the experimental time points. note 'ab' indicates b2 haplotype. the file contains a total of 362 rows including the header line containing field names (sample comparison-indicates the specific pair of time points for which gene expression changes were identified; gene set-indicates the specific set of differentially expressed genes, 'down' or 'up'; category-indicates the specific subset of terms that were used for the analysis; term -provides the gene ontology identifier for the identified gene ontology term/annotation; description-the specific enriched gene ontology biological process term; gene count-the number of genes within the differentially expressed genes that are mapped to the particular enriched gene ontology term; %-the corresponding percent associated with the specific number of genes; p-value-the p-value associated with the gene ontology term enrichment). (xlsx) s3 table. significant kegg pathway enrichment analysis results. since the b2 chickens exhibited the most robust macrophage phenotype these samples were used for the analysis as a means of characterizing the kegg pathways that were associated with the altered gene expression across the experimental time points. note 'ab' indicates b2 haplotype. the file contains a total of 110 rows including the header line containing field names (sample comparison-indicates the specific pair of time points for which gene expression changes were identified; gene set-indicates the specific set of differentially expressed genes, 'down' or 'up'; categoryindicates the specific subset of terms that were used for the analysis; term-provides the kegg pathway identifier for the identified pathway term/annotation; description-the specific enriched kegg pathway term; gene count-the number of genes within the differentially expressed genes that are mapped to the particular enriched kegg pathway term; %-the corresponding percent associated with the specific number of genes; p-value-the p-value associated with the kegg pathway term enrichment). (xlsx) s4 table. significant reactome pathway enrichment analysis results. since the b2 chickens exhibited the most robust macrophage phenotype these samples were used for the analysis as a means of characterizing the reactome pathways that were associated with the altered gene expression across the experimental time points. note 'ab' indicates b2 haplotype. the file contains a total of 110 rows including the header line containing field names (sample comparison -indicates the specific pair of time points for which gene expression changes were identified; gene set-indicates the specific set of differentially expressed genes, 'down' or 'up'; category -indicates the specific subset of terms that were used for the analysis; term-provides the reactome pathway identifier for the identified pathway term/annotation; description-the specific enriched reactome pathway term; gene count-the number of genes within the differentially expressed genes that are mapped to the particular enriched reactome pathway term; %-the corresponding percent associated with the specific number of genes; p-valuethe p-value associated with the reactome pathway term enrichment). association of inos, trail, tgf-beta2, tgf-beta3, and igl genes with response to salmonella enteritidis in poultry. genetics, selection, evolution: gse tumour necrosis factor-alpha-857t allele reduces the risk of hepatitis b virus infection in an asian population heterophils isolated from chickens resistant to extra-intestinal salmonella enteritidis infection express higher levels of pro-inflammatory cytokine mrna following infection than heterophils from susceptible chickens profiling pro-inflammatory cytokine and chemokine mrna expression levels as a novel method for selection of increased innate immune responsiveness association of the major histocompatibility complex with avian leukosis virus infection in chickens host age and major histocompatibility genotype influence on rous sarcoma regression in chickens 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polyunsaturated fatty acids increase the binding of lipopolysaccharide to macrophages toll-like receptor 2 and 4 surface expressions on human monocytes are modulated by interferon-gamma and macrophage colony-stimulating factor. immunology letters triggering of toll-like receptors modulates ifn-gamma signaling: involvement of serine 727 stat1 phosphorylation and suppressors of cytokine signaling 1 and icsbp control constitutive and ifn-gamma-regulated tlr9 gene expression in mouse macrophages development of monocytes, macrophages, and dendritic cells shaping of monocyte and macrophage function by adenosine receptors a(2b) adenosine receptors in immunity and inflammation an avian, oncogenic retrovirus replicates in vivo in more than 50% of cd4+ and cd8+ t lymphocytes from an endangered grouse a dna vaccine expressing env and gag offers partial protection against reticuloendotheliosis virus in the prairie chicken (tympanicus cupido) differential nitric oxide production by chicken immune cells non-replicating adenovirus vectors expressing avian influenza virus hemagglutinin and nucleocapsid proteins induce chicken specific effector, memory and effector memory cd8(+) t lymphocytes. virology profile of toll-like receptor expressions and induction of nitric oxide synthesis by toll-like receptor agonists in chicken monocytes transcriptome analysis reveals human cytomegalovirus reprograms monocyte differentiation toward an m1 macrophage gata6 regulates aspartoacylase expression in resident peritoneal macrophages and controls their survival trim family proteins: retroviral restriction and antiviral defence evolution of a cytoplasmic tripartite motif (trim) protein in cows that restricts retroviral infection a mechanistic basis for the co-evolution of chicken tapasin and major histocompatibility complex class i (mhc i) proteins we would like to thank the vivarium staff of western university of health sciences and the sequencing facilities staff at the university of delaware for their work. we would also like to thank rich upshaw and richard applebee at western university, and christopher sullivan, at oregon state university, who helped acquire, set up and maintain the computational hardware required to complete this project. additionally, the authors wish to thank paul gettler for his expertise and time in helping with figures for this paper.visualization: yd ki. writing -review & editing: yd ki. conceptualization: ki yd ec. key: cord-333522-zsdymkjd authors: gruse, jeannine; kanitz, ellen; weitzel, joachim m.; tuchscherer, armin; stefaniak, tadeusz; jawor, paulina; wolffram, siegfried; hammon, harald m. title: quercetin feeding in newborn dairy calves cannot compensate colostrum deprivation: study on metabolic, antioxidative and inflammatory traits date: 2016-01-11 journal: plos one doi: 10.1371/journal.pone.0146932 sha: doc_id: 333522 cord_uid: zsdymkjd immaturity of the neonatal immune system is causative for high morbidity in calves and colostrum intake is crucial for acquiring passive immunity. pathogenesis is promoted by reactive oxygen species accumulating at birth if counter-regulation is inadequate. the flavonol quercetin exerts antioxidative and anti-inflammatory effects that may enhance neonatal health. the aim of this work was to study effects of quercetin feeding on metabolic, antioxidative and inflammatory parameters in neonatal calves to investigate whether quercetin could compensate for insufficient colostrum supply. twenty-eight newborn calves were assigned to two dietary groups fed colostrum or milk-based formula on day 1 and 2 and milk replacer thereafter. from day 2 onwards, 7 calves per diet group were additionally fed quercetin aglycone (50 mg/(kg body weight × day)). blood samples were taken repeatedly to measure plasma concentrations of flavonols, glucose, lactate, total protein, albumin, urea, non-esterified fatty acids, triglycerides, cholesterol, insulin, glucagon, cortisol, immunoglobulins, fibrinogen, haptoglobin and serum amyloid a. trolox equivalent antioxidative capacity, ferric reducing ability of plasma, thiobarbituric acid reactive species and f2-isoprostanes were analyzed to evaluate plasma antioxidative status. expression of tumor necrosis factor, interleukin-1α, interleukin-1β, serum amyloid a, haptoglobin, fibrinogen, c-reactive protein, catalase, glutathione peroxidase and superoxide dismutase mrna were measured in liver tissue on day 8. plasma flavonol concentrations were detectable only after quercetin-feeding without differences between colostrum and formula feeding. plasma glucose, lactate, total protein, immunoglobulins, triglycerides, cholesterol, trolox equivalent antioxidative capacity and thiobarbituric acid reactive species were higher after colostrum feeding. body temperature, fecal fluidity and plasma concentrations of cortisol and haptoglobin were higher in formulathan in colostrum-fed groups. hepatic mrna expression of tumor necrosis factor was higher after quercetin feeding and expression of c-reactive protein was higher after formula feeding. data confirm that colostrum improves neonatal health and indicate that quercetin feeding cannot compensate for insufficient colostrum supply. calfhood diseases play a key role in the economy of dairy farms because they increase operating costs and reduce long-term productivity of the animal. incidence of disease is associated with increased mortality rates [1] , and enteritis is the most common diagnosis in young calves [2] , which, according to svensson, linder and olsson [3] , contributes to 23% of calf losses during the first 14 days of life. neonatal calves are prone to sickness because their immune system is immature. furthermore, the process of birth itself causes an elevated stress level for the newborn and exposure to an oxygen-rich environment leads to an increased generation of reactive oxygen species [4, 5] . reactive oxygen species induce peroxidation of lipids and other macromolecules, leading to alteration of cellular components, interaction with signaling cascades and modification of physiological cell functions [6] . if not properly counterbalanced by antioxidative defenses, excessive production of reactive oxygen species results in oxidative stress, which is a cofactor of disease in humans and farm animals [5, 7, 8] . adequate colostrum supply is vital to calves because colostrum ensures ingestion of nutrients and contains immunoglobulins (ig), peptides, antioxidants and other bioactive factors supporting maturation, antioxidative and immune defense as well as local intestinal immunity [9] . the ban on antibiotic performance promoters by the european union in 2006 increased efforts to establish natural alternatives to enhance health and productivity in breeding. special focus has been directed to phytochemicals because their use can be manifold according to the respective compound [10] . flavonoids are secondary plant metabolites that are widely distributed in the plant kingdom and are able to modulate inflammation and immune function and exert antioxidative activity [11] [12] [13] . quercetin, which belongs to the subclass of flavonols, is ubiquitous in most plants and is of interest for scientists for its beneficial use in humans and farm animals. its antioxidative capacity can ameliorate the acquisition of passive immunity in neonates, based on the finding that feeding antioxidant-enriched colostrum enhanced igg absorption and antioxidative status in newborn calves and piglets [14, 15] . similarly, retskii et al. [16] showed that correcting the antioxidative balance in newborn calves prior to first colostrum ingestion increases the acquisition of colostral immunity and reduces the incidence of enteric colibacillosis. another beneficial effect of quercetin is its local action in the gastrointestinal tract. in vitro studies of intestinal epithelium demonstrated that quercetin down-regulates the expression of genes related to inflammation in inflamed epithelium [17] , and lozoya et al. [18] showed in a clinical study that oral quercetin administration reduced abdominal pain in acute diarrheic disease in humans. in guinea pigs, mice and rats, the inhibitory action of quercetin on prostaglandin e 2induced ileal contractions and on castor-oil-induced diarrhea has been demonstrated [19, 20] . furthermore, quercetin acts as a prebiotic, thus inhibiting adhesion of enteropathogens to caco-2 cells without affecting the viability of probiotics [21] , and improves performance in hens by modulating cecal microflora populations [22] . although a multitude of research on quercetin has been performed in vitro or in animal models for medical conditions, studies of the effects in neonatal farm animals are scarce. the aim of the present work was to investigate the potential health-promoting effects of feeding quercetin to newborn calves during the first week of life and to evaluate whether the healthpromoting effects of quercetin compensate for initial colostrum deprivation in calves. we hypothesized that quercetin improves antioxidative balance and immune function and that local antibacterial and anti-inflammatory effects reduce the incidence of diarrhea and gastrointestinal dysfunctions. experimental procedures were conducted in compliance with the german animal protection regulations with approval of the authorities of the state mecklenburg-western pomerania, germany (landesamt für landwirtschaft, lebensmittelsicherheit und fischerei mecklenburg-vorpommern; lallf m-v/tsd/7221.3-1.1-044/12). liver biopsies were performed under local lidocaine anesthesia and all calves received metamizole post-operatively for pain relief. twenty-eight male holstein friesian calves were separated from their dams immediately after birth and were housed in single boxes during their first 8 days of life. before the trial started, separate colostrum pools were prepared from the first and third milkings after parturition. according to the colostrums' macronutrient compositions, milk-based formulas with comparable amounts of macronutrients [23] but without bioactive factors were provided (bergin mat-formula; bergophor futtermittelfabrik, kulmbach, germany). calves were randomly assigned to two dietary groups and were bucket-fed twice daily, receiving either colostrum (col, n = 14) or corresponding formula (for, n = 14) on days 1 and 2 of life (10% and 12% of body weight/day, respectively). if appetite was reduced, calves were tube fed to ensure complete ingestion of colostrum or formula. from day 3 until day 8, all calves were fed commercial milk replacer (12% of body weight/day; 150 g/l; salvalac mirapro 45; salvana tiernahrung, klein-offenseth sparrieshoop, germany). on day 2, the dietary groups were subdivided into control (colq-and forq-; n = 7 per group) and treatment groups (colq+ and forq+; n = 7 per group), the latter receiving quercetin aglycone twice daily with feeding (50 mg/(kg body weight × day); quercetin aglycone dihydrate 98%, carl roth, karlsruhe, germany). the control groups received no quercetin aglycone. treatment. navels were disinfected with 10% povidone iodine solution (vet-sept; animedica, senden-bösensell, germany). neonatal calves received an oral dose of 1 g iron dextran with their first meal on day 1 (ursoferran; serumwerk bernburg, bernburg, germany). to support immunological defense during the first 5 days of life, all calves received chicken eggderived immunoglobulins with the morning feeding (0.25 g/kg body weight; globigen life start 25%, ew nutrition, visbek, germany) [24] . to prevent cryptosporidiosis, calves were treated with halofuginone (0.1 mg/kg body weight per os; halocur, intervet, igoville, france) after the evening feeding from day 1 to day 7. colostrum-deprived calves (forq+, forq-) additionally received b-vitamins (100 mg nicotinamide/calf, 40 mg thiamin chloride hydrochloride/calf, s.c.; vitamin-b-komplex, serumwerk bernburg, germany) and bovine colostral immunoglobulins on days 1 (s.c.), 3 and 5 (per os) (2 g gammaglobulins/calf with antibodies against escherichia coli, rotavirus, coronavirus; aniserin orinject; animedica, seden-börsensell, germany). furthermore, formula-fed calves were treated metaphylactically with colistin sulfate from day 2 to day 8 (3 mg/kg body weight, i.m.; belacol; belapharm, vechta, germany). all calves were weighed immediately after birth and before evening meals on days 2 and 6. every morning, health status was examined and appetite, general condition, heart rate, respiratory rate, rectal temperature and gut motility were assessed. fecal fluidity was scored according to larson et al. [25] . calves with reduced vitality after the first 2 days of life were allowed one recovery day before further sample taking. in these cases, the times referred to as days 3, 4, 7 and 8 in the results section are days 4, 5, 8 and 9 after birth, respectively. due to gastrointestinal imbalances, two calves (one calf of group colq+ and one calf of group forq+) had to be excluded from the study. sample taking. basal blood samples were taken before the morning feeding on days 1, 2, 4 and 7 from the jugular vein using evacuated tubes containing either potassium-edta (1.2-2 mg/ml edta) for analyses of plasma metabolites, insulin, glucagon, immunoglobulins and acute-phase proteins or li-heparin (12-30 iu heparin) for the determination of the cortisol and flavonol concentrations and the antioxidative status in the plasma. for flavonol analysis, additional blood samples were taken before the morning feeding on days 3 and 8. after centrifugation (1,500 × g, 4°c, 20 min), plasma aliquots were stored at -20°c until analyses (-80°c for analyses of flavonol concentrations and antioxidative status, respectively). plasma flavonols, metabolites and hormones. plasma concentrations of flavonols (quercetin, isorhamnetin, tamarixetin and kaempferol) were measured via hplc as previously described [26] with a detection limit of 2 nmol/l and a recovery rate of 92 ± 2%. the intra-and inter-assay coefficients were 0.5 and 7.2%, respectively. plasma metabolites were analyzed using an automatic spectrophotometer (abx pentra 400; horiba abx, montpellier, france) and respective kits: glucose (#a11a01667), lactate (#a11a01721), albumin (#a11a01664) and triacylglycerides (#a11a01640) from horiba abx, montpellier, france; total protein (#553-412) and cholesterol (#553-127) from mti-diagnostics, idstein, germany; urea (#lt-ur 0010) from labor+technik, e. lehmann, berlin, germany; and non-esterified fatty acids (#434-91795, #436-91995) from wako chemicals, neuss, germany. plasma concentrations of insulin (#ria-1257) and glucagon (#ria-1258) were determined by ria using kits from drg instruments, marburg, germany, which were adapted to bovines [27] . intra-and inter-assay coefficients of variation were 3.7% and 5.5% for insulin and 3.4% and 22.5% for glucagon, respectively. plasma cortisol concentrations were analyzed in duplicate after extraction with diethylether using a commercially available elisa kit (#eia1887; drg instruments gmbh, marburg, germany) according to the instructions of the manufacturer. cross reactivities of the antibody to corticosterone and progesterone were 45% and 9%, respectively, and <2% to any further competing plasma steroids. the assay was validated for use with bovine plasma. the test sensitivity was 3.4 μg/l, and intra-and inter-assay coefficients of variation were 5.3% and 12.1%. immunoglobulins (ig) and acute-phase proteins. the concentrations of igg1, igg2 and igm, as well as acute-phase proteins (haptoglobin, serum amyloid a and fibrinogen), were measured in the edta plasma samples taken on days 1, 2, 4 and 7. igg1 was analyzed by radial immunodiffusion [28] (modified by gasowska and stefaniak [29] ) using bovine reference serum (rs10-103; bethyl laboratories inc., montgomery, usa) as standard. igg2 (#e10-117) and igm (#e10-101) were determined by elisa using kits from bethyl laboratories inc., montgomery, usa. intra-assay coefficients of variation were 10.9% and 4.0% for igg2 and igm, respectively. the detection limit of igg2 was 7.8 μg/l and that of igm was 15.6 μg/l. for detection of serum amyloid a (saa; #tp-802), we used a multispecies elisa kit from tridelta development, maynooth, ireland. the detection limit of saa was 9.4 mg/l. the intra-assay coefficient of variation was 12.0%. the haptoglobin concentration was analyzed using the guaiacol method developed by jones and mould [30] with human haptoglobin hp 2-2 (sigma #h9762) as a standard. the detection limit of haptoglobin was 0.01 g/l. plasma fibrinogen was determined by rapid heat precipitation according to millar, simpson and stalker [31] . antioxidative status. li-heparinized plasma samples taken on days 1, 4 and 7 were used to analyze the trolox-equivalent antioxidative capacity (teac, as trolox equivalents (te) in mmol/l) and ferric reducing ability of plasma (frap, as ascorbic acid equivalents, (asce) in μmol/l) as parameters of antioxidative capacity as well as thio-barbituric acid reactive species (tbars, as malondialdehyde equivalents (mdae) in μmol/l) and 8-iso-pgf 2α (f2-isoprostanes, in ng/l) as markers for oxidative stress. teac was analyzed as described by miller et al. [32] and modified by re et al. [33] . frap and tbars were determined according to luehring et al. [34] . for determination of f2-isoprostanes, a commercial elisa kit (#adi-900-091; enzo life sciences, lause, switzerland) was used. cross-reactivities of the assay to pgf 1α and pgf 2α were 4.6% and 1.85%, respectively, and <1% to any further eicosanoids. on day 8, a liver biopsy was conducted 2 h after the morning meal using a custom-made biopsy trocar [23] . biopsy tissue was immediately frozen in liquid nitrogen and stored at -80°c until further analysis. hippocalcin-like 1 (hpcal1; nm_001098964), low-density lipoprotein 10 (lrp10; bc149232) and rna polymerase ii (polr2a; nm_001206313.1) were used as reference genes (given accession numbers related to nih genbank). primer sequences, accession numbers and pcr conditions for target genes related to antioxidative status (catalase (cat); glutathione peroxidase (gpx1); superoxide dismutase (sod)) and inflammation (tumor necrosis factor (tnf); interleukin-1α and -1β (il1a, il1b); haptoglobin (hp); fibrinogen (fga); serum amyloid a2 (saa2); and c-reactive protein (crp)) are listed in table 1 . as recently described [23] , primer products were verified by sequencing using the bigdye terminator v1.1 cycle sequencing kit and an abi 3130 genetic analyzer (life technologies, carlsbad, usa). real-time pcr was performed using a lightcycler (roche molecular biochemicals, mannheim, germany); sybr green i was used as the fluorescent dye. melting curve analysis and agarose gel electrophoresis were used to confirm the specificity of the pcr products. quantification cycle values and amplification efficiencies obtained using linregpcr version 2013.0 [35] were imported into qbase+ version 2.6.1 (biogazelle, gent, belgium) for all subsequent calculations and quality controls. the geometric mean of the reference gene abundances was used for normalization. data are presented as the ratio of the copy numbers of genes of interest and the geometric mean of the reference genes' abundances. statistical analyses were conducted using sas software, version 9.3 for windows, sas institute inc., cary, usa. descriptive statistics and tests for normality were calculated using the uni-variate procedure of base sas software. body weight, average daily weight gain and data for hepatic gene expression were analyzed by anova with the mixed procedure of sas/ stat taking a model with the fixed factors diet (levels: col vs. for), quercetin (levels: q+ vs. q-) and the interaction diet×quercetin. feed intake, body temperature, heart and respiratory rate and plasma concentrations of metabolites, hormones, flavonols and markers of antioxidative status were analyzed by repeated measurement anova using the mixed procedure of sas/stat software and a model with the fixed factors diet, quercetin and day of life (repeated variable) and all interactions between the fixed factors. repeated measures on the same calf were taken into account using the repeated statement of the mixed procedure and a type for the block diagonal residual covariance matrix chosen in dependence on the levels of day of life. for concentrations of plasma metabolites, hormones and data on antioxidative status in plasma, an unstructured type was used. for the plasma concentrations of flavonols, acutephase proteins and ig, a compound symmetry type was used. for data on heart and respiratory rate, as well as body temperature and feed intake, an autoregressive (1) type was applied. leastsquares means (lsm) and their standard errors (se) were computed for each fixed effect in the models and all pairwise differences of lsm were tested by the tukey-kramer procedure. the slice statement of the mixed procedure was used to conduct partitioned analyses of the lsm for interactions. fecal score was analyzed by a generalized linear mixed model using the glimmix procedure of sas/stat and a poisson model with the fixed factors diet, quercetin and day of life (repeated variable) and all interactions between these fixed factors. repeated measurements on the same animal were taken into account by the residual option of the random statement of the glimmix procedure using a compound symmetry structure for the block diagonal residual covariance matrix. sick frequencies of calves with respect to diet and quercetin were analyzed with the freq procedure of sas/stat software using two-way tables of diet by sick and quercetin by sick and the exact pearson chi-square test. effects and differences were considered significant at p < 0.05. mean body weight at birth was 45.5 ± 1.9 kg and increased with age (p < 0.01) by 368 ± 140 g/ d, without group differences, respectively. all calves received their first meal 2.2 ± 0.1 h after birth. milk intake related to body weight did not differ among groups on days 1 and 2 but was lower on days 3 and 4 in the colostrum-deprived calves (p < 0.01; fig 1a) . on day 2, appetite was reduced in the formula-fed calves and the amount of tube-fed milk was higher than in colostrum-fed calves (p < 0.01; fig 1b) . heart rate decreased with age (p < 0.01) and respiratory rate tended to decrease (p = 0.08; fig 1c and 1d) . rectal temperature was highest on days 3 and 4 and subsequently decreased (p < 0.01). rectal temperature and fecal score were higher in formula-fed than in colostrum-fed calves (p < 0.01; fig 1e and 1f ). the number of calves with an allowed recovery day was similar among groups. with the exception of colq +, we treated one calf per group medically for navel infection. four calves in group forq + needed antispasmodic/analgesic treatment during a recovery day because of abdominal pain. thus, col-fed calves tended to be less susceptible to illness (p = 0.08), whereas quercetin treatment did not affect well-being (p = 0.38). due to severe disease, we had to remove two calves from the study on day 4 (forq+) and day 7 (colq+). preprandial concentrations of total flavonols in plasma on days 1 and 2 (before first quercetin supplementation) did not reveal significant differences among groups, but very low concentrations were detectable in five colostrum-fed calves. in the control groups forq-and colq-, the plasma concentrations did not change with age. in colq+ and forq+, the plasma flavonol concentrations changed with age (p < 0.01); they reached the maximum on day 3 and decreased subsequently but tended to differ among groups only on day 4 (p = 0.08; fig 2a) . comparing the flavonol fractions in the plasma, the quercetin fraction (60% of total flavonoids) was highest in both dietary groups. the concentrations of isorhamnetin and tamarixetin on day 3 were higher in colq+ than in forq+ (fig 2b) . plasma glucose concentrations were lowest on day 1, increased on day 2 in colq-, forq+ and forq-(p < 0.05) and decreased on day 4 only in forq+ and forq-(p < 0.05). the mean plasma concentration of glucose was higher in the colostrum-fed than in the formula-fed calves (p < 0.03; table 2 ). the plasma lactate and non-esterified fatty acid concentrations were highest on day 1 and decreased with age (p < 0.01) without any group differences ( table 2 ). the concentrations of total protein in the plasma increased only in the colostrum-fed calves (p < 0.01) and were higher in the colostrum-fed than in the formula-fed calves on day 2, day 4 and day 7 (p < 0.01; fig 3a) . plasma albumin decreased with age (p < 0.01) in all groups ( fig 3b) . for both total protein and albumin, the diet×age interaction was significant (p < 0.01). the plasma urea concentrations increased on day 2 in the formula-fed and on day 7 in the colostrum-fed calves (p < 0.01; table 2 ). the plasma triglyceride concentrations increased until day 4 only in the colostrum-fed calves (p < 0.05) and continuously decreased from day 2 to day 7 in the formula-fed calves (p < 0.01). the plasma cholesterol concentration increased with age (p < 0.01). the plasma triglyceride (day 7) and cholesterol concentrations (day 4 and day 7) were higher in the colostrum-fed than in the formula-fed calves (p < 0.01; table 2 ). the plasma insulin concentrations decreased with age (p < 0.01; table 2 ). glucagon increased to a maximum plasma concentration on day 2 and subsequently decreased in all calves (p < 0.01). the glucagon concentration on day 4 and day 7 was higher in the colostrumfed than in the formula-fed calves (p < 0.01; table 2 ). the cortisol concentrations decreased with age in all groups (p < 0.01) but decreased earlier in the colostrum-fed calves. the mean cortisol concentrations in plasma during the first week of life were lower in the colostrum-fed than in the formula-fed calves (p = 0.02; table 2 ). quercetin treatment did not affect the concentrations of metabolites nor hormones in the plasma. the plasma concentrations of igg1 and igg2 increased on day 2 only in colostrum-fed calves (p < 0.01; fig 3c and 3d) . the plasma concentrations of igm increased sharply until day 2 in colostrum-fed calves and then slowly decreased (p < 0.01). in the formula-fed groups, the igm concentration increased until day 7 (p < 0.01) but was still lower than in the colostrum-fed groups (p < 0.01) at the end of the trial (fig 3e) . the concentrations of haptoglobin in blood plasma were below the detection limit on day 1 in all calves and increased only in the formulafed calves (p = 0.01) and were highest in forq-on day 7 (fig 3f) . the mean haptoglobin concentration was higher in formula-fed than in the colostrum-fed calves (p = 0.03). the concentrations of serum amyloid a increased until day 2 and slowly decreased afterwards in all calves (p < 0.01; fig 3g) . the concentrations of fibrinogen increased until day 4 and then decreased (p < 0.01) without differences among groups (fig 3h) . however, the mean fibrinogen concentration tended to be higher after formula feeding (p = 0.10). plasma teac increased in all groups until day 4 of life (p < 0.01) but was higher in the colostrum-fed than in the formula-fed calves (p < 0.01; fig 4a) . frap decreased from day 1 to day 7 only in the formula-fed groups (p 0.01) and was higher (p < 0.05) on day 7 in the colostrum-fed groups (fig 4b) . although not statistically significant, the frap decrease was delayed in the quercetin-fed calves compared with the control groups. the mean concentrations of tbars in plasma were higher in the colostrum-fed than in the formula-fed calves (p = 0.01) and revealed a diet×age interaction (p = 0.01) with higher concentrations in the colostrum-fed than in the formula-fed calves on day 4 ( fig 4c) . the mean concentrations of f2-isoprostanes decreased from day 1 to day 4 (p < 0.01) in all calves and tended to increase on day 7 only in forq-(p = 0.08; fig 4d) . regarding inflammation markers, the relative mrna abundances of tnf was higher after quercetin feeding (p = 0.04; table 3 ). the relative mrna abundances of crp was higher (p = 0.03) and that of saa2 tended to be higher (p = 0.09) in the formula-fed than in the colostrum-fed groups (table 3) . for the antioxidative enzymes, the mrna abundances of cat tended to be higher in colq-than in colq+ (p = 0.06) but did not differ between forq-and forq+, revealing a diet×age interaction (p = 0.04; table 3 ). in this study, we were able to confirm the oral bioavailability of quercetin in colostrum-fed calves and further showed that after colostrum deprivation, quercetin is absorbed in equal amounts. as indicated earlier, oral bioavailability decreases with age due to intestinal maturation and therefore reduced permeability or more effective elimination processes [24] . the pattern of flavonol fractions in the plasma in this study was different from the patterns in 29-dayold calves or adult cattle but comparable to observations in 2-day-old calves [24, 36] . this result can be explained by the maturation of intestinal metabolism because it has been shown for rats and pigs [37, 38] that orally administered flavonols undergo complete metabolism inside the intestinal mucosa, where glucuronidation is of major importance [39] . although flavonols were present in the plasma of quercetin-fed calves, we failed to detect any effect on the metabolic parameters or hormone concentrations, which was also the case in previous studies in cattle [24, 36, 40] . however, we could confirm a variety of effects caused by colostrum-deprivation during the first 2 days of life. cortisol is crucial for initiation of parturition and catabolic activity, especially during hypoxia at birth. however, a decrease of the plasma cortisol concentration is delayed in formula-fed calves, as seen in previous studies [41, 42] . we assume that the delay was possibly caused by either abdominal pain or by reduced utilization of nutrients, which is proven to increase stress parameters in sheep [43, 44] . higher plasma glucose in colostrum-fed groups was most likely caused by enhanced intestinal maturation and therefore enlarged absorptive surface [45, 46] . additionally, colostrum feeding seems to accelerate maturation of the pancreas [47] because glucagon concentrations were also higher in colostrum-fed calves from day 4 onwards. although the basal insulin table 3 . hepatic mrna expression of inflammatory and antioxidative traits in calves. relative mrna expression of genes related to inflammation and antioxidative status in the liver of 8-d-old calves; data are given in arbitrary units and are presented as the means ± standard errors (se). q+, quercetinsupplemented; q-, control (no quercetin); n = 6 per group. cat, catalase; gpx1, glutathione peroxidase; sod, superoxide dismutase; tnf, tumor necrosis factor; il1a, interleukin-1α; il1b, interleukin-1β; hp, haptoglobin; fga, fibrinogen; saa2, serum amyloid a2; crp, c-reactive protein. concentrations were similar among dietary groups, we showed in a companion paper that the postprandial insulin response is more pronounced in colostrum-fed groups [23] . higher triglyceride and cholesterol concentrations in colostrum-fed calves are in accordance with observations in other studies [41, 42] and are due to the enhanced stimulation of intestinal differentiation by colostral growth factors and hormones. however, it must be considered that diarrhea in formula-fed calves accelerated intestinal transit and therefore reduced absorption time, which might also account for differences in the plasma triglyceride and cholesterol concentrations between dietary groups. similar concentrations of non-esterified fatty acids indicate that the energy balance in the dietary groups is equal and that lipid mobilization seems to be of minor importance to maintaining energy balance; previous studies also failed to demonstrate consistent effects in neonatal calves [41, 42, 48] . as we expected, the concentration of total protein in the plasma increased only in the colostrum-fed groups, although the protein content of the formula and colostrum was similar. the decrease of the albumin concentration from day 1 to day 2 was probably caused by hemodilution after first feed intake and coincides with previous findings [49, 50] . because only ig, but not the albumin concentrations in plasma were affected by colostrum feeding, differences in total protein are most likely caused by absorption of colostral immunoglobulins after colostrum intake [42] . colostrum intake is important not only for maturational processes but also to acquire passive immunity because the bovine placenta is impermeable to antibodies. as reviewed by weaver et al. [51] , calves with serum igg concentrations below 10 g/l (total protein < 52 g/l) 24 h after birth suffer from failure of passive transfer, which is associated with increased morbidity and mortality as well as reduced performance. in our study, colostrum-fed calves exceeded this threshold; however, failure of passive transfer was a relevant problem in formula-fed calves, although they were parenterally supplemented with bovine immunoglobulins on day 1. only formula-fed calves showed slight hyperthermia on the first days of life and a higher incidence of diarrhea, probably caused by gastrointestinal inflammation due to missing local immunity [52] , accompanied by abdominal pain and diminished appetite. obviously, parenteral and oral treatments with immunoglobulins could not prevent local or systemic infections in formula-fed calves, probably because of missing herd-specific immunoglobulins in the formula and the administered drugs. however, we did not perform microbiological analyses in the feces of the calves to determine pathogens that might have caused loose feces. the time course of plasma igm concentration in formula-fed calves indicates that the indigenous production of igm is evident as early as day 4 of life, but the concentrations are too low to effectively protect against infections [9] . we assume that inflammatory processes also activated the synthesis of acute phase proteins. although the increase of the plasma acute phase proteins in all calves emphasized the immunological burden of the new environment, higher haptoglobin concentrations in formula-fed calves suggest more severe inflammatory processes than in colostrum-fed calves [53, 54] , which was underlined by the greater incidence of gastrointestinal infections. because serum amyloid a is more susceptible to stress [53, 55] , including physical stress, we suppose that the plasma concentrations were equally high in all groups due to the experimental procedures (e.g., continued venipuncture, restricted feeding, single penning, temporal fixation and dietary changes). on the mrna level, we did not find differences in the acute phase proteins between dietary groups because hepatic transcription precedes translation and protein release into circulation. thus, we obviously missed the time point of elevated mrna abundances of hp. however, mrna abundance of crp, a moderate acute phase protein, was elevated in the formula-fed groups on day 8, which could indicate the importance of c-reactive protein as a major component of the bovine innate immune system [56] . hence, increased c-reactive protein production is compensative for the absence of immunoglobulins in colostrum-deprived calves. regarding pro-inflammatory cytokines, we found elevated hepatic mrna abundance of tnf in quercetin-supplemented calves but no differences between dietary groups. under immunocompetent conditions, the impact of various noxae leads to local production of proinflammatory cytokines, e.g., tumor necrosis factor (tnf) or interleukin-1, which in turn induce the systemic acute phase response, including synthesis of acute phase proteins [57] . within signal transduction, reactive oxygen species act as second messengers, thus enhancing tnf-induced gene expression, and oxidative conditions potentiate the activation of respective pathways [57, 58] . although high doses of quercetin were repeatedly shown to reduce mrna expression of tnf in vitro [59, 60] , application of prophylactic doses increased the ratio of pro-to anti-inflammatory cytokines in murine macrophages [60] . we assumed that the hepatic quercetin concentration in our experiment did not exceed the prophylactic dose; thus, quercetin might have increased the expression of tnf. unfortunately, we did not measure tnf protein concentration, but we suppose that posttranscriptional processes anticipated tnf signal transduction [17, 61] , because quercetin scavenged reactive oxygen species necessary for signal transduction. therefore, the observed quercetin effects on tnf gene expression could not have been forwarded on the expression of target genes, e.g., il1b or acute phase proteins, as would have been expected otherwise. concerning the antioxidative status in the plasma, previous findings in neonatal calves are inconsistent. inanami et al. [62] and stohrer, lutz and stangassinger [63] concluded from comparisons between calves and dams that the former are highly susceptible to oxidative stress due to immature defense systems, whereas gaál et al. [64] deduced from high frap values, despite the high reactive oxygen species, that calves are well-prepared to address oxidative stress. although the increase of teac in this and a previous study of our group [65] indicates rising antioxidative capacity during the first week of life, this result was not supported by determination of frap, another marker of antioxidative capacity. furthermore, the values of tbars, which serve as a proxy to measure the products of lipid peroxidation, were unaffected by age in this study, which contradicts earlier studies [62, 64, 66] . however, the reliability of tbars is criticized for low sensitivity and specificity, and the use of f2-isoprostanes is recommended as the most reliable approach to assess oxidative stress in vivo [67, 68] . the time courses of f2-isoprostanes were significantly decreased in all calves; thus, we support the theory that exposure to an oxygen-rich environment following hypoxia during birth results in severe oxidative stress in the newborn [5] . although quercetin is known to exert antioxidative effects, we did not find improved antioxidative status in the plasma of calves nor increased hepatic expression of antioxidative enzymes, which is in line with previous findings of our group in research conducted on neonatal calves and lactating dairy cows [65, 69] . orally administered quercetin is completely metabolized inside the intestinal mucosa; thus, the gastrointestinal tract is the first site of action for quercetin [39] , as it is for pathogens and gastrointestinal disorders commonly occurring during the first weeks of life. because we did not examine the intestinal tissue, we cannot exclude local effects of quercetin on antioxidative status. except for f2-isoprostanes, the parameters of the antioxidative system in the plasma were higher after colostrum feeding. colostrum is a source of reactive oxygen species, but it also contains a variety of antioxidative factors [66, 70] , the latter increasing with time after parturition [71] . the absorption of proand antioxidants present in colostrum [72] might have contributed to the rise of the respective parameters in the plasma of colostrum-fed calves and triggered immunological processes, which in turn caused elevated plasma levels. however, the hepatic mrna abundances of antioxidative enzymes seemed not to be affected by diet, but all groups were fed milk replacer from day 3 onwards and potential dietary effects on hepatic antioxidative status might not be longlasting. in conclusion, oral administration of quercetin aglycone at a daily dose of 50 mg/kg body weight to newborn calves during the first week of life is unable to compensate for inadequate colostrum supply. quercetin did not show any positive effect on neonatal antioxidative or antiinflammatory status, whereas colostrum feeding improves neonatal health status by supporting passive immunity and by promoting antioxidative/oxidative status. supporting information s1 table. complete data set of parameters regarding dry matter intake and health status as shown in fig 1. (pdf) s2 table. complete data set of flavonoid measurements in blood plasma as shown in fig 2. (pdf) s3 table. complete data set of plasma concentrations of metabolites and hormones as shown in fig 3 and table 2 . table. complete data set of immune and inflammatory status in blood plasma as shown in fig 3. (pdf) s5 table. complete data set of antioxidative status in blood plasma as shown in fig 4. (pdf) s6 table. complete data set of hepatic mrna expression of inflammatory and antioxidative traits as shown in table 3 . 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effects on lipopolysaccharide-treated mouse peritoneal macrophages ex vivo quercetin attenuates tnf-induced inflammation in hepatic cells by inhibiting the nf-κb pathway lipid peroxides and antioxidants in serum of neonatal calves vitamine und zusatzstoffe in der ernährung von mensch und tier 8 symposium free radicals, lipid peroxidation and the antioxidant system in the blood of cows and newborn calves around calving short communication: effects of oral flavonoid supplementation on the metabolic and antioxidative status of newborn dairy calves the comparison of antioxidative/oxidative profile in blood, colostrum and milk of early post-partum cows and their newborns insights into oxidative stress: the isoprostanes measurement of f 2-isoprostanes as an index of oxidative stress in vivo influence of 4-week intraduodenal supplementation of quercetin on performance, glucose metabolism, and mrna abundance of genes related to glucose metabolism and antioxidative status in dairy cows antioxidants in bovine colostrum physiological antioxidative/oxidative status in bovine colostrum and mature milk effect of colostrum redox balance on the oxidative status of calves during the first 3 months of life and the relationship with passive immune acquisition we gratefully thank c. fiedler for assistance in conducting the experimental procedures, o. bellmann for veterinary assistance and k.d. witt and b. stabenow for providing experimental facilities. we also thank w. kühl, p. schulz, c. reiko, p. müntzel and u. wiedemuth for excellent laboratory assistance. key: cord-353866-0r1b44id authors: sun, hongpeng; zhang, qiuju; luo, xiao; quan, hude; zhang, feng; liu, chang; liu, meina title: changes of adult population health status in china from 2003 to 2008 date: 2011-12-02 journal: plos one doi: 10.1371/journal.pone.0028411 sha: doc_id: 353866 cord_uid: 0r1b44id objectives: the purpose of this study was to examine the change in health status of china's adult population between the years of 2003 and 2008 due to rapid economic growth and medical system improvement. methods: data from the third and fourth chinese national health services surveys covering 141,927 residents in 2003 and 136,371 residents in 2008 who were aged >18 years were analyzed. results: chinese respondents in 2008 were more likely to report disease than in 2003. smoking slightly decreased among men and women, and regular exercise showed much improvement. stratified analyses revealed significant subpopulation disparities in rate ratios for 2008/2003 in the presence of chronic disease, with greater increases among women, elderly, the han nationality, unmarried and widow, illiterate, rural, and regions east of china than other groups. conclusions: chinese adults in 2008 had worse health status than in 2003 in terms of presence of chronic disease. china's reform of health care will face more complex challenges in coming years from the deteriorating health status in chinese adults. in recent years, the healthcare system in china has significantly improved [1, 2, 3, 4] . in 2001, beijing succeeded in the bid to host the 2008 olympic games. subsequently, chinese government and civil organizations launched extensive fitness programs for the general public to raise the level of physical activity and health. as a result, more sports facilities were built with free access in the community, there was increased parkland, and news media also participated in this campaign [5] . china suffered greatly in the 2003 when the worldwide prevalence of severe acute respiratory syndrome (sars) took a sharp rise. since that lesson, there is evidence that chinese people have improved their general and personal hygiene and diet, undertaken more intensive physical exercise, and increased the overall frequency of hand washing [6] . over the next 5 years, following 2003, chinese healthcare systems have made significant reforms, and reflected on the vulnerability of public health [7, 8] . available health resources in china have been continuously increasing along with china's dramatic economic development [9, 10, 11] . from 2003 to 2009, the medical insurance system in china underwent rapid development and the government input among total health expenditure increased by 17%. a new type of rural cooperative medical care system, a form of community-based health insurance for the rural population, was piloted in some counties in 2003; coverage rate reached 72.6% by december 2004, and would gradually cover all rural residents by 2010 [12, 13] . at the same time, urban resident health insurance scheme included 80% of cities by the end of 2007. urban employee basic health insurance scheme in china has covered all cities since 2008 [13, 14, 15] . with healthcare reform deepening, these systems made the proportion of out-of-pocket payments drop to 30% and will be further reduced in future [13] . during the last decade, the health status of china's population has been changing. chinese life expectancy increased from 71.4 years in 2000 to 73 years in 2005; infant mortality decreased from 25.5/1000 live births in 2003 to 15.3/1000 live births in 2007 while maternal mortality declined from 51.3/100,000 to 36.3/ 100,000 live births at the same time [16] . although infectious disease, malnutrition, child, and maternal mortality rates have decreased, chronic and noncommunicable disease mortality has gradually increased [17] . for example, cancer and stroke are the major causes of death, accounting for 44.8% of all deaths in 2008, followed by respiratory disease and heart disease; chronic and noncommunicable disease mortality increased from 76.5% of allcause death in 2003 to 82.5% in 2008 [18] . on the other hand, such indicators are insensitive to nonfatal conditions that contribute indirectly to death. whether chinese overall health status changed during the period from 2003 to 2008 remains unresolved. hence it is imperative to assess population health using indicators that reflect contemporary health issues. this study aimed to describe the male and female adult chinese population health status in multiple dimensions, including overall morbidity, presence of illness in the last 2 weeks and chronic disease in the last 6 months, and healthy behavior as regards smoking, alcohol consumption, and physical activity, using data from the most recent national health services surveys by the chinese government in 2003 and 2008. the main objective of these surveys was to measure performance of health systems and forecast health demand and long-term health problems. our study provided important information for furthering research questions for future studies. the china national survey employed a multistage cluster sampling to select the sample randomly. the mainland of china was clustered according to the government administrative geographic system [19] . both surveys were conducted in the same sampling areas, whereas all households were randomly selected again. first, 94 counties and cities were randomly selected from rural and urban areas (95 areas were selected in 2003). second, 5 towns were selected from each county and 5 communities from each city respectively, resulting in 470 towns or communities. third, 2 villages in each town and 2 neighborhoods in each community were randomly selected. fourth, 60 households were randomly selected in each village or neighborhood, resulting in about 56,400 households in 2008 (57,000 households in 2003). during the survey, candidate households that could not be contacted by interviewers on three calls on different days were replaced. survey completion rate was maintained at .95%. interviewers, who were trained, explained the purposes and confidentiality of the survey then invited family members to participate. residents could choose not to participate and their participation in the survey was accepted as oral consent. adult residents were themselves required to answer questions; if they were not at home at the time of the survey then their nearest relative could serve as proxy. as a result, the survey response rate for adults was 83% in 2008 (77.8% in 2003). completeness of questionnaires was checked by a district survey manager at the end of each day. if there was missing information and errors on the survey, probands could be re-surveyed the next day. a 5% sample of households was randomly selected and resurveyed to examine survey quality by telephone or visit again; the agreement was 95%. the quality of survey data and consistency check demonstrated a myer's index of 3.48; there was no age preference in the survey. the results of goodness-of-fit showed that the sample was not significantly different with the general population in age distribution. similarity coefficient and gini concentration ratio indicated that family size in the survey was consistent with the established national picture. demographic variables included sex, age, ethnicity, marital status, education level, rural/urban residency, and geographic region. ethnicity was grouped into han or minority. educational level was categorized into five categories such as illiterate, elementary school, junior high school, senior high school, and college or university or higher. china was geographically grouped into urban (city) or rural areas (town or village) after the governmental administration system, as well as eastern china, mid-china, and western china based on economic development status; eastern china is considered the most developed region, mid-china less developed, and western china the least. information about smoking, alcohol consumption, and physical exercise were collected. presence of illness in the previous 2 weeks and physician-diagnosed chronic diseases in the last 6 months was recorded. the same survey method was used in both the 2003 and 2008 surveys. definitions of variables have been described in detail elsewhere [20, 21] . all analyses were conducted separately for women and men, urban and rural, and total. descriptive statistics were used to test the statistical differences in sociodemographic characteristics, smoking, alcohol consumption, physical exercise, presence of illness (in the previous 2 weeks) and chronic disease between the two surveys. because of the large sample size and multiple categories in sociodemographic characteristics variables, p-values for differences between the two surveys were not reported. finally, multiple binomial regressions with a log link were used to generate adjusted p-values to examine whether chinese residents of 2008 had better health status compared with in 2003. clustering of individuals within family was adjusted for using generalized estimating equations in sas 9.1 proc genmod [22, 23] . stratified analyses by sociodemographic variables were conducted for chronic disease to determine whether adjusted rate ratio (rr) and 95% confidence interval (95%ci) for 2008/2003 differed across strata. demographic characteristics for residents are presented in table 1 . there was a difference in composition of some age groups between the two surveys: 18 presence of illness in the previous 2 weeks was 19 (table 3) . however, the proportion of drank alcohol frequently was similar between 2008 and 2003. chinese residents in 2008 were significantly more likely to perform regular exercise than in 2003 (14.6% vs. 11.3%). compared with men, women were less likely to smoke and frequently drink alcohol in 2003 and 2008, and had more likelihood to perform regular exercise. rural residents did less regular exercise than urban residents, and smoked more. after adjusting for independent variables listed in table 1 our review of data for the china third and fourth national health services surveys suggests conclusions in three broad areas. first, we found that chinese population in 2008 was more likely to report illness compared with 2003, but less likely to smoke and more likely to do regular exercise. second, chronic diseases in 2008 were more highly prevalent than in 2003, particularly hypertension, diabetes, heart disease, and stroke. third, the disparity in the prevalence chronic disease of the two surveys was distinctly evident in different subpopulations. in the current study, we found that prevalence rates of chronic diseases in the last 6 months before the surveys increased from 2003 to 2008. for example, the prevalence of hypertension and diabetes in 2008 approximately doubled since 2003, and that of stroke increased by 1.4 times. however, the prevalence of infectious disease did not change. there are several possible explanations for the above findings. first, china's population has been in very rapid transition from a youthful to an aging population as life expectancy has increased [24, 25, 26] . birth rates have fallen, and china's one-child family policy has been a strong driver of population aging [27] . the rapid decrease in china's birth rate, combined with stable or improving life expectancy, has led to an increasing proportion of elderly people. in 1982, only 7.6% of the population was aged .60 years; this proportion grew to 10.5% by 2000. in our study this proportion was 13.6% in 2003 and 15.4% in 2008. the un predicts that .453 million chinese will be aged .60 years by 2050 [28] . this so-called graying of china's population has increased the incidence rates of diseases associated with elderly populations, and will become more problematic in future [29] . second, many of the known risk factors for chronic diseases have dramatically increased as societal change progresses. these behavioral elements include changing diets, levels of physical activity, and alcohol and tobacco consumption and have accelerated shift at a historically unprecedented pace and scale in china. dietary grains intake decreased substantially, whereas that of meat, fat, and edible oil increased [30, 31, 32] . consequently, obesity in chinese people has increased as well as hypercholesterolemia [33, 34] . obesity is a major public health problem since it contributes to development and exacerbation of major chronic diseases [35, 36, 37] including heart disease, type 2 diabetes, and some cancers. in addition, the increase in overweight people and obesity can be attributed to physical inactivity [38] . physical activity helps a person maintain better posture and balance, stronger muscles and bones, more vitality, reduced stress, and continued independent living in later life [39] . although the present research revealed that chinese adults were more physically active in 2008 compared with 2003, only 14.6% of adult residents reported exercising $3 times per week. this may be due to improved access to physical activities or enhanced awareness of health. however, overall chinese adult population health status has not been improved due to short time and small proportion of residents performing frequent exercise; hence it seems that the prevalence and burden of chronic diseases will continue to grow. the third possible explanation is that the prevalence of hypertension in china has been rising rapidly during the period from 2003 to 2008. our results indicate that the prevalence of hypertension in the last 6 months before the survey has doubled from 3.6% in 2003 to 7.1% in 2008; and 17.5% of chinese people had been diagnosed with hypertension by medical doctors. however, the true prevalence of hypertension should be higher than reported because of respondents' unawareness of their condition. previous national studies suggest that the prevalence of hypertension in the chinese adult population has increased from 5.1% in 1959, to 7.7% in 1980, to 13.6% in 1991, and to 18.8% in 2002 [40, 41, 42] . nevertheless they should be compared cautiously owing to methodological differences in sampling and to differences in the criteria used to define hypertension. in addition, control of hypertension in china is far from optimum. according to the national nutrition survey of 2002, 18.8% of chinese adults had hypertension, and of the 24% affected individuals who were aware of their condition, 78% were treated and 19% adequately controlled [40, 42] . at the same time, 27.2% of diabetes patients took medication and 9.7% achieved controlled diabetes [43] . therefore a national education program that can eliminate the huge gaps among presence, awareness, treatment, and ability to control of hypertension and diabetes should be given to the public, clinicians, and healthcare decision makers. fourth, insurance coverage that has been increasing in china, leading to higher health services utilization [13] . previous national health services surveys revealed that the 2-week consultation rates were 11.8% of urban residents and 13.9% rural residents in 2003 [20] . the percentages increased to 12.7% of urban and 15.2% rural in 2008. therefore chinese adults in 2008 seemed more likely to detect disease. the increasing range in the prevalence of chronic disease was more significant in women, han chinese, elderly, widowed, illiterate, rural, and eastern china than other subpopulations. the presence of chronic disease is rapidly increasing among more affluent people. for example rural residents, who have benefited from china's economic development similarly to urban populations, have experienced dramatic lifestyle changes in the past two decades. they are doing less physical work owing to mechanization and eating more high-protein or -fat food; and are therefore more likely to be aware of the presence of chronic disease and insurance coverage, especially the new rural cooperative medical care scheme, more health service utilization, and better transport have enabled rural residents to visit physicians [12, 13] . as a result, they are more likely to report illness. social determinants of health have become important factors associated with the decline of health status. our findings suggest that to promote health status we should focus on elderly and widowed people and promote higher national educational level. prevention strategies such as reduction of tobacco use, exposure to second-hand smoke, less dietary intake of salt and fat, and promotion of increased physical activity should also be prioritized. there are four major limitations to this study. a major limitation is that data were collected through two cross-sectional surveys, whereas the two questionnaires did not have precisely the same structure. also, the cross-sectional nature allows for errors in recall. a second limitation is that the present study was based on observational data; therefore we cannot be completely sure of the association between demographic characteristics and health status, even after controlling for potential confounders. a third limitation is that we only analyzed seven major risk factors but were unable to assess other important risk factors of diet and obesity. the fourth major limitation is that the prevalence of chronic disease was likely underestimated because only physician-diagnosed chronic disease in the last 6 months was recorded. however, this study mainly explored relative change for presence of chronic disease from 2003 to 2008. some bias in both cross-sectional surveys could be offset to some extent. despite these limitations, our findings make a significant contribution to address the health characteristics of chinese adult population. additional research is needed to explore the reasons for the patterns we found, including how the outcomes examined in our study might differ for rural residents compared with urban residents. future studies also might explore how types of health insurance might be related to health status. in conclusion, our analysis demonstrates that chinese adults in 2008 had worse health status than 2003 in terms of presence of various chronic diseases. especially, prevalence of hypertension and diabetes in 2008 was twice that in 2003. however, smoking showed slight decrease among men and women, and regular exercise suggested much improvement. our results also indicate that prevalence of chronic illnesses increased more among women, elderly, han chinese, unmarried and widowed, illiterate, rural, and those in eastern china. public health policy should pay explicit attention to these issues. as economic development and environmental degradation continue, systems of disease prevention and health promotion in china will face ever-bigger difficulties and challenges. adjusted p value ,0.001 for 2008 versus 2003 after adjustment for age, minority, marital status, education, gender (or residence area) and geographic region prevalence of smoking, alcohol intake, and physical activity in respondents aged .18 years in china. men women urban rural total adjusted p value ,0.001 for 2008 versus 2003 after adjustment for age, minority, marital status, education, gender (or residence area) and geographic region the chinese health system at a crossroads the health 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insurance beijing: information centre, health ministry of china changing health in china: re-evaluating the epidemiological transition model major causes of death among men and women in china the third national health services survey design. beijing: china ministry of health rural and urban disparity in health services utilization in china male and female adult population health status in china: a cross-sectional national survey estimating the relative risk in cohort studies and clinical trials of common outcomes easy sas calculations for risk or prevalence ratios and differences ageing of the population in china: trends and implications trend of population aging in china and the strategy the sixth national population census main data communique in 2010 china's family planning policy: an overview of its past and future tackling the challenges to health equity in china emergence of chronic non-communicable diseases in china the nutritional status and dietary pattern of chinese adolescents a new stage of the nutrition trends of obesity and underweight in older children and adolescents in the united states obesity: preventing and managing the global epidemic is china facing an obesity epidemic and the consequences? the trends in obesity and chronic disease in china human body composition and the epidemiology of chronic disease who reassesses appropriate body-mass index for asian populations overweight and obesity in china prevalence, awareness, treatment, and control of hypertension in china: data from the china national nutrition and health survey hypertension prevalence and status of awareness, treatment and control in china a description on the chinese national nutrition and health survey in 2002 the authors thank the china ministry of health for providing the data for the analysis. conceived and designed the experiments: ml hs hq. analyzed the data: hs qz. wrote the paper: hs xl fz cl. key: cord-339026-eu11larc authors: ryals, renee c.; patel, siddharth; acosta, chris; mckinney, madison; pennesi, mark e.; sahay, gaurav title: the effects of pegylation on lnp based mrna delivery to the eye date: 2020-10-29 journal: plos one doi: 10.1371/journal.pone.0241006 sha: doc_id: 339026 cord_uid: eu11larc gene therapy is now an effective approach to treat many forms of retinal degeneration. delivery agents that are cell-specific, allow for multiple dosing regimens, and have low immunogenicity are needed to expand the utility of gene therapy for the retina. we generated eight novel lipid nanoparticles (lnps) ranging in size from 50 nm to 150 nm by changing the peg content from 5% to 0.5%, respectively. subretinal injections of lnp-mrna encoding luciferase revealed that 0.5% peg content within nanoparticles elicits the highest expression. similar injections of lnp delivered cre mrna into ai9 mice revealed cell-specific protein expression in the retinal pigment epithelium (rpe), confirmed by fundus photography and immunohistochemistry of whole globe cross-sections. to investigate mechanisms of lnp delivery to the eye, we injected mcherry mrna using the subretinal approach in apoe(-/-) and mertk(-/-) mice. rpe transfection was observed in both mouse models suggesting that lnp intracellular delivery is not solely dependent on apolipoprotein adsorption or phagocytosis. to investigate lnp penetration, particles were delivered to the vitreous chamber via an intravitreal injection. the 0.5% peg particles mediated the highest luciferase activity and expression was observed in the müller glia, the optic nerve head and the trabecular meshwork, but failed to reach the rpe. overall, particles containing less peg (~150 nm in size) mediated the highest expression in the eye. thus far, these particles successfully transfect rpe, müller cells, the optic nerve head and the trabecular meshwork based on route of administration which can expand the utility of lnp-mediated gene therapies for the eye. despite major advancements in restoring vision, there are still an estimated 285 million vision-impaired and 39 million blind people around the world [1] . in part, these large numbers exist because there are still no treatments for some of the most devastating blinding diseases including advanced glaucoma, atrophic macular degeneration, advanced diabetic retinopathy, myopia, and inherited retinal degeneration [1] . these disorders have become the focus of ocular gene therapy and effective treatments are being developed. for and internalization in the retina post-intravitreal injection are needed to rationally design enhanced nanotherapeutics. upon characterizing many of the intravitreal nanoparticles developed thus far and assessing their effectiveness, four main parameters continuously affect their intraocular distribution and elimination: size, charge, stability and ligands [17] . it has been demonstrated that size affects the half-life of intravitreally injected nanoparticles. specifically, for polystyrene nanoparticles smaller particles sizes~50 nm had the highest half-life [21] . furthermore, size impacts the process for internalization. in general, particles <500 nm are internalized via endocytosis, whereas particles >1000 nm utilize phagocytosis [22, 23] . stability and size are often linked as the particle size varies upon disassembly or aggregation of the particles. if nanoparticles are unstable, they will fall apart upon entry, and the dissociated components can be rapidly eliminated [24] . thus, particles need to be stable enough to travel to the cell of interest, but then have the ability to disassemble once internalized in the cell of interest. it has been shown that the hydrophilic polymer, polyethylene glycol (peg), which is a commonly used excipient to increase in vivo stability, evade recognition by the reticuloendothelial system, prolong circulation half-life and reduce immunogenicity, can be easily adjusted to impact particle size, stability and intracellular delivery [25] . to evaluate the important parameters of size and stability for intravitreal delivery, we generated 8 novel lnps that varied in size by changing mol % of peg. we found that larger particles (~150 nm), containing less peg-lipid (0.5%), mediated the highest levels of gene expression post-subretinal and intravitreal delivery. most intriguingly, these particles successfully transfect rpe, müller glia, the optic nerve head and the trabecular meshwork based on route of administration which can expand the utility of lnp-mediated gene therapies for the eye. dlin-mc3-dma (mc3) was purchased through biofine international inc. 1,2-distearoyl-snglycero-3-phosphocholine (dspc) was acquired from avanti polar lipids. cholesterol and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (dmg-peg2k) was purchased from mp biomedicals and nof america corporation, respectively. pierce d-luciferin was purchased from thermo fisher scientific. firefly luciferase (l-7202), cre (l-7211) and mcherry (l-7203) mrna were all purchased from trilink biotechnologies. albino balb/c (cat# 000651), ai9 (cat# 007909), apoe -/-(cat# 002052), mertk -/-(cat# 011122) and c57bl6 (cat# 000664) mice were either bred in house or purchased from the jackson laboratory (bar harbor, me, usa). ai9 is a cre reporter tool designed to have a loxp-flanked stop cassette preventing transcription of tdtomato under the control of a ubiquitous promoter. following cre-mediated recombination ai9 mice express robust tdtomato [26] . both male and female mice aged 1 to 6 months were used in experiments. all the experimental procedures followed the protocols approved by the institutional animal care and use committee at oregon health & science university and were in adherence to the association for research in vision and ophthalmology (arvo) statement for the use of animals in ophthalmic and vision research. lnps were formulated via microfluidic mixing of one-part ethanol phase (containing the lipids) and three parts aqueous phase (containing the mrna). the ethanol phase contained the mc3, dspc, dmg-peg2k, and cholesterol at a molar ratio of 50:10:1.5:38.5, respectively. for synthesizing novel particles, the percentage of dmg-peg2k was changed from 1.5% to 0.5%, 3% or 5% while cholesterol was changed from 38.5% to 39.5%, 37.0% or 35%, respectively. for the second set of particles, dmg-peg2k varied from 0.5% to 5% while dspc was changed from 10% to 11%, 8.5% or 6.5%, respectively ( table 1 ). the aqueous phase consisted of the mrna in 50 mm citrate buffer ph 4. following microfluidic mixing, the nanoparticle solution was subjected to buffer exchange with pbs (ph 7.2) and concentrated using amicon ultra-4100k mwco (emd millipore) centrifugal filters. the nanoparticles were stored at 4˚c until diluted for injections. the lnps were characterized for hydrodynamic radius (nm) and polydispersity index (pdi) using dynamic light scattering (zetasizer nano zsp, malvern instruments), while mrna encapsulation efficiency was measured using a modified quant-it ribogreen rna reagent (life technologies). prior to injections, mice were topically administered 0.5% proparacaine, 1% tropicamide, and 2.5% phenylephrine and anesthetized with ketamine (100 mg/kg)/xylazine (10 mg/kg). our subretinal injection procedure has been previously described elsewhere [13] . for intravitreal injections, 2.5% hypromellose was placed over the eye and a 30-gauge needle was used to make an incision in the limbus. going through the scleral incision in the limbus, using a hamilton syringe with a 33-gauge 20˚beveled needle, 1.5 μl of pbs, lnp-luciferase, lnp-cre or lnp-mcherry was delivered to the vitreous chamber. a 2% fluorescein solution was added to the pbs or lnps so injection quality could be observed and documented. for subretinal injections, 200 ng (1 μl, 200 ng/μl) of mrna was delivered. for intravitreal injections, a low dose of 300 ng (1.5 μl, 200 ng/μl) of mrna was evaluated first. to maximize mrna delivery, particles were concentrated to~1000 ng/μl and a high dose of 1.5 μg (1.5 μl, 1000 ng/μl) of mrna was delivered. mice were injected intraperitoneally with 150 mg of luciferin/kg body weight according to manufacturer's protocol [13] . bioluminescent imaging was conducted on the ivis spectrum in vivo imaging system (perkinelmer). image analysis for region of interest (roi) measurement was performed on living image software (perkinelmer) and was reported as average radiance (the sum of the radiance from each pixel inside the roi/number of pixels or super pixels (photons/sec/cm 2 /sr)). sample sizes were 4 to 6 per group. to quantify tdtomato expression, pixel intensity was measured for each image over a consistent area (640,000 pixels 2 ) using imagej (version 1.49; national institutes of health, bethesda, md). there were 3 to 6 images per group. at specified time points, mouse eyes were enucleated and fixed in 4% paraformaldehyde (ph 7.4) overnight at 4˚c. after fixation, oriented eyes were placed in cassettes, processed and embedded for sectioning (tissue-tek vip 1 6, tissue-tek 1 tec™ 5, sakura finetek usa, inc., ca). whole globe cross-sections were cut with a microtome to a thickness of 4 microns. to initiate staining, sections were dipped in 100% xylene, 100% ethanol, 95% ethanol, 80% ethanol, running water and deionized water to deparaffinize the tissue. all sections were then washed with phosphate buffered saline (pbs), permeabilized with 0.3% triton for 10 minutes and blocked with 1% bsa for 1 hour. a primary antibody consisting of an anti-rfp antibody (1:500, rabbit, ab62341) in 1% bsa covered the sections overnight at 4˚c. the next day, sections were washed with pbs and incubated in secondary antibody (donkey anti-rabbit alexa 594, 1:800, cat#a21207, life technologies, eugene, or). confocal images were obtained with the tcs sp8 x (leica microsystems, buffalo grove, il). z-stacks (spanned 10 μm with 1 μm interval) were collected using a 40x objective, and maximum intensity projections were reported. sample size was 3 to 5 per group. for in-vivo bioluminescent imaging measurements from balb/c mice, differences between groups at each time point were calculated using a two-way anova, tukey's multiple comparisons test (prism 8 software, graphpad software, la jolla, ca). an unpaired t-test was used for in-vivo bioluminescent imaging analysis comparing c57bl6 and apoe -/mice. for tdtomato quantification from fundus images, intensity values were transformed and reported as fold change to pbs-injected animals. an ordinary one-way anova, with tukey's correction for multiple comparisons test was used for comparisons between groups. data are presented as mean ± sd. a p < 0.05 was considered as statistically significant. lnps were generated with dspc (structural lipid), mc3 (ionizable lipid), cholesterol and dmg-peg2k (peg-lipid) ( fig 1a) . in order to vary lnp size, mol percentages of peg-lipid were increased from 0.5% -5% [25] . the increase in mol percentage of peg was off-set by a decrease in cholesterol (referred to as modulated against cholesterol) or phospholipid (referred to as modulated against dspc) in order to control for the impact of other components on delivery. peg-lipid variations from 0.5% to 5% generated particles sized 150 nm to 50 nm, respectively (fig 1b and 1c) . regardless of the modifications, particle formulations were successful as pdi values were <0.1 and encapsulation efficiency averaged 97% for the eight particles evaluated (fig 1 and table 1 ). since the subretinal injection places the lnps in close proximity to the rpe, we utilized this method to understand how pegylation and size may impact intracellular delivery ( in general, for all particles, luciferase activity in the eye was measurable at 4 hours post-injection, increased to a maximum level at 24 hours post-injection and decreased by 48 hours post-injection (figs 2b and 3b). at 24 hours post-injection, 0.5% peg particles with corresponding cholesterol modifications demonstrated significant 1.9-fold, 5.2-fold and 18.9-fold increases in luciferase activity compared to 1.5%, 3% and 5% peg particles, respectively (fig 2b, p<0 .05). at the same time point, 0.5% peg particles with corresponding dspc changes mediated significantly higher levels of luciferase activity compared to 1.5% and 5% peg particles (3.6-fold and 17.3-fold, respectively) (fig 3b, p<0 .05). although 0.5% peg particles showed at 2.3-fold increase in luciferase activity compared to 3% peg particles, this difference was not significant due to the variation in activity meditated by the 3% peg particles (fig 3b) . when comparing the luciferase expression between 0.5% peg particles modulated against cholesterol with 0.5% peg particles modulated against dspc, modifications in dspc mediated a 6-fold, 1.9-fold and 3.9-fold increase in expression compared to cholesterol at 4-, 24-and 48-hours post-injection, respectively. to determine the location of protein expression in the eye, cre mrna was subretinally injected in ai9 mice, which results in tdtomato fluorescence upon successful transfection and cre-mediated recombination [26] . for all eight particles, fundus images showed a cobble stone pattern of expression, traditional of rpe morphology (figs 2c and 3c). when tdtomato intensity was analyzed, 0.5% peg particles modulated against cholesterol had 3.4-fold increased tdtomato intensity compared to pbs control and 2-fold increased tdtomato intensity compared to 5% peg, which was statistically significant (fig 2d, p<0 .05). in contrast, regardless of the peg content, particles modulated against dspc averaged a 1.8-fold increase in tdtomato intensity compared to pbs controls, but no statistical differences between the groups were identified (fig 3d) . immunohistochemistry, performed with a red fluorescent protein (rfp) antibody, demonstrated that protein expression was localized to the rpe for all particles, regardless of modifications (figs 2e and 3e) . after systemic delivery of lnps, apolipoprotein e (apoe) binds lnps in the blood, which facilitates apoe-mediated uptake by low-density lipoprotein (ldl) receptors (ldlr) into hepatocytes. this was established by observing diminished lnp transfection efficiency in both apoe -/and ldlr -/mice [27] . to determine if this mechanism plays a role in lnp uptake in the rpe, the fda approved lnps (mc3, dspc, dmg-peg2k, and cholesterol at a molar ratio of 50:10:1.5:38.5) containing luciferase mrna were subretinally injected into apoe -/mice (fig 4a and 4b ). luciferase expression was evident in apoe -/mouse eyes at 24 hours post-injection and was not significantly different from expression measured in c57bl6 control animals (fig 4a and 4b, p>0.05) . these data suggested that apoe is not functioning as an endogenous targeting ligand for lnp uptake in the rpe. once a day, rpe cells phagocytose and digest the shedding rod and cone photoreceptor outer segment tips making rpe cells the the effects of pegylation on lnp based mrna delivery to the eye most actively phagocytic cells in the body [28] . we hypothesized that if phagocytosis was blocked, lnp entry in the rpe would diminish. lnps containing mcherry mrna were the effects of pegylation on lnp based mrna delivery to the eye subretinally injected in mertk -/mice, in which the main receptor responsible for phagocytosis is dysfunctional and leads to the accumulation of shed photoreceptor outer segments in the subretinal space [29] . immunohistochemistry, probing for mcherry, demonstrated robust protein expression localized to the rpe in mertk -/and apoe -/mice ( fig 4c) verifying that neither phagocytosis nor apoe adsorption are solely responsible for lnp intracellular delivery in the rpe. to determine the impacts of pegylation and reduced size on lnp penetration from the vitreous to the outer retina, lnps were delivered intravitreally at a high (figs 5 and 6) and low dose (s2 and s3 figs) . first, lnp kinetics were measured with a luciferase assay (figs 5a and 6a, s4 fig). in general, for all particles, luciferase activity in the eye was measurable at 4 hours post-injection, increased to a maximum level at 24 hours post-injection and decreased by 48 hours post-injection (figs 5b and 6b ). at 24 hours post-injection, 0.5% peg particles modulated against cholesterol demonstrated significant 2.7-fold, 3.0-fold and 17.5-fold increases in luciferase activity compared to 1.5%, 3% and 5% peg particles, respectively (fig 5b, p<0.05 ). at the same time point, 0.5% peg particles modulated against dspc mediated a less robust response, only showing a 1.5-fold and 5.2-fold increase in luciferase activity compared to 3% and 5% peg particles, respectively (fig 6b, p<0 .05). the 1.5% peg particles modulated against dspc showed similar magnitudes of activity to 0.5% peg particles and mediated a significantly higher level of luciferase activity compared to 5% peg particles (5.6-fold, fig 6b, p<0.05). when comparing the luciferase expression between 0.5% peg particles modulated against cholesterol with 0.5% peg particles modulated against dspc, modifications in cholesterol mediated a 2.9-fold and 8.4-fold increase in expression compared to dspc at 24-and 48-hours post-injection, respectively. for all eight particles, fundus images showed patchy patterns of expression localized around the optic nerve head (onh) and blood vessels, traditional of inner retinal cell expression (figs 5c and 6c ). when tdtomato intensity was analyzed, peg particles modulated against cholesterol showed no significant differences between the groups (fig 5d, p>0.05 ). in contrast, 0.5% peg particles modulated against dspc averaged a 2.4-fold increase in tdtomato intensity compared to pbs controls, which was a significant increase compared to the tdtomato intensity mediated by 1.5% and 5% peg particles (fig 6d, p<0.05) . immunohistochemistry, probing for rfp, demonstrated that protein expression was localized to the müller glia, the onh and the trabecular meshwork (tm), regardless of modifications (figs 5e and 6e) . when lnps were delivered at a low dose, rfp expression was localized to the müller glia and the tm (s2 and s3 figs) . the effects of pegylation on lnp based mrna delivery to the eye there is a critical need to develop non-viral gene delivery vehicles for the retina. even though there are over 20 gene therapy clinical trials utilizing viral vectors for retinal degenerations [4] , it is well understood that viral vectors are unable to solely treat all of the genetic forms of retinal degeneration due to cost, packaging capacity and immune response [5] . additionally, generating penetrating particles that can transfect the outer retina post-intravitreal delivery would generate a paradigm shift moving away from detaching fragile degenerating retinas during subretinal surgery. to optimize the existing fda approved lnps for the retina, studies are needed that investigate pharmacokinetics and intracellular mechanisms of lnps post-ocular delivery. our studies, first evaluating many cationic and ionizable lipids [13] , and now investigating the impacts of size and pegylation, aim to understand the critical lnp components needed for optimal retina internalization and penetration to the eye. in this paper, we show that particles with less peg (0.5%) and larger in size (~150 nm) facilitate the highest levels of protein expression post-subretinal and intravitreal delivery. furthermore, we demonstrate that neither apoe adsorption, nor mertk dependent phagocytosis are critical for lnp intracellular delivery to the rpe. finally, we demonstrate that our lnps can transfect müller glia, the onh and the tm post-intravitreal delivery which could have significant impacts for retinal degeneration and glaucoma. we utilized a subretinal injection, which places lnps in close proximity to the rpe to focus on lnp internalization. our data suggests that particles with 0.5% peg and~150 nm in size facilitate the best intracellular delivery in the rpe post-subretinal delivery (figs 2 and 3) . these data are consistent with previous studies that demonstrate 1) lnps with 0.5 mol % peg-dma mediated the highest fvii gene silencing in hepatocytes [25, 30] and 2) that while increasing the peg content makes particles smaller it also makes them more stable [31, 32] . we hypothesize that the 3% and 5% particles aren't shedding enough peg-lipid to allow for sufficient cellular uptake, which is inhibiting the transfection efficiency. in addition, the enhanced efficiency observed with 0.5% peg particles is most likely due to a combination of particle size and surface composition. larger particle size allows for more mrna encapsulation per particle, whereas the mol % of peg on the particle surface impacts protein adsorption and disassembly for endosomal escape [26, 27] . interestingly, when comparing dspc versus cholesterol modifications, increasing dpsc content on the particle surface increased expression by 6-fold. we hypothesize that by decreasing the mol % of peg and increasing surface dspc, protein adsorption is enhanced allowing for more efficient internalization [30] . previous studies established an apoe-ldlr dependent internalization pathway for lnp-mediated expression in the liver after systemic delivery [27] . successful rpe transfection in apoe -/mice after subretinal delivery of lnp-mcherry suggests that apoe, an essential endogenous targeting ligand for the liver, is not required for lnp internalization in the rpe. since the subretinal injection delivers the lnps to the rpe, it's possible that targeting ligands are unnecessary, or alternatively, there are still unknown rpe specific ligands facilitating cellular entry. we also hypothesized that phagocytosis could be playing a role in lnp uptake in the rpe. however, the lnp-mediated expression observed in mertk -/mice, which lack the main receptor required for phagocytosis [29] , post-subretinal injection did not support this hypothesis. this groups. n.s.-not significant, lnp-lipid nanoparticle, rfp-red fluorescent protein, onl-outer nuclear layer, inl-inner nuclear layer, gclganglion cell layer, onh-optic nerve head, tm-trabecular meshwork, cb-ciliary body. https://doi.org/10.1371/journal.pone.0241006.g005 finding is consistent with previous studies that demonstrate phagocytosis is dependent on nanoparticle size [23] . our particles are~150 nm in size, which is outside the range for phagocytosis (>1000 nm), but within the range for endocytosis (<500 nm). overall, the exact endocytic process for lnp uptake in the rpe still remains elusive. different internalization pathways as well as the lnp protein corona and adsorption in the retina needs further investigation. in order to evaluate lnp penetration, we utilized an intravitreal injection, which releases the lnps in the vitreous chamber. we hypothesized that smaller particles would allow for penetration from the vitreous chamber to the rpe. however, our data showed that larger particles (~80 nm to 150 nm in size) mediated a higher magnitude of expression than smaller particles (~50 nm to 60 nm in size) and transfection was limited to the inner retina and did not reach the outer retina (more discussion on cellular tropism below) (figs 5 and 6). as stated above, it's feasible that increasing the peg content increases stability and decreases cellular uptake, which is inhibiting the transfection efficiency. future studies can focus on generating smaller particles through microfluidic processing instead of increasing peg content to separate these confounding factors of size and stability. interestingly, when comparing surface modifications, increasing the mol % of cholesterol mediated an~3-fold (at 24 hours) and~8-fold (at 48 hours) increase in magnitude of expression compared to dspc. although cholesterol has been closely linked to enhancing endosomal escape [33] , our data suggest that in the retina having more cholesterol potentially on the surface could enhance penetration, which could be linked to protein adsorption as the particle moves through the retina. it is well understood that lnp size, stability, surface charge and ligands will all work together to generate penetrating lnps [17] . we now add that surface composition including mol % of cholesterol is an important determinant and that 1.5 mol % of peg or less is crucial for penetration in the eye. lnp modifications did not alter cellular tropism, but the route of delivery altered cell specific expression. post-subretinal delivery, all expression was observed in the rpe, which we have previously reported (figs 2-4) [13] . the olm, which has an~30 angström pore radius (in rabbits), is a junction between the müller glia and the outer nuclear layer (onl) which acts as a barrier to the inner retina post-subretinal delivery [16] . it is possible that our smallest particle (~50 nm in size) was still unable to traverse the olm to allow for transfection in the onl. when we transitioned to intravitreal delivery, the müller glia, the onh and the tm were successfully transfected regardless if particles were 150 nm or 50 nm. although the ilm prevents penetration into the retina, the müller glia endfeet sit at the interface of the vitreous humor and the ilm and are susceptible to lnp uptake. müller glia are a critical therapeutic target for retinal degeneration. with the recent success of reprogramming müller glia into functional rod photoreceptors, müller glia remain key targets for regenerating the retina [34, 35] . although the onh sits at the back of the eye, the ilm is not part of the onh composition and it becomes thinner as the retinal interface gets closer to the onh, which decreases the difficulty of transducing the onh. moving to the anterior part of the eye, the tm is located in are presented as mean ± sd. an ordinary one-way anova, with tukey's correction for multiple comparisons test was used for comparisons. � p<0.05. n = 3-6. (e) representative confocal images of immunohistochemistry showing rfp expression in the müller glia, the onh and the tm. n.s.-not significant, lnp-lipid nanoparticle, rfp-red fluorescent protein, onl-outer nuclear layer, inl-inner nuclear layer, gcl-ganglion cell layer, onh-optic nerve head, tm-trabecular meshwork, cb-ciliary body. https://doi.org/10.1371/journal.pone.0241006.g006 the angle of the eye and is responsible for regulating the outflow of aqueous humor [36] . the tm is most likely transduced as the particles get circulated out of the vitreous chamber. the tm and the onh are commonly under investigation to better understand and treat glaucoma [36] [37] [38] . specific to the tm, researchers have been investigating and developing gene therapy approaches that reduce ocular pressure [37] . as glaucoma is a multifaceted disease, researchers have also developed a method of small molecule delivery to the onh of rats that allows for exploration of molecular and cellular pathways involved in neuropathies and treatment screening [38] . in future studies our lnps could be used to target the müller glia, the onh and the tm to aid in treatment development for retinal degeneration and glaucoma. our long-term goal is to generate penetrating lnps that can transfect the outer retina postintravitreal delivery. coincidently, by generating better penetrating lnps, we will be able to enhance transfection efficiency after other important ocular delivery methods for retinal degeneration including subretinal and suprachoroidal delivery [39] . decreasing particle size by increasing peg content failed to improve lnp penetration. instead, the use of 0.5% or 1.5% peg is optimal for retinal transfection. in the future, we will generate smaller particles without the addition of peg. furthermore, solely adjusting the lnp composition (peg, cholesterol, dspc, ionizable lipid) may not be enough to alter the lnp transfection profile. the discovery and addition of rpe and photoreceptor targeted ligands may be required to generate lnps that can penetrate through the vitreous. our future studies will focus on the generate of these novel ligands and understanding the critical lnp characteristics required for effective retinal transfection. restoring vision. nature prospect of retinal gene therapy following commercialization of voretigene neparvovec-rzyl for retinal dystrophy mediated by rpe65 mutation voretigene neparvovec: an emerging gene therapy for the treatment of inherited blindness canadian agency for drugs and technologies in health retinal gene therapies in clinical trials a proliferation of clinical trials means hope for patients. retinal physician inherited retinal degenerations: current landscape and knowledge gaps subretinal injection: a review on the novel route of therapeutic delivery for vitreoretinal diseases lipid nanoparticle systems for enabling gene therapies lipid nanoparticles enabling gene therapies: from concepts to clinical utility an investigational rnai therapeutic for patients with hereditary transthyretin-mediated (hattr) amyloidosis: results from the phase 3 apollo study an evidence based perspective on mrna-sars-cov-2 vaccine development lipid nanoparticles for delivery of messenger rna to the back of the eye vitreous: a barrier to nonviral ocular gene therapy structure and composition of the inner limiting membrane of the retina. graefe's arch clin exp ophthalmol the outer limiting membrane (olm) revisited: clinical implications intravitreal nanoparticles for retinal delivery. drug discovery today structural recovery of the retina in a retinoschisin-deficient mouse after gene replacement therapy by solid lipid nanoparticles a novel cationic niosome formulation for gene delivery to the retina a novel penetratin-modified complex for noninvasive intraocular delivery of antisense oligonucleotides effect of particle size of polymeric nanospheres on intravitreal kinetics engineered nanoparticles interacting with cells: size matters cellular uptake of nanoparticles: journey inside the cell pharmacokinetic aspects of retinal drug delivery. progress in retinal and eye research effect of pegylation on biodistribution and gene silencing of sirna/lipid nanoparticle complexes a robust and high-throughput cre reporting and characterization system for the whole mouse brain targeted delivery of rnai therapeutics with endogenous and exogenous ligand-based mechanisms understanding photoreceptor outer segment phagocytosis: use and utility of rpe cells in culture an rcs-like retinal dystrophy phenotype in mer knockout mice successful reprogramming of cellular protein production through mrna delivered by functionalized lipid nanoparticles characterization of the inhibitory effect of peg-lipid conjugates on the intracellular delivery of plasmid and antisense dna mediated by cationic lipid liposomes wanted and unwanted properties of surface pegylated nucleic acid nanoparticles in ocular gene transfer boosting intracellular delivery of lipid nanoparticle-encapsulated mrna müller glia cell reprogramming and retina regeneration restoring sight with native cell reprogramming extracellular matrix in the trabecular meshwork: intraocular pressure regulation and dysregulation in glaucoma the pathway from genes to gene therapy in glaucoma: a review of possibilities for using genes as glaucoma drugs in vivo small molecule delivery to the optic nerve in a rodent model suprachoroidal gene transfer with nonviral nanoparticles. science advances we would like to thank the casey eye institute, leonard christenson eye pathology laboratory for their support with tissue processing and sectioning. conceptualization: renee c. ryals, siddharth patel, mark e. pennesi, gaurav sahay. key: cord-347014-88zmtky7 authors: esposito, susanna; di gangi, maria; cardinale, fabio; baraldi, eugenio; corsini, ilaria; da dalt, liviana; tovo, pier angelo; correra, antonio; villani, alberto; sacco, oliviero; tenero, laura; dones, piera; gambino, monia; zampiero, alberto; principi, nicola title: sensitivity and specificity of soluble triggering receptor expressed on myeloid cells-1, midregional proatrial natriuretic peptide and midregional proadrenomedullin for distinguishing etiology and to assess severity in community-acquired pneumonia date: 2016-11-15 journal: plos one doi: 10.1371/journal.pone.0163262 sha: doc_id: 347014 cord_uid: 88zmtky7 study design: this study aimed to evaluate the diagnostic accuracy of soluble triggering receptor expressed on myeloid cells-1 (strem-1), midregional proatrial natriuretic peptide (mr-proanp) and midregional proadrenomedullin (mr-proadm) to distinguish bacterial from viral community-acquired pneumonia (cap) and to identify severe cases in children hospitalized for radiologically confirmed cap. index test results were compared with those derived from routine diagnostic tests, i.e., white blood cell (wbc) counts, neutrophil percentages, and serum c-reactive protein (crp) and procalcitonin (pct) levels. methods: this prospective, multicenter study was carried out in the most important children’s hospitals (n = 11) in italy and 433 otherwise healthy children hospitalized for radiologically confirmed cap were enrolled. among cases for whom etiology could be determined, cap was ascribed to bacteria in 235 (54.3%) children and to one or more viruses in 111 (25.6%) children. a total of 312 (72.2%) children had severe disease. results: crp and pct had the best performances for both bacterial and viral cap identification. the cut-off values with the highest combined sensitivity and specificity for the identification of bacterial and viral infections using crp were ≥7.98 mg/l and ≤7.5 mg/l, respectively. when pct was considered, the cut-off values with the highest combined sensitivity and specificity were ≥0.188 ng/ml for bacterial cap and ≤0.07 ng/ml for viral cap. for the identification of severe cases, the best results were obtained with evaluations of pct and mr-proanp. however, in both cases, the biomarker cut-off with the highest combined sensitivity and specificity (≥0.093 ng/ml for pct and ≥33.8 pmol/l for proanp) had a relatively good sensitivity (higher than 70%) but a limited specificity (of approximately 55%). conclusions: this study indicates that in children with cap, strem-1, mr-proanp, and mr-proadm blood levels have poor abilities to differentiate bacterial from viral diseases or to identify severe cases, highlighting that pct maintains the main role at this regard. community-acquired pneumonia (cap), with viruses and bacteria as its main causes, is one of the leading causes of morbidity and mortality in young children worldwide [1] . early detection of bacterial cases that have the potential for a rapid negative evolution is essential to guide clinical management and to avoid prolonged hospitalization and the risk of death [2] . furthermore, the differentiation of viral from bacterial cap is necessary for the rational use of antibiotics and the consequent reduction in the emergence of bacterial resistance and drug-related adverse events [3] . unfortunately, both these goals are difficult to achieve, particularly in younger children in whom the collection of respiratory samples is difficult or impossible to obtain [4] . clinical signs and symptoms and radiological findings are frequently similar in cases of viral and bacterial disease [4] . moreover, in most cases, the results from routine laboratory tests, such as white blood cell (wbc) count and c-reactive protein (crp) serum level determination, tend to overlap, making the differentiation impossible [5] . this challenge also exists when using procalcitonin (pct) to define the etiology and severity of cap. pct was the latest biomarker to enter into routine clinical practice [6] . these limitations explain why several attempts to find more effective biomarkers of cap bacterial etiology and disease severity have been made in recent years. recently, it has been suggested that soluble triggering receptor expressed on myeloid cells-1 (strem-1), midregional proatrial natriuretic peptide (mr-proanp) and midregional proadrenomedullin (mr-proadm) could improve the determination of cap etiology and severity [7] [8] [9] . for all these biomarkers, data collected in adults seem to indicate that their concentrations in body fluids are increased in cases of bacterial infections, particularly in the most severe cases. however, the available data are limited and sometimes conflicting. moreover, to date no evaluation was performed in children. this study aimed to evaluate the diagnostic accuracy of these new biomarkers to distinguish bacterial from viral cap and to identify severe cap cases in children. the results were compared with those derived from wbc counts, neutrophil percentages, and serum crp and pct levels. this research was a prospective, multicenter study carried out in the 11 most important children's hospitals of italy (fondazione irccs ca' granda, ospedale maggiore policlinico, milan; di cristina hospital, palermo; ospedale giovanni xxiii, bari; padova hospital, padua; ospedale sant'orsola, bologna; treviso hospital, treviso; regina margherita hospital, turin; santobono hospital, naples; irccs bambino gesù hospital, rome; irccs giannina gaslini hospital, genoa, italy; and policlinico g.b. rossi, verona). the protocol was approved by the ethics committee of each center. written informed consent was obtained from either the parent(s) or legal guardian(s) of each study participant, and children aged >8 years provided their written assent. otherwise healthy children 4 months-14 years old consecutively hospitalized for clinical signs suggestive of cap, such as tachypnea and abnormal breath sounds, and a radiological confirmation of cap were recruited. exclusion criteria included the presence of an underlying chronic disease or an antibiotic treatment of any type in the 48 hours before the admission. in each center, all chest radiographs were evaluated by an expert radiologist who classified the findings as alveolar cap, non-alveolar cap or no cap in accordance with the world health organization (who) criteria for the standardized interpretation of pediatric chest radiographs for a diagnosis of pneumonia [10] . chest radiography characterized by presence of consolidation (defined as a dense or fluffy opacity that occupies a portion or whole of a lobe or of the entire lung, that may or may not contain air-bronchograms above) or pleural effusion in the lateral pleural space was considered indicative of alveolar cap. non-alveolar cap was diagnosed in case of linear and patchy densities (interstitial infiltrate) in a lacy pattern involving both lungs, featuring peribronchial thickening and multiple areas of atelectasis. the same diagnosis was made when minor patchy infiltrates not of sufficient magnitude to constitute primary consolidation and small areas of atelectasis that could not be distinguished from consolidation were evidenced. the cap severity of disease was established in all the participating hospitals using the criteria indicated for children by the british thoracic society (bts) [11] . in particular, features of severe disease in an infant were considered as follows: oxygen saturation <92%; cyanosis; respiratory rate >70 breaths/min; significant tachycardia for the fever level; prolonged central capillary refill time !2 s; difficulty in breathing; intermittent apnea; grunting; and not feeding. features of severe disease in an older child included the following: oxygen saturation <92%; cyanosis; respiratory rate >50 breaths/min; significant tachycardia for the fever level; prolonged central capillary refill time !2 s; difficulty in breathing; grunting; and signs of dehydration. both the evaluation of the chest radiograph and the classification of severity of each cap episode were blinded to all the studied biological criteria, including wbc count, and crp and pct serum levels. after enrollment, within minutes from hospitalization the demographic, clinical history and clinical disease characteristics of each child were recorded. moreover, a blood sample was drawn at admission to the hospital and divided in two parts: one sample was sent to the central laboratory of the hospital for the determination of routine tests including the wbc count, the percentage of neutrophils, and crp level; the second sample was used for the determination of pct, strem-1, mr-proanp, and mr-proadm levels as well as for pneumococcal and mycoplasma pneumoniae detection. finally, a nasopharyngeal swab was obtained from all the enrolled children using a pernasal nylon flocked swab and was stored in a tube of universal transport medium (kit cat. no. 360c, copan italia, brescia, italy) for respiratory virus, streptococcus pneumoniae, and mycoplasma pneumoniae detection. the serum of the blood samples that had to be used for new biomarkers' serum level determination and nasopharyngeal samples were conserved in freezer at -80˚c in each center and later sent to the laboratory of the pediatric high intensity care unit of the university of milan for centralized processing. biomarker determination. wbc counts, neutrophil percentages and serum crp levels were determined by the central laboratory of the hospital using routine methods. strem-1 concentrations were measured using an elisa according to the manufacturer's instructions (iq products, groningen, the netherlands) with a detection level of <7 pg/ml. an automated immunofluorescent assay was used for the determination of the levels of mr-proadm, mr-proanp and pct according to the manufacturer's instructions (bráaáhámás, germany). the functional assay sensitivity was previously assessed as being less than 0.25 nmol/l for mr-proadm, 10 pmol/l for mr-proanp, and 0.06 ng/ml for pct. the detection limit that was calculated using the imprecision profile was previously assessed as being 0.02 ng/ml with a probability of 95% for pct, 0.05 nmol/l for mr-proadm, and 2.1 pmol/l for mr-proanp. strem-1, mr-proadm, mr-proanp and pct were chosen due to the sensitivity and specificity showed in adults with cap for differentiating viral and bacterial cap or severe and nonsevere cap [7] [8] [9] . clinical and radiographic information, new biomarkers' serum level determinations and results on nasopharyngeal samples were not available for the central laboratory of the hospital where routine methods were performed. two different persons in the laboratory of the pediatric high intensity care unit of the university of milan performed the determination of the new biomarkers and the viral and bacterial analyses on nasopharyngeal samples without exchanging information and in absence of any clinical and radiographic information. respiratory virus detection. viral rna or dna was extracted from the respiratory secretions within 24 hours of collection using a nuclisens easymag automated extraction system (biomérieux, craponne, france) and was then tested using the luminex x tag respiratory virus panel fast assay (luminex molecular diagnostics inc., toronto, canada) to detect influenza a virus (subtype h1 or h3), influenza b virus, respiratory syncytial virus (rsv)-a and -b, parainfluenzavirus-1, -2, -3 and -4, adenovirus, human metapneumovirus (hmpv), coronaviruses 229e, nl63, oc43 and hku1, enterovirus/rhinovirus (rv) and human bocavirus in accordance with the manufacturer's instructions. the enterovirus/rv-positive samples were retested using a real-time polymerase chain reaction (pcr) assay using the iag-path-id one step rt-pcr kit (applied biosystems, foster city, ca) and the primers and probe sequences reported by lu et al. to identify rv cases [12] . streptococcus pneumoniae and mycoplasma pneumoniae detection. to identify pneumococcal cases, nucleic acid extracts from blood and swab samples were tested for the autolysin-a (lyta) and wzg (cpsa) genes of s. pneumoniae using real-time pcr as previously described [13] . each sample was tested in triplicate and was considered positive if at least 2 of the 3 tests were positive. to maximize sensitivity, no internal amplification control was used in the reaction, but there was an external control. m. pneumoniae was looked for in blood and nasopharyngeal swabs with validated, nested pcr, as described previously [14] . identification of probable bacterial and viral infection. chest radiographs with alveolar or non-alveolar findings were initially classified as of possible bacterial or of possible viral origin, respectively, according to the who indication [10] . then, radiological findings were coupled with results of the real-time pcr tests on blood samples and nasopharyngeal swabs. evidence of s. pneumoniae or m. pneumoniae in these samples further supported bacterial etiology. notably, s. pneumoniae can be detected in the nasopharyngeal secretions of children with viral cap [15] , but its presence in absence of viral detection in children with alveolar cap is suggestive of probable pneumococcal cap [16] [17] [18] . moreover, even if it has been recently reported that m. pneumoniae is frequently carried in otherwise healthy children [19] , it seems reasonable to think than when this pathogen is detected in children with cap in absence of s. pneumoniae or respiratory viruses, it is the real cause of the lower respiratory infection independent of the radiological characteristics [20] . finally, the presence of one or more respiratory viruses in the nasopharynx is commonly considered the etiologic agent of cap because carriage of viruses in healthy subjects is uncommon [21] . in practical terms, cap was considered to have a probable bacterial (pb) origin in the presence of 1) the detection of s. pneumoniae and m. pneumoniae in the blood with a chest radiograph indicative of any type of cap; 2) a nasopharyngeal swab positive for s. pneumoniae associated with chest radiograph suggesting alveolar cap; and 3) a nasopharyngeal swab positive for m. pneumoniae associated with chest radiograph suggesting any type of cap. probable viral (pv) cap was diagnosed in the presence of a nasopharyngeal swab that was positive for one or more respiratory viruses associated with a chest radiograph leading to the diagnosis of non-alveolar cap. cases that could not be included in these groups were considered undetermined. clinical information and blood test results were not available to the person that made this final classification of bacterial versus viral cap: a total sample size of 430 patients (assuming that about 60% of them have bacterial infection) achieves 80% power, with alpha = 0.05, to detect a change in sensitivity from 0.70 to 0.78 (and 83% power to detect a change in specificity from 0.7 to 0.8), using a two-sided binomial tests. sample size was computed using pass software v.11 (ncss, lcc, kaysville, utah, usa). all pb cases and all pv cases were evaluated together. continuous variables are presented as the mean ± standard deviation (sd), and categorical variables are presented as numbers and percentages. comparisons between groups (i.e., pb vs pv and severe vs non-severe cap) were performed using the χ 2 or fisher's exact test, as appropriate (for categorical variables), or a two-sided student's t-test after confirming that the data were normally distributed (based on the shapiro-wilk statistic) or a two-sided wilcoxon's rank-sum test otherwise (for continuous variables). diagnostic performances of the biomarkers were evaluated with receiver operating characteristic (roc) curves and the area under roc curve (auc). the best cut-off values for different biomarkers were obtained based on the highest sensitivity and specificity through the roctab function in stata. in case of indeterminate results for crp and new biomarkers, the lowest limit of detection of the various methods was considered. missing data were reported in the tables and the missing information was not included in the statistical analyses. all analyses were conducted using sas version 9.2 (cary, nc, usa) and stata version 11.0 (statacorp lp, college station, tex) statistical packages. a total of 433 children (males, 56.0%; mean age 4.2 ± 3.5 years) with radiologically confirmed cap were enrolled. their demographic, clinical and laboratory characteristics are reported in table 1 . results on respiratory viruses, s. pneumoniae, and m. pneumoniae were available for all the patients. cap was ascribed to bacteria in 235 (54.3%) children and to one or more viruses in 111 (25.6%) children. in 87 cases (20.1%), the etiology of the disease was undetermined. bacteremia was detected in 28 cases (6.5%): s. pneumoniae in 27 cases and m. pneumoniae one case. globally, s. pneumoniae was considered the probable etiologic agent in 195 (83.0%) pb cases, and m. pneumoniae was considered the probable etiologic agent in 40 (17%) cases. among pv cap, rsv and rv were the most common and were detected as single pathogens in 52 (46.8%) and 12 (10.8%) children, respectively. in the remaining cases (47, 42.4%), co-infections between these viruses and other viral agents were found. moreover, 312 (72.2%) children had severe disease. among them, 178 (57.0%), 78 (25.0%), and 56 (18.0%) had a pb, a pv or an undetermined infection, respectively. at admission, the percentage of neutrophils was significantly higher in children with pb than in those with pv cap (63.6% ± 20.7 vs 56.6 ± 19.7; p<0.05). similar results were observed for both crp and pct (crp, 21.3 ± 48.1 mg/l in pb and 8.0 ± 30.4 mg/l in pv cases, p<0.05; pct, 6.1 ± 17.0 ng/ml in pb and 1.1 ± 3.4 in pv cap, p<0.05). moreover, the levels of mrproadm found in pb cap cases were significantly higher than those in undetermined cap cases (0.50 ± 0.94 vs 0.35 vs ± 0.17, p< 0.05). for the evaluation of the diagnostic performance of the studied biomarkers, only children with defined pb or pv cap were considered. diagnostic performance of studied biomarkers at enrollment to predict bacterial and viral infections is reported in table 2 . all of them had low auc values. however, crp and pct had the best performances for both pb (auc of 0.66, 95% ci: 0.61-0.71, and 0.69, 95% ci: 0.63-0.75, respectively) and pv (auc of 0.68, 95% ci: 0.62-0.63, and 0.67, 95% ci: 0.60-0.64, respectively) cap identification. cut-off values with the highest sensitivity and specificity combination for the identification of pb and pv infections using crp were !7.98 mg/l and 7.5 mg/l, respectively. when pct was considered, the cut-off values with the highest combined sensitivity and specificity were !0.188 ng/ ml for pb cap and 0.07 ng/ml for pv cap. strem-1, mr-proanp, and mr-proadm had predictive values for both pb and pv infections that were lower than that evidenced for crp and pct but were not higher than that from the wbc count and neutrophil percentage, as evidenced by the auc value that was lower than 0.60. table 3 shows the biomarker levels at enrollment according to the severity of the disease. all the studied parameters, with the exception of mr-proadm, were significantly higher in severe cap compared with non-severe cap (p<0.01 for mr-proanp, wbc count, neutrophil percentage and pct; p<0.05 for strem-1 and crp). even for the identification of severe cases, the predictive value of all the studied biomarkers was poor ( table 4 ). the best results were obtained when pct (auc = 0.65, 95% ci: 0.57-0.63) and mr-proanp (auc = 0.65, 95% ci: 0.59-0.71) were evaluated. however, in both table 2 . diagnostic performance of soluble triggering receptor expressed on myeloid cells-1 (strem), midregional proatrial natriuretic peptide (proanp) and midregional proadenomedullin (proadm) biomarkers, as compared to white blood cell (wbc) count, neutrophils percentage, c reactive protein (crp) and procalcitonin (pct) at enrolment to predict bacterial and viral infections, according to biomarker cut-off with highest sensitivity and specificity. cases, the biomarker cut-off with the highest combined sensitivity and specificity (!0.093 ng/ ml for pct and !33.8 pmol/l for proanp) had relatively good sensitivity (higher than 70%) but limited specificity (of approximately 55%). other biomarkers were even less effective (all auc < 0.60). this study is the first to evaluate the utility of serum strem-1, mr-proanp, and mr-proadm concentrations in predicting the etiology and severity of pediatric cap in comparison to routine biomarkers. in this study, to overcome the clinical, radiological and laboratory problems that limit the definition of etiology and severity of cap in children, the identification of bacterial and viral cap was based on the criteria usually accepted by the international literature [16] [17] [18] . a combined evaluation of radiological findings and detection in the blood and in nasopharyngeal samples of the most important respiratory viral and bacterial agents of cap in children, was performed. moreover, to establish severity, the criteria suggested by bts were used. despite a certain number of cap cases, probably those due to mixed infection, remained 2.6 ± 9.1 * crp, c-reactive protein; pct, procalcitonin; sd, standard deviation; strem, soluble triggering receptor expressed on myeloid cells-1; mr-proanp, midregional proatrial natriuretic peptide; mr-proadm, midregional proadenomedullin; sd, standard deviation; wbc, white blood cell count. & one subject could not be categorized as severe or non-severe disease due to missing data. § p<0.05 for comparison between severe and non-severe disease groups. * p<0.01 for comparison between severe and non-severe disease groups. doi:10.1371/journal.pone.0163262.t003 table 4 . diagnostic performance of soluble triggering receptor expressed on myeloid cells-1 (strem), midregional proatrial natriuretic peptide (proanp) and midregional proadenomedullin (proadm) biomarkers, as compared to white blood cell (wbc) count, neutrophils percentage, c reactive protein (crp) and procalcitonin (pct) at enrolment to predict severity of disease, according to biomarker cutoff with highest sensitivity and specificity. undetermined, this method has probably lead to the identification of those caps which are more likely due only to bacteria or only to viruses. interestingly, as reported in several recent studies and probably thanks to the new molecular diagnostic methods that permit us to enlarge in comparison with the past possibilities for viral identification [4] , the prevalence of pb and pv among children with severe cap was similar. a global evaluation of the results of this study seemed to indicate that in children with cap, strem-1, mr-proanp, and mr-proadm blood levels are unable to differentiate bacterial from viral diseases or to identify severe cases. at admission, the mean values of all these biomarkers were similar in pb and pv cases. when attempts to evaluate the sensitivity and specificity of each of these biomarkers in defining the etiology and severity of the studied cap cases were made, either the sensitivity or the specificity was found to be very low, leading to a modest predictive value. this result was confirmed by the values of the auc, which were always below 0.70 for a single biomarker for both the definition of the etiology and the assessment of severity, a value that suggests poor accuracy of the studied tests. moreover, for the definition of the etiology of pediatric cap, the predictive ability of these biomarkers seemed to be lower than that of crp and pct, whereas for the identification of severe cases the best results were obtained with evaluations of pct and mr-proanp. the diagnostic relevance of strem-1 in bacterial diseases, including cap, has been studied in experimental animal studies and in adult humans with conflicting results. some studies have reported that in cases of sepsis or cap, strem-1 is a superior indicator of bacterial disease compared with crp and pct [22, 23] . moreover, it was demonstrated that strem-1 levels were significantly higher in neonates with sepsis than in healthy controls [24] . finally, in children with bronchiectasis, strem-1 sputum levels correlated with markers of neutrophilic inflammation but not necessarily with crp concentrations, suggesting that strem-1 may be more sensitive in detecting pulmonary neutrophilic inflammation than crp [25] . however, other studies have reported results similar to those found by our study. in adults with ventilator-associated pneumonia, strem1 was a poor predictor of vap among critically ill subjects undergoing direct bronchoscopy [26, 27] . different patient and sample characteristics used to measure strem-1 concentrations might explain the different results. in most of the cases showing a role for strem-1 in the diagnosis of bacterial infection, the data were collected in patients with a chronic underlying disease who were hospitalized in intensive care unit, whereas in this study, only otherwise healthy children with an acute cap episode were enrolled. moreover, the different criteria used to classify the etiology and severity of cap could explain the differences among the results presented in the literature. changes in mr-proanp have been associated with acute and chronic heart failure [28] , although the prognostic value of this biomarker in cap seems to be maintained independent of chronic heart failure [29] . moreover, in diseases associated with bacteremia, this biomarker is increased [30] . because cap with bacteremia is generally the most severe, it was concluded that this biomarker could simultaneously detect bacterial cap and identify the most severe cases. alan et al. reported that the addition of blood biomarkers, including mr-proanp, to clinical scores significantly improved the prognostic capabilities of the pneumonia severity index [31] . kruger et al. reported that mr-proanp was a good predictor of mortality risk in patients with cap [29] . however, the poor ability by mr-proanp to predict bacteremia in patients with cap was evidenced by guinard-barbier et al. [32] . additionally, in this case, differences in the characteristics of the studied patients might explain the results. none of the children in our study had chronic heart failure, and the number of bacteremia cases was very small. it is possible that the blood levels of this biomarker significantly increased and had a true prognostic value only in very severe bacteremic cases of cap. moreover, the criteria used in this study to measure severity were different from those used in the studies in which adults were enrolled. in those studies, severity was measured considering the final outcome of the disease, whereas in this study, consistent with pediatric guidelines, severity was considered only at admission. similar conclusions can be drawn for the mr-proadm evaluation. additionally, this marker has been tested mainly as a marker of severity, although there are data indicating that it can be of value in the diagnosis and prognosis of sepsis and bacterial cap [9] . once again, it seems highly likely that modifications of this biomarker occur only in very severe bacterial diseases that are relatively uncommon iamong children with cap. the problems that pediatricians have in differentiating bacterial from viral cap and in assessing the severity of the disease are highlighted by the poor predictive value evidenced in this study for wbc count, neutrophil percentage, and crp and pct levels. crp and pct were slightly better than wbc count and neutrophil percentage. however, the importance of crp and pct in the definition of the etiology and the severity of pediatric cap has been largely studied and discussed with conflicting results. crp as a type of acute-phase reaction protein is closely related with inflammatory reaction and tissue injuries and can be influenced by factors other than bacterial components. for many years, it has been shown that only extremely high crp serum levels are associated with bacterial disease and a negative prognosis, whereas in many cases, such values do not permit an estimate of the real etiology of the disease [32] [33] [34] . cohen et al. showed that pct and not crp was the only independent predictor of apyrexia in children hospitalized for cap [35] . however, recently, it has been reported that crp can predict outcomes of pediatric cap cases because crp levels are significantly associated with both fever duration and hospital length of stay [36] . for every 1 mg/dl increase in crp, the length of stay increased by 1 hour. moreover, it was shown that extremely elevated crp levels are associated with unfavorable outcomes, including death, in pediatric patients, highlighting the importance of very high levels, uncommonly assessed in daily practice [37] . similarly, the results reported for pct are inconsistent. nascimento-carvalho et al. showed that in 95 children, pct had a negative predictive value for differentiating bacteremic infections from viral infections, atypical bacterial infections, and nonbacteremic typical bacterial infections [38] . more recently, galetto-lacour et al. reported a positive role of pct in differentiating bacterial from viral cp, particularly when the disease was due to s. pneumoniae [39] . additionally, we previously used a pct cut-off value to identify pv cap and to guide antibiotic therapy [40] . conflicting results were reported in other studies. korpi et al. reported that pcr had a sensitivity in the identification of bacterial cap of only 55% [41] , whereas later, these authors found that high values of pct !5 ng/ml were associated with a high predictivity of bacterial cap [42] . however, it seems evident that only very high serum concentrations of pct are predictive of true bacterial cap. regarding pct and disease severity, a study by don et al. found that higher mean values were present in children who needed hospitalization; additionally, in this case, a non-marginal number of cases could not be classified because of similar results among children with mild and severe disease [43] . limitations of our study include the missing data for laboratory biomarkers in some patients, potential classification bias in the etiologic diagnosis, and the fact that patients with undetermined infection were excluded from the analysis. however, our evaluation has been done in a large study population with an appropriate calculation of sample size even excluding patients with undetermined infection. in addition, viral and bacterial results in nasopharyngeal aspirates and blood were available for all the study patients. moreover, a sophisticated statistical analyses has been performed, although further research on the role of the studied biomarkers in cap and other infectious diseases is recommended. in conclusion, most of the problems related to the evaluation of the pb or pv origin of a pediatric cap case together with the prevision of its outcome remain unsolved. no advantages are given by the use of strem-1, mr-proanp, and mr-proadm, whereas pct maintains its role. more studies are needed to assure a proper approach to cap therapy in children and to avoid the risk of useless antibiotic therapy. epidemiology and etiology of childhood pneumonia in 2010: estimates of incidence, severe morbidity, mortality, underlying risk factors and causative pathogens for 192 countries improving outcomes in community-acquired pneumonia antibacterial resistance worldwide: causes, challenges and responses unsolved problems in the approach to pediatric community-acquired pneumonia clinical features for diagnosis of pneumonia in children younger than 5 years: a systematic review and meta-analysis utility of serum procalcitonin and c-reactive protein in severity assessment of community-acquired pneumonia in children diagnostic value of strem-1 in bronchoalveolar lavage fluid in icu patients with bacterial lung infections: a bivariate meta-analysis direct comparison of three natriuretic peptides for prediction of short-and long-term mortality in patients with 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children with alveolar community-acquired pneumonia and respiratory syncytial virus or rhinovirus infection association between nasopharyngeal load of streptococcus pneumoniae, viral coinfection, and radiologically confirmed pneumonia in vietnamese children carriage of mycoplasma pneumoniae in the upper respiratory tract of symptomatic and asymptomatic children: an observational study differentiation between mycoplasma and viral communityacquired pneumonia in children with lobe or multi foci infiltration: a retrospective case study viral pneumonia soluble triggering receptor expressed on myeloid cells and the diagnosis of pneumonia surface and soluble triggering receptor expressed on myeloid cells-1: expression patterns in murine sepsis circulating soluble triggering receptor expressed on myeloid cells-1 (strem-1) as diagnostic and prognostic marker in neonatal sepsis expression of soluble triggering receptor expressed on myeloid cells-1 in childhood cf and non-cf bronchiectasis soluble triggering receptor expressed on myeloid cells-1 (strem-1) as a diagnostic marker of ventilator-associated pneumonia soluble triggering receptor expressed on myeloid cells-1 in bronchoalveolar lavage fluid is not predictive for ventilator-associated pneumonia mid-region pro-hormone markers for diagnosis and prognosis in acute dyspnea: results from the bach (biomarkers in acute heart failure) trial pro-atrial natriuretic peptide and pro-vasopressin for predicting short-term and long-term survival in community-acquired pneumonia: results from the german competence network capnetz is mid-regional pro-atrial natriuretic peptide (mrproanp) an accurate marker of bacteremia in pyelonephritis? clinical risk scores and blood biomarkers as predictors of long-term outcome in patients with community-acquired pneumonia: a 6-year prospective follow-up study bacteremia and mr-proanp changes in mild community-acquired pneumonia differential diagnosis of viral, mycoplasmal and bactaraemic pneumonias on admission to hospital diagnostic and prognostic value of interleukin-6 and c-reactive protein in community-acquired pneumonia procalcitonin predicts response to beta-lactam treatment in hospitalized children with community-acquired pneumonia association of white blood cell count and c-reactive protein with outcomes in children hospitalized for community-acquired pneumonia extremely elevated c-reactive protein levels are associated with unfavourable outcomes, including death, in paediatric patients procalcitonin is useful in identifying bacteraemia among children with pneumonia elevated inflammatory markers combined with positive pneumococcal urinary antigen are a good predictor of pneumococcal community-acquired pneumonia in children procalcitonin measurements for guiding antibiotic treatment in pediatric pneumonia serum procalcitonin in pneumococcal pneumonia in children the value of clinical features in differentiating between viral, pneumococcal and atypical bacterial pneumonia in children efficacy of serum procalcitonin in evaluating severity of community-acquired pneumonia in childhood key: cord-337795-khqx4t4q authors: pellecchia, umberto; crestani, rosa; decroo, tom; van den bergh, rafael; al-kourdi, yasmine title: social consequences of ebola containment measures in liberia date: 2015-12-09 journal: plos one doi: 10.1371/journal.pone.0143036 sha: doc_id: 337795 cord_uid: khqx4t4q introduction: in the ebola virus disease (evd) outbreak in liberia, two major emergency disease-control measures were cremation of bodies and enforcement of quarantine for asymptomatic individuals suspected of being in contact with a positive case. enforced by state-related actors, these were promoted as the only method to curtail transmissions as soon as possible. however, as with other harsh measures witnessed by liberian citizens, in many cases those measures elicited uncontrolled negative reactions within the communities (stigma; fear) that produced, in some cases, the opposite effect of that intended. methodology: the research has been conducted in two phases, for a total of 8 weeks. ethnography of local practices was carried out in 7 neighbourhoods in monrovia and 5 villages in grand cape mount county in liberia. 45 focus group discussions (432 participants) and 30 semi-structured interviews sustained the observing participation. randomly selected people from different social layers were targeted. the principal investigator worked with the help of two local assistants. perceptions and practices were both analysed. results: participants stressed how cremation perpetuated the social breakdown that started with the isolation for the sickness. socio-economical divides were created by inequitable management of the dead: those who could bribe the burial teams obtained a burial in a private cemetery or the use of funeral homes. conversely, those in economic disadvantage were forced to send their dead for cremation. state-enforced quarantine, with a mandatory prohibition of movement, raised condemnation, strengthened stigmatization and created serious socio-economic distress. food was distributed intermittently and some houses shared latrines with non-quarantined neighbours. escapes were also recorded. study participants narrated how they adopted local measures of containment, through local task forces and socially-rooted control of outsiders. they also stressed how information that was not spread built up rumours and suspicion. conclusions: populations experiencing an epidemic feel a high degree of social insecurity, in addition to the health hazards. vertical and coercive measures increase mistrust and fear, producing a counter-productive effect in the containment of the epidemic. on the other hand, local communities show a will to be engaged and a high degree of flexibility in participating to the epidemic response. efforts in the direction of awareness and community involvement could prove to be better strategy to control the epidemic and root the response on social participation. the recent ebola epidemic hit three countries in west africa (liberia, sierra leone and guinea) the most, while it was swiftly secured in other countries where it took hold. by the end of may 2015, the disease resulted in 11,162 deaths, on 27,781 cases globally declared [1] . the containment of the largest outbreak of ebola in human history strained international and local response capacity, distressing local populations, caught in the grip of a disastrous epidemic and a limited public health service. emergency coercive measures were adopted in liberia in august 2014, when the epidemic was finally officially declared. mainly enforced by state-related actors, in liberia the most significant measures were cremation of bodies and quarantine of contacts (hereinafter, state-imposed quarantine). the objectives of these measures were to quickly interrupt the transmission caused by funerals and by contacts of symptomatic persons. lifestyles, traditions, and an ill-defined concept of culture were held to be the main responsible of the circulation of the virus by the implementing actors [2] [3] [4] [5] [6] . this rationale, as well as the way the measures were imposed, and the communication messages sustaining the emergency actions, created social consequences for the liberian population. a body of literature exists on how public health measures negatively impact people without offering a substantial reduction of virus transmission [7] [8] [9] [10] . existing research focuses on theories of public health, their relations with ethics and human rights, and the practicability of measures in different settings and for different infectious diseases. amongst these, influenza, severe acute respiratory syndrome (sars) and few about ebola, are the most recent studied, although the effects of quarantine for example have been an issue dating back to the plague [11] . conversely, little consideration has instead been dedicated to a full analysis of cremation and to communities' direct experiences of coercive measures in public health. how do people perceive interventions to protect their health? what dynamics does coercion trigger? what social practices do communities activate to resist, or respond to, such interventions? to which extent are these measures effective in the light of the social consequences they generate? to address these questions, this study was conducted to assess liberian community perspectives on state-imposed ebola public health and outbreak containment measures implemented in 2014 and 2015. the research consisted of a qualitative approach based on ethnography of social practices and perceptions. the research took place in montserrado (greater monrovia and st. paul bridge districts) and grand cape mount (tewor and commonwealth districts) counties. specifically, the zones in montserrado were new kru town, congo town, paynesville, clara town, west point, and central monrovia; and in grand cape mount: bangoma, tiene, diah, camp 3, and augustine village. the areas were purposively selected, on the basis of different criteria: epidemiological incidence of ebola (cases per neighbourhood or village); presence of quarantined houses; and particular sociological or demographic features (i.e. large religious community; presence of market; bus stations; health facilities). the ethnic composition of montserrado county is mixed, while the included areas in grand cape mount county are mainly vai, mende, and to a limited extent fula. the governance of the areas is structured around the co-existence of two forms of power, with on the one hand a government-based political power, with superintendents, commissioners, and chairpersons, all affiliated to a greater or lesser extent to the political parties, and on the other hand the "traditional" system of chieftaincy. the research dealt with both types of leaders, with an emphasis on those who were based locally (i.e. chairpersons and chiefs) and who were most involved in the daily activities of their communities. other forms of administration of power and socio-political legitimacy included the "secret societies" of which poro and sande are the most important. the fieldwork was carried out in two phases: the first, in october and november 2014, focused on an analysis of the general social response toward the epidemic and in particular the modification of funeral and burial practices. an in-depth enquiry of the perceptions of cremation was conducted during this phase. data on quarantine was collected in the second phase in january and february 2015. participants were purposively recruited on a voluntary basis, after full explanation of the goals of the research. all demographic strata of society were included, both males and females, with the exclusion of children. specific attempts were made to cluster people into groups particularly affected by the epidemic, e.g. public transportation workers; market sellers; and healthcare workers. both political (government and traditional) and religious (christian, muslim, and traditional) leaders were involved as participants. representatives of the two main secret societies in liberia (poro and sande) were involved both as informants on the practices of their societies, and as opinion leaders in general. the principal investigator worked with two local assistants, trained as data collectors and interpreters. both assistants held university degrees and were selected on the basis of their background in research, their knowledge of the area and their local language competencies (especially vai and mende). one of the two assistants was originally from grand cape mount county. the researchers collected both social practices and narratives (opinions; perceptions; feeling; comments). the first were acquired through participant observation: the researchers visited, and participated in, social activities; gatherings; family reunions; meetings of local leaders; and religious and other daily activities. reflexivity on the positioning of the researchers into the research settings was permanently performed in order to contain misrepresenting dynamics possibly activated in the relationship between researcher and observed / respondent. narratives were collected through 45 focus group discussions (fgds) for a total of 432 participants and 30 semi-structured individual interviews. narratives were collected in liberian english, and were transcribed and translated in standard english if necessary. translations were checked for consistency by a third liberian native not involved in the research. question grids with starter questions were provided to the moderator of fgds and interviewers, in order to frame the discussion. the grids were organized according to three main themes: 1) general social perception of the epidemic and community's reactions; 2) funerary and burial practices before and during the epidemic, and opinions on cremation; and 3) health-seeking behaviours and perception of quarantine. each grid contained basic questions but was open to probes and contextual questions. social status, religious belonging, degree of involvement in the epidemic (e.g. family link with the sick or dead) were considered as patterns of discussion. narratives were linked with observation of practices and reciprocally used to frame new questions and targets of observation until saturation of information was reached. the following table (table 1) shows the characteristics of the study participants. data were analysed through a medical and social anthropological approach. narratives and observations were recorded and transcribed. data were compared through triangulation of methodological tools (observation / fgd / interview); time (e.g. during quarantine / after quarantine); space (different neighbourhood or villages); persons (e.g. leader / common person). themes rising from the interviews and fgds were compared, taking into consideration social status of the interviewed; kind of involvement in the epidemic (e.g. history of illness; experience of quarantined; experience of death of a relative); area and general social environment where the interviewed acted; the narrative logic of the themes emerging from his/her interview. no software was used: data analysis was carried out through assessment of transcriptions, with key-words and key-concept comparison. quotes excerpted and used as references in the following article are examples of general understanding. the research was conducted in a high risk environment, with a mandatory no-touch policy and obvious restrictions. participant observation was therefore organized without a full involvement of the researchers in the social practices, as common in an anthropological fieldwork, relying on observations and collection of narratives. informed consent was obtained verbally following explanation of the purpose of the research, the anonymity of the data gathered, and the voluntary basis of involvement. research was conducted on the basis of the principles of professional responsibility as declared in the statement of ethics of the american anthropological association [12] . the full study was approved by university of liberia-pacific institute for research & evaluation institutional review board (monrovia, liberia). forced quarantine of asymptomatic contacts of positive cases was the main state-imposed measure that transformed social perceptions and practices. in august 2014 the government of liberia commanded the quarantine of an entire neighbourhood in monrovia, called west point, considered a slum [13] . west point was quarantined through the intervention of military forces to respond to the protest of the residents. many people were injured, and one person died during the clashes. some students living in the area recalled that day in a fgd: west point's quarantine paved the way for forced isolation of communities and individuals in liberia [14] . all participants in the study had a relative, a friend or an acquaintance isolated. state-enforced quarantine has been enforced in several areas of monrovia and villages in rural counties. while the intensity of the enforcement differed, overall it manifested common characteristics. movements of the quarantined were restricted for the incubation period (21 days); a daily control of the temperature was planned; distribution of food and items were planned. the research records that few awareness messages were spread to explain the public health measure. the government imposed the measure through a formal mandate to commissioners, chairpersons, and chiefs of rural villages. however, the local leaders who participated in the research reported a lack of involvement in the decisional forums and felt the form of the state-enforced quarantine did not comply with local communities' dynamics. in addition, spiritual leaders were not consulted, despite their strong influence in the communities. state-enforced quarantine broke social networks of solidarity and was implemented with lots of gaps that raised social concern. study participants, both those under quarantine and neighbours, explained how food, water and other items were only intermittently distributed by the implementing agencies, creating harm and forcing residents to disobey the imposed isolation. in augustine village, in grand cape mount county, dwellers helped with food and water a woman under quarantine who for days was not receiving assistance. one of the man clearly stated that such help was a common habit among the residents of the small village, and that quarantine threatened this spirit (man, 38 years old, augustine village, 15/1/2015, s7). a second consequence was that state-enforced quarantine increased level of stigmatization, which instead was much lower where community managed contacts follow-up. quarantine triggered a process of publicly labelling those under forced isolation, which created panic, fear and disenfranchisement of minority groups. in zinc camp, st. paul bridge, monrovia, communities experienced several houses under quarantine connected to two positive cases. a local religious leader, during a fgd, remarked bitterly how the quarantine brought suspicion and mistrust to his community: he feared that the stigma associated with forced isolation would eventually undermine the mutual trust, forcing those who need help not to ask for it (pastor, 50 years old, st. paul bridge, monrovia, 16/2/2015, s8). stigma was reported frequently: people isolated were accused of being infected and judged for their alleged controversial social behaviours. the common labelling was "ebola people", despite the fact that they were not even infectious. indeed, quarantine fed rumours and gossip: families that happened to be under quarantine were said to belong to a different ethnic group or religion, and therefore dangerous for being alien. in tiene, a village in grand cape mount county, a young man conferred the reason of forced isolation of his neighbours on their belonging to the "fula" tribe. the village hosts a small fula community, mainly engaged in business activities and vehicle repair. despite being perfectly integrated and actually liberian citizens, a subtle popular belief holds that fula are nomads and therefore foreigners. due to this, people interviewed in tiene linked the spread of ebola-known to come from outside the country-to the fula. a third consequence of coercive quarantine can be identified in the obstacles to health seeking behaviour. being blocked into houses, and with the fear of being infectious, people under quarantine kept easily treatable non-ebola illnesses as a secret. this fostered "informal" medicine providers-such as drugs sellers-who were more mobile than "formal" medicine. the temperature control planned as a way to monitor quarantined, was actually very discontinuous, rooting a perception of a measure not beneficial and purely coercive. the state-enforced quarantine was imposed vertically, and overshadowed local isolation measures that were already organized by local leaders (such as chairpersons, village chiefs, and opinion leaders) and were more socially accepted. these local measures started as spontaneous and self-organized form of protection, but institutional levels paid very little attention to them, choosing the imposition of power. indeed, communities did understand that isolation was crucial in case of symptoms. local leaders, such as chairpersons, organized the limitation of movements in the communities for contacts, inviting them to be reachable, or for foreigners to be identifiable as soon they arrived in the neighbourhood. in case of isolation of contacts, leaders and community-based task forces provided food and water. in most cases, locally developed isolation of asymptomatic contacts was not total. the case of an area near mount barclay, monrovia, illustrates the differences between coercive quarantine and local management of contact follow-up. two houses with a total of 52 individuals, contacts of two positive cases, were requested to be available for contact tracing by the local chairperson, who later worked in close collaboration with the local contact tracer, visiting the contacts every day. food and water were provided by the community, but only minimally. in fact, no restriction of movement was imposed, since contacts agreed to be available daily. here through the words of the local leader and one of the individual contacts: â«i don't use the word quarantine, it is too harsh. i prefer surveillance or monitoring. . .i can't understand why everyone's using quarantine! when mr. b. [the positive case] died, all community participated to the mourning, he was part of us. we don't see his relatives as our enemies, they're our fellows. quarantine cannot work here, it drowned people in fear and economic hardship. we don't ban movements, they are free to go and get food. cremation of bodies was another measure taken to curtail transmission. participants acknowledged that during the months of august and september, the amount of bodies was so high that such a measure was needed. however, they criticized the way it was implemented and the duration of enforcement, continuing cremation even after the number of fatalities decreased, when safe and dignified burials were considered feasible again. health care workers explained that the rationale of cremation stands on the belief that funerals are locally dangerous and people handle bodies unsafely. however, the narratives of participants do show a high degree of flexibility within communities in changing practices. a significant decrease in unsafe handling of funerals-the so-called "traditional" funerals-was registered and recognized by all respondents, including the representatives of poro and sande secret societies, that were believed to be resistant to change. in early october, the leader of one of the biggest muslim communities in monrovia published an article in a local newspaper encouraging his fellows to handle deaths in a proper way to avoid the spread of ebola [15] . this contrasted with a general narrative in the media that claimed resistance among muslims to changing their practices. indeed, initial resistance was transversal amongst the different religions, and participant observation in greater monrovia showed how some pastors of charismatic churches preached the non-existence of the virus. the mandatory cremation and the disorganized body collection created an informal economy of dead bodies, uncontrolled by health authorities and international ngos. demands for proper funerals resulted in burials organized by non-official teams that, upon payment of a sum of money, took care of the removal of the body, finding a graveyard and burying the body. however, such a "service" parallel to the official one was not affordable to every family. research participants reported a sort of "price-list" that these teams presented each time: those who could afford it, paid for the whole process, while the poor where forced either to keep the body at home or to wait for the burial team and send the body to the crematorium. some participants even claimed that bribery and corruption was normal, and rich families could pay the official burial team to "close an eye" and let them contact a funeral home. these dynamics and social behaviours have been labelled by the media as "secret burials" although they were very well known by locals in monrovia. secret burials" was used especially by the media and ngos as a catchphrase to identify very different practices. these practices included the "secret burials" allegedly performed by the secret societies sande and poro and "secret" burials" which resulted as a form of resistance to the mandatory cremation,. the latter had little to do with the secret societies, and were "secret" in the meaning of being hidden to institutional governmentbased authorities. while they were performed "in secret", their existence was hardly hidden, and circulating around the city it was possible to observe burials performed in the small private cemeteries spread throughout. all study participants confirmed through empirical examples the following situations that frequently occurred in monrovia until the cemetery started its activities: -families calling the national hotline of the ebola task force to activate the burial teams for picking up a body and not receiving any answer; -families waiting for burial teams for hours or even days, with dead bodies in the streets or indoors; -long waits for the non-ebola certification in order to bury the non-ebola bodies and consequent development of an informal market of certificates, quite expensive for the average local income; -several cases of non-ebola deceased sent to cremation because of fear; -in the event of death, families were hesitant to involve the ebola task force to remove the body since they knew that it would be sent to the crematorium. the body was therefore kept at home and secretly buried in private cemeteries in town or sent in the villages of origins in the countryside; -upper classes of monrovia's society could afford the involvement of funeral homes to quickly obtain certificates of non-positivity to evd and therefore organize a funeral and burial. before the epidemic, funeral homes were normally used by all families despite their economic condition. during the epidemic such firms increased the prices of the different services operating a consequent, although indirect, selection of the customers. the following quote from a fgd held in paynesville, monrovia, exemplifies the communities' feeling around the situations listed: â«when i saw that body on the road i felt very bad. it stayed there for hours under the sun! and nobody came to pick up. . .we called, called, called. the chairman too he called the moh but nobody picked. and then-i tell you!-i understand those that keep the body in, which is unsafe, but at least they can do a proper funeral and cry for it. i'd do the same: just pay someone to dig the grave and you know where your beloved is.â» (man, 40 years old, red light, monrovia, 26/10/2014, s3) participants in fgds and interviews agreed that outbreak response managed by state-related actors created a climate of fear that did not help them to understand the causes, ways of transmission, and prevention strategies. they stressed how the awareness campaign relying on harsh messages-such as "ebola is a killer!", "ebola can kill anyone!" or "last warning: ebola is real!"-frightened them to the point that they believed contagion was unavoidable. this feeling guided their health behaviours, as the following quote evokes: although messages evolved to a less drastic tone throughout the outbreak, fear and insecurity continued to shape people's perceptions. the outbreak was described as exploiting social bonds and creating mutual distrust. containment measures duplicated such feelings and revived the time of civil war, with its climate of insecurity and suspicion towards neighbours, close acquaintances and foreigners. as during the war, all but the family were perceived as a potential enemy. a participant to a fgd pictures his experience as such: â«now you can't trust anyone-not your mother, not your father, not your wife. everyone's a danger. your dead too are a danger [. . .] this is a breakdown, a breakdown of our habits, of our traditions. we will never return back.â» (man, 34 years old, elwa, monrovia, 9/10/2014, s1). during an interview, a local leader said: â«ebola first kills and then stealsâ» (man, 49 years old, new kru town, monrovia,12/10/2014, s4), to describe the modus operandi of cremation that "steals" the beloved. cremation of bodies and forced quarantine, as ways to quickly reduce transmission of ebola outbreak for the benefit of the larger public, produced social dynamics of resistance in the same population that they wished to protect. on one hand, nonconformity to cremation was based on the socio-cultural and economic importance conferred by liberian society to funerals. unequal and drastic imposition of cremation contributed to condemnation of a measure that, at least initially, was understood as a containment strategy. on the other hand, forced quarantine of asymptomatic contacts tore up an already broken social material, fuelling fears and mutual mistrust that did not help promotion of health seeking behaviours. respondents attributed the main reason of the social resistance to coercive measures to the experience of war that fragmented the society as well as the epidemic [16] . scholars in law, public health, and bioethics show how enforcement of coercive measures in communicable diseases may possibly help contain an outbreak, but since they operate through command, they result in a huge social harm, which threatens the same outbreak control strategy [10, [17] [18] [19] . as the observations and interviews of this research reveal, similar observations can be seen in the case of ebola in liberia, where coercive "public" health measures produced the opposite effect of distancing individuals (the "public") to implementing agencies [20] [21] . an anthropological perspective allowed an understanding of socio-political implications of public health measures, analysing how these are practically experienced and performed by people [22] [23] [24] [25] . the set of dynamics that sprang from liberian society while facing the epidemic appeared to be coherent responses to imposed devices not perceived as helpful. the quarantine increased the level of insecurity and fear, forcing people under isolation to avoid seeking care, and neighbours to stigmatize and deny. the social response of liberians to state-enforced, vertical, quarantine, as described by participants, demonstrated a thin boundary between collective responsibility and respect of individual rights and civil liberties. the argument of interrupting transmission through vertical measures was not balanced by a guarantee of basics for living, social protection and individual freedom. although top-down quarantine has shown its public health limits in several past occasions [17] , and even though it has been strictly regulated in usa and europe, it was indiscriminately used in liberia [19, 26] , with means inconceivable in the western world, such as military force [27] . on the contrary, less invasive forms of isolation can be developed at community level, and can be practiced with the consensus of involved citizens, supported by close leaders and a network of acquaintances. cremation was for the first time widely performed as an outbreak control measure in liberia. despite a "technical" success in quickly eliminating bodies, its unintended consequence was the emergence of burials performed in secret, often with unsafe practices, indirectly promoting what can be termed an informal economy of death. then, sustained by the idea that traditions were wrong and funerals were the main cause, implementation of mandatory cremation crudely defined the transition from life to death as a simple biomedical passage of state, wiping out deep social links, and endangering the credibility of the measure itself. indeed, both measures seemed to underscore that the local population's health should be ruled instead of managed. three major implications emerge from this research. first, as stressed by the narratives of the informants, alienating the participation of populations and excluding their involvement, leads to non-compliance and increases risk of virus transmission. a community-based isolation of contacts, managed locally by leaders, health authorities, and community-based organizations, would likely have been a more acceptable alternative to vertical quarantine. second, a top-down militarized-like measure does not allow the population to understand the emergency situation, creating a distance between institutions and citizens [14] . third, coercion does not directly result in a change of practices or benefit health-seeking behaviours. on the contrary, enforcement of vertical measures is seen as a command, an injustice and a judgment of allegedly untoward behaviours. such mechanisms feed self-organization as a form or resistance, but also a creation of informal economies that could be hazardous for public health and increase socio-economic divides. throughout the epidemic, safe and dignified funerals should been sustained as a clear sign of respect and understanding of the complex socio-cultural and spiritual significance of the dead, even in times of crisis. cremation, for example, could have been promoted as a temporary measure and organized through the involvement of community leaders-in particular those more in touch with people, such as chairpersons, chiefs, pastors and imams. family of the deceased could have been involved and could have been returned the ashes. again, local patterns of funerals could have been identified, discussed with health promoters in collaboration with recognized local leaders, to plan a prevention of transmission within the families [28] . in postcolonial environments such as those mainly affected by ebola in 2014 and 2015 already characterized by inequalities, fear, insecurity and stigma constitute the social aspect of the disease outbreak. this research supports a comprehensive approach to outbreak control, where health is considered in its totality of shades, from the biomedical to the social. in this sense, an understanding of the drivers of fear and mistrust in the affected communities which ultimately result in behaviour that may increase disease transmission, appear to be a crucial and substantial part of an outbreak control. geneva: who kissing the corpses in ebola country smuggled bushmeat is ebola's back door to america bushmeat and the politics of disgust liberia's ebola epidemic: did the government fall asleep at the wheel? do funerals spread ebola? ethical and legal challenges posed by severe acute respiratory syndrome: implications for the control of severe infectious disease threats the journal of law, medicine & ethics: a journal of the american society of law containing pandemic influenza at the source from sars to ebola: legal and ethical considerations for modern quarantine fire and gallows: quarantine in history principles of professional responsibility ebola in urban slums: the elephant in the room press union of liberia. imam against bathing dead body ebola: the missing link factors influencing compliance with quarantine in toronto during the 2003 sars outbreak the ethics of quarantine. the virtual mentor: vm anthropology's contribution to public health policy development. mcgill journal of medicine: mjm: an international forum for the advancement of medical sciences by students ebola: limitations of correcting misinformation strengthening west african health care systems to stop ebola: anthropologists offer insights ten things that anthropologists can do to fight the west african ebola epidemic teaching anthropology to medical students the anthropology of an emerging disease. belmont using a tactic unseen in a century, countries cordon off ebola-racked areas the lancet infectious diseases. rationality and coordination for ebola outbreak in west africa social pathways for ebola virus disease in rural sierra leone, and some implications for containment the author wishes to thank rupa kanapathipillai for her valuable comments to this report. thanks to emilie venables, patrik kamara, alfred massaquoi, the msf field and coordination teams that have worked during the ebola outbreak in liberia, and msf-ocb ebola emergency task force, for their direct or indirect inputs to this work. conceived and designed the experiments: up. performed the experiments: rc. analyzed the data: td. contributed reagents/materials/analysis tools: rvdb. wrote the paper: yak. key: cord-339796-gccnvh0z authors: zhang, si min; jejcic, alenka; tam, james p.; vahlne, anders title: membrane-active sequences within gp41 membrane proximal external region (mper) modulate mper-containing peptidyl fusion inhibitor activity and the biosynthesis of hiv-1 structural proteins date: 2015-07-31 journal: plos one doi: 10.1371/journal.pone.0134851 sha: doc_id: 339796 cord_uid: gccnvh0z the membrane proximal external region (mper) is a highly conserved membrane-active region located at the juxtamembrane positions within class i viral fusion glycoproteins and essential for membrane fusion events during viral entry. the mper in the human immunodeficiency virus type i (hiv-1) envelope protein (env) interacts with the lipid bilayers through a cluster of tryptophan (trp) residues and a c-terminal cholesterol-interacting motif. the inclusion of the mper n-terminal sequence contributes to the membrane reactivity and anti-viral efficacy of the first two anti-hiv peptidyl fusion inhibitors t20 and t1249. as a type i transmembrane protein, env also interacts with the cellular membranes during its biosynthesis and trafficking. here we investigated the roles of mper membrane-active sequences during both viral entry and assembly, specifically, their roles in the design of peptidyl fusion inhibitors and the biosynthesis of viral structural proteins. we found that elimination of the membrane-active elements in mper peptides, namely, penta trp→alanine (ala) substitutions and the disruption of the c-terminal cholesterol-interacting motif through deletion inhibited the anti-viral effect against the pseudotyped hiv-1. furthermore, as compared to c-terminal dimerization, n-terminal dimerization of mper peptides and n-terminal extension with five helix-forming residues enhanced their anti-viral efficacy substantially. the secondary structure study revealed that the penta-trp→ala substitutions also increased the helical content in the mper sequence, which prompted us to study the biological relevance of such mutations in pre-fusion env. we observed that ala mutations of trp664, trp668 and trp670 in mper moderately lowered the intracellular and intraviral contents of env while significantly elevating the content of another viral structural protein, p55/gag and its derivative p24/capsid. the data suggest a role of the gp41 mper in the membrane-reactive events during both viral entry and budding, and provide insights into the future development of anti-viral therapeutics. the envelope protein (env) of human immunodeficiency virus type i (hiv-1) is a class i fusion glycoprotein [1] . it protrudes out of the viral envelope as homotrimers composed of non-covalently-linked gp120/gp41 heterodimers [2] [3] [4] . recognition of the viral receptor and co-receptor by the surface gp120 subunit activates the fusion machinery in the transmembrane (tm) gp41 subunit (fig 1) [5] [6] [7] [8] , resulting in the insertion of gp41 n-terminal fusion peptide region (fp) into the target cell membrane. this pre-fusion intermediate conformation of gp41 connects the cellular membrane and the viral envelope, exposing and extending the two heptad repeat (hr) regions, hr1 and hr2 [9] [10] [11] . the intermediate conformation quickly resolves into a stable six-helix bundle (6-hb) conformation, after hr2 folds back onto the central hr1 to form a coiled-coil trimer-of-dimers [12, 13] . this predisposes the opposing membranes into sufficient proximity for subsequent envelope fusion with the plasma membrane and viral content delivery [14] . the post-6-hb-formation lipid mixing and subsequent membrane fusion is mediated by the membrane proximal external region (mper) in gp41 (fig 1) , a hydrophobic region between hr2 and the tm domain [16, 17] . mper induces fusion-required membrane perturbation through a direct interaction with the membranes [18, 19] . sequential alignments have revealed a high conservation of mper among different groups of hiv-1 (fig 1) [15] . in particular, it contains two conserved sequence elements that contribute to its membrane perturbation function. one is an enrichment of aromatic amino acids, in particular trp (fig 1) , and the other is its cholesterol-interacting c-terminus. previous studies have shown that ala substitutions of the five trp residues abrogated the ability of mper-containing peptides to partition into and destabilize liposomal membranes [15] . sequences of the anti-hiv-1 first and second generation fusion inhibitor, t20 and t1498, respectively, are shown together with the mper-containing peptides tested in this study, ek37, el30, qk26, qt19, lk21 and lk21-5w5a, and all are aligned with the mper sequence. the mper sequence is highlighted in bold with its conserved residues shaded. peptide lk21-5w5a have all five tryptophan residues in mper sequence substituted by ala. [18, 19] . in the context of gp41, the trp!ala substitutions in mper inhibit membrane fusion events, such as the fusion pore expansion, during viral entry. [17] . furthermore, the mper cterminus (lwyik) shows sequence characteristics of the cholesterol recognition/interaction amino acid consensus (crac) motif,-l/v-(x)(1-5)-y-(x)(1-5)-r/k[20, 21] . cholesterol is enriched in viral envelopes and also in the cell membrane lipid rafts where viral receptors are enriched during viral entry, making the membranes rigid and counteracting viral entry [22, 23] . the c terminus of mper can facilitate the induction of membrane destabilization and subsequent fusion in the cholesterol-enriched liposomal membranes [24] [25] [26] . the membrane destabilizing ability of mper sequences has implication in designing viral fusion inhibitors. t20 and t1249, two of the first fusion inhibitors, are synthetic peptides containing sequences from both the hr2 and the n-terminus of mper (fig 1) [27, 28] . hr2 sequences enable these inhibitors to bind to the exposed hr1 of gp41 in the pre-fusion intermediate conformation, and thereby halt the formation of the 6-hb [9] . in addition, mper sequences act as inhibitors at a later stage of the viral entry, possibly through anchoring the peptide into the cellular membrane through their last four residues, wnwf [29] [30] [31] . the membrane anchoring abilities of the two fusion inhibitors correlate with their antiviral activity [32] . still, peptide fusion inhibitors, such as t20 and t1249, only included partial mper sequences without the c-terminal sequence [33] . due to the high variation and mutation rate of hiv-1 env protein, the fusion inhibitors were constantly challenged by drug resistance issues. the mper is a conserved, exposed and accessible region, and therefore it could be an additional target for the design of potential fusion inhibitors. apart from its role in viral entry, env also interacts with host cellular membranes during its biosynthesis and trafficking to the viral budding site. as a type i transmembrane protein, env is co-translationally translocated into the rough endoplasmic reticulum (er) and further transported to the golgi complex for maturation into gp160 and subsequent proteolytic processing [34, 35] . the resulting gp120/gp41 trimers are then transported to the cholesterol-rich plasma membrane regions (e.g. lipid rafts), following the secretory pathway [36] . studying sars-cov (severe respiratory syndrome associated corona virus) we observed that the trp residues in mper modulate the selective incorporation of the spike protein into lipid rafts (unpublished observations, see s1 and s2 figs). however, salzwedel and colleagues found that neither deletion nor trp substitution mutations in the hiv-1 mper affected env maturation, or steadystate levels, but had an effect on its incorporation into virus particles [17] . here we describe the roles of the trp residues in the membrane-active mper sequence in anti-hiv fusion inhibitor design and a surprising role in the biosynthesis of viral structural proteins. six peptides ranging from 19 to 37 amino acids (a.a.) were designed to contain the mper sequence in its full length, with c-terminal truncation, or with penta trp!ala substitution (fig 1) . their anti-viral activities were tested in a single-round infectivity assay using pseudovirus. dimerization of anti-viral peptides has been shown to enhance both their structural stability and the number of interaction sites and thus their anti-viral efficacy [37] [38] [39] [40] . therefore, we also tested our peptides as n-or c-terminal dimers. hiv-1 env with mutations of trp residues at the mper region were also constructed to examine the roles of the trp residues in the biosynthesis, maturation, trafficking, and viral incorporation of the viral structural proteins. hiv-1 gp41-derived monomeric peptides were custom synthesized by synpeptide co ltd (shanghai, china) and the dimeric peptides by pepscan presto bv (amsterdam, netherlands). the antibody to gp160/41 (chessie 8) and vif (#319) were obtained through the nih aids research and reference reagent program [41] . polyclonal rabbit anti-nef antibody was obtained from thermo fisher scientific, inc. monoclonal mouse anti-β-actin antibody was obtained from sigma. the antibody to p24 (ef7), has previously been described [42] . the secondary antibodies, hrp-conjugated polyclonal goat anti-rabbit immunoglobulins and hrpconjugated polyclonal rabbit anti-mouse immunoglobulins, were obtained from dako. hltat (cat. #1293) and tzm-bl (cat. #8129) cell lines were obtained through nih aids research and reference reagent program [43] [44] [45] [46] [47] and cultured in dulbecco's modified eagle medium (life technologies) supplemented with 10% fetal bovine serum (life technologies) and 1% penicillin-streptomycin (life technologies). the expression plasmid for env, nef (negative regulator factor) and vpu (virion protein u) from the hiv-1 strain nl4-3 (pnl1.5eu+) was kindly provided by dr. stefan schwartz (lund university, lund, sweden) [48] . pnlhivxδuδss contains a vpu-deficient hiv-1 genome with the signal sequence of env truncated and was constructed from pnl4-3 with quick change ii xl (stratagene) [49] . specifically, the fragment ecori-bamhi from pnl4-3 was cloned into puc18 to create puc18envxδu that has the vpu start codon mutated to ata and contains a xma i cleavage site immediately upstream of vpu, using the primer pair b(xδu): (forward) 5'-gcagtaagtagtacccggga tacaacctataatagtagcaatag-3', (reverse) 5'-ctattgctactattataggttg tatcccggtactacttactgc-3'. the same mutation was introduced into δnss-gp160 [50] , encoding vpu and a gp160 with a signal peptide of 11 a.a. length, using the primer pair a (xδu): (forward) 5'-gaagcgcgcacggagtacccgggatacaacctataatagtagc-3', (reverse) 5'-gctactattataggttgtatcccgggtactccgtgcgcgcttc-3'. the xmai-nhei fragment of the constructed mutant δnss-gp160 was then directionally inserted into pucenvxδu to create pucenvxδuss11, in which site-directed mutagenesis was carried out with primer pair(δss): (forward) 5'-ggatattgataatctgtagtgctatggaaaaattgtgg tc-3', (reverse) 5'-gacccacaatttttccatagcactacagattatcaatatcc-3' to introduce gp160 signal sequence mutation. the ecori-nhei fragment of the resulted mutant construct was then subcloned into p83-10 [51] , of which the saii-ncoi fragment was directionally transferred into pnl4-3, creating pnlhivxδuδss. pnl1.5eu+w5a, pnl1.5eu+w3a and pnl1.5+w2a were constructed from pnl1.5eu+, using quick change ii (agilent technologies). pnl1.5eu+w2a was constructed using the primer pair a(w676.678): (forward) 5'-gcaagtttgtggaattggtttaacataacaaa tgcgctggcgtatataaaattattcataatgatagtaggagg-3', (reverse) 5'-cc tcctactatcattatgaataattttatatacgccagcgcatttgttatgttaaacc aattccacaaacttgc -3'. pnl1.5eu+w3a was constructed using the primer pair b (w664.668.670): (forward) 5'-gaatgaacaagaattattggaattagataaagcggca agtttggcgaatgcgtttaacataacaaattggctgtggta -3', (reverse) 5'-tacc acaccacatttgttatgttaaacgcattcgccaaacttgccgctttatctaattc caataattcttgttcattc -3'. pnl1.5eu+w5a was constructed from pnl1.5eu+w3a using primer pair a (w676.678). the mutated pnl1.5eu+ plasmids were sequenced between nucleotide 7988 to 8533 with the sequencing primer (7988f-ctcctggggatttggggttg and 8533r-gtctctcaagcggtggtagc). hltat cells (5 x 10 5 ) were co-transfected with the hiv-1 proviral clone pnlhivxδuδss, and with the env-and vpu-expressing plasmid pnl1.5eu+, using fugenehd (roche). for the production of mutant pseudotyped hiv-1, we used pnl1.5eu+w5a, pnl1.5eu+w3a or pnl1.5eu+w2a during transfection, instead of pnl1.5eu+. the cell culture supernatant was collected 48 hours (h) post-transfection, clarified by centrifugation and 0.45-μm filtration, used for virion precipitation or stored at -80°c until further use. viral particles were precipitated from the clarified and 0.45μm-filtered cell culture supernatant at 4°c for 48h in 1:6 (vol:vol) polyethylene glycol 6000 containing 0.667m nacl. the precipitated particles were further concentrated by centrifugation at 16,000 times g for 20 min at 4°c. the virus pellets were dissolved in radio-immunoprecipitation assay (ripa) buffer containing 50 mm tris-hcl (ph = 7.4), 1% triton x-100, 1% deoxycholate, 150 mm nacl, 1 mm edta, and 0.1% sodium dodecyl sulfate (sds) and supplemented with complete protease inhibitor cocktail (roche). cell-free pseudotyped hiv-1 virus of 100tcid50 was either pre-incubated with peptides of various concentrations at 37°c for 1 h, or applied directly to infect 10 4 cd4+, ccr5+ tzm-bl cells. tzm-bl cells contain integrated copies of an ltr (long terminal repeats)-driven luciferase reporter gene. seventy-two h post-infection, the infectivity of pseudovirus was assessed as luciferase activity, using the one-glo luciferase assay system (promega). the tcid50 of pseudotyped hiv-1 was calculated based on the luciferase activity of the infected tzm-bl cells, using the reed-muench method and the cut-off value set at 3 times of the background signal [52] . effective concentrations of peptides inhibiting 50% (ic 50 ) and 80% (ic 80 ) of viral infectivity were estimated with graphpad prism. the ic50 and ic80 values were estimated from the dose-response curves that were curve-fitted with the sigmoidal dose-response non-linear regression model on prism graphpad software, using the percentage of inhibition data and the log values of peptide concentrations. the cytotoxicity effect of the peptides on tzm-bl cells were determined by prestoblue cell viability assay (lifetechnologies, singapore), according to the manufacturer's protocol. briefly, peptides of different concentrations were added to 10,000 tzm-bl cells seeded in 96-well plates. upon 24 h of incubation, prestoblue cell viability reagent was added to the cells and incubated for 30 min at 37°c. resulting absorbance values were recorded at 570nm and 600nm (baseline). final spectrum was obtained by normalizing the 570 nm values to the 600 nm values. circular dichroism spectroscopy analysis was performed to study the secondary structure of the monomeric peptides in trifluoroethanol (tfe). tfe was used to mimic the hydrophobic environment at the membrane fusion junction. the measurements were made on chirascan circular dichroism spectrometer (applied photophysics). fifty μm peptide was dissolved in 10%, 20%, or 40% tfe and subjected to the measurement with three repeats in a cell of 0.1mm pathlength (hellma uk ltd.) at 25°c. samples were measured between 190nm and 260nm, with a 0.5nm step resolution, a measurement speed of 60nm/min and a 1nm bandwidth. the baseline was measured with 10% tfe, with three repeats. the final spectrum was generated by subtracting the averaged sample spectrum with the baseline spectrum, followed by smoothing with a savitsky-golay filter. the secondary structures of the peptides were estimated from deconvoluting the respective circular dichroism spectra using the cdsstr deconvolution algorithm on dichroweb, with a cut-off nrmsd value set at 0.15 [53] [54] [55] . hltat cells (5 x 10 5 ) expressing wt or mutant pseudotyped hiv-1 were lysed in ripa buffer on ice, centrifuged and mixed with laemmli reducing buffer. precipitated pseudotyped hiv-1 viral particles dissolved in ripa buffer were also prepared in laemmli reducing buffer. cell lysates or virus lysates were resolved by sds-polyacrylamide gel electrophoresis (page), transferred to nitro-cellulous membranes and immunoblotted with anti-gp41 antisera and followed by hrp-conjugated anti-rabbit secondary antibody for env expression, or with anti-p24 antisera and followed by hrp-conjugated anti-rabbit secondary antibody for p24 and p55/gag expression, or with anti-nef antibody and followed by hrp-conjugated anti-rabbit secondary antibody for nef expression, or with anti-vif and followed by hrp-conjugated anti-mouse secondary antibody for vif expression, or with anti-β-actin and followed by hrp-conjugated anti-mouse secondary antibody for β-actin expression. the membranes were either exposed to film or analyzed with g:box chemi xx6 (syngene). the band intensities were quantified by imagej software. p24 levels in the cell-free lysate of the virus-producing hltat cells or the cell culture supernatants were quantified by the automated system architect (abbott). a standard curve was generated using p24 of known concentration and curve-fitted with the linear non-regression model on prism graphpad software. prior to each measurement, the samples were diluted to the concentrations within the linear range of the standard curve. six mper-containing peptides (fig 1) ranging from 19 to 39 a.a were prepared with acetylated n-termini and amidated c-termini and were tested as fusion/entry inhibitors against pseudotyped hiv-1(nl4-3). to ensure study the early events of the hiv-1 viral replication cycle a hiv-1 pseudovirus system allowing only a single replication cycle was employed. pseudovirus particles were produced by the co-transfection of the env-and vpu-expression-deficient proviral vector pnlhivδuδss and the env-, vpu-and nef-expressing vector pnl1.5eu+ [48] , which generates viral particles capable of entering and infecting target cells but not capable of giving rise to infectious second-generation viral particles. the pseudotyped hiv-1 (nl4-3) particles were pre-incubated with each of the six mper-containing peptides for 1 h and then added to the target cells, tzm-bl cells, which stably express a tat-responsive luciferase reporter gene allowing for the monitoring of successful hiv-1 entry and viral protein production. the inhibition of infection was monitored by measuring the luciferase expression at 72 h post-infection. peptide lk21, which contained the entire and exclusively the mper sequence, inhibited 50% and 80% of viral entry and infection at 8.0 μm and 12.3 μm, respectively ( fig 2a) . inclusion of five hr2 residues to lk21 n-terminus, generating the 26-a.a. peptide qk26, decreased the ic 50 and ic 80 values to 3.9 μm and 8.8 μm, respectively. however, further n-terminal extension with the addition of nine and sixteen hr2 residues to lk21, resulting in peptides ek30 and ek37, did not lead to any substantial increase in their anti-viral potency. however, adding the hr2 hydrophilic residues greatly enhanced the solubility and structural stability of the mper-containing peptides qk26, ek30 and ek37, allowing further application and design, such as peptide dimerization that will be elaborated on in section 2. concentrations of the peptides yielding a 50% and 80% reduction in luciferase activity were estimated with graphpad prism. results shown were summarized from three independent experiments with serial dilutions of peptides in replicates of two. b. no cytotoxicity effect was observed for the mper-derived peptides at 50 μm in tzm-bl cells. fifty μm of the peptides were incubated with 10,000 vero cells for 24 h. prestoblue cell viability reagent was subsequently added to the cells, and cytotoxicity effects were monitored as absorbance values (od) at 570 nm and 600 nm (baseline). c. circular dichroism spectra and the estimated secondary structure contents of the peptide lk21, qk26, ek30, ek37 and qt19 in 10% tfe. fifty μm of the peptides were dissolved in h 2 o supplemented with 10% tfe and were subjected to circular dichroism spectroscopy measurement. d. circular dichroism spectra and the estimated secondary structure contents of peptide lk21 in increasing concentrations of tfe. fifty μm of the peptides were dissolved in h 2 o supplemented with 10%, 20% or 40% tfe and were subjected to circular dichroism measurement. e. circular dichroism spectra and the estimated secondary structure contents of peptide lk21-5w5a in increasing concentrations of tfe, measured as described in c. to determine the necessity of the conserved mper c-terminal cholesterol-interacting motif (lwyik) for the antiviral effect of the peptides, the mper c-terminal sequence, nwlwyik, was deleted from the most active peptide, qk26, creating the peptide qt19. this deletion abrogated the antiviral activity, as qt19 failed to inhibit viral infection at concentration up to 33.3μm (fig 2a) . in addition, the importance of the enriched aromatic residue trp in the mper region was examined by mutating all the trp to ala in the exclusively mper-containing peptide, lk21, resulting in the peptide lk21-5w5a. interestingly, the substitutions of all the trp did not just abolish the antiviral activity of the peptide, but even enhanced viral infectivity in a dose-dependent manner (fig 2a) . at 26.6 μm, the lk21-5w5a increased the viral infectivity by 50%. the inhibitions of viral infectivity by the mper-derived peptides were not due to cytotoxicity, as incubating tzm-bl cells with 50 μm of the peptides for 24 h did not result in any statistical difference in cell viability between the control (dmso-treated) and the peptide-treated cells (fig 2b) . to investigate the structure-function relationship of mper-containing peptides in inhibiting the entry of pseudo-hiv-1, the secondary structures of the peptides were determined by circular dichroism (fig 2c) . the hr2 region of another class i viral fusion glycoprotein, the spike (s) protein of the severe acute respiratory syndrome associated coronavirus (sars-cov), has previously been shown to be largely α-helical and a synthetic peptide (hr2-s) derived from this region served as a control peptide in the following cd study [56] . the peptides were prepared in 10% tfe that mimics the lipidic environment at the juxtamembrane junction. the n-terminal extension of lk21 with hr2-derived residues generally increased the helicity in the mper-containing peptides. deleting the c-terminal sequence, nwlwyik, from qk26 did not change the secondary structure drastically (fig 2c) . to mimic the increasingly lipidic environment transition, which the mper undergoes during membrane fusion, the tfe concentration was increased from 10%, 20% and to 40%. with increasing tfe concentrations, lk21 gradually exhibited a more alpha-helical conformation, with the first minimum of its spectra shifted from 212 nm to 209 nm, and then to 208 nm, and the estimated α-helical content increased from 41% to 52% and then to 71% (fig 2d) . in contrast, lk21-5w5a, the peptide with all the trp replaced by ala, exhibited canonical alpha-helical spectra and a high α-helical content (84%) starting from 20% tfe (fig 2e) , which indicates the importance of trp residues in maintaining the structural plasticity of the mper sequence. we next investigated if the antiviral effect of our peptides could be enhanced by dimerization at either the n-or c-terminus. we hypothesized that n-terminally dimerized mper-containing peptides would mimic the fusion-active oligomerization state of gp41 mper, thus having an enhanced binding affinity with their interaction partners (e.g. fp), and thereby possess an improved viral inhibitory effect. dimeric peptides were constructed with parallel peptide chains with either two carboxylic termini or two amino termini using chemoselective ligation strategy [57] . to obtain the nterminal linked dimers (-dn), monomeric peptides were synthesized with an additional n-terminal cys residue, which was further ligated via a thiazolidine linkage to a linker molecule consisting of two ser branching from lys (fig 3) . the c-terminal peptide dimers (-dc) were synthesized on mbha resins, and ligated c-terminally to the linker via the amino functional groups on the linker (fig 3) . n-and c-terminal dimers were synthesized from the peptide ek30 and ek37. c-terminally linked qk26 dimer was also synthesized, but due to synthetic difficulties, its n-terminal dimer was not obtained. the dimeric peptides were tested for their ability to inhibit viral entry using the single-round infectivity assay with tzm-bl cells as described above for the monomeric peptides (fig 4a-4d) . the n-terminal dimerization of ek37 and ek30 enhanced their anti-viral potencies (fig 4b and 4c) . the ek37-dn and ek30-dn have ic 50 values of 1.2 μm and 1.1 μm, an increase of the potency by 5.2-and 1.9-fold compared to the monomeric ek30 and ek37, respectively (fig 4d) . in contrast, the c-terminal dimerization decreased the potency of ek37 and qk26, with ic 50 values of ek37-dc and qk26-dc elevated by 1.7 and 1.3 fold, respectively ( fig 4d) . meanwhile, the c-terminal dimerization had inconsistent effect on the antiviral potency of the ek30-dc, its ic50 value decreased and ic80 value significantly increased with respect to its monomeric form (fig 4d) . in summary these data indicate that the anti-viral activity is benefited by n-terminal dimerization of mper-containing peptides. no cytotoxicity effect of the monomeric nor dimeric peptides was observed in vitro in tzm-bl cells at concentrations up to 100 μm, as determined by the prestoblue cell viability assay (fig 4e) . our circular dichroism data suggest that the penta-trp!ala substitutions induces the mper peptide to commit to a predominantly helical structure regardless of the environmental lipidity fig 2e) . this poses the possibility that the same mutations may also affect the secondary structure of the mper sequence within the hiv-1 precursor env glycoprotein, gp160, which in turn may disturb its proper folding in the er, or affect its biosynthesis in other ways and eventually lead to viral defect. to investigate this, site-directed mutagenesis was performed on envexpressing plasmid pnl1.5eu+ [48] and generated three gp160 mutants, where all five trp, the three n-terminal trp (w664, w668 and w670), or the two c-terminal trp (w676 and w678) in the mper sequence were substituted with ala. the resulting constructs were termed w5a, w3a, and w2a, respectively. to examine the effect of the trp substitutions on env expression and viral maturation in the context of virus-producing cells and budding viral particles, hltat cells were co-transfected with the env-and vpu-expression-deficient proviral vector pnlhivxδuδss (described above) and with the wt or mutant pnl1.5eu+ to produce pseudo-typed hiv-1 (nl4-3) viral particles containing wt, w5a, w3a or w2a env. accordingly, the expressed pseudo-hiv-1 particles were termed hiv(wt), hiv(w5a), hiv(w3a) or hiv(w2a). at 48 h post-transfection, it was found that the env levels in cell lysates containing hiv(w5a), hiv(w3a) or hiv (w2a) were lower than that in lysate containing the hiv(wt) (fig 5b) , with the steady-state intracellular gp160 levels in cells producing hiv(w5a), hiv(w3a), hiv(w2a) were approximately 60% of that in hiv(wt) (p = 0.038 for w5a, p = 0.020 for w3a, p = 0.026 for w2a, n = 5) (fig 5c) . more striking effects of trp mutations were observed in the other viral structural proteins, specifically p55/gag and its derivative proteins. the hiv-1 genome encodes three structural proteins, which are the precursor protein env, p55/gag, and gag-pol. while env is translated into the er and is transported to the site of viral assembly at the plasma membrane via the secretory pathway, the p55/gag and gag-pol are expressed in the cytosol. the two viral cytosolic precursor structural proteins meet up with env at the plasma membrane where p55/gag directly or indirectly interacts with cytosolic tail of env to recruit it into the assembling viral particles [36] . during or after viral budding, the viral protease within gag-pol is activated leading to processing of gag-pol and p55/gag. among the p55/gag-derived proteins is the capsid protein, p24, which is commonly used to detect viral particle production [58, 59] . analysis of the p24 levels in cells producing wt or mutant pseudo-hiv-1 revealed that at 48 h post-transfection p24 in cells producing hiv(w3a) was approximately 45% (p<0.0001, n = 7) higher as compared to p24 in cells producing hiv(wt) (fig 5a) . however, the other two mutants, hiv(w5a) and hiv(w2a), showed no increased levels of intracellular p24 (fig 5a) . the analysis was performed using the automated system archiect (abbot) accreditated for detection of p24 in human serum. we also performed in-house validation experiments with hiv-1 infected cells in the presence of the protease inhibitor indinavir to confirm that the system did not detect the p24 within p55/gag but only the mature p24 once processed from its precursor protein. the corresponding cell lysates were therefore subjected to analysis by western blot and the findings clearly confirmed the elevated intracellular levels of p24 in the hiv(w3a) as compared to the hiv(wt) (fig 5b) . two other viral proteins, vif and nef were blotted and served as control for the transfection efficiency to which p55/gag and p24 expression was standardized. the results further indicated that the intracellular elevation of p24 by 93% (p = 0.01, n = 5) was a result of increased expression of p55/gag, as the p55/gag levels were 330% in the hiv(w3a) mutant as compared to that in the hiv(wt) (p = 0.007, n = 5) (fig 5b and 5c) . the extracellular p24 levels in the hiv(w3a)-expressing cultures, analyzed by the automated system archiect (abbot), were further found to be 67% higher than those of the hiv(wt) (p<0.0001, n = 7), consistent with the increased intracellular p24 expression levels from this mutant (fig 5d) . a smaller increase by 10% of extracellular p24 levels were detected in the hiv(w5a) (p = 0.0378, n = 7) expressing cultures (fig 5d) . in addition, the viral particles were isolated from equal volumes of the respective cell culture supernatants and subjected to analysis for env, p55/gag and p24 by western blot followed by densitometric analysis. as compared to that in hiv(wt), there was moderate decrease in env level among the mutant viral particles. in contrast, p55/gag and p24 contents in isolated hiv(w3a) virus particle were elevated by approximately 200% and 310%, respectively (fig 5e and 5f ), which is in line with elevated p24 levels detected in both the cell culture lysates and supernatants by the automated system, architect. it further indicates an increased hiv(w3a) viral particle production as were produced by co-transfecting hltat cells with pnlhivxδuδss and pnl1.5eu+, pnl1.5eu+w5a, pnl1.5eu+w3a, or pnl1.5eu+w2a, respectively. the p24 levels (ng) in cell lysates were quantified by the automated system architect (abbott). ***p < 0.001 as compared to wt by the unpaired student's t test. b. steady-state intracellular levels of viral proteins in pseudovirus-producing hltat cells. hltat cells from a were harvested 48 h post-transfection and the lysates were resolved by sds-page and immunoblotted with antibodies against gp41, p24, vif, nef and β-actin. vif and nef expression served as the transfection control. un-transfected hltat cells served as negative control. c. densitometric analysis of protein bands in blots from two independent experiments as described in in b was performed in imagej and presented as means ± sd, with gp160, p55/gag, and p24 levels in hiv(wt) standardized to 100%. *p < 0.05; **p < 0.01 as compared to wt by the unpaired student's t test. d. p24 levels (ng) in the culture supernatants of pseudovirus-producing hltat cells. p24 levels in the culture supernatant of hltat cells in a was quantified by the automated system architect (abbott). ****p < 0.0001 as compared to wt by the unpaired student's t test. e. env gp41, p55/gag and p24 levels in precipitated hiv(wt), hiv (w5a), hiv(w3a) and hiv(w2a). viral particles from the cell culture supernatant from a were precipitated, lysed, separated by sds-page and immunoblotted with antibodies against gp41 and p24. f densitometric analysis of the blot in e was performed in imagej and presented as means, with gp41, p55/gag and p24 a result of increased intracellular p55/gag expression. similar increase of p55/gag and p24 contents was observed in the hiv(w2a) particle isolate (fig 5e and 5f ). the influence of the trp substitutions to ala in the mper on viral entry and infectivity was also tested by adding equal volumes of the respective cell free culture supernatant to the tzmbl cells. forty-eight hours post-infection, the tat-activated expression of luciferase in the tzm-bl cells were measured and showed that in contrast to the hiv(wt), all the mutant particles hiv(w5a), hiv(w3a) and hiv(w2a) lost their ability to infect the tzm-bl cells (fig 5g) . these data collectively suggest that, while substitutions of the trp to ala in the mper sequence of gp160 decreased its intracellular expression levels and consequently moderately reduced the env incorporation into the viral particles, the mutations significantly influenced the intracellular expression levels of p55/gag. in particular, the substitutions of the trp664, trp668 and trp670 in the mper significantly elevated the intracellular p55/gag expression and subsequently the viral particle production. furthermore, the substitutions of the trp to ala rendered the viral particles non-infectious, in accordance with the previous literature [17] . the gp41 mper (hiv-1 nl4-3 : ldkwaslwnwfnitnwlwyik) induces membranefusion-required membrane perturbation in the viral envelope and cellular membranes, through its two conserved membrane-active sequence elements; the enrichment of aromatic residues (e.g. trp) and a c-terminal cholesterol interacting motif (lwyik) [17] . here we show that the membrane active sequential elements of gp41 mper are vital for mper-containing short peptidyl fusion inhibitors, as the omission of the c-terminal motif (lwyik) and the penta-trp!ala substitution abrogated their anti-hiv-1 activity. the peptide anti-viral activity could be enhanced through n-dimerization, but not c-dimerization. in this study, peptide lk21, containing the entire mper sequence, inhibited pseudotyped hiv-1(nl4-3) entry and infection with the ic 50 values at 8.0 μm. n-terminal extension of lk21 with five amphipathic residues derived from hr1-biding region of hr2 (628 a.a.-666 a. a.) enhanced the anti-viral effect and reduced the ic 50 by half. further n-terminal extension of lk21 did not enhance its anti-viral effect substantially, probably because this sequence does not include enough residues to mediate a stable interaction between the peptide and the viral hr1 as 6-hb formation. instead, the enhancement of the anti-viral efficacy through addition of five amphipathic residues correlated with the subtle increase in the helicity of the resulting peptides while maintaining the general structural profiles, as estimated from the respective circular dichroism spectrum. in the context of gp41, this amphipathic sequence upstream to the mper serves as an extension into hr2 and induces the n-terminus of mper in fusogenic gp41 to transform from an extended conformation to a helix upon increases in local lipidity [60] . this suggests that the addition of five residues of the n-terminal amphipathic sequence confers the peptides a more stable conformation and a capacity to interact with hr1, which lead to their enhanced anti-viral efficacy. the formation of 6-hb has been suggested to induce not only a secondary structural transformation in the mper n-terminus, but also to result in its quaternary structural rearrangement and oligomerization [60, 61] . meanwhile, the mper c-termini of the neighboring gp41 hiv-1 env mper in anti-viral design and viral life cycle stay monomeric and assume an extended platform to destabilize the cholesterol-enriched lipid bilayers, likely through its crac motif (lwyik) [24] [25] [26] . our data show that deletion of the c-terminal sequence including the conserved crac motif in the mper-containing peptide qk26 abrogated its anti-viral effect, suggesting that this membrane-active sequence plays an essential role in the anti-viral mechanism of the peptide. the peptide dimerization data further support that the inclusion of free c-termini for membrane interaction in the mper peptides are important for their inhibition of hiv-1 entry, as constraining the peptide c-termini through c-terminal resulted in the anti-viral effects of peptide ek37 and qk26 unenhanced. a previous study by nomura et.al also observed that the c-terminal trimerization of t20 failed to enhance its anti-viral efficacy significantly, presumably because the trimerization constrains its membrane-active mper sequence, offsetting the activity enhancement due to the potential cooperative interaction between its hr2 sequences and the viral gp41 hr1 [38, 62] . the enrichment of trp residues in mper is a second important membrane-active characteristic exhibited by class i fusion glycoproteins [63] . trp contains a large indole-ring side-chain that is preferred by the juxtamembrane interface of proteins, facilitating protein-membrane interaction and stabilizing protein structure [64] [65] [66] . in this study, we examined the influence of trp residues in the design of fusion inhibitors by substituting the indole-ring side-chains of the five trp residues in lk21 with the alkyl moieties of ala. surprisingly, the resulted lk21-w5a peptide promoted viral infectivity rather than inhibiting it, a phenotype that has been also observed when ala-substituting the trp residues of the sars-cov mper peptide [67] . the data indicate the significance of membrane-active elements in mper-containing peptides, both the crac motif and trp residues, for the inhibition of hiv-1 entry. this is in agreement with previous findings that increased membrane reactivity owing to inclusion of the mper n-terminal sequence (ldkwaslwnwf) as in t20 and t1249, correlated with an enhanced anti-viral effect of the peptides [29] [30] [31] [32] . of note, our data further demonstrate the involvement of the cterminal crac motif (lwyik) in facilitating viral inhibition for short mper-containing peptides with minimal inclusion of hr2 sequences. aside from its involvement in membrane fusion events, trp residues have also been shown to modulate the interaction between mper and other viral domain(s) during viral entry. specifically, gp41 mper interacts with fp to form a continuous hydrophobic track along with the 6-hb and promote membrane juxta-positioning [68, 69] . shortly before the interaction stabilizes and during the transition from the pre-fusion to post-fusion conformation, the mper and/or fp could be temporarily exposed and vulnerable to dominant-negative binding by peptidyl fusion inhibitors, such as lk21 of this study. we have recently shown that mper in the sars-cov spike protein interact with the internal fp (ifp) in a trp-dependent manner [67] . in the same study, trp!ala substitutions also resulted in mper-containing peptides to lose the dose-dependent inhibition of coronavirus entry, correlating with the disruption of the mper-ifp interaction. in gp41, trp670 has previously been shown to mediate the mper-fp interaction [70] . its ala substitution in the peptide lk21-5w5a could lead to a diminished affinity between the peptide and the fp in gp41, and hence contribute to the loss of the antiviral effect of the peptide. furthermore, penta-trp!ala substitutions in lk21 prematurely predisposes the peptide to the final helical conformation, losing the lipidity-induced structural plasticity. it suggests that the potential interactions between mper and other viral regions (such as fp) could also be conformation-dependent, and that the transition of mper from an extended to helical conformation could result in an intermediate species for such interaction to take place. whilst the c-terminal dimerization failed to enhance the anti-viral efficacy of peptides studied here, n-terminal dimerization lowered their ic 50 and ic 80 values up to 5-fold. our data would suggest that n-terminally dimerized mper-containing peptides mimic the fusion-active oligomerization state of gp41 mper, thus enhancing binding affinity with their interaction partners (e.g. fp and membrane), thereby having an improved anti-viral effect. the optimal length of the linker between the monomers could be explored to enhance the flexibility of the unit peptide, which may further enhance the cooperative interactions between the peptide multimer and gp41. the differential effects of n-and c-dimerization on the anti-viral effects of mper-containing peptides have been previously observed in our group, with sars-cov as the model virus ( s3 fig). n-and c-terminally dimerized peptides containing the s protein mper sequence were prepared as described in this paper, and enhanced and inhibited, respectively, the anti-viral effects against pseudotyped sars-cov compared to the monomeric peptide. the hiv-1 mper contains neighboring epitopes for broadly neutralizing antibodies, including 662-dkwa-665 for antibody 2f5 and 669-nwfnit-674 for antibody 4e10 [71, 72] . both antibodies have been shown to neutralize different strains of primary isolates of hiv-1 when administrated in cocktails in animal models [73] . despite being proven difficult, a tetramer peptide mimetic containing the mper sequence has elicited broadly neutralizing antibodies 4e10 and 2f5 in guinea pigs [74] . this rises the potential immunogenicity problem with the mper-derived anti-viral peptides. however, any such immune responses is not expected to induce major adverse effects in host, as any elicited anti-hiv-mper antibodies could probably be immunologically tolerated by the host, and may even further help to control the hiv replication. the direct evidence of the host immunological tolerance of 2f5 and 4e10 has been provided their recognition of two autoantigens, human kynureninase and splicing factor 3b subunit 3 [75] . furthermore, t20 which contains the epitopes for 2f5 and 4e10 has not only been clinically proven to be safe and effective in the presence of cross-reactive antibodies [76] , it has been shown to act synergistically with 4e10 in inhibiting viral infectivity [72] . in addition, the neutralizing capacities of the antibodies 2f5 and 4e10 require functional env trimers and probably would not neutralize the mper-derived peptides' anti-viral effect [73] . nevertheless, at the prospect of developing mper-derived peptidyl entry inhibitors, any potential elicited immunological responses should be examined and investigated [77] . finally, the peptides should be tested with different subtypes of hiv-1 to confirm the antiviral activity. env interacts with host membranes during both fusion/entry, and its biosynthesis and trafficking during viral budding. the substitution of all five trp to ala in the mper antiviral peptide resulted in it predominantly adopting a helical structure regardless of the environmental lipidity. this suggest that the trp residues could be equally vital for the secondary structure of the mper region within gp160, and consequently their substitution could hamper a proper folding and function of gp160. we further investigated the biological relevance of the mper trp residues in the biosynthesis of env and other viral proteins. previously, salzwedel et al. found that trp mutations in the mper affected incorporation of env into virions but no effect of the mutations was seen on the env levels in the cell lysate or on the plasma membranes [17] . here we observed that, in the transfected hltat cells expressing pseudotyped hiv-1, ala substitutions of all five trp, the n-terminal three trp (w664, w668, w670), and the c-terminal two trp (w676 and w678) residues, although moderately, lowered the steady-state intracellular gp160 and intraviral gp41 levels without affecting the migration patterns as compared to wt. the discrepancy between their and our findings may be due to differences in the assay used to quantify env, immunoprecipitation using patient serum versus immunoblotting. more interestingly, we observed that the mutations in env up-regulated the expression of the capsid protein p24 through up-regulating its precursor protein p55/gag, despite differential biosynthesis pathways between p55/gag and env. the results generally agreed with the previous understandings that env expression inhibits the steady-state intracellular level of p55/gag. it has been shown that the downregulation of p55/gag expression by env could be executed at both protein level through the env cytoplasmic tail, or at rna level through actions via the rev response element within the env gene [78, 79] . in this study, the upregulation of gag/p55 protein intracellular and intraviral levels were not proportional to the reduction of env protein levels and interestingly the pseudo-hiv-1 in which the n-terminal three trp or the c-terminal two trp were substituted gave a much higher increase of p55/gag than the pseudo-hiv-1 with all five trp replaced with an ala. hence it remains an open question if there is another distinctive mechanism through which the trp!ala mutations in env mper upregulated the gag expression, besides through lowering the intracellular env levels. while env is responsible for receptor/co-receptor recognition, membrane fusion and viral entry; p55/gag can independently induce the assembly and budding of virus-like particles in living cells and in vitro. its derivative protein, p24, dictates the proper maturation, size and morphology of the budding virions, which are essential for viral infectivity [80] . our data indicate that the trp residues in env mper are important for the biosynthesis of env and another major viral structural proteins p55/gag, which could collectively affect the viral fitness and be an additional factor, besides the absence of membrane-active trp indole ring sidechain, for the failed viral entry, as observed in a previous study [17] and confirmed in this study. in summary, our findings suggest active participation of membrane-active elements within mper (e.g. trp) in events that require protein-membrane interactions during both viral entry and assembly. these results indicate the importance of the five trp residues and c-terminal sequence (nwlwyik) in mper for the design of future mper-based fusion inhibitors and offer further insights into the understanding of viral structural proteins biosynthesis. the role of gp41 mper trp residues in modulating the viral contents of gag proteins might guide the discovery of potential therapeutic targets against hiv-1 infection. supporting information s1 fig. trafficking of the sars-cov spike protein to the lipid rafts. 293t cells were transiently transfected to express wild type spike protein (swt). twenty-four h post-transfection, cells were harvested and lysed on ice in 1% triton x-100 tne lysis buffer, and the cell postnuclear extracts were fractionated by 5%-30% sucrose gradient ultracentrifugation. eleven fractions were collected from top to bottom after centrifugation. samples were resolved by sds-page and western blot, with or without pngase f treatment. caveolin-1 serves as a positive marker for lipid raft. were detected in the lipid-raft-containing interfacial section between 5% sucrose and 30% sucrose, co-localizing with the lipid raft marker caveolin-1. both constructs contain two protein species with different sizes of 180 kda (mature) and 170 kda (immature), due to different glycosylation and maturation stages [81] . for both swt and s3w3a, n-deglycosylation via pngase f confirmed the gp180 and gp170 species originated from a common precursor but differed in glycosylation stage. the majority of swt gp180 was directed to lipid-raft containing fractions, while swt gp170 was predominantly retained in the bottom fractions. triple trp!ala substitutions resulted in an altered trafficking pattern of the mature form of the s protein. in s3w3a, both s3w3a gp180 and s3w3a gp170 were found in the upper and bottom fractions at equal amounts, suggesting that a lower percentage of mature s3w3a was recruited to the lipid raft. the data suggest that the trp residues function to fine-tune the clustering of fully mature s protein into lipid rafts during budding. (tif) s3 fig. effects of nand c-dimerization on the anti-viral effects of peptides containing sars-cov spike mper. peptide m sars, a peptide containing the sars-cov s protein mper sequence (kyeqyikwpwyvwlgf) and its n-and c-terminal dimers, n-m sars and c-m sars , were tested as fusion inhibitors against pseudotyped sars-cov. pseudotyped sars-cov was prepared by co-transfecting 293t cells using calcium phosphate transfection method with pnl4-3luc+env-vpr-and pcdna3.1-opt9-s mutant plasmids. pnl4-3luc +env-vpr-was kindly provided by prof. zhang linqi (aaron diamond aids research center, rockefeller university, new york 10016). peptides were incubated with the virus for 1 h under 5% co 2 at 37°c, prior to being added to vero e6 cells and incubated for another 72 h. inhibitory activities of the peptides were calculated from the luciferase activities of the vero e6 cells, determined by a td-20/20 luminometer (tuner designs viral membrane fusion a trimeric structural domain of the hiv-1 transmembrane glycoprotein electron tomography analysis of envelope glycoprotein trimers on hiv and simian immunodeficiency virus virions inhibition of furin-mediated cleavage activation of hiv-1 glycoprotein gp160 the t4 gene encodes the aids virus receptor and is expressed in the immune system and the brain hiv-1 entry cofactor: functional cdna cloning of a seventransmembrane, g protein-coupled receptor chemokine receptors as hiv-1 coreceptors: roles in viral entry, tropism, and disease capture of an early fusion-active conformation of hiv-1 gp41 dissection of human immunodeficiency virus type 1 entry with neutralizing antibodies to gp41 fusion intermediates conformational changes in hiv-1 gp41 in the course of hiv-1 envelope glycoprotein-mediated fusion and inactivation atomic structure of the ectodomain from hiv-1 gp41 atomic structure of a thermostable subdomain of hiv-1 gp41 hiv-1 envelope proteins complete their folding into six-helix bundles immediately after fusion pore formation comparative study of adaptive molecular evolution in different human immunodeficiency virus groups and subtypes role of the membrane-proximal domain in the initial stages of human immunodeficiency virus type 1 envelope glycoprotein-mediated membrane fusion a conserved tryptophan-rich motif in the membrane-proximal region of the human immunodeficiency virus type 1 gp41 ectodomain is important for env-mediated fusion and virus infectivity the pre-transmembrane region of the human immunodeficiency virus type-1 glycoprotein: a novel fusogenic sequence membrane interface-interacting sequences within the ectodomain of the human immunodeficiency virus type 1 envelope glycoprotein: putative role during viral fusion identification of a conserved domain of the hiv-1 transmembrane protein gp41 which interacts with cholesteryl groups peripheral-type benzodiazepine receptor function in cholesterol transport. identification of a putative cholesterol recognition/interaction amino acid sequence and consensus pattern hiv-1 assembly: viral glycoproteins segregate quantally to lipid rafts that associate individually with hiv-1 capsids and virions evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid-enriched membrane lipid rafts the c-and the n-terminal regions of glycoprotein 41 ectodomain fuse membranes enriched and not enriched with cholesterol, respectively the tryptophan-rich region of hiv gp41 and the promotion of cholesterol-rich domains cholesterol-dependent membrane fusion induced by the gp41 membrane-proximal external region-transmembrane domain connection suggests a mechanism for broad hiv-1 neutralization peptides corresponding to a predictive alpha-helical domain of human immunodeficiency virus type 1 gp41 are potent inhibitors of virus infection short-term safety and antiretroviral activity of t-1249, a second-generation fusion inhibitor of hiv hiv gp41 c-terminal heptad repeat contains multifunctional domains. relation to mechanisms of action of anti-hiv peptides mode of action of an antiviral peptide from hiv-1. inhibition at a post-lipid mixing stage different from the hiv fusion inhibitor c34, the anti-hiv drug fuzeon (t-20) inhibits hiv-1 entry by targeting multiple sites in gp41 and gp120 hiv fusion inhibitor peptide t-1249 is able to insert or adsorb to lipidic bilayers. putative correlation with improved efficiency updating the use of synthetic peptides as inhibitors of hiv-1 entry control of expression, glycosylation, and secretion of hiv-1 gp120 by homologous and heterologous signal sequences endoproteolytic cleavage of gp160 is required for the activation of human immunodeficiency virus hiv-1 envelope glycoprotein biosynthesis, trafficking, and incorporation increase of anti-hiv activity of c-peptide fusion inhibitors using a bivalent drug design approach multimerized chr-derived peptides as hiv-1 fusion inhibitors modular assembly of dimeric hiv fusion inhibitor peptides with enhanced antiviral potency covalent stabilization of coiled coils of the hiv gp41 n region yields extremely potent and broad inhibitors of viral infection epitope mapping and topology of baculovirus-expressed hiv-1 gp160 determined with a panel of murine monoclonal antibodies mapping of b-cell epitopes in rabbits immunised with various gag antigens for the production of hiv-1 gag capture elisa reagents sensitivity of human immunodeficiency virus type 1 to the fusion inhibitor t-20 is modulated by coreceptor specificity defined by hiv-1 env mper in anti-viral design and viral life cycle emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (t-20) monotherapy. antimicrob agents chemother identification of gammaretroviruses constitutively released from cell lines used for human immunodeficiency virus research evidence that ecotropic murine leukemia virus contamination in tzm-bl cells does not affect the outcome of neutralizing antibody assays with human immunodeficiency virus type 1 cloning and functional analysis of multiply spliced mrna species of human immunodeficiency virus type 1 env and vpu proteins of human immunodeficiency virus type 1 are produced from multiple bicistronic mrnas production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone small molecule targets env for endoplasmic reticulum-associated protein degradation and inhibits human immunodeficiency virus type 1 propagation construction and in vitro properties of hiv-1 mutants with deletions in "nonessential" genes estimation of protein secondary structure from circular dichroism spectra: comparison of contin, selcon, and cdsstr methods with an expanded reference set dichroweb, an online server for protein secondary structure analyses from circular dichroism spectroscopic data protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases sars-cov heptad repeat 2 is a trimer of parallel helices a facile ligation approach to prepare three-helix bundles of hiv fusion-state protein mimetics initial cleavage of the human immunodeficiency virus type 1 gagpol precursor by its activated protease occurs by an intramolecular mechanism sequential steps in human immunodeficiency virus particle maturation revealed by alterations of individual gag polyprotein cleavage sites structural and functional roles of hiv-1 gp41 pretransmembrane sequence segmentation structure of an hiv-1-neutralizing antibody target, the lipid-bound gp41 envelope membrane proximal region trimer a synthetic c34 trimer of hiv-1 gp41 shows significant increase in inhibition potency interfacial pre-transmembrane domains in viral proteins promoting membrane fusion and fission non-random distribution of amino acids in the transmembrane segments of human type i single span membrane proteins membrane proteins: from sequence to structure. annual review of biophysics and biomolecular structure the preference of tryptophan for membrane interfaces tryptophan-dependent membrane interaction and heteromerization with the internal fusion peptide by the membrane proximal external region of sars-cov spike protein the membrane proximal external region of the hiv-1 envelope glycoprotein gp41 contributes to the stabilization of the six-helix bundle formed with a matching n' peptide functional links between the fusion peptide-proximal polar segment and membrane-proximal region of human immunodeficiency virus gp41 in distinct phases of membrane fusion crystal structure of hiv-1 gp41 including both fusion peptide and membrane proximal external regions a conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1 anti-human immunodeficiency virus type 1 (hiv-1) antibodies 2f5 and 4e10 require surprisingly few crucial residues in the membrane-proximal external region of glycoprotein gp41 to neutralize hiv-1 the membrane-proximal external region of hiv-1 gp41: a vaccine target worth exploring multiple antigen peptide mimetics containing gp41 membrane-proximal external region elicit broad neutralizing antibodies against human immunodeficiency virus type 1 in guinea pigs identification of autoantigens recognized by the 2f5 and 4e10 broadly neutralizing hiv-1 antibodies. the journal of experimental medicine t-20) cross-reactive glycoprotein 41 antibody does not impair the efficacy or safety of enfuvirtide an ancestral hiv-2/simian immunodeficiency virus peptide with potent hiv-1 and hiv-2 fusion inhibitor activity downregulation of human immunodeficiency virus type 1 gag expression by a gp41 cytoplasmic domain fusion protein rre-dependent hiv-1 env rna effects on gag protein expression, assembly and release the retroviral capsid domain dictates virion size, morphology, and coassembly of gag into virus-like particles we thank dr. y. liao and dr. t. l. neo for the valuable discussions and technical support in this study. we also thank the original donors and the nih aids research and reference reagent program, division of aids, niaid for the cell lines hltat iii from barbara k. felber and dr. george n. pavlakis, and tzm-bl from john c. kabbes, xiaoyun wu, and transzyme, inc. we are grateful for the anti-gp41 antibody (chessie 8) from dr. george lweis and the plasmid pnl4-3 from dr. malcolm martin. we also thank jorma hinkula for kindly providing the anti-p24 (ef7) antibody. key: cord-356364-ipi81ce3 authors: ho, bo-lin; cheng, shu-chun; shi, lin; wang, ting-yun; ho, kuan-i; chou, chi-yuan title: critical assessment of the important residues involved in the dimerization and catalysis of mers coronavirus main protease date: 2015-12-14 journal: plos one doi: 10.1371/journal.pone.0144865 sha: doc_id: 356364 cord_uid: ipi81ce3 background: a highly pathogenic human coronavirus (cov), middle east respiratory syndrome coronavirus (mers-cov), has emerged in jeddah and other places in saudi arabia, and has quickly spread to european and asian countries since september 2012. up to the 1(st) october 2015 it has infected at least 1593 people with a global fatality rate of about 35%. studies to understand the virus are necessary and urgent. in the present study, mers-cov main protease (m(pro)) is expressed; the dimerization of the protein and its relationship to catalysis are investigated. methods and results: the crystal structure of mers-cov m(pro) indicates that it shares a similar scaffold to that of other coronaviral m(pro) and consists of chymotrypsin-like domains i and ii and a helical domain iii of five helices. analytical ultracentrifugation analysis demonstrated that mers-cov m(pro) undergoes a monomer to dimer conversion in the presence of a peptide substrate. glu169 is a key residue and plays a dual role in both dimerization and catalysis. the mutagenesis of other residues found on the dimerization interface indicate that dimerization of mers-cov m(pro) is required for its catalytic activity. one mutation, m298r, resulted in a stable dimer with a higher level of proteolytic activity than the wild-type enzyme. conclusions: mers-cov m(pro) shows substrate-induced dimerization and potent proteolytic activity. a critical assessment of the residues important to these processes provides insights into the correlation between dimerization and catalysis within the coronaviral m(pro) family. a highly pathogenic human coronavirus (cov) 1 , middle east respiratory syndrome coronavirus (mers-cov), emerged in jeddah and other places in saudi arabia in september 2012 and purification, the codons of the thrombin cutting recognition sequence and a ndei cutting site were removed and then inserted the codons of leu-arg-leu-lys-gly-gly into the above vector. the forward primer sequence for site-directed mutagenesis was 5'-catcacagcagcggcct gcgtctgaaaggcggcagcggtttggtgaaaatg-3' and the reverse primer was 5'-catttt caccaaaccgctgccgcctttc agacgcaggccgctgctgtgatg-3'. the reading frame of the final plasmid was confirmed by sequencing. the expression vector was transformed into e. coli bl21 (de3) cells (novagen). cultures were grown in 0.8 liters of lb medium at 37°c for 4 h, induced with 0.4 mm isopropyl-β-d -thiogalactopyranoside, and then incubated overnight at 20°c. after centrifuging at 6,000 x g at 4°c for 15 min, the cell pellets were resuspended in lysis buffer (20 mm tris, ph 8.5, 250 mm nacl, 5% glycerol, 0.2% triton x-100, and 2 mm β-mercaptoethanol) and then lysed by sonication. the crude extract was then centrifuged at 12,000 x g at 4°c for 25 min to remove the insoluble pellet. next the supernatant was incubated with 1-ml ni-nta beads at 4°c for 1 h and then loaded onto an empty column. after allowing the supernatant to flow through, the beads were washed with washing buffer (20 mm tris, ph 8.5, 250 mm nacl, 8 mm imidazole, and 2 mm β-mercaptoethanol). the sars-cov papain-like protease [12] (1 mg in 100 mm phosphate buffer (ph 6.5)) was then added and incubated for 3 h. the sars-cov papain-like protease digestion, which removed the 6 x his tag and leu-arg-leu-lys-gly-gly fragment, resulted in a native protein product with an authentic n-terminus. the digest was allowed to flow through and then loaded onto a s-100 gel-filtration column (ge healthcare) equilibrated with running buffer (20 mm tris, ph 8.5, 100 mm nacl, and 2 mm dithiothreitol). the purity of the fractions collected was analyzed by sds-page and the protein was concentrated to 30 mg/ml by amicon ultra-4 10-kda centrifugal filter (millipore). crystals of the mers-cov m pro were obtained at 295 k by the sitting-drop vapor-diffusion method. the protein solution was set up at 5 mg/ml and the reservoir solution consisted of 0.1 m tris, ph 8.4, 15% (w/v) peg 4000 and 0.2 m sodium acetate. clusters of needle crystals appeared in 2 days and were used for micro-seeding. single cystals of rectangle shape and with dimensions of 0.3-0.5 mm were obtained in less than a week. all crystals were cryoprotected in the reservoir solution with 15% glycerol and were flash-cooled in liquid nitrogen. data collection, structure determination and refinement x-ray diffraction data were collected at 100 k on the spxf beamline 13c1 at the national synchrotron radiation research center, taiwan, roc, using a adsc quantum-315r ccd detector (x-ray wavelength of 0.976 å). the diffraction images were processed and scaled using the hkl-2000 package [23] . the structure was solved by the molecular replacement method by phaser [24] using the structure of sars-cov m pro r298a mutant (pdb entry 4hi3; [25] ) as the search model. manual rebuilding of the structure model was performed using coot [26] . structure refinement was carried out using refmac [27] . the data-processing and refinement has been deposited in the protein data bank (pdb entry 5c3n). the colorimetry-based peptide substrate, tsavlq-para-nitroanilide (tq6-pna) (purity 95-99% by hplc; gl biochem ltd, shanghai, china), was used to measure the proteolytic activity of mers-cov m pro and its mutants throughout the course of the study as described previously [25, 28] . this substrate is cleaved at the gln-pna bond to release free pna, resulting in an increase in absorbance at 405 nm. the absorbance at 405 nm was continuously monitored using a jasco v-550 uv/vis spectrophotometer. the protease activity assay was performed in 10 mm phosphate (ph 7.6) at 30°c. the substrate stock solution was 1600 μm and the working concentrations were from 25 to 1200 μm. in the substrate titration assay, the concentration of mers-cov m pro and its mutants, v4r, t126s, e169a, m298r and t126s/m298r was 0.3, 0.4, 0.7, 1.2, 0.15 and 0.26 μm, respectively, while that of sars-cov m pro was 1.1 μm. steady state enzyme kinetic parameters were obtained by fitting the initial velocity (ν 0 ) data to the michaelis-menten eq (1) where k cat is the catalytic constant, [e] is the enzyme concentration, [s] is the substrate concentration and k m is the michaelis constant of the substrate. the program sigmaplot (systat software, inc., richmond, ca) was used for the data analysis. to assess the cooperativity effect, the kinetic parameters were obtained by fitting the initial velocities to the hill eq (2) where k' is a constant that is related to the dissociation constant and h is the hill constant. auc was performed on a xl-a analytical ultracentrifuge (beckman coulter) using an an-50 ti rotor [11, 12, 25, [28] [29] [30] . the sedimentation velocity experiments were carried out using a double-sector epon charcoal-filled centerpiece at 20°c with a rotor speed of 42,000 rpm. protein solutions of 0.05 to 0.5 mg/ml (330 μl) and reference (370 μl) solutions, both containing d 2 o, were loaded into the centerpiece. the absorbance at 280 nm was monitored in a continuous mode with a time interval of 300 s and a step size of 0.003 cm. multiple scans at different time intervals were then fitted to a continuous c(s) distribution model using the sedfit program [31] . additionally, the results with the various different protein concentrations were globally fitted to a monomer-dimer self-association model using the sedphat program to calculate the dissociation constant (k d ) [32] . to measure the substrate-induced dimerization, the active enzyme centrifugation (aec) [33] was performed. briefly, mers-cov m pro of 15 μl (1 mg/ml) was added into the small well of the band-forming centerpiece before the cell assembled. then 330 μl of peptide substrate at 0, 200 and 400 μm in d 2 o were respectively loaded into the bulk sample sector space. at a rotor speed of 42,000 rpm, the protein solution flowed into the substrate-containing channel and form a protein band. it can be detected by absorbance at 250 nm. during the centrifugation, the sediment protein continuously met and cleaved the substrate, which can be detected by absorbance change at 405 nm. the dataset from the multiple scans at 250 nm at various time intervals were fitted to a continuous c(s) distribution model using the sedfit program [31] , while the first five scans (0-30 min) at 405 nm were used to derive the product concentration and then initial velocity values. the protocol followed that of previous studies [28] with some modifications. apparent dissociation constants and stoichiometry of the enzyme-ligand interactions were measured by a thermal activity monitor 2277 from ta instruments (new castle, de). calorimetric titrations of the peptide substrate tq6-pna (0.5 mm in a 250-μl syringe) and m pro (6 μm in a 4-ml ampoule) were carried out at 25°c in 10 mm phosphate buffer (ph 7.6). the peptides were titrated into the enzyme using a 10-μl aliquot for each injection with a time interval of 20 min. a control experiment in the absence of enzyme was performed in parallel to correct for the dilution of heat. the data obtained was then analyzed by integrating the heat effects normalized against the amount of injected protein using curve-fitting based on a 1:1 binding model. this involved the use of digitam software (ta instruments, new castle, de). as part of the present study, an expression vector was constructed and the bl21 (de3) star (invitrogen) strain of e. coli were used to express mers-cov m pro . unlike sars-cov m pro [25, 28] , the mers-cov m pro with 6 x his-tag retained at the c-terminus cannot be expressed. instead, the bacteria are able to express the m pro when there is a n-terminal 6 x his-tag fusion that can be removed during the purification. however, thrombin digestion leaves two extra residues (gly-ser) at the n-terminus of m pro , resulting in protein with no proteolytic activity (data not shown). therefore we used sars-cov papain-like protease [12, 30, 34] , which is a highly active viral deubiquitinase and does not leave any residues at the n-terminus of m pro . after gel-filtration, the purity of authentic n-terminus m pro was about 99% (s2 fig). the size of the mers-cov m pro was found to be close to 30 kda, while any uncut protein was located at higher molecular weight position. the typical yield was about 10 mg after purification from 0.8 liter of e. coli culture. the structure of the mers-cov m pro was determined at 3.0 å resolution by x-ray crystallography (table 1 and fig 1a) . the crystal packing belonged to space group c222 1 , with unit-cell parameters a = 87.2, b = 94.0, c = 155.1 å and α = β = γ = 90°. the final atomic model containing two m pro molecules in a crystallographic asymmetric unit agrees well with the crystallographic data and the expected values of geometric parameters (table 1 ). there are no residues in the disallowed region of the ramachandran plot, while 81.3% of the residues are in the most favored region. the overall dimeric structure of m pro is similar to that of sars-cov m pro ; although a relative shift of 10°to 30°could be observed for the two domain iii within the dimer (s3a fig) . indeed, the r.m.s. distance between equivalent cα atoms of the domain i+ii of the two structures is 0.9 å, while that between the domain iii of the two structures is 3.1 å. compared with the structures of the ligand-bound complex (pdb entry 4ylu) and c148a mutant (pdb entry 4wme) [14, 15] , the r. m. s. distance is 0.8 and 0.7 å over 540 cα atom pairs, respectively (s3b fig) . this indicates that the dimeric structures show no significant difference; although the present structure is a free enzyme and the other two structures involve enzyme-ligand complexes and higher resolution. besides, there is minor difference between the present structure and bat-cov hku4 m pro (pdb entry 2yna), with 80% sequence identity, as the r. m. s. distance over 539 cα atom pairs is 0.8 å (s3b fig). interestingly, the dimerization interface situation with the mers-cov m pro was found to be different to that of sars-cov m pro where there are four amino acid pairs with intermolecular polar interactions (ser1. . .glu166, arg4. . .glu290, ser123. . .arg298 and ser139. . .gln299). there are only two pairs of intermolecular hydrogen bonds, ser1. . .glu169 and ser142. . .gln299 that are associated with the dimer surface of mers-cov m pro according to the current structure ( fig 1b) . this led us to compare the dimerization and catalytic activity of the two types of m pro . in the present study, in addition to using the wild-type mers-cov m pro , we also mutated several residues at the dimerization interface in order to evaluate their role in dimerization and catalysis of mers-cov m pro (see below). to compare catalysis between the two m pro , tq6-pna, a peptide substrate for sars-cov m pro [25, 28] , was used to measure the proteolytic activity. at first, the dependence of the initial additional allowed region 13.6 generously allowed region 5.2 disallowed region 0 a the numbers in parentheses are for the highest-resolution shell. where i hi is the integrated intensity of a given reflection and hi h i is the mean intensity of multiple corresponding symmetry-related reflections. velocity on enzyme concentration was analyzed and showed a nonlinear upward correlation (fig 2a) . the pattern is similar to that of sars-cov m pro , as the monomeric m pro may not have catalytic activity [28] . however, mers-cov m pro displayed a sigmoid curve for its rate constant pattern at various substrate concentrations (fig 2b, open circles) ; this contrast with sars-cov m pro , which exhibited a classical saturation curve (s4 fig). the results were then fitted to the hill equation (eq 2) in order to evaluate the kinetic parameters ( table 2 ). the k cat (2.33 s -1 ) of mers-cov m pro is close to that of sars-cov m pro (2.11 s -1 ), while the hill constant was 1.8, suggesting a significant degree of positive cooperativity among the m pro protomers. the comparable activity levels of the two m pro in the present study is dissimilar to the results obtained during a recent study in which the activity of the mers-cov m pro was found to be 5-fold lower than that of sars-cov [14] . using different substrates may cause the difference. tomar et al. [14] used a longer peptide substrate with residues present at both p and p' table 2 . site. however, in the present studies we used a peptide substrate that contains only p site residues. besides, they utilized fret substrate and could only be used at low substrate concentrations to prevent the inner-filter effect, while we are able to use higher substrate concentrations to capture the kinetic parameters. the cooperativity phenomenon associated with the mers-cov m pro is similar to that of the sars-cov m pro r298a/l monomer mutants; these were found to show monomer to dimer conversion during catalysis [28] . as a result of the above, we investigated the quaternary structure of the m pro by auc (fig 3) . the cumulative spectra (fig 3a) were analyzed using the continuous c(s) distribution model and the results suggested that mers-cov m pro is a monomer in phosphate buffer (s5 fig) and this contrasts with a distribution of 30% monomer and 70% dimer in the presence of 600 μm tq6-pna ( fig 3b) . we also measured the size distribution of m pro at various tq6-pna concentrations (s6 fig). the results indicated that the sedimentation coefficient of the major species was shifted as the substrate dosage changed (s6b fig). more substrate led to the major species moving close to the dimer position. however, before the centrifugation, the enzyme had been mixed with the substrate and the catalysis began, resulting in a mixture of substrate and product with enzyme. it is unable to confirm that our observation is a substrate-induced or substrate/product-induced dimerization. to solve this, a modified auc technique, aec [33] , was utilized to detect the quaternary structure change in the absence and presence of substrate (fig 4) . here the enzyme solution was put into the small well of band-forming centerpiece and then flowed into the substratecontaining channel when the centrifugation began. during the centrifugation, the protein layer gradually sediment and continuously met peptide substrates. not surprisingly, there was a broad distribution between the monomer and dimer species in the presence of 200 μm peptide substrate, while a major species shifted to the dimer in 400 μm substrate (fig 4b) . it suggests that mers-cov m pro acts as a rapid self-associated and substrate-induced dimerization. using different strategies, tomar et al. [14] confirmed that inhibitor binding can also induce and maintain the dimerization of m pro . on the other hand, we measured the velocity of the product formation during the centrifugation; although the rate (0.017 μm/s) is 10-fold lower than that by the spectrometric assay (fig 4b) . the values were derived from a global fit of the auc data to a monomer-dimer self-association model by sedphat [32] . the experiments for the assay were obtained at protein concentration of 1.5 to 30 μm. c the value was from our previous studies for comparison [28] . doi:10.1371/journal.pone.0144865.t002 to quantitatively characterize the monomer-dimer equilibrium of m pro in the absence and presence of substrate, the auc results at protein concentrations of 1.5, 6 and 15 μm were globally fitted to the monomer-dimer self-association model ( table 2 ). the analysis indicated that the k d value of mers-cov m pro in the absence of substrate was 7.7 μm and this decreased 11-fold in the presence of substrate, which brings it close to the value for sars-cov m pro (1.7 μm; table 2 ). thus it can be concluded that, like the sars-cov m pro r298a mutant [25] , the presence of substrate is able to induce the dimerization of mers-cov m pro . in addition, although the k d values of wild-type sars-cov m pro without or with substrates show no significant difference (table 2) , it was possible to detect substrate-induced dimerization at a protein concentration of 1 μm by aec [33] . previous studies have demonstrated that a conserved residue, glu166, plays a pivotal role in connecting the substrate binding site with the dimerization interface of sars-cov m pro [28] . here the equivalent residue, glu169, was mutated and its influence on mers-cov m pro evaluated ( fig 1b) . not surprisingly, compared to the wild-type enzyme, the activity (k cat ) of e169a showed a 6-fold decrease (fig 2b, open diamonds and table 2 ). furthermore, auc analysis suggested that this enzyme consisted of a single species close to monomeric form even in the presence of substrate (fig 3e) . the k d values of the e169a mutant without substrate was 14 μm, which is 2-fold higher than that of the wild-type and this did not decrease in the presence of substrate ( table 2 ). the results suggest that mutation of glu169 is able to block substrate-induced dimerization and that this results in a decrease in enzyme activity. unexpectedly, as well as the monomer, some octamer (6.3%) with a sedimentation coefficient of 4.9 s was observed in the presence of substrate (fig 3e) . previous studies have suggested that a super-active octamer of sars-cov m pro can be locked by 3d domain swapping [35] . in this study, the presence of the octamer form of the mers-cov m pro e169a mutant in the presence of substrate may explain why this mutant is not totally inactive. taking the above as a whole, the dual role of the conserved glu residue in catalysis and dimerization is consistent for both m pro . our results confirmed that there are fewer intermolecular polar interactions at the dimerization interface of mers-cov m pro and this results in the enzyme being in the monomer form in aqueous buffer; this contrasts with sars-cov m pro , which is mostly in the dimer form in similar circumstances due to the greater number of intermolecular interactions (figs 1b and 3b ). based on the sequence alignment, three residues in the dimerization interface vary in coronaviral m pro sequences (s1 fig). in sars-cov m pro , residues arg4, ser123 and arg298 are different to the equivalent ones in mers-cov m pro , which are val4, thr126 and met298, respectively. based on the above, three single mutants, v4r, t126s and m298r, were generated and their proteolytic activity and dimerization were assessed. unexpectedly, both the v4r and the t126s mutant showed a higher k d for the dimer to monomer and a lower level of activity than wild-type m pro (fig 2b and table 2 ). k d values were decreased by 1.5-fold to 2.4-fold for the two mutants in the presence of substrate, which suggests reduced substrate-induced dimerization (fig 3c and 3d) . based on the current structure, residue val4 showed a hydrophobic contact with another protomer's residue gly141 (fig 1b) . mutation of valine to arginine may lose the contact. furthermore, the side chain of residue val131 of mers-cov m pro is hydrophobic while that of the equivalent residue, cys128 of sars-cov m pro , is hydrophilic (fig 1b) . val131 is close to the arg4 and will disfavor the electrostatic interaction of arg4. . .glu290 in mers-cov m pro . these variance may result in v4r failed to form a stable dimer. for the t126s mutant, after compared with the other two mers-cov m pro structures (pdb entry 4ylu and 4wme), we found that the side chain of thr126 is free of rotation. it is able to make a hydrophobic interaction with the residue tyr121, resulting in a 180°-rotation of the phenol ring, and lead to a hydrogen-bond with the backbone amide of leu144 (fig 1b) . it further results in the side chain of leu144 toward another protomer's ile300 and make a hydrophobic contact for the two protomers. mutation of thr126 to serine will lose this contact and may disfavor the dimerization. by way of contrast, the m298r mutant resulted in a stable dimer form in phosphate buffer (fig 3f) . the k d value for the mutant in the absence and presence of substrate were 1.1 and 0.7 μm, respectively, which are very close to those for sars-cov m pro (table 2) . furthermore, the stable dimer form showed higher proteolytic activity and the mutation transformed the enzymes rate constant pattern at various substrate concentrations into a classical saturation curve (fig 2c, open triangles) . in addition, the k m of the m298r mutant is 5.8-fold lower than that of sars-cov m pro , which suggests a higher substrate binding affinity ( table 2 ). it can be concluded that mutation of residue met298 to arginine within the mers-cov m pro results in the stabilization of the dimer formation, which in turn gives rise to more efficient catalysis. based on our structure, arg298 is able to make a hydrogen bonding interaction with thr126. residue thr126 can be replaced by serine because the t126s/m298r double mutant also shows similar dimerization characteristics ( fig 3g) and saturated catalytic pattern to that of the m298r mutant (fig 2c, open hexagons and table 2 ). however, it can only be achieved in the presence of arg298, not met298. the sigmoid nature of the curve describing the rate constant pattern at various substrate concentrations ( fig 2b) means that it is not possible to obtain a k m value for this enzyme, which would allow us to evaluate the substrate binding affinity of the wild-type mers-cov m pro and its mutants; the exceptions being the m298r single mutant and the t126s/m298r double mutant (fig 2) . to further delineate the binding of substrate to the enzyme, itc was used to measure the k d for the substrate (or substrate/product)-enzyme complex and the binding stoichiometry (n) (fig 5) . during the titration, the enzymatic hydrolysis might produce additional heat, resulting in higher δh. so we can only compared the n and k d of the wild-type m pro with those of e169a and m298r mutants. the three enzymes exhibited similar n (0.89 to 1.06) and k d (14.2 to 20.3 μm) . this suggested that the monomeric and dimeric m pro show quite the same substrate binding affinity. such phenomenon is also found in monomeric and dimeric sars-cov m pro , whose n and k d for the same substrate were 0.97 to 1.05 and 29.9 to 33.8 μm, respectively [28] . with the k m ((k -1 +k cat )/k 1 ), k cat and k d (k -1 /k 1 ), we are able to calculate the k 1 and k -1 , the rate of the association and dissociation of the enzyme-substrate complex. the k 1 and k -1 for the m298r mutant and substrate is 0.049 s -1 μm -1 and 0.98 s -1 , while those for sars-cov m pro is 0.00246 s -1 μm -1 and 0.084 s -1 . the rate constants for m298r mutant showed 20-and 12-fold higher than those for sars-cov m pro . the more rapid association and relatively slower dissociation of enzyme-substrate complex may be used to explain why the catalytic efficiency (k cat /k m ) of the mers-cov m pro m298r mutant is higher than that of sars-cov m pro (table 2) . the crystal structure of authentic n-terminus mers-cov m pro was determined and this was found to involve a dimeric form with less intermolecular polar interactions. biochemical and auc studies indicated that mers-cov m pro shows almost the same proteolytic activity as sars-cov m pro ; although it is a monomer in aqueous buffer and displays substrate-induced dimerization (fig 6) . a conserved residue, glu169, plays an essential role in the substrateinduced dimerization of both mers-cov m pro and sars-cov m pro . moreover, mutation of a residue in the dimerization interface, m298r, was found to result in a more stable dimer form in aqueous buffer that had higher enzyme activity; while other two mutations, v4r and t126s, showed the reverse effect. critical assessment of the residues important to dimerization of and catalysis by mers-cov m pro provides valuable insights into the mechanism that controls the monomer-dimer switch of important and valuable enzyme. isolation of a novel coronavirus from a man with pneumonia in saudi arabia is the discovery of the novel human betacoronavirus 2c emc/2012 (hcov-emc) the beginning of another sars-like pandemic? emerging human coronaviruses-disease potential and preparedness in-vitro renal epithelial cell infection reveals a viral kidney tropism as a potential mechanism for acute renal failure during middle east respiratory syndrome (mers) coronavirus infection hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study close relative of human middle east respiratory syndrome coronavirus in bat ecology, evolution and classification of bat coronaviruses in the aftermath of sars isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor assessing activity and inhibition of middle east respiratory syndrome coronavirus papain-like and 3c-like proteases using luciferase-based biosensors structural and functional characterization of mers coronavirus papain-like protease structural basis for catalysis and ubiquitin recognition by the severe acute respiratory syndrome coronavirus papain-like protease thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of middle east respiratory syndrome coronavirus ligand-induced dimerization of mers coronavirus nsp5 protease (3clpro): implications for nsp5 regulation and the development of antivirals structures of the middle east respiratory syndrome coronavirus 3c-like protease reveal insights into substrate specificity the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs quaternary structure of the severe acute respiratory syndrome (sars) coronavirus main protease mechanism of the maturation process of sars-cov 3cl protease structures of two coronavirus main proteases: implications for substrate binding and antiviral drug design biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3c-like proteinase prediction and biochemical analysis of putative cleavage sites of the 3c-like protease of middle east respiratory syndrome coronavirus processing of x-ray diffraction data collected in oscillation mode phaser crystallographic software mechanism for controlling the monomerdimer conversion of sars coronavirus main protease features and development of coot refmac5 for the refinement of macromolecular crystal structures mutation of glu-166 blocks the substrate-induced dimerization of sars coronavirus main protease structural and functional characterization of human apolipoprotein e 72-166 peptides in both aqueous and lipid environments differential domain structure stability of the severe acute respiratory syndrome coronavirus papain-like protease size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling on the analysis of protein self-association by sedimentation velocity analytical ultracentrifugation applications of analytical ultracentrifugation to protein size-and-shape distribution and structure-and-function analyses thiopurine analogues inhibit papain-like protease of severe acute respiratory syndrome coronavirus three-dimensional domain swapping as a mechanism to lock the active conformation in a super-active octamer of sars-cov main protease espript: analysis of multiple sequence alignments in post-script this research was supported by grants from ministry of science and technology, taiwan (103-2320-b-010-025 and 104-2320-b-010-034) to cyc. we also thank nymu for its financial conceived and designed the experiments: cyc. performed the experiments: blh scc ls tyw kih. analyzed the data: blh tyw. contributed reagents/materials/analysis tools: scc. wrote the paper: cyc. key: cord-345695-5vi9wibk authors: hicks, lorin l.; schwab, nathan a.; homyack, jessica a.; jones, jay e.; maxell, bryce a.; burkholder, braden o. title: a statistical approach to white-nose syndrome surveillance monitoring using acoustic data date: 2020-10-22 journal: plos one doi: 10.1371/journal.pone.0241052 sha: doc_id: 345695 cord_uid: 5vi9wibk traditional pathogen surveillance methods for white-nose syndrome (wns), the most serious threat to hibernating north american bats, focus on fungal presence where large congregations of hibernating bats occur. however, in the western usa, wns-susceptible bat species rarely assemble in large numbers and known winter roosts are uncommon features. wns increases arousal frequency and activity of infected bats during hibernation. our objective was to explore the effectiveness of acoustic monitoring as a surveillance tool for wns. we propose a non-invasive approach to model pre-wns baseline activity rates for comparison with future acoustic data after wns is suspected to occur. we investigated relationships among bat activity, ambient temperatures, and season prior to presence of wns across forested sites of montana, usa where wns was not known to occur. we used acoustic monitors to collect bat activity and ambient temperature data year-round on 41 sites, 2011–2019. we detected a diverse bat community across managed (n = 4) and unmanaged (n = 37) forest sites and recorded over 5.37 million passes from bats, including 13 identified species. bats were active year-round, but positive associations between average of the nightly temperatures by month and bat activity were strongest in spring and fall. from these data, we developed site-specific prediction models for bat activity to account for seasonal and annual temperature variation prior to known occurrence of wns. these prediction models can be used to monitor changes in bat activity that may signal potential presence of wns, such as greater than expected activity in winter, or less than expected activity during summer. we propose this model-based method for future monitoring efforts that could be used to trigger targeted sampling of individual bats or hibernacula for wns, in areas where traditional disease surveillance approaches are logistically difficult to implement or because of human-wildlife transmission concerns from covid-19. introduction north american bat species face several contemporary conservation challenges throughout their range, including threats to habitat from loss and alteration of forested environments, and direct mortality from development of wind energy infrastructure [1, 2] . however, transmission of pseudogymnoascus destructans (pd), the pathogen that causes white-nose syndrome (wns), has emerged as the most serious threat to over a dozen species of north american bats that use caves and mines for hibernacula [3] . the pd fungus invades skin tissues (e.g., wing membrane) which disrupts homeostasis and causes dehydration that corresponds to an abnormally high frequency of arousals from torpor [4] . bats with wns increase arousal [5] and acoustic activity rates [6] above baseline levels during the normal hibernation period, which depletes critical energy reserves and can ultimately cause mortality. wns has caused precipitous declines in populations of hibernating bats and has contributed to the federal listing of the northern longeared bat (myotis septentrionalis) as threatened under the u.s. endangered species act [7] and endangered listings for three bat species in canada under the species at risk act [northern long-eared bat, little brown bat (myotis lucifugus), and tri-colored bat (perimyotis subflavus)] [8] . monitoring for wns is a critical management action and primarily has focused on detecting the pd pathogen either directly from bats captured in hibernacula or with environmental samples from occupied caves and mines [9] . since its emergence in eastern north america in 2006, wns has spread westward with some models predicting wns to arrive to montana by 2026 [10] . in june 2020, sampling conducted at bridges in 3 eastern montana counties confirmed the presence of pd, but not wns [11] . further, wns was detected in a little brown bat in washington state in 2016 and pd detection continues to increase among sites and across different species within washington [12] . thus, there is a second wns epicenter, which could affect known and previously unrecognized susceptible bat species and increase the spread of wns through western north america from both the west and the east [13] . the presence of wns in the pacific northwest opens another front in critical efforts to combat bat declines and shortens the timeline for implementing conservation strategies to protect vulnerable bat species. this effort is complicated by the fact that winter hibernation behavior for most western species is poorly understood [14] , yet this is when wns has the greatest negative impacts on bat populations [2, 15, 16] . further, little is known about winter roost sites and activity patterns of many bats in western north america. a recent review indicates that wns-susceptible species rarely congregate in large numbers in the western u.s. and that known winter roosts may be relatively uncommon or difficult to identify [17] . thus, typical surveillance approaches of estimating hibernating bat numbers and sampling for pd implemented in eastern north america where large hibernacula are more common does not translate well to all ecoregions [18] . therefore, alternate approaches to facilitate pathogen surveillance, monitor disease impacts, and conduct mitigation efforts for wns are urgently needed [13] . in addition to wns, bats can be influenced by habitat alteration from anthropogenic activities, such as forest harvesting [19] . forested areas provide essential habitat features that support bat diversity, including roost sites (e.g., trees) and foraging areas (e.g., riparian zones, wetlands) [20] [21] [22] . sustainable management of these forest resources may minimize the environmental stress on bats outside of the winter season (when wns most severely impacts bats). despite a robust network of public lands managed for preservation in western north america [23] , bat species richness is low in some protected areas, such as glacier national park [24] and lower bat activity is commonly reported in undisturbed habitats compared to disturbed [19] . managed forests support many species of bats not found or poorly represented in gov/reqapp/usermain.asp. these data were produced as part of a regional bat acoustic monitoring network and we had no special privileges to access these data and obtained them via request from the montana natural heritage program. funding: funding was provided by national council for air and stream improvement, inc., plum creek timber, weyerhaeuser, stimson lumber, f. h. stoltze land and timber. weyerhaeuser provided access and logistical support to collect empirical data and provided support in the form of salary for authors lorin hicks and jessica homyack. weyerhaeuser also contracted with tetratech for data collection and analysis, completed by author nathan schwab. the funder provided support in the form of salaries for authors [lh, jh], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. plum creek timber, stimson lumber, f. h. stoltze land and timber also provided funding and support in the form of salary for lorin hicks. weyerhaeuser also contracted with tetratech for data collection and analysis and nathan schwab received support in the form of salary. the specific roles of these authors are articulated in the 'author contributions' section. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. weyerhaeuser provided access and logistical support to collect empirical data and provided support in the form of salary for lorin hicks and jessica homyack. plum creek timber, stimson lumber, f. h. stoltze land and timber also provided funding and support in the form of salary for lorin hicks. weyerhaeuser also contracted with tetratech for data collection and analysis and nathan schwab received support in the form of salary. this does not alter our adherence to plos one policies on sharing data and materials. protected areas on public lands [1] and some forest management practices that reduce understory vegetation such as commercial thinning and prescribed burning can increase bat activity and occupancy [19, 25, 26] . nationally, public and private forestlands are managed under state forest practices rules, water quality best management practices, and forest sustainability programs (i.e., sustainable forestry initiative, forest stewardship council, american tree farm) to protect water quality and quantity, which includes riparian buffers that often provide habitat for bats. we quantified presence of bats across forested sites in montana, usa to document baseline levels of species diversity and activity rates prior to presence of wns. our objective was to evaluate long-term acoustic monitoring as a surveillance approach to monitor for potential wns presence in overwintering bat populations on both managed and unmanaged forest. specifically, we developed a statistical method to evaluate whether activity data occur outside the range of normal conditions, thus warranting more intensive sampling for the presence of pd on bats or in the environment. we predicted that bat populations in the western u.s. would have low levels of activity during winter months, high levels of activity during the summer, and that acoustic monitoring could be used as an effective approach for disease surveillance in remote areas of the intermountain west. our study area included forested areas across montana, usa (fig 1) . the forested areas in the western portion of the state were douglas fir (pseudotsuga menziesii)/snowberry (symphoricarpos albus) forest type, which generally characterized the region [27] and included locations of our acoustic detector stations. these forests had a mixed overstory, including douglas-fir, ponderosa pine (pinus ponderosa), grand fir (abies grandis), western larch (larix occidentalis), lodgepole pine (pinus contorta), and engelmann spruce (picea engelmannii), with understory vegetation comprised of snowberry, huckleberry (vaccinium spp.) and beargrass (xerophylum tenax). westside elevations ranged from 800 to 1150 meters. the climate in these ecoregions was continental-maritime with relatively low annual precipitation, with most precipitation occurring as snow [28] . the forested locations in eastern montana were characterized by limber pine (pinus flexilus) and ponderosa pine (pinus ponderosa) forest with understory of idaho fescue (festuca idahoensis) and bluebunch wheatgrass (agropyron spicatum). eastside sample site elevations ranged from 850-1520 meters. climate in the eastern ecoregions was continental with dramatic fluctuations in winter temperatures and longer, hotter, more arid summers than western ecoregions [29] . we selected 41 sampling sites that were near lentic or lotic systems and had �25% forest cover within a 1-kilometer buffer, estimated from the montana land cover data set in a geographic information system [30] (fig 1) . we did not randomly select sites for acoustic detectors but installed them in areas with potential for high bat activity that included proximity to standing live and dead trees, talus and rock cliffs, and waterbodies. we located four sites on intensively managed forest owned and managed by plum creek timber company (after 2016, weyerhaeuser) for production of sawtimber and other forest products. the remaining 37 sampling sites were selected from a regional bat acoustic monitoring network organized by the montana natural heritage program [31, 32] . these locations are primarily represented by federal and state landownership, but also include some tribal and private land. we we installed a single, full-spectrum acoustic recording unit (aru) at each sampling site. not all arus recorded data simultaneously or across the entire 2011-2019 study period, but we installed units for year-round recording of bat echolocation. each aru station consisted of a sm3bat or sm2bat+ acoustic detector (wildlife acoustics, maynard, massachusetts) and a microphone (u1 or smx-us) mounted on a pole at a height of approximately 3 meters. the arus operated on a 6-or 12-volt charging system (20-watt solar panel, 36-amp hour battery, and a charge controller) and housed in a weather-proof container on the ground below the microphone. we set the arus to record from sunset to sunrise to maximize battery life and balance with other logistical restraints of year-round sampling in remote locations. the units employed a sampling rate (samples per second) of 196,000, 256,000 or 384,000 khz. all arus used a minimum frequency filter of 8 khz, a trigger window of 2 seconds and a maximum file length of 5-8 seconds. most arus recorded in the wac0 compression format while four arus recorded directly to.wav format. the individual aru equipment and settings varied between the hardware platforms (sm3bat vs. sm2bat+), with detection distances of approximately 20-30 meters [33] , depending on the frequency of the source and other environmental variables (i.e., temperature and humidity) [34] . the equipment and settings were consistent for a given site throughout the study. the sensitivities of all microphones were within the manufacturer's specifications at the beginning of the deployment at each sampling site and replaced approximately annually. we examined equipment for functionality, checked microphone sensitivity, and exchanged data storage cards approximately once per month during summer and up to 6 months between visits during winter due to logistical constraints of access. each aru recorded a site-specific temperature approximately once every 1 or 5 minutes. at the end of our study, we parameterized acoustic files with sonobat 4.1.0 (sonobat, arcata, california usa) using a high-performance computing cluster at montana technological university. the parameters were then input into the sonobat montana [20160912] classifier to automatically assign a species classification to each bat pass. the montana classifier uses two regional species suites, each tuned to the species potentially present in that portion of the state. in cases where a bat pass was unable to be classified to species, a generic "bat" label was assigned. similar to the north american bat monitoring program [35] , we defined a bat pass as a sequence of echolocation pulses separated by more than 2 seconds of silence. we limited the maximum file duration to 5-8 seconds to reduce potential for recording more than one bat per sequence [36] . to quantify activity rates of bats, we analyzed the number of bat passes per detector-night (the number of operational detectors multiplied by the number of nights). for a night to be included in our estimates of detector-nights, the detector needed to register an internal temperature in the status file. we excluded data that did not meet this criterion from analysis. we manually verified a subset of diagnostic bat passes (approximately monthly for each species) with sonobat to ensure that species diversity of bats was accurate at all sampling stations [37] . we calculated the average number of passes per night over each month, by species, site and year using the ratio of total monthly qualified passes and the number of nights in the month. similarly, we calculated the average number of passes per night across all species, including passes without a species classification (total bat activity). a mean monthly temperature (˚c) was estimated from the mean of temperatures recorded at each site between sunset and sunrise across all nights in a month. we used a statistical model to describe variation in monthly-average nightly bat activity associated with several sources. to address our research questions, we aimed to characterize the extent to which activity varied among sampling sites, across years, by season, and in association with temperature. we fit a linear mixed-effects model with fixed effects for monthlyaverage mean temperature, season, and their interaction. we included random intercepts by site and year and random slopes for the temperature effect by site and year. thus, this model allowed for different overall levels of bat activity by site and year, and different temperature trends by site and year, while estimating population-average effects and quantifying the variation among sites and years. bat activity was log-transformed prior to analysis, and a constant of 0.01 was added prior to transformation to accommodate values of zero. we defined season by assigning each month to one of four calendar-based seasons (winter = dec, jan, feb; spring = mar, apr, may; summer = jun, jul, aug; fall = sep, oct, nov). we fit all models in r [38] using package lme4 [39] . we excluded sites with < ten months with at least one bat detection across all species from data used to fit the model. additionally, only data from 2012-2016 were used to fit the model, as limited data were available outside of this range. we note that both data restriction choices were performed to ensure sufficient data were available to estimate site-specific effects in the mixed effects model, but that no such restrictions were used in our raw data summaries. in addition to using the fitted model to describe variation in bat activity, we also used the model to generate prediction intervals [40] for new or future observations of bat activity in our region. we generated prediction intervals based on the model output using the 'predictinterval' function in package mertools [41] . we collected acoustic data used for our analysis between 28 sept 2011 and 4 august 2019, totaling 24,850 detector-nights at 41 sites. stations operated for an average of 606 (sd = 307) nights through the study. there were gaps in recordings because of equipment malfunction and wildlife encounters, but more than 5.37 million bat passes were recorded. after removing months with erroneous data due to equipment malfunction, 868 months of bat pass and temperature data were analyzed (per site mean: 21.2 months, sd = 10.7 months). we manually vetted acoustic files to confirm 13 species across the study, 11 of which were observed on managed forest sites (table 1) . three species [big brown bat (eptesicus fuscus), silver-haired bat (lasionycteris noctivagans), and western small-footed myotis (myotis ciliolabrum)] had confirmed activity in all calendar months, indicating that some bat species maintained some level of flight activity year-round. three presumed migratory species-spotted bat (euderma [2] . due to the high percentage of missing species information, our analyses include both estimates of total bat activity, i.e., total activity over all species, including bat passes not identified to species, and estimates for individual species where possible. across 24,850 detector-nights of sampling ambient temperatures, mean monthly temperatures ranged from -16.5˚c to 23.3˚c. mean monthly temperatures (± sd˚c) in montana varied across seasons [winter -1.7˚c (± 3.9˚c); spring 5.8˚c (± 4.3˚c); summer 16.2˚c (± 3.2˚c); and fall 6.7˚c (± 5.7˚c]). bat activity tended to be highest during summer months with most sites averaging >100 passes/night during july and august (fig 2) . additionally, most sites showed some activity during december and january, with one site averaging >10 passes/night. species-specific results show similar trends. we observed a strong positive association between average total bat activity and mean monthly temperature (fig 3) . the relationship was approximately loglinear for all 41 study sites, although the level of activity varied by site. results from our fitted model indicated a strong positive association between mean monthly temperature and total bat activity that varied by season (figs 4 and 5) . the estimated trends during the fall and spring indicated approximately 12.0-and 8.5-times greater numbers of bat passes per day, respectively, for every 5˚c increase in mean monthly temperature, on average, after adjusting for site and inter-annual differences. the association between bat activity and temperature was less pronounced during the winter and summer, where in both seasons we estimated approximately 2.8-fold greater activity, respectively, for 5˚c increases in temperature. overall levels of activity at a similar temperature also varied by season, although the difference was strongly dependent on temperature (fig 6) . we used the model random effects to estimate among-site and among-year variation in the mean response and temperature trends. we estimated considerable among-site variation in total bat activity after adjusting for temperature and seasonal effects, with site-specific means varying approximately 3.3-fold (1 sd) and among-year variation within a site of approximately 1.8-fold (fig 4) . estimated temperature trends among sites and among years were generally consistent, with variation in slopes of approximately 12% and 1% respectively. we fit this same model form to three individual species-eptesicus fuscus, myotis lucifugus, and m. californicus-to explore the possibility of seasonal differences in temperature associations among a species less vulnerable to wns [e. fuscus; [43] ], one highly vulnerable (m. lucifugus; [44] and one of unknown vulnerability (m. californicus). we note that since the majority of the bat passes were unidentified, the species-specific estimates may contain substantial unknown biases, as an unknown proportion of the unidentified bat passes consist of these species. our results suggest that winter activity for m. californicus is less strongly associated with mean monthly temperature than e. fuscus, while the reverse is suggested for the fall (fig 5) . we used the model fit to e. fuscus activity data (i.e., detections classified to the species e. fuscus) to illustrate a proposed monitoring approach with a statistical model for a single wnssusceptible species. the model was fit to data from all 41 sites to estimate among-site and among-year variation in activity, but we use the results to generate prediction intervals for four forest sites to depict how a landowner might implement this approach for wns surveillance monitoring. most observed activity data points fell within the 95% prediction interval, as expected (fig 7) . the prediction intervals were constructed to incorporate the estimated among-year variation in activity within a site. future observations of activity for these sites, along with model predictions, could be plotted to compare with prediction intervals. rapidly evolving technologies to passively observe and monitor wildlife concurrent with computational improvements in processing and analyzing large quantities of data are shifting management and research techniques from an animal-in-hand approach to indirect measurements of presence, abundance, diversity, and health [45, 46] . although acoustic monitoring has been implemented to investigate habitat relationships and community composition of bats [36] , uses of this technology are expanding into other objectives. here, we describe how yearround activity rates of bat species derived from acoustic monitoring can be used for surveillance monitoring for wns. we developed a simple and repeatable statistical modeling approach for wns surveillance monitoring with bat activity data collected from a broad geographic and temporal scale prior to known occurrence of wns in the intermountain west. this statistical approach can be applied elsewhere and may be particularly relevant for geographic locations where prominent hibernacula are inaccessible, rare, or unidentified [18] . further, our long-term and year-round collection of acoustic data for bats expands on limited knowledge of activity rates of species through time, seasons, across locations, and prior to wns. very little is known about the winter ecology of many species, specifically in western north america because of the limited sampling that has occurred during that season. technological advances in passive monitoring devices and identification software and an interest in wns has contributed to increased monitoring for bats in winter, when previously it was assumed they were rarely active [6, 47] . across our broad selection of 41 forested sites in montana, we documented a diverse community of bats (13 confirmed species), including at sites with a history of forest management. several species maintained some level of activity through winter, despite cold temperatures that averaged -1.0˚c in this season, which was below temperatures where at least some insect prey could sustain flight [48] . as expected, bat activity was positively related to warmer temperatures, although the strength of the relationship varied by season, with the strongest relationships with temperatures in spring and fall. in more temperate climates, other species of bats also displayed year-round activity that varied by season, sites, and species, including both migratory and non-migratory bats [2, 6] . the implications of winter activity of bats is not well-understood, particularly as it relates to body condition and energy stores or susceptibility to wns [6] . the ecology of bats infected with pd may lead individuals to change behaviors, such as higher activity rates during winter when bats typically use torpor to conserve energy [6] . aberrant behaviors by bats, including daytime activity out of roosts or hibernacula and during subfreezing temperatures increased after detection of wns in the southeastern u.s. [6] . however, the effects of winter bat activity on individual fitness have not been well-quantified. in north carolina, it was posited that year-round activity of typically migratory bats, including l. borealis, whose non-migratory behaviors could increase survival by reducing mortality from threats during migration, such as strikes from wind turbines [2] . alternatively, year-round activity by bats could be energetically costly if food resources do not meet metabolic demands and reduce reproductive output by individuals [2] . where underground caves or human structures are uncommon as hibernacula (e.g., the intermountain west), bats may congregate in smaller numbers in rock crevices during winter ( [49] ; unpublished data from this study). these wintering behaviors may provide protection against bat to bat transmission of pd. however, talus slopes can provide suitable temperature and humidity profiles for growth of pd, albeit below optimal levels [50] . the variability of relationships among prey availability, yearround activity, individual fitness, and susceptibility to wns remain a knowledge gap for north american bats [51, 52] . in addition to describing presence and seasonal activity rates of bats across forested sites in montana, we developed a process for using acoustic monitoring for disease surveillance that can be applied elsewhere. whereas approaches to incorporate uncertainty in estimates of population trends from bat count data in hibernacula are established, less information is available for managers to identify early-warnings from acoustic monitoring data [18] . here, we suggest the same basic framework that we used for the descriptive model of bat activity could be applied to an acoustic monitoring program aimed at identifying changes in bat activity consistent with wns. the approach could be used for single or multiple species and on individual sites or a collection of sites. additionally, the modeling approach is useful for detecting aberrant behaviors that could be associated with wns, including higher than normal activity rates during seasons where bats should be in torpor, or low activity rates due to population-level declines in bats. the general approach we consider is as follows: 1. collect baseline (pre-wns) data within a season or seasons of interest, and across several years and optionally across several sites. 2. fit a mixed-effects model to the data, incorporating variation with temperature, among years and among sites. optionally include other covariate information predictive of bat activity that can be easily gathered. 3. generate a population or site-level prediction interval that includes among-year variation. 4. monitor the collection of new data for excessive departures from the model prediction interval. in particular, look for bat activity in excess of the predicted range during winter months followed by bat activity below the predicted range during the following spring and summer [53] . we emphasize that our modeling approach was descriptive in nature and that we only recorded bat activity during nighttime. however, the approximately log-linear relationship with mean monthly temperature was strong in our dataset and may be useful in other investigations of bat activity. bat activity also showed approximately log-linear relationships with mean daily-max and daily-min temperatures. the non-random selection of sites included in our analysis limits the interpretation of the estimated model random effects or their application to other populations of sites. another limitation of species-specific results in this study relates to the large proportion of bat detections where no species label was assigned, where unknown biases could impact model estimates. we suggest that future site-specific surveillance models may be improved by including acoustic monitoring in daytime and by obtaining more high-quality species-specific call data with updated microphone technology [6] . we used prediction intervals to characterize expected ranges of among-year activity at the site level. one advantage of this approach in a monitoring program is that one set of intervals can be applied across multiple years without the need for annual refitting, provided suitable baseline data were used for model fitting. it is important to note that prediction intervals for mixed-effects models can be defined for any level of the random-effects structure [41] , with the appropriate level dependent on the end-use application. alternatives to prediction intervals could be employed in a monitoring program, including refitting a model each year to directly compare new data with a reference time period. currently, most conservation and management actions related to wns in north america are related to known hibernacula. for regions where large hibernacula are uncommon or rare, but where bats may still be susceptible to this threat, alternative approaches are necessary to ensure early detection of wns outbreaks and prevent further losses with targeted management actions. we propose a proactive and simple monitoring approach that could assist in managing risk of further loss of bat populations, which is especially relevant given current restrictions on field research due to concerns about transmission of covid-19 from humans to north american bats and among humans [54] . deploying passive arus to quantify bat activity, developing a predictive model of activity rates for one or more sites (including managed and unmanaged forest), and examining future data for deviations from these prediction intervals can all assist with a rapid response, including wns confirmation that could assist in recovery efforts. strong geographic and temporal patterns in conservation status of north american bats winter activity of coastal plain populations of bat species affected by white-nose syndrome and wind energy facilities white-nose syndrome: the devastating disease of hibernating bats in north america wing pathology of white-nose syndrome in bats suggests life-threatening disruption of physiology frequent arousal from hibernation linked to severity of infection and mortality in bats with white-nose syndrome winter behavior of bats and the progression of white-nose syndrome in the southeastern united states endangered and threatened wildlife and plants; 4(d) rule for the northern long-eared bat bats and white-nose syndrome 2020 determinants of pseudogymnoascus destructans within bat hibernacula: implications for surveillance and management of whitenose syndrome spread of white-nose syndrome on a network regulated by geography and climate fungus that causes white-nose syndrome found in eastern montana bat-killing disease white-nose syndrome confirmed east of the cascade range in washington first detection of bat white-nose syndrome in western north america. msphere activity following arousal in winter in north american vespertilionid bats western crevice and cavity roosting bats winter foraging of silver-haired and california myotis bats in western washington a review of bat hibernacula across the western united states: implications for white-nose syndrome surveillance and management improved analysis of long-term monitoring data demonstrates marked regional declines of bat populations in the eastern united states bently wigley t. site occupancy of foraging bats on landscapes of managed pine forest use of modified water sources by bats in a managed pine landscape bats in forests: what we know and what we need to learn bat activity in harvested and intact forest stands in the allegheny mountains us protected lands mismatch biodiversity priorities a macroecological perspective on strategic bat conservation in the u bat response to prescribed fire and overstory thinning in hardwood forest on the cumberland plateau effects of fire and its severity on occupancy of bats in mixed pineoak forests department of agriculture, forest service, intermountain forest and range experiment station ecoregions of the conterminous united states forest regions of montana land cover/land use theme montana natural heritage program montana bat and white-nose syndrome surveillance plan and protocols 2012 -2016 a directory of reports on long-term acoustic monitoring for bats at sites across the northern rocky mountains and great plains do you hear what i hear? implications of detector selection for acoustic monitoring of bats weather conditions determine attenuation and speed of sound: environmental limitations for monitoring and analyzing bat echolocation a plan for the north american bat monitoring program (nabat) on the importance of articulating assumptions when conducting acoustic studies of habitat use by bats montana bat and white-nose sydrome surveillance plan and protocols r: a language and environment for statistical computing. r foundation for statistical computing statistial intervals: a guide for practitioners mertools: tools for analyzing mixed effect regression models white-nose syndrome response team. bats affected by wns 2020 the resistance of a north american bat species (eptesicus fuscus) to white-nose syndrome (wns) an emerging disease causes regional population collapse of a common north american bat species computational sustainability: computing for a better world anda sustainable future how to learn to stop worrying and love edna monitoring winter acoustic activity of bats in montana effects of the environmental temperature on aedes aegypti and aedes albopictus mosquitoes: a review. insects winter bat activity in the canadian prairies unsuspected retreats: autumn transitional roosts and presumed winter hibernacula of little brown myotis in colorado frequent arousals from winter torpor in rafinesque's big-eared bat (corynorhinus rafinesquii) identifying research needs to inform white-nose syndrome management decisions. conservation science and practice going, going, gone: the impact of white-nose syndrome on the summer activity of the little brown bat (myotis lucifugus) fish & wildlife health committee and wildlife resource policy committee, bat working group, guidance for bat-related activities in response to covid-19. association of fish and wildlife agencies key: cord-353245-es7b1rs0 authors: song, deping; huang, dongyan; peng, qi; huang, tao; chen, yanjun; zhang, tiansheng; nie, xiaowei; he, houjun; wang, ping; liu, qinglan; tang, yuxin title: molecular characterization and phylogenetic analysis of porcine epidemic diarrhea viruses associated with outbreaks of severe diarrhea in piglets in jiangxi, china 2013 date: 2015-03-19 journal: plos one doi: 10.1371/journal.pone.0120310 sha: doc_id: 353245 cord_uid: es7b1rs0 porcine epidemic diarrhea (ped), caused by porcine epidemic diarrhea virus (pedv), is a highly contagious, acute enteric viral disease of swine characterized by vomiting, watery diarrhea, dehydration and death. to identify and characterize the field pedvs associated with the outbreaks of severe diarrhea in piglets in jiangxi, 2013, the complete genome sequences of two representative strains of pedv, designated ch/jx-1/2013 and ch/jx-2/2013, were determined and analyzed. the genome sequences of both emergent jiangxi pedv strains, ch/jx-1/2013 and ch/jx-2/2013, were 28,038 nucleotides in length excluding 3’ poly (a) tail. compared to the pedv cv777 strain, ch/jx-1/2013 and ch/jx-2/2013 had some unique genetic characteristics in the proximal region of the 5´-utrs. phylogenetic analysis of the complete genomes and the structural proteins revealed that ch/jx-1/2013 and ch/jx-2/2013 had a close relationship with post-2010 chinese pedv strains and us strains identified in 2013. the nucleotide identity between the two jiangxi strains (ch/jx-1/2013 and ch/jx-2/2013) and 30 strains of pedv identified ante-2010 and post-2010 ranged from 96.3–97.0% and 97.3–99.7%, respectively. multiple nucleotide and deduced amino acid mutations were observed in the orf1a/b, s, orf3, e, m and n genes among the current field pedv strains when compared to the cv777 strain. some of the mutations altered the amino acid charge and hydrophilicity, and notably, there was an amino acid substitution in the middle of one neutralizing epitope (l1371i) of the s gene of both ch/jx-1/2013 and ch/jx-2/2013. taken together, the accumulated genetic variations of the current field pedv strains might have led to antigenic changes of the viruses, which might confer the less effectiveness or failure of the cv777-based vaccines currently being widely used in jiangxi, china. the ethics committee of jiangxi agricultural university approved the animal protocol for this study (protocol number p-2013-03). all the procedures involving animals in this study were carried out in accordance with the care and use guidelines of experimental animals established by the ministry of agriculture of china. intestinal and fecal samples were collected according to the approved procedures. the intestinal samples used in this study were obtained from the dead piglets and the fecal samples were non-invasively collected immediately after excretion from healthy and diarrheal pigs from premises with ped outbreaks in jiangxi, china (s1 table) . fecal and intestinal samples (n = 125) were collected from < 10-day-old suckling piglets with severe watery diarrhea, vomiting and dehydration from different premises in jiangxi province, china (25-29°n and 114-118°e) in 2013. all the samples were examined by a rt-pcr established in our laboratory. briefly, the total rna was extracted from the samples using rnaplus reagent (takara, japan) following the manufacturer's instructions. the one-step rt-pcr for determination of pedv-positive samples was carried out using 50-200 ng of extracted rna, forward primer (5´-gtattggtggtgagcggaat-3´), reverse primer (5´-cctgttcc gccattctatca-3´) and onestep rt-pcr kit (qiagen, valencia, ca, usa) according to the manufacturer's protocol. to eliminate possible co-infections caused by porcine transmissible gastroenteritis virus (tgev), porcine rotavirus (porv), and other common pathogenic intestinal pathogens, differentiation assays were performed using standard protocols. ninety-six out of 125 samples were tested positive for pedv, and all samples had been confirmed to be free of tgev, porv and other common enteric pathogens (data not shown). to address the genetic/antigenic variations, and phylogenetic characteristics of pedv strains associated with 2013 jiangxi ped outbreaks, two representative pedv strains, designated ch/jx-1/2013 and ch/jx-2/2013 (the genbank accession numbers are kf760557 and kj526096, respectively), were used for sequencing the full-length genome. total rnas were extracted from the feces and small intestinal homogenates by rnaplus reagent (takara, japan) according to the manufacturer's instructions. the concentrations of the extracted rnas were measured by nanodrop 2000 spectrophotometer (thermo scientific, usa) and then stored at −80°c until use. the first-strand cdna synthesis was performed at 42°c for 50 min and then 95°c for 5 min to inactivate the m-mlv reverse transcriptase (takara, japan) and followed by 4°c for 5 min. the entire pedv genome was amplified by 33 pairs of primer designed with primer 3 software (http://primer3.ut.ee/) based on the conserved regions determined by a multiple alignment analysis of the reference strains and cv777 (s2 table) . fragments were amplified on the conditions of a denaturation at 94°c for 4 min, 35 cycles (94°c x 45 sec, 53°c x 45 sec, 72°c x 1.5 min), and then with a final extension at 72°c for 10 min. pcr products obtained were subjected to gel purification using a gel extraction kit (takara, japan), and afterwards cloned into pmd 18-t vectors (takara, japan) following the manufacturer's protocols. five positive clones of each amplicon were submitted to a commercial sequencing company (sangon biotech, shanghai, china) for sequencing at both directions by sanger sequencing methodology. the 5'-and 3'-race for the determination of the terminal sequences of both ch/jx-1/2013 and ch/jx-2/2013 were performed by using 5'/3' smarter race kit (clontech, beijing, china) following the manufacturer's instructions. the raw sequence fragments were imported to seqman in dnastar lasergene v 7.10 (dnastar, inc., madison, wi) for assembly and annotation. nucleotide and deduced amino acid (aa) sequences of both ch/jx-1/2013 and ch/jx-2/2013 and 30 reference pedv sequences retrieved from genbank were comparatively analyzed. a summary of the background information of pedvs used in this study is shown in table 1 . the complete genome sequences of ch/jx-1/2013 and ch/jx-2/2013 were deposited into genbank. phylogenetic trees based on the entire genomes, and deduced aa sequences of s, orf3, e, m, and n genes were constructed using the neighbor-joining method of mega 5.2.2 (http://www.megasoftware.net/) with a bootstrap of 1,000 replicate datasets. primary sequences of 5´-proximal region of 5´-utrs (nt 42 to 133) were pairwise compared between the two jiangxi strains (ch/jx-1/2013 and ch/jx-2/2013) and the reference strains. antigenicity and hydrophilicity analyses based on the aa sequence from 1 to 350 at n-terminal of the s proteins were carried out by protean software of dnastar lasergene v7.10 (dnastar, inc., madison, wi). table) . a total of 26 unique nucleotide substitutions were identified between two jiangxi pedv strains (ch/jx-1/2013 and ch/jx-2/2013) and 30 reference pedv strains, which resulted in 14-aa changes, and most of them were located in orf1a, orf1b and s gene. two of the nucleotide substitutions led to aa changes along with the charge variations ( table 2 ). the phylogenetic tree based upon the full-length genome sequence of ch/jx-1/2013, ch/jx-2/2013 and 30 reference pedvs indicated that the pedv strains could be divided into two groups, designated for group1 (g1) and group 2 (g2): g1 could be further divided into three subgroups, i.e., 1a, 1b and r, a tentative cluster consisting of virulent dr13 isolated in south korea in 1999 and ch/s isolated in china in 1986; and g2 was split into two subgroups, 2a and 2b (fig. 1a) . notably, all the strains identified from 2011 to 2013 were fallen into g2; while the prototype of cv777, together with three korean strains (sm98, virulent dr13 and attenuated dr13), and four earlier chinese strains (js2008, js2008new, lzc and ch/s) were fallen into another group, i.e., group 1. genetic characteristics were observed between the two groups: 1) compared to genome sequences of the members in g1, four insertions, 20803g, 20810cagggtgtcaa20820, 20830g, 21042aat21044 and two deletions, 20842a, 21097cgtgat21102, existed in the n-terminal domain (ntd) of the s protein in g 2 members; 2) the three field pedv strains of js2008, js2008new and sd-m together with two attenuated pedv strains, dr13 and vaccine_kc189944, were clustered into subgroup 1b. all of the five pedv strains aforementioned had 24-nt deletions in nsp3 of orf1a and 49-nt deletions in the c terminus of orf3; 3) ch/jx-1/2013 and ch/jx-2/2013 along with three chinese strains (gd-b, js-hz2012 and bj-2011-1) were clustered into an independent clade, and ch/jx-1/2013 and ch/jx-2/2013 had 99.7%, 99.7% and 99.6% nucleotide identity with the three chinese strains, respectively. these strains shared six additional unique nucleotide substitutions (t227a, c2342t, g2346t, t2724c, t6331c, c7233t) with the rest of reference pedv strains. of which, one led to an aa change (t682m in orf1a, from hydrophilic polarity t to hydrophobic polarity m). the full-length of s gene of ch/jx-1/2013 and ch/jx-2/2013 was 4,161 nt in size, which was 9nt longer than that of the prototype of pedv cv777 strain. the results from nucleotide sequence comparisons of the s gene of 34 strains of pedv, including ch/jx-1/2013, ch/jx-2/2013 and other 32 reference pedv strains from china, united states, uk, south korea, belgium, france and japan showed that the two jiangxi strains had a 99.8% nucleotide identity with each other, and 96.7% nucleotide identity with cv777. ch/jx-1/2013 and ch/jx-2/2013 shared 97.3-99.6% nt identity and 93.1-99.1% aa identity with the post-2010 pedv strains, respectively. by contrast, the two jiangxi strains showed only 93.5-95.0% nt identity, and 92.5-94.8% aa identity with the ante-2010 pedv strains and attenuated vaccine strains, respectively (s4 table) . the phylogenetic tree based on the aa sequences of the s protein of 34 strains of pedv showed that they could be classified into two major groups, and each group contained two subgroups (fig. 1b) . both ch/jx-1/2013 and ch/jx-2/2013 belonged to subgroup 2b, which also included five newly identified us strains (13-019349, co/13, ia1, isu13-22038-ia-homogenate and isu13-22038-ia-p9) and 13 chinese strains identified at the same period or later than 2010. compared to g1, there were two insertions (61vnst64 and 136n) and one deletion (155dg156) in the g2 members. amino acid differences were also present between g1 and g2, and most of them were located in the area of aa 1 to 350 at n-terminal of the s protein ( table 3 ). the analysis based on aa from positioned 1 to 350 of the s protein suggested that the antigenicity and hydrophilicity of s protein of ch/jx-1/2013, ch/jx-2/2013 might have already changed due to the mutated amino acids with changes of charges and polarities (fig. 2) . notably, an amino acid substitution was found in the middle of one neutralizing epitope phylogenetic trees based on the complete genome, aa sequences of structural proteins and orf3 of pedv strains. the trees were constructed by the distance-based neighbor-joining algorithm using mega 5.2.2 software. bootstrap was set in 1,000 replicates with a value >70% to assess the significance of the tree topology. a bar of 0.002/0.005 indicates nucleotide or amino acid substitutions per site. "•" indicates the strains identified in this study, "" indicates the strains from china, "◆" indicates the strains from belgium, "■"indicates the strains from usa, "▲"indicates the strains from south korea, "▼"indicates the strains from france, "◇"indicates the strains from japan. 1a: phylogenetic tree generated on the basis of nucleotide sequences of the complete genome of 33 pedvs. 1b to 1f: phylogenetic trees based on deduced amino acid sequences of s glycoprotein genes, orf3, envelope, membrane, and nucleocapsid genes, respectively. table 4 ). the phylogenetic tree based on the deduced aa sequences of the orf3 of the earlier european strains, cv777, korean strains, us strains and chinese strains identified ante or post-2010 revealed that those strains were grouped into two different groups (fig. 1c) . cv777 and two early strains sm98 and lzc, and two cell-adapted strains sd-m and attenuated dr13 were classified into group 1; while the 24 strains, including ch/jx-1/2013, ch/jx-2/2013, 15 chinese pedv strains, a korean strain (virulent dr13), five newly determined us strains, and (from negative charge/hydrophilic polarity e to neutral charge/ hydrophobic polarity q) of the n terminus of the m protein, which might have an impact on its antigenicity/immunogenicity. the entire n gene of ch/jx-1/2013 and ch/jx-2/2013 was 1,326 nt long, encoding a protein of 441-aa. sequence analyses revealed that the aa homology between ch/jx-1/2013 and ch/jx-2/2013 and other pedv strains used in this study varied from 95-98.9%. the nucleotide and amino acid sequences of n protein were highly conserved and neither insertion nor deletion was found in all these pedv strains analyzed in the study except a few sporadic mutations. multi-alignment results of the aa sequences of the pedv n proteins indicated that all the strains could be divided into two groups, i.e., group 1 and group 2. each group contained two subgroups (fig. 1f) . the phylogenetic topology of n protein was similar to that of orf3. three specific aa changes were present between group 1 and group 2, at the residue 142 (a142t), 242 (h242l, from hydrophilic polarity i to hydrophobic polarity t) and 397 (q397l). five aa substitutions at the residue 84 (g84a), 205 (n205k, from neutral charge n to positive k), 381(l381p), 395 (l395q, from hydrophobic l to hydrophilic q), and 398 (h398n, from hydrophilic h to hydrophobic n) existed in subgroup 2a when compared with other three subgroups, i.e., subgroup 2b, subgroup1a and subgroup 1b. the 5´utrs of ch/jx-1/2013 and ch/jx-2/2013 were 292 nt in length, which was 4-nt shorter than that of cv777, resulting from 5-nt deletion and one nt insertion in the proximal region of 5´-utrs of the two jiangxi strains (fig. 3) . the core sequence (cuaaac) of the pedv leader transcription-regulating sequence (trs) was extremely conserved with no nucleotide substitutions in all the pedv strains. an 'a' deletion was observed immediately following the core sequence of these pedv strains except the strains of cv777 and lzc. in comparison with cv777 and early isolated strains of sm98 and lzc, a 'u' insertion in loop 2 was found in both ch in other four pedv strains isolated earlier (cv777, attenuated vaccine, lzc, and sm98). compared to cv777, a 4-nt deletion (uucc) existed in the stem region of sl4 of ch/jx-1-/2013, ch/jx-2/2013 and other six pedv strains. however this deletion would not impact the secondary structure of the sl4 as demonstrated by an analysis (data not shown). the 3'utrs of both ch/jx-1/2013 and ch/jx-2/2013 were 334 nt in size, and no insertion and deletion was observed (table 2 ). ped affected massive pig farms in jiangxi, china in 2013, causing substantial economic losses in the pig industry of jiangxi. to elucidate the molecular characterization and phylogeny of the pedv field strains associated with 2013 jiangxi ped outbreaks, the entire genome sequences of two representative pedv strains confirmed in jiangxi were determined and analyzed. phylogenetic analyses demonstrated that ch/jx-1/2013 and ch/jx-1/2013 defined in this study along with the strains determined post-2010 were clustered into group 2, whereas the strains detected ante-2010 were clustered into group 1. the results of phylogenetic analyses of those pedv strains examined in this study were similar to that of described elsewhere [22] . ch/jx-1/2013 and ch/jx-1/2013 showed the highest nucleotide identity (>99%) with six newly confirmed us strains (ia1, co/13, usa/indiana, isu13-22038-ia-homogenate, isu13-22038-ia-p9, and 13-019349) and five chinese strains (gd-b, ah2012, bj-2011-1, ch/zmd/zy, and ch-hz/2012) identified post-2010, suggesting these pedv strains might have evolved from the same origin although the mechanisms of the evolution of the viruses are roughly unknown yet. three field pedv strains, js2008, js2008new and sd-m together with two attenuated pedv strains, dr13 and vaccine_kc189944, were clustered into an independent cluster and showed 24-nt deletions in nsp 3 of orf1a and 49-nt deletions in the c terminus of orf3. these unique characteristics suggested that the three field pedv strains might derive from the same source, and a recombination event might have occurred between these three viruses and the two vaccine strains in the same subgroup. interestingly, a comparison analysis revealed that ch/jx-1/2013 and ch/jx-1/2013 had 99.7% nucleotide identity to the strain of gd-b (genbank accession no.jx088695) isolated from guangdong, an adjacent province of jiangxi, in which the severe ped outbreaks emerged in october 2010 [11, 23] . pigs are frequently traded between jiangxi and guangdong, which might be one of important factors causing the cross dissemination of pedvs in this region. however, further study needs to be performed. the findings from this study demonstrated that the 5´-utr and orf3 protein of ch/jx-1/2013 and ch/jx-2/2013 and all other pedv strains analyzed were highly conserved, which were consistent with the studies reported previously [5, 9] . in general, strict conservation of these regions is essential for the pedv life cycle. it is known that the 5´-utrs of coronaviruses form conserved rna structural elements which are critical for viral replication, sgmrna transcription, and translation [24] . studies on the betacoronavirus have indicated that the stemloop 2 (sl2) in 5´utr is extremely conserved in all coronaviruses, and plays a cis-acting reaction on transcription and replication, and more importantly the replication of the virus requires the conservative sequence and a firm number of nucleotides with specific properties in sl2 in 5´utr [25] . although the 'u' insertion in sl2 does not alter the rna secondary structure, it might slightly affect the efficiency of virus replication, which needs to be addressed in the future studies. the core sequence (5´-cuaaac-3´) in trs of pedv, which was also present in ch/jx-1/2013 and ch/jx-2/2013, has reported to be a determinant factor in transcriptional regulation in coronavirus because the synthesis of sgmrna requires the appropriate tertiary structure of core sequence [26] . both pedv and tgev belong to the alphacoronavirus genus within the coronaviridae family and possess the conserved core sequence structure. studies based on the infectious genomic tgev cdnas have proved that the nucleotides immediately flanking the trs sequence could apparently affect the expression of the sgmrna. and the nucleotides adjacent to core sequences of the leader trs (cs-l) by the 3´region are more decisive for mrna synthesis than nucleotides in the 5´region. the motif of 5´-gaaa-3´within 3´cs-l (5´-cuaaacgaaa-3´) shows a higher expression than that of other motifs [27] . in respect of the pedv, an 'a' deletion immediately followed the cs-l sequence in the 5´utr of ch/jx-1/2013 and ch/jx-2/2013 as well as other post-2010 pedv strains analyzed in the study and thus, this deletion formed a four base oligonucleotide (5´-gaaa-3) adjacent to the cs-l by the 3´region, which might up-regulate the sgmrna expression in pedv [24] . the s protein of pedv is known to play pivotal roles in viral entry and inducing the neutralizing antibodies in natural hosts, and thus makes it become a primary target for the development of effective vaccines against pedv [28] [29] [30] [31] . it was demonstrated that there were significant genetic variations in the s gene between the newly determined pedv field strains and early isolates [20, 32] . the results in this study agreed with previous documentations. additionally, ch/jx-1/2013 and ch/jx-2/2013 showed extremely high nucleotide and aa identity with each other but less to cv777 and attenuated strains (attenuated vaccine_kc18944 and attenuated dr13). there were significant aa differences between the g1 and g2 members, and most of them were located in ntd of the s protein of pedvs. when compared to the group 1 strains of pedv, ch/jx-1/2013 and ch/jx-2/2013, members of group 2, showed significant antigenic and hydrophobic differences, and some of which were located in the neutralizing epitope regions. those genetic variations made the ch/jx-1/2013 and ch/jx-2/2013 and post-2010 field pedv strains different from the ante-2010 strains, especially the cv777. it might explain why the recent ped outbreaks were significantly different from the previous sporadic outbreaks. present ped became a devastating enteric viral disease causing substantial economic losses in the pig industry in major pig-raising countries in the world, and recently made it become a reportable disease by usda [33] . moreover, the present pedv field strains displayed considerable genetic variations from cv777-based vaccine strain, a strain being widely used for production of pedv vaccines in china for many years. notably, leucine, a highly hydrophobic aa residue in the middle of one neutralizing epitope (l1371i) of the s gene of cv777 was substituted by an isoleucine, a higher hydrophobic index aa residue in both ch/jx-1/2013 and ch/jx-2/2013 strains. this variation might provide a possible mechanism for a poor protection on swine vaccinated with the cv777-based vaccines. park et al. [29] have reported that a reduced cell-culture-adapted pedv strain (attenuated dr13, passage 100) has a truncated orf3 of a 51-nt deletion, suggesting that this gene may be involved in cell tropism and is essential for the virulence of pedvs. by contrast, ch/jx-1/2013 and ch/jx-2/2013, together with the post-2010 variant pedv strains had an intact orf3 of 675 nucleotides encoding a protein of 224 aa. the phylogenic analysis demonstrated that the orf3-based tree had a similar topology with the one generated from the entire genome sequences. and thus the orf3 of pedv may serve as a useful target gene for phylogenetic analysis of newly emerging field pedv strains since its small size. as recognized previously [34, 35] , there is a large deletion region existing in pedv isolates attenuated dr13 (51-nt at the position of 245-295), sm98 (210 nt at the position of 1-210) and sd-m (51-nt at the position of 245-295), a chinese strain propagated only four passages on cell lines. in summary, the findings obtained in this study provide some insight into the genetic-/phylogenetic variations and molecular characterizations of the jiangxi field pedv strains associated with the outbreaks in piglets in jiangxi, china 2013. the comparisons of the fulllength genome and the structural protein genes and deduced aa sequences revealed that two jiangxi strains ch/jx-1/2013 and ch/jx-2/2013 defined in this study had a close relationship with the recent prevailing field pedv strains in china and the united states. differences at the level of the nucleotides and deduced aa between the present field pedv strains and cv777, especially the aa substitutions in the neutralizing epitope of pedv field strains, might have conferred the less effectiveness of the vaccines currently widely being used in china. it might be urgently needed to develop improved efficacious and safe vaccines against field pedvs being currently circulating in china. a new coronavirus-like particle associated with diarrhea in 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replication a u-turn motif-containing stem-loop in the coronavirus 5' untranslated region plays a functional role in replication transcription regulatory sequences and mrna expression levels in the coronavirus transmissible gastroenteritis virus role of nucleotides immediately flanking the transcription-regulating sequence core in coronavirus subgenomic mrna synthesis identification of two novel b cell epitopes on porcine epidemic diarrhea virus spike protein sequence analysis of the partial spike glycoprotein gene of porcine epidemic diarrhea viruses isolated in korea phage-displayed peptides having antigenic similarities with porcine epidemic diarrhea virus (pedv) neutralizing epitopes identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus heterogeneity in spike protein genes of porcine epidemic diarrhea viruses isolated in korea deadly pig virus slips through us borders genetic variability and phylogeny of current chinese porcine epidemic diarrhea virus strains based on spike, orf3, and membrane genes cloning and further sequence analysis of the orf3 gene of wild-and attenuated-type porcine epidemic diarrhea viruses xiangbai hua and jiaxiang li are greatly appreciated for their help in sampling campaign. key: cord-311288-6ttux2uu authors: luo, chen; li, yuru; chen, anfan; tang, yulong title: what triggers online help-seeking retransmission during the covid-19 period? empirical evidence from chinese social media date: 2020-11-03 journal: plos one doi: 10.1371/journal.pone.0241465 sha: doc_id: 311288 cord_uid: 6ttux2uu the past nine months witnessed covid-19's fast-spreading at the global level. limited by medical resources shortage and uneven facilities distribution, online help-seeking becomes an essential approach to cope with public health emergencies for many ordinaries. this study explores the driving forces behind the retransmission of online help-seeking posts. we built an analytical framework that emphasized content characteristics, including information completeness, proximity, support seeking type, disease severity, and emotion of help-seeking messages. a quantitative content analysis was conducted with a probability sample consisting of 727 posts. the results illustrate the importance of individual information completeness, high proximity, instrumental support seeking. this study also demonstrates slight inconformity with the severity principle but stresses the power of anger in help-seeking messages dissemination. as one of the first online help-seeking diffusion analyses in the covid-19 period, our research provides a reference for constructing compelling and effective help-seeking posts during a particular period. it also reveals further possibilities for harnessing social media’s power to promote reciprocal and cooperative actions as a response to this deepening global concern. according to the world health organization (who), covid-19 has resulted in more than 14 million confirmed cases and nearly 0.6 million deaths worldwide by july 20th, 2020 [1] . due to the destructive power and highly infectious feature of this new coronavirus, many countries have taken various kinds of measures to prevent virus transmission. meanwhile, people use social media to acquire and exchange multiple types of information at a historic and unprecedented scale [2] . so far, some researches have concentrated on the effects of social media in this particular period. for example, harness the social media posts to predict infected case counts and inform timely responses under the infoveillance or infodemiology framework [3, 4] ; analyze the help-seeking posts to identify the characteristics of covid-19 patients [5] ; more retransmission means more users receiving the message, thus increasing the chance of getting help. secondly, from a practical point of view, facing the medical resources shortage and uneven distribution of medical facilities, widely disseminated help-seeking posts played the role of social warning, assisted official institutions in understanding urgent affairs as well as allocating supplies more effectively. what are the driving factors behind online help-seeking messages retransmission? most existing studies categorized driving forces into two dimensions: the sender factors and the content factors [11, 23, 27] . however, significant driving forces are varied by context. in this study, ordinary users are actively adopting the weibo platform to express their needs and compete for public attention. other types of users, including celebrities and organizations, are rarely asking for help online. in short, the main component of online help seekers is ordinaries, and their online identities are nearly the same. thus, it would be more reasonable and significant to explore further in the content factor. there is plenty of research providing conceptual references, such as the depth of self-disclosure [16, 28] , different types of support messages [16, 29] , physical and emotional proximity to the target [30] , the social capital stock of the help seeker [22] . enlightened by existing experience, we will summarize the impelling factors of help-seeking information diffusion into five dimensions: completeness, proximity, support typology, disease severity, emotion, and elaborate them in the following sections. under the dual-process perspective, the former four determinants stay close with the slow and deliberative process while the emotion is akin to the fast and intuitive cognitive process. in other words, retweeting behavior is propelled by "relying on reason" and "relying on emotion" simultaneously [14, 31] . completeness is bound up with credibility. credibility has always been seen as perceived quality and as a result of intertwined dimensions [32, 33] . previous studies usually concentrated on source credibility and stressed the significant influence of source credibility perception on retweeting behavior in sports news [34] and health information diffusion [35] , the importance of content credibility was overlooked to some extent. in brief, content with high credibility should be internally consistent and clearly presented. many scholars have elucidated the connotation of content credibility, including complete, in-depth, precise, reliable, accurate, unbiased, objective, factual, and fair [36] [37] [38] . in a systematic literature review, sbaffi and rowley [39] listed dozens of content features influencing trust judgments and credibility perception, which means credibility is a typical compound concept made up of various subdimensions [40] . as a result, it is impossible to investigate all elements of credibility in one study. the proper approach is to pick out the critical factor based on the specific research context. completeness is a pivotal part of content credibility, especially in online help-seeking during covid-19. as the name suggests, it means how much information does an individual disclose and the clarity of the provided information [41] . completeness contributes to the perception of health information quality on the internet [42] . firstly, more complete information means more additional information, which increases the "real world feel" [43] . more detailed information implies more communication cues, improves the social presence of communicators in nonverbally environments [44] . secondly, completeness is directly bound up with the amount and depth of self-disclosure. altman and taylor [28] argued that breadth is the amount of disclosed information, while depth is the intimacy of disclosed information. a complete self-disclosure can effectively convey individuals' needs and reveal who they are, also benefits the likelihood of receiving social support [45] . in this study, we extrapolate that online help seekers' willingness to divulge their personal information helps the audience comprehend their identities. besides, a complete expression of disease development or health condition usually demonstrates the seriousness of the current problem, improves credibility perception, and eventually leads to successful support provision. we propose the following: h1: completeness of individual information in online help-seeking posts is positively associated with retransmission. h2: completeness of disease status in online help-seeking posts is positively associated with retransmission. proximity can be interpreted as a form of perceived psychological distance bound up with the construal level theory (clt) proposed by trope and liberman [46] . the psychological distance is defined on spatial, temporal, social, and hypothetical dimensions-those distances further influence how people think about an object or event [46, 47] . substantial studies have scrutinized the impacts of proximity on an individual's responses to others' misfortune, and the main mechanisms can be elaborated in two aspects. firstly, high proximity implies low perceived distance, which increases empathy and compassion toward the victim, while low proximity weakens it [48, 49] . secondly, proximity links to the perceived trustworthiness of specific information or source. generally speaking, people often conceive a proximal source as more credible than a distant one [4, 35] . driven by those two threads, when engaged in online activities, high proximity promotes information sharing [50] and information adoption [51] . the work of huang et al. [30] also manifests the evident influences of physical and emotional proximity on online information seeking under the crisis context. based on the above rationale, in the supportive communication context, high proximity induces compassion and sympathy more efficiently, which has a potential impact on information diffusion. likewise, some studies noted that reciprocal social support behaviors frequently occur in close social proximity circumstances [50] and a higher social presence environment arousing psychological proximity [44] . thus, we propose the following hypothesis: h3: high proximity triggers more retransmission than low proximity. people give support to others in multiple ways, while how different types of social support are associated with the seeking-provision remains underexplored. a considerable number of studies summarized distinct social support types and put forward some conceptual frameworks [52, 53] . some scholars conceptually divided social support into four categories, including emotional support (e.g., expression of empathy, love, trust, and caring), informational support (e.g., advice-giving, providing suggestions, sharing information), appraisal support (i.e., offering information for self-evaluation), and instrumental support (i.e., providing tangible support) [52] [53] [54] . others extended the categories of social support by incorporating emotion, esteem, information, network, and tangible assistance [55] [56] [57] . moreover, the social support inventory of ucla (the university of california at los angeles) proposes a classification framework consists of three types: information or advice, tangible assistance or aid, and emotional support [58] . for concision and clarity, also following the previous operation [59] [60] [61] , we merged the existing frameworks into two major types: emotional support and instrumental support. the former one typically refers to needs regarding caring, empathy, love, and trust [59] [60] [61] [62] , while the latter one denotes the requirements of instrumental resources and practical help [53, 59] . the interplay between social support types and support provision is still under-investigated, particularly the comparison of different types' outcomes has not been thoroughly examined in the online context. this study intends to remedy the defects by discussing how distinct types of social support induce different feedbacks. therefore, we raise a research question: rq1: what is the relationship between support seeking types and retransmission? a patient's demands vary by the disease phase because each phase presents a new set of challenges and concomitant opportunities [63, 64] . covid-19 is a relatively novel virus with severe clinical manifestation, and it even incurs death [65] . however, bounded by the shortage of testing capacity in covid-19's early stage, not all infected patients can get diagnosed formally on time [4] . as a result, suspected cases and confirmed cases were treated differently, receiving different medical services. before the establishment of makeshift hospitals, patients who have mild symptoms but not get laboratory-confirmed often isolated themselves at home, while those who got confirmed were admitted to hospitals in a relatively short time. thus, it is reasonable to infer that help seekers in different disease phases would get dissimilar attention. and normally, the more serious the illness, the more urgent the help-seeking and the more likely for the patient to obtain support provision. accordingly, we posit: h4: disease severity expressed in online help-seeking posts is positively associated with retransmission. previous studies have proven different emotions affect social decisions differently [66, 67] . the logical cues and emotional cues always work in parallel, and emotions sometimes surpass factual information and deliberation [10] . recent studies regarding emotions and preventive practices (e.g., wear a face covering) during the covid-19 period indicated that negative emotions mediate the relationship between gender difference and face covering usage [68] . since rely on reasoning and rely on emotions often occur simultaneously [69] , we decide to dive deep into negative emotions in our study for positive feelings are rarely exist in help-seeking posts. among a plethora of emotional types concerning illness [70] , fear is the most prominent emotion during pandemic times, which is highly contagious and makes people feel imminent threats easily [10, 71] . lazarus [72] and dillard et al. [73] summarized primary discrete emotional kinds in health communication. apart from fear, anger and sadness are the other two dominating emotions. anger always connected with removing obstacles, while sadness denotes a manifestation of a sense of failure [72] [73] [74] . however, it is still not clear how distinct emotional types induce different retweeting effects. therefore, we formulate the following research question: rq2: what is the relationship between emotional types and retransmission? we choose china as our research field for the following reasons. firstly, wuhan, the capital of hubei province, is the earliest epicenter of the covid-19 outbreak [75] . however, the chinese government adopted a series of prevention and control measures to bring the virus under control, almost tamed the spread of covid-19 in mainland china [76] . china's experience can be an essential reference to other countries, especially in current global pandemic time. secondly, with the rapid development of internet facilities in china, internet penetration has reached 61.2% in china until june 2019, along with a continuous growing scale of social media users [77] . some social media platforms play the role of organizing campaigns, promoting the formation of civil society and the public sphere in china [78] . in today's crisis period, chinese social media exerts the potential to mobilize collective intelligence to overcome difficulties, inspiring other countries to utilize social media to solve health-related problems and cross tough barriers. sina weibo, one of the most popular social media platforms in china, was selected as the sample pool. according to weibo's user report in 2018, this social media service has accumulated more than 0.4 billion monthly active users and nearly 0.2 billion daily active users until q4 of 2018 [79] . weibo has been proved as a vibrant discussion platform and help-seeking space during major social events, especially in the covid-19 period [4, 5] . all authors went through relevant help-seeking posts on weibo and picked up pertinent keywords. after screening and merging, three pairs of keyword combinations were determined as search terms, including "pneumonia + ask for help" ("肺炎 + 求救" in chinese), "pneumonia + seek help" ("肺炎 + 求助" in chinese), and "pneumonia + help me" ("肺炎 + 救救" in chinese). date range starts from jan. 20th, 2020, which is the date that nanshan zhong confirmed human-to-human transmission [80] , and ends at mar. 1st, 2020, as the closing date of the first fang cang makeshift hospital in the epicenter wuhan city, indicating the epidemic had been roughly under control in china [81] . we retrieved and collected the data on june 3, 2020. for now, sina weibo doesn't prohibit automated data access explicitly. previous studies have adopted web crawling scripts to collect sina weibo posts related to covid-19 [4, 82] . besides, our study has been reviewed and approved by the ethics review committee of tsinghua university, china (no. thu202023). thus, we took a similar strategy to write a web crawler in python programming language to retrieve all qualified posts in the designated date range. 34,088 posts were collected in the first round, and 9,826 posted remained after removing duplicated and unqualified posts. we conducted a random sampling to extract a representative sample from the overall corpus because of the large amount of data. following the american association for public opinion research's (aapor) suggestion [83] , we set a 95% confidence level and a 3.5% sampling error, as typical thresholds in social science research. it turns out that the minimum sample size should be 726. thus, we randomly sampled 727 posts for further analysis. every single post acts as the analysis unit in our study, contains the user id, user name, user's registration time, post time, number of followers, number of retweets, content, images' links, authentication type, post's link. the measurement scheme was built on the literature review. emotion type is composed of fear, anger, sadness, and others. support typology contains emotional support seeking, instrumental support seeking, and no specific kind of support seeking. proximity was operationalized into reporting self-illness, reporting others' illness, and both. disease severity consists of three kinds: suspected case, confirmed case, and others. completeness is further decomposed into the completeness of individual information and disease status. detailed meanings of those categories are listed in table 1 . number of followers, posting frequency and some other indicators are incorporated into analysis as control variables according to existing research [15, 84] . a pilot study with 100 posts was conducted to test the coding scheme and train the coders. the pilot study validated the effectiveness of the proposed coding scheme and the accuracy of the corresponding classifications. three coders major in communication studies were recruited to code the posts for inter-coder reliability evaluation. the average krippendorff's alpha coefficient was 0.753 in the first round, which is slightly below the acceptable level. we then retrained the coders, resolved all discrepancies, and sampled another 114 posts from the corpus randomly for a new round trial coding. the average reliability coefficient improved to 0.873 in the second round, which means highly consistent among the coders. all coders then performed coding work on the remaining posts independently. since the dependent variable is the number of retweets, traditional linear regression models are inadequate for modeling this kind of highly skewed count variable. four count models: poisson regression, negative binomial regression, zero-inflated poisson regression, and zeroinflated negative binomial regression were compared to fit the data. firstly, the conditional variance of the outcome variable far exceeds the conditional mean on most categorical explanatory variables, which violates the underlying assumption of poisson regression. secondly, we compared the negative binomial regression with the zero-inflated negative binomial regression. fig 1 displays the residuals from the two tested models, and small residual distribution indicates the good-fitting of the corresponding model. besides, the bic indicator and the sum of pearson also approved the conclusion that the negative binomial regression model (bic = 243.908, sum of pearson = 7.887) is more preferred to the other one (bic = 265.635, sum of pearson = 27.138). table 2 shows the descriptive statistical results of all variables. table 3 summarized the results of negative binomial regression models. model a contains all control variables, while model b contains both control variables and principal independent variables. all coefficients are evaluated based on robust standard errors. with regard to the completeness, for each one-unit increase in disclosing individual information, the expected log count of the number of retweets increased by 0.565 (p < 0.001, irr = 1.759). however, the correlation between completeness of disease status and the number of retweets is insignificant. thus, h1 was supported but h2 was rejected. when it comes to proximity, reporting self-illness (b = 1.853, p < 0.05, irr = 6.377) and both self and others' illnesses (b = 1.634, p < 0.05, irr = 5.126) are expected to receive more retweets than only reporting others' illnesses. h3 was supported. for support seeking type, the expected log retweet count of instrumental support seeking is significantly more than emotional support seeking (b = 2.330, p < 0.001, irr = 10.279), no specific support seeking comes afterwards (b = 1.400, p < 0.01, irr = 4.054). rq1 was answered. h4 discusses the relationship between disease severity and retransmission. compared with other illness stages, posts about suspected cases receive more attention (b = 1.686, p < 0.001, irr = 5.398), confirmed cases afterwards (b = 1.583, p < 0.01, irr = 4.868). h4 was rejected. rq2 asks how emotional types affect help-seeking diffusion. the expected log retweet count for fear is lower than the expected log count for anger (b = -2.725, p < 0.001, irr = 0.066), followed by sadness (b = -2.749, p < 0.001, irr = 0.064). borrowing former scholars' experience [11] , we further conducted a sensitivity test using the manova analysis for cross-validation. the robustness check results (both coefficient size and significance) are close to the negative binomial regression, proving the accuracy of our estimation. emotional support seeking emotional support involves acting as a confidant for someone, providing empathy or other positive affection toward people who suffer from misfortune [59] [60] [61] . "i have a low fever these nights, so afraid. can someone give me some comfort?" instrumental support seeking instrumental support means offering assistance tangibly or physically, such as donating money, providing medical supplies to someone in sickness [59] [60] [61] . "my mother is in urgent need of a sickbed, can someone help me contact an available hospital?" no specific kind of support not mention any specific kind of support seeking. "who can help them?" severity stage of illness suspected patients with some covid-19 infection symptoms but not get laboratory-confirmed [87] . "i took a chest x-ray, and the doctor said i am a suspected case. but i haven't taken a nucleic acid test yet." patients who get laboratory-confirmed [87] . "the nucleic test is positive. it turns out i was infected with the coronavirus." others unknown status. illness status is not mentioned. emotional type fear an emotion experienced in anticipation of some specific pain or danger. fear is predicated on the belief that the individual faces impending danger over which he or she may have little or no control [73] . "at present, mr. zhao is in critical condition. he has been sent to the intensive care unit of central south hospital. he is in urgent need of blood plasma of type o from recovered patients." the health system and government may evoke anger due to their incapability to provide the necessary protection. particular individuals can also stir up anger for transmitting the disease [88] . "i'm furious at the work efficiency of local hospitals!!!" sadness sorrow and sadness are commonly experienced for loss [88] and irrevocable failure to meet the goal [73] . "i feel too sad and helpless in front of this pneumonia, will you help me?" other emotions except fear, anger, and sadness. "i hope everything will get better soon." a all examples come from posts in our research corpus (translated from chinese). first of all, the findings of the completeness part are in concert with one recent research. pan et al. [85] found that support-seeking posts with peripheral self-disclosure elicit lower perceived anonymity in the support-provider side, thus increasing the trustworthiness of the support-seeking messages and improving the quality of advice. peripheral self-disclosure denotes biographical data, including name, age, gender, geographical information [85] , which is precisely the "completeness of individual information" defined in our study. from the perspective of uncertainty reduction theory, message without clear source identity is more likely to be perceived as low credibility, further impeding support-providers' engagement in supportive communication [89, 90] . in other words, detailed personal information disclosure can effectively diminish perceived anonymity and demonstrate the vulnerability of help-seekers, which is pivotal in text-based online anonymous settings. on the contrary, the completeness of disease status fails to trigger retweet behavior. one possible explanation could be the high threshold of medical knowledge posed a high demand for laypersons, resulting in an invisible "communication gap" [91] . the diagnostic report, chest x-ray photo, or medical record requires expertise to understand, which seems impossible to most weibo users. however, this does not mean that the description of the disease is not important. to improve the communication effect, symptoms and development of the illness should be described in detail through simple words to make it easily understood by ordinary people. this rule could be verified by the positive correlation between the text length and the retransmission [92] . the impact of proximity on information transmission is consistent with the previous studies [30, 35] , which demonstrates that a high level of proximity triggers more retransmission. proximity was operationalized into reporting self-illness, reporting others' illness, and reporting both in our study. on the one hand, proximity can be interpreted as a type of constructed imaginary relationship with "others," or a perceived connection between individuals [48] . imaginary relationship plays a vital role in eliciting emotional or informational responses to suffering. reporting self-illness, reporting others' illness, and reporting both represent different levels of perceived psychological distance, lead to varying amounts of information retransmission. on the other hand, the rhetoric school emphasizes the effect of "narrative distance" on audiences' emotional involvement [93] . specifically, narrative perspectives (first-person versus third-person) can influence victim blame and supporting intention by affecting the perceived psychological distance [94] . follow this thread, reporting self-illness and reporting others' illness can be separately associated with first-person and third-person narrative perspectives, stimulating disparate psychological distances and leads to different responses toward the patients eventually. in other words, reporting self-illness usually creates a sense of telling firsthand experience, which fosters a perception of reliability and authenticity on the support-provider side. it is especially true in the health communication context because a direct expression always outperforms interpreting others' stories. thus, it is critical to add information about "myself" in a help-seeking message if one intends to gain extensive attention. although social network services generally offer substantial opportunities for social support transactions, their potential to provide emotional and instrumental support may differ [95] . in this study, more than half of posts contain instrumental support seeking intention, far exceed emotional support seeking and aimless support seeking. instrumental support seeking posts receive more retransmission. this result suggests that in the health communication field, especially during a severe pandemic, it is inevitable to face the explosive growth of information. under this circumstance, attention becomes a scarce but valuable resource [96] . compared with nihilistic emotional support seeking, instrumental support seeking focused on improving one's health condition, demonstrating direct material needs, and showing the willingness to receive immediate help. besides, based on vitak and ellison's work [97] , many people are reluctant to express emotional needs online because they do not want to appear "needy." emotional support also articulates with intimacy, a prerequisite for emotional communication between interactive partners [98] . the anonymity feature of online communication hinders help seekers' desire for emotional support, impedes the occurrence of in-depth emotional connection [99] . additionally, the dissemination effect of aimless support seeking also surpasses the emotional one. one possible reason is that there exist many similar posts with intense emotions (e.g., fear toward covid-19). the audience may immune to those contents after longlasting exposure, thus mitigate the desire to forward such posts. regarding severity, existing studies revealed that giving priority to the worst-case when allocating health resources in pandemics is a fundamental principle [100] [101] [102] . however, our findings go against this principle. one significant reason is that demands in posts about suspected cases are easier to meet than posts about confirmed cases. suspected patients are eager for testing kits and opportunities to visit doctors, while confirmed patients are craving for sickbeds, plasma, and even transfer to another hospital. the former type is more achievable, along with less probability of failure. impelled by difficulty comparison, retweeters incline to disseminate help-seeking messages which are easy to implement. when it comes to emotion, posts expressing anger received more retransmission than fear, sadness, and other kinds of emotions. based on our observation, anger mostly stems from hospitals' unfair treatment or official institutions' delayed responses. it is reasonable for social media users to stand on the "just side" to support the unfortunate patients. by retweeting the angry posts, retweeters vented their feelings, intended to get more attention, and urged relevant departments to take effective measures. in our research corpus, sadness is the second common emotion. however, it received the least retransmission. this can be attributed to sadness's intrinsic characteristics: posts with sadness mainly express disappointment toward reality but resist to advocate practical attempts to change reality. this finding implies that when seeking help online, one should stress the principal problem and avoid simple catharsis. similarly, ingeniously using prevailing emotion contributes to receiving sympathy and pragmatic feedback from others. covid-19 poses enormous threats to the whole world. due to the unbalanced distribution of medical resources and inadequate response at the early breakout stage, many patients (both suspected and confirmed), along with their relatives or friends, turned to social media to seek support. this study explores the driving forces behind online help-seeking post transmission in a global pandemic period by emphasizing the content characteristics. completeness, proximity, support typology, disease severity, and emotion composed the analytical framework. by employing content analysis, 727 randomly sampled help-seeking posts were analyzed based on a coding scheme we construct. negative binomial regression reveals that posts release anger, express instrumental support seeking intention, report self-illness, expound suspected cases' conditions, and have detailed individual information disclosure are likely to have more retransmission. coronavirus is still spreading fast around the world. as one of the first countries to restrain the spread of the epidemic, china provides a valuable experience to other countries stuck in a dilemma. as the first online help-seeking analysis research in the covid-19 period, this study offers some insights about health communication via social media, especially how to develop a potent help-seeking post in public health emergencies. however, a couple of limitations need to be mentioned. first, retransmission of post, as the core dependent variable in our study, is a multidimensional concept. for example, wang et al. [11] decomposed retransmission into "scale" and "structural virality." limited by research resources and time, we failed to elucidate information diffusion comprehensively. second, although china's experience is representative, whether this pattern can be generalized into other contexts worth carefully examining. future studies should conduct more explorations on other social media platforms (e.g., twitter) to verify the reliability and validity of our results and summarize effective online help-seeking strategies in diversified environments. conceptualization: chen luo, yuru li, anfan chen, yulong tang. formal analysis: chen luo, yuru li, anfan chen. methodology: chen luo, yuru li, anfan chen. project administration: chen luo. visualization: chen luo. writing -original draft: chen luo, yuru li, anfan chen, yulong tang. writing -review & editing: chen luo, anfan chen. coronavirus disease (covid-19) pandemic characterizing the propagation of situational information in social media 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satisfaction with social support from social network structure it's not that i don't have problems, i'm just not putting them on facebook: challenges and opportunities in using online social networks for health who should get influenza vaccine when not all can? principles for allocation of scarce medical interventions ethical considerations: care of the critically ill and injured during pandemics and disasters: chest consensus statement the authors want to thank prof. jing qian (department of psychology, tsinghua university) for her tremendous help in the ethical review process. key: cord-336364-2ust3qoq authors: artigas, laura; coma, mireia; matos-filipe, pedro; aguirre-plans, joaquim; farrés, judith; valls, raquel; fernandez-fuentes, narcis; de la haba-rodriguez, juan; olvera, alex; barbera, jose; morales, rafael; oliva, baldo; mas, jose manuel title: in-silico drug repurposing study predicts the combination of pirfenidone and melatonin as a promising candidate therapy to reduce sars-cov-2 infection progression and respiratory distress caused by cytokine storm date: 2020-10-02 journal: plos one doi: 10.1371/journal.pone.0240149 sha: doc_id: 336364 cord_uid: 2ust3qoq from january 2020, covid-19 is spreading around the world producing serious respiratory symptoms in infected patients that in some cases can be complicated by the severe acute respiratory syndrome, sepsis and septic shock, multiorgan failure, including acute kidney injury and cardiac injury. cost and time efficient approaches to reduce the burthen of the disease are needed. to find potential covid-19 treatments among the whole arsenal of existing drugs, we combined system biology and artificial intelligence-based approaches. the drug combination of pirfenidone and melatonin has been identified as a candidate treatment that may contribute to reduce the virus infection. starting from different drug targets the effect of the drugs converges on human proteins with a known role in sars-cov-2 infection cycle. simultaneously, guildify v2.0 web server has been used as an alternative method to corroborate the effect of pirfenidone and melatonin against the infection of sars-cov-2. we have also predicted a potential therapeutic effect of the drug combination over the respiratory associated pathology, thus tackling at the same time two important issues in covid-19. these evidences, together with the fact that from a medical point of view both drugs are considered safe and can be combined with the current standard of care treatments for covid-19 makes this combination very attractive for treating patients at stage ii, non-severe symptomatic patients with the presence of virus and those patients who are at risk of developing severe pulmonary complications. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the identification of host key points of intervention has been done through manual curation of scientific publications and gene expression analysis of data included in the gene expression omnibus (geo) database [20] . this has provided 3 sets of proteins related with the infection process: 1) coronavirus-host interaction set (including sars-cov-2 entry points), 2) lungcells infection set, and 3) acute respiratory distress (ard) set. the 'coronavirus-host interaction set' is composed of human proteins with a relevant role in sars-cov-2 infection and a set of human coronaviruses host interactome retrieved through manual curation of scientific publications (s1 table) . the 'lung-cells infection set' has been populated through differential expression of the gse147507 [21] gene expression dataset using edger [22] package (p-value <0,05 and log2fc>1 or <-1). the 'ard set' has been defined using microarray data sets included in geo that define the role of different important cell types in ard pathophysiology: gse89953 (alveolar macrophages and monocytes ard) [23], gse76293 (neutrophils in ard) [24] and gse18712 (lung cells at different ard stages) [25] . the first 2 sets have been analysed with the geo2r tool [20] , and the last one with a statistical analysis method based on significance analysis of microarrays (sam) [26, 27] to determine the differentially expressed proteins. additionally, ard cytokine storm has been characterized by manual curation of scientific literature (s1 table) . the tpms technology employed has been previously described [12] and applied in different clinical areas with different objectives [8, 9, [13] [14] [15] [16] [17] [18] . tpms uses a human biological network that incorporates the available relationships (edges or links) between proteins (nodes) from a regularly updated in-house database drawn from public sources: kegg [28], reactome [29] , intact [30] , biogrid [31] , hprd [32] , and trrust [33] . drug targets and indications were obtained from drugbank [34] . the molecular description of the indications was obtained from a hand-curated collection of associations between biological processes and molecular effectors (defined as bed, biological effectors database, from anaxomics biotech). the method uses an artificial neural network (ann) to measure the potential relationship between the nodes of a network (i.e. proteins), grouped according to their association with a phenotype. the ann algorithm provides a score (from 0 to 100%). each score is associated with a probability (p-value) that the protein or group of proteins being evaluated, drug targets with molecular description of pathological processes, are functionally associated. scores greater than 91% indicate a very strong relationship with a p-value below 0.01; scores between 76-91% have p-value between 0.01-0.05; scores between 40-76% have medium-strong relationship and p-value in the range 0.05-0.25; and a scores lower than 40% have p-values above 0.25. the mechanism of action (moa) of pirfenidone and melatonin has been simulated by using the tpms technology. on the basis of the human protein functional network described above, mathematical models have been built. the models simulate the activation/inhibition status in the human protein network. as input, the model takes the activation (+1) or inactivation (-1) of the drug target proteins (s2 table) , and as output the protein states of the pathology of interest (s1 table) . it then optimizes the paths between the two protein sets and computes the activation and inactivation values of the full human interactome. the resulting subnetwork of proteins with positive and negative values defines the moa of the drug. sampling methods are used to generate mathematical models with stratified ensembles that comply with a set of validated benchmarks [12] . guildify v2.0 web server to calculate the effect of the covid-19 sets of proteins and repurposed drugs to the network guildify v2.0 [19] web server is used to extend the information of sets of proteins through the human biological network. guildify scores proteins according to their proximity with the genes associated with a drug or phenotype (seeds). in this study, guildify has been employed to calculate the neighbourhoods of the human biological network that are associated with the host-key points (for sars-cov infection) and at the same time affected by specific drugs. in this way, the mechanism of action of the treatments proposed by tpms can be corroborated using a completely different setting. guildify v2.0 has been run for each drug, using their targets as seeds (by introducing the name of the drug in the search box). the same has been done for each set of proteins: the coronavirus-host interaction set, the entry points set (a subset of the coronavirus-host interaction set containing only 5 host proteins relevant in the infection of sars-cov-2), the lung cells infection set and the ard set (by uploading a file with the gene symbols of the proteins). we included the entry points subset in the analysis to have a more specific neighborhood of the sars-cov-2 infection mechanism. there are two additional parameters to be chosen by the user in guildify web server: the human biological network and the scoring function. the human biological network used has been the protein-protein interactions network of i2d [35], as it is the more complete network in the web server (with 13,568 proteins and 224,675 interactions). the scoring function selected (used to score the proteins of the network according to the proximity with the seeds) has been netcombo [36], an algorithm that combines the performance of three network-based prioritization algorithms: netshort (considers the number of edges connected to phenotype-associated nodes), netzcore (iteratively assesses the relevance of a node for a given phenotype by averaging the normalized scores of the neighbors) and net-score (considers the multiple shortest paths from the source of information to the target). the top 1% scored proteins of each test have been selected to proceed with the analysis of each subnetwork. the overlap between the selected subnetworks has been used to calculate the number of common proteins between the top-scoring proteins associated with the drugs and the topscoring proteins associated to the host-key points (results detailed in s3 table) . human key molecular enclaves in covid-19 that could be good target areas for treatment have been identified through manual curation of scientific publications and gene expression analysis, describing molecularly aspects of the virus infection process from the host perspective and aspects related with the respiratory problems associated to the infection. we have defined three different main protein sets: 1) coronavirus-host interaction set, 2) lung cells infection set and 3) ard set. 1) 'coronavirus-host interaction set' is composed of human proteins with a relevant role in sars-cov-2 infection process. this set of proteins has been obtained from a bibliographic revision and it is composed of proteins that potentially interact with sarscov . in addition, a list of proteins reported to have a direct interaction with coronaviruses, retrieved from zhou et al. [6] has been included. in this study, the authors define a human coronavirus (hcov)-host interactome network including 119 host proteins with the aim of identifying antiviral drugs through repurposing strategies. it integrates known human direct targets of hcov proteins or that are involved in crucial pathways of hcov infection using the available information from four known hcovs (sars-cov, mers-cov, hcov-229e, and hcov-nl63), one mouse cov (mhv), and one avian cov (ibv, n protein). the final set contains 122 human proteins, the full list is provided in s1 table. 2) 'lung cells infection set' describes the transcriptional response of human lung epithelial cells to sars-cov-2 infection. it has been defined through differential expression analysis of human lung epithelial cells to sars-cov-2 infection, geo series reference gse147507. this set includes 47 proteins, the full list is provided in s1 table. 3) 'ard set' is composed of 412 unique protein entries defined by 6 subsets. it includes gene differential expression data on alveolar macrophages, monocytes and neutrophils in ard (gse89953 and gse76293), gene differential expression data on lung changes induced in the intermediate and late stages of the pathophysiology (gse18712) and the key players in ard cytokine storm extracted from literature research. all ard set data can be retrieved from s1 table. we have centred the drug repositioning analysis in the coronavirus-host interaction set. but the potential effects of the drug combination selected have been evaluated for the 3 data sets defined. an overall depiction of the whole procedure is depicted in fig 1. tpms-ann approach has been used to identify drugs that can be repurposed for set 1. the potential moa for these repurposed drugs has been identified by guiildify and tpms approaches, as well as moas of this repurposed drug candidates for sets 2 and 3. a systems biology based strategy has been used to screen the predicted relationship between 6,605 different drugs, as defined in drugbank [34] , and the coronavirus-host interaction set previously defined. drug candidates for repurposing were sorted and selected by the probability of its relationship with the coronavirus-host interaction set, 12 approved drugs scored higher than 74% (p-value <0.05). all candidates were grouped in two drug categories: (1) anti-inflammatory and immunosuppressant agents (ibuprofen, phosphatidyl serine, prasterone, vitamin c, pirfenidone, tacrolimus and pimecrolimus); (2) cardiovascular agents (isosorbide, icatibant, moexipril, irbesartan, phenindione). among them, 4 out of 12, are currently being tested in covid-19 clinical trials, these are: vitamin c (nct04264533, nct04323514, nct04323514), pirfenidone (nct04282902), tacrolimus (nct04341038) and irbesartan (nct04330300). two others appear described as potential treatments by third authors, moexipril [41] and icatibant [42], or somehow related to the disease outcome, such as ibuprofen [43] . among these candidates, pirfenidone (with score 75%) was selected because of its reported association with furin [39] and a good safety profile. the s protein of sars-cov-2 has a furinlike domain for cleavage and some recent works have addressed the potential implication of this domain in the s protein maturation and, as a consequence, in the host cell entry mechanism of the virus. furin, a member of the subtilisin-like proprotein convertase family processes proteins of the secretory pathway, is a type 1 membrane-bound protease that is expressed in multiple organs, including the lungs [44]. sars-cov-2, differently from sars-cov, presents a furin cleavage site between the s1/s2 subunits of the s glycoprotein, which is cleaved during the biogenesis of the virus to create a mature s protein [45] . inhibiting the expression of furin could then be a possible approach to diminish sars-cov-2 infectivity by making difficult s protein maturation [46] . in recent years, furin has emerged as a promising target for therapeutic intervention in a variety of infectious diseases as influenza a virus [47] or newcastle disease virus [48] . given this evidence, pirfenidone stands out as one of the most interesting repurposing candidates to treat covid-19. taking into consideration the drug repurposing analysis and the supporting evidence we have selected pirfenidone for further studies. combining different drugs can be an advantageous option as we can affect a larger number of key points of intervention and/or increase the effects against disease pathophysiology. we have applied ann of tpms technology to screen drug combinations composed by pirfenidone and other approved drugs listed in drugbank database. among all, 214 non-synonymous repurposed drug candidates have shown a predicted relationship value � 90%. experimental drugs, withdrawn drugs and those with poor safety profile or with opposite mechanistic effect than the desired one were further filtered out. lastly, we selected the combination of pirfenidone (predicted value 92% p>0.05) with melatonin as it fulfilled all criteria. furthermore, there is also mounting evidence that melatonin could be a good candidate to treat covid-19, due to its anti-inflammatory and anti-oxidative properties protecting against ards caused by other pathogens including viruses [6, [49] [50] [51] . to further characterize at a molecular level the action of the selected drug combination on the coronavirus-host interaction set, we have used the modelling strategy based in sampling methods of tpms. the models predict the most probable molecular routes that transmit the effect from the drugs' molecular targets to proteins in coronavirus-host interaction set. the main protein interactors identified are depicted in fig 2. pirfenidone inhibits furin, a protein effector included in the virus-host network, with a potential implication for the entry of the virus in the host cell. the inhibition of furin also results in the inhibition of transforming growth factor-beta1 (tgf-β1) [52] a gatekeeper of the immune response and one of the key proteins in set 1, as it is upregulated by the papain-like protease (plpro) from severe acute respiratory syndrome (sars) coronavirus (sars-cov) [53] . the inhibition of furin (a tgf-β1 converting enzyme) by pirfenidone reduces its expression [52, 54] . on the other hand, melatonin, through the suppression of estrogen receptor alpha [55] regulates the expression of ubc9 [56] , another key protein of set 1 that interacts with sars-cov n protein [57] . it has also been described that melatonin can lead to the activation of stat3 in some cell types [58, 59] , and it could be useful to counteract the stat3 dephosphorylation induced by sars-cov [60] . in the previous section, the drug combination pirfenidone and melatonin was selected as a potential treatment against the infection of sars-cov-2. the selection is fundamentally based on the mechanism of action of the combination, targeting the entry mechanism of the virus and specially furin, a protein that may have a key role in the infection process [39] . to corroborate the effect of pirfenidone and melatonin to the proteins that are relevant for the infection, we have used an alternative approach also based on the use of networks. we used guildify v2.0 [19] , a web server that extends the information of sets of proteins through the human biological network. guildify scores proteins according to their proximity with the genes associated with a drug or phenotype (seeds). using this web server, we can identify a list of topscoring proteins that are critical on transmitting the perturbation of certain proteins through the network (also known as network propagation). thus, applied to this case, we: (i) identify the proteins of the human biological network that are key on transmitting the therapeutic effect of pirfenidone and melatonin, (ii) identify the proteins of the network perturbed by the infection of the sars-cov-2, and (iii) calculate the network overlaps between the treatment and the infection, reflecting the potential effect of the treatment over the infection. the network used by guildify, obtained from i2d [35], is completely independent from the network used in the tpms, becoming an ideal, independent context to test the results of tpms. we have observed a significant overlap of 32 proteins between the top-scoring proteins achieved with the targets of pirfenidone (pirfenidone-network) and the top-scoring proteins of the sars-cov-2 infection cycle set (a subset of the coronavirus-interaction set specific for sars-cov-2, conformed by tmprss2, ace2, grp78, furin and cd147). additionally, there are 10 common proteins between the top-scoring proteins achieved with the targets of melatonin (melatonin-network) and the top-scoring proteins of the 5 entry point proteins. there are also 7 common proteins between the top-scoring proteins of pirfenidone and the top-scoring proteins of ard set. however, the rest of the sets (coronavirus-host interaction set and lung infection set) do not have a significant overlap with any of the two drugs (results detailed in s3 table) . the highly significant overlap between pirfenidone and the top-scoring proteins of the 5 entry point proteins of sars-cov-2 is however biased, because furin is in this set and it's also a target of pirfenidone (fig 3) . the therapeutic effect of pirfenidone on furin has a clear source effect on the neighbourhood of proteins that surround the entry points of sars-cov-2. melatonin has also a significant overlap with the entry points of sars-cov-2. this effect is explained because it targets the proteins mtnr1a and mtnr1b (melatonin receptors). melatonin affects sars-cov-2 grp-78 entry point (also called hspa5) because mtnr1a and mtnr1b are direct interactors of grp-78 (fig 3) . melatonin also affects sars-cov-2 furin entry point because mtnr1b interacts with the membrane protein itm2c, which directly interacts with furin. by identifying the interacting partners of mtnr1a and mtnr1b, we can understand better the potential mechanism of action of melatonin towards the entry of the virus. apparently, both pirfenidone and melatonin target close subnetworks but in different areas. this could explain the interesting additive effect predicted by tpms. as mentioned above, the overlap between pirfenidone-network and the sars-cov-2 entry points is biased by the fact that both sets of proteins contain furin. therefore, we have repeated the same analysis but removing furin from the set of sars-cov-2 entry points. here, we have observed that the overlap with pirfenidone is not significant, with only 4 common proteins, which proves that pirfenidone targets the sars-cov-2 network specifically by targeting furin. in contrast, the melatonin-network has a significant overlap of 7 proteins, affecting mainly neighbours of grp-78. guildify also reflects the effect of pirfenidone over 7 proteins related with ard, leaded by other targets of the drug: tnf-alpha and mapk13. according to the findings by guildify, we confirm the effect of the combination of pirfenidone and melatonin in the entry points of the sars-cov-2 infection, specifically the neighbours of furin and grp-78, and some proteins associated with ard. to further evaluate the impact of pirfenidone and melatonin as a novel therapy for covid-19, the relationship of the drug combination against the associated respiratory problems has been evaluated using various systems biology approaches. in a first approach, the relationship between the protein drug targets and the 2 sets of proteins defining respiratory problems associated with covid-19 (sets 2 and 3) has been evaluated using the tpms ann modelling approach. this type of measurement provides a probability of a relationship between groups of proteins. a weak relationship has been obtained for the combination with lung cells infection set while we obtained a strong relationship between the drug combination and ard set. specifically, with the subsets related to alveolar macrophages, monocytes, late phase ard and ard cytokine storm. a second approach using the sampling methods-based models of tpms has been used to evaluate at the molecular level the moa of pirfenidone and melatonin individually and in combination for treating ard set, evaluating each subset individually. this modelling approach identifies a probable molecular pathway between the protein drug targets and the ard subsets. the ability of each treatment to reverse the protein alterations occurring in these pathological mechanisms has been assessed, i.e. the ability of each drug to activate proteins that are inhibited in ard subnetworks or vice versa (according to the molecular characterization). we have calculated the percentage of proteins theoretically affected respect to the total number defining the subset (fig 4) . table. the main effect, for both drugs, is over the cytokine storm subset. pirfenidone and melatonin are able to affect 86% of the proteins defining the set individually. the drug combination increases the % of proteins affected to a 91%, providing a potential synergy in this area. the protein-network overlap indicates that most of the proteins (82%) in ard cytokine storm subset are modulated by both drugs. while the proteins modulated by one drug alone are 5% in both cases. the second subset with largest % of affected proteins, 27% for the drug combination, is alveolar macrophages set. the main effect is registered for melatonin, pirfenidone contribution is negligible for this subset. we measure also a minor modulation for the rest of ard subsets, mostly contributed by melatonin with pirfenidone slightly potentiating the effect in some of them. the anti-inflammatory role of both melatonin and pirfenidone has been described in the literature. they are able to attenuate nlrp3 inflammasome and il-1β [61] [62] [63] , being a possible starting point for the cytokine storm. studies have shown the potential of melatonin in decreasing the levels of inflammatory cytokines by reducing the activation of nf-κb, thus becoming an interesting candidate to treat different types of viral infections [49, 64, 65] . other studies have also shown the anti-inflammatory role of pirfenidone, modulating inflammatory cytokines [66, 67] . the anti-inflammatory effect of both treatments could potentially be useful to reduce the effects caused by the cytokine storm. pirfenidone has also been described as antifibrotic, reducing the rate of lung damage by 50% in patients with idiopathic pulmonary fibrosis [68] . antifibrotic therapy has been highlighted as a potential strategy to treat covid-19 patients and prevent fibrosis after sars-cov-2 infection [69] . in addition, both pirfenidone [70, 71] and melatonin [72, 73] have been reported to provide antioxidant effect, which has an important role in ard pathophysiology. multiple studies have reported reduced levels of antioxidant agents in ard patients [74, 75] , and remark the positive effects of therapies that include antioxidants in the treatment of ard [76, 77] . therefore, the antioxidant effect of both drugs could be one of the factors that explains the positive effect in ard predicted by tpms. the proposed drug combination has also a relevant effect modulating the expression of proteins associated to the phenotype of alveolar macrophages in ard. it has been suggested that these cells are key players in the pathophysiology of ard. they seem to have a proinflammatory role in early stage and anti-inflammatory effect at the late stage [78] . the combined mechanism of action (moa) of pirfenidone and melatonin in ard have been identified at molecular level through the use of sampling methods-based models [12] . tpms sampling-based methods trace the most probable paths, both in biological and mathematical terms, which lead from a stimulus (e.g. drug) to a response (e.g. disease) through the biological human protein network. in other words, tpms identifies the set of possible moas that achieve a physiological response when the system is stimulated with a specific stimulus. there is a certain variability in these moas, generating several possible pathways that represent patient heterogeneity. however, when identifying a graphical representation of the moa, only the mean and most represented paths among the set of possible solutions is represented. the moa of the combination of pirfenidone and melatonin in ard has been studied. as shown in fig 5, and as previously predicted through other strategies, the combination of both drugs counteracts the cytokine storm, which is responsible of the disease severity. melatonin and pirfenidone may reduce the levels of proinflammatory chemokines and cytokines that have been detected at high levels in patients with covid-19 [79] and which are well known for their role in the cytokine storm and contribution to ard: il-1b, il-6, cxcl10, il-8, ccl5 and tnf-alpha [79] [80] [81] [82] . these molecules are involved in both, early and late phases of cytokine storm. these effects have already been previously described in the literature for both drugs individually [49, 66, [83] [84] [85] [86] reinforcing the potential use of the drug combination to improve patient outcomes. this drug combination could modulate tgf-β1 and vegfa involved in processes associated to lung fibrosis, a late complication of ard. tgf-β1 is a profibrotic mediator known to be associated with the fibroproliferative response responsible of this disease complication [87] . pirfenidone inhibits furin, one of the proprotein convertases that mediate the maturation/activation of tgf-β1 [88] . on the other hand, it has been described that melatonin supress tgfβ1 expression through the inhibition of erk phosphorylation [89, 90] supressing pulmonary fibrotic response. one key component of the infrastructure in the lung that is damaged in ard is the vasculature. vegfa is involved in the excessive angiogenic response that may contribute to the initial tissue injury and drive the fibroproliferative response [87] . according to our model, melatonin could be downregulating vegfa expression through the inhibition of oestrogen receptor alpha (er1). it has been described that melatonin inhibits er transactivation [55] and that vegf expression in some cell types is regulated by 17β-oestradiol in a er dependent process [91] . there are some experimental evidences suggesting the inhibitory role of melatonin on vegf activity reinforcing the hypothesis of our model, although the pathway involved on this effect has not been described [92] [93] [94] . in summary, we predict that this drug combination could modulate the vast majority of effectors involved in the cytokine storm. furthermore, as detailed in fig 4, the drugs also act on other pathophysiological subnetworks of ard. it has also been described in the literature the anti-oxidant effect of both, pirfenidone and melatonin [70, 72, 95] . as reactive oxygen species (ros) play a central role in inflammatory responses and viral replication [96] , such antioxidant effect could also be beneficial for improving the pathophysiology of ard patients. the current situation with covid-19 prompts for easy and fast ways to control and reduce the burthen of the disease. the systems biology-based drug repurposing screening method has proved to be a good option, identifying most of the treatments already in clinical evaluation and highlighting others that may have not got sufficient attention on their potential. our analysis has highlighted pirfenidone as one of the best candidates for treating the sars-cov-2 host cell entry and melatonin as combination theraphy to increase the measured effect. the identification of the potential moa for this drug combination against the coronavirushost interaction set indicates that the principal effect is registered by the inhibition of furin by pirfenidone and the suppression of estrogen receptor alpha by melatonin. the effect of the two drugs in the key proteins surrounding the sars-cov-2 entry mechanism, furin and grp-78, has been corroborated with the web server guildify v2.0. we have also measured a potential effect of the drug combination over ard set. screening in a deeper detail the potential moa we have measured the strongest modulation over cytokine storm subset of ard with pirfenidone and melatonin contributing in a similar manner. the other ard subsets are not so strongly modulated and most of the effect comes from melatonin. several authors have suggested both compounds individually to treat covid-19 or in combination with other compounds. pirfenidone is already in clinical trials (nct04282902) to treat covid-19 associated lung injury. the fact that our study has identified also an effect on sars-cov-2 infection cycle and thus possibly contributing to reduce the viral load makes this drug combination very attractive in the treatment of covid-19. from a medical point of view both drugs are considered safe and can be combined with the current standard of care treatments for covid-19. the pharmacokinetics of the combination therapy can be altered respect to the individual drugs due to their competition (substratesubstrate) over cyp1a2. this substrate competition can lead to the drugs remaining longer in the organism than in monotherapy, potentially increasing the known pharmacologic effects of the drugs that in turn could lead to dose-dependent adverse drug reactions. the design of any clinical trial to test this combination therapy has to take into consideration this possibility and compensate by reducing the dosage of the drugs compared to their regular use in monotherapy. this drug combination is foreseen to tackle two important issues in covid-19, the viral load and the pulmonary associated complications making it very appropriate for treating patients' at stage ii [97] , this are non-severe symptomatic patients with the presence of virus and at risk of evolving to severe pulmonary complications. a clinical study to evaluate this drug combination is foreseen. supporting information s1 table. characterization of the different sets of proteins associated to covid-19. 1) coronavirus-host interaction set (including sars-cov-2 entry points), 2) lung-cells infection set, and 3) acute respiratory distress (ard) set that is composed of 6 subsets (alveolar macrophages, monocytes, neutrophils, intermediate phase ard, late phase ard and ard cytokine storm). proteins are listed by their uniprotkb identifier and under effect the type of action of the protein is described: 1 (being the protein more active contributes to the pathological effect of the motive) 0 (being the protein less active contributes to the pathological effect of the motive). coronavirus incubation could be as long as 27 days, chinese provincial government says centers for disease control and prevention (cdc). symptoms of coronavirus disease 2019 centers for disease control and prevention (cdc) clinical trials on drug repositioning for covid-19 treatment hopes rise for coronavirus drug remdesivir a sars-cov-2 protein interaction map reveals targets for drug repurposing network-based drug repurposing for novel coronavirus 2019-ncov/sars-cov-2 the imex coronavirus interactome: an evolving map of coronaviridae-host molecular interactions novel neuroprotective multicomponent therapy for amyotrophic lateral sclerosis designed by networked systems neuroprotective drug for nerve trauma revealed using artificial intelligence repurposed analogue of glp-1 ameliorates hyperglycaemia in type 1 diabetic mice through pancreatic cell reprogramming proximal pathway enrichment analysis for targeting comorbid diseases via network endopharmacology in-silico simulated prototypepatients using tpms technology to study a potential adverse effect of sacubitril and valsartan unveiling the role of network and systems biology in drug discovery proteome-wide alterations on adipose tissue from obese patients as age-, diabetes-and gender-specific hallmarks sardó n t. systems biology applied to non-alcoholic fatty liver disease (nafld): treatment selection based on the mechanism of action of nutraceuticals mechanisms of action of sacubitril/valsartan on cardiac remodeling: a systems biology approach network medicine framework for identifying drug repurposing opportunities for covid-19 melatonin as a potential compound against sars-cov-2 evidence that furin is an authentic transforming growth factor-β1-converting enzyme sars coronavirus papain-like protease induces egr-1-dependent up-regulation of tgf-β1 via ros/p38 mapk/stat3 pathway processing of transforming growth factor beta 1 precursor by human furin convertase melatonin inhibits estrogen receptor transactivation and camp levels in breast cancer cells estrogen receptor alpha and nuclear factor y coordinately regulate the transcription of the sumo-conjugating ubc9 gene in mcf-7 breast cancer cells sumoylation of the nucleocapsid protein of severe acute respiratory syndrome coronavirus by interaction with ubc9 interleukin-6 autocrine signaling mediates melatonin mt(1/2) receptor-induced stat3 tyr(705) phosphorylation melatonin protects against ischemic stroke by modulating microglia/macrophage polarization toward anti-inflammatory phenotype through stat3 pathway the complex role of stat3 in viral infections melatonin attenuates airway inflammation via sirt1 dependent inhibition of nlrp3 inflammasome and il-1β in rats with copd pirfenidone ameliorates lipopolysaccharide-induced pulmonary inflammation and fibrosis by blocking nlrp3 inflammasome activation an open-label study with pirfenidone on chronic hypersensitivity pneumonitis melatonin possesses an anti-influenza potential through its immune modulatory effect melatonin: its possible role in the management of viral infections-a brief review antifibrotic activities of pirfenidone in animal models anti-inflammatory effect of pirfenidone in the bleomycin-hamster model of lung inflammation a phase 3 trial of pirfenidone in patients with idiopathic pulmonary fibrosis pulmonary fibrosis and covid-19: the potential role for antifibrotic therapy antioxidant activity mediates pirfenidone antifibrotic effects in human pulmonary vascular smooth muscle cells exposed to sera of idiopathic pulmonary fibrosis patients potent antioxidant role of pirfenidone in experimental cirrhosis melatonin as an antioxidant: biochemical mechanisms and pathophysiological implications in humans melatonin as an antioxidant: under promises but over delivers antioxidant status in patients with acute respiratory distress syndrome alveolar antioxidant status in patients with acute respiratory distress syndrome effect of enteral feeding with eicosapentaenoic acid, γ-linolenic acid, and antioxidants in patients with acute respiratory distress syndrome enteral nutrition with eicosapentaenoic acid, γ-linolenic acid, and antioxidants reduces alveolar inflammatory mediators and protein influx in patients with acute respiratory distress syndrome the role of macrophages in the pathogenesis of ali/ards. mediators inflamm cytokine storm in covid-19 and treatment the immunobiology of sars into the eye of the cytokine storm pathogenesis of covid-19 from a cell biologic perspective. the european respiratory journal. nlm (medline) proprotein convertase furin is preferentially expressed in t helper 1 cells and regulates interferon gamma furin-mediated protein processing in infectious diseases and cancer furin inhibition reduces vascular remodeling and atherosclerotic lesion progression in mice effect of pirfenidone (pfd) on cytokine/chemokine release from alveolar macrophages (ams) in interstitial lung diseases (ild): preliminary results the fibroproliferative response in acute respiratory distress syndrome: mechanisms and clinical significance regulation of tgfβ and related signals by precursor processing. seminars in cell and developmental biology beneficial effects of melatonin on nicotine-induced vasculopathy melatonin suppresses fibrotic responses induced by cigarette smoke via downregulation of tgf-β1 regulation of vascular endothelial growth factor (vegf) gene transcription by estrogen receptors α and β melatonin restricts the viability and angiogenesis of vascular endothelial cells by suppressing hif-1α/ros/vegf regulation of vascular endothelial growth factor by melatonin in human breast cancer cells melatonin modulates the expression of vegf and hif-1α induced by cocl2 in cultured cancer cells melatonin as a master regulator of cell death and inflammation: molecular mechanisms and clinical implications for newborn care. cell death and disease the cytokine storm of severe influenza and development of immunomodulatory therapy. cellular and molecular immunology covid-19 infection: the perspectives on immune responses. cell death and differentiation. springer nature; 2020 the authors acknowledge dr. pablo coto from the hospital vital alvarez-buylla (asturias, spain) for his collaboration about details about the covid-19 infection and specially the sars molecular characterization, and dr. esther ramírez for her contribution in the clinical trial design. key: cord-340656-ltd6ueoi authors: grant, michael c.; geoghegan, luke; arbyn, marc; mohammed, zakaria; mcguinness, luke; clarke, emily l.; wade, ryckie g. title: the prevalence of symptoms in 24,410 adults infected by the novel coronavirus (sars-cov-2; covid-19): a systematic review and meta-analysis of 148 studies from 9 countries date: 2020-06-23 journal: plos one doi: 10.1371/journal.pone.0234765 sha: doc_id: 340656 cord_uid: ltd6ueoi background: to limit the spread of sars-cov-2, an evidence-based understanding of the symptoms is critical to inform guidelines for quarantining and testing. the most common features are purported to be fever and a new persistent cough, although the global prevalence of these symptoms remains unclear. the aim of this systematic review is to determine the prevalence of symptoms associated with covid-19 worldwide. methods: we searched pubmed, embase, cinahl, amed, medrxiv and biorxiv on 5(th) april 2020 for studies of adults (>16 years) with laboratory test confirmed covid-19. no language or publication status restrictions were applied. data were independently extracted by two review authors into standardised forms. all datapoints were independently checked by three other review authors. a random-effects model for pooling of binomial data was applied to estimate the prevalence of symptoms, subgrouping estimates by country. i(2) was used to assess inter-study heterogeneity. results: of 851 unique citations, 148 articles were included which comprised 24,410 adults with confirmed covid-19 from 9 countries. the most prevalent symptoms were fever (78% [95% ci 75%-81%]; 138 studies, 21,701 patients; i(2) 94%), a cough (57% [95% ci 54%-60%]; 138 studies, 21,682 patients; i(2) 94%) and fatigue (31% [95% ci 27%-35%]; 78 studies, 13,385 patients; i(2) 95%). overall, 19% of hospitalised patients required non-invasive ventilation (44 studies, 6,513 patients), 17% required intensive care (33 studies, 7504 patients), 9% required invasive ventilation (45 studies, 6933 patients) and 2% required extra-corporeal membrane oxygenation (12 studies, 1,486 patients). the mortality rate was 7% (73 studies, 10,402 patients). conclusions: we confirm that fever and cough are the most prevalent symptoms of adults infected by sars-cov-2. however, there is a large proportion of infected adults which symptoms-alone do not identify. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the novel coronavirus (sars-cov-2; 2019-ncov; covid-19) pandemic is a global crisis. as of april 10 th 2020, there were over 1.5 million confirmed cases of whom over 92,000 have died [1] . in the absence of a vaccine or treatment with proven efficacy, limiting human-to-human transmission is critical [2-4]. self-isolation (or self-quarantine) is an effective global strategy for limiting transmission following the emergence of symptoms [5] and equally, the manifestation of symptoms is used to guide testing. coronavirus is most infectious in the early phase of the illness [6, 7] [8], so screening people with compatible symptoms [9] is fundamental to determining who should be quarantined and be tested [9] . several systematic reviews have considered the symptoms of covid-19 (amongst other parameters) [8,10-14] although all of them have major limitations. none systemically searched the grey literature (e.g. preprint archives such as medrxiv and biorxiv) and in the context of a pandemic, the quality and quantity of the literature is evolving at speed [15] . without incorporating all relevant preprints the findings of any systemic review will be weeksmonths out-of-date at the time of publication [16] . furthermore, with few included studies (30 in the largest and most recent [12] ), the range of symptoms were limited and the estimates of prevalence are likely to be upwardly biased because only unwell patients (largely those admitted to hospital) were tested in the early phase of the outbreak. to facilitate the rapid dissemination of high-quality open-science, there has been a surge of preprints related to covid-19 manuscripts uploaded to the online archives medrxiv and biorxiv [15] . the necessity to address deficiencies in current literature and potential to substantially improve the precision of estimates of symptom prevalence using both indexed and (the more voluminous and up-to-date) preprint literature from multiple geographical regions, represents the rational for this review. the aim of this systematic review is to determine the prevalence of symptoms associated with covid-19 worldwide. derived from oro-or naso-pharyngeal swabs. we excluded case reports, articles which failed to disaggregate symptoms in adult and paediatric cohorts, studies of patients with prior respiratory infections (e.g. tuberculosis) or co-infections with other viruses (e.g. similar viruses sars-cov-1 or hcov-emc/2012, etc) and articles which we are unable to translate to english in a timely fashion. the incubation period of covid-19 is typically 5 days, but reported to last a maximum of 7 days [20] [21] [22] [23] [24] . the illness typically lasts 8 days [21] . therefore, we will include any symptom(s) described up to 15 days before laboratory confirmed infection and during the illness. pubmed, embase, amed and cinahl, medrxiv [25] and biorxiv were interrogated according to our search strategy (s1 appendix). searches were limited to 1 st january onwards. no language restrictions were applied. after de-duplication, all unique citations were independently screened by three review authors (mg, lg and rgw). the full texts of all potentially relevant articles were obtained. the reference lists for included articles and other systematic reviews were also scrutinised. final lists of included articles were compared and disagreements resolved by consensus discussion between five authors (mg, lg, zk, elc and rgw). two authors (mg and lg) independently extracted data and three authors (zm, elc and rgw) checked the accuracy of the extracted data using a standardised spreadsheet. disagreements were resolved by discussion. we combined the following symptoms: "chest tightness" into the more prevalent symptom of wheeze; "shivers" and "chills" into rigors; malaise and "generalised weakness" (in the absence of any objective neurological deficit) into the more widely reported symptom of fatigue; conjunctivitis, conjunctival congestion and conjunctivital secretions into conjunctivitis. where studies reported one symptom "or" another (e.g. nausea or vomiting) we did not extract this information as it was impossible to disaggregate. where studies reported one symptom "and" another (e.g. nausea and vomiting) we extracted the prevalence of both. when studies grouped symptoms together (e.g. "respiratory symptoms") without further description or definition we were not able to extract this information. the risk of bias for included studies was not assessed for two main reasons: firstly, there is no consensus on ideal tool, nor one designed specifically for studies of prevelance [26] and secondly, such assessments would not change the approach to the modelling or presentation of the data, as per our protocol. given such assessments are also time-intensive, we have taken the pragmatic decision to not perform risk of bias assessments. of the prevalence was used to normalise variance. 95% confidence intervals (cis) were computed around the study-specific and pooled prevalence based on the score-test statistic. [28] . the variation in prevalence by country was assessed by subgroup meta-analyses and metaregression. statistical heterogeneity is assessed by i 2 which corresponds with the proportion of total variation due to inter-study heterogeneity and by p-values for inter-study heterogeneity within countries, between countries and overall [29] . given the use of a random-effects model, inter-study heterogeneity within countries was only assessable when at least three studies were available. a z-test (and the corresponding p-values) assessed whether the observed prevalence was different from zero percent. publication bias was not assessed. our search returned 2403 hits in pubmed, 2234 in embase, 310 in cinahl, 1 in amed, and 434 preprints in medrxiv and biorxiv on 5 th april 2020. following deduplication, the titles and abstracts of 851 unique records were assessed against the inclusion criteria. 743 of these were deemed to be potentially eligble. full text screening then resulted in 148 included articles (fig 1) . [166, 167] , the usa [168, 169] , singapore [170, 171] , italy [172, 173] , australia [174] , japan [175] , korea [176] and the netherlands [177] . the mean age of patients was 49 years (sd 11) and where sex data were available, the ratio of males:females was 1.2:1 (10,306:8593). the characteristics of the included studies are shown in s1 table. thirty-four studies reported that 845 of 7519 patients required non-invasive ventilation (pooled prevalence 17% [95% ci 11%-24%]; i 2 98%). forty-four studies (6513 patients) reported that 970 patients were admitted to an intensive care unit (pooled prevalence 19% [95% ci 13%-26%]; i 2 97%). forty-five studies (6933 patients) reported that 495 required invasive mechanical ventilation (pooled prevalence 9% [95% ci 6%-13%]; i 2 95%). twelve studies (1486 patients) reported that 2% of patients (36) required extra-corporeal membrane oxygenation (95% ci 0%-5%; i 2 95%). of the 73 studies that reported survival in 10,402 patients, there were 938 deaths (pooled prevalence 7% [95% ci 4%-11%]; i 2 98%) which were attributable to covid-19. table 1 shows the meta-analysed prevalence of symptoms, group by bodily system, and s2 table shows the meta-analytical prevalence estimates from studies grouped by geographical region. the most prevalent symptom in patients with laboratory confirmed covid-19 was a fever, experienced by 78% of patients (99% ci 75%-81%; fig 2 and s2-s5 figs) . whilst, there was substantial heterogeneity between countries (i 2 94%) with estimates ranging from 83% in singapore (99% ci 61%-98%) to 32% in korea (99% ci 15%-51%), there was no evidence of a statistically significant difference between countries (s2 table) . a cough was the second most prevalent symptom, reported by 57% of test-positive patients (95% ci 54%-60%; fig 3 and s6 -s10 figs). whilst there was substantial heterogeneity between countries (i 2 94%) with estimates ranging from 18% in korea (99% ci 8%-36%) to 76% in the netherlands (95% ci 66%-83%), there was no evidence of a statistically significant difference. this review describes 24,410 adults with laboratory test confirmed covid-19 from 9 countries. we confirm that the purported cardinal symptoms of fever and a new persistent cough are indeed the most prevalent symptoms of covid-19 worldwide. however, we also show that at approximately 1 in 5 test-positive adults were never febrile and fewer than 3 in 5 developed a cough. since the patients in the included studies are likely to have moderate-severe disease warranting hospitalisation and thus testing, it is likely that we over-estimate the true prevalence of symptoms in the population. consequently, the use of symptoms alone to screening adults for sars-cov-2 infection is likely to miss a substantial number of infected individuals. our point estimates of the prevalence of fever (78%) and cough (57%) are approximately 10% lower than the estimates from prior reviews [8,10-13] which we feel might explained by two specific factors. firstly, prior reviews [8,10-13] did not systematically search (or search at all [8,10,11,13]) for preprints uploaded to online repositories such as medrxiv or biorxiv [15], both of which have seen a surge in uploads related to the covid-19 pandemic [15] . this . therefore, it is likely that the prior reviews [8,10-13] had a higher proportion of adults with more severe disease (given that testing was limited to those admitted to hospital in the early phase of the outbreak) whereas more recent studies are likely to include adults with mild symptoms due to the wider availability of testing alongside natural progression of the disease. conversely, more-recent studies of realtime population-wide tracking of self-reported symptoms in subsequently test-positive patients are essentially identical to our point estimates for cough [179] ; however, data concerning fever [180] are less concordant but given the variability of core body temperature, methods of measurement and the definition of this symptom, variability is expected. we acknowledge that there is both within-country and between-country differences in the estimated prevalence of different symptoms, which presents issues with regards to generalising the findings. however, the unique strength of a meta-analysis is the ability to compare datasets from difference sources, identify patterns and discrepancies [181] , and no statistical technique is able to correct for weaknesses or idiosyncrasies of the original data. differences in the study designs, settings and what types of patients (mild, moderate or critically unwell) were sampled are all likely to be responsible for the observed heterogeneity. the sampling strategy is known to bias the prevalence of conditions and ideally, prevalence studies recruit a (non-probabilistic) consecutive sample because they are more likely to represent the target population. in comparison, convenience sampling (i.e. those with available data) and purposive sampling (e.g. reports of individuals with specific clinical features) which are common in the included studies, introduce selection bias and tend to upwardly bias estimates of symptom prevalence. equally, enrolling patients from hospital settings rather than the community is more likely to upwardly bias the estimates of prevalence. overall, we suspect that our results over-estimate the true prevalence of symptoms amongst test-positive adults. in some instances, it was impossible to ascertain whether different publications which originated from the same hospital or region included (some of) the same subjects because the recruitment timeframes and sampling strategies were not sufficiently described in the study methods. we recommend that future publications detail (where possible) if their sample is also represented in other works and describe their methods in accordance with relevant reporting guidelines. the way in which we extracted some of the data might bias the findings. we dichotomised fever (based on the definition in the parent study) and thresholds differed study-to-study (between 37˚c and 38˚c, s2 table) which limits the transferability of the findings to clinical practice. we also did not extract data on combinations of symptoms (such as fever and cough together, or diarrhoea and vomiting for example) which was on oversight in the protocol development phase though equally, this was poorly reported in the literature. future researchers who wish to build upon this dataset might consider extracting combinations of symptoms alongside isolated symptoms from the few studies were this is reported [174] . we confirm that fever and cough remain the most prevalent symptoms of adults infected by sars-cov-2. however, there is a large proportion of infected adults which symptoms-alone do not identify. to expedite future iterations of this work, our data is freely available in the open science framework repository. covid-19) situation report-81. world heal clinical 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washington state epidemiological and clinical predictors of covid-19 epidemiologic features and clinical course of patients infected with sars-cov-2 in singapore sudden hyposmia as a prevalent symptom of covid-19 infection use of siltuximab in patients with covid-19 pneumonia requiring ventilatory support non-severe vs severe symptomatic covid-19: 104 cases from the outbreak on the cruise ship "diamond princess early epidemiological and clinical characteristics of 28 cases of coronavirus disease in south korea sars-cov-2 infection in 86 healthcare workers in two dutch clinical characteristics of 50404 patients with 2019-ncov infection real-time tracking of self-reported symptoms to predict potential covid-19 a framework for identifying regional outbreak and spread of covid-19 from one-minute population-wide surveys can meta-analysis be salvaged? key: cord-351868-w4d45fue authors: zuwała, kaja; golda, anna; kabala, wojciech; burmistrz, michał; zdzalik, michal; nowak, paulina; kedracka-krok, sylwia; zarebski, mirosław; dobrucki, jerzy; florek, dominik; zeglen, sławomir; wojarski, jacek; potempa, jan; dubin, grzegorz; pyrc, krzysztof title: the nucleocapsid protein of human coronavirus nl63 date: 2015-02-20 journal: plos one doi: 10.1371/journal.pone.0117833 sha: doc_id: 351868 cord_uid: w4d45fue human coronavirus (hcov) nl63 was first described in 2004 and is associated with respiratory tract disease of varying severity. at the genetic and structural level, hcov-nl63 is similar to other members of the coronavirinae subfamily, especially human coronavirus 229e (hcov-229e). detailed analysis, however, reveals several unique features of the pathogen. the coronaviral nucleocapsid protein is abundantly present in infected cells. it is a multi-domain, multi-functional protein important for viral replication and a number of cellular processes. the aim of the present study was to characterize the hcov-nl63 nucleocapsid protein. biochemical analyses revealed that the protein shares characteristics with homologous proteins encoded in other coronaviral genomes, with the n-terminal domain responsible for nucleic acid binding and the c-terminal domain involved in protein oligomerization. surprisingly, analysis of the subcellular localization of the n protein of hcov-nl63 revealed that, differently than homologous proteins from other coronaviral species except for sars-cov, it is not present in the nucleus of infected or transfected cells. furthermore, no significant alteration in cell cycle progression in cells expressing the protein was observed. this is in stark contrast with results obtained for other coronaviruses, except for the sars-cov. coronaviruses cause a variety of diseases in animals, whereas human infections are almost exclusively associated with respiratory tract infections (rti). contemporary taxonomy divides the coronavirinae subfamily into four genera (alpha, beta, gamma, and delta). only the alpha cell culture llc-mk2 cells (atcc: ccl-7; macaca mulatta kidney epithelial cell line) were maintained in minimal essential medium (mem), containing 2 parts of hank's mem and 1 part of earle's mem (paa laboratories, austria) supplemented with 3% heat-inactivated fetal bovine serum (fbs) (paa laboratories, austria), penicillin (100 u/ml), and streptomycin (100 μg/ml). cells were cultured on t75 flasks (tpp, switzerland) at 37°c with 5% co 2 . 293t cells (ecacc: 12022001; human embryonic kidney sv40 transformed, genetically modified) were maintained in dulbecco-modified eagle's medium (dmem; paa laboratories, austria) supplemented with 3% heat-inactivated fetal bovine serum (fbs; paa laboratories, austria), penicillin (100 u/ml), and streptomycin (100 μg/ml). cells were cultured on t75 flasks (tpp, switzerland) at 37°c with 5% co 2 . 293t cells (atcc crl-3216) were transfected with the plko.1.-trc-ace2 plasmid using polyethylenimine (pei; sigma-aldrich, poland). the plasmid was based on the addgene plasmid 10878 [42] . at 24 h post-transfection, the cells were washed with sterile 1 × pbs and cultured at 37°c for 48 h in media supplemented with puromycin (2 μg ml -1 ) at 37°c with 5% co 2 . following selection, cells were passaged and the surviving clones were collected and analyzed. ace2-expressing (ace2 + ) cells were maintained in dulbecco's mem (paa laboratories, austria) supplemented with 10% fbs, penicillin (100 u ml -1 ), streptomycin (100 μg ml -1 ), ciprofloxacin (5 μg ml -1 ) and puromycin (1 μg ml -1 ). 293t_ace2 + cells were maintained as wild type cells. human tracheobronchial epithelial cells were obtained from airway specimens resected from patients undergoing surgery under silesian center for heart diseases approved protocols. this study was approved by the bioethical committee of the medical university of silesia in katowice, poland (approval no: knw/0022/kb1/17/10 dated on 16.02.2010) . a written informed consent was obtained from all patients (2 adult patients). primary cells detached from human bronchi and trachea with pronase e were expanded on collagen-coated (collagen type iv, sigma-aldrich) plastic in bronchial epithelial growth media (begm) to generate passage 1 cells and plated at density of 3×10 5 cells per well on permeable transwell supports (6.5-mmdiameter; corning transwell-clear) in begm. cells were cultured at 37°c in presence of 5% co 2 until confluence. human airway epithelium (hae) cultures were generated by changing the media to air liquid interface media (ali) and provision of an air-liquid interface for 6 to 8 weeks to form well-differentiated, polarized cultures that resemble the in vivo structure of pseudostratified mucociliary epithelium. all procedures were performed as previously described [43] . all cell cultures were routinely screened for mycoplasma spp. contamination using hoechst 33258 staining. hcov-nl63 (amsterdam i strain) stock was generated by infecting llc-mk2 cells. infected cells were lysed 6 days post-infection by two freeze-thaw cycles. the virus-containing fluid was cleared by centrifugation, aliquoted and stored at -80°c. a control from mock infected cells was prepared in the same manner as the virus stocks. virus yield was assessed by titration on fully confluent llc-mk2 cells, according to reed and muench formula [44] . cells on 96-well plates were incubated at 32°c for 6 days and the cytopathic effect was scored using an inverted microscope. all experimental procedures were conducted as previously described [45] . virus identity was confirmed by cdna sequencing. rna from viral and control cultures was extracted using genejet rna purification kit (thermo scientific, lithuania), according to manufacturer's protocol. isolated rna was stored at -80°c. dna fragments were synthesized by a third party (genomed, poland). isolated viral rna was reverse transcribed using high capacity cdna kit (life technologies, poland) and used as a template for subsequent amplification. in order to obtain eukaryotic expression vector, nl63-n gene was amplified using primers n_nl63_5hindiii (5 0 -gta caa gct tgc cac cat ggc tag tgt aaa ttg ggc c-3 0 ) and n_nl63_3bamhi (5 0 -gac tgg atc cgc atg caa aac ctc gtt gac aat-3 0 ) with marathon dna polymerase (a&a biotechnology, poland). resulting pcr product was subsequently gel purified using genejet gel extraction kit (thermo scientific, lithuania) and digested with hindiii and bamhi restriction enzymes. resulting fragment was cloned into the pmaxfp-green-n plasmid (lonza, switzerland) using corresponding restriction sites. plasmids (pmaxfp-green-n/nl63-n) were recovered in dh5α escherichia coli (life technologies, poland) and their identity and sequence were confirmed by dna sequencing (genomed, poland). expression plasmids for prokaryotic expression of the nl63-n protein were prepared using pmaxfp-green-n/nl63-n as a template. briefly, the fragments of n gene were amplified using primers given in parentheses: procnl63-n (5 0 -atg ccc atg ggc cat cac cat cat cac cac tct ggc gac gac gac gac aag gct agt gta aat tgg gcc gat g-3 0 and 5 0 -atg cct cga gtt aat gca aaa cct cgt tga caa t-3 0 ), procnl63-20/144-n (5 0 -cat agg atc cag aaa acc tgt att ttc agg gat cat ttt aca tgc ctc ttt tg-3 0 and 5 0 -cag caa gct ttt aag agc gat cct caa act caa c-3 0 ) and procnl63-221/340-n (5 0 -cat agg atc cag aaa acc tgt att ttc agg gat ctc aac cca ggg ctg ata ag-3 0 and 5 0 -cag caa gct ttt atg act gca ttt ctt tga tag-3 0 ) and cloned into the pet duet 1 plasmid (clonetech, usa). the element encoding 6 × his tag was introduced at the n-terminus of the gene. three plasmids for prokaryotic expression were generated: procnl63-n, procnl63-20/144-n (for expression of the n terminal-domain), and procnl63-221/340-n for expression of the cterminal domain. plasmid pmaxfp-green-n/nl63-n or control plasmids were transfected to 293t cells using cationic carrier (polyethylenimine, pei; sigma-aldrich, poland). briefly, 2 × 10 5 cells were seeded onto collagen-coated (purecol; advanced biomatrix, usa) glass coverslips in a 6-well plate. next day media was removed, cells were washed with 1 × pbs and overlaid with 2 ml of dmem supplemented with 4 μg of pei and 4 μg of plasmid. 24 h post-transfection coverslips were harvested for analysis. in order to test subcellular localization of the n protein in llc-mk2 cells, the maxfp-green-n/nl63-n encoding rna was prepared based on the original plasmid. briefly, the plasmid was used as a template with primers sp6_negfpmrna (5 0 -act gac tga ttt agg tga cac tat aga agn gaa gct tgc cac cat ggc tag tg -3 0 ) and egfpmr-na_r (5 0 -ttt ttt ttt ttt ttt ttt ttt cat taa tgc aaa acc tcg ttg ac -3 0 ), where the 5 0 primer carries the sp6 promoter. in vitro transcription was carried out using the mmessage mmachine sp6 kit (life technologies, poland). further, rna was polyadenylated using poly(a) tailing kit (life technologies, poland) and transfected into cells using transit-mrna transfection kit (mirus, usa), as advised by the manufacturer. rna encoding maxfp-green protein (control) was prepared and transfected in the same manner using primers sp6_gfpmrna (atg cat tta ggt gac act ata gat gga gag cga cga gag cgg cct gc) and gfpmrna_r (ttt ttt ttt ttt ttt ttt ttt ttt ttt ttt ttt ttt ttt ttc att att ctt cac cgg cat ctg cat c). in order to assess the influence of the n protein on cell cycle, the n-encoding rna was prepared based on the original plasmid and transfected in the same manner as described above. primers sp6_nmrna (5 0 -tcg gcc tcg tag gcc att tag gtg aca cta tag aag nct gag aga acc cac tgc tta c -3 0 ) and nmrna_r (5 0 -ttt ttt ttt ttt ttt ttt ttt cat taa tgc aaa acc tcg ttg ac -3 0 ) were used, where the 5 0 primer carries the sp6 promoter. cells transfected with mrna encoding the n protein or control cells were harvested 48 h posttransfection by trypsinization and pelleted in sterile 1 × pbs. after fixation in 70% etoh for 2 h on ice, cells were incubated in staining solution (50 μg/ml propidium iodide and 10 μg/ml rnase a in sterile 1 × pbs; sigma-aldrich, poland) for 30 min at 37°c. the n protein was visualized with monoclonal antibody specific to nl63-n (ingenasa, spain) and secondary alexa fluor 488 goat anti-mouse antibody (life technologies, poland). cells expressing the n-nl63 protein were analyzed by flow cytometry (facscalibur, becton dickinson) as previously described [46] . cells treated with nocodazole (sigma-aldrich, poland), a mitotic spindle poison, were sampled after 24 h and evaluated as positive control for cell cycle arrest. obtained data were analyzed using modfit lt software (verity software house, usa). all experiments were conducted independently at least three times. cells were fixed using 4% formaldehyde solution in sterile 1 × pbs for 15 minutes. subsequently, cells were washed three times with 1 × pbs and incubated with 0.1% triton solution in 1 × pbs to remove lipid fraction. further, cells were incubated in blocking buffer (10% of bsa; bio-shop, canada, 0.1% tween 20; bioshop, canada in 1 × pbs) for 60 minutes. for detection of hcov-nl63 n protein mouse monoclonal anti-hcov-nl63-n antibody (diluted 4000 ×, ingenasa, spain) was incubated with the sample for 1 hour at 4°c, followed by incubation with an anti-mouse alexa fluor 488 (dilution 400 ×, thermo fisher scientific, poland) for 1 hour at 4°c. for visualization of nucleic acids, dapi dye (1 μg/ml; sigma-aldrich, poland) was used. fluorescent images were acquired with leica tcs sp5 ii confocal microscope (leica microsystems gmbh, germany). images were pre-processed using leica application suite advanced fluorescence las af v. 2.2.1 (leica microsystems gmbh) and further deconvolved with huygens essential package ver. 4.4 (scientific volume imaging b.v., the netherlands). all experiments were conducted independently at least three times. the nl63-n ntd (amino acids 2-144) and ctd (amino acids 221-340) expression constructs were designed in silico by analysis of sequence alignments, comparative modeling and literature data. the sequences and structures of the homologous coronavirus nucleocapsid polypeptides used are listed in table 1 . the strategy is described in the supporting information section. sequence sets were prepared using blast and spdbv. the comparative modeling was performed with spdbv, coot and pymol [47] [48] [49] . procedure of gene amplification and plasmid preparation is described above. in order to express the n protein, ntd and ctd in e. coli, respective plasmids were transformed to bl21 cells and further cultured in lb media supplemented with ampicillin (100 μg/ml) at 37°c, until the optical density (λ = 600nm) reached 0.5-0.6. expression was induced by addition of iptg (1 mm) and continued overnight at 20°c. subsequently cells were pelleted by centrifugation and suspended in lysis buffer (50 mm tris, 500 mm nacl, 20 mm imidazol ph 8.0). for the full length protein, the buffer was supplemented with 5 mm β-mercaptoethanol. bacterial cells were lyzed by sonication, and cellular debris was removed by centrifugation. proteins of interest were recovered by affinity chromatography (ni sepharose 6 fast flow, ge healthcare, poland), ion-exchange chromatography (resource q, ge healthcare, poland) and size exclusion chromatography (superdex s75, ge healthcare, poland). protein was detected using western-blotting technique and anti-6 × his antibodies (life technologies, poland). samples for mass spectrometry were prepared by dialysis into 50 mm nh 4 hco 3 , ph 7.8. measurements were performed using the microtof-qii mass spectrometer (bruker, germany) in positive ionization mode, using appollo source esi sprayer. prior to measurements the device was calibrated with tunemix solution. the obtained ms spectra were analyzed using data analysis 4.0 software (bruker, germany). molecular weight of proteins was confirmed using maximum entropy algorithm for ms spectra deconvolution (bruker, germany). protein preparations were overlaid on the poly-l-lysine coated glass slides (diameter of 16 mm). samples were fixed with 2.5% glutaraldehyde in 0.1m sodium cacodylate buffer ph = 7.4 for 20 minutes. subsequently, samples were washed with the abovementioned buffer and gently dehydrated by using solutions of ethanol in a graded series of concentrations. preparations were dried in a critical point dryer (quorum technologies, united kingdom). slides were mounted on holders using self-adhesive carbon discs (taab laboratories, united kingdom) and sputter coated with gold (ion sputter jfc-1100e; jeol, japan). electron micrographs were prepared using scanning electron microscope jsm-5410 (jeol, japan). is heat capacity at the t m measured with respect to the chemical baseline. the ratio c exc;max p ∕ dhðt m þis sensitive to the shape (width) of the transition. if two-state model holds true, the van't hoff and calorimetric enthalpies are equal within the experimental uncertainty, and so the ration = δh(t m )/δh vh should be equal to unity. protein electrophoresis in denaturing conditions was carried out in schagger & von jagow system. electrophoretic separation was carried out at 75 v (stacking) / 135 v (separation). proteins were visualized using coomassie brilliant blue g-250 (serva, germany). page ruler plus (thermo scientific, lithuania) was used as a prestained protein size marker. for emsa assay 10 μg of rna or dna corresponding in sequence to the n-nl63 gene (prepared in the same manner as for the transfection of eukaryotic cells) was incubated in buffered solution (5 mm tris, 50 mm nacl, ph8.0) with 10 μg of the ntd or ctd for 30 minutes at room temperature. subsequently, samples were separated on agarose gels and signal from nucleic acids was visualized with ethidium bromide staining. all experiments were conducted independently at least three times. in order to assess whether the ntd or ctd are able to form oligomers, 50 μg of the protein was mixed with glutaraldehyde (0.007%; serva). following 15 minute incubation at room temperature 0.5 μl of 1m tris solution was added to the mixture, samples were mixed with protein sample buffer, denatured at 95°c and loaded onto the polyacrylamide gel. all experiments were conducted independently at least three times. the sequences of dna, rna and proteins used within the study correspond to those of hcov-nl63 isolate amsterdam 1 (genbank accession number: nc_005831). accession numbers for n proteins of different coronaviruses are provided in s1 file. in silico analysis of nl63-n nl63-n is a basic protein (predicted pi, 9.78) comprising 377 amino acids (aa). the predicted molecular weight is 42,252.47 da. literature data indicate that the full length coronaviral nucleocapsid protein consists of two folded domains linked by an unstructured region. in more details the n protein includes following elements: n-tail, n-terminal domain (ntd), r.s.a.g. rich linker, c-terminal domain (ctd) and c-tail [50] . the constructs of ntd and ctd used in this study were designed based on literature data, hcov-nl63 n protein amino acid sequence alignment with known homologs and on the comparative analysis of currently available crystal structures of these homologs. according to saikatendu et al. the ntd of hcov-nl63 n encompasses residues 17-141 [51] . our sequence alignment and structural analysis suggests that nl63-n 2-144 fragment better reflects the full n-terminal domain. nl63-n fragment encompassing its ctd was chosen exclusively on the basis of sequence alignment and structural analysis which suggests that fragment 221-340 contains full, structurally stable ctd. the sequences and structures of n proteins used in above analysis are listed in table 1 . the analysis strategy is summarized in s1 file and the amino acid sequences of the final constructs of ctd and ntd are presented in s2 file. in silico analysis conducted using psort ii revealed that two nuclear localization signals (nls) are buried within nl63-n: pat4 (aa 232-kkpr-235) and pat7 (aa 234-prwkrvp-240). no bipartite nls were detected. the complete nl63-n protein and its ctd and ntd were expressed in e. coli bl21 cells. ntd and ctd were purified to homogeneity whereas the full length n-protein was purified to about 80% homogeneity as demonstrated by sds-page (fig. 1) . the identity of purified proteins was confirmed with mass spectrometry (data not shown). we next used differential scanning calorimetry (dsc) to examine thermal stability of the proteins. the dsc curves for the first heating scans obtained for nl63-n, the ctd, and the ntd are shown in fig. 2 . nl63-n underwent irreversible denaturation and showed a broad transition curve. the denaturation temperature was estimated at 45.7°c, with an enthalpy change of approximately 80 kcal/mol ( table 2 ). however, due to low signal to noise ratio the resulting baseline was variable and these values can only be treated as rough estimates. nevertheless, the low enthalpy value suggests that nl63-n protein is relatively unstable. unfolding of the ntd was irreversible and accompanied by protein aggregation (indicated by the exotherm present in the high temperature region of the dsc curve). the transition temperature was 45°c and the δh cal was 104.4 kcal/mol. surprisingly, thermal transition of the ctd was fully reversible, showing t t of 55.7°c, a δh cal of 143.6 kcal/mol, and a δs cal of 0.44 kcal/ kmol. thermal transition of the ctd was cooperative, with a van't hoff enthalpy/calorimetric enthalpy ratio (δh van'hoff / δh cal ) of 1.03. the coronaviral nucleocapsid forms a protective scaffold around the viral rna [52] [53] [54] . the n protein forms oligomers via specific interactions between different regions within the protein [55] [56] [57] . to confirm this assumption for hcov-nl63, we performed mass spectrometry analyses. the results confirmed the presence of complete n protein dimers. furthermore, similar results were obtained for the ctd but not ntd, suggesting that ctd harbors the sites responsible for n protein dimerization ( table 3) . the mass spectrometry results were confirmed by protein crosslinking studies. incubating the ctd in the presence of glutaraldehyde followed by sds-page analysis revealed the presence of protein dimers and higher molecular weight oligomers (fig. 3a) . similar results were obtained using size exclusion chromatography, showing that~40% of the protein is present as dimers (fig. 3b) . dimerization was not observed for ntd. we next performed an electrophoretic mobility shift assay to determine whether the n protein interacts with nucleic acids. briefly, samples containing nucleic acids were separated in agarose gel under native conditions in the presence/absence of the ctd or the ntd. nucleic acids were detected by ethidium bromide staining. as shown in fig. 4 , the ntd binds nucleic acids (both dna and rna), as demonstrated by retarded rna and dna migration. the ctd did not bind nucleic acids. we also conducted similar analysis for the complete n protein (data not shown). obtained results suggested that the complete n protein has lower nucleic acid binding ability or is more specific compared to the ntd. however, due to rapid degradation of the complete n protein into separate domains and resulting presence of the free ntd in the solution these results were inconclusive. coronaviral n proteins localize to the cytoplasm, where they are involved in virus replication and assembly. however, the n proteins of almost all coronaviruses (except for sars-cov) also localize to the nucleus or to micronuclei [58] [59] [60] [61] . to examine the subcellular localization of the nl63-n, cultures of 293t_ace2 + , llc-mk2, and hae cultures were infected with the hcov-nl63 virus. subsequently, the cells were fixed and stained with antibodies specific for the n protein. in all cell types tested the protein localized exclusively in the cytoplasm (fig. 5, s3, s4, s5 files) . to test whether the observed lack of nuclear localization of nl63-n does not result from insufficient nuclear staining, 293t cells were also transfected with pmaxfp-green-n/nl63-n plasmid. llc-mk2 cells were transfected with maxfp-green/nl63-n encoding mrna due to poor transfection efficiency using conventional dna delivery methods. maxfp-green protein was used as a control. we then examined the subcellular localization of nl63-n using confocal microscopy ( fig. 6a and 6b ). maxfp-green-labeled nl63-n localized exclusively to the cytoplasm in all tested cell types and no staining was observed in the nucleus. overexpression of the n protein resulted in formation of large deposits of the n protein in the cytoplasm, which may be attributed to vast overexpression of the protein, as infection of these cells did not result in formation of such structures. similar patterns were previously seen for other coronaviruses [62] . previous reports show that expression of the coronaviral n protein results in delayed cellular growth. these observations were supported by biochemical studies showing that the n protein may actively participate in cell cycle regulation in infected cells by localizing to the nucleus and interacting with cyclins [36] [37] [38] 61] . to examine whether overexpression of the n protein affects cell cycle progression, we transfected llc-mk2 and 293t cells with rna encoding nl63-n. in this particular case, we used rna transfection because it was more efficient than transfecting cells with dna vectors (unpublished observations). only cells expressing nl63-n (as identified by staining with specific antibodies) were used for subsequent analyses. there was no significant difference in cell cycle progression in nl63-n-expressing and non-expressing cells (fig. 7) . since its discovery, hcov-nl63 was considered to be the closest relative of hcov-229e. however, later identification of novel bat coronavirus species revealed several alphacoronaviruses closely clustering with the two human pathogens. such an observation raised speculations on zoonotic origin of hcov-nl63 [63, 64] . considering that hcov-nl63 is rather an atypical alphacoronavirus (e.g., in terms of receptor usage and the predicted protease active site [26, 29] , it has been suggested that, despite the high similarity between hcov-229e and hcov-nl63 [65] , these viruses may represent two distinct species that evolved from a common ancestor in bats, and were then introduced into human population via two independent zoonotic transmission events [63, 64] . the nl63-n is a basic protein comprising of 377 aa. both the ntd and the complete n protein are unstable, as shown by the broad, irreversible dsc curves and obtained thermal parameters values: t m below 50°c and low δh value for complete n protein. in general, δh represents the energy amount of non-covalent bonds occurring within native protein. this is hcov-nl63 n protein consistent with our finding that the n protein rapidly degrades in aqueous solutions (data not shown). obtained results are consistent with previous reports [55, 66, 67] . however, the ctd was surprisingly stable; no protein aggregation was observed upon heating and thermal denaturation was fully reversible, moreover judging from δh(t m )/ δh vh ratio it undergoes cooperative transition. thermodynamic protein stability is defined as the gibbs energy difference between the denatured and native states but, for some practical purposes, the denaturation temperature, t m (where δg = 0 for two-state reversible transition) may be more useful to measure the protein stability. irreversibility is a common feature of dsc measurements when the large, multidomain proteins are considered. irreversibility limits the use of a standard equilibrium thermodynamic analysis. for such a process, the denaturation temperature measured in dsc experiment is significantly lower than that corresponding to δg = 0. in these cases, the t m (so called operational thermal stability) is kinetically controlled. the simplest model of irreversible unfolding can be described in the terms of kinetic analysis (rate equations) according to general lumry-erying scheme: where k is the equilibrium unfolding constant characterizing reversible unfolding step, k is the first order rate constant describing following irreversible step; n, u and d correspond to native, reversibly unfolded and irreversibly denatured monomer of the protein, respectively. therefore, d represents a modified denatured state existing in a quasi-two-state equilibrium with the native state [68, 69] . in fact, for irreversible denaturation the enthalpy and t m become only apparent values since both parameters are dependent on the scan rate. the existence of the exotherm on the downhill part of endotherm dsc peak is the obvious evidence for the presence of association-aggregation processes-the most common reason of the protein unfolding irreversibility. lower stability of ntd and the complete n protein in comparison to ctd could be attributed to the distinct structural flexibility of these proteins. in turn, higher flexibility allows ntd to bind the nucleic acids which has obvious biological relevance for ntd and complete n protein function. one of the major functions of the n protein is to bind viral rna to form a nucleoprotein, which is then packed into new virions. therefore, we next examined the ability of nl63-n to bind nucleic acids in an electrophoretic mobility shift assay. our results confirmed that the ntd efficiently bound rna and dna. however, previous studies show that coronaviral n protein has a higher affinity for the viral rna than non-viral nucleic acids and that only nucleoproteins with coronaviral genomic rna are efficiently incorporated into new virions [51, [70] [71] [72] . observed limited binding of the nucleic acids by the complete n protein (data not shown) hypothetically resulted from interaction between the ntd and ctd. however, rapid n-protein degradation at the conditions of the experiment did not allow us to obtain conclusive answer. this phenomena requires further studies, as we were not able to distinguish at this stage hcov-nl63 n protein the mechanism of ntd-ctd interaction that limits nucleic acid binding and most likely increases the binding specificity. previous studies demonstrated that coronaviral n proteins form dimers, and to lesser extent higher order oligomers [55] . the crystal structures of the sars-cov, infectious bronchitis virus (ibv), and mouse hepatitis virus (mhv)-n ctds revealed that all those domains are characterized by a similar polypeptide fold and are dimeric, strongly suggesting that the dimeric n protein is the unit that functions in vivo [56, 57] . furthermore, tang et al. compared the n proteins from sars-cov and hcov-229e [55] demonstrating that they formed oligomers and bound to nucleic acids. moreover, lo et al. showed that oligomerization of the 229e-n protein is most likely also mediated by the ctd [57] . here we show that the complete nl63-n protein and the ctd can self-associate to form dimers and higher order oligomers. the role of aforementioned n-n interaction is, however, debatable, as nl63-n strongly binds nucleic acids during formation of the ribonucleocapsid. it is therefore possible that this interaction may be important for stabilizing and shaping the ribonucleocapsid; however, it may also be important for other n-mediated processes [73] . n protein of most coronaviruses is abundantly present in the nucleus of the infected cell [58] [59] [60] [61] . analysis of the nl63-n protein using psort ii revealed the presence of two nls (pat4 and pat7), both of which are homologous to those observed in other coronaviral n proteins. for example, the ibv virus carries two very similar putative nls signals: pat4 rpkk [aa 359-362] and pat7 pkkekkl [aa 360-366]. one may, therefore, expect that the nl63-n protein would also localize to the nucleus. surprisingly, no sign of nl63-n localization to the nuclei of infected or transfected cells was detected, suggesting that these nls motifs are buried within the nl63-n structure, as previously proposed for sars-cov [58, 74] . to validate that the solely cytoplasmic localization of nl63-n is found not only in established cell lines, we tested a fully differentiated human airway epithelium cultures, mimicking the natural environment of the human airway epithelium; identical results were obtained. it is, however, possible that the lack of nuclear staining observed after natural infection or transfection with the nl63-n-encoding plasmid may be due to poor antibody staining within the nucleus [75] . to address this problem, we used a vector encoding maxfp-green-n/nl63-n fusion protein, but found no difference in the subcellular localization of the fusion protein and the native protein [60, 76, 77] . also, we found that overexpression of maxfp-green-n/nl63-n in the cells yielded a staining pattern identical to that shown by the native n protein. presented data show that the nl63-n protein does not localize to the nucleus (or does so in a very limited fashion), similarly to the sars-cov n protein and differently than other coronaviral n proteins [60, 76] . nuclear localization of the n protein may be important for several processes, including direct interference with the cell cycle. dysregulation of the cell cycle is a common strategy used by many dna and rna viruses, which enables them to hijack and exploit the host cell machinery for their own benefit. indeed, the n proteins of many coronaviruses (including sars-cov; although the n protein does not localize into the nucleus in this case) inhibit cell cycle progression [36] [37] [38] 61] . however, we found no difference in the proportion of cells in each phase of cell cycle for nl63-n protein-expressing and non-expressing cells. in conclusion, we demonstrated here that although nl63-n is largely similar to other coronaviral n proteins, it possesses some unique characteristics: it does not localize to the nucleus of the infected cell and its expression does not appear to affect the cell cycle progression. supporting information s1 file. design of the n-and c-terminal domains of hcov-nl63 n protein expression constructs was based on the sequence alignment with homologous n proteins together with the structural comparative analysis. upper panel: in order to predict polypeptide sequence encompassing the n-terminal domain of n protein from hcov-nl63 the following sequences of n proteins were aligned: q6q1r8 (uniprot id), hcov-nl63; p69596, avian infectious bronchitis virus (ibv, strain beaudette), sequence referring to the 2bxx crystal structure (protein data bank id); p69598, ibv (strain beaudette us), sequence referring to the 2c86 crystal structure; p32923, ibv (strain gray), sequence referring to the 2gec crystal structure; p03416, murine hepatitis virus (strain a59), sequence referring to the 3hd4 crystal structure; p59595, sars cov, sequence referring to the 2ofz crystal structure, p59595, sars cov, sequence referring to the 2og3 crystal structure. sequences referring to residues 1-210 of n protein from hcov-nl63 are presented as it is enough to reflect the full n-terminal domains of each sequence aligned. sequences reflecting residues defined by the electron density maps of the crystal structures (2bxx, 2c86, 2gec, 3hd4, 2ofz, 2og3) are colored green. the sequence of hcov-nl63 n protein predicted to constitute the structurally stable n-terminal domain is colored blue. to predict polypeptide sequence encompassing the c-terminal domain of n protein from hcov-nl63 the following sequences of n proteins were aligned: q6q1r8, hcov hcov-nl63 n protein nl63; p69596, avian infectious bronchitis virus (ibv, strain beaudette), sequence referring to the 2ca1 crystal structure; p32923, ibv (strain gray), sequence referring to the 2ge7 crystal structure; p32923, ibv (strain gray), sequence referring to the 2ge8 crystal structure; p59595, human sars coronavirus, sequence referring to the 2cjr crystal structure; p59595, sars cov, sequence referring to the 2jw8 solution structure. sequences referring to residues 194-377 of n protein from hcov-nl63 are presented as it is enough to reflect the full c-terminal domains of each sequence aligned. sequences reflecting residues defined by the electron density maps of the n protein ctd crystal structures (2ca1, 2ge7, 2ge8, 2cjr) and the residues of 2jw8 solution structure are colored violet. the sequence of hcov-nl63 n protein predicted to constitute the structurally stable c-terminal domain is colored red. lower panel: superposition of n protein ntd and ctd structures used in the comparative analysis. multiple molecules composing the asymmetric unit of given structure are colored the same. multialignment was performed in muscle [78] . comparative structural analysis was done in spdbv, coot and pymol [47, 79] . s5 file. subcellular localization of nl63-n protein in fully differentiated human airway epithelium cultures cells infected with hcov-nl63. virions were labelled with antibodies specific to the n-nl63 protein (green); dna was stained with dapi (blue). analysis was carried on with confocal microscopy using leica tcs sp5 ii confocal microscope. voxel size: 49.9 × 49.9 × 125.9 nm, step size: 130 nm, scale bar: 10 μm. taxonomy of viruses., king amq (2012) virus taxonomy: classification and nomenclature of viruses: ninth report of the international committee on taxonomy of viruses a new virus isolated from the human respiratory tract growth in suckling-mouse brain of "ibv-like" viruses from patients with upper respiratory tract disease cultivation of a novel type of common-cold virus in organ cultures the morphology of three previously uncharacterized human respiratory viruses that grow in organ culture identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome identification of a new human coronavirus identification of new human coronaviruses characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia isolation of a novel coronavirus from a man with pneumonia in saudi arabia detection of new viruses by vidisca. virus discovery based on cdna-amplified fragment length polymorphism croup is associated with the novel coronavirus nl63 epidemiological and clinical features of human coronavirus infections among different subsets of patients. influenza other respir viruses coronavirus infections in hospitalized pediatric patients with acute respiratory tract disease human coronavirus nl63 in children: epidemiology, disease spectrum, and genetic diversity the dominance of human coronavirus oc43 and nl63 infections in infants human coronavirus nl-63 infection in a brazilian patient suspected of h1n1 2009 influenza infection: description of a fatal case fatal lower respiratory tract disease with human corona virus nl63 in an adult haematopoietic cell transplant recipient clinical manifestations of human coronavirus nl63 infection in children in taiwan role of human coronavirus nl63 in hospitalized children with croup human aminopeptidase n is a receptor for human coronavirus 229e feline aminopeptidase n serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup i identification of a putative cellular receptor 150 kda polypeptide for porcine epidemic diarrhea virus in porcine enterocytes human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry replication-dependent downregulation of cellular angiotensin-converting enzyme 2 protein expression by human coronavirus nl63 genome structure and transcriptional regulation of human coronavirus nl63 molecular characterization of human coronavirus nl63 quantification of individual subgenomic mrna species during replication of the coronavirus transmissible gastroenteritis virus fields virology. philadelphia: wolters kluwer/lippincott williams & wilkins health gene n proximal and distal rna motifs regulate coronavirus nucleocapsid mrna transcription coronavirus nucleocapsid protein facilitates template switching and is required for efficient transcription m and n proteins of sars coronavirus induce apoptosis in hpf cells the sars coronavirus nucleocapsid protein induces actin reorganization and apoptosis in cos-1 cells in the absence of growth factors the nucleocapsid protein of severe acute respiratory syndrome coronavirus inhibits cell cytokinesis and proliferation by interacting with translation elongation factor 1alpha characterisation of cyclin d1 downregulation in coronavirus infected cells the nucleocapsid protein of severe acute respiratory syndrome-coronavirus inhibits the activity of cyclin-cyclin-dependent kinase complex and blocks s phase progression in mammalian cells sars-cov nucleocapsid protein antagonizes ifn-beta response by targeting initial step of ifn-beta induction pathway, and its c-terminal region is critical for the antagonism psort: a program for detecting sorting signals in proteins and predicting their subcellular localization a knowledge base for predicting protein localization sites in eukaryotic cells a lentiviral rnai library for human and mouse genes applied to an arrayed viral high-content screen well-differentiated human airway epithelial cell cultures a simple method of estimating fifty per cent endpoints infection with human coronavirus nl63 enhances streptococcal adherence to epithelial cells practical flow cytometry coot: model-building tools for molecular graphics automated protein modelling-the proteome in 3d basic local alignment search tool the nucleocapsid protein of coronavirus infectious bronchitis virus: crystal structure of its n-terminal domain and multimerization properties ribonucleocapsid formation of severe acute respiratory syndrome coronavirus through molecular action of the n-terminal domain of n protein isolation and morphology of the internal component of human coronavirus, strain 229e ribonucleoprotein-like structures from coronavirus particles ribonucleoprotein of avian infectious bronchitis virus biochemical and immunological studies of nucleocapsid proteins of severe acute respiratory syndrome and 229e human coronaviruses x-ray structures of the n-and c-terminal domains of a coronavirus nucleocapsid protein: implications for nucleocapsid formation oligomerization of the carboxyl terminal domain of the human coronavirus 229e nucleocapsid protein trafficking motifs in the sars-coronavirus nucleocapsid protein characterization of the nuclear export signal in the coronavirus infectious bronchitis virus nucleocapsid protein subcellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein localization to the nucleolus is a common feature of coronavirus nucleoproteins, and the protein may disrupt host cell division phosphorylation of the arginine/serine dipeptide-rich motif of the severe acute respiratory syndrome coronavirus nucleocapsid protein modulates its multimerization, translation inhibitory activity and cellular localization evidence supporting a zoonotic origin of human coronavirus strain nl63 distant relatives of severe acute respiratory syndrome coronavirus and close relatives of human coronavirus 229e in bats human coronaviruses 229e and nl63: close yet still so far high affinity interaction between nucleocapsid protein and leader/intergenic sequence of mouse hepatitis virus rna low stability of nucleocapsid protein in sars virus differential scanning calorimetry of proteins a differential scanning calorimetry study of tetracycline repressor specific interaction between coronavirus leader rna and nucleocapsid protein mass spectroscopic characterization of the coronavirus infectious bronchitis virus nucleoprotein and elucidation of the role of phosphorylation in rna binding by using surface plasmon resonance coronavirus nucleocapsid protein is an rna chaperone characterization of n protein self-association in coronavirus ribonucleoprotein complexes nuclear/nucleolar localization properties of c-terminal nucleocapsid protein of sars coronavirus a higher concentration of an antigen within the nucleolus may prevent its proper recognition by specific antibodies intracellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein: absence of nucleolar accumulation during infection and after expression as a recombinant protein in vero cells the coronavirus infectious bronchitis virus nucleoprotein localizes to the nucleolus muscle: multiple sequence alignment with high accuracy and high throughput swiss-model and the swiss-pdbviewer: an environment for comparative protein modeling coronavirus n protein n-terminal domain (ntd) specifically binds the transcriptional regulatory sequence (trs) and melts trs-ctrs rna duplexes structure of the sars coronavirus nucleocapsid protein rna-binding dimerization domain suggests a mechanism for helical packaging of viral rna solution structure of the c-terminal dimerization domain of sars coronavirus nucleocapsid protein solved by the sail-nmr method key: cord-334695-cjxlw1tu authors: kam, yiu-wing; okumura, yuushi; kido, hiroshi; ng, lisa f. p.; bruzzone, roberto; altmeyer, ralf title: cleavage of the sars coronavirus spike glycoprotein by airway proteases enhances virus entry into human bronchial epithelial cells in vitro date: 2009-11-17 journal: plos one doi: 10.1371/journal.pone.0007870 sha: doc_id: 334695 cord_uid: cjxlw1tu background: entry of enveloped viruses into host cells requires the activation of viral envelope glycoproteins through cleavage by either intracellular or extracellular proteases. in order to gain insight into the molecular basis of protease cleavage and its impact on the efficiency of viral entry, we investigated the susceptibility of a recombinant native full-length s-protein trimer (trispike) of the severe acute respiratory syndrome coronavirus (sars-cov) to cleavage by various airway proteases. methodology/principal findings: purified trispike proteins were readily cleaved in vitro by three different airway proteases: trypsin, plasmin and tmprss11a. high performance liquid chromatography (hplc) and amino acid sequencing analyses identified two arginine residues (r667 and r797) as potential protease cleavage site(s). the effect of protease-dependent enhancement of sars-cov infection was demonstrated with ace2 expressing human bronchial epithelial cells 16hbe. airway proteases regulate the infectivity of sars-cov in a fashion dependent on previous receptor binding. the role of arginine residues was further shown with mutant constructs (r667a, r797a or r797ar667a). mutation of r667 or r797 did not affect the expression of s-protein but resulted in a differential efficacy of pseudotyping into sars-covpp. the r667a sars-covpp mutant exhibited a lack of virus entry enhancement following protease treatment. conclusions/significance: these results suggest that sars s-protein is susceptible to airway protease cleavage and, furthermore, that protease mediated enhancement of virus entry depends on specific conformation of sars s-protein upon ace2 binding. these data have direct implications for the cell entry mechanism of sars-cov along the respiratory system and, furthermore expand the possibility of identifying potential therapeutic agents against sars-cov. proteolytic cleavage of the viral envelope glycoprotein into a receptor binding and a fusogenic transmembrane subunit is important to regulate virus entry and infectivity [1] . previous studies showed that viral glycoprotein activation is mediated by secreted proteases recognizing either monobasic or multibasic cleavage sites [2] . cleavage of viral glycoprotein has been demonstrated in retrovirus, ortho and paramyxoviruses to regulate virus entry and fusion [3, 4, 5] . the extracellular processing of the envelope glycoprotein has a major impact on the infectivity of virulent or avirulent strains of influenza viruses, sendai virus and newcastle disease virus [6, 7, 8] . a typical example is influenza a virus, where virus-cell fusion activity is induced by post-translational proteolytic cleavage of the envelope glycoprotein that is mediated by trypsin-like protease in the bronchial epithelium and airway secretion [9] . several proteases such as tryptase clara, mini-plasmin, ectopic anionic trypsin, mast-cell tryptase and tryptase tc30, which have been isolated from airway epithelial, can selectivity cleave the consensus cleavage motif of human influenza a virus envelope glycoprotein [10, 11, 12, 13] and determine the virus tropism and infectivity. recent advance of human genome studies identified a large number of transmembrane serine protease (tmprss). various tmprss members with known airway localization have been identified from the respiratory tract. tmprss11a, one of the newly identified members of type ii transmembrane serine proteases, is expressed in upper respiratory tract (pharynx and trachea) (unpublished data). however, less is known about tmprss that activate pneumotropic virus under natural infection. although enhancement of virus infection has been demonstrated for bovine (bcov) and rat (rcv) coronaviruses by treatment of cells with trypsin [14, 15] , the precise role of trypsin during coronavirus infection is still unknown. several members of coronavirus possess a protease cleavage site, which is essential for infectivity and virus-cell membrane fusion, and cleavage at these sites yields two non-covalently linked subunits s1 and s2 [16, 17] . the n-terminal s1 subunit is responsible for receptor binding whereas the membrane-anchored s2 subunit is important for fusion between viral and cellular membranes. evidence from mouse hepatitis virus (mhv) and bcov suggested the importance of s protein cleavage into two non-covalently linked s1 and s2 subunits that remain on the virus envelope surface during virus maturation [18, 19] . uncleaved s protein is functional but cleavage may enhance cell fusion activity and/or virus infectivity [20, 21] . susceptibility of s protein to cleavage depends on virus strains and host cell types. similar to group i coronaviruses, sequence analysis suggests that s protein from sars-cov is not expected to be cleaved since typical amino acid cleavage sites found in coronavirus group ii and iii (rrfrr, rrsrr, rsrr, rars and rarr) are not located in the sars s protein [22] . recent findings have suggested the importance of trypsin treatment in activating sars spike glycoprotein mediated cell-cell fusion [23] . syncytia formation was observed between sars spike glycoprotein expressing 293t cells and veroe6 cells after brief trypsin treatment [24] ; trypsin has been shown to induce cleavage of monobasic cleavage site and activate influenza viruses in cell culture system [4, 25] . it is not known whether the functionality of spike glycoproteins is dependent on the activity of trypsin inducing their proteolytic cleavage. nothing is known about the role of proteases that cleave/modify sars spike glycoprotein under natural infection. conformational reorganization of sars spike glycoprotein has been demonstrated from cryo-electron microscopic analysis whereby structural transition of the spike glycoprotein has been observed when irradiated sars-cov virion binds to the virus receptor, angiotensinconverting enzyme (ace2) [26] . these experiments showed that receptor-binding and subsequent membrane fusion occur with different phases of structural re-arrangements. possibly, the protease-modified sars spike glycoprotein is de facto the glycoprotein responsible for virus entry. to address this possibility we have investigated whether cleavage has any significant effect on sars-cov entry into airway epithelial cells by using s-pseudotyped lentiviral vectors (sars-covpp) encoding a luciferase reporter gene to mimic sars-cov entry. we observed that sars-cov spike glycoprotein can be efficiently cleaved by several airway proteases and that this processing enhances entry of sars-covpp. furthermore, we have identified the putative cleavage sites of airway proteases and, by site-directed mutagenesis, have determined the role of specific amino acid residue for proteolytic processing of the envelope glycoprotein, and for sars-covpp entry into human airway epithelial cells (16hbe) in vitro. while this manuscript was still in progress, one of the two natural cleavage sites described here, at position 797, was reported in a separate independent study using only trypsin for cleavage [27] . this study further supports and strengthens the demonstration of the critical role of receptor-dependent cleavage of spike protein by airway proteases, providing deeper insights into the exact mechanism of virus entry enhancement. the respiratory tract has been shown to be the primary site for sars-cov entry [28, 29] . in order to study the effects of airway protease on sars-cov entry mechanism, the susceptibility of various human airway epithelial cell lines were tested for sars-cov s-mediated infection. to monitor sars-cov entry, we pseudotyped a lentiviral vector with the sars-cov spike glycoprotein (referred to herein as sars-covpp). these recombinant viruses encoding the luciferase reporter gene and expressing the sars-cov spike glycoprotein at the virion surface have been shown to faithfully mimic the sars-cov entry process [30] . three different airway cell lines: 16hbe, beas-2b and a549 were analyzed for sars-covpp infection. we observed that only 16hbe cells (human bronchial epithelial cells) were efficiently infected by sars-covpp, with levels of luciferase activity similar to those measured in the positive control cell line veroe6 ( figure 1a ). by contrast, another human bronchial epithelial cell line, beas-2b, and a human alveolar cell line, a549 were nonpermissive to sars-covpp infection ( figure 1a) . the susceptibility to sars-s mediated infection correlated well with ace2 expression [31] , which was indeed detected by rt-pcr only in figure 1 . susceptibility of various human airway epithelial cells to sars-cov s-mediated infection. a, veroe6 (10,000 cells/well), 16hbe, beas-2b and a549 (20,000 cells/well) cells were seeded onto 96well plates 24 h before sars-covpp infection. pseudotypes (sars-covpp) were collected from culture medium and concentrated as described previously [33] . sars-covpp were incubated with various cell lines and transduction was measured by determination of the luciferase activity expressed as luminescence counts per second (lcps). veroe6 cells were used as positive control. all experiments were performed in triplicates and data are presented as means6se of two or three independent experiments. b, ace2 expression from various mammalian airway cell lines. cell lysates were collected and ace2 rna molecules were detected by rt-pcr. amplified ace2 cdna products from 16hbe, beas-2b and a549 are shown in lanes 2 to 4, respectively. veroe6 cell line (lane 1) was used as positive control for ace2 expression. the quantity of total rna templates was normalized to b-actin expression as shown from the lower panel. lane m represents the dna size marker and the size of dna bands are indicated on the right. doi:10.1371/journal.pone.0007870.g001 16hbe and veroe6 but not in a549 and beas-2b cells ( figure 1b) . these results support previous data showing ace2 is the functional cellular receptor for sars-cov and 16hbe cells were therefore chosen for the subsequent protease cleavage study. the susceptibility of purified recombinant trispike proteins to protease cleavage was investigated with airway proteases because sars-cov is pneumotropic. protease cleavage on purified trispike protein was performed by treating the trispike protein with airway proteases followed by amino acid sequence analyses. results indicated that different airway proteases recognize amino acid residues of spike as cleavage sites (figure 2a ). we also observed additional bands, migrating between 75 and 150 kda, which may represent either incomplete cleavage products or a contamination of the immunopurified trispike. amino acid sequences corresponding to the cleaved sars spike were identified and sequenced ( figure 2a ). the efficiency of protease cleavage activity was also studied by comparing the yields of cleavage products obtained from the hplc analysis using synthetic peptides corresponding to the cleaved sars spike sequence. our results showed that trypsin recognize and cleave peptides at both position r667 and r797 more efficiently than the other proteases. tmprss11a cleave peptides at the position r667 more efficiently than r797 whereas plasmin cleaves peptides at the position r797 more efficiently than r667 (data not shown). sequence analysis of cleavage products showed that trypsin, plasmin and tmprss11a proteases cleave the spike glycoprotein at positions 667 and 797 ( figure 2b ). interestingly, amino acid residue 667 has been previously proposed to be the potential cleavage site in spike generating the s1 and s2 subunits [32] . because protease processing of coronaviruses surface glycoproteins into s1 and s2 subunits is important to activate the fusion between the viral envelope and the cellular membrane during virus entry step [18, 21] , cleavage by trypsin, plasmin and tmprss11a proteases at amino acid residue 667 suggests the involvement of airway proteases during virus entry and fusion. amino acid residue 797 is also sensitive to cleavage by trypsin, plasmin and tmprss11a proteases. this site, which lies within the s2 subunit region, could be important for its further rearrangement, possibly bringing the virus envelope and cellular membrane in close proximity to facilitate virus-cell fusion. altogether, we have identified at least two amino acid residues which are sensitive to airway protease cleavage in vitro and may be important for in vivo virus entry and fusion. we next investigated the effects of airway protease treatment on sars-covpp entry into human airway epithelial cells (16hbe cells). firstly, sars-covpp was pre-treated with airway proteases and then incubated with 16hbe cells. we observed that proteolytic cleavage of spike protein before virus attachment to cells had no enhancing effects on virus entry ( figure 3a ). on the contrary, trypsin and plasmin treatment significantly reduced the infectivity of sars-covpp ( figure 3a ). according to this observation, it is most likely that protease treatment digested out the s1 region which is crucial for ace2 binding, thus resulting in reduced infectivity of sars-covpp. since attachment to virus receptor(s) may trigger critical conformational changes for proteolytic cleavage, we reasoned that it was necessary to investigate whether airway proteases would alter sars-covpp entry after virus-cell attachment. 16hbe cells were pre-incubated with sars-covpp ( figure 3b ) or lentiviral vector without surface glycoprotein (referred to herein as empp) ( figure 3c ) on ice, hence allowing virus attachment but not virus entry. unbound sars-covpp or empp was washed away and proteases were added. under these conditions airway protease treatment significantly enhanced the infection by trispike pseudotypes ( figure 3b ). to verify if the concentration of airway proteases had any enhancement effects on virus entry, unbound sars-covpp was washed away and various concentrations of proteases were added. the enhancement of sars-covpp entry increased with higher concentration of the proteases ( figure 3d -f). taken together, these observations clearly demonstrate that airway protease activity can modulate the efficiency of sars-cov entry into human ace2-expressing airway epithelial cells, in a manner that is step-dependent on virus-cell attachment. multiple sequence alignments were done on different isolates of the sars-cov spike glycoprotein to locate the airway protease cleavage sites ( figure 4a ). clearly, the potential airway protease cleavage motif is relatively conserved between the various isolates with over 90% sequence identity. in order to study the functional role of the protease cleavage site, 2 alanine substitutions (either single or double mutation) were generated by site-directed mutagenesis ( figure 4b ) and tested by pseudotyping lentiviral vectors with different mutants of sars-cov spike glycoprotein. expression of wild-type and mutant envelope proteins in transfected cells was tested by western blot analysis ( figure 4c ). cell lysates expressing wild-type spike showed the characteristic doublet of sars spike glycoprotein (upper panel, lane 1), indicating that sars spike glycoprotein is expressed and processed to incorporate high mannose or complex sugars [33] . in the case of the mutant plasmids, the expression levels and doublet patterns of sars spike glycoprotein (r667a or r797a) in cell lysates suggest that envelope protein expression and posttranslational modifications have not been significantly affected by any of the substitutions ( figure 4c; upper panel, lanes 2-3) . by contrast, double mutation (r797ar667a) plasmids exhibited greatly reduced levels of complex-sugar linked envelope expression in the cell lysates, without apparent altering the addition of highmannose sugars ( figure 4c; upper panel, lane 4) . possibly, the double mutation could have affected proper folding and oligomerization, which is important for the maturation of envelope glycoproteins. we then aimed to establish whether mutant sars spike glycoprotein can efficiently incorporate into sars-covpp by western blot analysis of pseudotyped particles purified (20% sucrose) from cells transfected with either wild-type or mutant plasmids. proper virion incorporation requires surface expression of envelope glycoprotein, and is dependent upon correct folding and oligomerization [34, 35] . as shown in figure 4c , assembly of co-transfected foreign proteins into bona fide pseudoparticles was tested by the presence of the hiv p24 protein of the lentiviral vector backbone in the same samples. the wild-type pseudotypes expressed the envelope glycoprotein as a single band ( figure 4c ; middle panel, lane 1), suggesting that the envelope glycoprotein was incorporated into pseudotypes. the detection of complexsugar (but not high-mannose) linked envelope in purified pseudotypes indicates that envelope glycoprotein incorporation requires the completion of all the quality-control processes along the secretory pathway [35] . in contrast, the double mutant (r797ar667a) exhibited a complete lack of pseudotype incorporation of envelope glycoprotein ( figure 4c ; middle panel, lane 4), illustrated by the presence of p24 in the absence of mature envelope glycoprotein ( figure 4c ; lower panel, lane 4). surprisingly, the single mutants (r667a and r797a) exhibited differential patterns of envelope glycoprotein incorporation into pseudotypes ( figure 4c ; middle panel, lanes 2-3). both constructs displayed a band pattern similar to that of wild-type, suggesting that the envelope glycoprotein was expressed in transfected cells. however, only r667a was detected in purified pseudotypes. the absence of r797a envelope glycoprotein incorporation into the pseudotypes may be due to a disruption of folding and/or oligomerization along the secretory pathway. the mutational effect on virus entry into 16hbe cells was then explored by measuring the activity of the luciferase reporter gene ( figure 4d ). the r667a mutant was functional and exhibited virus entry activity similar to that induced by wild-type sars-covpp, albeit at a slightly lower level ( figure 4d ). as expected, r797a and r797ar667a mutants were unable to enter cells ( figure 4d , cf. with the empty vector). this observation correlates with the absence of envelope glycoprotein incorporation into the pseudotypes. in an effort to directly demonstrate that airway protease mediated virus entry enhancement is due to the presence of cleavage site on the sars spike glycoprotein, 16hbe cells were pre-incubated with wild-type (sars-covpp) or mutant (r667app) pseudotypes on ice, which allowed virus attachment but not entry. unbound pseudotypes were washed away and proteases were added. our results showed that both the wild-type and mutant spike glycoproteins were functional and mediated virus entry into 16hbe cells ( figure 5a and 5b) . whereas airway protease treatment significantly increased the entry of sars-covpp into susceptible cells ( figure 5a ), this enhancing effect was completely abrogated with r667app ( figure 5b ). our data indicate that the arginine residue at position 667 is sensitive to airway protease cleavage which, in turn, significantly increases sars-cov infectivity. airway proteases could cleave the sars spike glycoprotein following the virus-cell attachment stage, thereby enhancing sars-cov entry into human ace2-expressing airway epithelial cells in vitro. the spike glycoproteins of many enveloped viruses, including various strain of human influenza viruses and sendai virus are cleaved by host-derived proteases into two non-covalently linked subunits [5, 36] . proteolytic modification of spike glycoproteins is the major determinant of virus tropism and pathogenicity as shown in pneumotropic viruses whose infectivity is determined by airway proteases [5, 36, 37] . a monobasic cleavage site has been identified in various viral glycoproteins and is recognized by proteases secreted by epithelial cells [2] . our data demonstrate that sars spike glycoprotein has two monobasic cleavage sites that are susceptible to airway protease cleavage. we show here that specific airway proteases secreted along the respiratory tract, such as trypsin, plasmin and tmprss11a, recognize and cleave the same two monobasic motifs on the sars spike glycoprotein. furthermore, we have taken advantage of the flexibility of pseudotyped particles to investigate the molecular mechanisms underlying initial steps of virus entry following cell attachment. we have observed that protease cleavage of spike glycoprotein greatly enhances entry but only after receptor-binding interaction at the cell surface. these observations suggest that proteolytic processing of bound spike results in a conformational change that favors entry of the virus. this is the first report which demonstrates the functional impact of disrupting cleavage at position 667, which had not been addressed before. more importantly, it clarifies the mechanism of virus entry enhancement by several airway proteases. our data are in agreement with the recent study showing that sars-cov s-mediated virus entry is dependent on the sequential proteolytic cleavage of monobasic sites [27] . it has been shown in clinical observations from sars-infected patients' samples that the respiratory tract is the major cellular target for infection and replication [28, 29] . similar to other coronaviruses, sars-cov utilizes the respiratory and gastrointestinal tract system as the primary entry site for infection. however, unexpected findings from several studies indicated that human airway epithelial cell lines were non-permissive to sars-cov [30, 38, 39] . we found that the permissiveness of sars-cov depended on ace2 expression, further confirming earlier studies which identified ace2 as the functional cellular receptor for sars-cov entry [40, 41] . sars-cov was also detected from lungs, gastrointestinal tract and kidney autopsy samples [42] , correlating tissue tropism with the pattern of ace2 expression in humans [43] . interestingly, ace2 expression on epithelial cell was found to correlate with the levels of cellular differentiation [31] , and primary cells grown under air-liquid interface stimulated cellular differentiation and apical surface expression of ace2 [31] . although 16hbe and beas-2b cells are derived from human bronchial epithelium [44, 45] , they showed unique morphological and biochemical characteristic according to their specific levels of cellular differentiation [46, 47] . our study demonstrate another unique characteristic within these two cell lines in terms of cell surface protein expression, further establishing 16hbe cells, which have ace2 expression under normal culturing system, as an in vitro model for sars-cov study. pseudotyped viruses have been useful for studying virus entry mechanism, cell tropism, neutralizing antibody, and receptor identification [48, 49, 50] . we have observed differential virion incorporation, which is dependent on surface expression of envelope glycoproteins, correlating with the proper folding and post-translational modification of the envelope glycoprotein [35] . our data is consistent with previous studies indicating that sars spike producing cells express both the high-mannose and complexsugar linked envelope glycoprotein [33] , and showed that complex-sugar linked sars spike glycoprotein is solely incorporated into sars-covpp. the sars spike double mutant (r797ar667a) had defects in virion incorporation, likely due to the impaired post-translational modification of the envelope glycoprotein. surprisingly, even mutant r797a showed a lack of virion incorporation. we postulate that the defect in this case is due to the impaired oligomerization of envelope glycoprotein. amino acid residue 797 is located within the s2 region of sars spike glycoprotein, which is important for trimeric envelope glycoprotein formation (unpublished observations). as a result, any mutations at this residue might generate localized folding defects of the envelope glycoprotein, and cause reduced levels of stable surface expression that prevent efficient incorporation into pseudotypes. a separate study has recently demonstrated the incorporation of a similar mutant construct (r797n) into a murine leukemia virus (mlv)-based pseudotyping vector [27] . the difference in mutant envelope incorporation with respect to our study might be attributed to the greater hydrophobicity of our substituted amino acid, which disrupts the oligomerization process. clearly, further investigation will be required to assess the role of this amino acid residue in sars spike oligomerization. previous studies have demonstrated the effects of intracellular proteases treatment (e.g. cathepsin l) in activating the membrane fusion property of sars spike [51, 52] . for example, an important role of r797 cleavage site has been shown by artificially inserting a furin cleavage site, which resulted in the production of cleaved spike glycoprotein pseudotype, and allowed the infection of cells in the presence of protease inhibitors [52] . however, wild-type sars spike has no putative furin cleavage motif/site. in addition, sars-cov has primarily respiratory tropism where a number of extracellular proteases are secreted by the airway epithelium. therefore, our results suggest that, extracellular airway proteases localized along the respiratory tract cleave and modify the wildtype sars spike during the very early stage of virus entry (viz., virus-cell attachment step). this modification enables the formation of a cleaved form of spike, and facilitates the activation of membrane fusion by subsequent intracellular protease treatment. it remains possible that the sequential activation of extracellular and intracellular proteases facilitates sars-cov entry along the respiratory tract. another aspect which should be considered is that the cleavage sites described in this report were identified using soluble spike protein, and it is conceivable that additional sites may be involved upon receptor binding. however, the impact of the r667a mutation clearly indicates the functional importance of cleavage at this position for virus entry. one study also showed an effect of very large concentrations of proteases in enhancing sars-cov infection on veroe6 cells [24] . however, our data demonstrates that the effective concentrations of airway proteases inducing sars-cov entry enhancement was at least 100-fold less than that previously reported [24] . this difference could be due to the duration of airway protease treatment onto virus-adsorbed cells, the cleavage efficiency of airway proteases or the sensitivity of the detection system used. of note, we found that the enhancing effect of airway proteases on sars-covpp entry is strictly dependent on the virus-cell attachment step. our findings are in contrast with earlier studies done on coronavirus mhv-a59 where proteolytic cleavage of envelope glycoprotein on virion surface, before binding to its cellular receptor, is necessary for entry and fusion process [18, 21] . we clearly observed that pre-treatment of sars-covpp with airway proteases dramatically reduced the infectivity of pseudotype particles, whereas the addition of airway proteases after virus-cell attachment enhanced virus-cell fusion. we postulate that receptorbound spike glycoprotein is required for cleavage mediated virus entry enhancement, as the spike glycoprotein undergoes conformational changes once it binds to the cellular receptor. it is believed that envelope glycoprotein undergoes multiple programmed conformational changes during cellular receptor interaction [53, 54, 55] . this process will expose the hydrophobic fusion peptide and mediate fusion of the viral envelope with the host cell membranes [56] . we have shown, however, that entry of sars-covpp could still complete without addition of airway proteases. several possibilities could account for this finding. first, sars-covpp could enter 16hbe cells through an endosomal pathway as described previously [23] ; alternatively, there could be a partial switch of spike glycoprotein from inactive to active conformation upon receptor binding. we hypothesize that, in the presence of airway proteases, cleavage of receptor-bound spike glycoprotein might reduce the threshold for conformational change of spike glycoprotein into its fusogenic form, thus facilitating the exposure of fusion peptide and its interaction with host cell membranes. current studies have suggested that sars-cov enters and exits preferentially via the apical surface of the epithelium [31] , and colocalization of airway proteases with sars-cov along the respiratory tract supports the positive feedback loop of virus infection in vivo. to conclude, we have found that cleavage of the receptor-bound spike glycoprotein by airway proteases enhances in vitro virus entry and fusion. therefore, identification of reagents that are able to suppress in vivo activity of airway proteases might provide additional antiviral strategy against sars-cov infection [57] , and possibly other viral respiratory infections such as human influenza a virus in the face of the current flu epidemic threat. hek293t and veroe6 cell lines (atcc) were cultured at 37uc, 5% co 2 , in dulbecco's modified eagle medium supplemented with 10% fbs, 100 u/ml penicillin, and 100 mg/ml streptomycin. transformed human alveolar basal epithelial a549 (atcc) and transformed bronchial epithelial beas-2b [50] cell lines were cultured at 37uc, 5% co 2 , in f12k medium supplemented with 10% fbs, 1% l-glutamine, and 1% antibiotic/antimycotic. transformed bronchial epithelial 16hbe cells [49] were cultured at 37uc, 5% co 2 , in dulbecco's modified eagle medium supplemented with 10% fbs, 1% l-glutamine and 1% antibiotic/antimycotic. complete medium was sterilized using 0.22 mm filtering units (corning). sars-cov spike cdna and tagging with the c-terminal m2-flag peptide sequence (s-flag) have been described previously [58] . for improved expression yield, a codon-optimized sars-s dna containing a flag sequence fused in-frame at the 39 end was produced using geneoptimizer technology (geneart). codon-optimized s-flag was subcloned into pcdna3.1 expression vector resulting in the expression plasmid pcdna-s-flag. cell lysates were collected from 293t producing cells in lysis buffer (20 mm tris-hcl ph 7.5, 150 mm nacl, 2 mm edta, 1% triton x-100). virus pellets were prepared by ultracentrifugation on a 20% sucrose cushion at 140,000 x g for 3 h using a beckman sw32ti rotor and resuspended in tne buffer (50 mm tris-hcl, 100 mm nacl, 0.5 mm edta; ph 7.4) before being subjected to sds-page (4-12%). proteins were transferred onto nitrocellulose membranes, which were first incubated in blocking solution (pbs, 0.1% tween 20, 5% milk powder, 3% normal goat serum), and then probed with anti-flag m2 monoclonal antibody (sigma). hiv-1 p24 was detected by hiv-1 p24 antibody (abcamh). peroxidase-conjugated goat anti-mouse igg (h+l) (zymed) was used as the secondary antibody. bands were visualized on x-ray films (kodak) using chemiluminescence (amersham biosciences). total mrna was isolated from veroe6, 16hbe, beas-2b and a549 cells using qiagen rneasy midi total rna extraction kit according to the manufacturer's instructions and dissolved in 60 ml of rnase free h 2 o. ace2 cdna was synthesized using the thermoscripttm rt-pcr kit with ace2 specific primers (forward primer: 59-gca ctc acg att gtt ggg act-39, reverse primer: 59-att agc cac tcg cac atc ctc-39) and amplified using redtaq dna polymerase (sigma). the quantity of total rna templates was normalized to b-actin expression (forward primer: 59-gct cgt cgt tcg aca acg gct c-39, reverse primer: 59-caa aca tga tct ggg tca tct tct c-39). airway protease cleavage site mutants were prepared from plasmid pcdna-s-flag with the stratagene quikchange sitedirected mutagenesis kit. site-specific mutagenesis were carried out by a single-step polymerase chain reaction using gene specific primers encompassing the site to be mutated and carrying the desirable mutated nucleotide(s). methylated, non-mutated parental dna template was removed by dpni (10 u/ml) treatment and plasmids carrying the mutated nucleotide(s) were transformed into dh5a by electroporation. positive clones carrying the mutated plasmids were screened by digesting the mutagenized pcdna-s-flag sequences with unique restriction enzymes (msci for r667a; stui for r797a; msci and stui for r797ar667a). mutations were verified by dna sequencing. sars spike glycoprotein (trispike) was prepared as described previously [33] . the amount and degree of purity of recombinant trispike protein was assessed by western blot and silver staining, respectively, as described previously [28] . trypsin was purified from rat lung as described [11] . plasmin was purchased from calbiochem. recombinant tmprss11a was kindly provided from dr. noboru yamaji and masako kagoshima (astellas pharma inc). protease cleavage on purified trispike protein was performed by treating the trispike protein with airway proteases followed by amino acid sequence analyses. purified trispike protein was first incubated with various proteases including trypsin (1 mg/ml), tmprss11a (0.1 mg/ml) and plasmin (0.1 mg/ml) (equivalent to 0.2 mu of each proteases) in the presence of 0.1 m tris-hcl, ph 7.5 at 37uc for 2 h. purified trispike protein incubated without proteases was included as control. samples were then subjected to sds-page, performed on gradient acrylamide gels (2-15%) under reducing conditions according to the method of laemmli [59] . proteins were then transferred to pvdf membranes, which were stained with amino black or cbb to visualize cleavage products. after staining, protein bands were cored out and analyzed by n-terminal amino acid sequence determination using the applied biosystems 492 gas-phase sequencer/140c system, according to the manufacturer's instructions. protease cleavage assays were performed by treating sars-covpp with airway proteases during the infection steps. recombinant sars-covpp lentiviral vectors expressing a luciferase reporter gene were produced from hek293t cells as described previously [60] using 10 mg of plasmid pnl4.3.luc.r -epro-viral genome [61, 62] and 10 mg of plasmid pcdna-s-flag. for virus entry assays, veroe6, 16hbe, beas-2b and a549 cells were infected with sars-covpp. veroe6 cells were seeded onto 96-wells plates at 10,000 cells/well in triplicates. 16hbe, beas-2b and a549 cells were seeded onto 96-wells plates at 20,000 cells/well, in triplicates and, the following day, 40 ml of sars-covpp was added to each well. cells were incubated for 1 h at 37uc in a 5% co 2 atmosphere, and then 160 ml of complete medium was added to each well for an overnight incubation. medium was renewed 16 h later and incubation was continued for additional 48 h, after which cells were washed and lysed. luciferase activity was measured by a microbeta jet counter (perkin elmer) according to the instructions provided by the supplier (promega). to test the effect of airway protease treatment on virus entry, 16hbe cells were used as the model cell line for sars-covpp infection. 16hbe cells were seeded onto 96-wells plates at 20,000 cells/well in triplicates. before infection, cells were pretreated with reaction buffer (dmem supplemented with 0.1 m tris-hcl, ph 7.5) for 30 min on ice. sars-covpp were added onto each well and incubated with the cells for another 30 min on ice. the mixture was removed and 100 ml of fresh reaction buffer was added to cells. diluted airway proteases were added into each well and incubated for 40 min at room temperature. the mixture was removed; cells were washed twice with complete dmem medium and incubated overnight at 37uc, in 5% co 2 . luciferase activity measurements were performed as described above. results are presented as means6se of the specified number of samples from 2-3 independent experiments. comparisons between two populations of data were made using the 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quantification by highresolution scanning and transmission electron microscopy folate receptor-alpha is a cofactor for cellular entry by marburg and ebola viruses identification of a major co-receptor for primary isolates of hiv-1 characterization of ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines cathepsin l functionally cleaves the severe acute respiratory syndrome coronavirus class i fusion protein upstream of rather than adjacent to the fusion peptide entry from the cell surface of severe acute respiratory syndrome coronavirus with cleaved s protein as revealed by pseudotype virus bearing cleaved s protein receptor specificity and receptor-induced conformational changes in mouse hepatitis virus spike glycoprotein conformational changes in the spike glycoprotein of murine coronavirus are induced at 37 degrees c either by soluble murine ceacam1 receptors or by ph 8 mechanisms of viral membrane fusion and its inhibition structural basis for membrane fusion by enveloped viruses secretory leukoprotease inhibitor and pulmonary surfactant serve as principal defenses against influenza a virus infection in the airway and chemical agents upregulating their levels may have therapeutic potential differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins s, m and e cleavage of structural proteins during the assembly of the head of bacteriophage t4 c-type lectins l-sign and dc-sign capture and transmit infectious hepatitis c virus pseudotype particles hepatitis c virus glycoproteins interact with dc-sign and dc-signr vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes we thank dr. francois kien (hku-prc) for helpful discussion, etsuhisa takahashi (university of tokushima graduate school), kid chu, patricia lau and josephine ng (hku-prc) for technical assistance. key: cord-348499-7ll7sd3o authors: manderstedt, eric; lind-halldén, christina; lethagen, stefan; halldén, christer title: genetic variation in the c-type lectin receptor clec4m in type 1 von willebrand disease patients date: 2018-02-01 journal: plos one doi: 10.1371/journal.pone.0192024 sha: doc_id: 348499 cord_uid: 7ll7sd3o von willebrand factor (vwf) levels in healthy individuals and in patients with type 1 von willebrand disease (vwd) are influenced by genetic variation in several genes, e.g. vwf, abo, stxbp5 and clec4m. this study aims to screen comprehensively for clec4m variants and investigate their association with type 1 vwd in the swedish population. in order to screen for clec4m variants, the clec4m gene region was re-sequenced and the polymorphic neck region was genotyped in 106 type 1 vwd patients from unrelated type 1 vwd families. single nucleotide variants (snv) and variable number tandem repeat (vntr) allele and genotype frequencies were then compared with 294 individuals from the 1000genomes project and 436 swedish control individuals. re-sequencing identified a total of 42 snvs. rare variants showed no accumulation in type 1 vwd patients and are not thought to contribute substantially to type 1 vwd. the only missense mutation (rs2277998, np_001138379.1:p.asp224asn) had a higher frequency in type 1 vwd patients than in controls (4.9%). the vntr genotypes 57 and 67 were observed at higher frequencies than expected in type 1 vwd patients (6.4% and 6.2%) and showed an increase in patients compared with controls (7.4% and 3.1%). strong linkage disequilibrium in the clec4m region makes it difficult to distinguish between the effect of the missense mutation and the vntr genotypes. in conclusion, heterozygous vntr genotypes 57 and 67 of clec4m were highly enriched and are the most likely mechanism through which clec4m contributes to disease in the swedish type 1 vwd population. von willebrand disease (vwd) is characterized by low levels of, or defective plasma von willebrand factor (vwf) and is classified into three different types depending on the nature of the disease. type 1 vwd is the least serious subtype accounting for approximately 70% of diagnosed cases and is defined as a partial deficiency of functionally normal vwf. in most cases it shows dominant inheritance [1] . even in well-defined type 1 vwd patient groups approximately 35% of all type 1 vwd patients do not have a mutation in the promoter, coding sequence or splice junctions of the vwf gene [1] . this suggests that other genes affect the level plos of vwf and contribute to the disease. the most important gene identified thus far is the abo blood group gene. the vwf levels of individuals with blood group o are reduced by 30% in comparison to non-o individuals and blood group o is more common in the type 1 vwd disease population in comparison with the type 2 vwd and normal populations [2] . patients with type o-blood exhibit an increase of vwf clearance leading to a reduction of the half-life for vwf [3] . a meta-analysis of several genome-wide association studies identified eight genes that contribute to plasma levels of vwf in the normal population [4] . the abo gene showed the strongest effect, but smaller effects were seen for the stxbp5, scara5, vwf, stab2, stx2, tc2n and clec4m genes. other studies have confirmed parts of the results above and identified an additional four genes [5, 6] . one linkage study identified a locus on chromosome 2 with an effect size on vwf variation comparable to the effect of the abo locus. detailed analysis identified a total of eight genes that may have an effect on vwf levels [7] . additional studies of stxbp5 and stx2 [8] and clec4m [9, 10] have confirmed that single nucleotide variants (snvs) in these genes are associated with the variation observed for plasma levels of vwf. clec4m (c-type lectin member 4 family m) is a lectin receptor with a cytoplasmic domain, a transmembrane domain, a highly polymorphic neck region and a carbohydrate recognition domain. the carbohydrate recognition domain binds to molecules or cells that are glycosylated and this function is dependent upon the exact number of repeat units that are present in the neck region. the neck region consists of a 23 amino acid long motif that is repeated from three to nine times in different variants of clec4m. the neck region stabilizes clec4m by tetramerization of single clec4m molecules and affects the conformation of the receptor [11] . previously genetic variation in the neck region of clec4m has been associated, for example, with susceptibility to infection by hiv [12] and sars [13] . clec4m also binds to vwf [9] and variants in this gene contribute to the variation in the vwf level observed both in normal individuals [4] and in type 1 vwd patients [9, 10] . clec4m is coded for by the clec4m gene that is located on chromosome 19 and has seven exons located over 6.4 kbp. exons 1-3 code for the cytoplasmic and transmembrane regions, exon 4 for the polymorphic neck region and exons 5-7 for the ligand binding domain of the clec4m protein. exons 1 and 7 have untranslated regions and all exons have less than 200 bp of coding sequence, except for exon 4 which codes for the neck region and is the largest exon. since the neck region consists of a 69-bp repeated sequence that is repeated from three to nine times, this region is difficult to sequence by conventional sanger sequencing and is instead commonly analyzed by determining the number of repeat units [14] . genetic diversity of the clec4m gene has been studied extensively by genotyping and re-sequencing in african and non-african populations [11, 15, 16] . the present study aimed to screen comprehensively for genetic variation in the clec4m gene in individuals from 106 unrelated type 1 vwd families by re-sequencing the gene region (excluding exon 4) and genotyping the polymorphic neck region (exon 4) of the gene. a first functional assessment of all variants was made using frequency comparisons between patients and controls and comparison with sequence data of the 1000genomes project. contrary to previous studies that have used tagsnps to investigate the effects of clec4m variation on vwf level, the present study also screened for rare variants. the overall goal of the study was to investigate whether genetic variants in clec4m are associated with type 1 vwd in the swedish population looking at both common and rare variants. the type 1 vwd study population was recruited at the department for coagulation disorders, malmö university hospital (malmö, sweden). the population consisted of consecutive patients and their relatives who attended the clinic between the years 1988-2005 and corresponded to approximately 1000 individuals belonging to 126 families. this population represented the majority of all families diagnosed with type 1 vwd in sweden during this time period. clinical and laboratory data were recorded for each patient and their bleeding phenotypes were classified [17] . we used historical vwf levels usually determined at the time of the original diagnostic work-up. there were no further analyses of vwf levels in this study. therefore, different phenotypical methods were used. vwf activity was measured with the traditional vwf:rco method based on aggregation of platelets or an automated vwf:rco assay based on the bcs coagulation analyzer using the bc von willebrand reagent (dade behring inc., newark, de, usa). vwf antigen levels (vwf:ag) were measured with electroimmunoassay (the laurell method) and irma, elisa, or lia. vwf levels are given as iu/dl and were vwf:ag median 39, min 10, max 63. the corresponding vwf activity values were vwf:rco median 44, min < 5, max 71 (table 1) . patients were characterized with respect to their vwf mutations in manderstedt et al. [18] . of these 126 families mutation analysis identified 20 families with type 2 mutations. the remaining 106 families were analyzed in the present study. in a strict sense, not all index cases fulfilled the modern definition of type 1 vwd, but at the time of diagnosis their bleeding symptoms in combination with lowered vwf levels were interpreted as reflecting type 1 vwd. more than two-thirds of the patients had a family history of bleeding, the remaining were individual patients with low vwf levels. since the present study investigated one of the additional factors associated with vwf level variation, individuals with bleeding symptoms and low vwf levels were included in the study regardless of whether they had bona fide vwf mutations or not. in addition, two swedish control populations were also analyzed: control population 1 (c 1 ) consisting of 225 unrelated individuals from the general population and control population 2 (c 2 ) consisting of 211 unrelated individuals from the general population with no history of bleeding [19] . these controls were recruited from the same geographical region as the patients included in the study. the present study was approved by the ethics committee of the medical faculty, lund university, and the swedish data inspection board according to lu 436-01. written informed consent was obtained from all subjects. dna from human whole blood was isolated using a qiagen blood dna kit (qiagen, hilden, germany) and dna concentrations were determined by fluorometry using picogreen1 (molecular probes, eugene, or, usa). the ensembl genome browser (http://www.ensembl.org) referring to the grch37.p13 version of the ncbi database contributed all dna sequences that were used for primer design. tandem repeats finder software was used to identify repeated sequences in the clec4m gene region (http://tandem.bu.edu/trf/trf.html) and masking of repeated sequences used the ensembl genome browser. primers were designed using primer-blast (http://www.ncbi.nlm.nih.gov/tools/ primer-blast/) and purchased from dna technology a/s (risskov, denmark). the vntr polymorphism in exon 4 of the clec4m gene was genotyped using the following pcr protocol: one cycle at 94˚c for 3 min, followed by 10 table) . the pcr products were visualized with gel electrophoresis on 30 x 40 cm 3% agarose gels (seakem le, lonza, rockland, me, usa) using separation for 2 h at 10 v/cm. the genotyping results were validated in several ways: 1) the proper inheritance of alleles was confirmed by the analysis of three sets of trios (mother, father and child), 2) control populations c 1 and c 2 were used to confirm that the overall allele frequencies were as expected for background populations compared with previous studies, and 3) the type 1 vwd population was analyzed twice and evaluated independently by two persons. a few uncertainties were resolved by repeated analysis. long range pcr (lrpcr) was used to amplify one amplicon including the clec4m promoter, exons 1-3 and intron 3 (promoter region) and another amplicon including intron 4 and exons 5-7 (the neck region and the ligand binding domain, s1 table) . the reactions used kapa taq extra hs pcr kit (kapa biosystems) as recommended by the manufacturer using the following pcr conditions: one cycle at 94˚c for 3 min, followed by 40 cycles of 94˚c for 15 s, 63˚c for 30 s, 68˚c for 4 min, and finally one cycle at 68˚c for 10 min. the lrpcr products were then used as substrates in dna sequencing reactions using a set of overlapping sequencing primers (s1 table) . big dye terminator sanger sequencing was performed in both directions using a 3130xl genetic analyzer (applied biosystems). primary pcr products were treated with exosap-it1 (applied biosystems) according to the manufacturer's instructions and dna sequencing was performed subsequently in a total volume of 5 μl containing 0.5x big dye sequencing ready reaction premix (big dye terminator v 2.0, applied biosystems), 0.5x big dye sequencing buffer and 3.2 pmol of the sequencing primer. the following pcr conditions were used: one cycle of 96˚c for 1 min, 25 cycles of 96˚c for 10 s, 50˚c for 5 s and 60˚c for 4 min. the sequencing reactions were purified using xterminator (applied biosystems) according to the manufacturer's instructions. sequences were interpreted and all variants were identified using seqscape ver. 2.5 and confirmed by manual inspection. both the type 1 vwd and the c 1 and c 2 control populations were genotyped for rs868875, rs2277998, rs8113469 and rs62128260 using taqman-assays. the reactions were set up according to the manufacturer's protocol and the genotyping was performed on a bio-rad cfx 384 with genotypes determined using cfx manager™ software (bio-rad laboratories inc., hercules, ca, usa). identified variants in the clec4m gene were compared to publicly available data in dbsnp (http://www.ncbi.nlm.nih.gov/snp/) and 1000genomes databases (http://www.1000genomes. org/). the 1000genomes database consisted of 26 populations from different geographic regions. we extracted information from three of these populations: utah residents of [20] using the 106 type 1 vwd index cases and the c 1 and c 2 individuals as controls. haplotypes, haplotype blocks and linkage disequilibrium (ld) plots were constructed using haploview 4.2 [21] . the functional consequences of missense variants were evaluated using sift [22] , polyphen-2 [23] and mutationtaster [24] . the promoter and all exons and introns of clec4m except exon 4 were screened for variants in 106 individuals from unrelated type 1 vwd families by sanger sequencing. the sequence data was generally of high quality with > 95% of bases having a phred score of 30 or higher in > 95% of individuals. a total of 42 variants were found, 10 in the promoter, four in exons and 28 in introns of the gene ( comparison of the results of the type 1 vwd population (106 individuals) with data from the similarly sized 1000genomes populations ceu, gbr, and tsi (99, 88 and 107 individuals, respectively) showed that both the total number of variants (42 versus 41, 40 and 38) and the number of variants present in only one or two chromosomes (10 versus 8, 8, and six) were similar for the data sets both in number and in their distribution along the gene ( table 2, s2 table and fig 1) . a total of 36 variants were common to the type 1 vwd and one or more of the 1000genomes populations. these had in general high mafs: 31 snps > 0.10, one snp 0.01-0.10 and four snp < 0.01. pairwise comparisons between the type 1 vwd population and the three 1000genomes populations identified similar number of variants unique to each population in all combinations. all of them were rare variants. thus, no significant accumulation of rare variants was detected in the type 1 vwd population. in the first part of the gene (promoter-intron 3) five snps had allele frequencies differing more than 5% between the type 1 vwd population and the sum of the 1000genomes populations, but the allele frequencies of these five snps also varied considerably between the three 1000 genomes populations. in contrast, the ligand binding domain of the gene (intron 4-exon 7) contained seven snps with allele differences above 5%. for rs868875, rs868876, rs2277998 rs874492 and rs12610506 the observed differences between the type 1 vwd and the 1000genomes populations were greater than two times the maximum internal difference observed for the three 1000genomes populations. there were two variation deserts in the clec4m gene, one encompassing the first half of intron 2 and one covering intron 3 ( table 2 and fig 1) . the only snp in the coding sequence of this gene was rs2277998, a g>a (asp > asn) missense mutation. the aallele had a higher frequency in type 1 vwd compared with the 1000genomes populations (0.364 versus 0.292). this missense mutation was predicted to be a tolerated variant by the three functional prediction programs: sift, polyphen and mutationtaster (s3 table) . the ligand binding domain of the gene was analyzed further to ascertain whether the observed allele frequency differences were truly associated with type 1 vwd or if they were a result of variation due to ethnicity. haplotyping using 1000genomes data revealed rather strong ld across the entire clec4m gene region and allowed the selection of four tagsnps: rs868875, rs2277998, rs8113469 and rs62128260. taqman genotyping was performed in the two swedish control populations (c 1 and c 2 ) and in the type 1 vwd population. the allele frequencies of all four snps were similar in the c 1 , c 2 and 1000genomes populations. all four snps showed allele frequency differences between the type 1 vwd and the control populations. the largest frequency difference and the lowest p-value was observed for rs868875 (5.8%, p = 0.10). the remaining snps showed a gradual decrease in their frequency differences with increasing distance from the rs868875 snp (table 2 ). the type 1 vwd population and the c 1 and c 2 control populations were genotyped for the vntr in exon 4 of clec4m. the alleles with five, six and seven repeat units were the most frequent and together explained > 94% of all alleles. only small allele frequency differences were observed between the two control populations, whereas the type 1 vwd population showed an increase of allele 5 (3.2%) and a decrease of allele 7 (3.0%) compared with the control populations (table 3 and fig 1) . the allele frequencies observed for each population were used to calculate the expected genotype frequencies and these were subsequently compared with the observed genotype frequencies. with seven different possible alleles (alleles 3-9), corresponding to a total of 28 different possible genotypes, this could potentially result in a very complex table. as a result of alleles 5, 6 and 7 in all possible pairwise combinations together explaining 89% of all genotypes, table 4 only shows the results for these six genotypes. the differences between the expected and observed genotype frequencies within each control population were generally small. the average deviation was 1.0%, with a maximum deviation of 2.6% for genotype 67 in c 2 . also, the observed differences between the c 1 and c 2 populations were small, with an average deviation of 1.0% and a maximum value of 1.4%. contrary to this pattern, the type 1 vwd population showed three larger differences between the observed and expected genotype frequencies: surpluses of genotypes 57 (6.4%; p = 0.27) and 67 (6.2%; p = 0.18) and a deficiency of genotype 77 (5.8%; p = 0.31). the average deviation for the remaining genotypes was 2.4%. comparing the observed genotype frequencies of the type 1 vwd population with the average of the c 1 and c 2 populations resulted in surpluses of genotypes 57 (7.4%; p = 0.13) and 67 (3.1%; genetic variation in clec4m in type 1 vwd patients p = 0.41) and a deficiency of genotype 77 (8.4%; p = 0.08) ( table 4 ). thus, the pattern of deviating genotype frequencies observed within the type 1 vwd population corresponds to the pattern observed for the comparison of the type 1 vwd population versus the control populations. the type 1 vwd, c 1 and c 2 populations were genotyped using rs868875, rs2277998, rs8113469 and rs62128260 and the vntr locus and the genotype data were used to construct combined snp and vntr haplotypes for all three populations. two single haplotypes, one containing vntr allele 3 and one with allele 8, were excluded. the remainder consisted of 19 haplotypes and six of these showed frequencies > 1% in the type 1 vwd population (table 5) . a number of observations can be made: 1) each vntr allele was primarily associated with one specific snp haplotype. the only exception to this pattern was vntr allele 7 that was associated with two relatively common snp haplotypes. 2) all haplotypes with vntr allele 4 had the same snp haplotype (gatt). this haplotype was also the dominating haplotype for vntr allele 5. 3) all haplotypes with vntr allele 9 (with one exception) had the same snp haplotype (agct). this haplotype was also one of the two dominating haplotypes for vntr allele 7. 4) all snp-vntr haplotypes present at frequencies > 1% were present in both the type 1 vwd population and the control populations at similar frequencies. 5) the major frequency differences between the type 1 vwd population and control populations were seen for the 5gatt (surplus of 3.5% in type 1 vwd population) and for the 7agtt haplotype (deficiency of 3.4% in type 1 vwd population). genetic variation in clec4m has been associated with variation in plasma levels of vwf in healthy individuals [4] . it has also been shown that the clec4m protein binds to and internalizes vwf, and variants in clec4m contribute to the variability of vwf plasma levels in type 1 vwd patients [9, 10] . family-based association analysis of 318 type 1 vwd patients and 173 unaffected family members showed excess transmission of vntr allele 6 to unaffected individuals and an association of this allele with increased vwf:rco [9] . the rs868875 variant in clec4m showed association with both vwf level and activity in 364 type 1 vwd patients from the netherlands [10] . thus, clec4m was suggested to be causally involved in the development of type 1 vwd. the authors suggested a mechanism where certain variants of this protein are more efficient in clearing vwf from the circulation than others. an explanation model where the protein is more efficient in clearing vwf from the circulation may mean that the clec4m gene exists in a number of forms with differences in their level of expression or with differences in their affinity for vwf. such differences may depend upon common mutations that are present for example in the promoter, the 3´utr, the neck region or the ligand binding domain of the protein. that is, certain haplotypes are expected to be more common in patients compared with controls if common variants are operating to cause type 1 vwd. if rare variants contribute to type 1 vwd, an accumulation of rare variants in patients relative to controls is expected. the present study analyzed a historical vwd population. this may have introduced a bias compared with the analysis of vwd populations using contemporary definitions. a recent study in the united states investigated 482 patients historically diagnosed with type 1 vwd [25] . when these patients were retested 172 patients did not meet the current diagnostic criteria for type 1 vwd or low vwf level (vwf:ag < 50 iu/dl or vwf:rco < 53 iu/dl). there was also no difference in bleeding score whether or not the current criteria were fulfilled. complete vwf resequencing showed that 45% of the 482 patients with historical type 1 vwd diagnosis carried a rare variant in the vwf gene compared to 62% of the 310 patients fulfilling the modern criteria. thus, this type 1 vwd population is very similar to our type 1 vwd population [18] . the five new variants detected in the promoter, introns and 3´utr were all rare (maf < 0.01), with three out of five variants detected in a single chromosome. they could therefore potentially explain only a very minor fraction of the occurrence of the disease, since the rare new variants have no obvious functional consequences and were all present in patients already carrying bona fide vwf mutations [18] . however, several earlier reports associate the common snv rs868875 with low vwf levels in healthy subjects and type 1 vwd patients. similarly to these reports, the maf of rs868875 was increased in the present study population (5.8%; p = 0.10) though the increase failed to reach significance. except for rs2277998, no missense or nonsense mutations were detected. this particular variant was also present at a higher frequency in the type 1 vwd population compared with the swedish control populations (0.364 versus 0.315; p = 0.21). assuming that this amino acid change may somehow alter the function of the ligand binding domain of the clec4m protein, resulting in a higher affinity for and more efficient binding of vwf molecules in the bloodstream, the increased allele frequency in the type 1 vwd population relative to the control populations (4.9%) was compatible with an explanation model where rs2277998 contributes to type 1 vwd. when the vntr allele and genotype frequencies were compared in two different swedish control populations only small frequency differences were observed, whereas the type 1 vwd population showed larger differences compared with the control populations for some alleles and genotypes; e.g. genotypes 57 (7.4%) and 77 (-8.4%). in addition, when the allele frequencies were used to calculate the expected genotype frequencies and these were then compared with the observed frequencies only small frequency differences were observed in the two control populations. again, the type 1 vwd population showed several distinct genotype frequency differences for genotypes involving the alleles 5 and 7, namely genotypes 57, 67 and 77 at frequency differences of 6.4%, 6.2% and -5.8%, respectively. thus, the frequency differences were increased when going from alleles to genotypes. in addition, both heterozygous genotypes were observed at higher frequencies than expected, whereas the homozygous genotype was observed at a lower frequency than expected. this strongly indicates an association between vntr heterozygosity and type 1 vwd. earlier reports have reported associations between rs868875 and type 1 vwd [9, 10] and between vntr allele 6 and type 1 vwd [9] . strong ld between rs868875, rs2277998 and the vntr locus complicates determining whether either or all of them contribute to type 1 vwd. from a naïve functional analysis, there is no obvious explanation indicating an effect of rs868875 since it is intronic, but both rs2277998 and the vntr might affect the affinity for vwf. previous biochemical studies have shown large effects on tetramerization and stability of tetramers due to the different sizes of vntr alleles. vntr alleles with six or more repeats readily tetramerize whereas allele 5 gives rise to equilibrium between monomers and tetramers. alleles of length four do not tetramerize at all [26] . other studies have shown differences in in vitro binding affinity due to differing neck length both with regard to carbohydrates [27] and to vwf itself [9] . several reports also link neck length variation to variation in infection rates for viruses such as hiv [12] and sars-cov [13] both on the allelic and genotypic levels; other reports indicate that the hiv transfection efficiency is due to the number of clec4m proteins on the cell surface [28] . specifically, the number of proteins on the cell surface depends on combinations of vntr (alleles 5, 7, 9) and snp (rs2277998; a, g) alleles. they showed that constructs containing 5a and 7g gives rise to the highest numbers of proteins on the cell surface. these combinations are in almost complete linkage disequilibrium in both cases and controls in the present study. the largest enrichment between patients and controls found in the present study is the higher frequency of the 57 vntr genotype (7.4%). in addition, this genotype is more increased than what would be expected from the increase in vntr allele frequencies alone (6.4%). unfortunately, the study by zhu et al. [28] did not include heterozygote combinations of these two alleles compared with the respective homozygotes. also, the strong ld makes it impossible to differentiate the effects of the vntr and the rs2277998 snp and there is a lack of studies of the functional effect of the asp > asn substitution caused by rs2277998 with regards to vwf. but since this missense variant was predicted to be tolerated by the three functional prediction programs sift, polyphen and mutationtaster it seems to be a less likely candidate than the vntr genotypes. earlier work involving clec4m and its relation to infectious disease has generated much debate regarding reported associations [13, 29, 30] . the analysis is complicated by the fact that the ligand binding domain shows large population-specific variation and seems to be under balancing selection [15] , which makes any analysis vulnerable to undetected population stratification and confounding factors such as selection driven by different microbial interactions. in this study two control populations were used to investigate whether there is any population stratification. both populations were collected from the same geographical area as the type 1 vwd population. the genetic variation in the two control populations was very similar. in contrast to the type 1 vwd population they showed no skewness with regards to the vntr genotypes, while the type 1 vwd population was highly skewed in favour of heterozygotes. nonetheless, the rather small effect size and the multi-allelic structure of the vntr require larger studies or a meta-analysis to determine the impact of the vntr polymorphisms on type 1 vwd. previous studies have shown that vwf levels of individuals with blood group o are reduced by 30% in comparison to non-o individuals and that blood group o is overrepresented in type 1 vwd patients [2] . in the present study 75 index cases (71% of 106 type 1 vwd index cases) were of type o, clearly higher than the swedish national average of~40% type o. compatible with other studies, no overrepresentation of type o was found in index cases carrying a type 2 vwd mutation (25%). thus, the patients analyzed had a higher incidence of blood group o as would be expected. this verifies that the patients being analyzed in the present study are enriched for factors other than bona fide vwf mutations that affect the vwf levels. since blood group o is by far the strongest of these factors the corresponding enrichment is obvious. the additional factors that have been identified and confirmed in a number of studies show considerably lower individual effects of~5%. one of these other factors is genetic variation in clec4m. our results are compatible with previous findings and confirm the role of certain vntr genotypes and the common missense variant. the resequencing in the present study could not detect any obvious sign of an additional contribution from rare variants that individually could have a larger effect on the phenotype. however, smaller effects cannot be excluded due to the small population size. this means that the clec4m effect on the vwf level in the majority of cases is limited to the contribution from the common haplotype. supporting information s1 von willebrand disease: advances in pathogenetic understanding, diagnosis, and therapy the genetic basis of von willebrand disease clearance of von willebrand factor novel associations of multiple genetic loci with plasma levels of factor vii, factor viii, and von willebrand factor: the charge (cohorts for heart and aging combined analysis of three genome-wide association studies on vwf and fviii plasma levels macrophage lrp1 contributes to the clearance of von willebrand factor linkage analysis identifies a locus for plasma von willebrand factor undetected by genome-wide association effect of genetic variation in stxbp5 and stx2 on von willebrand factor and bleeding phenotype in type 1 von willebrand disease patients the c-type lectin receptor clec4m binds, internalizes, and clears von willebrand factor and contributes to the variation in plasma von willebrand factor levels clec4m and stxbp5 gene variations contribute to von willebrand factor level variation in von willebrand disease the origin and evolution of variable number tandem repeat of clec4m gene in the global human population the vntr polymorphism of the dc-signr gene and susceptibility to hiv-1 infection: a meta-analysis homozygous l-sign (clec4m) plays a protective role in sars coronavirus infection determination of dc-sign and dc-signr repeat region variations the heritage of pathogen pressures and ancient demography in the human innate-immunity cd209/cd209l region dc-sign and dc-signr genetic diversity among different ethnic populations: potential implications for pathogen recognition and disease susceptibility genetic analysis of 31 swedish type 1 von willebrand disease families reveals incomplete linkage to the von willebrand factor gene and a high frequency of a certain disease haplotype genetic variation in the von willebrand factor gene in swedish von willebrand disease patients. th open origin of swedish hemophilia a mutations plink: a tool set for wholegenome association and population-based linkage analyses haploview: analysis and visualization of ld and haplotype maps predicting the effects of coding non-synonymous variants on protein function using the sift algorithm predicting functional effect of human missense mutations using polyphen-2 mutationtaster evaluates disease-causing potential of sequence alterations clinical and laboratory variability in a cohort of patients diagnosed with type 1 vwd in the united states all but the shortest polymorphic forms of the viral receptor dc-signr assemble into stable homo-and heterotetramers geometry and adhesion of extracellular domains of dc-signr neck length variants analyzed by force-distance measurements influence of polymorphism in dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin-related (dc-signr) gene on hiv-1 transinfection lack of support for an association between clec4m homozygosity and protection against sars coronavirus infection lack of support for an association between clec4m homozygosity and protection against sars coronavirus infection key: cord-347465-yu6oj30v authors: kurskaya, olga; ryabichenko, tatyana; leonova, natalya; shi, weifeng; bi, hongtao; sharshov, kirill; kazachkova, eugenia; sobolev, ivan; prokopyeva, elena; kartseva, tatiana; alekseev, alexander; shestopalov, alexander title: viral etiology of acute respiratory infections in hospitalized children in novosibirsk city, russia (2013 – 2017) date: 2018-09-18 journal: plos one doi: 10.1371/journal.pone.0200117 sha: doc_id: 347465 cord_uid: yu6oj30v background: acute respiratory infections (aris) cause a considerable morbidity and mortality worldwide especially in children. however, there are few studies of the etiological structure of aris in russia. in this work, we analyzed the etiology of aris in children (0–15 years old) admitted to novosibirsk children’s municipal clinical hospital in 2013–2017. methods: we tested nasal and throat swabs of 1560 children with upper or lower respiratory infection for main respiratory viruses (influenza viruses a and b, parainfluenza virus types 1–4, respiratory syncytial virus, metapneumovirus, four human coronaviruses, rhinovirus, adenovirus and bocavirus) using a rt-pcr kit. results: we detected 1128 (72.3%) samples were positive for at least one virus. the most frequently detected pathogens were respiratory syncytial virus (358/1560, 23.0%), influenza virus (344/1560, 22.1%), and rhinovirus (235/1560, 15.1%). viral co-infections were found in 163 out of the 1128 (14.5%) positive samples. we detected significant decrease of the respiratory syncytial virus-infection incidence in children with increasing age, while the reverse relationship was observed for influenza viruses. conclusions: we evaluated the distribution of respiratory viruses in children with aris and showed the prevalence of respiratory syncytial virus and influenza virus in the etiological structure of infections. this study is important for the improvement and optimization of diagnostic tactics, control and prevention of the respiratory viral infections. we detected 1128 (72.3%) samples were positive for at least one virus. the most frequently detected pathogens were respiratory syncytial virus (358/1560, 23.0%), influenza virus (344/1560, 22.1%), and rhinovirus (235/1560, 15.1%). viral co-infections were found in 163 out of the 1128 (14.5%) positive samples. we detected significant decrease of the respiratory syncytial virus-infection incidence in children with increasing age, while the reverse relationship was observed for influenza viruses. we evaluated the distribution of respiratory viruses in children with aris and showed the prevalence of respiratory syncytial virus and influenza virus in the etiological structure of plos acute respiratory infections (aris) pose a significant public health problem worldwide, causing considerable morbidity and mortality among people of all age groups [1] . children are on average infected two to three times more frequently than adults. [2] . there are more than 200 respiratory viruses that can cause aris. human respiratory syncytial virus (hrsv), human rhinovirus (hrv), human metapneumovirus (hmpv), human parainfluenza virus (hpiv), human enterovirus (ev), influenza virus (ifv), human coronavirus (hcov), adenovirus (hadv), and human bocavirus (hbov) are the most common viral agents associated with aris, accounting for around 70% of aris [3, 4] . the frequency of mixed respiratory viral detection varies from 10% to 30% in hospitalized children [5] [6] [7] . in addition, several new human respiratory viruses have been described in recent years, including human metapneumovirus [8, 9] , human bocavirus [10] , and novel human coronaviruses, including severe acute respiratory syndrome coronavirus (sars-cov) [11] , human coronaviruses nl63 (hcov-nl63), hku1 (hcov-hku1) [12] , and middle east respiratory syndrome coronavirus (mers-cov) [13] . although the majority of aris are associated with respiratory viruses, antibiotics are often used in the clinical treatment of aris. as children with aris often have similar clinical symptoms, studying the clinical hallmarks of children with virus-related aris and the spectrum of respiratory viruses will help in developing more accurate treatments for aris [14] . rapid diagnosis is important not only for timely treatment starting but also for the detection of a beginning influenza epidemic and the avoidance of unnecessary antibiotic treatment [15, 16] . western siberia plays a key role in ecology, epizootiology and epidemiology of emerging diseases. this territory was involved in the circulation of a/h5n1 and a/h5n8 avian influenza viruses in 2005-2017 [17, 18] . these viruses were spread by wild birds' migration. in western siberia migratory flyways of birds' wintering in different regions of the world: south east asia, central asia, middle east, hindustan, europe, and africa-cross. for this reason, there is high probability of the emergence of humans and animal influenza viruses reassortants, as well as emergence of local outbreaks of human morbidity caused by uncommon variants of influenza viruses. furthermore, novosibirsk is the largest transport hub in this part of russia with numerous international connections, that is important for the spread of aris [19, 20] . the prevalence of respiratory viruses among children with aris differs in different regions and varies over time [21] [22] [23] [24] [25] . thus, to better understand the epidemiology of acute respiratory infections in novosibirsk region, we investigated etiology of aris in children admitted to novosibirsk children's municipal clinical hospital in 2013-2017. all aspects of the study were approved by the ethics committee of the federal state budgetary institution "research center of clinical and experimental medicine" (2013-23). accordingly, written informed consent was obtained from parents prior to sample taking. children 0-15years of age were enrolled in the study within 3 days of illness onset and had at least two of the following symptoms: fever, sore throat, cough, rhinorrhea, nasal congestion, sputum, shortness of breath, lung auscultation abnormalities, tachypnea, and chest pain. paired nasal and throat swabs were collected from each patient admitted to novosibirsk children's municipal clinical hospital by hospital nurses. a total of 1560 samples collected during four epidemic seasons of 2013-2017 (october-april) were enrolled to the study. the epidemiological and clinical information including case history, symptoms, physical signs, and examination were included in a standardized questionnaire. the samples were placed immediately in viral transport medium (eagle mem, bsa and antibiotics) and stored at 4-8˚c prior transportation to the laboratory (not more than 24 hours). detection of respiratory viruses was performed immediately after delivery to the laboratory. all specimens were tested for 15 common respiratory viruses, including influenza virus types a, b (ifva and ifvb), human parainfluenza virus (hpiv) types 1-4, human respiratory syncytial virus (hrsv), human metapneumovirus (hmpv), four human coronaviruses (hco), human rhinovirus (hrv), human adenovirus (hadv) and human bocavirus (hbov), using a real-time pcr assay-kit. viral nucleic acids were extracted from nasal and throat swabs using rna/dna extraction kit «ribo-sorb» (interlabservice, russia) according to the manufacturer's instructions. the extracted viral nucleic acid was immediately used to perform the reaction of reverse transcription using commercial kit "reverta-l" (interlabservice, russia). detection of respiratory viruses, including hpiv 1-4, hrsv, hmpv, hcov-oc43, hcov-229e, hcovnl63, hcov-hku1, hrv, hadv, and hbov was performed using a rt-pcr kit «amplisens arvi-screen-fl» (interlabservice, russia), and ifva and ifvb virus detection was performed using a rt-pcr kit « amplisens influenza virus a/b-fl» (interlabservice, russia) according to the manufacturer's instructions. positive and negative controls were included in each run. two-tailed chi-square test (two by two table) was performed to compare the infection rates for respiratory viruses among different age groups. p-value <0.05 was considered to be statistically significant. totally, 1560 samples collected from patients with aris during the period from december 2013 to april 2017 were enrolled in the investigation. there were 824 males (52.8%) and 736 females (47.2%), and the patient's ages ranged from 3 months to 15 years. the most numerous age group (43.2%) was between 1 and 3 years old. the age distribution is shown in table 1 . the incidence of respiratory viral infection between boys (601/824; 72.9%) and girls (527/736; 71.6%) (χ 2 = 0.345, p>0.05). the respiratory virus positive rate appeared to decrease with age. the lowest positive rate was observed in the age group of more than 6 years old (167/302; 55.3%), while the positive rates in age groups less than 6 years old were more than 70% (table 1 ). statistically significant difference was observed between the age group of more than 6 years old and other age groups (χ 2 = 54.113, p<0.01). no statistically significant difference was observed among the age groups less than 1 year old, 1-3 years, and 4-6 years. among all the samples, single infections accounted for 61.9% (965/1560), while co-infections accounted for 10.4% (163/1560) with the lowest rate of incidence in children more than 6 years old compared to children younger than 6 years (χ 2 = 20.389, p<0.01) (fig 1) . hrsv and ifv were the most frequently detected viruses with high incidence of 23.0% (358/ 1560) and 22.1% (344/1560), respectively, among all patients with aris. hrv was detected in 15.1% (235/1560), followed by hmpv, hpiv and hbov with the detection rates higher than 5.0%. the positivity rates of hcov and hadv were lower than 5.0% (fig 2) . the data were analyzed with regard to the age and gender distribution of virus infection. no difference in the etiological distribution of viral pathogens was observed between males and females. among detected respiratory viruses, hrv, hpiv, hcov, and hadv did not have statistically significant difference in the distribution among the different age groups. hmpv was detected in age group less than 1 year old much more frequently than in children 1-3 years old (χ 2 = 4.986, p<0.05) and 7-15 years old (χ 2 = 8.174, p<0.05). hbov was significantly more frequently observed in children younger than 3 years old compare with children of 4-15 years old (χ 2 = 28.523, p<0.005). the incidence of hrsv decreased significantly with increasing age (p < 0.05) dropping from 35.1% in children younger than 1 year old to 5.3% in the school-age children (7-15 years old group), while the reverse relationship was observed for ifv (fig 3) . the data were analyzed with regard to the seasonality. overall, virus detection positive rate did not differ significantly between seasons, ranging from 71.3% to 73.3%. co-infections with two or more viruses were detected in 163 out of the 1128 (14.5%) positive samples (table 1) . dual infections accounted for 11.4% (129/1128) of all positive samples and acute respiratory infections are a serious health and economic problem, causing high morbidity and significant economic losses due to temporary disability payments and medical costs. children are the most vulnerable group to the development of the disease. as several authors mentioned previously [26] , aris can lead to serious diseases such as bronchiolitis and pneumonia and sometimes even cause death in infants and children worldwide. nevertheless, most of the data on the epidemiological features and etiological structure of aris were from moredeveloped countries, and less is known about the etiology of aris in russia. in the present study, we examined the viral etiology of aris in hospitalized children in novosibirsk by real-time rt-pcr assay. we detected at least one of the tested viruses in 72.3% (1128/1560) of the samples. in similar studies conducted in different regions of the world, the virus detection rate ranged from less than 50% to 75% [27] [28] [29] . for example, in studies performed in china, the proportion of positive samples in children with aris ranged from 37.6% to 78.7% [15, 17, 30, 31] . the previous study of respiratory infections among children in european part of russia revealed the 71.5% detection rate of respiratory viruses [32] . the percentage of the respiratory viruses' detection varies in different years in different regions, which may be associated with climatic and environmental factors, population distribution, economic status and diagnostic methods used [1] . in addition, seasonality of sampling can also lead to differences in the level of viruses' detection in various studies. thus, ju x. et al. carried out a study continuously from july 2011 through july 2013 and found 48.66% of samples to be positive for at least one respiratory virus [33] . in contrast, we collected samples only during epidemic seasons of aris, so the positivity rate in our study was considerably higher. furthermore, acute respiratory infections can be caused by viruses that are not yet known, as well as bacteria that have not been included in these studies [14] . we found that the prevalence of respiratory viruses did not differ between boys and girls (72.9% and 71.6% respectively), which confirms the absence of a gender-based susceptibility to respiratory viral infections [14] . however, we observed a decrease in the respiratory viruses' detection rate with age, with the lowest detection rate in school-age children compared to children under 7 years of age (55.3% versus 76.4%, respectively). these data are consistent with findings obtained in other regions of russia [32] and it may be due to decreased sensitivity to respiratory virus infections in older children. etiology of aris and the respiratory virus prevalence varies in different studies. in the united states, ifv, hrsv and hpiv were the most frequently detected [34] . studies conducted in france have shown that metapneumovirus and respiratory syncytial virus are the most common [35] . ifv, hrsv and hrv were the most commonly detected respiratory viruses among children with aris in most regions of china [14] . the study of aris etiologic structure showed that hrsv, hrv, hpiv and ifv were registered significantly often among children in western part of russia [32] . in our study the most common viruses detected were hrsv and ifv, followed by hrv. the age distribution of aris indicated that children under 3 years old were more likely to be infected by hrsv confirmed the importance of rsv in children with aris, especially in children < 4 years of age [36] [37] [38] [39] . we observed a high rate of hrsv-detections in 2013-2014 (44.4%), while in 2016-2017 it was less than 10% which could be due to the annual variation in the circulation pattern of hrsv. such year-to-year variation in the epidemiological patterns of viral infections confirms importance of the long-term study of the aris epidemiology [40] . influenza virus is one of the major causative agents of respiratory disease in humans and may lead to serious illness [41] . in temperate countries influenza outbreaks usually occur during the winter season. finally, in our study, ifv (344/1560, 22.1%) was the second most frequent detected pathogen with markable seasonality in winter months. during the 2013-2014 epidemic season, influenza virus detection rate was low-6.9% of all respiratory viruses, which was in accordance with the official influenza surveillance results of ministry of health, russia [42] . in our study in 2014-2015, influenza viruses were detected in 17.2% among all respiratory viruses. among those, the percentage of influenza a virus accounted for 73.3% and influenza b virus made up 26.7% of all detected influenza viruses. at the same time, in russia influenza b was the main etiological agent, accounting for 50.6% of all detected influenza viruses. in 2015-2016, influenza a(h1n1)pdm09 virus was predominant in russia, accounting for 79% of all influenza-positive samples consistent with our results (82%). in 2016-2017 influenza a(h3n2) virus was dominant in russia detected in 61.3% of all influenza virus cases [42] . in our study influenza a and b viruses were detected with approximately the same frequency (48% and 52% respectively). with the introduction of molecular techniques, the detection of multiple co-infecting viruses has become common, though the prevalence of each virus varies between studies [43]. in our study detection rate of viral co-infection was 14.5% among the positive samples. according to the previous reports, the incidence of viral co-infection in children can reach 30% [44] . co-infection is most often found in children under the age of 5, due to the immaturity of the immune system and, thus, greater susceptibility to infection [45] . in our study, we significantly more frequently detected cases of simultaneous infection with two or more viruses in children under 7 years of age compared with children of school age (12.2% versus 3.4%), while there was no significant difference in the incidence of viral co-infection between the age groups of 0-1 year, 1-3 years and 4-6 years. in conclusion, in our study we investigated the etiological structure of acute respiratory viral infections in hospitalized children in novosibirsk, russia, and evaluated age and seasonal distribution of the various respiratory viruses. systematic monitoring of respiratory viruses is necessary to better understand the structure of respiratory infections. such studies are important for the improvement and optimization of diagnostic tactics, as well as measures for the control and prevention of the respiratory viral infections. prevalence of respiratory viruses among children hospitalized from respiratory infections in shenzhen, china estimates of world-wide 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viral co-infections in pediatric patients hospitalized with lower tract acute respiratory infections clinical disease severity of respiratory viral co-infection versus single viral infection: a systematic review and meta-analysis surveillance of 16 respiratory viruses in patients with influenza-like illness in nanjing we thank the clinicians of novosibirsk children's municipal clinical hospital for their assistance with sample collection. we thank dr. galina skosyreva and elena timofeeva (department of propaedeutic of childhood diseases, novosibirsk state medical university, novosibirsk, russia) for their assistance with sample collection. key: cord-353596-8iqjugcx authors: bédubourg, gabriel; le strat, yann title: evaluation and comparison of statistical methods for early temporal detection of outbreaks: a simulation-based study date: 2017-07-17 journal: plos one doi: 10.1371/journal.pone.0181227 sha: doc_id: 353596 cord_uid: 8iqjugcx the objective of this paper is to evaluate a panel of statistical algorithms for temporal outbreak detection. based on a large dataset of simulated weekly surveillance time series, we performed a systematic assessment of 21 statistical algorithms, 19 implemented in the r package surveillance and two other methods. we estimated false positive rate (fpr), probability of detection (pod), probability of detection during the first week, sensitivity, specificity, negative and positive predictive values and f(1)-measure for each detection method. then, to identify the factors associated with these performance measures, we ran multivariate poisson regression models adjusted for the characteristics of the simulated time series (trend, seasonality, dispersion, outbreak sizes, etc.). the fpr ranged from 0.7% to 59.9% and the pod from 43.3% to 88.7%. some methods had a very high specificity, up to 99.4%, but a low sensitivity. methods with a high sensitivity (up to 79.5%) had a low specificity. all methods had a high negative predictive value, over 94%, while positive predictive values ranged from 6.5% to 68.4%. multivariate poisson regression models showed that performance measures were strongly influenced by the characteristics of time series. past or current outbreak size and duration strongly influenced detection performances. public health surveillance is the ongoing, systematic collection, analysis, interpretation, and dissemination of data for use in public health action to reduce morbidity and mortality of health-related events and to improve health [1] . one of the objectives of health surveillance is outbreak detection, which is crucial to enabling rapid investigation and implementation of control measures [2] . the threat of bioterrorism has stimulated interest in improving health surveillance systems for early detection of outbreaks [3, 4] reemergence of infectious diseases such as middle east respiratory syndrome due to new coronavirus (mers-cov) in 2012 [5] or ebola in west africa in 2014 [6] . nowadays, a large number of surveillance systems are computer-supported. the computer support and statistical alarms are intended to improve outbreak detection for traditional or syndromic surveillance [7, 8] . these systems routinely monitor a large amount of data, recorded as time series of counts in a given geographic area for a given population. they produce statistical alarms that need to be confirmed by an epidemiologist, who determines if further investigation is needed. one limitation of these detection systems is an occasional lack of specificity, leading to false alarms that can overwhelm the epidemiologist with verification tasks [9, 10] . it is thus important to implement statistical methods that offer a good balance between sensitivity and specificity in order to detect a large majority of outbreaks without generating too many false positive alarms. in the literature, a broad range of statistical methods has been proposed to detect outbreaks from surveillance data. the main statistical approaches have been reviewed by shmueli et al. [11] and unkel et al. [12] . by restricting these reviews to the methods that allow temporal detection of outbreaks without integrating the spatial distribution of cases, the general principle is to identify a time interval in which the observed number of cases of an event under surveillance (i.e. the number of reported cases) is significantly higher than expected. this identification is mainly based on a two-step process: first, an expected number of cases of the event of interest for the current time unit (generally a week or a day) is estimated and then compared to the observed value by a statistical test. a statistical alarm is triggered if the observed value is significantly different from the expected value. the main difference between statistical methods lies in how the expected value is estimated, which is most often done using statistical process control or regression techniques or combination of both [12] . a major constraint to the practical implementation of these methods is their capacity to be run on an increasing number of time series, provided by multiple sources of information, and centralized in large databases [3, 13, 14] . monitoring a large number of polymorphic time series requires flexible statistical methods to deal with several well-known characteristics observed in time series: the frequency and variance of the number of cases, secular trend and one or more seasonality terms [14] . even if some authors proposed to classify time series into a small number of categories and sought suitable algorithms for each category, in this automated and prospective framework, statistical methods cannot easily be fine tuned by choosing the most appropriate parameters adapted to each time series in an operational way, as explained by farrington et al. [15] . a key question for public health practitioners is what method(s) can be adopted to detect the effects of unusual events on the data. some authors have proposed a systematic assessment of the performances of certain methods in order to choose one reference algorithm [16] [17] [18] [19] [20] . they assessed these methods on a real dataset [16, 21] , a simulated dataset [18-20, 22, 23] or on real time series for which simulated outbreaks were added [24, 25] . simulating data offers the advantage of knowing the exact occurrence of the simulated outbreaks and their characteristics (amplitude, etc.). for example, lotze et al. developed a simulated dataset of time series and outbreak signatures [26] . in the same way, noufaily et al. [9] proposed a thorough simulation study to improve the farrington algorithm [15] . guillou et al. [27] compared the performance of their own algorithm to that of the improved farrington, using the same simulated dataset. this dataset was also used by salmon et al. to assess their method [28] . to our knowledge, no study has been proposed to thoroughly evaluate and compare the performance of a broad range of methods on a large simulated dataset. the objective of this paper is to evaluate the performance of 21 statistical methods applied to large simulated datasets for outbreak detection in weekly health surveillance. the simulated dataset is presented in section 2. the 21 evaluated methods and performance measures are described in section 3. evaluations and comparisons are presented in section 4. a discussion follows in the last section. we simulated data following the approach proposed by noufaily et al. [9] . first, simulated baseline data (i.e. time series of counts in the absence of outbreaks) were generated from a negative binomial model of mean μ and variance ϕμ, ϕ being the dispersion parameter !1. the mean at time t, μ(t), depends on a trend and seasonality modeled using fourier terms: time series were simulated from 42 parameter combinations (called scenarios and presented in table 1 in [9] ) with different values taken by θ, β, γ 1 , γ 2 , m and ϕ, respectively associated with the baseline frequency of counts, trend, seasonality (no seasonality: m = 0, annual seasonality: m = 1, biannual seasonality: m = 2) and the dispersion parameter. for each scenario, 100 replicates of the baseline data (time series with 624 weeks) were generated. we thus obtained 42 × 100 = 4200 simulated time series. the last 49 weeks of each time series were named current weeks. the evaluated algorithms were run on these most recent 49 weeks. performance measures described below were computed based on detection during these 49 weeks. secondly, for each time series, five outbreaks were simulated. four outbreaks were generated in baseline weeks. each outbreak started at a randomly drawn week and we generated the outbreak size (i.e. the number of outbreak cases) as poisson with mean equal to a constant k 1 times the standard deviation of the counts observed at the starting week. the fifth outbreak was generated in the current weeks in the same manner, using another constant noted k 2 . we chose the values of k 1 to be 0, 2, 3, 5 and 10 in baseline weeks and k 2 from 1 to 10 in current weeks as in [9] . finally, outbreak cases were randomly distributed according to a lognormal distribution with mean 0 and standard deviation 0.5. a total of 231,000 time series were generated from the 42 scenarios: 21,000 time series during the first step of simulation process (42 × 100 duplicates × 5 values for k 1 ), and 210,000 time series during the second step of simulation process (21,000 × 10 values for k 2 ), leading to a large simulated dataset including a great variety of time series, as observed in real surveillance data. at the end of the simulation process, 10,290,000 current weeks were generated, among which 6.2% were classified as outbreak weeks as they were included in an outbreak. we studied 21 statistical methods, 19 of which were implemented in the r package surveillance [29, 30] : • cusum variants: original cusum [29, 32] , a rossi approximate cusum [32] , a cusum algorithm for which the expected values are estimated by a glm model [29] , a mixed rossi approximate cusum glm algorithm [29] , • the original farrington algorithm [15] and the improved farrington algorithm [9] , • a count data regression chart (glrnb) [29, 33] and a poisson regression chart (glr poisson) [29, 34] , • the outbreakp method [35] , • ears c1, c2 and c3 algorithms [19, 36] for all simulated time series, we used the tuning parameters recommended by their authors for each algorithm when available and proposed by default in the package surveillance. the commands used from the r package surveillance and the control tuning parameters chosen for these 19 algorithms are presented in table 1 . we also proposed two additional methods not implemented in the package surveillance: • a periodic poisson regression where μ(t) is defined as in eq (1). the threshold is the 1 − α quantile of a poisson distribution with mean equal to the predicted value at week t. • a periodic negative binomial regression, also defined as in eq (1), where the threshold is the 1 − α quantile of a negative binomial distribution with mean equal to the predicted value at week t and a dispersion parameter estimated by the model. rki 3 algo.rki3() [29] glr negative binomial algo.glrnb() arl = 5, dir = "inc" [29, 33] glr poisson algo.glrpois() arl = 5, dir = "inc" [29, 34] ears c1 earsc() method = "c1", α 1 [19, 36] ears c2 earsc() method = "c2", α 1 [19, 36] ears c3 earsc() method = "c3", α 1 [19, 36] outbreakp algo.outbreakp() k = 100, ret = c("value") [35] these last two models were run on all the historical data. an alarm was triggered if the observed number of cases was greater than the upper limit of the prediction interval. these two methods are basic periodic regressions. the r code of these two algorithms is presented in the s24 appendix. we evaluated the performances of the methods with three different α values: α = 0.001, α = 0.01 and α = 0.05. we considered eight measures to assess the performance of the methods: • measure 1 is false positive rate (fpr). for each method and each scenario, we calculated the fpr defined as the proportion of weeks corresponding to an alarm in the absence of an outbreak, as in [9] . nominal fprs were 0.0005 for analyses with α = 0.001, 0.005 for analyses with α = 0.01 or 0.025 for analyses with α = 0.05. • measure 2 is probability of detection (pod). for each scenario and for each current week period, if an alarm is generated at least once between the start and the end of an outbreak, the outbreak is considered to be detected [9] . pod is an event-based sensitivity (i.e. the entire outbreak interval is counted as a single observation for the sensitivity measurement) and is thus the proportion of outbreaks detected in 100 replicates. • measure 3 is probability of detection during the first week (pod1week), which makes it possible to evaluate the methods' ability to enable early control measures. • measure 4 is observation-based sensitivity (se): outbreak weeks associated with an alarm were defined as true positive (tp), non-outbreak weeks without alarm as true negative (tn), outbreak weeks without alarm as false negative (fn) and non-outbreak weeks with alarm as false positive (fp). thus, se = tp/(tp+fn). • measure 5 is specificity (sp) defined as sp = tn/(tn+fp). unlike fpr which was calculated on current weeks without any simulated outbreak, specificity was calculated on the entire number of current weeks out of the 210 000 time series including current outbreaks. • measure 6 is positive predictive value (ppv) defined as: ppv = tp/(tp+fp). • measure 7 is negative predictive value (npv) defined as: npv = tn/(tn+fn). • measure 8 is f 1 -measure defined as the harmonic mean of the sensitivity and the ppv: in the result section, we proposed to calculate averaged performance measures, i.e. to calculate fpr on the overall 21,000 time series without outbreak during the current weeks, and to calculate the other performance measures on the overall 210,000 time series with simulated outbreaks during the current weeks. fpr was estimated prior to the simulation of current outbreaks, i.e. among the 49 current weeks for 21,000 (5 × 4,200) time series. other indicators (pod, pod1week, se, sp, ppv, npv) were estimated once outbreaks had been simulated, i.e. on the current weeks of all the time series (210,000 time series). for each α value, we proposed roc curve-like representation of these results with four plots representing sensitivity according to 1-specificity, pod and pod1week as functions of fpr, and sensitivity according to ppv. to identify the factors associated with the performance measures for α = 0.01 and assess the strength of associations, multivariate poisson regression models [38] were run, as in barboza et al. [39] or buckeridge et al. [40] . a set of covariates corresponding to the characteristics of the simulated time series was included: trend (yes/no), seasonality (no/annual/biannual), the baseline frequency coefficient θ, the dispersion coefficient ϕ and k 1 representing the amplitude and duration of past outbreaks. the last three covariates and k 2 were treated as continuous and modeled using fractional polynomials. the statistical methods were introduced as covariates to estimate performance ratios, i.e. the ratios of performances of two methods, adjusted for the characteristics of the time series represented by the other covariates. adjusted fpr, pod, pod1week, sensitivity, and specificity ratios were estimated with the improved farrington algorithm as reference. 95% confidence intervals were calculated with robust estimation of standard errors. for each continuous covariate modeled by fractional polynomials, ratios were presented for each value [41] . the simulation study, the implementation of the detection methods, and the estimations of performance were carried out using r (version 3.2.2), in particular using the package surveillance. poisson regression models used to identify the factors associated with the performance measures and to assess the strength of associations were run using stata 14. in this section, we present the averaged performances of each evaluated method, i.e. the performances irrespective of the scenario and of the characteristics of the time series. table 2 presents averaged fpr, specificity, pod, pod1week, sensitivity, negative predictive value, positive predictive value and f 1 -measure for all 42 scenarios and all past and current outbreak amplitude and duration and for α = 0.01. overall, fpr ranged from 0.7% to 59.9% and pod from 43.3% to 88.7%. methods with the highest specificity, such as the improved farrington method or the periodic negative binomial regression, presented a pod lower than 45% and a sensitivity lower than 21%. averaged measures for α = 0.001 and α = 0.05 are presented in s1 table and s2 table. rki 1-3, glr negative binomial, glr poisson, bayes 1-3 and outbreakp algorithms' performances do not vary with α values (see table 1 ). their performances are only reported in table 2 . for each method, a radar chart presenting the measures 1-7 for α = 0.01 is proposed in the s23 appendix. . two groups stand out from the rest. the first group consists of bayes 1, 2 and 3. these methods present the best pod (around 0.8) and pod1week with a fpr around 10%. the second group consists of the 4 cusum methods: cusum, cusum rossi, cusum glm, and cusum glm rossi. for α = 0.01, these methods present the best sensitivity (around 0.80) but the lowest specificity (0.55) and the highest fpr (0.40). note that while of the algorithm test statistics are based on the likelihood of single-week observations independent of recent ones, cusums are not, and they may be important for applications where detection of gradual events rather than one-week spikes is especially critical. the outbreakp method had the lowest specificity without having a better pod or pod1week than the first two groups. finally, a third group consists of the other methods that had good specificity (over 0.9) but a lower sensitivity, pod and pod1week than the first two groups. all 21 methods presented a high negative predictive value, greater than 94%. the ppv of outbreakp is very low (6.5%), while the periodic negative binomial glm method had the highest ppv (68.4%). a first attempt to visualize certain differences is to plot pod and fpr according to the scenario and the k 1 or k 2 values. to illustrate this, fig 2 shows the performances of the cdc method. the first row represents fpr for an increasing past outbreak constant k 1 = 0, 2, 3, 5 and 10 according to the 42 scenarios. the second row shows pod according to k 2 for the 42 scenarios (each curve corresponds to a simulated scenario) for an increasing past outbreak constant k 1 = 0, 2, 3, 5 and 10. it clearly shows that performance depends on the scenario. the same plots with tables presenting numerical values for each method and different α values are presented in the s2 appendix to s22 appendix. to better compare the 21 methods, we presented on a single display in the s1 appendix, their fpr according to the scenarios and their pod according to the k 2 values for k 1 = 5 and α = 0.01. to better understand which characteristics are associated with each performance and to compare each method with the improved farrington method, we present the results obtained from the multivariate poisson regression models in the next section. adjusted performance ratios and associated factors table 3 presents the adjusted performance ratios for performance measures 1 to 5 as described in the methods' section (α = 0.01 for improved farrington, original farrington, periodic poisson glm and neg binomial glm, cdc and ears c1-c3. α = 0.05 for bayes 1-3). evaluation and comparison of statistical methods for early temporal detection of outbreaks • adjusted fpr ratios decreased when the amplitude and duration (driven by k 1 in eq (1)) of past outbreaks increased. it is indeed more difficult to detect an outbreak when past outbreaks have occurred, especially when these outbreaks are large and when the method does not under-weight their influence to estimate the expected number of cases. adjusted fpr ratio was 2.75 times higher for time series with a secular trend than for the others. as we simulated time series with a non-negative trend (β ! 0 in eq (1)), it was expected that fpr would decrease with a trend, especially for methods which do not integrate a trend in the estimation of the expected number of cases. in the same way, annual seasonality-and biannual seasonality to an even greater extent-and overdispersion increased fpr. we observed a nonlinear relation between fpr and baseline frequency: fpr ratio increased from the lowest frequencies to 12 cases per week, then decreased for the highest frequencies, with no clear explanation. only periodic negative binomial glm presented a fpr lower than improved farrington fpr (fpr ratio = 0.71). adjusted fpr ratios of outbreakp and all cusum variants were higher than 40. another group of methods all presented fpr ratios below 10: cdc, rki variants, ears methods, periodic poisson glm, original farrington, bayes 2 and glr negative binomial. fpr ratios for other methods (bayes 1 and 3, and glr poisson) were between 10 and 17. • adjusted specificity ratios were almost all equal to 1 as the amplitude and duration of past outbreaks had little influence on specificity. they were significantly lower for time series with a secular trend (adjusted specificity ratio = 0.84) or with annual or biannual seasonality (respective ratios: 0.99 and 0.98). specificity decreased when dispersion increased but increased when the baseline frequency (θ in eq (1)) increased. only the periodic negative binomial glm presented a specificity as good as that of the improved farrington method (specificity ratio = 1.00). • the adjusted pod ratios significantly decreased when past outbreak amplitude and duration (k 1 ) increased, which is logical. they increased when current outbreak amplitude and duration (k 2 ) increased, which is also normal. pod was higher for time series with secular trends which can be explained by the positive trend. pod decreased when there was an annual or a biannual seasonality (respective pod ratio = 0.97 and 0.92). only the highest dispersion value (θ = 5) had an influence on pod (adjusted pod ratio = 1.09). bayes 1, 2 and 3, cusum variants and the glr poisson method presented the highest pod ratios, from 1.75 (glr poisson) to 1.95 (cusum glm). any method was less able to detect an outbreak than the improved farrington algorithm. • pod1week presented results that were similar to those of pod. adjusted pod1week ratios were significantly lower than those of pod for ears c3 (0.25 versus 1.25), for cdc (0.55 versus 1.04) and for glr negative binomial (1.17 versus 0.87). other methods presented ratios for pod1week that were similar to or greater than those of pod. • finally, similar results were observed for sensitivity and for pod. bayes 2 and 3 methods, outbreakp, rki 3, cusum variants and the glr poisson method presented the highest sensitivity ratios, from 2.04 (rki 3) to 3.89 (cusum glm). as observed in the pod model, any method was less able to detect an outbreak than the improved farrington algorithm. estimation from the multivariate regression models to explain ppv and npv are presented in s3 table. we presented a systematic assessment of the performance of 21 outbreak detection algorithms using a simulated dataset. one advantage of a simulation study for outbreak detection methods benchmarking is the a priori knowledge of the occurrence of outbreaks, which enables the developpment of a real "gold standard". some authors have already proposed that simulation studies be used to assess outbreak detection methods [18, 19, 23] , and others have suggested adding simulated outbreaks to real surveillance data baselines [16, 24, 25] , but without proposing a systematic assessment of the performance of a broad range of outbreak detection methods. choi et al. [20] proposed such a study design based on the daily simulation method proposed by hutwagner et al. [18] but do not study the influence of past outbreaks or time series characteristics (frequency, variance, secular trends, seasonalities, etc.), on methods performance. the simulated dataset we used to perform our study is large enough to include the considerable diversity of time series observed in real surveillance systems. we also simulated a high diversity of outbreaks in terms of amplitude and duration. in our opinion, this simulated dataset presents a high representativeness of real weekly surveillance data. to extend our results to daily surveillance data, it should be necessary to perform a similar study with daily surveillance data. these characteristics of the simulated dataset enabled us to propose simple intrinsic performance indicator estimations such as fpr and pod and sensitivity and specificity to compare the performance of the evaluated methods. furthermore, this allows us to compare our results to other studies based on the same dataset. negative predictive value and positive predictive value are proposed as operational indicators for decision making when an alarm is triggered, or not triggered, by an algorithm. a benefit of the addition of outbreaks to the baseline weeks is that outlier removal strategies considered by many authors may be objectively tested and evaluated. one limitation in the simulation process was the fact that only increasing secular trends were used. increasing secular trends would facilitate outbreak detection, while decreasing trends would hamper it. furthermore, our study was designed based on weekly surveillance, while syndromic surveillance systems are most often daily systems. in daily surveillance time series, other seasonalities such as the "day of the week" effect need to be taken into account, which is not the case in our study. the performance of the evaluated methods was only considered from a general perspective, in order to detect outbreaks in a large number of polymorphic weekly-based time series. in a pragmatic approach, it seems very difficult to adapt the tuning parameters of these methods for every time series. in france, public health agencies, such as the french national public health agency (santé publique france), the french agency for food, environmental and occupational health safety (anses) and the french armed forces center for epidemiology and public health (cespa) have deployed computer-supported outbreak detection systems in traditional or syndromic surveillance contexts [42] [43] [44] [45] . they monitor a broad range of time series on a daily or weekly basis without, however, having rigorously evaluated the algorithms implemented. in the same way, the performance of the methods varied according to different baseline profiles depending on trend, seasonality, baseline frequency and overdispersion. even if similar meta-models were already proposed by buckeridge et al. for example [40] , an original approach was to compare performance indicators adjusted for these parameters in a regression model. as expected, the adjusted performance of the 21 methods was penalized by increasing amplitude and duration in past outbreaks and by annual or biannual seasonality. conversely, performance was better for increasing amplitude and duration in current outbreaks to be detected. more generally, the methods' performance was highly dependent on simulation tuning parameters. we proposed various measures to monitor the performance of outbreak detection methods. false positive rate (fpr) and probability of detection (pod) were proposed by noufaily et al. [9] . we proposed an observation-based sensitivity measure and an event based sensitivity (pod). the concept of sensitivity based on alerting in each observation period is not applicable in some applications because signals of interest are intermittent and multimodal and may even be interpreted as multiple events. many of the algorithms are based on the likelihood of single-week observations independent of recent ones, but cusums are not, and the large sensitivity advantage in the cusums methods, diminished for pod and pod1week, may be a result of the way the outbreak effects are modeled. by contrast, the implementation of the pod measure is uniformly applicable. public health response to an outbreak depends on its early detection. in the pod definition, an outbreak was considered to be detected even if the first statistical alarm was issued during its last week. with the aim of estimating early detection performance, we also proposed pod during the first week, which cannot be considered alone, because even if it is done belatedly, an outbreak needs to be detected by the methods. while pod1week was an indicator of a method's ability to detect an outbreak early, we did not propose any measure of timeliness like salmon et al. [28] or jiang et al. [45] . this topic could be further explored in another study. to give some insight on the speed of detection, we calculated it for the improved farrington algorithm and the cusum glm rossi algorithm. on average, on the overall dataset, it took 1.23 weeks for the improved farrincton method to detect an outbreak or 1.16 weeks for the cusum glm rossi method. no method presented outbreak detection performances sufficient enough to provide reliable monitoring for a large surveillance system. methods which provide high specificity or fpr, such as the improved farrington or cdc algorithms, are not sensitive enough to detect the majority of outbreaks. these two algorithms could be implemented in systems that monitor health events to detect the largest outbreaks with the highest specificity. conversely, methods with the highest sensitivity and able to detect the majority of outbreaks-bayes 3 or cusum glm rossi for example-produced an excessive number of false alarms, which could saturate a surveillance system and overhelm an epidemiologist in charge of outbreak investigations. as a screening test in clinical activity, the aim of an early outbreak detection method is to identify the largest possible number of outbreaks without producing too many false alarms. the performances presented in this paper should be interpreted with caution as they depend both on tuning parameters and on the current implementation of the methods in the r packages. packages evolve with time and their default parameters may also change. so this work based on r available packages, may be viewed as a starting point for researchers to enhance the comparison of methods and/or to optimize the tuning according to their data. since no single algorithm presented sufficient performance for all scenarios, combinations of methods must be investigated to achieve predefined minimum performance. other performance criteria should be proposed in order to improve the choice of algorithms to be implemented in surveillance systems. therefore, we suggest that a study of the detection period between the first week of an outbreak and the first triggered alarm be conducted. table. fpr, specificity, pod, pod1week, sensitivity, negative predictive value, positive predictive value and f 1 -measure for 12 evaluated methods and α = 0.001 (for past outbreak constant k 1 = 0, 2, 3, 5, 10 and current outbreak k 2 = 1 to 10 for pod and sensitivity). (pdf) s2 table. fpr, specificity, pod, pod1week, sensitivity, negative predictive value, positive predictive value and f 1 -measure for 15 evaluated methods and α = 0.05 (for past outbreak constant k 1 = 0, 2, 3, 5, 10 and current outbreak k 2 = 1 to 10 for pod and sensitivity). (pdf) s3 table. other performance ratios, adjusted on past and current outbreak duration and amplitude, trend, seasonality, dispersion and baseline frequency (α = 0.01 for improved framework for evaluating public health surveillance systems for early detection of outbreaks: recommendations from the cdc working group. mmwr recommendations and reports: morbidity and mortality weekly report recommendations and reports / centers for disease control the emerging science of very early detection of disease outbreaks statistical issues and challenges associated with rapid detection of bio-terrorist attacks outbreak detection through automated surveillance: a review of the determinants of detection isolation of a novel coronavirus from a man with pneumonia in saudi arabia the next epidemic-lessons from ebola practical usage of computer-supported outbreak detection in five european countries a system for automated outbreak detection of communicable diseases in germany an improved algorithm for outbreak detection in multiple surveillance systems public health monitoring tools for multiple data streams. mmwr morbidity and mortality weekly report statistical challenges facing early outbreak detection in biosurveillance statistical methods for the prospective detection of infectious disease outbreaks: a review automated biosurveillance data from england and wales a statistical algorithm for the early detection of outbreaks of infectious disease an evaluation and comparison of three commonly used statistical models for automatic detection of outbreaks in epidemiological data of communicable diseases assessing surveillance using sensitivity, specificity and timeliness comparing aberration detection methods with simulated data comparing syndromic surveillance detection methods: ears' versus a cusum-based methodology comparison of various statistical methods for detecting disease outbreaks statistical algorithms for early detection of the annual influenza peak season in hong kong using sentinel surveillance data a simulation model for assessing aberration detection methods used in public health surveillance for systems with limited baselines evaluation of a method for detecting aberrations in public health surveillance data comparing early outbreak detection algorithms based on their optimized parameter values a simulation study comparing aberration detection algorithms for syndromic surveillance. bmc medical informatics and decision making yahav i simulating multivariate syndromic time series and outbreak signatures, social science research network an extreme value theory approach for the early detection of time clusters. a simulation-based assessment and an illustration to the surveillance of salmonella bayesian outbreak detection in the presence of reporting delays surveillance: an r package for the monitoring of infectious diseases surveillance: temporal and spatio-temporal modeling and monitoring of epidemic phenomena detection of aberrations in the occurrence of notifiable diseases surveillance data an approximate cusum procedure for surveillance of health events count data regression charts for the monitoring of surveillance time series discussion paper // sonderforschungsbereich 386 der ludwig-maximilians-universität münchen robust outbreak surveillance of epidemics in sweden the bioterrorism preparedness and response early aberration reporting system (ears) agreement, the f-measure, and reliability in information retrieval a modified poisson regression approach to prospective studies with binary data factors influencing performance of internet-based biosurveillance systems used in epidemic intelligence for early detection of infectious diseases outbreaks predicting outbreak detection in public health surveillance: quantitative analysis to enable evidence-based method selection the use of fractional polynomials to model continuous risk variables in epidemiology automated early warning system for the surveillance of salmonella isolated in the agro-food chain in france ten years experience of syndromic surveillance for civil and military public health value of syndromic surveillance within the armed forces for early warning during a dengue fever outbreak in french guiana in 2006. bmc medical informatics and decision making generalized amoc curves for evaluation and improvement of event surveillance the authors would like to thank angela noufaily and paddy farrington for providing them with simulated datasets and an r code to simulate outbreaks. conceptualization: gabriel bédubourg, yann le strat. key: cord-351918-pu7i1jfe authors: baek, yae jee; lee, taeyong; cho, yunsuk; hyun, jong hoon; kim, moo hyun; sohn, yujin; kim, jung ho; ahn, jin young; jeong, su jin; ku, nam su; yeom, joon-sup; lee, jeehyun; choi, jun yong title: a mathematical model of covid-19 transmission in a tertiary hospital and assessment of the effects of different intervention strategies date: 2020-10-26 journal: plos one doi: 10.1371/journal.pone.0241169 sha: doc_id: 351918 cord_uid: pu7i1jfe novel coronavirus (named sars-cov-2) can spread widely in confined settings including hospitals, cruise ships, prisons, and places of worship. in particular, a healthcare-associated outbreak could become the epicenter of coronavirus disease (covid-19). this study aimed to evaluate the effects of different intervention strategies on the hospital outbreak within a tertiary hospital. a mathematical model was developed for the covid-19 transmission within a 2500-bed tertiary hospital of south korea. the seir (susceptible-exposed-infectious-recovered) model with a compartment of doctor, nurse, patient, and caregiver was constructed. the effects of different intervention strategies such as front door screening, quarantine unit for newly admitted patients, early testing of suspected infected people, and personal protective equipment for both medical staff and visitors were evaluated. the model suggested that the early testing (within eight hours) of infected cases and monitoring the quarantine ward for newly hospitalized patients are effective measures for decreasing the incidence of covid-19 within a hospital (81.3% and 70% decrease of number of incident cases, respectively, during 60 days). front door screening for detecting suspected cases had only 42% effectiveness. screening for prohibiting the admission of covid-19 patients was more effective than the measures for patients before emergency room or outpatient clinic. this model suggests that under the assumed conditions, some effective measures have a great influence on the incidence of covid-19 within a hospital. the implementation of the preventive measures could reduce the size of a hospital outbreak. in late december 2019, an outbreak of an emerging disease (covid19) due to a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (sars-cov-2) originated in wuhan, china and rapidly spread across china and beyond. this outbreak began from a seafood and live animals whole-sale market in wuhan, but cases of patients suffering from the infection have been documented both in hospital and in family settings [1] . people become infected by respiratory droplets from coughing and talking but aerosol transmission is also possible in cases of protracted exposure to elevated aerosol concentrations in closed spaces [2] . transmission may occur indirectly through touching a contaminated surface, followed by touching their eyes, nose, or mouth. the coronavirus may also be unexpectedly transmitted by an asymptomatic carrier [3] . in fact, patients considered asymptomatic released large amounts of viruses at the early phase of the infection, which posed enormous challenges to contain the spread of covid-19 [4] , but 97.5% of patients with covid-19 developed symptoms within 11.5 days [5] . novel coronavirus can spread widely in confined settings, including hospitals, cruise ships, prisons, and places of worship [6] . in particular, a healthcare-associated outbreak could become the epicenter of covid-19. transmission in a hospital raises serious problems since many immunocompromised and aged patients live together and an outbreak in a hospital could paralyze its role of providing essential medical care within the healthcare system. therefore, effective strategies to contain covid-19 outbreaks in hospitals are required [7] . however, even for a well-established hospital, coping with the unprecedented covid-19 outbreak would be a complex challenge. in this study, we developed a mathematical compartment model to predict covid-19 transmissions over time in a tertiary hospital, and to evaluate the effectiveness of different intervention strategies. we divided individuals into four infection classes: susceptible, exposed, infectious, and removed. (i.e., recovered, or otherwise no longer infectious). the susceptible, s, represents the people who can be infected by sars-cov-2. the exposed, e, are those already infected but who did not recognize the disease and even front door screening could not detect it. the infectious, i, and removed, r, follow the usual immunizing infection [8] . the study hospital consists of three main categories: ward, outpatient clinic, and emergency room. we divided people who entered the hospital into four main compartments: doctor, nurse, patient, and caregiver. doctors and nurses are a part of the medical staff who are fixed in the hospital, while patients and caregivers are visitors who vary from day to day. we assumed doctors work across departments while nurses work in their own departments. therefore, individuals in the hospital were divided into 10 statuses: doctors as a whole and nurses, patients, and caregivers in each department (fig 1) . because there are three factors that divided the population, we represented the compartment in the form of x y z . x indicates 4 infection classes. y is the occupation and z represents the department to which the component belongs. we used the notations d for the doctors' group, n for the nurses' group, p for patients', and c for caregivers'. according to convention, we denoted adm as the ward, opd for the outpatient department, and er for the emergency room. the component of waifw (who-acquires-infection-from-whom) matrix w ði;i k þðj;j k þ represents the transmissibility from the (j,j k )-th infectious group to the (i,i k )-th susceptible group, where the index (i,i k ) indicates i-th occupation and i k -th department. note that the compartment for the doctors' group does not have the subscript z. usually, the exposed person does not participate in the infection, but this is not clear in case of covid-19. therefore, we assumed the exposed person is involved in the foi (force of infection) λ with transmissibility reduced by ε. in this research, we set this value as 0.1. public health authorities define a significant exposure to covid-19 as face-to-face contact with a symptomatic patient within six feet that is sustained for at least a few minutes. we estimate the contact rate matrix, c, based on the short survey and employ the reproductive number, r 0 , from the literature. setting the population vector η as the number of staff, and the stabilized number of inflow and outflow to each department for visitors, we construct the waifw matrix, w, by assuming that it is proportional to the contact rate matrix [8, 9] : the diagram for the seir (susceptible-exposed-infectious-recovered) model with compartments of doctor, nurse, patient, and caregiver. we assumed doctors work across departments while nurses work in their own departments; therefore, individuals in the hospital are divided into 10 statuses. blue arrows refer to the in-and out-flow of patients and caregivers in the opd and er, with only inbound arrows for those in adm. abbreviations:-adm: admission; opd: outpatient department; er: emergency room. https://doi.org/10.1371/journal.pone.0241169.g001 the proportionality factor q represents the transmission risk per contact, which can be calculated through the relation between waifw and r 0 . here f is the rate that at which the exposed becomes infectious, and the γ is the rate that the infectious would recover. the population vector η denotes the number of staff, and the stabilized number of inflow and outflow to each department for visitors and ρ(c�η) is the spectral radius of the resulting matrix multiplying each row of c by corresponding element of η. fig 1 shows the diagram for the seir (susceptible-exposed-infectious-recovered) model with compartments of doctor, nurse, patient, and caregiver. the patients and caregivers in opd and er come in and out, but adm does not have the inbound arrows, as we assumed that patients are not directly admitted to the ward from the outside but only from other departments. average contact duration matrix indicates the average hours of contact in a day among medical staff and visitors (fig 2) . the horizontal axis indicates the compartments having the contacts and the vertical one indicates the compartments that are contacted. the matrix meets the reciprocity of contacts which makes the contact rate matrix symmetric. the study site was severance hospital, a tertiary care hospital with 2500 beds in south korea. there are 74 wards for inpatients within the study hospital, and we assumed each unit had the same capacity of doctors and nurses. if two doctors work in a unit, there are 148 doctors working in wards daily. other numbers based on epidemiology are shown in table 1 . we retrieved data from the hospital administration department on the number of patients who were admitted from the er, hospitalized in wards, and had gone to outpatient clinics from february 24, 2020 to march 13, 2020. the hospital administration department provided us with the data without identifiable private information. before we began this study, we confirmed with the institutional review board (irb) of severance hospital that ethics approval was not needed, since we did not utilize any personal or identifiable information of the patients. authors who were affiliated to the hospital devised parameters through anonymized data and work experience in the study site. at outpatient clinics, some patients are accompanied by caregivers, and we assumed half of outpatients visit clinics with one caregiver. on the other hand, we assumed one caregiver was assigned per patient in each ward and the emergency center as a hospital policy. we arbitrarily assumed 10 exposed and 10 infectious people from the patient and caregiver group came into the er every three days, while the same number from the patient group and half of them from the caregiver group came into the opd every five days. we presumed they had not been diagnosed by any reason when they entered the study hospital, which meant they were not detectable with the known data. all other parameters for the model are shown in table 2 . impact of the exposed onto the infection the average inflow number of adm from the outside per day † -0 the average inflow number of opd from the outside per day † -11242.6 the average inflow number of er from the outside per day † -209.3 the average number from er to adm per day † -51.4 the average number from opd to adm per day † -314.6 the rate of outflow from the adm [1/days] † -0.1491 the rate of outflow from the opd [1/days] † -6 the rate of outflow from the er [1/days] † -4 � the incubation period and infectious period are from reference [13] , [17] × rate at which the exposed persons become infectious † an average of data collected from the hospital administration department in the study site abbreviation:-adm: admission; opd: outpatient department; er: emergency room. https://doi.org/10.1371/journal.pone.0241169.t002 the study site had implemented several controlling measures to prevent outbreaks within the hospital (table 3 ) but, in this mathematical model, we set up four intervention scenarios for the model (fig 3) . front door screening. according to one study, on admission, 43.8% of covid-19 patients presented a fever, 67.8% a cough, and 33.7% with sputum [1] . the sensitivity is the test's ability to correctly designate a subject with the disease as positive, and we calculated the sensitivity of front door screening at 0.5. however, if we sought an epidemiologic relationship to confirmed patients or travel history along with their current symptoms, the sensitivity would increase, which we assumed at 0.7. front door screening was performed on visitors of three different departments: amd, opd, and er. quarantine unit for newly admitted patients. even though the study hospital executed a pneumonia preemptive isolation unit, asymptomatic patients (usually in the exposed group) could be missed in this control. therefore, we assumed all patients who were admitted either from the er or outpatient clinics were sent to a quarantine unit for two weeks. early testing (within eight hours) of suspected people to detect the disease. we assumed the average time for diagnosis would be eight hours. as covid-19 patients in the hospital were confirmed, they were directed to isolated rooms (the removed group). as medical staff were aware of the clinical symptoms of covid-19 and patients' medical conditions were regularly and closely monitored, the groups were immediately tested when the related symptoms occurred. however, caregivers were not as attentive as patients were; therefore, we assumed they were tested three days later than other groups. personal protective equipment for both medical staff and visitors. the regulation of the study hospital specifies that all people in the hospital are required to wear masks. since sars-cov-2 is a respiratory virus like other coronaviruses or influenza, facial masks significantly reduce transmission of human coronavirus from symptomatic individuals, which could be a way to control of covid-19 [10] . however, because the chance of catching covid-19 table 3 . control measures to prevent covid-19 outbreak in the study site. inquiring about contact history and related symptoms (cough, sputum, sore throat) by a standard checklist triage clinics for high risk group separate clinics for persons who have either fever or respiratory illness, and who have a history of traveling abroad or visiting high risk areas, located in front of the entrance to the outpatient clinic and emergency room. the study site minimized the number of entrances to hospital buildings and restrict visiting to admitted patients universal mask wearing all healthcare workers, employees, patients, and visitors are obligated to wear masks in the hospital real-time reverse transcription polymerase chain reaction (rt-pcr) for any patients with related symptoms or suspected findings without specific causes the frequency of real-time rt pcr testing to diagnose covid-19 in the study hospital has increased from 1 time/day to 6 times/day during covid-19 outbreak since january 2020 the test results could be reported within four hours isolation ward with negative pressure isolation rooms in operation patients with either fever or pneumonia were preemptively isolated and treated within the ward healthcare workers for those patients are required to wear appropriate personal protective equipment (ppe). https://doi.org/10.1371/journal.pone.0241169.t003 from a passing interaction in a public space is minimal, some people raise the question of effectiveness of universal use of masks by all healthcare workers and visitors [11] . however, the effect of face masks, respirators and eye protection that result in reducing risk of outbreak had been verified [12] . we assumed the protection rate of transmission by masks would be 0.3, and the protection rates could reach 0.3, 0.6, or 0.9 as reinforcing personal protective equipment (ppe) with gloves, gowns, eye protection, etc. reproductive number. the vulnerability of results for each intervention required checking as the reproductive number varied since we set the waifw matrix from it. we employed , infectious class in seir diagram; green arrows indicate front door screening and brown arrows indicate testing the infectious patients. front door screening intervenes the inflow of the infectious patients and the tested patients are removed at the rate of 1/t. as we described, we assumed caregivers are tested three days later than other groups. (b), waifw diagram of each group; pink arrows indicate reduction of transmission by wearing a universal mask while red arrows are reinforcement of the protection device among medical staff. reinforcing the protection device among medical staff reduces the probability of transmission as in red arrows. (c), diagram for pre-isolating the patients who are to be admitted, which we named the quarantine unit. patients are usually admitted from opd and er (light gray arrows), but if all patients are directed to the isolated ward (iso) (dark gray arrow), isolated people are out of the dynamics for 14 days. abbreviations:-adm: admission; opd, outpatient department; er: emergency room; iso: quarantine unit. https://doi.org/10.1371/journal.pone.0241169.g003 the value 2.2 from the paper for early transmission analysis of covid-19 in wuhan [13] . however, over time, many studies have been performed regarding an estimation of the reproductive number in different circumstances with control measures [14, 15] . variability must be taken into consideration within the range of reproductive numbers for evaluating each intervention. therefore, we also simulated the impact of our interventions in case of a very high reproductive number (6.47) [14] . incubation period and serial interval. the incubation period has not been determined yet and we set it at 5.2 days [13] as a base case and 6.4 days [16] for sensitivity analysis. the serial interval has not been determined and we assumed 9.5 days [17] and 4.6 days for sensitivity, which is 2 times of 2.3 days-that is the difference between 7.5 days serial interval and 5.2 days incubation period [13] . note that these parameter values were to be fitted with different assumptions for distribution. however, in an average sense, they have few differences with other fitting results and can be used as parameters in our model. with these parameters, we set the base-, worst-, and best-case scenarios and performed the sensitivity analysis with them (see table 4 ). the model simulated the epidemic curve of covid-19 in the study site with base parameters. fig 4 shows the daily new incidence of covid-19 without any intervention for 60 days. the horizontal axis represents time (days) and the vertical axis indicates the number of people who are newly confirmed patients within the past 24 hours. this predicts daily new cases of infected people from four compartments over time within the hospital. since doctors work across departments, their epidemic is shown in fig 4a. other groups work or stay in separate departments, which indicate different epidemics (fig 4b) . the infected cases in er and opd are very small due to relative short duration of stay and low reproductive number, so the spread is r 0 6.47 [14] � we set the best-and worst-case scenario parameter sets in terms of curbing viral transmission. if the virus has a long infectious period and low reproductive number, the transmissibility is low, which is helpful in curbing the spread of disease. on the other hand, with a short infectious period and high reproductive number, it would lead to high transmissibility even in a restricted condition. † 1/f is the incubation period; a reversal of the rate at which the exposed patients become infectious ‡ 1/γ denotes the infectious period, a reversal of the rate at which the infectious patients would recover § r 0 denotes the reproductive number, an average number of secondary cases generated by a case in an entirely susceptible population. https://doi.org/10.1371/journal.pone.0241169.t004 restricted and there is no remarkable outbreak in the er or opd. however, when confirmed cases are in wards, they become more transmissible since visitors have a high contact duration matrix within the group. fig 5 indicates total epidemic curves of covid-19. the number of the exposed and infectious people among visitors grows in the early period but this reaches a plateau. the curve of the recovered patients represents a trend of a totally susceptible population in the hospital before the ourbreak being immune to the disease. the total number of infectious people is about 30 at the end of the dynamics. next, we simulated the model with various interventions. we set the sum of new incidence for 60 days as an outcome measure. "1-effectiveness of an intervention (%)" is defined as the ratio of the outcome with an intervention to one without any intervention, so effectiveness denotes the proportion of decrease of the confirmed cases due to an intervention. fig 6 shows the effectiveness of all intervention scenarios and the effectiveness of the detection of infectious patients, 80.7%, is the highest among the control measures. therefore, an examination of any suspected cases is the most important way to prevent an outbreak in the hospital. wearing universal masks by medical staff and visitors (see ppe dn 0.3 pc 0.3 in fig 6) shows about 66.4% of effectiveness. the impact of different protection rates of medical staff tested by three scenarios (0.3, 0.6, or 0.9) turned out to be insignificant (66.4%, 67.8%, or 68.9%, respectively). in other words, reinforcement of ppe for medical staff does not show an expected improvement of effectiveness. quarantine of new hospitalized patients is another effective way to prevent outbreaks. the quarantine unit is as effective as 65.7%, while front door screening shows less effectiveness, which is up to 43.1%. the screening of patients who are admitted to the ward is the most effective method, followed by er and opd (30.7%, 28.7%, and 2.2% with sensitivity of screening of 0.5). as expected, the more accurate the screening is, the more effective it is as a control measure. if the sensitivity of screening is 0.7, the effectiveness of front door screening for inward, er, and opd is 43.1%, 40.1%, and 3.3%, respectively. according to our mathematical model, screening of an opd is not a good measure to prevent an outbreak in a hospital setting. we performed a sensitivity analysis by taking various combinations of parameter values based on plausible ranges. the worst-case scenario has a short incubation period and high reproductive number, resulting in the biggest outbreak in the hospital. on the other hand, the reverse combination yields the best-case scenario. first, we estimated the outbreak in our hospital in the absence of control measures, which is shown in fig 7 with sensitivity analysis. the exposed people in the worst-case scenario (fig 7b) rises to a peak of 902 people, while the exposed people in the best-case scenario (fig 7a) only reaches 30 people. a highly transmissible case is hard to control with an exponentially increasing number of exposed and infectious patients. additionally, we evaluated the effectiveness of different interventions in three scenarios: base, best, and worst cases (fig 8) . the effectiveness of control is higher in the best-case scenario through front door screening and use of a quarantine unit. screening of inpatients through front door screening with a sensitivity of 0.7 shows effectiveness of 44.0% and 6.6% in best-and worst-case scenarios, respectively. regarding the er, the effectiveness is 41.3% in the best-case scenario and 5.6% in the worst-case scenario. effectiveness is about seven times higher in front door screening of admission wards and ers without reference to sensitivity of screening. in a low transmission setting, it is crucial to detect the patients with covid-19 from er and in wards before inflow to a hospital. a quarantine unit also helps prevent the outbreak in the hospital more in the best-case scenario than in the worst (64.1% vs 52.6%). a high transmission rate in the hospital offsets the effort of screening of infectious patients and on the other hand, the probability of control by testing and protection devices is higher in worst-case scenarios. ppe reinforcement decreases transmission effectively in highly transmissible conditions, which contributes to reducing the outbreak as with the worst-case scenario. strengthening protection has little impact on the new incidence of confirmed cases in sensitivity analysis. testing to detect and isolate confirmed cases is also more important in higher transmission cases (92.3%). sensitivity analysis shows diagnosis of the disease within eight hours and isolation is still the best intervention strategy in a hospital. since the global outbreak of covid-19, many control measures have been implemented to try and contain the pandemic: isolation of confirmed and suspected cases; contact tracing; social distancing; and, travel restrictions. suggestion of best strategies which offer greater benefits is difficult in the context of an epidemic. several mathematical models have been proposed to explain the system and help decision making beyond hospital settings [19] [20] [21] [22] . one study explored the spatial association of the early stages of the covid-19 pandemic in china [23] . a dynamic mathematical model estimated that the growth rate of covid-19 is about twice that of the sars and mers, and the doubling cycle is two to three days without intervention [24] . a stochastic transmission model assessed the potential for transmission in locations outside wuhan, if cases were introduced. it calculated that once there are at least four independently introduced cases, there is a more than a 50% chance that the infection will establish itself within that population [25] . in this study, we simplified the transmission of covid-19 in a hospital to construct a mathematical model that would enable us to estimate the outbreak and determine the effectiveness of control measures. we conducted a sensitivity analysis with different assumptions because of the uncertainty of the parameters. early testing of infected cases and monitoring the quarantine ward for newly hospitalized patients are effective ways to minimize the covid-19 outbreak within a hospital. detecting the patients with covid-19 from the er and in wards before inflow to a hospital is effective in low transmission settings; ppe is important to control transmissibility in high transmission settings. our results could expand to many interventions implemented in society. in high transmissible and short latent cases, transmission reduction interventions including wearing universal masks are more important than restriction of inflow of patients by screening and isolating suspected cases. above all, quarantine and isolation efficacy should be increased by means of proper hygiene and personal protection. to our knowledge, this study is the first model to estimate the epidemics in a hospital and evaluate the effects of control measures for covid19 . other studies about hospital outbreaks from infectious diseases were conducted with many different mathematical models: a multiagent model or seir transmission model [26] [27] [28] [29] . in the case of mers-cov, the emergency departments exercised great influence over the epidemic size for both patients and healthcare workers, and isolation and related strict measures (added ppe or environmental sanitation) suppressed the epidemics with the help of the seir compartmental model [26] . the seir compartmental model was similar to our model, which is deterministic, multi-type, and spatial in a hospital setting. our model set the reproductive number with sensitivity analysis, assumed the regular inflow of covid-19 patients to a hospital, and compared the control measures. there are some limitations of this model. our model does not include people who enter the hospital and do not belong to the four occupations specified (doctors, nurses, patients, and caregivers). weekends, when most of the medical staff are off duty were not taken into consideration. various units and situations of the hospital have not been included in this model, such as the intensive care unit, operation room, and confirmed cases of medical staff. while the model assumes the same inflow of exposed and infectious people from the patient and caregiver group, the prevalence of asymptomatic infection has not been clarified yet. we set the rate at which the exposed individuals become infectious at 0.1, which has also not been confirmed. in addition, we assumed that the sensitivity of the front door screening is low (0.5 or 0.7) and detects only infectious persons; however, thorough checklists and use of thermometers might detect more infectious or exposed people. therefore, it is cautious to assert that front door screening is effortless. more studies should be conducted on the outbreak and control measures from different perspectives. even though we did not include all the details, this study could improve our insights into epidemiological situations and identify which control measures are most efficacious in hospital settings. though this study was confined to one hospital, it can be tailored to the requirements of other hospitals, facilitating effective hospitalbased management during this rapidly evolving outbreak. clinical characteristics of coronavirus disease 2019 in china. the new england journal of medicine evaluation and treatment coronavirus (covid-19) transmission of sars-cov-2: implications for infection prevention precautions: scientific brief unique epidemiological and clinical features of the emerging 2019 novel coronavirus pneumonia (covid-19) implicate special control measures pathophysiology, transmission, diagnosis, and treatment of coronavirus disease 2019 (covid-19): a review transmission potential of the novel coronavirus (covid-19) onboard the diamond princess cruises ship effective strategies to prevent coronavirus disease-2019 (covid-19) outbreak in hospital. the journal of hospital infection an introduction to infectious disease modelling: oup oxford modeling infectious disease parameters based on serological and social contact data: a modern statistical perspective respiratory virus shedding in exhaled breath and efficacy of face masks universal masking in hospitals in the covid-19 era physical distancing, face masks, and eye protection to prevent person-to-person transmission of sars-cov-2 and covid-19: a systematic review and meta-analysis. the lancet early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia. the new england journal of medicine estimation of the transmission risk of the 2019-ncov and its implication for public health interventions time-varying transmission dynamics of novel coronavirus pneumonia in china incubation period of 2019 novel coronavirus (2019-ncov) infections among travellers from wuhan, china clinical characteristics of 24 asymptomatic infections with covid-19 screened among close contacts in nanjing, china serial interval of novel coronavirus (covid-19) infections. international journal of infectious diseases: ijid: official publication of the international society for infectious diseases transmission dynamics of the covid-19 outbreak and effectiveness of government interventions: a data-driven analysis feasibility of controlling covid-19 outbreaks by isolation of cases and contacts the effect of travel restrictions on the spread of the 2019 novel coronavirus (covid-19) outbreak short-term predictions and prevention strategies for covid-2019: a model based study spatial epidemic dynamics of the covid-19 outbreak in china. international journal of infectious diseases mathematical model of infection kinetics and its analysis for covid-19 early dynamics of transmission and control of covid-19: a mathematical modelling study. the lancet infectious diseases analysis of the healthcare mers-cov outbreak in king abdulaziz medical center role of fomites in sars transmission during the largest hospital outbreak in hong kong probable transmission routes of the influenza virus in a nosocomial outbreak the authors are grateful to all the medical staff who worked at the study site. key: cord-351387-i0zamkpd authors: witte, katrin; koch, egon; volk, hans-dieter; wolk, kerstin; sabat, robert title: the pelargonium sidoides extract eps 7630 drives the innate immune defense by activating selected map kinase pathways in human monocytes date: 2015-09-25 journal: plos one doi: 10.1371/journal.pone.0138075 sha: doc_id: 351387 cord_uid: i0zamkpd pelargonium sidoides is a medical herb and respective extracts are used very frequently for the treatment of respiratory tract infections. however, the effects of pelargonium sidoides and a special extract prepared from its roots (eps 7630) on human immune cells are not fully understood. here we demonstrate that eps 7630 induced a rapid and dose-dependent production of tnf-α, il-6, and il-10 by human blood immune cells. this eps 7630-induced cytokine profile was more pro-inflammatory in comparison with the profile induced by viral or bacterial infection-mimicking agents. the search for eps 7630 target cells revealed that t-cells did not respond to eps 7630 stimulation by production of tnf-α, il-6, or il-10. furthermore, pretreatment of t-cells with eps 7630 did not modulate their tnf-α, il-6, and il-10 secretion during subsequent activation. in contrast to lymphocytes, monocytes showed clear intracellular tnf-α staining after eps 7630 treatment. accordingly, eps 7630 predominantly provoked activation of map kinases and inhibition of p38 strongly reduced the monocyte tnf-α production. the pretreatment of blood immune cells with eps 7630 lowered their secretion of tnf-α and il-10 and caused an il-6 dominant response during second stimulation with viral or bacterial infection-mimicking agents. in summary, we demonstrate that eps 7630 activates human monocytes, induces map kinase-dependent pro-inflammatory cytokines in these cells, and specifically modulates their production capacity of mediators known to lead to an increase of acute phase protein production in the liver, neutrophil generation in the bone marrow, and the generation of adaptive th17 and th22 cells. today, plant secondary substances, especially polyphenols, get more and more into the focus of medical research. in south africa, polyphenol-rich herbal preparations made up from roots of pelargonium sidoides and pelargonium reniforme are traditionally used to treat respiratory and gastrointestinal infections, dysmenorrhea, and hepatic disorders [1] . inspired by the healing of his tuberculosis, charles henry stevens introduced this phytomedical drug to england already in 1897 [1] . more than seven decades later, a special ethanolic extract of pelargonium sidoides roots, eps 7630, was finally developed [eps 1 7630 is the active ingredient of the herbal medicinal product umckaloabo 1 (iso arzneimittel, ettlingen, germany)]. in germany, eps 7630 is approved today for the therapeutic use in patients with acute bronchitis. in addition, eps 7630 was shown to be effective in clinical trials with patients suffering from tonsillopharyngitis, rhinosinusitis, common cold or copd [2] [3] [4] [5] [6] . as an alternative to antibiotic treatments, eps 7630 has the advantage of not promoting microbial resistances [7] . the latter aspect is mainly explained by its characteristic not to interfere with the metabolism of viruses or bacteria. the main constituents of eps 7630 include coumarins (e.g., umckalin) and flavanoles (polyphenols) [8] . the latter comprise oligomeric proanthocyanidins, which are highly abundant (~40%) in eps 7630, especially oligo-and polymeric prodelphinidins. they are constructed mainly of gallocatechin and epigallocatechin components and are present with different interflavonoid bonds in pelargonium sidoides roots [9] . although the research of the last years has made substantial progress with respect to the broad antiviral and antibacterial efficacy of eps 7630, the exact mode of action of eps 7630 is not fully understood yet. in vitro, eps 7630 shows efficacy against cellular infections with influenza virus, hsv, emcv, rsv, coronavirus, parainfluenza virus, and coxsackie virus, and this appears to be mainly mediated indirectly by inhibition of virus attachment and spreading [7, [10] [11] [12] [13] . different modes of action underlying the antibacterial effects of eps 7630 have been proposed. eps 7630 inhibits the adherence of bacteria such as streptococcus pyogenes and helicobacter pylori to epithelial cells in vitro [14] [15] [16] [17] [18] . furthermore, ciliated cells isolated from the nasal epithelium enhanced their ciliary beat frequency in the presence of eps 7630, which should allow a better removal of excess mucus and bacteria [19] . regarding the infection with candida albicans, eps 7630 was shown to enhance the oxidative burst and intracellular pathogen killing by human blood phagocytes [15] . in leishmania major-infected murine macrophages, eps 7630 increased cellular nitric oxide production and mrna levels of inos and several cytokines (il-1β, il-10, il-12, il-18, tnf-α, ifn-α, ifn-γ) [13, [20] [21] [22] . however, our knowledge regarding the influence of pelargonium extract on human immune cells, in particular on their cytokine production, is still highly restricted. to gain insight into this matter we comprehensively studied the immunoregulatory effects of eps 7630 on human blood immune cells. eps 7630 is prepared from the roots of pelargonium sidoides with a drug to extract ratio of 1:8-10 using aqueous ethanol (11% w/w) as extraction solvent. dried extract of a single batch (no. psc2003/l01-11/sy06-041-a) was used to prepare a stock solution of 3 mg/ml. this solution was obtained by dissolving the powder in sterile pbs containing 10% ethanol, followed by ultrasound treatment and sterile filtration using a 0.2 μm filter unit. human peripheral blood mononuclear cells (pbmcs) were isolated from the blood of healthy donors by ficoll (biochrom) density gradient centrifugation as previously described [23, 24] . in the first setting, pbmcs were stimulated with eps 7630 (3 and 30 μg/ml), escherichia coli 0127:b8 lipopolysaccharide (tlr4 ligand; 100 ng/ml; sigma-aldrich), polyinosinic-polycytidylic acid [poly (i:c); 10 μg/ml; sigma-aldrich], a cytokine mixture of il-1β, il-2 and il-12 (10 ng/ml each; r&d systems), anti-cd3 (orthoclone; cilag) and anti-cd28 (r&d systems) monoclonal antibodies (1 μg/ml each), or were left without specific treatment (0.1% ethanol as solvent control) for 4 and 24 h, before cell culture supernatant was recovered for elisa cytokine production analysis. in another setting, pbmcs, after serum-starvation for 3.5 h, were stimulated with the same stimuli (for tcr stimulation cd3/28 coated dynabeads were used at a cell / bead ratio of 2:1) but for 10 and 30 min, and cells were recovered for western blot analysis. for eps 7630 dose-response analyses of cytokine production, isolated pbmcs were stimulated with eps 7630 at concentrations ranging from 0.1 to 10 μg/ml or were left without stimulation (0.1% ethanol control) for 48 h. in some of these concentration-response analyses, lipopolysaccharide (lps; 100 ng/ml) or poly (i:c) (10 μg/ml) was added after the first 24 h and incubation was continued for a further 24 h. to study the kinetics of cytokine production, isolated pbmcs were stimulated with 10 μg/ml eps 7630 or 0.1% ethanol (solvent control) for 4 to 72h. for intracellular tnf-α analysis, pbmcs were stimulated for 4 to 5 h with eps 7630 concentrations ranging from 1 to 30 μg/ml, with 0.1% ethanol solvent control and, if indicated, 25 ng/ml phorbol 12-myristate 13-acetate (pma)/1 μg/ml ionomycin before being prepared for flow-cytometry-based cytokine analysis. in some of these studies, pbmcs were pretreated with sp600125, pd98059, wedelolactone (all from sigma aldrich) and sb202190 (invivogen) at 10 μm each or with 0.1% dmso (solvent control, sigma aldrich) for 45 min before eps 7630 (10 and 30 μg/ml only) application. the possible influence of eps 7630 on pbmc proliferation and apoptosis was tested by stimulation of pbmcs with 0 (0.1% ethanol solvent control), 3, 10 and 30 μg/ml eps 7630 as well as with 1% dmso (positive control for apoptosis induction) for 24 h. afterwards, cells were recovered for flow cytometric analysis. cd4 + memory t-cells were purified from isolated pbmcs by negative selection using the macs system and the memory cd4 + t-cell isolation kit (miltenyi). for elisa-based cytokine production analysis, isolated t-cells were cultured in the presence or absence of eps 7630 for 48 h. for the last 24 h of culture, one part of these cells was stimulated with anti-cd3/anti-cd28-coated dynabeads (life technologies; cell/bead ratio 1:1). all cell cultures were performed using rpmi culture medium supplemented with 2 mm l-glutamin and 10% fetal bovine serum (biochrom). the study of eps 7630 effects, the collection and usage of the blood samples were approved by the clinical institutional review board of the university hospital charité berlin, and written informed consent was obtained from donors. culture supernatants were analyzed for tnf-α, il-6 and il-10 content by elisa using respective detection kits from r&d systems. in some settings, quantification of tnf-α was carried out using the immulite device and respective detection kit (dpc biermann). lysing of pbmcs, quantification of proteins in the cell lysates, sds page gel electrophoresis and blotting of the respective gel was carried out as published earlier [25, 26] . analysis of signal transduction elements was performed by incubation of blots with antibodies detecting phospho-jnk1/2, phospho-p38, phospho-erk1/2, phospho-akt, phospho-stat5 and phospho-p65 (all from cell signaling technology) and gapdh (merck millipore), followed by incubation with peroxidase-conjugated affinipure goat anti-rabbit or goat anti-mouse igg (h&l, dianova) and subsequent chemiluminescence detection using ecl reagent (lumigen). for analysis of intracellular cytokines, treated pbmcs were incubated with brefeldin a (sigma aldrich; 5 μg/ml) for the last 3 to 4 h of treatment. afterwards, cells were fixed and permeabilized using the cytofix/cytoperm kit (bd biosciences) and subsequently stained for 40 min with fluorescent antibodies detecting tnf-α (clone mab11, biolegend), cd14 (clone rmo52, beckmann coulter), and cd4 (clone sk3, bd biosciences). to assess the purity of isolated memory t-helper cells, cells were stained with fluorescent antibodies detecting the following cell surface markers as described previously [23, 24] : cd45ra (clone hi100), cd45ro (clone uchl1), cd3 (clone sk7), cd4 (clone sk3), cd56 (clone ncam16.2) (all from bd biosciences) as well as cd16 (clone 3g8), cd14 (clone rmo52), and cd19 (clone j4.119) (all from beckmann coulter). for analysis of pbmc proliferation, total cell numbers after treatment were assessed by flow cytometry. for analysis of pbmc apoptosis, treated cells were stained with fluorescent annexin v antibodies and propidium iodide using the annexin v apoptosis detection kit apc (ebioscience) according to the manufacturer's instructions. afterwards, the proportion of apoptotic cells (annexin v + cells) were evaluated. all data acquisitions and analyses were carried out using a facscalibur device and cell-quest software (bd biosciences). to test the significance of pairwise differences between the treatment groups, the wilcoxon matched-pairs signed-rank test was used (spss software, ibm). a p-value of p 0.05 was considered as indicator of significance. understanding the molecular effects of eps 7630 is key to take advantage of its medical potential and the identification of further indications. so far, data regarding its target cells and effects thereof within the human immune system are lacking. to address this issue, we first investigated whether eps 7630 influences resting human immune cells. therefore, pbmcs isolated from healthy donors were treated or not (control) with eps 7630 at two concentrations and were tested for cytokine production. as a comparison, these cells were also stimulated with toll-like receptor (tlr)3 [poly(i:c)] and tlr4 ligands (bacterial lipopolysaccharide), which mainly act on antigen-presenting cells (e.g., monocytes) and mimic viral and bacterial infection, respectively. as further positive controls a mix of cytokines (il-1β, il-2, il-12), able to activate nk-cells, as well as t-cell activating anti-cd3/anti-cd28 antibodies, were used. interestingly, cells responded to eps 7630 by secretion of tnf-α and il-6 with levels that were much higher than those induced by the mix of cytokines and tcr engagement (fig 1a) . furthermore eps 7630 slightly triggered il-10 production, and this production was clearly smaller (~3-to 5-fold) than that induced by cd3/cd28-mediated stimulation. importantly, the cytokine profile induced by eps 7630 in pbmcs was also different from that induced by tlr3 or tlr4 ligands (fig 1a) . in fact, the eps 7630-induced response was much more pro-inflammatory than that induced by the viral or bacterial infection-mimicking agents ( fig 1b) . as no relevant cytotoxicity or influence on pbmc proliferation were observed by eps 7630, the cytokine increase provoked by eps 7630 seems to be due to an induced production rate (s1 fig). a more detailed investigation revealed a concentration-dependent induction of cellular tnf-α, il-6, and il-10 secretion by eps 7630, with clear effects already seen at a concentration of 1 μg/ml (fig 1c) . additionally, a kinetic analysis of eps 7630-dependent cytokine production by pbmcs revealed, that especially tnf-α is produced very quickly (fig 1d) . afterwards, the tnf-α level continuously decline, probably as a result of binding and internalization of this cytokine by monocytes. in contrast to tnf-α, il-6 and, in particular, il-10 were produced more slowly after eps 7630 stimulation, with levels steadily increasing over time. the next study part aimed at identifying the cell type that accounts for the response observed in eps 7630-stimulated blood immune cells. since memory t helper cells are very important cytokine producers, we first isolated these cells from human pbmcs by magnet-based cell sorting (fig 2a) . surprisingly, no relevant tnf-α, il-6, or il-10 production was found by these cells stimulated with different eps 7630 concentrations (fig 2a) . in the next step, we tested whether eps 7630 might modulate the cytokine response in activated memory t helper cells. however, cd3/cd28 stimulation of these cells, pretreated with different doses of eps 7630, did not result in a relevant increase of tnf-α, il-6, or il-10 secretion (fig 2b) . moreover, flow-cytometric analysis of intracellular cytokine staining showed that no population of t helper cells was able to produce tnf-α after stimulation with eps 7630, whereas they strongly responded to pma/ionomycin activation (fig 2c and s2 fig) . these data suggest that, within pbmcs, not th-cells but other immune cells are major targets of eps 7630 action. to prove monocytes as being the cell population sensitive among pbmcs to the action of eps 7630, we analyzed intracellularly stained tnf-α in these cells after eps 7630 stimulation using flow cytometry. indeed, we observed a strong dose-dependent induction of tnf-α production in monocytes, whereby, similar to the effect on whole pbmc, this effect was already seen at 1 μg/ml (fig 3a and 3b) . to understand the action of eps 7630 at the molecular level we then investigated the signaling pathways activated by this drug. pbmcs were treated for 10 and 30 min with eps 7630 at two different concentrations (3 and 10 μg/ml) and, as comparison, with tlr3 and tlr4 ligands, the il-1β, il-2, and il-12 comprising cytokine mixture, and anti-cd3/anti-cd28 antibodies. because of the cytokine profile observed after pbmc incubation with eps 7630, we focused our analysis on map kinase, nf-κb, and pi3k pathways. respective western blot analyses revealed that eps 7630 activated the map kinases jnk1/2, p38, and erk1/2. additionally we observed a slight activation of the pi3k (phosphorylation of akt) and nf-κb (phosphorylation of p65) pathways, whereas stat5 expectedly remained unaffected (fig 3c) . in line with the different cytokine profiles induced by eps 7630 versus tlr3 and tlr4 ligands, cytokine mixture, and anti-cd3/anti-cd28 antibodies, the pattern of activated kinases by eps 7630 was unique. by pharmacological blocking using the selective inhibitors sp600125 (jnk1/ 2/3), pd98059 (mek; activator of erk1/2), sb202190 (p38α/β), and wedelolactone (iκb kinase), we observed that eps 7630-induced production of tnf-α in monocytes was strongly dependent on p38 activity and at least partly dependent on the activation of erk1/2 and nf-κb ( fig 3d) . eps 7630 is very frequently used for infection prevention, in particular in the autumn and winter. therefore, we asked whether eps 7630 pretreatment would influence the immune response in the context of viral and bacterial infections. to address this question, we pretreated pbmcs with increasing concentrations of eps 7630 (0.1-3 μg/ml) for 24 h followed by stimulation with tlr3 or tlr4 ligands (mimicking viral or bacterial infection, respectively) for another 24 h. surprisingly, we observed that eps 7630 pretreatment concentration-dependently inhibited the tlr3-and tlr4-mediated production of tnf-α and il-10 (fig 4) . in contrast, we detected eps 7630 concentration-dependent high il-6 secretion in this situation (fig 4) . these data suggest that eps 7630 pretreatment specifically modulates the cytokine production in subsequent viral and bacterial infection. the special extract of pelargonium sidoides roots, eps 7630 (umckaloabo 1 ) is therapeutically active against upper respiratory tract infections and currently approved for the application in acute bronchitis in germany. surprisingly, the large use of this preparation is not associated with a respective knowledge regarding its pharmacodynamic properties. in fact, besides the known anti-infective properties of eps 7630, directed against viruses and bacteria, little is known about its molecular effects and cellular targets. moreover, we do not understand the pathway(s), via which eps 7630 mediates its effects on cells. so far, the majority of published studies regarding the effects of pelargonium extracts concern tissue cells. only few studies involved immune cells, and most of them focused on murine macrophages [13, 15, [20] [21] [22] . especially, there are no studies that investigated the influence of eps 7630 on cytokine production by human primary immune cells. however, cytokines play an essential role in the defense against bacteria and viruses and in the intercellular communication between immune and tissue cells [27] [28] [29] [30] [31] [32] . therefore, the focus of our current study was to unravel the possible influence of eps 7630 on human immune cells. our data show that eps 7630 strongly and dose-dependently induced the production of the pro-inflammatory cytokines tnf-α and il-6 in human blood immune cells. moreover, a less prominent induction of the anti-inflammatory acting il-10 was observed. importantly, the cytokine profile induced by eps 7630 was completely different from that induced by viral or bacterial infection-mimicking agents that caused production of a more anti-inflammatory cytokine milieu. these observations suggest that eps 7630 acts as an immunostimulant and, before infection, may promote the innate immune defense and the ability of the body to quickly and efficiently eliminate potentially incoming microbes. underlying mechanisms may include in particular the activation of phagocytes by tnf-α, the induction of acute phase proteins in the liver, and the elevation of neutrophil generation in the bone marrow. within human pbmcs, monocytes but not cd4 + t cells were found to be targets of eps 7630 and to produce high amounts of tnf-α upon this treatment. this result is in line with previous data showing that pelargonium extract acted on murine myeloid cells, in which it induced the production of tnf-α and il-1β [13, [20] [21] [22] . when investigating the signaling cascades induced by eps 7630, we found a strong mapk kinase pathway activation, which included phosphorylation of jnk, erk1/2, and p38. furthermore, eps 7630 slightly provoked nf-κb and pi3k pathway activation. however, pharmacological blockade of only p38 resulted in a strongly decreased monocyte tnf-α production. the observation that this signaling pattern differed from that induced by tlr3 and tlr4 ligand, inflammatory cytokines, and cd3/cd28 engagement suggest that pelargonium extract affects monocytes via receptors different from those used by the mentioned stimuli. it has previously been reported that the majority of in vitro macrophage activation with immune stimulating botanicals is caused by lipoproteins and lipopolysaccharide derived from bacterial contamination of the herbal raw material or from endophytes colonizing these plants [33] . as described above, we observed that the eps 7630-activated pathway and cytokine pattern was very different from the response seen after stimulation of human pbmcs with bacterial lipopolysaccharide. furthermore, analysis of eps 7630 for the presence of contaminating bacterial lipopolysaccharide by a limulus amebocyte lysate (lal) assay revealed a very low content of less than 200 eu/mg, which is equivalent to about 20 ng/mg. currently, it is not known which constituents of eps 7630 are responsible for the observed effects on cytokine production. previous investigations have suggested that the antiviral activity of the extract is quantification of tnf-α, il-6, and il-10 in respective culture supernatants by elisa. mean (± sem) cytokine concentration data from 12 donors are given as percent of 0 μg/ml eps 7630 group (control). cytokine concentrations in the absence of eps 7630 in tl3l and tl4l groups were: 1267±414 and 1534±324 pg/ml (tnf-α), 1278±369 and 2135±650 pg/ml (il-6), 1011±127 and 1430±140 pg/ml (il-10), respectively. significant differences compared to 0 μg/ml eps 7630 group are indicated (* p<0.05, ** p<0.01, wilcoxon matched-pairs signed-rank test). doi:10.1371/journal.pone.0138075.g004 primarily due to its content of prodelphinidins [7] . as these complex molecules may not be systemically bioavailable, further studies are required to demonstrate whether the clinically observed therapeutic effect of eps 7630 is mediated by components different from prodelphinidins via stimulation of the immune system in the gastrointestinal tract. by our study, we also demonstrated that eps 7630 preincubation of immune cells influenced their subsequent, by viral or bacterial infection-mimicking agents induced cytokine production towards an il-6-dominated milieu, with lower tnf-α and il-10 content. this observation suggests that taking eps 7630 before infection might reduce flu-like symptoms during infection, which are associated with the action of tnf-α. furthermore, the eps 7630 caused elevated il-6 production during infection might strengthen the production of acute phase proteins, neutrophilic granulocyte generation in the bone marrow, and the establishment of adaptive th17 and th22 cells. in fact, the differentiation of th17 cells is, besides il-1β, il-23 and tgf-β, particularly dependent on the presence of il-6 [34] . furthermore, il-6 also cooperates with tnf-α in inducing the differentiation of th22 cells [32, 35] and both th17 and th22 cells play an essential role in the defense against microbes at body barriers [36, 37] . thus, by indirect induction of th17 and th22 responses, eps 7630 treatment of patients may promote the adaptive host defense against different viral, bacterial or fungal pathogens at respiratory epithelia. a historical, scientific and commercial perspective on the medicinal use of pelargonium sidoides (geraniaceae) pelargonium sidoides for acute bronchitis: a systematic review and meta-analysis treatment of acute rhinosinusitis with the preparation from pelargonium sidoides eps 7630: a randomized, double-blind, placebo-controlled trial efficacy of extract of pelargonium sidoides in children with acute non-group a beta-hemolytic streptococcus tonsillopharyngitis: a randomized, double-blind, placebo-controlled trial efficacy of a pelargonium sidoides preparation in patients with the common cold: a randomized, double blind, placebo-controlled clinical trial randomised, doubleblind, placebo-controlled trial of eps 7630 in adults with copd eps(r) 7630 (umckaloabo(r)), an extract from pelargonium sidoides roots, exerts anti-influenza virus activity in vitro and in vivo fascinating metabolic pools of pelargonium sidoides and pelargonium reniforme, traditional and phytomedicinal sources of the herbal medicine umckaloabo a detailed view on the constituents of eps 7630 the root extract of the medicinal plant pelargonium sidoides is a potent hiv-1 attachment inhibitor investigation of the influence of eps(r) 7630, a herbal drug preparation from pelargonium sidoides, on replication of a broad panel of respiratory viruses efficacy of an aqueous pelargonium sidoides extract against herpesvirus anti-infective activities of pelargonium sidoides (eps(r) 7630): effects of induced no production on leishmania major in infected macrophages and antiviral effects as assessed in a fibroblast-virus protection assay eps 7630, an extract from pelargonium sidoides roots inhibits adherence of helicobacter pylori to gastric epithelial cells extract of pelargonium sidoides (eps 7630) improves phagocytosis, oxidative burst, and intracellular killing of human peripheral blood phagocytes in vitro evaluation of an aqueous-ethanolic extract from pelargonium sidoides (eps(r) 7630) for its activity against group a-streptococci adhesion to human hep-2 epithelial cells anti-adhesive activities of flavan-3-ols and proanthocyanidins in the interaction of group a-streptococci and human epithelial cells an extract of pelargonium sidoides (eps 7630) inhibits in situ adhesion of helicobacter pylori to human stomach a new approach to pharmacological effects on ciliary beat frequency in cell cultures-exemplary measurements under pelargonium sidoides extract (eps 7630) tannins and related compounds induce nitric oxide synthase and cytokines gene expressions in leishmania major-infected macrophage-like raw 264.7 cells pharmacological profile of extracts of pelargonium sidoides and their constituents nitric oxide synthase and cytokines gene expression analyses in leishmania-infected raw 264.7 cells treated with an extract of pelargonium sidoides (eps 7630). phytomedicine cutting edge: immune cells as sources and targets of the il-10 family members? maturing dendritic cells are an important source of il-29 and il-20 that may cooperatively increase the innate immunity of keratinocytes il-22 and il-20 are key mediators of the epidermal alterations in psoriasis while il-17 and ifn-gamma are not il-22 regulates the expression of genes responsible for antimicrobial defense, cellular differentiation, and mobility in keratinocytes: a potential role in psoriasis tumor necrosis factor receptor signaling in keratinocytes triggers interleukin-24-dependent psoriasis-like skin inflammation in mice il-19 is a component of the pathogenetic il-23/il-17 cascade in psoriasis il-22 increases the innate immunity of tissues deficiency of il-22 contributes to a chronic inflammatory disease: pathogenetic mechanisms in acne inversa the th17 cytokine il-22 induces il-20 production in keratinocytes: a novel immunological cascade with potential relevance in psoriasis il-29 is produced by t(h)17 cells and mediates the cutaneous antiviral competence in psoriasis the majority of in vitro macrophage activation exhibited by extracts of some immune enhancing botanicals is due to bacterial lipoproteins and lipopolysaccharides the differentiation of human t(h)-17 cells requires transforming growth factor-beta and induction of the nuclear receptor rorgammat production of interleukin 22 but not interleukin 17 by a subset of human skin-homing memory t cells the role of th17 cytokines in primary mucosal immunity therapeutic opportunities of the il-22-il-22r1 system the authors acknowledge brigitte ketel and beate pust for their steady excellent technical assistance. conceived and designed the experiments: rs. performed the experiments: k. witte. analyzed the data: rs k. wolk. contributed reagents/materials/analysis tools: ek. wrote the paper: k. witte rs k. wolk hdv ek. key: cord-355874-nz6eqcdb authors: wang, le; zhao, mengchuan; shi, zhongren; feng, zhishan; guo, weiwei; yang, shuo; liu, lanping; li, guixia title: a gexp-based assay for simultaneous detection of multiple viruses in hospitalized children with community acquired pneumonia date: 2016-09-14 journal: plos one doi: 10.1371/journal.pone.0162411 sha: doc_id: 355874 cord_uid: nz6eqcdb the gexp-based assay has recently been developed for simultaneous detection of multiple pathogens. so far, the application of the gexp assay to test larger clinical samples has hardly been reported. community-acquired pneumonia (cap) is the leading cause of death in children worldwide and a substantial proportion of childhood cap is caused by viruses. rapid and accurate diagnosis of virus infection is important for the clinical management of cap. in this study, we explored the gexp assay for simultaneous detection of 20 types/subtypes of viruses in hospitalized children with cap. a total of 1699 nasopharyngeal swabs were prospectively collected and viral nucleic acid was extracted and assayed. using viral genomic dna or rna as template, we showed that at the concentration of 10(4) copies of dna or rna of each virus/μl, all 20 target viruses were simultaneously identified by the gexp assay. fifteen control microorganisms, in contrast, failed to be amplified by the assay. about 65% of cases tested in this study had viral infection, with patients aged <3 years having a 70% positive rate, significantly higher than that in patients aged > 3 years (40%). the most frequently detected virus was rsv followed by piv3, hrv, adv and hbov. seasonal distribution analysis revealed that rsv was the most predominant in autumn and winter, while in spring and summer piv3 and rsv were the most frequently identified with similar positive percentages. one hundred twenty randomly-chosen samples tested by the gexp assay were re-evaluated by mono-rt-pcr, the results showed 97.5% diagnosis agreement between these 2 methods. our findings suggest that the gexp assay could be a valuable diagnostic tool for virus infection in pediatric patients with cap. community acquired pneumonia (cap) in children can be potentially serious and often results in hospitalization [1] [2] [3] . it is the leading cause of respiratory morbidity and mortality in children worldwide [4] [5] [6] . etiologic agents of cap include bacteria and virus [6] [7] [8] [9] [10] [11] [12] . while bacterial infection appears to occur mostly in older children, viral infection is more prevalent in patients under the age of 5 years [9, 12] . viruses commonly detected in children with cap include respiratory syncytial virus (rsv), influenza a and b (flu a and b), parainfluenza viruses (piv), adenovirus (adv), human rhinovirus (hrv), and human metapneumovirus (hpmv) etc. [7, [9] [10] [11] [12] . multiplex reverse transcription polymerase chain reaction (multiplex rt-pcr) combined with automated capillary electrophoresis has provided a platform for multi-target genome analysis. an example of this technology is the genomelab gexp genetic analysis system (https://www.beckmancoulter.com/wsrportal/bibliography?docname=br-11776a.pdf) developed by beckman coulter (crea, ca, usa). the principle of the gexp multiplex amplification assay (the gexp-based assay) is that diverse genomic sequences are specifically amplified in one reaction, and the amplicons with different sizes labelled with fluorescence dye are then separated and distinguished by automated capillary electrophoresis. for multiplex pcr, two sets of primers are used: one set of forward and reverse primers that are chimeric (target primers) containing a target sequence and a universal tag at the 5'-end. the other set of forward and reverse primers (universal primers) targeting the tag is fluorescence labeled. with the use of a significantly higher concentration of the universal primers than target primers in the reaction system, the target sequence is amplified with the chimeric primers in first few cycles, and then the amplification is overtaken by the universal primers, eventually generating amplicons containing target sequence and fluorescence-labeled tag [13] . using the gexp-based assay, hu et al. was able to simultaneously genotype nine serotypes of enteroviruses [13] ; zhang et al. detected and differentiated 11 duck viruses in one reaction [14] ; and li et al simultaneously detected multiple human respiratory viruses in nasopharyngeal aspirates from patients with pneumonitis or bronchopneumonia [15] . so far, the application of the gexp assay to test larger clinical samples has rarely been reported. in this study, we used the gexp-based assay for simultaneous detection of 20 types/subtypes of viruses in 1699 nasopharyngeal specimens collected from hospitalized children with cap. sensitivity and specificity of the gexp assay were determined using genomic nucleic acid (rna or dna) from various pathogens as template. afterwards, nasopharyngeal swab samples from 1699 patients with cap were tested using the gexp assay. in view of commonly identified and newly emerged respiratory viruses [7, [9] [10] [11] [12] 16] , 20 types/subtypes of viruses were selected as the detection targets. the target panel contained following viruses: flu a, flu b, influenza h5n1 (h5n1), piv-1, -2, and -3, rsv, hrv, adv, hmpv, human bocavirus (hbov), coronavirus hku1/oc43 (hcov-hku1/oc43), coronavirus nl63/229e (hcov-nl63/229e), sars-associated coronavirus (sars), influenza a (h1n1) pdm09 (swh1n1), influenza n2 (n2, nomenclature of this strain is based on the neuraminidase gene, genbank access number: j02156.1), influenza n1 (n1), seasonal h1n1 (seh1n1), influenza h1 (h1) and influenza h3 (h3 alignment of the conserved genomic regions was performed and a pair of chimeric primers was designed for each target virus using the primer premier software version 6.0 (premier biosoft, palo alto, ca). each primer contained a gene-specific sequence with a universal tag at the 5'-end. a pair of primers targeting the universal tag was used with the forward primer labeled with cy5 fluorescent dye. information of all primers was listed in table 1 . all primers were synthesized by sangon biotech (shanghai, china). to evaluate the sensitivity of the gexp assay, nucleic acid from all 20 target viruses and the internal control pcdna3.1 (+) dna were mixed to make the template pool. the target pool was serially diluted (10-fold) to obtain dilutions containing 10 4 −10 1 copies of dna or rna of each virus/μl. for multiplex reverse transcription (rt), the mix of all reverse primers (rt primer) from 20 target viruses (table 1 ) was used. rt was performed in a total volume of 10 μl containing 1 μl of template pool dilution, 2 μl 5× rt buffer, 1 μl rt primer mix (1 pmol each primer), 0.5 μl (20 units) reverse transcriptase, 1 μl rnase inhibitor (4 units) and 4.5 μl nuclease-free water. rt was carried out as follows, 48°c for 1 min; and then 42°c for 60 min. the reaction was terminated by incubation at 95°c for 5 min. for multiplex pcr, the mix of all target virus primers including both forward and reverse, and the mix of universal primers including both forward and reverse were used. multiplex pcr was performed as follows, 3.0 μl rt product was mixed with 1 μl 10×pcr buffer, 2 μl mgcl 2 (25 mm), 1 μl viral primers mix (1.25 pmol each primer), 1 μl universal primer mix (12.5 pmol each primer), 1 μl solution x and 1.0 μl taq dna polymerase. the pcr was completed on a thermocycler (veriti thermal cycler, applied biosystems china, beijing, china) in following steps: step 1, 94°c for 1 min; step 2, 94°c for 30 s, 60°c for 30 s and 70°c for 30 s. step 2 was repeated for 40 cycles followed by incubation at 70°c for 1 min. the amplified products were then assessed using the genomelab gexp genetic analysis system (beckman coulter). to evaluate the specificity of the gexp assay, nucleic acid from all 15 control microbes and pcdna3.1 (+) dna were mixed to make the control pool. the control pool containing 1.0 ng of dna or rna from each microbe/μl was prepared and 1 μl was used for multiplex rt-pcr analysis as described above. multiplex rt-pcr products were analyzed with the genomelab gexp genetic analysis system as follow, 220 μl of separation buffer was added into the separation plate, and 38.5 μl loading solution, 0.5 ml dna size standard-400 and 1μl multiplex pcr product were added into the reader plate covered with a drop of mineral oil. the analysis was then performed in an automated manner following the established protocol and the data were compiled by the gexp system software provided by beckman coulter. in case the peak is low, a cut off value of 2000 was used for positive/negative judgement, and the test was repeated to confirm the consistency of the results. the underlined sequences were the universal tag sequences. doi:10.1371/journal.pone.0162411.t001 the research protocol, collection and use of clinical data were approved by the research ethics board, children's hospital of hebei province. informed written consent was obtained from the parent of all participants. from march 2013 to february 2014, a total of 1699 nasopharyngeal swab specimens were collected from hospitalized patients diagnosed with cap within 48 hours of admission after written informed consent was obtained from the parent of the participant. the diagnosis was based on cap diagnosis and management guidelines established by the respiratory society of chinese medical association [17] . the nasopharyngeal specimen was collected into a transport tube containing 1 ml dmem medium with 2% heat-inactivated fetal calf serum, 50 iu/ml of penicillin and 100 μg/ml of streptomycin (gibco, beijing, china). the sample was stored at 4˚c for the same day viral nucleic acid extraction. two hundred μl of nasopharyngeal sample was spiked with 5 ng pcdna3.1 (+) dna and the rest 800 μl was stored at a -80°c freezer. the spiked sample was used for total viral nucleic acid (both dna and rna) extraction using the easypure viral dna/rna kit (qsjbio, beijing, china) according to the manufacturer's instructions. the final viral nucleic acid was eluted in 30 μl nuclease-free water. afterwards, 3 μl of extracted nucleic acid was analyzed with the gexp-based assay as described above. positive percentages in male and female patients and positive percentages among different age groups were analyzed with chi-square test using the spss 13.0.1 statistics package (spss inc., chicago, usa). p<0.05 was considered statistically significant. sensitivity was determined using the serially diluted target pool, as shown in fig 1a, at the concentration of 10 4 copy/μl, all viruses in the target pool were detected as positive, while at 10 3 copy/μl, flua, adv, hbov, hrv, sars, n2, piv2, hmpv, swh1n1, h1 and hcov-hku1/ oc43 were tested positive (fig 1b) . at 10 2 copy/μl, only piv2, adv and hbov were detected as positive while at 10 1 copy/μl, none of the target viruses was amplified (both data not shown). specificity test showed that all templates from the control pool were tested negative ( fig 1c) . those tiny peaks shown in panel c were further confirmed to be background (see s1 fig) . nasopharyngeal specimens collected from 1699 hospitalized children (male: 878; female: 821) aged from 5 days to 13 years were tested with the gexp assay. demographics of all patients were listed in table 2 . a total of 1096 samples (64.51%, 1096/1699) were positive for at least one virus, among which, coinfection was observed in 165 cases. viruses discovered in the samples included rsv, piv3, hrv, adv, hbov, piv1, hmpv, flua, hcov, flub, n1 and n2, among which rsv was the most detected followed by piv3, hrv, adv, and hbov (table 3) . the positive rates in samples collected from males and females were 66.10% and 62.8%, respectively, the difference was not statistically significant (p = 0.16). sample positivity was further compared among 4 different age groups, i.e., <1 year, 1-3 years, 3-5 years and ˃5 years. as shown in table 4 , positive rates for the groups of <1 year and 1-3 years are significantly higher than those in groups of 3-5 years and ˃5 years, respectively. seasonal distribution analysis revealed that rsv was the most predominant in autumn and winter, while in spring and summer piv3 and rsv were the most frequently identified with similar positive percentages (table 5) . nucleic acid amplification methods such as pcr and rt-pcr have increasingly been explored for identification of pathogens including virus detection in infectious respiratory diseases [9, 16, 18] . so far, only a few reports have described the use of these techniques for pathogen detection in children with cap [7, 11, 12] . determination of viral etiology for pediatric cap in larger clinical series has not been reported. in the present study, we applied multiplex rt-pcr together with automated capillary electrophoresis, namely the gexp-based assay, to detect virus in 1699 nasopharyngeal specimens from hospitalized children with cap. we showed that the gexp-based assay had high sensitivity and specificity for simultaneous detection of multiple viruses, and about 65% of cases tested were positive for virus. we randomly chose 120 samples that had been tested by the gexp-based assay, redo the mono-rt-pcr and found 97.5% diagnosis agreement between these 2 methods (data not shown, and mono-rt-pcr was run as the multiplex reactions but used only the chimeric and universal primers for the single target virus that was tested positive in the sample). further analysis revealed that patients younger than 3 years had a significantly higher virus infection rate (positive rate) than those older than 3 years. seasonal distribution analysis saw rsv as the most predominant in autumn and winter, while in spring and summer piv3 and rsv were the most frequently identified with similar positive percentages. only a few reports have thus far described different assays for virus detection in pediatric cap patients [7, 11, 12, 19] . chen et al measured antibodies in 1204 serum samples collected from cap children and found only about 15% were positive for virus [19] , in sharp contrast to our findings and others' [7, 11, 12] . this discrepancy is likely attributed to 1) that the antibody assay targeted only a few types of viruses, i.e., influenza a and b, parainfluenza 1, 2 and 3 and rsv [19] ; and 2) that the antibody assay is less sensitive than pcr [7, 16] . juven et al explored several methods, i.e., cell culture, viral antigen detection, antibody assay and rt-pcr to determine the etiology of cap in 254 hospitalized children, and they discovered that 62% of the patients had viral infection with rsv (29%) and hrv (24%) being the most commonly detected [7] . cantais tested 9 types of viruses, i.e., rsv, adv, hmpv, hbov, coronavirus, flua, flub, hrv and pivs using the biplex commercially-available rt-pcr kit in 85 children with cap, and revealed that the viral infection was 62.4% [11] . rhedin et al employed real time pcr to detect virus in nasopharyngeal aspirates from children aged 5 years with cap, and reported that viruses were detected in 81% of the cases [12] . these data together with ours suggest that majority of pediatric cap cases have virus infection, especially in younger patients. to our knowledge, this study is the first to use the gexp-based assay for virus detection in pediatric cap patients. the assay covered a broad range of virus targets and a large size of samples were tested. due to its automation and multiplex procedure, the gexp-based assay is less laborious than mono-pcr. it also saves samples and avoids well-to-well and pipetting errors. in the present study, plasmid dna was used as the internal control, which monitors only pcr but not the reverse transcription step. interestingly, in a few published papers describing the gexp-based multiplex assays internal controls were not employed [13] [14] [15] . we showed that genomic rna from all rna viruses in the target panel was successfully amplified by the gexp-based assay (fig 1a) , indicating that reverse transcription is not a problem. however, we felt an rna template should be used as the internal control in future work. another limitation of this assay is that highly specialized laboratory equipment is needed, namely an automated capillary electrophoresis system. many clinical laboratories do not have this equipment, and outsourcing the fragment analysis would greatly increase the turnaround time. control template pool containing 1 ng nucleic acid of each control pathogen and 10 3 copies of internal control was used and the reaction was repeated. as shown in this scaled-down plot, all those tiny peaks were identified as background products. similar results were observed in 3 independent reactions. (tif) conceptualization: ll gl. epidemiology and clinical characteristics of community-acquired pneumonia in hospitalized children national hospitalization trends for pediatric pneumonia and associated complications community-acquired pneumonia requiring hospitalization among u.s. children who estimates of the causes of death in children global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since epidemiology and etiology of childhood pneumonia in 2010: estimates of incidence, severe morbidity, mortality, underlying risk factors and causative pathogens for 192 countries etiology of communityacquired pneumonia in 254 hospitalized children community-acquired pneumonia in children detection of 11 common viral and bacterial pathogens causing community-acquired pneumonia or sepsis in asymptomatic patients by using a multiplex reverse transcription-pcr assay with manual (enzyme hybridization) or automated (electronic microarray) detection association of human metapneumovirus with radiologically diagnosed community-acquired alveolar pneumonia in young children epidemiology and microbiological investigations of community-acquired pneumonia in children admitted at the emergency department of a university hospital respiratory viruses associated with community acquired pneumonia in children: matched case-control study simultaneously typing nine serotypes of enteroviruses associated with hand, foot, and mouth disease by a gexp analyzer-based multiplex reverse transcription-pcr assay gexp analyzer-based multiplex reversetranscription pcr assay for the simultaneous detection and differentiation of eleven duck viruses the development of a gexp-based multiplex reverse transcription-pcr assay for simultaneous detection of sixteen human respiratory virus types/ subtypes gexp-based assay for multiple virus detection in children with cap development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbead-based assay guidelines for the diagnosis and treatment of community-acquired pneumonia: learning and practicing simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays the aetiology of community associated pneumonia in children in nanjing, china and aetiological patterns associated with age and season key: cord-353200-5csewb1k authors: jehi, lara; ji, xinge; milinovich, alex; erzurum, serpil; merlino, amy; gordon, steve; young, james b.; kattan, michael w. title: development and validation of a model for individualized prediction of hospitalization risk in 4,536 patients with covid-19 date: 2020-08-11 journal: plos one doi: 10.1371/journal.pone.0237419 sha: doc_id: 353200 cord_uid: 5csewb1k background: coronavirus disease 2019 is a pandemic that is straining healthcare resources, mainly hospital beds. multiple risk factors of disease progression requiring hospitalization have been identified, but medical decision-making remains complex. objective: to characterize a large cohort of patients hospitalized with covid-19, their outcomes, develop and validate a statistical model that allows individualized prediction of future hospitalization risk for a patient newly diagnosed with covid-19. design: retrospective cohort study of patients with covid-19 applying a least absolute shrinkage and selection operator (lasso) logistic regression algorithm to retain the most predictive features for hospitalization risk, followed by validation in a temporally distinct patient cohort. the final model was displayed as a nomogram and programmed into an online risk calculator. setting: one healthcare system in ohio and florida. participants: all patients infected with sars-cov-2 between march 8, 2020 and june 5, 2020. those tested before may 1 were included in the development cohort, while those tested may 1 and later comprised the validation cohort. measurements: demographic, clinical, social influencers of health, exposure risk, medical co-morbidities, vaccination history, presenting symptoms, medications, and laboratory values were collected on all patients, and considered in our model development. results: 4,536 patients tested positive for sars-cov-2 during the study period. of those, 958 (21.1%) required hospitalization. by day 3 of hospitalization, 24% of patients were transferred to the intensive care unit, and around half of the remaining patients were discharged home. ten patients died. hospitalization risk was increased with older age, black race, male sex, former smoking history, diabetes, hypertension, chronic lung disease, poor socioeconomic status, shortness of breath, diarrhea, and certain medications (nsaids, immunosuppressive treatment). hospitalization risk was reduced with prior flu vaccination. model discrimination was excellent with an area under the curve of 0.900 (95% confidence interval of 0.886–0.914) in the development cohort, and 0.813 (0.786, 0.839) in the validation cohort. the scaled brier score was 42.6% (95% ci 37.8%, 47.4%) in the development cohort and 25.6% (19.9%, 31.3%) in the validation cohort. calibration was very good. the online risk calculator is freely available and found at https://riskcalc.org/covid19hospitalization/. limitation: retrospective cohort design. conclusion: our study crystallizes published risk factors of covid-19 progression, but also provides new data on the role of social influencers of health, race, and influenza vaccination. in a context of a pandemic and limited healthcare resources, individualized outcome prediction through this nomogram or online risk calculator can facilitate complex medical decision-making. based on the latest estimates from the centers for disease control (week ending in june 6, 2020), hospitalization rates in the united states due to coronavirus disease of 2019 (covid-19) range from 5.6/100,000 population in patients 4 years or younger and up to 273.8/100,000 population in those 65 years or older, posing a significant capacity challenge to the healthcare system. strategies to address this challenge have focused on imposing social distancing to reduce viral transmission and increasing hospital bed capacity by drastically reducing usual occupancy, eliminating elective surgical procedures, and creating makeshift surge hospitals [1] . social distancing practices have indeed helped in curbing the acute need for hospital bedsat least momentarily-but the long-term healthcare capacity requirements remain unclear as strategies for lifting restrictions and resuming normal activities are in flux. improving our understanding of the clinical outcomes of patients infected with covid-19 is therefore paramount. in addition, we need predictive algorithms that identify the covid-19 patients at highest risk of progressing to severe disease to develop alternative approaches to safely manage hospitalization risk prediction and outcomes in covid-19 plos one | https://doi.org/10.1371/journal.pone.0237419 august 11, 2020 2 / 15 ethical restrictions by the cleveland clinic regulatory bodies including the institutional review board and legal counsel. in particular, variables like the patient's address, date of testing, dates of hospitalization, date of icu admission, and date of mortality are hipaa protected health information and legally cannot be publicly shared. since these variables were critical to the generation and performance of the model, a partial dataset (everything except them) is not fruitful either because it will not help in efforts of academic advancement, such as model validation or application. we will make our data sets available upon request, under appropriate data use agreements with the specific parties interested in academic collaboration. requests for data access can be made to mascar@ccf.org. them. these predictive algorithms could also be used at a population level to guide social distancing and other risk limiting strategies in a focused fashion, rather than the blanket approaches of shelter-in-place for society. older age [2] [3] , smoking [4] , and medical co-morbidities such as diabetes, hypertension, cardiovascular disease, chronic kidney disease, chronic lung disease [5] , and cancer [5] [6] have been correlated with disease worsening in patients who are already hospitalized with covid-19. it is unclear how these comorbidities, or other patient characteristics, factor into clinical worsening that leads to hospitalization. translating their significance at an individual patient care level when faced with a decision to hospitalize patients presenting with symptoms of covid-19 is even more elusive. the end result is patients being told to go home from the emergency room only to return much more ill and be admitted days later, or patients hospitalized for observation for several days without any significant clinical deterioration. we present the clinical characteristics and outcomes of patients with covid-19, including a subset who were hospitalized. we also develop and validate a statistical model that can assist with individualized prediction of hospitalization risk for a patient with covid-19. this model allows us to generate a visual statistical tool (a nomogram) that can consider numerous variables to predict an outcome of interest for an individual patient [7] . we included all patients, regardless of age, who had positive covid-19 testing at cleveland clinic between march 8, 2020 and june 5, 2020. the study cohort included all covid positive patients, whether they were hospitalized or not, from across the cleveland clinic health system which includes >220 outpatient locations and18 hospitals in ohio and florida. as testing demand increased, we adapted our organizational policies and protocols to reconcile demand with patient and caregiver safety. prior to march 18, any primary care physician could order a covid-19 test. after that date, testing resources were streamlined through a "covid-19 hotline" which followed recommendations from the centers for disease control (recommending to focus on high risk patients as defined by any of the following: age older than 60 years old or less than 36 months old; on immune therapy; having comorbidities of cancer, end-stage renal disease, diabetes, hypertension, coronary artery disease, heart failure with reduced ejection fraction, lung disease, hiv/aids, solid organ transplant; contact with known covid 19 patients; physician discretion was still allowed). demographics, co-morbidities, travel and covid-19 exposure history, medications, presenting symptoms, socioeconomic measures, treatment, disease progression, and outcomes were collected. registry variables were chosen to reflect available literature on covid-19 disease characterization, progression, and proposed treatments, including medications thought to have benefits through drug-repurposing studies [8] . capture of detailed research data was facilitated by the creation of standardized clinical templates that were implemented across the healthcare system as patients were seeking care for covid-19-related concerns. outcome capture was facilitated by a home monitoring program whereby patients who tested positive were called daily for 14 days after-test result to monitor their disease progression. data were extracted via previously validated automated feeds [9] from our electronic health record (epic, epic systems corporation) and manually by a study team trained on uniform sources for the study variables. the covid-19 research registry team includes a "reviewer" group and a "quality assurance" group. the reviewers were responsible for manually abstracting and entering a subset of variables (signs and symptoms upon presentation) that cannot be automatically extracted from the electronic health record, and for verifying high-priority variables (co-morbidities) that have been automatically pulled into the database from the electronic health record. the quality assurance group provided an independent second layer of review. study data were collected and managed using redcap electronic data capture tools hosted at cleveland clinic [10] [11] . redcap (research electronic data capture) is a secure, web-based software platform designed to support data capture for research studies, providing 1) an intuitive interface for validated data capture; 2) audit trails for tracking data manipulation and export procedures; 3) automated export procedures for seamless data downloads to common statistical packages; and 4) procedures for data integration and interoperability with external sources. this research was approved by the cleveland clinic institutional review board (irb# 20-283). consent was waived by irb. nasopharyngeal and oropharyngeal swab specimens were both collected in all patients and pooled for testing by trained medical personnel. given previous beliefs that co-infection with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and other respiratory viruses is rare [12] [13] , a reflex testing algorithm was implemented to conserve resources. all patient specimens were first tested for the presence of influenza a/b and respiratory syncytial virus (rsv), and only those negative for influenza and rsv were subsequently tested for sars--cov-2. infection with sars-cov-2 was confirmed by laboratory testing using the centers for disease control and prevention (cdc) reverse transcription polymerase chain reaction (rt-pcr) sars-cov-2 assay that was validated in the cleveland clinic robert j. tomsich pathology and laboratory medicine institute. this assay uses roche magnapure extraction and abi 7500 dx pcr instruments. between march 8 and 13, the tests were sent out to lab-corp, burlington, north carolina. all testing was authorized by the food and drug administration under an emergency use authorization (eua), and in accordance with the guidelines established by the cdc. baseline data are presented as median [interquartile range [iqr] ) and number (%)]. continuous variables were compared using the mann-whitney u test, and categorical variables were compared using the chi-square test. the outcome of interest was hospitalization anytime within three days of a positive covid test. the model was built using a development cohort (patients with covid positive test resulted before may 1, 2020), and subsequently tested in a validation cohort (patients with covid positive test resulted between may 1 and june 5, 2020). this allowed us to test the model's validity over time. a full multivariable logistic model was initially constructed to predict hospital admission with covid-19 based on demographic variables, comorbidities, immunization history, symptoms, travel history, lab variables, and medications that were identified pre-admission. for modeling purposes, methods of missing value imputation for labs variables were compared using median values and using values from multivariate imputation by chained equations (mice) via the r package mice. restricted cubic splines with 3 knots were applied to continuous variables to relax the linearity assumption. a least absolute shrinkage and selection operator (lasso) logistic regression algorithm was performed to retain the most predictive features. a 10-fold cross validation method was applied to find the regularization parameter lambda which gave the minimum mean cross-validated concordance index. predictors with nonzero coefficients in the lasso regression model were chosen for calculating predicted risk. the final model was internally validated by assessing the discrimination and calibration with 1000 bootstrap resamples. discrimination was measured with the concordance index [14] . calibration was assessed visually by plotting the nomogram predicted probabilities against the observed event proportions over a series of equally spaced values within the range of the predicted probabilities. the closer the calibration curve lies along the 45˚line, the better the calibration. a scaled brier score, called the index of predictive accuracy (ipa) [15] , was also calculated, as this has some advantages over the more popular concordance index. the ipa ranges from -1 to 1, where a value of 0 indicates a useless model, and negative values imply a harmful model. we adhered to the tripod checklist for reporting the prediction model [16] . we calculated sensitivity, specificity, positive addictive value, negative predictive value at different cutoffs of predicted risk. we used r, version 3.5.0 (r project for statistical computing) [17] , with tidyverse [18] , mice [19] , caret [20] , and risk regression [21] packages for all analyses. statistical tests were 2-sided and used a significance threshold of p < .05. we included all covid positive patients during the study period in this model development and validation to optimize model performance: no specific sample size calculations were performed. an outcome of "hospitalized versus not" allows us to predict the likelihood that the patient is actually getting admitted to the hospital. this decision, however, is influenced by multiple "non-medical" factors including bed availability, regulatory systems, and individual physician preferences. to test the applicability of our model towards a determination of whether a patient should have been admitted or not, we subdivided patients included in our validation cohort and development cohorts into 4 categories: a-hospitalized and not sent home within 24 hours; b-sent home (not initially hospitalized) but ultimately hospitalized within 1 week of being sent home; c-not hospitalized at all; d-hospitalized but sent home within 24 hours. in this construct, categories a and c represent patients who were "correctly managed", at categories b and d represent those who were "incorrectly managed". we then tested the discrimination of our model in each one of those categories separately. no model recalibration was done. 4,536 patients tested positive during the study period, including 2,852 patient in the development cohort (dc) of whom 582 (20.4%) were hospitalized, and 1,684 patients in the validation cohort (vc) of whom 376 (22.3%) were hospitalized. table 1 provides demographic, exposure, clinical, laboratory, social characteristics, and medication history of covid-19 patients who were hospitalized versus those who completed their treatment on an outpatient basis in both the dc and vc. at the time of hospital admission, 260 patients were known to have covid-19, while the results of the (rt-pcr) sars-cov-2 nasopharyngeal assay were still pending on 698. six hundred and sixty five were admitted from the emergency room, 32 were transferred from other hospitals, and 261 were directly admitted from the outpatient areas. overall outcomes illustrated in fig 1 show the cumulative incidence of hospital discharge, transfer to intensive care unit, and death in our hospitalized cohort. imputation methods were evaluated with 1000 repeated bootstrapped samples. we found that models based on median imputation appeared to outperform those based on data from mice imputation, so median imputation was selected for the basis of the final model. variables that we examined and were not found to add value beyond those included in our final model for predicting hospitalization included exposure to covid 19, other family members with covid-19, fever, fatigue, sputum production, flu-like symptoms, recent international travel, coronary artery disease, heart failure, on immunosuppressive treatment, other heart disease, other lung disease, pneumovax vaccine, bun, on angiotensin converting enzyme inhibitor, angiotensin receptor blocker, toremifene, and paroxetine. model discrimination was excellent with an area under the curve of 0.900 (95% confidence interval of 0.886-0.914) in the development cohort, and 0.813 (0.786, 0.839) in the validation cohort. the scaled brier score was 42.6% (95% ci 37.8%, 47.4%) in the development cohort and 25.6% (19.9%, 31.3%) in the validation cohort. the nomogram is presented in fig 2, and an online version of the statistical (fig 3) is available at https://riskcalc.org/covid19hospitalization/. the calibration curves are shown in fig 4 and suggest that predicted risk matches observed proportions relatively well throughout the risk range. table 2 shows the sensitivity, specificity, negative predictive value, and positive predictive value at different cutoffs of predicted risk. appropriately managed patients represented the majority of the cohort: 750 patients were hospitalized with a length of stay that exceeded 24 hours (431 in dc and 319 in vc), and 3549 patients were not hospitalized at all (2258 in dc and 1291 in vc). a minority of patients (237 patients, 5.4%) fell in the category of inappropriate initial management: 208 had been initially sent home from the emergency room but were then admitted within 1 week of emergency room visit (151 in dc, 57 in vc), and 29 patients were hospitalized but then discharged within 24 hours (12 in dc, and 17 in vc). when tested in each one of those categories, the predictive model performed very well in the appropriately managed subgroup (area under the curve of 0.821), but its performance was inadequate in the 5.4% of patients who fell in the inappropriate initial management category. our results confirm a higher risk of hospitalization with older age (median age in hospitalized patients of 65.5 years compared to 48.0 years in non-hospitalized patients), male sex (56.9% of hospitalized vs 48.3% of non-hospitalized), and medical co-morbidities most prominently hypertension, diabetes, and immunosuppressive disease (variables significant on univariable analysis in table 1 , but also relevant in final model). the significant association of shortness of breath and diarrhea with hospitalization may reflect the need for inpatient supportive care with these symptoms, regardless of the etiology. beyond the expected, our results provide some insights that advance the existing literature: the world health organization warns of a higher morbidity for covid-19 in smokers, and proposes multiple possible mechanisms including frequent touching of face and mouth during the act of smoking, sharing cigarettes, and underlying lung disease [22] . we found that former smokers rather than current smokers are at higher risk of covidrelated hospitalization (table 1) , favoring the underlying lung disease mechanism. 2. medications: we found a higher risk of hospitalizations in covid-19 patients who were on angiotensin converting enzyme (ace) inhibitors, or angiotensin ii type-i receptor blockers (arbs) on univariable analysis [16, [23] [24] . however, being on these medications did not influence the final multivariable model, suggesting that prior associations between acei's and arbs with covid severity may be confounded by the underlying medical comorbidities (hypertension and diabetes) that are linked to highest covid hospitalization rates, and which are most often treated with these same drugs. ace2 can also be increased by thiazolidinediones and ibuprofen, potentially explaining the higher hospitalization risk seen in our patients on non-steroidal anti-inflammatory drugs (nsaids); in fact, the latest fda guidance cautions against the use of nsaids in covid patients [25] . overall, we recommend caution using retrospective data to draw robust conclusions assigning causation to drugs vs underlying co-morbidity vs genetically driven ace2 polymorphism. we highlight the need for carefully designed, large observational studies or randomized clinical trials to address these critical questions. 3. race: african american race was correlated with a higher hospitalization risk (36.2% of hospitalized vs 21% of non-hospitalized). this is consistent with a recent look at hospitalizations for covid-19 across 14 states from march 1 to 30 [26] . race data, which were available for 580 of 1,482 patients, revealed that african americans accounted for 33 percent of the hospitalizations, but only 18 percent of the total population surveyed [26] . the authors proposed explanations like higher rates of medical co-morbidities, higher exposure risks, and distrust of the medical community as a postulated rationale. our data, however, show that the effect of race on the individualized hospitalization risk prediction far outweighs that of any medical co-morbidity (fig 2) . it is already known that race influences the effectiveness of an immune response [27] . a deeper exploration of the underlying genetics and biology of race in the defense against and the response to a sars-cov-2 infection is needed. this should be paired with a deeper exploration of social influencers of health such as population per square kilometer, and population per household which were also relevant in our nomogram. in our online risk calculator, only the zip code entry is required: the relevant social influencers data are derived from the zip code by our program. given the multitude of risk factors discussed, the nomogram and online risk calculator assist with obviating challenges of translating complex information to patient-level clinical decisionmaking [28] . during a pandemic, with hospital beds in short supply, it is critical to empower front-line healthcare providers with tools that can supplement and support decision-making about who to admit. advances in tele-health can be leveraged for home monitoring to guide care delivery in an outpatient setting for those determined to be low risk based on the nomogram calculation. models like ours developed with data obtained through an automated abstraction from the electronic health record (ehr) offer the promise of integration within the ehr to facilitate rapid and efficient implementation into the clinical workflow. such a strategy is a pragmatic application of overdue calls for a learning health system [29]. model performance, as measured by the concordance index, is excellent (c-statistic = 0.900). this level of discrimination is clearly superior to a coin toss or assuming all patients are at equivalent risk (both c-statistics = 0.5). the calibration of the model is excellent in both the dc and vc (see fig 4) . the metric that considers calibration, the ipa value, confirms that the model predicts substantially better than chance or no model at all. overall, the model performs very well. our next step will be to integrate this model into the clinical workflow. manually abstracting data and inputting it in an online calculator is cumbersome in a busy clinical practice. interpreting the prediction without some frame of reference is complex. however, failing to see beyond these hurdles risks wasting opportunities to innovate and improve patient care. it is therefore imperative to develop a clear implementation strategy that aligns with the existing clinical needs and clinical operations of a health organization. one could start by identifying the clinical problems that would benefit from this prediction tool, and reference the information in table xx on sensitivity, specificity, positive predictive value, and negative predictive value at different prediction cutoffs to provide a framework for clinical application. an illustrative example now being explored from our own health system is the use of this calculator to tailor the intensity of home monitoring for covid positive patients. currently, every patient who tests positive for covid is being called daily for 14 days to check on their symptoms and identify disease progression early enough for intervention. with only 20-30% of covid positive patients progressing to the point of requiring hospitalization, the nurses can use our prediction tool to identify this high risk group and only call them daily, while reducing the intensity of follow-up with the rest. this is not a multicenter study. it is important to note though that it includes all hospitals and outpatient facilities of the cleveland clinic health system within the us (>220 outpatient locations and18 hospitals in ohio and florida) creating robust sampling of the covid-19 population. as with any other statistical model, other hospital systems may elect to validate this model internally for their specific patient populations as they contemplate options for integrating it in their workflow. given the alternative of no or constantly changing practice guidelines, implementation of this nomogram into our clinical workflow will allow prospective evaluation of its impact on patient care and outcomes. our model includes age as a predictor: this may mitigate our ability to identify risk factors for disease progression specific in the younger population, and may underestimate the risks in the younger population with less severe disease and less likely to seek medical care. lastly, although our model performs very well in the majority of covid positive patients, more research is needed to optimize it for the sub group (5.4% of the total cohort in our series) with either delayed or unnecessary admission. drivers of disease progression and worsening in covid-19 are multiple and complex. we developed a statistical model with excellent predictive performance (c-statistic of 0.926) to individualize the hospitalization risk assessment at the patient level. this could help guide clinical decision-making and resource allocation. supporting information s1 checklist. (pdf) conceptualization: lara jehi, steve gordon, michael w. kattan. data curation: alex milinovich. american hospital capacity and projected need for covid-19 patient care clinical progression of patients with covid-19 in shanghai clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study analysis of factors associated with disease outcomes in hospitalized patients with 2019 novel coronavirus disease characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention cancer patients in sars-cov-2 infection: a nationwide analysis in china network-based drug repurposing for novel coronavirus 2019-ncov/ sars-cov-2 extracting and utilizing electronic health data from epic for research research electronic data capture (redcap)-a metadata-driven methodology and workflow process for providing translational research informatics support the red-cap consortium: building an international community of software partners public health response to the initiation and spread of pandemic covid-19 in the united states rapid sentinel surveillance for covid-19 evaluating the yield of medical tests the index of prediction accuracy: an intuitive measure useful for evaluating risk prediction models transparent reporting of a multivariable prediction model for individual prognosis or diagnosis (tripod): the tripod statement r: a language and environment for statistical computing. r foundation for statistical computing tidyverse: easily install and load the 'tidyverse mice: multivariate imputation by chained equations in r caret: classification and regression training na). riskregression: risk regression models and prediction scores for survival analysis with competing risks receptor recognition by novel coronavirus from wuhan: an analysis based on decadelong structural studies of sars the vasoprotective axes of the renin-angiotensin system: physiological relevance and therapeutic implications in cardiovascular, hypertensive and kidney diseases hospitalization rates and characteristics of patients hospitalized with laboratory-confirmed coronavirus disease 2019-covid-net, 14 states genetic adaptation and neandertal admixture shaped the immune system of human populations complete blood count might help to identify subjects with high probability of testing positive to sars-cov-2 national academies press (us) key: cord-329999-flzqm3wh authors: buchanan, tom title: why do people spread false information online? the effects of message and viewer characteristics on self-reported likelihood of sharing social media disinformation date: 2020-10-07 journal: plos one doi: 10.1371/journal.pone.0239666 sha: doc_id: 329999 cord_uid: flzqm3wh individuals who encounter false information on social media may actively spread it further, by sharing or otherwise engaging with it. much of the spread of disinformation can thus be attributed to human action. four studies (total n = 2,634) explored the effect of message attributes (authoritativeness of source, consensus indicators), viewer characteristics (digital literacy, personality, and demographic variables) and their interaction (consistency between message and recipient beliefs) on self-reported likelihood of spreading examples of disinformation. participants also reported whether they had shared real-world disinformation in the past. reported likelihood of sharing was not influenced by authoritativeness of the source of the material, nor indicators of how many other people had previously engaged with it. participants’ level of digital literacy had little effect on their responses. the people reporting the greatest likelihood of sharing disinformation were those who thought it likely to be true, or who had pre-existing attitudes consistent with it. they were likely to have previous familiarity with the materials. across the four studies, personality (lower agreeableness and conscientiousness, higher extraversion and neuroticism) and demographic variables (male gender, lower age and lower education) were weakly and inconsistently associated with self-reported likelihood of sharing. these findings have implications for strategies more or less likely to work in countering disinformation in social media. disinformation is currently a critically important problem in social media and beyond. typically defined as "the deliberate creation and sharing of false and/or manipulated information that is intended to deceive and mislead audiences, either for the purposes of causing harm, or for political, personal or financial gain", political disinformation has been characterized as a significant threat to democracy [1, p.10] . it forms part of a wider landscape of information operations conducted by governments and other entities [2, 3] . its intended effects include political influence, increasing group polarisation, reducing trust, and generally undermining civil society [4] . effects are not limited to online processes. they regularly spill over into other parts of our lives. experimental work has shown that exposure to disinformation can lead to attitude change [5] and there are many real-world examples of behaviours that have been directly attributed to disinformation, such people as attacking telecommunications masts in response to fake stories about '5g causing coronavirus' [6, 7] . social media disinformation is very widely used as a tool of influence: computational propaganda has been described as a pervasive and ubiquitous part of modern everyday life [8] . once disinformation has initially been seeded online by its creators, one of the ways in which it spreads is through the actions of individual social media users. ordinary people may propagate the material to their own social networks through deliberate sharing-a core function of platforms such as facebook and twitter. other interactions with it, such as 'liking', also trigger the algorithms of social media platforms to display it to other users. this is a phenomenon known as 'organic reach' [9] . it can lead to false information spreading exponentially. as an example, analysis of the activity of the russian 'internet research agency' (ira) disinformation group in the usa between 2015 and 2017 concluded that over 30 million users shared and otherwise interacted with the ira's facebook and instagram posts, propagating them to their families and friends [4] . there is evidence that false material is spread widely and rapidly through social media due to such human behaviour [10] . when individuals share or interact with disinformation they see online, they have essentially been persuaded to do so by its originators. influential models of social information processing suggest there are different routes to persuasion [e.g . 11] . under some circumstances, we may carefully consider the information available. at other times, we make rapid decisions based on heuristics and peripheral cues. when sharing information on social media occurs, it is likely to be spontaneous and rapid, rather than being a considered action that people spend time deliberating over. for example, there are indications of people using the interaction features of facebook in a relatively unthinking and automatic manner [12] . in such situations, a peripheral route to persuasion is likely be important [13] . individuals' choices to share, like and so on will thus be guided primarily by heuristics or contextual cues [14] . three potentially important heuristics in this context are consistency, consensus and authority [15] . these are not the only heuristics that might possibly influence whether we share false material. however, in each case there is suggestive empirical evidence, and apparent realworld attempts to leverage these phenomena, that make them worth considering. consistency. consistency is the extent to which sharing would be consistent with past behaviours or beliefs of the individual. for example, in the usa people with a history of voting republican might be more likely to endorse and disseminate right-wing messaging [16] . there is a large body of work based on the idea that people prefer to behave in ways consistent with their attitudes [17] . research has indicated that social media users consider headlines consistent with their pre-existing beliefs as more credible, even when explicitly flagged as being false [18] . in the context of disinformation, this could make it desirable to target audiences sympathetic to the message content. consensus. consensus is the extent to which people think their behaviour would be consistent with that of most other people. in the current context, it is possible that seeing a message has already been shared widely might make people more likely to forward it on themselves. in marketing, this influence tactic is known as 'social proof' [19] . it is widely used in online commerce in attempts to persuade consumers to purchase goods or services (e.g. by displaying reviews or sales rankings). the feedback mechanisms of social networks can be manipulated to create an illusion of such social support, and this tactic seems to have been used in the aftermath of terror attacks in the uk [20] . bot networks are used to spread low-credibility information on twitter through automated means. bots have been shown to be involved in the rapid spread of information, tweeting and retweeting messages many times [21] . among humans who see the messages, the high retweet counts achieved through the bot networks might be interpreted as indicating that many other people agree with them. there is evidence which suggests that "each amount of sharing activity by likely bots tends to trigger a disproportionate amount of human engagement" [21, p.4] . such bot activity could be an attempt to exploit the consensus effect. it is relatively easy to manipulate the degree of consensus or social proof associated with an online post. work by the nato strategic communications centre of excellence [22] indicated that it was very easy to purchase high levels of false engagement for social media posts (e.g. sharing of posts by networks of fake accounts) and that there was a significant black market for social media manipulation. thus, if boosting consensus effectively influences organic reach, then it could be a useful tool for both those seeding disinformation and those seeking to spread counter-messages. authority. authority is the extent to which the communication appears to come from a credible, trustworthy source [23] . research participants have been found to report a greater likelihood of propagating a social media message if it came from a trustworthy source [24] . there is evidence of real-world attempts to exploit this effect. in 2018, twitter identified fraudulent accounts that simulated those of us local newspapers [25] , which may be trusted more than national media [26] . these may have been sleeper accounts established specifically for the purpose of building trust prior to later active use. factors influencing the spread of disinformation. while there are likely to be a number of other variables that also influence the spread of disinformation, there are grounds for believing that consistency, consensus and authority may be important. constructing or targeting disinformation messages in such a way as to maximise these three characteristics may be a way to increase their organic reach. there is real-world evidence of activity consistent with attempts to exploit them. if these effects do exist, they could also be exploited by initiatives to counter disinformation. not all individuals who encounter untrue material online spread it further. in fact, the great majority do not. research linking behavioural and survey data [16] found that less than 10% of participants shared articles from 'fake news' domains during the 2016 us presidential election campaign (though of course when extrapolated to the huge user base of social network platforms like facebook, this is still a very large number of people). the fact that only a minority of people actually propagate disinformation makes it important to consider what sets them apart from people who don't spread untrue material further. this will help to inform interventions aimed at countering disinformation. for example, those most likely to be misled by disinformation, or to spread it further, could be targeted with counter-messaging. it is known that the originators of disinformation have already targeted specific demographic groups, in the same way as political campaigns micro-target messaging at those audience segments deemed most likely to be persuadable [27] . for example, it is believed that the 'internet research agency' sought to segment facebook and instagram users based on race, ethnicity and identity by targeting their messaging to people recorded by the platforms as having certain interests for marketing purposes [4] . they targeted specific communications tailored to those segments (e.g. trying to undermine african americans' faith in political processes and suppress their voting in the us presidential election). research has found that older adults, especially those aged over 65, were by far the most likely to spread material originally published by 'fake news' domains [16] . a key hypothesis advanced to explain this is that older adults have lower levels of digital media literacy, and are thus less likely to be able to distinguish between true and false information online. while definitions may vary, digital media literacy can be thought of as including ". . . the ability to interact with textual, sound, image, video and social medias . . . finding, manipulating and using such information" [28, p. 11] and being a "multidimensional concept that comprised technical, cognitive, motoric, sociological, and emotional aspects" [29, p.834] . digital media literacy is widely regarded as an important variable mediating the spread and impact of disinformation [e.g. 1]. it is argued that many people lack the sophistication to detect a message as being untruthful, particularly when it appears to come from an authoritative or trusted source. furthermore, people higher in digital media literacy may be more likely to engage in elaborated, rather than heuristic-driven, processing (cf. work on phishing susceptibility [30] ), and thus be less susceptible to biases such as consistency, consensus and authority. educating people in digital media literacy is the foundation of many anti-disinformation initiatives. examples include the 'news hero' facebook game developed by the nato strategic communications centre of excellence (https://www.stratcomcoe.org/news-hero), government initiatives in croatia and france [8] or the work of numerous fact-checking organisations. the effectiveness of such initiatives relies on two assumptions being met. the first is that lower digital media literacy really does reduce our capacity to identify disinformation. there is currently limited empirical evidence on this point, complicated by the fact that definitions of 'digital literacy' are varied and contested, and there are currently no widely accepted measurement tools [28] . the second is that the people sharing disinformation are doing so unwittingly, having been tricked into spreading it. however, it is possible that at least some people know the material is untrue, and they spread it anyway. survey research [31] has found that believing a story was false was not necessarily a barrier to sharing it. people may act like this because they are sympathetic to a story's intentions or message, or they are explicitly signalling their social identity or allegiance to some political group or movement. if people are deliberately forwarding information that they know is untrue, then raising their digital media literacy would be ineffective as a stratagem to counter disinformation. this makes it important to simultaneously consider users' beliefs about the veracity of disinformation stories, to inform the design of countermeasures. personality. it is also known that personality influences how people use social media [e.g. 32]. this makes it possible that personality variables will also influence interactions with disinformation. indeed, previous research [24] found that people low on agreeableness reported themselves as more likely to propagate a message. this is an important possibility to consider, because it raises the prospect that individuals could be targeted on the basis of their personality traits with either disinformation or counter-messaging. in a social media context, personalitybased targeting of communications is feasible because personality characteristics can be detected from individuals' social media footprints [33, 34] . large scale field experiments have shown that personality-targeted advertising on social media can influence user behaviour [35] . the question of which personality traits might be important is an open one. in the current study, personality was approached on an exploratory basis, with no specific hypotheses about effects or their directions. this is because there are a number of different and potentially rival effects that might operate. for example, higher levels of conscientiousness may be associated with a greater likelihood of posting political material in social media [36] leading to a higher level of political disinformation being shared. however, people higher in conscientiousness are likely to be more cautious [37] and pay more attention to details [38] . they might therefore also be more likely to check the veracity of the material they share, leading to a lower level of political disinformation being shared. the overall aim of this project was to establish whether contextual factors in the presentation of disinformation, or characteristics of the people seeing it, make it more likely that they extend its reach. the methodology adopted was scenario-based, with individuals being asked to rate their likelihood of sharing exemplar disinformation messages. a series of four studies was conducted, all using the same methodology. multiple studies were used to establish whether the same effects were found across different social media platforms (facebook in study 1, twitter in study 2, instagram in study 3) and countries (facebook with a uk sample in study 1, facebook with a us sample in study 4). data were also collected on whether participants had shared disinformation in the past. a number of distinct hypotheses were advanced: h1: individuals will report themselves as more likely to propagate messages from more authoritative compared to less authoritative sources. h2: individuals will report themselves as more likely to propagate messages showing a higher degree of consensus compared to those showing a lower degree of consensus. h3: individuals will report themselves as more likely to propagate messages consistent with their pre-existing beliefs compared to inconsistent messages. h4: individuals lower in digital literacy will report a higher likelihood of sharing false messages than individuals higher in digital literacy. other variables were included in the analysis on an exploratory basis with no specific hypotheses being advanced. in summary, this project asks why ordinary social media users share political disinformation messages they see online. it tests whether specific characteristics of messages or their recipients influence the likelihood of disinformation being further shared online. understanding any such mechanisms will both increase our understanding of the phenomenon and inform the design of interventions seeking to reduce its impact. study 1 tested hypotheses 1-4 with a uk sample, using stimuli relevant to the uk. the study was completed online. participants were members of research panels sourced through the research company qualtrics. participants were asked to rate their likelihood of sharing three simulated facebook posts. the study used an experimental design, manipulating levels of authoritativeness and consensus apparent in the stimuli. all manipulations were between, not within, participants. consistency with pre-existing beliefs was not manipulated. instead, the political orientation of the stimuli was held constant, and participants' scores on conservative political orientation were used as an index of consistency between messages and participant beliefs. the effects of these variables on self-rated likelihood of sharing the stimuli, along with those of a number of other predictors, were assessed using multiple regression. the primary goal of the analysis was to identify variables that statistically significantly explained variance in the likelihood of sharing disinformation. the planned analysis was followed by supplementary and exploratory analyses. all analyses were conducted using spss v.25 for mac. for all studies reported in this paper, ethical approval came from both the university of westminster research ethics committee (eth1819-1420) and the lancaster university security research ethics committee (buchanan 2019 07 23). consent was obtained, via an electronic form, from anonymous participants. a short questionnaire was used to capture demographic information (gender; country of residence; education; age; occupational status; political orientation expressed as right, left or centre; frequency of facebook use). individual differences in personality, political orientation, and digital / new media literacy were measured using established validated questionnaires. ecologically valid stimuli were used, with their presentation being modified across conditions to vary authoritativeness and consensus markers. personality was measured using a 41-item five-factor personality questionnaire [38] derived from the international personality item pool [37] . the measure provides indices of extraversion, neuroticism, openness to experience, agreeableness and conscientiousness that correlate well with the domains of costa and mccrae's [39] five factor model. conservatism was measured using the 12-item social and economic conservatism scale (secs) [40] , which is designed to measure political orientation along a left-right; liberal-conservative continuum. it was developed and validated using a us sample. in pilot work for the current study, mean scores for individuals who reported voting for the labour and conservative parties in the 2017 uk general election were found to differ in the expected manner (t (28) = -2.277, p = .031, d = 0.834). this provides evidence of its appropriateness for use in uk samples. while the measure provides indices of different aspects of conservatism, it also provides an overall conservatism score which was used in this study. digital media literacy was measured using the 35-item new media literacy scale (nmls) [29] . this is a theory-based self-report measure of competences in using, critically interrogating, and creating digital media technologies and messaging. in pilot work with a uk sample, it was found to distinguish between individuals high or low in social media (twitter) use, providing evidence of validity (t (194) = -3.847, p < .001, d = .55). while the measure provides indices of different aspects of new media literacy, it also provides an overall score which was used in this study. participants were asked to rate their likelihood of sharing three genuine examples of 'fake news' that had been previously published online. an overall score for their likelihood of sharing the stimuli was obtained by summing the three ratings, creating a combined score. this was done, and a set of three stimuli was used, to reduce the likelihood that any effects found were peculiar to a specific story. the stimuli were sourced from the website infowars.com (which in some cases had republished them from other sources). infowars.com has been described [41] as a high-exposure site strongly associated with the distribution of 'fake news'. rather than full articles, excerpts (screenshots) were used that had the size and general appearance of what respondents might expect to see on social media sites. the excerpts were edited to remove any indicators of the source, metrics such as the numbers of shares, date, and author. all had a right-wing orientation (so that participant conservatism could be used as a proxy for consistency between the messages and existing beliefs). this was established in pilot work rating their political orientation and likelihood of being shared. the three stories were among seven rated by a uk sample (n = 30) on an 11-point scale asking "to what extent do you think this post was designed to appeal to people with right wing (politically conservative) views?" anchored at "very left wing oriented" and "very right wing oriented". all seven were rated statistically significantly above the politically-neutral midpoint of the scale. of the three stimuli selected for use in this study, a one-sample t-test showed that the least right-wing was statistically significantly higher than the midpoint, (t (39) = 4.385, p < .001, d = 0.70). one of the stimuli was a picture of masked and hooded men titled "censored video: watch muslims attack men, women & children in england". one was a picture of many people walking down a road, titled "revealed: un plan to flood america with 600 million migrants", with accompanying text describing a plan to "flood america and europe with hundreds of millions of migrants to maintain population levels". the third was a picture of the swedish flag titled "'child refugee' with flagship samsung phone and gold watch complains about swedish benefits rules", allegedly describing a 19 year-old refugee's complaints. the authoritativeness manipulation was achieved by pairing the stimuli with sources regarded as relatively high or low in authoritativeness. the source was shown above the stimulus being rated, in the same way as the avatar and username of someone who had posted a message would be on facebook. the lower authoritativeness group were slight variants on real usernames that had previously retweeted either stories from infowars.com or another story known to be untrue. the original avatars were used. the exemplars used in this study were named 'tigre' (with an avatar of an indistinct picture of a female face), 'jelly beans' (a picture of some jelly beans) and 'chucke' (an indistinct picture of a male face). the higher authoritativeness group comprised actual fake accounts set up by the internet research agency (ira) group to resemble local news sources, selected from a list of suspended ira accounts released by twitter. the exemplars used in this study were 'los angeles daily', 'chicago daily news' and 'el paso top news'. pilot work was conducted with a sample of uk participants (n = 30) who each rated a selection of 9 usernames, including these 6, for the extent to which each was "likely to be an authoritative source-that is, likely to be a credible and reliable source of information". a within-subjects t-test indicated that mean authoritativeness ratings for the 'higher' group were statistically significantly higher than the 'lower' group (t (29) = -11.181, p < .001, d z = 2.04). the consensus manipulation was achieved by pairing the stimuli with indicators of the number of shares and likes the story had. the indicators were shown below the stimulus being rated, in the same way as they normally would be on facebook. in the low consensus conditions, low numbers of likes (1, 3, 2) and shares (2, 0, 2) were displayed. in the high consensus conditions, higher (but not unrealistic) numbers of likes (104k, 110k, 63k) and shares (65k, 78k, 95k) were displayed. the information was presented using the same graphical indicators as would be the case on facebook, accompanied by the (inactive) icons for interacting with the post, in order to maximise ecological validity. procedure. the study was conducted completely online, using materials hosted on the qualtrics research platform. participants initially saw an information page about the study, and on indicating their consent proceeded to the demographic items. they then completed the personality, conservatism and new media literacy scales. each of these was presented on a separate page, except the nmls which was split across three pages. participants were then asked to rate the three disinformation items. participants were randomized to different combinations of source and story within their assigned condition. for example, participant a might have seen story 1 attributed to source 1, story 2 attributed to source 2, and story 3 attributed to source 3; while participant b saw story 1 attributed to source 2, story 2 attributed to source 1, and story 3 attributed to source 3. each participant saw the same three stories paired with one combination of authoritativeness and consensus. there were 24 distinct sets of stimuli. each participant saw an introductory paragraph stating "a friend of yours recently shared this on facebook, commenting that they thought it was important and asking all their friends to share it:". below this was the combination of source, story, and consensus indicators, presented together in the same way as a genuine facebook post would be. they then rated the likelihood of them sharing the post to their own public timeline, on an 11-point scale anchored at 'very unlikely' and 'very likely'. this was repeated for the second and third stimuli, each on a separate page. having rated each one, participants were then shown all three stimuli again, this time on the same page. they were asked to rate each one for "how likely do you think it is that the message is accurate and truthful" and "how likely do you think it is that you have seen it before today", on 5-point scales anchored at 'not at all likely' and 'very likely'. after rating the stimuli, participants were asked two further questions: "have you ever shared a political news story online that you later found out was made up?", and "and have you ever shared a political news story online that you thought at the time was made up?", with 'yes' or 'no' response options. this question format directly replicated that used in pew research centre surveys dealing with disinformation [e.g. 31] . finally, participants were given the opportunity once again to give or withdraw their consent for participation. they then proceeded to a debriefing page. it was only at the debriefing stage that they were told the stories they had seen were untrue: no information about whether the stimuli were true or false had been presented prior to that point. data screening and processing. prior to delivery of the sample, qualtrics performed a series of quality checks and 'data scrubbing' procedures to remove and replace participants with response patterns suggesting inauthentic or inattentive responding. these included speeding checks and examination of response patterns. on delivery of the initial sample (n = 688) further screening procedures were performed. sixteen respondents were identified who had responded with the same scores to substantive sections of the questionnaire ('straightlining'). these were removed, leaving n = 672. these checks and exclusions were carried out prior to any data analysis. where participants had missing data on any variables, they were omitted only from analyses including those variables. thus, ns vary slightly throughout the analyses. participants. the target sample size was planned to exceed n = 614, which would give 95% power to detect r 2 = .04 (a benchmark for the minimum effect size likely to have realworld importance in social science research [42] ), in the planned multiple regression analysis with 11 predictors. qualtrics was contracted to provide a sample of facebook users that was broadly representative of the uk 2011 census population in terms of gender; the split between those who had post-secondary-school education and those who had not; and age profile (18+). quotas were used to assemble a sample comprising approximately one third each self-describing as left-wing, centre and right-wing in their political orientation. participant demographics are shown in table 1 , column 1. descriptive statistics for participant characteristics (personality, conservatism, new media literacy and age) and their reactions to the stimuli (likelihood of sharing, belief the stories were likely to be true, and rating of likelihood that they had seen them before) are summarised in table 2 . all scales had acceptable reliability. the main dependent variable, likelihood of sharing, had a very skewed distribution with a strong floor effect: 39.4% of the participants indicated they were 'very unlikely' to share any of the three stories they saw. this is consistent with findings on real-world sharing that indicate only a small proportion of social media users will actually share disinformation [e.g. 16] , though it gives a dependent variable with less than ideal distributional properties. to simultaneously test hypotheses 1-4 a multiple regression analysis was carried out. this evaluated the extent to which digital media literacy (nmls), authority of the message source, consensus, belief in veracity of the messages, consistency with participant beliefs (operationalised as the total secs conservatism scale score), age and personality (extraversion, conscientiousness, agreeableness, openness to experience and neuroticism), predicted self-rated likelihood of sharing the posts. this analysis is summarised in table 3 . checks were performed on whether the dataset met the assumptions required by the analysis (absence of collinearity, independence of residuals, heteroscedasticity and non-normal distribution of residuals). despite the skewed distribution of the dependent variable, no significant issues were detected. , and with likelihood of having seen the stimuli before (r = .107, n = 672, p = .006). selfreported belief that respondents had seen the stories before also correlated significantly with likelihood of sharing (r = .420, n = 672, p < .001), and a number of other predictor variables. accordingly, a further regression analysis was performed, including these additional predictors (gender, education, level of facebook use, belief they had seen the stories before). given inclusion of gender as a predictor variable, the two respondents who did not report their gender as either male or female were excluded from further analysis. the analysis, summarised in table 4 , indicated that the model explained 43% of the variance in self-reported likelihood of sharing the three disinformation items. neither the authoritativeness of the story source, nor consensus information associated with the stories, was a significant predictor. consistency of the items with participant attitudes (conservatism) was important, with a positive and statistically significant relationship between conservatism and likelihood of sharing. the only personality variable predicting sharing was agreeableness, with less agreeable people giving higher ratings of likelihood of sharing. in terms of demographic characteristics, gender and education were statistically significant predictors, with men and less-educated people reporting a higher likelihood of sharing. finally, people reported a greater likelihood of sharing the items if they believed they were likely to be true, and if they thought they had seen them before. participants had also been asked about their historical sharing of untrue political stories, both unknowing and deliberate. 102 out of 672 participants (15.2%) indicated that they had ever 'shared a political news story online that they later found out was made up', while 64 out of 672 indicated they had shared one that they 'thought at the time was made up' (9.5%). predictors of whether or not people had shared untrue material under both sets of circumstances were examined using logistic regressions, with the same sets of participant-level predictors. having unknowingly shared untrue material (table 5 ) was significantly predicted by lower conscientiousness, lower agreeableness, and lower age. having shared material known to be untrue at the time (table 6 ) was significantly predicted by lower agreeableness and lower age. the main analysis in this study (table 4 ) provided limited support for the hypotheses. contrary to hypotheses 1, 2, and 4, neither consensus markers, authoritativeness of source, nor new media literacy were associated with self-rated likelihood of sharing the disinformation stories. however, in line with hypothesis 3, higher levels of conservatism were associated with higher likelihood of sharing disinformation. this finding supports the proposition that we are more likely to share things that are consistent with our pre-existing beliefs, as all the stimuli were right-wing in orientation. an alternative explanation might be that more conservative people are simply more likely to share disinformation. however, as well as lacking a solid rationale, this explanation is not supported by the fact that conservatism did not seem to be associated with self-reported historical sharing (tables 5 and 6 ). the strongest predictors of likelihood of sharing were belief that the stories were true, and likelihood of having seen them before. belief in the truth of the stories provides further evidence for the role of consistency (hypothesis 3), in that we are more likely to share things we believe are true. the association with likely previous exposure to the materials is consistent with other recent research [43, 44] that found that prior exposure to 'fake news' headlines led to higher belief in their accuracy and reduced belief that it would be unethical to share them. of the personality variables, only agreeableness was a significant predictor, with less agreeable people rating themselves are more likely to share the stimuli. this is consistent with previous findings [24] that less agreeable people reported they were more likely to share a critical political message. lower education levels were associated with a higher self-reported likelihood of sharing. it is possible that less educated people may be more susceptible to online influence, given work finding that less educated people were more influenced by micro-targeted political advertising on facebook [45] . finally, gender was found to be an important variable, with men reporting a higher likelihood of sharing the disinformation messages than women. this was unanticipated: while there are a number of gender-related characteristics (e.g. personality traits) that were thought might be important, there were no a priori grounds to expect that gender itself would be a predictor variable. study 1 also examined predictors of reported historical sharing of false political information. consistent with real-world data [16] , and past representative surveys [e.g. 31], a minority of respondents reported such past sharing. unknowingly sharing false political stories was predicted by low conscientiousness, low agreeableness, and lower age, while knowingly sharing false material was predicted only by lower agreeableness and lower age. the effect of agreeableness is consistent with the findings from the main analysis and from [24] . the finding that conscientiousness influenced accidental, but not deliberate, sharing is consistent with the idea that less conscientious people are less likely to check the details or veracity of a story before sharing it. clearly this tendency would not apply to deliberate sharing of falsehoods. the age effect is harder to explain, especially given evidence [16] that older people were more likely to share material from fake news sites. one possible explanation is that younger people are more active on social media, so would be more likely to share any kind of article. another possibility is that they are more likely to engage in sharing humorous political memes, which could often be classed as false political stories. study 2 set out to repeat study 1, but presented the materials as if they had been posted on twitter rather than facebook. the purpose of this was to test whether the observed effects applied across different platforms. research participants have reported using 'likes' on twitter in a more considered manner than on facebook [12] , raising the possibility that heuristics might be less important for this platform. the study was completed online, using paid respondents sourced from the prolific research panel (www.prolific.co). the methodology exactly replicated that of study 1, except in the case of details noted below. the planned analysis was revised to include the expanded set of predictors eventually used in study 1 (see table 4 ). measures and materials were the same as used in study 1. the key difference from study 1 was in the presentation of the three stimuli, which were portrayed as having been posted to twitter rather than facebook. for the authoritativeness manipulation, the screen names of the sources were accompanied by @usernames, as is conventional on twitter. for the consensus manipulation, 'retweets' were displayed rather than 'shares', and the appropriate icons for twitter were used. participants also indicated their level of twitter, rather than facebook, use. procedure. the procedure replicated study 1, save that in this case the nmls was presented on a single page. before participants saw each of the three disinformation items, the introductory paragraph stated "a friend of yours recently shared this on twitter, commenting that they thought it was important and asking all their friends to retweet it:", and they were asked to indicate the likelihood of them 'retweeting' rather than 'sharing' the post. data screening and processing. data submissions were initially obtained from 709 participants. a series of checks were performed to ensure data quality, resulting in a number of responses being excluded. one individual declined consent. eleven were judged to have responded inauthentically, with the same responses to all items in substantive sections of the questionnaire ('straightlining'). twenty were not active twitter users: three individuals visited twitter 'not at all' and seventeen 'less often' than every few weeks. three participants responded unrealistically quickly, with response durations shorter than four minutes (the same value used as a speeding check by qualtrics in study 1). all of these respondents were removed, leaving n = 674. these checks and exclusions were carried out prior to any data analysis. participants. the target sample size was planned to exceed n = 614, as in study 1. no attempt was made to recruit a demographically representative sample: instead, sampling quotas were used to ensure the sample was not homogenous with respect to education (pre-degree vs. undergraduate degree or above), age (under 40 vs. over 40) and political preference (left, centre or right wing orientation). additionally, participants had to be uk nationals resident in the uk; active twitter users; and not participants in prior studies related to this one. each participant received a reward of £1.25. participant demographics are shown in table 1 (column 2) . for the focal analysis in this study, the sample size conferred 94.6% power to detect r 2 = .04 in a multiple regression with 15 predictors (2-tailed, alpha = .05). descriptive statistics are summarised in table 7 . all scales had acceptable reliability. the main dependent variable, likelihood of sharing, again had a very skewed distribution with a strong floor effect. to simultaneously test hypotheses 1-4, a multiple regression analysis was carried out using the expanded predictor set from study 1. given inclusion of gender as a predictor variable, the three respondents who did not report their gender as either male or female were excluded from further analysis. the analysis, summarised in table 8 , indicated that the model explained 46% of the variance in self-reported likelihood of sharing the three disinformation items. neither the authoritativeness of the story source, nor consensus information associated with the stories, nor new media literacy, was a significant predictor. consistency of the items with participant attitudes (conservatism) was important, with a positive and statistically significant relationship between conservatism and likelihood of sharing. no personality variable predicted ratings of likelihood of sharing. in terms of demographic characteristics, gender and education were statistically significant predictors, with men and less-educated people reporting a higher likelihood of sharing. finally, people reported a greater likelihood of sharing the items if they believed they were likely to be true, and if they thought they had seen them before. participants had also been asked about their historical sharing of untrue political stories, both unknowing and deliberate. 102 out of 674 participants (15.1%) indicated that they had out ever 'shared a political news story online that they later found out was made up', while 42 out of 674 indicated they had shared one that they 'thought at the time was made up' (6.2%). predictors of whether or not people had shared untrue material under both sets of circumstances were examined using logistic regressions, with the same sets of participant-level predictors. having unknowingly shared untrue material (table 9 ) was significantly predicted by higher extraversion and higher levels of twitter use. having shared material known to be untrue at the time (table 10 ) was significantly predicted by higher neuroticism and being male. for the main analysis, study 2 replicates a number of key findings from study 1. in particular, hypotheses 1, 2 and 4 were again unsupported by the results: consensus, authoritativeness, and new media literacy were not associated with self-rated likelihood of retweeting the disinformation stories. evidence consistent with hypothesis 3 was again found, with higher levels of conservatism being associated with higher likelihood of retweeting. again, the strongest predictor of likelihood of sharing was belief that the stories were true, while likelihood of having seen them before was again statistically significant. the only difference was in the role of personality: there was no association between agreeableness (or any other personality variable) and likelihood of retweeting the material. however, for self-reports of historical sharing of false political stories, the pattern of results was different. none of the previous results were replicated, and new predictors were observed for both un-knowing and deliberate sharing. for unintentional sharing, the link with higher levels of twitter use makes sense, as higher usage confers more opportunities to accidentally share untruths. higher extraversion has also been found to correlate with higher levels of social media use [32] so the same logic may apply for that variable. for intentional sharing, the finding that men were more likely to share false political information is similar to findings from study 1. the link with higher neuroticism is less easy to explain: one possibility is that more neurotic people are more likely to share falsehoods that will reduce the chances of an event that they worry about (for example, spreading untruths about a political candidate who one is worried about being elected). given that these questions asked about past behaviour in general, and were not tied to the twitter stimuli used in this study, it is not clear why the pattern of results should have differed from those in study 1. one possibility is that the sample characteristics were different (this sample was younger, better educated, and drawn from a different source). another realistic possibility, especially given the typically low effect sizes and large samples tested, is that these are simply 'crud' correlations [46] rather than useful findings. going forward, it is likely to be more informative to focus on results that replicate across multiple studies or conceptually similar analyses. study 3 set out to repeat study 1, but presented the materials as if they had been posted on instagram rather than facebook. instagram presents an interesting contrast, as the mechanisms of engagement with material are different (for example there is no native sharing mechanism). nonetheless, it has been identified as an important theater for disinformation operations [47] . study 3 therefore sought to establish whether the same factors affecting sharing on facebook also affect engagement with false material on instagram. the study was completed online, using paid respondents sourced from the prolific research panel. the methodology exactly replicated that of study 1, except in the case of details noted below. the planned analysis was revised to include the expanded set of predictors eventually used in study 1 (see table 4 ). materials. measures and materials were the same as used in study 1. the only difference from study 1 was in the presentation of the three stimuli, which were portrayed as having been posted to instagram rather than facebook. for the consensus manipulation, 'likes' were used as the sole consensus indicator, and the appropriate icons for instagram were used. procedure. the procedure replicated study 1, save that in this case the nmls was presented on a single page. before participants saw each of the three disinformation items, the introductory paragraph stated "imagine that you saw this post on your instagram feed:" and they were asked to indicate the probability of them 'liking' the post. data screening and processing. data submissions were initially obtained from 692 participants. a series of checks were performed to ensure data quality, resulting in a number of responses being excluded. four individuals declined consent. twenty-one were judged to have responded inauthentically, with the same scores to substantive sections of the questionnaire ('straightlining'). five did not indicate they were located in the uk. ten were not active instagram users: three individuals visited instagram 'not at all' and seven 'less often' than every few weeks. two participants responded unrealistically quickly, with response durations shorter than four minutes (the same value used as a speeding check by qualtrics in study 1). all of these respondents were removed, leaving n = 650. these checks and exclusions were carried out prior to any data analysis. participants. the target sample size was planned to exceed n = 614, as in study 1. no attempt was made to recruit a demographically representative sample: instead, sampling quotas were used to ensure the sample was not homogenous with respect to education (pre-degree vs. undergraduate degree or above) and political preference (left, centre or right-wing orientation). sampling was not stratified by age, given that instagram use is associated with younger ages, and the number of older instagram users in the prolific pool was limited at the time the study was carried out. additionally, participants had to be uk nationals resident in the uk; active instagram users; and not participants in prior studies related to this one. each participant received a reward of £1.25. participant demographics are shown in table 1 (column 3) . for the focal analysis in this study, the sample size conferred 93.6% power to detect r 2 = .04 in a multiple regression with 15 predictors (2-tailed, alpha = .05). descriptive statistics are summarised in table 11 . all scales had acceptable reliability. the main dependent variable, probability of liking, again had a very skewed distribution with a strong floor effect. to simultaneously test hypotheses 1-4, a multiple regression analysis was carried out using the expanded predictor set from study 1. given inclusion of gender as a predictor variable, the three respondents who did not report their gender as either male or female were excluded from further analysis. the analysis, summarised in table 12 , indicated that the model explained 24% of the variance in self-reported likelihood of sharing the three disinformation items. neither the authoritativeness of the story source, consensus information associated with the stories, nor consistency of the items with participant attitudes (conservatism) was a statistically significant predictor. extraversion positively and conscientiousness negatively predicted ratings of likelihood of sharing. in terms of demographic characteristics, men and younger participants reporting a higher likelihood of sharing. finally, people reported a greater likelihood of sharing the items if they believed they were likely to be true, and if they thought they had seen them before. participants had also been asked about their historical sharing of untrue political stories, both unknowing and deliberate. eighty five out of 650 (13.1%) participants who answered the question indicated that they had out ever 'shared a political news story online that they later found out was made up', while 50 out of 650 indicated they had shared one that they 'thought at the time was made up' (7.7%). predictors of whether or not people had shared untrue material under both sets of circumstances were examined using logistic regressions, with the same sets of participant-level predictors. having unknowingly shared untrue material (table 13 ) was significantly predicted by higher extraversion, lower conscientiousness and male gender. having shared material known to be untrue at the time (table 14) was significantly predicted by higher new media literacy, higher conservatism, and higher neuroticism. as in studies 1 and 2, results were not consistent with hypotheses 1, 2 and 4: consensus, authoritativeness, and new media literacy were not associated with self-rated probability of liking the disinformation stories. in contrast to studies 1 and 2, however, conservatism did not predict liking the stories. belief that the stories were true was again the strongest predictor, while likelihood of having seen them before was again statistically significant. among the personality variables, lower agreeableness returned as a predictor of likely engagement with the stories, consistent with study 1 but not study 2. lower age predicted likely engagement, a new finding, while being male predicted likely engagement as found in both in study 1 and study 2. unlike study 1 and study 2, education had no effect. with regard to historical accidental sharing, as in study 3 higher extraversion was a predictor, while as in study 1 so was lower conscientiousness. men were more likely to have shared accidentally. deliberate historical sharing was predicted by higher levels of new media literacy. this is counter-intuitive and undermines the argument that people share things because they know no better. in fact, in the context of deliberate deception, motivated individuals higher in digital literacy may actually be better equipped to spread untruths. conservatism was also a predictor here. this could again be a reflection of the consistency hypothesis, given that there are high levels of conservative-oriented disinformation circulating. finally, as in study 3, higher neuroticism predicted deliberate historical sharing. study 4 set out to repeat study 1, but with a us sample and using us-centric materials. the purpose of this was to test whether the observed effects applied across different countries. the study was completed online, using as participants members of research panels sourced through the research company qualtrics. the methodology exactly replicated that of study 1, except in the case of details noted below. the planned analysis was revised to include the expanded set of predictors eventually used in study 1 (see table 4 ). social media disinformation measures and materials were the same as used in study 1. the only difference from study 1 was in the contents of the three disinformation exemplars, which were designed to be relevant to a us rather than uk audience. two of the stimuli were sourced from the website infowars.com, while a third was a story described as untrue by the fact-checking website politifact.com. in the same way as in study 1, the right-wing focus of the stories was again established in pilot work where a us sample (n = 40) saw seven stories including these and rated their political orientation and likelihood of being shared. all were rated above the mid-point of an 11-point scale asking "to what extent do you think this post was designed to appeal to people with right wing (politically conservative) views?" anchored at "very left wing oriented" and "very right wing oriented". for the least right-wing of the three stories selected, a one-sample ttest comparing the mean rating with the midpoint of the scale showed it was statistically significantly higher, t (39) = 6.729, p < .001, d = 1.07). one of the stimuli, also used in study 1-3, was titled "revealed: un plan to flood america with 600 million migrants". one was titled "flashback: obama's attack on internet freedom", subtitled 'globalists, deep state continually targeting america's internet dominance', featuring further anti-obama, china and 'big tech' sentiment, and an image of barack obama apparently drinking wine with a person of east asian appearance. the third was text based and featured material titled "surgeon who exposed clinton foundation corruption in haiti found dead in apartment with stab wound to the chest". the materials used to manipulate authoritativeness (facebook usernames shown as sources of the stories) were the same as used in studies 1-3. these were retained because pilot work indicated that the higher and lower sets differed in authoritativeness for us audiences in the same way as for uk audiences. a sample of 30 us participants again each rated a selection of 9 usernames, including these 6, for the extent to which each was "likely to be an authoritative source-that is, likely to be a credible and reliable source of information". a within-subjects ttest indicated that mean authoritativeness ratings for the 'higher' group were statistically significantly higher than the 'lower' group (t (29) = -9.355 p < .001, d z = 1.70). procedure. the procedure replicated study 1, save that in this case the nmls was presented across two pages. data screening and processing. prior to delivery of the sample, qualtrics performed a series of quality checks and 'data scrubbing' procedures to remove and replace participants with response patterns suggesting inauthentic or inattentive responding. these included speeding checks and examination of response patterns. on delivery of the initial sample (n = 660) further screening procedures were performed. nine respondents were identified who had responded with the same scores to substantive sections of the questionnaire ('straightlining'), and one who had not completed any of the personality items. twelve respondents were not active facebook users: six reported using facebook 'not at all' and a further six less often than 'every few weeks'. all of these were removed, leaving n = 638. these checks and exclusions were carried out prior to any data analysis. participants. the target sample size was planned to exceed n = 614, as in study 1. qualtrics was contracted to provide a sample of active facebook users that was broadly representative of the us population in terms of gender; education level; and age profile (18+). sampling quotas were used to assemble a sample comprising approximately one third each self-describing as left-wing, centre and right-wing in their political orientation. sampling errors on the part of qualtrics led to over-recruitment of individuals aged 65 years, who make up 94 of the 160 individuals in the 60-69 age group. as a consequence, the 60-69 age group is itself over-represented in this sample compared to the broader us population. participant demographics are shown in table 1 , column 4. for the focal analysis in this study, the sample size conferred 92.6% power to detect r 2 = .04 in a multiple regression with 15 predictors (2-tailed, alpha = .05). descriptive statistics are summarised in table 15 . all scales had acceptable reliability. the main dependent variable, likelihood of sharing, again had a very skewed distribution with a strong floor effect. to simultaneously test hypotheses 1-4 a multiple regression analysis was carried out using the expanded predictor set from study 1. given inclusion of gender as a predictor variable, the one respondent who did not report their gender as either male or female was excluded from further analysis. the analysis, summarised in table 16 , indicated that the model explained 56% of the variance in self-reported likelihood of sharing the three disinformation items. neither the authoritativeness of the story source, consensus information associated with the stories, nor consistency of the items with participant attitudes (conservatism) was a statistically significant predictor. extraversion positively predicted ratings of likelihood of sharing. in terms of demographic characteristics, age was a significant predictor, with younger people reporting a higher likelihood of sharing. finally, people reported a greater likelihood of sharing the items if they believed they were likely to be true, and if they thought they had seen them before. participants had also been asked about their historical sharing of untrue political stories, both unknowing and deliberate. of the 638 participants, 185 (29.0%) indicated that they had ever 'shared a political news story online that they later found out was made up', while 132 out of 638 indicated they had shared one that they 'thought at the time was made up' (20.7%). predictors of whether or not people had shared untrue material under both sets of circumstances were examined using logistic regressions, with the same sets of participant-level predictors. having unknowingly shared untrue material (table 17 ) was significantly predicted by higher new media literacy, lower conscientiousness, higher education, and higher levels of facebook use. having shared material known to be untrue at the time (table 18 ) was significantly predicted by higher extraversion, lower agreeableness, younger age, and higher levels of facebook use. again, the pattern of results emerging from study 4 had some similarities but also some differences from studies 1-3. once again, hypotheses 1, 2 and 4 were unsupported by the results. similarly to study 3, but unlike studies 1 and 2, conservatism (the proxy for consistency) did not predict sharing the stories. belief that the stories were true, and likelihood of having seen them before, were the strongest predictors. higher levels of extraversion (a new finding) and lower ages (as in study 3) were associated with higher reported likelihood of sharing the stimuli. for historical sharing, for the first time-and counterintuitively-new media literacy was associated with higher likelihood of having shared false material unknowingly. as in studies 1 and 2, lower conscientiousness was also important. counterintuitively, higher education levels were associated with higher unintentional sharing, as were higher levels of facebook use. for intentional sharing, higher extraversion was a predictor, as was lower agreeableness, younger age and higher levels of facebook use. when interpreting the overall pattern of results from studies 1-4, given the weakness of most of the associations, it is likely to be most useful to focus on relationships that are replicated across studies and disregard 'one off' findings. tables 19-21 provide a summary of the statistically significant predictors in each of the studies. it is clear that two variables consistently predicted self-rated likelihood of sharing disinformation exemplars: belief that the stories were likely to be true, and likely prior familiarity with the stories. it is also clear that three key variables did not: markers of authority, markers of consensus and digital literacy. hypothesis 1 predicted that stories portrayed as coming from more authoritative sources were more likely to be shared. however, this was not observed in any of the four studies. one interpretation of this is that the manipulation failed. however, pilot work (see study 1, study 4) with comparable samples indicated that people did see the sources as differing in authoritativeness. the failure to find the predicted effect could also be due to use of simulated scenarios-though care was taken to ensure they resembled reality-or weaknesses in the methodology, such as the distributional properties of the dependent variables. however, consistent relationships between other predictors and the dependent variable were observed. thus, the current studies provide no evidence that authoritativeness of a source influences sharing behaviour. hypothesis 2 predicted that stories portrayed as having a higher degree of consensus in audience reactions (i.e. high numbers of people had previously shared them) would be more likely to be shared. in fact, consensus markers had no effect on self-reported probability of sharing or liking the stories. therefore, the current studies provide no evidence that indicators of 'social proof' influence participant reactions to the stimuli. hypothesis 3 was that people would be more likely to share materials consistent with their pre-existing beliefs. this was operationalised by measuring participants' political orientation (overall level of conservatism) and using stimuli that were right-wing in their orientation. in studies 1 and 2, more conservative people were more likely to share the materials. further evidence for hypothesis 3 comes from the finding, across all studies, that level of belief the stories were "accurate and truthful" was the strongest predictor of likelihood of sharing. this is again in line with the consistency hypothesis: people are behaving in ways consistent with their beliefs. the finding from study 3 that more conservative people were more likely to have historically shared material they knew to be untrue could also be in line with this hypothesis, given that a great many of the untrue political stories circulated online are conservativeoriented. hypothesis 4, that people lower in digital literacy would be more likely to engage with disinformation, was again not supported. as noted earlier, measurement of digital literacy is problematic. however, pilot work showed that the new media literacy scale did differentiate between people with higher and lower levels of social media use in the expected manner, so it is likely to have a degree of validity. in study 4, higher nmls scores were associated with having unwittingly shared false material in the past, which is counterintuitive. however, this may be due to the fact that more digitally literate people should be more able to see that something was false in hindsight. higher nmls scores were also associated with deliberately sharing falsehoods in study 3. this could be attributable to greater ease with which digitally literate individuals can do such things, if motivated to do so. a number of other variables were included on an exploratory basis, or for the purpose of controlling for possible confounds. of these, the most important was participants' ratings of the likelihood that they had seen the stimuli before. this variable was originally included in the design so that any familiarity effects could be controlled for when evaluating the effect of other variables. in fact, rated likelihood of having seen the materials before was the second strongest predictor of likelihood of sharing it. it was a predictor in all four studies, and for the facebook studies (1 and 4) it was the second most important variable. this is consistent with work on prior exposure to false material online, where prior exposure to fake news headlines increased participants' ratings of their accuracy [44] . furthermore, it has been found that prior exposure to fake-news headlines reduced participants' ratings of how unethical it was to share or publish the material, even when it was clearly marked as false [43] . thus, repeated exposure to false material may increase our likelihood of sharing it. it is known that repeated exposure to statements increases people's subjective ratings of their truth [48] . however, there must be more going on here, because the regression analyses indicated that the familiarity effect was independent of the level of belief that it is true. when considering work that found that amplification of content by bot networks led to greater levels of human sharing [21] , the implication is that repeated actual exposure to the materials is what prompts people to share it, not metrics of consensus such as the number of likes or shares displayed beside an article. of the five dimensions of personality measured, four (agreeableness, extraversion, neuroticism and conscientiousness were predictors of either current or historical sharing in one or more studies. consistent with findings from [24] , studies 1 and 3 found that lower agreeableness was associated with greater probability of sharing or liking the stories. it was also associated with accidental historical sharing in study 1, and deliberate historical sharing in studies 1 and 4. in contrast to this, past research on personality and social media behaviour indicates that more agreeable people are more likely to share information on social media: [49] reported that its role in this was mediated by trust, while [32] found that higher agreeableness was associated with higher levels of social media use in general. given those findings, it is likely that the current results are specific to disinformation stimuli rather than social sharing in general. agreeableness could potentially interact with the source of the information: more agreeable people might conceivably be more eager to please those close to them, however, while it is possible that agreeableness interacted in some way with the framing of the material having been shared by 'a friend' in study 1, study 3 had no such framing. more broadly, the nature of the stories may be important: disinformation items are normally critical or hostile in their nature. this may mean they are more likely to be shared by disagreeable people, who themselves may be critical in their outlook and not concerned about offending others. furthermore, agreeableness is associated with general trusting behaviour. it may be that disagreeable people are therefore more likely to endorse conspiracist material, or other items consistent with a lack of trust in politicians or other public figures. lower conscientiousness was associated with accidental historical sharing of false political stories in studies 1, 3 and 4. this is unsurprising, as less conscientious people would be less likely to check the veracity of a story before sharing it. the lack of an association with deliberate historical sharing reinforces this view. higher extraversion was associated with probability of sharing in study 4, with accidental historical sharing in study 2 and 3, and with deliberate historical sharing in study 4. higher neuroticism was associated with historical deliberate sharing in studies 2 and 3. all these relationships may simply reflect a higher tendency on the part of extraverted and neurotic individuals to use social media more [32] . there are clearly some links between personality and sharing of disinformation. however, the relationships are weak and inconsistent across studies. it is possible that different traits affect different behaviours: for example low conscientiousness is associated with accidental but not deliberate sharing, while high neuroticism is associated with deliberate but not accidental sharing. thus, links between some personality traits and the spread of disinformation may be context-and motivation-specific, rather than reflecting blanket associations. however, lower agreeableness-and to a lesser extent higher extraversion-may predict an overall tendency to spread this kind of material. demographic variables were also measured and included in the analyses. younger individuals rated themselves as more likely to engage with the disinformation stimuli in studies 3 and 4, and were more likely to have shared untrue political stories in the past either accidentally (study 1) or deliberately (studies 1 and 4) . this runs counter to findings that older adults were much more likely to have spread material from 'fake news' domains [16] . it is possible that the current findings simply reflect a tendency of younger people to be more active on social media. people with lower levels of education reported a greater likelihood of sharing the disinformation stories in studies 1 and 2. counterintuitively, more educated people were more likely to have accidentally shared false material in the past (study 4). one possible explanation is that more educated people are more likely to have realised that they had done this, so the effect in study 4 reflects an influence on reporting of the behaviour rather than on the behaviour itself. in each of studies 1, 2 and 3, men reported a greater likelihood of sharing or liking the stimuli. men were also more likely to have shared false material in the past unintentionally (study 3) or deliberately (study 2). given its replicability, this would seem to be a genuine relationship, but one which is not easy to explain. finally, the level of use of particular platforms (facebook, twitter or instagram) did not predict likelihood of sharing the stimuli in any study. level of use of twitter (study 2) predicted accidental sharing of falsehoods, while facebook use predicted both accidental and deliberate sharing (study 4). for historical sharing, this may be attributable to a volume effect: the more you use the platforms, the more likely you are to do these things. it should be noted that the level of use metric lacked granularity and had a strong ceiling effect, with most people reporting the highest use level in each case. in all four studies, a minority of respondents indicated that they had previously shared political disinformation they had encountered online, either by mistake or deliberately. the proportion who had done each varied across the four studies, likely as a function of the population sampled (13.1%-29.0% accidentally; 6.2%-20.7% deliberately), but the figures are a similar magnitude to those reported elsewhere [31, 16] . even if the proportion of social media users who deliberately share false information is just 6.2%, the lowest figure found here, then that is still a very large number of people who are actively and knowingly spreading untruths. the current results indicate that a number of variables predict onward sharing of disinformation. however, most of these relationships are very small. it has been argued that the minimum effect size for a predictor that would have real-world importance in social science data is β = .2 [42] . considering the effect sizes for the predictors in tables 4, 8, 12 and 17, only belief that the stories are true exceeds this benchmark in every study, while probability of having seen the stories before exceeded it in studies 1 and 4. none of the other relationships reported exceeded the threshold. this has implications for the practical importance of these findings, in terms of informing interventions to counteract disinformation. some of the key conclusions in this set of studies arise from the failure to find evidence supporting an effect. proceeding from such findings to a firm conclusion is a logically dangerous endeavour: absence of evidence is not, of course, evidence of absence. however, given the evidence from pilot studies that the manipulations were appropriate; the associations of the dependent measures with other variables; and the high levels of power to detect the specified effects, it is possible to say with some confidence that hypotheses 1, 2 and 4 are not supported by the current data. this means that the current project does not provide any evidence that interventions based on these would be of value. this is particularly important for the findings around digital literacy. raising digital media literacy is a common and appealing policy position for bodies concerned with disinformation (e.g. [1] ). there is evidence from a number of trials that it can be effective in the populations studied. however, no support was found here for the idea that digital literacy has a role to play in the spread of disinformation. this could potentially be attributed to the methodology in this study. however, some participants-288 in total across all four studies-reported sharing false political stories that they knew at the time were made up. it is hard to see how raising digital literacy would reduce such deliberate deception. trying to raise digital literacy across the population is therefore unlikely to ever be a complete solution. there is evidence that consistency with pre-existing beliefs can be an important factor, especially in relation to beliefs that disinformation stories are accurate and truthful. this implies that interventions are likely to be most effective when targeted at individuals who already hold an opinion or belief, rather than trying to change people's minds. while this would be more useful to those seeking to spread disinformation, it could also give insights into populations worth targeting with countermessages. targeting on other variables-personality or demographic-is unlikely to be of value given the low effect sizes. while these variables (perhaps gender and agreeableness in particular) most likely do play a role, their relative importance seems so low that the information is unlikely to be useful in practice. alongside other recent work [43, 44] , the current findings suggest that repeated exposure to disinformation materials may increase our likelihood of sharing it, even if we don't believe it. the practical implication would be that to get a message repeated online, one should repeat it many times (there is a clear parallel with the 'repeat the lie often enough' maxim regarding propaganda). social proof (markers of consensus) seems unimportant based on current findings, so there is no point in trying to manipulate the numbers next to a post as sometimes done in online marketing. what might be more effective is to have the message posted many times (e.g. by bots) so that people had a greater chance of coming across it repeatedly. this would be true both for disinformation and counter-messages. as a scenario-based study, the current work has a number of limitations. while it is ethically preferable to field experiments, it suffers from reduced ecological validity and reliance on selfreports rather than genuine behaviour. questions could be asked, for example, about whether the authoritativeness and consensus manipulations were sufficiently salient to participants (even though they closely mirrored the presentation of this information in real-life settings). beyond this, questions might be raised about the use of self-reported likelihood of sharing: does sharing intention reflect real sharing behaviour? in fact, there is evidence to suggest that it does, with recent work finding that self-reported willingness to share news headlines on social media paralleled the actual level of sharing of those materials on twitter [50] . the scenarios presented were all selected to be right-wing in their orientation, whereas participants spanned the full range from left to right in their political attitudes. this means that consistency was only evaluated with respect to one pole of the right-left dimension. there are a number of other dimensions that have been used as wedge issues in real-world information operations: for example, support for the black lives matter movement; climate change; or for or against britain leaving the european union. the current research only evaluated consistency between attitudes and a single issue. a better test of the consistency hypothesis would be to extend that to evaluation of consistency between attitudes and some of those other issues. a key issue is the distributions of the main outcome variables, which were heavily skewed with strong floor effects. while they still had sufficient sensitivity to make the regression analyses meaningful, they also meant that any effects found were likely to be attenuated. it may thus be that the current findings underestimate the strength of some of the associations reported. another measurement issue is around the index of social media use (facebook, twitter, instagram). as table 1 shows, in three of the studies over 60% of respondents fall into the highest use category. again, this weakens the sensitivity of evaluations of these variables as predictors of sharing disinformation. in order to identify variables associated with sharing disinformation, this research programme took the approach of presenting individuals with examples of disinformation, then testing which of the measured variables was associated with self-reported likelihood of sharing. a shortcoming of this approach is that it does not permit us to evaluate whether the same variables are associated with sharing true information. an alternative design would be to show participants either true or false information, and examine whether the same constructs predict sharing both. this would enable identification of variables differentially impacting the sharing of disinformation but not true information. complexity arises, however, from the fact that whether a story can be considered disinformation, misinformation, or true information, depends on the observer's perspective. false material deliberately placed online would be categorized as disinformation. a social media user sharing it in full knowledge that it was untrue would be sharing disinformation. however, if they shared it believing it was actually true, then from an observer's perspective this would be technically categorised as misinformation (defined as "the inadvertent sharing of false information" [1, p.10] ). in fact, from the user's perspective, it would be true information (because they believe it) even though an omniscient observer would know it was actually false. this points to the importance of further research into user motivations for sharing, which are likely to differ depending on whether or not they believe the material is true. in three of the four studies (studies 1,2, 4) , the stimulus material was introduced as having been posted by a friend who wanted them to share it. this is likely to have boosted the rates of self-reported likelihood of sharing in those studies. previous work has shown that people rate themselves as more likely to engage with potential disinformation stories posted by a friend, as opposed to a more distant acquaintance [24] . to be clear, this does not compromise the testing of hypotheses in those studies (given that the framing was the same for all participants, in all conditions). it is also a realistic representation of how we may encounter material like this in our social media feeds. however, it does introduce an additional difference between studies 1, 2 and 4 when compared with study 3. it would be desirable for further work to check whether the same effects were found when messages were framed as having been posted by people other than friends. finally, the time spent reading and reacting to the disinformation stimuli was not measured. it is possible that faster response times would be indicative of more use of heuristics rather than considered thought about the issues. this could profitably be examined, potentially in observational or simulation studies rather than using self-report methodology. a number of priorities for future research arise from the current work. first, it is desirable to confirm these findings using real-world behavioural measures rather than simulations. while it is not ethically acceptable to run experimental studies posting false information on social media, it would be possible to do real-world observational work. for example, one could measure digital literacy in a sample of respondents, then do analyses of their past social media sharing behaviour. another priority revolves around those individuals who knowingly share false information. why do they do this? without understanding the motivations of this group, any interventions aimed at reducing the behaviour are unlikely to be successful. as well as being of academic interest, motivation for sharing false material has been flagged as a gap in our knowledge by key stakeholders [7] . the current work found that men were more likely to spread disinformation than women. at present, it is not clear why this was the case. are there gender-linked individual differences that influence the behaviour? could it be that the subject matter of disinformation stories is stereotypically more interesting to men, or that men think their social networks are more likely to be interested in or sympathetic to them? while the focus in this paper has been on factors influencing the spread of untruths, it should be remembered that 'fake news' is only one element in online information operations. other tactics and phenomena, such as selective or out-of-context presentation of true information, political memes, and deliberately polarising hyperpartisan communication, are also prevalent. work is required to establish whether the findings of this project related to disinformation, also apply to those other forms of computational propaganda. related to this, it would be of value to establish whether the factors found here to influence sharing of untrue information, also influence the sharing of true information. this would indicate whether there is anything different about disinformation, and also point to factors that might influence sharing of true information that is selectively presented in information operations. the current work allows some conclusions to be drawn about the kind of people who are likely to further spread disinformation material they encounter on social media. typically, these will be people who think the material is likely to be true, or have beliefs consistent with it. they are likely to have previous familiarity with the materials. they are likely to be younger, male, and less educated. with respect to personality, it is possible that they will tend to be lower in agreeableness and conscientiousness, and higher in extraversion and neuroticism. with the exception of consistency and prior exposure, all of these effects are weak and may be inconsistent across different populations, platforms, and behaviours (deliberate v. innocuous sharing). the current findings do not suggest they are likely to be influenced by the source of the material they encounter, or indicators of how many other people have previously engaged with it. no evidence was found that level of literacy regarding new digital media makes much difference to their behaviour. these findings have implications for how governments and other bodies should go about tackling the problem of disinformation in social media. conceptualization: tom buchanan. formal analysis: tom buchanan. investigation: tom buchanan. methodology: tom buchanan. disinformation and 'fake news': final report the global disinformation order: 2019 global inventory of organised social 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willingness to share political news articles in online surveys correlates with actual sharing on twitter writing -review & editing: tom buchanan. key: cord-329223-f84gjxm1 authors: kouokam, joseph calvin; huskens, dana; schols, dominique; johannemann, andrew; riedell, shonna k.; walter, wendye; walker, janice m.; matoba, nobuyuki; o'keefe, barry r.; palmer, kenneth e. title: investigation of griffithsin's interactions with human cells confirms its outstanding safety and efficacy profile as a microbicide candidate date: 2011-08-02 journal: plos one doi: 10.1371/journal.pone.0022635 sha: doc_id: 329223 cord_uid: f84gjxm1 many natural product-derived lectins such as the red algal lectin griffithsin (grft) have potent in vitro activity against viruses that display dense clusters of oligomannose n-linked glycans (nlg) on their surface envelope glycoproteins. however, since oligomannose nlg are also found on some host proteins it is possible that treatment with antiviral lectins may trigger undesirable side effects. for other antiviral lectins such as concanavalin a, banana lectin and cyanovirin-n (cv-n), interactions between the lectin and as yet undescribed cellular moieties have been reported to induce undesirable side effects including secretion of inflammatory cytokines and activation of host t-cells. we show that grft, unlike cv-n, binds the surface of human epithelial and peripheral blood mononuclear cells (pbmc) through an exclusively oligosaccharide-dependent interaction. in contrast to several other antiviral lectins however, grft treatment induces only minimal changes in secretion of inflammatory cytokines and chemokines by epithelial cells or human pbmc, has no measureable effect on cell viability and does not significantly upregulate markers of t-cell activation. in addition, grft appears to retain antiviral activity once bound to the surface of pbmc. finally, rna microarray studies show that, while cv-n and cona regulate expression of a multitude of cellular genes, grft treatment effects only minimal alterations in the gene expression profile of a human ectocervical cell line. these studies indicate that grft has an outstanding safety profile with little evidence of induced toxicity, t-cell activation or deleterious immunological consequence, unique attributes for a natural product-derived lectin. hiv-1 is the prototype example of a virus that utilizes an oligomannose-rich ''glycan shield'' to occlude functionally important domains of the envelope glycoproteins from antibodies, and evade the immune response [1, 2] . recently doores et al. [3] showed that previous measurement of the proportion of oligomannose nlg relative to complex nlg on recombinant hiv envelope glycoproteins underestimated the representation of oligomannose nlg on the native envelope spikes of hiv-1, which appear to display nlg that are almost exclusively mannoseterminal man 5-9 -glcnac 2 structures. it is likely that limited access to the high density of nlg presented on the hiv-1 trimeric glycoprotein spike by golgi and endoplasmic reticulum (er) a1r2 mannosidases results in an atypical preponderance of oligomannose glycans rather than complex nlg on hiv-1 surface glycoproteins [3, 4, 5] . given that man 5-9 -glcnac 2 structures are present on less than 4% of the normal human n-glycome [6, 7] , dense clusters of oligomannose nlg appear to be a feature specific to viral envelope glycoproteins, particularly those of hiv-1 and other immunodeficiency lentiviruses [3] . consequently, clusters of oligomannose nlg may be attractive molecular targets for antiviral drugs and vaccines that act to interrupt hiv-1 infection of target cells by: (i) binding on the virus envelope and thereby interfering with the structural transitions involved in receptor and co-receptor docking and virus entry into t-cells and (ii) blocking access to viral envelope oligomannose nlg targeted by c-type lectin receptors dc-sign and mmr on dendritic cells and macrophages [5, 8, 9] . it has long been known that a variety of oligomannose-specific lectins have potent in vitro hiv-1 inhibitory activities, and therefore have been proposed as microbicide candidates for topical prophylaxis of hiv-1 infection, and as potential therapeutics [8, 9] . however many lectins possess lymphocyte mitogenic activities incompatible with their use as pharmaceuticals, and some are known human and animal toxins, although the pharmacological basis for their toxicity is poorly characterized [10, 11] . the antiviral potency of lectins has been correlated to their capacity to bind multiple glycans simultaneously, often facilitated by their ability to form dimers and higher order multimers [12, 13, 14] . the most extensively characterized antiviral lectin is cv-n, a small protein that exists in both monomeric and homodimeric configurations and has exceptionally potent anti-hiv activity, in the low nanomolar range [15] . cv-n targets the mana1r2man terminating glycans displayed in the man 6-9 glcnac 2 structures on the surface of many viral envelope glycoproteins. each monomer of cv-n has the capacity to bind two oligomannose structures [16] . when formulated into a carboxyethylcellulose gel matrix, cv-n provided almost complete protection against a single high dose intrarectal or intravaginal challenge with a pathogenic simian-human immunodeficiency virus (shiv) [17, 18] . however, subsequent in vitro toxicity studies have raised concerns about the safety of cv-n based microbicides, finding that in vitro, cv-n has the capacity to promote secretion of pro-inflammatory cytokines and chemokines from human peripheral blood mononuclear cells (pbmc), activate quiescent cd4 + t-cells, and promote t-cell proliferation [19, 20, 21, 22] . similar results were also reported for other lectins such as microvirin (mvn) (17) and concanavalin a (con a) (18) . it should be noted that the toxicities of cv-n were much milder in treated cervical explants in comparison with pbmc [20, 21] . the possible pathogenic consequences associated with these off-target activities have raised concerns about all other members of the natural product-based class of antiviral lectins [19, 20, 21] . grft has the most potent and broad spectrum hiv-1 inhibitory activity yet described for any antiviral lectin [15, 23, 24, 25] . it is a 25 kda domain-swapped homodimer, with the first 16 amino acids of each 12.7 kda monomer completing the b-prism fold of the other [26] . the homodimer has six carbohydrate binding pockets, 3 located at each of the opposite ends of the double-prism homodimer. atomic resolution crystal structures of an engineered monomeric grft showed that each monomer can bind to two different nonamannoside molecules through all three carbohydrate binding sites [13, 26] . the antiviral activity of monomeric grft is substantially lower than that of the homodimeric form, confirming that the grft potency is dependent on its ability to bind multiple oligomannose structures simultaneously, with strong avidity [13] . we showed recently that grft causes no mitogenic stimulation of pbmc exposed to the drug [23] . grft is fully active in the presence of macaque vaginal secretions [25] , and was shown to have a good safety profile in the rabbit vaginal irritation model, the gold standard preclinical safety test for vaginal products [23] . moreover, treatment of human cervical explants with grft induced minimal alterations in the expression profile of a panel of proinflammatory chemokines and cytokines. grft also strongly inhibited hiv-1 infection of the cervical explants, and dissemination of hiv-1 infection from cells resident in the explants to donor t-cells [23] . in the present study, we performed a comprehensive set of experiments to interrogate the molecular response of cultured human cervicovaginal cells and pbmc to grft exposure. our investigations employed comparisons between the biological activities of grft, which binds mannose-terminal man 5-9 -glcnac 2 , with other lectins of well-defined carbohydrate binding specificity: (1) cv-n, which binds mana1r2man terminating glycans on man 6-9 -glcnac 2 structures; (2) phytohemagglutinin a (pha), targeting d-galactose and n-acetyl-d-galactosamine on gycan structures; (3) cona, specific for terminal (tri) mannose on high mannose glycans and (4) pokeweed agglutinin (pkm), which binds n-acetylglucosamine. our studies reveal clear distinctions in biological and toxicological properties of these lectins, and confirm grft's superior safety profile for use as a topical microbicide. grft and cv-n binding to human cervical epithelium, cultured human cervicovaginal cells, and pbmc we used paraffin-embedded cervical epithelial sections from a 21year old donor to evaluate the binding pattern of fluorescently labeled grft and cv-n to human mucosal epithelia. grft lec-, a mutant form of grft where we eliminated the lectin activity through mutation of all six mannose binding sites, was used as a control to help distinguish binding mediated by the grft carbohydrate binding pockets versus binding associated with other grft structures. the light micrograph in figure 1a shows an h&e stained cervical tissue section to orient the observer to the microanatomy of the human cervical epithelium. different layers of the squamous epithelium starting at the basement membrane (basal, parabasal, intermediate, superficial) are evident; cervical connective tissue or stroma is beneath the basement membrane. tissues incubated with labeled grft, grft lecand cv-n are shown in fig. 1b , c and d, respectively. minimal fluorescence seen in tissues exposed to grft lec(fig. 1c ) compared with grft-stained tissues (fig. 1b) confirmed that the binding of grft to the outermost layer of the squamous epithelium was via its carbohydrate binding activity. there were distinct differences evident in the binding pattern of grft ( fig. 1c ) relative to cv-n (fig. 1d) , which bound far more extensively than grft throughout all layers of the squamous epithelium, basement membrane and underlying stromal tissue. additional fluorescence micrographs are provided in fig. s1 . in cultured cervicovaginal epithelial cells we also observed binding of both grft and cv-n, but not grft lecto ect1/e6e7 cells (compare fig. 1e through h), as well as end1/e6e7 and vk2/e6e7 cells (data not shown). we used flow cytometry to evaluate binding of fluorescently labeled grft, grft lecand cv-n to human pbmc. clear shifts in fluorescence intensity show that grft (fig. 1 i) and cv-n ( fig. 1k ) efficiently bind the surface of human pbmc relative to grft lec(fig. 1j ), for which we observed only minimal binding. binding of grft to the surface of pbmc was significantly reduced when occluding the glycan binding pockets by pre-incubation with yeast mannan (fig. 1i) . interestingly, the binding of cv-n to pbmc was reduced, but not eliminated, by mannan binding, which implies a second mode of binding between cv-n and the cell surface (fig. 1k ). we assume that distinct populations of differentially labeled cells seen in the flow histograms reflect differences in the amount of labeled protein that binds different subpopulations of leukocytes in the unfractionated pbmc samples. when freshly-isolated pbmc were pre-incubated for 24 hrs with grft at various concentrations, washed and then infected with hiv-1 r5 strain bal (without adding new compound), grft inhibited viral replication for 9 days of cell culture (fig. 2) . as a control maraviroc (mvr) at 2 mm was included and this also showed anti-hiv activity after 9 days in culture, as this compound is known to bind specifically to the ccr5 receptor. however, lower concentrations of maraviroc (at 0.4 mm) did not retain its antiviral activity in this assay protocol (data not shown). an mtt assay was used to assess the effects of grft on end1/ e6e7, ec1/e6e7 and vk2/e6e7 cell viability by measuring the metabolic activity of treated cells. in these experiments (fig. 3) we observed no loss in cell viability after a 3 day exposure of the endocervical and ectocervical cell lines to concentrations of up to 1 mg/ml (84 mm) grft, at least 10-times more concentrated than a likely microbicide formulation [23] . high doses of grft did, however, slightly reduce viability of the vaginal keratinocyte (vk2) cells. in marked contrast to grft, the mannose-specific mitogenic lectin cona showed clear concentration-dependent cytotoxicity towards all three cell lines. as shown in fig. 3 , treatment with 1 mm cona killed approximately 94% of the ectocervical cells and vaginal keratinocytes, and 80% of the endocervical cells. pha, another lectin known to be mitogenic in vivo and in vitro also caused doseresponsive cell death, but to a lesser extent than cona. we previously showed that grft exhibits no mitogenic stimulatory effect in human pbmc, using incorporation of tritiated thymidine as a marker for cell proliferation [23] . in the present study we investigated the mitogenic activity of grft on pbmc by flow cytometry, evaluating changes in size and morphology of cells treated with grft in comparison with cells treated with the vehicle (pbs). cells treated with 1 or 4 mm grft had flow cytometry profiles similar to the control cells (fig. 4a , b and c). in contrast treatment with lectins cona and pha at doses (0.37 mm cona and 10 mg/ml pha) that do not negatively affect cell viability, resulted in completely different flow cytometric plots, with emergence of a subpopulation composed of larger cells (higher forward scatter fsc) with perceptibly higher side scatter (ssc) values gated in region r2, as shown in fig. 4d and e. in addition, a clear decrease in cell number was observed in region r1 after treatment with cona and pha, as quantified in fig. 4f . in fig. 5 , we show that grft also does not induce cell proliferation in any of the three cultured human cervical and vaginal epithelial cells. cell division was assessed by monitoring brdu incorporation in newly synthesized dna of actively dividing cervicovaginal cell lines. treatment with 1 or 8 mm grft did not induce proliferation of any of the cell lines since brdu counts were not elevated in comparison with control cells treated with vehicle alone (pbs). in contrast pokeweed agglutinin (pkm), a well characterized mitogen, caused concentration-dependent increase in cell division, especially in the cervical cell lines end1/e6e7 and ect1/e6e7. to evaluate the effect of grft on cell surface markers of immune activation, we measured expression of the following membrane proteins: (i) cd69, a marker of activated tlymphocytes, considered an ''early'' marker of t-cell activation; (ii) cd25, the alpha chain of the il-2 receptor, upregulated on activated t-cells are highly susceptible to hiv-1 infection, and hence induction of these markers indicates an undesirable side effect of lectin treatment of pbmc. pbmc were incubated in the presence of the test compounds for 72 hours. the vehicle control was pbs, and positive controls were 10 mg/ml pha [20] , as well as 0.37 mm cona, chosen as this concentration was not cytotoxic. in pbs treated cells, 1.2% pbmc in average were double positive (cd4 + /cd25 + ) and a non significant increase of this population was observed after incubation in presence of 1 or 4 mm grft (fig. 6 , left panel and fig. s2a ). treatment with pha and cona resulted in an impressive increase in the number of cd4 + /cd25 + cells (fig. 6 , left panel and fig. s2a ). in addition, the numbers of cd4 2 /cd25 + cells were elevated when pbmc were cultured in presence of pha or cona compared to their pbs and grft counterpart (fig. 6 , left panel and data not shown). similarly, grft treatment did not affect significantly the proportion of cd4 + /cd69 + pbmc compared with pbs treatment, whereas cona and pha treatment resulted in an increase of more than 10 times in this sub-population (fig. 6 , middle panel and fig. s2b ). the numbers of cd4 2 /cd69 + were also elevated after treatment with cona and pha (fig. 6 , middle panel, and data not shown). in unstimulated cells, about 15% of cd4 positive pbmc expressed the late activation marker hla-dr and treatment with grft and cona did not affect this number in a significant fashion (fig. 6 , right panel and fig. s2c ). pha treatment of cells yielded more than twice the number of cd4 + /hla-dr + cells (33.4%) compared with unstimulated pbmc. activation of pbmc may also be reflected by the production of cytokines and chemokines. to investigate potential activating properties of grft more thoroughly, pbmc were cultured in the presence of 10 mg/ml grft (788 nm) for 72 h. in the culture supernatant, the concentrations of il-1a, il-1ra, il-2, il-4, il-5, il-6, il-7, il-8, il-9, il-10, il-12, il-13, il-15, il-17, eotaxin, fgf, g-csf, gm-csf, ifn-c, ip-10, mcp-1, mip-1a, mip-1b, pdgf-bb, rantes, tnf-a, and vegf were determined. a detailed overview of the cytokine profiles of grft-treated pbmc from multiple blood donors is given in fig. 7 . for reference, we provide previously published data where pbmc with the same donor origins were treated with cv-n at 2 mg/ml (182 nm), lower than the grft concentration tested, since higher concentrations of cv-n proved too toxic to pbmc [19] . the concentration of the separate cytokines was compared with that of the untreated pbmc and calculated as a fold increase value. in the previous studies on pbmc treatment with cv-n and mvn, considerable variability in the lectin-induced cytokine profile was observed between the different pbmc donors. therefore, the fold increase values obtained from the different donors were divided over different ranking groups (i.e. 1-3-, 3-10-, 10-100-, 100-500-, and .500-fold increase), and the number in each rank is given as a percentage of the total and indicated by a specific color (fig. 7) . confirming our data in fig. 6 that grft has very little effect on lymphocyte activation markers, we also see minimal alterations in the cytokine and chemokine release for the majority of markers, in most donor pbmc. this profile indicates that grft induces significantly less response from pbmc than has been previously reported for cv-n, mvn and cona [19] . the only chemokine induced weakly by grft in the majority of donors (75%) was mcp-1 [19] . although these studies were performed at a later date than the previously published cv-n and mvn studies, we used exactly the same donor panel, and the assays were performed in the same laboratory (d. schols), justifying comparison between the experiments. in our previously published studies, we showed that treatment of human cervical explants with a range of grft concentrations had no significant effects on expression of a panel of chemokines and cytokines [23] . in the present studies, we used cultured human endocervical, ectocervical and vaginal cell lines for more detailed analysis of potential ''off-target'' effects of grft treatment, since the cell lines are easier to procure than fresh human cervical tissues, and experiments with the cell lines are more easily reproduced without question of variability in genetic background and physiological conditions of the donor. to validate the utility of these cell lines (which are immortalized by induction of papillomavirus e6 and e7 oncogenes) for analysis of any ''off-target'' effects of grft treatment, we wished to confirm that the cell lines behaved similarly to cervical explants after treatment with grft, and cona, which induced inflammatory responses in our pbmc studies. we assessed the secretion of many key mediators of inflammatory responses, including il-1b, il-2, il-6 and il-8 by all three cervico-vaginal cell lines. elisa kits specific for these cytokines were used for their detection in end1/e6e7, ect1/ e6e7 and vk2e6e7 cell culture supernatants after 24 h of incubation in presence of the test compounds. after treatment with 8 mm grft, the supernatants showed barely detectable levels of il-1b and il-2, similar to the cells treated with pbs ( fig. 8a and b) . in contrast, all three cell lines produced impressive amounts of these cytokines upon incubation in presence of cona ( fig. 8a and b) . treatment of cervical epithelial cell lines end1/e6e7and ect1/ e6e7 with grft (8 mm), cona (2 mm) or pbs resulted in similar levels of il-6 secretion (fig. 8c ), but in vk2/e6e7, il-6 release was approximately three times higher after cona treatment compared to pbs, whereas grft did not induce the secretion of this cytokine (fig. 8c ). in the case of il-8, each of the three cell lines showed a distinct pattern of secretion of this cytokine, although no induction was observed after treatment with 8 mm grft: vk2/ e6e7 showed barely detectable levels of il-8 regardless of the treatment; il-8 levels in ect1/e6e7 were relatively high after pbs and grft treatment and these amounts were about ten times reduced after treatment with cona; grft treatment resulted in a slight decrease of il-8 in end1/e6e7 cell culture supernatants whereas the concentrations of this cytokine were significantly increased following treatment with cona (fig. 8d ). in addition, grft treatment did not alter the production of il-10, ip-10, mip-1b and tgf-a in the cervico-vaginal cells studied (data not shown). these data indicated that all 3 cell lines behaved as predicted in response to grft and cona treatment. since the ectocervical epithelium is an important point of mucosal transmission of hiv-1, we focused on the ect1/e6e7 cell line to derive a more comprehensive assessment of the effect of grft treatment on cytokine production by the cultured ectocervical cells. a multiplexed luminex assay of the ect1/e6e7 cell line treated with grft confirmed that the ect1 cells behaved very similarly to human cervical explants (data not shown), and validated our use of these cells in our microarray studies. gene expression in ect1/e6e7 cells in response to treatment with grft was compared with vehicle (pbs), grft lec-, cv-n and cona treatments (all qualified with endotoxin levels less than 0.05 eu per milligram). this was studied using a microarray experiment in which the whole human genome was represented by the 41,000 known genes and transcripts [27] . the microarray data were deposited in the gene expression omnibus database, under accession number gse28584. the heat map shown in fig. 9a indicates that cells exposed for 24 hours to grft lec-(1 and 8 mm), and low concentrations of grft (0.1 mm ) and cv-n (0.05 mm) showed comparable gene expression profiles to those that were incubated in presence of pbs alone. treatment of ect1/ e6e7 with 1 mm grft resulted in minor changes in the expression profile while 4 mm grft appeared to alter the expression of many genes but to a much lesser extent compared to cv-n (0.5 and 4 mm) and cona at 1 mm (fig. 9) . thus, the microarray studies confirm the data showing that grft has substantially lower ''off-target'' effect in comparison with cv-n and cona, although there are clearly several genes that are regulated by the carbohydrate binding activity of grft (compare with grft lec-). as predicted from the heat map (fig. 9a) , no gene was identified as regulated by 0.1 mm grft, even when the cutoff was brought to 1.0. however, a first analysis using benjamini-hochberg low stringency correction with a cutoff of 2.0 yielded 107 and 35 entries as differentially expressed in samples treated with 1 and 4 mm grft, respectively (fig. 9b) . treatment with cv-n (0.5 and 4 mm) or cona (1 mm) resulted in regulation of impressive numbers of human genes (fig. 9b) . we then employed stricter criteria by keeping only the positive entries that showed grft concentration dependent gene expression. this yielded 2 and 32 genes for 1 and 4 mm grft, respectively. the entries which showed an increased gene expression after treatment with 1 mm grft included a gene annotated as immunoglobulin-like and fibronectin type iii domain containing 1 (igfn1). the entries that were found to be affected by 4 mm grft are summarized in table s2 . among the 26 genes mapped by the ingenuity database, there was overrepresentation of genes in the following canonical pathways: nrf2-mediated oxidative stress response (maf, hmox1, sod2), phospholipid degradation, glycerophospholipid metabolism and endothelin-1 signaling (hmox1, wisp2), camp mediated signaling (calml5, pkib) and acute phase response signaling (hmox1, sod2). furthermore using the ingenuity software we identified five toxicological functions with an overrepresentation of genes including liver hyperbilirubinemia and steatohepatitis, cardiac arteriopathy, renal and liver necrosis (data not shown). none of these is relevant to mucosal treatment with grft. we used quantitative rt-pcr (q-rt-pcr) to validate the microarray results. expression of 14 genes was studied using 18s rna and b-actin mrna as controls. with the exception of mycn, all genes studied showed comparable expression levels in both experimental systems including microarrays and q-pcr (table s3 ). natural product lectins have received considerable attention as potential antiviral drugs, particularly in the context of prevention of hiv-1 transmission via mucosal surfaces (reviewed recently in [8] ). although the volume of literature supporting their use as antivirals in vivo is dwarfed by a comprehensive set of data showing potent in vitro antiviral activity, there are reports of impressive in vivo efficacy of cv-n in animal models of hiv-1 prevention [17, 18] , influenza prevention and treatment [28] , and ebola virus prophylaxis and treatment [29] , and of grft in prevention of sars-cov infection [30] . despite the myriad of potential prophylactic and therapeutic applications of antiviral lectins, enthusiasm for their development as pharmaceuticals is tempered by a long history of research into natural product lectins, which characterizes many members of this broad class as erythrocyte agglutinins, lymphocyte mitogens, and potentially lethal toxins [10, 11] . the pharmacological basis of natural product lectin toxicity is generally poorly understood, but at a basic mechanistic level is thought to reside in the lectins' capacity for multivalency of binding to cell surface glycans, resulting in cell agglutination and/ or cross-linking of cell surface receptor molecules with consequent activation of signaling pathways. why different lectins that bind identical glycan moieties have quite distinct biological effects in vitro remains a paradox that is well illustrated by our data which, show very different in vitro activity profiles of four different oligomannose-binding lectins: grft, cv-n, mvn [19] and cona. characterization of grft is the primary focus of our studies, but understanding the molecular pharmacology and toxicology of this potent antiviral lectin is informed by comparison to cv-n, for which a rich set of in vitro and in vivo data is available. both molecules have comparable hiv-1 neutralization activities, with mean ic 80 values against clade c viruses of 42.764.4 nm for grft and 77.0618.2 for cv-n [15] . both proteins bind oligomannose glycans, with grft targeting terminal mannose residues found on man 5-9 -glcnac 2 [13] and cv-n specific for the mana1r2man linkages found on man 6-9 -glcnac 2 [16, 31, 32] . they thus share overlapping binding specificities, and should target identical cell surface and viral glycans. if anything, grft would be predicted to bind to a larger number of glycan targets than cv-n since it can bind pentamannose structures that lack the a1r2 mannose linkages that cv-n targets [13] . in this context, it is surprising that cv-n appears to bind more promiscuously than grft throughout the cervical epithelium and sub-epithelial stroma (fig. 1a-d, and fig. s1 ). given that grft lec-(the carbohydrate-binding deficient form of grft) hardly bound the epithelial sections or cultured cervical and vaginal epithelial cells (fig. 1) , and grft binding to cultured epithelial cells was blocked by mannan, we conclude that grft's binding activity to the cell surface is exclusively via its carbohydrate binding activity. it is very interesting to note that cv-n binding to the surface of pbmc is not entirely eliminated by blocking its carbohydrate binding sites with mannan (fig. 1k ). this implies that its capacity to bind and induce signaling in cells may not reside entirely in its lectin activities, and supports prior studies with cv-n [21] . another important result showed that grft seems to bind somewhat selectively to terminally differentiated keratinocytes on the epithelial surface, presumably reflecting presence of optimal glycoprotein binding targets on the surface of those cells only in the intact epithelium ( fig. 1c and d; and fig. s1 ). the data presented in fig. 2 strongly support grft's candidacy as a microbicide and antiviral, since they show that grft retains antiviral activity even when complexed to the surface of pbmc. this contrasts with many other lectins or compounds we have tested in this assay. the most plausible mechanism that might explain grft's retention of hiv-1 entry inhibition activity is that fewer than all six carbohydrate binding sites are occupied when the lectin is docked on cell surface glycoprotein/s, leaving sites available for binding to the viral envelope glycoprotein. the crystal structure of grft suggests that the three glycan-binding pockets of each grft monomer are on opposite ends of the double prism homodimer [13] , suggesting that only one monomer of the homodimer is engaged in binding the cell surface, leaving the ''free'' monomer competent to bind and cross-link two oligomannose structures on the surface of hiv-1. since the pbmc were washed extensively 24 hours after treatment and prior to addition of the hiv-1 inoculum, this suggests that cell surface-bound grft irreversibly inactivated the inoculum, with no evidence of breakthrough infection at 9 days post infection. this duration of antiviral activity is unprecedented. conventionally, antiviral potencies of virus-targeted entry inhibitor drugs are measured without washing the cells prior to addition of viruses. in traditional antiviral assays the ic 50 of grft against hiv-1 bal is 0.2 nm in pbmc, and 0.1 nm against hiv-1 bal in monocytes/macrophages (data not shown). remarkably, the ic 50 for grft in the washed pbmc assay (fig. 2) , when the test agent is applied 24 hours prior to cell washing and infection, is 0.78 nm, showing quite exceptional antiviral activity of grft. in the same assay we show that activity of ccr5 antagonist maraviroc persists for 24 hrs, although a high concentrations, but grft's activity persists substantially longer, a property that may facilitate non-coitally linked administration of microbicides containing grft as an active ingredient. the cardinal rule in development of an anti-hiv-1 microbicide must be ''first, do no harm''. randomized controlled preclinical and clinical studies with detergent-based microbicides such as nonoxynol-9 showed a trend towards evidence of harm, with increased incidence of not only hiv-1 infection, but also hsv-2 and hpv seen in the experimental arms [33, 34, 35, 36, 37] . the microbicides field therefore requires stringent and extensive in vitro and in vivo safety studies before human trials initiate. initial studies showed that grft was not cytotoxic; had no mitogenic activity; did not induce secretion of chemokine and cytokine-mediators of inflammation in treated cervical explants; and showed a good safety profile in the rabbit vaginal irritation test [23, 24, 25] . the first tissues that grft would be exposed to in the human vagina are keratinocytes in the outer surface of the vaginal and cervical epithelia. grft would also encounter hiv-1 target cells such as dendritic cells and macrophages resident in the epithelium and submucosal stroma as well as the primary hiv-1 target cells, mucosal cd4+ t-cells. in this work we expanded the safety studies to include analyses of off-target effects derived from grft binding cell surface oligomannose glycans on human pbmc as well as characterized ectocervical, endocervical and vaginal keratinocyte cell lines end1/e6e7, ect1/e6e7 and vk2e6e7, which were originally established from normal human endocervical, ectocervical, and vaginal epithelia, respectively, and immortalized by expression of human papillomavirus 16/e6e7 [38] . the immortalized cell lines were shown to have close resemblance to those of their respective tissues of origin and primary cultures in their morphological and immunocytochemical characteristics and therefore proposed for studies dealing with testing pharmacological agents for intravaginal application [38] . first we evaluated the effects of grft on cell viability and we showed that up to 84 mm grft did not decrease cell viability in the cervical cell lines end1/e6e7and ect1/e6e7 whereas vk2/ e6e7 was somewhat sensitive to grft at high concentrations of 8 mm and above. these results are consistent with the pioneering work of mori and coworkers in which no significant cellular toxicity was found when a variety of human cell types were treated with grft at concentrations of up to 0.783 mm [24] . since many lectins have been reported to be mitogenic [39, 40] , we examined if grft would have an effect on the cervico-vaginal cell proliferation. using a colorimetric assay for detection of brdu in newly synthesized dna, we showed that high concentrations of grft did not increase the proliferation of either end1/e6e7, ect1/e6e7 or vk2/e6e7. we then evaluated the concentrations of a panel of cytokines and chemokines in cervico-vaginal cell culture supernatants using elisa. we found that high concentrations of grft had little to no effect on the secretion of cytokines/chemokines studied including il-1b, il-2, il-6, il-8, il-10, ip-10, mip-1b and tgfa in all the three cervico-vaginal cell lines including end1/e6e7, ect1/e6e7 and vk2/e6e7. therefore, our results, taken together suggest that grft at these concentrations does not alter the secretion of the immune system mediators by cervico-vaginal cells in a significant fashion. using the ect1/e6e7 cell model, we tested grft in order to evaluate its effects on gene expression after over night incubation. to our knowledge, this is the first study evaluating the effect of an anti-hiv lectin on gene expression using human genome microarrays. our data revealed that exposure to grft at 0.1 mm did not affect gene expression whereas treatment of ect1/ e6e7 with 1 mm grft resulted in minor alteration of the gene expression profile, showing only 2 genes that were considered as significantly upregulated (cutoff of 2.0). of note our working concentrations were very high. for instance, 1 mm corresponds to about 1,600 times and more than 23,000 times the ec 50 found for the most resistant and the most sensitive hiv-1 strain, respectively. at higher concentrations of 4 mm grft, 32 genes were considered as differentially expressed (cutoff = 2.0). the fold changes found were less or equal to 3.2. q-rt-pcr evaluation of the expression of selected genes validated the microarray data presented here. it is unclear what is the biological relevance and significance of this level of regulation. using the same cell line (ect1/e6e7) sharkey et al. classified a gene as differentially expressed when the fold change was more than 2.0 and found a total of 444 probe sets that fell in this category after treating the cells 12 h with 10% human seminal plasma [41] . this is in sharp contrast to treatment with 4 mm grft, a level at least 1,000 fold greater than the average antiviral ec 50 , which regulated expression of only 32 genes. in summary, our data provide further evidence that grft, an exceptionally potent antiviral lectin, has very minor effects on the molecular physiology of human cells. at this point, the molecular basis for the distinct biological activities of different antiviral lectins is uncharacterized, we propose that the specific spatial arrangement of the carbohydrate binding sites may determine the nature and extent of cross-linking of cell surface glycoproteins. grft clearly has superior binding and cross-linking activity with the hiv-1 envelope glycoprotein, which displays dense clusters of oligomannose nlg, but does not induce off-target cellular signaling to the extent that other lectins do. we believe this provides further data in strong support of focused clinical development of hiv-1 microbicides containing grft as an active ingredient. recombinant grft was produced in nicotiana benthamiana plants. recombinant cv-n was produced in escherichia coli. methods for expression and purification of both products have been described previously [23, 42] . a synthetic cdna encoding a lectin activity-deficient mutant of grft, termed grft lec-, was designed with a conservative amino acid substitution of aspartic acid to asparagine in each of the 3 carbohydrate binding pockets identified in the primary amino acid sequence and crystal structures of grft [24, 26] . grft lecwas expressed in n. benthamiana and purified exactly as described for grft [23] . proteins were purified to .99% purity, and formulated in phosphate buffered saline (pbs), ph 7.4 at 10 mg/ml protein concentration. endotoxin was removed from grft, grft lecand cv-n protein samples using detoxi-gel endotoxin-removing gel gravity flow columns (thermo scientific). endotoxin levels were measured using the toxinsensor tm chromogenic lal endotoxin assay kit from genscript (piscataway, nj). only products with final endotoxin readings less than 0.05 endotoxin units (eu) per milligram were used in the in vitro studies, and all dilutions were performed in endotoxin-free buffers. grft, grft lecand cv-n were fluorescently-labeled with amine-reactive alexa fluor 488 carboxylic acid, succinimidyl ester using a kit from molecular probes/invitrogen, according to the manufacturer's specifications. control lectins concanavalin a (cona), phytohemagglutinin a (pha) and pokeweed agglutinin (pkm) were purchased from sigma. lectin activity measurements using hiv-1 gp120-binding elisa immobilized hiv-1 gp120 (protein sciences corporation) was used to measure the lectin activity of purified grft, cv-n and fluorescently labeled conjugates thereof, and to confirm that grft leclacked gp120 binding activity. nunc maxisorp elisa plates were coated overnight with 1 mg/ml gp120 (strain iiib, protein sciences) diluted in pbs. the wells were blocked with 5% (w/v) non-fat dry milk in pbs+0.05% tween (pbs-t; immunowash, bio-rad) and washed before addition of serial dilutions of lectin analyte (grft, grft lec-, cv-n or alexa-fluor 488-labelled conjugates thereof) diluted in 16 pbs for 1 h. after three washes with pbs-t, a primary polyclonal antiserum (rabbit anti-grft or cv-n or alexa-fluor 488 (invitrogen), as appropriate) diluted 1:10,000 in pbs was added for 1 h at room temperature. the wells were again washed before goat anti-rabbit igg-hrp (southern biotech) was added at a 1:10,000 dilution. colorimetric values reflecting hrp activity were derived using kpl sureblue tmb microwell peroxidase substrate, with the reaction stopped by addition of 1 n h 2 so 4 . the plates were read at 450 nm on a biotek synergy ht reader with data collected using gemini software. we confirmed that the labeled products retained lectin activities comparable to the unlabeled product by gp120-binding elisa. we used elisa with anti-alexafluor 488 detection to measure the total amount of label conjugated to grft, grft lecand cv-n, which displayed quantitatively similar labeling efficiency. end1/e6e7, ect1/e6e7 and vk2/e6e7 are well characterized immortalized cell lines derived from normal human endocervical, ectocervical and vaginal epithelia, respectively [38] . all 3 cell lines were purchased from the american type culture collection (atcc, rockville, md). the cervico-vaginal cell lines were grown as previously described [38] in keratinocyte serum-free medium (ksfm) supplemented with recombinant human epidermal growth factor (0.1 ng/ml), bovine pituitary extract (50 mg/ml), calcium chloride (0.4 mm) and an antibiotic cocktail composed of penicillin and streptomycin at final concentrations of 100 u/ml and 100 mg/ml, respectively. reagents were obtained from invitrogen (sandiego ca, or from sigma chemical company). cryopreserved human pbmc used in assays of inflammatory cytokines and chemokines were purchased from seracare life sciences inc. (milford, ma) and were immediately cultured for the experiments in rpmi 1640 supplemented with 10% fetal bovine serum (fbs) and the penicillin-streptomycin antibiotic cocktail (to 100 u/ml and 100 mg/ml final concentrations, respectively). slides with paraffin embedded human cervical tissue sections from a healthy 21 year old female (us biomax, inc.) were deparaffinized and rehydrated. alexa-fluor 488-labelled proteins of interest were added to the slides and incubated overnight at 4uc in a humidity chamber. the slides were rinsed with pbs twice for 10 minutes and coverslipped using vectashield mounting media with dapi (vector laboratories, burlingame, ca). for light microscopy, tissue sections were stained with hematoxylin and eosin by standard methods. ect1/e6e7, end1/e6e7 and vk2/ e6e7 cultured cells were seeded onto eight-well lab-tek chamber slides (nalgene nunc) in duplicate at 10,000 cells per well and allowed to incubate at 37uc with 5% co 2 . after eight hours the fluorescently labeled proteins of interest were added to the wells and incubated overnight at 37uc with 5% co 2 . the slides were washed twice for 10 minutes with pbs and coverslipped using vectashield mounting media with dapi (vector laboratories, burlingame, ca). slides were visualized using the axio observer z1 microscope with apotome assembly (carl zeiss, thornwood, ny). human pbmc (seracare life sciences, md) were thawed and seeded onto 48-well culture plates (celltreat, ma) at 2.5610 5 cells per well. three dilutions of each alexa-fluor 488-labeled protein were added to the cells for overnight incubation at 37uc with 5% co 2 . samples were also prepared with 5 and 10 mg of mannan (sigma, st. louis mo) at each protein dilution. all samples were analyzed in duplicate. following incubation, cells were briefly trypsinized (tryple express, gibco) and placed in 5 ml polystyrene tubes (bd falcon, ma). cells were washed twice with pbs and analyzed using the bd facsaria (bd biosciences, nj) flow cytometer. freshly isolated pbmc were cultured in the presence of grft, cv-n and maraviroc for 24 hrs. then the cells were collected, washed in culture medium, suspended in rpmi medium with 2 ng/ml il-2 and seeded in a 48-well flat bottom plate (5610 5 cells in 450 ml) and 50 ml of the ccr5-tropic clade b hiv-1 bal stock was added at 100 tcid 50 . the supernatant of each sample was collected after 9 days and viral replication measured by a specific p24 ag elisa (perkin elmer, zaventem, belgium). viability of cervico-vaginal cell lines was measured using a colorimetric mtt [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide] assay kit from biotium inc. (hayward, ca) following the manufacturer's instructions. briefly, 10 4 cells per well were seeded (96 well plates), the test reagents added and the cultures incubated for three days in a humid environment with 5% co 2 at 37uc. afterwards, 10 ml of mtt solution were added to each well followed by 4 hours incubation at 37uc. then the medium was gently removed and the insoluble purple formazan product dissolved in dmso to yield a colored solution which absorbance was read at 570 nm with a background at 630 nm. proliferation of cervicovaginal cell lines was evaluated by bromodeoxyuridine (brdu, a thymidine analog) incorporation in newly synthesized dna using a cell proliferation elisa kit from roche, according to the manufacturer's instructions. for human pbmc, cells were treated with grft for three days and analyzed by flow cytometry for any changes in size and/or morphology using forward scatter (fsc) and side scatter (ssc) with a facscalibur (bd, san jose, ca) counting 10,000 events per sample. data were acquired and analyzed using cellquest pro from bd. cona (0.37 mm), pha (10 mg/ml) and pbs were used as controls. three day old pbmc were analyzed flow-cytometrically after dual fluorescent staining with anti-mouse antibodies purchased from bd pharmingen (san diego, ca). briefly, cell cultures were transferred from plates to a 5 ml round bottom tubes and washed with pbs containing 5% inactivated fbs (washing solution). after 10 min blocking with purified rat anti-mouse cd16/cd32 (mouse bd fc block), cells were incubated in dark with fitcconjugated anti-cd4 mab in combination with pe-conjugated anti-cd25, anti-cd69 or anti hla-dr mab for 30 min on ice. finally pbmc were washed and analyzed with a facscalibur (bd, san jose, ca), counting 10 000 events per sample. data were acquired and analyzed using cellquest pro from bd. cona (0.37 mm) and pha (10 mg/ml), and pbs were used as positive and negative controls, respectively. multiplex immunoassays were carried out on a bio-plex instrument (biorad) using a milliplex human cytokine/chemo-kine 42-plex luminex bead-based assay (millipore), following the manufacturer's instructions. each luminex immunoassay was repeated three times. the kit allows simultaneous quantification of human-epidermal growth factor (egf), eotaxin, fibroblast growth factor-2 (fgf-2), fms-like tyrosine-kinase 3 ligand (flt-3 ligand), fractalkine, granulocyte colony-stimulating factor (g-csf), granulocyte-macrophage-csf (gm-csf), gro, interferon-a2 (ifn-a2) , ifn-c, interleukin-10 (il-10), il-12 (p40), il-12 (p70), il-13, il-15, il-17, il-1a, il-1b, il-2, il-3, il-4, il-5, il-6, il-7, il-8, il-9, il-1 receptor antagonist (il-1ra), interferoninducible protein-10 (ip-10), monocyte chemoattractant protein-1 (mcp-1), mcp-3, macrophage-derived chemokine (mdc), macrophage inflammatory protein-1a (mip-a), mip-1b, plateletderived growth factor-aa (pdgf-aa), pdgf-ab/bb, regulated upon activation normal t-cell expressed and secreted (rantes), soluble cd40 ligand (scd40l), soluble il-2ra (sil-2ra), transforming growth factor a (tgf-a), tumor necrosis factor-a (tnf-a), tnf-b and vascular endothelial growth factor (vegf). supernatants collected from ect1/e6e7 after 24, 48 and 72 hours of culture in presence of test compounds were studied in the multiplex-immuno assays and data were analyzed using the luminex 6ponent r software. individual elisa experiments were performed on end1/e6e7, ect1/e6e7 and vk2e6e7 cell culture supernatants collected after 24 hours of incubation in presence of grft or controls. elisa ready-set-go! kits designed for accurate and precise measurement of human il-1b, il-2, il-6 and il-10 were purchased from ebioscience, inc. quantikineh elisa kits from r&d systems, inc. were used for the detection of il-8, ip-10 and tgf-a. mip-1b was quantified using an elisa kit from cellsciences. all elisas were performed according to the manufacturer's specifications. bio-plex cytokine assay in human pbmc supernatants pbmc from multiple blood donors [19] were cultured for 72 h in presence of 10 mg/ml grft (788 nm). in the culture supernatants, the concentrations of il-1a, il-1ra, il-2, il-4, ilgene expression analysis in ect1/e6e7 cells cultured ect1/e6e7 cells were incubated with dilutions of grft, grft lec-, cv-n, cona, or vehicle only (pbs, ph 7.4) for 16 hours. the cells were lysed using a qiagen qiashredder, and total rna was extracted using a qiagen rneasy mini kit. total rna was quantified spectrophotometrically and the sample quality was checked on an agilent 2100 bioanalyzer (agilent technologies, wilmington, de). 200 ng rna were used to generate cyanine 3 (cy3) crna with the aid of low rna input linear amplification kit, one-color (agilent technologies, wilmington, de) according to the manufacturer's instructions. 1.65 ug of each labeled crna sample were fragmented at 60uc for 30 min using an agilent gene expression hybridization kit (agilent technologies, wilmington, de) followed by hybridization to a whole human genome agilent oligonucleotide slide containing four high-definition 44 k microarray (agilent technologies, wilmington, de) at 65uc for 17 hours. after hybridization, the slides were washed with agilent gene expression wash buffers (agilent technologies, wilmington, de) and scanned using an agilent g2565ba microarray scanner system (agilent technolo-gies, wilmington, de). then, one-color microarray images were extracted with the feature extraction software v 9.5.1 (agilent technologies, wilmington, de) and the raw data imported into genespring gx 10 software (agilent technologies, wilmington, de) for normalization and further analysis to yield a list of differentially expressed genes. benjammini and hocheberg correction controlling the false discovery rate fdr [43, 44, 45] was used and we considered only a fold change of at least 2.0 compared to the pbs treated samples to represent a meaningfully altered gene expression [41, 46, 47] . the raw microarray data were uploaded into the gene expression ominbus (geo) database, a miame-compliant database as detailed on the mged society website http://www.mged.org/workgroups/miame.html. the geo accession number is gse28584. further analysis of the regulated genes was carried out using the ingenuity knowledge database (ingenuity systems inc., redwood city, ca) yielding putative toxicological functions, and canonical pathways. quantitative rt-pcr was carried out in order to validate the microarray results. first strand cdna was reverse transcribed from 250 ng rna employing a high capacity rna-to-cdna kit (applied biosystems) following the manufacturer's instructions. optimal amounts of template cdna were added to a reaction mixture containing 10 ml of 26taqmanh gene expression master mix (applied biosystems) and water to 20 ml and this mixture was used to set the pcr reactions in taqmanh array standard 96 well plates (applied biosystems). these plates contain pre-spotted individual taqmanh gene expression assays for detection of human cysteine-aspartic acid protease (caspase14, casp14), beta defensin 103a (defb103a), immunoglobulin-like and fibronectin type iii domain containing 1 (igfn1), interleukin-1b (il-1b), il-2, il-6, il-8, il-10, il-33, ip-10, v-myc myelocytomatosis viral related oncogene (mycn), thyroglobulin (tg ), tgfa, tripartite motif-containing 63 (trim63) as well as the house keeping genes 18 s and beta actin (actb). individual probes used in these experiments are described in table s1 . pcr amplification was performed in an 7900ht fast real-time pcr system (appliedbiosystems) as follows: initial activation step (95uc, 20 min), 40 cycles (95uc, 1 min) and 20 min at 60uc. sds 2.3 or rq manager 1.2 software from applied biosystems was used to evaluate the cycle threshold (ct) for each reaction, which is the cycle number at which 50% maximal amplicon synthesis is achieved. ratios were derived as proposed elsewhere [48] . group means and standard deviations were derived from the values obtained in three individual replicates. statistical signifi-cance was analyzed by a one-way analysis of variance (anova) and student's t-test unless otherwise stated, using graphpad software (san diego, ca). differences were considered statistically significant if p,0.05. for microarray data, the statistical analysis was carried out as described in the gene expression analysis methods above. figure s1 analysis of binding specificity of grft in comparison with grft lecand cv-n. in the first column we depict the identical fluorescence micrographs to fig. 1 antibody neutralization and escape by hiv-1 hiv-1 gp120 mannoses induce immunosuppressive responses from dendritic cells envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens glycosylation: heterogeneity and the 3d structure of proteins exploiting the defensive sugars of hiv-1 for drug and vaccine design ) variability, heritability and environmental determinants of human plasma nglycome profile of native n-linked glycan structures from human serum using high performance liquid chromatography on a microfluidic chip and time-of-flight mass spectrometry potential of carbohydrate-binding agents as therapeutics against enveloped viruses carbohydrate-binding agents cause deletions of highly conserved glycosylation sites in hiv gp120: a new therapeutic concept to hit the achilles heel of hiv history of lectins: from hemagglutinins to biological recognition molecules anti-hiv activity of defective cyanovirin-n mutants is restored by dimerization monomerization of viral entry inhibitor griffithsin elucidates the relationship between multivalent binding to carbohydrates and anti-hiv activity actinohivin: specific amino acid residues essential for anti-hiv activity mannose-rich glycosylation patterns on hiv-1 subtype c gp120 and sensitivity to the lectins, griffithsin, cyanovirin-n and scytovirin multivalent interactions with gp120 are required for the anti-hiv activity of cyanovirin cyanovirin-n inhibits aids virus infections in vaginal transmission models cyanovirin-n gel as a topical microbicide prevents rectal transmission of shiv89.6p in macaques microvirin, a novel alpha(1,2)-mannose-specific lectin isolated from microcystis aeruginosa, has anti-hiv-1 activity comparable with that of cyanovirin-n but a much higher safety profile safety concerns for the potential use of cyanovirin-n as a microbicidal anti-hiv agent mutational pathways, resistance profile, and side effects of cyanovirin relative to human immunodeficiency virus type 1 strains with n-glycan deletions in their gp120 envelopes cyanovirin-n potently inhibits human immunodeficiency virus type 1 infection in cellular and cervical explant models scaleable manufacture of hiv-1 entry inhibitor griffithsin and validation of its safety and efficacy as a topical microbicide component isolation and characterization of griffithsin, a novel hiv-inactivating protein, from the red alga griffithsia sp griffithsin, a potent hiv entry inhibitor, is an excellent candidate for anti-hiv microbicide domain-swapped structure of the potent antiviral protein griffithsin and its mode of carbohydrate binding igfbp-3 hypermethylation-derived deficiency mediates cisplatin resistance in non-small-cell lung cancer treatment of influenza a (h1n1) virus infections in mice and ferrets with cyanovirin-n cyanovirin-n binds to the viral surface glycoprotein, gp1,2 and inhibits infectivity of ebola virus broad-spectrum in vitro activity and in vivo efficacy of the antiviral protein griffithsin against emerging viruses of the family coronaviridae dissecting carbohydrate-cyanovirin-n binding by structure-guided mutagenesis: functional implications for viral entry inhibition crystal structure of cyanovirin-n, a potent hiv-inactivating protein, shows unexpected domain swapping genital transmission of hpv in a mouse model is potentiated by nonoxynol-9 and inhibited by carrageenan in vitro preclinical testing of nonoxynol-9 as potential anti-human immunodeficiency virus microbicide: a retrospective analysis of results from five laboratories safety study of nonoxynol-9 as a vaginal microbicide: evidence of adverse effects a comprehensive murine model to evaluate topical vaginal microbicides: mucosal inflammation and susceptibility to genital herpes as surrogate markers of safety the impact of the use of col-1492, a nonoxynol-9 vaginal gel, on the presence of cervical human papillomavirus in female sex workers generation of papillomavirus-immortalized cell lines from normal human ectocervical, endocervical, and vaginal epithelium that maintain expression of tissue-specific differentiation proteins inhibition of hiv entry by carbohydrate-binding proteins molecular cloning of the mitogenic mannose/maltose-specific rhizome lectin from calystegia sepium seminal plasma differentially regulates inflammatory cytokine gene expression in human cervical and vaginal epithelial cells recombinant production of cyanovirin-n, a potent human immunodeficiency virus-inactivating protein derived from a cultured cyanobacterium controlling the false discovery rate in behavior genetics research identifying differentially expressed genes using false discovery rate controlling procedures biological microarray interpretation: the rules of engagement a new strategy to identify and annotate human rpe-specific gene expression gene expression profiling in autoimmune noninfectious uveitis disease analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method key: cord-340763-cxnu9g8y authors: grimm, sebastian k.; battles, michael b.; ackerman, margaret e. title: directed evolution of a yeast-displayed hiv-1 sosip gp140 spike protein toward improved expression and affinity for conformational antibodies date: 2015-02-17 journal: plos one doi: 10.1371/journal.pone.0117227 sha: doc_id: 340763 cord_uid: cxnu9g8y design of an envelope-based immunogen capable of inducing a broadly neutralizing antibody response is thought to be key to the development of a protective hiv-1 vaccine. however, the broad diversity of viral variants and a limited ability to produce native envelope have hampered such design efforts. here we describe adaptation of the yeast display system and use of a combinatorial protein engineering approach to permit directed evolution of hiv envelope variants. because the intrinsic instability and complexity of this trimeric glycoprotein has greatly impeded the development of immunogens that properly represent the structure of native envelope, this platform addresses an essential need for methodologies with the capacity to rapidly engineer hiv spike proteins towards improved homogeneity, stability, and presentation of neutralizing epitopes. we report for the first time the display of a designed sosip gp140 on yeast, and the in vitro evolution of derivatives with greatly improved expression and binding to conformation-dependent antibodies. these efforts represent an initial and critical step toward the ability to rapidly engineer hiv-1 envelope immunogens via directed evolution. broadly neutralizing antibodies (bnabs) are widely thought to represent the best means of preventing hiv infection, making development of an immunogen capable of raising bnabs a cornerstone and priority of vaccine development efforts [1, 2] . unfortunately, various factors relating both to the characteristics of known bnabs as well as the hiv virus itself have pointed toward substantial obstacles to the realization of this goal. the sequence diversity and instability of the trimer, its heavily glycosylated structure, low surface density on the viral particle, and limited access to functionally critical epitopes have confounded efforts to induce bnabs by vaccination [1, 3] . furthermore, cues from natural infection suggest that monoclonal bnabs are uncommon, arise after years of infection and high viral load, fail to control established infection, must have precisely oriented binding interactions, and often have unusual properties [4] [5] [6] [7] [8] [9] [10] [11] , indicating that without a fundamental breakthrough in immunogen design, the generation of such bnabs by vaccination is likely to remain a daunting challenge [12, 13] . even so, exciting progress has been achieved recently in characterizing the neutralizing capacity of antibodies generated in the course of natural infection [11, [14] [15] [16] [17] , as well as in identifying novel bnabs [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] . these and other bnabs have greatly informed immunogen design, highlighting new regions of the envelope trimer, variable loops, envelope glycan, the membrane proximal region, novel quaternary epitopes, and receptor and co-receptor binding sites as epitopes with a combination of sufficient conservation and functional relevance to be key targets of an effective antibody response. it is anticipated that with the continued use of high-throughput b-cell screening methods, the set of bnabs with different fine-epitope specificities and viral coverage will continue to grow and provide a rich set of probes to reinvigorate and diversify immunogen design efforts. however, a high-throughput and adaptable platform is required to ensure these findings are efficiently translated into candidate immunogen development. in the context of natural infection, bnabs have tended to be isolated from individuals with high viral loads, persistent antigen exposure, and progressive disease. in the absence of replicating vectors, it is difficult to envision how similar levels of antigen exposure could be accomplished via vaccination. additionally, envelope diversity may be a key driver in the generation of neutralization breadth, posing another fundamental challenge. together, the antigen exposure associated with natural infection likely represents both orders of magnitude greater levels and diversity than can be achieved by current strategies, leading to the discouraging conclusion that a successful immunogen may need to possess an orders of magnitude improved capacity to elicit bnabs over natural envelope. with these technical and immunological gaps in mind, we sought to establish a yeast surface display (ysd) platform to apply directed molecular evolution principles to the development of hiv envelope variants with fundamentally improved biophysical properties. ysd allows the display of millions of sequence variants and selection based on flexible design criteria to allow efficient and deep coverage of the envelope structure:function landscape, representing a potentially enabling technology for the rapid translation of findings from basic studies to the development of novel candidate immunogens. as such, ysd has been established as a powerful method to engineer diverse proteins for a broad range of functional improvements, including stability, specificity, affinity, catalysis, and enantioselectivity [28] [29] [30] [31] . routinely, variants with million-fold improvements can be isolated from large libraries, and repeated cycling of mutagenesis and selection has resulted in evolution of some of the highest affinity synthetic interactions ever observed [32] . indeed, promising efforts aimed at the development of scaffolded epitopes and gp120 cores with desirable properties such as enhanced recognition of germline antibody families have routinely relied upon such combinatorial approaches and ysd-based directed evolution [33, 34] . however, yeast expression and display of complete gp120, much less gp140, has not been described. here we investigated yeast as a host for the display of hiv spike protein variants and report for the first time the display of full-length gp140 on s. cerevisiae. a jr-fl gp140 variant with the sosip mutations served as starting point because it has previously been engineered for stability and homogeneity, was well-expressed in mammalian cells [35] [36] [37] and because the sosip mutations proved key to enabling structural insights into the native hiv-1 envelope trimer [38] [39] [40] [41] . we have applied random mutagenesis and dna shuffling in combination with stringent vrc01 driven selections to isolate gp140 sosip variants with significantly enhanced expression and that bind to conformational hiv bnabs, suggesting proper antigenicity. the yeast-displayed gp140 sosip variants described here demonstrate the feasibility of production of hiv envelope in yeast, and the advantages of this robust platform in evolving variant envelopes. if trimeric presentation can be confirmed or further engineered, ysd may prove a robust platform for the rapid evolution and selection of promising vaccine immunogens. influence of n-glycosylation on binding of conformational hiv antibodies to yeast-produced gp120 since hiv spike proteins are highly glycosylated, proteolytically processed, and contain numerous disulfide bonds, only eukaryotic cells with appropriate protein processing machinery can serve as hosts for the display of functional protein. whereas mammalian cell lines are relatively slow growing, and achieving the single genotype-phenotype linkage requires viral transfections or other rather cumbersome and time-consuming methodologies, the yeast s. cerevisiae is a robust and well-described eukaryotic host for cell-surface display [42] . yeast are capable of displaying complex mammalian glycoproteins such as antibody fragments, full-length antibodies, peptide-mhc molecules, or growth factor receptors [42] [43] [44] [45] , as well as a growing number of viral envelope proteins of fragments thereof such as hemagglutinin, the gp120 core, dengue e protein, sars-cov, west nile virus envelope, and the hepatitis c virus envelope protein e2 [46] [47] [48] [49] [50] [51] , suggesting that the display of hiv envelope protein is feasible in principle. nevertheless, in s. cerevisiae, n-linked glycans are extended and mannose-rich [52] , though p. pastoris yeast strains with human or human-like n-linked glycosyation have been reported to overcome this limitation [53, 54] . to investigate whether heterologous n-glycosylation in yeast has a major effect on the recognition of gp120 by conformational antibodies, we compared hiv-1 jr-fl gp120 produced in either s. cerevisiae strain yvh10 or glycan-engineered p. pastoris [53] for binding to a panel of conformational hiv-1 bnabs. the mannose-rich n-glycosylation in yvh10 is trimmed to a more human-like 5-mannose stem in engineered pichia, which may promote proper protein folding and conformational ab recognition. however, we found that pichia-produced gp120 did not display a clearly improved binding profile across a panel of bnabs (s1 fig.) . furthermore, p. pastoris-produced gp120 appeared somewhat more fragmented on a western blot, suggesting more proteolytic degradation as compared to s. cerevisiae. therefore, and because of the well-established and characterized aga1/2-based display system, s. cerevisiae was chosen as host for hiv spike protein display. design of a codon-optimized gp140 for expression in s. cerevisiae since the secretion of a full-length jr-fl gp140 including the stabilizing sos and i559p substitutions (sosip jr-fl gp140 [36, 37] ) in s. cerevisiae yvh10 had failed, we fragmented the gene and compared the level of secreted protein between a gp120 stripped core construct described previously [47] , gp120 trimmed at n-and c-termini, full-length gp120, a full-length sosip gp140, as well as gp41, and gp41 lacking its hydrophobic fusion peptide (fig. 1a) . notably, proteins were not secreted as aga2 fusions in this study. firstly, both gp120 fragments and fulllength gp120 could be secreted at clearly detectable levels, but not full-length sosip gp140. secondly, the omission of the fusion peptide clearly enhanced secretion of gp41, suggesting that its omission or modification may also improve full-length gp140 secretion. thirdly, gp120 collapsed near its expected mw of 52 kda after treatment with n-glycosidase pngase f, suggesting extensive n-glycosylation ( fig. 1b and 1c ). based on these findings, we rationally designed a jr-fl sosip-based gp140 (designed, or d-sosip) with some modifications: codons were optimized for s. cerevisiae [55] ; hydrophobic amino acids within the fusion peptide were replaced by structurally related but more hydrophilic residues, which if possible were found among circulating viral sequences (fig. 1d) ; and lastly the furin protease cleavage site was substituted with a yeast kex2 recognition site, and a predicted internal kex2 site was removed. we then compared secreted protein content in cell culture supernatant and found that d-sosip, but not the initial jr-fl sosip gp140 could be detected at significant levels. the d-sosip variant was significantly n-glycosylated, and collapsed after pngase f-treatment (s1c fig.) . the rationally designed d-sosip variant and a mutant with disrupted cd4 binding site (cd4bs)-specific ab binding (d-sosip d368r) were displayed as aga2 fusion proteins on yeast ( fig. 2a ), and compared for display level and binding to a panel of hiv bnabs together with the well-characterized and folded yu2 gp120 core [47] and an unrelated viral envelope protein (e2) derived from hepatitis c virus (hcv e2) as positive and negative controls fig. 2) . notably, gp120 core epitopes are included in d-sosip, while the gp120 core lacks many of the epitopes of d-sosip, including variable loop and gp41-associated epitopes (fig. 1a) . induced yeast cells always included a population that did not display the heterologous protein, which is a feature of the aga2 display system [42] . the d-sosip constructs showed extremely low display levels as compared to the gp120 core (fig. 2b) , and only somewhat above background binding of the cd4bs-specific vrc01 ab (fig. 2c) . however, standardizing the binding of hiv-specific abs when variable display level was taken into account lead to detectable binding across a wide range of ab specificities, including conformational, linear, and glycan-dependent sites. yeast-displayed d-sosip bound well to the v3-specific ab 447-52d and to mper abs, as might be expected due to their ability to bind to unconstrained peptides. surprisingly, low but reproducible signal was observed, suggestive of recognition by glycan and trimer-preferring antibodies such as pgt121/126 [56] and pg9/16 [24] (fig. 2d and s2 table) . furthermore, the d-sosip d368r substitution was observed to reduce binding of cd4bs antibodies. nonetheless, the low signal relative to gp120 core for many of the antibodies tested clearly indicated significant room for improvement, which might be achieved by coupling the rational design modifications made thus far with directed evolution strategies. to select for properly folded sequence variants of d-sosip, a library of 2.0x10 7 members was prepared, with each library member containing an average of about 6.7 random amino acid mutations (table 1) . conformation-dependent mabs that specifically bind to discontinuous epitopes served as molecular probes for properly folded antigens. here, the d-sosip library was extensively screened for improved binding to the conformation-dependent cd4bs bnab vrc01, which mimics the natural ligand cd4 in its mode of binding [57] . the pool of clones selected was further diversified to up to 7.7x10 7 members during two additional cycles of error prone pcr and repeated facs selection for improved binding to either vrc01 (track 1), alternating enrichment based on binding to hiv cd4bs abs vrc01, ch31 or pgv04 (track 2), or against a pooled mix of the five cd4bs mabs vrc01, pgv04, ch31, b12 and b6 (track 3), as described in table 1 . these mabs were chosen because they all bind to the conformational, conserved and functionally critical cd4bs, while being different in sequence and paratope, so as to minimize the risk of selecting specific affinity-enhancing contact residues instead of general structurally stabilizing mutations. a comparative binding analysis of the initial d-sosip and the selected pools from cycle 2 onward showed enhanced binding not only to target bnab vrc01, but also to non-target cd4bs mab b6, suggesting a generally improved structural representation of the conformational cd4bs epitope (fig. 3b) , as vrc01 and b6 significantly differ in their epitope footprints within the cd4bs [58, 59] . dna sequencing revealed that two dominating and distinct clusters of sequences had been enriched (s3a fig.) . to select for combinatorial mutants with further improved binding properties among those clusters, and to remove structurally disruptive mutations that likely had been co-selected, d-sosip and the 3.3 pool variants were dna-shuffled [60] at a 1:1 ratio, and then subjected to an additional three rounds of facs following selection tracks 1, 2 and 3. the selected 4.3 population pools showed both greatly enhanced expression and binding to cd4bs mabs vrc01 and b6 as compared to the 3.3 pool selected before dna shuffling (fig. 3) , while there was no difference in binding observed between tracks 1, 2 and 3 (s4 fig.) . a total 16 clones from each selection track were evaluated for binding to vrc01 and absence of binding to two negative control mabs. using an aggregated ranking analysis, the 12 clones with the best vrc01 and least control mab binding were further screened for broad recognition of conformational cd4bs mabs pgv04 and b6, as well as the natural ligand cd4 and gp41 membrane-proximal extracellular region mab 2f5. f425 b4e8 and mper ab 2f5, as compared to the initial d-sosip. perhaps surprisingly, improved binding to natural ligand cd4-fc was not detected, but binding was maintained at a comparable level with d-sosip (fig. 4) . to determine whether the enhancements observed in displayed variants were generalizable to secreted protein, the clone 4.3.d01 was expressed solubly. the evolved variant was subsequently compared to yeast-secreted gp120 for binding to envelope-specific antibodies. notably, reference d-sosip could not be secreted at sufficient quantities in yeast for binding analyses, in line with the extremely low display level of d-sosip observed on yeast cells (figs. 2 and 3 ). beyond enabling expression, the 4.3.d01 clone exhibited improved binding to a number of mabs, notably nih45-46 g54w, relative to gp120. (s7 fig.) , suggesting that the improved binding properties observed on yeast cells correlate with improved binding properties of soluble gp140. mapping selected mutations onto the structure of bg505 sosip.664 mapping of the mutations on the crystal structure of bg505 sosip.664 gp140 [39] showed that the mutations found in 4.3.b01 and 4.3.d01 cluster in a relatively narrow region within the v1/v2 loop region of gp120, but proximal to the cd4bs (fig. 5a) . their side chains are buried within the protein or located in small cavities and do not appear to directly contact cd4bs mabs (fig. 5b) , suggesting that structurally stabilizing mutations have been selected. interestingly, mutated residues h66q and w112r may form hydrogen bonds stabilizing helices within the inner domain of gp120. similarly, i424n may form a novel hydrogen bond with y384 (fig. 5c) . mutation n156i disrupts an n-glycosylation motif located within the v1/v2 loop region. three additional gp120 mutations that were lost after shuffling (t50a, l122f, and s209t) were all surface located, suggesting that surface-located mutations may not be essential to improved protein folding in yeast. the mutations found within gp41 could not be mapped due to disordered electron density in this particular region of the sosip crystal structure. since 4.3.b01 and 4.3.d01 showed virtually identical binding profiles and expression levels (fig. 4) , these gp41 mutations do not appear to strongly contribute to either improved protein expression or ab recognition. a key attribute of most effective anti-viral vaccine responses is development of antibodies to viral envelope proteins which result in the ability to prevent or clear viral infection [61] . to this end, induction of neutralizing antibodies capable of blocking viral infection has been a cornerstone of hiv vaccine efforts. however, hiv has an arsenal of ways to evade antibody recognition, including multiple conformational states such as closed and open, cleaved or uncleaved trimer and various monomeric spike protein forms, which are thought to distract and dilute the adaptive immune response to the infectious "native" envelope trimer [11, [62] [63] [64] . immunogen design efforts aimed to circumvent these barriers stem back to the discovery of the hiv bnab b12, and began with the identification of epitope mimetic peptides that were capable of binding to b12 with high affinity. however, vaccination with mimetic peptides failed to induce abs that bound to gp120 [65] . other more recent approaches have been based on a subtractive principle, in which the immunogenic features of the antigen which lead to nonneutralizing but immunodominant antibody responses were removed or masked with glycans [58, 66, 67] , or even more dramatically, on the direct grafting of functionally relevant sites onto alternative scaffolds [68] [69] [70] , with the goal of focusing the immune response on functionally relevant epitopes. the subtraction of alternative epitopes, however, may not actively promote a response against the desired epitope, and may rather result in a blunted immune response, in which the titer of ab recognizing the selected epitope is not enhanced, but the total antibody response is decreased. while mixed success has recently been reported from these efforts in the setting of hiv [71] , the subtraction of epitopes may unnecessarily constrain the immune response-strongly limiting potentially protective recalled t cell responses, and non-productively decreasing the polyclonality of the antibody response, resulting in reduced recruitment of effector mechanisms reliant on avid interactions with antibody opsonized cells or virus. because these effector mechanisms have a demonstrated importance in preventing acquisition, decreasing viral burden, and delaying progression [72] , the possible immunologic outcome of vaccines with reduced epitopic coverage may be dividing rather than conquering. extensive studies utilizing soluble gp120 have generally failed to provide protective ab responses, and numerous full-length and trimeric gp140-based antigens that may better resemble the structure of the native spike have also been tested [73] [74] [75] , following the rationale that any antibody recognizing the native, trimeric spike should neutralize virus [41] . sosip trimers fall into this class, and a growing number of envelope sequences with sos and ip mutations have been individually constructed and tested, culminating with the bg505 sosip.664 gp140 trimer [39] . a complementary approach to the strictly rational development of stable, uniform and wellbehaved proteins is combinatorial protein engineering, involving screening of large libraries of protein mutants displayed on viruses or cells for improved properties such as affinity, stability or protein expression. toward this end, hiv virus particles have been exposed to temperature or chemical denaturant stress and screened for retained infectivity of mammalian cells. more homogeneous and stable viruses could be selected following this procedure [76] . however, cellular protein display methods, yeast display in particular, have standing as the gold-standard for evolution of complex and glycosylated proteins based on advantages in speed, library construction, scalable selections, and ease of use. our application of this system to a designed variant of jr-fl sosip gp140 represents the first use of a high-throughput platform for whole envelope evolution. the ease with which variants with improved expression and binding to conformational hiv antibodies could be evolved from repeated rounds of random mutagenesis and flow-cytometric selection points toward promising utility. while the mannose-rich glycosylation of s. cerevesiae in theory reduces enthusiasm about this host, major classes of bnabs, including glycan-specific abs, bound to yeast-produced gp140 variants, and loss of only one n-glycosylation motif (n156i) was observed after selection. while improved binding to cd4 was not observed, the selected variants exhibited similar cd4 binding as gp120 core, and showed broadly improved binding across multiple cd4bs mabs, suggesting a generally improved structural representation of this epitope. whether the selected d-sosip variants could elicit improved neutralizing immunity when applied as immunogens is unclear. because improved antigenicity does not equate to improved immunogenicity, immunization studies would be required to evaluate their ability to elicit neutralizing antibodies. differences in glycosylation can also have a large impact on antigenicity [17] , a factor that has not been optimized in our study. importantly, while yeast utilize the same glycosylation motif, the composition of the glycan incorporated differs significantly, and could be engineered. we rather consider the methodology described here and gp140 variants as an enabling platform for future investigations towards development of improved immunogens. a panel of diverse, stabilized gp140 variants is thought to likely be required to cover circulating virus diversity, minimize immune escape by mutation and glycan masking, and elicit broad neutralization activity [15] . the nine mutations within gp120 that contributed to the enhanced properties in yeast are located in a relatively narrow region, either in proximity of the cd4bs or within the v1/v2 loop domain, as determined by homology modeling. interestingly, three of the nine mutations (h66q, w112r and i424n) may form novel hydrogen bonds. the predominantly buried locations of the mutations identified and the predicted presence of additional, buried hydrogen bonds in selected clones suggest that the poor surface display level and limited binding of yeast-displayed gp140 to conformational abs observed initially were related to poor folding of gp140 in s. cerevisiae, and that improved gp120 core packing and hydrogen bonding within the core may have helped to stabilize the protein, enhancing folding and resulting in higher display levels. in fact, the display level of proteins fused to aga2p was shown previously to correlate with their thermal stability and soluble expression levels [77] , and yeast is known to contain a chaperone machinery that can retain and degrade misfolded protein [78] . further, the accumulation of mutations within the v1/v2 loop domain suggest that this region, in addition to the gp120 core itself, had a critical impact on obstructing proper folding of gp120. this finding is further supported by the notion that yeast produced gp120 core lacking this domain binds well to cd4bs conformational abs, while full-length gp120 containing v1/v2 did not. the more hydrophilic gp41 fusion peptide has provided a critical handle to rationally improve expression of d-sosip gp140 in s. cerevisiae, however no additional expression enhancing or apparently structurally stabilizing gp41 mutations were identified in the evolved d-sosip gp140 variants. evidence as to the utility of these rational mutations can be found in the absence of reversions observed among selected clones. notably, this study does not provide any evidence for the display of gp140 trimers on yeast, which from a steric perspective seems unlikely due to clashes of the aga2 cell wall anchoring fusion partners. further, the engineering of the fusion peptide towards reduced hydrophobicity may, while being as conservative as possible, have an effect on an overall trimer structure. to address this, in future studies, a secretion-capture-based display system could be developed [79] , allowing for the evolution of trimeric gp140 spike protein libraries that are non-covalently linked to aga2. the engineered fusion peptide could gradually be mutated back to wild-type if it affects trimer structure, or fusion peptide variants could be selected from yeast-displayed libraries that improve trimer-specific antibody recognition. in summary, the d-sosip gp140 variants evolved and the platform and approach described here provide a step forward toward the development of promising novel hiv immunogens, which could be rapidly and iteratively evaluated in immunization studies in animal models. such evaluation could help elucidate the value of whole envelope strategies relative to subtractive approaches focusing on a restricted set of epitopes. a less hydrophobic fusion peptide proved to be a critical feature to achieve an initial detectable expression level allowing for yeast display-based cell sorting. the shuffling of accumulated mutations and backcrossing to wild type greatly boosted binding to the target hiv mabs and potentially eliminated deleterious mutations. variants with further improved binding to certain hiv bnabs or their germline precursors, following the concept of b-cell-lineage immunogen design [80, 81] may be evolved relatively quickly using the approach described here. glyco-engineered yeast strains [82] could serve as hosts for future yeast-based display systems to select spike protein variants homogeneously decorated with human-like glycan that is involved in binding of many bnab-classes such as pg9/16 [24] or pgt121-123 [56] . such evolved yeast-produced spike protein derivatives may also be well suited for solving additional spike protein structures, facilitating rational vaccine design approaches, and further contributing to the immunogen pipeline. general pcr products were prepared using proofreading phusion dna polymerase (finnzymes) and oligonucleotide primers from integrated dna technologies, unless otherwise stated. dna restriction and modifying enzymes were from new england biolabs. pcr products were purified and gel-extracted using kits from either qiagen or geneaid. dna sequences of all constructs were verified using an applied biosystems model 3100 dna sequencer. e. coli strain dh5 alpha was used as host for molecular cloning. yeast strains s. cerevisiae yvh10 or p. pastoris superman5 (research corporation technologies) were used as hosts for soluble protein secretion and s. cerevisiae eby100 for cell surface display. plasmids pbgp1-jrfl-gp120, pbgp1-jrfl-gp41 and pbgp1-jrfl-gp41dfp for soluble protein secretion in p. pastoris strains were prepared as follows. hiv-1 jr-fl gp120, gp41 and gp41dfp were amplified from pppi4-jrfl-sosipopt_r6 gp140 [36] using primers jrfl-for2 and jrflgp120-rev2, jrflgp41-for2 and jrfl-rev3 or jrflgp41dfp-for and jrfl-rev3 and inserted between the ecori and xbai sites of pbgp1 [83] . plasmids prs-jrfl-gp140, prs-jrfl-gp120h and prs-jrfl-gp120 for soluble protein secretion in s. cerevisiae yvh10 were prepared by amplification of gp140, gp120h or gp120 using primers jrfl-for and jrfl-rev2, jrfl-for and jrflgp120h-rev or jrfl-for and jrflgp120-rev and insertion between eagi and bamhi sites of prs-4g [47] . for plasmid prs-d-sosip, a yeast codon-optimized and designed hiv-1 jrfl gp140, denoted d-sosip, based on jrfl-sosipopt_r6 gp140 [36] was constructed (dna2.0) and inserted between eagi and bamhi sites of prs-4g. the dna sequence of d-sosip is shown in s6 fig. plasmid pcha was obtained as a kind gift from jordi mata-fink [47] . plasmids pcha-d-sosip and pcha-jrfl-gp120 for s. cerevisiae cell surface display were prepared by amplification of gp140 or gp120 from prs-d-sosip using primers gp120co-for and gp140co-rev or gp120co-rev and insertion of pcr products between nhei and bamhi sites of pcha, resulting in an orientation of ha tag, d-sosip, c-myc tag, followed by aga2p from the n to c termini, respectively. prs-d-sosip-d368r was prepared using primers jrfld368r-for and jrfld368r-rev, following the quikchange site-directed mutagenesis procedure (agilent). pcr primers are listed in s1 table. protein production and purification for secretion of soluble hiv spike protein variants lacking any cell-wall anchoring domain from p. pastoris superman5, electro-competent yeast cells were prepared as described previously [84] , transformed with pbgp1 constructs, plated on ypd plates containing 100 î¼g/l zeocin (invivo gen) and incubated at 30â°c for 3 days to obtain single clones. typically 2 to 200 ml ypd cultures containing 100 î¼g/l zeocin were inoculated to an od600 of 0.1 from over-night pre-cultures and grown for 6 h at 30â°c followed by 5 days at 20â°c for constitutive protein expression. for secretion of soluble hiv spike protein variants lacking any cell-wall anchoring domain from s. cerevisiae yvh10, chemically competent yeast cells were prepared and transformed with prs constructs using the frozen-ez yeast transformation ii kit (zymo), plated onsd-caa plates supplemented with 40 mg/l l-tryptophan and incubated at 30â°c for 3 days to obtain single clones. typically, 2 to 50 ml sdcaa cultures supplemented with 40 mg/l l-tryptophan were inoculated from over-night pre-cultures to an od600 of 0.2 and grown for 6 h at 30â°c. for induction of protein production, cells were spun down, suspended in 200 ml sgcaa medium containing 40 mg/l l-tryptophan and grown for 5 days at 20â°c. media compositions have been described previously [84] . secreted spike protein variants were either detected directly in culture supernatant or imac-purified and buffer-exchanged to pbs prior to further analysis. yeast cell surface display and flow-cytometric analysis hiv spike protein variants were displayed on s. cerevisiae eby100 as decribed previously [47] . briefly, s. cerevisiae was transformed with pcha constructs using the frozen-ez yeast transformation ii kit (zymo) and clones were grown on sdcaa plates at 30â°c. typically 2 ml sdcaa medium was inoculated to an od600 of 0.2 from an over-night pre-culture, grown at 30â°c for 6 h, transferred to sgcaa medium and grown for 12 to 18 h at 30â°c. induced cells were analyzed for cell surface display on a macsquant analyzer (miltenyi biotec). typically 10 7 yeast cells were suspended in 200 î¼l pbs supplemented with 0.1% bsa (pbsf) in 96-well plates (greiner), spun down for 4 min at 2500 g and supernatant was removed by vacuum aspiration. cells were the suspended in 50 î¼l pbsf containing 1:200 dilutions of mouse monoclonal anti-ha tag antibody ha.11 clone 16b12 (covance) or chicken polyclonal anti-cmyc tag antibody (gallus immunotechnology) for detection of display levels, and human hiv antibody at various concentration and incubated for 1 h at ambient temperature while shaking at 1000 rpm. cells were then washed twice with 200 î¼l pbsf, suspended in 50 î¼l pbsf containing 1:200 dilutions of polyclonal goat anti-mouse or goat anti-chicken antibody and goat anti-human antibody conjugated to alexa488 or alexa647 (life sciences) and incubated for 20 min at ambient temperature while shaking. cells were again washed once, suspended in 200 î¼l pbsf, respectively, and kept cold for flow-cytometric analyses. routinely, cells were gated using forward-and side scatters and median fluorescence intensities (mfis) were determined from 10,000 scattergated events that were then gated for display signal. the gate for display level was set using a non-displaying control sample, assuring that non-displaying cells are excluded. mfi values associated with antibody binding were then normalized to display level by division with the mfi associated with expression tag (nmfis). all analyses were performed using macsquantify software (miltenyi). yeast-secreted and imac purified gp120 was denatured for 5 min at 95â°c, loaded on a nupage novex 3-8% tris-acetate gel (life scienes), and 150 v were applied for 1 h in mops buffer (life sciences). the gel was stained using gelcode blue stain reagent (pierce) and analyzed using a gel doc xr system (bio-rad). for western blotting analyses of hiv spike protein variants, typically 20 î¼l of denatured and reduced crude culture supernatant were loaded and the gel was blotted for 1.5 h at 30 v on a 0.45 î¼m pore-size pvdf membrane (life sciences). the membrane was washed twice for 10 min in pbs, blocked for 1 h in pbs supplemented with 5% bsa and 0.1% v/v tween 20, washed twice for 10 min in pbs supplemented with 0.05% v/v tween 20 (pbst) and once in pbs. the washed membrane was incubated with 1 ml pbs containing 3% bsa, 0.1% v/v tween 20 and 5 î¼l goat polyclonal anti-gp120 antibody hrp conjugate ab53840 (abcam) for gp120 detection or 1 î¼l mouse monoclonal anti-his antibody hrp conjugate 34460 (qiagen) for his-tag detection while covered by a transparency. the membrane was then washed as before and incubated for 1 min with enhanced chemiluminescence substrate (pierce) and analyzed using the gel doc xr system (bio-rad). jr-fl gp140 library insert was prepared based on a protocol by fromant et al. [85] , aiming for 5 amino acid mutations per gene. a 100 î¼l reaction containing 230 î¼m datp, 200 î¼m dctp, 420 î¼m dgtp, 2.9 mm dttp, 512 pm pcha-d-sosip template dna, 500 nm primer epyd-for2 and 500 nm primer epyd-rev2, 5 î¼g/ml bsa, 3.95 mm mgcl2, 500 î¼m mncl2, 5 u taq dna polymerase (neb) in 50 mm kcl and 10 mm tris-hcl ph 8.7 at 25â°c. thermo-cycling conditions were 94â°c for 5 min, followed by 16 cycles of 94â°c for 1 min, 65â°c for 1 min and 72â°c for 4 min, and finally 72â°c for 10 min. pcr products were column-purified and 200 to 800 ng were used as template for amplification in 1000 to 4000 î¼l of total pcr reaction volume using phusion dna polymerase and primers epyd-for3 and epyd-rev3. thermocycling conditions were 98â°c for 1 min, followed by 35 cycles of 98â°c for 10 s, 60â°c for 30 s and 72â°c for 1 min, and finally 72â°c for 10 min. library vector was prepared by cleaving 250 î¼g of pcha-sosip-jrfl-gp140-design5 with 400 u of bamhi and nhei, respectively, in 1 ml final volume for 8 h at 37â°c, followed by the addition of another 400 u of each enzyme, dtt to 1 mm and incubation over night at 37â°c. the reaction was heat-inactivated for 20 min at 65â°c. vector was gel-extracted using a dna extraction maxi kit (geneaid). both insert and vector preparations were ethanol-precipitated, washed and concentrated with pellet paint co-precipitant (millipore) and suspended in a final volume of approximately 200 î¼l (insert) and 80 î¼l (vector). a total of 1200 î¼l electrocompetent s. cerevisiae eby100 were prepared essentially as described previously [84] , mixed with all insert and vector, split in eight 2 mm cuvettes and transformed at 1.2 kv and 25 uf using a gene pulser xcell (bio-rad). transformed cells were grown in typically 500 ml sdcaa medium and passaged at least once prior to induction and facs. library size was determined by plating dilutions of the transformation on sdcaa plates and counting colonies. dna encoding the enriched and designed gp140 variants was shuffled based on a protocol by stemmer [86] . briefly, 2.5 î¼g of gp140 pcr products were incubated with 1 u dnasei in 50 î¼l for exactly 2 min at 24â°c, respectively, followed by immediate heat-inactivation at 75â°c for 10 min. reactions were separated on a 2% agarose gel and fragments between 25 bp and 1 kb were gel-extracted. approximately 80 ng of digested, selected gp140 pool was assembled with 80 ng of digested, initial d-sosip in 20 î¼l reactions using phusion dna polymerase and 30 s at 98â°c followed by 40 cycles of 10 s at 98â°c, 30 s at 55â°c, 45 s at 72â°c and finally 10 min at 72â°c. exactly 2 î¼l of assembly products were amplified in 50 î¼l reactions using primers epyd-for3, epyd-rev3 and phusion dna polymerase during 1 min at 98â°c, 30 cycles of 10 s at 98â°c, 30 s at 60â°c and 1 min at 72â°c and finally 5 min at 72â°c. the assembled pcr product was ethanol-precipitated, washed, concentrated and used for library preparation as described in the previous section. freshly grown yeast libraries were diluted to an od600 of 0.2 while oversampling the library size at least 10-fold and grown for at least 6 h at 30â°c while shaking at 250 rpm. cells were sedimented, suspended in sgcaa medium and induced for at least 12 h at 30â°c. induced cells were filtered using a 40 î¼m mesh (bd biosciences) and typically 2x10 8 cells were washed 3 times with 1 ml pbsf and incubated for 1 h at ambient temperature with varying concentrations of hiv antibodies (table 1 ) and optionally a 1:200 dilution of chicken polyclonal anti-cmyc tag antibody (gallus immunotechnology) in 500 î¼l pbsf. cells were washed twice with 1 ml pbsf and stained for 20 min on ice with 1:200 dilutions of goat anti-human polyclonal antibody conjugated to alexa647 (life sciences) and optionally goat anti-chicken polyclonal antibody conjugated to alexa488 (life sciences), while shielded from light. cells were washed twice, split in four aliquots and kept as pellets on ice and shielded from light until sorting. facs was performed using an icyt sy3200 cell sorter system (sony). briefly, aliquots of typically 4x10 7 stained yeast cells were suspended in 2 ml pbsf and sorted at around 10,000 events/s using 0.1% to 5% sorting gates in recovery mode during initial and ultra purity mode during later sort cycles. typical sorting gates for initial and later selection rounds are shown in s2 fig.. sorted cells were suspended in sdcaa medium and grown for 12 to 36 h for subsequent sorting rounds or analyses of selected pools. all binding analyses were performed at 25â°c using an octet red96 system (fortebio), 1000 rpm shaking speed and pbs containing 0.1% bsa, 0.02% v/v tween20 as sample buffer. protein a-coated biosensor tips were loaded for 10 min with 10 î¼g/ml igg, dipped in buffer and probed with 500 nm gp120 for 8 min, followed by a dissociation phase of 5 min in buffer. a trace obtained from igg probed with buffer only was subtracted as reference. the gp120 crystal structure used for modeling was taken from 4nco chain a. the cd4-binding site was determined using pymol interface residues script on gp120-cd4 co-crystal structure pdb id: 1gc1 and mapped to the corresponding side chains on 4nco. mutations of d-sosip picked up during selections were mapped onto the bg505 gp140.664 sosip structure via sequence alignment. side chain rotamers with minimal clashes were chosen. hydrogen bonds were designated at distance of <4 ã� and >2 ã�. mutation d180n and those in gp41 were not mapped due to lack of resolution in the crystal structure. toward an antibody-based hiv-1 vaccine rational antibody-based hiv-1 vaccine design: current approaches and future directions induction of immunity to human immunodeficiency virus type-1 by vaccination role of complex carbohydrates in human immunodeficiency virus type 1 infection and resistance to antibody neutralization a fusion-intermediate state of hiv-1 gp41 targeted by broadly neutralizing antibodies 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display direct random mutagenesis of gene-sized dna fragments using polymerase chain reaction dna shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution the authors thank dr. rogier sanders for providing the construct encoding for jr-fl sosip gp140, dr. jordi mata-fink and dr. dane wittrup for providing yeast strains and dna constructs, dr. laura walker and dr. dennis burton, and iavi for providing hiv mabs, bryan t. rogers, weijie lin, and morgan gilman for assisting experimentally, austin boesch for producing and providing target bnab vrc01, the nih aids reagent program for providing hiv mabs, dr. karl griswold and dr. sarah dostal for access to a cell sorter and technical assistance, dr. tom scanlon for valuable discussions and dr. benjamin hackel for providing the protocol for yeast library preparation. this study was supported by a grant from the national institute of health (nih), national institutes of allergy and infectious diseases (niaid) nih 1r01ai102691 to mea. key: cord-352447-bc1pf272 authors: nishida, yu; hosomi, shuhei; yamagami, hirokazu; fujimoto, koji; nakata, rieko; itani, shigehiro; nadatani, yuji; fukunaga, shusei; otani, koji; tanaka, fumio; nagami, yasuaki; taira, koichi; kamata, noriko; watanabe, toshio; iseki, yasuhito; fukuoka, tatsunari; shibutani, masatsune; nagahara, hisashi; ohfuji, satoko; fujiwara, yasuhiro title: novel prognostic biomarkers of pouchitis after ileal pouch-anal anastomosis for ulcerative colitis: neutrophil-to-lymphocyte ratio date: 2020-10-26 journal: plos one doi: 10.1371/journal.pone.0241322 sha: doc_id: 352447 cord_uid: bc1pf272 objectives: pouchitis is a major complication after restorative proctocolectomy with ileal pouch-anal anastomosis (ipaa) in patients with ulcerative colitis (uc). although there have been many investigations of the neutrophil-to-lymphocyte ratio (nlr) in various diseases, its role in predicting the development of pouchitis remains unclear. we aimed to evaluate the clinical utility of the nlr for predicting the development of pouchitis after ipaa in uc patients. materials and methods: uc patients who underwent ipaa at osaka city university hospital between may 2006 and march 2019 were included. the incidence of pouchitis was estimated using the kaplan-meier method. potential preoperative, intraoperative, and postoperative predictors for pouchitis, including various demographic and clinical variables, were analyzed. the combined impact of the nlr and other known prognostic factors were investigated using cox proportional hazard regression with inverse probability of treatment weighting (iptw). results: forty-nine patients with uc who underwent ipaa were included. the median follow-up period was 18.3 months (interquartile range: 10.7–47.2 months). eighteen patients (36.7%) developed pouchitis. the incidence of pouchitis was 19.2%, 32.6%, and 45.9% at 1, 2, and 5 years, respectively. nlr was significantly associated with the development of pouchitis in the univariate cox regression analysis (hazard ratio (hr), 1.14; 95% confidence interval (ci), 1.01–1.28; p = 0.03). the nlr cutoff value of 2.15 was predictive of the development of pouchitis according to receiver operating characteristic analysis (specificity: 67.7%, sensitivity: 72.2%). the incidence of pouchitis was significantly lower in the low nlr group than that in the high nlr group (p = 0.01, log-rank test). cox regression analyses using iptw also identified nlr as a prognostic factor for the development of pouchitis by statistically adjusting for background factors (hr, 3.60; 95% ci, 1.31–9.89; p = 0.01). conclusions: nlr may be a novel and useful indicator for predicting the development of pouchitis after ipaa in uc and should be introduced in clinical practice. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 restorative proctocolectomy with ileal pouch-anal anastomosis (ipaa) has become a standard surgical procedure for medically intractable ulcerative colitis (uc), colitis-associated dysplasia, and cancer [1] . the most common long-term complication of this surgery is pouchitis, with a cumulative prevalence of 15%-50% [2] [3] [4] , which is one of the major factors reducing the postoperative quality of life (qol) in patients with uc [5, 6] . although the main cause of pouchitis is unclear, it is more prevalent in patients with uc than in those with familial adenomatous polyposis [1, 7] . despite conflicting results, the development of pouchitis in patients with uc, as reported in several studies, has been linked to various factors, including primary sclerosing cholangitis [2, 4] , other extraintestinal manifestations of inflammatory bowel disease [4, 8, 9] , young age at uc diagnosis [4] , preoperative terminal ileal inflammation [10, 11] , extensive colonic disease [10], presence of interleukin-1 receptor antagonist gene allele 2 [12] , total steroid dose of > 10000 mg [13] , use of infliximab [14] , neutrophil percentage of > 65% [13] , and presence of perinuclear antineutrophil cytoplasmic antibodies [15, 16] . interleukin-1 receptor antagonist gene allele 2 and perinuclear antineutrophil cytoplasmic antibodies have been reported to be useful markers for predicting the development of pouchitis; however, measuring these markers is costly and not covered by medical insurance in japan. easily accessible and low-cost markers are required to predict pouchitis in patients with ipaa. a growing body of evidence has suggested that the neutrophil-to-lymphocyte ratio (nlr) is not only an easily accessible laboratory test, but also a useful predictive parameter for various types of cancer [17] [18] [19] [20] , rheumatoid arthritis [21] , coronavirus disease 2019 [22] , and coronary heart disease [23, 24] . regarding inflammatory bowel disease, several studies have reported an association between the nlr and uc disease activity [25, 26] . we previously reported the utility of nlr as a useful prognostic marker for predicting the long-term outcomes in patients with uc treated with infliximab or tacrolimus therapy [27, 28] . lorenzo et al. reported that the nlr played a promising role as an early predictor of therapeutic response to anti-tumor necrosis factor (tnf) therapy in patients with uc [29] . however, no studies have tested the value of this parameter in predicting the development of pouchitis after ipaa in patients with uc. therefore, our study aimed to evaluate the clinical utility of the nlr for predicting the development of pouchitis after ipaa in patients with uc. rectal inflammation, vital signs, anemia, nutritional status, and age. the two-stage operation starts with total proctocolectomy and ipaa, along with a diversion ileostomy construction in the first stage and ends with ileostomy closure in the second stage. the three-stage operation starts with subtotal colectomy, along with ileostomy construction and sigmoid mucous fistula formation, in the first stage, followed by remnant proctocolectomy and ipaa, with a reconstruction of the ileostomy in the second stage. the procedure is completed with ileostomy closure in the third stage [30] . patients with one-stage surgery were excluded because we analyzed postoperative nlr. all patients were followed up with a physical examination and a blood test. the differential white blood cell (wbc) count was analyzed using an xe-5000 hematology analyzer (sysmex, kobe, japan), as per the manufacturer's protocol. in patients undergoing one-stage surgery, the nlr was calculated from a blood sample measured before ipaa by dividing the absolute neutrophil count by the absolute lymphocyte count. patients were followed up from the time of ipaa to the onset of pouchitis, loss to follow-up, or until the end of march 2020. the nlr was calculated from a blood sample measured before stoma closure. patients were followed up from the time of stoma closure to the onset of pouchitis, loss to follow-up, or until the end of march 2020. the pouchitis disease activity index (pdai) is a 19-point index of pouchitis activity based on clinical symptoms as well as endoscopic and histologic findings [31] . in this study, pouchitis was diagnosed based on the modified pouchitis disease activity index (mpdai) score using a combination of clinical symptoms and endoscopic examination. an mpdai score of � 5 points was used to define pouchitis in this study [32] . the primary outcome measure of this study was the onset of pouchitis. potential preoperative, intraoperative, and postoperative predictors for pouchitis, including various demographic and clinical variables, were analyzed. continuous variables are presented as medians and interquartile ranges. the differences in clinical characteristics were compared using either the chi-square test or fisher's exact test for categorical variables and the mann-whitney u-test for continuous variables. receiver operating characteristic (roc) curves were plotted so that the area under the roc curve could be calculated. optimal cutoff values were determined according to the youden criterion, which marks the point on a roc curve where "sensitivity + specificity-1" is maximal [33] . the cumulative incidence of pouchitis was illustrated using a kaplan-meier plot. differences in the survival curves were assessed using the log-rank test. furthermore, continuous values of laboratory data were evaluated using the cox proportional hazard model. data are presented as hazard ratios (hrs) with 95% confidence intervals (cis). an iptw analysis was applied to each observation in the cox model to assess the relationship between the nlr and the development of pouchitis. the iptw analysis was derived using propensity scores on all observations before matching to reduce selection bias by statistically adjusting for background factors [34] . variables included in the iptw analysis were age, sex, disease location, and history of anti-tnf therapy. a p-value of < 0.05 was considered statistically significant. all statistical analyses were performed with ezr (saitama medical center, jichi medical university), a graphical user interface for r (the r foundation for statistical computing, version 2.13.0). more precisely, it is a modified version of r commander (version 1.6-3) that includes statistical functions frequently used in biostatistics. this study was approved by the osaka city university hospital certified review board; (no. 4291), which waived the requirement for written informed consent because the analysis used anonymized clinical data that were retrospectively obtained after each patient agreed to receive the treatment. all data were fully anonymized before we accessed them. nevertheless, all patients were notified of the content and information of this study and given the opportunity to refuse participation. none of the patients refused participation. this study followed the ethical guidelines for medical and health research involving human subjects established by the ministry of education, culture, sports, science and technology and the ministry of health, labor and welfare in japan. overall, we included 79 patients who underwent ipaa for uc during the study period. twenty-nine patients were excluded due to a lack of data on differential wbc count. one patient with one-stage surgery was excluded. forty-nine patients were retrospectively reviewed. of those patients, 30 patients underwent ipaa for disease refractory to medication, 9 patients for dysplasia or cancer, 3 patients for perforation, 3 patients for toxic megacolon, 2 patients for colonic strictures, and 2 patients for massive bleeding. the median follow-up period was 18.3 months (interquartile range: 10.7-47.2 months). eighteen patients (36.7%) developed pouchitis. the incidence of pouchitis was 19.2%, 32.6%, and 45.9% at 1, 2, and 5 years, respectively (fig 1) . the demographic characteristics of the patients are summarized in table 1 . nlr was significantly associated with the development of pouchitis according to univariate cox regression analysis (hr, 1.14; 95% ci, 1.01-1.28; p = 0.03). no other clinical variables such as age, sex, disease duration, age at onset, or disease location have shown a statistically significant association with pouchitis development (table 2) . when the nlr was examined as a dichotomous variable, a cutoff value of the nlr for the risk of pouchitis was determined using roc analysis. from the youden index, the roc analysis showed that the best cutoff value for the nlr was 2.15 (specificity: 67.7%, sensitivity: 72.2%) (fig 2) . therefore, a cutoff value of 2.15 was chosen for further study. twenty-six (53.1%) patients had an nlr of < 2.15 (low nlr group), whereas 23 (46.9%) patients had an nlr of � 2.15 (high nlr group). table 3 shows a comparison of the baseline characteristics between the low nlr (< 2.15) and high nlr (� 2.15) groups. no significant differences were noted in the background characteristics between the two groups. fig 3 shows a comparison of the cumulative incidence of pouchitis between the low nlr group and the high nlr group. the incidence of pouchitis was significantly lower in the low nlr group than in the high nlr group (p = 0.01, log-rank test). univariate cox regression analysis also identified high nlr as a significant risk factor for the onset of pouchitis (unadjusted hr, 3.45; 95% ci, 1.23-9.71; p = 0.02). therefore, to elucidate the influence of reported risk factors, cox regression analysis was performed using the iptw method to identify factors associated with the development of pouchitis. variables included in the iptw analysis were age, sex, disease location, and history of anti-tnf therapy. cox regression analyses using the iptw method identified nlr as a prognostic factor for the development of pouchitis by statistically adjusting for background factors (adjusted hr, 3.60; 95% ci, 1.31-9.89; p = 0.01). as drug discontinuation during the course may occur in a number of situations, including a history of anti-tnf therapy as an adjusting background factor in iptw analysis might not be appropriate in this study due to several biases. therefore, we also performed iptw analysis without a history of anti-tnf-therapy as an adjusting background factor. cox regression analyses using the iptw method, excluding the history of anti-tnf therapy as an adjusting background factor, also identified nlr as a prognostic factor for the development of pouchitis (adjusted hr, 3.67; 95% ci, 1.38-9.79; p = 0.01). in this study, we investigated the utility of nlr for the development of pouchitis after ipaa in patients with uc. our results suggest that a high nlr is strongly associated with an increased risk of developing pouchitis, and patients with high nlr should be followed up carefully. the mechanism by which the nlr predicts the development of pouchitis is unclear. regarding neutrophils count, neutrophils are important leukocytes that can cause inflammation in uc [35] . neutrophil accumulation and abscess formation within the intestinal crypts at the apical epithelial surface are typical pathological features of uc [36] . patients with high peripheral neutrophil count may be prone to pouchitis. indeed, patients who had higher neutrophil counts tended to develop pouchitis in this study, in line with a previous report by koike et al. that identified a neutrophil percentage of > 65% before ipaa surgery as a risk factor for pouchitis [13] . regarding lymphocyte count, hirata et al. reported significantly increased numbers of cd19 + cd138 + cells in the pouchitis mucosa of patients with uc compared to non-inflamed uc pouches. the proliferation of this cell population suggests the possibility of involvement of a uc-derived abnormality in the pathogenesis of pouchitis [37] . the number of cd19 + cd20 -cd138 + cells, which are an immature subset of igg-producing plasma cells, was also significantly higher in the peripheral blood and inflamed colon of patients with active uc [38] [39] [40] . this cell subset was reported to have a feature of plasma cells; eccentrically located nuclei and abundant rough endoplasmic reticula, by immunoelectron microscopy [39] . therefore, this cell population could be identified as a highly fluorescent population distinct from lymphocytes by a hematology analyzer, implying that uc patients with abundant cd19+cd138+ cells would have a relatively low percentage of lymphocytes in peripheral blood. this might be one of the possible mechanisms to explain how the nlr could predict the development of pouchitis. another possible mechanism is that peripheral wbcs might affect the gut microbiome. the role of the gut microbiome in pouchitis development is indicated by the effectiveness of antibiotics in the treatment of pouchitis, the use of probiotics for its prevention [41, 42] , and the influence of innate lymphoid cells on the microbiome [43] . taken together, patients with higher nlr might consist of a unique subset whose microbiome predisposes them to pouchitis. johnson et al. have reported that fecal calprotectin correlates with the severity of pouchitis and is a useful marker for diagnosing pouchitis [44] . measuring fecal calprotectin is non-invasive, relatively cheap, and its sensitivity and specificity are very high. although the fecal calprotectin test is a potentially useful screening tool for estimating the presence and severity of pouch inflammation, no evidence has been reported for its predictive role in inflammation. regarding nlr, the nlr has been found to predict pouchitis before the operation. regarding medications before the operation, only a total steroid dose of > 10000 mg [13] and the use of infliximab [14] was reported to be a risk factor for pouchitis, to the best of our knowledge. in this study, although the use of immunomodulators, corticosteroids, anti-tnf-α antibody therapy, and calcineurin inhibitor therapy was not identified a risk factor for pouchitis, we could not say that these medications were not associated with the development of pouchitis because of the relatively small sample size and several biases such as drug discontinuation during the course. as the sample size of this study was relatively small, we calculated the required sample size. sample size calculation as a post-hoc analysis indicated that a total of 38 patients were required to detect a significant association between high nhr and the development of pouchitis with the following assumptions: an α level of 0.05, a β level of 0.20, half of the patients were allocated to the high nlr group, the incidences of pouchitis in patients with high and low nlr group were 54% and 19%, registration period was 13 years, and the follow-up period was 1.5 years. therefore, the sample size in this study was satisfied to examine the association between nlr and the development of pouchitis. this study has some limitations. first, this was a retrospective study with a relatively small cohort that is susceptible to bias in data selection and analysis. second, we could not evaluate the predictive value of preoperative nlr since differential wbc count is not routinely measured in all patients just before the operation. only 39 of 49 patients included in this study had their differential white blood cell count evaluated before the operation. among these 40 patients, univariate cox regression analysis did not identify nlr as a predictor of pouchitis development (hr, 0.98; 95% ci, 0.93-1.04; p = 0.56). a possible reason why preoperative nlr did not predict the development of pouchitis is that preoperative nlr would be affected by infection or treatment and, therefore, would not accurately reflect the situation. the other possible reason is that the number of patients analyzed for preoperative nlr was statistically too small. therefore, we may not be able to conclude that the preoperative nlr is meaningless for pouchitis. third, we were unable to evaluate the reported pouchitis predictive factors, such as extraintestinal manifestations of inflammatory bowel disease, preoperative terminal ileal inflammation, the presence of interleukin-1 receptor antagonist gene allele 2, total steroid dose of >10000 mg, or presence of perinuclear antineutrophil cytoplasmic antibodies owing to the retrospective design of the study. furthermore, nlr can be influenced by concurrent infections and concomitant drugs. therefore, further large prospective studies will help confirm the nlr as a key predictor for pouchitis after ipaa in patients with uc. despite these limitations, our study suggests that the nlr could be associated with the development of pouchitis after ipaa in patients with uc. nlr should, therefore, be introduced in clinical practice. conceptualization: yu nishida, yasuhiro fujiwara. ileal pouch-anal anastomoses complications and function in 1005 patients pouchitis after ileal pouchanal anastomosis for ulcerative colitis occurs with increased frequency in patients with associated primary sclerosing cholangitis prospective study of the incidence, timing and treatment of pouchitis in 104 consecutive patients after restorative proctocolectomy pouchitis following pelvic pouch operation for ulcerative colitis. incidence, cumulative risk, and risk factors. diseases of the colon and rectum comprehensive evaluation of inflammatory and noninflammatory sequelae of ileal pouch-anal anastomoses. the american journal of gastroenterology quality of life after ileal pouchanal anastomosis: an evaluation of diet and other factors using the cleveland global quality of life instrument diagnosis and management of pouchitis pouchitis and extraintestinal manifestations of inflammatory bowel disease after ileal pouch-anal anastomosis perinuclear anti-neutrophil cytoplasmic antibody and refractory pouchitis. a case-control study. digestive diseases and sciences assessment of neutrophil-lymphocyte ratio in ulcerative colitis: a promising marker in predicting disease severity. clinics and research in hepatology and gastroenterology pubmed central pmcid: pmc5226844 faculty members of a course sponsored by eisai co., ltd. the other authors declare that they have no conflicts of interest, and that there were no external funding sources for this study pretreatment neutrophil-to-lymphocyte ratio predicts clinical relapse of ulcerative colitis after tacrolimus induction pubmed central pmcid: pmc6405082 were previously faculty members of a course sponsored by eisai co., ltd, up to last year. they are now no longer faculty members of this course. this does not alter our adherence to plos one policies on sharing data and materials novel prognostic biomarkers of mucosal healing in ulcerative colitis patients treated with anti-tnf: neutrophil-to-lymphocyte ratio and platelet-to-lymphocyte ratio. inflammatory bowel diseases history of and current issues affecting surgery for pediatric ulcerative colitis pouchitis after ileal pouch-anal anastomosis: a pouchitis disease activity index modified pouchitis disease activity index: a simplified approach to the diagnosis of pouchitis regret graphs, diagnostic uncertainty and youden's index. statistics in medicine survival analysis using inverse probability of treatment weighted methods based on the generalized propensity score the role of phagocytes in inflammatory bowel disease inflammatory bowel disease: prevalence and level of activation of circulating t-lymphocyte subpopulations mediating suppressor/cytotoxic and helper function as defined by monoclonal antibodies proliferation of immature plasma cells in pouchitis mucosa in patients with ulcerative colitis. inflammatory bowel diseases increased numbers of immature plasma cells in peripheral blood specifically overexpress chemokine receptor cxcr3 and cxcr4 in patients with ulcerative colitis infiltration of cd19+ plasma cells with frequent labeling of ki-67 in corticosteroid-resistant active ulcerative colitis. virchows archiv: an international journal of pathology mucosal cxcr4+ igg plasma cells contribute to the pathogenesis of human ulcerative colitis through fcγr-mediated cd14 macrophage activation writing -review & editing: shuhei hosomi, yasuhiro fujiwara. key: cord-349911-dx8wvqkm authors: dahl, viktor; tegnell, anders; wallensten, anders title: communicable diseases prioritized according to their public health relevance, sweden, 2013 date: 2015-09-23 journal: plos one doi: 10.1371/journal.pone.0136353 sha: doc_id: 349911 cord_uid: dx8wvqkm to establish strategic priorities for the public health agency of sweden we prioritized pathogens according to their public health relevance in sweden in order to guide resource allocation. we then compared the outcome to ongoing surveillance. we used a modified prioritization method developed at the robert koch institute in germany. in a delphi process experts scored pathogens according to ten variables. we ranked the pathogens according to the total score and divided them into four priority groups. we then compared the priority groups to self-reported time spent on surveillance by epidemiologists and ongoing programmes for surveillance through mandatory and/or voluntary notifications and for surveillance of typing results. 106 pathogens were scored. the result of the prioritization process was similar to the outcome of the prioritization in germany. common pathogens such as calicivirus and influenza virus as well as blood-borne pathogens such as human immunodeficiency virus, hepatitis b and c virus, gastro-intestinal infections such as campylobacter and salmonella and vector-borne pathogens such as borrelia were all in the highest priority group. 63% of time spent by epidemiologists on surveillance was spent on pathogens in the highest priority group and all pathogens in the highest priority group, except for borrelia and varicella-zoster virus, were under surveillance through notifications. ten pathogens in the highest priority group (borrelia, calicivirus, campylobacter, echinococcus multilocularis, hepatitis c virus, hiv, respiratory syncytial virus, sarsand mers coronavirus, tick-borne encephalitis virus and varicella-zoster virus) did not have any surveillance of typing results. we will evaluate the possibilities of surveillance for the pathogens in the highest priority group where we currently do not have any ongoing surveillance and evaluate the need of surveillance for the pathogens from the low priority group where there is ongoing surveillance in order to focus our work on the pathogens with the highest relevance. national public health agencies need to prioritize their activities as there are limited resources for surveillance of on communicable diseases. established routines, personal interests and short-term political agendas can lead to a situation where resources are spent on surveillance of pathogens with less relevance for public health. the public health agency of sweden identified the need to use a structured method that takes relevant aspects into account in order to rationally prioritize between different pathogens when allocating resources for surveillance. in the literature we found several attempts to create frameworks and procedures for prioritization in public health [1] [2] [3] [4] [5] . some attempts have also been made to prioritize among pathogens causing communicable diseases [6, 7] . the method we identified to be most appropriate to our needs was one that was developed at the robert koch institute in germany in 2011 [8] [9] [10] . the robert koch institute started by generating a list of pathogens to prioritize and included pathogens that were a) notifiable according to german law, b) reportable within the european union, c) reportable to the world health organization under the international health regulations, d) agents with potential for deliberate release, and e) pathogens represented in the "control of communicable disease manual" by heymann et al. [11] occurring in germany. some pathogens were then grouped together when biologically and clinically plausible. the robert koch institute invited ten senior external experts and ten internal experts and asked them to score the pathogens with -1, 0 or 1 for ten variables"incidence", "work and school absenteeism", "health care utilization", "chronicity of illness or sequelae", "case fatality rate", "proportion of events requiring public health actions", "trend", "public attention", "prevention and treatment possibilities") ( table 1 ). the scoring was done in a modified delphi process consisting of two rounds. first, the panel of experts received the list of pathogens they should score. they then gave their score and motivation for the score given. the scores and motivations were anonymized and sent out in a second round so each participant could change their answers in light of the other experts' replies. this was done so that the panel could reach a consensus without letting "group dynamics" affect the process. internal and external experts were then asked to give a weight of 0 to 10 for each criterion and the median value was then used to give each criterion a weight. the score for each pathogen was multiplied by the weight and re-scaled from 0-100. the pathogens were divided into four priority groups (the highest, high, medium and low) based on their final score. the cut-off limits for the groups were 76-100, 51-75, 26-50, and 0-25. we made some modifications to the prioritization method and then applied it in sweden in order to generate a prioritized list of pathogens. we then measured the current allocation of resources for surveillance at the public health agency of sweden in order to examine to what degree resources were spent after public health relevance. we used the methodology developed at the robert koch institute [8] , with some modifications as described below. we started with the list of pathogens that had been used during the prioritization procedure in germany. we then distributed the list among experts at the public health agency of sweden (at the time "the swedish institute for communicable disease control"). experts at each unit at the institute were allowed to make additions to the list if they deemed it to be necessary. after additions we then removed pathogens where spread is not ongoing, or deemed not likely to occur, in sweden. this gave us the final list of pathogens to prioritize. we used the same scoring criteria as in the original method with some modifications. the criterion for incidence was changed to include not only symptomatic infections but also asymptomatic infections (but not asymptomatic colonization with bacteria). we also changed the table 1 . prioritization variables. the ten variables that pathogens were scored for during the prioritization and the criteria for each score. scoring values need for medical treatment is established but currently no effective treatment is available or amr limits treatment options amr = anitimicrobial resistance note 1. all criteria apply to the geographical settings where the prioritization is conducted; the time-frame applicable to the requested epidmiological data should be defined prior to the process initiation and depends on the frequency with which pathogens are planned to be re-scored. indicated numercial thresholds apply to the country where the prioritization is conducted: in other geographical settings different thresholds might need to be considered. note 2. event is defined as the occurrence of a disease that is unusual with respect to a particular time, place or circumstances. for certain infectious diseases one case may be sufficient to constitute an event (e.g. polio virus). public health actions are any kind of targeted actions aiming to identify the nature of the event and/or to apply control measures in response to the event occurrence. *assessed against the total burden of infectious diseases. **assessed for each particular pathogen in question, e.g., the criterion "treatment possibilities and needs" refers to availability and adequacy of treatment for each case of an illness caused by a particular pathogen but does not take into account the incidence of illnesses or the availability of preventive measures. doi:10.1371/journal.pone.0136353.t001 criteria of "chronicity of illness and sequelae" from the contribution of a pathogen to the total amount of chronicity or sequelae to "the risk for each pathogen to cause chronicity and sequelae" (table 1) . five experts were invited to participate in the scoring procedure. one expert from the national board of health and welfare, one from the public health agency of sweden and three experts who were county communicable disease control officers. we sent out the pathogens for scoring in batches of around ten pathogens in each batch. the weighting step was removed. this was the other modification of the german method in addition to those mentioned in the "scoring" paragraph. as in the original method, the scores for each variable for each pathogen were summarized and the score for the pathogen was then re-scaled to 1-100 in order to reach the final score. we then compared the outcome of the prioritization process in sweden to the outcome in germany by examining which (if any) pathogens were prioritized more than one group higher or lower in the swedish list compared to the german list. for those pathogens we examined how the scores for each variable differed. in order to examine how resources are currently allocated in relation to the outcome of the prioritization procedure, we selected three key indicators. first, we compiled a list of all pathogens that are currently under surveillance by the public health agency of sweden through mandatory and/or voluntary notifications of cases by clinicians and/or laboratories. we did not include systems for "event-based surveillance". even if only one manifestation of the pathogen (e.g. invasive infection or strains with antibiotic resistance) was under surveillance, that pathogen was considered to be under surveillance. in addition, according to swedish law, all cases of viral meningoencephalitis are notifiable. thus we considered the viruses that can cause viral meningoencephalitis to be under surveillance. second, we asked all epidemiologists responsible for surveillance of a particular pathogen at the national public health agency to estimate how many full-time equivalents (ftes) (1 year of full time work = 1 fte) that were spent on surveillance activities for each pathogen, as an average over a year (if several people worked with one pathogen their work time was combined). time spent on surveillance of typing results was not included in this estimation. third, we compiled a list of all pathogens that were under surveillance through typing (e.g. sequencing, pulsed-field gel electrophoresis and resistance patterns for antibiotics) of all isolates or a sample of isolates depending on the pathogen. either through collection of typing results from the laboratories at the county level or where isolates were collected and typed at the public health agency of sweden. this work was considered to be part of the duties of the public health agency of sweden and clearance was given by the public health agency of sweden before the study was started. the study did not involve human subjects. experts in infectious diseases were consulted as part of their job as communicable disease officers. using the process described in the method section "selection of pathogens" we generated a list of 106 pathogens. this list of 106 pathogens were distributed in batches to the experts for scoring. after scoring and re-scaling of the scores 21 pathogens were in the highest priority group, 28 in the high priority group, 37 in the medium priority group and 20 in the low priority group ( table 2 and s1 table) . in sweden 106 pathogens were prioritized compared to 127 in germany. twelve pathogens were prioritized in sweden but not in germany and 31 pathogens were prioritized in germany but not in sweden. three pathogens differed by more than one group when comparing the swedish to the german list ( table 2) . borrelia was placed in the highest priority group in sweden but in the medium priority group in germany. staphylococcus epidermidis (coagulase-negative staphylococcus) was placed in the medium priority group in sweden but in the highest priority group in germany. hepatitis e virus was placed in the lowest priority group in sweden but in the high priority group in germany. borrelia differed in the scores for "chronicity of illness or sequelae", s. epidermidis differed in both "incidence" and "chronicity of illness or sequelae" and hepatitis e differed in "incidence" when comparing the swedish to the german scoring for each variable (data not shown). at the national public health agency of sweden we currently conduct surveillance based on mandatory notifications for 59 of the pathogens and on voluntary notifications for 5 pathogens, of the 106 pathogens that were prioritized ( table 2) . of the 63 pathogens under surveillance through notification of cases 19 (30%) were in the highest priority group, 19 (30%) in the high priority group, 17 (27%) in the medium priority group and 8 (13%) in the low priority group ( table 2) . two pathogens, (borrelia and varicellazoster virus) were in the highest priority group but did not have any ongoing surveillance through notification of cases. eight pathogens under surveillance through notifications of cases were placed in the low priority group (atypical mycobacteria, chlamydiophila psittacci, entamoeba histolytica, hepatitis e virus, jc-virus, salmonella typhi and paratyphi, vibrio cholerae and vibrio non-cholerae). work-time for epidemiologist for surveillance corresponded to 7.0 fte per year (this was however distributed among more epidemiologist than 7 since some work part-time and for some not all of their work time is dedicated to surveillance related activities) (s2 table) . 4.5 fte:s (63%) of the ftes were spent on pathogens in the highest priority group. 2.2 fte:s (31%) were spent on pathogens in the high priority group and 0.3 fte:s (4%) were spent on pathogens in the medium priority group. of the 28 pathogens under surveillance through typing 11 (39%) where in the highest priority group, 13 (46%) were in the high priority group, 3 (11%) in the medium priority group and 1 (4%) were in the low priority group ( table 2 ). ten pathogens in the highest priority group did not have any surveillance through typing (borrelia, campylobacter, calicivirus, e. multilocularis, hepatitis c virus, human immunodeficiency virus, respiratory syncytial virus, sarsand mers coronavirus, tick borne encephalitis virus and varicella-zoster virus). in the low priority group there was only surveillance through typing for salmonella typhi and paratyphi. we used a standardized procedure developed at the robert koch institute to generate a list of pathogens prioritized for surveillance to be used by the public health agency of sweden. before applying the robert koch institute prioritization procedure in sweden we made two modifications in order to make the procedure less time consuming. first, we excluded pathogens where there was no ongoing spread in sweden or where spread in sweden was not deemed likely. second, we excluded the weighting step described in the original procedure. the reasons for excluding the weighting step was that in the experience from germany for some variables, such as "public attention" there was a high variation in the weight given by different experts. in addition for some variables the weight given differed between different groups of experts in germany. for example "incidence" was given a high weight by epidemiologists and public health experts but a low weight by clinicians. the relationship was reversed for the criteria "work and school absenteeism" and "health care utilization". the number of experts invited from each category therefore affected the weighting score. due to these limitations and in order to make the procedure less time consuming we decided to remove the weighting step. we also made modifications to the scoring criteria for two of the ten variables. we changed the criteria for "incidence" so that it would also include asymptomatic infections. we made this change since even asymptomatic infections can require interventions such as contact tracing and vaccination of those exposed. in addition, the swedish sureveillance system for notifiable diseases does not include data on whether the infection was symptomatic or not. thus, there is a lack of epidemiological data in sweden that differentiates between symptomatic and asymptomatic infections. we also modified the criteria for chronicity of illness and sequelae from the contribution of a pathogen to the total amount of chronicity or sequelae for all pathogens combined to the individual risk for each pathogen to cause chronicity and sequalae. this was done in order to give the incidence of a pathogen less influence as incidence was already included as a separate variable. only three of the pathogens (borrelia, s. epidermidis and hepatitis e virus) that were included in the prioritization procedure in both sweden and germany differed by more than one priority group. to some extent this difference could have been due to changes in the scoring criteria for the variables "incidence" and for "chronicity of illness or sequelae", but this was likely not the entire explanation to the differences in priority groups for these pathogens. true differences in incidence could explain why "work and school abseentism" and "health care utilization" was scored lower in sweden than in germany for s. epidermidis. the incidence for borrelia could also be higher in sweden which could have affected the scoring for "incidence" and "chronicity of illness or sequelae". public and media attention probably also differ between countries, which could explain why "public attention" for s. epidermidis and hepatitis e scored lower in sweden than in germany. lower scores for "prevention possibilities and needs" and "treatment possibilities and needs" for hepatitis e in sweden than in germany more likely reflect a difference in the interpretation of the available evidence on if and how this infection should be treated than a real difference in the available interventions for prevention or treatment between sweden and germany. we compared the ongoing surveillance at the public health agency of sweden to the prioritized list of pathogens which gave a useful indication on how well resources were currently allocated. when comparing the existing surveillance through notifications in sweden to the priority groups we found that the public health agency of sweden did not have any surveillance through notifications for two pathogens in the highest priority groups (borrelia and varicellazoster virus). we found that for eight pathogens in the low priority group (mycobacterium non-tuberculosis, chlamydophila psittaci, entamoeba histolytica, hepatitis e virus, jc-virus, salmonella typhi and paratyphi, vibrio cholerae and vibrio non-cholerae) there was ongoing surveillance through notifications. this could partly be explained by the law requiring all viral meningoencephalitis to be under surveillance, thus jc virus is under surveillance. for salmonella our method divided salmonella spp and salmonella typhi and non-typhi but the law requires surveillance for all forms of salmonella which could explain why salmonella typhi and non-typhi was under surveillance although it was considered to be of low relevance to public health. when we estimated ftes for surveillance through notifications per pathogen we found that 95% of ftes were spent on surveillance through notifications for pathogens in the highest and the high priority group. we take this as an indication that surveillance through notifications at the public health agency of sweden is already focused on the most important pathogens. we also compared the existing surveillance through typing results in sweden to the different priority groups. the surveillance through typing was to a large extent focused on the highest and the high priority group. however, ten pathogens in the highest priority group lacked programs for surveillance through typing (borrelia, campylobacter, calicivirus, e. multilocularis, hepatitis c, human immunodeficiency virus, respiratory syncytial virus, sars-and mers coronavirus, tick borne encephalitis virus and varicella-zoster virus). reasons for this could be that in the past, suitable methods for typing for some of the pathogens were lacking, but as new techniques such as whole-genome sequencing become less expensive, this might change. another reason for lack of surveillance through typing be due to the pathogenesis of the disease caused by the pathogen under surveillance. for example, when the symptoms for tick-borne encephalitis virus are seen, the virus is usually no longer detectable and isolation for typing is therefore not possible. discrepancies between ongoing surveillance through notifications and surveillance through typing and the outcome of the prioritization procedure should be interpreted with caution since there might be good reasons for not having surveillance for the pathogens in the highest priority group or for having surveillance for pathogens in the low priority group. the results should mainly function as an indication that the need for surveillance, or lack of surveillance, for certain pathogens should be evaluated. a key aspect to evaluate would be the availability of actions that could be triggered by the data from the surveillance system. this study had limitations. despite the modifications to the procedure, the delphi process was very time consuming and took over a year to carry out. apart from the associated costs, it could have led to "expert fatigue" and a changing interpretation of the ranking criteria over time. another limitation of this study is that we estimated full-time equivalents for surveillance through notifications per pathogen by self-assessment. this might not accurately reflect the actual time spent on surveillance through notifications for over a year. for example surveillance activities with a yearly variation (e.g. for influenza virus) might be overestimated if the assessment takes place during the influenza season and vice versa. in addition to that, ftes per pathogen also depend on the efficiency of the surveillance system. a poorly designed surveillance system might require more time spent than several well designed systems. we did not have the possibility to assess ftes for surveillance of typing results. furthermore, we did not measure the resources spent on research activities at the public health agency of sweden. it would be of interest to find out how they are distributed among the priority groups. in conclusion we have generated a list of pathogens prioritized for surveillance that can be used by the public health agency of sweden. the list could also be used when prioritizing among public health research projects on communicable diseases. we have made the prioritization method less time consuming when comparing the prioritized list to the current activities at the public health agency of sweden we found that to a large extent our activities are already focused on the pathogens with higher priority. we did however identify pathogens where surveillance (or discontinuation of surveillance) should be evaluated. the public health agency of sweden will consider surveillance for the pathogens in the highest priority group where we currently do not have any ongoing surveillance and to evaluate the possibilities to stop the surveillance for the pathogens from the low priority group where there is ongoing surveillance. in addition the results indicate that other countries with a similar panorama of communicable diseases and the same level of health care as in sweden and germany could consider using the list from either sweden or germany in order to save time. for countries who plan to conduct the prioritization method we recommend to consider additional modifications of the prioritization method in order to make it less time consuming, such as replacing the delphi procedure with a "mini-delphi" that can take place during a one-day workshop, which has previously been done in other settings [12] . supporting information s1 table. scores for each pathogen and all variables. individual score, total score, re-scaled score and priority group for all pathogens prioritized. sweden, 2013. (docx) s2 table. full-time equivalents spent on surveillance for each pathogen. self-estimated time spent on surveillance of notifications at the public health agency of sweden for all pathogens that were prioritized. sweden, 2013. (docx) setting priorities for research. health policy on being a good listener: setting priorities for applied health services research. the milbank quarterly evaluating priority setting success in healthcare: a pilot study. bmc health services research priority setting: what constitutes success? a conceptual framework for successful priority setting. bmc health services research a checklist for health research priority setting: nine common themes of good practice establishing priorities for national communicable disease surveillance. the canadian journal of infectious diseases strategic emphases for tropical diseases research: a tdr perspective communicable diseases prioritized for surveillance and epidemiological research: results of a standardized prioritization procedure in germany prioritisation of infectious diseases in public health-call for comments. euro surveillance: bulletin europeen sur les maladies transmissibles = european communicable disease bulletin how can infectious diseases be prioritized in public health? a standardized prioritization scheme for discussion control of communicable disease manual a standardized process for developing a national notifiable diseases list in a pacific island setting. asia-pacific journal of public health / asia-pacific academic consortium for public health we want to express our gratitude to drs leif dotevall, anders lindberg, gunnar nylã©n and arne runehagen who participated in the scoring and to dr marion muehlen for valuable comments on the manuscript. we also want to than dr yanina balbanova from the robert koch institute, germany, for kindly providing us with data from the prioritization procedure carried out in germany. conceived and designed the experiments: vd at aw. performed the experiments: vd at aw. analyzed the data: vd aw. wrote the paper: vd at aw. key: cord-354052-x4ckzw64 authors: li, chunhua; li, zhen; zou, yong; wicht, oliver; van kuppeveld, frank j. m.; rottier, peter j. m.; bosch, berend jan title: manipulation of the porcine epidemic diarrhea virus genome using targeted rna recombination date: 2013-08-02 journal: plos one doi: 10.1371/journal.pone.0069997 sha: doc_id: 354052 cord_uid: x4ckzw64 porcine epidemic diarrhea virus (pedv) causes severe economic losses in the swine industry in china and other asian countries. infection usually leads to an acute, often lethal diarrhea in piglets. despite the impact of the disease, no system is yet available to manipulate the viral genome which has severely hampered research on this virus until today. we have established a reverse genetics system for pedv based on targeted rna recombination that allows the modification of the 3′-end of the viral genome, which encodes the structural proteins and the orf3 protein. using this system, we deleted the orf3 gene entirely from the viral genome and showed that the orf3 protein is not essential for replication of the virus in vitro. in addition, we inserted heterologous genes (i.e. the gfp and renilla luciferase genes) at two positions in the viral genome, either as an extra expression cassette or as a replacement for the orf3 gene. we demonstrated the expression of both gfp and renilla luciferase as well as the application of these viruses by establishing a convenient and rapid virus neutralization assay. the new pedv reverse genetics system will enable functional studies of the structural proteins and the accessory orf3 protein and will allow the rational design and development of next generation pedv vaccines. porcine epidemic diarrhea virus (pedv) causes diarrhea and dehydration in newborn piglets. the virus infects the epithelial cells of the small intestine resulting in severe mucosal atrophy and consequent malabsorption. pedv is common and the cause of serious problems, particularly in pigs in asia. the disease usually appears in winter during which it can cause high fatalities in suckling piglets (see for a recent review [1] ). from 2010, an outbreak of pedv has swept china with over 1 million fatalities among newborn piglets causing substantial economic losses in the swine industry [2] . the characteristics of the infection and its epidemiology were quite dramatic with morbidity and fatality approaching 100% in one-week old piglets, despite the use of commercial, inactivated vaccines. virus transmission occurs via the fecal-oral route and possibly also by vertical transmission through lactation [2] . currently there is no efficient way of treatment of the disease. prevention of the infection usually relies on vaccination with cell culture adapted live-attenuated or inactivated viruses although the efficacy of current vaccines has been questioned [2, 3] . pedv belongs to the alphacoronavirus genus within the coronavirinae subfamily of the coronaviridae family. coronaviruses are important pathogens of concern for human and animal health. they occur in almost any species, usually causing respiratory or intestinal infections. interest in these viruses has increased significantly as a result of the sars epidemic in 2002 and 2003. coronaviruses are enveloped viruses and possess a positive-sense rna genome ranging from 26 to 32 kilobases, which is the largest viral rna genome known (fig. 1a) . the 59 two-third of the viral genome contains two large open reading frames (orfs), 1a and 1b, which encode two non-structural polyproteins, pp1a and pp1ab, that direct genome replication and transcription. the remaining part of the genome contains orfs specifying structural and non-structural proteins. they are expressed via a 39-terminal nested set of subgenomic messenger rnas, the transcription of which is regulated by conserved six-nucleotides transcriptionregulating sequences (trss; in pedv xua(a/g)ac [4] ). these subgenomic mrnas encode at least four structural proteins, three membrane anchored proteins called the spike (s), membrane (m) and envelope (e) protein, and the nucleocapsid (n) protein that encapsidates the genomic rna. the non-structural proteins expressed from the subgenomic mrnas encode one or more accessory proteins, which are specific for each coronavirus genus. the genome structures of alphacoronaviruses including pedv and related members such as the human coronavirus (hcov) strains 229e and nl63 show the typical set of essential core genes but they share only one accessory gene, orf3, located between the s and the e gene (fig. 1a) . the pedv orf3 gene encodes a 224 amino acids (aa) long protein with three to four predicted transmembrane domains [5] . entry of coronaviruses into their host cells is mediated by the approximately 200 kda large s glycoprotein. trimers of s form the characteristic spikes on the viral surface which interact with the host receptor and mediate membrane fusion. pedv was reported to utilize the porcine aminopeptidase n as a receptor [6] . yet, pedv is usually propagated in vero cells, which are derived from the african green monkey kidney, indicating that pedv can utilize non-porcine receptors for cell entry. propagation of pedv in cell culture requires addition of trypsin which is believed to prime or activate the s protein for membrane fusion during virus cell entry and syncytia formation [7] recently it was demonstrated that trypsin cleavage may also play a role in detachment of the virus from infected cells [8] . interestingly, a cell culture adapted strain was reported to replicate in the absence of trypsin [9] , which suggests that the virus acquired mutations in the s protein conferring its trypsin-independence. the s protein also stimulates the induction of neutralizing antibodies and hence is an important target in developing effective vaccines. research on the molecular biology and pathogenicity of pedv has been severely hampered by the lack of a reverse-genetic system. here we report the first reverse genetic system for pedv based on targeted rna recombination. establishment of the reverse genetic system included two stages (fig. 1b) . one was the generation of the chimeric virus mpedv, a pedv derivative carrying spikes derived from the murine coronavirus mouse hepatitis virus (mhv), hence growing only in murine cells. in the second stage the mpedv virus was used as a recipient virus to reintroduce the pedv spike along with other genome alterations, in casu the deletion of the orf3 gene or the insertion of foreign, reporter genes. the generated pedv derivatives now carrying again pedv spikes could be easily selected by their regained tropism for non-murine cells. to set up a targeted rna recombination system for pedv we first created a recombinant pedv virus carrying mhv spikes (mpedv). to this end a transfer vector p-mpedv was construced ( fig. 2a) that was composed of a 59-terminal genomic cdna fragment ligated to a cdna representing the entire 39-terminal part of the genome starting within orf1b, except for the s gene. this gene was replaced by a hybrid gene encoding a chimeric s protein composed of the 1,263 aa long ectodomain from mhv s and the transmembrane domain plus cytoplasmic tail (61 aa) from pedv s. rna was transcribed from the t7 promotor of this vector and electroporated into pedv-infected vero cells after which the cells were overlaid onto a murine cell (l cells) monolayer. the recombinant mpedv virus generated during subsequent incubation was cloned by two rounds of plaque selection on l cells. the identity of purified mpedv viruses was checked at a genetic level by rt-pcr sequencing of the orf1b-s gene junction (data not shown) and at the protein level by an immunofluoresence assay (fig. 3a ). all mpedv infected cells stained positive both with the polyclonal mhv serum and with the monoclonal antibody directed against the pedv nucleocapsid protein confirming the purity and the identity of the chimeric virus. in contrast to the parental virus, mpedv displayed the ability to induce syncytia in the absence of trypsin (fig. 3a) . as predicted, cell-cell fusion mediated by mpedv could be inhibited by a mhv s specific, peptidic fusion inhibitor (fig. 3b) . the generated mpedv virus was used as a recipient virus to reintroduce by similar procedures the pedv spike along with other genome modifications by targeted rna recombination. candidate recombinant viruses carrying the pedv spikes can be selected by their regained ability to replicate in vero cells. apart from the wild-type recombinant virus (r-wtpedv) we aimed at constructing a virus lacking the orf3 gene (pedv-dorf3). a number of cell culture adapted viruses including the strain used in this study have each acquired during passaging an identical 51 nucleotide in-frame deletion in the orf3 gene, giving rise to a 17 amino acid deletion (aa 82-98) in their orf3 protein [10] . we constructed a transfer vector (ppedv-dorf3, fig. 2a ) from which the entire orf3 gene was deleted. donor rnas transcribed from the ppedv and ppedv-dorf3 transfer vectors were electroporated into mpedv-infected l cells after which we were able to recover and purify the r-wtpedv and pedv-dorf3 viruses in vero cells. rt-pcr analysis confirmed the intended loss of the orf3 gene from the viral genome ( fig. 4a ) and the genetic identity of the orf3 lacking virus was further verified by sequencing of the rt-pcr product (data not shown). the pedv-dorf3 grew unimpaired in cell culture (fig. 4b) , demonstrating that the orf3 gene product is not required for virus propagation in vitro. in addition, the successful deletion of the orf3 gene from the viral genome demonstrated the feasibility of the mpedv-based targeted rna recombination system to manipulate the 39 end of the viral genome. we next explored the possibilities of expressing heterologous proteins from the pedv genome by inserting reporter genes at different genomic positions. transfer vectors were made with the renilla luciferase gene (936 nt) and the gfp gene (720 nt) at the position of orf3, creating the ppedv-dorf3/rluc and ppedv-dorf3/gfp vectors ( fig. 2a) . these marker genes are under the transcriptional control of the trs of orf3 (ctagac) which is located in the 39end of the s gene, 46 nucleotides upstream of the orf3 gene. the renilla luciferase gene was also inserted as an extra expression cassette between the orf1b and s gene, creating the ppedv-rluc vector. to this end the otherwise overlapping orfs 1b and s were first separated and a unique bamhi restriction site was introduced (p-rpedv vector, fig. 2a and b), which did not hamper the generation of a viable virus (data not shown). the renilla luciferase gene was subsequently cloned into the bamhi site of the p-rpedv vector under control of the trs in orf1b (gtaaac) originally driving s gene expression, whereas the s gene was provided with a new trs (gtaaac; fig. 2b ). the pedv-dorf3/gfp, pedv-dorf3/rluc and pedv-rluc recombinant viruses were successfully recovered by the targeted rna recombination procedure. rt-pcr analyses confirmed the insertion of both reporter genes at the intended positions ( fig. 5a) , which was further confirmed by sequencing. we studied the luciferase expression by the 2 recombinant viruses carrying a rluc gene as well as the expression kinetics of one of these viruses, pedv-rluc, upon infection of vero cells at three different moi's. the result shows (fig. 5b ) that luciferase expression levels were linearly related to the moi during the early phase of infection until 12 hours p.i. whereas at 24 hours p.i. luciferase values converged due to reinfections. similar kinetics of luciferase expression, but to higher levels, was observed for the pedv-dorf3/rluc recombinant virus (fig. 5b ). next we studied the gfp expression of the pedv-dorf3/gfp virus upon infection of vero cells at two moi's. gfp expression in pedv-dorf3/gfp virus infected cells could be seen starting from 9 hours p.i. and became clearly evident at 12 hours p.i. (fig. 5c ). the cell adapted pedv dr13 p100 strain can propagate in the absence of trypsin in the growth medium but does not form syncytia when trypsin is absent. yet the clustered appearance of gfp-positive cells suggests that the virus predominantly spreads locally from cell to cell which may correlate with the reported cell surface attachment of progeny viruses released from infected cells in the absence of trypsin [11] . the early detection of the luciferase and gfp reporter proteins during infection can be applied to develop a more rapid pedv neutralization diagnostic test. the readout of the classical virus neutralization assay with wild-type pedv is based on the visual inspection of cytopathic effect and can only be done after a multicycle infection which takes at least 2-3 days. thus, the pedv-dorf3/gfp and pedv-dorf3/rluc virus were preincubated with dilutions of serum obtained from an experimentally pedv-infected pig and control serum, and the mixtures were subsequently added to vero cells and incubated after which the gfp and renilla luciferase expression was recorded at 9 and 6 hours p.i., respectively (fig. 5d) . in contrast to the control serum, the pedv antibody-positive serum was able to neutralize pedv infection as reflected by the reduction of gfp positive cells and luciferase activity. the results demonstrate that neutralization of the pedv-dorf3/gfp and pedv-dorf3/rluc virus can already be scored within a single replication cycle, thereby significantly speeding up the assay time. this type of assay is additionally preferred as it avoids the subjectivity that is associated with scoring of cytopathic effects. here we describe the first reverse genetics system for pedv. as we illustrate, this system now enables the manipulation of the 39proximal ,8 kilobases of the pedv genome including the structural protein genes. generation of pedv recombinants was based on the well-known high efficiency of rna recombination of coronaviruses in combination with host cell tropism switching for selection of the recombinant viruses. similar recombination systems have been successfully developed for mhv and fipv coronaviruses by the masters and rottier laboratories [12, 13] . for a number of coronaviruses genetic engineering of the full length genome has also become accomplished by the development of infectious cdna clones [14] [15] [16] [17] [18] [19] [20] . the ability to manipulate the pedv genome will be extremely valuable to study the molecular and biological features of pedv infections as well as to develop new tools and strategies for prevention and therapy of this important veterinary pathogen. unlike most other coronaviruses, the pedv genome contains only a single accessory gene, the orf3 gene, which encodes a multispanning 224-aa long membrane protein. intriguingly, propagation of pedv isolates in tissue culture cells readily leads to deletions within orf3 suggesting a dispensable role, at least for the parts deleted from the orf3 protein, for viral replication in vitro. in all these adapted viruses a shorter orf3 gene product is still translated with a minimal size of 91 amino acids [10] . the orf3 gene of the cell-adapted dr13 vaccine strain (genbank accession no.: jq023162.1) employed in our study has a 49 nucleotide deletion compared to that of the parental dr13 virus (genbank accession no.: jq023161.1), but still encodes the nterminal 81 residues long orf3 protein part including the first transmembrane domain, after which it gets out of frame due to the deletion. the deletion of the entire orf3 gene from the genome the function of the pedv orf3 product remains enigmatic. recently it was shown that the protein exhibits ion channel activity and modulates virus production [5] . sirna knockdown of orf3 gene in pedv infected cells reduced the number of particles released from the cells [5] . the question remains here why passaging of pedv in cell culture would lead to the functional loss of a gene beneficial for virus propagation in vitro, unless the 91residue truncated protein still provides that function. homologues of the orf3 protein are found in all other alphacoronaviruses. the orf3 protein of hcov-nl63 was shown to be nglycosylated at the amino terminus and incorporated into virions [21] . yet, deletion of the orf3 gene from the viral genome had little effect on virus replication in cell culture [22] . like for pedv, loss of orf3 genes of the alphacoronaviruses tgev and hcov-229e (here named orf4) is associated with unimpaired virus passaging in cell culture [23, 24] . despite a non-essential role in cell culture, the maintenance of the orf3 gene in alphacoronavirus field isolates strongly points to an important role of the orf3 protein in natural infection in the animal host. consistently, the loss of virulence of life-attenuated pedv vaccine strains has been associated with mutations in the orf3 gene resulting from cell culture adaptation [10, 25] although a contribution of the numerous additionally acquired mutations in other genes such as the spike gene can obviously not be excluded [26, 27] . the specific function of the orf3 protein (and other viral proteins in the 39 genome region) in pedv replication and pathogenesis can now be further investigated using the reverse genetics system. the introduction of foreign genes at different genomic positions without apparent great fitness loss of the virus in vitro (data not shown) once more illustrates the remarkable genome plasticity of the coronavirus genome [28, 29] . the insertion of reporter genes like for gfp and luciferase will be very useful for the study of various molecular and virological aspects of pedv infection. in addition, as we demonstrate here, these reporter properties may also be exploited for applications such as the establishment of convenient virus neutralization assays that provide answers within hours rather than days. furthermore, genomic insertion of genes encoding foreign antigens using the reverse genetics system opens avenues to the development of pedv as a vaccine vector for protection against other relevant porcine pathogens in addition to pedv. l [12] and vero ccl81 cells (purchased from atcc) were maintained as monolayer cultures in dulbecco's modified eagle medium containing 10% fetal calf serum, 100 iu of penicillin/ml, and 100 mg of streptomycin/ml (all from life technologies, ltd., paisley, united kingdom). pedv (isolated from a commercial vaccine of greencross, south korea) was propagated in vero cells in the absence of trypsin. virus was harvested by three cycles of freeze-thawing the infected cells and supernatant followed by removal of cell debris by centrifugation at 3,0006g for 20 minutes. virus infectivity in the supernatant was measured by an end-point dilution assay on vero cells and 50% tissue culture infectious doses (tcid 50 ) were calculated. mhv (strain a59) was propagated in mouse l cells as described previously [12] . the rabbit anti-mhv serum k135 raised against purified mhv has been described elsewhere [30] . the monoclonal antibody (mab) 3f12 recognizing the pedv nucleocapsid protein was obtained from bionote, korea. polyclonal pedv serum from a pig experimentally infected with pedv (strain cv777) was kindly provided by dr. kristin van reeth (gent university). pedv antibody-negative control serum was obtained from a newborn piglet deprived of colostrum. ppedv vector. a cdna clone encompassing the 39-terminal 7,832 nt part of the pedv genome starting within orf1b was obtained by reverse transcription-pcr (rt-pcr) with viral genomic rna isolated from virions as a template and primers 4922 and 4815 as plus-and minus-strand primers (for primer sequences see table 1 ), respectively. the overhang of primer 4922 and primer 4815 contained a bglii and a paci restriction site, respectively. the bglii-paci digested fragment was cloned into the bamhi-paci digested pmh54 vector [12] , creating the plasmid ppedv-1b-3t. the 59-terminal 605 nt of orf1a was amplified using primers 4884 and 4885. primer 4884 contains a t7 polymerase recognition site, as well as a bglii restriction site and primer 4885 contained a bamhi restriction site. the bglii-bamhi digested fragment was ligated into the bamhi site of the ppedv-1b-3t plasmid, resulting in the ppedv vector. p-rpedv vector. a transfer vector was constructed in which the partly overlapping orf1b and s gene were separated by introduction of a unique bamhi site to facilitate further cloning. the stop codon of orf1b was mutated to taa to knock out the overlapping atg start codon of the spike gene. first, the forward primer 5127 containing the bamhi site and a trs (taaac), and the reverse primer 4815 containing a unique paci site were used to amplify the 39 proximal 7,332 nt of the pedv genome starting with the spike gene. this fragment was cloned into the bamhi-paci site of pmh54 vector, creating the ppedv-s-3t vector. second, primers 4884 and 4885 containing a bglii and bamhi site, respectively, were used to rt-pcr amplify the orf1a fragment which was introduced into the bamhi digested ppedv-s-3t vector creating the ppedv-1a-s-3t plasmid. third, primers 4922 and 4923 that contain a bglii and bamhi in the overhang, respectively, were used to amplify the orf1b fragment by rt-pcr. this fragment was cloned into the bamhi site of the ppedv-1a-s-3t vector, creating the p-rpedv vector. p-mpedv vector. first, the plasmid ptums [31] encoding the mhv spike was used as an intermediate vector to construct a chimeric spike composed of the ectodomain of mhv and the transmembrane and cytoplasmic domain of pedv. for the construction of the hybrid gene, a styi restriction site was used that is located in both s genes at the transition between the protein's ectodomain and transmembrane domain. the forward primer 4814 (styi site in overhang) and reverse primer 4924 (eagi site in overhang) were used to amplify the 39 end of the pedv s gene and downstream sequences and cloned into the styi-eagi digested ptums plasmid, creating the ptums(mp) vector. second, to create the p-mpedv vector, the pedv s gene in the p-rpedv 0.1 and fluorescence images were taken at different times p.i. nuclei of cells were stained with dapi (blue). (d) a rapid virus neutralization assay based on recombinant pedvs expressing reporter proteins. pedv-dorf3/rluc and pedv-dorf3/gfp (8,000 tcid50) were mixed with subsequent dilutions of serum positive for pedv antibodies and a negative control serum (n.c.) for 30 minutes at room temperature. mixtures were incubated with vero cells and renilla luciferase (left panel) or gfp (right panel) expression was measured at 8 and 9 hours p.i., respectively. doi:10.1371/journal.pone.0069997.g005 vector was replaced by the chimeric mhv-pedv spike gene by cloning the bamhi-pmli digested fragment of ptums(mp) into the bamhi-pmli digested p-rpedv vector. ppedv-dorf3 vector. primers 5300 and 5301 were used to amplify the e gene and downstream sequences using the ppedv vector as a template. the forward primer 5300 contained a pmli and an ecorv restriction site and the reverse primer 5301 contained an restriction econi site to facilitate further cloning. the pmli-econi digested pcr fragment was cloned into the pmli-econi digested ppedv vector to create the ppedv-dorf3 vector. ppedv-rluc and ppedv-dorf3/rluc vector. the renilla luciferase gene was excised from the prlnull vector (promega) using enzymes nhei and xbai, blunted with dnapolymerase i large (klenow) fragment and ligated into the bamhi digested and blunted p-rpedv vector or the ecorv digested ppedv-dorf3 vector, resulting in the ppedv-rluc and ppedv-dorf3/rluc transfer vector, respectively. ppedv-dorf3/gfp vector. the gfp gene was excised from the pegfp-n1 plasmid (clontech) with enzymes ncoi and noti, blunted with dna-polymerase i large (klenow) fragment and ligated into the ecorv digested ppedv-dorf3 vector yielding the ppedv-dorf3/gfp transfer vector. the identity of all generated transfer vectors was verified by sequencing. a targeted recombination system was established for pedv in a two-stage process as outlined in fig. 1b . stage 1 generation of mpedv. introduction of the hybrid mhv-pedv s gene into the pedv genome by targeted rna recombination was carried out essentially as described previously for mhv and fipv [12, 13] . briefly, capped runoff donor rna transcripts were synthesized from the paci-linearized p-mpedv vector using a t7 rna polymerase kit (ambion) as specified by the manufacturer. donor rna was electroporated (gene pulser electroporation apparatus [bio-rad]; two consecutive pulses of 0.3 kv/975 mf) into pedv-infected (multiplicity of infection [moi] of 0.4) vero cells (2610 7 cells) at 8 hours post infection (p.i.). the electroporated cells were co-cultured in a 25-cm 2 flask with 5610 6 murine l cells. after 48-60 h of incubation at 37uc, when syncytia could be detected in the murine l cells, progeny virus in the culture supernatant was harvested and mpedv recombinant virus was purified by two consecutive cycles of plaque purification on l cells at 37uc. stage 2 generation of recombinant pedvs. the construction of pedv recombinant viruses that had regained the pedv s gene was carried out in a reverse process by using ppedv-derived donor rnas and mpedv as the recipient virus. capped runoff transcripts were synthesized from paci-linearized ppedv, ppedv-rluc, ppedv-dorf3, ppedv-dorf3/rluc, or ppedv-dorf3/gfp, respectively, with a t7 rna polymerase kit (ambion) as specified by the manufacturer. the donor transcripts were electroporated (as specified above) into murine l cells (2610 7 cells) that had been infected 4 h earlier with mpedv (moi = 1). these cells were then plated onto a monolayer of vero cells. after 4-5 days of incubation at 37uc progeny virus in the culture supernatant was harvested by freeze-thawing and candidate recombinant viruses were purified by two rounds of end-point dilutions on vero cells. recombinant genotypes were confirmed by rt-pcr on purified genomic rna and subsequent sequencing. (immuno)fluorescence microscopy l cells and vero cells were inoculated with mhv, mpedv or pedv (moi = 0.05). after 2 hours of incubation the cells were washed with pbs and incubated in culture medium. at 6.5 hours p.i., the cells were rinsed with pbs and fixed with 3.7% formaldehyde for 20 min at room temperature. the cells were washed three times with pbs and incubated with the k135 rabbita-mhv serum and the 3f12 mouse mab a-pedv-n. after 30 min at room temperature, the cells were rinsed three times with pbs and stained with goat a-rabbit fitc-conjugated and donkeya-mouse cy3 conjugated secondary antibodies (cappel). nuclei were stained with dapi (molecular probes) for 10 min at room temperature. finally, the cells were washed three times with pbs and fluorescence was viewed with an evos-fl fluorescence microscope (advanced microscopy group) at 106 magnification. the evos-fl was also used to view gfp fluorescence from pedv-dorf3/gfp infected cells after paraformaldehyde fixation. vero cell monolayers were infected as described above with the pedv-rluc and pedv-dorf3/rluc viruses at indicated moi's. at indicated times post infection, cell lysate samples were assayed for luciferase activity using the renilla luciferase assay system (promega) according to the manufacturer's instructions, and the relative light units (rlu) were determined with a berthold centro lb 960 plate luminometer. pedv-dorf3/rluc or pedv-dorf3/gfp were mixed with serial dilutions of positive or negative piglet serum or with cell culture medium. the inoculum was incubated for 30 minutes at room temperature to allow virus neutralization before inoculating vero cell monolayers as described above. cells were either lysed at 8 hours post infection and assayed for renilla luciferase activity as described above or subjected to fluorescence microscopy as described above at 9 hours post infection. porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines. virus genes outbreak of porcine epidemic diarrhea in suckling piglets molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field isolates in korea pedv leader sequence and junction sites pedv orf3 encodes an ion channel protein and regulates virus production porcine aminopeptidase n is a functional receptor for the pedv coronavirus propagation of the virus of porcine epidemic diarrhea in cell culture role of proteases in the release of porcine epidemic diarrhea virus from infected cells derivation of attenuated porcine epidemic diarrhea virus (pedv) as vaccine candidate cloning and further sequence analysis of the orf3 gene of wild-and attenuated-type porcine epidemic diarrhea viruses enhanced cell fusion activity in porcine epidemic diarrhea virus adapted to suckling mice retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier switching species tropism: an effective way to manipulate the feline coronavirus genome engineering the largest rna virus genome as an infectious bacterial artificial chromosome reverse genetics system for the avian coronavirus infectious bronchitis virus infectious rna transcribed in vitro from a cdna copy of the human coronavirus genome cloned in vaccinia virus a reverse genetics approach to study feline infectious peritonitis reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus systematic assembly of a fulllength infectious cdna of mouse hepatitis virus strain a59 strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model human coronavirus nl63 open reading frame 3 encodes a virion-incorporated n-glycosylated membrane protein systematic assembly of a full-length infectious clone of human coronavirus nl63 human coronavirus 229e encodes a single orf4 protein between the spike and the envelope genes efficacy of a transmissible gastroenteritis coronavirus with an altered orf-3 gene oral efficacy of vero cell attenuated porcine epidemic diarrhea virus dr13 strain cloning and further sequence analysis of the spike gene of attenuated porcine epidemic diarrhea virus dr13 mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo coronaviruses as vectors: position dependence of foreign gene expression coronaviruses maintain viability despite dramatic rearrangements of the strictly conserved genome organization translation of three mouse hepatitis virus strain a59 subgenomic rnas in xenopus laevis oocytes nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex key: cord-345717-ktajrf7d authors: monagin, corina; paccha, blanca; liang, ning; trufan, sally; zhou, huiqiong; wu, de; schneider, bradley s.; chmura, aleksei; epstein, jonathan; daszak, peter; ke, changwen; rabinowitz, peter m. title: serologic and behavioral risk survey of workers with wildlife contact in china date: 2018-04-03 journal: plos one doi: 10.1371/journal.pone.0194647 sha: doc_id: 345717 cord_uid: ktajrf7d we report on a study conducted in guangdong province, china, to characterize behaviors and perceptions associated with transmission of pathogens with pandemic potential in highly exposed human populations at the animal-human interface. a risk factor/exposure survey was administered to individuals with high levels of exposure to wildlife. serological testing was performed to evaluate prior infection with several wildlife viral pathogens. follow up serology was performed on a subset of the cohort as well as close contacts of individuals. 1,312 individuals were enrolled in the study. contact with a wide range of wildlife species was reported in both occupational and occasional contexts. the overall proportion of individuals seropositive to any of the tested wildlife pathogens was approximately 4.0%. however, persons employed as butchers demonstrated a seropositivity of 9.0% to at least one pathogen of interest. by contrast, individuals working as hunters had lower rates of seropositivity. among the study population, a number of other behaviors showed correlation with seropositivity, including contact with particular wildlife species such as field rats. these results demonstrate the need to further explore zoonotic risks of particular activities regarding wildlife contact, and to better understand risks of persons working as butchers with wildlife species. the majority of human infectious diseases have an animal origin, therefore understanding the human-animal interface as it relates to disease emergence and risk is of upmost importance [1] . the increasing frequency and variety of human-wildlife interactions in china provide opportunities for the transmission of zoonotic pathogens from animals to humans [2] . a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 handling, transporting, and butchering of hunted or farmed wildlife poses a risk of pathogen spillover into humans [3] . in southern china provinces, including guangdong, a significant percentage of the population obtains fresh meat for consumption from wet markets, community markets that specialize in selling and butchering live animals, including animals that are rare and endangered. research has demonstrated that human-animal interfaces, such as within these wet markets, provide an ideal environment for infectious disease emergence, transmission, and amplification [4] [5] [6] [7] . wet markets and restaurants that butcher and sell wild animals are common in china and south east asia, creating a high-risk interface where humans come into regular contact with the blood and bodily fluids of wild and domesticated animals. intermingling of wildlife in wet markets can lead to inter-species transmission of pathogens, amplifying and maintaining these pathogens, and may result in spillover into a species that can more efficiently transmit the pathogen to humans [8] . furthermore, increasing numbers of individuals are traveling to urban-rural interface areas to eat at "wildlife" restaurants. hunted or farmed wildlife are transported and kept alive at these restaurants until consumption, increasing the risk of pathogen spillover. the wildlife trade has played a role in the emergence of severe acute respiratory syndrome (sars), avian influenza, ebola, and other diseases of wildlife origin [2, 9] . guangdong province in china was the site of the first cases of sars in 2002. now known to have originated in bats [10] , sars emerged in humans and other mammals in wet markets, such as himalayan palm civets (paguma larvata) and raccoon dogs (nyctereutes procyonoides) [11] . the disease spread to 26 countries and infected over 8,000 people; killing nearly 800 [12] . sars shut down trade of domestic animals and resulted in nearly $40 billion in losses to the global economy [13] . h5n1 outbreaks during 1999-2008 and h7n9 in 2013 have demonstrated the realities of the risk of disease transmission and spread in china and the detriments to animal and human health, as well as human livelihoods [9, 14] . these epidemics shed new light on how quickly and how far newly emerged zoonotic diseases can spread in today's world. these outbreaks continue to challenge scientists, policymakers, and public health systems, exemplifying the need for a better understanding of the factors leading to emergence, and clarity regarding emerging infectious disease policy, particularly as it concerns high-risk human-animal interfaces. although mounting evidence implicates illegal trade and behavioral and cultural factors as influencing disease emergence, it is clear that more research is needed to identify these drivers [2, 4, 6, 7, 15] . a key aspect of understanding human-wildlife interfaces is the characterization of risk behaviors and perceptions of individuals who have wildlife contact. specifically, there is a need to evaluate whether particular behaviors are associated with increased risk of transmission. as well, serological studies may be helpful to evaluate exposure, either current or prior, to zoonotic agents. for example, when guan et al. (2003) surveyed animals in markets for evidence of sars-like coronavirus [11] , they also performed serological analysis on market employees. seroprevalence in their study ranged widely between traders (wild-animal to vegetable), butchers and community controls. the present study focuses on the potential for zoonotic viral transfer through contact with wildlife in guangdong prefectures in china, and seeks to augment our understanding and identification of risky populations, occupations, and behaviors, as well as the perceptions of risk at these interfaces. we performed a serological survey and concurrent behavioral questionnaire of individuals with wildlife contact in guangdong province, china, in order to better characterize occupations and community-level behavioral risks that contribute to zoonotic transmission of various wildlife pathogens with pandemic potential. this study took place in guangdong province (23˚24'n, 113˚30'e), china from 2009-2013. guangdong is located on the south china sea coast, bordering guangxi province, hunan province, jiangxi province and fujian province. it neighbors hong kong and macau, and has over 50 million residents living in 9 cities: guangzhou, shenzhen, dongguan, foshan, zhongshan, zhuhai, jiangmen, huizhou and zhaoqing. enormous consumption of animal and animal products in this region is believed to serve an economical incentive for butchers, cooks and hunters in less developed guangdong prefectures, e.g. qingyuan, yunfu and meizhou. guangdong province has consistently reported the largest population in china since 2005, with the highest gdp rankings for the last 25 years. its economy is highly dependent on the pearl river delta region, which contributes around 80.0% total provincial gdp while supporting 29.0% of the provincial population. the study was conducted in 12 prefectures within the guangdong province: dabu, jiaoling, pingyuan, lianping, heping, lianshan, lianzhou, yunfu, yunan, xinyi, deqing and fengkai (fig 1) . we chose field sites within the identified prefecture areas that have wet-markets, wildanimal restaurants, and other environments with a higher risk of animal-to-human pathogen transmission. these environments, particularly the markets, were deliberately chosen due to their size and character. similar species of wildlife, both live and butchered, were present, with similar butchering methods being utilized. populations in these environments with high-risk animal-to-human pathogen transmission activities, such as hunting, and butchering in wetmarkets and wild-animal restaurants were selected. we targeted high-risk individuals, defined as individuals with high levels of exposure to wildlife (wild animal blood or bodily fluids)-primarily hunters, persons working in wet markets and restaurants that butcher wild game, who could be followed over a period of time. inclusion criteria included participant age between 18-65, and individuals that reported previously hunting, selling, butchering, or eating wild animals. individuals who did not provide informed consent and/or did not agree to biological sample collection were excluded. individuals were residents of the site area so that they were available for follow up visits. more than 20 potential field sites were visited to gauge level of risk at the site (number of high-risk animal-human interfaces). local prefecture-level chinese center for disease control (cdc) teams were asked to pre-enroll individuals who fit the inclusion criteria and would be available for follow up visits for up to three years. based on pre-enrollment numbers and human-animal interfaces identified at each site, 12 field sites that were identified in the previously named prefectures: dabu, jiaoling, pingyuan, lianping, heping, lianshan, lianzhou, yunfu, yunan, xinyi, deqing and fengkai were chosen to participate in the study (fig 2) . study participation was voluntary and respondents were given the equivalent of 5 usd (approximately 30 cny) as compensation for their time. informed consent was obtained from all enrolled participants prior to enrollment and specimen collection. in total, 1,267 participants were enrolled in the first round of the study. the prefecture-level cdc research teams were trained in all enrollment procedures (biological and behavioral questionnaire). ten (10) ml of peripheral venous blood was collected using pre-labeled vacuum edta blood collection tubes. all blood samples were chilled immediately at 4˚c, and transferred to participating cdc laboratories at surveillance sites within 2 hours after sampling. upon arrival of blood samples into the laboratories, sera were isolated by 1,500 g centrifugation for 15 min. serum was aliquoted, flash frozen in liquid nitrogen, and then stored at -80˚c. all study participants responded to a detailed questionnaire including questions about demographics, occupation, rural vs. urban habitation, zoonotic disease risk perceptions, and contact with particular types of wildlife through hunting, butchering, or eating. the questionnaire was based on a previous study in cameroon and is available in the s1 file [16, 17] . it was adapted in english for the study environment in china and then translated into simplified chinese. during the training of the local cdc research teams, pretesting of the questionnaire was done to ensure the intelligibility of the questions and proper translation. pretesting was also done with the study population during the pre-enrollment phase for the study sites. during enrollment, a trained member of the local cdc team who was fluent in the local language of the study site administered the questionnaire. in guangdong province, both mandarin and cantonese are spoken in the study sites. the questionnaire was administered in either mandarin or cantonese depending on the respondent. all follow up study respondents were contacted by a member of the trained research team regarding their test results. seropositive individuals or those with indeterminate results were referred to a local clinic/hospital where they could follow up on their results. this study did not provide additional financial or medical support for respondents who tested positive. all results were kept confidential and only reported to the respondent themselves. 95 individuals who tested seropositive from the first enrollment were asked to participate in an additional follow up sampling visit. they were also asked to invite up to three of their close contacts to enroll in the follow up study. we aimed to continue behavioral information and biological sample collection towards gauging virus transmissibility. prior to sampling, we re-consented seropositive individuals and consented their close contacts. due to geographic reallocation of some respondents, we were unable to locate 52 individuals with indeterminate or seropositive results for inclusion in our follow up study. we successfully re-enrolled 43 seropositive respondents and 45 of their close contacts in our follow-up study (fig 2) . the serological investigation focused on detection of hantavirus, sars cov, and severe fever with thrombocytopenia syndrome (sfts) bunyavirus. this study aimed to demonstrate viral transfer associated with exposure to wildlife and the virus targets for this study were selected based on prevalence and infection associated with animal/wildlife contact as well as an interest by the gdcdc [18] [19] [20] . to determine hantavirus specific antibodies in study respondents, stored human sera were tested using hantavirus hantaan igg/igm elisa in a single well for each sample (ibl, hamburg, germany) [21] . microtiter plates were pre-coated with recombinant nucleo-capsidprotein of hantaan virus. according to the manufacturer's instructions, control groups and experiment group sera were diluted at 1: 100 using 1×pbs, ph 7.4, and incubated at 37˚c. coated wells were washed, and then incubated with 100 μl diluted peroxidase conjugated anti-igg enzyme at 37˚c. color development reaction was initiated by adding a chromogenic substrate, tetramethylbenzidine, followed by a 10 minute incubation at room temperature. reaction was quenched by adding 100 μl 0.5 m h 2 so 4 , and then measured at 450 nm to obtain its optical density. human sera were also tested for sars cov using the sars coronavirus igg diagnostic kit (bgi, shenzhen, china) [22] [23] [24] . microtiter plates were pre-coated with vero e6 cell lysate. cells were previously infected by sars coronavirus that was isolated during the sars outbreak in 2002 in china. sera preparation and indirect elisa were conducted as described above. the average optical density (od) of negative control sera plus 0.13 was used as the cut off value as suggested by the manufacturer. we adopted recombinant coating antigen in the diagnosis of sfts bunyavirus using indirect elisa. gene encoding 6×his tagged sfts bunyavirus hb29 nucleocapsid (n) protein was expressed in e. coli, and purified using nickel affinity chromatography. recombinant anti-sftsv igg was immobilized in 96-well plate as coating antigen. sera were diluted at 1: 100 using 1×pbs, ph 7.4, and incubated at 37˚c. we used horseradish peroxidase (hrp)-conjugated anti-human igg antibody (sigma, saint louis, mo) as the secondary antibody for color development. od was measured at 450 nm, with a reference wavelength of 620 nm. cut-off value was determined by adding 3× sd to the mean of od values resulting from analyses of negative control sera. moreover, a microneutralization assay was performed to detect neutralizing antibodies against sfts bunyavirus [25] . this was done to ensure the highest level of certainty given the newness of the sfts virus assays. fifty (50) μl serially diluted human sera were mixed with an equal volume of 100 tcid50 /0.1ml of sfts bunyavirus hb29, and then incubated at 37˚c for 1.5 h. incubated mixture was added to cultured vero cells in a 96-well plate in quadruplicate. subsequently, the plates were incubated at 37˚c with 5.0% co 2 for 7 d. viral infection was assessed via an immunofluorescence assay using mouse polyclonal anti-sfts bunyavirus antibody. end-point titer was then expressed as the reciprocal of the highest dilution of serum with viral infection prevention capability. laboratory results and behavioral data from the questionnaire were manually entered twice (double entry system) and compared for entry error. hand checks were also done on 10.0% of the database to further reduce chances of human entry error. questionnaire responses and serological test results were then analyzed and compared using sas (version 9.4; sas institute inc., cary, nc, usa). we assigned primary occupation to the categories of hunter, agriculture worker, butcher, market worker, and restaurant worker based on questionnaire responses. we also aggregated reported behaviors to assign an "ever" status to each individual in terms of eating, butchering, or hunting wildlife. basic descriptive univariate statistics were carried out on the data set. we then examined bivariate associations between serological status for the three viruses of interest and independent demographic and risk behavior variables. variables showing bivariate association with seropositivity at a p value of 0.1 or less were included in multivariate logistic models for seropositivity to individual viruses or any of the three viruses using firth's penalized likelihood adjustment to address small sample size and possible collinearity [26] . since butchers were the only occupational group showing increased risk of seropositivity, we also performed a subanalysis of specific behaviors performed by butchers, again including behaviors with bivariate association of p = 0.1 or less in a multivariate logistic model. a total of 1,267 individuals provided a baseline blood sample for serological testing and completed a behavioral risk factor survey. the demographics of these individuals are shown in table 1 . the study population was predominantly younger than age 60, with a mean age of 36.97 ±11.03 years. junior high school was most frequently reported as the highest level of education attained. most of the respondents were married or living with their partner and had between 1-2 children, with a total of 3-6 people living in the house. traveling to a forest at least once a month, which may provide additional exposure to wild animals, was reported by 67.0% of respondents (data not shown). some of the most common reasons for traveling to the forest were fieldwork (56.0%), gathering of fruit and vegetables (21.0%), collecting firewood (11.0%), and hunting (10.0%). more respondents reported ever living in a city than not, with the most common urban occupations being restaurant work followed by "other" and factory jobs. table 2 describes type and location of occupational activities. restaurant-related occupations such as cook or kitchen worker were the most commonly reported primary occupation, followed by agriculture, butcher, and jobs working in a market. table 3 summarizes reported exposures to wild animals. almost all respondents indicated that they had ever touched any live or dead wild animal. having butchered and eaten wild animals were the most common types of animal interactions, and injury was experienced by less than a quarter of the respondents. table 4 shows the beliefs and reported protective behavior regarding contact with animals and zoonotic disease transmission. less than one third of respondents reported believing that they could get infected from contact with blood or other types of contact with wild animals. a smaller proportion reported taking at least one type of protective measure to prevent infection in such circumstances. among those who reported taking protective measures, use of gloves, and avoiding touching the animal were the most commonly reported protective practices. risk factors for seropositivity. table 5 summarizes the results of baseline testing of blood for evidence of antibodies against zoonotic wildlife viruses. the overall rate of seropositivity to any of the other three tested pathogens (hantavirus, sars cov, or sfts bunyavirus) was low among the study population (approximately 4%). seropositivity in respondents was only found in the first phase of enrollment. among contacts of these seropositive respondents (n = 45), all individuals were seronegative for the pathogens of interest. the most common seropositivity was for hantavirus (2.3% of the study population). however, among the 112 persons reporting that their occupation was a butcher, the rate of seropositivity to at least one pathogen was higher (9.0%), and this elevated risk was statistically significant (fisher's exact test, p = 0.005). butchers had elevated seropositivity rates to all three of the viruses compared to the overall study population. no other occupational group showed an increased risk of seropositivity. in a multivariate model of the butchers, those who reported butchering porcupines showed an elevated risk of seropositivity (data not shown). there was no increased risk among the butchers associated with not wearing gloves or taking fewer protective measures. by contrast, the 38 persons reporting hunting as a profession showed a decreased risk for seropositivity as did those reporting their profession as housework. no other occupational groups showed significantly increased or decreased risk. persons who reported ever hunting also had a lower risk of seropositivity compared to those who never hunted. age and gender did not show a significant association with seropositivity, nor did reporting living in a city or going frequently into the forest. while 111 persons reported butchering as a profession, a larger number (966) reported ever butchering a wild animal; this was associated with an elevated risk of seropositivity, however when the professional butchers were removed from this group, the risk was no longer significant. as shown in the table s1 table, reported contact with certain wildlife species such as civets (for hanta and bunyavirus) and field rats (for sars) showed elevated risk for certain viruses in bivariate analyses. in multivariate models of specific wildlife contact, (table 6) , the principal association remaining significant was eating civets (associated with hantavirus seropositivity). the risk estimate for bunyavirus was elevated but did not reach statistical significance. this study, examining the association between reported wildlife contact and seropositivity for wildlife zoonotic viruses, found detectable levels of antibodies for several pathogens in the population surveyed. overall, the population had contact with multiple species of animals. among reported primary occupations, working as a butcher was associated with an increased risk of infection with wildlife pathogens, while professional hunters were at decreased risk, and no increased risk was seen for market or restaurant workers. contact with certain animals, such as civets and field rats, showed evidence of increased risk of seropositivity. few people in the study reported using protective measures such as gloves when working with wild animals, and less than a third of individuals reported believing that they could become infected through wildlife contact. when seropositive individuals were followed over time, no evidence of infection of close contacts was seen, although the number of contacts enrolled was small and there are limited conclusions that can be draw about alternative transmissions risks. further investigation is warranted to gauge transmissibility of the viruses studied. these findings have a number of implications for prevention of zoonotic disease transmission and outbreaks. those individuals at the animal-human interface (such as persons butchering wild animals) that are highly exposed to wild animals are influential in disease amplification and dissemination and reduction of risk factors can play a large role in mediating potential outbreaks. while we identified butchers as an occupational group at increased risk, better understanding of the specific exposures driving this increased risk would require more in-depth study. among butchers, we did find an elevated risk of virus seropositivity associated with butchering porcupines, but due to small numbers, such a finding must be considered preliminary. this study also identified an increased risk of seropositivity with contact with civets and field rats which could be due to multiple factors. civets and field rats are among the most popular types of wild animals found in markets and restaurants and prolific contact may result in higher risk. there is also a bias related to the viruses that the study targeted with two of the three (sars cov and hantavirus) associated with both civets and rats. this study had a number of limitations, including selection bias. we deliberately targeted high risk individuals, making our sample less representative of the general population. conversely, it is possible that there could be differential participation rates between high risk and lower risk individuals, further limiting any extrapolation to community levels. we focused on wildlife contact, and therefore were unable to assess the risk impact of contact with other animal species including livestock and companion animals. further research should aim to include larger sample sizes from more geographically diverse regions, and assess seroprevalence rates in random population samples. underreporting of risk activities on the behavioral survey may have occurred, since many activities related to wild animals are illegal in china. in addition, since this was a hypothesis generating study, the analysis involved multiple comparisons. therefore, any reported association should be viewed with caution. while this study involved serological testing for more than one potential wildlife pathogen, there are numerous other wildlife pathogens, including bacterial and viral agents, which the study did not investigate. it is therefore possible that this study failed to detect other significant transmission events of human health relevance. future studies should consider such possibilities. the finding that less than one third of the surveyed population, most of whom reported wild animal contact, believed that they could become infected through such contact, indicates the need for targeted educational programs for prevention, especially among those involved in butchering wildlife for consumption. targeted education programs should aim to increase knowledge of disease risk, perception of risk, and risky behaviors in the identified high-risk sub groups. previous studies indicate the need to target the high-risk sub group of hunters [16, 17] . in this study, however, hunters and persons reporting ever hunting or hunting as a profession exhibited a lower rate of seropositivity compared with the overall population. it is possible that hunting activities may be underreported, but the results of this study indicate some of the difficulties of relying on self report to identify individuals at increased risk. due to the huge demand by the local residents and immigrants in the larger pearl river delta area, which includes guangdong, hong kong and macau, increasing number of individuals engaged in animal production and value chain systems are believed to continue to come into contact with wild animals and engage in risky behaviors increasing the risk of a possible infectious disease outbreak that could have a potentially severe impact. wild animals are an important part of southern chinese economy and culture, and pragmatic policy changes should be implemented to control the risks of possible disease outbreaks. these policy changes should aim to increase risk perception and uptake of precautionary behaviors without risking the cultural and financial security of the community. results of the analyses of these research findings can be used to inform public health strategies such as communication and educational campaigns, targeting those behaviors that place individuals at higher risk of disease. our study provides evidence towards recommendations that include: an increase in the level of collaboration between animal and human health programs targeting human-animal interfaces to increase efforts to control and prevent outbreaks; development of new educational campaigns to increase knowledge and awareness of infectious diseases that target specific sub-groups, such as butchers, that are at increased risk; and increase monitoring of highrisk groups to detect continued transmission of zoonotic diseases in a timely manner. supporting information s1 file. study questionnaire for persons hunting, butchering, eating and/or keeping wild animals as pets. (pdf) s1 table. prevalence of seropositivity for tested viruses, by reported wildlife exposure. (pdf) global trends in emerging infectious diseases wildlife trade and global disease emergence. emerging infectious disease bushmeat hunting, deforestation, and prediction of zoonotic disease wet markets-a continuing source of severe acute respiratory syndrome and influenza? the lancet animal movements and the spread of infectious diseases disease and destiny-mystery and mastery infectious diseases emerging from chinese wet-markets: zoonotic origins of severe respiratory viral infections the role of wildlife in emerging and re-emerging zoonoses emergence and predominance of an h5n1 influenza variant in china bats are natural reservoirs of sars-like coronaviruses isolation and characterization of viruses related to the sars coronavirus from animals in southern china sars-beginning to understand a new virus learning from sars: preparing for the next disease outbreak: workshop summary. the national academies collection: reports funded by national institutes of health human infection with a novel avian-origin influenza a (h7n9) virus emerging angiostrongyliasis in mainland china emergence of a novel and highly divergent htlv-3 in a primate hunter in cameroon new directions in conservation medicine: applied cases of ecological health hantavirus infections in humans and animals distribution of tick-borne diseases in china a national assessment of the epidemiology of severe fever with thrombocytopenia syndrome a global perspective on hantavirus ecology, epidemiology, and disease the life cycle of sars coronavirus in vero e6 cells establishment of vero e6 cell clones persistently infected with severe acute respiratory syndrome coronavirus diagnosis of severe acute respiratory syndrome (sars) by detection of sars coronavirus nucleocapsid antibodies in an antigen-capturing enzyme-linked immunosorbent assay fever with thrombocytopenia associated with a novel bunyavirus in china. the new england journal of medicine the application of firth's procedure to cox and logistic regression university of vienna, section of clinical biometrics domcs we thank prefecture cdcs at dabu, jiaoling, pingyuan, lianping, heping, lianshan, lianzhou, yunfu, yunan, xinyi, deqing and fengkai for their efforts in participant enrollment and specimen collection. there are no competing interests in this scientific work. key: cord-354547-eomm1sl5 authors: wang, jibin; fang, shouguo; xiao, han; chen, bo; tam, james p.; liu, ding xiang title: interaction of the coronavirus infectious bronchitis virus membrane protein with β-actin and its implication in virion assembly and budding date: 2009-03-16 journal: plos one doi: 10.1371/journal.pone.0004908 sha: doc_id: 354547 cord_uid: eomm1sl5 coronavirus m protein is an essential component of virion and plays pivotal roles in virion assembly, budding and maturation. the m protein is integrated into the viral envelope with three transmembrane domains flanked by a short amino-terminal ectodomain and a large carboxy-terminal endodomain. in this study, we showed co-purification of the m protein from coronavirus infectious bronchitis virus (ibv) with actin. to understand the cellular factors that may be involved in virion assembly, budding and maturation processes, ibv m was used as the bait in a yeast two-hybrid screen, resulting in the identification of β-actin as a potentially interacting partner. this interaction was subsequently confirmed by coimmunoprecipitation and immunofluorescence microscopy in mammalian cells, and mutation of amino acids a159 and k160 on the m protein abolished the interaction. introduction of the a159-k160 mutation into an infectious ibv clone system blocks the infectivity of the clone, although viral rna replication and subgenomic mrna transcription were actively detected. disruption of actin filaments with cell-permeable agent cytochalasin d at early stages of the infection cycle led to the detection of viral protein synthesis in infected cells but not release of virus particles to the cultured media. however, the same treatment at late stages of the infection cycle did not affect the release of virus particles to the media, suggesting that disruption of the actin filaments might block virion assembly and budding, but not release of the virus particles. this study reveals an essential function of actin in the replication cycle of coronavirus. enveloped viruses acquire their envelope by budding from the host cell. in this process, viral envelope proteins gather at a special membranous structure and cooperate with other viral components to induce budding [1] . for example, some viruses including human immunodeficiency virus bud from the plasma membrane and release the virion from host cells by pinching-off. others are budding at intracellular membranes [2, 3] . in this way, virions are wrapped within intracellular membrane-bound compartments, such as the endoplasmic reticulum (er) and golgi apparatus, and the newly budded viruses exit the cell by using the cellular secretory pathway [2] . however, the detailed mechanisms of viral assembly and budding, especially the host factors that are involved in these processes, are yet to be revealed for many viruses. in this study, we report that interaction between coronavirus membrane protein (m) and actin with functional implication in facilitating virion assembly and budding. coronavirus is an enveloped virus with a large, positivestranded rna genome of about 27 to 31 kilobases in length. the avian coronavirus infectious bronchitis virus (ibv) belongs to the third group of coronaviruses genus. like many other coronavriuses, ibv virion is built from four structural proteins, including the nucleocapsid (n) protein with which the genomic rna is packed, the spike (s) protein that forms the prominent coronavirus spikes, the m protein which is the most abundant component of coronavirus, and the envelope (e) protein which is a minor but yet critical component in virion assembly [4] . some group ii coronaviruses also encode an additional structural protein, the hemagglutinin-esterase (he). coronaviruses are known to assemble and bud at membranes of the intermediate compartment (ic) , locating between the er and golgi complex [5] . the m protein is a type iii membrane protein and a key player in coronavirus assembly. it spans the membrane bilayer three times, leaving a short amino-terminal domain on the virion exterior surface (or exposed luminally in intracellular membranes) and a large carboxy-terminal tail in the virion interior (or in the plasma) [6] . lateral interactions between m proteins are thought to mediate the formation of the virion envelope [7] . when expressed alone, m protein accumulates in the golgi complex in the form of homomultimeric complexes [8, 9] . however, in combination with the e protein, m is retained in the budding compartment and incorporated into virus-like particles (vlps) with similarity in size and shape to authentic virions, demonstrating that the m and e proteins are the minimal requirements for envelope formation for most coronaviruses, [10] . the m protein appears to interact with s and he proteins, and the s-m-he protein complexes can be detected in cells infected with the bovine coronavirus [6] . the m protein was also shown to interact with the mouse hepatitis virus (mhv) nucleocapsid consisting of the genomic-size mrna 1 and n protein in a pre-golgi compartment, probably at the er membrane. it may interact directly with the genomic rna through the packaging signal, initiating the m-nucleocapsid interaction [11] . there is also a detectably direct interaction between m and n proteins in the nucleocapsid, which may further stabilize the m-genomic rna interaction [11] . actin is the most abundant protein in a typical eukaryotic cell, accounting for about 15% in some cell types [12] . the protein is highly conserved, differing by no more than 5% between species as diverse as algae and humans. it polymerizes in a helical fashion to form actin filaments (or microfilaments) that form the cytoskeleton, a three-dimensional network inside a eukaryotic cell. actin filaments provide mechanical support for the cell, determine the cell shape, enable cell movements (through lamellipodia, filopodia, or pseudopodia), and participate in certain cell junctions, in cytoplasmic streaming and in cell contraction during cytokinesis [13] . in the present study, actin was shown to be co-purified with the ibv particles and was identified as a potential interacting protein of the ibv m protein. the interaction was subsequently confirmed by coimmunoprecipitation and immunofluorescent staining. mutation of amino acids a159 and k160 in the m protein abolished the interaction. introduction of the a159-k160 mutation into an infectious ibv clone system showed no infectious virus could be recovered, although viral rna replication and subgenomic mrna transcription were detected. furthermore, treatment of the infected cells with cell-permeable agent cytochalasin d at early, but not late, stages of the replication cycle showed no release of the virion to the cultured media, suggesting that disruption of actin filaments might block virion assembly and budding. yeast two-hybrid screening yeast two-hybrid screening was performed with the pretransformed matchmaker human bone marrow cdna library (clontech) according to the instructions of the manufacturer. in brief, the bait plasmid was transformed into yeast strain ah109, and transformants containing the bait plasmid were mated with the pretransformed cdna library. candidates were initially selected on sd-ade/-his/-leu/-trp plates, and plasmid dna was isolated from positive clones and sequenced according to the instructions of the manufacturer. h1299 and vero cells were cultured in rpmi-1640 and complete dulbecco's modified eagle's medium (invitrogen), respectively, supplemented with 10% new born calf serum (sterile) and 1% penicillin/streptomycin (invitrogen) and maintained at 37uc in humidified 5% co 2. constructs containing plasmid dna under the control of a t7 promoter were transiently expressed in mammalian cells using a vaccinia virus-t7 system. briefly, 90% monolayers of h1299 cells were infected with 10 plaque forming units (pfu)/cell of the recombinant vaccinia virus (vtf-3), which express the t7 rna polymerase gene, for 2 hours at 37uc prior to transfection. the plasmid dna was transfected into vtf-3 infected cells using lipofectamine 2000 reagent according to the instructions of the manufacturer (invitrogen). hela cells transfected with appropriate plasmids were lysed with tntg lysis buffer (30 mm tris-hcl ph 7.4, 150 mm nacl, 1% np40, and 10% glycerol) in the presence of 16protease inhibitor mixture (sigma) at 24 hours post-transfection. total cell lysates were immunoprecipitated with appropriate antibodies for 2 hours at 4uc and further incubated for 2 hours at 4uc after adding buffer-balanced protein a agarose beads. the beads were washed three times and subjected to electrophoresis on sds-12% polyacrylamide gel. ibv m protein was transiently expressed in h1299 cells grown on 4-well chamber slides (iwaki). after rinsing with phosphatebuffered saline (pbs), cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature and permeabilized with 0.2% triton x-100, followed by incubating with specific antibodies diluted in fluorescence dilution buffer (pbs with 5% newborn calf serum) at room temperature for 2 hours. cells were then washed with pbs, incubated with fitc-conjugated anti-rabbit secondary antibodies (dako) in fluorescence dilution buffer at 4uc for 1 hour and with alexa fluor 488 phalloidin (molecular probe) at rt for 20 minutes before mounting. confocal microscopy was performed on a zeiss microscope. samples were lysed with 26sds loading buffer and subjected to 10% sds-page. proteins were transferred to pvdf membrane (bio-rad) by using a semi-dry transfer cell (bio-rad, trans-blot sd), and blocked overnight at 4uc with 10% nonfat milk in pbs-t. the membranes were probed with specific primary antibodies followed by anti-mouse or anti-rabbit secondary antibodies conjugated with harderadish peroxidase (sigma). membranebound antibodies were detected with the enhanced chemiluminescence (ecl) detection reagents (amersham, uk). the virus was layered onto 20% tne-buffered sucrose solution (tne buffer: 50 mm tris-hcl, ph 7.4, 100 mm nacl, 1 mm edta) and centrifuged at 175,0006g for 3 hours at 4uc (cushion). the resulting virus pellets were resuspended in tne buffer, layered onto 10-50% linear sucrose gradient prepared with tne buffer, and centrifuged at 175,0006g for 18 hours at 4uc. aliquots of fractions starting from the top of the gradient were analyzed by sds-page. in vitro assembly and transcription of full-length cdna clones were carried as previously described [14] . briefly, plasmids were digested with either bsmbi (fragment a) or bsai (fragments b, c, d and e). bands corresponding to each of the fragments were cut from the gels and purified with qiaquick gel extraction kit (qiagen inc.). all the fragments were ligated with t4 dna ligase at 16uc overnight. the final ligation products were extracted with phenol/chloroform/isoamyl alcohol (25:24:1), precipitated with ethanol, and detected by electrophoresis on 0.8% agarose gels. full-length transcripts and n transcripts (using a linearized pkt0-ibvn containing ibv n gene and the 39-utr region as templates) were generated in vitro using the mmessage mmachine t7 kit (ambion, austin, tex) according to the manufacturer's instructions. vero cells were grown to 90% confluence, trypsinized, washed twice with cold pbs, and resuspended in pbs (14) . rna transcripts were added to 400 ml of vero cell suspension in an electroporation cuvette, and electroporated with one pulse at 450 v, 50 mf with a bio-rad gene pulser ii electroporator. the transfected vero cells were cultured overnight in 1% fbscontaining dmem in a 60 mm dish and further cultured in dmem without fbs. reverse transcription, rt-pcr, and real-time pcr viral rna was reverse-transcribed to cdna using superscript iii (invitrogen) with modification to the protocol as follows: random hexamers (300 ng) and total rna (5 mg) were incubated for 10 minutes at 70uc. the remaining reagents were added according to the manufacturer's recommendation and the reaction was incubated at 55uc for 1 hour followed by 20 minutes at 70uc to inactivate the rt. for rt-pcr, a forward primer in the leader sequence and a reverse primer were used to generate a product by pcr. quantitative real time rt-pcr was conducted using smart cycler ii (cepheid) with sybr green (cepheid) to detect subgenomic cdna with primers (7.5 pm) optimized to detect a product spanning the leader sequence to the 59 end of m gene or genomic cdna with primers (7.5 pm). the cdna from the rt reaction of each virus was diluted 1:10, and 1 ml was used for each pcr, with a total reaction volume of 25 ml. m gene was cloned by digesting pibvm with ncoi and ecori, and inserted into ecori/ncoi digested pgbkt7 to generate pgbkt7-m. the pcr fragment for actin was amplified by using primers 59-cgcggatccatggatgatgatatcgccgcg-39 and 59-ccgctcgaggaagcatttgcggyggacgat-39. the pcr fragment was digested with bamhi and xhoi and ligated into bamhi and xhoi digested pxj-myc to generate pxj-myc-actin. the deletion constructs md1, md2, md3, md4 and md5 were made by two rounds of pcr as described previously [15] . the primer used for md1 is 59-aactgcagttaagc-aagccactgaccctc-39. the primers used for md2 are 59-tcttttgtaggttataagtgtgaaccagac-39 and 59-gtctggttcacactta taacctacaaaaga-39. the primer for md3 is 59-aactgcagccgcttt ggtcaccag-39. the primers for md4 are 59-tgtgagggtccagac-cacttg-39 and 59-caagtggtctggaccctcaca-3. the primers for md5 are 59-ggtcagtggc tttgtgaa-ccagac-39 and 59-gtctggttcacaaagccactgacc-3. all constructs were confirmed by automated nucleotide sequencing. in a previous study, it was observed that a cellular protein with migration properties on sds-page similar to actin was consistently co-purified with highly purified ibv particles [16] . however, as no suitable antibodies against actin were readily available at the time, the identity of this protein was not established. this issue was revisited in this study. confluent monolayers of vero cells were infected with ibv at a multiplicity of infection of approximately 2 pfu/cell. the supernatants were collected at 18 hours post-infection and centrifuged at 4,000 rpm for 20 minutes to remove cell debris. the virus particles were spun down by ultracentrifugation through a 2 ml sucrose cushion (20%), and purified using a 10-50% sucrose gradient. following ultracentrifugation, 11 fractions were collected from top to bottom, and the presence of viral proteins was checked by western blot with anti-n antibodies, showing that the ibv n protein was detected in fractions 7-11 with majority of the protein located in fractions 8-10 ( fig. 1, top panel) . analysis of the same fractions by western blot with anti-actin antibodies showed that actin appeared in the same fractions as n protein, with the majority of the protein detected in fractions 8-10 ( fig. 1 , middle panel). fractionation of total lysates from cells without ibv infection under the same conditions showed that actin was mainly detected in fractions 3-4 ( fig. 1, bottom panel) . these results demonstrate that actin could indeed be co-purified with the virus particles. however, it is currently uncertain if actin could be incorporated into the virions. in an attempt to search for ibv proteins that may interact with b-actin, ibv structural and nonstructural proteins were used as baits to screen a human cdna library in yeast two-hybrid screening. among all the ibv proteins used, only the c-terminal cytoplamsic portion of the ibv m protein was able to interact with b-actin. to confirm the interaction by co-immunoprecipitation, the fulllength cdna for b-actin was amplified by rt-pcr from hela cells, cloned into an expression vector with a c-myc tag at its nterminus (myc-actin), and co-transfected into hela cells with the ibv m. analysis of cells expressing the myc-tagged actin either on its own or together with the m protein by western blot with anti-myc monoclonal antibody showed the detection of the myctagged actin (fig. 2, lanes 1 and 3) . similarly, analysis of cells expressing the m protein either on its own or together with the myc-tagged actin by western blot with anti-m polyclonal antibodies showed the detection of the full-length glycosylated (two upper bands) and unglycosylated (25 kda) forms of the m protein (fig. 2, lanes 5 and 6) . the same cell lysates were then subjected to immunoprecipitation with anti-myc antibody. western blot analysis of the precipitates with the same anti-myc antibody showed the detection of the myc-tagged actin expressed either on its own or together with the m protein (fig. 2, lanes 7 and 9) . western blot analysis of the same precipitates with anti-m antibodies showed the detection of the m protein only in cells coexpressing the two proteins (fig. 2, lane 12) . no detection of the m protein was found in cells expressing either the m protein or mycactin alone (fig. 2, lanes 10 and 11) . these results confirm that the ibv m protein could indeed interact with actin. to map the region in the m protein responsible for the interaction with actin by yeast two-hybrid screening, five deletion and one mutation constructs were made (fig. 3a) . in figure 3a , md1 contains the amino acid sequence from 104 to 159 (with deletion of amino acids 160 to 225); md2 contains the deletion of amino acids 104-159; md3 contains the amino acid sequence from 104 to 192 (with deletion of amino acids 193 to 225); md4 contains the deletion of amino acids 155-162; and md5 contains the deletion of amino acids 159-160 (fig. 3a) . the mutant construct mm1 contains the mutation of amino acids 159 and 160 from alanine and lysine to proline and glutamic acid, respectively (fig. 3a) . introduction of individual deletion and mutant constructs into the yeast stain ah109 together with pact-actin showed growth of wild type and md3 constructs on sd-trp/-leu/-his/ -ade selective plates (fig. 3b) . however, none of the other deletion and mutant constructs could grow on the same plate (fig. 3b) . western blot analysis showed similar expression levels of wild type and mutant constructs in yeast (data not shown). as md5 contains the minimal deletion of two amino acids, this study maps the actinbinding site on the m protein to the region containing amino acids a159 and k160. co-immunoprecipitation was then carried out by expressing the myc-tag wild type m (c-terminal domain from amino acid 104 to 225, myc-m) and myc-mm1 in cells. western blot analysis with anti-myc monoclonal antibody showed the expression of myc-m and myc-mm1 at a similar level (fig. 3c, upper panel, lanes 1 and 2) . the same cell lysates were then subjected to immunoprecipitation with anti-actin antibody, and western blot analysis of the precipitates with anti-myc antibody showed the presence of myc-m but not myc-mm1 (fig. 3c, lower panel, lanes 1 and 2) . the detection of myc-m in the immunoprecipitates was greatly enhanced by co-expression of myc-m and actin (fig. 3b, lane 4) . colocalization of the m protein with actin in cells treated with cytochalasin d as a type iii membrane protein with three transmembrane domains, the ibv m protein is mainly localized to the golgi apparatus in virus-infected cells and in cells expressing the m protein [8] . cytochalasin d is a cell-permeable fungal toxin. it can bind to the barbed end of actin filaments and inhibit both the association and dissociation of actin subunits, resulting in the disruption of actin filaments and inhibition of actin polymerization. to test the subcellular localization of the m protein in cells treated with cytochalasin d, h1299 cells were transfected with m construct and 12.5 mg/ml of cytochalasin d was added to the cells at 4 hours post-transfection. cells were permeabilized with 0.2% triton x-100 and stained with anti-m antibodies at 48 hours posttransfection. the same cells were also stained with alexa fluor 488 phalloidin, a specific dye for actin (molecular probe). staining of cells treated with dmso and alexa fluor 488 phalloidin showed a normal three-dimensional network (fig. 4, upper panels) . phalloidin showed that the regular cell actin filaments were destroyed (fig. 4, lower panels) , displaying random distribution of actin dots in cells without the m protein expression. in cells expressing the m protein, both m protein and actin were found mainly in the golgi area with fairly well overlapping of the two staining patterns (fig. 4, lower panels) . effects of deletion and mutation of amino acid residues a159 and k160 on the replication, budding and release of ibv two approaches were used to test the effect of deletion and mutation of amino acid residues a159 and k160 in the m protein on the replication, budding and release of ibv. first, the a159-k160 deletion and mutation were introduced into a full-length ibv infectious clone, and the effect of this minimal deletion and mutation on the replication of viral rna and the recovery of infectious ibv was analyzed. introduction of full-length transcripts derived from wild type (wt), the a159-k160 deletion construct (md5) and a159-p/k160-e mutation construct (mm1) into vero cells by electroporation showed efficient recovery of infectious ibv from cells transfected with wild type transcripts. however, it was consistently observed that no infectious virus could be recovered from cells transfected with either md5 or mm1 transcripts. as no infectious virus was recovered from cells transfected with md5 and mm1 transcripts, rt-pcr amplification of the negative strand rna was performed to check if rna replication occurred in the transfected cells. the primer pair (59-14931 gcttatc-cact agtacatc 14949 -39 and 59-15578 cttctcgcacttc-tgcactagca 15600 -39) was chosen to amplify the negative strand ibv sequence from nucleotides 14931 to 15600 by the rt-pcr. if replication of viral rna occurred, a 670 bp pcr fragment would be expected. as shown in fig. 5a , the 670 bp rt-pcr fragments were obtained from cells transfected with both wild type and mutant transcripts at 24 and 72 hours posttransfection, respectively (upper panel, lanes 2-7). interestingly, the amounts of the fragment detected from cells transfected with md5 and mm1 transcripts were increased at 72 hours posttransfection (fig. 5a , upper panel, lanes 3-4 and 6-7), demonstrating the replication of the transfected mutant transcripts. no rt-pcr fragment was detected in cells without transfection (fig. 5a, upper panel, lane 1) . rt-pcr amplification of subgenomic mrnas was then carried out to check if subgenomic mrna synthesis could occur in cells transfected with the mutant transcripts at 24 and 72 hours postelectroporation. the forward primer (59-acttaagataga-tatta a-39) used in this reaction corresponds to the leader sequence from nucleotides 26-46 in the genomic rna and the downstream primer (59-ctctggatccaataacctac-39) covers the ibv sequence from nucleotides 24784 to 24803. if transcription of subgenomic mrnas occurs, a 415 bp pcr product corresponding to the 59-terminal region of the subgenomic mrna4 would be detected. as shown in fig. 5a , a dominant 415 bp was observed in cells electroporated with wild type full-length transcripts at two time points (lower panel, lanes 2 and 5). in cells transfected with md5 and mm1 transcripts, a much weaker band was detected in cells transfected with the two mutant transcripts at 24 hours post-transfection (fig. 5a, lower panel, lanes 3-4) . similarly to the results described above, the amounts of the subgenomic rna 4 fragment detected from cells transfected with md5 and mm1 transcripts were increased approximately 3 to 5 fold, respectively, at 72 hours posttransfection based on real time pcr assay (fig. 5a, lower panel, lanes 3-4 and 6-7) . as a negative control, no detection of the amplified fragments was obtained from a mixture of rna templates containing the in vitro transcribed rna and total rna extracted from mock infected cells (fig. 5a, lower panel, lane 1) . in the second approach, plasmids containing wild type m (pibvm) and the a159-k160 deletion m sequences (pmd5) were transfected into h1299 cells using the vaccinia/t7 recombinant antibodies, respectively (fig. 5b) . in cells transfected with both wild type and md5 constructs with or without ibv infection, detection of similar amounts of m protein was observed (fig. 5b, top two panels, lanes 1 and 2) , suggesting that both constructs were expressed at similar efficiencies. however, much more n protein was detected in the infected cells transfected with wild type m construct than that in cells transfected with the md5 mutant (fig. 5b, middle panel, lanes 1 and 2) . as a loading control, similar amounts of actin were detected in cells transfected with both constructs (fig. 5b, bottom panel) . these results confirm that expression of the a159-k160 deletion m protein significantly reduces the infectivity of ibv, suggesting that expression of this mutant protein could inhibit the production of infectious virus in a dominant-negative manner. to rule out the possibility that the deletion and mutation may render detrimental effect on the overall structure of the m protein and its interaction with other viral proteins that are essential for virion assembly, interaction of the mm1 mutant with the e protein was tested by co-immunoprecipitation. as shown in fig. 5c , western blot analysis with anti-ibv e protein antibodies showed the presence of e protein in cells expressing the protein on its own or together with wild type or mm1 mutant protein (top panel). immunoprecipitation of the same lysates with anti-m antibodies and subsequent analysis of the precipitates by western blot with anti-m antibodies led to the detection of wild type or the mm1 mutant proteins in cells expressing m forms either alone or together with the e protein (fig. 5c, middle panel) . western blot analysis of the same precipitates with anti-ibv e antibodies, however, showed the presence of e protein only if it was coexpressed with either wild type or the mm1 mutant protein (fig. 5c, bottom panel) . these results confirm that the a159-p/ k160-e mutations did not affect the interaction between ibv m and e proteins. to define more precisely the stage of the viral replication cycle that is facilitated by the interaction between m protein and actin, the effects of disrupting actin filaments by cytochalasin d on the replication, budding and release of ibv were tested by detailed time-course experiments. confluent monolayers of vero cells in six-well plates were infected with ibv at a multiplicity of infection of approximately 3 pfu/cell. at 0, 4, 8, 12 and 16 hours postinfection, either 12.5 mg/ml of cytochalasin d or equal volume of dmso was added to the cells. the supernatants and cell pellets were separately harvested at 24 hours post-infection and the presence of ibv n protein was checked by western blot with anti-n antibodies. typical n protein profiles, including the full-length and posttranslationally modified forms of the protein, were detected in the total cell lysates (fig. 6, top panel) . in cells treated with cytochalasin d, slightly less amounts of the n protein were detected when cytochalasin d was added to the cells at 0, 4, 8 and 12 hours post-infection, respectively (fig. 6, top panel, lanes 1-4) . no obvious difference in the expression of n protein was seen when cytochalasin d was added at 16 hours post-infection (fig. 6 , top panel, lane 5). western blot analysis of actin was included as a loading control (fig 6, middle panel) . analysis of the n protein in the clarified supernatants showed the detection of a single n protein species that represents the n protein incorporated into the virion particles in cells treated with dmso alone (fig. 6, bottom panel, lanes 6-10) . in supernatants collected from cells treated with cytochalasin d, much reduced amounts of the n protein were detected in cells treated with the reagent at 12 and 16 hours postinfection, respectively (fig. 6, bottom panel, lanes 4 and 5) . no n protein was detected in supernatants harvested from cells treated with cytochalasin d at 0, 4 and 8 hour post-infection, respectively (fig. 6, bottom panel, lanes 1-3) . coronavirus m protein plays essential roles in virion assembly and budding [18, 19] . the protein itself cannot bud. however, with the help of e protein, vlps may be formed at the ic membranes. as coronaviruses do not contain a matrix protein that underlines the membrane, the m protein may serve as a matrixlike protein. it is proposed that the formation of coronavirus envelope is dominated by lateral interaction between m molecules that form a two-dimensional lattice in intracellular membranes [20] . the m-m interaction is essential but not sufficient for coronavirus envelope assembly. free energy is needed to generate and stabilize membrane curvature, suggesting that interaction of m protein with host proteins would be a must [21] . to support this speculation, the results present in this study demonstrate that interaction of the ibv m protein with b-actin is essential for virion assembly and budding. the interacting region in the m protein was pinpointed by deletion and mutation studies. as md3 is able to interact with actin, but md1 and md2 could not, it was reasoned that the domain responsible for this interaction may be located in the region covering amino acids 159-160. this region was chosen for further deletion and mutational analysis, confirming that it is indeed involved in the interaction with actin. however, the two amino acids are not conserved in other coronavirus m proteins. at present, we do not know whether interaction between coronavirus m protein and actin is a general phenomenon for all coronaviruses, or is just ibv-specific. available evidence suggests that the host cytoskeleton, especially actin, is involved in the budding process of several animal viruses [22] . actin has been identified in many enveloped viruses, such as rous sarcoma virus, mouse mammary tumor virus, sendai virus and measles virus. in the case of measles virus, actin was originally thought as a cellular contaminant, but was later demonstrated to be an internal component of the virus [23] [24] [25] . actin was found in the purified paramyxovirus particles and actin filaments were seen in association with the budding virions by electron microscopy [24] [25] [26] . polymerized actin was also found in hiv preparations [27, 28] . the functional implication for the incorporation of actin into these virus particles remains unclear. one possibility is that actin polymerization serves as an additional force for membrane bending during budding [1, 29] . as an illustrative example, actin was shown to control the movement of measles virus envelope proteins on the surface of infected cells [30] . interaction of actin with viral components was also reported in several other viral systems. one example is the interaction of the nuclocapsids of murine mammary tumor virus with actin, which was required for extruding the virus particles from virus-infected cells [31] . the m protein of newcastle disease virus could interact with actin in vitro [1, 32] . the sars-cov n protein could aggregate elongation factor 1a through direct interaction, leading to the inhibition of protein translation and cytokinesis by blocking f-actin bundling [33] . it was also reported that the sars-cov n protein could induce apoptosis and actin reorganization in mammalian cells under stress conditions [34] . the results shown in this study demonstrate that actin could be co-purified with ibv virions and that the ibv m protein is associated with actin, suggesting that actin is likely to be incorporated into ibv virions through its interaction with the m protein. however, more conclusive data, such as immunogold labeling of highly purified virions, are currently lacking. it is interesting that ibv replication cycle was disturbed in cells treated with cytochalasin d. cytochalsin d could prevent actin from polymerization, leading to malfunction of actin. when actin was disrupted, much less ibv virions were released from the cells into the culture medium, demonstrating that actin plays a very important role in the ibv replication cycle. two possible mechanisms were considered. first, actin may be involved in the formation of the m lattice that may promote and stabilize its curving, resulting in the enhancement of some late processes, such as virion budding and release. second, the mhv m protein was shown to be recycled from the golgi complex back to early compartments, such as the er and ic, which are functionally important in coronavirus infected cells [21] . therefore, interaction of actin with m protein may help the retrograde transport of m protein from golgi apparatus to some early compartments and provide more m protein for virion assembly and budding. the actin-binding proteins are grouped into 60 distinct classes based on primary structures [35] . the sequence of the actinbinding sites ranges from 10-30 residues, which shows no obvious homology among different classes [36] . for example, aldolase, a glycolytic enzyme, binds actin filaments to concentrate enzyme and substrate [37] . the sequence 32-adestgsiakrlqsig-tente-52 of aldolase has been identified as the actin-binding motif [38] . furthermore, carcinoembryonic cell adhesion molecule 1 (ceacam1) was found to bind f-actin. the actin binding site is flhfgktgssgplq, which is not similar to other existing actin-binding sequences [39] . coronavirus m protein is an essential and predominant component of the virions. it plays pivotal roles in virion assembly and budding by interaction with other viral components. firstly, the monomers of m could interact with each other particularly via the transmembrane domains [21] . besides, the m-n interaction region was narrowed to the 16 residues adjacent to the carboxy terminus of tgev m protein [40] . however, the specific amino acids on m protein responsible for m-s and m-e interactions are still enigmatic. in the present study, deletion or mutation of amino acids a159 and k160, which are essential for the interaction of m protein with actin, is detrimental to the recovery of ibv from an infectious ibv clone due to a defect in late stages of the ibv replication cycle. although, the deletion or mutation of amino acids a159 and k160 could also interact with another structural protein e, we cannot exclude the possibility that this region is essential for maintaining the integrity of the m protein and involved in the interactions with other viral components. nevertheless, it lends support to the conclusion that interaction of the ibv m protein with actin is essential for the completion of the ibv infection cycle. more evidence is derived from the study using cytochalasin d. similar amount of viral proteins were detected in the total cell lysates but were not detected in supernatants when cytochalasin d was added early in the viral replication cycle suggests that actin filaments are essential for some early events, such as virion assembly and budding, during the assembly and maturation processes of ibv. as ibv proteins can be efficiently detected in the supernatants when cytochalasin d was added at 12 and 16 hours post-infection, respectively, it suggests that actin filaments are unlikely involved in the release of the ibv particles. instead, our data support that actin filaments may be involved in the virion assembly and budding process of the coronavirus replication cycles. as coronavirus m protein could bind to the viral genomic rna through packaging signal [41] and rna replication in the cytoplasm would rely on the support of actin [42] , the interaction of m protein with actin may serve as a platform to promote the binding of m protein to viral rna as well as viral rna replication. alternatively, coronavirus m protein could bind to the n protein-rna complex to stabilize the nucleocapsid [11] and extra energy would be needed in this process [21] . the interaction of m protein with actin may serve as a power station to provide energy for completion of the viral replication cycle. virus maturation by budding cell biology of viruses that assemble along the biosynthetic pathway cargos and genes: insights into vesicular transport from inherited human disease coronavirus particle assembly: primary structure requirements of the membrane protein characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the rer to 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infectious bronchitis virus 3c protein with the virion envelope cell cycle arrest and apoptosis induced by the coronavirus infectious bronchitis virus in the absence of p53 structural analysis of virion proteins of the avian coronavirus infectious bronchitis virus isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid envelope glycoprotein interactions in coronavirus assembly assembly of the coronavirus envelope: homotypic interactions between the m proteins cellular proteins bound to immunodeficiency viruses: implications for pathogenesis and vaccines chromatographic and electrophoretic analysis of viral proteins from hamster and chicken cells transformed by rous sarcoma virus the synthesis of sendai virus polypeptides in infected cells structural polypeptides of measles virus structural polypeptides of mumps virus cytoskeletal proteins inside human immunodeficiency virus type 1 virions rotavirus spike protein vp4 binds to and remodels actin bundles of the epithelial brush border into actin bodies dynamics of membranes driven by actin polymerization polar appearance and nonligand induced spreading of measles virus hemagglutinin at the surface of chronically infected cells the mechanisms of appearance of viral glycoproteins at cell surface membrane interaction of cellular tubulin with sendai virus m protein regulates transcription of viral genome the nucleocapsid protein of severe acute respiratory syndrome coronavirus inhibits cell cytokinesis and proliferation by interacting with translation elongation factor 1alpha the sars coronavirus nucleocapsid protein induces actin reorganization and apoptosis in cos-1 cells in the absence of growth factors xin repeats define a novel actin-binding motif structural relationships of actinbinding proteins opposite effects of alpha-actinin and of fructose 1,6 -bisphosphate aldolase on the microfilament network. the role of orthophosphate revisited identification of an actin binding region in aldolase carcinoembryonic antigen cell adhesion molecule 1 directly associates with cytoskeleton proteins actin and tropomyosin the membrane m protein carboxy terminus binds to transmissible gastroenteritis coronavirus core and contributes to core stability nucleocapsid-independent specific viral rna packaging via viral envelope protein and viral rna signal cellular factors in the transcription and replication of viral rna genomes: a parallel to dna-dependent rna transcription key: cord-352219-z245sb3s authors: tallam, aravind; perumal, thaneer m.; antony, paul m.; jäger, christian; fritz, joëlle v.; vallar, laurent; balling, rudi; del sol, antonio; michelucci, alessandro title: gene regulatory network inference of immunoresponsive gene 1 (irg1) identifies interferon regulatory factor 1 (irf1) as its transcriptional regulator in mammalian macrophages date: 2016-02-12 journal: plos one doi: 10.1371/journal.pone.0149050 sha: doc_id: 352219 cord_uid: z245sb3s immunoresponsive gene 1 (irg1) is one of the highest induced genes in macrophages under pro-inflammatory conditions. its function has been recently described: it codes for immune-responsive gene 1 protein/cis-aconitic acid decarboxylase (irg1/cad), an enzyme catalysing the production of itaconic acid from cis-aconitic acid, a tricarboxylic acid (tca) cycle intermediate. itaconic acid possesses specific antimicrobial properties inhibiting isocitrate lyase, the first enzyme of the glyoxylate shunt, an anaplerotic pathway that bypasses the tca cycle and enables bacteria to survive on limited carbon conditions. to elucidate the mechanisms underlying itaconic acid production through irg1 induction in macrophages, we examined the transcriptional regulation of irg1. to this end, we studied irg1 expression in human immune cells under different inflammatory stimuli, such as tnfα and ifnγ, in addition to lipopolysaccharides. under these conditions, as previously shown in mouse macrophages, irg1/cad accumulates in mitochondria. furthermore, using literature information and transcription factor prediction models, we re-constructed raw gene regulatory networks (grns) for irg1 in mouse and human macrophages. we further implemented a contextualization algorithm that relies on genome-wide gene expression data to infer putative cell type-specific gene regulatory interactions in mouse and human macrophages, which allowed us to predict potential transcriptional regulators of irg1. among the computationally identified regulators, sirna-mediated gene silencing of interferon regulatory factor 1 (irf1) in macrophages significantly decreased the expression of irg1/cad at the gene and protein level, which correlated with a reduced production of itaconic acid. using a synergistic approach of both computational and experimental methods, we here shed more light on the transcriptional machinery of irg1 expression and could pave the way to therapeutic approaches targeting itaconic acid levels. immune cells specifically respond to various inflammatory environments based on the nature and type of external stimuli. macrophages trigger defensive pathways upon stimulation of pattern-recognition receptors, such as toll-like receptors (tlrs) [1, 2] . previous studies have shown that mouse macrophages under pro-inflammatory conditions, such as bacterial infections or lipopolysaccharide (lps) stimulation, highly express immunoresponsive gene 1 (irg1) [3, 4] . recently, we demonstrated the active role of irg1 in antimicrobial responses linking metabolism to immunity [5] : immune-responsive gene 1 protein/cis-aconitic acid decarboxylase (irg1/cad) catalyzes the decarboxylation of cis-aconitic acid to itaconic acid (also known as methylenesuccinic acid) during the tca cycle. itaconic acid is an organic compound that inhibits isocitrate lyase, the first enzyme of the glyoxylate shunt, a savior pathway for bacterial growth under nutrient-deprived conditions. for the first time, we also demonstrated irg1 expression and itaconic acid production in lps-treated human peripheral blood mononuclear cells (pbmcs)-derived macrophages [5] . thus, irg1/cad contributes to the host immune response against bacterial invasion providing an additional support to the innate immune system. in addition to our findings, irg1 was also previously shown to be expressed in mouse macrophages under different tlr ligand stimulations [3, 6] and microbial infections [4, 7] . high upregulation of irg1 was observed in the lungs of mice when infected with influenza a virus, thus showing its induction also under viral infections [8] . in a different context, irg1 was found to be highly expressed in the uterine luminal epithelium of the mouse during the early stages of pregnancy due to the synergistic regulation by progesterone and estradiol mediated by the protein kinase c pathway [9, 10] . irg1 was also reported to be highly expressed in pbmcs of septic patients where it fosters endotoxin tolerance by enhancing a20 expression via reactive oxygen species (ros) signaling [11] . apart from mouse and human, irg1 is also expressed in other species under microbial infections. in zebrafish (danio rerio), when infected with salmonella species, irg1 is specifically expressed by macrophage-lineage cells and is cooperatively regulated by glucocorticoid and jak/stat signaling pathways [12] . furthermore, it was shown that irg1 is a key component responsible for the production of mitochondrial ros augmenting the bactericidal activity of macrophages. hence, in zebrafish, irg1/cad additionally contributes to immune responses by the production of ros [12] . taken together, these findings demonstrate a pivotal role of irg1/cad in the immune metabolism axis connecting the immune system with cellular metabolism through the production of itaconic acid and ros. despite the profound biological importance of irg1/cad, the molecular mechanisms that induce irg1 expression have not yet been investigated. the regulation of irg1 expression was reported on several findings, which are rather contrasting, such as de novo protein synthesis independent [3] and dependent [13] , myd88-independent [14] and dependent [7] , trif-independent [15] , tlr2-and tlr4-independent [13] . interestingly, irg1 was highly induced when stimulated with ifnγ alone or in combination with tnfα and it was shown that the vast majority of the murine irg1/cad protein was found in the mitochondrial fraction [6] . all the above experiments differ by cell types, the nature of the stimuli and perturbing compound concentrations. it is highly likely that irg1 is regulated by a complex transcriptional machinery responding to cellular environments and external stimuli. hence, it is important to unravel its transcriptional machinery to understand its role and expression under specific inflammatory conditions. gene regulatory networks (grns) capture the dependency between transcription factors, genes, proteins and small molecules underlying cellular processes [16, 17] . inferring regulatory interactions linked to irg1 can contribute to identify the major regulatory elements involved in the induction of irg1. there are three main grn inference approaches, which are widely used. the first is based on existing literature information which use natural language processing algorithms and manual curation to mine scientific articles [18] [19] [20] . the second approach is purely data-driven without any prior information. these category of methods infer the regulatory networks directly from multi-omics data based on the underlying conditional dependency structures such as co-expression [16] , mutual information [21] or regression [22, 23] among the genes. the third category of methods uses predictive modeling and experimental approaches, such as chip-seq, to infer transcription factor-dna (tf-dna) binding sites (tfbs) and motifs [24, 25] . all the three category of methods have their own advantages and disadvantages, while some require high dimensional data (i.e. high number of replicates and/or perturbation conditions), others are skewed with most studied interactions or biological processes. however, due to the course of dimensionality, most of them suffer from a high number of false positive interactions, thus making the subsequent network analysis of grns more elusive. to this end, in this context, we used an integrated algorithm, which leverages on the information from all the three different category of methods (i.e. literature, tfbs and data-driven) to infer the regulatory network of irg1. this algorithm, with some minor modifications from [26] , is used to infer the grn of irg1 from literature and tfbs, and to prune inconsistent interactions by contextualizing the model to predict differential expression from genome-wide expression arrays. putative transcriptional regulators of irg1 were hypothesized from the resulting grn and tested using sirna-mediated gene silencing experiments in mouse and human macrophages under lps stimulation. the murine raw264.7 macrophage cell line [27] was cultured in dmem medium (invitrogen, life technologies, carlsbad, california) with 10% fetal bovine serum (fbs), 0.1mg/ml streptomycin (invitrogen, life technologies, carlsbad, california) and 100u/ml penicillin (invitrogen, life technologies, carlsbad, california). for the experiments, lipopolysaccharide (lps from escherichia coli 055:b5, sigma) was used at a final concentration of 10ng/ml. primary monocytes were extracted from the blood samples of anonymous healthy male donors, donated by the luxembourgish red cross (http://www.croix-rouge.lu/). human blood samples in the present study were obtained under a mutual agreement between the university of luxembourg and the luxembourgish red cross for blood donation to non-therapeutic purposes. the institutional review board waived the need for consent. the comité national d'ethique de recherche (cner) (http://www.cner.lu/) approved this study. the components of the blood (peripheral blood mononuclear cells-pbmcs -, plasma and erythrocytes) were separated by ficoll density gradient separation. for this purpose, the blood was diluted 1:1 with phosphate buffered saline (pbs, invitrogen, life technologies, carlsbad, california) in falcon tubes and was transferred to leucosep tubes (greiner bio one, kremsmünster, austria) filled with 15ml of ficoll (vwr, radnor, pennsylvania). after a 10 minute centrifugation (1000 g at room temperature without break), the pbmcs layer was collected and the cd14+ monocytes were isolated by using the macs1 technology (magnetic separation) from miltenyi biotec (bergisch gladbach, germany). pbmcs were mixed with 200μl of cd14 microbeads and incubated for 30min at 4°c on a rotating platform followed by magnetic separation using lscolumn. isolated cd14+ monocytes were plated at 4x10 6 cells per well in 6-well plates (or 2x10 6 cells per well in 12-well plates, 1x10 6 cells per well in 24-well plates, 2.5x10 5 cells per well in 96-well plates) and differentiated for 11 days into macrophages using rpmi 1640 medium (vwr, radnor, pennsylvania) supplemented with 10% human serum, off the clot, type ab (a&e scientific, paa, pasching, austria, lot number: c02108-1021), 0.1mg/ml streptomycin, 100u/ml penicillin and 0.1mm l-glutamine (invitrogen, life technologies, carlsbad, california). during the differentiation, the medium was replaced with fresh medium on day 4 and day 7. alternatively, cd14+ monocytes were plated at 2x10 6 cells per well in 12-well plates and differentiated for 8 days into dendritic cells using human serum supplemented with 20ng/ml of both granulocyte-macrophage colony-stimulating factor (gm-csf, r&d systems europe ltd., united kingdom) and interleukin-4 (il-4, r&d systems europe ltd., united kingdom). for the experiments, lps was used at 10μg/ml, while tumor necrosis factor alpha (tnfα, r&d systems europe ltd., united kingdom) and interferon gamma (ifnγ, r&d systems europe ltd., united kingdom) were used at a final concentration of 50ng/ml. total rna was purified from cultured cells using the qiagen rneasy mini kit (qiagen) according to manufacturer's instructions. first-strand cdna was synthesized from 0.5 to 2μg of total rna using superscript iii (invitrogen) with 1μl (50μm)/reaction oligo(dt)20 as primer. individual 20μl sybr green real-time pcr reactions consisted of 2μl of diluted cdna, 10μl of 2×iq sybr green supermix (bio-rad), and 0.5μl of each 10μm optimized forward and reverse primers in 7μl rnase free water. primer sequences designed using beacon designer software (bio-rad), provided by eurogentec, or directly designed by thermo scientific and are shown in s1 table. for the human irg1 primers, the ncbi/primer-blast tool available at http://www.ncbi.nlm.nih.gov/tools/primer-blast/ was used. the pcr was carried out on a light cycler 480 (roche diagnostics), using the following program: 10min at 95°c and 40 cycles of 30s at 95°c, 30s at 60°c and 30s at 72°c followed by 10s 70-95°melting curves. all experiments, including three no template controls, were performed in triplicates for each sample. for normalization, l27 was amplified simultaneously. the on-targetplus smartpool, containing four different sirna sequences specifically targeting each of murine irf1 (sirna irf1, l-046743-01-0005), murine cebpb (sirna cebpb, l-043110-00-0005), human irf1 (sirna irf1, l-011704-00-0005) and the corresponding non-targeting control (sirna neg, d-001810-10-05), were designed and synthesized by thermo scientific dharmacon. murine raw264.7 macrophages were transfected with amaxa 4d nucleofector device, xunit (lonza) using the amaxa sg cell line 4d nucleofector kit for thp-1 cells according to the manufacturer's instructions. briefly, transfection with sirna complexes was carried out from pelleted and resuspended cells (1x10 6 cells per condition). transfection reagent and sirna were prepared according to the manufacturer's instructions (amaxa). specific sirnas were added at a final concentration of 100nm. after nucleofection using the program "raw264.7 (atcc)", the cells were seeded at a density of 1x10 6 cells per well in 12-well plates in dmem supplemented with 10% fbs and incubated for 24h. human pbmcs-derived macrophages were transfected using the amaxa 4d nucleofector device, y-unit, which was specifically designed for transfection of adherent cells. for these experiments, cells were seeded at 1x10 6 cells in 24-well plates for 11 days and the medium was then replaced with nucleofection reagent and specific sirnas (2μm) according to the manufacturer's protocol. the reagent solution was removed and the medium was added to the cells which were then incubated for 24h and stimulated with lps. for immunofluorescence the cells were grown on cellcarrier 96 well plates (perkin elmer), washed with pbs, and fixed with 4% paraformaldehyde (ph 7.4) for 30min at ambient temperature. after washing in pbs, the cells were permeabilized for 5min with 0.5% triton x-100 in pbs. permeabilization was followed by 3 x 10min washing steps in pbs. for blocking, samples were incubated with 5% goat serum in pbs for 1h at room temperature. primary antibodies against irg1/cad (anti-irg1 antibody produced in rabbit, sigma, 1/200) and a mitochondrial surface antigen (mab1273, millipore, 1/100) were diluted in pbs + 1% bsa and bound for 1h at room temperature. after three washing steps in pbs, secondary antibodies (a-21428, a-11001, invitrogen, both 1/500) were added and incubated at dark for 1h at room temperature. for staining of nuclei and stabilization of fluorescent signals the samples were covered with fluoroshield mounting medium containing dapi (f6057, sigma). image acquisition started after 10min of incubation. image stacks with 11 planes were acquired on a confocal opera qehs high content screening microscope (perkin elmer), using a 60x water immersion objective (na 1.2). the antibody-labeled mitochondrial channel was excited with a 488nm laser and detected with a 520/ 35 band-pass filter. antibody-labeled irg1/cad channel was excited with a 561nm laser and detected with a 600/40 band-pass filter. dapi was excited with a 405nm laser and detected with a 450/50 band-pass filter. automated image analysis was performed in matlab 2015a. nucleus segmentation was following the rule that pixels of low pass filtered dapi images, convolved with a gaussian filter of size 20 and sigma 5 have to be at least 25% brighter within nuclei as compared to local surroundings as defined by an average filter of size 100. the minimum size of nuclei was set to 1000 pixels. for the detection of cell covered regions, the three channels were summed up and low pass filtered with a gaussian filter of size 50 and sigma 20. resulting images were thresholded by the first quartile of pixel values. to detect single cell areas, a watershed algorithm was applied to the euclidian distance transform of the nucleus mask. mitochondria were segmented via a combination of local thresholding and segmentation of non-uniformly corrected mitochondria images. according to the local thresholding algorithm, mitochondrial pixel assignment requires a raw mitochondrial image, low pass filtered with a gaussian filter of size 5 and sigma 2 to be brighter than the same image convolved with an average filter of size 5. for non-uniform correction mitochondria channel images average filtered with a structuring element of size 50 were subtracted from original mitochondria images. for segmentation these corrected images were thresholded to a pixel intensity of 10. for connected components confirmed by both rules the minimum volume was set to 10 pixels. irg1/cad positive pixels were defined by the same algorithm as mitochondrial pixels, in this case applied to the irg1/cad channel. subcellular localization of irg1/cad was evaluated by computing single cell proportions of irg1/cad positive pixels in nuclei and mitochondria. to lyse the cells. cells were then scraped out from the plates and the lysate was shaken at 4°c for 20min at maximum speed. the lysate was then centrifuged at 4°c for 5min at maximum speed. the supernatant was collected and stored at -80°c. the extracted protein samples were then quantified using a bradford assay and the measurements were used for subsequent western blotting. heat-denatured protein samples (20μg) were separated on a 10% sds-polyacrylamide gel electrophoresis followed by transfer to nitrocellulose membranes 0.2μm (sigma). affinity-purified goat anti-mouse irf1 antibody (catalogue #: af4715) and rabbit anti-goat igg secondary antibody (catalogue #: haf017) were obtained from r&d systems europe ltd., united kingdom, while rabbit anti-human irg1 antibody (catalogue #: hpa040143) and normal rabbit igg (catalogue #: sc-2027) were purchased from sigma and santa cruz biotechnology, respectively. goat anti-β-actin (catalogue #: sc-1616) and anti-goat secondary antibodies (catalogue #: rpn1025) were purchased from santa cruz biotechnology and ge healthcare, respectively. after blocking with 5% (wt/vol) dry milk in pbs, the membrane was incubated overnight at 4°c with primary antibody in 1% bsa/pbs (dilution 1:2500 for irf1 and irg1) on a rotating platform. after three washing steps with pbs containing 0.1% tween-20, the membrane was incubated with secondary antibodies (dilution 1:5000) coupled to horseradish peroxidase and revealed by chemiluminescence using the amersham ecl detection reagents (ge healthcare) and odissey imaging system. cells seeded in 6-well plates were washed with 1ml of saline solution (0.9% nacl) and quenched with 0.4ml cold methanol. after adding an equal volume of cold water, cells were collected with a cell scraper and transferred in tubes containing 0.4ml cold chloroform. the extracts were vortexed at 1400rpm for 20min at 4°c and centrifuged at 13500rpm for 5min at 4°c. a volume of 0.3ml of the upper aqueous phase was collected in specific gc glass vials and evaporated under vacuum at -4°c using a refrigerated centrivap concentrator. extractions of metabolites from cells grown in 12-well plates were performed using half of the volumes. the interphase was centrifuged with 50μl cold methanol at 13500rpm for 5min at 4°c. when needed, the pellet was stored at -80°c for subsequent rna isolation. metabolite derivatization was performed using a multi-purpose sampler (gerstel). dried samples were dissolved in 15μl pyridine, containing 20mg/ml methoxyamine hydrochloride, at 40°c for 60 minutes by shaking. after adding 15μl n-methyl-n-trimethylsilyl-triflouroacetamide (mstfa), samples were incubated at 40°c for 30min with continuous shaking. gc-ms analysis was performed by using an agilent 7890a gc coupled to an agilent 5975c inert xl msd. a sample volume of 1μl was injected into a split/splitless inlet operating in splitless mode at 270°c. the gas chromatograph was equipped with a 30m agilent j&w db-35ms capillary column + 5m duraguard capillary in front of the analytical column. helium was used as carrier gas with a constant flow rate of 1.2ml/minute. the gc oven temperature was held at 90°c for 1min and then increased to 320°c at 15°c/minute. the final temperature was held for 8min. the transfer line temperature was set constantly to 280°c. the msd was operating under electron ionization at 70ev. the ms source was held at 230°c and the quadrupole at 150°c. the gc-ms was operated in selected ion monitoring (sim) mode (m/z 215.1, m/z 230.1, m/z 259.1). the total run time of one sample was 24.3min. all gc-ms chromatograms were processed using metabolitedetector [28] for targeted data analysis. the software package supports automatic deconvolution of all mass spectra. the obtained mass spectra were matched against a reference library (including the mass spectrum of the authentic standard "itaconic acid"). compounds were annotated by retention time and mass spectrum (overall similarity: 0.95 or higher). for quantification, fragment ion m/z 259 was used. total rna from pbmcs-derived macrophages was extracted using trizol reagent (invitrogen) while total rna from raw264.7 cells was harvested using the qiagen rneasy mini kit (qiagen), according to the manufacturer's protocols. rna purity and integrity were monitored using nanodrop1 nd-1000 spectrophotometer and agilent 2100 bioanalyzer with rna 6000 nano assay kit. only rnas with no sign of contamination or marked degradation (rin > 9) were considered good quality and used for further analysis. genechip human gene 1.0st and genechip mouse gene 2.0st arrays (affymetrix) were used to determine the genome-wide expression profiles of pbmcs-derived macrophages and raw264.7 cells, respectively. total rnas (250ng) were processed using the affymetrix whole transcriptome (wt) expression kit according to the manufacturer's instructions (user manual p/n 4425209 rev. c 09/2009). microarrays were hybridized, washed and stained using the affymetrix genechip wt terminal labeling and hybridization kit following the microarrays were washed, stained and scanned according to manufacturer's standard procedures (user manual p/n 702808 rev. 6). genechips were scanned using an affymetrix genechip scanner 3000 generating cel files containing hybridization raw signal intensities which were preprocessed and normalized using the genechip robust multiarray averaging (gcrma) algorithm [29] from bioconductor in r. redundant probe sets were merged by considering mean values, resulting in a list of unique annotated genes mapped based on entrez gene identifiers. using the limma package and the ebayes function from affy library in bioconductor, genes whose expression values between any two conditions having a difference with a log fold change (logfc) ! +/-1 and a p-value<0.01 were considered as differentially expressed genes (degs). microarray expression data are available at gene expression omnibus (geo) database (http://www.ncbi.nlm.nih.gov/geo/) under the accession number gse76563. grn inference was performed using three distinct approaches: (a) from transcription factor-dna (tf-dna) binding models: upstream regulation of irg1 was inferred using match™ algorithm of transfac 1 database from the biobase international corporation [25, 30] . match™ algorithm is a weight-matrix based algorithm which searches for potential transcription factor binding sites (tfbs) on any given genomic sequence using the position weight matrices (pwms) library from transfac 1 database. the pwms for transcription factors (tf) in transfac 1 are constructed based on the consensus of its dna binding sequences across the genome. each pwm consists of the nucleotides and their frequency at respective position on the binding sequence. for our analysis, immune cell specific profiles of match™ consisting of pwms of tf that are involved in the immune responses in t-cells, bcells, mast cells, myeloid cells, natural killer cells and macrophages were used. transcription start site (tss) information of genes from refseq and genome sequences of range 2000bp upstream and 1000bp downstream with respect to tss from hg19 (human), mm10 (mouse) from ucsc website were obtained. taking these sequences as input, match™ algorithm searches for potential binding sites of tfs on the sequence using its collection of pwms. the output consists of information about the transcription factor, binding position, genomic strand and the two quality scores (core and matrix similarity), sequence motif of each binding site found. only the transcription factor binding predictions that have both the similarity scores ! 0.90 were considered for further analysis (s2 table) . (b) from published literature: on the other hand, downstream regulation of lps stimulus in macrophages was inferred using pathway studio desktop 10 from elsevier [20] . pathway studio [20] is the knowledge base of ontologies, taxonomies and biological relationships derived based on the text mining algorithms and the expert curation of published scientific literature. medscan1 text mining technology was used to scan all the pubmed abstracts (%30 million) and relevant sentences were collected based on the manually curated dictionaries with the synonyms of biological terms. for this network, the extraction of published literature was restricted as follows: species: human/mouse; cell type: macrophages; interactions: direct regulations in the network building settings of the pathway studio software. (c) combining and contextualizing literature and tfbs networks: by virtue, the grn inferred above are from different cell, tissue and/or organism. also, for some of the tf-dna predictions and literature inferences, the mode of action is unspecified (i.e. activation or inhibition). therefore, in order to find context specific interactions (i.e. that are specific to irg1 expression in mouse macrophages and/or human pbmcs), we prune the inconsistencies in the inferred grn with an improved version of the previously developed contextualization algorithm [31] . in gist, using feedback regulations as basic building blocks, this algorithm tries to predict differential expression from genome-wide arrays, using a boolean modeling framework, by removing as well as assigning mode of actions to the interactions in the grn [32] . this method was implemented in matlab using the genetic algorithm (ga) function with the assumption that each cell phenotype represents a stable steady state (attractor) of the network. the pbn-matlab-toolbox (http://code.google.com/p/pbn-matlab-toolbox/downloads/list) with the synchronous updating scheme was employed in this algorithm for the boolean simulation. in order to identify the transcriptional regulators for irg1 under lps stimulation, the contextualised grn was used to hypothesize experimentally testable predictions. all simple paths connecting lps stimulation to irg1 from the inferred grn were computed using the graph k shortest paths algorithm of matlab. these paths provide the basis set of putative regulators and pathways for translating the lps signal to induce irg1. further, using the simple paths, the importance of tfs were established using a gene essentiality metric score. this score defines the importance of a particular gene in propagating the signal from the ligand stimulus to the target gene. the score ranges from 0 to 1 with 1 being the most essential gene and 0 being nonessential. however, the score cannot be 0 for any gene as all the genes are at least participating in one of the paths between lps-irg1. these genes were then ranked according to their essentiality metric in these paths. the score was calculated according to the formula: where em j denotes the essentiality metric for gene j, n tp denotes the number of total paths, n jp denotes the number of paths in which gene j is absent. hence, if the number of paths in which a particular gene is absent is low, its essentiality metric will be high. the top ranked tfs were then chosen to perform sirna-mediated gene silencing experiments to analyze the effect on irg1 expression. overall, the workflow from the inference of grns to validation studies is depicted in fig 1. fig 1. workflow for the identification of transcriptional regulators for a specific gene. the scheme shows the workflow of the different steps followed to identify potential transcriptional regulators for a given gene under lps exposure. the upstream network was initially constructed using the pwms from transfac 1 and the prediction algorithm match™. the literature network downstream of lps was inferred using the pathway studio knowledge database. these networks were merged and the merged network was contextualised using the booleanised genome-wide expression data. finally, the ranking scheme with simple paths and essentiality metric resulted in a set of potential transcriptional regulators which were tested using sirna mediated gene silencing experiments. otherwise mentioned, p-values were calculated according to the student t-test with the twotailed distribution assuming two-sample equal variance. we first analysed irg1 expression in human pbmcs-derived monocytes (fig 2a) , pbmcsderived macrophages (fig 2b) and pbmcs-derived dendritic cells (fig 2c) under various inflammatory conditions induced either by lps (10μg/ml), tnfα (50ng/ml), ifnγ (10ng/ ml), lps with tnfα or ifnγ exposures. after 6 hours, the expression levels of irg1 were highly increased following the different pro-inflammatory treatments in all the analysed immune cells compared to the untreated cells, with the highest levels obtained after their exposure to lps in combination with ifnγ (fig 2) . in order to investigate irg1 expression over time following a pro-inflammatory stimulus, we analysed its expression levels at different time points following lps activation. the results show that irg1, similarly to the corresponding mouse gene [5] , is expressed at the highest levels at 6-9 hours in both monocytes and macrophages (s1 fig) and that its expression is already significantly induced after 40 minutes in human pbmcs-derived macrophages as well as after 20 minutes in the murine raw264.7 macrophage cell line (s2 fig). taken together, these results show that irg1 is expressed by human monocytes, macrophages and dendritic cells and that it is rapidly induced in both human and murine macrophages under inflammatory conditions. it was previously shown that the murine irg1/cad protein associates with mitochondria [6] . however, the sub-cellular localization of the corresponding human protein was not described until now. thus, to elucidate irg1/cad compartmentalization in human macrophages, we applied immunofluorescence in combination with confocal microscopy to human pbmcsderived macrophages under inflammatory conditions. results showed that, similarly to the murine protein, human irg1/cad is accumulating in mitochondria (fig 3a and 3b) . indeed, treatments with lps alone or in combination with ifnγ caused significant mitochondrial accumulation of irg1/cad (wilcoxon rank sum test, p < 0.001) (fig 3c) . following our results at the gene and protein level, we investigated in more details the transcriptional machinery, which is responsible for irg1 expression in both murine and human macrophages. using biobase resources, such as transfac 1 and match tm , we inferred the tf-dna interactions (directed and unsigned) with irg1 as the starting node. we adopted a similar approach for both the murine and human genes. the mouse analysis resulted in an irg1 upstream network containing 70 nodes and 3936 edges. in parallel, the interactions (signed and directed) related to the biological process of lps stimulation in macrophages were inferred from pathway studio and resulted in a lps downstream network with 615 nodes and 1278 edges. as expected, we did not obtain irg1 as one of the nodes in this network reflecting the lack of specific information about the transcriptional regulation of irg1 following an lps treatment. taking the union of all the interactions, these two networks were then merged into a single network. the merged network with 663 nodes and 5214 edges established the connection between lps and irg1 with common nodes from both networks. similarly to the analysis performed in mouse macrophages, the match tm algorithm with the human irg1 promoter sequence and the immune cell specific profile yielded an irg1 upstream network with 75 nodes and 4235 edges, which passed the quality threshold. the literature network downstream of lps inferred from pathway studio resulted in a network of 1458 nodes and 2857 edges. as for the mouse analysis, these two networks were then merged with a total of 1490 nodes and 7092 edges. since the murine and human merged networks were obtained from heterogeneous data, we generated genome-wide gene expression data in the murine raw264.7 macrophage cell line and human pbmcs-derived macrophages after 6 hours of lps stimulation for network contextualization. the differentially expressed genes (degs) with their respective fold changes and pvalues are included in the supplementary material (s3 and s4 tables). from these genes, we identified the top 100 degs (log2 fc!|1| and p-value <0.01) between untreated and lps-activated conditions for both mouse raw264.7 macrophages (s3 fig) and human pbmcs-derived macrophages (s4 fig), respectively. the results show that both mouse and human cells are highly affected by lps displaying a classical pro-inflammatory signature were the classical proinflammatory markers, such as il1β, ccl5 and il6, are among the top 100 degs in both the species. gene ontology (go) analysis reveals up-regulation of biological processes that are reflecting a pro-inflammatory response, such as "response to molecule of bacterial origin" or "regulation of cytokine production" in both mouse and human cells (s3 fig and s4 fig) . (fig 4a and 4b) . the classical pro-inflammatory-associated transcription factors, such as nfκb, junb and irfs, are highly up-regulated in lps conditions when compared to untreated cells. in human cells, 1051 genes were up-regulated in lps conditions when compared to control and, among them, 64 were transcription factors, while a total of 90 genes were down-regulated and 5 were transcription factors (fig 4c) . in mouse macrophages, a total of 564 genes were up-regulated and, among them, 28 were transcription factors, while 397 genes were down-regulated and 33 were transcription factors (fig 4d) . the relevance of these genome-wide gene expression data to prune the networks allowed us to remove the interactions that were inconsistent with the gene expression data as well as to infer the signs to the previously unsigned interactions from irg1 upstream networks. from the contextualised networks, we then aimed to identify the potential transcriptional regulators of irg1. for this, all the possible paths that connect lps and irg1 were calculated using the k shortest simple (loopless) paths-"graphkshortestpaths"-implementation from matlab and the representation of the resultant networks is shown in fig 5. in the mouse network, irg1 has direct connections with the transcriptional regulators irf1, prdm1, cebpd and stat1, while it has indirect connections with junb and cebpb (fig 5a) . similarly, in the human network, irg1 is directly connected to the transcriptional regulators irf1, vdr, stat4, runx1, rara and ets2, with rara and ets2 be predicted to have an inhibitory effect on irg1 expression. moreover, irg1 has one indirect interaction with fos, which, according to the network, transcriptionally regulates irg1 in multiple ways (fig 5b) . given the topology of these simple paths networks, we then calculated the gene essentiality metric for all the transcriptional regulators to rank their importance in the network ( table 1 ). the essentiality metric scores were calculated as described in materials and methods. the total paths represent the number of paths in the simple paths networks from lps to irg1. the paths present after perturbation of transcriptional regulators is the number of paths where a specific regulator is not present and does not disturb the signal transduction between lps and irg1. a higher essentiality metric score value corresponds to a higher importance of the specific node between lps and irg1. based on these scores, we were then interested to investigate the effect of the transcriptional regulator irf1 on irg1 expression, since from our ranking, irf1 resulted to be the top scoring transcriptional regulator in both the mouse and human networks. gene interference mediated by sirnas is widely used to study the effects caused by gene silencing in functional genomics and therapeutic applications [33, 34] were transfected with sirna targeting irf1 or with a non-targeting sirna as control. after 24 hours of transfection, mouse cells were stimulated with lps and rna was extracted after 2 hours. as a proof of concept, we first silenced cebpb, our second top scoring transcriptional regulator. indeed, cebpb was already previously described to be a transcription factor responsible for irg1 induction in zebrafish [12] . silencing of cebpb resulted in 55% and 45% decrease of the cebpb mrna levels when compared to the non-targeting sirna in unstimulated and lps-treated cells, respectively (s7 fig). correspondingly, irg1 expression was decreased by 69% and 16% in cebpb silenced cells as compared to non-targeting sirna in unstimulated and lps treated cells, respectively (s7 fig). thus, these results confirm the previous findings in zebrafish, showing that cebpb is a transcriptional regulator of irg1 in mouse macrophages, as also predicted by our analysis. silencing of irf1, our first top scoring transcription factor, resulted in 55% and 45% decrease of the irf1 mrna levels when compared to the non-targeting sirna in unstimulated and lpstreated cells, respectively (fig 6a) . correspondingly, irg1 expression was significantly decreased by 66% and 26% in irf1 silenced cells as compared to non-targeting sirna in unstimulated and lps treated cells, respectively (fig 6b) . in parallel, proteins were extracted from the cells 4 hours after lps stimulation. as expected, irf1 and irg1/cad proteins were not detectable in control conditions, while they were detected in lps-stimulated cells in both sirna irf1 and non-targeting sirna conditions. in accordance with gene expression results, irf1 and irg1/cad expression levels both decreased in irf1 silenced cells as compared to non-targeting sirna transfected cells following lps stimulation ( fig 6c) . as irg1/cad has been recently described to enzymatically convert cis-aconitic acid into itaconic acid [5] , we next measured itaconic acid levels in irf1 silenced cells. indeed, itaconic acid amounts in sirna irf1 cells were decreased by 11% in unstimulated cells and by 20% in lps-treated cells when compared to non-targeting sirna treated cells (fig 6d and 6e) . similarly to the mouse macrophages, human pbmcs-derived macrophages were transfected for 24 hours with sirna specifically targeting irf1 or a non-targeting sirna as control. transfected macrophages were then stimulated with lps and rna was extracted after 6 hours. differently from mouse macrophages, human primary macrophages do not have a detectable basal level of irg1 expression, thus we only analysed gene expression levels following lps exposure. as a result of irf1 silencing, which held a decrease of its expression levels, as an average of four donors, by 54% (fig 7a) , irg1 expression levels were correspondingly reduced by approximately 50% under lps conditions (fig 7b) in sirna irf1 transfected cells when compared to non-targeting sirna treated cells. in order to gain additional support for irf1 as a direct regulator of the irg1 locus, we overlaid the putative top scoring irf1 binding motifs identified in our match™ analysis (s2 table) with open chromatin regions in the human irg1 locus in pbmcs and blood cd14+ monocytes using publicly available encode data on ucsc genome browser (s8 fig). the results show that, in the human monocyte lineage, irf1 binding sites in the irg1 locus are associated with active chromatin marks, such as h3k4me1 and h3k27ac, which mark active/poised enhancers and which could represent putative irf1 binding sites responsible for irg1 expression. taken together, these results show that irf1 is a transcriptional regulator of irg1 in both mouse and human macrophages and that our combination of computational and experimental approaches represents an efficient method to identify transcriptional regulators of a given inducible gene. we have recently revealed that irg1 codes for the enzyme irg1/cad which catalyses the decarboxylation of cis-aconitate, a tca cycle intermediate, to itaconic acid [5] . this metabolite inhibits isocitrate lyase, the key enzyme of the glyoxylate shunt [35, 36] . we demonstrated that silencing irg1 in murine macrophages decreases itaconic acid levels resulting in a defective antimicrobial activity towards different bacterial infections. thus, we showed that irg1/cad, by the production of itaconic acid from a tca cycle intermediate, plays an essential role in innate immune responses. here, we additionally show that irg1 is not only expressed by human macrophages, but also by monocytes as well as by dendritic cells. thus, due to the important role of irg1/cad at the interface between the innate immune system and the central carbon metabolism, we aimed to shed more light into its expression and transcriptional regulation under inflammatory conditions. for this, we designed an integrative approach of computational and experimental methods to identify the transcriptional regulators of irg1 in mammalian macrophages, where the notion "co-expression implies co-regulation" is satisfied [37] . transcription factors play a pivotal role in modulating gene expression and thus contribute to the overall regulation of biological processes. in order to identify the transcriptional regulators responsible for irg1 induction, we used a weight matrix-based tool for searching putative transcription factor binding sites in dna sequences: match tm [25] . this tool uses the matrix library collected in transfac 1 , thus providing the possibility to search for a large variety of different transcription factor binding sites [25] . the main bottleneck in using these tools is the high amount of false positives generated due to the short and degenerate sequence motifs of transcription factors. a comprehensive method is hence needed to increase the specificity. with our algorithm, we eliminated non-cell type/condition specific transcription factors, thus rendering the biological validation appropriate. though our method could potentially identify the transcriptional regulators of any given gene, it presents some limitations. for instance, to contextualize our networks, we exclusively considered degs from microarrays data, although some transcription factors are phosphorylated in order to be activated and shuttled into the nucleus to subsequently act as transcription factors. thus, in follow-up studies it would be valuable to add an additional layer of information on top of the transcriptomic data, such as phosphoproteomics data. this could allow contextualizing the generated networks not only taking into account gene expression data, but also the phosphorylation data of various regulating proteins. from our contextualized murine and human networks, we identified potential transcriptional regulators of irg1 in macrophages. of interest, our mouse and human analysis provide insights for the identification of species-specific regulators for irg1 induction under lps activation. although the human data were obtained from primary cells, while the mouse analysis was conducted using the macrophage cell line raw264.7, it is tempting to speculate that the transcriptional machinery inducing irg1 expression, with the exception of irf1, is mostly species-specific, as highlighted by the different transcriptional regulators identified in the two species (table 1) . experimentally, we confirmed that cebpb in mouse and irf1 in both species represent positive targets which modulate irg1 expression. we used cebpb as a proof of concept for our method, since we predicted it in our ranking and it has been previously shown to be a transcriptional regulator of irg1 in zebrafish (danio rerio). cebpb, as a primary response gene, is known as a transcriptional regulator of the acute phase response [38] . in our experimental conditions in murine macrophages, cebpb is highly up-regulated under lps stimulation and, when silenced, irg1 expression levels are decreased. these results are in agreement with those obtained in zebrafish, thus highlighting cebpb as a transcriptional regulator of irg1 in both the species. irf1 is referred to as a positive transcription factor as it induces several genes, which are important in regulating several immunological and physiological functions in mammalian cells. irf1 is also transcriptionally activated by several pro-inflammatory cytokines and pathogens [39, 40] . irf1 selectively regulates specific sets of genes depending on the cell type and the appropriate response needed to counter the external stimuli, thus having its function driven by cell-type specific factors. irf1 mainly activates interferons (ifns) and ifn inducible genes. some of the targets of irf1 which are known to play a role in host defence are ifn (α, β), inducible nitric oxide synthase (inos), interleukin 12 (il12), cyclooxygenase 2 (cox2), il15, caspase 1-7 and lysyl oxidase [17, 41] . among these, inos is one of the most studied downstream targets of irf1. the enzyme inos catalyses the production of nitric oxide (no) which is essential for the antimicrobial and anti-tumorigenic properties of macrophages. no is a biologically active intermediate produced by the cells using l-arginine, an urea cycle intermediate, as the substrate [17, 42] . increased no production correlates with resistance towards invading pathogens [43] . several studies showed that the silencing of irf1 diminishes the expression of inos and the production of no, thereby attenuating the antibacterial and antiviral activities and thus worsening the severity of the disease [43] [44] [45] . inos is induced upon stimulation by il1, il12, tnf, ifnγ and lps via irf1 [46] . nevertheless, ifnγ and lps are the major and necessary stimulants to induce inos expression and no production in murine macrophages [47] . its transcriptional induction following lps stimulation occurs after the synthesis of ifns and the jak/stat signalling pathway [48] . inos promoter requires the dimeric binding of irf1 for the full cytokine activation. hence, other transcription factors, like nfκb, that are induced by other cytokines, may cooperate with irf1 to induce the full transcriptional activity of inos [49] . irf1 protein is highly unstable with a half-life of 30 minutes [50] and lpsinduced no production can be abrogated by inhibiting the translation of irf1 by 10-hydroxytrans-2-decenoic acid, a medium chain fatty acid [51] . irf1 is serine-phosphorylated by protein kinase a (pka) and protein kinase c (pkc). mutation of these tyrosine residues inhibit the induction of irf1 expression [52, 53] . it was reported earlier that lps induction of irg1 in macrophages is mediated via the pkc pathway [3] . thus the pkc pathway may be responsible for irg1 induction through irf1. irg1 was shown to be co-regulated and co-expressed with inos when murine cells were either infected with mycobacterium [13] or stimulated with lps and was also reported as a family member of ifn inducible genes [14, 54] . of interest, mycobacteria infection studies in irf1 knock-out mice revealed that irf1 is required for the mycobacteria induced granuloma necrosis. gene expression analysis of infected mice resulted in the grouping of differentially expressed genes into different clusters based on the dependence of gene expression on ifnγ or irf1. irg1 was classified to the cluster of genes whose expression is mostly dependent on irf1, but less on ifnγ [55] . in line with the results obtained with mycobacteria infections, our results show that irf1 expression is highly up-regulated under lps exposure and that irf1 silencing induces a significant decrease of irg1 expression levels in both human and murine macrophages. upon tlr activation by external stimuli, such as lps, irf1 was shown to interact with myd88 adaptor molecule to translocate into the nucleus and induce the expression of several genes to mediate immune responses [56] . the analysis of transcription start site (tss) distributions of immune related genes by liang et al. classified irf1 as a gene dependent on myd88 pathway along with irg1 which was stated as an interferon stimulated gene (isg) element [57] . however, irg1 was previously shown to be expressed independently from myd88 or trif adaptor molecules [14, 15] , thus demonstrating that additional pathways regulating irg1 expression independent from irf1 and myd88 could exist. it is already known that irf1 has a significant role in antibacterial and antiviral responses, induction of apoptosis and tumorigenesis, with several of these processes known to be mediated by inos and no [58] . thus, from our results, it is tempting to conclude that irf1 regulates these processes also through the induction of irg1 and itaconic acid production. to this end, inos and irg1 can be considered as the gene twins, regulated by irf1, which contribute to the host protection towards pathogen invasion through the production of effector molecules, such as no and itaconic acid, respectively. of interest, in contrast to mouse, inos expression and no production in human cellular systems have been an argument of discussion by cellular biologists and immunologists since a long time [59] . even though there were reports showing low levels of inos and no in activated human cells [60] [61] [62] , it was argued that these results neither show the functional pathway of no production, as through l-arginine in mouse, nor describe the precise experimental details, such as cell types and culture conditions [63] [64] [65] . in accordance with this argument, accordingly to our microarrays data, we did not detect inos expression in lps-stimulated human macrophages, while its expression was highly up-regulated in mouse cells. however, taking the side of the discussion that human macrophages do not express inos, irf1 plays a crucial role in mediating antimicrobial responses through irg1 gene regulation in human immune cells. finally, in a recent study irf1 and irg1 expression levels were correlated when neurons were infected with several viruses such as saint louis encephalitis virus (slev) and a coronavirus (mouse hepatitis virus, mhv). irg1 had higher basal and also ifn-β-induced expression levels in granule cell neurons when compared to cortical neurons. viral replication was increased in granule cell neurons when transduced with lentiviruses expressing shrna targeting irg1 [66] , thus postulating the role of irg1 in inhibiting viral replication in neurons. irf1 induction by interferons is already known as an antiviral response against certain viruses [45] . even though the role of irg1 in antiviral responses is not yet reported, future studies could reveal that irf1 mediates antiviral actions through the induction of irg1. here we provide evidence for a method integrating computational and experimental approaches as a tool to successfully identify transcriptional regulators of a given inducible gene, thereby stepping towards the understanding of its gene regulatory network. improved knowledge about irg1 transcriptional regulation provides a better understanding of the molecular mechanisms that are involved in specific inflammatory and infectious diseases. to this end, we identified irf1 as a transcriptional regulator of irg1 expression in both human and mouse macrophages under inflammatory conditions. understanding the mechanisms of irg1 induction during immune responses could lead to novel therapeutic approaches, which aim to modulate the intrinsic host response. show the mean of 3 technical replicates (± sem) of irg1 mrna levels measured by real-time pcr normalised with l27 as the housekeeping gene. ããã p < 0.001, ã p < 0.05. b) rna was extracted from raw264.7 macrophages at 20, 40, 60, 80, 100, 120 minutes after treatment with lps (10ng/ml). the bars show the mean of 3 biological replicates (± sem) of irg1 mrna levels measured by real-time pcr normalised with l27 as the housekeeping gene. ããã p < 0.001, ãã p < 0.01, ã p < 0.05. toll-like receptor signaling combined chromatin and expression analysis reveals specific regulatory mechanisms 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to irf-1 synthesis inhibitory effect of 10-hydroxydecanoic acid on lipopolysaccharide-induced nitric oxide production via translational downregulation of interferon regulatory factor-1 in raw264 murine macrophages a role for casein kinase ii phosphorylation in the regulation of irf-1 transcriptional activity mutational analysis of interferon (ifn) regulatory factors 1 and 2. effects on the induction of ifn-beta gene expression 2-aminopurine inhibits lipopolysaccharide-induced nitric oxide production by preventing ifn-beta production mycobacteria-induced granuloma necrosis depends on irf-1 evidence for licensing of ifngamma-induced ifn regulatory factor 1 transcription factor by myd88 in toll-like receptor-dependent gene induction program analysis of changes in transcription start site distribution by a classification approach effect of cycloheximide on the expression of lps-inducible inos, ifn-beta, and irf-1 genes in j774 macrophages of mice and not men: differences between mouse and human immunology evidences for inos expression and nitric oxide production in the human macrophages expression of the inducible no synthase in human monocytic u937 cells allows high output nitric oxide production human mononuclear phagocyte inducible nitric oxide synthase (inos): analysis of inos mrna, inos protein, biopterin, and nitric oxide production by blood monocytes and peritoneal macrophages macrophage biology and immunology: man is not a mouse species differences in macrophage no production are important nitric oxide production and nitric oxide synthase type 2 expression by human mononuclear phagocytes: a review differential innate immune response programs in neuronal subtypes determine susceptibility to infection in the brain by positive-stranded rna viruses we thank dr. elisabeth john and dr. lasse sinkkonen for helping with human rna samples for microarrays experiments. we are grateful to françois bernardin and dr. tony kaoma for excellent assistance with microarrays platforms and analysis. key: cord-353410-tbmtg88k authors: sharma, shreela v.; haidar, amier; noyola, jacqueline; tien, jacqueline; rushing, melinda; naylor, brittni m.; chuang, ru-jye; markham, christine title: using a rapid assessment methodology to identify and address immediate needs among low-income households with children during covid-19 date: 2020-10-01 journal: plos one doi: 10.1371/journal.pone.0240009 sha: doc_id: 353410 cord_uid: tbmtg88k objective: brighter bites is a school-based health promotion program that delivers fresh produce and nutrition education to low-income children and families. due to covid-19-related school closures, states were under “shelter in place” orders, and brighter bites administered a rapid assessment survey to identify social needs among their families. the purpose of this study is to demonstrate the methodology used to identify those with greatest social needs during this time (“high risk”), and to describe the response of brighter bites to these “high risk” families. methods: the rapid assessment survey was collected in april 2020 across houston, dallas, washington dc, and southwest florida. the survey consisted of items on disruption of employment status, financial hardship, food insecurity, perceived health status and sociodemographics. the open-ended question “please share your greatest concern at this time, or any other thoughts you would like to share with us.” was asked at the end of each survey to triage “high risk” families. responses were then used to articulate a response to meet the needs of these high risk families. results: a total of 1048 families completed the covid-19 rapid response survey, of which 71 families were triaged and classified as “high risk” (6.8% of survey respondents). during this time, 100% of the “high risk” participants reported being food insecure, 85% were concerned about their financial stability, 82% concerned about the availability of food, and 65% concerned about the affordability of food. a qualitative analysis of the high-risk group revealed four major themes: fear of contracting covid19, disruption of employment status, financial hardship, and exacerbated food insecurity. in response, brighter bites pivoted, created, and deployed a framework to immediately address a variety of social needs among those in the “high risk” category. administering a rapid response survey to identify the immediate needs of their families can help social service providers tailor their services to meet the needs of the most vulnerable. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 on march 11, 2020, the world health organization declared the sars cov-2 (covid-19) a pandemic [1] . on march 13, 2020 the united states (u.s.) declared a national emergency concerning the covid-19 outbreak, leading to numerous social distancing measures including, school closures, cancellation of public gatherings, and remote working [2] . starting the week of march 16 th 2020, through the week of march 23 rd 2020, states began ordering schools to close for the academic year and issuing statewide stay-at-home orders, with many companies implementing work from home policies [3] . these social distancing measures had the detrimental consequences of disrupting food systems, businesses, and economies throughout the u.s. [4] [5] [6] . rapid epidemiological assessment (rea) refers to post-disaster assessment methods that attempt to accurately assess a population by using the fewest resources in the shortest time [7] . these measures have included surveys, door-to-door assessments, surveillance methods, and screening and individual risk assessment using qualitative and quantitative methods [8] . typically, during an outbreak investigation, these methods may be used to assess symptoms of disease to contain and prevent further outbreaks [7] . however, the application of a rapid assessment could extend to identifying social determinants of health needs during a disaster such as food insecurity, financial instability, unemployment, housing insecurity, and access to healthcare among vulnerable populations. this is particularly important to covid-19, given that reducing risk of covid-19 complications is related to maintaining optimal immune function and health, all of which are linked to these social determinants of health [4] [5] [6] . brighter bites is a non-profit, school-based health promotion program implemented across six cities (houston, dallas, austin, new york city, washington, d.c., and southwest florida areas) in the u.s., with the goal of delivering fresh produce and nutrition education to lowincome children and families in underserved communities to mitigate food insecurity and improve dietary habits. brighter bites currently has an ongoing partnership with university of texas health science center at houston (uthealth) school of public health for research and evaluation. during the month of april 2020, amid the covid-19 pandemic, while states were under "shelter-in-place" orders, brighter bites conducted a rapid assessment survey of families across four of the six cities (houston, dallas, washington dc, and florida). the survey screened families for food security, housing security, financial stability, employment status, transportation needs, access to childcare, and access to healthcare, to better understand the immediate needs of families, identify those with the greatest need, and provide them with critical resources during this time of crisis. the purpose of this study is to demonstrate the methodology used to conduct the rapid assessment of needs using qualitative and quantitative measures among low-income households with children during the covid-19 pandemic to identify those in greatest need, to present our findings, and describe the strategies taken to meet the participants' needs. the rapid assessment survey was collected in april 2020 across houston, dallas, washington dc, and southwest florida. the self-report survey was administered electronically using formsite (vroman systems inc., illinois, usa) in english and spanish to 16,500 bb families who were enrolled in the program in the 2019-2020 school year and had provided their contact information to the program. the participants in our study were predominately hispanic (87%), while 7% were african american, and 5% were white/other. this is significantly higher than the sampled cities where the hispanic population makes up at most 45% of the population in houston, 42% in dallas, 27% in florida, and 11% in washington d.c. completion of the survey was voluntary and informed consent was obtained from all participants prior to the start of the study. data is collected by brighter bites non-profit organization, and then is de-identified and shared with the university of texas health science center (uthealth) for analysis as part of a data sharing agreement and approved by the uthealth committee for protection of human subjects. data obtained in the first wave of survey responses, while cities were under "shelter-in-place" orders, were used for this paper. the self-reported rapid assessment survey consisted of a 30-item questionnaire on covid-19 related concerns, social determinants of health, and sociodemographics. the 5-10 minute in length survey was administered in english and spanish. sociodemographic variables included child gender, respondents' relationship to child, race/ ethnicity of both parent and child, parent education level, parent employment status, and government assistance program enrollment. program options included the special supplemental nutrition program for women, infants, and children (wic), supplemental nutrition assistance program (snap), double dollars, medicaid, medicare, national school lunch and/or breakfast programs, and children's health insurance program. household food security status was self-reported by the participants using the two-item hunger vital sign™ screening questionnaire developed and validated by hager et al. [9] . if the participant responded "often true" or "sometimes true" to either of the two questions, then the household was considered food insecure. if a participant answered "never true" to both questions, then the household was considered food secure. participants were asked about their concerns regarding various social determinants of health including financial stability, employment status, access to food, affordability of food, availability and affordability of housing, access to reliable transportation, access to childcare, access to clinic/doctor, or other concerns, which was open-ended for participants to fill in. participants could check all that applied. participants were asked to rate their health status. responses included poor, fair, good, very good, and excellent. stata 15.0 (statacorp college station, tx) was used to perform all data analysis. means, standard deviations, and frequency distributions were computed. significance was set at p<0.05. the open-ended question "please share your greatest concern at this time, or any other thoughts you would like to share with us." was asked at the end of each survey. the responses to this question were collated and analyzed for themes. two trained uthealth project staff conducted a thematic analysis to analyze the responses using an inductive approach, where codes and themes are derived based on the content from the survey data [10, 11] . the coding of the data was done initially through microsoft word using an open coding method by each coder. to ensure reliability of codes, coders collectively re-coded the data in microsoft excel. a codebook of codes and definitions was created, and codes were used to search for patterns and to identify possible themes. triaging for "high risk" families: "high risk" families were defined as the following: 1) if indicated in the open-ended question "please share your greatest concern at this time, or any other thoughts you would like to share with us "the following response": a) running out of food, b) diagnosed with covid-19 and/or living with someone who has been diagnosed with covid-19 and experiencing challenges, c) is ill and needs assistance, d) is about to lose their place of living, e) is about to lose their utilities, or f) no one at home is making an income; or 2) if indicated on the survey as "poor" on the question when they were asked to rate their current health status. if they met any of these categories, they were classified as "high risk". subsequently, these data were used to articulate a response to meet the needs of these high risk families. the sociodemographic characteristics of the high-risk families are presented in table 1 . a total of 1048 families completed the covid-19 rapid response survey, of which 28 families were triaged and classified as high risk (2.8% of survey respondents). overall, the mean age of highduring this time frame, 100% of the high risk participants reported being food insecure, 85% were concerned about their financial stability, 82% concerned about the availability of food, and 65% concerned about the affordability of food. they were also concerned about the availability/affordability of housing (50%), while 45% were concerned their employment status would change in the near future. other concerns were access to reliable transportation (11%), access to childcare (13%), and access to a clinic/doctor (35%). almost half the participants (49%) reported being of fair or poor health status during this time. while overall results of the qualitative thematic analysis for the n = 1048 participants are presented elsewhere (sharma et al., under review) , a qualitative analysis of participant responses in the high-risk group revealed four major themes presented in table 2 : fear of contracting covid-19, disruption of employment status, financial hardship, and exacerbated food insecurity. fear of contracting covid-19 was an immediate threat for the families as is witnessed in one of the comments "my daughter was diagnosed with coronavirus and i am very scared for all the members of my family". moreover, loss of employment was of major concern leaving parents with little or no resources to meet their family's needs. one parent said, "i don't have enough work, i'm a single mother, i have two children and i no longer have enough for food for my children and i'm behind with the rent." table 2 "my daughter was diagnosed with coronavirus and i am very scared for all the members of my family." {{e.g. bb produce vouchers provided, grocery drop off}} • provided language-appropriate information and resources on safety measures-hand washing/sanitizer, wearing masks (talked about various items that could be a mask if they did not have access to any) social distancing and staying home. • encouraged to seek testing if there was a concern of potential covid-19 infection. • provided information on community resources for clinics, doctors and testing sites by way of family resource links "we are worried that my husband does not have a job and we do not know how we will be able to buy food, pay the mortgage, electricity, water, the internet, what will happen to our future, we are very afraid, regarding the virus, of taking our children to the doctor, to the dentist, etc." "i don't want to go to the store and i no longer have food, afraid of going out." "i was left without work, and i don't have for supplies, or bills, or food." • provided information on community resources for employment and unemployment benefit services. • provided electronic $50 gift card to a local retail store. if they did not have access to an email, the $50 gift card was printed and handdelivered to them. "i'm very worried because i haven't had work for three weeks and i don't have money to pay rent, electricity, or water. my work has been reduced by 90%, i'm an assistant housecleaner." "i don't have enough work, i'm a single mother, i have two children and i no longer have enough for food for my children and i'm behind with the rent." "there's no work, and you still have to pay rent, and there's not enough money for food." financial hardship "my biggest worry is not being able to pay next month's rent and not knowing where to go." • provided information on community resources for assistance programs, housing, transportation, child care and other services such as free or reduced internet access by way of family resource links • cross-checked that they were receiving the produce vouchers to local grocery retail store and updated contact information accordingly. • sent electronic $50 gift card to local retail store. multiple electronic $50 gift cards were sent to families that communicated continued hardship. if they did not have access to an email, the $50 gift card was printed and hand-delivered to them. "i am worried about not being able to pay my rent next month (may_2020) because we are out of work." "i don't have enough food for my children." • provided dates and times for community food distributions and brighter bites food distributions. • sent electronic $50 gift card to local retail store • if they did not have access to an email, the $50 gift card was printed and hand-delivered to them. • if they could not go shopping due to barriers such as lack of transportation, groceries were bought and delivered to them. (family was asked to share food preferences) • families received a $25 produce voucher to the local grocery retail store. "i am worried about how to feed the children because there is no work right now, i am worried about paying my debts" "my biggest worry at the moment is food for my family. loss of employment further led to financial instability leaving parents unable to pay their bills or buy food. one parent commented, "my biggest worry is not being able to pay next month's rent and not knowing where to go." this financial uncertainty also left parents concerned about their food security, and about how they were going to access healthy foods with the limited produce available and the increasing prices in the grocery stores. one parent described their concerns by saying, "i feel worried because i'm not giving enough vegetables and fruit to my children since my husband only works 3 days, and i'm not working because my baby was just born, so there are 4 children and 2 adults and i'm short of food and diapers for my baby, but what i need is food for the family." brighter bites response to high risk families: in response to these aforementioned needs, brighter bites pivoted and created an infrastructure and a standardized framework to meet the needs of these high-risk families (see fig 1) . a core, centralized group of brighter bites staff was assigned specifically to address these families' immediate concerns. following the triage, trained brighter bites staff made follow up phone calls to each high-risk family and obtain more details regarding their concerns and assistance needed. a tracking database was created, in which all phone calls and family concerns were tracked for each family. this information was then relayed back to the brighter bites leadership where a tailored response was generated for each family. brighter bites partnered with public health and medical schools' faculty and students to develop educational materials and informational resources to provide to the families (haidar et al., under review). responses ranged from a) providing immediate financial relief in the form of gift cards to local retail stores, b) grocery drop off to families unable to leave home, and c) providing area-specific resources via text, email and phone regarding food distributions, financial assistance, safety from eviction due to inability to pay rent, covid-19 testing sites near their homes, participation in government assistance programs etc. all responses were documented in the tracking database for each family. a 100% of the high-risk families were reached through this process. the covid-19 pandemic has had a major impact on the u.s. economy leading to a worldwide economic crisis [6] . the most vulnerable populations in our communities have been disproportionality affected by the direct and indirect health, social, and financial hardships of covid-19 [12, 13] . low-income, vulnerable populations contribute the most to healthcare costs, are more likely to suffer from health disparities, have increased risk factors, and have fewer resources for promoting health and treating illness [14] . it is therefore imperative that pertinent health, financial, and critical needs resources are provided in a timely matter to families and communities in need. this study provided insight into how organizations could potentially identify and respond to the needs of communities in times of crisis and demonstrates how to effectively reach families that are struggling and need support. social factors play a critical role in determining health outcomes [15, 16] . addressing the social determinants of health of underserved communities through rapid response is important to reduce health disparities and improve health outcomes associated with covid-19 and other comorbidities [12] . minority populations, specifically african-american and hispanic have been disproportionately affected by covid-19, with increased risk of complications and mortality [17] [18] [19] . these same populations also suffer the most from chronic diseases such as obesity, diabetes, respiratory disorders, and other pre-disposing conditions, accounting for the majority of healthcare costs [20] . often times, these diseases are also rooted in unequitable social and structural needs surrounding employment, transportation, housing, poverty, food, education, and health literacy [21, 22] . conducting a covid-19 rapid response survey was a purposeful decision on part of brighter bites to identify those with highest need during this time of crisis and develop a framework to immediately address a variety of social needs among those in the "high risk" category. due to the rapid transmission of coronavirus, many cities including the four in our study were rapidly brought to a halt with many states issuing statewide stay-at-home-orders, closing schools, restaurants, and businesses [3] . for low-income families this resulted in loss of wages and increased financial instability adding further strain to previous economic hardships. administering a rapid response survey during such times is important to help identify the immediate needs of these families can help social service providers re-direct their services to meet the needs of this vulnerable population. in response to the covid-19 pandemic, brighter bites pivoted rapidly to invest its network into addressing urgent needs of their participating families. brighter bites used the expertise of public health faculty and students of their partner institutions to create and rapidly provide resources for their families. for example, if a parent indicated that they were fearful of contracting covid-19, the team discussed safety measures such as handwashing, social distancing, and various items that could serve as a mask. if a parent was concerned about paying for rent or food, then bright bites team provided information on community resources for food, bill relief, and ensured they were receiving vouchers, and gift cards to local grocery stores. details regarding these strategies and their dissemination is provided elsewhere (haidar et al., under review). by having a triaging and tracking system, along with a centralized response team and strategic partners, in place, brighter bites was able to have a targeted response to meet the needs of 100% of families with the highest needs during this time. social service organizations can use similar strategies to identify and address the needs of their populations rapidly during times of disaster. this study has some limitations and strengths. the study sample is a sub-sample of families who participated in brighter bites in the 2019-2020 school year. the sample size varied across each city as family enrollment in the brighter bites program is not proportional to size of the city, and likely those families who needed the most help responded to the survey. finally, survey was conducted to families electronically which introduces a selection bias as a limitation due to the fact that only families that had access electronically could participate. however, in this phase of the pandemic, given closures of schools and businesses, this was the most effective way to reach people. finally, the qualitative themes observed in our study cannot be sorely attributed to the pandemic, and could be a result of other multitude of factors which remain to be seen. the purpose of this study was to identify the families with the highest need for social services during a time when all cities surveyed were in "shelter-in-place" and deploy an immediate response, which was successfully achieved. finally, the timeline from the pandemic occurring to our response to these families is one of the major strengths of this paper. our study provides the methodology and framework to screen at-risk low-income families for social needs during the time of the covid-19 pandemic, and to provide a timely response to these critical needs. in the future, this framework could be used for other pandemics and times when an immediate response to screen at-risk families will be needed. world health organization. who director-general's opening remarks at the media briefing on covid-19-11 the covid-19 pandemic in the usa: what might we expect? map: coronavirus and school closures the early food insecurity impacts of covid-19 feeding low-income children during the covid-19 pandemic the socio-economic implications of the coronavirus and covid-19 pandemic: a review rapid epidemiological assessment of health status in displaced popula-tionsâ€"an evolution toward standardized minimum essential data sets development of rapid epidemiologic assessment methods to evaluate health status and delivery of health service development and validity of a 2-item screen to identify families at risk for food insecurity using thematic analysis in psychology transforming qualitative information: thematic analysis and code development.: sage centers for disease control and prevention. covid-19 in racial and ethnic minority groups impacts of covid-19 on vulnerable children in temporary accommodation in the uk vulnerability and vulnerable populations. foundations for population health in community/public health nursing-e-book addressing social determinants of health and health inequalities the social determinants of health: it's time to consider the causes of the causes racial health disparities and covid-19â €"caution and context ná poles am, pé rez-stable ej. covid-19 and racial/ethnic disparities covid-19 and african americans multiple chronic conditions in the united states prevalence of single and multiple leading causes of death by race/ethnicity among us adults aged 60 to 79 years social determinants of health the authors would also like to acknowledge the families who participated in this study. conceptualization: shreela v. sharma, christine markham. key: cord-353869-l53ms3q8 authors: friesen, robert h. e.; koudstaal, wouter; koldijk, martin h.; weverling, gerrit jan; brakenhoff, just p. j.; lenting, peter j.; stittelaar, koert j.; osterhaus, albert d. m. e.; kompier, ronald; goudsmit, jaap title: new class of monoclonal antibodies against severe influenza: prophylactic and therapeutic efficacy in ferrets date: 2010-02-08 journal: plos one doi: 10.1371/journal.pone.0009106 sha: doc_id: 353869 cord_uid: l53ms3q8 background: the urgent medical need for innovative approaches to control influenza is emphasized by the widespread resistance of circulating subtype h1n1 viruses to the leading antiviral drug oseltamivir, the pandemic threat posed by the occurrences of human infections with highly pathogenic avian h5n1 viruses, and indeed the evolving swine-origin h1n1 influenza pandemic. a recently discovered class of human monoclonal antibodies with the ability to neutralize a broad spectrum of influenza viruses (including h1, h2, h5, h6 and h9 subtypes) has the potential to prevent and treat influenza in humans. here we report the latest efficacy data for a representative antibody of this novel class. methodology/principal findings: we evaluated the prophylactic and therapeutic efficacy of the human monoclonal antibody cr6261 against lethal challenge with the highly pathogenic avian h5n1 virus in ferrets, the optimal model of human influenza infection. survival rates, clinically relevant disease signs such as changes in body weight and temperature, virus replication in lungs and upper respiratory tract, as well as macroand microscopic pathology were investigated. prophylactic administration of 30 and 10 mg/kg cr6261 prior to viral challenge completely prevented mortality, weight loss and reduced the amount of infectious virus in the lungs by more than 99.9%, abolished shedding of virus in pharyngeal secretions and largely prevented h5n1-induced lung pathology. when administered therapeutically 1 day after challenge, 30 mg/kg cr6261 prevented death in all animals and blunted disease, as evidenced by decreased weight loss and temperature rise, reduced lung viral loads and shedding, and less lung damage. conclusions/significance: these data demonstrate the prophylactic and therapeutic efficacy of this new class of human monoclonal antibodies in a highly stringent and clinically relevant animal model of influenza and justify clinical development of this approach as intervention for both seasonal and pandemic influenza. a novel class of human monoclonal antibodies against influenza has been recently discovered [1] . these antibodies bind to the membrane-proximal stem of haemagglutinin, the major viral surface protein, and neutralize the influenza virus by blocking its fusion with the host cell [2] . a panel of antibodies with a similar mode of action was reported subsequently by sui et al. [3] . due to the high conservation of their recognition site, this class of antibodies has shown the ability to neutralize a broad spectrum of influenza subtypes, including h1, h2, h5, h6 and h9 [1] , and can be expected to also neutralize viruses from subtypes h4, h8, h11-h14 and h16, as well as their future antigenic drift variants [2] . one of these antibodies, cr6261, was investigated in mice and shown to be protective when given before and after lethal challenges with h1n1 and h5n1 virus, suggesting that it has potential as the first-ever broad-spectrum monoclonal antibody for prophylaxis and treatment of influenza virus infections [1] . according to the world health organization, seasonal influenza causes up to 500,000 deaths worldwide each year [4] . immunologically naïve infants, immunocompromised individuals and the elderly are particularly susceptible to illness caused by seasonal influenza viruses, with 90% of deaths occurring in the latter group [4, 5] . in addition, subtypes of influenza a viruses that have not previously circulated among humans occasionally cross from animal reservoirs, raising the spectre of a pandemic. over the past decade, the most prominent pandemic threat appeared to be posed by highly pathogenic avian influenza viruses of subtype h5n1. however, it was a new strain of human h1n1 that emerged in mexico and the united states in march and april 2009 and rapidly spread across the globe that caused the who to declare a pandemic on june 11th [6, 7] . although the virulence of this virus is currently moderate, particularly compared to the case fatality rate of over 60% of human h5n1 infections, this may change over time. meanwhile, the threat from highly pathogenic avian h5n1 viruses persists as they continue to circulate and evolve in bird populations. preventive vaccination has historically been the primary means of influenza control, but this approach has important limitations. vaccines typically elicit a potent neutralizing antibody response only to the specific viral strains they contain, and closely related viruses [8, 9] . furthermore, influenza vaccines have suboptimal immunogenicity and efficacy in the groups at highest risk of severe disease: the very young, the elderly and immunocompromised individuals [10] . the current therapeutic regimen for influenza a is limited to two classes of drugs: the adamantanes (amantadine and rimantadine) and the neuraminidase inhibitors (oseltamivir and zanamivir). adamantanes rapidly elicit viral resistance, and resistance rates are high among h3n2 viruses and certain clades of h5n1 viruses [11] [12] [13] [14] . the use of oseltamivir, the leading antiviral influenza drug, has been limited by the sudden and widespread emergence of resistance among circulating h1n1 influenza strains [15, 16] . oseltamivir resistance has also been observed during treatment of h5n1 infection [17, 18] . zanamivir is still effective against h1n1 viruses and resistance to this drug is less likely to arise [19] . however, its use is limited to patients who can actively use an inhaled drug, which excludes young children, impaired older adults, or patients with underlying airway disease [16] -once again, the group of patients most vulnerable to serious complications from influenza infection. in the absence of reliable antiviral drugs and vaccines, development of alternative strategies for influenza prophylaxis and therapy is urgently required. in order to assess whether cr6261 may be a viable option for human treatment, we evaluated the prophylactic and therapeutic efficacy of this human monoclonal antibody in a highly stringent lethal h5n1 influenza ferret model. the ferret is the most suitable disease model for human influenza infection as it displays very human-like disease [20] [21] [22] . four groups of 6 ferrets each received 30, 10, 3 or 1 mg/kg cr6261 via an intravenous injection and were challenged the next day with the highly pathogenic avian a/indonesia/5/2005 (h5n1) virus. a control group of 6 ferrets received 30 mg/kg of the irrelevant isotype-matched antibody cr3014. all ferrets that received 30 or 10 mg/kg cr6261 survived, compared to only 33.3% of the control animals ( figure 1a) . a further reduction of the dose to 3 mg/kg was clearly correlated with a lower survival rate of 66.7%. though not statistically significant different from survival observed in control animals, there is a 50% mortality reduction. the lowest dose of 1 mg/kg cr6261 was not associated with survival benefit compared to control animals. survival times differed significantly between groups receiving 30 or 10 mg/kg cr6261 and the control group (p = 0.020). moribund animals showed general depression, anorexia and lethargy, and exhibited clinical signs of respiratory disease, including dyspnoea. animals treated at efficacious dose levels (30 and 10 mg/kg) did not loose body weight, whereas the mean weight loss in the control group was 10.5% by the time the ferrets died or were euthanized ( figure 1b ). an exception was one control ferret that succumbed to infection within 48 hours after challenge and lost hardly any weight before. ferrets that received 3 or 1 mg/ kg cr6261 showed similar declines in body weight as the control animals. one day after challenge the maximum body temperature was observed; for each ferret the maximum body temperature is depicted in figure 1c . the groups treated with 30 or 10 mg/kg had mean temperatures of 39.4uc and 40.7uc, respectively, which were significantly lower than the mean of 41.7uc observed in the control group (p,0.001 and p = 0.015, respectively). the mean temperature observed in animals treated with the lower doses of cr6261 (41.3uc and 41.5uc for 3 and 1 mg/kg, respectively) did not differ significantly from the control group. the mean temperature in animals observed 3 days before challenge was similar across the 5 groups, ranging from 37.6uc to 38.4uc. individual body temperature varied considerably within one healthy ferret over 24 hours (standard deviation 0.7uc). ferrets treated with 30 or 10 mg/kg cr6261 did not shed infectious virus in the upper respiratory tract at any time, whereas animals in the control group did ( figure 1d ). treatment with 3 and 1 mg/kg did not prevent shedding, but reduced the proportion of ferrets with infectious virus in nasal and/or throat swabs. after necropsy, all ferrets were assessed for viral load in the lungs (figure 1e), and the animals that received 30 mg/kg cr6261 or control antibody were also assessed for viral load in the brain, liver, spleen, blood and kidney. the levels of virus replication in the lungs of ferrets treated with 30 and 10 mg/kg cr6261 were 3.9 and 2.9 log 10 tcid 50 /g lower than that in the control group (both p,0.001). no infectious virus was detected in any of the other organs of the ferrets that received 30 mg/kg cr6261, whereas infectious virus was found in the brain of 2, the liver of 3, and the spleen of 5 of the 6 control animals (data not shown). there was no significant difference in lung viral load in the groups receiving 3 or 1 mg/kg cr6261 compared to the control group (p = 0.74 and p = 0.91, respectively). histopathological results were in accordance with the findings described above; animals that received 30 and 10 mg/kg cr6261 showed much less pulmonary lesions such as primary atypical pneumonia, subacute bronch(iol)itis, emphysema and congestion, or showed such lesions at a lower grade of severity, compared to animals from the other groups. bronchiolitis obliterans was not observed in any animal that received 30 mg/kg cr6261. compared to this higher-dose group, animals that received 10 mg/kg cr6261 showed more regenerative response (diffuse grey/red area and bronchioloalveolar hyperplasia) in the lungs, and more inflammatory changes in the trachea. pulmonary oedema was not observed in animals that received 30 or 10 mg/kg cr6261, but was observed frequently in all other groups. these findings were in agreement with mean lung weights, which were lowest in the animals treated with 30 or 10 mg/ kg cr6261 (6.3 g and 7.6 g, respectively) and significantly lower in this group than in control animals (15.0 g; both comparisons p,0.001). no significant difference in lung weight was found between the groups receiving 3 or 1 mg/kg cr6261 (14.1 g and 15.4 g, respectively) and the control group (15.0 g). these findings show that, in a dose dependent way, prophylactically administered cr6261 confers protection against lethal h5n1 challenge, prevents morbidity and viral dissemination and reduces pulmonary pathology. to assess the therapeutic efficacy of the monoclonal antibody cr6261, two groups of 10 ferrets were challenged as above and given 30 mg/kg of cr6261 either 4 or 24 hours later. a comparator group of 10 ferrets received 30 mg/kg of the control antibody 4 hours after challenge. survival rates in the groups receiving cr6261 at 4 and 24 hours after challenge were 100%, whereas only 20% of the animals in the control group survived (p,0.001) (figure 2a ). mean decline in body weight at the end of the experiment was 6.2% in the group of ferrets that received cr6261 4 hours after challenge ( figure 2b) , which was significantly less (p = 0.025) than the 10.1% observed in control animals. animals treated 24 hours post challenge showed a mean body weight loss of 8.4%, which was not significantly different from the control animals (p = 0.427). the group of ferrets treated with cr6261 4 hours post challenge had a mean maximum temperature of 40.0uc, compared to 41.8uc in the control group (p,0.001). in line with the rapid rise in temperature after challenge observed in the prophylaxis experiment ferrets treated with cr6261 24 hours after challenge showed a mean maximal temperature of 41.5uc before cr6261 was administered (p = 0.15 versus 41.8uc of the control group, figure 2c ). ferrets treated with cr6261 at 4 hours post challenge did not shed infectious virus in the upper respiratory tract throughout the study ( figure 2d ). in the group treated with cr6261 at 24 hours post challenge, one ferret had a low concentration of infectious virus (2.8 log 10 tcid 50 ) in the throat on day one, but no virus was detected on subsequent days. in contrast, all animals in the control group shed virus during one or more days. accordingly, the mean viral loads in the lungs of ferrets treated with cr6261 at 4 and 24 hours post challenge were considerably lower than that in the control group (differences were 3.9 and 4.5 log 10 tcid 50 /g, respectively, both comparisons p,0.001; figure 2e ). the lungs of animals that received cr6261 at 4 hours post challenge showed less pulmonary lesions (alveolar oedema, bronchiolitis obliterans, congestion, emphysema, bronchioloalveolar hyperplasia and primary atypical pneumonia), or showed such lesions at a lower grade of severity, compared to the lungs of animals from the other two groups. animals of the control group were most affected by primary atypical pneumonia. these findings were in accordance with the observation that the mean lung weights of ferrets treated with cr6261 at 4 hours post challenge were lower compared to the control group (5.7 g versus 14.9 g, p,0.001; figure 2f ). animals that received cr6261 at 24 hours post challenge showed most regenerative response (bronchioloalveolar hyperplasia) in the lungs, suggesting damage to the lung parenchyma with subsequent regenerative response. the mean lung weight in this group was significantly higher than that of the group receiving cr6261 at 4 hours post challenge (8.4 g versus 5.7 g), but lower than that of the control group (p,0.001). from the study outset, one animal had been added to each of the two treatment groups to be sacrificed for gross-pathology and histology on lungs as soon as 50% of the control animals died. the purpose was to test for possible bias due to differences in the timing of death. infectious virus titres in the lungs of the treated ferrets sacrificed at day 3 were identical to those in treated animals sacrificed at the end of the study (open circles in figure 2e) . similarly, there were no differences in lung weight and pathology between animals sacrificed at day 3 or after day 5 ( figure 2f ). this indicates that the results were not biased by differences in the timing of euthanasia or spontaneous death. cr6261 represents a new class of human monoclonal antibodies that exhibits immediate and potent efficacy for the prevention or treatment of influenza in a clinically relevant model for severe disease. the results presented here also confirm previously reported data demonstrating the prophylactic and therapeutic efficacy of cr6261 in mice challenged with h5n1 influenza viruses. together with the h1n1 challenged mice data published earlier [1] these findings indicate that cr6261 is effective across a broad spectrum of influenza viruses, including seasonal and potentially pandemic strains. passive immunotherapy would be particularly beneficial for the groups at highest risk of severe disease due to seasonal influenzathe elderly and immuno-compromised-but may also be indispensable for the general public in the event of a pandemic disease outbreak caused by high-risk pandemic candidates such as h2, h5, h6 and h9. monoclonal antibodies against influenza viruses have been studied for decades, but their potential-and thus development-as 'passive' immunotherapy for influenza has been inhibited by the lack of monoclonal antibodies with broad neutralizing activity. this lack is due to the tolerance of influenza virus for genetic changes in the most immunogenic regions on its surface. the recent discovery of broadly neutralizing human monoclonal influenza antibodies [1] and the demonstrated efficacy of a representative of this novel class of antibodies against lethal viral challenge in a clinically relevant model, as presented in this paper, create an opportunity for the prevention and treatment of influenza infections, regardless of the causal strain. the possibility of a potent and broadly neutralizing agent that would equip clinicians and public health workers to deal effectively with the influenza viruses of the future represents a paradigm shift in the approach to influenza control. influenza illness observed in the ferrets infected with h5n1 virus in this study closely resembles influenza in humans with h5n1 infection, who present with fever, cough, shortness of breath, and radiological evidence of pneumonia [23] . besides respiratory symptoms, gastrointestinal symptoms such as diarrhoea, vomiting and abdominal pain are often present. in severe cases, the pneumonia rapidly progresses to acute respiratory distress syndrome and multiorgan failure. high viral loads, extrapulmonary virus dissemination and hypercytokinaemia are associated with fatal outcome (reviewed in [24] [25] [26] ). autopsies of patients who succumbed to influenza a (h5n1) virus infection have shown diffuse alveolar damage, patchy interstitial lymphoplasmacytic infiltrates, bronchiolitis with squamous metaplasia and pulmonary congestion with various degrees of haemorrhage [27] [28] [29] . the clinical signs observed in the control animals of this ferret study correspond to the most severe influenza pathology in humans [23] . the efficacy of cr6261 in preventing these clinical signs in the prophylactic and therapeutic ferret model presented here, together with the efficacy shown in mice after lethal challenges with different viruses, strongly indicate that cr6261 can be expected to be efficacious against disease caused by the other, less virulent viruses it neutralized in vitro [1] . in a meta-analysis of experimental influenza infection of placebo-treated and untreated healthy volunteers, carrat et al. [30] studied 1280 participants who were challenged with either influenza type h1n1, h3n2 or b. interestingly, viral shedding was highly correlated with the presence of clinical symptoms such as fever, runny nose, sore throat, sneezing, cough and shortness of breath. the authors concluded from their meta-analysis that subjects with symptomatic illness shed virus in amounts 100 to 1000 fold higher than subjects who were not ill. the fact that cr6261 after intravenous administration instantaneously reduces viral shedding indicates that in man cr6261 might reduce clinical symptoms in subjects infected with influenza virus. in addition, studies in guinea pigs showed that a reduction in nasal wash titers correlate with a decreased efficiency of viral transmission by aerosol [31] . the ability of cr6261 to abolish shedding of virus in pharyngeal secretions strongly suggests that antibodies like cr6261 might prevent or reduce virus spreading at the onset of an epidemic influenza outbreak in nursing homes or in case of a pandemic. in this study, we assessed the efficacy of the human monoclonal antibody cr6261 against a highly pathogenic avian h5n1 virus, as this provides a stringent disease model for severe influenza. however, the potential use of this antibody is not limited to viruses of this subtype or to other avian strains that may pose a pandemic threat. cr6261 has been shown to recognize h1 viruses that have emerged over a time span of 90 years from the h1n1 virus which caused the 1918 'spanish flu' pandemic to the latest brisbane viruses. since the epitope is conserved cr6261 is predicted to bind to future antigenic drift variants. this means that cr6261 could be used to protect against all h1 influenza viruses, including the ones resistant to oseltamivir. use of cr6261 in combination with effective medication against h3, such as oseltamivir or zanamivir, would effectively protect against all seasonal influenza viruses, without a need for prior knowledge of the virus subtype or strain. in the present study ferrets were challenged with an inoculum which is probably much higher compared to the viral exposure in naturally infection in humans. the rapid deterioration in ferrets with death occurring within 3 days underpins this hypothesis since humans exposed to h5n1 develop the first symptoms 2-4 days after the last exposure and even periods of up to 8 days have been reported [25] . the clinical signs in this model are quite extreme, but important for establishing the efficacy of the antibody as proof of concept. the data of these two experiments demonstrate the prophylactic and therapeutic efficacy of this new class of human monoclonal antibodies in a highly stringent and clinically relevant ferret model of human influenza. the human monoclonal antibody cr6261 was isolated from the igm+, cd27+ b cell repertoire of a healthy individual who was recently vaccinated with the seasonal influenza vaccine, using phage display selection on recombinant h5 haemagglutinin [1] . cr3014, an isotype-matched antibody with the ability to neutralize sars corona virus-which has similar tissue/organ tropism as that of h5 viruses-was used as a control antibody [32] . both antibodies were produced on per.c6h cells. the study was performed with outbred ferrets (mustela putorius furo, female, age approximately 8 months, schimmel farms, uddel, the netherlands). ferrets were screened for the presence of serum antibodies against aleutian disease virus, circulating seasonal influenza virus strains (a/h1n1, a/h3n2 and b) and the challenge virus (h5n1, a/indonesia/5/2005), and only seronegative animals were used in the study. the animals were housed in study groups of 6 (prophylactic experiment) or 10 (therapeutic experiment). antibodies were administered by intravenous injection in the jugular vein. viral challenge was performed intratracheally with 10 5 tcid 50 a/indonesia/05/2005 in 3 ml of pbs [33] . clinical observations were performed twice a day on days of intervention and once daily on other days. in the prophylactic experiment, animals were weighed 2 weeks before viral challenge (day 214), immediately prior to antibody administration (day 21), and after challenge (days 2, 4, and 5 or on the day of premature death). in the therapeutic experiment, animals were weighed on day 214 and day 22, immediately prior to challenge and antibody administration, and on days 2, 4 and the last study day. body temperature was recorded every 15 minutes throughout both experiments using a device (dst micro-t, star-oddi, reykjavik, iceland) implanted in the peritoneal cavity 14 days before challenge. the animal experiments were carried out in the central animal facilities of the netherlands vaccine institute (nvi, bilthoven) under conditions that meet the dutch legal requirements for animal experimentation and are in accordance with the 'guide for the care and use of laboratory animals', the recommendations of the institute for laboratory animal research (us national institutes of health), and association for assessment and accreditation of laboratory animal care international (aaa-lac) standards. viral titrations were performed as described elsewhere [34] . briefly, pharyngeal and nasal swabs were collected from all animals at day 21, day 2, day 4, and day 5. individual swabs were homogenized and resuspended in 3 ml medium and stored at 280uc until analysis. viral titres were determined by virus titration on madine darby canine kidney (mdck) cells. viral shedding from the upper respiratory tract was analysed by calculating the proportion of ferrets with detectable levels of infectious virus in nasal and/or throat swabs relative to the number of living ferrets. after necropsy, the cranioventral, craniodorsal, caudoventral and caudodorsal sections of the right lung were collected from each animal, weighed, homogenized and resuspended in 3 ml medium, and stored at 280uc until analysis. viral titres were determined after thawing of the tissue sections followed by homogenization and resuspension by virus titration on mdck cells. in addition, viral titres in tissues of brain, spleen, liver, kidney, and plasma from animals that received either 30 mg/kg of cr6261 or cr3014 one day prior to challenge were determined using the same method. a complete macroscopic post-mortem examination was performed on all animals. this included examination of the external surfaces and all orifices; the thoracic, abdominal and pelvic cavities with their associated organs and tissues; and the neck with its associated organs and tissues. lungs were weighed and all lung lobes were inspected and lesions described. the left lung (including trachea) was collected during autopsy, inflated with 10% neutral buffered formalin for fixation/histology and microscopic examination. paraffin embedded tissue sections (left cranial lobe, left caudal lobe, right cranial-, middle-and caudal lobes and accessory lobe) were stained with haematoxylin and eosin and assessed by light microscopy for aspects like congestion, emphysema, presence of foreign body, haemorrhagy, bronchioloalveolar hyperplasia and inflammation, and oedema. prior to blood sampling, the taking of nose and throat swabs and euthanasia, the ferrets were anaesthetised with ketamin (25 mg/ kg; i.m.). for implantation of temperature sensors, antibody administration and viral challenge, the animals were anaesthetized with a mixture of ketamin (12.5 mg/kg; i.m.) and domitor (7.5 mg/ kg; i.m.), followed by antisedan (0.5 mg/kg; i.m.). survival times after viral challenge were analysed using the logrank test and survival proportions using the fisher's exact test. body weight expressed as percentage change from baseline was calculated at the end of the study period (or earlier in the event of earlier death). maximum body temperatures were observed during day one and these values were subsequently used for calculating the mean maximum body temperature for each group. variables were analysed in an one-way analysis of variance (anova) with post-hoc testing to compare to the control group using dunnett's adjustment for multiple comparisons. lung weight and lung viral titres were compared across arms using anova, with day of necropsy entered as a covariate. differences between treatment groups were estimated using marginal means, with sidak's adjustment for multiple comparisons. statistical analyses were performed using spss version 15.0 (spss inc. usa). statistical significance level was set at a = 0.05. heterosubtypic neutralizing monoclonal antibodies cross-protective against h5n1 and h1n1 recovered from human igm+ memory b cells antibody recognition of a highly conserved influenza virus epitope structural and functional bases for broad-spectrum neutralization of avian and human influenza a viruses fact sheet 211: influenza. world health organization vaccines against epidemic and pandemic influenza swine influenza a (h1n1) infection in two children-southern california world now at the start of 2009 influenza pandemic the contents of the syringe interim within-season estimate of the effectiveness of trivalent inactivated influenza vaccine-marshfield, wisconsin, 2007-08 influenza season prevention and control of influenza; recommendations of the advisory commitee on immunization practices (acip) surveillance of resistance to adamantanes among influenza a(h3n2) and a(h1n1) viruses isolated worldwide amantadine-resistance among h5n1 avian influenza viruses isolated in northern china adamantane resistance among influenza a viruses isolated early during the 2005-2006 influenza season in the united states high levels of adamantane resistance among influenza a (h3n2) viruses and interim guidelines for use of antiviral agents-united states, 2005-06 influenza season drug resistance. a 'wimpy' flu strain mysteriously turns scary global transmission of oseltamivir-resistant influenza crystal structures of oseltamivir-resistant influenza virus neuraminidase mutants oseltamivir resistance during treatment of influenza a (h5n1) infection medical management of influenza infection animal models for the study of influenza pathogenesis and therapy ferrets and the challenges of h5n1 vaccine formulation animal models in influenza vaccine testing factors associated with case fatality of human h5n1 virus infections in indonesia: a case series update on avian influenza a (h5n1) virus infection in humans human infection with highly pathogenic h5n1 influenza virus human h5n1 influenza: current insight into pathogenesis h5n1 infection of the respiratory tract and beyond: a molecular pathology study the comparative pathology of severe acute respiratory syndrome and avian influenza a subtype h5n1-a review apoptosis and pathogenesis of avian influenza a (h5n1) virus in humans time lines of infection and disease in human influenza: a review of volunteer challenge studies blocking interhost transmission of influenza virus by vaccination in the guinea pig model human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets crossprotection against lethal h5n1 challenge in ferrets with an adjuvanted pandemic influenza vaccine comparison of rna hybridization, hemagglutination assay, titration of infectious virus and immunofluorescence as methods for monitoring influenza virus replication in vitro we thank andrea dingemans for her critical review of the manuscript, willem van aert and geert van amerongen for excellent technical assistance, james simon for supervising the viroclinics input, vera teeuwsen for preparing the protocols, and elina van herwijnen for independent data check, and jaco klap for statistical support. key: cord-354763-odzrco6q authors: drake, john m.; chew, suok kai; ma, stefan title: societal learning in epidemics: intervention effectiveness during the 2003 sars outbreak in singapore date: 2006-12-20 journal: plos one doi: 10.1371/journal.pone.0000020 sha: doc_id: 354763 cord_uid: odzrco6q background: rapid response to outbreaks of emerging infectious diseases is impeded by uncertain diagnoses and delayed communication. understanding the effect of inefficient response is a potentially important contribution of epidemic theory. to develop this understanding we studied societal learning during emerging outbreaks wherein patient removal accelerates as information is gathered and disseminated. methods and findings: we developed an extension of a standard outbreak model, the simple stochastic epidemic, which accounts for societal learning. we obtained expressions for the expected outbreak size and the distribution of epidemic duration. we found that rapid learning noticeably affects the final outbreak size even when learning exhibits diminishing returns (relaxation). as an example, we estimated the learning rate for the 2003 outbreak of severe acute respiratory syndrome (sars) in singapore. evidence for relaxation during the first eight weeks of the outbreak was inconclusive. we estimated that if societal learning had occurred at half the actual rate, the expected final size of the outbreak would have reached nearly 800 cases, more than three times the observed number of infections. by contrast, the expected outbreak size for societal learning twice as effective was 116 cases. conclusion: these results show that the rate of societal learning can greatly affect the final size of disease outbreaks, justifying investment in early warning systems and attentiveness to disease outbreak by both government authorities and the public. we submit that the burden of emerging infections, including the risk of a global pandemic, could be efficiently reduced by improving procedures for rapid detection of outbreaks, alerting public health officials, and aggressively educating the public at the start of an outbreak. rapidly spreading outbreaks of infectious diseases are an increasing concern for global public health [1, 2] and security [3] . emerging infections, which are typically defined as infectious diseases that have newly appeared in a population or are rapidly increasing in incidence or geographic range [4] , are a particular concern because at the time of emergence little is known about their epidemiology, particularly pathology, symptomatology, and transmissibility. thus, the crucial tasks of assessing epidemic risk and determining what public health interventions should be taken are complicated by uncertainty that borders on complete ignorance. of course, this uncertainty is rapidly reduced as the outbreak progresses and information concerning symptoms of infection, the biology of the infectious agent, the epidemiology of transmission, and the effectiveness of health precautions and intervention is collected and disseminated. this learning process has not been considered in theories of outbreak control [5, 6] or in near real-time models of emerging infections [7, 8] (compare correspondence in refs [9, 10] ). here, we study the collective effects of various processes (including possibly unidentified phenomena) on the change in the rate at which infectious persons are isolated. we refer to this set of processes collectively as ''societal learning''. a partial list of the processes contributing to societal learning includes isolation and identification of the infectious agent, development of tests for clinical diagnosis, disseminating information to public health and medical personnel, disseminating information to the public, and implementing public health policies including restrictions on individual movement or quarantine. disease control theory focuses on an quantity called the reproductive ratio, designated here as r 0 at the start of the outbreak and, if changing over time, r t at time t. outbreaks are considered to be under control when r t ,1, implying that outbreak conditions are such that on average disease prevalence will decline. most research in theoretical epidemiology has focused on how r t is related to disease and population parameters in order to understand how to induce the change from r 0 .1, during emergence, to r t ,1. recent developments include techniques for estimating r 0 from the initial stages of an outbreak [11, 12] and a model to ascertain the effect of a delay between the onset of an outbreak and the implementation of public health policies aimed at controlling disease spread [13] . here, we contribute to this developing toolbox for disease forecasting a model to understand how societal learning affects the expected final size and duration of disease outbreak. though some computational disease-specific models have recognized the importance of time-varying rates in disease spread, particularly with respect to the outbreak of sars in 2003 [14, 15] (compare [16] ), we believe this is the first analytical treatment of the concept. we also retrospectively explore the effect of societal learning during the 2003 outbreak of sars in singapore, using weekly data on the time between onset of symptoms and removal of infectious individuals. we speculate that societal learning will generally exhibit diminishing returns because increasing the removal rate becomes more difficult as individual isolation approaches a theoretical maximum rate. in such a case, the rate of societal learning is said to relax. we introduce statistical models to distinguish between relaxing and non-relaxing learning and test for relaxation during this outbreak. finally, we discuss societal and epidemiological factors that might affect societal learning, we observe that a difficult task during the early stages of an outbreak is to estimate the learning rate and suggest that the rate estimated here might be used as prior information in future outbreaks, and we conclude by recommending rapid investment in research at the time of initial detection when actions taken to reduce disease spread can be most efficient and cost effective. public health officials routinely make judgments whether or not to raise alarms about developing outbreaks. this decision is complicated by severe uncertainty during the early phases of an outbreak. further, bureaucratic inertia and the ignorance that necessarily accompanies emerging infections discourage rapid response. by contrast, false alarms resulting from hasty and premature assessment of outbreak risk can be very costly, and must be avoided if possible. understanding the role of societal learning in disease outbreaks is important for properly balancing these competing objectives. our concept of societal learning is characteristically reflected in outbreak dynamics as an increase over time in the rate at which infectious individuals are removed from circulating in the population. that is, we expect that as information about clinical symptoms, modes of transmission, the duration of incubation, etc., is collected and disseminated, the average time between the onset of symptoms and individual self-removal from the population (for instance by admission to hospital) or forced isolation (e.g., quarantine) will decline. from a dynamical perspective, we represent the average removal rate of individual cases as a function of time since the outbreak began, marked by the time at which the index case became infectious. for emerging diseases we assume that direct transmission between infected persons is the primary source of infection and that development of immunity and removing infectious individuals have negligible impact on the susceptible population. these assumptions are reasonable for outbreaks that ultimately do not infect more than a small fraction of the total population, i.e., emerging infections with relatively low prevalence. finally, we assume that transmission is a markov process, an approximation that amounts to assuming that individual infectious contacts are independent (compare [17] ). thus, representing the individual rate of infection by the constant parameter b 0 and the rate of removal as a function of time c(t), these assumptions imply that the growth of the epidemic is a timeinhomogeneous stochastic birth-death chain [18] [19] [20] . accordingly, the change over time in the probability distribution of the number of infected individuals x is given by this model has been previously studied and applied to problems ranging from population dynamics to astronomy [18, 19, 21] . in particular, the expected final epidemic size for this model is [18] : where and i 0 is the initial number of infected individuals. further, the distribution function for the duration of the outbreak with i 0 = 1 is: this is a very general model, as we have only specified that the transmission rate b 0 is constant and that the rate of removal c(t) changes over time, consistent with the concept of societal learning. conceptually, we decompose the removal rate, c(t), into two components. the first component represents removal in the absence of societal learning (i.e., through unexceptional health procedures or natural recovery) and is referred to as the base removal rate. the second component is an effect of societal learning and is assumed to be additive to the base removal rate. consequently, we represent the total removal rate as function of time c(t) = a(t)+b where b is the base removal rate and a(t) is a function for the additional effect of societal learning. (refer to table 1 for biological interpretations of parameters discussed in this section.) next, we consider two different learning scenarios. first, we suppose that societal learning is constant, i.e., that over any interval a doubling in time since the outbreak began corresponds to a doubling in the learned component of the removal rate. then, the effect due to learning can be represented as a line a(t) = a 0 t, where a 0 is called the 'basic learning rate', and the removal rate is linear: c 1 (t) = a 0 t+b. special cases of this model have a 0 = 0, where there is no effect of societal learning (resulting in the simple stochastic epidemic), and b = 0 where there is no natural recovery. the model with linear removal rate implies that the average time between infection and removal over time follows a hyperbola, g(t) = (c 1 (t)) 21 = (a 0 t+b) 21 , and that there is no upper bound to the rate at which infected individuals can be isolated; effectively, we suppose that the average time between infection and removal can be brought arbitrarily close to 0. for most (perhaps all) diseases this is an unreasonable assumption in the long run (though it may be a reasonable approximation at the start of an outbreak). in particular, the effect of societal learning probably decreases as the removal rate gets high and the interval between the onset of symptoms and isolation approaches a minimum biologically plausible quantity. this is a scenario in which cumulative number of removed patients is a decelerating function of time marked by diminishing returns. to incorporate such relaxation in our model we should generalize a(t) for instance a(t) = a 0 t a1 , with a 1 #1. where a 1 = 1 this model is equivalent to the linear model discussed above. of course, there is no principled theoretical reason why a 1 cannot be greater than 1. such a case is unlikely, however, and would imply acceleration not only in removals, but in the removal rate. in either case we have the general model for the removal rate c 2 (t) = a 0 t a1 +b and the associated model of the duration of the interval between onset of symptoms and removal g 2 (t) = c 2 (t) = (a 0 t a1 +b) 21 . in this case g(t) is approximately a power law with respect to time. we remark that learning relaxation could also result from diminishing returns on methods for disseminating information. for instance, if diagnostic information is transmitted by word-of-mouth, models for the spread of a rumor suggest that the fraction of the population which remains uninformed declines roughly logistically: first approximately proportional to the number of people who are in possession of the rumor but declining constantly over time as uninformed individuals become increasingly rare [6, 22] . examples of c 1 and c 2 and the associated g 1 and g 2 are shown in figure 1 . substituting the above model for societal learning in eqns (2) and (4) obtains two quantities of special interest: the expected outbreak size, and the distribution of extinction times, from which the probability density of the duration of outbreaks is obtained as the derivative with respect to time, finally, in this representation of the epidemic process, the concept of the reproductive ratio (designated by r 0 at the beginning of the outbreak, r t thereafter) is deterministic and is given by setting this equation to one and solving for t obtains the time until the outbreak is brought under control. for the case c(t) = c 1 (t), the time to control is given by t c = (b 0 2b)/ a 0 .still more models could be considered. however, we report below that the final epidemic size is affected mostly by the parameter a 0 , the rate of societal learning at the beginning of the outbreak, so that the precise shape of the removal function does not greatly matter. to test for societal learning in the 2003 outbreak of sars in singapore, we used the mean number of days between the onset of clinical symptoms and removal, by week, to fit different models for the removal process c. these data are slightly different than those that appeared previously as figure 1 in [15] and include some reclassified cases based on serological tests (s. ma, unpublished data). societal learning models were fit to the reciprocal of the mean of observed lags between onset of symptoms and removal c i = 1/g i for each week i, using nonlinear least squares regression. model fit was assessed using akaike's information criterion (aic) assuming the observations are drawn from a normal distribution with mean c i and homogeneous variance. we tested three hypotheses: (i) the null hypothesis of no base removal rate corresponding to b = 0; (ii) the null hypothesis of no saturation in learning corresponding to a 1 = 1; finally, (iii) the null hypothesis of no societal learning at all is given by a 1 = 1 for a 0 = 0. to represent the full epidemic process for sars the societal learning theory developed above must be modified to account for a significant latent period [12] . accordingly, we adopt the familiar s-e-i-r modeling framework (figure 2a in [5] ), modified to represent stochastic (markov) dynamics with time-inhomogeneous parameters. as before, we adopt the reasonable assumption that the population is large compared to the eventual size of the outbreak so that s remains constant throughout. thus, by substituting b 0 = as and ignoring the dynamics of removed individuals, we obtain the two-compartment model in figure 2b , where x and y designate the classes that were formerly e and i. finally, consistent with our earlier definition of societal learning, we allow the removal rate c to be a function of time, designated c(t). we assume that each state variable x and y can take only integer values (demographic stochasticity) and that individual transitions between classes are markovian. this model is a pair of coupled birth-death chains and is a generalization of the model studied in the earlier part of this paper. we obtained parameter values for these simulations as follows. using a bayesian approach, lipsitch et al. [15] determined that the basic reproductive ratio (r 0 ) for this outbreak was in the range [2.2, 3.6] . these values accord well with the likelihood-based estimate of wallinga and teunis [23] , who report a point estimate ofr 0~3 :1 and 95% confidence interval [2.3, 4 .0]. interpreting the estimates of lipsitch et al. [15] as the rate of secondary infection in a wholly susceptible population, r 0 is related to our parameters through the relation b 0 = r 0 6c 0 . recognizing that uncertainty in both r 0 and c will affect the accuracy of model projections we obtain an upper limit on b 0 (not a confidence interval because the parameters are not independent) from b + = r 0 + 6c + and a lower limit from b 0 2 = r 0 2 6c 2 , where (+) and (2) indicate the upper and lower limits on the estimate intervals for the respective parameters. to obtain a central (''best'') estimate of b 0 we take the midpoint of the range [2.2, 3.6] = 2.9 and multiply by the point estimate of our regressionä� 0~0 :12 to obtainb~0:35. throughout, we used the point estimate from the regression analysis above (0.046, see also results) for the basic learning rate after dividing by seven to convert from weeks to days: a 0 = 0.0066. as the learning rate never declined over the course of this outbreak, no relaxation was included in the model. finally, the transition rate between latent and infectious individuals (g) is approximately equal to the reciprocal of the duration of the incubation period. we used a transition rate of 0.15 d 21 , corresponding to an average incubation period of approximately 6.7. days. this is roughly consistent with, e.g., the ranges of estimates compiled by the world health organization (table 1 in [24] ) and the estimate (6.37 d) and 95% confidence interval [5.29, 7 .75] reported by donnelly et al. [25] , but slightly larger than the estimate of 4.8 d (95% confidence interval: [4.37, 5.29] ) obtained by kuk and ma [26] under the assumption that incubation times are drawn from a weibull distribution. retrospectively comparing model-based estimates of the expected outbreak size with the 238 observed cases (a partially circular comparison to begin with) is complicated by the fact that the number of initially infected individuals (the initial condition) is not defined by the model but must be asserted. one possibility is to assume that the outbreak begins with the index patient (i 0 = 1), but then the outbreak size of the theoretical model is biased by a significant portion of outbreaks that fail due to stochastic fadeout [27] . an alternative is to compare the observed outbreak size with the theoretical distribution of outbreak sizes for outbreaks initialized at i 0 = 1 conditioned on a 'major' or 'observable' outbreak occurring. however, this simply pushes back the problem of specifying the initial condition as some number of cases must be in four compartments corresponding to susceptible, exposed, infectious, and removed (or recovered) individuals. the rate at which individuals move from susceptible to exposed is according to massaction dynamics with proportionality constant a. individuals move from exposed to infectious at rate g and from infectious to removed at rate c. (b) by assuming that the number of susceptible individuals is approximately constant (an appropriate approximation for outbreaks in which prevalence is never a large fraction of the total population) we introduce the new variable b = as and reduce the four-compartment s-e-i-r model to a two-compartment model, designated here by the state variables x and y. doi:10.1371/journal.pone.0000020.g002 specified to correspond with 'major' or 'observable'. we adopted a third alternative. we reasoned that the first time medical personnel are alerted to the fact that there might be an emerging outbreak is the time that the index patient is observed to be infectious, corresponding to the removal of the patient from the population. at this time, the patient has infected an expected additional r 0 individuals (by the definition of r 0 ) and these infectious, or soon-to-be-infectious individuals are circulating in the susceptible population. we refer to this as the 'second generation initialization'. alternatively, the hospitalization of one individual with an anomalous infection is unlikely to attract significant attention. consideration of a possible outbreak more likely corresponds to the admittance in quick succession of several patients with anomalous infections, that is when the second generation of infected individuals is isolated and a third generation of individuals is infected. this is the 'third generation initialization'. accordingly, we simulated two distributions of final outbreak sizes. first we initialized at i 0 = 3, which is the midpoint of the estimated interval for r 0 identified by lipsitch et al. [15] , i 2~r0~2 :9, rounded to the nearest integer, corresponding to second generation initialization. second, we initialized at i 0 = 8, which is the rounded value of the expected number of infected individuals in the third generation, i 3~r 2 0 &8:4. to understand the importance of societal learning during the actual outbreak in singapore, we simulated 10,000 iterations of the stochastic s-e-i-r model described above using gillespie's direct method [28] with double and half the estimated basic learning rate while all other parameters were set to their best estimates and with initial condition i 0 = 8. empirical quantiles and the coefficient of variation (a measure of dispersion, the ratio of the standard deviation to the mean) were used to summarize the distributional properties of simulations. to look at the effects of societal learning and relaxation on outbreak control, we studied the average outbreak size over a range of scenarios (figure 3 ). for simplicity, we assumed b 0 = 1 throughout and compared different versions of the removal and learning process by tuning the parameters for the basic learning rate (a 0 ) and the relaxation rate (a 1 ). the temporal resolution of this model is therefore not explicit. thus, for concreteness assume that all rates are in units of days and that the baseline infectious period (g = c 21 ) is 3 d. then, the basic reproductive ratio is r 0~b0 =c~3 and we obtained the average epidemic size from eqn these ranges illustrate the range of cases between extremes in which societal learning is slow and relaxation is rapid (practically no effect of societal learning) and where societal learning is fast and no relaxation occurs at all (similar to the outbreak of sars). figure 3 shows that a 0 , the basic rate of societal learning, can be important for controlling outbreaks. the effect of relaxation can be examined by comparing the average outbreak size at various values of a 1 ,1 with the value at a 1 = 1, where there is no relaxation. evidently, relaxation must be extremely rapid (around a 1 = 0.5) for the effect to be noticeable. of course, this phenomenon is accentuated by its interaction with the basic societal learning rate so that if learning is extremely slow the effect of relaxation becomes more important. the observed removal rate increased consistently over the course of the 2003 sars outbreak in singapore (figure 4) . we found no effect of relaxation in the rate of societal learning, although there was strong evidence for both a baseline removal rate and an effect of learning (table 2) . we first fit the full model, but failed to reject the null hypothesis of no relaxation. consequently, we fit the reduced model with a constant learning rate, which is equivalent to the full model with exponential parameter a 1 = 1. in this model, both the base removal and learning parameters were highly significantly different than zero (base: p = 0.002; learning rate: p,0.0001). we remark that the reciprocal of the estimated base removal rate (b) can be interpreted as the duration of the infectious period in the absence of special intervention. accordingly, we obtained an estimate of 8.3 d (95% confidence interval: [5.8, 14.3] , obtained by inverting the confidence limits reported in table 2 ). inspection of the plots in figure 4 suggests that the observation in week 8 may be of exceptional importance to the final model. in terms of regression diagnostics, it may have high leverage (greatly affecting the uncertainty in parameter estimates) and high influence (greatly affecting the estimates themselves). a plot of standardized residuals versus leverage for the reduced model shows that this point is indeed matched by only one other point (week 0) for leverage ( figure 5 ). overlaying contour intervals for cook's distance, a measure of influence, shows that this point also has high influence. accordingly, so that the reader may compare we re-fit both the full and reduced models after dropping this point ( table 2 ). in this case the aic difference is less than two, so that neither model is better supported by the data. further while the maximum likelihood estimate for a 1 is quite low (a 1 = 0.676; to be interpreted as considerable relaxation), the confidence interval barely fails to include 1, so the evidence is not conclusive. to study the effect of the duration of the latent period on average outbreak size, we simulated 500 iterations of the model at each of 13 different durations for the average latent period. the average outbreak size decreased with the duration of the latent period as shown in figure 6 . the average size of simulated outbreaks initiated with the second generation initialization condition (i 0 = 3) was 102 cases. the 2.5% and 97.5% quantiles were 4 and 321 cases, respectively. the coefficient of variation in the final outbreak size was 0.85. the average size of simulated outbreaks initiated with the third generation initialization condition (i 0 = 8) was 278 cases. the 2.5% and 97.5% quantiles were 56 and 611 cases, respectively, with coefficient of variation 0.52. thus, the observed total outbreak size (238 cases; [29] ) is consistent with either the second or the third generation initialization conditions. outbreak simulations in which learning occurred at half the observed rate had average final outbreak size of 799 cases while outbreak simulations in which learning occurred at twice the observed rate had average final outbreak size of 116 cases. we found little evidence for relaxation in the learning rate for sars in singapore. first, restricting our discussion to the analysis with all data, we find that the maximum likelihood estimate of the relaxation parameter is extraordinarily close to one (differing by 0.07%), perfect non-relaxation. admittedly, the confidence interval on this parameter is large. one interprets this to mean that the vigilance of the public health community as a whole continued throughout the outbreak and that improvement in intervention effectiveness continued unabated. however, we also found that one relatively uncertain data point was important to this analysis (week 8). whether this point should be excluded from interpretation is unclear. on one hand, it is a real observation and (because of its high influence) is known to contain a great deal of information. therefore, one is inclined to allow this observation considerable weight. on the other hand, its importance, especially at the end of the data series is suspicious. if we exclude this point from analysis post hoc, we find that we are unable to make any strong conclusions at all. what most likely occurred is that the distribution of average infectious period at the point where the outbreak was rapidly brought under control was highly dispersed (high variance) and highly skewed. accordingly, the mean removal rate probably does relax, but the data that were available to this study are too aggregated to make this inference conclusively. it is unknown if the rate of learning estimated in this study is unique to this outbreak or if it might be more representative. we remark that both parameters in the learning rate model are readily interpreted, and that theoretical effects of improvement in surveillance, mechanisms for informing public health personnel and the public, and rapid research response could be studied by extending this simple model to represent more realistically the effects of alternative policies as covariates. the final size of an outbreak is greatly affected by transmission events early during the outbreak process. outbreaks can be curtailed when public health interventions are rapid and efficient. but the severity of an outbreak is often unclear during these initial stages of transmission when intervention can be most effective [11, 27] . further, there are limits to how quickly diagnostic information about an emerging infection can be obtained and disseminated to health care providers. this is not the first model to consider the effect of changes in the removal rate (e.g., [14, 15] ). however, in contrast to earlier studies, we first explicitly considered societal learning parametrically in a theoretical model. our model also more realistically represents the ramping up of intervention in contrast to models that simply have ''before control'' and ''after control'' regimes (e.g., [13] ). we showed that the final outbreak size decreases rapidly with a modest investment in learning. we also found strong evidence of learning in data from the 2003 outbreak of sars in singapore. public health interventions for sars include encouragement to report to hospital rapidly after the onset of clinical symptoms, contact tracing for confirmed and suspected cases, and quarantine, monitoring, and restricting the travel of contacts [25, 30] . we believe these interventions were highly effective at reducing the final size of the sars outbreak. a limitation of this analysis is that we only consider temporal changes in removal, though information dissemination and public concern almost certainly led to a decline in transmission (b 0 ) too [15] . unfortunately, this effect is much more difficult to independently estimate and must instead be inferred from the information provided by the epidemic curve together with observations of the onset-of-symptoms to removal interval. in general, however, the model studied here (eqn 1) and its solution (eqn 2) will also apply to this situation and can be used wherever such data are available. the effects of biological and social factors that might bring about changes in transmissibility is an important area for further theoretical research. our estimate of the duration of the infectious period (8.3 d, 95% ci: [5.8, 14.3] ) is consistent with measures of viral shedding, obtained by peiris et al. [31] using quantitative reverse transcriptase on sequential nasopharyngeal aspirates/throat and nose swabs (npa/tns), in which maximum virus excretion occurs around the tenth day of illness (compare also [24] ). indeed, only about 35% of npa/tns continued to test positive by the third week since the onset of symptoms [24] . figure 6 . relationship between the average latent period (x-axis) and average total outbreak size in simulations (y-axis). latent period is log 2 transformed (to illustrate a wide range of possible values) and ranges from 1 d to 4096 d (,11 y). the approximate location of sars is indicated by the arrow. doi:10.1371/journal.pone.0000020.g006 these results underscore the value of immediate action at the start of an outbreak (high a 0 ). the processes considered to contribute to societal learning include such publicly visible actions as declaring a state of emergency, global health alert, or (minimally) disseminating information to the public. the societal and economic costs of mistakenly declaring a state of emergency can be tremendous, but are probably small in comparison to the costs of failing to intervene in a major preventable outbreak. thus, we echo anderson et al. [32] in concluding that the major lessons of the 2003 outbreak of sars are to improve surveillance and detection, including real-time data collection; develop capability for rapid response by the research community; and devise mechanisms for immediate implementation of effective interventions. important topics for research include estimating the effect of learning on transmission (the parameter b 0 in the model), and identifying the different activities that contribute to learning (a 0 ) and relaxation (a 1 ) and their costs. then, a cost sensitive model should be developed to balance the competing goals of raising unnecessary alarm and preventing a major outbreak. such a model would be most useful if it had reference points that would trigger alerts at different levels (i.e., to function as an early warning system) and could guide intervention efforts. such a model would not need to be purely economical, but could incorporate loss of human life and well-being as constraints on the decision set. of course, learning rates (and possibly relaxation) will vary geographically reflecting different societal conditions, research institutions, levels of emergency preparedness, etc. further, these phenomena may also differ among emerging diseases, for instance depending on their similarity to diseases that are well understood or their resistance to laboratory isolation and characterization. despite these limitations, we suggest that our estimate of the basic learning rate (0.0066 d 21 ; 95% confidence interval [0.0051, 0.0081]) could be used as prior information during future outbreaks. the difficulty of forecasting the total epidemic curve at an early stage is well appreciated [7] . by eliminating the need to simultaneously estimate highly correlated parameters, a good understanding of the dynamical consequences of public health response would enable real-time modeling to focus on estimating disease parameters like transmission rates [11] . then, estimated disease components and known or conjectured models for response, including models of societal learning, could be integrated in a single modeling framework for projections. a systematic analytic approach to pandemic influenza preparedness planning heading off an influenza pandemic factors in the emergence of infectious diseases infectious diseases of humans: dynamics and control epidemic modelling: an introduction sars, lay epidemiology, and fear severe acute respiratory syndrome epidemic in asia sars epidemiology modeling (reply) estimation and inference of r 0 of an infectious pathogen by a removal method appropriate models for the management of infectious diseases limits to forecasting precision for outbreaks of directly transmitted diseased transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions transmission dynamics and control of acute severe respiratory syndrome a branching mode for the spread of infectious animal diseases in varying environments realistic distributions of infectious periods in epidemic models: changing patterns of persistence and dynamics on the generalized birth-and-death process the elements of stochastic processes with applications to the natural sciences uncertainty in sars epidemiology stochastic processes and population growth all-or-none elements and mathematical models for sociologists different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures consensus document on the epidemiology of severe acute respiratory syndrome (sars) epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong the estimation of sars incubation distribution from serial interval data using a convolution likelihood superspreading and the effect of individual variation on disease emergence a general method for numerically simulating the stochastic time evolution of coupled chemical reactions summary of probable sars cases with onset of illness from 1 public health measures implemented during the sars outbreak in singapore clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study epidemiology, transmission dynamics and control of sars: the 2002-2003 epidemic we thank members of the penn state center for infectious disease dynamics for helpful discussion of this study. a referee suggested fitting the model to the data without the observation at week 8. key: cord-351185-3y3gou6v authors: buckles, thomas c.; ziemba, brian p.; djukovic, danijel; falke, joseph j. title: rapid exposure of macrophages to drugs resolves four classes of effects on the leading edge sensory pseudopod: non-perturbing, adaptive, disruptive, and activating date: 2020-05-29 journal: plos one doi: 10.1371/journal.pone.0233012 sha: doc_id: 351185 cord_uid: 3y3gou6v leukocyte migration is controlled by a membrane-based chemosensory pathway on the leading edge pseudopod that guides cell movement up attractant gradients during the innate immune and inflammatory responses. this study employed single cell and population imaging to investigate drug-induced perturbations of leading edge pseudopod morphology in cultured, polarized raw macrophages. the drugs tested included representative therapeutics (acetylsalicylic acid, diclofenac, ibuprofen, acetaminophen) as well as control drugs (pdgf, gö6976, wortmannin). notably, slow addition of any of the four therapeutics to cultured macrophages, mimicking the slowly increasing plasma concentration reported for standard oral dosage in patients, yielded no detectable change in pseudopod morphology. this finding is consistent with the well established clinical safety of these drugs. however, rapid drug addition to cultured macrophages revealed four distinct classes of effects on the leading edge pseudopod: (i) non-perturbing drug exposures yielded no detectable change in pseudopod morphology (acetylsalicylic acid, diclofenac); (ii) adaptive exposures yielded temporary collapse of the extended pseudopod and its signature pi(3,4,5)p(3) lipid signal followed by slow recovery of extended pseudopod morphology (ibuprofen, acetaminophen); (iii) disruptive exposures yielded long-term pseudopod collapse (gö6976, wortmannin); and (iv) activating exposures yielded pseudopod expansion (pdgf). the novel observation of adaptive exposures leads us to hypothesize that rapid addition of an adaptive drug overwhelms an intrinsic or extrinsic adaptation system yielding temporary collapse followed by adaptive recovery, while slow addition enables gradual adaptation to counteract the drug perturbation in real time. overall, the results illustrate an approach that may help identify therapeutic drugs that temporarily inhibit the leading edge pseudopod during extreme inflammation events, and toxic drugs that yield long term inhibition of the pseudopod with negative consequences for innate immunity. future studies are needed to elucidate the mechanisms of drug-induced pseudopod collapse, as well as the mechanisms of adaptation and recovery following some inhibitory drug exposures. leukocytes, including macrophages and neutrophils, possess a sophisticated chemosensory system that controls cellular migration up primary attractant gradients to sites of infection, injury, or tumor formation during the innate immune response [1] [2] [3] [4] [5] [6] [7] [8] . at the target site, recruited leukocytes and other cells can release secondary attractants that recruit additional leukocytes, but excessive secondary attractant signaling and recruitment may lead to local inflammation and, in extreme cases, toxic effects (reviewed in [9] [10] [11] [12] [13] [14] ). the chemosensory pathway that directs leukocyte migration up primary and secondary attractant gradients is localized on the broad sensory pseudopod at the leading edge of the polarized cell, where pathway components assemble on the cytoplasmic leaflet of the plasma membrane (reviewed in [3-5, 9, 15-17] ). the leukocyte chemosensory pathway is highly specialized, conferring unique properties to leukocyte chemotaxis not observed in other cell types. in particular, the leukocyte pathway possesses a positive feedback loop that is able to maintain the stability and ruffling activity of the leading edge pseudopod even in the absence of an attractant gradient, enabling the pseudopod to rapidly detect and direct migration up a newly appearing gradient [3, 4, 16, [18] [19] [20] [21] [22] [23] [24] [25] [26] . the feedback loop is comprised of multiple essential signaling proteins, cytoskeletal elements, and second messengers, including: phosphoinositide-3-kinase (pi3k); protein kinase c (pkc); small g-proteins (ras, rac); actin filaments; the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (pip 3 ); and ca 2+ ions [2, 7, 16, 18, 19, [21] [22] [23] [24] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] . activation of any loop component leads to a characteristic co-activation of other loop components, increased activity of the lipid kinase pi3k and accumulation of its product pip 3 signaling lipid, and expansion of the leading edge pseudopod. by contrast, inhibition of any loop component yields inactivation of other loop components, decreased pi3k activity and pip 3 levels, and contraction of the pseudopod. the leading edge chemosensory pathway also possesses an intrinsic adaptation mechanism that enables the leading edge pseudopod to adapt as needed to changes in its chemical environment. adaptation is a ubiquitous feature of chemosensory pathways in archaea, bacteria, and eukaryotes [41] [42] [43] [44] [45] . such intrinsic adaptation adjusts pathway gain and dampens background signals, thereby preventing signal saturation and maintaining sensory function as the cell migrates up an attractant gradient in a complex chemical environment with competing stimuli. in leukocytes, intrinsic pseudopod adaptation is provided by a hardwired adaptation branch of the chemosensory pathway that regulates the net activity of specific pathway components, often by covalent modification [41] [42] [43] [44] [45] . different types of leukocytes, such as macrophages and neutrophils, possess different classes of cell surface receptors and other specializations, but appear to share similar mechanisms of chemosensing and intrinsic adaptation [15, 20, 32, 46] . such intrinsic pseudopod adaptation could in principle counteract perturbations triggered by drug exposure, thereby restoring normal pseudopod morphology and function. alternatively, extrinsic intracellular or extracellular processes besides intrinsic chemosensory adaptation could counteract drug-induced perturbations; such extrinsic mechanisms could extrude, sequester, or chemically modify a drug that perturbs the leading edge pseudopod [47] [48] [49] . the leading edge pseudopod is clearly visualized in live, cultured leukocytes by differential interference contrast microscopy (dicm) or by fluorescence microscopy [26, 30] . prior studies have shown that drugs that target key components of the leading edge positive feedback loop yield dramatic changes in the size of the pseudopod and its leading edge pip 3 signal [26, 30, [50] [51] [52] [53] [54] . well characterized inhibitors of the leading edge positive feedback loop that collapse the leading edge pseudopod include wortmannin, a covalent inhibitor of pi3k that directly inhibits leading edge pip 3 production, and gö6976, an inhibitor of pkc protein kinase activity that indirectly inhibits pip 3 production by blocking a key component of the feedback loop [26] . natural attractants for leukocytes include platelet-derived growth factor (pdgf), a powerful macrophage attractant that activates a receptor tyrosine kinase (rtk, in particular the pdgf receptor) that stimulates pi3k and pip 3 production. pdgf has been observed to expand the leading edge pseudopod, increase its pip 3 signal, and drive increased levels of cell polarization [26, 55] . the present study begins by developing a novel combination of live cell assays and applying them to cultured, polarized raw macrophages in order to quantify the effects of four therapeutic and three control drugs on leading edge pseudopod morphology. the four selected therapeutic drugs possess well established clinical safety and are investigated within their standard clinical dosages (i.e. total plasma concentrations, [56] [57] [58] [59] [60] [61] [62] ). three are non-steroidal antiinflammatory drugs (nsaids) that inhibit cyclooxygenase enzymes (cox) and thereby inhibit the production of inflammatory signals [63] [64] [65] : acetylsalicylic acid (aspirin), ibuprofen and diclofenac. the fourth, acetaminophen (paracetamol), is not an nsaid and relieves pain via unknown mechanisms. the three control drugs, wortmannin, gö6976, and pdgf each target a known pathway component (see above) and are employed at their standard experimental concentrations [26, 30, [50] [51] [52] [53] [54] . the findings demonstrate that the present approach can resolve drug effects on the leukocyte leading edge pseudopod of polarized, cultured macrophages into four operational classes: (i) non-perturbing exposures, (ii) adaptive exposures, (iii) disruptive exposures, and (iv) activating exposures. when any of the four therapeutic drugs are added slowly to mimic the slow rise of drug levels in the plasma of patients following standard oral dosages [56] [57] [58] [59] [60] [61] [62] 66] , these drugs are found to have little or no effect on leading edge morphology, as consistent with their well-established clinical safety. however, rapid addition of the therapeutic and control drugs reveals the four operational classes. acetylsalicylic acid and diclofenac are classified non-perturbing because they have little or no detected effect on the macrophage leading edge pseudopod and its pip 3 signal. for these drugs, either rapid or slow addition retains normal pseudopod morphology in single cell and population studies. in contrast, ibuprofen and acetaminophen are classified as adaptive because rapid addition of either drug to polarized cells yields short-term collapse of the leading edge pseudopod and loss of the pip 3 signal, followed by slow recovery. in contrast to the four therapeutic adaptive drugs, the two non-clinical control inhibitors wortmannin and gö6976 are each known to directly inhibit key components of the leading edge positive feedback loop and rapid addition is observed herein to trigger long term collapse of the pseudopod with no detected recovery as previously observed [26, 30, [50] [51] [52] [53] [54] . at the other extreme, pdgf is a well studied attractant and rapid addition yields leading edge expansion and increased cell polarization over the three hour observation period as predicted [26, 55] . overall, the findings provide impetus for future studies probing the inhibitory mechanisms underlying therapeutic drug-triggered pseudopod collapse, as well as the adaptive mechanisms that enable pseudopod recovery following rapid exposure to some perturbing drugs. raw 264.7 macrophages were obtained from the american type culture collection (atcc tib-71, manassas, va; lots 70000171, 70012232 with cell identity confirmed by atcc via growth properties, morphology, and coi assay). cell media (supplemented dmem) was composed of dmem obtained from thermo fischer scientific (waltham, ma), fetal bovine serum (fbs, contains~400 μm bsa as specified by manufacturer) from millipore-sigma (st. louis, mo), penicillin, streptomycin and glutamineplus (l-alanyl-l-glutamine) from atlanta biologicals (flowery branch, ga). dulbelcco's phosphate buffered saline (d-pbs) was from gibco (gaithersburg, md). all cell media was regularly subjected to mycoplasma testing to ensure lack of infection. matriplate 96 well imaging plates were from matrical, inc. (spokane, wa). acetylsalicylic acid (lot slbv2290), ibuprofen (lot slbx6584), acetaminophen (lot slbr2060v), diclofenac (lot bcbs5961v), wortmannin (lot 086m4026v), pdgf (lot slb63548v), and hepes (free acid) were obtained from millipore-sigma (st. louis, mo). gö6976 (lot 6a/174381) was purchased from tocris biosciences (minneapolis, mn). cellmask deep red and green membrane stains were purchased from thermofischer scientific (waltham, ma). cell-culture grade dmso was purchased from mp biomedicals (santa ana, ca). akt-ph-mrfp mammalian expression plasmid was subcloned by john h. evans [26, 30] . cell culture techniques used herein have been previously described [26, 30] . briefly, starting from cryogenic stocks, raw macrophages were grown as per atcc recommendation at 37˚c under 5% co 2 in supplemented dmem media containing 2 mm glutamineplus, 20 mm hepes ph 7.2 with naoh, 100 u/ml penicillin, 100 μg/ml streptomycin, and supplemented with 10% fbs (where undiluted fbs contains~400 μm bsa as specified by manufacturer), resulting in a final albumin concentration of~40 μm. cells were washed in d-pbs ph 7.2 with hcl and passaged upon reaching~80% confluency, as recommended by atcc. single cell or population experiments used raw cells following at most 14 or 5 such passages, respectively. for studies of cells transfected with akt-ph-mrfp to monitor pip 3 lipid, the transfection protocol using the neon electroporation system has been previously described [26] . live-cell microscopy methods have been previously described [26] . cells were counted by hemocytometer and plated at known density in 96 well imaging plates, then subsequently incubated at 37˚c under 5% co 2 for~24 hours to facilitate cell adhesion and generate conditions of steady state cell polarization. in the bottom of each well, cells attach to a flat glass surface and many spontaneously polarize in the absence of an attractant gradient. two microscopes were employed. (i) images for figs 1a and 1b and 4c and 4d and s1 and s3 were captured using a nikon tie microscope equipped with a 40x, 0.95 n.a. objective and a hamamatsu orca-flash 4.0 v3 digital cmos camera. (ii) images for figs 1c and 1d and 4e-4l and s2 were acquired with a nikon tie spinning-disc confocal microscope equipped with a yokogawa csu-x1 scanning unit, an andor ixon 888 emccd camera, and a 60x, 1.3 n.a. water-immersion objective (fig 1c and 1d ) or a 40x, 0.95 n.a. objective (figs 4e-4l and s2). for both microscopes, the imaging stage was enclosed in an environmental chamber maintaining humidity, 5% co 2 , and 37˚c. imaging studies monitored a single cell, or a population of cells, following the addition of drug or its carrier as a control. two types of addition protocols were employed. in rapid addition the drug or carrier was added (0.5 μl into 500 μl) at t = 0 via a 2 μl micropipettor (gilson) with immediate, gentle pump-mixing with the same micropipettor and tip to yield the final concentration within 10 sec. this pump-mixing procedure slowly drew and expelled 100 μl of media into and out of the tip, and then repeated for a total of 5 pump-mixing cycles during the 10 sec mixing time. in slow addition the drug or carrier was added via a stepwise procedure over a period of 90 min to generate a more gradual rise up to the same final total drug concentration as rapid addition. this slow addition protocol added the drug in four equal increments using a 1 μl pre-calibrated hamilton syringe (each 0.25 μl into 500 μl) at t = 0, 30, 60, 90 min with immediate, gentle pump-mixing as above to generate a more gradual, stepwise increase in concentration. final total drug concentrations were pdgf ββ 2.1 μm, wortmannin 500 nm, gö6976 1.0 μm, acetylsalicylic acid 1.1 mm, diclofenac 17 μm, ibuprofen 240 μm, acetaminophen 170 μm. all drugs were added in carrier dmso with the exception of pdgf ββ, which was added in supplemented dmem. care was taken to ensure that drug effects observed after addition were due to the intended global increase in the total drug concentration, not due to locally high drug concentrations prior to complete mixing. the microscopic field observed following drug addition in single cell and population measurements represented a small fraction (< 0.2% and < 6%, respectively) of the 8 x 8 mm 2 glass surface area in the observation well. in single cell and population studies, cells appropriate for imaging were selected at random locations in three of the four quadrants of the surface, then drug was added at the outside corner of the remaining quadrant (always the outside corner of the far-left quadrant) followed by pump mixing at that same location. no correlation was observed between drug effects on cells and the distance of the cells from the drug addition. moreover, in single cell measurements each cell observed exhibited approximately the same drug effect, while in population measurements all four quadrants of the large grid showed approximately the same drug effects. such independence of drug effects from the distance to the point of addition indicated the effects were global, not local. a novel cell population analysis was developed to quantify both initial and long-term drug effects, including pseudopod adaptation and recovery following an initial drug-induced perturbation. timecourses of cell populations were measured in a large field containing 1200 ± 100 cells (figs 3, 4a and 4b and s1), which was scanned as either 8 x 8 (fig 4ab) or 6 x 6 ( fig 3) unit areas. the unit areas, which served as individual data points, were stitched together into a full field super-image using nikon elements software. for each full field, an initial super-image was captured at t = 0, then subsequent super-images were captured at the specified timepoints after drug or control addition. analysis of population timecourse data was as follows. at each timepoint, the number of polarized cells with extended leading edge pseudopods was counted in fiji (imagej) yielding an average over the 64 (fig 4ab) or 36 ( fig fig 1. imaging highly polarized, actively ruffling cultured raw macrophages and drug-induced collapse of the leading edge pseudopod. raw macrophages were plated on glass at 37˚c in the absence of an attractant gradient. individual, spontaneously polarized cells exhibiting extended, ruffling, leading edge pseudopods were imaged as described in methods. these cells migrate slowly on glass and are thus well suited for quantitative imaging of event timecourses at their leading edges. drugs were added at t = 0 to the total concentration indicated in fig 2. panels a through d images show representative effects of drug addition on the leading edge pseudopod: (a,b) dicm images of a single cell at t = 0 and 5 min after ibuprofen addition, respectively, illustrating the drug-triggered loss of leading edge pseudopod area; (c,d) fluorescence images of the pip 3 sensor aktph-mrfp in a single cell at t = 0 and 5 min after wortmannin addition, respectively [26, 30] illustrating the drug-triggered loss of the signaling lipid pip 3 on the leading edge pseudopod. https://doi.org/10.1371/journal.pone.0233012.g001 3) unit areas of that super-image. for each super-image, the resulting mean timepoints were normalized to the mean initial value of the t = 0 timepoint. finally, the final global mean timecourse was determined by averaging the corresponding mean normalized timepoints from different super-images (experiments), yielding the global mean timepoints and their sem for n = the number of super-images. the resulting global mean timecourses represent averages over 3-6 super-images (experiments) each containing approximately 1000 cells and collected from independent wells over 3-6 separate days for therapeutic drug treatments, or 2-6 separate days for control treatments. single-cell timecourses ( fig 5) were measured and analyzed as previously described for individual, polarized cells with extended, actively ruffling leading edge pseudopods [26] . briefly, for each cell the value of the initial t = 0 timepoint was used to normalize all timepoints. drugs employed in the present study and key parameters. shown are the four representative therapeutic drugs employed (acetylsalicylic acid, acetaminophen, diclofenac, ibuprofen) as well as the three control drugs (gö6976, wortmannin, pdgf ββ). information provided for each drug includes: chemical structure, molecular weight, pka, therapeutic peak total concentration in human plasma following clinical dosage, total concentration employed in the present cultured cell experiments, target (where known), and percent bound to proteins in human plasma. notes: a recommended therapeutic range of total drug concentration in human plasma; b experimental total drug concentration in cell culture medium (this study); c references [73, 74] ; d references [57, 59] ; e reference [58] ; f references [75] [76] [77] [78] . https://doi.org/10.1371/journal.pone.0233012.g002 subsequently, for a group of at least 3 cells in an experiment carried out on the same day, corresponding normalized timepoints were averaged to generate a mean value for each timepoint. finally, the resulting mean timepoints for individual experiments were averaged over all experiments to generate a global mean and its standard error of the mean (sem) for n = the number of experiments. final global averages represented 17-32 total cells imaged in at least 3 effects of gradual drug addition on populations of polarized macrophages. raw macrophages were plated on glass at 37˚c in the absence of an attractant gradient, then images of cell populations containing large numbers of individual, spontaneously polarized cells exhibiting extended, ruffling, leading edge pseudopods were acquired as described in methods. subsequently, drug or carrier was added in four equal increments at 0, 30, 60, 90 min summing to yield the total drug concentration indicated in fig 2. images of the cell population were collected 5 min after each incremental addition at the indicated timepoints over a 2 hour observation period (filled symbols). the number of cells in the population displaying extended leading edge pseudopods was counted for each timepoint and normalized to the initial number prior to drug addition. data collected for multiple populations was averaged, yielding the illustrated mean timecourse for each treatment. as an internal control, rapid addition at t = 0 of the full total drug concentration was also carried out for two of the four therapeutics (ibuprofen fast and acetaminophen fast, open symbols; these data can be directly compared with more extensive, independent rapid addition data in fig 4 below) . panel a summarizes the resulting timecourses. panel b compares the dose dependence of drug effects measured 5 min after addition at t = 0 of either 1x total drug concentration (fig 2) or 0.25x total drug concentration. error bars are sem for n = 3 (fast acetaminophen) or 4 (others) experiments carried out on at least three days. error bars smaller than their data point symbol are not visible. cell cultures were prepared for mass spectrometry analysis of drug stability using exactly the same procedures described above for long-term studies of drug effects on cell populations (above). 50 μl of media were taken from the culture immediately after drug addition and mixing, then snap frozen in ln 2 . cultures were allowed to incubate 180 minutes before another 50 μl sample was withdrawn and snap frozen. images of the cell population in each culture were analyzed to ensure the drug effects on the cells were within their characteristic ranges. the number of replicate samples, each prepared from separate cultures, ranged from 3 (acetylsalicylic acid and diclofenac) to 6 (acetaminophen and ibuprofen). means and standard deviations were calculated for each set of replicates. after storage at -80˚c and thawing, samples were diluted into 250 μl meoh containing 50 μm meloxicam as an internal sample standard. samples were then stored at -20˚c for 20 minutes and subsequently spun at 15,000 x g for 15 minutes. while not disturbing the precipitated pellet, 200 μl of supernatant was extracted and dried via speedvac. prior to lc-ms analysis, dried study samples containing each drug were reconstituted in 200 μl (ibuprofen, acetaminophen and aspirin) or 100 μl (diclofenac) of 20 mm ammonium acetate in 60% water/35% acetonitrile/ 5% methanol + 0.1% formic acid. except for acetaminophen samples, the reconstitution solvent also contained 20 μm 4-amino-salicylic acid (4-asa) that was used as a post-reconstitution internal standard to monitor final sample preparation and mass spec performance (4-asa overlapped the acetaminophen peak). the lc system was composed of an agilent 1100 binary pump, an agilent 1110 auto-sampler and an agilent 1100 temperature-controlled column compartment (agilent technologies, santa clara, ca). 10 μl of each sample was injected into the lc. chromatography was performed in reverse phase (rp) on agilent eclipse xdb-c8 column (150 x 4.6 mm, 5.0 μm particle size, agilent technologies, santa clara, ca). the flow rate was 0.400 ml/min, autosampler temperature was kept at 4˚c, and the column compartment was set at 40˚c. chromatography time was 35 min, and all 6 peaks eluted off the column between 10 and 20 min without any overlapping except for acetaminophen and 4-asa. after the chromatographic separation, ms ionization and data acquisition was performed using ab sciex qtrap 4000 mass spectrometer (ab sciex, toronto, on, canada) equipped with methods) , then images of cell populations containing large numbers of spontaneously polarized cells with extended leading edge pseudopods were acquired as described in methods. drugs were added rapidly at t = 0 to the total concentration indicated in fig 2. shown are timecourses following drug addition illustrating the changing number of cells in the full population that display extended leading edge pseudopods, normalized to the initial number prior to drug addition. panels a and b summarize findings for the four therapeutic drugs (a) and for the three control drugs and two carriers (b), respectively. data collected for multiple population measurements was averaged, yielding the illustrated mean timecourse for each treatment. error bars are sem for n = 6 experiments carried out on at least five days for experimental treatments, or at least two days for control treatments. error bars smaller than their data point symbol are not visible. panels c through l show representative cell images in these studies, as follows. (c,d) small subsection of a dicm super-image used to quantify the number of cells with extended leading edge pseudopods in a population before and 5 min after ibuprofen addition, respectively. dividing cells are occasionally observed as illustrated by the pair at lower left. (e-l) expanded sequential images showing the leading edge pseudopod of a single polarized macrophage within a super-image, illustrating temporary pseudopod collapse after rapid ibuprofen addition at t = 0, followed by pseudopod adaptation and recovery. panels e-l correspond to timepoints 0, 2.5, 5, 9, 30, 60, 120, 180 min after ibuprofen addition, respectively. scale bars indicate 10 μm. https://doi.org/10.1371/journal.pone.0233012.g004 fig 5. effects of rapid drug addition on polarized macrophages: single cell analysis. raw macrophages were plated on glass at 37˚c in the absence of an attractant gradient. individual, spontaneously polarized cells exhibiting extended, ruffling, leading edge pseudopods were imaged for 5 min as previously described ( [26] and methods). drugs were added rapidly at t = 0 to their standard total concentration (fig 2) . panels a and c show the timecourses of leading edge pseudopod area loss following the addition of the four therapeutic drugs (a), and the three control drugs or media controls (c). panels b and d show the timecourses of leading edge pseudopod pip 3 loss following the addition of the four therapeutic drugs (b), and the three control drugs or media controls (d). all timecourses were quantified as previously described [26] . briefly, a box of uniform dimensions and placement was used to define the leading edge region of each cell, then the leading edge area or fluorescence was quantified at the indicated timepoints. note these parameters do not decay to zero, even for the strongest pseudopod inhibitors wortmannin and gö6976, because the box contains both the pseudopod and the tip of the cell body at t = 0. thus, the final timepoints at t = 5 min show the loss of area and fluorescence due to the pseudopod collapse, but retain the signals arising from the cell body remaining within the box, respectively. error bars are sem for n = 17 to 32 cells over at least three experiments on at least two different days. error bars smaller than their data point symbol are not visible. the extracted mrm peaks were integrated using multiquant 2.1 software (ab sciex, toronto, on, canada). the two spiked internal standards (meloxicam and 4-aminosalicylic acid) were used to monitor sample preparation and lc-ms assay performance. a standard mixture of all four study drugs was used as a quality control to measure signal reproducibility. intra-and interday reproducibility for each measured standard was under 10% without data normalization. replicates suitable for statistical analysis were carried out for all measurements, as described in detail above and reported in the relevant figure legend. no outliers were removed during data analysis and calculation of reported means. data were normalized to the initial t = 0 measurement for individual cells and for populations to facilitate comparisons between different cells and different populations, respectively, given standard biological variability. statistical significance of differences between means was calculated using student's unpaired t-test [67] a onetailed test was employed for significance of a predicted increase or decrease, while a two-tailed test was used for significance of a possible difference of either sign. the threshold for significance is defined as 97% (p � 0.03). to characterize the effects of drugs on the leukocyte leading edge chemosensory pathway, we utilized both cell population and single cell assays to monitor the leading edges of raw macrophages plated at controlled, low density on clean glass in standard media at 37˚c. plating on clean glass significantly reduces cell movement and facilitates long-term pseudopod imaging [26, 30] . no global attractant was added (excepting experiments with global pdgf addition), and no attractant gradient was imposed. in the absence of added attractant, the leading edge positive feedback loop maintains spontaneous polarization of a sizable subset (9 ± 2%) of the macrophage population. this protocol yields large numbers of relatively immobile, easily imaged polarized cells with well-developed leading edges displaying a ruffling sensory pseudopod (see figs 1a and 4c below) [26, 30] . activators and inhibitors of the positive feedback loop are known to expand and contract the leading edge pseudopod, respectively, via a signaling mechanism that involves increased or decreased levels of the signaling lipid pip 3 in the cytoplasmic leaflet of its plasma membrane, respectively [16, 26, 30, 68] . the leading edge pseudopod also possesses an intrinsic adaptation mechanism that damps out fluctuations in the chemical environment as required for movement in an attractant gradient [42, 69, 70] . a novel cell population analysis was developed to quantify both short-and long-term effects of drugs on the leading edge pseudopods of large numbers of polarized raw macrophages over a timescale of 2-3 hours. these studies employed dicm to record high resolution images of macrophage populations containing~1200 total cells, including~100 polarized cells with well-defined leading edge pseudopods, captured in subpopulations of~20-40 total cells per unit area in a grid containing 36-64 such areas per superimage (s1 fig). the number of cells with extended leading edge pseudopods per unit area was quantified before drug addition, and at selected timepoints after drug addition for up to 3 hours. finally, the mean timecourses for 3-6 superimages were averaged to generate an overall mean timecourse for effect of each drug on hundreds of polarized cells across all experiments. single cell imaging studies were also carried out to probe the short-term effects of drugs on both the size (area) and pip 3 signal of the leading edge pseudopod in individual raw macrophages. previous studies have shown that leading edge area and pip 3 levels are sensitive indicators of pseudopod perturbation by drugs [26, 30, 71, 72] . each single cell imaging experiment employed differential interference contrast microscopy (dicm) and z-stacked spinning disk confocal fluorescence microscopy to record high resolution images of the leading edge pseudopod of a polarized, actively ruffling macrophage before addition of each drug, and at selected timepoints for 5 min after drug addition. the resulting dicm images were used to quantify the timecourse of drug effects on pseudopod area, while the fluorescence images quantified levels of a standard fluorescent pip 3 sensor (akt1 ph domain fused to mrfp [26, 30] ) on the pseudopod membrane. for a given drug treatment, timecourses obtained from observations of 17 to 32 single cells were averaged to generate a mean timecourse for drug effects on the leading edge pseudopod area and pip 3 signal. the population and single cell assays are complementary. the population analysis monitors the effects of drugs on the leading edge pseudopods of at least~300 polarized cells over a period of hours, making it possible to quantify how drugs impact the frequency of cells with deployed, extended pseudopods, and to ascertain whether the perturbed pseudopods exhibit long term recovery following drug treatment. the single cell analysis provides a higher timeresolution view of the initial effects of drugs on the leading edge pseudopod and its pip 3 signal for at least 17 individual, polarized macrophages over a period of 5 minutes. in both the population and single cell assays, selective observation of polarized, ruffling cells ensures that all studied cells are healthy and possess an active leading edge pseudopod and signaling pathway. representative cell images and drug effects are shown in figs 1 and 4 and s2 and s3 for all drugs. fig 1 shows representative dicm and fluorescence images of the leading edge, both before and 5 min after addition of an inhibitory drug, illustrating drug-triggered collapse of both the leading edge area (fig 1a and 1b ) and its pip 3 signal (fig 1c and 1d) . the drugs employed in the present study, their total concentrations, structures, and other key parameters are summarized in fig 2. the four therapeutic drugs employed (acetylsalicylic acid, acetaminophen, diclofenac, ibuprofen) are each studied at the same total peak drug concentration reached in human plasma following maximum clinical oral dosage. the three control drugs employed (gö6976, wortmannin, pdgf ββ) are each studied at their standard total concentrations in cultured cell studies [26, 30] . to measure drug effects on populations of polarized raw macrophages, populations of~1200 well-resolved cells plated on glass in standard media were imaged at 37˚c in each experiment (see methods and approach). a target subpopulation of~100 cells displayed a readily observed extended, ruffling leading edge pseudopod [26, 30] and were counted before drug addition, and at multiple timepoints during a 2-3 hour observation period following drug addition at t = 0. altogether, 3 to 6 experiments were carried out for each treatment on at least 3 days, yielding average timecourses summarizing observations of~300 to~600 polarized cells, respectively. fig 3 first examines the effects of gradual addition of the four therapeutic drugs on leading edge morphology. in the clinic, these therapeutic drugs are typically administered orally, which yields gradually increasing drug levels in the plasma over a period of 30-90 min [79, 80] . here, gradual addition of therapeutic drugs was carried out by adding four equal increments over 90 min that sum to yield the maximum total drug concentration achieved at peak plasma levels in patients (fig 2) . fig 3a shows that such gradual, incremental addition of each of the four therapeutics or carrier has no significant effect on the number of polarized cells with extended leading edges per unit observation area (0.91 > p > 0.21 for the full range of treatments and timepoints, respectively). in contrast, however, rapid addition of two therapeutics at t = 0, yielding the same final, maximum drug concentration achieved by incremental addition above, triggers significant collapse of the leading edge subpopulation within 5 min. specifically, rapid addition of ibuprofen or acetaminophen decreased the number cells with extended leading edges 70 ± 5% (p < 0.002) and 26 ± 6%, (p < 0.03), respectively, followed by gradual recovery within 2 hrs. as illustrated in fig 3, gradual and rapid addition of ibuprofen or acetaminophen yields dramatically different effects on the leading edge pseudopod, thus the effects of rapid drug addition were examined more carefully. fig 4 compares timecourses observed following rapid addition of the four therapeutic drugs, as well as three control drugs for which the effects of rapid addition are well documented in the literature. fig 4a summarizes the effects of the therapeutic drugs, each added rapidly at t = 0 to their standard peak clinical concentration (fig 2) , on the relative number of cells possessing extended leading edge pseudopods per unit observation area. rapid acetylsalicylic acid or diclofenac addition had no significant effect on the number of cells with extended pseudopods over a 3 hour observation period, yielding timecourses indistinguishable from that of control vehicle addition (fig 4a and 4b ). in contrast, within 5 min of addition, ibuprofen and acetaminophen each triggered significant reduction of the number of cells with extended pseudopods (decreased 74 ± 14%, p < 0.001 and 27 ± 5%, p = 0.008, respectively; see also figs 3a and 4c-4l and s2. again, as observed in fig 3a, within 2 hrs of ibuprofen or acetaminophen addition the number of cells with extended pseudopods returned to the initial level, indicating that the macrophage population recovered from each drug perturbation by an unidentified mechanism (fig 4a; see also figs 4e-4l and s3). figs 4b and s2 show the effects of the three control drugs, rapidly added at t = 0 to their standard working concentration (fig 2) , on the normalized number of pseudopod-possessing cells in the raw macrophage population. global addition of the pathway activator (pdgf ββ), a known macrophage chemoattractant, triggered a significant increase (by 52 ± 9%, p = 0.002) in the number of pseudopod-possessing cells within 3 hours. at the other extreme, addition of a control pathway inhibitor (either wortmannin or gö6976 to inhibit pi3k or pkc, respectively) triggered a major decrease (by 90 ± 1%, p < 0.001 or 62 ± 3%, p < 0.001 respectively) in the number of observed pseudopods within 5 min, with little or no recovery during the 3 hour observation period as expected for standard inhibitors of essential components of the leading edge pathway. to further investigate the effects of rapid drug addition on leading edge pseudopod morphology and its signature pip 3 lipid signal, single cell imaging was carried out. such single cell measurements provide enhanced spatial and time resolution of within the 5 min observation period following drug addition, but observations could not be extended over a period of hours for technical reasons (on this timescale, even slowly moving cells plated on glass must be imaged periodically to maintain single cell identity, which bleaches the akt-ph-mrfp pip 3 sensor). for each drug or control treatment, an average timecourse was obtained based on observations of 17-32 individual, highly polarized raw macrophages with extended, ruffling leading edge pseudopods, as illustrated above in fig 1a and 1c . fig 5 displays average single cell timecourses following rapid addition at t = 0 of the four therapeutic drugs to their standard total concentrations (fig 2) . rapid addition at t = 0 of acetylsalicylic acid or diclofenac has little or no detectable effect on the pseudopod area ( fig 5a) and pip 3 levels (fig 5c) . by contrast, rapid addition of ibuprofen or acetaminophen triggers significant loss of pseudopod area (decreased 49 ± 10%, p < 0.001, or 53 ± 9%, p < 0.001, respectively; fig 5a) and significant loss of pseudopod pip 3 (decreased 27 ± 7%, p < 0.001, or 32 ± 9%, p < 0.001, respectively; fig 5c) within 5 min. the observation that rapid ibuprofen or acetaminophen addition triggers both collapse of pseudopod area and decreased pip 3 levels is consistent with pseudopod destabilization via inhibition of the leading edge signaling pathway. fig 5 also summarizes the effects of the three control drugs on the leading edge pseudopod. addition of the known pathway activator (pdgf ββ) led to a significant pseudopod area expansion (increased 43 ± 12%, p < 0.001) and increased pip 3 levels (by 17 ± 5%, p < 0.001) during the 5 min observation period (fig 5b and 5d ). by contrast, the two control pathway inhibitors wortmannin and gö6976 each triggered significant losses of both the pseudopod area (decreased 38 ± 6%, p < 0.001 and 39 ± 7%, p < 0.001, respectively) and the pip 3 signal (decreased 52 ± 16%, p < 0.001 and 55 ± 15%, p < 0.001, respectively) (fig 5b and 5d ). the figure also shows that carriers alone had no detectable effect on the pseudopod. the observation that a subset of the tested compounds significantly perturb pseudopod area and pip 3 levels in single polarized cells raises the possibility these drugs may directly and specifically modulate the enzyme activity of pi3k lipid kinase. control studies summarized in supplemental s4 fig confirm that wortmannin, a known irreversible pi3k inhibitor, directly blocks the pip 3 production of purified pi3k in vitro as previously observed [26, 81] . in contrast, the other drugs have little or no direct effect on the pip 3 production of purified pi3k, indicating that only wortmannin directly inhibits pi3k activity in vitro. the finding that two therapeutic drugs had no detectable effect on the leading edge pseudopod, while two other therapeutic drugs triggered an initial pseudopod collapse followed by recovery, raised the possibility that drugs might be unstable in the media. quantitative mass spectrometry analysis was carried out to ascertain whether each of the four target drugs was present at the same total concentration during three hours in culture. fig 6 shows that each of the four therapeutic drugs was present in the cell culture media at the intended clinical concentration at the first timepoint after addition, and exhibited relatively little change during the three hour incubation. thus, each of the four drugs remained at an approximately fixed, standard clinical concentration during the timecourses described in this study. the present findings reveal that gradual exposure of cultured raw macrophages to four representative therapeutic drugs (acetylsalicylic acid, diclofenac, ibuprofen, acetaminophen) has little or no effect on the native morphology of the leading edge sensory pseudopod. thus, when the concentration of each drug is increased in four equal, stepwise increments up to its standard clinical total concentration (fig 2) over a period of 90 min, no significant loss of polarized cells with extended pseudopods is detected. these findings are consistent with the well-documented safety of these therapeutics in the clinic, where standard oral dosage also yields a gradually increasing drug level in the plasma up to the total concentration employed herein (fig 2) within~90 min [79, 80, [82] [83] [84] . strikingly different results are observed, however, when the same four therapeutic drugs (acetylsalicylic acid, diclofenac, ibuprofen, acetaminophen) and three control drugs (pdgf, gö6976, wortmannin) are added rapidly to cultured raw macrophages. such rapid drug addition, in one step at t = 0 that raises the drug up to its standard total concentration (fig 2) within 10 sec, yields at least four operational classes of drug effects on the leading edge pseudopod and its signature pip 3 lipid signal. (i) non-perturbing rapid exposures yield little or no effect on pseudopod area, pip 3 levels and stability (as observed for acetylsalicylic acid, diclofenac). (ii) adaptive rapid exposures trigger temporary collapse of the pseudopod followed by recovery (observed for ibuprofen, acetaminophen). (iii) disruptive rapid drug exposures yield long-term pseudopod collapse with little or no adaptation over the 3 hour observation period (gö6976, wortmannin). (iv) activating exposures yield pseudopod expansion and increased cell polarization (pdgf). to analyze the molecular mechanisms of these four different classes, it is useful to begin by noting the significant drug buffering capacities of human plasma and cell culture media. this buffering capacity is dominated by drug binding to the highly homologous proteins human serum albumin (hsa) and bovine serum albumin (bsa), respectively. both hsa and bsa possess two high affinity drug binding sites and multiple low affinity sites [49, [85] [86] [87] . as a result, in human plasma the four well-studied therapeutic drugs employed herein each show significant binding to hsa at their standard total concentrations, ranging from 24 to 99% bound (fig 2) . drug binding to serum albumins is rapid, often on the msec timescale depending on the drug and albumin concentrations. while the drug binding properties of hsa and bsa are extremely similar [87, 88] , their concentrations are quite different in human plasma (hsa, 500-800 μm) [89, 90] vs cell culture medium (bsa, 30-50 μm; provided by 10% fbs containing 300-500 μm bsa (methods)). as a representative example, we can estimate the rate of ibuprofen binding to albumin binding sites under these conditions since its hsa equilibrium association constant (k a = 2.3 x 10 6 m -1 ) and off-rate constant (k off = 0.055 sec -1 ) are known [87, 91] . assuming the standard total clinical concentration of ibuprofen (240 μm) and the relevant hsa (~500 μm) or bsa (~40 μm) concentration, the initial on-rate for drug binding to protein is~10 mm per sec or~1 mm per sec in human plasma or cell culture media, respectively. it follows that the binding of 240 μm ibuprofen to bsa in cultured cell experiments will occur in less than a second, or during the 10 sec mixing time of our drug addition protocol. two mechanisms are possible for the lack of pseudopod perturbation observed upon rapid addition of the non-perturbing drugs acetylsalicylic acid and diclofenac: either the free drug could be non-perturbing, or serum albumin buffering in the media could ensure that little free drug is available for cell binding. for acetylsalicylic acid, the high total drug concentration (1.1 mm, fig 2) relative to the bsa concentration in cell culture media (30-50 μm) ensures that high levels of free drug exceeding 1 mm will be present immediately upon addition to cells. moreover, mass spec analysis shows the total drug concentration does not change significantly over the 3 hour observation period (fig 6) . it follows that high levels of free acetylsalicylic acid in the millimolar range do not perturb the leading edge pseudopod morphology. for diclofenac, however, the total drug concentration (17 μm) is significantly lower than the bsa concentration in the cell media, and the affinity of this drug for serum albumin is very high (99% bound in human plasma). thus the free diclofenac concentration in the cell media is likely sub-micromolar, and the lack of pseudopod perturbation could arise from strong drug buffering by bsa that ensures the free drug concentration remains below its unknown pseudopod perturbation threshold. two mechanisms are also possible for the temporary collapse of the macrophage pseudopod and its signature pip 3 lipid signal upon rapid addition of ibuprofen or acetaminophen. mechanism (i), which is favored by the available evidence, proposes that the pseudopod is strongly perturbed by appearance of a relatively constant level of free drug triggered by rapid drug addition at t = 0, collapses within 5 min, and then recovers due to cell adaptation to the constant free drug concentration. in contrast, mechanism (ii) proposes that the pseudopod is strongly perturbed by a transient, high local concentration of free drug during addition and mixing that exceeds a drug threshold for pseudopod perturbation. in this model, the transient, high local concentration of drug rapidly decreases as mixing is completed and drug binds to bsa; subsequently, the pseudopod gradually recovers from the initial drug shock simply because the free drug has fallen below its pseudopod perturbation threshold with no contribution from cell adaptation. multiple lines of evidence support mechanism (i) for both drugs as follows. the total concentrations of ibuprofen (240 μm) and acetaminophen (170 μm) added to cells are significantly higher than the total bsa concentration in the cell media (30-50 μm) , ensuring that substantial free concentrations of drug are present throughout the observation period. moreover, the affinity of acetaminophen for bsa is low, such that only 24% of the drug is bound even in human plasma where the serum albumin concentration is~10-fold higher. we cannot formally rule out a version of mechanism (ii) in which rapid addition of the drug stock solution yields a transient spike of high drug concentration; however both mixing and bsa buffering should be complete within~10 sec and 1 sec, respectively (see methods and binding rate calculation above). evidence disfavoring this transient high local concentration model is provided by the observed strong reproducibility with which each drug triggers pseudopod collapse. a mechanism requiring high local drug concentrations during mixing would likely yield variable results between replicates, and between cells in different regions of the same coverslip, with cells close to the addition point more strongly perturbed than distal cells. spatial heterogeneity is not observed in the large fields of~1200 cells scanned for population studies (figs 3 and 4 and s1), and reproducibility is high in studies monitoring 17-32 single cells (fig 5) . to fully exclude the high local concentration model, future studies will compare the addition of drugs as a 100x stock solution and as a 2x stock solution in cell media. (such a comparison was planned but prevented by the covid 19 pandemic and lab shut-down). the cellular mechanism(s) by which rapid addition of ibuprofen or acetaminophen triggers the initial collapse of leading edge pseudopod and pip 3 levels remain unknown, but the findings rule out the possibility that the drugs act as intrinsic pi3k inhibitors since neither blocks pip 3 production by purified pi3k in a lipid kinase activity assay (s4 fig). thus, the perturbing drugs must directly or indirectly modulate as yet unidentified component(s) of the leading edge signaling pathway, or trigger a nonspecific intracellular perturbation (likely not a ph change, since the drug perturbations are not correlated with drug pkas (fig 2) ). similarly, assuming that mechanism (i) accurately describes ibuprofen and acetaminophen, multiple possibilities exist for the cellular adaptation mechanism(s) that drive pseudopod recovery following the initial drug-triggered collapse. the simplest hypothesis is that the same unknown adaptation mechanism(s) are acting both to drive pseudopod recovery after rapid drug addition, and to prevent pseudopod collapse during slow drug addition. this hypothesis proposes that rapid drug addition overwhelms the adaptation process which is unable to counteract the perturbation in real time, but gradually eliminates the perturbation within 3 hours. in contrast, gradual drug addition allows concurrent gradual adaptation that is able to counteract the drug perturbation in real time. adaptation does not involve extracellular chemical or enzymatic degradation of drugs in the media surrounding the cultured cells, since mass spectrometry analysis indicates that each drug is stable in the media during the three hour cell incubation (fig 6) . instead, the adaptation mechanism is hypothesized to be intracellular. such intracellular adaptation could be provided by the intrinsic adaptation mechanisms of the leading edge chemosensory pathway which, like other sensory pathways, must adapt to changing background stimuli and attractant concentrations as the cell moves through different environments [41, 42, 92, 93] . other possible adaptive mechanisms could include activation of an intracellular sink, pump, degradation or modification reaction that eliminates free drug in the cytoplasm, but has a negligible effect on the bulk extracellular drug concentration. such mechanisms have been reported for other drugs (reviewed in [47, 48] ). the mechanisms of the three control drugs are well defined by previous studies. exposures to the non-clinical control inhibitors wortmannin and gö6976 are disruptive, yielding long term collapse of the leading edge pseudopod and its pip 3 lipid signal. these drugs are known to directly inhibit essential components of the leading edge positive feedback loop (pi3k and pkc, respectively), and their disruption of the leading edge pseudopod upon rapid addition has been previously described [26, 30, [50] [51] [52] [53] [54] . rapid exposure to pdgf yields pseudopod expansion and increased cell polarization, as previously observed and commensurate with its known importance as a strong macrophage attractant in vitro and in vivo [26, 55] . in closing, the present findings suggest a number of directions for future research. further studies are needed to determine the molecular mechanisms by which rapid ibuprofen or acetaminophen addition trigger macrophage leading edge pseudopod collapse, and by which the pseudopod adapts (or recovers) from these drug exposures. given the~10-fold higher concentrations of albumins in plasma compared to cell culture media, the greater drug buffering capacity of plasma will help protect macrophages and other cell types from drug perturbations, (fig 4) to ascertain the stability of the total drug concentration in the media surrounding the cells. aliquots of media were removed from cell imaging wells immediately after rapid drug addition at t = 0 to the total concentration indicated in fig 2, and again at t = 3 hr after completion of imaging, then snap frozen in liquid n 2 . later, the samples were prepared for quantitation with addition of internal standards (methods), then quantitative mass spectrometry was carried out to compare the initial and final total drug concentrations. error bars are sd for n = 3 (acetylsalicylic acid, diclofenac) or 6 (ibuprofen, acetaminophen). https://doi.org/10.1371/journal.pone.0233012.g006 but the sensitivity of the cultured macrophage leading edge pseudopod to drugs makes it an excellent model system for detecting and classifying drugs capable of inhibiting macrophages, either transiently or permanently. transient macrophage inhibition could be useful in controlling extreme inflammation events, while long-term inhibition that blocks macrophage participation in the innate immune response would be highly detrimental. it will be important to carry out future studies that directly test whether drug-induced pseudopod collapse causes inhibition of macrophage motility and chemotaxis in vitro and in vivo [94] . more broadly, we hypothesize that the same four classes of drug effects observed herein for cultured macrophages will also be observed for primary macrophages and other leukocytes such as neutrophils which possess similar chemosensory pathways [15, 20, 32, 46] , but this prediction also remains to be tested. finally, the effects of other therapeutic and experimental drugs on the leading edge pseudopod remain to be determined. in short, the present study opens several new research paths with major implications for a basic understanding of drug-induced perturbation and adaptation in leukocyte signaling pathways. a single molecule tirfm assay was employed to measure the specific kinase activity of purified pi3k molecules on a supported lipid bilayer by counting the number of pi3k molecules, as well as the number of product pip 3 molecules they produce as previously described in detail [20] [21] [22] [23] . this assay utilizes physiological concentrations of class i pi3ka and saturating concentrations of fluorescently-labeled grp-ph domain, a high-affinity pip 3 product lipid binder, in order to count each product lipid produced. drug is added to therapeutic dose identified in fig 2 prior to the start of the assay. enzyme rates are calculated as the slope of product lipid created vs. time. bars indicate the lipid kinase activity of pi3k in the presence of the indicated drug. error bars are sd where n = 3 for acetylsalicylic acid and diclofenac, or n = 6 for ibuprofen and acetaminophen. (tiff) temporal and spatial regulation of chemotaxis signaling mechanisms for regulation of chemotaxis feedback signaling controls leading-edge formation during chemotaxis moving towards a better understanding of chemotaxis new paradigms in the establishment and maintenance of gradients during directed cell migration self-organization of protrusions and polarity during eukaryotic chemotaxis rac2 functions in both neutrophils and macrophages to mediate motility and host defense in larval zebrafish neutrophil plasticity in the tumor microenvironment signaling to migration in neutrophils: importance of localized pathways. the international journal of biochemistry & cell biology live imaging of wound inflammation in drosophila embryos reveals key roles for small gtpases during in vivo cell migration calcium flickers steer cell migration role of platelets in leukocyte recruitment and resolution of inflammation triggering the resolution of immune mediated inflammatory diseases: can targeting leukocyte migration be the answer? front pharmacol lipid droplets in leukocytes: organelles linked to inflammatory responses moving in the right direction: how eukaryotic cells migrate along chemical gradients interplay between phosphoinositide lipids and calcium signals at the leading edge of chemotaxing ameboid cells signaling networks that regulate cell migration signaling mechanisms for chemotaxis calcium gradients underlying cell migration molecular players in neutrophil chemotaxis-focus on pi3k and small gtpases single-molecule studies reveal a hidden key step in the activation mechanism of membrane-bound protein kinase c-alpha regulation of pi3k by pkc and marcks: single-molecule analysis of a reconstituted signaling pathway regulation of a coupled marcks-pi3k lipid kinase circuit by calmodulin: single-molecule analysis of a membrane-bound signaling module single-molecule study reveals how receptor and ras synergistically activate pi3kalpha and pip3 signaling neutrophil chemotaxis a pkc-marcks-pi3k regulatory module links ca2+ and pip3 signals at the leading edge of polarized macrophages activation of phosphatidylinositol 3-kinase is sufficient for cell cycle entry and promotes cellular changes characteristic of oncogenic transformation specific translocation of protein kinase calpha to the plasma membrane requires both ca2+ and pip2 recognition by its c2 domain unusual interplay of two types of ras activators, rasgrp and sos, establishes sensitive and robust ras activation in lymphocytes ca2+ influx is an essential component of the positive-feedback loop that maintains leading-edge structure and activity in macrophages mechanism of specific membrane targeting by c2 domains: localized pools of target lipids enhance ca2+ affinity eukaryotic chemotaxis: a network of signaling pathways controls motility, directional sensing, and polarity phosphoinositides: tiny lipids with giant impact on cell regulation pi3k signalling in inflammation exploring cells with targeted biosensors chemoattractant receptors activate, recruit and capture g proteins for wide range chemotaxis mutually inhibitory ras-pi(3,4) p2 feedback loops mediate cell migration understanding phosphoinositides: rare, dynamic, and essential membrane phospholipids signal transduction: principles, pathways and processes the microtubule-associated protein eb1 maintains cell polarity through activation of protein kinase c the two-component signaling pathway of bacterial chemotaxis: a molecular view of signal transduction by receptors, kinases, and adaptation enzymes integrating conflicting chemotactic signals. the role of memory in leukocyte navigation a model for direction sensing in dictyostelium discoideum: ras activity and symmetry breaking driven by a gbetagamma-mediated, galpha2-ric8-dependent signal transduction network bacterial pep-dependent carbohydrate: phosphotransferase systems couple sensing and global control mechanisms cellular memory in eukaryotic chemotaxis moving towards a paradigm: common mechanisms of chemotactic signaling in dictyostelium and mammalian leukocytes the different mechanisms of cancer drug resistance: a brief review mechanisms of antibiotic resistance determination of rate constants and equilibrium constants for solution-phase drug-protein interactions by ultrafast affinity extraction regulation of chemotaxis by the platelet-derived growth factor receptor-beta the chemotactic response to pdgf-bb: evidence of a role for ras pi-3-kinase and mapk regulate mesangial cell proliferation and migration in response to pdgf current clinical recommendations for use of platelet-rich plasma platelet-rich plasma: review of current literature on its use for tendon and ligament pathology platelet-derived growth factor-bb and transforming growth factor beta 1 selectively modulate glycosaminoglycans, collagen, and myofibroblasts in excisional wounds non-steroidal anti-inflammatory drugs. current status and rational therapeutic use ibuprofen: plasma concentrations in man clinical pharmacokinetics of diclofenac. therapeutic insights and pitfalls drug levels: therapeutic and toxic serum/plasma concentrations of common drugs evolution of nonsteroidal anti-inflammatory drugs (nsaids): cyclooxygenase (cox) inhibition and beyond trends in the use of aspirin and nonsteroidal anti-inflammatory drugs in the general u.s. population use of non-steroidal anti-inflammatory drugs in us adults: changes over time and by demographic mechanism of action of nonsteroidal anti-inflammatory agents mechanism of action of nonsteroidal anti-inflammatory drugs effects of nonsteroidal anti-inflammatory drugs at the molecular level the effect of nonsteroidal anti-inflammatory drugs on tissue healing mutual inhibition between pten and pip3 generates bistability for polarity in motile cells sensory adaptation of leukocytes to chemotactic peptides adaptation of human neutrophil responsiveness to the chemoattractant n-formylmethionylleucylphenylalanine. heterogeneity and/or negative cooperative interaction of receptors essential role of phosphoinositide 3-kinase delta in neutrophil directional movement live imaging reveals distinct modes of neutrophil and macrophage migration within interstitial tissues kinetics and metabolism of paracetamol and phenacetin nonsteroidal antiinflammatory drugs and uncoupling of mitochondrial oxidative phosphorylation the binding of ibuprofen to plasma proteins in vitro protein binding of diclofenac sodium in plasma and synovial fluid studies on paracetamol binding to serum proteins aspirin binding and the effect of albumin on spontaneous and enzyme-catalysed hydrolysis effects of food on pharmacokinetics of immediate release oral formulations of aspirin, dipyrone, paracetamol and nsaids-a systematic review an overview of clinical pharmacology of ibuprofen structural determinants of phosphoinositide 3-kinase inhibition by wortmannin, ly294002, quercetin, myricetin, and staurosporine pharmgkb summary: pathways of acetaminophen metabolism at the therapeutic versus toxic doses emerging evidence in nsaid pharmacology: important considerations for product selection nsaids and aspirin: recent advances and implications for clinical management kinetics and mechanism of bilirubin binding to human serum albumin interactive association of drugs binding to human serum albumin species differences of serum albumins: i. drug binding sites a comprehensive spectroscopic and computational investigation to probe the interaction of antineoplastic drug nordihydroguaiaretic acid with serum albumins the correlation of plasma proteins binding capacity and flavopiridol cellular and clinical trial studies on the molecular interaction between albumin and ibuprofen: an afm and qcm-d study mechanisms of gradient detection: a comparison of axon pathfinding with eukaryotic cell migration incoherent feedforward control governs adaptation of activated ras in a eukaryotic chemotaxis pathway a high-throughput, multi-cell phenotype assay for the identification of novel inhibitors of the authors gratefully acknowledge helpful conversations with expert colleagues, including: prof. anna huttenlocher from university of wisconsin, madison; and drs. ken hunt, rachel frank, and sourav poddar from the university of colorado sports medicine clinic. furthermore, we gratefully acknowledge resources and experts at university of colorado, boulder integral to the completion of this work: the biofrontiers advanced light microscopy core (dr. joseph dragavon), the biochemistry cell culture facility (dr. theresa stines nahreini, nicole kethley), and the biochemistry mass spectrometry laboratory at the central analytical laboratory (thomas lee). we thank the national institutes of health and the nih/cu molecular biophysics training program for support. finally, we thank the editor and reviewers for helpful comments that enabled us to significantly improve this contribution. conceptualization: thomas c. buckles, brian p. ziemba, joseph j. falke. key: cord-350398-w75flrwv authors: sampath, rangarajan; mulholland, niveen; blyn, lawrence b.; massire, christian; whitehouse, chris a.; waybright, nicole; harter, courtney; bogan, joseph; miranda, mary sue; smith, david; baldwin, carson; wolcott, mark; norwood, david; kreft, rachael; frinder, mark; lovari, robert; yasuda, irene; matthews, heather; toleno, donna; housley, roberta; duncan, david; li, feng; warren, robin; eshoo, mark w.; hall, thomas a.; hofstadler, steven a.; ecker, david j. title: comprehensive biothreat cluster identification by pcr/electrospray-ionization mass spectrometry date: 2012-06-29 journal: plos one doi: 10.1371/journal.pone.0036528 sha: doc_id: 350398 cord_uid: w75flrwv technology for comprehensive identification of biothreats in environmental and clinical specimens is needed to protect citizens in the case of a biological attack. this is a challenge because there are dozens of bacterial and viral species that might be used in a biological attack and many have closely related near-neighbor organisms that are harmless. the biothreat agent, along with its near neighbors, can be thought of as a biothreat cluster or a biocluster for short. the ability to comprehensively detect the important biothreat clusters with resolution sufficient to distinguish the near neighbors with an extremely low false positive rate is required. a technological solution to this problem can be achieved by coupling biothreat group-specific pcr with electrospray ionization mass spectrometry (pcr/esi-ms). the biothreat assay described here detects ten bacterial and four viral biothreat clusters on the niaid priority pathogen and hhs/usda select agent lists. detection of each of the biothreat clusters was validated by analysis of a broad collection of biothreat organisms and near neighbors prepared by spiking biothreat nucleic acids into nucleic acids extracted from filtered environmental air. analytical experiments were carried out to determine breadth of coverage, limits of detection, linearity, sensitivity, and specificity. further, the assay breadth was demonstrated by testing a diverse collection of organisms from each biothreat cluster. the biothreat assay as configured was able to detect all the target organism clusters and did not misidentify any of the near-neighbor organisms as threats. coupling biothreat cluster-specific pcr to electrospray ionization mass spectrometry simultaneously provides the breadth of coverage, discrimination of near neighbors, and an extremely low false positive rate due to the requirement that an amplicon with a precise base composition of a biothreat agent be detected by mass spectrometry. technology for detecting biothreat agents requires accurate identification of a broad array of bacterial and viral organisms that can cause severe disease and/or death, whether they occur as a result of a biological attack or from a natural source in the environment. the national institute of allergy and infectious diseases (niaid) has compiled a list of priority pathogens for biodefense (http://www.niaid.nih.gov) and several of these are also defined as select agents (http://www.selectagents.gov/) by various agencies such as health and human services (hhs) and the united states department of agriculture (usda) (some of the vaccine and live attenuated strains are, however, excluded from the select agents list: http://www.selectagents.gov/select%20 agents%20and%20toxins%20exclusions.html). these bioagents are often virtually indistinguishable from a group of phylogenetically related species or subspecies often referred to as ''near neighbors'' [1] . near neighbors to biothreat agents may be human pathogens or harmless environmental organisms. the biothreat agent along with its near neighbors can be thought of as a biothreat cluster or biocluster for short. when monitoring for biothreat agents, it is important to determine whether any organisms from the biothreat clusters are present and to precisely identify the organism as a biothreat agent or a near neighbor. in some cases, the near neighbors are commonly found in the environment, and it is possible that a pathogenic near neighbor of a biothreat agent might deliberately be chosen for use in a biological attack. thus, effective biosensor technology must be capable of identifying a broad array of biothreat agents and distinguishing these threats from their near neighbors unambiguously. this requirement presents a problem for conventional molecular methods where specific pcr is used in conjunction with probes to detect specific bioagents. not only are potentially pathogenic near neighbors present in a specimen often not distinguished, but the near neighbors sometimes react to produce false positives for the biothreat agent. to overcome these limitations, we have developed a new strategy for biothreat identification that couples biothreat cluster-specific pcr amplification to electrospray ionization/mass spectrometry (pcr/esi-ms) [2] [3] [4] . the biothreat assay is performed on a hardware platform with prototypes known as tiger [4] and as the ibis t5000 [2, 5] that is now marketed commercially as the abbott plex-id [6] . in the pcr/esi-ms approach, pcr primers are designed to amplify regions of the genomes of all species from the entire biothreat cluster, encompassing groups of organisms that include the biothreat and the associated near-neighbor organisms. the primers are designed to target genomic regions sufficiently conserved such that amplification occurs comprehensively within a biothreat cluster, but not outside of the cluster. the amplification products are then analyzed by mass spectrometry, which weighs the amplicons with sufficient mass accuracy that the base composition of a, g, c, and t nucleotides that make up the amplicon can be accurately counted. the base composition serves as a signature of each organism and enables identification and discrimination of the biothreat agents and their near neighbors with equal facility. in addition, previously undiscovered or newly emerging organisms from within these biothreat clusters are also detected. the database of signatures against which multiple additional pathogens could be identified increases over time as newer strain variants are archived and tested. an example of this was the discovery of the 2009 h1n1 virus by the naval health research center [7, 8] ; this offered the first characterization of a previously unrecognized influenza strain, demonstrating the capability of the plex-id in identification of a real-world case of novel pathogen emergence. because the mass spectrometer weighs all amplicons presented to it, the amplicons from unexpected or new organisms are detected and identified [2, 5, 9, 10] . using this strategy, we designed a comprehensive assay to detect ten bacterial and four viral biothreat clusters. the assay identified the major biothreat organisms and differentiated these from their near neighbors and from thousands of other bacteria and viruses, providing a seamless net of biosurveillance for these clusters in a comprehensive biothreat assay ( figure 1 ). in this manuscript, we provide a detailed description of the methodology and the results of formal validation experiments with a variety of biothreats, near neighbors, and specimen types. we also describe several examples of how the assay has been used in real-world biothreat scenarios. biothreat clusters targeted by this assay are shown in figure 1 . these organisms make up the majority of the niaid category a, b, and c priority pathogens and hhs/usda select agents (http://www.niaid.nih.gov/topics/biodefenserelated/biodefense/ research/pages/cata.aspx). as shown in figure 1 , these biothreat organisms are phylogenetically related to a number of other ubiquitous organisms, making correct identification of these organisms a challenge. in addition to detecting the threat organisms, the biothreat assay described here also detects virulence factors associated with three of the agents: bacillus anthracis (pxo1 and pxo2), yersinia pestis (pla and caf), and vibrio cholera (ctx1). pcr primers were designed to conserved regions within the selected target genes such that the targeted threat agent was clearly identified and differentiated from its near-neighbor species ( table 1) . many of the primer pairs used in this assay have previously been used in other assays on the biosensor system described here [2, 3, [11] [12] [13] [14] [15] [16] . a complete analysis of each biocluster and the resolution provided by the assay is described below. bacillus anthracis. bacillus anthracis, which causes anthrax in animals and humans, is closely related to b. cereus (which causes human food poisoning), b. thuringiensis (an insect pathogen and a biological insecticide), and b. mycoides (considered a harmless saprophyte). members of the this group, known as the bacillus cereus clade, are environmentally ubiquitous. classical microbiological methods can only differentiate b. anthracis from other nearneighbor species when the unknown isolate is shown to cause anthrax in laboratory animal models. classical molecular phylogenetic tools, such as the analysis of rrna gene sequences, cannot distinguish among members of the b. cereus clade. we demonstrate here that two primer pairs, bct352 targeting the translation initiation factor if-4 (infb) gene and bct355 targeting the small acid-soluble spore protein (sspe) gene, provide signatures that can distinguish b. anthracis from all of the nearneighbor species within the b. cereus clade. full virulence of b. anthracis requires the presence of two plasmids, pxo1 and pxo2, containing three toxin components (protective antigen, lethal factor, and edema factor) and an anti-phagocytic capsule. the primers used for the detection of these two plasmids (bct2379 and bct2381) were chosen to capture a previously described single-nucleotide polymorphism (snp) [17] . this region in each of the plasmids provides two different alleles that can be used to distinguish b. anthracis ames and other ames-like strains from non-ames strains. thus, the positive identification of the b. anthracis chromosome using the infb and sspe targets, combined with detection of the virulence plasmid signatures, can be used to differentiate non-pathogenic, vaccine, fully virulent, and genetically modified strains of b. anthracis. to demonstrate the resolving capabilities of these genomic signatures, we obtained a collection of 34 bacilli from the united states army medical research institute for infectious diseases (usamriid). these include fully virulent, partially virulent, and avirulent b. anthracis strains and an assortment of near-neighbor bacilli (table s1 ). each isolate was correctly identified by comparison of base composition signatures obtained in the biothreat assay with genomic sequence data obtained from genbank ( table 2 ). the infb locus provided identical signatures across all b. anthracis strains tested, whereas the sspe locus provided two different allelic signatures. the majority of the b. anthracis strains tested had a base composition signature of ''a42g23c23t21,'' whereas the b. anthracis strains from the western north american region showed a snp at this locus and had the signature of ''a41g24c23t21.'' as predicted, the ameslike strains were different from the rest of the b. anthracis strains in the pxo1 and pxo2 loci. an additional 89 strains of b. anthracis were obtained from the keim genetics lab; these strains have various phylogenetic variations (table s2 ). the diverse strains in this collection were correctly identified in our assay. the a1.a clade showed the snp pattern at the sspe locus previously associated with the western north american lineage (table s3) . these strains were previously reported to be distinct from other clade a strains as they contain a 153-bp allele in the cg3 locus [17] . representative strains of the b. cereus clade, including 24 b. cereus strains and 12 b. thuringiensis strains were also tested ( table s2) . none of these carried the virulence plasmids pxo1 or pxo2 (data not shown). futher, the base compositions observed for the two genomic markers showed distinct signatures compared to b. anthracis. the amplicon for sspe showed a 6-bp deletion compared to the b. anthracis signatures, whereas the infb signature was the same length but had a different base composition. based on the combined compositions of these two primer regions, the b. cereus/ b. thuringiensis biocluster could be divided into 29 distinct genotypes. some of the b. thuringiensis species have individual clusters but most are related to b. cereus species. several studies over the past two decades have looked at the fine structure of the b. cereus clade and have reported similar findings [18] . nucleic acids from species outside the b. cereus clade, such as b. subtilis and b. megaterium, were not amplified with the sspe primer pair. the advantage of measuring multiple signatures across the genome and in associated plasmids is that this provides sufficient information to characterize the biothreat agent (i.e., vaccine vs. virulent strains of b. anthracis) as well as distinguish it from its near-neighbors. yersinia pestis. the bubonic plague caused by yersinia pestis is a highly contagious disease that persists endemically in many countries in the world with unpredictable resurgences [19] . y. pestis is classified as a biothreat agent. it is a nonmotile, capsulated, gram-negative bacterium transmitted to humans and susceptible animals through flea bites or aerosols. other species in this biocluster include y. enterocolitica (a diarrheagenic pathogen), y. frederiksenii, y. ruckeri, and y. pseudotuberculosis (an enteric pathogen). y. pseudotuberculosis exhibits more than 90% genomic homology with y. pestis, making much of the y. pestis genome unsuitable for amplification as base composition signatures do not resolve y. pestis and y. pseudotuberculosis [20, 21] . two specific markers within the y. pestis genome were identified that provide differentiation from y. pseudotuberculosis. one of these is located in the valyl-trna synthetase gene (vals). this region (table 1 ) has a snp that is retained in all the y. pestis genomes studied to date and that is distinct from the y. pseudotuberculosis signature for this region ( table figure 1 . biothreat clusters of interest. ten bacterial and four viral clusters identified in the biothreat assay are shown. in each cluster the key biothreat agent and its near neighbors are indicated. the hhs/usda select agent and niaid a, b, c pathogen lists are reflected by symbols shown in the legend. attenuated or live vaccine strains of some of these organisms are, however, excluded from the select agent list (http://www.selectagents. gov/select agents and toxins exclusions.html). b. anthracis and y. pestis plasmid markers are explicitly annotated. organisms with names given within brackets cannot be distinguished from each other within this assay. primer pairs used for the detection of each biocluster are indicated. doi:10.1371/journal.pone.0036528.g001 s4). a second primer pair was designed to target the invasin gene inva, a surface-expressed protein that is reponsible for cellular penetration and invasion. although both y. pseudotuberculosis and y. enterocolitica contain intact invasin genes, the central region of the y. pestis inv gene is disrupted by a 708-bp is200-like element [22] . primers targeting this region allow for the unambiguous detection of y. pestis (table s4 ). in addition, y. pestis harbors plasmids that are required for the expression of virulence [23] [24] [25] [26] . primer pairs targeted against the pla gene from the ppcp1 plasmid of y. pestis and the caf1 gene from the pmt1 plasmid of y. pestis were included in the assay to provide specific detection of virulence plasmidcarrying y. pestis. genomic data from the completely sequenced y. pestis genomes were used to verify the expected signatures for these primer pairs (table s4 ). all y. pestis strains for which relevant sequence was available in genbank showed identical base composition signatures. partially virulent y. pestis strains such as y. pestis angola (pla+/caf2) and pestoides f (pla2/caf+) were correctly identified. these results were further confirmed by experimental testing a collection of seven y. pestis strains with known phenotypes obtained from the usamriid culture collection (table s5 ). in addition to the four virulent phenotypes containing pla+/caf+, two strains (nairobi and java 9) lacking the caf gene and pestoides f lacking the pla gene were analyzed. all of the results showed data consistent with the expected molecular signatures. finally, a set of 15 near neighbors of y. pestis were tested using the assay; each was correctly identified and differentiated from the y. pestis signatures (table s6 ). in particular, this assay clearly distinguished the y. pestis signatures from those of y. pseudotuberculosis, which is often the confounder in molecular assays. francisella tularensis. the francisella tularensis biothreat cluster is comprised of the select agent f. tularensis and the nearneighbor species f. philomiragia and f. novicida. f. tularensis is the causative agent of tularemia, a disease that affects humans and other mammals; the natural reservoir is thought to be lagamorphs and rodents with ticks as the primary vector [27, 28] . f. tularensis has been divided into three subspecies: f. tularensis subsp. tularensis (type a), which is divided into subtypes a.i and a.ii., is the most virulent and is found primarily in north america and europe [29] . f. tularensis subsp. holarctica (type b) is less virulent and found in europe and in asia [28] , f. tularensis subsp. mediaasiatica has been isolated only in central asia and is considered to be of lower virulence [30] . the latter two subspecies can cause an incapacitating infection, however [30] . the near-neighbor species f. novicida (considered by some investigators to be another subspecies of f. tularensis) and f. philomiragia are reportedly of of low pathogenicity and cause disease only in immunocompromised humans [30] . because of the potential of each of these species and subspecies to cause disease of varying severity, it is important to both detect and distinguish these species and subspecies. in the biothreat assay, the francisella biocluster is identified by two genus-specific primer pairs targeting the asd (bct2328) and gale (bct2332) genes ( table 1) . use of gale for identification of francisella genus has been previously described [31] . based on inspection of the sequence alignments for these genes of all available francisella sequences, both of these primer pairs are expected to generate amplicons from all known members of the francisella genus. base composition analysis of the expected amplicons shows that francisella species and subspecies are distinguishable from each other (table s7 ). f. tularensis base counts are characterized by a homogenous signature for all type a.i strains. schu s4 is the type strain, and the rest of the francisella signatures are defined here as variations compared to this reference strain. f. tularensis subsp. tularensis str. wy96-3128 (type a.ii) had a t to c snp in the gale primer pair region. the same signature was observed for f. tularensis subsp. mediaasiatica fsc217 strain. this is in agreement with genome-wide snp analysis, which indicates that differentiation of these particular strains likely predated the acquisition of the asd or gale mutations that characterize the subspecies. further, the subspecies mediaasiatica is reported to be closer to the type a.ii lineage, even though its pathogenicity is characteristic of the type b strains [29] . f. tularensis subspecies novicida was distinguished by a g to a mutation from the consensus seen in f. tularensis signature from primer pair bct2332. to demonstrate the specificity of the assay for the francisella biocluster, we tested a collection of 57 reference isolates obtained from the usamriid (table s7 ). included were members of all phylogenetic lineages within the francisella biocluster. francisella species were correctly identified and grouped into phylogenetic clades. all 34 type a.i subspecies yielded identical base compositions for both primer pairs consistent with the predicted amplicons from the schu s4 genome. twelve of the type b, subspecies holarctica strains had signatures that differed from the type a.i subspecies in both the primer pairs, clearly differentiating the two major groups of the francisella genus. type a.ii strain signatures were different from the type a.i signatures by a single t to c snp in the gale primer amplicon, consistent with the genome sequence of francisella subsp. tularensis strain wy96-3128. however, two of the type b strains tested, fran041 (strain jap-cincinnati) and fran011 (strain lr) could not be distinguished from the type a.ii subspecies using these two primer pairs. as described above, this signature appears to be consistent with subsp. mediaasiatica strains as well. sequence similarities between the type b japanese strains, f. tularensis mediaasiatica and type a.ii lineages were previously noted ( [29] , duncan, manuscript in preparation). the f. novicida strain (fran003) differed from type a.i by a single snp in the gale region, whereas the species outlier, f. philomiragia, was clearly different from the rest of the f. tularensis biocluster in both primer regions. the asd primer pair produced the expected amplicon for all strains of f. philomiragia. in contrast, the gale locus primer pair did not yield an amplicon for all strains. this region in a recently sequenced f. philomiragia strain (atcc 25017) has mismatches to the primer regions (genbank accession number nc_010336). even without data from the asd primer pair, the differentiation of this species from other f. tularensis was unambiguous. based on the two primer analysis done here, francisella strains could be categorized into five groups: types ''a.i,'' ''b,'' ''novicida,'' ''philomiragia,'' and a fifth group that contains strains within the type a.ii/mediaasiatica/holarctica/ japanese lineages that might have diverged before the acquisition of the asd and gale mutations. vibrio cholerae. the genus vibrio, within the family vibrionaceae, represents a diverse group of gram-negative bacteria that contain at least 65 described species, most of which are found exclusively in aquatic environments. of these, at least 12 species are known human pathogens, and several other species are known to be pathogenic to marine mammals and fish. members of this genus include vibrio cholerae, the causative agent of cholera. although large outbreaks of cholera are caused by toxigenic strains of the serogroups o1 and o139, non-toxigenic strains cause sporadic cases of disease. other important pathogens in this group include v. parahaemolyticus and v. vulnificus, which cause significant morbidity worldwide [32] . we previously described a high-throughput pan-vibrio assay for simultaneous identification of all known pathogenic vibrio species [14] . the assay included broad-range pcr primers that targeted conserved sites in several housekeeping genes and the v. choleraespecific toxin genes ctxa and ctxb. base compositions from these regions were able to distinguish the various species tested and provided sub-species differentiation within the v. cholerae isolates. for the biothreat assay, three of the primer pairs from the pan-vibrio assay were used ( table 1) . one of these primer pairs was exclusive to v. cholerae species detection (ompu). one amplifies the toxin gene ctxa. the third primer pair, targeted to gapa gene, was designed to amplify most of the known species in this family and the resulting base compositions provide species-level resolution (table s8) . genomic data analysis and experimental analysis of 42 well-characterized strains representing the phylogeny of this biocluster was used to demonstrate assay specificity. to demonstrate the ability to detect and identify vibrio spp. from natural aquatic samples, a subset of samples collected in 2006 from freshwater lakes and sites along the georgian coast of the black sea were tested [14] . six different vibrio species were detected and identified in 13 of the 19 natural water samples collected from both freshwater and seawater sites spanning the seasons summer to winter in this study [14] . more than one vibrio species were also detected in some samples [14] . these detections were confirmed by 16s rrna clone library analysis. burkholderia mallei/pseudomallei. the burkholderia biocluster is identified in the biothreat assay by two primer pairs, bct1070 and bct1071 ( table 1 ). the primer sets amplify conserved regions of the rna component of ribonuclease p (rnase p) and regions cr-iv and cr-v that bracket the highly variable extension p19. these primer pairs have homology to this gene from other proteobacteria; however, the length and base composition of the resulting amplicons are highly discriminating. the information content within amplicons from bct1070 and bct1071 is basically the same, and this feature is meant as a builtin redundancy check for speciation calls. some b. pseudomallei strains provide the same signature as b. mallei (table s9 ), but the next closest relative, b. thailandensis, is clearly segregated. analysis of known sequences indicates that the assay will resolve most known burkholderia species (with members of the b. cepacia biocluster being further distinguished through their distinct amplicon lengths. a notable exception is the polychlorinated biphenyl reducer b. xenovorans, which shows the same mass signature as b. pseudomallei str. 668. while this is a source of potential false positive reporting of b. pseudomallei, the occurrence of b. xenovorans, which occupies a distinct ecological niche and is not pathogenic, is quite rare. brucella. several extremely dangerous pathogens that can infect humans and animals are found in the brucella biocluster. these bacteria are easily transmitted by ingestion of unsterilized milk or meat from infected animals or close contact with their secretions or by inhalation of aerosols. brucella species have slightly different preferred host specificities: b. melitensis infects goats and sheep, b. abortus infects cattle, b. suis infects pigs, b. ovis infects sheep, b. canis infects dogs, and b. neotomae infects wood rats. taxonomists have alternated between individual species naming and naming as a single species b. melitensis, containing b. melitensis 16m and five other biovars: abortus, canis, neotomae, ovis, and suis. recently, four additional species of brucella have been described, including two that infect marine mammals. the current convention adopted by the international committee on systematics of prokaryotes, subcommittee on brucella (http://www.the-icsp.org/ subcoms/brucella.htm) recommends re-approval of the classical brucella species with their recognized biovars. in the biothreat assay, members of the brucella biocluster are identified by two primer pairs, bct1111 and bct1112, that amplify two non-overlapping regions of the rna component of ribonuclease p gene (table 1 ). these two regions were chosen for their ability to amplify all brucella species and to distinguish these from other near-neighbor alphaproteobacteria species. table s10 shows the base compositions expected from the two brucella primer pairs used in the assay; signatures were derived from genbank data for the sequenced brucella species. clostridium botulinum/perfringens. the genus clostridium consists of relatively large, gram-positive, rod-shaped bacteria in the phylum firmicutes. most clostridia are opportunistic pathogens that are anaerobic, but spores are able to survive long periods of exposure to air. most of the clostridia are saprophytes, but a few are pathogenic in humans, primarily clostridium botulinum, c. perfringens, c. difficile, and c. tetani. botulism is an acute neurological disease caused by a neurotoxin produced by c. botulinum. eight c. botulinum neurotoxin types have been identified: types a, b, c1, c2, d, e, f, and g [33, 34] . types a, b, e, and f cause human botulism. types c and d cause most cases of botulism in animals. c. perfringens is classified into five types on the basis of its ability to produce one or more of the major lethal toxins, alpha, beta, epsilon, and iota. c. tetani is another clostridium that can be highly toxigenic to humans. other clostridia can be highly invasive under certain circumstances. in the biothreat assay, the clostridia are identified using two primer pairs, bct1075 and bct1076, targeting rnase p. these two primer pairs are capable of amplifying all members of the genus clostridium and differentiate the major species (table s11 ). the c. botulinum strains form three base composition clusters, differing from each other by one or more snps in each of the two amplified regions. types a, b1, and f form a unique group and are distinguishable from all other clostridia. the second group consists of types a2, a3, and ba4 have base compositions that overlap with c. sporogenes. the third c. botulinum group comprises types b and e and some strains of c. perfringens as well as the recently sequenced c. ljungdahlii. most strains of c. tetani and the opportunistic clinical pathogen c. difficile form unique groups. other clostridia that are rarely human pathogens are clearly differentiated from all the above, thus providing a rapid means of detection of the pathogenic clostridia. coxiella burnetii. q fever is a zoonosis caused by coxiella burnetii, an obligate gram-negative intracellular bacterium [35] . c. burnetii infects various hosts, including humans, ruminants, and pets. because of its highly infectious nature, c. burnetii is recognized as a biothreat agent. the bacterium can exist in a spore-like life cycle and remain viable and virulent for many months. phylogenetically it occupies a unique niche, with very few near neighbors. the closest known organism based on genomic and 16s rrna analysis is legionella pneumophila [36] . in the biothreat assay, we use primers targeting isocitrate dehydrogenase (icd) and insertion sequence is1111a transposase for unambiguous detection of c. burnetii (bct1079 and bct1080, respectively). the latter occurs in multiple copies in the bacterium (from 5 to 31 copies) [35, 37] . base composition analysis of the expected products showed that all sequenced coxiella genomes (nm, dugway, k, and q) share identical signatures in both amplified regions (table s12 ). these primer pairs do not amplify legionella (data not shown) and should not amplify nucleic acids from any of the other proteobacteria. the c. burnetii g (q212) sequenced genome showed two different base counts at the is111a locus, suggesting a snp variant in this region, similar to operonic diversities often seen in 16s rrna sequences for other bacteria. rickettsia prowazekii. the rickettsiaceae are a family of obligate intracellular small gram-negative coccobacilli that infect humans chiefly through insect vectors, mostly from animal hosts [38] . the rickettsial fevers are acute bacteremic illnesses charac-terized by headache, mental confusion, and, in severe cases, meningoencephalitis. the genus rickettsia is divided into three main groups: r. prowazekii, the agent of classical epidemic typhus; r. typhi, the causal agent of endemic typhus; and the ''spotted fever'' group of rickettsiae, which contains a large number of species transmitted from rodents, dogs, and wild animals by ticks. the latter group includes r. rickettsii, the agent of rocky mountain spotted fever; r. conorii, the cause of tick typhus in the mediterranean area and in india; r. africae, which is found in the african veld; r. japonica, r. australis, and a variety of other similar organisms that are widely distributed in asia and australia. organisms in the rickettsia biocluster are identified by two primer pairs, bct1083 and bct1084, which prime different regions of the rna component of the rnase p. since the primers target rickettsia-specific sequences, no amplification is expected outside the rickettsiaceae family. using these two primer pairs, members of the rickettsia biocluster can be distinguished from each other. in particular, r. prowazekii, r. typhi, and r. rickettsii species yield distinct plex-id base composition clusters (table s13) . enterobacteriaceae (escherichia coli o157:h7, shigella, salmonella enterica). diarrheagenic enterobacteria, such as e. coli, salmonella, and shigella species, are closely related organisms that are ubiquitous food and water-borne pathogens. these agents have the potential to cause significant damage to the food supply and are high-risk clinical pathogens. there are over 3,500 salmonella subtypes, and all are human pathogens. the majority of these serotypes belong to a single salmonella species, salmonella enterica, which includes six subspecies (subsp. enterica, subsp. salamae, subsp. arizonae, subsp. diarizonae, subsp. houtenae, and subspecies indica). for shigella, there are four species (shigella dysenteriae, s. flexneri, s. boydii, and s. sonnei); all can cause enteric illnesses. there are at least five pathotypes of e. coli: enteroinvasive e. coli (eiec), enterotoxigenic e. coli (etec), enteropathogenic e. coli (epec), enterohemorrhagic e. coli (ehec), and enteroaggregative e. coli (eaec). symptoms caused by these organisms are often similar, and these organisms are difficult to differentiate by genomic analysis. in the biothreat assay, a primer pair targeting the valine synthetase (bct358) gene provides identification of all of the above species in the enterobacteriaceae family. in addition, this primer pair also provides species resolution of yersinia as discussed previously. the base count clusters shown in table s14 demonstrate the ability of this primer pair to amplify members of the family enterobacteriaceae and to provide species-level differentiation of salmonella from e. coli and shigella, although the shigella species base counts are indistinguishable from a group of the e. coli strains using this primer pair (plex-id cluster 5, table s14 ). this is consistent with the previously described phylogenetic relationship of these bacteria [39] . importantly, the pathogenic e. coli o157:h7 species is clearly differentiated from all other enterobacteriaceae except enteropathogenic e. coli o55:h7, strain 5905 (bc cluster 1). this strain is considered the immediate ancestor of the e. coli o157:h7 lineage and contains the shiga toxin, which is atypical for other e. coli o55:h7 strains [40] . the salmonella species are divided into several clusters, but cannot be grouped according to the subspecies nomenclature based on data from this assay. these pathogens can be resolved in a more targeted food-borne bacteria assay (manuscript in preparation). in order to provide additional separation between e. coli and shigella species, we added two more primer pairs targeting two different regions of invasion plasmid antigen h (ipah). as shown in table s15 , these two primer pairs amplify all shigella species but do not amplify e. coli or salmonella (data not shown). thus, the four major shigella species can be clearly identified as a group distinct from e. coli using information from the three primer pairs. this assay, however, does not distinguish among the various shigella species. alphaviruses. the genus alphavirus, of the family togaviridae, contains at least 37 species and subtypes or varieties and at least 23 have been associated with human illness. some new world alphavirus, such as the eastern equine encephalitis viruses (eeev) and venezuelan equine encephalitis virus (veev) complex, are considered potential bioweapons [41] . other important members of this virus group include the western equine encephalitis virus (weev) complex viruses that include sindbis virus. the old world cluster includes chikungunya virus and semiliki forest virus complex among others. we have previously described an assay for pan-alphavirus detection and demonstrated the utility of this with field-collected mosquito and clinical samples [12] . this assay detects a wide variety of alphavirus in naturally occurring biological backgrounds and was used to identify a virus that was a novel subtype iiid in the veev complex [12] . two of the three primer pairs described previously were used in the biothreat assay and have been tested against a broad panel alphavirus isolates representing both the old world and new world alphavirus (table s16 ). both are targeted to the ns1 region on the 59-end of alphavirus genome. primer pair vir966 exhibited the greatest breadth of coverage. base counts from this primer pair amplicon alone were sufficient to distinguish most of the isolates at the species and strain level. the second primer pair (vir2499) used in the study contains inosine (i) nucleotides at selected sites in both the forward and reverse primers to enhance hybridization, but despite this, did not amplify several of the old world alphavirus. all the alphavirus samples tested were detected with at least the vir966 pair and most were identified to strain or subtype level. orthopoxvirus. the genus orthopoxvirus contains several species of related viruses including the causative agent of smallpox (variola virus). in addition to smallpox, several other members of the genus are capable of causing human infection, including monkeypox, cowpox, and other zoonotic rodent-borne poxviruses. we have previously described a pan-orthopoxvirus assay for identification of all members of the genus based on four pcr reactions targeting orthopoxvirus dna and rna helicase and polymerase genes. the assay can detect and identify diverse orthopoxviruses, provide sub-species information, and characterize viruses from the blood of rabbitpox virus-infected rabbits [11] . in the biothreat assay, we used two of these four primer pairs (vir979 and vir985). the two provide species-level resolution of the genus orthopoxvirus and, in particular, differentiate the variola and monkeypox viruses from each other and from vaccinia, rabbitpox, and ectromelia viruses (table s17) . influenza virus. influenza a viruses are important respiratory pathogens that cause annual epidemics and occasional pandemics. influenza viruses cause serious global economic and public health burdens. emergence of new influenza a virus strains can be caused by ''antigenic shift,'' resulting from reassortment of gene segments (including h and/or n types), by ''antigenic drift'' resulting from the continuing accumulation of mutations in the h and n genes, or by species jump by a virus that acquires the ability to infect and be transmitted among humans as has happened in the influenza pandemics over the last century [42, 43] . in april 2009, a previously unseen virus emerged and rapidly spread globally leading to the first influenza pandemic of the 21 st century. the continuous evolution of influenza genomes together with reassortment events pose challenges to the effective monitoring of influenza viruses. we previously described an rt-pcr/esi-ms assay for the detection and characterization of influenza viruses [8, 10, 15, 16, 44, 45] . identification of each influenza a virus is based on the summation of base composition signatures obtained from the six to eight primer pairs. in the biothreat assay, two of the previously described sets of primer pairs (vir2798 and vir1266) were included for rapid detection of presence of influenza a or b virus. base composition data from the amplified regions of over a 1000 influenza a h5n1 strains from genbank were analyzed. the majority of these could be grouped into the six base composition clusters as shown in table s18 . these clusters were unique and distinct from other avian and non-avian signatures (data not shown), with the exception of two instances of avian h9n2 sharing base composition overlap with one or more of the avian h5n1 clusters. in both these instances, however, all the overlapping strains were from local outbreaks (shantou 2003 and guangxi 2006) and were not widely distributed. similar correlations were found for pandemic 2009 h1n1, seasonal h3n2, and seasonal h1n1 viruses as previously described [44] . in the biothreat assay, the non-avian h5n1 subtypes will be reported only at the species level as influenza a virus. further differentiation of the sub-species may be achieved using the broader pcr/esi-ms influenza assay previously described [13] . filovirus. filoviridae is a viral family of negative-strand rna viruses that include two major genera, ebolavirus and marburgvirus, both of which contain highly pathogenic and potential biowarfare agents. some of the species in these groups include sudan, reston, zaire, and cote d'ivoire ebolavirus and several strain variants of the lake victoria marburgvirus species. we have developed an assay for the detection of all members of this family using two primer pairs targeting the rna-dependent rna polymerase region of the genome. there is significant sequence diversity among the filoviruses and in order to ensure primer hybridization to all the above viruses, we used modified nucleotides in the pcr primer pairs as previously described for the detection of the sars coronavirus [9] . we tested these primers with samples obtained from the cdc special pathogens branch (dr. stuart nichol, personal communication). in all cases, we obtained the base composition signatures expected based on sequenced genomes of these viruses (table s19 ). due to the highly pathogenic nature of these viruses, these viruses were not used in any additional analytical characterization studies described below. the above sections describe in detail the primer pairs used in the biothreat assay and the ability of the individual groups of primer pairs to detect the targeted biothreat cluster. all of these primer pairs were assembled into a single assay kit containing groups of two to three primer pairs per well for screening for all the listed biothreat agents simultaneously. the assay layout is shown in figure s1 . sixteen wells of a 96-well microtiter plate were utilized for analysis of each sample. up to six samples may be screened per plate. importantly, each pcr well included a synthetic dna calibrant that was amplified by one of the target primer pairs. this calibrant served as a pcr positive control and allowed relative determination of the quantity of the target organism as previously described [2, 4] . data analysis and reporting for this assay were optimized for detecting the targeted biothreat clusters, and detection of organisms outside this group are not reported. two different types of report are currently available. the first is a summary that reports detection and lack thereof for each of the 14 groups described in the previous sections for each sample ( figure s2 ). the criteria for inclusion in this report are the detection of the biothreat cluster organisms in one or more primer pairs targeting the individual clusters. the primer pairs targeting the plasmid markers are reported separately. in the specific example shown, the test organism was a b. anthracis strain containing both virulence plasmids. the report indicates detection of the genome and the two plasmids. the approximate genome equivalents per well for the target organism are based on relative amplification compared to the calibrant. at higher target organism concentrations, the calibrant is often outcompeted in the pcr well; therefore, the reported levels at higher loads could be inaccurate. another more in-depth analysis of the data is available that provides base composition details to support the calls reported in the summary ( figure s3 ). analytic sensitivity. the limit of detection (lod) of the biothreat assay in analysis of environmental aerosol samples, one of its intended uses, was determined by analyzing serial 10-fold dilutions of nucleic acids from a number of test agents with air filter nucleic acid extract from a biodefense monitoring program (''dirty air''). in parallel, samples were also tested in dna elution buffer (te buffer) alone. fivefold serial dilutions of nucleic acid samples (with a high concentration of 1000 ge/well for all targets except veev which was 5000 ge/well) of the nucleic acid extracts from all the target organisms were prepared and 10 replicates of each dilution of each agent were analyzed. the presumptive lod was ascribed to be the lowest concentration resulting in 10 correct identifications and detections in all primer pairs targeting each biothreat cluster ( figure s4 ). additional replicates were analyzed (as many as 105) to allow for sufficient data to determine the lod with a 95% confidence interval. these measured lods were used for subsequent analysis. nucleic acids from test organisms were paired to reduce overall sample numbers (for example, b. anthracis and vaccinia virus nucleic acids were spiked together and analyzed in the same sample). these lod studies were preceded by analysis of synthetic constructs, plasmids containing dna sequences similar to those of the biothreat agents. these constructs were carefully measured using real-time pcr assays targeting the plasmid backbone (data not shown). using this synthetic approach, the lod for the various target primer pairs in the biothreat assay were determined to be between 7 and 250 genome equivalents (ge) per well, with most organisms detected at between 15 and 62.5 ge/well ( figure s4 ). the outliers were the three rna virus groups, which showed either 125 or 250 ge/well lod. the lods and the false negative rates for all threat agents tested are summarized in table 3 . the lod for the threat agents tested in the context of environmental air background ranged from 40 to 1000 ge/well. it was found that 37.5% (6/16) of the threat agents tested had lods of 40 ge/well, 50% (8/16) had lods of 200 ge/well, and 12.5% (2/16) had lods of 1000 ge/well. at the lods, false negative rates were less than 5% for 14 of the agents used to challenge the biothreat assay kit and less than 10% for two of the agents. detection and identification of all the threat agents relies on more than one primer pair; in the case of b. anthracis, it relies on four primer pairs. for calculation of lod, all targeted primer pairs were expected to amplify and produce results. however, in routine operation, any primer pair producing a result would result in an organism identification. the false negative rates were calculated based on a failure to detect the threat organism. specificity. it is critical that an assay used for biodefense monitoring be capable of detecting threats and, perhaps as importantly, of not falsely identifying a threat. the sample preparation methods employed by environmental monitoring programs result in samples containing significant amounts of nucleic acids from a variety of species. two methods were employed to determine the specificity of the biothreat assay. first, over 1000 samples containing environmental background without a specific target agent were analyzed and used to calculate false positive rates for each agent. second, the ability of the biothreat assay kit to detect a threat when the sample contained both the targeted threat and a near neighbor but not when the sample contained only the near neighbor was assessed. a total of 1,353 samples in an environmental air background were analyzed during the determination of sensitivity. each of these samples contained only two of the agents under investigation. because the biothreat assay simultaneously analyzes each sample for every threat agent, the results from those samples not containing a particular threat agent were used to determine the false positive rates of that agent. for example, in the lod determination of b. anthracis, the sample contained nucleic acids extracted from the environmental collection and from b. anthracis and vaccinia virus. because those samples did not contain any of the other 14 threat agents, the data from the analysis of those samples could be used to calculate the false positive rate. the false positive rates for each of the threat agents tested was 0%, except for rickettsia prowazekii, which was 14% ( table 4 ). the matrix that was used as background had high loads of environmental bacterial signatures (data not shown), including alphaproteobacteria such as rickettsia and mesorhizobium species. these might account for the observed higher false positive rates for rickettsial species compared to other organisms. higher background prevalence of some of the potential biothreat agents or their near neighbors in certain regions of the world might similarly affect other detections. for instance, burkholderia pseudomallei, which is described as a highly distributed soil saprophyte in southeast asia would result in higher rates of reporting of this organism. near-neighbor nucleic acids were added to the sample in excess (fivefold higher than the lod) of the target biothreat nucleic acids (added at twofold the lod). the results are presented in table 5 . for each of the threat agents tested, the threat organism was correctly identified by the plex-id system even when excess near-neighbor nucleic acids were present in the sample. as expected for the brucella melitensis cluster, the plex-id biothreat assay did not discriminate between b. melitensis, b. abortus, b. suis, and b. ovis, which are all considered to be potential threat organisms. when rickettsia canadensis was included during testing of the rickettsial primer pairs, coxiella burnetii was also identified as a false positive. this detection was reported by the coxiella primer pairs, not the rickettsial primer pairs, indicating that there was a possible contamination of the r. canadensis with coxiella burnettii. thus, the plex-id clearly demonstrated the ability to detect threat agents in the presence of both background and excess nearneighbor nucleic acids. breadth of coverage. to determine the ability of the biothreat assay to distinguish between threat agents and near neighbors, the biothreat assay was challenged with nucleic acids purified from a panel of organisms. these samples were diluted in tris-edta (ph 8.0) to allow for analysis on the biothreat assay plate at 1000 ge/well for each organism individually. in every case, the challenge organism was correctly identified. when possible, additional strains or sub-species were also identified. however, the intended use of the biothreat assay is to alarm the end user when a threat is present. the data presented in table 6 clearly demonstrate the ability of the assay and the instrument to discriminate between threat agents and near neighbors. linearity. the ability of an instrument to provide measurements that are directly proportional to the concentration of the test analyte is referred to as linearity. data obtained from experiments used to determine the lod within an environmental matrix were analyzed for linearity. the total ge reported by the plex-id was plotted against the actual concentration; linear trend lines were generated to determine linearity within the challenge concentrations. figure 2 shows results for b. anthracis linearity test, demonstrating linearity for the entire range of concentrations tested. other organisms were linear up to the maximum concentration tested. the data are summarized in table 7 . the biothreat assay described here identifies ten bacterial and four viral biothreat clusters included in the niaid priority pathogen (category a: seven agents, category b: 18; category c: three) and hhs/usda select agent (18 agents) lists. the assay also identifies a broad range of near-neighbor organisms that may cause severe disease in humans or animals or that may be harmless environmental organisms. the biothreat cluster analysis strategy using pcr/esi-ms addresses several fundamental design requirements for biothreat protection. first, biothreat agents and near-neighbor organisms are identified unambiguously and equally. closely related organisms often cause false alarms in conventional pcr approaches to detect biothreat agents. perhaps more importantly, it enables identification of unexpected pathogens within the biothreat clusters that might be used in a biological attack. second, the genetic targets for amplification in the biothreat assay are universally conserved, essential to microbial life. this lowers the risk of failed detections because these targets cannot be dispensed with by the microbe and would be difficult to modify by engineering to avoid detection. third, the comprehensive nature of the biothreat assay enables very broad surveillance of the potential biothreat landscape, including the detection of the virulence plasmids where appropriate. fourth, the pcr/esi-ms instrumentation enables very high-throughput sample analysis; the theoretical maximum throughput of the biothreat assay on a current-generation plex-id instrument is approximately 180 specimens over 24 hours. some of the component primer pairs of the comprehensive biothreat assay were validated before the biothreat assay was assembled. the orthopoxvirus primers have been shown to detect and identify a diverse collection of over 30 isolates of orthopoxviruses and to identify sub-species and characterize viruses from the blood of rabbitpox-infected rabbits [11] . the alphavirus primers were used to amplify a panel of 36 virus isolates representing characterized old world and new world alphavirus [12] . base compositions from the resulting amplicons were used to unambiguously determine the species or subtypes of 35 of the isolates. in addition, the assay was used to identify alphavirus directly in mosquitoes and detected an unanticipated mucambo virus species [12] . the francisella biocluster primers were used in an investigation to understand why the u.s. government biowatch sensors were triggered by an apparent false alarm on september 24-25, 2005 during a large public gathering along the capital mall area in washington, dc. the sensors signaled low-level detections of f. tularensis. uncertainty as to whether or not there was a biological attack led the cdc to issue an official national health advisory via the health alert network to alert healthcare personnel to possible tularemia exposure (cdchan-00238-05-09-30-adv-n). specimens analyzed by the primer pairs that comprise the f. tularensis biothreat cluster component of the biothreat assay were used to analyze the air specimens. the results showed that the base composition signature in the extracted air samples had the signature of f. tularensis subsp. novicida, a naturally occurring organism with significantly lower virulence than f. tularensis subsp. tularensis, the main bioagent in the cluster [46] . thus the detections of f. tularensis observed in these air samples most likely represented detection of a naturally occurring organism. the biothreat assay described here would immediately identify this organism as a near neighbor of a biothreat agent. the v. cholerae biocluster primers were previously field-tested using natural water samples from both freshwater lakes and the georgian coastal zone of the black sea. of the 278 total water samples screened, nine different vibrio species were detected, 114 samples were positive for v. cholerae, and five samples were positive for the cholera toxin a gene (ctxa) [14] . the results were confirmed with conventional pcr. the y. pestis primers were used to identify the first reported case of plague in afghanistan where the illness is associated with consumption of camel meat [47] . in late december 2007, an outbreak of acute gastroenteritis occurred in nimroz province of southern afghanistan. of the 83 patients, 17 died. molecular testing of patient clinical samples and of tissue from the camel using pcr/esi-ms revealed dna signatures consistent with y. pestis. confirmatory testing using real-time pcr and immunological seroconversion of one of the patients confirmed that the outbreak was caused by plague with a rare gastrointestinal presentation. assembly of this comprehensive collection of biothreat cluster primers into a single assay on the plex-id has the potential to serve a variety of biosecurity needs. first, the biothreat assay can be used for environmental surveillance 2 the application that was experimentally demonstrated in this manuscript. another potential use of the assay is as a reflex test to the pan-bacterial plex-id assay intended to identify all bacteria in normally sterile bodily fluids (e.g., blood, cerebral spinal fluid) [6] . in this concept of operation, the pan-bacterial assay would be used routinely in clinical diagnostics and, if potential biothreat agents were detected, the biothreat assay would be used to provide detailed analysis. although several of the primer pairs used in this assay were studied previously, the biothreat assay is a multiplexed version: thirty-six primer pairs are combined into 16 pcr reactions. the multiplexed assay was tested with environmental air from biowatch filters. feasibility studies were performed at 1000 ge of each target organism spiked into background matrix. the environmental background did not inhibit the ability of plex-id to correctly detect the target organisms. additional studies were conducted to demonstrate the analytical performance of the assay. these included sensitivity, specificity, linearity, and breadth of coverage of the biothreat clusters. analytical sensitivity of the target organisms varied between 40-1000 ge/well with no false positive detections, and false negative rates below 5% for most organisms tested. the matrix that was used as background had high loads of environmental bacterial signatures (data not shown), including alphaproteobacteria such as rickettsia and mesorhizobium species. these might account for the observed higher false positive rates for rickettsial species compared to other organisms. breadth of coverage and specificity for detection of biothreat agents were determined with critical reagents program (crp; http://www. jpeocbd.osd.mil/packs/default.aspx?pg = 1205) reagents. there was an excellent correlation between plex-id identifications and the identity of the spiked strains. presence of two-to five-fold excess of a near-neighbor organism did not interfere with the detection of the biothreat agent. plex-id assays in general are semi-quantitative at best and have a limited dynamic range for reporting genome levels of detected organisms [4] . the levels of organisms determined using the biothreat assay indicate the approximate genome equivalents/well for the target organism based on relative amplification compared to the calibrant. at higher target organism concentrations, the calibrant is often outcompeted in the pcr reaction, therefore the reported levels at the higher loads were inaccurate. this, however, does not interfere with the ability of the assay to detect the threat agent. in contrast, a specific rt-pcr assay targeting a single organism might be able to provide a measurement over a much larger linear range. in summary, the plex-id biothreat assay kit was evaluated for detection of biothreat agents in environmental air samples. the data presented demonstrate the capability of the pcr/esi-ms method to accurately detect and identify organisms from ten bacterial and four viral biothreat clusters. the assay discriminated between target agent and near neighbors with high specificity and sensitivity. the method accurately reported each organism with which it was challenged and accurately identified threat species as a threat; species and/or strains that are not considered a threat were not reported as such. the assay is capable of simultaneous detection of most niaid category a, b, and c priority pathogens and hhs/usda select agents and thus provides a means for comprehensive coverage using a high-throughput assay. the validation data support use of the ibis plex-id and the biothreat assay for detection of biological warfare agents in complex environmental matrices. additional testing of this assay with an eu validation panel is described in a companion manuscript (grunow et al. [49] ). the nucleic acid samples used in this study were obtained from the critical reagents program (crp), bei resources, atcc, keim genetics lab, or were prepared from mri culture collections. environmental collections were obtained on dry filter unit (dfu) filters from a variety of locations in the washington, d.c. region. nucleic acids were eluted from the environmental matrix by placing dfus in 20 ml pbs/0.2% triton x-200 and manually shaking. the eluent was then shaken in a biospec mini beadbeater-96 with atl buffer (qiagen), herring sperm dna, proteinase k, and antifoam for 5 min. samples were incubated for 21 min at 16uc and then centrifuged. al buffer (qiagen) was added to the supernatants and incubated for 1 min at 70uc. ethanol was added to the sample, which was then loaded onto a qiagen spin column, centrifuged, and sequentially washed with al, aw2, and aw4 buffers (qiagen). the sample was eluted from the spin columns in 200 ml of ae buffer (qiagen). the primer pairs that make up the biothreat assay (table 1) were selected to provide the desired resolving capability for the assay as described in detail in the results section. thirty-six primer pairs were used in the assay; primer pairs were grouped two to three per well to occupy 16 wells of a 96-well microtiter plate. the groupings were chosen to eliminate primer interactions and target overlap. the assay layout is shown in figure s1 . each assay plate can be used to screen up to six samples. internal positive controls similar to the amplicon expected from one of the primer pairs in each of the multiplexed reactions were made from cloned synthetic dna (blueheron biotechnology, bothell, wa) and were included in each pcr reaction at 100 copies per reaction. the internal controls were designed to be identical to the expected target priming regions with the exception of five-base pair deletions to enable the control to be distinguished from the target-derived amplicon. pcr was performed in a 50 ml reaction volume containing 5 ml nucleic acid extract in a reaction mix as previously described [48] . the plate was heat-sealed with foil on a thermo scientific alps microplate heat sealer (rockford, il). each sealed plate was loaded onto a mastercycler pro thermocycler (eppendorf, hauppauge, ny) and pcr-amplified under the following conditions: 95uc for 10 min; then 8 cycles of 95uc for 30 s, 48uc for 30 s, and 72uc for 30 s; then 37 cycles of 95uc for 15 s, 56uc for 20 s, and 72uc for 20 s; followed by 72uc for 2 min, and 99uc for 20 min. one-step rt-pcr was performed in wells with primers designed for viral detection. since all reactions for a sample were run in the same 96-well plate rt-pcr cycling conditions were used for both the rt-pcr and pcr reactions as previously described [12] . after thermocycling, plates were stored at 240uc until the samples could be analyzed by esi/ms on the plex-id. mass spectrometry was performed on a plex-id biosensor (abbott molecular, des plaines, il). after pcr amplification, 30 ml aliquots of each pcr reaction were desalted and analyzed by mass spectrometry as previously described [2, 4] . briefly, the plex-id platform is capable of analyzing nearly 3000 pcr reactions in 24 hours and integrates a novel sample purification scheme with a high throughput fluidics/robotics platform. the plex-id instrument is comprised of an input plate stacker which accommodates fifteen 96-well microtiter plates, an automated purification module which desalts and purifies amplicons with a magnetic-bead-based weak anion exchange method, and an autosampler coupled to a novel dual electrospray head which injects analytes into a perkin elmer esi-tof mass spectrometer. the platform analyzes one pcr reaction every 30 seconds in a fully automated modality. the plex-id platform utilizes a novel carousel-based design for the rapid and efficient purification of pcr amplicons. the carousel is comprised of 22 identical spin cuvette modules in which the analyte solution is purified prior to esi-ms analysis. every 30 seconds the carousel rotates by one position facilitating the aspiration/or dispensation of the requisite reagents. data analysis and results reporting was performed in an automated fashion using on-board computer on the ibis plex-id system. for this assay, a customized reporting rule set was designed that allowed rapid and accurate detection of the biothreat targets. the biothreat assay report has 21 primer groups or threat clusters as shown in figure s2 . these groups consist of primer pairs used to identify the target biothreat organisms. each of the threat clusters is treated independently and the results are reported for each cluster separately. thus, the presence or absence of each of the target biothreat clusters is directly reported. mixed detections of two or more threats or a threat with an unrelated near neighbor in another group are also reported. further, two additional metrics (q-score and level) are provided in the report to assess the quality of the reported detection. the q-score is a rating between 0 (low) and 1 (high) of the confidence in the identification of the organism. the q-score is based on a number of different parameters such as, the multi-primer joint log likelihood ratio, which is an indicator of how well the hypothesized organisms as a group represent the observed data; the multiprimer single log likelihood ratio, which is an indicator of how significant the contribution of a single organism is to the solution; the fraction of missed detections, which represents the percentage of primers for a detected organism that should have produced known base count compositions, but did not; and, finally, the fraction of no data, which indicates the percentage of primers for a detected organism for which no known data exists within the plex-id system. for the biothreat assay described here, a qscore $0.85 is considered a reportable result. the level is an indication of the amount of the amplicon present in the sample reported as genome equivalents/well. this is calculated with reference to the internal calibrant as described previously [4] . the normal range for reporting these levels is between 0.16 and 106 the levels of internal controls in the assay, which in the case of the plex-id biothreat assay represents a working range of ,10 ge/ well to 1000 ge/well. in addition to the summary style report shown in figure s2 , the system is capable of reporting organism/strain level matches based on the genomic sequence data in the plex-id database ( figure s3 ). this is provided as a research utility tool in a separate analysis workstation. additional details for each of the matches, including the detected base compositions and levels, are available using this report. figure s1 biothreat assay plate layout. left panel: sample wells and target biothreat cluster for each primer pair are indicated. each well contains two or three multiplexed primer pairs. letters a through h represent 8 rows of a 96-well plate, whereas numbers 1 through 12 represent the columns. each sample is analyzed in 16 pcr reaction wells and six samples can be tested per pcr plate. details of the primer pairs are given in table 1 . right panel: the 96-well pcr plate layout. each pcr well includes a synthetic nucleic acid template that serves as a calibrant. in multiplexed wells, this calibrant provides an amplicon similar to the amplicon expected for the organism shown in red. (docx) figure s2 example of plex-id summary report. each biothreat cluster is listed separately. detection of an organism within a cluster is listed at the species level. if no organism within the cluster is detected, the cluster is marked as ''not detected''. the microbial rosetta stone database: a common structure for microbial biosecurity threat agents the ibis t5000 universal biosensor -an automated platform for pathogen identification and strain typing rapid identification and strain-typing of respiratory pathogens for epidemic surveillance tiger: the universal biosensor ibis t5000: a universal biosensor approach for microbiology new technology for rapid molecular diagnosis of bloodstream infections rapid-test sensitivity for novel swine-origin influenza a (h1n1) virus in humans initial identification and characterization of an emerging zoonotic influenza virus prior to pandemic spread rapid identification of emerging pathogens global surveillance of emerging influenza virus genotypes by mass spectrometry rapid and high-throughput pan-orthopoxvirus detection and identification using pcr and mass spectrometry direct broad-range detection of alphaviruses in mosquito extracts global surveillance of emerging influenza virus genotypes by mass spectrometry identification of pathogenic vibrio species by multilocus pcr-electrospray ionization mass spectrometry and its application to aquatic environments of the former soviet republic of georgia reverse transcription polymerase chain reaction and electrospray ionization mass spectrometry for identifying acute viral upper respiratory tract infections rapid identification of viruses from nasal pharyngeal aspirates in acute viral respiratory infections by rt-pcr and electrospray ionization mass spectrometry multiplelocus variable-number tandem repeat analysis reveals genetic relationships within bacillus anthracis population structure and evolution of the bacillus cereus group microevolution and history of the plague bacillus, yersinia pestis intraand interspecies relatedness of yersinia pestis by dna hybridization and its relationship to y. pseudotuberculosis yersinia pestis, the cause of plague, is a recently emerged clone of yersinia pseudotuberculosis invasin production by yersinia pestis is abolished by insertion of an is200-like element within the inv gene intraspecific diversity of yersinia pestis genome sequence of yersinia pestis, the causative agent of plague yersinia pestis pfra shows biovar-specific differences and recent common ancestry with a salmonella enterica serovar typhi plasmid genome sequence of yersinia pestis kim the immunologically distinct o antigens from francisella tularensis subspecies tularensis and francisella novicida are both virulence determinants and protective antigens comparative genomic characterization of francisella tularensis strains belonging to low and high virulence subspecies worldwide genetic relationships among francisella tularensis isolates determined by multiple-locus variable-number tandem repeat analysis genetic fingerprinting of biodefense pathogens for epidemiology and forensic investigation manual of clinical microbiology preliminary evaluation of a simple in vitro test for the diagnosis of type c botulism in wild birds botulism in the united states: a clinical and epidemiologic review comparative genomics reveal extensive transposon-mediated genomic plasticity and diversity among potential effector proteins within the genus coxiella reassessment of the taxonomic position of rickettsiella grylli is1111 insertion sequences of coxiella burnetii: characterization and use for repetitive element pcr-based differentiation of coxiella burnetii isolates rickettsial diseases: the typhus group of fevers-a review bergey's manual of systematic bacteriology escherichia coli 0157:h7 and other shiga toxinproducing e. coli strains evolutionary relationships and systematics of the alphaviruses pandemic influenza's 500th anniversary what is a pandemic? genomic signature-based identification of influenza a viruses using rt-pcr/electrospray ionization mass spectrometry (esi-ms) technology rt-pcr/electrospray ionization mass spectrometry approach in detection and characterization of influenza viruses microbial forensic analysis of trace and unculturable specimens outbreak of gastroenteritis caused by yersinia pestis in afghanistan genotypic variation and mixtures of lyme borrelia in ixodes ticks from north america and europe rapid and high-throughput detection of highly pathogenic bacteria by ibis plex-id technology the authors thank dr. matthew davenport from the department of homeland security and keim genetics lab for providing b. anthracis strains. the authors acknowledge dr. jackie wyatt, jl-sciedit, for editorial assistance in preparation of the manuscript. key: cord-352522-qnvgg2e9 authors: langille, morgan g. i.; eisen, jonathan a. title: biotorrents: a file sharing service for scientific data date: 2010-04-14 journal: plos one doi: 10.1371/journal.pone.0010071 sha: doc_id: 352522 cord_uid: qnvgg2e9 the transfer of scientific data has emerged as a significant challenge, as datasets continue to grow in size and demand for open access sharing increases. current methods for file transfer do not scale well for large files and can cause long transfer times. in this study we present biotorrents, a website that allows open access sharing of scientific data and uses the popular bittorrent peer-to-peer file sharing technology. biotorrents allows files to be transferred rapidly due to the sharing of bandwidth across multiple institutions and provides more reliable file transfers due to the built-in error checking of the file sharing technology. biotorrents contains multiple features, including keyword searching, category browsing, rss feeds, torrent comments, and a discussion forum. biotorrents is available at http://www.biotorrents.net. the amount of data being produced in the sciences continues to expand at a tremendous rate [1] . in parallel, and also at an increasing rate, is the demand to make this data openly available to other researchers, both pre-publication [2] and post-publication [3] . considerable effort and attention has been given to improving the portability of data by developing data format standards [4] , minimal information for experiment reporting [5] [6] [7] [8] , data sharing polices [9] , and data management [10] [11] [12] [13] . however, the practical aspect of moving data from one location to another has relatively stayed the same; that being the use of hypertext transfer protocol (http) [14] or file transfer protocol (ftp) [15] . these protocols require that a single server be the source of the data and that all requests for data be handled from that single location (fig. 1a ). in addition, the server of the data has to have a large amount of bandwidth to provide adequate download speeds for all data requests. unfortunately, as the number of requests for data increases and the provider's bandwidth becomes saturated, the access time for each data request can increase rapidly. even if bandwidth limitations are very large, these file transfer methods require that the data is centrally stored, making the data inaccessible if the server malfunctions. many different solutions have been proposed to help with many of the challenges of moving large amounts of data. bio-mirror (http://www.bio-mirror.net/) was started in 1999 and consists of several servers sharing the same identical datasets in various countries. bio-mirror improves on download speeds, but requires that the data be replicated across all servers, is restricted to only very popular genomic datasets, and does not include the fast growing datasets such as the sequence read archive (sra) (http://www.ncbi.nlm.nih.gov/sra). the tranche project (https://trancheproject.org/) is the software behind the proteome commons (https://proteomecommons.org/) proteomics repository. the focus of the tranche project is to provide a secure repository that can be shared across multiple servers. considering that all bandwidth is provided by these dedicated tranche servers, considerable administration and funding is necessary in order to maintain such a service. an alternative to these repository-like resources is to use a peer-to-peer file transfer protocol. these peerto-peer networks allow the sharing of datasets directly with each other without the need for a central repository to provide the data hosting or bandwidth for downloading. one of the earliest and most popular peer-to-peer protocols is gnutella (http://rfcgnutella.sourceforge.net/) which is the protocol behind many popular file sharing clients such as limewire (http://www. limewire.com/), shareaza (http://shareaza.sourceforge.net/), and bearshare (http://www.bearshare.com/). unfortunately, this protocol was centered on sharing individual files and does scale well for sharing very large files. in comparison, the bittorrent protocol [16] handles large files very well, is actively being developed, and is a very popular method for data transfer. for example, bittorrent can be used to transfer data from the amazon simple storage service (s3) (http://aws.amazon.com/ s3/), is used by twitter (http://twitter.com/) as a method to distribute files to a large number of servers (http://github.com/lg/ murder), and for distributing numerous types of media. the bittorrent protocol works by first splitting the data into small pieces (usually 514 kb to 2 mb in size), allowing the large dataset to be distributed in pieces and downloaded from various sources (fig. 1b) . a checksum is created for each file piece to verify the integrity of the data being received and these are stored within a small ''torrent'' file. the torrent file also contains the address of one or more ''trackers''. the tracker is responsible for maintaining a list of clients that are currently sharing the torrent, so that clients can make direct connections with other clients to obtain the data. a bittorrent software client (see table 1 ) uses the data in the torrent file to contact the tracker and allow transferring of the data between computers containing either full or partial copies of the dataset. therefore, bandwidth is shared and distributed among all computers in the transaction instead of a single source providing all of the required bandwidth. the sum of available bandwidth grows as the number of file transfers increases, and thus scales indefinitely. the end result is faster transfer times, less bandwidth requirements from a single source, and decentralization of the data. torrent files have been hosted on numerous websites and in theory scientific data can be currently transferred using any one of these bittorrent trackers. however, many of these websites contain materials that violate copyright laws and are prone to being shut down due to copyright infringement. in addition, the vast majority of data on these trackers is non-science related and makes searching or browsing for legitimate scientific data nearly impossible. therefore, to improve upon the open sharing of scientific data we created biotorrents, a legal bittorrent tracker that hosts scientific data and software. the most basic requirement of any torrent server software is the actual ''tracker'' that individual torrent clients interact with to obtain information about where to download pieces of data for a particular torrent. in order to minimize any possible transfer disruptions arising from the biotorrents tracker not being accessible, a secondary tracker is added automatically to all new torrents uploaded to biotorrents. currently this backup tracker is set to use the open bittorrent tracker (http://openbittorrent. com/). also, many bittorrent clients support a distributed hash table (dht) for peer discovery, which often allows data transfer to continue in the absence of a tracker, further enhancing the reliability over traditional client-server file transfers. in addition to the basic tracker, biotorrents has several features supporting the finding, sharing, and commenting of torrents. relevant torrents can be found by browsing categories (genomics, transcriptomics, papers, etc.), license types (public domain, creative commons, gnu general public license, etc.) and by using the provided text search. also, torrents are indexed by google (http://www.google.com) allowing users searching for datasets, but unaware of biotorrents existence, to be directed to their availability on biotorrents. information about each dataset on biotorrents is supplied on a details page giving a description of the data, number of files, date added, user name of the person who created the dataset, and various other details including a link to the actual torrent file. to begin downloading of a dataset, the user downloads and opens the torrent file in the user's previously installed bittorrent client software ( table 1 ). the user can then control many aspects of their download (stopping, starting, download limits, etc.) through their client software without any further need to visit the biotorrents webpage. the bittorrent client will automatically connect with other clients sharing the same torrent and begin to download pieces in a non-random order. the integrity of each data piece is verified using the original file hash provided in the downloaded torrent ensuring that the completed download is an exact copy. the bittorrent client contacts the biotorrents tracker frequently (approximately every 30 minutes) to obtain the addresses of other clients and also to report statistics of how much data they have downloaded and uploaded. these statistics are linked to the user's profile (default is the guest account), to allow real-time display on biotorrents of who is sharing a particular dataset. the choice of bittorrent client will depend on the operating system and options that the user requires. for example, some bittorrent clients (see table 1 ) have a feature called local peer discovery (lpd), that searches for other computers sharing the same data on their local area network (lan), and allows rapid direct transfer of data over the shared network instead of over the internet. this situation may arise often in research institutions where lans are often quite large and multiple researchers are working on similar datasets. another significant feature of the bittorrent client, utorrent, is the addition of a newly designed transfer protocol called utp [17] , that is able to monitor and adapt to network congestion by limiting its transfer speeds when other network traffic is detected. this functionality is important for system administrators and internet service providers (isps) that figure 1 . illustration of differences between traditional and peer to peer file transfer protocols. a) traditional file transfer protocols such as http and ftp use a single host for obtaining a dataset (grey filled black box), even though other computers contain the same file or partial copies while downloading (partially filled black box). this can cause transfers to be slow due to bandwidth limitations or if the host fails. b) the peer-to-peer file transfer protocol, bittorrent, breaks up the dataset into small pieces (shown as pattern blocks within black box), and allows sharing among computers with full copies or partial copies of the dataset. this allows faster transfer times and decentralization of the data. doi:10.1371/journal.pone.0010071.g001 may have previously attempted to block or hinder bittorrent activity due its bandwidth saturating effects. sharing data on biotorrents is a simple three step process. first, the user creates a torrent file on their personal computer using the same bittorrent client software that is used for downloading ( table 1) . the only piece of information the user needs to create the torrent, is the biotorrents tracker announce url which is personalized for each user (see below), and is located on the biotorrents upload page. second, this newly created torrent file is uploaded on the ''biotorrents -upload'' page along with a user description, category, and license type for the data. third, the user leaves their computer/server on with their bittorrent client running so that other users can download the data from them. it should be noted that only users that have created a free account with biotorrents are able to upload new torrents. this is to limit any possible spamming of the website as well as provide accountability for the data being shared. biotorrents enforces this and tracks users by giving each user a passkey. this passkey is automatically embedded within each torrent file that is downloaded from biotorrents and is appended to the biotorrents tracker's announce url. although, we would hope that most users create an account on biotorrents, we still allow anyone to download torrents without doing so. an alternative upload method is provided for more advanced users that have many datasets to share and/or are sharing data from a remote linux based server. this method uses a perl (http://www.perl.org) script that takes the dataset to be shared as input and returns a link to the dataset on biotorrents along with the torrent file; therefore, allowing torrents to be created for numerous datasets automatically. this feature would be useful for institutions or data providers that would like to add a bittorrent download option for their datasets. considering that many datasets in science are often updated, biotorrents allows torrents to be optionally grouped into versions. this functionality allows improved browsing of biotorrents by providing links between torrents. more importantly, this versioning classification allows users interested in certain software or datasets to be notified via a really simple syndication (rss) feed that a new version is available on biotorrents. in addition, this rss feed can be used to obtain automated updates for datasets that are often changing, such as genomic and protein databases. for example, a user could copy the rss feed for a dataset that is being updated often on biotorrents (weekly, monthly, etc.) into their bittorrent rss capable client. when a new version is released on biotorrents the bittorrent client automatically downloads the torrent file, checks to see what parts of the data have changed, and downloads only pieces that have been updated. the speed and effectiveness of the bittorrent protocol depends on the number of peers; in particular, those peers that have a complete copy of the file and can act as ''seeds''. therefore, it is important that individuals or institutions act as seeds to achieve full potential. currently, all newly added data is automatically downloaded and shared from the biotorrents server. this is to ensure that each dataset always has at least one server available for downloading. as the number of datasets and users of biotorrents increases, and to improve on transfer speeds on a geospatial scale (i.e. across countries and continents), we would encourage other institutions to automatically download and share all or some of the data on biotorrents. any logged in biotorrents user can write comments or questions about a particular torrent directly on its details page. this can provide useful feedback both to the creator of the dataset as well as to other users downloading it. alternatively, researchers wanting to discuss more general questions about biotorrents, particular datasets, or science, can use the provided ''biotorrents -forums''. comments and discussion posts can be read by all visitors, but a free account is necessary to post to either of these. users that would like to be updated on newly uploaded datasets can use the biotorrents rss web feed. the rss feeds can be configured for certain categories, license types, users, and search terms, and can also be used with many bittorrent clients to automatically download all or some of the datasets on biotorrents without human intervention. finally, the ''biotorrents -faq'' (frequently asked questions) page provides users with information about bittorrent technology and general help for using biotorrents for both downloading and sharing of data. bittorrent technology can supplement and extend current methods for transferring and publishing of scientific data on various scales. large institutions and data repositories such as genbank [18] , could offer their popular or larger datasets via biotorrents as an alternative method for download with minimal effort. the amount of data being transferred by these large institutions should not be underestimated. for example, in a single month ncbi users downloaded the 1000 genomes (8981 gb), bacteria genomes (52 gb), taxonomy (1gb), genbank (233 gb), and blast non-redundant (nr) (3 gb) datasets; 100000, 30000, 15000, 10000, and 7000 times, respectively (personal correspondence). if bittorrent technology was implemented for these datasets then the data supplier would benefit from decreased bandwidth use, while researchers downloading the data, especially those not on the same continent as the data supplier, enjoy faster transfer times. small groups or individual researchers can also benefit from using biotorrents as their primary method for publishing data. although, these less popular datasets may not enjoy the same speed benefits from using the bittorrent protocol due to the lack of data exchange among simultaneous downloads, the lower barrier of entry to providing data compared with running a personal web server, and the ability to operate behind routers employing network address translation (nat) makes the use of biotorrents for less popular datasets still beneficial. in addition, biotorrents allows researchers to make their data, software, and analyses available instantly, without the requirement of an official submission process or accompanying manuscript. this form of data publishing allows open and rapid access to information that would expedite science, especially for time-sensitive events such as the recent outbreaks of influenza h1n1 [19] or severe acute respiratory syndrome (sars) [20] . no matter what the circumstance, biotorrents provides a useful resource for advancing the sharing of open scientific information. the source code for biotorrents.net was derived from the tbdev.net (http://tbdev.net) gnu general public licensed (gpl) project. the dynamic web pages are coded in php with some features being implemented with javascript. all information, including information about users, torrents, and discussion forums are stored in a mysql database. the original source code was altered in various ways to allow easier use of biotorrents by scientists; the most significant being, that anyone can download torrents without signing up for an account. in addition, torrents can be classified by various categories and license types, and grouped with other alternative versions of torrents. the biotorrents web server along with the source code is available freely under the gnu general public license at http:// www.biotorrents.net. big data: how do your data grow? prepublication data sharing postpublication sharing of data and tools the functional genomics experiment model (fuge): an extensible framework for standards in functional genomics the minimum information required for reporting a molecular interaction experiment (mimix) the minimum information about a proteomics experiment (miape) taking the first steps towards a standard for reporting on phylogenies: minimum information about a phylogenetic analysis (miapa) minimum information about a microarray experiment (miame)-toward standards for microarray data omics data sharing a system for management of information in distributed biomedical collaboratories computational representation of biological systems mimas 3.0 is a multiomics information management and annotation system hypertext transfer protocol -http/1.1. internet engineering task force rfc959, file transfer protocol (ftp) incentives build robustness in bittorrent low extra delay background transport (ledbat) emergence and pandemic potential of swine-origin h1n1 influenza virus the genome sequence of the sars-associated coronavirus the authors would like to thank elizabeth wilbanks and aaron darling for reading and editing of the manuscript, and mike chelen and reviewer andrew perry for suggesting several improvements for the biotorrents website. conceived and designed the experiments: ml jae. performed the experiments: ml. wrote the paper: ml jae. key: cord-353730-owcapg8h authors: dietrich, jes; andreasen, lars vibe; andersen, peter; agger, else marie title: inducing dose sparing with inactivated polio virus formulated in adjuvant caf01 date: 2014-06-23 journal: plos one doi: 10.1371/journal.pone.0100879 sha: doc_id: 353730 cord_uid: owcapg8h the development of new low cost inactivated polio virus based vaccines (ipv) is a high priority, and will be required to eradicate polio. in addition, such a vaccine constitutes the only realistic polio vaccine in the post-eradication era. one way to reduce the cost of a vaccine is to increase immunogenicity by use of adjuvants. the caf01 adjuvant has previously been shown to be a safe and potent adjuvant with several antigens, and here we show that in mice ipv formulated with caf01 induced increased systemic protective immunity measured by binding and neutralization antibody titers in serum. caf01 also influenced the kinetics of both the cellular and humoral response against ipv to produce a faster, as well as a stronger, response, dominated by igg2a, igg2b, and igg2c isotypes as well as ipv specific t cells secreting ifn-γ/il-2. finally, as intestinal immunity is also a priority of polio vaccines, we present a vaccine strategy based on simultaneous priming at an intradermal and an intramuscular site that generate intestinal immune responses against polio virus. taken together, the ipv-caf01 formulation constitutes a new promising vaccine against polio with the ability to generate strong humoral and cellular immunity against the polio virus. poliomyelitis is caused by the polio virus, an rna virus that can colonize the gastroenteral tract which may lead to an acute, viral, infectious disease that spreads from person to person, primarily via the fecal-oral route. in 1988, the world health assembly resolved to globally eradicate poliomyelitis (polio) [1] . the initial objective, the end of polio by 2000, has proven more difficult than originally envisioned and polio still exist in countries such as afghanistan, nigeria and pakistan. however, due to great efforts the number of polio cases has decreased to a level where full eradication within a decade or two is a realistic goal. two vaccines exist against polio; inactivated polio vaccine (ipv) and trivalent live oral polio virus (topv). topv with attenuated sabin strains of poliovirus types 1, 2 and 3, has been the vaccine of choice for polio vaccination in most countries because it induces both systemic and intestinal immunity, can immunize or boost immunity of close contacts through secondary spread, and is inexpensive and easy to administer. however, one problem with opv is that on rare occasions opv can cause vaccine-associated paralytic poliomyelitis (vapp) and/or can revert to a neurovirulent form of poliovirus which is believed to be as transmissible and virulent as wild polioviruses [1] [2] [3] . therefore, steps have been taken to discontinue opv as a vaccine against polio, rendering ipv the only realistic polio vaccine in the post-eradication era. when opv is withdrawn, several challenges concerning ipv have to be dealt with. one such challenge is that the high purchase costs for ipv potentially can lead to limited supplies of ipv in many countries. another challenge concerns the immunity induced by ipv, and how to achieve intestinal immunity with this vaccine. ipv protects the vaccine recipient from paralysis, but compared to opv it provides less protection against re-infection. furthermore ipv does not reduce fecal excretion following reinfection as much as opv because it provides weaker intestinal immunity [4] [5] [6] [7] [8] . there are however studies that demonstrated that ipv can induce some intestinal immunity [5] [6] [7] . one way to reduce the cost of a vaccine is to use adjuvants [9] . in the field of pandemic influenza vaccines the use of adjuvants has permitted dose reduction, increased the availability and reduced cost of the vaccine [10] [11] [12] [13] [14] . therefore, it has been speculated that an adjuvanted vaccine formulation of ipv would reduce cost and also increase the number of available ipv doses worldwide. in support of this, it was recently shown that the potency of sabin inactivated polio vaccines is increased when adjuvanted with aluminium hydroxide or cpg [15, 16] . caf01 is a novel adjuvant composed of cationic liposomes dda (dimethyldioctadecylammonium) stabilized with the synthetic immunomodulator tdb (trehalose 6,69-dibehenate) [17] . caf01 has proven to enhance both humoral and cell-mediated memory immune responses to a number of different experimental vaccine candidates [17] [18] [19] in preclinical models. caf01 has furthermore already been tested in three phase-i trials with an excellent safety and immunogenicity profile (eag, personal communications and [20] [21] [22] ). additionally, caf01 was also found to provide dose-sparing when used in a combination with the trivalent influenza split vaccine in ferrets [23] . we therefore tested the suitably of the caf01 adjuvant for inducing protective immunity against an infection with polio virus. it is well known that mucosal immunity can be achieved through vaccination at the mucosal site in question and that a vaccine given by the intramuscular route primarily produce systemic immunity [24, 25] . mucosal sites are not isolated immunological sites, and priming of an immune response at one mucosal site may also induce protection at another mucosal site [26] . although the skin is not traditionally regarded as a mucosal site, previous results from other groups have nevertheless indicated that an intradermal administration may confer intestinal/mucosal protection [27, 28] . in addition, intradermal administration is a well-known delivery route for non-adjuvanted ipv vaccines in humans, and has been shown to be a safe delivery route with no adverse effects [29, 30] . we therefore decided to test different vaccine strategies involving both intradermal and intramuscular administration of caf01 adjuvanted ipv in order to achieve intestinal immunity with an ipv vaccine. taken together, the aim for the present work was twofold: 1) to achieve dose sparing with caf01, measured by neutralization titers, and 2) to induce intestinal immunity against ipv with an ipv-caf01 vaccine. studies were performed with 6-to 8-wk-old female cb6f1 c57bl/6xbalb/c mice from harlan, scandinavia. animals were housed in appropriate animal facilities at statens serum institut. the ipv vaccines applied throughout this study was commercial gmp grade material manufactured at statens serum institut (ssi). the trivalent ipv was formulated in the ratio 40:8:32 for polio virus type 1 (brunhilde strain, ipv1), type 2 (mef-1, ipv2) and type 3 (saukett, ipv3), respectively. the trivalent ipv for the vaccines was in this study always formulated to a trivalent stock, ipv tp (400:80:320 du/ml), from individual concentrated bulk materials of ipv1, ipv2 and ipv3. monovalent ipvs were stored in m199 buffer. dilution of ipv to the respective concentration used for immunization was performed with 10 mm tris buffer, ph 7.6. the trivalent stock ipv tp of 400:80:320 du/ml was diluted to the appropriate concentration by adding the ipv stock solution to a given volume of buffer. indicated dosage units in the all experiments correspond to ipv1 d antigen units and all vaccine are trivalent unless otherwise stated. the caf01 adjuvant suspension was manufactured at two lipid concentrations: 3 mg/ml (caf01 2500/500 mg of dda and tdb) and 12 mg/ml (caf01 10000/2000), respectively. dda and tdb were manufactured by niels clauson-kaas a/s, farum, denmark. in brief, a lipid film for 80 ml caf01 10000/2000 was formed by dissolving dda (0.80 g) in 10 ml chloroform/ methanol (9:1, v/v) and mixing it with tdb (0.16 g) dissolved in 10 ml chloroform/methanol (9:1, v/v) in a 100 ml bluecap flask. the organic solvent was removed using a gentle stream of n 2 forming a thin lipid film at the bottom of the flask. the lipid film was dried under vacuum over night to remove trace amounts of the organic solvent. the lipid film was hydrated 80 ml 10 mm tris-buffer, ph 7.6 by high speed high-shear mixing (heidolph silent crusher m) at 60-80uc for 15 minutes. adjuvants were stored at 2-8uc until use. for the formulation of caf01 2500/ 500, the same procedure is applied, with smaller amounts of dda and tdb, 0.20 g and 0.04, respectively in 80 ml. all adjuvants were stored at 2-8c until use. the ipv-caf01 vaccines were formulated by add-mixing 1:1 v/v of an ipv solution into a solution of caf01 under moderate stirring using magnetic stirring bars. the vaccines were rested for 15 minutes to allow for adsorption of ipv to the cationic caf01 liposomes. for ipv-caf01 vaccines intended for mice, the caf01 10000/ 2000 mg was applied in order to formulate a mouse dose caf01 (250/50 mg) into 50 ml dose volume for im vaccination after 1:1 v/v mixing with ipv antigen solution. caf01 2500/500 was applied to formulate vaccines for physic-chemical analyses. all vaccine formulations were performed in laf units. all vaccines were stored at 2-8c until use. manufacturing of both the caf01 adjuvants, ipv vaccines and ipv-caf01 vaccines was performed in laf units. the final concentration of caf01 in all the mouse studies was 250/50 ug dda/tdb. mice were immunized two or three times at two-week intervals intramuscularly (im in the caudal thigh) with 50 ml ipv or ipv formulated with caf01. alternatively the mice were immunized with 50 ml via the intradermal (id, on the back) route. the ipv doses used in the experiments are indicated in the figures. two weeks after the second or third vaccination the immune response against ipv was analyzed. in some experiments the immune response was also analyzed 8 and 11 weeks after the second vaccination. lymphocytes were cultured and purified as described previously [31] . briefly, pbmcs were purified on a density gradient and splenocyte cultures were obtained by passage through a cell strainer (bd pharmingen). ipv for stimulation were all used at 2 mg/ml. supernatants from triplicate cultures (2610 5 cells per well) were harvested from cultures after 72 h of incubation for the investigation of cytokines. a sandwich elisa was used to determine the concentration of ifn-c in culture supernatants as previously described [31] . alternatively, the cytokines were analyzed by multiplex cytokine assay. the th1/th2 cytokine 9-plex assay and the il-17 singleplex assay (meso scale discovery, gaithersburg, md) were performed according to the manufacturer's instructions. the plates were read on the sector imager 2400 system, and calculation of cytokine concentrations in unknown samples was determined by 4parameter logistic non-linear regression analysis of the standard curve. ipv d-antigen elisa (du elisa) ipv d-antigen was quantified using a sandwich elisa. the du elisa detects the d-unit activity of ipv. in brief, immuno plates (maxisorp, nunc) were coated with polyclonal antibodies (raised in rabbits by immunization with inactivated poliovirus) of type 1 (brunhilde strain), type 2 (mef strain), or type 3 (saukett strain)) against polio type 1, 2, or 3 diluted in pbs with phenol-red for 2-3k hours in a humidified atmosphere at room temperature. after washing and blocking of the wells, standards and samples diluted in pbs containing nacl, kcl, triton x-100, bsa and phenol-red were added to the wells followed by an overnight incubation in a humidified atmosphere at 2-8uc. the wells were washed and subsequently hrp-conjugated polyclonal antibodies against polio type 1, 2, or 3 diluted in fbs with phenol-red were added to the wells and incubated for 2 hours in a humidified atmosphere at room temperature. the wells were washed and opd substrate was added followed by a 30 minute incubation period in the dark at room temperature. the reaction was stopped by the addition of h 2 so 4 and plates were read at od 490 nm with non-specific absorption at 630 nm subtracted. briefly, neutralizing antibody titers to the three poliovirus serotypes 1, 2 and 3 were measured using brunhilde, mef-1, and saukett virus stocks on a vero cells. dilutions of the sera in 50 ml were made in duplicate starting at 1:8 with serial 2-fold dilutions. 50 ml of approximately 100 ccid 50 (cell culture infectious dose 50%) of poliovirus type 1, 2 or 3 was added to each well (in a 96 well plate), and incubated for 4k-6 hours at 37 (36) (37) (38) uc and 5% co 2 , and at 2-8uc till the next day. vero cells were prepared as a cell suspension of 6610 4 cells/ml and 50 ml are added to each well containing the polio virus/serum mixture. plates were incubated at 37uc and 5% co 2 for 7 (6-8) days. the wells showing neutralizing/cytopathogenic activity are recorded. the result from a single dilution series is given as 1/!2 times the lowest dilution factor with dead vero-cells and the final titre calculated as the geometric mean of the results from two independent dilution series. samples were tested in triplicate using a standard microneutralization assay for antibodies to poliovirus types 1, 2, and 3 according to established protocols at the global polio specialized laboratory, centers for disease control and prevention (cdc). briefly, 80-100 ccid50 of each poliovirus serotype and two-fold serial dilutions of serum were combined and pre-incubated at 35uc for 3 hours before addition of hep-2(c) cells. after incubation for 5 days at 35uc and 5% co2, plates were stained with crystal violet and cell viability measured by optical density in a spectrophotometer. each specimen was run in triplicate, with parallel specimens from one study subject tested in the same assay run, and the neutralization titers estimated by the spearman-kä rber method and reported as the reciprocal of the calculated 50% endpoint. each run contained multiple replicates of a reference antiserum pool starting at a 1:32 dilution to monitor performance variation. a serum sample was considered positive if antibodies were present at $1:8 dilution. specimens with antibody titers ,1:8 were considered seronegative and specimens with titers .1:8 were considered seropositive. mice were bled for the collection of serum following the vaccination 1-3. maxisorp micro titer plates (nunc, maxisorp, roskilde, denmark) were coated with ipv tp (trivalent inactivated polio virus, see materials and methods) or ipv type 1, 2 or 3 (1 ug/ml) in pbs over night at 4uc. free binding sites were blocked with 2% skimmed milk in pbs. individual mouse sera were analyzed in duplicate in five-fold dilutions in pbs containing bovine serum albumin starting with a 100-fold dilution. horseradish peroxidase (hrp)-conjugated secondary antibodies (rabbit anti-mouse igg, igg1, igg2a, igg2b, igg2c and iga; zymed) diluted 1/2000 in pbs with 1% bovine serum albumin were added. after 1 h of incubation, antigen-specific antibodies were detected by tmb substrate as described by the manufacturer (kem-en-tec, copenhagen, denmark). to stop the reaction, 100 ul of 4 n h 2 so 4 was added, and the optical density (od) was measured at 450 nm. the absorbance values were plotted as a function of the reciprocal dilution of serum samples. fecal pellets were collected from mice two weeks following each immunization. the mice were placed in individual cages and fresh fecal pellets (5-6 pellets per mouse) were collected into microfuge tubes containing 600 ml of ice-cold buffer: pbs with soybean trypsin inhibitor (sigma; 0.1 mg/ml), bovine serum albumin (bsa; 1% w/v), ethylenediaminetetraacetic acid (edta; 25 mm), glycerol (50% v/v) and phenylmethylsulfonylfluoride (pmsf; 1 mm). the fecal pellets were broken up to form a suspension and then incubated on ice for 4 hours. after incubation, fecal pellet solutions were clarified by centrifugation at 15,500 g for 10 minutes at 4uc, and the supernatants transferred to microfuge tubes that had been blocked overnight with pbs containing 1% (w/v) bsa. supernatants were then frozen and analysed at a later date by elisa. the z-average diameter, z avg , of the liposomes was determined by dynamic light scattering using the photon correlation spectroscopy (pcs) technique. the measurements were performed at 25uc using a malvern nanozs (malvern instruments, worcestershire, uk) equipped with a 633 nm laser and 173u detection optics. malvern dts v. 5.10 software was used for data acquisition and analysis. nanospheretm size standards 60 nm (duke scientific corp., duke, nc) was used to verify the performance of the instrument. the samples were diluted with 10 mm trisbuffer at ph 7.4 to achieve the optimal vesicle concentration. surface charge on the vesicles was measured indirectly via analysis of zeta-potential at 25uc using a malvern nanozs (using monomodal analysis model) (malvern instruments, worcestershire, uk) of a 1/10-1/400 dilution in milli-q water. a significant difference in immune responses measured by elisa, or a difference in neutralization titers, was evaluated using a one-way anova, and tukey's multiple comparison test for multiple comparisons. a value of p,0.05 was considered significant. prism version 5 software (graphpad) was used for analysis. the trivalent ipv was analyzed with or without caf01. the trivalent ipv vaccine was formulated from individual ipv1, ipv2 and ipv3 materials. both ipv3 and the stock ipv tp showed a z avg (hydrodynamic diameter) of 35 nm. the z avg of 35 nm is in accordance with values for polio virus size determined by electron microscopy (30 nm) indicating that the size did not change upon mixing of three ipv monovalents into a trivalent ipv (fig. 1a) . furthermore, the analysis of ipv content measured as d-unit activity confirmed that ipv d-unit activity was maintained after mixing of ipv1-3 into ipv tp (fig. 1b) . on formulation with caf01, the particle size distribution for an ipv-caf01 vaccine (z avg, d.nm ) was found to be 267 nm compared to 203 nm for the caf01 adjuvant. this indicated a slight increase in particle size on ipv binding to the caf01, which however was within an acceptable range. we also measured the zeta potential of ipv, caf01 and ipv-caf01 formulations (fig. 1c) . the zeta potential for the cationic caf01 of 80 mv was in accordance with previous observations [32] . monovalent ipv showed a slightly negative zeta potential (2 13 mv), whereas the adjuvanted ipv-caf01 vaccine showed a positive zeta potential of 54 mv. therefore, in ipv vaccines formulated with caf01, a binding between antigen and adjuvant is electrostatically favorable. to measure the degree of association between caf01 and ipv we assumed that since caf01 is a suspension of cationic liposomes, it is possible to separate an ipv-caf01 vaccine formulation into a vaccine pellet fraction and a supernatant fraction by centrifugation, and subsequently measure ipv (by du elisa) in both fractions. ipv bound to caf01 will be expected to be located in the vaccine pellet. three mono-ipv-caf01 vaccines were formulated to evaluate the binding of each ipv type 1-3. the results of the du elisa from centrifuged samples are shown in fig. 1d . no ipv was found in the supernatant fraction of the three mono-ipv-caf01 formulations, which indicated that all ipv serotypes are indeed bound to caf01. this was in agreement with the zeta potential analysis that suggested a favorable interaction of caf01 and ipv. on analyzing the resuspended vaccine pellet a recovery rate of 30-75% ipv was obtained (fig. 1d) . the reason for a recovery of less than 100% is most probably due to steric hindrance (caused by caf01) of the binding of the detecting antibodies in the ipv du elisa to ipv. however, we cannot fully exclude that some ipv was partly inactivated in the caf01 formulation. we next examined the ipv-caf01 formulation in mice. the first objective was to determine the ability of caf01 to increase the neutralizing antibody titer against polio virus in the blood of vaccinated animals. based on neutralization titers obtained from mice vaccinated with a range of doses from 30 du (d-units) to 0.1 du (data not shown) we choose 20 du as a full mouse dose and 2 du as the dose formulated into the caf01 adjuvant (indicated dose units in the all experiments correspond to polio virus type-1 d antigen units). thus, the aim was to achieve 10 times dose sparing with caf01. mice were immunized with 20 du ipv or with the lower 2 du ipv, the latter with or without caf01. the mice received two immunizations spaced by two weeks and neutralizing antibody titers were determined in the sera two weeks after the last immunization. three independent experiments were performed and two assays (performed at statens serum institut, ssi, and center for disease control, atlants, cdc) were used to determine the neutralization titers. figure 2 represents all three experiments, whereas the individual experiments are shown in figure s1 . the results showed that caf01 induced a significant increase in neutralization titers to all serotypes (type 1, type 2 and type 3, p,0.05) (fig. 2) . neutralization titers to type 1 increased from 7.1+/21.8 (log2 transformed titers) in the 2 du ipv group to 10 (fig. 3) . however, two vaccinations with ipv 2 du + caf01 gave neutralization titers that were not statistically different from 3 vaccinations with ipv 2du, although there was a trend towards higher titers against type 1 in the ipv alone group that received three vaccinations (fig. 3) . in conclusion, addition of the adjuvant caf01 allowed for a 10 times dose sparing. moreover, only two vaccinations with the caf01 formulated vaccine were required to achieve the same neutralization titers as observed after three vaccinations with nonadjuvanted ipv. we also examined if the observed increase in neutralization titers correlated with an increase in the antibody binding titer. two weeks after the second vaccination, sera from individual mice were analyzed for ipv specific antibody titers measuring both the different ipv specific igg isotypes (igg1, igg2a, igg2b, igg2c), as well as the total igg level against the three virus serotypes. in the ipv 20 du group we observed higher igg titers than in sera from the ipv 2 du against trivalent ipv, or against serotypes 1 and 2. binding titers against type 3 were not different between the ipv 20 and 2 du groups. importantly, the titers in sera from mice vaccinated with ipv 2 du + caf01 were higher than both the ipv 2 du group against all the serotypes, and at least as high as the ipv 20 du group (fig. 4a) . the igg isotypes responsible for the binding titer observed in figure 4a was also examined. the results showed that caf01 led to increased antibody titers for the igg isotypes igg2a, igg2b, and igg2c (fig. 4b) . the increased igg titer observed in the caf01 group was also observed 5 and 8 weeks following the second vaccination, demonstrating that it was not merely a transient increase (fig. 4c ). to further characterize the ipv-caf01 formulation we also analyzed the cellular response against ipv. cellular immunity towards polio virus has received less attention due to compelling evidence that humoral immunity is the most important effector mechanism against polio. however, there are numerous examples that cellular immunity, besides being an effector mechanism in itself, can be important for humoral immunity [33] [34] [35] [36] [37] [38] [39] , even humoral immunity against polio virus [40, 41] . the cellular response was characterized by analyzing the cytokine profile of ipv-specific t cells induced by vaccination with ipv alone or ipv adjuvanted with caf01. pbmcs were obtained from mice vaccinated with 2 or 20 du ipv alone or ipv 2 du + caf01 two and five weeks following the booster vaccination. non-vaccinated mice were included as control. thereafter, the cells were stimulated in vitro with ipv for 72 hours and the supernatant was collected for multiplex cytokine analysis. at week 2 post vaccination, in the ipv non-adjuvanted group, multiplex cytokine analysis showed very low responses for both doses (fig. 5a) . the highest response was observed with il-2 (5256165 and 667665 pg/ml in the ipv 2 du and 20 du groups, respectively). in contrast, in the ipv 2 du + caf01 group we observed a significant increase for the cytokines ifn-c, il-17, il-5, il-10, il-2 and tnf-a but not il13. in particular, at week 2 post vaccination caf01 led to strong induction of ifn-c (2379+/2490 pg/ml versus 153+/286 pg/ml in the ipv 2 du group) and il-10 (5024+/2927 pg/ml versus 232+/2138 in the ipv 2du group). at week 5 post vaccination, in the ipv alone groups we observed an increased secretion of cytokines ifn-c (to 2536+/2 443 pg/ml in ipv 2 du mice and 5018+/2394 pg/ml in the ipv 20du group) and il-2 (928+/235 pg/ml in the ipv 2du mice and 1046+/2255 pg/ml mice). in the ipv 2 du + caf01 we observed an increase in ifn-c (to 7492+/21896 pg/ml) and il-2 (to 2432+/2383 pg/ml), but a significant (p,0.05) decrease in secretion of il-17, il-5, il-10, and tnf-a compared to week 2 post vaccination. thus, at week 5 the major secretion of cytokines was observed with ifn-c and il-2 in all the groups with the strongest responses observed in the caf01-adjuvanted group (fig. 5) . taken together, the use of caf01 induced a faster and stronger cellular response compared to non-adjuvanted ipv. interestingly, the cytokine profile of the caf01 group changed from week 2 to week 5 to resemble a th1 profile similar to the profile induced by ipv alone (fig. 5) . shedding of polio virus is considered the principal marker for protection and studies have implicated intestinal iga as one of the effector mechanisms to reduce shedding of virus [42] . we therefore examined different vaccine strategies with caf01 with the aim of generating intestinal iga. more specifically, as previous experiments have indicated that an intradermal vaccine administration may induce intestinal iga [27, 28] we decided to first compare intramuscular (im) and intradermal (id) administration. we first examined the iga production in the intestine two weeks following two vaccinations with either ipv 2 du + caf01 given either id or im. to avoid the risk of blood contamination of the intestinal samples we only examined fecal pellets taken from the colon of sacrificed animals. neither of the administration forms induced intestinal iga (fig. 6a) . in contrast, both vaccine strategies induced igg in the serum with the im administration being the highest igg inducer (fig. 6b) . this correlated well with the serum neutralization titers (fig. 6c) . thus, ipv-caf01 induced a significant increase in titers compared to both the ipv id group and the ipv im group. this was observed with all the virus serotypes. furthermore, the id administration gave a reduced binding and neutralization titer compared to im administration, suggesting that the id administration is less efficient at priming a protective humoral immune response compared to the im administration. we next decided to test a strategy combining im administration and id administration. of practical importance the animals received 50 ml of the same vaccine in the im and id dose (in contrast to two individually formulated vaccines). as booster vaccination the mice were given an im administration (or another id+im co-administration, data not shown). the results showed that supplementing an id administration with an im administration in the first vaccination induced a strong increase in the iga titer in the feces (fig. 7a) . in contrast, we did not observe increased fecal igg levels (fig. 7b) . we next examined if the id+im administration also affected the systemic humoral or cellular response. at week 2 post the final vaccination the animals were sacrificed and blood was analyzed for igg binding titers, neutralization titers, and for cellular immunity. igg titers in both the adjuvanted groups were increased compared to the non-adjuvanted ipv 2du group and reached levels comparable to the ipv 20 du group (fig. 7c) . cellular immunity was measured by ifn-c elisa on supernatants from pbmcs stimulated with ipv in vitro. the results showed that the ifn-c response in the ipv 2 du + caf01 id+im group was as high (6071+/21404 pg/ml) as in the ipv 2 du + caf01 im group (4960+/21029 pg/ml) and significantly higher than in the non-adjuvanted group (fig. 7d) . finally, the neutralization titers in the ipv 2 du + caf01 id+im group was as high as in sera from the ipv 2 du + caf01 im group, and significantly higher that the non-adjuvanted ipv 2 du group (fig. 7e) caf01 id+im group were even significantly higher than titers from the ipv 20 du group demonstrating a dose sparing effect of more than 10 for the ipv 2 du + caf01 id+im group. taken together, compared to im administration a side by side id+im administration with a caf01-adjuvanted vaccine followed by an im administration did not negatively affect systemic immunity, measured by antibody binding and neutralization titers and t cell ifn-c secretion (fig. 6) . importantly, in contrast to the ipv 2 du id or ipv 2 du im groups, in the ipv 2 du id+im group we observed a significant increase in fecal iga whereas fecal igg was not observed in either of the vaccine groups. as the id+im group received twice the antigen amount in the first vaccination, we also tested a group that received two im vaccinations at the same time (at two different sites). however, this did not lead to fecal iga (data not shown). finally, we did not observe any significant adverse effects at the id site of administration. the primarily goal of the present study was to examine if the adjuvant caf01 could be used to achieve dose sparing with ipv, and a subsequent goal was to achieve intestinal immunity with an ipv-caf01 vaccine. it is well known that a prerequisite for worldwide replacement of opv with ipv is the availability of affordable ipv in low and middle-income countries. lower cost options being pursued include reducing the number of doses and the amount of antigen per dose [15, 16, 43, 44] . in addition, lowering the manufacturing cost for ipv in developing countries is a priority. our results showed that with the adjuvant caf01 we were able to achieve a 10 times dose sparing effect in mice measured by the virus neutralization titers in the blood (fig. 2) . importantly, the results also showed that two vaccinations with ipv 2 du + caf01 gave neutralization titers that either equaled those induced by 3 vaccinations with ipv 2 du (type 2 and 3 titers), or were slightly reduced (type 1 titers) (fig. 3) . thus, addition of the adjuvant caf01 induced a significant dose sparing effect, or allowed for a vaccine schedule with one less dose. the potential of applying only two vaccinations compared to three will have significant economical and practical impact. presently, ipv vaccination schedules involve up to 5 doses commonly administered at week 6, 10, 14 and at month 9 and 15. a recent report concluded that children in resource-poor settings probably are protected against paralytic poliomyelitis with as few as two doses of ipv in primary vaccines [45] . the study was based on ipv vaccines given at month 6 and 9, i.e. when maternal transferred antibodies have declined and consequently were not expected to inhibit the ipv vaccines. in addition, children receiving a previous opv vaccination were protected with just a single ipv dose most probably due to boosting of a prominent opv induced memory b cell response [45] . thus, at least with a booster vaccine, the study indicated that one or two vaccinations should protect children in resource-poor settings against paralytic poliomyelitis [46] . our results indicated that the inclusion of an adjuvant will affect both the required dose (allowing for a 106 reduced ipv dose) and the number of vaccinations (allowing for a vaccine schedule with less vaccine doses) required to induce proper protection against infection with polio virus. the response to ipv and several other vaccines are highly sensitive to the maternal antibody level [47] [48] [49] [50] [51] [52] . there were significantly lower rates of seroconversion for all three poliovirus types in a study performed in thailand and for types 1 and 3 in an oman study including babies from birth and 24 weeks onwards and with children with baseline maternal antibody titres of $64. in addition, previous studies have shown that ipv appears to be more sensitive than opv to the effect of high maternal antibody titers on reducing the neutralizing antibody response to vaccine given in the first months of life [28] [29] [30] [31] [32] . however, previous studies have highlighted two important points concerning the antibody response against ipv. one study showed that maternal antibodies inhibited vaccine-induced antibody responses but not t cell responses, and that the latter allowed for high antibody titers following a booster vaccine [53] . in addition, krishnan et al. [54] showed that the negative effect of maternal antibodies on the response to ipv may be reduced by increasing the intervals between doses. taken together, and in combination with our results, we suggest that with the inclusion of an adjuvant, such as caf01, combined with a booster vaccine given at a longer interval than the commonly used 4 weeks (e.g. 8 weeks as proposed previously [54] ), a 5-10-fold reduction of the ipv vaccine dose is indeed feasible, even in the presence of maternal antibodies. we also characterized the influence of caf01 on the immune response against ipv. firstly, the antibody binding titers at week 2 post vaccination 2 were increased to levels that in most cases were higher than the levels induced by a 10 times higher ipv dose (20 du) (fig. 4) . this was observed against all three polio virus types (fig. 4a) . interestingly, in contrast to for igg2a, igg2b, and igg2c, we only observed a minor igg1 response in both adjuvanted and non-adjuvanted groups (fig. 4b) . in contrast, previous observations with the caf01 adjuvant formulated with recombinant proteins showed a strong igg1 response [55] . this suggested that even in the presence of a strong adjuvant, ipv can influence the igg response. as the igg isotype profile of the ipv-caf01 vaccine resembled that of ipv alone, this suggested that it is ipv that determines the igg profile even in the presence of caf01. the increased antibody response induced by caf01 also correlated with an increased cellular response measured by multiplex cytokine analysis. although the cellular immune response is not considered a direct correlate of protection for polio vaccines it may play an indirect role by supporting a memory b cell response [56, 57] . our analysis of anti-ipv t cell immunity revealed that caf01 induced both a faster and stronger response against ipv. at week 2 post vaccination only a minor response was observed in the non-adjuvanted ipv groups in contrast to the caf01-adjuvanted groups that showed increased levels against all the cytokines tested (fig. 5a) . the caf01 adjuvanted group also showed elevated cytokine responses at week 5 post vaccination 2. interestingly our results also showed that the cytokine profile of the ipv-caf01 group changed significantly from week 2 to week 5 to resemble the profile induced by ipv alone, a profile dominated by ifn-c and il-2 secretion (fig. 5b) . this indicates that ipv as an antigen can also influence the type of cellular immunity induced against it. furthermore, as ifn-c is known to induce isotype switch to igg2a, these results also explains the observed induction of igg2a, and the lack of igg1. thus, both regarding the igg response and the cellular t cell response the role of caf01 in the formulation with ipv is to induce a response that is quantitatively different from the response induced by ipv but not qualitatively different. the observed t cell profile presented in our study is in agreement with a previous study showing the same cytokine profile following immunization with sabin ipv +/2 alum [40] . interestingly, in that study it was also demonstrated that the t cells were required for the protection against a polio infection, most probably by acting as helper t cells for b cells, and through the stimulation of neutralizing antibody production of the igg2a type. in addition, cd4 t cells have also been shown to be required for the generation of optimal antibody responses following infection with coronavirus [33] , vaccinia virus [34, 35] , yellow fever virus [36] or vesicular stomatitis virus (vsv) [37] , supporting that the role of t cells against a viral infection such as polio virus should not be underestimated. we speculate that t cell immunity mediated by the ifn-c/il-2 expressing th1 t cells may be important for the protection against the polio virus due to their ability to 1) induce isotype switching to igg2a and 2) induce innate immunity to contributes to antiviral immunity [40, 41, 58] . it is accepted that the major port of entry for the polio virus is the intestinal tract, and that fecal iga is important in preventing entry of the pathogen [42, 59] . therefore, inducing intestinal iga is a priority of any polio vaccine. several publications indicate an immunological connection between the intradermal and intestinal site [27, 28] . however, in humans, the results obtained with id administration of ipv are not entirely clear. thus, one large-scale pediatric study conducted in cuba by the who initiative for vaccine research [60] failed to meet non-inferiority criteria, whereas another showed that three immunizations with one-fifth of the dose of ipv given id resulted in similar seroconversion rates compared to three full doses of ipv given im in infants [61] although the antibody were significantly lower in infants given the decreased id dose of ipv [61] . thus, the usefulness of the id administration route for ipv is still being debated. the results presented in this paper indicated that as an injection site to prime an immune response, the intradermal administration is not as efficient as the im administration in mice. this was based on experiments showing reduced binding/neutralization titers in the id group compared to the im group, receiving the same vaccine (fig. 6 ). in addition, the id administration did not induce iga in the intestine or in fecal samples ( fig. 6 and data not shown) . however, compared to im or id alone administration, a side-byside id+im administration with a caf01 adjuvanted vaccine followed by an im administration induced significant levels of fecal iga and, of importance, without compromising serum binding and neutralization titers. although a subject for future investigations, it may be suggested that t cells primed systemically by ipv-caf01 can be recruited to other sites, e.g. the intestine, and act as helper t cells to potentiate the response at these sites. this may be of some importance as it suggests that a strong intestinal priming may not be entirely required for intestinal protection as long as an intestinal challenge can effectively boost a pre-existing minor intestinal immunity and/or lead to fast recruitment of immunity residing in other (systemic) locations. in fact, in this respect it is of particular interest that some studies have shown that ipv (administered via the im route) may indeed 'prime' substantial protective intestinal immunity [7] . furthermore, in tropical developing countries, a supplemental dose of ipv administered to children previously exposed to opv boosts humoral and intestinal immunity more effectively than a supplemental dose of opv [5] [6] [7] 45, 62] , which is supported by a study performed in the netherlands [63] . thus, it seems reasonable to assume that an im vaccination with ipv will induce immunity with some ability to interact with the intestinal site and that a simultaneous priming at both the im and id site with the same vaccine will increase the intestinal response. in conclusion, we have for the first time characterized the formulation of ipv with the caf01 adjuvant. the formulation proved to constitute a stable formulation where the majority of ipv was associated with caf01 and maintained its d-unit activity. ipv-caf01 induced a strong systemic protective immunity in mice and also influenced the kinetics of the ipv immune response to produce a faster response. finally, a simultaneous priming at an intradermal and an intramuscular site with the ipv-caf01 vaccine led to intestinal immunity. caf01 has successfully completed several phase i studies with other vaccine candidates, and further studies are now ongoing to support the development the ipv-caf01 towards a human trial. figure s1 virus neutralization titers from 3 individual experiments. virus neutralization antibody titers from mice (n = 8/ group) 14 days following the second or the third immunization with inactivated polio vaccine (with or without adjuvant). 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titers. thanks to dr morten ruhwald for critically reading the manuscript. we thank center for disease control, atlanta (cdc) for performing polio virus neutralization assays.